PHYTOCHEMICAL SCREENING AND IN VITRO ANTIMICROBIAL ACTIVITY OF BUTEA MONOSPERMA (L) BARK ETHANOLIC AND AQUEOUS EXTRACT

PHYTOCHEMICAL SCREENING AND IN VITRO ANTIMICROBIAL ACTIVITY OF BUTEA MONOSPERMA (L) BARK ETHANOLIC AND AQUEOUS EXTRACT

Pattari Lohitha*, V. Ravi Kiran, K. R. Mohan Babu, K. Nataraj, P. Alankritha Rani, N. Madhavi, M. Chaitanya and N. Divya

Vishnu Institute of Pharmaceutical Education and Research (VIPER) - Vishnupur, Narsapur, Medak Dist., Hyderabad (AP), India

ABSTRACT

Infectious diseases are the second leading cause of death worldwide. Treatment of infections continues to be problematic in modern time because of the severe side effects of some drugs and the growing resistance to antimicrobial agents. Butea monosperma (L) bark ethanolic and aqueous extract has shown good efficacy against Bacillus cereus. Pseudomonas aerugenosa and Escherichia Coli in concentration dependent manner which were grown on MH- Agar medium. 1000mg/ml has shown better zone of inhibition (ZOI) in all the cases of above micro organisms

INTRODUCTION: The use of medicinal plants as a source for relief from illness can be traced back over five millennia to written documents of the early civilization in China, India and the Near east,   but   it   is doubtless an art as old as mankind ¹. Antibiotics are one of our most important weapons in fighting bacterial infections and have greatly benefited the health-related quality of human life since their introduction. However, over the past few decades these health benefits are under threat as many commonly used antibiotics have become less and less effective against certain illnesses not only because many of them produce toxic reactions but also due to emergence of drug-resistant bacteria.  It is essential to investigate newer drugs with lesser resistance ².

The use of herbs and medicinal plant as the first medicines is a universal phenomenon. Every culture on the earth, through written or oral tradition, has relied on the vast variety of natural chemistries found in plants for their therapeutic properties. All drugs from the plant are substances with a particular therapeutic action extracted from plants ³. Butea monosperma (Lam) is a deciduous tree, belongs to family fabaceae, which grows up to 15 m in height and 1.5- 1.8m in girth, with a crooked trunk. Bark is light- brown or bluish grey, yielding a ruby-red vitreous gum. Wood white or yellowish–brown, often becoming grey or grayish- brown, leaves 3- foliolate, large, unequal, 10.2-20.4cm. Flowers are borne in racemes, brilliant orange red, 3.8-5.1cm long, lower calyx-teeth deltoid, pods silvery- white, broad dehiscing by one suture. Seeds are flat, elliptic, reddish- grey, 3.2cm ⁴.

MATERIALS AND METHODS

Plant Material: Bark of Butea monosperma plant was collected from local region of Narsapur, District of Medak, Andhra Pradesh, India in the month of June, 2010. The botanical identity was confirmed by a botanist Prof T. Mohana Department of Botany, Government Mehbubia Junior College, Gunfoundry, Hyderabad. (Reference No: 3/2010)

Preparation of Extracts: 5 Kg of bark was crushed to coarse powder and passed through sieve # 44. The sieved powder was stored in air tight, high density poly ethylene containers before extraction.   Extraction was performed by using soxhlet apparatus (12 hours), carried out first with petroleum ether (60-80 ^oC) to defat the material. The defatted material was then extracted with ethanol to get ethanolic extract and finally with water and small quantity of Chloroform (as 6.5ml/litre) as preservative to get aqueous extract. The aqueous and ethanolic extracts were concentrated for further studies at reduced pressure and temperature in a rotary evaporator and tested for presence of secondary metabolites by different phytochemical tests ⁵.

Preliminary Phytochemical Analysis: The  bark extract  was  screened  for  the  phytochemical  components using the  standard  Method ^{5, 6}. The phytochemical components analyzed were alkaloids, steroids, starch, proteins, anthraquinone glycosides, saponins, flavonoids, tannins, and cardiac glycosides.

Preparation of Bacteria: The bacteria Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Escherichia Coli were purchased from M.T.C.C Institute of Microbial Technology, Chandigarh, India (Invoice No. 9/7/5790). The   ability   of    the   various   extracts to  inhibit  growth  of  clinical bacteria  and  fungi  isolates  was  determined  using  the  Agar disc diffusion method.  Sterile filter paper discs, 11 mm in diameter were impregnated with each extract concentration and dried at 30° C in the static incubator. They were  then  carefully  placed  aseptically with  a  forceps  on  the surface  of  the Mueller-Hinton (MH) agar  plates ⁸ that   were   pre-inoculated   with    the   24 hr culture   of bacteria  and  0.1 ml  spore  suspension  (1 x  10⁵ spores/ml).  The control antibiotics disc containing gentamicin (40 µg/ml) was placed on each of the inoculated plates of nutrient agar. The plates were left on the bench undisturbed for few minutes, after which the bacterial culture plates were incubated    at   37 ^oC    for   24   h. The external  diameters  of  visible  zones  of  growth  inhibition  were  measured  after incubation ⁷.

RESULTS: From the Figure 1 and 2 and table 1 and 2, both ethanolic extract and aqueous extract of Butea monosperma (L) has shown significant effect at 1000mg/ml as compared with the standard gentamicin. It is more effective against Bacillus aureus and E. coli in Concentration dependent manner. From table 3 we can confirm that presence of alkaloids, tannins, phenolics, carbohydrates and saponins in both extracts.

FIGURE 1: INDICATES ZONE OF INHIBITION WITH THE ETHANOLIC EXTRACT

Indicates 125mg/ml of Ethanolic extract

Indicates 250mg/ml of Ethanolic extract

Indicates 500mg/ml of Ethanolic extract

Indicates 1000mg/ml of Ethanolic extract

Indicates 40µg/ml of Gentamicin

FIGURE 2: INDICATES ZONE OF INHIBITION WITH THE AQUEOUS EXTRACT

Indicates 125mg/ml of aqueous extract

Indicates 250mg/ml of aqueous extract

Indicates 500mg/ml of aqueous extract

Indicates 1000mg/ml of aqueous extract

Indicates 40µg/ml of Gentamicin

TABLE 1: PHYTOCHEMICAL SCREENINGS OF BUTEA MONOSPERMA BARK ETHANOLIC EXTRACT

+ indicates presence of active constituents

- indicates absence of active constituents

TABLE 2: DIAMETERS (MM) OF ZONES OF INHIBITIONS PRODUCED BY BUTEA MONOSPERMA (L) BARK ETHANOLIC EXTRACT

TABLE 3:  DIAMETERS (MM) OF ZONES OF INHIBITIONS PRODUCED BY BUTEA MONOSPERMA (L) BARK AQUEOUS EXTRACT

Following pictures indicates Microbial Zone of inhibition of cultured Bacillus cereus at different concentrations of ethanolic extract of Butea monosperma (L).

Following pictures indicating Microbial Zone of inhibition of cultured Escherichia coli at different concentrations of ethanolic extract of Butea monosperma (L).

Following pictures indicating Microbial Zone of inhibition of cultured Pseudomonas aerugenosa at different concentrations of ethanolic extract of Butea monosperma (L).

Following pictures indicates Microbial Zone of inhibition of cultured Bacillus cereus at different concentrations of aqueous extract of Butea monosperma (L).

Following pictures indicates Microbial Zone of inhibition of cultured E. coli at different concentrations of aqueous extract of Butea monosperma (L).

Following pictures indicates Microbial Zone of inhibition of cultured Pseudomonas aerugenosa at different concentrations of aqueous extract of Butea monosperma (L).

DISCUSSION: Plants are important source of potentially useful structures for the development of new chemotherapeutic agents. The first step towards this goal is the in vitro antibacterial activity assay. Many reports are available on the antiviral, antibacterial, antifungal, antihelmintic, antimolluscal and anti-inflammatory properties of plants. Some of these observations have helped in identifying the active principle responsible for such activities and in the developing drugs for the therapeutic use in human beings ¹. However, not many reports are available on the tannins have been traditionally used for protection of  Inflamed surfaces of  the mouth and  treatment  of  catarrha,  wounds, haemorrhoids,  and  diarrhea,  and  as antidote  in  heavy  metal  poisoning. Flavonoids are naturally occurring phenols which possess numerous biological activities including anti-inflammatory, antiallergic, antithrombotic and vasoprotective effects. Cyanogenetic glycosides are reported to possess antimicrobial activity. The observed antimicrobial  activity  against  the  tested organisms  could  be  due  to  the  presence  of tannins  and  cyanogenetic  glycosides  in  the extract  as  these  have  previously  been reported  to  possess  antimicrobial  activities. These could explain the rationale for the use of the plant in the treatment of the various conditions in traditional medical practice ⁹.

CONCLUSION: In conclusion ethanolic and aqueous extract of Butea monosperma (L) bark was assessed in this study.  The results seem to justify their continued use in the treatment of microbial infections. The  inhibition  of  growth  of  the  test  organisms  that  are  known  to  cause  nosocomial infections  and displaying multidrug  resistance  to most  antibiotics  and non-antibiotic antimicrobial  agents  justify  the  continued use of  these plants  in  folk  and  traditional medical  practice.  Further investigation on the isolation and identification of anti microbial component(s) in the plant may lead to chemical entities with potential for clinical use.

ACKNOWLEDGEMENTS: We are grateful to our Principal Dr. V. H. K. Verma, staff members, Director and our honorable chairman Sri. K. V. Vishnu Raju garu  of  Vishnu  Institute of Pharmaceutical  Education  and  Research (VIPER),  for providing us necessary  facilities  to carry out the research project.

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