Isolation and structure elucidation of anti-inflammatory and cytotoxic compounds from Valeriana phu and V. tuberosa species

This thesis aimed to isolate the anti-inflammatory and cytotoxic compounds from Valeriana phu and V. tuberosa via anti-inflammatory activity-directed fractionation and to elucidate their mechanisms of action using in vitro and in silico methods. The crude EtOH extracts, subextracts, and fractions were assessed for their NO inhibition activities in LPS-induced RAW 264.7. From the active fractions, 28 secondary metabolites, including nine new ones, namely phuosides I-III (2, 7, 10) valephuoside (4), phuol (14), and valtuberosides IIV (21, 22, 24, 27), were purified and characterized by NMR and HRESIMS. Compounds 1, 2, 5, 7, 11, 12, 14-17, 19, 20, 24, 25, 28 reduced nitrite release with IC50 14.94-95.71 µM. Furthermore, underlying mechanisms were elucidated by ELISA, Western blot, qRT-PCR, immunostaining and supported by molecular modelling studies. Compounds 1, 15-17, 19, 25, and 28 exhibited significant decrease in TNF-α, IL-1β, IL-6, PGE2, and COX-2, while 1, 15, and 25 reduced iNOS expression in RAW 264.7. Moreover, 25 significantly inhibited mRNA expression of cox2, tnfα, il1b, and il6, and translocation of NF-κB to nuclear region. Additionally, it exhibited the strongest binding affinity to iNOS in in silico assays. Compounds with notable anti-inflammatory activities were further evaluated for their cytotoxicity potentials against MCF-7, MDA-MB-23, U87, A172, MIA PaCa-2, PANC-1, Mahlavu, Hep3B cancer cell lines and their cell death mechanisms were elucidated. Compounds, 14-16, and 19 exhibited significant cytotoxicity against A172, Hep3B, MIA PaCa-2, and PANC-1 cell lines (IC50 18.3-25.4 µM) via inducing cell cycle arrest, especially at G2/M phase, and initiating the apoptotic pathway.