Comparative Evaluation of in vitro Antioxidant Activity of Root of Blue and...

[Full title: Comparative Evaluation of in vitro Antioxidant Activity of Root of Blue and White Flowered Varieties of Clitoria ternatea Linn.]

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Life International Journal of Pharmacology ISSN 1811-7775 Life science alert ansinet Asian Network for Scientific Information

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International Journal of Pharmacology 7 (4): 485-491. 2011 ISSN 1811-7775 DOI: 10.3923/ijp.2011.185.191 2011 Asian Network for Scientific Information Comparative Evaluation of in vitro Antioxidant Activity of Root of Blue and White Flowered Varieties of Clitoria ternatea Linn. A.P. Patil and V.R. Patil TVES's Honorable Loksevak Madhukarrao Chaudhari, College of Pharmacy, Faizpur. India Abstract: Pet ether, chloroform and methanol extracts of roots of blue and white flowered varieties of Clitoria ternated were studied for their antioxidant potential. DPPII free radical scavenging assay, reducing power assay, hydroxyl radical scavenging assay were studied for evaluation of antioxidant potential. Pet ether, chloroform and methanol extracts Tools of blue and white flowered varieties of Clitoria ternatea (CT) significantly inhibited the DPPH free radical at the concentrations ranging from 50-600 μg mL Pet ether. chloroform and methanol extracts roots of blue flowered variety of CT showed highest inhibition i.e., 19.11, 35.42 and 70.67% at 600 µg mL −1, respectively. Pet ether, chloroform and methanol extracts roots of white Nowered variety of (CT) showed highest inhibition i.e., 54.48, 39.21 and 78.13% al 600 µg m1.-1, respectively. Methanol extracts of blue and white flowered varieties of CT showed a very powerful antioxidant activity in DPPII radical-scavenging assay. Methanol extracts of CT also showed significant reductive ability as well as hydroxyl radical scavenging activity. Methanol extract of white flowered variety of CT showed more significant antioxidant activity as compared to blue flowered variety of CT. All the concentrations of methanol extract of CT (MECT) antioxidant activity when compared to control and these differences were statistically significant (p<0.001). Clitoria ternatea Linn. could be considered as a potential source of natural antioxidants. Key words: Clitoria ternated, antioxidant, DPPH, reducing power, hydroxyl INTRODUCTION Cell damage caused by free radicals appears to be a major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular disease, cataracts. immune system decline and brain dysfunction. Reactive Oxygen Species (ROS) have been found to play an important role in the initiation and/or progression of various diseases such as atherosclerosis, inflammatory injury, cancer and cardiovascular disease (Halliwell, 1997). Thus, present studies have investigated the potential of plant products as antioxidants against various diseases induced by free radicals (Hou et al., 2003). Earliest description of curative properties of medicinal plants is found in Rig-Veda. Charaka, Samhita and Sushrusha Samhita which give extensive description on various medicinal herbs. Information on medicinal plants in India has been systematically organized (Satyavati et al., 1976, Satyavati et al., 1987). Clitoria ternatea L. (CT) a perennial twining herb, found throughout India in tropical areas. CT is commonly known as Bullerily pea belong to Family: Fabaceae. CT has two flowered varieties one is white flowered variety and second blue flowered variety. CT has been traditionally used as a remedy for various disease like urinogenital disorder, bronchilis, purgative, diuretic. anthelmintic rheumatism. demulcent, anticancer, antidote for animal stings (Kapoor, 2005; Parrotta, 2001; Prajapati et al., 2003; Khare, 2001). CT has been used as an ingredient in Medhya Rasayana a rejuvenating recipe used for treatment of neurological disorders (Kamkaen and Wilkinson 2009). CT has been scientifically studied for various pharmacological activities like local anesthetic (Mukherjee et al., 2008), anthelmintic (Khadatkar et al., 2008; Nirmal et al., 2008). antipyretic, anti-inflammatory, analgesic (Devi et al.. 2003), anxiolytic, antidepressant, anticonvulsant, sedative (Jain et al., 2003), hypoglycemic (Sharma and Majumdar, 1990), anticancer (Balachandran and Govindarajan, 2005) also enhances acetylcholine content in rat hippocampus (Rai et al., 2002). A wide range of chemical constituents are present in CT. Still the comparative evaluation of antioxidant activity of blue and white flowered variety of CT has TOI been carried out. Aim of this stukly to explore the antioxidant potential both varieties of Clitoria ternatea L. MATERIALS AND METHODS Plant material: The roots of blue and white flowered variety of Clitoria ternatea were collected from local habilal The plant specimens were authenticated Corresponding Author: A.P. Patil, TYES's Honorable Loksevak Madhukarrao Chaudhari, College of Pharmacy, Faizpur, India 485

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Int. J. Pharmacol., 7 (4): 485-491, 2011 (Specimen No 9492, 9493) Botany Department Rashtrasant Tukdoji Maharaj, Nagpur University, Nagpur. Extraction of plant material: The roots of blue and white flowered variety of Clitoria ternated were cut into small pieces and dries at room temperature. The dried rools were subjected to size reduction to coarse powder by using dry grinder. This powder is packed into soxhlet apparatus and extracted with peeler (60-80°C). chloroform, methanol (Sharma et al., 2009). The extracts were evaporated to dryness at 10°C (White flowered variety yields 9, 11, 12% w/w, respectively and blue flowered variety yields S, 11, 10% w/w, respectively). A phytochemical screening of residues revealed the presence of phenolic compounds, triterpenoid sterol and protein (Egwuche et al., 2011). In-vitro antioxidant assay Chemicals: DPPH solution (200 µM) Phosphate buffer (0.2M, pH 6.6) (Potassium dihydrogen phosphate 0.2M): (NaOII 0.2M): Potassium ferricyanide (1%), TCA (10%), Ferric chloride (0.1%). Dpph free radical scavenging assay: DPPII free radical scavenging assay was performed according to method of (Rajeshwar et al., 2005; (till et al., 2011a). Percent. inhibition of DPPII free radical scavenging activity was calculated using the following formula: DPPII radical scavenged (%) = (A cont-A les) A coal x100 Where, Acont is the absorbance of the control reaction. Atest is the absorbance in the presence of the sample of the extracts. Determination of reducing power: The total reducing power of roots of blue and white flowered variety of Clitoria ternatea was determined according to the method of Oyaizu (Oyaizu, 1986; Wong and Kilts, 2003). IIydroxyl radical scavenging assay: The hydroxyl radical scavenging activity of MECT was performed according to Halliwell and Londonkar. (Halliwell et al., 1987; Londonkar and Kamble, 2009) The hydroxyl radical scavenging activity of MECT is reported as %inhibition of deoxyribose degradation and is calculated as follows: (A cont - A test). %Inhibition X100 A cont where, A cont is the absorbance of control at 532 nm and A test is the absorbance of BMS samples al 532 mm. Statistical analysis: All the values were expressed as Mean-Standard Error of Means (SEM). Statistical analysis was performed by one-way Analysis of Varience (ANOVA). Further comparison WHS done by Tukey's multiple range tests using prism 4.0 software. A probability value was considered to be statistically (Krishna et al., 2010). RESULTS graphpad of p<0.05 significant DPPII radical scavenging assay: Pet ether, chloroform and methanol extracts Tools blue flowered variety of CT showed significant inhibition of DPPII radical i.e., 49.11% (Fig. 1), 35.12% (Fig. 2) and 70.67% (Fig. 3) at 600 μg mL¯¯¯ Pct. cther, chloroform and methanol extracts roots of white flowered variety of (CT) showed highest inhibition of DPPH radical i.c., 54.48% (Fig. 4), 39.21% (Fig. 5) and 78.13% (Fig. 6) at 600 μg mL MECT of blue and white Powered varieties showed significant. DPPH radical scavenging activity as compared to other extracts. The IC; values of methanol extract of blue flowered is 492 μg mL. The ICs, values of methanol extract of white flowered is 312 ug mL Ascorbic acid was used as reference standard for the DPPH free radical scavenging assay; it significantly inhibits DPPH free radical at the concentrations ranging from 2-20 μg mL, showing highest % inhibition i.e., $1.58% at 20 μg mL (Fig. 7). The IC value obtained was found to be 9.38 µg mL 50- 40- 30- 50 100 200 300 400 500 600 Concentration (μg mL) +1 Fig. 1: DPPH free radical scavenging activity of Pet. ether extract of blue flowered variety of CT (PEECT). Values ate expressed HS Mean Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, when compared with control, n = 3. **Represents no statistical significance: p>0.05, n-3 486

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Int. J. Pharmacol., 7 (4): 485-491, 2011 100000000 50 100 200 300 400 500 600 Concentration (μg mL) Fig. 2: DPPII free radical scavenging activity of chloroform extrael of blue flowered variety of CT (CECT). Values are expressed as a Mean Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, when compared with control, n = 3 50 100 200 Fig. 5: DPPH 300 400 500 600 Concentration (μg mL) free radical scavenging activity of choloform extract of white flowered variety of CT (CECT). Values are expressed as a Mean+Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, when compared with control, n = 3 % inhibition of DPPH radical 60- 50- % inhibition of DPPH radical 50 100 200 300 400 Concentration (μg mL) 500 600 Fig. 3: DPPII free radical scavenging activity of methanol extract of blue flowered variety of CT(MECT). Values are expressed as a Mean standard error of mcan of the 3 observations. *Represents statistical significance: p<0.001, when compared with control, n = 3 50 100 200 300 400 Concentration (μg mL) 500 600 Fig. 6: DPPH free radical scavenging activity of methanol extract of white flowered variety of CT (MECT). Values are expressed as a Mean+Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, when compared with control. 11-3 100- 40- % inhibition of DPPH radical 90- 80- 2 5 10 15 20 50 100 300 200 400 Concentration (μg mL) 500 600 Fig. 4: DPPII free radical scavenging activity of pet ether extract of white flowered variety of CT (PEECT). Values are expressed as a Mean+Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, when compared with control. n = 3 Methanol extracts of both flowered varieties of CT were further used for reducing power assay and hydroxyl radical scavenging assay. Concentration (μg mL) Fig. 7: DPPH Free radical scavenging activity of Lascorbic acid: Values are expressed as a Mean Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, when compared with control, n = 3. **Represents no statistical significance: p>0.05, 11-3. Reducing power assay: For the measurements of the reductive ability, the Fe³-Fe* transformation in the presence of methanol and aqueous extracts 487

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0.87 Int. J. Pharmacol., 7 (4): 485-491, 2011 0.7 0.6 0.5- 0.4- 0.3- 0.2- 0.1Control 50 100 200 300 000 400 500 600 Concentration (μg mL) Fig. 8 Reducing power of methanol extract of blue flowered variety of CT al 700 m. Values are expressed as a Mean Standard error of mean of observations. *Represents statistical significance: p<0.001, n the 3 3 + 50 100 200 300 400 500 600 Concentration (μg mL) Fig. 11: Hydroxyl radical scavenging activity of methanol extract of blue flowered variety of CT (MDCT). Values are expressed as Mean+Standard 3 observations error a of *Represents mean of the statistical 1.0 0.9 0.8- 0.7- 0.6 0.5- 0.4- 0.3- 0.2 Control 50 100 200 90- 80- 70- 60- 50significance: p<0.001, when compared with control, n-3 00000000 Concentration (μg mL) Fig. 9: Reducing power of methanol extract of white flowered variety of CT at 700 nm. Values are expressed as a Mean±Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, n − 3 Absorbance at 700 nm 1.2- 1.0- 0.8- 0.6Lo0000 0.4- 0.2Control 10 20 40 60 Concentration (μg mL) 80 100 Fig. 10: Reducing power of L-ascorbic acid at 700 nm. Values are expressed as a Mean±Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, n = 3 using the method of oyaizu was studied. The reducing capacity of a compound may serve as a significant indicator of its CxITHCl. founkl Lo be significant (p<0.001). The antioxidant activity has been reported to be concomitant with development of reducing power. The reducing power of methanol extracts both 488 40 30. 50 100 200 300 Concentration (μg mL) a Fig. 12: Lydroxyl radical scavenging activity of methanol extract of white flowered variety of CT (MECT). Values ate expressed as Mean Standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, when compared with control, n = 3 zlowered varieties of CT were found to be increase with increasing amount of extraels concentration. All the concentrations of methanol extracts of both flowered varieties of CT were showed significant activity when compared to control and these differences were statistically significant (p<0.001) (Fig. 8-10). Methanol extract white flowered variety of CT showed significant reducing power as compared to methanol extract bluc flowered variety of CT. Hydroxyl Radical scavenging assay: The hydroxyl radical scavenging activity of MECT is reported as %inhibition of deoxyribose degradation. Methanol extract white flowered variety of CT showed significant hydroxyl radical scavenging activity as compared to blue flowered variety of CT. Methanol extract white flowered variety of CT showed 75.50% (Fig. 11) and methanol extract blue flowered variety of CT showed 67.37% (Fig. 12) inhibition of hydroxyl radical at 600 μg mL. Ascorbic acid was

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% inhibition of radicals Int. J. Pharmacol., 7 (4): 485-491, 2011 5 10 20 40 60 80 100 Concentration (ug mL) +1 Fig. 13: Hydroxyl radical scavenging activity of LAscorbic acid. Values are expressed us mean standard error of mean of the 3 observations. *Represents statistical significance: p<0.001, when compared with control, n = 3 used as reference standard for this assay. It significantly inhibits hydroxyl radical at the concentrations ranging from 5-100 μg mL¯¹, showing highest. %inhibition i.c.. 83.16% at 100 μg mL (Fig. 13). J DISCUSSION It has been studied that damages caused by free radical induced oxidative stress is the major causative agent of many disorders including cancer, tissue injury and rheumatoid arthritis (Govindarajan et al., 2005) neurodegenerative diseases, aging (Khlifi et al., 2006). When oxidative stress reaches a certain limit, a defence mechanism become insufficien led ko decrease intracellular concentration of GSII and antioxidant enzymes (Bashandy and AIWasel, 2011). Oxidative stress major pathological factor for many disease. The chronic consumption of high fructose diet contribute to excessive formation of reactive oxygen species. This leads to induce oxidative stress (Bakoma et al., 2011). The human body counteract oxidative stress by producing antioxidants which are either natuarllu produced in situs. or externally supplied through food and supplements (Gill et al., 2011b). Flower of CT was previously used for gel preparation (Kamkaen and Wilkinson, 2009), CT has been traditionally used as a remedy for various disease like urinogenital disorder, bronchitis, purgative, diuretic, anthelmintic, rheumatism, demulcent, anticancer and antidote for animal stings. Root of the CT also used in preparation of medhya rasayana, still antioxidant activity in comparisonof both varictics was not carried out yet. So this was attempt to explore antioxidant potential. DPPH is a stable radical that has been used widely to evaluate the antioxidant activity of various natural products. DPPH is one of a few stable and commercially available organic nitrogen radicals and has a UV-vis absorption maximum at 517 nm. Upon reduction, the solution color fades: the reaction progress is conveniently monitored by a spectrophotometer. The free radical scavenging activities of test compounds were examined based on their ability to bleach the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Thus, the absorbance of DPPII solution decreases when kept in contact with antioxidant. test. sample and free radical scavenging activity is inversely proportional to the absorbance of DPPH solution (Blois, 1958). The methanol extract of white flowered variety showed significant antioxidant activity as compared to all other extracts of CT. Methanol extracts of Both flowered varieties of CT were fiuther used for Reducing power assay and hydroxyl radical scavenging assay. The reducing power assay resembles the redox titration in classical chemical analysis. The degree of the color change is proportional to the antioxidant concentrations. The reaction end point is reached when color change stops. The antioxidant activity has been reported to be concomitant with development of reducing power. The reducing power of MECT was found to be ITCTCHSC with increasing amount of extract. concentration. All the concentrations of MECT showed significant activities when compared to control and these differences were statistically significant (p<0.001). The potentially reactive hydroxyl radical can cause oxidative damage to DNA, lipids and proteins. The effect of Clitoria ternated on inhibition of free radical was significant (Jornot et al., 1998). In recent years, antioxidants derived from natural resources, mainly from plants, have been intensively used to prevent oxidative damages (Ali et al., 2008). Polyphenolic compounds like flavonoids. Tannins, Phenolic acids commonly found in the plant. have been reported to have multiple biological effects, including antioxidant activity (Rahman et al., 2011). Natural antioxidants have also some advantages over synthetic ones. They can be obtained easily and economically and have slight or negligible side effects. announced Many plants have been posses antioxidant activity etc. Natural antioxidants have also some advantages over synthetic ones. The methanol extract of white flowered variety showed significant antioxidant activity in DPPII assay, reducing power assay and hydroxyl radical scavenging assay. Root of Clitoria ternatea would be good source for antioxidants. to 489

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CONCLUSION Int. J. Pharmacol., 7 (4): 485-491, 2011 The present study showed prominent, antioxidant. activity of Clitoria ternatea Linn. Methanolic extract of white flowered variety of root of CT showed good antioxidant activity as compared to blue flowered variely of CT. The white flowered variety may be the good source of natural antioxidant. Antioxidant activity may be due to presence of phenolic compounds in MECT. However, the exact components responsible for the antioxidant activity of MECT are not clear. Therefore, further work is posekkitu lo isolate characterize those constituents. ACKNOWLEDGMENT The authors wish to thank the management of the college for encouraging and providing research facilities. The authors also wish to thanks Botany Department Rashtrasant Tukdoji Maharaj, Nagpur University, Nagpur was authenticated the plant specimens. Authors would like to thanks the coauthor for their continuous and prestigious guidance till the end of the study. REFERENCES Ali, S.S.. N. Kasoju A. Luthra, A. Singh. 11. Sharanabasava, A. Sahu and L. Bora, 2008. Indian medicinal herbs as sources of antioxidants. Food Res. Trl 41: 1-15. Bakoma, B., K. Eklu-Gadegkeku, A. Agbonon. K. Aklikokou, E. Bassene and M. Gbeassor, 2011. Preventive effect of Bridelia ferruginea against highfructose diet induced glucose intolerance, oxidative stress and hyperlipidemia in male wistar rals. J. Pharmacol Toxicol., 6240-257. Balachandran, P. and R. Govindarajan, 2005. Cancer-an ayurvedic perspective. Pharmacol. Res., 51: 19-30. Bashandy, S.A. and S.H. AlWasel 2011. Carbon tetrachloride-induced hepatotoxicity and nephrotoxicity in rals: Protective role of vitamin C. J. Pharmacol. Toxicol., 6: 283-292. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature, 181: 1199-1200. Devi, B.P., R. Boominathan and S.C. Mandal, 2003. Antiinflammatory, analgesic and antipyretic properties of Clitoria ternated rool. Fitoterapia, 74: 345-349. Egwuche, R.U., A.A. Ödetola and O.L. Erukainure, 2011. Preliminary investigation into the chemical properties of Peperomia pellucida L. Res. J. Phytochem.. 5: 48-53. Gill N.S., J. Bajwa, K. Dhiman, P. Sharma and S. Sood et al., 2011a. Evaluation of therapeutic potential of traditionally consumed Cucumis melo seeds. Asian J. Plant Sci., 10: 86-91. Gill NS, J. Bajwa, P. Sharma, K. Dhiman and S. Sood et al., 2011. Evaluation of antioxidant and antiulcer activity of traditionally consumed Cucumis melo seeds. J. Pharmacol. Toxicol., 6: 82-89. Govindarajan, R., M. Vijayakumar and P. Pushpangadan, 2005. Antioxidant approach to disease management and the role of 'Rasayana' herbs of Ayurveda. J. Ethnopharmacol., 99: 165-178. Halliwell, B., 1997. Antioxidants and human diseases: A general introduction. Nutr. Rev., 55: $11-$19. Halliwell, B., J.M.C. Gutteridge and O.I. Amoma, 1987. The deoxyribose method: A simple "test-tube" assay for determination of rate constant for reactions of hydroxyl radicals. Anal. Biochem., 165: 215-219. Ilou, W.C., R.D. Lin, K.T. Cheng, Y.T. IIung and CH. Cho et al., 2003. Free radical-scavenging activity of Taiwanese native plants. Phytomedicine. 10: 170-175. Jain NN, CC. Ohal, S.K. Shroff. R.H. Bhutada. R.S. Somani, V.S. Kasture and S.B. Kasture, 2003. Clitoria ternated and the CNS. Pharmacol. Biochem. Behav., 75: 529-536. Jornot, L., H. Peterson and A.F. Junod, 1998. Hydrogen peroxide induced DNA damage is independent of nuclear calcium but dependent on redox active ions. Biochemistry, 33.5: 85-94. Kamkaen, N. and J.M. Wilkinson, 2009. The antioxidant activity of Clitoria ternatea flower petal extracts and eye gel. Phytother. Res., 23: 1624-1625. Kapoor, L.D., 2005. IIandbook of Ayurvedic Medicinal Plants. CRC Press, Boca Raton, FL, USA.. PP: 126-127. Khadatkar, S.N., J.V. Manwar and N.S. Bhajipale, 2008. In vitro anthelmintic activity of root of Clitoria ternatea Linn. Pharmacogn. Mag.. 4: 148-150. Khare, C.P., 2004. Encyclopedia of Indian Medicinal Plants. Springer Publishing Company, New York, pp: 153-154. Khlifi S. Y. El-Hachii, A. Khalil, N. Es-Safi, A. Belahyani R. Tellal and A. El-Abbouyi, 2006. In vitro antioxidant properties of Salvia verbenaca L. hydromethanolic extrael. Indian J. Pharmacol., 38: 276-280. Krishna, K.L., K. Muthunjaya and J.A. Patel, 2010. Antioxidant and hepatoprotective potential of stem methanolic extract of Justicia gendarussa buin. Int. J. Pharmacol., 6: 72-80. 490

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