FORMULATION AND EVALUATION OF ANTIBACTERIAL AND ANTIOXIDANT POLYHERBAL LOTION

There is increased scientific evidence that plants possess a vast and complex arsenal of active ingredients which have the ability to calm or smooth the skin as well as restore actively, heal and protect the skin. The present work deals with the development and evaluation of the poly herbal lotion containing Trigonella foenum-graecum, Citrus lemon, Matricaria chamomilla and Cymbopogon citrates. Different types of formulations oil in water (O/W) herbal lotions namely F1 to F13 were formulated by incorporating different concentrations of stearic acid and Triethanolamine. Formulation of Triethanolamine and stearic acid was optimized as 2.52:9.35. The prepared lotion was evaluated for its antimicrobial, antioxidant and pharmaceutical parameters. The lotion formulation showed no redness, edema, inflammation and irritation during sensitivity test indication that it is safe to use. Stability studies of the lotion showed that the lotion was stable after three months.


Phytochemical screening
Phytochemical screening of all extracts was carried out according to standard method (Trease & Evans 1989).The extracts were analyzed for presence of alkaloids, carbohydrates, glycosides, saponin, phytosterol, phenols, tannins and flavonoids.

Antibacterial property
Antibacterial property was evaluated by well diffusion method (Collins et. al 1995).The microorganisms used were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumonia.Total 20 µl of test solutions of the sample and standard prepared (2%, 4%, and 6%) were then pipetted with the help of the micropipette and poured in the prepared bore in each nutrient agar plate swabbed with different bacteria with great care.Similarly, 20 µl of DMSO (10%) solution was also poured in another bore as control.The nutrient agar plates were allowed to stand for about 1 hour till the test solution completely diffuses in the media.The plates were then incubated at the temperature of 37 ο C for 48 hours.After 48 hours, zone of inhibition was measured (Junaid et al. 2006).Minimum Inhibitory Concentration (MIC) test was carried out by serial dilution method (Mhatre et al. 2014).The extracts and EO were serially diluted to obtain the concentration from 200 mg/ml to 6.25 mg/ml.A loop full of microbial suspension which was already prepared was added to each test tube.The test tubes were then incubated at 37 ο C for 24 hours.The MIC is the concentration of the highest dilution tube in which the bacterial growth was absent, hence no turbidity observed.

Determination of DPPH free-radical scavenging activity
The antioxidant activity of the extracts and the essential oil based on the scavenging activity of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical was determined by the method described by Mensor et al. (2001) with a slight modification.In the DPPH radical scavenging assay, antioxidants react with DPPH, and convert it to the yellow colored а, а-diphenyl-β-picryl hydrazine.Ascorbic acid was used as standard.The DPPH radical scavenging activity percentage was calculated by using the formula: DPPH radical scavenging activity (%) = (A control -A sample ) / A control ×100 Where, A control is the absorbance of a DPPH solution without sample, and A sample is the absorbance of the sample.

Formulation of lotion
Topical lotion formulation was designed using central composite design (CCD) with two factor and 3 levels.Stearic acid and Triethanolamine were chosen as independent variables while spreadability, pH and viscosity as dependent factors.The lotion was prepared by adding non-polar phase to the polar phase with rapid stirring to avoid separation of water and oil phase.Non-polar phase was first melted together and slowly added to the preheated mixture of polar phase.Triethanolamine and stearic acid when mixed, undergo an acid-base reaction to yield an ionic compound (a salt).This salt has a nonpolar section and a charged section, so it is a good emulsifying agent.Cetosteryl alcohol in combination with other ingredients in the formulation such as Triethanolamine and stearic acid forms an emulsion.This stops oil and water from separating.

Statistical analysis
Mathematical relationships for the measured dependent variable (response) and the independent variables were developed using statistical software Minitab.Thirteen tests were conducted as per the software.The three output variables (responses), such as viscosity (Y1), pH (Y2), spreadability (Y3), were evaluated.The experimental results were fitted to a polynomial quadratic model by applying multiple regression analysis for pH, viscosity and spreadability.The effect of formulation variables on different dependent or response variables was assessed by the generated regression coefficients and r 2 values.The fitted quadratic equations relating the responses such as viscosity (Y1), pH (Y2), spreadability (Y3), to the transformed factor are given in equations respectively below: Y 1 = +5656.6-101.1 X 1 +1755.Where; X 1 is concentration of Triethanolamine, X 2 is the concentration of stearic acid.

Optimization of Formulation
The formulation was optimized using response optimizer in Minitab Software.The excipients used as variables were Triethanolamine and Stearic acid.Desirability function was calculated for pH (Y1), viscosity (Y2), and spreadability (Y3) using the software.On the basis of the ratio obtained from the software final optimized formulation was developed.

Evaluation of Pharmaceutical parameters of lotion
Preliminary Pharmaceutical evaluation of lotion formulations was carried out as follows: PH: Lotion pH was measured with a digital pH meter.10% solution of lotion was prepared and the solution was immersed in the pH meter and the measured pH was recorded (Namita & Nimisha 2013).
Viscosity: viscosity was evaluated in Brookfield viscometer using LV-64 spindle.The rotation rate was adjusted to 25 RPM.The formulated lotion was directly immersed into the spindle and the viscosity was measured (Kumar et al. 2011, Neuwald 1966).Spreadability: The spreadability of lotion was determined by the parallel plate method.Two glass slides of 20/20 cm were selected.About 1 g of the lotion formulation was placed over one of the slides.The other slide was placed upon the top of the lotion such that the lotion was sandwiched between the slides and 125 g weight was placed upon the upper slide so that lotion between the two slides was pressed uniformly to form a thin layer.
The weight was removed and the spread diameter was measured (Garg et al. 2002).

Stability Test:
The formulated lotion was stored at different temperatures and humidity conditions of 25±2 ο C / 60±5% RH (at room temperature), 40±2 ο C / 75±5% RH (accelerated temperature) for a period of three months and studied for pH, viscosity and spreadability (Negi et al. 2012).Sensitivity Test: A portion of lotion was applied on the forearms of 6 volunteers and left for 20 minutes.After 20 minutes any kind of irritation if occurred was noted (Draize et al. 1944).Washability Test: A portion of lotion was applied over the skin of hand and allowed to flow under the force of flowing tap water for 10 minutes.The time when the lotion completely removed was noted.
Appearance: The color, odor and homogeneity of the lotion were visually determined.Type of emulsion test: Dye solubility and dilution test was conducted to determine the type of emulsion formed (Tharwat 2013).

Phytochemical screening
The plants were undertaken for phytochemical screening of alkaloids, carbohydrates, glycosides, saponins, phytosterols, phenols, tannins, flavonoids.It was investigated that tannin, phenols and phytosterols were commonly found.Alkaloid was absent in Allium sativum and Citrus lemon.Carbohydrate and glycosides were absent in all samples.Flavonoids, exhibits the large number of biological activities like anti-inflammatory, antioxidant and antimicrobial properties (Ojala, 2000).

Antibacterial activity study
Ethanol extract of the plants were undertaken for antibacterial activity.All of the extracts showed significant antibacterial activity at 6 percent concentration.Among five extracts Allium sativum showed less activity against bacteria while Citrus lemon showed highest activity.Afterwards, five different formulations were prepared using four of the five plant extract excluding Allium sativum since it possesses minimum antibacterial activity.Against Escherichia coli the best MIC (50 mg/ml) was shown by Cymbopogan citrates as compared to other extracts and essential oil.Against Klebsiella pneumonia all of the extracts and essential oils showed MIC of 100 mg/ml which is not very effective.Since lower MIC is indication of better antibacterial property, it was concluded that Citrus lemon is best antibacterial agent as compared to other extracts and oils which was followed by Cymbopogon citratus.

Effect of independent variables on lotions Effect on viscosity of lotion
It was observed that Triethanolamine have negative effect on the viscosity whereas, the stearic acid have positive effect on the viscosity.The increase in the concentration of Triethanolamine decreases viscosity and the increase in concentration of stearic acid increase the viscosity.

Effect on pH of lotion
The positive magnitude of the coefficient of Triethanolamine represents the positive effect between the Triethanolamine and the pH.The increase in concentration of Triethanolamine leads to the increase in the pH of the formulation.
Similarly, the negative magnitude of the coefficient of stearic acid denoted the inverse relation between the pH of the formulation and the stearic acid.The increase in concentration of stearic acid causes the decrease of the pH of the formulation.

Evaluation of pharmaceutical parameters of lotions
The pH of the lotion was within the limit which indicated that the lotion is safe to use for skin and is stable.The pH of lotion influences the stability.
The stability of lotion increases with an increase in viscosity of the medium.Among thirteen different formulations the value of viscosity differed from 4908 cps to 10187 cps.The range of spreadability for lotion was from 7 to 13 cm.The measured minimum spread diameter of the lotion was 8.2 cm and the maximum spread diameter was 10.3 cm.The lotion was not irritating to the skin and it was water removable.The final appearance of lotion was found to be yellowish white in color.Emulsion formed during the formulation of lotion was evaluated for the type of emulsion.Oil miscible dye Sudan III was mixed with the sample and observed under microscope.Small red droplets of oil can be observed surrounded by water phase.This helps to identify that the lotion was oil-in water emulsion.
The formed lotion was oil-in water type.

Optimization of formulation
The range of pH suitable for hand lotion was 4 to 7 and for spreadability the desired range was 7 to 13 cm.The viscosity should be such that the lotion can be easily spread over the skin surface.The optimized formulation was evaluated for the response parameters and the value pH of optimized formulation was 5.6, value of viscosity was 5561 cps and the value of spreadability was 9.8.All the three parameters were within the limit and suitable for application over the skin.

Fig. 6. Response Optimization plot.
The desirability results showed (2.52:9.35)ratio of Triethanolamine and Stearic acid as optimum.So, on the basis of the ratio obtained from the software final optimized formulation was developed.

Stability test
The stability test of final optimized lotion was carried out and it was found that the lotion was stable in room temperature and accelerated temperature for at least three months.The value of pH, viscosity and spreadability all lay within the required range.There was no major difference in values of pH, viscosity and spreadability as compared to the initial value.[2.5286] [9.3572] different plant extracts it is possible to improve as well as synergize the cosmetic properties of prepared products compare to individual extracts.
From the above study it has been revealed that that the lotion was stable in room temperature and accelerated temperature for at least three months.

Fig 1 :
Fig 1: Comparison of IC 50 value of antioxidant property of different plant extracts, oils and ratios.
Thirteen tests were conducted as per the software.The three output variables (responses), such as viscosity (Y1), pH (Y2), spreadability (Y3), were evaluated.The effect of formulation variables on different dependent or response variables was assessed by the generated regression coefficients and r 2 values.The fitted quadratic equations relating the responses such as viscosity (Y1), pH (Y2), spreadability (Y3), to the transformed factor are given in equation 1 to 3 respectively.

Fig. 5 .
Fig. 5.Estimated contour plot and response surface plot elucidating the relationship between the spreadability and the independent variables (Triethanolamine and stearic acid).

Table 7 . Stability test at room temperature and accelerated temperature (of optimized formulation)
Oil in water type lotion was formulated using formulation of Trigonella foenum-graecum, Citrus lemon, Matricaria chamomilla and Cymbopogon citratus at the ratio of 1:1:1:2.This optimum formulation was designed with 2.52:9.35ratios of triethanolamine and stearic acid.The lotion formulation showed no redness, edema, inflammation and irritation during sensitivity test indicating it is safe to use.By combining the