PHYTOTHERAPY RESEARCH Phytother Res (2012) Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.3710 Xanthine Oxidase Inhibitors from Vietnamese Blumea balsamifera L Mai Thanh Thi Nguyen* and Nhan Trung Nguyen Faculty of Chemistry, University of Science, Vietnam National University, HoChiMinh City, Vietnam From the MeOH extract of the aerial part of Blumea balsamifera L., a new dihydroflavonol, (2R,3S)-(À)-4′-Omethyldihydroquercetin (1), together with seven known compounds has been isolated Their structures were elucidated on the basis of spectroscopic data Compounds 1–4 and 6–8 displayed significant xanthine oxidase inhibitory activity in a concentration-dependent manner, and compounds 1, and showed more potent inhibitory activity, with IC50 values ranging from 0.23 to 1.91 mM, than that of a positive control allopurinol (IC50 2.50 mM) Copyright © 2012 John Wiley & Sons, Ltd Keywords: Blumea balsamifera; Asteraceae; (2R,3S)-(À)-4′-O-methyldihydroquercetin; xanthine oxidase inhibition Supporting information may be found in the online version of this article (Supplementary Material) INTRODUCTION MATERIAL AND METHOD Xanthine oxidase (XO) is a key enzyme that catalyses the last step in the conversion of purines to uric acid, and plays a vital role in producing hyperuricemia and gout Allopurinol, the medication prescribed for gout prevention, is a xanthine oxidase inhibitor (Oettl and Reibnegger, 1999) However, due to the unwanted side effects of allopurinol, such as hepatitis, nephropathy and allergic reactions, new alternatives with increased therapeutic activity and fewer side effects are desired Moreover, superoxide anion radicals generated by XO are involved in various pathological states such as hepatitis, inflammation, ischemia-reperfusion, carcinogenesis and aging (Cos et al., 1998) Thus, the search for novel XO inhibitors would be beneficial not only to treat gout but also to combat various other diseases Blumea balsamifera L (Asteraceae) has been used in Vietnamese traditional medicine for the treatment of rheumatism and inflammatory diseases (Do, 2001) Several studies on the chemical constituents of B balsamifera have been reported and a number of flavonoids, monoterpenes and sesquiterpenes have been isolated from this plant (Nessa et al., 2004; Fazilatun et al., 2001) Our preliminary screening study revealed that the methanol extract of the aerial part of B balsamifera exhibited significant XO inhibitory activity with an IC50 value of 6.0 mg/mL (Nguyen et al., 2004) Therefore, activityguided fractionation of the MeOH extract was carried out and a new dihydroflavonol (1) isolated together with seven known compounds (2–8) In the present study, the isolation and structure elucidation of the new compound by spectroscopic techniques is reported, together with the XO inhibitory activity of the isolated compounds General experimental procedures Optical rotations were recorded on a Perkin-Elmer 241 digital polarimeter The IR spectra were measured with a Shimadzu IR-408 spectrophotometer in CHCl3 solutions The NMR spectra were taken on a Bruker Avance III 500 spectrometer (Bruker Biospin) with tetramethylsilane (TMS) as an internal standard, and chemical shifts are expressed in d values The HR-ESI-MS was performed on a Micro O-QIITOF mass spectrometer (Bruker Daltonics) The CD spectra were measured in a Jasco J-805 spectropolarimeter Analytical and preparative TLC were carried out on precoated Merck Kieselgel 60F254 or RP-18F254 plates (0.25 or 0.5 mm thickness) * Correspondence to: Dr Mai Thanh Thi Nguyen, Faculty of Chemistry, University of Science, Vietnam National University, HoChiMinh City, Vietnam E-mail: nttmai@hcmus.edu.vn Copyright © 2012 John Wiley & Sons, Ltd Plant material The aerial part of B balsamifera was collected at Lam Dong province, Vietnam, in October 2008 and was identified by Ms Hoang Viet, Faculty of Biology, University of Science, National University Ho Chi Minh City A voucher sample of the aerial part (AN-2962) has been deposited at the Department of Analytical Chemistry of the University of Science, National University Ho Chi Minh City, Vietnam Extraction and isolation The aerial part of B balsamifera was cut into pieces and then part (2.1 kg) was extracted with MeOH (12 L, reflux, h  3) to yield a MeOH extract (180 g; IC50, 6.0 mg/mL) The MeOH extract was suspended in H2O and partitioned successively with hexane, CHCl3, EtOAc and H2O to yield hexane (57 g; IC50 > 100 mg/mL), CHCl3 (68 g; IC50, 5.0 mg/mL), EtOAc (32 g; IC50, 1.0 mg/mL) and H2O (15 g; IC50, > 100 mg/mL) fractions, respectively The CHCl3 fraction (62 g) was subjected to silica gel column (9  40 cm) chromatography eluted with MeOH/CHCl3 (0–30%) to give four fractions: fr.1 (5.7 g; IC50, > 100 mg/mL), fr.2 (9.1 g; IC50 1.4 mg/mL), fr.3 (11.2 g; IC50, 10.7 mg/mL) and fr.4 (15.7 g; IC50, 71.4 mg/ mL) Fraction was rechromatographed on silica gel with MeOH–CHCl3, followed by reversed-phase preparative TLC with CH3CN: MeOH: H2O = 1: 1: 3, to give Received 29 June 2011 Revised 13 October 2011 Accepted 17 October 2011 M.T.T NGUYEN AND N.T NGUYEN (6.7 mg) and (8.3 mg) Fraction was further separated by silica gel column chromatography, followed by reversed-phase preparative TLC with CH3CN: MeOH: H2O = 1: 1: 3, to give (9.2 mg) The EtOAc fraction (32 g) was subjected to silica gel column (7  40 cm) chromatography eluted with MeOH/CHCl3 (0–30%) to give six fractions: fr.1 (5.2 g; IC50, > 100 mg/mL), fr.2 (4.4 g; IC50, 3.5 mg/mL), fr.3 (4.6 g; IC50, 1.0 mg/mL), fr.4 (3.5 g; IC50, 0.5 mg/mL), fr.5 (13 g; IC50 4.7 mg/mL) and fr.6 (2 g; IC50, 55.9 mg/mL) Fraction was subjected to silica gel column chromatography with MeOH–CHCl3, followed by reversed-phase preparative TLC with CH3CN: MeOH: H2O = 1: 1: 4, to give (5.3 mg) and (14.6 mg) Fraction was separated by silica gel column chromatography with MeOH–CHCl3, followed by reversed-phase preparative TLC with CH3CN: MeOH: H2O = 2: 1: 3, to yield (2.5 mg) Fraction was separated by silica gel column chromatography with MeOH–CHCl3, followed by reversed-phase preparative TLC with CH3CN: MeOH: H2O = 1: 2: 3, to yield (7.2 mg) Fraction was separated by silica gel column chromatography with MeOH–CHCl3, followed by reversed-phase preparative TLC with MeOH: H2O = 1: 1, to yield (17.4 mg) and (7.2 mg) (2R,3S)-(À)-4′-O-methyldihydroquercetin (1) [a]25 D – 12.5 (c 0.5, CH3OH) IR (CHCl3) cmÀ1: 3400, 1660, 1605, 1450 1H- and 13C-NMR (CD3OD, 500 MHz), see Table HR-ESI-MS m/z: 319.0830 [M + H]+ (Calcd for C16H15O7: 319.0818) CD[θ]264 0, [θ]295 –29146, [θ]317 0, [θ]336 +13160 (c 0.5, CH3OH) (For further information, see supplementary data) XO inhibitory assay The XO inhibitory activity was assayed spectrophotometrically at 290 nm under aerobic conditions by using 96-well plates as described previously (Nguyen et al., 2004) The XO inhibitory activity was expressed as the percentage inhibition of XO in the above assay system, calculated as B 1À  100; A where A and B are the activities of the enzyme without and with test material The IC50 values were calculated from the mean values of data from four determinations Table 1H- and 13C-NMR data for compound in CD3OD (J values in parentheses) Position 10 1′ 2′ 3′ 4′ 5′ 6′ 4′-OCH3 dH 5.39 (1H, d, J = 3.5 Hz) 4.42 (1H, d, J = 3.5 Hz) 5.98 (1H, d, J = 2.0 Hz) 6.01 (1H, d, J = 2.0 Hz) 7.03 (1H, d, J = 2.0 Hz) 6.84 (1H, d, J = 8.5 Hz) 6.94 (1H, dd, J = 2.0, 8.5 Hz) 3.89 (3H, s) Copyright © 2012 John Wiley & Sons, Ltd dC 80.6 71.5 194.4 163.9 95.9 167.2 96.6 162.4 100.7 128.0 113.9 145.6 147.1 110.7 118.9 55.8 RESULTS AND DISCUSSION The dried aerial part of B balsamifera was extracted with refluxing MeOH, and the MeOH extract was suspended in H2O and partitioned successively with hexane, CHCl3, EtOAc and H2O to yield hexane, CHCl3 and H2O fractions The CHCl3 and EtOAc-soluble fractions showed XO inhibitory activity with IC50 values of 5.0 and 1.0 mg/ mL, respectively Further separation and purification of these fractions led to the isolation of a new dihydroflavonol (1), together with seven known compounds (2–8) The known compounds were identified as (2R,3R)-(+)-4′-Omethyldihydroquercetin (2) (Islam and Tahara, 2000), (2R,3R)-(+)-4′,7-di-O-methyldihydroquercetin (3) (Nessa et al., 2004), (2R,3R)-(+)-7-O-methyldihydroquercetin (4) (Harborne and Mabry, 1982); 5,7,3′,5′-tetrahydroxy flavanone (5) (Nessa et al., 2004), quercetin (6) (Nessa et al., 2004), quercetin-3,7,3′-trimethyl ether (7) (Kumari et al., 1986) and quercetin-3,3′,4′-trimethyl ether (8) (Urbatsch et al., 1976) (Fig 1) based on the spectroscopic analysis and comparison with literature data Compound showed a quasimolecular ion at m/z 319.0830 [M + H]+, corresponding to the molecular formula C16H15O7 in HR-ESI-MS Its IR spectrum displayed the absorbance of hydroxyl (3400 cm–1), phenyl (1605, 1450 cm–1) and carbonyl (1660 cm-1) groups The 1H NMR spectrum of compound (Table 1) revealed the presence of one methoxy group at d 3.89 (3H, s) and two oxymethine groups at d 5.39 (1H, d, J = 3.5 Hz, H-2) and 4.42 (1H, d, J = 3.5 Hz, H-3) In addition, it displayed three ABX type protons at d 7.03 (1H, d, J = 2.0 Hz, H-2′), 6.84 (1H, d, J = 8.5 Hz, H-5′) and 6.94 (1H, dd, J = 2.0, 8.5 Hz, H-6′) together with two singlets of a 1,2,4,5-tetrasubstituted benzene ring at d 5.98 (1H, d, J = 2.0 Hz, H6) and 6.01 (1H, d, J = 2.0 Hz, H-8) Moreover, the 13 C and DEPT NMR spectra exhibited signals for an methoxy, two oxymethines, five oxygenated aromatic quaternary carbons, an aromatic quaternary carbon, five aromatic carbons and one carbonyl carbon On the basis of the analysis of the COSY and HMQC spectra (Fig 2), was suggested to be a dihydroflavonol The location of methoxy group was deduced to be at C-4′, based on the HMBC correlations between methoxy protons and C-4′ On the other hand, in 2, the H-2 and H-3 protons each resonated as a doublet with J = 11.5 Hz reflecting the transdiaxial orientation normally found in naturally occurring 3-hydroxyflavanones (dihydroflavonols) However, both H-2 at d 5.39 and H-3 at d 4.42 of had a coupling constant of 3.5 Hz which suggests that in this compound the C-ring protons are related in a cis axial-equatorial fashion (Ingham et al., 1986) Moreover, the negative and positive CD bands at 295 and 336 nm in were similar to those of (2R,3S)-3′,5dihydroxy-4′,7-dimethoxydihydroflavonol suggested that the absolute configuration of this compound should be ‘2R, 3S’ (Islam and Tahara, 2000) Thus, the structure of was deduced as (2R,3S)-(À)-4′-O-methyldihydroquercetin The isolated compounds were tested for their XO inhibitory activity (Table 2) The assay was carried out at five different concentrations ranging from 0.2– 100 mM Compounds 1–4 and 6–8 possessed significant XO inhibitory activity in a concentration-dependent manner, and compounds 1, and showed more potent inhibitory activity, with IC50 values ranging from 0.23 to 1.91 mM, than that of a positive control allopurinol (IC50, 2.5 mM), a well known XO inhibitor used clinically for treatment of gout (Oettl and Reibnegger, 1999) (Fig 3) Phytother Res (2012) XANTHINE OXIDASE INHIBITORS FROM B BALSAMIFERA L OR3 OH OR4 R R1O O H R2 R1O R5 O OR2 R3 OH O OH O R1 = R2 = H, R3 = OH, R4 = OCH3, R5 = H R1 = H, R2 = OH, R3 = H, R4 = OCH3, R5 = H R1 = CH3, R2 = OH, R3 = H, R4 = OCH3, R5 = H R1 = CH3, R2 = OH, R3 = H, R4 = OH, R5 = H R1 = R2 = R3 = R4 = H, R5 = OH R = R2 = R3 = R4 = H R1 = R2 = R3 = CH3, R4 = H R1 = H, R2 = R3 = R4 = CH3 Figure Structures of the isolated compounds from B balsamifera 100 OH OCH3 Inhibition (%) HO 80 O OH OH 60 40 20 O Figure Connectivity (bold line) deduced by the COSY spectrum and significant HMBC correlations (arrow) observed for 0 0.2 20 50 100 Concentration (µM) Table Xanthine oxidase inhibitory activity of the isolated compounds Compound IC50 (mM) Allopurinol 0.23 Ỉ 0.01 3.04 Ỉ 0.12 6.08 Ỉ 0.08 5.72 Ỉ 0.13 > 100 1.91 Æ 0.02 9.53 Æ 0.14 1.28 Æ 0.01 2.50 Æ 0.01 Each value represents the mean Ỉ SD of four determinations The activity of these compounds greatly depended upon the nature of the substitution in the flavonoids The compounds having the hydroxyl groups at C-5 and C-7 and the double bond between C2 and C3 in the ring C (6 and 8) displayed stronger XO inhibitory activity than those of the other compounds (2–5, 7), except These results are in agreement with the previous report on the structure–activity relationship of flavonoids and their XO inhibitory activity (Cos et al., 1998) All isolated flavonols (6–8) possessed stronger activity than that of All Figure Dose-dependent inhibition of XO by 1, 6, and allopurinol the positive control allopurinol and the presence of methoxyl groups at C-3, C-7, C-3′/C-4′ (7 and 8) instead of hydroxyl group in showed slightly improved activity Among the five flavanones, compounds having a hydroxyl group at C-3 (1–4) displayed strong activities with IC50 values smaller than that of without the hydroxyl group at C-3 (Table 2) In addition, the presence of an a-OH group at C-3 (1) showed more potent activity than that of the b-OH group at the same position (2) In conclusion, the traditional use of B balsamifera for the treatment of rheumatism and inflammatory diseases in Vietnam may be attributable to the XO inhibitory activity of the flavonoid constituents Acknowledgements This work was supported by grant 104.01.68.09 from Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) Conflict of Interest The authors declare no conflict of interest REFERENCES Cos P, Ying L, Calomme M et al 1998 Structure-activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers J Nat Prod 61: 71–76 Do TL 2001 Vietnamese Medicinal Plant Medicine Publisher: Hanoi Copyright © 2012 John Wiley & Sons, Ltd Fazilatun N, Zhari I, Nornisah M, Rosemal HMH 2001 Phytochemical investigation on Blumea balsamifera DC J Trop Med Plants 2: 17–22 Harborne JB, Mabry TJ 1982 The Flavonoids: Advances In Research Chapman and Hall: London, 70–107 Phytother Res (2012) M.T.T NGUYEN AND N.T NGUYEN Ingham JL, Tahara S, Dziedzic SZ 1986 New 3-hydroxyflavanone (dihydroflavonol) phytoalexins from the Papiolionate legume Shuteria vestita J Nat Prod 49: 631–638 Islam MT, Tahara S 2000 Dihydroflavonols from Lannea coromandelica Phytochemistry 54: 901–907 Kumari GN, Rao LJM, Rao NSP 1986 13C NMR data of flavonol methyl ethers of Solanum pubescens Chem Sci 97: 171–176 Nessa F, Ismail Z, Mohamed N, Haris MRHM 2004 Free radicalscavenging activity of organic extracts and of pure Copyright © 2012 John Wiley & Sons, Ltd flavonoids of Blumea balsamifera DC leaves Food Chem 88: 243–252 Nguyen MTT, Awale S, Tezuka Y, Tran LQ, Watanabe H, Kadota S 2004 Xanthine oxidase inhibitory activity of Vietnamese medicinal plants Biol Pharm Bull 27: 1414–1421 Oettl K, Reibnegger G 1999 Protein structure and molecular enzymology Biochim Biophys Acta 1430: 387–395 Urbatsch LE, Mabry TJ, Miyakado M, Ohno N, Yoshioka H 1976 Flavonol methyl ethers from Ericameria diffusa Phytochemistry 15: 440–441 Phytother Res (2012) ... calculated as B 1À  100; A where A and B are the activities of the enzyme without and with test material The IC50 values were calculated from the mean values of data from four determinations... al 1998 Structure-activity relationship and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers J Nat Prod 61: 71–76 Do TL 2001 Vietnamese Medicinal Plant Medicine... smaller than that of without the hydroxyl group at C-3 (Table 2) In addition, the presence of an a-OH group at C-3 (1) showed more potent activity than that of the b-OH group at the same position