Discovery of anti-inflammatory phytoconstituents from Lactuca indica L. based on its traditional uses

The arial parts of Lactuca indica have been traditionally used to relieve skin inflammation or to alleviate stomach-ache. This study aims to evaluate pharmacological activities relating to traditional usage of the plant, with a special focus on anti-inflammatory, anti-bacterial and cytotoxic effects of extracts, followed by bio-activity-guided isolation of phytochemicals. The ethanolic extract was prepared by means of percolation, from which 5 sub-extracts were obtained by sequential liquid-liquid partition, including n-hexane, chloroform, ethyl acetate, n-butanol and aqueous sub-extracts. Through preliminary screening, the n-hexane, chloroform and ethyl acetate sub-extracts showed noteworthy inhibition on NO excessive synthesis in LPS-stimulated RAW 264.7 cells. Only the n-hexane sub-extract demonstrated anti-bacterial effect. None of the sub-extracts expressed cytotoxic effects on human acute leukemia (HL-60), hepatocellular (Hep-G2) or gastric (MKN-7) carcinoma cell lines. Phytochemical investigations of anti-inflammatory constituents were carried out for n-hexane, chloroform and ethyl acetate sub-extracts, leading to isolation of 12 compounds: stearic acid (1), glyceryl monostearate (2), β-amyrin acetate (3), oleanolic acid (4), lupeol (5), 4α(15),11β(13)-tetrahydroridentin B (6), 5-hydroxymethylfurfural (7), p-hydroxymethylbenzoic acid (8), protocatechuic acid (9), succinic acid (10), cynaroside (11) and lynarin (12). Molecular docking was then performed to investigate the underlying mechanism, revealing that four compounds 3, 4, 11, 12 exhibited excellent binding affinities to the iNOS enzyme. This study suggests that L. indica may be the source of promising phytochemicals against inflammation mediated by excessive production of NO. This could be a mechanism contributing to partly explain for traditional applications of this herb.