STUDY ON THE ANTIMICROBIAL ACTIVITY OF THE CRUDE EXTRACT OBTAINED FROM THE ROOTS OF PLUMBAGO ZEYLANICA AND EVALUATION OF ITS MICROSPHERES

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1 ISSN: Research Article Journal of Global Trends in Pharmaceutical Sciences Vol.2, Issue 2, pp , April-June 2011 STUDY ON THE ANTIMICROBIAL ACTIVITY OF THE CRUDE EXTRACT OBTAINED FROM THE ROOTS OF PLUMBAGO ZEYLANICA AND EVALUATION OF ITS MICROSPHERES Srujana panga*, Sheema Meenaz shaik Mother Theresa post graduate and research institute of health sciences, Pondicherry, India. *Corresponding Author ABSTRACT The plant Plumbago Zeylanica is native of south East Asia whose roots are known for its significant anti microbial activity. The ethanolic crude root extract is extracted using Soxhlet apparatus and anti microbial assay is performed using disc diffusion method (saboround medium for fungi and Muller Hinton medium for bacteria).the highest zone of inhibition is seen in Vibrio cholerae of bacteria. Percentage inhibition of mycelial growth in fungi is seen in Curvularia lunata. The microspheres obtained using solvent evaporation method and coaservation phase technique from the extract is evaluated. Key words: Plumbago Zeylanica, Anti microbial activity, microspheres, solvent evaporation method and coaservation phase technique. INTRODUCTION: The plant species Plumbago zeylanica (known vernacularlychitraka, Chitramulamu, Tellac hitramulamu, Agnichela, Agnimaala or by its trade or popular names of Lead wortwhite flowered and Ceylon Lead wort ) of the Plumbaginaceae, is distributed as a weed in throughout the tropical and subtropical countries of the world. The family Plumbaginaceae consists of 10 genera and 280 species. The genus Plumbago includes 3 species, namelyplumbago indica L.(P. rosea L.) P. c apensis L., and P. zeylanica L., which are distributed in several parts of India. Among these species Plumbago zeylanica grows all districts of plains in Andhra Pradesh, common, wild or in cultivation due to its 212

2 more therapeutic uses. The natural climatic conditions in Chittoor district of Andhra Pradesh not only famous for world popular piligrim center as a holyshire of the Lord Sri Venkateswara or Balaji is situated intirumala Tirupati but also famous for treasure house of rich medicinal plants and providing more favourable conditions for an extensive growth of P. zeylanica To obtain the crude extract from the roots of Plumbago Zeylanica through Soxhlet apparatus and its conversion to microspheres. To estimate the antimicrobial activity of crude extract obtained from the roots of Plumbago Zeylanica. To evaluate the microspheres prepared from crude extract obtained from Plumbago Zeylanica MATERIALS AND METHODS: COLLECTION OF SAMPLES: Roots of the medicinal plant Plumbago Zeylanica were collected from the local retail shop of vaithialingam modaliyar siddha marutuva kadai at puducherry.the collected plant materials were identified at the Rapinat Herbarium, St.joseph s college, Tiruchirappalli, south india.the roots were shade - dried at room temperature for 10 days. MATERIALS: Glass wares (thoroughly cleaned and dried).sample bottles, beakers, conical flasks, measuring cylinders, pipettes, volumetric flasks (Borosil R Grade) Heating source electrical hot plate Single and triple glass distilled water Packaging materials (for sample collection)- clean LDPE bags Electronics weighing machine Chemicals and reagents (Excel R Grade from Qualigens) Samples METHODOLOGY: ACQUEOUS-ALCOHOLIC EXTRACTION: The dried and powdered plant material (100g) was extracted successively with 600 ml water, ethanol, or methanol with a Soxhlet extractor for 48 hr at temperature not exceeding the boiling point of the solvent. The extracts were filtered through Whatman No.1 filter paper and then concentrated in a vacuum at 40ºC using a rotary evaporator. Each extract was transferred to glass vials and kept at 4ºC before use. MICROSPHERES PREPARATION: PREPARATION OF MICROSPHERES THROUGH COASERVATION PHASE TECHNIQUE: 4 g sodium alginates were dissolved in 40 g of ethyl acetate at room temperature. The organic phase was cooled 213

3 to 4ºC. The aqueous phase was homogenized into the organic phase. This W/O preparation was poured into 680 g of aqueous phase containing 20 %( w/w) of polyoxyethylene sorbitan fattyacid ester (Tween 80) and 7g of sodium chloride and having a temperature of 4ºC. The homogenization was performed and the drug (30%W/W) was dispersed uniformly in aqueous mucilage of sodium alginate (2%W/V) using mechanical stirrer maintaining the speed at rpm.to this dispersion the desired polymer was mixed in suitable proportions and the entire mixture was stirred for 30 minutes. The pellets were formed by dropping the bubble free dispersions through a glass syringe into a gently agitated calcium chloride (5%W/V) solution 100 ml. The gelled pellets were cured for 30 min before being filtered and washed thoroughly with distilled water. They are then oven dried for 6 hr at 60ºC, were collected by filtration. The micro particles were then vacuum dried at room temperature. PREPARATION OF MICROSPHERES THROUGH SOLVENT EVAPORATION: Microspheres were prepared using solvent evaporation technique. Drug (0.500g) and E.C,HPMC(constianta)(1:1) ratio in a beaker magnesium stearate were dispersed into this solution, the mixture was stirred at 10ºC over 20minutes at 750 rpm and then poured drop wise into 100ml liquid paraffin (previously cooled to 10ºC with continuously stirring the mixture was continuously stirred at 35ºC until the solvent was removed (AAP 3 hrs)by evaporation the 20ml of cyclohexane was added to this suspension to dissolve liquid paraffin microspheres were separated by filtration, were washed twice with 60ml cyclohexane and the dried at room temperature. ANTI MICROBIAL ACTIVITY OF CRUDE EXTRACT OF PLUMBAGO ZEYLANICA: PREPARATION OF INOCULUM: Bacterial strains preserved in nutrient agar at 4ºC were revived in nutrient broth (liquid medium) and incubated at 37 ± 1ºC overnight, and the suspensions were checked to provide 105 cfu/ml. ANTIMICROBIAL ASSAY: In vitro antibacterial and antifungal activities of the crude extract of the plant were determined by disc diffusion method respectively Mueller- Hinton medium (agar and broth) was used for culture of bacteria and sabouraud medium (agar and broth) was used for culture of fungi. Aqueous ethanolic solution (5%) of the crude extract was used 214

4 as the test antimicrobial agent against 6 human pathogenic bacteria and 3 phytopathogenic fungi. The results were compared with the standard antibacterial antibiotic ampicillin (20µg/disc, and antifungal antibiotic nystatin (100µg/ml) medium. Antimicrobial activity of the crude extract from P.Zeylanica against the pathogenic bacteria is summarized in Table using the disc diffusion method. RESULTS AND DISCUSSION: ANTIMICROBIAL ACTIVITY: Ethanolic extract of Plumbago Zeylanica root was investigated for its antimicrobial activities against 6 human pathogenic bacteria and 3 phytopathogenic fungi using diffusion method respectively. The extract exhibited good antibacterial and antifungal activities against the test organisms. ANTIBACTERIAL ACTIVITY: Antimicrobial activity of the crude extract from P.Zeylanica against the pathogenic bacteria is summarized in Table 1.Using the disc diffusion method, The extract exhibited zone of inhibitions ranged from 5 to 16 mm in diameter with 250 µg/disc and mm in diameter with 500 µg/disc concentration against the test bacteria. The highest zone of inhibition (27 mm) was recorded against Vibrio cholera. The extract at a concentration 500µg/disc showed larger zone of inhibition as compared to that formed by the standard ampicillin disk. It was apparent that the crude extract was equally active against Gram-positive and Gram-negative bacteria, or enteric and nonenteric pathogens. Among the test bacteria, vibrio cholerae was found to be the most sensitive to the extract showing the highest diameter of zone of inhibition.the extract was also very effective against Escherichia coli and Bacillus subtilitis. Table-1: Anti bacterial activity of crude extract from Plumbago Zeylanica 215

5 ANTIFUNGAL ACTIVITY: Antifungal activity of the crude extract against 3 phytopathogenic fungi was studied and the results were compared to that of standard antifungal antibiotic nystatin. The results of the inhibition of fungal radial mycelial growth are summarized in Table 2. It appeared that the crude extract of P.Zeylanica inhibited radial mycelial growth of all the test fungi at a concentration of 100 µg/ml medium to varying degrees that ranged from about 17% in case of Macrophomina phaseolina and 60% in case of curvularia lunata. This indicates that root extract of P.Zeylanica could be used as an eco-friendly antifungal agent in the control of fungal diseases. The extract could also be used by farmers for the control of seedborne fungal pathogens. Among the phytopathogenic 3 fungi tested, Curvularia lunata exhibited the highest sensitivity to the extract, which was followed next by Fusariumequiseti and Macrophomina phaseolina. The root extract from P.Zeylanica seems promising since it showed both antibacterial and antifungal activities. 216

6 MICROSPHERES BY COASERVATION PHASE TECHNIQUE AND SOLVENT EVAPORATION METHOD: The microspheres were prepared in an Smaller particle can be prepared by environment free from organic solvents by dropping a mixtures of colloidal copolymer dispersion drug Plumbago Zeylanica and adjusting the height of the syringe from the level of counter ion solution, compression force on the plunger of the syringe. The mucilage of sodium alginate in calcium gelled particles were cured to get chloride solution, which acted as a sufficiently hardened and then filtered and counterion. The droplets instantaneously dried. The colloidal polymer particles fused formed gelled spherical beads due to cross linking of calcium ion with the sodium ion into the polymer matrix during drying with the drug being dispersed in the latex at room which remained ionized in the solution. temperature.microspheres were prepared 217

7 using solvent evaporation technique were separated by filtration. SEM RESULTS: Particle size of particles was determined by scanning electron microscopy(sem) Sizes were determined with 9 parts of samples for each product. Each part at an average is made of 10 particles and mean the particle size of the 10 particles is given in micro meters.(µm) 218

8 CONCLUSION: Among the test bacteria, vibrio cholerae was found to be the most sensitive to the extract showing the highest diameter of zone of inhibition. The extract was also very effective against Escherichia coli and Bacillus subtilitis. Here particles developed by solvent evaporation reveal highly uniform structure. Among the ACKNOWLEDGEMENT: phytopathogenic 3 fungi tested, Curvularia lunata exhibited the highest sensitivity to the extract, which was followed next by Fusariumequiseti and Macrophomina phaseolina. The root extract from P.Zeylanica seems promising since it showed both antibacterial and antifungal activities. I am thankful to the Dr.K.V.RAMAN, M.B.B.S Dean of MTPG&RIHS, Puducherry for providing necessary facilities and assistance. I consider it a privilege to record me respect and indebtedness to siddha research center for providing us information regarding the plant sources and its availability. 219

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