CN106924655A

CN106924655A - A kind of preparation method of the Chinese medicine composition for treating AMD

Info

Publication number: CN106924655A
Application number: CN201511035171.9A
Authority: CN
Inventors: 夏忠庭; 缪兴龙; 何毅; 颜璐璐; 张莉华; 马晓慧
Original assignee: Tasly Pharmaceutical Group Co Ltd
Current assignee: Tasly Pharmaceutical Group Co Ltd
Priority date: 2015-12-31
Filing date: 2015-12-31
Publication date: 2017-07-07
Anticipated expiration: 2035-12-31
Also published as: CN106924655B

Abstract

The present invention relates to a kind of preparation method of the Chinese medicine composition for treating AMD, methods described step is as follows：1. fritillaria thunbergii,Radix Angelicae Sinensis,2~8 times of 50~95% ethanol of amount of root tuber of aromatic turmeric are extracted,Extract 1~3 time,0.5~2.5h every time,Extract solution is filtered,70 DEG C of filtrate be concentrated into 50~80 DEG C at relative density d=1.10-1.40,70 DEG C of dryings,Obtain alcohol extracting dry extract,Crush,Cross 80 mesh sieves,2. astragalus root,Poria cocos,Charred RADIX ET RHIZOMA RHEI,6~12 times of amount water of Pollen Typhae of decocting a drug wrapped are extracted,Extract 1~3 time,0.5~2.5h every time,Extract solution is filtered,Filtrate be concentrated into 50~80 DEG C at relative density d=1.05-1.25,Add 95% ethanol to 50-90%,Stand more than 12h,Supernatant concentration is to relative density d=1.10-1.40 at 50~80 DEG C,70 DEG C of dryings,Obtain water extraction dry extract,Crush,Cross 80 mesh sieves,3. pseudo-ginseng is crushed,Cross 50~150 mesh sieves,Starch.

Description

A kind of preparation method of the Chinese medicine composition for treating AMD

Technical field：

The present invention relates to a kind of preparation method of Chinese medicine composition, more particularly to one kind treats AMD Chinese medicine composition preparation method.

Background technology：

AMD (age-related macular degeneration, AMD) be mainly in 55 years old with Upper the elderly.AMD be with macular area retinal pigment epithelium (RPE), Bruch ' s films, CC degenerative disease, AMD points is dryness and moist two types.Especially moist AMD is shown in CNV under macula area retina The formation of (choroidal neovascularization, CNV), is the basic reason of this sick blinding.

AMD is ophthalmology difficult disease, and its pathogenesis is unclear so far, and current treatment method is exploration sex therapy. Mainly include：PDT, through pupil thermotherapy, laser photocoagulation, radiotherapy, operative treatment, drug therapy, gene treat Method, Chinese medicine etc..Photodynamic therapy is to confirm relatively effective treatment method at present, but its curative effect be with eyesight not after It is continuous to be reduced to evaluation criterion, and this therapy is expensive, and need repetitive therapy；Laser therapy is to the new life below central fovea of macula Blood vessel light coagulates, it will usually cause the direct decline of central vision, and the recurrence rate of laser photocoagulation treatment new vessels is also high；West The current focus of medicine medicinal treatment in the research and development of anti-neovascular endothelium growth factor receptor inhibitors, such as Eylea (VEGF Trap), Lucentis (promise is fitted), Conbercept (Compaq is western general) etc..

Chinese patent CN102058845A discloses a kind of Chinese medicine composition for treating macular degeneration, the raw material of said composition Medicine is constituted：

Radix Astragali 10-40 weight portion Radix Angelicae Sinensis 10-40 weight portions

Poria cocos 10-40 weight portion fritillaria thunbergii 5-20 weight portions

Pseudo-ginseng 3-18 weight portion charred RADIX ET RHIZOMA RHEI 1-10 weight portions

Pollen Typhae 10-40 weight portion root tuber of aromatic turmeric 10-40 weight portions.

Preferably, the bulk drug composition of the pharmaceutical composition is：

Radix Astragali 10-30 weight portion Radix Angelicae Sinensis 10-30 weight portions

Poria cocos 12-30 weight portion fritillaria thunbergii 6-15 weight portions

Pseudo-ginseng 4-14 weight portion charred RADIX ET RHIZOMA RHEI 2-4 weight portions

Pollen Typhae 10-30 weight portion root tuber of aromatic turmeric 10-30 weight portions.

Most preferably, the bulk drug composition of the pharmaceutical composition is：

The weight portion of 20 weight portion Radix Angelicae Sinensis of the Radix Astragali 20

The weight portion of 20 weight portion fritillaria thunbergii of Poria cocos 10

The weight portion of 9 weight portion charred RADIX ET RHIZOMA RHEI of pseudo-ginseng 3

The weight portion of 20 weight portion root tuber of aromatic turmeric of Pollen Typhae 20

Or

The weight portion of 15 weight portion Radix Angelicae Sinensis of the Radix Astragali 25

The weight portion of 28 weight portion fritillaria thunbergii of Poria cocos 8

The weight portion of 6 weight portion charred RADIX ET RHIZOMA RHEI of pseudo-ginseng 4

The weight portion of 24 weight portion root tuber of aromatic turmeric of Pollen Typhae 15

Or

The weight portion of 26 weight portion Radix Angelicae Sinensis of the Radix Astragali 16

The weight portion of 18 weight portion fritillaria thunbergii of Poria cocos 12

The weight portion of 12 weight portion charred RADIX ET RHIZOMA RHEI of pseudo-ginseng 2

The weight portion of 15 weight portion root tuber of aromatic turmeric of Pollen Typhae 25

The Chinese medicine composition has treatment wet age related macular degeneration and choroidal neovascularization lesion Purposes.

The preparation method of the Chinese medicine composition is also disclosed in the patent Example：

Above-mentioned raw materials medicine is taken, extraction is added water to cook, or adds alcohol dipping or seepage pressure effects, add customary adjuvant, pressed More solito technique, is made various preparations.

But above-mentioned preparation method is excessively simple, the complexity of traditional Chinese medicine ingredients is not accounted for, using a kind of extracting method, and The active ingredient of various medicinal materials in prescription can not completely be extracted, need to respectively be had according to the chemical composition species of each flavour of a drug Extracting method is targetedly designed, best process flow is screened with pharmacodynamic results, and related process parameters need to be determined, it is ensured that product Stability, it is therefore necessary to find a kind of more perfect preparation method.

The content of the invention：

The present invention provides a kind of preparation method of Chinese medicine composition, and the Chinese medicine composition is bulk drug by following Chinese medicine It is prepared from：

Poria cocos 10-40 weight portion fritillaria thunbergii 5-20 weight portions

Preferably, the Chinese medicine composition, by following Chinese medicine for bulk drug is prepared from：

Poria cocos 12-30 weight portion fritillaria thunbergii 6-15 weight portions

Described Chinese medicine material medicine, the indication of each taste medicine is as follows：

The Radix Astragali, is the dry root of legume astragalus mongolicus or the pod membrane Radix Astragali.Its indication is tonifying Qi and lifting yang, Gu table stops Sweat, inducing diuresis for removing edema, blood-nourishing of promoting the production of body fluid, the stagnant logical numbness of row, pus draining and toxin expelling, expelling pus and promoting granulation.Main component includes flavones, saponins and the Radix Astragali The compositions such as polysaccharide, such as Astragalus Saponin I, second, third, soyasaponin I, different astragaloside II, onocerin, calycosin etc. Deng.

Radix Angelicae Sinensis, is the dry root of umbelliferae angelica.Its indication is replenishing and activating blood, and menstruction regulating and pain relieving relaxes bowel. Radix Angelicae Sinensis contains volatile oil component.Neutral oil component in volatile oil have butylidenephthalide, nopinene, australene, amphene, to poly- umbrella spend Element, β-phellandrene, laurene etc., other compositions also containing stigmasterol, sitosterol, stigmasterol-D-Glucose glucoside, tetradecyl alchohol -1, Elegant jessamine luciferin etc..Additionally, also containing sucrose, fructose, glucose；Vitamin A, vitamin B12, vitamin E, 17 kinds of amino acid And sodium, potassium, calcium, magnesium etc. more than 20 plants inorganic elements.

Poria cocos, is the dry sclerotia of On Polyporaceae Poria cocos.Its indication is clearing damp and promoting diuresis, invigorating the spleen, calming heart.Mainly Composition contains polysaccharide, triterpene, aliphatic acid, sterol, enzyme etc..Polysaccharide such as β-pachyman, pachymaran, xylitol, hydroxyl first Base pachymaran etc..Triterpenes includes the dilute type triterpene compounds of wool steroid -8-, (11)-diene type triterpenes of wool steroid -7,9 Compound etc..Other compositions also have octanoic acid, undecanoic acid, laurate, lauric acid/dodecanoic acid, histidine, chitin, choline etc..

Fritillaria thunbergii, alias Zhejiang shellfish, Bulbus Fritillariae Thunbergii, big fritillaria, pearl shellfish are the bulb of liliaceous plant fritillaria thunbergii.Primary efficacy is Strongly fragrant dissipating bind is opened in facilitaing lung heat-clearing, preventing phlegm from forming and stopping coughing.Cure mainly wind-heat or phlegm-heat cough, abscess of throat, lung carbuncle, goiter and tumor, sore and toxic.It is main Composition is wanted for alkaloid, such as contains peimine, peiminine, peimine glycosides, different peimine, fritillaria Buddhist nun's fourth alkali, Different fritillaria Buddhist nun fourth alkali, fritillaria sterol, choline etc..

Pseudo-ginseng, is the dry root and rhizome of panax araliaceae plant.Its indication is dissipate stasis of blood hemostasis, detumescence ding-tong.It is main Composition is wanted for arasaponin, including 20 (S) protopanaxadiol-types, 20 (S) Protopanaxatriol types, oleanolic acid type.Additionally Contain flavone compound, dencichine, carbohydrate content, amino acid and volatile oil etc..

Charred RADIX ET RHIZOMA RHEI, the Preparation process product of Chinese medicine rheum officinale.Its indication be heat and toxic materials clearing away, clearing heat-fire, removing pattogenic heat from the blood and toxic material from the body, Dispelling stasis of blood and stimulating the menstrual flow, removing dampness through diuresis and removing jaundice.Main component be anthraquinone derivatives for example aloe-emodin, chrysaron, Chrysophanol, rheum emodin, Reidin, palm leaf dianthrone etc., glycosides compound such as the rhaponticin,-O- β-D- of Resveratrol alkene -4 ' (6 ' - O- nutgall acyls) glycoside etc., and the composition such as tannin class and organic acid.

Cattail pollen, is the dry pollen of Typhaceae plant raupo cattail, typha orientalis or congener.Untill its indication It is blood, stagnation resolvation, treating stranguria.Main such as typhaneoside, Quercetin, Kaempferol, Isorhamnetin, naringenin etc. containing flavones ingredient, also contains There are sterol constituents, cupreol, cupreol glycoside, cupreol palmitate etc., and polysaccharide and amino acid etc. Composition.

Root tuber of aromatic turmeric, the dried root of zingiberaceous plant RADIX CURCUMAE, turmeric, Guangxi zedoary or zedoary.For chest side of body shouting pain, the obstruction of qi in the chest Pained, closed dysmenorrhea, swollen breasts, pyreticosis coma, epilepsy is gone mad, blood-head tells nosebleed, jaundice urine is red.Mainly contain volatile oil into Divide such as curcumene, turmerone, fragrant zingiberone, curcumin chemical compounds, such as curcumin, demethoxycurcumin and double de- family's epoxides Curcumin etc..The invention reside in, there is provided a kind of new preparation method, the present invention is according to chemical composition in each flavour of a drug in above-mentioned prescription The difference of property, while considering the property of medicine and production cost, effective component extracting is distinguished using different solvents, is retained as far as possible Active ingredient in extract, removes invalid components, the pharmaceutical composition optimal to obtain drug effect.

Preparation method of the present invention, comprises the following steps：

1. fritillaria thunbergii, Radix Angelicae Sinensis, 2~8 times of 50~95% ethanol of amount of root tuber of aromatic turmeric are extracted, extraction 1~3 time, every time 0.5~2.5h, Extract solution is filtered, and 70 DEG C of filtrate is concentrated into relative density d=1.10-1.40 (50~80 DEG C), and 70 DEG C of dryings obtain alcohol and promote leaching Cream, crushes, and crosses 80 mesh sieves.

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 6~12 times of Pollen Typhae (decocting a drug wrapped) amount water are extracted, and extract 1~3 time, every time 0.5~ 2.5h, extract solution filtration, filtrate is concentrated into relative density d=1.05-1.25 (50~80 DEG C), adds 95% ethanol to 50- 90%, more than 12h is stood, supernatant concentration to relative density d=1.10-1.40 (50~80 DEG C), 70 DEG C of dryings obtain water extraction and do Medicinal extract, crushes, and crosses 80 mesh sieves.

3. pseudo-ginseng is crushed, and crosses 50~150 mesh sieves, starch.

Preferably, preparation method of the invention, comprises the following steps：

1. fritillaria thunbergii, Radix Angelicae Sinensis, 3~6 times of 70~95% ethanol of amount of root tuber of aromatic turmeric are extracted, and are extracted 1~3 time, and 1~2h, extracts every time Liquid is filtered, and 70 DEG C of filtrate is concentrated into relative density d=1.10-1.40 (50~80 DEG C), and 70 DEG C of dryings obtain alcohol extracting dry extract, powder It is broken, cross 80 mesh sieves.

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 8~11 times of Pollen Typhae (decocting a drug wrapped) amount water are extracted, and extract 1~3 time, every time 0.5~ 1.5h, extract solution filtration, filtrate is concentrated into relative density d=1.05-1.25 (50~80 DEG C), adds 95% ethanol to 70- 90%, more than 12h is stood, supernatant concentration to relative density d=1.10-1.40 (50~80 DEG C), 70 DEG C of dryings obtain water extraction and do Medicinal extract, crushes, and crosses 80 mesh sieves.

3. pseudo-ginseng is crushed, and crosses 60~120 mesh sieves, starch.

Most preferably, preparation method of the invention, comprises the following steps：

1. fritillaria thunbergii, Radix Angelicae Sinensis, 4 times of 80% ethanol of amount of root tuber of aromatic turmeric are extracted, and are extracted 3 times, each 1.5h, extract solution filtration, filtrate 70 DEG C are concentrated into relative density d=1.28-1.30 (60 DEG C), and 70 DEG C of drying under reduced pressure obtain alcohol extracting dry extract, crush, and cross 80 mesh sieves.

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 10 times of amount water of Pollen Typhae (decocting a drug wrapped) are extracted, and are extracted 3 times, each 1h, extract solution filter Cross, filtrate is concentrated into relative density d=1.16-1.18 (60 DEG C), add 95% ethanol to 80%, stand more than 12h, supernatant Relative density d=1.28-1.30 (60 DEG C) is concentrated into, 70 DEG C of drying under reduced pressure obtain water extraction dry extract, crushed, cross 80 mesh sieves.

3. pseudo-ginseng is crushed, and crosses 80-100 mesh sieves, starch.

Through drug ingedient prepared by the method for the present invention, by obtaining pharmaceutically active substance after mixing, can further use Galenic pharmacy routine techniques is prepared into pharmaceutical preparations composition.Pharmaceutical preparations composition of the invention, exists in a unit, The unit dosage form refers to the unit of preparation, such as every of tablet, every capsule of capsule, every bag of granule etc..This hair Bright Chinese medicine preparation can be any pharmaceutically useful formulation, and these formulations include：Tablet, capsule, granule, pill, powder, Paste, sublimed preparation, pulvis, solution, injection, suppository, spray, drops, patch.Preparation of the invention, preferably orally Formulation, such as：Capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, paste etc..

Chinese medicine composition of the invention, the preparation that it is administered orally can contain conventional excipient, such as adhesive, filling Agent, diluent, lubrication prescription, disintegrant, colouring agent, flavor enhancement and wetting agent.Applicable filler include starch, sucrose, cellulose, Mannitol, the lactose filler similar with other.Suitable disintegrant includes that starch, polyvinylpyrrolidone and starch derive Thing, such as sodium starch glycollate.Suitable lubricant includes, such as magnesium stearate,.Can be filled by mixing, compressing tablet etc. is often Method prepares solid oral composition.Conventional adjunct ingredient includes：Mannitol, sorbierite, sodium pyrosulfite, sulfurous acid Hydrogen sodium, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, injection Vitamin B_6 DTA disodiums, Ethylenediaminetetraacetic Acid Calcium Salt, monoacidic base The carbonate of metal, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, chlorination Potassium, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, Silicon derivative, cellulose and its derivates, alginates, gelatin, polyvinylpyrrolidone, glycerine, POLYSORBATE 80, agar, calcium carbonate, Calcium bicarbonate, surfactant, polyethylene glycol, cyclodextrin, beta-schardinger dextrin, phospholipid material, kaolin, talcum powder, stearic acid Calcium, magnesium stearate etc..

Preparation of the invention determines usage and dosage according to development status when in use, can take 1-3 times daily, each takes list Dosage or multiple dose.

In preparation method of the invention, fritillaria thunbergii, Radix Angelicae Sinensis, principle active component such as alkaloid, volatile oil, the turmeric of root tuber of aromatic turmeric The polarity of the compounds such as element is smaller, is relatively adapted to alcohol extracting；Astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, the main component of Pollen Typhae are soap The class compound such as glycosides, polysaccharide, flavones, polarity is larger, is adapted to water and extracts, while being purified by alcohol precipitation, pectin, mucus can be removed again The impurity components such as matter, further improve the content of active ingredient；Pseudo-ginseng is rare traditional Chinese medicine, and its main component is saponins, Huang Ketone and polysaccharide compound, the pharmacological activity of these compounds are stronger, suitably retain full composition, while avoiding waste of resource. The product obtained using the preparation method has optimal drug action.

Tests prove that, the effect by traditional Chinese medicine composition for treating macular degeneration obtained in preparation method of the invention is obvious Better than the method disclosed in patent CN102058845A.And with patent CN102058845A in technique obtained in Chinese medicine composition Compare, the Chinese medicine composition for the treatment of AMD of the invention can obviously reduce takes crude drug amount day.

Beneficial effects of the present invention are illustrated below by way of experimental data.

Experiment 1：

Test objective

By using CNV rat model, in the treatment AMD that observation is prepared by distinct methods The sample of drug composition, to the preventive and therapeutic effect of choroidal neovascularization in rat, is to select suitable process exploitation treatment macula lutea from now on The medicine of denaturation provides experimental basis.

Experiment material

1st, by reagent and dose design：

The Chinese medicine composition sample message of 1.1 treatment AMD

1) sample 1：Method according to the embodiment of the present invention 1 is prepared, content 4.13g crude drugs/g powder, lot number 2014061501

2) sample 2：Method (full side's water extraction) according to embodiment 1 in patent CN102058845A is prepared, content 3.34g crude drugs/g powder, lot number 2014061502

3) sample 3：Method (full side's alcohol extracting) according to embodiment 2 in patent CN102058845A is prepared, content 4.54g crude drugs/g powder, lot number 2014061503

1.2 positive drugs

1) (Lucentis parenteral solution), lot number are fitted to obtain in promise：S0018, specification 10mg/ml, 0.2ml/ bottle, Novartis Pharma Schweiz AG。

1.3 dose designs：

1) sample 1, sample 2, sample 3, people's body weight are calculated with 70kg, and rat dose,equivalent is 10g crude drugs/kg, using big Mouse dose,equivalent is middle dosage (clinical equivalent dosage), and 1/2 is low dose group, and 2 times of dose,equivalents are high dose, the agent of this secondary design Amount 20,10g, 5g crude drug/kg.

2) promise is fitted, and this product is administered through intravitreal injection, and clinical recommended dose is each 0.5mg (equivalent to 0.05ml Injection volume), be monthly administered.In terms of 200g, people 70kg, the μ g/kg of dose,equivalent 50 add up to 5 μ l/kg, rat to rat 250g or so, 1.25 μ l of injection in every vitreum.

2nd, medicine is prepared：

3 samples of this experiment are weighed in right amount, ground with distilled water be made into required concentration respectively, are given for animal gavage Medicine, administered volume：Sample 1, sample 3 are respectively 1ml/kg body weight, and sample 2 is 1.5ml/kg body weight, administration number of times：Daily gavage It is administered once.

Promise in 1 week is suitable that intravitreal is administered 1.25 μ l after laser photocoagulation, is administered once.

3rd, reagent：

Yellow Jackets, lot number：120505, Merck companies.

Oxybuprocaine hydrochloride eye drops, lot number：B1026, Santen Pharmaceutical Co. Ltd..

Compound tropicamide eye drops, lot number：131201, Shenyang Sinqi Eye Pharmaceutical Co., Ltd..

Fluorescein sodium, lot number：F8140, sigma company.

FITC-D, lot number：SLBB6384V, sigma company.

Paraformaldehyde, lot number：20130415, Tianjin chemical reagent supply and marketing company.

Laboratory apparatus

Multi-wavelength argon laser machine, model：Novus omni, producer：Japanese olympus companies

Fundus fluorescein angiography instrument, Japanese TOPCON companies

TS100 fluorescence inverted microscope models, laser emission wavelength：488nm, collects fluorescent signals wavelengths：530nm, day This NIKON companies

ML203 electronic balances：Mettler-Toledo Instrument (Shanghai) Co., Ltd..

E3000-0.5 electronic balances：Shuan Jie testers factory of Changshu City.

Microscope：OLYMPUS BX51, Japanese Olympus Optical Co., Ltd.

Photomicrography：OLYMPUS DP71, Japanese Olympus Optical Co., Ltd.

Dewaterer：Oriental cherry closed tissue dehydrating machine VIP5J-F2, Sakura Finetek Japan Co., Ltd..

Embedding machine：Leica EG-1150H+C tissue paraffin embedding machines, German Leica companies.

Slicer：Oriental cherry IVS-410 type sliding microtomes, Japanese big and ray machine Industrial Co., Ltd.

Overflow dyeing machine：Oriental cherry automatic staining device DRS-2000J-D2, Sakura Finetek Japan Co., Ltd..

Mounting machine：The automatic mounting machine Glas-J2 of oriental cherry, Sakura Finetek Japan Co., Ltd..

HPX-90502MBE digital display electric heating incubators：Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd..

Experimental animal

BN rats, SPF grades, male, 180-200g of body weight, 98, Beijing Vital River Experimental Animals Technology Co., Ltd. There is provided, production unit licensing numbering：SCXK (capital) 2012-0001, Quality of Experimental Animals quality certification numbering： 11400700063232, invoice number：01073883, date received：20140926.

Laboratory Animal Facility and feeding and management

1. animal facility

Facility name：Tianjin new drug Research on Safety Assessment central animals laboratory building (barrier environment).

Facility address：Tianjin strand high and new technology industrial development zone strand Technology Park Hui Ren roads 308.

Experimental animal uses credit number：SYXK (Tianjin) 2011-0005.

Sign and issue unit：Tianjin Science and Technology Commission.

2. animal feed

Feed title：The big mouse of SPF maintain feed.

Sterilization method：⁶⁰Co is irradiated.

Supplying unit：Tianjin Huarong Animal Science Co., Ltd provides.

Animal feed production licence：SCXK (Tianjin) 2012-0001.

Rat feed lot number：2014108.

Sign and issue unit：Tianjin Science and Technology Commission.

Feed is detected：The every batch of feed has the Self-Check Report that the certification of fitness and supply of forage unit are provided, quarterly by Supply of forage unit provides a third party's feed examining report in the recent period, and detection project includes that feed nutrient and physics and chemistry refer to Mark, examination criteria is with reference to GB14924.2-2001《Experimental animal mixed feed sanitary standard》、GB14924.3‐2010《Experiment is dynamic Thing mixed feed nutritional ingredient》.

Feed testing result：Every Testing index meets the requirements.

3. drinking water

Drinking water is originated：Sterilized water (sterilizing of level Four filters ultra violet) prepared by the multiple filtering with microporous membrane system of 1T/h types.

Water way：With drinking bottle directly filling supply, animal free water.

Drinking water is detected：Barrier environment animal drinking water quarterly by this center self-inspection microbiological indicator, send Tianjin every year Municipal Disease Control and Prevention Center carries out hygienical evaluation detection, including microbiological indicator and biochemical indicator detection.Center self-inspection ginseng It is GB14925-2010 that sighting target is accurate《Experimental animal environment and facility》, censorship reference standard is GB-5749-2006《Life is drunk Water hygiene standard》.

Drinking water testing result：Every Testing index meets the requirements.

4. rearing conditions

In polypropylene mouse box, mouse box specification is length × width × height=48cm × 35cm × 20cm, male and female point to rat feeding Support, raised with 5 animals of sex per box.

Temperature：20 DEG C -26 DEG C of temperature range.

Humidity：Humidity range 40%~70%.

Rate of ventilation：No less than 15 all-fresh air/hours

Illumination：Secretly replace within bright 12 hours 12 hours

5. feeding and management

In addition to particular/special requirement (such as fasting), animal free choice feeding, free water.Animal drinking water is daily in drinking bottle and bottle Change, it is impossible to reuse, reused after pulsation vacuum sterilizer autoclave sterilization using rear drinking bottle.

Animal feeding cage tool chassis is changed 1 time daily, and cage for rearing poultry is changed weekly 1 time, and cage lid is changed 1 time for every 2 weeks, is owned Animal feeding cage tool is used by after pulsation vacuum sterilizer autoclave sterilization into barrier environment；Animal feeding cage is clear weekly Clean, sterilization is wiped 1 time.Animal feeding observation ward carries out cleaning-sterilizing daily, and scope includes：Ground, table top, desktop, door and window and Enter exhaust outlet.The thimerosal that barrier environment is used includes：0.1% bromogeramine solution, 0.1% peracetic acid soln and 1:250 84 thimerosals, three kinds of thimerosals are used in turn, and one kind is used weekly.

6. animal receives and inspection

After experimental animal is arrived, testing crew, animal doctor and animal ensure that department together receives.Transport is first looked at during reception Whether instrument meets the requirements, then checks the animal verification of conformity that animal supplying unit is provided, and confirms quality certification content with application The animal species of purchase, rank, quantity are consistent with sex.Then check whether external packing meets the requirements and whether is animal external packing There is breakage.Animal external packing is through the incoming quarantine room of the 3rd passing cabinet.Test toolss and experimental record are incoming through the 4th passing cabinet.

In quarantine chamber opening animal external packing, verification Animal Sex and quantity and the contained item of animal verification of conformity whether one Cause.Animal quarantine number is marked in rat-tail with marking pen, visual examination (including sex, body weight, head are then carried out to animal one by one Portion, trunk, afterbody, four limbs, fur, spirit, activity etc.), and fill in《Experimental animal receives single》With《Animal acceptance inspection list》. Be put into animal husbandry cage after animal test, in cage tool hang quarantine label, be then placed on quarantine room in carry out the laundering period Raise.Unqualified animal removes that to be put into the temporary refrigerator-freezer of corpse medium pending through buffering area after putting to death.

Animal adapts to feeding period and is limited to 7 days.Animal is observed daily, including body weight, head, trunk, afterbody, four Limb, fur, spirit, activity etc..Laundering period no abnormal animal.

Test method and result

1st, test method

1.1st, model is set up

Male BN rats 98, body weight 200-220g, observation ward raises 7 days, and it is control group, remaining rat penta to reserve 10 Barbital sodium is anaesthetized, the abundant mydriasis of compound Tropicamide, after Oxybuprocaine table fiber crops, before being placed in Kr laser, by three mirror contact lens It is placed on mydriasis eye, is focused on hot spot is aimed on retina by slit-lamp microscope, big blood vessel is avoided, with krypton laser (work( Rate 300mW, 50 μm of diameter, light coagulates 0.1 second time), do ring light away from the outer 2-3PD of optic disk and coagulate, light is solidifying 10 points, to break up Bruch Film is degree.2 eyes of every rat carry out the solidifying modeling of light.

1.2nd, it is grouped situation

Modeling rat is divided into model group, the high dose group of sample 1, the middle dose group of sample 1, the low dose group of sample 1, sample at random The high dose group of product 2, the middle dose group of sample 2, the low dose group of sample 2, the high dose group of sample 3, the middle dose group of sample 3, low dose of sample 3 Amount group, positive drug promise are fitted and obtain group, every group of 8 animals, successive administration 4 weeks after modeling.Promise is suitable to obtain group 1 week glass after laser photocoagulation Body cavity drug administration by injection 1 time.

1.3rd, detection method

1.3.1 underwent eye-ground vascular fluorescence visualization：After being administered 4 weeks, anesthetized rat and mydriasis, the fluorescein sodium of intraperitoneal injection 10% (l.0mL/kg) 5 minutes 1, photos after radiography, are shot.

1.3.2 Flat mounts of choroidal neovascularization：After being administered 4 weeks, with HMW FITC-dextran (FITC-D), Choroidal artery is irrigated.Method for filling：After anesthetized rat, rat thoracic cavity is cut off, 23G scalps are needled into the left heart Room, it is slow to continue to inject 0.1MPBS (pH7.4) through heart.Flow out when most blood, after efflux loses color, made into use The slow perfusion fixation rats of FITC-D Lactated Ringer liquid 20mL (including FITC-D100mg, gelatin 2g).After perfusion is fixed, Ice cube freezes eyeball 10min, and left eye ball, fixation, materials are extractd immediately, obtains RPE- choroids-sclera complex sample.Utilize Laser confocal microscope is observed and measured to it.With 488mn laser excitation samples, 530nm fluorescence signals are collected, computer is adopted Collection digital picture.Image input Image-proplus6.0 professional image softwares are analyzed.

1.3.3 HE dyes om observation：After being administered 4 weeks, to carrying out the rat after choroidal artery perfusion, right side is won Eyeball, fixation, tissue carry out histological examination through repairing block, dehydration, embedding, serial section, HE dyeing, mounting under light microscopic.

2nd, result of the test

2.1 pairs of influences of underwent eye-ground vascular fluorescence visualization

Result shows (Fig. 1), the radial traveling of normal rat retinal vessel, and retinal blood vessels caliber is uniform, tube wall is complete Fluorescein filling in whole, tube chamber, choroidal artery fills and is fused into the background fluorescence for diffusing, unstressed configuration element seepage.

4 weeks after light is solidifying, there is discoid Fluorescein Leakage in model light coagulation zone, is administered that each group light coagulation zone is visible fluorescence Plain seepage, fluorescence leakage scope is significantly less than model group；By the display (table 1), sample 1 of scoring light coagulation zone Fluorescein Leakage High, medium and low dosage group, the high dose group of sample 2 can substantially reduce optical fundus blood vessel Fluorescein Leakage, and its effect is not less than positive drug Promise is fitted；In sample 2, low dose group have no the effect for significantly reducing optical fundus blood vessel Fluorescein Leakage.

Table 1 to Fundus angiography Fluorescein Leakage influence (N=16)

Compare with Normal group^ΔΔΔp<0.001

Administration group compares * p with model group<0.05**p<0.01***p<0.001

Fig. 1 underwent eye-ground vascular fluorescence visualization photos

2.2 pairs of influences of Flat mounts of choroidal neovascularization

Result shows (Fig. 2 of table 2), and the visible model group new vessels such as flower sample of light coagulation zone is with laser spot under fluorescence microscope For substrate grows upwards, the obvious new vessels net of formation, the administration visible netted fluorescent spot of each group light coagulation zone, the height of sample 1, In, low dose group, the high dose group fluorescence intensity of sample 2 and scope be significantly less than model group.

Table 2 to Flat mounts of choroidal neovascularization CNV influence (N=8)

Administration group compares * p with model group<0.05**p<0.01***p<0.001

Fig. 2 choroidal arteries digest tile

2.3 pairs of choroids, the influences of retinal histopathology

Eyeball tissue checks discovery, control group through repairing block, gradient alcohol dehydration, FFPE, HE dyeing under mirror：Each Animal retina internal limiting membrane, nerve fibre layer, ganglion-cell layer, inner molecular layer, inner nuclear layer, external plexiform layer, outer nuclear layer, the external world Film visual cell layer, retinal pigment epithelium (RPE) layer, Bruch films and choroid structural integrity, clear layer, each confluent monolayer cells knot Structure, form have no substantially change.Compare this test model group light coagulation zone major lesions with control group for outer nuclear layer and RPE Layer local failure, and see a small amount of RPE cells divided a word with a hyphen at the end of a line, wherein a small amount of or Some Animals inner nuclear layer arrangement disorder, visible early stage CNV Formed and a small amount of pigmentosa macrophage and fibroblast, show that krypton laser has to the retina and choroid of BN rat eyes Minor injury.Suitable must the group of sample 1,2,3 high, medium and low dosage groups and positive drug promise also has above-mentioned lesion, but lesion size of animal And degree compare with model group and each group between to compare difference not clear aobvious (being shown in Table 3, Fig. 3-14).

In sum, krypton laser can cause retina and the choroid minor injury of BN rat eyes；Sample 1, sample 2, sample The high, medium and low dosage group of product 3 be administered 4 weeks after the BN rat retinas caused by krypton laser, choroidal injury are compared with model group and Compare between each group, the size of animal and degree of lesion have no significant difference.

Table 3：Lesion statistics administration 4 weeks

Note：- have no lesion；± Minimal change；+ slight lesion；++ moderate lesion；+++ severe lesion is similarly hereinafter

Brief description of the drawings：

Fig. 1 underwent eye-ground vascular fluorescence visualization photos

Fig. 2 choroidal arteries digest tile

Fig. 3 is administered 4 weeks eyeballs of control rats (HE 20x)

Retina, the tela chorioidea of normal eyeball

Fig. 4 is administered 4 weeks eyeballs of model group rats (HE 20x)

Light coagulation zone outer nuclear layer, RPE layers of local failure, it is seen that the RPE cells divided a word with a hyphen at the end of a line, pigmentosa macrophage, a small amount of into fibre Dimension cell and early stage CNV are formed

Fig. 5 is administered 4 weeks eyeballs (HE 20x) of the high dose group rat of sample 1

Light coagulation zone inner nuclear layer arrangement disorder, outer nuclear layer, RPE layer local failure, it is seen that a small amount of pigmentosa macrophage, into fibre Dimension cell and CNV are formed

Fig. 6 is administered 4 weeks eyeballs (HE 20x) of the middle dose group rat of sample 1

The inside and outside stratum nucleare of light coagulation zone, RPE layers of local failure, normal configuration disappear, the obvious hyperplasia of CNV, and proliferation of fibrous tissue will New vessels is wrapped, RPE cells, the pigmentosa macrophage see also divided a word with a hyphen at the end of a line on a small quantity

Fig. 7 is administered 4 weeks eyeballs (HE 20x) of the low dose group rat of sample 1

Light coagulation zone inner nuclear layer arrangement disorder, outer nuclear layer, RPE layer local failure, it is seen that CNV is formed and a small amount of RPE for dividing a word with a hyphen at the end of a line Cell, pigmentosa macrophage, fibroblast

Fig. 8 is administered 4 weeks eyeballs (HE 20x) of the high dose group rat of sample 2

Light coagulation zone inner nuclear layer arrangement disorder, outer nuclear layer, RPE layer local failure, it is seen that a small amount of CNV is formed and a small amount of divided a word with a hyphen at the end of a line RPE cells, pigmentosa macrophage, fibroblast

Fig. 9 is administered 4 weeks eyeballs (HE 20x) of the middle dose group rat of sample 2

The inside and outside stratum nucleare local failure of light coagulation zone, it is seen that CNV formation, proliferation of fibrous tissue and a small amount of RPE cells divided a word with a hyphen at the end of a line, Pigmentosa macrophage

Figure 10 is administered 4 weeks eyeballs (HE 20x) of the low dose group rat of sample 2

Light coagulation zone outer nuclear layer, RPE layers of local failure, it is seen that a small amount of RPE cells divided a word with a hyphen at the end of a line

Figure 11 is administered 4 weeks eyeballs (HE 20x) of the high dose group rat of sample 3

Figure 12 is administered 4 weeks eyeballs (HE 20x) of the middle dose group rat of sample 3

Figure 13 is administered 4 weeks eyeballs (HE 20x) of the low dose group rat of sample 3

Figure 14 is administered once the suitable eyeball (HE 20x) that must organize rat of positive drug promise

The inside and outside stratum nucleare of light coagulation zone, RPE layers of local failure, it is seen that a small amount of CNV is formed, proliferation of fibrous tissue and divide a word with a hyphen at the end of a line on a small quantity RPE cells, pigmentosa macrophage

Specific embodiment：

Embodiment 1

3. pseudo-ginseng is crushed, and crosses 100 mesh sieves, starch.

Embodiment 2

1. fritillaria thunbergii, Radix Angelicae Sinensis, 8 times of 50% ethanol of amount of root tuber of aromatic turmeric are extracted, and are extracted 2 times, each 2.5h, extract solution filtration, filtrate 70 DEG C are concentrated into relative density d=1.35-1.40 (70 DEG C), and 70 DEG C of dryings obtain alcohol extracting dry extract, crush, and cross 80 mesh sieves.

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 12 times of amount water of Pollen Typhae (decocting a drug wrapped) are extracted, and are extracted 3 times, each 0.5h, extract solution Filtration, filtrate is concentrated into relative density d=1.20-1.25 (50 DEG C), adds 95% ethanol to 90%, stands more than 12h, supernatant Liquid is concentrated into relative density d=1.35-1.40 (50 DEG C), and 70 DEG C of dryings obtain water extraction dry extract, crushes, and crosses 80 mesh sieves.

3. pseudo-ginseng is crushed, and crosses 150 mesh sieves, starch.

Embodiment 3

1. fritillaria thunbergii, Radix Angelicae Sinensis, 2 times of 95% ethanol of amount of root tuber of aromatic turmeric are extracted, and are extracted 1 time, 1h, and extract solution filtration, 70 DEG C of filtrate is dense Relative density d=1.10-1.15 (50 DEG C) is reduced to, 70 DEG C of dryings obtain alcohol extracting dry extract, crushed, cross 80 mesh sieves.

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 6 times of amount water of Pollen Typhae (decocting a drug wrapped) are extracted, and are extracted 3 times, each 0.5h, extract solution Filtration, filtrate is concentrated into relative density d=1.15-1.25 (60 DEG C), adds 95% ethanol to 50%, stands more than 12h, supernatant Liquid is concentrated into relative density d=1.20-1.30 (70 DEG C), and 70 DEG C of dryings obtain water extraction dry extract, crushes, and crosses 80 mesh sieves.

3. pseudo-ginseng is crushed, and crosses 50 mesh sieves, starch.

Embodiment 4

1. fritillaria thunbergii, Radix Angelicae Sinensis, 3 times of 70% ethanol of amount of root tuber of aromatic turmeric are extracted, and are extracted 2 times, each 2h, extract solution filtration, filtrate 70 DEG C relative density d=1.25-1.30 (70 DEG C) is concentrated into, 70 DEG C of dryings obtain alcohol extracting dry extract, crushed, cross 80 mesh sieves.

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 8 times of amount water of Pollen Typhae (decocting a drug wrapped) are extracted, and are extracted 3 times, each 1h, extract solution filter Cross, filtrate is concentrated into relative density d=1.20-1.25 (80 DEG C), add 95% ethanol to 90%, stand more than 12h, supernatant Relative density d=1.35-1.40 (70 DEG C) is concentrated into, 70 DEG C of dryings obtain water extraction dry extract, crushed, cross 80 mesh sieves.

4. pseudo-ginseng is crushed, and crosses 80 mesh sieves, starch.

Embodiment 5

1. fritillaria thunbergii, Radix Angelicae Sinensis, 6 times of 85% ethanol of amount of root tuber of aromatic turmeric are extracted, and are extracted 1 time, 2h, and extract solution filtration, 70 DEG C of filtrate is dense Relative density d=1.10-1.40 (50 DEG C) is reduced to, 70 DEG C of dryings obtain alcohol extracting dry extract, crushed, cross 80 mesh sieves.

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 11 times of amount water of Pollen Typhae (decocting a drug wrapped) are extracted, and are extracted 2 times, each 1.5h, extract solution Filtration, filtrate is concentrated into relative density d=1.15-1.25 (50 DEG C), adds 95% ethanol to 70%, stands more than 12h, supernatant Liquid is concentrated into relative density d=1.10-1.20 (70 DEG C), and 70 DEG C of dryings obtain water extraction dry extract, crushes, and crosses 80 mesh sieves.

5. pseudo-ginseng is crushed, and crosses 120 mesh sieves, starch.

Claims

1. a kind of preparation method of Chinese medicine composition, by following Chinese medicine for bulk drug is prepared from：

Poria cocos 10-40 weight portion fritillaria thunbergii 5-20 weight portions

Pollen Typhae 10-40 weight portion root tuber of aromatic turmeric 10-40 weight portions,

The preparation method, comprises the following steps：

1. fritillaria thunbergii, Radix Angelicae Sinensis, 2~8 times of 50~95% ethanol of amount of root tuber of aromatic turmeric are extracted, and are extracted 1~3 time, and 0.5~2.5h, extracts every time Liquid is filtered, and relative density is d=1.10-1.40 at 70 DEG C of filtrate is concentrated into 50~80 DEG C, and 70 DEG C of dryings obtain alcohol extracting dry extract, Crush, cross 80 mesh sieves,

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 6~12 times of amount water of Pollen Typhae of decocting a drug wrapped are extracted, extraction 1~3 time, every time 0.5~2.5h, Extract solution filter, filtrate be concentrated into 50~80 DEG C at relative density d=1.05-1.25, add 95% ethanol to 50-90%, it is quiet More than 12h is put, supernatant concentration to relative density d=1.10-1.40 at 50~80 DEG C, 70 DEG C of dryings obtain water extraction dry extract, powder It is broken, 80 mesh sieves are crossed,

3. pseudo-ginseng is crushed, excessively 50~150 mesh sieves, starch,

4. will more than 1. -3. step gained, be well mixed, you can.

2. preparation method according to claim 1, comprises the following steps：

1. fritillaria thunbergii, Radix Angelicae Sinensis, 3~6 times of 70~95% ethanol of amount of root tuber of aromatic turmeric are extracted, extraction 1~3 time, every time 1~2h, extract solution filter Cross, 70 DEG C of filtrate be concentrated into 50~80 DEG C at relative density d=1.10-1.40,70 DEG C of dryings obtain alcohol extracting dry extract, crush, mistake 80 mesh sieves,

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 8~11 times of amount water of Pollen Typhae of decocting a drug wrapped are extracted, extraction 1~3 time, every time 0.5~1.5h, Extract solution filter, filtrate be concentrated into 50~80 DEG C at relative density d=1.05-1.25, add 95% ethanol to 70-90%, it is quiet More than 12h is put, supernatant concentration to relative density d=1.10-1.40 at 50~80 DEG C, 70 DEG C of dryings obtain water extraction dry extract, powder It is broken, 80 mesh sieves are crossed,

3. pseudo-ginseng is crushed, excessively 60~120 mesh sieves, starch,

4. will more than 1. -3. step gained, be well mixed, you can.

3. preparation method according to claim 1, comprises the following steps：

1. fritillaria thunbergii, Radix Angelicae Sinensis, 4 times of 80% ethanol of amount of root tuber of aromatic turmeric are extracted, and are extracted 3 times, each 1.5h, extract solution filtration, 70 DEG C of filtrate Relative density d=1.28-1.30 at being concentrated into 60 DEG C, 70 DEG C of drying under reduced pressure obtain alcohol extracting dry extract, crush, and cross 80 mesh sieves,

2. astragalus root, Poria cocos, charred RADIX ET RHIZOMA RHEI, 10 times of amount water of Pollen Typhae of decocting a drug wrapped are extracted, and are extracted 3 times, each 1h, extract solution filtration, filter Liquid be concentrated into 60 DEG C at relative density d=1.16-1.18, add 95% ethanol to 80%, stand more than 12h, supernatant concentration Relative density d=1.28-1.30 to 60 DEG C, 70 DEG C of drying under reduced pressure obtain water extraction dry extract, crush, and cross 80 mesh sieves,

3. pseudo-ginseng is crushed, excessively 80-100 mesh sieves, starch,

4. will more than 1. -3. step gained, be well mixed, you can.

4. a kind of preparation method of Chinese medicine composition, by following Chinese medicine for bulk drug is prepared from：

Poria cocos 12-30 weight portion fritillaria thunbergii 6-15 weight portions

Pollen Typhae 10-30 weight portion root tuber of aromatic turmeric 10-30 weight portions,

The preparation method, comprises the following steps：

3. pseudo-ginseng is crushed, excessively 50~150 mesh sieves, starch,

4. will more than 1. -3. step gained, be well mixed, you can.

5. preparation method according to claim 4, comprises the following steps：

3. pseudo-ginseng is crushed, excessively 60~120 mesh sieves, starch,

4. will more than 1. -3. step gained, be well mixed, you can.

6. preparation method according to claim 4, comprises the following steps：

3. pseudo-ginseng is crushed, excessively 80-100 mesh sieves, starch,

4. will more than 1. -3. step gained, be well mixed, you can.

7. the preparation method according to any one of claim 1 or 4, further includes the drug ingedient that will be prepared, By after mixing, pharmaceutical preparations composition being prepared into galenic pharmacy routine techniques.

8. preparation method according to claim 7, the pharmaceutical preparations composition, can be any pharmaceutically useful formulations, These formulations include：Tablet, capsule, granule, pill, powder, paste, sublimed preparation, pulvis, solution, injection, suppository, Spray, drops, patch.

9. the preparation method according to any one of claim 1 or 4, by following Chinese medicine for bulk drug is prepared from：

The weight portion of 20 weight portion root tuber of aromatic turmeric of Pollen Typhae 20.

10. the preparation method according to any one of claim 1 or 4, by following Chinese medicine for bulk drug is prepared from：

The weight portion of 28 weight portion fritillaria thunbergii of Poria cocos 8

Or

The weight portion of 15 weight portion root tuber of aromatic turmeric of Pollen Typhae 25.