INTRODUCTION:

Plants contain numerous biologically active compounds, many of which have been shown to have antibacterial properties^[1].So they have been used for the treatment of various diseases all over the world before the advent of modern clinical drugs and are known to contain substances that can be used for therapeutic purposes or as precursors for the synthesis of useful drugs^[2]. Even though pharmacological industries have produced a number of new antibiotics in the last three decades, resistance to these drugs by microorganisms has increased. In general, bacteria have the genetic ability to transmit and acquire resistance to drugs, which are utilized as therapeutic agents^[1,2]. Such a fact is cause for concern, because of the number of patients in hospitals who have suppressed immunity, and due to new bacterial strains, which are multi-resistant. Consequently, new infections can occur in hospitals resulting in high mortality. Diseases spread by microbes are the number one cause of death worldwide. Microbial resistance to almost all antibacterial agents has been reported^[3]. This resistance is largely due to indiscriminate use of antibacterial drugs commonly used in treatment of these diseases.

A part from resistance, some antibiotics has side effects which limit their usage. So there is a urgent need to discover new spectrum of antibacterial agents with minimal side effects, and higher plants are potential source of novel antibacterial prototypes ^[4,5].The present study describes the evaluation and phytochemical screening of antibacterial potential plant species Sapindus saponaria against four common human pathogens.

Sapindus saponaria (family: Sapindaceae) with alternative Botanical Names Sapindus thurstoniiI and commonly known as Manele, A'e , Hawaiian Soapberry is a known medicinal plant. Sapindus saponaria is a tall deciduous tree, growing up to 80 feet in height. It has pale brown bark that falls off in large patches on mature trees. The leaves are made up of 3 to 6 pairs of leaflets. The upper surface of the leaves is shiny green and the lower surface is fuzzy ^[6]. As a part of search for antibacterial compounds from these plants we extracted and screened the leaves for antibacterial activity. It was found that the alcoholic extract showed the antibacterial activity.

MATERIAL AND METHODS:

Plant material:

The matured healthy leaves of plant material Sapindus saponari was obtained from the local area in and around Narasaraopet, Guntur district (India). The plant was indentified based on its floral description given in the literature. The plant leaves were air dried under shade and made into fine powder by using hand homogenizer and sieved through sieve no. 40 and the fine powder was used for extraction procedure and other evaluation.

Chemicals:

All the solvents used in this study were purchased from Merck Chemicals, India, of analytical grade.

Preparation of extract:

The leaves were washed in tap water, shade dried for 20 days and made into a fine powder of 40 mesh size using the laboratory mill. Following that, 100g of the powder was filled in the thimble and extracted using 500 ml of distilled petroleum ether, ethanol and chloroform in soxhlet apparatus for 8 – 10 hours. The extract was filtered through Whatman No.1 filter paper to remove all unextractable matter, including cellular materials and other constitutions that are insoluble in the extraction solvent. The entire extract was concentrated to dryness using rotary flash evaporator under reduced pressure. The dried extract was redissolved in petroleum ether, ethanol and chloroform to yield solutions containing 50, 100, 200 and 300mg of leaf extract per ml solvent^[7,8]. (Table1).

Table 1 Extractive value of petroleum ether, ethanol and chloroform extracts of Sapindus saponaria

S. No	Type of extract	Extractive Value (in grams)
1	Petroleum ether	10
2	Ethanol	7.9
3	Chloroform	6.4

Table 2 Phytochemical screening of leaf extracts of Sapindus saponaria

S. No	Plant constituents	Petroleum ether extract	Ethanol extract	Chloroform extract
1	Test for Alkaloids	+	+	+
2	Test for Volatile oils	-	-	-
3	Test for Carboxylic acids	-	-	-
4	Test for Fixed oils	-	-	-
5	Test for Saponins	+	+	+
6	Test for Tannins	-	+	-
7	Test for flavonoids	+	+	+
8	Test for Phenols	-	+	-
9	Test for Carbohydrates	-	-	-
10	Test for Fatty acids	+	-	-
11	Test for Resins	-	-	-
12	Test for Quinones	-	-	-
13	Test for Terpenes	-	+	-
14	Test for Cardinolides	-	-	-
15	Test for Glycosides	-	+	+

+ Indicates the presence of the constituents.

- Indicates the absence of the constituents.

Preliminary Phytochemical Analysis:

Phytochemical screening of plant extracts was done following the standard procedure by Santaram (1983), Chhabra et al (1984) and Harbone (1998). All the prepared plant leaf extracts were subjected to preliminary phytochemical screening for the presence of alkaloids, quinines, resins, tannins, fixed oils, flavanoids, fats, saponins, phenolic compounds, Proteins and carboxylic acids ^{[9, 10]}. The results were shown in Table 2.

Antibacterial Screening:

The four different concentrations of the leaf extracts were tested for antibacterial activity using agar disc diffusion assay according to the method of Bauer et al., 1966 ^[10]. The strains of microorganisms obtained were inoculated in conical flask containing 100 ml of nutrient broth. These conical flasks were incubated at 37º C for 24 h and were referred to as seeded broth. Media were prepared using Muller Hinton Agar (Himedia, Mumbai, India), poured on Petri dishes and inoculated with the test organisms from the seeded broth using cotton swabs. Sterile discs of six millimeter width had been impregnated with 20 μl of test extract and introduced onto the upper layer of the seeded agar plate. The plates were incubated overnight at 37º C. Antibacterial activity was assigned by measuring the inhibition zone formed around the discs. The experiment was done three times and the mean values were presented. Streptomycin (10μg/disc) as standards.

a) Micro organisms used:

The screening of the antibacterial activity of crude extracted from the leaf of Sapindus saponaria were carried out individually on active cultures of Streptococcus faecalis, Streptococcus aureus, Bacillus subtilis and Bacillus peritolis. All the strains were procured from the Microbial Type Culture and collection, Chandigarh, India.

b) Preparation of media:

Muller Hinton Agar (MH, Hi media) was used. The formula (gm/liter) Beef extract 2g, casein acid hydrolysate 17.5g, starch 1.5 g and agar 17g; pH 7.4 ± 0.2.About 38g of MH agar was weighed and dissolved in 1000 ml of distilled water and adjusted to pH 7.4 ± 0.2, sterilized by autoclaving at 121 °C for 15 minutes at 15 psi pressure and was used for sensitivity tests ^[11-13].

RESULT AND DISCUSSION:

The petroleum ether, ethanol and chloroform extracts of Sapindus saponaria leaf having extractive value 10 gm, 7.9 gm and 6.4 gm (Table 1) on phytochemical screening showed the presence of alkaloids, saponins, steroids, flavonoids, glycosides and phenols as chemical constituents. The results are shown in Table 2. The antibacterial activities of various extracts like petroleum ether extract, ethanol extract, chloroform extract are evaluated and compared using disc diffusion method. All the test extracts of Sapindus saponaria possess significant antibacterial activity against these common human pathogens. Among the three extracts, the ethanol extract (Fig 2) showed a higher activity than other extracts (Fig -1 and 3). This may be due to the solvent extract containing different constituents having antibacterial activity. Ethanol was proved as the most effective solvent for extracting broad spectrum of antibacterial compounds from plants.

Figure no 1 Antibacterial activity of leaf petroleum ether extracts of Sapindus saponaria

Figure no 2 Antibacterial activity of leaf ethanolic extracts of Sapindus saponaria

Figure no 3 Antibacterial activity of leaf chloroform extracts of Sapindus saponaria

CONCLUSION:

The result of this work suggested that the leaf extracts of Sapindus saponaria has potent antibacterial activity against common human pathogens. Therefore the results justify the use of the leaf extract in treating these pathogenic strains and these may serve as leads for the development of new pharmaceuticals that address hither unmet therapeutic needs. . It is essential that research should continue to isolate and purify the bio active components of this natural plant and use in drug discovery.

REFERENCES:

1) COWAN MM. Plant products as antibacterial agents. Clin Microbiol Rev.12;1999: 564-582.

2) Sofowora A. Medicinal Plants and Traditional Medicine in Africa. John Willey and Sons ltd., Ibadan. 1982: 8-14.

3) Cohen, M.L. Epidemiology of drug resistance: implications for a postantibacterial era. Science. 257; 1992:1050-1055.

4) Truiti MCT, Sarragiotto MH, Filho BAA, Nakamura CV and Filho BPD. Mem Inst Oswaldo Cruz .98(2); 2003:283-286.

5) Maureer – Grimes B, Macbeth DL, Hallihan B and Delph S. Int. Journal of pharmacognosy. 34; 1996: 243 – 248.

6) Wagner, Warren L, Darrel R, Herbst and Sohmer S.H. Manual of the flowering plants of Hawai'i. 2 vols, Bishop Museum Special Publication 83. Honolulu: University of Hawaii Press and Bishop Museum Press.1990: 1229.

7) Kokate CK. Practical Pharmacognosy, Vallabh Prakashan, New Delhi. 2005.

8) Bakshu LMd. Ethnomedicobotanical and Phytochemical evaluation of certain rare, endemic and endangered medicinal plants from Eastern Ghats, Andhra Pradesh, India. 2002.

9) Harbone, JB. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis, Chapman and Hall London. 1998.

10) Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. American journal of clinical pathology. 45; 1966: 493-496.

11) Agnese AM, Perez C, Cabrera JL. Phytomedicine. 8; 2001: 389-394.

12) Acar JF and Goldstein FW. Disc susceptibility test: Antibiotics in laboratory medicine, 4^th edition, edited by L. victor, Williams and Wilkins publishers. 1998.

13) National Committee for clinical laboratory standards, performance standards for antibacterial disc susceptibility testing, twelfth information supplement (M100-S12), Wayne, PA: NCCLS, 2002.

Received on 12.12.2011 Modified on 14.01.2012

Research J. Pharm. and Tech. 5(2): Feb. 2012; Page 273-276