Material Foundation Study Of Drug Pair Of Radix Rehmanniae And Cornus Officinalis On Inhibiting The Diabetic Nephropathy Glycosylation Products

PART I The option of the best Radix Rehmanniae and Cornus officinalisObjective:In order to identity the best Radix Rehmanniae and Cornus officinalis, and prepare for the following experiments, we choosed Radix Rehmanniae and Cornus officinalis of different fields and batches, and determined the content of the catalpa, acteoside and loganin according to the 2010 China pharmacopoeia.Methods:1. Take some Radix Rehmanniae, and dry 24h at 80℃ through decompression process. Make them into the thick powder, weigh 0.8g precisely from them into the round bottom flask, and weigh after adding 50mL methanol. Weigh again through a hot reflux extraction about 1.5h. The weight loss is replaced by methanol, shake up and filtrate it. Take the filtrate 10mL precisely and concentrate it to nearly dry. The residual is dissolved by the mobile phase and transferred into lOmL measuring flask which is diluted to the scale by the mobile phase. Shake up and filtrate it. Take the filtrate to the volume by the mobile phase, and each sample is paralleled 3 times; Take the filtrate lOmL precisely and concentrate it to nearly dry through decompression process. The residual is dissolved by the mobile phase and transferred into 5mL measuring flask which is diluted to the scale by the mobile phase. Shake up and filtrate it. Take the filtrate to the volume by the mobile phase, and each sample is paralleled 3 times. The content is determined using HPLC.2. Weigh the powder of Cornus officinalis 0.1g precisely into a tapered plug bottle and weigh after adding 25mL 80%methanol precisely. Weigh again through a hot reflux extraction about 1h. The weight loss is replaced by 80%methanol, shake up and filtrate it. Each sample is paralleled 3 times. The content is determined using HPLC.Results:1. The content of catalpol and acteoside in Radix Rehmanniae are 1.44% and 0.02% from Henan, batch for 110201; The content of catalpol and acteoside in Radix Rehmanniae are 1.69% and 0.01% from Henan, batch for 100816; The content of catalpol and acteoside in Radix Rehmanniae are 2.52% and 0.02% from Henan, batch for 20110202; The content of catalpol and acteoside in Radix Rehmanniae are 2.27% and 0.01% from Shanxi.2. The content of loganin in Cornus officinalis is 0.44% from Henan, batch for 100031; The content of loganin in Cornus officinalis is 0.47%from Henan, batch for 1006041; The content of loganin in Cornus officinalis is 0.59% from Henan, batch for 100031; The content of loganin in Cornus officinalis is 0.6% from Zhejing; The content of loganin in Cornus officinalis is 0.5% from Zhejiang, batch for 101116; The content of loganin in Cornus officinalis is 0.65% from Henan, batch for 110301.Conclusions:Radix Rehmanniae from Henan, batch for 20110202 is the best in four samples; Cornus officinalis from Zhejiang, batch for 110301 is best in six samples, so we will choose these for the following experiments.PART Ⅱ The preparation and content determination of the effective-partsObjective:To extract, detach and purify the effective-parts of Radix Rehmanniae and Cornus officinalis, and determine the content of them while preparation for the material foundation study of effective-parts of Radix Rehmanniae, Cornus officinalis and their compatibility.Methods:1. Take some Radix Rehmanniae after smashed over 40 mesh, and extract through 3 times. First time is adding 10 times volume of water, extract about 2h, filtrate to get the extraction liquid Ⅰ; the second time is adding 6 times volume of water into the herbal residue, extract about 1h, and get the extraction liquid Ⅱ; the third time is adding 4 times volume of water into the herbal residue, extract about 1 h, and get the extraction liquid Ⅲ. Combine each time of the extarction liquid, concentrate until 1g crude drug/ml, and add alcohol to concentration 70%. Place four 24h and filtrate it. Oligosaccharide:The supernatant liquid is made with no alcohol and place the volume to 0.5g crude drug/ml. Place it on macroporous resin, and wash untile the molish reaction becoming negative with distilled water. Collect the water eluent, concentrate and spray drying to get the powder of oligosaccharide. Iridiod glycoside:Wash by 30% alcohol with the velocity of 1.8BV/h. Collect the alcohol eluent, concentrate and spray drying to get the powder of iridiod glycoside. Polysaccharide:The filter cake after alcohol precipitation is dissolved with distilled water to 0.25g crude drug/ml, and centrifuge to remove insoluble. The supernatant liquid is made with no alcohol, and spray drying to get the powder of polysaccharide. Determine the purity of iridiod glycoside using the catalpol as an indicator by HPLC; determine the purity of oligosaccharide using the stachyose as an indicator by HPLC; determine the purity of polysaccharide using the glucose as an indicator by UV.2. Plus 12 times the amount of Cornus officinalis water extraction 2 times, each time 2h, 70% aclcihol and alcohol precipitation, standing more than 24 hours, filtered. Iridoid glycoside: Place the supernatant liquid on macroporous resin with 10% ethanol 3.5BV/h elution. polysaccharide:The filter cake after alcohol precipitation is dissolved with distilled water, and centrifuge to remove insoluble. Concentrate and spray drying to get the powder of polysaccharide. Triterpenic acid:After the water put the dregs drained, by 1:6(solvent:water extraction before the medicine is not the quality) 95% alcohol at 85℃, extracted 2 times, rotary evaporator to remove the solvent, vacuum drying dry extract. The dry extract washed with 10% NaOH, and then other times the amount of precipitation was acidic aqueous extract Triterpenic acid. To loganin and morroniside, used as the indicators of iridoid glycoside purity determined by HPLC. Glucose as an indicator of polysaccharide determined by UV. To ursolic acid and oleanolic acid, used as the indicators of triterpenic acid purity determined by HPLC.Results and Conclusions:The catalpol content of Radix Rehmanniae iridoid glycosides is 1.91%; the stachyose content of Radix Rehmanniae oligosaccharide is 26.01%; the polysaccharide content of Radix Rehmanniae polysaccharide is 68.52%; the loganin and morroniside content of Cornus officinalis iridoid glycoside is 44.8%; the polysaccharide content of Cornus officinalis polysaccharide is 68.52%; the ursolic acid and oleanolic acid content of Cornus officinalis triterpenic acid is 20.89%.PART Ⅲ The effects of Radix Rehmanniae, Cornus officinalis and their compatibility on diabetic nephropathy glycosylation productsObjective:To screen Radix Rehmanniae, Cornus officinalis and their compatibility water extraction, medicated serums, effective-parts and their compatibilities, effective constituents using the generation model of diabetic nephropathy glycosylation products in vitro, and research the Material foundation of Radix Rehmanniae and Cornus officinalis on inhibiting the generation of diabetic nephropathy glycosylation products in vitroMethods:Make the reaction system with 200M glucose and bovine serum albumin(BSA), and Radix Rehmanniae, Cornus officinalis and their compatibility water extraction, medicated serums, effective-parts and their compatibilities, effective constituents are added in them respectively, using aminoguanidine as positive control. At the same time also set other control groups, for example (1) the whole non-enzyme saccharification systems with no drugs; (2) the systems with no drugs and glucose; (3) the systems with no drugs and BSA; (4) the systems with drugs but no BSA; (5) the systems with drugs but no glucose. They are incubated at 37℃. After two weeks, the early saccharification protein(fructosamine) are measured at 530nm by automatic microplate reader; after four weeks, fluorescence degrees of AGEs are measured at excitation wavelength 360 and emission wavelength 460, and calculate their inhibitory rate and IC50 or IC25.Results:1. The compatibility water extraction of Radix Rehmanniae and Cornus officinalis is stronger in inhibiting fructosamine and AGEs than others, and its IC50 is 0.077g/L and 0.076 g/L; the effect of its medicated serums is as same as the compatibility water extraction, and IC50 is 31.6% and 73.2%.2. The oligosaccharide of Radix Rehmanniae and the iridoid glycoside of Cornus officinalis are stronger in inhibiting fructosamine than others, and IC25 are 0.0002 g/L and 0.0001 g/L; but the iridoid glycoside and polysaccharide of Radix Rehmanniae are stronger in inhibiting AGEs than others, and IC25 are 0.0108 g/L and 0.0103 g/L.3. The effective-parts of Radix Rehmanniae and Cornus officinalis are composed through orthogonal design which have varying degrees in inhibiting fructosamine and AGEs. (Iridoid glycoside of Radix Rehmanniae:oligosaccharide of Radix Rehmanniae:polysaccharide of Radix Rehmanniae:iridoid glycoside of Cornus officinalis:triterpenic acid ratio 1:1:1:2:1) is confirmed as the optimum component group in inhibiting fructosamine in vitro; and (iridoid glycoside of Radix Rehmanniae:polysaccharide of Radix Rehmanniae:iridoid glycoside of Cornus officinalis: triterpenic acid ratio 2:2:2:1) is confirmed as the optimum component group in inhibiting AGEs in vitro.4. Catalpol, stachyose and ursolic acid are stronger in inhibiting fructosamine than others, and IC50 are 4.2×10-8mol/L,9.4×10-8mol/L and 5.7×10-8mol/L; but loganin is stronger in inhibiting AGEs than others, its IC50 is 6.1 × 10-7mol/L.Conclusions:1. There is synergistic effect between Radix Rehmanniae and Cornus officinalis in inhiiting fructosamine and AGEs.2. Effective-parts and effective constituents have varying degrees in inhibiting fractosamine and AGEs, but there are different on action time and action segment.3. The best compatibility ratio of effective-parts in inhibiting fructosamine and AGEs are confirmed.