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Chemical Constituents from the Root Bark of Rodgersia sambucifolia

Hao-Bin Hu* ( Hong Cao ( ), Yu-Feng Jian ( ) and Xu-Dong Zheng ( ), )

Department of Chemistry, Longdong University, Qingyang 745000, P.R. China

A new demethylacetovanillochromene glycoside, 2,2-dimethyl-2H-(8-hydroxy-6-acetyl)-[2,3-b] pyran-8-O-b-D-apiofuranosyl-(16)-b-D-glucopyranoside (1), together with twenty-three known compounds, have been isolated from the root bark of Rodgersia sambucifolia. These known compounds include two diterpenoids, three flavonoids, two catechins, four lignans, two benzenoids, two isocoumarins, two steroids and six monoterpene glycosides, which were determined by means of spectral analyses. Keywords: Rodgersia sambucifolia; Saxifragaceae; Root bark; Demethylacetovanillochromene glycoside.

INTRODUCTION There are about 5 species and 3 variations in the genus Rodgersia (Saxifragaceae), which are mainly distributed in East Asia. Some of them are used as traditional medicines which have anti-bacterial, anti-virus, antalgic, anti-inflammatory, hemostatic, anti-rheumatic and antitussive activities.1,2 Previous studies have indicated that tannins, monoterpenoids and related polyphenols are the main constituents in this genus.3 Rodgersia sambucifolia (Chinese name xinanguidengqing) is a traditional Chinese folk medicine for treating rheumatism, diarrhoea and having a strong effect on antiphlogistic, analgesic, and astringent conditions and on stopping bleeding, etc. It is mainly distributed in Yunnan, Guizhou and Sichuan provinces of China.4 However, very little is known about its chemical constituents.5 To systematically investigate the chemical constituents, we examined the root bark of R. sambucifolia and finally isolated twenty-four compounds, including a new one (1), from the methanolic extract. The present paper deals with the isolation and structural identification of the new compound.

RESULTS AND DISCUSSION The methanolic extract of the root bark of R. sambucifolia was fractionated to provide twenty-four compounds. Compound 1 was obtained as white amorphous powder from acetone and fluoresced under UV lamp. The FABMS spectrum exhibited a molecular ion peak at m/z 513

[M+H]+, and the molecular formula was determined as C24H32 O12 by high-resolution FAB-MS at m/z 512.1904 (calcd. for C24H32O12, 512.1894), giving 1 an unsaturation index of 9. The Molisch test showed it to be a glycoside, and this was also indicated by its IR spectrum which exhibited the absorption bands at 3018 (Ar-H), 1658 (>C=O), 1610, 1595, 1485 (aromatic ring), 1088, 1070, 1036 (pyranose C-H), 1270, 1126 and 880 (vinyl ether), 1370 and 1384 (gem-dimethyl), and 870 cm-1 (tetrasubstituted aryl). The 13C NMR and DEPT spectra of 1 clearly exhibited 24 carbon signals (3 CH3, 3 CH2, 11 CH, 7 C). The 1H NMR signals at the range of 3.10 to 5.30 and eleven 13C NMR signals at the range of 64 to 112 (dC 104.6, 75.4, 78.1, 71.2, 77.2, 68.2 for glucose unit, and dC 111.1, 77.2, 80.1, 75.5, 64.9 for apiose unit) suggested the existence of disaccharides. Besides signals for sugars, thirteen additional signals assigned to the aglycone moiety, including an acetyl (dC 196.8 and 25.7) and a gem-dimethylchromene ring (dC 125.8, 115.8 for olefinic carbons, dC 120.9, 131.2, 116.7, 146.7, 158.9 and 105.7 for aromatic carbons, dC 76.8 for saturated quaternary carbon, and 25.7 2 for gem-dimethyl carbons) were recognized in 13C NMR and DEPT (90 and 135). The 1H NMR spectrum of 1 also revealed an orthocoupled doublets at dH 5.64 and 6.62 (1H each, d, J = 10.0 Hz, olefonic protons) and 6H singlet at dH 1.46 (3H, s, dimethyl protons), indicating the presence of a 2,2-dimethylchromene system.6 Moreover, the 1H NMR spectrum showed that no long range coupling existed between any of the chromenic and aryl protons except for 2H meta-coupled protons at dH 7.21 and 7.42 (1H each, d, J = 2.0 Hz, aro-

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matic protons) flanked the acetyl function (dH 2.48, 3H, s), indicating 8 position of 1 is substituted. The 1H and 13 C NMR spectra of the aglycone showed a very close similarity with those of acetovanillochromene,7 the only differences being the presence of the signals for the HO-8 of the aglycone instead of the signals for the MeO-8 of acetovanillochromene. Thus, the aglycone moiety of 1 was presumed to be demethylacetovanillochromene previously reported from Tithonia diversifolia.8 The acid hydrolysis of compound 1 gave an aglycone and two sugars identified as D-glucose and D-apiose on the basis of co-TLC with authentic sample, showing the presence of D-glucose and D-apiose unit. In addition, it was also deduced from the FAB-MS spectral observation of 381 [M+H-132]+ and 219 [M+H-132-162]+ fragment ions, arising from the elimination of a glucose and an apiose unit, further indicating the apiose was terminal sugars, and glucose was attached to aglycone. The 1H NMR spectrum showed signals at dH 5.28 (1H, d, J = 2.1 Hz, H-1, api. anomeric H) and 5.07 (1H, d, J = 7.8 Hz, H-1 , glu. anomeric H) due to anomeric protons of two sugars. The configuration of the glucopyranosyl unit was deduced to be b from the J value (7.8 Hz) of the anomeric proton, and of the apiofuranosyl unit to be b by compairing the 13C NMR spectra data of 1 with those of a-D-(dC 104.5) and b-Dapiofuranosides (dC 111.5), respectively.9 The HMBC experiment of 1 showed long-range correlations (Table 1) between the H-1 (dH 5.28) of apiose with the C-6 (dC 68.2) of glucose as well as between the H-6 (dH 3.72) of glucose with the C-1 (dC 111.1) of apiose, suggesting the linkage of apiose-(16)-glucose. Additionally, the HMBC correlation between the H-1 (dH 5.07) of glucose and the C-8 (dC 146.7) suggested that the disaccharide chain was located at C-8 of the aglycone. Easy hydrolysis showed that the glycoside linkage was C-O-C type, and the 13C NMR data also support this conclusion. Based on the above observations, compound 1 was determined as 2,2-dimethyl-2H-(8-hydroxyl-6-acetyl)-[2,3b] pyran-8-O-b-D-apiofuranosyl-(16)-b-D-glucopyranoside. The known compounds included three flavonoids [5,4-dihydroxy-7,8-[(3,4-dihydro-3,4-dihydroxy)-2, 2-dimethylpyran]-flavone (2),10 5,4-dihydroxy-3,5-dimethoxy-6,7-(2,2-dimethylpyran)-flavone (3),11 quercetin (4)12], two diterpenoids [14a-hydroxy-7a-ethoxy11,16-diketo-apian-8-en-(20,6)-olide (5),13 7a-hydroxy-

11,16-diketo-apian-8,14(15)-dien-(20,6)-olide (6)13], two catechins [(+)-catechin (7),14 procyanidin B-2 monogallate ( 8)15 ], four lignans [(7S,8R)-2,4-dihydroxy-7,3 -epoxy8,4-oxyneolign-7-ene (9),16 (7S,8S)-2,4-dihydroxy-7,3epoxy-8,4 -oxyneolign-7 -ene (10),16 (7S,8R)-2,4-dihydroxy-7,3-epoxy-8,4-oxyneolign-7-ine (11),16 (7S,8S)2,4-dihydroxy-7,3-epoxy-8,4-oxyneolign-7-ine (12)16], two benzenoids [p-hydroxybenzoic acid (13),17 methyl paraben (14)18], two isocoumarins [bergenin (15),19 7-methoxy bergenin (16)20], two steroids [ergosterol (17),21,22 stigmast-5-en-3b-ol (18)20,23], six monoterpene glycosides [(E)-3,7-dimethyl-1-O-[a-L-rhamnopyranosyl-(16)-bD-glucopyranosyl]-oct-2-en-7-ol (19),24 (E)-3,7-dimethyl1-O-[a-L-arabinofuranosyl-(16)-b-D-glucopyranosyl]oct-2-en-7-ol (20),25 geranyl-1-O-a-L-arabinofuranosyl(1 6) - b -D-glucopyranoside (21),26 geranyl-1-O-a -Lrhamnopyranosyl-(1 6) - b-D-glucopyranoside (22),26 geranyl-1-O-b-D-xylopyranosyl-(16)-b-D-glucopyranoside (23),27 geranyl-1-O-a-L-arabinopyranosyl-(16)-bD-glucopyranoside (24)28]. These known compounds were identified by comparison of relevant data (UV, IR, NMR, MS) with the literature values or with authentic samples. In addition to compound 1, two known compounds 2 and 3 have been isolated for the first time from the Rodgersia species.

EXPERIMENTAL SECTION General Experimental Procedures Melting points were determined on an XT-4A melting point apparatus and are uncorrected; UV spectra on a Shimadzu UV-300 (double beam) spectrophotometer; IR spectra (KBr disks) were obtained on an Alpha-Centari FT-TR spectrometer; 1H and 13C NMR spectra were scanned on a Bruker AM-500 spectrometer operating at 500 and 125 MHz (chemical shifts in d downfield from TMS internal standard), and mass spectra on a VG Auto Spec-3000 spectrometer; Elemental analysis was carried out on a 1106 elemental analysis apparatus. Silica gel (100-200, 200-300 mesh) used for column chromatography and GF254 for TLC were from Qingdao Marine Chemical Factory, China. Gel sephadex LH-20 and RP-18 were the products of the Pharmacia AB Laboratory Separation Division, Uppsala, Sweden, and Merck Company, Germany, respectively. The

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Table 1. 1H and 13C NMR data of compounds 1 (500 MHz & 125 Hz, JHz, acetone-d6, TMS) Position 2 3 4 5 6 7 8 9 10 11 12 Me Glucose 1 2 3 4 5 6 Apiose 1 2 3 4 5 dH 5.64 (1H, d, J = 10.0 Hz) 6.62 (1H, d, J = 10.0 Hz) 7.42 (1H, d, J = 2.0 Hz) 7.21 (1H, d, J = 2.0 Hz) dC 76.8 125.8 115.8 120.9 131.2 116.7 146.7 158.9 105.7 196.8 25.7 25.7 104.6 75.4 78.1 71.2 77.2 68.2 DEPT C CH CH CH C CH C C C C CH3 CH3 CH CH CH CH CH CH2 HMBC 6.62, 5.64, 1.46 6.62 5.64, 7.42 6.62, 7.42, 7.21 7.42, 7.21 7.42 5.07 7.21 6.62, 7.42 7.21, 2.48

2.48 (3H, s) 1.46 (6H, s) 5.07 (1H, d, J = 7.8 Hz) 3.31 (1H, dd, J = 9.0, 7.8 Hz) 3.36 (1H, dd, J = 9.0, 8.3 Hz) 3.28 (1H, dd, J = 9.9, 8.3 Hz) 3.10 (1H, ddd, J = 9.9, 5.8, 1.6 Hz) 3.48 (1H, dd, J = 11.3, 5.8 Hz) 3.72 (1H, dd, J = 11.3, 1.6 Hz) 5.28 (1H, d, J = 2.1 Hz) 3.88 (1H, d, J = 2.1 Hz) 3.65 (1H, d, J = 9.2 Hz), 3.91 (1H, d, J = 9.2 Hz) 3.52 (2H, s)

5.28

111.1 77.2 80.1 75.5 64.9

CH CH C CH2 CH2

3.58, 3.72

spots were visualized by UV (254 nm) and EtOH-H2SO4. Plant Material The root bark of R. sambucifolia was collected from the Dali area of Yunnan province of China in August 2003, and were identified by Prof. Yun-Shan Lian, Department of Biology. A voucher specimen (No. 100813) was deposited at the Herbarium of the Botany Department, Northwest Normal University, Lanzhou, 730070, China. Extraction and Isolation The fresh and shade-dried root bark (5 kg) was chipped, extracted three times with MeOH (15 L 7 d each) at room temperature, and concentrated in vacuo. The MeOH extract (252 g) was partitioned between CHCl3-H2O (1:1, V/V) to divide into a CH3Cl-soluble fraction (fraction A, 80 g) and an H2O-soluble fraction. Fraction A was subjected to silica gel column chromatography and eluated with n -hexane, CHCl 3 , CHCl 3 -Me 2 CO (5:1-1:5, V/V), Me2CO and Me2CO-MeOH (4:1-1:10, V/V) gradient elution to give 16 fractions (A1-A16). Fractions A2, A5, A8, A10

and A11 were repeatedly rechromatographed over silica gel and purified by recrystallization or preparative TLC to afford 2 (18.5 mg), 3 (12.3 mg), 4 (25.0 mg), 5 (18.5 mg) and 6 (8.2 mg), respectively. Fraction A14 was fractionated over a silica gel column with CHCl3-MeOH gradient to give 8 (9.5 mg), 13 (18.6 mg), and 14 (12.8 mg), respectively. The H2O-soluble fraction was partitioned with EtOAc, then with n-BuOH to give EtOAc fraction (fraction B, 75 g), the n-BuOH-soluble fraction (fraction C, 35 g) and the H2Osoluble fraction. Fraction B was chromatographed over silica gel column, eluting with CH2Cl2, and then polarity was gradually increased with EtOAc to obtain 13 fractions (B1B18). From fractions B1-B4, rechromatographed on silica gel with CHCl3-MeOH (5:1-1:5, V/V) as eluent and preparative TLC, to yield 7 (10.0 mg), 9 (11.2 mg), 10 (8.4 mg), 11 (15.6 mg), and 12 (12.7 mg), respectively. Then from fraction B5-B10, through rechromatographed on silica gel with EtOAc-MeOH (5:1-1:10, V/V) as eluent, preparative TLC and recrystallization, respectively, 15 (21.0 mg), 16 (13.2 mg), 17 (12.0 mg) and 18 (11.5 mg) were obtained. Fraction B11-B18 rechromatographed on silica gel, eluted

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with CHCl3-MeOH (5:1, V/V), and by gradually increase the polarity with MeOH and preparative TLC, afforded 1 (5.6 mg), 19 (8.5 mg), 20 (11.6 mg), 21 (7.5 mg), 22 (12.8 mg), 23 (15.4 mg) and 24 (10.2 mg). 2,2-Dimethyl-2H-(8-hydroxy-6-acetyl)-[2,3-b] pyran-8O-b-D-apiofuranosyl-(16)-b-D-glucopyranoside 1 White amorphous powder (acetone), mp. 218-220 C; -1 KBr UV l MeOH max (nm): 248 and 312; IR n max (cm ): 3018, 1658, 1610, 1595, 1485, 1384, 1370, 1270, 1126, 1088, 1070, 1036, 880, 870; HR-FAB-MS: m/z 512.1904 (M+, calcd for C24H32O12, 512.1894); FAB-MS: m/z 513 [M+H]+, 381 [M+H-132]+ and 219 [M+H-132-162]+; for 1H and 13 C NMR data, see Table 1. Acid hydrolysis of compound 1 Compound 1 (8 mg) was treated with 50% MeOHHCl (5 mL) under reflux at 95 C for 1.5 hr in a water bath. The reaction mixture was cooled, then concentrated under pressure to give a syrup which was partitioned between EtOAc/H2O. The aqueous layer was neutralized with NH3 H2O and concentrated in vacuo. The sugars were identified by co-TLC (GF254 silica gel plate) with authentic samples using a solvent system [CHCl 3 -MeOH-H 2 O (70:30:3)], visualization by 0.9% C6H6NH2-C2H2O4 spray and then heating at 100 C for 5 min; the spot of glucose displayed brown (Rf: 0.26), and apiose showed red (Rf: 0.64). The EtOAc extract was dried over anhydrous Na2SO4, and recrystallized from (CHCl 3-Me 2CO) to give aglycone of 1 (3 mg). Aglycone of 1 was colourless crystals (CHCl3/ Me2CO), mp. 140-142 C (lit.8 142 C); UV l MeOH (nm): max -1 246 and 310; IR n KBr (cm ): 3246, 1660, 1608, 1483, 1384, max 1 1372, 1269, 1124, 880, 870; H NMR (acetone-d 6, 500 MHz) dH: 1.45 (6H, s, CH3 2), 2.50 (3H, s, H-12), 5.64 (1H, d, J = 10.0 Hz, H-3), 6.58 (1H, d, J = 10.0 Hz, H-4), 7.26 (1H, d, J = 2.0 Hz, H-7), 7.45 (1H, d, J = 2.0 Hz, H-5); 13 C NMR (acetone-d6, 125 MHz) dC: 77.2 (C-2, C), 126.7 (C-3, CH), 115.9 (C-4, CH), 121.7 (C-5, CH), 130.6 (C-6, C), 115.1 (C-7, CH), 152.2 (C-8, C), 153.9 (C-9, C), 105.9 (C-10, C), 194.9 (C-11, C), 26.4 (C-12, CH3), 27.1 (CH3 2). EI-MS: m/z 218 [M]+, 203, 175, 160, 157, 129, 94. (Found: C, 71.42; H, 6.56. calcd for C13H14O3: C, 71.53; H, 6.47). 5,4-dihydroxy-7,8-[(3,4-dihydro-3,4-dihydroxy)2,2-dimethylpyran]-flavone 2 White amorphous powder (acetone), mp. 234-236 C; 1 UV l MeOH max (nm): 268, 315, 340; H NMR (acetone-d6, 500

MHz) dH: 7.98 (2H, d, J = 8.1 Hz, H-2/6), 7.02 (2H, d, J = 8.1 Hz, H-3/5), 6.84 (1H, s, H-6), 6.31 (1H, s, H-3), 4.75 (1H, d, J = 4.8 Hz, H-4), 4.36 (1H, d, J = 4.8 Hz, H-3), 1.28 (3H, s, Me-2), 1.16 (3H, s, Me-2); 13C NMR (acetone-d6, 125 MHz) dC: 162.8 (C-2, C), 103.7 (C-3, CH), 182.9 (C-4, C), 163.7 (C-5, C), 100.3 (C-6, CH), 165.8 (C-7, C), 104.2 (C-8, C), 152.9 (C-9, C), 108.1 (C-10, C), 120.9 (C-1, C), 128.4 (C-2, CH), 116.4 (C-3, CH), 161.8 (C-4, C), 115.9 (C-5, CH), 128.4 (C-6, CH), 69.9 (C-2, C), 94.2 (C-3, CH), 70.5 (C-4, CH), 25.5 (CH3-2, CH3), 25.2 (CH3-2, CH3). EI-MS: m/z 370 [M]+, 334, 294, 216, 118. (Found: C, 64.78; H, 4,98. Calcd for C20 H18O7: C, 64.85; H, 4.90). 5,4-dihydroxy-3,5-dimethoxy-6,7-(2,2-dimethylpyran)-flavone 3 Yellow prisms (CHCl3), mp. 230-232 C; UV l MeOH max (nm): 246, 297, 312, 358; 1H NMR (CDCl3, 500 MHz) dH: 6.54 (1H, s, H-3), 6.43 (1H, s, H-8), 7.05 (2H, s, H-2/6), 5.60 (1H, d, J = 9.8 Hz, H-3), 6.68 (1H, d, J = 9.8 Hz, H-4), 4.01 (6H, s, MeO-3/5), 1.45 (6H, s, Me-2), 5.98 (1H, s, HO-4), 12.98 (1H, s, HO-5); 13C NMR (CDCl3, 125 MHz) dC: 163.5 (C-2, C), 104.1 (C-3, CH), 182.3 (C-4, C), 156.1 (C-5, C), 105.1 (C-6, C), 158.9 (C-7, C), 98.5 (C-8, CH), 156.9 (C-9, C), 105.7 (C-10, C), 122.1 (C-1, C), 112.4 (C-2, CH), 148.9 (C-3, C), 146.1 (C-4, C), 148.7 (C-5, C), 103.8 (C-6, CH), 76.5 (C-2, C), 129.7 (C-3, CH), 115.2 (C-4, CH), 28.4 (CH3-2, CH3), 56.7 (CH3O-3/5, CH3). EI-MS: m/z 396 [M]+, 381, 365, 203, 69. (Found: C, 66.58; H, 5.12. Calcd for C22H20O7: C, 66.65; H, 5.09).

ACKNOWLEDGEMENTS We are grateful to Prof. Yun-Shan Lian for identifying the plant and Prof. Shang-zhen Zheng for kind help with our work. The work was kindly supported by the Education Department, Foundation of Gansu Province (Grant No. 2003039-04), China.

Received February 9, 2006.

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