ABSTRACT
OBJECTIVE: To explore the protective effect of mullein glycoside polysaccharide of Cistanche deserticola Ma on PC12 nerve-cell model induced by D-galactose. METHODS: The cell survival rate was determined by MTT assays, which provided the basis for selecting mullein glycoside polysaccharide dose and estimated the dose and action time of D-galactose for inducing PC12 nerve cell damage model. After mullein glycoside polysaccharide incubation of PC12 cells, western blotting was used to detect the levels of CREB and p-CREB protein expression. ELISA Kit was used to detect the levels of cyclic adenosine monophosphate(cAMP), cAMP dependent protein kinase(PKA) and brain derived neurotrophic factor(BDNF). The content of MDA, activities of SOD and LDH were measured by their respective kits. RESULTS: (1)After the exposure of the PC12 cells to 16 g·L-1 D-galactose for 40 h, the cell survival rate was (46.67±6.59)%, which has a significant difference compared with the control group(P<0.05), indicating that successful cell aging model was established. (2)Compared with those in model group, mullein glycoside polysaccharide could significantly increase p-CREB expression in dose-dependent manner(r=0.989, P<0.01), content of PKA, cAMP, BDNF and SOD and decrease the levels of MDA and LDH(rMDA=0.875, P<0.05);(rLDH=0.834, P<0.05). However, blockers H-89 could significantly decrease p-CREB expression, PKA, cAMP, SOD and BDNF content(P<0.05), and increase the levels of MDA and LHD(P<0.05). CONCLUSION: The mullein glycoside polysaccharide of Cistanche deserticola Ma has obvious protective effect on PC12 nerve-cell damage model induced by D-galactose and its mechanism relates to the upregulation of cAMP/PKA/ CREB signaling pathways.
ABSTRACT
Objective To establish an optimum DNA extraction method for Chinese traditional medical herbs in order to meet necessary for DNA barcoding research.Methods Four Chinese traditional herbs, Glycyrrhiza uralensis Fisch, Phellodendron Chinensis Cortex, Cistanche tubulosa Wight and Cistanche deserticola Ma were chosen as the experimental materials, the DNA was extracted by 6 different kinds of DNA extraction method, including the improved method of high-salt combined low-pH,the improved method of SDS,CTAB method,PVP method,PlantZol Kit and Ezup Kit, the quality of DNA was investigated by ultraviolet spectrophotometry,agarose gel electrophoresis and PCR amplification by using specific primers of ITS2 and psbA-trnH. ResuIts The quality of the DNA was better than other four kinds of methods by the improved method of high-salt combined low-pH and Ezup Kit, the value of OD260/OD280 was between 1.7 ~1.9,the yield of DNA was the highest by the PlantZol kit , followed by the improved method of high-salt combined low-pH( P <0.05 ) , but the purity of DNA was poor by the PlantZol kit.The DNA electrophoresis tests showed that the DNA integrity of Glycyrrhiza uralensis Fisch and Cistanche tubulosa Wight were better with the improved method of high-salt combined low-pH, the improved SDS method, the CTAB method and the PlantZol kit.The DNA of Phellodendron Chinensis Cortex and Cistanche deserticola Ma were extracted by the six methods appeared diffuse status in the lanes.But only the improved method of high-salt combined low-pH could make the PCR amplification of the success rate 100% by using specific primers of ITS2 and trnH-psbA.ConcIusion The DNA extraction method of high-salt combined low-pH can be used to establish the Chinese DNA barcoding which has the advantages of lower cost, simpler procedure and less time.