1. INTRODUCTION:

In the ancient times, plants parts were used for treatment of health illness¹. They serve as source of medicines, consists of antimicrobial agents with therapeutic properties. Despite the development of various antimicrobial agents, microbial infections especially by bacteria are still prevailing². One main reason for this is development of resistance to already existing therapeutic agents³. Thus there is need of novel medicines and plants are the safest and cheapest alternative sources. Phytochemical and antimicrobial properties of plants can be of significant importance in treating various illness⁴.Hence, screening and antimicrobial studies of plant extracts gained importance in the last few years.

In South India, plants have been used as traditional medicines as they are the good source of therapeutic agents⁵. Only 30 % of the population depend on commercial drugs and remaining depend exclusively on medicines of natural origin^{6, 7}.

Thus there is a need to investigate the properties, safety and efficacy of plants. The secondary metabolites like alkaloids, saponins, flavonoids, tannins, steroids, carbohydrates etc., produced by the plants function as bioactive compounds and serves as therapeutic agents⁸. Their inhibitory effects over various microorganisms were also reported in the past^{9, 10}. Microorganisms like Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Bacillus subtilis, Pseudomonas aeruginosa etc., are the most common and prevailing microorganisms causing microbial infections in India¹¹. E. coli can cause serious health problems like abdominal pain diarrhea and also produce toxins, which can cause acute renal failure¹². S. aureus can cause common infections like abscesses, furuncles and cellulitis. Also these species can cause certain serious infections such as blood stream infections, pneumonia and bone and joint infections¹³. B. subtilis was found to cause meningitis and urinary tract infections¹⁴. Therefore, present study mainly focused on microorganisms like E. coli, S. aureus, B. subtilis and P. aeruginosa. In the present study, four different plants viz., A. vasica, T. chebula, C. angustifolia and A. racemosus, which are commonly present in south India having medicinal value were investigated for presence of phytochemical compounds and antibacterial property against four kinds of bacterial microorganisms viz., E. coli, S. aureus, P. aeruginosa and B. subtilis. Further, the antibacterial activity of plant extracts was compared with commercial antibiotic ampicillin. The Minimum Inhibitory Concentration (MIC) of all the plant extracts was also studied

2. MATERIAL AND METHODS:

2.1. Plant materials:

Grounded materials of A. vasica, T. chebula, C. angustifolia and A. racemosus were obtained from commercial source, Guntur, India. All the samples were air dried at room temperature overnight before use.

2.2. Chemicals:

All the solvents used in the study were obtained from Sigma Aldrich. DMSO used in the study was obtained from Sigma. Antibiotic ampicillin was obtained from Guntur Drug House Pvt. Ltd., Guntur, India.

2.3. Microorganisms:

Microorganisms viz., E. coli (MTCC 40), S. sureus (MTCC 3160), P. aeruginosa (MTCC 424) and B. subtilis (MTCC 121) were obtained from MTCC – IMTECH- Chandigarh, India. The bacterial strains were sub cultured on nutrient agar at 37°C overnight from stock cultures maintained on agar slants at 4°C before use.

2.4. Preparation of extracts:

50 g of powdered samples were dissolved in 250 ml of respective solvents used in the study and were refluxed using reflux condenser at corresponding boiling temperatures for 8 h. The residue was filtered by using two layered muslin cloth and the filtrate was centrifuged at 5000 rpm for 10 min. Further, supernatant was collected and left to evaporate under fume hood. A constant dry weight of extract was measured and dissolved in DMSO at 1 mg/ml and stored at 4°C until further use. In case of aqueous extract, the supernatant was boiled until the concentration was reduced to 1 mg/ml and stored at 4 °C until further use.

2.5. Phytochemical Analysis:

Phytochemical screening was carried out by using standard procedures as described by Harborne¹⁵.

2.6. Determination of antibacterial activity by agar diffusion method:

Agar well diffusion method was used to determine antimicrobial activity. Test microorganisms with inoculum density of 3.5 x 10⁶ colony forming units were swabbed on to 20 ml of sterile nutrient agar plates by using cotton swab. Wells of 10 mm diameter were made in each of the plates using cork borer. About 100 µl of plant extracts (1 mg/ml) were added by using sterile syringe and allowed to diffuse for 1 h at room temperature. The plates were incubated at 37 °C for 24 h. the activity of plant extracts were determined by measuring the zone of inhibition. Each extract was analyzed in triplicate and average values was reported. Control sample without any plant extract served as a negative control.

2.7. Minimal Inhibitory Concentration (MIC):

Plant extracts were diluted to different concentrations ranging from 100 to 3.21 µg/ml were prepared and inoculated with overnight grown test cultures having inoculum density of 3.5 X 10⁶ colony forming units. The samples were incubated at 37 °C for 24 h at 100 rpm. The MIC values were determined basing on the turbidity of sample, which was analyzed by measuring the absorbance at 600 nm by using UV-Vis Spectroscopy.

3. RESULTS:

3.1. Phytochemical screening:

Phytochemical analysis of selected medicinal plants was shown in Table 1. It was clear that all the plant extracts were showing carbohydrates and saponins. Tannins were found to be present only in A. vasica. Flavanoids were found to be present in extracts of C. angustifolia and A. racemosus. Alkaloids were found to be present in extracts of T. chebula, C. angustifolia and A. vasica respectively. Steroids were found to present in A. vasica and T. chebula.

Table 1. Phytochemical analysis of plants used in the study

Constituents	A. vasica	T. chebula	C. angustifolia	A. racemosus
Carbohydrates	+	+	+	+
Steroids	+	+	-	-
Alkaloids	+	+	+	-
Tannins	+	-	-
Saponins	+	+	+	+
Flavanoids	-	-	+	+

Key: + = Present; - = Absent

3.2. Antimicrobial assays:

Table 2 shows the antibacterial activity of four medicinal plants used in the study. Four kinds of solvent plant extracts viz., aqueous, methanol, petroleum ether and ethyl acetate were tested against five different bacterial microorganisms (E. coli, S.aureus, P.aeruginosa and B. subtilis).

All the plant extracts showed antibacterial activity, however the activity of plant extracts was different for different microorganisms. Ethyl acetate, petroleum ether and methanol extracts of Adathoda vesica showed highest zone of inhibition towards E. coli (35 mm), S. aureus (33 mm) and B. subtilis (33 mm) when, compared to extracts of other medicinal plants. The aqueous extracts showed poor activity against all microorganisms with zone of inhibition ranging from 9 to 11 mm. Ethyl acetate extract of T. chebula showed highest zone of inhibition towards P.aeruginosa (34 mm) when, compared to other extracts. Clearly the antibacterial activities of used plant extracts were comparable to commercial antibiotic ampicillin. Further, ethyl acetate extracts of A. vasica showed maximum zone of inhibition when compared to Ampicillin.

Table 2. Zones of inhibition induced by various plant extracts (1 mg/ml) against microorganisms used in the study

Extracts		Zone of inhibition (mm)
Extracts		E.coli	S. aureus	P.aeruginosa	B. subtilis
A. vasica	Aqueous	11 ±0.15	10 ±0.28	11 ±0.31	11 ±0.44
	Ethyl acetate	35 ±0.20	33 ±0.12	32±0.54	33 ±0.22
	Methanol	32±0.33	28±0.53	33±0.23	18±0.44
	Petroleum ether	16 ±0.25	18 ±0.58	16 ±0.22	13 ±0.42
T. chebula	Aqueous	10 + 0.12	11 ±0.29	11 ±0.37	9 ±0.84
	Ethyl acetate	30 ±0.28	27 ±0.15	32 ±0.62	30 ±0.41
	Methanol	25 ±0.33	26 ±0.38	22 ±0.18	24±0.29
	Petroleum ether	15±0.26	13±0. 49	13±0.45	15±0.33
C. angustifolia	Aqueous	11 ±0.42	11 ±0.24	9 ±0.58	10 ±0.13
	Ethyl acetae	25 ±0.82	25 ±0.37	17 ±0.22	15 ±0.29
	Methanol	20±0.23	21±0.55	17±0.51	15±0.44
	Petroleum ether	18 ±0.33	14 ±0.19	12±0.19	13±0.20
A. racemosus	Aqueous	7 ±0.72	8 ±0.18	7 ±0.18	7±0.32
	Ethyl acetate	20 ±0.23	19 ±0.11	13 ±0.28	14 ±0.49
	Methanol	20 ±0. 42	17 ±0.28	13±0.81	17 ±0.11
	Petroleum ether	11±0.22	12 ±0.55	10 ±0.27	9 ±0.42
Ampicillin (5 µg/ml)		32	28	30	32

Table 3. MIC of various plant extracts for microorganisms used in the study

Extracts		Minimal Inhibitory concentration (µg/ml)
Extracts		E.coli	S. aureus	P.aeruginosa	B. subtilis
A. vasica	Ethyl acetate	3.21	6.25	12.5	6.25
	Methanol	6.25	12.5	12.5	12.5
	Petroleum ether	12.5	12.5	25	25
T. chebula	Ethyl acetate	6.25	6.25	6.25	12.5
	Methanol	12.5	12.5	12.5	25
	Petroleum ether	25	25	25	50
C. angustifolia	Ethyl acetate	12.5	12.5	12.5	12.5
	Methanol	12.5	25	50	50
	Petroleum ether	50	25	100	100
A. racemosus	Ethyl acetate	50	50	50	50
	Methanol	50	100	100	100
	Petroleum ether	100	100	100	100

4. Discussion:

Solvent and aqueous extracts of medicinal plants used in the study exhibited different range of antibacterial activity. A. vasica extracts exhibited highest antibacterial activity in the range of 10 to 35 mm. maximum inhibition was observed against E. coli (35 mm) and minimum inhibition against S. aureus (10 mm). Also the extracts of A. vasica showed MIC values in the range of 25 to 3.21 µg/ml. Previously, it was reported that diethyl ether extract of A. vasica (500 µg/ml) showed maximum zone of inhibition (10 mm) against Staphylococcus aureus¹⁶. In another work, methanolic extracts of A. vasica showed 19 mm and 17 mm zones of inhibition against S. aureus and E. coli respectively¹⁷. Similarly, in another report the methanolic extracts of A. vasica containing saponins, carbohydrates, alkaloids and flavonoids exhibited wide range of antimicrobial activity¹⁸.

The extracts of T. chebula exhibited antibacterial activity against all bacterial strains used in the study. The range of inhibition was found to be 9 to 34 mm, with maximum inhibition towards P. aeruginosa (34 mm) and minimum inhibition towards B. subtilis (9 mm). Further, the plant extracts showed MIC values in the range of 50 to 6.25 µg/ml. This kind of activity of T. chebula was also previously reported¹⁹. Aneja and his coauthors reported that, ethyl acetate fruit extracts of T.chebula exhibited zone of inhibition of 33 mm against S. aureus and further, MIC value was found to be 12.5 mg/ml²⁰. In another report on antimicrobial activity of Terminalia bellerica, the ethyl acetate extract was found to show zone of inhibition of 30 mm and 28 mm against S. aureus and E. coli. Further, the extracts were found to rich in alkaloids and flavonoids²¹.

The extracts of C. angustifolia showed zone of inhibition range (9 to 25 mm). All the extracts showed good antibacterial activity with MIC values in the range of 100 to 12.5 µg/ml. This antibacterial activity might be due to presence of saponins, flavonoids and alkaloids in the extracts²². Mahalingam and his coauthors reported that, ethyl acetate extract of C.angustifolia exhibited maximum zone of inhibition of 12 mm against B. subtilis²³. In another work methanol leaf extract of C. angustigolia showed maximum inhibition zone of 18 mm and MIC value of 0.65 µg/ml against S.aureus²⁴. Ethyl acetate and methanol extracts of A. racemosus showed high antibacterial activity towards all bacterial organisms used in the study, when compared to petroleum ether extract. Further, aqueous extract showed very poor antibacterial activity with zone of inhibition in the range of 7 to 8 mm. This difference in antibacterial activity is due to the phytochemical compounds they contain. Ethyl acetate and petroleum ether extracts rich in flavonoids, saponins and alkaloids, whereas in methanol and aqueous extracts they are absent²⁵. The MIC values of A. racemosus extracts were in the range of 100 to 50 µg/ml. Previously, it was reported that ethyl acetate and petroleum ether extracts (100 µg/ml) of A. racemosus exhibited maximum zone of inhibition of 10 and 8 mm respectively towards B. subtils. Similarly, the same extracts were also found to show maximum inhibition of 13 mm towards E. coli²⁶. The antimicrobial properties of medicinal plant extracts used in the study were because of due to bioactive compounds present in them. These bioactive compounds includes flavonoids, tannins, alkaloids, saponins, steroids etc. the extraction of these compounds depends on their solubility in solvents. From the results obtained from Table 1, ethyl acetate and methanol extracts were found to dissolve most of the bioactive compounds when compared to petroleum ether and aqueous solvents²⁷. The antimicrobial activity of aqueous extract was due to the sulfates, sulfites, nitrides and other water soluble salts, which are naturally present in plants²⁸.

5. CONCLUSIONS:

Based on our results, plant extracts of A. vasica and T. chebula were found to be potent source of antimicrobial agents. Our results also confirmed that ethyl acetate and methanol serve as better solvents for extraction of bioactive compounds from plants. Comparison studies with commercial antibiotic, ampicillin also confirms that extracts of A. vasica and T. chebula serves as better antibacterial agents than ampicillin. The MIC tests also proved that, the plant extracts used in the study were effective even at minimal concentrations.

6. ACKNOWLEDGEMENTS:

The authors would like to thank Bapatla Engineering College and JNTU- Kakinada for their support in research

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Received on 13.09.2014 Modified on 19.09.2014

Research J. Pharm. and Tech. 7(11): Nov. 2014 Page 1300-1304