Optimizing Extraction Of Saponins Of Rhizoma Polygonati And Its Effects On Behaviors Of Rats With Chronic Stress Depression And Its Possible Mechanism

OBJECTIVE1. To select optimum extraction procedure of Saponins of Rhizoma Polygonati (SRP) and determine its content which was elute by macroporous resin (D101).2. Via establish model of rats with chronic stress depression. To explore the anti-depression effect and its mechanism of SRP, the ethology, the number of neuron in hippocampus and cerebral cortex and hypothalamus, expressions of brain derived neurotrophic factor (BDNF) and tyrosine kinase receptor (TrkB) in hippocampus and cerebral cortex of rats with chronic stress depression. METHODS1. The optimum extraction procedure with the content of SRP by orthogonal test design based on the extract time (2 hours), the extract frequency (two times) and solid-liquid ratio (1:15). The three main factors were established for the optimization factors, namely, A (temperature), B (ethanol concentration), C (pH). each of factors set three levels :A(40℃, 60℃, 80℃),B(70%, 75%, 80%),C(8, 7, 6). The content of SRP was determined by vanillin-perchloric acid colorimetric at 546nm with dioscin as control article. Ethanol extract was obtained at condition of optimization of the extraction. Alcoholic extract pass macroporous resin with distilled water 10%, 30%, 50%, 75% and 95% alcohol. SRP was gained by collecting and vacuum concentrating the eluent of 30%, 50% and 75% alcohol.2. The rats with chronic stress depression was made by the chronic unpredictable stress and solitary custody (d1^d21). Rats were divided into six guoups and were administrated intragastrically SRP (30, 60, 120 mg·kg^-1, d1^d21) and Fluoxetine (Flu) (2mg·kg^-1, ig, d1^d21) as control groups. And for the groups of normal and model group, rats were given an equal volume of vehicle (CMC-Na) at the same time.To record the weight of each rat in the experimental section first, 21th day and comparie the weight gain of rat in each group. The changes of behaviors in rat were observed by determing autonomous activity, sugar water consumption, immobility time in tail suspension and spatial learning and memory capacity.Using HE staining to observe changes of the number of neurons in hippocampus, cerebral cortex and hypothalamus of rats. The expression level of BDNF and TrkB in hippocampus and cerebral cortex was analyzed by immunohistochemistry determination. RESULTS1. The result of extractionVarious factors have different effects on the rate of the extraction of SRP via result of orthogonal experiment, RB>RA>RC. Namely, the Alcoholic density is Maximum factor, the temperature is more important than solid-liquid ratio. The best optimum extraction procedure of SRP: temperature (60℃), alcoholic content (75%) and pH (6).SRP was obtained by extracting with optimal extraction program and passing macroporous resin, its content is 51.8%.2. The pharmacodynamic action of SRP2.1 The effects of SRP on body weight of rats with chronic stress depression. On the 1st day the weight of all groups was no significance on the 21st day, the weight gain of model rats were markedly less than that of normal group, the weight gain of rats in three doses of the SRP all increased compared with model control.2.2 The effects of SRP on behaviors of rats with chronic stress depression(1) The number of autonomic activities of model rats decreased significantly compared with normal control, while that of SRP groups all increased.(2) The immobility time in tail suspension of model rats were obviously longer than that of normal group, and SRP groups all increased.(3) The consumption of sugar water of model rats less than normal group, while that of SRP groups rose.(4) In the water maze test, the escape latent period of model rats were longer than normal group on the fourth day, the explore time of original platform area of model rats were significantly shorter than normal group. The three doses of SRP could shorten the escape latent period and prolong the explore time of original platform area of rats with chronic stress depression. 2.3 The effects of SRP on the number of neuron in hippocampus and cerebral cortex and hypothalamus of rats with chronic stress depressionThe number of neuron in hippocampus and cerebral cortexa of model rats decreased significantly compared with normal control, while that of SRP groups all increased. The number of neuron hypothalamus of all group rats is few, but have many lymphocyte, astrocyte and gliocyte.2.4 The effects of SRP on the expression of BDNF and TrkB in hippocampus and cerebral cortex of rats with chronic stress depressionThe number of BDNF and TrkB positive cells and their gray scales in hippocampus and cerebral cortexa of model rats decreased significantly compared with normal control, while that of SRP groups and Flu group all increased.BDNF and TrkB positive cells were observed in each section of hippocampus and cerebral cortex, the most locatedⅡ^Ⅵlayers of cerebral cortexa, pyramidal cell and granular cell layer (gcl) of CA1 and CA3 in hippocampus.CONCLUSIONS1. Various factors have differenteffects on the rate of the extraction of SRP via result of orthogonal experiment, RB>RA>RC. Namely, the Alcoholic content is Maximum factor, the temperature is more important than solid-liquid ratio. The best optimum extraction procedure of SRP: temperature (60℃), alcoholic content (75%), pH (6), the extract time (2 hours), the extract frequency (two times) and solid-liquid ratio (1:15).2. The SRP could improve behaviors of rats with chronic stress depression. It suggested that SRP may prevent and improve depressive symptoms. 3. To play the role of antidepressant, SRP can be effective in restraining downregulation of neurons of hippocampus and cerebral cortex to express BDNF and TrkB in model rats with chronic stress depression. SRP can relieve the injury from the chronic stress stimulation by restraining downregulation of the expression of BDNF and TrkB.This is possible mechanism of antidepressant of SRP.