INTRODUCTION:

80% of the world population relies on medicinal plants for their primary health care. Such herbal medicines that are easily available, cheaper, time tested and considered safer than most of modern synthetic drugs. Over 50% of the best selling pharmaceuticals in use today were derived from natural products.^[1][2] Plants provide a bank of rich, complex and highly varied structures, which are unlikely to be synthesized in laboratories. Ginger (Zingiber officinale) is a flowering plant whose rhizome, ginger root or simply ginger, is widely used as a spice or a folk medicine.^[3][4] It is a herbaceous perennial which grows annual pseudostems (false stems made of the rolled bases of leaves) about meter tall bearing narrow leaf blades. The inflorescences bear pale yellow with purple flowers and arise directly from the rhizome on separate shoots. Ginger is in the family Zingiberaceae to which also belong turmeric (Curcuma longa), cardamom (Elettaria cardamomum), and galangal.^[5][6]

The disadvantages associated with topical formulations include stickiness on skin causing uneasiness to the patient when applied. They also have less spreadability and need to apply with rubbing. In order to overcome those limitations, an emulsion based approach was attempted. When gels and emulsions are used in combination, the dosage forms are referred as emulgel.^[7][8] Emulgels are emulsions, either of the oil-in-water or water in-oil type, which are gelled by mixing with a gelling agent. They have a high patient acceptability since they possess the previously mentioned advantages of both emulsions and gels. Therefore, they have been recently used as vehicles to deliver various drugs to the skin.^[9][10] They are also called as creamed gel, quassi emulsion, gelled emulsion. Emulgel have several favourable properties such as being thixotropic, greaseless, easily spreadable, easily removable, emollient, non-staining and pleasing appearance^.In the present study, topical Emulgel formulations of ginger powder extract Oil were prepared using Sodium CMC as gelling agent of varying concentrations^.[11][12]

MATERIALS:

Na CMC was obtained from Vikash pharma Mumbai, Light liquid paraffin, Tween 80 and PEG 200 was obtained from Loba chemie Mumbai Emulsifying wax was obtained from Research chem. Fine lab industry Islampur, Ethanol, Methanol, and Water

Plant material and Preparation of extract:

Ginger rhizome was purchased from local market washed with clean water, cut, and dried by keeping it and then fine powder was made. 10g ginger rhizome powder was thoroughly mixed with 100 ml organic solvent (methanol). The mixture was placed at room temperature for 24h under magnetic stirring. Solution was filtered throw muslin cloth and then re-filtered through Whatman No.1. The filtrate thus obtained was concentrated by completed evaporation of solvent at room temperature to yield the pure extracts.

FORMULATION DEVELOPMENT:

The composition of ginger extract emulgel formulations was shown in Table 1. Different formulations were prepared using varying amount of gelling agent. The method only differed in the process of making gel in different formulations. The preparation of emulsion was same in all the formulations The gel bases were prepared by dispersing Na CMC, in heated purified water (80°C) separately, and the dispersion was cooled and left overnight. The oil phase of the emulsion was prepared by dissolving Emulsifying wax in light liquid paraffin while the aqueous phase was prepared by dissolving Tween 80 in purified water. Propyl paraben was dissolved in PEG 200 whereas ginger extract was dissolved in ethanol, and both the solutions were mixed with the aqueous phase. Both the oily and aqueous phases were separately heated to 70° to 80°C; then the oily phase was added to the aqueous phase with continuous stirring until cooled to room temperature. The obtained emulsion was mixed with the gel in 1:1 ratio with gentle stirring to obtain the emulgel.

Table No 1: Composition of emulgel formulations containing methanolic extract of Zingiber officinale

INGREDIENTS	F1	F2	F3	F4
Ginger extract(g)	05	05	05	05
Na CMC(g)	0.5	1.0	1.5	2.0
Light liquid paraffin(mL)	02	02	02	02
Tween 80(mL)	0.5	0.5	0.5	0.5
Emulsifying wax(g)	0.20	0.20	0.20	0.20
PEG 200(mL)	2.5	2.5	2.5	2.5
Ethanol	05	05	05	05
Water	qs	qs	qs	qs

Preformulation study:

It needed to ensure the development of stable, effective and safe dosage form.

Solubility analysis:

Solubility analysis is made by adding solute in small incremental amount to fixed volume of solvents such as distilled water, ethanol, chloroform and acetone. Then it is examined for undissolved particles.

Melting point:

The melting point of the obtained extract was estimated by the standard melting point determination method.

Confirmation of drug using UV-VIS Spectrophotometric method:

Phosphate buffer 6.8 was selected for preparation of calibration curve.

100mg of crude extract was dissolved in phosphate buffer and diluted upto 100ml to get concentration of 1000ppm which is treated as stock solution. This stock solution was diluted further to get different concentrations. Resultant solutions were scanned for λmax in the range of 200-400 nm using UV spectrophotometer.

Calibration curve of ginger extract:

In phosphate buffer l Stock solution of ginger extract were pipette out in to series of in volumetric flasks and diluted with phosphate buffer to get final concentration of 20-100mg /ml. The absorbance of resultant solution was measured at 248 nm.

EVALUATION OF EMULSION:

Centrifuge Stress Test:

Emulsions were centrifuged at for 30 min and then examined for phase separation.

Type of emulsion:

Little amount of water is mixed with emulsion. If water distributes uniformly it is O/W type of emulsion and if water separates out as a layer than it is W/O type of emulsion.

EVALUATION OF EMULGEL:

Physical Examination:

The prepared emulgel formulations are inspected visually for their colour, and appearance.

pH Evaluation:

pH evaluation is the important criteria especially for the topical formulation. The pH of the emulgel should be between 5-7 to mimic the skin condition. If the pH of the prepared emulgel is acidic or basic, it may cause irritation to the patient. pH of the prepared emulgel was measured using digital pH meter (ELICO LI 613). 1gm of gel was dissolved in 100 ml of distilled water and it was placed for 2 hr and then dip the glass electrode into an emulgel. The measurement of pH of each formulation was done in triplicate and average values were calculated

Spreadability:

Spreadability denotes the extent of area to which the emulgel readily spreads on application to skin or the affected part. The bioavailability efficiency of an emulgel formulation also depends on its spreading value. The Spreadability was expressed in terms of time in seconds taken by two slides to slip off from the emulgel which was placed in between the slides, under certain load. Lesser the time taken for separation of the two slides, better the spreadability. Two sets of glass slides of standard dimensions were taken. The herbal emulgel formulation was placed over one of the slides. The other slide was placed on the top of the emulgel, such that the emulgel was sandwiched between the two slides in an area occupied by a distance of 7.5 cm along the slide. 100gm weight was placed upon the upper slides so that the emulgel between the two slides was pressed uniformly to form a thin layer. The weight was removed and the excess of emulgel adhering to the slides was scrapped off. The two slides in position were fixed to a stand without slightest disturbance and in such a way that only the upper slide to slip off freely by the force of weight tied to it. A 10 gm weight was tied to the upper slide carefully. The time taken for the upper slide to travel the distance of 6.8 cm and separated away from the lower slide under the influence of the weight was noted. The experiment was repeated by three times and the mean time was taken for calculation. Spreadability was calculated using formula:

S = M. L / T

Where,

M= weight tied to upper slide (10g)

L = length of glass slide (6.8cm)

T = Time taken to separate the slides

Drug Content Determination:

0.1g was taken and was diluted with 10 mL phosphate buffer 6.8 and shaken to dissolve the drug in buffer. The solution was filtered through whattmann filter paper; 5 ml of the filtrate was taken out and diluted to 50 ml with buffer. The content of the drug was estimated spectrophotometrically and using slope and intercept using standard curve plotted at 248 nm

Compatibility studies by FT-IR:

FTIR Spectroscopy was carried out to check the compatibility between drug and polymer .The IR spectra of ginger extract, Sodium CMC and physical mixtures of drug and polymer was carried out by using FTIR, Bruker. The scanning range was 400 to 4000 cm-1 and the resolution was 1cm-1. The wave numbers of characteristic peaks of physical mixtures were compared with the pure samples and interpreted

In-vitro Release/Permeation Studies:

In vitro release studies were carried out using Franz diffusion cell of 10ml capacity. Egg membrane was isolated and used for the study. Pre-weighed emulgel was spread evenly on the egg membrane. The egg membrane was clamped between donor and receptor compartment. The receptor compartment was filled with 10 ml of pH 6.8 phosphate buffer maintained at 37oC and stirred by using magnetic stirrer. 1 ml sample was collected at suitable time intervals (i.e., for every 30mins until complete drug was released) and replaced with fresh buffer. The collected samples were analyzed for drug content by UV –Visible Spectrophotometer at 248nm.

Antimicrobial study:

Bacterial strains selected for susceptibility Assay :

The two bacteria namely Escherichia coli (gram-negative bacteria) and Staphylococcus aureus (gram-positive bacteria) were selected for the current study. These bacterial cultures were preserved in nutrient agar slants at 37°C. Individually of the bacteria was reenergized before to sensibility testing by moving them into a distinct test tube having nutrient broth and keep warm overnight at 37°C.

Antibacterial testing:

Antibacterial activities was studied for the best proved formulation (F3) by standard agar well diffusion assay. Petri dishes were prepared by pouring agar (red MacConkey ager medium for Escherichia coli and nutrient ager for Staphylococcus aureus) and were seeded with inoculum of bacterial strain. Media was allowed to get hard and then specific petri dishes were marked for the each bacteria. Bores of 6mm width were scratch in hardened ager media with assistance of sterilized cup-borer. Little amount of the formulation was poured in the wells and plates were incubated at 37C˚ for 24h. The zone of inhibition were measured. Result of the studies is given in the table no 6.

Stability Studies:

Stability studies were carried out for the optimized formulation (F3) according to International Conference on Harmonization (ICH) guidelines. Short term accelerated stability studies were carried out for the period of 3 months for the formulations. The samples were stored at different temperature conditions i.e., refrigeration temperature (4-8°C), room temperature (25 ±2°C) and oven maintained at (45°C ± 2°C). Sample was withdrawn on monthly interval and analyzed for visual appearance, clarity, pH, and drug content. Sample was withdrawn for three months and assessed for various parameters. At the end of third month they were evaluated for physical parameters and integrity of the product.

Kinetic studies and release mechanism:-

In vitro drug release kinetics:

The kinetics were estimated by fitting the data in various kinetic models like zero order, first order and the results were observed.

Release mechanisms:

By incorporating release data in Higuchi and Hixson Crowell model, the R2 values of all the formulations were found. To further confirm the exact mechanism of drug release, the data was incorporated into koresmeyer Peppas model and the mechanism of drug release was indicated according to the value of exponent ‘n’.

RESULTS AND DISCUSSION:

Preformulation studies results:

Solubility analysis:

Table 2:- Solubility of the Ginger extract in various solvent

Sr No	Solvent	Solubility behaviour
1	Distilled water	Soluble
2	Acetone	Soluble
3	Chloroform	Soluble
4	Ethanol	Soluble

Determination of melting point:-

The melting point of the Ginger extract was found to be 32^oC.

Confirmation of drug using UV-VIS Spectrophotometric method:

Fig 1: UV spectra of Zingiber officinale extract

The λmax value was found to be 248nm.

Calibration curve for the Ginger Extract:

Calibration curve of methanolic extract of Zingiber officinale in phosphate buffer of pH 6.8:

Standard plot of Zingiber offiinale was plotted as per the procedure in experimental methods and its linearity was shown in Table 3 and Fig 2. The standard graph of Zingiber officinale shows good linearity with R² value of 0.986, which indicates that it obeys Beer’s-Lambert’s Law in the concentration range of 0-100 mg/ml.

Table No 3:- Calibration curve of methanolic extract Zingiber officinale in phosphate buffer of pH 6.8

Concentration (mg/mL)	Absorbance (248 nm)
10	0.102
20	0.242
30	0.318
40	0.412
50	0.491

Fig No 2: Calibration curve of methanolic Zingiber officinale in phosphate buffer of pH 6.8.

EVALUATION OF EMULSIONS:

No phase separation was seen for any of the formulation. The two layer remains intact after centrifugation. No phase separation was seen for any of the formulation.

Type of emulsion:

Table 4: Determination of the emulsion type

EMULSION	EMULSION TYPE
F1	O/W
F2	O/W
F3	O/W
F4	O/W

Evaluation of Emulgel:

Physical appearance:

All the formulations were evaluated for their colour and appearance. The physical appearances of all the formulations were found to be brown, and translucent (Table no 5)

Table 5: Characterization of Zingiber officinale Emulgel Formulations for Colour, Appearance

FORMULATION	APPEARANCE	COLOUR
F1	Translucent	Brown
F2	Translucent	Brown
F3	Translucent	Brown
F4	Translucent	Brown

pH determination:

Skin compatibility is the primary requirement for a good topical formulation. It was found that the pH of all formulations were in the range of pH 6.4 to 7.0 (Table 6) which indicates skin compatibility i.e., ginger extracts emulgels can be applied to the skin without any discomfort or irritation.

Spreadability studies:

Spreadability study is one of the criteria for an emulgel to meet the ideal qualities that it should possess good spreadability. If spreadability value is more, it would be properly spread over the skin which is more beneficial as per patient compliance concern. All the formulations were checked for the spreadability and the data was given in the table no 6 F3 shows best spreadibility.

Drug content determination:

Drug content of all the formulations was found as per the method mentioned in the methodology section. Drug content of all the formulations were found to be in the range of 95-100% as indicated in the table no 6

Table 6: Characterization of Zingiber officinale Emulgel Formulations for pH, Spreadability, Drug content.

FORMULATION	Ph	SPREADIBILITY (cm/sec)	DRUG CONTENT (%)
F1	6.5	11.33	95.2
F2	6.5	8.5	95.54
F3	6.9	13.6	98.22
F4	6.8	6.8	96.7

Compatibility studies:

From the Fig 3 and 4 FT-IR spectra of physical mixture of the drug, polymer, and other ingredients, it was revealed that there was no chemical interaction of plant extract with other excipients.

Table 7: In-vitro drug release profiles of Zingiber officinale emulgel formulations (F1-F4)

TIME	% DRUG RELEASE
TIME	F1	F2	F3	F4
0	0	0	0	0
1	11.61	10.5	21.2	19
2	30.61	23.22	38.7	39.05
3	44.33	43.27	52.2	46.44
4	52.77	64.38	62.27	53.83
5	60.16	72.83	72.83	62.27
6	80.22	73.88	73.94	65.44
7	84.44	85.5	93.94	83.38
8	97.11	96.05	97.11	95

In vitro drug permeation for formulations:

The emulgel formulations of Zingiber officinale (F1-F4) were tested for their drug diffusion study through a egg membrane. The release of ginger extract from the prepared emulgels was performed in order to study the effect of different concentration of Sodium CMC. (Table No 7 and fig no 5

Fig 5: Drug release profiles of Zingiber officinale emulgel formulations (F1-F4)

Table No 8: Zone of inhibition showed by F3 (optimized formulation) for selected bacteria

Antibacterial assay

Bacteria	Zone of inhibition
S.aureus( gram positive)	7mm
E.coli (gram negative)	10mm

Stability studies:

Stability study was performed on optimized formulation F3 at ambient conditions. The results obtained after 3 month time period are shown in table 8. The result revealed that there was no significant change in the colour, pH, and drug content in optimized formulation. The results conclude that the optimized formulation is stable for 3 months and can be applied topically.

In vitro drug release kinetics:

The kinetics were studied by fitting the data in various kinetic models like zero order, first order and the results were shown in Table 10. When the R² values of regression plots for first order and zero order were considered, R² values of first order plots were found to be higher than zero order plots. Hence it is evident that the drug release from all emulgel formulations followed zero order kinetics.

CONCLUSION:

Studies have shown that ginger has many components like gingerol and shagols which have many properties including antibacterial activity. So in this work the attempt was made to use this ginger extract into topical emulgel.

The confirmation of the component present in the extract was done by finding λmax, running TLC, and determining melting point and the results were compared with standard result found during literature survey. All the formulations were almost neutral falling n the pH range suitable for skin.(6.4-7). Drug content was found to be in the range of 90-100%. Comparing the results obtained for all formulation F3 was found to have highest spreadability. Maximum release was shown by F3 which follows zero order kinetics and higuchi diffusion model. Stability studies showed that there was no significant change in the parameters. Also the in vitro studies showed that F3 releases maximum amount of drug over the time period of 8 hours. Also the F3 formulation inhibits the growth of bacteria selected for the assay. It was finally concluded that the F3 formulation was best among the others. Future research work will include evaluation of other parameters and some other tests using animal models

ACKNOWLEDGEMENT:

Authors would like to thank DM’s college for providing chemicals and required infrastructure to carry out this research work. Also the authors would like to thank all the people who boosted authors with their support and encouragement.

REFERENCES:

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Received on 28.09.2018 Modified on 03.11.2018

Research J. Pharm. and Tech. 2019; 12(4):1559-1565.

DOI: 10.5958/0974-360X.2019.00258.0

Initial			After 3 months
Appearance	pH	Drug content	Appearance	pH	Drug content
Brown and translucent	6.9	98.22	Brown and translucent	6.9	98.19

Formulation	Zero order R²	First order R²	Higuchi R2	Hixson Crowell R²	Korseymeyer peppas R²	Korseymeyer peppas ‘n’
F3	0.965	0.973	0.972	0.936	0.958	0.717