Studies On Extraction, Purification Of Flavonoids And Autitumor Effects From Juglans Mandshurica Maxim Bark

Chinese medicine, which has a long history and extensive clinical use in the folk, is an important part of traditional medicine of our country. The security and the effectiveness are the two necessary elements for the development of Chinese medicine. Therefore we researched the bark of Juglans mandshurica Maxim (JMM) in these two aspects in this paper.As the member of Juglandaceac, JMM is the important Phytotherapic drugs of our country, mainly distributed over northeast of China (Lesser Khingan Mountains), and also founded in Henan, Beijing, inner Mongolia and Shanxi. Its unripe epicarp (known as pericarpium juglandis), tegument of root and branch, shells, leaves and seeds all can be used for medical purpose, which have the effectiveness of anti-bacteria, anti-inflammatory, abirritation, anti-oxidation and antineoplastic, and can be used in clinical treatment, for example to treat enteritis, bacillary dysentery, bone tuberculosis and stye etc. The treatment effect will be improved when used with other antineoplastic drugs together.The chemical composition and pharmacologic action of bark of JMM(JMMB) have been profoundly researched in the folk, however, the security of the bark and the workmanship, which distill, separate and sublimate the active ingredient of antineoplastic drugs, are lake of study. We made use of the special toxicity to test the security of bark of JMM, and do more studies on its active ingredient—flavonoids, in order to make full use of the rich resources of medicinal herbs in north of China, and provide a scientific basis for the development of new antineoplastic drugs of our country.The main research contents and conclusions in the paper are as follows:First.Research the toxicity of JMMB. There were eight groups in micronuclei assay of mouse. Negative controlled with saline water, and positive controlled with 0.04 g/kg CP. There mutagenesis groups composed by 1/8LD50 g/kg, 1/4LD50 g/kg, and 1/2LD50 g raw durg/kg, and the does given to the three groups are 26.18 g/kg, 13.09 g/kg, 6.55g/kg. three anti-mutagenesis groups composed by 1/8LD50g/kg+CP, 1/4LD50g/kg+CP, 1/2LD50g/kg+CP. Made decisions by observing the rate of the change of micronuclei and the ratio of PEC and NEC. The result showed that the ratio of micronuclei in negative group is 2.65%, and 28.82% in positive group, there was a significant difference, which had statistic significance, between the two groups (p<0.01). It also showed that the experiment system was reliable. There was no significant difference among all does groups of the three extracts and negative control (p>0.05). The result imply that: the bark of JMM had no mutagenesis effect, and cannot control the bone marrow cells effectively. There was no significant difference among three mutagenesis groups and negative group (p>0.05). And there was a significant difference among anti-mutagenesis groups and negative group (p<0.05). The result imply that: the bark of JMM had no mutagenesis effect, and cannot control the bone marrow cells effectively, on the contrary, it had anti-mutageneicity.Second. The active ingredient, flavonoids, are mainly studied. It is studied amply in this dissertation. The spectrophotometry is used to analyze flavonoids content. The recovery is 100.25%. Use ultrasonic technology to distill, and the optimal distilling parameter is worked out by univariate analysis of variance and orthogonal test: ethanol concentration is 60%, ultrasound time is 30 minutes, ratio of solid and liquid is 1/15, ultrasound temperature is 75℃, and extract one time. Under this condition, the ratio of the flavonoids is 4.76mg/g, and the recovery weight of the medicine is 51.31mg/g.Third. The purifieation of flavonoids is studied after extraeting it from The JMMB. And D101resin had been established to separate and purify flavonoids. The technology is feasible, simple, reasonable, and applicable to industrial production. Based on the research of eluent solvent,the velocity of initial solution and eluent,the optimalconditions are as follows: the sample solution speed is2BV/h, the flow rate is3BV/h, the elution solvent 4BV of water and 5BV 50% ethanol,and the Purity of flavonoids could reach the height of 48.98%,which is approximately 5.25 times higher than the Previous 9.33%, while the yield of flavonoids could reach 78.33%.Finally,The experiments were studied the medical effectiveness of the flavonoids sublimate product of JMMB by pharmacodynamic.In Vitro experiments using CCK-8 assay to evaluate the growth inhibition of K562 cells and BGC-823 cells,flow Cytometry to detect cell cycle arrest,and apoptosis was detected by AnnexinⅤ-FITC&PI kit. The result showed that the purified samples have inhibitory effect on growth of K562 cells and BGC-823 cells in dose dependent manner IC50 of JMMB are 0.0223mg/mL and 0.0648mg/mL,respectively.There issignificant statistical difference in term of cell proliferation between JMMB and vehicle treatment group(P<0.05). Flow Cytometry revealed k562 cell cycle arrest in S Phase and BGC-823 cell arrest in G0/G1 Phase by Medium dose of JMMB. Compared with Control, the total apoptosis rate of the different doses (1/2 IC50,IC50,2IC50) of K562 cells increased Significantly( P<0.01),Late apoptosis ratio was significant (P<0.05), That drug-induced apoptosis in K562 cells significantly, the occurrence of late in the cell, with the attendant increase in drug concentration increased the rate of apoptosis.for BGC-823 cell, Compared with Control, High dose group was significantly increased apoptosis(P<0.01). Early apoptosis rate increased significantly(P<0.05). Description JMMB flavonoids on BGC-823 cells induced apoptosis occurred in cells in the early days, the drug concentration corresponding increase in apoptosis rate increased.