In-Vitro Antibacterial Activity of Two Medicinal Plants Neem (Azadirachta indica) and Peppermint

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1 Research Paper In-Vitro Antibacterial Activity of Two Medicinal Plants Neem (Azadirachta indica) and Peppermint Dr. Saba Irshad *, Muneeba Butt and Hira Younus * Assistant Professor, Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, Pakistan. s: irshad.saba@gmail.com, sabairshad3@yahoo.com Abstract The use of medicinal plants is a universal phenomenon. Every culture on earth has relied on the huge variety of natural chemistry found in healing plants for their therapeutic properties. All drugs of the past were substances with a particular therapeutic action and they were extracted from plants. Thus, medicinal plants may define as any plant that may have medicinal use. The objective of the present study was to evaluate the antibacterial activity of Neem (Azadirachta indica) and Peppermint (Mentha piperita) by using agar diffusion assay and gel filtration chromatography. The bacterial strains used in this research work were Escherichia coli, subtilius, Salmonella typhi,, Staphylococcus aureus, Klebsiella pneumoniae, Staphylococcus epidermitis. The Neem leaves (Azadirachta indica) were macerated in different organic solvents including pure, ethanol, acetone and methanol whereas Peppermint leaves (Mentha piperita) were macerated in acetone, methanol and ethanol. Then, by using agar diffusion assay antibacterial activity of these medicinal plants was estimated. The zones of inhibition were measured by scaling and represented by tables and graphs. The Neem extract of and Peppermint Oil showed the maximum antibacterial activity as compared to other solvent extracts. Then, distilled water macerated form of Neem and Peppermint were used for gel filtration chromatography technique in order to determine the fraction containing the active components. 8 of both Neem and Peppermint showed maximum antibacterial activity against all above mentioned bacterial strains. Furthermore, this study recommended for the isolation and separation of bioactive compounds responsible for the antibacterial activity which would be done by using thin layer chromatography (TLC) and High-performance liquid chromatography (HPLC). Keywords: Neem (Azadirachta indica), Peppermint (Mentha piperita), Antibacterial Activity, Agar Diffusion Assay, Gel Filtration Chromatography 1. Introduction Neem has extensive utilization in Ayurveda, Unani and Homeopathic medicine (Kausik et al, and Girish & Shankara, 8). Neem is a member of the Mahogany family (Rembold, 1996). Neem and its leaves used for the treatment of various diseases including eczema, ringworm, acne, anti-inflammatory activities, anti-hyperglycemic and also treat chronic wounds, diabetic foot and gangrene. It also removes toxins from the body; neutralize the free radicals present in body and used as blood purifier. Recently it is reported as anticancer and used for hepatorenal protective activity and hypolipidemic effects (Kumar & Gupta, ). Neem is distributed widespread in the world. The Chemical constituents of Neem contain many bioactive compounds including alkaloids, flavonoids, triterpenoids, phenolic compounds, carotenoids, steroids and ketones. Azadirachtin is a mixture of seven isomeric compounds (Verkerk & Wright, 1993). To clean wounds, soothes, swellings and erases skin problems, boiled Neem is used. Neem leaves have been demonstrated to have vast properties like as immunomodulatory, antiinflammatory, antihyperglycaemic, antiulcer, antimalarial, antifungal, antibacterial, antiviral, antioxidant, antimutagenic and anticarcinogenic (Hoque et al, 7). El-Mahmood et al (1) observed the anti bacterial effects of crude extract Available online at 9

2 of Neem seed against pathogens involved in eyes and ear infections. Peppermint (Mentha piperita) belongs to family Labiate and is of huge importance medicinally (Clark & Menory, 198). Peppermint leaves contains about.5-4% volatile oil. This oil is composed of 5-78% free menthol, monoterpene, menthofurane and traces of jasmine (.15%) to improve the quality (Diaz et al, 1988). Peppermint oil and menthol possesses antibacterial effects against both gram-positive and gram negative bacteria (Chaumont & Senet, 1978). Peppermint also has antiviral and fungicidal activity. Menthol is virucidal against influenza, herpes and other viruses (Scavroni et al, 5). Peppermint is very unique and beneficial plant. It is used in food, cosmetics and medicines (Samarth et al, 6). It is chemopreventive and antimutagenic (Gardiner, ). It is also used as an analgesic and to treat headache (Tunon et al, 1993). It is mosquito repellent and has antinematodal, antiviral, antifungal and antibacterial properties (Walker & Melin, 1996). Sandasi et al (1) observed that microbial biofilms have become increasingly problematic in the food processing and medicinal industries, where they cause contamination. The inhibitory effects of herbal extracts on the development of microbial biofilms were investigated. Mentha piperita was the only extract that showed some antibiofilm activity against both pathogens. Saeed et al (6) observed the antibacterial activities of different forms of peppermint (Mentha piperita) viz., aqueous infusion, decoction, juice and essential oil. Essential oil of peppermint exhibited highest antibacterial activity with mm mean zone of inhibition.. Materials and Methods The present research work was designed to evaluate antibacterial activity of medicinal plants including Neem and Peppermint. This study was performed at Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore. In order to see the antibacterial activity of Neem and Peppermint, the following main steps were involved:.1. Collection and Identification of Medicinal Plants and The medicinal plants Neem and Peppermint were collected from different areas in Lahore, Punjab. The bacterial strains used in this project were Escherichia coli, subtilius, Salmonella typhi,, Klebsiella pneumoniae, Staphylococcus epidermitis. These bacterial strains were identified, characterized and collected from Jinnah Hospital, Sheikh Zayed Hospital and the Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore. These bacterial strains were preserved in glycerol stock solution at -7 C in the Institute of Biochemistry and Biotechnology. The medicinal plants used in this research were identified by Dr. Abdul Rehman Niazi (taxonomist) from the Department of Botany, University of the Punjab, Lahore... Crude Extracts Preparation Leaves of Neem and Peppermint were washed and crushed by pestle and mortal. The 1 g of each leaves was dissolved in 1 ml of different organic solvents including pure, ethanol, acetone and methanol and their crude extracts were prepared by maceration. The crushed macerated parts of these medicinal plants were placed at magnetic stirrer and were equally mixed in organic solvents by using magnet. The conical flasks of these extracts were covered by cotton plugs to avoid the solvent evaporation. The extracts were placed in shaking incubator at 5 rpm for 48 hours. After shaking, they were filtered with muslin cloth. The filtered extracts of both the medicinal plants were centrifuged at 8 rpm for minutes. The supernatants were collected in sterile flask. The filtered extracts were stored at 4 C..3. Antibacterial Activity Assay The agar disc-diffusion assay was performed to evaluate antibacterial activity of crude extracts. Briefly, 1 ml of L.B broth was inoculated with each test organism and incubated at 37 C for overnight. The 1 ml of M.H agar was mixed well and poured on the sterile Petri plates. The agar media on Petri plates were allowed to set and harden for few minutes. The small autoclaved discs of Whatmann filter paper were used. The 1 µl of nutrient L.B broth culture was poured on each agar Petri plates. The nutrient broth culture was spread by using sterilized glass spreader. During agar-disc diffusion assay, the sterile discs were dipped in the different crude extracts of medicinal plants with sterilized forceps and placed at the centre of the Petri plates. The antibiotic Ampicillin drug was loaded on Petri plates as a control to check the comparison of antibacterial activity with different crude extracts of medicinal plants. The maximum antibacterial activity observed by Ampicillin against E. coli was mm. The Petri plates were sealed and left at room temperature for hours, to allow the diffusion of the test sample and then incubated at 37 o C for overnight. The diameter of the zones of inhibition was measured in mm..4. Gel Filtration Chromatography Gel filtration chromatography was used to isolate the particular fractions involved in the inhibition of bacterial growth. The swollen gel Sephadex G-1 was packed in a Available online at 1

3 column (.5x3cm) and equilibrated with 5 mm Tris-Cl buffer (ph 7.5). Supernatants (ml each) containing extracts of Neem and Peppermint were loaded onto a gel filtration column individually. The flow rate of column was 1. ml/minute. Total 1 fractions ( ml of each) were collected. The fractions were stored at 4 C for further analysis. The antibacterial activity of these fractions was checked by agar disc-diffusion assay. The zones of inhibition were measured by scaling. The fraction containing maximum zone of inhibition in (mm) was further analyzed by thin layer chromatography. 3. Results and Discussion We conducted a prospective observational study of antibacterial activity of Neem and Peppermint extracts. The Pure,, and extracts of Neem were screened against bacterial strains i.e. Escherichia coli, subtilius, Salmonella typhi,, Staphylococcus aureus, Klebsiella pneumoniae and Staphylococcus epidermitis by using agar disc-diffusion assay and fractions obtained from gel filtration chromatography are shown in Tables (1 & ). Similarly, zones of inhibition of Peppermint by agar discdiffusion assay and fractions by gel filtration chromatography were measured as shown in Tables 3 and 4. 1 and could not be tested further because these fractions were colourless and contained buffer only. The fractions (6-9) of Neem and fractions (6-8) were screened against seven bacterial strains as mentioned above. Graphical presentation of the average of zones of inhibition of Pure,, and extracts of Neem and Peppermint are shown in Figures 1-8. The antibiotic Ampicillin drug was loaded as a control to check the comparison of antibacterial activity with different crude extracts of medicinal plants. The maximum antibacterial activity observed by ampicillin against was mm. Table 1. Pure,, and Extracts of Neem Showing Measurement of Zone of Inhibition. Pure E. coli mm 1 mm mm 3 mm B. subtillus 5 mm 1 mm mm mm S. typhi.5 mm 3 mm 15 mm mm 3 mm mm 3 mm mm S. aureus 3 mm mm mm 1 mm K. pneumoniae 3 mm 3 mm 4 mm 1 mm S. epidermitis mm.5 mm.5 mm mm Neem has universally been accepted as a wonder tree because of its diverse utility (Brahmachari, 4). For the determination of antibacterial activity, Neem leaves are usually used. The medicinal plant Neem showed its antibacterial activity against bacterial strains i.e. Escherichia coli, Staphylococcus aureus. Table. Antibacterial activity of Neem s, Isolated by Gel Filtration Chromatography Bacterial Strains E. coli 3 mm 1 mm 8 mm mm B. subtillus 1 mm 1 mm 5 mm 1 mm S. typhi.1 mm.1 mm 1 mm 1 mm.8 mm mm 3 mm 1 mm S. aureus.5 mm.6 mm mm 1 mm K. pneumoniae.1 mm mm mm 1 mm S. epidermitis.1 mm mm mm 1 mm Table 3. Bacterial Strains Peppermint Oil,, and Extracts of Peppermint Extracts Showing Measurement of Zone of Inhibition. Peppermint Oil E. coli 1 mm mm 4 mm 1 mm B. Subtillus mm 3 mm 4 mm 1 mm S. typhi 1 mm mm 3 mm 3 mm 1 mm 3 mm 5 mm mm S. aureus 5 mm mm 1 mm mm K. pneumoniae 11 mm 3 mm 5 mm 1 mm S. epidermitis 7 mm 1 mm mm mm Table 4. Antibacterial Activity of Peppermint s, Isolated by Gel Filtration Chromatography E. coli 1 mm 1 mm 1 mm B. Subtillus 1 mm 1 mm mm S. typhi 1 mm 1 mm 1 mm 1 mm 1 mm mm S. aureus 1 mm 1 mm 1 mm K. pneumoniae 1 mm 1 mm 1 mm S. epidermitis mm 3 mm 4 mm This data of present study correlates with the study of El- Mahmood et al (1). In this study, we used four macerated forms pure, ethanol, acetone and methanol of Neem leaves as shown in Table 1. The data came from our study showed that acetone extracts of Neem leaves are more effective than other macerated extracts as observed in Figures 1 and. The ethanol extract of Neem is very useful orally to treat many diseases caused by bacteria Available online at 11

4 (Awasthy et al, 1999). The fractions obtained from gel filtration chromatography as shown in Table. The maximum antibacterial activity observed in # 8 as shown in Figures 3 and 4. Pure Average zone of inhibitions (mm #6 #7 #8 #9 Different column fractions of Azadirachtra indica 1 Zone of Inhibition (mm) Figure 4. Average of Zone of Inhibition Different Column s of Neem (Azadirachta indica) Figure 1. Comparison of Zone of Inhibition of Neem with Different Different Organic Solvents Pure Average of Zone of Inhibitions (mm) Figure. Average Zone of Inhibition of Neem Leaves Extracts in Different Organic Solvents Zone of Inhibition (mm) #6 #7 #8 #9 Figure 3. Comparison of Zone of Inhibition of Different Column Chromatographic s of Neem (Azadirachta indica) Zone Inhibitions (mm) Figure 5. Comparison of Zone of Inhibition of Peppermint with Different Organic Solvents & Oil oil Oil Average of Zone of Inhibitions (mm) Figure 6. Average Zone of Inhibition of Peppermint Oil in Different Organic Solvents The plant Peppermint which possesses antibacterial activity against seven bacterial strains as shown in Table 3. Our observation about Peppermint is correlated with the Available online at 1

5 study of Bupesh et al (7) in which he studied the antibacterial activity of Mentha piperita (Peppermint) from leaf extracts against subtilis, and Streptococcus aureus. Peppermint oil or peppermint tea is often used to treat gas and indigestion; it may also increase the flow of bile from the gall bladder (Mimica et al, 3). Molecular Genetics, University of the Punjab, Lahore for providing bacterial strains and their identification. Special thanks to Dr. Abdul Rehman Niazi (taxonomist) from the department of Botany, University of the Punjab, Lahore. References Zone of Inhibition (mm) #6 #7 #8 Awasthy, K., Chaurasia, O., and Sinha, S. (1999) Prolonged murine genotoxic effects of crude extracted from Neem. Journal of Phytotherapy Research, 13, pp Brahmachari, G. (4) Neem-an omnipotent plant: retrospection. Journal of Chembiochem., 5(4), Bupesh, G., Amutha, C., Nandagopal, S., Ganeshkumar, A., Sureshkumar, P., and Saravanamurali, K. (7) Antibacterial activity of Mentha piperita L. (peppermint) from leaf extracts-a medicinal plant. Actaagriculturae Slovenica, 89 (1), pp Figure 7. Comparison of Zone of Inhibition of Different Column s of Peppermint Average zone of Inhibition #6 #7 #8 Different Column s Obtained Figure 8. Comparison of Zone of Inhibition of Different Column Chromatographic s of Peppermint It is cleared from the present study that the Peppermint oil and its acetone extract are more effective against bacterial strains as compared to its other crude extracts. The maximum value of zone of inhibition is obtained by Peppermint Oil as shown in Figures 5 and 6. The leaves of Peppermint were used to check antibacterial activity. The fractions obtained from gel filtration chromatography as shown in Table 4. The maximum antibacterial activity observed in # 8 as shown in Figures 7 and 8. Acknowledgements Authors wish to thank the staff of Jinnah Hospital, Sheikh Zayed Hospital, Department of Microbiology and Chaumont, J.P, and Senet, J.M. (1978) Antagonistic properties of higher plants against fungal parasites of man from food contaminants: screening of fungi. Plant. Med. Phytother., 1, pp Clark, R.K., and Menory, R.C. (198) Environmental effects or peppermint (Mentha piperita). Australian Journal of Plant Physiology, 7, pp Diaz, R., Quevedo-Sarmiento, J., Ramos-Cormenzana, A., Cabo, P., and Cabo, J. (1988) Phytochemical and antibacterial screening of some species of Spanish Lamiaceae. Fitoterapia, 59, pp El-Mahmood, A.M., Ogbonna, O.B., and Raji, M. (1) The antibacterial activity of Azadarichta indica (neem) seeds extracts against bacterial pathogens associated with eye and ear infections. Journal of Medicinal Plants Research, 4(14), pp Gardiner, P. (). Peppermint (Mentha piperita) [Internet]. Available from: < cis.pdf> [Accessed 8th August 11]. Girish, K., and Shankara, B.S. (8) Neem-A Green Treasure. Electronic Journal of Biology, 4(3), pp Kausik, B., Chattopadhyey, I., Benerjee R.K., and Bandyopdyey, U. () Biological activities and medicinal properties of Neem. Current Science, 8(11), pp Kumar, R.V., and Gupta, V.K. () Thrust on Neem is need of today. Chembiochemistry, 5, pp Available online at 13

6 Hoque, M.M.D., Bari, M.L., Inatsu, Y., Juneja, V.K., and Kawamoto, S. (7) Antibacterial activity of guava (Psidiumguajava L.) and Neem (Azadirachtaindica A. Juss.) extracts against foodborne pathogens and spoilage bacteria. Foodborne Pathogens and Disease, 4(4), pp Mimica, D.N., Bozin, B., Sokovic, M., Mihailovic, B., and Matavulj, M. (3) Antimicrobial and antioxidant activities of three Mentha species essential oils. Planta Medica, 69, pp Rembold, H. (1996) Neem and its general development for pest control. Neem and Environment. New Delhi, Oxford and IBH Publishing Co. Pvt. Ltd. Sandasi, M., Leonard, C.M., Van-Vuuren, S.F., and Viljoen, A.M. (11) Peppermint (Mentha piperita) inhibits microbial biofilms in vitro. South African Journal of Botany, 77(1), pp Samarth, R.M., Panwar, M., Kumar, M., and Kumar, A. (6) Protective effects of Mentha piperita Linn on benzo[a]pyrene-induced lung carcinogenicity and mutagenicity in Swiss albino mice. Mutagenesis, 1(1), pp Saeed, S., Naim, A., and Tariq, P. (6) In vitro antibacterial activity of peppermint. Pakistan Journal of Botany, 38(3), pp Scavroni, J., Boaro, C.S.F., Marques, M.O.M., and Ferreira, L.C. (5) Yield and composition of the essential oil of Mentha piperita (Lamiaceae) grown with bio solid. Brazilian Journal of Plant Physiology, 17(4), pp Tunon, H., Thorsell, W., and Bohlin, L. (1993) Mosquito repelling Activity of Compounds Occurringin Achilleamillefolium. Economic Botany, 48, pp Verkerk, R.H.J., and Wright, D.J. (1993) Biological activity of Neem seed kernel extract and synthetic azadirachtin against larvae of Plutellaxylostellal. Pesticide Science, 37, pp Walker, J.T., and Melin, J.B. (1996) Mentha piperita, Menta spicata and effects of their essential oils on meloidogyne in soil. Journal of Nem atology, 8, pp Available online at 14