HPLC Fingerprint Data Establishment For Pueraria Lobata (Willd) Ohwi In DABIE Mountains And Study On The Protection Effects Of Its Phytochemicals Against Ethanol-induced Neurotoxicity

Herbal medicines have important roles in clinical therapy in Asian countries such as Korea, Japan, and China. Over the past decade, herbal medicine has become an increasingly important topic all over the world such as European Community and America. However, with the worldwide application of the Herbal medicines, the safety has been worried seriously. The reason lies in lack of the standard and method that evaluate the quality of the traditional Chinese medicines(TCM). Quality of many TCM is the cause of concern. At present, Fingerprint analysis of traditionalChinese medicines has been a way of identifying and evaluating the safety, stability and quality and has become the most effective method of controlling the quality of traditional Chinese medicines and natural medicines recognized internationally.As death report data of 2001 in America shown, the chronic causes of death related with alcohol lie in ahead, such as alcohol abuse, alcohol dependence syndrome, alcohol polyneuropathy, alcohol-induced chronic pancreatitis, alcoholic gastritis, alcoholic liver disease, et al. While ,the acute causes of death in second place is alcohol poisoning. Alcohol abuse and alcohol dependence have been a serious public health problem in modern world. Traditional Chinese medicines plays an important role in prevention and therapy of alcohol-related diseases.There is a long history that Pueraria is used to alleviate a hangover.Nowadays ,most of the research of alcohol-related disease concentrate on one or several components of traditional Chinese medicines.For example,Wang Qing-rui,et al concluded that total flavonoids of Pueraria has the inhibiting effect on nerve center of mouse.However,more and more people realize that thepharmacodynamic actions of traditional Chinese medicines depend on cooperation of all active components instead of one of the active chemical components in traditional Chinese medicines. Therefor,on the basis of establishing fingerprint of TCM,it is necessary to reveal the corresponding relation between characteristic absorption band and pharmacodynamic action ,that is spectum-effiency relation.lt is essential to construct the standard of controlling the quality of the traditional Chinese medicines and its treatment.lt is critical of the modernization and globlization of traditional Chinese medicines.PartiEstablishment of HPLC fingerprint data for PuerariaLobatafflilH) Ohwi in DABIE MountainsThe traditional herbal medicines (HM) and their preparations have been widely used for thousands of years in many oriental countries, such as in China, Korea, Japan, etc. However, quality control of oriental herbal drugs is more difficult than that ofwestern drug. As pointed in "General Guidelines for Methodologies on Research and Evaluation of Traditional Medicines (World Health Organization,2000)",Despite its existence and continued use over many centuries, traditional medicine has not been officially recognized in most countries. Consequently, education, training and research in this area have not been accorded due attention and support. The quantity and quality of the safety and efficacy data on traditional medicine are far from sufficient to meet the criteria needed to support its world-wide usage. The reasons for the lack of research data are due not only to health care policies, but also to a lack of adequate or accepted research methodology for evaluating traditional medicine. Pueraria Lobata^Rillti) Ohwi is one of the earliest medicinal plants used in traditional Chinese medicine. It has many profound pharmacological actions.Radix Puerariae is one of the most popular drugs to safeguard public health, commonly employed for acute febrile disease and used as an anti-inebriation agent for the management of alcohol abuse. At present, establishing the fingerprint data of tradition Chinese medicine hasbeen the most effective method of quality control of tradition medicine which is accepted world-widely.Chapter I HPLC fingerprint analyses ofPueraria Lobata(Willd) Ohwi This study is designed to develop fingerprint of Pueraria loata by HPLCIt could provide a method of quality control for Pueraria Lobata(H illd) Ohwi and an experimental basis for harvesting and exploiting the Pueraria Lobata(Willd) Ohwi in the experiment. Method:The samples were analyzed by HPLC on an Zorbax XDB-Cjg column, with mixture of methanol and water as mobile phase in a gradient mode at flow rate 0.5mL/min and detection at wavelenth of 250nm, column temperature of 25 V. Result:1. The method has good linear relationship, exactitude,stability and high recovery rate.2. There are high separation degrees among the flavonoids in Pueraria.HPLC Fingerprint show that there are 10 commonpeaks. The RSD% of relative retention time of different common peaks in different samples is less than 5%,while the average percentage of common peaks total area in total peak area is 95.68%,RSD% is 1.22%. Discussion1. The method of methanol-water gradient elution could be used in evaluating and controlling the quality of Pueraria LobataQRilU) Ohwi.2. It is an excellent way for controlling the qulity of traditional Chinese medicines to improve and stabilize crude medical material and to establish HPLC fingerprint of TCM.Chapter II establishment of HPLC fingerprint data for PuerariaLobatatyilld) Ohwi in DABIEMountainsThis study is designed to develop HPLC fingerprint data ofPueraria Lobata(Wrilld) Ohwi with different seasons,differentparts,different annual rings and different extracting methods thatcould provide a method of quality control for PuerariaLobata(Willd) and an experimental basis for harvesting andexploiting the Pueraria Lobata(Hilid) in the experiment.Method:To estabilsh fingerprint of Pueraria loata with differentseasons,different parts,different annual rings and differentextracting methods by methods of Chapter I.Result:1. The HPLC fingerprint of Pueraria Lobata(RI lid) Ohwi in different seasons is relatively similar .All the same common peaks exist.But the relative peak area of different common peaks is different,and the range of RSD% is 0.92%-17.42%; In spring,there are the most pecentage of common peaks area and most average of purerarin content ,that is 94.73% and 4.01%.2. Comparing the HPLC fingerprint of peri-root vine and apo-root vine with that of the root ,there are certain similarity. All the same common peaks exist.But the relative peak area ofdifferent common peaks is different,and the range of RSD% is 0.58%-16.09%. Total pecentage of common peaks area and average of puerarin content in root is obviously more than other parts of Pueraria Lobata(fHUd) Ohwi,that is 95.68% and 4.78%.3. The HPLC fingerpring is relatively similar between 2 years and 3 years of Pueraria Lobata(WHid) Ohwi. All the common peaks exist.But the relative peak area of different common peaks is different,and the range of RSD% is 0-14.58%.Total pecentage of common peaks area of 2 years' Pueraria Lobata(Willd) Ohwi is more than that of 3 years';While the average of puerarin content of 2 years'Pueraria Lobata is less than that of 3 years'.4. HPLC Fingerprints are very similar between Pueraria Lobata(7iI lid) Ohwi in southern hillside and that in northern hillside. All the same common peaks exist. But the relative peak area of different common peaks is different, and the range of RSD% is 0-7.07%.Total percentage of common peaks area of Pueraria Lobata (Willd) Ohwi in southern and northernhillsides respectively are 95.30% and 96.02%; While average of puerarin content between them is different deeply ,that is 5.70% and 3.87%.5. There are certain difference among HPLC fingerprint of Pueraria Lobata (Willd) Ohwi from different extracting technologies.The common peaks is not completely same .The extractive by ethanol has the most common peaks total area,puerarin peak area and puerarin content.Discussion:1. The quality of Pueraria Lobata (Willd) Ohwi is different with different seasons. The best harvest season is in spring.2. The value of different parts ofPueraria Lobata (Willd) Ohwi is obviously different .In the process of exploiting the natural resources, radix is regarded as chief available material.3. The growing time has much influence on the puerarin content. Pueraria Lobata (Willd) Ohwi that has long growing years is chosen to be material in order to improve the availability of the natural resources.4. The puerarin content of Pueraria Lobata (Willd) Ohwi fromsouthern hillside is obviously more than that from northern hillside. Pueraria Lobata (Wi 1 Id) Ohwi from southern hillside is chosen as material which will be exploited in order to improve the availability of the natural resources.5. The method of extraction by dehydrated alcohol is a better way of establishment of HPLC fingerprint and processing of the natural resources.Chapter IE Research on resource availability in traditional processing of Pueraria by HPLC fingerprintThe utilization of natural resources is related to national economy , people's livelihood, resources saving and social harmony. At present, the ecology is in danger. The study is designed to research the natural resources availability intraditional processing of Pueraria by HPLC fingerprint in older to provide scientific evidences for reasonable utilization and exploitation of Pueraria Lobata^Hilid) Ohwi.Method:Develop the HPLC fingerprint of the wasteful serous fluid and pueraria starch in the traditional processing of Pueraria by the method of Chapter I.Results:There are 10 common peaks in the HPLC fingerprint of Pueraria in DABIE Mountains, while there are 8 common peaks in the HPLC fingerprint of the wasteful serous fluid in the traditional processing of PuerariaThe percentage of common peaks area of the wasteful serous fluid reaches 97.83% and that of pueraria peak area reaches 71.08%.Howerver, there is only pueraria peak in HPLC fingerprint of pueraria starch, and the percentage of pueraria content is little, only 0.05%.Discussion:It is unscientific for pueraria utilization in the traditional processing of Pueraria.The product of this processing is pueraria starch which has only one component-puerarin that is litte.The composition of the wasteful serous fluid is similar to that of the Pueraria. So the wasteful serous fluid is rather valuable. And the discarding pueraria fibers is good paper-making materials and ecological building materials. We should utilize and exploit the natural resources on basis of scientific development and circular economicsPart II Study on the protection effects of PuerariaLobata(Wilid) Ohwi' s phytochemicals againstethanol-induced neurotoxicitychapter I Study on ethanol-induced neurotoxicityeffectPrenatal exposure to ethanol can result in Fetal AlcoholSyndrome (FAS),which is characterized by facial dysmorphology, growth retardation, CNS developmental anomalies and behavioral and cognitive deficits.Nervous system especially hippocampus tissue in development stage is sensible to ethanol-induced neurotoxic effect particularly. The method of whole embryo culture or embryo hippocampus cell culture is used in research of its mechanism in vitro. The ethanol of toxic dosage will affect the nervous system in development stage by the way of the influence on proliferative viability of nerve cell .Ethanol is the micromolecule that provoke cell stress reaction which adjust the oxidative process of boby quickly and alter the expression and physiological activity of some functional protein.This study is designed to research influence of ethanol on proliferation viability of hippocampus cell and the adjustment of expression of Hsp 70 and SOD by ICR fetus mouse in the pregnance of 18 days in the sensitive development period of hippocampus tissue in order to research mechanism of ethanol-induced neurotoxic effect. Method:Hippocampus cultures were prepared from gestational day 18mice. Cell viability was measured by MTT assay. RT-PCR and Western Blot were employed to determine mRNA and protein expression of HSP70 and SOD. Results:1. As measured by MTT assay, Influence on proliferation viability of hippocampus cell by ethanol had a better dose-effect relationship in the range of 50-350mol/L.2. As measured by RT-PCR,influencet of fetus mouse hippocampus cell by ethanol could improve expression of mRNA of Hsp70 and SOD obviously in the range of 50 ~ 350mol/L.3. As measured by Western-blot,ethanol could improve the expression of protein of the Hsp 70 and SOD in the range of 50 ~ 350mol/L.Discussion:1. There is obvious ethanol-induced neurotoxic effect in the range oflOO-350mol/L.2. Hippocampus is the sensiitve area of ethanol-induced neurotoxic effect;The nervous system damage due to Prenatalexposure to ethanol has close relationship with thehippocampus development.HSP could protect agaist damageby ethanol. 3. The ethanol could cause the expression of the mRNA andprotein of SOD.chapter II Study on the protection effects ofPuerariaLobataddiWd) Ohwi' s phytochemicalsagainst ethanol-induced neurotoxicityThe damage of nervous system caused by ethanol is related to cell's redox state disequilibrium.There are lots of reactive oxygen species(ROS) that cause neurotoxicity and strengthen lipid peroxidation in the metabolism of ethanol.The extractive of pueraria has comparatively effective antioxygen activity.ItS active components is mainly flavonoids which have many pharmacological action that is related to its antioxygen activity.This study was designed to examine whether Chinesemedical herb Puerara crude extract (CP) and standard of pure puerarin (SP) posses the similar neuroprotective effects during concomitant ethanol (EtOH) treatment. Methods:Hippocampus cultures were prepared from gestational day 18 mice. Cell viability was measured by MTT assay. RT-PCR and Western Blot were employed to determine mRNA and protein expression of HSP70 and SOD. Results:1. Supplementation with 15 mg/L CP or 10 mg/L SP afford neuroprotection against all EtOH concentrations (50, 200 and 350 mol/1, respectively) in embryonic hippocampal culture system.2. As measured by MTT assay, there were no significant variations within vehicle control, 15 mg/L CP and 10 mg/L SP. CP and SP possesses almost equal neuroprotective effects. Both 15 mg/L CP and 10 mg/L SP could partly improve cell viability from 5O~35O mol/l EtOH and increase by 12.5% and 17.1% at 50 mol/1 EtOH treatment, 15.5% and 16.8% at 200mol/1 EtOH treatment, 13.7% and 15.2% at 350 mol/1 EtOHtreatment, respectively. 3. both 15 mg/L CP and 10 mg/L SP could decrease oxidativestress induced by ethanol treatment, as shown by thedecreased expression of HSP70 and SOD at mRNA andprotein level. Conclusions:1. Both CP and SP could lower ethanol-induced neurotoxic effect through improving cell viability .2. Both CP and SP could lower the expression of Hsp70 and SOD by ethanol,that confirm and explain the same neuroprotective effect of CP and SP.3. The Puerara crude extract (CP) and standard of pure puerarin (SP) posses the same effect of improving ethanol-induced neurotoxic effect...