Formulation, Optimization and Evaluation of Glycerogelatin In Situ Film Containing Ethanolic Leaf Extract of Calotropis gigantea for Arthritis

Reshma Thampy and AJ Chacko*

Department of Pharmaceutical Sciences, Centre for Professional and Advanced Studies, Kottayam, Kerala, India

*Corresponding Author:
AJ Chacko
Department of Pharmaceutical Sciences
Centre for Professional and Advanced Studies
Kottayam, Kerala, India
Tel: +9562705203
E-mail: [email protected]

Received date: October 16, 2018; Accepted date: October 24, 2018; Published date: October 30, 2018

Citation: Thampy R, Chacko AJ (2018) Formulation, Optimization and Evaluation of Glycerogelatin In Situ Film Containing Ethanolic Leaf Extract of Calotropis gigantea for Arthritis. Int J Drug Dev & Res 10: 1-7.



The aim of the study was to design, optimize and evaluate transdermal glycerogelatin in situ film containing Calotropis gigantea leaves extract for the treatment of Arthritis. Skin is considered as an important route of administration for both local and systemic effects. Topical film forming systems are developing drug delivery system ment for topical application to the skin, which adhere to the body, forming a thin transparent elastic film which provide delivery of active ingredient to the body tissue. The formulation was optimized by mixture design (design expert software, version 11.03) with glycerin, gelatin, water as the factors and spreadability, elasticity, drying time, and tensile strength as the responses. Calotropis gigantea having significant anti-inflammatory potential and its ethanolic extract was incorporated into the optimized formula of glycerogelatin in situ film. The optimized formula contained 1.5% of drug extract and showed a drug release of 79% at 8th hour, and 96% in 24 hour time period.


Arthritis; In situ film; Calotropis gigantean; Optimization; Mixture design


Arthritis is a chronic inflammatory disease that affects people of age ≥ 60. It is reported to affect 14-47% of Indian population [1,2]. Hormonal, genetic, aging, metabolic and mechanical factors regulate the biology of the articular cartilage by complex molecular mechanisms [3]. Rheumatoid arthritis is both an extravascular immune complex disease and a disorder of cell-mediated immunity leads to chronic inflammation, granuloma formation and joint destruction. Calotropis gigantea R.Br (Asclepiadaceae) known as Arka and Jayanti in Ayurveda, have been widely documented in the Ayurveda and traditional medical literature for various therapeutics applications. Traditionally extracts and preparations from roots and leaves are used against rheumatism, wounds, piles, tuberculosis and cancer.

Film Forming Systems (FFS) are novel approach which can be used as an alternative to the conventional topical and transdermal formulations. The polymeric solution applied to the skin as a liquid turn to a film in situ by solvent evaporation with in few minutes [4-6].

Transdermal Drug Delivery System (TDDS) can provide some desirable performances, such as avoiding gut and hepatic first-pass metabolism, improving drug bioavailability, reducing dose frequency and stabilizing drug delivery profiles, easy application, avoid fluctuation of drug level spreads easily. Glycerogelatins are melted before application, cooled to slightly above body temperature and applied to the affected area [7-9]. Following application, the glycerogelatin hardens, is usually covered with a bandage. The formulation was optimized by design expert (11.03) with gelatin, glycerin, water is factors spreadability, elasticity, drying time, and tensile strength is responses. 14 formulations were prepared and evaluated for tensile strength, percentage moisture content, percentage moisture uptake, spreadability, elasticity, drying time. One of the formulae suggested by the software having desirability=1 as optimal and was prepared and evaluated to conform the responses. To the optimized formula 1.5% drug extract was incorporated and further evaluated for drug content and in vitro drug release study.

Materials and Methods


The Tween 80 was obtained from chemdynes corporation, Rajkot, Gujarat. The other chemicals and reagents used were analytical grade.

Collection of medicinal plant

The Indian medicinal plant Calotropis gigantea was collected from the medicinal garden of DPS CPAS Puthuppally, Kottayam, Kerala. India. The plant was authenticated at the Department of botany, CMS College Kottayam, Kerala.

Preparation of plant extract

The ethanolic extract of dried leaves of Calotropis gigantea was used in the study. The leaves were separated, freed from adhering moisture, dried in sunshade and powdered. The powdered material (32 gm) was packed in soxhlet apparatus and extraction was done using 450 ml of ethanol (100%) at 60 to 70`c for 56 hours. The extracts was filtered using whatman filter paper (No.1) while hot, concentrated in vacuum under reduced pressure using rotary flask evaporator, and dried under vaccum. The ethanolic extract yielded was a dark greenish semi solid residue. The extract was then kept in sterile bottle, under refrigerated conditions at 2 to 4°C until further use [10,11].

Preparation of standard curve

Accurately measured Calotropis gigantea equivalent to 100 mg and was dissolved in 1 ml of ethanol and made-up to 100 ml with phosphate buffer pH 7.4 from that 10 ml was taken and made up to 100 ml using phosphate buffer from that 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml were pippeted out and made up to 10 ml using phosphate buffer to contain 2, 4, 6, 8, 10 mcg extract/milliliter of solutions [12].

UV scanning revealed a prominent peak at 318 nm along with other peak. Components phytol, octadecatrienoic acid (GCMS) has the UV absorbance at 318 nm. Further two components have high antiinflammatory activity. Therefore 318 nm was kept as the λ max for further studies.

Design of experiments (Mixture design)

Mixture design was the technique used to determine the combination of constituent that deliver the desired response with minimum number of runs. The key attribute of mixture design is that proportions of ingredients are used and sum of which is always one. 14 runs with 11 different combinations of gelatine, glycerin and water, 3 out of 11 run were duplicated. Observed responses spreadability, elasticity, drying time, tensile strength was then applied to the model to get optimum levels of combination. The design was generated by design expert 11.03 software, the design region for mixture proportion is a simplex i.e., with 3 factor, simplex is a triangle [13].

Preparation of glycerogelatin in situ film

The required quantities of ingredients were measured accurately as per the table. The gelatin was hydrated in warm water at 50○C to get a clear solution to this glycerine was added. The solution was evaluated for various properties are given below [14,15].

Evaluation of in situ film

Spreadability: A grounded glass plate of 15 × 30 cm was fixed on the work table and excess of the prepared gel (2 gms) was placed on the grounded slide and was then sandwiched with smaller grounded glass slide (3 × 10 cm). 1 kg weight was placed on the top of the slide for 3 minutes to expel the air and to get a uniform film of gel between the slides. The excess of gel was craped from the edges. The top slide was hooked horizontally and pulled by 80 grams weight over a pulley. The time taken in seconds by the top slide to cover a distance of 7.5 cm was noted. Spreadibility was calculated using the following formula. Shorter time indicates better spreadability.

Spreadibility was calculated using the following formula:

Spreadibility=M × L/T

where, S=Spreadibility, M=Weight in the pan to pull the slide, L=Length moved by the glass slide and T=Time (in sec.) taken to cover the distance of 7.5 cm [16,17].


Preparation of film

The molten formula was poured into different moulds to get films of uniform size and thickness. Circular moulds of thickness 2.5 mm was used for the preparation.

Percentage drying

A definite volume of film forming formula (7.5 ml) was poured into the mould fixed on a small glass plate. The initial weight was determined and the loss of weight was further determined at regular intervals for 3 hr. on an electronic balance [18].

Elasticity/ percentage elongation

Elasticity was determined using custom designed elongation testing apparatus. The dried film of 1 cm width was cut out. The film was held strongly between two clips and the upper clip was fixed permanently on a vertical wooden board. The weight was added to the weight pan which was fixed to the lower clip and the weight was gradually increased to effect the elongation of the film. The film length at any time could be read from the scale attach on the wooden board. The initial length between the clips was noted the weight was gradually added until the film was broken. The elongated length was read at from the scale just before breaking the film.

The percentage elongation was determined using formula

% elongation=[L2-L1]/L1 × 100

where, L1 is the initial length and L2 is the final length of the film before breaking.

Tensile strength

Tensile strength was determined similar way as elasticity. It is determined using the formula:

Tensile strength=(break force/a × b) × (1+L/I) (2)

where, “a” is width, “b” is thickness, “L” is length, and “I” is elongation of the films [19,20].

Film thickness

A definite volume of film (7.5 ml) was poured into the mould fix on a small glass plate. Film was left overnight for drying and then the film was peeled off and the thickness was determined from three different points on the film. Film thickness was measured by screw gauge.

Weight uniformity

The prepared patches were dried at room temperature for 4 hrs before testing. A specified area of patch were cut in different parts of the patch and weighed on digital balance. The average weight and standard deviation values were calculated from the individual weights.

Folding endurance

The folding endurance was determined manually by taking a strip of patch (4 × 2 cm) and repeatedly folding it at the same place till it breaks. Folding endurance is considered as the number of times the film is folded at the same place without breaking/cracking.

Drug content

Film of specific area (1 cm2) was cut and placed in a 50 ml volumetric flask. To this 25 ml of pH 7.4 was added; gently heated to 45°C for 30 minutes and kept for 24 hours; with occasional shaking the volume was made up to 50 ml with phosphate buffer pH 7.4. The solution was filtered and suitable dilutions were made against blank solution which was prepared by following same procedure containing film without drug [21,22].

In vitro drug permeation study

An in vitro drug diffusion study was performed using modified Franz diffusion cell. It consists of a donor and receptor compartment. The receptor compartment filled with 25 ml of phosphate buffer pH 7.4 as diffusion medium. The cellophane membrane was mounted between the donor and receptor compartment of the diffusion cell. The prepared film 1 cm2 was placed in the donor compartment. The whole assembly was fixed on a hot plate magnetic stirrer and the solution in the receptor compartment was continuously stirred at 100 rpm using magnetic beads and the temperature was maintained at 37 ± 2°C. 1 ml of sample of the receptor fluid was withdrawn at predetermined time intervals through the sampling port and replaced immediately with same volume of phosphate buffer. Similar in vitro drug diffusion study was also carried out for a similar composition but with added with 0.5% tween 80. The samples were analysed for drug content at 318 nm using UV spectrophotometer. The suitable dilution with phosphate buffer pH 7.4 and cumulative amount of drug permeated was calculated and plotted against time [23,24].


Folding endurance

Folding endurance of film was (265 ± 2) and lowest (234 ± 40).

Film thickness

All the film has uniform thickness throughout. The thickness was found in range of 0.18 mm to 0.29 mm.

Percentage drying

The percentage drying at 15 minutes is 0.86 and 3 hrs ranged from 9.7 to 53.

Tensile strength

The tensile strength of the film varied with the polymer concentration. It increased proportionately and ranged between 2146 to 4695 g/cm2.


The elasticity of film ranged from 184 to 388.89%.

Drug content and percentage entrapment

The drug content was 5.95 mg/cm2 and the percentage entrapment was 97.86% (Tables 1-6; Figures 1-13).


Figure 1: Calibration Curve of Calotropis gigantea.


Figure 2: Schematic representation of spread ability.


Figure 3: Schematic representation of elasticity.


Figure 4: Spreadability.


Figure 5: Contour plot for spreadability.


Figure 6: Elasticity.


Figure 7: Contour plot for elasticity.


Figure 8: Percentage drying.


Figure 9: Contour plot for percentage drying.


Figure 10: Tensile strength.


Figure 11: Contour plot for tensile strength.


Figure 12: The formula suggested by design expert software 11.03 with expected responses.


Figure 13: In vitro drug release profile for two formulations.

Serial number Formulation code Gelatin grams Glycerin Grams Water grams
1 F1 22.5 12.5 65
2 F2 25 15 60
3 F3 20 10 70
4 F4 27.5 10 62.5
5 F5 20 25 55
6 F6 22.5 20 57.5
7 F7 35 10 55
8 F8 35 10 55
9 F9 27.5 17.5 55
10 F10 20 10 70
11 F11 20 25 55
12 F12 30 12.5 5.5
13 F13 20 17.5 62.5
14 F14 27.5 17.5 55

Table 1: Formulation code.

Source Sum of squares Df Mean square F value P Value  
Model 58155.49 8 7269.44 36.55 0.0005 Significant
Linear mixture 13292.30 2 6646.5 33.41 0.0013  
AB 4476.82 1 446.82 22.51 0.0051  
AC 5453.15 1 5453.15 27.42 0.0034  
BC 26210.13 1 26210.13 131.77 <0.000  
A2BC 2503.41 1 2503.41 12.59 0.0164  
AB2C 17352.35 1 17352.35 87.24 0.0002  
ABC2 164.61 1 164.61 0.8276 0.4047  
Residual 994.55 5 198.91      
Lack of fit 8.55 1 8.55 0.00347 0.8613 Not significant

Table 2: ANOVA summary of response-spreadability (Y1).

Source Sum of squares Df Mean square F value P Value  
Model 71270.29 2 35635.14 8.45 0.0060 Significant
Linear mixture 71270.29 2 35635.14 8.45 0.0060  
Residual 4636.14 11 4215.19      
Lack of fit 20635.64 7 294.95 0.4583 0.8270 Not significant

Table 3: ANOVA summary of response- elasticity Y2.

Source Sum of squares Df Mean square F value P Value  
Model 3.91 5 0.7817 14.86 0.0007 Significant
Linear mixture 2.57 2 1.29 24.48 0.0004  
AB 0.5655 1 0.5655 10.5 0.0112  
AC 0.5277 1 0.5277 10.03 0.0132  
BC 0.1649 1 0.1649 3.14 0.1146  
Residual 0.4208 8 0.0526      
Lack of fit 0.2122 4 0.0530 1.02 0.4936 Not significant

Table 4: ANOVA summary of response-percentage drying Y3.

Source Sum of squares Df Mean square F value P Value  
Model 9.665E+06 8 1.208E+06 8.57 0.0150 Significant
Linear mixture 4.842E+05 2 2.42E+05 1.72 0.2706  
AB 6.82E+05 1 6.82E+05 4.84 0.0790  
AC 1.925+05 1 1.925E+05 1.37 0.2952  
BC 2.224E+05 1 2.224E+05 1.58 0.2646  
A2BC 9114.20 1 9114.20 0.0647 0.8094  
AB2C 30277.6 1 30277.76 0.2148 0.6625  
ABC2 3.434E+06 1 3.434E+06 24.36 0.0043  
Residual 7.048E+05 5 1.410E+05      
Lack of fit 2560.46 1 2560.46 0.0146 0.9097 Not significant

Table 5: ANOVA summary of response-tensile strength (Y4).

Evaluation Predicted Observed
Spreadability 177.6 gm/sec 175 gm/sec
Elasticity 358.806% 375%
Drying time 3.59846% 4.80%
Tensile strength 5125.35 gm/cm2 4552 gm/cm2

Table 6: Evaluation results of optimized formula.


Calotropis gigantea using glycerogelatin in situ films were successfully developed and evaluated. In situ film was prepares by solvent evaporation method using gelatin, glycerin, water, tween 80, it was evaluated for the spreadability, elasticity, drying time, tensile strength. The results were fed to the software and it suggested n-number of compositions and corresponding responses. One among the different compositions with desirability one was selected as optimum. That was then prepared and the drug extract was incorporated into it. The prepared in situ film was evaluated and the responses were very close to the predicted. Further the permeation was also evaluated with and without permeation enhancers. The optimized in situ film showed good permeation.


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