Soumya et al.

Res Rev Biomed Biotech 1(1); 2010 Research and Reviews in Biomedicine and Biotechnology Volume [1], Issue [1], 2010, 31-37 www.rrbb.in

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DPPH radical scavenging and Insecticidal activity of seeds of Abrus pulchellus Wall (Fabaceae)
Soumya K.V 1*, Kiran R 1, Ashwini S.K 1, Prashith Kekuda TR 1, Raghavendra HL 2, Vinayaka KS 3 1 Department of Microbiology, S.R.N.M.N College of Applied Sciences, NES Campus, Balraj Urs Road, Shivamogga-577201, Karnataka, India. 2 Department of Biochemistry, Kuvempu University, Shivagangothri, Davangere-577 002, Karnataka, India. 3 Department of Studies and Research in Applied Botany, Jnana Sahyadri, Shankaraghatta577451, Karnataka, India.
* Corresponding author Email: soumya.abhime@gmail.com Article received on 03.08.2010; Revised article accepted on 16.10.2010 Copyright: 2010 rrbb.in

ABSTRACT The present study was carried out to determine free radical scavenging and insecticidal activity of methanol extract of seeds of Abrus pulchellus Wall (Fabaceae). The insecticidal activity of extract was tested against second instar larvae of Aedes aegypti. Antioxidant potential in terms of free radical scavenging activity was determined using DPPH radical scavenging assay. The larval mortality was 100% at extract concentration 15 mg/ml and higher. The extract exhibited marked antioxidant activity by scavenging free radical and the activity was found to be dose dependent. The scavenging activity of ascorbic acid was slightly greater than that of extract. The preliminary phytochemical analysis of methanol extract revealed the presence of flavonoids, saponins, glycosides and alkaloids. The presence of these phytoconstituents might account for the insecticidal and antioxidant efficacy of extract. Further studies on isolation of active constituents from the extract and their biological activity are under investigation. Keywords: Abrus pulchellus Wall, Antioxidant activity, DPPH radical scavenging assay, Insecticidal activity, Aedes aegypti, Larval mortality
INTRODUCTION

Mosquitoes are considered as a major health menace as they serve as disease transmitting vectors to humans and animals. Several mosquito species belonging to the genera Anopheles, Aedes and Culex are vectors of pathogens of various diseases such as malaria, filariasis, Japanese encephalitis, dengue, chickungunya etc. The approach to combat these diseases largely relied on interruption of the disease transmission cycle

by either targeting the mosquito larvae through spraying of stagnant water breeding sites or by killing the adult mosquitoes using insecticides. The control of mosquito borne diseases is becoming difficult nowadays due to increasing resistance to pesticides, lack of vaccines and drugs to treat diseases transmitted by them. Hence, an alternative approach to control mosquitoes is the use of plant extracts. Search for natural insecticides, which are easily degradable and do not have

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Soumya et al. any ill effects on the non-target population, remains one of the top priority issues for many countries [1-4]. Free radicals are believed to be involved in bacterial and parasitic infections, lung damage, inflammation, reperfusion injury, cardiovascular disorders, atherosclerosis, ageing and neoplastic diseases [5]. These free radicals, also known as reactive oxygen species (ROS), include oxygen and nonoxygen radicals. Among the oxygen radicals are singlet oxygen, superoxide anion, hydrogen peroxide and hydroxyl radicals. In biochemical systems, H2O2 generates extremely reactive hydroxyl radicals in the presence of certain transition metal ions (e.g. iron and copper) or by ultraviolet (UV) photolysis [6]. Hydroxyl radicals can attack DNA molecules, cause lipid peroxidation [7], tissue damage, protein denaturation and glutathione depletion [8]. Although organisms have endogenous antioxidant defences produced during normal cell aerobic respiration against ROS, other antioxidants are taken both from natural and synthetic origin [9]. Antioxidants that can inhibit or delay the oxidation of an oxidizable substrate in a chain reaction, therefore, appear to be very important [10]. Synthetic antioxidants are widely used but their use is being restricted nowadays because of their toxic and carcinogenic effects. Thus, interest in finding natural antioxidants, without any undesirable effect, has increased greatly [9]. Abrus pulchellus Wall (Fabaceae) synonym A. fruticulosus Wall. Ex Wight and Arn., and A. laevigatus E. May. (Shveta Gunjaa) is a twinning shrub commonly known as Bili gulaganji in Kannada and Rosary pea in English. It is also used for the same medicinal purposes as A. precatorius. Leaves are pinnately compound, leaflets 9 to 12 pairs, oblong, leaf rachis 12 cm long, stipulate, adnate or free lateral stipules are present, entire margin, leaf apex obtuse, reticulate venation. Flowers are in axillary long racemes, calyx 5 lobed, fused, corolla rose/white. Fruit is a pod, flat appressed and

Res Rev Biomed Biotech 1(1); 2010 pubescent. Seeds are pale yellow/white (Figure 1) [11, 12]. The present study was carried out to determine insecticidal and free radical scavenging activity of methanol extract of seeds of Abrus pulchellus Wall (Fabaceae).

Figure 1: Seeds of A. pulchellus Wall

MATERIALS AND METHODS

Collection and identification of seed materials The seeds of A. pulchellus (Voucher no. PK/SRNMN/Ap-301) were collected from the botanical garden of department of Botany, S.R.N.M.N College of Applied Sciences, Shivamogga and the voucher specimen was deposited for future reference. Extraction and Phytochemical analysis For extraction, about 50g of the dried and powdered seed material was taken and added to 100 ml of methanol. The mixture was sonicated for 30 min, and then left at room temperature overnight. The extracts were filtered over Whatman No 1 filter paper, and the filtrates were concentrated under reduced pressure to pasty mass. The methanol extract was subjected to chemical tests to screen the presence of various secondary metabolites [13,14] . Insecticidal activity solvent extracts For determining insecticidal activity, the extract was dissolved in 10% Dimethyl sulfoxide (DMSO) to get different concentrations of extract namely 5, 10, 15, 20 and 25mg/ml. The insecticidal efficacy of methanol extract was determined against second instar larvae of Aedes aegypti.

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Soumya et al. Twenty larvae were placed separately into beakers containing different concentrations of extract. A beaker containing water without extract serves as control. The larvicidal effect of extracts was determined by counting the number of dead larvae after 24 hours, 48 hours and 72 hours. Dead larvae were identified when they failed to move after probing with a needle in siphon or cervical region. The test was repeated thrice and the percentage of larval mortality was calculated [3]. Antioxidant activity of methanol extract by DPPH free radical scavenging activity The antioxidant activity, in terms of radical scavenging ability, of different concentrations of extract and the standard (Ascorbic acid) was tested on the basis of the radical scavenging effect of the stable 1,1diphenyl-2-picryl-hydrazyl (DPPH)-free radical activity [15]. Different concentrations namely 25, 50, 100, 200 and 400 g/ml of methanol extract and standard were prepared in methanol. 0.002% of DPPH was prepared in methanol. In clean and labeled test tubes, 2 ml of DPPH solution was mixed with 2 ml of different concentrations of solvent extracts and standard separately. The tubes were incubated at room temperature in dark for 30 minutes and the optical density was measured at 517 nm using UV-Vis Spectrophotometer. The absorbance of the DPPH control (containing no sample) was also noted. The degree of stable DPPH* decolorization to DPPHH (reduced form of DPPH) yellow indicated the scavenging efficiency of the extract. The scavenging activity of the extract against the stable DPPH* was calculated using the following equation. Scavenging activity in % = A B / A x 100 Where A is the absorbance of control and B is the absorbance of test/standard.
RESULTS

Res Rev Biomed Biotech 1(1); 2010 seeds of A. pulchellus was tested against second instar larvae of A. aegypti. The insecticidal activity was determined after 24 hours. All the concentrations of extract tested produced over 50% mortality of larvae. The methanol extract caused 100% mortality of larvae at concentration of 15mg/ml and higher (Table-1). DPPH free radical scavenging activity of different concentrations of methanol extract and Ascorbic acid is presented in Table-2. The extract exhibited marked antioxidant activity by scavenging DPPH* (free radical) and converting into DPPHH. A dose dependent radical scavenging activity was observed in this study. Over 75% scavenging of free radical was observed in all concentrations of the extracts. The scavenging activity of ascorbic acid was slightly greater than that of extract.
DISCUSSION

Preliminary phytochemical analysis showed the presence of flavonoids, alkaloids, saponins and glycosides in A. pulchellus. Insecticidal activity of methanol extract of

Mosquitoes are the most important single group of insects acting as vector for many tropical and subtropical diseases [16]. The large-scale use of chemical pesticides in agriculture and public health leads to adverse effects such as development of pesticide resistance, frequent pest out breaks, emergence of new pests, pollution and health hazards. In order to search an environmentally safe alternative, scientists considered the pesticides of biological origin (biopesticides) in the place of synthetic insecticides [17]. It is observed that the carbohydrates, saponins, phytosterols, phenols, flavonoids and tannins are having mosquito larvicidal activity. Prenylated xanthones, tetracyclic phenols and saponins are reported to be effective in controlling mosquito A. aegypti, the vector of yellow fever [3, 18]. In this study, the crude extracts have exhibited potent activity in terms of causing mortality of larvae. The presence of phytoconstituents such as saponins, flavonoids and others was detected in this study which might be responsible for the mortality of larvae. The death of larvae was observed within short period of time and thus

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Soumya et al. could be used to control mosquito vectors and diseases transmitted by them. Reactive oxygen species (ROS), such as superoxide anion, hydrogen peroxide, hydroxyl radical, nitric oxide, and peroxynitrite radical play an important role in oxidative stress related to the pathogenesis of various diseases [19]. In healthy individuals, endogenously produced free radicals are neutralised by antioxidants. Thus, oxidative stress is generated when equilibrium favours free radicals generation as a result of a depletion of antioxidant levels. The oxidation of lipids, DNA, proteins, carbohydrates, and other molecules, and glutathione depletion by toxic ROS may cause DNA mutation and/or severe damage to target cells or tissues, and this often results in cell senescence and death [6]. There are several methods available to assess antioxidant activity of compounds. An easy, rapid and sensitive method for the antioxidant screening of plant extracts is free radical scavenging assay using 1,1, diphenyl2-picryl hydrazyl (DPPH) stable radical spectrophotometrically. In presence of an antioxidant, DPPH radical obtains one more electron and the absorbance decreases [20]. In this study, the scavenging activity of methanol extract was found to be dose dependent i.e., higher the concentration, more was the scavenging activity. Though the DPPH radical scavenging abilities of the extracts were less than that of ascorbic acid, the study showed that the extract has the proton-donating ability and could serve as free radical inhibitors or scavenger, acting possibly as primary antioxidant. Various herbs and spices have been reported to exhibit antioxidant activity, the majority of the antioxidant activity is due to the flavones, isoflavones, flavonoids, anthocyanin, coumarin lignans, catechins and isocatechins [21]. It has been mentioned the antioxidant activity of plants might be due to their phenolic compounds [22]. Phenolics are the largest group of phytochemicals and have been said to account for most of the antioxidant activity

Res Rev Biomed Biotech 1(1); 2010 of plant extracts [23]. Flavonoids are a group of polyphenolic compounds with known properties, which includes free radical scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory action [24]. The preliminary phytochemical analysis of the methanol extract of both seeds showed the presence of flavonoids and this may account for the radical scavenging potential of the extracts. The species of Abrus are characterized by slender woody twinners with pinnate leaves, rachis ending in a bristle, stamens connate in a sheath with racemose flowers. Abrus pulchellus type-2 RIP, or pulchellin, is a heterodimeric glycoprotein found in A. pulchellus seeds. These chimerolectins are characterized as highly toxic proteins with enzymatic and lectin properties performed by two separate polypeptide subunits [25]. In a study, Kavya et al. [26] compared the proximate composition and antifungal activity of seeds of A. precatorius and A. pulchellus. The moisture content was low in A. precatorius as compared to A. pulchellus. Abrus species were quite rich in fibre content. The ash content of A. pulchellus (3.75%) was found to be slightly higher than A. precatorius (2.75%). Both the seed varieties exhibited more or less similar percentage of carbohydrate content. The crude protein content of A. pulchellus and A. precatorius were 24% and 25.2% respectively. The aqueous extracts of A. precatorius and A. pulchellus were subjected to antifungal activity against Aspergillus niger, A. oryzae and Mucor sp by poison food technique. Among extracts, A. pulchellus extract was more effective against A. niger and Mucor sp. The ethanol extract of A. pulchellus was investigated for hepatoprotective activity against CCl4 induced hepatic damage in experimental rat model by Hasan et al. [27]. The animal group treated with ethanol extract showed significant decrease in the levels of serum markers and significant increase in total protein which is comparable to the values recorded in the animal groups treated with

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Soumya et al. standard drug sylimarin, indicating the potency of ethanol seed extract in recovery of hepatic cells against carbon tetra chloride induced hepatic damage. Kekuda et al. [28] studied antibacterial and antifungal activity of methanolic extract of Abrus pulchellus Wall and Abrus precatorius Linn. The antibacterial activity was tested by Agar well diffusion method. Among extracts, A. pulchellus inhibited test bacteria to more extent than A. precatorius. Antifungal activity of extracts revealed 100% inhibition of fungal growth on seeds. In a previous study, we have reported antioxidant, antibacterial and insecticidal potential of different concentrations of methanolic extract of A. pulchellus leaves. The extract exhibited marked insecticidal and antioxidant activity in a dose dependent manner. The extract was found to inhibit Gram positive bacteria to more extent when compared to Gram negative bacteria [29].
CONCLUSION

Res Rev Biomed Biotech 1(1); 2010 fertility. The results of the present study are in justification of this and the extract of A. pulchellus could be used in control of arboviral infections transmitted by Aedes aegypti. Further studies on isolation of active constituents from the extract and their biological activities are under investigation.
ACKNOWLEDGEMENT

Authors are thankful to HOD, Dept. of Microbiology, Principal, S.R.N.M.N College of Applied Sciences, Shivamogga for their support. Authors also express thanks to N.E.S, Shivamogga for the moral encouragement.
REFERENCES 1. El Hag EA, El Nadi AH and Zaitoon AA. Toxic and growth retarding effects of three plant extracts on Culex pipens larvae (Diptera: Culicidae). Phyto. Res. 1999; 13(5): 388-392. 2. Redwane A, Lazrek HB, Bouallam S, Markouk M, Amarouch H and Jana M. Larvicidal activity of extracts from Quercus lusitania var infectoria galls (oliv). J. Ethnopharmacol. 2002; 79: 261263. 3. Khanna VG and Kannabiran K. Larvicidal effect of Hemidesmus indicus, Gymnema sylvestre, and Eclipta prostrata against Culex qinquifaciatus mosquito larvae. Afr. J. Biotech. 2007; 6: 307311. 4. Joseph CC, Ndoile MM, Malima RC and Nkuniya MHM. Larvicidal and mosquitocidal extracts, a coumrin, isoflavonoids and pterocarpans from Neorautanenia mitis. Trans. R. Soc. Trop. Med. Hyg. 2004; 98: 451455. 5. Burdon RH. Free radical damage and its control. Elsevier Science B. V. Netherland; 1994. 6. Shyur L.F., Tsung J.H., Chen J.H., Chiu C.Y., Lo C.P.. Antioxidant properties of extracts from medicinal plants popularly used in Taiwan. Int. J. App. Sci. Eng. 2005; 3(3): 195202. 7. Getteridge JM and Halliwell B. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 1995; 41: 18191828. 8. Nelson DL and Michael UC. Role of mitochondria in oxidative stress. Lehninger Principles of Biochemistry. 4th edition. W. H. Freeman Company: 2003. 9. Rechner AR, Kuhnle G, Bremmer P, Hubbard GP, Moore KP and Rice-Evans CA. Free Rad. Biol. Med. 2002; 33: 220-235. 10. Halliwell B, Gutteridge JM and Cross CE. Free radicals, antioxidants, and human disease: where are we now? J. Lab. Clin. Med. 1992; 119: 598620.

Currently available synthetic antioxidants like butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), tertiary butylated hydroquinones and gallic acid esters have been suspected to cause or prompt negative health effects. Hence, strong restrictions have been placed on their application and there is a trend to substitute them with naturally occurring antioxidants. In this study, the extract has shown promising results in terms of scavenging activity. Thus, the extract in suitable form could be exploited as the natural antioxidants that could protect the body against damage caused by free radicals. Higher plants are a rich source of novel substances that can be used to develop environmental safe methods for insect control as the chemical agents have caused some ill effects and also the mosquitoes developed resistance against them. The deleterious effects of plant extracts or pure compounds from plants on insects can be manifested in several manners including toxicity, mortality, antifeedant growth inhibitor, suppression of reproductive behavior and reduction of fecundity and

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11. Khare CP. Indian Medicinal Plants: An Illustrated Dictionary. Springer Verlag, Berlin: 2007. 12. Keshawamurthy K.R.. Medicinal Plants of Karnataka. J. Exp. Biol. 2005; 43: 722-727. 13. Kekuda TRP, Vinayaka KS, Kumar SVP and Sudharshan SJ. Antioxidant and Antibacterial activity of Lichen extracts, honey and their combination. J. Pharm. Res. 2009; 2(12): 18751879. 14. Parekh J and Chanda SV. In vitro Antimicrobial Activity and Phytochemical Analysis of Some Indian Medicinal Plants. Turk. J. Biol. 2007; 31: 53-58. 15. Khalaf NA, Shakya AK, Al-Othman A, El-Agbar Z and Farah H. Antioxidant activity of some common plants. Turk. J. Biol. 2008; 32: 51-55. 16. Service MW. Management of vectors. In: Youdeowei A, Service MW, editors. Pest Vector Management in Tropics, 2nd edn. Longman group Ltd., England; 1983. 17. Saxena RC. Botanical pest control. In: Dhaliwal GS, Heinrichs (Eds.), Critical issues in insect pest management. Commonwealth Publisher, New Delhi, India; 1998. 18. Marston A, Maillard M and Hostettmann K. Search for antifungal, molluscicidal and larvicidal compounds from African medicinal plants. J. Ethnopharmacol. 1993; 38: 215-223. 19. Halliwell B and Gutteridge JMC. Free radicals in Biology and Medicine. 3rd edition. Oxford University Press: Oxford, UK, 1999. 20. Koleva II, Vanbreek TA, Linssen JPH, Groot ADE and Evstatieva LN. Screening of plant extracts for antioxidant activity: A comparative study on the three testing methods. Phytochem. Anal. 2002; 13: 8-17. 21. Aqil F, Ahmed I and Mehmood Z. Antioxidant and free radical scavenging properties of twelve traditionally used Indian medicinal plants. Turk. J. Biol. 2006; 30: 177-183. 22.

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Scholer P. Natural antioxidants exploited commercially. In: Hudson BJF, Food antioxidants. Elsevier; London, 1990. Thabrew MI, Hughes RD and McFarlane IG. Antioxidant activity of Osbeckia aspera. Phyto. Res. 1998; 12: 288-290. Finkel T and Holbrook NJ. Oxidants, oxidative stress and the biology of aging. Nature 2000; 408: 239247. Goto LS, Beltramini LM, de Moraes DI, Moreira RA and de Araujo AP. Abrus pulchellus type-2 RIP, pulchellin: heterologous expression and refolding of the sugar-binding B chain. Protein Expr Purif 2003; 31(1):12-18. Kavya R, Shrungashree RM, Suchitra SV, Divakara R, Kekuda TRP. Comparative study on Antifungal activity and Proximate composition of Abrus pulchellus Wall and Abrus precatorius Linn. Res. J. Pharmacog. Phytochem. 2010; 2(4): Hasan AT, Kavya R, Nandini H, Shrungashree RM and Suchitra SV. Studies on Phytochemical and Hepatoprotective activity of Abrus pulchellus. Project Dissertation submitted to Dept. of Botany, S.R.N.M.N College of Applied Sciences, Shivamogga for the award of Bachelor of Science by Kuvempu University, Shankaraghatta, Shivamogga, Karnataka, INDIA, 2007. Kekuda TRP, Vinayaka KS, Soumya KV, Ashwini SK and Kiran R. Antibacterial and Antifungal activity of methanolic extract of Abrus pulchellus Wall and Abrus precatorius Linn - A comparative study. International Journal of Toxicological and Pharmacological Research 2010; 2(1): 284-287 Vinayaka KS, Swarnalatha SP, Preethi HR, Surabhi KS, Kekuda TRP and Sudharshan SJ. Studies on In vitro Antioxidant, Antibacterial and Insecticidal Activity of Methanolic Extract of Abrus pulchellus Wall (Fabaceae). Afr. J. Bas. Appl. Sci. 2009; 1(5-6): 110-116

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Table-1: Insecticidal activity of methanol extract of A. pulchellus concentration (mg/ml) Total no. of larvae No. of dead larvae % mortality of larvae 5 20 12 60.00 10 20 18 90.00 15 20 20 100.00 20 20 20 100.00 25 20 20 100.00 Results are average of three trials Table-2: DPPH free radical scavenging activity of methanol extract of A. pulchellus Conc. ( g/ml) 400 200 100 50 25 Radical scavenging activity (%) Methanol extract Ascorbic acid 93.440.98 96.291.18 91.052.08 95.690.92 83.681.05 94.962.10 80.162.89 92.691.07 76.694.24 89.362.39 Results are average of three trials

Sources of support: Nil Conflicts of interest: Nil

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