CN103599340A - Pharmaceutical composition used for treating and preventing diabetes and eye complications thereof, and applications of the pharmaceutical composition - Google Patents
Pharmaceutical composition used for treating and preventing diabetes and eye complications thereof, and applications of the pharmaceutical composition Download PDFInfo
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Abstract
The invention discloses a pharmaceutical composition used for treating and preventing diabetes and eye complications thereof, and applications of the pharmaceutical composition. The pharmaceutical composition is prepared from following components by weight: 1-5 parts of fried sophorae flower bud, 1-5 parts of radix scutellariae, 1-5 parts of salty rhizoma anemarrhenae and 1-5 parts of dried tangerine peel. The pharmaceutical composition can be prepared by an alcohol extraction method or an aqueous extraction method. The pharmaceutical composition has significant effects for treating and preventing the diabetes and the eye complications thereof.
Description
Technical field
The invention belongs to technical field of Chinese medicines, be specifically related to pharmaceutical composition and the application thereof of a kind for the treatment of and prevent diabetes and ocular complication thereof.
Background technology
In every 20 adults in the whole world, just have a people for diabetics, China's diabetics 3,980 ten thousand people, occupy the world the 2nd, and sickness rate improves constantly.
Diabetes main cause lethal and that disable is not hyperglycemia itself, but its complication.Wherein ocular complications has become the first cause of adult's blinding.10 years above diabetics ocular complications sickness rate of the course of disease are up to more than 60%.
At present Western medicine hypoglycemic medicine action pathway is single, and life-time service hypoglycemic effect declines but the double complication of controlling simultaneously; Diabetics ocular complications operative treatment has certain risk; At present complication medicine is if calcium dobesilate and XUEMINGMU PIAN are without remarkable hypoglycemic effect.So such medicament research and development of the medicine for the treatment of and prevent diabetes and ocular complication thereof has practical significance
Chinese medicine compound has the feature of multicomponent, many target spots, and exploitation has probability and space at hypoglycemic compound recipe for the treatment of diabetic eye complication simultaneously.
Summary of the invention
The pharmaceutical composition that the object of this invention is to provide a kind of effectively treatment and prevent diabetes and ocular complication thereof.
Another object of the present invention is to provide the preparation method of aforementioned pharmaceutical compositions.
The object of the invention is to realize in the following manner:
An effective pharmaceutical composition for treatment and prevent diabetes and ocular complication thereof, said composition is made by the component of following weight portion:
Stir-baked FLOS SOPHORAE IMMATURUS 1-5 part, Radix Scutellariae 1-5 part, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1-5 part, Pericarpium Citri Reticulatae 1-5 part.
Said composition is preferably made by the component of following weight portion:
Stir-baked FLOS SOPHORAE IMMATURUS 1-3 part, Radix Scutellariae 1-3 part, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1-3 part, Pericarpium Citri Reticulatae 1-3 part.
Said composition is further preferably made by the component of following weight portion:
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 2 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 1 part of Radix Scutellariae, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 2 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 1 part of Radix Scutellariae, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 2 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 1 part of Radix Scutellariae, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 1 part of Radix Scutellariae, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 1 part of Radix Scutellariae, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 1 part of Radix Scutellariae, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 2 parts of Radix Scutellariaes, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 2 parts of Radix Scutellariaes, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae.
Said composition is most preferably made by the component of following weight portion: 1 part of stir-baked FLOS SOPHORAE IMMATURUS, 2 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae.
Described compositions and pharmaceutically acceptable carrier are made preparation.Described preparation can be oral liquid, pill, tablet, powder, granule, capsule, drop pill or injection.
Above-mentioned composition can prepare by alcohol extracting method, concrete preparation method comprises the following steps: take stir-baked FLOS SOPHORAE IMMATURUS, Radix Scutellariae, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution and Pericarpium Citri Reticulatae, adding medical material gross weight 8-12, doubly to measure concentration be that the alcohol heating reflux of 10%-90% extracts 1-3 time, extracts 1-3 hour, filtration at every turn, merging filtrate, concentrated.The mass concentration of the ethanol preferably adopting is 60%-70%.
Above-mentioned composition also can prepare by water extraction method, and concrete preparation method comprises the following steps: take stir-baked FLOS SOPHORAE IMMATURUS, Radix Scutellariae, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution and Pericarpium Citri Reticulatae, add 8-12 times of water gaging of medical material gross weight, heating extraction 1-3 time is extracted 0.5-1 hour at every turn, filter, merging filtrate, concentrated.
Pharmaceutical composition of the present invention can be in the medicine of preparation treatment or prevent diabetes and ocular complication thereof application.The ocular complication that described diabetes cause is retinopathy or cataract.
The dry flower that the Flos Sophorae Immaturus that the present invention uses is leguminous plant Chinese scholartree Sophora japonica L..Function cooling blood for hemostasis, purging liver-fire.Stir-baked FLOS SOPHORAE IMMATURUS, for being used as medicine with the micro-stir-fry of slow fire, slows down the property of bitter cold, can strengthen the effect of cooling blood for hemostasis.Stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution is put the micro-stir-fry of use slow fire in pot for getting Rhizoma Anemarrhenae sheet, spray saline, and fried dry is taken out, and cools.(100 jin of every Rhizoma Anemarrhenae sheets add certain amount of boiling water with 2 jin 8 liang of salt and melt clarification).Cure mainly premature ejaculation.Red tongue, thin fur, thready pulse and number person.Pericarpium Citri Reticulatae, often shells into several lobes, and base portion is connected, and what have is irregular lamellar, thick 1-4mm.Bitter in the mouth, pungent, warm, regulating qi-flowing for strengthening spleen, drying dampness to eliminate phlegm.Radix Scutellariae (Scutellaria baicalensis Georgi), real name " a kind of reed mentioned in ancient books ", another name Radix Scutellariae, tsuchikane tea root, because careless yellow skin has popular name " Radix Scutellariae ".For labiate, with root, be used as medicine, bitter in the mouth, cold in nature, energy heat clearing and damp drying, eliminating fire and detoxication, hemostasis, antiabortive, have heat clearing and damp drying, and removing heat from blood is antiabortive, detoxicating functions.The advantage of the comprehensive above-mentioned raw materials medical material of inventor, in conjunction with modern new drug research means, develops the pharmaceutical composition of effectively treatment and prevent diabetes and ocular complication thereof, and this pharmaceutical composition also has safe, the rapid-action feature of using.
Beneficial effect of the present invention compared with the prior art: the present invention has significant reduction blood glucose and treatment of diabetic retinopathy becomes and cataractous effect; With positive control drug metformin, calcium dobesilate and compare overall drug effect with XUEMINGMU PIAN and there is optimal efficiency; This prescription has prescription novelty and active novelty simultaneously; Its hypoglycemic mechanism is relevant with increase insulin sensitivity with increase amount of insulin secretion; It is relevant with antioxidation, inhibition sorbitol pathway, PKC path and AGEs path with the mechanism of cataract pathological changes that its treatment of diabetic retinopathy becomes.
By concrete test example, above-mentioned effect is further described below:
The model experiment of test example 1.HepG2 cell tryptase insulin resistance
The pharmaceutical composition for the treatment of and prevent diabetes and ocular complication thereof is comprised of stir-baked FLOS SOPHORAE IMMATURUS, Radix Scutellariae, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, Pericarpium Citri Reticulatae 4 herbal medicines.Four kinds of medicines can be within the scope of the part by weight of 1-5:1-5:1-5:1-5 combination in any, now list the proportionate relationship of pharmaceutical composition and sequence number for the experiment of vitro inhibition α glycosidase, insulin sensitivity enhancing experiment, promote insulin secretion experiment and repair high microvascular endothelial injury experiment and diabetes rat model experiment in specifically.
1 group of prescription: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=1:2:3:1
2 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=2:3:1:1
3 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=3:1:2:1
4 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=1:2:3:2
5 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=2:3:1:2
6 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=3:1:2:2
7 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=1:2:3:3
8 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=2:3:1:3
9 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=3:1:2:3
10 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=1:1:1:1
11 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=1:1:1:2
12 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=1:1:1:3
13 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=2:2:2:1
14 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=2:2:2:3
15 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=3:3:3:2
16 groups of prescriptions: stir-baked FLOS SOPHORAE IMMATURUS: Radix Scutellariae: stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution: Pericarpium Citri Reticulatae=3:3:3:1
1. experiment material:
Cell: human liver cancer cell (HepG2, FROM ATCC, The National Center for Drug Screening of China Medicine University).
Reagent: DMEM in high glucose culture medium (Gibco, lot number: 914292); Low sugar DMEM culture medium (Gibco, lot number: 914292); Acarbose tablet (Bayer HealthCare Co, lot number: 117893); Glibenclamide (Tianjin Pacific Pharmaceutical Co., Ltd., lot number: 120108); Metformin (Beijing JingFeng Pharmaceutical Co., Ltd, lot number: 120915); Calcium dobesilate (Xi'an Li Jun pharmaceutical Co. Ltd, lot number: 1202023); And XUEMINGMU PIAN (Beilin Pharmaceutical Co., Ltd., Xi'an, 201205009).
2. experimental technique
By HepG
2cell is by 8 * 10
4concentration is inoculated on 96 orifice plates, every hole 200 μ L, and modeling group working concentration is 10
-6after the insulin-induced liquid induction of mol/L 24h, changing insulin concentration is 10
-9mol/L culture fluid continues to hatch 12h, measures the glucose utilization of respectively organizing cell.
2.1HepG2 cell culture and fishplate bar
HepG2 cell is inoculated in Tissue Culture Flask, and every bottle adds about 12mL containing the DMEM in high glucose culture medium of 10% hyclone, is placed in 37 ℃ and contains 5%CO
2incubator in cultivate, approximately every 3 days one time 0.25% trypsin-0.01%EDTA solution had digestive transfer culture, fishplate bar after trypsinization when cell state is better.
2.2 the preparation of medicinal liquid
2.2.1 prescription sample liquid 1(2.5mg crude drug/ml) preparation:
The extraction of prescription 1 medicine: in the ratio of 1:2:3:1, take stir-baked FLOS SOPHORAE IMMATURUS 1g, Radix Scutellariae 2g, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3g, Pericarpium Citri Reticulatae 1g, is placed in 250mL round-bottomed flask, adds the alcoholic solution that 10 times of amount mass concentrations of medical material gross weight are 70%, soaks after 1h, 85 ℃ of reflux, extract, 1h, 16 layers of filtered through gauze, obtain filtrate.With method, extract 3 times, merge filtrate 3 times, 70 ℃ of reclaim under reduced pressure, to without alcohol taste, are transferred in evaporating dish and are placed on 70 ℃ of water-baths and steam to nearly dry rear 60 ℃ of drying under reduced pressure, dried cream powder end 3.4949g that must this prescription sample, yield 49.93%.
Prescription analyte sample fluid 1(2.5mg crude drug/ml) preparation: take prescription sample 0.0012g in 2mlEP pipe, add 2ml low sugar DMEM culture fluid vortex and dissolve, cross 0.22 μ m filter membrane degerming in super-clean bench, obtain the mother solution that concentration is 2.5mg crude drug/ml.
2.2.2 prescription sample liquid 2-16(2.5mg crude drug/ml) preparation: respectively in the regulation ratio under 1 of test example, that gets prescription 2-16 group respectively organizes medical material, then by " prescription 1 sample liquid (2.5mg crude drug/ml) preparation " method, prepare respectively prescription 2-16 sample liquid (2.5mg crude drug/ml).
2.2.3 prescription sample liquid 17(2.5mg crude drug/ml) preparation:
The extraction of prescription 17 medicines: the ratio in the medical material 1:2:3:1 of prescription 1, takes stir-baked FLOS SOPHORAE IMMATURUS 1g, Radix Scutellariae 2g, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3g, Pericarpium Citri Reticulatae 1g, is placed in 250mL round-bottomed flask, adds 10 times of water gagings of medical material gross weight, soaks after 1h, 1h is extracted in water-bath, and 16 layers of filtered through gauze, obtain filtrate.With method, extract 3 times, merge filtrate 3 times, on 80 ℃ of water-baths, steam to nearly dry rear 60 ℃ of drying under reduced pressure, dried cream powder end 3.57g that must this prescription sample, yield 51%.
Prescription sample liquid 17(2.5mg crude drug/ml) preparation: take prescription sample 0.0013g in 2mlEP pipe, add 2ml low sugar DMEM culture fluid vortex and dissolve, cross 0.22 μ m filter membrane degerming in super-clean bench, obtain the mother solution that concentration is 2.5mg crude drug/ml.
2.3HepG2 cell grouping, modeling and administration
When HepG2 Growth of Cells to 80～90% merges, outwell culture medium, with PBS, clean 2 times; Cell, with 0.25% the about 40s of trypsinization, is poured out to pancreatin, add the culture medium containing 10% serum to stop digestion, outwell culture medium, add the new culture medium containing 10% hyclone, blow and beat gently cell to evenly, by cell dilution to 8 * 10
4individual/mL, is inoculated in 96 orifice plates, and 200 μ L are inoculated in every hole, and around every hole adds 200 μ LD-Hank's solution.96 orifice plates are placed in to 5%CO
2, cultivate in 37 ℃ of constant incubators, when Growth of Cells merges to 80-90%, sucking-off culture fluid, is grouped as follows, ，Ge hole, 6 every group multiple holes continues to cultivate after adding following culture medium, induced liquid and medicinal liquid: blank group: 200 μ L DMEM in high glucoses; Model group: 180 μ L DMEM in high glucoses and 20 μ L10
-5the insulin-induced liquid of mol/L; Sample sets: 160 μ L DMEM in high glucoses, 20 μ L induced liquids, 20 μ L0.025mg/ml glibenclamide solution, 20 μ L436 μ g/mL phenolsulfonic acid calcium solutions, 20 μ L2.5mg/ml and XUEMINGMU PIAN sample liquid, 20 μ L400 μ g/ml metformin sample liquid, 20 μ L400 μ g/mL acarbose sample liquid and each 20 μ L prescription sample liquid 1-17.
In 2.4 cell culture fluids, glucose content is measured
After inducing culture 16-20h, pastille culture fluid is taken out, every hole adds fresh in 10
-9mol/L insulin also, containing the low sugar culture-medium 200 μ L of 1%FBS, is drawn 20 μ l culture fluid in 1.5mLEP pipe after cultivation 12h from each hole, add the glucose concentration determination working solution of 1000 μ L, fully mixes, and is placed in 37 ℃ of water-bath 15min.After colour developing, at wavelength 492nm place, measure the light absorption value of each sample, before and after calculating cultivation, in culture medium, the content difference of glucose is and respectively organizes glucose utilization.It is as shown in the table for experimental result.
Table 1. different pharmaceutical and the impact of prescription sample liquid 1-17 group on HepG2 cell insulin sensitivity
Note: with the comparison of blank group,
#represent P<0.05,
##represent P<0.01; With model group comparison, * represents P<0.05, and * * represents P<0.01.
Experimental result shows: prescription sample liquid 1-17 group, metformin group all can extremely significantly increase the insulin sensitivity (P<0.01) of HepG2 cell; Glibenclamide group also has certain sensitization, relatively has significant difference (P<0.05) with model group, but effect is not as good as metformin group and prescription sample liquid 1-17 group; Other positive drug have no remarkable effect of enhanced sensitivity.
Test example 2. α glycosidase inhibition experiments
1. experimental technique
By 1mgmL
-1alpha-glucosidase PBS solution 100 μ L and each sample 50 μ L add in 96 orifice plates, shake up, and add the PNPG solution 50 μ L of concentration 10mmol/L in 37 ℃ of water-baths after hatching 10min, shake up, and after 37 ℃ of reaction 45min, add 0.3molL
-1na
2cO
3100 μ L, cessation reaction.In 405nm place, measure and record light absorption value and calculate suppression ratio.
1.1 grouping and administrations
Reaction is carried out on 96 orifice plates, considers to get rid of medicinal liquid color and disturbs, and experiment is divided into model group and the blank group of model, positive drug acarbose group and the blank group of positive drug, sample sets and sample matched group, and each organizes sample with test example 1.Establish 6 multiple holes for every group.
The mensuration of 1.2 alpha-glucosidase suppression ratio
After alpha-glucosaccharase enzymatic solution 100 μ L are mixed with each sample solution 50 μ L, shake up, add 10mmolL hatch 10min in 37 ℃ of water-baths after
-1pNPG solution 50 μ L, shake up, and 37 ℃ of reaction 45min, add 0.3molL
-1na
2cO
3100 μ L, cessation reaction.In 405nm place, measure and record light absorption value.Experimental result is as shown in table 2.
According to following formula, calculate and respectively organize the suppression ratio of medicine to alpha-glucosidase:
Table 2. different pharmaceutical and the suppression ratio of prescription sample liquid 1-17 group to alpha-glucosidase
Note: with the comparison of blank group,
#represent P<0.05,
##represent P<0.01; With model group comparison, * represents P<0.05, and * * represents P<0.01.
Experimental result shows, prescription sample liquid 1-17 group is 17.59%-22.22% to the suppression ratio of alpha-glucosidase, with model group comparison, has utmost point significance inhibitory action (P<0.01); With XUEMINGMU PIAN to the suppression ratio of α glycosidase, be 8.333%, there is significance inhibitory action (P<0.05); The two is all not as good as the suppression ratio of acarbose 86.11%; Other positive drug do not show the inhibitory action to α glycosidase; Except acarbose group, prescription sample liquid 1-17 group and other positive control drug group comparison, have effectiveness.
The model experiment of test example 3.RIN-m5F cell insulin secretagogue
1. experiment material: RIN-M5F rat Langerhans islet oncocyte (Chinese Academy of Medical Sciences's tumor cell storehouse).
2. test method
The RIN-m5F cell DMEM culture fluid of the hyclone that contains 10%, is placed in 37 ℃ and contains 5% CO
2incubator in cultivate, 0.25% trypsin-0.01%EDTA solution had digestive transfer culture.When cell state is better, after trypsinization, adjusting cell density is 5 * 10
5～1 * 10
6individual mL
-1fishplate bar, cultivates after 36h sucking-off culture fluid, the administration of dividing into groups.Blank group and positive drug group are established in experiment.The every hole of blank group adds 200 μ L low sugar DMEM culture fluid, and the every hole of sample sets adds 180 μ L low sugar DMEM culture fluid and 20 μ L concentration to be, 6 every group multiple holes.
Continue to cultivate after 24 hours, using concentration of glucose instead is the DMEM culture medium culturing 4h of 15mmol/L, and sucking-off culture fluid, measures wherein insulin content.Draw culture fluid 10 μ L in every hole, according to test kit description, use insulin ELISA kit measurement respectively to organize insulin content in culture fluid.
2.1RIN cell culture and fishplate bar
RIN-M5F cell is inoculated in Tissue Culture Flask, and every bottle adds about 12mL containing the low sugar DMEM culture medium of 10% hyclone, is placed in 37 ℃ and contains 5%CO
2incubator in cultivate, approximately every 4 days one time 0.25% trypsin-0.01%EDTA solution had digestive transfer culture, fishplate bar after trypsinization when cell state is better.
2.2RIN cell fishplate bar, grouping and administration
When RIN cell culture merges to approximately 80%, outwell culture medium, with PBS, clean 2 times, add 0.25% trypsin-0.01%EDTA solution to digest about 30-40s, outwell pancreatin, add the culture medium containing 10% serum to stop outwelling culture medium after digestion, add the new culture medium containing 10% hyclone, piping and druming cell is to evenly, by cell dilution to 5 * 10
5-1 * 10
6individual/mL, is inoculated in 96 orifice plates, and 200 μ L are inoculated in every hole, and 96 orifice plates are placed in to 5%CO
2, in 37 ℃ of constant incubators, cultivate.
Cultivate after 36h, sucking-off culture fluid, is divided into blank group, glibenclamide group, calcium dobesilate group and XUEMINGMU PIAN group, metformin group, acarbose group and prescription sample liquid 1-17 group, and each organizes sample with test example 1.The every hole of blank group adds 200 μ L low sugar DMEM culture fluid, and the every hole of sample sets adds 180 μ L low sugar DMEM culture fluid and 20 μ L sample liquid, 6 every group multiple holes.
In 2.3 cell culture fluids, insulin content is measured
Continue to cultivate after 24 hours, using concentration of glucose instead is the low sugar DMEM culture medium culturing 4h of 15mmol/L, and sucking-off culture fluid, measures wherein insulin content.Draw culture fluid 10 μ L in every hole, according to test kit description, use insulin ELISA kit measurement respectively to organize insulin content in culture fluid.Experimental result is as shown in table 3.
Table 3. different pharmaceutical and the impact of prescription sample liquid 1-17 group on RIN cell amount of insulin secretion
Note: with the comparison of blank group,
#represent P<0.05,
##represent P<0.01.
Experimental result shows: only have glibenclamide group and prescription sample liquid 1-17 group can significantly increase RIN cell amount of insulin secretion (P<0.01), other group medicines all can not increase RIN emiocytosis insulin.
The high sugar of test example 4. causes the model experiment of HUVEC endothelial cell damage
1. experiment material: Human umbilical vein endothelial cells, (HUVEC, The National Center for Drug Screening of China Medicine University).
2. experimental technique
When HUVEC cell state is better, use trypsinization, by cell dilution to 3 * 10
4individual/mL, is inoculated in 96 orifice plates, and 200 μ L are inoculated in every hole.96 orifice plates are placed in to 5%CO
2in 37 ℃ of constant incubators, cultivate, when Growth of Cells to 90% fusion, the low sugar DMEM culture medium culturing 24h using instead containing 1% hyclone makes cell synchronization, use again concentration of glucose instead and be 33mmol/L's and the DMEM culture medium culturing 72h that contains 1%FBS, obtain the endothelial cell damage model of high sugar induction.
2.1HUVEC cell culture and fishplate bar
HUVEC cell is inoculated in Tissue Culture Flask, and every bottle adds about 12mL containing the low sugar DMEM culture medium of 10% hyclone, is placed in 37 ℃ and contains 5%CO
2incubator in cultivate, approximately every 4 days one time 0.25% trypsin-0.01%EDTA solution had digestive transfer culture, fishplate bar after trypsinization when cell state is better.
The grouping of 2.2HUVEC cell, modeling and administration
When HUVEC cell grows to 80～90% fusion in culture bottle, outwell culture medium, with PBS, clean 2 times; Cell, with 0.25% the about 1min of trypsinization, is outwelled to pancreatin, add the culture medium containing 10% serum to stop digestion, outwell culture medium, add the new culture medium containing 10% hyclone and blow and beat cell to evenly, by cell dilution to 3 * 10
4individual/mL, is inoculated in 96 orifice plates, and 200 μ L are inoculated in every hole.96 orifice plates are placed in to 5%CO
2, cultivate in 37 ℃ of constant incubators, when Growth of Cells merges to 80-90%, use instead containing after the low sugar DMEM culture medium synchronized culture 24h of 1% hyclone by cell by dividing into groups under 3.1.3 item, establish 5 multiple holes for every group, continue to cultivate after adding corresponding culture medium, induced liquid and medicinal liquid according to following grouping: blank group: add 200 μ L containing the low sugar DMEM culture medium culturing of 1%FBS; Model control group: adding 200 μ L concentration of glucose is 33mmol/L's and the DMEM culture medium that contains 1%FBS; Administration group: adding 180 μ L concentration of glucose is 33mmol/L and the DMEM culture medium that contains 1%FBS, 20 μ L difference medicinal liquid to be tested, each group medicinal liquid sample to be tested is with test example 1.
The mensuration of 2.3HUVEC cell viability
Cultivate after 72h, pastille culture fluid is taken out, every hole adds low sugar DMEM culture medium 200 μ L, the MTT that adds again 20 μ L5mg/mL, continue to cultivate 4h, then liquid in hole is absorbed, in every hole, add 200 μ L DMSO jolting 10min, under 492nm, detect the light absorption value in every hole, and record result.Experimental result is as shown in table 4.
The protective effect to endothelial cell damage due to high sugar of table 4. different pharmaceutical and prescription sample liquid 1-17 group
Note: with the comparison of blank group,
#represent P<0.05,
##represent P<0.01; With model group comparison, * represents P<0.05, and * * represents P<0.01.
Experimental result shows, prescription sample liquid 1-17 group, calcium dobesilate and and XUEMINGMU PIAN all can extremely significantly increase HUVEC cell viability (P<0.01), prescription sample liquid 1-17 group effect is better than and XUEMINGMU PIAN, maintains an equal level with calcium dobesilate effect.Metformin also has the effect of certain reparation endothelial cell damage, but effect is not as good as prescription sample liquid 1-17 group, calcium dobesilate and and XUEMINGMU PIAN.
Weight analysis result
Each medicine comprehensive drug weight method is as follows:
Resultant effect=repair harm with * short the secreting effect * 20%+ sensitization * 20%+ of 40%+ but enzyme effect * 20%
By weight analysis, the comprehensive function of prescription sample liquid 1 and each positive drug is as shown in table 5 below
Table 5 prescription and each positive drug comprehensive drug result table
From result, the drug effect of calcium dobesilate and XUEMINGMU PIAN, metformin, glibenclamide, acarbose is comparatively single, mainly concentrate in a certain respect, and prescription sample liquid 1-17 group have comparatively balanced shortly secrete, enhanced sensitivity, inhibition α glycosidase and the drug effect of repairing endothelial cell damage, wherein repairing endothelial cell damage effect and sensitization and positive drug remains basically stable, and final result shows that the comprehensive drug of prescription sample liquid 1-17 group is optimum, show that prescription sample liquid 1-17 group compares and have optimal efficiency with other positive drug.
Brief summary:
Distinguish by experiment study group's quadrat sampling product 1-17 group and different positive drug to the effect of RIN cell insulin secretagogue, HepG2 cell Insulin Resistance, alpha-glucosidase inhibitory action, the repair of HUVEC cell injury.Select the comprehensive function comparison of prescription sample liquid 1 and different positive drug.Result shows that the comprehensive drug of prescription sample liquid 1-17 group is better than other five kinds of positive drug, has verified the optimal efficiency of prescription sample liquid 1-17 group from external drug effect angle.
Test example 5STZ lumbar injection causes the model experiment of rat diabetes ocular complications
1, experiment material: 176 of SD rats (male, 180-220g).
2, improving and grouping administration of rat diabetes ocular complication model:
Modeling agent STZ solution is 1%STZ citric acid solution.
Rat once divides into groups: 176 male SD rat average weights reach about 220g, is divided at random 22 groups.Be respectively blank group, model group, calcium dobesilate group and XUEMINGMU PIAN group, metformin group, prescription sample liquid 1-17 group.
The modeling of STZ diabetes rat model: water 14h is can't help in rat fasting, left lower quadrant injection modeling agent 1%STZ solution carries out modeling.Dosage 60mg/kg.4h after injection STZ, gavage gives 50% D/W 10mL/kg, to prevent that rat is because of hypoglycemia death.
The mensuration of rat modeling blood glucose and secondary grouping:
After modeling the 3rd day, since 8 of mornings, rat is carried out to fasting, 6 pm cuts tail and gets blood, and blood glucose meter is measured blood sugar level.Blood glucose is greater than 16.7mmol/L and thinks formation hyperglycemia model.Because later stage testing index needs rat eye sample, so can not get blood by eye socket, select to cut tail and get blood, use blood glucose meter to measure blood glucose.The rat feeding that does not form hyperglycemia carries out modeling for the second time after fasting 14h after 3 days, complement STZ dosage is 60mg/kg.Within after complement the 3rd day, measure fasting glucose, each rat blood sugar, higher than 16.7mmol/L, is divided into 22 groups at random.
Through SPSS statistics, except blank group, all the other 21 groups of blood glucose values think overall from same sample, can carry out parallel laboratory test research relatively, and concrete blood glucose value is at 23 ± 4mmol/L.
Administration: each is organized rat and forms the beginning administration in the 2nd day after hyperglycemia grouping, and it is as follows that each organizes dosage: blank group: gavage gives 0.5%CMC-Na solution once a day, administration volume 20mL/kg; Model group: gavage gives 0.5%CMC-Na solution once a day, administration volume 20mL/kg; Positive group (the 8mg/mL calcium dobesilate group) dosage of Western medicine is decided to be 160mg/kg, administration volume 20mL/kg; The positive group (24mg/mL and XUEMINGMU PIAN) of Chinese medicine: and XUEMINGMU PIAN dosage is 480mg/kg, administration volume 20mL/kg; 7.5mg/mL metformin hydrochloride group: metformin dosage is 150mg/kg, administration volume 20mL/kg; Prescription sample 1-17 group: dosage is 3.6g crude drug/kg/ days.
The preparation of prescription sample liquid 1: in the ratio of 1:2:3:1, take stir-baked FLOS SOPHORAE IMMATURUS 10g, Radix Scutellariae 20g, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 30g, Pericarpium Citri Reticulatae 10g, be placed in 2500mL round-bottomed flask, add the alcoholic solution that 10 times of amount mass concentrations of medical material gross weight are 70%, soak after 1h 85 ℃ of reflux, extract, 1h, 16 layers of filtered through gauze, obtain filtrate.With method, extract 3 times, merge filtrate 3 times, 70 ℃ of reclaim under reduced pressure, to small size, are 389mL with CMC-Na solution standardize solution, and concentration is 0.18g/mL.Administration volume is 20mL/kg, and dosage is 3.6g/kg.
The preparation of prescription sample liquid 2-16: respectively in the regulation ratio under 1 of test example, get the sample medical material of prescription 2-16, then by " preparation of prescription sample medicinal liquid 1 " method, preparation prescription sample medicinal liquid 2-16.Concentration is respectively 0.18g/mL.Administration volume is 20mL/kg, and dosage is respectively 3.6g/kg.
The preparation of prescription sample liquid 17: the ratio in the medical material 1:2:3:1 of prescription 1, take stir-baked FLOS SOPHORAE IMMATURUS 1g, Radix Scutellariae 2g, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3g, Pericarpium Citri Reticulatae 1g, is placed in 250mL round-bottomed flask, adds 10 times of water gagings, soaks after 1h, and 1h is extracted in water-bath, and 16 layers of filtered through gauze, obtain filtrate.With method, extracting 3 times, merge filtrate 3 times, on 80 ℃ of water-baths, steam to small size, is 389mL with CMC-Na solution standardize solution, and concentration is 0.18g/mL.Administration volume is 20mL/kg, and dosage is 3.6g/kg.
3, sample collection:
Serum sample gathers: every rat femoral is got blood 4mL, after being placed in the standing 2h of 5ml centrifuge tube, the centrifugal 10min sucking-off of 3500rpm upper serum is placed in ℃ freezing preservation of 1.5mL centrifuge tube-20, for measuring wherein SOD vigor, blood sugar content and insulin level etc.
Eyeball, retina and crystalline lens collection: rapid separated left and right eyeball after every rat extracting blood, left eye is placed on ice making platform, with pin, insert in eyeball surrounding tissue fixing, along corneoscleral junction, peel off cornea, separated crystalline lens, crystalline lens is placed on graph paper and takes pictures rapidly, for scoring; All the other right eye balls are placed in ℃ freezing preservation of 2mL centrifuge tube-20, for measuring wherein aldose reductase vigor.
Use operating scissors rip cutting optic cup, use tweezers softly to peel off retina after upset, isolated retina is placed in ℃ freezing preservation of 2mL centrifuge tube-20, for measuring wherein VEGF, ICAM-1 and AGEs protein content.
Each index of correlation is measured:
Rat blood sugar and body weight determination method: diabetes rat model is set up and started and within every 2 weeks, measures a body weight after administration, within every 4 weeks, uses Johnson & Johnson's blood glucose meter (TouchEasy, johnson & johnson, USA) to measure blood glucose one time.
The assay method of VEGF, ICAM-1, AGEs content in rat retina:
The acquisition method of sample: every rat femoral is got rapid separated left and right eyeball after blood, left eye on ice platform pin fixedly tailing edge corneoscleral junction peel off cornea, rip cutting optic cup after separated crystalline lens, separated retina after upset, isolated retina is placed in ℃ freezing preservation of 2mL centrifuge tube-20, wherein VEGF to be determined, ICAM-1 and AGEs protein content.
The preparation of retina analyte sample fluid: every experimental rat retinoid-like is originally placed in respectively to 1mL glass homogenizer, use liquid-transfering gun to add respectively 120 μ L to organize lysate, the beaker ice bath that homogenizer is placed in to filled with ice slag is ground to evenly translucent, then sucking-off homogenate, put in 1.5mL centrifuge tube, in 4 ℃ of centrifugal 10min (16000rpm), supernatant is testing sample.By supernatant sucking-off and be sub-packed in the Dorf pipe of 3 200 μ L, every group of rat is contained in a little valve bag for measuring the homogenate of same protein content, 12 groups of rats for measure same protein content homogenate be divided in a large valve bag, be divided into and fill 3 large valve bag pipes (measuring total protein content for measuring 3 hatching egg Pseudobulbus Bletillae (Rhizoma Bletillae)), freezing standby in-20 ℃.
According to each ELISA test kit explanation, measure respectively VEGF, ICAM-1 in each sample, AGEs content, and use total protein content in each sample of BCA kit measurement, after correction, obtain above each factor protein expression content (pg/mg histone) in retinal tissue Unit Weight.
Determining the protein quantity in sample:
Protein standard curve plotting: standard substance are pressed to 0,1,2,4,8,12,16,20 μ L are added in 96 orifice plates, and each hole adds standard substance diluent and supplies 20 μ L, each hole adds 200 μ L BCA working solutions, 37 ℃ of reaction 30min, measure each hole light absorption value under 570nm, take OD value as abscissa, standard protein concentration is that vertical coordinate is done regression equation, obtains standard curve.
Determining the protein quantity in sample: get and respectively organize each 2 μ L of rat retina homogenate sample liquid, be placed in 96 orifice plates, each hole adds standard substance diluent and supplies 20 μ L, each hole adds 200 μ L BCA working solutions subsequently, 37 ℃ of reaction 30min, measure each hole light absorption value under 570nm, substitution standard curve equation, obtains and respectively organizes histone concentration in sample.
Aldose reductase vitality test in large rathole: the method for pressing test kit regulation is measured.
SOD vigor, insulin content and blood-sugar level measuring in serum: blood serum acquisition method is as described under sample collection method item, and centrifugal gained serum is used for measuring SOD vigor, blood glucose value and insulin content.Assay method is undertaken by test kit regulation.
Insulin content determination step: get and respectively organize rat blood serum 10 μ L, use ELISA kit measurement serum insulin content.
Blood-sugar level measuring step: 12 weeks rat fasting blood-glucose values of administration adopt glucose kit to measure.
Crystalline lens is taken pictures and is marked: because color can change to some extent after crystalline lens separation, therefore be placed in immediately on graph paper after respectively organizing rat lens separation, under fixed-illumination, use CANNON1000D photographing unit fixed aperture and aperture time to take pictures, according to each crystalline lens clarification degree, adopt LOCS III marking system that the crystalline lens of each group rat is marked and added up.
Standards of grading are as follows:
0 minute: transparent clarification; 1 minute: a small amount of point turbidity (I phase); 2 minutes: cortex muddy (II phase); 3 minutes: kernel muddy (III phase); 4 minutes: cortex and kernel completely muddy (IV phase).
Each organize numerical value all with
represent, between each group, crystalline lens turbidity is analyzed with Ridit, and other each group difference adopts t-test to carry out statistical analysis, and P<0.05 thinks significant difference, and P<0.01 thinks utmost point significant difference.
(1) each prescription sample liquid 1-17 group and the influence result of different positive control drug to blood glucose
Experimental result is as shown in table 6, and diabetes rats, more than duration of test blood glucose maintains 16mmol/L all the time, is compared remarkable rising (p<0.01) with normal group.Metformin group rat blood sugar all significantly decline administration 4 weeks, 8 weeks, 12 weeks time (p<0.01); Calcium dobesilate group rat is at administration blood glucose significantly decline (p<0.05) in the time of 4 weeks; With XUEMINGMU PIAN group rat blood sugar at administration blood glucose significantly decline (p<0.05) in the time of 12 weeks; Prescription sample liquid 1-17 group (3.6g crude drug/kg) administration is all extremely significantly decline (p<0.01) of blood glucose in the time of 4 weeks, 8 weeks, 12 weeks.
Above result shows that the prescription sample liquid 1-17 of diabetic ophthalmopathy compound recipe can significantly reduce blood glucose in diabetic rats, its hypoglycemic effect be better than this sulfoacid calcium of hydroxyl and and XUEMINGMU PIAN, but not as good as metformin.
Table 6. different pharmaceutical and prescription sample liquid 1-17 group are to 12 weeks rear change of blood sugar of rat
Note: with the comparison of blank group,
#represent P<0.05,
##represent P<0.01; With model group comparison, * represents P<0.05, and * * represents P<0.01.
(2). each prescription sample liquid 1-17 group and the affect result of different positive control drug on retina VEGF
Experimental result is as shown in table 7, and in diabetic model group rat retina, vegf expression is compared remarkable rising (p<0.01) with normal group; In calcium dobesilate group and XUEMINGMU PIAN group, prescription sample liquid 1-17 group rat (3.6g/kg) retina, vegf expression significantly declines; Metformin group rat vegf expression slightly declines than normal group, but without significant difference.
The drug effect that prescription sample liquid 1-17 group suppresses vegf expression is better than and XUEMINGMU PIAN, metformin, suitable with calcium dobesilate.
Table 7. different pharmaceutical and prescription sample liquid 1-17 group are expressed measurement result to vegf protein in rat retina
Note: with the comparison of blank group,
#represent P<0.05,
##represent P<0.01; With model group comparison, * represents P<0.05, and * * represents P<0.01.
(3). each prescription sample liquid 1-17 group and different positive control drug are on the diabetes complicated cataractous result that affects
There is cataract in diabetes rat, after administration 12 weeks, sampling is respectively organized rat take pictures one by one the previous day successively since the 8th week, serious cataract appears in the normal and model group rat of blank group rat eye.Administration gathers and respectively organizes rat lens while sampling after 12 weeks, use CANNON1000D photographing unit fixed aperture and aperture time to be placed in and on graph paper, take pictures and according to each crystalline lens clarification degree employing LOCS III marking system, the crystalline lens of each group rat marked and added up, the results are shown in Table 8.
Table 8. different pharmaceutical and prescription sample liquid 1-17 group change experimental result to rat cataract crystalline lens
Note: with the comparison of blank group,
#represent P<0.05,
##represent P<0.01; With model group comparison, * represents P<0.05, and * * represents P<0.01.
From experimental result, there is serious cataract in diabetic model group group rat, and scoring total points is 10 minutes; Metformin and calcium dobesilate group respectively total points are respectively 7 minutes and 8 minutes; The scoring total points of prescription sample liquid 1-17 group is 0 minute; Ridit analyzes and shows prescription sample liquid 1-17 group and and with XUEMINGMU PIAN group, the formation of diabetic cataract had to certain prevention and therapeutical effect.
(4). each prescription sample liquid 1-17 group and different positive control drug affect result to rat blood serum insulin content
It is to destroy rat Langerhans islet tissue that this experiment adopts streptozotocin to cause the cardinal principle of Hyperglycemia In Stz-induced Diabetic Rats, cause hypoinsulinism, in order to inquire into YSXP compound recipe, whether there is the effect that promotes insulin secretion, each group rat blood serum insulin content is measured.
From experimental result, model group rat blood serum insulin content compared with normal group significantly decline (p<0.01); Compare with model group, prescription sample liquid 1-17 can significantly improve insulin content in rat blood serum (p<0.05); Metformin, calcium dobesilate and and XUEMINGMU PIAN to serum insulin content do not make significant difference (p>0.05).
Table 9. different pharmaceutical and prescription sample liquid 1-17 group change experimental result to rat blood serum insulin content
Note:
#expression has significant difference (P<0.05) with blank group ratio,
##expression has utmost point significant difference (P<0.01) * to represent there is significant difference (P<0.05) with model group ratio with blank group ratio.
(5). each prescription sample liquid 1-17 group and different positive control drug affect result to insulin resistance index HOMA-IR
HOMA-IR is that computational methods are as follows for evaluating the index of insulin resistant level:
HOMA-IR=fasting glucose (mmol/L) * fasting insulin level (Mu/l)/22.5
[11]
By experimental result known (table 10), model group Insulin Resistance of Rats index compared with normal group significantly raise (p>0.01); Compare with model group, metformin group Insulin Resistance of Rats index significantly reduces (p<0.01); Prescription sample liquid 1-17 Insulin Resistance of Rats index (3.6g crude drug/kg) also significantly reduces (p<0.05); Calcium dobesilate and and XUEMINGMU PIAN group Insulin Resistance of Rats index without significant change (p>0.05).
Table 10. different pharmaceutical and prescription sample liquid 1-17 group are to Insulin Resistance of Rats index HOMA-IR
Note:
#expression has significant difference (P<0.05) with blank group ratio,
##expression has utmost point significant difference (P<0.01) * to represent there is significant difference (P<0.05) with model group ratio with blank group ratio, and * * represents there is utmost point significant difference (P<0.01) with model group ratio.
(6). each prescription sample liquid 1-17 group and the different positive control drug result that affects on retinitis inflammation factor ICAM-1, TNF-α, IL-1 β expression
Experimental result is as shown in table 11, and in rat model retina, ICAM-1, TNF-α and IL-1 β compare all significantly raise (p<0.01) with normal group; Calcium dobesilate group and and XUEMINGMU PIAN group rat retina in above 3 kinds of factor expressions all significantly decline (p<0.01); All significantly decline (p<0.01) of expression of above 3 factors in prescription sample liquid 1-17 group rat (3.6g/kg) retina; Metformin group rat ICAM-1, IL-1 β content significantly decline (p<0.05, p<0.05), and TNF-alpha content slightly declines but compares with model group without significant difference (p>0.05).
Table 11. different pharmaceutical and prescription sample liquid are to cytokine protein expression measurement result in rat retina
Note:
#expression has significant difference (P<0.05) with blank group ratio,
##expression has utmost point significant difference (P<0.01) * to represent there is significant difference (P<0.05) with model group ratio with blank group ratio, and * * represents there is utmost point significant difference (P<0.01) with model group ratio.
(7). each prescription sample liquid 1-17 group and the affect result of different positive control drug on retina nonenzymatic glycosylation product (AGEs) content
Result is as shown in table 12, and in rat model retina, AGEs content is compared remarkable rising (p<0.01) with normal group; With all significantly decline (p<0.01) of AGEs content in XUEMINGMU PIAN group rat retina; In prescription sample liquid 1-17 group rat (3.6g/kg) retina, the expression of AGEs content all significantly declines; In calcium dobesilate group and metformin group rat retina, AGEs content slightly declines but compares with model group without significant difference (p>0.05).
Table 12. different pharmaceutical and prescription sample liquid 1-17 group are to AGEs assay result in rat retina
Note:
#expression has significant difference (P<0.05) with blank group ratio,
##expression has utmost point significant difference (P<0.01) * to represent there is significant difference (P<0.05) with model group ratio with blank group ratio, and * * represents there is utmost point significant difference (P<0.01) with model group ratio.
(8). each prescription sample liquid 1-17 group and different positive control drug are on the result that affects on large rathole aldose reductase
Diabetes rat eye aldose reductase vitality test experimental result is as shown in table 13, compare with normal group, the large rathole aldose reductase of model group vigor significantly raise (p<0.01), calcium dobesilate group and the large rathole aldose reductase of XUEMINGMU PIAN group vigor significantly reduce (p<0.01, p<0.05); The large rathole aldose reductase vigor of prescription sample liquid 1-17 group (3.6g crude drug/kg) also significantly reduces (p<0.01); The large rathole aldose reductase of metformin group vigor is compared with model group without significant difference (p>0.05).
Table 13. different pharmaceutical and prescription sample liquid 1-17 group are to large rathole aldose reductase vitality test result
Note:
#expression has significant difference (P<0.05) with blank group ratio,
##expression has utmost point significant difference (P<0.01) * to represent there is significant difference (P<0.05) with model group ratio with blank group ratio, and * * represents there is utmost point significant difference (P<0.01) with model group ratio.
(9). each prescription sample liquid 1-17 group and different positive control drug affect result to diabetes rat antioxidative
From table 14 experimental result, model group rat SOD value compared with normal group significantly decline (p<0.01); Compare with model group, in each administration group rat blood serum, SOD vigor all has remarkable rising (p<0.05, p<0.01), its prescription sample liquid 1-17 group and and XUEMINGMU PIAN effect be better than metformin and calcium dobesilate.
Table 14. different pharmaceutical and prescription sample liquid 1-17 group are to rat blood serum SOD vitality test result
Note:
#expression has significant difference (P<0.05) with blank group ratio,
##expression has utmost point significant difference (P<0.01) * to represent there is significant difference (P<0.05) with model group ratio with blank group ratio, and * * represents there is utmost point significant difference (P<0.01) with model group ratio.
(10). each prescription sample liquid 1-17 group and different positive control drug are to organizing after weight in the comparison of body comprehensive drug
In order to compare more intuitively the optimal efficiency of prescription carrier drug effect, take prescription sample liquid 1 as the comprehensive drug comparative analysis of representative from different positive drug, the comparative approach with reference to external comprehensive drug, carries out weight to the drug effect of prescription sample liquid 1 and different positive drug.By following 2 kinds of methods, carry out weight: method 1 is carried out weight with final pharmacodynamics index (fasting glucose, retinal thickness and VEGF content, the muddy degree of crystalline lens); Method 2 is with pharmacodynamics index (the same) and the common weight of mechanism (insulin content, insulin resistance index, AR vigor, antiinflammatory index, AGEs content, Antioxidant Indexes).Concrete grammar is as follows:
Method 1: comprehensive drug=reduction fasting glucose effect * 40%+ treatment diabetic retinal tissue in rat pathological changes effect (thicken retina and reduce VEGF effect) * 30%+ reduces muddy degree * 30% of diabetes rat crystalline lens
Method 2: comprehensive drug=reduction fasting glucose effect * 20%+ increases insulin secretion effect * 10%+ increase insulin sensitivity effect * 10%+ treatment diabetic retinal tissue in rat pathological changes effect (reducing VEGF effect) * 10%+ reduction diabetes rat crystalline lens muddy degree * 10%+ reduction AR vigor effect * 10%+ and reduces AGEs content effect * 10%+ antiinflammatory action * 10%+ antioxidation * 10%
Method 1 weight result is as shown in Table 15.Result shows to be better than metformin, calcium dobesilate and and XUEMINGMU PIAN by YSXP comprehensive drug after method 1 weight.
1 group of table 15. prescription sample liquid to and different positive control drug weight after comprehensive drug (the first evaluation method)
Method 2 weight results are shown in table 16.Result shows to be better than metformin, calcium dobesilate and and XUEMINGMU PIAN by prescription sample liquid 1 comprehensive drug after method 2 weights.
1 group of table 16. prescription sample liquid to and different positive control drug weight after comprehensive drug (the second evaluation method)
Experiment adopts lumbar injection STZ (60mg/kg) to cause Hyperglycemia In Stz-induced Diabetic Rats model, selecting metformin is blood sugar lowering positive drug, select simultaneously calcium dobesilate and and XUEMINGMU PIAN as treatment diabetic complication Western medicine and Chinese medicine positive drug, curative effect by comparable group side sample liquid 1-17 group with above positive drug, the blood sugar lowering of overall merit diabetic ophthalmopathy compound recipe and treatment ocular complications effect ，Bing study group side sample liquid 1-17 group compare whether have optimal efficiency with above positive drug.
More than experimental session model group rat blood sugar is stabilized in 16mmol/L, show to be hyperglycemia state experimental session rat, and not because there is spontaneous decline compared with length experimental period in blood glucose.
Experiment conclusion:
1) dosage is that 3.6g crude drug/kg prescription sample liquid 1-17 group can significantly reduce blood glucose in diabetic rats, and the rat blood sugar of each prescription group is in administration i.e. significantly decline (0<0.01) of blood glucose after 4 weeks; Prescription sample liquid 1-17 organizes its hypoglycemic effect lower than metformin, but is better than calcium dobesilate and and XUEMINGMU PIAN.
2) compare with normal group, model group rat lens cortex and kernel are completely muddy, through scoring, be judged to be IV phase cataract, metformin group and calcium dobesilate group rat all have III phase or IV phase Cataractogenesis, prescription sample liquid 1-17 group and and XUEMINGMU PIAN group rat have no III phase and IV phase cataract; Appraisal result and Ridit analyze and show that the scoring of prescription sample liquid 1-17 group rat is significantly lower than model group (P<0.05), show that compound recipe can stop the cataractous formation of diabetes rat, and metformin and calcium dobesilate to diabetic cataract without obvious effect.
3) compare with normal group, diabetic model group rat blood serum insulin content significantly reduces (p<0.01), HOMA-IR significantly raises (p<0.01), show that diabetes rat amount of insulin secretion reduces, and insulin sensitivity degree declines.Through treatment, prescription sample liquid 1-17 group rat blood serum insulin content significantly raises (p<0.05), and HOMA-IR significantly reduces (p<0.05); Metformin group rat HOMA-IR significantly reduces (p<0.01), shows that prescription sample liquid 1-17 group is at the content of rising diabetes rat serum insulin, and has reduced insulin resistant degree.
4) compare with normal rats, in diabetic retinal tissue in rat, VEGF, ICAM-1, TNF-α, IL-1 β, AGEs express significantly and raise, and full eye aldose reductase vigor also significantly raises simultaneously; Metformin group rat through treatment after only ICAM-1, IL-1 β content significantly reduce (p<0.05), other indexs have no significant change (p>0.05); Calcium dobesilate group rat VEGF, ICAM-1, TNF-α, IL-1 β content and aldose reductase vigor after treatment all significantly reduce (p<0.01), but AGEs content is without significant change; Above index after treatment all significantly reduces (p<0.05, p<0.01) with XUEMINGMU PIAN and prescription sample liquid 1-17 group rat, and prescription sample liquid 1-17 group curative effect is better than and XUEMINGMU PIAN group.
5) compare diabetes rats serum activity of SOD significantly decline (p<0.01) with normal group; SOD vigor in prescription sample liquid 1-17 group rat blood serum significantly raises (p<0.01), effect be better than calcium dobesilate and and XUEMINGMU PIAN; SOD vigor in metformin group rat blood serum significantly raises (p<0.05), but the effect of metformin antioxidative is inferior to prescription sample liquid 1-17 group.
In sum, prescription sample liquid 1-17 group has the effect of significant treatment diabetes and ocular complications thereof, and its curative effect is with metformin, calcium dobesilate and compare and have optimal efficiency with XUEMINGMU PIAN; Prescription sample liquid 1-17 group treatment DR and cataractous mechanism are by increasing amount of insulin secretion, and thereby the body that raises reduces blood glucose to the sensitivity of insulin, control on this basis oxidative damage simultaneously, and suppress the activation of sorbitol pathway, AGEs path and PKC path.
The specific embodiment
By specific embodiment, further illustrate the present invention below.But the detail of embodiment only, for explaining the present invention, should not be construed as limited overall technical solution.
Embodiment 1
Stir-baked FLOS SOPHORAE IMMATURUS 1 weight portion, Radix Scutellariae 2 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3 weight portions, Pericarpium Citri Reticulatae 1 weight portion.
Preparation method: take stir-baked FLOS SOPHORAE IMMATURUS, Radix Scutellariae, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, Pericarpium Citri Reticulatae extracts three times with the alcohol heating reflux of concentration 60%, each extraction 2,2,1.5 hours is respectively 12,10,8 times of weight of medical material at every turn by the amount of ethanol, filter, merge 3 times filtrate, decompression recycling ethanol, concentrated.
Embodiment 2
Stir-baked FLOS SOPHORAE IMMATURUS 2 weight portions, Radix Scutellariae 3 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1 weight portion, Pericarpium Citri Reticulatae 1 weight portion.Preparation method is with embodiment 1.
Embodiment 3
Stir-baked FLOS SOPHORAE IMMATURUS 3 weight portions, Radix Scutellariae 1 weight portion, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 2 weight portions, Pericarpium Citri Reticulatae 1 weight portion.Preparation method is with embodiment 1.
Embodiment 4
Stir-baked FLOS SOPHORAE IMMATURUS 1 weight portion, Radix Scutellariae 2 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3 weight portions, Pericarpium Citri Reticulatae 2 weight portions.Preparation method is with embodiment 1.
Embodiment 5
Stir-baked FLOS SOPHORAE IMMATURUS 2 weight portions, Radix Scutellariae 3 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1 weight portion, Pericarpium Citri Reticulatae 2 weight portions.Preparation method is with embodiment 1.
Embodiment 6
Stir-baked FLOS SOPHORAE IMMATURUS 3 weight portions, Radix Scutellariae 1 weight portion, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 2 weight portions, Pericarpium Citri Reticulatae 2 weight portions.Preparation method is with embodiment 1.
Embodiment 7
Stir-baked FLOS SOPHORAE IMMATURUS 1 weight portion, Radix Scutellariae 2 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3 weight portions, Pericarpium Citri Reticulatae 3 weight portions.Preparation method is with embodiment 1.
Embodiment 8
Stir-baked FLOS SOPHORAE IMMATURUS 2 weight portions, Radix Scutellariae 3 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1 weight portion, Pericarpium Citri Reticulatae 3 weight portions.Preparation method is with embodiment 1.
Embodiment 9
Stir-baked FLOS SOPHORAE IMMATURUS 3 weight portions, Radix Scutellariae 1 weight portion, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 2 weight portions, Pericarpium Citri Reticulatae 3 weight portions.Preparation method is with embodiment 1.
Embodiment 10
Stir-baked FLOS SOPHORAE IMMATURUS 1 weight portion, Radix Scutellariae 1 weight portion, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1 weight portion, Pericarpium Citri Reticulatae 1 weight portion.Preparation method is with embodiment 1.
Embodiment 11
Stir-baked FLOS SOPHORAE IMMATURUS 1 weight portion, Radix Scutellariae 1 weight portion, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1 weight portion, Pericarpium Citri Reticulatae 2 weight portions.Preparation method is with embodiment 1.
Embodiment 12
Stir-baked FLOS SOPHORAE IMMATURUS 1 weight portion, Radix Scutellariae 1 weight portion, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1 weight portion, Pericarpium Citri Reticulatae 3 weight portions.Preparation method is with embodiment 1.
Embodiment 13
Stir-baked FLOS SOPHORAE IMMATURUS 2 weight portions, Radix Scutellariae 2 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 2 weight portions, Pericarpium Citri Reticulatae 1 weight portion.Preparation method is with embodiment 1.The medicinal liquid obtaining adds the acceptable adjuvant of conventional pharmaceutical to make capsule according to conventional method.
Embodiment 14
Stir-baked FLOS SOPHORAE IMMATURUS 2 weight portions, Radix Scutellariae 2 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 2 weight portions, Pericarpium Citri Reticulatae 3 weight portions.Preparation method is with embodiment 1.The medicinal liquid obtaining adds the acceptable adjuvant of conventional pharmaceutical according to the agent of conventional method granulation.
Embodiment 15
Stir-baked FLOS SOPHORAE IMMATURUS 3 weight portions, Radix Scutellariae 3 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3 weight portions, Pericarpium Citri Reticulatae 2 weight portions.Preparation method is with embodiment 1.The medicinal liquid obtaining adds the acceptable adjuvant of conventional pharmaceutical to be pressed into tablet.
Embodiment 16
Stir-baked FLOS SOPHORAE IMMATURUS 3 weight portions, Radix Scutellariae 3 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3 weight portions, Pericarpium Citri Reticulatae 1 weight portion.Preparation method is with embodiment 1.The medicinal liquid obtaining adds conventional adjuvant to make pill according to conventional method.
Embodiment 17
Stir-baked FLOS SOPHORAE IMMATURUS 3 weight portions, Radix Scutellariae 3 weight portions, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 3 weight portions, Pericarpium Citri Reticulatae 1 weight portion.
Preparation method: according to aforementioned proportion, take stir-baked FLOS SOPHORAE IMMATURUS, Radix Scutellariae, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, Pericarpium Citri Reticulatae, water heating extraction three times is extracted 1,1,0.5 hour at every turn, and the amount of each water is respectively 12,10,8 times of weight of medical material.Filter, merge filtrate 3 times, concentrated.
Claims (10)
1. a pharmaceutical composition for treatment and prevent diabetes and ocular complication thereof, is characterized in that said composition made by the component of following weight portion:
Stir-baked FLOS SOPHORAE IMMATURUS 1-5 part, Radix Scutellariae 1-5 part, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1-5 part, Pericarpium Citri Reticulatae 1-5 part.
2. the pharmaceutical composition for the treatment of according to claim 1 and prevent diabetes and ocular complication thereof, is characterized in that said composition made by the component of following weight portion:
Stir-baked FLOS SOPHORAE IMMATURUS 1-3 part, Radix Scutellariae 1-3 part, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution 1-3 part, Pericarpium Citri Reticulatae 1-3 part.
3. the pharmaceutical composition for the treatment of according to claim 1 and prevent diabetes and ocular complication thereof, is characterized in that said composition made by the component of following weight portion:
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 2 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 1 part of Radix Scutellariae, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 2 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 1 part of Radix Scutellariae, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 2 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 1 part of Radix Scutellariae, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 1 part of Radix Scutellariae, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 1 part of Radix Scutellariae, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
1 part of stir-baked FLOS SOPHORAE IMMATURUS, 1 part of Radix Scutellariae, 1 part of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 2 parts of Radix Scutellariaes, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae;
2 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 2 parts of Radix Scutellariaes, 2 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 3 parts of Pericarpium Citri Reticulataes;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 2 parts of Pericarpium Citri Reticulataes;
3 parts of stir-baked FLOS SOPHORAE IMMATURUSs, 3 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae.
4. pharmaceutical composition according to claim 1, is characterized in that said composition made by the component of following weight portion: 1 part of stir-baked FLOS SOPHORAE IMMATURUS, 2 parts of Radix Scutellariaes, 3 parts of stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution, 1 part of Pericarpium Citri Reticulatae.
5. according to the pharmaceutical composition described in claim 1,2,3 or 4, compositions and pharmaceutically acceptable carrier described in it is characterized in that are made preparation.
6. pharmaceutical composition according to claim 5, is characterized in that described preparation is oral liquid, pill, tablet, powder, granule, capsule, drop pill or injection.
7. the preparation method of the pharmaceutical composition described in a claim 1,2,3 or 4, it is characterized in that the method comprises the following steps: take stir-baked FLOS SOPHORAE IMMATURUS, Radix Scutellariae, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution and Pericarpium Citri Reticulatae, adding medical material gross weight 8-12, doubly to measure concentration be that the alcohol heating reflux of 10%-90% extracts 1-3 time, extracts 1-3 hour, filtration at every turn, merging filtrate, concentrated.
8. the preparation method of the pharmaceutical composition described in a claim 1,2,3 or 4, it is characterized in that the method comprises the following steps: take stir-baked FLOS SOPHORAE IMMATURUS, Radix Scutellariae, stir-baked RHIZOMA ANEMARRHENAE before sprinking salt solution and Pericarpium Citri Reticulatae, add 8-12 times of water gaging of medical material gross weight, heating extraction 1-3 time, each 0.5-1 hour that extracts, filter, merging filtrate, concentrated.
9. the application of the pharmaceutical composition described in claim 1,2,3 or 4 in the medicine of preparation treatment or prevent diabetes and ocular complication thereof.
10. application according to claim 9, is characterized in that the ocular complication that described diabetes cause is retinopathy or cataract.
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| CN109030804A (en) * | 2018-08-16 | 2018-12-18 | 青岛大学 | The insulin secretion rating model of glucose stimulation |
| CN114831310A (en) * | 2022-04-13 | 2022-08-02 | 华南农业大学 | Composition for improving eyesight |
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| CN107753767A (en) * | 2017-11-25 | 2018-03-06 | 衡阳县华盛达农林科技有限公司 | A kind of sophora bud traditional Chinese medicine oral liquid and preparation method thereof |
| CN109030804A (en) * | 2018-08-16 | 2018-12-18 | 青岛大学 | The insulin secretion rating model of glucose stimulation |
| CN114831310A (en) * | 2022-04-13 | 2022-08-02 | 华南农业大学 | Composition for improving eyesight |
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| CN103599340B (en) | 2017-01-18 |
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