Study On The Preparation Of Effective Fraction Of SKS And Their Effect On The Proliferation Of Glomeruli Mesangial Cells

Chronic glomerulus nephritis is very difficult to treat in modern medicine, and effective methods of treatment and drug are very deficient. In most cases,the method of treatment is complex or suit in case.. In Chinese tradtional medicine, Syndrome differentiation-based treatment- displays its superiority. Based on the argument of -BuShen, QingLi, HuoXue the specialist prepared an effective prescription which contains Astragalus membranaceus (Fisch) Bge, Radix Salvia miltiorriaz., Angelica sinensis polysaccharide, Fructus corni and et al. We think it is reasonable to develop preparation of effective fraction (EF) after analyzing of the chemical composition and pharmacological action of single medicinal herb in the prescription. So we begin to study the preparation of new drug and the subject was endowed with fund of military new drug.The main subject of this thesis is to extract, isolate and prurify the EF from the medicine materials, then study their quality standard and effect on the proliferation of glomeruli mesangial cells.This thesis includes seven parts, its methods and results describe as follows.1 Study on the preparation technology(PT) of total saponins and total polysaccharides from Astragalus membranaceus (Fisch) Bge.Methods: (1) Establish the determination method of total saponins and total polysaccharides: respectively it is vanillin-sulfuric acid color comparison and phenol-sulfuric acid color comparison. Then compared the contents of effectivecomponent in Astragalus membranaceus (Fisch) Bge from different producing areas. (2) About the preparation technology of total saponins: compare the extracting rate of the alcohol reflux, water boil out and ultrasound extract; study the condition of alcohol reflux by L4(23) orthogonal test; study the process of purification by macroporous resin: containing resin type, absorption capacity, liquor density,absorb flow rate, eluation flow rate and the category and quantity of eluant. (3) In the system of preparation technology of polysaccharide: compare the extracting rate of the water reflux, water boil out and ultrasound extract; investigate the parameter of the water reflux method; compare different methods of alcohol precipitation. (4) About the quality standard: determinate the content of total saponins and total polysaccharide; described the UV absorbance of polysaccharide; determinated the content of astragaloside IV with HPLC-ELSD.Result: (1) Astragaloside IV shows a good linear relationship(r=0.9991) at a range of 0.0988-0.494 mg and the same of glucose in 8⁴8u g ? ml/1 (r=0.9985).At last we choose the Astragalus membranaceus (Fisch) Bge.var.mongholicus (Bge) Hsiao that produced in province Shanxi. (2) The extract technology of saponins is: making 20 cohort dust, extractting two hours with alcohol reflux after adding six factor 95% alcohol, then adding six factor 80% alcohol to extract two hours. (3) Made sure the paramater of purification process with resin: use resin of D101, the density of liquor is 8 mg ? ml/1,and the absorb flow rate is 1¹.5 mL ? min"1, then the resin is eluated orderly with water, 30%, 50%, 70% alcohol, and the dosage in order is 1.5 BV, 0.7 BV, 2².5 BV, 0.7^l BV. (4) The extract technology of polysaccharide is: extractting two times with water reflux and every time is two hours. First time eight factor water will be added, and second time six factor water will be added. At last the extracting liquor will be concentrated and then the density of alcohol precipitation is 75%. (5)The content of total saponins and total polysaccharide is 56.5% and 53.2% respectively, and the content of Astragaloside IV is 0.6%.2 Study on the PT of Salvianolic acids from Radix Salvia miltiorriaz.Methods: (1) Establish the methods of determination of Salvianolic acids with UV spestrophotometry, and the reference substanse is protocatechuic. (2) Study the condition of extraction and isolation of Salvianolic acids with water boil out. (3) Study the process of purification by macroporous resin: containing resin type,absorption capacity, liquor concentration, salt effect, the category and quantity of eluant. (4) Study the quality standard of Salvianolic acids with UV spestrophotometry and HPLC chromatogram.Result: (1) Protocatechuic shows a good linear relationship(r=0.9995) at a range of 2.572 15.432 u g ? mU' . (2) The paramater of water boil out is : extracting two times after adding ten factor water and the time is 2 h and 1.5 h respectively. And the extracting liquor will be isolated with centrifugation in high speed. (3) Make sure the paramater of purification process with resin: use resin ofADF8, the density of liquor is 10 mg ? ml/1 and 2% sodium chloride will be added, then the resin is eluated orderly with water and 30% alcohol, and the dosage in order is 2.5 BV and 4BV.3 Study on the PT of Angelica sinensis polysaccharide.Methods: (1) Compare the contents of polysaccharide in Angelica sinensis(Oliv.) Diels from different producing areas. (2) Compare the extracting rate of the water reflux, water boil out and ultrasound extract. (3) Study the paramater of water boil out by I₄(23) orthogonal test, then studyed the method of alcohol precipitation. (4) Research the technology of purification with ultrafiltration technique: containing gradocol membranes, liquor density and time. (5) Study the quality standard of Angelica sinensis polysaccharide.Result: (1) Make sure the producing area is LeKang in province GanSu. (2) The paramater of water boil out is : extracting three times after adding ten factor water and the time is 1.5 h every time. At last the extracting liquor will be concentrated to 1:1 1:1.2 and then the density of alcohol precipitation is 75%. (3) About the ultrafiltration: the molecular weight of membranes is 10000, the density of liquor is 5.0 m g-mLT1, the time changed with quantity of liquor. (4) The result of determination of ASP is 60.6%.4 Study on the PT of the polysaccharide in Fructus corni.Method: (1) Study the technology of water reflux with Lj(23) orthogonal test. (2) Compare different method of alcohol precipitation. (3) Compare the alcohol reflux and alcohol precipitation for second to purify the polysaccharide.Result: (1) Make sure the paramater of water reflux, which is: at first time, adding eight factor water and the time is 2 h, secondly, adding six factor water and the time is 1.5 h. At last the extracting liquor will be concentrated to 1: 1 and then thedensity of alcohol precipitation is 70%. (2) The method of purification is alcohol precipitation for second. (3) The result of determination is 50.7%.5 Study on the PT of flavanones in P.petiolosa (Christ) Ching.Method: (1) Establish the determination method of flavanones with UV spestrophotometry, and the reference substanse is naringenino (2) Study the condition of alcohol reflux with Lg(34) orthogonal test. (3) Study the process of purification by macroporous resin: containing resin type, absorbing quantity, liquor concentration, the category and quantity of eluant. (4) Study the quality standard of total flavanones.Result: (1) The maximize wavelength of reference substanse and sample is about 286 nm. Naringenin shows a good linear relationship(r=0.9994) at a range of 7.36³6.8u g ? mL"1. (2) The condition of extraction is: adding eight factor 80% alcohol to reflux two times, and time is 2h and 1.5 h respectively. (3) Make sure the purification technology with resin: use resin of AB8, the density of liquor is 6 mg ? mL"1, then the resin is eluated orderly with water, 30% and 50% alcohol, and the dosage in order is 1.5-2 BV, 2².5 BV and 1.5 BV. (4) The result of determination is 32.8%.6 Study on the effect of EF on the proliferation Glomeruli Mesangial Cells.Method: (1) Extract and isolate GMsC from SD rat of 150 to 180 g, and culture them with RPMI-1640 containing 20% FCS. (2) Passage the cells and identify them . (3) Research the effect of EF in different density on the Proliferation of GMsC with MTTmethod.The liquor density is 1, 10, 100, 500, 1000 u g -ml/1. According the absorbance, calculate the inhibition rate of the different drug. Then,make the inhibition rate curve with inhibition rate as ordinateand and density as abscissa.Result: Every EF has some inhibition activity in some density. Astragalan Polysaccharide and Fructus corni polysaccharide can stimulate the cells to grow.But Astragaloside, Salvianolic acids, Angelica sinensis polysaccharide and flavanones in F.petiolosa (Christ) Ching all have some inhibition activity.Conclusions from this thesis:Made sure the preparation method and technology of Astragalan Polysaccharide, Fructus corni polysaccharide, Astragaloside,Salvianolic acids, Angelica sinensis polysaccharide and flavanones in P.petiolosa (Christ) Ching. And made their quality standard preliminarily. All methods are practical and simple.By researching the effect on the GMsC, we can obtain the range of effective density to the cells. In one word, the result will be a good foundation for next research.