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Journal of American Science 2010;6(10)

Studies on Anti-oxidant activity of Tinospora cordifolia (Miers.)

Leaves using in vitro models Ramya Premanath and N. Lakshmidevi*

Department of Microbiology, Manasagangotri, University of Mysore, Mysore 570 006, Karnataka, India [email protected]

Abstract: Plants produce a diverse range of bioactive molecules, making them a rich source of different types of medicines. A regular and widespread use of herbs throughout the world has increased serious concern over their quality, safety and efficacy. Thus, a proper scientific evidence or assessment has become the criteria for acceptance of herbal health claims. In the present study, we examined the anti-oxidant effects of leaves of Tinospora cordifolia. Dried and powered leaves of T. cordifolia were extracted with hexane, chloroform, methanol, ethanol and water. Total phenolic and flavonoid contents of different solvent extracts were determined. Of the different solvent extracts, ethanol extract had the highest phenol and flavonoid content of 5.1±0.25 mg/g and 0.52±0.02 mg/g respectively. Antioxidant assays were carried out by using different in vitro models such as total reducing power, total antioxidant activity, lipid peroxidation inhibitory activity, DPPH radical scavenging activity and superoxide radical scavenging activity. Ethanol extract showed the highest total antioxidant activity of 41.4±0.45 µM Fe(II)/g. The EC50 values of ethanol extract for lipid peroxidation inhibitory activity and DPPH radical scavenging activity was found to be 0.1 and 0.5 mg/ml respectively. The anti-oxidant activities of other solvent extracts were poor when compared to the ethanol extract. These results suggest that, the active antioxidant compounds are better extracted in ethanol and there is a direct correlation between the total polyphenols extracted and its anti-oxidant activity. The in vitro anti-oxidant activity of T. cordifolia justifies the ethno medical use of this plant. [Journal of American Science 2010;6(10):736-743]. (ISSN: 1545-1003). Key words: Medicinal plant; Tinospora cordifolia; solvent extracts; anti-oxidant activity

1. Introduction Plants have been a source of medicine in the past centuries and today scientists and the general public recognize their value as a source of new or complimentary medicinal products. Recently, wide array of research investigations highlight the potential health beneficial principles from phytal sources. Over the past twenty years, interest in medicinal plants has grown enormously from the use of herbal products as natural cosmetics and for self-medication by the general public to the scientific investigations of plants for their biological effects in human beings. Beyond this pharmaceutical approach to plants, there is a wide tendency to utilize herbal product to supplement the diet, mainly with the intention of improving the quality of life and preventing the diseases of elderly people (Maffei, 2003). The WHO estimates that up to 80% of people still rely mainly on traditional remedies such as herbs for their medicine (Tripathi and Tripathi, 2003). India has been identified as a major resourceful area in the traditional and alternative medicines globally. Multi-factorial health beneficial activity of these plant extracts has been attributed to multi-potent anti-oxidant, anti-microbial, anti-cancer, anti-ulcerative and anti-diabetic properties. Generally, anti-oxidants

have been identified as major health beneficial compounds reported from varieties of medicinal plants and are sources for alternative medicines (Daniel, 2005). Free radicals or reactive oxygen species (ROS) are formed in our body as a result of biological oxidation. The over production of free radicals such as hydroxyl radical, super oxide anion radical, hydrogen peroxide can cause damage to the body and contribute to oxidative stress (Diplock, 1994; Thomson, 1995). Oxidative damage of proteins, DNA and lipid is associated with chronic degenerative diseases including cancer, coronary artery disease, hypertension, diabetes etc (Lee et al., 2000) and compounds that can scavenge free radicals have great potential in ameliorating these disease processes (Kris-Etherton et al., 2002; Di Malteo and Esposito, 2003; Behera et al., 2006). Most of the reactive oxygen species are scavenged by endogenous defense systems such as catalase, superoxide dismutase and peroxidase-glutathione system (Rice-Evans and Bourdan, 1993). But these systems may not be completely efficient requiring them to depend on exogenous anti-oxidants from natural sources. Medicinal plants constitute one of the main sources of new pharmaceuticals and health care

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products. A whole range of plant derived dietary supplements, phytochemicals and pro-vitamins that assist in maintaining good health and combating disease are now being described as functional ingredients and neutraceuticals. The role of medicinal plants in disease prevention or control has been attributed to antioxidant properties of their constituents (Ivanova et al., 2005). The protective effect of plant products are due to the presence of several components such as enzymes, proteins, vitamins (Halliwell, 1996), carotenoids (Edge et al., 1997), flavonoids (Zhang and Wang, 2002) and other phenolic compounds (Argolo et al., 2004). Since synthetic anti-oxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have restricted use in foods, the search for natural anti-oxidants has greatly increased in the recent years. The researchers have focused on natural anti-oxidants and numerous crude extracts and pure natural compounds have been recognized to have beneficial effects against free radicals in biological systems as anti-oxidants (Reena et al., 2004; Hazra et al., 2008; Demiray et al., 2009). Tinospora cordifolia Miers. is a widely used shrub in folk and Ayurvedic systems of medicine. It is a large, glabrous, deciduous climbing shrub belonging to the family menispermaceae. It is distributed throughout tropical Indian subcontinent and China. It is reported to possess anti-spasmodic, anti-inflammatory, anti-allergic, anti-diabetic, anti-oxidant properties (Singh et al., 2003). The objective of the present study was to determine the anti-oxidant activity of T. cordifolia leaves in different solvent extracts using standard methods. The findings from this work may add to the overall value of the medicinal potential of the shrub, since most of the studies have focused on antioxidant activities of root and stem of T. cordifolia. 2. Materials and Methods 2.1. Plant material Fresh and healthy leaves of T. cordifolia were collected from local growers. The leaves were washed thoroughly in distilled water and the surface water was removed by air drying under shade. The leaves were subsequently dried in a hot air oven at 40 0C for 48h, powdered and used for extraction. 2.2. Preparation of aqueous extract Fifty grams of powdered leaves of T. cordifolia was macerated with 100 ml sterile distilled water in a blender for 10 min. The macerate was first filtered through double layered muslin cloth and centrifuged at 4000 g for 30 min. The supernatant was filtered through Whatman No.1 filter paper and heat sterilized at 120 0C for 30 min. The extract was

preserved aseptically in a brown bottle at 4 0C until further use. 2.3. Preparation of solvent extract Fifty grams of shade dried powered leaf material was extracted successively with chloroform, hexane, methanol and ethanol until the plant material became colorless. It was then filtered with sterile Whatman filter paper into a clean conical flask and the filtrate was transferred into the sample holder of the rotary flash evaporator. The extracts so obtained was weighed and preserved at 4 0C in airtight bottles until further use. 2.4. Total phenolic content Total soluble phenolic content was estimated by Folin-Ciocalteau reagent method (Malick & Singh, 1980) using gallic acid as a standard phenolic compound. One ml of stock solutions of different solvent extracts was prepared (1g/ml) from which different aliquots were pipetted out into test tubes. The volume was made up to 3 ml with distilled water to which freshly prepared Folin-Ciocalteau reagent was added. After 3 min, 2 ml of 20% sodium carbonate solution was added to each tube and mixed thoroughly. The tubes were placed in boiling water for one minute, cooled and the absorbance was measured at 650 nm in a spectrophotometer against a reagent blank. The concentrations of the total phenolic compounds in the extracts were obtained by extrapolating the absorbance of gallic acid on standard gallic acid graph. The experiment was repeated thrice and concentration of total phenols was expressed as mg /g of dry extract. 2.5. Total flavonoid content The total soluble flavonoid content was estimated by aluminium chloride colorimetric method for both aqueous and solvent extracts (Woisky & Salatino, 1998). 0.5ml of stock solution (1g/ml) of the extract, 1.5 ml methanol, 0.1ml potassium acetate (1M) was added to reaction test tubes and volume was made up to 5 ml with distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm. Total flavonoid content was calculated by extrapolating the absorbance of reaction mixture on standard curve of rutin. The experiment was repeated thrice and the total flavonoid content was expressed as equivalent to rutin in mg/ g of the extracts. 2.6. Antioxidant activity assays 2.6.1. Total reducing power The determination of reducing power was performed as described by Yen and Duh (1993). Various extracts (0.1 - 0.9 mg/ml) were mixed with


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phosphate buffer (500 µl, 20 mM, pH 6.6) and 1% potassium ferricyanide (500 µl), and incubated at 50 0C for 20 min; 500 µl of 10% trichloro acetic acid were added, and the mixture was centrifuged at 2500 rpm for 10 min. The supernatant was mixed with distilled water (1.5 ml) and 0.1% ferric chloride (300 µl) and the absorbance was read at 700 nm. The experiment was repeated thrice. Increase in the absorbance of the reactions mixture indicated increase in the reducing power. 2.6.2. Ferrous reducing antioxidant power assay (Total antioxidant activity assay) The method employed was a modification method of Benzie & Strain (1996) method. The stock solutions included 300 mM acetate buffer (pH 3.6), 10 mM 2, 4, 6-tripyridyl-s-tri-azine solution in 40 mM HCl, and 20 mM FeCl3.6H2O solution. The fresh working solution was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ and 2.5 ml FeCl3.6H2O. The temperature of the solution was raised to 37 0C before using. Plant extracts (150 µl) were allowed to react with 2850 µl of the FRAP solution for 30 min in the dark condition. Readings of colored product (ferrous tripyridyltriazine complex) were taken at 593 nm. The experiment was repeated thrice. Results were expressed in µM Fe (II)/g dry mass and compared with that of BHT. 2.6.3. Lipid peroxidation inhibitory activity The lipid peroxidation inhibitory activity of the leaf extracts was determined according to the method of Duh & Yen (1997). Egg lecithin (3 mg/ml phosphate buffer, pH 7.4) was sonicated in an ultrasonic sonicator for 10 min to ensure proper liposome formation. Test samples (100 µl) of different concentrations (0.1 - 0.9 mg/ml) were added to liposome mixture (1 ml); the control was without test sample. Lipid peroxidation was induced by adding ferric chloride (10 µl, 400 mM) and L-ascorbic acid (10 µl, 200 mM). After incubation for 1 h at 370 C the reaction was stopped by adding hydrochloric acid (2 ml, 0.25 N) containing trichloroacetic acid (150 mg/ml) and thiobarbutyric acid (3.75 mg/ml). The reaction mixture was subsequently boiled for 15 min, cooled, centrifuged at 1000 rpm for 15 min and the absorbance of the supernatant was measured at 532 nm and compared with that of BHA. Percentage radical scavenging was calculated using the following formula: % Inhibition = [(Acontrol – (Asamaple - Asampleblank / Acontrol] x 100 2.6.4. DPPH radical scavenging activity The free radical scavenging activity of the leaf extracts was assayed using a stable free radical, 1, 1-

diphenyl-2-picryl hydrazyl (DPPH). The DPPH scavenging assay employed in the present study was a modification of the procedure of Moon & Terao (1998). 0.1 ml of test sample at different concentration (0.1 - 0.9 mg/ml) was mixed with 0.9 ml of Tris-HCl buffer (pH 7.4); then 1 ml of DPPH (500 µM in ethanol) was added. The mixture was shaken vigorously and left to stand for 30 min. The absorbance of the resulting solution was measured at 517 nm in a spectrophotometer and compared with that of BHA. The experiment was repeated thrice. The percentage of DPPH scavenging was calculated using the following formula: % Scavenging = [(Acontrol – (Asamaple - Asampleblank / Acontrol] x 100 2.6.5. Superoxide radical scavenging activity The measurement of superoxide anion scavenging activity was based on the method by Fontana, Mosca, & Rosei (2001). Superoxide radical is generated in phenazine methosulfate-nicotinamide adenine dinucleotide (PMS-NADH) systems by oxidation of NADH and assayed by the reduction of nitroblue tetrazolium (NBT) to a purple formazan. The 1 ml reaction mixture contained phosphate buffer (20 mM, pH 7.4), NADH (73 µM), NBT (50 µM), PMS (15 µM) and various concentrations of sample solution. After incubation for 5 min at ambient temperature, the absorbance at 562 nm was measured against an appropriate blank to determine the quantity of formazan generated. The experiment was repeated thrice. The results were compared with that of quercetin. The % inhibition of superoxide anion generation was calculated using the following formula: % Scavenging = [(Acontrol – (Asamaple - Asampleblank / Acontrol] x 100 3. Results In this study, some of the biological activities of T. cordifolia leaves have been investigated, whereby; hexane, chloroform, methanol, ethanol and aqueous extracts were assayed for their total phenolic and flavonoid contents and anti-oxidant activities using different in vitro models. 3.1. Total phenolic and flavonoid content Results obtained in the present study revealed that the level pf polyphenols in the ethanol extract was 5.1±0.25 mg/g which was higher when compared to methanol, chloroform, hexane and aqueous extracts of T. cordifolia. Ethanol extract of the leaves had a flavonoid content of 0.52±0.02 mg/g. The flavonoid content of other extracts tested was lower than the ethanol extract. Aqueous extract had the least polyphenol and flavonoid content (Table 1).


Table 1: Polyphenol and Flavonoid content of Tinospora cordifolia leaves in different solvent extracts

Solvents Total phenols (mg/g) Flavonoid content (mg/g) Ethanol 5.1±0.25 0.52±0.02

Methanol 4.2±0.30 0.45±0.03 Chloroform 2.1±0.25 0.25±0.02

Hexane 1.5±0.35 0.19±0.04 Aqueous 1.1±0.05 0.12±0.02

3.2. Antioxidant activity assays 3.2.1. Total reducing power The reducing power of different solvent extracts using the potassium ferricyanide method is shown in Figure 1. The result indicates that the

reducing ability of the extracts increased with the concentration. Among all the extracts tested for their reducing abilities ethanol extract of T. cordifolia showed better reducing power as shown by the increasing optical density at 700 nm.

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Figure 1: Reducing power of Tinospora cordifolia leaf extract at different concentrations (mg/ml). 3.2.2. Total antioxidant activity The ability of the plant extracts to reduce ferric ions was determined by FRAP assay (Figure 2). An anti-oxidant capable of donating a single electron to the ferric-TPTZ (Fe (II)-TPTZ) complex would cause the reduction of the complex into the blue ferrous TPTZ (Fe (II)-TPTZ) complex which absorbs

strongly at 593 nm. The FRAP values for the extracts were lower than that of BHT (63±0.35 µm/g fw). Among the extracts tested, ethanol extract had a total anti-oxidant activity of 41.4±0.45 µm/g fw followed by methanol 33.9±0.49 µm/g fw. Aqueous extract had the least reducing ability of 4.8±0.30 µm/g fw.

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Figure 2: Total Antioxidant activity of Tinospora cordifolia solvent extracts (µM Fe (II)/g).


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3.2.3. Lipid peroxidation inhibitory activity The anti-oxidative action of T. cordifolia leaf extracts in the liposome model, induced by ferric chloride plus ascorbic acid and determined by thiobarbutyric acid method is shown in Figure 3. Ethanol extract had an EC50 value of 0.1 mg/ml which showed an inhibition of 57.5%. EC50 value could be achieved only with methanol extract at a

concentration of 0.7 mg/ml. As with other extracts, 50% inhibition could not be achieved even at 0.9 mg/ml. BHA showed very strong lipid peroxidation inhibitory activity with an EC50 value 12 µg/ml. Lipid peroxidation inhibitory activity of ethanol extract gradually decreased with an increase in concentration.

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inh

ibit

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Methanol

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Figure 3: Lipid peroxidation inhibitory activity of Tinospora cordifolia leaf extract at different concentrations (mg/ml). 3.2.4. DPPH radical scavenging activity The DPPH radical scavenging activity of T. cordifolia leaf extracts is shown in Figure 4. Among the extracts tested, ethanol extract had better

scavenging activity (EC50 value of 0.5 mg/ml) followed by methanol (EC50 value of 0.9 mg/ml). When compared to BHA which had an EC50 value of 5.3 µg/ml, the e EC50 value of ethanol was quite high.

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% D

PP

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Figure 4: DPPH radical scavenging activity of Tinospora cordifolia leaf extract at different concentrations (mg/ml). 3.2.5. Superoxide radical scavenging activity The superoxide radical generated from dissolved oxygen by PMS-NADH coupling can be measured by their ability to reduce NBT. The decrease in absorbance at 562 nm with the plant extracts and the reference compound quercetin indicates their abilities to quench superoxide radicals in the reaction mixture. As shown in Figure 5 the

quenching ability generally was low with all the solvent and aqueous extracts. As with ethanol extract, even at 0.7 mg/ml concentration the percentage radical scavenging was 40.1%. Percent radical scavenging abilities of other extracts were lower than ethanol extract. Quercetin was found potent with an EC50 value of 155 µg/ml.


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Figure 5: Superoxide radical scavenging activity of Tinospora cordifolia leaf extract at different concentrations (mg/ml). 4. Discussion 4.1. Total phenolic and flavonoid content Medicinal plants are an important source of antioxidants (Rice-Evans, 2004). Natural anti-oxidants increase the anti-oxidant capacity of the plasma and reduce the risk of certain diseases (Prior and Cao, 2000). Polyphenols are the major plant compounds with anti-oxidant activity. Typical phenolics that possess anti-oxidant activity are known to be mainly phenolic acids and flavonoids (Demiray et al., 2009). It is reported that the phenolics are responsible for the variation in the anti-oxidant activity of the plant (Luo et al., 2004). They exhibit anti-oxidant activity by inactivating lipid free radicals or preventing decomposition of hydro peroxides into free radicals (Pokorny 2001; Pitchaon et al., 2007). Flavonoids are phenolic acids which serve as an important source of anti-oxidants found in different medicinal plants and related phytomedicies (Pietta, 1998). The anti-oxidant activity of flavonoids is due to their ability to reduce free radical formation and to scavenge free radicals. 4.2. Antioxidant activity assays 4.2.1. Total reducing power Reducing power is associated with its anti-oxidant activity and may serve as a significant reflection of the anti-oxidant activity (Oktay et al., 2003). Compounds with reducing power indicate that they are electron donars and can reduce the oxidized intermediates of lipid peroxidation processes, so that they can act as primary and secondary anti-oxidants (Yen and Chen, 1995). 4.2.2. Total antioxidant activity FRAP assay is based on the ability of anti-oxidants to reduce Fe3+ to Fe2+ in the presence of 2, 4, 6-tri (2-pyridyl)-s-triazine (TPTZ) forming an intense blue Fe2+ -TPTZ complex with an absorption

maximum at 593 nm. This reaction is PH dependent (optimum PH 3.6). The absorbance decrease is proportional to the anti-oxidant content (Benzie and Strain, 1996). In the present study there was an increase in the anti-oxidant activity which was proportional to the polyphenol content. Ethanol extract exhibited high anti-oxidant activity due to its high polyphenol content. Similar results have been observed in Calpurnia aurea leaf and stem extracts, where in methanolic stem extract has shown high anti-oxidant activity than the leaf extract due to high polyphenol content (Adedapo et al., 2008). 4.2.3. Lipid peroxidation inhibitory activity To evaluate the lipid peroxidation inhibitory activity of the leaf extracts of T. cordifolia, a liposome model was used. Anti-oxidant effect of polyphenols (flavonoids) on lipid peroxidation is the result of scavenging of hydroxyl radicals at the stage of initiation and termination of peroxyl radicals has been reported by Hussain et al. (1987). Earlier study by Prasad et al. (2005) has shown the lipid peroxidation inhibitory activity of a flavonoid isolated from Ipomea aquatica leaf. From the present study, it was found that the percent inhibition of lipid peroxidation by ethanol leaf extract decreased after a certain concentration which may be due to the degradation or peroxidation of the source. 4.2.4. DPPH radical scavenging activity The stable radical DPPH has been used widely for the determination of primary anti-oxidant activity (Brand-Williams et al., 1995; Katalinic et al., 2004). The DPPH anti-oxidant assay is based on the ability of DPPH a stable free radical, to decolorize in the presence of anti-oxidants (Ara and Nur, 2009). The present study indicates that the IC50 value of ethanol leaf extract is quite high when compared to the standard which may be attributed to its poor proton


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donating ability (Voravuthikunchai et al., 2009). A study carried out by Hasan et al. (2009) has shown the DPPH radical scavenging activity of T. cordifolia aerial parts with an EC50 value of 0.02 mg/ml. The difference in the EC50 value can be attributed to the distribution of secondary metabolites that may fluctuate between different plant organs (Lissiewska et al., 2006). 4.2.5. Superoxide radical scavenging activity Although superoxide anion is a weak oxidant, it gives rise to generation of powerful and dangerous hydroxyl radicals as well as singlet oxygen, both of which contribute to oxidative stress (Meyer and Isaker, 1995). Among the extracts of T. cordifolia, ethanol extract showed mild scavenging activity and EC50 value could not reached even at 0.9 mg/ml concentration. The result supports the earlier study by Mathew and Kuttan (1997), which showed that the EC50 value for superoxide scavenging could be as high as 6 mg/ml. Acknowledgement The authors acknowledge the assistance of Dr. S. M. Aradhya (Senior Scientist, Fruit and Vegetable Technology, CFTRI, Mysore) for his help in carrying out the experiments. Corresponding author: Dr. N. LAKSHMIDEVI DOS in Microbiology, University of Mysore Mysore, Karnataka, India-570 006. Mail: [email protected] 5. References 1. Adedapo AA, Jimoh OF, Koduru S, Afolayan JA,

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8/30/2010

Ramya and Devi, IJPSR, 2011; Vol. 2(8): 2091-2099 ISSN: 0975-8232

Available online on www.ijpsr.com 2091

IJPSR (2011), Vol. 2, Issue 8 (Research Article)

Received on 22 April, 2011; received in revised form 13 July, 2011; accepted 28 July, 2011

ANTIBACTERIAL, ANTIFUNGAL AND ANTIOXIDANT ACTIVITIES OF ANDROGRAPHIS PANICULATA NEES. LEAVES

Ramya Premanath and N. Lakshmi Devi*

Department of Studies in Microbiology, Manasagangotri, University of Mysore, Mysore, Karnataka, India

ABSTRACT

Andrographis paniculata Nees. is an important medicinal plant in India which is used in traditional medicine. Although, its antibacterial property has been shown by some researchers, there are very few studies showing its antioxidant activity and there are no reports of its antifungal activity against dermatophytes. So, in this study we evaluate the antibacterial, antifungal and antioxidant activities of A. paniculata leaves in different solvent extracts. A. paniculata leaves were extracted with chloroform, hexane, methanol, ethanol and water separately and phenol and flavonoid contents were estimated in these extracts. The antibacterial and antifungal activity studies were carried out by using disc diffusion method and mycelial dry weight method respectively. Ferrous reducing antioxidant power assay, 1, 1-diphenyl-2-picryl hydrazyl (DPPH) scavenging activity, lipid peroxidation inhibitory activity and superoxide scavenging activity were used for in vitro antioxidant activity studies. A. panicualta ethanol leaf extract showed the highest phenol and flavonoid contents of 64.82 mg/g and 0.87 mg/g respectively. The highest antibacterial activity was recorded in the ethanol extract with Minimum Inhibitory Concentration (MIC) values of 0.75 mg/ml for Pseudomonas aeruginosa and 1.0 mg/ml for Staphylococcus aureus. MIC values of 1.75 mg/ml and 3.0 mg/ml were recorded for Epidermophyton floccosum and Trichophyton rubrum in ethanol extract. Antioxidant activity was more in ethanol extract, which showed Inhibitory Concentration (IC50) values of 0.5 mg/ml, 0.1 mg/ml and 0.9 mg/ml for DPPH scavenging activity, lipid peroxidation inhibitory activity and superoxide scavenging activity respectively. The result indicates that ethanolic leaf extract of A. paniculata shows potent antibacterial, antifungal and antioxidant activities.

INTRODUCTION: Plants have been an important source of medicine for thousands of years. Plants are considered not only as dietary supplement to living organism but also traditionally used for treating many health problems. The use of plants for the treatment of many diseases dated back to prehistory and people of all continents have this old tradition. Despite the remarkable progress in the preparation of synthetic drugs, over 25% of prescribed medicines in industrialized countries are derived directly from

plants 1. The use of medicinal plants in developing countries is increasing, which offer a new source of antibacterial, antifungal and antioxidants agents. The higher plants are used to treat a number of infectious diseases around the world. The World Health Organization (WHO) also considers phytotherapy in its health programs and suggested basic procedures for validation of drugs from plant origin in developing countries 2. The development of drug resistance as well as appearance of undesirable side effects of

Keywords:

Andrographis paniculata,

Solvent extracts,

Antibacterial activity,

Antifungal activity,

In vitro antioxidant activity

Correspondence to Author:

Dr. N. Lakshmidevi

Reader, Department of studies in Microbiology, Manasagangotri, University of Mysore, Mysore, Karnataka, India



certain drugs has led to the search of new antimicrobial agents in particular from medicinal plants. Literature indicates that medicinal plants are the backbone of traditional medicine. Indigenous plants are reservoirs of various metabolites and provide unlimited source of important chemicals that have diverse biological properties 3.

Almost all organisms are well protected against free radical damage by the presence of enzymes or compounds such as ascorbic acid, tocopherols and glutathione. When the mechanism of antioxidant protection becomes unbalanced by several internal and external factors, deterioration of physiological functions may occur requiring the system to depend on exogenous antioxidants from natural sources. Several synthetic antioxidants such as Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT) have restricted use in foods as they are suspected to be carcinogenic 4. Therefore, the importance of search for natural antioxidants has greatly increased in the recent years.

Andrographis paniculata Nees. belongs to the family Acanthaceae and is an important medicinal plant in India, China and Thailand. It is an annual, branched, erect herb whose aerial parts (stem and leaves) are used in traditional medicine. The plant is claimed to possess immunological, antibacterial, anti-inflammatory, antithrombotic, hepatoprotective, hypoglycaemic and hypotensive properties 5.

The importance of search and exploitation of natural antimicrobials and antioxidants of plant origin has greatly increased in recent years. Though, in traditional medicine A. paniculata has been claimed to possess antimicrobial and antioxidant properties, very few studies have shown the antibacterial and antioxidant activity and to our knowledge no study has been carried out to know its antifungal property against dermatophytes. In all the studies carried out till now on A. paniculata, the whole plant has been used. So, the aim of the present study was to investigate the antibacterial, antifungal and antioxidant activities of A. paniculata leaves in various solvent systems and to determine the solvent that best extracts the compounds responsible for these activities.

MATERIALS AND METHODS:

Plant Material: Fresh and healthy leaves of A. paniculata were obtained from local growers during 2009. The sample specimen was identified based on the taxonomical characteristics and deposited in the herbarium of department of Applied Botany, University of Mysore. The leaves were washed thoroughly in distilled water and the surface water was removed by air drying under shade. The leaves were subsequently dried in a hot air oven at 40oC for 48 h, powdered and used for extraction.

Test Microorganisms: The bacteria used were clinical isolates causing urinary tract infections (UTI) namely, Escherichia coli, Proteus mirabilis, Klebsiella spp., Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacter aerogens. The test fungi used were dermatophytes, namely, Trichophyton rubrum and Epidermophyton floccosum. Both the bacteria and the fungi were obtained from Department of Microbiology, JSS Medical College, Mysore. The bacterial and the fungal cultures were maintained on nutrient agar medium and saborauds dextrose agar (SDA) medium respectively.

Preparation of Aqueous Extract: Fifty grams of powdered leaves of A. paniculata were macerated with 100 ml sterile distilled water in a blender for 10 min. The macerate was first filtered through double layered muslin cloth and centrifuged at 4000 g for 30 min. The supernatant was filtered through Whatman No. 1 filter paper and heat sterilized at 120oC for 30 min. The extract was preserved aseptically in a brown bottle at 4oC until further use.

Preparation of Solvent Extract: Fifty grams each of the powdered material was extracted initially with 300 ml of chloroform, hexane, methanol and ethanol separately for 24 h at 23±2oC. The extract was filtered with sterile whatman filter paper into a clean conical flask. Second extraction was carried out with same amount of solvent for another 24 h at 23±2oC and filtered. The extracts were later pooled and transferred into the sample holder of the rotary flash evaporator for the evaporation of the solvents.

The evaporated extract so obtained was weighed and preserved at 4oC in airtight bottle until further use. 10



mg each of dried solvent extract was dissolved in 1 ml of respective solvent and used for the antibacterial assay.

Determination of Total Phenolic Content: Total soluble phenolic content was estimated by Folin-Ciocalteau method 6. The extracts were oxidized with Folin Ciocalteau reagent and were neutralized with sodium carbonate. The absorbance of the blue color was measured at 650 nm in a spectrophotometer against a reagent blank. The concentrations of the total phenolic compounds in the extracts were obtained by extrapolating the absorbance of gallic acid on standard gallic acid graph. The concentration of total phenols was expressed as mg/g of dry extract.

Determination of Total Flavonoid Content: The total soluble flavonoid content was estimated by Spectrophotometeric method 7. 0.5 ml of stock solution (1g/ml) of the extract, 1.5 ml methanol, 0.1 ml potassium acetate (1M) was added to reaction tubes and volume was made up to 5 ml with distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm. Total flavonoid content was calculated by extrapolating the absorbance of reaction mixture on standard curve of rutin. The total flavonoid content was expressed as equivalent to rutin in mg/g of the extracts.

Antibacterial Screening: The antibacterial activity was carried out by disc diffusion method. Bacterial cultures (adjusted to 1 x 106 CFU/ml using spectrophotometer) were used to lawn nutrient agar plates evenly using a sterile swab. The plates were dried for 15 min and sterile discs (5 mm in diameter, Whatman No.1) impregnated with 10 µl of the plant extracts were placed on the nutrient agar surface. 10 µl of the respective solvent served as the negative control. Streptomycin standard antibiotic disc served as the positive control (10 µg/disc). The plates were then incubated at 37 0C for 18 – 24 h. After overnight incubation the plates were examined for the zone of inhibition.

Determination of Minimum Inhibitory Concentration (MIC): Broth dilution method was used to find out the MIC values. The test organisms were grown in nutrient broth medium to a concentration of 1 x 106 CFU/ml.

0.5 ml of extract (0.25 - 2 mg/ml) was mixed with 4 ml of nutrient broth inoculated with 0.5 ml of bacterial suspension. The tubes containing 4.5 ml of broth and 0.5 ml of bacterial suspension served as bacterial control and 5 ml of un-inoculated broth served as blank. The tubes were incubated at 37oC for 18 h. Inhibition of bacterial growth was determined by measuring the absorbance at 600 nm in a colorimeter. The lowest concentration of the compound that inhibits the growth of the organism was determined as the MIC. The percentage of growth inhibition was calculated according to the formula:

Percent growth inhibition = [(Acontrol – Atest) / Acontrol ] x 100

Antifungal activity assay: The ethanol extract was selected for antifungal activity assay by mycelial dry weight method 8. The dermatophytes grown on SDA medium for a week were flooded with 0.85% saline. After settling of the larger particles, conidia were counted with a haemocytometer and diluted in saborauds dextrose broth to a final spore concentration of 1x106 spores/ml.

For anti-dermatophytic assay in broth, 5 ml of sterile saborauds dextrose broth medium taken in screw capped tubes were inoculated with 20 µl of fungal suspension and 1-5 mg/ml concentration of the extract. The tubes were incubated for a week at 30oC. The visible mycelia growth in the tubes expressed the degree of activity of the extract. Fungal mycelia from the above tubes were separated by passing through Whatmann No. 1 filter paper. The filter paper was allowed to dry at 60 0 C to reach a constant weight. Fungal growth inhibition was calculated by considering the control and sample mycelial dry weights. The percentage of growth inhibition was calculated according to the formula:

% growth inhibition = [(Acontrol – Atest ) / Acontrol ] x 100

Anti-oxidant Activity Assays:

Total Antioxidant Activity Assay: Total antioxidant activity was carried out by using the ferrous reducing antioxidant power (FRAP) assay 9. The FRAP solution contained 25 ml acetate buffer (300 mM, pH 3.6), 2.5 ml 2, 4, 6-tripyridyl-s-tri-azine (10 mM solution in 40 mM HCl) and 2.5 ml Ferric Chloride (20 mM). The temperature of the solution was raised to 37 0C before



use. 0.15 ml of plant extracts were allowed to react with 2.85 ml of the FRAP solution for 30 min in the dark condition. Readings of coloured product (ferrous tripyridyltriazine complex) were taken at 593 nm. Results were expressed in µM Fe (II)/g dry mass and compared with that of a synthetic antioxidant Butylated Hydroxytoluene (BHT).

DPPH radical scavenging activity: The free radical scavenging ability of the extracts was determined using DPPH assay 10. Briefly, 0.1 ml of test sample at different concentration (0.1 - 0.9 mg/ml) was mixed with 0.9 ml of Tris-HCl buffer (pH 7.4) and 1 ml of DPPH (500 µM in ethanol). The mixture was shaken vigorously and left to stand for 30 min. The absorbance of the resulting solution was measured at 517 nm in a spectrometer and compared with that of Butylated Hydroxyanisole (BHA). Radical scavenging potential was expressed as IC50 values, which represents the sample concentration at which 50% of the radicals are scavenged. The percentage of DPPH scavenging was calculated using the following formula:

Percent Scavenging = [(Acontrol – (Asample - Asample blank / Acontrol] x 100

Lipid Peroxidation Inhibitory Activity: The lipid peroxidation inhibitory activity was determined in a liposome model 11. In brief, egg lecithin (3 mg/ml phosphate buffer, pH 7.4) was sonicated in an ultrasonic sonicator for 10 min to ensure proper liposome formation. 0.1 ml of test samples of different concentrations (0.1- 0.9 mg/ml) was added to 1 ml of liposome mixture, the control was without test sample. Lipid peroxidation was induced by adding 10 µl of ferric chloride (400 mM) and 10 µl of L-ascorbic acid (200 mM).

After incubation for 1 h at 37oC the reaction was stopped by adding 2 ml of hydrochloric acid containing 15% trichloroacetic acid and 0.375% of thiobarbutyric acid. The reaction mixture was subsequently boiled for 15 min, cooled, centrifuged at 1000 rpm for 15 min and the absorbance of the supernatant was measured at 532 nm and compared with that of BHA. Inhibitory activity was expressed as IC50 value, which represents the sample concentration at which 50% lipid peroxidation inhibition takes place. Percentage radical scavenging was calculated using the following formula:

Percent Inhibition = [(Acontrol – (Asample - Asample blank / Acontrol] x 100

Superoxide radical scavenging activity: The superoxide scavenging ability was assessed according to the method of Nishikime et al. 12 with slight modifications. The reaction mixture contained Nitroblue tetrazolium (0.1 mM) and Nicotinamide adenine dinucleotide (0.1 mM) with or without sample to be assayed in a total volume of 1 ml of Tris-HCl buffer (0.02 M, PH 8.3). The reaction was started by adding Phenazine methosulphate (10 µM) to the mixture, and change in the absorbance was recorded at 560 nm every 30 sec for 2 min. The percent inhibition was calculated against a control without test sample. Radical scavenging potential was expressed as IC50 value, which represents the sample concentration at which 50% of the radicals are scavenged. The results were compared with that of quercetin. The percentage inhibition of superoxide anion generation was calculated using the following formula:

Percent Scavenging = [(Acontrol – (Asample - Asample blank / Acontrol] x 100

Statistical Analysis: The experimental results are expressed as mean ± standard deviation (SD) of triplicate measurements. The data was subjected to One Way Analysis of Variance (ANOVA) and the significance of differences between the sample means was calculated by Turkey’s post hoc test. Data was considered statistically significant at P value ≤ 0.001. Statistical analysis was performed using Graph Pad statistical software.

RESULTS AND DISCUSSION:

Total Phenol and Flavonoid Content: The result of total phenolic and flavonoid content of A. paniculata leaves in different solvent extracts is shown in Table 1. Ethanol extract had the highest phenol and flavonoid content of 64.82±1.35 mg/g and 0.87±0.06 mg/g respectively followed by methanol. The lowest phenol and flavonoid content was found in the chloroform extract. Polyphenols and flavonoids are the plant secondary metabolites and are very important by virtue of their antimicrobial 13 and antioxidant activity 14.

The result from the present study indicates that the phenolic compounds and flavonoids are better extracted with ethanol than with other solvents.



Phenolic acids and flavonoids are generally extracted using alcohols, water or a mixture of water and alcohols 15.

The result obtained from our study may vary from the earlier report which has shown lower phenolic content in A. paniculata plant 16. Significant differences between the plants are likely due to genotypic and environmental differences within species, choice of parts tested, time of taking samples and determination methods.

TABLE 1: POLYPHENOL AND FLAVONOID CONTENT OF ANDROGRAPHIS PANICULATA LEAVES IN DIFFERENT SOLVENT EXTRACTS

Solvents Total phenols

(mg/g) Flavonoid content

(mg/g)

Aqueous 17.53 ± 0.68a

0.13 ± 0.01ab

Ethanol 64.82 ± 1.35d 0.87 ± 0.06d

Methanol 47.84 ± 2.05c

0.49 ± 0.02c

Hexane 23.23 ± 2.27b 0.21 ± 0.03b

Chloroform 14.96 ± 1.19a 0.11 ± 0.02a

Values are means of three independent replicates. Mean values with different superscripts are significantly different from each other as indicated by Tukey’s HSD (α = 0.001)

Antibacterial activity of solvent extracts: The antibacterial activity of different solvent extracts

against the pathogenic bacteria showed varied levels of inhibition. As shown in Table 2, among the solvent extracts tested, ethanol extract had a broad spectrum of activity and showed the highest zone of inhibition against P. aeruginosa (15.0±1.74 mm) and S. aureus (13.0±2.64 mm). The least zone of inhibition for all the tested bacteria was observed with the chloroform extract. The results obtained in the present study indicate that the ethanol extract is more active against the pathogenic bacteria and has a broad spectrum activity.

This indicates the involvement of more than one active principle of biological significance 17. In the present study, we have shown the highest polyphenol and flavonoid content in ethanol leaf extract. As polyphenols and flavonoids are to known to exhibit antibacterial activity, the antibacterial activity of ethanol extract can be said to be due to the presence of these compounds 18. The variation in the antimicrobial activity of different solvents can be rationalized in terms of the polarity of the solvents used, polarity of the compounds being extracted by each solvent and, in addition to their extrinsic bioactivity and by their ability to dissolve or diffuse in the media used in the assay 19.

TABLE 2: ZONE OF INHIBITORY ACTIVITY (IN MILLIMETER) OF DIFFERENT SOLVENT EXTRACTS OF ANDROGRAPHIS PANICULATA LEAVES AGAINST UTI CAUSING BACTERIA

Extract E. coli E. aerogenes Klebsiella sp. P. vulgaris S. aureus P. aeruginosa

Aqueous

Ethanol

Methanol

Hexane

Chloroform

Streptomycin10µg/disc

2.0 ± 2.0a

11.0 ± 1.0bc

8.0 ± 2.64b

3.0 ± 1.0a

1.0 ± 0.0a

15.0 ± 2.0c

0.0 ± 0.0a

10.0 ± 1.73c

6.0 ± 1.73b

1.0 ± 1.0a

0.0 ± 0.0a

14.0 ± 1.73d

0.0 ± 0.0a

8.0 ± 3.0bc

5.0 ± 1.0b

0.0 ± 0.0a

0.0 ± 0.0a

11.0 ± 1.72c

1.0 ± 1.0a

9.0 ± 1.70bc

7.0 ± 2.0b

2.0 ± 1.0a

0.0 ± 0.0a

12.0 ± 1.73c

3.0 ± 1.73a

13.0 ± 2.64b

9.0 ± 1.0b

4.0 ± 1.73a

2.2 ± 0.26a

11.0 ± 2.0b

4.0 ± 1.72a

15.0 ± 1.74c

10.5 ± 1.80b

4.5 ± 1.32a

3.0 ± 1.0a

14.0 ± 1.70bc


Determination of MIC: As ethanol extract showed potent antibacterial activity against both gram positive and gram negative organisms, the determination of MIC values was carried out only with ethanol extract. MIC values for P. aeruginosa and S.aureus were found to be 0.75 mg/ml and 1.0 mg/ml respectively (Table 3). MIC values for other bacteria tested were found to exceed 2.0 mg/ml. The result from the present study indicated that, P. aeruginosa was more susceptible to the ethanol extract followed by S. aureus. This is in

contrary with the earlier reports, which have shown that, most antibacterial medicinal plants attack gram positive strains than gram negative strains because of their permeability differences 20, 21. The possible mechanism for their broad spectrum activity against both gram positive and gram negative bacteria may due to their ability to complex with cell wall 22.



TABLE 3. MINIMUM INHIBITORY CONCENTRATION (MIC) OF ANDROGRAPHIS PANICULATA LEAF ETHANOL EXTRACT USING BROTH DILUTION METHOD

Bacteria Control Concentration (mg/ml)

0.25 0.5 0.75 1.0 2.0

E. coli 0.27 ± 0.05c 0.27 ± 0.01c 0.17 ± 0.02b 0.14 ± 0.03ab 0.13 ± 0.00ab 0.11 ± 0.00a

E. aerogenes 0.30 ± 0.02d 0.29 ± 0.01d 0.24 ± 0.01c 0.19 ± 0.02bc 0.15 ± 0.01ab 0.14 ± 0.01a

Klebsiella sp. 0.25 ± 0.00c 0.24 ± 0.01bc 0.20 ± 0.02ab 0.20 ± 0.02ab 0.18 ± 0.02a 0.16 ± 0.01a

P. vulgaris 0.25 ± 0.01c 0.24 ± 0.00c 0.20 ± 0.00b 0.19 ± 0.01b 0.16 ± 0.00a 0.15 ± 0.01a

S. aureus 0.22 ± 0.04e 0.21 ± 0.01de 0.15 ± 0.00cd 0.09 ± 0.02bc 0.03 ± 0.01ab 0.02 ± 0.00a

P. aeruginosa 0.14 ± 0.03b 0.13 ± 0.02b 0.06 ± 0.01a 0.02 ± 0.01a 0.02 ± 0.00a 0.01 ± 0.01a

Values are absorbance at 600 nm. Values are means of three independent replicates. Mean values with different superscripts are significantly different from each other as indicated by Tukey’s HSD (α = 0.001)

Antifungal activity assay by mycelial dry weight method: Table 4 represents the MIC values of A. paniculata leaf ethanol extract against two dermatophytes, T. rubrum and E. floccosum. Among the two fungi, E. floccosum was found to be more susceptible to the ethanol leaf extract with an MIC value of 1.75 ± 0.05mg/ml (74.6% growth inhibition) than T. rubrum which had an MIC value of 3.0 ± 0.04 mg/ml (70.9% growth inhibition). Aerial parts of A. paniculata have been known to possess diterpenoids,

flavonoids and polyphenols 23. In the present study, we have shown that the ethanol leaf extract of A. paniculata has the highest polyphenol and flavonoid content. As these polyphenols and flavonoids are known to possess antimicrobial activity, the antifungal activity of the extract could be related to the effect of these compounds. Based on our review of current literature, there are no previous reports showing the antifungal activity of A. paniculata leaf ethanol extract on dermatophytes.

TABLE 4: MINIMUM INHIBITORY CONCENTRATION (MIC) OF ANDROGRAPHIS PANICULATA LEAF ETHANOL EXTRACT AGAINST DERMATOPHYTES USING MYCELIAL DRY METHOD METHOD

T. rubrum E. floccosum

MIC (mg/ml) % inhibition MIC (mg/ml) % inhibition

Ketaconazole 0.4 ± 0.01a 90.3 0.25 ± 0.02a 93.0

Extract 3.0 ± 0.04b 70.9 1.75 ± 0.05b 74.6


Antioxidant Activity Assays: Ferrous reducing antioxidant power assay: The ferrous ion reducing ability of A. paniculata leaf extract in different solvents is summarised in Table 5. The ethanol extract showed a total antioxidant activity of 59.01 ± 1.06 µM Fe (II)/g which was higher than the other solvent extracts tested but lower than that of BHT (63.25 ± 0.35 µM Fe (II)/g).

From the results obtained, it was found that ethanol had the highest antioxidant activity which may be due to the increased concentration of polyphenols than the other solvent extracts. There was a direct correlation between the polyphenol content and total antioxidant activity. The results are in agreement with an earlier study which has shown the better efficiency of polyphenols in reducing ferric ions 24.

TABLE 5: TOTAL ANTIOXIDANT ACTIVITY OF ANDROGRAPHIS PANICULATA SOLVENT EXTRACTS

Solvents Total antioxidant activity (µM Fe(II) / g)

Aqueous

Ethanol

Methanol

Hexane

Chloroform

BHT

15.71 ± 0.59a

59.01 ± 1.06d

43.70 ± 1.71c

21.83 ± 0.77b

16.10 ± 1.49a

63.25 ± 0.35e


DPPH Scavenging Activity: Figure 1 represents the DPPH radical scavenging activity of A. paniculata leaf extracts in different solvents. Ethanol extract showed the highest scavenging ability of 53.16% at 0.5 mg/ml (IC50 value). Methanol extract showed a scavenging ability of 53.86% at a higher concentration of 0.9 mg/ml (IC50 value).



The synthetic antioxidant BHA had an IC50 value of 0.0053 mg/ml. IC50 values could not be achieved with other solvent extracts tested in this assay. The underlying principle in this assay is the relatively stable DPPH radical is reduced in an alcoholic solution by the presence of hydrogen donating antioxidants. The ethanol extract recorded the highest phenolic and flavonoid content and also had the highest scavenging activity. There was a linear correlation between the antioxidant activity and total phenolic and flavonoid content of A. paniculata leaves. This result was consistent with the earlier study which reported such positive correlations between total phenolic content and antioxidant activity 25.

FIG. 1: DPPH SCAVENGING ACTIVITY OF A. PANICULATA LEAF EXTRACTS AT DIFFERENT CONCENTRATIONS (MG/ML)

Lipid Peroxidation Inhibitory Activity: The lipid peroxidation inhibitory activity of A. paniculata leaf extracts in the liposome model is shown in Figure 2. Ethanol extract had an IC50 value as low as 0.1 mg/ml and showed a lipid peroxidation inhibition of 58.73% which was followed by methanol with an IC50 value of 0.7 mg/ml. BHA showed a very strong lipid peroxidation inhibitory activity with an IC50 of 0.012 mg/ml. As with other extracts tested 50% inhibition could not be reached even at higher concentrations.

Antioxidant effect of flavonoids on lipid peroxidation is the result of scavenging of hydroxyl radicals at the stage of initiation and termination of peroxyl radicals. It is reported that lipid peroxidation can be inhibited by flavonoids, possibly through their activity as strong oxygen scavengers and singlet oxygen quenchers 26. The inhibitory activity of the ethanol extract in this

study was better than other solvent extracts tested even at lower concentration. This indicates that, the phenols and flavonoids in the ethanol extract were responsible for this inhibitory activity. There was a gradual decrease in the inhibition of lipid peroxidation even with the increase in the concentration of the extract which may be due to degradation or peroxidation of the source itself 27.

FIG. 2: LIPID PEROXIDATION INHIBITORY ACTIVITY OF A. PANICULATA LEAF EXTRACTS AT DIFFERENT CONCENTRATIONS (MG/ML)

Superoxide Scavenging Activity: The superoxide radical scavenging ability of A. paniculata leaf extracts in different solvents is depicted in Figure 3. There was a scavenging of 51.06% of superoxide anions with ethanol extract at a concentration of 0.9 mg/ml (IC50

value). Quercetin was found to be more potent having an IC50 value 0.155 mg/ml.

FIG. 3: SUPEROXIDE SCAVENGING ACTIVITY OF A. PANICULATA LEAF EXTRACTS AT DIFFERENT CONCENTRATIONS (MG/ML).



Even though other solvent extracts showed superoxide scavenging, IC50 values could not be achieved with the concentration used in this assay. Superoxide radicals are generated during the normal physiological process mainly in mitochondria. Although, superoxide anion is by itself a weak oxidant, it gives rise to the powerful and dangerous hydroxyl radicals as well as singlet oxygen both of which contribute to oxidative stress 28. Therefore, superoxide radical scavenging by antioxidants has physiological implications.

From the present study, it was shown that IC50 values could be reached only with ethanol extract at a higher concentration. But there was a positive correlation with the flavonoid content and superoxide scavenging activity of the extract which may be due to the presence of flavonoids 29.

CONCLUSION: In the present study, ethanolic extract of A. paniculata leaves gave the highest phenolic and flavonoid contents and showed potent antibacterial and antifungal activity against UTI causing bacteria and dermatophytes respectively. This explains the use of A. paniculata in folk medicine for treatment of infectious diseases. It was evident from the present study that, ethanolic extract of A. paniculata leaves showed strong lipid peroxidation inhibitory activity, DPPH scavenging activity and superoxide scavenging activity. On the basis of the results obtained, it can be concluded that ethanol is the best solvent for extracting antimicrobial and antioxidant bioactive compounds (phenols and flavonoids). Further research is needed to identify the nature and number of individual phenolic compounds and the existence of possible synergism if any, among these compounds.

ACKNOWLEDGEMENTS: The authors wish to thank Dr. Vijay Kumar, Head, Department of Microbiology, JSS Medical College, Mysore, India for the clinical bacterial and fungal isolates.

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