International Journal of Drug Development and Research

Keywords

Acacia catechu willd, Anti bacterial evaluation, Mac Farland’s standard, Zone of inhibition, Enteric pathogens.

Introduction

The use of natural products has been one of the most successful strategies for the discovery of new drugs[1]. Natural products have been used for thousands of years in folk medicine; they were believed to be the new source of antimicrobial agents[2]. Global, regional and national estimates clearly place diarrhoeal diseases as a major, albeit a substantially neglected, public health problem. Deaths of children aged<5 years owing to diarrhoea was estimated to be 1.87 million at the global level (uncertainty range from 1.56 to 2.19 million), which is approximately 19% of total child deaths[3]. In the south-east Asian region, almost 48% of the estimated 3.07 million deaths annually are attributed to acute respiratory infections and diarrhoeal diseases with the highest burden of diarrhoeal disease in 5 countries: Bangladesh, India, Indonesia, Myanmar and Nepal where these diseases caused 60,000 deaths annually[4,5].

Diarrhoea is a syndrome that can be caused by different bacterial, viral and parasitic pathogens. Accurate understanding of the cause of diarrhoea in a given setting is an onerous task that requires systematic monitoring of the various pathogens. The availability of a well equipped clinical microbiology laboratory is a prerequisite to undertake such studies. Clinical studies conducted at the National Institute of Cholera and Enteric Diseases (NICED), which includes hospital and community, based surveillance for diarrhoea was focused on common enteric pathogens using conventional assays[6-8].

Acacia catechu Willd (Family: Fabaceae and subfamily: Mimosoideae.) is widely used in Ayurveda for many diseases and mainly for skin diseases[9]. Most of the people in Kerala use boiled Khadira water (karingali water) for drinking purpose.

There are a number of ayurvedic taila (oil) formulations which contain Khadira as one of the active ingredients[10]. Acacia catechu is highly valuable for its powerful astringent and antioxidant activities. It is commonly known as Katha which is an indispensable ingredient of Pan that is beetle leaf preparation chewed in India. It is useful in Dental, oral, throat infections and as an astringent for reducing oozing from chronic ulcers and wounds. The concentrated aqueous extract known as Khair gum or cutch is an astringent, cooling and digestive, beneficial in cough and diarrhea, applied externally to ulcer, boils and skin eruptions and is used extensively in Ayurvedic formulations[11]. The extracts of Acacia catechu exhibits.

various pharmacological effects like antipyretic, antiinflammatory, antidiarrhoeal, hypoglycaemic, hepatoprotective, antioxidant and antimicrobial activities[12,13-24]. Main chemical constituents of Acacia catechu willd are catechin, (-) epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallete rocatechin,phloroglucin,protocatechuic acid, quercetin, poriferasterol glucosides, poriferasterol acyglucosides, lupenone, lupeol, procyanidin AC, kaempferol, dihydrokaemferol, taxifolin, (+)-afzelchin gum and mineral[12,25-30]. The chief phytoconstituents of the heartwood are catechin and epicatechin. Catechins have siginificant antioxidant and antimicrobial effects[31]. It is considered to be the best antioxidant.

Acacia catechu is useful as a topical agent for sore gums and mouth ulcers.The bark of Acacia catechu in combination with other drugs is prescribed for snake bite[32]. Acacia catechu also shows hypotensive effect[33]. The decoction of bark mixed with milk is taken to cure cold and cough[34,35]. The bark is said to be effective against dysentery, diarrhoea and in healing of wounds. The seeds have been reported to have an antibacterial action[36].

Traditional medicines are cheaper, with minimal side effect and safe. To ensure the safety of its products and practices standardization is of vital importance. The knowledge of medicinal plants came from our ancient literature. Due to over and subclinical use of antibiotics lead pathogens to gain resistance against new generation antibiotics. More over in the Indian system of medicine, most herbal practitioners formulate and dispense their own recipes; this requires proper documentation and research[37]. Hence an attempt was carried by us to evaluate the in-vitro Antibacterial activity of Ethanolic Bark extract of Acacia catechu willd against enteric pathogens.

Materials and Methods

Plant material

The Ethanolic Bark extract of Acacia catechu willd was obtained from Mr.R.Rajendran. Green Chem. Herbal Extract & Formulations. Bangalore.

Test microorganisms

Bacterial strains used were Shigella dysentriae [Gram negative bacilli-GNB], E.coli[GNB], Klebsiella pneumoniae[GNB], Vibrio cholerae[GNB], Pseudomonas aeruginosa[GNB],and Staphylococcus aureus,[GPC]. The organisms were obtained from department of Microbiology, Saveetha Dental College and maintained in nutrient agar slope at 4°C

Methodology

The extracts were prepared in the following concentrations in sterile water. 2mg/ml, 4mg/ml and 6mg/ml. 50μl of extract of different concentrations were loaded on sterile filter paper discs measuring 6mm in diameter, so that the concentration of the extract on each disc was 100μg, 200μg and 300 μg respectively. The discs were dried and kept aseptically.

Screening Of Antibacterial Activity [Disc Diffusion Technique]

Broth culture of the bacterial strains compared to Mac Farland’s standard 0.5 was prepared38,39. Lawn culture of the test organisms were made on the Muller Hinton agar [MHA-Hi media M1084] plates using sterile cotton swab and the plates were dried for 15 minutes. Filter paper discs loaded with different concentrations of the extract were placed on the respective plates. The plates were incubated at 37°C overnight and the zone of inhibition of growth was measured in millimeters. Standard antibiotic discs of amoxicillin (30mcg/disc) and Penicillin G (30mcg/disc) were used as positive control. All the tests were done in triplicate to minimize the test error.

Minimum Inhibitory Concentration(MIC)

Macro broth dilution or tube dilution method was done to determine the Minimum inhibitory concentration (MIC) of the extracts. A series of two fold dilution of each extract ranging from 8mg/ml to 0.125mg/ml was made in Muller Hinton broth as specified by National Committee for Clinical Laboratory Standards (NCCLS, 1990). 100μl of standard inoculum of the bacterial strains matched to 0.5 Mac Farland’s standard was seeded into each dilution. Two control tubes were maintained for each test batch. These included antibiotic control (tube containing extract and growth media without inoculum) and organism control (tube containing the growth medium and the inoculum) .The tubes were incubated at 37°C for 24 hours and checked for turbidity. MIC was determined as the highest dilution (that is, lowest concentration) of the extract that showed no visible growth[39-41].

Table 1: Pharmacological Activity of Acacia Catechu Willd.

Minimum Bactericidal Concentration (Mbc)

The MBCs were determined by selecting tubes that showed no growth during MIC determination; a loop full from each tube was sub cultured onto Muller Hinton agar plates and incubated for further 24 hours at 37°C. The least concentration, at which no growth was observed, was noted as the MBC.

Result And Discussion

The antibacterial activity of the Ethanolic bark extract of Acacia catechu willd at different concentrations was screened by disc diffusion technique and the zone of inhibition was measured in mm diameter. The results are given in the Table 2. The minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC] were also determined for the extracts and the results are given in Table 3.

B1-Shigella dysenteriae, B2-E.coli, B3-Vibrio cholerae, B4-Klebsiella pneumoniae, B5-Psuedomonas aerugenosa, B6- Staphylococcus aureus.

Table 2: Anti Bacterial activity of Acacia catechu willd bark Extract. (Disc Diffusion Method)

Table 3: Anti Bacterial Activity of Acacia catechu willd bark Extract. (MIC& MBC)

Fig: 1 Anti Bacterial Activity of Acacia catechu willd Bark extract
B1-Shigella dysenteriae, B2-E.coli, B3-Vibrio cholerae, B4-Klebsiella pneumoniae, B5- Psuedomonas aerugenosa, B6- Staphylococcus aureus

B1-Shigella dysenteriae, B2-E.coli, B3-Vibrio cholerae, B4-Klebsiella pneumoniae,B5- Psuedomonas aerugenosa, B6- Staphylococcus aureus.

Fig: 2 Anti Bacterial Activity of Acacia catechu willd Bark extract (MIC & MBC).

The extract at different concentrations exhibited antibacterial activity against all bacterial strains tested. The ethanolic bark extract exhibited a high degree of activity against the organism tested. The ethanolic bark extract was more effective against Staphylococcus aureus, Shigella dysentriae, and E.coli with a zone of inhibition of 22mm, 20mm and 19 mm diameter (at conc300 μg.) respectively. With Vibrio cholerae and Pseudomonas aeruginosa the zone of inhibition was found to be 17mm (at conc. 300 μg.) Klebsiella pneumoniae showed comparatively less inhibition zone of 14mm diameter (at conc. 200 μg.).

The ethanolic bark extract of Acacia catechu willd was found to have low MIC and MBC values of 1mg/ml & 1mg/ml for Staphylococcus aureus and for Shigella dysenteriae it was 2mg/ml & 2mg/ml. With Pseudomonas aeruginosa, E.coli, and Vibrio cholerae the MIC and MBC value were found to be 4 mg/ml & 4mg/ml and for Klebsiella pneumoniae it was 4mg/ml & 8mg/ml. The lower MIC and MBC value is an indication of high effectiveness of the extract whereas higher MIC and MBC indicates the less effectiveness of the extract.

Conlcusion

Majority of the diarrhoeal patients were infected with more than one pathogen. Polymicriobial infection (20- 50%) seems widespread in many developing countries .This trend in diarrhoea is a clear indication that the source of infection is related to grossly contaminated food and water, affecting the patients from low-income group and those who are living in unhygienic environments. If a vaccine is available against V. cholerae O1, rotavirus and shigellae, the overall hospitalization due to diarrhoea can be considerably reduced. Rotavirus and cholera vaccines are now available as prescription product in India for the first time after a hiatus of 30 years. Not much progress has, however, been made with a Shigella vaccine. It would be interesting to see how these vaccines would ameliorate the burden of enteric infections in settings of diarrhoea in endemic areas all over India.

Hence Our finding suggest that the antibacterial effects of the ethanolic bark extract of Acacia catechu willd, is an indication of its broad spectrum antibacterial potential which may be helpful in the management of enteric infections. However, further studies are necessary to isolate and reveal the active compound(s) contained in the refined extract Acacia catechu willd and to establish the mechanism of action.

Acknowledgement

My Heartfelt thanks to Mr.R.Rajendran CEO of Green Chem Herbal Extracts & Formulations, Bangalore, India for providing us the Ethanolic Bark Ext

ract of Acacia Catchu Willd as a Gift sample to conduct this In vitro

Study.

Conflict of Interest: NIL

Conflict of Interest: NIL

Source of Support: NONE

References

1. Mau JL, Chen CP, Hsieh PC. Antimicrobial effect of extracts from Chinese chive, cinnamon, and corni fructus. J. Agric. Food Chem. 49, 2001, 183-188.
2. Cowan, M.M. Plant Products as Antimicrobial Agents. Clin.Microbiol. Rev 1999. 12(4), 564-582
3. Lewis, K.; Ausubel, F.M. Prospects for Plant- derived Antibacterials. Nat. Biotechnol 2006. 24(12), 1504-1507
4. Bissett NG. Herbal drugs and phytopharmaceuticals. Stuttgart: Med harm CRC Press, 1994:566.
5. Fleming T. PDR for herbal medicines. Montvale, NJ: Medical Economics Company, Inc., 1998.
6. Peirce A. The American Pharmaceutical Association practical guide to natural medicines. New York: William Morrow and Company, Inc., 1999
7. Schilcher H. Phytotherapy in paediatrics: handbook for physicians and pharmacists: with reference to commission E monographs of the Federal Department of Health in Germany: includes 100 commission E monographs and 15 ESCOP monographs. Stuttgart:  medpharm Scientific Publishers, 1997:181.
8. Evans WC, Saponins, cardioactive drugs and other steroids, Trease and Evans Pharmacognosy, 15th ed. London, WB Saunders 2002, page 304
9. Sirtori CR, Aescin: pharmacology, pharmacokinetics and therapeutic profile, Pharmacol Res. 2001 Sep; 44 (3):183 93.
10. Robbers JE, Tyler VE. Tyler's herbs of choice: the therapeutic use of phytomedicinals. New York: Haworth Herbal Press, 1999: x, 287
11. Newall CA, Anderson LA, Phillipson JD. Herbal medicines: a guide for health-care professionals. London: Pharmaceutical Press, 1996: ix, 296.
12. Schulz V, Hansel R, Tyler VE. Rational Phytotherapy: A Physicians' Guide to Herbal Medicine. Berlin: Springer, 1997:306.
13. Connie R.Mahon., George Manuselis., Saunder’s Diagnostic Microbiology 2nd Edition.
14. Collins, CH and Lyne, P.M 1976.Microbiological methods, London, Butterworths and co.288p
15. J.G.Colle., A.G.Faster., B.P.Marmion., A.Simmons. Practical Microbiology [Mackie & Mc Cartney] 14th edition page851 – 852.
16. Betty A.Forbes., Daniel F.Sahm., Alice S.Weissfeld. Bailey & Scott’s Diagnostic Microbiology 11th edition Mosby page 229 – 257