| Objective:1. To detect the content of Pb, Cd, Cu, As and Hg 5 heavy metal elements and the ginkgo acid in the Ginkgo biloba exocarp extracts(GBEE) so as to provide a reference for evulating the safety of drug use and establishing the quality standard;2. To study the anti-tumor immune escape of GBEE so as to provide a scientific basis for the further revelation of the anti-tumor effect of GBEE.Methods:1. GBEE components analysisThe Ginkgo Biloba was collected from Taixing Jiangsu in 2013, GBEE was prepared according to the invention patent (Patent No.:201010251050.9). The content of ginkgolic acid was detected by the high performance liquid chromatography(HPLC) method; The Pb, Cd, Cu, As and Hg 5 kinds of heavy metal elements were detected by the inductively coupled plasma mass spectrometry(ICP-MS) and the atomic fluorescence spectrometry(AFS).2. GBEE anti-tumor immune escape assaysThe C57BL/6J mouse Lewis lung cancer(LLC) transplanted tumor model was established, the tumor bearing mouse were randomly divided into different groups:tumor control group, GBEE(50,100,200) in low, middle and high 3 dosage groups, with 10 mouse in each group, in addition to normal group that were not inoculated with tumor cells. During the experiment, the tumor volume in tumor bearing mice and the mice body weight and food intake were detected dynamically. After 15 days of drug administration, the tumors were stripped, the quality of the tumors were weighed and the tumor inhibition rate was calculated. H&E staining method was used to observe the infiltration of lymphocytes in tumor tissues; RT-qPCR method was adopted to detect the mRNA expression of Fas, Fas ligand(FasL) and decoy receptor 3(DcR3) in tumor tissues; Flow cytometry was used to detect the proportion of regulatory T cell(Treg) in the spleen and tumor tissue of mice. The ELISA method adopted to detect the expression of transforming growth factor beta 1(TGF-β1) in tumor tissue.Results:1. The results of component analysis showed:GBEE was prepared by water extraction and alcohol precipitation of Ginkgo biloba that collected from Taixing, Jiangsu, and the yield ofwhich was 2.05%. HPLC results showed that GBEE did not contain any compent of ginkgo acid. ICP-MS/AFS results showed that the content of Pb, Cd, Cu, As and Hg in GBEE were all lower than the limit of the 2010 edition of Chinese Pharmacopoeia.2. The results of anti-tumor immune escape showed:The body weight of each group increased with the increase of tumor volume; Compared with the control group, GBEE (50,100, 200 mg·kg-1) groups all improved the food intake of mice in different degree; After 15 days of drug administration, the tumor mass of GBEE each group were all lower than the control group (P<0.01 or P＜0.001) and the inhibition rates were 32.33%,34.79% and 46.89%, respectively. H&E staining results showed that, compared with the control group, the middle and high dose groups saw more lymphocytes infiltrating in the tumor stroma; lymphocytes of GBEE low dose group distributed scatterly in the tumor space. RT-qPCR results indicated that, GBEE middle and high dose groups could significantly upregulate the Fas mRNA expression rate of tumor cell (P<0.05 or P<0.01), GBEE high dose group could significantly downregulate the FasL mRNA expression rate (P<0.01) and the DcR3 mRNA expression rate (P<0.05). Compared with the control group, GBEE each dose group could significantly decrease the proportion of Treg cell in spleen of mice (P<0.05 or P<0.01), GBEE middle and high dose groups could significantly decrease the proportion of Treg cell in tumor tissue of tumor bearing mice (P<0.05 or P<0.01), GBEE each dose group could significantly decrease the expression of TGF-β1 in tumor tissue (P<0.05 or P<0.01).Conclusions:1. GBEE prepared from Taixing, Jiangsu did not contain any component of ginkgo acids; the contents of Pb, Cd, Cu, As and Hg 5 kinds of heavy metal elements were all complied with the limited edition of 2010 edition of the Chinese Pharmacopoeia.2. GBEE could interfere with the immune escape of LLC cell, whose mechanism may be related to regulating the tumor itself Fas/FasL system and DcR3 pathway, reducing the ratio of Treg cell in the immune system and the tumor microenvironment. |
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