Studies On Bioactive Polysaccharide Production In Liquid Culture Of Protocorm-Like Bodies From Dendrobium Huoshanense, An Endangered Medicinal Plant

Dendrobium species are used as the traditional Chinese medicine herbs for centries. Much attention has been focused on its resource regenesis, chemical components and pharmacological activities. Polysaccharides were the major active constituent from Dendrobium plants. Dendrobium huoshanense C. Z. Tang et S. J. Cheng, from which the Chinese medicine was most originally prepared, was studied in this paper. After the high expression system of polysaccharides from protocorm-like bodies (PLBs) in suspension culture was researched, water-soluble polysaccharides were extracted, isolated and purified from PLBs of D. huoshanense, with the advanced methods of isolation and purification, chemical and pharmacological instrument analysis technique of biomacromolecular. Some pharmacological action and mechanisms were investigated. The chemical properties and structural features of main polysaccharides of HPS-1B23 from PLBs of D. huoshanense (HPS) were identified. The main results were obtained as follows:An efficient procedure was developed for PLBs formation from stem segments of D. huoshanense and subsequent subculture for a long time. The highest frequency (79.9%) of PLB formation occurred on MS medium supplemented with 7.5 μmol L^-1 NAA and 0.5 μmol L^-1 KIN. Growth-regulator free MS medium at half-strength was suitable for PLB proliferation. The obtained PLBs were stable in growth, plantlet regeneration, polymorphic DNA of genome and polysaccharide synthesis including content and monosaccharide ratio.Pharmacological studies testified that PLBs have the same potential of synthesizing active polysaccharide as that of wild plants. The crude HPS from wild plants significantly stimulated interferon γ (IFN-γ) release in the supernatant of murine splenocytes and tumor necrosis factor alpha (TNF-α) release in the supernatant of peritoneal macrophages. The critical concentration of HPS was 800 μg mL^-1 for IFN-γ and 200 μg mL^-1 for TNF-α release. RT-PCR analysis indicated those responses were attributed to the up-regulation of the expression of theirown genes in cells. Different HPS fractions showed different capability in stimulating IFN-γ and TNF-α release. By DEAE-Cellulose chromatography, five fractions were all obtained from wild plants and PLBs, in which HPS-1 eluted by distilled water was the main constituent (79%). These analytical methods of polysaccharide bioactivities could be applied to monitor the liquid culture system of PLBs.The carbon and nitrogen metabolism was analysed and the relationship between PLB growth,...