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Journal of Ethnopharmacology 133 (2011) 1117–1120

Contents lists available at ScienceDirect

Journal of Ethnopharmacology

journa l homepage: www.e lsev ier .com/ locate / je thpharm

thnopharmacological communication

etrahydroamentoflavone (THA) from Semecarpus anacardium as a potentnhibitor of xanthine oxidase

anjith Arimboor, Meena Rangan, S.G. Aravind, C. Arumughan ∗

groprocessing and Natural Products Division, National Institute for Interdisciplinary Science and Technology (CSIR), Trivandrum 695019, India

r t i c l e i n f o

rticle history:eceived 23 July 2008eceived in revised form 9 September 2010ccepted 8 October 2010vailable online 20 October 2010

eywords:emecarpus anacardiumanthine oxidase

a b s t r a c t

Ethnopharmacological relevance: Seed of Semecarpus anacardium L. is widely used in Indian traditionalmedicine; Ayurveda and Sidha, for treatment of inflammatory disorders and gout.Aim of the study: The present study was aimed at isolation of a compound for its potential to inhibitxanthine oxidase (XO), over expression of which lead to inflammation and gout.Materials and methods: Activity guided fractionation of S. anacardium seed was conducted usingliquid–liquid partition and preparative HPLC. The fractions were evaluated for their XO inhibition andantioxidant activity. The ethyl acetate fraction with the highest XO activity yielded a biflavonoid com-pound tetrahydroamentoflavone (THA). Lineweaver–Burk (LB) plot for the XO inhibition of THA and

outiflavonoidetrahydroamentoflavone (THA)

allopurinol was constructed from the kinetic data.Results: IC50 values of THA and allopurinol for XO inhibition were 92 and 100 nM respectively and theircorresponding 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 chemopre-ventive agent, after establishing its pharmacological and clinical evaluation. The study results appear to

tradit

support the claim of theinflammation and gout.

. Introduction

Semecarpus anacardium L. is a deciduous tree belonging tonacardiaceae family and is growing in tropical and temperateegions of South East Asian countries. Its seed, commonly knowns ‘marking nut’ is largely used in Indian traditional medicine

Ayurveda’ for the treatment of rheumatoid arthritis, gout andther inflammatory diseases, tumours, asthma, epilepsy, psoria-is and leprosy (Khare, 2007). A wide spectrum of physiologicalctivities including anti-inflammatory (Ramprasath et al., 2004),ntiarthritic (Singh et al., 2006) and anticancer (Mathivadhani et al.,007) properties have been attributed to the seed and its prepara-ions. The phytochemical investigations of the seed revealed theresence of a variety of bioactive compounds such as phenolics,iflavonoids, bhilawanols and sterols (Gedam et al., 1974). A uniquerray of biflavonoid compounds including semecarpuflavanone,eediflavanone, galluflavanone, semecarpetin and anacarduflavoneMurthy, 1983, 1984, 1985, 1988) has been reported in the seeds.

etrahydroamentoflavone (THA) has been reported as the majornti-inflammatory compound in the seed extract (Selvam andachak, 2004).

∗ Corresponding author. Tel.: +91 471 2492901; fax: +91 471 2495050.E-mail address: carumughan@yahoo.com (C. Arumughan).

378-8741/$ – see front matter © 2010 Published by Elsevier Ireland Ltd.oi:10.1016/j.jep.2010.10.027

ional medicine with respect to the efficacy of S. anacardium seed against

© 2010 Published by Elsevier Ireland Ltd.

Though the preparations of S. anacardium seed are recom-mended in Indian traditional medicines for gout, no studies havebeen conducted to identify the active compounds and mechanismof action. It was in this context that the present study was designedto investigate the S. anacardium seeds for its XO inhibitory activ-ity and to isolate and identify the active principles responsible.Since the oxidative stress and reactive oxygen species may alsocontribute to inflammation and gout, the seed extracts were alsoevaluated for their antioxidant (AO) potency using various in vitromethods.

2. Materials and methods

2.1. Plant material

Dried S. anacardium seeds were collected from authorised herbalsuppliers in Thiruvananthapuram, India with help of an ‘Ayurveda’medicinal practitioner on January 2008 and were authenticated byDepartment of Indian System of Medicine, Government of India,Delhi. The samples were stored in air tight containers at 4 ◦C tillthey were used.

2.2. Chemicals and reagents

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

1118 R. Arimboor et al. / Journal of Ethnopharmacology 133 (2011) 1117–1120

tate fr

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Fig. 1. Semi-preparative HPLC chromatogram of ethyl ace

igma Aldrich (St. Louis, MO, USA). Other reagents and solventssed were of analytical grade.

.3. Preparation of seed extracts and isolation of THA

Dried seeds of S. anacardium (500 g) were crushed well andefatted with hexane (2 L) and then extracted with methanol2 L) in a Soxhlet apparatus. The extract was filtered and desol-entised under vacuum at 40 ◦C. The extract was suspended inater (1 L) and sequentially partitioned with hexane (3 × 200 mL),

thyl acetate (3 × 200 mL) and butanol (3 × 200 mL). These fractionsere dried and evaluated for their XO inhibitory capacity. Further

ractionation of the active ethyl acetate extract was achieved byemi-preparative HPLC. A Shimadzu HPLC (LC 8A) equipped withPDA (SPD 10AV) detector and a C18 Phenomenex column (15 �,50 mm × 21.20 mm) was used for the separation. The separationas performed with a gradient of methanol (solvent B) in water

solvent A) as mobile phase with a flow rate of 25 mL/min. This wasrogrammed as 0–5 min 30% B, 5–20 min 50% B, 35–60 min 100% Bnd 60–90 min 100% B. The chromatogram (Fig. 1) was monitoredt 280 nm and F1 to F5 fractions were collected. The fraction F2ith the highest XO inhibition capacity yielded an off white pow-er which was further purified by recrystallizing from methanolnd was identified as THA by comparing the spectral data with anarlier report (Selvam and Jachak, 2004).

Spectral data. UV (Shimadzu UV–VIS 2450) �max (methanol):90 nm; FAB-MS (The MS Route JMS 600H, JEOL) (m/z): 543.2,07.4, 176.2 and 154.2; FTIR (Perkin Elmer 100 series) (cm−1): 3436nd 637 and 1H NMR (Avance DPX 300, Bruker) ı(ppm) (CD3OD,00 MHz): 7.1(4H, m), 6.78 (1H, d J = 7.80), 6.6 (2H, d, J = 7.83), 5.91H, s), 5.7 (2H, s), 5.2 (2H, m), 2.9 (2H, br m), 2.75 (2H, br m).

.4. XO Inhibitory activity

XO inhibition capacity was measured by monitoring uric acidormation at 290 nm in xanthine-XO system (Osada et al., 1993).he assay system consisted of 1 mL reaction mixture containing

.1 U/mL XO with 0.2 mL xanthine (0.26 �M) in 50 mM phosphateuffer (pH = 7.4). The reaction was initiated by adding the enzymeith or without inhibitors and the change in absorbance of the mix-

ure at 290 nm for 10 min was measured against a reagent blank.ontrol without test compounds was prepared in order to have

action of S. anacardium seed methanol extract at 280 nm.

the maximum uric acid formation in the reaction system. Allop-urinol was used as a positive control. The substrate concentrationwas kept less than 1 �M to avoid the substrate inhibition. The IC50values for the inhibition were calculated from the % inhibition.

2.5. Lineweaver–Burk (LB) plots

In order to determine the mode of inhibition, kinetic studieswere carried out in the absence and presence of THA with varyingconcentrations of xanthine (0.1–0.7 �M) as substrate. The reac-tion mixture contained 200 �L xanthine, 100 �L THA (10–50 �g),50 �L XO (0.1 U/mL) and 50 mM phosphate buffer in a total volumeof 1 mL. The enzyme activity was determined spectrophotomet-rically by measuring the uric acid formation at 290 nm. Thedata obtained from the kinetic studies were transformed into LBplot.

2.6. AO activity assays

ABTS radical scavenging activity was estimated according to themethod by Re et al. (1999) and the radical scavenging activity ofthe samples was expressed as trolox equivalent antioxidant capac-ity (TEAC). DPPH radical scavenging activity was measured by themethod by Brand-Williams et al. (1995) following the slight modifi-cations suggested by Benherlal and Arumughan (2007). Superoxideradical anion scavenging assay was performed by method of Parejoet al. (2002) following the slight modifications as suggested byBenherlal and Arumughan (2007). The DPPH and superoxide rad-ical scavenging activities of samples were compared in terms oftheir IC50 (�g/mL) values. Fe(III) reducing power was estimated bypotassium ferricyanide-ferrous chloride method (Zhu et al., 2002)and the reducing powers of samples were expressed as vitamin Cequivalents. Metal chelation efficacy of extracts/compounds wasestimated as their ability to chelate ferrous ions by the method ofDinis et al., 1994 and the efficacy of the samples was expressed interms of EDTA.

2.7. Statistical analysis

All the experiments were carried out five times and the resultswere expressed as mean ± SD. One way analysis of variance wasperformed by ANOVA procedures. Significant differences between

R. Arimboor et al. / Journal of Ethnopharmacology 133 (2011) 1117–1120 1119

Table 1XO inhibition, radical scavenging, Fe(II) chelation and Fe(III) reduction efficiencies of S. anacardium seed extracts and THA (Mean ± SD, n = 5).

Extract/compound XO inhibition IC50

(�g/mL)DPPH IC50

(�g/mL)TEAC (nM) SRS IC50

(�g/mL)Fe(II) chelation/100 �g (nM of EDTA)

Fe(III) reduction/100 �g (nM of Vit. C)

Methanol extract 253 ± 9 9.2 ± 0.2 2.09 ± 0.07 25.5 ± 1.0 13 ± 1 48 ± 1Hexane fraction 891 ± 12 N.S N.S 75.7 ± 5.9 N.S N.SEthyl acetate fraction 156 ± 5 25.3 ± 1.9 0.06 ± 0.01 22.7 ± 2.1 N.S 10 ± 1Butanol fraction 493 ± 10 14.2 ± 1.2 0.44 ± 0.08 65.3 ± 4.7 7 ± 0 31 ± 2Water fraction 378 ± 8 4.9 ± 0.9 5.16 ± 0.93 38.7 ± 1.7 19 ± 1 119 ± 7Allopurinol 14 ± 1 N.S N.S 3.6 ± 0.8 N.S N.S

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THA 50 ± 3 344.3 ± 10.2 0.11 ± 0.02Gallic acid 129 ± 4 1.2 ± 0.0 3.91 ± 0.10

.S, not significant.

eans were determined by Student’s t-test and p < 0.05 wasegarded as significant.

. Results

XO inhibitory capacity of seed methanol extract and its frac-ions was evaluated and the results are shown in Table 1. Amonghe seed extracts, methanol extract (IC50 = 253 �g) had the highestp ≤ 0.05) XO inhibitory capacity. Further fractionation of methanolxtract resulted in the enrichment of the XO inhibitory activityn ethyl acetate fraction (IC50 = 156 �g) (p ≤ 0.05). This fraction onreparative HPLC separation yielded a biflavonoid compound ashe major XO inhibitory principle in the seed. The compound thussolated was further purified and confirmed as THA by comparing

ith the reported spectral data (Selvam and Jachak, 2004) (Fig. 1).HA (IC50 = 92 nM (50 �g)) inhibited XO in a dose dependent man-er with an efficacy close to that of a standard XO inhibitor drugllopurinol (IC50 = 100 nM (14 �g)). Kinetic studies were carried outnd LB plot was drawn in order to determine the mode of XOnhibition of THA and allopurinol. In LB plot THA revealed a lin-ar non-competitive inhibitory activity with Ki value of 0.982 �M,hereas, allopurinol had competitive XO inhibitory nature which

s in agreement with the earlier report (Lin et al., 2002). Allopurinolhowed a lower (p ≤ 0.05) Ki value (0.612 �M) than that for THA.

The AO efficacy of the methanol extract and its fractions andHA was also evaluated using various in vitro models and comparedith that of a reference phenolic AO gallic acid and the results are

iven in Table 1. Seed Methanol extract showed significantly highPPH, ABTS and superoxide radical scavenging, Fe(II) chelation ande(III) reduction capacities. The AO capacity values shown by galliccid under the experimental conditions were significantly higherp ≤ 0.05) than those for methanol extract. Further fractionation of

ethanol extract resulted in the enrichment of AO activities exceptuperoxide radical scavenging in its water fraction (p ≤ 0.05). Sim-lar to XO inhibition, the superoxide radical scavenging activitynriched in its ethyl acetate fraction (p ≤ 0.05). THA showed poorPPH and ABTS radical scavenging, metal chelation and Fe(III)

eduction efficacies. The low radical scavenging capacities showny THA, allopurinol and the ethyl acetate fraction indicated thatheir high superoxide radical scavenging activity in xanthine-XOystem could be mainly due to their XO inhibitory potential ratherhan their radical scavenging efficacy.

. Discussion

Gout is a hyperurecemic condition associated with the deposi-ion of sodium urate crystals in the joints. Hyperurecemic condition

aused by the over expression of xanthine oxidase (XO) and sub-equent excess production of uric acid could be controlled by thenhibition of increased XO activity (Terkeltaub et al., 2006). Allop-rinol, a widely recommended drug for gout is a potent competitiveO inhibitor. Allopurinol and similar analogues are reported to have

2.9 ± 0.5 N.S N.S11.9 ± 0.2 33 ± 3 946 ± 27

severe side effects like hepatitis, nephropathy and drug intoler-ance, therefore search for alternatives with least side effects hasbeen actively pursued (Osada et al., 1993). In the present study,activity guided fractionation of S. anacardium seed yielded a non-competitive XO inhibitory compound, THA with an efficacy close tothat of allopurinol. There are reports related to flavonoids such asapigenin, quercetin, myricetin, isovitexin, and genistein as poten-tial inhibitors of XO with Ki values varying from 0.6 to 5.0 �M (Linet al., 2002, Nagao et al., 1999). Structure function correlation for XOinhibition has been demonstrated for some flavonoids (Cos et al.,1998, Lin et al., 2002). According to these authors, hydroxyl groupsat C5 and C7 and CO at C4 positions in flavonoids facilitate XO inhibi-tion by electrostatic interaction and hydrogen bonding at the activesite and therefore could be competing with the substrate, xanthine.In this study, THA was found to be a strong non-competitive XOinhibitor. THA possesses six, OH groups with two occupying at C5and C7 and CO at C4 positions (Fig. 1) meeting the requirementsreported for the XO inhibiting flavonoids. However, THA differsfrom other flavonoids in terms of its high molecular weight (Mol.Wt. 542), higher number of hydroxyl groups (six), bulkiness andnon-planarity. Such structural differences from other flavonoidscould be attributed to its non-competitive nature of XO inhibi-tion as observed here. The AO activity evaluation showed that XOinhibitiory efficacy of seed extracts and THA was independent oftheir radical scavenging capacities. The high XO inhibitory poten-tial of THA along with its considerable abundance in S. anacardiumseed (1% in dry seeds) (Aravind et al., 2008), made this compound apotential drug candidate or a chemopreventive of commercial sig-nificance for controlling gout. A process has been developed by theauthors to isolate THA with an yield of 0.5% on seed weight (dataunpublished).

5. Conclusion

XO inhibitory activity guided fractionation of S anacardiumseeds yielded a biflavonoid THA as the active principle. Kineticstudies indicated that THA was a non-competitive XO inhibitorwith an inhibitory potency close to that of the standard drugallopurinol. Having developed a process to isolate THA in largequantity, further studies related to its bio-availability, toxicityincluding clinical trials using the active principle, THA are underway.

Acknowledgements

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

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