CN100594913C - Medicine combination for curing diabetes and complication thereof and the preparing method and purpose - Google Patents
Medicine combination for curing diabetes and complication thereof and the preparing method and purpose Download PDFInfo
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- CN100594913C CN100594913C CN200610021191A CN200610021191A CN100594913C CN 100594913 C CN100594913 C CN 100594913C CN 200610021191 A CN200610021191 A CN 200610021191A CN 200610021191 A CN200610021191 A CN 200610021191A CN 100594913 C CN100594913 C CN 100594913C
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Abstract
The present invention provides one kind of medicine composition for treating diabetes and its complication and its preparation process and use. The medicine composition is prepared with astragalus root, kudzu vine root and fleabane as active components, and consists of the effective parts of the said Chinese medicinal materials. It has the functions of promoting blood circulation, dredging meridian and improving eyesight, and possesses determined curative effect.
Description
Technical field
The invention provides a kind of pharmaceutical composition for the treatment of diabetes and complication thereof, particularly, is to be the pharmaceutical composition that feedstock production forms with the Chinese crude drug, belongs to drug world.
Background technology
Diabetic retinopathy (diabetic retinopathy, DR) be diabetes (diabetes mellitus, DM) the most common, have a strong impact on vision and the most thorny ocular complications of treatment, be one of common cause of blinding.Along with the raising of social economy's level, the quick growth of population and the modernization and the social aging of life pattern, the sickness rate and the prevalence of diabetes rise year by year.Show that according to WHO diabetes epidemiologic data in 2002 prevalence of diabetes has reached 6.0%～7.6% in western countries, global diabetics has reached 1.77 hundred million, predict to the year two thousand thirty with double surge to 3.66 hundred million.Diabetes have leapt to one of five kinds of the highest diseases of whole world M ﹠ M.
The sickness rate of diabetes has the trend that increases gradually in recent years, the survey showed that for 1997 11 provinces and cities of China, the onset diabetes rate of China rises to 3.26% from the less than 1% at the beginning of the eighties, in the part urbanite even surpassed 5%, estimate existing 4,000 ten thousand diabeticss of China.It is predicted that by 2015, the diabetics of China will reach 6,000 ten thousand.DR is the chronic blood capillary neural complication that DM occurs the earliest.According to the literature, 30～50% merge DR among the DM crowd, and wherein 1/4 has obvious visual disorder, and life quality and health level seriously descend, and its blind rate is 8～12%.Along with the rising year by year of DM sickness rate, be that the neural microvascular complication of the DM of representative endangers more and more serious for numerous patients DM with DR.
U.S. ETDRS studies show that blood sugar control, blood pressure are useful to the DR treatment, development process, preservation central vision that the optical fundus laser light is coagulated DR capable of blocking be one of treatment means of generally acknowledging, but laser light can be damaged peripheral visual field with fixed attention, the vision of forfeiture also is difficult to recover, and has limited its application.Medicine is unsatisfactory to the therapeutic effect of DR at present simultaneously, makes the control of DR become a difficult problem of present puzzlement medical circle.
The treatment of DR is a complicated system engineering, blood sugar control is the basic principle of treatment DR, but blood sugar control can not stop all DR patients' development fully, has report to show through after the strict glycemic control, still has part DR patient's retina pathological changes increasing the weight of in various degree
[1,2]Treatment measure at DR mainly comprises medicine, and developing of DR retinopathy can not prevented or control to laser and vitrectomy etc. but still have a kind of active drug or Therapeutic Method so far.Doxium (Doxium), Difrarel curative effect in clinical practice are not satisfactory, the methyl vitamin B
12(Methycobal, methycobal), aldose reductase inhibitor (aldose reductase inhibitor, ARI such as Sorbinil)
[3,4], pkc inhibitor
[5], the VEGF inhibitor
[5], the protein non-enzyme glycosylation inhibitor
[6](as aminoguanidine) etc. still be in clinical before or the clinical research stage.(the Early TreatmentDiabetic retinopathy Study ETDRS) confirms the aspirin anti-inflammatory treatment to DR to no effect in the early diabetic retinopathy treatment research of the U.S.
[7]For propagation phase DR, at present main treatment means is that laser light is coagulated and vitrectomy, though certain effect is arranged, also has certain limitation, also can the infringement of the concurrent visual field, serious consequence such as detachment of retina
[8]
Chinese medicine thinks that the basic pathogenesis of DR is: deficiency of both QI and YIN, caused by liver and kidney deficiency, the resistance of the order network stasis of blood, wherein non-propagation phase DR is based on deficiency of both QI and YIN, deficiency of the liver and kindey, the retardance of order network, enter the propagation after date, then deficiency of both YIN and YANG occurs and be its outstanding feature with obstruction of collaterals by blood stasis, the turbid interior life of expectorant and deficiency of YIN affecting YANG.
The empty stasis of blood is also controlled the method for treatment that Chinese medicine compound adopts the tonify deficiency blood stasis dispelling, carries out integrated control at the basic pathogenesis of the non-propagation of DR phase, and curative effect is better than Western medicine Doxium capsule, and can improve patient's vision, significantly improves DR patient's General Symptoms.
In recent years, the research and development of Radix Puerariae receive publicity gradually.Puerarin is one of effective ingredient of separating from Radix Puerariae, has the expansion coronary vasodilator, reduces effects such as peripheral vascular resistance, blood pressure lowering, blood sugar lowering, beta-receptor retardance, and gradually be extensive in the application of ophthalmology
[9-14], use puerarin for clinical ophthalmology foundation is provided, be the effective site of treatment of diabetic retinopathy change based on the osajin composition of puerarin.
The extract formulation of the Radixs Astragali such as Radix Astragali injection contains triterpenoid saponin, flavonoid, polysaccharide, aminoacid and trace element.Radix Astragali extract is not only clinical to be used for treatment of diabetes, and shows on treatment of eye disorders and good prospects for application from number of ways diabetic renal papillary necrosis is played certain therapeutical effect
[15-20]From Radix Astragali injection stock solution, isolate 14 chemical compounds, through NMR and MS technical appraisement their structure, comprising 6 isoflavone compounds, 1 Lignum pterocarpi indici hydride compounds, 6 Radix Astragali saponin compounds, the main chemical compositions of Radix Astragali injection includes in above chemical compound.
In recent years, Herba Erigerontis has been carried out big quantity research, and this medicine tried out in the multiple disease of ophthalmology, obtain promising result, Herba Erigerontis and preparation thereof show good prospects for application on treatment of eye disorders, and can early stage and concurrent pathological changes plays certain therapeutical effect to diabetic nephropathy from number of ways
[20-23]
Because the natural medicinal ingredients complexity, compatibility of drugs uses issuable effect difference.At present, still the no-trump Radix Astragali, Radix Puerariae, Herba Erigerontis are the relevant report of material combination treatment diabetes and complication thereof, also do not have active component is wherein extracted the relevant report that compatibility is treated diabetes and complication thereof.
Summary of the invention
Technical problem to be solved by this invention has provided a kind of pharmaceutical composition for the treatment of diabetes and complication thereof, and the present invention also provides this preparation of drug combination method and purposes.
The invention provides a kind of pharmaceutical composition for the treatment of diabetes and complication thereof, it is by containing the medicament that the following weight proportion raw material is prepared from:
10～40 parts of the Radixs Astragali, 10～30 parts of Radix Puerariaes, 10～30 parts of Herba Erigerontiss.
Further, it is the medicament that is formed by the following weight proportion raw material:
20～30 parts of the Radixs Astragali, 20～40 parts of Radix Puerariaes, 20～30 parts of Herba Erigerontiss.
Further, it is the medicament that is prepared from by the following weight proportion raw material:
20 parts of the Radixs Astragali, 30 parts of Radix Puerariaes, 20 parts of Herba Erigerontiss.
Pharmaceutical composition of the present invention is to be active component by the raw material of the Radix Astragali, Radix Puerariae, Herba Erigerontis or water or extractive with organic solvent, adds the medicament that acceptable accessories or complementary composition are prepared from.
Puerarin, scutellarin in the quality control assay selective extraction thing, astragaloside, total saponins, total flavones, total isoflavone.Described Radix Puerariae extract contains total isoflavone must not calculate with puerarin and is lower than 60%, contains puerarin and must not be lower than 20%; Radix Astragali extract contains total saponins must not calculate with astragaloside and is lower than 50%, contains astragaloside and must not be lower than 5%; Herba Erigerontis extract contains total flavones must not calculate with rutin and is lower than 35%, contains scutellarin and must not be lower than 3%.
Wherein, the weight proportion of described active component is:
1～10 part of Radix Astragali extract, 50～125 parts of Radix Puerariae extracts, 20～60 parts of oil lamp extracts.
Further, the weight proportion of described active component is:
1 part of Radix Astragali extract, 70 parts of Radix Puerariae extracts, 25 parts of oil lamp extracts.
Described medicament is granule, tablet, capsule, pill, mixture.
The present invention also provides a kind of method for preparing this pharmaceutical composition, comprises the steps:
A, take by weighing the following weight proportion raw material:
20～30 parts of the Radixs Astragali, 10～30 parts of Radix Puerariaes, 10～30 parts of Herba Erigerontiss;
B, powder of Radix Puerariae are broken into coarse powder, with 60～95% ethanol extractions, filter, merging filtrate reclaims ethanol, concentrates, by macroporous adsorptive resins, water elution discards water elution liquid, reuse 10～95% ethanol elutions are collected ethanol elution, reclaim ethanol, be condensed into thick paste, drying under reduced pressure is ground into fine powder, gets Radix Puerariae extract;
C, astragalus membranaceus powder are broken into coarse powder, extract with 60～95% alcohol heating reflux, filter, merging filtrate reclaims ethanol, concentrates, hydro-oxidation sodium to alkalinity is 2～5%, adds the n-butyl alcohol dynamic extraction 2～5 times, merges n-butyl alcohol liquid, the washing of 2～5% sodium hydroxide solutions washes with water again, discards washing liquid, merge n-butyl alcohol liquid, be evaporated near doing, add the acetone grinding and wash to color shallow, the taking precipitate drying under reduced pressure is ground into fine powder, gets Radix Astragali extract;
D, Herba Erigerontis decoct with water, and collecting decoction filters, and concentrate the back by macroporous adsorptive resins, water elution, discard water elution liquid, continue to collect ethanol elution, reclaim ethanol with 10～95% ethanol elutions, be condensed into thick paste, drying under reduced pressure is ground into fine powder, gets Herba Erigerontis extract;
E, the Radix Astragali extract with b, c, the preparation of d step, Radix Puerariae extract, Herba Erigerontis extract mixing add acceptable accessories or complementary composition and are prepared into preparation pharmaceutically commonly used.
The present invention also provides the purposes of described each materials of weight proportions in the medicine of preparation treatment diabetes and complication thereof.
Further, described medicine is the medicine of treatment diabetic retinopathy.
The proportioning raw materials of medicine of the present invention is according to motherland's Chinese medicine theory, forms in conjunction with the pharmacology proportioning of diabetes.Radix Astragali benefiting vital QI and blood in the raw material, the Radix Puerariae tonifying YIN promotes the production of body fluid, and promotes blood circulation with the Herba Erigerontis blood stasis dispelling again, and three medicines share the effect of playing supplementing QI and nourishing YIN, blood circulation promoting and blood stasis dispelling altogether, the effect of performance Synergistic.Determine the effective ingredient in the medicine material of the present invention simultaneously, and the effective ingredient compatibility used to have the effect of arteries and veins all of invigorating blood circulation of supplementing QI and nourishing YIN for the pathological state of the non-propagation of diabetic renal papillary necrosis phase deficiency in origin and excess in superficiality, simulataneous insufficiency and excessive.By the empty stasis of blood and control symptoms such as obviously to improve blurring of vision, improve patient's vision; Medicine of the present invention also has the effect of promoting blood circulation to remove obstruction in the collateral, can promote retinal hemorrhage and the absorption of oozing out.Obviously be better than Western medicine " Doxium " aspect raising vision, the various condition of illness of improvement.This medicine is the preparation of pure Chinese medicine simultaneously, adopts advanced technology to make, and effective ingredient is clear and definite, and is easy to carry and use.Toxicological test proof safe without toxic side effect can be taken for a long time.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
The specific embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
The pharmacodynamics screening of Fig. 1 compound recipe extraction process route preparation flow of sample
The pharmacodynamics screening of Fig. 2 Radix Puerariae extraction process route preparation flow of sample
The pharmacodynamics screening of Fig. 3 Radix Astragali extraction process route preparation flow of sample
The pharmacodynamics screening of Fig. 4 Herba Erigerontis extraction process route preparation flow of sample
The specific embodiment
The preparation of embodiment 1 medicine capsule of the present invention
Take by weighing: Radix Puerariae 30kg, Herba Erigerontis 20kg, Radix Astragali 20kg, powder of Radix Puerariae is broken into coarse powder, extracts secondary with 60% alcohol heating reflux, each 1 hour, filter merging filtrate, reclaim ethanol, suitably concentrate, be added on the macroporous adsorptive resins of having handled well, water elution discards water elution liquid, continues to use 70% ethanol elution, collect ethanol elution, concentrating under reduced pressure becomes thick paste, drying under reduced pressure, be ground into fine powder, get Radix Puerariae extract;
Astragalus membranaceus powder is broken into coarse powder, extracts three times with 90% alcohol heating reflux, each 1 hour, filters, merging filtrate reclaims ethanol, suitably concentrates, hydro-oxidation sodium to alkalinity is 5%, adds the n-butyl alcohol dynamic extraction 2 times, merges n-butyl alcohol liquid, 5% sodium hydroxide solution washing 1 time, water washing 2 times discards washing liquid, merge n-butyl alcohol liquid, be evaporated near doing, add the acetone grinding and wash to color shallow, the taking precipitate drying under reduced pressure is ground into fine powder, gets Radix Astragali extract;
Herba Erigerontis decocts with water three times, and each 1 hour, collecting decoction, filter, suitably concentrate, be added on the macroporous adsorptive resins of having handled well, water elution discards water elution liquid, continues to use 70% ethanol elution, collect ethanol elution, reclaim ethanol, be condensed into thick paste, drying under reduced pressure, be ground into fine powder, get Herba Erigerontis extract;
Add above-mentioned fine powder and carboxymethylstach sodium, hydroxypropyl cellulose, micropowder silica gel, magnesium stearate, mixing incapsulates, promptly.
The preparation method of other dosage forms is, the dry fine powder of the three flavor medicine main components that will obtain according to preceding method adds adjuvant by the preparation method of Chinese medicine regular dosage form and makes different dosage forms, as dosage forms such as tablet, granule, pill, soft capsule and mixture.
The preparation of embodiment 2 bolus of drug of the present invention
A, take by weighing Radix Astragali 10kg, Radix Puerariae 10kg, Herba Erigerontis 10kg;
B, powder of Radix Puerariae are broken into coarse powder, use 95% ethanol extraction, filter, merging filtrate reclaims ethanol, concentrates, by macroporous adsorptive resins, water elution discards water elution liquid, reuse 10～95% ethanol elutions are collected ethanol elution, reclaim ethanol, be condensed into thick paste, drying under reduced pressure is ground into fine powder, gets Radix Puerariae extract;
C, astragalus membranaceus powder are broken into coarse powder, extract with 95% alcohol heating reflux, filter, merging filtrate reclaims ethanol, concentrates, hydro-oxidation sodium to alkalinity is 5%, adds the n-butyl alcohol dynamic extraction 5 times, merges n-butyl alcohol liquid, the washing of 5% sodium hydroxide solution washes with water again, discards washing liquid, merge n-butyl alcohol liquid, be evaporated near doing, add the acetone grinding and wash to color shallow, the taking precipitate drying under reduced pressure is ground into fine powder, gets Radix Astragali extract;
D, Herba Erigerontis decoct with water, and collecting decoction filters, and concentrate the back by macroporous adsorptive resins, water elution, discard water elution liquid, continue to use 95% ethanol elution, collect ethanol elution, reclaim ethanol, be condensed into thick paste, drying under reduced pressure is ground into fine powder, gets Herba Erigerontis extract;
The said extracted thing is mixed, add adjuvant pharmaceutically commonly used, as starch, granulation, granulate get tablet.
The preparation of embodiment 3 medicinal tablets of the present invention
A, take by weighing Radix Astragali 40kg, Radix Puerariae 30kg, Herba Erigerontis 30kg;
B, powder of Radix Puerariae are broken into coarse powder, use 60% ethanol extraction, filter, merging filtrate reclaims ethanol, concentrates, by macroporous adsorptive resins, water elution discards water elution liquid, reuse 10～95% ethanol elutions are collected ethanol elution, reclaim ethanol, be condensed into thick paste, drying under reduced pressure is ground into fine powder, gets Radix Puerariae extract;
C, astragalus membranaceus powder are broken into coarse powder, extract with 60% alcohol heating reflux, filter, merging filtrate reclaims ethanol, concentrates, hydro-oxidation sodium to alkalinity is 2%, adds the n-butyl alcohol dynamic extraction 2 times, merges n-butyl alcohol liquid, the washing of 2% sodium hydroxide solution washes with water again, discards washing liquid, merge n-butyl alcohol liquid, be evaporated near doing, add the acetone grinding and wash to color shallow, the taking precipitate drying under reduced pressure is ground into fine powder, gets Radix Astragali extract;
D, Herba Erigerontis decoct with water, and collecting decoction filters, and concentrate the back by macroporous adsorptive resins, water elution, discard water elution liquid, continue to use 10% ethanol elution, collect ethanol elution, reclaim ethanol, be condensed into thick paste, drying under reduced pressure is ground into fine powder, gets Herba Erigerontis extract;
The preparation of embodiment 4 other dosage forms of medicine of the present invention
Take by weighing Radix Astragali 30kg, Radix Puerariae 40kg, Herba Erigerontis 30kg, directly beat powder, get powder.
In the zone of reasonableness that medicine material of the present invention is selected,, can be prepared into preparation pharmaceutically commonly used by extract drugs processing method of the present invention.
The preparation of embodiment 5 medicinal tablets of the present invention
Press Radix Astragali extract 1kg, Radix Puerariae extract 50kg, the oil lamp extract 30kg of the method preparation of embodiment 1, mix, add starch, granulation, granulate add magnesium stearate, tabletting, promptly.
The preparation of embodiment 6 medicine capsules of the present invention
Radix Astragali extract 10kg, the Radix Puerariae extract 125kg, the oil lamp extract 60kg that press the method preparation of embodiment 1 mix, and add starch, and granulation, granulate are directly encapsulated.
The preparation of embodiment 7 medicinal granules of the present invention
Radix Astragali extract 2kg, the Radix Puerariae extract 80kg, the oil lamp extract 40kg that press the preparation of embodiment 1 method mix, and add starch, and granulation, granulate are directly encapsulated.
The preparation of embodiment 8 medicinal tablets of the present invention
Press Radix Astragali 30kg, Radix Puerariae 40kg, Herba Erigerontis 30kg, Radix Rehmanniae 20kg, Fructus Lycii 20kg, Semen Cassiae 15kg, the Fructus Leonuri 20kg of the method preparation of embodiment 1, wherein the Radix Astragali, Radix Puerariae, Herba Erigerontis are pressed the extraction of embodiment 1 method, other medicines directly decoct with water, concentrate, mix, add starch, granulation, granulate, add magnesium stearate, tabletting, promptly.
The quality control of embodiment 9 extract drugs raw materials of the present invention
The capsule of embodiment 1 preparation is carried out quality control, and described " this product " be the capsule that embodiment 1 prepares, and method of quality control wherein is not only at the quality control of medicinal raw material, also at the quality control of raw extract.
The quality control assay of 1 crude drug
(1) Radix Astragali
Statutory standards come from one one the 212nd page of Pharmacopoeia of the People's Republic of China version in 2005.
(2) Radix Puerariae
Statutory standards come from one one the 233rd page of Pharmacopoeia of the People's Republic of China version in 2005.
(3) Herba Erigerontis
Statutory standards come from one one the 100th page of Pharmacopoeia of the People's Republic of China version in 2005.
2 quality standards
(1) Astragaloside content is measured in the finished product
Index and method determine that astragaloside is the important activity composition of the Radix Astragali, are the index components of assay so select astragaloside.The assay of the Radix Astragali, Pharmacopoeia of the People's Republic of China version (an one) in 2000 employing tlc scanning determination, but this method complicated operation.This test adopts the HPLC-ESLD under Pharmacopoeia of the People's Republic of China version (an one) in 2005 Radix Astragali item to detect.The evaporation light detection method can be got rid of evaluated error and the interference that causes because of saponins material uv absorption low (about 200nm), and the method is quick, easy, accurate, sensitivity and favorable reproducibility, lessly influenced by anthropic factor.
Content limit this product is according to 4 batch sample measurement results, and the astragaloside average content is the 0.225mg/ grain in the finished product, because pilot scale Milkvetch Root Astragaloside content is about 0.048%, according to calculating, medical material is about 50% to the rate of transform of not considering yield rate of finished product.Consider and receive cream rate and the Correlative Influence Factors of medicine in storing, transporting, so every of this product contains the Radix Astragali with astragaloside (C
41H
68O
14) meter, must not be less than 0.18mg.
(2) puerarin content is measured in the finished product
Index and method determine that this product adopts the high effective liquid chromatography for measuring puerarin, because puerarin is the relevant main effective ingredient of this product effect, high performance liquid chromatography is quick, easy, accurate, sensitivity and favorable reproducibility, lessly influenced by anthropic factor.
This experimental basis of content limit 4 batch sample measurement results, puerarin (C in the finished product
21H
20O
9) average content is the 67.1mg/ grain, pilot scale Radix Puerariae medical material puerarin content is about 6.24%, and according to calculating, medical material is 63.32% to the mean transferred rate of not considering yield rate of finished product.Consider and receive cream rate and the Correlative Influence Factors of medicine in storing, transporting, therefore tentative every of this product contains Radix Puerariae with puerarin (C
21H
20O
9) meter, must not be less than 50.0mg.
(3) scutellarin assay in the finished product
Index and method are determined Herba Erigerontis in this product prescription, cure mainly closely related with the function of medicine of the present invention; Scutellarin is the important activity composition of Herba Erigerontis, is the index components of assay so select scutellarin.This standard adopts the scutellarin in the rp-hplc determination sample, and this method is quick, easy, accurate, sensitivity and favorable reproducibility, lessly influenced by anthropic factor.
Content limit this product is according to 4 batch sample measurement results, scutellarin (C in the finished product
21H
18O
12) average content is the 3.0mg/ grain, pilot scale Herba Erigerontis medical material scutellarin content is 0.41%, according to calculating, medical material is 64.83% to the not consideration yield rate mean transferred rate of finished product.Consider and receive cream rate and the Correlative Influence Factors of medicine in storing, transporting, therefore tentative every of this product contains Herba Erigerontis with scutellarin (C
21H
18O
12) meter, must not be less than 2.5mg.
(4) total saponin content is measured in the Radix Astragali semi-finished product extract
The preparation of reference substance solution
Precision takes by weighing 80 ℃ of drying under reduced pressure, and (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides to the astragaloside reference substance of constant weight; Lot number: 0781200210) 10.31mg, put in the 25ml measuring bottle, add methanol to scale, precision is measured 3ml and is put in the 10ml measuring bottle, adds methanol to scale, shakes up, and promptly gets (every 1ml contains astragaloside 0.1237mg).
The preparation of standard curve
Precision is measured reference substance solution 0.6,0.8,1.0,1.2,1.4,1.6ml, put respectively in the tool plug rub oral examination tube, put in the water-bath and heat, solvent evaporated adds vanillin-glacial acetic acid 0.2ml of 5%, add perchloric acid 0.8ml, close plug is put 70 ℃ of constant temperature water bath heating 20 minutes, takes out, with the flowing water cooling, add glacial acetic acid 5.0ml and shake up immediately; With corresponding solution is blank.According to colorimetry (Pharmacopoeia of the People's Republic of China version (an one) in 2000 appendix VB), measure trap at 560nm wavelength place, be that vertical coordinate, concentration (mg/ml) they are abscissa with trap (A), the drawing standard curve calculates regression equation.
The result shows that in 0.0124 ~ 0.0330mg/ml scope, astragaloside reference substance x (mg/ml) and trap (A) y linear relationship be (r=0.9999) well, and regression equation is y=21.252x+0.0039.
The need testing solution preparation
Get the about 50mg of this product powder, the accurate title, decide, and adds methanol 50ml, reflux 60 minutes filters, and filtrate is reclaimed methanol and is concentrated into dried, residue adds water-saturated n-butanol 20ml, slight fever makes dissolving, sodium hydroxide jolting washing 3 (10ml, 10ml with 1%, 10ml), discard caustic lye of soda, n-butyl alcohol liquid washs 3 (10ml, 10ml with the n-butyl alcohol saturation water, 10ml), discard water layer, n-butyl alcohol liquid evaporate to dryness, residue adds dissolve with methanol, be transferred in the 25ml measuring bottle, add methanol to scale, shake up, as need testing solution.
Sample determination
Get lot number and be each 25mg of sample (each 3 parts) of 040401,050701,050702,050703, accurately claim surely, measure by the total saponin content assay method.
The formulation of content limit: consider factor and many crowdes of assay results such as the places of origin of raw materials, collecting season, storage, the Radix Astragali extract total saponin content must not be less than 50% in astragaloside.
(5) total isoflavone assay in the Radix Puerariae semi-finished product extract
1), instrument condition
Lambda35 ultraviolet-uisible spectrophotometer (Perkin Elmer company); Cuvette: quartz, 1cm; CQ-250 type supersound process ripple washer (the letter supersound process company limited in Shanghai); Electronic balance BP121S, BP211D (Sai Duolisi Instr Ltd.); W201 thermostat water bath (Shen, Shanghai is along bio tech ltd).
2), the preparation of reference substance solution: precision takes by weighing the puerarin reference substance, and (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides; For assay usefulness, lot number: 110752-200209) 17.8mg, put in the 50ml measuring bottle, add methanol to scale, shake up, in contrast product solution (containing puerarin 0.356mg among every 1ml).
3), the preparation of standard curve: precision is measured reference substance solution (0.356mg/ml) 1.0,1.5,2.0,2.5,3.0ml, put respectively in the 100ml measuring bottle, add methanol to scale, shake up, making concentration is 3.56 μ g/ml, 5.34 μ g/ml, 7.12 μ g/ml, 8.9 μ g/ml, 10.68 the reference substance solution of μ g/ml, according to ultraviolet spectrophotometry (appendix VA of Pharmacopoeia of People's Republic of China version in 2000), measure trap at 250nm wavelength place, with the trap is vertical coordinate, the concentration of reference substance (μ g/ml) is abscissa, the drawing standard curve, and calculate regression equation.
The result shows that in 3.56 ~ 10.68 μ g/ml scopes, the linear relationship of puerarin trap y and reference substance concentration x (μ g/ml) is (r=0.9999) well, and regression equation is y=0.0736x+0.003.
Algoscopy: precision is measured sample solution, measures trap in accordance with the law, calculates, promptly.
4), sample determination
Get the about 20mg of different lot number samples (lot number 040401,050701,050702,050703) (each 3 parts), the accurate title, decide, and measures by the total isoflavone assay method.
The formulation of content limit: consider factor and many crowdes of assay results such as the places of origin of raw materials, collecting season, storage, Radix Puerariae extract total isoflavone content must not be less than 60% in puerarin.
(6) determination of total flavonoids in the Herba Erigerontis semi-finished product extract
The preparation precision of reference substance solution takes by weighing 110 ℃ of drying under reduced pressure, and (Chinese pharmaceutical biological product check institute provides to the control substance of Rutin 10.0mg of constant weight, lot number: 0080-9705), put in the 50ml measuring bottle, it is an amount of to add 60% ethanol, puts that slight fever makes dissolving in the water-bath, puts cold, with 60% ethanol dilution to scale, shake up, lucifuge cold preservation is as stock solution.(concentration is 0.204mg/ml)
The preparation precision of the preparation standard curve of standard curve is measured rutin solution 0,1.0,1.5,2.0,2.5,3.0ml, put respectively in the 10ml measuring bottle, respectively add 30% ethanol to 5.0ml, precision respectively adds sodium nitrite (1-＞20) 0.3ml, shakes up, place 6min, add aluminum nitrate solution (1-＞10) 0.3ml, shake up, place 6min again, hydro-oxidation sodium test solution 4ml, be diluted with water to scale respectively, shake up, leave standstill 15min, wavelength place at 510nm measures trap respectively, with the trap is vertical coordinate, and concentration is abscissa, the drawing standard curve.The calculating regression equation is y=10.951x-0.0178 (R=0.999)
The accurate absorption of algoscopy need testing solution is a certain amount of, and the method under the sighting target directrix curve preparation is measured absorbance from " adding 30% ethanol to 5.0ml " in accordance with the law, calculates.
Sample determination
Get each 50mg of Herba Erigerontis pilot scale extract (lot number is 040401,050701,050702,050703) (each 3 parts), the accurate title, decide, and measures by the determination of total flavonoids method.
The formulation of content limit: consider factor and many crowdes of assay results such as the places of origin of raw materials, collecting season, storage, the Herba Erigerontis extract general flavone content must not be less than 35% in rutin.
(7) reference substance source
Scutellarin reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute is for assay usefulness, by 96.4% conversion, lot number 842-200102); Puerarin reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute is for assay usefulness, lot number 110752-200209); Astragaloside is that (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides reference substance, for assay usefulness, lot number: 0781-200311); (Chinese pharmaceutical biological product check provides lot number: 0080-9705) to control substance of Rutin.
Extract to embodiment 1 carries out quality control as stated above simultaneously, and the result is as follows:
According to 4 batches of measurement results of pilot-scale sample, the technological standards limit of tentative intermediate extract (inner quality standard, with the content average 80%～90% as limit):
Extract contains total isoflavone and must not calculate with puerarin and be lower than 60% in the middle of the Radix Puerariae; Contain puerarin and must not be lower than 20%;
Extract contains total saponins and must not calculate with astragaloside and be lower than 50% in the middle of the Radix Astragali; Contain astragaloside and must not be lower than 5%;
Extract contains total flavones and must not calculate with rutin and be lower than 35% in the middle of the Herba Erigerontis; Contain scutellarin and must not be lower than 3%;
Below prove beneficial effect of the present invention by pharmacodynamics test.
Test example 1 Radix Astragali, Radix Puerariae and the test of Herba Erigerontis extract craft screening
Below by the Radix Astragali, Radix Puerariae and Herba Erigerontis extract are tested the influence that external high sugar is cultivated the bovine retina perivascular cell respectively, thereby determine best extraction process.
One, the screening test of compound recipe united extraction process route
Water and ethanol be present laboratory and produce in the most frequently used solvent: general concentration is at the suitable lower composition of polarity of flavonoid glycoside that extracts of the ethanol 90% or more, and the ethanol of concentration about 60% is suitable to extract that flavonoid glycoside and triterpene saponin isopolarity are medium to reach bigger composition; The suitable big composition of polysaccharide isopolarity that extracts of water.According to the physicochemical property of prescription drug chemical constituent, select the extract of above three kinds of solvent gained to supply test agent as pharmacodynamics.(preparation flow sees Fig. 1 for details)
Two, Radix Puerariae extracts the medicine efficacy screening test of route
What the Radix Puerariae extracting method was made solvent with ethanol is many, be because the puerarin content that extraction process by water extracts is low on the one hand, there is research data to show its effect and being proportionate property of puerarin content on the one hand, adopt ethanol extraction technology that the activity of medicine is produced appreciable impact, alcohol extracting method is the puerarin content height not only, and the amount of total solid is few in the extracting solution, has improved the percentage rate of puerarin in the unit mass medicine.
The main chemical position of Radix Puerariae is: 1. osajin comprises with big legumin and is the chemical compounds such as daiazi, puerarin of aglycon with it; 2. polysaccharide comprises starch based.Wherein 1. constituents is dissolved in ethanol, uses alcohol extraction process; 2. constituents is water-soluble, is insoluble to ethanol, can use extraction process by water after alcohol extraction.According to the absorption property of macroporous adsorbent resin, 1. constituents can be adsorbed, and can be adsorbed by ethanolysis, can initial gross separation, therefore draft Radix Puerariae extraction process route.(preparation flow sees Fig. 2 for details)
Three, the Radix Astragali extracts the medicine efficacy screening test of route
At present to Milkvetch Root and the quality evaluation of Chinese patent medicine that contains the Radix Astragali all with the index of Radix Astragali saponin (astragaloside) as evaluation.Therefore the Radix Astragali saponin reference literature drafts Radix Astragali extraction process route.(preparation flow sees Fig. 3 for details)
Four, Herba Erigerontis extracts the medicine efficacy screening test of route
From the Herba Erigerontis plant, separate, identify flavone compound, caffeic acid ester compounds, phenolic acid compound and other compounds.The main component of Herba Erigerontis injection and tablet is the flavonoid based on scutellarin (scutellarin), and the flavonoid chemical constituent is mainly to reach one of effective ingredient in the Herba Erigerontis, so reference literature is drafted Herba Erigerontis extraction process route.(preparation flow sees Fig. 4 for details)
Five, process route screening conclusion
The selection result shows that sample TA, TB, GB, HC, DB have facilitation to the propagation of the pericyte that high sugar is cultivated, and these samples have comprised the main effective site in the primary crude drug.
On the basis that preliminary extraction process route is determined, carried out extraction, separation, concentrated, technical study such as purification, determined optimized parameters, the intermediate semi-finished product (lot number 040401) that prepared each flavour of a drug of writing out a prescription, wherein Radix Puerariae intermediate semi-finished product yield is 12.49%, Radix Astragali intermediate semi-finished product yield is 0.27%, Herba Erigerontis intermediate semi-finished product yield is 6.17%.
According to clinical use experience and and the medication usage ratio, in the pharmaceutical decocting piece ratio of Radix Puerariae, the Radix Astragali, Herba Erigerontis, adopt each medical material intermediate semi-finished product to carry out the pharmacodynamic study of compound recipe active component.
Table 1 compound recipe extracts the compatibility proportioning test design of the active component of purification respectively
Results of pharmacodynamic test shows that sample TA and TB biased sample T, sample P have facilitation to the propagation of the pericyte that high sugar is cultivated, and the effect of sample P is obvious.
Six, concrete experimental data:
1, experiment material
1, experimental drug
Radix Astragali extract: make different samples (HA ~ HD), press the described method preparation of Fig. 2 through series of process by pharmaceutical college of Chengdu University of Traditional Chinese Medicine by Chinese medicine astragalus;
Radix Puerariae extract: make different samples (GA ~ GD), press the described method preparation of Fig. 3 through series of process by pharmaceutical college of Chengdu University of Traditional Chinese Medicine by Chinese medicine astragalus.
Herba Erigerontis extract: make sample (DA ~ DD), press the described method preparation of Fig. 4 through series of process by pharmaceutical college of Chengdu University of Traditional Chinese Medicine by Herba Erigerontis.
Dimethyl sulfoxide (DMSO): worker's biological engineering company limited is given birth in Shanghai.
2, experimental technique:
Adopt mtt assay to measure (sample GA ~ GD), the Herba Erigerontis extract (protective effect of the bovine retinal microvascular pericytes that sample DA ~ DD) cultivates external high sugar of Chinese medicine astragalus extract (sample HA ~ H D), Radix Puerariae extract.Adopting concentration of glucose according to first's experimental result is DMEM culture fluid, incubation time 2 days culture fluid concentration and incubation times as drug screening of 20mmol/.
Radix Astragali extract (sample HA ~ HD), Radix Puerariae extract (sample GA ~ GD), Herba Erigerontis extract (sample DA ~ DD) begins concentration with 1/2 maximum as screening sample concentration of the saturated concentration of each sample, more successively with 5 times spacing as screening sample concentration.
Get the 3rd generation pericyte, behind the digestion counting, be inoculated in 96 well culture plates, cultivating and using concentration of glucose after 24 hours instead is that the culture fluid of 20mmol/L was cultivated 48 hours, grouping, the various Radix Astragali extract solution that add variable concentrations, take out 96 well culture plates after continuing to cultivate 48h, add MTT solution, cultivate 4h, observation of cell form and number under inverted phase contrast microscope, supernatant then exhausts, every hole adds 150 μ l DMSO vibration several minutes, measures each hole absorbance value (OD value) immediately, and calculates cell survival rate according to the OD value.Cell survival rate=(test group OD value-matched group OD value)/matched group OD value * 100%.
3, statistical method
Adopt SPSS11.0 version statistical package and analyze, relatively use one factor analysis of variance between many groups, relatively check (L-S-D method) in twos between many groups with q.
4, experimental result and analysis
Table 2 shows: HA, HB, HC do not have facilitation to the propagation of the pericyte that high sugar is cultivated.0.773mg/ml, the HD of 0.1546mg/ml, 0.0309mg/ml, 0.00618mg/ml, 0.00124mg/ml, 0.00025mg/ml, 0.00005mg/ml, 0.00001mg/ml, 0.000002mg/ml has facilitation to the propagation of the pericyte that high sugar is cultivated.
Because HA1, the HA2 color of high concentration are denseer, influential to the OD value, former according to the observation finding under the inverted phase contrast microscope: pericyte's number of HA1, HA2 group obviously reduces, the cell shape irregular, it is big that the part cell volume becomes, the nuclear membrane profile is unclear or disappear cracked, the pyknosis of karyon, visible cell fragment.Do not think that HA1, HA2 have short proliferation function to pericyte.
Table 3 shows: GA, GC, GD do not have facilitation to the propagation of the pericyte that high sugar is cultivated.0.00816mg/ml, the GB of 0.00163mg/ml, 0.00033mg/ml, 0.000066mg/ml, 0.000013mg/ml, 0.0000026mg/ml has facilitation to the propagation of the pericyte that high sugar is cultivated, difference has statistical significance (P＜0.05).
Because GA1, GC1, the GD1 color of high concentration are denseer, influential to the OD value, former according to the observation finding under the inverted phase contrast microscope: pericyte's number of GA1, GC1, GD1 group obviously reduces, the cell shape irregular, it is big that the part cell volume becomes, the nuclear membrane profile is unclear or disappear cracked, the pyknosis of karyon, visible cell fragment.Do not think that HA1, HA2 have short proliferation function to pericyte.
Table 4 shows: DA, DC, DD do not have facilitation to the propagation of the pericyte that high sugar is cultivated.0.1249mg/ml, the DB of 0.02498mg/ml, 0.004996mg/ml, 0.0009992mg/ml has facilitation to the propagation of the pericyte that high sugar is cultivated, difference has statistical significance.
Because DB 1 color of high concentration is denseer, influential to the OD value, former according to the observation finding under the inverted phase contrast microscope: pericyte's number of HA1, HA2 group obviously reduces, the cell shape irregular, it is big that the part cell volume becomes, the nuclear membrane profile is unclear or disappear cracked, the pyknosis of karyon, visible cell fragment.Do not think that there is short proliferation function in 1 pair of pericyte of DB.
5, conclusion
The extract sample HD of 1 Chinese medicine astragalus, external high sugar is cultivated pericyte short proliferation function.
The extract sample GB of 2 Chinese medicine Radix Puerariaes cultivates pericyte to external high sugar short proliferation function.
3 Herba Erigerontis extract sample DB cultivate pericyte to external high sugar short proliferation function.
The main effective site of each medical material and main constituent type thereof: the effective site of Radix Puerariae is total isoflavone class and total saponins class (based on total isoflavone); The effective site of the Radix Astragali is total isoflavone class and total saponins class (based on total saponins); Herba Erigerontis effective site is the total flavones glycoside.
Therefore, determine that according to above-mentioned pharmacodynamics The selection result the basic technology route of medicine material of the present invention is:
(1) the preliminary extraction process route of Radix Puerariae: ethanol extraction, extracting solution concentrate, and by macroporous adsorptive resins, are washed to colourlessly, and ethanol elution is collected ethanol elution, concentrates drying.
(2) the preliminary extraction process route of the Radix Astragali: ethanol extraction, extracting solution concentrates, alkalization, n-butanol extraction, washing concentrates acetone precipitation, collecting precipitation, drying.
(3) the preliminary extraction process route of Herba Erigerontis: decocting extracts, and extracting solution concentrates, and by macroporous adsorptive resins, is washed to colourlessly, and ethanol elution is collected ethanol elution, concentrates drying.
The influence that test example 2 Radix Puerariaes, the Radix Astragali, the different proportionings of three kinds of extracts of Herba Erigerontis are cultivated the bovine retina perivascular cell to high sugar
Adopt mtt assay to measure the protective effect of Chinese medicine astragalus extract (press the method for embodiment 1 preparation), Radix Puerariae extract (press the method for embodiment 1 preparation), Herba Erigerontis extract (press the method for embodiment 1 preparation) to the bovine retinal microvascular pericytes of the sugared cultivation of external height.Adopting concentration of glucose according to first's experimental result is DMEM culture fluid, incubation time 2 days culture fluid concentration and incubation times as drug screening of 20mmol/.
Radix Astragali extract (by the method for embodiment 1 preparation), Radix Puerariae extract (by the method for embodiment 1 preparation), Herba Erigerontis extract (method for preparing by embodiment 1) begin concentration with 1/2 maximum as screening sample concentration of the saturated concentration of each sample, more successively with 5 times spacing as screening sample concentration.Three kinds of different proportionings (1～No. 25) of extract: the extract by Herba Erigerontis, the Radix Astragali, Radix Puerariae is mixed with proportioning 1～No. 25 by all possible compatible combination.
Get the 3rd generation pericyte, behind the digestion counting, be inoculated in 96 well culture plates, cultivating and using concentration of glucose after 24 hours instead is that the culture fluid of 20mmol/L was cultivated 48 hours, grouping, the various Radix Astragali extract solution that add variable concentrations, take out 96 well culture plates after continuing to cultivate 48h, add MTT solution, cultivate 4h, observation of cell form and number under inverted phase contrast microscope, supernatant then exhausts, every hole adds 150 μ l DMSO vibration several minutes, measures each hole absorbance value (OD value) immediately, and calculates cell survival rate according to the OD value.Cell survival rate=(test group OD value-matched group OD value)/matched group OD value * 100%.
Table 5 compound recipe extracts the compatibility proportioning test design of the active component of purification respectively
The influence of (optical density value) is bred by the pericyte that table 6 proportioning 1-25 cultivates high sugar
With add PBS liquid matched group relatively: " ★ " P＜0.05.
Last table shows: all there are short proliferation function in proportioning 1, proportioning 2, proportioning 3, proportioning 4, proportioning 9, proportioning 10, proportioning 11, proportioning 12, proportioning 13, proportioning 14, proportioning 17, proportioning 18, proportioning 20, proportioning 21, proportioning 22, proportioning 23, proportioning 24,25 pairs of external high sugared pericyte of cultivating of proportioning, wherein, the effect that No. 4, proportioning is the strongest, and the effect of proportioning 21 is taken second place.The prescription ratio of having verified medicine of the present invention by the pharmacodynamics screening test is: Radix Puerariae: the Radix Astragali: Herba Erigerontis=18: 12: 12=3: 2: 2.
The compatibility of different pharmaceutical brings out the influence of diabetes rat blood-retina barrier in test example 3 medicines of the present invention to STZ
Various combination by three main components in the medicine of the present invention is to the influence of blood-retina barrier, and whether the effect of comparative study the present invention three flavor recurrence due to taking drug side compatibilities is better than other compatibilities.
Adopt streptozotocin (Streptozotocin, STZ) lumbar injection is made diabetes rat model, after modeling, animal was divided into 9 groups in 3 months, be respectively: D, G, H, DG, DH, GH, DGH group and blank, model group (D: Herba Erigerontis, G: Radix Puerariae, H: the Radix Astragali), each group is irritated stomach treatment (be expressed as D single to use Herba Erigerontis, DG be that Herba Erigerontis and Radix Puerariae share) with three flavor singles of medicines or various combination extract respectively, result according to test example 1 and 2, the ratio that DGH group adopted 3: 2: 2 is made up a prescription, and dosage 300mg/kg body weight is 10 times of adult's consumption every day.The medicine of other each groups prepares to irritate the stomach medication with reference to said ratio, and dosage also is 300mg/kg body weight every day with the DGH group.When treatment finished in three months, adopt azovan blue perfusion spike to show STZ diabetes rat blood retina barrier (Blood-retina barrier, seepage situation BRB) studied.The result: STZ diabetes model rat BRB can occur at blood sugar increasing and significantly damage after 6 months, obviously increases (P＜0.01) than the vascular leakage of normal rats.The vascular leakage that the infringement of the obvious BRB of compatibility use can the minimizing of medicine three flavor medicine DGH of the present invention causes, than single medicinal material D and G to improve blood vessel function obvious, difference has significant statistical significance (P＜0.01) and single H and DG compatibility comparing difference that statistical significance (P＜0.05) is arranged.
Conclusion: under same materials consumption condition, the present invention's three flavor medicine compatibilities can obviously improve the seepage of the retinal vessel of STZ diabetes rat, and effect is better than the independent use of three flavor medicines of the present invention and compatibility use in twos.
Table 7 medicine various combination of the present invention is to the influence of STZ diabetes rat blood-retina barrier (BRB)
Annotate: "
△" P＜0.05, "
△ △" P＜0.01, compare with normal group
"
▲" P＜0.05, "
▲ ▲" P＜0.01, compare with negative control group
" * " P＜0.05, compare with the DGH group " * * " P＜0.01
Test example 4 medicines of the present invention bring out the influence of diabetes rat blood-retina barrier to STZ
Each time point adopts azovan blue perfusion spike to show continuous 8 times in 6 months after modeling, studied STZ diabetes rat blood-retina (Blood-retina barrier, BRB) seepage situation, and after modeling, began in 3 months the treatment group is irritated stomach treatment 3 months with medicine of the present invention, matched group with the Doxium capsule, observe medicine to BRB.The result: comparing difference has significant statistical significance (P＜0.01) before can occurring comparatively tangible seepage and modeling after two weeks of modeling.Along with the increase of the course of disease, the continuing of STZ rat hyperglycemia state, the leakage of azovan blue also increases gradually, the trend of the leakage showed increased of azovan blue 3 months time the after modeling.To 3 months STZ diabetes models of modeling rat continuous irrigation stomach Drug therapy of the present invention 3 months, results suggest: medicine of the present invention had significant protective effect to STZ diabetes BRB, can obviously reduce the seepage of retinal vessel; The effect of blood-retina barrier under the protection hyperglycemia state.Conclusion: the infringement of BRB and visual function can appear in STZ diabetes model rat in early days, and increases the weight of along with continuing of hyperglycemia.Medicine of the present invention can reduce the vascular leakage that the infringement of BRB causes, and is better than contrasting Western medicine Doxium capsule.The result is as follows:
Table 8 medicament capsule of the present invention is to the influence of STZ rat retina blood vessel azovan blue seepage
Annotate: "
△" P＜0.05, "
△ △" P＜0.01, compare with normal group
"
▲" P＜0.05, "
▲ ▲" P＜0.01, compare with negative control group
" * " P＜0.05, compare with the Doxium group " * * " P＜0.01
" ● " P＜0.05, " ● ● " P＜0.01, compare with low dose group
Test example 5 medicines of the present invention bring out the influence of diabetic retinal tissue in rat function (mfERG) to STZ
(Streptozotocin, STZ) lumbar injection is made diabetes rat model, the visual function situation of change of continuous 8 the how burnt electroretinograies of employing of each time point (mfERG) evaluation STZ diabetes rat in 6 months after modeling to adopt streptozotocin.And after modeling, began in 3 months the treatment group is irritated stomach treatment 3 months with medicine of the present invention, matched group with the Doxium capsule, observe of the influence of medicine medicine of the present invention to the rat visual function.
After STZ modeling rat in March carried out continuous 3 months treatment, find that at treatment mfERG at 3 the end of month record each organizes N
1The peak latency of ripple and reaction density difference are not remarkable, not statistically significant.And negative control group and normal group compare P
1The peak latency of ripple then obviously prolongs, and reaction density obviously reduces, and difference has significant statistical significance (P＜0.01); And the P of each treatment group of medicine of the present invention and Doxium group
1The ripple peak latency shortens again and the negative control group comparing difference has statistical significance (P＜0.05), and and normal group comparing difference not statistically significant (P＞0.05).The P of each treatment group of medicine of the present invention and Doxium group
1The ripple reaction density increases, but with the lower difference of normal group statistical significance (P＜0.05) is arranged; But the P of medicine high dose group of the present invention, middle dosage group and Doxium group
1The ripple reaction density increases more obvious and negative group comparing difference remarkable statistical significance (P＜0.01), and the reaction density of the middle and high dosage group of medicine of the present invention increases degree and be better than matched group, and difference has statistical significance (P＜0.05).
Table 9 medicament capsule of the present invention is to the total waveform N of STZ rat mfERG
1, the peak latency of P1 ripple and the influence of reaction density
Annotate: "
△" P＜0.05, "
△ △" P＜0.01, with normal group comparison in June
"
▲" P＜0.05, "
▲ ▲" P＜0.01, compare with modeling negative control group in June
" * " P＜0.05, compare with the Doxium group " * * " P＜0.01
The diabetic retinal tissue in rat morphology damage effect that test example 6 medicines of the present invention bring out STZ
The diabetes rat model of selecting for use STZ to bring out was observed after 3 months successive administration 3 months again.The diabetes rat that research STZ brings out is continuing the change aspect the morphology of retina pathology under the hyperglycemia situation; And on this basis, observe the influence of medicine of the present invention to the morphology infringement.Result of study shows: the retinal capillary of STZ diabetes rat the capillary basement membrane segmental can occur and thicken, structure is unclear, endotheliocytic swelling propagation, and vascular permeability increases, the tangible material of blood oozes out, and the visible capillary lumen of weight person is the full cut-off plug fully or not.Basement membrane is the sections sample and obviously thickens bulge, and dyeing is uneven, and obscurity boundary is unclear, or visible capillary wall fracture.Each dosage group of medicine of the present invention and Difrarel group (DIfrare, the billows pharmaceutical factory of French Le Kangmei produces) take a turn for the better than the blank group, but still visible blood capillary.The inner nuclear layer pathological changes is more obvious, visible neuronal apoptosis, and karyorrhexis, dyeing is uneven, and edge blurry is unclear, and Chinese medicine medicine of the present invention suppresses thickening of capillary basement membrane, alleviates the swelling of endotheliocyte.Medicine height of the present invention, middle dosage can lower diabetic rat retina capillary basement membrane thickness, and is approximate with Western medicine Difrarel effect.
Table 10 medicine capsule of the present invention is to the influence of STZ rat retina capillary basement membrane thickness
Annotate: " * " P＜0.05, compare with normal group " * * " P＜0.01
" ▲ " P＜0.05, " ▲ ▲ " P＜0.01, compare with the blank group
" △ " P＜0.05, compare with the Difrarel group " △ △ " P＜0.01
Each group of P＜0.01, " ☆ " P＜0.05 " ☆ ☆ " and medicament capsule of the present invention relatively
The diabetic retinal tissue in rat blood capillary damage effect that test example 7 medicines of the present invention bring out STZ
Select for use STZ successfully to duplicate the diabetes rat model that experimental STZ brings out, observed after 3 months again successive administration 3 months.Diabetes rat retinal tissue under lasting hyperglycemia situation that STZ is brought out digests the shop sheet, and the change of research retinal capillary morphology aspect is studied; And observation medicine of the present invention is to the influence of STZ diabetic rat blood capillary infringement.Result of study shows: negative control group rat retina blood capillary segmental is expanded encystation sample or be twisted into and is stumbled, and pericyte's number obviously reduces, and microangioma forms, visible shadow week the capillary blood vessel; Caliber inequality, tube chamber diminish or are inaccessible, and no perfusion area forms.The obvious apoptosis of contrast medicine Difrarel group rat retina pericapillary cells, but still as seen blood capillary differentially expanding, bulge, microangioma formation and permeability change.Chinese medicine part compound drug particles height of the present invention, middle dosage group pericapillary cells decreased number, chromatin concentrates, karyopycnosis or densely dye dot or irregular shape, but the blood capillary tube chamber is normal substantially, and it is straight that blood vessel moves towards.Analyze and calculate the ratio (E/P) of endotheliocyte and pericyte, the result shows: Chinese medicine medicine of the present invention can obviously reduce the E/P ratio of diabetes rat.
Table 11 medicament capsule of the present invention is to the influence of STZ rat retina pericapillary cells and endotheliocyte
Annotate: " * " P＜0.05, compare with normal group " * * " P＜0.01
" ▲ " P＜0.05, " ▲ ▲ " P＜0.01, compare with the blank group
" △ " P＜0.05, compare with the Difrarel group " △ △ " P＜0.01
Each group of P＜0.01, " ☆ " P＜0.05 " ☆ ☆ " and medicament capsule of the present invention relatively
The influence that the pericyte that table 2 Radix Astragali extract is cultivated high sugar breeds
Compare with PBS liquid group: " ★ " P＜0.05 (proliferation function); " ☆ " P＜0.05 (inhibitory action).
(annotating :) because medicinal liquid adopts dissolving of PBS liquid and dilution, so each concentration group of medicinal liquid is all organized in contrast with PBS liquid group.
The influence that the pericyte that table 3 Radix Puerariae extract is cultivated high sugar breeds
The influence that the pericyte that table 4 Herba Erigerontis extract is cultivated high sugar breeds
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Claims (10)
1, a kind of pharmaceutical composition for the treatment of diabetic retinopathy is characterized in that: it is the medicament that is prepared from by the following weight proportion raw material:
10～40 parts of the Radixs Astragali, 10～30 parts of Radix Puerariaes, 10～30 parts of Herba Erigerontiss.
2, the pharmaceutical composition of treatment diabetic retinopathy according to claim 1 is characterized in that: it is the medicament that is prepared from by the following weight proportion raw material:
20～40 parts of the Radixs Astragali, 20～30 parts of Radix Puerariaes, 20～30 parts of Herba Erigerontiss.
3, the pharmaceutical composition of treatment diabetic retinopathy according to claim 1 and 2 is characterized in that: it is the medicament that is prepared from by the following weight proportion raw material:
20 parts of the Radixs Astragali, 30 parts of Radix Puerariaes, 20 parts of Herba Erigerontiss.
4, the pharmaceutical composition of treatment diabetic retinopathy according to claim 1 and 2, it is characterized in that: it is to be active component with the water of the raw material of the Radix Astragali, Radix Puerariae and Herba Erigerontis or the Radix Astragali, Radix Puerariae and Herba Erigerontis or extractive with organic solvent, adds the medicament that acceptable accessories is prepared from.
5, the pharmaceutical composition of treatment diabetic retinopathy according to claim 4 is characterized in that: Radix Puerariae extract contains total isoflavone must not calculate with puerarin and is lower than 60%, contains puerarin and must not be lower than 20%; Radix Astragali extract contains total saponins must not calculate with astragaloside and is lower than 50%, contains astragaloside and must not be lower than 5%; Herba Erigerontis extract contains total flavones must not calculate with rutin and is lower than 35%, contains scutellarin and must not be lower than 3%.
6, the pharmaceutical composition of treatment diabetic retinopathy according to claim 5 is characterized in that: the weight proportion of described active component is:
1～10 part of Radix Astragali extract, 50～125 parts of Radix Puerariae extracts, 20～60 parts of Herba Erigerontis extracts.
7, the pharmaceutical composition of treatment diabetic retinopathy according to claim 6 is characterized in that: the weight proportion of described active component is:
1 part of Radix Astragali extract, 70 parts of Radix Puerariae extracts, 25 parts of Herba Erigerontis extracts.
8, the pharmaceutical composition of treatment diabetic retinopathy according to claim 1 is characterized in that: described medicament is granule, tablet, capsule, pill or mixture.
9, a kind of method for preparing the pharmaceutical composition for the treatment of diabetic retinopathy is characterized in that step is:
A, take by weighing the following weight proportion raw material:
20～30 parts of the Radixs Astragali, 10～30 parts of Radix Puerariaes, 10～30 parts of Herba Erigerontiss;
B, powder of Radix Puerariae are broken into coarse powder, with 60～95% ethanol extractions, filter, merging filtrate reclaims ethanol, concentrates, by macroporous adsorptive resins, water elution discards water elution liquid, reuse 10～95% ethanol elutions are collected ethanol elution, reclaim ethanol, be condensed into thick paste, drying under reduced pressure is ground into fine powder, gets Radix Puerariae extract;
C, astragalus membranaceus powder are broken into coarse powder, extract with 60～95% alcohol heating reflux, filter, merging filtrate reclaims ethanol, concentrates, hydro-oxidation sodium to alkalinity is 2～5%, adds the n-butyl alcohol dynamic extraction 2～5 times, merges n-butyl alcohol liquid, the washing of 2～5% sodium hydroxide solutions washes with water again, discards washing liquid, merge n-butyl alcohol liquid, be evaporated near doing, add the acetone grinding and wash to color shallow, the taking precipitate drying under reduced pressure is ground into fine powder, gets Radix Astragali extract;
D, Herba Erigerontis decoct with water, and collecting decoction filters, and concentrate the back by macroporous adsorptive resins, water elution, discard water elution liquid, continue to collect ethanol elution, reclaim ethanol with 10～95% ethanol elutions, be condensed into thick paste, drying under reduced pressure is ground into fine powder, gets Herba Erigerontis extract;
E, the Radix Astragali extract with b, c, the preparation of d step, Radix Puerariae extract, Herba Erigerontis extract mixing add acceptable accessories and are prepared into preparation pharmaceutically commonly used.
10, the purposes of the described pharmaceutical composition of claim 1 in the medicine of preparation treatment diabetic retinopathy.
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| CN104940220B (en) * | 2015-07-07 | 2019-03-26 | 广州康臣药物研究有限公司 | The pharmaceutical composition and preparation method thereof for preventing and treating diabetic complication |
| CN110538214A (en) * | 2018-05-28 | 2019-12-06 | 重庆太极实业（集团）股份有限公司 | Composition containing astragalus root, kudzu vine root and erigeron breviscapus and application thereof |
| CN110302191A (en) * | 2019-07-09 | 2019-10-08 | 西安乐健生物科技有限公司 | A kind of pharmaceutical composition based on diplopia ' Yanming ' capsules for clearing and its application in treatment diabetic eye diseases |
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