RESEARCH ARTICLE

ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL ESTIMATION OF MICROPROPAGATED Andrographis paniculata (Burm.f) NEES

Ankita Kataky* and Handique, PJ.

Department of Biotechnology, Gauhati University, Guwahati-781 014, Assam, India

Received 8th August, 2010; Received in revised form; 24th August, 2010; Accepted 30thAugust, 2010; Published online 4th September, 2010

Antimicrobial activity of eight-months old micropropagated plantlets of Andrographis paniculata was evaluated with various organic and aqueous extract against gram negative (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa), gram positive (Staphylococcus aureus and Bacillus subtilis) bacteria and the fungal pathogen (Candida albicans). Agar well diffusion method was used to assess the antimicrobial activity of Andrographis paniculata. Chloroform extract showed strong inhibitory activity with all the microbes tested. Out of the five microbial test organisms Staphylococcus aureus was the most susceptible. The minimal inhibitory concentration (MIC) of the chloroform extract ranged from 15.625µg/ml to 31.5µg/ml. Phytochemical test reveals the presence of carbohydrates, proteins, flavonoids, phenolics, saponin and alkaloids in the dried powder.

Key words: Antimicrobial activity, Andrographis paniculata, bacteria, fungi, MIC, phytochemical test.

INTRODUCTION

Antimicrobials of plant origin have enormous therapeutic potential. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials. The beneficial medicinal effects of plant materials typically result from the combinations of secondary metabolites such as alkaloids, steroids, tannis, and phenol compounds, flavonoids, resins fatty acids gums which are capable of producing definite physiological action on body (Joshi et al., 2009). Nowadays multiple drug resistance has developed due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious disease. In addition to this problem, antibiotics are sometimes associated with adverse effects on the host including hypersensitivity, immune-suppression and allergic reactions. This situation forced scientists to search for new antimicrobial substances. Giving the alarming incidence of antibiotic resistance in bacteria of medical importance, there is a constant need for new and effective therapeutic agents. Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases from medicinal plants (Agarwal et al., 1996).

A. paniculata is in demand in terms of its medicinal properties. It has been used for centuries in Asia to treat gastro-intestinal tract and upper respiratory infections, fever, herpes, sore throat, and a variety of other chronic and infectious diseases (Mishra et al., 2007). *Corresponding Author: ankita_lon03@rediffmail.com

Mostly the leaves and roots have been traditionally used over the centuries for medicinal purposes in Asia and Europe as a folklore remedy for a wide spectrum of ailments or as an herbal supplement for health promotion. The Indian Pharmacopoeia narrates that it is a predominant constituent of at least 26 Ayurvedic formulations, Zhang (1978). In Traditional Chinese Medicine, it is an important “cold property” herb used to get rid the body heat, as in fevers, and to dispel toxins of the body, Deng (1997). In Scandinavian countries, it is commonly used to prevent and treat common cold (Caceres et al., 1997). In Thailand, this plant was selected by the Ministry of Public Health as one of the medicinal plants to be included in “The National List of Essential Drugs A.D. 1999” (List of Herbal Medicinal Products) (Pholphana et al., 2004; Kanokwan and Nobuo, 2008).

So far antimicrobial activities of Andrographis paniculata are reported with aqueous, methanol, ethanol, hexane and chloroform extracts (Prajjal et al., 2003; Xu et al., 2006; Mishra et al., 2009; Bobbarala et al., 2009). However, the antimicrobial activities of ethyl acetate, acetone, DMSO and petroleum ether with in vitro raised 8 months old plants were not made yet. Thus, an attempt was made to evaluate the antimicrobial potential of the micropopagated plants with common human pathogenic microbes.

MATERIALS AND METHOD

Plant extract preparation

The field established micropropagated plants of A. paniculata were collected from the green house of

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Asian Journal of Science and Technology Vol. 5, pp.091-094, September, 2010

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092 Asian Journal of Science and Technology, Vol. 5, pp.091-094, September, 2010 Department of Biotechnology, Gauhati University, Assam, India. 10g of dried plant material (leaf, stem and roots) was extracted with various organic (chloroform, acetone, ethyl acetate, DMSO, petroleum ether) and aqueous solvents in a soxhlet extractor until the solvent was colorless. The extract was collected and filtered through eight layers of muslin cloth. The filtrate was evaporated to dryness in vacuo and weighed. Microbial strains tested

Five microbial strains were used from the Microbial Type Culture Collection (MTCC) Chandigarh, India. They were gram negative Klebsiella pneumoniae (MTCC 432), Escherichia coli (MTCC 739), Pseudomonas aeruginosa (MTCC 443), gram positive Staphylococcus aureus (MTCC 96) and Bacillus subtilis (MTCC 441) bacteria and the fungal pathogen Candida albicans (MTCC 227).

Antimicrobial assay

Antimicrobial activity was determined by the well diffusion method (Shanab et al., 2004) with minor modifications. Petri plates containing 25 ml of Nutrient agar/Potato dextrose medium were seeded with a 24 hours culture of the microbial strains. Wells (6 mm diameter) were cut into the agar and 100µl of the plant extracts were tested in a concentration of 50µg/ml. The inoculum size was adjusted so as to deliver a final inoculum of approximately 106 colony-forming units (CFU)/ml. Incubation was performed at 37ºC for 24 hours for bacterial strains and 72 hours at 28ºC - 30ºC for the fungal pathogen, C. albicans. Microbial growth was determined by measuring the diameter of zone of inhibition in millimeters (mm). For each bacterial and fungal strains control were maintained where pure solvents were used. Broad spectrum antibiotics viz. ampicillin, tetracycline and fluconazole at 1mg/ml concentrations were used as standards.

Minimal inhibitory concentration (MIC)

Minimal inhibitory concentration (MIC) was done by two fold serial dilution. 2000µg of the plant extract was dissolved in 1ml of the solvent which gives maximum zone of inhibition in the antimicrobial sensitivity test. The test tubes were taken and labeled as per concentrations taken. 2ml nutrient broth was added to all the test tubes. Serial dilution of the plant extract was done by transferring 2ml from each test tube to obtain two fold dilutions (1000µg/ml, 500µg/ml, 250µg/ml, 125µg/ml, 62.5µg/ml, 31.25µg/ml, 15.625µg/ml, 7.81µg/ml, 3.90µg/ml and 1.95µg/ml). 50µl of the test organism(s) were added to all the test tubes except the negative control. The tubes were then incubated at 37°C for 24 hours for bacterial cultures and 28°C for yeast for 2-3 days. MIC was determined by choosing the lowest concentration in which no growth occurs.

Phytochemical estimation

The A. paniculata extracts were subjected to various biochemical tests to determine the total carbohydrate (Sadasivam and Manickam, 1991a), total protein (Sadasivam and Manickam, 1991b) and total phenolics (Sadasivam and Manickam, 1991c), total flavonoid (Pourmorad et al., 2006), total saponin (Obadoni and Ochuko, 2001; Edeoga et al., 2005) and total

alkaloid, Harborne (1973) present in the crude extract. All the data expressed as mean ± standard deviation (SD), where n = 5.

RESULT AND DISCUSSION

Antimicrobial assay

The antimicrobial activities of the various extracts of Andrographis paniculata (leaves, stems and roots) exhibited different degrees of antimicrobial activity against the test organisms. The traditional healers use primarily water as a solvent. The aqueous extract in the present study showed inhibitory effect against all the gram positive, gram negative bacteria (except P. aeruginosa) and the fungal pathogen C. albicans. The antimicrobial activities of the plant extracted in different solvents varied greatly because there are many factors influence the active compounds present in the plant. Here the polarity of the extracting solvents are different and greatly influenced the antimicrobial properties. This may be due to the better solubility of the active compounds in organic solvents (De Boer et al., 2005; Parekh et al 2005). In almost all the test, the crude chloroform extract showed better inhibition against all the tested microbial strains, indicating active ingredients in plant material could be extracted into chloroform. However, highest antimicrobial activity was observed against B. subtilis and C. albicans. Ethyl acetate and acetone extracts showed inhibitory effects against all the gram positive bacteria but no inhibitory effect was observed with gram negative bacteria and the fungal pathogen C. albicans. Whereas, the DMSO extract showed inhibitory effect against the gram positive bacteria S. aureus. Petroleum ether extract does not have any inhibitory effects on the microorganisms tested. However, no inhibition was observed in control, which proves that solvents could not act as antimicrobial agents (Table 1). Amongst the gram positive and gram negative bacteria, gram positive bacterial strains were more susceptible to the extracts as compared to the gram negative bacteria. Generally gram negative bacteria are more resistant than gram positive bacteria (Rabe and van Staden, 1997; Kelmanson et al., 2000). This could be due to several possible reasons; one is the presence of the double membrane surrounding each bacterial cells. Their outer membrane excludes certain drugs and antibiotics from penetrating the cell, partially accounting for why gram negative bacteria are generally more resistant than gram positive bacteria (Yao and Moellering, 1995; Tortora and Funke, 2001; Lotfipour et al., 2008). Secondly the basis of their differences in susceptibility might be due to the cell wall composition of gram positive and gram negative bacteria (Grosvenor et al., 1995). The minimal inhibitory concentration (MIC) values with respect to chloroform extract for Klebsiella pneumoniae, Escherichia coli, Psuedomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and Candida albicans were found to be 15.6255µg/ml to 31.5µg/ml, 15.6255µg/ml to 31.55µg/ml, 15.6255µg/ml, 15.6255µg/ml, 15.6255µg/ml and 15.6255µg/ml respectively (Table 2).

Phytochemical estimation

The phytochemical estimation was done for analyzing primary and secondary metabolites. The A. paniculata

Ankita Kataky and Handique., Antimicrobial activity and phytochemical estimation of micropropagated Andrographis paniculata (Burm.f) nees 088

extracts were subjected to various biochemical tests to determine the active constituents present in the crude extract and it is depicted in (Table 3). The biochemical estimation results revealed the presence of carbohydrates, proteins, phenolics, flavonoids, saponins and alkaloids. Flavonoids protect plants from attacks by microbes and insects. Flavonoids have been referred to as “nature’s biological response modifiers” because of strong experimental evidence of their inherent ability to modify the body’s reaction to allergens, viruses and carcinogens. They show anti-allergic, anti-inflammatory (Yamamoto and Gaynor 2008), antimicrobial (Cushnie and Lamb, 2005), anti-neoplastic, anti-viral, anti-thrombic (Ayoola et al., 2008) and anti-cancer activity. Phenols, the aromatic compounds with hydroxyl groups are widespread in plant kingdom. They occur in all parts of the plant. Phenols are

said to offer resistance to diseases and pest in plants (Sadasivam and Manickam c, 1991). Saponin protects the plant against microbes and fungi. Alkaloids usually have marked the physiological action on human or animals (Adedapo et al., 2009). Whereas, carbohydrates and proteins determine the nutritive value of A. paniculata. Therefore, it was reasonable to determine the total primary and secondary metabolite content in the plant extract. In the present investigation, the screening of micropropagated plantlets of A. paniculata with chloroform, ethyl acetate, DMSO and acetone extracts gives better response than the methanol, ethanol or hexane extracts in the preliminary studies. The phytochemical estimation of Andrographis paniculata revealed that the antibacterial activity is due to the presence of the secondary metabolites. And carbohydrates and proteins

Table 1. Antimicrobial activity of A. paniculata and the standard antibiotics.

Plant extract K. pneumoniae E.coli P.aeruginosa S.aureus B.subtilis C.albicans Aqueous leaf Aqueous stem Aqueous roots Chloroform leaf Chloroform stem Chloroform roots Ethyl acetate leaf Ethyl acetate stem Ethyl acetate roots Acetone leaf Acetone stem Acetone roots DMSO leaf DMSO stem DMSO roots Pet ether leaf Pet ether stem Pet ether roots

12±0.344 12±0.216 14±0.297 12±0.283 16±0.295 10±0.329

7±0.428 8±0.179

10±0.173 14±0.241 15±0.313 10±0.134

8±0.444 12±0.215 10±0.163

12±0.447 12±0.568 10±0.328 15±0.335 18±0.001 15±0.006 14±0.357 8±0.303 7±0.356

13±0.416 16±0.374 15±0.055 15±0.141 16±0.332 14±0.187

12±0.084 13±0.373 12±0.363 22±0.071 24±0.219 18±0.055 15±0.152 8±0.327

10±0.626 10±0.283 13±0.365 12±0.251

8±0.227 12±0.336 12±0.311 34±0.297 37±0.503 24±0.305

Ampicillin 17±0.426 Tetracyclin 14±0.218 16±0.315 15±0.167 17±0.376 17±0.283 Fluconazole 21±0.403

Data reported as mean ± SD, where n = 5

Table 2. Minimal inhibitory concentrations of the chloroform extract against the 6 test organisms

Minimal Inhibitory Concentration (µg/ml) Tested Organisms Chloroform Leaf Chloroform Stem Chloroform Roots

K. pneumoniae E. coli P. aeruginosa S. aureus B. subtilis C. albicans

31.5 15.625 31.5

15.625 15.625 15.625

15.625 15.625

31.5 15.625 15.625 15.625

31.5 31.5 31.5

15.625 15.625 15.625

Table 3: Phytochemical constituents of A. paniculata

Plant material

Phytochemicals (mg/g) Leaf Stem Leaf/stem Roots Total carbohydrates Total proteins Total phenolics Total flavonoids Total saponin Total alkaloids

58.00±0.002 0.56 ±0.006 0.50 ±0.002 4.35 ±0.007

274.60±0.015 598.80±0.002

57.00 ±0.007 0.37 ±0.003 0.70 ±0.012 3.68 ±0.006

179.20±0.133 299.00±0.017

60.00 ±0.002 0.78 ±0.005 2.50 ±0.002 6.82 ±0.005

200.60±0.007 622.60±0.002

54.00 ±0.004 0.25 ±0.005 3.20 ±0.004 3.41 ±0.001

175.45±0.004 268.40±0.003

Data reported are the mean of 5 replications ± SD, where n = 5

094 Asian Journal of Science and Technology, Vol. 5, pp.091-094, September, 2010 give the nutritive value of A. paniculata. These results indicated the possibility of using Andrographis paniculata for medicinal uses and food preservation.

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