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Antibacterial, antisecretory and antihemorrhagic activity of Azadirachta indica used to treat cholera and diarrhea in India

Prarthana Thakurta a , Poulami Bhowmik a , Souryadeep Mukherjee a , Tapas K. Hajra a , Amarendra Patra b , Prasanta K. Bag a,
b a Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, India Department of Chemistry, University of Calcutta, 92 Acharya Prafulla Chandra Road, Kolkata 700009, India

Abstract Indigenous uses of Azadirachta indica A. juss (Maliaceae) (locally known as neem) leaves in different parts of India for curing gastrointestinal disorder such as diarrhea and cholera is wide spread. The objective of the present study was to evaluate the antibacterial and antisecretory activity of neem extract against Vibrio cholerae, a causative agent of watery diarrhea such as cholera. The methanol extract of neem leaf was tested for its antibacterial, antisecretory and antihemorrhagic activity against Vibrio cholerae. Azadirachta indica extract had signicant antibacterial activity against the multi-drug-resistant Vibrio cholerae of serotypes O1, O139 and non-O1, non-O139. The minimum inhibitory concentration reached by 50% (MIC50 ) and 90% (MIC90 ), and minimum bactericidal concentration for the extract were 2.5, >5, and 10 mg/ml, respectively. Neem extract showed antisecretory activity on Vibrio cholerae induced uid secretion in mouse intestine with inhibition values of 27.7%, 41.1%, 43.3%, 57.0%, and 77.9% at doses of 100, 200, 300, 450 and 1800 mg/kg, respectively. Oral administration of the extract inhibited hemorrhage induced by Vibrio cholerae in mouse intestine at a dose 300 mg/kg. The results obtained in this study give some scientic support to the uses of neem employed by the indigenous people in India employed for the treatment of diarrhea and dreadful disease cholera.
Keywords: Antibacterial; Antihemorrhagic; Antisecretory; Azadirachta indica; Cholera; Indigenous

1. Introduction Acute watery diarrhea accounts for 80% of the cases (death account for 50%) in the developing world (Tullock and Richards, 1993). Among the diarrheal diseases, cholera is a serious epidemic disease caused by the gram-negative bacterium Vibrio cholerae (Cholera Working Group, 1993; Nair et al., 1994). Vibrio cholerae, serotypes O1 and O139 has ability to produce an enterotoxin, cholera toxin (CT) that is a major determinant of virulence for cholera. Cholera toxin acts on mucosal cells and decreases the net ow of sodium ion into tissue producing a net ow of chloride (and water) out of tissue and into the lumen. This results in massive diarrhea and electrolyte imbalance. A person with full-blown cholera can lose 20 l of water daily (Lencer et al., 1992). Among the other virulence factors, ElTor hemolysin pro-

Corresponding author. Tel.: +91 33 2461 4981; fax: +91 33 2461 4419. E-mail address: pkbbioc@caluniv.ac.in (P.K. Bag).

duced by Vibrio cholerae is also reportedly a potent toxin with both enterotoxic and cytotoxic activities (Honda and Finkelstein, 1979; Ichinose et al., 1987; Ramamurthy et al., 1993). Treatment with oral rehydration solution (ORS) has reduced the levels of mortality in children and adult by dehydration, but not morbidity for diarrhea (Amstrong and Cohen, 1999; Turvill et al., 2000). Some drugs such as racecadotril and loperamide are used to treat the secretary diarrhea. However, these drugs have side effects such as bronchopasm, vomiting and fever, and loperamide should not be administrated to children below 6 years of age, patients with constipation, and intestinal obstruction (Brown, 1979; Rog et al., 1993; Salazar et al., 2000). e Different antibiotics are used to treat diarrheal patient. However, reports of drug-resistant Vibrio cholerae strains are appearing with increasing frequency (Mukhopadhyay et al., 1996). Emergence of resistance to multiple drugs is a serious clinical problem in the treatment and containment of the disease, as reected by the increase in the fatality rate from 1% to 5.3% after the emergence of drug-resistant strains in Guinea-Bissau

during the 19961997 epidemic of cholera (Dalsgaard et al., 1999). The appearance of nalidixic acid resistance among Vibrio cholerae O1 strains is also a disturbing event. This is probably related to the wide spread use of nalidixic acid and in the treatment of multiply resistant Shigella dysenteriae type I cases in Kolkata (Mukhopadhyay et al., 1996). The therapeutic and prophylactic treatment of cholera with antibiotics viz. tetracycline, noroxacin, etc. has probably contributed to the sporadic appearance of drug resistance strains in different geographical areas. Ethnopharmacology and natural product drug discovery remains a signicant hope in the current target-rich, lead-poor scenario (Patwardhan, 2005). Many modern drugs have origin in traditional medicine and ethnopharmacology. Ayurveda, the science of life, is a comprehensive medical system that has been the traditional system of healthcare in India for more than 5000 years (Mishra et al., 2001). This comprehensive medical system has been prescribed many preparations from Indian medicinal plants. Ayurveda remains a vital system of medicine and drug therapy in India and elsewhere (Borchardt, 2003). In the traditional system of healthcare in India, the uses of medicinal plant to treat diarrheal diseases such as watery diarrhea (atisar) are wide spread (Nautiyal et al., 2000). Leaves of Azadirachta indica A. juss (Maliaceae) (locally known as neem) are used by indigenous people in different parts of India for curing gastrointestinal disorder such as diarrhea and cholera is wide spread (Nayar and Sastry, 1987; Jain, 1991; Maikhuri et al., 2000; Nautiyal et al., 2000; Kala et al., 2004). However, this has not investigated from a pharmacological point of view to demonstrate its antisecretory properties, which could lead to support their use as antidiarrheal and anticholera drug in traditional medicine. The phytochemical analysis and antibacterial activity of neem has been documented previously (Okpaniyi and Ezeukwu, 1981; Chawla et al., 1994; Fabry et al., 1998; Pai et al., 2004). The present study was to evaluate the antibacterial and antisecretory activity of neem leaf extract against Vibrio cholerae that was prescribed by the Indian traditional healthcare system for the treatment of cholera and diarrheal patients. Vibrio cholerae produce the hemolysin which hemolysed the erythrocytes and a potent toxin having contribution for diarrhea. We also investigated in the present study to assess the antihemorrhagic activity of neem against Vibrio cholerae using mice model.

2.2. Crude extract preparation Plant extracts were prepared by macerating air dried neem leaves (500 g) with 2500 ml of methanol in a Soxhlet apparatus for 18 h. The extract was then ltered through Whatman No. 42 lter paper and lyophilized yielding 21.84 g (4.36% yields) for neem leave extract. Lyophilized powder was stored at 20 C. Lyophilized powder was re-suspended in methanol to a concentration of 500 mg/ml and stored as stock sample at 20 C. Test samples were prepared by diluting further with methanol. 2.3. Bacterial strains Among the clinical strains of Vibrio cholerae used in this study, strains NB2 and SG24 belonged to O1 and O139 serotypes, respectively (these strains were kindly provided by Dr. G. Balakrish Nair, NICED, Kolkata, present address ICDDRB, Dhaka, Bangladesh). All these strains were able to produce cholera toxin and hemolysin. The other strains used in this study were Vibrio cholerae belonged to non-O1, nonO139 serotypes (strains PC4, PC9, PC11 and PC14) (these strains were isolated from aquatic environment and identied in our laboratory using API-20E system). These strains were positive for hemolysin production but negative for CT production. 2.4. Antibiotic susceptibility test Antimicrobial susceptibility testing was performed by the disc diffusion method (Bauer et al., 1996) with commercially available disks (HiMedia, Mumbai, India) of ampicillin (10 g); chloramphenicol (30 g); co-trimoxazole (25 g); ciprooxacin (5 g); furazolidone (100 g); gentamicin (10 g); neomycin (30 g); nalidixic acid (30 g); noroxacin (10 g); streptomycin (10 g); tetracycline (30 g). Culture suspensions were obtained after incubation at 37 C in 5 ml MuellerHinton broth (MHB) (HiMedia) for 45 h and spread on MuellerHinton agar (MHA) (HiMedia) at 0.5 McFarland. The plates were incubated at 37 C for 24 h. Isolates were considered susceptible, reduced susceptible, or resistant to a particular antimicrobial agent on the basis of the diameters of the inhibitory zones that matched the criteria of the manufacturers interpretive table, which followed the recommendations of the National Committee for Clinical Laboratory Standards (NCCLS, 2002). The American Type Culture Collection (ATCC) strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used for quality control. 2.5. Antibacterial activity 2.5.1. Screening for antimicrobial activity Antibacterial activity of the extract was determined by agardiffusion assay (Reeves, 1989). Bacterial strains were rst grown in MHB under shaking condition for 4 h at 37 C and after the incubation period 1 ml of culture were spread on MHA. The wells were made using sterile 6 mm cork borer in the inocu-

2. Materials and methods 2.1. Plant material and crude extract preparation Leaves of Azadirachta indica A. juss (Maliaceae) were collected by the authors from local area (Kolkata, West Bengal, India). These leaves were thoroughly washed with distilled water to remove dirt. They were shade dried. The botanical identication of the leaves (with ower) was done by Prof. N.D. Paria (Department of Botany, University of Calcutta, Kolkata, India). The voucher specimen is conserved at Calcutta University Herbarium, Department of Botany, University of Calcutta, India, under the accession number CUH2395.

lated MHA plate. The wells were lled with 200 l of the plants extracts (re-suspended in methanol) and blanks (methanol). The concentrations of extract employed were 10, 25, 50, 100, and 200 mg/ml. Tetracycline (150 g/ml, 200 l) was used as antibacterial positive control and the ATCC strain Escherichia coli ATCC 25922 was included for quality assurance. Zone diameter was measured after 24 h incubation at 37 C. The photograph was taken in Gel documentation system (Vilber Lourmat, France). 2.5.2. Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) MIC and MBC of neem extract were assessed using the broth microdilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS, 1997, 1999). An inoculum of the microorganism was prepared from 24 h MHB cultures and suspensions were adjusted with a turbidity equivalent to that of a 0.5 McFarland standard. Suspensions were further diluted 1:10 in sterile MHB to obtain a nal inoculum of 5 105 CFU/ml. The 96-well round bottom sterile plates were prepared by dispensing 180 l of the inoculated broth into each well. A 20 l aliquot of the plant extract was added. The concentrations of plant extract tested were 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 20, and 40 mg/ml. Dilutions of tetracycline served as positive control, while broth with 20 l of methanol was used as negative control. The ATCC strain Escherichia coli ATCC 25922 was included for quality assurance purposes. Plates were covered and incubated for 24 h in ambient air at 37 C. After incubation, minimum inhibitory concentrations (MIC) were read visually; all wells were plated to nutrient agar (Hi-Media) and incubated. The minimal bactericidal concentration (MBC) was dened as a 99.9% reduction in CFU from the starting inoculum after 24 h incubation interval. 2.6. Toxicity assay BalB/C mice (2225 g body weight) were used in the present study. The animals kept in wire-mesh cages were acclimated to laboratory conditions (12 h dark:12 h light cycles; 24 1 C) and had been free access to food and water ad libitum. Male mice were treated with a dose of 1800 mg/kg of crude extract given orally (PO). The mortality rate within 72 h period was determined. 2.7. Antisecretory and antihemorrhage assay The effect of the extract on uid secretion and hemorrhage in intestine induced by Vibrio cholerae was studied using mouse model. In traditional medicine infusions or decoctions are usually taken three to four times (one cup of plant tea each time) per day when diarrhea occurs. Neem methanolic extract was tested at an oral dose of 450 mg/kg because the used dose is approximately three cups of plant tea which is recommended by Indian people to treat diarrheal diseases (Bhattacharya, 1977). In vivo activity was also tested at oral doses of 100, 200, 300, and 1800 mg/kg. Mice were fasted for 24 h before administration of bacterial inoculums and extract. For the preparation of

bacterial inoculums, Vibrio cholerae strains were grown in MH broth overnight at 37 C under shaking condition. After harvesting by centrifugation, cells were washed with 10 mM phosphate buffered saline (PBS), pH 7.2. The cells were then re-suspended in same buffer and cell numbers are adjusted to 2 109 CFU/ml. Each mouse was fed with 2% NaHCO3 prior to administering either bacterial inoculums or extract or both. Mice were divided into two groups. Mice of each of groups 1 and 2 (n = 2 per group in duplicated) were administered 500 l of bacterial inoculums (2 109 CFU/ml) orally. After 1 h mice were fed either with 200 l of methanol (group 1) or crude extract (group 2; 100, 200, 300, 450, and 1800 mg/kg in 200 l of methanol) orally. Mice were sacriced after 24 h incubation. The uid accumulation and hemolytic activity of Vibrio cholerae in intestine were observed. Photograph was taken, scanned with a Hewlett-Packard ScanJet 2400 scanner, and the image was arranged for the gure and labeled with Adobe Photoshop Version 6. Fluid accumulation ratio (the weight of intestine/rest of the body weight of mouse) was measured and the antisecretory activity was expressed in percentage of inhibition. 2.8. Statistical analysis Values are expressed as mean S.D. Statistical signicance was determined using Students t-test. Values with p < 0.05 were considered signicance. 3. Results and discussion We tested the strains of Vibrio cholerae belonged to serogroups O1, O139 and non-O1, non-O139 to evaluate the antibacterial activity of neem which used in Indian traditional medicine for the treatment of diarrhea and cholera. Antibiotic susceptibility test was performed to conrm their multi-drug resistance patterns. All the Vibrio cholerae strains included in this study showed multi-drug resistance (Table 1). However, crude methanol extract from neem leaves showed inhibitory activity against Vibrio cholerae belonged to serotypes O1, O139 and non-O1, non-O139 by agar-diffusion assay with signicance (p < 0.05) (Table 2 and Fig. 1). Thus the active plant extracts in this study showed antibacterial activity against multiply drug-

Table 1 Antibiotic susceptibility patterns of the strains of Vibrio cholerae used in this study Strain NB2 SG24 PC4 PC9 PC11 PC14 Serotype O1 O139 Non-O1, non-O139 Non-O1, non-O139 Non-O1, non-O139 Non-O1, non-O139 Resistant A, T, Na, Fr, Co, S A, Co, S A A, T, Co A, T, Fr, Na A, T Reduced susceptible T N, Fr S Fr

A, ampicillin; C, chloramphenicol; Co, co-trimoxazole; Cf, ciprooxacin; Fr, furazolidone; G, gentamicin; N, neomycin; Na, nalidixic acid; Nx, noroxacin; S, streptomycin; T, tetracycline.

Table 2 Antibacterial activity of Azadirachta indica leaves extract on Vibrio cholerae O1, O139 and non-O1, non-O139 Strains of Vibrio cholerae Zone of inhibition diameter (mm) Crude methanol extract (200 l)/well 200 mg/ml SG24 (O139) NB2 (O1) PC4(non-O1, non-O139) PC9(non-O1, non-O139) PC11(non-O1, non-O139) PC14(non-O1, non-O139) 16.5 16.7 18.0 15.3 16.7 14.5 1.0 1.2 2.3 1.2 0.8 0.5 100 mg/ml 14.25 14.70 14.80 14.30 14.50 12.70 0.90 0.80 0.40 1.50 1.30 0.30 50 mg/ml 12.75 12.80 12.70 11.20 12.70 10.20 1.90 1.20 1.30 3.00 1.50 0.30 25 mg/ml 10.0 11.0 9.3 9.5 9.7 8.8 1.0 1.5 0.4 1.3 1.0 0.7 10 mg/ml 0.0 0.0 0.0 0.0 0.0 0.0 Control methanol (200 l/well) 0.0 0.0 0.0 0.0 0.0 0.0

Antibacterial activity was expressed in terms of diameter of zone of inhibition (mean S.D., n = 3). p < 0.05 compared to control (methanol) is considered signicant.

Fig. 1. Determination of effect of Azadirachta indica leaf methanol extract on Vibrio cholerae by agar-diffusion assay method. Vibrio cholerae strain PC14 (A) and strain NB2 (B) were spread on MHA. In each case, 200 l of 200, 100, 50, and 25 mg/ml of neem extract (in methanol), 200 and 100 l of methanol were added to the wells 1, 2, 3, 4, c1 and c2, respectively (Section 2).

resistant Vibrio cholerae that is a serious clinical problem in the treatment and containment of the disease. For determination of MIC and MBC for the neem extract against Vibrio cholerae, the concentration ranges tested were from 0.025 to 40 mg/ml. The minimum inhibitory concentration reached by 50% (MIC50 ) and 90% (MIC90 ) of the strains for the extracts were 2.5 and >5 mg/ml, respectively. The minimum bactericidal concentration for the extract was 10 mg/ml. Administration of leaves extract of Azadirachta indica (1800 mg/kg) did not produce any sign of toxicity in mice and none of the mice died. Mice (group 1) which were administered with Vibrio cholerae inoculums at a dose of 1 109 CFU plus 200 l of methanol (vehicle control) orally showed uid accumulation and hemorrhage (Fig. 2) in the intestines. Fluid accumulation ratios in these control experiments were between 0.11 0.01 and 0.16 0.02 (n = 3 for each strain of bacteria). However, it was found that the extract at an oral dose of 450 mg/kg inhibited the uid secretion (57.0% inhibition) and hemorrhage in the intestine induced by the strains of Vibrio cholerae for group 2 mice. In traditional medicine since infusions or decoctions are usually taken three to four times per day when diarrhea occurs, our results can be related with its traditional use because the used dose is approximately two to three cups of plant tea which is recommended by Indian people to treat diarrheal diseases (Bhattacharya, 1977). The

antisecretory activity of extract tested is shown in Table 3. Neem extract showed activity with inhibition values of 27.7%, 41.1%, 43.3%, 57.0%, and 77.9% at doses of 100, 200, 300, 450, and 1800 mg/kg, respectively. Oral administration of the extract inhibited hemorrhage induced by Vibrio cholerae in mouse intestine at a dose 300 mg/kg (visually observed). Fluid secretion and hemorrhage was almost completely inhibited at oral dose of 1800 mg/kg (Fig. 2). These results indicated that methanolic extract was able to signicantly reduce the uid secretion and
Table 3 Antisecretory activity of neem extract on uid secretion induced by Vibrio cholerae in mice intestine Bacterial inoculum (CFU) administered 1 109 1 109 1 109 1 109 1 109 Extract administered at a dose (mg/kg)a 100 200 300 450 1800 % inhibitionb 27.7 41.1 43.3 57.0 77.9 7.8 3.4 1.3 5.9 7.2

a Mouse was orally administered with bacterial inoculum followed by neem extract (Section 2). b The antisecretory activity was expressed as the percentage inhibition (mean S.D., n = 4) compared to control (methanol). p < 0.05 is considered to be signicant.

Fig. 2. Antisecretory and antihemorrhagic effect of Azadirachta indica leaf crude extract on uid secretion and hemolytic activity induced by Vibrio cholerae in intestine of mice: (A) mouse administered with 1 109 CFU of bacterial inoculums plus 200 l of methanol orally; (B) mouse administered with 1 109 CFU of bacterial inoculums and after 1 h followed by inoculation with 200 l of crude neem extract (1800 mg/kg in methanol) orally; (C) mouse administered with 200 l of methanol orally.

hemorrhage induced by the strains of Vibrio cholerae in mice intestines. 4. Conclusion This study showed that methanolic extract of Azadirachta indica leaves was an effective antibacterial and antisecretory agent against Vibrio cholerae, the causative agent of dreadful disease cholera. Additionally, it was also found that neem extract had antihemorrhagic activity. The active extract found in this work may be employed for the treatment of the patient infected with Vibrio cholerae belonged to serotypes O1, O139 and nonO1, non-O139. Emergence of multiply drug-resistant Vibrio cholerae is a serious clinical problem in the treatment and containment of the disease. However, the active leaves extract in this work had bactericidal activity against different strains of multidrug-resistant Vibrio cholerae. This could be suggested that the active Azadirachta indica leaves extract found in our work may be used as potential source to develop novel antimicrobial compound and antisecretory drug useful to treat cholera and diarrheal patients. The results obtained in this study give some scientic support to the indigenous uses of neem in India employed for the treatment of diarrhea and cholera. Further efforts should be directed at investigating the principle in this extract which has these antibacterial, antisecretory and antihemorrhagic activities against Vibrio cholerae.

Acknowledgements The authors are grateful to Prof. N.D. Paria, Department of Botany, University of Calcutta, Kolkata, India, for authentication of the plant materials. This work was supported by grants from Indian Council of Medical Research, New Delhi, India. Fellowship of Prarthana Thakurta was provided by Council for Scientic and Industrial Research, India. References
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