Chemical Constituents Of Three Species Of Hypericum In Qinba Mountains And Their Bioactive

Natural products are an important resource of discovery for drugs or lead molecules.The plants of genus Hypericum have been of great interest due to their unique chemical composition and diverse pharmacological activities.The phloroglucinol derivatives and pharmacological activities,such as anti-inflammatory and anti-tumor activities have become research hotspots.In order to find structurally unique and highly active compounds from the Hypericum genus,and to provide valuable template structures for the development of new drugs and the search for new disease targets,this paper focuses on studying the chemical composition of Hypericum elatoides R.Keller,Hypericum przewalskii,and Hypericum hubeiense,which are characteristic plants in the Qinba Mountain area.Various chromatographic and spectroscopic techniques were used to systematically analyze their chemical composition,as well as to investigate the pharmacological activities and related molecular mechanisms of the isolated compounds.The main research results are as follows:（1）Chemical constituents of Hypericum elatoides and their bioactive（1）A total of 78 compounds were isolated and identified from Hypericum elatoides R.Keller,including 36 new compounds[31 PPAPs and 5 lactone compounds（1 semisynthetic lactone derivative）].New PPAPs compounds include various novel polycyclic compounds,such as 6/6/7/9/6 polycyclic compounds,6/8/6 novel skeleton compounds,B-type PPAPs compounds containingα-aminonitrile fragments for the first time,diamondane and diamondane-like compounds,and B-type PPAPs compounds.Two new skeleton compounds,δ-lactone ring compounds andγ-lactone ring compounds,were also identified.（2）The 6/8/6 polycyclic compounds isolated from Hypericum elatoides,which have a taxol-like skeleton,are well-known as anticancer drugs that inhibit microtubule depolymerization.Therefore,this paper further screened the taxane-like compounds isolated from Hypericum elatoides for their anticancer activity.Compound 2-2 was identified as showing significant inhibitory activity against human cervical cancer cells（C-33A）.Subsequently,the effects of compound 2-2 on microtubules in C-33A cells were investigated.The results showed that compound 2-2 induced abnormal spindle division by inhibiting microtubule depolymerization,thereby interfering with cell mitosis and inhibiting the proliferation of C-33A cells.It arrested C-33A cells in the S phase and induced apoptosis by activating Caspase-3 through the mitochondrial pathway.The inhibitory effect of compound 2-2 on C-33A cells was further validated in vivo.A zebrafish xenograft model of cervical cancer was established to investigate the effects of compound2-2 on proliferation,metastasis,angiogenesis,and brain tissue lesions of C-33A cells in vivo.The results showed that compound 2-2 significantly inhibited the proliferation and brain metastases of C-33A cells,as well as brain tissue lesions.It also significantly suppressed tumor-induced neovascularization in zebrafish.（3）The isolated compounds were screened for their inhibitory activity against nitric oxide（NO）using an LPS-induced BV-2 microglial cell inflammation model,and twelve compounds（2-10,2-11,2-12,2-13,2-18,2-19,2-20,2-23,2-27,2-28,2-42,2-44,2-45,2-48,and 2-78）showed inhibitory activity against NO.Among them,four compounds（2-11,2-20,2-27,and 2-45）exhibited significant inhibitory effects on NO(IC₅₀=5.44±0.86～5.74±0.27μM).Through structure-activity relationship analysis,it was found that the size of the anti-inflammatory activity in adamantane and homoadamantane-type PPAPs might be determined by the side chain isoprenyl group.In compounds withδ-lactone andγ-lactone ring structures,δ-lactone ring may be more favorable for anti-inflammatory activity.（4）In an H₂O₂-induced PC-12 cell model of oxidative damage,the neuroprotective activity of the isolated compounds under oxidative stress was evaluated using the MTT assay.Twelve compounds（2-1,2-4,2-20,2-25,2-27,2-29,2-30,2-31,2-45,2-46,2-49,and 2-57）showed significant protective effects against H₂O₂-induced PC-12 cell damage and significantly increased cell viability.Compound 2-27 increased cell viability from61.78%in the model group to 90.51%.Compounds 2-20,2-27,and 2-45 exhibited excellent dual anti-neuroinflammatory and neuroprotective activities.（5）Analyzing the structural characteristics of the above active compounds,we further investigated the potential molecular mechanisms of the unique lactone ring compound（2-10）and the PPAPs compound containingα-aminonitrile fragment（2-11）,aiming to discover novel anti-neuroinflammatory targets or mechanisms different from conventional ones using structurally unique compounds.The results showed that compound 2-10 could inhibit the expression of TLR-4 protein and prevent NF-κB nuclear translocation by inhibiting the phosphorylation of IKK and thus reducing the degradation of IκB,which prevented the release of p50/p65 and exhibited anti-inflammatory pharmacological activity.Compound 2-11 could concentration-dependently decrease the expression of i NOS and COX-2 proteins in LPS-induced BV-2 cells,thereby inhibiting the release of NO.It also exerted its anti-inflammatory pharmacological activity by inhibiting the expression of NF-κB protein in the cytoplasm and nucleus.（2）Chemical constituents of Hypericum przewalskii and their anti-neuroinflammatory activity.Ten compounds were isolated and identified from Hypericum przewalskii,including 2new compounds and 8 known compounds.Based on the analysis of compound structures,the new compounds were determined to be dihydrochalcone glycosides.The anti-neuroinflammatory activity evaluation experiment revealed that compounds 5-2,5-4,5-7,and 5-8 exhibited significant anti-neuroinflammatory effects(IC₅₀=0.61±0.12～1.28±0.15μM).Structure-activity relationship analysis indicated that the position of the benzoyl group on the sugar moiety of the new dihydrochalcone glycosides influenced their anti-neuroinflammatory activity.（3）Chemical constituents of Hypericum hubeiense and their anti-neuroinflammatory and neuroprotection activity.Twelve known compounds were isolated and identified from Hypericum hubeiense,ten of which were compounds belonging to the sesquiterpene class.The isolated compounds were evaluated for their anti-neuroinflammatory and neuroprotective activities.The results showed that compound 5-1 exhibited strong inhibitory activity against LPS-induced NO production in BV-2 cells(IC₅₀=5.96±0.33μM).Compounds 5-7 and 5-12demonstrated varying degrees of protective effects against H₂O₂-induced oxidative damage in PC-12 cells,with compound 5-7 showing the most significant activity（cell viability:84.62±2.07%）.