Flavones and Lignans from the Stems of Wikstroemia scytophylla Diels.

BACKGROUND: The genus Wikstroemia has about 70 species, but only a limited number of species have been studied chemically. Wikstroemia indica has long been used as a traditional crude drug in China. However, there is no report about the bioactivity of Wikstroemia scytophylla.
OBJECTIVE: This paper reports the chemical investigation and biological evaluation of the W. scytophylla.
MATERIALS AND METHODS: The EtOAc extraction of W. scytophylla was isolated using chromatographic methods, and the compounds were analyzed by spectroscopic methods. The in vitro antitumor activities against five human cancer cell lines were performed according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.
RESULTS: The chemical investigation of the stems of W. scytophylla resulted in the isolation of 12 compounds mainly including one biflavone (1), five flavones (2-6) compounds, and six lignans (7-12), in which compound 8 was a new natural product. Compounds 1 and 7-12 were evaluated for their antitumor activities while these compounds showed weak cytotoxicity with the half maximal inhibitory (IC50) values more than 40 μM.
CONCLUSION: All of these compounds were isolated from this plant for the first time, and compounds 2-12 were first reported from genus Wikstroemia, in which compound 8 was a new natural product. Compounds 1 and 7-12 exhibited weak antitumor activities (IC50>40 μM). The chemotaxonomic significance of all the isolations was summarized.
SUMMARY: The chemical investigation of the stems of W. scytophylla resulted in the isolation of 12 compoundsThe 12 compounds including six lignans (7-12), in which compound 8 was a new natural productThe isolated compounds 1 and 7-12 were evaluated for their antitumor activitiesThe chemotaxonomic significance of all the isolations was summarized. Abbreviations used: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC50: Half maximal inhibitory; HL-60: Human leukemia cell line; SMMC-7721: Human hepatocellular carcinoma cell line; A549: Human lung tumor cell line; MCF-7: Human breast cancer cell line; SW480: Human colon cancer cell line; MS: Mass spectrometry; NMR: Nuclear Magnetic Resonance.

INTRODUCTION

The genus Wikstroemia (Thymelaeaceae) comprising approximately 70 species is widely distributed in northern Asia through the Himalayas, Malaysia, Oceania, Polynesia to the Hawaiian Islands.[1] In China, about 44 species have been found mainly in the south of the Yangtze River. Wikstroemia scytophylla Diels is regionally distributed in the provinces of Yunnan, Sichuan, and Tibet in China. There is no report about the bioactivity of W. scytophylla. Wikstroemia indica, the same genus with W. scytophylla, has long been used as a traditional crude drug for the treatment of pneumonia, rheumatism, and bronchitis in China.[2]

Previous phytochemical studies on W. scytophylla, which has been investigated only by our group, have resulted in the isolation and identification of flavones (including biflavones), coumarins, sesquiterpene, and diterpene esters.[34]

MATERIALS AND METHODS

Plant material

The stems of W. scytophylla were collected in May, 2015, in Diqing Tibetan Autonomous Prefecture, Yunnan Province, China, and identified by Professor Liang-Ke Song (Southwest Jiaotong University). Voucher specimen (JHZ-201505) was deposited at the Natural Products Chemistry Laboratory, Southwest Jiaotong University, China.

Extraction and isolation

The dried and powdered stems of W. scytophylla (5.0 kg) were extracted with 95% EtOH under reflux for three times (3 × 15 L). The extract was concentrated and suspended in water followed by successive partition with petroleum ether (3 × 5 L), EtOAc (3 × 5 L), and n-BuOH (3 × 5 L).

The EtOAc extract (380 g) was chromatographed over silica gel (3.8 kg, 200–300 mesh) using a gradient solvent petroleum ether/acetone (100:1-1:1) to afford nine fractions. Fraction 3 (42 g) was subjected to silica gel column using a gradient solvent petroleum ether/EtOAc (20:1-0:1) to afford fractions 3-1 - 3-8. Fraction 3-3 (2.9 g) was separated on Sephadex LH-20 (CHCl3/MeOH 1:1) to give compounds 3 (19 mg) and 7 (15 mg). Fraction 3-4 (14.1 g) gel filtrated on Sephadex LH-20 (CHCl3/MeOH 1:1) to give six subfractions (3-4-1 – 3-4-6). Fraction 3-4-3 (4.4 g) was further purified on repeated RP-18 (MeOH/H2O, 40%–100%) and Sephadex LH-20 (CHCl3/MeOH 1:1) to give compounds 6 (4 mg), 8 (28 mg), and 9 (143 mg). Fraction 3-5 (22.5 g) gel filtrated on Sephadex LH-20 (CHCl3/MeOH 1:1) to give five subfractions (3-5-1 – 3-5-5). Fraction 3-5-3 (13.7 g) was further purified on repeated RP-18 (MeOH/H2O, 40%-100%) and Sephadex LH-20 (CHCl3/MeOH 1:1) to give compounds 2 (5 mg) and 4 (12 mg). Fraction 4 (118 g) was chromatographed on silica gel column using a gradient solvent petroleum ether/EtOAc (15:1-0:1) to give nine fractions (4-1 - 4-9). Fraction 4-4 (27.8 g) was further separated on repeated RP-18 (MeOH/H2O, 40%-100%) and Sephadex LH-20 (CHCl3/MeOH 1:1) to afford compounds 5 (11 mg), 10 (5 mg), and 11 (5 mg). Fraction 4-5 (16.5 g) was separated on silica gel column using a gradient solvent CHCl3/MeOH (30:1-1:1) to give seven fractions (4-5-1 - 4-5-7). Fraction 4-5-1 (302 mg) was further purified on Sephadex LH-20 (CHCl3/MeOH 1:1) to give 12 (2 mg). Further purification of 4-5-6 (1.58 g) was submitted to Sephadex LH-20 (CHCl3/MeOH 1:1) to yield 1 (6 mg).

Cytotoxicity bioassay

The isolated compounds 1 and 7-12 were evaluated for their cytotoxicities against five human cell lines HL-60, SMMC-7721, A549, MCF-7, and SW480, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method[5] with cisplatin (Sigma) and culture solution as positive and negative controls, respectively. Half maximal inhibitory (IC50) values were calculated by Reed and Muench's method.

RESULTS

The structures of compounds 1-12 [Figure 1] were identified as wikstrol B (1),[67] apigenin 7-O-methyl ether (2),[8] apigenin 5, 7-dimethyl ether (3),[9] luteolin 7, 4’-dimethyl ether (4),[10] 5, 7-di-O-methylquercetin(5),[11] 3’-hydroxy-4’, 5, 7-trimethoxy-flavone (6),[12] 3-(1, 3-benzodioxol-5-ylmethyl)-4-[(3, 4-dimethoxyphenyl)methyl]dihydro-, (3S-cis)-2(3H)-furanone (7),[1314] 4-[(R)-1, 3-benzodioxol-5-ylhydroxymethyl]-3-(1, 3-benzodioxol-5-ylmethyl)dihydro-, (3S, 4R)-2(3H)-furanone (8),[1415] (-)-dihydrosesamin (9),[16] phenol, 4, 4’-[(2R, 3S, 4S)-tetrahydro-2-methoxy-3, 4-furandiyl]bis(methylene)]bis[2-methoxy- (10),[17] 4, 4’-dihydroxy-3, 3’, 9-trimethoxy-9, 9’-epoxylignan (11),[17] (+)-1-hydroxypinoresinol (12),[1819] respectively, by analysis of mass spectrometry and nuclear magnetic resonance (NMR) data, and comparison with those in the literature.

Figure 1

Structures of compounds 1-12 isolated from Wikstroemia scytophylla

As a continuation of our previous phytochemical investigation[34] of W. scytophylla, the present study reports a new natural product (8) along with 11 known compounds, including one biflavone (1), five flavones (2-6), and five lignans (7, 9-12). All these compounds were isolated from this plant for the first time, and compounds 2-12 were first reported from the genus Wikstroemia. According to the cytotoxicity bioassay results, compounds 1 and 7-12 exhibited weak antitumor activities (IC50 >40 μM).

DISCUSSION

The chemical constituents of the family Thymelaeaceae especially the genus Daphne and Aquilaria are characterized by terpenoids (sesquiterpenoids, daphnane-type diterpene ester), coumarins, flavonoids (flavones, isoflavones, biflavones), and lignans.[2021222324] The genus Wikstroemia has about 70 species, but only a limited number of species (W. indica, W. chamaedaphne, etc.) have been studied chemically, and Wikstroemia is also characterized by the occurrence of terpenoids, coumarins, flavonoids, and lignans, and the types of these reported compounds are very similar with those in the genus Daphne.[34252627] Compounds 1-12 isolated in the current research were classified as biflavone (1), flavones (2-6), and lignans (7-12). The structural types of these isolates confirm the relationship between W. scytophylla and other species of Wikstroemia. The phytochemical study suggests that there is a close chemotaxonomic relationship between the genus Wikstroemia, Aquilaria, and Daphne in the family Thymelaeaceae. As flavonoids were widely present in W. scytophylla metabolites and isolated as the main type of components in the current study, while terpenoids, coumarins, and lignans were the main bioactive compositions in Thymelaeaceae, lignans could be used as chemotaxonomic markers for the species W. scytophylla according to this study. Moreover, it can also be concluded that lignans could be considered as chemotaxonomic markers of the genus Wikstroemia. The result of the present study also supports the conclusion of Zhang et al.[20] that biflavonoids are characteristic constituents of the Thymelaeaceae family.

SUPPLEMENTARY DATA

Flavones and lignans from the stems of Wikstroemia scytophylla Diels

:

Wikstrol B (1): C30H22O10, yellow powder, [α]20 D-31.4° (c = 0.16, MeOH); 1H NMR (400 MHz, CD3COCD3) δ: 7.56 (2H, d, J = 8.8 Hz, H-10’’, H-14’’), 6.86 (2H, d, J = 8.8 Hz, H-11’’, H-13’’), 6.83 (2H, d, J = 8.6 Hz, H-2’, H-6’), 6.72 (2H, d, J = 8.6 Hz, H-3’, H-5’), 6.38 (1H, d, J = 2.4 Hz, H-8’’), 6.20 (1H, d, J = 2.4 Hz, H-6’’), 6.16 (1H, s, H-6), 4.57 (1H, d, J = 7.8 Hz, H-2), 3.75 (1H, m, H-3), 2.81 (1H, dd, J = 16.0 Hz, 5.2 Hz, H-4a), 2.58 (1H, dd, J = 16.0 Hz, 8.4 Hz, H-4b); 13 C NMR (100 MHz, CD3COCD3) δ: 181.2 (s, C-4’’), 164.8 (s, C-7’’), 163.3 (s, C-2’’), 163.3 (s, C-5’’), 160.2 (s, C-12’’), 158.6 (s, C-8a), 157.7 (s, C-4’), 157.1 (s, C-5), 156.0 (s, C-8a), 154.1 (s, C-7), 131.2 (s, C-1’), 131.1 (d, C-10’’, C-14’’), 129.1 (d, C-2’, C-6’), 125.5 (s, C-9’’), 115.6 (d, C-11’’, C-13’’), 115.5 (d, C-3’, C-5’), 113.7 (s, C-3’’), 104.8 (s, C-4’’a), 100.7 (s, C-4a), 99.3 (s, C-8), 99.3 (d, C-6’’), 96.2 (d, C-6), 94.1 (d, C-8’’), 82.3 (d, C-2), 68.5 (d, C-3), 28.8 (t, C-4).

Apigenin 7-O-methyl ether (2): C16H12O5, yellow powder; 1H NMR (500 MHz, CD3COCD3) δ: 7.96 (2H, d, J = 8.6 Hz, H-2’, H-6’), 7.13 (2H, d, J = 8.6 Hz, H-3’, H-5’), 6.74 (1H, s, H-3), 6.72 (1H, d, J = 1.6 Hz, H-8), 6.32 (1H, d, J = 1.5 Hz, H-6), 3.92 (3H, s, 7-OCH3); 13C NMR (100 MHz, CD3COCD3) δ: 182.5 (s, C-4), 166.4 (s, C-7), 165.2 (s, C-2), 162.9 (s, C-4’), 161.5 (s, C-5), 158.0 (s, C-9), 129.2 (d, C-2’, C-6’), 119.5 (s, C-1’), 116.9 (d, C-3’, -5’), 104.7 (s, C-10), 103.9 (d, C-3), 98.7 (d, C-6), 93.2 (d, C-8), 56.4 (q, 7-OCH3).

Apigenin 5, 7-dimethyl ether (3): C17H14O5, yellow needle crystal; 1H NMR (500 MHz, C5D5N) δ: 7.97 (2H, d, J = 8.8 Hz, H-2’, H-6’), 7.12 (2H, d, J = 8.8 Hz, H-3’, H-5’), 6.97 (1H, s, H-3), 6.71 (1H, d, J = 2.0 Hz, H-8), 6.62 (1H, d, J = 2.0 Hz, H-6), 3.77 and 3.75 (6H, 2s, 2×-OCH3); 13C NMR (125 MHz, C5D5N) δ: 182.9 (s, C-4), 166.0 (s, C-7), 164.3 (s, C-2), 163.1 (s, C-4’), 162.8 (s, C-5), 158.2 (s, C-9), 128.7 (d, C-2’, C-6’), 115.0 (d, C-3’, -5’), 106.0 (s, C-10), 104.8 (d, C-3), 98.7 (d, C-6), 93.0 (d, C-8), 56.0 and 55.6 (q, 7-OCH3).

Luteolin 7, 4’-dimethyl ether (4): C17H14O6, brown needle crystal; 1H NMR (500 MHz, CD3COCD3) δ: 7.59 (1H, d, J = 8.5 Hz, 1.8 Hz, H-6’), 7.52 (1H, d, J = 1.8 Hz, H-2’), 7.14 (1H, d, J = 8.6 Hz, H-5’), 6.73 (1H, d, J = 1.8 Hz, H-8), 6.69 (1H, s, H-3), 6.32 (1H, d, J = 1.7 Hz, H-6), 3.95 and 3.93 (6H, 2s, 2×-OCH3); 13C NMR (150 MHz, CD3COCD3) δ: 183.2 (s, C-4), 166.7 (s, C-7), 165.1 (s, C-5), 163.0 (s, C-2), 158.8 (s, C-9), 151.9 (s, C-4’), 147.9 (s, C-3’), 124.8 (s, C-1’), 119.9 (d, C-6’), 113.8 (d, C-5’), 112.5 (d, C-2’), 106.1 (s, C-10), 104.8 (d, C-3), 98.8 (d, C-6), 93.3 (d, C-8), 56.5 and 56.4 (q, 2×-OCH3).

5, 7-Di-O-methylquercetin (5): C17H14O7, yellow crystal; 1H NMR (500 MHz, C5D5N) δ: 8.10 (1H, d, J = 8.5 Hz, H-6’), 7.23 (1H, s, H-2’), 7.06 (1H, d, J = 8.6 Hz, H-5’), 6.70 (1H, d, J = 1.7 Hz, H-8), 6.33 (1H, d, J = 1.7 Hz, H-6), 3.85 and 3.74 (6H, 2s, 2×-OCH3); 13C NMR (125 MHz, C5D5N) δ: 169.2 (s, C-4), 161.3 (s, C-7), 159.3 (s, C-5), 158.8 (s, C-9), 144.8 (s, C-2, -4), 143.1 (s, C-3’), 124.6 (s, C-3), 120.9 (s, C-1’), 118.0 (d, C-6’), 117.5 (d, C-5’), 114.4 (d, C-2’), 108.7 (s, C-10), 96.1 (d, C-6), 94.9 (d, C-8), 56.4 and 55.6 (q, 2×-OCH3).

3’-Hydroxy-4’, 5, 7-trimethoxy-flavone (6): C18H16O6, brown needle crystal; 1H NMR (500 MHz, C5D5N) δ: 8.03 (1H, d, J = 8.5 Hz, 1.8 Hz, H-6’), 7.91 (1H, d, J = 2.3 Hz, H-2’), 7.30 (1H, d, J = 8.6 Hz, H-5’), 7.03 (1H, s, H-3), 6.63 (1H, d, J = 2.2 Hz, H-8), 6.61 (1H, d, J = 2.2 Hz, H-6), 3.80, 3.76 and 3.60 (9H, 3s, 3×-OCH3); 13C NMR (125 MHz, C5D5N) δ: 182.8 (s, C-4), 165.9 (s, C-7), 164.7 (s, C-5), 162.7 (s, C-2), 158.1 (s, C-9), 152.1 (s, C-4’), 148.6 (s, C-3’), 129.0 (d, C-6’), 124.6 (s, C-1’), 118.9 (d, C-5’), 117.0 (d, C-2’), 112.2 (s, C-10), 103.8 (d, C-3), 98.6 (d, C-6), 92.8 (d, C-8), 60.4, 56.5 and 56.4 (q, 3×-OCH3).

3-(1, 3-benzodioxol-5-ylmethyl)-4-[(3, 4-dimethoxyphenyl)methyl]dihydro-, (3S-cis)-2(3H)-Furanone (7): C21H22O6, colorless oil, [α]20 D+67.8° (c = 0.11, MeOH); 13C NMR (100 MHz, CDC13) δ: 178.5 (s, C-9), 149.0 (s, C-3’), 147.84 (s, C-3), 147.80 (s, C-4’), 146.4 (s, C-4), 131.3 (s, C-1), 130.4 (s, C-1’), 122.2 (d, C-6), 120.6 (d, C-6’), 111.6 (d, C-5’), 111.2 (d, C-5), 109.4 (d, C-2’), 108.1 (d, C-2), 101.0 (t, C-1’’), 71.2 (t, C-9’), 55.9 (q, 3’-OCH3), 55.7 (q, 3-OCH3), 46.4 (d, C-8), 41.2 (d, C-8’), 38.2 (t, C-7’), 34.7 (t, C-7).

4-[(R)-1, 3-benzodioxol-5-ylhydroxymethyl]-3-(1, 3-benzodioxol-5-ylmethyl) dihydro-, (3S, 4R)-2(3H)-Furanone (8): C20H18O7, colorless needle crystal, [α]20 D+54.7° (c = 0.23, CHCl3); 1H NMR (500 MHz, C5D5N) δ: 7.07 (1H, s, H-2’), 6.93 (1H, d, J = 7.8 Hz, H-5’), 6.86 (1H, d, J = 8.0 Hz, H-5), 6.78 (1H, d, J = 7.8 Hz, H-6’), 6.73 (1H, d, J = 7.9 Hz, H-2), 6.01 ~ 5.91 (4H, m, 2×-OCH2O-), 4.96 (1H, d, J = 4.9 Hz, H-7’), 4.31 ~ 4.20 (2H, m, H-9’), 3.26 ~ 3.04 (3H, m, H-7, H-8), 2.78 (1H, m, H-8’); 13C NMR (125 MHz, C5D5N) δ: 179.6 (s, C-9), 148.3 (s, C-3’), 148.1 (s, C-3), 147.3 (s, C-4’), 146.7 (s, C-4), 138.1 (s, C-1’), 132.4 (s, C-1), 123.2 (d, C-6), 119.6 (d, C-6’), 110.3 (d, C-5), 108.4 (d, C-5’), 108.3 (d, C-2), 107.0 (d, C-2’), 101.6 and 101.4 (2t, 2×-OCH2O-), 74.3 (d, C-7’), 69.7 (t, C-9’), 46.1 (d, C-8), 43.5 (d, C-8’), 35.8 (t, C-7).

(-)-Dihydrosesamin (9): C20H20O6, colorless powder, [α]20 D+21.3° (c = 0.12, MeOH); 1H NMR (400 MHz, CDC13) δ: 6.80 ~ 6.58 (6H, m, H-2’, H-5’, H-5, H-6’, H-2), 5.91 ~ 5.89 (4H, m, 2×-OCH2O-), 4.75 (1H, d, J = 6.1 Hz, H-7), 4.00 ~ 3.62 (4H, m, H-9’, H-9), 2.80 (1H, dd, J = 13.5 Hz, 5.0 Hz, H-7’b), 2.45 (1H, m, H-7’a), 2.45 (1H, dd, J = 13.4 Hz, 10.8 Hz, H-8), 2.26 (1H, m, H-8’); 13C NMR (100 MHz, CDC13) δ: 179.6 (s, C-9), 148.3 (s, C-3’), 148.1 (s, C-3), 147.3 (s, C-4’), 146.7 (s, C-4), 138.1 (s, C-1’), 132.4 (s, C-1), 123.2 (d, C-6), 119.6 (d, C-6’), 110.3 (d, C-5), 108.4 (d, C-5’), 108.3 (d, C-2), 107.0 (d, C-2’), 101.6 and 101.4 (2t, 2×-OCH2O-), 74.3 (d, C-7’), 69.7 (t, C-9’), 46.1 (d, C-8), 43.5 (d, C-8’), 35.8 (t, C-7).

Phenol, 4, 4’-[(2R, 3S, 4S)-tetrahydro-2-methoxy-3, 4-furandiyl]bis (methylene)]bis[2-methoxy- (10): C21H26O6, colorless oil, [α]20 D+71.4° (c = 0.15, MeOH); 1H NMR (400 MHz, CDCl3) δ: 6.82 (1H, d, J = 8.0 Hz, H-5’), 6.77 (1H, d, J = 7.2 Hz, H-5), 6.62 (1H, d, J = 1.6 Hz, H-2), 6.57 (1H, dd, J = 8.0, 1.6 Hz, H-6’), 6.53 (1H, d, J = 1.6 Hz, H-2’), 6.44 (1H, dd, J = 7.2, 1.6 Hz, H-6), 4.74 (1H, d, J = 1.0 Hz, H-9’), 4.04 (1H, t, J = 8.2 Hz, H-9b), 3.83 (3H, s, 3-OCH3), 3.82 (3H, s, 3’-OCH3), 3.67 (1H, t, J = 8.2 Hz, H-9a), 3.34 (3H, s, 9’-OCH3), 2.72 (1H, dd, J = 13.1, 6.9 Hz, H-7’b), 2.57 (1H, m, H-7b), 2.55 (1H, m, H-7a), 2.42 (1H, dd, J = 13.1, 6.9 Hz, H -7’a), 2.15 (1H, m, H-8), 2.15 (1H, m, H-8’); 13C NMR (125 MHz, CDC13) δ: 146.5 (s, C-4’), 146.3 (s, C-4), 144.0 (s, C-3), 143.8 (s, C-3’), 132.9 (s, C-1’), 132.2 (s, C-1), 121.4 (d, C-6), 121.3 (d, C-6’), 114.3 (d, C-2’), 114.2 (d, C-5), 111.5 (d, C-2), 111.1 (d, C-5’), 105.6 (d, C-9’), 72.3 (t, C-9), 55.9 (q, 3-OCH3), 55.8 (q, 3’-OCH3), 54.5 (q, 9’-OCH3), 52.2 (d, C-8’), 43.3 (d, C-8), 39.3 (t, C-7’), 33.5 (t, C-7).

4, 4’-dihydroxy-3, 3’, 9-trimethoxy-9, 9’-epoxylignan (11): C21H26O6, colorless oil, [α]20 D-23.7° (c = 0.14, MeOH); 1H NMR (400 MHz, CDCl3) δ: 6.83 (1H, d, J = 8.0 Hz, H-5), 6.81 (1H, d, J = 7.8 Hz, H-5’), 6.68 (1H, dd, J = 7.8, 1.5 Hz, H-6’), 6.67 (1H, d, J = 1.5 Hz, H-2’), 6.64 (1H, d, J = 8.0 Hz, H-6), 6.60 (1H, s, H-2), 4.66 (1H, d, J = 3.6 Hz, H-9’), 3.99 (1H, t, J = 6.6 Hz, H-9b), 3.86 (3H, s, 3-OCH3), 3.86 (3H, s, 3’-OCH3), 3.59 (1H, t, J = 6.6 Hz, H-9a), 3.32 (3H, s, 9’-OCH3), 2.76 (1H, m, H-7’b), 2.73 (1H, m, H-7b), 2.52 (1H, m, H -7’a), 2.46 (1H, m, H-7a), 2.39 (1H, m, H-8’), 2.01 (1H, m, H-8); 13C NMR (125 MHz, CDC13) δ: 146.5 (s, C-4’), 146.4 (s, C-4), 144.0 (s, C-3), 143.8 (s, C-3’), 132.5 (s, C-1’), 131.6 (s, C-1), 121.6 (d, C-6), 121.2 (d, C-6’), 114.1 (d, C-2’), 114.0 (d, C-5), 111.1 (d, C-2), 110.9 (d, C-5’), 110.1 (d, C-9’), 72.2 (t, C-9), 55.7 (q, 3-OCH3), 55.7 (q, 3’-OCH3), 54.7 (q, 9’-OCH3), 52.4 (d, C-8’), 45.8 (d, C-8), 39.3 (t, C-7’), 38.7 (t, C-7).

(+)-1-Hydroxypinoresinol (12): C20H22O7, yellow powder, [α]20 D+33.6° (c = 0.10, MeOH); 1H NMR (400 MHz, CD3COCD3) δ: 7.16 (1H, d, J = 8.0 Hz, H-5’), 7.11 (1H, d, J = 2.0 Hz, H-2’), 7.08 (1H, dd, J = 8.0, 1.9 Hz, H-6’’), 6.92 (1H, d, J = 8.0 Hz, H-5’’), 6.91 (1H, dd, J = 8.0, 2.0 Hz, H-6’), 6.88 (1H, d, J = 1.9 Hz, H-2’’), 4.88 (1H, s, H-2), 4.56 (1H, d, J = 6.8 Hz, H-6), 4.31 (1H, m, H-8a), 3.87 (1H, dd, J = 8.8, 7.5 Hz, H-4a), 3.85 (3H, s, 3-OCH3), 3.82 (3H, s, 3’-OCH3), 3.78 (1H, d, J = 8.9, 4.1 Hz, H-4b), 3.70 (1H, m, H-8b), 3.50 (1H, m, H-5); 13 C NMR (100 MHz, CD3COCD3) δ: 151.8 (s, C-3’’), 148.5 (s, C-3’), 147.9 (s, C-4’, C-4’’), 132.9 (s, C-1’’), 129.5 (s, C-1’), 120.5 (d, C-6’), 119.2 (d, C-6’’), 115.15 (d, C-5’’), 115.06 (d, C-5’), 111.4 (d, C-2’), 110.8 (d, C-2’’), 91.1 (s, C-1), 88.4 (d, C-2), 86.6 (d, C-6), 73.7 (t, C-8), 71.4 (t, C-4), 61.8 (d, C-5), 56.2 and 56.1 (q, -OCH3).

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China (NSFC) (No. 81374022, 81274184, and 81473337), the research project of the Administration of Traditional Chinese Medicine of Sichuan (2016Q037), the Fundamental Research Funds for the Central Universities (the Medicine thematic research projects, 2682016YXZT05), the Foundation of Science and Technology Agency of Chengdu (2015-HM01-00311-SF), and the Student Research Training Program (SRTP, 2016089 and 201610613073). The authors are grateful to the members of the analytical group of the Chengdu Institute of Biology, and the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, for the spectral measurements.

Conflicts of interest

There are no conflicts of interest.