Antimicrobial Activity of Root Extracts of Cyperus rotundus (Linn) using Diarrhoea Inducing Microbes

Shivakumar S. I.*, Amreen Begum, Syeda Sana, Ansari Firdous

Department of Pharmacology, HKE’S Matoshree Taradevi Rampure Institute of Pharmaceutical Sciences Gulbarga, Sedam Road, Gulbarga-585105, Karnataka, India.

ABSTRACT:

Due to the development of antibiotic resistance and the outbreak of infectious diseases caused by resistant pathogenic bacteria, the pharmaceutical companies and the researchers are now searching for new unconventional antibacterial agents. Hence, there is an ever-growing need to develop new antimicrobial compounds. In the present study various medicinal plants were selected to check the antimicrobial activity and Cyperus rotundus (linn) was found to have the best activity against microbes. Pet ether extract, chloroform extract and methanol extract were screened for their antimicrobial activity against various microbes which are responsible for inducing diarrhoea viz. Staphylococcus aureus, bacillus cereus, Escherichia coli and Campylobacter jejunir using agar well diffusion method. Methanolic extract at the dose of 100 µg, have shown good antibacterial activity where as Methanolic extract at the dose of 80 µg have shown moderate activity, using Loperamide as a standarad drug, against Staphylococcus aureus bacillus cereus, Escherichia coli and Campylobacter jejuni with zones of inhibition ranging from 19 mm to 24 mm. It might be concluded from the present study that the potential of this herb to produce useful antimicrobial compounds is great and must be better explored

KEYWORDS: Cyperus rotundus (linn), pet ether, chloroform and Methanolic extract Loperamide antimicrobial activity.

INTRODUCTION:

Cyperus rotundus (linn) grows all over India upto 2000 meters altitude, especially on the banks of streams and rivers. The spiklets in compound umbels of this herb are 5-20 cm long. The rhizomes are blackish, hard, fragrant tubers.¹ The decoction of the roots and tubers are excellent antidote to all poisons. A paste of the fresh tubers applied to the breast acts as an effective galactagogue. The root is often used for developing high memory. This herb also harmonizes the liver, spleen, and pancreas. It has anthelmintic, anti-fungal, anti-parasitic, anti-rheumatic, antispasmodic, aphrodisiac and astringent. It enjoys an important place among medicinal herbs in India since ancient times, the drug of choice for any type of fever, an appetizer, digestant and anti–diarrhoeal, anti-saturative, thirst relieving, reducing herb, anti-pruritic and lactodepurant herb. It is also well known for its digests and diaphoretic properties. It cures dyspepsia, vomiting, indigestion, thirst, worm troubles, cough, bronchitis, dysuria, and poisonous affections.

Cyperus rotundus (linn) belongs to family Cyperaceae. The essential oil from the plant contains at least 27 components comprising sesquiterpene hydrocarbons, epoxides, ketones, monoterpene and aliphatic alcohols and some unidentified constituents, The rhizomes contain sitosterol, cyperene, seniline, cyperenone and sesquiterpenes²cyperone is obtained from the tubers. The bulbous roots of C. rotundus have great medicinal value and are used for medicinal purpose.

It is used both, internally as well as externally. C. rotundus is used in vast range of diseases. It is one of the best herbs, useful in digestive disorders. It is a keen stimulant for appetite, digestion, and is also vermicide, astringent, sedative.³ C. rotundus is highly praised as the best panacea for dental diarrhea in children. It is also one of the most effective menstrual regulators. (Shamkuwar et. al., 2012). The effect of this herb against most of the microbes is largely unexplored. So in the present study an attempt was made to see the antimicrobial activity of this herb against microbes causing diarrhoea. Diarrhoea is induced by various microorganisms like Staphylococcus aureus, Bacillus cereus, Escherichia coli and Campylobacter jejuni⁴ etc. Few studies have found that the efficacy of antimicrobial agents can be improved by combining them with crude plant extracts against different microbes with crude plant extracts against different microbes. This alternative approach has been evaluated using in vitro interaction between all plant extracts of C. rotundus (PEECR, CECR and MECR)⁵ and known antibiotic like Loperamide using agar well diffusion method against various microbes.

Diarrhoea is the most common infectious disease worldwide.⁶ Gastrointestinal infections kill 1.8 million people globally each year, mainly children in developing countries.⁷Acute, watery, bloody diarrhoea may be due to a variety of pathogens- bacterial (e.g., Staphylococcus aureus, Bacillus cereus, Escherichia coli, and Campylobacter jejuni ), agents. These organisms disrupt intestinal functions and cause diarrhoea through several mechanisms. These include microbial attachment to the intestinal epithelium and localised effacement, production of toxin(s), and penetration and invasion of intestinal epithelial cells that result in alteration of absorption due to the rearrangement in cytoskeletal structure.⁸

MATERIALS AND METHODS:

Microorganism

Bacillus cereus Gram positive bacteria

Staphylococcus aureus Gram positive bacteria

Campylobacter jejuni Gram negative bacteria

Escherichia coli Gram negative bacteria

PLANT MATERIAL

The plant Cyperus. rotundus (linn) (Family cyperaceae) was collected from the all over india. In the month of July 2012.The plant was identified by Prof. S.B. Beknal, Department of Pharmacognosy, H.K.E.S’s MTRIPS, Gulbarga. The herbarium specimen is preserved in the college museum, and air-dried for two weeks at room temperature (25±2°C) and pulverized with a grinder into smooth powder for solvent extractions. Solvent extractions were carried out in the following solvents in the given sequence viz. Petroleum ether, Chloroform and methanol. This sequence was used to analyse the efficacy of various solvents.

PREPARATION OF MEDIA:

Nutrient agar media was prepared by dissolving (40 g), bacteriological peptone (1 g), beef extract (5 g) and sodium chloride (5 g) in distilled water 1000 ml. The pH of the solution was adjusted to 7 to 7.4 by using NaOH solution (40% approximately 0.25 ml for 100 ml of nutrient broth) and then sterilized for 30 minutes at 15 lbs pressure in an autoclave.⁹

PREPARATION OF SUB CULTURE:

One day prior to test, the microorganisms were inoculated into the sterilized nutrient broth and incubated at 37⁰C for 24 h. On the day of testing the organisms were sub-cultured into sterile nutrient broth. After incubating for 3 h, the growth thus obtained was used as inoculums for the test.

STERILIZATION OF MEDIA AND GLASS WARES:¹⁰

The media used in the present study, nutrient agar and nutrient broth were sterilized in a conical flask of suitable capacity by autoclaving the same at 15 lbs pressure for 20 minutes. The cork borer, petridishes, test tubes and pipettes, were sterilized by employing hot air oven at 160⁰C for 1 h.

PREPARATION OF SOLUTION OF TEST COMPOUND:

The test compounds (5mg each) was dissolved in freshly distilled DMF (5 ml) in serially labeled sterile test tubes, thus giving a final concentration of 100 µg/0.1 ml.

METHOD OF TESTING ¹¹:

The method depends on the diffusion of an antibiotic from a cavity through the solidified agar layer in a petridish to an extent such that growth of the added microorganisms is prevented entirely in a circular area or zone around the cavity containing a solution of an antibiotic. About 15-20 ml of molten nutrient agar was poured into each of the sterile petridishes. The cups were made by scooping out nutrient agar with a sterile cork borer.

Table no.1 Antimicrobial activity of root extracts of Cyperus rotundus (Linn) against diarrhoea inducing Microbes

S No	Treatment	DOSE µg/ml	Diameter of Inhibition of Zones (mm)
S No	Treatment	DOSE µg/ml	E.C	E.C	E.C	E.C
1	Control	-	24±0.9	24±0.3	24±0.2	23±0.9
2	Standard	3	10±0.8	12±0.6	11±0.6	13±0.6
3	PEECR	20	24±0.1	21±1.0	22±0.09	23±0.4
4	CECR	40	22±0.10	23±0.20	23±0.14	20±0.6
5	MECR	80	19±0.5	21±0.4	19±0.23	20±0.18
6	MECR	100	18±0.15	19±0.5	21±0.78	19±0.35

The agar plates so prepared are divided into different set and each set of the plates were inoculated with the suspension of particular organism by spread plate technique. The cups of inoculated plates were then filled with 0.1 ml of the test solution; the plates were then incubated at 37⁰C for 24 h. The zone of inhibition developed, if any, was then measured for the particular compound with each organism. The solvent DMF was used as negative-control to know the activity of the solvent. The results of antibacterial testing are summarized in the Table No.1

STATISTICAL ANALYSIS

The correlation coefficient is calculated for zones of inhibition of extract alone and for the zones of inhibition due to both extracts and antibiotic. The standard deviation, variance, z-test, t-test, Critical 2-sided T-value (5%) and Critical 1-sided T-value (5%) are calculated to analyze the data statistically. The result of conducted experiments using well-diffusion method. The results are shown in table no. 1.The experiments were conducted in triplicates and the mean was recorded. The Zones of inhibition were observed in the range of 19-24 mm.

RESULTS:

According to the World Health Report 2004, diarrhoea is the cause of 3.3% of all deaths.¹² The past decade has witnessed several attempts towards the management of diarrhoea. These include improved formulations of oral rehydration solution (ORS) and the development of a feasible vaccine. Although ORS has contributed to reduction in diarrhoeal mortality rates, it is often less efficient in high stool output state. In addition, response to vaccines in developing countries is not encouraging.^13,14 With the threat of drug resistance, a definite niche exists for the development of an alternative approach to treat infectious diarrhoea. Medicinal plants can fill this niche. This study was an attempt to explore the antimicrobial activity of PEECR, CECR and MECR extract of Cyperus rotundus (Linn). It was observed that The MeOH extract affected colonisation.

DISCUSSION:

All the Plant extracts were screened for antimicrobial activity. Among those MECR 100 µg, have shown good antibacterial activity where as MECR 80 µg have shown moderate activity. However, the CECR 40 µg were weakly active. Where as PEECR 20 µg have shown no activity on tested gram + ve and gram – ve organisms in comparison with standard loperamide at the concentrations 20, 40, 80 and 100 µg/ml.

The extracts have bioactive compound which inhibits the growth of various pathogens. The spectrum of the antimicrobial compound present is found to be broad as it is inhibiting bacterial spicies¹². So this herb has potential to produce useful antimicrobial compounds and it must be better explored.

CONCLUSION:

PEECR, CECR and MECR inhibited the adherence of E. coli, Staphylococcus aureus, Bacillus cereus and Campylobacter jejuni. The decrease in colonisation was observed in the above study. suggesting that as mentioned earlier, Cyperus rotundus (Linn) had antimicrobial activity.

ACKNOWLEDGMENT:

The authors are thankful to Prof. S.B. Beknal, department of Pharmacognosy, H.K.E.S’s MTRIPS, Gulbarga for authenticating the collected plant material. The authors also wish to thank the authority of MTR IPS Gulbarga Karnataka, and Department of Pharmacology, Rajiv Gandhi University for providing necessary facilities to carry out this research work.

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Received on 09.05.2013

Modified on 05.06.2013

Accepted on 11.06.2013

Research J. Pharmacology and Pharmacodynamics. 5(4): July–August 2013, 244-246