Aloe-Emodin standard compound was received as a gift from Prof. Masaki, (Kobe University, Japan).

The Aloe sinkatana plants were collected, from the Sinkat area, Red Sea state, Sudan, they were authenticated by the Department of Chemistry, Medicinal & Aromatic Plants Research Institute and Traditional Medicine, National Research Center, Khartoum, Sudan.

Mature, healthy, and fresh leaves of Aloe sinkatana having a length of approximately 75 to 90 cm were washed with fresh water.

The inner gel is scrapped and cut into pieces. A traditional hand filleting method of processing Aloe leaves was used. In this method, the lower leaf base, the tapering point at the leaf top, and the short spines located along the leaf margins were removed by sharp blades. The blade was then introduced into the mucilage layer below the green rind avoiding the vascular bundles, and the top rind was removed. The epidermis of the leaves was peeled off, and the colorless, solid mucilaginous gel was cut into pieces. Then 250 gm of gel was loaded into a 1000ml flask and 500 ml solvent (Ethanol) was added. Ultrasound-assisted extraction was performed at 60° C for 60 min. After that the solution was filtered and the solvent was removed under reduced pressure in a Rotary evaporator until it became completely dry 17.

To obtain the "best" or an "optimized product" eight different formulations were generated using two levels, three-factor, full factorial design.

A 2³ factorial design for three factors at two levels each was selected to optimize the varied response variables. The three factors, amount of gelling agent X1, amount of emulsifying agent X2, and amount of liquid paraffin X3 were selected as independent formulation variables, and the factor levels were suitably coded (Table 1). Viscosity and Spreadability were taken as dependent variables or response variables. Experimental trials were performed at all 8 possible combinations (Table 2). All other formulation variables and processing variables were kept invariant throughout the study 18.

Design-Expert_DX 8 Software was used for the generation and evaluation of the statistical experimental design.

Table 3

Ingredients (%) Coded formula	F1	F2	F3	F4	F5	F6	F7	F8
Carbopol 940	1	2	1	2	1	2	1	2
Liquid Paraffin	5	5	5	5	7.5	7.5	7.5	7.5
Tween20	0.6	0.6	1	1	0.6	0.6	1	1
Span20	0.9	0.9	1.5	1.5	0.9	0.9	1.5	1.5
Extract	5
Ethanol	2.5
DEMSO	5
Propylene glycol	5
Triethanolamine	Q.s to Adjust pH 6-6.5
Distilled Water	Q.S

The composition of emulgel formulations is shown in Table 3.

First, the gel was prepared by dispersing Carbopol 934 in heated purified water (80 °C), and the dispersion was cooled and left overnight. The oil phase of the emulsion was prepared by dissolving Span20 in liquid paraffin while the aqueous phase was prepared by dissolving Tween20 in purified water.

Propylene glycol was dissolved in ethanol whereas plant extract was dissolved in DMSO, and both 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 at room temperature. The obtained emulsion was mixed with the gel in a 1:1 ratio with gentle stirring to obtain the emulgel. Finally, the pH of the emulgel was adjusted by using triethanolamine 19, 20.

The 8 batches of the emulgel formulae were viewed under a microscope to study their color, texture, homogeneity, consistency, and phase separation 21, 22.

Centrifugation study is a useful means to check the stability of the prepared emulgel formulae. These studies were performed one week after the preparation of emulgels. Centrifugation was carried out using a Mini centrifuge at 3000 rpm for 30 minutes 23.

The pH value of 1% aqueous solutions of the prepared A. sinkatana emulgel was measured using a pH meter (Max Instruments Chandigarh, India), which was calibrated before each reading, with buffered solution at pH4.0 and 7.0. The measurement of the pH of each formulation was done in triplicate and average values were calculated. 24, 25, 26

Measurements of viscosity were done by Viscometer VR 3000 (Viscotech, Spain) by choosing the appropriate spindle number (L4) and rpm (100 rpm) at room temperature. An appropriate amount of each emulgel formulation was kept in a suitable beaker, the spindle groove was dipped, and the rpm was set. Viscosity measurements were started, and the readings were measured after 1 minute, and the viscosity of each formulation was calculated 27, 28.

The Spreadability was determined by the parallel plate method, which is widely used, by measuring the spreading diameter of 1gm of emulgel between two horizontal plates (30cm x 30cm) after one minute 29.

The emulgel formulae were diluted either with oil or water. If the emulsion of the emulgel is o/w type and it is diluted with water, it will remain stable as water is the dispersion medium but if it is diluted with oil the emulsion will break as oil and water are not miscible with each other 30.

Accurately weighed Aloe-emodin standard material (0.01g) was, transferred to a 100 ml volumetric flask and dissolved by ultrasonic shaker with methanol to get a standard solution of 100µg/ml (stock solution). Various concentrations (2 µg /ml, 4 µg / ml, 6 µg /ml, 8 µg /ml, 10 µg /ml, and 12 µg /ml) of stock solution were made and the absorbance of various dilutions was taken at wavelength 430 nm using a UV spectrophotometer. The calibration curve was determined by plotting the absorbance, against the concentration.

0.5gm of dried extracts were dissolved by ultrasonic shaker in 100ml of methanol (0.5%). The absorbance of the resultant solution was read at wavelength 430 nm using a UV spectrophotometer.

For determination of the aloe emodin compound content in the different formulas of Aloe sinkatana extract was weighted 5gm of emulgel formula and dissolved by ultrasonic shaker in 50ml of methanol (10%). The absorbance of the resultant solution was read at wavelength 430 nm using a UV spectrophotometer. The percentage drug content was calculated.

The data obtained was statistically analyzed by Design-Expert_ DX 8 Software statistical program.