Preliminary Phytochemical Analysis and Antimicrobial Activity of In -vitro Condition Asparagus racemosus Willd. leaf 

 

L. S. Patel¹ and R. S. Patel²

¹Jagdishprasad Jhabarmal Tibrewala University, Jhunjhunu, Rajasthan.

 ²Maninagar Science College, Ahmadabad, Gujarat.

 

 

ABSTRACT:

This study scientifically supports the usage of plant as a remedy for various superficial bacterial and fungal infections in traditional medicine. The findings of the current study also can be useful to progress further scientific investigation on the leaf of this species. The present study deals with preliminary phytochemical analysis and antimicrobial activity of in vitro condition Asparagus racemosus Willd. leaf with various extracts like petroleum ether, methanol, chloroform, acetone, ethyl acetate and water. The presence and absence of the primary and secondary phytoconstituents like Alkaloids, Glycosides, Cardiac Glycosides, Saponins, Tannins and Phenolic Compounds, Flavonoids, Anthroquinone, Proteins and Amino Acids, Sterols and Triterpenoids and Carbohydrates were detected by usual prescribed methods. The effect of different extracts were tested on Gram positive bacteria like Bacillus subtilis, Staphylococcus aureus and Gram negative bacteria E coli, Pseudomonas aeruginosa  and the yeast Candida utilis by agar well diffusion method. In the present study showed the presence of primary and secondary metabolites like alkaloids, tannins, phenolic compound, Flavonoids, steroids, Saponins, glycosides and carbohydrates and proteins in the Methanolic extracts than other extracts like chloroform, Acetone, Petroleum ether, Ethyl acetate and Water. The maximum antimicrobial activity at 100µg/ml was found for Staphylococcus aureus (20mm) and Escherichia coli (20mm) in methanol and minimum 8 mm in Escherichia coli in petroleum ether leaf extracts of A.racemosus In vitro condition and no activity of fungi was observed against some extracts.

 

 

 

INTRODUCTION:

Asparagus racemosus Willd, is commonly known as Shatavari, belongs to family Liliaceae (Madhavan et al., 2010) which is distributed in tropical and subtropical forest and in central parts of India. Medicinal plants are the nature’s gift to human being to make disease free healthy life. It plays a vital role to preserve our health. Medicinal plants are believed to be much safer and proved elixir in the treatment of various various disorders such as Antiulcer (Sairam et al., 2003), Antitussive, Antioxidant (Mandal et al., 2000A; Kamat et al., 2000; Wiboonpun et al., 2004), Antitumour, Anticancer (Senna et al., 1993; Shao et al., 1996; Diwanay et al., 2004), immunostimulant properties, Antiulcerogenic (Datta et al., 2002), Antiinfammatory (Mandal et al., 1998), Antimicrobial (Mandal et al., 2000), immunomodulator (Rege et al., 1999), Antistress (Kamat et al., 2000), Antiamoebic, Antifungal (Nair and chanda, 2006) and Antiviral (Rajbhandari et al., 2001), treatment of menopause and immune system modulator.

 

MATERIALS AND METHODS:

Plant Material:

The leaf extracts of in vitro Asparagus racemosus (Family: Liliaceae) was collected. The collected leaves were washed; shade dried and was pulverized with mechanical pulveriser for size reduction. It was then passed through whattman filter; the fine powder was collected and stored in air tight container for the further study.

 

Preparation of extracts:

The dried powder of leaf was successively extracted with Petroleum ether, Acetone, Chloroform, Methanol, Ethyl acetate and Water in soxhlet apparatus. The extract was stored at 4ºC and used for phytochemical analysis and antibacterial activity.

 

Phytochemical analysis:

 In the Preliminary Phytochemical analysis, presence and absence of the primary and secondary phytoconstituents like Alkaloids, Glycosides, Cardiac Glycosides, Saponin, Tannins and Phenolic Compounds, Flavonoids, Anthroquinone, Proteins and Amino Acids, Sterols and Triterpenoids, and Carbohydrates were detected by usual prescribed methods (Khandelwal, 1996 and 2006; Harborne, 1998; Evans and Trease, 2002; Mukherjee, 2002; Mali et al., 2010; Kokate et al., 2003, 2008 and 2009; Sofowara, 1993).

 

Antimicrobial activity:

The effect of different extracts were tested on Gram positive bacteria like Bacillus subtilis, Staphylococcus aureus and Gram negative bacteria E coli, Pseudomonas aeruginosa and the yeast Candida utilis by agar well diffusion method to detect the presence of anti-bacterial and anti-fungal activities (Perez et al., 1990; Popoola et al., 2007). Nutrient Agar (Hi-media) for bacteria and Sabouraud’s Agar (Hi-media) for fungus were prepared according to the manufacturer’s instructions. Nutrient agar slants after solidification was inoculated with the test microorganisms, by spreading the bacterial inoculums under aseptic conditions. Wells of 5mm diameter were punched in the agar medium with sterile cork borer and filled with plant extract. Control wells containing neat solvents (negative control) or standard antibiotic solution (positive control) viz., Streptomycin used (Valsaraj et al., 1997). The plates were incubated at 37⁰C (fungal plates at 28⁰C) for 24 hrs. The negative control was added without adding the cultures to know the sterile conditions. The antibacterial activity was assessed by measuring the diameter of the zone of inhibition for the respective plant extract and antibiotic. The relative antibacterial potency of the given preparation was calculated by comparing its zone of inhibition with that of the standard drug Streptomycin for bacteria and Fluconazole for fungi. The readings were taken in all 3 replicates (Battu and Kumar, 2010).

 

Table.1 Phytochemical screening of In vitro A.racemosus leaf extracts.

Sr. No.	Name of the Test	Petroleum ether extract	Methanol extract	Chloroform extract	Acetone extract	Ethyl acetate extract	Water extract
1	Test for Alkaloids
A.	Dragendroff’s Test	+	-	+	-	+	+
B.	Mayer’s Test	-	+	-	-	+	-
C.	Wagner’s Test	+	+	-	+	-	+
D.	Hager’s Test	-	-	-	-	-	-
2	Test for Glycosides
A.	Legal’s Test	+	+	-	-	+	-
B.	Baljet’s Test	-	-	+	+	-	+
C.	Raymond’s Test	-	+	-	-	-	-
D.	Modified Borntrager's test	+	-	+	+	+	+
3	Test for Saponins
A.	Forth Test	-	+	-	-	+	+
B.	Hemolytic Test	+	-	+	-	+	-
4	Test for Flavanoids
A.	Shinoda Test	-	+	-	+	-	-
B.	Zn-HCl Test	-	-	+	-	-	+
C.	Alkaline reagent Test	+	+	-	+	-	-
D.	Lead acetate test	-	+	+	+	-	+
E.	Ammonia Test	+	+	-	-	-	-
5	Test for Tannins
A.	Gelatin Test	-	+	-	+	-	-
B.	Ferric Chloride Test	+	-	+	-	+	+
C.	Vanillin-HCl Test	-	+	-	-	+	-
D.	Lead acetate Test	-	-	+	+	-	+
6	Test for Sterols
A.	Libermann- Buchard test	+	+	-	-	+	+
B.	Salkowaski Test	-	+	+	+	+	-
7	Test for Anthroquinone	+	+	+	+	+	+
8	Test for Triterpinoids
A.	Libermann- Buchard test	-	-	-	-	-	-
B.	Salkowaski Test	-	+	-	-	+	-
9	Test for Proteins
A.	Xanthoproteic Test	-	-	-	+	-	-
B.	Millon's Test	+	-	-	-	+	-
C.	Biuret Test	-	-	+	-	-	-
D.	Ninhydrin Test	-	+	-	-	-	+
10	Test for Carbohydrates
A.	Molisch's Test	+	+	-	+	+	-
B.	Barfoed's Test	-	+	+	-	-	+
C.	Benedict's Test	+	+	-	-	+	-
D.	Fehling's Test	-	-	-	+	-	-

+ indicates the presence of active constituents       

- indicates the presence of active constituents

 

 

RESULTS:

Different extract like Petroleum ether, Benzene, Chloroform, Ethyl acetate, Water, Methanol was subjected to preliminary phytochemical analysis for their presence of the constituents. It showed the presence of primary and secondary metabolites like alkaloids, tannins, phenolic compound, Flavonoids, steroids, Saponins, glycosides and carbohydrates and proteins. All the results are tabulated in table 1.

 

In the present study, we evaluated the antibacterial and antifungal investigations were carried out from the leaf of in vitro Asparagus racemosus Willd. using different solvents like Petroleum ether, Methanol, Chloroform, Acetone, Ethyl acetate and Water were subjected to antimicrobial activity against Bacillus subtilis, Staphylococcus aureus,, Escherichia coli, Pseudomonas aeruginosa and candida utilis. The results are showed in Table 2. 

 

DISCUSSION:

Phytochemical screening Showed in vitro condition that leaf extracts of A.racemosus was rich with different chemical constituents like Alkaloids, Glycosides, Saponins, Flavonoids, Tannins, Sterols, Anthraquinones, triterpenoids, Proteins and carbohydrates.

 

Many compounds were found in the Methanolic extracts than other extracts like chloroform, Acetone, Petroleum ether, Ethyl acetate and Water. This is because methanol is much polar than chloroform, Acetone, Petroleum ether, Ethyl acetate and Water hence extracting many of the active ingredients.

Antimicrobial activity of in vitro condition that leaf extracts like Petroleum ether, Methanol, Chloroform, Acetone, Ethyl acetate and Water of A. racemosus was studied for antimicrobial activity using agar well diffusion method to detect the presence of antibacterial activity against Bacillus subtilis, Staphylococcus aureu, Escherichia coli, Pseudomonas aeruginosa and antifungal activity against Candida utilis for evaluation of its antimicrobial efficiency.

The antibacterial activity of different extracts like Petroleum ether, Methanol, Chloroform, Acetone, Ethyl acetate and Water of leaf extracts of A. racemosus plant with different concentration 100µg/ml, 50µg/ml, 25µg/ml was very well comparable with same concentration of standard reference drug streptomycin 5µg/ml for antibacterial activity and fluconozole 5µg/ml for antifungal activity.

 

The maximum antimicrobial activity at 100µg/ml was found for Staphylococcus aureus (20mm) and Escherichia coli (20mm) in methanol and minimum 8 mm in Escherichia coli in petroleum ether leaf extracts of A.racemosus In vitro condition and no activity of fungi was observed against some extracts.

 

ACKNOWLEDGEMENTS:

Author thanks to Dr. R.S. Patel, Assistant Professor, Biology Department, K.K. Shah Jarodwala Maninagar Science College, Ahmadabad (Gujarat) for taxonomic identification of the plant and his support for providing necessary facilities.

 

Table.2 Antimicrobial activity of In vitro A.racemosus leaf extract

Zone of inhibition (mm)
	Concentration (µg/ml)	Bacillus subtilis	Staphylococcus aureus	Escherichia coli	Pseudomonas aeruginosa	Candida utilis
Methanol	25	10	14	17	15	-
	50	11	17	19	16	-
	100	13	20	20	18	-
	control	0	0	0	0	0
Chloroform	25	9	7	11	9	-
	50	11	8	14	10	-
	100	12	9	16	12	-
	control	0	0	0	0	0
Water	25	9	9	8	6	7
	50	9	10	9	7	9
	100	10	10	11	9	10
	control	0	0	0	0	0
Ethyl Acetate	25	9	10	9	8	-
	50	11	12	10	8	-
	100	12	13	10	9	-
	control	0	0	0	0	0
Petroleum ether	25	10	9	8	9	-
	50	11	10	8	10	-
	100	13	10	8	11	-
	control	0	0	0	0	0
Acetone	25	8	7	9	10	6
	50	9	8	10	12	8
	100	10	9	12	12	9
	control	0	0	0	0	0
Standard Antibiotic		15	18	22	12	20

Value is mean of three replicates.

Standard Antibiotic Streptomycin 5 μg/ml for Bacterial Strain.

Standard Antibiotic Fluconazole 5 μg/ml for Fungi.

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Received on 15.10.2013       Modified on 22.10.2013

Accepted on 27.10.2013      © RJPT All right reserved