Identification And Study On The Activity Of Natural Compounds From The Seeds Of Iris Pallasii Fisch. Var.Chinensis Fisch

Ma lin zi(MLZ) are seeds of Iris pallasii Fisch.Var.chinensis Fisch., MLZ is widely used in traditional Chinese medicine (TCM) for the treatment of such diseases as jaundice, secretary diarrh(o)ea, haematemesis, flooding, pharyngitis and carbuncle. MLZ is the main constituent of the well known TCM formula "shiliu wei malinzi wan". Because MLZ is rich in flavonoids and quinonoids that have extensive biological activities, it is generally regarded that these flavonoid and quinonoid compounds account for the so many biological activites of MLZ. Precise molercular pharmacological basis of MLZ is still under investigated. Aim of the present study is to isolate chemical constituents from MLZ, which will not only optimize systemalic single compounds purification method, help us to find new compounds from MLZ, our study also provide chemical basis for further pharmacological study of the multiple bioactivities of MLZ.In the present paper, crude extracts of MLZ was prepared by using continuously reflux with95%ethnol. The water soluble phase was extracted with different solvent got petroleum ether, ethyl acetate and water. The petroleum ether and ethyl acetate extracts were fractionated by silica gel column chromatography, which generated200fractions (MLZ1to MLZ200, from petroleum ether phase) and321frctions (MLZ201to MLZ521, from ethyl acetate phase). Similar fractions were mixed according to TLC results. The fractions were further fractionated by using preparative HPLC methods, determination the purity of compounds by analytical HPLC, and chemical structures of purified compounds were deciphered by using mass spectrometry (MS), nuclear magnetic resonance (NMR) and infrared spectrum (IR) methods. Five compounds ((-)-catechin, trans-s-viniferin, r-2-viniferin,2-methoxy-6-pentadecyl-1,4-benzoquinone,3-hydroxyirisquinone respectively) were isolated so far, the purities of the compounds are98%,100%,100%,99%,99%, respectively. Remarkable, except for3-hydroxyirisquinone, the other four compounds were proved to be the first time isolated from MLZ.The anti-cancer activity of the above five compounds were tested by using MTT method found that r-2-viniferin can resistance the HepG2cell line. Flow cytometry method was used for detecting the apoptosis, the mitochondrial membrane potential and intracellular reactive oxygen species (ROS) levels of r-2-viniferin. It showed that r-2-viniferin inhibited HepG2cell proliferation in a dose-dependent manner, compared with the control group, the ROS was increased and mitochondrial membrane potential was reduced. Therefore it can be concluded that induction of apoptosis of HepG2cells associated with cell cycle arrest atG2/M phase and increase ROS.