INTRODUCTION:

New antibiotics were produced by pharmacological industries in the last three decades¹. However, these antibiotics have failed to discourage the growth of many bacteria that have the genetic ability to transmit and acquire resistance to drugs².In addition, many researches have established the side effects of overuse and misuse of antibiotics which can harm vital organs like liver, kidneys and the cells of pancreas and spleen as well as their impact on the immune system³. Medicinal plants are rich in wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, flavonoids, phenols and quinones⁴. These compounds have been used worldwide in traditional medicine to treat several diseases and infection⁵. Annona reticulata L. belonging to family annonaceae is a highly apparent plant in Ayurvedic system of medicine for the treatment of various ailments⁶. Annona reticulata was selected to determine the antimicrobial activity as it contains phytochemicals such as tannic acid, flavonoids, phenolic compounds etc. The bark has been reported as powerful astringent and used in diarrhoea and dysentery⁷. It is also used as antidysentric and vermifuge. Leaves are used as anthelmintic. The unripe fruit is considered astringent, anthelmintic, antidysentric and used in treatment of fever. The fruits are used in treatment of diarrhoea⁸.

It is reported that seeds contain alkaloids, flavonoids and tannins. Phytochemical screening of plant has shown the presence of tannins, flavonoids and saponins. The present study was carried out to evaluate the antimicrobial activity of aqueous and ethanolic extract of bark and seeds of Annona reticulata.

MATERIALS AND METHODS:

Collection:

The bark and seeds of the plant Annona reticulata were collected in bags from Shirur during the month of January 2014.

Authentication:

The plant was authenticated from Botanical Survey of India, Pune.

Microbial strains used:

Microbial strains were obtained from National Collection of Industrial Microorganisms

Table 1: Microbial strains with their NCIM number

Name	NCIM number
E. coli	2065
S. aureus	2079
Salmonella abony	2257
P. aeruginosa	2200

Methods:

Preparation of extracts:

Stem bark and seeds of Annona reticulata were collected, cleaned and shade dried. Twenty grams of shade dried powder of stem bark was used for extraction with 200 ml ethanol for 24 hours. The solvent was removed in rotary evaporator and the crude extract was dried at room temperature in steady air current and stored at 4⁰ C. The dried seeds and bark were pulverised by a mechanical grinder and passed through a 20 mesh sieve. Powdered seeds and bark were extracted with chloroform water by cold maceration process for 7 days. At the end of 7^th days it was filtered through muslin cloth and the filtrate was concentrated. The remaining solution was evaporated by heating on a water bath. The brown coloured residue was obtained. The residue was then stored in desiccator⁹.

Screening for Phytochemical Constituents:

Aqueous and ethanolic extracts were subjected to preliminary phytochemical investigation for presence of secondary metabolites such as flavonoids, tannins, saponins etc.

Table 2: Phytochemical Analysis of Annona reticulata bark and seeds

Chemical constituents	Aqueous extract		Ethanolic extract
Chemical constituents	Bark	Seeds	Bark
Carbohydrates	+	+	+
Tannins	+	+	+
Flavonoids	+	+	+
Saponins	+	+	+

+ Indicates presence

Determination of Antimicrobial Activity:

The various strains of Gram-positive and Gram-negative bacteria were used for testing the antimicrobial activity of aqueous extracts of seeds and bark of Annona reticulata. Antimicrobial activity of ethanolic extract of bark of Annona reticulata was also determined. The assay was conducted by agar cup method.

Muller-Hinton medium was used as a nutrient medium for testing the antimicrobial activity¹⁰. It was sterilized by autoclaving at 121⁰ C for 15 min.⁶(15lb/sq.inch)¹¹. About 30ml of Muller-Hinton medium inoculated with the respective strains of bacteria was transferred aseptically into each sterile Petri plate. The plates were left at room temperature to allow solidification. In each plate wells of 8mm were made using a sterile cork borer. The extracts were freshly reconstituted with suitable solvent and tested at various concentrations. The test sample and the control (0.2 ml) were placed in 8mm well. Streptomycin was used as positive control. These plates were kept in the refrigerator for half an hour for pre-diffusion of the extract into agar layer. After pre-diffusion these plates were incubated at 37 ⁰C for 24 hours.

RESULTS AND DISCUSSION:

Preliminary phytochemical screening of the extracts showed presence of secondary metabolites such as tannins, flavonoids, carbohydrates and saponins. Tannins and flavonoids have been reported to possess antimicrobial activity. The aqueous and ethanolic extracts of seeds and stem bark of Annona reticulata were tested for antimicrobial activity against E. coli, P. aeruginosa, Salmonella abony and Staphylococcus aureus. Results showed that aqueous and ethanolic extracts of seeds and bark have significant antimicrobial activity against Staphylococcus aureus. E. coli was found to be less sensitive to aqueous and ethanolic extracts of seeds and bark. P. aeruginosa was found to be more sensitive to aqueous extract of stem bark than ethanolic extract. Salmonella abony was found to be resistant to all the extracts. The insensitivity of Salmonella abony may be due to the insufficient concentration of phytoconstituents required for antimicrobial activity.

Table 2: Antimicrobial Activity of Annona reticulata Bark and Seeds

Extracts	Concentrations ( mg/ml )	Zone of inhibition in mm for following bacterial strains
Extracts	Concentrations ( mg/ml )	E. coli	S. aureus	P. aeruginosa	Salmonella abony
Bark (aqueous extract)	10	-	18mm	11mm	-
	12	12mm	19mm	12mm	-
	14	13mm	20mm	16mm	-
Seeds (aqueous extract)	10	-	12mm	-	-
	12	-	13mm	12mm	-
	14	12mm	16mm	13mm	-
Bark (ethanolic extract)	10	-	10mm	-	-
	12	-	12mm	11mm	-
	14	11mm	13mm	13mm	-

-Indicates absence

CONCLUSION:

From the results it was concluded that aqueous extracts of bark of Annona reticulata showed better antimicrobial activity than other extracts. Further work on the profile and chemical constituents of seeds and bark of Annona reticulata will provide valuable information on the therapeutically active constituents responsible the pharmacological properties.

REFERENCES:

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Received on 02.07.2014 Modified on 25.07.2014

Res. J. Pharmacognosy & Phytochem. 6(3): July-Sept.2014; Page 129-131