| Part1Research on the separation of FRSH with inorganic ceramic membraneBackground:Rabdosia serra (Maxim.) Hara(RSH), a labiatae and perennial herbaceous plant, contains flavonoids, phenols and other chemical compositions. It is widely used for the treatment of acute hepatitis, enteritis and diarrhea in south China and provides main raw material for health care products. So, this plant is worthy of extensive study and development. However, existing preparation adopts the traditional water extract-alcohol sedimentation process; the process is complex and contains certain shortcomings. Application of membrane separation technology to the extraction process of traditional Chinese medicine (TCM) is a desirable choice and it has advantages of high efficiency, low pollution and low consumption of energy. Inorganic ceramic membrane has the characteristics of high mechanical strength and corrosion resistance; it has unique advantages on the extraction of traditional Chinese herb and purification in removing impurities. This study focused on the most optimal parameters for ceramic membrane microfiltration, it provides methodological support for the extraction and purification of total flavonoids from RSH.Methods:Water extract of RSH was filtrated through inorganic ceramic membrane filtration system with cross-flow microfiltration. the pore size of ceramic membrane, the total content of flavonoids, transfer rate and removal rate of solid impurities were measured, so as to determine the best condition for ceramic membrane microfiltration; the cleaning effect on membrane flux recovery was determined by measuring the flux recovery rate in each step; effects of six various temperatures on the components transfer rate, membrane flux, flavonoids content were measured, so as to determine the best microfiltration temperature; transfer rate and membrane flux of active ingredients with four operating pressure were investigated respectively to investigate the optimum operating pressure; water solution of RSH was filtrated under the determined condition of microfiltration, the transfer rate of flavones was evaluated, so as to determine the best washing condition.Results:1. The best ceramic membrane pore size:The filtrate rate of various pore size of ceramic membrane (0.1μm,0.2μm,0.5μm,0.8μm) was93%,92%,86%,90%and the total cumulative flavonoids transfer rate was82%,87.1%,82%and87.2%respectively, in addition to0.8μm membrane, the removal rate of solid impurities was about20%,0.2μm aperture in the process of the ceramic microfiltration membrane flux attenuation of the membrane with0.2μm pore is small and the membrane flux is the largest in stable condition.2. The optimum microfiltration temperature:the membrane flux is about300L·h-1·m-2when the temperature set at45℃-75℃25℃, the flavonoids transfer rate is about95%when the temperature was25℃-45℃.3. The best operating pressure:In a constant microfiltration temperature (25℃), flavonoids transfer rate was96%under0.15MPa.4. The determination of optimum washing conditions:The cumulative total flavonoids transfer rate was99.79%when washed twice with1.5times the amount of residual liquid.5. The influence of washing on the recovery of membrane flux:For the membrane with small pore (0.1μm and0.2μm), besides pure water, acid and alkaline cleaning membrane flux can be achieved about90%of the pure water flux, flux can be recovered to100%after soaking with NaClO and nitric acid overnight.Conclusions:microfiltration can be most efficiently obtained for FESH with a suitable membrane (0.2μm pore), at a suitable temperatyre (25℃), under a suitable pressure (0.15MPa) and washed twice with1.5times residual liquid. Ceramic membrane can effectively remove insoluble or macromolecular impurities in the water extract of RSH and it is suitable for the purification and refine of the total flavonoids in RSH. Part2Effect of flavonoids from Rabdosia serra (Maxim.) Hara on liver fibrosisBackgrounds:Liver fibrosis (hepatic fibrosis, HF) is a necessary stage to develop liver cirrhosis, the main causes of LF including liver inflammation caused by a variety of pathogenic factors and compensatory responses during the process of tissue repair after liver injury. During the course, the synthesis of extracellular matrix (ECM) is greater than the degradation of ECM, this response leads to the excessive deposition of ECM and the formation of LF. Studies have shown that pathological changes of liver fibrosis could be reversed or slowed down after effective treatment. So far, even many anti fibrosis drugs have been developed under the efforts of pharmacologists from all over the world, however, the ideal compounds/drugs have not yet been synthesed or found. In the present study, we aimed to identify effective natural ingredients for the treatment of liver fibrosis from the natural plants in China. Three different kinds of commonly used animal models of hepatic fibrosis were adopted and the protective effects of flavonoids from Rabdosia serra (Maxim.) Hara (FRSH) against acute liver injury were evaluated. Our research provided the experimental basis for its clinical application.Methods1. Healthy SD rats were randomly divided into6groups, normal control group, model group, Silibinin group (50mg/kg), FRSH group (9mg/kg), FRSH group (18mg/kg) and FRSH group (36mg/kg). 2. Animal models of liver fibrosis were constructed through ligation of common bile duct, intraperitoneal injections of pig serum or subcutaneous injection of CCl4、the levels of TBIL、DBIL、ALT、AST、HA、LN、PC-Ⅲ、TNF-α、ALB/GLB in the serum were detected, the activities or amount of SOD、MDA、GSH-Px、Hyp in the homogenate of liver tissue were analyzed, ELISA assay was used to identify the expression of MMP-1and TIMP-1protein in liver tissue, the structure of liver cell and the degree of liver fibrosis was observed by HE staining and the protein level of a-SMA was obtained by immunohistochemical method.Results:1. Effect of FRSH against liver fibrosis induced by Bile duct ligation (BDL) Compared with the Sham group, the levels of TBIL、DBIL、ALT、AST were increased significantly in the model group(P=0.000), Compared with the model group, levels of TBIL、DBIL、ALT、AST in the rats treated with FRSH were decreased significantly(P<0.05or0.01). Compared with the Sham group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01).Compared with the Sham group, the activities of SOD and GSH-Px were decreased in model group (P=0.000). Meanwhile, the content of MDA、TNF-α were increased significantly (P=0.000). Compared with the model group, the activities of SOD and GSH-Px were increased and the content of MDA, TNF-α were decrease significantly (P<0.05) in three kind of FRSH group. Compared with the Sham group, the protein expression of TIMP-1were increased (P<0.01), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.2. Effect of FRSH against liver fibrosis induced by porcine serum Compared with the control group, the levels of ALT、AST were increased significantly in model group(P=0.000), the injury of liver decreased the ratio of ALB/GLB(P=0.000). Compared with the model group, The index of liver function were decreased significantly in three kind of FRSH group(P<0.05or0.01). Compared with the control group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group (P=0.000), Compared with the model group, the index of liver fibrosis were decrease(P<0.05or0.01). Compared with the control group, the protein expression of TIMP-1were increased (P=0.000), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.3. Effect of FRSH against liver fibrosis induced by CCl4compounded other factors Compared with the control group, the levels of TBIL、DBIL、ALT、AST were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01), Compared with the control group, the content of PC-Ⅲ、HA、LN、Hyp were increased significantly in model group(P=0.000), Compared with the model group, the rats treated with FRSH were decreased significantly(P<0.05or0.01). Compared with the control group, the activities of SOD and GSH-Px were decreased in model group (P=0.000). Meanwhile, the content of MDA、TNF-α were increased significantly (P=0.000). Compared with the model group, the activities of SOD and GSH-Px were increased and the content of MDA、TNF-α were decrease significantly (P<0.05) in three kind of FRSH group. Compared with the control group, the protein expression of TIMP-1were increased (P<0.01), However, there is no marked alteration was observed concerning the content of MMP-1(P>0.05). Compared with the model group, the protein expression of TIMP-1were decreased (P<0.01) after FRSH treatment.4. liver HE staining showed that hepatic lobule structure was clear and intact in the normal control group, no cellular proliferation of the small bile ducts and fibrous tissue was observed as well; In model group, There was no discernable normal lobular architecture, obvious edema and focal necrosis were observed in liver cells, small bile duct proliferation with radial extension within the liver parenchyma could be seen under an electron microscope, fibrous tissue hyperplasia and inflammatory cell infiltration were formed around the bile duct, hepatic lobule structure in the silymarin groups was clear, however, lobules in focal liver cells exhibited spotty necrosis, the small bile ducts in the liver portal area were centered with mild hyperplasia, mild hyperplasia of fibrous tissue and a small amount of inflammatory cell infiltration could be observed. Based on these findings, we further investigated the protective effect of FRSH against liver injury in these animal models, we found that treatment with middle dose of FRSH attenuated the pathological characteristics of the liver cells, the hepatic lobule structure was still clear, small duct in portal area was suffered with mild hyperplasia and around with proliferating fibrous tissues.5. Immunohistochemical staining of a-SMA protein expression in the liver showed that a-SMA proteins in the liver tissue were mainly distributed in portal area. In normal control group, only a few positive expression of a-SMA were visible in liver tissue portal area; while, in model control group, a-SMA positive expression in the liver tissue area were greatly increased; a-SMA positive expression in the liver tissue area of silymarin group was less than that in the model control group; FRSH protected the structure of liver cells and attenuated the expression of a-SMA in the liver tissue.Conclusions:FRSH has a therapeutic effect on hepatic fibrosis in the animal model of common bile duct ligation, intraperitoneal injections of pig serum and subcutaneous injection of CCl4; liver functions and liver fibrosis index are remarkably improved in all cases, the protective role of FRSH may be related to its alleviation of oxidative stress and inflammatory response in rats. Part3The Preventive effects of flavonoids from Rabdosia serra (Maxim.) Hara on acute liver injuryBackground:Liver injury is a common disease in clinic, which refers to the hepatic lesion caused by invasion of external factors. According to its etiology, it can be divided into biological liver injury, autoimmune liver injury and chemical liver injury. Common causes of liver damage including drinking too much, critical fatigue or exhaustion, environmental pollution, alcoholic hepatitis and fatty liver disease. In recent years, with the enhanced rate of drug abuse, the incidence of drug-induced liver injury is increasing year by year. Prevention and treatment of liver injury have become a hot research topic and also the core of this thesis. We focused on the total flanovoids, which is an important extracted part from Rabdosia serra (Maxim.) Hara. We intend to examining the protective effects of FRSH against liver injury in various acute liver injury models, further study on the preliminary mechanisms will also be performed.Methods:1. Healthy mice were randomly divided into6groups, namely, control group, model group, Bifendate group (100mg/kg)、FRSH group(13mg/kg)、FRSH group(26mg/kg) and FRSH group(52mg/kg).2. liver injury models were constructed based on treatment with CCl4、BCG-LPS or Alcohol; Blood samples were taken to measure the activities of AST and ALT. The contents of MDA、GS、SOD、NOS in liver were measured by chemical colorimetry method; Pathological examinations were conducted to investigate the severity of liver injury.Results1. Effect of FRSH against acute liver injury induced by CCl4Compared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH (P<0.05or P<0.01). Compared with normal control group, the content of MDA was increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH downregulated the expression of MDA and enhanced the activities of GSH and SOD significantly (P<0.05or P<0.01).2. Effect of FRSH against acute liver injury induced by combined treatment with bccillus calmette guerin and lipopolysaccharideCompared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH and positive control group (P<0.05or P<0.01). Compared with normal control group, the content of MDA and NOS were increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH reduced the levels of both MDA and NOS, on the other hand, the activities of GSH and SOD were increased significantly after treatment with FRSH or bifendate group (P<0.05or P<0.01).3. Effect of FRSH against acute liver injury induced by alcohol.Compared with normal control group, levels of ALT and AST in the serum of rats in model control group were significantly increased (P<0.01); compared with model control group, the activities of ALT and AST in blood decreased remarkably and showed a dose effect relationship in each dosage group of FRSH (P<0.05or P<0.01). Compared with normal control group, the content of MDA was increased, while the levels of GSH and SOD in the liver tissue were decreased in model control group; both bifendate and FRSH downregulated the expression of MDA and enhanced the activities of GSH and SOD significantly (P<0.05or P<0.01).4. Histopathologic examinationLivers in the mice of normal control group mice were ruddy, luster and elastic, the structure of lobules was intact and normal, the cytoplasm of liver cells was homogeneous and the nucleus was round in shape. Cells around central vein were were basically complete, and neatly arranged as well. On the contrary, hepatocyte showed multiple spotty necrosis in the model group, the main pathologic changes including yellowish gray dot necrosis, slightly crispy texture; By light microscopy, the hepatocytes were necrotic with fuzzy boundary around the central vein, the cytoplasm was light color, the nucleus was shrink and the intercellular space was increased, around central vein, inflammatory cell infiltration is also evident. Dotted necrosis in the hepatic lobule and inflammatory cells infiltration was also observed in the positive control group; however, the changes described above were markedly abated. After treatment with FRSH, the lobular architecture was relatively complete and the liver shows only mild necrosis,Conclusions:FRSH has markedly protective effect on liver injury induced by CCl4or combined treatment with bccillus calmette guerin and lipopolysaccharide or alcohol, this effect may be attributed to the property that FRSH could improve the resistance to oxidative stress, reduce the cytoplasm membrane lipid peroxidation, so as to fix the cytoplasm membrane and reduce liver damage. |
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