Applied Basal Studies On Epimedium Plants And Rabdosia Rubescens (Hemls.) Hara

The aim of natural products chemistry is to discover new biologically activecompounds, to combine biologically active compounds and look for suitable productionmethod. In this thesis, we we made a thorough pharmaceutical-chemistry investigationand study on the chemical constituents, production method and HPLC-NMRfingerprints of the flavonol glycoside active fractions of Epimedium, together with thethe production method of oridonin encapsulated in long-circulating liposome.1. the applied basal studies of Epimedium plantsThe aerial parts of Epimedium plants (Berberidaceae), which have long been usedas a tonic, aphrodisiac and antirhermatic in chinese medicinal medicine under the name"yinyanghuo", have been shown to be rich of flavonoid glycosides with aγ,γ-dimethylallyl group. Much work has benn done on the chemical constituents,pharmacological effects, new medicinal plant resources and quality assessment ofEpimedium plants. But few work has benn done on the systemic production method,active fraction fingerprints and completely NMR assignment of chemical constituents.Moreover, only 20 of the toally 50 species have been studied and the chemicalconstituents of the same speciess may be biggishly changed with the change ofproducing area. So it is necessary to make continuously applied basal study on the theEpimedium plants. Our short-term achievements are established as follows:Two novel flavonol glycosides, 3"'-carbonyl-2"-β-L-quinovosyl icarisideâ…¡and3'"-carbonyl-2"-β-L-quinovosyl icariin, together with known flavonol glycosides,icarisideâ…¡, icariin, epimedin K, epimedo-koreanoside I, caohuoside A, was isolatedfrom the flavonol glycoside active fractions of the aerial parts of Epimedium koreanum(producing area: Dandong, Jilin, China). The structrue of the first new compound waselucidated as 4'-Methylether, 3-O-[3-one-β-L-quinovosyl-(1→2)-α-L-rhamno-pyranoside], 5,7-dihydroxy-2-(4-hydroxyphenyl)-8-(3-methyl-2-butenyl)-4H-1-benzopyran-4-one. The structrue of the second new compound was elucidated as 4'-Methylether, 3-O-[3-one-β-L-quinovosyl-(1→2)-α-L-rhamnopyranoside], 7-O-D-glucopyranoside, 5,7- dihydroxy-2-(4-hydroxyphenyl)-8-(3-methyl-2-butenyl)-4H-1-benzopyran-4-one.On this basis, we discussed the establishment of HPLC-NMR(¹³CNMR,¹HNMR) fingerprints of active fractions and reckoned (deduced) thecomputing formula of active fractions. Knowing the quantitative content of icariin, wecan directly calcaluted the the quantitative content of other main compounds from thequantitative ¹³CNMR or ¹HNMR fingerprints.After making optimum choice between Diaion HP20 and Sepabeds SP825macroporous absorption resin, we finally selected HP20 macroporous absorption resinand cc (silica gel) to isolate and purificate Epimedium koreanum and optimized thetechnological conditions of the whole isolation and purification process together withthe former treatment method. Using only one extraction solvent (EtOAc: 95ï¼…EtOH10:1), the icariin content in the silica gel active fractions can be heightened to two ormore times than that in the resin active fractions. Through macroporous absorption resinand silica gel, we gained series products of diffient recovery ratios and different icariincontents. Using HPLC methods, the content of the two selected standards, icariin(themain compound) and icarisideâ…¡(one of the lesser compounds which had the longestretention time), was exactly determained. On all this basis, the HPLC-NMR fingerprintsin relation to icariin was establisted, among which the ¹³CNMR quantitative fingerprintswas established for the first time in China.Epimedium sagittatum Maxim. Var. glabratum T. S. Ying is a species with a closerelationship with Epimedium sagittatum which is an important medical species. Itsmedical use has not been officially developed and its chemical constituents has not beenstudied to date. Studies on the chemical constituents of rhizome of this plant resulted inthe isolation of eight known compounds, (7S,8R, 8'R)-3,3'-dimethoxy-4,4'-dihydroxy-9-acetoxy-7,9-exopylignan, daucosterol, epimedoside C, 2"-O-rhamnosyl icariside A,icariin, epimedoside A, epimedin C and rouhuoside. Studies on the chemicalconstituents of leaves of this plant resulted in the isolation of four known compounds,sutchnenoside A, thalictoside, icariside D₂ and kaemferitrin. The stereo-structure of(7S,8R, 8'R)-3,3'-dimethoxy-4,4'-dihydroxy-9-acetoxy-7,9-exopylignan in original literature was wrong and too little NMR data was offered. Sutchuenoside A was anflvone which was found to have inhibitory effects on three different activities of HumanImmunodeficiency Virus-1 Reverse Transcriptase, its sturcture have not completelyassigned. Using a combination of 1D and 2D NMR techniques, their ¹H and ¹³C NMRspectra were completely assigned and analyzed.Icarisideâ…¡, icariin and epimedin C were three important flavonoid glycosidesisolated from different Epimedium speices. Using a combination of 1D (¹H, ¹³C NMRand DEPT) and 2D NMR (¹H-¹H COSY, HSQC and HMBC) techniques, their ¹H and¹³C NMR spectra were completely assigned and analyzed. We studied the difference andsimilarity of the linkage of the three flavonoid glycosides, synthesized the basic NMRcharacter of flavonoid glycosides with aγ,γ-dimethylallyl group at 8 position, made upthe ¹HNMR data of sugar groups and the hydroxy of the sugar groups, summarized theNMR character which had not been noticed in earlier literatures and corrected thewrong data existed in earlier literatures.2. the applied basal studies ofRabdosia rubescensRabdosia rubescens, commonly known as "Dong Ling Cao" in China, is a traditionalChinese medicine used as remedies for various diseases. Oridonin, an ent-kauranediterpenoid first isolated from Isodon trichocarpus,is an main active component ofRabdosia rubescens. Oridonin has various pharmacological and physiological effects suchas anti-inflammation, anti-bacteria and anti-tumor effects, and has been used for thetreatment of human cancers, especially esophageal carcinoma and prostate carcinoma.But because of its poor watersolubility, perfect targeted drug delivery system has not beenexcogitated.Firstly, we use a simple method to achieve much oridonin. After decolourized byactive charcoal, the EtOH extraction of dried and powdered leaves was directlysubmitted to cc (silica gel) eluting with increasing proportions of Petroleumether-Me₂CO to stepply give a large number of oridonin crystals. During this process,other 8 compounds,β-sitosterd, Oleanolic acid, ursolic acid, ponicinin, rabdoternin A,rabdoternin B, wikstroemioidin B and lasiodonin acetonide were isolated. In which lasiodonin acetonide was a new compound and its structure was determined as 1α, 6β,7β,11β-tetrahydroxy-cyclic, 1,11-(1-methyl-ethylidene acetal)-7,20-epoxy-Kaur-16-en-15-one by 1D and 2D NMR techniques.Secondly, using an advanced method (proposed by Hope M J, et al.), oridoninlong-circulating liposome was successfluly developed. The efficiency of oridonin inliposome was more than 98ï¼…and the mean particle size of oridonin long-circulatingliposome was 120nm. The results of animal experiments showed that oridoninlong-circulating liposome has higher antitumoral activity and lower toxicity than themasculine standard...