Formulation of a novel emulgel incorporated with Alpinia calcarata essential oil and assessment of its anti-inflammatory activity

Introduction
Inflammation is a complex biological response involving immune cells, cytokines, and vascular changes, initiated to eliminate pathogens or resolve injury. While acute inflammation is protective, chronic inflammation can contribute to diseases such as arthritis, cardiovascular disorders, and cancer. Natural products, including essential oils, have long been used in traditional medicine for their therapeutic properties, and there is growing interest in developing plant-based anti-inflammatory medicines.

Alpinia calcarata (A. calcarata) Roscoe, a perennial herb of the Zingiberaceae family, is cultivated in tropical regions for medicinal use. Its rhizomes have demonstrated antibacterial, analgesic, antioxidant, and anti-inflammatory activities. Although the essential oil of A. calcarata is known to suppress inflammatory mediators such as reactive oxygen species, cytokines, and prostanoids in vitro and reduce edema and pain in animal models, it has not been widely developed into pharmaceutical dosage forms. This study aimed to formulate a topical emulgel incorporating A. calcarata rhizome essential oil and to investigate its anti-inflammatory mechanisms using in vitro assays. Emulgels are particularly advantageous for topical delivery as they combine hydrophilic and lipophilic phases, allowing incorporation of diverse phytochemicals, while the gel matrix ensures good spreadability, patient comfort, and enhanced skin penetration.

Materials and Methods
Study Design and Ethical Approval:
This was a laboratory-based in vitro study. Ethical approval was obtained from the Ethics Review Committee of the Faculty of Medical Sciences, University of Sri Jayewardenepura (B.Pharm/01/18), and written informed consent was obtained from all blood donors. Protocols adhered to the Declaration of Helsinki (2024 revision).

Plant Material and Essential Oil Extraction:
Fresh rhizomes of A. calcarata were collected from Navagiri Indigenous Clinic and Herbal Garden, Jaffna, Sri Lanka, and authenticated by the Bandaranaike Memorial Ayurvedic Research Institute. Rhizomes were shade-dried, diced, and hydro-distilled for 4 hours using a Clevenger-type apparatus. The essential oil was extracted with n-pentane and ether (1:1 v/v), dried over anhydrous sodium sulfate, and collected after solvent evaporation.

Thin-Layer Chromatography (TLC):
The essential oil was dissolved in dichloromethane:methanol (1:1 v/v), and TLC was performed using methanol:dichloromethane:hexane (0.2:8.8:1 v/v/v) as the mobile phase. Plates were visualized under UV at 254 nm and after spraying with vanillin sulphate.

Emulgel Formulation:
Carbopol 940 was dispersed in purified water, and triethanolamine was added to adjust pH to 6.0–6.5. An oil phase was prepared by dissolving Tween 20 in light liquid paraffin, then adding the essential oil (dissolved in ethanol) and methyl paraben (dissolved in propylene glycol). An aqueous phase was prepared by dissolving Tween 80 in purified water. Both phases were heated to 70–80°C, then the aqueous phase was added to the oil phase with stirring. After cooling to 25°C, the emulsion was mixed 1:1 with the Carbopol gel base. A placebo emulgel was prepared identically, substituting ethanol for the essential oil.

Physical Characterization:
The emulgel was evaluated for color, consistency, homogeneity, pH (at 25°C), spreadability (glass plate method), viscosity (Brookfield viscometer, spindle 5 at 10 rpm), syneresis (at 25°C and 35°C for 1 hour), and phase separation after centrifugation (1,000 rpm for 5 minutes). All tests were performed in triplicate.

In Vitro Anti-inflammatory Activity:
Blood was collected from 12 healthy, non-smoking volunteers who had not taken any medication for seven days.

1. Heat-Induced Hemolysis: Erythrocyte suspension (10% w/v) was incubated with A. calcarata emulgel (6.25–50 μg/mL), placebo (50 μg/mL), or indomethacin (positive control) at 56°C for 30 minutes. Hemoglobin release was measured spectrophotometrically at 540 nm.

2. Hypotonicity-Induced Hemolysis: Erythrocyte suspension (40% w/v) was incubated with hypotonic saline (0.25% w/v NaCl) and test samples (100–400 μg/mL), placebo (400 μg/mL), or indomethacin (200 μg/mL) at 37°C for 30 minutes. Hemolysis was measured similarly.

3. Thrombolytic Activity: Pre-weighed blood clots were incubated with test samples (100–400 μg/mL), streptokinase (positive control), or placebo (400 μg/mL) at 37°C for 90 minutes. Clot lysis percentage was calculated from weight loss.

Statistical Analysis:
Data were expressed as mean ± standard error of the mean (SEM) of triplicates. One-way ANOVA followed by Dunnett's post-hoc test (SPSS 25.0) was used, with significance set at p < 0.05.

Results and Discussion
Extraction Yield and Phytochemical Profile:
The essential oil yield was 0.61% (v/w). TLC fingerprinting revealed multiple bands with distinct retention factor (Rf) values under UV and after vanillin sulphate spraying, confirming the presence of diverse phytochemicals. This aligns with previous studies identifying compounds such as α-terpineol, 1,8-cineole, berberine, phytol, and various terpenes in A. calcarata.

Emulgel Formulation and Physical Properties:
The formulated emulgel was smooth, white, viscous, and homogeneous, with no phase separation. Its pH was 6.30 ± 0.00 at 25°C, within the recommended physiological range for skin (5.0–6.5), minimizing irritation risk. Spreadability was 118.0 ± 0.3 g·cm/min, indicating easy application and uniform coverage. Viscosity was 4.322 ± 0.124 Pa·s, demonstrating pseudoplastic (shear-thinning) behavior suitable for topical use. No syneresis or phase separation was observed under thermal or centrifugal stress, confirming good stability.

Anti-inflammatory Activity:

• Heat-Induced Hemolysis: The A. calcarata emulgel significantly inhibited heat-induced hemolysis in a concentration-dependent manner (r² = 0.99). The highest inhibition (87.68 ± 0.35%) was observed at 50 μg/mL, significantly greater than placebo (p < 0.05). This suggests membrane-stabilizing activity, likely due to interaction of essential oil constituents (e.g., 1,8-cineole, α-pinene) with erythrocyte membranes, protecting against thermal stress.

• Hypotonicity-Induced Hemolysis: The emulgel showed modest, dose-dependent inhibition of hypotonic hemolysis, with maximum inhibition (7.29 ± 0.19%) at 400 μg/mL, which was significant compared to placebo (p < 0.05). However, indomethacin (200 μg/mL) exhibited far greater protection (51.28 ± 0.15%). The lower efficacy against hypotonic stress suggests that the essential oil's protective mechanism is primarily directed against thermal rather than osmotic membrane disruption, possibly by stabilizing membrane proteins rather than countering osmotic imbalance.

• Thrombolytic Activity: None of the tested doses showed significant thrombolytic activity compared to placebo (p > 0.05), whereas streptokinase produced marked clot lysis. This indicates that A. calcarata essential oil does not activate fibrinolytic pathways, consistent with its primary mechanism of membrane stabilization rather than enzymatic clot degradation.

Interpretation and Implications:
The membrane-stabilizing effect observed in the heat-induced hemolysis assay is significant because erythrocyte membranes are structurally similar to lysosomal membranes. Stabilization of lysosomal membranes is a key anti-inflammatory mechanism, preventing the release of hydrolytic enzymes that exacerbate tissue damage. The concentration-dependent activity and high r² value (0.99) support a specific interaction between essential oil phytochemicals and membrane components.

The lack of significant thrombolytic activity suggests that the anti-inflammatory effects of A. calcarata are not mediated by fibrin dissolution but rather by direct membrane protection and potentially by inhibition of inflammatory mediators, as reported in prior studies. The favorable physicochemical properties of the emulgel—appropriate pH, good spreadability, shear-thinning viscosity, and stability—indicate its suitability as a topical formulation.

Limitations and Future Directions
This study was limited to in vitro assays, which, while providing mechanistic insights, do not capture the complexity of in vivo inflammatory responses. Future research should include:

• In vivo anti-inflammatory studies (e.g., animal models of edema or dermatitis).

• Investigation of specific molecular mechanisms, such as cyclooxygenase (COX) and lipoxygenase (LOX) inhibition, cytokine modulation, and nuclear factor kappa-B (NF-κB) pathway involvement.

• Optimization of essential oil concentration and formulation variables through dose-response and stability studies.

• Evaluation of skin irritation, sensitization potential, and clinical efficacy in human subjects.

Conclusions
A novel topical emulgel incorporating A. calcarata rhizome essential oil was successfully formulated and demonstrated concentration-dependent anti-inflammatory activity through membrane stabilization in vitro. The formulation exhibited favorable physicochemical properties, including appropriate pH, good spreadability, pseudoplastic rheology, and stability. These findings support the potential of A. calcarata essential oil emulgel as a promising natural topical anti-inflammatory agent, warranting further in vivo and clinical investigation.

Full text:

https://www.xiahepublishing.com/2835-6357/FIM-2025-00029

The study was recently published in the Future Integrative Medicine.

Future Integrative Medicine (FIM) is the official scientific journal of the Capital Medical University. It is a prominent new journal that promotes future innovation in medicine.It publishes both basic and clinical research, including but not limited to randomized controlled trials, intervention studies, cohort studies, observational studies, qualitative and mixed method studies, animal studies, and systematic reviews.

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