| Inula is an important genus of the Asteraceae family. There exist about 100 species in Europe, Africa, Asia, and predominantly, in the Mediterranean, with more than 20 species being distributed in China. Plants belonging to this genus show high diversity in their pharmacological effects, such as antitumor, anti-inflammatory, antibacterial, hepatoprotective, pre-diabetic and anti-diabetic activities. This genus has been reported to be a rich source of sesquiterpenes, especially those with eudesmanes, germacranes, pseudoguaines, guaianes, and xanthenes frameworks. As part of an ongoing research program aimed at the isolation, structural characterization, and pharmacological evaluation of new natural products, we have focused on the phytochemical investigation of four genus Inula plants, including two widespread species, I. japonica and I. lineariifolia, and two endemic species in China, I. hupehensis and I. helianthus-aquatica.Part 1. Phytochemical InvestigationBy detailed chemical investigations with various chromatographic methods, 209 compounds were isolated from the aerial parts of above four medicinal plants. Their structures were fully determined on the basis of spectroscopic analysis, including 108 sesquiterpenes, 31 sesquiterpene dimers, 16 monoterpenes, 4 diterpenes, 1 triterpene, 4 sterols, 9 apocarotenoids, 5 anthranilic acid derivatives, 11 flavonoids, 6 coumarins, 1 phenylpropanoid, 2 lignans, 3 alkaloids, and 8 other compounds.Among the 108 sesquiterpenes, 53 compounds were identified as new ones, which belonged to nine different frameworks, including eudesmanes, 1,10-secoeudesmanes, pseudoguaines, guaianes, xanthanes, germacranes, carabrones, elemanes, and eremophilanes. These nine frameworks contain even all the sesquiterpene frameworks discovered from Inula genus before. These results strongly support for the classical taxonomy of Inula from the view of phytochemistry and investigation of the relationships between chemical constituents and plant groups of Asteraceae family.Among the 31 sesquiterpene dimers, 29 compounds were identified as new ones. All of these sesquiterpene dimers are Diels-Alder-type condensation products of two sesquiterpenes, and exhibit diverse chemical structures, comprised by eudesmane, 1,10-secoeudesmane, pseudoguaine, guaiane, xanthane, and germacrane sesquiterpenes (moiety B) along with a guaiane sesquiterpene (moiety A). Except for IJ-56, the other dimers have a bridged ring system, a norbornene moiety, and their linkage mode is C-11-C-3?. Compound IL-21 was considered as the first exo-adduct of such xanthane-guaiane dimers from natural source. Compound IJ-56 was identified to be the first example of a sesquiterpene dimer comprised by 1,10-secoeudesmane and guaiane sesquiterpenes, which were connected by an unique valerolactone system (Linkage mode: C-13-C-3?).During the identification of these sesquiterpene and their dimers, six single-crystal X-ray structures with different frameworks, including two novel sesquiterpene dimers, were obtained. Furthermore, the modified Mosher reaction, CD spectrum, and computational approaches were performed to determine the absolute configurations of some novel sesquiterpenes and their dimers.In addition, one new thymol derivative, one new ent-kaurane type diterpenoid glycoside, and three new anthranilic acid derivatives were isolated from I. japonica. Aliphatic monoterpenes, apocarotenoids, anthranilic acid derivatives, coumarins, phenylpropanoids, lignans, and alkaloids were isolated from this genus for the first time, and most of the 122 known compounds also were reported firstly from this genus.Based on the great similarity between chemical constituents of I. hupehensis and I. helianthus-aquatica, the phytochemical analysis of I. helianthus-aquatica was realized by HPLC/DAD/ESI-MS~n and tracking isolation. Finally, 61 peaks were found out and 40 of them were identified by comparison with standard compounds from the aerial parts of I. hupehensis. The structures of other 21 unknown compounds were deduced from their UV and fragmentation behaviour. Furthermore, 10 of these unknown compounds were purified and identified by HPLC/DAD/ESI-MS~n-guided isolation, including 3 new compounds.Part 2. Study on the Fragmentation Behavior of Sesquiterpene DimersIn this part, electrospray ionization multi-stage mass spectrometry (ESI-MS~n) was performed to study the fragmentation behavior of 29 sesquiterpene dimers (IJ-34-IJ-54, IL-18-IL-25), which have been classified into five major core groups. Their fragmentation pathways haven't been previously reported. In positive ion mode, these five groups of sesquiterpene dimers showed very different characteristic ions and these fragments were used to deduce their mass spectral fragmentation mechanisms. Furthermore, the chloroform fraction of the 95% ethanolic extracts of the aerial part of I. japonica was investigated by using HPLC/DAD/ESI-MS~n. As a result, four 1,10-secoeudesmanolides (IJ-18-IJ-21) and 21 sesquiterpene dimers (IJ-34-IJ-54, IJ-47 and IJ-48 are inseparable) were unambiguously identified from 32 selected peaks by comparing with standard compounds. Furthermore, the other eight selected peaks (1, 4, 10, 11, 12, 19, 23, and 26) were tentatively indentified or deduced according to their ESI-MS~n data. Compound 1 and 23 were found to be sesquiterpenes and compounds 4, 10, 11, 12, 19, and 26 to be sesquiterpene dimers. In addition, the chemical structures of compounds 4, 10, and 11 were deduced by comparing their characteristic fragment ions. Given the important structural information of known and unknown sesquiterpene dimers in crude herbal extracts, this study is useful for identifying these five types of sesquiterpene dimers in crude materials rapidly and selectively.Part 3. Summarization on NMR Spectroscopy Rules of Sesquiterpenes and Their DimersIn this part, the NMR spectroscopy rules of sesquiterpene lactones and their dimers from Inula genus were summarized. A cyclic or bicyclic sesquiterpene nucleus coupled with the NMR characteristic chemical shifts of methyl groups were presented to deduce the frameworks of these sesquiterpenes for the first time.Part 4. Biological Activities Evaluation of Sesquiterpenes and Their DimersThese four plants studied in our course have long been used in Chinese folk medicine, most frequently employed for their anti-inflammatory and antitumor properties. Modern pharmacological study also has exhibited the diversified effects of their extracts, such as anti-inflammation and antitumor, but the active ingredient is not clear. Based on above, all the sesquiterpenes and sesquiterpenes dimers were tested for their anti-inflammatory and antitumor effects, including a series of biological evaluation of these compounds on NO production in LPS-stimulated RAW264.7 cells, NF-?B activation, and cytotoxicities against six human tumor cell lines. The results showed that even all the sesquiterpene lactones exhibited significant inhibitory effects, and the sesquiterpene dimers revealed stronger activities than sesquiterpene lactones. Among them, japonicone A (IJ-34) showed the most potent cytotoxicities against two human tumor cell lines, LOVO and CEM, with IC50 values of 0.478 and 0.002?M, respectively, which were even stronger than the positive control doxorubicin. According to their structural features, the structure-activity relationships were concluded, and these SARs would be meaningful for designing anti-inflammatory and antitumor agents in the future.In the previous study, japonicone A (IJ-34) exhibited strong inhibitory activity on the LPS-induced NF-?B activation, but the targets which are associated with its inhibitory effect remains elusive. Therefore, a panel of computational, chemical and biological approaches, including reverse docking method, surface plasmon resonance measurements, ELISA assays, and bioactive assays in vitro and in vivo were carried out and revealed that TNF-?was a crucial target of japonicone A (IJ-34). All the findings also support the anti-inflammatory activity of japonicone A (IJ-34) targeting of TNF-?for the inhibitory effect on NF-?B activation.Even the extract of Inula plants showed strong activity, the active ingredient is not clear. By detailed chemical investigations, this thesis further confirmed the effective fractions, and revealed effective substances for anti-inflammatory and antitumor effects. All these will provide important scientific foundation for rational development and utilization of resources of the genus Inula, and supplied information for the future design of anti-inflammatory and antitumor agents. |
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