The therapeutic potential of plant extracts has attracted significant interest, especially regarding indigenous species with health-promoting properties. Gymnema inodorum, native to Northern Thailand, is recognized for its rich phytochemical profile; however, the impact of various extraction techniques on its phenolic composition and bioactivity remains underexplored. Optimizing extraction methods is essential to enhance the pharmacological efficacy of this plant’s bioactive compounds. This study investigated the influence of four extraction methods—ethanol maceration, ethanol reflux, aqueous decoction, and microwave-assisted extraction—on the bioactive profile of G. inodorum leaves, with a focus on the phenolic content and biological activities. Antioxidant activities were evaluated using DPPH, ABTS, and FRAP assays, while the total phenolic and flavonoid contents were quantified by colorimetric methods. High-Performance Liquid Chromatography (HPLC) quantified gymnemic acid and key phenolic compounds. Among the methods, ethanol reflux yielded the highest antioxidant activities (DPPH and ABTS scavenging), with a total phenolic content of 82.54 mg GAE/g and flavonoid content of 31.90 mg QE/g. HPLC analysis identified sinapic acid, myricetin, and p-hydroxybenzoic acid as major phenolics. Furthermore, the ethanol reflux extract displayed potent anti-diabetic activity, with IC₅₀ values of 13.36 mg/mL for α-amylase and 7.39 mg/mL for α-glucosidase, as well as strong anti-inflammatory activity (IC₅₀ of 1.6 mg/mL) and acetylcholinesterase inhibition (IC₅₀ of 1.2 mg/mL). These findings suggest that ethanol reflux extraction is a highly effective method for producing bioactive-rich G. inodorum extracts, with substantial pharmacological potential for developing herbal remedies and nutraceuticals, particularly in enhancing therapeutic approaches for diabetes and other health-related conditions.

In recent years, growing attention has been directed towards the potential of plant extracts as great therapeutic agents in controlling oxidative stress and inflammation. Gymnema inodorum (Lour.) Decne, an indigenous plant locally grown in Northern Thailand, is known for its anti-diabetic activity. This study aims to explore the antioxidant properties and phenolic profile of column chromatographic fractionated G. inodorum leaf extract. The ethanolic-reflux extracts of G. inodorum leaves were subjected to column chromatography for the isolation of bioactive molecules. Total phenolic and flavonoid content were assessed using Folin–Ciocalteu and aluminium chloride colorimetric methods, respectively. Total antioxidant activity was evaluated by employing different antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic)acid (ABTS) scavenging assay and ferric reducing antioxidant power (FRAP) assay. High-Performance Liquid Chromatography (HPLC) with a C18 column was employed to determine the level of gymnemic acid, while Liquid Chromatography Quadrupole Time-of-Flight/Mass Spectrometry (LC-QTOF/MS) was used to identify potential phenolic compounds. The three separated fractions indicating A, B, and C, exhibited remarkable antioxidant activity, with total phenolic content of 0.294 ± 0.056, 0.085 ± 0.039, and 0.227 ± 0.041 mg QE/mL, and total flavonoid content of 0.274 ± 0.050, 0.309 ± 0.110, and 0.384 ± 0.007 mg QE/mL, respectively. Fraction A exhibited the highest DPPH and ABTS scavenging activity, while fraction C demonstrated significantly higher ferric-reducing power. HPLC analysis revealed a highest concentration of gymnemic acid in fraction C (0.393 ± 0.003 mg/g) compared to fraction A (0.159 ± 0.003 mg/g) and fraction B (0.142 ± 0.000 mg/g). The LC-QTOF/MS phenolic screening showed seven potential phenolic compounds in fraction A, seven compounds in fraction B, and four compounds in Fraction C. A significant relationship is found between antioxidant potential and total phenolic content, suggesting that phenolic compounds are the major contributors to the antioxidant activity of G. inodorum.