Studies On The Chemical Constituents And Bioactivities Of Hypericum Japonicum

Hypericum japonicum Thunb. ex Murray, as a type of annual or perennial herb, belongs to the genus Hypericum (family Guttiferae), which was termed Diercao, Tianjihuang, Xiaoyuanbaocao, Sifangcao, and Qianchonglou, etc., and prosperously distributed from Liaoning and Shandong Provinces to the Southern Provinces of the Yangtze River. As a type of folk medicines, Hypericum japonicum was mainly applied to cure bacterial diseases, infectious hepatitis, acute and chronic hepatitis, gastrointestinal disorder, and internal hemorrhage, and was applied in detumescence for relieving pain. In Europe and Americas, H. japonicum also has been recorded as medical folk medicine. Modern pharmacological research showed that the extract of Tianjihuang had the potency of antibacterial, hepatoprotective, antitumor, and prevention of cardiovascular disease, which has already developed into injection, and clinically used to treat acute and chronic hepatitis. In this study, the aerial parts of H. japonicum were collected from Da-Bie Moutain, Qichun County, Hubei Province and Lu Moutain, Jiangxi Province, which were extracted with ethanol solvent by percolation procedure and then experienced isolations and pharmacological studies.In order to discover more structurally unique and biologically active metabolites from Hypericum japonicum, extensive chromatographies were performed such as silica gel column chromatography (CC), ODS reversed phase chromatography, Sephadex LH-20 chromatography, semi-preparative High-Performance Liquid Chromatography (HPLC), and semipreparative enantioseparation HPLC to afford sixty-eight secondary metabolites (including thirty-four new compounds). Comprehensive spectra like HRESIMS, NMR, UV, and IR were performed to determine their planar structures. The absolute configurations of these new compounds were established via the application of the modified Mosher's method, ECD of the [Rh2(OCOCF3)4] complex, X-ray diffraction (XRD) crystallographic analysis, quantum-chemical predictions (QCP) on I3C NMR data, and time-dependent density functional theory (TD-DFT) methods on ECD calculation. The new isolates possess the structure properties:(1) ten compounds belong to the chemical class of phloroglucinols with the form of filicinic acid fused sesquiterpenoid (we termed this type of phloroglucinols as filicinic acid-sesquitrpenoid adducts), possessing novel ring systems, namely,6/6/11, 6/6/7/5, and 6/6/10 ring systems, therein, six compounds were isolated as three pairs of enantiomers from their racemates and two compounds were separated as epimers; (2) sixteen compounds were isolated as eight pairs of enantiomers, and classified to the type of phloroglucinol-monoterpenoid derivatives. Thereof, three enantiomeric pairs of compounds possess unusual ring systems, viz.,2-oxabicyclo[3.3.1]nonane, pyrano[3,2-b]pyran, and benzo[b]cyclopenta[e]oxepine; (3) four compounds with unusual 5,5-spiroketal cores, viz. 1,6-dioxaspiro[4.4]non-2-en-4-ones, were also obtained as two pais of enantiomers via enantioseparation procedures; (4) one compound belongs to megastigmane sesquiterpenoid; (5) Two compounds were sorted into new pyranones; (6) one compound was regarded as a new lignin.As our exhaustive investigations on discovering more effective lead drugs/compounds from traditional Chinese medicine, several bioactivity screenings, such as cytotoxicity (including five common human cancer cell lines, viz., colon cancer cell line SW480, breast cancer cell line MCF-7, lung cancer cell line A-549, hepatocellular carcinoma cell line SMMC-7721, and leukaemia cell line HL-60, and a type of noncancerous human pulmonary epithelial cell line BEAS-2B.), BACE1 inhibitor assessment, inhibitory activities against NO production, inhibitory effects on the lytic replication of the Epstein-Barr virus (EBV), and inhibitory effects on kaposi's sarcoma associated herpesvirus (KSHV) infection were carried out to evaluate all new compounds. Intriguingly, the results revealed that most filicinic acid-sesquiterpenoid adducts exhibited potential anti-EBV activities, and especially, compounds 3 and 7 exhibited significant inhibitory effects on the lytic replication of the Epstein-Barr virus (EBV) in vitro, with EC50 values of 0.57 and 0.49?M and selectivity indexes of 52.63 and 106.78, respectively, approximately 5 times more potency than positive control, ganciclovir (EC50,2.86?M; selectivity index,104.50), which is a first discovery for phloroglucinols with the potential inhibitory activities on lytic EBV DNA replication. Furthermore, an initiate discussion on structure-activity relationships (SARs) was also proposed as follows:meroterpenoids featuring dextro rotation and a macrocyclic sesquiterpenoid moiety without an exo-ethenyl group (?5,14), and fusion of the isobutyryl-substituted filicinic acid via C-5'-O-C-1 could be structurally optimized lead compounds for designing potential drugs with enhanced anti-EBV activity. Regarding phloroglucinol-monoterpenoids, most of which exhibited potential activities on anti-KSHV, thereof, compounds 28 and 34 exhibited noteworthy effects with EC508.30 and 4.90 ?M and selectivity indexes 23.49 and 25.70, respectively. Meanwhile, it is a first assessment for phloroglucinols of finding the definite activities on anti-KSHV infections, which could provide a good clue to conceive or develop more selective lead drugs of K.SHV inhibitors.Secondary metabolites were reported to be mostly of phloroglucinols in Guttiferae. In the current study, we isolated three types of filicinic acid-sesquiterpenoid adducts with novel skeletons from H. japonicum, which means a first discovery in Guttiferae. Furthermore, we assessed, for the first time, the inhibitory effects of phloroglucinols on EB viral genomic DNA replication and lytic KSHV infection, and most of these phloroglucinols exhibited potential anti-EBV and anti-KSHV activities. Moreover, we further investigated the underlying molecular mechanisms through a small scale inverse docking and an assessment of compound-DNA polymerase interactions by microscale thermophoresis (MST) analyses in vitro. In addition, we acquired the enantioseparation of thirteen new pairs of racemates and each pair of them gave the approximate equal mass, which might provide a new clue for exploring nonenzymatic reactions in natural products.