Formulation and Evaluation of a Polyherbal Topical Gel Containing Camellia sinensis, Boswellia serrata, and Glycyrrhiza glabra for Anti-inflammatory Activity

Inflammation is a complex and essential physiological response to tissue injury, infection, or irritation. While acute inflammation serves a protective role, chronic inflammation is implicated in a wide array of diseases, including arthritis, dermatitis, and various musculoskeletal disorders. Standard pharmaceutical anti-inflammatory treatments, although effective, often come with undesirable side effects when used long-term. This concern has driven significant interest in herbal alternatives that are both effective and safer for prolonged use. ^{[1, 2, 3]} Polyherbal formulations, which combine multiple plant-based extracts, are gaining popularity for their synergistic therapeutic effects and lower side effect profiles. In this context, the present research aims to formulate and evaluate a polyherbal topical gel incorporating Camellia sinensis (green tea), Boswellia serrata (Indian frankincense), and Glycyrrhiza glabra (licorice)—each known for their distinct yet complementary anti-inflammatory properties.^[20] Camellia sinensis is rich in epigallocatechin-3-gallate (EGCG), a powerful antioxidant that inhibits pro-inflammatory cytokines and downregulates COX-2 expression and NF-κB activation. Boswellia serrata contains boswellic acids, which are known inhibitors of 5-lipoxygenase, reducing leukotriene-mediated inflammation. Glycyrrhiza glabra possesses glycyrrhizin and liquiritin, compounds with corticosteroid-like effects that help in reducing skin irritation and supporting wound healing. ^{[4, 5, 6]} The formulation of these herbs into a topical gel is advantageous due to its ability to deliver active constituents directly to the site of inflammation, ensuring localized action with minimal systemic exposure. The gel form enhances bioavailability, ease of application, and patient compliance, making it a suitable alternative to conventional treatments.^[7] This study is therefore designed with the following objectives: to screen the phytochemical constituents of the selected herbal extracts, formulate a stable gel using suitable excipients, evaluate its physicochemical and rheological properties, and assess its in vitro anti-inflammatory efficacy in comparison with a standard drug. By combining traditional knowledge with modern pharmaceutical techniques, this research aims to offer a natural, effective, and patient-friendly alternative for managing inflammatory conditions.^[8]

MATERIALS AND METHODS:

Collection of Plant Material and Chemicals

The leaves of Camellia sinensis and Boswellia serrata were collected from the tea gardens of Palampur, Kangra, Himachal Pradesh, and the local market of Bhopal, respectively. The aerial parts of Glycyrrhiza glabra were also sourced from Bhopal’s local market. All plant materials were authenticated prior to use. Chemicals and reagents, including Carbopol 940, Propylene glycol, Ethanol, Methyl paraben, Propyl paraben, EDTA, and other analytical grade reagents were procured from Central Drug House (P) Ltd., Alpha Chemika, Changshu Hongsheng Fine Chemical Co. Ltd., and other certified suppliers.

Preparation of Plant Extracts

Dried plant materials were coarsely powdered and defatted using petroleum ether (60–80°C) in a Soxhlet apparatus. The defatted residues were then extracted with methanol. The extracts were concentrated via distillation and stored in desiccators until use.

Formulation of Polyherbal Gel

Polyherbal gels (CSBSGG-1 to CSBSGG-6) were prepared using Carbopol-940 as the gelling agent, along with Propylene glycol, Ethanol, Methyl and Propyl parabens, and EDTA. Extract concentrations ranged from 1% to 10%. A gel of Diclofenac sodium (0.5%) was used as the standard. Composition details are listed in Table 1. ^[9-15]

TABLE 1: Composition Of Polyherbal Gel

*CSBSGG: Combination of methanolic extract of Boswellia serrata, Camellia sinensis and Glycyrrhiza glabra

Evaluation of Gel Formulations

pH: Measured using a digital pH meter.^[16]

Appearance & Homogeneity: Assessed visually. ^[17]

Spreadability: Measured by the diameter of gel spread under a standard weight. ^[18]
Extrudability: Evaluated by extruding gel from aluminium tubes and calculating percentage output. ^{[19, 20]}

Viscosity: Measured using a Brookfield viscometer (Model RVTDV II) with spindle no. 6 at 100 rpm. ^[21]

In-vitro Diffusion Study: Conducted using a cellophane membrane diffusion setup in phosphate buffer (pH 6.8) at 37 ± 0.5°C. Samples were analysed using UV-VIS spectrophotometry. ^{[22, 23]}

In-vitro Anti-inflammatory Activity

Protein denaturation inhibition method was used to evaluate anti-inflammatory activity. Egg albumin was mixed with phosphate buffer and various concentrations of gel, and absorbance was recorded at 660 nm post incubation and heating. ^{[24, 25]}

In-vivo Anti-inflammatory Activity

Animals: Wistar rats (150–200 g) were maintained under standard laboratory conditions. Ethical approval was obtained from the Institutional Animal Ethics Committee (Approval No. 648/02/6/CPCSEA). Carrageenan-induced Paw Edema: Inflammation was induced via subplantar injection of 0.1 mL carrageenan (1% w/v). Gels were applied topically 1 hour before induction. Paw thickness was measured at 0, 30, 60, 120, 180, 240, and 300 minutes using a digital vernier caliper. ^{[26, 27]}

RESULT AND DISCUSSION

Evaluation of Polyherbal Gel

All gel formulations demonstrated satisfactory physical properties, as summarized in Tables 2 and 8. Among all, CSBSGG-6 emerged as the optimal formulation with a favourable pH (6.17), excellent homogeneity, light green colour, and high spreadability (55 mm), indicating ease of application. Its extrudability (89%) and viscosity (4500 cp) were within ideal ranges for topical delivery. The formulation showed performance metrics closely matching the standard Diclofenac gel (0.5%), which had a pH of 6.15, spreadability of 59 mm, extrudability of 92%, and viscosity of 4600 cp.

Table 2: Evaluation Parameters of Prepared Polyherbal Gels

In-vitro Diffusion Study

The in-vitro diffusion studies revealed that drug release was inversely proportional to the concentration of Carbopol-940 used as the gelling agent. CSBSGG-6 exhibited the highest drug release among the test formulations, comparable to the marketed Diclofenac gel. This suggests optimized release characteristics likely due to balanced viscosity and excipient composition.

Fig 1: In-vitro diffusion study of polyherbal gel (CSBSGG-1 to CSBSGG-6) compared with marketed formulation

In-vitro Anti-inflammatory Activity

The CSBSGG-6 formulation exhibited a concentration-dependent inhibition of protein denaturation. At 50 µg/mL, it achieved an 86.27% inhibition rate, closely approaching that of Diclofenac sodium (88.63%). This confirms the gel’s strong potential to counteract inflammatory protein denaturation pathways in vitro.

Fig 2: Effect of polyherbal formulation (CSBSGG-6) on heat induced protein denaturation

Chronic Dermal Toxicity Study

In vivo irritation testing in rats indicated that the herbal gel was well-tolerated. Only minor erythema was observed in two animals at early time points (PDII = 1.0), which resolved within 48 hours. No edema or severe irritation was recorded. This suggests the polyherbal gel is safe for chronic dermal application with negligible irritation.

In-vivo Anti-inflammatory Activity (Carrageenan-Induced Paw Edema)

Topical application of CSBSGG-6 significantly reduced carrageenan-induced paw edema in rats. The gel demonstrated anti-inflammatory effects comparable to the standard Diclofenac formulation across both early and late inflammatory phases. These results validate the polyherbal gel’s efficacy in modulating inflammation mediated by histamine, bradykinin, and prostaglandins.

Fig 3: Anti-inflammatory effect of topical application of single extract gels and polyherbal gels in combination on the first phase and late phase of carrageenan-induced paw edema in rat.