INTRODUCTION:

The practice of herbal medicine has existed since prehistoric times as the primary form of medicine. In this space age where the technology has very much advanced, herbal medicines still flourish and are finding exceptional acceptance in both the developing and the developed countries due to their natural origin and lesser side effects.

Earlier plant products were used as a starting material for the preparation of semi synthetic drugs, but now a days modern herbal technology delivers medicinal plant extracts in a dosage form which can be adsorbed readily into the system using appropriate vehicle and excipients. The herbal extracts can be prepared in various formulations such as cream, lotions and gels. Safe and effective formulations deliver active substances to the desired site of action with negligible discomfort and side effects¹.

Polycarpaea aurea belonging to family Caryophyllaceae, the genus Polycarpaea is comprised up of 133 species of plants dispersed crossover Asia, Mauritania, Morocco, North America and available species in India P.nivea, P.gnaphalodes, P.candid, P.lancifolia, P.robusta, P.microphylla. Polycarpaea aurea commonly known as Rathirajuma (Telugu), Areshina saasuvae (Kannada)². Traditionally it was claimed as anti diabetic, anti dysentery, diaphoretic, anti microbial, anti fungal, anti diarrhoeal³. In the present study an effort has been made to exact identity and standardisation of whole plant of Polycarepaea aurea Weight and Arn. Herbal extracts or molecules are known for their potent therapeutical action and fewer side effects but they have draw backs like low bioavailability, high molecular weight and low lipophilicity. To overcome such difficulties novel drug delivery systems are employed for herbal extracts/drugs. One of such an approach is emulgel. Gel formulations commonly offer faster drug release than conventional dosage forms. The present study focused on the exploration of active plant metabolites present in whole herb and involves the preparation of emulgel of Polycarpaea aurea extract by using different natural penetrants, followed by the evaluation for physical appearance, consistency, odour, extrudability, pH, viscosity and invitro anti bacterial studies.

MATERIALS AND METHODS:

Collection of plant material:

The plant Polycarpaea aurea wight and Arn was collected from Tirumala hills, Tirupati, India and it was identified and authenticated. The taxonomical identification and authentication of the plant was done by Dr. P. Jayaraman, Botanist, Director for Institute of Plant Anatomy Research Center (PARC), Chennai, India.

Preparation of extracts:

About 100gm of the whole plant powder was successively extracted with solvents like petroleum ether, chloroform, and methanol in Soxhlet apparatus. The extracts were concentrated and traces of the solvent were completely removed under reduced pressure and stored in vacum desiccators for the further use. Aqueous extract was prepared by macerating the dried powder in distilled water. The extract was concentrated in water bath and stored in desiccators. The concentrated extracts were subjected to preliminary phytochemical screening for the identification of the various active constituents as per the standard procedures⁴.

Preparation of Emulgel:

Chemicals:

All the chemicals and reagents used were of analytical grade and purchased from Sd fine chemicals, Himedia and Rajesh chemicals and are follows carbopol 934, liquid paraffin, tween 80, span 80, ethanol, propylene glycol, methyl paraben, propyl paraben, triethanolamine, DMSO, nutrient agar, lavender oil, eucalyptus oil, orange oil, and mentha oil.

Procedure:

Different formulations were prepared using constant amount of gelling agent and by varying natural penetration enhancers. The method is same in process of making gel in different formulation. The preparation of emulsion was same in all the formulations. The gel phase in the formulations was prepared by dispersing Carbopol 934 in purified water with constant stirring at a moderate speed using mechanical shaker, then the pH was adjusted to 6–6.5 using tri ethanol amine (TEA). The oil phase of the emulsion was prepared by dissolving span 80 in light liquid paraffin while the aqueous water. Methyl and propyl parabens were dissolved in propylene glycol, whereas Polycarpaea aurea methanol extract was dissolved in ethanol, and both solutions were mixed with the aqueous phase. Orange oil, eucalyptus oil, lavender oil and mentha oil^5,6,7,8 were mixed in oil phase. Both the oily and aqueous phases were separately heated to 70–80⁰C, and then the oily phase was added to the aqueous phase with continuous stirring until it got cooled to room temperature. The obtained emulsion was mixed with the gel in1:1 ratio with gentle stirring to obtain the emulgel⁹ and various emulgel formulations are shown in table 1.

Table1. Composition of different emulgel formulation batches (%w/w)

Ingredients	F1	F2	F3	F4
Polycarpaea aurea methanol extract	0.5	0.5	0.5	0.5
Carbopol 934	1.0	1.0	1.0	1.0
Liquid paraffin	3.7	3.7	3.7	3.7
Tween 80	0.25	0.25	0.25	0.25
Span 80	0.5	0.5	0.5	0.5
Propylene glycol	2.5	2.5	2.5	2.5
Ethanol	1.25	1.25	1.25	1.25
Methyl paraben	0.015	0.015	0.015	0.015
Propyl paraben	0.005	0.005	0.005	0.005
Lavender oil	5	-	-	-
Eucalyptus oil	-	5	-	-
Orange oil	-	-	5	-
Mentha oil	-	-	-	3
Water	q.s	q.s	q.s	q.s

* q.s. - a sufficient quantity

Evaluation of Emulgels:

Physical appearance:

The prepared emulgel formulations were inspected visually for their color, homogeneity, consistency, grittiness and phase separation.

p^H:

The pH of emulgel formulations was determined by using digital pH meter. 1gm of gel was dissolved in 100 ml of distilled water and it was placed for 2 hr. The measurement of pH of each formulation was done in triplicate and average values were calculated¹⁰.

Viscosity:

Viscosity of the emulgels was determined using Brookfield viscometer. Spindle type; model LVDV-E at 10 rpm. 200 grams of the gel was taken in a beaker and the spindle was dipped in it for about 5 minutes and then the reading was taken.¹⁰.

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 7.5cm 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. 20gm weight was tied to the upper slide carefully. The time taken for the upper slide to travel the distance of 7.5cm 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 taken for calculation¹¹.

Spreadability was calculated by using the following formula:

S = M. L / T

Where,

S – Spreadability

m – Weight tied to the upper slide (20gm)

l - Length of the glass (7.5 cm)

t - Time taken in seconds

Drug content:

Polycarpaea aurea wight and Arn crude extract content in emulgel was measured by dissolving known quantity of emulgel in solvent (ethanol) by Sonication. Filtration of resulting solution was done by using whatman filter paper no.41. Absorbance was measured after suitable dilution at 319 nm using UV/VIS spectrophotometer¹².

In vitro release/ Permeation studies:

The in vitro drug release studies were carried out using a modified vertical Franz diffusion cell (with effective diffusion area 1.44cm² and 15.5ml cell volume). The formulations were applied on Nylon membrane 0.45μm (which was previously soaked in Phosphate buffer pH 7.4 for 24 hours); which was sandwiched between donor and receptor compartment of the Franz diffusion cell. Phosphate buffer pH 7.4 + ethanol (80:20) was used as a dissolution media. The temperature of the cell was maintained at 37±0.2⁰ C by keeping it in water bath. This whole assembly was kept on a magnetic stirrer and the solution was stirred continuously using a magnetic bead at 50 rpm. The samples (1.0 ml aliquots) were withdrawn at suitable time interval and analysed for drug content by UV visible spectrophotometer at 321 nm after appropriate dilutions¹³.

Extrudability:

The extrudability of formulations were determined using aluminium collapsible tubes filled with 10gm gel. Tubes were held between two clamps. A tube was compressed and extrudability of the formulation was determined in terms of weight in grams required to extrude a 0.5cm ribbon of gel in 10 seconds ¹⁴. The extrudability was calculated by using the following formula.

Extrudability = Applied weight to extrude emulgel from tube (in gm) / area (in cm²)

Swelling index:

To determine the swelling index of the prepared topical emulgel, 1gm of emulgel is taken on porous aluminium foil and then placed separately in a 500ml beaker containing 10ml 0.1 NaOH. Then samples were removed from the beakers at different time interval’s and put it on dry place for some time after it reweighed¹⁵. Swelling index is calculated as follows:-

Swelling index (%) = [(W_t - W_e)]/W_e X 100

Where,

Swelling index =equilibrium per cent swelling,

W_e= original weight of emulgel at zero after time t,

W_t=weight of swollen emulgel.

In vitro anti bacterial evaluation

Diffusion assay method:

The four emulgels were tested against bacterial strains namely gram +ve microorganism (Enterococus and Staohylococus aureus) and gram –ve microorganism (Escherichia coli and Pseudomonas). A loopful of the pure bacterial culture was suspended in nutrient broth and incubated for 24 hours. Nutrient agar media was sterilized and poured into petri plates. After solidification, 0.1ml of the inoculum was spread over the agar evenly using a rod. 6mm diameter cavity was prepared and formulated gel is placed in the cavity. A standard antibiotic was used as the control. The inoculated plates are incubated for 24 hours. Later, the zone of inhibition around the disc was measured and recorded¹⁶.

RESULTS AND DISCUSSION:

Phytochemical screening: The chief phytochemicals present in the different extracts whole plant of Polycarpaea aurea were flavanoids, tannins, alkaloids, glycosides, steroids, terpenoids and shown in shown in Table 2.

Table 2. Preliminary Phytochemical analysis of various extracts of Polycarpaea aurea

S.No.

Test

Pet. Ether

Chloroform

Ethyl acetate

Methanol

Water

Carbohydrates

Alkaloids

Glycosides

Tannins

Steroids

Triterpenoids

Flavanoids

Saponins

“+” represent presence “_” represent absence

In general, gel formulation is more preferred, among the other topical semisolid preparations, since it has long residence time on the skin, high viscosity, moisturising effect on flaky skin due to their occlusive properties, more bio adhesiveness, less irritation, independent of water solubility of active ingredient, ease of application and better release characters¹⁷. Many studies have indicated that poly phenolic plant metabolites, alkaloids and glycosides possess anti bacterial activity. Further, these active constituents in herbs reported that they can penetrate the human skin and hence a topical herbal emulgel formulation was designed containing these active metabolites with some natural penetrant enhancers such as orange oil, lavender oil, mentha oil and eucalyptus oil ¹⁸.

Physical appearance:

All the formulations were evaluated for their color and appearance. The physical appearances of all the formulations were found to be cream coloured, slightly orange (F3), clear and transparent. (Table 3)

Extrudability studies:

Extrudability studies of all formulation were carried out as per standard procedure stated in methodology section. The results were shown in Table 3. It was found that formulation F3 had excellent extrudability; formulations F1 and F4 showed good extrudability where as formulation F2 exhibited poor extrudability.

Table 3. Characterization of Emulgel Formulations for Color, Appearance and Extrudability

Formulation	Colour	Appearance	Extrudability
F1	Cream coloured	Clear, Transparent	Good
F2	Cream coloured	Thick, opaque	Poor
F3	Light Orange coloured	Clear, Translucent	Excellent
F4	Cream coloured	Clear, Transparent	Good

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 6.8 to 7.1 as shown in Table 4 which indicates skin compatibility and signifies that P. aurea emulgels can be applied to the skin without any discomfort or irritation.

Spreadability studies:

Spreadability study is one of the major 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 4. The values of the spreadability indicated that the emulgels were easily spreadable by small amount of shear. The order of increasing spreadability was F3> F2> F4> F1. By taking the data into consideration, it was observed presence of different permeation enhancers makes the difference in the spreadability. Among all the formulations F3 emulgel formulation containing Orange oil (1%) has shown highest spreadability value (9.4 cm/sec).

Rheological study:

The viscosities of all the formulations were measured using Brookfield viscometer, spindle 42 (DV++) at 0.5 and 1 rpm and the viscosities for all the formulations were given in the Table 5. It was found that all the formulations followed shear thinning effect with thixotropic property. It was observed that the viscosity of the formulation varied with type of penetrant employed. Among all the formulations F2 emulgel formulation containing Eucalyptus oil in minimum concentration (1%) has shown low viscosity (148.6 cps) while F1 emulgel formulation containing Lavender oil (2%) has shown high viscosity (236 cps).

Table 4. pH, Spreadability, Viscosity values of emulgel formulations

Formulation	pH	Spreadability (cm/sec)	Viscosity (cps)
F1	6.8	6.2	236
F2	7.1	8.4	148.6
F3	6.9	9.4	172.3
F4	7.0	7.3	180.4

Calibration curve of methanolic extract of Polycarpaea aurea in phosphate buffer of pH 6.8:

Standard plot of P. aurea was plotted as per the procedure in experimental methods and its linearity was shown in Table 5 and Figure 1. The standard graph of Polycarpaea aurea shows good linearity with R2 value of 0.999, which indicates that it obeys Beer’s-Lambert’s Law in the concentration range of 0-100 μg/ml.

Drug content determination:

Drug content of all the formulations was carried out as per standard procedure. Drug content of all the formulations were found to be in the range of 76.04-86.2% as indicated in the Table 7. The result indicates that uniform amount of drug is present in all the emulgel formulations.

Table 5. Calibration curve of Methanolic extract of Polycarpaea aurea

S. No	Concentration (mcg/ml)	Absorbance
1	20	0.12
2	40	0.24
3	60	0.36
4	80	0.47
5	100	0.68

Figure 1. Standrad Calibration Curve

Table 6. Drug content in emulgel formulations

Formulation	Drug content (%)
F1	84.6
F2	83.1
F3	86.2
F4	76.04

Figure 2: Drug content in emulgel formulations

In-vitro drug permeation for Polycarpaea aurea emulgel formulations:

The emulgel formulations of Polycarpaea aurea were characterized for their drug diffusion study using Franz diffusion cell through a membrane. The release of P. aurea from the prepared emulgels was performed in order to study the effect of different penetrants on the release of P. aurea aiming to select the best formula.

The percentage of Polycarpaea aurea released from emulgel formulations containing 1% of different penetrants were reported in Table 6 and Figure 3.

These results suggested that F3 is effective for topical application as highest percentage of drug released after 270min (96%). Whereas formulations F1, F2 and F4 exhibited drug release of 88.03- 95.4%. It was observed that the most influenced factor in the Polycarpaea aurea release is the type of penetrant employed.

Table 6. Percentage Drug release of emulgels

Time (min)	Percentage Drug release/ Permeation studies
Time (min)	F1	F2	F3	F4
0	0	0	0	0
15	4	3.3	8	3.4
30	12.2	15.4	19.2	11.3
60	23.6	26.8	25.6	22.4
90	31.3	34.3	31.4	30.2
120	52.4	49.5	40.6	33.3
150	61.1	60.4	58.2	41.4
180	76.0	71.3	71.0	53.2
210	81.3	79.7	82.4	64.4
240	89.4	86	89.3	75.9
270	95.5	93.4	96	88.03

Figure 3: Percentage drug release/ Permeation of emulgel formulations

In vitro Anti bacterial studies:

In vitro antibacterial activity of Polycarpaea aurea was assessed by the disc diffusion method against four strains of bacteria i.e., E.coli, Pseudomonas aeroginosa, Staphylococcus aureus, and Enterococcus was expressed as zone of inhibition. Among all the formulations, formulation F3 (containing orange oil) has exhibited good broad spectrum antibacterial activity against all the strains employed with a zone of inhibition of 16- 18mm, which is attributed due to better diffusion of the penetrant. The zones of inhibition are depicted in the Figure 3.