Optimizing Anti-inflammatory Activities of Arabica Coffee Ground (Coffea arabica L.) Nanoparticle Gel

3.1. Ethical Approval

This study was approved by the Ethics Committee of Health Research, Medical School, Syiah Kuala University, District Public Hospital dr. Zeinoel Abidin Banda Aceh, Aceh Province, Indonesia No. 320/EA/FK-RSUDZA/2019.

3.2. Formulation of Arabica Coffee Ground Nanoparticles Gel

Carbopol 940 with the variation of 0.5; 0.75 to 1.00% was respectively dissolved in 100 mL distilled water, heated to 80°C for 30 min, and stirred with a magnetic stirrer. After the Carbopol had expanded, it was placed in the mortar, TEA was added with the variation of 0.40; 0.50 to 0.60%, and 7.5 mL glycerin; it was ground until it reached a homogeneity. Afterward, 0.1 g methylparaben was added and stirred constantly; subsequently, nanoparticles of arabica coffee grounds were added with a variation of 1.50; 2.25 to 3.00%, followed by another grinding until it turned homogenous (27). Variations of Carbopol 940, TEA, and nanoparticles of arabica coffee grounds were chosen because these materials could affect the viscosity, pH, and anti-inflammatory properties of the gel produced.

3.3. Preparation of the Experimental Animals

The experimental animals used were male mice (Mus musculus) from strain Balb/c, aged 2 - 3 months, with 20 - 30 g body weight. All 20 mice were acclimatized for one week through feed and water intake. After one week, the mice were fasted for 8 h before the experiment.

3.4. Anti-inflammatory Activities Test

During the test, each mice was cleaned, and its left foot was marked. Then, the marked foot was measured with plethysmometer and noted as initial volume (Vo); the foot volume before the intervention. The foot sole was given an interplanar injection of 0.1 mL carrageenan suspension 1%. The foot volume was re-measured 30 min after the injection. Next, each group was given topical intervention according to the treatment group (arabica coffee ground nanoparticles gel-treated test group, positive control group, and negative control group). After 60 min, the marked foot was re-measured as t-time volume (Vt). The measurement of the mice’s foot was conducted until 360th min with a 30 min time difference (1).

3.5. Data Analysis

Edema volume was calculated from the mice’s foot measurement using Equation 1, the Area Under Curve (AUC) was calculated using Equation 2, and inflammatory inhibition was calculated using Equation 3 (3). The optimization of anti-inflammatory activities of arabica coffee ground nanoparticles gel was assessed using Box-Behnken design of the response surface methodology in Design Expert Version 10.0.3.0 software as follows:

I. Generating the formulation design and response based on the experimental design.

II. Formulation step; carrying out the experiment according to the determined formula condition.

III. Analyzing the resulted response.

IV. Conducting an optimization, followed by verification as the evidence against the predicted response value of optimum formula solution.

Information:

Vu: Edema foot volume

Vt: t-time foot volume

Vo: Initial foot volume

Vun-1: Average edema volume at tn-1-time

Vun: Average edema volume at tn-time

AUCk: AUC of edema volume curve for negative control group

AUCp: AUC of edema volume curve for test group

4.1. Design Formulation of Coffee Grounds Nanoparticle Gel

Box-Behnken Design on Design Expert Version 10.0.3.0 software used to obtain a combination of gel formulation treatment with variations of Carbopol 940 (0.5; 0.75 and 1.00 %), TEA (0.40; 0.50 and 0.60 %) and arabica coffee ground nanoparticles (1.50; 2.25 and 3.00 %), resulting in the design as shown in Table 1 (27).

The design model selected in this research is quadratic model owing to the high score of R-square and low score of Prediction Residuals Error Sum of Square (PRESS), as seen in Table 2. Pred R-Squared value of 0.7390 and Adj R-Squared value of 0.7758 with a difference of less than 0.2. The expected ratio is higher than 4 and the ratio of 7.647 can produce an adequate signal (29, 30). This design model can be used to control the design space.

4.2. Anti-inflammatory Activities

The anti-inflammatory activities or inflammatory inhibition of arabica coffee ground nanoparticles gel are determined by measuring the edema volume of the carrageenan-induced mice foot. The increase of edema volume of the mice’s foot from the first to 180th min was caused by the release of inflammation mediator such as histamine, prostaglandins, bradykinins, and serotonin (31, 32). Meanwhile, at the 240th min, the edema of the mice’s foot experienced a decrease due to the inhibition of prostaglandins synthesis (1, 5). The measurement of the mice’s foot edema in this study exhibits the average inflammatory inhibition values of 27.75%, 27.11%, 0.00% for arabica coffee ground nanoparticles gel (Run 1-17), nanoparticle powder, and negative control, respectively (see Table 3). It suggests that the inflammatory inhibition of arabica coffee ground nanoparticles gel has ½ times ability of the positive control.

Table 4 represents that the quadratic model is significant as shown by the F-value of 7.15 and the value of "Prob> F" with a value of less than 0.0500. The nanoparticle factor (C and C2) has a value of less than 0.0500, so the factor is significant. "Lack of Fit F-value" has a value of 0.11, which means it is not significant relative to pure error. There is a 95% chance that a "Lack of Fit F-value" of this magnitude can occur due to noise. There is a relationship between inflammatory gel inflammation with a factor (x) of the coefficient values, as seen in the Equation 4 (1).

Figure 1 shows the graph of the normal plot of residuals, which can be used to identify the correlation of actual and predicted values. The picture shows that inflammatory inhibition values are close to the normality line, suggesting a normal distribution of the data. It further suggests, the actual result will be found to be close to the program-predicted values. The 3D plot of the correlation of inflammatory inhibition factors, as shown in Figure 2. Moreover, a significant effect of arabica coffee ground nanoparticles is seen on the inflammatory inhibition, according to the curved graph. However, Carbopol 940 and TEA did not affect the inflammatory inhibition, according to the flat-shaped graph. It is corroborated by the inflammatory inhibition value of the negative control, which is the base of the preparations gel.

Figure 1.

Normal plot of residuals of inflammatory inhibition

Figure 2.

A 3D plot of the relationship of (A) Carbopol 940, TEA and the Inflammatory inhibition, (B) TEA, nanoparticles and the Inflammatory inhibition, and (C) Carbopol 940, nanoparticles and the inflammatory inhibition

4.3. Optimization of Arabica Coffee Ground Nanoparticles

General combination of the formulation of the arabica coffee ground nanoparticles gel using Box-Behnken Design of Design Expert Version 10.0.3.0 software is 0.500% Carbopol 940, 0.400% TEA, and 2.313% nanoparticles, giving 29.360% and 29.670% predicted and experimental inhibition inflammatory values, respectively. Meanwhile, the desirability value is 0.812, which is seen in Table 5. The 3D plot graph of the desirability can be seen in Figure 3, where the desirability value is observable at point 0.8122229.

Figure 3.

A 3D plot of desirability value of the optimum formulation