B-2806 [1-5] Indian J. Anim. Res.,
AGRICULTURAL RESEARCH COMMUNICATION CENTRE
Print ISSN:0367-6722 / Online ISSN:0976-0555
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Comparative evaluation of antibacterial efficacy of plants traditionally used as antidiarrheal against enteropathogenic Escherichia coli Ashish Srivastava* and D.B. Mondal Division of Veterinary Medicine, Indian Veterinary Research Institute, Izatnagar, Bareilly-243 122, Indian Received: 09-09-2014 Accepted: 13-12-2014
DOI:10.5958/0976-0555.2015.00133.8
ABSTRACT The study was conducted to investigate the antibacterial efficacy against enteropathogenic E. coli of plants commonly used to treat calf-diarrhoea. Methanolic extracts of six plants (Aegle marmelos, Curcuma longa, Dalbergia sissoo, Mangifera indica, Psidium guajava and Punica granatum) were screened for their antibacterial property against enteropathogenic E. coli by standard disc diffusion method. Minimum inhibitory concentration (MIC) and of the extract exhibiting highest antibacterial activity was estimated by broth dilution method and minimum bactericidal concentration (MBC) was measured by streaking the contents of MIC tubes on nutrient agar plates. Among the six extracts tested, only extracts of Curcuma longa, Psidium guajava and Punica granatum exhibited antibacterial activity against E. coli. Out of these three, Punica granatum extract was found to be most effective with a mean inhibition zone of 14.67±0.577 mm followed by Psidium guajava (9.67±0.577 mm) and Curcuma longa (8.67±0.577 mm), produced by the disc containing 8.00 mg of respective extract. Minimum inhibitory concentration and minimum bactericidal concentration of the Punica granatum extract were estimated to be 02.00 mg/mL and 03.00 mg/mL respectively. These findings suggest that methanolic extracts of Curcuma longa, Psidium guajava and Punica granatum possess antibacterial activity against enteropathogenic E.coli Keywords: Antibacterial, Curcuma longa, Diarrhoea, Escherichia coli, Punica granatum, Psidium guajava INTRODUCTION Diarrhoea is one of the most common disease complexes of animals, particularly in newborn farm animals like calves under 30 days of age. It is a significant cause of economic loss to cattle industry (Radostits et al.., 2000). Colibacillosis, commonly referred as calf scours, is the principal cause of neonatal calf diarrhoea leading to calf mortality (Haggard, 1985). Regarding the therapy of calf diarrhoea, various antimicrobial agents have been the drug of choice for the clinicians throughout the world. Oral administration of various antibiotics, such as amoxicillin, chlortetracycline, neomycin, oxytetracycline, streptomycin, sulfachloropyridazine, sulfamethazine, and tetracycline have been labelled currently in the United States for the treatment of calf diarrhoea (Constable, 2004). It has been observed that oral administration of antibiotics for prolonged period is potentially detrimental to the intestinal mucosa of the neonatal calves and may result in malabsorption and diarrhoea due to direct modification of the intestinal mucosa (Mero et al.., 1985). Also, modern medicines like the latest generation of
antibiotics due to their high cost are becoming out of reach of common people. The other limiting factors for the utility of these drugs are the associated side effects, problems of drug resistance and non-availability in rural areas. The use of herbal preparations among the rural folks is gaining importance because of their therapeutic value, local availability and cost effectiveness. A large number of plants commonly growing in plains of northern India have traditionally been used by rural people as non specific anti-diarrhoeal agents (Laloo and Hemalatha, 2011). For enteropathogenic E. coli being one of the most common causes of diarrhoea in new born calves, the present study was conducted to comparatively evaluate the antibacterial activity of five most commonly used plants to treat diarrhoea (Aegle marmelos, Dalbergia sissoo, Curcuma longa, Punica granatum, Psidium guajava and Mangifera indica) against enteropathogenic E. coli. MATERIALS AND METHODS Identification of common plant materials and preparation of extracts: Plant materials were selected as per the existing
*Corresponding author’s e-mail:
[email protected] and address: Department of Veterinary Clinical Medicine, Ethics and Jurisprudence, U.P. Pandit Deen Dayal Upadhyaya Veterinary University, Mathura-281 001, India
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scientific literature as well as easy availability of that particular plant in the adjoining locality. Indigenous technical knowledge (ITK) and traditional myths / beliefs were also considered for selection and identification of plant materials. On these bases, six different plants materials were selected for the study viz. semi ripe fruit of Aegle marmelos, leaves of Dalbergia sissoo, rhizome of Curcuma longa, rind of Punica granatum, leaves of Psidium guajava and bark of Mangifera indica. A specimen of these plant materials was deposited in the Botany Department of Bareilly College, Bareilly for proper identification and cataloguing for future reference. The study was conducted at Indian veterinary Research Institute, Izatnagar, Bareilly.
dilution method. For estimating the MIC, varying amount of extract (20.0, 18.0, 16.0, 14.0, 12.0, 10.0, 8.0, 6.0, 4.0, 2.0 and 1.0 mg) was added to 11 different tubes containing 2 ml of Mueller Hinton Broth. To these tubes 10 µl of saline suspension of E. coli whose turbidity is adjusted equivalent to 0.5 McFarland standards was added. A tube containing Mueller Hinton Broth only was seeded with the test organism as described above to serve as control. Tubes were then incubated at 37°C for 24 h. After incubation the tubes were examined for microbial growth by observing for turbidity. The lowest concentration of the extract at which no visible growth (turbidity) could be detected by visual inspection was considered the MIC of the extract.
Selected plant materials were washed thoroughly with distilled water, shade dried and then grinded to a powdered form. Then 50-100 grams of powder was extracted in Soxhlet apparatus with methanol (100 %) as extractant. The extraction process was carried up to 12-15 cycles. The extract thus obtained was dried under reduced pressure to almost powdery consistency.
To determine the MBC, for each test tube in the MIC determination, a loopful of broth was collected and inoculated on sterile nutrient agar. Inoculated plates were then incubated at 37°C for 24 hours. After incubation, the lowest concentration at which no visible bacterial growth was seen was noted as the MBC.
In vitro screening of plant extracts for their antibacterial property against enteropathogenic E.coli: Relevant enteropathogenic strain of E. coli was obtained from the Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar. A standard disc diffusion method (Bauer et al.., 1966) was used for this test. Methanolic extract (Prasanth et al.., 2001) of plant materials were screened for antibacterial property and compared with ciprofloxacin (HiMedia® discs, 6 mm) as a standard antibiotic against enteropathogenic E.coli. Sterile filter paper discs of 6 mm diameter (Hi Media®) were soaked with 20 µl of extract diluted in 1 % tween-20 solution in three different concentrations (100, 200 and 400 mg/ml), so that for each plant extract three discs were impregnated with 2 mg, 4 mg and 8 mg of the extract respectively. All the extracts were filtered by millipore syringe filter prior to use. These discs were dried overnight at 37°C and then applied to the surface of Mueller Hinton Agar (MHA) plates seeded with saline suspension of E. coli whose turbidity is adjusted equivalent to 0.5 McFarland standards. Antibiotic susceptibility disc containing ciprofloxacin was used as control. These plates were then incubated at 37°C for 24 hrs. The antibacterial activity was evaluated by measuring the diameter of inhibition zone. The experiment was conducted in triplicate and mean of the three zones was calculated. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC): MIC of the plant extract exhibiting highest antibacterial activity during in vitro screening was measured using broth
Statistical analysis: Statistical analysis of the data obtained was done by one way analysis of variance (ANOVA) at 95 % level of significance and results are displayed as mean ± standard error of mean using the methods described by Snedecor and Cochran, (1994). RESULTS AND DISCUSSION Among the 6 extracts tested for their antibacterial property, only extracts of Curcuma longa, Psidium guajava and Punica granatum produced inhibition zones against the tested strain of enterotoxigenic E.coli whereas other 3 plant species viz. Aegle marmelos, Dalbergia sissoo and Mangifera indica did not exhibit any antibacterial activity. The maximum mean inhibition zone of antibacterial effect was demonstrated by the methanolic extract of Punica granatum (14.67±0.577 mm), followed by Psidium guajava (9.67±0.577 mm) and Curcuma longa (8.67±0.577 mm) (Table 1). Standard antibiotic i.e. Ciprofloxacin (HiMedia® discs @ 5µg/disc) produced consistent inhibition zone of 34 mm and served as positive control whereas discs impregnated with aqueous tween-20 solution (1%) didn’t exhibit any antibacterial activity in the form of inhibition zone and served as negative control. During in vitro screening of plant extracts for antibacterial property, Punica granatum exhibited maximum zone of inhibition and therefore considered for further evaluation of MIC and MBC. MIC of the Punica granatum extract was calculated by broth dilution method. After 24 hours of incubation, turbidity appeared in tubes containing the extract @ 0.5 and 1.0 mg/ml whereas no growth was
Vol. Issue , () observed in all the other tubes (containing Punica extract @ 2, 3,4,5,6,7,8,9 and 10 mg/ml) on visual inspection. Luxuriant growth was observed in the tube not containing any amount of the extract. On this basis the MIC of Punica granatum extract was considered to be 2 mg/ml. During MBC trial, good growth was observed on nutrient agar plates streaked with the inoculum taken from MIC tubes having P. granatum extract @ 0.5 and 1.0 mg/ml whereas significant reduction in bacterial growth was seen at 2 mg/ml (MIC value). No bacterial growth occurred at concentrations of 3-10 mg/ml. On this basis, MBC of Punica granatum extract was considered to be 3 mg/ml. In another study, Psidium guajava and Punica granatum were demonstrated to have antibacterial activity against E. coli O157:H7 (Voravuthikunchai et al.., 2004). Type of extractant used in the process of extraction may also change the antibacterial property of the extract. Plants which are found to have antimicrobial properties against variety of micro-organisms including enteropathogenic strains of E. coli exhibited much more pronounced activity when methanol or acetone was used as extractant in comparison to ethanol or water (Rani and Khullar, 2004; Eloff, 1998). Based on these observations, all the plant materials used in the present study were extracted with methanol (100%) for further investigation of their antimicrobial property. Some studies have reported antibacterial activity of Aegle marmelos against a variety of enteropathogenic bacteria such as V. cholerae, Salmonella typhi, Shigella spp. and E. coli (Mazumder et al.., 2006; Rani and Khullar, 2004). However, in the present study no antibacterial activity was found in the methanolic extract of semi-ripe fruit of Aegle marmelos. This difference in results could be due to use of different portions of the plant or due to different in vitro methods used in earlier studies. The antibacterial activity of plant extracts may be indicative of the presence of some metabolic toxins or broadspectrum antimicrobial compounds. Punica granatum
possesses high amount of tannin (25%). Antimicrobial property of this substance is well-established (Cowan, 1999). Tannins are water soluble polyphenols having astringent activity. One of the molecular actions of tannins is to complex with proteins. Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesins, enzymes, cell envelope transport proteins etc. (Cowan, 1999). It has also been proposed that tannins may affect the integrity of bacterial cell membranes in a way which is similar to that of synthetic phenols compounds which are commonly used as disinfectants (Scalbert, 1991). Complex formation with metal ions such as iron may be another mechanism. Antimicrobial activity through iron depletion is well known e.g. infection of humans by E. coli is inhibited by the iron chelating lactoferrin present in human milk (Scalbert, 1991). It has earlier been observed that hydro-ethanolic extract of Punica granatum rind had MIC value of more than 1000 µg/ml against E.coli (Holetz et al.., 2002). In the present study, methanolic extract of Punica granatum rind presented a MIC value of 2 mg/ml and a MBC value of 3 mg/ml. Differences in the result of this study and previous studies may be due to several factors. The most important one is in the methodology, as in the present study the concentration difference was of 1mg/ml (MIC tubes had extract @ 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mg/ml). A smaller difference would have provided a more accurate value. Also most other workers have employed the agar dilution technique whereas broth dilution technique has been used here to measure the MIC of the extract. Although, there are several reports which attribute the antimicrobial property of plants to tannins present in them but in the present study no antimicrobial activity was observed in the methanolic extracts of Mangifera indica bark and Dalbergia sissoo leaves which contain significant amount of tannins. Therefore, there is a need to evaluate other phytochemicals also which are present in the plant extracts exhibiting significant antibacterial activity. In a study, antibacterial and antiviral potential of some plant flavonoids including quercetin has been assessed
TABLE 1: In vitro antibacterial activity of plant extracts against enteropathogenic E. coli Plant Aegle marmelos Curcuma longa Dalbergia sissoo Mangifera indica Psidium guajava Punica granatum Ciprofloxacin (5 µg/ disc) Aq. Tween-20 solution (1%)
Zone of inhibition (diameter in mm) Mean ± S.E. 2 mg/disc
4 mg/disc
8 mg/disc
NIL 8.00±0.000aA NIL NIL 9.00±0.000aA 8.33±0.667aA 34.00±0.000 00.00±0.000
NIL 8.33±0.577aA NIL NIL 9.00±0.000aA 11.67±0.333bB
NIL 8.67±0.577aA NIL NIL 9.67±0.577aA 14.67±0.577cB
Values with different superscripts across the row (a, b, c) and different superscripts across the column (A, B) differ significantly at P d” 0.05
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and found effective against E. coli and several other bacteria (Ozcelik et al.., 2006). Since quercetin is a major phytochemical present in the leaves of Psidium guajava (Gutierrez et al.., 2008), whose methanolic extract has been found quite effective against enteropathogenic E. coli in the present study, it may be due to presence of quercetin in it. Antimicrobial and anti-diarrhoeal property of Curcuma longa is well documented. Rhizome part of this plant is used to treat indigestion, diarrhoea including colibacillosis, loss of appetite and colic (Duke et al.., 2000). Antintibacterial activity against various strains of E. coli have been reported in aqueous extract of Curcuma longa which is rich in curcumin (Niamsa and Sittiwet, 2009) as well as in turmeric oil which is a by-product from curcumin manufacture and contains ar-turmerone, turmerone and curlone as active ingredients (Negi et al., 1999). In the present trial, Curcuma longa exhibited less anti-bacterial activity than Punica granatum and Psidium guajava but it is worth noting that its dried methanolic extract was not readily soluble in the selected
solvent (1% Tween-20 in water) and its diffusibility from filter paper disc on Mueller Hinton Agar was apparently less than the extracts of other plants. Overcoming these constraints could have resulted in different picture. As evident from the existing scientific literature, antibacterial activity of different plants tested could not be attributed entirely to presence of any singular chemical constituent. It is need of the hour to further elucidate different components of the crude extract in terms of their mechanism of action and possible interaction responsible for their antibacterial activity. This study tends to validate the use of Punica granatum, Psidium guajava and Curcuma longa as sole or adjunct therapy against colibacillosis in animals. ACKNOWLEDGEMENTS Assistance by the Indian Council of Agricultural Research to the corresponding author in the form of junior research fellowship is duly acknowledged.
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