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Research ArticleISSN: 0974-6943 Available online through

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*Corresponding author.Dr. Varaprasad BobbaralaPlot No. 92, Phase 1, IDA, CherlapallyHyderabad, A.P 500051 IndiaTel.: + 91-9949129539E-mail:phytodrugs@gmail.com

INTRODUCTIONMedicinal plants continue to be an important therapeutic aid for alleviatingthe ailments of humankind. The search for perpetual health and prolongedexistence for remedies to mitigate pain and discomfort drove early man toexplore his immediate natural surroundings and led to the use of many plants,animal products, and minerals, etc. and the development of an array of thera-peutic agents. Today, there is a transformed interest in traditional medicineand an increasing demand for more drugs from the plant sources. This resur-gence of interest in plant-derived drugs is mainly due to the current widespreadbelief that “green medicine” is safe and more dependable than the costlysynthetic drugs, many of which have adverse side effects. Nature has bestowedupon us a very rich botanical affluence and a large number of diverse types ofplants grow wild in different parts of our country. In India, the use of differentparts of several medicinal plants to cure specific ailments has been in trendfrom ancient times1. India is rich in medicinal plant diversity. All known typesof agro-climatic, ecologic and edaphic conditions are met within India. India isrich in all three levels of biodiversity, as species diversity, genetic diversity andhabitat diversity2.

In order to promote the use of medicinal plants as potential sources of anti-bacterial compounds, it is pertinent to thoroughly investigate their bioactiv-ity and thus validate their potential use3-5. Natural products are known to playan important role in both drug discovery and chemical biology. In fact, manyof the current drugs either mimic naturally occurring molecules or have struc-tures that are fully or in part derived from natural motifs6. Natural Antibacte-rials can be derived from barks, stems, leaves, flowers and fruits of plants,various animal tissues or from microorganisms7-9.

Some phytochemicals produced by plants have antibacterial activity allowingthese plants to be studied and used for the development of new Antibacterialdrugs10. The effectiveness of phytochemicals in the treatment of variousdreadful diseases may lie in their antioxidant effects 11. In the ancient times,plant metabolites were generally considered as sources of antinutritional fac-tors. According to World Health Organization, medicinal plants are the bestsources to obtain a variety of newer herbal drugs. In many developing coun-tries, traditional medicine is one of the primary health care systems12-14. Herbsare widely exploited in the traditional medicine and their curative potentials

Antibacterial activity screening of few medicinal plants from the Southern Region of IndiaSunilbabu Koppula1, K. Ammani1, Varaprasad Bobbarala2* and P.V.Bramhachari 3

1Department of Microbiology, Acharya Nagarjuna University, AP, India2Department of Biotechnology, Krishna University, AP, India3Vivimed Labs Ltd., Veeranag Towers, Habsiguda, Hyderabad, AP.Received on: 04-07-2010; Revised on: 09-08-2010; Accepted on:20-09-2010

ABSTRACTAntibacterial activity of fifteen medicinal plants, Andrographis paniculata Nees, Azadirachta indica A. Juss., Butea monosperma (Lam.) Taub., Calotropisprocera (Ait.) R.Br., Cassia occidentalis Linn., Catharanthus roseus Linn., Hyptis sauveolens (L.) Poit., Morus alba L., Ocimum sanctum L., Physalis minimaLinn., Piper nigrum L., Plumbago zeylanica L., Terminalia chebula L., Tinospora cordifolia (Willd.) and Vernonia cinerea (L.) Less., were screened againstmedically important pathogenic bacterial strains, Klebsiella pneumonia MTCC 4032, Staphylococcus aureus MTCC 3160, Staphylococcus epidermis MTCC3615 and Micrococcus luteus MTCC 7527. The antibacterial activity was determined in aqueous and methanol extracts using both agar disc diffusion and agar welldiffusion methods. The methanol extracts were more potent than aqueous extracts of all the plants studied. Micrococcus luteus was the most resistant strain whilethe more susceptible bacterial strain was Staphylococcus epidermis. Terminalia chebula L., Morus alba L. and Azadirachta indica A. Juss., showed strong activityagainst all the tested bacterial strains. Hence, these medicinal plants can be used to discern bioactive natural products and new pharmaceutical molecules that servein the development of unmet therapeutic needs.

Key words: Antibacterial activity, Methanol extract, Micrococcus luteus,Staphylococcus epidermis, Terminalia chebula L., Morus alba L. and Azadirachtaindica A. Juss.,

are well documented15. Large scale evaluation of the local flora exploited intraditional medicine for various biological activities is a necessary first step inthe isolation and characterization of the active principle and further leadingto drug development. In view of these few medicinal plants were studied ex-haustively for its potential against important four human pathogenic bacteria.

MATERIALS AND METHODSPlant material collection:The plant materials were collected in Jan 2009 from in and around AcharyaNagarjuna Univesity (ANU), Guntur, A.P, India. The taxonomic identities ofthese plants were identified by their vernacular names and later validated byProf. T. Pullaiah, Sri Krishna Devaraya University, AP, India. The voucherspecimens were deposited at ANU. The ethno botanical information of thescreened plants is given in (Table 1). A total of fifteen plants were screened(Table.1) Different parts of the plants were collected, shade dried, and pow-dered in a homogenizer, and 10g of each plant was used for aqueous andmethanol extraction and finally stored in airtight bottles.

Preparation of plant extracts:Plant parts were air dried at room temperature at constant weights. The driedplant material was separately ground to powder. The powdered was then weighedand extracted with aquaous and methanol with reflux on water bath at 40° Cfor three consecutive days. The extract was filtered and evaporated undervacuum to dryness with rotary evaporator and then placed in and over at 60°C until constant weight was obtained and the solutions were prepared withconcentrations 1mg/ml using DMSO.

Determination of Antibacterial activity:The test organisms used were obtained from Microbial Type Culture Collec-tion & Gene Bank (MTCC), (Institute of Microbial Technology (IMTECH),Chandigarh, India. The tested organisms used in this study were as follows:Micrococcus luteus (MTCC 5198), Staphylococcus aureus (MTCC B96), Kleb-siella pneumonia (MTCC 109) and Staphylococcus epidermis (MTCC 435).The medium used for the activation of the microorganisms was soybean caseinbroth (SBCB). All the culture media were prepared and treated according to themanufacturer guidelines (Himedia laboratories, Mumbai, India).

The microorganisms were inoculated into SBCB and incubated at 35 ± 2°C for4 h. The turbidity of the resulting suspensions was diluted with SBCB to obtaina transmittance of 25.0 % at 580 nm. That percentage was found spectropho-tometrically comparable to 1 McFarland turbidity standard. This level ofturbidity is equivalent to approximately 3.0 × 108 CFU/ml. The Shimadzu

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Table 1. Ethnobotanical information of some medicinal plant species of Southern India selected for antibacterial activity.

S.No. Botanical Name Family Parts used Uses / Ailments treated

1. Andrographis paniculata Nees Acanthaceae Whole plant, leaves Anti-biotic, anti-viral, anti-parasitic and immune System stimulant2. Azadirachta indica A. Juss. Meliaceae Fruits and leaves Effective against fungi, bacteria, viral, antimalarial and antihelmintic.3. Butea monosperma (Lam.) Taub. Fabaceae Bark, flowers &leaves Leprosy, antihelmintic and tumerous4. Calotropis procera (Ait.) R.Br. Asclepiadaceae Root bark Leprosy, diarrhea, dysentery, cough and asthma. 5. Cassia occidentalis Linn. Caesalpinaceae Whole plant Boils, Spasm. Hysteria, Whooping cough6. Catharanthus roseus Linn. Apocynaceae Leaves and roots Anti-mitotic and Anti-microtubule agents7. Hyptis sauveolens (L.) Poit. Lamiaceae Leaves Stimulant, carminative and used for healing wounds.8. Morus alba L Moraceae Bark and fruit Asthma, bronchitis, cold and constipation9. Ocimum sanctum L. Lamiaceae Leaves Anthelmintic, bronchitis and genitor-urinary diseases10. Physalis minima Linn. Solanaceae Leaves, fruit & flower Laxative, abdominal troubles and spleen enlargement.11. Piper nigrum L. Piperaceae Fruits Asthma, fever, arthritis and hemorrhoids.12. Plumbago zeylanica L. Plumbaginaceae Roots Ulcers, piles, skin diseases and influenza.13. Terminalia chebula Retz. Combritaceae Fruit Antibacterial, digestive problems, mouthwash/gargle, astringent and douche for vaginitis.14. Tinospora cordifolia (Willd.) Asclepiadaceae Stem Analgesic and anti-inflammatory.15. Vernonia cinerea (L.) Less Asteraceae Leaves Elephantiasis, cough, asthma, bronchitis and gastrointestinal disorders.

Figure.1. Antibacterial activity of selected medicinal plant extracts (aqueous and methanol) against Micrococcus luteus

Figure.2. Antibacterial activity of selected medicinal plant extracts (aqueous and methanol) against Staphylococcus aureus

spectrophotometer was used to adjust the transmittance of the working sus-pensions.Antibacterial Activity:The antibacterial activity of different plant species was evaluated by agar discdiffusion method 16-17 for aqueous extracts and agar well diffusion method18-19

for solvent extract using Mueller Hinton agar No. 2 medium for the antibac-terial assay. The test microorganism was activated by inoculating a loop full of

the strain in the SBC broth (30 ml) and incubated on a rotary shaker. Thenbacterial cultures was inoculated into the molten Muller Hinton agar media andafter proper homogenization it was poured into the Petri plate (Hi-media)using pour plate technique. For agar disc diffusion method, the test compound(0.1 ml) was introduced on the disc (0.7 cm) (Hi-Media) and then allowed todry. Then the disc was impregnated on the seeded agar plate. For agar welldiffusion method, a well was made in the seeded plates with the help of a cup-

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Figure.3. Antibacterial activity of selected medicinal plant extracts (aqueous and methanol) against Staphylococcus epidermis

Figure.4. Antibacterial activity of selected medicinal plant extracts (aqueous and methanol) against Klebsiella pneumonia.

borer (0.6 cm). The test compound was introduced into the well and the plateswere incubated at 370C for 24 h. Microbial growth was determined by measur-ing the diameter of zone of inhibition. For each bacterial strain, controls weremaintained in which pure solvents were used instead of extract. The experi-ment was done in three replicates and the mean values are presented.

RESULTS AND DISCUSSIONThe antibacterial activity of both aqueous and methanolic extracts of thedifferent plants against Micrococcus luteus is shown in (Figure 1). Of aqueousextracts, only that of Azadirachta indica showed maximum activity againstMicrococcus luteus. Methanolic extracts of 6 of the 15 plants, Terminaliachebula L., Azadirachta indica, Plumbago zeylanica, Piper nigrum , Morusalba, and Butea monosperma showed considerable antibacterial activity. Maxi-mum activity was hown by Terminalia chebula followed by Piper nigrum andAzadirachta indica. Minimum antibacterial activity was shown by Vernoniacinerea.

The antibacterial activity of the 15 plants against S. aureus, is shown in(Figure 2). The methanolic extracts of all the plants showed antibacterialactivity while the aqueous extracts of only 4 plants showed antibacterialactivity. The antibacterial activity of methanolic extracts was more than thatof aqueous extracts. Maximum antibacterial activity was shown by Terminalia

chebula followed by Morus alba However, minimum activity was shown byTinospora cordifolia extract.

The antibacterial activity of both aqueous and methanolic extracts of the 15plants against S. epidermis is shown in (Figure 3). Of the 15 plants, 7 showedantibacterial activity against this bacterial strain. Maximum activity ofmethanolic extract was shown by Morus alba followed by Azadirachtaindica, Piper nigrum and Terminalia chebula . Aqueous extracts of only 3 ofthe 15 plants showed antibacterial activity against S. epidermis; however,maximum activity was shown by Azadirachta indica, followed by Andrographispaniculata and Terminalia chebula extracts. Antibacterial activity of bothaqueous and methanolic extracts of the 15 plants against K. pneumonia isshown in (Figure 4). Methanolic extracts of all the plants showed considerableantibacterial activity against this bacterial strain. Maximum activity ofmethanolic extract was shown by Terminalia chebula and Plumbago zeylanica.However excepting Vernonia cinerea, methanolic extracts of all the remain-ing plants showed antibacterial activity to a certain extent. Of the 15 plants,aqueous extracts of 3 showed antibacterial activity against K. pneumonia .Maximum activity of aqueous extract was shown by Terminalia chebula .

All plants showed greater inhibitory activity in the methanolic when com-pared to aqueous extracts. Aqueous plant extracts of Morus alba and Tinospora

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cordifolia had no antibacterial activity against any of the four bacterial strainsinvestigated. Of the investigated 4 bacterial strains, Micrococcus luteus wasthe most resistant strain, whereas the more susceptible bacterial strain wasStaphylococcus epidermis. Terminalia chebula , Morus alba and Azadirachtaindica showed strong activity against all the tested bacterial strains. Based onour results, it is concluded that plant extracts have great potential as Antibac-terial compounds against microorganisms and they can be used in the treat-ment of infectious diseases caused by resistant microorganisms. Several pub-lished reports show the effectiveness of traditional herbs against microorgan-isms, as a result, plants are one of the bedrocks for modern medicine to attainnew principles20. Acquired resistance to various antibiotics in bacteria over 25years now constitutes a serious threat to public health21-22. Nearly all of theidentified components from plants active against microorganisms are aro-matic or saturated organic compounds, they are often obtained through initialethanol or methanol extraction23. Terminalia chebula showed stronger activ-ity than the other plants against all the tested bacterial strains. Therefore,Terminalia chebula could be selected for further analysis. Similar results werereported in the ethanolic extracts of T. chebula fruit, which showed highestactivity against various human pathogens such as Salmonella typhi SSFP 4S,Staphylococcus epidermidis MTCC 3615, Staphylococcus aureus ATCC 25923,Bacillus subtilis MTCC 441 and Pseudomonas aeruginosa ATCC 27853.Conversely, the alcoholic extracts of black myrobalan (Teminalia chebulaRetz) on Helicobactor pylori also showed significant antibacterial activity24.Based on these results, it can be concluded that plant extracts have a greatpotential Antibacterial compounds against microorganisms that can be used intreatment of infectious diseases caused by a range of resistant microorgan-isms. Therefore screening of various natural organic compounds and identifi-cation of active agents is the need of the hour, because successful prediction oflead molecules and drug-like properties at the inception of drug discovery willpay off later in drug development. It is vital that research strategies be ori-ented towards discovery and development of Antibacterial agents urgentlyrequired in the future.

ACKNOWLEDGEMENTS:The authors acknowledge the assistance of the technicians Prasad and Nagraju.We also acknowledge Professor T. Pullaiah for assisting us with the identifica-tion of plant materials. This work was supported by grants from the RajivGandhi National fellowship as Senior Research Fellowship, UGC Grants, India.

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Source of support: Nil, Conflict of interest: None Declared