Study On Chemical Constituents And Biological Activity Of Glechoma Longituba And Euonymus Fortunei

In the human circulatory system,there is a dynamic equilibrium process between coagulation and anticoagulation under normal condition.Coagulation factors and platelets play important roles in the balance of this equilibrium process.Increased platelet aggregation or disturbance of plasma coagulation will trigger the tendency of"high coagulation"of blood,leading to embolic disease in the heart and brain,including coronary artery disease,angina pectoris,and myocardial infarction.The incidence of thrombotic diseases has increased year by year and has become one of the leading causes of death in low-and middle-income countries.Similarly,blood clotting dysfunction caused by blood coagulation factor deficiency or thrombocytopenia,such as hemophilia,also seriously affects people’s health.Some medicinal plants including panax pseudo,thyme,licorice,grape,and galangal with good hemostatic effects,have been promoted as clinic used hemostatic drugs.In the prevention and treatment of cardiovascular diseases,Chinese herbal medicine has demonstrated the synergistic effect of multi-component and multi-target,and has shown unique advantages in exerting the function of regulation.Therefore,screening cardiovascular drug leading compounds from medicinal plants has become a hotspot in recent research.The chemical constituents of Glechoma longituba（Nakai）Kupr.and Euonymus fortunei（Turcz.）Hand.-Mazz,two medicinal plants in Guangxi were systematically studied in this thesis,and their procoagulant/anticoagulant activities and antitumor activity were also evaluated.Sixty-one compounds were identified using infrared,UV-visible,mass spectrometry and nuclear magnetic spectroscopy techniques,hydrolysis,and other spectral and chemical methods,including twenty-nine triterpenoids and its saponins,six rosmarinic acids,one lignan,two phenylpropanoids,sixteen flavonoids,seven simple organic acid compounds,among them ten compounds are new compounds.The antithrombotic activity and antitumor activity of new compounds and some known compounds were evaluated using in vitro and in vivo models.Glechoma longituba（Nakai）Kupr.is a medicinal plant of family Labiatae,which is widely distributed in Fujian,Guangdong,Guangxi,Hunan,Zhejiang,Jiangxi,Taiwan,and Philippines.G.longituba is a characteristic“Lao-Ban”Yao medicine in Guangxi by the name of“Zuan-Di-Feng”,one of“seventy-two FENGs”in Yao medicine.It has the effects of heat-clearing and detoxifying,diuretic and stone-removing,dilated phlegm and swelling,promoting blood circulation,relieving pain and relieving itching.The chemical constituents and pharmacological activities of the family Labiatae in the world have been extensively studied and reported.However,the related research on the blood-staining plants is obviously lacking.The chemical constituents of G.longituba were systematically studied,and forty-one compounds,including ten new compounds and thirty-one known compounds were isolated,purified and identified.The new compounds I-1^I-8 possess a rare 11α,12α-epoxy pentacyclic triterpenoid lactone structure,and a structure fraction of glucuronic acid which is easily methylated under acidic conditions.Therefore,the four compounds of Compounds I-1 and I-3^I-5 are new artifacts obtained during the separation and purification process,and their corresponding unmethylated compounds I-1a and I-3a^I-5a are also new compounds.Compound I-9 is urethane triterpenoid saponin with an aldehyde group at C-28.Compound I-10 is a ursane type triterpenoid with 13α,27-cyclopropane skeleton.These skeletons haven’t been reported in this genus.The absolute configuration of the sugar of new componds,except for compound I-10 was determined by sugar hydrolysis in combination with ¹H NMR.Euonymus fortunei（Turcz.）Hand.-Mazz is a medicinal plant of family Euonymus,widely distributed in China,North Korea and Japan.E.fortunei is also one of the commonly used Yao medicines with the name of“Fei-Gui-Qing”in Guangxi,normally used for the treatment of lumbar muscle strain,rheumatic pain,bruises,various types of blood disorder（such as hemoptysis,blood stasis,irregular menstruation,traumatic bleeding）.The chemical constituents and pharmacological activities of the same family such as E.alatus,E.japonicus and E.europaeus have been studied at home and abroad.However,there are less than 60 compounds been isolated from E.fortunei in the previous chemical constituents’study to date.Furthermore,pharmacological activities of E.fortunei have also rarely been reported.We systematically studied the chemical constituents of the aerial part of E.fortunei,and twenty-two compounds were isolated and twenty of them were identified.These compounds are included fourteen flavonoids,three triterpenes,one lignin,and two phenylpropanoids.The aglycons of flavonoids are mainly kaempferol,a small amount of quercetin and apigenin.The literature reports that kaempferol-3,7-O-α-L-dirhamnopyranoside（I-43）,one of the most abundant compounds in E.fortunei,has anti-arthritic activity,which may be related to the plant used in China for the treatment of arthritis.Finally,based on the basic usages of G.longituba and E.fortunei as folk medicine plants,the anticoagulation of G.longituba and procoagulant of E.fortunei were evaluated in vitro.The results showed that compound I-1^I-5 in G.longituba could prolong TT and APTT in the blood coagulation activity of rabbits,and inhibit platelet aggregation in rabbits,indicating that these compounds have anticoagulant activity.In vitro cell experiments were performed to screen the antitumor activities of compounds I-1^I-6.As a result,it was found that these triterpenoid saponins derived from the n-butanol fraction showed weak in antitumor activity.However,compound I-10 from ethyl acetate fraction has a good inhibitory effect on a series of human tumor cells,including NCI-H460,T24,SKOV3,HepG2 and MGC-803,with an IC₅₀ value of4-11μM.In particular,the IC₅₀ values for NCI-H460 cells were 13.41±1.45μM（24 h）,4.11±0.49μM（48 h）,and 2.28±0.17μM（72 h）,respectively.The mechanism of action of this compound on NCI-H460 was further explored.Our results showed that compound I-10 exerted strong antiproliferative activity against NCI-H460 cells in a concentration-and time-dependent manner.Flow cytometry demonstrated compound I-10 arrested the cell cycle at G1 phase,induced cellular apoptosis,accompanied by ROS generation and mitochondrial membrane potential reduction.Mechanistic studies revealed that compound I-10 treatment inhibited IKKα/βphosphorylation and IκBαphosphorylation,which subsequently caused the blockage of NF-κB p65 phosphorylation and nuclear translocation.In conclusion,these results suggested that compound I-10 from G.longituba showed potential anticancer efects against lung cancer cells via inducing cell cycle arrest and apoptosis,at least partly,through NF-κB signaling pathways.