Studies On Isolation And Identification Of Constituents From Turkish Galls And Their Pharmacological Activities

Turkish galls is produced by larvae of Gallic bee insects (Cynips gallae-tinctoriae Olivier), who are parasiticed in young branches of the Fagaceae Gallic tree (Quercus infectoria Olivier). Their female ovipositor stabs Gallic tree bud to make vegetation grow, in which its eggs are hatched into larvae. The larvae can secret enzyme-contained liquid, which can induce rapid transformation from starched to sugar in somatic cells of plants, and stimulate the plant meristem cells, then the neoplasm gradually grows into Turkish galls. Turkish galls is mainly produced in Mediterranean Sea, Turkey, Arabia, Iran, India, Greece and other places, especially in the largest production in Asia Minor; Turkish galls in Xinjiang province is mainly imported from Pakistan. Turkish galls has a long history of medicinal use, with a solid-gas, arresting seminal emission, astringing lung, the hemostasis. Previous studies in our laboratory indicated that its extract had a strong inhibitory function in vitro.At present, the study on Turkish galls is limited to its pharmacology and its extracts. Research showed that Turkish galls has a wide range of pharmacological effects, for example the crude extract had a better antimicrobial effect on Staphylococcus aureus, Salmonella choleraesuis and E. coli in vitro. At present, some researches reported the chemical composites of Turkish galls, but they were particularly prevalent in the small and medium-polarity polar pars. There is few study on its active composites. Based on the previous related studies, we systematically studied the chemical composition of Turkish galls, determined the structure of some monomer of and did some pharmacological experiments on them. Our study provides scientific evidence for the development and utilization of Turkish galls, and its quality standards.In this study, we overviewed the progress of chemical constituents and pharmacological research about Turkish galls. The chemical constituents of Turkish galls were separated by using silica gel, SephadexLH-20 column chromatography, preparative thin-layer chromatography and HPLC. After that, we obtained 12 compounds and their structures were identified by physiochemical properties and 1H-NMR, 13C-NMR, HMQC, HMBC and other spectroscopic methods. The compounds obtained were identified to be gallic acid (1);m-digallic acid (2); p-digalloyl acid (3); methyl gallate (4);ethyl gallate (5);1,2,3, 6-tetra-0-galloyl-β-D-glucose(6);1,2,3,4,6-penta-0-galloyl-β-D-glucose(7),1-0-galloyl-β-D-glucose(8),and1,6-di-0-galloyl-β-D-glucose(9), p-digalloyl acid, Ethyl gallate, 1-0-galloyl-β-D-glucose,and 1,6-di-0-galloyl-β-D-glucose were isolated from this plant for the first time. Some pharmacological activities of 1,2,3, 6-tetra-0-galloyl-β-D-glucose, gallic acid, methyl gallate, and ethyl gallate were studied, and the results of pharmacological experiments indicated that four kinds of compounds had antibacterial activity on four Gram-positive bacteria, three kinds of gram-negative bacteria and fungi Candida albicans (ATCC10231) strains in vitro. The inhibitory concentration was 7.27-29.09μg/mL. Among them, gallic acid compounds, methyl gallate, ethyl gallate against Staphylococcus aureus (ATCC25923), and Salmonella paratyphi piglets (S.C 500) had strong antibacterial activity, and the minimum inhibitory concentration was 7.27μg/mL. All compounds had strong anti-tumor activity to small mouse myeloma SP2 / 0 cells in vitro. They significantly inhibited the growth of mouse cell, and the effect of inhibition was concentration-dependent.The IC50 of compound1,4, 5, 6 were1.838mg/mL, 1.221mg/mL, 3.658mg/mL, and 3.035mg/mL, respectively. The acute toxicity test results showed that the LD50 value of compound gallic acid, ethyl gallate, and 1, 2, 3, 6-d-0-galloyl-β-D-glucose could not be obtained. The maximum tolerated dose in multiples of security were the 137,151 and 157, which were 100 times higher than the dose of adults commonly used (0. 08g/kg ? d - 1), so they are safe and feasible. LD50 of methyl gallate was 1.9004 (g / kg), and confidence limit of LD50 was 1.821±1.984 (g / kg). The average confidence limit LD50 was the (1.9004±0.0815) g / kg, which indicated that the compounds were slightly toxic, so attentions should be paid in its clinical application. Its mechanism of toxicity remains to be further studied.