PHARMA SCIENCE MONITOR

PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES ANTIBACTERIAL ACTIVITY OF PHYLLANTHUS MADERASPATENSIS LEAVES EXTRACT Karthikeyan. M, Jeethu Anu Mathews and A. Annamalai* Department of Biotechnology, School of Biotechnology and Health Sciences, Karunya University, Coimbatore-641114, Tamil Nadu, India. ABSTRACT Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources; many of these isolations were based on the uses of the agents in traditional medicine. This plant-based, traditional medicine system continues to play an essential role in health care, with about 80% of the world s inhabitants relying mainly on traditional medicines for their primary health care. According to World Health Organization, medicinal plants would be the best source to obtain a variety of drugs. Therefore, such plants should be investigated to better understand their properties, safety and efficacy. Phyllanthus maderaspatensis commonly known as a Madras Leaf Flower have promising therapeutic value with its various phytochemical constituents such as tannins, alkaloids, steroids, flavonoids, terpenoids, fatty acids, phenols, minerals, carbohydrates and vitamins. The present study was done to determine the antibacterial activity of P. maderaspatensis leaves extract against clinical isolates of some bacteria using well diffusion method. The extracts showed inhibitory activity against clinical isolates of gram negative bacteria such as Salmonella typhi, Shigella dysentriae, Klebsiella, Pseudomonas and Escherichia coli and gram positive bacteria such as Bacillus subtilis and Staphylococcus. The results showed that the acetone extracts were more potent than the other extracts. The acetone extracts of Phyllanthus maderaspatensis showed best activity against Klebsiella with a minimum inhibitory concentration(mic) of 10 mg/ml. Keywords: Phyllanthus maderaspatensis, antibacterial activity, MIC. INTRODUCTION India is one of the nations blessed with a rich heritage of traditional medical systems and rich biodiversity to complement the herbal needs of the treatment administered by these traditional medical systems. The recognized Indian Systems of Medicine are Ayurveda, Siddha and Unani, which use herbs and minerals in the formulations. India, which has 15 agro-climatic zones, 47000 plant species of which 15000 are reported to have medicinal properties varying degrees. The World Health www.pharmasm.com IC Value 4.01 1

Organization (1980) has also recommended the evaluation of the effectiveness of plants in conditions where there is lack of safe synthetic drugs. [1] During the last ten years pace of development of new antimicrobial drugs has slowed down while the prevalence of resistance has increased astronomically. [2] The problem of microbial resistance of growing and the outlook for the use of antimicrobial drugs in the future is still uncertain. Therefore, actions must be taken to reduce this problem, such as controlling the use of antibiotics and carrying out research for the better understanding of the genetic mechanism of resistance. This prompted us to evaluate plants as the source of potential chemotherapeutic agent, antimicrobial agent and their ethno medicinal use. [3] P. maderaspatensis is an erect or spreading sub shrub, growing to only 50 cm tall, well-branched and hairless. It is also called as Madras Leaf flower as it is originated from the Madras region of India. P. maderaspatensis occurs in deciduous wood land, wooded savanna and grass land, on beaches and dunes, and also along streams and ponds in cultivated and distributed localities, from sea level up to 1400 m altitude. It grows on a wide variety of soils, usually on heavy clay and alluvial soils of low altitude river valleys on river banks and in flood plains. Outside tropical Africa P. maderaspatensis is often considered a weed. It shows a marked preference for calcareous sites in humid tropical areas. [4] The active constituents of P. maderaspatensis are essential oil, Maderin, mucilage, β- Sitosterol. A clear deep yellow oil can be extracted from the seeds of Phyllanthus maderaspatensis. The seeds contain myristic, palmitic,stearic, oleic and linolenic acids and β- Sitosterol. The deffated seed cake contains a fibrous mucilage which can be hydrolysed to galactose, arabinose, rhamnose, and aldobionic acid. [5] Entire plant of P. maderaspatensis such as seed and leaves used in folk medicine. The Indications of Leaves of P. maderaspatensis Linn. are Ophthalmia, Griping, Liver Affections, Cough,sAscites,tEarache,jHeadache,bIncipient, blindness.vthenindications ofnseedlof Phyllanthus maderaspatensis Linn. are Cough, Earache, Sores, Hard Swellings, Ulcers, Stomachache,bInflammations,nIntestinalbSpasms. The Indications of Decoction (seed) of P. maderaspatensis Linn. are Gonorrhoea, Internal Inflammations. www.pharmasm.com IC Value 4.01 2

The Indication of Infusion (leaves) of P. maderaspatensis is headache. The Indications of Seeds (Extract) of P. maderaspatensis Linn, are Dysentery, Diarrhoea. [6] The seeds have confirmed laxative, carminative and diuretic properties. Many of the medicinal uses of P. maderaspatensis are related to the astringent action of tannins. Though short term effects may be beneficial, the frequent systemic use of tannins might be dangerous because of their anti nutrient effects. In India a herbal medicine called Bhumyamlaki is sold which may be pure P. amarus or pure P. maderaspatensis or a mixture with P. fraternus.it is marketed as a medicine especially for liver troubles. [5] Having known the bioactive potency of P. maderaspatensis this study was done to explore the antibacterial activity present in P. maderaspatensis leaves. MATERIALS AND METHODS COLLECTION OF PLANT MATERIAL Fresh leaves of P. maderaspatensis free from disease were collected from Karunya University campus. The leaves were washed thoroughly two to three times with running water and then dried under shade and grinded into powder form. SOLVENT EXTRACTION Thoroughly washed fresh leaves of the plant material were dried in shade and powdered with the help of a grinder. 25 grams of shade dried powder was filled in the thimble and extracted successively with 250 ml of solvents such as water, methanol, acetone, ethyl acetate and benzene using soxhlet apparatus for 24-48 hours at their respective boiling points such as 100, 65, 56, 72 and 80ºC. The extracts were then vacuum dried using rotary vacuum evaporator to evaporate the solvents and to yield a semisolid mass whose mass was then determined. The extracts were then stored in a refrigerator at 4ºC until further use. WELL PREPARATION The prepared extract was weighed and mixed with known volume of water, methanol, acetone, ethyl acetate and benzene. Wells were punched on the petri plate and the extract of different concentrations such as 25mg/ml, 50mg/ml, 75mg/ml and 100mg/ml were added. Chloramphenicol (30mg/disc) was used as the positive control against all the microorganisms except Pseudomonas aeruignosa it showed a range of zone of inhibition from 19-25 mm. And Imipenum was used against Pseudomonas www.pharmasm.com IC Value 4.01 3

which gave a zone of inhibition of 26 mm. The respective solvents are used as negative control. MICROORGANISMS The gram negative bacteria such as Salmonella typhi, Pseudomonas, Escherichia coli, Klebsiella e and Shigella dysentriae and gram positive bacteria such as Bacillus subtilis, and Staphylococcus were obtained from Microbiology lab of Kovai Medical Centre Hospital, Coimbatore. All the test strains were maintained on nutrient agar slopes (Hi-Media) and were sub-cultured once in every two weeks. These bacteria served as test pathogens for antibacterial activity assay. ANTIBACTERIAL TEST PREPARATION OF CULTURE MEDIUM AND INOCULATION The petri plates and Muller Hinton Agar medium were sterilized for 20 minutes at 120ºC. The rest of the procedure was carried out in laminar air flow. Approximately 20ml of the media was poured into the sterile petri plates and allowed to get solidified. After the media gets solidified the bacterial organisms were swabbed on the medium using sterile cotton swabs. AGAR WELL DIFFUSION METHOD Microorganisms were inoculated over the dried surface of Muller Hinton Agar plate by spreading with the swab over the entire surface of the sterile agar plate. This procedure was repeated two or more times and the plates were rotated 60º each time to ensure an even distribution of inoculums. Appropriate wells were punched evenly (not closer than 24mm from centre to centre) on the surface of the agar plate. The extracts of different concentrations were added into the wells carefully using micropipettes. The plates were then incubated at 35ºC within 15 mins after the extracts were added. The plates were incubated aerobically. After 16 to 18 hours of incubation each plate was tested for activity by checking the zone of inhibition. MINIMUM INHIBITORY CONCENTRATION (MIC) OF LEAVES EXTRACTS: The MIC method was applied on extracts that proved their high efficacy against microorganisms by the disc diffusion method. The MIC of the plant extracts was determined on Muller Hinton Agar medium by means of well diffusion method. A lawn of culture was made on Muller Hinton Agar plates and to the wells different www.pharmasm.com IC Value 4.01 4

concentrations of plant extracts such as 5, 10, 15 and 20 mg/ml were added. The plates were then incubated at 37ºC for 24 hrs. The lowest concentration inhibiting the visible growth of the organism was considered as MIC. RESULTS AND DISCUSSION Ethanobotanical approach is one of the common methods that are employed in choosing the plants for pharmacological study. [7] India is one of the twelve mega biodiversity centres having more than 45000 plant species. Its diversity is unmatched due to the presence of sixteen different agro climatic zones, ten vegetative zones and fifteen biotic provinces. [8] Use of plants as a source of medicine has been inherited and is an important component of the health care systems. Approximately 20% of the plants found in the world have been submitted to pharmacological or biological tests. [9] The systemic screening of plant extracts for anti bacterial activity is a continuous effort to find new anti bacterial components. In the present study, the antibacterial activities were performed with water, methanol, acetone, ethyl acetate and benzene extracts of the P. maderaspatensis.the study was made against five gram negative pathogenic bacteria and two gram positive bacteria using the well diffusion method. The aqueous, methanolic, acetonic, and ethyl acetate extracts showed activity against human pathogens namely Salmonella typhi, Shigella dysentriae, Escherichia coli, Klebsiella e, Pseudomonas, Bacillus subtilis and Staphylococcus. Thus the activity of P. maderaspatensis against the human pathogens was thus proved. While all the extracts were active but benzene extract showed no sensitivity against human pathogens (Table 1). www.pharmasm.com IC Value 4.01 5

TABLE 1: ANTIBACTERIAL ACTIVITY OF VARIOUS SOLVENT EXTRACTS OF P. MADERASPATENSIS Sl.No. Microorganisms Water Methanol Acetone Ethyl acetate Benzene 1. Salmonella + + + + _ paratyphi 2. Shigella dysentriae + + + + _ 3. Escherichia coli + + + + _ 4. Klebsiella + + + + _ 5. Pseudomonas + + + + _ 6. Bacillus subtilis + + + + _ 7. Staphylococcus + + + + _ The zones of inhibitions were produced by water, methanol, acetone and ethyl acetate extract except benzene against all the test organisms.bacetone extracts were more active than the other extracts against all the microorganisms. The zones of inhibition were ranging from 3-27mm in diameter. The highest zone of inhibitions (27mm) was noted in acetone extract against Klebsiella pnuemoniae in 100 mg/ml concentration (Table 2). The extracts of higher plants can be very good source of antibiotics against various bacterial pathogens. Plant having antimicrobial compounds have enormous therapeutic potential as they can act without any side effect as often found with synthetic antimicrobial products. TABLE 2: ANTIBACTERIAL ACTIVITY OF DIFFERENT SOLVENT EXTRACTS USING WELL DIFFUSION METHOD (Inhibition zones) Solvent Microorganisms Zone of inhibition Control mg/ml 25 mg/ml 50 mg/ml 75 mg/ml 100 mg/ml (mm) (mm) (mm) (mm) (mm) Water Salmonella typhi 24 7 13 15 18 www.pharmasm.com IC Value 4.01 6

Shigella dysentriae 23 6 11 13 16 Escherichia coli 22 9 13 15 18 Klebsiella 20 5 8 10 14 Pseudomonas 26 8 12 14 17 Bacillus subtilis 20 6 8 9 13 Staphylococcus 21 10 14 16 18 Methanol Acetone Ethyl acetate Salmonella typhi 24 8 11 14 16 Shigella dysentriae 23 13 16 20 25 Escherichia coli 22 16 20 23 26 Klebsiella 20 17 22 24 26 Pseudomonas 26 4 9 10 15 Bacillus subtilis 20 5 10 12 14 Staphylococcus 21 3 7 9 12 Salmonella typhi 24 13 15 20 25 Shigella dysentriae 23 12 14 21 24 Escherichia coli 22 4 6 10 14 Klebsiella 20 16 19 22 27 Pseudomonas 26 3 5 10 14 Bacillus subtilis 20 12 17 19 24 Staphylococcus 21 11 16 18 22 Salmonella typhi 24 10 12 15 18 Shigella dysentriae 23 8 10 13 17 Escherichia coli 22 7 11 14 18 Klebsiella 20 6 9 11 12 Pseudomonas 26 8 10 12 16 Bacillus subtilis 20 5 8 10 12 Staphylococcus 21 9 11 13 16 www.pharmasm.com IC Value 4.01 7

The MIC values confirmed the significant activity against the tested microorganisms as shown in table 3. The MIC values of Phyllanthus maderaspatensis ranged from 5 to 25 mg/ml for acetone extracts. The MIC value were found to be 10mg/ml against Klebsiella pnuemoniae, 15 mg/ml against Salmonella typhi and Shigella dysentriae,20 mg/ml against Bacillus subtilis and Staphylococcus and 25mg/ml against E. coli and Pseudomonas. TABLE 3: MINIMAL INHIBITORY CONCENTRATION OF PLANT EXTRACTS WITH ANTIBACTERIAL ACTIVITY (MG/ ML) Microorganisms Acetone Salmonella typhi 15 Shigella dysentriae 15 Escherichia coli 25 Klebsiella 10 Pseudomonas 25 Bacillus subtilis 20 Staphylococcus 20 Most antimicrobial medicinal plant are more effective against gram-positive than gram-negative bacteria.[10,11] However, the current findings showed a remarkable activity against gram-negative bacteria including multi-resistant gram-negative strains. The antimicrobial activity of the P. maderaspatensis leaves methanol and aqueous extract may be due to tannins and other phenolic constituents. Tannins are considered nutritionally undesirable because they precipitate proteins, inhibit digestive enzyme and affect the absorption of vitamins and minerals. However, some kinds of tannins can reduce the mutagenicity of a number of mutagens and display anticarcinogenic, antimicrobial and antioxidant activities. A special feature of higher angiospermic plants is their capacity to produce a large number of organic chemicals of high structural diversity hence P. maderaspatensis have www.pharmasm.com IC Value 4.01 8

been screened for their anti bacterial activity against human pathogenic bacteria. P. maderaspatensis possess broad spectrum of activity and a high degree of activity against the test pathogens. the results of the present investigation such as that this plant is an important candidate plant for further investigation on isolation and characterization of the bioactive principle responsible for anti bacterial activity. The result obtained in this study suggests a potential application of P. maderaspatensis leaves for treatment of Ophthalmia, Griping, Liver Affections, Cough, Ascites, Earache, Headache, Incipient Blindness and further investigations should be conducted in order to explore their applications. [6] Other medicinal plants containing phenolic compounds, including tannins, as major constituents are used topically for care and repair of skin wounds. The advantage of the use of topical antimicrobials is their ability to deliver high local concentrations of antibiotic irrespective of vascular supply. Further benefits include the absence of adverse systemic effects, and a low incidence of resistance. CONCLUSION P. maderaspatensis seems under exploited, but it will probably continue to be regarded as an important medicinal plant species. There is no commercial seed production, although this aspect deserves more attention given the ease of propagation. In the present study antibacterial activity of P. maderaspatensis extracts towards drug resistant / clinically significant microbes has been investigated. Considering the many medicinal uses, there is a large scope for future research and further phytochemical and pharmacological investigation need to be done. A number of compounds may provide interesting leads for pharmacological evaluation and therefore merit future research. P. maderaspatensis deserves to be a part of germ plasm collection. ACKNOWLEDMENTS The authors convey their thanks to the HOD and the Director, School of Biotechnology and Health Sciences, Karunya University, Coimbatore for providing lab facilities. www.pharmasm.com IC Value 4.01 9

REFERENCES 1. Upadhayay VP, Pandey K.: In Ayurvedic approach to diabetes mellitus and its management by indigenous resources. Diabetes Mellitus in Developing Countries, Interprint, New Delhi, 1984. 375-377. 2. Akinpelu DA, Onakoya ZTM.: Antimicrobial activities of medicinal plants used in folklore remedies in South-Western. Afr. J. Biotechnol, 2006. 5:1078-1081. 3. Prashanth KN, Neelam S, Chauhan S, Harishpadhi B, Ranjani M.: Search for antibacterial and antifungal agents from selected Indian medicinal plants. J. Ethanopharmacol, 2006. 107:182-188. 4. http://www.flowersofindia.net/catalog/slides/madras%20leaf-flower.html 5. Medicinal plants by Gabriëlla Harriët Schmelzer, Ameenah Gurib-Fakim.426 6. http://www.druginfosys.com/herbal/herb.aspx?code=407&name=phyllanthus%2 0maderaspatensis%20Linn.&type=3 7. Cox PA, MJ Balick.: The ethnobotanical approach to drug discovery. Sci. Amer, 1994. 270: 60-65. 8. Anonymous.Drugs and Pharmaceuticals Industry Highlights Incorporating Patent Information. CDRI, Lucknow, India, 1998. 21: 33-34. 9. Suffredini JB, HS Sader, AG Goncalves, AO Reis, AC Gales, AD Varella, RN Younes.: Screening of antimicrobial extracts from plants native to the Brazilian Amazon rainforest and Atlantic forest. Braz. J. Med. Biol. Res, 2004. 37: 379-384. 10. Lin J Opoku AR, Geheeb-Keller M.Hutchings AD, Terblanche SE, Jagar AK, Van Staden J.: Preliminary screening of some traditional zulu medicinal plants for anti-inflammatory and antimicrobial activities. J.Ethanopharmacol, 1999. 68:267-274. 11. Scrinivasan D, Nathan S, Suresh T, Perumalsamy O.: Antimicrobial activity of certain Indian medicinal plants used in folkloric medicine. J. Ethanopharmacol, 1989. 217-220. For Correspondence: Dr. A. Annamalai Email: aannamalai2001@yahoo.com www.pharmasm.com IC Value 4.01 10