Preliminary Phytochemical Screening and Antibacterial Activity of Ethyl Acetate Extract of Cuscuta reflexa Roxb.

 

Manore D.*, Pillai S., Joshi A. and Punashiya R.

GRY Institute of Pharmacy, Borawan (Khargone) MP – 451 228

Corresponding author: deepmala30pharm@gmail.com

 

ABSTRACT:

The plant Cuscuta reflexa Roxb. belonging to family Convolvulaceae was selected for the project, on the basis of ethnobotanical information and easy availability. The whole plant of Cuscuta reflexa Roxb. were successively extracted with Petroleum ether and Ethyl acetate by soxhlet extraction. Preliminary Phytochemical screening of Ethyl acetate extract of whole plant of Cuscuta reflexa Roxb. resulted in the identification of various chemical constituents such as - Cardiac glycosides, Phenols and Tannins. Further the Ethyl acetate extract were vacuum dried and subjected for Antibacterial activity by Agar well diffusion method. Zone of inhibition produced by Ethyl acetate extract in dose 25, 50, 100, 150mg/ml against human pathogenic bacteria stains gram positive and gram negative respectively Mycobacterium tuberculosis (MTCC 300) and Salmonella typhimurium (MTCC 98) were measured and compared with standard antibiotics Ciprofloxacin (10µg/ml). The present study demonstrated that the Ethyl acetate extract of Cuscuta reflexa Roxb. shows potent Antibacterial activity against MTCC 98 and MTCC 300 at concentration 150mg/ml respectively. So on the basis of the zone of inhibition we are concluded that Ethyl acetate extract shows more potent Antibacterial activity against gram negative Salmonella typhimurium (MTCC 98) bacterial stains and studies revealed that the Ethyl acetate extract of Cuscuta reflexa Roxb. having Antibacterial activity.

 

 

INTRODUCTION:

Many plants were found to contain compounds which are used as natural medicines to treat common bacterial infection. Indian medicinal plants are regularly used in various system of medicine because of minimal side effect and cost effectiveness. The potential for developing antibacterial from plants appears rewarding as it may lead to the development of phytomedicine against bacteria. Hence the sensitivity study of bacterial stains to the plant Cuscuta reflexa Roxb. was evaluated. Cuscuta reflexa Roxb. belonging to family Convolvulaceae popularly known as “Amarbela”¹ in Hindi and “Dodder”² in English ; is a holoparasite, parasitizing a large number of Angiospermic plants.

 

The continuity of parasite with the host plant is maintained by means of special organs called Hausteria through which it absorb organic and inorganic solute. Cuscuta reflexa Roxb. is a rootless, leafless plant². Comprised of twining stem³ and white waxy flowers^2,4. This plant is found in all over India, Sri Lanka, Afghanistan, Pakistan, Bhutan, Nepal, Malaysia etc^2,3. Cuscuta reflexa Roxb. is a folk remedy for Cough,  Eye infection, Eczema, Constipation, Heart disease ,Chronic Diarrhea, Epilepsy, Paralysis, Insanity, Vomiting, Myalgia, Headache, Pain of Joints, Vertigo, Fever, Skin disease, Bronchitis, Jaundice etc. Seed are carminative and anthelmintic and also used along with sarsaparilla to purify blood. Stems are also used in bilious disorders. Ayurvedics believing the stem to be – Demulcent, Diaphoretic, Hepatoprotactive, Ophthalmic, Tonic, Purgative, Bitter, Expectorant, Anthelmintic, Diuretic, Antipyretic, Antifungal, Antimicrobial, Antioxidant and  Hair growth promoting activity etc. Various chemical constituents are reported in Cuscuta reflexa Roxb. like – Plant contains - Alkaloids, Proteins, Flavonids, Resin, Tannins, Glycosides, Carbohydrates, Cuscutin, Cuscatalin, β-sitosterol, Reducing sugar, Ferulic acid etc. Seeds contain – Fixed Oil, Pigment, Bitter Resin, Reducing sugar etc^1,4,5.6,7.

 

MATERIALS AND METHODS:

PLANT MATERIAL: The details regarding the description and identification of plant were already given. The plant Cuscuta reflexa Roxb. parasitizing on Ficus bengalensis was collected from forest of Simrol, Mhow; district (M.P.), India in month of Sep. 2010. The plant   was   identified and authenticated by Dr. C. L. Dulkar, M.Sc., Ph D., Asstt. Professor of Botany, Govt. P.G. College, Khargone district, (M.P.), India. The specimen copy submitted to GRY Inst. of Pharmacy, Borawan.

 

EXTRCTION OF PLANT MATERIAL:

The whole plant of Cuscuta reflexa  Roxb. was dried in shade and subjected to size reduction to get coarse powder. The dried coarse powder (900gm) of Cuscuta reflexa Roxb. plant was defatted with petroleum ether at 60-80˚C by continuous hot percolation method. The defatted marc was successively extracted with ethyl acetate by continuous hot percolation by using soxhlet apparatus for 72 hr. The ethyl acetate extract was taken and then filtered and concentrated to the dry semi-solid mass. A greenish black residue (3.18%) table no. 2 was obtained it is used for Preliminary Phytochemical Screening and Antibacterial activity of plant.

 

PRELIMINARY PHYTOCHEMICAL SCREENING^8-11:

The qualitative chemical test of ethyl acetate extract of Cuscuta reflexa Roxb. was performed as per standardized  procedure and various phytoconstituents were identified in the extract are cardiac glycosides, phenols and tannins.

 

IN VITRO ANTIBACTERIAL ASSAY:

Test organisms: The antibacterial activity of the ethyl acetate extract was studied against gram positive and gram negative bacterial strains. The test organisms, Salmonella typhimurium (MTCC 98) are gram negative and Mycobacterium tuberculosis (MTCC 300) are gram positive were obtained from the Institute of Microbial Technology, Chandigarh, India.

 

Nutrient Medium¹²: Culture of the test organism were maintained on nutrient agar slants and were sub cultured in Petri dishes to testing. Three replicates were maintained for each treatment. The antibacterial activity was performed using agar well diffusion technique. Nutrient agar (1.2%) medium was weighed and suspended in distilled water and heated to boiling until it is dissolved completely.

 

Table 1: Composition of media

Nutrient Broth
Beef extract	10 gm
Peptone	10 gm
Sodium chloride	5 mg
Water up to	1000 ml

For nutrient broth the above ingredients are dissolving with the aid of heat. Adjust the pH   8.0 to 8.4 with 5M Sodium hydroxide and boil for 10 minutes. Filter sterilizes by maintaining temp. at 115°C for 30 minutes and adjust the pH  7.3 ±0.1.

 

Sterilization:

1. Sterilization of media: The sterilization of media was done by autoclaving at 15 lb/sq. inch pressures for 30 min.

2. Sterilization of other material: The sterilization of culture tubes, pipettes and other materials was done by hot air oven at 160°C for 60 min.

 

Bacterial Stock Culture: A loopful of bacterial strain were transferred to nutrient medium and incubated for 24hr. at 37 °C. The number of colony forming unit were found to be 10³ per ml.

 

Standard Drug: Ciprofloxacin (10µg/ml).

Preparation of Drug Sample: Ethyl acetate extract was dissolved in DMSO solvent and make different concentration sample such as 25mg/ml, 50 mg/ml, 100mg/ml and 150mg/ml.

 

Method employed for antibacterial studies:

Agar well diffusion method^13,14,15: Antibacterial  study was determined by using agar well diffusion plate method. The nutrient agar plates were prepared by pouring 20 ml of molten media into sterile Petri plates under aseptic condition in a laminar flow chamber. The plates were allowed to solidify for 5 minutes and 0.5 ml of test culture was inoculated and uniformly spread over the agar surface with the help of a sterile L- shaped bent glass rod. After inoculation, wells were scooped out with 6 mm sterile cork borer and the lids of the dishes were replaced. To each well, different concentrations of test solutions were added separately and controlled were same maintained. The treated plates were kept for incubation at 37°C for 24 hr. At the end of incubation, inhibition zones formed around the discs were measured and diameter was calculated in millimeter.

 

RESULT AND DISCUSSION:

The exhaustive successive extractions of the plant material were done with the Ethyl acetate and characteristics of extract are shown in table no.2 then the extract was screened for the presences of the medicinally active Phytoconstituents.

 

The Preliminary Phytochemical Screening of ethyl acetate extract of Cuscuta reflexa Roxb. revealed the presence of cardiac glycosides, phenols and tannins. The observed result of Preliminary Phytochemical Screening of ethyl acetate extract of Cuscuta reflexa Roxb. are listed in table no.3.

 

Table 2: Characteristics of C.reflexa Roxb. whole plant extract.

Extract	Yield (%w/w of powdered drug)	Physical Appearance	Color	Odor
Ethyl acetate extract	3.18	Semi solid Mass	Greenish black	Aromatic

The ethyl acetate extract of whole plant of Cuscuta reflexa Roxb. used in the study to investigate their antibacterial potential by using both gram positive Mycobacterium tuberculosis (MTCC 300) (fig.-2) and gram negative Salmonella typhimurium (MTCC 98) (fig.-1) bacterial stains as test organism. Ciprofloxacin used as positive control. The ethyl acetate extract showed antibacterial activity against both the tested bacterial stains and it gives maximum activity at the higher concentration 150mg/ml respectively. So on the basis of the zone of inhibition (table no.4) we concluded that ethyl acetate extract of whole plant - Cuscuta reflexa Roxb. was shows more potent antibacterial activity against gram negative bacteria Salmonella typhimurium (MTCC 98)  and studies revealed that the ethyl acetate extract of Cuscuta reflexa Roxb. having antibacterial activity.

 

Table 3: Phytochemical present in whole plant extract of C.reflexa Roxb.

Sr. No.	Phytoconstituents	Ethyl acetate extract
1.	Alkaloids	-
2.	Carbohydrates	-
3.	Glycosides a. Anthraquinone  glycosides b. Saponins glycosides c. Cardiac glycosides	- - +
4.	Phenols and tannins	+
5.	Steroids	-
6.	Terpenoids	-
7.	Saponins	-
8.	Flavonids	-
9.	Fats and fixed oil	-
10.	Protein and amino acids	-

(+) = present, (-) = absent.

 

(A)

 

 

(B)

 

(C)

Fig. 1: Antibacterial studies of Ethyl acetate extract of whole plant of Cuscuta reflexa Roxb. with MTCC 98 bacteria  at (A)50mg/ml, (B)100mg/ml, (C)150mg/ml concentration.

 

(A)

 

(B)

 

(C)

Fig. 2: Antibacterial studies of Ethyl acetate extract of whole plant of Cuscuta reflexa Roxb. with MTCC 300 bacteria  at (A)50mg/ml, (B)100mg/ml, (C)150mg/ml concentration .

Table 4: Antibacterial activity of Ethyl acetate extract of whole plant of C. reflexa Roxb. on various bacterial strains  by Agar well diffusion method.

SN.	Bacterial Strains	*Standard (10µg/ml)	Zone of inhibition in mm
			*DMSO	Ethyl acetate extract
				25 mg/ml	50 mg/ml	100 mg/ml	150 mg/ml
1.	MTCC 98	40	--	0	12	15	20
2.	MTCC 300	34	--	0	9	12	17

 

*Standard = Ciprofloxacin (10µg/ml); DMSO = Dimethyl sulphoxide. MTCC 98 = Salmonella typhimurium; MTCC 300 = Mycobacterium tuberculosis.

 

CONCLUSION:

The Preliminary Phytochemical Screening of ethyl acetate extract of Cuscuta reflexa Roxb. revealed the presence of cardiac glycosides, phenols and tannins. The ethyl acetate extract showed antibacterial activity against both the tested bacterial stains we concluded that ethyl acetate extract of whole plant - Cuscuta reflexa Roxb. was shows more potent antibacterial activity against gram negative bacteria Salmonella typhimurium (MTCC 98)  and studies revealed that the ethyl acetate extract of Cuscuta reflexa Roxb. having antibacterial activity.

 

REFERENCES:

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2.        Chopra, R. N., Nayar, S. L. and Chopra, I. C.  Glossary of Indian Medicinal Plants, Council of Scientific and Industrial Research New Delhi, 1956:218.

3.        Nadkarni, K. M. and Nadkarni, A. K., Indian Materia Medica, Popular Prakashan  Bombay, 1976:207.

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5.        The Wealth of India, Raw Materials, Publications and Information Directorate, CSIR,   Hillside road, New Delhi, India, IX, 1972:177.

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10.     Harbone, J. B., Phytochemical methods- A guide to modern techniques of plant analysis. 3^rd Edition Springer, 2005:1-32,291-293.

11.     Raaman., N., Phytochemical techniques, New India publishing agency New Delhi. 2006:25.

12.     Indian Pharmacopoeia, Government of India, 6th ed., Vol. I, Ministry of Health and Family Welfare, 2007:39.

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Received on 30.08.2011          Modified on 20.09.2011

Accepted on 03.10.2011         © RJPT All right reserved