Studies on Antioxidant, Antibacterial and Insecticidal ...

African Journal of Basic & Applied Sciences 1 (5-6): 110-116, 2009 ISSN 2079-2034 © IDOSI Publications, 2009

Studies on In vitro Antioxidant, Antibacterial and Insecticidal Activity of Methanolic Extract of Abrus pulchellus Wall (Fabaceae) 1

K.S. Vinayaka, 2S.P. Swarnalatha, 2H.R. Preethi, 2K.S. Surabhi, 2 T.R. Prashith Kekuda and 3S.J. Sudharshan 1

Department of Studies and Research in Applied Botany, Jnana Sahyadri, Shankaraghatta-577451, Karnataka, India 2 Department of Microbiology, S.R.N.M.N College of Applied Sciences, NES Campus, Balraj Urs Road, Shivamogga-577201, Karnataka, India 3 Department of Veterinary Physiology and Biochemistry, Govt. Veterinary College, Shivamogga-577204, Karnataka, India Abstract: Abrus pulchellus Wall is a twinning shrub belonging to the family Fabaceae. The present investigation was carried to evaluate antioxidant, antibacterial and insecticidal potential of different concentrations of methanolic extract of A. pulchellus leaves. DPPH radical scavenging assay and Fe+3 reducing assay were carried to determine antioxidant activity of extract. Insecticidal efficacy of extract was tested using second instar larvae of Aedes aegypti. The extract exhibited marked antioxidant activity by scavenging DPPH free radical in a concentration dependent manner. In Fe+3 reducing assay, increase in the absorbance revealed the reducing power of extracts. The extract was found to inhibit Gram positive bacteria to more extent when compared to Gram negative bacteria. The minimum inhibitory concentration of extract was found to be less for Gram positive bacteria. A dose dependent mortality of second instar larvae of A. aegypti was observed in this study. The larval mortality was recorded as 100% in the case of 30mg/ml and higher concentrations of methanolic extract. Preliminary phytochemical analysis of methanol extract showed the presence of tannins, saponins, steroids and glycosides. The efficacy of extract of A. pulchellus may be attributed to the phytochemicals present in the solvent extract. Further studies on isolation of active constituents and their biological activities are to be carried out. Key words: Abrus pulchellus % DPPH radical scavenging assay % Fe+3 reducing assay % Agar well diffusion % Minimum inhibitory concentration % Insecticidal activity % Aedes aegypti INTRODUCTION

hold great promise in clinical therapy due to their potential to reduce side effects associated with chemotherapy or radiotherapy and significant advantages in reducing the health care cost [4]. The history of plants being used for medicinal purpose is probably as old as the history of mankind. Extraction and characterization of several active phyto-compounds from these green factories have given birth to some high activity profile drugs. The potential natural anticancer drugs like vincristine, vinblastine and taxol can be the best examples [5]. Free radicals are found to be a product of normal metabolism. Although oxygen is essential for aerobic forms of life, oxygen metabolites are highly toxic. As a consequence, reactive oxygen species (ROS) are known

Obtaining adequate nutrients from various foods plays a vital role in maintaining normal function of the human body. With recent advances in medical and nutrition sciences, natural products and health-promoting foods have received extensive attention from both health professionals and the common population. New concepts have appeared with this trend, such as nutraceuticals, nutritional therapy, phytonutrients and phytotherapy [1-3]. These functional or medicinal foods and phytonutrients or phytomedicines play positive roles in maintaining well being, enhancing health and modulating immune function to prevent specific diseases. They also

Corresponding Author: T.R., Prashith Kekuda, Department of Microbiology, S.R.N.M.N College of Applied Sciences, NES Campus, Balraj Urs Road, Shivamogga-577201, Karnataka, India

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African J. of Basic & Appl. Sci., 1 (5-6): 110-116, 2009

to be implicated in many cell disorders and in the development of many diseases including cardiovascular diseases, atherosclerosis, chronic inflammation etc [6, 7]. Although organisms have endogenous antioxidant defences produced during normal cell aerobic respiration against ROS, other antioxidants are taken both from natural and synthetic origin [8]. Antioxidants that can inhibit or delay the oxidation of an oxidizable substrate in a chain reaction, therefore, appear to be very important [9]. 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 [8]. Mosquitoes are the most important single group of insects acting as vector for many tropical and subtropical diseases such as dengue fever, yellow fever, malaria, filariasis, Japanese encephalitis and others [10]. 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 [11]. 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. Throughout history, plant products have been successfully exploited as insecticides, insect repellents and insect antifeedants [12]. Infectious diseases caused by bacteria, fungi, viruses and parasites remain a major threat to public health, despite tremendous progress in human medicine. Their impact is particularly great in developing countries because of the relative unavailability of medicines and the emergence of widespread drug resistance [13]. Interest in natural products with antimicrobial properties has revived as a result of current problems associated with the use of antibiotics [14]. Abrus pulchellus (Fabaceae) is a twinning shrub commonly known as Bili gulaganji in Kannada and Rosary pea in English. 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 pubescent. Seeds are pale yellow/white [15]. The objective of the present investigation was to evaluate antioxidant,

antibacterial and insecticidal potential of different concentrations of methanol extract of Abrus pulchellus leaves. MATERIALS AND METHODS Collection and Identification of Plant Material: The plant material was collected from the botanical garden of department of Botany, S.R.N.M.N College of Applied Sciences, Shivamogga, authenticated and the voucher specimen (voucher no. SRNMN/MB/Ap/101) was deposited in the department for future reference. Extraction and Phytochemical Analysis: For extraction, about 50g of the shade dried and powdered leaf material was taken and added to 100ml of methanol. The mixtures were 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 [16]. The methanol extract was subjected to preliminary phytochemical screening to screen the presence of various secondary metabolites [17, 18]. Antioxidant Activity of Methanolic Extract by DPPH Free Radical Scavenging Assay: DPPH free radical scavenging assay was performed to determine the antioxidant activity of different concentrations of methanol extract and ascorbic acid (0.125, 0.250, 0.500 and 1.000mg/ml) [19, 20]. DPPH (0.002%) was used as free radical. Equal volume of different concentrations of methanol extracts and DPPH were mixed in clean and labeled test tubes separately and the tubes were incubated at room temperature in dark for 30 minutes. The optical density was measured at 517nm using UV-Vis Spectrophotometer. 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 (%) = A – B / A x 100 Where A is absorbance of DPPH and B is absorbance of DPPH and extract combination. Antioxidant Activity of Methanolic Extract by Fe+3 Reducing Power Assay: Different concentrations of Methanolic extracts and tannic acid (0.125, 0.250, 0.500 and 1.000mg/ml) in 1ml of methanol were mixed with 2.5ml of phosphate buffer (200mM, pH 6.6) and 2.5ml of 1%

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potassium ferricyanide separately. The mixtures were placed in a water bath for 20 min at 50°C, cooled rapidly, mixed with 2.5ml of 10% trichloroacetic acid and 0.5ml of 0.1% Ferric chloride. The intensity of iron (II)- ferricyanide complex was determined by measuring the formation of Perl’s Prussian blue at 700nm after 10 min. The higher absorbance of the reaction mixture indicates increased reducing power [21,22].

RESULTS Preliminary phytochemical analysis of methanolic extract revealed the presence of tannins, saponins, steroids and glycosides. The result of antioxidant activity of different concentrations of methanolic extract and standard (ascorbic acid) is shown in Table 1. The extract exhibited marked antioxidant activity by scavenging DPPH* (free radical) and converting into DPPHH. The extract has shown concentration dependent radical scavenging activity. The scavenging activity of standard was higher than that of methanol extract. Fe+3 reducing power assay was carried out for the measurements of reductive ability of different concentrations of methanolic extract and standard (Tannic acid). An increase in the absorbance revealed the reducing power of extracts. In this study, the reducing power of methanolic extract was found to increase with the dose (Table 2). The result of antibacterial activity of methanolic extract of A. pulchellus is shown in Table 3. The results were recorded as presence or absence of zones of inhibition around the well. The inhibitory zone around the well indicated the absence of bacterial growth and it as reported as positive and absence of zone as negative [26]. It was found that the crude solvent extract showed antibacterial activities against Gram-positive bacteria to more extent than Gram negative bacteria. Standard antibiotic also exhibited marked activity against Gram positive bacteria than Gram negative bacteria. Among Gram negative bacteria, E. coli showed more susceptibility than P. aeruginosa whereas, in case of Gram positive bacteria, C. perfringens exhibited high susceptibility to methanol extract than S. aureus. Among bacteria, Gram positive bacteria were inhibited by low concentrations of extract as compared to Gram negative bacteria. The MIC of extract was found to be least for C. perfringens (125µg/ml) followed by S. aureus (250µg/ml), P. aeruginosa (500µg/ml) and E. coli (550µg/ml). The mortality (in %) of the second instar larvae of A. aegypti in different concentrations of various solvent extracts is shown in Table 4. Concentration dependent mortality was observed. Over 50% larval mortality was observed in all concentrations of the extract. The larval mortality was recorded as 100% in the case of 30mg/ml and higher concentrations of methanol extract.

Antibacterial Activity of Methanolic Extract: The antibacterial activity was tested against Staphylococcus aureus MTCC-902, Clostridium perfringens MTCC-450, Escherichia coli MTCC-405 and Pseudomonas aeruginosa MTCC-1934 by Agar well diffusion method [23]. The test bacteria were obtained from IMTECH, Chandigarh, INDIA Twenty four hours old Muller-Hinton broth cultures of test bacteria were aseptically swabbed on sterile Muller-Hinton agar plates. Wells of 6 mm diameter were made aseptically in the inoculated plates and the methanol extract (20mg/ml of 10% DMSO), Standard (Chloramphenicol, 1mg/ml) and Control (10% DMSO) were added into the respectively labeled wells. The plates were incubated at 37°C for 24 hours in upright position. The experiment was carried in triplicates and the zone of inhibition was recorded. Determination of Minimum Inhibitory Concentration (MIC): Sterile nutrient broth tubes containing different dilutions of extract (0.05mg to 1.0mg/ml) were specifically inoculated with 0.1 ml of standardized inoculum (107cfu/ml). The tubes were incubated aerobically at 37°C for 18-24 h. Two control (tube containing the growth medium, saline and the inoculum) tubes for each organism were maintained. The lowest concentration (highest dilution) of the extract that produced no visible bacterial growth (no turbidity) when compared with the control tubes were regarded as MIC [24]. Insecticidal Activity of Methanolic Extract: Insecticidal activity of different concentrations of methanolic extract was tested on Second instar larvae of Aedes aegypti mosquito. Twenty larvae were placed in each of the beakers containing different concentrations of extracts (10, 20, 30, 40 and 50mg/ml). A control was kept without adding extract. The larvicidal effect of extracts was determined by counting the number of dead larvae after 24 hours. Dead larvae were identified when they failed to move after probing with a needle in siphon or cervical region. Each test was repeated thrice; the percentage of larval mortality was determined [25].

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African J. of Basic & Appl. Sci., 1 (5-6): 110-116, 2009 Table 1: Antioxidant activity of methanolic extract of A. pulchellus by DPPH free radical scavenging assay Radical scavenging activity (in %) -----------------------------------------------------------Concentration (mg/ml)

Methanolic extract

Ascorbic acid

0.125

68.49

88.69

0.250

76.37

92.52

0.500

83.16

95.12

1.000

88.68

97.55

Table 2: Antioxidant activity of methanolic extract of A. pulchellus by Fe+3 reducing assay Absorbance at 700nm ----------------------------------------------------------Concentration (mg/ml)

Methanolic extract

Tannic acid

0.125

0.159

0.264

0.250

0.298

0.331

0.500

0.388

0.469

1.000

0.514

0.668

Table 3: Antibacterial activity of methanolic extract of A. pulchellus Zone of inhibition in cm ------------------------------------------Test bacteria

Methanolic extract

Standard

MIC (µg/ml)

E. coli

12

2.6

550

P. aeruginosa

11

2.4

500

S. aureus

16

2.9

250

C. perfringens

18

3.2

125

Table 4: Insecticidal activity of methanolic extract of A. pulchellus Concentration (mg/ml)

Larval mortality (%)

0 (Control)

0.00

10

55.00

20

70.00

30

100.00

40

100.00

50

100.00

DISCUSSION Plants produce a diverse range of bioactive molecules, making them rich source of different types of medicines. Higher plants, as sources of medicinal compounds, have continued to play a dominant role in the maintenance of human health since ancient times. Over 50% of all modern clinical drugs are of natural plant origin and natural products play an important role in drug development programs in the pharmaceutical industry 113

[27]. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids and phenolic compounds [28]. Phytomedicines derived from plants have shown great promise in the treatment of various diseases including viral infections. Single and poly herbal preparations have been used throughout history for the treatment of various types of illness [29]. Plant derived natural products have received considerable attention in recent years due to their diverse pharmacological activities [30]. Free radicals are chemical species containing one or more unpaired electrons that makes them highly unstable and cause damage to other molecules by extracting electrons from them in order to attain stability [31]. Free radicals contribute to more than one hundred disorders in humans including atherosclerosis, arthritis, ischemia and reperfusion injury of many tissues, central nervous system injury, gastritis, cancer and AIDS [32,33]. In recent years much attention has been devoted to natural antioxidant and their association with health benefits [31]. There are several methods available to assess antioxidant activity of compounds. DPPH free radical scavenging assay is an easy, rapid and sensitive method for the antioxidant screening of plant extracts. In presence of an antioxidant, DPPH radical obtains one more electron and the absorbance decreases [34]. In this study, the scavenging activity of methanol extract was found to be dose dependent. Though the DPPH radical scavenging abilities of the extract was 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. In the Fe+3 reducing assay, the reducing power of crude methanol extract was found to increase with the dose. The reducing capacity of compound may serve as significant indicator of its potential antioxidant activity [35]. The antioxidant activities have been reported to be the concomitant development of reducing power [36]. The results of antibacterial activity of methanolic extract of A. pulchellus are consistent with previous reports regarding Gram-positive bacteria. The resistance of Gram-negative bacteria to plant extracts was not unexpected as, in general, this class of bacteria is more resistant than Gram-positive bacteria. Such resistance could be due to the permeability barrier provided by the cell wall or to the membrane accumulation mechanism [37].


It appears that overall the microorganisms were found to be sensitive to solvent extract. The reasons for this could be that the components from the plant active against microorganisms are most often obtained through solvent extraction. Antimicrobial activity of tannins [38,39], flavonoids [40,41], saponins [42,43], terpenoids [44], alkaloids [45,46] have been documented. The methanol extract of the A. pulchellus was found to possess most of these phytoconstituents. The antibacterial activity of solvent extract could be chiefly due to the presence of these phytoconstituents. Killing larvae of mosquitoes is a successful way of minimizing mosquito densities in breeding grounds before they reach adult stage. It largely depends on the use of synthetic chemical insecticides. But their repeated use has caused environmental problems and widespread development of resistance. Plants offer an alternative source of insect-control agents because they contain a range of bioactive chemicals, many of which are selective and have little or no harmful effect on non-target organisms and the environment. It is observed that the carbohydrates, saponins, phytosterols, phenols, flavonoids and tannins are having mosquito larvicidal activity [25]. Prenylated xanthones, tetracyclic phenols and saponins are reported to be effective in controlling mosquito A. aegypti, the vector of yellow fever [47]. The insecticidal activity of methanol extract in terms of larvicidal effect could be mainly due to the presence of phytoconstituents.

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CONCLUSION A marked antioxidant, antibacterial and insecticidal activity by methanolic extract of A. pulchellus has been observed in this study. Thus, herbal preparations involving A. pulchellus could be used to prevent damage caused by free radicals, infections caused by pathogenic bacteria and diseases such as dengue and others transmitted by mosquitoes. Further investigations are to be carried out to isolate active constituents from methanolic extract and determine the biological efficacy.

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12. ACKNOWLEDGEMENT Authors are thankful to Head of the dept. of Microbiology and 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.

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