PREPARATION AND EVALUATION OF ANTIMICROBIAL HERBAL FORMULATION OF PTEROSPERMUM ACERIFOLIUM WILLD

PREPARATION AND EVALUATION OF ANTIMICROBIAL HERBAL FORMULATION OF PTEROSPERMUM ACERIFOLIUM WILLD

Ashis Kumar Manna^*, Upendranath Nanda and Subrat Kar

Seemanta Institute of Pharmaceutical Sciences, Jharpokharia, Mayurbhanj - 757086, Odisha, India.

ABSTRACT: The present work deals with formulation of topical gels containing aqueous extract of P. acerifolium seeds and also evaluation of prepared gels against some Gram-positive and Gram-negative bacteria. The antibacterial activity of various P. acerifolium seed extracts was measured through determining minimum inhibitory concentration (MIC) by cube dilution method and zone of inhibition by disc diffusion method against E. coli, P. aeruginosa and S. aureus. Aqueous P. acerifolium seed extracts exhibited comparatively higher antibacterial potential in comparison with that of other extracts and employed to formulate topical herbal gels using 4.5% sodium carboxymethyl cellulose as gel base. Prepared herbal gels were tested for pH, viscosity, extrudability and spreadability. All the prepared gels were subjected to antibacterial study by determining zone of inhibition by disc diffusion method. Herbal gels containing 15% aqueous P. acerifolium seed extract exhibited better zones of inhibition against E. coli, P. aeruginosa and S. aureus in comparison with that of 7.5% and 10%. From the results of this study, it can be concluded that herbal gels containing 15% aqueous P. acerifolium seed extract is found suitable for topical use against bacterial infections.

Pterospermum acerifolium, Antimicrobial, Minimum inhibitory concentration (MIC), Gel formulation

INTRODUCTION: In recent years, considerable research efforts have been directed towards the use of naturally derived materials in various medicinal, biomedical as well as pharmaceutical applications ^1-11. Amongst these naturally derived materials, herbal medicines have been recognized as the most important remedy in the traditional medicine system all over the world by the people to treat various diseases since antiquity ^{12 - 18}.

According to World Health Organization (WHO 1993), a major fraction (almost 80%) of the global population is dependent on the traditional medicine systems with the use of extracts derived from plant parts like leaves, pods, bark, fruits, seed, roots, exudates, etc. ^{12, 19 - 20}. Moreover, these herbal medicines are cheap, safe and devoid of side effects on long term use ^{18 - 21}.

Extensive survey of the ethno-medicinal and phytochemical literature reveals that numerous herbal extracts have already been studied for their anti-microbial activities against many microbial infections ^22-25. Pterospermum acerifolium willd (family, Sterculiaceae, commonly known as ‘Kanak champa’) is a shrub distributed in tropical Asia ²⁶.

It is a popular ornamental plant having wide range of medicinal properties ²⁷. Various part of P. acerifolium plant is used for the treatment of different diseases like conjunctivitis, anorexia, dysmenorrheoea and fever. P. acerifolium is also reported as bitter, acidic, anti-inflammatory, analgesic, sedative, carminative, digestive, antimicrobial, anthelmintic ^{26, 28 - 33}. Extensive survey of literature reveals the fact that very little work has been done in the direction of its use as antibacterial through topical gels. No topical gel formulation yet is prepared using this herbal drug towards the antibacterial dermatological treatment. Therefore, in the present work, attempt has been taken to develop topical gels containing aqueous extract of P. acerifolium seeds and also to evaluate the prepared gels against some Gram-positive and Gram-negative bacteria.

MATERIALS AND METHODS:

Collection and Authentification of Plant Material: Seeds of P. acerifolium were collected in the month of January, 2009 from the hill area near the bank of Subarnarekha River in the district of Mayurbhanj, Odisha. The collected plant with complete herbarium was authenticated at Botanical survey of India, Central National Herbarium, Botanical Garden, Howrah, Kolkata (vide Letter No CNH/1-1(15)/2009/Tech11/413). Also a sample specimen was deposited there.

Preparation of Extract: 1kg of the air dried seeds of P. acerifolium was reduced to fine powder and extracted using Soxhelet apparatus with petroleum ether, ethanol and purified water up to 30 siphons each one after another. Each extract was concentrated using a Rota evaporator. The marc left after extraction was dried in an air oven below 30 °C.

TABLE 1: DETAILS ABOUT THE MICRO-ORGANISMS USED IN THE CURRENT STUDY

Antimicrobial Screening:Microorganism: The microorganisms were obtained from the microbiological laboratory of Department of Pharmaceutical Technology, Jadavpur University, Kolkata. The details about the micro organism are shown in Table 1.

Determination of Minimum Inhibitory Concentration (MIC): MIC of each extract was determined by cube dilution method. 10 Test tubes for each extract per microorganism were thoroughly washed and sterilized. 0.5ml of test solutions (7.8 mg/ml to 1000 mg/ml) was taken in 8 test tubes. 0.5ml of freshly prepared and sterilized nutrient broth and 0.5ml 6% DMSO was added to each of 8 test tubes. Test tubes were thoroughly shaken. To each test tube, 50mg of 24 hours sub culture of bacteria was added and shaken properly. The 9^th test tube contained 0.5ml of nutrient broth and 0.5ml 6% DMSO and 50mg of sub culture of bacteria to act as positive control and 10^th test tube contained 0.5ml of nutrient broth and 1ml of 6% DMSO to act as negative control. All the test tubes were incubated in an incubator (Remi Corporation, India) at 37 °C for 24 hours and observed for turbidity comparing with both the controls. Same procedure was adopted for all extracts and all the three bacteria.

Determination of Zone of Inhibition: Zone of inhibition was determined for each extract at a concentration of 1000mg/ml and compare to that of standard ciprofloxacin 5mg/disc by disc diffusion method. Agar plates were made by pouring nutrient agar suspension (sterilized) in cleaned petridish to get 4mm thickness (approx). Plates were kept under laminar air flow.

Stock solution of each extract 100mg/ml were prepared by dissolving the extract in 6% DMSO. Paper disc (Whatman^® No. 1) of 6mm diameter were cut and sterilized in hot air oven. 10ml (1000mg/disc) of each extract was taken in a sterile pipette and soaked in paper disc. The Discs were dried. The discs were carefully kept on the agar media and slightly pressed for proper fixing. Standard disc of ciprofloxacin (5mg/disc) was taken and placed on the media and fixed properly. The plates were incubated at 37 °C for 24 hours. Plates were observed for zone of inhibition.

Formulation and Preparation of Herbal Gels: Accurately weighed quantity of Sodium Carboxymethyl Cellulose was soaked in distilled water and was allowed to swell. Accurate quantity of aqueous extract was weighed and dispersed in distilled water. The drug dispersion was added to the soaked polymer with stirring, until a gel was formed. The prepared gel was filled in collapsible tube and sealed by crimping the ends. Different formulations for the herbal gels by using sodium carboxymethyl cellulose are given in Table 2.

TABLE 2: FORMULA OF HERBAL GELS BY USING SODIUM CARBOXYMETHYL CELLULOSE

Evaluation of Herbal Gels:

pH measurements: The pH of the gels was measured by using digital pH meter (Systronics Instruments, India). The glass electrode was completely dipped into the gel system so as to cover the electrode and the pH of gels was measured within 5 min.

Determination of Viscosity: The viscosity of formulated gels was determined using Brookfield viscometer (DV-III Units Programmable Rheometer, USA) using the spindle No. 4 at 1.5 rpm.

Determination of Extrudability: It is a useful empirical test to measure the force required to extrude the material from a bottle or tube since the passing of gels have gained a considerable importance in the delivery of desired quantity of gel from collapsible tubes. Therefore, the measurement of extrudability becomes an important criterion for gels. While not strictly a test of product characteristics due to inclusion of force necessary to deform the container, the method applied is for the determination of applied shear in the region of the rheogram corresponding to the shear rate exceeding. The yield value was exhibiting. Consequent plug flow on such apparatus is described by Wood et al., ³⁵. The gels were filled in standard capped collapsible tubes and sealed by crimping the ends. The weight of tube was recorded. The tube was placed between two glass slides and was clamped. A 500gm weight was placed over the glass slide and then the cap was removed. The amount of gel extruded was collected and weighed.

Determination of Spreadability: Two glass slides 6cm long were used containing gel in between. Lower slide were fixed on a wooden plate and the upper one was tied to a hook having a balance at the other end in which a weight was kept in order to pull that slide. Five gram of gel was uniformly placed on the lower slide and the upper slide was placed on it. A one kilogram weight was kept on the slides for five minutes to expel the entrapped air. The excess discharged gel was carefully scrapped off. A weight of 80gm was kept on the balance. The time in seconds required to separate the slides completely was noted. Less time indicates more slip and better spreadability. The experiments were repeated thrice and mean value was taken. Spreadability is calculated using the formula:

S = M x L/t

Where, S = Spreadability of gel; M = weight tied to the upper slide; L = length of glass slide; t = time taken.

RESULTS AND DISCUSSION: Topical gels are semisolid formulations, which have gained a greater deal of importance in the medical field for their topical effect in the treatment of pain, strain, inflammation, infections and other diseases ^36-40. In the current work, topical herbal gels containing aqueous extract of P. acerifolium seeds was developed using sodium carboxymethyl cellulose. The antibacterial potential of these formulated herbal gels were also investigated against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.

MIC of P. acerifolium seed extracts was determined by cube dilution method. It was observed that all the seed extracts showed MIC ranging from 31.25 to 1000mg/ml (Table 3). The aqueous extract showed lowest MIC value against all three bacteria. The aqueous extract was found to possess MIC value of 31.25mg/ml, against E. coli and S. aureus. Against P. aeruginosa, it was 125 mg/ml. From the above observation, it was clearly concluded that aqueous extract of seeds showed lowest MIC value.

TABLE 3: MIC OF P. ACERIFOLIUM SEED EXTRACTS BY DILUTION METHOD

In order to evaluate the antibacterial activity of extracts having MIC up to 1000mg/ml, disc diffusion method was adopted and zone of inhibition was recorded. The antibacterial sensitivity of extracts and that of standard (ciprofloxacin) were compared. Aqueous extract of P. acerifolium seeds at 1000mg/disc showed higher zone of inhibition against P. aeruginosa (22mm) than that of S. aureus (19mm) and E. coli (17mm) while comparing to ciprofloxacin (standard, 5mg/disc) (32 – 36mm). Other extracts showed fewer zones of inhibitions. Thus, antibacterial screening of P. acerifolium seed extracts by disc diffusion method finally showed comparatively higher antibacterial potential in case of aqueous extract of seeds and hence, it was taken for further formulation study.

TABLE 4: ZONE OF INHIBITION OF P. ACERIFOLIUM SEED EXTRACTS AND CIPROFLOXACIN (STANDARD) BY DISC DIFFUSION METHOD

Prepared herbal gels of aqueous P. acerifolium seed extract prepared using 4.5% sodium carboxymethyl cellulose were tested for pH, viscosity, extrudability and spreadability. pH of these gels was within the range between, 6.28 - 7.30. Viscosities of these herbal gels were measured within 42000 - 52000 cps. It was observed that gel containing 15% aqueous P. acerifolium seed extract showed good extrudabilty in comparison with other formulated gels.

The spreadability of these gels was found almost similar. From these results, it can be suggested that the gel containing 15% aqueous extract exhibited satisfactory extrudability and spreadability. The extrusion of gels from the collapsible tube is important during application; whereas, spreadability plays an important role in helping uniform application of gels. A good gel takes less time to spread and should have high spreadability.

TABLE 5: pH, VISCOSITY, EXTRUDABILITY AND SPREADABILITY OF HERBAL GELS CONTAINING AQUEOUS P. ACERIFOLIUM SEED EXTRACT

* +++ = Good; ++ = Average; + = Poor

All the prepared gels were subjected to antibacterial study by determining zones of inhibition by disc diffusion method. Zones of inhibition against E. coli, P. aeruginosa and S. aureus were compared with the standard gels containing clarithromycin (1%). Clarithromycin gel (1%) exhibited zones of inhibition in the range of 28 – 37mm for all these 3 bacteria. It was observed that herbal gel containing 15% aqueous P. acerifolium seed extract exhibited better zones of inhibition against E. coli, P. aeruginosa and S. aureus in comparison with that of 7.5% and 10%. Zones of inhibitions of herbal gel containing 15% aqueous P. acerifolium seed extract against E. coli, P. aeruginosa and S. aureus is shown in Fig. 1. The antibacterial potential of these herbal gels were found to be increased with the increasing percentage content of the aqueous P. acerifolium seed extract present in herbal gels.

TABLE 6: ZONE OF INHIBITION OF HERBAL GELS CONTAINING AQUEOUS P. ACERIFOLIUM SEED EXTRACT AND CLARITHROMYCIN GEL (1%) AS STANDARD BY DISC DIFFUSION METHOD

FIG. 1: ZONES OF INHIBITIONS OF HERBAL GEL CONTAINING 15% AQUEOUS P. ACERIFOLIUM SEED EXTRACT AGAINST (A) E. COLI, (B) P. AERUGINOSA AND (C) S. AUREUS

CONCLUSION: Extracts of P. acerifolium seeds were tested for antibacterial activity against some Gram-positive and Gram-negative bacteria like E. coli, P. aeruginosa and S. aureus. MIC (by cube dilution method) and zone of inhibition (by disc diffusion method) of aqueous P. acerifolium seed extracts exhibited comparatively higher antibacterial potential in comparison with that of other extracts. On the basis of it, topical herbal gels containing aqueous extract of P. acerifolium seeds were formulated using 4.5% sodium carboxymethyl cellulose as gel base and evaluated for their antibacterial potential against E. coli, P. aeruginosa and S. aureus. pH and viscosities of these gels were within the range, 6.28 - 7.30 and 42000 - 52000 cps, respectively. Gel containing 15% aqueous extract exhibited satisfactory extrudability and spreadability.

All the prepared gels were subjected to antibacterial study by determining zones of inhibition by disc diffusion method. Herbal gels containing 15% aqueous P. acerifolium seed extract exhibited better zones of inhibition against E. coli, P. aeruginosa and S. aureus in comparison with that of 7.5% and 10%. From the results of this study, it can be concluded that herbal gels containing 15% aqueous P. acerifolium seed extract was found suitable for topical use against bacterial infections.

It can be suggested that before its commercialization, the gel formulations should be subjected to detailed clinical studies using animals as well as human subjects. Also, extensive accelerated stability studies should be performed.

ACKNOWLEDGEMENT: We are very grateful to BPUT, Rourkela, Governing body SIPS for providing facilities and thankful to Dr. Sanmoy Karmakar, Professsor Jadavepur University, Kolkata and Dr. Amit Kumar Nayak, Associate Professor SIPS, Jharpokharia for their suggestions.

CONFLICT OF INTEREST: None.

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    How to cite this article:

    Manna AK, Nanda U and Kar S: Preparation and evaluation of antimicrobial herbal formulation of Pterospermum acerifolium willd. Int J Pharm Sci Res 2017; 8(11): 4788-94.doi: 10.13040/IJPSR.0975-8232.8(11).4788-94.

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