Resource Of Shaanxi Huaijiao, Quality, Chemical Composition And Biological Activity Studies

As one of the most commonly used Chinese traditional medicine, Sophorae Fructus refers to the fruits from Sophora japonica L. which belongs to Leguminosae sp. After being dried, Sophorae fructus has the function of clearing heat-fire and cooling blood and hemostasis, and it is widely used to cure enteric fever hemafecia, bleeding hemorrhoids and vertigo red eye and so on. The fruit, as a kind of medicine, has been written in every edition of Pharmacopoeia of The People's Republic of China. Containing several ingredients of flavones and isoflavones, the medicine has shown an outstanding pharmacological action and has been widely used in clinic, which indicates its value in future research and application. Shaanxi Province is the main producing area of sophorae fructus. As the plant usually grows in a natural way with features of wide distribution and complicated sources, the related biological characteristics, sources, quality, ingredients and biological activity of the medicine still remain unclear. Therefore, the study of this medicine will be based on the existed results to deepen the research on its Physical effect of foundation and biological activity, so as to enlarge the field of application and provide scientific prove for the rational use of sophorae fructus. This thesis is to apply several technologies and methods including plant resources, phytochemistry, pharmacology, and molecular biology, in order to conduct a systematic research on Shaanxi sophorae fructus from four aspects of source, quality, chemical ingredients and biological activity. The research also includes the investigation on the wild sophorae fructus in Shaanxi. Through analyzing the quality of the plant from different sources, we tested the antiphlogistic hemostatic activity of the sophorae fructus flavonoids suppository, observed the extraction, separation, purification, and structure analysis process of sophoricoside, and found out its pharmacokinetic characteristics, the protection effect of genistein on hippocampal neurons, and the influence of quercetin on insulin fibrils. This thesis will discuss on the overall level of animal, cell and molecular level the function and medicine process of the active constituent of sophorae fructus and finally to provide experimental evidence for further development of Shaanxi's sophorae fructus.The contents and results are following: 1. Sophorae Fructus Source Investigation:The relevant literature and data of Sophorae Fructus were scrutinized and herbarium specimens were compared, interview and field survey methods were carried out. According to the investigation, the sources of sophorae fructus are widely distributed in Shaanxi Province and remain in wild state, which include natural herbs found mainly in farmland, uncultivated land, balk, or along the roads. Its associated plants are artificial cultivated crops, forest tree in villages, and a few herbs of M.sativa, artemisia gmelinii, and bothriochloa ischaemum. There are white and black Sophora japonica. L divided by their quality type in which the white one is considered the main seeding tree for sophorae fructus. In the sample area, the age of the tree is over twenty years, and the average production of each plant is over200kilogram. The natural reserved quantity of the fruit is estimated to reach2100ton per year. With the speed up of the urbanization, the original source of the fruit has changed in many ways including the transplant of a great amount of adult trees, sharply declining of the production (the annual declining rate is13%), the increasing of the varietas of the tree. These factors are affecting the quality of sophorae fructus and we need to make efforts to collect and preserve the fine quality seed resource of the plant and protect the existing sources.2. Quality Assessment of Sophorae Fructus:the application of the methodology of UV and RP-HPLC makes possible the determination of the total flavonoid content and the sophoricoside content as well as the examination of the ash content and the heavy metal content in the sophorae fructus samples elicited from different regions of Shaanxi province. The results of the determination and examination together with that of the investigation of the estrogen-like effect in mice formed the foundation for the quality assessment of sophorae. The research results show that the optimal process for the extraction of total flavonoids is adding eight-time-amount of ethanol in the reflux extraction for three times (one hour per time). In this case, the average yield of the total flavonoid content ranges from10.92%to20.18%, while the sophoricoside content is in-between4.21%and8.05%. The quality of sophorae fructus varies in different regions. Amongst the samples, the maximum sophoricoside content resides in the sophorae fructus in Bao Ji, while the minimum impurity and heavy metal exists in the sophorae fructus in Yichuan County in Yan'an. Comparatively speaking, higher level of total falvonoid content can be found in the sophorae fructus samples obtained in September from the gully regions of the Loess Plateau in Northern Shaanxi and of the hilly areas to the north of Weihe River, and in those obtained in October and November from the flatland and hilly regions to the north of Weihe River. Compared with the blank control group, the sophorae fructus extract shows certain weak estrogen-like effect in mouse. The number of the mouse's vaginal opening has been increased in the first three days of the experiment, and the proportion amounted to54%on the seventh day. Meanwhile, the weight of the mouse's ovary and uterus has also been increased significantly (*P<0.05or**P<0.01). The research has it that the sophorae fructus extract obtained from Yichuan County in Yan'an and Pucheng County in Weinan has a comparatively strong estrogen-like effect.3. Evaluation of Biological Activity of Sophorae Fructus Flavonoids Suppository:Sophorae Fructus Flavonoids Suppository (SFFS) can release on some extent the swelling auricle of the mice caused by xylol. specifically,0.60g/kg and0.30g/kg medicine were given to mice6d,9d,14d and showed an obvious restrain effect on the swelling auricle, and the result was different compared with the model (**P<0.01). It also reflected certain relations between the dosage and the effect. Every dose group caused the lightening of the filter paper granulation tissue (**P<0.01), in which the high dose groups have an inhibition ratio of over50%whose effect is close to positive drug aspirin group (0.50g/kg). The entire SFFS dose groups showed clear restrains effect for capillary permeability caused by acetic acid on mice cavum abdominis (*P<0.05or**P<0.01), and reflected certain dose-effect relationship, which is weaker than the aspirin bolt group. The SFFS in the medicine can release the foot swelling of the rats caused by carrageenin and means valuable in statistics as well compared to model group (*P<0.05or**P<0.01). The experiment took effect in2hours after the inflammation and the restrain effect became more obvious after4hours with an inhibition ratio of over40%. The medicine can also shorten the bleeding and coagulating time of the normal mice. The difference (*P<0.05or**P<0.01) between the group and the blank group indicates the medicine has the effect on stanching bleeding. The bolt can reduce the rectal exudates of rats caused by10%acetic acid, reduce the ulcer area, which showed an obvious difference with the model group (*P<0.05or**P<0.01). These experiments proved that the SFFS has pharmacological action to control intestinal ulcer and protect the wound, therefore, it can be developed as a new medicine for haemorrhoids.4. Separation, purification, testing, and pharmacokinetics study of sophoricoside: We established a method to measure the contents of sophoricoside in sophorae fructus under the chromatographic condition, the mobile phase was composed of methanol-acetonitrile-10mL-L-1glacial acetic acid(40:5:55); at a flow rate of1.0mL/min, The detector wavelength was260nm, and sample size was20μL, the lowest detectable concentration was0.015μg/mL,This method has been proved to be reliable and accurate. Extraction processes were studied by orthogonal design, the optimal process was performed by1.5-hour extraction for three times, with8-fold volume of95%ethanol (W/V), the raw sophoricoside will be obtained after alcohol extraction, water sedimentation, ethyl acetate purification and washing the1000g sophorae fructus in three batches of samples. the extraction rate of sophoricoside was1.326%,1.258%,1.203%, and the purity was85.21%,85.08%,84.46%respectively. Based on this experiment, we applied macroporous resin adsorption to purify the raw sophoricoside and tested the adsorption condition. As a result, the eluant potency is80%ethanol with the flow speed of2BV/h, and the sample solution potency is1.0mg/mL. Under the above condition, the purity of the raw sophoricoside was improved from84.92%to91.98%. Then we used methods of methanol hot dissolve, acetone precipitation, and repeated crystallization to extract sophoricoside monomer with the purity of over97%. Through physicochemical property and spectral identification, the monomer was proved to be5,7-Dihydroxy isoflavones-4'-O-glucoside which is sophoricoside. For the first time we established HPLC, a method of quantitative analysis to measure the amount of sophoricoside in the rats'blood plasma, then found out the rules of blood drug level change through tail intravenous injection. The results indicate that the prototype drug potency in female rats'plasma is higher than that in male rats at every sampling point-Cmax=2.531mg/L (female rats) and Cmax=1.648mg/L (male rats). Tmax of sophoricoside in female rats is in10minutes, while in male rats the time is7minutes. The uptake speed of female rats to the medicine are higher than the male. The metabolic process of prototype drug in blood plasma meets a criterion with one compartment model. The sophoricoside in rats has a half-life period of t1/2=55.70min (female) and t1/2=23.14min (male), which means the absorbing and metabolizing of sophoricoside in female rats are slower than that in male rats.5. The Antagonism and Mechanism Research on Genistein in Fructus Sophorae to Cure Damage of Hippocampus Neurons Caused by Aβ25-35:With obtaining the sophoricoside monomer at first place, hydrolyzing the50%alcohol (with10%HC1 inside) in90℃thermal reflux AW (acid water) for4hours, adjusting the pH of NaOH liquor of10mol/L to7, extracting the alcohol, washing, desiccating in80℃and making methanol recrystallization, we will get genistein monomer (5,7,4'-trihydroisoflavone through the physical and chemical properties and spectral identification and the HPLC purity test is above98%). As the research object to the hippocampus neurons of primary cultured rat, after using Aβ25-35to deal with hippocampus neurons and observing the antagonism of hippocampus neurons extracting form sophorae fructus to cure cytotoxic of Aβ25-35, it shows that genistein can effectively improve the morphology change of hippocampus neurons caused by inducing of Aβ25-35, reduce the damage of cell membrane, resist the activity reduction of hippocampus neurons caused by Aβ25-35, improve the survival rate of cells and reduce the apoptosis caused by oxidative injury. Comparing with the estradiol, both have the similar effect for resisting toxic effect of A(3. Genistein can restrain the enzyme activities of caspase-3, improve the impression of Inhibitory molecules apoptosis Bcl-2, and resist the change of ROS and MDA. The protection function of genistein may connect with its antioxidant effect, In addition, genistein can resist the downregulation of nAChR subunit a7mRNA and protein expression caused by Aβ, maintain the normal metabolization activity of hippocampus neurons, and thus exert neuroprotective effects.6. The Effect of Quercetin in Sophorae to restrain Bovine Insulin Protein Amyloid Fibrosis: The experiment applied bovine insulin as model protein to detect with ThT fluorescence, observe from transmission electron microscope, test the protein deposition rate, and conduct the hemolytic test and protein gel electrophoresis. Through vitro incubation, we studied the mechanism and effect of quercetin anti protein amyloid fibrils extracted from sophorae fructus. The experiment displayed that when insulin and quercetin (potency of25μg/mL or50μg/mL) were incubated together, the ThT fluorescence became weaker with the change of the dose and the S curve of insulin fibrils faded, which indicates the effect of quercetin to restrain the formation of insulin amyloid fibrils. However the quercetin of different potencies (10,25,50μg/mL) can damage the appearance of the insulin fibrils when processing it. Observed from the electric mirror, it became granulum and amorphous polymers without specific pattern. Bred by40μg/mL quercetin,13%insulin fibrils turned in to amorphous sediments within4hours. The deposition effect showed an obvious dependency on time and dosage. After the incubation, hemolysis caused by insulin fibrils of the same dosage was released with the dosage. According to the results of gel electrophoresis, insulin fibrils may lead to the gathering of spectrin proteins, while the quercetin weakened the formation of heavy polymer caused by insulin fibrils. The motivations of quercetin to develop its effect to resist amyloid fibrosis and destroy the fibrils formation might be hydrophobic interaction, aromatic amino acid overlap and hydrogen bonding.