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Antibacterial, antisecretory and antihemorrhagic activity ofAzadirachta 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,∗

a Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkata 700019, Indiab Department of Chemistry, University of Calcutta, 92 Acharya Prafulla Chandra Road, Kolkata 700009, India

bstract

Indigenous uses of Azadirachta indica A. juss (Maliaceae) (locally known as neem) leaves in different parts of India for curing gastrointestinalisorder such as diarrhea and cholera is wide spread. The objective of the present study was to evaluate the antibacterial and antisecretory activityf neem extract against Vibrio cholerae, a causative agent of watery diarrhea such as cholera. The methanol extract of neem leaf was tested for itsntibacterial, antisecretory and antihemorrhagic activity against Vibrio cholerae. Azadirachta indica extract had significant antibacterial activitygainst the multi-drug-resistant Vibrio cholerae of serotypes O1, O139 and non-O1, non-O139. The minimum inhibitory concentration reached by0% (MIC50) and 90% (MIC90), and minimum bactericidal concentration for the extract were 2.5, >5, and 10 mg/ml, respectively. Neem extracthowed antisecretory activity on Vibrio cholerae induced fluid secretion in mouse intestine with inhibition values of 27.7%, 41.1%, 43.3%, 57.0%,

nd 77.9% at doses of 100, 200, 300, 450 and 1800 mg/kg, respectively. Oral administration of the extract inhibited hemorrhage induced by Vibrioholerae in mouse intestine at a dose ≥300 mg/kg. The results obtained in this study give some scientific support to the uses of neem employed byhe indigenous people in India employed for the treatment of diarrhea and dreadful disease cholera.

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eywords: Antibacterial; Antihemorrhagic; Antisecretory; Azadirachta indica;

. Introduction

Acute watery diarrhea accounts for 80% of the cases (deathccount for 50%) in the developing world (Tullock and Richards,993). Among the diarrheal diseases, cholera is a serious epi-emic disease caused by the gram-negative bacterium Vibrioholerae (Cholera Working Group, 1993; Nair et al., 1994). Vib-io cholerae, serotypes O1 and O139 has ability to produce annterotoxin, cholera toxin (CT) that is a major determinant ofirulence for cholera. Cholera toxin acts on mucosal cells andecreases the net flow of sodium ion into tissue producing a netow of chloride (and water) out of tissue and into the lumen. This

esults in massive diarrhea and electrolyte imbalance. A personith full-blown cholera can lose 20 l of water daily (Lencer et al.,992). 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).

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uced by Vibrio cholerae is also reportedly a potent toxin withoth enterotoxic and cytotoxic activities (Honda and Finkelstein,979; Ichinose et al., 1987; Ramamurthy et al., 1993).

Treatment with oral rehydration solution (ORS) has reducedhe levels of mortality in children and adult by dehydration, butot morbidity for diarrhea (Amstrong and Cohen, 1999; Turvillt al., 2000). Some drugs such as racecadotril and loperamide aresed to treat the secretary diarrhea. However, these drugs haveide effects such as bronchopasm, vomiting and fever, and lop-ramide should not be administrated to children below 6 yearsf age, patients with constipation, and intestinal obstructionBrown, 1979; Roge et al., 1993; Salazar et al., 2000).

Different antibiotics are used to treat diarrheal patient.owever, reports of drug-resistant Vibrio cholerae strains are

ppearing with increasing frequency (Mukhopadhyay et al.,

996). Emergence of resistance to multiple drugs is a seriouslinical problem in the treatment and containment of the disease,s reflected by the increase in the fatality rate from 1% to 5.3%fter the emergence of drug-resistant strains in Guinea-Bissau

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uring the 1996–1997 epidemic of cholera (Dalsgaard et al.,999). The appearance of nalidixic acid resistance among Vibrioholerae O1 strains is also a disturbing event. This is proba-ly related to the wide spread use of nalidixic acid and in thereatment of multiply resistant Shigella dysenteriae type I casesn Kolkata (Mukhopadhyay et al., 1996). The therapeutic androphylactic treatment of cholera with antibiotics viz. tetracy-line, norfloxacin, etc. has probably contributed to the sporadicppearance of drug resistance strains in different geographicalreas.

Ethnopharmacology and natural product drug discoveryemains a significant hope in the current target-rich, lead-poorcenario (Patwardhan, 2005). Many modern drugs have originn traditional medicine and ethnopharmacology. Ayurveda, thecience of life, is a comprehensive medical system that has beenhe traditional system of healthcare in India for more than 5000ears (Mishra et al., 2001). This comprehensive medical systemas been prescribed many preparations from Indian medicinallants. Ayurveda remains a vital system of medicine and drugherapy in India and elsewhere (Borchardt, 2003). In the tradi-ional system of healthcare in India, the uses of medicinal plant toreat diarrheal diseases such as watery diarrhea (atisar) are widepread (Nautiyal et al., 2000). Leaves of Azadirachta indica A.uss (Maliaceae) (locally known as neem) are used by indige-ous people in different parts of India for curing gastrointestinalisorder such as diarrhea and cholera is wide spread (Nayarnd Sastry, 1987; Jain, 1991; Maikhuri et al., 2000; Nautiyal etl., 2000; Kala et al., 2004). However, this has not investigatedrom a pharmacological point of view to demonstrate its anti-ecretory properties, which could lead to support their use asntidiarrheal and anticholera drug in traditional medicine. Thehytochemical analysis and antibacterial activity of neem haseen documented previously (Okpaniyi and Ezeukwu, 1981;hawla et al., 1994; Fabry et al., 1998; Pai et al., 2004). Theresent study was to evaluate the antibacterial and antisecre-ory activity of neem leaf extract against Vibrio cholerae thatas prescribed by the Indian traditional healthcare system for

he treatment of cholera and diarrheal patients. Vibrio choleraeroduce the hemolysin which hemolysed the erythrocytes and aotent toxin having contribution for diarrhea. We also investi-ated in the present study to assess the antihemorrhagic activityf neem against Vibrio cholerae using mice model.

. Materials and methods

.1. Plant material and crude extract preparation

Leaves of Azadirachta indica A. juss (Maliaceae) were col-ected by the authors from local area (Kolkata, West Bengal,ndia). These leaves were thoroughly washed with distilled watero remove dirt. They were shade dried. The botanical iden-

ification of the leaves (with flower) was done by Prof. N.D.aria (Department of Botany, University of Calcutta, Kolkata,ndia). The voucher specimen is conserved at Calcutta Univer-ity Herbarium, Department of Botany, University of Calcutta,ndia, under the accession number CUH2395.

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.2. Crude extract preparation

Plant extracts were prepared by macerating air dried neemeaves (500 g) with 2500 ml of methanol in a Soxhlet appara-us for 18 h. The extract was then filtered through Whatmano. 42 filter paper and lyophilized yielding 21.84 g (4.36%ields) for neem leave extract. Lyophilized powder was storedt −20 ◦C. Lyophilized powder was re-suspended in methanolo a concentration of 500 mg/ml and stored as stock sample at

20 ◦C. Test samples were prepared by diluting further withethanol.

.3. Bacterial strains

Among the clinical strains of Vibrio cholerae used in thistudy, strains NB2 and SG24 belonged to O1 and O139erotypes, respectively (these strains were kindly provided byr. G. Balakrish Nair, NICED, Kolkata, present address ICD-RB, Dhaka, Bangladesh). All these strains were able toroduce cholera toxin and hemolysin. The other strains usedn this study were Vibrio cholerae belonged to non-O1, non-139 serotypes (strains PC4, PC9, PC11 and PC14) (these

trains were isolated from aquatic environment and identi-ed in our laboratory using API-20E system). These strainsere positive for hemolysin production but negative for CTroduction.

.4. Antibiotic susceptibility test

Antimicrobial susceptibility testing was performed by theisc diffusion method (Bauer et al., 1996) with commerciallyvailable disks (HiMedia, Mumbai, India) of ampicillin (10 �g);hloramphenicol (30 �g); co-trimoxazole (25 �g); ciprofloxacin5 �g); furazolidone (100 �g); gentamicin (10 �g); neomycin30 �g); nalidixic acid (30 �g); norfloxacin (10 �g); strepto-ycin (10 �g); tetracycline (30 �g). Culture suspensions were

btained after incubation at 37 ◦C in 5 ml Mueller–Hinton brothMHB) (HiMedia) for 4–5 h and spread on Mueller–Hinton agarMHA) (HiMedia) at 0.5 McFarland. The plates were incubatedt 37 ◦C for 24 h. Isolates were considered susceptible, reducedusceptible, or resistant to a particular antimicrobial agent on theasis of the diameters of the inhibitory zones that matched theriteria of the manufacturer’s interpretive table, which followedhe recommendations of the National Committee for Clinicalaboratory Standards (NCCLS, 2002). The American Type Cul-

ure Collection (ATCC) strains Escherichia coli ATCC 25922nd Staphylococcus aureus ATCC 25923 were used for qualityontrol.

.5. Antibacterial activity

.5.1. Screening for antimicrobial activityAntibacterial activity of the extract was determined by agar-

iffusion assay (Reeves, 1989). Bacterial strains were first grownn MHB under shaking condition for 4 h at 37 ◦C and after thencubation period 1 ml of culture were spread on MHA. Theells were made using sterile 6 mm cork borer in the inocu-

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samsrtcactivity against Vibrio cholerae belonged to serotypes O1, O139and non-O1, non-O139 by agar-diffusion assay with significance(p < 0.05) (Table 2 and Fig. 1). Thus the active plant extracts inthis study showed antibacterial activity against multiply drug-

Table 1Antibiotic susceptibility patterns of the strains of Vibrio cholerae used in thisstudy

Strain Serotype Resistant Reducedsusceptible

NB2 O1 A, T, Na, Fr, Co, S –SG24 O139 A, Co, S –PC4 Non-O1, non-O139 A TPC9 Non-O1, non-O139 A, T, Co N, FrPC11 Non-O1, non-O139 A, T, Fr, Na S

ated MHA plate. The wells were filled with 200 �l of the plantsxtracts (re-suspended in methanol) and blanks (methanol).he concentrations of extract employed were 10, 25, 50, 100,nd 200 mg/ml. Tetracycline (150 �g/ml, 200 �l) was used asntibacterial positive control and the ATCC strain Escherichiaoli ATCC 25922 was included for quality assurance. Zoneiameter was measured after 24 h incubation at 37 ◦C. Thehotograph was taken in Gel documentation system (Vilberourmat, France).

.5.2. Determination of minimum inhibitory concentrationMIC) and minimum bactericidal concentration (MBC)

MIC and MBC of neem extract were assessed using the brothicrodilution method recommended by the National Commit-

ee for Clinical Laboratory Standards (NCCLS, 1997, 1999). Annoculum of the microorganism was prepared from 24 h MHBultures and suspensions were adjusted with a turbidity equiv-lent to that of a 0.5 McFarland standard. Suspensions wereurther diluted 1:10 in sterile MHB to obtain a final inoculum of× 105 CFU/ml. The 96-well round bottom sterile plates wererepared by dispensing 180 �l of the inoculated broth into eachell. A 20 �l aliquot of the plant extract was added. The concen-

rations of plant extract tested were 0.025, 0.05, 0.1, 0.25, 0.5,, 2.5, 5, 10, 20, and 40 mg/ml. Dilutions of tetracycline serveds positive control, while broth with 20 �l of methanol was useds negative control. The ATCC strain Escherichia coli ATCC5922 was included for quality assurance purposes. Plates wereovered and incubated for 24 h in ambient air at 37 ◦C. Afterncubation, minimum inhibitory concentrations (MIC) were readisually; all wells were plated to nutrient agar (Hi-Media) andncubated. The minimal bactericidal concentration (MBC) wasefined as a 99.9% reduction in CFU from the starting inoculumfter 24 h incubation interval.

.6. Toxicity assay

BalB/C mice (22–25 g body weight) were used in the presenttudy. The animals kept in wire-mesh cages were acclimatedo laboratory conditions (12 h dark:12 h light cycles; 24 ± 1 ◦C)nd had been free access to food and water ad libitum. Maleice were treated with a dose of 1800 mg/kg of crude extract

iven orally (PO). The mortality rate within 72 h period wasetermined.

.7. Antisecretory and antihemorrhage assay

The effect of the extract on fluid secretion and hemorrhage inntestine induced by Vibrio cholerae was studied using mouse

odel. In traditional medicine infusions or decoctions are usu-lly taken three to four times (one cup of plant tea each time)er day when diarrhea occurs. Neem methanolic extract wasested at an oral dose of 450 mg/kg because the used dose ispproximately three cups of plant tea which is recommended by

ndian people to treat diarrheal diseases (Bhattacharya, 1977).n vivo activity was also tested at oral doses of 100, 200, 300,nd 1800 mg/kg. Mice were fasted for 24 h before administra-ion of bacterial inoculums and extract. For the preparation of

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acterial inoculums, Vibrio cholerae strains were grown in MHroth overnight at 37 ◦C under shaking condition. After harvest-ng by centrifugation, cells were washed with 10 mM phosphateuffered saline (PBS), pH 7.2. The cells were then re-suspendedn same buffer and cell numbers are adjusted to 2 × 109 CFU/ml.ach mouse was fed with 2% NaHCO3 prior to administeringither bacterial inoculums or extract or both. Mice were dividednto two groups. Mice of each of groups 1 and 2 (n = 2 per groupn duplicated) were administered 500 �l of bacterial inoculums2 × 109 CFU/ml) orally. After 1 h mice were fed either with00 �l of methanol (group 1) or crude extract (group 2; 100, 200,00, 450, and 1800 mg/kg in 200 �l of methanol) orally. Miceere sacrificed after 24 h incubation. The fluid accumulation andemolytic activity of Vibrio cholerae in intestine were observed.hotograph was taken, scanned with a Hewlett-Packard ScanJet400 scanner, and the image was arranged for the figure andabeled with Adobe Photoshop Version 6. Fluid accumulationatio (the weight of intestine/rest of the body weight of mouse)as measured and the antisecretory activity was expressed inercentage of inhibition.

.8. Statistical analysis

Values are expressed as mean ± S.D. Statistical significanceas determined using Student’s t-test. Values with p < 0.05 were

onsidered significance.

. Results and discussion

We tested the strains of Vibrio cholerae belonged toerogroups O1, O139 and non-O1, non-O139 to evaluate thentibacterial activity of neem which used in Indian traditionaledicine for the treatment of diarrhea and cholera. Antibiotic

usceptibility test was performed to confirm their multi-drugesistance patterns. All the Vibrio cholerae strains included inhis study showed multi-drug resistance (Table 1). However,rude methanol extract from neem leaves showed inhibitory

C14 Non-O1, non-O139 A, T Fr

, ampicillin; C, chloramphenicol; Co, co-trimoxazole; Cf, ciprofloxacin; Fr,urazolidone; G, gentamicin; N, neomycin; Na, nalidixic acid; Nx, norfloxacin;, streptomycin; T, tetracycline.

Table 2Antibacterial 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 Control methanol(200 �l/well)

200 mg/ml 100 mg/ml 50 mg/ml 25 mg/ml 10 mg/ml

SG24 (O139) 16.5 ± 1.0 14.25 ± 0.90 12.75 ± 1.90 10.0 ± 1.0 0.0 0.0NB2 (O1) 16.7 ± 1.2 14.70 ± 0.80 12.80 ± 1.20 11.0 ± 1.5 0.0 0.0PC4(non-O1, non-O139) 18.0 ± 2.3 14.80 ± 0.40 12.70 ± 1.30 9.3 ± 0.4 0.0 0.0PC9(non-O1, non-O139) 15.3 ± 1.2 14.30 ± 1.50 11.20 ± 3.00 9.5 ± 1.3 0.0 0.0PC11(non-O1, non-O139) 16.7 ± 0.8 14.50 ± 1.30 12.70 ± 1.50 9.7 ± 1.0 0.0 0.0PC14(non-O1, non-O139) 14.5 ± 0.5 12.70 ± 0.30 10.20 ± 0.30 8.8 ± 0.7 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 significant.

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Table 3Antisecretory activity of neem extract on fluid secretion induced by Vibriocholerae in mice intestine

Bacterial inoculum(CFU) administered

Extract administeredat a dose (mg/kg)a

% inhibitionb

1 × 109 100 27.7 ± 7.81 × 109 200 41.1 ± 3.41 × 109 300 43.3 ± 1.31 × 109 450 57.0 ± 5.91 × 109 1800 77.9 ± 7.2

ig. 1. Determination of effect of Azadirachta indica leaf methanol extract on Vtrain NB2 (B) were spread on MHA. In each case, 200 �l of 200, 100, 50, andhe wells 1, 2, 3, 4, c1 and c2, respectively (Section 2).

esistant Vibrio cholerae that is a serious clinical problem in thereatment and containment of the disease.

For determination of MIC and MBC for the neem extractgainst Vibrio cholerae, the concentration ranges tested wererom 0.025 to 40 mg/ml. The minimum inhibitory concentrationeached by 50% (MIC50) and 90% (MIC90) of the strains forhe extracts were 2.5 and >5 mg/ml, respectively. The minimumactericidal concentration for the extract was 10 mg/ml.

Administration of leaves extract of Azadirachta indica1800 mg/kg) did not produce any sign of toxicity in mice andone of the mice died. Mice (group 1) which were admin-stered with Vibrio cholerae inoculums at a dose of 1 × 109

FU plus 200 �l of methanol (vehicle control) orally showeduid accumulation and hemorrhage (Fig. 2) in the intestines.luid accumulation ratios in these control experiments wereetween 0.11 ± 0.01 and 0.16 ± 0.02 (n = 3 for each strain ofacteria). However, it was found that the extract at an oral dosef 450 mg/kg inhibited the fluid secretion (57.0% inhibition)nd hemorrhage in the intestine induced by the strains of Vib-io cholerae for group 2 mice. In traditional medicine sincenfusions or decoctions are usually taken three to four times

er day when diarrhea occurs, our results can be related withts traditional use because the used dose is approximately twoo three cups of plant tea which is recommended by Indianeople to treat diarrheal diseases (Bhattacharya, 1977). The

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cholerae by agar-diffusion assay method. Vibrio cholerae strain PC14 (A) andg/ml of neem extract (in methanol), 200 and 100 �l of methanol were added to

ntisecretory activity of extract tested is shown in Table 3.eem extract showed activity with inhibition values of 27.7%,1.1%, 43.3%, 57.0%, and 77.9% at doses of 100, 200, 300, 450,nd 1800 mg/kg, respectively. Oral administration of the extractnhibited hemorrhage induced by Vibrio cholerae in mouse intes-ine at a dose ≥300 mg/kg (visually observed). Fluid secretion

a Mouse was orally administered with bacterial inoculum followed by neemxtract (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 toe significant.

Fig. 2. Antisecretory and antihemorrhagic effect of Azadirachta indica leaf crude extract on fluid secretion and hemolytic activity induced by Vibrio cholerae ini 9 lums 9

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emorrhage induced by the strains of Vibrio cholerae in micentestines.

. Conclusion

This study showed that methanolic extract of Azadirachtandica leaves was an effective antibacterial and antisecretorygent against Vibrio cholerae, the causative agent of dreadful dis-ase cholera. Additionally, it was also found that neem extractad antihemorrhagic activity. The active extract found in thisork may be employed for the treatment of the patient infectedith Vibrio cholerae belonged to serotypes O1, O139 and non-1, non-O139. Emergence of multiply drug-resistant Vibrio

holerae is a serious clinical problem in the treatment and con-ainment of the disease. However, the active leaves extract in thisork had bactericidal activity against different strains of multi-rug-resistant Vibrio cholerae. This could be suggested that thective Azadirachta indica leaves extract found in our work maye used as potential source to develop novel antimicrobial com-ound and antisecretory drug useful to treat cholera and diarrhealatients. The results obtained in this study give some scientific

upport to the indigenous uses of neem in India employed forhe treatment of diarrhea and cholera. Further efforts should beirected at investigating the principle in this extract which hashese antibacterial, antisecretory and antihemorrhagic activitiesgainst Vibrio cholerae.

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cknowledgements

The authors are grateful to Prof. N.D. Paria, Department ofotany, University of Calcutta, Kolkata, India, for authentica-

ion of the plant materials. This work was supported by grantsrom Indian Council of Medical Research, New Delhi, India.ellowship of Prarthana Thakurta was provided by Council forcientific and Industrial Research, India.

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