Studies On Gingko Kernel Allergic Protein And The Allergic Mechanism

Taking the ginkgo kernel, Dafozhi cultivar, harvested from Taixing, Jiangsu province, as material, allergicity and the mechanism of ginkgo protein were studied. The research would provide some basic materials for the ginkgo kernel safety assessment and product development. The main findings were as follows:(1) The extract technology of protein was conducted after the comparing of material treatment and extract methds. pI of the crude protein was 4.62. It was also ascertained no cyanide by assessing the possible toxins.(2) Allergicity of ginkgo kernel protein was studied in mice and guinea pigs. There was no death with the thickest concentration of protein in acute toxicity with mice. Guinea pigs were sensitized orally at days 1, 3 and 5, and challenged intraperitoneally. Allergy symptoms were showed after guinea pigs were treated with ginkgo kernel crude protein. Swelling in foot pads and a positive reaction emerged. IgE and IgG level from guinea pigs treated with ginkgo kernel protein were higher than those from CK, extremely significantly. There was inflammatory cell soakage. Taking into account, ginkgo kernel protein may be allergic to guinea pigs.(3) Ginkgo kernel protein allergy mouse model was evaluated and on the basis, allergy mechanism was studied. The established animal model was as follows. With Kunming mice, 100 mg/mLof ginkgo kernel protein extract was exposed orally at 1,7,14 d to sensitize and 200 mg/mL was treated intraperitoneallyto challenge with 0.3 mg/10g weight of both dose. According to this mouse model, animals was conducted as 4 groups, negative control (Tris-HCl buffer), ginkgo kernel protein of thin concentration, ginkgo kernel protein of thick concentration and positive control (OVA). The result showed IgE and IgG level of mice from each group during the immune time. The antibody level from 3 protein groups all was higher than the negative control significantly. After challenged in vitro, degranulation of mast cell was observed in 3 protein groups. Challenge in vitro also could result to histamine release typtase activity increased. Histamine content in plasma increased. Histopatholgic result indicated inflammatory manifestation in intestines, livers and lungs of mice from two ginkgo kernel protein groups. It could be deduced ginkgo kernel protein could resultⅠtype allergy reaction and the mouse model could be used in allergy researh. (4) Allergens of ginkgo kernel protein were detected with Western blotting. With ginkgo kernel proptein extract as antigen, sera from mice immunized with the protein extract as antibody, western blotting was producted. There were 3 proteins in the gel, and the molecular 21 kDa,32 kDa,36 kDa. The purification technology and parameters for 32 kDa were researched with ion exchange chromatography and gel chromatography, detected with SDS-PAGE. From it ginkgo kernel allergic protein from the above technology was a glycoprotein with molecular 32.12 kDa and the ratio of protein and sugar was 20.56:1.(5) Biochemistry and characteristics of the allergy protein were studied. The protein was not stable to heat, but stable in acid, alkali and salt solution, and anti-digested. Such stability perhaps resulted to its allergicity.(6) Undirective ELISA was used to detect the IgE titer of mice immunited with ginkgo kernel protein as 1:6400. Double-antibodies-sandwich-ELISA was established to assess antigen, with it, the least quantity of ginkgo kernel protein was 1.87μg/mL.