Antioxidant potential of hydro-methanolic extract of seed of Caesalpinia bonduc: An in vitro study
Identifieur interne : 001002 ( Pmc/Corpus ); précédent : 001001; suivant : 001003Antioxidant potential of hydro-methanolic extract of seed of Caesalpinia bonduc: An in vitro study
Auteurs : Kishalay Jana ; Kausik Chatterjee ; Kazi Monjur Ali ; Abhinandan Ghosh ; Tushar Kanti Bera ; Debidas GhoshSource :
- Journal of Advanced Pharmaceutical Technology & Research [ 2231-4040 ] ; 2011.
Abstract
It is well known that the over production of reactive oxygen species is harmful for living organisms and it damages major cellular constituents such as DNA, protein, and lipid. At present, searching of new plant sources having free radical scavenging activity is an important field of research in phytomedicine as natural products are safe and relatively low cost. In this respect, attention has been focused to evaluate the antioxidant potential of hydro-methanolic extract of seed of
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DOI: 10.4103/2231-4040.90884
PubMed: 22247894
PubMed Central: 3255348
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Antioxidant potential of hydro-methanolic extract of seed of <italic>Caesalpinia bonduc</italic>: An <italic>in vitro</italic> study</title><author><name sortKey="Jana, Kishalay" sort="Jana, Kishalay" uniqKey="Jana K" first="Kishalay" last="Jana">Kishalay Jana</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author><author><name sortKey="Chatterjee, Kausik" sort="Chatterjee, Kausik" uniqKey="Chatterjee K" first="Kausik" last="Chatterjee">Kausik Chatterjee</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author><author><name sortKey="Ali, Kazi Monjur" sort="Ali, Kazi Monjur" uniqKey="Ali K" first="Kazi Monjur" last="Ali">Kazi Monjur Ali</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author><author><name sortKey="Ghosh, Abhinandan" sort="Ghosh, Abhinandan" uniqKey="Ghosh A" first="Abhinandan" last="Ghosh">Abhinandan Ghosh</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author><author><name sortKey="Bera, Tushar Kanti" sort="Bera, Tushar Kanti" uniqKey="Bera T" first="Tushar Kanti" last="Bera">Tushar Kanti Bera</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Ghosh, Debidas" sort="Ghosh, Debidas" uniqKey="Ghosh D" first="Debidas" last="Ghosh">Debidas Ghosh</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">22247894</idno><idno type="pmc">3255348</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255348</idno><idno type="RBID">PMC:3255348</idno><idno type="doi">10.4103/2231-4040.90884</idno><date when="2011">2011</date><idno type="wicri:Area/Pmc/Corpus">001002</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Antioxidant potential of hydro-methanolic extract of seed of <italic>Caesalpinia bonduc</italic>: An <italic>in vitro</italic> study</title><author><name sortKey="Jana, Kishalay" sort="Jana, Kishalay" uniqKey="Jana K" first="Kishalay" last="Jana">Kishalay Jana</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author><author><name sortKey="Chatterjee, Kausik" sort="Chatterjee, Kausik" uniqKey="Chatterjee K" first="Kausik" last="Chatterjee">Kausik Chatterjee</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author><author><name sortKey="Ali, Kazi Monjur" sort="Ali, Kazi Monjur" uniqKey="Ali K" first="Kazi Monjur" last="Ali">Kazi Monjur Ali</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author><author><name sortKey="Ghosh, Abhinandan" sort="Ghosh, Abhinandan" uniqKey="Ghosh A" first="Abhinandan" last="Ghosh">Abhinandan Ghosh</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author><author><name sortKey="Bera, Tushar Kanti" sort="Bera, Tushar Kanti" uniqKey="Bera T" first="Tushar Kanti" last="Bera">Tushar Kanti Bera</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Ghosh, Debidas" sort="Ghosh, Debidas" uniqKey="Ghosh D" first="Debidas" last="Ghosh">Debidas Ghosh</name><affiliation><nlm:aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Advanced Pharmaceutical Technology & Research</title><idno type="ISSN">2231-4040</idno><idno type="eISSN">0976-2094</idno><imprint><date when="2011">2011</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>It is well known that the over production of reactive oxygen species is harmful for living organisms and it damages major cellular constituents such as DNA, protein, and lipid. At present, searching of new plant sources having free radical scavenging activity is an important field of research in phytomedicine as natural products are safe and relatively low cost. In this respect, attention has been focused to evaluate the antioxidant potential of hydro-methanolic extract of seed of <italic>Caesalpinia bonduc</italic> (Caesalpenacae) using different <italic>in vitro</italic> models. To evaluate the antioxidant activity, extract was examined on 2, 2-diphenyl-1-picrylhydrazyl radical scavenging effect, scavenging of hydrogen peroxide, hydroxyl radical scavenging potential, and anti-lipid peroxidation activity by biochemical methods. Total phenol and flavonoids contents in the said extract were measured biochemically as per standard methods. Results were compared with butylated hydroxyl toluene and α-tocopherol. Results indicated that hydro-methanolic extract has strong scavenging activity on 2, 2-diphenyl-1-picrylhydrazyl radical with IC<sub>50</sub> value 157.4 μg/ml, hydroxyl radical with IC<sub>50</sub> value 61.9 μg/ml and hydrogen peroxide with IC<sub>50</sub> value 64.32 μg/ml. Hydro-methanolic extract also showed notable inhibition in lipid peroxidation having IC<sub>50</sub> value 58.87 μg/ml. Phytochemical study focused that the extract is rich in phenolic compounds (24.66 mg gallic acid equivalent/g dried extract) and flavonoids (136.65 mg quercetin equivalent/g dried extract). Findings of the experiment indicated that the hydro-methanolic extract of seed of <italic>Caesalpinia bonduc</italic> is a source of natural antioxidants.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Van De Vijver, Lp" uniqKey="Van De Vijver L">LP Van de Vijver</name></author><author><name sortKey="Kardinaal, Af" uniqKey="Kardinaal A">AF Kardinaal</name></author><author><name sortKey="Grobbee, De" uniqKey="Grobbee D">DE Grobbee</name></author><author><name sortKey="Princen, Hm" uniqKey="Princen H">HM Princen</name></author><author><name sortKey="Van Poppel, G" uniqKey="Van Poppel G">G Van Poppel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Smith, Ma" uniqKey="Smith M">MA Smith</name></author><author><name sortKey="Perry, G" uniqKey="Perry G">G Perry</name></author><author><name sortKey="Pryor, Wa" uniqKey="Pryor W">WA 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Adv Pharm Technol Res</journal-id><journal-id journal-id-type="publisher-id">JAPTR</journal-id><journal-title-group><journal-title>Journal of Advanced Pharmaceutical Technology & Research</journal-title></journal-title-group><issn pub-type="ppub">2231-4040</issn><issn pub-type="epub">0976-2094</issn><publisher><publisher-name>Medknow Publications & Media Pvt Ltd</publisher-name><publisher-loc>India</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">22247894</article-id><article-id pub-id-type="pmc">3255348</article-id><article-id pub-id-type="publisher-id">JAPTR-2-260</article-id><article-id pub-id-type="doi">10.4103/2231-4040.90884</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Antioxidant potential of hydro-methanolic extract of seed of <italic>Caesalpinia bonduc</italic>: An <italic>in vitro</italic> study</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Jana</surname><given-names>Kishalay</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chatterjee</surname><given-names>Kausik</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ali</surname><given-names>Kazi Monjur</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ghosh</surname><given-names>Abhinandan</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Bera</surname><given-names>Tushar Kanti</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Ghosh</surname><given-names>Debidas</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"></xref></contrib></contrib-group><aff id="aff1">Department of Bio-Medical Laboratory Science and Management (U.G.C. Innovative Programme) Andrology, Endocrinology and Molecular Medicine Laboratory, Vidyasagar University, Midnapore, India</aff><aff id="aff2"><label>1</label>Department of Herbal Drug Development, Pharmaceutical Division, Southern Health Improvement Samity (SHIS), Bhangar, West Bengal, India</aff><author-notes><corresp id="cor1"><bold>Address for correspondence:</bold> Prof. Debidas Ghosh, Department of Bio-Medical Laboratory Science and Management (U.G.C Innovative Department), Vidyasagar University, Midnapore - 721 102, West Bengal, India. E-mail: <email xlink:href="debidas_ghosh@yahoo.co.in">debidas_ghosh@yahoo.co.in</email></corresp></author-notes><pub-date pub-type="ppub"><season>Oct-Dec</season><year>2011</year></pub-date><volume>2</volume><issue>4</issue><fpage>260</fpage><lpage>265</lpage><permissions><copyright-statement>Copyright: © Journal of Advanced Pharmaceutical Technology & Research</copyright-statement><copyright-year>2011</copyright-year><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><abstract><p>It is well known that the over production of reactive oxygen species is harmful for living organisms and it damages major cellular constituents such as DNA, protein, and lipid. At present, searching of new plant sources having free radical scavenging activity is an important field of research in phytomedicine as natural products are safe and relatively low cost. In this respect, attention has been focused to evaluate the antioxidant potential of hydro-methanolic extract of seed of <italic>Caesalpinia bonduc</italic> (Caesalpenacae) using different <italic>in vitro</italic> models. To evaluate the antioxidant activity, extract was examined on 2, 2-diphenyl-1-picrylhydrazyl radical scavenging effect, scavenging of hydrogen peroxide, hydroxyl radical scavenging potential, and anti-lipid peroxidation activity by biochemical methods. Total phenol and flavonoids contents in the said extract were measured biochemically as per standard methods. Results were compared with butylated hydroxyl toluene and α-tocopherol. Results indicated that hydro-methanolic extract has strong scavenging activity on 2, 2-diphenyl-1-picrylhydrazyl radical with IC<sub>50</sub> value 157.4 μg/ml, hydroxyl radical with IC<sub>50</sub> value 61.9 μg/ml and hydrogen peroxide with IC<sub>50</sub> value 64.32 μg/ml. Hydro-methanolic extract also showed notable inhibition in lipid peroxidation having IC<sub>50</sub> value 58.87 μg/ml. Phytochemical study focused that the extract is rich in phenolic compounds (24.66 mg gallic acid equivalent/g dried extract) and flavonoids (136.65 mg quercetin equivalent/g dried extract). Findings of the experiment indicated that the hydro-methanolic extract of seed of <italic>Caesalpinia bonduc</italic> is a source of natural antioxidants.</p></abstract><kwd-group><kwd>Antioxidant</kwd><kwd><italic>Caesalpinia bonduc</italic></kwd><kwd>free radicals</kwd><kwd>lipid peroxidation</kwd></kwd-group></article-meta></front><body><sec id="sec1-1"><title>INTRODUCTION</title><p>Majority of the diseases or disorders including diabetes mellitus, arthritis, cancer, ageing processes are often connected with reactive oxygen species (ROS) and lipid peroxidation.[<xref ref-type="bibr" rid="ref1">1</xref><xref ref-type="bibr" rid="ref2">2</xref>] The main contributor of oxidative stress is uncontrolled generation of free radical together with reduced levels of antioxidative vitamins and enzymes.[<xref ref-type="bibr" rid="ref3">3</xref>] These free radicals interfere with biochemical processes and represent an essential part of aerobic life and metabolism.[<xref ref-type="bibr" rid="ref4">4</xref>] Therefore, antioxidant with free radical scavenging activities may have great relevance for the management and therapeutics of free radical inducing diseases. Polyphenolic compounds like flavonoids and phenolic acid commonly found in plant have multiples biological effects including antioxidant activity, free radical scavenging abilities, anti inflammatory, and anti carcinogenic activities.[<xref ref-type="bibr" rid="ref5">5</xref>] Currently, available synthetic antioxidants like butylated hydroxytoluene (BHT), tertiary butylated hydroquinone, and gallic acid esters have suspected to cause or prompt negative health effects.[<xref ref-type="bibr" rid="ref6">6</xref>] Hence, strong restrictions have been placed on their application and there is a trend to substitute them with phyto-antioxidants. Neutraceuticals having antioxidant properties are non toxic or may have minimum side effects than synthetic compounds. In this concern, our attempt is to search out the neutraceuticals for substitute of synthetic antioxidant drug. Therefore, the aim of the present study is to investigate the antioxidant activity of hydro-methanolic extract of seed of <italic>Caesalpinia bonduc</italic> (<italic>C. bonduc</italic>) on different <italic>in vitro</italic> models such as 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide, hydroxyl radical, and lipid peroxidation inhibition activity. The antioxidant activities of the said plant part have been expressed in term of percentage of inhibition on free radical generation on the different <italic>in-vitro</italic> experimental models.</p><p><italic>C. bonduc</italic> is a medicinal plant belonging to the family of Caesalpenacae. In Indian traditional plant medicine, it has been considered as an important remedy for the treatment of filarial infection, tumor, asthma, and diabetes.[<xref ref-type="bibr" rid="ref7">7</xref>] This plant part also has a remedial effect on hyperglycemic and hyperlipidemic state in diabetic rats which was noted in our previous report.[<xref ref-type="bibr" rid="ref8">8</xref>]</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Chemicals</title><p>Chemicals like 2, 2-diphenyl-1-picrylhydrazyl (DPPH), trichloroacetic Acid (TCA), thiobarbituric Acid (TBA), gallic acid, ascorbic acid, α-tocopherol, butylated hydroxytoluene (BHT), and folin-ciocalteu (FC) reagent were used in this experiment and these were purchased from Himedia Laboratories Pvt. Ltd, Mumbai, India. Ethylene diamine tetra acetic acid (EDTA), hydrogen peroxide, sodium hydroxide (NaOH), sodium carbonate (Na<sub>2</sub>CO<sub>3</sub>), ferric chloride (FeCl<sub>2</sub>), and sodium nitrite (NaNO<sub>2</sub>) were purchased from Sisco Research Laboratories (SRL) Pvt. Ltd. Mumbai, India. Aluminium chloride (AlCl<sub>3</sub>) was obtained from Sd Fine Chemicals Ltd, Mumbai, India.</p></sec><sec id="sec2-2"><title>Collection of Plant Material</title><p>The dried seeds of <italic>C. bonduc</italic> were collected from village area of Paschim Medinipur district, West Bengal, India, in the months of July-September. The plant was identified by the taxonomist, Prof. R. K. Bhakat, Department of Botany and Forestry, Vidyasagar University, Midnapore, West Bengal, and the voucher specimen was deposited having the Reference No. Bio-Med/V.U/C.B/24/10.</p></sec><sec id="sec2-3"><title>Preparation of Hydro-Methanolic Extract of Seed of <italic>C. bonduc</italic></title><p>Hydro-methanolic extract of seeds of <italic>C. bonduc</italic> was prepared as per the standard method.[<xref ref-type="bibr" rid="ref9">9</xref>] In brief, fresh seeds of <italic>C. bonduc</italic> were dried in an incubator for 2 days at 40°C, then crushed in an electric grinder and pulverized. From this powder, 50 g was suspended in the mixture solvent consisting of 80 ml of water and 120 ml methanol in a container for 48 hrs at room temperature and then the supernatant was filtered through No. 3 Whatman filter paper. The filtrate was concentrated and the collected residue was preserved in a refrigerator at 2-8°C for use in the experiments.</p></sec><sec id="sec2-4"><title>Phytochemical Screening</title><p>Qualitative tests for phytochemicals of the seeds of <italic>C. bonduc</italic> were performed as per the standard methods.[<xref ref-type="bibr" rid="ref10">10</xref>]</p></sec><sec id="sec2-5"><title>Determination of Total Flavonoid Content</title><p>The total flavonoid content was determined with the AlCl<sub>3</sub> method[<xref ref-type="bibr" rid="ref11">11</xref>] using quercetin as a standard. The seed extract (0.25 ml) was added to 1.25 ml of distilled water followed by addition of 75 μl of 5% NaNO<sub>2</sub>. The preparation was allowed to incubate at room temperature for 5 minutes, and then AlCl<sub>3</sub> (0.15 ml, 10%) was added. After a further incubation for 6 min at room temperature, the reaction mixture was treated with 0.5 ml of 1mM NaOH. Finally, the reaction mixture was diluted with 275 μl of distilled water. Further incubation for 20 min at room temperature was performed and the absorbance was measured at 510 nm. All tests were performed in triplet. The flavonoid content was expressed as mg of quercertin equivalents (QE) per gram (g) of dried extract.</p></sec><sec id="sec2-6"><title>Determination of Total Phenolic Content</title><p>Total phenolic content was determined using the Folin-Ciocalteu (FC) reagent method[<xref ref-type="bibr" rid="ref12">12</xref>] with slight modification. Briefly, the seed extract (0.5 ml) was mixed with 0.5 ml of FC reagent (previously diluted with 1:1 with distilled water) and incubated for 5 min at room temperature, and then 1 ml of 2% Na<sub>2</sub>CO<sub>3</sub> solution was added. After incubation at room temperature for 10 min, the absorbance was noted. Gallic acid monohydrate was used as the standard. The phenolic content was expressed as mg of gallic acid equivalents (GAE) per g of dried extract.</p></sec><sec id="sec2-7"><title>Assessment of <italic>in-vitro</italic> Antioxidant Activity of Hydro-Methanolic Extract</title></sec><sec id="sec2-8"><title>DPPH radical scavenging assay</title><p> The radical scavenging activity of <italic>C. bonduc</italic> against DPPH was determined spectrophotometrically.[<xref ref-type="bibr" rid="ref13">13</xref>] DPPH reacts with an antioxidant compound that can donate hydrogen and thereby DPPH is reduced. Change in color of the solution, (from deep violet to light yellow) was measured. The intensity of the yellow color depends on the amount and nature of radical scavenger present in the sample and standard compounds. The reaction mixture containing 1 ml of 0.1 mM 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) and various concentrations of extract (50, 100, 150, 200 and 250 μg) were made up to 3 ml with water. Then the tubes were incubated for 10 minutes. Once the blue color chromophore was formed, the absorbance of this solution was measured at 517 nm, against reagent blank containing water in place of extract. BHT was used as the standard for the comparison. The ability to scavenge the DPPH radical in terms of percentage of inhibition was calculated according to the following equation: % inhibition = {(A<sub>0</sub>–A <sub>1</sub>)/A<sub>0</sub> × 100} where A<sub>0</sub> is the absorbance of the control (without extract) and A<sub>1</sub> is the absorbance in the presence of the extract.</p></sec><sec id="sec2-9"><title>Hydrogen peroxide scavenging assay</title><p>A solution of hydrogen peroxide (20 mM) was prepared in phosphate buffer saline (PBS) (pH-7.4). Various concentrations (20, 40, 60, 80 and 100 μg) of extract or standard in hydro-methanol (1 ml) were added to 2 ml of hydrogen peroxide solution in PBS. After 10 min of incubation, the absorbance was measured at 230 nm against a blank solution containing phosphate buffer without hydrogen peroxide.[<xref ref-type="bibr" rid="ref14">14</xref>] The result was compared with alpha tocopherol as a standard. The percentage of inhibition was calculated using the formula given before.</p></sec><sec id="sec2-10"><title>Hydroxyl radical scavenging assay</title><p>Hydroxyl radical inhibitory activity was performed as per deoxyribose method.[<xref ref-type="bibr" rid="ref15">15</xref>] To the reaction mixture containing deoxyribose (3 mM, 0.2 ml), FeCl<sub>2</sub> (0.1 mM, 0.2 ml), EDTA, disodium salt (0.1 mM, 0.2 ml), ascorbic acid (0.1 mM, 0.2 ml), and hydrogen peroxide (2 mM, 0.2 ml) in PBS (pH, 7.4, 20 mM), various concentrations (20, 40, 60, 80 and 100 μg) of 0.2 ml of the extract or standard in DMSO were added to give a total volume of 1.1 ml. The solutions were then incubated for 30 min at 37°C. After incubation, ice-cold TCA (0.2 ml, 15% w/v) and TBA (0.2 ml, 1% w/v) in 0.25 N hydrochloric acid (HCl) were added. The reaction mixture was kept in a boiling water bath for 30 min, cooled in room temperature, and the absorbance was measured at 532 nm with reagent blank containing water in the place of extract. Alpha tocopherol was used as the standard for the comparison. The percentage of inhibition was calculated using the formula given before.</p></sec><sec id="sec2-11"><title>Lipid Peroxidation Inhibitory Activity</title><p>The lipid peroxidation inhibitory activity of hydro-methanolic extract was studied <italic>in-vitro</italic> following the modified method.[<xref ref-type="bibr" rid="ref16">16</xref><xref ref-type="bibr" rid="ref17">17</xref>] Rats were killed by cervical dislocation (NIH, 1985), the liver tissue was excised, rinsed in ice-cold saline solution, and blotted dry. Then, 0.5 gm of the liver was sliced and homogenized with 10 ml of 150 mM KCL-Tris-HCl buffer (pH-7.2). The reaction mixture was composed of 0.25 ml of liver homogenate, Tris-HCl buffer (pH-7.2), 0.1 mM ascorbic acid (AA), 4 mM FeCl<sub>2</sub>, and 0.05 ml of various concentration of extract (25, 50, 75, 100 and 150 mg). The mixture was incubated at 37°C for 1 hr in capped tubes. Then, 0.5 ml of 0.1 N HCl, 0.2 ml of 9.8% SDS, 0.9 ml of distilled water, and 2 ml of 0.6% TBA were added to each tube and the tubes were vigorously shaken. All the tubes were then placed in boiling water bath at 100°C for 30 minutes. The tubes were allowed to keep at room temperature and centrifuged at 3000 rpm for 20 minutes. The absorbance of the supernatant was measured at 532 nm against reagent blank containing water in place of extract. The result was compared with BHT as a standard. The percentage inhibition of lipid peroxidation was calculated by comparing the results of test with those of controls not treated with the extract as per the formula:</p><p>% inhibition = {(A<sub>0</sub>–A<sub>1</sub>)/A<sub>0</sub> × 100} where A<sub>0</sub> is the absorbance of the control (without extract) and A<sub>1</sub> is the absorbance in the presence of the extract.</p></sec><sec id="sec2-12"><title>Statistical Analysis</title><p>Statistical analysis was performed by software (Origin-8.1). Data was expressed as means ± SD of three measurements. Data was analyzed using analysis of variance (ANOVA) followed by multiple comparison two tail ‘<italic>t</italic>-test’. The results obtained were considered statistically significant if the <italic>P</italic>-value was <0.05. The amount of extract needed to inhibit free radicals concentrations by 50%, IC<sub>50</sub> was performed by software (STATISTICA) based on the percentage of inhibition in different doses or concentration.</p></sec></sec><sec id="sec1-3"><title>RESULTS AND DISCUSSION</title><p>Oxidative stress has been implicated in the pathology of many diseases and conditions including diabetes, cardiovascular disease, inflammatory condition, cancer, ageing etc.[<xref ref-type="bibr" rid="ref18">18</xref><xref ref-type="bibr" rid="ref19">19</xref>] Antioxidants may offer resistance against the oxidative stress by scavenging the free radicals, inhibiting the lipid peroxidation, and by many other mechanisms and thus prevent diseases.[<xref ref-type="bibr" rid="ref20">20</xref>] Consequently, we studied the antioxidant activities of hydro-methanolic extract by a series of <italic>in-vitro</italic> protocols using some biologically relevant models.</p><p>The findings of the phytochemical screening indicated that the seeds of <italic>C. bonduc</italic> are rich in flavonoids, phenols, and saponins which may be responsible for the antioxidative efficacy as these phytochemicals act as antioxidants.[<xref ref-type="bibr" rid="ref21">21</xref>–<xref ref-type="bibr" rid="ref23">23</xref>]</p><p>Phenolic compounds may contribute directly to the antioxidative action. The total phenolic content was 24.66 mg GAE/g dried extract. The total flavonoids content of the hydro-methanolic extract was 136.65 mg QE/g dried extract [<xref ref-type="table" rid="T1">Table 1</xref>]. Due to redox properties, phenolic compounds play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen or decomposing peroxides.[<xref ref-type="bibr" rid="ref24">24</xref><xref ref-type="bibr" rid="ref25">25</xref>] It has been also recognized that flavonoids show antioxidant activity through scavenging or chelating process and their effects on human nutrition and health are considerable.[<xref ref-type="bibr" rid="ref26">26</xref>]</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Total phenolic and flavonoids contents of hydro-methanolic extract of seed of <italic>C. bonduc</italic></p></caption><graphic xlink:href="JAPTR-2-260-g001"></graphic></table-wrap><p>The scavenging ability of hydro-methanolic extract on DPPH radical is represented by line diagram [<xref ref-type="fig" rid="F1">Figure 1</xref>] and compared with BHT. The scavenging activity of the investigated extract varied widely from 23.32% to 75.92% (IC<sub>50</sub> value 157.4 mg/ml) and in standard 26.78% to 79.88% (IC<sub>50</sub> value 145.89 mg/ml). From the result, we say that DPPH antioxidant assay is based on the ability of DPPH, a stable free radical, to decolorize in the presence of antioxidants. The DPPH radical contains an odd electron which is responsible for the absorbance at 517 nm and also for a visible deep purple color. When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which can be quantitatively measured from the changes in absorbance.[<xref ref-type="bibr" rid="ref27">27</xref>] It was observed that the radical scavenging activity is increasing with the increase of phenolic compound content.[<xref ref-type="bibr" rid="ref28">28</xref>] The two separate studies were also reported a high concentration between DPPH radical scavenging potential and total phenolic content.[<xref ref-type="bibr" rid="ref29">29</xref><xref ref-type="bibr" rid="ref30">30</xref>]</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Inhibition in DPPH radical by hydro-methanolic extract of seed of <italic>C. bonduc</italic> and standard BHT. The IC<sub>50</sub> value of the extract was 157.4 μg/ml</p></caption><graphic xlink:href="JAPTR-2-260-g002"></graphic></fig><p>Hydrogen peroxide neutralization ability of the extract [<xref ref-type="fig" rid="F2">Figure 2</xref>] varied from 11.22% to 80.53% (IC<sub>50</sub> value 64.32 mg/ ml) and in standard 18.26% to 85.16% (IC<sub>50</sub> value 57.06 mg/ml). The ability of the said extract to neutralize hydrogen peroxide was dose dependent. Hydrogen peroxide is important because of its ability to penetrate biological membranes. Hydrogen peroxide itself is not very reactive, but it can sometimes be toxic to cell because it may give rise to hydroxyl radical in the cells.[<xref ref-type="bibr" rid="ref31">31</xref>] Scavenging of hydrogen peroxide by <italic>C. bonduc</italic> may be attributed to their phenolic compound which could donate electron to hydrogen peroxide, thus it is neutralizing to water.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Scavenging of hydrogen peroxide by hydro-methanolic extract of seed of <italic>C. bonduc</italic> and standard α-tocopherol. The IC<sub>50</sub> value of the extract was 64.32 μg/ml</p></caption><graphic xlink:href="JAPTR-2-260-g003"></graphic></fig><p>Hydroxyl radical scavenging ability of hydro-methanolic seed extract was shown in [<xref ref-type="fig" rid="F3">Figure 3</xref>] and was compared with a-tocopherol. The extract inhibited the degradation of deoxyribose in dose dependent manner. Thereby, hydroxyl radical neutralization values ranges from 14.21% to 87.82% (IC<sub>50</sub> value 61.9 mg/ml) and in standard from 17.98% to 92.66% (IC<sub>50</sub> value 54.97 mg/ml). Hydroxyl radical is an extremely reactive free radical formed in biological systems and has the capacity to cause DNA strand breakage which contributes to carcinogenesis, mutagenesis, and cytotoxicity.[<xref ref-type="bibr" rid="ref32">32</xref>] Like many free radicals, hydroxyl radical can be neutralized if it is provided with hydrogen atoms. Oxygen radical may attack DNA either in sugar or in base giving rise to a large number of products. Phytochemical study of seed extract revealed the presence of phenolic compounds which may responsible for the hydroxyl radical scavenging activity.[<xref ref-type="bibr" rid="ref33">33</xref><xref ref-type="bibr" rid="ref34">34</xref>]</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Hydroxyl radical scavenging ability of hydro-methanolic extract of seed of <italic>C. bonduc</italic> and a-tocopherol as a standard. The IC<sub>50</sub> value of hydro-methanolic extract was 61.9 μg/ml</p></caption><graphic xlink:href="JAPTR-2-260-g004"></graphic></fig><p>Inhibition percentage in lipid peroxidation varies widely in different doses ranges from 27.23% to 91.26% (IC<sub>50</sub> value 58.87 mg/ ml) in hydro-methanolic extract and 32.18% to 94.54% (IC<sub>50</sub> value 51.34 mg/ml) in case of standard [<xref ref-type="fig" rid="F4">Figure 4</xref>]. Lipid peroxidation has been broadly defined as the oxidative deterioration of polyunsaturated fatty acids and involves formation of lipid radicals leading to membrane damage. Free radicals induced lipid peroxidation mainly occurs in brain and liver due to presence of polyunsaturated lipid.[<xref ref-type="bibr" rid="ref35">35</xref>] Increased lipid peroxidation is a salient characteristic of chronic diabetes which impairs membrane function by reducing the activity of enzymes as well as receptors.[<xref ref-type="bibr" rid="ref36">36</xref>] Results focused that the hydro-methanolic extract of the seeds of <italic>C. bonduc</italic> inhibit lipid peroxidation under <italic>in vitro</italic> conditions indicating the anti-lipid peroxidant effect of the seed of <italic>C. bonduc</italic>.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Inhibition in lipid peroxidation by hydro-methanolic extract of seed of <italic>C. bonduc</italic> and standard BHT. The IC<sub>50</sub> value of the extract was 58.87 μg/ml</p></caption><graphic xlink:href="JAPTR-2-260-g005"></graphic></fig></sec><sec id="sec1-4"><title>CONCLUSION</title><p>The results of the study clearly indicate that hydro-methanolic extract of seed of <italic>C. bonduc</italic> possess <italic>in vitro</italic> antioxidant activity. The encouraging results of this extract in various <italic>in vitro</italic> tests proved that the plant seeds act as a reducing agent, its hydrogen donating ability and effectiveness as scavengers of hydrogen peroxide and hydroxyl radical. Hence, it is worthwhile to isolate and elucidate the bioactive principle(s) responsible for the antioxidant activity of the extract which is underway in our laboratory.</p></sec></body><back><ack><title>ACKNOWLEDGMENTS</title><p>The authors are thankful to the Ayurvedic division, Southern Health Improvement Samity (SHIS), Bhangar, South 24-Paraganas, West Bengal, India, for providing the necessary help, support and information.</p></ack><fn-group><fn fn-type="supported-by"><p><bold>Source of Support:</bold> Nil</p></fn><fn fn-type="conflict"><p><bold>Conflict of Interest:</bold> Nil.</p></fn></fn-group><ref-list><title>REFERENCES</title><ref id="ref1"><label>1</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Van de 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