ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF LEAF EXTRACT ...

Int. J. Drug Res. Tech. 2012, Vol. 2 (3), 273-278

ISSN 2277 - 1506

International Journal of Drug Research and Technology Available online at http://www.ijdrt.com/ Original Research Paper ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF LEAF EXTRACT OF ADHATODA VASICA AGAINST THE BACTERIA ISOLATED FROM THE SPUTUM SAMPLES OF ASTHMATIC PATIENTS Inderjit Kaur1*, P.K. Chauhan2, Munish Jaryal1, Shubham Saxena3 and Kanishak1 1

Department of Microbiology, Himachal Institute of Life science Paonta Sahib, Himachal Pradesh, India 2 Department of Biochemistry, Himachal Institute of Life science Paonta Sahib, Himachal Pradesh, India 3 Department of Pharmacognosy Institute of Pharmacy and Emerging Sciences Baddi, Himachal Pradesh, India ABSTRACT In the present study antioxidant and antimicrobial activity of aqueous and methanolic extracts of Adhatoda vasica were evaluated against the bacteria isolated from the sputum samples of asthmatic patients. From antioxidant study the SOD activity was observed to maximum in methanolic extract as compared to aqueous extract of Adhatoda vasica. Among the two extracts of Adhatoda vasica, the highest activity of catalase was observed in aqueous extract and lowest in methanolic extract. Adhatoda vasica showed a broad spectrum of antibacterial activities against Gram-positive (Staphylococcus aureus and Streptococcus pneumoniae) bacterial species in comparison to the Gram-negative (E.coli and Klebsiella pneumoniae) bacterial species. On the basis of the results obtained in the present study, we concluded that the aqueous and methanolic extract of Adhatoda vasica has significant amounts of antioxidant and antimicrobial agents.

Keywords: Adhatoda vasica, Staphylococcus aureus, Antioxidant and antimicrobial activity, Catalase. INTRODUCTION Asthma is one of the most common chronic diseases in the world. It is estimated that around 300 million people in the world currently have asthma. Considerably higher estimates can be obtained with less conservative criteria for the diagnosis of clinical asthma. The international patterns of asthma prevalence are not explained by the current knowledge of the causation of asthma. Research into the causation of asthma, and the efficacy of primary and secondary intervention strategies, represent key priority areas in the field of asthma research. Asthma has become more common in both children and

adults around the world in recent decades. The increase in the prevalence of asthma has been associated with an increase in atopic sensitisation, and is paralleled by similar increases in other allergic disorders such as eczema and rhinitis. The rate of asthma increases as communities adopt western lifestyles and become urbanized. With the projected increase in the proportion of the world's population that is urban from 45% to 59% in 2025, there is likely to be a marked increase in the number of asthmatics worldwide over the next two decades. It is estimated that there may be an additional 100 million persons with asthma by 2025. In

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many areas of the world persons with asthma do not have access to basic asthma medications or medical care. Increasing the economic wealth and improving the distribution of resources between and within countries represent important priorities to enable better health care to be provided (Matthew Masoli, Denise Fabian, Shaun Holt, Richard Beasley, 2002).

MATERIAL AND METHOD

Adhatoda vasica is a well-known plant drug in Ayurvedic and Unani medicine (Manjunath, 1948). The medicinal properties of Adhatoda vasica, called Vasa or Vasaka in Sanskrit. The plant has been used in the indigenous system of medicine in India for over 2000 years (Atal, 1980). Adhatoda leaves have been used extensively in Ayurvedic Medicine primarily for respiratory disorders including cough, cold, asthma, bronchitis etc. Infectious diseases remain the leading cause of death worldwide and infections due to antibiotic resistant microorganisms have become more widespread in recent years.

The plant material taken for the study was stored under refrigerated condition till use. The samples were prepared by grinding one gram of sample in 10 ml of aqueous and methanol separately, in a pre-chilled mortar and pestle and the extract were centrifuged at 10,000 rpm at 4o C for 10 minutes. The supernatant thus obtained were used within four hours for various enzymatic and non-enzymatic assays.

In addition to this problem, antibiotics are sometimes associated with adverse effects on the host, including hypersensitivity, immune suppression and allergic reactions (Ahmad et al, 1998). This situation forced scientists to search for new antimicrobial substances. Given the alarming incidence of antibiotic resistance in bacteria of medical importance (Monroe and Polk, 2000), there is a constant need for new and effective therapeutic agents (Bhavnani and Ballow, 2000). Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases from medicinal plants (Cordell, 2000). Several screening studies have been carried out in different parts of the world. There are several reports on the antimicrobial, antioxidant activity of different herbal extracts in different regions of the world (De Boer et al, 2005). In the present study Antioxidant and Antimicrobial activity of aqueous and methanolic extracts of Adhatoda vasica were evaluated against the bacteria isolated from the sputum samples of Asthmatic patients.

Collection of plant material The plant material used is the dried leaves of Adhatoda vasica which were collected from forest region of Paonta Sahib and were identified by Botanical Survey of India, Dehradun. Extraction of plant material

Phytochemical analysis The extracts were used for preliminary screening of phytochemicals such as alkaloids (Wagner and Dragendorff’s tests), flavonoids (Shinda and Lead acetate tests), Phenols (ellagic acid and Fecl3 tests), tannins (gelatine tests), saponins (foam tests), sterols (Liberman‐Burchard and Salkowski tests) carbohydrates (Molishs test, Benedicts) by Dey and Harborne method (Helen B. Florido and Fe F, et al, 1999) Antioxidant activity of Adhatoda vasica leaf extract Assay of Superoxide Dismutase (SOD) activity The assay of superoxide dismutase was done according to the procedure of (Das K et al., 2000). Assay of catalase activity Catalase activity was assayed by the method of (Sinha A. K, 1972). Assay of Peroxidase activity The assay was carried out by the method of (Addy and Goodman, 1972). Assay Procedure for Ascorbate Oxidase Based on that of (Vines and Oberbacher, 1965) in which the decrease of absorbance due to


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oxidation of Ascorbate by AO (Ascorbate Oxidase) is measured at 265nm and 25°C. Quantification of vitamins The determination of ascorbic acid was carried out by the procedure given by (Sadasivam and Manickam, 1997). Collection of Bacterial strains

The total four bacterial strains were isolated from the sputum samples of asthmatic patients from Paonta sahib region i.e., Staphylococcus aureus, Staphylococcus pneumoniae, E. coli and Klebsiella pneumonia.

RESULTS Antioxidant analysis

Table 1.1: Enzymatic Antioxidant analysis in both the extracts of Adhatoda vasica Samples

Superoxide dismutase

Catalase

Peroxidase IU/L

Ascorbate oxidase µ mole/ml

Unit/mg protein

µ/moles of H2O2 decomposed/min/g protein

Aqueous extract of Adhatoda vasica

1.40

112.40

1.929X 106

0.021

Methanolic extract of Adhatoda vasica

2.10

110.03

2.787X 106

0.123

1 unit = Inhibition of 50% nitrite formation

1 unit = µ/moles of H2O2 decomposed/min/g protein

1 unit = µ moles pyrogallol oxidized/min

1 unit = 0.01 O.D change/min

The concentration of different non-enzymatic antioxidants in Adhatoda vasica extracts was also assessed and the results are represented in Table 1.2

Table 1.2: Non-Enzymatic Antioxidant Activity (Ascorbic acid) Samples

Vitamin C (mg/g)

Aqueous extract

1.455

Methanolic extract

2.232

Vitamin C content was low in aqueous extract (1.455mg/ g tissue), whereas in methanolic extract it is (2.232mg/ g tissue). Ascorbate has been found in the chloroplast, cytosol, vacuole and extracellular compartments of the plant cells and shown to function as a reductant for many free radicals (Foyer C, 1993). Flavonoids and tocopherol levels have been documented extensively in plant tissue (Hess J.L, 1993) and their concentration shown to vary among plant tissues from 200 mg/g fresh weight to 5 mg/g in oil palm leaflets. The antioxidant properties of tocopherol are the result of its ability to quench

both singlet oxygen and peroxides (Freyer M J, 1992).

Antimicrobial Assay The in-vitro antibacterial activity of aqueous and methanolic extract of Adhatoda vasica against two of pathogenic bacteria isolated from the Sputum sample of asthmatic patients were examined by Agar well diffusion method and their potency was assessed by the presence or absence of inhibition zones and zone diameters against Gram +ve and Gram -ve bacterial species (Table-4.3).The working concentration of the extracts was 100mg/ml each.


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Table 2.1: Zone of inhibition of Aqueous and Methanolic extracts of Adhatoda vasica against Gram positive & negative bacterial species. Bacterial species

Aqueous extract (mg/ml)

Methanolic extract (mg/ml)

Staphylococcus aureus

17mm

19mm

Staphylococcus pneumonia

15mm

18mm

E.coli

15mm

17mm

Kiebsiella pneumonia

12mm

14mm

Gram positive

Gram negative

From the present study it has been found that both the extracts of Adhatoda vasica showed a broad spectrum of antibacterial activities against Grampositive (Staphylococcus aureus and Staphylococcus pneumonia) bacterial species in comparison to the Gram-negative (E. coli and Klebsiella pneumonia) bacterial species.

DISCUSSION The antibacterial activity of methanolic extract of Adhatoda vasica against all tested microorganisms was greater than the antibacterial activity of aqueous extract of Adhatoda vasica. Adhatoda’s antibacterial properties have been clinically evaluated by Brantner AH and Chakraborty A, 1998. A leaf extract was investigated for antibacterial activity using agar well diffusion method. The present study was conducted to investigate the in vitro antibacterial activity of Adhatoda vasica. Generally, plants extracts are usually more active against gram positive bacteria than gram negative bacteria. In-vitro screening showed a strong activity of Adhatoda’s alkaloids against the bacteria Staphylococcus aureus. Significant antibacterial activity against the Gram-positive and other Gram-negative bacterial strains Streptococcus faecalis, Staphylococcus aureus, Staph epidermidis, Bacillus cereus and E. coli were also noted by Patel VK and VenkatakrishnaBhatt H, 1984. In another study, Adhatoda was

tested for its capacity to inhibit the growth of bacteria in untreated water. At pH 7, growth of the bacterial population was inhibited by 82%. At pH 6.5, several coli form bacterial strains were also inhibited. These findings suggest a possible application of Adhatoda in the improvement of drinking water quality (Kumar S and Gopal K, 1999). But over all methanol extract of Adhatoda vasica showed maximum photochemical, antioxidant enzymes and antimicrobial assay as compared to aqueous extract of Adhatoda vasica . When we compare the work done by Shiv Shanker Gautam, Navneet, Sanjay Kumar, Prabhat 2012, antibacterial potential of aqueous and methanolic extracts of Nepeta ciliaris against S. aureus, S. pneumonia showed antibacterial activity 13mm,13mm and 12mm,13mm respectively while our studied aqueous and methanolic leaf extract of Adhatoda vasica showed 17mm,15mm and 19mm,18mm respectively. On the other hand work done by Aibinu et al. 2007, aqueous and Methanol extract of Bryophyllum pinnatum leaves showed antibacterial activity against E.coli and Klebsiella was 0mm,0mm and 16mm,12mm respectively. While our observed aqueous and methanolic leaf extract of Adhatoda vasica showed antibacterial activity against E.coli and Klebsiella is 15mm, 12mm and 17mm, 14mm respectively. Thus our studied plant is more effective and can


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be used as drug formulation with other medicinal plant or individually in the treatment of asthma.

CONCLUSION The number of disability-adjusted life years (DALYs) lost due to asthma worldwide has been estimated to be currently about 15 million per year. Worldwide, asthma accounts for around 1% of all DALYs lost, which reflects the high prevalence and severity of asthma. The number of DALYs lost due to asthma is similar to that for diabetes, cirrhosis of the liver, or schizophrenia. The burden of asthma in many countries is of sufficient magnitude to warrant its recognition as a priority disorder in government health strategies. Particular resources need to be provided to improve the care of disadvantaged groups with high morbidity, including certain racial groups and those who are poorly educated, live in large cities, or are poor. Resources also need to be provided to address preventable

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