INTRODUCTION:

Inflammation^[1] is a defense reaction caused by tissue damage or injury characterised by redness, heat, swelling and pain. The primary objective of inflammation is to localise and eradicate the irritant and repair the surrounding tissue. Inflammation aids disposal of microbes, toxins or foreign material at the site of injury, prevents their spread to other organs and prepares the site for tissue repair. Thus it helps to restore tissue homeostasis. There are three basic stages of inflammation: Vasodilatation and increased permeability of blood vessels, Phagocyte migration, and tissue repair. Signs of inflammation: rubor(redness), tumor(swelling), calor (heat), dolor (pain),function laesa (loss of function).

Inflammation may causes due to Physical agents, Chemical agents and Infective agents. Mainly two types of inflammation , acute and chronic. Polymers are used to give the structural network, which is essential for the preparation of gels. Gel forming polymers are classified as follows , natural polymers, semi synthetic polymer and synthetic polymers. Advantages of topical gels are to avoid the first pass effect, they are less greasy and can be easily removed from the skin, reduction of doses as compared to oral dosage forms and having localised effect with minimum side effect. The oleo gum resin obtained from Boswellia serrata has been used for variety of the therapeutic purposes such as cancer, colitis, Crohn’s disease and hyperlipidemia^[8]. Its alcoholic extract was reported to possess anti inflammatory, anti arthritic activities in animals which were due to boswellic acids which are pentacyclictriterpenes^[7]. Boswellic acid selectively inhibits leukotriene synthesis by inhibiting 5-lipoxygenase in an enzyme directed mechanism. The effectiveness of any herbal medication^[2] is dependent on the delivery of effective level of the therapeutically active compound. Phytovesicles^[3] are recently introduced herbal formulations that are better absorbed and as a result produced better bioavailability and actions than the conventional botanical extract. Phytovesicles are produced by a process whereby the standardized plant extract or its constituents are bound to phospholipid mainly phosphatidylcholine producing a lipid compatible molecular complex. Phytoconstituents including formulations has a lot of stability and bioavailability problems. Phytovesicular drug delivery systems have more advantageous over these problems. Phyto means plant and vesicles mean cells. Phospholipids used for the preparation of phytovesicles bound with the plant extract producing a lipophilic form of the drug. Because of its high lipid profile, it penetrates well through the skin and thus enhances the anti inflammatory activity of the Boswellia serrata gum extract. As compared to the conventional formulation, phytovesicles has a lot of advantages as well as application. Herbal anti-inflammatory gels which is non-toxic, safe, and effective and improves patient compliance by the utilization of herbal extracts would be highly acceptable.

MATERIAL AND METHODS:

Collection and authentification:

Gum resin was collected from the local market of Ernakulam and authentified by Dr. Elsamma Joseph Arackal, Associate Professor and HOD, Department of Botany, Maharaja’s College, Ernakulam

Apparatus and chemicals:

Soxhlet apparatus, rotary vacuum evaporator, Linomat V applicator, Cammag Twin Trough Glass chamber.

Boswellia serrata gum resin^[6], ethanol, vanillin sulphuric acid, Anisaldehyde sulphuric acid, phosphate buffer, Diclofenac.

Extraction of Boswellia serrata gum^[4][9]

500gm of Boswellia serrata gum resin was extracted with ethanol 95% in a Soxhlet apparatus and the extract was concentrated under reduced pressure on a rotary vacuum evaporator at 40⁰C to get the thick brown residue (75g).The extract obtained was stirred with 3% sodium hydroxide solution for approximately 15min and then filtered. The filtrate was extracted with n-hexane: ethyl acetate (95:5) in a separation funnel. The aqueous portion was then acidified with 1N hydrochloric acid to precipitate the total organic acid. The filtered acid was washed several times with distilled water to remove hydrochloric acid completely.

Thin Layer Chromatography^[5]

10 mg of Boswellia serrata extract obtained above was weighed by an electronic balance and dissolved in 10 ml methanol. Spot of above solution was applied on the TLC plate with the help of thin capillary tube. Solvent system is Chloroform: methanol (90:10). Detection: After drying, spot of both the samples are identified by spraying Vanillin sulphuric acidsolution on the TLC plate.

High performance Thin Layer Chromatography:

TLC Aluminium pre coated plate with Silica gel60 GF254 (20x10 cm2; 0.2 mm thick) was used with Toluene: Ethyl acetate: Methanol (8:2:1) V/V as mobile phase. N‐hexane extract of samples and β‐Boswellic acid standard solution applied on plate by using Linomat V applicator. Cammag Twin Trough Glass Chamber (20x10cm2) with SS lid was used for development of TLC plate. The Twin Trough Glass Chamber was saturated with mobile phase for 20 minutes. TLC plate was developed to 8 cm distance above the position of the sample application. The plate was removed from the chamber and air dried at room temperature. This plate was sprayed (derivertized) with Anisaldehyde‐ Sulphuric acid. HPTLC profile was snapped by Cammag Reprostar III, before derivertization under UV 254 nm, 366 nm and after derivertization. The derivertized plate was scanned immediately using Camag TLC Scanner III at wavelength 530nm. Wincats an integrated Software 4.02 was used for the detection as well as for the evaluation of data.

Solubility:

Solubility of Boswellia serrata extract gum resin was determined in different solvents such as water, chloroform, ethanol, methanol and acetone.

λ _max of the Boswellia serrata gum resin extract:

An absorption maximum of extract was determined using ethanol. Solutions ranging from 2.5-20µg/ml were scanned from 200-400nm using UV spectrometer

Formulation of conventional gel:

3% of carbopol 934 is mixed with sufficient quantity of distilled water. After complete dispersion, the prepared carbopol mixture was kept in dark for 24 hours for complete swelling. Add a drop of triethanolamine in order to maintain the pH. Accurately weighed amount of Boswellia serrata gum extract was dissolved in ethanol. The extract solution was added slowly to the aqueous dispersion of carbopol gel base with the help of a magnetic stirrer taking precaution that air did not entrap. Finally the remaining ingredients were added to obtain a homogeneous dispersion of gel.

Formulation of phytovesicular gel:

Solvent evaporation method is adopted for the preparation of phytovesicle. Phytovesicles was prepared by reacting with a natural phospholipid, phosphatidylcholine with the Boswellia serrata gum extract in a ratio 1:1.The reaction is carried out in an aprotic solvent chloroform for the solubilisation or the complex formation of the extract with the phytovesicles.After the completion of solubilisation, the complex compound was isolated by removing the solvent under rotary vacuum evaporator. The dried film of drug – lipid complex deposited in the round bottom flask was hydrated using ethanol, a polar solvent. This method is called solvent evaporation or thin film hydration technique.

Vesicular suspension obtained was added to the prepared carbopol gel base with the help of a magnetic stirrer in order to obtain a homogenous phytovesicular gel.

Evaluation of gels^[10]

Clarity:

The clarity of the formulations were determined by visual inspection and it was graded as follows; turbid: +, clear:++,very clear:+++.

Homogenecity:

All developed gels were tested for homogeneity by visual inspection after the gels have been set in the container for their application and presence of any aggregate.

Colour and odour:

Colour and odour was examined by visual examination

Spreadability:

Spreadability was determined by applying weight above the slides in which the formulation was placed and time in seconds, required to separate the slides was noted. Spreadability of the formulation was reported in seconds. As the time for spreading reduces it shows better spreadability. Spreadability was calculated using the formula:

S=M.L/T

Where S= Spreadability

M= weight to upper slide

L= length of glass slide

T= time taken to separate the slide completely from each other

Extrudability:

Extrudability was measured using a closed collapsible tube. Weight in gram required to extrude a 0.5 cm ribbon of the formulation in 10 seconds was determined.

Measurement of pH:

The pH of gel was determined using digital pH meter. The measurement of pH was done in triplicate and average values were calculated.

Drug release study

Phosphate buffer of pH of 6.8 was prepared according to the standard procedure given in IP. The solution was filled in the receptor compartment. Egg membrane was tied between the donor and receptor part and weighed amount (3gm) gel was filled in the donor compartment. Using a magnetic stirrer the medium was agitated in the receptor compartment and temperature was maintained at 37⁰C±1.Samples (5ml) from the receptor compartment were taken at various intervals over a 1hour period of time and assayed for drug released.

Stability studies:

Stability is defined as the extent to which a product retains its efficacy within specified limits throughout the period i.e., shelf life. Both the formulations were subjected to a stability testing for 6 weeks at room temperature (30±2ºC), at refrigerator temperature (4±2ºC).Then analysed for the change in pH, homogenecity, spreadability etc by procedure stated earlier.

TEST FOR IN VITRO ANTI INFLAMMATORY ACTIVITY ^[4][13]

1. Inhibition of Albumin denaturation:

The reaction mixture consisting of test gel solution and 1% aqueous solution of Bovine Serum Albumin (BSA)^[11], pH of the reaction mixture was adjusted using small amount of 1N Hydrochloric acid. The mixture was then incubated at 37⁰C for 20 minute and then heated to 51⁰ C for 20 minutes, after cooling the sample was kept a room temperature for 12 hrs. The absorbance was measured at 660nm.The test was similarly performed for control where no drug was added, for standard diclofenac and for Boswellia serrata extract. Each experiment was performed in triplicate and average was taken. The percentage inhibition of protein denaturation was calculated as follows:

Percentage Inhibition= (Abs_control – Abs _sample) 100/Abs _control

2. Anti proteinase action^[12]:

This test was performed based on the fact that the proteinase activity inherent in leukocytes play an important role in the development of tissue damage during inflammatory reactions. A significant level of protection was therefore provided by proteinase inhibitors. The reaction mixture (2ml)comprises of Tripsin (0.06mg), PH 7.4 Tris Hydrochloridebuffer(1ml) and test sample (1ml). The mixture was incubated at 37⁰C for 5 min and then 0.8% w/v casein(1ml)was added. The mixture was incubated for an additional 20 min and 70% perchloric acid (2ml) was added to arrest the reaction. Cloudy suspension was centrifuged and the absorbance of supernatant was read at 210nm against buffer as blank. Repeated the experiment for standard Diclofenac and test sample. The experiment was performed in triplicate. The percentage of proteinase inhibitory action was calculated.

Percentage Inhibition= (Abs _control– Abs _sample) 100/Abs _control

3. Membrane stabilization:

Heat induced haemolysis:

Blood (10ml)is collected from a healthy volunteer who has not taken any NSAIDs for two weeks prior to the experiment and transferred to centrifuge tubes. The tubes are then centrifuged at 3000rpm for 10min and washed thrice with equal volume of normal saline. The volume of blood is measured and reconstituted as 10%v/v suspension with normal saline. The reaction mixture (2ml)consisting of 1ml of either Boswellia serrata phytovesicular suspension, Boswellia serrata extract or standard drug diclofenac and 1ml of 10%v/v human red blood suspension were collected in centrifuging tubes and were incubated in a water bath at 56⁰C for 30 min. The tubes were cooled by running in the tap water after the incubation time and centrifuged at 2500rpm for 5 min and absorbance of the supernatant was taken at 560nm.The experiment was performed in triplicates for all test samples

Percentage = (Abs _control- Abs _sample) 100/Abs _control

Inhibition of haemolysis

Hypotonicity induced haemolysis:

Different concentration of extract (100-500mcg/ml), standard drug Diclofenac(100mcg/ml), control (normal saline) were prepared. To each of these preparations added 1ml phosphate buffer, 2ml hyposaline, 0.5ml human red blood cell suspension. The assay mixture was incubated at 37⁰C for 30 minute and then centrifuged at 3000rpm. Supernatant was decanted and Hb contents estimated at 560nm.

Percentage = 100 – (OD sample / OD control) × 100

protection

RESULT:

The presence of Boswellic acid was determined by TLC. The Rf value 0.58 obtained was compared with the standard value 0.61, it is shown in table 1.Further the presence of Boswellic acid in Boswellia serrata gum extract was confirmed by HPTLC and the RF value was found to be 0.57 , is shown in Figure 1.The Extract was found to be soluble in chloroform , ethanol , methanol and acetone. And insoluble in water. Calibration curve of Boswellic acid was plotted (Figure 2), the λmax of the extract was found to be 241 nm. The gel was evaluated and the value of spreadability indicates that both formulations of gel are easily spreadable. The extrudability of both gel was found to be good and compatible. The pH value of conventional gel was found to be 5.65 and phytovesicular gel was 5.68 which has slight increase is considered acceptable to avoid the risk of irritation upon application to the skin because adult skin pH is in range of 4.0-7.0. Both the gels were free from presence of particles. The prepared phytovesicular gel is more transparent and clear. These values are shown in table 2.Percentage drug release of both the gels was calculated. (table 3) and the graph was plotted shows that the phytovesicular gel has enhanced drug release, which is shown in Figure 3.Based upon the stability studies the phytovesicular gel shows stability problems at room temperature and given in Figure 4 .The percentage inhibition by different in- vitro anti inflammatory methods shows phytovesicular gel has an increased inhibitory action than conventional gel. The result is tabulated in table 4 and represented graphically in the Figure 5 respectively.

Table 1: TLC analysis

Distance travelled by the solute (cm)	Distance travelled by the solvent (cm)	RF value obtained
3	12	0.25
6.9	12	0.58
9.0	12	0.75
10.5	12	0.88


UV 366 nm	UV 254 nm	After derivertiztion

Figure 1 : HPTLC chromatogram of Boswellic acid

Figure 2: Calibration curve of Boswellia serrata gum extract in ethanol

Figure 3: Comparison of drug release study

Figure 4: Comparative stability study of conventional gel v/s phytovesicular gel

Figure 5: Comparison of anti inflammatory activity

DISCUSSION:

The results from the study shows that, the phytovesicular gel containing the gum extract of Boswellia serrata possess potent anti inflammatory activity.The stability profile shows that phytovesicular gel is stable at a low temperature . The ethanolic extract shows maximum anti inflammatory activity. In future, the phytovesicular drug delivery system can be used to target the drug at site of inflammation.

Table 2: Values of evaluation parameters of developed gels

Parameters	Conventional Gel	Phytovesicular Gel
Spreadability	23.08	22.92
Extrudability	++	+++
pH	5.65	5.68
Clarity	++	+++

Table 3: In vitro drug release study

Time(min)	% Drug Release
Time(min)	Conventional gel	Phytovesicular gel
0	0	0
10	12.4978	14.2302
20	18.7997	22.5438
30	25. 4902	28.2521
40	30. 9039	37.2479
50	37.6951	45.6204
60	43.9028	52.4954

Table 4: Comparison of in vitro anti inflammatory activity

Sl. No.	in- vitro methods	Conventional gel	Phytovesicular gel
1	% Proteinase inhibition	84.9%	96.5%
2	% Haemolysis inhibition	66.7%	75.2%
3	Hypotonicity induced haemolysis	76%	99.2%
4	% Albumin inhibition	73.52%	78.5%

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Received on 09.07.2014 Modified on 20.07.2014

Research J. Pharm. and Tech. 7(11): Nov. 2014 Page 1305-1310