Integrating Herbal Science and Nanostructured Emulgel Technology for Enhanced Topical Anti-Inflammatory Action

Inflammation is the response of living tissue to damage. Any assaults to living tissue, whether due to physical, chemical, or microbiological origin result in inflammation, manifested by redness(rubor), heat(carol), pain(dolor), swelling (tomour) and loss of function (functio laesa) in acute phase. The inflammatory process involves series of events that can be elicited by numerous stimuli (eg. Infectious agents, ischemia, antigen-antibody interaction and thermal or other physical injury.) Each type of stimulus provokes a characteristic pattern of response that represents a relatively minor variation of them. At the macroscopic level, the response is usually accompanied by the familiar clinical signs of erythema, edema, tenderness and pain. ^[1-2] Emulgels are emulsions, either of oil in water type or water in oil type, which are gelled by mixing with a gelling agent. This research aims for the preparation and evaluation of polyherbal Emulgel containing potential herbal anti-inflammatory agent viz., tea tree oil, lemongrass oil, ginger oleoresin & capsaicin. The research scheme also focusses on the evaluation of prepared polyherbal Emulgel and its comparison with the marketed synthetic or herbal anti-inflammatory gel. Regional delivery involves the application of drug to the skin for purpose of treating diseases or alleviating symptoms in deep tissue beneath the application. Here the intent is effectuating the pharmacological action of drug at muscular, vasculature, joints, etc. ^[3-4]

MATERIAL AND METHODS:

Plant material: ^[5-7]

Tea tree oil, lemon grass oil, ginger oleo resin extract and capsaicin extract has been selected for the development of anti-inflammatory formulation. Tea tree oil (Venus’s chemicals, Pune) Lemon grass oil (Venus’s chemicals, Pune) Linseed oil (Venus’s chemicals, Pune)

Chemicals:

Menthol (S.D. Fine Chem. limited, Mumbai) Sodium lauryl sulfate (S. D. Fine Chem. limited, Mumbai) Methyl Salicylate (Nice Chemicals, Kerala, India), Glycerol monostearate (Loba Chemie. Pvt. ltd. Mumbai) Triethanolamine (Fisher scientific) Carrageenan sodium salt (Fine chemical industries) Carbopol 940 (Molychem, Mumbai)

 Animals:

Male wistar albino rats (200-300g each) were maintained under standard animal housing conditions, and used for the anti-inflammatory activity of prepared emulgel formulation. All the animal study was carried out at S.N. Institute of Pharmacy, Pusad having Institutional Animal Ethics Committee, Register No. CPCSEA/729/O2/a/CPCSEA.

Method of preparation:

Step 1.:

Aqueous gel base was prepared by dissolving menthol in sufficient quantity of alcohol. This solution was added in 100 ml of purified water. The weighed amount of Carbopol 940 was sprinkled slowly in solution while stirring SLS is added. This solution was kept overnight for complete hydration of Carbopol.

 Step 2.:

 Oil phase was prepared by dissolving all plant material extract in methyl salicylate. The glyceryl monostearate (GMS) was dissolved into warm linseed oil, and this solution was added into above solution to get final oil phase.

 Step 3.:

This oil phase was then slowly added in the above aqueous Carbopol gel base with continuous stirring with using homogenizer. Finally, Triethanolamine was added to the solution with constant stirring, which cause gelling of polymer solution as neutralization of carbopol takes place to get emulgel formulation F1. Cow ghee was used as permeation enhancer added in oil phase of formulation F2. 

Evaluation of gel:

1. Appearance:

The prepared gel was inspected visually for clarity, colour and presence of any particle. The test is important regarding patient compliance.⁸

2. Ph:

pH of gel was determined using pH meter. About 1 gm of gel was stirred in neutralized alcohol till a uniform suspension effected. The volume was made up to 10ml and pH of the solution was measured.

3. Rheological properties:

1. Determination of viscosity

Viscosity of emulgel was determine by using (LV) Brookfield viscometer (Dial type).As the system is non –Newtonian spindle no.4 is used.⁸

Viscosity was calculated using formula-

Dial reading × factor = viscosity (cps)

2. Spreadability:

Ideal gel must have low spreadability values but good consistency. Spreadability of formulation was determined by an apparatus suggested by Multimer et al,⁹ which was fabricated itself in laboratory and used for the study. It consisted of wooden block provided with two glass slides. Lower slide fixed on wooden block and upper slide with one end tied to glass slide and other end tied to weight pan. An excess of gel (2.5gm) between two glass slides and 1000g weight was placed on slides for 5 min to compress the sample to a uniform thickness. Weight was added to pan. The time (seconds) requires to separate the two slides, was taken as a measure of spreadability.

It was calculated using formula:

S = m.l / t

Were,

S = Spreadability

M = weight tied to upper slide

t = Time taken

l = length of slide 7 cm

Shorter time interval, to cover distance of 7 cm, indicates better Spreadability.

3. In vitro drug diffusion study:

Topical gel formulations were expected to release the drug quickly, when they are applied to the skin for quick relief. To test the pattern of release of drug from formulation in formulation in vitro diffusion studies were carried out.^10-

In vitro drug diffusion study was carried out as:

1) In vitro drug diffusion through cellulose membrane:

All formulations were subjected to in vitro diffusion through cellulose membrane by using diffusion cell type apparatus. Cellulose membrane was socked in distilled water over night. The membrane was mounted on the diffusion cell apparatus. The system was kept such for 15min to achieve uniformity in temperature.  

4. Skin irritation:

Ten healthy male and female volunteers were selected for skin irritation testing. 100 mg gel was applied on area of 2 cm² for 6 hours, on the interior surface of upper arm and covered with cotton bandage. After 6 hr the sites were cleaned with acetone and reading are made according to the scale given by Draize et.al¹⁴

5. Anti-inflammatory study:¹⁵

“Carrageenan induced rat paw edema method” suggested by chi S.C. and Jun H.W.was employed to study the effectiveness of gel formulations. These gels were also compared against the marketed polyherbal gel formulation. Albino rats (strain –wistar) of either sex, weighing about 200-300gm were used. Make a mark on both the hind paws (right and left). Just beyond tibiotarsi junction, so that every time the paw is dipped in the mercury column up to fixed mark to ensure constant paw volume. Note the initial paw volume is (both right and left) of each rat by mercury displacement method. Divide the animals into groups of each of 5 animals. Each animal was subjected to carrageenan challenge (o.1 ml of 1% carrageenan solution injected sublunary) and injection time was noted. Formulations were applied to inflamed paw (0.5 gm of emulgel) by gently rubbing. Rats of control group received only the gel base without drug by the same mode of applications. Paw volumes were noted hourly for 3 hours using paleothermometer. The percent inflammation inhibition was determined using formula.

Where, V = paw volume (average after 3hr)

Vi = initial volume

The average paw volume in the group of the drug treated rats was compared with that of control rats and % inhibition of edema was determined using following equation.

RESULT AND DISCUSSION:

The objective of the present work is to develop safe and effective anti-inflammatory poly-herbal formulation that could be used in the treatment of inflammation without any side effects. Preliminary studies have indicated that Carbopol 940 can be used as gelling agent. Tea tree oil, lemon grass oil, linseed oil & capsaicin were incorporated for its topical delivery system in the gel form for its anti-inflammatory activity and cow ghee was used as a permeation enhancer.  Two different formulations of with and without permeation enhancer were screened at preliminary level on the basis if solubility of drug, drug release, spreadability, Extrudability, rheological behavior and anti-inflammatory potential of prepared emulgel. Also, best formulation that contains cow ghee as a permeation enhancer was compared with marketed preparation for its anti-inflammatory activity. The gels were subjected for evaluation on the basis of appearance, pH, rheological behavior, in vitro release performance, anti-inflammatory study. In appearance the prepared gel was inspected visually for clarity, colour and presence of any particle, after evaluating both these formulation does not show the presence of particles with clear appearance and orange in colour.

Table No. 1 Appearance of emulgel

The pH of gel was determined using pH meter both the formulation shows the neutral pH.

TableNo.2   pH of formulations

Viscosity of Emulgel was determined by using Brookfield viscometer, As the system is non- Newtonian spindle no.4 is used.

Table No.3   Viscosity of formulation (F1) and (F2)

Table No.4 Spreadability study for formulation (F1) and (F2)

Table No.5   Drug diffusion study of formulation (F1) and (F2)