Studies on the activity of Cyperus rotundus Linn. tubers against infectious diarrhea.

To study the antidiarrheal activity of the decoction of Cyperus rotundus Linn. tubers using representative assays of diarrheal pathogenesis and understand its mechanism of action.Antibacterial, antigiardial and antirotaviral activities were studied. Effect on adherence of enteropathogenic Escherichia coli (EPEC) and invasion of enteroinvasive E. coli (EIEC) and Shigella flexneri to HEp-2 cells was evaluated as a measure of effect on colonization. Effect on enterotoxins such as enterotoxigenic E. coli (ETEC) heat labile toxin (LT), heat stable toxin (ST) and cholera toxin (CT) was also assessed. The decoction showed antigiardial activity, reduced bacterial adherence to and invasion of HEp-2 cells and affected production of CT and action of LT. The decoction of C. rotundus does not have marked antimicrobial activity and exerts its antidiarrheal action by mechanisms other than direct killing of the pathogen.

Introduction

Diarrheal diseases, one of the most common infectious worldwide, are predicted to remain a leading health problem.[1] Oral rehydration therapy has been the key strategy for effective case management. However, it often fails in high stool output state. With contraindications of antimotility agents in infectious diarrhea and an increasing threat of drug resistance, various attempts for developing vaccines against diarrheal pathogens have been made.[23] However, the response to vaccines in developing countries has not been encouraging.[3] In the recent past, attempts have been made to treat infectious diarrhea with supportive therapy such as probiotics; but these are still under development.[4] Therefore, medicinal plants may provide a cost-effective alternative for treatment of diarrhea.

Most of the studies on antidiarrheal medicinal plants have focused on intestinal motility and/or antibacterial activity.[5] Hence, there is limited information on their mechanism(s) of action against pathogenicity of infectious diarrhea. In this study, we have evaluated the effect of crude decoction of tubers of Cyperus rotundus Linn. (family Cyperaceae) on various parameters, viz., bacterial adherence to and invasion of epithelial cells and production and action of enterotoxins, in addition to their antimicrobial activity.

Materials and Methods

Plant Material

Tubers of C. rotundus were collected from the Parinche valley near Pune, Maharashtra, India, and authenticated by Dr. P. Tetali. A voucher specimen has been deposited at Botanical Survey of India (Western Circle), Pune, India, under herbarium number 124666. Tubers were shade dried and stored at 4°C until further use. All experiments were performed with the same dried material.

Preparation of the Crude Aqueous Extract (Decoction)

The decoction was prepared by boiling 1 g of the powdered dried plant material in 16 mL double-distilled water till the volume was reduced to 4 mL.[6] To replicate field conditions, the decoction was freshly prepared every time. To minimize variability, similar boiling conditions were maintained for each preparation and the dry weight was recorded. The decoction was centrifuged and filtered through a membrane of 0.22-μm pore size before use. The decoction was diluted 1:100, 1:20 and 1:10 in appropriate media for each experiment (referred to as 1%, 5% and 10%, respectively, throughout the text).

Phytochemistry

The qualitative phytochemical analysis of the decoction was carried out using standard methods.

Microorganisms Used

Six bacteria, viz., enteropathogenic Escherichia coli (EPEC) strain B170, serotype 0111:NH; enterotoxigenic E. coli (ETEC) strain B831-2, serotype unknown (heat labile toxin, LT, producer) and strain TX1, serotype 078:H12 (heat stable toxin, ST, producer); enteroinvasive E. coli (EIEC) strain E134, serotype 0136:H-; Vibrio cholerae C6709 El Tor Inaba, serotype 01 (cholera toxin, CT, producer) and Shigella flexneri M9OT, serotype 5 were used. Giardia lamblia P1 trophozoites and simian rotavirus SA-11 were also studied.[78]

Biological Assays

The assays briefly described below were undertaken using methods and positive controls described previously.[78] Each experiment was done in duplicate/triplicate and repeated at least three times.

Antimicrobial Action

The decoction was assessed for its antibacterial, antigiardial and antirotaviral activities using agar dilution, trypan blue and neutral red assay, respectively. Ofloxacin (1 μg/mL) and metronidazole (10 μg/mL) were used as controls for the former two assays, respectively.

Effect on Bacterial Colonization

Adherence of EPEC and invasion of EIEC and S. flexneri to HEp-2 cells was assessed. HEp-2 cells were incubated in the absence (control) and presence of various dilutions of the decoction, either 18–20 hours prior to infection (pre-incubation) or simultaneously with infection (competitive inhibition). Results were compared with that of lactulose, a prebiotic oligosaccharide.

Effect on Bacterial Enterotoxins

The production of LT/CT and their binding to ganglioside monosialic acid receptor were assessed by ganglioside monosialic acid enzyme linked immunosorbent assay GM1-ELISA. Results were compared with those of 2-mercaptoethanol and gallic acid, respectively. The production and action of ST was assessed by suckling mouse assay.

Approval from the Institutional Ethics Committee and the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, registration No. 424/01/a/CPCSEA, June 20, 2001) was obtained for the study.

Statistical Analysis

Data were expressed as mean ± standard error of the percentage values from three independent experiments. The percentage in each experiment was calculated using the formula (C or T)/C × 100, where C is the mean value of the duplicate/triplicate readings of the control group and T is mean value of the duplicate/triplicate readings of the test (dilutions of the decoction) groups. Hence, the values of the test groups were represented as percentages relative to control (100%). Data were analyzed by analysis of variance (ANOVA) and Dunnett's post-test using the software Prism 4.0 (GraphPad, Inc., San Diego, CA, USA). P ≤ 0.05 was considered statistically significant.

Results

Dry Weight/Yield

The variability between different preparations of the decoction was within acceptable limits as indicated by the standard error of the dry weights. The average dry weight was 52 ± 2.8 mg/mL (n = 25), the yield being 21 ± 1.12% (w/w) with respect to the starting material. Thus, the concentrations of the different dilutions used in the biological assays were 0.52 ± 0.028 mg/mL (1%), 2.6 ± 0.14 mg/mL (5%) and 5.2 ± 0.28 mg/mL (10%).

The decoction contained carbohydrates, reducing sugars, proteins, amino acids, flavonoids, tannins and saponins, whereas glycosides, alkaloids, and phytosterols were absent.

Antimicrobial Activity

C. rotundus did not exhibit antibacterial and antirotaviral activity (data not shown). The multiplication of G. lamblia was restricted at all the concentrations tested in a dose-dependent manner with maximum inhibition (43.81 ± 2.54%) at 10% concentration [Figure 1]. However, the decrease was less than that caused by metronidazole.

Figure 1

Antigiardial activity of the decoction of C. rotundus [C: Control, trophozoites in medium alone; M: trophozoites incubated in medium with metronidazole (10 μg/mL)]. Values represent mean ± standard error (n= 3) of percentage viable trophozoites relative to control (100%); ∗P < 0.05

The adherence of E. coli B170 and invasion of E. coli E134 and S. flexneri to HEp-2 cells was significantly affected when the HEp-2 cells were incubated with the decoction either prior to or simultaneously with the infection [Figure 2a–c]. In comparison to lactulose, the decoction showed greater decrease in bacterial colonization.

Figure 2

Effect of the decoction of C. rotundus on bacterial colonization to HEp-2 cells: (a) Adherence of EPEC strain B170; (b) Invasion of EIEC strain E134; (c) Invasion of S. flexneri (C: Control, bacterial adherence/invasion to HEp-2 cells in medium alone; L1: adherence/invasion to HEp-2 cells in medium with 2.5 mg/mL lactulose; L2: adherence to HEp-2 cells in medium with 15 mg/mL lactulose in the competitive protocol). Values represent mean ± standard error (n = 3) of percentage adherence/invasion relative to respective control (100%); ∗P < 0.05

The decoction showed an overall increase (statistically insignificant) in the production of LT by E. coli B831-2 at all the concentrations tested. The binding of LT to GM1 was marginally decreased at 10% concentration. The decrease, however, was lesser than that observed with gallic acid [Figure 3a].

Figure 3

Effect of the decoction of C. rotundus on bacterial enterotoxins: (a) Production of heat labile toxin (LT) by E. coli B831-2 and its binding to GM1; (b) Production of cholera toxin (CT) by V. cholerae and its binding to GM1 (C: Control, toxin in medium alone; M1: LT in medium with 5 mM 2-mercaptoethanol; M2: CT in medium with 1 mM 2-mercaptoethanol; G: LT/CT in medium with 50 mM gallic acid). Values represent mean ? standard error (n = 3) of percentage production/binding relative to respective control (100%); ∗P < 0.05

The decoction significantly decreased CT production by V. cholerae at 5 and 10% concentrations which was comparable to that of 2-mercaptoethanol. However, there was no effect on the binding of CT to GM1 at any of the concentrations tested [Figure 3b].

The production and action of ST was not affected at any concentration (data not shown).

Discussion

C. rotundus Linn., commonly known as nut grass and locally as Musta, is said to possess antidiarrheal, anti-inflammatory and antipyretic activities.[910] The tubers are used in Ayurvedic medicine and have been mentioned in ancient texts for various ailments.

Our previous study conducted on C. rotundus tubers collected from Madhya Pradesh, India, had reported selective action against ETECs with no direct killing of bacteria.[11] We expanded the study with this plant to include multiple pathogens and parameters. In the present study, tubers from Parinche, Maharashtra, were used. Though the antibacterial profile of the two batches was similar, differential effects were seen on adherence of EPEC, LT and ST. Despite the mode of extract preparation being similar, the difference in the results of the two studies can be attributed to different ecotypes and the time of collection.

Some other studies have also reported antidiarrheal activity of C. rotundus. Antidiarrheal action in castor oil-induced diarrhea and in irritable bowel syndrome in animal models has been demonstrated.[1213] However, there is a dearth of information regarding its mechanism(s) of action in controlling infectious diarrhea. We, therefore, undertook the present study.

The present study was intentionally restricted to crude extract as it is our belief that the different biological activities may not be due to a single constituent. This has also been highlighted in recent studies on Psidium guajava and Alchornea cordifolia.[814] Previous studies with the essential oil of C. rotundus showed it to be more bactericidal against Gram-positive bacteria.[1516] In this study, however, C. rotundus showed no antibacterial activity which could be due to a difference in the extract used and/or a difference in the test strains, all the strains being Gram negative. The major constituents present in C. rotundus are essential oil, triterpenes, polyphenol, alkaloids and flavonoids. However, none of these have been attributed with antidiarrheal activity.[10] The decoction used herein showed the presence of carbohydrates, reducing sugars, proteins, amino acids, tannins, flavonoids and saponins. Tannins and flavonoids, in general, have been reported to have antidiarrheal activity.[17-19] Thus, these compounds may be responsible for the observed activity. However, it may be noted that since tannins and flavonoids have not been studied for their activities vis-à-vis colonization of enteric pathogens to the gut epithelium and/or production and action of enterotoxins, further investigations with isolated constituents are necessary.

The results show that C. rotundus has limited antimicrobial action. Since bacterial colonization was reduced when the HEp-2 cells were incubated with the decoction prior to and simultaneously with infection, it is likely that C. rotundus affects metabolism of HEp-2 cells and/or modifies its receptors to prevent bacterial adherence/entry. Since the decoction did not kill V. cholerae, the suppression of CT production could be due to its effect on bacterial metabolism. LT production was, however, not affected. Since LT and CT have antigenic similarities, the differential effect on binding of these toxins (inhibiting the binding of only LT) suggests that the decoction may not be affecting the common antigenic moiety of these toxins.[20]

To conclude, the results suggest that C. rotundus has limited activity against different forms of infectious diarrhea due to its selective activity against diarrheal pathogens. In the absence of a marked antimicrobial activity, this plant seems to exhibit the antidiarrheal action because of its action on some features of bacterial virulence viz., bacterial colonization, production of CT and action of LT.