Ⅰ. Studies On The Bioactive Constituents Of Rubus Chingii Ⅱ. Studies On The Stability Of Stilbenoids From The Roots Of Caragana Sinica

The unripe fruits of Rubus chingii Hu(Rosaceae),referring to "Fu-pen-zi" in Chinese, have been used in China as a tonic medicine.The pharmacological studies indicated the EtOAc soluble fraction of the crude extract of the Rubus chingii has a strong phytoestrogenic activity and chloroform soluble fraction has a strong activity in inhibiting TNF-α-induced NF-κB activation.Thus,chromatographic separations of the two fractions of Rubus chingii were carried out under the guide of bio-assay in vitro respectively.22 pure compounds were isolated and 18 of them were determined using various modern spectroscopic analysis and chemical methods:p-hydroxybenzoic acid(R1),ellagic acid(R2),di-n-butyl phthalate (R3),di(2-ethylhexyl)phthalate(R4),ethyl gallate(R5),aromadendrin(R6),kaempferol (R7),quercetin(R8),quercitrin(R9),hirsutrin(R10),hyperin(R11),tiliroside(R12), cis-tiliroside(R13),2-hydroxyquinoline-4-carboxylic acid(R14),β-sitosterol(R15), daucosterol(R16),ursolic acid(R17),oleanolic acid(R18).Among them,R3～R6,R9,R13 and R14 were isolated from this plant for the fisrt time.Among 17 compounds isolated from EtOAc fraction,6 compounds can stimulate the proliferation of MCF-7 cells.Hirsutrin(R10),hyperin(R11) and ursolic acid(R17) can stimulate the proliferation of cells in lower concentrations and show inhibitory activity in higher concentrations.Ellagic acid(R2) and 2-hydroxyquinoline-4-carboxylic acid(R14) can stimulate the proliferation of MCF-7 cells in higher concentrations.From chloroform fraction,two active compounds ursolic acid(R17) and oleanolic acid(R18) were isolated.They can inhibit TNF-α-induced NF-κB activation with IC₅₀ values 5.79μg/mL and 6.23μg/mL,respectively.2-Hydroxyquinoline-4-carboxylic acid(R14),an unfrequent alkaloid in the plants, shows moderate phytoestrogenic activity in MCF-7 bio-assay.A capillary electrophoresis with electrochemical detection(CE-ED) method has been established to determine it in 8 Rubus chingii fruit samples collected from 8 different places.The result shows that R14 exists in all samples but varies in concentration.A GC-MS method had been established to study organic acids of the water-soluble fraction of crude extract.9 organic acids were determined. Primary study of chemical consistents of the roots of Rubus chingii Hu reveals that they are rich in triterpenoids.7 compounds were isolated and 5 of them were determined:β-sitosterol(R15),daucosterol(R16),ursolic acid(R17),euscaphic acid(R19) and 11α-euscaphic acid(R20).R20 was isolated from this plant for the fisrt time.The roots of Caragana sinica(Buc'hoz) Rehd.(Chinese name:Jinquegen) have been used in China as a folk medicine for the treatment of asthenia syndrome,vascular hypertension,leukorrhagia,bruises and contused wounds.In our previous study,we found that the EtOAc extract of the roots contained many oligostilbenes which had multifaceted bioactivities.The major stilbenoids are kobophenol A(1),carasinol B(2) and (+)-α-viniferin(3).They can stimulate the proliferation of osteoblasts and have been developed as a type of medicine for the treatment of osteoporosis.During previous studies,the content of some stilbenoids including 1 would change in the plant materials and extracts when stored as an alcohol syrup.This instability caused great difficulties for the quality control of this traditional Chinese herb medicine.In order to inspect the stability of oligostilbenes and feasibility of their structure modification,we studied kobophenol A(1),carasinol B(2) and(+)-α-viniferin(3) under different conditions with acid,base,light and hot,respectively.The result indicated that all 3 compounds are stable under base or light condition but unstable in acid and hot condition.Kobophenol A(1) can convert to carasinoi B(2) with a high proportion when refluxing in a hydrochloric acid solution and carasinol B(2) can convert to carasinol D(4) with a moderate proportion in the same condition.Kobophenol A also can convert to carasinol D when prolonging the reaction time.(+)-α-Viniferin(3) is also unstable when refluxing in a hydrochloric acid solution:it can convert with a high proportion to caragaphenol A(5) and a new stilbene caragaphenol B(6).