In vitro anti-inflammatory activity of nabhi oil constituent oils by protein denaturation inhibition and erythrocyte membrane stabilisation assays

Background: Ayurvedic Nabhi Chikitsa employs polyherbal oil formulations combining neem oil (Azadirachtaindica), clove essential oil (Syzygiumaromaticum), ginger essential oil (Zingiberofficinale), peppermint essential oil (Mentha × piperita) and sesame oil (Sesamumindicum) - each with individual ethnopharmacological claims of anti-inflammatory activity. While computational evidence (OR3, OR4) and published in vitro studies support specific molecular anti-inflammatory mechanisms (COX-2 inhibition, NF-κB suppression, TACE inhibition), direct in vitro anti-inflammatory quantification for these oils under standardised assay conditions against a reference standard has not been systematically reported. Protein denaturation inhibition (PDI) and erythrocyte membrane stabilisation (EMS) are established, reproducible, low-cost in vitro surrogates for anti-inflammatory screening that measure the ability of test substances to inhibit heat-induced protein unfolding and to stabilise erythrocyte membranes against osmotic haemolysis- both pathological processes relevant to inflammatory tissue damage. Methods: Five Nabhi oil constituent oils (neem oil, clove EO, ginger EO, peppermint EO, sesame oil) were evaluated at four concentrations (25, 50, 100, 200 µg/mL) against Diclofenac sodium reference (100 µg/mL) in two validated in vitro anti-inflammatory assays. PDI assay: BSA (0.2 mg/mL in phosphate buffer pH 6.4) incubated with each oil at 72 °C for 5 min; turbidity (optical density at 660 nm) measured spectrophotometrically; % protein denaturation inhibition calculated from OD reduction versus heated control. EMS assay: washed erythrocyte suspension in hypotonic saline (0.5% NaCl) incubated with each oil at 37 °C for 30 min; haemolysis measured at 540 nm; % membrane stabilisation calculated. IC₅₀ values were determined by non-linear sigmoidal regression. Synergy for selected oil combinations was assessed by the Chou–Talalay Combination Index (CI) and fractional inhibitory concentration index (FICI). n = 3; mean ± SD; one-way ANOVA with Tukey post-hoc. Results: Clove EO demonstrated the strongest anti-inflammatory activity across both assays (PDI IC₅₀ = 38.2 ± 2.8 µg/mL; EMS IC₅₀ = 44.6 ± 3.2 µg/mL), followed by neem oil (PDI IC₅₀ = 68.4 ± 4.2 µg/mL), ginger EO (84.6 ± 5.4 µg/mL), peppermint EO (112.4 ± 6.8 µg/mL) and sesame oil (185.2 ± 10.2 µg/mL). Diclofenac sodium reference showed PDI IC₅₀ 32.8 ± 2.4 µg/mL. A strong linear correlation was observed between PDI and EMS IC₅₀ values across all five oils (R² 0.976–0.991), confirming assay consistency. All five oils showed concentration-dependent, statistically significant inhibition at all four concentrations. Chou–Talalay Combination Index analysis confirmed synergism (CI < 0.5) for neem oil + clove EO (CI = 0.42), clove EO + peppermint EO (CI = 0.48) and the complete VS Nabhi Oil blend (CI = 0.38 - strongest synergy), supporting the pharmacological rationale for polyherbal formulation. Conclusions: All five VS Nabhi oil constituent oils demonstrate concentration-dependent anti-inflammatory activity in PDI and EMS assays. Clove EO is the most potent direct anti-inflammatory active; neem oil and ginger EO show intermediate potency; sesame oil shows the weakest direct in vitro activity, consistent with its primarily intracellular (NF-κB/Nrf2) anti-inflammatory mechanism confirmed by OR4 docking. The VS Nabhi Oil polyherbal blend demonstrates the strongest synergistic anti-inflammatory activity (CI = 0.38), validating the Ayurvedic multi-herb formulation approach. These standardised in vitro anti-inflammatory data constitute a direct evidence base for VS Nabhi Oil anti-inflammatory product claims in regulatory dossiers.