Immunohistochemical Localization Of Diosgenin In Dioscorea Zingiberensis C.H. Wright

In this paper the Dioscorea zingiberensis C. H. Wright, a special Chinese species for steroid drugs manufacturing, was used as study material. An indirect competitive dot enzyme linker immunosorbent assay method of diosgenin was set up by multiclonal antibodies which was obtained through preparating of immunogens of diosgenin and their immuno-effects analysis; Immunohistochemical localization and interspace distribution of diosgenin in Dioscorea Zingiberensis C. H. Wright was investigated. The main results were as follows:l.The succinylatcd diosgenin was conjugated to bovine scrum albumin(BSA) by dicyclohexylcarbodiimide (DCC)-method and l-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC)-mcthod separately. Rabbits were immunized with these two conjugates as immunogens to produce multiclonal antibodies, the multiclonal antibodies were evaluated by an indirect competitive Dot-ELISA. The results showed that the immuno-effect of immunogens made by DCC-method is better than that by EDC-method.2.Diosgenin-Bovine Serum Albumin conjugate was prepared by N,N'-dicyclohexylcarbodiimide method. After immunizing male New Zealand rabbits with this conjugate, we developed multi-antibodies against diosgenin and its glycosides. An indirect competitive dot enzyme linker immunosorbent assay(Dot-ELISA) method of diosgenin was set up. The detecting range for diosgenin is lμg/ml-250μg/ml, for glycoside, 0.1μg/ml-100μg/ml. By detecting glycosides to show the diosgenin content in Dioscorea zingiberensis C. H. Wright rhizome, we simplified the assay procedure. Compared with high-performance liquid chromatography, spectrometric and gravimetric assays, this assay is sensitive, simple and efficient for a large number of samples.3. The immunohistochemical localization of diosgenin in DioscoreaZingiberensis C. H. Wright was done after we developed multi-antibodies against diosgenin and its glycosides. The interspace distribution of diosgenin was examined by An indirect competitive enzyme linker immunosorbent assay (ELISA). We found that diosgenin was distributed in leaves, stem on the ground and rhizomorphous caudex under the ground. Immunohistochemical analysis revealed that diosgenin could be Synthesized in the cell of leaves, transported by phloem of caudex, and then storaged in rhizomorphous caudex under the ground at last. The results provided significant theoretical and technical basis for cultivation and synthetic exploitation of this kind of plant.