Study On The Efficacy And Composition Of Tianzhu Powder In The Treatment Of Vascular Dementia

Objective:The model of vascular dementia rats was established by a permanent ligation of the bilateral common carotid arteries. The model was used to evaluate the pharmacological effects of Tianzhusan(TZS) on vascular dementia. Combined with network pharmacology, LC-MS and chromatographic separation, this study separated and purified the effective ingredients which were used to treat Vascular Dementia (VD) in TZS.Methods:1. FTIR was used to identify the compound type of TZS.2. The model of VD rats was prepared by a permanent ligation of the bilateral common carotid arteries.3. This study detected the indexes of food intake, aggressive behavior, tactile response, the sucrose preference degree and Morris water maze to evaluate the influence of TZS on the behavior of VD rats.4. The pathological changes in hippocampus of VD rats were detected by HE staining and Nissl staining. The mechanism of the changes was explored by immunohistochemistry with the expression of MAP-2 and SYN-1.5. The effective constituents acting on VD in TZS were predicted with network pharmacology. The effective constituents in TZS were inferred by LC-MS. Guided by the result of network pharmacology and LC-MS, the effective constituents in Rhizoma et Radix Trillii were separated and purified using the method of silica gel column chromatography, thin layer silica gel column chromatography, ODS reversed-phase chromatography, and semi-preparative liquid chromatography. The construction of the effective constituents were identified by LC-MS and NMR techniques.6. The content determination of effective constituents in Rhizoma et Radix Trillii was tested by HPLC-ELSD.Results:1. The FTIR results of TZS and its single medicine showed that they were in high similarity and all of them had glycosyl, carbonyl, hydroxy, methyl and amide.2. Compared with the control group rats, the response time of model group rats increased obviously, and TZS could reduce the response time. The scores of aggressive behaviour of VD rats in TZS group were higher than model group rats (P<0.01). However, the indexes of food intake and the sucrose preference degree had no difference among different groups. Morris water maze training found that the latency period of the model group rats was significantly longer than control group rats (P<0.05), showing a decline of learning and memory abilities of VD rats. Compared with the rats in the model group, the latency period of VD rats in Nimodipine and TZS group was shortened in the Morris water maze training (P<0.05). The rats in TZS group got a larger number of searching across the original platform position (P<0.05).3. HE staining showed that the nerve cells in hippocampus of rats in control group were tightly packed with intact structure. However, the nerve cells in hippocampus of rats in model group were irregular, and breakage were observed obviously. The number of intact cells in hippocampal of CA1 and CA3 region of model rats was significantly reduced (P<0.01 or P<0.05), compared with the control group rats. The number of intact cells in hippocampal of CA1 and CA3 region in Nimodipine group rats (P<0.01) and TZS group rats (P<0.05) were significantly increased compared with the control group rats.4. Nissl staining showed that the cells in hippocampus of model group rats were in scattered accumulation, and some of them were ruptured. The IOD of Nissl bodies in model group was significantly reduced (P<0.01) compared with control group. On the other hand, Nimodipine and TZS could increase the content of Nissl bodies in hippocampus compared with the rats in model group.5. Compared with the rats in control group, the expression area and IOD of MAP-2 were both decreased in hippocampus of CA3 (P<0.05), Dentate Gyrus (DG, P<0.01) and CA1 region of rats in model group. However, compared with the rats in model group, Nimodipine could significantly increase the expression of MAP-2 (P<0.05) in CAl, and TZS could also improve the expression of MAP-2.6. The expression area and IOD of SYN-1 in CA1 region in the model rats were higher than the rats in control group, but lower in CA3 region. Compared with the rats in model group, Nimodipine and TZS could increase the expression of SYN-1. TZS (P<0.05) could significantly increase the expression IOD of SYN-1 in DG region compared with the rats in model group.7.16 chemical ingredients acting on VD in TZS were predicted with network pharmacology.13 chemical ingredients were from Gastrodiae Rhizom and 3 chemical ingredients were from Rhizoma et Radix Trillii. The chemical ingredients played different roles in treating VD by acting on different proteins.23 chemical ingredients were found in TZS by LC-MS and 19 of them were from Rhizoma et Radix Trillii. Guided by the results of network pharmacology and LC-MS, two chemical ingredients were separated and purificated from Rhizoma et Radix Trillii. And they were respectively identified as polyphyllin VI and Pennogenin-3β-O-α-L-rhamnopyranosyl-(1→4)-[O-α-L-rhamnopyranosyl-(1→2)]-O-β-D-glucopyranoside.Conclusion:TZS can effectively decrease the damage in brain tissue of VD rats caused by cerebral ischemia. TZS can reduce the rupture and swell of nerve cells in hippocampus and arrange the cells closely and regularly. TZS can also protect the brain by increasing the content of Nissl bodies and promoting the synthesis of neurotransmitter.The injure of nerve cells can change the expression of MAP-2 and SYN-1. TZS can improve the expression of MAP-2 in hippocampus of VD rats caused by cerebral ischemia. TZS can also promote the development of neurocyte and the regeneration of axon, and it can promote the repairing of the damaged tissue in hippocampal of VD rats. And then, it improves the learning and memory abilities of space in the water maze experiments. Through regulating the expression of SYN-1 in the CA3 and DG region, TZS can increase the content of synaptophysin, reduce the injure of brain tissue caused by ischemia, and furtherly improve the learning and memory ability of VD rats.FTIR of TZS showed that TZS contain many constituents with glycosyl and carbonyl, which are inferred as saponin and flavonoids (or flavonoid glycosides). The constituents in TZS can be accurately predicted and identified with the guidance of network pharmacology and LC-MS. Then the effective constituents of TZS will be discovered efficiently by considering the structural features of constituents and designing a reasonable purification process.