| OBJECTIVEMorinda officinalis is one of the forth best-known herbs in China, expecially be deemed highly in SouthChina. Because of the harvest time was more then five years and it economical value was underestimated with no product accessory, the planting area and yield was reducing, especially it genuine producing areas such as Deqin and Gaoyao. For lifted the economical value of Morinda officinalis significantly by researching and developping a new drug, we should research the effective components deeply. Bajijiasu a dimeric fructose isolated from Morinda officinalis. Previous studies have demonstrated that Bajijiasu protects against the ischemia-induced neuronal damage or death. So this project mainly researched the best de-coloration conditions of bajijiasu from the polysaccharide of Morindae Officinalis purified on D-900ion exchange resins, to improve the industrial prepared process and quality control system, and the determining the nature and quantitative differentiation method of bajijiasu in Morinda Officinalis. At the same time, study the effect and pharmacological mechanism of bajijiasu on Alzheimer’s disease model on PC12cells induced by Aβ25-35and SD rats treated by Aβ25-35, to elucidate the effect and pharmacological mechanism of bajijiasu on Alzheimer’s disease, and provide basic information for the new drug development.METHODSStatic and dynamic adsorption experiments were carried out to study the de-coloration conditions of Morinda officinalis How polysaccharide on D-900Resins. The factors of temperature, amount of resins, adsorption flow rate and pH which affect adsorption process were investigated. And then purify the bajijiasu using the recrystallization method by different concentrations mixture of acetonitrile and water sevsral times from the ploysaccharide.The qualitative way was established by thin layer choromatography, and the quantitative analytical method by HPLC-ELSD, for measuring the bajiajisu of Morinda Officinalis How.Training PC12cell in vitro, the cell toxicity of bajijiasu is measured by MTT assay. Cells damage model preparated by Aβ25-35and use MTT and LDH method to detect cell survival rates, TUNEL test and flow cytometry to detect apoptosis rate. Using the fluorescence spectrophotometer detection of [Ca2+]i, mitochondrial membrane potential, reactive oxygen species (ROS), and Kit microplate method for the determination of intracellular SOD, MDA and GSH-Px. RT-PCR, Western-blot methods be used to detect mRNA and proten such as Caspase-3, BAX, p21, E2F1, CDK4, NF-κB and JAK2/STAT3. The effect and mechanism studies of baijijiasu which can protect PC12cell induced by Aβ25-35be detected by those methods.In the experiment we use acetylcholinesterase (AChE) which role can decompose acetylthiocholine (ATCh) and generate thiocholine (Thiocholine), thiocholine interact with chromogenic reagent DTNB rapidly will product a yellow substance which has a characteristic absorption at412nm. At the steady-state phase of enzyme-catalyzed reaction, the absorbance values can represent the initial reaction rate theory and it can evaluate the ACHE inhibition ability of bajijiasu in vitro.The dementia modes of SD rats’ were prepared by injected Aβ25-3510μg into bilateral hippocampal. High, medium, low-dose group of bajijiasu, morinda group, blank group, sham group and the positive group will be made at the same time. After25days’ Intragastrically administered, Morris water maze be used to test behavior, Kit microplate method for the determination of brain tissue super oxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione reductase (GSH-Px), and the content of acetylcholine (Ach), acetylcholinesterase (TchE) and Na+/K+-ATPase. HPLC-ECD was detected in brain tissue monoamine neurotransmitter levels and related indicators in hippocampal pyramidal cells in brain tissue and cerebral cortex neuron cells by the HE staining.RESULTSThe results demonstrated that the optimum adsorption parameters for polysaccharide of Morindae Officinalis was that the extracted solution at temperature35℃, adsorption flow rate1Bv·h-1and pH6.0. and then add five times amout of the mixture80% acetonitrile and water in the polysaccharide of Morindae Officinalis purified on D-900ion exchange resins, and then reflux and extract for30min and filter the hot solution in time, get the filter residue and add five times amout of the mixture55%acetonitrile and water refluxing and extracting for30min and filtered the hot solution and totally extracting for2or3times, gte the filtrate add the acetonitrile to the concentration of75%and then crystallize at4℃for24h, which can get the purity raw medicine of bajijiasu above95%.The qualitative way was established by thin layer choromatography is that weigh the cribble of Morindae Officinalis and extract in50ml80%acetone and water for1h, then extract the filter residue by15ml40%acetonitrile and water for0.5h, get the filtrate3μL onto the silica gel G thin layer plate, with n-butanol-ethanol-water-phosphoric acid (3:2.6:2:0.3) used as developer and10%sulfate-ethanol as color-developing agent at105℃for the spot of bajijiasu was clear and the bajijiasu solution1mg/ml of control.The quantitative analytical method of the bajijiasu in Morindae Officinalis by HPLC-ELSD with the column of Rezex RCM (Phenomenex,300mmx7.8mm) at35℃, the mobile phase was ultrapure water at0.6mL·min-1, and the temperature of drift tube was100℃with air flow rate of was2.0L-min-1.MTT assay results showed that:low-dose group of bajijiasu can promote cell growth, and there is a dose-effect relationship. Under the range of100μg·mL-1, the cells proliferation is accelerating increasing with the bajijiasu’s concentration. Over this concentration the cells growth began to slow inhibition, it shows that bajijiasu has a certain degree of cytotoxicity.MTT and LDH assay results showed that Aβ25-35interact with an IC50on PC12cells is21.46μmol·L-1, at the concentration, cells apoptosis rate of [Ca2+]i, mitochondrial membrane potential, reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione reductase (GSH-Px), and the mRNA and protein of Caspase-3, BAX, P21, E2F1, CDK4, NF-κB, JAK2/STAT5is significant different from the control group. While deal with the Aβ25-35and bajijiasu simultaneity, the above indicators are returned to normal levels, it suggests that in the effective dose range, bajijiasu has the ability of protection on Aβ25-35-induced PC12cells.The ACHE inhibition ability of bajijiasu in vitro depend on the reaction time, the inhibition rate was higher with the reaction time longer in50min, but out50min the inhibition rate kept invariant. And the inhibition ability depend on the concentration of the bajijiasu, the inhibition rate was higher with the concentration added. Treated by infusing the stomach with the bajijiasu25days, the Morris water maze test showed that the latency and total route of place navigation were longer than those of blank group, while the treated groups were shortened. The results of spatial probe test showed that the swimming time (27.36±3.38) s was significantly longer (P<0.01) in the first quadrant where the platfromhad been during the place navigation test than in other three quadrant. Compared with blank group, the swimming time (20.77±5.63) s was significantly shortened (P<0.01) in the first quadrant of the model group, while the high treated group (31.93±3.39) s was significantly longer (P<0.01), and the other treated groups were not obvious (P>0.05). Compared with model group, the swimming time of the treated group was significantly longer. The results show that neurotoxic mechanism of Aβ on neurons can lead the rats to be postnatal ability of learning and memory as the dementia symptoms, and the bajijiasu have the ability to recover that.The organ coefficients of model group were low compared with that of blank group, the results showed that the Aβ influence the function of brain, spleen, kidney and testis. While treated by bajijiasu the organ coefficients were higher showed that the bajijiasu have the therapy for invigoration and promoting the organ.Compared with model group, the levels of oxidative stress had been ameliorated after treated by infusing the stomach with the bajijiasu, showed that the levels of SOD, CAT and GSH-Px were higher, while the levels of MDA were lower in brain tissue of treated group, and the high dose group was more obvious (P<0.01). Also the activities of Na+/K+-ATPase and the levels of neurotransmitter were higher, while the inhibit rate of the neuron apoptosis was obvious lower of the treated group than that of model group.CONCLUSIONThe results are significant in researching de-coloration conditions. The retention rate of polysaccharide is reasonable and the process is stabilize and easy-handing. And then purify the bajijiasu from the polysaccharide by using different concentration of acetonitrile and water. The study is offer a good, stable, simple and save2/3time than the previous, and the process can be used for the industrial preparation production of bajijiasu.The thin layer choromatography can qualitativly and the HPLC-ELSD can quantitativly measure the bajiajisu of Morinda Officinalis.The study is obout the effect and pharmacological mechanism of bajijiasu on Aβ25-35-induced PC12cells at cellular and molecular level. We found that the bajijiasu can inhibit apoptosis. The mechanism is that the bajijiasu can decrease the [Ca2+]i which can lead the cells injured indirectly or directly, increase the mitochondrial membrane potential, level of the cells energy metabolism and oxidative stress. And inhibit the pro-apoptosis factors such as NF-κB, JAK2/STAT5and so on can cut off the necessary choice of cascade reaction apoptosis enforced by Caspase-3from activating. At the same time, it can correct the cell cycle to ensure the cells differentiate normally by activating the cyclin of P21, inhibiting the cyclin of CDK4, E2F1.The ACHE inhibition ability of bajijiasu in vitro showed that the bajijiasu have the ACHE inhibition ability with dose-effect relationship.The dementia modes of SD rats’were prepared by injected Aβ25-3510μ.g into bilateral hippocampal. Evaluate the effect on the model rats therapied of bajijiasu by testing behavior, oxidative stress, cholinergic system, energy metabolism, neurotransmitter and neuron apoptosis. The results showed that the bajijiasu can imporve the dementia symptoms, and the mechanism is that the bajijiasu can increase the level of oxidative stress and energy metabolism, imporve the demage cholinergic system and inhibit the neuron apoptosis.The innovative points of this study1The study showed the effect and pharmacological mechanism of bajijiasu on AP25-35-induced PC12cells at cellular and molecular level, it is that decrease the [Ca2+]i, increase the mitochondrial membrane potential and inhibit the pro-apoptosis factors such as NF-κB, JAK2/STAT5, correct the cell cycle to ensure the cells differentiate normally by activating the cyclin of P21, inhibiting the cyclin of CDK4, E2F1at the same time.2The results of the experiments in vitro and on animal showed that the bajijiasu can imporve the dementia symptoms, and the mechanism is that the bajijiasu can increase the level of oxidative stress and energy metabolism, imporve the demage cholinergic system and inhibit the neuron apoptosis. |
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