New Phenolic Compounds from Coreopsis tinctoria Nutt. and Their

Article pubs.acs.org/JAFC

New Phenolic Compounds from Coreopsis tinctoria Nutt. and Their Antioxidant and Angiotensin I‑Converting Enzyme Inhibitory Activities Wei Wang,†,∥ Wei Chen,†,§,∥ Yingshi Yang,†,§ Tianxing Liu,† Haiyan Yang,*,§ and Zhihong Xin*,† †

Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic China § College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi, People’s Republic China ABSTRACT: Three new phenolic compounds, coretinphenol (1), coretincone (2), and coretinphencone (3), were isolated from the buds of Coreopsis tinctoria Nutt., together with nine known compounds, including butein (4), okanin (5), isoliquiritigenin (6), maritimetin (7), taxifolin (8), isookanin (9), marein (10), sachalinoside B (11), and 2-phenylethyl-β-Dglucoside (12). The chemical structures of these compounds were elucidated by extensive spectroscopic analysis and on the basis of their chemical reactivity. This work represents the first recorded example of the isolation of compounds 1−3, 6, 7, 9, 11, and 12 from C. tinctoria. Compounds 5−9 showed strong diphenyl(2,4,6-trinitrophenyl)iminoazanium (DPPH) radical-scavenging activity, with IC50 values of 3.35 ± 0.45, 9.6 ± 2.32, 4.12 ± 0.21, 6.2 ± 0.43, and 7.9 ± 0.53 μM, respectively. Compounds 2 and 8 exhibited angiotensin I-converting enzyme inhibitory activity, with IC50 values of 228 ± 4.47 and 145.67 ± 3.45 μM, respectively. The activities of phenolic compounds isolated from C. tinctoria support the medicinal use of this plant in the prevention of cardiovascular diseases. KEYWORDS: Coreopsis tinctoria, structural identification, flavonoids, antioxidant activity, angiotensin I-converting enzyme inhibitory activity

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INTRODUCTION Cardiovascular diseases (CVDs) are the leading cause of death worldwide, causing 17.3 million deaths per year.1,2 It has been estimated that by 2030, there will be more than 23.6 million deaths annually resulting from CVDs all over the world.2 Hypertension, one of the major risk factors related to CVDs, may be induced by many causes, for example, the imbalance of the renin angiotensin aldosterone system (RAAS), the kallikrein kinin system, and the sympathetic nervous system and genetic influences.3 Among these causes, the angiotensin I-converting enzyme (ACE) plays a significant role in the RAAS, by converting the precursor angiotensin I into angiotensin II, a potent vasoconstrictor that is responsible for increasing blood pressure.4 Therefore, finding potential ACE inhibitors from diverse natural resources has become a major target.5 In particular, edible and medicinal plants have played important roles in functional food and drug development and continue to represent a promising source of new and interesting ACE inhibitors against hypertension. Coreopsis tinctoria Nutt., which belongs to the Asteraceae/ Compositae family, is a small, glabrous, aromatic annual plant distributed all over the world.6,7 In China, it grows in the Karakorum Mountains at altitudes above 3000 m in southern Xinjiang. It is known as “snow chrysanthemum” or “snow tea” locally8 and is traditionally used not only as a tea-like beverage but also as a folk medicine for the treatment of hypertension and hyperlipidemia in Uyghur folk medicine.9 According to historical records, the decoction of C. tinctoria has been described by Huá Shòu as a traditional Chinese formula for diabetes as early as during the Yuan Dynasty (1271−1368 AD).6 In North America © XXXX American Chemical Society

and Romania, C. tinctoria was commonly cultivated in a variety of garden settings as an ornamental plant,10 whereas the indigenous people of North America employed it as a therapeutic agent for several diseases, for example, diarrhea, internal pain, and bleeding, to strengthen blood, and as an emetic.6 In Portugal, an infusion of two cups per day of C. tinctoria flowering tops, known as “Estrelas-do-Egipto”, has been traditionally used to control diabetes.10 Previous studies have shown that C. tinctoria contains a diverse range of bioactive phytochemicals, including flavonoids,6,11 phenolics,12 phenylpropanoids,13 polyacetylene glycosides,8 and sterols,14 with flavonoids being the major phytochemical present.15 Although several investigations have been conducted on C. tinctoria, the precise nature of all of its bioactive components in antioxidant and antihypertensive activities remains unclear. It is reported that the alcoholic extract of C. tinctoria buds showed the strongest antioxidant and antihypertensive effects.9 Furthermore, in our preliminary studies, the methanol extract of C. tinctoria buds exhibited significant DPPH radical-scavenging activity and ACE inhibitory activity. It was for this reason that methanol was selected as an extraction solvent for the buds of C. tinctoria. An investigation of the chemical constituents in the bioactive extract led to the isolation of three new phenolic glycosides, together with nine other known compounds, and Received: September 7, 2014 Revised: December 14, 2014 Accepted: December 17, 2014

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DOI: 10.1021/jf504289g J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry

Fraction C (2.5 g) was separated into two subfractions (C-1 and C-2) by silica gel CC using petroleum ether/acetone (100:1, v/v) as an eluent. Fraction C-1 was subsequently purified by silica gel CC using petroleum ether/acetone (50:1, v/v) as an eluent to afford compound 6 (9.3 mg). Purification of fraction C-2 by silica gel CC eluting with a cyclohexane/EtOAc (10:1, v/v) gave compound 7 (11.3 mg). Fraction D (5.8 g) was purified by normal-phase CC over silica gel, eluting with a petroleum ether/acetone eluent (15:1, v/v), to produce two subfractions, D-1 and D-2. Fraction D-1 was subjected to CC over silica gel with a petroleum ether/acetone eluent (100:1, v/v) to give compound 8 (10.5 mg), and fraction D-1-1. Fraction D-1-1 was subsequently purified over a Sephadex LH-20 column with a CHCl3/ MeOH eluent (1:1, v/v) to give compound 9 (13.4 mg). Fraction D-2 was purified by reversed-phase chromatography over silica gel using a stepwise gradient elution of MeOH/H2O (80:20−0:100, v/v) to give fraction D-2-1, which was further purified using a Sephadex LH-20 column with a CHCl3/MeOH eluent (1:1, v/v) to give compound 10 (51.9 mg). Fraction E (2.0 g) was subjected to CC over silica gel using a stepwise gradient elution of petroleum ether/acetone (100:1 to 0:100, v/v) to provide two subfractions, E-1 and E-2. Fraction E-1 was subsequently purified by CC over a silica gel column using a petroleum ether/acetone eluent (10:1, v/v) to afford faction E-1-1, which was purified using a Sephadex LH-20 column with a CHCl3/MeOH eluent (1:1, v/v) to give compound 11 (8.9 mg). Fraction E-2 was purified by silica gel CC, eluting with petroleum ether/acetone (60:1, v/v) to give compound 12 (21.5 mg). Acid Hydrolysis of the Saponins and Determination of the Absolute Configuration of the Monosaccharides. The assay used to determine the absolute configuration of the monosaccharides was performed as described in the literature with minor modifications.15 Briefly, a solution of the pure compound (2 mg) in 2 M HCl (2 mL) was heated at 85 °C for 15 h, and the resulting mixture was then evaporated under vacuum to give a residue, which was repeatedly distilled to dryness from H2O under vacuum to produce a neutral residue. Then, the residue obtained and 2 mg of L-cysteine methyl ester hydrochloride were dissolved in 1 mL of anhydrous pyridine, and the resulting mixture was stirred at 60 °C for 1 h. A 3:1 mixture of HMDS−TMCS (hexamethyldisilazane−trimethylchlorosilane) was then added (300 μL), and the solution was stirred for 30 min. Hexane (3 mL) and water (1 mL) were then added to the solution. The hexane layer was dried over anhydrous Na2SO4 and subjected to GC-MS analysis under the following conditions: capillary column, HP-5MSi (30 m × 0.25 mm, with a 0.25 μm film, Agilent, USA); detection, FID; injection temperature, 250 °C; initial temperature, 160 °C, raised to 250 °C at 15 °C/min; final temperature maintained at 250 °C for 10 min. Assay of DPPH Radical Scavenging. The DPPH radicalscavenging assay was performed according to the method described by Rivero-Pérez et al.16 with slight modifications. Briefly, 1 mL of 0.2 mM DPPH ethanol solution was added to 2.5 mL of the sample solutions at different concentrations, and the resulting mixtures were shaken vigorously in the absence of light for 30 min. The absorbance values were then measured at 517 nm. Vitamin E (VE) was employed as a positive control. The antioxidant activity of the test compounds was expressed as IC50, with the IC50 being defined as the concentration of test compounds required to inhibit the formation of DPPH radicals by 50%. All of the samples were analyzed in triplicate. Measurement of Inhibitory Activity toward ACE. The measurement of inhibitory activity toward ACE was performed according to the method described by Cushman and Cheung,17 which was slightly modified. The enzyme was added last to initiate the reaction, and tubes were incubated, usually for 10 min at 37 °C. One milliliter of ethyl acetate was added to the tubes and mixed by vortex for 15 s to extract hippuric acid from the acidified solution. The hippuric acid was redissolved in 3 mL of water, and the amount formed was determined by its absorbance at 228 nm. Captopril was employed as a positive control. The inhibitory activity of the test compounds was expressed as IC50, with the IC50 being defined as the concentration of test compounds required to inhibit the ACE activity by 50%. All of the samples were analyzed in triplicate.

evaluations of the antioxidant and ACE inhibitory activities of these compounds were performed. Herein, we describe the experimental details of the separation process, as well as provide information pertaining to the elucidation of the structures of these compounds based on their spectroscopic properties and chemical reactivity.

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MATERIALS AND METHODS

Chemicals. Dimethyl sulfoxide-d6 (DMSO-d6) and CDCl3 were obtained from Merck (Darmstadt, Germany). Diphenyl(2,4,6trinitrophenyl)iminoazanium (DPPH), ACE (EC 3.4.15.1), hippuric acid, and captopril were purchased from Sigma-Aldrich (St. Louis, MO, USA). All of the chemicals and solvents used in the current study were of analytical grade. Materials. The aerial parts of C. tinctoria were purchased in September 2012 from the Beiyuanchun farmers market in Urumchi city of the Xinjiang Uygur Autonomous Region, China. The herbariums of C. tinctoria were deposited in the Key Laboratory of Food Processing and Quality Control, Nanjing Agricultural University, under number XJ8126. Thin-layer chromatography (TLC) analysis was performed on plates precoated with silica gel GF254 (10−40 μm). Column chromatography (CC) was performed over silica gel (300−400 mesh, Qingdao Marine Chemical Factory, Qingdao, China), reversed phase C18 (octadecylsilyl, ODS) silica gel (Silicycle, 50 μm, Canada), or Sephadex LH-20 (Sigma-Aldrich, St. Louis, MO, USA). Instruments. UV spectra were recorded on a Beckman DU640 spectrophotometer (Beckman Coulter, Beijing, China). IR spectra were taken from KBr disks on a Nicolet Nexus 470 spectrophotometer (Thermo Scientific, Beijing, China). All of the 1H and 13C NMR spectra were recorded on Bruker Avance 300, 400, and 500 spectrometers (Bruker BioSpin GmbH, Beijing, China), using tetramethylsilane (TMS) as an internal standard. Two-dimensional NMR spectra include correlation spectroscopy (COSY), heteronuclear singular quantum correlation (HSQC), and heteronuclear multiple-bond correlation (HMBC). The chemical shifts in the NMR spectra were recorded as δ values. Electrospray ionization mass spectrometry (ESI-MS) analyses were measured on a Q-Tof Ultima Global GAA076 LC mass spectrometer (Waters Asia, Ltd., Singapore). Extraction and Isolation. Dried buds of C. tinctoria (3 kg) were homogenized using a Polytron homogenizer prior to extracting three times with 80% acetone (1:6, v/v) at ambient temperature for 24 h. The resulting solution was then filtered, and the filtrate was combined and concentrated under vacuum at 30 °C until approximately 90% of the solvent had been evaporated. The remaining solution was then extracted sequentially with equal volumes of ethyl acetate (EtOAc) and methanol (three times), and the methanol solution was collected and concentrated under vacuum to give the methanol extract (15.8 g). The methanol extract was separated into five fractions (A−E) by normal-phase silica gel column chromatography (CC) (100 g of silica gel, 300−400 mesh) using a stepwise gradient elution of cyclohexane/ EtOAc/MeOH (from 100:0:0 to 0:100:0 to 0:0:100, v/v/v). Fracton A (2.1 g) was separated into two subfractions (A-1 and A-2) on a normal-phase silica gel column using a stepwise gradient elution of petroleum ether/acetone (from 100:0 to 0:100, v/v). Fraction A-1 was then passed through a Sephadex LH-20 column with a CHCl3/MeOH eluent (1:1, v/v) to yield compound 1 (3.5 mg). Fraction A-2 was purified on a silica gel column with a cyclohexane/EtOAc eluent (50:1, v/v) to produce compound 2 (3.7 mg). Fraction B (3.4 g) was also purified by silica gel column chromatography with a cyclohexane/EtOAc eluent (50:1, v/v) to give three subfractions, B-1, B-2, and B-3. Fraction B-1 was passed through a Sephadex LH-20 column using a CHCl3/MeOH eluent (1:1, v/v) to yield compound 3 (3.6 mg). Fraction B-2 was purified by silica gel column chromatography with a cyclohexane/EtOAc eluent (95:1, v/v) to give fraction B-2-1, which was separated through a Sephadex LH-20 column using a CHCl3/MeOH eluent (1:1, v/v) to produce compound 4 (8.3 mg). Fraction B-3 was purified by column chromatography over silica gel with a cyclohexane/EtOAc eluent (200:1, v/v) to afford compound 5 (12.3 mg). B


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Journal of Agricultural and Food Chemistry Table 1. NMR Spectral Data for Compound 1 in DMSO-d6 at 400 (1H) and 100 MHz (13C) H (J, Hz)

position 1 2 3 3-CH3 4 5 6 1′ 2′ 3′ 4′ 5′ 6′ 6′-CH3 7′ 1″ 2″ 3″ 4″ 5″ 6″

2.50 (s, 3H) 6.70 (s, 1H) 1.59 (d, J = 6.84 Hz, 3H) 4.07 (m, 1H) 1.95 (dd, J = 7.68 Hz, 15.32 Hz, 2H) 2.35 (m, 2H) 5.84 (t, J = 7.24 Hz, 1H) 1.98 (s, 3H) 4.34 (s, 2H) 4.89 (d, J = 7.08 Hz, 1H) 3.99 (m, 1H) 3.99 (m, 1H) 3.99 (m, 1H) 3.80 (m, 1H) 4.30 (m, 1H), 4.22 (m, 1H)

HSQC 137.0 148.4 108.7 8.0 152.8 102.1 138.1 21.8 30.2 37.8 25.4 124.6 135.0 12.8 67.6 106.7 70.3 76.9 74.4 76.9 61.6

Statistical Analysis. All of the assay results reported in the present work were analyzed according to Duncan’s multiple-comparison test (p < 0.05), using version 18.0 of the SPSS software package. The resulting data were presented as the mean ± standard deviation.

HMBC (H→C)

1

H−1H COSY

C-2, C-3, C-4 C-3, C-4, C-6, C-2′ C-2′, C-3′, C-6 C-5, C-6 C-1′, C-2′, C-4′, C-5′, C-6

H-1′, H-3′ H-3′ H-4′

C-5′, C-6′, C-7′ C-4′, C-5′ C-1

H-2″ H-2″ H-3″, H-5″ H-5″

sample of the same material that had been prepared in the same manner. The results revealed that the acid hydrolysate of pure compound 1 contained a D-glucose derivative. This result was confirmed through a comparison of the retention time of this derivative with that of the authentic D- (tR, 20.254) and L-glucose derivatives (tR, 20.705), respectively, with both samples providing a retention time of 20.254 min. Consequently, the structure of compound 1 was determined to be 2,4-dihydroxy-3methyl-6-(7-hydroxy-6-methylhep-5-en-2-yl)-β-D-glucopyranoside and informally named coretinphenol (Figure 1). Compound 2 was isolated as a yellow amorphous powder, and its molecular formula was determined to be C21H22O10 on the basis of HRESIMS analysis of the pseudomolecular ion peak at m/z 433.1142 [M − H]− (calculated as 433.1135). The UV spectrum of compound 2 revealed strong absorption bands at 212, 267, and 383 nm. The IR spectrum displayed strong absorption bands at 3421, 3254, 1656, 1399, and 1062 cm−1, which are characteristic of flavonoid-glycoside type structures. Analysis of its 1D NMR data suggested the presence of an aglycone moiety and a sugar unit (Table 2). The aglycone resonances showed a β-unsaturated ketone olefinic proton at δ 7.78 (d, J = 8.88 Hz, 1H, H-β), an ABX spin system at δ 6.60 (d, J = 2.44 Hz, 1H, H-3′), 6.65 (dd, J = 2.44 Hz, 8.92 Hz, 1H, H-5′), and 8.22 (d, J = 8.96 Hz, 1H, H-6′), and an ABCX spin system at δ 7.40 (d, J = 2.08 Hz, 1H, H-2), 7.78 (d, J = 8.88 Hz, 1H, H-4), 6.93 (d, J = 8.20 Hz, 1H, H-5), and 7.26 (dd, J = 2.08 Hz, 8.16 Hz, 1H, H-6), as well as a broad proton singlet at δ 13.50 (s, 1H) attributable to 2′-OH. The 13C NMR of compound 2 revealed the presence of 21 carbon signals, with 6 of these being assigned to the sugar moieties, 15 to the aglycone including 12 aromatic ring carbons, 2 olefinic carbons, and a characteristic carbonyl group at δ 192.3. These data revealed that the structure of compound 2 closely resembled that of compound 4, which has been previously isolated from this plant.18 A direct comparison of the 1H and 13C NMR data of these two compounds revealed that they share the same skeleton. The 1H NMR spectrum of compound 2 revealed that the resonance signals corresponding to the H-4 at δ 7.78 and α-OH protons in compound 2 had been

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RESULTS AND DISCUSSION Elucidation of the Structures of the New Phenolics. Compound 1 was isolated as a yellow amorphous powder. Its molecular formula was determined to be C21H32O9 on the basis of HRESIMS analysis of the pseudomolecular ion peak at m/z 451.1952 [M + Na]+ (calculated as 451.1944), indicating six degrees of unsaturation. The UV spectrum of compound 1 revealed three strong absorption bands at 207, 230, and 280 nm. The IR spectrum displayed strong absorption bands at 3421, 1650, 1399, and 1047 cm−1, corresponding to hydroxyl, olefinic, alkyl, and glycosidic functionalities, respectively. The 1H NMR and 13C NMR spectra of compound 1 revealed the presence of one benzene ring, one double bond, and one sugar unit in the molecule. Analysis of the 1H−1H COSY, HSQC, and HMBC spectra of compound 1 allowed for the complete assignment of the 1H and 13C NMR spectral data (Table 1). The sugar unit sequence from an anomeric proton H-1″ to oxymethylene proton H-6″ through oxymethine protons H-2″, H-3″, H-4″, and H-5″, as well as H-1′ to H-5′, was established by 1H−1H COSY. HMBC analysis of compound 1 displayed several key correlations, including the 3-CH3 group with the C-2, C-3, and C-4 carbons, H-1′ with the C-2′, C-3′, and C-6 carbons, H-2′ with the C-5 and C-6 carbons, and the 6′-CH3 group with the C5′, C-6′, and C-7′ carbons. Key correlations from H-1″ to C-1 were observed in the HMBC experiments, indicating that the sugar moiety was connected directly to the C-1 position of the benzene ring. The anomeric configuration of the pyranosyl sugar unit was identified as a β-glucopyranosyl group on the basis of its 3 JH‑1″, H‑2″ coupling constant of 7.08 Hz. Acid hydrolysis of compound 1 with 2 M HCl liberated aglycone and D-glucose, which were identified by gas chromatography−mass spectrometry (GC-MS) analysis of the corresponding trimethylsilyl Lcysteine derivative and a direct comparison with an authentic C


Article


Figure 1. Chemical structures of the 12 compounds isolated from the methanol extract of C. tinctoria: 1, coretinphenol; 2, coretincone; 3, coretinphencone; 4, butein; 5, okanin; 6, isoliquiritigenin; 7, maritimetin; 8, taxifolin; 9, isookanin; 10, marein; 11, sachalinoside B; 12, 2-phenylethyl-β1 1 D-glucoside. Key H− H COSY and HMBC correlations for compounds 1, 2, and 3 are also shown.

replaced by a hydroxyl group and one olefinic proton at δ 7.65 in compound 4. This difference was also supported by the presence of a methine carbon at δ 117.3 (C-4) and a quaternary carbon at δ 145.5 (C-α) in the 13C NMR spectrum of compound 2. These signals were replaced by the carbon signals at δ 148.8 and 117.3 in the spectrum of compound 4, respectively. This assignment was further supported by HMBC correlations from the 2′-OH to C1′, C-2′, and C-3′ and H-β to C-1, C-2, C-6, and C-β′. The full structure of compound 2 was further confirmed by 1H−1H COSY, HSQC, and HMBC experiments (Table 2). The anomeric proton of the glucopyranosyl sugar moiety at δ 5.15 (d, J = 7.52 Hz, 1H, H-1″) coupling to the C-4′ at δ 163.9 suggested the sugar moiety connected directly to C-4′ of aglycone. The anomeric configuration of the sugar unit was determined as a β-glucopyranosyl group from its 3JH‑1″, H‑2″ coupling constant of 7.52 Hz. Following the hydrolysis of compound 2 with 2 M HCl and its subsequent trimethylsilylation

for GC-MS analysis, the retention time of the sugar was found to be the same as that of an authentic sample of D-glucose. On the basis of this evidence, the structure of compound 2 was characterized as α,3,2′-trihydroxy-4′-O-β-D-glucopyranosylchalcone and informally named coretincone (Figure 1). Compound 3 was isolated as a colorless amorphous powder. Its molecular formula was established as C14H18O9 on the basis of HRESIMS analysis of the pseudomolecular ion peak at m/z 329.0881 [M − H]− (calculated as 329.0873), indicating six degrees of unsaturation. The UV spectrum showed strong absorption bands at 217, 283, and 343 nm. The IR spectrum displayed strong absorption bands at 3415, 1638, 1508, and 1074 cm−1, corresponding to hydroxyl, carbonyl, aromatic, and glycosidic functionalities, respectively. The 1H and 13C NMR spectra were typical of a glycosidic type compound. The 1H NMR spectrum revealed the presence of an AB spin system at δ 7.39 (d, J = 9.05 Hz, 1H) and 6.75 (d, J = 8.95 Hz, 1H), an D


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Journal of Agricultural and Food Chemistry Table 2. NMR Spectral Data for Compound 2 in DMSO-d6 at 400 (1H) and 100 MHz (13C) position 1 2 3 4 5 6 1′ 2′ 2′-OH 3′ 4′ 5′ 6′ α β β′ 1″ 2″ 2″-OH 3″ 3″-OH 4″ 4″-OH 5″ 6″

H (J, Hz)

HSQC 127.2 115.2 148.5 117.3 115.5 122.7

7.40 (d, J = 2.08 Hz, 1H) 7.78 (d, J = 8.88 Hz, 1H) 6.93 (d, J = 8.20 Hz, 1H) 7.26 (dd, J = 2.08 Hz, 8.16 Hz, 1H)

1

HMBC (H→C)

H−1H COSY

C-3, C-6, C-β C-2, C-5, C-6 C-1, C-3 H-5

115.0 166.1 13.50 (s, 1H) 6.60 (d, J = 2.44 Hz, 1H)

103.8 163.9 108.1 131.9 145.5 145.3 192.3 100.1 73.6

6.65 (dd, J = 2.44 Hz, 8.92 Hz, 1H) 8.22 (d, J = 8.96 Hz, 1H) 7.78 (d, J = 8.88 Hz, 1H) 5.15 (d, J = 7.52 Hz, 1H) 3.51 (m, 1H) 4.76 (d, J = 4.08 Hz, 1H) 3.57 (m, 1H) 4.48 (d, J = 3.92 Hz, 1H) 3.49 (m, 1H) 4.40 (d, J = 4.32 Hz, 1H) 3.57 (m, 1H) 3.74 (m, 1H), 3.97 (m, 1H)

C-1′, C-2′, C-3′ C-1′, C-5′ C-1′, C-4′ C-2′, C-4′, C-β′

H-5′

C-1, C-2, C-6, C-β′ C-4′

H-2″

C-1″, C-3″

H-2″ H-2″, H-4″ H-3″

77.1 70.3

H-4″ H-4″ H-5″

76.9 61.6

anomeric proton at δ 4.87 (d, J = 7.15 Hz, 1H), and a methyl at δ 2.57 (s, 3H). The 13C NMR spectrum of compound 3 revealed the presence of 14 carbon signals, with 8 of these signals being assigned to the aglycone and 6 to the sugar moieties. The signals at δ 150.5, 134.2, and 151.1, connecting to three oxygen atoms, were attributed to the C-1, C-2, and C-3 carbons, respectively, and a signal at δ 203.8, corresponding to a carbonyl group, was assigned to the C-7 carbon. This assignment was further confirmed by HMBC correlations from H-5 to C-1, C-3, and C-7, from H-6 to C-1, C-2, and C-4, and from H-8 to C-4 and C7. The full structure of compound 3 was further confirmed by 1 H−1H COSY, HSQC, and HMBC experiments (Table 3). Key correlations from H-1′ to C-1 were observed in the HMBC experiments, indicating that the sugar unit was connected directly to the C-1 position of the aromatic ring. The anomeric configuration of the sugar unit was determined as a βglucopyranosyl group from its 3JH‑1′, H‑2′ coupling constant of 7.15 Hz. The β-glucopyranosyl group for compound 3 was also identified as D-glucose by the same procedures as for compounds 1 and 2. Consequently, the structure of compound 3 was characterized as 2,3-dihydroxy-4-formoxyl-1-O-β-D-glucopyranoside and informally named coretinphencone (Figure 1). Nine known compounds (Figure 1) were identified as butein (4),18 okanin (5),19 isoliquiritigenin (6),18 maritimetin (7),20 taxifolin (8),21 isookanin (9),22 marein (10),11 sachalinoside B (11),23 and 2-phenylethyl-β-D-glucoside (12),24 respectively, by comparing their physical and spectroscopic data with those reported in the literature. Antioxidant Activity of the Pure Compounds Isolated from C. tinctoria. The antioxidant activities of the 12 pure compounds were tested using the DPPH radical-scavenging assay. The results of these experiments are shown in Figure 2A. DPPH radical-scavenging activity of compounds 1−12 ranged

Table 3. NMR Spectral Data for Compound 3 in DMSO-d6 at 400 (1H) and 100 MHz (13C) 1

position 1 2 3 4 5 6 7 8 1′ 2′ 3′ 4′ 5′ 6′

H (J, Hz)

7.39 (d, J = 9.05 Hz, 1H) 6.75 (d, J = 8.95 Hz, 1H) 2.57 (s, 3H) 4.87 (d, J = 7.15 Hz, 1H) 3.34 (m, 1H) 3.36 (m, 1H) 3.17 (t, J = 8.95 Hz, 1H) 3.30 (m, 1H) 3.47 (m, 2H) 3.71 (d, J = 11.75 Hz, 1H)

HSQC

HMBC (H→C)

150.5 134.2 151.1 115.4 122.0

C-1, C-3, C-7

106.5

C-1, C-2, C-4

203.8 26.8 100.9

C-4, C-7 C-1

73.1 77.2 69.6 75.8 60.6 60.6

H−1H COSY

H-6

H-2′

C-1′ H-2′, H-4′ H-4′ H-5′

from 13.2 to 89.4% at the concentration of 100 μM. Compounds 5−9 displayed powerful DPPH radical-scavenging activity compared with the positive control vitamin E, followed by compounds 1−4 and 10, whereas compounds 11 and 12 had the lowest activity. These compounds were further tested to calculate their IC50 values at different concentrations ranging from 1 to 100 μM, and the results are shown in Table 4. The IC50 values of compounds 5−9 were 3.35 ± 0.45, 9.6 ± 2.32, 4.12 ± 0.21, 6.2 ± 0.43, and 7.9 ± 0.53 μM, respectively, and were much lower than E


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Table 4. IC50 Values of the 12 Compounds Isolated from the Methanol Extract of C. tinctoria and Positive Control for DPPH Radical-Scavenging Activity and ACE Inhibitory Activity IC50 (μM)

a

compd

DPPH assay

ACE inhibitory assay

1 2 3 4 5 6 7 8 9 10 11 12 VEa captopril

92.51 ± 1.17 33.49 ± 0.47 51.25 ± 0.51 61.24 ± 0.41 3.35 ± 0.45 9.6 ± 2.32 4.12 ± 0.21 6.2 ± 0.43 7.9 ± 0.53 48.35 ± 0.36 >100 >100 24.73 ± 0.97

>300 228 ± 4.47 >300 >300 >300 >300 >300 145.67 ± 3.45 >300 >300 >300 >300 >300 0.009 ± 2.32

VE, vitamin E.

determined to be 0.009 ± 2.32 μM, similar to the value previously reported in the literature.29 Previous studies have demonstrated that flavonoids have the potential to inhibit ACE activity in vitro by developing complexes with metal ions, which is an active center of ACE. Therefore, the metal ions of ACE are reduced and ACE activity decreases.30,31 It has been reported that the ability of flavonoids to chelate metal ions varies depending on the position of the hydroxyl group in the molecular structure, where a hydroxyl group attached to the 3,5- and 3′,4′positions are crucial to the inhibition of ACE activity.32,33 The structure of compound 8 is consistent with this characteristic; therefore, compound 8 showed potent ACE inhibitory activity. NMR and ESI-MS Spectroscopic Data. Coretinphenol (1): yellow amorphous powder; 1H NMR and 13C NMR data, see Table 1; ESI-MS m/z 451.1952 [M + Na]+. Coretincone (2): yellow amorphous powder; 1H NMR and 13 C NMR data, see Table 2; ESI-MS m/z 433.1142 [M − H]−. Coretinphencone (3): colorless amorphous powder; 1H NMR and 13C NMR data, see Table 3; ESI-MS m/z 329.0881 [M − H]−. Butein (4): yellow amorphous powder; 1H NMR (300 MHz, acetone-d6, TMS) δ 7.27 (d, J = 1.71 Hz, 1H, H-2), 6.82 (d, J = 8.13 Hz, 1H, H-5), 7.20 (dd, J = 1.74, 8.19 Hz, 1H, H-6), 6.27 (d, J = 2.22 Hz, 1H, H-3′), 6.40 (d, J = 2.16, 8.82 Hz, 1H, H-5′), 8.14 (d, J = 8.91 Hz, 1H, H-6′), 7.65 (s, 2H, H-α, β); 13C NMR δ 126.6 (C-1), 115.7 (C-2), 145.5 (C-3), 148.8 (C-4), 115.7 (C-5), 122.3 (C-6), 113.0 (C-1′), 164.8 (C-2′), 102.5 (C-3′), 165.6 (C4′), 108.0 (C-5′), 132.7 (C-6′), 117.3 (C-α), 144.6 (C-β), 191.4 (C-β′); ESI-MS m/z 271.1 [M − H]−. Okanin (5): yellow amorphous powder; 1H NMR (400 MHz, DMSO-d6, TMS) δ 7.26 (d, J = 1.95 Hz, 1H, H-2), 6.82 (d, J = 8.13 Hz, 1H, H-5), 7.20 (dd, J = 2.01, 8.22 Hz, 1H, H-6), 6.44 (d, J = 8.88 Hz, 1H, H-5′), 7.68 (d, J = 8.22 Hz, 1H, H-6′), 7.65(s, 2H, H-α, β); 13C NMR δ 126.2 (C-1), 115.7 (C-2), 145.5 (C-3), 148.8 (C-4), 115.6 (C-5), 122.2 (C-6), 113.3 (C-1′), 153.5 (C2′), 132.4 (C-3′), 152.5 (C-4′), 107.6 (C-5′), 117.3 (C-6′), 122.2 (C-α), 144.4 (C-β), 191.9 (C-β′); ESI-MS m/z 287.1 [M − H]−. Isoliquiritigenin (6): yellow amorphous powder; 1H NMR (400 MHz, acetone-d6, TMS) δ 7.77 (dd, J = 2.44, 6.64 Hz, 2H, H-2, 6), 6.96 (d, J = 8.64 Hz, 2H, H-3, 5), 6.50 (dd, J = 2.44, 8.88

Figure 2. DPPH radical-scavenging activity (A) and ACE inhibitory activity (B) of the isolated compounds at concentrations of 100 and 400 μM, respectively. Coretinphenol (1), coretincone (2), coretinphencone (3), butein (4), okanin (5), isoliquiritigenin (6), maritimetin (7), taxifolin (8), isookanin (9), marein (10), sachalinoside B (11), and 2phenylethyl-β-D-glucoside (12). Each value has been presented as the mean ± standard deviation of three experiments. (a−i) Results with a different letter differ significantly (p < 0.05).

those of vitamin E; these results are consistent with the values previously reported.22,25−28 ACE Inhibitory Activity of the Pure Compounds Isolated from C. tinctoria. The ACE inhibitory activities of the 12 pure compounds at the concentration of 400 μM were assessed, and the results are reported in Figure 2B. Among the pure compounds isolated from C. tinctoria, compound 8 exhibited strong ACE inhibitory activity, followed by compounds 2, 4−7, 9, and 10, whereas other compounds had weak ACE inhibitory activity. To further characterize the ACE inhibitory effect of these compounds, all of the compounds at different concentrations, ranging from 10 to 400 μM, were tested. Compounds 2 and 8 in particular possessed ACE inhibitory activity with IC50 values of 228 ± 4.47 and 145.67 ± 3.45 μM, respectively, whereas other compounds exhibited no inhibitory effect on ACE (Table 4). The IC50 value of captopril was F


Article


2H, H-8), 4.36 (d, J = 7.72 Hz, 1H, H-1′), 2.95 (m, 1H, H-2′), 3.39 (m, 1H, H-3′), 3.40 (m, 1H, H-4′), 3.41 (m, 1H, H-5′), 3.68 (m, 1H, H-6′α), 3.85 (m, 1H, H-6′β); 13C NMR δ 139.0 (C-1), 128.2 (C-2, 6), 128.9 (C-3, 5), 126.0 (C-4), 36.0 (C-7), 70.0 (C8), 103.5 (C-1′), 73.9 (C-2′), 76.6 (C-3′), 70.8 (C-4′), 77.0 (C5′), 62.1 (C-6′); ESI-MS m/z 283.3 [M − H]−. Purification of the methanol extract of C. tinctoria allowed for the isolation of three new phenolics, coretinphenol (1), coretincone (2), and coretinphencone (3), and nine known compounds, including butein (4), okanin (5), isoliquiritigenin (6), maritimetin (7), taxifolin (8), isookanin (9), marein (10), sachalinoside B (11), and 2-phenylethyl-β-D-glucoside (12). The structures of these compounds were elucidated using a combination of spectroscopic methods and chemical analysis. To the best of our knowledge, this is the first report concerning the isolation of compounds 1−3, 6, 7, 9, 11, and 12 from C. tinctoria. Compounds 5−9 exhibited strong antioxidant activity against DPPH radical, and compounds 2 and 8 showed ACE inhibitory activity. It is worth mentioning, however, the bioactivities of these compounds in vitro are not necessarily equal to their actual beneficial effects in vivo. These mainly depend on the bioaccessibility and bioavailability in vivo, where they are mostly mixed with biomacromolecule such as carbohydrates, lipids, and proteins to form the complex matrix. Several factors interfere with the real effects of a compound, such as food source and chemical interactions with other phytochemicals and biomolecules present in the food matrix.34 Further studies should be carried out to assess the bioactivities of these compounds in animal experiments, as well as to validate their real effects in the human body in the future. In conclusion, 12 compounds have been identified from the buds of C. tinctoria, and 8 of these compounds have been isolated for the first time. In particular, three of these compounds have been identified as novel phenolics: coretinphenol (1), coretincone (2), and coretinphencone (3). The findings in the current research may explain, in part, the antioxidant function and antihypertensive effect of C. tinctoria as a tea beverage and a folk medicine. Further studies are warranted to confirm the antihypertensive effect of C. tinctoria in vivo and elucidate the mechanism of ACE inhibition.

Hz, 1H, H-3′), 6.38 (dd, J = 2.24, 8.88 Hz, 1H, H-5′), 8.16 (d, J = 8.80 Hz, 1H, H-6′), 7.84 (d, J = 11.96 Hz, 2H, H-α, β); 13C NMR δ 126.6 (C-1), 130.9 (C-2, 6), 115.8 (C-3, 5), 160.1 (C-4), 113.6 (C-1′), 164.7 (C-2′), 102.8 (C-3′), 166.7 (C-4′), 107.7 (C-5′), 132.4 (C-6′), 117.3 (C-α), 144.2 (C-β), 191.9 (C-β′); ESI-MS m/z 255.1 [M − H]−. Maritimetin (7): yellow amorphous powder; 1H NMR (500 MHz, acetone-d6, TMS) δ 5.42 (dd, J = 2.84, 12.88 Hz, 1H, H-2), 2.67 (dd, J = 2.88, 16.80 Hz, 1H, H-3), 3.04 (dd, J = 12.88, 16.76 Hz, 1H, H-3), 7.33 (d, J = 8.60 Hz, 1H, H-5), 6.62 (d, J = 8.64 Hz, 1H, H-6), 7.06 (d, J = 1.80 Hz, 1H, H-2′), 6.88 (d, J = 8.80 Hz, 1H, H-5′), 6.89 (dd, J = 1.76, 8.80 Hz, 1H, H-6′); 13C NMR δ 145.9 (C-2), 182.4 (C-3), 116.4 (C-4), 112.2 (C-5), 155.4 (C-6), 130.6 (C-7), 154.6 (C-8), 115.0 (C-9), 113.0 (C-10), 123.9 (C1′), 118.8 (C-2′), 146.3 (C-3′), 148.3 (C-4′), 115.6 (C-5′), 124.9 (C-6′); ESI-MS m/z 287.1 [M − H]−. Taxifolin (8): yellow amorphous powder; 1H NMR (400 MHz, acetone-d6, TMS) δ 5.05 (d, J = 11.44 Hz, 1H, H-2), 4.64 (d, J = 11.44 Hz, 1H, H-3), 5.96 (d, J = 2.16 Hz, 1H, H-6), 6.00 (d, J = 2.16 Hz, 1H, H-8), 7.08 (d, J = 2.08 Hz, 1H, H-2′), 6.88 (d, J = 8.08 Hz, 1H, H-5′), 6.94 (dd, J = 2.08, 8.16 Hz, 1H, H-6′); 13C NMR δ 83.6 (C-2), 72.2 (C-3), 197.3 (C-4), 166.9 (C-5), 96.1 (C-6), 164.1 (C-7), 95.1 (C-8), 163.8 (C-9), 100.6 (C-10), 128.8 (C-1′), 114.8 (C-2′), 144.8 (C-3′), 145.6 (C-4′), 114.8 (C-5′), 119.9 (C-6′); ESI-MS m/z 303.1 [M − H]−. Isookanin (9): yellow amorphous powder; 1H NMR (300 MHz, DMSO-d6, TMS) δ 7.41 (s, 1H, H-2′), 7.36 (d, J = 8.46 Hz, 1H, H-6′), 7.10 (d, J = 8.46 Hz, 1H, H-4), 6.84 (d, J = 8.34 Hz, 1H, H-5′), 6.72 (d, J = 8.10 Hz, 1H, H-5), 6.58 (s, 1H, H-10); 13C NMR δ 145.9 (C-2), 182.4 (C-3), 116.4 (C-4), 112.2 (C-5), 155.4 (C-6), 130.6 (C-7), 154.6 (C-8), 115.0 (C-9), 113.0 (C10), 123.9 (C-1′), 124.9 (C-2′), 115.6 (C-3′), 148.3 (C-4′), 146.3 (C-5′), 118.8 (C-6′); ESI-MS m/z 285.1 [M − H]−. Marein (10): yellow amorphous powder; 1H NMR (500 MHz, DMSO-d6, TMS) δ 7.29 (d, J = 2.05 Hz, 1H, H-2), 6.81 (d, J = 8.15 Hz, 1H, H-5), 7.22 (dd, J = 2.10, 8.25 Hz, 1H, H-6), 6.77 (d, J = 9.20 Hz, 1H, H-5′), 7.73 (d, J = 9.20 Hz, 1H, H-6′), 13.12 (s, 1H, 2′-OH), 4.90 (d, J = 7.30 Hz, 1H, H-1″), 3.33 (m, 1H, H2″), 3.20 (m, 1H, H-3″), 3.39 (m, 1H, H-4″), 3.50 (dd, J = 5.95, 11.85 Hz, 1H, H-5″), 3.49 (dd, J = 5.95, 11.85 Hz, H, H-6″), 3.73 (dd, J = 2.00, 11.85 Hz, 1H, H-6″), 7.66(s, 2H, H-α, β); 13C NMR δ 126.1 (C-1), 115.7 (C-2), 145.5 (C-3), 148.9 (C-4), 115.8 (C-5), 122.5 (C-6), 115.9 (C-1′), 152.4 (C-2′), 134.4 (C3′), 150.4 (C-4′), 106.3 (C-5′), 121.4 (C-6′), 101.0 (C-1″), 73.2 (C-2″), 69.8 (C-3″), 75.8 (C-4″), 77.3 (C-5″), 60.5 (C-6″), 145.9 (C-α), 117.4 (C-β), 192.6 (C-β′); ESI-MS m/z 449.0 [M − H]−. Sachalinoside B (11): colorless amorphous powder; 1H NMR (500 MHz, acetone-d6, TMS) δ 4.94 (dd, J = 1.60, 10.80 Hz, 1H, H-1α), 5.21 (dd, J = 1.28, 17.40 Hz, 1H, H-1β), 6.01 (dd, J = 10.80, 17.40 Hz, 1H, H-2), 1.24 (s, 3H, 3-CH3), 4.06 (t, J = 7.00 Hz, 1H, H-4), 1.77 (m, 2H, H-5), 1.85 (m, 1H, H-6α), 1.99 (m, 1H, H-6β), 1.23 (s, 3H, H-8), 1.20 (s, 3H, H-9), 4.60 (d, J = 7.70 Hz, 1H, H-1′), 3.10 (t, J = 8.40 Hz, 1H, H-2′), 3.39 (d, J = 8.35 Hz, 1H, H-3′), 3.29 (m, 1H, H-4′), 3.29 (m, 1H, H-5′), 3.64 (m, 1H, H-6′α), 3.80 (m, 1H, H-6′β); 13C NMR δ 111.3 (C-1), 145.5 (C-2), 79.0 (C-3), 84.2 (C-4), 38.3 (C-5), 27.5 (C-6), 83.5 (C-7), 23.8 (C-8), 22.4 (C-9), 97.9 (C-1′), 75.2 (C-2′), 78.1 (C3′), 71.8 (C-4′), 77.2 (C-5′), 63.1 (C-6′); ESI-MS m/z 331.2 [M − H]−. 2-Phenylethyl-β-D-glucoside (12): colorless amorphous powder; 1H NMR (500 MHz, acetone-d6, TMS) δ 7.18−7.30 (m, 5H, H-2, 3, 4, 5, 6), 3.23 (t, J = 8.56 Hz, 2H, H-7), 4.06 (m,

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AUTHOR INFORMATION

Corresponding Authors

*(H.Y.) E-mail: [email protected]. *(Z.X.) Phone/fax: +86 25 8439 5618. E-mail: xzhfood@njau. edu.cn. Author Contributions ∥

W.W. and W.C. contributed equally to this work and should be considered co-first authors. Funding

This work was financially supported by the Chinese National Natural Science Fund (Grant 31260377), the science and technology program of Urumqi city (Grant Y121120008), the Fundamental Research Funds for the Central Universities (Grant KYZ201118), the project funded by special funds of agro-product quality safety risk assessment of Ministry of Agriculture of the People’s Republic of China (GJFP2014011), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions. Notes

The authors declare no competing financial interest. G


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New Phenolic Compounds from Coreopsis tinctoria Nutt. and Their

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