Marc Mr

Litaudon

Christiane Mme

Garbay

Sylvie Mme

Michel

M Chavalit

M Uthai Sotanaphun

Michel Koch

Brigitte Deguin

Marie-Christine Lallemand

Hanh Dufat

Sabrina Boutefnouchet

Marina Kritsanida

Raphaël Grougnet

François-Hugues Poree

Thomas Gaslonde

Chouaha Bouzidi

Maryse Rouxel

Oumar Kante

Olivier Hermet

Elise Prost

Keywords: Dipterocarpaceae, Hopea odorata, Dipterocarpus costatus, terpenoids, cytotoxicity, 3, 4-seco-cycloartanes, lupane, norlupane, dammarane, nordammarane, 2, 3seco Chrysophanol Antimicrobial [76] Genus Cotylelobium, Hopea, Cotylelobium, Hopea, Takhian thong, Takhian

Preliminary study of the extracts from Hopea odorata leaves and Dipterocarpus costatus wood showed that the hexane extracts of both plants exhibited cytotoxicity against breast cancer (MCF-7) and small cell lung cancer (NCI-H187), in conjuction with potent anti-malarial (against Plasmodium falciparum K1 strain) activity of D.costatus wood hexane extract.

Phytochemical studies of H. odorata leaves hexane extract led to the isolation of 16 terpenoids in the series lupane (n = 8), 3,4-seco-cycloartane (n = 4), friedelane (n = 2) and oleanane (n = 2). Among lupanes, 3,30-dioxolup-20(29)-en-28-oic acid was isolated for the first time from a natural source. Cytotoxicity of lupanes triterpenes against four human cancer cell lines (PC3, MDA-MB-231, HT-29 and HCT116) was evaluated and showed the structure-activity relationship related to the oxidation degree at position 3, 28 and 30. Among cycloartanes two new 3,4-seco-cycloartanes were identified. They are saturated fatty acid esters of (24S,25S,26)-trihydroxy-3,4-seco-cycloart-4(29)-en-3-oic acid.

Isolation of D. costatus wood hexane extract discovered 30 terpenoid of which 12 triterpenes are new, including 5 norlupanes, 3 dammaranes, 2 nordammaranes and 2 secodammaranes. The biological activities of all isolated compounds were evaluated through cytotoxicity against human cancer cell lines mentioned above and rat myoblast-derived cells L-6, as well as antimalarial activity against Plasmodium falciparum FcB1 strain. Interestingly, the norlupane 36, possessing an endoperoxide group, showed a strong antiplasmodial activity associated with low cytotoxicity.

LIST OF TABLES

2 Discovery of lupane-type triterpenes in some plants. [START_REF]Are the number of cancer cases increasing or decreasing in the world? Accessed[END_REF] Cytotoxicity of betulonic acid against various cell lines 4

Cytotoxicity of other lupane-type triterpenes 5

Anti-inflammatory activity of some lupanes (effect on NO and PGE2 production)

6

Anti HIV-1 activity of some lupanes 7

Discovery of dammarane-type triterpenes in some plants. [START_REF] Saroyobudiono | Oligostilbenoids from Shorea gibbosa and their cytotoxic properties against P-388 cells[END_REF] In vitro antiviral activity of dammaranes. 9

In vitro bioactivity assays of H.odorata leaves extract 10

Fractionation and isolated compounds from a small portion of H.odorata leaves hexane extract.

11

Fractionation of H.odorata leaves hexane extract isolation by MPLC. [START_REF] Sahidin | Cytotoxic properties of oligostilbenoids from the tree barks of Hopea dryobalanoides[END_REF] Fractionation of Fraction B23 isolated by MPLC. [START_REF] Syah | Two oligostilbenes, cis-and trans-diptoindonesin B, from Dryobalanops oblongifolia[END_REF] Cytotoxic activity against HCT116, HT29, MDA-MB231 and PC3 cell lines.

14

The 1 H NMR data for (S)-MTPA and (R)-MTPA ester of compound 18a.

15

The 1 H-NMR (400 MHz) and 13 C-NMR (75 MHz) data of 1 and 4 (in CDCl 3 )

16

The 1 H-NMR (400 MHz) and 13 C-NMR (75 MHz) data of 2 and 6 (in CDCl 3 )

17

The 1 H-NMR (400 MHz) and 13 C-NMR (75 MHz) data of 3, 7 and 8 (in CDCl 3 )

18

The 1 H-NMR (400 MHz) data of 5, 10, 11, 12 and 13 (in CDCl 3 ) 19

The 1 H-NMR (400 MHz) data of 14, 16 and 17 (in CDCl 3 ) 20

The 13 C-NMR (75 MHz) data of 5, 10, 11, 12, 13, 14, 16 and 17 (in CDCl 3 )

21

The 1 H-NMR (400 MHz) and 13 C-NMR (75 MHz) data of 9 (in CDCl 3 )

22

The 1 H-NMR (400MHz) and 13 C-NMR (75 MHz) data of 15 (in CDCl 3 [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] The 1 H-NMR (300 MHz) of 18, 18a, 18b and 19 (in CDCl 3 ). 24

The 13 C-NMR (75 MHz) of 18, 18a, 18b and 19 (in CDCl 3 ). 25

The 1 H-NMR (300 MHz) and 13 C-NMR spectrum (75 MHz) The 1 H-NMR (400 MHz) and 13 C-NMR spectrum (75 MHz)

The 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz) of 45 (in acetone) 37

The 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz) of 29, 32 and 38 (in CDCl 3 )

38

The 1 H-NMR (300 MHz) of 30, 34, 37 and 47 (in CDCl 3 ) 39

The 13 C-NMR (300 MHz) of 30, 34, 37 and 47 (in CDCl 3 ) 40

The 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz) of 27, 28 and 35 (in CDCl 3 )

41

The 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz)

LIST OF FIGURES

Structure of some reported bioactive terpenoids and phenolic compounds in the family Dipterocarpaceae. 9 2 Some reported lupane-type triterpenes structures. 18 3 Some reported dammarane-type triterpenes structures. 27 4 Two Mosher conformers showing the (S)-MTPA and (R)-MTPA derivatives proposed by Mosher.

5

The (S)-MTPA ester presenting the H A,B,C,X,Y,Z instead of group R 1 and R 2 . 31 6

Some tetrazolium salts for cell proliferation and cell viability measurement. 13 C NMR (75 MHz) spectrum of 2 and 6 (in 200 75 The expanded HMBC correlation of 22 and 31 (in CDCl 3 ) and expanded spectrum in the range of δ H 0.7-1.8 ppm and δ C 15-58 ppm.

76

The 1 H NMR spectrum (300 MHz) of 24 (in CDCl 3 ) and expanded spectrum in the range of 0.7-2.1 ppm.

77

The 13 C NMR spectrum (300 MHz) of 24 (in CDCl 3 ) and expanded spectrum in the range of 13-56 ppm. (with trace of dipterocarpol)

78

The HSQCedoted correlation of 24 (in CDCl 3 ) and expanded spectrum in the range of δ H 0.7-3.8 ppm and δ C 13-72 ppm.

79

The HMBC correlation of 24 (in CDCl 3 ) and expanded spectrum in the range of δ H 2.9-3.8 ppm, δ C 10-50 ppm and δ H 1.2-1.8 ppm, δ C 81-84ppm.

80

The 1 H NMR spectrum (300 MHz) of 25 (in CDCl 3 ) and expanded spectrum in the range of 0.95-1.10 ppm.

81

The 13 C NMR spectrum (75 MHz) of 25 (in CDCl 3 ) and expanded spectrum in the range of 16-60 ppm.

82

The expanded HSQCedited correlation of 25 (in CDCl 3 ) in the range of δ H 0.8-4.9 ppm and δ C 16-110 ppm.

83

The HMBC correlation of 25 (in CDCl 3 ) and expanded spectrum in the range of δ H 4.7-4.9 ppm, δ C 20-55 ppm and δ H 0.9-2.0 ppm, δ C 18-57ppm.

Figure page 84

The expand 1 H NMR spectrum (300 MHz) of 26 (in CDCl 3 ). 85

The 13 C NMR spectrum (75 MHz) of 26 (in CDCl 3 ) and expanded spectrum in the range of 13-58 ppm.

86

The expanded HSQCedited correlation of 26 (in CDCl 3 ) in the range of δ H 0.8-2.7 ppm and δ C 13-58 ppm.

87

The expanded HMBC correlation of 26 (in CDCl 3 ) in the range of δ H 0.7-2.7 ppm, δ C 13-59 ppm and δ H 1.0-2.7 ppm, δ C 211-219 ppm.

88

The expand 1 H NMR spectrum (300 MHz) of 27 and 28 (in CDCl 3 ) δ H 0.7-2.7 ppm.

89

The 13 C NMR spectrum (75 MHz) of 27 and 28 (in CDCl 3 ). [START_REF] Gonzalez | Triterpenes of Hippocratea celastroides (Celastraceae)[END_REF] The expanded 13 C NMR spectrum (75 MHz) of 27 and 28 (in CDCl 3 ) in the range of δ H 10-60 ppm.

91

The expanded HSQCedited correlation of 27 (in CDCl 3 ) in the range of δ H 0.8-2.8 ppm and δ C 13-58 ppm.

92

The HMBC correlation of 27 (in CDCl 3 ) and expanded spectrum in the range of δ H 0.9-2.8 ppm, δ C 15-57 ppm.

93

The 1 H NMR spectrum (300 MHz) of 29 (in CDCl 3 ) and expanded spectrum in the range of 0.85-1.20 ppm.

94

The 13 C NMR spectrum (75 MHz) of 29 (in CDCl 3 ). 95

The HSQCedited correlation of 29 (in CDCl 3 ) in the range of δ H 4. 31-4.36 ppm, δ C 89.00-90.50 ppm and δ H 5.00-5.11 ppm, δ C 113.00-115.50 ppm.

96

The HMBC correlation of 29 (in CDCl 3 )and expanded spectrum in the range of δ H 1.78-1.82 ppm, δ C 86.00-150.00 ppm and δ H 1.17-1.22 ppm, δ C 32.00-80.00 ppm 97

The 1 H NMR spectrum (300 MHz) of 32 (in CDCl 3 ) and expanded spectrum in the range of 0.7-2.2 ppm.

98

The 13 C NMR spectrum (75 MHz) of 32 (in CDCl 3 ). [START_REF] Bhakuni | Constituents of Cornus capitata[END_REF] The HSQCedited correlation of 32 (in CDCl 3 ). 100 The HMBC correlation of 32 (in CDCl 3 ). 101 The 1 H NMR spectrum (300 MHz) of 36 (in CDCl 3 ) and expanded spectrum in the range of 0.8-2.6 ppm. 102 The 13 C NMR spectrum (300 MHz) of 36 (in CDCl 3 ) and expanded spectrum in the range of 11-58 ppm. 257 103 The HSQCedited correlation of 36 (in CDCl 3 ) and expanded spectrum in the range of δ H 0.9-2.6 ppm and δ C 10-56 ppm.

258 Figure page 104 The HMBC correlation of 36 (in CDCl 3 ) and expanded spectrum in the range of δ H 0.9-2.1 ppm, δ C 13-58 ppm and δ H 3.5-4.3 ppm, δ C 19.5-22.0 ppm.

105 The 1 H NMR spectrum (300 MHz) of 39 (in CDCl 3 ) and expanded spectrum in the range of 0.7-2.7 ppm.

Figure page 123

The HMBC correlation of 45 (in acetone) and expanded spectrum in the range of δ H 0.6-4.2 ppm and δ C 10-85 ppm.

124 The 1 H NMR spectrum (300 MHz) of 46 (in CDCl 3 ) and expanded spectrum in the range of 0.7-2.7 ppm. 4.4.1 Friedelin (1) and Epifriedelanol (4) 4.4.2 β-Amyrin [START_REF] Antimonova | Synthesis of betulonic acid amides[END_REF] and Saturated Fatty Acid Ester of -Amyrin (6) 4. , -Sitosterol Palmitate [START_REF] Ge | Hopeahainol A: an acetylcholinesterase inhibitor from Hopea hainanensis[END_REF] and Mixture of Saturated and Unsaturated Fatty Acid Ester of -Sitosterol (8) 4.4.4 Betulonic Acid (5), Epibetulinic Acid [START_REF] Ito | Two novel trimeric resveratrol derivatives from Cotylelobium lanceolatum[END_REF], Betulone [START_REF] Liu | New resveratrol oligomers from the stem bark of Hopea hainanensis[END_REF], -ene [START_REF] Sahidin | Cytotoxic properties of oligostilbenoids from the tree barks of Hopea dryobalanoides[END_REF], Betulinic Acid ( 13), [START_REF]Are the number of cancer cases increasing or decreasing in the world? Accessed[END_REF]*, [START_REF] Ashton | Dipterocarpaceae[END_REF]-en-3-one ( 16)** and Messagenic Acid G (17)** 4.4.5 Caryophyllene oxide (9) 4.4.6 Mangiferonic acid (15) 4.4.7 26-Fatty acid ester of (24S,25S)- 24,25,26-trihydroxy-3,4-seco-cycloart-4(29)-en-3-oic acid [START_REF] Tanaka | Oligostilbenoids in stem bark of Vatica rassak[END_REF]***, its 3methyl ester (18a) and its triterpene moiety (transesterification product, 18b) 4.4.8 C-24 Fatty acid ester of (24S,25S)- 24,25,26trihydroxy-3,4-seco-cycloart-4(29)-en-3-oic acid [START_REF] Tanaka | Stilbenoids in the stem bark of Hopea parviflora[END_REF]*** and its , 24-diene-3,26-dioic acid (20)

LIST OF SCHEMES

INTRODUCTION

It has been long known that plants are a rich source of bioactive compounds that mankind has learned to use throughout history. Natural products have been used by man since antiquity for a number of applications, such as drugs, pigments and flavours. However, mankind was not aware of the specific importance and distinct chemical nature of such compounds until the end of the 19 th century, and it has only been during the last century that natural products have experienced a great surge in phytochemistry. Many factors have led to this change, including the development of new and more powerful spectroscopic and chromatographic techniques and the change in the perception of society about chemicals. During the 20 th century, phytochemical studies and, subsequently, natural products studies led to the discovery of an enormous number of compounds with a variety of chemical structures (many of them are bioactive), all of which are presented in the chemical composition of living organisms (from unicellular to higher organisms) [START_REF] Macias | Evolution and current status of ecological phytochemistry[END_REF]. Plants provide a broad spectrum of potential drug substances. Several researches reported about phytochemistry of various plants to investigate their bioactivity. The active molecules isolated from traditional medicinal plants might not only provide valuable drugs but are also valuable as "lead molecule" which might be modified chemically, or serve as a template for the design of synthetic molecules incorporating the pharmacophore responsible for the activity [START_REF] Antimonova | Synthesis of betulonic acid amides[END_REF] The situation of cancer in the world is seriously concerned. WHO [START_REF]Are the number of cancer cases increasing or decreasing in the world? Accessed[END_REF] indicated that cancer was a leading cause of death worldwide and the total number of cases globally was increasing. The number of global cancer deaths is projected to increase 45% from 2007 to 2030 (from 7.9 million to 11.5 million deaths), influenced in part by an increasing and aging global population. New cases of cancer in the same period are estimated to jump from 11.3 million in 2007 to 15.5 million in 2030. In most developed countries, cancer is the second largest cause of death after cardiovascular disease, and epidemiological evidence points to this trend emerging in the less developed world. This is particularly true in countries in "transition" or middle-income countries, such as in South America and Asia. Already more than half of all cancer cases occur in developing countries. On the other hand, Malaria is one of the diseases that cause morbidity and mortality in the world for many decades. Although world malaria report 2011 of WHO showed continued progress for the malaria control and the incidence of disease was reduced more than 50% between 2000 to 2010 in 43 of the 99 countries, the resistance to artemisinins -a vital component of drugs used in the treatment of Plasmodium falciparum malaria -has been reported in a growing number of countries in South-East Asia [START_REF]World malaria report[END_REF]. This will cause the problem in malaria control in the future. Hence, many researchers worked on prevention and control of these diseases. Scientists are interested to find bioactive agents from natural sources, especially from traditional herbal medicine.

During recent decades, the biodiversity awareness in Thailand is growing to improve and sustain management of natural resources and to support the knowledge of traditional herbal medicine. Diptercarpaceae, constituted dominant and economically important trees of lowland forest of the country, was known as a family which was enrich of resveratrol oligomer. A number of researches have reported about resveratrol oligomers in this family [START_REF] Atun | Oligostilbenoids from Hopea Mengarawan (Dipterocarpaceae)[END_REF][START_REF] Atun | Resveratrol derivatives from stem bark of Hopea and their biological activity test[END_REF][START_REF] Ge | Hopeahainol A: an acetylcholinesterase inhibitor from Hopea hainanensis[END_REF][START_REF] Saroyobudiono | Oligostilbenoids from Shorea gibbosa and their cytotoxic properties against P-388 cells[END_REF][START_REF] Atun | Oligostilbenoids from Vatica umbonata (Dipterocarpaceae)[END_REF][START_REF] Ito | Two novel trimeric resveratrol derivatives from Cotylelobium lanceolatum[END_REF][START_REF] Liu | New resveratrol oligomers from the stem bark of Hopea hainanensis[END_REF][START_REF] Sahidin | Cytotoxic properties of oligostilbenoids from the tree barks of Hopea dryobalanoides[END_REF][START_REF] Syah | Two oligostilbenes, cis-and trans-diptoindonesin B, from Dryobalanops oblongifolia[END_REF][START_REF] Ito | New resveratrol oligomers in the stem bark of Vatica pauciflora[END_REF][START_REF] Tanaka | Hopeafuran and a C-glucosyl resveratrol isolated from stem wood of Hopea utilis[END_REF][START_REF] Aminah | Diptoindonesin A, a new C-glucoside of [var epsilon]-viniferin from Shorea seminis (Dipterocarpaceae)[END_REF][START_REF] Ito | A new resveratrol octamer, vateriaphenol A, in Vateria indica[END_REF][START_REF] Tanaka | Oligostilbenoids in stem bark of Vatica rassak[END_REF][START_REF] Tanaka | Stilbenoids in the stem bark of Hopea parviflora[END_REF][20]. Since resveratrol oligomers possess polyphenol in the structure, they are in more polar extract. Yet, the nonpolar hexane extracts of Hopea odorata Roxb. leaves and Dipterocarpus costatus Gaertn.f. wood were also interesting because of their cytotoxic activities against several cancer cell lines [START_REF] Sittisombut | Investigation of anti-cancer activity of plants in families of Rutaceae and Dipterocarpaceae in Thailand[END_REF].

H. odorata is a folk medicine which its wood has been used for treatment of yaws, blood disorder, fever, and as expectorant. Its dried stem latex was ground and used for wound healing [START_REF] Chuakul | Medicinal Plants in Thailand[END_REF]. As well as, the previous study of dammar resin from D. costatus found many dammaranes that possessed anti-HIV activity [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF]. All of these made the inspiration for the investigation in this project.

In this thesis, we describe the isolation and structure elucidation of isolated compounds from the hexane extract of H.odorata leaves and D. costatus wood. A number of new compounds in different triterpenoid types, as well as several known triterpenoids, were discovered. Some isolated compounds were then evaluated for cytotoxic activity.

Dipterocarpus (the characteristics: valvate sepals in fruit, solitary vessels, scattered resin canals, and basic chromosome number x =11).

• Imbracate-Shoreae group (tribe Shoreae) consists of genus Shorea, Parashorea, Hopea, Neobalanocarpus, Dryobalanops (the characteristics: imbricate sepals in fruit, grouped vessels, resin canals in tangential bands, and basic chromosome number x= 7).

The plants in subfamily Monotoideae have basi-versatile anthers, 4-5 celled ovary, 2-4 ovules in each cell and small and caducous stipules. The petals are longer than the sepals, and the wood rays are uniseriate. The wood, flowers, and leaves of these trees do not produce a resin or have secretory ducts. The plants in genus Monotes have secretory cavities instead of resin canals as found in the Dipterocarpoideae plants.

The morphological characteristics of Pakaraimoideae plants, the third subfamily, in term of the anthers (basi-versatile), stipules (small and caduceus), and the lack of resin ducts are similar to those of the Monotoideae. The petals are shorter than the sepals, and the wood rays are dominantly biserate. The subfamily Pakaraimoideae seems to be closer to the monotoideae than the Dipterocarpoideae. The lack of resin canals in wood of both the Monotoideae and the Pakaraimoideae constitutes a major distinction from the Dipterocarpoideae.

Diversity of opinions still exists for generic divisions, especially with the genus Shorea and the group of genera Vatica and Cotylelobium. However, certain well defined genera exist, such as Dryobalanops, Dipterocarpus, Anisoptera and Upuna.

Thai Dipterocarpaceae [START_REF] Pooma | Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis[END_REF][START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF][START_REF] Pooma | Checklist of Dipterocarpaceae in Thailand[END_REF] Dipterocarpaceae in Thailand was first described in "Florae Siamensis Enumeratio" by Craib (1925), followed by Smitinand (1954) who published his "Identification Keys to Genera and Species of the Dipterocarpaceae of Thailand" and continued the work with his co-worker, Satisuk and Plengklai, by publishing "The Manual of Dipterocarpaceae of Mainland South-East Asia".

In year 2001, Pooma reviewed "Checklist of Dipterocarpaceae in Thailand" and included this paper in his thesis report "Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis" in 2003.

Thai Dipterocarpaceous plants, deciduous or semi-evergreen trees, consists only in the subfamily Dipterocarpoideae which is represented by about 65 species in 8 genera; Valvate-Dipterocarpi group (Anisoptera, Cotylelobium, Dipterocarpus, Vatica) and Imbricate-Shoreae group (Hopea, Neobalanocarpus, Shorea and Parashorea). Scheme 1. Taxonomic classification of genus Hopea and Dipterocarpus [20,[START_REF] Pooma | Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis[END_REF][START_REF] Ashton | Dipterocarpaceae[END_REF][START_REF]The free encyclopedia[END_REF] Kingdom : Plantae

• Vateria • Vateriopsis • Stemonoporus • Upuna • Vatica • Cotylelobium • Anisoptera • Dipterocarpus • Dryobalanops • Shorea • Parashorea • Hopea • Neobalanocarpus
Valvate sepals in fruit, solitary vessels, scattered resin canals, and basic chromosome number x=11

Imbricat sepals in fruit, grouped vessels, resin canals in tangential bands and basic chromosome number x=7

Bioactive components in Dipterocarpaceous plants

The review of Seo and Kinghorn [20] stated that, in addition to oligostilbenoids, the bioactive constituents in Dipterocarpaceous plants, especially in Dipterocarpoideae subfamily, were terpenoids, coumarins, ellagic acid derivatives, flavonoids, phenolics and quinones. The study of several Dipterocarpaceaous plants is in the following table. The researchers were interested in oligostilbenoids rather than other groups of compounds. Thus, most studies worked on high polarity extract, not in hexane extract as in this research.

The plants belonging to the family Dipterocarpaceae have been known to produce oligostilbenoids (or resveratrol oligomers), which are limited in only a number of families, namely, the Cyperaceae, Dipterocarpaceae, Gnetaceae, Leguminosae, and Vitaceae. They occur in subfamily Dipterocarpoideae as di-, tri-, tetra-, hexa-, hepta-, and octastilbenoids, containing various molecular frameworks as a result of different condensation of the resveratrol monomer. Some of these polyphenol compounds show interesting biological activities, such as antioxidant [START_REF] Atun | Resveratrol derivatives from stem bark of Hopea and their biological activity test[END_REF], anti-inflammatory [START_REF] Tabata | Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation[END_REF], antibacterial [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF], antiviral [START_REF] Dai | HIV-inhibitory and cytotoxic oligostilbenes from the leaves of Hopea malibato[END_REF], and cytotoxic effects [START_REF] Saroyobudiono | Oligostilbenoids from Shorea gibbosa and their cytotoxic properties against P-388 cells[END_REF][START_REF] Ito | Antitumor effect of resveratrol oligomers against human cancer cell lines and the molecular mechanism of apoptosis induced by vaticanol C[END_REF], as well as acetylcholinesterase inhibitor activity [START_REF] Ge | Hopeahainol A: an acetylcholinesterase inhibitor from Hopea hainanensis[END_REF]. Hopeaphenol, the first resveratrol oligomer to be structurally characterized from the subfamily Dipterocarpapoideae, was isolated initially from the heartwood of Hopea odorata and Balanocarpus heimii and its structure was determined in 1966. Hopeaphenol strongly inhibited murine leukemia P-388 cells [START_REF] Muhtadi | Cytotoxic resveratrol oligomers from the tree bark of Dipterocarpus hasseltii[END_REF].

A number of resins from some genera in the subfamily Dipterocarpoideae were investigated and found to contain several sesquiterpenes and triterpenes.

• The resin from five species of the genus Doona (Shoreae group) were examined (1966) [START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF] and reported to contain sesquiterpene such as humulene, -elemene, caryophyllene, and copaene, and triterpenes such as -amyrin (largely predominated among all triterpenes), -taraxasterol, dammarenediol-I, hydroxydammarenone-I, and ursolic acid. Moreover, asiatic acid, dipterocarpol and dihydroxyolean-12-en-28-oic acid were also isolated from the D.congestiflora resin of the work of [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF] [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF].

• Chemical analysis of the neutral fraction of the resin from the genus Dipterocarpus was revealed by Bisset et al and Bandaranayake et al [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF]. Several sesquiterpenes and triterpenes were identified: sesquiterpenehumulene, caryophyllene, copaene, -gurjunene, calerene, γ-gurjunene, alloaromadendrene, cyperene, caryophyllene oxide, farnesane, dehydrofarnesane; triterpene -diptercarpol, dammarenediol-II, dammaradienone, and ocotillone, together with asiatic acid and 2,3dihydroxyurs-12-en-28-oic acid. The triterpenes showed little variation with the most abundant -dipterocarpol (ca 48% of the neutral fraction) while the sesquiterpene were much more variable. The mixture of caryophyllene, humulene and alloaromadendrene (ca 24%) was the next most abundant and the compounds were present in the ratio 7:10:3, respectively.

• The following polyterpenes from the resin of thirty-five species of the genus Shorea were characterized [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF]: the sesquiterpene hydrocarbon -elemene, caryophyllene, -gurjunene and cyperene; the sesquiterpene alcohol -spathulenol; the triterpenes - -Amyrin, ursolic aldehyde, dipterocarpol, dammarenediol II (20S), dammaradienone and hydroxyhopanone, including acid constituents as shoreic acid and dammarenolic acid. In genus Shorea, the sesquiterpene hydrocarbon showed only little variation; the variable occurrence of the sesquiterpene alcohol and the triterpenes enabled the sub-genus Anthoshorea to be distinguished from the sub-genera Shorea, Richetia and Rubroshorea. The copaene, -elemene, and caryophyllene association was a feature peculiar to the genus and the occurrence of shoreic acid and oxygenated sesquiterpenes appeared to be specific to the genus Shorea. Further study of Shorea robusta resin [START_REF] Hota | Triterpenoids from the resin of Shorea robusta[END_REF][START_REF] Misra | Triterpenoids from Shorea robusta resin[END_REF] led to finding of more ursene and oleanene triterpenes eg. asiatic acid (the largest amount), -amyrin, ursolic acid and uvaol, together with mangiferonic acid.

• Triterpenes from Dryobalanops aromatica (Shoreae group) resin were investigated by Cheung and Wong [START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF]. Asiatic acid was found as one of the most abundant components, accompanying oleanolic acid, hedragonic acid, dryobalanonolic acid, dryobalanolide, [START_REF] Burger | Archaeological resinous samples from Asian wrecks: taxonomic characterization by GC-MS[END_REF] established taxonomic characterization of dammar resin by GC-MS [START_REF] Burger | Archaeological resinous samples from Asian wrecks: taxonomic characterization by GC-MS[END_REF]. Dammar resin displayed a similar characteristic profile containing ursane and oleanane derivatives ; 2,3-dihydroxyolean-12-en-28-oic acid, 2,3-dihydroxyurs-12-en-28oic acid, 2,3-dihydroxyoleanadien-28-oic acid and 2,3-dihydroxyursadien-28-oic acid (in large quantity). Betulonal was reported as a biomarker characteristic of genus Dipterocarpus but not found in the Shorea one.

The triterpenes isolated from 8 species of bark and timber of Stemonoporus (Dipterocarpi group) belong only to the ursene or oleanene series [START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF]. All the species examined had the pentacyclic triterpenes δ-amyrenone, -amyrin and ursolic acid, sitosterol and sitosteryl-o-methoxybenzoate. The most outstanding feature of the chemistry of the genus was the absence of the tetracyclic dammarane skeleton which was otherwise widespread in the family. δ-Amyrenone, -amyrin along with ursolic acid presented in all the species of Stemonoporus studied and could be considered to be the triterpenoid marker for this genus [START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF]. Moreover, bergenin (isocoumarin), was found in large quantity of bark extracts from 5 studied species, whereas timber extractives contained two other aromatic compounds, 4-hydroxybenzaldehyde and methyl 2,4dihydroxybenzoate. The chemical constituent study of Dipterocarpus hispidus bark found betulinic acid, dipterocarpol and dammarenediol II whilst the timber contained dipterocarpol and asiatic acid [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF]. A study of Geevanada et al [START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF] illustrated phenolic compounds and triterpenes from bark and timber of 11 species belonging to the genera Cotylelobium, Hopea, Shorea, Vateria and Vatica. The result yielded fatty acid ester of sitosteryl, -amyrin acetate, -amyrin, dipterocarpol, ursolic acetate, lupeol, sitosterol, ursolic acid, betulinic acid, hexamethyl-coruleoellagic acid, tetramethylellagic acid, chrysophanol (anthraquinone), and scopoletin (coumarin).

Isolation of ethyl acetate extract of Vatica diospyros stem and chloroform extract of Vatica cinerea leaves and twigs (Dipterocarpi group) [START_REF] Seo | Resveratrol tetramers from Vatica diospyroides[END_REF][START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF] yielded mangiferonic acid, betulin, betulinic acid as the major triterpenoids which exhibited mild anti-HIV activity.

Most of the isolated terpenoids have been reported to be biologically active as indicated in Table 1, although such work has occurred on these compounds isolated from plants of other family [20,[START_REF] Adio | (-)-trans--Elemene and related compounds: occurrence, synthesis, and anticancer activity[END_REF][START_REF] Cho | Glioblastoma cell death induced by asiatic acid[END_REF][START_REF] Kim | Synthesis of betulinic acid derivatives with activity against human melanoma[END_REF]. For example, the common triterpenoids, betulonic acid and betulinic acid, were reported having cytotoxicity against several cell lines (see lupane triterpene).

The characteristic triterpenes in family Dipterocarpaceae could be summarized in pentacyclic (lupanes, ursanes and oleananes) and tetracyclic (dammaranes and cycloartanes) triterpenes.

Furthermore, a lot of flanovoids are already known as antioxidants. A flavonoid aglycone survey [START_REF] Joshi | Leaf flavonoid aglycone patterns in the species of Dipterocarpaceae in Sri Lanka[END_REF][START_REF] Joshi | Leaf flavonoid patterns in Dipterocarpus and Hopea (Dipterocarpaceae)[END_REF] was carried out on the leaves of 7 genera of the family Dipterocarpaceae: Cotylelobium, Dipterocarpus, Hopea, Shorea, Stemonoporus, Vateria and Vatica. After acid hydrolysis of the leaves material, the main flavonoid aglycones found were the flavonols quercetin and kaempferol, and the flavones apigenin. The flavones luteolin was defined as a chemotaxonomic significance in the genus Shorea because it was present only in this genus and absent in the rest of the species of the family. The genus Shorea and Dipterocarpus were considered as the most primitive with respect to the presence of myricetin and proanthocyanidins (cyanidin and delphinidin) and the other five genera have more advanced patterns, recognizing Stemonoporus as the most advanced with an increase of apigenin. Anti-inflammatory, antibiotic, antioxidant, anticarcinogenic and local anaesthetic activities [START_REF] Legault | Potentiatine Effect of -Caryophyllene on anticancer activity of -humulene, isocaryophyllene and paclitaxel[END_REF] Doona sp. (re) [START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF], Shorea sp. (re) [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF] Copaene Male medfly attractant [START_REF] Flath | Male Lures for Mediterranean Fruitfly (Ceratitis capitata wied.): Structural Analogs of -Copaene[END_REF] Doona sp. (re) [START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF], Shorea sp. (re) [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF] Humulene Anticancer [START_REF] Legault | Potentiatine Effect of -Caryophyllene on anticancer activity of -humulene, isocaryophyllene and paclitaxel[END_REF] Doona sp. (re) [START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF] Caryophyllene oxide Anti-microbial [START_REF] Ulubelen | Terpenoids from Salvia sclarea[END_REF], antimalarial [START_REF] Kanokmedhakul | Bioactive constituents of the roots of Polyalthia cerasoides[END_REF] Dipterocarpus sp. (re) [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF],

Resveratrol oligomers (=oligostilbenoids) Steroid

Spathulenol Immunomodulator [START_REF] Ziaei | Identification of Spathulenol in Salvia mirzayanii and the Immunomodulatory Effects[END_REF], Antimicrobial [START_REF] Ulubelen | Terpenoids from Salvia sclarea[END_REF],Antifungus [START_REF] Joycharat | Terpenoid constituents and antifungal activity of Aglaia forbesii seed against phytopathogens[END_REF] Shorea sp. (re) [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF] Triterpenes -Amyrin, -amyrin Antinociception [START_REF] Otuki | Antinociceptive properties of mixture of α-amyrin and β-amyrin triterpenes: evidence for participation of protein kinase C and protein kinase A pathways[END_REF], hepatoprotectant [START_REF] Oliveiraa | Protective effect of -and -amyrin, a triterpene mixture from Protium heptaphyllum (Aubl.) March. trunk wood resin, against acetaminophen-induced liver injury in mice[END_REF] Doona sp. (re) [START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF], Stemonoporus sp. (b,t) [START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF]

Ursolic acid

Antiangiogenesis [START_REF] Kanjoormana | Antiangiogenic activity of ursolic acid[END_REF] Stemonoporus sp. (b,t) [START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF], Doona macrophylla (re) [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF]

Ursonic acid

Antitumor [START_REF] Kim | Antitumor Activity and Inhibitory of Topoisomerase-I by ursonic acid[END_REF], Anti-HSV [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] Stemonoporus affinis, S.lancifolius (b,t) [START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF] Dryobalanolide Antimicrobial [START_REF] Tsiri | Triterpenoids from Eucalyptus camaldulensis Dehnh. tissue cultures[END_REF] Dryobalanops aromatic (re) [START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF]

Asiatic acid

Anti-inflammatory [START_REF] Yun | Inhibition of LPS-induced NO and PGE2 production by asiatic acid via NF-$B inactivation in RAW 264.7 macrophages: Possible involvement of the IKK and MAPK pathways international[END_REF], cytotoxic [START_REF] Cho | Glioblastoma cell death induced by asiatic acid[END_REF] Dipterocarpus pilosus (re) [START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF], D.hispidus (re, t), D.zeylaicus (re), Doona macrophylla and D.congestiflora (re) [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF], Dryobalanops aromatic (re) [START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF] -Taraxasterol Antioedema [START_REF] Zitterl-Eglseer | Anti-oedematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.)[END_REF] Doona sp. (re) [START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF] -Sitosterol Analgesic [START_REF] Villaseñor | Bioactivity studies on β-sitosterol and its glucoside[END_REF] Stemonoporus sp. (b,t) [START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF] hydroxyhopanone Anti-HSV [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] Shorea sp. (re) [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF], Dammar resin [START_REF] Mills | The constitution of the neutral, tetracyclic triterpenes of dammar resin[END_REF] Hydroxydammarenone I Anti-HSV [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] Dammar resin [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF], Doona sp. (re) [START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF]

Betulinic acid

Cytotoxic [START_REF] Kim | Synthesis of betulinic acid derivatives with activity against human melanoma[END_REF], anti-HIV [START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF] Dipterocarpus hispidus (t, b) [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF], genera Cotylelobium, Hopea, Shorea, Vateria and Vatica (t,b) [START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF][START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF]

Betulonic acid

Cytotoxic [START_REF] Kim | Synthesis of betulinic acid derivatives with activity against human melanoma[END_REF], anti-HIV [START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF] Vatica cinerea (l) [START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF] Hydroxydammarenone II (=dipterocarpol) Anti-HSV [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] Shorea sp. (re) [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF], Dammar resin [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF][START_REF] Mills | The constitution of the neutral, tetracyclic triterpenes of dammar resin[END_REF],Dipterocarpus glandiflorus (w) [START_REF] Mclean | Extractives from the Dipterocarpaceae[END_REF] Dammarenediol II Anti-HSV [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] Shorea sp. (re) [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF], Dammar resin [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF][START_REF] Mills | The constitution of the neutral, tetracyclic triterpenes of dammar resin[END_REF]

Dammarenolic acid

Anti-HSV [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF], Anti-HIV [START_REF] Esimone | Dammarenolic acid, a seco-dammarane triterpenoid from Aglaia sp. shows potent anti-retroviral activity in vitro[END_REF] Shorea sp. (re) [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF], Dammar resin [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF]

Shoreic acid

Anti-HSV [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] Shorea sp. (re) [START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF], Dammar resin [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] Ocotillone Cytotoxic [START_REF] Makino | Tirucallane-type triterpenes from Juliana adstringens[END_REF] Dipterocarpus sp. (re) [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF]

Coumnarins

Bergenin Antibacteria and Antioxidant [START_REF] Bizimenyera | The potential role of antibacterial, antioxidant and antiparasitic activity of Peltophorum africanum Sond. (Fabaceae) extracts in ethnoveterinary medicine[END_REF] Stemonoporus sp. (b) [START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF] Scopoletin Acetylcholenesterase inhibitor [START_REF] Rollinger | Acetylcholinesterase Inhibitory Activity of Scopolin and Scopoletin Discovered by Virtual Screening of Natural Products[END_REF]

Biosynthesis of lupane and dammarane-type triterpenes in plants

The terpenoids constitute the largest family of natural products derived from C 30 precursors; over 22,000 individual compounds of this class have been described since 1991 [START_REF] Mcgarvey | Terpenoid metabolism[END_REF]. Because various triterpenes are an increasing promising group of plant metabolites, the utilization of different plants as their sources is of interest [START_REF] Laszczyk | pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy[END_REF]. Nearly 200 different triterpene skeletons are known from natural sources or enzymatic reactions that are structurally consistent with being cyclization products of squalene, oxidosqualene, or bis-oxidosqualene [START_REF] Xu | On the origins of triterpenoid skeletal diversity[END_REF]. Scheme 2. Biosynthesis pathway of dammarane and lupanes triterpene [START_REF] Xu | On the origins of triterpenoid skeletal diversity[END_REF] Biosynthesis of most 3 -OH-triterpenes is proposed to be arisen from oxidosqualene, although squalene cyclization followed by oxidation at C-3 is also Then, most of the widespread pentacyclic triterpene alcohols in plants such as lupeol and -amyrin could be synthesized from the dammarenyl cation after D-ring expansion via C-16 migration followed by 18 E-ring cyclization and sometimes, further E-ring expansion. The lupyl cation, with trans-D,E ring junction, is generated from either 17 -or 17 -dammarenyl cation by D-ring expansion (C-16 migration) to form baccharenyl cation followed by E-ring closure to the -face of C-18. Direct deprotonation without rearrangement provides lupeol, a precursor of a variety of lupane derivatives. Lupyl cation could also undergo E-ring expansion and finally produce ursane, oleanane and taraxastane-type triterpenes.

Lupane-type triterpenes

Pentacyclic triterpenes are one group of promising secondary plant metabolites for cancer treatment with multifaceted effects and targets. The pentacyclic triterpenes such as lupeol, betulin, betulinic acid, oleanolic and ursolic acid are proposed to be multitarget agents. They fit to the concept of modern cancer therapy, by treating cancer from different sides, including the tumour environment and the immune system [START_REF] Laszczyk | pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy[END_REF]. Lupane triterpenoids are pentacyclic compounds with 30 carbon atoms, biosynthetically derived from the cyclization of squalene, and a vast class of natural products whose structural diversity includes a wide array of functional groups. Lupane-containing plants are systematically widespread within the angiosperms and are found predominantly among trees and bushes. They were found wide spread distribution in several family; Betulaceae, Celastraceae, Apocyaceae, Euphorbiaceae, Anacardiaceae and Leguminoseae etc. Not many norlupanes were also found in family Myrtaceae and Betulaceae. Only one study obtained 2,3-seco-lupane, a rare structure, from Microtropis jokienensis (Cealastraceae) and very few obtained 3,4-seco-lupane.(Table 2.)

In addition to cytotoxicity, compounds of this class are reported to be bioactive with antitumor-promoting, antiviral, and anti-inflammatory activities [START_REF] Monaco | Isoprenoids from the leaves of Quercus suber[END_REF]. Rhus chinensis (stem) betulin; betulonic acid [START_REF] Gu | A new benzofuranone and anti-HIV constituents from the stems of Rhus Chinensis[END_REF] Apocyaceae Nerium oleander (leaves) (=lupa-12,20(29)-dien-3 ,27,28-triol); betulin; betulinic acid [START_REF] Siddiqui | Oleanderol, a new pentacyclic triterpene from the leaves of Nerium oleander[END_REF] Betula alleghaniensis (outer bark) lup-20 (29)-ene-28-ol-3-one-30-al; 29-norlupan-3,20-dione; 29norlupan-28-ol-3,20-dione; lupan-20,28-diol-3-one; 29-norlupan-3 -ol-20-one; lupeol; lupenone; betulone; betulin; betulonic acid; lupenyl formate; lup-20(29)-ene-30-ol-3-one; lup-20(29)ene-3 ,30-diol; lup-20(29)-ene-28-ol-30-al; lupan-20-ol-3-one; lupan-3 ,20-diol; lupan-3 -ol-29-oic acid. [85] B. platyphylla var.japonica (floral spike)

Oleanderol

Betulonic acid [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF] B. pubescens (bark) betulin; lupenone; betulonic acid; lupeol; betulinic acid [START_REF] Abyshev | Studies of the chemical composition of birch bark extracts (cortex betula) from the Betulaceae family[END_REF] Betulaceae B. verrucosa (bark) betulinol; lupeol; betulinic acid; lupan- 3 -,20-diol; lupan-3 ,20,28triol [88] Cassine papillosa (stem bark) 3-oxolup-20-en-30-ol; lup-20-en-3 ,30-diol [89] Hippocratea celastroides (aerial part) 3-oxolup-20-en-30-ol; lup-20-en-3 ,30-diol [90] Maytenus Canariensis (aerial part) [START_REF]Are the number of cancer cases increasing or decreasing in the world? Accessed[END_REF][START_REF] Ashton | Dipterocarpaceae[END_REF][START_REF] Ashton | Dipterocarpaceae[END_REF] M.imbricate (stem) -en-3 ,29-diol; [START_REF] Ribeiro | Lupane pentacyclic tricyclic triterpenes isolated from stems and branches of Maytenus imbricata (Celastraceae)[END_REF] M.chiapensis (leaves) 3 ,6 -dihydroxylup-20(29)-ene; 6 ,28-dihydroxy-3-oxolup-20(29)ene; 6 -hydroxy-3-oxolup-20(29)-en-

Family

Plant

Lupanes found Reference M.nemerosa (wood and stem) 3-oxo-20(29)-lupen-30-al; 30hydroxy-20(29)-lupen-3-one; lup-20(29)-ene-3 ,30-diol, [94] Microtropis jokienensis (stem) 7 -hydroxy-methyl betulinate; 7senecioyl betulinic acid; 30-hydroxy-2,3-seco-lup-20(29)-ene-2,3-dioic acid; 3-epi-thurberogenin [START_REF] Chen | Lupane-type triterpenoids from Microtropis fokienensis and Perrottetia arisanensis and the apoptotic effect of 28-hydroxy-3-oxo-lup-20(29)-en-30-al[END_REF] Perrottetia arisanensis (stem) 3-epi-thurberogenin; 3-epithurberogenin-22 -dodecanoate; 3epi-thurberogenin-22 -tetradecanoate; 3-epi-thurberogenin-22hexadecanoate; [95] Microtropis jokienensis (stem) + Perrottetia arisanensis (stem) 28-hydroxy-3-oxo-lup-20(29)-en-30al; betulinic acid; 3-epibetulinic acid; 3-epibetulin; 30-hydroxylupeol; 30hydroxybetulin; 30-hydroxylup-20(29)-en-3-one; 28,30-dihydroxy-3oxolup-20(29)-ene;

3-methoxybetulinic acid; lupeol; 3epibetulinaldehyde; thurberonenin; thurberogenone [START_REF] Chen | Lupane-type triterpenoids from Microtropis fokienensis and Perrottetia arisanensis and the apoptotic effect of 28-hydroxy-3-oxo-lup-20(29)-en-30-al[END_REF] Rzedowskia tolantonguensis (aerial part) 3-oxo-lup-20-en-30-ol; lup-20-en-3 ,30-diol, betulin [96] Solacia chinensis (leaves)  betulin; betulinic acid [START_REF] Zhang | The absolute stereostructures of three rare d:bfriedobaccharane skeleton triterpenes from the leaves of Salacia chinensis[END_REF] S. cordata (stem bark) [START_REF] Tanaka | Hopeafuran and a C-glucosyl resveratrol isolated from stem wood of Hopea utilis[END_REF]-en-3-one; betulin [START_REF] Tinto | Lupane triterpenoids of Salacia cordata[END_REF]

Cornaceae

Cornus capitata (leaves)

Lupeol; betulin; epibetulin; betulinic acid; epibetulinic acid (not so much lupanes) [START_REF] Bhakuni | Constituents of Cornus capitata[END_REF] Euphorbiaceae Bischofia javanica (bark)

Betulinic acid; betulonic acid [100] Hamamelidaceae Liquidamber styraciflua (cone) [START_REF] Atun | Resveratrol derivatives from stem bark of Hopea and their biological activity test[END_REF]-en-28-oic acid; Acacia mellifera (stem bark) 28-hydroxy-3-oxo-lup-20(29)-en-30al; 3-oxo-lup-20(29)-en-30-al; 3hydroxy-lup-20(29)-en-30-al; 28hydroxy-lup-20(29)

Moraceae

Ficus microcarpa (aerial root) acetylbetulinic acid; betulonic acid (only small quantity of lupanes)

[106]

Melaleuca ericifolia (leaves) 28-norlup-20(29)-en-3 -hydroxy-17 -hydroperoxide; 28-norlup-20(29)en-3 -hydroxy-17 -hydroperoxide; 20S-17 ,29-epoxy-28-norlupan-3ol*; betulinic acid, betulin, betulinaldehyde [107] M.leucadendron (leaves) 3 ,27-dihydroxy lup-20( 29)-en-28oic acid methyl ester; 3 -acetoxy-27hydroxylup-20( 29)-en-28-oic acid methyl ester; betulinic acid; 3acetoxy-betulinic aicd; epibetulinic acid; 3-acetoxy-epibetulinic acid [115] Vatica cinerea (leaves and stem) betulinic acid; betulin; betuonic acid [START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF]

Dipterocarpaceae

Vatica diospyros (stem) betulin, betulinic acid [START_REF] Seo | Resveratrol tetramers from Vatica diospyroides[END_REF] The underlined compounds demonstrated the major lupane in the extract. * There was argument that it was impossible to have the configuration at C-17 and C-19 as described in this paper [116].

Cytotoxicity

Betulinic acid and betulonic acid had been known as potent antiproliferative agents (Table 3) [START_REF] Kim | Synthesis of betulinic acid derivatives with activity against human melanoma[END_REF][START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF]106,[117][118][119][120]. DNA Topoisomerases II (Topo II) are target enzymes for anticancer chemotherapeutic drug development. Wada et al suggested that betulinic acid and its derivatives were catalytic inhibitors of Topo II activities [100]. Zhang et al [121] reported that the antitumor action of betulonic acid might be inducing apoptosis through inhibiting the PI3K, AKT pathways and promoting caspase-3,9 activities. The modification of betulonic acid and betulinic acid at C-3, C-28 and isopropenyl group could produce a number of potential antimelanoma activity [START_REF] Kim | Synthesis of betulinic acid derivatives with activity against human melanoma[END_REF] and anti-HIV activity [122,123]. In a study on relationships between structure and activity of lupane triterpenes, a carboxylic group at C-28 was an important part for cytotoxicity [106]. Hata works [124,125] showed that the keto group at C-3 and carbonyl group at C-17 were necessary for antiproliferative effect against some cancer cells. The structural similarities of the lupane groups from Mutai work [101] indicated that the presence of at least one hydroxyl group is important for cytotoxic activity. Furthermore, the position of the hydroxyl on C-3 is more important than on C-28, the presence of the conjugated carbonyl influences the activity very slightly and finally the presence of two hydroxyls (on C-3 and C-28) results in a reduction of activity. A number of amino acid conjugates of betulinic acid at C-28 carboxylic acid position showed improved water solubility as well as selective cytotoxicity [126]. Since the finding that betulinic acid was a highly promising anticancer drug after inducing apoptosis in melanoma cell lines in 1995, various experimental works focused on the apoptosis inducing mechanisms of betulinic acid and other triterpenes (Table 4). The antitumor effects were subsequently confirmed in a series of cancer cell lines from other origins, for example breast, colon, lung and neuroblastoma. Many studies have shown further effects that justify the expectation that lupane triterpenes are useful to treat cancer by several modes of action [START_REF] Laszczyk | pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy[END_REF]. Lupane-type triterpenes, such as betulin, betulinic acid and lupeol, displayed anti-inflammatory activity which often accompanied by immune modulation. Although up to now no clinical trial has been published using these triterpenes in cancer therapy, the pharmacological potential of the lupane seems high. [START_REF] Chen | Lupane-type triterpenoids from Microtropis fokienensis and Perrottetia arisanensis and the apoptotic effect of 28-hydroxy-3-oxo-lup-20(29)-en-30-al[END_REF] 28, ene HeLa (4.0 µg/mL), Hep-2 (7.1 µg/mL) [START_REF] Nunez | Lupane triterpenoids from Maytenus Species[END_REF] NSCLC-N6 (15 µg/mL)

[101] HL60 (1.6 µg/mL) Ca9-22 (1.4 µg/mL) MCF-7 (2.9 µg/mL) MDA-MB231 (7.9 µg/mL) -en-30-al Hep3B (4.7 µg/mL) [START_REF] Chen | Lupane-type triterpenoids from Microtropis fokienensis and Perrottetia arisanensis and the apoptotic effect of 28-hydroxy-3-oxo-lup-20(29)-en-30-al[END_REF] HL60 (1.7 µg/mL) MCF-7 (16.0 µg/mL) A549 (15.7 µg/mL) Hep3B (9.3 µg/mL) Betulin HepG2 (6.7 µg/mL) [START_REF] Chen | Lupane-type triterpenoids from Microtropis fokienensis and Perrottetia arisanensis and the apoptotic effect of 28-hydroxy-3-oxo-lup-20(29)-en-30-al[END_REF] (20R)-28-Hydroxylupan-30-al-3-one NSCLC-N6 (39.5 µM) [102] 3-Oxo-lup-20( 29

Anti-inflammatory activity

A number of lupane triterpenes from Maytenus sp were evaluated for potential antiinflammatory activity [127], and several compounds exhibited in vitro potent inhibitory e ects on NO and prostaglandin E2 production in mouse macrophages (RAW264.7) stimulated with bacterial endotoxin. Compounds able to reduce the excessive production of these mediators had a potential for the prevention and treatment of different inflammatory pathologies. The structure-activity relationship was discussed. The substitution of a C-28 methyl by a hydroxyl methyl group (betulin, betulone) increased the potency mainly on NO, whereas the presence of a C-28 carboxyl group (betulonic acid, epibetulinic acid) increased both the potency on NO and PGE2 production and the cytotoxicity. In contrast, the introduction of C-28 carbonyl aldehyde (betulinic aldehyde) or C-20 carbonyl ketone group (28-hydroxy-3,20-dioxo-29-norlupane) was detrimental. On the other hand, the presence of the -hydroxyl group at C-11 (11--hydroxyglochidone) resulted in a higher inhibitory activity, especially for NO. The acetylation of betulin at C-28 increased the potency and reduced the cytotoxicity of this compound, although the double acetylation at C-28 and C-3 strongly reduced the activity. Also, the acetylation at C-11 or the chlorination at C-30 of rigidenol increased the potency of the compound. b Percentages of inhibition for NO and PGE2 production were obtained at 5 µM. c Values represent the concentration required to produce 50% inhibition of the response, along with the 95% confidence limits.

In accordance with 30% in EPP-induced rat ear oedema inhibition of 0.5 mg/ear betulinic acid [128], Danstan et al reported 52% inhibition of prostaglandin biosynthesis by betulinic acid [110].

Antiviral activity

Some lupanes expressed antiviral activity with low selectivity to virus. Betulin, betulinic acid and betulonic acied were reported as anti-HIV agent, however, selectivity indexes, concentration ratio of 50% cytotoxic response of media cell (CC 50 ) to 50% inhibition of HIV-1 replication (IC 50 ) were low [START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF]. Sun et al suggest that the modification at C-3 and C-28 of betulin and betulinic acid (by addition of 3',3'dimethylglutaryl group) could enhance the activity with the higher TI value [122]. It was suggested that the carboxylic group at C-28 of betulinic acid analogous was essential for anti-HIV-1 while a hydroxyl or ketone group at C-3 has little effect on the anti-HIV-1 activity [START_REF] Gu | A new benzofuranone and anti-HIV constituents from the stems of Rhus Chinensis[END_REF]. Betulin, betulonic acid and betulinic acid were also active against herpes simplex and ECHO 6 virus with low TI too [130].

Miscelleneous activity

Antimalarial activity -Santos's work [131] studied the antimalarial activity of betulinic acid and its derivative compounds, betulonic acid, betulinic acid acetate, betulinic acid methyl ester, and betulinic acid methyl ester acetate was evaluated. These substances showed potent antiplasmodial activity against chloroquine-resistant Plasmodium falciparum parasites in vitro, with IC 50 values of 9.89, 10.01, 5.99, 51.58, and 45.79 µM, respectively. Antimicrobial activity -Mutai's work [103] discovered antimicrobial activity of 2 lupanes against different test organism. They reported that 3-(Z)-cis coumaroylbetulin exhibited a very strong activity against Pseudomonas aeruginosa and a strong activity against Staphylococcus aureus at a concentration of 0.1 mg/disc while 30-hydroxylup-20(29)-en-3 -ol also showed pronounced effects against Pseudomonas aeruginosas, Staphylococcus aureus, Microsporum gypseum, and Trichophyton mentagrophytes. The latter compound presented higher antifungal activity against Microsporum gypseu at a concentration of 0.1mg/disc. The hydroxyl substituent at C-3 of 30-hydroxylup-20(29)en-3 -ol was important for the ability to inhibit microorganism growth by comparison to inactive 30-hydroxylup-20(29)-en-3-one. The result also confirmed the important of conformation of molecule because 3-E-trans coumaroylbetulin showed lower activity.

Antituberculosis -Epibetulinic acid showed antituberculosis activity with MIC 50 µg/mL. This agreed with the finding that low polarity pentacyclic triterpenes with a hydroxyl or keto group in the A or B rings and an acid group in the E ring possess moderate antitubercular activity. In addition, the lipophilicity of these triterpenes is likely to allow them to rapidly penetrate the lipid-rich mycobacterial cell wall [114].

Allelopathic activity -The allelopathic activity of lupane derivatives (lupeol, betulinic acid, betuladehyde, betulin and messagenin) has been reported in Macias' work [104]. These triterpenes possessed potential allelopathic activity in particular over dicotyledon species.

Protective effect against the cytotoxicity of Cadmium -Betulone exhibited protective effect against Cd cytotoxicity at a high concentration (20µM) without any cell damage [101].

Dammarane-type triterpene

Dammarane triterpene is a characteristic triterpene in Dipterocarpaceae family. However, it also appears as the main triterpene in Betulaceae, Compositae, Meliaceae and Oleaceae families [START_REF] Joycharat | Terpenoid constituents and antifungal activity of Aglaia forbesii seed against phytopathogens[END_REF][START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF][132][133][134][135][136][137][138][139][140][141][142][143][144][145]. Dammarenyl cation was a cyclization product of oxidosqualene and was proposed to be the precursor of most triterpene alcohols in lupane, ursane and oleanane skeletons [START_REF] Xu | On the origins of triterpenoid skeletal diversity[END_REF]. Many publications have reported bioactivity of dammarane triterpenes especially antiviral activity [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF]. 20,24-dihydroxy-3,4-seco-dammara-4(28),25dien-3-oic acid, (23E)(20S)-20,25-dihydroxy-3,4-seco-dammara-4(28),23-dien-3-oic acid, (23E)(20S) -20,25,26-trihydroxy-3,4-secodammara-4(28), 23-dien-3-oic acid, (23E)(12R,20S) -12,20,25-trihydroxy-3,4-secodammara-4(28),23-dien-3-oic acid.

[134] Betula platyphylla var.japonica (floral spikes)

3-epiocotillol II (and its acetate, 3-Omethylmalonyl, 3-O-malonyl), ocotillone, cabraleone (only a little), 12 -acetoxy-20(S),24(R)-epoxy-3 ,25-diOH-dammarane (and its 3 -acetoxy ester), papyriferic acid and its methyl ester¸ methyl shoreate, betulafolientriol oxide, cabraleahydroxylactone, 20(S),24(R)-diOHdammara-26-en-3-one, dipterocarpol [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF]135] Betulaceae Betula manschurica (leaves) Aglaia forbesii (seed) isoeichlerialactone, isoeichlerianic acid, isocabralealacone, aglinin A, spathulenol [START_REF] Joycharat | Terpenoid constituents and antifungal activity of Aglaia forbesii seed against phytopathogens[END_REF] Aglaia foveolata (bark)

Moraceae

Ficus pumila (fruit) 3 -acetoxy-22,23,24,25,26,27- hexanordammarane-20-one, 3 -acetoxy-20,21,22,23,24,25,26,27-octanordammarane-17ol, 3 -acetoxy-(20R,22E,24RS)-20,24dimethoxydammaran-22-en-25-ol, 3 - 20,24-dimethoxydammaran-22-en-25-ol 3 -acetoxy-22,23,24,25,26,27hexanordammarane-20-one 3 -acetoxy-20,21,22,23,24,25,26,27octanordammarane-17 -

Cytotoxicity

Cabralealactone exhibited cytotoxic activity against P388 leukemia cells with IC 50 3.8 µg/mL [161]. However the relationship between structure and cytotoxic activity could not be deduced. Nevertheless, it also presented multidrug resistance-reversing effect [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF]. It presented moderate cytotoxic activity against KB-C2 cells (multidrug resistant human epidermoid carcinoma of the nasopharynx cells), with IC 50 =28.5 µM in the presence of 2.5 µM colchicine although cabralealactone itself was not cytotoxic (IC 50 >100 µM). The report also showed the recovery of cytotoxicity of 2.5 µM colchicine against KB-C2 in the presence of cabralealactone 2.5 and 5 µg/mL with IC 50 5.7 and 3.0 µM, respectively, compared to 18.2 µM when cabralealactone was absent [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF].

According to MTT cytotoxic assay, 20(S),24(S)-dihydroxydammar-25-en-3-one showed weak cytotoxic activity and showed enhanced cytotoxicity against KB-C2 in the presence of 2.5µM (non toxic concentration) colchicine as compared with those in the absence of colchicine [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF].

With the inhibitory effect against EBV-EA (Epstein-Barr virus-early antigen) activation, dammarenediol II (IC 50 = 300 mol ratio/32 pmol TPA compare to 397 mol ratio/32 pmol TPA of -carotene as reference compound) was suggested to be a valuable antitumor promoter (potential cancer chemopreventive agent). The intensively study showed that epoxidation at C-20 -C-24 of the side chain leads to a decrease in the activity as observed for 3-epicabraleahydroxylactone, 3-epicabraleadiol, ocotillol II and ocotillol I, with IC 50 = 587, 553, 571 and 525 mol ratio/32 pmol TPA, respectively [160].

Cell growth inhibitory activity of ocotillol II was examined. It showed strong cytotoxicity to WI-38 (normal human lung cell) with IC 50 1.3 µM but moderate to weak cell growth inhibitory activities to VA-13 (malignant lung tumor) and HepG2 cells (human liver cancer) with IC 50 15 and 136 µM [147], then it is not suitable for anticancer.

Ocotillone showed moderate cytotoxicity against leukemia cells (L-1210, IC 50 20µg/mL) [START_REF] Makino | Tirucallane-type triterpenes from Juliana adstringens[END_REF] Wang et al [151] performed the experiments to show that the caffeoyl derivative of 3 ,20(S),24(S)-trihydroxydammar-25-ene, fouquierol and 3 ,20(S),25-trihydroxydammar-23(Z)-ene could increase the cytotoxicity of these compounds against human cervical squamous carcinoma (Hela) cell line (with IC 50 6.4, 5.3 and 6.5 µg/mL, respectively.) The hydrolysates of three compounds gave IC [START_REF] Adio | (-)-trans--Elemene and related compounds: occurrence, synthesis, and anticancer activity[END_REF] 26.4, 35.3 and 5.6 µg/mL, respectively.

Anti-inflamatory Activity

Dammarenediol-II showed anti-inflammatory activity, with a 50% inhibitory dose (ID 50 ) of 0.3 mg/ear, which was more inhibitive than quercetin (ID 50 =1.6 mg/ear), a known inhibitor of TPA-induced inflammation in mice [162].

Inhibitory Effects on Production of NO in LPS (lipopolysaccharide)-Activated Macrophages

The inorganic free radical NO has been implicated in physiologic and pathologic processes, such as vasodilation, nonspecific host defense, ischemia-reperfusion injury, and chronic or acute inflammation. NO is produced by the oxidation of L-arginine by NO synthase (NOS). In the family of NOS, inducible NOS (iNOS) is specifically involved in pathologic aspects with the overproduction of NO and can be expressed in response to proinflammatory agents such as interleukin-1 , tumor necrosis factor-, and LPS in various cell types including macrophages, endothelial cells, and smooth muscle cells. Isofouquierol (100µM) showed 86.6% inhibitory effect on NO production induced by LPS in mouse peritoneal macrophages [149].

Antiviral activity

In vitro antiviral activity of some dammarane was initially investigated by Poehland et al [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF]. The result showed potent antiviral activity against HSV-1 and HSV-2 (table 8). Isofouquierol from twigh, leaves and flower of Gierocarpus intricarpus (Rosaceae) was also reported as a major anti-herpes active [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF].

Antifungal activity

Terpenoid constituents and their antifungal activity of Aglaia forbesii seed were studied. The 3,4-seco-dammarane triterpenes, isoeichlerialactone, isoeichlerianic acid and aglinin A, showed a broader antifungal spectrum against Phytophthora botryosa, P.palmivora, and Rigidoporus microporus whereas the dammaran-3-one derivative, isochlerialactone, and spathulenol were only active against R.microporus [START_REF] Joycharat | Terpenoid constituents and antifungal activity of Aglaia forbesii seed against phytopathogens[END_REF]. It was proposed that the 3,4-seco-3-acid skeleton could account for antifungal property.

Insect antifeedant

Ocotillone exhibited insect antifeedant and growth-regulating activities against Spodoptera litura [163].

Determination of the absolute configuration by modified Mosher's method.

The high-field FT NMR application of modified Mosher's method has been reliably utilized for determining the absolute configuration of secondary alcohols and primary amines [149,[164][165][166][167][168][169]. This method involoves derivatization of the secondary alcohol with both (R)-MTPA-Cl and (S)-MTPA-Cl affording two diastereoisomeric ester [(S)-and (R)-ester, respectively].

The ester derivatives of -methoxy-trifluoromethylphenylacetic acid (MTPA) have proved especially useful in this regard. Mosher proposed that, in solution, the carbinyl proton, ester carbonyl and trifluoromethyl groups of the MTPA moiety were in the same plane (Figure 4). The 1 H NMR signal of R 1 of the (R)-MTPA ester will appear upfield relative to that of the (S)-MTPA ester due to the diamagnetic effect of the benzene ring [It is important to be noted that (R)-MTPA-Cl yields (S)-MTPA ester]. The chemical shif differences of the ester proton at R 1 and R 2 were then calculated (∆δ=δ S -δ R ) (R 1 :∆δ>0 and R 2 :∆δ<0) [168]. extended this method to longer chain secondary alcohols (Figure 5). The values of ∆δ (=δ S -δ R ) could be measured for protons residing within group R 1 and R 2 (as many proton signals as possible with respect to each of the (S)-MTPA and (R)-MTPA ester). The H A,B,C… NMR signals of the (R)-MTPA ester were relatively more shielded and should appear upfield relative to those of the (S)-MTPA ester. The reverse should be true for the H X,Y,Z… . Therefore, the values of ∆δ (=δ S -δ R ) of the H A,B,C… would be positive value (∆δ>0) while those of the H X,Y,Z… would be negative values (∆δ<0). This method was applied to determine the absolute configuration at position 24 of compound 18a. The use of tetrazolium salts, such as MTT (3-(4,5-dimethylthiazole-2-yl)-2,5diphenyltetrazolium bromide) and WST-1, is based on the fact that live cells reduce tetrazolium salts into highly colored formazan compounds whose absorbance is in direct proportion to the number of viability cells. The biochemical procedure is based on the activity of the succinate-tetrazolium reductase, a mitochondrial enzyme, which is inactivated shortly after cell death. So, this method was found to be very efficient in assessing the viability of cells. An increase in number of living cells results in an increase in the overall activity of mitochondrial dehydrogenases in the sample. This increase directly correlates to the amount of formazan formed, as monitored by the absorbance. However, due to neutrality of the solution, the use of MTT produces a water-insoluble formazan compound which is disadvantage when spectrophotometric measurement of formazan products is required. It needs an extra step to solubilize this insoluble product. The compound producing highly water-soluble formozan was developed. WST-1, containing two sulfonate groups to improve water-solubility, was proved to be more sensitive than MTT and less cytotoxic than XTT (a former water-soluble tetrazolium salt, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamono)carbonyl]-2H-tetrazolium hydroxide), in the proliferation assay using P388 cell lines. Therefore, WST-1 is of value as a viability indicator in cell proliferation assay.

CF 3 O O R 1 R 2 H OCH 3 Ph (S)-MTPA ester CF 3 O O R 1 R 2 H Ph H 3 CO (R)-MTPA ester
3 O O H OCH 3 H C H B H A H Z H Y H X

WST-1 MTT XTT

Cytotoxic evaluation by REMA (resazurin microplate assay) method [173][174][175] Resazurin is an oxidation-reduction indicator used for the evaluation of cell growth, particularly in various cytotoxicity assays. It has also been used for decades to demonstrate bacterial and yeast contamination of milk and provided a convenient index of cell proliferation following irradiation. Resazurin is a blue non-fluorescent and non-toxic dye that becomes pink and fluorescent when reduced to resorufin that could be further reduced to hydroresorufin (colorless and non-fluorescent). This conversion is intracellular, facilitated by mitochondrial, microsomal and cytosolic oxidoreductases, then, the reduced product could be excreted into the medium. Resorufin, the result of resazurin bioreduction, is measured colorimetrically or fluorometrically. Reduction of the fluorescent resorufin into a further reduced non-fluorescent product may lead to aberrant results in which living cells produced a weak signal and dying cells, which could not sustain further reduction, yielded a high fluorescent signal.

Resazurin is non-toxic to cells and stable in culture medium, allowing continuous measurement of cell proliferation in vitro as either a kinetic or endpoint assay. Toxic insult that impairs cell viability and proliferation also affects the capacity of cultures to reduce resazurin, and the rate of dye reduction is directly proportional to the number of viable cells present. The certain precaution should be taken in REMA method. First, it is worth checking the cross-reactivity of resazurin with any compound to be tested, without any cells in the medium. Second, the reduction rate by cells in culture should also be checked so that conditions for resazurin concentration and incubation time are optimized in order to avoid over-reduction of resazurin into the colourless and non-fluorescent hydroresorufin. Third, resazurin is more valuable as an endpoint measurement for cytotoxicity rather than as a kinetic measure for monitoring cell growth. All these initial steps may be seen as cumbersome and lengthening a process which is supposed to be quick and simple. However, once established for routine screening, high throughput screening is probably the most obvious application for this test, since this reagent is not as expensive as the reagents of other method.

I. BOTANICAL STUDY

Genus Hopea [START_REF] Pooma | Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis[END_REF][START_REF] Ridley | The Flora of the Malay Peninsula[END_REF][START_REF] Pooma | Checklist of Dipterocarpaceae in Thailand[END_REF] The genus Hopea is distributed from Sri Lanka, to Bangladesh, India, Myanmar, South China, Hainan, Indochina and Malaysia. There are [START_REF] Ito | New resveratrol oligomers in the stem bark of Vatica pauciflora[END_REF][START_REF] Tanaka | Hopeafuran and a C-glucosyl resveratrol isolated from stem wood of Hopea utilis[END_REF]

II. PREVIOUS CHEMICAL WORK ON GENUS HOPEA

Aqueous and ethanolic crude extracts of Hopea utilis screened for antibacterial and cytotoxic activities were studied. Antibacterial activity of ethanolic extracts of H. utilis were more successful with the pathogens Salmonella typhi and Streptococcus aureus (MIC = 25 mg/mL and 36 mg/L, respectively). The results of both extracts (aqueous and ethanolic) of H. utilis showed the brine shrimp lethality assay LD 50 values at 1.64 µg/mL and 1.34 µg/mL, respectively [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF].

Oligostilbenoids from Hopea hainanansis, hopeahainol A, a dimer, and neohopeaphenol A, a tetramer, were found to be acetylcholinesterase inhibitors with an IC 50 value of 4.33 µM, and 7.66 µM, respectively [START_REF] Ge | Hopeahainol A: an acetylcholinesterase inhibitor from Hopea hainanensis[END_REF][START_REF] Liu | New resveratrol oligomers from the stem bark of Hopea hainanensis[END_REF].

Hopeanolin, an unusual resveratrol trimer with an ortho-quinone nucleus, was isolated and characterized from the stem bark of Hopea exalata. Also obtained were six known stibenoids, shoreaphenol, vaticanol G, -viniferin, pauciflorol A, vaticanol A, and trans -3,5,4'-trihydroxystilbene-2-C-glucoside. Hopeanolin demonstrated antifungal activity in the MIC value range 0.1-22.5 g/mL [178]. [START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF] contained sitosterol, lupeol, ursolic acid, β-amyrin, betulinic acid, dipterocarpol (in the order of quantity from high to low). On the other hand, it showed that there was more lupane triterpene in genus Hopea than dammarane triterpene.

The isolation of the extract from the stem bark of Hopea odorata, H. mengarawan and H. nigra, found seven known resveratrol derivatives, named balanocarpol, heimiol A, vaticanol G, vaticanol B, hopeaphenol, ampelopsin H, and hemlesyanol C. Hopeaphenol was more active in antioxidant than ascorbic acid whereas vaticanol B and ampelopsin H were very active against HeLa-S3 (human epithelial carcinoma cell line) and Raji cell (human Burkitt's lymphoma cell line) [START_REF] Atun | Resveratrol derivatives from stem bark of Hopea and their biological activity test[END_REF]. Nguyen et al [179] also isolated two oligostilbene, hopeaphenol and malibatol A, from the methanolic extract of the stem bark of Hopea odorata Roxb. Malibatol A had been reported to have significant effect against cancer cell line CEM-SS (human T lymphoblastoid cell line) with the IC 50 = 21 g/mL.

Quercetin, kaempferol, apigenin and quercetin-3-glucoside were isolated in the study of flavanoid pattern of H. odorata leaves [START_REF] Joshi | Leaf flavonoid patterns in Dipterocarpus and Hopea (Dipterocarpaceae)[END_REF].

Hopeanolin

Hopeahainol A neohopeaphenol A

III. EXPERIMENTAL PART

METHOD AND APPARATUS

1D AND 2D NMR spectra were recorded on Bruker AC300 (300 MHz MPLC (medium pressure liquid chromatography) BUCHI684 Fraction collector was used for rough fractionation.

Silica gel 60A C.C.20-45µm chromagel sds from CARLOERBA was used in column chromatography

Lipophilic Sephadex ® LH20100-100G (10 g for 100 mg extract)

TLC was carried on a Merck ® aluminium sheet 20x20 cm silica gel 60 F 254 . Spots were detected under UV (254 and 366 nm) before spraying with vanillin-sulfuric acid solution followed by heating the plate at 110 o C for 5 minute.

PRELIMINARY STUDY

Preliminary extraction by soxhlet extraction

The 50.4128 g dried and powdered leaves was first extracted by soxhlet apparatus. The extraction gave the 1.7908 g (3.55%) and 10.6608 g (21.15%) of the hexane and ethanol extracts, respectively. Then, both extracts were examined for bioactivity (Scheme 3).

In vitro bioactivity assays

The in vitro cytotoxic and antifungal activities of both extracts were performed by the Resazurin Microplate assay (REMA) [173], while antituberculosis and antimalarial activity were measured by green fluorescent protein microplate assay (GFPMA) and microculture Radioisotope Technique, respectively. All samples were tested by BIOTEC, Thailand.

The results were shown in the following table.

EXTRACTION AND ISOLATION OF H. odorata LEAVES HEXANE EXTRACT.

Extraction by maceration

The dried and powdered leaves (1,077.30 g) of H.odorata was sequentially extracted by maceration with hexane and ethanol (3L*48h*3, each). Each extract was concentrated in reduced pressure to dark green residues of both hexane extract (19.52

Bioactivity assay Cytotoxicity

• + against MCF-7

• + against NCI-H187 Antifungal activity

• -Antimalarial activity

• -Antituberculosis

• -

Bioactivity assay Cytotoxicity

• -against MCF-7

• -against NCI-H187 Antifungal activity

• Not tested Antimalarial activity

• Not tested Antituberculosis

• Not tested Scheme 4. Extraction of H.odorata leaves for phytochemical study.

3.4.2.

Isolation of major compounds with a small portion of hexane extract.

Isolation with silica gel column chromatography

A small portion (2.03 g) of the hexane extract was fractionated by column chromatography (column ID 5 cm) on silica gel (35-70 µm, 98.83 g) and was eluted with hexane, hexane-CH 2 Cl 2 mixture (9:1, 7:3, 6:4, 5:5, 3:7, 1:9, 5:95), CH 2 Cl 2 and methanol. Fractionation was achieved by comparison of each fraction on TLC (hexane-CH 2 Cl 2 1:9). Then, 19 fractions were collected (A01-A19). The fractions of interest were isolated by SiO 2 cc with various solvent systems (Scheme 5, Table 10). The isolated compounds had been determined based on spectroscopic data and comparison to the previous data.

Study of fraction A07

The existing stain on the wall of fraction A07 was not dissolve in hexane but had to be dissolved with CH 2 Cl 2 . After drying, it provided a small white needle crystal that was characterized by spectroscopic data as friedelin (1, 8 mg).

Study of fraction A10

Fraction A10 was subjected through silica gel (20-45 µm) cc and eluted with cHex-EtOAc 6:1. A small white crystal of β-amyrin (2, 20 mg) was found.

Study of fraction A11

Fraction A11 was purified in silica gel (20-45 µm) cc with silica gel cc using cHex-EtOAc

(5:1) as eluent. A white powder of β-sitosterol (3, 35 mg) was obtained.

Study of fraction A17

Fraction A17 (15.1 mg) was purified with a small silica gel (20-45 µm) cc and eluted with cHex -EtOAc 10 : 1 system. Friedelin (1, 3 mg)

Isolation with sephadex column chromatography

A portion of hexane extract (100 mg) was subjected to sephadex (10 gm sephadex swelled in dichloromethane) cc and eluted with CH 2 Cl 2 (Scheme 5). Each fraction was examined on TLC (cH-EtOAc = 5:1). Chlorophyll (green band) and carotene (yellow band) were removed from the column quickly. Continuing collection obtained 12 mg of betulonic acid ( 5) which was further purified by a silica gel cc (cHex-EtOAc = 5:1 as eluent).

Scheme 5. Isolation of main compounds from H. odorata leaves hexane extract.

Isolation of a large portion by MPLC.

The hexane extract of H. odorata leaves (10.35 g) was deposited on 10 g dry silica gel and then subjected to medium pressure column chromatography (MPLC) (sample compartment column: ID 3 cm length 25 cm, isolation column: ID 7.5 cm and length 45 cm) over silica gel; eluted with cHex, cHex-CH 2 Cl 2 gradient mixture (with 5% increasing in each 500 mL portion), CH 2 Cl 2 and methanol. The eluated liquid was collected in 250-ml tubes. The tubes were then combined according to their TLC patterns (system cHex-CH 2 Cl 2 20:1 and 10:1) (Scheme 6, Table 11). Then, 24 fractions (B01-B24) were collected. Fraction B11-

Study of fraction B11 and B12

Fraction B11 and B12 were pooled (50 mg) and chromatographed over silica gel column and eluted with mixture of cHex-CH 2 Cl 2 (10:1). It yielded a white amorphous solid (25 mg) which showed the characteristic of saturated fatty acid ester of -amyrin (6, 25 mg) by spectroscopic consideration.

Study of fraction B14

Fraction B14 (217 mg) was submitted through silica gel cc using cHex-EtOAc 80:1 as mobile phase. The elution yielded 155 mg of white solid which was sequentially purified with sephadex column (System: CHCl 3 -MeOH = 1.5:0.5). A white wax of fatty acid ester of -sitosterol (76 mg) was obtained and then transesterification of the ester was perfomed Transesterification of fatty acid ester [180] An aliquot of the ester (45 mg) was refluxed in dry MeOH (20 mL) with sodium methoxide (20 mg) overnight. The reaction product was extracted with H 2 O and CH 2 Cl 2 . The organic phase was separated, dried over Na 2 SO 4 and evaporated. Dried CH 2 Cl 2 phase was then subjected to silica gel cc using cHex-EtOAc 5:1 as eluent. The eluted compounds were analyzed by GCMS. Methyl ester of saturated fatty acid; palmitic acid (main) with a little of stearic acid, together with -sitosterol were obtained. Then, the main ester was determined as -sitosterol palmitate [START_REF] Ge | Hopeahainol A: an acetylcholinesterase inhibitor from Hopea hainanensis[END_REF].

Study of fraction B15

Fraction B15 (404 mg) was submitted to cc on SiO 2 eluted with cHex/EtOAc (80:1). The fractions were grouped. The fraction of interest was sequentially submitted to purification through sephadex column using CHCl 3 -MeOH = 1.5:0.5 as moblile phase and gave fatty acid ester compound 8 (133 mg). Transesterification (as same as fraction B14) followed by identification with GC-MS of compound 8 gave -sitosterol [START_REF] Ge | Hopeahainol A: an acetylcholinesterase inhibitor from Hopea hainanensis[END_REF] and the mixture of saturated fatty acid (palmitic acid) and unsaturated fatty acid (linoleic and oleic acid). The result was in agreement with NMR determination.

Study of fraction B16

Fraction B16 (137 mg) was further purified by SiO 2 cc using cHex/EtOAc (70:1) system as mobile phase. After fractionation, the first isolated fraction (25 mg) gave the same pattern of 1 H NMR and HSQCedit spectrum as compound 8 from fraction B15.

Study of fraction B21

Fraction B21 (110 mg) was submitted through column chromatography on silica gel using cychlohexane-CH 2 Cl 2 1:2 as mobile phase. The elution yielded 10 mg small white needle crystal of friedelin [START_REF] Macias | Evolution and current status of ecological phytochemistry[END_REF]. Scheme 6. Isolation of H.odorata leaves extract by MPLC (SiO 2 ).

Purification of fraction B23

Fraction B23 (7.28 g) was deposited on 7 g dry silica gel and was isolated over silica gel medium pressure liquid chromatography (MPLC) (column for sample compartment 3 cm ID, 10 cm length and isolation column 7.5 cm ID and 45 cm length), which was H.odorata leaves hexane extract (10.35 successively eluted with dichloromethane, dichloromethane-ethyl acetate gradient mixtures, ethyl acetate and methanol (Scheme 7, Table 12). The flow rate of applied eluent was set at 50 mL/min. Then, 48 fractions were collected based on TLC patterns using system hexane-dichloromethane (1:10) and hexane-ethyl acetate (5:2). The results of the chromatography were shown in Table 12. Crystals of -Sitosterol (3, 324 mg) and betulonic acid (5, 424 mg) were obtained from fraction C12 and C22-C25, respectively.

Scheme 7. Further purification of fraction B23

Fraction B23 (7.28 g)

Study of fraction C02

Fraction C02 (75 mg) was further rechromatographed over SiO 2 cc with cHex-CH 2 Cl 2 (1:3) and further purified using SiO 2 cc. Elution with cHex-EtOAc (14:1) yielded friedelin (1, 1.6 mg)

Study of fraction C05

Fraction C05 (36 mg) was subjected to SiO 2 cc using cHex-CH 2 Cl 2 (2:1) system as eluent. After elution, caryophyllene oxide (9, 16 mg) was obtained.

Study of fraction C07

Fraction C07 (176 mg) was resubmitted through column chromatography on silica gel (cHex-EtOAc 20:1). The elution yielded 45 mg -amyrin [START_REF] Antimonova | Synthesis of betulonic acid amides[END_REF].

Study of fraction C20

Fraction C20 (268 mg) was chromatographed over silica gel (cHex-EtOAc 6.5:1) and yielded epibetulinic acid (10, 26 mg) and betulone (11, 20 mg).

Study of fraction C21

Fraction C21 (140 mg) was further purified by SiO 2 cc using the mixture of cHex-EtOAc 10:1 as eluent to obtain betulonic acid (5, 95 mg).

Study of fraction C27

Fraction C27 (90 mg) was chromatographed over SiO 2 cc and eluted with the mixture of cHex-EtOAc 10:1 to yield betulonic acid (5, 31 mg) and30-hydroxy-3-oxolup-20(29)-ene (12, 18 mg).

Study of fraction C29

Further separation of fraction C29 (96 mg) by SiO 2 cc (cHex-EtOAc 10:1) provided betulinic acid (13, 20 mg).

Study of fraction C32

Compound 14 (3,30-dioxo-lup-20(29)-en-28-oic acid, 3 mg) was crystallized after successive chromatography of fraction C32 (295 mg) over SiO 2 cc with system cHex-EtOAc 10:1.5.

Partial systhesis of 14 Selective SeO 2 allylic oxidation of 5, as previously described by F.A. Macias et al [181,182], afforded compounds 14 and 17 in 22 and 29% yield, respectively. Briefly, 5 (28 mg) was stirred in 20 ml CHCl 3 , (3 mM), with 1.6 mg of SeO 2 , and 0.05 ml of tert-butyl hydroperoxide (t-ButOOH) during 24 hr at room temperature. The crude reaction was filtered and washed through silica gel using CHCl 3 , and EtOAc. Polar EtOAc fraction was then purified using silica gel cc. The elution was begun with the system cHex-EtOAc 10:1 and followed by 5:2.

Study of fraction C35

Fraction C35 (129 mg) afforded compound 15 (mangiferonic acid, 3 mg) by further purification with silica gel cc eluting with system cHex-EtOAc 5:0.7.

Study of fraction C38

From fraction C38 (96 mg), repeating purification on SiO 2 cc with cHex-EtOAc 5:2 obtained green oil of 28,30-dihydroxy-3-oxolup-20(29)-ene (16, 31 mg).

Study of fraction C39

Fraction C39 (218 mg) was applied to a silica gel cc and eluted with cHex-EtOAc gradient system to give 30 subfractions (C39-01 to C39-30) in increasing polarity (Scheme 8). Subfraction C39-24 and C39-25 were combined and further purified by SiO 2 cc with cHex-EtOAc 2:1 system to obtained messagenic acid (17, 12 mg). Subfraction C39-18 and C39-19 were pooled and subjected to SiO 2 cc with cHex-EtOAc 5:1 to yield 26-fatty acid ester of (24S,25S)- 24,25,26-trihydroxy-3,4-seco-cycloart-4(29)-en-3-oic acid (18, 3 mg).

Study of fraction C41

Fraction C41 (228 mg) was rechromatographed over a silica gel cc and eluted with cHex-EtOAc gradient system to obtain 10 mg of 18, 19 (very few amount), 32.8 mg of the mixture of 18 and 19 (24-fatty acid ester of (24S,25S)- 24,25,26-trihydroxy-3,4-seco-cycloart-4(29)-en-3-oic acid), and 64.5 mg of the mixture of 18 and 20 ((24E)-3,4-secocycloart-4(29),24-diene-3,26-dioic acid) (Scheme 9).

Methylation of the mixture of 18+19 This mixture was dissolved in 3.5 mL anhydrous DMF under argon in a 10-mL flask. Sodium bicarbonate (2 eq, 0.2 mmol, 16.8 mg) was added. Methyl iodide (3 eq, 28.4 mg, 12.5 L) was added dropwise at room temperature and magnetic stirring was maintained 24 h. The reaction could be monitored by TLC (cHex-EtOAc 2:1). After this period, 10 mL EtOAc and 5 mL of distilled water were added. The organic phase was separated. The residual aqueous phase was extracted with 10 mL EtOAc (2 times). The organic phases were combined and washed with distilled water (2 x 5mL), then dried over MgSO 4 , filtered and evaporated under reduced pressured [183]. The residue was chromatographed on silica gel cc (cHex-EtOAc 5:1) to isolate 3-methyl ester of compound 18 (=18a) and 3-methyl ester of 19 (=19a). The NMR experiments of both compounds were performed.

Hydrolysis of 18a In a 10 mL flask, compound 18a (10.4 mg) was dissolved in 3 mL of anhydrous dichloromethane. A small catalytic amount of sodium methoxide (2 mL) was added to the solution. The reaction mixture was stirred and refluxed for 4 h at 50°C. The reaction was monitored by TLC (cHex-EtOAc 2:1). The solvent was evaporated under vacuum. The residue was dissolved in CH 3 COOH-H 2 O 3:1. The reaction products were extracted with CH 2 Cl 2 to obtain a mixture of two acids. The solvent was again evaporated under vacuum. To isolate 2 compounds, the mixture was extracted with MeOH-H 2 O (3:1) and cHex. Methyl arachidate and (24S,25S)- 24,25,26-trihydroxy-3,4-seco-cycloart-4(29)en-3-oic acid (=18b) were obtained.

Methylation of the mixture of 18+20 Methylation was performed as aboved procedure. The reaction products were isolated by silica gel cc (cHex-EtOAc 5:1) to obtain 3-methyl ester of 18 (18a, 19 mg) and 3,26-dimethyl ester of 20 (20a, 6 mg). Compound 18a was reacted with MTPA to determine the absolute configuration (Scheme 10) whereas compound 20a was pooled with 20a from fraction C42 and saponified for structural elucidation (Scheme 12)

Preparation of (R)-and (S)-MTPA ester of 18a. Compound 18a was dissolved in 0.5 mL anh CH 2 Cl 2 . Then, 1.36 mg triethyl amine (Et 3 N, 2µL), 4.6 mg 4dimethylaminopyridine (4-DMAP) and 5.36 mg (+)-S-MTPA-Cl (4 µL) were subsequently added. The reaction mixture was stirred at room temperature under nitrogen for 24 h (Scheme 10). The reaction was monitored by TLC (cHex-EtOAc 5:1). The solvent was evaporated under vacuum system and the residue was chromatographed on a SiO 2 cc (cHex-EtOAc 9:1) to isolate (R)-MTPA ester of 18a. Reaction of 18a with 5.36 mg (-)-R-MTPA-Cl (4 µL) according to the same procedure yielded the (S)-MTPAester of 18a [168,184]. The absolute stereostructure of 18a was determined by the application of modified Mosher's method. The proton chemical shift differences of both esters (∆δ=δ S -δ R ) at adjacent positions to C-24 were measured (Table 14). (S)-MTPA ester of 18a EtOAc 5:1 to give 3-methyl ester of 19 (19a, 7.5 mg) and 3,26-dimethyl ester of 20 (20a, 4.4 mg).

• 10 mg of 18a • 2 µL Et 3 N • 4 µL (R)-MTPA-Cl • 0.5 mL CH 2 Cl 2 (R)-MTPA ester of 18a • 10 mg of 18a • 2 µL Et 3 N • 4 µL (S)-MTPA-Cl • 0.5 mL CH 2 Cl 2 Fraction C41 ( 228
Hydrolysis of 20a. Within a 10-mL flask, 3,26-dimethyl ester of 20 (=20a) from fraction C41 and C42 were pooled and dissolved in 3 mL of THF-H 2 O (1:2) with a small amount of LiOH.H 2 O (3 mg) as a catalyst (Scheme 12). The reaction was stirred for 24 h at room temperature under N 2 . The reaction was monitored via TLC (cHex-EtOAc 2 :1). The products were neutralized by 3%HCl to pH6, then extracted with EtOAc.

The solvent was evaporated by reduced pressure to give 20 (8 mg).

CYTOTOXIC EVALUATION OF 8 ISOLATED LUPANES.

The effect of the isolated lupanes on various cell lines was evaluated using WST-1 method (Table 13). The assay principle is based upon the reduction of the tetrazolium salt WST-1 to formazan by cellular mitochondrial succinate tetrazolium reductase. Difference cell lines; prostate cancer cell line (PC3), human breast adenocarcinoma cell line (MDA-MB-231), colorectal adenocarcinoma cell line (HT-29) and colorectal carcinoma cell line (HCT 116), were selected and seeded onto 96-well plates at a cell density of 5,000 cells/well in 5% CO 2 incubator at 37°C. After 24 h incubation, cells were exposed to various concentrations of compound 5, 10, 11, 12, 13, 14, 16 and 17 (3 wells for each dilution) for 24 h. Then, the tetrazolium salt WST-1 solution was added and cultured for further 4 h.

To determine the cell survival, Optical Density (OD) was measured with a Bio-Rad Coda microplate analyzer at a wavelength of 450 nm (reference wavelength: 600 nm). Cisplatin was used as a positive control. Results in Table 13 were expressed as IC 50 , the concentration required for 50% inhibition cell growth of treated cells compared to untreated controls.

Statistical analysis: IC 50 are shown as mean ± standard deviation (SD) of triplicates from each independent experiment. Cell proliferation form WST-1 activities were analyzed using the student's t-test. P-values less than 0.05 were considered statistically significant. method, b colorectal carcinoma cell line, c colorectal adenocarcinoma cell line, d human prostate cancer cell line, e human breast adenocarcinoma cell line, f Inactive compounds: less than 50% inhibition at the threshold concentration of 200 µg/mL. * P-values less than 0.05 compared between tested compound and cisplatin.

PHYSICAL CHARACTERISTICS AND SPECTRUM OF ISOLATED COMPOUNDS

Friedelin [START_REF] Macias | Evolution and current status of ecological phytochemistry[END_REF] Caryophyllene oxide (9) [START_REF] Kanokmedhakul | Bioactive constituents of the roots of Polyalthia cerasoides[END_REF]191,192 1 H-and 13 C NMR-see Table 21 Epibetulinic acid [START_REF] Ito | Two novel trimeric resveratrol derivatives from Cotylelobium lanceolatum[END_REF] [START_REF] Monaco | Isoprenoids from the leaves of Quercus suber[END_REF][START_REF] Siddiqui | Oleanderol, a new pentacyclic triterpene from the leaves of Nerium oleander[END_REF]113,127,185,193] and betulinic acid ( 13) [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF][START_REF] Siddiqui | Oleanderol, a new pentacyclic triterpene from the leaves of Nerium oleander[END_REF]111] Epibetulinic acid [START_REF] Ito | Two novel trimeric resveratrol derivatives from Cotylelobium lanceolatum[END_REF] Betulinic acid ( 13 1 H NMR -see Table 17, 13 C NMR -see Table 19 Betulone (11) [125, 194, 195 1 H NMR -see Table 17, 13 C NMR -see Table 19 30-Hydroxy-3-oxolup-20(29)-ene (12) [START_REF] Ribeiro | Lupane pentacyclic tricyclic triterpenes isolated from stems and branches of Maytenus imbricata (Celastraceae)[END_REF][START_REF] Tinto | Lupane triterpenoids of Salacia cordata[END_REF] Molecular 1 H NMR -see Table 18, 13 C NMR -see Table 19 Mangiferonic acid (15) [START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF]196,197] 1 H NMR -see Table 23, 13 C NMR -see Table 24 3-Methyl ester of 18 (=18a)

Molecular
Specific rotation 20 ] [ D α +74 (CHCl 3 , 0.10)

IR spectrum

ν max (film) 3700-2500 (br), 2924, 2861, 1740, 1457 cm -1 NMR spectrum 1 H NMR -see Table 23, 13 C NMR -see Table 24 Table 14.

PLANT MATERIAL

H.odorata leaves were collected in Chiangmai, a province with biodiversity in plants. Chiangmai is in the northern part of Thailand. It is located among the highest mountains in the country including the deciduous forest association of lowlands and the evergreen forest of the upland. Plant sample was prepared for the study by drying and chopping.

PRELIMINARY STUDY

From preliminary study, H. odorata leaves were extracted by soxhlet apparatus. Plant sample was first extracted with hexane, then, extraction was continued with ethanol. After drying using reduced-pressure evaporator, the in vitro cytotoxic and antifungal activities of both extracts were performed by the Resazurin Microplate assay (REMA), while antituberculosis and antimalarial activity were measured by green fluorescent protein microplate assay (GFPMA) and microculture radioisotope technique, respectively. The hexane extract showed stronger cytotoxic activity against both cell lines, especially against NCI-H187. Hence, it was chosen to be separated by repeated column chromatography.

EXTRACTION AND ISOLATION OF H. odorata LEAVES HEXANE EXTRACT.

After repeated column chromatography of H.odorata leaves hexane extract, two new fatty acid esters of 3,4-seco-cycloartanes (18 and 19) were obtained together with one lupane [START_REF] Ito | New resveratrol oligomers in the stem bark of Vatica pauciflora[END_REF] which was found for the first time in the nature. Phytochemical study of this extract also led to the isolation of other seventeen known compounds. The structures of isolated compounds were established conclusively based on UV, IR, MS and extensive 1 Hand 13 C-NMR spectra analysis. The spectra of known compounds were compared to literature data. One sesquiterpene and five types of triterpenes were identified. Isolated terpenoids are as follows:

Sesquiterpene : caryophyllene oxide [START_REF] Atun | Oligostilbenoids from Vatica umbonata (Dipterocarpaceae)[END_REF] Triterpenes :

• Friedelane type : friedelin (1) and epifriedelanol (4) • Oleanane type : -amyrin ( 2) and its saturated fatty acid ester (6) • Steroidal type : -sitosterol (3) and its saturated and unsaturated fatty acid esters (7, 8) • Lupane type : betulonic acid ( 5), epibetulinic acid [START_REF] Ito | Two novel trimeric resveratrol derivatives from Cotylelobium lanceolatum[END_REF], betulone [START_REF] Liu | New resveratrol oligomers from the stem bark of Hopea hainanensis[END_REF], 30hydroxylup-20(29)-en-3-one [START_REF] Sahidin | Cytotoxic properties of oligostilbenoids from the tree barks of Hopea dryobalanoides[END_REF], betulinic acid (13), 3,30dioxolup-20(29)-en-28-oic acid (14), 28,30-dihydroxy-3oxolup-20(29)-ene ( 16), messagenic acid G (17) • Cycloartane and seco-cycloartane type : mangiferonic acid ( 15), 26-arachidic acid ester of (24S,25S)-24,25,26-trihydroxy-3,4-seco-cycloart-4(29)-en-3-oic acid [START_REF] Tanaka | Oligostilbenoids in stem bark of Vatica rassak[END_REF], 24-stearic acid ester of (24S,25S)- 24,25,26-trihydroxy-3,4-seco-cycloart-4(29)-en-3oic acid [START_REF] Tanaka | Stilbenoids in the stem bark of Hopea parviflora[END_REF], (24E)-3,4-seco-cycloartane-4(29), 24-diene-3,26-dioic acid (20) Friedelane-, oleanane-and steroidal-types triterpenes in this study are wide-spread triterpenes found in plants. However, it should be noted that fatty acid esters ofsitosterol and -amyrin were also obtained.

When the polarity of mobile phase was increased, the elution of isolated compound was in the order of fatty acid ester of -amyrin ( 6) and fatty acid esters of -sitosterol [START_REF] Ge | Hopeahainol A: an acetylcholinesterase inhibitor from Hopea hainanensis[END_REF][START_REF] Saroyobudiono | Oligostilbenoids from Shorea gibbosa and their cytotoxic properties against P-388 cells[END_REF], friedelin (1), caryophyllene oxide, -amyrin ( 2), -sitosterol (3), lupane triterpenes with less polarity [START_REF] Atun | Oligostilbenoids from Hopea Mengarawan (Dipterocarpaceae)[END_REF][START_REF] Ito | Two novel trimeric resveratrol derivatives from Cotylelobium lanceolatum[END_REF][START_REF] Liu | New resveratrol oligomers from the stem bark of Hopea hainanensis[END_REF][START_REF] Sahidin | Cytotoxic properties of oligostilbenoids from the tree barks of Hopea dryobalanoides[END_REF][START_REF] Syah | Two oligostilbenes, cis-and trans-diptoindonesin B, from Dryobalanops oblongifolia[END_REF][START_REF] Ito | New resveratrol oligomers in the stem bark of Vatica pauciflora[END_REF][START_REF] Aminah | Diptoindonesin A, a new C-glucoside of [var epsilon]-viniferin from Shorea seminis (Dipterocarpaceae)[END_REF], mangiferonic acid, more polar lupane (messagenic acid G, 17), fatty acid ester of 3,4-seco-cycloartane-3-oic acid [START_REF] Tanaka | Oligostilbenoids in stem bark of Vatica rassak[END_REF][START_REF] Tanaka | Stilbenoids in the stem bark of Hopea parviflora[END_REF], and finally, 3,4seco-cycloartane dioic acid (20). From the previous literatures [20,[START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF][START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF], the common terpenenoids such as -amyrin, -sitosterol, friedelin and caryophyllene oxide, as well as betulinic acid, betulonic acid and mangiferonic acid have been reported in Dipterocarpaceous plants. The major compounds in this extract were betulonic acid,sitosterol and -amyrin.

It has been reported that -amyrin and its palmitate ester exhibited antidepressant and antinociceptive properties [START_REF] Otuki | Antinociceptive properties of mixture of α-amyrin and β-amyrin triterpenes: evidence for participation of protein kinase C and protein kinase A pathways[END_REF]198,199] as well as anti-inflammatory activity [200].

-Sitosterol is one of the most prevalent phytosterols which is ubiquitous throughout the plant kingdom. It is structurally related to cholesterol. -Sitosterol has an amazing array of scientifically acknowledged benefits for key areas of health in immune dysfunction, inflammatory disorder and rheumatoid arthritis, hypercholesterolemia, breast cancer, colon cancer and, especially, benign prostatic hypertrophy [201]. Caryophyllene oxide showed a variety of bioactivity such as a potent antimalarial, mild cytotoxic [START_REF] Kanokmedhakul | Bioactive constituents of the roots of Polyalthia cerasoides[END_REF] and anti-inflammatory activity [202].

All lupanes gave blue-violet spot with vanillin-sulfuric TLC reagent. The TLC trace and the amount of each fraction showed that betulonic acid [START_REF] Atun | Oligostilbenoids from Hopea Mengarawan (Dipterocarpaceae)[END_REF] was the most abundant compound in this extracts. Among the family containing lupanes as major triterpenoids, this H. odorata leaves hexane extract gave much more betulonic acid, an oxidation product at C-3, than betulinic acid. Accompanied by oxidation at C-28 and C-30, it showed higher degree of oxidation in the metabolism system. Moreover, the higher oxidized derivatives at C-30 normally exhibited a narrower distribution and this is the first report of compound 10, 11, 12, 14, 16 and 17 in the family Dipterocarpaceae. In addition to cytotoxic activity, they also possessed anti HIV [START_REF] Gu | A new benzofuranone and anti-HIV constituents from the stems of Rhus Chinensis[END_REF], antimalarial [131], antiinflammatory [127] activities.

Epibetulinic acid [START_REF] Ito | Two novel trimeric resveratrol derivatives from Cotylelobium lanceolatum[END_REF] was not found as much as 5 and 13 and not normally distributed in the same family as 13. It has been isolated from Simaroubaceae [113], Verbenaceae [114], Zygophyllaceae [115] and Cornaceae [START_REF] Bhakuni | Constituents of Cornus capitata[END_REF]. It is interesting to note the co-occurrence of betulinic and epibetulinic acid in H. odorata. This incident was found only in Cornus capitata leaves (Cornaceae) [START_REF] Bhakuni | Constituents of Cornus capitata[END_REF] and the aerial part of Peganum nigellastrum (Zygophyllaceae) [115]. The cytotoxic and anti-inflammatory activity of this compound were reported [START_REF] Nunez | Lupane triterpenoids from Maytenus Species[END_REF]127] together with moderate anti-tuberculosis activity [114].

Betulone [START_REF] Liu | New resveratrol oligomers from the stem bark of Hopea hainanensis[END_REF] has been achieved from the plants of Betulaceae [START_REF] Cole | Triterpenoid constituents in the outer bark of Betula alleghaniensis (yellow birch)[END_REF], Celastraceae [START_REF] Zhang | The absolute stereostructures of three rare d:bfriedobaccharane skeleton triterpenes from the leaves of Salacia chinensis[END_REF]127] and Leguminoseae [101,105]. It exhibited only mild cytotoxicity in Mutai's work, however, it possessed protective effect against Cd cytotoxicity at a high concentration (20µM) without any cell damage. [194] Compound 12 (30-hydroxy-3-oxo-lup-20(29)-ene) was one of the major compounds of plants in family Celastraceae [START_REF] Ribeiro | Lupane pentacyclic tricyclic triterpenes isolated from stems and branches of Maytenus imbricata (Celastraceae)[END_REF][START_REF] Zhang | The absolute stereostructures of three rare d:bfriedobaccharane skeleton triterpenes from the leaves of Salacia chinensis[END_REF][START_REF] Tinto | Lupane triterpenoids of Salacia cordata[END_REF] This was the first finding of 3,30-dioxolup-20( 29)-en-28-oic acid ( 14) as a natural product in plant. It was a co-product in the synthesis of messagenic acid G from betulinic acid [181]. Since this compound was obtained only in small amount, in order to confirm the proposed structure, a partial synthesis was performed using betulonic acid, which was isolated in large amounts from H. odorata, as starting material. Selective SeO 2 allylic oxidation of betulonic acid, as described by Macias et al.[181,182], gave 3,30-dioxolup-20( 29)-en-28-oic acid, 14, (22%) and messagenic acid G, 17, (29%). The NMR data of synthesized product were in good agreement with the proposed structure. The incomplete 1 H NMR data was reported in the study of Macias et al [181], however no 13 C-NMR data has been provided. The complete NMR data were provided in this research.

Compound 16 (28,30-dihydroxylup-20(29)-en-3-one) is one of rare lupanes in the nature. Until now, there are only 3 papers about this compound from plants in family Celastraceae) [START_REF] Gonzalez | Triterpene from Maytenus canariensis and synthesis of a derivative from Betulin[END_REF][START_REF] Nunez | Lupane triterpenoids from Maytenus Species[END_REF][START_REF] Chen | Lupane-type triterpenoids from Microtropis fokienensis and Perrottetia arisanensis and the apoptotic effect of 28-hydroxy-3-oxo-lup-20(29)-en-30-al[END_REF] and never been reported in Dipterocarpaceous plant. However, the structure elucidation in previous study was based mainly on the comparison to the 1 H-NMR spectrum of 3β, 28,30-lup-20(29)-ene triol and the data obtained from chemical reaction. Only incomplete NMR spectra has been reported [START_REF] Gonzalez | Triterpene from Maytenus canariensis and synthesis of a derivative from Betulin[END_REF]. The complete 1D and 2D NMR data of 28,30-dihydroxy-3-oxolup-20(29)-ene were reported for the first time in this study. Nunez et al [START_REF] Nunez | Lupane triterpenoids from Maytenus Species[END_REF] reported its moderate cytoxicity against HeLa (human carcinoma of cervix, IC 50 4.0 µg/mL=8.75 µM) and Hep-2 (human carcinoma of larynx, IC 50 7.1 µg/mL=15.55 µM).

Compound 17 (messagenic acid G) has been reported only in Melilotus messanensis (Leguminosae) [181] and Liquidamber styraciflua (Hamamelidaceae) [204]. It could be synthesized from oxidization of betulinic acid in which compound 14 was a coproduct. It showed the effect on the germination and growth of dicotyledons [181]. Compound 15 (mangiferonic acid) is one of common cycloartane triterpenes in Dipterocarpaceae [START_REF] Misra | Triterpenoids from Shorea robusta resin[END_REF][START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF]205,206]. It has been reported in Tillandsia sp. (Bromeliaceae) [207] and Mangifera sp (Anacardiaceae) [197,208].

In fraction C41 and C42, the use of silica gel as stationary phase did not allows satisfactory separation. The compounds were eluted in mixtures such as the mixtures of 18 + 19, 18 + 20 and the mixture of 19 + 20. Only a little amount of each compound was obtained as pure and it was not possible to get NMR spectrum of sufficient quality. (However, it should be noted that the revelation of TLC by the vanillin-sulfuric reagent is very sensitive, much more than the sensitivity of NMR analysis.) To facilitate the purification, the mixtures with carboxylic acid group in the structure were subjected to methylation with sodium bicarbonate and methyl iodide in anhydrous DMF (Scheme 13). Then, after reaction, methyl ester of each compound (18a, 19a, 20a) was succesfully isolated through silica gel column chromatography. Compound 18a was performed transesterification at C-26 with small quantity of CH 3 ONa (as a catalyst) and hydrolysis of the carboxymethylate at C-3 was suddenly taken place after redissolved in CH 3 COOH-H 2 O 3:1, an aqueous acidic condition (Scheme 14). The reaction with (S)-and (R)-MTPA-Cl by means of modified Mosher's method was carried out to detect the absolute =CHO 3,-en-28-oic acid, 14 Overall reaction 24 of 18a from modified Mosher's reaction seemed to be R-form with some ambiguity in the raw data, finally, the actual configuration was clearly determined as 24S,25S. The detail was discussed under the topic of compound 18, 18a, and 18b in section 4.4 "Identification and structural study of isolated compounds". The breakage of C-3 and C-4 bond of 3,4-seco-cycloartane-3-oic acid (18, 19 and 20) was explained through the oxidation process. Compound 20 could be proposed as the oxidation product of mangiferonic acid [START_REF] Tanaka | Hopeafuran and a C-glucosyl resveratrol isolated from stem wood of Hopea utilis[END_REF], a known cycloartane presented in this extract, as same as oxidation of schizandronic acid in Schisandra micrantha (Schisandraceae) [214]. During the course of this work, compound 20 has been isolated from the aerial parts of Abies georgei (Pinaceae) [217] and named abiestrine J, while its dimethyl ester (20a) had been isolated from Tillandsia usneoides (Bromeliaceae) [216]. Up to now, compound 18 and 19 have never been discovered from any other sources. The amount of 3,4-seco-cycloartane triterpenes was not sufficient for cytotoxic assay. The starting point for the assignments of compound 4 was the hydrogen (δ H 3.78) attached to the oxygenated C-3 (δ C 72.8). From COSY spectrum, this hydrogen (H-3) showed a correlation with two hydrogen atoms of H-2 (δ H 1.67 and 1.93) and H-4 (δ H 1.30). The proton signal at H-23 (δ H 0.98, d) also showed a correlation with the signal at H-4. The δ C of C-23 (δ C 11.6) was shifted upfield than other methyl groups but downfield compared to friedelane with -OH configuration on C-3 (δ C 10.0) [190] and 3-oxo friedelane. The remaining δ C and δ H were compared to the literature data [189,190] and the structure was identified as epifriedelanol (synonym: 3 -friedelinol). The EI-MS spectrum confirmed this conclusion.

R 1 ,R 2 =O, R 3 =COOH, R 4 =CH 3 Betulonic acid, 5 R 1 =OH, R 2 =H, R 3 =COOH, R 4 =CH 3 Betulinic acid, 13 R 1 =H, R 2 =OH, R 3 =COOH, R 4 =CH 3 Epibetulinic acid, 10 R 1 ,R 2 =O, R 3 =CH 2 OH, R 4 =CH 3 Betulone, 11 R 1 ,R 2 =O, R 3 = CH 3 , R 4 = CH 2 OH 30-Hydroxylup-20(29)-en-3-one, 12 R 1 ,R 2 =O, R 3 =COOH, R 4
R 1 ,R 2 =O, R 3 =CH 2 OH, R 4 =CH 2 OH 28,30-Dihydroxylup-20(29)-en-3-one, 16 R 1 ,R 2 =O, R 3 = COOH, R 4 =
The 1 H and 13 C-NMR data of both compounds were compared in Table 15.

1 4
Table 15 The 1 H-NMR (400 MHz) and 13 C-NMR (75 MHz) data of 1 and 4 (in CDCl 3 ); δ in ppm; J in Hz . This structure was then characterized as β-amyrin.

It was confirmed by the EI-MS spectrum that was compatible with the reference spectrum.

The identification of the triterpene moiety of 6 as being -amyrin was possible by direct comparison of the NMR data of 6 with the spectra of compound 2 (Table 16). The 1 H NMR spectrum was very close to that of -amyrin (Figure 41). The 13 C NMR spectrum of 6 corresponded to that of -amyrin with methine and quarternary carbon signals (δ C-12 121.7 and δ C-13 145.2), a characteristic of oleanene triterpenes. Detail analysis of the extra δ C in this spectrum indicated that this triterpene was esterified with fatty acid. This observation was supported by the presence of an additional methyl (δ C 14.2), many methylenes (δ C [START_REF] Smitinand | The manual of Dipterocarpaceae of mainland South-East Asia[END_REF][START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF] and acyl group (δ C 173.7). In addition, the oxymethine carbon (C-3) appeared at higher chemical shift (δ C 80.6) than that observed for -amyrin (δ C 79.0).

This phenomenon also appeared in δ C-3 of 3-fatty acid ester of -sitosterol (7 and 8) (Table 17, Figure 42) and implied esterification at C-3. Moreover, there was no additional signal of carbon with double bond then this fatty acid should be saturated fatty acid. However, its amount was not enough for transesterification to establish the complete structure.

6

Table 16 The 1 H-NMR (400 MHz) and 13 C-NMR (75 MHz) data of 2 and 6 (in CDCl 3 ); δ in ppm; J in Hz This was supported by the ESI-MS spectrum ([M+Na] + m/z at 437) and GC-EI-MS spectrum (comparison to the reference spectrum).

The 1 H-NMR of 7 showed protons of H-3 and H-6 at δ H 4.10 and δ H 5.41, respectively.

The position of H-3 was shifted down field compare to that of -sitosterol (Figure 42). Transesterification of 7 yielded -sitosterol and methyl palmitate (m/z 270) which were characterized by GC-MS analysis. Although small amount of methyl stearate was also found in MS spectrum, the main ester was determined as -sitosterol palmitate [START_REF] Ge | Hopeahainol A: an acetylcholinesterase inhibitor from Hopea hainanensis[END_REF].

8

The 1 H-NMR and 13 C-NMR spectra of compound 8 were consistent with those of 7, except more δ C signals of double bond (127.9, 128.0 and 130.2 ppm) (Figure 42). After transesterification, methyl palmitate, methyl linoleate and methyl oleate were obtained by means of GC-MS determination. However, the amount of palmitate ester was still more than the other 2 unsaturated fatty acid esters. The isolated compounds in this group were lupane triterpenes. All lupanes gave blueviolet spot with vanillin-sulfuric TLC reagent.

IR spectrum of compound 5 obviously showed the presence of carboxylic (3500-2500 cm -1 , broad band) and carbonyl (about 1705 cm -1 , sharp) groups. The HSQCedited correlation of compound 5 indicated 2 terminal olefenic protons at 4.75 and 4.62 ppm attached on the same δ C 109.8 ppm that confirmed the terminal methylene structure. The HMBC spectrum showed the correlation between these 2 olefinic protons and a methyl carbon at 19.36 ppm (C-30) as well as a quaternary carbon at 46.9 ppm (C-19) (Figure 12). Lupane group normally had 7 methyl groups. However, one methyl group was oxidized to carboxylic group that shown at 180.8 ppm of 13 C-NMR spectrum.

The oxidation at C-28 made δ C of C-16 and C-22 upfield shift. There were also the correlations between ketone carbon (δ C 218.2) and the protons of 2 methyl groups (δ H 1.08, 1.03) that indicated carbonyl group on ring A at position 3.

After searching for chemical constituents of Dipterocarpaceaous plant and comparison to previous NMR data [129], compound 5 was identified as betulonic acid. It was supported by the MS (ES-: m/z 453 of [M-H] -). The TLC trace and the amount of each fraction showed that betulonic acid ( 5) was the most abundant compound in this extracts.

Both compound 10 and 13 exhibited the equal mass [M-H] -m/z at 455 amu representing molecular formula of C 30 H 48 O 3 . Their IR spectrum obviously showed the presence of carboxylic (3500-2500 cm -1 , broad band) and hydroxyl (about 3400 cm -1 ) groups. The NMR spectra of both compounds were very close to betulonic acid [START_REF] Atun | Oligostilbenoids from Hopea Mengarawan (Dipterocarpaceae)[END_REF] and indicated that a hydroxyl group was substituted at C-3 instead of a keto group. Selected HMBC correlations of both compounds were shown in Figure 16. The difference between 10 and 13 was only the stereochemistry of this position. H-3 of 13 resonated at δ H 3.21. Its splitting pattern (dd, J= 11.2, 4.8 Hz) indicated it was arranged in axial orientation [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF][START_REF] Siddiqui | Oleanderol, a new pentacyclic triterpene from the leaves of Nerium oleander[END_REF]111]. On the other hand, H-3 of 10 resonated at δ H 3.40 and its equatorial orientation was suggested based on the splitting pattern (t, J=2.7 Hz) [START_REF] Monaco | Isoprenoids from the leaves of Quercus suber[END_REF][START_REF] Siddiqui | Oleanderol, a new pentacyclic triterpene from the leaves of Nerium oleander[END_REF]113,127,185,193].

Furthermore, δ C of CH 3 -24 which was the axial methyl group at the C-4 was strongly influenced by the stereochemistry of the hydroxyl group at C-3. Indeed, δ C-24 of 13 showed upperfield shift (∆+7 ppm) compared to that of 10. Moreover, δ C of C-1 and C-5 of 13 shifted to lower field as compared to those of 10 on account of γ-e ect. The δ C of C-2, C-3 and C-4 of 13 also shifted lower field, compared to those of 10, but with lesser degree. From all of these data, compounds 10 and 13 were elucidated as epibetulinic and betulinic acid, respectively. 16). The physical properties and spectroscopic data were compared to previous literature [125,194,195]. Then, it was identified as betulone.

The MS of compound 12 showed the same mass as compound 11. The 13 C-NMR spectrum exhibited the presence of 30 carbon signals which were assigned by the 2D HSQCedited experiment as 6 methyl, 10 methylene, 5 methine, 5 quatenary carbons, two olefinic carbons, 1 hydroxy methylene and 1 carbonyl carbons. Its NMR data were close to compound 11. In the 1 H-NMR spectrum, the signals at δ H 4.17 showed the hydroxyl group, as well as two olefinic protons at δ H 4.91 and 4.95. The HMBC correlations between δ H-29 4.91/4.95 and δ C-30 64.9 and -δ C-19 43.8 together with between δ H-30 4.17 and δ C-20 154.7 (Figure 16) allowed to the assignment of the OH group at C-30. With the comparison to other lupanes in this study [START_REF] Atun | Oligostilbenoids from Hopea Mengarawan (Dipterocarpaceae)[END_REF][START_REF] Ito | Two novel trimeric resveratrol derivatives from Cotylelobium lanceolatum[END_REF][START_REF] Liu | New resveratrol oligomers from the stem bark of Hopea hainanensis[END_REF][START_REF] Syah | Two oligostilbenes, cis-and trans-diptoindonesin B, from Dryobalanops oblongifolia[END_REF], C-28 of this compound was a methyl carbon. This methyl group significantly affected C-17 (upfield) and C-16 as well as C-22 (comparable downfield shift). The C-28 methyl group also was suggested by the HMBC correlations of δ H-28 at 0.80 ppm with δ C at 35.4, 43.3 and 48.8 ppm of C-16, C-17 and C-18, respectively. With all of this spectroscopic data and comparison to the literature [START_REF] Ribeiro | Lupane pentacyclic tricyclic triterpenes isolated from stems and branches of Maytenus imbricata (Celastraceae)[END_REF][START_REF] Tinto | Lupane triterpenoids of Salacia cordata[END_REF], compound 12 was identified as 30-hydroxy-3-oxolup-20( 29 16). These olefinic H-29 signals were presented at lower field because of the effect of hydroxyl group at C-30. From HSQCedited spectrum, the 2 hydroxyl protons (δ H-28 at 3.33 and 3. 16). The other assignment of carbon and proton positions was consistent to those of 16. Finally, it was identified as 29-hydroxy-3-oxolup-20(30)-en-28-oic acid or messagenic acid G.

Caryophyllene oxide (9)

Figure 18 Structure of caryophyllene oxide [START_REF] Atun | Oligostilbenoids from Vatica umbonata (Dipterocarpaceae)[END_REF] Caryophyllene oxide was identified by using GC-EI-MS ([M] + ion at m/z 220 and high matching quality with caryophyllene oxide spectrum). [START_REF] Kanokmedhakul | Bioactive constituents of the roots of Polyalthia cerasoides[END_REF]191,192]. Figure 19 Structure and Selected HMBC correlations of compound 15

The 1 H-NMR spectrum of compound 15 indicated the presence of a cycloartane skeleton.

The typical doublets at δ H 0.63 and 0.84 (J=4.1 Hz) were the characteristic of a cyclopropane methylene (H 2 -19). A keto group at C-3 was suggested based on δ C at 216.8

which showed HMBC correlations with H 3 -28 and H 3 -29 (Figure 19). Its [M-H] -ion at m/z 453 corresponded to the molecular formula C 30 H 46 O 3 . A trisubstituted olefin group was indicated based on an olfinic proton (δ H-24 6.95) and two olefinic carbons (δ C-24 145.9 and δ C-25 126.5). A carboxylic and a methyl were suggested for substitution on C-25 since there were HMBC correlations between the methyl protons (δ H-27 1.90) and the olefinic carbons together with the carboxylic carbons (δ C-26 172.7) (Figure 19). The conjugated double bond was established to be in the E-conformation due to the downfield shifted of the double bond proton caused by the close proximity of the carbonyl carbon (δ H 6.09 for Z-form of schizandronic acid) [196]. It was clearly supported by the chemical shift of the CH 3 -27 (δ C 12.00), since it was shielded by steric interaction with the C-23 methylene (δ C 20.6 for Z-form) [196].

The data of compound 15 were in agreement with the reported data [196] of mangiferonic acid which was one of common cycloartane triterpenes in Dipterocarpaceae [START_REF] Misra | Triterpenoids from Shorea robusta resin[END_REF][START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF]205,206]. Compound 18 exhibited a characteristic pair of doublets at δ H 0.42 (J=4 Hz) and 0.73 (J=4 Hz), corresponding to the methylene protons of the cyclopropane ring of a cycloartane triterpene. Position of these two protons at C-19 was concluded from their HMBC correlations to C-1, C-5, C-8 and C-11 (δ C 28.8, 45.9, 47.7 and 26.9, respectively) (Figure 22). One carboxylic group (C-3) at δ C 178.20 ppm showed long range correlation to H-2

(δ H 2.57) which subsequently correlated to C-1(δ C 28.8). The HMBC correlations were also observed among protons and carbons at position 4, 5, 28 and 29, but no long range correlations from these protons and carbons to those at positions 1, 2 and 3 (Figure 22). This suggested the clevaging of ring A at positions 3-4. Then, a 3,4-seco-cycloartane skeleton was suggesteded for this compound. However, this compound possessed more 20 carbons than normal cycloartane. Based on NMR data, a C20 fatty acid side chain was suggested.

The chemical shifts δ C at 34.3, 31.9, 29.1-29.7, 22. According to Ohtani et al.[169], the stereochemistry of the substituents adjacent to the secondary hydroxyl group was determined by difference in their 1 H NMR resonances in the (S) and (R)-MTPA esters (∆δ=δ S -δ R ). The absolute configuration of 18a at position 24 created using modified Mosher's method was supposed to be R-form due to positive values of ∆δ obtained for H-20, H-21, and H-22b, as well as opposite results for H-26a, H-26b, and H-27. However, there were some difficulties in the rate of reaction, quantity of reaction product and some unclear 1 H-NMR data. First, esterification reactions with both S-and R-MTPA-Cl were not clean and/or conversion rates were low. Only 2 mg of ester was isolated from the reaction with S-MTPA-Cl and most of starting molecules were still unchanged. The low yields of esterification might be resulted from the hindered reaction due to long chain fatty acid at position 26. In case of reaction with R-MTPA-Cl, the reaction took very long time and the conversion was very low too. Furthermore, 1 H-NMR spectrum of product fraction showed a mixture of two compounds (in a 1:2.45 ratio). This suggested epimerization of the product and calculation was made on the major compound. Secondly, very low difference values, ∆δ = +0.0008 and +0.0036, were calculated for H-21 and H-20, respectively. Other signals on the left of side chain, H-22b and H-23, were difficult to be assigned because of low amount of the ester product. It is consistent with the results from Banskota et al's work . They found that the advanced Mosher's method could not be applied to determine the configuration of a cycloartane which hydroxylated at C-24 and C-25. The presence of C-25 hydroxylated group made unusual conformation and caused dehydration into olefin (double bond between C-25 and C-27). Then, in our work, the presence of ester group at both C-24 and C-26 may promote epimerization at C-25. A better way to determine the actual configuration has recently been reported through an asymmetric synthesis of four isomers of ganodermanotriol [219] (a lanostane-type triterpene bearing a triol side chain similar to compound 18b). By comparison to the 13 C-NMR data of those four synthetic isomer (recorded in CDCl 3 ), the absolute configuration was clearly determined as 24S,25S.

(R) (S) H 3 COOC (S) (R) (R) (R)

C-24 Fatty acid ester of (24S,25S)-24,25,26-trihydroxy-3,4-secocycloart-4(29)-en-3-oic acid (19) and its 3-methyl ester (19a)

Compound 19 was always obtained in the mixture with 18 or 20. Methylation was performed to enable them to separate from each other by SiO 2 cc. Pure compound 19 from fraction C41 and C42 was so few that we cannot run with enough quality of NMR spectrum. However, it was possible to establish the spectrum of methylation product of 19 (=19a). NMR assignments of compound 19a were compared to those of 18a, with significant differences noted only for resonances associated with position 22 to 27. As same as compound 18 and 18a, there were the HMBC correlations of H-21 ( δH 0.88) with C-17 ( δC 52.3), C20 ( δC 35.6) and C-22 ( δc 32.7), indicating side chain at C-17 (Figure 22). The

19a

R=C 17 H 35 HMBC spectrum also showed the correlatations of H-24 ( δH 4.73) to C-25 ( δC 73.2) and C-27 ( δC 17.6), and the correlations of H-27 to C-25 and C-26. However, the resonances of H-22, H-23 and H-24 were downfield shift whereas those of H-26 and H-27 were undergone upfield. Another interesting difference from 18 and 18a was that the HMBC correlations of carbonyl carbon of fatty acid ( δC 175.7) with methine proton H-24 ( δH 4.73) instead of methylene protons H-26 ( δH 3.38 and 3.23). These data indicated that esterification was on hydroxy group of C-24 instead of C-26. The difference between the HRESI-MS of 19a and 18b suggested stearic acid as the fatty acid of compound 19. Hence, the proposed structure of 19 was proposed as 24-stearic acid ester of (24S,25S)-24,25,26-trihydroxy-3,4-seco-cycloart-4(29)-en-3-oic acid. Nevertheless, the amount of 19a was too low to investigate more reaction. Compound 20 was obtained from the mixtures of 18+20 and 19+20 which were methylated to be able to separate each compound and then pure methyl ester product of 20 (20a) was hydrolyzed to its original molecule by LiOH. However, the amount of pure separated compound 20 was too low for extensive NMR study. -26). On the basis of the above spectroscopic studies the structure of 20 was identified as (24E)-3,4seco-cycloartane-4( 29), 24-diene-3,26-dioic acid [217].

20a 20a

CYTOTOXIC EVALUATION OF 8 ISOLATED LUPANES.

In the present study, cytotoxic evaluation of 8 lupane triterpenes using WST-1 antiproliferative method revealed that all compounds were inactive against HT29 and MDA-MB231cell lines. Betulonic acid ( 5) exhibited cytotoxicity against both HT116 and PC3 cell lines, with IC 50 values of 85.60+37.61 and 139.00+3.64 µM, respectively. Compounds 14 displayed weak cytotoxicity against PC3 cells (IC 50 282+17.78 µM). Compounds 13, 11, 17 were shown slightly cytotoxic effects against PC3 cell line. Interestingly, messagenic acid G (17, IC 50 43.5+16.26 µM), which differs from betulonic acid ( 5) by the presence of an additional hydroxyl group at the C-30 position, was about 2 fold more potent in inhibiting the growth of HCT116 cells than 5. In contrast, C-30 aldehyde diminished this activity. These results showed that 3-keto and 17-COOH groups were necessary for cytotoxicity against PC-3 cell line whereas the hydroxyl group at C-30 could enhance this activity.

CONCLUSION

Phytochemical study of H.odorata leaves hexane extract presented the major triterpenes as betulonic acid, -sitosterol and -amyrin, in the order from high to low quantity. Compound 18 and 19 are new, while compound 14 is isolated for the first time in the nature. Within this investigation, H.odorata provides various different triterpene compositions. Lupane triterpenes are the main triterpenes in this plant with the minor of cycloartanes and 3,4-seco-cycloartanes.

The chemotaxonomic marker in this extract should be lupane-and secocycloartane-type triterpenes. This work is the first research that studied deeply about triterpenes in H. odorata leaves.

Besides compound 5 and 13, other lupanes are characterized for the first time in family Dipterocarpaceae. Most of lupanes in this study have been found to be major components in plants from family Celastraceae [START_REF] Ribeiro | Lupane pentacyclic tricyclic triterpenes isolated from stems and branches of Maytenus imbricata (Celastraceae)[END_REF]127], Leguminosae [101] and Betulaceae [220].

On the other hand, this study shows an interesting relationship among these families. Purified lupanes exhibit closed structural similarities and differed from each other in the degree of oxidation, particularly at the positions C-3, C-28 and C-30. These findings underlined the chemotaxonomic significance of lupane triterpenes in H. odorata Roxb. This results are not in accordance with the conclusion of Geevananda et al [START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF] indicating lupeol as a marker for the genus Hopea, but it is betulonic acid.

The proposed of biosynthesis of 18, 19 and 20 was based on the oxidation between C-3 and C-4 bond. The relationship between compound 20 and mangiferonic acid [START_REF] Tanaka | Hopeafuran and a C-glucosyl resveratrol isolated from stem wood of Hopea utilis[END_REF], found in this extract as well, was observed through the similarity of the side chain. Since compound 18, 19 and 20 were isolated from higher polarity fraction than the others, more free triterpenes of 18, 19 and 20 might be obtained from the ethanol extract because of their higher polarity (compared to other isolated compounds).

In this research, we evaluated the cytotoxic activity of betulonic acid and its derivatives against four cell lines: PC3, MDA-MB-231, HT-29 and HCT 116. Cytotoxicity of this hexane extract might be from betulonic acid that obtained about 5% of dried extract. The result supports a specific activity against prostate cancer cells PC3, as previously described for betulonic acid [221]. The structure-activity realationship of lupanes is in agreement with the previous literature. The potency of activity was mainly based on the degree of oxidation at positon 3, 28 and 30. Moreover, messagenic acid G, 17, was found to display a significant cytotoxic activity against HCT116 cells. More extensive investigations are therefore needed for a better understanding of the structureactivity relationships of cytotoxic lupanes and to elucidate their mechanism of action. Because of higher oxidation degree at C-3 of seco-cycloartanes, in case it is possible to obtain more quantity, it is interesting to assess bioactivity of cycloartane and secocycloartane groups too.

PART II

Phytochemical Study of Dipterocarpus costatus wood.

I. BOTANICAL STUDY

Genus Dipterocarpus [START_REF] Pooma | Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis[END_REF][START_REF] Ridley | The Flora of the Malay Peninsula[END_REF][START_REF] Gardner | A Field Guide to Forest Trees of Northern Thailand[END_REF]177] The genus Dipterocarpus is widely distributed from Sri Lanka, India, South China to Indochina and Malaysia but not east of Wallace's Line. The resin takes the form of wood-oil and is obtained by cutting a large hole in the tree-trunk and burning it within. In Thailand, fourteen species have been recorded.

Genus description: Small to large evergreen or deciduous trees, usually buttressed in evergreen species. Bark scaly in evergreen species, roughly cracked or V-shape fissured in deciduous species. Stipule enlarged into protecting bud, lanceolate, caducous, with distinct ring-like stipular scar on twigs. Leaves coriaceous, symmetrical at base, distinctly plicate. usually large, sometimes sinuate-crenate; Straight, parallel side veins, bending just before margin. Domatia not present. Flowers white or pink, very fragrant. 5 Petals, twisted together into an open-mouthed funnel, fused at base & falling as one piece. Calyxtube free. Stamens numerous with long pointed projections on top of anthers. Anther narrowly-lanceolate, with 4 subequal pollen sacs. Filament compressed in upper half, broad at lower half. Ovary 3-celled, 2 ovules in each. Inflorescences usually raceme (unbranched panicle), axillary or terminal, with 3-4 large flowers, zig-zag arrangement. Flower buds almost sessile or with short pedicels. Fruit large with 2 long and 3 much shorter wings (calyx lobes), fused together at base and completely covering the nut. Nut usually with apically conical stylopodium. Cotyledon thin, convolute.

Dipterocarpus costatus

Gaertn.f. [25-27, Diagnostic characters: Deciduous tree to 40 m with very tall, straight trunk and rather open spherical crown. Bark pale brown peeling in thin rounded flakes leaving a distinctive swirling pattern, reminiscent of temple motifs. Indumentum dark brown tufted hairs on young twigs, buds, petioles and racemes. Leaves 8-14 * 4-8 cm, usually elliptic to ovate with slightly pointed tip and blunt or slightly heart-shaped base, more or less hairy on both surface, secondary nerves 10-18 pairs. Young leaves densely covered with starshaped hairs, mature leaves with scattered short hairs on veins and lower surface. Stalks 1.5-2.7 cm, stout, usually with long shaggy hairs and fairly persistent narrow stipules, 5 mm. Flowers 2 cm, pale orange, in short unbranched clusters of 3-6 flowers at axils of young leaves. Calyx narrowly ridged, coarsely hairy, 8-12 stamens. Fruits 2 long wings, 8-12 cm, 3-5 main veins, 3 short wings <1cm, rounded and deeply folded. Body of fruit 1.2-1.5 cm globose with 5 narrow ridges, <2 mm wide, roughly hairy. Young fruits bright red, standing out clearly against the dark green leaves, often produced in great profusion Distribution: A widespread species, distributed from the Andaman Islands, Bangladesh to lower Myanmar, Indochina except North Vietnam, and Peninsular Malaysia (from Negri Sembilan northwards). Found throughout Thailand, occasionally by streams, 50-1,300 m altitude. It also occurs in association with Pinus merkusii in disturbed lowland semi-evergreen forest and in typical deciduous dipterocarp forest at Khong Chiam, Ubon Ratchathani. (Figure 24) Phenology Flowering: January-December Fruiting: January-December Putative hybrids : Dipterocarpus costatus x D. obtusifolius has been reported several times, especially at high elevation in deciduous dipterocarp forest, Doi Suthep, and also in Myanmar, but can also be found in lowland areas, under 100 m altitude, in the same area as the hybrid D.alatus x D. costatus. The leaves resemble those of D.obtusifolius, the bark and fruits are closer to D. costatus in size and shape.

II. PREVIOUS CHEMICAL WORK ON GENUS DIPTEROCARPUS

Though some studies have been conducted on the bark and timber, the phytochemical study of genus Dipterocarpus have been mostly based on the resin which plays an important economical role in South Eastast Asian countries. The major constituents are in terpenoids especially, dammarane and ursane [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF][START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF][START_REF] Mclean | Extractives from the Dipterocarpaceae[END_REF]222]. The most abundant terpene of the extracts from the genus Dipterocarpus is dipterocarpol.

The main neutral triterpenes of dammar resin from the early study were dammadienone, dammadienol, two isomeric hydroxydammarenones and their related diols, and a third ketol, hydroxyhopanone. Based on acid triterpenes, in addition to dammarolic acid, three acids were isolated ursolic acid, dammarenolic acid and dammarenonic acid, the latter obtained only in small yield as its methyl ester (1955) [222]. The work of [START_REF] Mclean | Extractives from the Dipterocarpaceae[END_REF] demonstrated dipterocarpol from light petrolatum extract of D.verrucosis and D.glandiflorus wood [START_REF] Mclean | Extractives from the Dipterocarpaceae[END_REF].

The following chemical analysis of the resin from 42 different species of the genus Dipterocarpus by [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF] [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF] revealed the presence of the following sesquiand triterpenoids: humulene, caryophyllene, copaene, -gurjunene, calarene, γ-gurjenene, alloaromadendrene, cyperene, caryophyllene oxide, farnesane, dehydrofarnesane, dipterocarpol (hydroxydammarenone-II), dammarenediol-II, dammaradienone, and ocotillone. The triterpene of the various species showed little variation. The sesquiterpenes were much more variable, and caused defining of six groups in the genus, on the basis of the composition of the sesquiterpene fraction of their resins. [START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF] [START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF] worked on the triterpene fraction of oleoresin from Dipterocarpus pilosus and found dipterocarpol with several dammaranes (dammara-20,24dien-3-one, dammara-24-ene-3,20-diol, ocotillone-II, hollongdione, and dipterocarpolic acid), accompanying two ursane derivatives -asiatic acid (with two of its acetyl derivatives), and 2 -hydroxyursolic acid. [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF] [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF] isolated terpenoids from resin and bark of 2 Dipterocarpus species (D.hispidus and D.zeylanicus). Dipterocapol was found in large quantities accompanied by asiatic acid, ocotillone, dammarenediol 20S, betulinic acid, humulene and alloaromadendrene. It has been shown that dipterocarpol, the major component of the neutral fraction of D. costatus resin, was present to the extent of ca 48%.

The mixture of sesquiterpenoids, caryophyllene, humulene and alloaromadendrene was the next most abundant (ca 24%) and the compounds were present in the ratio 7:10:3, respectively. Ocotillone 20R and ocotillone 20S were isolated in yields of ca 0.05%, and ca 0.04% respectively. The petrol insoluble fraction of the resin was shown to contain 2 component ; 2 ,3 -dihydroxyurs-12-en-28-oic acid (minor, 0.13%) and asiatic acid (major, ca 15%, 2 ,3 ,23 -trihydroxyurs-12en-28-oic acid).

A resveratrol tetramer, named diptoindonesin E, was isolated from the acetone extract of the tree bark of Dipterocarpus hasseltii, together with (-)--viniferin, laevifonol, (-)--viniferin, vaticanol B, (-)-hopeaphenol, and a coumarin, scopoletin. Hopeaphenol strongly inhibited murine leukemia P-388 cells [START_REF] Muhtadi | Cytotoxic resveratrol oligomers from the tree bark of Dipterocarpus hasseltii[END_REF].

A study of flavonoid pattern of D. costatus leaves found the same aglycone as H.odorata (quercetin, kaempferol and apigenin) but different flavonoid glycoside (quercetin 3-rutinoside) [START_REF] Joshi | Leaf flavonoid patterns in Dipterocarpus and Hopea (Dipterocarpaceae)[END_REF].

III. EXPERIMENTAL PART

METHOD AND APPARATUS

As same as those for H. odorata. • -against NCI-H187 Antifungal activity

PRELIMINARY EXTRACTION

• Not tested Antimalarial activity

• -Antituberculosis

• Not tested

In vitro bioactivity assays

The hexane and the ethanol extracts were assayed for biological activity with the same method as applied for H.odorata extract. The result was shown in Table 26. • maceration (EtOH, 3L*48h*3)

• filtering and drying D02 and isofouquierone (40, 119 mg) for fraction D50. When fraction D16 was dried, its characteristic was a white flake which showed a broad single spot on TLC (cHex-EtOAc 2:1, sprayed with H 2 SO 4 /vanillin reagent) with some pale spots of impurity. The spectroscopic data indicated the structure of -sitosterol (3, 200 mg). Other fractions were purified by rechromatography over SiO 2 column chromatography. 20,25,26-trihydroxydammar-23-en-3-one (46, 8

Study of fraction D11

Fraction D11 (375 mg) was further chromatographed over SiO 2 , using cHex-EtOAc 30:1 as mobile phase, to yield caryophyllene oxide (9, 150 mg)

Study of fraction D22

Fraction D22 (502 mg) was subjected to SiO 2 cc using cHex-EtOAc (8:1) system as eluent. After elution, dipterocarpol (22, 400 mg) and isofouquierone peroxide (23, 20 mg) were obtained.

Study of fraction D42 and D43

Fraction D42 and D43 were combined (360 mg) and chromatographed over SiO 2 cc, eluting with cHex:EtOAc (3:1) to give (20R) -20-hydroxy-17 ,29-epoxy-28-norlupan-3one (42, 17 mg) and (20S,24S) -20,24-dihydroxydammar-25-en-3-one (38, 73 mg).

Study of fraction D44

Compound 38 (15 mg) was crystallized in fraction D44 and was characterized as (20S,24S) -20,24-dihydroxydammar-25-en-3-one (38).

Study of fraction D53

Fraction D53 (98 mg) was chromatographed by SiO 2 cc with cHex-EtOAc (1:1) and further purified using GC-MS to give an unidentified sesquiterpene and clovane-2,9-diol ( 43)

Study of fraction D55

Fraction D55 (163 mg) was chromatographed over SiO 2 cc, eluting with cHex-EtOAc (1.5:1) to give isofouquierol (44, 54 mg).

Study of fraction D58 and D59

Fraction D58 (59 mg) and D59 (48 mg), upon further chromatography over SiO 2 cc using cHex-EtOAc (1:1), furnished (20S,22E,24R)- 20,24,25-trihydroxydammar-22-en-3-one (45, 6 and 5mg, respectively).

Study of fraction D64 and D65

Chromatography of fraction D64 (80 mg) and D65 (58 mg) over SiO 2 cc with cHex-EtOAc (1:8) a orded (20S,23E) -20,25,26-trihydroxydammar-23-en-3-one (46, 8 and 4 mg, respectively).

Study of fraction D66

Fraction D66 (286 mg) was subjected to silica gel column chromatography with CH 2 Cl 2 :MeOH (30:1) and addition of a little of acetic acid gave (20S,24R)-20,24-epoxy-25-hydroxy-2,3-seco-dammarane-2,3-dioic acid (47, 16 mg) and (20S,23E)- 25hydroperoxy-20-hydroxy-2,3-seco-dammar-23-en-2,3-dioic acid (48, 5 mg).

Study of fraction D68

Fraction DML68 (138 mg) was rechromatographed over silica gel (cHex-Acetone-MeOH = 20:4:1) to give (20S) -20-hydroxy-3-oxo-24,25,26,27-tetranordammar-23-oic acid (49, 5 mg) and (20S,24R) -20,24-epoxy-25-hydroxy-2,3-seco-dammarane-2,3-dioic acid (47, 8 mg).

* the compound found for the first time in the nature. ** the rare compound in the nature. *** New compounds. Scheme 20. Purification of D. costatus wood hexane extract (continued).

CYTOTOXIC EVALUATION OF ISOLATED TRITERPENES

The cytotoxic effect of the isolated triterpenes against various cell lines was evaluated using WST-1 method as same as that of lupane triterpenes. The result was shown in Table 20. The cytotoxic activity against prostate cancer cell line (PC3), human breast adenocarcinoma cell line (MDA-MB-231), colorectal adenocarcinoma cell line (HT-29) and colorectal carcinoma cell line (HCT 116) were determined. Briefly, cells were seeded in 96-well plates and incubated at 37°C for 24 h. Cells were then treated with the isolated compounds (about 7-10 µM). After 72 h incubation in 5%CO 2 and 37 o C, WST-1 reagent was added to the test wells and further incubated at 37 o C for 1 h. The absorbance of each culture was read at 455 nm. The results were compared to negative control (no compound

IN VITRO ANTIPLASMODIAL ACTIVITY OF ISOLATED TRITERPENES

In vitro antiplasmodial activity

Theory -The antiplasmodial activity of various triterpenes was determined according to Desjardins et al 1979 [223-225]. To evaluate the in vitro growth of Plasmodium falciparum FcB1 strain, [ 3 H]hypoxanthine was added to parasite microcultures. Inhibition of uptake of a radiolabeled nucleic acid precursor ([ 3 H]hypoxanthine) by the parasite served as the indicator of antimalarial activity. [ 3 H]hypoxanthine incorporation was directly proportional to the number of parasitized erythrocytes in culture. The isolated triterpenes were dissolved in dimethylsulfoxide (DMSO) and tested at a concentration of 10 µM for screening assay. The compounds showing significant inhibition rates were submitted to serial dilution with culture medium before being added to parasite cultures (1% parasite and 2% hematocrite) in 96-well microplates. The growth inhibition for each concentration was determined by comparing the radioisotope incorporated in the treated cultured with that in the control culture maintained on the same plate. The concentrations causing 50% and 90% inhibition of parasite growth (IC 50 , IC 90 ) were calculated from the drug concentration-response curves. All assays were done in triplicate. The experiments were prepared using strict aseptic techniques inside a laminar flow hood in the following procedure.

Screening test -A 96-well plate, with 8 rows and 12 columns, were used to prepared 300 µL of the mixture of 20 µM tested triterpenes and culture medium in DMSO. Take 100 µL of each mixture to the second and third plates (The experiments were triplicate in 3 plates). Then, add 100 µL of parasite culture to make total 200 µL each well. The plates were placed in the incubator at 37 o C for 24 h in the atmosphere without oxygen. After the 24-h incubator period, the plates were removed and 25 µL of [ 3 H]hypoxanthine was added to each well. The plates were then returned to the incubutor with the same temperature for an additional 24 h in the atmosphere with oxygen. The experiment was stopped by freezing at -25 o C. Incorporation of [ 3 H]hypoxanthine into DNA of parasites was measured using 1450 Microbeta trilux (Wallac) liquid scientillation and luminescence counter. An untreated parasite control was included. The result was shown in Table 29. [START_REF] Gonzalez | Triterpenes of Hippocratea celastroides (Celastraceae)[END_REF] -The obviously potent triterpenes were prepared in serial dilution from 100 to 0.195 µM with the parasite culture as above. After incubation of 48 h at 37 o C under anaerobic atmosphere, [ 3 H]hypoxanthine was added to all wells and the plates were again incubated for 24 h at 37 o C under aerobic condition. The reaction was stopped by freezing the plate at -25 o C. The result was expressed as the IC 50 and IC 90 values of the selected triterpene (Table 30). Chloroquine was used as a positive control.

Determination of IC 50 and IC

Cytotoxicity test on mammalian cells

Rat myoblast-derived cells L-6 was seeded into 96-well microplates and treated with 10 and 1µM of isolated triterpenes from antiplasmodial screening test for 24 h, at 37 o C under a 5% CO 2 atmosphere. Cytotoxicity was determined using the colorimetric MTT assay. After incubation sterile MTT solution was added to each well and incubated for another 2 h. The absorbance reduction percentages at 540 nm for the treated cultures and the untreated control culture were obtained and compared. The % growth inhibition of L-6 cell was then calculated. All assays were done in triplicate. The result was shown in Table 29.

(R) (R) (R) (R) (R) (R) (S) (S) O (S)
H

-Sitosterol (3) and caryophyllene oxide (9)

The obtained data were the same as those obtained from H.odorata leaves hexane extract.

-Elemene ( 21 Dipterocarpol ( 22) [39, 67, 71, 227- Cabralealactone (20S) (28) [START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]133,161,229,231] Synonym 25,26,27-trinordammaran-3-one-20(S) Cabraleone (20S,24S) (30) [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]138,141,148]

Molecular

NMR spectrum

1 H-NMR -see Table 38, 13 C-NMR -see Table 39 Dammarenediol II (31) [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF][START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]132,159,227

NMR spectrum

1 H-NMR -see Table 33, 13 C-NMR -see Table 35 Ocotillone (20S,24R) (34) [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]138,141,148] 1 H-NMR -see Table 34, 13 C-NMR -see Table 35 Ocotillol II (20S,24R) (37) [START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]135,147,153,154]

Molecular
Molecular

PLANT MATERIAL

D. costatus wood was also collected in Chiangmai and was prepared for the study by drying and chopping.

PRELIMINARY STUDY

The plant sample was extracted in soxhlet apparatus, with hexane following by ethanol, and yielded 1.0367 g (2.56%) hexane and 1.3755 g (2.73%) ethanol extracts, respectively. Bioactivity of both extracts was evaluated by BIOTEC. The hexane extract showed cytotoxic activity against MCF7 (breast cancer with IC 50 25.55 µg/mL) and NCI-H187 (small cell lung cancer with IC 50 9.06 µg/mL). Although the potency of in vitro cytotoxicity looked lower than that of the H.odorata leaves hexane extract, it was interesting that this extract expressed potent anti-malarial activity with IC 50 3.20 µg/mL against Plasmodium falciparum K1 strain. This result harmonized with Chavalit's work [START_REF] Sittisombut | Investigation of anti-cancer activity of plants in families of Rutaceae and Dipterocarpaceae in Thailand[END_REF] that only the hexane extract of D. costatus wood exhibited cytotoxicity against P-388 (murine lymphocytic leukemia), KB (humnan pharyngeal carcinoma), BCA-1 (human breast cancer), Lu-1 (human lung cancer) and Col-2 (human colon cancer) but was not observed from the ethanol extract.

EXTRACTION AND ISOLATION OF D. costatus WOOD HEXANE EXTRACT

Phytochemicalstudy of the hexane extract of Dipterocarpus costatus led to the isolation of 31 compounds. These compounds are as follows

Steroid

: -sitosterol (3) Sesquiterpene : caryophyllene oxide ( 9), β-elemene (21), clovane-2,9-diol [START_REF] Misra | Triterpenoids from Shorea robusta resin[END_REF] Triterpenes :

Dammarane and seco-dammarane type : dipterocarpol [START_REF] Chuakul | Medicinal Plants in Thailand[END_REF], dammarenediol II [START_REF] Pooma | Checklist of Dipterocarpaceae in Thailand[END_REF], isofouquierone peroxide [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF], isofouquierol peroxide [START_REF] Tabata | Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation[END_REF], isofouquierone [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF], isofouquierol ( 44), (20S,23E)-20,25,26-trihydroxydammar-23-en-3-one ( 46), (20S,23E)-25hydroperoxy-20-hydroxy-2,3-seco-dammar-23-en-2,3-dioic acid ( 48), (20S,22E)-20,24,25-trihydroxydammar-22-en-3-one ( 45), (20S,24S)-20,24-dihydroxydammar-25-en-3-one ( 38), (20S)-20-hydroxy-24-perhydroxy-dammar-25-en-3-one [START_REF] Smitinand | The manual of Dipterocarpaceae of mainland South-East Asia[END_REF], cereotagaloperoxide [START_REF]The free encyclopedia[END_REF], cabraleone [START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF], ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF], ocotillol II [START_REF] Muhtadi | Cytotoxic resveratrol oligomers from the tree bark of Dipterocarpus hasseltii[END_REF], (20S,24R)-20,24-epoxy-25-hydroxy-2,3-secodammarane-2,3-dioic acid [START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF] Nordammarane type : Isocabralealactone [START_REF] Gardner | A Field Guide to Forest Trees of Northern Thailand[END_REF], Cabralealactone ( 28) and 3-Epicabraleahydroxylactone [START_REF] Dai | HIV-inhibitory and cytotoxic oligostilbenes from the leaves of Hopea malibato[END_REF], Octanordammarane-3,17-dione ( 26) (20S,23E)-20-Hydroxy-27-nordammar-23-ene-3,25-dione ( 39), (20S) [START_REF] Pooma | Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis[END_REF][START_REF] Ridley | The Flora of the Malay Peninsula[END_REF] Norlupane type : 17 -hydroxy-28-norlupan-20( 29)-en-3-one ( 25), 17hydroxy-28,29-dinorlupan-3,20-dione (41), (20R)-17 ,29epoxy-28-norlupan-3-one ( 24), (20S)-29-hydroxy-17 ,20peroxy-28-norlupan-3-one ( 36), (20R)- 20-hydroxy-17 ,29epoxy-28-norlupan-3-one (42) The result is in agreement with the previous reported [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF]. The most abundant compound in this extract is dipterocarpol [START_REF] Chuakul | Medicinal Plants in Thailand[END_REF], following by ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF] and isofouquierone [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF]. Based on TLC trace, dipterocarpol [START_REF] Chuakul | Medicinal Plants in Thailand[END_REF] was found as a major constituent in fraction D21-D23. The total amount was more than 1 g. The TLC and NMR data showed that betulonic acid always contaminated in the subfraction of dipterocarpol. Ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF] presented totally almost 1 g in fraction D28-D29 while isofouquierone [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF] was obtained totally more than 500 mg in fraction D49-D51. Cabraleone [START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF], 24R-epimer of ocotillone, was also isolated from this extract although in less amount. This suggested cyclization and oxidation on the side chain of dipterocarpol, the basic dammarane. Although both C-3 keto and hydroxyl group were found in this extract, 3-oxodammaranes were major derivatives according to their higher amount.

The high amount of dammarane derivatives is interesting from a pharmacological point of view because of their antitumour activities [132]. Dammarenediol II [START_REF] Pooma | Checklist of Dipterocarpaceae in Thailand[END_REF] was suggested to be a valuable antitumor promoter (potential cancer chemopreventive agent) [160], an anti-inflammatory agent [162] and antiviral agent [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF]. Ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF], the second largest amount of triterpenes in this extract, showed moderate cytotoxicity against leukeamia cells (L-1210, IC 50 20µg/mL) [START_REF] Makino | Tirucallane-type triterpenes from Juliana adstringens[END_REF], and insect antifeedant and growth-regulating activities against Spodoptera litura [163]. Cabralealactone [START_REF] Ashton | Dipterocarpaceae[END_REF] exhibited potent cytotoxicity against P388 leukemia cells [161], as well as multidrug resistance-reversing effect [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF]. With in the same report, the potency of colchicine was also increased with the presence of (20S,24S) -20,24-dihydroxydammar-25-en-3-one (38). Isofouquierol [START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF] at the concentration of 100 µM showed 86.6% inhibitory effect on nitric oxide (NO) production induced by LPS in mouse peritoneal macrophages [149] that related to its antiinflammatory activity. In contrast, ocotillol II [START_REF] Muhtadi | Cytotoxic resveratrol oligomers from the tree bark of Dipterocarpus hasseltii[END_REF], which exhibited moderate to weak cell growth inhibitory activities to VA-13 (malignant lung tumor) and HepG2 cells (human liver cancer), displayed strong cytotoxicity to WI-38 (normal human lung cell) [147]. Then, it is not suitable to be developed for anticancer.

Dammarenediol-II ( 31) is, in plants, the primary biosynthetic products of the tetracyclization of (S)-2,3-oxidosqualene to be dammaranes, lupanes, ursanes and oleananes [START_REF] Xu | On the origins of triterpenoid skeletal diversity[END_REF]147]. The TLC showed the presence of dammarenediol II as a major component in fraction D26-D27, and its amount should be totally about 400 mg. The dammarenediols were first isolated by Mills in 1956 [222] from dammar resin produced by various Dipterocarpus species (in 0.05% yield). Two Dammarenediols have since been found esterified with fatty acids in the seed oil of Cacalia atriplicifola L.(Asteracea) [232] and as components of extracts from Cowania mexicana (Rosaceae) [233]. Dammarenediol-II was found in 3-acetate form in edible Chrysanthemum morifolium (Compositeae) [162]. Moreover, it appeared in 0lea madagascariensis (Oleaceae) [132], in seed oil of Camellia japonica (Theaceae) [160] and in the fruit of Ceriops tagal (Rhizophoraceae) [158].

Dipterocarpol [START_REF] Chuakul | Medicinal Plants in Thailand[END_REF], an oxidation product at C-3 of dammarenediol II and first isolated from Dipterocarpus species [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF]222,229], is a constituent of dammar resins and Ginseng sapogenins [227] and is abundant in galls of Pistacia terebinthus [234]. It is a common composition in genus Dipterocarpus. It was found to be the most abundant triterpene in the neutral fraction of oleoresin of various Dipterocarpus species [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF][START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]229]. The configuration of ring D was reported as 13 , 17 -H. octanordammarane -3,17-dione (26) Scheme 22. Semisynthesis of cabralealactone ( 28), 3-epicabraleahydroxylactone ( 35) and 3 -acetoxy-17-oxo-octanordammarane from dipterocarpol [START_REF] Chuakul | Medicinal Plants in Thailand[END_REF] [START_REF] Mills | The constitution of the neutral, tetracyclic triterpenes of dammar resin[END_REF]229,235] Although octanordammarane -3,17-dione (26) has previously been semisynthesized from oxidation reaction of 20(S),24(R)-epoxydammarane -3 ,17 ,25-triol (scheme 21) [230], to the best of our knowledge, this is the first instance of its isolation from a natural source. Compound 26 could be reduced to be octanor-13 -dammarane by the Wolff-Kishner reduction. Furthermore, Biellmann et al [235] reported 3 -acetoxy-17-oxooctanordammarane, which was possible to be converted to 26, as an oxidation product of 3 ,17-diacetoxy-octanordammarane, synthesized from dipterocarpol (scheme 22). In this work, octanordammarane-3,17-dione was supposed to be from the second pathway (Biellmann's work) because no 17-hydroxy dammarane was isolated from this extract. Scheme 22 also showed that cabralealactone ( 28) and 3-epicabraleahydroxylactone [START_REF] Dai | HIV-inhibitory and cytotoxic oligostilbenes from the leaves of Hopea malibato[END_REF] (lactone group) were the oxidative degradation products from dipterocarpol/dammarenediol. It has been proposed that ocotillone-group dammaranes (ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF], cabraleone [START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF] and ocotillol) were the intermediates in this biosynthesis pathway [133,154]. Warnhoff et al [154] suggested that ocotillol was probably formed in the plant from a dammarenediol derivative by the formation of 1,2-epoxidation at C 24 =C 25 with subsequent oxide opening at C-24 by the C-20 hydroxyl group (scheme 23). The work of Cascon demonstrated oxidation of cabraleone to cabralealactone by Jones reaction and then reduction to 3-epicabraleahydroxylactone with NaBH 4 (scheme 24) [133].

! " # ! " $ % % ! & $ " # ! # " & " # " "
Scheme 23. Proposed mechanism of ocotillol and 3-epicabraleahydroxylactone formation from dammarenediol derivative (in plant)

According to the previous literatures [START_REF] Joycharat | Terpenoid constituents and antifungal activity of Aglaia forbesii seed against phytopathogens[END_REF]141], the 20S and 20R of dammarane triterpenes with a tetrahydrofurryl or lactone sidechain can be easily distinguished by the resonance of C-21, which resonated at about C 23.6-27.2 ppm and at about 21.7-22.0 ppm for the 20S isomer and 20R isomer, respectively. Consequently, this fact was used to assign either 20S or 20R configuration of dammarane triterpenes accumulated in this extract. In our study, the stereochemistry at C-20 of all isolated dammaranes except compound 27 was characterized as S configured by comparison of the carbon chemical shifts at C-17, C-20, and C-21 with the corresponding reported dammaranes. Isocabralealactone ( 27), 20R-epimer of cabralealactone [START_REF] Ashton | Dipterocarpaceae[END_REF], has just been reported for the first time in the nature by Joycharat, N. in 2010 [START_REF] Joycharat | Terpenoid constituents and antifungal activity of Aglaia forbesii seed against phytopathogens[END_REF]. It was a rare compound and only found in Aglaia forbesii seed (Meliaceae). Compound 28 was obtained more than 27 so it agreed with the normal configuration (S) at C-20 of dammaranes in this extract. Both isomers exhibited the difference in the chemical shift at position 21 and 22, as well as the Specific rotation value. Cabralealactone ( 28) was isolated from family Betulaceae [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF], Meliaceae [133,139,143] and Capparaceae [150,161]. was isolated from the seed oil of the camellia (Theaceae) [160].

Ocotillone group was one of the major triterpene intermediates in family Meliaceae [133,137,139,141,144,145,[236][237][238][239], Betulaceae [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF]135] and Burseraceae [148]. Ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF] has been isolated from family Dipterocarpaceae [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF][START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF], Meliaceae [133,141,145,236], Betulaceae [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF]135], Julianiaceae [START_REF] Makino | Tirucallane-type triterpenes from Juliana adstringens[END_REF] and Simaroubaceae [240]. Cabraleone, its 24S-epimer, appeared in Meliaceae (especially genus Cabralea) [133,137,139,141,144,145,[236][237][238][239] In addition to dipterocarpol, fouquierol and isofouquierol [START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF], acyclic side chain of 3-hydroxydammaranes, have been reported to be biological precursors of 3hydroxycabralealactone and ocotillol II [START_REF] Muhtadi | Cytotoxic resveratrol oligomers from the tree bark of Dipterocarpus hasseltii[END_REF], respectively [155]. Fouquierone [START_REF]The free encyclopedia[END_REF][START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF] and isofouquierone groups [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF][START_REF] Tabata | Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation[END_REF][START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF][START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF] were commonly found in Burseraceae, Betulaceae, Fouquieriaceae, Meliaceae and Anacardiaceae plants [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF]136,140,146,148,242] but have never been isolated from Dipterocarpaceous plant.

Isofouquierone group is the third most abundant triterpenes in this study. Isofouquierone [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF], isofouquierone peroxide [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF], isofouquierol [START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF] and isofouquierol peroxide [START_REF] Tabata | Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation[END_REF] (in the order from high to low quantity in the extract) are known compounds.

Isofouquierone [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF] and isofouquierol [START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF] were proposed to be alternate pathways besides ocotillone group (cabraleone, cabraleadiol, ocotillone and ocotillol), if the 20,24,25-triol dammarane was viewed as a precursor [148] (figure 27). 23-ene-3 ,25,28-triol in literature [140, 148] that the reaction might have been catalyzed by traces of hydrochloric acid from the CDCl 3 solvent. The precise interpretation of these corresponding NMR spectra, with the presence of additional conjugated olefin and disappearance of hydroxyl C-25 group, confirmed this degradation. From this study, isofouquierone was proposed to be the precursor of the compounds in this group. The oxidation of isofouquierone will result in the other 3 compounds [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF][START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF][START_REF] Seo | Resveratrol tetramers from Vatica diospyroides[END_REF]. The compounds of this group with the reduction on C-3 carbonyl group [START_REF] Tabata | Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation[END_REF][START_REF] Cheung | Structures of triterpenes from Dryobalanops aromatica[END_REF] were less seen in this extract.

Compound 29, 32 and 38 were classified as fouquierone group but have not been determined the configuration on C-24 except 38. They were position isomers of double bond and hydroxyl (or hydroperoxide) group on the side chain of isofouquierone peroxide [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] and isofouquierone [START_REF] Tabata | Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation[END_REF]. Compound 29 and 38 were 3-oxodammarane while C-3 carbonyl group of 32 was substituted by hydroxyl group. On the other hand, hydroperoxy group were on C-24 of compound 29 and 32 with unidentified configuration, while 24Shydroxyl group appeared in compound 38. No NMR data have been reported to indicate configuration on hydroperoxy (C-24) carbon of the sidechain of 29 and 32. Compound 29 should be 20(S)-hydroxy-24-perhydroxy-dammar-25-en-3-one with unidentified configuration on C-24 rather than fouquierone as described in literature [146] because of downfield shift of C-24 as well as the MS spectrum at m/z 497.05 representing C 30 H 50 O 4 Na of the [M+Na] + ion. There was no any reported NMR data for this compound before. Although compound 32 was characterized as cereotagaloperoxide, its proposed structure was closer to fouquierol peroxide because the functional group at C-29 was CH 3 instead of -CH 2 OH as in cereotagalol A. Compound 32 has just been found only in Ceriops tagal (Rhizophoraceae). In this study, compound 29 and 32 were found only a little quantity and contaminated with isofouquierone peroxide [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF] and isofouquierol peroxide [START_REF] Tabata | Vaticanol B, a resveratrol tetramer, regulates endoplasmic reticulum stress and inflammation[END_REF], respectively. Each pair of compounds possessed the silimar IR spectrum, exact masses, and R f value and were difficult to be separated on the column. Compound 38 was found much more than the former 2 compounds as same as its isomer, isofouquierone [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF], that appeared with much larger amount than the peroxide derivatives. Compound 38 has been isolated from genus Betula (family Betulaceae) [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF]136,140] and was found to exhibit mild cytotoxicity with MDR effect [START_REF] Kashiwada | Triterpenoids from the floral spikes of Betula platyphylla var. japonica and their reversing activity against multidrug-resistant cancer cells[END_REF].

Compound 45 and 46 were characterized as new acyclic dammarane compounds. The structure of 46 was very close to isofouquierone except C-26 which was oxidized to be hydroxyl methylene group. Adversely, compound 45 looked like fouquierone with the oxidation of C-25 to quaternary hydroxyl carbon and dehydrogenation at C-22 and C-23. The influence of the 2 adjacent hydroxyl group make downfield shift of C-24 and C-25, compared to compound 38 and 40. Compound 45 was first isolated from fraction D58 but, unfortunately, it degraded in CDCl 3 solvent within 24 hours during NMR experiments. The NMR data of degradation product showed an additional double bond with terminal methylene group and the loss of water at position 20 to make double bond. It might be the same reason as compound 40 that traces of hydrochloric acid in CDCl 3 solvent caused this degradation. However, it was isolated again in fraction D59. The NMR solvent was changed to deuterated-acetone.

Compound 39 and 49 are new nor-dammarane triperpenes while compound 26 is a nor-dammarane that has been isolated for the first time from plants and has already been discussed on its biosynthesis from the oxidation of dipterocarpol and lactone groups. The side chain of 39 is very close to that of isofouquierone excluding the loss of one C-27 methyl and the substitution of C-25 hydroxyl with a carbonyl carbon. It showed that isofouquierone was possible to be oxidized to compound 39. Compound 49 was the only tetranordammarane monoacid found in this extract. It was supposed to be produced from the cleavage at C-23 and C-24 of isofouquierone.

In general, ring-A opened triterpenoids have a cleavage between C-3 and C-4, while 2,3-seco-triterpenoids, as compound 47 and 48 are rare in the nature. This is the first time that 2,3-seco-dammarane derivatives have been isolated from a Dipterocarpaceous plant. They appeared in high polarity fractions. However, 2,3-seco-acids have been synthesized by Zorina et al and Wei et al [243,244] through oxidative scission of A-ring, with the aid of either potassium bichromate in the presence of sulfuric acid or hydrogen peroxide in potassium hydroxide and methanol (H 2 O 2 /KOH/MeOH). The proposed structure of 47 and 48 revealed that they are 2,3-seco-dioic acid derivatives of ocotillone and isofouquierone peroxide, compounds with high quantity in this extract. Compound 48 was then performed NMR experiment in acetone to avoid dehydration.

A group of norlupane derivative was isolated. The plausible pathway for the formation of 17 -hydroxy-28-norlupan-20(29)-en-3-one ( 25) might be from C-28 ( -orientation) decarboxylation of betulonic acid (found in trace amount with dipterocarpol subfraction) and the attachment of 17 -hydroxyl group to be the new norlupane. The NOE correlations of compound 41 confirmed the C-17 -hydroxyl configuration, that were consistent with the suggestion of previous assumption [107,108]. This result was refered to C-17 of compound 25 as well. Compound 25 was assigned to be the precursor of the other isolated norlupanes. Cyclization between C-29 and 17 -hydroxyl group of C-17 produced compound 24. Compound 36 and 42 were the oxidation products at C-20 of 25 to be hydroxyl group instead of terminal olefin, nevertheless cyclization took place on different position. Cyclization of compound 42 happened between one of methyl group and hydroxyl group of C-17 while one of methyl group after C-20 oxidation of 36 was continually oxidized to be methylene alcohol and cyclization was formed between two hydroxyl groups of C-17 and C-20. Moreover, oxidation of C-29 olefin of 25 to make keto group on C-20 generated compound 41. The NOE correlations of compound 42 showed that the assumption of 17-hydroxyl configuration from the chemical shifts of C-13 as described in literature [107,108] could not applied to the ring side chain at C-17. As the result, the -configuration was assigned on C-17 and C-19 of compound 24, 36 and 42.

The 3 -OH derivative of compound 24 showed stronger potency of antiproliferative activity against the malignant SA mouse mammary epithelial cells than that of betulinic acid [107]. Hence, cytotoxicity of this norlupane group is interesting to be investigated. 32) suggested the 3-oxodammarane skeleton. The side chain consisted of a hydroxyl group as well as one double bond between quaternary carbon and methine carbon. In the HMBC spectrum, the correlations of the methyl proton at δ H 1.17 As the result of a detailed spectroscopic analysis and comparison with the published data, the structure of 22 was identified as dipterocarpol, a common triterpene in Dipterocarpaceous plants.

The NMR data of 31 corresponded to those of dipterocarpol except the 1 H and 13 C resonance of position 2, 3, 4 and 29. The chemical shift indicated the presence of C-3 methine alcohol in stead of carbonyl group. The HMBC spectrum also exhibited the similar correlations with those of dipterocarpol such as the correlation of H-21 with C-17, C-20 and C-22, including that of H-26 and H-27 with C-25 and C-24 (figure 29).

GC-EI-MS investigation for 31 became available for the present study. It was significant that the [M]+ ions in this compound was not visible in the spectra [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF]. MS spectra of 31 showed the tetracyclic dammarane fragments at m/z 317, 299, 207 and 189 (Figure 30). Peak at m/z !" indicated the A, B and C rings of the tetracyclic skeleton limiting the choice to the dammarane ring system and excluding euphane or lanostane type skeletons. Peak at m/z # " was due to the wreckage of the bond between C-" and C-! A number of peaks at m/z 109, 95 and 69 represented the side chain fragments. The fragment m/z 109 corresponded to water loss from the side chain (C 8 H 15 O) [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF] which lose one more methylene in the fragment at m/z 95. From the literature [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF]148,159] and NMR data, compound 31 was considered to be dammaranediol-II.

e ( 208) a ( 69 The shift resulted from anisotropic deshielding effect of both carbonyl groups in 3D conformation. The H and C of the rest positions were consistently with compound 23.

On the basis of the above spectroscopic data, the structure of compound 48 was assigned as (20S,23E)-25-hydroperoxy-20-hydroxy-2,3-seco-dammar-23-en-2,3-dioic acid, a new 2,3-seco-dioic dammarane derivative. . The HRESI-MS of 45 at m/z 497.3602 [M+Na] + ion, represented the molecular formula of C 30 H 50 O 4 Na (calc mass 497.3607). IR absorption showed C=O stretching and O-H stretching at 1701 and 3405 cm -1 , respectively. Thirty carbon signals in the 13 C-NMR spectrum corresponded to 3-oxodammarane skeleton as dipterocarpol [START_REF] Chuakul | Medicinal Plants in Thailand[END_REF] [227] but with differences in the signals of the side chain. Three oxygenated carbons, which were one methine and two quaternary carbons, together with three methyl carbons and two olefinic carbons, were presented for the side chain in the HSQCedited spectrum. The HMBC spectrum showed the correlations of the olefinic protons (δ H-22 5.85, δ H-23 5.78) with 2 oxygenated carbons (δ C-20 74.5 and δ C-24 79.2). This suggested the structure of a double bond between 2 oxygenated carbons. This double bond was assigned to be at position 22-23 based on HMBC correlations from methyl protons at position 21 (δ H-21 1.24) to a olifinic carbon (δ C-22 127.1) and from a olefinic proton (δ H-22 5.85) to C-20 (δ C-20 74.5) (Figure 32). The configuration of the double bond was E-form because of the large coupling constant (J=15.8 Hz) between the olefinic protons.

Thus, the structure of compound 45 was determined to be a new dammarene triterpene with a acyclic trihydroxyl side chain, (20S,22E)-20,24,25-trihydroxydammar-22-en-3-one. 4.98, 4.86; δ C 111.0) were observed for instead. The HMBC correlations from H 3 -27 to these oxymethine and methylene carbons (Figure 33) suggested their positions to be at 24 and 26, respectively. The oxymethine at position 24 was also confirmed by the HMBC correlations from H-24 to C-27, C-25 and C-22. Compound 38 was then elucidated as (20S,24S)-20,24-dihydroxydammar-25-en-3-one or 24S-epimer of fouquierone. Its 24Sconfiguration was determined by comparing its NMR data and specific rotation to the literature [136,151,158].

Compound 29 showed very similar NMR data to those of 38 except for the chemical shift around C-24. The δ C 89.8 was assigned to be the signal of C-24 carbon according to its HMBC correlation to H-27 (δ H 1.80) (Figure 33). Downfield shift of δ C-24 at 89.8 ppm suggested the presence a hydroperoxyl substitution rather than a hydroxyl one. This was supported by the ESI-MS of the [M+Na] + ion at m/z 497, representing C 30 H 50 O 4 Na, which showed one more oxygen atom than that of 38. The stereochemistry of C-24 could not be investigated because very small quantity with impurity of compound 29 was isolated. From the above information, compound 29 was characterized as (20S)-20-hydroxy-24perhydroxy-dammar-25-en-3-one. With the best of our knowledge, this compound was new. 33). With comparison to the reported data [146,158], this compound was determined as (20S)-3 ,20-dihydroxy-24-perhydroxydammar-25-ene or cereotagaloperoxide that has just been found only from Ceriops tagal (Rhizophoraceae) [158]. 34). The NMR data of 30 and 34 were compared to the data from the previous reports [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]138,141,148] and they were identified as cabraleone and ocotillone, respectively. From previous study [141], stereochemistry at C-20 was determined as S-configuration. These two compounds were 24-epimers. The signal of 24S epimer [START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF] at δ C 86.4 was more downfield than that of 24R epimer [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF] at δ C 83.3. Comparison to the data of 34, instead of C-3 keto, NMR spectra of compound 37 exhibited an oxymethine (δ H 3.21, δ C 79.0) of which C-3 gave the HMBC correlations to H-28 (δ H 0.99), and H-29 (δ H 0.79). As described in the identification of 11 and 13, the 3β-OH configuration was assigned by upfield shift of δ H-3 at 3.21 and δ C-29 at 15.4. Splitting pattern of H-3 as doublet of doublet (J=10.9, 5.2 Hz) also confirmed the stereochemistry of 3β-OH. Remaining NMR data was close to that of ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF]. From these spectroscopic evidences and comparison to the previous literatures [START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]135,147,153,154] 37 was identified as ocotillol II.

The molecular formula of 47 was deduced to be C 34). These indicated that the carboxylic groups were at C-2 and C-3. Two carboxyl groups and a germinal dimethyl group at C-4 suggested a 2,3-seco-dammarane structure. The structure of 47 was thus concluded to be a new 2,3-seco-dammarane derivative with the same side chain as ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF] and was named as (20S,24R)-20,24-epoxy-25-hydroxy-2,3-seco-dammarane-2,3-dioic acid. The molecular formula of compounds 27 and 28 was assigned as C 27 H 42 O 3 based on the [M+Na] + ions at m/z 437 from ESI-MS and 437.3023 from HRESI-MS, respectively. Their 13 C NMR spectra showed the characteristic of 3-keto tetracyclic dammarane skeleton and were in good agreement with that of ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF] and cabraleone [START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF] except for the downfield signals of the side chain at δ C 176. 8-177.0 (C-24) and disappearance of C-25 to C-27. A lactone ring was indicated based on their strong IR absorption band at 1770 cm -1 . The assignment of carbons on the lactone ring was proved through the HMBC correlations (Figure 35). The correlations of H-22 and H-23 to oxygenated C-20 and carbonyl C-24, together with the usual correlations of H-21 to C-17, C-20 and C-22, demonstrated the γlactone moiety attached to C-17. Difference between compounds 27 and 28 was only the stereochemistry at C-20. The data from other 20-epimers of this study and the literatures [START_REF] Joycharat | Terpenoid constituents and antifungal activity of Aglaia forbesii seed against phytopathogens[END_REF][START_REF] Gupta | Higher Isoprenoids-I, : Triterpenoids from the Oleoresin of Dipterocarpus pilosus: Hollongdione and dipterocarpolic acid[END_REF]133,161,229,231] indicated that the chemical shifts of proton and carbon at position 21 of 20S epimer were more downfield than those of 20R epimer (δ H 1.34, δ C 22.08). Chemical shifts of 27 were at δ H 1.34, δ C 22.1 whereas those of 28 were at δ H 1.39, δ C 25.4. Therefore, the structures of 27 and 28 were assumed to be isocabralealactone and cabralealactone, respectively. The HRESI-MS at m/z 449.3400 of 25 was consistent with a molecular formula of C 29 H 46 O 2 with seven degree of unsaturation. IR absorptions at 3441 and 1704 cm -1 were attributable to a hydroxyl and a carbonyl group, respectively. The 13 C and HSQCedited data showed 6 methyl, 10 methylene, 5 methine groups and 8 quaternary carbons. The 1 H NMR spectrum showed the 5 methyl groups, appearing as singlets, bonded to quaternary carbons of pentacyclic ring.

In addition, an isopropenyl group was evident from a downfield methyl signal (H- The HRESI-MS at m/z 449.3399 [M+Na] + of 24 suggested the molecular formula of C 29 H 46 O 2 . The 1 H and 13 C NMR data of 24 were consistent with a 3-oxonorlupane skeleton. The chemical shifts of 13 C NMR data were close to those of the known compound, (20S) -17 ,29-epoxy-28-norlupan-3 -ol [107] except the values of δ C on rings A and B, especially at C-2, the carbonyl C-3 and C-24.

The carbonyl carbon C-3 (δ C 218.3) was assigned on the basis of its HMBC correlations with the methyl protons (H-23 and H-24) and the methylene protons (H-1 and H-2) (Figure 37). The structure of the isopropyl side chain at C-19 was elucidated based on COSY spectrum which showed the sequential vicinal couplings of H 39). If the structure was 20S configuration, the splitting patterns of both protons at position 29 would be changed to doublet of doublet. Thus, the structure of 24 was determined to be (20R)-17 ,29-epoxy-28-norlupan-3-one.

CYTOTOXIC EVALUATION OF SOME ISOLATED TRITERPENES

The cytotoxic activities of the isolated compounds were evaluated against a variety of human cancer cell lines, namely, PC3, MDA-MB-231, HT-29 and HCT 116. The results were shown in Table 43. Among these compounds, 17 -hydroxy-28,29dinorlupan-3,20-dione (25) exhibited more than 50% cell proliferation inhibition against HCT 116 at the concentratin of 8.67 µM (3.7 µg/mL). Although antitumor promoter activity of dammarenediol II [START_REF] Pooma | Checklist of Dipterocarpaceae in Thailand[END_REF] and cytotoxicity against P-388 leukemia cells of ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF] and cabralealactone [START_REF] Ashton | Dipterocarpaceae[END_REF] had been reported [START_REF] Makino | Tirucallane-type triterpenes from Juliana adstringens[END_REF]160,161], in this study, they seemed to possess only weak cytotoxicity at the concentration of 10 µM owing that different cell lines were tested.

IN VITRO ANTIPLASMODIAL ACTIVITY OF ISOLATED TERPENOIDS

The antiplasmodial activity of the triterpenes was tested in vitro against Plasmodium falciparum FcB1 strain, as well as, toxicity against the cell line L-6 of rat (rat skeletal muscle cell line) was evaluated. The growth inhibition of each compound against P. falciparum was summarized in Table 29. Antiplasmodial activity of all triterpenes in this study has not previously been reported. Of the 25 compounds tested, the most active specie was compound 36 with 90.94% P.falciparum growth inhibition at the concentration of 10µM. The IC 50 and IC 90 values of compound 36 against P.falciparum were 3.74 and 10.31 µM (1.72 and 4.73 µg/mL), respectively (Table 30). Other 5 triterpenes with the structure in nordammarane [START_REF] Ridley | The Flora of the Malay Peninsula[END_REF][START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF] and fouquierone [START_REF]The free encyclopedia[END_REF][START_REF] Diaz | Etude chimio-taxonomiques dans la famille des Dipterocarpaceaes-I: Introduction generale. Constutuants du genre Doona thw[END_REF][START_REF] Burger | Archaeological resinous samples from Asian wrecks: taxonomic characterization by GC-MS[END_REF] groups showed mild antiplasmodial activity with more than 20% growth inhibition at the concentration of 10 µM. Cytotoxic assay against L-6 cell showed that all compounds exhibited only low activity against normal cell at the concentration of 10 µM.

The presence of endoperoxide group and lupane structure of compound 36 might support our result. Artemisinin is a sesquiterpene lactone with endoperoxide antimalarial drug while antimalarial activity of some lupanes such as betulinic acid has been reported [131]. artemisinin

CONCLUSION

Among 31 isolated compounds, twelve compounds were new. They were (20R)-17 ,29-epoxy-28-norlupan-3-one ( 24), (20S)-20-hydroxy-24-perhydroxy-dammar-25-en-3-one ( 29), (20S)- 29-hydroxy-17 ,20-peroxy-28-norlupan-3-one (36), (20R) -20-hydroxy-17 ,29-epoxy-28-norlupan-3-one (42), 17 -hydroxy-28-norlupan-20(29)-en-3-one (25), 17 -hydroxy-28,29-dinorlupan-3,20-dione (41), (20S,23E) -20,25,26-trihydroxydammar-23-en-3-one (46), (20S,22E)- 20,24,25-trihydroxydammar-22-en-3-one (45), (20S,23E)- 20hydroxy-27-nordammar-23-ene-3,25-dione (39), (20S) -20-hydroxy-3-oxo-24,25,26,27tetranordammar-23-oic acid (49), (20S,23E)-25-hydroperoxy-20-hydroxy-2,3-secodammar-23-en-2,3-dioic acid [START_REF] Seo | Resveratrol tetramers from Vatica diospyroides[END_REF], and (20S,24R)-20,24-epoxy-25-hydroxy-2,3-secodammarane-2,3-dioic acid [START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF]. Octanordammarane-3,17-dione ( 26) was found for the first time in the nature whereas two dammaranes; cereotagaloperoxide [START_REF]The free encyclopedia[END_REF] and isocabralealactone [START_REF] Gardner | A Field Guide to Forest Trees of Northern Thailand[END_REF], were rare compounds.

Five different side chain groups of dammarane triterpenes were found in this work. Dipterocarpol and ocotillone groups were the common types in this family while isofouquierone, fouquierone and lactone sidechain groups were hitherto never reported in Dipterocarpaceous plants. Dipterocarpol 22, dammarenediol-II 31, and ocotillone 34 (no cabraleone) were previously isolated from some species in genus Dipterocarpus [START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF]. It is interesting that the works on genus Betula (Betulaceae) also found these 3 dammaranes, as well as lupane triterpene. This finding showed their closely related taxonomy. However, the dammarane derivatives from this extract were close to those found from plants in Meliaceae too.

The presence of 28-norlupane in genus Dipterocarpus may be due to the presence of an oxidative enzyme system specific to the conversion of lupanes such as betulonic acid to C-17 oxy-28-norlupane. This is the first report of norlupane in this family.

The isolation of norlupanes, acyclic side chain of fouquierone and isofouquieronegroup, 2,3-seco-dammaranes together with trihydroxy sidechain dammaranes [START_REF] Burger | Archaeological resinous samples from Asian wrecks: taxonomic characterization by GC-MS[END_REF][START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF], represented a significant departure from the patterns of triterpene production previously noted [20, 38-40, 43, 44, 46, 47, 205, 206] in the Dipterocarpaceae. Their occurrence is also notable in highlighting the chemotaxonomic significance of plants in the Dipterocarpaceae.

In this investigation, the relationship in the biosynthesis of dammaranes in dipterocarpol, ocotillone and lactone group has been proposed, as well as the biosynthesis of octanordammarane-3,17-dione. It is attractive to make more investigation on synthesis pathway of the compounds with higher oxidation such as compound 45-49 (scheme 25). The dammarane derivatives were supposed to be formed by enzymatic oxidation and degradation of the tetracyclic triterpenoid precursor, dipterocarpol. Moreover, the relationship among fouquierone, isofouquierone and dipterocarpol and the stereochemistry at C-24 of compound 29 and 32 should be examined.

In cytotoxic assay, it was interesting that compound 25, a norlupane triterpene, was active against HCT116 cell line. It showed lower growth inhibition to L-6 cell, a normal mammalian cell, than HCT116 cell, a tumor cell.

The investigation of in vitro antiplasmodial activity of new triterpene 36 (an endoperoxide norlupane) demonstrated an important finding for the treatment of malaria with the low cytotoxicity against L-6 of rat, a normal mammalian cell. To ensure the safety of compound 36 usage, its selectivity index (SI) should be carried out. (selectivity index is defined as the ratio of cytotoxicity to antiplasmodial activity, and is determinded by dividing the IC 50 values for the L-6 cells by the IC 50 for P.falciparum.) In addition, the relationship between structure and activity should be studied intensively.

Moreover, it was found in some study that dammarene triterpenes from dammar resin possesed potent antiviral activity against Herpes simplex [START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF]. Then, it is interesting to investigate this activity of the isolated dammaranes and related compounds from this research too.

GENERAL CONCLUSION

Lupane and dammarane are the major triterpenes found in the present study. The result is in agreement with the previous report of chemical constituent in Dipterocarpaceous plants. The relationship in biosynthesis among these triterpenes has been reported [START_REF] Xu | On the origins of triterpenoid skeletal diversity[END_REF] and it could explain the triterpenes in this research.

The isolation of H.odorata leaves hexane extract yielded 2 new fatty acid esters of 3,4-seco-cycloartane triterpenes [START_REF] Tanaka | Oligostilbenoids in stem bark of Vatica rassak[END_REF][START_REF] Tanaka | Stilbenoids in the stem bark of Hopea parviflora[END_REF] and 1 lupane first found in the nature [START_REF] Ito | New resveratrol oligomers in the stem bark of Vatica pauciflora[END_REF]. The chromatography of D. costatus wood hexane extract gave the finding of 5 new norlupanes [START_REF] Ashton | Dipterocarpaceae[END_REF][START_REF] Pooma | Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis[END_REF][START_REF] Ito | Antitumor effect of resveratrol oligomers against human cancer cell lines and the molecular mechanism of apoptosis induced by vaticanol C[END_REF][START_REF] Bisset | Constituants Sesquiterpeniques et Triterpeniques des Resines du Genre Shorea[END_REF][START_REF] Hota | Triterpenoids from the resin of Shorea robusta[END_REF], 2 new nordammaranes [START_REF] Bandaranayake | Tepenes of Dipterocarpus and Doona species[END_REF][START_REF] Zhang | Natural Anti-HIV Agents. Part IV: Anti-HIV Constituents from Vatica cinerea[END_REF], 3 new dammaranes [START_REF] Smitinand | The manual of Dipterocarpaceae of mainland South-East Asia[END_REF][START_REF] Burger | Archaeological resinous samples from Asian wrecks: taxonomic characterization by GC-MS[END_REF][START_REF] Bandaranayake | Triterpenoid taxonomic markers for Stemonoporus and other genera of the Dipterocarpaceae[END_REF], 2 new 2,3-seco-dammaranes [START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF][START_REF] Seo | Resveratrol tetramers from Vatica diospyroides[END_REF], 1 nordammarane first found in the nature [START_REF] Ridley | The Flora of the Malay Peninsula[END_REF] as well as 3 rare dammaranes [START_REF] Gardner | A Field Guide to Forest Trees of Northern Thailand[END_REF][START_REF] Smitinand | The manual of Dipterocarpaceae of mainland South-East Asia[END_REF][START_REF]The free encyclopedia[END_REF].

Dammaranes are the most abundant triterpens in D. costatus wood hexane extract whereas H.odorata leaves hexane extract is composed of many lupanes. It is interesting that the first tricyclic rings of triterpenes in both extracts are very close to each other. Lupane triterpenes from H.odorata extract are pentacyclic ring while D. costatus hexane extract contained both norlupane and teracyclic dammarane triterpene with side chain instead of cyclic ring E.

Although both species are in the same family, the major triterpens found are in different groups. However, it should be noted that the close triterpene structure between dammarane and cycloartane are found as well. It means a close metabolism happened in both species. The occurrence of 3,4-seco-cycloartane in H.odorate extract together with rare 2,3-seco-dammarane, trihydroxy sidechain of dammarane, and nordammarane in D. costatus extract are of interest and have never been reported from Dipterocarpaceous plants. Furthermore, the finding of a number of dammarane with epoxide and peroxide derivatives make it interesting to further investigation of antimalarial activity in other plants of this family.

Comparison to dammaranes with the same method of assay, lupane triterpenes exhibited higher potency of cytotoxicity. Messagenic acid G showed an attractive potency. The modification at C-3, C-28 and isopropenyl groups could produce the difference potency. Compound 25, a norlupane triterpene with hydroxyl group at position 17 instead of carboxylic group (compared to betulonic acid), was found potent cytotoxic active against HCT116 cell line. In addition, compound 36, an endoperoxide norlupane, gave a satisfied antiplasmodial activity. Dammarane triterpenes were less active at the tested concentration. However, it is fascinating to further antiviral activity of all compounds as there are a number of publications reported both activities of these two groups.

Figure 1 .Figure 1 .Figure 1 .

 111 Figure 1. Structure of some reported bioactive terpenoids and phenolic compounds in the family Dipterocarpaceae.

  -en-3-one; (20R)-3-oxolupan-30-al; (20S)-3-oxolupan-30-al; (20R)-28-hydroxylupan-30-al-3-one; (20S)-3 -hydroxylupan-30-al; 30-hydroxylup-20(29)-en-3-one; 30hydroxylup-20(29)-en-3 -ol; lupenone; lupeol; betulin; betulinic acid; betulonic acid; 3

Figure 2 .Figure 2 .Figure 2 .

 222 Figure 2. Some reported lupane-type triterpenes structures.

  carcinoma of cervix Hep-2 = human carcinoma of larynx HL60 = human leukemia cell Ca9-22 = gingival cancer MDA-MB231, MCF7 = breast cancer HepG2, Hep3B = hepatoma cancer A549 = lung cancer Malignant +SA = Malignant +SA mouse mammary epithelial cell NSCLC-N6 = human non-small-cell bronchopulmonary carcinoma.

CC 50 =

 50 Concentration mediating a 50% cytotoxic response IC 50 = Concentration mediating a 50% inhibition of HIV-1 replication TI = CC 50 / IC 50 HIV-1 = human immunodeficiency virus type 1 a media = human osteosarcoma cell (HOS) b media = T cell leukemia lymphocyte (C-8166) c media = T lymphoid cell line (H-9)

Figure 4 .

 4 Two Mosher conformers showing the (S)-MTPA and (R)-MTPA derivatives proposed by Mosher.

CF

Figure 5 .Figure 6 .Figure 7 .

 567 Figure 5. The (S)-MTPA ester presenting the H A,B,C,X,Y,Z instead of group R 1 and R 2

Figure 8 .

 8 Figure 8. The principle of REMA method

Figure 9 .

 9 Figure 9. Characteristics of H.odorata Roxb. A. from reference [176] B. from reference[START_REF] Gardner | A Field Guide to Forest Trees of Northern Thailand[END_REF]

Figure 10 .

 10 Figure10. Distribution of H.odorata in Thailand[START_REF] Pooma | Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis[END_REF]

  of Hopea cordifolia and H. jucunda

  ) and Avance 400 (400 MHz). o Chemical shifts were given in parts per million (ppm,δ) relative to solvent peaks as internal standards (δ: CDCl 3 : 7.27 ppm ( 1 H), 77.0 ppm ( 13 C)). o Coupling constants (J) were expressed in hertz (Hz). o Multiplicity of 1 H-NMR spectrum: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, dt = doublet of triplet, m = multiplet. MS o Low resolution MS was run on Thermo Finnergan LCQ Advantage (ESIion trap) o High resolution MS was run on LCT Premier Waters® (ESI-TOF) o GC/MS analyses for sesquiterpenes and fatty acid analysis were carried out in a Hewlett Packard 6890 GC coupled to a 5975 quadrupole MS. IR spectrum was recorded using Nicolet 510 FT-IR spectrophotometer as film on NaCl pellets. The positions were presented in wave number (ν, cm -1 a Perkin-Elmer Model 341 polarimeter with the D-ray of Na (always at 589 nm) at 20 o C. The concentration was expressed in g/100mL.

Scheme 8 .Scheme 9 .

 89 Scheme 8. Further purification of Fraction C39

Scheme 11 .Scheme 12 .

 1112 Scheme 11. Purification of fraction C42

  and appeared here just as a minor component. It has never been reported in family Dipterocarpaceae. It exhibited weak cytotoxic activity against A2780 (Human ovarian cancer cell line) with the IC 50 value of 16.4 µg/mL [112] as well as antimalarial activity against P.falciparum [203]. Mambu's work suggested that in comparison to the C-30 aldehyde and the C-3 ketone, a C-30 hydroxyl group possed lower antimalarial activity and with hydroxyl at both C-3 and C-30 the activity diminished considerably.

Figure 11 .

 11 Figure 11. Structure of lupane triterpenes from H.odorata leaves hexane extract

Scheme 14 .Scheme 16 .

 1416 Scheme 14. Reaction mechanism of transesterification at C-26 and hydrolysis at C-3 of 18a.

Scheme 17 .Figure 12

 1712 Scheme 17. Proposed biosynthesis of 20 from mangiferonic acid

Figure 14 8 The 13 C

 14813 Figure 14 Structures of compounds 3, 7 and 8The 13 C NMR spectrum of 3 showed the presence of 29 carbon signals that were contributed to 6 methyl, 11 methylene, 9 methine, and 3 quaternary carbons. From the HSQCedited spectrum, one methine carbon signal at 71.8 ppm correlated to δ H-3 3.58 and an olefin carbon at δ C-6 121.7 ppm correlated to δ H-6 5.40. The 13 C-NMR data of 3 was then compared to the previous literature[188] and then it was identified as β-sitosterol.

The 13 C

 13 NMR spectrum also showed characteristics of -sitosterol (C-3 oxymethine at δ C 73.66 and quaternary and methine carbon signals at δ C 139.7 and 122.6), with additional carbon signals of fatty acid ester (methylenes at δ C 29-30 and acyl group at δ C 174.0).

  Figure 15 Structures of the isolated lupanes

  )-ene. The molecular formula C 30 H 44 O 4 of compound 14 was suggested on the basis of the [M-H] -ion at m/z 467.3146 (calcd C 30 H 43 O 4 at m/z 467.3161). The 13 C-NMR data showed the presence of 30 signals which were attributed (by HSQCedited correlation) to 5 methyl, 11 methylene, 6 methine and 8 quaternary carbons. The NMR spectrum of 14 was very close to those of betulonic acid (5), except an aldehyde signals at δ H 9.53 and δ C 195.0 instead of the methyl signal of C-30. The presence of aldehyde functional group was confirmed by IR band at 1731 cm -1 . The HMBC correlations from the aldehyde proton (δ H 9.53) to C-20 (δ C δ C 156.2) and from deshield H-29 (δ H 5.93 and 6.31 ppm) to the aldehyde carbon (194.9 ppm) (Figure 16) suggested the position of this functional group at C-30. The presence of dienone system ( ) caused this compound detected under UV at 254 nm on TLC. Then, compound 14 was defined as 3,30-dioxolup-20,29-en-28-oic acid. The ESI-MS spectrum of 16 showed a [M+Na] + at m/z 479 corresponding to the molecular formula C 30 H 48 O 3 . The difference between the NMR data of 16 and betulonic acid (5) was obviously seen through the chemical shift at position 30 and 28. The signal at δ H 4.12 ppm (s, 2H) represented the protons at C-30 while the germinal coupling protons at δ H 3.33 and 3.80 ppm were the protons at C-28. The HMBC spectrum presented the correlations between 2 olefinic protons (δ H 4.96 and 4.91 ppm) and hydroxy C-30 (δ C 65.0) as well as C-20 and C-19 (Figure

Figure 20 Compound 18 ,

 2018 Figure 20 Structures of compounds 18, 18a and 18b and selected HMBC correlations of 18

  7 and 14.2 ppm revealed the methylene and methyl groups of fatty acid. No additional data of double bond in fatty acid chain was observed. The oxy-methylene protons (δ H 4.29/4.03) at position 26 of 3,4-secocycloartane displayed HMBC correlation to the carbonyl carbon of this fatty acid (δ C 174.3) (Figure20). These evidences suggested saturated fatty acid ester at C-26.Examining the NMR spectrum of 18a gave the correlation of methyl proton (δ H 3.65) with C-3 (δ C 178.2). Finally, after transesterification, the HRESI-MS indicated the molecular formula of triterpene as C 30 H 50 O 5 (18b) and of fatty acid as C 20 H 40 O 2 . Analysis of the data enabled compound 18 to be proposed as 26-arachidic acid ester of24,25,26-trihydroxy- 3,4-seco-cycloart-4(29)-en-3-oic acid, a new natural product.

Figure 21 .

 21 Figure 21. Proposed configuration of 18 a) The proton chemical shift differences (δ S -δ R ) of protons around C-24 of 18a-MTPA ester b) δ C-24 to δ C-26 (in parenthesis) of ganodermanotriol (24S,25R) and its stereoisomers (reference [219]) c) proposed configuration of 18b according to four isomers in b) and related δ C .

Figure 22 .

 22 Figure 22. Structures of compounds 19 and 19a and Selected HMBC correlations of 19a

Figure 23 .

 23 Figure 23. Structures and selected HMBC correlations of compound 20 and 20a.

Figure 24 .Figure 24 .

 2424 Figure 24. Distribution of D. costatus in Thailand[START_REF] Pooma | Dipterocarpaceae in Thailand: Taxonomic and Biogeographical Analysis[END_REF]

  found for the first time in the nature. ** the rare compound in the nature. *** New compounds.

  expressed as % inhibition of cell proliferation. Cisplatin (1 and 18 µM) was used as positive control.

Figure 26 .Figure 25 .

 2625 Figure 26. Proposed structure of isolated compounds from D. costatus wood hexane extract

Figure 25 .

 25 Figure 25. Proposed structure of isolated compounds from D. costatus wood hexane extract. (continued) * the compound found for the first time in the nature. ** the rare compound in the nature. *** New compounds.

Scheme 21 .

 21 Scheme 21. Semisynthesis of octanordammarane-3,17-dione (26) from (20S,24R)-20,24epoxydammarane-3 ,17 ,25-triol.[230] CrO 3 = chromium trioxide, NH 2 NH 2 .H 2 O = hydrazine hydrate

POCl 3 =

 3 phosphorous oxychloride, O 3 = ozone, BH 4 K = potassium borohydride, CF 3 COOH = trifluoroacetic acid, Py = pyridine, CrO 3 = chromium trioxide, Di = dioxane, AcOH = acetic acid, LiAlH 4 = lithium aluminium hydride, THF = tetrahydrofuran, Ac 2 O = acetic anhydride, Na 2 Cr 2 O 7 = sodium dichromate

Scheme 24 .

 24 Scheme 24. Oxidation reaction of cabraleone[START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF] to cabralealactone[START_REF] Ashton | Dipterocarpaceae[END_REF]; R/R' = H/Ac, LAH = lithium aluminium hydride

  , Fagaceae [241], and Burseraceae [148]. Ocotillol II (37), a reduction product of ocotillone (20S,24R), was found in family Dipterocarpaceae [152], Rhizophoraceae [158], Euphorbeaceae [153], Theaceae [160], and Apocyanaceae [147].

Figure 27 .

 27 Figure 27. The possible degradation products of the 20,24,25-triol dammarane. During the NMR experiments (in CDCl 3 ), isofouquierone (40) could decomposed

Figure 29 .

 29 Figure 29. Selected HMBC correlations of 22 and 31 Compound 22 was obtained as a white shiny flakes with the highest amount among all triterpenes in the extract. Its molecular formula of C 30 H 50 O 2 was deduced by a combination of ESI-MS (m/z 465 of the [M+Na] + ion) and NMR data. Analysis of the 1 H and 13 C NMR data (Tables 32) suggested the 3-oxodammarane skeleton. The side chain consisted of a hydroxyl group as well as one double bond between quaternary carbon and

  (H-21) with δ C49.8 (C-17), δ C75.4 (hydroxyl quaternary carbon C-20) and δ C40.4 (C-22) were dicisive to establish a hydroxyl group and a methyl group attached on C-20. Indeed, the methyl protons (δ H-26 1.71 and δ H-27 1.64) also showed correlations with quaternary C-25 (δ C 131.7) and methine C-24 (δ C 124.7) of double bond. In addition, HMBC correlations of methylene protons at δ H1.54 (H-22) with the carbon signals at δ C49.8 (C- 17),75.4 (C-20), 22.6 (C-23) and 124.7 (C-24) confirmed the presence of a double bond with 2 terminal methyl groups, together with 2 methylenes between hydroxyl carbon (C-20) and methine carbon of double bond (C-24).

Figure 30 .

 30 Figure 30 .Fragmentation of dammarenediol II[START_REF] Pooma | Checklist of Dipterocarpaceae in Thailand[END_REF]

Figure 31 .

 31 Figure 31. Selected HMBC correlations of compounds 23, 33, 40, 44, 46 and 48

Figure 32 .

 32 Figure 32. Selected HMBC correlations of compound 45

  Comparison to the13 C NMR spectrum of 22, compound 38 showed 30 carbon resonances, which the carbon signals agreed well with 3-oxodammarane skeleton. The difference was on acyclic side chain. The signals of olefinic methine protons and one methyl group were disappeared. Signals of oxymethine (δ H 4.06, δ C 76.5), together with terminal methylene(δ H

29 32 38 Figure 33 .

 293833 Selected HMBC correlations of compounds 29, 32 and 38 The 1D and 2D NMR spectrum of 32 were very close to those of 29 except for the chemical shift around C-3. The ESI-MS revealed a pseudomolecular ion peak at m/z 499. [M+Na] + , corresponding to the molecular formula C 30 H 52 O 4 which was two more hydrogen atoms than 29. The presence of δ H 3.22 and δ C 79.0, as well as the absence of δ C 218.3 made it more likely to be a hydroxyl rather than a keto group at C-3. It was supported by the chemical shifts of C-2 (δ C 27.4), C-4 (δ C 39.0) and C-29 (δ C 15.4) together with the HMBC correlations of H-28 (δ H 1.02) and H-29 (δ H 0.82) to C-3 (δ C 79.0) (Figure

Figure 34

 34 Figure 34Seleted HMBC correlations of compounds 30, 34, 37 and 47

Figure 35 .

 35 Seleted HMBC correlations of compounds 27, 28 and 35

Figure 37 .

 37 Figure 37. Seleted HMBC correlations of compounds 24, 25, 36, 41 and 42 The 13 C-NMR and 2D NMR data of 41 indicated that this compound was dinorlupane with 28 carbon signals; 6 methyl, 10 methylene, 5 methine and 7 quaternary carbons. The HRESI-MS at m/z 521.3486 [M-H] -of 41 suggested the molecular formula C 28 H 44 O 3. IR spectrum at 1704 cm -1 indicated the presence of carbonyl group. Its NMR data were close to compound 25 except that one carbonyl carbon (δ C 214.5), instead of an olefinic group, on the side chain was observed. The HMBC correlation of the terminal methyl proton H-30 (δ H 2.25) to this carbonyl carbon and the methine carbon C-19 (δ C 55.8) supported a keto group at C-20. The 17 configuration of the hydroxyl group was suggested by the comparison of the chemical shifts of C-13 (δ C 41.9) and C-19 (δ C 55.8) to those previously reported [107, 108] (δ C-13 38 ppm and δ C-1948 ppm, respectively, in -configuration). The stereochemistry at C-19 was assigned by the NOESY spectrum that showed the correlations of H-19 with H-12a and H-21a. The structure of compound 41 was then proposed to be17 -hydroxy-28,29-dinorlupan-3,20-dione.

Figure 38 .

 38 Figure 38. Selected NOE and stereoscopic view of compound 41.

  C-17 should be in the opposite way, in other words, the bonds between C-19 and C-20, together with C-17 and O, should be in the same plane. That made it conform to the configuration at C-17 and C-19 of compound 25. The 19α-orientation of isopropyl group was fixed by lupane skeleton and was supported based on the splitting pattern of δ H-18 (d, J=11.7 Hz). From this configuration, the 90 o of the dihedral angle between H-19 and H-18 was possible and it caused H-18 to couple with only H-13. If the 19β-orientation was occurred, this angle would be around 30 o and resulted in doublet of doublet signal of H-18 (Figure 39). The bonds between C-19 and C-20, together with C-17 and oxygen atom, might be in the same plane, therefore, oxygenated substitution at C-17 was also designed as α-orientation. The stereochemistry at C-20 was determined through splitting patterns of H-29. Beside the germinal coupling (J=11.4 Hz) between each proton of H-29 the δ H-29 at 3.25 ppm possessed another axialaxial coupling (J=11.4 Hz) with H-20, whereas the other δ H-29 at 3.66 ppm displayed equatorial-axial coupling (J=5.6 Hz) with H-20 (Figure

Figure 39 .

 39 Figure 39. Comparison of 3D model of 19α-and 19β-orientation of compound 24 (by program Chem3D ultra 9.0) Compound 36 was inferred to have the molecular formula C 29 H 46 O 4 by the HRESI-MS at m/z 481.3294 [M+Na] + . Its 13 C-NMR and DEPT data, clearly showed 29 carbon signals; 6 methyl, 11 methylene, 5 methine and 7 quaternary carbons. The 13 C NMR data were close to those of 24, especially, carbon signals of ring A-C. As general 3-oxolupane, there were correlations between C-3 (δ C 218.1) and H-1(δ H 1.45, 1.94), H-2 (δ H 2.47), H-23 (δ H 1.09) and H-24 (δ H 1.04). Two oxygenated quaternary carbons and one oxygenated methylene

  2) indicated a linkage through an ether bridge between C-29 and C-17 of the 28-norlupan nucleus. The α-orientations of both substitutions at C-17 and C-19 were designed by the same reason as compounds 24 and 36. From NOESY experiment, NOEs of H-30 to H-19 and H-18 indicated the α-configuration of CH 3 -30. The structure of 42 was then described as 20-hydroxy-17 ,29-epoxy-28-norlupan-3-one.

Figure 40 Selected

 40 Figure 40 Selected NOE and stereoscopic view of compound 42 Compound 24, 25, 36, 41 and 42 were classified to 28-norlupane triterpenes. All of them were found herein for the first time.

  possibility ??? = It should be proven.

Figure 42 .

 42 Figure 42. The 1 H (300 MHz) and 13 C NMR (75 MHz) spectrum of 3, 7 and 8 (in CDCl 3 ).

Figure 43 .

 43 Figure 43. The 1 H (300 MHz) and 13 C NMR (75 MHz) spectrum of 14 (in CDCl 3 ).

Figure 44 .

 44 The HMBC correlation of 14 (in CDCl 3 ). a) whole spectrum b.) expanded HMBC spectrum in the range of d H 0.7-2.6 ppm and d C 33-62 ppm.

Figure 45 .Figure 46 .

 4546 Figure 45. IR spectrum of 14. (dry film)

Figure 47 .

 47 Figure 47. Expanded HSQCedited spectrum of 16 (in CDCl 3 ) in the range of d H 0.7-5.1 ppm and d C 10-110 ppm.

Figure 48 .

 48 Figure 48. The HMBC correlation of 16 (in CDCl 3 ).

Figure 49 .Figure 50 .

 4950 Figure 49. The expanded NOESY spectrum of 16 (in CDCl 3 ) in the range of d H 0.8-4.0 ppm.

Figure 51 .

 51 Figure 51. Expanded HSQCedited spectrum of 17 (in CDCl 3 ) in the range of d H 0.7-5.2 ppm and d C 0-160 ppm.

Figure 52 .

 52 Figure 52. The HMBC correlation of 17 (in CDCl 3 ).

Figure 53 .

 53 Figure 53. The 1 H (300 MHz) and 13 C NMR (75 MHz) spectrum of 18 (in CDCl 3 ).

Figure 54 .

 54 Figure 54. Expanded HSQCedited spectrum of 18 (in CDCl 3 ) in the range of d H 0.4-2.8 ppm and d C 10-55 ppm.

Figure 55 .

 55 Figure 55. Expanded HSQCedited spectrum of 18 (in CDCl 3 ) in the range of d H 3.2-5.0 ppm and d C 60-120 ppm.

Figure 56 .

 56 Figure 56. The HMBC correlation of 18 (in CDCl 3 ).

Figure 57 .

 57 Figure 57. Expanded HMBC correlation of 18 (in CDCl 3 ) in the range of a) d H 0.3-2.8 ppm and d C 10-80 ppm. b) d H 1.5-4.5 ppm and d C 10-80 ppm. c) d H 3.2-5.0 ppm and d C 10-80 ppm.

Figure 59 .

 59 Figure 59. The 13 C NMR spectrum (75 MHz) of 18,18a and 18b (in CDCl 3 ).

Figure 62 .

 62 Figure 62. The HSQCedited spectrum of 19a (in CDCl 3 ).

Figure 63 .

 63 Figure 63. The HMBC spectrum of 19a (in CDCl 3 ).

Figure 65 .Figure 66 .

 6566 Figure 65. The 13 C NMR spectrum (75 MHz) of 20 (in CDCl 3 ) and expanded spectrum in the range of 10.0-54.0 ppm.

17

 17

Figure 67 .

 67 Figure 67. The HMBC correlation of 20 (in CDCl 3 ) and expanded spectrum in the range of d H 0.3-7.0 ppm and d C 10-56 ppm.

17

 17

Figure 69 .

 69 Figure 69. The 13 C NMR spectrum (75 MHz) of 20 and 20a (in CDCl 3 ).

Figure 70 .

 70 Figure 70. The HSQCedited correlation of 20a (in CDCl 3 ) and expanded spectrum in the range of d H 0.3-2.6 ppm and d C 0-56 ppm.

Figure 71 .

 71 Figure 71. The HMBC correlation of 20a (in CDCl 3 ) and expanded spectrum in the range of d H 0.4-2.6 ppm and d C 12-59 ppm.

Figure 73 .

 73 Figure 73. The 13 C NMR spectrum (75 MHz) of 22 and 31 (in CDCl 3 ).

Figure 75 .Figure 76 .Figure 77 .Figure 78 .

 75767778 Figure 75. The expanded HMBC correlation of 22 and 31 (in CDCl 3 ) and expanded spectrum in the range of d H 0.7-1.8 ppm and d C 15-58 ppm.

Figure 79 .

 79 Figure 79. The HMBC correlation of 24 (in CDCl 3 ) and expanded spectrum in the range of d H 2.9-3.8 ppm, d C 10-50 ppm and d H 1.2-1.8 ppm, d C 81-84 ppm.

Figure 80 .

 80 Figure 80. The 1 H NMR spectrum (300 MHz) of 25 (in CDCl 3 ) and expanded spectrum in the range of 0.95-1.10 ppm.

Figure 81 .

 81 Figure 81. The 13 C NMR spectrum (75 MHz) of 25 (in CDCl 3 ) and expanded spectrum in the range of 16-60 ppm.

Figure 82 .

 82 Figure 82. The expanded HSQCedited correlation of 25 (in CDCl 3 ) in the range of d H 0.8-4.9 ppm and d C 16-110 ppm.

29

 29

Figure 83 .

 83 Figure 83. The HMBC correlation of 25 (in CDCl 3 ) and expanded spectrum in the range of d H 4.7-4.9 ppm, d C 20-55 ppm and d H 0.9-2.0 ppm, d C 18-57 ppm.

Figure 84 .

 84 Figure 84. The expand 1 H NMR spectrum (300 MHz) of 26 (in CDCl 3 ).

Figure 85 .

 85 Figure 85. The 13 C NMR spectrum (75 MHz) of 26 (in CDCl 3 ) and expanded spectrum in the range of 13-58 ppm.

Figure 86 .

 86 Figure 86. The expanded HSQCedited correlation of 26 (in CDCl 3 ) in the range of d H 0.8-2.7 ppm and d C 13-58 ppm.

Figure 87 .

 87 Figure 87. The expanded HMBC correlation of 26 (in CDCl 3 ) in the range of d H 0.7-2.7 ppm, d C 13-59 ppm and d H 1.0-2.7 ppm, d C 211-219 ppm.

Figure 88 .

 88 Figure 88. The expand 1 H NMR spectrum (300 MHz) of 27 and 28 (in CDCl 3 ) d H 0.7-2.7 ppm.

Figure 90 .

 90 Figure 90. The expanded 13 C NMR spectrum (75 MHz) of 27 and 28 (in CDCl 3 ) in the range of d H 10-60 ppm.

Figure 91 .

 91 Figure 91. The expanded HSQCedited correlation of 27 (in CDCl 3 ) in the range of d H 0.8-2.8 ppm and d C 13-58 ppm.

Figure 92 .

 92 Figure 92. The HMBC correlation of 27 (in CDCl 3 ) and expanded spectrum in the range of d H 0.9-2.8 ppm, d C 15-57 ppm.

Figure 93 .Figure 94 .

 9394 Figure 93. The 1 H NMR spectrum (300 MHz) of 29 (in CDCl 3 ) and expanded spectrum in the range of 0.85-1.20 ppm.

Figure 96 .

 96 Figure 96. The HMBC correlation of 29 (in CDCl 3 ) and expanded spectrum in the range of d H 1.78-1.82 ppm, d C 86.00-150.00 ppm and d H 1.17-1.22 ppm, d C 32.00-80.00 ppm.

Figure 97 .

 97 Figure 97. The 1 H NMR spectrum (300 MHz) of 32 (in CDCl 3 ) and expanded spectrum in the range of 0.7-2.2 ppm.

Figure 98 .

 98 Figure 98. The 13 C NMR spectrum (75 MHz) of 32 (in CDCl 3 ).

Figure 99 .

 99 Figure 99. The HSQCedited correlation of 32 (in CDCl 3 ).

Figure 100 .

 100 Figure 100. The HMBC correlation of 32 (in CDCl 3 ).

Figure 101 .

 101 Figure 101. The 1 H NMR spectrum (300 MHz) of 36 (in CDCl 3 ) and expanded spectrum in the range of 0.8-2.6 ppm.

Figure 102 .

 102 Figure 102. The 13 C NMR spectrum (75 MHz) of 36 (in CDCl 3 ) and expanded spectrum in the range of 11-58 ppm.

Figure 103 .

 103 Figure 103. The HSQCedited correlation of 36 (in CDCl 3 ) and expanded spectrum in the range of d H 0.9-2.6 ppm and d C 10-56 ppm.

Figure 104 .Figure 105 .

 104105 Figure 104. The HMBC correlation of 36 (in CDCl 3 ) and expanded spectrum in the range of d H 0.9-2.1 ppm, d C 13-58 ppm and d H 3.5-4.3 ppm, d C 19.5-22.0 ppm.

Figure 106 .

 106 Figure 106. The 13 C NMR spectrum (75 MHz) of 39 (in CDCl 3 ) and expanded spectrum in the range of 10-58 ppm.

Figure 107 .

 107 Figure 107. The HSQCedited correlation of 39 (in CDCl 3 ) and expanded spectrum in the range of d H 0.9-2.9 ppm and d C 12-58 ppm.

Figure 108 .

 108 Figure 108. The HMBC correlation of 39 (in CDCl 3 ) and expanded spectrum in the range of d H 0.6-2.7 ppm and d C 10-80 ppm.

Figure 109 .

 109 Figure 109. IR spectrum of 39. (dry film).

Figure 110 .

 110 Figure 110. The 1 H NMR spectrum (300 MHz) of 41 (in CDCl 3 ).

Figure 111 .

 111 Figure 111. The 13 C NMR spectrum (75 MHz) of 41 (in CDCl 3 ) and expanded spectrum in the range of 10-59 ppm.

Figure 112 .

 112 Figure 112. The expanded HSQCedited correlation of 41 (in CDCl 3 ) in the range of d H 0.8-3.0 ppm and d C 11-57 ppm.

Figure 113 .

 113 Figure 113. The HMBC correlation of 41 (in CDCl 3 ) and expanded spectrum in the range of d H 0.7-2.6 ppm and d C 10-59 ppm.

Figure 114 .

 114 Figure 114. The NOE correlation of 41 (in CDCl 3 ).

Figure 115 .Figure 116 .

 115116 Figure 115. The 1 H NMR spectrum (300 MHz) of 42 (in CDCl 3 ) and expanded spectrum in the range of 0.8-2.6 ppm.

Figure 117 .

 117 Figure 117. The expanded HSQCedited correlation of 42 (in CDCl 3 ) in the range of d H 0.8-3.6 ppm and d C 11-80 ppm.

17

 17

Figure 118 .

 118 Figure 118. The HMBC correlation of 42 (in CDCl 3 ) and expanded spectrum in the range of d H 0.7-3.7 ppm and d C 13-90 ppm.

Figure 119 .

 119 Figure 119. The NOE correlation of 42 (in CDCl 3 ) and expanded spectrum in the range of d H 0.7-2.7 ppm.

Figure 120 .

 120 Figure 120. The 1 H NMR spectrum (300 MHz) of 45 (in acetone) and expanded spectrum in the range of 0.8-2.6 ppm.

Figure 121 .Figure 122 .

 121122 Figure 121. The 13 C NMR spectrum (75 MHz) of 45 (in acetone) and expanded spectrum in the range of 10-58 ppm.

Figure 123 .

 123 Figure 123. The HMBC correlation of 45 (in acetone) and expanded spectrum in the range of d H 0.6-4.2 ppm and d C 10-85 ppm.

Figure 124 .Figure 125 .

 124125 Figure 124. The 1 H NMR spectrum (300 MHz) of 46 (in CDCl 3 ) and expanded spectrum in the range of 0.7-2.7 ppm.

Figure 126 .

 126 Figure 126. The HSQCedited correlation of 46 (in CDCl 3 ) and expanded spectrum in the range of d H 0.8-2.6 ppm and d C 10-58 ppm.

Figure 127 .

 127 Figure 127. The HMBC correlation of 46 (in CDCl 3 ) and expanded spectrum in the range of d H 0.6-4.2 ppm and d C 10-85 ppm.

Figure 128 .

 128 Figure 128. The 1 H NMR spectrum (300 MHz) of 47 (in CDCl 3 ) and expanded spectrum in the range of 0.6-2.8 ppm.

  20S,24S contaminated in the compound of 20S,24R[START_REF] Geevananda | Distribution of some triterpenes and phenolic compounds in the extractives of endemic Dipterocarpaceae species of Sri Lanka[END_REF].

Figure 129 .

 129 Figure 129. The 13 C NMR spectrum (75 MHz) of 47 (in CDCl 3 ) and expanded spectrum in the range of 10-52 ppm.

Figure 131 .

 131 Figure 130. The HSQCedited correlation of 47 (in CDCl 3 ) and expanded spectrum in the range of d H 0.8-2.6 ppm and d C 10-58 ppm.

Figure 132 .

 132 Figure 132. The 1 H NMR spectrum (300 MHz) of 48 (in acetone) and expanded spectrum in the range of 0.8-2.8 ppm.

Figure 133 .

 133 Figure 133. The 13 C NMR spectrum (75 MHz) of 48 (in acetone) and expanded spectrum in the range of 10-55 ppm.

Figure 134 .

 134 Figure 134. The HSQCedited correlation of 48 (in acetone) and expanded spectrum in the range of d H 0.9-2.7 ppm and d C 13-52 ppm.

Figure 135 .

 135 Figure 135. The HMBC correlation of 48 (in acetone) and expanded spectrum in the range of d H 0.8-2.7ppm and d C 15-53 ppm.

Figure 136 .

 136 Figure 136. The 1 H NMR spectrum (300 MHz) of 49 (in CDCl 3 ).

Figure 138 .

 138 Figure 138. The expanded HSQCedited correlation of 49 (in CDCl 3 ) in the range of d H 0.9-2.8 ppm and d C 12-58 ppm.

Figure 139 .

 139 Figure 139. The expanded HMBC correlation of 49 (in CDCl 3 ) in the range of d H 0.9-2.7ppm, d C 15-53 ppm and d H 1.1-2.8ppm, d C 170-220 ppm.

Table page 1

 page Some reported bioactive terpenoids and phenolic compounds in low polarity fraction of the family Dipterocarpaceae extracts.

  of 20 and 20a (in CDCl 3 ).

	26	In vitro bioactivity assays of D.costatus wood extract	108
	27	Fractionation of D.costatus wood hexane extract isolation by MPLC.	110
	28	% Cell proliferation inhibition of the isolated compounds	117
	Table		Page
	29	Screening test for in vitro antiplasmodial activity and cytotoxicity of	119
		the isolated triterpenes
	30	Antiplasmodial potency of compound 36	120
	31

  NMR (300 MHz) of 23, 33 (in CDCl 3 ) and 48 (in acetone) 34 The 1 H-NMR (300 MHz) of 40, 44 and 46 (in CDCl 3 ) 35 The 13 C-NMR (75 MHz) of 23, 33, 40, 44 and 46 (in CDCl 3 ) and 48 (in acetone)

	33	The 1 H-
		146
		CDCl 3 )

data of 21 (in CDCl 3 ) 143 32

The 1 H-NMR (300 MHz) and

13

C-NMR (75 MHz) of 22 and 31 (in

, 10, 11, 12, 13, 14, 16, and 17

			32
	7	Reduction of WST-1by NADH.	32
	8	The principle of REMA method.	33
	9	Characteristics of H.odorata Roxb.	37
		Distribution of H.odorata in Thailand.	38
		Structure of lupane triterpenes from H.odorata leaves hexane extract.	69
		Structures of compounds 1 and 4	73
		Structures of compounds 2 and 6	75
		Structures of compounds 3, 7 and 8	77
		Structures of the isolated lupanes	79
		Selected HMBC correlations of 580
		Selected NOE correlations of compound 16	86
		Structure of caryophyllene oxide (9)	87
		Structure and Selected HMBC correlations of compound 15	88
		Structures of compounds 18, 18a and 18b and selected HMBC	89
		correlations of 18
		Proposed configuration of 18	91
		Structures of compounds 19 and 19a and Selected HMBC correlations of	94
		19a
		Structures and selected HMBC correlations of compound 20 and 20a.	95
		Characteristics of D.costatus Gaertn.f.	102
		Distribution of D.costatus in Thailand	103
		Proposed structure of isolated secondary metabolites from D.costatus	121
		wood hexane extract.
		The possible degradation products of the 20,24,25-triol dammarane.	140
		Selected HMBC correlations of 21	143
		Selected HMBC correlations of 22 and 31	144
		Fragmentation of dammarenediol II	145
		Selected HMBC correlations of compounds 23, 33, 40, 44, 46 and 48	147
	Figure		page
		Selected HMBC correlations of compound 45	151
		Selected HMBC correlations of compounds 29, 32 and 38	153
		Seleted HMBC correlations of compounds 30, 34, 37 and 47	155
		Seleted HMBC correlations of compounds 27, 28 and 35	158
		Seleted HMBC correlations of compounds 26, 39 and 49	160
		Seleted HMBC correlations of compounds 24, 25, 36, 41 and 42	163
		Selected NOE and stereoscopic view of compound 41	163
		Comparison of 3D model of 19α-and 19β-orientation of compound 24	166
		Selected NOE and stereoscopic view of compound 42	167
		The 1 H (300 MHz) and

TABLE OF CONTENTS

 OF Extraction and Isolation of H. odorata leaves hexane extract 4.4 Identification and structural study of isolated compounds

	IV. Personnel work
	4.1 Plant material	page
	REMERCIEMENT 4.2 Preliminary study	i
	ACKNOWLEDGEMENT ABSTRACT 4.3	ii iii
	LIST OF TABLES	iv
	LIST OF FIGURES	vi
	LIST OF SCHEMES	xii
	ABBREVIATIONS	xiii
	TABLE OF CONTENTS	xv
	INTRODUCTION	1
	LITERATURE REVIEWS	3
	Dipterocarpaceae	3
	Thai Dipterocarpaceae	4
	Bioactive components in Dipterocarpaceous plants.	6
	Biosynthesis of lupane and dammarane-type triterpenes in plants
	Lupane triterpenoids
	Dammarane triterpenoids
	Determination of the absolute configuration by modified Mosher's
	method.
	Cytotoxic evaluation by WST-1 method.
	Cytotoxic evaluation by REMA method.
	PART I : Phytohemical study of Hopea odorata.
	I. Botanical study
	Genus Hopea.
	Hopea odorata Roxb.
	II. Previous chemical work on genus Hopea
	III. Experimental part
	3.1 Plant material
	3.2 Method and apparatus
	3.3 Preliminary study
	3.3.1 Preliminary extraction by soxhlet extraction
	3.3.2 In vitro bioactivity assays
	3.4 Extraction and isolation of H. odorata leaves hexane extract
	3.4.1 Extraction by maceration
	3.4.2 Isolation of major compounds with a small portion of
	hexane extract.
	3.4.2.1 Isolation by silica gel column chromatography	page
	3.4.2.2 Isolation by sephadex column
	chromatography
	3.4.3 Isolation of a large portion of hexane extract
	3.4.4 Purification of HML10
	3.5 Cytotoxic evaluation of 8 isolated lupanes
	3.6 Physical characteristics and spectrum of isolated compounds

Table 1 .

 1 Some reported bioactive terpenoids and phenolic compounds in low polarity fraction of the family Dipterocarpaceae extracts.

	Terpenoids	Bioactivity [reference]	Plant source (part)[reference]
	Sesquiterpenes
	-Elemene	Antitumor [50]	Doona sp. (re) [38], Shorea sp. (re)
			[41]
	Caryophyllene

  ]. A large variety of triterpene alcohols arise from one of two epimeric dammarenyl cations, 17 -dammarenyl cation or 17 -dammarenyl cation, which are 6-6-6-5 tetracycles with all-chair configurations. Initial cyclization might form a 6-6-5 tricyclic ring, and follow by ring expansion and D-ring annulations. Addition of a hydroxyl group at C-20 to 17 -dammarenyl cation without rearrangement generated dammarenediol, which was a precursor for dammarane triterpene.

	plausible [80
	Addition of a hydroxyl group at C20	D-ring expansion (C16 migration)
	(20S) and (20R) dammarenediol	Baccharenyl cation
		18 E-ring cyclization
	Dammarane triterpene group
		Lupyl cation
		Deprotonation
	Lupane triterpene group
		Lupeol

Table 2 .

 2 Discovery of lupane-type triterpenes in some plants

	Family	Plant	Lupanes found	Reference
	Anacardiaceae	Ozoroa insignis	betulonic acid; betulinic acid	[82]
		(root)

Table 3 .

 3 Cytotoxicity of betulinic acid and betulonic acid against various cell lines

	Cell	Cell type	IC 50 (µM)	references
			Betulonic	Betulinic
			acid	acid
	A549	Alveolar basal epithelial	15	146	[117]
		cells		2.8 µg/mL	[95]
	HT29	Human colon	17	89	[117] [106]
		adenocarcinoma grade II	>10
		cell line
	DU145	Human prostate carcinoma	36	196	[117]
		epithelial-like cell line
	PC-3	Human prostate cancer cell	15	91	[117]
		line
	MEL-1	Human melanoma cell	16.0 µg/mL	3.3 µg/mL [118]
	MEL-2	Human melanoma cell	0.9 µg/mL	1.2 µg/mL	[52]
			0.1 µg/mL	1.0 µg/mL	[118]
	SK-Mel2	Human melanoma cell	26	21	[117]
	K562	Human erythroleukenia cell	6	56	[117] [86]
		line	16.1
	MCF-7	Human breast	29	143	[117] [86]
		adenocarcinoma cell line	49		[95]
				4.0 µg/mL
	MDA-	Human breast cancer		3.5 µg/mL [95]
	MB231
	CEM	Human T-Lymphoblastoid	17	27	[117]
		CEM cell line
	K562 tax	Leukemia cell	17	112	[117]
	HL60	Human leukemia cell		2.0 µg/mL [95]
	COLO205 Colon carcinoma	36.8		[86]
	KB	Epidermoid carcinoma	3.8		[86] [106]
			8.2		[52]
			2.5 µg/mL	>20 µg/mL
	KB-C2	Colchicine-resistant KB	2.3		[86]
	HONE-1	Nasopharyngeal carcinoma	4.9		[106]
	SGC7901 Human gastric cancer cell	68.14		[120]
	HepG-2	Human liver cancer cell	110.77		[120]
				3.1 µg/mL	[95]
	Hep3B	Hepatoma cell		1.7 µg/mL [95]
	FS-5	Human foreskin fibroblast		20.7 µg/mL [119]
	OVCAR-3	Human ovarian		0.9 µg/mL [119]
		adenocarcinoma
	HeLa	Cervical carcinoma		0.8 µg/mL [119]

Table 4 .

 4 Cytotoxicity of other lupane-type triterpene

	compound	Tested cell lines (IC 50 )	Reference
	Epibetulinic acid	HeLa (2.1 µg/mL), Hep-2 (3.1 µg/mL)	[93]
		HL60 (2.3 µg/mL)
		A549 (9.7 µg/mL)
		MCF-7 (9.7 µg/mL)
		MDA-MB231 (9.2 µg/mL)
		Hep3B (8.1 µg/mL)
		HepG2 (9.3 µg/mL)

Table 5 .

 5 Anti-inflammatory activity of some lupanes (effect on NO and PGE2 production)[START_REF] Nunez | Lupane triterpenoids from Maytenus Species[END_REF]

	NO	PGE 2

a Compounds were assayed at 10 µM.

Table 6 .

 6 Anti HIV-1 activity of some lupanes

	Betulin	CC 50 19.5 µM	IC 50 13.8 µM	TI 1.4	[49] a	reference
		2.14 µg/mL	No suppression	-	[129] c
	Betulinic acid	36.0 µM	32.5 µM	1.1	[49] a
	Betulonic acid	105.1 µM	21.4 µM	4.9	[49] a
		30.66 µM	5.81 µM	5.27	[83] b
		1.8 µg/mL	0.22 µg/mL	8	[129] c

Table 7 .

 7 Discovery of dammarane-type triterpenes in some plants

	Family	Plant	dammaranes found	Reference
	Anacardiaceae	Rhus javanica (stem bark)	semialactone, semialatic acid, isofouquierone peroxide, fouquierone	[146]
	Apocyanaceae	Nerium oleander	ocotillol II, 24-epi-ocotillol	[147]
		(leaves)
		Alnus javanica (flower)	Me (24E)-3,4-seco-dammara-4(28),20,24-trien-26-oic acid-3-oate, (24E)-3,4-seco-dammara-4(28),20,24-trien-3,26-dioic acid, (20S,24S)-

Table 8 .

 8 In vitro anti-herpes activity of dammar resin[START_REF] Poehland | In vitro anitiviral activity of dammar resin triterpenoids[END_REF]

	Compound	IC 50 (µg/mL)
		HSV-1	HSV-2
	Dammaradienol	2.5	3.0
	Hydroxydammarenone I	2.0	5.0
	Damarenediol II	7.0	7.0
	Dammarenolic acid	3.0	2.0
	Shoreic acid	7.0	8.0
	Eichlerianic acid	7.0	8.0
	HSV-1=McIntyre stain (ATCC VR-539), HSV-2=MS stain (ATCC VR-540)

  species in Thailand.

	Genus description : Big or vast trees resinous, with transparent resin. Stipule
	oblong to linear-lanceolate, small, caducous. Leaves not plicate, symmetrical or
	asymmetrical at base, chartaceous or coriaceous; secondary nerves pinnate or
	dryobalanoid, arched near to margin; intermediate nerves present in dryobalanoid type;
	tertiary nerves scalariform or reticulate. Domatia frequently present. Flowers rather small
	in panicled unilateral racemes. Sepal 2 external, 3 internal, imbricate. Petal silky or
	tomentose on the part exposed in bud only, contort, narrow, yellow, red or white. Stamen
	usually 15, rarely 10. Anther ovate with 4 pollen sac. Ovary 3 celled, 2-ovuled each. Fruit
	surrounded by 5 enlarged sepal, free to base, 2 outer wings long linear, 3 inner ones not
	longer than fruit, connate in a cup at base, closely appressed to the small nut. Cotyledon
	fleshy, bilobed, unequal. Bark usually smooth, paper flaky, or distantly fissured. Timber
	light red and rather soft.

  Sample of the leaves of H. odorata was collected from Chiang Mai (Maerim district), a northern province of Thailand in September, 2004, and identified by Dr.Chavalit Sittisombut. A voucher specimen was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand.

	3.1 PLANT MATERIAL
	3.1	Plant material
	3.2	Method and apparatus
	3.3	Preliminary study
		3.3.1 Preliminary extraction by soxhlet extraction
		3.3.2 In vitro bioactivity assays
	3.4	Extraction and isolation of H. odorata leaves hexane extract
		3.4.1 Extraction by maceration
		3.4.2 Isolation of major compounds with a small portion of hexane extract.
		3.4.2.1 Isolation by silica gel column chromatography
		3.4.2.2 Isolation by sephadex column chromatography
		3.4.3 Isolation of a large portion of hexane extract
		3.4.4 Purification of HML10
	3.5	Cytotoxic evaluation of 8 isolated lupanes
	3.6	Physical characteristics and spectrum of isolated product

Table 9 .

 9 In vitro bioactivity assays of H.odorata leaves extract

	IC 50 (µg /mL)

Inact. = inactive at the level of 50 µg/mL.

The method for each assay was as follows: Cytotoxicity against MCF-7 (breast cancer)

Cytotoxicity against NCI-H187 (small cell lung cancer)

	Method	Resazurin Microplate assay (REMA)
	Negative control	0.5% DMSO
	IC 50 of positive control	Ellipticine=0.441 µg/mL, Doxorubicine = 0.065 µg/mL
	Maximum final concentration of tested sample	50 µg/mL
	Antifungal activity against Candida albicans
	Method	Resazurin Microplate assay (REMA)
	Negative control	0.5% DMSO
	IC 50 of positive control	Amphotericin B = 0.034 µg/mL
	Maximum final concentration of tested sample	50 µg/mL
	Antituberculosis (

anti-TB) against Mycobacterium tuberculosis H37Ra strain

	Antimalaria against Plasmodium falciparum K1 strain
	Method	Microculture Radioisotope Technique
	Negative control	0.1% DMSO
	IC50 of positive control	Dihydroartemisinine 4.5 nM
	Maximum final concentration of tested sample	10 µg/mL
	Method	Green fluorescent protein microplate assay (GFPMA)
	Negative control	0.5% DMSO
	MIC of positive control

Rifampicin = 0.003-0.012 µg/mL, Streptomycin= 0.156-0.313 µg/mL, Isoniazid = 0.023-0.046 µg/mL, Ofloxacin = 0.391-0.781 µg/mL Maximum final concentration of tested sample 50 µg/mL Scheme 3. Extraction and bioactivity assay of preliminary study of H. odorata leaves.

Table 10 .

 10 Fractionation and isolated compounds from a small portion of H. odorata leaves hexane extract.

	Dried and powdered H.odorata leaves (1,077.30 g)
		extracted with hexane
		(3L*48h*3)
		residue
		extracted with ethanol
		(3L*48h*3)
	19.52 g hexane extract (1.81%)	216.63 g ethanol extract (20.10%)

and epifridelanol (4, 3 mg) were isolated.

Table 11 .

 11 Solid on the surface of the tube B12, B14, B15-B16 and B21 were rechromatographed on silica gel, while B23 was subjected to MPLC (SiO 2 ) again for more purification (Scheme 7). Fractionation of H.odorata leaves hexane extract isolation by MPLC.

	Hexane extract
	hexane extract (2.03 g)	hexane extract (100 mg)
	SiO 2 cc
	Hexane-CH 2 Cl 2	Sephadex cc
	gradient system	(CH 2 Cl 2 )
	19 fractions
	(A01-A19)	Compound 5 (12 mg)
	Fraction A07 (61 mg)	Compound 1 (8 mg)
	Fraction A10 (108 mg)	Compound 2 (20 mg)
	SiO 2 cc
	cHex-EtOAc 6:1
	Fraction A11 (184 mg)	Compound 3 (35 mg)
	SiO 2 cc
	cHex-EtOAc 5:1
	Fraction A17	Compound 1 (3 mg)
	(15 mg)	Compoumd 4 (3 mg)
	SiO 2 cc
	cHex-EtOAc 10:1

  MPLC (cHex, cHex-CH 2 Cl 2 , CH 2 Cl 2 , MeOH)

			g)
	24 fraction B01-B24 Isolation by SiO 2 Fraction B11+B12
	(50 mg)		Compound 6 (25 mg)
		SiO 2 cc
		cHex-CH 2 Cl 2 10:1
	Fraction B14 (217 mg)		Compound 7 (45 mg)
		SiO 2 cc
		cHex-EtOAc 80:1
	Fraction B15 (404 mg)	SiO 2 cc	Compound 8 (133 mg)
		cHex-EtOAc 80:1
	Fraction B16 (137 mg)	SiO 2 cc	Compound 8 (25 mg)
		cHex-EtOAc 70:1
	Fraction B21 (110 mg)		Compound 1 (10 mg)
		SiO 2 cc
		cHex-CH 2 Cl 2 1:2
	Fraction B23 (7.43 g)		To MPLC (SiO 2 )

Table 12 .

 12 Cl 2 , CH 2 Cl 2 -EtOAc, EtOAc, MeOH) Fractionation of Fraction B23 isolated by MPLC

		Isolation by SiO 2 MPLC
	48 fractions C01-C48 (CH 2 Fraction C02 (75 mg)	Compound 1 (1.6 mg)
		SiO 2 cc
		cHex-CH 2 Cl 2 1:3
	Fraction C05 (36 mg)	cHex-EtOAc 14 :1 SiO 2 cc	Compound 9 (16 mg)
	Fraction C07 (176 mg)	cHex-CH 2 Cl 2 2:1 SiO 2 cc	Compound 2 (45 mg)
	Fraction C20 (268 mg)	cHex-EtOAc 20:1	Compound 10 (26 mg) Compound 11 (20 mg)
		SiO 2 cc
	Fraction C21 (140 mg)	SiO 2 cc cHex-EtOAc 6.5:1	Compound 5 (95 mg)
	Fraction C27	cHex-EtOAc 10:1	Compound 5 (31 mg)
	(90 mg)	SiO 2 cc	Compound 12 (10 mg)
		cHex-EtOAc 10:1
	Fraction C29 (96 mg)	SiO 2 cc	Compound 13 (23 mg)
		cHex-EtOAc 10:1
	Fraction C32 (295 mg)	SiO 2 cc	Compound 14 (3 mg)
		cHex-EtOAc 10:1.5
	Fraction C35 (129 mg)	SiO 2 cc	Compound 15 (3 mg)
		cHex-EtOAc 5:0.7
	Fraction C38 (96 mg)		Compound 16 (31 mg)
		SiO 2 cc
	Fraction C39, C41, C42	cHex-EtOAc 5:2	To be continued

28,30-dihydroxy-3-oxolu-20(29)

-ene

(16, 31 mg

)**

Table 12 .

 12 Fractionation of Fraction B23 isolated byMPLC (continued)

	Fraction	Eluent	Weight	Isolated compounds
		(CH 2 Cl 2 -EtOAc)	(mg)
	C39	70-65 : 30-35	218.2	C-26 fatty acid ester of 24,25,26-
				trihydroxy-3,4-seco-cycloart-4(29)-
				en-3-oic acid (18, 3 mg)***
				Messagenic acid G (17, 12 mg)**
	C40	65 : 35	123.1
	C41	60-55 : 40-45	228.7	18 (10 mg)
				18+19 (C-24 fatty acid ester of
				24,25,26-trihydroxy-3,4-seco-
				cycloart-4(29)-en-3-oic acid) (32
				mg)
				18+20 (3,4-seco-cycloart-4(29), 24-
				diene-3,26-dioic acid) (64 mg)
	C42	55-25 : 45-75	324.3	18 (3.8 mg)
				19 (only a little) ***
				20 (only a little)
				19+20 (26 mg)
	C43	25-20 : 75-80	52.6
	C44	20-0 : 80-100	229.5
	C45	100-97 : 0-3	134.0
		EtOAc : MeOH
		(from this fraction)
	C46	97-95 : 33-5	178.1
	C47	95-93 : 5-7	220.3
	C48	90-60 : 10-40	443.3

* the compound found for the first time in the nature. ** the compounds whose complete NMR data were first reported. *** New compounds.

Table 13 .

 13 Cytotoxic activity against HCT116, HT29, MDA-MB231 and PC3 cell lines

	Cpd	HCT 116 b	IC 50 (µM) a HT-29 c	PC3 d	MDA-MB-231 e
	5	85.60±37.61*	/ f	139.00±3.64*	/ f
	13	/ f	/ f		>500	/ f
	10	/ f	/ f		/ f	/ f
	11	/ f	/ f		>300	/ f
	12	/ f	/ f		/ f	/ f
	14	/ f	/ f	282±17.78*	/ f
	16	/ f	/ f		/ f	/ f
	17	43.50±16.26*	/ f		>500	/ f
	cisplatin	4.74±2.01	17.50±17.87	19.93±10.30	21.20±23.36
	a Concentration required for 50% cell growth inhibition. Determined according to WST-1

Table 16

 16 [185, 186]

	Molecular formula C 30 H 50 O		MW 426.72
	Description	small needle white crystal
	Retention factor	R f =0.65 (cHex-EtOAc =5:1)
	Specific rotation	23 [ D ] α -21 (CHCl 3 , c 0.3) (lit.[186] -21.5 (CHCl 3 , c=1, 22 o C))
	IR spectrum	ν max (film) 2926, 2862, 1715, 1389 cm -1
	Mass spectrum	GC-EI-MS		m/z: 426 [M] + , 273, 205, 125, 123, 109
		ESI-MS (ES+)	m/z: 449 [M+Na] + , 413, 236
	NMR spectrum	1 H-and 13 C NMR-see Table 14
	β β β β-amyrin (2) [187]
	Molecular formula C 30 H 50 O		MW 426.72
	Description	a small white crystal
	Retention factor	R f =0.41 (cHex-EtOAc 5:1)
	Specific rotation	20 [ D ] α +47.87 (CHCl 3 , c 0.175)
	IR spectrum	ν max (film) 3353, 2917, 2844, 1463, 1385, 1035, 759 cm -1
	Mass spectrum	GC-EI-MS	m/z : 426 [M] + , 218 (100%), 203
	NMR spectrum	1 H-and 13 C NMR-see Table 15
	β β β β-sitosterol (3) [188]
	Molecular formula C 29 H 50 O		MW 414.72
	Description	white powder
	Retention factor	R f = 0.20 (cHex-EtOAc =5:1)
	Specific rotation	20 [ D ] α -14.44 (CHCl 3 , c 0.18)
	IR spectrum	ν max (film) 3390, 2934, 2864, 1464, 1378, 1061, 757 cm -1
	Mass spectrum	ESI-MS	m/z (ES+): 437 [M+Na] +
		GC-EI-MS m/z: 414[M] + , 396, 381, 329, 303, 273, 255, 213
	NMR spectrum	1 H-and 13 C NMR-see Table 16
	Epifriedelanol (4) [189, 190]
	Molecular formula C 30 H 52 O		MW 428.73
	Description	white needle crystal
	Retention factor	R f =0.21 (cHex-EtOAc =5:1)
	Specific rotation	20 [ D ] α +4 (CHCl 3 , c 0.025)
	IR spectrum	ν

max (film) 2921, 2844 cm -1 Mass spectrum GC-EI-MS m/z: 428 [M] + , 413, 275, 165, 125, 109, 96, 95 NMR spectrum

1 H-and

13

C NMR-see Table

14

  ]

	Molecular formula C 15 H 24 O	MW 220.35
	Description	yellow oil
	Retention factor	R f =0.42 (cHex-EtOAc =10:1)
		R f =0.15 (cHex-CH 2 CL 2 =1:1)
	Specific rotation	20 [ D ] α -34.83 (CHCl 3 , c 0.29) (lit.[58] -46.40 (CHCl 3 , c 5.60, 29 o C))
	IR spectrum	ν max (film) 3066 (=CH 2 ), 2960, 2856, 1732, 1456, 1289, 1123(C-
		O), 1073 (C-O), 743 cm -1

Mass spectrum GC-EI-MS m/z: 220 [M] + , 109, 93, 79 (100%) NMR spectrum

  ]

	Synonym	3-oxobetulin
	Molecular formula C 30 H 48 O 2	MW 440.71
	Description	white wax
	Retention factor	R f =0.47 (cHex-EtOAc =5:2)
	Specific rotation	20 [ D ] α +21.67 (CHCl 3 , c 0.12) (lit.[125] +52.7 (CHCl 3 , c 0.3, 25 o C))
	IR spectrum	ν max (film) 3435, 3065, 2917, 2847, 1705, 1463, 1376, 1025 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 463 [M+Na] +
	NMR spectrum

26-Arachidic acid ester of (24S,25S)-24,25,26-trihydroxy-3,4-seco- cycloart-4(29)-en-3-oic acid (18)***

	IR spectrum	ν max (film) 3700-2500 (br), 2924, 2854, 1713, 1456, 1376 cm -1
	Mass spectrum	HRESI-MS m/z (ES+) : 807.6495 (C 50 H 88 O 6 +Na calc 807.6479)
		HRESI-MS m/z (ES+) triterpene after transesterification 18b :
			513.3572 (C 30 H 50 O 5 +Na calc 513.3556)
	NMR spectrum
	Molecular formula C 30 H 48 O 3	MW 456.71
	Description	green oil
	Retention factor	R f =0.29 (cHex-EtOAc =1:1)
	Specific rotation	20 [ D ] α +3.03 (CHCl 3 , c 0.33) (lit.[95]+7.6 (MeOH, c 0.19))
	IR spectrum	ν max (film) 3434, 2937, 1698, 1456 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 479 [M+Na] +
	NMR spectrum
	Synonym	30-hydroxy-3-oxolup-20(29)-en-28-oic acid
	Molecular formula C 30 H 46 O 4	MW 470.70
	Description	amorphous solid
	Retention factor	R f =0.40 (cHex-EtOAc =1:1)
	Specific rotation	20 [ D ] α +14.29 (CHCl 3 , c 0.14) (lit.[181] +22 (CHCl 3 , c 0.50))
	IR spectrum	ν max (film) 3700-2500 (br), 3074, 2938, 2864, 1695 cm -1
	Mass spectrum	ESI-MS m/z (ES-) : 469 [M-H] -
	NMR spectrum
	Molecular formula C 50 H 88 O 6	MW 785.23
	Description	White semisolid
	Retention factor R Specific rotation [ D 20 ] α +26.32 (CHCl 3 , c 0.19)

28,30-Dihydroxylup-20(29)-en-3-one (16)**

[START_REF] Gonzalez | Triterpene from Maytenus canariensis and synthesis of a derivative from Betulin[END_REF]

1 H NMR -see Table

18

,

13

C NMR -see Table

19

Messagenic acid G (17)** [181] 1 H NMR -see Table

18

,

13

C NMR -see Table

19

f =0.

Methyl ester of 24-Stearic acid ester of (24S,25S)-24,25,26- trihydroxy-3,4-seco-cycloart-4(29)-en-3-oic acid (19a)*** Specific rotation

	The 1 H NMR data for (S)-MTPA and (R)-MTPA ester of compound 18a
	Position	(S)-MTPA ester	(R)-MTPA ester	∆δ=δ S -δ R (ppm)
	H-20	1.4012	1.3976	+0.0036
	H-21	0.8400	0.8392	+0.0008
	H-22a	1.4800	1.3741	+0.1059
	H-22b	1.0000	1.0000	0.0000
	H-26a	3.9782	4.3252	-0.3470
	H-26b	3.8741	4.1466	-0.2725
	H-27	1.1620	1.2041	-0.0421
	3-20 ] [ D α +31.3 (CHCl 3 , c 0.15)
	IR spectrum	ν max (film) 2924, 1739, 1456 cm -1
	Mass spectrum	HRESI-MS m/z (ES+) : 793.6293 (C 49 H 86 O 6 +Na calc 793.6322)
	NMR spectrum

1

H NMR -see Table

23

,

13

C NMR -see

Table 24 (24E)-3,4-Seco-cycloart-4(29), 24-diene-3,26-dioic acid (20) [217]

 24

	Molecular formula C 30 H 46 O 4	MW 470.70
	Description	White solid
	Retention factor	R f = 0.5 (cHex-EtOAc =1:1)
	Specific rotation	20 [ D ] α +31 (CHCl 3 , c 0.07) (lit.[217] +23 (MeOH, c 0.36))
	IR spectrum	ν max (film) 2927, 1695, 1262, 1021, 801 cm -1
	Mass spectrum	EI-MS m/z 469 [M-H] -; HRESI-MS : found m/z 469.33333 ;
		calculated for C 30 H 45 O 4 ([M-H] -) :469.3318.
	NMR spectrum	1 H and 13 C NMR NMR -see Table 25

3,26-dimethyl ester of 20 (20a)

	Molecular formula C 32 H 50 O 4 4.1 Plant material	MW 498.75
	Description Specific rotation 4.2 Preliminary study colorless liquid 20 ] [ D α +43 (CHCl 3 , c 0.07)
	Mass spectrum 4.3 Extraction and isolation of H. odorata leaves hexane extract ESI-MS m/z 499 [M+H] +
	NMR spectrum 4.4 Identification and structural study of isolated compounds 1 H and 13 C NMR NMR -see Table 25
	4.5	Cytotoxic evaluation of 8 isolated lupanes
	4.6	Conclusion

  Pure methyl ester product of 20 (20a) was saponified to its original molecule by LiOH. The mechanism diagrams were shown in Scheme 15.

		-DMF
		-NaHCO 3
	RCOOH	-CH 3 I		RCOOCH 3			overall reaction
	RCOOH + HCO 3	-		RCOO -+ H 2 O + CO 2	1
	RCOO -+ CH 3 -I			RCOOCH 3 + I -		2
	Scheme 13. Reaction mechanism of methylation of carboxylic acid
		OH	O
				OH
		CH 2	O	C 19 H 39
					CH2	OH
		OH	CH 3 ONa CH 3 COOH-H 2 O 3:1	OH	O
	H 3 COOC				+	H 3 CO	C 19 H 39	+	CH 3 OH
				HOOC

CH 2 OH Messagenic acid G, 17 configuration at C-24 of 18a (Scheme 16). After reaction, the (R)-MTPA and (S)-MTPA ester were produced, respectively.

and Saturated Fatty Acid Ester of -Amyrin (6) Figure 13

  Structures of compounds 2 and 6The carbon multiplicities of compound 2 were determined by the13 C NMR spectrum and the HSQCedited correlation. The presence of 8 methyl, 10 methylene, 5 methine, and 7 quaternary carbons was thus established. The signals in the13 C NMR spectrum for oxymethine (δ C-3 79.0), double bond with methine and quaternary carbons (δ C-12 121.7 and δ C-13 145.2) in conjunction with the signals in the 1 H NMR spectrum at δ H-3 3.25 and δ H-12 5.19 were in good agreement with the presence of β-amyrin. The rest of assignment was compared to the literature data[187]

	4.4.2 β β β β-Amyrin (2)
	position	1		4
		δ H	δ C	δ H	δ C
	1	2.02 (m)	22.3	1.58 (m)	15.8
		1.74 (dd, J=13.0, 5.3)		1.49 (d, J=7.3)
	2	2.44 (m)	41.5	1.67 (m)	35.3
		2.36 (m)		1.93 (m)
	3		213.3	3.78 (br.s)	72.8
	4	2.32 (m)	58.2	1.30 (m)	49.2
	5		42.2		37.8
	6	1.80 (m)	41.3	1.78 (dd, J=13.1, 2.8)	41.7
		1.32 (m)		1.03 (d, J=10.3)
	7	1.53 (m)	18.2	1.44 (d, J=2.5)	17.5
		1.44 (m)		1.41 (d, J= 2.5)
	8	1.44 (m)	53.1	1.33 (dd, J=10.4, 3.5)	53.2
	9		37.4		37.1
		1.60 (m)	59.5	0.95 (dd, J=2.3, 11.3)	61.3
		1.52 (m)	35.6	1.59 (m, 1H)	35.5
				1.26 (m, 1H)
		1.39 (m)	30.5	1.39 (m, 1H)	30.6
				1.35 (m, 1H)
			39.7		39.7
			38.3		38.4
		1.32 (m)	32.4	1.56 (dd, J=10.1,4.2)	32.3
				1.33 (dd, J=10.4,3.5)
		1.40 (m)	36.0	1.50 (m)	36.1
				1.17 (m)
			30.0		30.0
		1.62 (dd, J=1.8, 6.0)	42.8	1.59 (m)	42.8
		1.27 (m)	35.3	1.39 (m)	35.2
				1.24 (m)
			28.2		28.2
		1.54 (m)	32.8	1.51 (d, 10.9)	32.8
				1.29 (dt, J=6.6,3.2)
		1.56 (d, J=4.3)	39.3	1.54 (m)	39.3
		1.00 (m)		0.97 (m)
		0.94 (s)	6.8	0.99 (d, J= 6.4)	11.6
		0.77 (s)	14.7	1.01 (s)	16.4
		0.92 (s)	18.0	0.91 (s)	18.2
		1.06 (s)	20.3	1.04 (s)	20.1
		1.10 (s)	18.7	1.06 (s)	18.7
		1.23 (s)	32.1	1.22(s)	32.1
		1.01 (s)	35.0	1.00 (s)	35.0
		1.06 (s)	31.8	1.05 (s)	31.8

Table 17

 17 The 1 H-NMR (400 MHz) and13 C-NMR (75 MHz) data of 3, 7 and 8 (in CDCl 3 ); δ in ppm; J in Hz

	position	3		7	8
		δ H	δ C	δ C	δ C
	1	1.90 (dd, J=9.6,3.6)	37.3	37.0	37.0
		1.13 (dd, J=6.4,3.5)
	2	2.02 (m)	31.7	31.9	31.5
		1.58 (m)
	3	3.58 (m)	71.8	73.7	73.7
	4	2.31 (m)	42.3	42.3	42.3
	5		140.8	139.7	139.7
	6	5.40 (d, J=4.8)	121.7	122.6	122.6
	7	1.59 (m)	31.9	31.9	31.9
	8	1.49 (d, J=4.6)	31.9	31.9	31.9
	9	0.95 (s)	50.1	50.0	50.0
			36.5	36.5	36.6
		1.54 (d, J=1.6)	21.1	21.0	21.0
		2.07 (m)	39.8	39.7	39.7
		1.20 (d, J=4.2)
			42.3	42.3	42.3
		1.03 (s)	56.8	56.7	56.7
		1.64 (m)	24.3	24.3	24.3
		1.33 (s)
		1.92 (m)	28.3	28.3	28.2
		1.30(s)
		1.16 (s)	56.0	56.0	56.0
		0.73 (s)	11.9	11.9	11.9
		1.06 (s)	19.4	19.3	19.3
		1.39 (m)	36.2	36.2	36.2
		0.97 (d, J=6.5)	18.8	18.8	18.8
		1.36 (m)	33.9	33.9	33.9
		0.93 (s)
		1.21 (m)	26.0	26.1	26.0
		0.95 (s)	45.8	45.8	45.8
		1.71 (m)	29.1	29.1	28.7
		0.88 (d, J=1.8)	19.8	19.8	19.8
		0.86 (s)	19.0	19.0	19.0
		1.27 (s)	23.1	23.1	23.1
		0.89 (s)	12.0	12.0	12.0
	-OC(=O)(CH 2 ) 14 CH 3			174.0
	-OC(=O)(CH 2 ) 14 CH 3			29.2-29.6
	-OC(=O)(CH 2 ) 14 CH 3			14.1
	-OC(=O)R				173.3
	CH 2 of fatty acid				22.7,
					29.1-29.7,
					31.9
	double bond of fatty				127.9, 128.0,
	acid				130.2
	terminal methyl of				14.1
	fatty acid

5 11 12 14 16 17 10 13

  The 13 C-NMR and HSQCedited spectra of compound 5 showed 30 carbon siganls representing 6 methyl, 11 methylene, 5 methine and 8 quaternary carbons. The δ C value displayed one ketone (δ C 218.2), one carboxylic group (δ C 180.8) and one double bond (δ C 150.3 (Civ) and 109.8 (CH 2 ))

Table 18 .

 18 The 1 H-NMR (400 MHz) data of 5, 10, 11, 12 and 13 (in CDCl 3 ); δ in ppm; J in

	Hz
	position	5	10	11	12	13
	1	1.38 (m)	1.28 (m)	1.42 (d, J=2.4)	1.38 (m)	0.98 (m)
		1.91 (m)	1.46 (m)	1.94 (t, J=5.1)	1.90 (m)	1.73 (m)
	2	2.42 (m) 2.50 (m)	1.56 (m)	2.45 (m) 2.55 (m)	2.43 (m) 2.50 (m)	1.66 (m)
	3		3.40 (t, J=2.7)			3.21(dd, J=11.2,4.8)
	5	1.35 (m)	1.27 (m)	1.38 (m)	1.32 (m)	0.70 (d, J=9.3)
	6	1.52 (m)	1.41 (m)	1.38 (m) 1.52 (d, J=2.8)	1.47 (m)	1.42 (m) 1.67 (m)
	7	1.44 (m)	1.45 (m)	1.50 (m)	1.45 (m)	1.42 (m)
	9	1.38 (m)	1.45 (m)	1.45 (m)	1.38 (m)	1.33 (m)
		1.34 (m) 1.44 (m)	1.52 (m)	1.47 (m)	1.28 (m) 1.42 (m)	1.49 (m)
		1.06 (m) 1.73 (m)	2.00 (m)	1.69 (m)	1.13 (m) 1.36 (m)	1.78 (m)
		2.23 (m)	2.23 (dt, J= 12.4, 3.4)	1.70 (m)	1.68 (m)	2.23(m)
		1.42 (m) 1.99 (m)	1.46 (m) 2.05 (m)	1.77 (m)	1.06 (m) 1.71 (m)	1.46 (m) 2.06 (m)
		1.44 (m) 2.28 (m)	1.47 (m)	1.47 (m) 2.00 (m)	1.40 (m) 1.52 (m)	1.47 (m)
		1.64 (m)	1.66 (m)	1.65 (m)	1.47 (m)	1.66 (m)
		3.02 (td,	3.02 (dt,	2.47 (dd, J=7.4,	2.34 (td,	3.04 (dt,
		J=10.7,4.8)	J=10.5,4.9)	4.7)	J=10.8,5.3)	J=10.7,4.8)
		1.22 (m) 1.54 (m)	1.25 (m) 1.58 (m)	1.98 (m)	1.33 (m) 2.08 (m)	1.58 (m)
		1.47 (m) 1.99 (m)	1.55 (m) 2.04 (m)	1.92 (m)	1.28 (m) 1.40 (m)	1.55 (m) 2.04 (m)
		1.08 (s)	0.94 (s)	1.08 (s)	1.08 (s)	0.96 (s)
		1.03 (s)	0.82 (s)	1.03 (s)	1.03 (s)	0.78 (s)
		0.94 (s)	0.84 (s)	0.93 (s)	0.93 (s)	0.82 (s)
		0.99 (s)	0.94 (s)	1.07 (s)	1.07 (s)	0.92 (s)
		1.00 (s)	1.00 (s)	1.00 (s)	0.96 (s)	0.98 (s)
				3.36 (d, J=10.8) 3.81 (d, J=11.0)	0.80 (s)
		4.62 (s)	4.62 (s)	4.59 (s)	4.91 (s)	4.62 (s)
		4.75 (s)	4.75 (s)	4.69 (s)	4.95 (s)	4.75 (s)
		1.70 (s)	1.70 (s)	1.70 (s)	4.17 (s, br)	1.71 (s)

Table 19 .

 19 The 1 H-NMR (400 MHz) data of 14, 16 and 17 (in CDCl 3 ); δ in ppm; J in Hz

	position	14	16	17
	1	1.37 (m)	1.38 (m)	1.39 (m)
		1.89 (m)	1.88 (m)	1.90 (m)
	2	2.41 (m)	2.42 (m)	2.43 (m)
		2.47 (m)	2.49 (m)	2.50 (m)
	5	1.31 (m)	1.33 (m)	1.33 (m)
	6	1.46 (m)	1.47 (m)	1.47 (m)
	7	1.43 (m)	1.45 (m)	1.43 (m)
	9	1.35 (m)	1.38 (m)	1.38 (m)
		1.31 (m)	1.27 (m)	1.32 (m)
		1.38 (m)	1.43 (m)	1.45 (m)
		0.91 (m)	1.02 (m)	1.48 (m)
		1.34 (m)	1.41 (m)
		2.22 (m)	1.67 (m)	2.21 (m)
		1.23 (m)	1.12 (m)	1.23 (m)
		1.55 (m)	1.73 (m)	1.54 (m)
		1.51 (m)	1.13 (m)	1.46 (m)
		2.32 (m)	1.90 (m)	2.31 (m)
		2.02 (m)	1.72 (m)	1.77 (t, J =11.5)
		3.35 (td, J=11.2,4.6)	2.31 (m)	2.90 (td, J=11.0,4.5)
		1.42 (m)	1.31 (m)	1.42 (m)
		2.16 (m)	1.96 (m)	2.11 (m)
		1.75 (m)	1.43 (m)	1.57 (m)
		2.00 (m)	2.12 (m)	1.98 (m)
		1.07 (s)	1.07 (s)	1.08 (s)
		1.01 (s)	1.02 (s)	1.02 (s)
		0.91 (s)	0.92 (s)	0.92 (s)
		0.96 (s)	1.06 (s)	0.97 (s)
		0.96 (s)	0.99 (s)	1.02 (s)
			3.33 (d, J= 10.8)
			3.80 (d, J=10.8)
		5.93 (s)	4.91 (s)	4.94 (s)
		6.31 (s)	4.96 (s)	4.99 (s)
		9.53 (s)	4.12 (m)	4.14 (s)

Table 20 .

 20 The 13 C-NMR (75 MHz) data of 5

, 10, 11, 12, 13, 14, 16 and

  17 (in CDCl 3 ); δThe molecular formula of C 30 H 48 O 2 of compound 11 was suggested by the MS m/z at 463 amu [M+Na] + . The NMR data of compound 11 were similar to betulonic acid[START_REF] Atun | Oligostilbenoids from Hopea Mengarawan (Dipterocarpaceae)[END_REF]. The 13 C-NMR and HSQCedited spectrum displayed 6 methyl, 12 methylene, 5 methine, 7 quaternary carbons with the presence of 1 hydroxy methylene (δ C60.5; δ H 3.36 and 3.81), 1 pair of terminal olefin carbons (δ C 150.4 and 109.8) and 1 carbonyl carbon (δ C 218.3). The 1 H-NMR showed signals ofH-23, -24, -25, -26, -27, -30 of methyl groups at δ 1.08, 1.03, 0.93, 1.07, 1.00 and 1.70 ppm, respectively. A hydroxyl methylene, in stead of carboxylic group, at position 28 was suggested based on two downfield δ H3.36 (d) and 3.81 (d) which attached on a downfield δ C at 60.51. It also showed δ H of terminal methylene group C-29 at 4.69 and 4.59. The HMBC correlations: H-29 (δ H 4.59, 4.69) and C-19 (δ C 47.8), C-30 (δ C 19.1) confirmed the presence of terminal olefin group, whereas those from H-28 (δ H 3.36, 3.81) to C-16 (δ C 29.6) and C-22 (δ C 34.0) as well as from H-18 (δ H 1.65) to C-28 (δ C 60.5) confirmed the presence of a hydroxyl group at C-28 (Figure

	in ppm
	position	5	10	11	12	13	14	16	17
	1	39.6	33.2	39.6	39.6	38.7	39.6	39.6	39.6
	2	34.1	25.4	34.1	34.2	27.4	34.1	34.1	34.1
	3	218.2	76.3	218.3	218.3	79.0	218.2	218.2	218.1
	4	47.3	37.5	47.4	47.3	38.9	47.3	47.3	47.3
	5	54.9	49.0	54.9	54.9	55.3	54.9	54.9	54.9
	6	19.6	18.2	19.7	19.7	18.3	19.6	19.6	19.6
	7	33.6	34.2	33.5	33.6	34.3	33.6	33.5	33.6
	8	40.6	40.9	40.9	40.8	40.7	40.6	40.9	40.6
	9	49.9	50.3	49.7	49.7	50.5	49.7	49.7	49.8
		36.9	37.3	36.9	36.9	37.2	36.9	36.8	36.9
		21.4	20.7	21.4	21.5	20.8	21.3	21.4	21.5
		25.4	25.5	25.2	26.7	25.5	27.2	26.8	26.8
		38.5	38.4	37.4	38.1	38.4	38.4	37.3	38.4
		42.5	42.5	42.8	42.8	42.4	42.4	42.7	42.4
		30.5	30.6	27.0	27.4	30.5	29.6	27.0	29.7
		32.1	32.2	29.3	35.4	32.1	31.8	33.8	32.0
		56.3	56.4	47.8	43.0	56.3	56.4	47.8	56.3
		49.2	49.2	48.7	48.8	49.2	50.5	49.3	49.9
		46.9	47.0	47.8	43.8	46.9	38.4	43.5	42.5
		150.3	150.5	150.4	154.7	150.4	156.2	154.4	154.6
		29.7	29.6	29.6	31.8	29.7	31.8	31.7	32.3
		37.0	37.1	34.0	39.8	37.1	36.9	29.1	36.8
		26.6	28.3	26.6	26.7	28.0	26.6	26.7	26.7
		21.0	22.1	21.1	21.1	15.4	21.3	21.0	21.0
		16.0	15.9	15.8	16.0	16.1	15.9	16.0	16.0
		15.8	16.0	16.0	15.8	16.1	15.8	15.8	15.8
		14.6	14.8	14.7	14.5	14.7	14.5	14.7	14.6
		180.8	181.2	60.5	17.7	180.5	181.7	60.2	180.7
		109.8	109.7	109.8	106.9	109.7	134.2	107.2	107.0
		19.4	19.4	19.1	64.9	19.4	195.0	65.0	65.3

  80) attached on the same carbon at δ C 60.2 ppm and, from HMBC, correlated with C-22 (δ C 31.7), C-16 (δ C 33.8) and C-18 (δ C 49.3) (Figure16). From NOESY spectrum, the stereochemistry of C-28 was comfirmed from the correlation between H 28a and H-13, H-15 and H-26 as well as the correlation of H 28b with H-19, H 21b and H 22b . The remaining signals were in agreement with those of betulonic acid[START_REF] Atun | Oligostilbenoids from Hopea Mengarawan (Dipterocarpaceae)[END_REF]. It was finally deduced that this compound was 28,30-dihydroxylup-20(29)-en-3one[START_REF] Gonzalez | Triterpene from Maytenus canariensis and synthesis of a derivative from Betulin[END_REF]. at 469 representing the molecular formula of C 30 H 46 O 4. The spectrums of HSQCedited and 13 C NMR showed 30 carbons: 5 methyl, 12 methylene, 5 methine and 8 quaternary carbons. The NMR data of 17 were close to 16 except the chemical shift at position 28. The downfield shift of δ C-17 to 56.3 indicated the position of the acidic group at C-28. The C-28 (δ C 180.7) of carboxylic group exhibited the HMBC correlations with H-18 (δ H 1.77) and H-22 (δ H 1.98) (Figure

Figure 17. Selected NOEs of compound 16

The ESI (-) MS of 17 showed [M-H] -

  The 13 C-NMR and HSQCedited spectrum showed 15 carbon signals representing 3 methyl, 6 methylene, 3 methine and 3 quaternary carbons. The δ C values suggested 1 oxy-quaternary carbon (δ C 59.9), 1 oxymethine carbon (δ C 63.8) and two terminal olefinic carbons (δ C 151.8 and 112.8). Based on HSQCedited spectrum, The 1 H and 13 C-NMR spectrum agreed with caryophyllene oxide in previous literature

Table 21 The 1

 211

	position	δ H	δ C
	1	1.80 (dd, J=19.8, 10.3)	50.8
	2	1.72 (m), 1.48 (m)	27.2
	3	2.13 (dd, J=7.9, 4.7), 1.05 (s)	39.1
	4		59.9
	5	2.93 (dd, J=10.5, 4.2)	63.8
	6	2.30 (td, J=16.3, 8.1, 4.1), 1.36 (m)	30.2
	7	2.40 (td, J=12.5, 8.1, 4.5), 2.17 (m)	29.8
	8		151.8
	9	2.67 (dd, J=18.8, 9.4)	48.7
	10	1.73 (d, J=8.3), 1.68 (d, J=10.5)	39.8
	11		34.0
	12	1.25 (s)	17.0
	13	5.03 (s), 4.91 (s)	112.8
	14	1.04 (s)	29.9
	15	1.06 (s)	21.6

H-NMR (400 MHz) and

13

C-NMR (75 MHz) data of 9 (in CDCl 3 ); δ in ppm; J in Hz

Table 22 The 1

 221

	position	δ H	δ C	position	δ H	δ C
	1	1.60 (m)	33.4	16	1.35 (m)	28.2
		1.95 (m)
	2	2.34 (m)	37.5	17	1.86 (d, J=8.1)	52.2
		2.77 (m)
	3		216.8	18	1.05 (s)	18.1
	4		50.3	19	0.63 (d, J=4.1)	29.5
					0.84 (d, J=4.1)
	5	1.76 (d, J=4.4)	48.4	20	1.48 (s)	36.0
	6	1.60 (m)	21.5	21	0.98 (s)	18.1
	7	1.42 (m)	25.9	22	1.20 (m)	26.0
	8	1.62 (d, J=3.8)	47.9	23	2.40 (m)	34.7
	9		21.1	24	6.95 (t, J=7.0)	145.9
	10		26.0	25		126.5
	11	1.24 (m)	26.7	26		172.7
	12	1.71 (m)	32.8	27	1.90 (s)	12.0
	13		45.4	28	1.10 (s)	22.2
	14		48.7	29	1.15 (s)	20.8
	15	1.36 (m)	35.5	30	0.96 (s)	19.3

H-NMR (400MHz) and

13

C-NMR (75 MHz) data of 15 (in CDCl 3 ); δ in ppm; J in Hz

Table 23 .

 23 The 1 H-NMR (400MHz) of 18, 18a, 18b and 19a (in CDCl 3 ) ; δ in ppm ; J in Hz.

	position		δ H
		18	18a	18b	19a
	1 CH 2	2.07 (m)	2.05 (m)	2.12 (m)	2.05 (m)
		1.39 (m)	1.38 (m)	1.35 (m)	1.38 (m)
	2 CH 2	2.57 (m)) 2.37 (m)	2.51 (m) 2.27 (m)	2.57 (m) 2.35 (m)	2.51 (m)
	3 -C(=O)-O-
	4 Civ db
	5 CH	2.43 (m)	2.43 (m)	2.46 (m)	2.43 (m)
	6 CH 2	1.50 (m) 1.08 (m)	1.53 (m) 1.50 (m)	1.61 (m)	1.53 (m) 1.09 (m)
	7 CH 2	1.31 (m) 1.10 (m)	1 .31 (m) 1.10 (m)	1.10 (m)	1 .33 (m) 1.11 (m)
	8 CH	1.57 (m)	1.57 (m)	1.60 (m)	1.57 (m)
	9 Civ
	10 Civ
	11 CH	2.11 (m)	2.11 (m)	2.11 (m)	2.10 (m)
		1.23 (m)	1.23 (m)	1.11 (m)	1.25 (m)
	12 CH	1.66 (m)	1.66 (m)	1.51 (m)	1.66 (m)
	13 Civ
	14 Civ
	15 CH	1.30 (m)	1.30 (m)	1.27 (m)	1.29 (m)
	16 CH	1.93 (m) 1.29 (m)	1.90 (m) 1.29 (m)	1.32 (m)	1.89 (m) 1.29 (m)
	17 CH	1.60 (m)	1.59 (m)	1.63 (m)	1.58 (m)
	18 CH	0.97 (s)	0.97 (s)	0.99 (s)	0.97 (s)
	19 CH	0.73 (d, J=4)	0.73 (d, J=4.1)	0.76 (d, J=4.3)	0.73 (d, J =4.3)
		0.42 (d, J=4)	0.41 (d, J=4.1)	0.43 (d, J=4.3)	0.41 (d, J= 4.3)
	20 CH	1.43 (m)	1.41 (m)	1.41 (m)	1.42 (m)
	21 CH	0.90 (d, J=3.3)	0.93 (d, J=4.2)	0.90 (d, J=6.2)	0.88 (d, J=4.6)
	22 CH	1.49 (m) 1.30 (m)	1.48 (m) 1.31 (m)	1.29 (m)	1.74 (m) 1.40 (m)
	23 CH	1.47 (m) 1.41 (m)	1.46 (m) 1.41 (m)	1.33 (m)	1.80 (m) 1.68 (m)
	24 CH-O-	3.43 (d, J=9.2)	3.42 (d, J=8.8)	3.55 (m)	4.73 (overlap with H 29b )
	25 Civ -O-
	26 CH -O-	4.29 (d, J=11.5)	4.28 (d, J=11.4)	3.87 (d, J=11.6)	3.38 (d, J=12)
		4.03 (d, J= 11.5)	4.04 (d, J=11.4)	3.50 (d, J=11.6)	3.23 (d, J=12)
	27 CH	1.21 (s)	1.20 (s)	1.13 (s)	1.06 (s)
	28 CH	1.75 (s)	1.69 (s)	1.70 (s)	1.69 (s)
	29 CH db	4.83 (s) 4.75 (s)	4.81 (s) 4.74 (s)	4.87 (s) 4.79 (s)	4.81 H29a (s) 4.73 (overlap with H-24)
	30 CH	0.94 (s)	0.94 (s)	0.96 (s)	0.93 (s)
	3-OCH 3		3.65 (s)		3.65 (s)
	1'-(CH 2 ) n -CH 3		2.37 (t, J=7.5) 1.30 (m)		2.38 (t, J=7.5) 1.26 (m)
	1'-(CH 2 ) n -CH 3		0.93 (s)		0.87 (s)

Table 25 .

 25 The 1 H-NMR (400 MHz) and13 C-NMR (75 MHz) data of 20 and 20a (in CDCl ); δ in ppm; J in Hz 24 and C-25 was assigned as E-form according to the downfield shift of H-24 of 20 (δ H 6.9 instead of 6.0). Dimethyl esters of 20a at position 3 and 26 were supported by the HMBC correlation of δ H 3.64 to δ C 174.4 (C-3) and δ H 3.73 to δ C 168.8 (C

	position	δ H		δ C
		20	20a	20	20a
	1 CH 2	2.08 (m)	2.05 (m)	28.8	29.0
		1.37 (m)	1.38 (m)
	2 CH 2	2.55 (m)	2.52 (m)	31.4	31.4
		2.30 (m)	2.27 (m)
	3 C(=O)-O			180.2	174.4
	4 Civ db			149.4	149.5
	5 CH	2.44 (dd, J=10.9, 5.3)	2.43 (dd, J=11.0, 4.8)	45.8	45.8
	6 CH 2	1.52 (m)	1.52 (m) ; 1.09 (m)	27.7	27.7
		1.08 (m)
	7 CH 2	1.31 (m)	1.31 (m)	25.0	25.0
		1.10 (m)	1.12 (m)
	8 CH	1.56 (m)	1.58 (m)	47.7	47.7
	9 Civ			21.3	21.3
	10 Civ			26.9	27.0
	11 CH	2.10 (m)	2.10 (m)	26.9	26.9
		1.26 (m)	1.26 (m)
	12 CH	1.66 (m)	1.66 (m)	33.0	33.0
	13 Civ			45.2	45.2
	14 Civ			49.0	49.0
	15 CH	1.30 (m)	1.30 (m)	35.6	35.6
	16 CH	1.90 (m)	1.91 (m)	28.1	28.1
		1.29 (m)	1.29 (m)
	17 CH	1.60 (m)	1.60 (m)	52.2	52.1
	18 CH	0 .97 (s)	0.96 (s)	18.1	18.0
	19 CH	0.75 (d, J=4)	0.73 (d, J=4)	30.0	29.9
		0.42 (d, J=4)	0.41 (d, J=4)
	20 CH	1.42 (m)	1.43 (m)	36.0	36.0
	21 CH	0.92 (d, J=6.4)	0.91 (d, J=6.4)	18.1	18.1
	22 CH	1.57 (m)	1.58 (m)	34.7	34.9
		1.18 (m)	1.17 (m)
	23 CH	2.26 (m)	2.24 (m)	26.0	25.7
		2.13 (m)	2.10 (m)
	24 CH db	6.91 (t, J=6.9)	6.77 (t, J=7.0)	146.0	143.2
	25 Civ db			126.6	127.1
	26 C(=O)-O			173.6	168.8
	27 CH	1.84 (s)	1.84 (s)	12.0	12.4
	28 CH	1.69 (s)	1.69 (s)	19.7	19.7
	29 CH db	4.82(s)	4.81(s)	111.6	111.5
		4.74(s)	4.73 (s)
	30 CH	0.94 (s)	0.93 (s)	19.3	19.3
	3-OCH 3		3.64 (s)		51.5
	26-OCH 3		3.73 (s)		51.7
	Compound 20 established the molecular formula of C 30 H 46 O 4 by ESI-MS at m/z 469 [M-
	H] -. Twenty eight amu increasing mass of methylation product (20a) indicated two
	carboxylic groups were presented in the structure. This information was campatible with

two additional methoxyl proton signals at δ H 3.64 and 3.73. More information of the side chain was obtained from the HMBC experiment which gave a good agreement with the side chain of mangiferonic acid

[START_REF] Tanaka | Hopeafuran and a C-glucosyl resveratrol isolated from stem wood of Hopea utilis[END_REF]

as shown in Figure

23

. The stereochemistry of olefin at C-

  177]

	Synonyms: Dipterocarpus angustifolius, Dipterocarpus insularis, Dipterocarpus
	artocarpifolius, Dipterocarpus parvifolius
	Vernacular name: Yang pai	and Yang phrai	are both preferred
	names. Yang kaen (	and Yang hi		Yang bai
	iat	hua waen

  Dipterocarpus costatus wood was collected from Chiang Mai (Maerim district), a northern province of Thailand in September, 2004, and identified by Dr.Chavalit Sittisombut. A voucher specimen was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand.

	3.1 PLANT MATERIAL
	3.1	Plant material
	3.2	Method and apparatus
	3.3	Preliminary extraction
		3.3.1 Preliminary extraction by soxhlet extraction
		3.3.2 In vitro bioactivity assays
	3.4	Extraction and isolation of D. costatus wood hexane extract
		3.4.1 Extraction by maceration
		3.4.2 Isolation
	3.5	Cytotoxic evaluation of isolated triterpenes
	3.6	In vitro antiplasmodial activity of isolated triterpenes
	3.7	Physical characteristics and spectrum of isolated compounds

Table 26 .

 26 In vitro bioactivity assays of D. costatus wood extract

	IC 50 (µg /mL)

anti-TB) against Mycobacterium tuberculosis H37Ra strain

	3.4 EXTRACTION AND ISOLATION OF D. costatus WOOD
	HEXANE EXTRACT
	3.4.1 Extraction by maceration
	The dried and chopped heartwood (2,645 gm.) of D. costatus was macerated with hexane
	(3L*48h*3), then maceration continued with dichloromethane, ethyl acetate and ethanol
	(3L*48h*3, each), one by one, at room temperature. All extracts were separately filtered
	and concentrated under vacuum. (Scheme 19)
	Dried and chopped D. costatus wood (2,645 g)
	• maceration (hexane, 3L*48h*3)
	• filtering and drying
	16.95 g hexane extract (0.64%)	residue
	(yellow brown gum)
		• maceration (CH 2 Cl 2 , 3L*48h*3)
		• filtering and drying
	8.50 g CH 2 Cl 2 extract (0.32%)	residue
	(dark brown flake)	• maceration (EtOAc, 3L*48h*3)
		• filtering and drying
	3.51 g EtOAc extract (0.13%)	residue
	(red brown solid)
		53.05 g EtOH extract (2.01%)
		(red brown resin)
	Scheme 19. Extraction of D. costatus wood for phytochemical study
	3.4.2 Isolation.
	The hexane extract (12 g) was adsorbed on silica gel (12 g), then, fractionated by silica gel
	Method medium pressure liquid chromatography (MPLC), and successively eluted with gradually Green fluorescent protein microplate assay (GFPMA)
	Negative control increasing polarity of eluents; cyclohexane, gradient mixtures of cyclohexane-0.5% DMSO
	MIC of positive control dichloromethane, dichloromethane, gradient mixtures of dichloromethane-ethyl acetate, Rifampicin = 0.003-0.012 µg/mL, Streptomycin= 0.156-
	0.313 µg/mL, Isoniazid = 0.023-0.046 µg/mL, Ofloxacin = 0.391-0.781 µg/mL ethyl acetate, gradient mixtures of ethyl acetate-methanol, and methanol. The elution
	Maximum final concentration of tested sample afforded 70 fractions (D01-D70, as described in table 19) on the basis of their TLC. 50 µg/mL

Fraction D02 (colorless semisolid) and fraction D50 (yellow gum) showed only one spot on TLC (cHex-EtOAc 100:1 and 1:1, respectively, sprayed with H 2 SO 4 /vanillin reagent). After spectroscopic analysis, they were identified as -elemene

(21, 10.6 mg)

for fraction

Table 27 .

 27 Fractionation of D. costatus wood hexane extract isolation by MPLC.

	Fraction	Eluent	Weight (mg)	Isolated compounds
		(cHex-CH 2 Cl 2 -EtOAc-MeOH, %)
	D01	cHex-CH 2 Cl 2 100-90:0-10	91.1
	D02	cHex-CH 2 Cl 2 85:15	10.6	β-elemene (21, 10.6 mg)
	D03	cHex-CH 2 Cl 2 80-70:20-30	11.9
	D04	cHex-CH 2 Cl 2 65-55:35-45	15.2
	D05	cHex-CH 2 Cl 2 50-45:40-55	118.9
	D06	cHex-CH 2 Cl 2 40-35:60-65	24.6
	D07	cHex-CH 2 Cl 2 30-25:70-75	21.2
	D08	cHex-CH 2 Cl 2 20-10:80-90	18.1
	D09	cHex-CH 2 Cl 2 5-0:95-100	143.6
	D10	cHex-CH 2 Cl 2 0:100	58.6
	D11	cHex-CH 2 Cl 2 0:100	375.5	caryophyllene oxide (9, 150 mg)
	D12	cHex-CH 2 Cl 2 0:100	164.6
	D13	cHex-CH 2 Cl 2 0:100	117.8
	D14	cHex-CH 2 Cl 2 0:100	35.1
	D15	cHex-CH 2 Cl 2 0:100	57.5
	D16	cHex-CH 2 Cl 2 0:100	200	-sitosterol (3, 200 mg)
	D17	CH 2 Cl 2 -EtOAc 100-96:0-4	62.1
	D18	CH 2 Cl 2 -EtOAc 94:6	227.5
	D19	CH 2 Cl 2 -EtOAc 94:6	102.6
	D20	CH 2 Cl 2 -EtOAc 94:6	38.4
	D21	CH 2 Cl 2 -EtOAc 94:6	242.0
	D22	CH 2 Cl 2 -EtOAc 92:8	502.5	dipterocarpol (22, 400 mg)
				isofouquierone peroxide (23, 20 mg)
	D23	CH 2 Cl 2 -EtOAc 92:8	962.8
	D24	CH 2 Cl 2 -EtOAc 92:8	539.7	(20R)-17 ,29-epoxy-28-norlupan-3-one
				(24, 20 mg) ***
				dipterocarpol (22, 70 mg)
				17 -hydroxy-28-norlupan-20(29)-en-3-one
				(25, 72 mg) ***
				octanordammarane-3,17-dione (26, 16 mg)
				*
				isocabralealactone (20R) (27, 42 mg) **
				cabralelactone (28, 131 mg)
				isofouquierone peroxide (23, 11 mg)
				(20S)-20-hydroxy-24-perhydroxy-
				dammar-25-en-3-one (29, 6 mg) ***
	D25	CH 2 Cl 2 -EtOAc 90:10	179.4
	D26	CH 2 Cl 2 -EtOAc 90:10	270.7

Table 27 .

 27 Fractionation of D. costatus wood hexane extract isolation by MPLC.

	(continued)
	Fraction	Eluent	Weight (mg)	Isolated compounds
		(cHex-CH 2 Cl 2 -EtOAc-MeOH, %)
	D27	CH 2 Cl 2 -EtOAc 88:12	617.6	cabraleone (30, 350 mg)
				dammarenediol II (31, 233 mg)
				cereotagaloperoxide= (20S)-3 ,20-
				dihydroxy-24-perhydroxydammar-25-ene
				(32, 3 mg) **
				isofouquierol peroxide (33, 11 mg)
	D28	CH 2 Cl 2 -EtOAc 88:12	656.7	cabraleone (30, 28 mg)
				ocotillone (34, 456 mg)
				cabralealactone (28, 14 mg)
				3-epicabraleahydroxylactone (35, 20 mg)
				isofouquierol peroxide (33, 8 mg)
	D29	CH 2 Cl 2 -EtOAc 86:14	432.2
	D30	CH 2 Cl 2 -EtOAc 86-84:14-16	252.8
	D31	CH 2 Cl 2 -EtOAc 84:16	136.9
	D32	CH 2 Cl 2 -EtOAc 82:18	143.0	(20S)-29-hydroxy-17 ,20-peroxy-28-
				norlupan-3-one (36, 4 mg) ***
	D33	CH 2 Cl 2 -EtOAc 82:18	129.0
	D34	CH 2 Cl 2 -EtOAc 82:18	60.2
	D35	CH 2 Cl 2 -EtOAc 80:20	265.2	ocotillol II (37, 40 mg)
				(20S,24S)-20,24-dihydroxydammar-25-en-
				3-one (38, 5 mg)
	D36	CH 2 Cl 2 -EtOAc 80:20	50.6
	D37	CH 2 Cl 2 -EtOAc 80:20	473.7	isofouquierone peroxide (23, 80 mg)
				(20S,23E)-20-hydroxy-27-nordammar-23-
				ene-3,25-dione (39, 8 mg) ***
				isofouquierone (40, 37 mg)
	D38	CH 2 Cl 2 -EtOAc 80-78:20-22	191.0	ocotillol II (37, 5 mg)
				isofouquierone peroxide (23, 6 mg)
				17 -hydroxy-28,29-dinorlupan-3,20-dione
				(41, 6 mg) ***
				(20S,23E)-20-hydroxy-27-nordammar-23-
				ene-3,25-dione (39, 40 mg) ***
	D39	CH 2 Cl 2 -EtOAc 78:22	136.8
	D40	CH 2 Cl 2 -EtOAc 78-76:22-24	109.2
	D41	CH 2 Cl 2 -EtOAc 76:24	143.7
	D42	CH 2 Cl 2 -EtOAc 74-72:26-28	267.3	(20R)-20-hydroxy-17 ,29-epoxy-28-
	D43	CH 2 Cl 2 -EtOAc 72:28	93.8	norlupan-3-one (42, 17 mg) ***
				(20S,24S)-20,24-dihydroxydammar-25-en-
				3-one (38, 73 mg)
	D44	CH 2 Cl 2 -EtOAc 72:28	112.9	(20S,24S)-20,24-dihydroxydammar-25-en-
				3-one (38, 15 mg)
	D45	CH 2 Cl 2 -EtOAc 70:30	92.0
	D46	CH 2 Cl 2 -EtOAc 70-68:30-32	58
	D47	CH 2 Cl 2 -EtOAc 68:32	116.4
	D48	CH 2 Cl 2 -EtOAc 66:34	90.2
	D49	CH 2 Cl 2 -EtOAc 66-62:34-38	549.2
	D50	CH 2 Cl 2 -EtOAc 62:38	119.3	isofouquierone (40,119 mg)
	D51	CH 2 Cl 2 -EtOAc 60:40	97.5
	D52	CH 2 Cl 2 -EtOAc 60-58:40-42	69.9
	D53	CH 2 Cl 2 -EtOAc 58-56:42-44	98.2	clavone-2,9-diol (43)

Table 27 .

 27 Fractionation of D. costatus wood hexane extract isolation by MPLC.

	(continued)
	Fraction	Eluent	Weight (mg)	Isolated compounds
		(cHex-CH 2 Cl 2 -EtOAc-MeOH, %)
	D54	CH 2 Cl 2 -EtOAc 56:44	62.6
	D55	CH 2 Cl 2 -EtOAc 54-50:46-50	163.2	isofouquierol (44, 54 mg)
	D56	CH 2 Cl 2 -EtOAc 50-48:50-52	41.4
	D57	CH 2 Cl 2 -EtOAc 48-44:52-56	36.2
	D58	CH 2 Cl 2 -EtOAc 44-40:56-60	59.6	(20S,22E,24R)-20,24,25-
				trihydroxydammar-22-en-3-one (45, 6 mg)
				***
	D59	CH 2 Cl 2 -EtOAc 40-35:60-65	48	(20S,22E,24R)-20,24,25-
				trihydroxydammar-22-en-3-one (45, 5 mg)
				***
	D60	CH 2 Cl 2 -EtOAc 35-30:65-70	26.4
	D61	CH 2 Cl 2 -EtOAc 25:75	33.5
	D62	CH 2 Cl 2 -EtOAc 25-20:75-80	37.7
	D63	CH 2 Cl 2 -EtOAc 15:85	17.5
	D64	CH 2 Cl 2 -EtOAc 15-0:85-100	80.0	(20S,23E)-

Table 28 .

 28 % Cell proliferation inhibition of the isolated triterpenes

	% cell proliferation inhibition

Table 29 .

 29 Screening test for in vitro antiplasmodial activity and cytotoxicity of the isolated triterpenes

		Antiplasmodial activity	Cytotoxicity against L-6 cells
	Compound	% growth inhibition at 10 µM	% cell proliferation inhibition at 10 µM	% cell proliferation inhibition at 1 µM
		14.21±11.36	0.00±0.00	0.65±7.75
		17.98±2.28	5.53±2.83	3.58±15.81
		0.00±0.00	8.67±10.84	0.14±8.92
		7.76±8.78	4.23±5.62	2.55±5.43
		26.67±0.93	9.61±2.69	11.69±6.00
		7.49±3.40	33.85±6.83	1.59±11.32
		13.90±0.89	10.72±10.29	6.20±6.97
		19.44±16.59	2.89±12.13	0.00±0.00
		11.35±10.18	0.33±13.26	6.33±4.39
		27.80±5.26	0.00±0.00	6.10±5.71
		4.15±10.92	0.83±8.73	24.13±5.06
		17.85±12.01	0.00±0.00	3.16±5.81
		0.00±0.00	3.81±3.23	0.00±0.00
		90.94±1.48	15.57±2.90	13.94±5.38
		0.00±0.00	0.75±0.34	0.00±0.00
		22.95±18.43	0.00±0.00	0.00±0.00
		27.81±4.87	0.00±0.00	0.00±0.00
		17.52±6.06	0.00±0.00	7.28±5.78
		0.00±0.00	2.88±10.47	12.18±4.67
		14.83±2.26	0.80±2.65	12.91±5.90
		1.26±3.78	8.01±2.43	0.92±3.05
		23.71±9.72	4.33±2.30	4.59±0.61
		14.38±9.36	13.53±6.15	12.03±4.22
		7.62±4.25	5.24±0.69	9.84±1.84
		3.65±1.48	0.34±6.52	8.67±5.03

Table 30 .

 30 Antiplasmodial potency of compound 36

	Antimalarial activities against
	P. falciparum (FcB1 strain)
	IC 50 (µM)	IC 90 (µM)
	3.74±0.76	10.31±0.62
	IC 50 for chloroquine was 72.6±7.4 nM

  )[226]

	Molecular formula C 15 H 24	MW 204
	Description	colorless resin
	Retention factor	R f =0.73 (cHex-EtOAc =100:1)
	Specific rotation	20 [ D ] α +3.2 (CHCl 3 , c 0.125) (lit.[226] +15.4 (CHCl 3 , c 0.59, 23 o C))
	IR spectrum	ν max (film) 3082(=C-H stretching), 2926, 2851, 1644,1455,1378 cm -1
	Mass spectrum	GC-EI-MS m/z: 204 [M] + , 189, 175, 147, 107, 93 (100%), 81, 67
	NMR spectrum	1 H and 13 C-NMR -see Table 31

Table 35 (20R)-17 ,29-Epoxy-28-norlupan-3-one (24) ***

 35 229] +74.1 (CHCl 3 , c 0.135) (lit.[71] +67 (CHCl 3 , c 1.09, 26 o C))

	Molecular formula C 30 H 50 O 2	MW 442.72
	Description	white shiny flakes
	Retention factor	R f =0.50 (cHex-EtOAc =3:1)
	Specific rotation α IR spectrum [ D 20 ] ν max (film) 3505, 2949, 2864, 1704, 1457, 1377, 1109 cm -1
	Mass spectrum	ESI-MS m/z (ES+): 907 [2M+Na] + , 465 [M+Na] +
	NMR spectrum	1 H and 13 C-NMR -see Table 32
	Isofouquierone peroxide (23) [146]
	Molecular formula C 30 H 50 O 4	MW 474.72
	Description	white powder
	Retention factor	R f =0.17 (cHex-EtOAc =3:1), =0.57 (cHex-EtOAc =1:1)
	Specific rotation	20 [ D ] α +41.0 (MeOH, c 0.105) (lit.[146] +67 (CHCl 3 , c 0.29))
	IR spectrum	ν max (film) 3412, 2960, 2862, 1695, 1455, 1384 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 971[2M+Na] + , 497 [M+Na] +
	NMR spectrum 1 H-NMR -see Table 33, 13 C-NMR -see Molecular formula C 29 H 46 O 2 MW 426.67
	Description	white needle crystal
	Retention factor	R f =0.60 (cHex-EtOAc =3:1)
	Specific rotation	20 [ D ] α +11.3 (CHCl 3 , c 0.115)
	IR spectrum	ν max (film) 2926, 2864, 1707, 1456, 1380 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 875[2M+Na]

+ , 449 [M+Na] + HRMS (ESI-TOF) m/z (ES+) : 449.3399 [M+Na] + (calcd for C 29 H 46 O 2 Na, 449.3396) NMR spectrum 1 H-NMR -see Table 42, 13 C-NMR -see Table 43

17 -Hydroxy-28-norlupan-20(29)-en-3-one (25) ***

	Molecular formula C 29 H 46 O 2	MW 426.67
	Description	colorless resin
	Retention factor	R f =0.42 (cHex-EtOAc =3:1)
	Specific rotation	20 [ D ] α +41.3 (CHCl 3 , c 0.155)
	IR spectrum	ν max (film) 3441 (OH), 2948, 2868, 1704 (C=O), 1642, 1454, 1384,
		756 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 875[2M+Na] + , 449 [M+Na] +
		HRMS (ESI-TOF) m/z (ES+) : 449.3400 [M+Na] + (calcd for
		C 29 H 46 O 2 Na, 449.3396)
	NMR spectrum	1 H-NMR -see Table 42, 13 C-NMR -see Table 43
	Octanordammarane-3,17-dione (26) *[230]
	Molecular formula C 22 H 34 O 2	MW 330.50
	Description	colorless resin
	Retention factor	R f =0.33 (cHex-EtOAc =3:1)
	Specific rotation	20 [ D ] α +94.0 (CHCl 3 , c 0.065)
	IR spectrum	ν max (film) 2947, 2869, 1738 (C-17 ketone), 1705 (C-3 ketone),
		1455, 1384, 755 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 683 [2M+Na] + , 353 [M+Na] +
		HRMS (ESI-TOF) m/z (ES+) : 353.2466 [M+Na] + , (calcd for
		C 22 H 34 O 2 Na, 353.2457) and 331.2645 [M+H] + (calcd for C 22 H 35 O 2 ,
		331.2637)
	NMR spectrum	1 H and 13 C-NMR -see Table 41
	Isocabralealactone (20R) (27)** [60, 67, 161, 229, 231]
	Synonym	25,26,27-trinordammaran-3-one-20(R),24-olide
	Molecular formula C 27 H 42 O 3	MW 414.31
	Description	white amorphous powder
	Retention factor	R f =0.23 (cHex-EtOAc =3:1)
	Specific rotation	20 [ D ] α +71.0 (CHCl 3 , c 0.100)
	IR spectrum	ν max (film) 2949, 2862, 1767 (lactone), 1704 (ketone), 1460, 1383,
		1245, 1193, 754 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 851 [2M+Na] + , 437 [M+Na] + , 415 [M+H] +
		HRMS (ESI-TOF) m/z (ES+) : 437.3023 [M+Na] + (calcd for
		C 27 H 42 O 3 Na, 437.3032)
	NMR spectrum	1 H and 13 C-NMR -see Table 40

  ]

	Cereotagaloperoxide (32) [146, 158]
	Synonym	20(S)-3 ,20-dihydroxy-24-perhydroxydammar-25-ene
	Molecular formula C 30 H 52 O 4	MW 476.73
	Description	white solid
	Retention factor	R f =0.15 (cHex-EtOAc =5:2)
	Specific rotation	20 [ D ] α +20.0 (CHCl 3 , c 0.070) (lit.[158] +54.1 (MeOH, c 0.04))
	IR spectrum	ν max (film) 3405, 2943, 2861, 1453, 1377, 756 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 975 [2M+Na] + , 499 [M+Na] +
	NMR spectrum	1 H and 13 C-NMR -see Table 37
	Isofouquierol peroxide (33) [157]
	Molecular formula C 30 H 52 O 4	MW 476.73
	Description	white solid
	Retention factor	R f =0.15 (cHex-EtOAc =3:1)
	Specific rotation	20 [ D ] α +21.0 (CHCl 3 , c 0.105) (lit.[157] -0.08 (CHCl 3 , c 0.05))
	IR spectrum	ν max (film) 3404, 2943, 2867, 1453, 1377, 1028, 757 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 975 [2M+Na]
	Molecular formula C 30 H 52 O 2	MW 444.73
	Description	colorless oil
	Retention factor	R f =0.38 (cHex-EtOAc =5:2)
	Specific rotation	20 [ D ] α +12.9 (CHCl 3 , c 0.155) (lit.[23] +32.8 (c 1.05))
	IR spectrum	ν max (film) 3444, 2944, 2870,1699,1455,1378, 1216, 1044, 757 cm -1
	Mass spectrum	EI-MS m/z : 317 [M-side chain] + , 299 (100%), 207, 189, 109, 95, 69
	NMR spectrum	1 H and 13 C-NMR -see Table 32

+ , 499 [M+Na] + , 477 [M+H] +

Table 39 3-Epicabraleahydroxylactone (20S) (35) [160]

 39

	formula C 30 H 50 O 3	MW 458.72
	Description	white needle crystal
	Retention factor	R f =0.44 (cHex-EtOAc =3:1)
	Specific rotation	20 [ D ] α +58.6 (CHCl 3 , c 0.145) (lit.[39] +59 (CHCl 3 , 26 o C))
	IR spectrum	ν max (film) 3473, 2965, 2871, 1705, 1456, 1384, 1080, 756 cm -1
	Mass spectrum	EI-MS m/z (ES+) : 939 [2M+Na] + , 481 [M+Na] +
	NMR spectrum
	Synonym	(20S)-3 -hydroxy-25,26,27-trinordammaran-20,24-olide
	Molecular formula C 27 H 44 O 3	MW 416.33
	Description	colorless resin
	Retention factor	R f =0.13 (cHex-EtOAc =3:1)
	Specific rotation	20 [ D ] α +34.8 o (CHCl 3 , c 0.115) (lit.[160] +6.7 (CHCl 3 , c 0.21))
	IR spectrum	ν max (film) 3473, 2946, 2871, 1761, 1455, 1379, 1191, 1075 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 855 [2M+Na] + , 439 [M+Na] +
	NMR spectrum	1 H and 13 C-NMR -see Table 40

1

H-NMR -see Table

38

,

13

C-NMR -see

(20S)-29-Hydroxy-17 ,20-peroxy-28-norlupan-3-one (36) ***

	Molecular formula C 29 H 46 O 4	MW 458.67
	Description	white star crystal
	Retention factor	R f =0.41 (cHex-EtOAc =2:1)
	Specific rotation	20 [ D ] α +23.5 (CHCl 3 , c 0.085)
	IR spectrum	ν max (film) 3474 (OH), 2948, 2864, 1703 (C=O), 1455, 1384,
		1047 (C-O), 755 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 939 [2M+Na] + , 481 [M+Na] + ,
		HRMS (ESI-TOF) m/z (ES+) : 481.3274 [M+Na] + (calcd for
		C 29 H 46 O 4 Na, 481.3294)
	NMR spectrum

Table 37 (20S,23E)-20-Hydroxy-27-nordammar-23-en-3,25-dione (39) ***

 37

	formula C 30 H 52 O 3	MW 460.73
	Description	white needle crystal
	Retention factor	R f =0.52 (cHex-EtOAc =1:1)
	Specific rotation	20 [ D ] α +24.6 (CHCl 3 , c 0.065) (lit.[135] +39.63 (CHCl 3 , c 1.04,
		22 o C))
	IR spectrum	ν max (film) 3439, 2946, 2862, 1462, 1376, 1082, 1043, 754 cm -1
	Mass spectrum	EI-MS m/z (ES+) : 943 [2M+Na] + , 483 [M+Na] +
	NMR spectrum	1 H-NMR -see Table 38, 13 C-NMR -see Table 39
	(20S,24S)-20,24-dihydroxydammar-25-en-3-one (38) [136, 151, 158]
	Molecular formula C 30 H 50 O 3	MW 458.72
	Description	white amorphous powder
	Retention factor	R f =0.33 (cHex-EtOAc =1:1)
	Specific rotation	20 [ D ] α +43.6 (CHCl 3 , c 0.110)
	IR spectrum	ν max (film) 3307, 2952, 2867, 1704, 1454, 1368 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 939 [2M+Na] + , 481 [M+Na] +
	NMR spectrum	1 H and 13 C-NMR -see

1

H-NMR -see Table

34

,

13

C-NMR -see Table

35

17 -Hydroxy-28,29-dinorlupan-3,20-dione (41) ***

	Molecular formula C 28 H 44 O 3	MW 428.65
	Description	colorless resin
	Retention factor	R f =0.42 (cHex-EtOAc =1:1)
	Specific rotation	20 [ D ] α +24.7 (CHCl 3 , c 0.170)
	IR spectrum	ν max (film) 3439 (OH), 2948, 2864, 1704 (C=O), 1455, 1383, 755
		cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 879 [2M+Na] + , 451 [M+Na] +
		HRMS (ESI-TOF) m/z (ES+) : 451.3191 [M+Na] + , (calcd for
		C 28 H 44 O 3 Na, 451.3188)
	NMR spectrum	1 H-NMR -see Table 42, the 13 C-NMR -see Table 43

(20R)-20-Hydroxy-17 ,29-epoxy-28-norlupan-3-one (42) ***

	Molecular formula C 29 H 46 O 3	MW 442.67
	Description	colorless resin
	Retention factor	R f =0.34 (cHex-EtOAc =1:1)
	Specific rotation	20 [ D ] α +85.3 (CHCl 3 , c 0.075)
	IR spectrum	ν max (film) 3440 (OH), 2954, 2869, 1704 (C=O), 1455, 1377, 1140,
		1066, 756 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 465 [M+Na] + , 443 [M+H] +
		HRMS (ESI-TOF) m/z (ES+) : 443.3533 [M+H] + , (calcd for
		C 29 H 47 O 3 , 443.3525)
	NMR spectrum	1 H-NMR -see Table 42, the 13 C-NMR -see Table 43
	Clovane-2,9-diol (43)
	Molecular formula C 15 H 26 O 2	MW 238.37
	Mass spectrum	GC-EI-MS m/z : 238 [M] + , 220, 182, 164 (100%), 150, 135
	The identification was achieved by GC-MS analysis and comparison to the reference
	spectrum.
	Isofouquierol (44) [148, 155, 158]
	Molecular formula C 30 H 52 O 3	MW 460.73
	Description	yellow gum
	Retention factor	R f =0.45 (cHex-EtOAc =1:5)
	Specific rotation	20 [ D ] α +28.5 (MeOH, c 0.365) (lit.[148] +22 (CHCl 3 , c0.1))
	IR spectrum	ν max (film) 3381, 2943, 2867, 1453, 1376, 1031, 983 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 943 [2M+Na] + , 483[M+Na] +
	NMR spectrum

1

H-NMR -see Table

34 and 13

C-NMR -see

Table 35 (20S,22E)-20,24,25-Trihydroxydammar-22-en-3-one (45)

 35

	Molecular formula C 30 H 50 O 4	MW 474.72
	Description	colorless resin
	Retention factor	R f =0.26 (cHex-EtOAc =1:4)
	Specific rotation	20 [ D ] α +45.0 (MeOH, c 0.140)
	IR spectrum	ν max (film) 3405, 2936, 2862, 1701, 1457, 1377 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 971 [2M+Na] + , 497 [M+Na] +
		HRMS (ESI-TOF) m/z (ES+) : 497.3602 [M+Na] + (calcd for
		C 30 H 50 O 4 Na, 497.3607)
	NMR spectrum

*** 1 H and the 13 C-NMR -see

Table 36 (20S,23E)-20,25,26-Trihydroxydammar-23-en-3-one (46

 36

	Molecular formula C 30 H 50 O 4	MW 474.72
	Description	colorless resin
	Retention factor	R f =0.23 (cHex-EtOAc =1:15)
	Specific rotation	20 [ D ] α +32.0 (MeOH, c 0.125)
	IR spectrum	ν max (film) 3406, 2939, 2862, 1701, 1456, 1380 cm -1
	Mass spectrum	ESI-MS m/z (ES+) : 513 [M+K] + , 497 [M+Na] +
		HRMS (ESI-TOF) m/z (ES+) : 497.3592 [M+Na] + (calcd for
	NMR spectrum	C 30 H 50 O 4 Na, 497.3607) 1 H-NMR -see Table 34 and 13 C-NMR -see Table 35
	(20S,24R)-20,24-Epoxy-25-hydroxy-2,3-seco-dammarane-2,3-dioic
	acid (47) ***
	Molecular formula C 30 H 50 O 6	MW 506.71
	Description	colorless resin
	Retention factor	R f =0.50 (CH 2 Cl 2 -MeOH=10:1-a little amount of acetic acid)
	Specific rotation	20 [ D ] α +29.4 (CHCl 3 , c 0.085)
	IR spectrum	ν max (film) 3650-2500 (br), 2966, 2871, 1699, 1455, 1377, 1216,
		1166, 757 cm -1
	Mass spectrum	ESI-MS m/z (ES-) : 1011 [2M-H] -, 505 [M-H] -
		HRMS (ESI-TOF) m/z (ES-) : 505.3533 [M-H] -(calcd for
		C 30 H 49 O 6 , 505.3529)
	NMR spectrum

) *** 1 H-NMR -see Table

38

,

13

C-NMR -see Table

39

(20S)-20-Hydroxy-3-oxo-24,25,26,27-tetranordammar-23-oic acid (49) ***

	4.1	Plant material
	Molecular formula C 26 H 42 O 4 Description colorless resin 4.2 Preliminary study	MW 418.61
	Retention factor 4.3 Extraction and isolation of D. costatus wood hexane extract R f =0.05 (cHex:acetone:MeOH =50 :15 :2.5)
	Mass spectrum	ESI-MS m/z (ES-) : 835 [2M-H] -, 417 [M-H] -
	4.4	NMR spectrum Identification and structural study of isolated compounds 1 H and the 13 C-NMR -see Table 41
	4.5	Cytotoxic evaluation of some isolated triterpenes
	4.6	In vitro antiplasmodial activity of isolated triterpene
	4.7	Conclusion

Table 32 .

 32 The 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz) of 22 and 31 (in CDCl 3 ) ; δ

	in ppm ; J in Hz (dipterocarpol group -dammarane triterpene)
	position	δ H		δ C
		22	31	22	31
	1	1.50 (m) 1.97 (m)	1.00 (m) 1.70 (m)	39.9	39.0
	2	2.50 (m) 2.57 (m)	1.62 (m)	34.1	27.4
	3		3.21 (dd, J=10.9, 5.2)	218.3	79.8
	4			47.4	39.0
	5	1.42 (d, J=11.2)	0.75 (d, J=11.1)	55.3	55.8
	6	1.53 (m) 1.60 (m)	1.46 (m) 1.51 (m)	19.6	18.3
	7	1.36 (m) 1.61 (m)	1.30 (m) 1.55 (m)	34.5	35.2
	8			40.3	40.4
	9	1.48 (m)	1.31 (m)	50.0	50.6
				36.8	37.1
		1.32 (m) 1.55 (m)	1.27 (m) 1.51 (m)	22.0	21.5
		1.55 (m) 1.80 (m)	1.50 (m) 1.75 (m)	24.8	24.8
		1.71 (m)	1.62 (m)	42.4	42.3
				50.3	50.3
		1.13 (m) 1.51 (m)	1.47 (m) 1.07 (m)	31.1	31.2
		1.33 (m) 1.91 (m)	1.27 (m) 1.82 (m)	27.5	27.5
		1.80 (m)	1.75 (m)	49.8	49.8
		0.96 (s)	0.86 (s)	16.0	16.2
		1.02 (s)	0.97 (s)	15.2	15.5
				75.4	75.4
		1.17 (s)	1.15 (s)	25.5	25.4
		1.54 (m)	1.48 (m)	40.4	40.5
		2.10 (m)	2.05 (m)	22.6	22.6
		5.12 (tt, J=7.1, 1.4)	5.13 (t, J=7.1)	124.7	124.7
				131.7	131.6
		1.71 (s)	1.70 (s)	25.8	25.7
		1.64 (s)	1.64 (s)	17.7	17.7
		1.10 (s)	0.99 (s)	26.7	28.0
		1.06 (s)	0.79 (s)	21.0	15.4
		0.91 (s)	0.89 (s)	16.3	16.5

Table 33 .

 33 The 1 H-NMR (300 MHz) of 23, 33 (in CDCl 3 ) and 48 (in acetone) ; δ in ppm ; J

	in Hz (isofouquierone group -dammarane triterpene)
	position		δ H
		23	33	48
	1	1.48 (m) 1.97 (m)	1.02 (m) 1.73 (m)	2.46 (d, J=17.3) 2.66 (d, J=17.3)
	2	2.48 (m)	1.61 (m) 1.67 (m)
	3		3.22 (dd, J=11.1, 5.2)
	4
	5	1.42 (m)	0.78 (d, J=10.7)	2.57 (m)
	6	1.48 (m) 1.53 (m)	1.48 (m) 1.55 (m)	1.49 (m) 1.70 (m)
	7	1.32 (m) 1.57 (m)	1.32 (m) 1.57 (m)	1.25 (m) 1.63 (m)
	8
	9	1.44 (m)	1.36 (m)	2.53 (m)
	10
	11	1.51 (m) 1.32 (m)	1.55 (m) 1.27 (m)	1.64 (m)
	12	1.76 (m) 1.54 (m)	1.77 (m) 1.53 (m)	1.68 (m)
	13	1.73 (m)	1.68 (m)	1.82 (m)
	14
	15	1.13 (m) 1.48 (m)	1.13 (m) 1.51 (m)	1.07 (m) 1.48 (m)
	16	1.32 (m) 1.93 (m)	1.29 (m) 1.85 (m)	1.20 (m) 1.92 (m)
	17	1.77 (m)	1.78 (m)	1.78 (m)
	18	0.96 (s)	0.97 (s)	1.00 (s)
	19	1.02 (s)	0.86 (s)	1.03 (s)
	20
	21	1.16 (s)	1.14 (s)	1.14 (s)
	22	2.26 (dd, J=7.1, 3.2)	2.25 (dd, J=7.0, 3.1)	2.21 (d, J=7.2)
	23	5.82 (dt, J=15.8, 7.1)	5.79 (dt, J=15.7, 7.1)	5.76 (dt, J=15.9, 7.2)
	24	5.64 (d, J=15.8)	5.63 (d, J=15.8)	5.62 (d, J=16.0)
	25
	26	1.37 (s) a	1.37 (s) a	1.29 (s)
	27	1.36 (s) a	1.36 (s) a	1.29 (s)
	28	1.10 (s)	0.99 (s)	1.26 (s)
	29	1.06 (s)	0.79 (s)	1.29 (s)
	30	0.90 (s)	0.89 (s)	0.91 (s)
	a interchangeable

Table 34 .

 34 The 1 H-NMR (300 MHz) of 40, 44 and 46 (in CDCl 3 ) ; δ in ppm ; J in Hz (isofouquierone group -dammarane triterpene)

	position		δ H
		40	44	46
	1	1.49 (m) 1.96 (m)	1.02 (m) 1.73 (m)	1.52 (m) 1.97 (m)
	2	2.50 (m)	1.61 (m) 1.67 (m)	2.49 (dd, J=14.8, 9.0) 2.57 (dd, J=14.8, 9.0)
	3		3.25 (dd, J=11.1, 4.8)
	4	1.41 (m)	0.78 (d, J=11.2)	1.42 (d, J=10.7)
	5	1.51 (m) 1.59 (m)	1.48 (m) 1.56 (m)	1.52 (m) 1.60 (m)
	6	1.35 (m) 1.60 (m)	1.32 (m) 1.57 (m)	1.38 (m) 1.61 (m)
	7	1.47 (m)	1.36 (m)	1.47 (t, J=11.9)
	8	1.32 (m) 1.54 (m)	1.29 (m) 1.55 (m)	1.35 (m) 1.58 (d, J=2.5)
	9	1.55 (m) 1.77 (m)	1.55 (m) 1.78 (m)	1.58 (m) 1.80 (d, J=2.8)
		1.72 (m)	1.69 (m)	1.72 (m)
		1.13 (m)	1.09 (m)	1.17 (m)
		1.50 (m)	1.48 (m)	1.54 (dd, J=22.6,10.6)
		1.30 (m)	1.29 (m)	1.31 (dd, J=10.3,5.6)
		1.90 (m)	1.88 (d, J=7.6)	1.90 (m)
		1.78 (m)	1.78 (m)	1.79 (d, J=5.5)
		0.98 (s)	089 (s)	1.05 (s)
		1.04 (s)	1.01 (s)	0.99 (s)
		1.17 (s)	1.18 (s)	1.20 (s)
		2.24 (broad)	2.25 (s)	2.29 (d, J=8.1)
		5.73 (m)	5.74 (m)	5.86 (m)
		5.73 (m)	5.74 (m)	5.62 (dd, J=15.8, 6.9)
		1.36 (s)	1.37(s)	3.52 (dd, J=24.0, 10.4)
		1.36 (s)	1.37(s)	1.32 (m)
		1.12 (s)	1.08 (s)	1.13 (s)
		1.07 (s)	0.82 (s)	1.09 (s)
		0.91 (s)	0.92 (s)	0.93 (s)
		1.49 (m)	1.02 (m)	1.52 (m)
		1.96 (m)	1.73 (m)	1.97 (m)
		2.50 (m)	1.61 (m) 1.67 (m)	2.49 (dd, J=14.8, 9.0) 2.57 (dd, J=14.8, 9.0)
			3.25 (dd, J=11.1, 4.8)
		1.41 (m)	0.78 (d, J=11.2)	1.42 (d, J=10.7)
		1.51 (m)	1.48 (m)	1.52 (m)
		1.59 (m)	1.56 (m)	1.60 (m)
		1.35 (m)	1.32 (m)	1.38 (m)
		1.60 (m)	1.57 (m)	1.61 (m)
		In acetone
		5.74 (m)
		5.64 (d, J=15.7)

Table 35 .

 35 The 13 C-NMR (75 MHz) of 23

, 33, 40, 44, 46

  (in CDCl 3 ) and 48 (in acetone); δ in ppm; J in Hz (isofouquierone group -dammarane triterpene)

	position	23	33	40	44	46	48
	1	39.9	39.0	39.9	39.0	39.9	42.1
	2	34.1	27.4	34.1	27.4	34.1	172.3
	3	218.1	79.0	218.3	79.0	218.2	180.4
	4	47.4	39.0	47.4	39.0	47.4	46.4
	5	55.3	55.8	55.3	55.8	55.3	48.1
	6	19.6	18.3	19.6	18.6	19.6	21.2
	7	34.5	35.2	34.5	35.2	34.5	34.3
	8	40.3	40.4	40.3	40.4	40.3	40.0
	9	50.0	50.6	50.0	50.6	50.0	42.2
	10	36.8	37.1	36.8	37.1	36.8	41.8
	11	22.0	21.5	22.0	21.5	22.0	22.3
	12	24.9	24.9	24.8	24.8	24.8	24.5
	13	42.6	42.5	42.5	42.4	42.6	42.1
	14	50.3	50.3	50.3	50.3	50.3	50.6
	15	31.1	31.1	31.1	31.1	31.1	31.0
	16	27.5	27.5	27.5	27.5	27.5	27.7
	17	50.1	50.2	49.8	49.9	50.0	49.5
	18	16.0	16.2	16.0	16.2	15.2	19.5
	19	15.2	15.5	15.2	15.5	16.1	14.7
	20	75.0	75.2	75.0	75.1	75.0	73.9
	21	25.8	25.7	25.7	25.9	25.5	25.7
	22	43.4	43.4	43.4	43.4	43.5	44.3
	23	127.2	127.2	122.4	122.3	125.9	126.4
	24	137.4	137.4	142.1	142.0	137.7	137.2
	25	82.1	82.1	70.8	70.8	73.3	80.7
	26	24.1 a	24.1 a	29.9	29.9	70.0	24.0 a
	27	24.4 a	24.5 a	29.9	29.9	24.4	24.1 a
	28	26.7	28.0	26.7	28.0	26.7	26.4
	29	21.0	15.4	21.0	15.4	21.0	24.2
	30	16.3	16.4	16.3	16.4	16.4	16.0
	a interchangeable

Table 36 .

 36 The 1 H-NMR (300 MHz) and13 C-NMR (75 MHz) of 45 (in acetone); δ in

	ppm; J in Hz
	position	δ H	δ C	position	δ H	δ C
	1	1.50 (m) 1.93 (m)	39.6	16	1.31 (m) 2.03 (m)	27.4
	2	2.41 (dd, J=15.4, 8.1) 2.50 (dd, J=15.4, 8.1)	33.6	17	1.79 (m)	50.8
	3		215.6	18	0.96 (s)	15.5
	4		46.9	19	1.01 (s)	14.8
	5	1.47 (m)	55.0	20		74.5
	6	1.50 (m) 1.61 (m)	19.4	21	1.24 (s)	28.0
	7	1.31 (m) 1.62 (m)	34.5	22	5.85 (d, J=15.8)	137.8
	8		40.2	23	5.78 (dd, J=15.8, 6.1)	127.1
	9	1.52 (m)	50.0	24	3.88 (d, J=6.1)	79.2
	10		36.7	25		71.9
	11	1.31 (m) 1.51 (m)	21.8	26	1.15 (s) a	25.6 a
	12	1.49 (m) 1.71 (m)	25.1	27	1.13 (s) a	24.0 a
	13	1.78 (m)	42.9	28	1.05 (s)	26.1
	14		50.0	29	1.01 (s)	20.4
	15	1.04 (m) 1.50 (m)	31.0	30	0.92 (s)	15.9

Table 37 .

 37 The 1 H-NMR (300 MHz) and13 C-NMR (75 MHz) of 29, 32 and 38 (in CDCl 3 ) ; δ in ppm ; J in Hz (fouquierone group -dammarane triterpene)

	position		δ H			δ C
		29	32	38	29	32	38
	1	1.46 (m) 1.94 (m)	0.94 (m) 1.71 (m)	1.50 (m) 1.97 (m)	39.9	39.2	39.9
	2	2.48 (m)	1.61 (m)	2.50 (m) 2.55 (m)	34.1	27.4	34.1
	3		3.22 (dd, J=10.9,5.2)		218.3	79.0	218.2
	4				47.4	39.0	47.4
	5	1.43 (m)	0.75 (d, J=10.8)	1.40 (m)	55.3	55.8	55.3
	6	1.52 (m) 1.61 (m)	1.47 (m) 1.51 (m)	1.52 (m) 1.60 (m)	19.6	18.3	19.6
	7	1.38 (m) 1.63 (m)	1.28 (m) 1.51 (m)	1.38 (m) 1.60 (m)	34.5	35.2	34.5
	8				40.3	40.4	40.3
	9	1.45 (m)	1.31 (m)	1.48 (m)	50.0	50.6	50.0
	10				36.8	37.1	36.8
	11	1.34 (m) 1.57 (m)	1.26 (m) 1.50 (m)	1.35 (m) 1.58 (m)	22.0	21.5	22.0
	12	1.55 (m) 1.80 (m)	1.49 (m) 1.73 (m)	1.55 (m) 1.80 (m)	24.9	24.8	24.9
	13	1.72 (m)	1.63 (m)	1.70 (m)	42.5	42.3	42.5
	14				50.3	50.3	50.3
	15	1.15 (m) 1.53 (m)	1.06 (m) 1.48 (m)	1.16 (m) 1.53 (m)	31.1	31.2	31.1
	16	1.33 (m) 1.90 (m)	1.27 (m) 1.85 (m)	1.33 (m) 1.91 (m)	27.5	27.5	27.5
	17	1.80 (m)	1.74 (m)	1.81 (m)	49.9	50.1	50.1
	18	0.96 (s)	1.00 (s)	0.96 (s)	16.0	16.2	16.0
	19	1.01 (s)	0.89 (s)	1.01 (s)	15.2	15.5	15.2
	20				75.1	75.3	75.1
	21	1.16 (s)	1.17 (s)	1.17 (s)	25.3	25.2	25.5
	22	1.53 (m) 1.63 (m)	1.48 (m) 1.60 (m)	1.55 (m) 1.63 (m)	36.4	36.1	36.6
	23	1.76 (m)	1.65 (m)	1.67 (m)	26.9	25.0	29.2
	24	4.31 (t, J=6.4)	4.33 (t, J=6.3)	4.06 (t, J=5.8)	89.8	89.8	76.5
	25				143.1	143.7	147.6
	26	5.05 (br s)	5.04 (br s)	4.86 (s) 4.98 (s)	114.1	114.1	111.0
	27	1.80 (s)	1.80 (s)	1.75 (s)	17.6	17.6	17.8
	28	1.10 (s)	1.02 (s)	1.10 (s)	26.7	28.0	26.7
	29	1.06 (s)	0.82 (s)	1.05 (s)	21.0	15.4	21.0
	30	0.90 (s)	0.92 (s)	0.90 (s)	16.3	16.5	16.4

Table 38 .

 38 The 1 H-NMR (300 MHz) of 30, 34, 37 and 47 (in CDCl 3 ) ; δ in ppm ; J in Hz (ocotillone group -dammarane triterpene)

	δ H

Table 39 .

 39 The 13 C-NMR (75 MHz) of 30, 34, 37 and 47 (in CDCl 3 ) ; δ in ppm ; J in Hz (ocotillone group -dammarane triterpene)

	δ C

  [START_REF] Yuwa-Amornpitak | Molecular phylogeny of diptercarpaceae in Thailand using trnL-trnF and atpB-rbcL intergenicspacer region in chloroplast DNA[END_REF] H 50 O 6 on the basis of HRESI-MS (m/z 505.3533 [M-H] -, calcd C 30 H 49 O 6 505.3529). The six degrees of unsaturation were implied by the molecular formula. The presence of two carbonyl signals ( C 177.9 and 186.7) without any olefin, suggested 4 cyclic rings. The 13 C-NMR and HMBC spectra indicated the same side chain structure as ocotillone[START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF]. Prominent differences in the 1 H and13 C NMR data between 47 and 34 were observed only in those spectroscopic data pertaining to ring A. In the HMBC spectrum of 47, the correlations were observed from H-1 at H 2.51 and 2.66 to the carbon signals at C 177.9 (C-2) and48.8 (C-5) together with the correlations of the proton signals at H 1.27 (H-28) and1.22 (H-29) to the carbon signals at C 186.7 (C-3) and 48.8 (C-5) (Figure

Table 40 .

 40 The 1 H-NMR (300 MHz) and13 C-NMR (75 MHz) of 27, 28 and 35 (in CDCl 3 ) ; δ in ppm ; J in Hz (lactone group -dammarane triterpene)The molecular formula C 27 H 42 O 3 of compound 35 was deduced from ESI-MS at m/z 439 of the [M+Na] + ion. The appearance of δ H3.22 (dd, J=10.8, 5.3 Hz) and δ C 79.0, as well as the HMBC correlations of H-1 (δ H 1.00, 1.74), H-2 (δ H1.60, 1.67), H-28 (δ H 0.99) and H-29 (δ H 0.79) with δ C 79.0 (Figure35) indicated the 3 -hydroxy structure. The

	position		δ H			δ H
		27	28	35	27	28	35
	1	1.48 (m) 1.96 (m)	1.47 (m) 1.95 (m)	1.00 (m) 1.74 (m)	39.8	39.8	39.0
	2	2.50 (m)	2.49 (m)	1.60 (m) 1.67 (m)	34.1	34.1	27.4
	3			3.22 (dd, J=10.8, 5.3)	218.1	218.0	79.0
	4				47.4	47.4	39.0
	5	1.42 (m)	1.40 (m)	0.73 (d, J=2.1)	55.3	55.2	55.8
	6	1.52 (m) 1.60 (m)	1.46 (m) 1.53 (m)	1.55 (m)	19.6	19.6	18.2
	7	1.36 (m) 1.62 (m)	1.33 (m) 1.60 (m)	1.29 (m) 1.57 (m)	34.5	34.5	35.2
	8				40.3	40.2	40.3
	9	1.46 (m)	1.44 (m)	1.31 (m)	50.0	49.8	50.5
					36.8	36.7	37.1
		1.31 (m) 1.56 (m)	1.31 (m) 1.54 (m)	1.26 (m) 1.54 (m)	21.8	21.9	21.4
		1.87 (m)	1.86 (m)	1.87 (m)	25.0	25.0	25.0
		1.74 (m)	1.64 (m)	1.60 (m)	42.7	43.2	43.2
					50.1	50.1	50.2
		1.18 (m) 1.55 (m)	1.15 (m) 1.54 (m)	1.15 (m) 1.53 (m)	31.0	31.0	31.2
		1.30 (m) 1.97 (m)	1.29 (m) 1.75 (m)	1.25 (m) 1.76 (m)	26.4	26.8	26.8
		2.00 (m)	2.03 (m)	2.00 (m)	49.4	49.2	49.3
		0.95 (s)	0.91 (s)	0.96 (m)	16.0	16.0	16.2
		1.01 (s)	0.97 (s)	0.97 (m)	15.2	15.2	15.5
					89.9	90.1	90.2
		1.34 (s)	1.39 (s)	1.38 (m)	22.1	25.4	25.4
		1.99 (m) 2.09 (m)	1.96 (m) 2.10 (dd, J=22.5, 9.7)	1.97 (m) 2.13 (m)	33.2	31.1	31.1
		2.61 (m)	2.55 (m)	2.58 (m)
			2.66 (dd, J=18.1,	2.66 (dd, J=18.7,	28.6	29.2	29.2
			9.8)	8.9)
					177.0	176.8	176.8
		1.08 (s)	1.05 (s)	0.99 (s, 3H)	26.7	26.7	28.0
		1.04 (s)	1.01 (s)	0.79 (s, 3H)	21.0	21.0	15.4
		0.89 (s)	0.87 (s)	0.90 (s, 3H)	16.1	16.1	16.2

Table 41 .

 41 The 1 H-NMR (300 MHz) and13 C-NMR (75 MHz) of 26, 39 and 49 (in CDCl 3 ) ;The molecular formula of compound 39 was suggested as C 29 H 46 O 3 with the aid of the HRESI-MS of the [M+Na] + ion at m/z 465.3359. Its 13 C-NMR spectrum showed 29 carbon signals with the characteristic of 3-oxodammarane skeleton. Compared to isofouquierone[START_REF] Bisset | Etudes chimio-taxonomiques dans la famille des Dipterocarpaceaes-II. Constituants du genre Dipterocarpus Gaertn.F. Essai de classification chimio-taxonomique[END_REF], the signal of a methyl group at position 27 was missing and an additional carbonyl carbon ( C-25 198.5) on the side chain was observed. This carbonyl group was assigned to be at position 25 with conjugated to double bond at position23 and 24. UV absorption ability at 254 nm on TLC and IR absorption band at 1663 cm -1 confirmed the presence of this , unsaturated carbonyl moiety. The HMBC correlations ofH-22 (δ H 2.41 and 2.48) to C-23 (δ C 144.6), C-24 (δ C 133.9) and C-20 (δ C75.34), of H-23 (δ H 6.96) and H-24 (δ H 6.16) to C-25 (δ C 198.5) and of H-24 (δ H 6.16) to C-26 (δ C 27.0) or vice versa concluded the structure of the side chain of compound 39 as shown in Figure36. Detailed comparison of the 13 C-NMR data of 39 showed that the chemical shifts of C-20 to C-26 were very similar to those of rhombenone[245]. The stereochemistry of the double bond was determined as E-form based on large coupling constant (J=15.9 Hz) of the olefinic protons. All of these spectroscopic data characterized compound 39 as (20S,23E)-20-hydroxy-27-nordammar-23-ene-3,25-dione, which was a new nordammarane. The ESI-MS of compound 49 corresponded to the molecular formula of C 26 H 42 O 4 . Its NMR data were very close to compound 39 with 3 less carbons. Two olefinic carbons and a methyl carbon on the side chain were disappeared. The HMBC spectrum showed correlations of H-21 ( H 1.30) with C-17 ( C 50.38), C-20 ( C 74.39) and C-22 ( C 43.06) as same as other dammaranes with 20S configuration. Examination of molecular formula and the value of C-23 , carbonyl carbon on C-23 should be carboxylic carbon. The HMBC correlations between this carbon and H-22 ( H 2.50 and 2.65) confirmed its position at C-23. Identification of the side chain of 49 was supported by the comparision of C to those of the side chainof 3 ,20S-dihydroxy-23-norchol-5-enoic acid [246] . Therefore, compound 49 was defined as (20S)-20-hydroxy-3-oxo-24,25,26,27tetranordammar-23-oic acid, a new nordammarane.

	δ in ppm ; J in Hz (nordammarane triterpene)
	position		δ H			δ C
		26	39	49	26	39	49
	1	1.48 (m) 1.96 (m)	1.50 (m) 1.97 (m)	1.50 (m) 1.95 (m)	40.0	39.9	39.9
	2	2.50 (m)	2.54 (m)	2.50 (m)	34.0	34.1	34.1
	3				217.8	218.2	218.0
	4				47.4	47.4	47.4
	5	1.45 (m)	1.42 (m)	1.41 (m)	55.2	55.3	55.3
	6	1.48 (m) 1.56 (m)	1.52 (m) 1.60 (m)	1.50 (m) 1.60 (m)	19.6	19.6	19.6
	7	1.56 (m)	1.35 (m) 1.60 (m)	1.35 (m) 1.63 (m)	33.7	34.5	34.5
	8				40.1	40.3	40.3
	9	1.45 (m)	1.47 (m)	1.46 (m)	50.5	49.9	49.9
	10				37.0	36.8	36.8
	11	1.23 (m)	1.33 (m) 1.57 (m)	1.34 (m) 1.58 (m)	20.6	21.9	21.9
	12	1.64 (m) 1.94 (m)	1.53 (m) 1.81 (m)	1.55 (m) 1.78 (m)	20.9	24.9	24.9
	13	2.22 (m)	1.74 (d, J=11.9)	1.66 (m)	53.9	42.6	42.8
	14				46.5	50.3	50.3
	15	1.52 (m) 1.97 (m)	1.17 (d, J=8.0) 1.53 (m)	1.15 (m) 1.54 (m)	27.8	31.1	31.0
	16	2.23 (m)	1.32 (m) 1.90(d, J= 6.6)	1.93 (m)	34.9	27.5	27.2
	17		1.80 (m)	1.86 (m)	218.1	50.5	50.4
	18	0.98 (s)	0.98 (s)	0.96 (s)	16.3	16.0	16.0
	19	1.11 (s)	1.04 (s)	1.02 (s)	15.5	15.2	15.2
	20					75.3	74.4
	21		1.22 (s)	1.30 (s)		26.3	25.7
	22		2.41 (d, J=8.1) 2.48 (d, J=8.1)	2.50 (d, J=15.9) 2.65 (d, J=15.9)		43.6	43.1
	23		6.96 (dt, J=15.9, 8.1)			144.6	174.6
	24		6.16 (d, J=15.9)			133.9
	25					198.5
	26		2.31 (s)			27.0
	27
	28	1.11 (s)	1.12 (s)	1.10 (s)	26.8	26.7	26.7
	29	1.07 (s)	1.08 (s)	1.06 (s)	21.0	21.0	21.0
	30	0.92 (s)	0.92 (s)	0.91 (s)	16.7	16.3	16.3

  30) at δ H 1.79 and two terminal methylene protons (H-29) at δ H 4.71 and 4.86. The HMBC data showed the correlations between C-3 (δ C 218.3) and H-

	1 (δ H 1.47, 1.92), H-2 (δ H 2.47), H-23 (δ H 1.08) and H-24 (δ H 1.03) (Figure 37). These
	data suggested the structure of 3-oxolupane triterpene. An oxygenated quaternary carbon
	signal at δ C 81.1 was assigned to C-17 based on the HMBC correlations between this
	carbon and H-15 (δ H 1.30), H-16 (δ H 1.83, 1.90), H-21 (δ H 1.73, 1.79) and H-22 (δ H 1.49,
	1.89). This information indicated that the structure of this compound was 28-norlupane

with 17-OH substitution. The -orientation of the hydroxyl group was supported by the chemical shift of C-13 (δ C 44.1) and C-19 (δ C 52.7). These signals were around

38 and 48 ppm, respectively, for -configuration [107, 108]

. Thus, the structure of 25 was determined as

17 -hydroxy-28-norlupan-20(29)

-en-3-one, a new norlupane.

Table 42 .

 42 The 1 H-NMR (300 MHz) of 24, 25, 36, 41 and 42 (in CDCl 3 ) ; δ in ppm ; J in

		Hz (norlupane triterpenes)
	position			δ H
		24	25	36	41	42
	1	1.46 (m) 1.94 (m)	1.47 (m) 1.92 (m)	1.45 (m) 1.94 (m)	1.47 (m) 1.94 (m )	1.44 (m) 1.93 (m)
	2	2.47 (m)	2.47 (m)	2.47 (m)	2.47 (m)	2.46 (m)
	3
	4
	5	1.37 (m)	1.40 (m)	1.38 (m)	1.38 (m)	1.36 (m)
	6	1.47 (m)	1.49 (m)	1.48 (m)	1.49 (m)	1.48 (m)
	7	1.46 (m)	1.44 (m) 1.49 (m)	1.47 (m)	1.41 (m) 1.48 (m)	1.47 (m)
	8
	9	1.44 (m)	1.47 (m)	1.42 (d, J=2.5)	1.48 (m)	1.43 (m)
		1.29 (m)	1.28 (m)	1.29 (m)	1.30 (m)	1.29 (m)
		1.52 (m)	1.54 (m)	1.53 (m)	1.56 (m)	1.52 (m)
		1.64 (m) 0.96 (m)	1.71 (m)	1.56 (m) 0.97 (m)	1.64 (m) 1.04 (m)	1.60 (m) 0.94 (m)
		1.22 (m)	1.17 (m)	1.26 (m)	1.14 (m)	1.23 (m)
		1.27 (m) 1.51 (m)	1.30 (m)	1.35 (m) 1.59 (m)	1.29 (m) 1.46 (m)	1.30 (m) 1.52 (m)
		1.63 (m)	1.83 (m) 1.90 (m)	1.56 (m)	1.72-1.84 (m)	1.66 (m)
		1.39 (d, J=11.7)	1.58 (d, J=2.4)	1.91 (d, J=5.4)	1.71 (m)	1.57 (d, J=11.0)
		2.03 (m)	2.40 (m)	2.02 (m)	2.92 (m)	2.00 (m)
		1.83 (m)
		1.58 (m)	1.73 (m) 1.79 (m)	1.74 (m) 1.99 (m)	1.98 (m)	1.65 (m) 1.91 (m)
		1.48 (m) 1.91 (m)	1.49 (m) 1.89 (m)	1.94 (m)	1.56 (m) 1.97 (m)	1.55 (m) 1.91 (m)
		1.09 (s)	1.08 (s)	1.09 (s)	1.09 (s)	1.09 (s)
		1.03 (s)	1.03 (s)	1.04 (s)	1.04 (s)	1.03 (s)
		0.94 (s)	0.94 (s)	0.94 (s)	0.94 (s)	0.94 (s)
		0.99 (s)	0.98 (s)	1.00 (s)	0.98 (s)	0.99 (s)
		0.99 (s)	0.98 (s)	0.99 (s)	0.97 (s)	0.98 (s)
		3.25 (t, J=11.4,) 3.66 (dd, J=11.5, 5.6)	4.70 (s) 4.85 (s)	3.54 (d, J=11.4) 4.18 (d, J=11.4)		3.42 (d, J=11.4) 3.49 (d, J=11.4)
		0.75 (d, J=6.7)	1.80 (s)	1.16 (s)	2.23 (s)	1.38 (s)

Table 43 .

 43 The 13 C-NMR (75 MHz) of 24, 25, 36, 41 and 42 (in CDCl 3 ) ; δ in ppm ; J in

	Hz (norlupane triterpenes)
	position			δ C
		24	25	36	41	42
	1	39.8	39.8	39.8	39.8	39.7
	2	34.1	34.0	34.0	34.0	34.0
	3	218.3	218.3	218.1	218.1	218.2
	4	47.3	47.2	47.3	47.3	47.3
	5	54.8	54.8	54.8	54.8	54.8
	6	19.6	19.6	19.6	19.6	19.6
	7	33.2	33.1	33.1	33.1	33.2
	8	40.5	40.4	40.6	40.4	40.5
	9	50.1	50.5	49.9	50.4	50.0
	10	36.9	36.9	36.9	36.9	36.9
	11	21.7	21.9	21.4	21.8	21.5
	12	25.6	26.8	24.9	27.1	25.2
	13	35.9	44.1	35.5	41.9	35.7
	14	40.6	40.8	40.6	41.2	40.6
	15	28.1	28.3	27.9	28.1	28.0
	16	28.4	28.2	24.3	30.5	27.9
	17	82.7	81.1	90.2	80.7	83.2
	18	54.2	52.2	45.3	52.2	49.2
	19	42.5	52.6	42.1	55.8	49.2
	20	36.6	150.9	85.3	214.5	70.8
	21	21.2	32.2	29.5	25.4	22.2
	22	30.0	37.6	23.3	36.8	28.7
	23	26.8	26.9	26.8	26.9	26.8
	24	21.0	20.9	21.0	20.9	21.0
	25	16.3	16.4	16.3	16.4	16.3
	26	15.5	15.3	15.5	15.3	15.5
	27	13.6	14.5	13.4	14.1	13.5
	28
	29	66.9	108.5	64.6		70.4
	30	15.2	21.7	20.8	29.7	26.5

  3 -30, H-20 and H 2 -29. The HMBC spectrum showed the correlations of the methylene H-29 (δ H3.25, 3.66) to the quaternary oxygenated C-17 (δ C82.71). This suggested a cyclic structure through an ether bridge between C-29 and C-17 of the 28-norlupane nucleus. A review of Connelly and Hill[116] noted that it is impossible to have the stereochemistry of the cyclic side chain as the proposed structure of 20S-17 ,29-epoxy-28-norlupan-3-ol from the work of Abdel-Bar et al[107].

  The resonance at unusual low field of both oxygenated quaternary carbons and HMBC correlation suggested the structure of an endoperoxide ring. The assignment of the oxygenated quaternary carbon (δ C85.3) at position 20 and an oxygenated methylene carbon (δ C 64.6) at position 29 were based on the correlations of these carbons to H-30 (δ H1.17) and the correlation between H-21(δ H1.99) and C-20 (δ C 85.3) (Figure 37). The position of the other oxy-quaternary carbon at δ C 90.2 (C-17) was indicated by the correlations with H-19 (δ H 2.02). As the result, the quaternary carbon should be the head of endoperoxide. The 17 -and 19 -configuration of the substitution on ring E were designed by using the same reason as compound 24. The 20S-configuration was suggested based on downfield shift of δ H-18 (δ H 1.91) (compared to those of compound 24 and 42), resulted from the inductive effect of the oxygen on C-29. Hence, the structure of 36 was then determined as (20S)-29-hydroxy-17 ,20-peroxy-28-norlupan-3-one. The HRESI-MS data of 42 at m/z 443.3533 [M+H] + suggested the molecular formula of C 29 H 46 O 2 with seven degrees of unsaturation. The 1 H and 13 C NMR data were consistent with compound 24 and 36 for the linkage between C-17 and C-19. The HSQCedited data showed 6 methyl, 11 methylene, 6 methine and 6 qurternary carbons. The HMBC correlations of H-30 (δ H 1.42) to C-20 (δ C 70.8), C-19 (δ C 49.2) and C-29 (δ C 70.4) indicated the attachment of an isopropyl group at C-19. Furthermore, the HMBC correlations between H-29 (δ H 3.49) and the quaternary oxygenated C-17 (δ C 83.

		19
		18
	19	20
	18
		29
	19α	19β

1) and H-1(δ H

1.45, 1.94)

, H-2 (δ H 2.47), H-23 (δ H 1.09) and H-24 (δ H 1.04). Two oxygenated quaternary carbons and one oxygenated methylene carbon were observed at δ C

90.2 (C-17)

,

85.3 (C-20)

and

64.6 (C-29)

ppm, respectively.

, 38 49 23, 33, 40, 44, 48 22, 31 30, 34, 37, 47 27, 28, 35

  The possible biosynthesis pathway of dammaranes found in D. costatus (based on derivatization on the sidechain).

	45
	29, 32???	(OOH)	???	???	???	(OOH)
	Scheme 25.

Table 44 .

 44 Compounds isolated from Hopea odorata leaves hexane extract and Dipterocarpus costatus wood hexane extract

	Compound Number	Name (source)	Structure
	1	Friedelin
		(H. odorata)
	2	β-Amyrin
		(H. odorata)
	3	β-Sitosterol
		(H. odorata and D.
		costatus)
	4	Epifriedelanol
		(H. odorata)
	5	Betulonic acid
		(H. odorata)

  The 1 H NMR spectrum (300 MHz) of 18,18a and 18b (in CDCl 3 ).

																																		FA
	178.366	178.209	4.834 177.925	4.756 176.780	174.360	4.313	4.275 149.467	4.072	4.033	3.457	3.455	3.448	3.447 112.169 3.441 111.804 3.436 111.613	3.427 110.814	3.421 76.394	73.947	68.474	52.362	49.160	48.998	47.769	45.908	45.164	35.897	1.703 34.338 33.058	1.445 31.972	1.270 31.053	1.205 29.753	0.984 29.525 29.414 0.951 29.318	0.909 29.204	0.898 28.866	0.890 27.777 27.717 0.760 26.951 0.746 25.040 22.743	0.443 0.428 21.374 20.716 19.771 19.368 18.203 18.144 14.185
																															28			27	30
																																cHex
																																		18
				18																													FA
																																		21
				29	29		26		26					24																			19	19
								150													100											50
		ppm (t1)
			4.861 174.486	4.780 174.348			4.334	4.305 149.525 4.097	4.068	3.693 111.552	77.481	3.481 77.057	3.457 76.634 76.384	73.915	68.463	52.334	51.571	48.999	47.732	45.852	45.159	35.871	35.620	34.337	33.244	1.730 33.072 31.972	31.468	30.001	1.346 29.752	1.233 29.524	1.012 29.414	0.979 29.318 29.203	0.937 29.043	0.926 28.200 27.790 0.777 27.672 27.050 0.766 26.957 25.039 22.742 0.460 21.334 20.714 19.787 0.449 19.355 18.204 18.119 14.186
	ppm (f1) ppm (t1) 4.841 4.838 4.837 4.835 18a 4.763 18b 29 29 ppm (t1) ppm (t1) Figure 58. Me at C3 methylation 3.0 4.0 3.0 4.0 4.759 3.885 3.847 3.846 3.548 3.544 3.539 3.537 3.532 3.522 3.510 3.506 3.483 26 24,26 150 150 179.036 4.843 149.458 111.616	100 100			77.260	74.285	67.435	2.0 2.0 52.455 49.010	1.706 47.788	45.839	45.164	35.989	1.128 50 50 33.290	0.989 33.083	1.0 1.0 0.956 0.915 31.329 28.847	0.894 28.259	0.764 27.779	0.758 26.978	0.750 26.939	0.442 25.054 21.365 0.434 0.427 19.780 19.368 18.203

  The 1 H NMR spectrum (300 MHz) of 20 (in CDCl 3 ) and expanded spectrum in the range of 0.3-2.7 ppm.

	6.929	6.926 180.228	173.566			149.449	146.051			4.841	4.835 126.608	4.757		111.624							52.164		48.989	47.709	45.878	1.857 45.190	1.704 45.165	36.029	1.310 35.613	1.304 34.777	1.271 33.054	0.980 31.459	0.949 29.750	0.940 26.969	0.918 25.985	0.764 21.356 19.773 0.751 0.442 0.428 19.345 18.123 11.989
											29
	ppm (f1) ppm (f1) ppm (f1) Figure 64. 24 2.50 2.571 2.443 2.368 50 52.164 48.989	2.343 47.709	2.336	2.329 23 2.320 150 45.878 45.190	2.197 45.165	2.069 23	2.00 40	1.857	27 36.029	1.704 35.613	28 34.777	33.054	1.50 1.443 1.408 cHex 1.405 100 30 31.459 29.750 28.819	1.403 28.130	1.310 27.750	1.304 26.969	1.271 25.985	2	1.00 0.980 0.949 18 30 0.940 5 2 20 21 0.918 50 21.356 19.773 19.345	0.764 18.123	0.751 19	0.50 0.442 11.989	0.428 19	10
																	220

  The 1 H NMR spectrum (300 MHz) of 20 and 20a (in CDCl 3 )

	6.929 180.228	6.926	173.566		149.449	146.051		4.841	4.835 126.608	4.757	111.624									52.164	48.989	1.857 47.709 1.704 45.878	1.310 45.190 45.165 1.304	1.271 36.029	0.980 35.613	0.949 34.777 33.054 0.940 31.459	0.918 29.750	0.764 26.969 0.751 0.442 0.428 25.985	21.356 19.773 19.345 18.123 11.989
	ppm (t1) 6.766 24 ppm (t1) ppm (t1) Figure 68. 20 20a 174.459 168.825	6.0	150 150 149.508	143.280	5.0 4.810 127.131 4.732 29	111.562	4.0	1'	100 100 1''	52.165	3.0 51.729 51.564	48.988	2 47.696	5 45.846	2.0 1.841 11,1,23 1.683 2 27 45.178 36.006 35.611 34.934	1.0 0.932 6 29.038 0.963 7 11 1.253 15 31.467 1.291 28 50 50 33.056 29.986 28.112 0.916 19 0.900 27.771 27.045 0.733 26.947 0.722 0.413 0.402 19 25.711 25.027 21.312 19.779 19.329 18.121 12.381
														224

  The 1 H NMR spectrum (300 MHz) of 22 and 31 (in CDCl 3 ).

	5.170 5.165	5.161	5.156	5.151	5.146		5.142	5.137	5.127	5.122	5.118	5.114																			1.712	1.647	1.168	1.099	1.057	1.017	0.963	0.906
		216.794																					131.721	124.680			75.405	55.355	50.289	50.014	49.817	47.472	42.399		40.462	40.291	39.919	36.852	34.556	34.164	31.187	27.552	26.724	19 29 25.804 25.509	24.834	22.591 22.053 21.053	19.675 17.769 16.375 16.078 15.240
																																				18	30
				22
																																				27
																																				26
				1551.392	1550.067	1548.669	1547.300	1545.793		1544.332	1542.971	1541.571	1538.586	1537.218	1535.818	1534.589
			1555.0	1550.0	1545.0	1540.0	1535.0	1530.0
		Hz (f1)				24
								200													150							100						50
		ppm (f1)
	5.161	5.157	5.153	5.148	5.138	5.133	5.129		5.119	5.114	5.110	5.105						131.635	3.240	3.222 124.730	3.203	3.186 78.966	75.444	55.859	50.643	50.319	49.848	42.293	40.494	40.373	39.052	1.704 39.001	1.636 37.135	35.239	31.213	1.154 28.024 27.562 0.986 27.422	0.971 26.946	0.890 25.800	0.856 25.423	0.785 24.847 22.583	21.560 18.300 17.761 16.477 16.259 15.521 15.402
		ppm (f1) Hz (f1) 1548.823 31 1550 1547.589 1546.311 ppm (f1) Figure 72. cHex 2.0 3.0 4.0 5.0 1530 1540 1544.926 1541.821 1540.491 1539.174 1536.161 1534.780 1533.397 1532.116 Hz (f1) 950.0 955.0 960.0 965.0 970.0 975.0 972.194 966.942 961.245 956.033 24 3 27 26 100 150 200	50	1.0 28	29 18 30 19	5
																											228

  The13 C NMR spectrum (75 MHz) of 27 and 28 (in CDCl 3 ).

		16.11 16.06 15.21					21.03 19.60 16.15 16.03 15.18
	1.083 1.040 1.005 0.946 0.897 0.892	34.11 29 33.18 28 31.02 28.66 26.74 26.44 24.99 22.11 21.79 21.05 19.63	19	18	30	50	34.08 1.049 1.006 0.970 29 31.17 31.07 29.19 0.909 0.866 26.91 26.81 26.70 25.46 25.01 21.90	28 18 19	30	1.00
		34.57					34.48
		36.85					36.79
		39.86					39.83
	1.426 1.374 1.345	89.93 55.29 49.99 49.46 47.42 42.76 40.33		21		100	90.08 55.24 50.12 49.87 1.393 49.25 1.341 43.25 21 40.23			1.50
							120.02
										2.00
						150
		176.99				176.79
						200				2.50
	27 Figure 89. ppm (f1) 218.08	217.97		28	ppm (f1) ppm (f1)
						244

  The13 C NMR spectrum (75 MHz) of 49 (in CDCl 3 ) and expanded spectrum in the range of 10-60 ppm.

	218.020			174.593						55.369	50.330	49.927	47.442	42.827	40.316	39.884	36.851	34.520	34.112	31.059	29.726	27.273	26.727	25.725	24.970 21.918 21.040	19.651 16.296 16.057 16.019 15.189
	0.911				30
	0.965 1.018	29		18	19																					1.00
	1.059	28
	1.101
	1.275			21
	1.305
															1											1.50
		200			150						100									50
	2.673 2.624 2.622 2.525 2.508 2.501 2.473 2.188 ppm (f1) 60 Figure 137. ppm (f1) 55.369	50.330	50 49.927	47.442	acetone 42.827	40.316	40 39.884	36.851	34.520	34.112	31.059	30 29.726	27.273	26.727	25.725	22 24.970	21.918	2 21.040	22 20 19.651	1 16.296	16.057	16.019	15.189	2.00 2.50	ppm (f1) 10
	2.675
									292

 ocotillol II, ocotillol I, dammarenediol II

7

ACKNOWLEDGEMENTS

It is a great pleasure to acknowledge the guidance and support of Prof. Sylvie MICHEL and Dr.Xavier CACHET throughout this work. I am deeply indebted to them for their enthusiasm patience, and friendship. I am extremely grateful to Assoc. Prof. Sindhchai KEOKITICHAI and Prof. Marie-Jose FOGLIETTI for their great assistance at the beginning of this collaboration, Prof. Christiane GARBAY for her cytotoxicity laboratory support as well as Assoc. Prof. Jurairat NUNTHANID for her continuing support from Silpakorn University. I am grateful to Assist. Prof. Chavalit SITTISOMBUT for his supply of the plant material, solvents for extraction and cytotoxic assays in preliminary study, Assoc. Prof. Uthai SOTANAPHUN for his guidance of the study plan and Prof.Marc LITAUDON together with Assoc. Prof. Busaban SIRITHUNYALUG for their commentation. I would like to thank all the people who have contributed information and supplies for my work: Dr.Perayot

APPENDIX

Spectroscopic spectrum of new compounds 198 199 [160] The underlined compounds demonstrated the major dammarane in the extract.

PART I

Phytochemical Study of Hopea odorata Roxb leaves.

Study of fraction D24

Fraction D24 (539 mg) was purified by SiO 2 cc with cHex-EtOAc 8:1 and 4:1 to afford 8 compounds; (20S) -17 ,29-epoxy-28-norlupan-3-one (24, 20 mg), dipterocarpol (22, 70 mg), 17 -hydroxy-28-norlupan-20(29)-en-3-one (25, 72 mg), octanordammarane-3,17dione (26, 16 mg), isocabralealactone (20R) (27, 42 mg), cabralelactone (28, 131 mg), isofouquierone peroxide (23, 11 mg) and (20S)-20-hydroxy-24-perhydroxydammar-25-en-3-one (29, 6 mg).

Study of fraction D27

Fraction D27 (617 mg) was subjected to SiO 2 cc using cHex:EtOAc = 8:1, followed by 5:1 and 3:1, as mobile phase. The elution gave 4 compounds: cabraleone (30, 350 mg), dammarenediol II (31, 233 mg), cereotagaloperoxide (32, 3 mg) and isofouquierol peroxide (33, 11 mg).

Study of fraction D28

Fraction D28 (656 mg) was subjected to SiO 2 cc with cHex-EtOAc 6:1 and 3:1 as eluent to give 5 compounds: cabraleone (30, 28 mg), ocotillone (34, >250 mg), cabralealactone (28, 14 mg), 3-epicabraleahydroxylactone (35, 20 mg) and isofouquierol peroxide (33, 8 mg).

Study of fraction D32

Fraction D32 (142 mg) was purified by SiO 2 cc with cHex-EtOAc (4:1) system. A star crystal of (20S)-29-hydroxy-17 ,20-peroxy-28-norlupan-3-one (36, 4 mg) was obtained and washed with cold EtOAc.

Study of fraction D35

Fraction D35 (265 mg) was separated chromatographically over SiO 2 cc with cHex-EtOAc (3.5:1) system. The elution provided ocotillol II (37, 40 mg) and (20S,24S)-20,24dihydroxydammar-25-en-3-one (38, 5 mg).

Study of fraction D37

Fraction D37 (473 mg) was further chromatographed over SiO 2 cc using cHex-EtOAc (4:1) to yield isofouquierone peroxide (23, 80 mg), (20S,23E)-20-hydroxy-27-nordammar-23-ene-3,25-dione (39, 8 mg) and isofouquierone (40, 37 mg).

Study of fraction D38

Fraction D38 (191 mg) was purified by SiO 2 cc using cHex-EtOAc (4:1) to afford 4 compounds: ocotillol II (37, 5 mg), isofouquierone peroxide (23, 6 mg), 17 -hydroxy-28,29-dinorlupan-3,20-dione (41, 6 mg) and (20S,23E)-20-hydroxy-27-nordammar-23ene-3,25-dione (39, 40 mg).

Scheme 20. Purification of D. costatus wood hexane extract. The spectroscopic data of 3 and 9 were campared to the data from phytochemical study of H.odorata leaves hexane extract. Caryophyllene oxide was again confirmed by GC-MS analysis. More caryophyllene oxide was found in D. costatus than in H.odorata. Clovane-2,9-diol [START_REF] Misra | Triterpenoids from Shorea robusta resin[END_REF] was found in trace amount and was identified by GC-EI-MS.

-Elemene (21)

Figure 28. Selected HMBC correlations of 21

This compound was obtained as a colorless resin which was characterized by GC-MS analysis with comparison to reference data. The identification was supported by the 1D NMR data and the HMBC correlations. The 1 H and 13 C NMR spectrum showed the presence of methyl, metylene and methine carbon with olefin moiety. The HRESI-MS of compound 36). This carbonyl functional group strongly influenced on the chemical shifts of the surrounding atoms. Compared to dipterocarpol [START_REF] Chuakul | Medicinal Plants in Thailand[END_REF] and ocotillone [START_REF] Maridass | Antimicrobial and cytotoxic activities of Hopea utilis fruits[END_REF], the 13 C resonance of the C-13 showed obviously down field shift (about 8 ppm), as well as that of C-16 with lesser degree, whereas those of C-