INTRODUCTION:

Ointment is a viscous semisolid preparation used topically on a variety of body surfaces. An ointment may or may not be medicated. Medicated ointments contain a medicament dissolved, suspended or emulsified in the base. Ointments are used topically for several purposes, e.g. as antiseptics, emollients, antipruritic, keratolytics and astringents. Ointment bases are mainly anhydrous and generally contain one or more medicaments in suspension or solution or dispersion. [1].

Many of the currently available drugs were derived either directly or indirectly from the plants. The plant kingdom represents a rich source of organic compounds, many of which have been used for medicinal and other purposes [2].

Butea frondosa belongs to family Fabaceae is a deciduous tree with a somewhat crooked trunk, up to15m [3]. The plant occurs and distribute commonly throughout the greater parts of India, Burma and outer Himalaya [4].

The various parts used in the traditional medicine are gum, seeds, flowers, bark and leaves of the plant. The bark of B. frondosa is used as appetizer, aphrodisiac, laxative, anthelmintic, antidysentery, to cures ulcers and tumors [4-6]. The stem bark, leaf and flower of Butea frondosa is used as an antimicrobial and anti-inflammatory agent [7-10]. On the basis of literature survey the present study was designed to carry out evaluation of antibacterial properties of the methanolic extract of Butea frondosa in ointments formulation.

MATERIALS AND METHODS:

Plant material Butea frondosa collected from the forest of Mayurbhanj, Orissa in the month of August. The collected material was cleaned of unwanted foreign materials, sun-dried for a week.

Test microorganisms:

The microorganisms used for the study were Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The bacterial strains were grown and maintained on agar at 37 °C.

Preparation of extracts:

The collected material was cleaned, dried under shade and powdered by a mechanical grinder. Hundred grams of the pulverized material was extracted with petroleum ether, chloroform and methanol successively in a soxhlet apparatus. Petroleum ether was used in initial step of extraction for defatting the plant materials. The successive extracts were separately filtered and concentrated at reduced temperature on a rotary evaporator.

Evaluation of antibacterial activity of Extract:

The antibacterial activity of the methanolic extract of Butea frondosa at concentrations of 50mg/ml, 100mg/ml, 150mg/ml and 200mg/ml were determined using the cup plate method. Agar stabilized at 45 °C was seeded with 0.1 ml of a 24 h broth culture of the test organism (B. subtilis, E. coli, P. aeruginosa and S. aureus) containing approximately 10⁸cfu / ml in a sterile petri dish and allowed to set. Wells of 6mm diameter were created with a sterile cork borer and filled to about three-quarters full with solutions of the methanolic extract of Butea frondosa. The plates were pre-incubated for 1 h at room temperature to allow for diffusion of the solution and then incubated for 24 h. The zones of inhibition were measured (mean, n=2). Streptomycin and propylene glycol were used as positive and negative controls respectively (Table 1).

Table 1: Preliminary in vitro antibacterial activity of methanolic extract of Butea frondosa (Zone of inhibition in mm)

Test organism	Concentration of Extract
Test organism	50mg/ml	100mg/ml	150mg/ml	200mg/ml
S. aureus	17.20± 0.53	18.00±0.2	20.22± 1.11	21.00± 1.43
B. subtilis	11.52± 0.50	12.00±1.19	13.11± 0.51	15.22± 0.51
E. coli	5.31± 1.2	7.01± 1.32	8.01± 1.11	8.45± 1.23
P. aeruginosa	0.00	0.00	0.00	0.00

Preparation of Ointments:

Two topical ointment bases of varying degrees of aqueous/anhydrous character (Table 2), namely: simple ointment and emulsifying ointment were prepared by fusion method. In this method the constituents of the base were placed together in a melting pan and allowed to melt together at 70°C. After melting, the ingredients were stirred gently maintaining temperature of 70°C for about 5 minutes and then cooled with continuous stirring. Formulation of ointment was done by incorporating 10 g of the semisolid methanolic extract of Butea frondosa into the various bases by triturating in a ceramic mortar with a pestle to obtain 100 g of herbal ointments containing 10 % w/w of Butea frondosa extract.

Evaluation of formulated Ointments:

Physical assessments were carried out on the ointments over a period of 30 days using the following parameters: Appearance, Odour, Texture and Colour. The pH of various formulations was determined by using Digital pH meter. 0.5g of the weighed formulation was dispersed in 50

Homogeneity: All the developed ointments were tested for homogeneity by visual inspection. They were tested for their appearance with no lumps.

Table 2: Preparation of medicated formulations with methanolic extract of Butea frondosa

Formulations	Ingredients	Concentration (%w/w)
Simple Ointment	Extract	5
	Wool fat	5
	Cetostearyl alcohol	5
	Hard paraffin	5
	White soft paraffin	80
Emulsifying Ointment	Extract	5
	Liquid paraffin	20
	Emulsifying wax	25
	White soft paraffin	50

In vitro antibacterial efficacy of formulated ointments:

The cup-plate method was used to assess the relative antibacterial efficacy of the formulated herbal ointments prepared with the extract of Butea frondosa. Agar stabilized at 45 °C, seeded with 0.1 ml of a 24 h broth culture of the test organism (Bacillus subtilis and Staphylococcus aureus) and containing approximately 10⁸ cfu / ml was used. Wells of 6mm diameter were created and filled to three-quarters full with the topical products of Butea frondosa extract. The plates were pre-incubated for 1 hr at room temperature to ensure adequate diffusion and finally incubated at 37 °C for 24 h. A commercial brand of Gentamicin ointment was used as standard while blank ointment base was used as control. The experiments were run in duplicate and the zones of inhibition were determined and recorded (mean± SD, n = 2).

RESULTS AND DISCUSSION:

The prepared formulations show a smooth and homogeneous appearance. The pH values of all the prepared formulations ranged from 6.3 to 7.1, which are considered acceptable to avoid the risk of irritation upon application to the skin. The pH values of the formulations are within the normal pH range of the human skin (6.8 ± 1). From the study, the ointments showed no changes in pH, consistency and phase separation after keeping for 30 days (Table 3).

Table 3: pH evaluation of different formulation of ointments

Time period (days)	Ointment formulation	pH
0	Simple Ointment	6.1±0.14
15^th		6.4±1.22
30^th		6.1±0.62
0	Emulsifying Ointment	6.2±0.11
15^th		6.2±0.19
30^th		6.2±0.31

The preliminary in vitro antimicrobial activity of the methanolic extract of Butea frondosa presented in Table 1 showed excellent activity against Staphylococcus aureus and Bacillus subtilis. The in vitro antimicrobial activity of the methanolic extract of Butea frondos based herbal ointments presented in Table 4.

They demonstrated excellent antibacterial activity. In the preliminary antimicrobial sensitivity screening, the methanolic extract of Butea frondos showed excellent activity against Staphylococcus aureus and Bacillus subtilis (p < 0.05) (Table 1). Most of these organisms are natural flora of the skin and also known etiologic agents of several skin and mucous membranes infections of man. The order of antibacterial activity of Butea frondos in the topical bases was as follows: emulsifying ointment > simple ointment (Figure 1). The results also revealed that the extracts incorporated into the ointment bases showed better activity than that of the crude extract of Butea frondos. This implied that there might have been better diffusion of drug for the herbal ointments than for the crude extract. The activity against Staphylococcus aureus is of significant interest because it is commonly found on the hands, face and in deep layers of the skin and is perhaps the most widely encountered and very undesirable.

Figure 1. Comparative representation of antimicrobial activity of extract and ointments

CONCLUSION:

This study shows that Butea frondos has antibacterial activity and has high potential as antibacterial agent when formulated as ointment for topical use and could therefore explain the successes claimed in the folk use of the plant in the treatment of microbial infection. This investigation suggests that ointments containing Butea frondos can be used as antibacterial agents for many infectious disease caused by the tested microorganism.

REFERENCES:

1. Carter SJ. Cooper and Gunn’s Dispensing for Pharmaceutical Students: Ointments, Pastes and Jellies. 12^th Edition, CBS Publishers and Distributors, India, 1987; 192-210.

2. Yaheya M, Ismail M., “Antiasthematic Herbal drug-A Review”, Int J Pharmacy Pharm Sci, 2010; 2: 28-9.

3. The wealth of India. A dictionary of Indian Raw Materials and Industrial Product. Revised edition, CSIR. New Delhi, 1988; 2 (B): 341-347.

4. Kirtikar KR, Basu BD., Indian Medicinal Plants., 2^nd Edition, Lalit Mohan Basu, Allahabad, 1998; 1: 784-789.

5. Nadkarni A K., Indian Materia Medica., Popular Prakashan, Bombay, 1976; 1: 222-225.

6. Mokkhasmit M, Ngarmwathana W, Sawasdimongkol K, Permphiphat U., “Pharmacological evaluation of Thai medicinal plants”, J Med Ass Thailand, 1971; 54(7): 490-504.

7. Mishra US, Dutta NK, Mazumdar K, Mahapatra SK, Chakroborty P, Dastidar SG., “Anti-Salmonella activity of a flavonone from Butea frondosa Bark in mice”, Oriental pharmacy and Experimental Medicine (OPEM), Kyung Hee University press, 2008; 8(4): 339-348.

8. Mishra US, Chakroborty P, Dasgupta A, Dastidar SG, Martins M, Amaral L., “Potent Bactericidal Action of a Flavonoid fraction isolated from the stem Bark of Butea frondosa”, in vivo, International Institute of Anticancer Research, 2009; 23: 29-32.

9. Mengi SA, Deshpande SG., “Antiinflammatory activity of Butea frondosa leaves”, Fitoterapia, 1999; 70 (5): 521 - 522.

10. Nazimuddin SK, Khaleefathullah S., “Antiinflammatory effect of Gul-E-Tesu (Butea monosperma lam flowers)”, Bull Islamic Med, 1982; 2: 522- 527.

Received on 01.11.2013 Modified on 30.11.2013

Research J. Pharm. and Tech. 7(2): Feb. 2014; Page 181-183

Test organism	Simple Ointment		Emulsifying Ointment
Test organism	100mg/ml	150mg/ml	100mg/ml	150mg/ml
S. aureus	17.33±0.13	18.25±0.2	21.24±1.51	22.05±1.23
B. subtilis	11.12±0.53	12.42±1.29	13.12±0.54	15.12±0.11
E. coli	5.11±1.1	7.11±1.12	8.11±1.01	8.66±1.26
P. aeruginosa	0.00	0.00	0.00	0.00