XANTHINE OXIDASE INHIBITORY ACTIVITY of the HYDRO-ETHANOLIC EXTRACTS of SELECTED INDIAN MEDICINAL PLANTS B.Meera 1, 2, Rajeswary Hari1, P.K

Journal of Xi'an University of Architecture & Technology Issn No : 1006-7930

XANTHINE OXIDASE INHIBITORY ACTIVITY OF THE HYDRO-ETHANOLIC EXTRACTS OF SELECTED INDIAN MEDICINAL PLANTS B.Meera 1, 2, Rajeswary Hari1, P.K. Siva3 1Department of Biotechnology, Dr.MGR Educational & Research Institute, Maduravoyal, Chennai, India. 2Department of Biochemistry, Soka Ikeda College of Arts and Science, Madhanangkuppam, Chennai, India. 3PG and Research Department of Botany, J.J.College of Arts and Science, Pudukkottai, Tamil Nadu - 622 422. Abstract Introduction : The search for novel xanthine oxidase (XO) inhibitors using an optimized protocol we have screened the Hydro - Ethanolic extracts of 15 medicinal plants belonging 10 families, regardless of their claimed ethanopharmacological and /or food uses . Methods : The Super oxide, Nitric oxide, hydrogen peroxide radical scavenging activity and Total antioxidant activity of these extracts were investigated employing various established in vitro systems. Total phenolic and flavanoid content were also determined. Results : The Xanthine oxidase enzyme inhibitory and the antioxidant activity of Hydro -Ethanolic plant extract were found to be in the following order, Trigonella foenum > Nigella sativa > Piper longum> Brassica juncea > Piper nigrum > Cuminum cyminum > Cinnamomum zeylanicum > Zingiber officinale > Foeniculum vulgare > Cinnamomum tamala > Coriandrum sativum > Murraya koenigii > Syzygium aromaticum > Elettaria cardamomum >Prunus amygdalus. Among them the extracts such as Trigonella foenum , Nigella sativa , Piper longum, Brassica juncea , Piper nigrum and Cuminum cyminum showed above 80% inhibition of Xanthine oxidase. The quantitative estimation of these plant extracts revealed the considerable amount of phenols and flavanoids which may be attributed for its antioxidant activity through the inhibition of Xanthine oxidase enzyme. Conclusion : The study showed that many of the tested plant species are potential sources of natural XO inhibitors that can be developed, upon further investigation, into successful herbal drugs for treatment of gout and other XO-related disorders.

KEY WORDS: Free radical scavenging, Total antioxidant, Xanthine oxidase.

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INTRODUCTION

The homodimer xanthine oxidase consists of catalytically independent subunits each having a molecular mass of 150 KD1. The enzyme catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid, which are the terminal biochemical reactions of the purine degradation pathway 2. Therefore, any defect in purine metabolism results in an increase in the level of uric acid. This eventually leads to the deposition of sodium hydrogen urate monohydrate crystals in joints and other tissues as a result of extracellular urate super saturation due to hyperuricemia which results from the overproduction and/or under excretion of uric acid and is greatly influenced by a high dietary intake of nucleic acids.The disease associated with this phenomenon is known as gout 3. Hyperuricemia, which is present in 5-30% of the general population, seems to be increasing worldwide and is considered an important risk factor in serious disorders 4,5.Moreover, increase in the level of XO also causes renal stone formation and ischemic myocardium and reactive oxygen species (ROS) induced diseases 6. Urinary excretion of uric acid is enhanced by uricosuric drugs or XO inhibitors which lower the plasma uric acid concentration by inhibiting the final step of uric acid biosynthesis, and there by it can be used in the treatment of gout 7. Allopurinol is a clinically used Xanthine oxidase inhibitor in the treatment of gout, but this drug suffers from many side effects such as hepatitis, nephropathy and allergic reactions 8. Moreover allopurinol and its active metabolite oxypurinol is catalyzed by Xanthine oxidase itself, resulting in the generation of reactive oxygen species such as Superoxide anion (O2-) is involved in various pathological states such as hepatitis, inflammation, ischemia, reperfusion, carcinogenesis and aging 9.

Superoxide anion will be subsequently converted to hydrogen peroxide (H2O2) by superoxide dismutase in

10 2+ most normal tissues .In the presence of transition metals, (fe ) H2O2 will become hydroxyl radical (OH) that are more reactive than super oxide or hydrogen peroxide 11. Besides the direct toxicity of O2- or its derivatives ,O2- reacts more rapidly with nitric oxide (NO), becoming a more toxic species such as peroxynitrite (ONOO-) by which tissue injury will be exacerbated . Hence Xanthine oxidase (XO) serves as an essential biological source of oxygen-derived free radicals which contribute to the oxidative damage of living tissues 12.Thus search for novel xanthine oxidase inhibitors having greater therapeutic activities and lesser side effects are not only capable of treating gout, but also used in therapy of many diseases associated with activity of xanthine oxidase. Several plants extracts from China, 13 Brazil, 14 Vietnam and Australia 15 have shown inhibitory effects on Xanthine oxidase. In the present investigation an attempt was made to find a new herbal derived material with potential Xanthine oxidase inhibitory activities, using 15 medicinal plants belonging 10 families, regardless of their claimed ethanopharmacological and /or food uses. Materials and Methods The chemicals used in the study such as Ascorbic acid, Gallic acid, Catechin, Vitamin E, Sodium nitroprusside, Ferric chloride and Potassium thiocynate were obtained from SD Fine Chemicals Ltd.,

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India. Xanthine oxidase from bovine milk was purchased from Sigma (×4500). All other chemicals and reagent used were of analytical grade. Plant Materials Plant materials, of the selected species (n = 15), proposed for the present study were collected from Sairam Siddha Medical College Herbal Garden and were authenticated by Dr.Sankaranarayanan, Assistant Director, Dept of Research and Development, Sairam Siddha Medical College and Research Centre, Chennai, India. The voucher specimen is also available in herbarium file of the same Centre. Plant Extraction The parts of plants mentioned in Table -1 (100gms each) were washed thoroughly in tap water, shade dried and powdered. The powder was passed through 40-mesh sieve and exhaustively extracted with 50% (v/v) ethanol in distilled water in Soxhlet apparatus at 60oC.All the solvent in the extract is removed trough evaporation under pressure and by freeze drying. Residues were collected and yield was calculated and stored in refrigerator for further analysis. Xanthine Oxidase Inhibitory Assay The Xanthine oxidase inhibitory activity with xanthine as the substrate was measured spectrophotometrically using the procedure of Owens and Johns, 16 with the following modifications. Total volume of the assay mixture is 3.4 ml and consists of the different concentrations of plant extract under study, 0.15M phosphate buffer (pH7.5) and 100μl of 0.03U/ml Xanthine oxidase enzyme solution. After pre-incubation of the test solution at 25°C for 10 min, the reaction was initiated by addition of 1ml of 0.6 mM substrate solution of xanthine, mixed thoroughly, and monitored through absorption increments read after 10 min at 295nm indicating the formation of uric acid using Shimadzu UV-1604 series spectrophotometer. Allopurinol was used as a positive control. The Xanthine oxidase inhibitory activity was estimated by comparing the absorbance value of control. The percent Xanthine

oxidase inhibitory activities of the assayed samples were calculated by using the formula [(A0–A1/A0) x

100], Where A0 is the absorbance of the control and A1 is the absorbance of plant extract or the standard sample. Xanthine oxidase generated Superoxide anion scavenging assay The Superoxide radical scavenging activity generated by the Xanthine/Xanthine oxidase system was determined by the NBT reduction method as described by Valentao,et al 17 The assay mixture consisted of a 50mM sodium carbonate buffer (pH 7.8), 50 mM xanthine, 50 mM nitro blue tetrazolium (NBT), and 0.1mM EDTA in the presence or absence of a tested compounds. The reaction was initiated by adding 5 ml of Xanthine oxidase (20 mM) and the increase in absorbance at 560 nm was measured after 5 min against the corresponding blank solution. L-Ascorbic acid was used as the positive control. The decrease of NBT reduction measured by the absorbance of the reaction mixture correlates with the supeoxide radical scavenging activity of the plant extracts. The inhibition percentage of superoxide

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radical = [(A0 –A1/A0) x100], Where A0 is the absorbance of the control, and A1 is the absorbance of sample. Nitric oxide scavenging activity Nitric oxide generated from sodium nitroprusside was measured by the Griess reagent by the method of Marcocci et al 18 .Various concentrations of the different extracts were mixed with sodium nitroprusside (5mM) in PBS and a final volume of 3 ml was incubated at 25ºC for 150min. After incubation, samples (0.5ml) were removed and diluted with 0.5ml of Griess reagent(1%sulphanilamide, 2%O-phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the chromophore formed was read at 546nm.The inhibition of nitric oxide generation was estimated by comparing the absorbance value of control with that of treatments. Vitamin E was used as standard. Hydrogen Peroxide Scavenging Activity

19 The ability of samples to quench H2O2 was determined by Ruch et al . The different concentrations of the plant extract samples were dissolved in 3.4 ml of phosphate buffered saline (PBS) and mixed with

0.6 ml of 2 mM solution of H2O2. The ability of the plant extracts to scavenge the H2O2 was calculated

by measuring the absorbance of H2O2 at 230 nm after10 min in a spectrophotometer. A separate blank sample was used for each concentration. Ascorbic acid was used as the standard under the same assay

conditions. The inhibition of H2O2 by the extracts was calculated as follows. Hydrogen peroxide radical

scavenging activity (%) = [(A0 –A1/A0) x100], where A0 is the absorbance of the control, and A1 is the absorbance of Ascorbic acid or the sample.

Total Antioxidant Activity The Total antioxidant activity of the extracts was measured by use of a linoleic acid system by the method of Mitsuda et al 20. The linoleic acid emulsion was prepared by mixing 0.2804 g of linoleic acid in Tween 20 and 50 ml of phosphate buffer (pH 7.0). The mixture was then homogenized. A 0.5ml of different concentration of the extracts and standard sample (in ethanol) was mixed with 2.5 ml of linoleic acid emulsion and 2 ml phosphate buffer. The reaction mixture was incubated at 37°C in the dark to accelerate the peroxidation process. The levels of peroxide formation were determined according to the thiocyanate method by sequentially adding 5ml of 75% ethanol 0.1ml of ammonium thiocyanate, 0.1ml sample solution and 0.1ml ferrous chloride. In the same way vitamin E sample was treated. After mixing for 3 min, the peroxide values were determined by reading the absorbance at 500 nm. Determination of total phenolic content Total phenolic content in the lyophilized extracts were determined with the Folin–Ciocalteu’s reagent (FCR) according to a published method of Slinkard and Singleton 21.100 mg of the sample dissolved in

0.5 ml of water was mixed with 2.5 ml FCR (diluted1:10, v/v) followed by 2 ml of Na2CO3 (7.5%, v/v)

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solution. The absorbance was then measured at 765 nm after incubation at 30°C for 90 min. Results were expressed as gallic acid equivalents (mg gallic acid/100g dried extracts). Determination of Total Flavonoid Content The total flavanoid content of the extracts were determined by a colorimetric method as described in the literature of Zhishen et al 22. Aliquots of sample containing 100mg of test drugs were mixed with 2 ml of distilled water and subsequently with 0.15 ml of sodium nitrite solution were added. After 6 min, 0.15 ml of aluminium chloride solution was added and allowed to stand for 6 min, then 2 ml of NaOH solution (4%) was added to the mixture. Immediately, water was added to bring the final volume to 5ml and the mixture was thoroughly mixed and allowed to stand for another 15 min. Absorbance of the mixture was then determined at 510 nm versus prepared water blank. Results were expressed as catechin equivalents (mg catechin/100 g dried extract). Statistical Analysis All data was analyzed statistically with Statistica/Macsoftware (Prism, USA). The experimental results

were mean ± SD of three parallel measurements. IC50 values were obtained through linear regression analysis the plot of concentration (200,400, 800 and1000μg/ml) against percent inhibition. Results In this study, the Hydro-Ethanolic extracts of 15 different plants belonging to 10 different families were investigated as potential XO inhibitors. Their free radical scavenging activities were also studied. The selected plants and their yield in Hydro- Ethanolic extract are summarized in (Table 1).Among the different plant extracts the extracts of Trigonella foenum, Murraya koenigii Nigella sativa, Piper longum and Brassica juncea showed the maximum yield. Whereas the yield was minimum for the Prunus amygdalus and Elettaria cardamomum extracts.

Table I : Selected parts of the plants for the proposed study and their percentage yield

Botanical name of plant Family Parts Common name Hydro-Ethanolic used extract Yield expressed in % (w/w)

Brassica juncea Brassicaceae seed Brown mustard 7.62 Elettaria cardamomum Zingiberaceae seed Cardamom 3.78 Cinnamomum tamala Lauraceae leaf Cinnamon 5.32 tamala Cinnamomum Lauraceae Inner bark Cinnamom 5.28 zeylanicum 5 Syzygium aromaticum Myrtaceae Flower Clove 6.60 Volume XII, Issue III, 2020 bud Page No: 4009 Coriandrum sativum Apiaceae Seed Coriander 6.02 Journal of Xi'an University of Architecture & Technology Issn No : 1006-7930

The Xanthine oxidase inhibitory activity of different plant extracts with their IC50 values were shown in the Table II.Their XO inhibitory activity was found to be in the following order, Trigonella foenum > NigellaBotanical sativa name > Piperof Xanthine longum> oxidase BrassicaSuper juncea oxide > Piper nigrumNitric oxide> Cuminum Hydrcyminumogen > Cinnamomumplant zeylanicum > Zingiber officinale > Foeniculum vulgare > Cinnamomumperoxide tamala > Coriandrum sativum > Murraya% koenigiiIC50 > Syzygium % arIComaticum50 > Elettaria % IC car50 damomum >Prunus% IC 50 amygdalus. Among theseinhibitio plant extractsvalue the inhibitiofirst seven valueextracts hadinhibitio greater valuethan 80%inhibitio inhibition valueof Xanthine oxidase at 1000μn l concentrationinµg/ againstn the positiveinµg/ controln allopurinolinµg/ which nshowed aboutin 93.39% inhibition at the same Table-IIml depicts the Xanthineml oxidase generated mlSuper oxide scavengingµg/ml activity of the different plant extracts along with the positive control Ascorbic acid. In the present investigationBrassica juncea all the plant84.59±0. extracts scavenged173.67 74.16±1. the super oxide143.58 radical78.42±0. considerably250.65. But84.59±0. in correlation158.86 with the Xanthine oxidase52 inhibitory activity 01here also the plant extracts64 Trigonella foenum52 , Nigella sativaElettaria , Piper longum and63.53±0. Brassica 180.56juncea 41.56±0.scavenged the295.76 super oxide74.43±0. radicals281.32 to a maximum63.17±1. extent243.23 cardamomum 61 43 55 02 with the IC50 values corresponds to114.68,126.32,132.70 and143.58 µg/ml respectively. TCinnamomumable-II also explains the73.58±0. Nitric oxide189.12 scavenging65.08±1. activity209.46 of plant61.45±0. extracts which310.39 is compared66.66±0. with236.32 standardtamala Vitamin E. In contrast70 with the above66 activities all the selected60 plants extracts scavenged64 nitric oxideCinnamomum radicals considerably 80.12±0. (60 to 80183.67 %) except64.35±0. Prunus amygdalus163.43 76.34±0. , when comparable263.56 to75.50±0. the standard170.33 vitamin-E,zeylanicum which showed45 89.43% inhibition of68 nitric oxide radicals.36 50 TheSyzygium hydrogen peroxide scavenging65.53±0. activity265.78 was49.33±0. shown in the270.21 Table 72.49±0.–II. Among 295.98all, the Hydro65.53±0. Ethanolic276.23 extractsaromaticum of Coriandrum sativum,64 Trigonella foenum,57 Nigella sativa,55 Piper longum Brassica63 juncea and MurrayaCoriandrum koenigii inhibited72.49±0. the hydrogen245.34 peroxide59.34±0. radicals219.23 to a maximum78.37±0. extent.289.42 At the89.17±0. concentration124.32 ofsativum 1mg/ml the percentage56 inhibition of hydrogen76 peroxide radicals61 by these extracts were64 found to be 89.43,Cuminum 86.78, cyminum 85.58, 84.78,84.5982.10±0. and225.90 81.67% 72.41±0.respectively154.64. In concurrence72.54±0. with293.75 the scavenging73.58±0. activity214.32 21 53 31 70 of these extracts the IC50 values were also found to minimum. Foeniculum vulgare 73.95±0. 184.00 66.48±0. 205.92 64.39±0. 298.78 68.89±0. 245.89 43 64 30 64 Nigella sativa 88.47±0. 158.95 77.42±0. 126.32 87.30±0. 213.45 85.58±0. 140.76

Murraya koenigiiTable II: Effect71.15±0. of Hydr67254.75o-Ethanolic53.83±0. (1mg/ml)63238.59 of plant71.31±0. extracts on29264.38 different81.67±0. Antioxidant67164.65 models Trigonella foenum44 89.56±0. 38152.89 80.62±0. 61114.68 90.30±0. 14189.98 86.78±0. 136.23 (Each value represents the mean SEM. (n=3)) Zingiber officinale 76.74±1. 40216.89 67.90±0. 13174.67 73.71±0. 66287.96 69.20±0. 44227.90 Piper longum42 87.45±0. 93164.78 76.89±0. 16132.70 85.78±0. 29229.90 84.78±0. 150.78 Allopurinol 93.39±0. 45- - 48- - 56- - 49- 6 Piper nigrum54 83.69±0. 176.66 72.45±0. 149.87 76.70±0. 234.70 78.47±0. 196.25 Volume XII, Issue III, 2020 96 59 51 54Page No: 4010 Journal of Xi'an University of Architecture & Technology Issn No : 1006-7930

Since we had made an attempt to study the individual free radical scavenging property of our selected plants extracts we wanted to know the total antioxidant activity also. The total antioxidant activity of plant extracts was measured using ferric thiocyanate test, which determines the amount of peroxide produced at the initial stage of lipid peroxidation. Antioxidant scavenging activity of different plant extracts and standard Vitamin E were shown in the Table-III. The Total antioxidant activity of the plant extract were almost similar to the Xanthine oxidase inhibitory activity, with Trigonella foenum showing the maximum and Prunus amygdalus showing the minimum peroxide scavenging activity .

Table III: Effect of Hydro-Ethanolic (1mg/ml) of plant extracts on Total antioxidant activity, phenol and flavanoid content (Each value represents the mean SEM. (n=3))

Botanical name of plant Total Antioxidant activity Total phenol Total flavanoid % inhibition of peroxide content content formation (mg/100g) (mg/100g)

Brassica juncea 83.70±1.00 263.40±0.25 63.10±0.49 Elettaria cardamomum 59.38±0.55 50.84±0.32 4.52±0.78 Cinnamomum tamala 74.81±0.77 145.58±0.23 21.14±0.53 Cinnamomum 79.35±0.57 215.26±0.49 37.74±0.19 zeylanicum Syzygium aromaticum 60.12±0.45 43.10±0.49 14.14±0.97 Coriandrum sativum 68.83±0.87 105.60±0.32 18.70±0.34 Cuminum cyminum 79.97±0.88 223.24±0.51 50.80±0.37 Foeniculum vulgare 75.79±0.32 165.00±0.33 22.75±0.19 Nigella sativa 86.58±0.97 282.24±0.16 74.58±0.31 Trigonella foenum 87.97±0.37 312.024±0.16 90.72±0.11 Piper longum 85.43±0.77 273.44±0.51 68.93±0.24 Piper nigrum 80.92±0.36 254.00±0.37 68.58±0.76 Prunus amygdalus 56.78±0.17 34.00±0.67 02.14±0.90 Murraya koenigii 63.43±0.84 94.00±0.17 16.14±0.33 Zingiber officinale 76.56±0.23 184.00±0.67 26.14±0.53

Total phenol content was expressed as mg gallic acid equivalent/100 g dried extract

Total flavonoid content was expressed as mg catechin equivalent/100g dried extract.

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The total phenol and flavanoid content of the different plants extract was depicted in Table-III. All the plant extracts possessed considerable quantity of phenols and flavanoids. The highest free radical scavenging potential of Trigonella foenum , Nigella sativa , Piper longum and Brassica juncea may due to their presence of high phenols and flavanoids contents, with a inhibitory potential of Xanthine oxidase enzyme which is responsible for the production of above said oxidants. Discussion

In human a given amount of oxygen taken in by the body is always converted to Reactive oxygen

- 23 species (ROS) such as O2 , H2O2 and hydroxyl radical (OH) by various enzymatic metabolism system . ROS affects various molecular components of the cell, like fatty acids, proteins and DNA and an excess production of ROS leads to cell degeneration and death 24 .When the amount of ROS exceeds the limit of defence mechanism of the body, many serious diseases may induced such as cancer, arteriosclerosis, gout, alzheimer’s disease and various age–related diseases. Among the enzymes concerning oxidation, Xanthine oxidase (XO) has been recognized as a key enzyme in pathology such as ischemia reperfusion and hypertension. XO, an enzyme widely distributed in mammal tissues, catalyzes metabolism of hypoxanthine to Xanthine (XA), and then Xanthine to uric acid in the presence of molecular oxygen,

- this being accompanied with super oxide (O2 ) generation and plays a vital role in producing hyperuricemia and gout. The uric acid is one of the major contributors of plasma antioxidant capacity; however under condition of increased oxidative stress there occurs depletion of local antioxidants like superoxide dismutase, glutathione peroxidase and catalase. This pathophysiology results in increased production of ROS especially super oxide ions as the endothelial nitric oxide synthase uncouples to produce superoxide ions instead of nitric oxide, increasing subsequently favouring antioxidant – pro oxidant urate redox shuttle 25. When serum uric acid levels are elevated the uric acid which was previously physiologically antioxidant paradoxically becomes pro- oxidant. In our investigation all the Hydro Ethanolic plant extracts inhibited the Xanthine oxidase enzyme considerably and also scavenged the Xanthine oxidase generated super oxide radicals. Among them the extracts such as Trigonella foenum , Nigella sativa , Piper longum, Brassica juncea , Piper nigrum and Cuminum cyminum showed above 80% inhibition. Shang et al., 26 have identified five different flavanoids namely vitexin, tricin, naringenin, quercetin and tricin-7-O-b-Dglucopyranoside to be present in fenugreek seeds. Priyadarsini et al.,27 has reported that the antiradical and antioxidant activities of fenugreek seeds are due to phytochemicals present in them . Chopra et al., 28 found that thymoquinone (TQ) is the main active constituent of the volatile oil of the Nigella sativa which has diverse pharmacological activites. The amide type of phytochemicals

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namely N-isobutyl-eicosa-2,4-dienamide, N-isobutyl-eicosa-2,4,14-trienamide,N-isobutyl-ocatadeca- 2,4,12-trienamide,guineensine,pipernonaline, pellitorine, piperine, piperanine, and piperlonguminine were reported by Wu, et al., 29from Piper longum . Reshmi,et al.,30 has isolated several piperdine compounds from the methonolic extract of piper nigrum and showed its antiproliferative activity. According to Hyun Ah Jung, et,al.,31 the leaves from Brassica juncea containing kaempferol glycosides, such as kaempferol-3-O-(2-O-sinapoyl)-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside-7- O-β-d-glucopyranoside,kaempferol-3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside-7-O-β-d- glucopyranosyl-(1→6)-β-d-glucopyranoside kaempferol-3-O-(2-O-sinapoyl)-β-d-glucopyranosyl- (1→2)-β-d-glucopyranoside-7-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside possessed the antioxidant activity. Lee,32 have isolated a component cumminaldehyde from aqueous extract of Cuminum cyminum seed which possesses Aldose reductase and alpha-Glucosidase inhibitory property. The maximum XO inhibitory activity and Super oxide scavenging potential of the above said plants may be due to the presence of these phyto chemical compounds present in the extracts. According to Hemnani and Parihar, the potential mechanism of oxidative damage is the nitration of tyrosine residues of proteins, peroxidation of lipids, degradation of DNA and oligonucleosomal

33 fragments . Reactive nitrogen species like NO2, N2O4, N3O4, nitrate and nitrite due to reaction with oxygen or with superoxide are very reactive. These compounds change the structure and function of many cellular components. The current study revealed the scavenging action of the selected plant extracts against nitric oxide radicals. This is attributed to the fact that all the Indian medicinal plants contain many important phytochemical constituents which can protect the body from the ill effects of oxidants.

Hydrogen peroxide is formed by two-electron reduction of O3 which is not a free radical, but an

oxidizing agent. In the presence of O3 and transition metal ions, the H2O2 can generate OH radical via

34 Fenton reaction. Mallakckakron et al., reported, that in addition, H2O2 can easily cross the cell membrane and exerts an injurious effect on tissues through a number of different mechanisms such as, perturbing intracellular Ca2+ homeostasis, increasing intracellular ATP, inducing DNA damage, and cell

apoptosis .So the removal of H2O2 is important for the antioxidant defence mechanism. Although all the

plant extracts effectively scavenged the H2O2 radicals, the maximum scavenging activity was exhibited by Coriandrum sativum. The presence aromatic acids containing compounds such as 2-decenoic acid, E-11-tetradecenoic acid, capric acid, undecyl alcohol tridecanoic acid and undecanoic acid in Coriandrum sativum as reported by Md. Nazrul Islam Bhuiyan, et al.,35 may be responsible for the maximum Hydrogen peroxide scavenging activity observed in the present study .

In correlation with the above investigations the Total antioxidant potential of the plant extracts followed the same sequence as that of the XO inhibitory activity. The flavanoid and phenol contents were also

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found to be high of those plant extracts which is showing maximum inhibitory activity. According to Jiang et al., 36 flavanoids generally occur as O-glycosides in which one or more of the hydroxyl groups are bound to sugars. As stated by Robards and Antolovvich this glycosylation renders flavanoids more water soluble, making them store readily in the cell vacuole where they are commonly found 37.The above-mentioned effects might be expected for our plant extracts in reducing the lipid peroxidation. The presence of significant quantities of flavanoid in these plant extracts are considered as the major contributors towards the antioxidant activity in our present study. The reactive oxygen species are stabilized by flavanoids by reacting with the reactive compound of the radical. According to Pietta, flavanoids are a class of secondary plant phenolics with powerful antioxidant properties38.

Conclusion

The obtained results reveals that the studied plants can form a good source of effective crude inhibitors for Xanthine Oxidase enzyme which can be used in the treatment of gout and other XO-related disorders. However, to verify the reported inhibitory activities, further biological investigations are needed, particularly using animal models, under in vivo conditions.

In future research, these active medicinal plants will be further investigated in order to isolate, identify, and evaluate the potentially phytoactive compounds responsible for the XO inhibitory activities reported in the present study. Undoubtedly, the XO inhibition, observed in the present study, would be an additional mechanism that can explain their antioxidant activities.

References

1. Hille R, Nishino T, Flavoprotein structure and mechanism.4.Xanthine oxidase and xanthine dehydrogenase. FASEB J 1995;9: 995.

2. Krenitsky T A, Spector T , Hall WW, Xanthine oxidase from human liver: purification and characterization, Arch Biochem Biophys 1986; 247:108. 3. Boss G R , Seegmiller J E, Hyperuricemia and gout. Classification, complications and management, N Engl J Med 1979; 26: 1459. 4. Forbes J M, Coughlan M T , Cooper M E, Oxidative stress as a major culprit in kidney disease in diabetes, Diabetes 2008; 57: 1446. 5. Watanabe S, Kanellis J, Nakagawa T, Han L, Ohashi R , Lan H et al., Reducing uric acid as a means to prevent cardiovascular and renal disease, Expert Opin Ther Pat 2002; 12: 193. 6. McCord J M, Oxygen-derived free radicals in post ischemic tissue injury, N Engl J Med 1985 ; 312 :159. 7. Terkeltaub R, Gout. Novel therapies for treatment of gout and hyperuricemia, 10

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Arthritis Res Ther 2009; 11: 236. 8. Osada Y, Tsuchimoto M, Fukushima H, Takahashi K, Kondo S, Hasegawa M , Komoriya K, Hypouricemic effect of the novel Xanthine oxidse inhibitor,. TEI-6720, in rodents, Euro J Pharmacol 1993; 241 : 183. 9. Akaike T , Maeda H, Pathophysiological effects of high-output production of nitricOxide, In: Ignarro LJ, ed. (Academic Press, San Diego) 2000; 733. 10. Cos P, Ying L, Calomme M, Hu J P, Cimanga K, Van Poel B, Pieters L, Vlietinck A J , Berghe DV, Structure-activity relationship and classification of flavanoids as inhibitors of Xanthine oxidase and superoxide scavengers, J Nat Prod 1998; 61: 71. 11. Berlett B S , Stadtman E R, Protein oxidation in aging, disease, and oxidative stress, J Biol Chem 1997; 272: 20313. 12. Valko M, Leibfritz D, Moncol J, Cronin M T, Mazur M , Telser J, Free radicals and antioxidants in normal physiological functions and human disease, Int J Biochem Cell Biol 2007; 39: 44. 13. Kong L D, Cai Y, Huang W, Cheng C , Tan R, Inhibition of xanthine oxidase by some Chinese medicinal plants used to treat gout, J Ethnopharmacol 2000; 73: 199. 14. Filha Z S, Vitolo I F, Fietto L G, Lombardi J A , Saude- Guimaraes D A, Xanthine oxidase inhibitory activity of Lychnophora species from Brazil (“Arnica”), J Ethnopharmacol 2006; 107: 79. 15. Sweeney A P, Wyllie S G, Halliker R A , Markham J L, Xanthine oxidase inhibitory activity of selected Australian native plants, J Ethnopharmacol 2001; 75: 273. 16. Owen P , Johns T, Xanthine oxidase inhibitory activity of north eastern North American plant remedies used for gout, J Ethnopharmacol 1999; 64: 149. 17. Valentao P, Frenandes E, Carvalho F, Andrade B P, Seabra R M , Bastos M L,Studies on the antioxidant activity of Lippia citriodora infusion: scavenging effect on super oxide radical, hydroxyl radical and hypochlorous acid, Biol Pharm Bull 2002; 25 : 1324. 18. Marcocci L, Maguire J J, Droy-Lefaix M T & Packer L, The nitric oxide scavenging Properties of Ginkgo biloba extract EGb761,Biochem Biophy Res Commun 1994; 201 : 748. 19. Ruch K J, Cheng S J , Klauning J E, Prevention of cytotoxicity and inhibition of intercellular communication by antioxidant catechin isolated from Chinese green tea, arcinogenesis 1989; 10 : 1003. 20. Mitsuda H, Yasumodo K , Iwami F, Antioxidant action of indole compounds during the autoxidation of linoleic acid, Eiyo to Shokuryo 1996; 19: 210. 21. Slinkard J , Singleton V L, Total phenol analysis: automation and comparison with manual methods, Am J Enol Vitic 1977; 28 : 49. 22. Zhishen J, Mengcheng T , Jianming W, The determination of flavonoid contents in mulberry and

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their scavenging effects on superoxide radicals, Food Chem 1999; 64 : 555.

23. H. Ukeda, In “Recent Research Developments in Analytical Biochemistry,Ed, Transworld Research Network, India 2001: 56. 24. Remacle J, Raes M, Toussaint O, Renard P , Rao G , Low levels of reactive oxygen species as modulators of cell function, Mutat Res 1995; 316: 103. 25. Hayden M R , Tyagi S C, Uric acid - A new look at an old risk marker for cardiovascular disease, metabolic syndrome and Type II D.M, Nutr Metab 2004; 1: 10.

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36. Jiang P, Burczynski C, Campbell G, Pierce J A, Austria C J , Briggs, Rutin and flavonoid contents in three buckwheat species Fagopyrum esculentum, F. tataricum, and F. homotropicum and their protective effects against lipid peroxidation, Food Res Int 2007; 40: 356. 37. Robards K , Antolovvich M, Analytical chemistry of fruit bio-flavonoids. A review, Analyst 1997; 122: 11. 38. Pietta P G, Flavonoids as antioxidants, J Nat Prod 2000; 63 : 1035.

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