INTRODUCTION:

Numerous investigations have been carried out in individual plants and the respective antimicrobial agents have been identified in a gratifying number of cases. From various studies, it is clear that the chemical structures of these agents belong to the most commonly encountered classes of higher plant secondary metabolites⁽¹⁾. Up till now, however, all antimicrobial substances from higher plants have been found either to be toxic to animals or not competitive therapeutically with the products of microbial origin, due to their low potency and narrow spectrum.

Therefore, no antimicrobial compound from a higher plant has yet come into significant clinical use⁽¹⁾.

Research, however, continues in the hope of finding plant antimicrobials that are effective for the systemic or topical treatment of human or agricultural infections. Occurrence of infrequent variation in concentrations within species and related organisms suggest that resistance to the individual extracts, when it occurs, is due to the intrinsic properties of the species involved rather than acquired characters. For this reason, it would be more effective and much more useful if the plant extract or its active constituents can be exploited in the development of antimicrobial chemotherapeutic agents⁽¹⁾. This can be considered in the line with the current search for such substances to augment or replace the antibiotics in current clinical uses which because of the spread of resistance are less useful than before. In the present study focused on antibacterial activity of plant extract and to find out possible secondary metabolite responsible for that.

Ipomoea aquatica Forsk (syn. Ipomoea reptans Linn.) of family Convolvulaceae; is perennial herb and distributed throughout India. According to Unani, it is carminative, lessens inflammation; useful in fever, jaundice, biliousness, bronchitis, liver complaints^(2,3).

MATERIAL AND METHOD:

Plant material and preparation of extracts:

The leaves and flowers of the plant Ipomoea aquatica were collected from Rajkot district of Gujarat, India. The plant material was identified by Botany Dept., Kotak Science College, Rajkot and a voucher specimen (Voucher No. ARGH-10) has been retained in Department of Pharmacognosy, A. R. College of Pharmacy, Vallabh Vidyanagar, Gujarat. The collected plant materials were dried under shade and then powdered with a mechanical grinder and stored in an air-tight container, the dried powder material of both leaves and flower were extracted successively with petroleum ether, toluene, chloroform, acetone, methanol and water in a Soxhlet apparatus. The solvents were completely removed under reduced pressure and residues were obtained.

Antibacterial activity:

The antibacterial activity was carried out using agar cup method⁽⁴⁾ on the gram positive bacteria Staphylococcus aureus. All the extracts petroleum ether, toluene, chloroform, acetone, methanol and water of both leaves and flowers of I. aquatica were subjected for this study. Different concentrations (50 mcg/ml, 100mcg/ml, 150 mcg/ml, 200mcg/ml, 250mcg/ml, 300mcg/ml and 350mcg/ml) of all the extracts were prepared and used in the study. Ciprofloxacin (5 mcg/ml) was used as standard.

Agar cup method⁽⁴⁾: 0.2 ml of young test culture (Staphylococcus aureus culture was prepared in sterile distilled water) was inoculated in melted top agar (which was previously sterilized by autoclaving) previously cooled to 50^oC. It was mixed well and poured over the nutrient agar (which was previously sterilized by autoclaving) and was allowed to solidify. The cup-borer was sterilized by dipping it in alcohol followed by flaming it. Four cups were made with the help of cup-borer, one in each quadrant, at equal distance in nutrient agar plate previously seeded with culture. Each cup was filled with definite quantity of different dilutions of extracts. The plates were incubated in refrigerator at 4-5^oC for 30 minutes so as to allow diffusion of extracts. The plates were incubated in upright position at 37^oC for 24 hours in incubator. After 24 hours the plates were observed for zone of inhibition. The zone of inhibition was measured using a ruler. The experiment was performed in triplicates for all the test samples.

RESULT AND DISCUSSION:

The extracts of both leaves and flowers of I. aquatica were subjected for the antibacterial study. The petroleum ether, toluene, chloroform, acetone and water extracts of both leaves and flowers were not showed antibacterial activity, while the methanol extracts of both leaves and flowers were showed the antibacterial activity. The zone of inhibition obtained at different concentration of the methanol extracts of leaves and methanol extracts of flowers of I.aquatica are shown in table 1. The zone of inhibition obtained of the methanol extracts of leaves of I.aquatica at concentrations 250mg/ml, 300mg/ml and 350mg/ml is shown in Figure 1. The zone of inhibition obtained of the methanol extracts of flowers of I.aquatica at concentrations 250mg/ml, 300mg/ml and 350mg/ml is shown in Figure 2. The results show that the zone of inhibition was increased with increase of the concentration of the extracts in both leaves and flowers of I. aquatica. The results revealed that the leaves were somewhat more active than that of the flowers of I. aquatica.

Table 2: The results of preliminary phytochemical screening with the extracts obtained by successive solvent extraction of leaves of Ipomoea aquatica

No.

Chemical tests

Extracts

Test for Alkaloids:

(a) Dragendorff’s test

(b) Wagner’s test

^_ve

Test for Carbohydrates:

(a) Molish’s test

(b) Fehling’s test

+ve

Test for Saponin Glycosides:

(a) Foam test

(b) Heamolytic test

^_ve

Test for Flavonoids:

(a) Shinoda test

(b) Lead acetate test

+ve

^_ve

Test for Phenolic compounds and Tannins:

(a) Ferric chloride test

(b) Lead acetate test

^_ve

Test for Anthocyanins:

(a) With 2M

(b) With 2M NaOH

^_ve

Test for Phytosterols:

(a) Liebermann’s test

(b) Liebermann- Burchard’s test

+ve

^_ve

Test for Fixed oils and Fats:

(a) Spot test

(b) Tincture alkana test

^_ve

Test for Proteins and Amino acids:

(a) Biuret test

(b) Million’s test

+ve

^_ve

PE: Petroleum ether (60-80^oC) extract; T: Toluene extract, C: Chloroform extract; A: Acetone extract; M: Methanol extract; W: Water extract; +ve: positive; -ve: Negative; *: Not done

Table 3: The results of preliminary phytochemical screening with the extracts obtained by successive solvent extraction of flowers of Ipomoea aquatica

No.

Chemical tests

Extracts

Test for Alkaloids:

(a) Dragendorff’s test

(b) Wagner’s test

^_ve

Test for Carbohydrates:

(a) Molish’s test

(b) Fehling’s test

+ve

Test for Saponin Glycosides:

(a) Foam test

(b) Heamolytic test

^_ve

Test for Flavonoids:

(a) Shinoda test

(b) Lead acetate test

+ve

Test for Phenolic compounds and Tannins:

(a) Ferric chloride test

(b) Lead acetate test

^_ve

Test for Anthocyanins:

(a) With 2M HCl

(b) With 2M NaOH

+ve

^_ve

Test for Phytosterols:

(a) Liebermann’s test

(b) Liebermann- Burchard’s test

+ve

^_ve

Test for Fixed oils and Fats:

(a) Spot test

(b) Tincture alkana test

^_ve

Test for Proteins and Amino acids:

(a) Biuret test

(b) Million’s test

^_ve

+ve

PE: Petroleum ether (60-80^oC) extract; T: Toluene extract, C: Chloroform extract; A: Acetone extract; M: Methanol extract; W: Water extract; +ve: positive; -ve: Negative; *: Not done

Test sample	Concentration (mcg/ml)	Zone of Inhibition (mm±SEM) includes the diameter of disc (6 mm)
Ciprofloxacin	5	28±0.52
Methanol extract of leaves of Ipomoea aquatica	50	NI
	100	14±0.45
	150	16±0.64
	200	18±0.33
	250	20±0.57
	300	23±0.25
	350	26±0.53
Methanol extract of flowers of Ipomoea aquatica	50	NI
	100	13±0.47
	150	16±0.38
	200	18±0.54
	250	20±0.40
	300	22±0.24
	350	25±0.68