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

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.

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 of bacteria is perpetual among the flavonoids, which are atoxic or less toxic.

Over the years, several deleterious outbreaks by the bacterial enteric pathogens 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

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Table 1.

Medicinal Uses of Oxalis Corniculata (Oxalidaceae)

Native population	Region	Medicinal uses
Mullu Kurama tribes	Wayanad District, Kerala, India	Used for the treatment of dysentery and diarrhea.⁸
Konda Reddi tribes and Koyas tribes	Khammam District, Andhrapradesh, India	Used for the treatment of dysentery and diarrhea.⁹
Marma tribes	Bangladesh	Used for the treatment of dysentery.¹⁰
Village communities	Dir Kohistan Valley NWFP, Pakistan	Used against diarrhea and dysentery.¹¹
Karens tribes	Andaman, India	Used as a drug for stomach disorder.¹²
Tamang tribes	Kabhrepalanchok District, Nepal	Used for the treatment of muscular swelling caused by some bruises, refrigerant and ant-scorbutic, and are also used as stomachics.¹³
Apatani tribes	Eastern Himalayan, India	Used as an appetizer and analgesic.¹⁴
Native people	Philippines, Southeast Asia	The juice of the leaves is used for the treatment of wounds, itches, fever, and dysentery. The fresh juice of the leaves relieves Datura intoxication. The leaves are also used in Tonga to cure convulsions in infants.¹⁵
Ethnic and rural people	Eastern Sikkim Himalayan Region, India	Herb leaf juice is being used to cure dysentery, and fever, anemia and for appetite/digestion.¹⁶
Ethnic people	West and south districts of Tripura, India	Used for the treatment of dysentery, stomach disorders, rheumatism, toothache.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. The in vivo anti-colonizing effect of extract from Oxalis corniculata (Oxalidaceae) in 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 Diseases (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 Laboratories Pvt. Ltd., Vadhani Ind.Est, LBS Marg, Mumbai, India. Antibiotic discs and 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.

Animals

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 25°C 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 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. Experiments were carried out under the supervision of Institutional Animal Ethical Committee.

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 −20°C 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 −60°C for 4 h in a LSL Secfroid lyophilizer (Lyolab BII, Aclens-Lausanne, Switzerland).¹⁸ The lyophilized material was then stored at −20°C for further use.

Determination of polyphenols

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 10-fold), 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 (GAE) per gram of extract.

Determination of flavonoids

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

Agar well diffusion assay

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 (HIMEDIA) was used for E. coli, S. aureus, S. dysenteriae 1 (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 μL (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 30°C 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 >7 mm indicated that the bacteria were susceptible²² to the extract from Oxalis corniculata (Oxalidaceae),whereas zones of inhibition formed by antibiotics were compared with the zone size interpretative chart supplied by HIMEDIA.²³

Table 2.

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

	Diameter of inhibition zone (mm)
Microorganisms	Extract	A	C	Cf	At	Do	Nx	Na	Of	S	T	DMSO
Escherichia coli ATCC 25922	19±0.5	17±0^S	21±0.5^S	25±0.3^S	22±0^S	18±0^S	24±0.3^S	20±0.3^S	27±0.3^S	12±0.3^I	20±0.3^S	0
Staphylococcus aureus ATCC 25923	15±0.5	28±0.5^S	20±0.5^S	24±0^S	25±0^S	23±0.3^S	17±0.3^S	0±0^R	24±0.5^S	14±0.3^I	25±0.3^S	0
Shigella flexneri 2a (2457T)	13±0.5	23±0.5^S	26±0.3^S	28±0.3^S	22±0.5^S	25±0.3^S	27±0.5^S	24±0.5^S	26±0.3^S	14±0.5^I	24±0.5^S	0
Shigella boydii 4 (BCH612)	12±0.5	20±0.3^S	26±0.5^S	28±0.5^S	21±0.5^S	0±0^R	25±0.3^S	25±0.3^S	24±0^S	0±0^R	0±0^R	0
Shigella sonnie phase I (IDH00968)	12±0	20±0.3^S	24±0.3^S	13±0^S	18±0.3^S	18±0^S	0±0^R	0±0^R	12±0.3^R	13±0^I	20±0.5^S	0
Shigella dysenteriae 1 (NT4907)	16±0	0±0^R	0±0^R	10±0^R	22±0.3^S	11±0^R	10±0.3^R	0±0^R	0±0^R	0±0^R	0±0^R	0

Values are expressed as mean ± SEM; n = 3.

The responsiveness of each microorganism to the various commercially available antibiotics is indiacted: ^Ssensitive, ^Rresistant, or ^Iintermediate.

A, ampicillin (30 μg/disc); C, chloramphenicol (30 μg/disc); Cf, ciprofloxacin (5 μg/disc); At, azithromycin (15 μg/disc); Do, doxycycline (30 μg/disc); Nx, norfloxacin (10 μg/disc); Na, nalidixic acid (30 μg/disc); Of, ofloxacin (5 μg/disc); S, streptomycin (10 μg/disc); T, tetracycline (30 μg/disc); DMSO, dimethyl sulfoxide.

Minimum inhibitory concentration

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 (37°C) for 24 and 48 h and the results were noted. Tube 11 (TSB+Extract/Antibiotic) and tube 12 (TSB+inoculum) were considered as controls.

Minimum bactericidal concentration

The minimum bactericidal concentration (MBC) values were evaluated by removing 100 μL of bacterial suspension from the MIC tubes that did not show any growth and subcultured into MHA plates and incubated at 37°C 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 per day. 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 dysenteriae 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, Franklin Lakes, NJ, USA). After 4 h of incubation, at 37°C 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 (PBS; 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). 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 body weight of extract was administered. We selected eight experimental groups of mice. Two control groups were infused with two test bacteria and PBS after 3 h of bacterial inoculums infusion, two experimental groups were treated with bacterial inoculum and extract supplemented after 3 h of bacterial inoculums infusion, two control groups were treated with two test bacteria and PBS simultaneously, and two experimental groups were treated with the same dose of extract simultaneously along with bacterial inoculum.

To assess colonization activity of bacteria, the whole intestine was placed in PBS and 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 90°C for 2 h with constant stirring. 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 (Nova-Pak C₁₈, 3.9 mm×150 mm). Wavelengths of 280 and 370 nm were selected for the detection. The flow rate of the mobile phase was 0.8 mL/min, and the injection volume was 20 μL. 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 μg/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 statistical analyses were performed by using SPSS version 18 (SPSS Inc., Chicago, IL, USA).

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.

Agar well diffusion assay

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 (BCH612) and 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

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).

Table 3.

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of Extract from Leaves of Oxalis Corniculata (Oxalidaceae)

	MIC (mg/mL)			MBC (mg/mL)
Organism	Extract	Chloramphenicol	Ampicillin	Extract	Chloramphenicol	Ampicillin
Escherichia coli ATCC 25922	0.08±0.001	0.04±0.001	0.04±0.007	0.1±0.001	0.05±0.006	0.05±0.001
Staphylococcus aureus ATCC 25923	0.1±0.002	0.04±0.003	0.045±0.005	0.12±0.001	0.05±0.005	0.05±0.001
Shigella flexneri 2a (2457T)	0.13±0.005	0.045±0.003	0.15±0.001	0.14±0.005	0.05±0.005	0.16±0.003
Shigella boydii 4 (BCH612)	0.1±0.001	0.08±0.007	0.09±0.003	0.12±0.007	0.1±0.001	0.1±0.001
Shigella sonnie phase I (IDH00968)	0.1±0.007	0.08±0.001	0.08±0.003	0.12±0.001	0.1±0.001	0.1±0.001
Shigella dysenteriae 1 (NT4907)	0.110±0.001	0.15±0.001	0.14±0.001	0.12±0.005	ND	ND

Values are expressed as mean±SEM; n=3.

MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; ND, not determined.

Minimum bactericidal concentration

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).

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).

FIG. 1.

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

In vivo anti colonization assay in suckling mice model

The colonization of S. flexneri 2a (2457T) and Shigella dysenteriae 1 (NT4907) in absence (control) and presence of 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 dysenteriae 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 anticolonization activity of the extract was more pronounced in Shigella dysenteriae 1 (NT4907) than the reference strain S. flexneri 2a (2457T).

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 dysenteriae 1 (NT4907). Values are expressed as mean±SEM; n=6. Statistically significant (P < .05) differences are indicated: *control vs. experimental CFU of S. flexneri; ^†control vs. experimental CFU of S. dysenteriae; ^#experimental CFU of S. flexneri vs. experimental CFU of S. dysenteriae.

FIG. 3.

Effect of supplementation of extract (E) of O. corniculata after 3 h of bacterial inoculation on colonization ability of S. flexineri and S. dysenteriae. Values are expressed as mean±SEM; n=6. Statistically significant (P < .05) differences are indicated: *control vs. experimental CFU of S. flexneri; ^†control vs. experimental CFU of S. dysenteriae; ^#experimental CFU of S. flexneri vs. experimental CFU of S. dysenteriae.

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>p-hydroxybenzoic acid>ferulic acid.

FIG. 4.

HPLC chromatogram of Oxalis corniculata (Oxalidaceae) at 280 nm showing peaks: (1) p-hydroxybenzoic acid and (2) ferulic acid.

FIG. 5.

HPLC chromatogram of Oxalis corniculata (Oxalidaceae) at 370 nm showing peaks: (1) rutin.

Table 4.

Relative Abundance of Identified Compound at 370 nm

	Parameters
Identified compound at 370 nm	Retention time (min)	Mean peak area of Standard (μV.sec)	Standard deviation of peak area of standard	Mean peak area of Sample (μV.sec)	Standard deviation of peak area of sample	% Conc.	Conc. (μg/mL)
Rutin	25.797	519,022	2390	278,040	963.52	53%	130 μg/mL

n=3.

Table 5.

Relative Abundance of Identified Compound at 280 nm

	Parameters
Identified compound at 280 nm	Retention time (min)	Mean peak area of Standard (μV.sec)	Standard deviation of peak area of standard	Mean peak area of Sample (μV.sec)	Standard deviation of peak area of Sample	% Conc.	Conc. (μg/mL)
p-Hydroxybenzoic acid	6.46	1,906,654	3193.6	657,467	1953.68	34%	34 μg/mL
Ferulic acid	26	15,881,636	831.93	229,876	2981.33	1.44%	1 μg/mL

n=3.

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 dysenteriae 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 spp., whereas our extract showed significant antibacterial activity against all Shigella spp.²² It was reported that green tea extract showed MICs at 0.8 mg/mL against E. coli ATCC 25922,³¹ 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 diarrhea 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 bacteria.³⁹ Moreover, it has already been reported that rutin not only has potent antimicrobial properties itself but also is found to enhance the antimicrobial properties 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.

Footnotes

Acknowledgment

The work was funded by Department of Science & Technology, New Delhi, India.

Author Disclosure Statement

No competing financial interests exist for any of the authors.

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