Xu Yang

Wenjie Zhang

Xixiang Ying

Didier Stien

New flavonoids

Keywords: Portulaca oleracea L. Flavonoids Isolation Antioxidant activity Anticholinesterase activity DPPH, 1, 1-diphenyl-2-picrylhydrazyl BHA, butylated hydroxyl anisole ATCI, acetylthiocholine iodide AChE, acetylcholinesterase DTNB, 5

HAL is

Introduction

Portulaca oleracea L. a herbaceous succulent annual plant, of which stems and leaves are edible with a slightly acidic and salty taste [START_REF] Lim | Antioxidant properties of different cultivars of Portulaca oleracea Food Chem[END_REF], belonging to the family of Portulacaceae, is widely distributed in tropical and subtropical areas [START_REF] Zhou | a review of phytochemistry and pharmacological effects[END_REF]. P. oleracea, cold in nature and acid in flavor [START_REF] Lei | Separation and identification of four new compounds with antibacterial activity from Portulaca oleracea[END_REF], is a nutritious vegetable crop, rich in essential ω-3 fatty acids, α-linolenic acid, α-tocopherol, ascorbic acid, linoleic acid, β-carotene, glutathione [START_REF] Palaniswamy | Bible Stage of harvest and polyunsaturated essential fatty acid concentrations in purslane (Portulaca oleraceae) leaves[END_REF], and highest dietary minerals, such as K, Ca, Mg, P, Fe and Zn [START_REF] Uddin | Purslane weed (Portulaca oleracea): a prospective plant source of nutrition, omega-3 fatty acid, and antioxidant attributes[END_REF], which are essential for normal human healthy growth. As a result, the vigorous, invasive, yet edible plant P. oleracea is widely used in folk medicine or for other applications [START_REF] Liu | Lan Two antioxidant alkaloids from Portulaca oleracea L Helv[END_REF]. In view of its popularity, more and more pharmacological effects have been studied, including anti-inflammatory [START_REF] Meng | The anti-inflammation and pharmacokinetics of a novel alkaloid from Portulaca oleracea[END_REF], analgesic [START_REF] Chan | Attas The analgesic and anti-inflammatory effects of Portulaca oleracea L. subsp. sativa (Haw.)[END_REF], antibacterial [START_REF] Zhang | Experimental studies on antibiotic functions of Portulaca oleracea L. in vitro[END_REF], antioxidant [START_REF] Sirithon | Maitree Microchemical components and antioxidant activity of different morphological parts of Thai wild purslane (Portulaca oleracea)[END_REF], and neuroprotective [START_REF] Moneim | Al-Quraishy The potential role of Portulaca oleracea as a neuroprotective agent in rotenone-induced neurotoxicity and apoptosis in the brain of rats Pestic[END_REF] ones. More recently, P. oleracea being a crassulacean acid metabolism (CAM) photosynthetic plant [START_REF] Kennedy | Characteristics of Crassulacean acid metabolism in the succulent C₄ dicot, Portulaca oleracea[END_REF], many scholars also concentrated on its constituents, such as phenolic acids [START_REF] Erkan | Antioxidant activity and phenolic compounds of fractions from Portulaca oleracea L Food Chem[END_REF], terpenes [START_REF] Elkhayat | a new diterpene from Portulaca oleracea L J[END_REF], coumarins [START_REF] Rasheed | Investigation of the active constituents of Portulaca oleraceae L. (Portulacaceae) growing in Jordan Pak[END_REF], flavonoids [START_REF] Xu | Determination of flavonoids in Portulaca oleracea L. by capillary electrophoresis with electrochemical detection[END_REF] and alkaloids [START_REF] Li | Ying Three novel alkaloids from Portulaca oleracea L. and their anti-inflammatory effects[END_REF], in which flavonoids can be produced via its photosynthesis [START_REF] Akhtar | Photosynthetic redox imbalance influences flavonoid biosynthesis in Lemna gibba[END_REF] that can also effectively prevent oxidative damage to the plant cells to enhance its antioxidant constituents [START_REF] Demmigadams | Antioxidants in photosynthesis and human nutrition[END_REF]. In the study, three new flavonoids including oleracone C (1), oleracone D [START_REF] Zhou | a review of phytochemistry and pharmacological effects[END_REF] and oleracone E (3) were found, especially, the absolute configuration of oleracone C was elucidated, (2R, 4R)-5,7-dimethoxy-4-O-2′-cycloflavan being the new natural product whose absolute configuration was also determined for the first time (Fig. 1). Flavonoids, with high antioxidation was related to many pharmacological effects, such as cardiovascular disease, senile dementia, cancer, etc. [START_REF] Stohs | The role of free radicals in toxicity and disease[END_REF]. At the same time, flavonoids as acetylcholinesterase (AChE) inhibitor, can promote the production of acetylcholine effectively [START_REF] Bensouici | Kabouche Compounds from Sedum caeruleum with antioxidant, anticholinesterase, and antibacterial activities[END_REF]. Therefore, the antioxidant and anticholinesterase activities of three new compounds isolated from P. oleracea, including oleracone C (1), D (2) and E (3) were studied.

Experimental

Chemical and reagents

1,1-Diphenyl-2-picrylhydrazyl radical (DPPH • ) (purity ≥ 99%) was purchased from Sigma Co. (USA). Butylated hydroxyanisole (BHA) (purity ≥ 98%) was purchased from Shanghai Xiangrui Biological Technology Co., Ltd. (Shanghai, China). Methanol and formic acid are all of HPLC grade provided by Damao Chemical Reagent Plant (Tianjin, China, purity ≥ 99.9%). All other reagents are of analytical grade purchased from Jinfeng Chemical Factory (Tianjin, China), and the water is WAHAHA purified water (Shenyang, China). Acetylthiocholine iodide (ATCI) (purity ≥ 99%) and acetylcholinesterase (AChE) (vitality ≥ 200 units/mg protein) were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). Eserine (purity ≥ 98%) was purchased from Shanghai Hanxiang Biotechnology Co., Ltd. (Shanghai, China). 5,5dithiobis-2-nitrobenzoic acid (DTNB) (purity ≥ 99%) was purchased from Shanghai Jinshui Biotechnology Co., Ltd. (Shanghai, China). Na 2 HPO 4 and NaH 2 PO 4 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) (purity ≥ 99.0%).

Plant material

The whole herbs of P. oleracea were collected in Shijiazhuang (Hebei, China) in June 2014, and identified by Prof. Xixiang Ying. Voucher specimens (No. 20140312) were deposited at School of Pharmacy, Liaoning University of Traditional Chinese Medicine.

General experimental procedures

In the separation process, column chromatography (CC) included silica gel (100-220 and 200-300 mesh, Qingdao Marine Chemical Co., Qingdao, China), polyamide resin (80-100 mesh, Taizhou Luqiao Sijia Biochemical Plastic Factory, Zhejiang, China) and ODS (20-40 μm, GE Healthcare, Marlborough, MA). TLC was performed on silica gel GF 254 (Qingdao Marine Chemical Co., Qingdao, China).

Equipment

The UV spectrum and data of absorbance were determined by using a HITACHI U-3010 spectrophotometer (Hitachi Ltd., Tokyo, Japan). The IR spectrum was obtained by using an IR200 spectrophotometer (Thermo Electron Corporation, Waltham, MA). The Circular dichroism spectra were obtained using J-810 Circular Dichroism spectrometer (JASCO Corporation, Japan). Optical rotations were measured with Autopol I automatic polarmeter (Rudolph Research Analytical, Hackettstown, NJ). The NMR spectra were recorded using an AVANCE 500 MHz instrument (Bruker Corporation, Switzerland). All compounds were dissolved in DMSO-d 6 or CDCl 3. Relative molecular mass were recorded by using a 6520 quadrupole-time-of-flight mass spectrometer (Agilent, Palo Alto, CA). Purity was detected on a Nexera X2 UHPLC LC-30A system (Shimadzu, Kyoto, Japan), using a Kromasil C18 column (150 mm × 4.6 mm, 5 μm, Dalian Johnsson Separation Science and Technology Corporation). The oven temperature was maintained at 40 °C. Water containing 0.1% formic acid served as solvent system A, and methanol served as solvent system B. The flow rate was 1.0 mL/min, and injection volume was 5 μL. 96-well microplate reader (HBS-1096A) was purchased from Nanjing Detie Experimental Equipment Co., Ltd. (Nanjing, China).

Extraction and isolation

The dried whole herb of P. oleracea (150 kg) were extracted twice with a 10-fold amount 50% ethanol for 2 h each time, and then the ethanol extract was concentrated under reduced pressure reflux, obtaining the extract (15 kg), which was latter absorbed on a 100-200 mesh silica-gel column chromatography (82 × 59 cm, approximately, 118.5 kg) with a 2-fold amount ethyl acetate added aqueous ammonia (100:1, v/v) 3 times as the isocratic elution, affording the fraction evaporated (510 g). Then, the fraction, via the a 80-100 mesh polyamide resin column chromatography (120 × 8 cm, approximately, 2.5 kg), was eluted with water, 30%, 50%, 70% and pure ethanol to obtain 5 fractions (4 L each). The fraction of 50% extracting was condensed under the reflux to obtain the P. oleracea extract of 100 g, which was subjected to a 200-300 mesh silica-gel column (120 × 8 cm, approximately, 2.5 kg) chromatography with ethyl acetate, ethyl acetate and methanol (5:1, 2:1, 1:2, v/v) as the gradient eluent in proper order, obtaining 4 fractions (Frs. 1-4, 500 mL each). Then the fractions were spotted on a thin-layer chromatography (TLC) plate and sprayed with ferric chloride reagent. Frs. 1 (62 g) turned cyan and afterwards passed through the 20-40 μm ODS (25 × 3 cm, approx. 150 g, Ultimate XB-C18) under medium pressure, with a gradient of methanol/water (84:16, 93:7, 97:3, v/v), acquiring 3 fractions (G1-G3, 200 mL each). Then the fractions were repeatedly spotted on a thin-layer chromatography (TLC) plate and sprayed with ferric chloride reagent. G1 (15 g) turned cyan and then was prepared by with ultra-high-performance liquid chromatography (UHPLC), using MeOH -0.1% formic acid different ratio as the mobile phase, with flow rate of 1.0 mL/min, and resulted in compound 1 (40 mg, purity of > 99% with UHPLC, MeOH -0.1% formic acid, 61:39, v/v), compound 2 (30 mg, purity of >98% with UHPLC, MeOH -0.1% formic acid, 58:42, v/v), compound 3 (3 mg, purity of >98% with UHPLC, MeOH -0.1% formic acid, 52:48, v/v), compound 4 (10 mg, purity of >98% with UHPLC, MeOH -0.1% formic acid, 57:43, v/v), compound 5 (4 mg, purity of >97% with UHPLC, MeOH -0.1% formic acid, 52:48, v/v), compound 6 (3 mg, purity of >96% with UHPLC, MeOH -0.1% formic acid, 56:44, v/v).

Oleracone

Assay of scavenging DPPH radicals

An assay of DPPH radical scavenging activity was performed by the modified method described formerly [START_REF] Liu | Hu DPPH radical scavenging activity of ten natural p-terphenyl derivatives obtained from three edible mushrooms indigenous to China[END_REF]. For each compound, a dilution into five different concentrations in methanol was prepared and the methanol was used to zero the spectrophotometer. A methanolic solution of the sample (1 mL) was mixed with a 126.80 μM DPPH (1 mL) dissolved in methanol. The reaction mixture was allowed to stand for 10 min at room temperature, measured at 517 nm, and then followed at 30 min. The DPPH solution (1 mL) added to methanol (1 mL) were measured as the blank control. At the same time, the sample solution (1 mL), with methanol (1 mL) was operated as the control group. BHA (butylated hydroxyanisole) was tested as a positive control as the same series of concentrations as other samples. Each sample was measured three times in parallel, and the percentage of DPPH radical scavenging was calculated in the following way [START_REF] Xu | Antioxidant activity of a water-soluble polysaccharide purified from Pteridium aquilinum[END_REF]:

where, A control is the absorbance of the control group, A sample is the absorbance of the test compound, and A blank is the absorbance of the blank control. The antioxidant capacity was evaluated by the value of IC 50 , and values are expressed as the means ± SD for n = 3.

Assay of anticholinesterase activity

Anticholinesterase activity was determined against AChE, using the modified method according to Ellman [START_REF] Ellman | Featherstone A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF]. The 20 μL sample solutions in methanol diluted into five different concentrations and 15 μL AChE solution in buffer of PBS (pH 8.0, containing 0.1 mol/L Na 2 HPO 4 and NaH 2 PO 4 ) were mixed in wells a 96-well microplate and incubated for 10 min at 37 °C, and 10 μL DTNB in buffer was added. Then 10 μL ATCI was added to initiate the reaction and the absorbance was monitored spectrophotometrically at 405 nm. Eserine, as the same concentration as samples, was used as the positive control. Instead of samples, methanol was included in the blank control. Each sample was measured three times in parallel, and the percentage of inhibition of AChE was calculated using the following formula [START_REF] Dirmenci | Topcu Investigation of anticholinesterase activity of a series of salvia extracts and the constituents of Salvia staminea Nat[END_REF]:Anticholinesterase activity%=Ablank-Asample/Ablank×100%where, A sample is the absorbance of the test compound, and A blank is the absorbance of the blank control. The anticholinesterase activity was evaluated by the value of IC 50 , and values are expressed as the means ± SD for n = 3. The signals at δ H 6.06/δ C 93.6 and δ H 6.07/δ C 94.8 are typical of a tetrasubstituted aromatic A-ring of a flavonoid. Both protons correlate with the methoxyl at δ H 3.79 in NOESY, indicating that the methoxy substituent is in C-7 (at δ C 167.4). The relative position of these two protons was difficult to ascertain. Both correlate with carbons at δ C 102.2 (C-4a) and 167.4 (C-7). Eventually, it was found that proton at δ H 6.06 correlates with carbon at δ C 162.9 (C-8a) while proton at δ H 6.07 correlates with carbon at δ C 163.6 (C-5). These correlations allowed us to assign δ H 6.06/δ C 93.6 at position 8 and δ H 6.07/δ C 94.8 at position 6.

Results and discussion

Compound 1 also contained a CH 2 -CH-CH 2 system identified by the sequence of COSY correlations H-2a, b/H-3/H-9a, b and coupling constants in the system. Both protons H-2 correlate with C-8a at δ C 162.9 through oxygen 1, and also correlate to carbonyl moiety at δ C 198.7 which had to be linked to C-3, forming the C-ring of an isoflavonoid. However, in the isoflavonoids, an aromatic ring is usually linked to C-3. Here, C-9 is inserted between C-3 and an aromatic ring as shown by correlations of protons H-9 with nearby carbons C-1′, C-6′ (and C-2′ for H-9a). Carbons C-1′ to C-6′ describe a disubstituted phenyl moiety as confirmed by the coupling figures and COSY correlations of protons H-3′ to H-6′, along with respective HMBC correlations (Fig. 2). It should be mentioned that H-6′ also correlates to C-9 further confirming the C-9-C-1′ link. The OH signals were attributed based on NOE correlation H-3′/HO-C2′. Finally, the absolute configuration at C-3 was assigned as S on the basis of the positive Cotton effect at 288 nm [START_REF] Adinolfi | Absolute configuration of homoisoflavanones from muscari species[END_REF][START_REF] Dai | Kingston Antiproliferative Homoisoflavonoids and Bufatrienolides from Urginea depressa[END_REF]. (Fig. S41-42, Supporting information).

Overall, compound 1 was identified as the (3S)-5-hydroxy-3-(2-hydroxybenzyl)-7methoxychroman-4-one (or (3S)-5,2′-dihydroxy-7-methoxyhomoisoflavanone) and was given the trivial name oleracone C. An olefinic proton at δ H 8.05 (1H, s), a methylene proton at δ H 3.61 (2H, s), a methoxy group at δ H 3.84 (3H, s), and two hydroxyl groups at δ H 9.66 (1H, s) and 12.76 (1H, s) were also seen. Seventeen carbons were detected in the 13 C NMR and DEPT 135 spectra. These include one carbonyl (δ C 181.8) and 13 other sp 2 carbons, one methylene (δ C 24.4,), one methine (δ C 154.9), and one methoxy group (δ C 56.0) (Table 1). Compound 7 Hz), one methylene with non-equivalent protons at δ H 2.11 (1H, dt, J = 14.1, 2.8 Hz) and 2.18 (1H, dt, J = 14.1, 2.8 Hz), two methine protons at δ H 5.38 (1H, brq, J = 2.5 Hz), 5.56 (1H, brq, J = 1.9 Hz), and two methoxy groups at δ H 3.65 (3H, s) and 3.79 (3H, s). In the 13 C NMR and DEPT 135 spectrum, there were 17 carbon signals, including 12 sp 2 carbons, one methylene carbon (δ C 26.0), two methine carbons (δ C 61.2, 66.6), two methoxy groups (δ C 55.1, 55.6) (Table 1). The COSY and HMBC data, along with comparison with compounds 1 and 2 provide evidence that this compound also contain the same di-and tetrasubstituted aromatic rings (B-and A-ring, respectively, Fig. 2). The methoxy substituents were located based of HMBC correlations between the methoxy groups at δ H 3.79 and 3.66 with C-5 (δ C 158.7) and C-7 (δ C 161.4), respectively. A CH-CH 2 -CH sequence was also identified based on 1 H-1 H couplings and COSY experiment. Unfortunately, it was not possible to detect any HMBC correlations for these protons. However, the methines chemical shifts at δ H 5.38 (H-2) and 5.56 (H-4) indicate that both CH are deshielded presumably by an oxygen and an aromatic ring. Interestingly, H-2 and H-4 are coupled to each other (J ≈ 1.9-2.5 Hz). This is specific to systems constrained in a Wshaped conformation, typical of the 4-O-2′-cycloflavan skeleton. In the closely related 3′hydroxy-5,7-dimethoxy-4-O-2′-cycloflavan isolated from Iris tenuifolia, H-2 and H-4 are reported at δ H 5.34 and 5.62 in dmso-d 6 , respectively [START_REF] Cui | Flavans from Iris tenuifolia and their effects on β-amyloid aggregation and neural stem cells proliferation in vitro Bioorg[END_REF]. These values are very similar to those of compound 3. Overall, it can be concluded that 3 is the 5,7-dimethoxy-4-O-2′cycloflavan. This compound has been obtained in the past by synthesis but its analytical data have not been described [START_REF] Nair | Stereochemistry of cyanomaclurin[END_REF][START_REF] Bhatia | Synthesis of cyanomaclurin-I: synthesis of some model compounds[END_REF]. It is isolated for the first time as a natural product. The circular dichroism spectrum of 3 was compared with the one reported for the (2R,4R)-4′-hydroxy-3′,5′-methyl-6,7-methylenedioxy-4-O-2′-cycloflavan [START_REF] Peng | Chen Two new flavans from the trunk and leaves of Horsfieldia glabra[END_REF]. This compound is the only 4-O-2′-cycloflavan, not hydroxylated in C-3 as in the cyanomaclurin series, for which the CD spectrum has been reported. Compound 3 presented 3 positive maxima at 217, 242 and 281 nm (Fig. S41-42, Supporting information), while the reported CD spectrum had negative Cotton effect at these wavelengths. This indicated that the absolute configuration of compound 3 is 2S and 4S, as represented in Fig. 1 1). Comparison with compounds 1-3 provided straightforward identification of the tetrasubstituted A-ring and the ortho-disubstituted B-ring. However in this case, it was eventually found that 4 was a dihydrochalcone, with both aromatic rings linked by a propane-1-one core. This linkage was identified based on the H-2-H-3 coupling and the HMBC correlations from H-3 (δ H 2.80) to C-1′ (δ C 127.3), C-2′ (δ C 155.3), C-6′ (δ C 129.7), C-2 (δ C 43.5), C-1 (δ C 204.8), and from H-2 (δ H 3.19) to C-1 (δ C 204.8), C-3 (δ C 25.1), and C-1′ (δ C 127.3). The HMBC correlations revealed that the methoxy groups (δ H 3.79) was coupled to the C-4′′ (δ C 165.4), and another methoxy groups (δ H 3.82) was coupled to the C-6′′ (δ C 162.3), which implied that two methoxy groups are affiliated with C-4′′ and C-6′′, respectively (Fig. 2). As a result, compound 4 was determined as the 1-(2-hydroxy-4,6-dimethoxyphenyl)-3-(2-hydroxyphenyl)propan-1-one and was given the trivial name oleracone E.

Compounds 5 and 6 had been described before and were identified as the (2S)-5,2′dihydroxy-7-methoxyflavanone and 2′,4′-dihydroxy-4,6′-dimethoxychalcone respectively, according to the NMR data and comparison with the literature [START_REF] Seidel | Rel)-1β,2α-di-(2,4-dihydroxy-6-methoxybenzoyl)-3β, 4α-di-(4-methoxyphenyl)cyclobutane and other flavonoids from the aerial parts of Goniothalamus gardneri and Goniothalamus thwaitesii[END_REF][START_REF] Jayaprakasam | ChemInform abstract: dihydroechioidinin, a flavanone from Andrographis echioides[END_REF].

Previously, the antioxidant activity of extracts was assayed by using the DPPH radical scavenging method [START_REF] Brandwilliams | Use of a free radical method to evaluate antioxidant activity[END_REF], which is now applied to evaluate the oxidation resistance of the three new compounds. Phenolic compounds are good hydrogen donors and are usually effective DPPH radical quenchers [START_REF] Cao | Prior Antioxidant and prooxidant behavior of flavonoids: structure-activity relationships Free Radic[END_REF]. Indeed, oleracones C (1), D (2) and E (4) were found more effective than reference compound butylated hydroxyanisole (BHA) (Fig. S43-44, Supporting information). Their antioxidant potential was dose-dependent with IC 50 values of 11.7, 13.2 and 23.3 μM, respectively (Table 2). The biological activity profiles were somewhat similar at 30 min, with IC 50 values of 12.0, 13.7 and 23.5 μM, respectively (Table 2). Oleracone D (2) remained the most effective. Overall, the compared radical scavenging activities on DPPH were oleracone D (2)>E ( 4)>C (1)>BHA.

Acetylcholinesterase (AChE) inhibitors, inhibiting the production of choline, play an significant role in the treatment of Alzheimer disease (AD) [START_REF] Akkol | Yeşilada Anticholinesterase and antioxidant effects of the ethanol extract, ethanol fractions and isolated flavonoids from Cistus laurifolius L. leaves[END_REF]. As a result, antioxidant activities of phenolic compounds can be implicated in the neuroprotective effect at the same time [START_REF]Ramassamy Emerging role of polyphenolic compounds in the treatment of neurodegenerative diseases: a review of their intracellular targets[END_REF], of which flavonoids are responsible to be the multipotent agents to fight against AD [START_REF] Ji | Theoretical evaluation of flavonoids as multipotent agents to combat Alzheimer's disease[END_REF]. Consequently, the anticholinesterase activities of three new compounds, oleracone C (1), D (2) and E (3), were determined. All of three new compounds had different degree of inhibitory activities against the AChE (Fig. S45, Supporting Information). Table 2 indicated that although the anticholinesterase activity of compound 2 was lower than that of eserine at the same concentration, it had the highest anticholinesterase activity with IC 50 , the lowest was that of compound 3.

Fig. 1 .

 1 Fig. 1. Chemical structures of compounds isolated from P. oleracea L.

Compound 2

 2 was obtained as yellowish powder, which turned cyan after spraying ferric chloride. Its molecular formula was inferred as C 17 H 14 O 5 based on a molecular ion peak at m/z 299.0911 [M + H] + in positive-ion HR-ESI-MS (calcd. for C 17 H 15 O 5 , 299.0914). The degree of unsaturation of compound 2 is 11. The 1 H NMR spectrum contains six aromatic proton signals including the two protons at δ H 6.38 (1H, d, J = 1.9 Hz) and 6.59 (1H, d, J = 1.9 Hz) attributable to H-6 and H-8 although they appear more downfield than in compound 1. Other aromatic signals are at δ H 6.82 (1H, dd, J = 7.9, 1.1 Hz), 6.70 (1H, brtd, J = 7.4, 1.1 Hz), 7.03 (1H, brtd, J = 7.6, 1.6 Hz), and 7.08 (1H, dd, J = 7.4, 1.6 Hz). These correspond to the sequence of protons H-3′ to H-6′, with chemical shifts almost identical to those of the same protons in 1.

2

 2 was clearly related to 1 with 1 more double bond. The HMBC correlations from H-9 (δ H 3.61) to C-1′ (δ C 124.5), C-2′ (δ C 155.2), C-6′ (δ C 129.8), C-2 (δ C 154.9), C-3 (δ C 121.0), C-4 (δ C 181.8), and from H-2 (δ H 8.05) to C-3 (δ C 121.0), C-4 (δ C 181.8) revealed direct connection of C-3 with C-4, and C-1′ with C-9. HMBC correlations from the CH 3 at δ H 3.84 (3H, s) to C-7 (δ C 165.1) indicated that the methoxy group should be linked to C-7, like in 1 (Fig. 2). As a result, compound 2 was identified as the 5-hydroxy-3-(2-hydroxybenzyl)-7-methoxy-4H-chromen-4-one (or 5,2′dihydroxy-7-methoxy-homoisoflavone) and was given the trivial name oleracone D (2). Compounds 3, [α] D 20 -14 (c 0.01, MeOH), was obtained as yellowish powder. Its molecular formula was identified as C 17 H 16 O 4 based on a molecular ion peak at m/z 285.1118 [M + H] + in its positive-ion HR-ESI-MS spectrum (calcd. for C 17 H 17 O 4 , 285.1121). The degree of unsaturation of the compound 3 is 10. The 1 H NMR spectrum contained six aromatic proton signals at δ H 5.96 (1H, d, J = 2.3 Hz), 6.09 (1H, d, J = 2.3 Hz), 6.78 (1H, brd, J = 8.2 Hz), 6.87 (1H, brtd, J = 7.6, 1.1 Hz), 7.19 (1H, ddd, J = 8.2, 7.3, 1.7 Hz) and 7.35 (1H, dd, J = 7.7, 1.

Table 1 .

 1 and Table S1. in Supplementary material); HR-ESI-MS m/z 299.0930 [M -H] -(calcd. for C 17 H 15 O 5 , 299.0925). UV (CH 3 OH) λmax 286, 254 nm; IR (KBr) v max 3428, 2925, 2853, 1659, 1634, 1596, 1384 cm -1 . 1 H and 13 C NMR data (Table 1. and Table S2. in Supplementary material); HR-ES-IMS m/z 299.0911 [M + H] + (calcd. for C 17 H 15 O 5 , 299.0914). MeOH); 1 H and 13 C NMR data (Table 1. and Table S3. in Supplementary material); HR-ESI-MS m/z 285.1118 [M + H] + (calcd. for C 17 H 17 O 4 , 285.1121). H and 13 C NMR data (Table 1. and Table S4. in Supplementary material); HR-ESI-MS m/z 303.1229 [M + H] + (calcd. for C 17 H 19 O 5 , 303.1227).

	C (1)
	Yellowish-brown powder; [α] D 20 + 0.8 (c 0.1, MeOH); UV (CH 3 OH) λ max 288, 214 nm; IR (KBr) v max 3425, 2928, 1650, 1603, 1506, 1456, 1303, 1231, 1193, 1162 cm -1 . 1 H and 13 C NMR data
	(2.5.2. Oleracone D (2)
	Yellowish powder; 2.5.3. 5,7-Dimethoxy-4-O-2′-cycloflavan (3)
	Yellowish powder; [α] D 20 -14 (c 0.01, 2.5.4. Oleracone E (4)
	Yellowish powder; UV (CH 3 OH) λ max 290, 212 nm; IR (KBr) v max 3446, 2925, 2862 1653, 1559, 1540, 1506, 1459 cm -1 .

1

  Compound 1, [α] D 20 + 0.8 (c 0.1, MeOH), was obtained as yellowish-brown powder turning cyan upon spraying ferric chloride. Its molecular formula was deduced as C 17 H 16 O 5 based on a molecular ion peak at m/z 299.0930 [M-H] -in the HR-ESI-MS spectrum (calcd. for C 17 H 15 O 5 , 299.0925). The degree of unsaturation of compound 1 is 10.

	The 1 H NMR
	spectrum exhibited six aromatic protons at δ H 6.06 (1H, d, J = 2.3 Hz), 6.07 (1H, d, J = 2.3 Hz),
	6.73 (1H, td, J = 7.4, 1.2 Hz), 6.82 (1H, dd, J = 8.4, 1.2 Hz), 7.05 (1H, brtd, J = 7.4, 1.6 Hz), and
	7.06 (1H, brd, J = 7.4 Hz), a methoxy group at δ H 3.79 (3H, s), and two hydroxyl groups at δ H 9.59 (1H, brs) and 12.12 (1H, brs) (Table 1). According to the 13 C NMR and DEPT 135
	spectrum, 17 carbon signals were detected, including 7 quaternary carbons with one
	carbonyl at δ C 198.7, 7 methines, two methylenes (δ C 27.2, 69.4), and one methoxy group
	(δ C 56.0).

.

  Compound 4 was obtained as yellowish powder, which became cyan after spraying Ferric chloride. Its molecular formula was identified as C 17 H 18 O 5 based on a molecular ion peak at m/z 303.1229 [M + H] + in its positive-ion HR-ESI-MS spectrum (calcd. for C 17 H 19 O 5 , 303.1227). The degree of unsaturation of compound 4 is 9. The 13 C NMR and DEPT 135 spectrum indicated that 17 carbon signals, including one carbonyl (δ C 204.8) and 12 other sp 2 carbons, two methylene carbons (δ C 25.1, 43.5), and two methoxy groups (δ C 55.6, 56.0) (Table

	The 1 H NMR spectrum exhibited
	six aromatic proton signals at δ H 6.08 (1H, d, J = 2.3 Hz), 6.09 (1H, d, J = 2.3 Hz), 6.68 (1H, td,
	J = 7.4, 1.0 Hz), 6.78 (1H, dd, J = 8.0, 1.0 Hz), 6.98 (1H, brtd, J = 7.6, 1.6 Hz) and 7.05 (1H, dd,
	J = 7.4, 1.6 Hz), two methylene protons at δ H 2.80 (2H, brt, J = 7.7 Hz), 3.18 (2H, brt,
	J = 7.7 Hz), two methoxy groups at δ H 3.79 (3H, s), 3.81 (3H, s), and two hydroxyl groups at
	δ H 9.57 (1H, s) and 13.59 (1H, s).

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant 81573546).

Conflict of interest statement

The authors declare no competing financial interest.