Formulation and Evaluation of Anthocyanin Gel for Anti-Inflammatory Activity in Rheumatoid Arthritis

Figure 1: Comparison of a healthy joint and a rheumatoid arthritic joint showing synovial inflammation, pannus formation, and cartilage/bone erosion.

MATERIALS AND METHODS

Drug and Excipient Profile

Anthocyanin was extracted in-house from fresh eggplant peels: molecular formula C15H11O6; MW 207.25 g/mol; soluble in water and alcohols; dose 180–215 mg/day; stored at 2–8°C.10 Carbopol-93411 (carboxypolymethylene; MW ~3×106; pH of 1% dispersion 2.5–3.0; bulk density 1.76 g/cm³) was used as primary gelling agent. SCMC11 (MW 262.19 g/mol; pH 6.0–8.5) served as thickener. HPMC K4M, propylene glycol, ethanol, methyl paraben, propyl paraben, and triethanolamine were of pharmaceutical grade.

Extraction of Anthocyanins

Eggplant peels were macerated in methanol containing 3% HCl (v/v) at room temperature for 24 h.10 Saturated lead acetate was added until precipitation was complete; the precipitate was re-dissolved in methanol/2% HCl and treated with diethyl ether. The brown precipitate was collected and the filtrate evaporated at 55°C. The crude anthocyanin powder was stored at 4°C.

Characterisation

Confirmatory tests:10 (i) AlCl3 addition gave a 12 nm bathochromic UV shift; (ii) UV absorbance at 200–700 nm; (iii) heating with 2 M HCl at 100°C for 5 min maintained stable purple colour (Figure 2); (iv) 2 M NaOH produced a green colour (Figure 3). FTIR was performed on KBr pellets (1:100, 10 t/in²) scanned at 450–3900 cm⁻¹19, UV λmax was established at 516 nm19 (Table 2; Figure 4).

Formulation of Anthocyanin Gels

Six formulations (F1–F6) were prepared using Carbopol-934, SCMC, and HPMC K4M individually or in combination (Table 1). Carbopol-934 gel: polymer dispersed in water at 40°C, 1200 rpm/30 min; pH adjusted with triethanolamine.20 SCMC gel: dissolved at 50°C, 1200 rpm/30 min.20 HPMC K4M gel: hot/cold technique—dissolved at 80°C, cold water added gradually, refrigerated overnight.20 Combination gels: individual polymer gels blended before anthocyanin incorporation.

Evaluation Parameters

pH: Digital pH meter at 24 h, 48 h, 1 week, 2 weeks, 1 month, and 3 months.19Homogeneity: Visual inspection.20Centrifuge stability: 2000 rpm for up to 60 min.  Temperature stability: Storage at 2–8°C, 25°C, and 40–45°C for 3 months.20

Viscosity: Brookfield viscometer, spindle no. 64, 50 rpm.19Spreadability: 1 g gel between 20×20 cm glass plates; 1000 g weight for 1 min; diameter in triplicate.18% Drug content: 2 g gel in 100 mL phthalate buffer (pH 5) for 2 h; absorbance at 516 nm.

In vitro drug release: Modified diffusion apparatus; semipermeable membrane; 100 mL phthalate buffer (pH 5, 37±2°C, 50 rpm); 5 mL aliquots at 30, 60, 120, 180, 240, 300, 360 min; absorbance at 516 nm.20

Anti-inflammatory study: RA patients randomised into anthocyanin-gel and placebo groups. Applied at 20 g/h once daily (weeks 1–4), then 10 g/h once daily (weeks 5–8); clinical parameters assessed at baseline and week 8.

Antimicrobial/antifungal activity: Macrobroth dilution against ATCC strains of S. aureus, E. coli, E. faecalis, P. aeruginosa and fungi C. albicans, C. tropicalis; serial two-fold dilutions 6.25–100 µg/mL; ciprofloxacin and ketoconazole as reference standards.15,16

Table 1: Composition of Anthocyanin Gel Formulations F1–F6

All quantities per 100 g gel; TEA added q.s. to adjust pH; Water q.s. to 100 g

Figure 2: Confirmatory test for anthocyanins using HCl — stable purple colouration at 100°C.

Figure 3: Confirmatory test for anthocyanins using NaOH — characteristic green colour.

RESULTS AND DISCUSSION

Identification and Characterisation of Anthocyanin

All four confirmatory tests were positive, confirming the identity and purity of the extracted material as anthocyanin.10 The UV absorbance at 516 nm and calibration linearity (r²=0.998; slope=0.001; intercept=0.006; Table 2) provide a reliable quantitative method (Figure 6). The pH of the crude extract was stable at 3.75±0.01 throughout 3 months of storage at 4°C. FTIR spectra of pure anthocyanin, pure polymers, and physical mixtures F1 and F4 showed that all characteristic anthocyanin peaks were retained without new peaks or significant shifts confirming complete physicochemical compatibility between the drug and excipients.19 The drug exists in its original form and is available for pharmacological action.

Physical Evaluation of Gel Formulations

All formulations maintained stable pH of 5.0–5.4 over 3 months (Table 3), within the physiologically safe skin pH range of 4.5–6.5,19 with no observed irritation. All gels were homogeneous with no aggregate formation, showed no sedimentation on centrifugation, and remained physically unchanged across all storage temperatures.20 HPMC K4M formulations (F3, F6) exhibited the highest viscosity (11,950–11,990 cps); Carbopol-934-based gels (F1, F4, F5) were least viscous (10,220–10,850 cps) and most spreadable (5.69–5.95 cm), a property beneficial for patient comfort.18,19 Results are shown in Table 4.

Percentage Drug Content

All formulations yielded drug content above 96% (Table 5), within pharmacopoeial acceptance limits. F1 (98.5%) and F4 (98.0%) were superior, reflecting uniform anthocyanin distribution within Carbopol-934 matrices and confirming reproducibility of the formulation process.

In Vitro Drug Release

All gel formulations exhibited sustained drug release over 6 h (Table 6; Figure 5), compared with the pure drug which released ~97.9% within 180 min alone. Formulations with lower viscosity released drug faster yet in a sustained manner: F1 (94.1%) and F4 (93.4%) were best at 6 h, following the rank F1 > F4 > F5 > F2 > F3 > F6—directly inversely proportional to viscosity.20

Anti-Inflammatory Activity in Human Volunteers

All anthocyanin gel formulations produced clinically observable reduction in RA-associated joint inflammation after 8 weeks compared with placebo. This aligns with anthocyanins' known inhibition of NF-κB-mediated COX-2 expression and suppression of Th17-cell-dependent cytokine production.13,14 The sustained release of F1 and F4 likely underpins their clinical efficacy by maintaining adequate dermal drug concentrations.5,7,12

Antimicrobial and Antifungal Activity

F1 and F2 showed the lowest MICs against gram-positive organisms (E. faecalis, S. aureus: 25–50 µg/mL; Table 7). Activity against gram-negative bacteria was generally weaker (>100 µg/mL), consistent with their outer-membrane barrier.15 F1, F5, and F6 showed moderate antifungal activity against C. albicans (MIC 50 µg/mL; Table 8). These properties are clinically relevant given RA patients' increased infection risk.16

Figure 4: Calibration curve of anthocyanin at λmax 516 nm (r² = 0.998).

Table 2: Calibration Curve Data for Anthocyanin At 516 Nm

r² = 0.998; slope = 0.001; intercept = 0.006; Beer–Lambert range: 100–600 µg/mL

Table 3: PH of Anthocyanin Gel Formulations Over 3 Months

Table 4: Viscosity and Spreadability Of Anthocyanin Gel Formulations

Table 5: Percentage Drug Content and Cumulative % Drug Release AT 6 H

Table 6: Cumulative % Drug Release Vs. Time for All Formulations

Figure 5: Comparative cumulative % drug release profiles of pure drug and formulations F1–F6 over 360 min.

Table 7: Antimicrobial Mic Values (ΜG/ML) Of Anthocyanin Gel Formulations

MIC values in µg/mL; Ciprofloxacin = reference standard

Table 8: Antifungal MIC Values (ΜG/ML) Of Anthocyanin Gel Formulations