Oxalis corniculata (Oxalidaceae) Leaf Extract Exerts In Vitro Antimicrobial and In Vivo Anticolonizing Activities Against Shigella dysenteriae 1 (NT4907) and Shigella flexneri 2a (2457T) in Induced Diarrhea in Suckling Mice

JOURNAL OF MEDICINAL FOOD J Med Food 16 (9) 2013, 1–9 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2012.2710

FULL COMMUNICATION

Oxalis corniculata (Oxalidaceae) Leaf Extract Exerts In Vitro Antimicrobial and In Vivo Anticolonizing Activities Against Shigella dysenteriae 1 (NT4907) and Shigella flexneri 2a (2457T) in Induced Diarrhea in Suckling Mice Sayani Mukherjee,1 Hemanta Koley,2 Soumik Barman,2 Soma Mitra,2 Sanjukta Datta,3 Santinath Ghosh,3 Debjyoti Paul,1 and Pubali Dhar1 1

Food and Nutrition Division, Laboratory of Food Science and Technology, University of Calcutta, Kolkata, India. 2 Division of Bacteriology, National Institute of Cholera and Enteric Disease (NICED), Kolkata, India. 3 Department of Chemical Technology, University of Calcutta, Kolkata, India. ABSTRACT In this study, the extract of a green leafy vegetable Oxalis corniculata (Oxalidaceae) was evaluated for its in vitro antibacterial and in vivo anti colonizing effect against common intestinal pathogenic bacteria. Methanolic extract (80%) of Oxalis corniculata (Oxalidaceae) leaf contained a polyphenol content of 910 mg Gallic acid equivalent per gram of dry weight and the yield was 8%. The flavonoid content was 2.353 g quercetin equivalent per 100 g of the extract. In vitro studies indicated that the extract inhibited numerous pathogenic bacteria like Staphylococcus aureus (ATCC 25922), Escherichia coli (ATCC 25923), Shigella dysenteriae 1 (NT4907), Shigella flexneri 2a (2457T), Shigella boydii 4 (BCH612), and Shigella sonnie phase I (IDH00968). The minimum inhibitory concentration (MIC) against E. coli (ATCC 25923) was minimal (0.08 mg/mL), whereas MIC against S. flexneri 2a (2457T) was higher (0.13 mg/mL). A suckling mouse model was developed which involved challenging the mice intragastrically with S. flexneri 2a (2457T) and S. dysenteriae 1 (NT4907) to study the anticolonization activity. It was revealed that the extract was more potent against S. dysenteriae 1 (NT4907) as compared to S. flexneri 2a (2457T). It was also found that simultaneous administration of extract along with bacterial inoculums promoted good anticolonization activity. Significant activity was observed even when treated after 3 h of bacterial inoculation. KEY WORDS:  anti-bacterial  anti-colonizing  flavonoids  Oxalis corniculata (Oxalidaceae)  polyphenols

of bacteria is perpetual among the flavonoids, which are atoxic or less toxic. Over the years, several deleterious outbreaks by the bacterial enteric pathogens viz., Vibrio cholerae, Salmonella spp. and Shigella spp. have been reported.5 The recent increase in antibiotic resistant strains and antibiotic complications have lead to an urgent necessity in finding alternative medicine remedies. India is blessed with diverse climatic conditions which enable the growth of a wide variety of plants. More than 13,000 plants are considered to possess miscellaneous pharmacological properties.6 Oxalis corniculata Linn. (Oxalidaceae) is consumed as green leafy vegetable in India. Traditionally this plant has been characterized by multifarious therapeutic applications.7 Additionally, use of Oxalis corniculata (Oxalidaceae) has been in practice by various other ethnic population (Table 1).8–17 Our study evaluates the in vitro anti-bacterial effects of extracts from the leaves of Oxalis corniculata (Oxalidaceae) against different strains of intestinal pathogenic drug resistant shigellosis causing bacteria and in vivo anti-colonizing effect in a suckling mouse model. In vivo anti-colonizing effect of extract from Oxalis corniculata (Oxalidaceae) in

INTRODUCTION

S

higella species are invasive organisms that clearly present a serious challenge for providing effectual antimicrobial therapy. They have increasingly become resistant to most of the widely used and inexpensive antimicrobials.1 Extensive studies have been done on the antimicrobial properties of polyphenols on the pathogenic intestinal bacteria.2 One of the largest class of naturally occurring polyphenolic compounds are the flavonoids. More than 4000 flavonoid compounds have been identified and categorised according to chemical configuration.3 The flavonoids may be classified into seven types: flavones, flavonols, flavonones, chalcones, xanthones, isoflavones, and biflavones. A number of flavones, flavonols, flavanones, and isoflavones, as well as some of their methoxy, isoprenyl, and acylated derivatives, show antibacterial activity.4 Presently, the search for naturally occurring compounds, active against pathogenic strains Manuscript received 4 December 2012. Revision accepted 24 May 2013. Address correspondence to: Pubali Dhar, PhD, Food and Nutrition Division, Laboratory of Food Science and Technology, University of Calcutta, 20B Judges Court Road, Kolkata 700027, West Bengal, India. E-mail: [email protected]

1

2

MUKHERJEE ET AL. Table 1. Medicinal Uses of Oxalis Corniculata (Oxalidaceae)

Native population

Region

Medicinal uses

Mullu Kurama Tribes Wayanad District, Kerala, India Konda Reddi Tribes and Koyas Tribes Khammam District, Andhrapradesh, India Marma Tribes Bangladesh Village Communities Dir Kohistan Valley NWFP, Pakistan Karens Tribes Andaman, India Tamang Tribes Kabhrepalanchok District, Nepal Apatani Tribes Native People

Eastern Himalayan, India Philippines, South East Asia

Ethnic and Rural People

Eastern Sikkim Himalayan Region, India

Ethnic People

West and South district of Tripura, India

suckling mice model has been explored using this approach to gain insights into its possible mechanism(s) of antidiarrhoeal activity. MATERIALS AND METHODS Bacterial strains Shigella dysenteriae 1 (NT4907), Shigella flexneri 2a (2457T), Shigella boydii 4 (BCH612), Shigella sonnie phase I (IDH00968) were used in our present studies. The strains were obtained from National Institute of Cholera and other Enteric Disease (NICED), Kolkata, India. All the strains were cultured in Trypticase soy broth (TSB). Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used as control strains. Bacterial media, and other chemicals TSB, Muller hinton agar (MHA), Hektoen enteric agar (HEA), and agar powder were purchased from HIMEDIA, HIMEDIA laboratories Pvt. Ltd., Vadhani Ind.Est, LBS Marg, Mumbai, India. Antibiotic discs, powder were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Folin and Ciocalteau’s Phenol Reagent AR, dimethyl sulfoxide, methanol extrapure AR were purchased from Merck Specialties Pvt. Ltd., Mumbai, India.

Used for the treatment of dysentery and diarrhea.8 Used for the treatment of dysentery and diarrhea.9 Used for the treatment of dysentery.10 Used against diarrhea and dysentery.11 Used as a drug for stomach disorder.12 Used for the treatment of muscular swelling caused by some bruises, refrigerant and ant-scorbutic, and are also used as stomachics.13 Used as appetizer and analgesic.14 The juice of the leaves is used for the treatment of wounds, itches, fevers and dysentery. The fresh juice of the leaves relieves Datura intoxication. The leaves is also used in Tonga to cure convulsions in infants.15 Herb Leaf juice is being used to cure dysentery & fever, anemia and for appetite, digestion.16 Used for the treatment of dysentery, stomach disorders, rheumatism, toothache.17

the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. The experimental protocols had the approval of the Institutional Animal Ethics Committee, Department of Physiology, University of Calcutta. Plant collection The leaf samples of Oxalis corniculata (Oxalidaceae) were collected from the university campus. The voucher specimen (No: CNH/93/2012/Tech.II/911) has been deposited at the Botanical Survey of India, Howrah. The leaves were then separated, sun dried, powdered, and stored in a refrigerator at - 20C for further studies. Methanolic extraction of Oxalis corniculata leaf Leaf extract was prepared by homogenizing the air-dried leaf (30 g) with 500 mL of 80:20 methanol:water at room temperature and kept in shaking condition for 18 h. The extract was then filtered and concentrated in a rotary vacuum evaporator (Eyela, Tokyo, Japan) and freeze-dried at 0.05 mbar pressure and - 60C for 4 h in a LSL Secfroid lyophilizer (Lyolab BII, Aclens-Lausanne, Switzerland).18 The lyophilized material was then stored at - 20C for further use.

Animals

Determination of polyphenols

Swiss albino mice were purchased from M/S Chakraborty Enterprise, 3/1D Girish Vidya Ratna Lane, Regn. No.:1443/ PO/b/11/CPCSEA and maintained at 25C with 75% humidity and fed sterile food and water. Gut sterilization of the animals were performed by treatment with a mixture of antibiotic solution containing metronidazole (400 mg), ciprofloxacin (500 mg), erythromycin (500 mg), and albendazole (400 mg) for 5 days .The animals were handled and all the experiments were carried out as per the guidelines of

Total polyphenol content of the leaf extracts were determined by the method described by Matthaus.19 Briefly, 0.2 mL of different concentrations of the extracts were taken, to which 1 mL of Folin-Ciocalteau reagent (diluted to 10-folds), 0.8 mL of 2% Na2CO3 were added and volume made up to 10 mL with methanol:water (6:4). After 30 min, the absorbance was read at 740 nm. The results were expressed as gallic acid equivalents per gram of extract.

3 values are expressed as mean – SEM n = 3. Ampicillin (30 lg/disc), cChloramphenicol (30 lg/disc), dCiprofloxacin (5 lg/disc), eAzithromycin (15 lg/disc), fDoxycycline (30 lg/disc), gNorfloxacin (10 lg/disc), hNalidixic Acid (30 lg/disc), i Ofloxacin (5 lg/disc), jStreptomycin (10 lg/disc), k Tetracycline (30 lg/disc). S, sensitive; R, resistant; I, intermediate. b

17 – 0(S) 28 – 0.5(S) 23 – 0.5(S) 20 – 0.3(S) 20 – 0.3(S) 0 – 0(R)

a

0 0 0 0 0 0 20 – 0.3(S) 25 – 0.3(S) 24 – 0.5(S) 0 – 0(R) 20 – 0.5(S) 0 – 0(R) 12 – 0.3(I) 14 – 0.3(I) 14 – 0.5(I) 0 – 0(R) 13 – 0(I) 0 – 0(R) 24 – 0.3(S) 20 – 0.3(S) 27 – 0.3(S) 17 – 0.3(S) 0 – 0(R) 24 – 0.5(S) 27 – 0.5(S) 24 – 0.5(S) 26 – 0.3(S) 25 – 0.3(S) 25 – 0.3(S) 24 – 0(S) 0 – 0(R) 0 – 0(R) 12 – 0.3(R) 10 – 0.3(R) 0 – 0(R) 0 – 0(R) 18 – 0(S) 23 – 0.3(S) 25 – 0.3(S) 0 – 0(R) 18 – 0(S) 11 – 0(R) 22 – 0(S) 25 – 0(S) 22 – 0.5(S) 21 – 0.5(S) 18 – 0.3(S) 22 – 0.3(S) 25 – 0.3(S) 24 – 0(S) 28 – 0.3(S) 28 – 0.5(S) 13 – 0(S) 10 – 0(R)

Sj Of i Nah Nxg Dof Ate Cf d Cc

19 – 0.5 15 – 0.5 13 – 0.5 12 – 0.5 12 – 0 16 – 0

The minimum bactericidal concentration (MBC) values were evaluated by removing 100 lL of bacterial suspension

Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 25923 Shigella flexneri 2a (2457T) Shigella boydii 4 (BCH612) Shigella sonnie phase I (IDH00968) Shigella dysenterie1 (NT4907)

Minimum bactericidal concentration

Ab

Minimum inhibitory concentration (MIC) was determined by the method of successive dilution method.24 TSB (1 mL) medium was added to 12 numbered screw tubes (10 · 100 mm) except for number 1. For the first and the second tubes of the series, 1 mL of extract (0.05–0.55 mg/ mL) or antibiotic (0.02–0.05 mg/mL) was added; tube 2 was stirred and 1 mL was withdrawn and transferred to tube 3. This successive transference was repeated until tube 11. All the tubes except tube number 11 were inoculated with 0.1 mL of inoculum (108 CFU/mL) at known concentration. The tubes were incubated at optimal temperature (37C) for 24 and 48 h and the results were noted. Tube 11 (TSB + EXTRACT/Antibiotic) and tube 12 (TSB + inoculum) were considered as controls.

Extract

Minimum inhibitory concentration

Microorganisms

The agar well-diffusion method21 was used to evaluate the inhibitory spectrum of extract against test microorganisms. A freshly grown culture was serially diluted, and 0.1 mL of diluted inoculums (108 CFU/mL) of test organism was spread on agar plates. MHA (Hi media) was used for E. coli, S. aureus, S. dysenteriae1 (NT4907) S. flexneri 2a (2457T), S. boydii 4 (BCH612) and S. sonnie phase I (IDH00968). Wells (6 mm in diameter) were made on agar plates using a sterilized stainless steel borer. Each well was filled with 50 lL (1 mg/mL) of diluted extracts. The plates were left at room temperature for 30 min to allow diffusion of materials in media. Different antibiotics as shown in Table 2 were used as positive control. Negative controls (DMSO) were also used under the same conditions. Plates were incubated at 30C for 24 h. Inhibition zones in mm (including well diameter) around wells were measured. The antimicrobial activity was expressed as the diameter of inhibition zones created by the extracts against test microorganisms. The experiment was repeated thrice. A zone size, greater than 7 mm indicated that the bacteria were susceptible22 to the extract from Oxalis corniculata (Oxalidaceae),whereas zone of inhibition formed by antibiotics were compared with the zone size interpretative chart supplied by HIMEDIA.23

Diameter of inhibition zone (mm)a

Agar well diffusion assay

Table 2. Antimicrobial Activity of Extract from Oxalis Corniculata (Oxalidaceae) Leaves Using Agar Well Diffusion Method

Total flavonoid content was evaluated by the aluminum chloride colorimetric assay. An aliquot of appropriately diluted (0.05 and 0.1 mg/mL) extract or standard solution of quercetin (0.02–0.1 mg) mixed with 4 mL of distilled H2O and subsequently with 0.3 mL 5% NaNO2 solution. After 5 min, 0.3 mL 10%AlCl3 were added and further the solution was appended with 2 mL 1 M NaOH. Final volume was made upto 10 mL with distilled water. The solution was mixed well and the absorbance was measured against prepared reagent blank at 510 nm.20

Tk

Determination of flavonoids

21 – 0.5(S) 20 – 0.5(S) 26 – 0.3(S) 26 – 0.5(S) 24 – 0.3(S) 0 – 0(R)

DMSO

ACTIVITY OF OXALIS CORNICULATA AGAINST SHIGELLOSIS

4

MUKHERJEE ET AL.

from the MIC tubes that did not show any growth and subcultured into MHA plates and incubated at 37C for 24 h. After incubation, the concentration at which no visible growth was seen was recorded as the MBC.25 In vivo toxicity assay The toxicity assay was carried out according to the World Health Organisation guideline for the evaluation of safety and efficacy of herbal medicines.26 The doses studied were 50, 40, 30, 20 mg$kg - 1$d - 1. Normal controls received PBS as placebo treatment. The toxic effects was expressed as LD50.27 In vivo anti colonization assay in suckling mice model S. flexneri 2a (2457T) was used as a reference strain and Shigella dysenterie 1 (NT4907) as a multi drug resistant strain for the in vivo experiment. The glycerol stocks were used to inoculate the bacteria in 2 mL of tryptic soy broth (TSB, Difco). After 4 h of incubation, at 37C with shaking, 1 mL of the preculture was added to 25 mL of TSB contained in 500 mL Erlenmeyer flask. After 18 h of growth, the culture was harvested by centrifugation at 8013 g for 15 min. The pellet was dispersed in sterile phosphate buffered saline (pH 7.4) and the bacterial mass was estimated using a spectrophotometer at 600 nm, and was aseptically diluted with sterile PBS upto 109 cells for use as the inoculum. Viable plate counts were made on nutrient agar (Difco, Franklin Lakes, New Jersey, USA). Neonate mice were inoculated intragastrically with 5 · 109 CFU of virulent Shigella strain without any starvation or antibiotic treatment. After oral infection, mice were separated from their mothers.28 20 mg/kg of body weight extract was administered. We selected eight experimental groups of mice. Two control groups infused with two test bacteria and PBS after 3 h of bacterial inoculums infusion, two experimental groups treated with bacterial inoculums and extract supplemented after 3 h of bacterial inoculums infusion, two control groups treated with two test bacteria and PBS simultaneously and two experimental groups treated with same dose of extract simultaneously along with bacterial inoculum. To assess colonization activity of bacteria, the whole intestine was placed in PBS homogenized and serial dilutions were plated onto HEA.

HPLC analysis of the Oxalis corniculata (Oxalidaceae) leaf extract The hydrolyzed sample was prepared for HPLC detection of the plant polyphenols.29 In brief, 0.5 g of the dried plant material was taken in a flask along with BHA (2 g/L) and sonicated for 5 min in 40 mL of 65% methanol. 10 mL of 6 N HCl was added and nitrogen purged for 60 sec. The mixture was then heated in a water bath at 90C for 2 h with stirring constantly. It was then cooled, filtered, and sonicated for 5 min, and then used for HPLC analysis. In brief, a gradient elution was employed for flavonoids other than catechins with a mobile phase consisting of 50 mM H3PO4, pH 2.5 (solution A), and acetonitrile (solution B), which is as follows: isocratic elution 95% A/5% B, 0–5 min; linear gradient from 95% A/5% B to 50% A/50% B, 5–55 min; isocratic elution 50% A/50% B, 55–65 min; linear gradient from 50% A/50% B to 95% A/5% B, 65– 67 min; post-time 6 min before the next injection. The WATERS 2487 was equipped with a C-18 column (NovaPak C18, 3.9 · 150 mm). About 280 and 370 nm wavelength were selected for the detection. The flow rate of the mobile phase was 0.8 mL/min, and the injection volume was 20 lL. The analysis were repeated thrice and the peaks were identified by comparing with authentic standards.30 The concentrations of flavonoids and phenolic acid standards were 250 and 100 lg/mL, respectively. The amounts of identified flavonoids and phenolic acids in the extract of the sample were calculated from the concentration of their respective standards. Statistical analysis Data are expressed as the mean – SEM. Statistical comparisons between experimental groups were performed using the Student t-test. All the statistical analysis were performed by using SPSS18. RESULTS Total phenolic and flavanoid contents The total polyphenol content of the extract was 910 mg of GAE/g of dry wt. and total flavanoid content was 2.353 g/ 100 g of extract.

Table 3. Minimum inhibitory Concentration and Minimum Bactericidal Concentration of Extract from Leaves of Oxalis Corniculata (Oxalidaceae) MIC (mg/mL)a Organism Escherichia coli ATCC 25922 Staphylococcus aureus ATCC 25923 Shigella flexneri 2a (2457T) Shigella boydii 4 (BCH612) Shigella sonnie phase I (IDH00968) Shigella dysenterie1 (NT4907) a

MBC (mg/mL)a

Extract

Chloramphenicol

Ampicillin

Extract

Chloramphenicol

Ampicillin

0.08 – 0.001 0.1 – 0.002 0.13 – 0.005 0.1 – 0.001 0.1 – 0.007 0.110 – 0.001

0.04 – 0.001 0.04 – 0.003 0.045 – 0.003 0.08 – 0.007 0.08 – 0.001 0.15 – 0.001

0.04 – 0.007 0.045 – 0.005 0.15 – 0.001 0.09 – 0.003 0.08 – 0.003 0.14 – 0.001

0.1 – 0.001 0.12 – 0.001 0.14 – 0.005 0.12 – 0.007 0.12 – 0.001 0.12 – 0.005

0.05 – 0.006 0.05 – 0.005 0.05 – 0.005 0.1 – 0.001 0.1 – 0.001 ND

0.05 – 0.001 0.05 – 0.001 0.16 – 0.003 0.1 – 0.001 0.1 – 0.001 ND

values are expressed as mean – SEM n = 3. MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; ND, not determined.

ACTIVITY OF OXALIS CORNICULATA AGAINST SHIGELLOSIS

5

(BCH612), S. sonnie phase I (IDH00968). The extract of Oxalis corniculata (Oxalidaceae) showed significant zone of inhibition against Shigella dysenterie (16 mm) and S. boydii 4 (BCH612) (12 – 0.5 mm). It also showed significant zone of inhibition against S. sonnie (12 mm) and S. flexneri 2a (2457T) (13 – 0.5 mm). Shigella dysenterie 1 (NT4907) is a multi-drug resistant strain and the effect of commercially available antibiotics were compared with the extract from Oxalis corniculata (Oxalidaceae) (Table 2). Minimum inhibitory concentration

FIG. 1. In vivo toxicity assay in different groups. Values are expressed as mean – SEM, n = 6.

Extract from Oxalis corniculata (Oxalidaceae) showed the lowest MIC against E. coli (0.08 mg/mL) and highest against S. flexneri 2a (2457T) (0.13 mg/mL). The extract showed intermediate MIC against S. aureus (0.1 mg/mL), S. dysenteriae 1 (NT4907) (0.11 mg/mL), S. sonnie phase I (IDH00968) (0.1 mg/mL), S. boydii 4 (BCH612) (0.1 mg/ mL) (Table 3).

Agar well diffusion assay

Minimum bactericidal concentration

The antimicrobial potency of the Oxalis corniculata (Oxalidaceae) extract against five intestinal pathogenic microorganisms were presented in Table 2. DMSO (negative control) was inactive against tested microorganisms. The extract of Oxalis corniculata (Oxalidaceae) showed a range of diameter of inhibition zone of 12 to 19 mm. The extract showed highest the zone of inhibition against E. coli and showed lowest zone of inhibition against S. boydii 4

The extract showed considerable bactericidal activity against the tested organisms (Table 3). It showed the lowest MBC against E. coli (0.1 mg/mL), highest MBC against S. flexneri 2a (2457T) (0.14 mg/mL), and intermediate MBC against S. aureus (0.12 mg/mL), S. dysenteriae 1(NT4907) (0.12 mg/mL), S. sonnie phase I (IDH00968) (0.12 mg/mL), and S. boydii 4 (BCH612) (0.12 mg/mL).

FIG. 2. Effect of simultaneous addition of extract (E) of Oxalis corniculata (Oxalidaceae) and bacterial inoculum on colonization ability of Shigella flexineri 2a (2457T) and Shigella dysenteri 1 (NT4907). Control Group: (Simultaneous addition of PBS + Bacteria). Experimental group: (Simultaneous addition of E + Bacteria). *aControl group versus exp. group C.F.U. of Shigella flexneri (P < .05). *bControl group versus exp. group C.F.U. of Shigella dysenteriae (P < .05). *cexp. group versus exp. group C.F.U. of S. flexneri and S. dysenteriae (P < .05). Values are expressed as mean – SEM n = 6.

FIG. 3. Effect of supplementation of extract (E) of Oxalis corniculata (Oxalidaceae) after 3 h of bacterial inoculation on colonization ability of Shigella flexineri 2a (2457T) and Shigella dysenteri 1 (NT4907). Control Group: (Bacteria + PBS after 3 h). Experimental group: (Bacteria + E after 3 h). *aControl group versus exp. group C.F.U. of S. flexneri (P < .05). *bControl group versus exp. group C.F.U. of S. dysenteriae (P < .05). *cexp. group versus exp. group C.F.U. of S. flexneri and S. dysenteriae (P < .05). Values are expressed as mean – SEM n = 6.

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MUKHERJEE ET AL.

FIG. 4. HPLC chromatogram of Oxalis corniculata (Oxalidaceae) at 280 nm showing peaks (1) p-hydroxybenzoic acid and (2) Ferulic Acid.

In vivo toxicity assay Extract from Oxalis corniculata (Oxalidaceae) exhibited an LD50 value of 50 mg/kg of body weight compared to 100% survival of PBS placebo control (Fig. 1). In vivo anti colonization assay in suckling mice model The colonization of S. flexneri 2a (2457T) and Shigella dysenterie 1 (NT4907) in absence (control) and presence extract of Oxalis corniculata (Oxalidaceae) were evaluated in suckling mice. The extract of Oxalis corniculata (Oxalidaceae) was supplemented along with the inoculums and after 3 h of administration of the inoculums as presented in Figures 2 and 3, respectively. The suckling

mice were all infected with the test inoculums and the colonization of both the strains were diminished by the extract of Oxalis corniculata (Oxalidaceae). The anticolonization activity of extract from Oxalis corniculata (Oxalidaceae) was significantly greater against Shigella dysenterie 1 (NT4907) than S. flexneri 2a (2457T) with simultaneous application of extract and the bacterial inoculums. When extract supplement was administered after 3 h of infusion of inoculums the colony forming ability still decreased significantly in the experimental groups than the control groups of mice. The anti colonization activity of the extract was more pronounced in Shigella dysenterie 1 (NT4907) than the reference strain S. flexneri 2a (2457T).

FIG. 5. HPLC chromatogram of Oxalis corniculata (Oxalidaceae) at 370 nm showing peaks (1) Rutin.

7

ACTIVITY OF OXALIS CORNICULATA AGAINST SHIGELLOSIS Table 4. Relative Abundance of Identified Compound at 370 nm Parameters Identified compound Retention Mean peak area of Standard deviation Mean peak area Standard deviation of % at 370 nm time (min) Standard (lV.sec) of peak area of standard of Sample (lV.sec) peak area of sample Conc. Rutin

25.797

519022

2390

278040

963.52

Conc. (lg/mL)

53% 130 lg/mL

n = 3.

HPLC analysis of the Oxalis corniculata (Oxalidaceae) leaf extract Seven standards were used to identify the peaks obtained from HPLC analysis of the analyte. This included comparison with standard peaks of kaempherol, catechin, myricetin, quercetin, rutin, ferulic acid and p-hydroxybenzoic acid. This resulted in identification of three major peaks of the HPLC chromatogram. At 280 nm, two peaks corresponding to p-hydroxybenzoic acid and ferulic acid were found, respectively (Fig. 4). The maximum peak at 370 nm was found to be rutin (Fig. 5). The retention time of the standards, area under standard peaks, as well as sample peaks and concentration of various identified compounds are presented in Tables 4 and 5. The order of abundance of identified flavonoid and phenolic acids in the extract of Oxalis corniculata (Oxalidaceae) was found to be: rutin > phydroxybenzoic acid > ferulic Acid. DISCUSSION Diarrhea caused by intestinal pathogens is a global health concern and one of the dominant causes of infant mortality especially in developing countries. The Oxalis corniculata extract showed phenomenal antibacterial activity against Shigella dysenterie 1(NT4907), the most vulnerable species when compared with other antibiotics (Table 2). S. boydii 4 (BCH612) showed resistance against doxycycline, tetracycline and streptomycin, but was susceptible to the extract. Extract from Oxalis corniculata (Oxalidaceae) also showed a significant zone of inhibition against S. sonnie, which was resistant to ciprofloxacin, norfloxacin, nalidixic acid. The reference strain S. flexneri 2a (2457T) was found to be less sensitive to extract. Oxalis corniculata (Oxalidaceae) showed potent MBC (0.14 mg/mL) against reference strain of Shigella flexneri 2a (2457T). Previous studies failed to prove the antibacterial effect of some plant extracts against Shigella sp., whereas our extract showed significant anti-

bacterial activity against all Shigella sp.22 It was reported that green tea extract showed MICs at 0.8 mg/mL against E. coli ATCC 2592231, whereas in our experiment the MIC was observed to be 0.08 mg/mL of extract. The process responsible for phenolic toxicity to bacteria involves the inhibition of enzymes by the oxidized compounds, it is likely through reaction with sulfydryl groups or more nonspecific interactions with proteins. Polyphenols which can form heavy soluble complexes with proteins is likely to bind with bacterial adhesions thereby hindering the availability of receptor on the cell surface.32 In the in vivo toxicity study, our extract exhibited an LD50 of 50 mg/kg body weight, whereas it was already documented that the chloroform extract of the Streblus asper root causes death of canines and rats at LD50 of 10.5 and 4.8 mg/kg body weight.33 LD50 also differs in different stages of development of mice.34 Ingested polyphenols are concentrated in the gastrointestinal tract and the high luminal concentrations achieved support a potential for therapeutic uses in the gastrointestinal tract.35 While many published reports demonstrated the failure of antibiotic treatment during the outbreak of diahorrea caused by S. dysenteriae type 1 strain,36,37 the extract effectively reduced the bacterial colonization in intestine of suckling mice. The HPLC analysis of Oxalis corniculata (Oxalidaceae) leaf demonstrated the presence of three different phenolic compounds: rutin, p-hydroxybenzoic acid, and ferulic acid (Fig. 4). The ipso facto results of HPLC analysis were found maximum for rutin (53%). It was documented that topoisomerase IV-dependent decatenation activity was inhibited by rutin and it also induced the SOS response of a permeable E. coli strain. Hydrophobic nature of ferulic acid exhibited substantial bioactivity against species of corynebacteria, enterococci, staphylococci, and streptococci.38 Previous report suggests that p-hydroxybenzoic acid inhibits the growth of both gram positive and gram negative

Table 5. Relative Abundance of Identified Compound at 280 nm Parameters Identified compound at 280 nm p-hydroxybenzoic acid Ferulic Acid n = 3.

Retention Mean peak area of Standard deviation of Mean peak area of Standard deviation of % time (min) Standard (lV.sec) peak area of standard Sample (lV.sec) peak area of Sample Conc. 6.46 26

1906654 15881636

3193.6 831.93

657467 229876

1953.68 2981.33

Conc. (lg/mL)

34% 34 lg/mL 1.44% 1 lg/mL

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bacteria.39 Moreover, it has already been reported that rutin not only has potent antimicrobial property itself but also is found to enhance the antimicrobial property of other flavonoids.40,41 Hence, all these identified compounds (flavonoid and phenolic acids) might be responsible for the antimicrobial properties of the extract reported in the present study. In conclusion, this research provides convincing evidence that a flavonoid rich extract from Oxalis corniculata (Oxalidaceae) could prevent bacterial colonization of the gut epithelia to control infectious diarrhoea due to pathogens, such as S. flexneri and multi-drug resistant S. dysenteriae. The hindrance of antimicrobial resistance in diarrheal diseases causing microorganisms will continue to be ongoing in both developed and developing countries. Currently, the continued discovery of new antimicrobials, particularly those for treatment of shigellosis in children, is extremely important. A comparison of the antimicrobial activity of extract from Oxalis corniculata (Oxalidaceae) to that of different antibiotics provides substantial support for the use of the extract as a substitute or as an adjuvant to well recognized chemotherapeutic agents.

ACKNOWLEDGMENT The work was funded by Department of Science & Technology, New Delhi, India. FUNDING Department of Science and Technology, New Delhi, India. ETHICAL APPROVAL Experiment was carried out under the supervision of Institutional Animal Ethical Committee of the Dept. of Physiology, University of Calcutta, India. AUTHOR DISCLOSURE STATEMENT No competing financial interests exist for any of the authors. REFERENCES 1. Bennish ML, Salam MA, Hossain MA, Myaux J, Khan EH, Chakraborty J, et al.: Antimicrobial resistance of Shigella isolates in Bangladesh, 1983–1990: increasing frequency of strains multiply resistant to ampicillin, trimethoprim-sulfamethoxazole, and nalidixic acid. Clin Infect Dis 1992;14:1055–1060. 2. Cowan MM: Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564–582. 3. Middleton E: Biological Properties of plant flavonoids: an overview. Pharm Biol 1996;34:344–348. 4. Bylka W, Matlawska I, Pilewski NA: Natural flavonoids as antimicrobial agents. JANA 2004;2:24. 5. Palpasa K, Pankaj B, Sarala M, Gokarna RG: Antimicrobial resistance surveillance on some bacterial pathogens in Nepal: a technical cooperation. J Infect Dev Ctries 2011;5:163–168.

6. Saravanakumar A, Vanitha S, Ganesh M, Jayaprakash J, Ramaswamy NM: Hypolipidemic activity of Sesbania grandiflora in triton wr-1339 induced hyperlipidemic rats. Int J of Phytomed 2010;2:52–58. 7. Das AK, Hui T: Ethnomedicinal studies of the Khamti tribe of Arunachal Pradesh. IJTK 2006;5:317–322. 8. Silja VP, Varma KS, Mohanan KV: Ethnomedicinal plant Knoledge of the Mullu Kuruma tribe of Wayanad district, Kerala. IJTK 2008;7:604–612. 9. Raju VS, Reddy KN: Ethnomedicine for dysentery and diarrhea from Khammam district of Andhra Pradesh. IJTK 2005;4:443–447. 10. Alam MK: Medical ethnobotany of the Marma tribe of Bangladesh. Econ Bot 1992;46:330–335. 11. Jan G, Khan MA, Gul F: Ethnomedicinal plants used against diarrhea and dysentery in dir kohistan valley (NWFP), Pakistan. Ethnobot Leaflets 2008;12:620–637. 12. Sharief MU, Kumar S, Diwakar PG, Sharma TVRS: Traditional phytotherapy among karens of middle Andaman. IJTK 2005;4: 429–436. 13. Manandhar NP: Medicinal Plant-Lore of Tamang Tribe of Kabhrepalanchok District. Econ Bot 1991;45:58–71. 14. Kala CP: Ethnomedicinal botany of the Apatani in the Eastern Himalayan region of India. J Ethnobiol Ethnomed 2005;1:11. 15. Michael A: Ethnomedicine in Tonga. Econ Bot 1971;25:423–450. 16. Das T, Mishra SB, Saha D, Agarwal S: Ethnobotanical Survey of medicinal plants used by ethnic and rural people in Eastern Sikkim Himalayan Region. Afr J Basic App Sci 2012;4:16–20. 17. Sen S, Chakraborty R, De B, Devanna N: An ethnobotanical survey of medicinal plants used by ethnic people in West and South district of Tripura. IJFR 2011;22:417–426. 18. Rauha JP, Remes S, Heinonen M, et al.: Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds. Int J Food Microbiol 2000;56:3–12. 19. Mattha¨us B: Antioxidant activity of extracts obtained from residues of different oilseeds. J Agric Food Chem 2002;50:3444–3452. 20. Ebrahinzadeh MA, Pourmorad F, Bekhradnia AR: Iron chelating activity screening, phenol and flavanoid content of some medicinal plants from Iran. Afr J Biotech 2008;32:43–49. 21. Perez C, Paul M, Bazerque P: Antibiotic assay by agar well diffusion method. Acta Biol Med Exp 1990;15:113–115. 22. Nascimento GGF, Locatelli J, Freitas PC, Silva GL: Antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. Braz J Microbiol 2000;31:247–256. 23. Bauer AW, Kirby WMM, Sherris JC, Turck M: Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493–496. 24. Mazzola PG, Jozala AF, Novaes LCL, Moriel P, Penna TCV: Minimal inhibitory concentration (MIC) determination of disinfectant and/or sterilizing agents. Braz J Pharm Sci 2009;45:241–248. 25. Andrews JM: Determination of minimum inhibitory concentrations. J Antimicrob Chemother 2001;48:5–16. 26. WHO: Research Guidelines for Evaluating the Safety and Efficacy or Herbal Medicines. Regional office for the western pacific. Working group on the safety and efficacy on herbal medicine. Manila, Philippines 1992. 27. Schorderet M: Pharmacology. The fundamental concepts for therapeutics applications Geneva, Slatkine, Paris Frison-Roche. 1992. 28. Shim DH, Suzuki T, Chang SY, et al.: New animal model of shigellosis in the Guinea pig: its usefulness for protective efficacy studies. J Immunol 2007;178:2476–2482.

ACTIVITY OF OXALIS CORNICULATA AGAINST SHIGELLOSIS 29. Michael GL, Hertog PC, Hollman H, Venema DP: Optimization of a quantitative HPLC determination of potentially anticarcinogenic flavonoids in vegetables and fruits. J Agric Food Chem 1992;40:1591–1598. 30. Siddhuraju P, Becker K: Antioxidant properties of various solvent extracts of total polyphenolic constituents from three different agroclimatic origins of drumstick tree (Moringa olifera Lam.) leaves. J Agric Food Chem 2003;51:2144– 2155. 31. Su P, Henriksson A, Nilsson C, Mitchell H: Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, Staphylococcus aureus and Streptococcus pyogenes. World J Microbiol Biotechnol 2008;24:1837–1840. 32. Samy RP, Gopalakrishnakone P: Therapeutic potential of plants as anti-microbials for drug discovery. Evid Based Complement Alternat Med 2010;7:283–294. 33. Sopit W, Pisamai L, Keskaew P, Premjai A, Chaisiri W, Tipawan T: Antimicrobial activity of Streblus asper leaf extract. Phytother Res 2001;15:119–121. 34. Edwin IG: A compilation of LD50 values in newborn and adult animals. Toxicol Appl Pharmacol 1971;l:185–207.

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35. Dryden GW, Song M, McClain C: Polyphenols and gastrointestinal diseases. Curr Opin Gastroenterol 2006;22:165–170. 36. Jahan Y, Hossain A: Multiple Drug-resistant Shigella dysenteriae Type 1 in Rajbari District, Bangladesh. J Diarrhoeal Dis Res 1997;15:17–20. 37. Bradley RS, Mahbubur R, Yunus M: Antimicrobial resistance in organisms causing Diarrheal Disease. Clin Infect Dis 1997;24: S102–S105. 38. Naz S, Ahmad S, Rasool SA, Sayeed SA, Siddiqi R: In vitro Antibacterial activity of the extract derived from Terminallia catappa. Res Microbiol 2007;2:180–184 39. Cho JY, Moon JH, Seong KY, Park KH: Antimicrobial activity of 4-hydroxybenzoic acid and trans 4-hydroxycinnamic acid isolated and identified from rice hull. Biosci Biotechnol Biochem 1998;62:2273–2276. 40. Singh M, Govindarajan R, Rawat AKS: Antimicrobial flavonoid rutin from Pteris vittata L. against Pathogenic Gastrointestinal Microflora. Am Fern J 2008;98:98–103 41. Arima H, Ashida H, Danno G-I: Rutin-enhanced antimicrobial activities of flavonoids against Bacillus cerus and Salmonella enteritidis. Biosci Biotechnol Biochem 2002;66:1009–1014.