Journal of Ethnopharmacology 133 (2011) 11171120

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Ethnopharmacological communication

Tetrahydroamentoavone (THA) from Semecarpus anacardium as a potent inhibitor of xanthine oxidase

a r t i c l e

i n f o

a b s t r a c t
Ethnopharmacological relevance: Seed of Semecarpus anacardium L. is widely used in Indian traditional medicine; Ayurveda and Sidha, for treatment of inammatory disorders and gout. Aim of the study: The present study was aimed at isolation of a compound for its potential to inhibit xanthine oxidase (XO), over expression of which lead to inammation and gout. Materials and methods: Activity guided fractionation of S. anacardium seed was conducted using liquidliquid partition and preparative HPLC. The fractions were evaluated for their XO inhibition and antioxidant activity. The ethyl acetate fraction with the highest XO activity yielded a biavonoid compound tetrahydroamentoavone (THA). LineweaverBurk (LB) plot for the XO inhibition of THA and allopurinol was constructed from the kinetic data. Results: IC50 values of THA and allopurinol for XO inhibition were 92 and 100 nM respectively and their corresponding values for Ki were 0.982 and 0.612 M respectively. Conclusion: THA was a potent XO inhibitor which could be considered as a drug candidate or chemopreventive agent, after establishing its pharmacological and clinical evaluation. The study results appear to support the claim of the traditional medicine with respect to the efcacy of S. anacardium seed against inammation and gout. 2010 Published by Elsevier Ireland Ltd.

Article history: Received 23 July 2008 Received in revised form 9 September 2010 Accepted 8 October 2010 Available online 20 October 2010 Keywords: Semecarpus anacardium Xanthine oxidase Gout Biavonoid Tetrahydroamentoavone (THA)

1. Introduction Semecarpus anacardium L. is a deciduous tree belonging to Anacardiaceae family and is growing in tropical and temperate regions of South East Asian countries. Its seed, commonly known as marking nut is largely used in Indian traditional medicine Ayurveda for the treatment of rheumatoid arthritis, gout and other inammatory diseases, tumours, asthma, epilepsy, psoriasis and leprosy (Khare, 2007). A wide spectrum of physiological activities including anti-inammatory (Ramprasath et al., 2004), antiarthritic (Singh et al., 2006) and anticancer (Mathivadhani et al., 2007) properties have been attributed to the seed and its preparations. The phytochemical investigations of the seed revealed the presence of a variety of bioactive compounds such as phenolics, biavonoids, bhilawanols and sterols (Gedam et al., 1974). A unique array of biavonoid compounds including semecarpuavanone, jeediavanone, galluavanone, semecarpetin and anacarduavone (Murthy, 1983, 1984, 1985, 1988) has been reported in the seeds. Tetrahydroamentoavone (THA) has been reported as the major anti-inammatory compound in the seed extract (Selvam and Jachak, 2004).

Though the preparations of S. anacardium seed are recommended in Indian traditional medicines for gout, no studies have been conducted to identify the active compounds and mechanism of action. It was in this context that the present study was designed to investigate the S. anacardium seeds for its XO inhibitory activity and to isolate and identify the active principles responsible. Since the oxidative stress and reactive oxygen species may also contribute to inammation and gout, the seed extracts were also evaluated for their antioxidant (AO) potency using various in vitro methods. 2. Materials and methods 2.1. Plant material Dried S. anacardium seeds were collected from authorised herbal suppliers in Thiruvananthapuram, India with help of an Ayurveda medicinal practitioner on January 2008 and were authenticated by Department of Indian System of Medicine, Government of India, Delhi. The samples were stored in air tight containers at 4 C till they were used. 2.2. Chemicals and reagents

Corresponding author. Tel.: +91 471 2492901; fax: +91 471 2495050. E-mail address: carumughan@yahoo.com (C. Arumughan). 0378-8741/$ see front matter 2010 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2010.10.027

ABTS, DPPH, ferrozine, NBT, allopurinol, xanthine, XO, trolox, ferrous chloride, ascorbic acid and gallic acid were purchased from

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R. Arimboor et al. / Journal of Ethnopharmacology 133 (2011) 11171120

Fig. 1. Semi-preparative HPLC chromatogram of ethyl acetate fraction of S. anacardium seed methanol extract at 280 nm.

Sigma Aldrich (St. Louis, MO, USA). Other reagents and solvents used were of analytical grade. 2.3. Preparation of seed extracts and isolation of THA Dried seeds of S. anacardium (500 g) were crushed well and defatted with hexane (2 L) and then extracted with methanol (2 L) in a Soxhlet apparatus. The extract was ltered and desolventised under vacuum at 40 C. The extract was suspended in water (1 L) and sequentially partitioned with hexane (3 200 mL), ethyl acetate (3 200 mL) and butanol (3 200 mL). These fractions were dried and evaluated for their XO inhibitory capacity. Further fractionation of the active ethyl acetate extract was achieved by semi-preparative HPLC. A Shimadzu HPLC (LC 8A) equipped with a PDA (SPD 10AV) detector and a C18 Phenomenex column (15 , 250 mm 21.20 mm) was used for the separation. The separation was performed with a gradient of methanol (solvent B) in water (solvent A) as mobile phase with a ow rate of 25 mL/min. This was programmed as 05 min 30% B, 520 min 50% B, 3560 min 100% B and 6090 min 100% B. The chromatogram (Fig. 1) was monitored at 280 nm and F1 to F5 fractions were collected. The fraction F2 with the highest XO inhibition capacity yielded an off white powder which was further puried by recrystallizing from methanol and was identied as THA by comparing the spectral data with an earlier report (Selvam and Jachak, 2004). Spectral data. UV (Shimadzu UVVIS 2450) max (methanol): 290 nm; FAB-MS (The MS Route JMS 600H, JEOL) (m/z): 543.2, 307.4, 176.2 and 154.2; FTIR (Perkin Elmer 100 series) (cm1 ): 3436 and 637 and 1 H NMR (Avance DPX 300, Bruker) (ppm) (CD3 OD, 300 MHz): 7.1(4H, m), 6.78 (1H, d J = 7.80), 6.6 (2H, d, J = 7.83), 5.9 (1H, s), 5.7 (2H, s), 5.2 (2H, m), 2.9 (2H, br m), 2.75 (2H, br m). 2.4. XO Inhibitory activity XO inhibition capacity was measured by monitoring uric acid formation at 290 nm in xanthine-XO system (Osada et al., 1993). The assay system consisted of 1 mL reaction mixture containing 0.1 U/mL XO with 0.2 mL xanthine (0.26 M) in 50 mM phosphate buffer (pH = 7.4). The reaction was initiated by adding the enzyme with or without inhibitors and the change in absorbance of the mixture at 290 nm for 10 min was measured against a reagent blank. Control without test compounds was prepared in order to have

the maximum uric acid formation in the reaction system. Allopurinol was used as a positive control. The substrate concentration was kept less than 1 M to avoid the substrate inhibition. The IC50 values for the inhibition were calculated from the % inhibition. 2.5. LineweaverBurk (LB) plots In order to determine the mode of inhibition, kinetic studies were carried out in the absence and presence of THA with varying concentrations of xanthine (0.10.7 M) as substrate. The reaction mixture contained 200 L xanthine, 100 L THA (1050 g), 50 L XO (0.1 U/mL) and 50 mM phosphate buffer in a total volume of 1 mL. The enzyme activity was determined spectrophotometrically by measuring the uric acid formation at 290 nm. The data obtained from the kinetic studies were transformed into LB plot. 2.6. AO activity assays ABTS radical scavenging activity was estimated according to the method by Re et al. (1999) and the radical scavenging activity of the samples was expressed as trolox equivalent antioxidant capacity (TEAC). DPPH radical scavenging activity was measured by the method by Brand-Williams et al. (1995) following the slight modications suggested by Benherlal and Arumughan (2007). Superoxide radical anion scavenging assay was performed by method of Parejo et al. (2002) following the slight modications as suggested by Benherlal and Arumughan (2007). The DPPH and superoxide radical scavenging activities of samples were compared in terms of their IC50 ( g/mL) values. Fe(III) reducing power was estimated by potassium ferricyanide-ferrous chloride method (Zhu et al., 2002) and the reducing powers of samples were expressed as vitamin C equivalents. Metal chelation efcacy of extracts/compounds was estimated as their ability to chelate ferrous ions by the method of Dinis et al., 1994 and the efcacy of the samples was expressed in terms of EDTA. 2.7. Statistical analysis All the experiments were carried out ve times and the results were expressed as mean SD. One way analysis of variance was performed by ANOVA procedures. Signicant differences between

R. Arimboor et al. / Journal of Ethnopharmacology 133 (2011) 11171120 Table 1 XO inhibition, radical scavenging, Fe(II) chelation and Fe(III) reduction efciencies of S. anacardium seed extracts and THA (Mean SD, n = 5). Extract/compound Methanol extract Hexane fraction Ethyl acetate fraction Butanol fraction Water fraction Allopurinol THA Gallic acid N.S, not signicant. XO inhibition IC50 ( g/mL) 253 891 156 493 378 14 50 129 9 12 5 10 8 1 3 4 DPPH IC50 ( g/mL) 9.2 0.2 N.S 25.3 1.9 14.2 1.2 4.9 0.9 N.S 344.3 10.2 1.2 0.0 TEAC (nM) 2.09 0.07 N.S 0.06 0.01 0.44 0.08 5.16 0.93 N.S 0.11 0.02 3.91 0.10 SRS IC50 ( g/mL) 25.5 75.7 22.7 65.3 38.7 3.6 2.9 11.9 1.0 5.9 2.1 4.7 1.7 0.8 0.5 0.2 Fe(II) chelation/ 100 g (nM of EDTA) 13 1 N.S N.S 70 19 1 N.S N.S 33 3 Fe(III) reduction/ 100 g (nM of Vit. C) 48 1 N.S 10 1 31 2 119 7 N.S N.S 946 27

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means were determined by Students t-test and p < 0.05 was regarded as signicant. 3. Results XO inhibitory capacity of seed methanol extract and its fractions was evaluated and the results are shown in Table 1. Among the seed extracts, methanol extract (IC50 = 253 g) had the highest (p 0.05) XO inhibitory capacity. Further fractionation of methanol extract resulted in the enrichment of the XO inhibitory activity in ethyl acetate fraction (IC50 = 156 g) (p 0.05). This fraction on preparative HPLC separation yielded a biavonoid compound as the major XO inhibitory principle in the seed. The compound thus isolated was further puried and conrmed as THA by comparing with the reported spectral data (Selvam and Jachak, 2004) (Fig. 1). THA (IC50 = 92 nM (50 g)) inhibited XO in a dose dependent manner with an efcacy close to that of a standard XO inhibitor drug allopurinol (IC50 = 100 nM (14 g)). Kinetic studies were carried out and LB plot was drawn in order to determine the mode of XO inhibition of THA and allopurinol. In LB plot THA revealed a linear non-competitive inhibitory activity with Ki value of 0.982 M, whereas, allopurinol had competitive XO inhibitory nature which is in agreement with the earlier report (Lin et al., 2002). Allopurinol showed a lower (p 0.05) Ki value (0.612 M) than that for THA. The AO efcacy of the methanol extract and its fractions and THA was also evaluated using various in vitro models and compared with that of a reference phenolic AO gallic acid and the results are given in Table 1. Seed Methanol extract showed signicantly high DPPH, ABTS and superoxide radical scavenging, Fe(II) chelation and Fe(III) reduction capacities. The AO capacity values shown by gallic acid under the experimental conditions were signicantly higher (p 0.05) than those for methanol extract. Further fractionation of methanol extract resulted in the enrichment of AO activities except superoxide radical scavenging in its water fraction (p 0.05). Similar to XO inhibition, the superoxide radical scavenging activity enriched in its ethyl acetate fraction (p 0.05). THA showed poor DPPH and ABTS radical scavenging, metal chelation and Fe(III) reduction efcacies. The low radical scavenging capacities shown by THA, allopurinol and the ethyl acetate fraction indicated that their high superoxide radical scavenging activity in xanthine-XO system could be mainly due to their XO inhibitory potential rather than their radical scavenging efcacy. 4. Discussion Gout is a hyperurecemic condition associated with the deposition of sodium urate crystals in the joints. Hyperurecemic condition caused by the over expression of xanthine oxidase (XO) and subsequent excess production of uric acid could be controlled by the inhibition of increased XO activity (Terkeltaub et al., 2006). Allopurinol, a widely recommended drug for gout is a potent competitive XO inhibitor. Allopurinol and similar analogues are reported to have

severe side effects like hepatitis, nephropathy and drug intolerance, therefore search for alternatives with least side effects has been actively pursued (Osada et al., 1993). In the present study, activity guided fractionation of S. anacardium seed yielded a noncompetitive XO inhibitory compound, THA with an efcacy close to that of allopurinol. There are reports related to avonoids such as apigenin, quercetin, myricetin, isovitexin, and genistein as potential inhibitors of XO with Ki values varying from 0.6 to 5.0 M (Lin et al., 2002, Nagao et al., 1999). Structure function correlation for XO inhibition has been demonstrated for some avonoids (Cos et al., 1998, Lin et al., 2002). According to these authors, hydroxyl groups at C5 and C7 and CO at C4 positions in avonoids facilitate XO inhibition by electrostatic interaction and hydrogen bonding at the active site and therefore could be competing with the substrate, xanthine. In this study, THA was found to be a strong non-competitive XO inhibitor. THA possesses six, OH groups with two occupying at C5 and C7 and CO at C4 positions (Fig. 1) meeting the requirements reported for the XO inhibiting avonoids. However, THA differs from other avonoids in terms of its high molecular weight (Mol. Wt. 542), higher number of hydroxyl groups (six), bulkiness and non-planarity. Such structural differences from other avonoids could be attributed to its non-competitive nature of XO inhibition as observed here. The AO activity evaluation showed that XO inhibitiory efcacy of seed extracts and THA was independent of their radical scavenging capacities. The high XO inhibitory potential of THA along with its considerable abundance in S. anacardium seed (1% in dry seeds) (Aravind et al., 2008), made this compound a potential drug candidate or a chemopreventive of commercial signicance for controlling gout. A process has been developed by the authors to isolate THA with an yield of 0.5% on seed weight (data unpublished).

5. Conclusion XO inhibitory activity guided fractionation of S anacardium seeds yielded a biavonoid THA as the active principle. Kinetic studies indicated that THA was a non-competitive XO inhibitor with an inhibitory potency close to that of the standard drug allopurinol. Having developed a process to isolate THA in large quantity, further studies related to its bio-availability, toxicity including clinical trials using the active principle, THA are under way.

Acknowledgements This study was conducted as a part of a National Programme for the standardisation and validation of medicinal plants and their formulations. The grant received for this study from AYUSH (Ayurveda, Yoga, Unani, Sidha and Homeopathy), Government of India is gratefully acknowledged.

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