FORMULATION AND EVALUATION OF ANTIMICROBIAL HERBAL GEL OF VITEX NEGUNDO
Shailesh Sharma*, Rajesh Khathuriya 1 , Abhishek Bhanot 2 , Vivek Kumar Gupta 3
1 Pacefic College of Pharmacy Udaipur, India.
2 Venus LifeSciences, Baddi (HP)
3 Dreamz College of Pharmacy, Sundernangar, HP
The present investigation was aimed to evaluate antimicrobial activity of various extracts of leaves and stems of Vitex negundo Linn. by Agar Cup method. The extracts of the plant (Vitex negundo Linn.) under investigation, were separately suspended in DMSO. The activity was compared with the standard Ampicillin (antibacterial) and Fluconazole (antifungal). Petroleum Ether extracts of leaves and stems showed more significant antimicrobial activity against tested species and comparable with standard. Transdermal hydrogel was prepared by cold fusion method with carbapol 940 and evaluated for its physiochemical properties and in-vitro diffusion studies. Promised hydrogels were also subjected to in-vitro anti microbial evaluation. It was found both of them possessed significant antimicrobial activity and may serve as promising antimicrobial gel formulations. Keywords: Antimicrobial activity, Microbial evaluation, Hydrogels, phytomedicine
Many plants are containing natural anti-bacterial compounds, which are used as to treat common bacterial infections as natural medicine. Indian medicinal plants are regularly used in various system of medicine because of minimal side effect and cost effectiveness. The potential for developing antimicrobials from higher plants appears rewarding as it may lead to the development of phytomedicine against microbes. 
Today mostly all pharmaceutical companies, pharmacists most popular R&D’s and Researchers are increasingly turning their attention to folk medicine, looking for new leads for developing better drugs against microbial infections.  The increasing failure of chemotherapeutics coupled with antibiotic resistance exhibited by pathogenic microbial infectious agents has led to the screening of several medicinal plants for their potential antimicrobial activity.  Vitex negundo (Verbanaceae) commonly known as Nirgundi. It is a large, aromatic shrub, sometimes a small slender tree found throughout the greater part of the India.  Vitex negundo Linn. have several types of compounds such as volatile oils, lignans, flavonoids, flavone glycosides and triterpenoids.  Essential oil contains α-pinene, camphene, citral and β-caryophyllene. 
Vitex negundo Linn. is used for treatment of eye-disease, toothache, inflammation, leucoderma, enlargement of the spleen, skin-ulcers, in catarrhal fever, rheumatoid arthritis, gonorrhoea, and bronchitis. It is also used as tonics, vermifuge, lactagogue, emmenagogue, antibacterial, antipyretic and antihistaminic agents. Oil prepared with it, is applied to sinuses and scrofulous sores. Its extract has also shown anticancer activity against Ehrlich ascites tumour cells. 
The present study was designed to develop the anti-microbial herbal gel formulation containing extract of Vitex negundo L. leaves.
MATERIALS AND METHODS
Carbapol was obtained as a gift sample from Sun Pharm, Ahmadabad, India. Propylene Glycol and propylene Paraben was purchased from the SD Fine–chemical Ltd., Mumbai. Triethanolamine was purchased from Universal Lab. Pvt. Ltd, Mumbai. All the strains of microorganism were obtained from MTCC, Institute of Microbial Technology, Chandigarh. All other chemicals or ingredients used in extraction of leaves and formulation of gel were used of analytical grades.
2.2.1 Collection and Authentication of Plant Material
Leaves and stems of Vitex negundo Linn. were collected from Kasauli, Himachal Pradesh in month of August, 2009 and were authenticated and identified by Dr. H.B. Singh, Scientist & Head, Raw Materials, Herbarium & Museum, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, (NISCAIR/RHMD/Consult/-2009-10/1260/64). The voucher specimen (VN/RK/1/2010) has been deposited in Department of Pharmacognosy, ASBASJSM College of Pharmacy, Bela (Ropar), for future reference. The leaves and stems were shade dried, coarsely powdered and processed for further studies.
2.2.2 Preparation of Crude Extract: The shade dried powdered of leaves and stems were subjected to sequential soxhlet extraction using various solvents of different polarity such as petroleum ether, chloroform, methanol and water to get respective extracts. The extracts were filtered individually, evaporated to dryness and the percent yields of all the extracts were determined.
2.2.3 In Vitro Testing of Extracts for Antimicrobial Activity
All the strains of microorganism were obtained from MTCC, Institute of Microbial Technology, Chandigarh. Table 1 is showing Gram-Positive, Gram-negative and fungal microorganisms used for the study.
Table 1: Details of Microorganisms (Bacteria, Fungus) Used for Study
|S.No.||Name of micro organism||Strain||Code|
|1||Staphylococcus aureus||Gram-positive bacteria||MTCC 87|
|2||Bacillus subtilis||Gram-positive bacteria||MTCC *121|
|3||Escherichia coli||Gram-negative bacteria||MTCC 40|
|4||Pseudomonas aeruginosa||Gram-negative bacteria||MTCC 424|
|5||Candida albicans||Fungus||MTCC 183|
|6||Fusarium solani||Fungus||MTCC 2935|
Preparation of Standard Bacterial and Fungal Suspensions
The strains of E. coli, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aerugenosa were maintained on Nutrient broth at 37°C and Candida albicans, Fusarium solani were maintained on Saboraud dextrose agar, incubated at 25ºC for 4 days. The fungal growth was harvested and washed with sterile normal saline and finally suspended in (100 ml) of sterile normal saline and the suspension was stored in refrigerator till used. 
Antibacterial and Antifungal Activity by Agar Cup Method
The agar cup method was adopted for determination of antibacterial activity of the prepared extracts. 1.2 ml of standardized bacterial stock suspensions was thoroughly mixed with 120 ml of sterile Nutrient agar. 40 ml of the inoculated Nutrient agar was distributed into sterile Petri dishes. The agar was left to set and in each of these plates 4 cups, 4 mm in diameter, was cut using a sterile borer and the agar discs were removed. Alternate cups were filled with 0.1 ml of each extracts dissolved in dimethylsulphoxide (20 mg/ml) using microtiter-pipette and allowed to diffuse at room temperature for two hours. The plates were then incubated in the upright position at 37ºC for 18 h. After the incubation period, the wells were evaluated for formation of inhibition zone. The same procedure was followed for each strain and extract. Each experiment was carried out in triplicates. Ampicillin (10 μg/ml) was used as standard drug. 
For antifungal activity, Sabouraud dextrose agar was used instead of nutrient agar. The inoculated medium was incubated at 25ºC for two days for the Candida albicans and Fusarium solani. Fluconazole (30 μg/ml) was used as standard drug. The mean of the inhibition zone was taken for evaluating the antimicrobial activity of the extracts and standard drug.
2.2.4 Hydrogel prepration by Cold dispersion Method
Hydrogels were prepared by mixing of the water and gelling agent (Carbopol 940) in specified concentration under the mechanical stirrer. This homogenous dispersion was transferred to motor and amount of propylene glycol previously mixed with propyl paraben was added to the polymeric dispersion. Triethanolamine was added drop wise with continuous stirring until the homogenous hydrogel was formed. Solution of petroleum ether extract of Vitex negundo Linn. was prepared in ethanol. This solution was added in the hydrogel. The entrapment of air bubbles were removed by keeping the hydrogel in vacuum for 2 h.  Formulation of Vitex negundo Linn. hydrogel have been shown in Table 2.
Table 2: Composition of Various Formulation of Vitex negundo Linn. Hydrogel
|Ingredients||Types of Formulation|
|Carbopol 940 (%w/v)||0.5||1.0||1.5||2.0||0.5||1.0||1.5||2.0|
|Propylene glycol (ml)||10||10||10||10||10||10||10||10|
|Propyl Paraben (g)||0.5||0.5||0.5||0.5||0.5||0.5||0.5||0.5|
|Distilled Water qs (ml)||100||100||100||100||100||100||100||100|
VLPE: Petroleum ether extract of Vitex negundo leaf;
VSPE: Petroleum ether extract of Vitex negundo stem
2.2.5 Evaluation of Hydrogel
Homogeneity and Grittiness
0.5 g of hydrogel was applied on the skin area 1 inch2 and rubbed with uniform stress. By the sensory examination homogeneity of the hydrogel was observed after 30 min hydrogel was wash out by normal water tap. Following examination were recorded nature and sensation of hydrogel.
Spreadability of Hydrogel
The spreadability of prepared hydrogel was determined using the following technique: 0.5 g test formulation was placed within a circle of 1 cm diameter pre-marked on a glass plate over which a second glass plate was placed. A weight of 500 g was allowed to rest on the upper glass plate for 5 min. The increase in the diameter due to spreading of the test formulation was noted. 
Measurement of pH
The pH of hydrogel formulations was determined by using digital pH meter. One gram of hydrogel was dissolved in 100 ml of distilled water (1% w/v). The instrument was standardized by using pH 4.0, pH 7.0 and pH 10.0 standard buffer solutions before use. 
Brookefield Viscometer (Model DV-E) with helipath stand was used for rheological studies. The sample (50 g) was placed in a beaker and was allowed to equilibrate for 5 min before measuring the digital reading using a spindle No. 63 at 10 rpm. At this speed, the corresponding reading on the viscometer was noted [13, 14]
Drug content was determined by dissolving accurately weighed (1 g) quantity of gel in methanol (2 ml) and diluted upto 10 ml with and phosphate buffer pH 7.4 in 10 ml of volumetric flask (5000 μg/ml). Take 1 ml from this solution and diluted up to 10 ml in volumetric flask (500 μg/ml). After that take 5 ml and diluted up to 10 ml in volumetric flask (50 μg/ml). After suitable dilution absorbance was recorded by using UV-Visible spectrophotometer.
In-vitro Diffusion Study
In-vitro diffusion studies were carried out using Keshary-Chein diffusion cell using cellophane membrane. In Keshary-Chein diffusion cell 50 ml of phosphate buffer was used as receptor compartment, then 1 g of gel was spread uniformaly on the cellophane membrane.The entire surface of membrane was in contact with the receptor compartment containing 50 ml of phosphate buffer pH 7.4. The receptor compartment was continuously stirred (100 rpm) using a magnetic stirrer. The temperature maintained at 37±1ºC. The study was carried out for 6 h with the interval of 1, 2, 3, 4, 5, 6 h. 5 ml of each sample was withdrawn at predetermined time interval and same volume was replaced with fresh phosphate buffer pH 7.4. The absorbance of withdrawn sample was measured spectrophotometrically against blank. [15, 16]
In vitro Testing Antimicrobial Activity of Hydrogel Formulation
Antimicrobial activity of hydroel formulation (F2, F6) was determined by Agar Cup Method. F2, F6 formulation were selected because they having optimum viscosity, consistency and drug release. Zone of inhibition were measured in mm (Including bore diameter 4 mm). Antimicrobial activity of F2 formulation was better than F6 formulation for Staphylococcus aureus, Bacillus subtilis, E. Coli, Candida albicans.
RESULT AND DISCUSSION:
3.1 In Vitro Testing of Extracts for Antimicrobial Activity
Antimicrobial activity of various extracts was determined by Agar Cup Method. Zone of inhibition were measured in mm (Including bore diameter 4 mm). Most of the extracts of leaves and stems of Vitex negundo Linn. have considerable antibacterial and antifungal activity against all microbial strains but VLPE and VSPE showed maximum antimicrobial activity against tested species. VLPE had comparative antimicrobial activity with VLSE. Results are shown in Table 3 and agar paltes are shown in Fig. 2.
Table 3: Antimicrobial Activity of Extracts of Vitex negundo Linn.
|S.No.||Name of micro organism||VLPE||VLSE||Standard|
Values are mean inhibition zone (mm) ± SD of three replicates, Inhibition zone including 4 mm bore diameter
(A) – Antimicrobial activity of leaf extracts
(B) – Antimicrobial activity of stem extracts
3.2 Evaluation of Hydrogel
All gel formulation was elegant in appearance. A thin and smooth film was formed on application to the skin and easily washable with the water. The pH of all formulation lies between 5.72 to 6.36 which lies in the normal pH range of the skin i.e. the pH at which no irritation to the skin was observed. The rheological behavior of the gel formulations were studied by Brookfield Viscometer using spindle no. 63 at 10 rpm. The viscosity of all formulations lies between 42600 to 49300 CPs. The result for spreadibility lies between 3.7 to 4.5 cm. A comparative study of viscosity and spreadibility showed that as viscosity of formulation increases spreadibility decreases and vice versa. Results are shown in Table 4.
The absorption maximum of petroleum ether extract in phosphate buffer (pH 7.4) with ethanol (2% w/v) as co-solvent was observed at 413 nm for leaf and 402 for stem when scanned between 200-500 nm. The data of the standard curve were linearly regressed. The slope and correlation coefficient values for petroleum ether extract of leaf in phosphate buffer were found to be 0.004 and 0.997. The slope and correlation coefficient values for petroleum ether extract of stem in phosphate buffer were found to be 0.001 and 0.996. The drug content for all gel formulations lies between 95.33 to 103.49% [Table 4]. It showed that drug uniformly distributed throughout the gel formulations. Drug content was determined using these standard curves.
Table 4: Evaluation of Prepared Hydrogel
|Drug Content (%)||97.72± 3.22||102.64± 1.40||99.53± 2.09||98.59± 1.32||95.33± 5.88||103.49± 1.29||97.86± 0.88||100.25± 2.59|
- represent good; (-) represent absent
In-vitro Diffusion Study
All the gel formulations were subjected to in-vitro Diffusion Study. Study was carried out using Keshary-Chein diffusion cell using cellophane membrane. In all formulations the VLPE concentration was kept constant (5% w/w) and the concentration of carbopol 940 was varied. At 37°C, F1 formulation was stable but the consistency was not too good and F3, F4 were too viscous but F2 formulation was having optimum viscosity and consistency. The release of F1 formulation was 77.29% whereas for F2 was 72.41%, F3 was 57.89% and F4 was 45.13%. So the F2 formulation was selected for further antimicrobial activity. Result observed in Table 5 and Figure 3.
In the next four formulations of VSPE, extract concentration was kept constant (5% w/w) and the concentration of carbopol 940 was varied. At 37°C, F5 formulation was stable but the consistency was not too good and F7, F8 were too viscous but F6 formulation was having optimum viscosity and consistency. The release of F5 formulation was 67.25% whereas for F6 was 61.2%, F7 was 48.22% and F3 was 42.8%. So the F6 formulation was selected for further antimicrobial activity. Result observed in Table 5 and Figure 4.
Table 5: In-vitro Diffusion of VLPE& VLSE from Hydrogels
|Time (h)||Cumulative Percent Drug Released|
In vitro Testing Antimicrobial Activity of Hydrogel Formulation
Antimicrobial activity of F6 formulation was better than F2 formulation for P. aeruginosa, Fusarium solani. The results are shown in the Table 6 and Fig. 5. It was observed that antimicrobial activity of F2 formulation was comparable with F6 formulation.
Table 6: Antimicrobial Activity of Gel Formulation F2, F6
Values are mean inhibition zone (mm) ± SD of three replicates, Inhibition zone including 4 mm bore diameter
The past three decades have noticed a dramatic increase in microbial resistance to antimicrobial agents that lead to repeated use of antibiotics and insufficient control of the disease. New prototype antimicrobial agents are required to overcome this situation. The plant Vitex negundo Linn. have been successively used in many disease (inflammation, leucoderma, enlargement of spleen, bronchitis, asthma etc.) by the traditional communities and quite familiar in Ayurveda.
In-vitro antimicrobial activity was performed by Agar Cup method. The various types of extracts (petroleum ether) of Vitex negundo Linn. leaves and stems were investigated for its antibacterial and antifungal activity on four bacterial species [Staphylococcus aureus (Gram positive, MTCC 87), Bacillus subtilis (Gram positive, MTCC *121), E. coli (Gram negative, MTCC 40), Pseudomonas aerugenosa (Gram negative, MTCC 424)] and two fungal species [Candida albican (Fungus, MTCC 183), Fusarium solani (Fungus, MTCC 2935)]. The antimicrobial effect of various extracts of leaves and stems were compared with Ampicillin (antibacterial) and Fluconazole (antifungal), which served as reference compounds. The mean of the inhibition zone was taken for evaluating the antimicrobial activity of the extracts and reference drug. An antimicrobial study by Agar Cup method has shown that most of the extracts of leaves and stems of Vitex negundo Linn. have considerable antibacterial and antifungal activity against the microbial strains. However, petroleum ether extract of leaves and stems of the plant possessed more significant activity against Bacillus subtilis, Escherichia coli and Candida albicans strains when compared with the reference drug. Hence, petroleum ether extract of leaves and stems were selected for gel formulation. Four types of gel formulations (F1, F2, F3 and F4) were prepared for leaf extract and another four types of gel formulations (F5, F6, F7 and F8) were prepared for stem extract In all formulations the extract concentration was kept constant (5% w/w) and the concentration of carbopol 940 was varied (0.5%, 1.0%, 1.5%, 2.0%). The various gel characteristics viz. homogeneity, grittiness, pH, viscosity, spreadibility, in-vitro release of drug were also studied. It was found that formulation F2 and F6 (5% drug extract in 1% carbopol 940) exhibited more significant and in-vitro drug release as compare to other developed formulation (F1, F3, F4, F5, F7 and F8). Therefore, F2 and F6 formulation were selected for evaluation of antimicrobial activity. It was found both of them possessed significant antimicrobial activity and may serve as promising antimicrobial gel formulations.
The present study supports the traditional use in the treatment of some diseases as broad spectrum antimicrobial agent. However, the crude extracts of the plant need to be further purified through isolation and identification of the responsible compounds for antimicrobial activity. To explore the precise mechanism of action may be another interested area to work in future.
- AJ Modi; SS Khadabadi; IA Farooqui; DS Ghorpade. Der Pharmacia Lettre, 2010; 2, 1, 102-105.
- N Benkeblia. Lebensm-Wiss u-Technol, 1996, 37, 127-134.
- ML Colombo; E Bosisio. Ethanopharmacol Res, 1996, 33, 157-166.
- GR Mallavarapu; S Ramesh; RN Kaul; AK Bhattacharya; BR Rajeswara Rao. Planta Med, 1994, 60, 583-584.
- LN Gautam, SL Shrestha, P Wagle, BM Tamrakar. Scientific World, 2008, 6, 6: 27-32.
- B Das; R Das. Indian Drugs, 1994, 31, 9, 431-435.
- OP Tiwari; YB Tripathi. Food Chem, 2007,100, 1170–1176.
- D Sharma; AA Lavania; A Sharma. Asian J Exp Sci, 2009, 23, 1, 169-172.
- F Kavanagh. Analytical Microbiology. 1st, Academic Press, New York, 1972; pp 2-11.
- GK Muruthy; KV Sai. Indian J Pharm Edu Res, 2008, 42, 3, 272-276.
- RM Sreenivasa; S Mutulik. Indian J Pharm Sci, 2006, 68,4, 432-436.
- V Sankar; AK Chandrasekaran; S Durga. Indian J Pharm Sci, 2005, 67, 4, 473-476.
- PA Sonawane; UB Hadker. Indian Drugs, 1994; 32, 7, 353-355.
- JY Kima; JY Song; EJ Lee; SJ Park. Colloid Polymer Sci, 2003, 281, 614-623.
- K Ruckmani; B Jayakar; S Durgamani; TS Easwari; S Hurmathunisea. Indian Drugs, 1998, 35, 5, 303-305.
- S Sarasija; HP Joshi. Indian Drugs, 2005; 42, 3, 383-385.