ANTI-MICROBIAL ACTIVITY OF THE CRUDE DRUGS AND THE POLYHERBAL FORMULATION (RVSPHF567) BY STANDARDIZED CUP AND PLATE METHOD C.S. Kandasamy 1,2*, Suman Nath 2, P. Arulraj 1,2, V. Gopal 3, P. Muthusamy 4, R. Venkatanarayanan 2. 1 SASTRA University, Tanjore. 2 R.V.S College of Pharmaceutical Sciences, Sulur, Coimbatore. 3 College of Pharmacy, Mother Theresa Post Graduate and Research Institute of Health Sciences, Puducherry. 4 Department of Pharmacognosy, College of Pharmacy, Madras Medical College, Chennai. ABSTRACT Antimicrobial study of crude drugs (Ajowan, Cardamom, Cumin, Mace, Clove, and Nutmeg) and the Polyherbal formulation RVSPHF567 was assayed by standardized cup and plate method by using bacterial and fungal culture. The microorganisms used are Aspergillus niger (fungus), Streptococcus aureus (G +ve bacteria) and Escherchia coli (G -ve bacteria). These microbes were cultured in a suitable nutritional media for their growth. The extractions of crude drugs were achieved by dissolving their powdered drugs in the suitable solvent. The solvents used for extracting crude drugs were alcohol and water, the solvent used for the preparation of the Polyherbal formulation RVSPHF567 was red tender coconut water. These extracted crude drugs and the Polyherbal formulation RVSPHF567 were then transferred to the pits on the solidified agar media, previously incubated with the different micro-organism separately. Then the plates were streaked with bacterial cultures and kept in an incubator at 37 o C for 24hrs for growth of bacterial cultures, and plates streaked with fungal culture were incubated at 25 C for 24hrs. After the incubation period the growth of microbes and their zone of inhibition surrounding the extracts of the crude drugs and the Polyherbal formulation RVSPHF567 were observed and measured. All the crude drugs and the Polyherbal formulation RVSPHF567 were found to be specifically effective against bacterial and fungal strain. Ajowan, Cardamom, Clove, Cumin, and Mace is highly active against Aspergillus niger. Nutmeg is highly active against Streptococcus, Polyherbal formulation RVSPHF567 is highly active against all the three organisms namely Aspergillus niger, Streptococcus aureus, Escherchia coli. KEY WORDS Antimicrobial activity, Crude drug extraction, Zone of Inhibition, Culture Media, RVSPHF567. 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 1. Whereas these side effects can be minimized by using natural crude drugs. A successful chemotherapeutic agent must have selective toxicity. It must kill or inhibit the microbial pathogen while damaging the host as little as possible. The degree of selective toxicity may be expressed in following terms. 1. The therapeutic dose, the drug level required for clinical treatment of a particular infection. ISSN : 0975-9492 189
2. The toxic dose, the drug level, at which the agent becomes too toxic for the host. 3. The therapeutic index is the ratio of the dose to the therapeutic dose. The larger the therapeutic index, the better the chemotherapeutic agent. Drugs vary considerably in their range of antimicrobial effectiveness 2. The antimicrobial activities of the crude drugs were determined by Cup and Plate method. The antimicrobial activity was determined against bacteria and fungus by taking 3 different dilution 3. In recent years antimicrobial properties of Indian medicinal plants have been increasingly reported. However a majority of traditionally used Indian medicinal plants have not yet been systematically screened against various microbes. In the present study, we have selected 6 crude drugs; these drugs are used in Indian traditional system of medicine against various ailments including infectious diseases 4. There is an increasing need for search of new compounds with antimicrobial activity as the treatment of microbial infection with the available drugs is often unsatisfactory due to the problem of microbial latency and conflicting efficacy in recurrent infection in immunocompromised patient. Herbal plants are used as medicine for asthma, vomiting, hypoglycemia, and bilious matter phlegmatic rheumatism and anti-mitotic, antioxidant and antitumor 5. MATERIALS AND METHODS The crude drugs were powdered well by grinding and avoiding the moisture contamination. These powdered drug passed through sieve for obtaining uniform particle size Then these powdered crude drugs was dissolved in several solution to determine their optimum solubility. The powdered drugs were dissolved in their appropriate solvent. Table No. I & II ANTIMICROBIAL STUDIES Microorganisms used Aspergillus niger (Fungus) Streptococcus aureus (G +ve ) Escherchia coli (G -ve ) Muller Hinton agar petri plates were swabbed with broth culture of each test bacteria is separate plate by sterile swab. Over this prepared antimicrobial disc were placed under aseptic conditions. Discs of each extract were placed in triangles. Control sets with standard sets were simultaneously maintained. The plates were then incubated at 37 0 c for 24 hours and zone of inhibition was measured and recorded 6. Ingredients of the medium Peptone - 5gm L -1 Sodium chloride - 5gm L -1 Beef extract - 1.5gm L -1 Yeast extracts - 1.5gm L -1 ph (at 25ºC) - 7.2 ± 0.2 EXPERIMENTAL DESIGN All the ingredients were dissolved in Milli-Q purified water. The prepared medium was sterilized by autoclaving at 15 pound pressure for 15 minutes at 121ºC. The Petri dishes were sterilized by means of hot air oven at 110ºC for 1hr. Agar gelled nutrient was poured into separate Petri- dishes and allowed to solidified, holes about 8 mm in diameter are cut in the medium with cork borer. ISSN : 0975-9492 190
Crude drugs were dissolved in several solvent(s)[table I]. Sample was prepared by dissolving crude drugs with their respective solvent [Table II]. Each sample of different dilution (Dil-I, Dil-II & Dil-III) along with the solvent control are transferred to the pits which made on the solidified agar media which is previously incubated with the different micro organism separately. The microbial cultures are obtained from Department of Biotechnology, R.V.S College of Pharmaceutical Sciences, Sulur, Coimbatore. The plates that streaked with bacterial cultures were kept in an incubator at 37 o C for 24hr for growth of bacterial cultures, and plates streaked with fungal culture were incubated at 25 C. After the incubation period, the zone of inhibitions were measured and tabulated. RESULTS & DISCUSSION The Antimicrobial study of the extract of crude drugs and Polyherbal formulation (RVSPHF567) were studied using cup and plate method (Figure-1) with following strains i.e., Streptococcus aureus, Escherichia coli and Aspergillus niger. This shows that they possess antimicrobial activity. The zone of inhibition had been observed in the nutrient agar media (Table 3). Ajowan, Cardamom, Clove, Cumin and Mace are highly active against Aspergillus niger. Nutmeg is highly active against Streptococcus, but Ajowan and mace is less active against Streptococcus aureus. Cardamom, Clove, Nutmeg and Cumin is less active against Escherichia coli. Whereas the Polyherbal formulation (RVSPHF567) is found to be highly active against all the three organisms namely Aspergillus niger, Streptococcus aureus, Escherchia coli. CONCLUSION The antimicrobial activity of each crude drug and the Polyherbal formulation is specific and used wisely for their therapeutic uses. All the crude drugs and the Polyherbal formulation (RVSPHF567) can inhibit the growth of microbes to a certain limit by producing zone of inhibition. ACKNOWLDGEMENT The authors are sincerely thankful to all those who helped us for carrying out the antimicrobial evaluation of the crude drugs and the polyherbal formulation (RVSPHF567). REFERENCES: [1] Nilesh Gawade, Supriya kore, Rachna Pandey, Madhukar B. Khetmalas, (2010), Antibacterial Activity of Coriandrum sativum (seeds) Against Drug Resistant Human Pathogenic Bacteria, Herbal Tech Industry, P. No. 19. [2] N.K Jain, Practical Microbiology, 5 th edition, P. No 33 [3] Synthesis Of Some New Thiophene Derivatives And Evaluation For Their Antimicrobial And Antitubercular Activities. Indian Drugs 2009 Vol 46(11) P. No. - 51 [4] Archana N. Sah, Neha Karki, Neelakshi Singh (July 2010) Antibacterial Activity of Some Traditionally Used Medicinal Plants, Herbal Tech Industry P. No 07 [5] Ramya S, Rajasekaran R, Muthuselvam M, Senthil kumar S (2010) Studies on Phytochemical Screening and Antimicrobial Activity of Solanum trilobatum Linn. Herbal Tech Industry. P. No. 08 [6] Senthilkumar R, Rajasekaran R, (March 2010) Antibacterial Effect of Sollanum Nigrum Linn. Herbal Tech Industry. P. No. 12-14 ISSN : 0975-9492 191
Table I SOLUBILITY OF THE CRUDE DRUGS IN DIFFERENT SOLVENTS DRUGS ALCOHOL BENZENE CHLORO-FORM WATER AJOWAN CARDAMOM CLOVE CUMIN MACE NUTMEG DRUGS CARBON TETRA CHLORIDE DICHLORO- ETHANE DICHLORO-METHANE AJOWAN CARDAMOM CLOVE CUMIN MACE NUTMEG ISSN : 0975-9492 192
Table - II EXTRACTS OF CRUDE DRUGS USED FOR HPTLC ANALYSIS: SAMPLE NO : CRUDE DRUGS SOLVENT EXTRACT 1 AJOWAN Water Extract 2 CARDAMOM Alcoholic Extract 3 CLOVE Water Extract 4 CUMIN Alcoholic Extract 5 MACE Alcoholic Extract 6 NUTMEG Water Extract Table - III ANTIMICROBIAL STUDY REPORTS S. NO Name of the Drug Microorganism Streaked 1 Ajowan 2 Cardamom 3 Clove 4 Cumin 5 Mace 6 Nutmeg 7 Polyherbal Formulation Zone of inhibition(in cm) Dil-I Dil-II Dil-III Control E- coli 1.0 0.6 0.5 0.0 Streptococcus 0.8 0.6 0.0 0.0 Aspergillus niger 1.2 0.7 0.5 0.0 E- coli 0.9 0.6 0.6 0.6 Streptococcus 1.0 0.8 0.6 0.6 Aspergillus niger 1.1 0.7 0.6 0.6 E- coli 0.9 0.7 0.6 0.0 Streptococcus 1.3 0.6 0.0 0.0 Aspergillus niger 1.7 0.9 0.6 0.0 E- coli 0.8 0.7 0.6 0.6 Streptococcus 0.9 0.8 0.7 0.6 Aspergillus niger 1.5 0.8 0.8 0.6 E- coli 1.0 0.8 0.6 0.0 Streptococcus 0.8 0.7 0.5 0.0 Aspergillus niger 1.2 1.1 0.8 0.0 E- coli 1.0 0.8 0.7 0.6 Streptococcus 1.5 1.4 1.2 1.0 Aspergillus niger 1.3 0.9 0.8 0.7 E- coli 0.7 0.6 0.5 0.2 Streptococcus 0.8 0.6 0.4 0.3 Aspergillus niger 1.2 0.9 0.7 0.6 ISSN : 0975-9492 193
FIGURE I ANTIMICROBIAL STUDIES BY CUP AND PLATE METHOD ISSN : 0975-9492 194
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