Vol - 4, Issue - 1, Jan 2013 ISSN: Sharma et al PHARMA SCIENCE MONITOR

Transcription

1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES COMPARATIVE ANTI BACTERIAL ACTIVITY OF LAGENARIA SICERARIA, MOMORDICA CHARANTIA AND CHENOPODIUM AMBROSIOIDES AGAINST CERTAIN HUMAN PATHOGENS Sharma S.*, Upadhyay S., Mistry S., Shukla K., Upadhyay U., Mahajan S. *Asst. Professor BIP, BITS Edu. Campus, Vadodara Mumbai NH-8, Varnama , Gujarat, India. ABSTRACT Petroleum ether, Chloroform, ethanol and water extracts of Lagenaria siceraria, Momordica charantia and Chenopodium ambrosioides were prepared separately and evaluated for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebisella species by cup-plate method (lawn and pour) and Disc diffusion method. Ethanol and Chloroform extracts (100 μ/ml) extracts showed significant activity against the tested microorganism in comparison with the standard Streptomycin and Amikacin. Various tests for physico chemical evaluation and phyto-chemical screening were carried out. Determination of foreign matter, ash value, extractive values, swelling index, foaming index, loss on drying and fluorescence analysis, bulk density and tapped density to assure the purity of drugs were evaluated.the extracts showing the positive activity can be formulated individually or in combination as internal or external dosage forms. In this regard there is great scope for developing new herbal formulation for treating bacterial infections using the above mentioned herbal combination. Further scope exists for research with formulation and development, safety and efficacy. This kind of study can generate more such ideas for re-inviting and using well known herbs in combination to treat more diseases. Keywords: Antibacterial, L. siceraria, M. charantia,c. Ambrosioides. INTRODUCTION Lagenaria siceraria, Momordica charantia and Chenopodium ambrosioides are some of the very common Indian herbs having various medicinal properties for treatment of IC Value

2 different kind of diseases. The aim of the study is to know most effective anti bacterial herb against the selected test organisms. [1] MATERIALS AND METHODS Plant material Lagenaria siceraria, Momordica charantia and Chenopodium ambrosioides were collected from local regions of Ujjain (MP) and were identified from Department of Botany, Vikram University, Ujjain (MP) and voucher specimen (MIPS/L/008/2008), (MIPS/M/009/2008), (MIPS/C/010/2008). Preparation of Extracts Extraction was carried out at room temperature under normal condition. Dried fruits of Lagenaria siceraria, Momordica charantia and leaves of Chenopodium ambrosioides were powdered, sieved and weighed accurately and subjected to extraction in a soxhlet apparatus at room temperature using petroleum ether, chloroform, ethanol and water successively. Before extraction with the next solvent the powder was air dried to remove the traces of adhering solvent. The extracts obtained were filtered, dried and percent yield of extracts were calculated and the dried extracts were stored in air tight container for further studies. Physico Chemical Analysis Physico chemical values such as percentage of ash value and extractive values were determined according to official methods prescribed. Determination of foreign matter,extractable matter, ash value, extractive values, swelling, index, foaming index, loss on drying and fluorescence analysis, bulk density,tapped density, carr s index were carried out to assure the purity of drugs. [2] Preliminary phytochemical screening Preliminary phyto chemical screening was carried out by using standard procedure. The phyto chemical screening showed the presence of alkaloids, glycoside, carbohydrates, saponins, fats and oils, flavonids, tannins and resins in the respective drugs. [3] IC Value

3 Test organisms Bacterial strains of (ATCC No.) i.e S.aureus (ATCC No.2267), E.coli (ATCC No ), P.aeruginosa, Klebsiella spieces and Candida albicans (ATCC No ) were obtained from R.D. Gardi Medical College, Ujjain (MP). Preparation of Inoculum [4] Suspension of organism was prepared as per Mcfarland standard (0.05%) 24 hours old culture (for bacteria) and 48 hours old culture (for fungi). Suspension of organism was made in the sterile isotonic solution of sodium chloride (0.9% w/v) and turbidity was adjusted such that it contains approximately 1x10 3 Cells/ ml. It was obtained by adjusting the optical density of bacteria suspension to that of a solution of 0.05 ml of 1.175% of Barium Chloride and 9.95 ml of 1% sulphuric acid. Preparation of culture media A. Nutrient agar media (NA) Direction Suspend 28.0 gms in 1000 ml distilled water. Heat to boiling to dissolve the media completely. Sterilize by autoclaving at 15 lbs. pressure (121 o C.) for 15 minutes. TABLE 1: INGREDIENT OF NUTRIENT AGAR MEDIA S.No. Ingredients Gms/ltr 1 Peptic digest of animal tissue Nacl Beef extract Yeast extract Agar PH at 25 o C Uses A general culture media which may be used as an enrichment medium by incorporating 10 % blood or other biological fluids. B. Muller Hinton Agar Media (MHA) Direction Suspend 38.0 gms in 1000 ml distilled water. Heat to boiling to dissolve the media completely. Sterilize by autoclaving at 15 lbs. pressure (121 o C.) for 15 minutes. Mix well before pouring. IC Value

4 TABLE 2: INGREDIENT OF MHA S.No. Ingredients Gms/ltr 1 Beef infusion Casein acid hydrolysate Starch Agar PH at 25 o C Uses For cultivation of Neisseria and for determination of susceptibility of microorganism and antimicrobial agents. Preparation of seeded media [5] 100 ml of sterile molten agar media was seeded by organism (about 6 ml according to Mcfarlands standard). In semi hot condition (40 o C) was poured aseptically in sterile petri plate and allowed to solidify at RT (room temperature). Assessment of invitro Antibacterial activity [6] Evaluation of activity was carried out by agar (lawn and pour) method. Antibacterial activity was measured in terms of zone of inhibition (ZOI) and minimum inhibitory concentration. I. Agar well method (lawn method) Preliminary antibacterial activity was studied by agar well method by slight modifications on the solidified agar. Wells of 6 mm diameter were punched with sterile borer. Stock culture was prepared by taking 1x10 5 organisms of 24 hours old culture into 5 ml normal saline. After 24 hours incubation these were firmly swept over the agar plate using sterile cotton swab to make uniform culture lawns. The extracts (10,50,100,200,300, µ/ ml.) were poured in wells and incubated to 18 to 24 hours. Next day these plates were obtained for clear zone around the wells if any. Amikacin and Gentamicin were used as a standard. % inhibition =AIC AIT / AIC x 100 Where, AIC - Area of inhibition of control AIT Area of inhibition of extracts. IC Value

5 Agar well method (pour method) - The medium was prepared by dehydrated media but dissolving in distilled water and subjected to sterilization in autoclave at 121 o for 15 minutes. The petri plates were washed thoroughly and sterilized in hot air over at 160 o C for two hours. 30 ml of sterile molton agar media was seeded by organism (about 2 ml according to Mcf. Standard) in semi hot condition was poured aseptically in sterile petri plates and allowed to solidify at RT. Bores of 6 mm diameter were made on medium using sterile borer and extracts were added to respective bores. Amikacin 30 mg and Gentamicine 10 mg were taken as standards. The petri plates seeded with organism containing extracts and the standard were kept in room temperature for one hour to facilitate the diffusion of extracts and the standards into the media. After diffusion the plates were incubated at 37 o C ( + 1 ) for 24 hours incubation and zone of inhibition was observed. Disc diffusion method A comparison of extract was done with the standard in terms of zone of inhibition. 10,50,100,200,300 µ/ ml solutions of respective extracts were prepared by using DMSO. Paper disc of 6 mm diameter (Wattman Filter paper No. 1) were sterilized impregnated with extracts, were dried and placed on the surface of inoculated petri plates for (MHA plates were used for bacterial isolation and sterilized Plates were inoculated using corresponding broth culture of bacteria) and are pressed down gently to ensure even contact. Whereas Amikacin 30 mg and Gentamycin 10 mg were used as antibacterial standard was similarly placed on respective plates. The plates were kept in refrigerator for two hours to allow diffusion followed by incubation for bacteria 24 hours, (28 o C + 2 o C ). Diameter of zone of inhibition was measured using scale and recorded in table. All above tests were carried out in aseptic environment and average values were recorded. Above tests were carried out in aseptic environment and average values were recorded. RESULTS AND DISCUSSION IC Value

6 The physic chemical evaluation, preliminary phyto chemical screening and anti bacterial activity of Lagenaria siceraria, Momordica charantia and Chenopodium ambrosioides were carried out. Foreign matter was absent in L.siceraria, M.charantia and chenopodium leaves. The results are tabulated in Table No. 3. TABLE 3: FOREIGN MATTER (IN GM) S.No. Name of herbs Quantity taken Foreign Matter (in g) (in g) 1. L.siceraria M.charantia C. ambrosioides All values are mean + S.D. (where n= 3) L.siceraria extractive value with different solvents Alcohol soluble extractive value obtained-4.75%, Water soluble extractive value obtained-6.50%, Petroleum ether extractive value obtained-1.75% Chloroform extractive value obtained-1.75%. M.charantia with different solvents- Alcohol soluble extractive value obtained-3.77%, Water soluble extractive value obtained-6.64%, Petroleum ether extractive value obtained-2.14%, Chloroform extractive value obtained- 5.37%. Chenopodium ambrosioides with different solvents - Alcohol soluble extractive value obtained-8.0%, Water soluble extractive value obtained-15.0%, Petroleum ether extractive value obtained-2.0%, Chloroform extractive value obtained -2.0%. The water soluble extractive value was maximum as compared with other solvent in all of the three extracts. The results are tabulated in Table No IC Value

7 TABLE 4: EXTRACTIVE VALUES OF DIFFERENT EXTRACTS (%W/W) S. No Name of the herbs Pet ether soluble components% Chloroform soluble components% Ethanol soluble components % M.charantia were 6% total ash, 0.4% acid insoluble ash. In the ash value determination various parameters are used (total ash, acid insoluble ash, water soluble ash and sulphated ash. The results of these are tabulated in Table No. 5. All values are in limit as compared with the standard. TABLE 5: ASH VALUES (%W/W) OF DIFFERENT HERBS Water soluble components% 1 L.siceraria M.charantia C.ambrsioides The ash values of L.siceraria were 9% total ash,4% acid insoluble ash.the ash values of S.No. Name of herb Total ash% Water soluble ash% Acid insoluble ash% 1 L.siceraria M.charantia Chenopodium No foam was observed in L.siceraria, foaming index of M.charantia and foaming index of C.ambrosioides was less than 1 cm. The foam in the M.charantia and the C.ambrosioides indicate the presence of saponin. The results are tabulated in Table No IC Value

8 TABLE 6: FOAMING INDEX (IN CMS) OF DIFFERENT HERBS S.No No swelling was observed in L.siceraria, M.charantia and C.ambrosioides Leaves swelling index is 8ml. Swelling in the C.ambrosioides shows the presence of mucilage. Swelling Index of C.ambrosioides was 8 ml. The results are tabulated in Table No. 7. S.No. Height of foam (in cm) M.charantia TABLE 7: SWELLING INDEX (IN ML) OF DIFFERENT HERBS Name of herbs Height of foam (in cm) L.siceraria Height of material before swelling (in ml) Height of foam (in cm) Chenopodium Height of material after swelling (in ml) 1 L.siceraria ±0.0 2 M.charantia C.ambriosioides Loss on drying for L.siceraria 2.5%, M.charantia 2.02%, Chenopodium Ambrosioides 1.3%. The L.siceraria has maximum LOD due to presence of moisture. The results are tabulated in Table No IC Value

9 TABLE 8: LOSS ON DRYING OF DIFFERENT HERBS S.No Name of herbs Quantity taken Loss on drying (in g) (%) 1 L.siceraria ± M.charantia ± C.ambriosioides ±0.02 Fluorescence analysis was observed in L.siceraria and M.charantia but absent in C.ambrosioides. The L.siceraria and the M.charantia powder exhibit fluorescence due to the presence of chromophore. The results are tabulated in Table No.9. S.No. TABLE 9: FLUORESCENCE ANALYSIS OF DIFFERENT HERBS Reagents 1 1 N Aq. NAOH 2 1 N Alc NAOH 3 1 N HCl 4 1 N HNO N H 2 SO 4 Brown 6 NH 4 Brown L.siceraria M.charantia Chenopodium Day UV Day UV Day UV Blackish Dark Dark Black Brown Brown Green Green Green Dark Dark Dark Dark Brown Green Yellow Green Green Green Yellowis Dark Yellow h Brown Green Green Green Green Buff Dark Brown Grey Yellow Green Green Green Dark Dark Yellow Green Grey Green Green Dark Green Yellow ish Brown Green Dark Green Green Yellowis Dark Dark Dark 7 FeCl 3 h Brown Green Brown Green Green Green Dark Dark 8 Picric Acid Brown Grey Green Yellow Green Green Blackish Dark 9 Iodine Grey Yellow Brown Green Green Green Percentage yield of L.siceraria with the Petroleum ether -1.5% Chloroform-3.7%, Ethanol -3.5%, Water-2.8% (magnetic stirrer with hot plate). The percent yield was maximum with water and minimum in pet ether. M.charantia with the Petroleum ether - IC Value

10 4.4%, Chloroform -8.0%, Ethanol -1.4%, Water-14.4% (magnetic stirrer with hot plate). The percent yield was maximum with water and minimum in ethanol. TABLE 10: PERCENTAGE YIELD (W/W) OF DIFFERENT HERBS S.No. Name of herbs % yield (w/w) Pet ether Chloroform Ethanol Water 1 L.siceraria M.charantia C.ambriosioides Physical parameters of L.siceraria, Bulk density 0.46 g/ml, Tapped density 0.61g/ml, Carr s index 24.59% poor flow of the powder, M.charantia Bulk density 0.39 g/ml, Tapped density 0.54g/ml Carr s index % poor flow of the water. Results are tabulated in Table No. 11. TABLE 11: PHYSICAL PARAMETER OF DIFFERENT HERBS S.No. Name of Bulk Density Tapped Carr s Index herbs gm/ml Density gm/ml % 1 L.siceraria M.charantia Screening of extracts (Pet. Ether, Ethanol, Chloroform and water) was carried out in the L.siceraria fatty oil, carbohydrate, proteins and phenols, M.charantia-alkaloid, glycosides, saponin, carbohydrate, proteins, tannin and flavonoids, C.ambrosioides - volatile oils, saponin and glycosides. Results are tabulated in Table No IC Value

11 TABLE 12: PHYTO CHEMICAL SCREENING OF DIFFERENT HERBS Phyto L.siceraria M.charantia C.ambrosioides constituents PE Ch Et Aq PE Ch Et Aq PE Ch Et Aq 1 Alkaloids Carbohydrates Glycosides Saponins Triterpenes Fats & Oils Resin Phenol Tannins Flavonoids Proteins * (+) Present (-) Absent The in-vitro anti bacterial studies were carried out by Disc diffusion and Agar well (lawn and pour) method. Initial screening was done in terms of zone on inhibition (ZOI) and further minimum inhibitory concentration (MIC) determined against test microorganisms. Disc, were pressed down gently to ensure even contact. Disc of standard Amikacin and Gentamycin were similarly placed on respective plates. Diameter of ZOI and MIC was measured using zone reader and recorded in table no 13 &14. All the above tests were carried out in aseptic environment and in triplicate and average values were recorded. TABLE 13: ASSESSMENT OF ANTIBACTERIAL ACTIVITY IN TERMS OF ZONE OF INHIBITION BY DISC DIFFUSION METHOD Bacterial strains Zone of inhibition (mm) L.siceraria (100µ/ml) M.charantia (100µ/ml) Chenopodium (100µ/ml) PE Ch Et Aq PE Ch Et Aq PE Ch Et Aq E.coli S.aureus Pseudomonas Klebsillea Gentamicin 35 * (-) shows no inhibition, mean value of 6 independent experiments, Gentamicin is used for positive control. ND Not Done. IC Value

12 Table 14: Assessment of anti bacterial activity in terms of Minimum inhibitory concentration (MIC) by disc diffusion Bacterial Strain E.coli S.aureus Conc. µ/ml Diameter of zone in mm L.siceraria M.charantia Chenopodium PE Ch Et Aq PE Ch Et Aq PE Ch Et Aq Klebsi-llea Pseudomonas Gentamicin 10 N.D. N.D. N.D. 50 N.D. N.D. N.D Amikac-in 10 N.D. N.D. N.D. 50 N.D. N.D. N.D * (-) shows no inhibition, mean value of 6 independent experiments, Gentamicin and Amikacin is used for positive control. ND Not Done IC Value

13 Figure 1 Comparison of Zone of inhibition of L.Scieraria, M.Charantia, C.ambrosioides with standard drugs against selected microorganisms. Figure 2 Assessment of antibacterial activity by disc diffusion * 1 - Chenopodium ethanol extract 2. Chenopodium pet. extract, 3. Chenopodium Chloroform extract, 4. Chenopodium water extract, G- Gentamycin, A- Amikacin. Pseudomonas aeruginosa, Klebsiella species, Escherichia coli, Streptococcus aureus IC Value

14 Figure 3 Assessment of antibacterial activity by disc diffusion *5. L.siceraria ethanol extract, 6. L.siceraria pet extract, 7. L.siceraria chloroform extract, 8. L.siceraria water extract. Pseudomonas aeruginosa, Klebsiella species, Escherichia coli, Streptococcus aureus. Figure 4 Assessment of antibacterial activity by disc diffusion * 9. M.charantia ethanol extract, 10. M.charantia pet. extract, 11. M.charantia Chloroform extract, 12. M.charantia water extract. Pseudomonas aeruginosa, Klebsiella species, Escherichia coli, Streptococcus aureus IC Value

15 Maximum inhibition was observed in ethanol and chloroform extracts among the other extracts of these drugs. It may be concluded that Lagenaria siceraria, Momordica charantia and Chenopodium ambrosioides were the most effective and potent inhibitor of bacterial growth. Seprate zone of inhibition was studied in case of ethanol and chloroform. The result of comparative study revealed that Chenopodium ambrosiodies was a most effective inhibitor of bacterial growth and Agar well (lawn) method appears to be most effective for studying antibacterial activity [7]. REFERENCES 1. Nayak S.H. Development and evaluation of cosmeceutical hair styling gel of ketoconazole. Indian Journal of Pharmaceutical Science Krishnamurthy D. Herbal Solution for Dandruff. African Journal of Biotechnology 2005; 5 : Quality Control Method for medicinal plant material. Published by the World Health Organization; Nanda U.N. Anti microbial activity of Luwigia octavalvis against selected human dermatological pathogens Roopashri T.S. Anti bacterial Activity of anti psoriatic herbs: Cassia tora, Momordica Charantia and Calendula Officinalis. International Journal of applied research in natural products 2008; I: Jarald E. Anti microbial activity of leaves of Lantana camara and Calotropis Procer. R.B.R., Indian Drugs 2008; 45: Sharma S. Formulation and evaluation of herbal shampoo. M. Pharm Thesis. Rajiv Gandhi Proudyogiki Vishwavidyalaya. Bhopal. (M.P.) For Correspondence: Shalini Sharma shalineesharma29@gmail.com IC Value