CN101273994A - Pharmaceutical composition for curing diabetic retina pathological changes and method of preparing the same - Google Patents

Abstract

The invention relates to a composition containing 50 to 99 parts by weight of arctiin with the purity of more than 90 percent or 1 to 50 parts by weight of arctigenin and a preparation method thereof, and the functions of the composition are the treatment and the prevention of diabetic retinopathy. In the invention, by extracting the arctiin and the arctigenin, the pharmacological, toxicological and clinical trial results show that the composition of arctiin or arctigenin of the invention has the advantages of strong treatment effect and small toxicity and side effects in the treatment of the diabetic retinopathy.

Description

Be used for the treatment of pharmaceutical composition of diabetic retinopathy and preparation method thereof

Technical field

The present invention relates to the vegetable material extract is the pharmaceutical composition of active component, specifically relates to one group of composition and method of making the same that is used for the treatment of diabetic retinopathy.

Background technology

At present whole world onset diabetes rate is about 4.6%, and accumulative total reaches 200,000,000 people.Its sickness rate rises year by year, estimates to 2025, and the onset diabetes rate is about 5.4%, reaches 300,000,000 people.

(diabetic retinopathy is the most common and serious ocular complications of diabetics DR) to diabetic renal papillary necrosis, is one of three big blinding diseases in the present world wide.The ratio of the concurrent retinopathy of China's diabetics is up to 47% (the international average level is 45%).Diabetic renal papillary necrosis there is no obvious eye symptom in early days in morbidity, and along with visual deterioration or rapid drawdown can appear in disease progression, severe patient can be as blind as a bat, has a strong impact on patient's quality of life.

Because the DR pathogenesis is very complicated, up to now, the pathogenesis of diabetic renal papillary necrosis is not illustrated as yet fully, but more consistent suggestion is: under diabetic disease states, retina is exposed in the plasma glucose of high concentration, and the endocellular liberation glucose is piled up, and sorbitol is synthetic to quicken, sorbitol is piled up in the retinal capillary wall, causes capillary wall to be in hyperosmotic state for a long time; The deposition of glycosylation dead end product, protein kinase intensifies, and causes vascular endothelial cell damage, stimulates carrying out property of basement membrane to thicken, and malfunction causes pericyte's death, and microaneurysm forms and the retinal vessel permeability increases; Pericyte's death widely can cause regional retinal blood flow regulating action forfeiture, destroy the integrity of blood capillary, the blood capillary that no cell structure occurs, cause the retinal capillary obturation, no hemoperfusion causes circumvascular retinal ischemia, and ischemia stimulates the activation of new vessels multiplicaiton factor, then cause that new vessels forms, vitreous body congestion and edema, fiber propagation and secondary detachment of retina finally cause losing one's sight.Though the pathogenesis of DR is complicated, its initiating link is diabetic retina blood vessel endothelium (Vascular endothelial Cells, VECs) damage that high sugar causes.Diabetes can cause endothelial cell damage and functional defect, cause that inflammatory cell immerses, vascular smooth muscle cell proliferation, increase cell death, cause blood vessel hyperplasia, finally cause atherosis, plaque rupture and vascular restenosis.Diabetic renal papillary necrosis is because due to vascular endothelial cell damage or the dysfunction to a great extent.

It is the pathologic basis of vascular lesion that vascular endothelial function changes, and the unusual process of also having quickened diabetes and complication thereof of endothelial function.Vascular endothelial cell also is one of target organ of insulin, and insulin is with after the endothelial cell surface Insulin receptor INSR combines, and (nitrogen oxide NO) increases and realizes its vasodilative physiological function to impel it to discharge nitric oxide.The cell membrane damage that comprises endotheliocyte causes that Insulin receptor INSR distributes and changes of function, thereby produces insulin resistant, impels the inner skin cell function progressive injury again, makes dysfunction of blood vessel take place, develop.Vascular endothelial function just exists at prediabetes unusually, and along with blood sugar increasing, vascular endothelial function is impaired can be increased the weight of gradually.

The NO that endothelium discharges has a kind of vasodilator effect and suppresses hematoblastic gathering, leukocyte to the adhesion of endothelium and the propagation of intimal smooth muscle cells.It is vital in the cardiovascular mechanism of many cruxs.

Western medicine also lacks the medicine of specific treatment diabetic retinopathy at present, and also has certain toxic and side effects.And the Chinese medicine prescription exists that effective ingredient is indeterminate, content is on the low side, be unfavorable for quality of production control, and curative effect is on the low side.So also need to seek a kind of safe, effective, and the little Therapeutic Method method of side effect.

Fructus Arctii (Fructus arctii) is the dry mature fruit of Fructus Arctii, and " Chinese pharmacopoeia is recorded, and has the effect of dispelling wind and heat pathogens, lung qi dispersing rash, resolving toxin and disinhibiting the throat in order to go through version.The tradition tcm clinical practice is used for anemopyretic cold, cough with copious phlegm, measles, rubella, laryngopharynx swelling and pain, mumps erysipelas etc. more.Modern pharmacology studies have shown that Fructus Arctii has effects such as antitumor, blood sugar lowering, anti-HIV and anticoagulant.Remarkable with Fructus Arctii treatment diabetes and complication diabetic nephropathy effect thereof clinically.Pharmacological evaluation proves that also Fructus Arctii has proteinic effect in blood sugar lowering and the urine.But all adopt the Fructus Arctii live part to carry out the blood sugar lowering experiment at present both at home and abroad, and its active component and indeterminate.

In recent years, domestic Fructus Arctii water extract, the ethanol extract of studies show that can significantly and enduringly reduce rat blood sugar, and carbohydrate tolerance is increased, and toxicity is less; Fructus Arctii extract has some improvement to the diabetes rat nephropathy, and its mechanism of action may be with to alleviate the intracellular protein nonenzymatic glycosylation relevant.

Mainly contain arctiin and arctigenin in the composition in the Fructus Arctii extract, shown in arctiin (arctiin) and the following structural formula of aglycon (arctigenin) (I) thereof, the structural formula (II):

Structural formula (I) structural formula (II)

Patent ZL02133954.6 discloses a kind of Fructus Arctii total lignans glycoside extracts that is used for the treatment of diabetic nephropathy or diabetes.Its technical scheme is: extract with organic solvents such as ethanol, refining, dry, acquisition contains 50～90% (using determined by ultraviolet spectrophotometry) Fructus Arctii total lignans glycoside material, or the extract that contains arctiin 15～70% (measuring with high-efficient liquid phase technique) is the pharmaceutical preparation that the crude drug preparation is used for the treatment of diabetic nephropathy or diabetes.

It is the Chinese medicine with the effect of blood sugar lowering kidney tonifying that raw material is made with the Fructus Arctii total lignans glycoside extracts that patent application CN200610013120.0 discloses a kind of, and wherein between the content 50～89% of Fructus Arctii total lignans, the content of arctiin is more than 30%.

Patent application CN200510025834.9 discloses a kind of Fructus Arctii extract and preparation method thereof and has used, comprise total lignin compounds and arctiin in this extract based on arctigenin, the weight content of total lignin compounds and arctiin accounts for 52%-80%, and the weight content of other compositions accounts for 20%-49%.

More than Fa Ming deficiency is: only find the hypoglycemic effect of Fructus Arctii extract, and medicinal ingredient is mixture, is unfavorable for the raising of quality of production control and drug effect; Arctiin purity is not high.

In addition, patent ZL 200410015465.0 discloses a kind of method for preparing antiviral and antitumor natural drug arctigenin; Patent application CN200510035169.1 discloses the application of arctiin in the preparation anti-inflammatory drug.

The inventor finds that through the research back arctiin and arctigenin can be used for the treatment of diabetic retinopathy, and arctiin and arctigenin have protective effect to the retinal blood endothelial tube.

Summary of the invention

The objective of the invention is to propose a kind of to the diabetic retinopathy treating both the principal and secondary aspects of a disease, and good effect, medicine that toxic and side effects is low.

In order to realize the invention target, the inventor has carried out the research to the protective effect of retinal blood endothelial tube of arctiin and arctigenin.Found that arctiin and arctigenin can significantly increase the expression of endotheliocyte endothelial nitric oxide synthase; Significantly the high sugar of antagonism improves inner skin cell function to the effect of endotheliocyte inhibition of proliferation; Significantly carry endotheliocyte and under the sugared condition of height, secrete the ability of NO; Significant protection endotheliocyte is avoided the damage due to the high sugared condition.

On this basis, the inventor has carried out wider and deeper study, has realized this target.For achieving the above object, the present invention is by the following technical solutions:

The compositions of a kind of treatment and prevent diabetes retinopathy is characterized in that stating compositions and comprises: (a) arctiin, medical additive and/or pharmaceutical carrier;

Or (b) arctigenin, medical additive and/or pharmaceutical carrier;

Or (c) arctiin, arctigenin, medical additive and/or pharmaceutical carrier.

The content of arctiin is the 50-99 weight portion in the wherein said compositions (c), and the content of arctigenin is the 1-50 weight portion; Be preferably arctiin 75-95 weight portion, arctigenin 5-25 weight portion.

Arctiin purity is more than 90% (high effective liquid chromatography for measuring) in the described compositions, and the purity of arctigenin is more than 90% (high effective liquid chromatography for measuring).

The content of medical additive and/or pharmaceutical carrier is the 100-20000 weight portion;

Medical additive and pharmaceutical carrier in the described compositions comprise: filler, disintegrating agent, binding agent, lubricant, wetting agent, stabilizing agent, emulsifying agent, dispersant, antiseptic, sweeting agent, coloring agent, flavoring agent, aromatic, thickening agent, diluent, buffer substance, solvent, solubilizing agent, the reagent that is intended to obtain storage effect, the salt that is intended to change osmotic pressure, smears or antioxidant.

Described compositions can be prepared into tablet, pill, capsule, injection, eye drop, eye drop or suppository;

Below the weight of each component in weight portion, have specified otherwise except;

Wherein contain in the label of tablet or pill: compositions (a) contains arctiin 1-100, at least a 250-350 that from lactose, starch, dextrin, hydroxypropyl cellulose, selects, at least a 5-10 that from Pulvis Talci, magnesium stearate, selects, coating pre-mixing agent Opadry OY-C-7000A 3-5; Compositions (b) contains arctigenin 1-100, at least a 250-350 that selects from lactose, starch, dextrin, hydroxypropyl cellulose, at least a 5-10 that selects from Pulvis Talci, magnesium stearate, coating pre-mixing agent Opadry OY-C-7000A 3-5; Arctiin 50-99 in the compositions (c), arctigenin 1-50, at least a 250-350 weight portion of from lactose, starch, dextrin, hydroxypropyl cellulose, selecting, at least a 5-10 that from Pulvis Talci, magnesium stearate, selects, coating pre-mixing agent Opadry OY-C-7000A 3-5;

Wherein contain in the label of the double-layer sustained release tablet of release layer or pill and contain: arctiin 1-100 in the compositions (a), from lactose, starch, hydroxypropyl cellulose, at least a 80-150 that selects in the polyvinyl pyrrolidone, at least a 5-10 that from Pulvis Talci, magnesium stearate, selects; Arctigenin 1-100 in the compositions (b), from lactose, starch, hydroxypropyl cellulose, at least a 80-150 that selects in the polyvinyl pyrrolidone, at least a 5-10 that from Pulvis Talci, magnesium stearate, selects; Arctiin 50-99 in the compositions (c), arctigenin 1-50, from lactose, starch, hydroxypropyl cellulose, at least a 80-150 that selects in the polyvinyl pyrrolidone, at least a 5-10 that from Pulvis Talci, magnesium stearate, selects; Contain in the coating solution of described double-layer sustained release tablet or pill: hydroxypropyl cellulose 70-100, Polyethylene Glycol-400 1-5, Tween-80 1-5, at least a 1-5 that from lemon yellow, titanium dioxide, selects.

Wherein contain in the capsule: arctiin 1-100 in the compositions (a), at least a 200-300 that from lactose, starch, dextrin, hydroxypropyl cellulose, selects, at least a 1-4 that from Pulvis Talci, magnesium stearate, selects; Arctigenin 1-100 in the compositions (b), at least a 200-300 that from lactose, starch, dextrin, hydroxypropyl cellulose, selects, at least a 1-4 that from Pulvis Talci, magnesium stearate, selects; Arctiin 50-99 in the compositions (c), arctigenin 1-50, at least a 200-300 that from lactose, starch, dextrin, hydroxypropyl cellulose, selects, at least a 1-4 that from Pulvis Talci, magnesium stearate, selects;

Wherein contain in the injection: arctiin 1-100 in the compositions (a), sodium chloride 18, water for injection is settled to 2000; Arctigenin 1-100 in the compositions (b), sodium chloride 18, water for injection is settled to 2000; Arctiin 50-99 in the compositions (c), arctigenin 1-50, sodium chloride 18, water for injection is settled to 2000;

Wherein contain in the suppository: arctiin 1-100 in the compositions (a), semi-synthetic fatty acid ester 600-800; Arctigenin 1-100 in the compositions (b), semi-synthetic fatty acid ester 600-800; Arctiin 50-99 in the compositions (c), arctigenin 1-50, semi-synthetic fatty acid ester 600-800;

Wherein contain in the eye drop: arctiin 1-100 in the compositions (a), AMSP 128.6, disodium hydrogen phosphate,anhydrous 37.8, sodium chloride 84, ethyl hydroxybenzoate 6, water for injection is settled to 20000; Arctigenin 1-100 in the compositions (b), AMSP 128.6, disodium hydrogen phosphate,anhydrous 37.8, sodium chloride 84, ethyl hydroxybenzoate 6, water for injection is settled to 20000; Arctiin 50-99 in the compositions (c), arctigenin 1-50, AMSP 128.6, disodium hydrogen phosphate,anhydrous 37.8, sodium chloride 84, ethyl hydroxybenzoate 6, water for injection is settled to 20000.

Described weight portion can be gram, liang, kilogram, jin, ton etc.

The present invention's arctiin and the arctigenin of purity of having purified respectively greater than 90% (high-performance liquid chromatogram determination), arctiin, the independent medication of arctigenin can act on retinal endothelial cell treatment and prevent diabetes retinopathy respectively, and arctiin and arctigenin composite reagent are had collaborative therapeutic effect for the treatment diabetic retinopathy.

Fructus Arctii after the purification and the impurity in the arctigenin comprise: arctigenin-4'-gentiobioside B (lappaol B), arctigenin-4'-gentiobioside A (lappaol A), arctigenin-4'-gentiobioside F (lappaol F), martairesinol (matairesinol), 8-hydroxyl pinoresinol (8-hydroxypinoresinol), (+)-Cortex Fraxini resinol ((+)-Fraxiresinol), kaempferol (kaempferol), acanthoside B (acanthoside B), Quercetin (quercetin) and isoquercitin (isoquercitrin) etc.

Being used for the treatment of with the preparation of compositions method of prevent diabetes retinopathy that the present invention takes is as follows:

1, the process for separation and purification of arctiin, arctigenin is:

(1) preparation of arctiin

1. slightly carrying of arctiin: Fructus Arctii is put in the apparatus,Soxhlet's, through boiling range 60-90 ℃ of petroleum ether backflow defat 2-4 time after pulverizing, take out after the defat and dry, 8 times of alcohol reflux of 60%-80% 2-4 time, each 0.5-2h, 60 ℃ of-80 ℃ of concentrating under reduced pressure of extracting solution get dark brown extractum;

2. macroporous resin is refining: dark brown extractum is added 15-35% ethanol, supersound process 15-35 minute, make its exactly fully dissolving, filtration transfers to into the solution that concentration is about 5-7mg/mL, and by AB-8 type macroporous adsorbent resin, the macroporous adsorbent resin volume is 1-2 a times of medicine liquid volume, flow speed control is 1.5-2.5BV/h, the post blade diameter length ratio is controlled to be 1: 5, washes 3 times of resin volumes, washing speed 0.5-3BV/h; Change the ethanol elution with 4-6BV50% again, alcohol is washed speed 1-3BV/h, collects washing liquid, and concentrate drying gets the arctiin crude product;

3. the separation of arctiin: the Fructus Arctii crude product is with the thick silica gel mixed sample of 60 orders, the 1-2 that thick silica gel volume is the arctiin crude product doubly, through 200-300 order silica gel column chromatography, silicagel column is 10-50 a times of arctiin crude product, 95: 1 eluting of chloroform-methanol, thin layer chromatography trace flow fluid, the stream part of collecting the arctiin single-point, merging concentrates, and gets arctiin;

(2) arctigenin preparation

1. arctigenin is slightly carried: after the Fructus Arctii pulverizing medicinal materials, cross 80 mesh sieves, put in the apparatus,Soxhlet's, through the oily ether backflow defat of 60～90 ℃ of boiling ranges 3 times, dry the back and extract 2-4 time, at every turn 1.5-2.5h with ethyl acetate backflow, 70 ℃ of concentrating under reduced pressure of extracting solution get brown extractum;

2. arctigenin is refining: with brown extractum with the thick silica gel mixed sample of 60 orders, the 1-2 that thick silica gel volume is the arctigenin crude product doubly, water-bath is dried, sample on the dry method, through 200-300 order silica gel column chromatography, 98: 1 eluting of chloroform-methanol, eluent concentrate to merge and obtain arctigenin.

Preferred version when wherein macroporous resin is made with extra care the arctiin extract is: get the arctiin ethanol extract, add 15-30% ethanol, fully stir, supersound process 15-30 minute, make its exactly fully dissolving, filtering and transferring to last sample liquor strength is the solution of 3-6.5mg/mL, use AB-8 type macroporous adsorbent resin, the macroporous adsorbent resin volume is 1-2 a times of medicine liquid volume, and last sample flow speed control is 2BV/h, and the post blade diameter length ratio is controlled to be 1: 5.Wash 2-3 times of resin volume, washing speed 1-2BV/h; Change the ethanol elution with 3.5-4.5BV50% again, alcohol is washed speed 1-2BV/h, collects pure washing liquid, and concentrate drying gets the Fructus Arctii crude product.

2, the proportional arrangement of pressing arctiin, arctigenin, medical additive and/or pharmaceutical carrier is mixed.

The compositions that the present invention relates to can give by any suitable approach, for example, by under oral, eye part, intravenous, intramuscular, intradermal, the epidermis, rectum, percutaneous or through the lung administration.

The compositions that the present invention relates to can include but not limited to pill by any oral form of medication that meets above-mentioned composition, conventional tablet, bilayer tablet, multilayer tablet, slow releasing tablet, the single chamber controlled release tablet, two chambers controlled release tablet, the pore type controlled release tablet, sublingual lozenge, oral cavity quick disintegrating slice, dispersible tablet, enteric coatel tablets, granule, delayed-release tablet, regularly/the position releasing piece, conventional capsule, enteric coated capsule, slow releasing capsule, controlled release capsule, the capsule that contains micropill or small pieces, the pH dependent form capsule that contains micropill or small pieces, granule, membrane or patch, aqueous solution, alcoholic solution or oil solution, Emulsion or suspensoid; The form of eye topical includes but not limited to eye drop, ophthalmic ointment, eye patch; The form of rectally includes but not limited to suppository; The form of drug administration by injection includes but not limited to solution, solid powder or block, Emulsion or suspensoid.Other form of medication that is suitable for also comprises and being not limited to through skin or topical, for example with the form of ointment, tincture, spraying or transdermal therapeutic agent system, or with the inhalation of the form of nasal spray or aerosol mixture, or take microcapsule, implant or implant the form administration of rod.This preferred form of medication is the form of oral, drug administration by injection form and eye topical.

The preparation method of pharmaceutical administration form waits to determine according to the character of its concrete form, compositions practical situation and additive usually.The method that is used to prepare pharmaceutical administration form of the present invention is well known in the art, and is conspicuous for those of ordinary skill in the art.At this on the one hand, combine with arctiin, arctigenin or arctiin and arctigenin and one or more solids or liquid medicine carrier and/or medical additive and with other chemical compounds, make suitable form of medication or dosage form with pharmaceutically active with treatment or prophylactic function.

The form of medication of the compositions that the present invention relates to also can comprise the conventional additives that those skilled in the art all know, for example filler, disintegrating agent, binding agent, lubricant, wetting agent, stabilizing agent, opacifier, emulsifying agent, dispersant, antiseptic, sweeting agent, coloring agent, flavoring agent, aromatic, thickening agent, diluent, buffer substance, solvent, solubilizing agent, the reagent that is intended to obtain storage effect, the salt that is intended to change osmotic pressure, smears or antioxidant.The selection of the carrier that the present invention relates to or the selection of additive and consumption thereof waits to determine according to concrete application form, oral cavity composition practical situation, preparation method and subjective demand.

The oral administration form comprises inert diluent or edible carrier usually.They can be encapsulated in the gelatine capsule or be pressed into tablet.For oral administration treatment, can be with reactive compound or its prodrug derivatives with mixed with excipients and with tablet, the use of the form of lozenge or capsule.Also can be included in pharmaceutically compatible binding agent and/or adjuvant material, as the part in the compositions.

Tablet, pill, capsule, lozenge or the like can comprise any following component or kin chemical compound: binding agent such as microcrystalline Cellulose, hydroxypropyl cellulose, methylcellulose or gelatin; Excipient such as starch, mannitol or lactose; Plasticizer is glycerol, propylene glycol or Polyethylene Glycol; Dispersant such as alginic acid or corn starch; Opacifier such as titanium dioxide; Antiplastering aid such as magnesium stearate or Pulvis Talci; Lubricant such as colloidal silica; Sweetener such as glucide, aspartame or steviosin; Correctives such as Herba Menthae, orange peel oil.When dosage unit form was capsule, except that the material of mentioned kind, it can comprise liquid-carrier such as fatty oil.In addition, dosage unit form can comprise various other and can change the material of dosage unit physical aspect or performance, for example coating materials.The carrier of Perle and suppository be for example fat, wax, semisolid and liquid polyol, natural or the sclerosis wet goods.The carrier that is applicable to microcapsule, implant or implants rod is the copolymer of hydroxyacetic acid and lactic acid for example.

Also can be with the lyophilized products of arctiin, arctigenin or arctiin and arctigenin, for example use it for preparation and be used for injecting preparation with infusion.

Be used for non-intestinal, intradermal, solution or suspension subcutaneous or topical can comprise following component: the diluent of sterilization such as water for injection, saline solution, fixing oil, Polyethylene Glycol, glycerol, propylene glycol or other synthetic; Antibacterial such as benzyl alcohol or nipagin; Antioxidant such as ascorbic acid or sodium sulfite; Intercalating agent such as ethylenediaminetetraacetic acid; Buffer agent such as acetate, the reagent of citrate salt or phosphate and adjustment of tonicity such as sodium chloride.The preparation of parenterai administration can be encapsulated in the ampoule of being made by glass or plastics, can handle in syringe or the multiple dose vials.

If by intravenous administration, preferred carrier is the saline of normal saline or phosphate-buffered.

The amount of the compositions that the present invention relates in pharmaceutical preparation is generally every dose of 0.1～1000mg (in arctiin, arctigenin or arctiin and arctigenin, down together), preferred 0.5～500mg, particularly 1～200mg.Typical topical dosage is 0.01～3%wt/wt (in suitable carrier).

The compositions that the present invention relates to obtains the peak serum concentration of reactive compound, and its value is 0.00001～10mmol/L, is preferably 0.001～300 μ mol/L, more preferably 0.01～30 μ mol/L.For example, this can be by the solution or the preparation of intravenous injection active component, and dispensable saline or aqueous vehicles perhaps can be rolled into a ball by the medicine that gives active component and obtained.The blood drug level of the reactive compound in the compositions (arctiin, arctigenin or arctiin and arctigenin) depends on the absorption of medicine, distributes deactivation and discharge rate and other factors well known by persons skilled in the art.

In the compositions that the present invention relates to, arctiin, arctigenin or arctiin and arctigenin and other can not weakened the active substance of required effect yet, or with the material that replenishes required effect, as other identical active medicine, antidiabetic drug, antioxidant, aldose reductase inhibitor, endothelial growth factor receptor inhibitors, platelet aggregation inhibitor or their mixture.

The invention still further relates to the purposes of above-mentioned compositions.It can be used for treating medicine, health product or food additive with the prevent diabetes retinopathy.

The invention still further relates to the compositions of a kind of treatment and prevent diabetes retinopathy, comprising:

I. arctiin, arctigenin or arctiin and arctigenin,

II. other curative, described curative is selected from: other identical active medicine with arctiin, antidiabetic drug, antioxidant, aldose reductase inhibitor, endothelial growth factor receptor inhibitors, platelet aggregation inhibitor or their mixture.

In the above-mentioned method, the dosage of using the administration of arctiin, arctigenin or arctiin and arctigenin depends on each example, thereby and according to usage reaches best effect according to each situation adjustment.

The dose value of using the administration of the compositions that the present invention relates to also produces heavy alleviating of degree along with disease and changes.Further will be appreciated that, with regard to any specific treatment target, specific dosage should be according to individual professional judgment of stopping the people that needs and concrete course of treatment or supervision give compositions, change in time and regulate, and the concentration range that this paper proposed only illustrates scope or the application that does not limit disclosed compositions as an example.Thus, above-mentioned dosage depends on the character and the order of severity of pending disease, the sex and the sign that also depend on the patient, depend on arctiin and aglycon action time thereof, whether depend on that treatment is acute secular or preventative, perhaps depending on has other reactive compound by administration except that arctiin, arctigenin or arctiin and arctigenin.Generally speaking, in order to obtain desirable effect, suitable dosage every day is 0.01～500mg/kg, preferred 0.05～200mg/kg, particularly 0.1～100mg/kg.Every day, dosage can use in single administration, perhaps was divided into several times (for example twice or three times) administration.In some cases, depend on individual reaction, may need to adjust up or down from dosage every day that provides.

The advantage of the relative prior art of the present invention:

1, the present invention has found that arctiin and arctigenin for the therapeutical effect of retina endotheliocyte, can significantly improve endotheliocyte is secreted NO under the sugared condition of height ability, significantly protects endotheliocyte to avoid damage due to the high sugared condition.

2, compare with the chemicals for the treatment of diabetic retinopathy in the past, the compositions that the present invention relates to has the effect for the treatment of both the principal and secondary aspects of a disease, promptly has very strong therapeutic effect at diabetes, improves the retina endotheliocyte again.Thereby have multiple curative effect, and toxicity is extremely low, and side effect is minimum.

3, compare with traditional prescription, the Chinese patent medicine for the treatment of diabetic retinopathy in the past, it is clear and definite that medicine that the present invention relates to or pharmaceutical composition have effective ingredient, the purity height; Help quality of production control, help implementing the modernization of Chinese medicine; Curative effect is higher, and toxic and side effects does not have advantages such as obviously increase.

Described the present invention thus in detail, obviously also various changes can have been arranged within the scope of the invention to those skilled in the art, the present invention is not subjected to the described restriction of description.

The specific embodiment

Described the present invention thus in detail, obviously also various changes can have been arranged within the scope of the invention to those skilled in the art, the present invention is not subjected to the described restriction of description.

The preparation method of embodiment 1 arctiin and arctigenin

(1) preparation of arctiin

1. slightly carrying of arctiin: Fructus Arctii is put in the apparatus,Soxhlet's after pulverizing, through boiling range 60-90 ℃ of petroleum ether backflow defat 2 times, take out after the defat and dry, 60% 8 times of alcohol reflux 2 times, each 0.8h, 60 ℃ of concentrating under reduced pressure of extracting solution, dark brown extractum;

2. macroporous resin is refining: dark brown extractum is added 15% ethanol, supersound process 15 minutes, make its exactly fully dissolving, filtration transfers to into the solution that concentration is about 5mg/mL, and by AB-8 type macroporous adsorbent resin, the macroporous adsorbent resin volume is 1 times of a medicinal liquid, flow speed control is 1.5BV/h, the post blade diameter length ratio is controlled to be 1: 5, washes 3 times of resin volumes, washing speed 0.5BV/h; Change the ethanol elution with 4BV50% again, alcohol is washed speed 1BV/h, collects washing liquid, and concentrate drying gets the arctiin crude product;

3. the separation of arctiin: the arctiin crude product is with the thick silica gel mixed sample of 60 orders, thick silica gel volume is 1 times of arctiin crude product, through 200-300 order silica gel column chromatography, silicagel column is 10 times of arctiin crude product, 95: 1 eluting of chloroform-methanol, thin layer chromatography trace flow fluid, collect stream part of arctiin single-point, merging concentrates, and gets arctiin, and high-performance liquid chromatogram determination purity is 90.3%;

(2) arctigenin preparation

1. arctigenin is slightly carried: after the Fructus Arctii pulverizing medicinal materials, cross 80 mesh sieves, put in the apparatus,Soxhlet's,, dry the back and extract 2 times with ethyl acetate backflow through the oily ether backflow defat of 60～90 ℃ of boiling ranges 3 times, each 1.5h, 70 ℃ of concentrating under reduced pressure of extracting solution, brown extractum;

2. arctigenin is refining: brown with extractum with the thick silica gel mixed sample of 60 orders, thick silica gel volume is 1 times of Fructus Arctii crude product, the water-bath oven dry, sample on the dry method, through 200-300 order silica gel column chromatography, 98: 1 eluting of chloroform-methanol, eluent concentrate to merge and obtain arctigenin, and high-performance liquid chromatogram determination purity is 90.5%.

The preparation method of embodiment 2 arctiins and arctigenin

(1) preparation of arctiin

1. slightly carrying of arctiin: Fructus Arctii is put in the apparatus,Soxhlet's after pulverizing, through boiling range 60-90 ℃ of petroleum ether backflow defat 4 times, take out after the defat and dry, 80% 8 times of alcohol reflux 4 times, each 2h, 80 ℃ of concentrating under reduced pressure of extracting solution, dark brown extractum;

2. macroporous resin is refining: dark brown extractum is added 35% ethanol, supersound process 35 minutes, make its exactly fully dissolving, filtration transfers to into the solution that concentration is about 7mg/mL, and by AB-8 type macroporous adsorbent resin, the macroporous adsorbent resin volume is 2 times of medicinal liquid, flow speed control is 2.5BV/h, the post blade diameter length ratio is controlled to be 1: 5, washes 3 times of resin volumes, washing speed 3BV/h; Change the ethanol elution with 6BV50% again, alcohol is washed speed 3BV/h, collects washing liquid, and concentrate drying gets the arctiin crude product;

3. the separation of arctiin: the arctiin crude product is with the thick silica gel mixed sample of 60 orders, thick silica gel volume is 2 times of arctiin crude product, through 200-300 order silica gel column chromatography, the silicagel column volume is 50 times of arctiin crude product, 95: 1 eluting of chloroform-methanol, thin layer chromatography trace flow fluid, collect stream part of arctiin single-point, merging concentrates, and gets arctiin, and high-performance liquid chromatogram determination purity is 90.7%;

(2) arctigenin preparation

1. arctigenin is slightly carried: after the Fructus Arctii pulverizing medicinal materials, cross 80 mesh sieves, put in the apparatus,Soxhlet's,, dry the back and extract 4 times with ethyl acetate backflow through the oily ether backflow defat of 60～90 ℃ of boiling ranges 3 times, each 2.5h, 70 ℃ of concentrating under reduced pressure of extracting solution, brown extractum;

2. arctigenin is refining: with brown extractum with the thick silica gel mixed sample of 60 orders, thick silica gel volume is 2 times of arctigenin crude product, the water-bath oven dry, sample on the dry method, through 200-300 order silica gel column chromatography, 98: 1 eluting of chloroform-methanol, eluent concentrate to merge and obtain arctigenin, and high-performance liquid chromatogram determination purity is 90.8%.

The preparation method of embodiment 3 arctiins and arctigenin

(1) preparation of arctiin

1. slightly carrying of arctiin: Fructus Arctii is put in the apparatus,Soxhlet's after pulverizing, through boiling range 60-90 ℃ of petroleum ether backflow defat 3 times, take out after the defat and dry, 70% 8 times of alcohol reflux 3 times, each 1h, 70 ℃ of concentrating under reduced pressure of extracting solution, dark brown extractum;

2. macroporous resin is refining: dark brown extractum is added 20% ethanol, supersound process 20 minutes, make its exactly fully dissolving, filtration transfers to into the solution that concentration is about 6mg/mL, and by AB-8 type macroporous adsorbent resin, the macroporous adsorbent resin volume is 1.5 times of medicine liquid volume, flow speed control is 2BV/h, the post blade diameter length ratio is controlled to be 1: 5, washes 3 times of resin volumes, washing speed 2BV/h; Change 50% ethanol elution with 5BV again, alcohol is washed speed 2BV/h, collects washing liquid, and concentrate drying gets the arctiin crude product;

3. the separation of arctiin: the arctiin crude product is with the thick silica gel mixed sample of 60 orders, thick silica gel volume is 2 times of arctiin crude product, through 200-300 order silica gel column chromatography, the silicagel column volume is 25 times of arctiin crude product, 95: 1 eluting of chloroform-methanol, thin layer chromatography trace flow fluid, collect stream part of arctiin single-point, merging concentrates, and gets arctiin, and high-performance liquid chromatogram determination purity is 91.3%;

(2) arctigenin preparation

1. arctigenin is slightly carried: after the Fructus Arctii pulverizing medicinal materials, cross 80 mesh sieves, put in the apparatus,Soxhlet's,, dry the back and extract 3 times with ethyl acetate backflow through the oily ether backflow defat of 60～90 ℃ of boiling ranges 3 times, each 2h, 70 ℃ of concentrating under reduced pressure of extracting solution, brown extractum;

2. arctigenin is refining: with brown extractum with the thick silica gel mixed sample of 60 orders, thick silica gel volume is 1.5 times of arctigenin crude product, the water-bath oven dry, sample on the dry method, through 200-300 order silica gel column chromatography, 98: 1 eluting of chloroform-methanol, eluent concentrate to merge and obtain arctigenin, and high-performance liquid chromatogram determination purity is 91.7%.

The preparation method of embodiment 4 arctiins and arctigenin

(1) preparation of arctiin

1. slightly carrying of arctiin: Fructus Arctii is put in the apparatus,Soxhlet's after pulverizing, through 80 ℃ of petroleum ether backflows of boiling range defat 3 times, take out after the defat and dry, 75% 8 times of alcohol reflux 3 times, each 1h, 70 ℃ of concentrating under reduced pressure of extracting solution, dark brown extractum;

2. macroporous resin is refining: dark brown extractum is added 25% ethanol, fully stir, supersound process 25 minutes makes its exactly fully dissolving, and filtering and transferring to last sample liquor strength is the solution of 5mg/mL, use AB-8 type macroporous adsorbent resin, the macroporous adsorbent resin volume is 1.5 times of medicine liquid volume, and last sample flow speed control is 2BV/h, and the post blade diameter length ratio is controlled to be 1: 5, wash 3 times of resin volumes, washing speed 1.5BV/h; Change the ethanol elution with 4BV50% again, alcohol is washed speed 1.5BV/h, collects pure washing liquid, and concentrate drying gets the arctiin crude product;

3. the separation of arctiin: the arctiin crude product is with the thick silica gel mixed sample of 60 orders, thick silica gel volume is 2 times of arctiin crude product, through 200-300 order silica gel column chromatography, the silicagel column volume is 40 times of arctiin crude product, 95: 1 eluting of chloroform-methanol, thin layer chromatography trace flow fluid, the stream part of collecting the arctiin single-point, merge to concentrate arctiin, high-performance liquid chromatogram determination purity is 90.8%;

(2) arctigenin preparation

1. arctigenin is slightly carried: after the Fructus Arctii pulverizing medicinal materials, cross 80 mesh sieves, put in the apparatus,Soxhlet's,, dry the back and extract 3 times with ethyl acetate backflow through the oily ether backflow defat of 60～90 ℃ of boiling ranges 3 times, each 2h, 70 ℃ of concentrating under reduced pressure of extracting solution, brown extractum;

2. arctigenin is refining: with brown extractum with the thick silica gel mixed sample of 60 orders, thick silica gel volume is 2 times of arctigenin crude product, the water-bath oven dry, sample on the dry method, analyse layer through 200-300 order silicagel column, 98: 1 eluting of chloroform-methanol, eluent concentrate to merge and obtain arctigenin, and high-performance liquid chromatogram determination purity is 91.2%.

Embodiment 5 capsules

Prescription: compositions (a)

Arctiin (active component) 100g

Lactose (excipient) 130g

Starch (excipient) 120g

Magnesium stearate (antiplastering aid) 2.5g

Make 1000 altogether

Compositions (b)

Arctigenin (active component) 100g

Lactose (excipient) 130g

Starch (excipient) 120g

Magnesium stearate (antiplastering aid) 2.5g

Make 1000 altogether

Compositions (c)

Arctiin (active component) 50g

Arctigenin (active component) 50g

Lactose (excipient) 130g

Starch (excipient) 120g

Magnesium stearate (antiplastering aid) 2.5g

Make 1000 altogether

Method for making: active ingredient and pharmaceutic adjuvant are all crossed 100 mesh sieves,, in mixer, mix 10-15min, add magnesium stearate 2.5g by prescription accurate weighing respectively, mix 2min after, pack 1000 capsules into promptly.

Embodiment 6 injections

Prescription: compositions (a)

Arctiin (active component) 100g

Sodium chloride (regulating the salt of osmotic pressure) 18g

Water for injection (pharmaceutical carrier) is to 2000ml

Make 1000 altogether

Compositions (b)

Arctigenin (active component) 100g

Sodium chloride (regulating the salt of osmotic pressure) 18g

Water for injection (pharmaceutical carrier) is to 2000ml

Make 1000 altogether

Compositions (c)

Arctiin (active component) 85g

Arctigenin (active component) 15g

Sodium chloride (regulating the salt of osmotic pressure) 18g

Water for injection (pharmaceutical carrier) is to 2000ml

Make 1000 altogether

Preparation method: the active ingredient that takes by weighing recipe quantity, the water for injection that adds full dose 90% stirs and makes dissolving, 0.1% (W/V) ratio in the injection water yield adds active carbon, stirred 20 minutes, after buchner funnel is spread two-layer aseptic filter paper sucking filtration, again with coarse filtration liquid through sterilized 0.22 μ m microporous filter membrane fine straining.Add water for injection to capacity, making arctiin and arctigenin content is 95.0%～105.0% of labelled amount, and fill promptly.

Embodiment 7 tablets or pill

Label prescription: compositions (a)

Arctiin (active component) 100g

Dextrin (excipient) 162.5g

Lactose (excipient) 137.5g

Low-substituted hydroxypropyl cellulose (binding agent) 18.75g

50% ethanol (binding agent) 112.5ml of 4% hydroxypropyl cellulose

Pulvis Talci (antiplastering aid) 3.75g

Magnesium stearate (antiplastering aid) 3.75g

Make 1250 altogether

Compositions (b)

Arctigenin (active component) 100g

Dextrin (excipient) 162.5g

Lactose (excipient) 137.5g

Low-substituted hydroxypropyl cellulose (binding agent) 18.75g

50% ethanol (binding agent) 112.5ml of 4% hydroxypropyl cellulose

Pulvis Talci (antiplastering aid) 3.75g

Magnesium stearate (antiplastering aid) 3.75g

Make 1250 altogether

Compositions (c)

Arctiin (active component) 75g

Arctigenin (active component) 25g

Dextrin (excipient) 162.5g

Lactose (excipient) 137.5g

Low-substituted hydroxypropyl cellulose (binding agent) 18.75g

50% ethanol (binding agent) 112.5ml of 4% hydroxypropyl cellulose

Pulvis Talci (antiplastering aid) 3.75g

Magnesium stearate (antiplastering aid) 3.75g

Make 1250 altogether

The coating solution prescription:

Coating pre-mixing agent (Opadry OY-C-7000A) 4.37g

Pure water 30g

Make 1250

Preparation method: active component and other pharmaceutic adjuvants are all crossed 100 mesh sieves, take by weighing the arctiin of recipe quantity, arctigenin, lactose, dextrin and account for half low-substituted hydroxypropyl cellulose of recipe quantity, by the equivalent method mix homogeneously that progressively increases, 50% ethanol with 4% hydroxypropyl cellulose is wetting agent system soft material, 18 mesh sieves are granulated, wet granular is in 45～50 ℃ of dryings, 20 mesh sieve granulate, the low-substituted hydroxypropyl cellulose that adds surplus, the micropowder silica gel of recipe quantity, the magnesium stearate mixing, after measuring active ingredient content, determine that sheet is heavy, in 10.0mm shallow concave punch tabletting.Wrap with the white film clothing coating weightening finish 1～2% according to a conventional method.

The preparation of coating solution: under agitation coating pre-mixing agent (Opadry OY-C-7000A) is added in the pure water, add in 5 minutes, continue to stir 45 minutes promptly.

Embodiment 8 eye drops

Prescription: compositions (a)

Arctiin (active component) 50.0g

AMSP (adjusting pH value) 128.6g

Disodium hydrogen phosphate,anhydrous (adjusting pH value) 37.80g

Sodium chloride (regulating the salt of osmotic pressure) 84.0g

Ethyl hydroxybenzoate (antibacterial antiseptic) 6.0g

Water for injection (pharmaceutical carrier) adds to 20000ml

Make 4000 altogether

Compositions (b)

Arctigenin (active component) 50.0g

AMSP (adjusting pH value) 128.6g

Disodium hydrogen phosphate,anhydrous (adjusting pH value) 37.80g

Sodium chloride (regulating the salt of osmotic pressure) 84.0g

Ethyl hydroxybenzoate (antibacterial antiseptic) 6.0g

Water for injection (pharmaceutical carrier) adds to 20000ml

Make 4000 altogether

Compositions (c)

Arctiin (active component) 40.0g

Arctigenin (active component) 10.0g

AMSP (adjusting pH value) 128.6g

Disodium hydrogen phosphate,anhydrous (adjusting pH value) 37.80g

Sodium chloride (regulating the salt of osmotic pressure) 84.0g

Ethyl hydroxybenzoate (antibacterial antiseptic) 6.0g

Water for injection (pharmaceutical carrier) adds to 20000ml

Make 4000 altogether

Preparation method: the active component that takes by weighing recipe quantity, add in about 80 ℃ hot waters for injection of temperature of about 30%, stirring makes its dissolving, again AMSP, disodium hydrogen phosphate,anhydrous, sodium chloride and oxybenzene second vinegar are progressively added wherein, stirring makes its dissolving, add 0.05% needle-use activated carbon, in 70～80 ℃ of absorption of temperature 15 minutes, take off charcoal, benefit adds to the full amount of water for injection, stir evenly, it is qualified after 0.22 μ m filtering with microporous membrane is in 100 ℃ of sterilizations of temperature 30 minutes, aseptic subpackaged to measure its pH value and content, every 5ml, promptly.

Embodiment 9 suppositorys

Prescription: compositions (a)

Arctiin (active component) 80g

Semi-synthetic fatty acid ester (pharmaceutical carrier) 700g

Make 400

Compositions (b)

Arctigenin (active component) 80g

Semi-synthetic fatty acid ester (pharmaceutical carrier) 700g

Make 400

Compositions (c)

Arctiin (active component) 85g

Arctigenin (active component) 15g

Semi-synthetic fatty acid ester (pharmaceutical carrier) 700g

Make 400

Preparation method: take by weighing active ingredient and cross 100 mesh sieves, other gets semi-synthetic fatty acid ester 700g heating and melting, when treating that temperature is reduced to below 60 ℃, active ingredient medicated powder is added, and stirs, and waters and is molded as 400, promptly.

Embodiment 10 contains the double-layer sustained release tablet of release layer

Slow release label prescription: compositions (a)

Arctiin (active component) 100.0g

Hydroxypropyl cellulose (binding agent) 28.7g

Lactose (excipient) 58.0g

Polyvinyl pyrrolidone (disintegrating agent) 8.0g

Stearic acid (antiplastering aid) 5.3g

Make 667

Compositions (b)

Arctiin (active component) 100.0g

Hydroxypropyl cellulose (binding agent) 28.7g

Lactose (excipient) 58.0g

Polyvinyl pyrrolidone (disintegrating agent) 8.0g

Stearic acid (antiplastering aid) 5.3g

Make 667

Compositions (c)

Arctiin (active component) 85.0g

Arctigenin (active component) 15.0g

Hydroxypropyl cellulose (binding agent) 28.7g

Lactose (excipient) 58.0g

Polyvinyl pyrrolidone (disintegrating agent) 8.0g

Stearic acid (antiplastering aid) 5.3g

Make 667

The coating solution prescription:

Hydroxypropyl cellulose (binding agent) 86g

Polyethylene Glycol-400 (solubilizing agent) 3ml

Polyoxyethylene sorbitan monoleate (emulsifying agent) 2ml

Titanium dioxide (coloring agent) 3g

Lemon yellow (coloring agent) 6mg

60% ethanol 3200ml

Make 3200ml

Preparation method:

1, pretreatment: active component is crossed sieve No. 7, and adjuvant hydroxypropyl cellulose, polyvinyl pyrrolidone, stearic acid are crossed sieve respectively No. 7.

2, nicotinic acid tablet slug particle preparation: take by weighing the hydroxypropyl cellulose of active ingredient, polyvinyl pyrrolidone and 31% recipe quantity of recipe quantity, mix homogeneously, as mixed powder, water is granulated.Wet grain is put 60 ℃ of aeration-dryings, and dried granule adds the stearic acid of recipe quantity and the HPMC of surplus, mixing behind No. 2 sieve granulate.Survey the granule content of dispersion, basis of calculation sheet is heavy.

3, tabletting: the stamping of 13mm scrobicula, tablet hardness are controlled at 5～6 kilograms (kgf).

4, the preparation of coating solution: take by weighing hydroxypropyl methylcellulose, Polyethylene Glycol-400 and the tween 80 of recipe quantity, be dissolved in 60% ethanol of recipe quantity, and grind the titanium dioxide of recipe quantity, get supernatant and promptly get blank coating solution with this liquid.

5, coating: get label and weigh, put into coating pan, temperature is controlled at 40 ± 2 ℃, wraps a few minutes in advance, and the arctiin of crossing No. 7 sieves is distributed in the coating solution, sprays into coating solution continuously, to tablet weightening finish 15%.

The pharmacodynamic experiment of embodiment 11 arctiins and arctigenin

One, test material

(1), trial drug

Trial drug: arctiin, arctigenin, purity all require greater than 90.0%, measured value: arctiin 94.2% (HPLC method), arctiin 95.2% (HPLC method).

(2), diabetic renal papillary necrosis rat model method for building up

120 of male Wistar rats (body weight 180-200g) after adaptability raised for 1 week, are divided into 2 groups with rat by body weight at random, wherein normal group (Normal, N) 20 give conventional feed; 100 rats of modeling group give high lipid food.After modeling group high lipid food continues to raise for 4 weeks, the beginning modeling.

Modeling method: (1) modeling group streptozotocin (STZ) medication: rat fasting 12h before the experiment, STZ face with preceding and are mixed with 5% solution with 0.1mmol citric acid-sodium citrate buffer (pH4.6), press 45mgkg ^-1Intraperitoneal injection STZ; (2) normal group medication: rat fasting 12h before the experiment, the isopyknic 0.1mmol citric acid-sodium citrate buffer of lumbar injection (pH 4.6).

The injection streptozotocin is measured 2 hours after the meal blood glucose after 1 week, selects blood glucose value at 11.1-20mmolL ^-1, and have polydipsia, polyuria, polyphagia characteristic person and be defined as the diabetic renal papillary necrosis model.The diabetic renal papillary necrosis model group continues high glucose and high fat raises, and carried out visual electrophysiology and detect after 3 months: animal dark adaptation 20min, pentobarbital sodium is pressed 50mgkg ^-1Intraperitoneal injection of anesthesia, the abundant mydriasis of Tropicamide, by the standard recording flash electroretinogram (FERG) of International Society for Clinical Electrophysiology of Vision, comprise retina oscillatory potential (Ops), filter out and diabetic renal papillary necrosis rat that visual function damages occurs as the DR rat model.

(2), experimental animal grouping:

60 diabetic renal papillary necrosis rat models getting the modeling success carry out random packet, the grouping situation:

1. matched group (n=20): give normal drinking-water and feedstuff;

2. model group (n=20): high-sugar-fat-diet is raised, and does not carry out pharmaceutical intervention;

3. arctiin group (n=20): the high glucose and high fat forage feed, press 50mgKg every day ^-1Arctiin is irritated stomach;

4. arctiin tuple (n=20): the high glucose and high fat forage feed, press 50mgKg every day ^-1Arctigenin is irritated stomach.

Two, index determining

(1) during the administration, the general situation of laboratory animal is observed

Observe every day in the administration process, and record general situation of animal and death condition; Per 2 weeks weigh 1 time, and calculate dosage according to body weight; Every month from tail vein extracting vein blood detection blood sugar concentration; Connect urine with metabolic cage per February, detects the urine amount.

(2) change of detection electroretinogram: the variation of a of detection electroretinogram (ERG), b ripple, retina oscillatory potential (Ops) wave amplitude

2% pentobarbital sodium is pressed 30mgKg ^-1Intraperitoneal injection of anesthesia, the abundant mydriasis of Tropicamide carries out the record of mfERG to the method for the guide of the how burnt electroretinogram of clinography (mfERG) with reference to International Society for Clinical Electrophysiology of Vision.Waveform with RetiScan (examination of visual electro physiology system) the intermediate range ordered pair mfERG of system is analyzed automatically, when hide in the peak that analyzing total waveform, 5 rings, four resemble district and each regional N1 ripple and P1 ripple, amplitude and calculate degree of reaction.

(3) the routine biochemistry index relevant with diabetes

Serum biochemistry index: after the last administration, fasting 3 hours, heart extracting blood, separation of serum detects serum total cholesterol (TC), glycerol three (TG), HDL-C (HDL-ch), low-density lipoprotein cholesterol (LDL-ch), blood urea nitrogen (BUN), serum creatinine (CREA) and glycolated hemoglobin concentration with biochemistry analyzer;

With the endothelial injury index of correlation: the concentration that detects nitric oxide (NO), superoxide dismutase (SOD), Endothelin (ET) and malonaldehyde (MDA) with test kit; Collect urine in 24 hours metabolic cages, detect twenty-four-hour urine albumen output and twenty-four-hour urine albumin output;

Hemorheology index: get 4ml blood EDTA anticoagulant, detect high and low whole blood viscosity, plasma viscosity, packed cell volume and the high and low erythrocyte deformability of cutting of cutting, use Contraves low share 30 type hemorheology instrument; Fibrinogen adopts the biuret turbidimetry, uses ultraviolet spectrometry degree instrument to measure.

(4) blood-retina barrier (BRB) is thought the permeability of indigo plant (EB) to the Ivan

Pentobarbital sodium intraperitoneal injection of anesthesia rat exposes right jugular vein, EB 45mgkg ^-1In 1min, slowly inject by vein.Behind the circulation 120min, the ligation postcava, left ventricle is poured into 1% paraformaldehyde buffer, cuts off left auricle simultaneously.After perfusion finishes, take out eyeball immediately, separate retina, 4 ℃ are spent the night and dry the back and claim dry weight.Retina and 150 μ L Methanamides are hatched 8h under 70 ℃.Then with 4 ℃ of 6000rmin of extracting solution ^-1High speed centrifugation 90min.Get supernatant 120 μ L and survey its absorbance, measure poor (the clean absorbance) of the absorbance of 620nm and 740nm2 kind wavelength sample, set up the standard curve of EB dye strength in Methanamide with microplate reader.With retina dry weight (mg) standardization EB (ng) content, the result is expressed as: ngmg ^-1

(5) the active measurement of glutathion peroxidase (GSH-Px)

1, GSH-Px vitality test

Crystalline lens added 10: 1 (mg/ml) normal saline is made 15000rpm10 ℃ of centrifugal 15min after the homogenate, get supernatant 100 μ l, make determining the protein quantity with the Lowry method, get supernatant 400 μ l and make GSH-Px vitality test (adopting the Hafeman improved method), unit of activity is represented with Hafeman unit/g pr albumen.

2, type glutathion (GSH) assay of redeeming a vow to a god

Standard pipe adds 1.0mM GSH; Measure pipe and add GSH-Px vitality test crystalline lens homogenate centrifugal liquid; Blank pipe adds phosphate buffer PBS, and (0.4M pH7.8) adds Polymeric sodium metaphosphate. solution respectively, shakes up the centrifugal 10min of back 3000rpm room temperature, gets supernatant 2ml, adds Na ₂HPO ₄(0.4M) solution 2ml, DTNB1ml compare survey OD412 value with blank pipe behind the mixing, and content is represented with nmol/g wt.

3, aldose reductase vitality test

Adopt the Gabbay improved method, represent enzymatic activity with unit of activity (u)/g wt.

4, crystalline lens cortex and nuclear weight ratio measurement

After taking by weighing the crystalline lens full weight, crystalline lens is placed in the clean tube, separate nucleus lentis, and take by weighing nuclear weight, deduct nucleus lentis weight with crystalline lens weight and be crystalline lens cortex weight, be calculated as follows: cortex/nuclear (w/w)=cortex weight/nuclear weight.

(6) pathologic finding: pentobarbital sodium intraperitoneal injection of anesthesia rat, get retina immediately and carry out sample preparations.The situation of change that the main blood vessel of diabetic renal papillary necrosis rat retina is observed in om observation: HE dyeing.Endotheliocyte and pericyte's form are observed in periodic acid one snow husband's reagent-haematoxylin (PAS) dyeing.

Three, result of the test

(1) arctiin, arctigenin are observed the general situation of laboratory animal

1, the rats in normal control group body weight gain is obvious, and mental status is good, moves freely, and is quick on the draw, and fur is glossy.The activity of model group rat reduces, obviously becomes thin, and the polydipsia polyuria, bradykinesia, hair is perpendicular matt, and when grasping animal, phenomenons such as dysphoria appear in animal.Each treatment group mental status of Fructus Arctii is good, moves freely, and compares with the normal control group, and reaction sensitivity is poor slightly.

Experimental session is dead 6 rats altogether, and wherein model group is dead 4, each dead 1 of arctiin, arctiin tuple.Compare with model group, show that arctiin, arctiin tuple all can improve the quality of life of artificial diabetes retinopathy rat, reduce mortality rate.

2, to the influence of diabetic renal papillary necrosis rat urine amount:

The influence of table 1 pair diabetic renal papillary necrosis rat urine amount

Annotate: compare with the normal control group: ^*P＜0.05, ^*P＜0.01; Compare with model control group: ^△P＜0.05, ^{△ △}P＜0.01

Presentation of results: diabetic renal papillary necrosis model group and normal control group relatively, the twenty-four-hour urine amount of diabetic renal papillary necrosis model group rat is apparently higher than the normal control group, and utmost point significant difference (p＜0.01) is all arranged.Compare with model group, arctiin, arctigenin treatment group can reduce diabetic renal papillary necrosis rat twenty-four-hour urine eliminating amount, and significant difference (p＜0.01) is all arranged.

3, to the influence of diabetic renal papillary necrosis rat retina electrograph

Normal rat right and left eyes ERGa, b wave number do not have significant difference, but there is some difference between the individuality.Compare with normal group, model group a, b wave-amplitude value all obviously reduce (p＜0.01), compare with model group, and each time point a, b wave-amplitude value obviously raise (p＜0.01) are organized in arctiin, arctigenin treatment.

4, to the influence of diabetic renal papillary necrosis rat blood sugar, saccharifying blood gp

The influence of table 2 pair rat model blood glucose, glycolated hemoglobin

Annotate: compare with the normal control group: ^*P＜0.05, ^*P＜0.01; Compare with model control group: ^△P＜0.05, ^{△ △}P＜0.01, ^#P＞0.05 glycolated hemoglobin rate=saccharification hemoglobin content/content of hemoglobin

Presentation of results: diabetic renal papillary necrosis rat model group and normal control group compare, and the blood glucose of diabetic renal papillary necrosis model group rat, saccharifying blood glucose egg concentration have significant difference (p＜0.05) apparently higher than the normal control group.With model group relatively, arctiin, arctigenin treatment group are not obvious to the blood glucose effect of diabetic renal papillary necrosis rat model, the two there was no significant difference (p＞0.05).

5, to the influence of diabetic renal papillary necrosis rat T-CHOL (TC), triglyceride (TG), HDL-C (HDL-ch) and low-density lipoprotein cholesterol (LDL-ch)

The influence of table 3 couple diabetic renal papillary necrosis rat TC, TG, LDL-ch and HDL-ch

Annotate: compare with the normal control group: ^*P＜0.05, ^*P＜0.01; Compare with model control group: ^△P＜0.05, ^{△ △}P＜0.01

Presentation of results: diabetic renal papillary necrosis rat model group and normal control group are relatively, TC, TG of diabetic renal papillary necrosis rat model group rat and LDL-ch concentration are apparently higher than the normal control group, HDL-ch concentration is starkly lower than the normal control group, and significant difference (p＜0.05) is all arranged.Compare with model group, arctiin, arctigenin treatment group all can reduce diabetic renal papillary necrosis Serum TC, TG and LDL-ch concentration, rising HDL-ch concentration, and the two all has significant difference (p＜0.05).

6. to the influence of diabetic renal papillary necrosis rat blood serum nitric oxide (NO), malonaldehyde (MDA), Endothelin (ET) and superoxide dismutase (SOD)

The influence of table 4 couple diabetic renal papillary necrosis rat blood serum MDA, NO, ET and SOD

Annotate: compare with the normal control group: ^*P＜0.05, ^*P＜0.01; Compare with model control group: ^△P＜0.05, ^{△ △}P＜0.01

Presentation of results: diabetic renal papillary necrosis model group and normal control group are relatively, MDA, ET content are apparently higher than the normal control group in the diabetic renal papillary necrosis model group rat blood serum, NO content and SOD activity are lower than the normal control group, and significant difference (p＜0.05) is all arranged.Compare with model group, arctiin, arctigenin treatment group all can reduce diabetic renal papillary necrosis rat blood serum MDA, ET content, elevation of NO content, and SOD activity improving, and significant difference (p＜0.05) is all arranged; But all example hydrochloric acid calcium dobesilate treatment group is not remarkable.

7, to the influence of diabetic renal papillary necrosis rat model blood urea nitrogen, serum creatinine

The influence of table 5 pair diabetic renal papillary necrosis rat model blood urea nitrogen (BUN), serum creatinine (Crea)

Annotate: compare with the normal control group: ^*P＜0.05, ^*P＜0.01; Compare with model control group: ^△P＜0.05, ^{△ △}P＜0.01

Presentation of results: diabetic renal papillary necrosis model group and normal control group relatively, BUN and Crea content and all have significant difference (p＜0.05) apparently higher than the normal control group in the diabetic renal papillary necrosis model group rat blood serum.Compare with model group, arctiin, arctigenin treatment group can reduce diabetic renal papillary necrosis rat blood serum BUN and Crea content, and significant difference (p＜0.05) is all arranged.

8, diabetic renal papillary necrosis rat retina barrier (BRB) is thought the permeability of indigo plant (EB) to the Ivan

Table 6 retinal barrier is to the permeability influence of EB

Group standardization EB content (ngmg ^-1)

Normal control 12.24 ± 3.24

Model group 25.66 ± 3.05 ^*

Arctiin group 16.98 ± 4.41 ^{△ △}

Arctiin tuple 15.73 ± 3.27 ^{△ △}

Annotate: compare with the normal control group: ^*P＜0.05, ^*P＜0.01; Compare with model control group: ^△P＜0.05, ^{△ △}P＜0.01

Presentation of results: diabetic renal papillary necrosis model group and normal control group compare, and diabetic renal papillary necrosis model group rat retina barrier (BRB) increases the permeability that the Ivan thinks indigo plant (EB), and significant difference (p＜0.05) is arranged.Compare with model group, arctiin, arctigenin treatment group can suppress retinal barrier (BRB) Ivan is thought the permeability of indigo plant (EB), and significant difference (p＜0.05) is all arranged.

9, to the influence of diabetic renal papillary necrosis rat model glutathione reductase activity

The table 7 pair active influence of diabetic renal papillary necrosis rat model glutathion peroxidase (GSH-Px)

Annotate: compare with the normal control group: ^*P＜0.05, ^*P＜0.01; Compare with model control group: ^△P＜0.05, ^{△ △}P＜0.01

Presentation of results: diabetic renal papillary necrosis model group and normal control group are relatively, diabetic renal papillary necrosis model group rat suppresses glutathion peroxidase (GSH-Px) vigor, reduces reduced glutathion (GSH) content and cortex/nuclear value, significantly reduce aldose reductase (AR) vigor, and significant difference (p＜0.05) is all arranged.Compare with model group, arctiin and arctigenin can significantly strengthen GSH-Px vigor, GSH content and cortex/nuclear value, significantly reduce the AR vigor, and significant difference (p＜0.05) is all arranged.

(7) om observation result

Retinopathy form of science shows:

The visible more complete vasoganglion of normal group group rat, the dyeing of tremulous pulse trunk is darker, caliber is thinner, vein dyeing is more shallow, caliber is thicker, arteriovenous alternately, capillary network between arteriovenous, the vasoganglion densification of back utmost point portion, periphery is more sparse.Tremulous pulse peripheral vessels net obviously is less than around the vein, uniform diameter, interconnection reticulating.Retinal capillary is arranged evenly rule, and traveling is more straight, caliber even thickness unanimity.Endotheliocyte is light to be dyed, and is spindle shape or ellipse, is positioned at lumen of vessels, and its major axis is how parallel with blood capillary; Pericyte's engrain, triangular in shape or spheroidal protrudes in blood vessel wall.The ratio of endotheliocyte and pericyte is about 2: 1, and bridge between the blood capillary of visible acellular nuclear.

Model group rat retina edema is obvious, and detachment of retina takes place, basement membrane thickened under the retina, hemorrhage, the fibrosis of matter, hyaline degeneration between the companion; Blood capillary caliber thickness differs, and blood vessel moves towards irregular, arrangement disorder, and visible leukocyte thromboembolism blood capillary tube chamber, endotheli ocytosis, pericyte obviously reduces.Pericyte and endothelial cell apoptosis, karyopycnosis, apoptotic body forms; The small artery obturation; Pericyte and endotheliocyte forfeiture; Acellular blood capillary forms.

The visible similar pathological change of arctiin and arctiin tuple, the visible Mild edema of retina is not seen new vessels, focal hemorrhage changes.

The acute toxicity test of embodiment 12 arctiins and arctigenin

Arctiin of the present invention and arctigenin are used for the acute toxicity test of mice, the oral median lethal dose(LD 50) LD of mice of arctiin ₅₀Be 139.8g/kg, get the oral median lethal dose(LD 50) LD of the mice of arctigenin ₅₀Be 64.6g/kg, be higher than the afore-mentioned test effective dose far away, show that this Fructus Arctii extract's toxic and side effects is little, having a extensive future aspect the exploitation preparation clinical application.

One, test material

Laboratory animal: Kunming mouse, SPF level, age in 6-8 week, 18-22g.Raise in totally-enclosed Animal House, temperature 20-22 ℃, relative humidity 50-60%, secretly 12 hours were bright in 12 hours in illumination, and central air-conditioning is concentrated ventilation 8-15 time/hour.Freely ingest and drink water, change drinking water every day once.Behind the adaptability breeding observing 2 days, fasting be can't help water and is spent the night, and weighs next day, numbers.

Two, experimental program

1. animal grouping: with the administration component is 5 dosage groups, every group of 10 mices, male and female half and half.

2. dosage: with the principle administration of " not isoconcentration isometric(al) ", Arctiin and aretigenin are made into for irritating the suspension that stomach is used with 0.5% sodium carboxymethyl cellulose, and each is organized mice and respectively irritates stomach at 1: 0.5 by geometric progression and be subjected to the reagent thing.Later on conventional the raising observed the hair color, spontaneous activity, diet, body weight of 7 days animals etc. continuously, writes down dead symptom and time, puts to death animal after 7 days, and each tissue of anatomic observation mice, internal organs have or not ANOMALOUS VARIATIONS.Fructus Arctii extract of the present invention is used to carry out the acute toxicity test of mice, gets the oral median lethal dose(LD 50) LD of the mice of arctiin ₅₀Be 139.8g/kg, get the oral median lethal dose(LD 50) LD of the mice of arctigenin ₅₀Be 64.6g/kg, be higher than the afore-mentioned test effective dose far away, show that this Fructus Arctii extract's toxic and side effects is little, having a extensive future aspect the exploitation preparation clinical application.

The mouse death rate of table 8 arctiin

The male female general mortality rate of dosage (g/kg)

401.44 5/5 5/5 100％

200.72 3/5 4/5 70％

100.36 1/5 1/5 20％

50.18 1/5 0/5 10％

25.09 0/5 0/5 0％

Annotate: in the table is " death toll/number of animals ".

Table 9 arctiin to the influence of male mice body weight (X ± s, g)

Dosage (g/kg) the 1st day the 7th day

401.44 18.2±0.74(5)

200.72 21.5±1.01(5) 33.2±1.27(2)

100.36 17.2±0.82(5) 32.7±1.08(4)

50.18 17.8±1.27(5) 31.9±0.85(4)

25.09 21.7±1.55(5) 30.9±1.27(5)

Annotate: in " () " is number of animals, down together.

Table 10 arctiin to the influence of female mice body weight (X ± s, g)

Dosage (g/kg) the 1st day the 7th day

401.44 19.1±0.73(5)

200.72 18.8±0.29(5) 28.1±0.99(1)

100.36 20.3±1.26(5) 29，6±1.65(4)

50.18 19.9±0.84(5) 30.1±1.31(5)

25.09 20.7±1.04(5) 29.5±0.87(5)

The mouse death rate of table 11 arctigenin

The male female general mortality rate of dosage (g/kg)

240.48 5/5 5/5 100％

120.24 3/5 4/5 70％

60.12 1/5 1/5 20％

30.06 1/5 0/5 10％

15.03 0/5 0/5 0％

The former influence to the male mice body weight of table 12 arctiin (X ± s, g)

Dosage (g extractum/kg) the 1st day the 7th day

240.48 17.5±0.74(5)

120.24 19.4±1.15(5) 32.6±1.32(2)

60.12 18.5±0.095(5) 31.8±1.24(4)

30.06 18.1±1.17(5) 31.7±0.98(4)

15.03 21.4±0.86(5) 30.7±1.32(5)

Table 13 arctigenin to the influence of female mice body weight (X ± s, g)

Dosage (g/kg) the 1st day the 7th day

401.44 19.5±0.91(5)

200.72 18.4±0.59(5) 27.8±1.15(1)

100.36 19.8±1.16(5) 28.5±1.55(4)

50.18 20.2±0.78(5) 30.7±0.71(5)

25.09 20.6±1.14(5) 29.6±0.77(5)

Conclusion: the oral LD of the mice of arctiin ₅₀Be 139.8g/kg, LD ₅₀The 95% credible 99.8～195.9g/kg that is limited to.The oral LD of arctigenin mice ₅₀Be 64.6g/kg, LD ₅₀The 95% credible 44.6～93.7g/kg that is limited to.The mice LD of arctiin and aglycon ₅₀Be higher than its effective dose far away, show that its toxic and side effects is little, having a extensive future aspect the exploitation preparation clinical application.

Embodiment 13 clinical experiments

The research case: Chinese medical discrimination belongs to type 2 diabetes mellitus simple retinopathy patient 60 examples (with reference to " new Chinese medicine clinical research guideline " version in 2002) of syndrome of deficiency of both qi and yin to be determined, with the simple randomization method patient is divided into 4 groups.The patient all uses three mirror contact lens, fundus photography and fundus fluorescein angiography inspection behind mydriasis.(Liu Jiaqi, Li Fengming chief editor, People's Health Publisher 2003:2) press six grades by stages to diabetic renal papillary necrosis according to " practical ophthalmology ".

Therapeutic Method: adopt at random the controlled observation method will be selected in object by being divided into three test group (a at 1: 1 at random, b, c), matched group, test group a takes arctiin capsule (100mg/ grain, embodiment 5a sample), test group b uses arctiin Yuan capsule (100mg/ grain, embodiment 5b sample), test group c to use arctiin, arctigenin mixture capsule (100mg/ grain, embodiment 5c sample), each 2 (every 0.1g), 1 day 3 times; Matched group import calcium hydrophenyl sulfonate capsule (Doxium, the big pharmaceutical factory of Austrian Yi Biwei produces specification 500mg/ grain), each 0.5g (1), 1 day 3 times, 3 months is 1 course of treatment; Each group is all given Primary Care: the duration of test experimenter is according to the best principle of medication that is fit to individual need, each group all simultaneously oral administration of metformin make experimenter's blood glucose reach control criterion or keep stable level with blood sugar control.

Observation index and evaluation criteria:

1, lab testing

The three big routines, fasting glucose (FBS), post-prandial glycemia (PBS), blood fat, hepatic and renal function, blood endothelial cell growth factor (ECGF) (VEGF), nitric oxide (NO) content, hemorheology and the retinal centre arteriovenous blood flow parameter that all carry out system after finishing the preliminary investigation and the course of treatment in March detect, take medicine and then only looked into FBS in 1 month and 2 months.

2, be divided into produce effects, effective and invalid according to vision and optical fundus variation

Improve: fluorescence fundus angiography microangioma, petechia area, leakage area reduce more than 10% (2 minutes, 2 minutes, 3 minutes); Vision improves 2 row above or vision 〉=1.0 (3 minutes).

Stable: fluorescence fundus angiography microangioma, petechia area, leakage area do not have variation or intensity of variation less than 10% (1 minute, 1 minute, 1.5 minutes); The vision fluctuation is gone with interior (1 minute) 1.

Worsen: fluorescence fundus angiography microangioma, petechia area, leakage area enlarge more than 10% (0 minute, 0 minute, 0 minute); Vision reduces by 2 row above (0 minute).

Overall merit: produce effects: These parameters total mark 〉=7 minute; Effectively: These parameters total mark 〉=4 minute; Invalid: These parameters total mark＜4 minute.Effective percentage=[(produce effects example number+effective routine number)/total routine number] * 100%.

3, the traditional Chinese medical science is evaluated according to the syndrome integration

Cardinal symptom: blurring of vision, dryness with foreign body sensation in the eyes, fatigue and weakness, breathe hard lazy speech, dysphoria with feverish sensation in the chest palms and soles, dry mouth and throat, every by do not have, light, in, weigh 4 grades, be respectively 0,2,4,6 fen; Minor symptom: thirst and liking drink, cardiopalmus, red, the constipation of having a sleepless night, urinate, every by do not have, light, in, weigh 4 grades, be respectively 0,1,2,3 fen; Picture of the tongue, pulse condition are only noted down, and do not score.

Therapeutic index (n)=[integration before (integration before the treatment-treatment back integration) ÷ treatment] * 100%.

Recovery from illness: primary symptom and sign all disappear in the syndrome, effective percentage n 〉=95%; Produce effects: the primary symptom overwhelming majority disappears effective percentage n 〉=70% in the syndrome; Effectively: the primary symptom in the syndrome disappears substantially, effective percentage 30%≤n＜70%; Invalid: the primary symptom in the syndrome has certain improvement, effective percentage n＜30%.

4, observe side effect.

Experimental result:

1) before and after the treatment fasting glucose (FBG), post-prandial glycemia (PBG), glycolated hemoglobin (HbAlc) the results are shown in Table 14,15.

FBG, PBG, HbAlc are relatively before and after table 14 (a) test group (a) treatment

Annotate: * represents p＜0.05

FBG, PBG, HbAlc are relatively before and after table 14 (b) test group (b) treatment

Annotate: * represents p＜0.05

FBG, PBG, HbAlc are relatively before and after table 14 (c) test group (c) treatment

Annotate: * represents p＜0.05

The result shows: each group all has certain hypoglycemic activity.

FBG, PBG, HbAlc are relatively before and after table 15 treatment of control group

Annotate: * represents p＜0.05, and # represents p＞0.05

2) each group treatment front and back serum VEGF (VEGF) the results are shown in Table 16.

Table 16 (a) VEGF (VEGF) level changes relatively

Annotate: * represents p＜0.05, and # represents p＞0.05

Table 16 (b) VEGF (VEGF) level changes relatively

Annotate: * represents p＜0.05, and # represents p＞0.05

Table 16 (c) VEGF (VEGF) level changes relatively

Annotate: * represents p＜0.05, and # represents p＞0.05

The result shows: the VEGF level all significantly descends before and after each group treatment.

3) change evaluation result in optical fundus sees Table 17.

Table 17 (a) test group (a) and matched group eye disease total effects situation

Annotate: * represents p＜0.05

Table 17 (b) test group (b) and matched group eye disease total effects situation

Annotate: * represents p＜0.05

Table 17 (c) test group (c) and matched group eye disease total effects situation

Annotate: * represents p＜0.05

The result shows: the curative effect of each group all is better than the Doxium curative effect.

4) the tcm syndrome efficacy result sees Table 18.

Table 18 (a) test group (a) is improved situation with the matched group tcm syndrome

Annotate: * represents p＜0.05

Table 18 (b) test group (b) is improved situation with the matched group tcm syndrome

Annotate: * represents p＜0.05

Table 18 (c) test group (c) is improved situation with the matched group tcm syndrome

Annotate: * represents p＜0.05

The result shows: the tcm syndrome curative effect of each group all is better than Doxium tcm syndrome curative effect.

5) content of nitric oxide the results are shown in Table 19.

Nitric oxide relatively before and after two groups of patient treatments of table 19 (a)

Annotate: compare before and after the group internal therapy, ^{★ ★ ★}P＜0.01, ^{★ ★}P＜0.05; Compare the treatment back between group, ^{★ ★ ★}Compare before the treatment between group p＜0.01. ^★P＞0.05.

Nitric oxide relatively before and after two groups of patient treatments of table 19 (b)

Annotate: compare before and after the group internal therapy, ^{★ ★ ★}P＜0.01, ^{★ ★}P＜0.05; Compare the treatment back between group, ^{★ ★ ★}Compare before the treatment between group p＜0.01. ^★P＞0.05.

Nitric oxide relatively before and after two groups of patient treatments of table 19 (c)

Annotate: compare before and after the group internal therapy, ^{★ ★ ★}P＜0.01, ^{★ ★}P＜0.05; Compare the treatment back between group, ^{★ ★ ★}Compare before the treatment between group p＜0.01. ^★P＞0.05.

The result shows: each group all can effectively suppress content of nitric oxide and descend.

6) the whole blood height cut viscosity (η Hb), whole blood low cut viscosity (η Lb), plasma viscosity (η P), erythrocyte aggregation index (EAI), Fibrinogen (Fb) the results are shown in Table 20.

Hemorheology changes before and after table 20 (a) treatment

Annotate: * represents p＜0.05

Hemorheology changes before and after table 20 (b) treatment

Annotate: * represents p＜0.05

Hemorheology changes before and after table 20 (c) treatment

Annotate: * represents p＜0.05

The result shows: control each group and treat back patient's whole blood height and cut low viscosity (η Lb), plasma viscosity (η P), erythrocyte aggregation index (EAI), the Fibrinogen (Fb) cut of viscosity (η Hb), whole blood and have obviously before than treatment and subtract (p＜0.05).

7) the shrinkage peak blood flow rate (PSV) of Zinn's artery, acceleration (A), blood flow maximal rate diastasis (EDV), central vein of retina back-flow velocity (CRV) the results are shown in Table 21

Retinal centre arteriovenous blood flow parameter changes before and after table 21 (a) treatment

Annotate: ^*Expression p＜0.05

Retinal centre arteriovenous blood flow parameter changes before and after table 21 (b) treatment

Annotate: ^*Expression p＜0.05

Retinal centre arteriovenous blood flow parameter changes before and after table 21 (c) treatment

Annotate: ^*Expression p＜0.05

The result shows: shrinkage peak blood flow rate (PSV), acceleration (A), blood flow maximal rate diastasis (EDV) of each group treatment back patient's Zinn's artery wait significantly increase, central vein of retina back-flow velocity (CRV) obviously slow down (p＜0.05).

8) safety relatively

Each test group and matched group period in a medicine hematuria routine, liver function index (alanine aminotransferase ALT, aspartate transaminase AST) and renal function index (blood urea nitrogen BUN, creatinine Cr) be display abnormality not all.But have 2 routine patients side effect such as slight heating, arthralgia, gastrointestinal disturbance to occur in the matched group, and each test group is not seen apparent side effect.

More than each result show that arctiin, arctiin, arctiin and arctigenin mixture can the sugared characteristic of disease retinopathys of safe and effective control.

Claims (8)

1. the compositions of treatment and prevent diabetes retinopathy is characterized in that, described compositions contains that purity is more than 90%, the arctiin of 50-99 by weight or the arctigenin of 1-50.

2. according to the described compositions of claim 1, it is characterized in that, contain the medical additive or the pharmaceutical carrier of 100-20000 weight portion in the described compositions.

3. the described compositions of claim 1 is characterized in that described preparation of compositions is become tablet, pill, capsule, injection, eye drop or suppository.

4. described compositions as claimed in claim 3, it is characterized in that tablet that described compositions makes or pill contain by weight arctiin 1-100 or the mixture of arctigenin 1-100 or arctiin 50-99 and arctigenin 1-50; The 250-350 weight portion from lactose, starch, dextrin and hydroxypropyl cellulose, select at least a, the 5-10 weight portion from Pulvis Talci and magnesium stearate, select in a kind of, the coating pre-mixing agent Opadry OY-C-7000A of 3-5 weight portion.

5. compositions as claimed in claim 3 is characterized in that double-layer sustained release tablet that contains release layer that described compositions makes or pill contain arctiin 1-100 or the two mixture of arctigenin 1-100 or arctiin 50-99 and arctigenin 1-50 by weight; The 80-150 weight portion from lactose, starch, hydroxypropyl cellulose and polyvinyl pyrrolidone, select at least a; The 5-10 weight portion from Pulvis Talci and magnesium stearate, select at least a; The hydroxypropyl cellulose of 70-100 weight portion, the Tween-80 of the Polyethylene Glycol of 1-5 weight portion-400,1-5 weight portion, the 1-5 weight portion from lemon yellow and titanium dioxide, select at least a.

6. compositions as claimed in claim 3, it is characterized in that capsule that described compositions makes contains arctiin 1-100 or the two mixture of arctigenin 1-100 or arctiin 50-99 and arctigenin 1-50 by weight, the 200-300 weight portion from lactose, starch, dextrin and hydroxypropyl cellulose, select at least a, the 1-4 weight portion from Pulvis Talci and magnesium stearate, select at least a.

7. method for compositions for preparing above-mentioned arbitrary claim, said method comprises:

(1) preparation of arctiin

1. slightly carry

After the Fructus Arctii pulverizing, put in the apparatus,Soxhlet's, through boiling range 60-90 ℃ of petroleum ether backflow defat 2-4 time, take out after the defat and dry, use 8 times of 60%-80% to measure alcohol reflux 2-4 time, each 0.5-2h in 60 ℃ of-80 ℃ of following concentrating under reduced pressure, gets dark brown extractum;

2. macroporous resin is refining

Dark brown extractum is added 15-35% ethanol, supersound process 15-35 minute, dissolving is filtered, and transfers to into the solution that concentration is 5-7mg/mL, by AB-8 type macroporous adsorbent resin, the macroporous adsorbent resin volume is 1-2 a times of medicine liquid volume, and flow speed control is 1.5-2.5BV/h, and the post blade diameter length ratio is controlled to be 1: 5, wash 3 times of resin volumes, washing speed 0.5-3BV/h; Change the ethanol elution with 4-6BV50% again, alcohol is washed speed 1-3BV/h, collects washing liquid, and concentrate drying gets the arctiin crude product;

3. separate

The arctiin crude product is with the thick silica gel mixed sample of 60 orders, the 1-2 that thick silica gel volume is the arctiin crude product doubly, analyse layer through 200-300 order silicagel column, the silicagel column volume is 10-50 a times of arctiin crude product, 95: 1 eluting of chloroform-methanol, thin layer chromatography trace flow fluid, the stream part of collecting the arctiin single-point, merging concentrates, and gets arctiin;

(2) arctigenin preparation

1. slightly carry

After the Fructus Arctii pulverizing medicinal materials, cross 80 mesh sieves, put in the apparatus,Soxhlet's, through the oily ether backflow defat of 60～90 ℃ of boiling ranges 3 times, dry the back and extract 2-4 time with ethyl acetate backflow, each 1.5-2.5h in 70 ℃ of following concentrating under reduced pressure, gets brown extractum;

2. refining

Brown with extractum with the thick silica gel mixed sample of 60 orders, the 1-2 that thick silica gel volume is the arctigenin crude product doubly, the water-bath oven dry, sample on the dry method, through 200-300 order silica gel column chromatography, 98: 1 eluting of chloroform-methanol, eluent concentrate to merge and obtain arctigenin;

(3) proportional arrangement of pressing arctiin, arctigenin, medical additive and/or pharmaceutical carrier is mixed.

8. preparation method as claimed in claim 7, it is characterized in that said arctiin in refining is: get the arctiin ethanol extract, add 15-30% ethanol, fully stir supersound process 15-30 minute, make its exactly fully dissolving, it is the solution of 3-6.5mg/mL that filtration transfers to last sample liquor strength, uses AB-8 type macroporous adsorbent resin, and the macroporous adsorbent resin volume is 1-2 a times of medicine liquid volume, last sample flow speed control is 2BV/h, and the post blade diameter length ratio is controlled to be 1: 5.Wash 2-3 times of resin volume, washing speed 1-2BV/h; Change the ethanol elution with 3.5-4.5BV50% again, alcohol is washed speed 1-2BV/h, collects pure washing liquid, and concentrate drying gets the Fructus Arctii crude product.

9. the described purposes that is used for the treatment of and prevents preparation diabetic retinopathy medicine of arbitrary compositions of claim 1-6, it is characterized in that, other curative of described compositions and other mixed use, described curative is selected from: identical active medicine with arctiin, antidiabetic drug, antioxidant, aldose reductase inhibitor, endothelial growth factor receptor inhibitors, platelet aggregation inhibitor or their mixture.