Glycosides and Xanthine Oxidase Inhibitors from Conyza Bonariensis

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Phytochemistry 58 (2001) 645–651 www.elsevier.com/locate/phytochem

Glycosides and xanthine oxidase inhibitors from Conyza bonariensis

L.D. Konga, Z. Ablizb, C.X. Zhoua, L.J. Lib, C.H.K. Chengc, R.X. Tana,* aInstitute of Functional Biomolecule, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing 210093, People’s Republic of China bDepartment of Instrumental Analysis for Determination of Chemical Structures, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China cDepartment of Biochemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong

Received in revised form 12 December 2000

Abstract Fractionation of the xanthine oxidase inhibitory methanol extract of Conyza bonariensis aﬀorded three glycosides, in addition to nine known compounds including amyrin, b-sitostero1 daucosterol, syringic acid 3-hydroxy-5-methoxybenzoic acid, eugenol 4-O- glucopyranoside, and luteolin, apigenin and takakin 8-O-glucuronide. The structures of the glycosides were established by a com- bination of spectroscopic methods (IR, MS, 1Hand 13C NMR, DEPT, COSY, HMQC and HMBC) as 4-hydroxypyridin-3-carboxylic acid 4-O-glucopyranoside, 8-hydroxy-6,7-dihydrolinalool 8-O-glucopyranoside and bonaroside [viz. 1,3,4,12-tetrahydroxy- 2-(9-hexadecenoylamino)octadecane 1-O-glucopyranoside]. The in vitro enzyme assay showed that syringic acid and takakin 8-O- glucuronide displayed weak inhibitory activity against xanthine oxidase with IC50 values of 50041 mM and 17012 mM, respectively. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Conyza bonariensis; Asteraceae; 4-Hydroxypyridin-3-carboxylic acid 4-O-glucopyranoside; 8-Hydroxy-6,7-dihydrolinalool 8-O-glucopyranoside; Bonaroside; Syringic acid; Takakin 8-O-glucuronide

1. Introduction three new glycosides (1–3), in addition to the known constituents amyrin, b-sitosterol, daucosterol, apigenin, Conyza bonariensis (L.) Crong (Asteraceae) has been luteolin, takakin 8-O-glucuronide, syringic acid and 3- phytochemically investigated (El-Karemy et al., 1986; hydroxy-5-methoxybenzoic acid. The in vitro evaluation Rizk et al., 1986; Ferraro et al., 1988; Sanz and Marco, of these isolates demonstrated that the enzyme inhibi- 1991). However, the species growing in China has not tors present in the title species were syringic acid and the been examined to date. In continuation of our investi- three flavonoids. gations of plant-derived xanthine oxidase inhibitors (Li The identification of amyrin, b-sitosterol and daucos- et al., 1999; Zhou et al., 1999), the methanol extract was terol was accomplished by direct comparisons (co-TLC, found to be active against the enzyme which is closely IR and EIMS) with authentic samples available in our related to the hyperuricemia and gout (Tsutomu et al., laboratory. The spectral data (IR, EIMS, 1Hand 13C 1991; Cos et al., 1998). We therefore analyzed the NMR) of the two acids revealed that they were syringic extract in detail in order to characterize the xanthine acid (Cuenca et al., 1992) and 3-hydroxy-5-methoxy- oxidase inhibitor(s). benzoic acid (Majumder et al., 1996), respectively. Syr- ingic acid was previously found to have antioxidant (Yoshiki et al., 1995), antibacterial (Fernandez et al., 2. Results and discussion 1996), antifungal (Lattanzio et al., 1994), properties, as well as being protective against cell damage induced by Repeated chromatography of the methanol extract of superoxide anion radicals in murine dermal fibroblasts C. bonariensis on silica gel and Sephadex LH-20 afforded (Masaki et al., 1995). The structures of apigenin and luteolin were ascertained by their IR, EIMS, 1Hand 13C * Corresponding author. Tel./fax: +86-25-359-3201. NMR spectra (Markham et al., 1978; Noro et al., 1983; E-mail address: [email protected] (R.X. Tan). Tosun and Akyuz, 1997). Both flavones were previously

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investigated biologically and reported to be antioxidant the attachment of the glucopyranosyl group, the chemi- and anti-inflammatory (Maokn et al., 1996; Silvan et al., cal shift of the anomeric proton doublet (J=7.8 Hz) at 1996; Yokozawa et al., 1997). Furthermore, apigenin 4.91 supported the formulated glycosidation pattern if was found to inhibit tumor necrosis factor-induced compared with that of carboxylic ( 6.20) and phe- intercellular adhesion molecule-1 upregulation in vivo nolic (4.90) glucosides described previously (Jaku- (Panes et al., 1996), and to have anti-TPA-induced povic et al., 1991; Tan et al., 1993). Finally, the transformation, anti-protein kinase C activation and structure of glycoside 1 was reinforced by its FABMS anti-TPA-induced c-jun expression activities (Lee and spectrum which gave an intense protonated molecular Lin, 1997), whereas luteolin had cancer chemopreven- ion at m/z 302, accompanied by a strong fragment at tive and antiplatelet effects (Cai et al., 1997; Lin et al., m/z 140 produced through the elimination of the gluco- 1997). Furthermore, luteolin and apigenin have been syl moiety from the quasimolecular ion. reported to be xanthine oxidase inhibitors (Cos et al. The structure of glycoside 2 followed from its spectral 1998, Kasai et al., 1999). The flavone glycoside was data. The El mass spectrum of 2 was not informative. identified as takakin 8-O-glucuronide by the IR, EIMS, However, an intense quasimolecular ion was observed 1Hand 13C NMR spectral data (Chen et al., 1994). The at m/z 335 ([M+H]+) in its FABMS spectrum. Besides second glycoside was demonstrated to be eugenol 4-O- a set of proton resonances attributable to a glucopyr- glucopyranoside by its spectral data (FABMS, IR, 1H anosyl moiety, the 1HNMR spectrum of glycoside 2 and 13C NMR), characterized previously as the corre- exhibited a methyl singlet at 1.20, a three-proton sponding peracetate from Artemisia dracunculus (Jaku- doublet (J=6.7 Hz) at 0.91, a pair of double doublets povic et al., 1991). at 3.82 and 3.66 due to an oxygenated methylene In the 1HNMR spectrum of compound 1, a broa- group, and a set of signals at 5.88 (1 H, dd, J=17.4, dened singlet at 8.34 and a pair of mutually coupled 10.8 Hz), 5.15 (IH, dd, J=17.4, 1.5 Hz) and 4.98 (1H, doublets (J=5.6 Hz) at 8.15 and 6.61 indicated that it dd, J=10.8, 1.5 Hz) arising from a vinyl function. These was most likely a pyridine derivative carrying pre- 1HNMR spectroscopic data showed that compound 2 sumably 3-carbonyl and 4-hydroxyl (or amino) groups was most probably a 8-hydroxy-6,7-dihydrolinalool (Rasala, 1993). This hypothesis was subsequently con- glucoside (Tan et al., 1991; Schulz et al., 1997). This firmed by the 13C NMR spectrum of 1, which exhibited proposal was substantiated by its 13C NMR and DEPT a total of twelve carbon resonance lines consisting of one spectra. All carbon signals could be assigned by com- methylene, eight methine and three quaternary carbons. paring them with those of 6,7-dihydrolinalool analogs Subtracting a set of carbon signals due to a glucopyr- (Yu et al., 1989). The glucopyranosyloxy group was anosyl group, the rest was ascribable to the 4-hydroxy- shown to be at C-8 by the glycosidation induced down- pyridin-3-carboxylic acid nucleus (Yu et al., 1989). As to field shift ( 76.2) of the 8-oxygenated methylene carbon 中国科技论文在线______www.paper.edu.cn

L.D. Kong et al. / Phytochemistry 58 (2001) 645–651 647 signal. In conclusion, glycoside 2 was 8-hydroxy-6,7- 316. The 9,10-double bond was assigned to the hexa dihydrolinalool 8-O-glucopyranoside. decenoyl group on the basis of the typical fragment ion The molecular formula of compound 3 was found to be at m/z 500 which was formed by elimination of pentene 1 13 C40H77NO10 by its FABMS, Hand CNMR,andDEPT from that at m/z 570 through McLafferty rearrangement data and the HREIMS spectrum of the corresponding (Fig. 1). Obviously, the other aliphatic chain should peracetate (3a). The 1Hand 13C NMR spectra of 3 consist of eighteen carbons. Moreover, the presence of a indicated that it was also a glucopyranoside (Table 1). total of seven hydroxyl groups in the molecule was Furthermore, a carbonyl IR absorption at 1720 cm1, deduced by comparison between the molecular ions and the six-proton triplet (J=7.0 Hz) at 0.86 and a pair appearing in the FABMS spectra of 3 and 3a. Accord- of olefinic proton signals at 5.34 and 5.42 in its 1HNMR ingly, after subtraction of the four hydroxyls involved in spectrum demonstrated that it possessed presumably two the glucopyranosyl group, the other three had to be 1 1 aliphatic chains, one of which carried a double bond. positioned in the C18 chain. The H– HCOSY spectrum Thus, the three unsaturations of the molecular formula of 3 showed that a pair of double doublets of an oxy- could be readily assigned to a double bond, a carbonyl genated methylene at 4.01 (dd, J=10.5, 6.2 Hz) and and a glucopyranosyl group. This hypothesis was sub- 3.76 (dd, J=10.5, 3.5 Hz) coupled to the oxygen-bearing sequently confirmed by the 13C NMR spectrum and methine signal at 4.24 (ddd, J=6.5, 6.2, 3.5 Hz). This FABMS of 3. In addition to the quasimolecular ion at resonance showed a correlation with the double doublet m/z 754 [M+Na]+, the FABMS spectrum of 3 exhib- (J=6.5, 6.0 Hz) at 3.56 which coupled further to the ited an intense fragment peak at m/z 570 which was signal at 3.48 (ddd, J=6.2, 6.5, 3.5 Hz). In the HMQC produced by elimination of the glucosyl residue from the spectrum of 3, the signal at 4.24 correlated to the protonated molecular ion. The loss of hexadecenamide methine carbon at 51.6. These observations required from the molecular ion gave rise to the fragment at m/z the given 1,3,4-trioxygenated and 2-nitrigenated pattern. The glycosidation at C-1 was indicated by the downfield shifted signal of C-1 at 70.0 (Kasai et al., Table 1 1999). The 9-hexadecenoylamino group was found to be 1Hand 13C NMR spectral data of glycoside 3a at C-2 by the resonance of H-2 at 4.24 which was b b Position C (DEPT) H , multiplicity J (Hz) shifted remarkably downfield owing to acylation. In addition to the 3,4-diol system, the final hydroxyl group 1 70.0 (CH2) 4.01 dd (10.5, 6.2), 3.76 was found to be at C-12 by diagnostic fragment ions in dd (10.5, 3.5) 2 51.6 (CH) 4.24 ddd (6.5, 6.2, 3.5) FABMS spectrum of 3a. The characteristic fragment at 3 75.5 (CH) 3.56 dd (6.5, 6.0) m/z 157, which yielded the peak at m/z 115 due to the 4 72.8 (CH) 3.48 ddd (6.2, 6.0, 3.5) loss of ketene, was produced through the cleavage of the 5 32.7 (CH ) 1.48 m (2H) 2 C11–C12 bond (Fig. 2). In conclusion, glycoside 3 was 1, 6, 10, 14 30.5 (2CH2), 3,4,1 2-tetrahydroxy-2-(9-hexadecenoylamino)octadecane 30.3 (CH ) d 2 1-O-glucopyranoside. The configuration at the chiral 16 33.1 (CH2) 1.25 m (2H) 17 23.7 (CH2) 1.25 m (2H) centers could not be established without chemical 18 14.5 (CH3) 0.86 t (7.0) transformations that would require much more mate- 10 104.7 (CH) 4.24 d (7.8) rial. We have named this glycoside bonaroside. 0 2 75.0 (CH) 3.13 dd (9.0,7.8) All isolates were tested for the xanthine oxidase inhi- 30 77.8 (CH) 3.33 dd (9.l, 9.0) 40 71.5 (CH) 3.26 m bitory activity as described elsewhere (Li et al., 1999; 50 78.0 (CH) 3.26 m Zhou et al., 1999). The xanthine oxidase inhibitors pre- 0 6 62.6 (CH2) 3.83 dd (11.9, 2.7), 3.63 sent in the methanol extract of C. bonariensis were dd (11.9, 4.8) revealed to be syringic acid, luteolin, apigenin and 100 177.1 (C) d 00 takakin 8-O- - -glucuronide. In addition to the known 2 35.7 (CH2) 1.58–1.62 m (2H) 00 xanthine oxidase inhibitory flavone luteolin and api- 3 26.1 (CH2) 1.26 m (2H) 00 00 8 ,11 27.1 (CH2), 1.95–2.01 m (4H) genin (Kasai et al., 1999; Cos et al., 1998), syringic acid 26.1 (CH2) and takakin 8-O-glucuronide were displayed for the first 900 131.4 (CH) 5.36 br dt (16.8, 6.5) time to be active against the enzyme with IC50 values of 1000 131.5 (CH) 5.42 br dt (16.8, 6.5) 00 50041 and 17012 mM, respectively. The IC50 value 14 33.1 (CH2) 1.25 m (2H) 00 of allopurinol co-assayed in the study as positive control 15 23.7 (CH2) 1.25 m (2H) 00 16 14.5 (CH3) 0.86 t (7.0) was shown to be 10. The present phytochemical investigation of C. bonar- a 1 1 Assigned by spin decoupling, H– HCOSY and HMQCexperi- iensis growing widely in China has led to the character- ments. b The chemical shifts of C-7–C-9, C–15, C-400–C-700, C-1200 and C- ization of three new glycosides and four xanthine 00 13 : 30.9–30.6 (10 CH2), and those of protons thereon: 1.25–1.32 m oxidase inhibitory natural products. However, except (20 H). widespread compounds such as amyrin, b-sitosterol, 中国科技论文在线______www.paper.edu.cn

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Fig. 1. Diagnostic fragments of glycoside 3.

Fig. 2. Diagnostic FABMS fragmentions of 3a. daucosterol, luteolin and apigenin, the constituents of and Marco, 1991). This is presumably due to different C. bonariensis growing in China are quite different from phytochemical strategies and/or geographical deviation, those of the same species collected abroad (El-Karemy the latter affecting putatively the biosynthesis and accu- et at., 1986; Rizk et al., 1986; Ferraro et al., 1988; Sanz mulation of plant secondary metabolites. 中国科技论文在线______www.paper.edu.cn

L.D. Kong et al. / Phytochemistry 58 (2001) 645–651 649

3. Experimental l, F-5-2 and F-5-3). Repeated gel filtration of F-5-1 on Sephadex LH-20 with CHCl3–MeOH(1:1) afforded 2 3.1. General (18 mg) and 3 (15 mg). Gel filtration of F-5-2 over Sephadex LH-20 with CHCl3–MeOH(1:9) yielded Optical rotations were measured on a DXP-118 again 3 (7 mg) and takakin 8-O-glucuronide (34 mg). instrument. IR spectra were recorded in KBr disks on a CC of F-5-3 over silica gel with CHCl3–MeOHgradient Perkin-Elmer 577 instrument. All NMR experiments (50:1!2:1) gave daucosterol (120 mg) and eugenol 4-O- were performed on a Jeol JNM-A 500 FT–NMR spec- glucopyranoside (26 mg). Separation of F-6 and F-7 trometer using TMS as the internal standard. El and following the same procedure gave again eugenol 4-O- FABMS experiments were run on either VG-ZAB-HS glucopyranoside (16 mg), glycoside 1 (220 mg) and 3- or Jeol JMX-HX110 mass spectrometers, respectively. hydroxy-5-methoxybenzoic acid (38 mg). Silica gel (200–300 mesh) for column chromatography and GF254 (30–40 m) for TLC were produced by Qing- 3.4. Xanthine oxidase assay dao Marine Chemical Factory, Qingdao, China. Sepha- dex LH-20 was from Pharmacia Biotech, Sweden. Other The xanthine oxidase inhibitory activity was mea- chemicals used in this study were of analytical grade. sured according to a slight modification of the previously reported method (Li et al., 1999; Zhou et al., 3.2. Plant material 1999). Xanthine oxidase from cow milk, xanthine and the standard inhibitor allopurinol were purchased from The aerial parts of C. bonariensis was collected on 19 Sigma Chemical Co. (St. Louis, MO, USA). The reac- June 1996 from the Xuanwu Lake Park, and identified tion mixture contains 80 mM sodium pyrophosphate by Professor Z.P. Wang, School of Life Science, Nanj- buffer (pH=8.5), 120 mM xanthine and 0.1 unit of xan- ing University, Nanjing 210093, China. A voucher spe- thine oxidase. The absorption at 295 nm indicating the cimen, registered under the number Y 96619B, was formation of uric acid at 25C was monitored and the preserved in the Herbarium of Nanjing University, initial rate was calculated. The test materials, initially Nanjing, China. dissolved in DMSO and then diluted with the same buffer, were incorporated in the enzyme assay to assess 3.3. Extraction and isolation the inhibitory activity. Extracts, fractions and isolates were tested at 100 mg/ml, and those inhibiting the The powdered air-dried plant material (900 g) was enzyme >50% were further tested to determine IC50 extracted twice (24 h each) with MeOHat room tem- values. perature. Evaporation of solvent from the combined extracts in vacuo at 55C gave a dark residue (57 g) 3.5. 4-Hydroxypyridyl-3-oic acid 4-O-glucopyranoside (1) which was dissolved in MeOHfollowed by filtration. 25 The filtrate was concentrated to a black gum (32 g) Colourless needles, mp 203–205 C, ½a D 250.4 under reduced pressure and subsequently separated by (MeOH; c 1.35). FABMS m/z: 302.0889 [M+H]+ + CC (silica gel, 900 g, 200–300 mesh). Elution with petrol– ([C12H15NO8+H] requires 302.0878), 40 + 1 acetone gradient (9:1!10:1) was followed by acetone [M+Hglucosyl] . HNMR (D 2O, 500 MHz): 8.34 and acetone-MeOHmixtures (100:5 !1:10). According to (1H, brs, H-2), 8.15 (1H, br d, J=5.6 Hz, H-6), 6.61 TLC monitoring and xanthine oxidase assay, seven (1H, d, J=5.6 Hz, H-5), 4.91 (1H, d, J=7.8 Hz, H-10), fractions were collected (F-1: 3.2 g, F-2: 2.1 g, F-3: 3.4 3.45–3.60 (4H, m,H-20–H-50), 3.93 (1H, dd, J=12.2, 0.3 g, F-4: 2.6 g, F-5: 4.6 g, F-6: 3.5 g and F-7: 2.5 g). F-1 Hz, H-60a), 3.75 (1H, dd, J=12.2, 5.0 Hz, H-60b). 13C and F-3 contained nothing of interest. F-4 and F-5 NMR (D2O, 125 MHz) (multiplicities by the DEPT showed significant inhibitory activity against xanthine pulse sequences): 146.7 (d, C-2), 126.5 (s, C-3), 147.2 oxidase. CC of F-2 over silica gel (200 g, 200–300 mesh) (s, C-4), 116.6 (d, C-5), 158.6 (d, C-6), 176.6 (s, C-7), with a gradient of petrol-acetone mixtures (25:1!1:9) 102.3 (d, C-10), 73.8 (d, C-20), 76.2 (d, C-30), 70.2 (d,C- gave b-sitosterol (96 mg) and amyrin (39 mg). CC of F-4 40), 77.4 (d, C-50), 61.4 (t, C-60). on silica gel with a CHCl3–MeOHgradient (100:1!+2:1) yielded 2 fractions (F-4-1 and F-4-2). 3.6. 8-Hydroxy-6,7-dihydrolinalool 8-O-glucopyranoside Gel filtration of F-4-1 on Sephadex LH-20 with CHCl3– (2) MeOH(1:1) yielded syringic acid (28 mg) and pigments. 1 Luteolin (35 mg) and apigenin (26 mg) were obtained Gum. IR (KBr) max cm : 3300 (OH), 1650 (C¼C). + + from F-4-2 by column chromatography on silica gel (50 FABMS m/z: 335.2101 [M+H] ([C16H30O7+H] 1 g) using CHCl3–MeOHmixtures of a growing polarity. requires 335.2072). HNMR (CD 3OD, 500 MHz): 5.8 F-5 was applied to a silica gel column (800 g) eluted 8 (1H, dd, J=17.4, 10.8 Hz, H-2), 5.15 (1H, dd, with CHCl3–MeOHgradient to afford 3 fractions (F-5- J=17.4, 1.5 Hz, H-1a), 4.98 (1H, dd, J=10.8, 1.5 Hz, 中国科技论文在线______www.paper.edu.cn

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H-1b), 3.82 (1H, dd, J=12.1, 3.3 Hz, H-8a), 3.66 (1H, El-Karemy, Z.A.R., Mansour, R.M.A., Frayed, A.A., Saleh, N.A.M., dd, J=12.1, 6.9 Hz, H-8b), 1.72 (1H, m, H-7), 1.44–1.30 1986. The flavonoids of local members of Astereae (Compositae). (2H, m, H-4), 1.20 (3H, s, H-10), 1.18–0.88 (4H, m,H- Biochemical Systematics and Ecology 15, 53–55. Fernandez, M.A., Garcia, M.D., Saenz, M.T., 1996. Antibacterial 5–H-6), 0.91 (3H, d, J=6.7 Hz, H-9), 4.20 (1H, d, activity of phenolic acid fractions of Scrophulariafrutescens and S. 0 0 J=7.8 Hz, H-1 ), 3.14 (1H, dd, J=9.0, 7.8 Hz, H-2 ), sambucifolia. Journal of Ethnopharmacology 53, 11–14. 3.21–3.37 (3H, m,H-30–H-50), 3.84 (1H, dd, J=12.2, 2.1 Ferraro, G.E., Broussalis, A.M., Van Baaren, C.M., Muschietti, L.V., Hz, H-60a), 3.64 (1H, dd, J=12.2, 5.0 Hz, H-60b). 13C Coussio, J.D., 1988. Polyphenolic compounds isolated from Conyza NMR (CD OD, 125 MHz) (multiplicities by the DEPT bonariensis (Compositae). Rev. Latinoam. Quim. 19, 141–143. 3 Jakupovic, J., Tan, R.X., Bohlmann, F., Jia, Z.J., Huneck, S., 1991. pulse sequences): 110.4 (t, C-1), 145.1 (d, C-2), 72.5 (s, Acetylenes and other constituents from Artemisia dracunlus. Planta C-3), 42.2 (t, C-4), 21.3 (t, C-5), 34.1 (t, C-6), 33.1 (d,C- Medica 57, 450–453. 7), 76.2 (t, C-8), 16.0 (q, C-9), 26.2 (q, C-10), 103.1 (d,C- Kasai, R., Sasaki, A., Hashimoto, T., Kaneko, T., Ohtani, K., l 0), 73.7 (d,C-20), 76.5 (d, C-30), 70.2 (d, C-40), 76.7 (d, Yamasaki, K., 1999. Cycloartane glycosides from Trichosanthes tri- C-50), 61.4 (t,C-60). cuspidata. Phytochemistry 51, 803–808. Lattanzio, V., De Cicco, V., Di Venere, D., Lima, G., Salerno, M., 1994. Antifungal activity of phenolics against fungi commonly 3.7. Bonaroside (3) encountered during storage. Italian Journal of Food Sciences 6, 23– 30. 25 Lee, S.F., Lin, J.-K., 1997. Inhibitory effects of phytopolyphenols on White powder, mp 172–174 C, ½a D +178.6 (MeOH; TPA-induced transformation, PKC activation, and c-jun expression c 0.14). IR (KBr) max 3350 (OH), 1720 (C¼O). FABMS 1 in mouse fibroblast cells. Nutrition and Cancer 28, 177–183. data and fragmentation were illustrated in Fig. 1. H Li, H., Meng, J.C., Cheng, C.H.K., Higa, T., Tanaka, J., Tan, R.X., 13 and C NMR, see Table 1. Treatment of compound 3 1999. New guaianolides and xanthine oxidase inhibitory flavonols with Ac2O–pyridine (1:1) at room temperature for 36 h from Ajania fruticulosa. Journal of Natural Products 62, 1053–1055. Lin, C.N., Kuo, S.H., Chung, M.I., Ko, F.N., Teng, C.M., 1997. A followed by preparative TLC with CHCl3–MeOH (25:1), gave the corresponding peracetate 3a. FABMS new flavone C-glycoside and antiplatelet and vasorelaxing flavones + + from Gentiana arisanensis. Journal of Natural Products 60, 851–853. m/z: 1026.6380 [M+H] ([C54H91 NO17+H] requires Majumder, P.L., Banerjee, S., Sen, S., 1996. Three stilbenoids from the + + 1026.6367), 984 [M+H–ketene] , 966 [M+H–AcOH] , orchid Agrostophyllum callosum. Phytochemistry 42, 847–852, and + + 772 [M–9-hexadecenamide] , 331 [glu(OAc)4] , 157 related references cited therein. + Maokn, T., Sakushima, A., Coskun, M., Ito, Y., 1996. Antioxidant [CH3CH2CH2CH2CH2CH2–CH¼O Ac], 115 [CH3 CH CH CH CH CH –C¼O+H]. Other spectral mea- activity of phenolic compounds of Boreava orientalis. Nihon Yuka- 2 2 2 2 2 gakkaishi 45, 671–673. surements were not permitted due to very limited Markham, K.R., Ternai, B., Stanley, R., Geiger, H., Mabry, T.J., amount of the derivative. 1978. Carbon-13 NMR studies of flavonoids–III naturally occurring flavonoid glycosides and their acylated derivatives. Tetrahedron 34, 1389–1397. Acknowledgements Masaki, H., Atsumi, T., Sakurai, H., 1995. 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