Timea Varjas

Ghodratollah Nowrasteh

Ferenc Budán

Gábor Horváth

József Cseh

Zoltán Gyöngyi

Sándor Makai

István Ember

The Effect of Fenugreek on the Gene Expression of Arachidonic Acid Metabolising Enzymes

Keywords: Fenugreek, gene expression, chemoprevention, lipoxygenase, cyclooxygenase

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

is available in most Asian countries especially in India and it is used as one of the most common spices [START_REF] Sur | Trigonella foenum graecum (Fenugreek) Seed Extract as an Antineoplastic Agent[END_REF].

The seeds and leaves (folium) of fenugreek are edible and are used as condiments and as ayurvedic medicine in treatment of diabetes, hypercholesterolemia, wound healing, inflammation, and gastrointestinal diseases [START_REF] Raju | Diosgenin, a Steroid Saponin of Trigonella foenum graecum (Fenugreek), Inhibits Azoxymethane-Induced Aberrant Crypt Foci Formation in F344 Rats and Induces Apoptosis in HT-29 Human Colon Cancer Cells[END_REF]. Bioactive compounds of fenugreek seed modulate the expression of several enzymes, especially those which involved in glucose and lipid metabolism. Fenugreek seed consists of 75% seed coat (testa) and 25% protein (albumen). The seed coat functions as spice and contains essential oils (17 kinds) that give the specific fragrance, saponins, steroidal saponins like diosgenin, yamogenin and proteins. Fenugreek seed coat contains 10-15% of saponin and approximate 32% of crude protein. On the other hand, albumen consists of 80% water soluble substances (e.g. viscous polysaccharide and viscous polymer) and 20% water insoluble substances (e.g. alkaloides) (Table 1.) [START_REF] Noor | Comparative Study on the Corrosion Inhibition of Mild Steel by Aqueous Extract of Fenugreek Seeds and Leaves in Acidic Solutions[END_REF]. The main bioactive compounds isolated from fenugreek seeds are protodioscin, trigoneoside, diosgenin, yamogenin [START_REF] Raju | Diosgenin, a Steroid Saponin of Trigonella foenum graecum (Fenugreek), Inhibits Azoxymethane-Induced Aberrant Crypt Foci Formation in F344 Rats and Induces Apoptosis in HT-29 Human Colon Cancer Cells[END_REF].

Generally speaking, few researches have been carried out on the effect of this herb on cancer. Extracts of fenugreek seeds have been found to have anticarcinogenic potency in different settings, such as proven antineoplastic effect in Ehrlich ascites carcinoma (EAC) model in Balb/C mice more than 70% inhibition of tumour cell growth with respect to the control were detected [START_REF] Sur | Trigonella foenum graecum (Fenugreek) Seed Extract as an Antineoplastic Agent[END_REF]. Moreover in vitro experiments indicated that diosgenin inhibits cell growth and induces apoptosis in HT-29 human colon cancer cell line in a dose-dependent manner. Furthermore, diosgenin induced [START_REF] Raju | Diosgenin, a Steroid Saponin of Trigonella foenum graecum (Fenugreek), Inhibits Azoxymethane-Induced Aberrant Crypt Foci Formation in F344 Rats and Induces Apoptosis in HT-29 Human Colon Cancer Cells[END_REF].

Certain phytochemical compounds, eg. flavonoids (flavans, flavanones, flavones, flavanonols, flavonols, catechins, anthocyanidins and isoflavone) are proven to exhibit antioxidant, anti-inflammatory, chemopreventive, antiviral and antibacterial, as well as a direct cytoprotective effect on coronary and vascular systems, the pancreas and the liver [START_REF] Burnett | A medicinal extract of Scutellaria baicalensis and Acacia catechu acts as a dual inhibitor of cyclooxygenase and 5lipoxygenase to reduce inflammation[END_REF][START_REF] Amin | Chemopreventive activities of Trigonella foenum graecum (Fenugreek) against breast cancer[END_REF][START_REF] Devasagayam | Immune system and antioxidants, especially those derived from Indian medicinal plants[END_REF]. The cellsignalling pathways inhibited by curcumin alone include NFκB, AP-1, STAT3, Akt, Bcl2, BclX, caspases, PARP, IKK, EGFR, HER2, JNK, MAPK, COX2, and ALOX5 [START_REF] Aggarwal | Molecular targets of dietary agents for prevention and therapy of cancer[END_REF]. Diosgenin as a precursor of steroid hormones like progesterone and cortisone has an anti-inflammatory effect [START_REF] Norton | Useful plants of dermatology. III. Corticosteroids, strophanthus, and dioscorea[END_REF]. Moreover Moalic et al. and Corbiere et al. reported that diosgenin inhibits cell proliferation in the human osteosarcoma 1547 cell line by the induction of apoptosis by cell cycle arrest in G1 phase as well principally by increasing the expression of the tumour suppressor TP53 [START_REF] Moalic | A plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma cells[END_REF][START_REF] Corbiere | Different contribution of apoptosis to the antiproliferative effects of diosgenin and other plant steroids, hecogenin and tigogenin, on human 1547 osteosarcoma cells[END_REF]. Other authors have described that diosgenin suppressed the TNF-induced NFκB activation as determined by DNA binding, activation of ΙκBα kinase, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, and p65 nuclear translocation through the inhibition of Akt activation. NF-κB-dependent reporter gene expression was also abrogated by diosgenin. Diosgenin suppresses proliferation, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-κB-regulated gene expression and enhances apoptosis induced by cytokines and chemotherapeutic agents (Shishodia and Aggarwal, 2005). On the basis of these data listed above, diosgenin and other fenugreek seed constituents possess anticarcinogenic properties, suggesting their potential as appropriate phytochemicals for cancer prevention. It is known that steps of carcinogenesis can be influenced by many compounds of plant origin [START_REF] Raju | Diosgenin, a Steroid Saponin of Trigonella foenum graecum (Fenugreek), Inhibits Azoxymethane-Induced Aberrant Crypt Foci Formation in F344 Rats and Induces Apoptosis in HT-29 Human Colon Cancer Cells[END_REF]. More publications have dealt with the complex inhibitory effects of rezveratol, curcumin, quercetin on tumour formation induction of suppressor genes and apoptosis [START_REF] Lin | Molecular targets of curcumin[END_REF][START_REF] Burnett | A medicinal extract of Scutellaria baicalensis and Acacia catechu acts as a dual inhibitor of cyclooxygenase and 5lipoxygenase to reduce inflammation[END_REF]. Moreover in animal experiment Bhatia et al. highlighted the possible protective effect of fenugreek in cyclophosphamide treatment, which causes toxicity by its reactive metabolites such as acrolein and phosphoramide mustard. Fenugreek is known for its hypoglycemic, anti-inflammatory and immunomodulatory activity, promising protective medicinal herb for consideration in complementary therapy for cancer patients under chemotherapeutic interventions [START_REF] Bhatia | Aqueous extract of Trigonella foenum-graecum L. ameliorates additive urotoxicity of buthionine sulfoximine and cyclophosphamide in mice[END_REF].

The COXs and ALOXs are two important enzyme classes that metabolize polyunsaturated fatty acids and affect carcinogenesis. The ALOXs convert arachidonic acid and linoleic acid and other polyunsaturated fatty acids into biologically active metabolites, those influence cell signalling, structure, and metabolism [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF]. According to the literature, prostate, the lungs, and other cancer cell lines express ALOX5, resulting the formation of a variety of metabolically active products with different roles in carcinogenesis [START_REF] Burnett | A medicinal extract of Scutellaria baicalensis and Acacia catechu acts as a dual inhibitor of cyclooxygenase and 5lipoxygenase to reduce inflammation[END_REF]. Both of COX1 and COX2 isoforms are responsible for the synthesis of prostaglandin E 2 (PGE 2 ), and there is evidence for correlation between increased level of PGE 2 and carcinogenesis presented [START_REF] Janakiram | Molecular markers and targets for colorectal cancer prevention[END_REF].

In summary the biologically active compounds of fenugreek may be tools of chemopreventive strategies as well, such as the possible inhibition of prostaglandin of metabolic pathway with lipoxygenase (ALOX) and cyclooxygenase (COX) enzymes. induced hamster model at the post-initiation stage through topical application [START_REF] Li | Overexpression of 5-lipoxygenase and cyclooxygenase 2 in hamster and human oral cancer and chemopreventive effects of zileuton and celecoxib[END_REF].

In the literature the effect of fenugreek seed extract on the expression of ALOXs and COXs genes is not reported yet. Nor the effect of DMBA exposure on the expression of ALOXs and COXs genes were examined yet. Therefore the chemopreventive effects of fenugreek by influencing the expression of genes involved in fatty acid metabolism (ALOXs and COXs) in AKR/J H-2 k inbred mice animal model exposed to carcinogenic dimethylbenz[α]antracene (DMBA) was examined in this study. Special feed preparation: Special food preparation. Special food was prepared as follows: the ground form of normal pet food granules were made, then the same quantity (50% mass) of ginseng dried fenugreek seed power power (obtained from University of West Hungary, Agriculture Science Faculty, Department of Medicinal and Aromatic Plants) was mixed in. By adding a small quantity of water, rod-shape objects were formed and dried at room temperature.

The treatment of the AKR/J H-2 k mice. The first group of mice was fed with normal nutriment; the second group was fed with the special nutriment for 7 days. A third group was fed with normal nutriment and DMBA was administered on day 6. The fourth group was fed with special nutriment, and similarly to the third group, DMBA was administered intraperitoneally (20 mg/bw kg doses DMBA) on day 6 (Table 2). [START_REF] Perjési | Effect of E-2-(4'-methoxybenzylidene)-1benzosuberone on the 7,12-dimethylbenz[a]anthracene-induced onco/suppressor gene action in vivo II: A 48-hour experiment[END_REF].

Isolation of mRNA.

All the groups were autopsied on day 7, 24 h postcarcinogenic exposure, with subsequent isolation of mRNA from lung, liver, spleen and kidney, using MagNA Pure Compact Instrument (Roche Applied Science).

Quantitative RT-PCR. Quantitative RT-PCR was performed using the Gene Amp® 5700 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. All the primers were designed by Primer Express™ Software (Applied Qualitative variables were summarized by means of absolute frequencies and percentages. For all calculations, X 2 tests were performed, where the level of significance was set at 5% (95% Cl -confidence interval).

License: All legal licenses needed to perform this study were in our possession.

Results

The mRNA expressional profiles of enzymes playing part in arachidonic acid metabolism and prostaglandin synthesis are demonstrated in Figures 12345. In recent years, genetic information about cancer, molecular signalling and metabolic pathways has permitted to target therapies and detecting specific molecules for prevencion [START_REF] Janakiram | Molecular markers and targets for colorectal cancer prevention[END_REF]. The arachidonic acid pathways are possible targets of prevention. Lipoxygenases leads to the formation of a variety of metabolically activated products from linolenic and arachidonic acids which have different roles in carcinogenesis [START_REF] Burnett | A medicinal extract of Scutellaria baicalensis and Acacia catechu acts as a dual inhibitor of cyclooxygenase and 5lipoxygenase to reduce inflammation[END_REF]. Arachidonic acid is converted by ALOX5 to 5(S)-HETE (5(S)-Hydroxyeicosatetraenoic Acid), which in turn is converted to LTA 4 (Leukotriene A 4 ) and then to LTB 4 (Leukotriene B 4 ) by LTA 4 hydrolase [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF]. Several studies have suggested a link between 5(S)-HETE formation and carcinogenesis in prostate and the lungs as well derived cancer cell lines expressing ALOX5 and FLAP mRNA [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF][START_REF] Anderson | E. 5-Lipoxygenase inhibitors reduce PC-3 cell proliferation and initiate nonnecrotic cell death[END_REF]. ALOX5 overexpression recently has been documented in human prostate cancer tissue [START_REF] Gupta | Lipoxygenase-5 is overexpressed in prostate adenocarcinoma[END_REF], and 5(S)-HETE formation and inhibition the growth of prostate cancer cells has been detected as well [START_REF] Ghosh | Arachidonic acid stimulates prostate cancer cell growth: critical role of 5-lipoxygenase[END_REF]. Several lines of evidence indicate that the ALOX12 product 12(S)-HETE (12(S)-hydroxyeicosatetraenoic acid) contributes substantially to carcinogenesis [START_REF] Tang | 12(S)-HETE in cancer metastasis[END_REF]. The expressions of ALOX12 mRNA and encoded protein have been detected in prostate, melanoma, and some other cancer cell lines [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF]Lippman, 2001, Timar et al., 2000). ALOX12 in tumour cells [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF].

Arachidonate-15-lipoxygenase-1 and Arachidonate-15-lipoxygenase-2 are two isoenzymes of ALOX15 that appear to exert important anticarcinogenic effects through the metabolism of polyunsaturated fatty acids. The preferred substrate for Arachidonate-15-lipoxygenase-1 is linolenic acid and for Arachidonate-15-lipoxygenase-2 is arachidonic acid [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF]. The main oxidative metabolite of linolenic acid in human cells is 13(S)-HODE (13(S)-hydroxyoctadecadienoic acid) [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF]. Studies have suggested that 13(S)-HODE enhances cell proliferation, potentially the mitogenic response to EGF in fibroblasts and in Syrian hamster embryo cells, and breast cancer cells [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF][START_REF] Glasgow | Modulation of the epidermal growth factor mitogenic response by metabolites of linoleic and arachidonic acid in Syrian hamster embryo fibroblasts. Differential effects in tumor suppressor gene (+) and (-) phenotypes[END_REF]. The transfection of C-erbB-2 (a protooncogene similar to EGF receptor) into normal fibroblasts increases 13(S)-HODE production. The activity level of 13(S)-HODE dehydrogenase, which metabolizes 13(S)-HODE to 13-oxo-octadecadienoic acid, decreases the malignant transformation of colonic epithelial cells undergo [START_REF] Shureiqi | Lipoxygenase Modulation to Reverse Carcinogenesis[END_REF]. These data assume that overexpression of ALOX enzymes could enhance oncogenesis and through the ability of suppressing them could be chemopreventive.

Cyclooxygenase-1 and Cyclooxygenase-2 (COX1 and COX2) are the two isoforms of the COX enzyme [START_REF] Janakiram | Molecular markers and targets for colorectal cancer prevention[END_REF][START_REF] Brink | International Union of Pharmacology XLIV. Nomenclature for the Oxoeicosanoid Receptor[END_REF]. COX1 and COX2 catalyze the conversion of arachidonic acid to eicosanoids, (like prostaglandins and thromboxanes) via formation of endoperoxides. The COX1 isoform synthesizes prostaglandins that are required for normal physiologic function like gastrointestinal cytoprotection and platelet activity. COX2 is not detectable in most of the normal tissues; however, it is induced at sites of inflammation by cytokines, growth factors, tumour promoters and other agents. Both isoforms are also responsible for the synthesis of prostaglandin E 2 (PGE 2 ), and there are evidences for correlation between increased level of PGE 2 and tumorigenesis [START_REF] Janakiram | Molecular markers and targets for colorectal cancer prevention[END_REF][START_REF] O'neill | Expression of RNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues[END_REF][START_REF] O'neill | Expression of RNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues[END_REF][START_REF] Bengmark | Curcumin, an atoxic antioxidant and natural NFΚB, cyclooxygenase-2, lipooxygenase, and inducible nitric oxide synthase inhibitor: a shield against acute and chronic diseases[END_REF]. Regarding the biological effect, its enzyme modulating ability has been detected by the expression of enzymes involved in lipid metabolism. Moreover, the results of this experiments suggests that bioactive components (protodioscin, trigoneoside, diosgenin, yamogenin) of fenugreek have tumour-preventive and anticarcinogenic effect [START_REF] Raju | Diosgenin, a Steroid Saponin of Trigonella foenum graecum (Fenugreek), Inhibits Azoxymethane-Induced Aberrant Crypt Foci Formation in F344 Rats and Induces Apoptosis in HT-29 Human Colon Cancer Cells[END_REF][START_REF] Hibasami | Protodioscin isolated from fenugreek (Trigonella foenum graecum L.) induces cell death and morphological change indicative of apoptosis in leukemic cell line H-60, but not in gastric cancer cell line KATO III[END_REF][START_REF] Sur | Trigonella foenum graecum (Fenugreek) Seed Extract as an Antineoplastic Agent[END_REF].

In our experiments the increased expression of ALOX12 mRNA in response to DMBA was suppressed by fenugreek containing nutriment only in the kidneys. Increased level of expression of ALOX15 gene was detected in all of the four examined tissues after DMBA treatment. Normal levels of mRNA concentrations were detected in the liver, lung and kidney tissues of mice kept on fenugreek containing diet. Similar results were found for the key enzyme of arachidonic metabolism (ALOX5). The expressions of the genes hich play role in prostaglandin biosynthesis (COX1, COX2) were down regulated as a result of fenugreek containing nutriment.

Comparing our results to other studies on different cell lines, it seems that the biologically active components of fenugreek are able to exert their chemopreventive effects via inhibiting the arachidonic acid metabolism. Studying the molecular targets of the herbs used for hundreds of years in eastern medicine is one of the important tasks of the modern medico-biological researches in the recent days. Fenugreek with its high saponin content (in admission to its pleasant taste) can be a supplementary factor in our strategy for preventing tumorigenesis. Moreover fenugreek seed extract has decreased the expression of genes taking part in inflammatory processes (eg. in the kidneys ALOX15 in comparison to the control group) considering its healing effect in ayurvedic medicine (for treatment of diabetes, hypercholesterolemia, wound healing, inflammation, and gastrointestinal diseases) [START_REF] Raju | Diosgenin, a Steroid Saponin of Trigonella foenum graecum (Fenugreek), Inhibits Azoxymethane-Induced Aberrant Crypt Foci Formation in F344 Rats and Induces Apoptosis in HT-29 Human Colon Cancer Cells[END_REF]. On the other hand chronic inflammatory processes elevate the risk of malignant transformation through increased cell necrosis, cytokine activity consequent stimulation of proliferation supporting regenerative processes. According to our results and data found in the literature, fenugreek seed and its biologically active compounds could have chemopreventive effect due to the suppression of ALOX and COX genes.

.

F

  foenum graecum L.) seed (semen), popularly known as "Methi"

  -29 cells at least in part by inhibition of bcl-2 and by induction of caspase-3 protein expression

Li

  et al. examined ALOX5 expression in DMBA-induced hamster and human oral cancer tissues by immuno histochemistry. In addition, COX2 expression was investigated in hamster oral tissues using in situ hybridization, as well. Evidence were presented for the chemopreventive effects of a specific ALOX5 inhibitor (Zileuton) and a specific COX2 inhibitor (celecoxib), either alone or in combination on the DMBA-

  In this experiment six mice (6 week female AKR/J H-2 k inbred) were used in each group. (The mice were bred in the animal test laboratory of the University of Pécs, Faculty of Medicine, Institute of Public Health).

  Biosystems) and synthesized by Integrated DNA Technologies. Relative gene expression values were calculated by the comparative C T (threshold cycle) method (∆∆C T method, Applied Biosystems). The comparative C T method gives the amount of to an endogenous reference gene and to a relative calibrator sample. Primers for the genes and endogenous reference gene are listed in Table3.Statistical anatysis.Mean values and ranges were derived from quantitative results of mRNA gene expression measurement of three independent parallel experiments.

Figure 1 .

 1 Figure 1. represents the gene expression pattern of arachidonate-12-lipoxygenase in

Figure 3 .

 3 Figure 3. demonstrates the expression pattern of the key gene of arachidonic acid

  benefit from observations linked to the identification of intermediate/surrogate biomarkers giving evidence for usefulness of various nutrients/drugs with specific cancer related molecular targets. As the early stages of tumour promotion and progression rate limiting, successful chemopreventive targeting of early molecular events inhibiting carcinogenesis or enhancing tumour suppression can have high impact on outcomes.

  S)-HETE, and 12(S)-HETE promotes invasion and metastasis

Figure 1 .:Figure 2 .:Figure 3 .:Figure 4 .:Figure 5 .:

 12345 Figure1.: The ALOX12 gene expression pattern and confidences interval of mRNA isolated from AKR/J mice liver, lung, spleen and kidney tissues (the arbitrary unit is gene expression % of HPRT1 gene)

Table 1 .

 1 Fenugreek seed contains (Average yields %)

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 .

 2 The treatment of the AKR/J mice

		1 to 7 th day	6 th day	7 th day
	Group 1	normal nutriment
			-
	Group 2	special nutriment
				autopsy
	Group 3	normal nutriment	20 mg/bwkg
	Group 4	special nutriment	DMBA i.p.

Table 3 .

 3 Listed the primers for the genes and endogenous reference gene

	Gene name	Symbol	Forward Primer / Reverse Primer
	Prostaglandin-endoperoxidase syntase 1 (cyclooxigenase 1)	COX1	5' CCAGAACCAGGGTGTCTGTGT 3' 5' GTAGCCCGTGCGAGTACAATC 3'
	Prostaglandin-endoperoxidase syntase 2 (cyclooxigenase 2	COX2	5' TGGTGCCTGGTCTGATGATG 3' 5' GTGGTAACCGCTCAGGTGTTG 3'
	Arachidonate omega-6-lipoxygenase	ALOX15	5′-GACCGAGGGTTTCCTGTCTC-3′ 5′-TGTCTCCAGCGTTGCATCC-3′
			5' TTTTATTTTTAAATGCCTAAACAAG 3'
	Arachidonate 12-lipoxygenase ALOX12	5' ACACTCCACATTAGTATTCTAACTACA
			3'
	Arachidonate 5-lipoxygenase	ALOX5	5' GGACCTCAGCATGTGGTATG 3' 5' GCTGGGTCAGGGGTACTTTA 3'
	Hypoxanthine guanine phosphoribosyl transferase 1	HPRT1	5' CAGGACTGAAAGACTTGCTC 3' 5' TCATAGGAATGGACCTATCAC 3'

http://mc.manuscriptcentral.com/ptr Phytotherapy Research


David D Haines

Balazs Varga

Istvan Bak

Bela Juhasz

Fadia Fouad Mahmoud

Hejbatullah Kalantari

Rudolf Gesztelyi

Istvan Lekli

Attila Czompa

Arpad Tosaki
email: tosaki@king.pharmacol.dote.hu

Fadia F Mahmoud

Prof Arpad Tosaki

Summative Interaction Between Astaxanthin, Ginkgo biloba Extract (EGb761) and Vitamin C in Suppression of Respiratory Inflammation: A comparison with Ibuprofen

Keywords: Inflammation, Ibuprofen, Astaxanthin, Ginkgolide, cAMP, cGMP Inflammation, Astaxanthin, Ginkgolide, ibuprofen, cAMP, cGMP

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

This report describes progress toward creation of dietary phytochemical formulations ("functional foods") containing bioactive components that interact summatively to strengthen host immunoregulatory mechanisms which prevent inflammation from becoming pathological; and augment drug therapies for age-related illnesses such as arthritis, cardiovascular syndromes, dementia, autoimmunity and many other severe conditions with underlying inflammatory pathogenesis. The products are designed to mediate therapeutic and preventive clinical outcomes equivalently or superior to commonly-used non-steroidal anti-inflammatory drugs (NSAIDs), immunosuppressants, corticosteroids and other pharmaceuticals, but with negligible adverse effects and at low cost to patients. Beyond their medical use it is anticipated that such products may be consumed for general health as food supplements and additionally provide protection against debilitating effects of a diverse range of illness. Here the antiinflammatory capacity of the NSAID ibuprofen is compared with formulations incorporating three naturally-occurring products: the dietary carotenoid astaxanthin (ASX), Ginkgo biloba whole leaf extract (EGb761) and vitamin C, for the ability to independently or interactively affect major indicators of inflammatory disease severity in ovalbumin-sensitized asthmatic guinea pigs.

Asthma was used as a disease model in this study, although this disorder was not our focus. Asthma pathogenesis is a cascade of progressively severe inflammatory processes initiated by an immune imbalance [START_REF] Umetsu | Asthma: an epidemic of dysregulated immunity[END_REF]) and provides a very good general paradigm for severe chronic inflammation. Moreover, the commonality of The emphasis here on general inflammatory, rather than asthma-specific processes, led to the choice of ibuprofen for comparison with the other agents in the context of anti-inflammatory potency. Ibuprofen is a widely-used NSAID that acts by strong inhibition of cyclooxygenases (COX), enzymes which metabolize membrane phospholipids into mediators called prostanoids, small molecules regulating a diverse range of homeostatic functions including inflammation [START_REF] Soberman | The organization and consequences of eicosanoid signaling[END_REF].

The drug acts on both major isoforms of the enzyme: COX-1 which is constitutively expressed in most cells and acts to protect tissues such as lining of small airways and the gastrointestinal tract [START_REF] Chandrasekharan | COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression[END_REF][START_REF] Soberman | The organization and consequences of eicosanoid signaling[END_REF][START_REF] Warner | Cyclooxygenase-3 (COX-3): filling in the gaps toward a COX continuum[END_REF]; and an inducible variant, COX-2, responsible for production of inflammatory mediators in macrophages, neutrophils and other cells participating in inflammation [START_REF] Grosser | Variability in the response to cyclooxygenase inhibitors: toward the individualization of nonsteroidal anti-inflammatory drug therapy[END_REF].

Ibuprofen has clinical utility in many diseases for which inflammation is a major component [START_REF] Hawkey | COX-1 and COX-2 inhibitors[END_REF][START_REF] Wallace | Distribution and expression of cyclooxygenase (COX) isoenzymes, their physiological roles, and the categorization of nonsteroidal anti-inflammatory drugs (NSAIDs)[END_REF]) and therefore, serves as a valuable benchmark against which the therapeutic potential of other NSAIDs may be compared.

However, unlike the phytochemical products used in these experiments, ibuprofen cannot be administered long-term as prophylaxis against inflammatory disease owing to its toxicity [START_REF] Bjarnason | Ibuprofen and gastrointestinal safety: a dose-duration-dependent phenomenon[END_REF][START_REF] Moore | Ibuprofen: a journey from prescription to over-the-counter use[END_REF]. For this reason, formulations with antiinflammatory potency in the same range as ibuprofen, but without adverse side effects, Activation of many cell types during inflammation typically results in a cascade of processes in which autocrine and paracrine expression of platelet activating factor (PAF) is a major event. This molecule interacts with PAF receptors (PAFR) to cause release of cytosolic calcium from cellular stores, constituting a positive feedback process that amplifies immune cell activation and the severity of associated inflammatory pathologies [START_REF] Asako | Reduction of leukocyte adherence and emigration by cyclosporine and L683,590 (FK506) in postcapillary venules[END_REF][START_REF] Sasakawa | FK506 suppresses neutrophil chemoattractant production by peripheral blood mononuclear cells[END_REF][START_REF] Wenzel-Seifert | Partial inhibition of human neutrophil activation by FK-506 at supratherapeutic concentrations[END_REF].

Compounds in Ginkgo extract (ginkgolides) that block PAFR thereby inhibiting increases of intracellular calcium, interrupt this process [START_REF] Brochet | Effects of single intraperitoneal injections of an extract of Ginkgo biloba (EGb 761) and its terpene trilactone constituents on barbital-induced narcosis in the mouse[END_REF], thereby allowing lower doses of drug to suppress calcium-dependent activation events resulting in enhanced therapeutic outcomes [START_REF] Bagnis | Prevention of cyclosporin nephrotoxicity with a plateletactivating factor (PAF) antagonist[END_REF][START_REF] Brochet | Effects of single intraperitoneal injections of an extract of Ginkgo biloba (EGb 761) and its terpene trilactone constituents on barbital-induced narcosis in the mouse[END_REF][START_REF] Haines | Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts[END_REF].

The present investigation builds on previous work by the authors demonstrating synergism between EGb761 and the immunosuppressant FK506 in an ex vivo rat heart model [START_REF] Haines | Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts[END_REF]; and between ASX and Ginkgolide B (GB) a purified, PAFRinhibitory ginkgolide, in a cultured human peripheral blood mononuclear cell (PBMC) model [START_REF] Mahmoud | In vitro effects of ginkgolide B combined with cyclosporin A on T-lymphocyte activation and IL-5 expression in peripheral blood mononuclear cells from asthmatic subjects[END_REF][START_REF] Mahmoud | In vitro effects of astaxanthin combined with ginkgolide B on T lymphocyte activation in 24 peripheral blood mononuclear cells from asthmatic subjects[END_REF]). Neither ASX nor EGb761 are independently potent enough to be used as stand-alone treatment of serious inflammatory disease. However, it is here hypothesized that both compounds summate when coadministered, to mediate therapeutic effects in the same range as currently available single-molecule drugs, with ibuprofen used in these experiments as a comparative model. The therapeutic effects of ginkgolides are due substantially to their ability to block PAF-PAFR-dependent calcium signaling, leading to prevention of inflammatory tissue damage [START_REF] Bagnis | Prevention of cyclosporin nephrotoxicity with a plateletactivating factor (PAF) antagonist[END_REF][START_REF] Brochet | Effects of single intraperitoneal injections of an extract of Ginkgo biloba (EGb 761) and its terpene trilactone constituents on barbital-induced narcosis in the mouse[END_REF]. ASX-mediated cytoprotection occurs through a different mechanism, involving highly efficient scavenging of toxic reactive oxygen compounds by ASX, with the additional capacity to induce host antioxidant defenses [START_REF] Kang | Effect of astaxanthin on the hepatotoxicity, lipid peroxidation and antioxidative enzymes in the liver of CCl4-treated rats[END_REF][START_REF] Wang | Astaxanthin-rich algal meal and vitamin C inhibit Helicobacter pylori infection in BALB/cA mice[END_REF].

The biological action of both ginkgolides and ASX result in several common antiinflammatory effects, including down-regulation of potentially pathological T cell activity [START_REF] Mahmoud | In vitro effects of ginkgolide B combined with cyclosporin A on T-lymphocyte activation and IL-5 expression in peripheral blood mononuclear cells from asthmatic subjects[END_REF][START_REF] Mahmoud | In vitro effects of astaxanthin combined with ginkgolide B on T lymphocyte activation in 24 peripheral blood mononuclear cells from asthmatic subjects[END_REF]). Thus, ginkgolides and ASX potentially exhibit additive and possibly synergistic interaction, an outcome seen in many drug combinations that act via different physiological pathways resulting in therapeutic benefits greater than that of each drug acting independently [START_REF] Evans | Drug synergies and the potential for combination therapy in onychomycosis[END_REF][START_REF] Haines | Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts[END_REF][START_REF] Mahmoud | In vitro effects of ginkgolide B combined with cyclosporin A on T-lymphocyte activation and IL-5 expression in peripheral blood mononuclear cells from asthmatic subjects[END_REF][START_REF] Mahmoud | In vitro effects of astaxanthin combined with ginkgolide B on T lymphocyte activation in 24 peripheral blood mononuclear cells from asthmatic subjects[END_REF]).

MATERIALS AND METHODS

Animals (ethical background and institutional policy):

Male Hartley guinea pigs weighing between 250-350 grams and kept under pathogen-free conditions were used for all experiments in this study. Illinois, USA) -hereafter for the purpose of brevity referred to as "drugs". Drugs were given orally with feed to groups of 6 animals, segregated on the basis of dosage and type of drug. Five studies were conducted, each defined by administration of a particular drug.

The treatment groups included animals fed: (1) astaxanthin;

(2) ginkgo extract/EGb761;

(3) vitamin C; (4) combinations of the previous 3 agents; and (5) ibuprofen. Controls included one drug-free, non-asthmatic control group; and one drug-free asthmatic cohort.

Animals in both control groups were OA-sensitized and given the same feed vehicle as treatment groups, but non-drug supplemented. One of these was subsequently challenged with inhaled OA to induce an asthmatic response. The second received no OA challenge and remained asthma free.

In initial studies of the effect of each agent on asthma-associated disease indicators, independent of the action of the other drugs, guinea pigs were administered a daily regimen of 5-200 mg astaxanthin per kg body weight; 5-100 mg/kg of ginkgo

Induction of Asthmatic response:

Asthmatic responses were induced in animals by sensitization to ovalbumin (OA)

and subsequent aerosol challenge with this antigen by the method of Underwood et al, and Santing et al, [START_REF] Santing | A novel method to assess airway function parameters in chronically instrumented, unrestrained guinea-pigs[END_REF][START_REF] Underwood | Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram[END_REF]. Briefly, each animal was sensitized by intramuscular injections of 0.35 ml of a 5% (W/V) ovalbumin (OA)/saline solution into each thigh on days 1 and 4 of each study. On day 25, the animals were challenged with ovalbumin aerosol in a specially designed animal cage, in which the guinea pigs may move freely. Habituation of each animal to the cage environment was initiated 2 days before exposure to OA. Challenge with the allergen was performed by inhalation of increasing aerosol concentrations containing 1.0, 3.0, 5.0 and 7.0 mg/ml ovalbumin in saline for 3 min, separated by 7-min intervals. Only those animals displaying prominent asthmatic symptoms were selected for further evaluation. These included: i. obvious wheezing, ii. copious nasal discharge of clear, viscous fluid. Average and combinations thereof, then was measured as described below compared with treatment groups receiving 10-1,000 mg/kg ibuprofen per day.

Evaluation of lung inflammatory cells:

Animal sacrifice and collection of biological samples was conducted according to the methods described in Underwood et al [START_REF] Underwood | Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram[END_REF]. Briefly, twenty-four hours after aerosol OA challenge (day 26 of each study), guinea pigs were sacrificed by decapitation conducted rapidly to minimize pain and discomfort (a departure from the method used by Underwood et al, who used cervical dislocation [START_REF] Underwood | Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram[END_REF].

Within a timeframe of no more than 8 seconds so as to prevent contamination of the lungs with peripheral blood, the chest was opened, trachea severed directly above the bronchi and lungs were removed and lavaged with 50 ml of DulBecco's phosphatebuffered saline (aliquots of 10 ml), which were aspirated after gentle massage of the organ. Bronchoalveolar lavage (BAL) fluid was collected and centrifuged at 2200 rpm (1100 x g) for 10 min, supernatant was aspirated, and pellets were resuspended in 5 ml 0.25% NaCl to lyse residual erythrocytes. This dispersion was centrifuged at 2200 rpm (1100 x g) for 10 min, supernatant was aspirated, and pellets were resuspended 5 ml 0.9%

NaCl. Total cell counts were done by hemocytometry using trypan blue stain. Slides were prepared on a Shandon Cytospin 2 (Pittsburgh, PA) at 300 rpm for 5 min, fixed and stained. Differential cell counts were performed using standard morphologic criteria to classify cells as eosinophils, neutrophils, or macrophages, and the results were expressed in cell numbers. Biopsies from lung tissues were harvested for measurement of cyclic nucleotide content. cAMP and cGMP were measured using commercially available radioimmunoassay kits (Amersham). Immediately after sampling, lung biopsies were frozen by means of a Wollenberger clamp pre-chilled in liquid nitrogen. Samples were powdered with a pestle and mortar in liquid nitrogen and trichloroacetic acid (TCA) was added to the powdered frozen samples (10 ml to every mg of tissue). Samples were further homogenized in frozen TCA using a drying mortar and then centrifuged at 14,000

x g for 10 min at 4 o C. The supernatants were extracted 6 times in water-saturated diethyl ether, evaporated and assayed for cAMP and cGMP by radioimmunoassay using liquid scintillation counter (Packard, Tri-Carb 2100TR).

Dosage selection for drug combination studies:

After evaluating the changes mediated by each drug independently on BAL inflammatory cells and lung tissue cAMP and cGMP content, the effect of drug coadministration on these parameters was measured. As shown in Table 1 and Figures 12345, three different combinations of astaxanthin, ginkgo extract and vitamin C were used to estimate the minimum dosage of each agent necessary to maximally suppress each disease-associated parameter. The dose ranges used in these studies (shown in Table 1 and Figures 12345) was in excess of what would reasonably be expected to correspond to veterinary clinical use; and would correspond to unrealistically high dosages if scaled linearly to the average weight of a human [START_REF] Feldman | The 3/4 mass exponent for energy metabolism is not a statistical artifact[END_REF]. However, 11 small animals with high metabolic rates typically metabolize a drug much more rapidly than larger animals; hence the effects seen at the dosages used in this study are expected to be reasonable predictors of the responses in humans [START_REF] Feldman | The 3/4 mass exponent for energy metabolism is not a statistical artifact[END_REF]. In planning these experiments it was also anticipated that response trends of disease indicators that may be difficult to detect at lower dosage (but are nonetheless of potential therapeutic significance), are better-defined at the dosages used here [START_REF] Feldman | The 3/4 mass exponent for energy metabolism is not a statistical artifact[END_REF].

Statistics:

The data were expressed as the mean ± SEM. One-way analysis of variance test was first carried out to test for any differences between the mean values of all groups. If differences were established, the values of the treated groups were compared with those of the drug-free control group by multiple t-test followed by Bonferroni correction. A change of p<0.05 between the drug-free control and treated groups was considered to be significant.

RESULTS

No unexpected deaths or other adverse effects occurred during the preconditioning period. This was somewhat surprising in the case of animals administered ibuprofen since there is toxicity associated with its chronic use [START_REF] Bjarnason | Ibuprofen and gastrointestinal safety: a dose-duration-dependent phenomenon[END_REF][START_REF] Moore | Ibuprofen: a journey from prescription to over-the-counter use[END_REF]) and doses administered were high. It is likely that the animals administered this drug suffered some degree of gastrointestinal damage, however an assessment for this was not made and none of the ibuprofen-treated animals exhibited obvious adverse effects.

Following preconditioning, the 5 inflammation-associated indicators of asthma severity described above in Methods were evaluated in the treated animals and compared with outcomes in two control groups: non asthmatic guinea pigs; and a group of OAchallenged asthmatic animals that had received no drug pre-treatment. As shown in Table 1 andFigures 1A); Ginkgo biloba leaf extract (Table 1B); vitamin C (Table 1C); combined administration of ASX, Ginkgo and vitamin C (Table 1D); and ibuprofen (Table 1E). As

shown in Figures 12345, when compared with drug-free asthmatic controls, animals pretreated with each agent or their combinations showed significant suppression of the indicator to the same range as ibuprofen (Table 1, Figures 12345), demonstrating that under the conditions of the present study, the three natural products (ASX, Ginkgo and vitamin C) mediated an anti-inflammatory effect equal or superior to that of ibuprofen.

Summative enhancement of drug effects between ASX, EGb761 and vitamin C was observed (Table 1) but was not an easily identified feature of the experimental outcomes. 1D).

DISCUSSION

The present study demonstrates that in comparison with untreated controls, animals fed astaxanthin, Ginkgo biloba extract and vitamin C alone or in combination exhibited significantly lower BAL fluid inflammatory cell numbers and enhancement of lung tissue content of cAMP and cGMP (Figures 12345). The magnitude of these antiinflammatory effects mediated by each phytonutrient product independently was observed to be in the same range as outcomes in ibuprofen-treated animal groups (Figures 12345).

The data suggest that the anti-inflammatory effects of ASX, EGb761 and vitamin C acting in concert counteracts pathological inflammation to a more significant degree than any component acting independently (Table 1). Although summative enhancement of drug effects was not a striking characteristic of the experimental outcomes, the significantly reduced dosage of each agent needed to achieve therapeutic effects when co-administered is clinically meaningful since products for human use in place of, or augmenting ibuprofen may be configured to contain lower dosage of each agent, thereby reducing risk of adverse reaction to persons who may be sensitive to any of the three agents.

The independent effect of ASX, EGb761, vitamin C and ibuprofen on selected disease indicators was expected based on the known mechanisms of inflammation underlying the pathogenesis of asthma and the pharmacology of each of the products. The particularly when it occurs in the lungs is observed to activate antioxidant/reactive oxygen signaling processes that favorably alter major indicators of asthma and related respiratory syndromes [START_REF] Fan | Does astaxanthin protect Haematococcus against light damage?[END_REF][START_REF] Kirkham | Oxidative stress in asthma and COPD: antioxidants as a therapeutic strategy[END_REF][START_REF] Kurashige | Inhibition of oxidative injury of biological membranes by astaxanthin[END_REF]).

The experimental endpoints chosen for this study were selected on the basis of their relevance to asthma-associated inflammation, which is a complex process involving activation of cytokine networks tissue damage and many other pathological features.

Nevertheless BAL content of inflammatory cells and lung tissue cyclic nucleotide levels are established indicators of the severity of inflammatory lung disease [START_REF] Busse | Leukotrienes and inflammation[END_REF][START_REF] Hai | Airway smooth muscle cell as therapeutic target of inflammation[END_REF][START_REF] Hamad | Guanylyl cyclases, nitric oxide, natriuretic peptides, and airway smooth muscle function[END_REF][START_REF] Underwood | Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram[END_REF]Wenzel 1996) and changes in the magnitude of these indicators provide a simple and direct picture of the effectiveness of a particular therapy.

The ability of EGb761 to mediate the same favorable changes in major disease indicators as ASX is most likely due primarily to components of the mixture that block the PAF receptor, principally the terpene trilactones such as ginkgolide B (GB) [START_REF] Bagnis | Prevention of cyclosporin nephrotoxicity with a plateletactivating factor (PAF) antagonist[END_REF][START_REF] Brochet | Effects of single intraperitoneal injections of an extract of Ginkgo biloba (EGb 761) and its terpene trilactone constituents on barbital-induced narcosis in the mouse[END_REF]. Systemic inhibition of PAF-dependent processes is expected to interrupt the pathogenesis of asthma at several checkpoints. Of particular importance is the probable effect on pathogenic T lymphocytes that act as triggers for the disease. These cells express high levels of IL-5 which act in an autocrine and paracrine manner to induce expression of PAF and its cognate receptor on T cells, inflammatory cells and several other categories of tissue involved in asthmatic inflammation 16 [START_REF] Calabresse | Induction of high-affinity paf receptor expression during T cell activation[END_REF]). PAF thus acts as an amplifier of a wide range of inflammationassociated processes [START_REF] Calabresse | Induction of high-affinity paf receptor expression during T cell activation[END_REF]), hence blockade of its receptor is expected to significantly attenuate the two major indicators of disease studied here, and a host of others. Other components of EGb761 undoubtedly also contributed to the observed effects. Ginkgo biloba leaf extract, contains bioflavones which inhibit COX-2 and cAMP-phosphodiesterase [START_REF] Saponara | Inhibition of cAMP-phosphodiesterase by biflavones of Ginkgo biloba in rat adipose tissue[END_REF], the latter property possibly acting as a contributor to EGb761's ability to enhance cyclic nucleotide levels in lung tissue. Here cAMP and cGMP content of lung tissue were evaluated as asthma-associated biomarkers, reflecting efficacy of drug treatments. This is a consequence of the role of both cAMP and cGMP as second messengers in airway smooth muscle cells (SMC) that act to decrease contractility thus relaxing the airways and contributing to lessening of disease severity [START_REF] Hai | Airway smooth muscle cell as therapeutic target of inflammation[END_REF][START_REF] Hamad | Guanylyl cyclases, nitric oxide, natriuretic peptides, and airway smooth muscle function[END_REF][START_REF] Underwood | Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram[END_REF]. It is therefore likely that phosphodiesterase inhibitors present in EGb761 contributed to antiinflammatory effects by preservation or enhancement of SMC cyclic nucleotide levels.

Vitamin C was included in our formulations as a water-soluble dietary antioxidant, expected to act as a physiologic relay, allowing scavenging and urinary elimination of reactive oxygen metabolites. It has been shown to augment the activity of astaxanthin in suppression of oxidative damage secondary to helicobacter pylori infection [START_REF] Wang | Astaxanthin-rich algal meal and vitamin C inhibit Helicobacter pylori infection in BALB/cA mice[END_REF], a result which suggested it would interact well with ginkgolides and astaxanthin in suppression of asthma-associated inflammation.

The decision to use ibuprofen as a reference drug against which to compare the anti-inflammatory capacities of the other agents was made with some reservation. Its therapeutic properties are due substantially to its ability to strongly inhibit COX-2. However, the compound also inhibits COX-1, a feature which contributes to disregulated platelet aggregation and gastric damage [START_REF] Kakuta | Cyclooxygenase-1-selective inhibitors are attractive candidates for analgesics that 23 do not cause gastric damage. design and in vitro/in vivo evaluation of a benzamide-type cyclooxygenase-1 selective inhibitor[END_REF][START_REF] Rao | Evolution of nonsteroidal anti-inflammatory drugs (NSAIDs): cyclooxygenase (COX) inhibition and beyond[END_REF]. COX-1 inhibition additionally promotes bronchoconstriction in asthmatics [START_REF] Harrington | COX-1, and not COX-2 activity, regulates airway function: relevance to aspirin-sensitive asthma[END_REF], thereby contributing to lung inflammation and potentially adding a built-in confounder to the study given the use of an asthma model. Rofecoxib (brand name Vioxx) was also considered for use as a reference drug, since as a strongly antiinflammatory, specific inhibitor of COX-2, with no associated effect on COX-1 [START_REF] Kose | Effects of selective Cox-2 inhibitor, rofecoxib, alone or combination with furosemide on renal functions and renal Cox-2 expression in rats[END_REF]) it was expected to favorably alter asthma inflammation-associated indicators without counteracting these outcomes through exacerbation of the disease process. Two other COX-2 selective NSAIDs, celecoxib (brand name Celebrex) and valdecoxib (brand name Bextra) were also considered for use in the study.

Although Vioxx, Celebrex, Bextra or other COX-2-selective NSAIDs would have been more appropriate to our model, using any of these as a reference drug would not have been consistent with the major aim of this research, which is to compare the anti-inflammatory capacity of phytochemical formulations that may be consumed regularly for general health, with a widely used NSAID that can be taken only for brief periods as a therapeutic measure. The three aforementioned drugs are associated with occurrence of serious, potentially lethal side effects [START_REF] Chan | Do COX-2 inhibitors raise blood pressure more than nonselective NSAIDs and placebo? An updated meta-analysis[END_REF][START_REF] Dajani | Cardiovascular and gastrointestinal toxicity of selective cyclo-oxygenase-2 inhibitors in man[END_REF][START_REF] El | Celecoxib-induced cholestatic liver failure requiring orthotopic liver transplantation[END_REF][START_REF] Ray | Beyond debacle and debate: developing solutions in drug safety[END_REF]) and only Celebrex remains in clinical use, making them irrelevant to the aims of this study due to their very limited use in healthcare. Antiasthmatic drugs were likewise not considered for the present investigation since the objective was not to develop an asthma-specific therapy, but establish groundwork for a phytochemical-based NSAID with clinical application in multiple disorders. World Health Organization. 2005). Moreover, despite its potential to exacerbate bronchoconstriction, it has known clinical utility in preventing asthma-associated morbidity in children [START_REF] Mazur | Ibuprofen was more protective against asthma morbidity than acetaminophen in asthmatic children with fever[END_REF], raising the possibility that use of ibuprofen as a reference for these studies might yield new insight into the ability of the drug to complement existing strategies for management of asthma.

CONCLUSIONS:

Results of the present study suggest that a product based on the combined activity of ASX, EGb761 and vitamin C would be of substantial benefit for a large percentage of the World's population at risk of or suffering from chronic inflammatory disease. The product would be as potent as ibuprofen, but could be consumed prophylactically for general health to decrease the overall risk of chronic inflammatory disease; and would also be valuable for augmenting the effects of other therapies for persons who become ill.

ASX and ginkgo extracts are minimally toxic [START_REF] Bagnis | Prevention of cyclosporin nephrotoxicity with a plateletactivating factor (PAF) antagonist[END_REF][START_REF] Haines | Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts[END_REF][START_REF] Mahmoud | In vitro effects of ginkgolide B combined with cyclosporin A on T-lymphocyte activation and IL-5 expression in peripheral blood mononuclear cells from asthmatic subjects[END_REF][START_REF] Mahmoud | In vitro effects of astaxanthin combined with ginkgolide B on T lymphocyte activation in 24 peripheral blood mononuclear cells from asthmatic subjects[END_REF][START_REF] Wang | Astaxanthin-rich algal meal and vitamin C inhibit Helicobacter pylori infection in BALB/cA mice[END_REF] and are marketed in the U.S. without prescription as components of food or food supplements. The present study demonstrates that oral administration of these phytonutrients to asthmatic animals along with vitamin C, significantly improved major indicators of asthma severity. Specifically, the therapeutic agents reduced lung infiltration in asthma [START_REF] Busse | Leukotrienes and inflammation[END_REF][START_REF] Hai | Airway smooth muscle cell as therapeutic target of inflammation[END_REF][START_REF] Hamad | Guanylyl cyclases, nitric oxide, natriuretic peptides, and airway smooth muscle function[END_REF]Wenzel 1996). These observations suggest that the phytochemical formulations evaluated here, have potential to decrease risk of, and improve therapy for a diverse spectrum of inflammatory pathologies and could greatly improve quality-of-life for millions of people Worldwide.

ACKNOWLEDGMENT

The OA-sensitized Guinea pig groups (6 animals in each group) treated as described previously with 0-200 mg/kg astaxanthin (ASX), 0-100 mg/kg Ginkgo biloba leaf extract (EGb761), 0-400 mg/kg vitamin C (VitC), combinations of ASX, Ginkgo and vitamin C and 0-1,000 mg/kg ibuprofen (IBP Numbers of inflammatory cells in bronchoalveolar lavage (BAL) fluid and levels of the cyclic nucleotides cAMP and cGMP in lung tissue were measured in ovalbumin (OA)-sensitized guinea pigs, segregated into dosage groups of 6 animals each and administered feed containing the following supplements (referred to here as "drugs): astaxanthin (1A), Ginkgo biloba leaf extract (EGb761) (1B), vitamin C (1C), selected combinations of each (1D), or ibuprofen (1E), daily for 26 days. Two groups, designated as drug-free controls were given feed without phytochemicals or ibuprofen. One of these two control groups was exposed to vehicle aerosol (nebulized saline) only, without OA challenge and is designated below: "OA-neg, non-asthma, no drugs". The other control group which received an OA challenge is designated below: "OA+ asthma+, no drugs". Twentyfour hours following the 26-day drug treatment period, asthma was induced by OA inhalation, animals were sacrificed and BAL inflammatory cell levels and lung tissue cyclic nucleotide content was measured in each animal. Data is reported as mean ± standard error of means (SEM) of measurements taken in the 6 animals constituting each treatment group.

  to tissue damage in asthma and most other inflammatory syndromes means that treatment strategies affecting major indicators of asthmaassociated inflammation are likely to be relevant for many conditions in which inflammation plays an underlying role.

  prophylaxes and therapy for persons at-risk of, or diagnosed with illness such as asthma, allergies, arthritis and other chronic inflammatory conditions.

  indicators in animals in groups receiving ASX, EGb761, vitamin C

  1-5, significant inhibition of disease indicators was observed in animals administered each product independently and in combination. Comparison of the average magnitude of indicators in treated animal groups with the untreated asthmatic group demonstrated significantly favorable effects (p<0.05) of all products and their combinations in the upper dosage range tested. The indicators: levels of BAL inflammatory cell infiltrate and lung tissue cyclic nucleotide content exhibited significantly favorable dose-responsive correlation to pre-treatment of animals with ASX (Table

  : BAL fluid inflammatory cell content (Figures 1-3); and lung tissue cyclic nucleotides (Figures 4 and 5) (p<0.05). Treatment of animals with ASX, Ginkgo and vitamin C or their combinations resulted in suppression of each disease

  Figures 1-5show average magnitudes of disease indicators in treatment groups administered the lowest dose of each agent or combination of agents significantly different (p<0.05) from the untreated asthmatic control group. At the dosages used, average disease indicator levels in animals receiving combined treatments do not appear obviously improved versus groups treated with single agents (Figures1-5). Indeed, qualitative comparison of inflammatory cell infiltrate numbers between treatment groups shown in Figures1-3suggest that some of the single-agent treated groups experienced better improvement than those treated with 10 mg/kg each of and EGb761 plus 200 mg/kg of vitamin C. Statistical analysis did however reveal significant additive interaction between these three agents suggesting benefits to co-administration in prevention or treatment of asthma. Animals receiving 10 mg/kg ASX (

  radical scavenging properties of ASX is expected to systemically suppress the intensity of oxidative processes in ASX-treated animals. Such a shift,

  decided that the advantages of ibuprofen outweighed its drawbacks. The drug is available without prescription, is one of the most widely-used anti-inflammatory compounds ever manufactured and in 2005 was designated by the World Health Organization (WHO) as an essential public health resource for all nations (WHO Model List of Essential Medicines.

  ; and increased levels of cyclic nucleotides in lung tissue. Both effects correlate strongly with reduced inflammation and improved prognosis

Figure 2 .

 2 Figure 2. Magnitude of inflammation-associated responses in animals treated with phytonutrients or ibuprofen: Bronchoalveolar lavage (BAL) fluid content of neutrophils.OA-sensitized Guinea pig groups (6 animals in each group) treated as described previously with 0-200 mg/kg astaxanthin (ASX), 0-100 mg/kg Ginkgo biloba leaf extract (EGb761), 0-400 mg/kg vitamin C (VitC), combinations of ASX, Ginkgo and vitamin C and 0-1,000 mg/kg ibuprofen (IBP). Data shown is restricted to outcomes from treatment groups receiving the lowest dose of each product resulting in values of each inflammation indicator significantly different (p<0.05) than results from asthmatic, drug-free control animals.

Figure 3 .

 3 Figure 3. Magnitude of inflammation-associated responses in animals treated with phytonutrients or ibuprofen: Bronchoalveolar lavage (BAL) fluid content of macrophages.OA-sensitized Guinea pig groups (6 animals in each group) treated as described previously with 0-200 mg/kg astaxanthin (ASX), 0-100 mg/kg Ginkgo biloba leaf extract (EGb761), 0-400 mg/kg vitamin C (VitC), combinations of ASX, Ginkgo and vitamin C and 0-1,000 mg/kg ibuprofen (IBP). Data shown is restricted to outcomes from treatment groups receiving the lowest dose of each product resulting in values of each inflammation indicator significantly different (p<0.05) than results from asthmatic, drug-free control animals.

Figure 4 .

 4 Figure 4. Magnitude of inflammation-associated responses in animals treated with phytonutrients or ibuprofen: enhanced lung tissue content of cAMP.OA-sensitized Guinea pig groups (6 animals in each group) treated as described previously with 0-200 mg/kg astaxanthin (ASX), 0-100 mg/kg Ginkgo biloba leaf extract (EGb761), 0-400 mg/kg vitamin C (VitC), combinations of ASX, Ginkgo and vitamin C

Figure 5 .

 5 Figure 5. Magnitude of inflammation-associated responses in animals treated with phytonutrients or ibuprofen: enhanced lung tissue content of cGMP.OA-sensitized Guinea pig groups (6 animals in each group) treated as described previously with 0-200 mg/kg astaxanthin (ASX), 0-100 mg/kg Ginkgo biloba leaf extract (EGb761), 0-400 mg/kg vitamin C (VitC), combinations of ASX, Ginkgo and vitamin C and 0-1,000 mg/kg ibuprofen (IBP). Data shown is restricted to outcomes from treatment groups receiving the lowest dose of each product resulting in values of each inflammation indicator significantly different (p<0.05) than results from asthmatic, drug-free control animals.

Table 1A

 1A

	combined administration of these agents at the dosages stated, correlated with significant
	improvement in all disease indicators (p<0.05) (Table
	F o
	r
	P
	e e r
	R e v i e w
	), or 10
	mg/kg EGb761 (Table 1B), or 200 mg/kg vitamin C (Table 1C), or less independently of
	treatment with other agents did not exhibit BAL inflammatory cell numbers or lung tissue
	cyclic nucleotide levels significantly different from asthmatic controls. However,
	14

  authors acknowledge the efforts of Stephanie C. Fox, President, QueenBeeEdit in formatting proofreading and editing this manuscript in preparation for publication. This

	Wenzel SE. 1996. Abnormalities of cell and mediator levels in bronchoalveolar lavage Figure 1. Magnitude of inflammation-associated responses in animals treated with
	phytonutrients or ibuprofen: Bronchoalveolar lavage (BAL) fluid content of
	fluid of patients with mild asthma. J Allergy Clin Immunol 98: S17-21; discussion eosinophils.
	OA-sensitized Guinea pig groups (6 animals in each group) treated as described
	S33-40. previously with 0-200 mg/kg astaxanthin (ASX), 0-100 mg/kg Ginkgo biloba leaf extract
	(EGb761), 0-400 mg/kg vitamin C (VitC), combinations of ASX, Ginkgo and vitamin C
	and 0-1,000 mg/kg ibuprofen (IBP). Data shown are restricted to outcomes from
	WHO Model List of Essential Medicines. World Health Organization. 2005 [cited. treatment groups receiving the lowest dose of each product resulting in values of each
	inflammation indicator significantly different (p<0.05) than results from asthmatic, drug-
	Available from <http://whqlibdoc.who.int/hq/2005/a87017_eng.pdf free control animals.
	* Inflammation indicator magnitudes significantly different (p<0.05) than results from asthmatic, drug-free
	control animals.
	F o F o F o F o F o F o F o F o
	r r r r r r r r
	P P P P P P P P
	study was supported by grants from OTKA 72315, OTKA 78223, GVOP-3.2.1.-2004-04-0269/3.0, TAMOP-4.2.2-08/1-2008-0007, and TAMOP-4.2.1.B-09/1. e e r e e r e e r e e r e e r e e r e e r e e r
	R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w
	20 26

Table 1 . Lung inflammation-associated parameter values in treatment groups (basic data).

 1 ). Data shown is restricted to outcomes from treatment groups receiving the lowest dose of each product resulting in values of each inflammation indicator significantly different (p<0.05) than results from asthmatic, drug-free control animals.

		Figure 1.
	Bronchoalveolar lavage (BAL) eosinophil content
	BAL Eosinophils x 10 6 per animal	F o r P e e r R e e w Treatments i v Non-asthmatic, drug-free Asthmatic, drug-free ASX 30mg/kg Vit. C 400 mg/kg EGb761 30 mg/kg ASX 10, Vit. C 200, EGb 10 mg/kg Ibuprofen 500 mg/kg 0 2 4 * * * * * * 6 8

1A. Astaxanthin (ASX)-treated animals.

	Cohort size:	OA-neg	OA+	OA+	OA+	OA+	OA+	OA+
	n = 6	non-asthma	asthma+	5mg/kg	10mg/kg	30mg/kg	100mg/kg	200mg/kg
		no drugs	no drugs	ASX	ASX	ASX	ASX	ASX
	Eosinophils (cells	1.4±0.2	5.4±0.8	5.0±0.7 4.6±0.6	3.1±0.5*	2.4±0.6*	2.3±0.5*
	x 10 6 /animal).
	Neutrophils (cells	1.1±0.3	5.8±0.7	5.4±0.8 5.1±0.6	3.3±0.8* 2.4±0.5*	2.0±0.4*
	x 10 5 /animal)
	Macrophages	1.7±0.5	9.9±1.1	9.6±1.0 8.0±0.8	3.7 ±0.7* 2.70±0.7*	2.6±0.5*
	(cells x
	10 6 /animal)
	cAMP pMol/mg	10.9±0.98	6.7±0.58	6.9±0.5 8.1±0.8	9.6±0.8* 12.6±0.8*	12.1±0.5*
	lung protein
	cGMP pMol/mg	2.6±0.26	1.1±0.17	1.2±0.1 1.4±0.1	1.8±0.3* 2.8±0.3*	2.7±0.2*
	lung protein
	1B.

Ginkgo biloba (EGb761)-treated animals. Cohort size: n = 6 OA-neg non- asthma no drugs OA+ asthma+ no drugs OA+ 5mg/kg EGb761 OA+ 10mg/kg EGb761 OA+ 30mg/kg EGb761 OA+ 100mg/kg EGb761 Eosinophils (cells x 10 6 /animal).

	1C. Vitamin C-treated animals.
	Cohort size:	OA-neg non-	OA+	OA+	OA+		OA+	OA+
	n = 6	asthma	asthma+	50mg/kg	100mg/kg	200mg/kg	400mg/kg
		no drugs	no drug	Vit. C	Vit. C		Vit. C	Vit. C
	Eosinophils (cells x	1.4±0.2	5.4±0.8	5.5±0.5	5.5±0.8		4.8±1.1	3.5±0.9*
	10 6 /animal).
	Neutrophils (cells x	1.1±0.3	5.8±0.7	5.4±0.6	5.6±0.8		5.0±0.8	4.7±0.9
	10 5 /animal)
	Macrophages (cells x	1.7±0.5	9.9±1.1	9.5±0.9	9.8±0.9		9.2 ±0.8	7.2±1.5*
	10 6 /animal)
	cAMP pMol/mg lung	10.9±0.98	6.7±0.58	6.8±0.5	7.1±0.8		7.9±0.9	7.9±0.8
	protein
	cGMP pMol/mg lung	2.6±0.26	1.1±0.17	1.1±0.2	1.0±0.2		1.2±0.2	1.5±0.2*
	protein
	1D. Combination treatments: Animals simulataneously administered astaxanthin, Gingko biloba (EGb761) and vitamin C F Cohort size: n = 6 OA-neg non-asthma OA+ asthma+ OA+ 5 mg/kg ASX + OA+ 10 mg/kg ASX OA+ 30 mg/kg ASX o no drugs no drug 5 mg/kg + 10 mg/kg + 30 mg/kg r EGb761 EGb761 EGb761
		P	+ 50 mg/kg Vit C	+ 200 mg/kg Vit C	+ 400 mg/kg Vit C
	Eosinophils (cells x 10 6 /animal). Neutrophils (cells x 10 5 /animal) Macrophages	1.4±0.2 1.1±0.3 1.7±0.5	5.4±0.8 e 5.8±0.7 e r 5.07±0.63 5.27±0.74 9.9±1.1 9.40±0.96		3.44±0.84* 3.65±0.65* 5.78±1.18*	2.95±0.62* 2.60±0.69* 2.98±0.71*
	Neutrophils (cells x 10 5 /animal) (cells x 10 6 /animal) cAMP pMol/mg lung protein cGMP pMol/mg lung protein 1E	1.4±0.2 1.1±0.3 10.9±0.98 2.6±0.26	5.4±0.8 5.8±0.7 6.7±0.58 1.1±0.17	5.1±0.9 5.6±0.6 7.17± 0.61 R 1.17± 0.19 e e 5.0 ±0.8 5.4±0.6 9.28±0.94* 3.5±0.8* 3.5±0,7* 2.09±0.48* w i v	3.1±0.5* 3.3±0.6* 13.00±0.76* 2.84±0.28*
	Macrophages (cells x	1.7±0.5	9.9±1.1	9.8±1.1	9.4±1.1	4.9 ±1.5*	4.2±0.6*
	10 6 /animal)
	cAMP pMol/mg lung	10.9±0.98	6.7±0.58	6.9±0.6	8.0±0.9	9.8±0.7*	12.0±1.3*
	protein
	cGMP pMol/mg lung	2.6±0.26	1.1±0.17	1.1±0.2	1.1±0.2	1.5±0.2*	1.8±0.3*
	protein

. Ibuprofen-treated animals. Cohort size: n = 6 Asthma-free control (OA neg) OA+ asthma, no drug OA+ IB 10mg/kg OA+ IB 100mg/kg OA+ IB 500mg/kg OA+ IB 1000mg/kg Eosinophils (cells x 10 6 /animal).

  *p<0.05 compared to corresponding values of OA antigen-challenged, asthmatic drug-free control group.

		1.4±0.2	5.4±0.8	5.4±0.8	4.7±0.6	3.0±0.8*	2.9±0.5*
	Neutrophils (cells x	1.1±0.3	5.8±0.7	5.7±0.7	5.0±0.7	3.1±0.9*	3.1±0.5*
	10 5 /animal)
	Macrophages (cells x	1.7±0.5	9.9±1.1	9.5±0.8	9.0±1.3	4.7±1.3*	4.3±0.9*
	10 6 /animal)
	cAMP pMol/mg lung	10.9±0.98	6.7±0.58	7.1±0.6	7.3±0.9	8.2±0.8*	8.1±0.9*
	protein
	cGMP pMol/mg lung	2.6±0.26	1.1±0.17	1.1±0.2	1.1±0.2	1.4±0.2	1.6±0.2*
	protein

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This study was supported by grants from OTKA 72315, OTKA 78223, GVOP-3.2.


Andreja Sinkovic
email: andreja.sinkovic@guest.arnes.si

David Suran

Lidija Lokar

Eva Fliser

Mojca Skerget

Zoran Novak

Zeljko Knez

Zeljko Knezd

Rosemary extracts improve flow-mediated dilatation of the brachial artery and plasma PAI-1 activity in healthy young volunteers

Keywords: Rosemary extracts, endothelial dysfunction, flow-mediated dilatation, plasminogen-activator-inhibitor- Rosemary extracts, endothelial dysfunction, flow-mediated dilatation, plasminogen-activator-inhibitor-1

Introduction

Moderate consumption of red wine and green tea polyphenols has been associated with decreased risk of cardiovascular morbidity and mortality [START_REF] Sato | Cardioprotection with alcohol: role of both alcohol and polyphenolic antioxidants[END_REF][START_REF] Grassi | Tea, flavonoids, and nitric oxide-mediated vascular reactivity[END_REF]. Recent clinical studies have demonstrated an improvement in flow-mediated dilatation (FMD) of the brachial artery in healthy young people after administration of polyphenols in green tea as well as in patients with documented atherosclerosis after administration of polyphenol isoflavone in soya beans [START_REF] Nishioka | Pycnogenol®, French maritime pine bark extract, augments endothelium-dependent vasodilation in humans[END_REF][START_REF] Alexopoulos | The acute effect of green tea consumption on endothelial function in healthy individuals[END_REF]Chan et al., 2008].

In vitro and in vivo studies demonstrated strong antioxidant and anti-inflammatory activity of rosemary extracts (RE) attributed to their diphenolic constituents, carnosic and rosmarinic acid in particular [START_REF] Aruoma | An evaluation of the antioxidant and antiviral action of extracts of rosemary and provencal herbs[END_REF][START_REF] Zeng | Antioxidant properties of phenolic diterpenes from Rosmarinus officinalis[END_REF][START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF]. As antioxidants, RE are scavengers of free radicals, they decrease consumption of NO due to depletion of free radicals, decrease the injury of endothelial cells and LDL oxidation by free radicals [START_REF] Petersen | Rosmarinic acid[END_REF][START_REF] Moreno | Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition[END_REF][START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF].

In experimental animals, superoxide dismutase (SOD) and glutathione peroxidase (GPX), being the most important antioxidant defense systems, are most often increased after consumption of polyphenols as a consequence of diminished oxidative stress [Chan et al., 2008;[START_REF] He | Neuroprotective effects of (-)-epigallocatechin-3-gallate on aging mice induced by D-galactose[END_REF]. For the same reason, the serum lipid profile is improved, resulting in decreased total and LDL-cholesterol [START_REF] Lecumberri | A diet rich in dietary fiber from cocoa improves lipid profile and reduces malondialdehyde in hypercholesterolemic rats[END_REF].

In comparison to other non-phenolic antioxidants, the advantages of RE are their more extensive effect upon the nuclear factor kappa B (NF-κB) pathway, resulting in more pronounced decrease in inflammation [START_REF] Aggarwal | Suppression of the Nuclear FactorkB Activation Pathway by Spice-Derived Phytochemicals. Reasoning for Seasoning[END_REF][START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF]. Their activity involving the NF-κB pathway is important in the control of the immune and inflammatory response, cellular proliferation and survival [START_REF] Aggarwal | Suppression of the Nuclear FactorkB Activation Pathway by Spice-Derived Phytochemicals. Reasoning for Seasoning[END_REF] [START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF]. Inflammatory activity of the NF-κB pathway, exerting its effect by expressing cytokines, chemokines, cell adhesion molecules, immunoreceptors, etc., was studied by evaluating serum markers such as high-sensitivity C-reactive protein (hs-CRP), fibrinogen, tumor necrosisi factor-α (TNF-α) and serum plasminogen-activator-inhibitor type 1 (PAI-1) activity [START_REF] Pasten | Polyphenols downregulate PAI-1 gene expression in cultured human coronary artery endothelial cells: molecular contributor to cardiovascular protection[END_REF]Chan et al., 2008;[START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF]. Studies even demonstrated that diphenols in RE decrease PAI-1 activity by several pathways, in addition to NF-κB transcription, TNF-α reduction and human peroxisome proliferator-activated receptor gamma activation [START_REF] Rau | Carnosic acid and carnosol, phenolic diterpene compounds of the labiate herbs rosemary and sage, are activators of the human peroxisome proliferator-activated receptor gamma[END_REF]].

Arterial endothelial dysfunction (ED) is an early event in atherogenesis, preceding structural atherosclerotic changes by years. It can be evaluated by ultrasound measurement of the flow-mediated dilatation (FMD) in the brachial artery and by measuring serum markers such as vascular cell adhesion molecule 1 (VCAM-1) and inter-cellular adhesion molecule 1 (ICAM-1) [START_REF] Celermajer | Relevance of phenolic diterpene constituents to antioxidant activity of supercritical CO(2) extract from the leaves of rosemary[END_REF][START_REF] Hwang | Circulating adhesion molecules VCAM-1, ICAM-1, and Eselectin in carotid atherosclerosis and incident coronary heart disease cases: the atherosclerosis risk in communities (ARIC) study[END_REF][START_REF] Jadhav | Noninvasive assessment of endothelial dysfunction by brachial artery flow-mediated dilatation in prediction of coronary artery disease in Indian subjects[END_REF].

Our aim was to evaluate prospectively the effect of 21-day oral RE supplementation with active substances carnosol, carnosic and rosmarinic acid, upon FMD and serum markers ICAM-1, VCAM-1, PAI-1, hs-CRP, fibrinogen, SOD, GPX, TNF-α, total serum cholesterol, triglycerides, LDH-cholesterol and HDL-cholesterol level in healthy young volunteers. We studied 19 healthy young volunteers, 7 men and 12 women, mean age 34.3 ± 7.7 years. 8 (42.1%) were smokers and 3 (15.8%) with the family history of symptomatic atherosclerosis. Inclusion criteria: more than 18 years old, negative urine-β-HCG test in young women, written informed consent. A week before the start and during the study we recommended discontinuation of all previously used antioxidants, vitamins and alcohol intake. We also recommended the use of a well-balanced diet of 1500 -2000 kcal/day with approximately 30% proteins, 60% carbohydrates and 10% fat with at least one daily ration of meat, but only up to 2 apples per day [START_REF] Chun | Serum C-reactive protein concentrations are inversely associated with dietary flavonoid intake in U.S. adults[END_REF].

Materials and methods

Subjects studied

Exclusion criteria: any known chronic, malignant disease and/or the use of any medication, pregnancy, breast-feeding and simultaneous participation in another clinical study.

Study design.

Observational prospective monocenter study performed at the University Clinical

Study protocol

On day 0, volunteers were clinically examined and screened for baseline demographic data, family history and history of prior diseases. Blood pressure and pulse were measured and standard ECG was recorded. When all the inclusion criteria were fulfilled and contraindications were excluded, first written informed consent was obtained. For [START_REF] Chun | Serum C-reactive protein concentrations are inversely associated with dietary flavonoid intake in U.S. adults[END_REF].

On day 7, inclusion and exclusion criteria were rechecked and second written informed consent was obtained. Blood pressure and pulse were measured. Fasting blood samples were drawn from the antecubital vein to estimate serum levels of PAI-1, SOD, ICAM-1, VCAM-1, fibrinogen, total serum cholesterol, triglycerides, GPX, LDH-cholesterol and HDL-cholesterol levels between 8:00 and 9:00 a.m. Vascular ultrasound examination was performed to estimate FMD of the brachial artery between 9:00 and 11:00 a.m.

Starting on day 7 the volunteers took oral supplementation (tablets) of RE each day for the next next 21 days. The tablets consisted of 77.7 mg of RE with the active substances carnosol (0.97 mg), carnosic acid (8.60 mg) and rosmarinic acid (10.30 mg), gained by high performance liquid chromatography (HPLC) and supplied by Vitiva d.o.o., Slovenia [Škrinjar et al., 2007]. The specific dosage was chosen on the basis of previous studies, showing good tolerance without side effects [START_REF] Samman | Green tea or rosemary extract added to foods reduces nonheme-iron absorption[END_REF].

Each week (day 14, day 21 and day 28) the volunteers were interviewed for any side effects.

On day 28, between 8:00 and 9:00 a.m., blood samples were redrawn. Between 9:00 and 11:00 a.m. FMD was re-evaluated by ultrasound measurements of the brachial artery.

Vascular ultrasound examination

FMD was studied using a high-resolution B mode Phillips ultrasound system with a 7

MHz linear array transducer. The subjects rested in the supine position for ten minutes before haemodynamic measurements were performed. The right brachial artery was scanned in the longitudinal section 2 to 15 cm above the elbow to find the clearest images of the anterior and posterior wall layers. The mean arterial diameter was Hyperaemic flow was recorded for the first 15 seconds and diameter measurements were taken 45-60 seconds after cuff deflation. In this way FMD was defined as the endothelium-dependent dilatation, expressed as the percentage change of the diameter after reactive hyperaemia, relative to the baseline scan. FMD, expressed in percentages, was calculated from the brachial artery baseline diameter (D 1 ) and 60-second postdeflation diameter (D 2 ), measured as described previously. The formula to calculate is following: FMD (%) =(D 2 -D 1 )/D 1 . The same investigator, who was blind to the subjects' characteristics, carried out all measurements [START_REF] Celermajer | Relevance of phenolic diterpene constituents to antioxidant activity of supercritical CO(2) extract from the leaves of rosemary[END_REF]. The FMD levels of less than 4.5% were defined as ED as demonstrated by clinical studies [Jadhaw et al., 2003]. Serum fibrinogen was estimated by the modified Clauss method (automatic analyser Dade Behring, Marburg GmbH, Germany); normal levels were 2.2 g/l -4.2 g/l.

Blood samples and laboratory methods

Statistical analysis

Statistics was performed by IBM PC, SPSS for Windows. The values were expressed as means ± standard deviations or proportions where necessary. The differences among the groups were tested by the two-sided Student's t-test for means ± standard deviations and by the chi-square test for proportions. The P value < 0.05 was statistically significant.

Results

During 21 days of oral RE supplementation, any side effects were registered.

Comparisons of mean levels of tested variables before and after 21 days of RE supplementation are displayed in Table 1. After 21 days, mean FMD of the brachial artery increased nonsignificantly (5.1 ± 5.3% vs. 7.1 ± 3.8%, P = 0.189). However, the rate of ED decreased significantly (68.4% vs. 15.9%, RR 4.3, 95% CI 1.468 to 12.791, P = 0.0038). Mean PAI-1 level decreased significantly (4.4 ± 1.3 U/ml vs. 3.3 ± 0.7 U/ml, P = 0.0025) as well. Median levels of FMD increased from 3.76% to 6.7% (Figure 1) and median PAI-1 levels decreased from 4.9 U/ml to 3.3 U/ml (Figure 2). 21 days of RE supplementation did not have any significant effect upon serum lipid profile, mean serum SOD, ICAM-1, VCAM-1, GPX, hs-CRP, TNF-α or fibrinogen level (Table 1). Impaired endothelial vascular function precedes atherosclerosis and its improvement by pharmacological agents would delay the onset of symptomatic atherosclerosis such as acute myocardial infarction or stroke, which are among the most prevalent causes of cardiovascular morbidity and mortality [START_REF] Celermajer | Relevance of phenolic diterpene constituents to antioxidant activity of supercritical CO(2) extract from the leaves of rosemary[END_REF][START_REF] Jadhav | Noninvasive assessment of endothelial dysfunction by brachial artery flow-mediated dilatation in prediction of coronary artery disease in Indian subjects[END_REF].

In vivo and in vitro studies demonstrated that the chronic and progressive nature of atherosclerosis is driven by several oxidative and inflammatory mechanisms such as inactivation of NO, oxidative alteration of DNA, proteins and lipids, activation of redox-sensitive genes, etc. [START_REF] Moreno | Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition[END_REF][START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF][START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF]. In vitro, animal and human studies suggest that cellular antioxidant enzymes such as SOD and GPX may protect against atherosclerosis [START_REF] Blankenberg | Glutathion Peroxidase 1 activity and cardiovascular events in patients with coronary artery disease[END_REF]. Nevertheless, meta-analysis of studies with antioxidant vitamin supplements and Selenium does not support a possible role of their supplementation in reducing the risk of cardiovascular disease. However, in this meta-analysis studies with phenolic antioxidants were not included and, at present, no definite conclusion can be drawn to justify the use of antioxidants for the prevention of atherosclerotic events [START_REF] Bjelakovic | Antioxidant supplements for prevention of mortality in healthy participants and patients with various diseases[END_REF][START_REF] Katsiki | Is there a role for supplemented antioxidants in the prevention of atherosclerosis?[END_REF].

The key event in the development and progression of atherosclerosis is migration of smooth muscle cells from media to the subendothelial region. In rosemary, carnosic [START_REF] Petersen | Rosmarinic acid[END_REF][START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF]. It inhibits the activation and expression of matrix metalloproteinase-9 through down-regulation of NF-κB, preventing the migration of human vascular smooth muscle cells [START_REF] Lin | Carnosic acid prevents the migration of human aortic smooth muscle cells by inhibiting the activation and expression of matrix metalloproteinase-9[END_REF]. Major active components in our oral RE were carnosic and rosmarinic acid, which both possess significant antioxidant and anti-inflammatory activity. Both have been successfully used as antioxidants in the food industry, but their anti-inflammatory and antimicrobial effects were also demonstrated in human studies [START_REF] Martinez-Tome | Antioxidant properties of mediterranean spices compared with common food[END_REF][START_REF] Samman | Green tea or rosemary extract added to foods reduces nonheme-iron absorption[END_REF][START_REF] Osakabe | Anti-inflammatory and anti-allergic effect of rosmarinic acid (RA); inhibition of seasonal allergic rhinoconjunctivitis (SAR) and its mechanism[END_REF][START_REF] Lukaczer | A pilot trial evaluating Meta050, a proprietary combination of reduced isoalpha acids, rosemary extracts and oleanolic acid in patients with arthritis and fibromyalgia[END_REF].

In our study serum PAI-1 activity was most significantly affected. Our findings are consistent with a study on the cultured human coronary artery cells, demonstrating downregulation of PAI-1 gene expression in arterial endothelial cells [START_REF] Pasten | Polyphenols downregulate PAI-1 gene expression in cultured human coronary artery endothelial cells: molecular contributor to cardiovascular protection[END_REF]. Decreasing serum PAI-1 activity may reflect molecular contribution to cardiovascular protection by the use of polyphenols [START_REF] Pasten | Polyphenols downregulate PAI-1 gene expression in cultured human coronary artery endothelial cells: molecular contributor to cardiovascular protection[END_REF]. Increased PAI-1 levels are associated with impaired endogenous fibrinolysis and increased risk of thrombotic events and progression of atherosclerosis. Decreasing PAI-1 by RE could prevent atherosclerosis and its complications [START_REF] Pasten | Polyphenols downregulate PAI-1 gene expression in cultured human coronary artery endothelial cells: molecular contributor to cardiovascular protection[END_REF]. However, further randomised studies with RE in established atherosclerosis would be necessary to demonstrate prevention of further thrombotic complications.

In our healthy volunteers, mean baseline levels of serum SOD, ICAM-1, VCAM-1, GPX, TNF-α, hs-CRP and fibrinogen were within normal limits and therefore, not surprisingly, RE supplementation had only a nonsignificant impact on their mean levels as estimated after 21 days. However, after 21 days, ED rate improved significantly (from 68.4% to 15.9%, p = 0.0038), suggesting an improvement of the balance between endothelial vasoconstriction and vasodilatation. This seems to correlate with improved endogenous fibrinolysis, characterised by decreased PAI-1 activity and with nonsignificant increase in SOD and nonsignificant decrease in VCAM-1, ICAM-1, fibrinogen, hs-CRP and TNF-α [START_REF] Nishioka | Pycnogenol®, French maritime pine bark extract, augments endothelium-dependent vasodilation in humans[END_REF]Chan et al., 2008;[START_REF] He | Neuroprotective effects of (-)-epigallocatechin-3-gallate on aging mice induced by D-galactose[END_REF][START_REF] Nieuwdorp | Tumor necrosis factor-α inhibition protects against endotoxin-induced endothelial glycocalyx perturbation[END_REF].

Our results suggest that oral RE supplementation in young and apparently healthy individuals is safe and has the potential to improve endothelial function, impaired endogenous fibrinolysis, characterised by a decrease in serum PAI-1 activity and resulting atherosclerosis. However, further randomised clinical studies are needed, particularly in patients with symptomatic atherosclerosis, arterial hypertension or diabetes, dyslipidemia and smoking to confirm their beneficial antioxidant and antiinflammatory effect upon arterial endothelial function and symptomatic atherosclerosis. Mean hs-CRP ± SD (mg/l) 1,6 ± 0.4 1.6 ± 0.9 0.459 ED (%) 13 (68.4) 3 (15.9) 0.0038

F

Mean red blood cell count ± SD (10 12 /l) 4.5 ± 0.5 4.5 ± 0.5 1.000

Mean platelet count ± SD (10 9 /l) 247.3 ± 55.9 237.5 ± 48.6 0.567

Mean white blood cell count ± SD (10 9 /l) 6.7 ± 2.1 6.8 ± 1.9 0.878 TNF-α ± SD (pg/ml) 9.3 ± 2.7 8.0 ± 2.

  Centre, Maribor, approved by the national ethics comittee (Number 58/06/06). Written informed consents were obtained before the start of the study from each participant. The study protocol conformed to the ethical guidelines of the Declaration of Helsinki. The study was also approved by the National Agency for Medical Products and Medical Devices (Number 297-2006-001894-12) and registered at EMEA (EudraCT number 2006-001894-12; Protocol Code Number ROS-001).

  weeks the volunteers discontinued the use of any vitamin and antioxidant supplementation and started the recommended diet

  end of diastole. At least three cardiac cycles were analysed for each scan and the measurements averaged. The flow velocity was measured at a fixed incident angle of 68° to the vessel with the range gate of 1.3 mm located in the centre of the artery. The baseline blood flow was estimated by multiplying the velocity time integral of the Doppler flow signal (corrected for incident angle) with the vessel crosssectional area. Hyperaemic flow increase was induced by inflation of a blood pressure tourniquet placed around the forearm to a pressure of 200 mmHg for 4 minutes.

  Total serum cholesterol, HDL-cholesterol and triglycerides were estimated by the colorimetric method (Ektachem 250 Analyzer, Eastman Kodak Company, Rochester, USA). LDL-cholesterol level was calculated by means of the Friedewald's equation (LDL-cholesterol = Total cholesterol -HDL-cholesterol -4.5. Triglicerides). The lipid profile was estimated at the start and 3 weeks after treatment. estimate PAI-1, SOD, ICAM-1, VCAM-1, GPX, hs-CRP, TNF-α and fibrinogen were drawn in fasting state between 8:00 and 10:00 a.m. to avoid circadian variations. Blood samples were centrifugated and plasma was frozen and stored at -70°C. Estimations were performed a few weeks later and were blinded to other investigators.Serum PAI-1 activity was estimated by the chromogenic method (Berichrom PAI by Dade Behring, Marburg, Germany). 1 unit (U) of PAI-1 activity is defined as the amount of PAI-1 that inhibits one international unit of human single tissue-plasminogen activator. Normal levels are 0.3-3.5 U/ml Serum SOD was estimated by an in-house method with xanthine, INT / 2-(4iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride and xanthine oxidase (Randox, Crumlin-UK). Serum SOD activity determines indirectly the grade of inhibition of generation of superoxide radicals by xanthine and xanthine oxydase. Normal values were 164-240 U/ml. Serum ICAM-1 was estimated by ELISA H uman sVCAM-1 Immunoassay (R&D Systems, Inc., Minneapolis, USA, normal levels 115-306 ng/ml). Serum VCAM-1 was estimated by ELISA Human sICAM-1 Immunoassay (R&D Systems, Inc., Minneapolis, USA, normal levels 395 -714 ng/ml). Serum GPX was estimated by an in-house method with glutathione (GSH), cumene hydroperoxide and glutathione reductase (GR) (Randox, Crumlin-UK). GPX determines indirectly the rate of formation of oxidized glutathione; normal values were 4171-10881 U/l. Serum TNF-α was estimated by quantitative immunometric chemoluminiscence IMMULITE ® TNF-α test, performed by automatic analyzer IMMULITE ® , DPC Corporation, Los Angeles, United States of Amerika, normal levels up to 8.1 pg/ml). Serum hs-CRP was estimated by immunoturbidimetric method with latex particles (PETIA) (Siemens Healthcare Diagnostics, former Dade Behring, Newark, USA); normal levels were < 3.0 mg/l.

  oral RE supplementation with the active substances carnosol, carnosic and rosmarinic acid resulted in significant decrease in mean serum PAI-1 activity, significant improvement of ED, characterised as FMD < 4.5% and in nonsignificant increase in mean FMD. To our knowledge, this was the first clinical study, evaluating the effect of oral RE supplementation on endothelial vascular function.

  major phenolic constituent. It is a typical diphenol with potent chainbreaking antioxidant activity

  , mean FMD changed nonsignificantly after RE supplementation.

  deviation; LDL, low density lipoproteins; HDL, high-density lipoproteins; PAI-1, plasminogen activator inhibitor type 1; U, unit; SOD, sodium dysmutase; VCAM-1, Vascular cell adhesion molecule 1; ICAM-1, Inter-Cellular Adhesion Molecule 1; GPX, Glutathione peroxidase 1; hs-CRP, high-sensitivity C-reactive protein; ED, endothelial dysfunction; TNF-α, tumor necrosis factor α

Table 1 :

 1 Comparisons of mean levels of tested variables before and after treatment with oral formulation of rosemary extracts for 21 days

	o r
	P
	e
	e r
	R
	e
	v i e
	w

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Acknowledgment

The work was supported by a grant from the Centre of Excellence -Supercritical Fluids in the frame of the Slovenian Ministry of Higher Education, Science and Technology and the European Regional Development Fund.