Evaluation of the pH-Dependent Anti-Inflammatory Potential of Mirabilis jalapa Linn. Root Extract Using Protein Denaturation and Membrane Stabilisation Assays

Abstract

Background: Inflammation is a biological response characterised by redness, oedema, fever, pain, and loss of function. Medicinal plants have long served as a valuable source of anti-inflammatory compounds, and Mirabilis jalapa Linn. (Four O’clock plant) is traditionally used in folk medicine to treat inflammatory conditions. Aims: This study evaluated the anti-inflammatory potential of the aqueous root extract of M. jalapa and investigated the influence of pH on its activity, with particular consideration of the traditional preparation in which the plant extract is combined with lime extract. Study Design: An in vitro study assessed the pH-dependent anti-inflammatory potential of the aqueous root extract of M. jalapa. Fresh extracts were prepared and used immediately to minimise potential changes in the stability and bioactivity of their phytochemical constituents. Place and Duration of Study: The study was conducted in the Microbiology Laboratory, Faculty of Allied Health Sciences, General Sir John Kotelawala Defence University, Sri Lanka. Methodology: Anti-inflammatory activity was assessed using two in vitro assays: inhibition of egg albumin denaturation and stabilisation of the Human Red Blood Cell (HRBC) membrane. The extract was tested across a range of concentrations and pH levels, with hydrochloric acid, citric acid, and lime extract used as pH-adjusting agents. A General Linear Model (GLM) was employed to assess the effects of pH, extract concentration, and pH-adjusting agent, as well as the interaction between extract concentration and pH-adjusting agent, on percentage inhibition. Results: The aqueous root extract showed substantial concentration-dependent inhibition of protein denaturation and membrane-stabilising activity that was comparable to diclofenac sodium at higher concentrations. In both assays, GLM analysis identified pH as the most influential factor affecting percentage inhibition. Increasing the pH from 2.6 to 5.0 was associated with a marked reduction in inhibitory activity, with the highest activity observed at pH 2.6. A decrease in activity was detected when the pH is increased from 2.6 to 5.0. In the egg albumin denaturation assay, pH exerted a highly significant effect on percentage inhibition (F = 6877.96, P < 0.001, partial η² = 0.917, df = 1), representing the largest effect among the factors examined. A similar pattern was observed in the HRBC membrane stabilisation assay, in which pH had a highly significant effect on percentage protection (F = 8881.415, P < 0.001, partial η² = 0.934, df = 1), again representing the largest effect in the model. A statistically significant interaction between extract concentration and pH-adjusting agent was also observed in both assays, indicating that the concentration–response relationship varied according to the agent used for pH adjustment. Lime extract enhanced the anti-inflammatory potential at lower concentrations but was associated with reduced activity at higher concentrations, whereas citric acid showed a minimal effect across the tested concentration range. In contrast, hydrochloric acid suppressed the activity at higher concentrations. Conclusion: These findings support the ethnomedicinal properties of the root extract of M. jalapa for treating inflammatory conditions and identify pH as an important parameter in determining its anti-inflammatory efficacy. In particular, the results provide a pharmacological rationale for the traditional practice of combining M. jalapa root paste with lime extract.