CN104784419A

CN104784419A - Traditional Chinese medicine composition for treating immune thrombocytopenic purpura and preparation method of traditional Chinese medicine composition

Info

Publication number: CN104784419A
Application number: CN201510208044.8A
Authority: CN
Inventors: 郝其军; 郝彬
Original assignee: SHAANXI HAO QI JUN PHARMACEUTICAL CO Ltd
Current assignee: SHAANXI HAO QI JUN PHARMACEUTICAL CO Ltd
Priority date: 2015-04-24
Filing date: 2015-04-24
Publication date: 2015-07-22

Abstract

The invention discloses a traditional Chinese medicine composition for treating immune thrombocytopenic purpura and a preparation method of the traditional Chinese medicine composition. The traditional Chinese medicine composition is prepared by processing the following raw materials: astragalus membranaceus, white paeony root, dioscorea nipponica makino, salviae miltiorrhizae, clinopodium polycephalum and radix rehmanniae praeparata in a certain proportion. The traditional Chinese medicine composition has the efficiacies of activating blood circulation, enriching blood, tonifying qi and controlling blood, is suitable for the immune thrombocytopenic purpura. According to the traditional Chinese medicine differentiation, hemorrhage syndromes are caused by qi deficiency and blood stasis, qi dysfunction in blood control and escape of blood from vessels and channels. Compared with the prior art, the traditional Chinese medicine composition is significant in clinical pharmacodynamic experiment result, high in bioavailability and free of a toxic or side effect.

Description

A kind of Chinese medicine composition for the treatment of immunologic thrombocytopenic purpura and preparation method thereof

Technical field

The present invention relates to a kind of Chinese medicine composition for the treatment of immunologic thrombocytopenic purpura and preparation method thereof, belong to technical field of traditional Chinese medicine pharmacy.

Technical background

Immunologic thrombocytopenic purpura (immune thrombocytopenic purpura, ITP) with hemorrhage, thrombocytopenia, Significance of Megakaryocyte Number is normal or increase, and is Clinical symptoms with dysmaturity.Majority is fallen ill since the childhood, symptom recurrent exerbation, and delay is difficult, finally develops into refractory idiopathic thrombocytopenic purpura.The first-selected glucocorticoid of western medical treatment ITP, though it is very fast to promote peripheral blood platelet number, holds time short, easily recur, long-term remission rate about 10 ~ 20%, and side effect is more.Cut spleen or immunosuppressant treatment to the many employings of hormone Ineffective Cases, the former is traumatic measure, is not easily accepted by patient, though latter agent is many, remission rate at a specified future date is unsatisfactory, about about 10%, and has more side effect.Danazol, ampeptide elemente, coenzyme A, plasmapheresis, platelet transfusion etc. have also been widely used in clinical, but curative effect is still unstable, and also failing that ITP is treated has basic change.In recent years, the medicines such as the gamma globulin of Clinical practice, interferon and interleukin-11 (huge and grain), although can improve rapidly patient peripheral's platelet numerical value in a short time, curative effect is difficult to lasting, and there are the medical expenses of great number, make most of patients be difficult to bear.Because ITP belongs to clinical frequently-occurring disease and difficult treatment, up to the present, also lacking pointed Chinese medicine and western medicine can make platelet maintain safer level.

Summary of the invention

The object of the invention is to for the defect existing for state of the art, by excavating the abundant Chinese medicine resource of motherland, in conjunction with a large amount of Study on clinical pharmacodynamics, a kind of scientific formula, evident in efficacy is provided, and a kind of Chinese medicine composition being used for the treatment of immunologic thrombocytopenic purpura to human non-toxic's side effect.

Another object of the present invention is to provide the preparation method of Chinese medicine composition of the present invention.

For achieving the above object, the technical solution used in the present invention is:

The present invention treats the Chinese medicine composition of immunologic thrombocytopenic purpura, and its formula consists of:

Radix Astragali 720-780g Radix Paeoniae Alba 470-530g Rhizoma Dioscoreae Nipponicae 720-780g

Radix Salviae Miltiorrhizae 470-530g Herba Clinopodii 470-530g Radix Rehmanniae Preparata 220-280g.

Chinese medicine composition of the present invention, its optimization formula consists of:

Radix Astragali 750g Radix Paeoniae Alba 500g Rhizoma Dioscoreae Nipponicae 750g

Radix Salviae Miltiorrhizae 500g Herba Clinopodii 500g Radix Rehmanniae Preparata 250g.

The preparation method that the present invention treats the Chinese medicine composition of immunologic thrombocytopenic purpura is as follows:

1, the preparation method of Chinese medicine composition of the present invention:

Above Six-element, Rhizoma Dioscoreae Nipponicae, Radix Salviae Miltiorrhizae, Herba Clinopodii are by 60-80% soak with ethanol after 12 hours, and heating and refluxing extraction secondary, 2 hours first times, adds alcohol 10 times amount, and second time 1.5 hours, adds alcohol 8 times amount, filters, merging filtrate; Medicinal residues add the Radix Paeoniae Alba, the Radix Astragali, Radix Rehmanniae Preparata decoct with water secondary, and first time adds water 8 times amount, decocts 2 hours, add water 6 times amount for the second time, decoct 1.5 hours, collecting decoction, filter, filtrate records the thick paste that relative density is 1.10 ~ 1.20 when being concentrated into 60 DEG C, let cool, add ethanol and make alcohol content reach 60-80%, leave standstill 48 hours, get supernatant, filter; Merge above-mentioned filtrate, reclaim ethanol, record the thick paste that relative density is 1.20 ~ 1.30 when being concentrated into 60 DEG C, dry, pulverize into fine powder, add each dosage form proper auxiliary materials, mixing, granulate, dry, make each dosage form.

2, the preparation method of capsule of the present invention:

Above Six-element, Rhizoma Dioscoreae Nipponicae, Radix Salviae Miltiorrhizae, Herba Clinopodii are by 70% soak with ethanol after 12 hours, and heating and refluxing extraction secondary, 2 hours first times, adds alcohol 10 times amount, and second time 1.5 hours, adds alcohol 8 times amount, filters, merging filtrate; Medicinal residues add the Radix Paeoniae Alba, the Radix Astragali, Radix Rehmanniae Preparata decoct with water secondary, and first time adds water 8 times amount, decocts 2 hours, add water 6 times amount for the second time, decoct 1.5 hours, collecting decoction, filter, filtrate record when being concentrated into 60 DEG C relative density be 1.10 ~ 1.20 thick paste, let cool, add ethanol and make alcohol content reach 70%, leave standstill 48 hours, get supernatant, filter; Merge above-mentioned filtrate, reclaim ethanol, record the thick paste that relative density is 1.20 ~ 1.30 when being concentrated into 60 DEG C, dry, pulverize into fine powder, granulate, dry, coating, encapsulated, make 1000, obtain capsule.

3, the preparation method of Tablets:

Above Six-element, Rhizoma Dioscoreae Nipponicae, Radix Salviae Miltiorrhizae, Herba Clinopodii are by 70% soak with ethanol after 12 hours, and heating and refluxing extraction secondary, 2 hours first times, adds alcohol 10 times amount, and second time 1.5 hours, adds alcohol 8 times amount, filters, merging filtrate; Medicinal residues add the Radix Paeoniae Alba, the Radix Astragali, Radix Rehmanniae Preparata decoct with water secondary, and first time adds water 8 times amount, decocts 2 hours, add water 6 times amount for the second time, decoct 1.5 hours, collecting decoction, filter, filtrate record when being concentrated into 60 DEG C relative density be 1.10 ~ 1.20 thick paste, let cool, add ethanol and make alcohol content reach 70%, leave standstill 48 hours, get supernatant, filter; Merge above-mentioned filtrate, reclaim ethanol, record the thick paste that relative density is 1.20 ~ 1.30 when being concentrated into 60 DEG C, dry, pulverize into fine powder, add proper auxiliary materials, granulate, dry, make 1000, obtain tablet.

4, the preparation method of granule of the present invention:

Above Six-element, Rhizoma Dioscoreae Nipponicae, Radix Salviae Miltiorrhizae, Herba Clinopodii are by 70% soak with ethanol after 12 hours, and heating and refluxing extraction secondary, 2 hours first times, adds alcohol 10 times amount, and second time 1.5 hours, adds alcohol 8 times amount, filters, merging filtrate; Medicinal residues add the Radix Paeoniae Alba, the Radix Astragali, Radix Rehmanniae Preparata decoct with water secondary, and first time adds water 8 times amount, decocts 2 hours, add water 6 times amount for the second time, decoct 1.5 hours, collecting decoction, filter, filtrate record when being concentrated into 60 DEG C relative density be 1.10 ~ 1.20 thick paste, let cool, add ethanol and make alcohol content reach 70%, leave standstill 48 hours, get supernatant, filter; Merge above-mentioned filtrate, reclaim ethanol, record the thick paste that relative density is 1.20 ~ 1.30 when being concentrated into 60 DEG C, dry, pulverize into fine powder, add proper auxiliary materials, granulate, dry, make 1000g, obtain granule.

Beneficial effect theory of Chinese medical science of the present invention is thought: in side, Radix Astragali nature and flavor are sweet, warm, return spleen, lung meridian, the gas of the large tonifying the spleen and stomach of effect, and kindly control all deficiency of vital energys and damage, for mending first of all deficiency of vital energy medicines, thriving, blood can be inspired, the effect of existing elimination blood stasis, has again the merit of nourishing qi to stop, is monarch drug in side.Radix Paeoniae Alba bitter in the mouth, acid, is slightly cold, returns Liver Channel, have suppressing liver-YANG, nourishes blood and receives effect of the moon; Rhizoma Dioscoreae Nipponicae nature and flavor are sweet, bitter, warm, GUIXIN, lung meridian, have effect of promoting blood circulation to remove obstruction in the collateral, promoting blood circulation by removing blood stasis, and this two tastes medicine is ministerial drug altogether in side.Radix Salviae Miltiorrhizae nature and flavor are bitter, be slightly cold, GUIXIN, Liver Channel, have blood circulation promoting and blood stasis dispelling, effect that removing heat from blood nourishes blood, and have the merit of hemopoietic of enriching blood, and thus Gu has saying of " SIWU TANG being contained by Radix Salviae Miltiorrhizae simply "; Herba Clinopodii mildly bitter flavor, pungent, cool, return Liver Channel, primary efficacy stops blooding; Two medicines coordinate one promoting blood circulation and hemostasis, its two blood stasis dispelling blood, tissue regeneration promoting blood, coordinate monarch-minister drug to strengthen the merit of blood circulation promoting and blood stasis dispelling, adjuvant drug in side.Radix Rehmanniae Preparata sweet in the mouth slightly warm in nature, returns liver, kidney channel, has nourishing the blood and yin, and effect of vital essence replenishing and marrow benifiting, is applicable to syndrome of deficiency of blood, makes medicine at Fang Zhongwei.The all medicines of full side share, and have nourishing qi to stop, blood circulation promoting and blood stasis dispelling, blood stasis dispelling hemopoietic, treat or alleviate hemorrhage.The all medicines of prescription share blood circulation promoting competent silt and motionless blood, qi-supplementing, blood-engendering and not stagnant blood.Can be used for immunologic thrombocytopenic purpura (i.e. ITP) Chinese medical discrimination is blood stasis due to qi deficiency internal resistance, deficiency-weakness of spleen-QI, QI failing to control blood, escape of blood from meridian cause various go out blood disorder.

(1) Radix Astragali: come from Shennong's Herbal, Gu makes Radix Astragali.For the dry root of leguminous plant Radix Astragali Asrtagalus membranaceus. and astragalus mongholicus Bge Asrtagalus membranacevs (Fisch.) Bge.Nature and flavor are sweet, warm.Return lung, spleen channel.Effect tonifying Qi and lifting yang, benefit defends consolidating superficial resistance, promoting pus discharge and tissue regeneration strengthening, diuretic detumescent." main carbuncle loses skin ulcer for a long time, evacuation of pus pain relieving, strong wind leprosy disease, five hemorrhoid scrofula, tonify deficiency, children's's all kinds of diseases and ailments in " herbal classic " record." " record not " have and " mend husband deficient, five kinds of over strain weakness and emaciation.Quench the thirst, stomachache, dysentery, QI invigorating ..." " Japan hanako materia medica " claim the Radix Astragali to have the effect of " help gas, strengthening bone and muscle, put on flesh, enrich blood "." pearl sac " thinks the sweet Wen Chunyang of the Radix Astragali, and " it is with having five: mend all empty not enough, one also; Benefit vigour, two also; Strong taste, three also; Go flesh heat, four also; Evacuation of pus pain relieving, hemopoietic of invigorating blood circulation, expelling the toxic substance from the body with tonic carbuncle of yin nature, be persons particularly liable to develop skin infection's panacea, five also." " Amplification on Materia Medica addendum " title " large benefit spontaneous perspiration due to yang insufficiency of the Radix Astragali.If exterior deficiency has heresy, send out no sweating person, taking this again can spontaneous perspiration ".Comprehensively above-mentioned, it is weak that the Radix Astragali can be used for the deficiency of vital energy, anorexia and loose stool, sinking of QI of middle-JIAO, chronic diarrhea proctoptosis, metrorrhagia of having blood in stool, exterior deficiency spontaneous perspiration, deficiency of vital energy edema, and carbuncle difficulty is burst, or burst for a long time and do not hold back, blood deficiency and yellow complexion, and interior-heat such as to be quenched one's thirst the disease.

(2) Radix Paeoniae Alba: the Radix Paeoniae Alba comes from Collective Notes to the Canon of Materia medica, bitter in the mouth, acid, is slightly cold, returns Liver Channel.There is suppressing liver-YANG, nourish blood and receive the effect of the moon." the lunar guiding drug of brothers enters liver, spleen blood system to " Bencao Jingshu " Radix Paeoniae Alba." " herbal classic ": " and main pathogen stomachache, except arthralgia due to stagnation of blood, broken hard long-pending, control cold and heat hernia abdominal mass, pain relieving, diuresis, QI invigorating." " not Lu ": " clear and coherent blood vessels, in slow, dispelling malignant blood, expelling extravasated blood, anhydrate gas, sharp bladder, intestine and small intestine, subduing inflammation, row cold and heat time (controlling), and attacked by pestiferous factors is suffered from abdominal pain, lumbago." " property of medicine opinion ": " control pathogen of the lung gas, Fu Zhong Xu pain, vim and vigour are gathered, and logical a surname's internal organs gather around gas, control evil pain deteriorated blood, and disease bone heat time main, strong the five internal organs, the kidney invigorating gas, controls trusted subordinate's heavily fortified point swollen, and married woman's blood closes obstructed, and repercussive blood can lose pus." " medicine origin ": " peace spleen channel, controls stomachache, receives gastric qi, antidiarrheal profit, and blood, fixing striae, eliminating pathogen in the liver, tonifying the spleen and stomach." Wang Haogu: " gas in reason, controls abdominal flatulence due to deficiency of the spleen, epigastric oppression, pain over the hypochondrium, frequent eructation, and lung is anxious swollen against breathing with cough, sun rhinallergosis and epistaxis, and order is puckery, deficiency of liver-blood, and sun dimension suffering of illness cold and heat, dai channel suffering of illness stomachache is full, and waist is broad as sit in water.”

(3) Rhizoma Dioscoreae Nipponicae: Rhizoma Dioscoreae Nipponicae is come from " Zhiwu Mingshi Tukao ".Nature and flavor are sweet, bitter, warm, GUIXIN, lung meridian.Effect promoting blood circulation to remove obstruction in the collateral, promoting blood circulation by removing blood stasis.Rhizoma Dioscoreae Nipponicae different name is more, and there is notable difference [2] each department." will planted by northeast medicine " is referred to as to wear bone dragon, Dioscorea nipponica Mak. Ningpo Yam Rhizome; " Hebei medical material " is called Canis familiaris L. Rhizoma Dioscoreae; " Shandong Chinese medicine " called after mountain Radix Dichroae; " China's economic flora " is named Rhizoma Dioscoreae Hypoglaucae, Rhizoma Curcumae Longae, native mountain potato; " illustrated handbook planted by middle traditional Chinese medicines " meaning wear mountain bone; " Shaanxi Chinese herbal medicine " gives a name fiery rattan root; " Chinese herbal medicine handbook is commonly used in northeast " renames string Eumeces Chinensis; " Zhejiang conventional medical herbs among the people " records bamboo root potato, ferrum root potato, male Rhizoma Zingiberis Recens, yellow whip; " Hebei Chinese medicine handbook " cry ground joist, mountain flower, Rhizoma Smilacis Bockii.But at the dry rhizome of its trade name system Dioscoreaceae plant Dioscorea nipponica Mak. Ningpo Yam Rhizome (Discorea nipponica Mak) of national most area and bavin Dioscorea zingiberensis (Discorea nipponica Mak.var.rosthornii Pr.et Burk).And Fujian Province is always using the dry root of Celastraceae plant Celastrus orbiculatus Thunb. (Celastrus orbiculatus Thunb. [C.articulatusThunb]) as Rhizoma Dioscoreae Nipponicae medical raw material, practises and claim " Fujian Rhizoma Dioscoreae Nipponicae " [4].The medical value of Rhizoma Dioscoreae Nipponicae is day by day subject to researcher and payes attention to.Be accustomed to according to local medication, Rhizoma Dioscoreae Nipponicae effect has some difference, and " will planted by northeast medicine " is recorded: " relaxing muscles and tendons to promote blood circulation, controls lumbago and skelalgia, and muscles and bones is numb." " Shaanxi plant medicine investigation " think that Rhizoma Dioscoreae Nipponicae can " be made cruel, relieve the pain, detumescence." " Shaanxi Chinese herbal medicine " describe " control cough, rheumatic arthritis, Kaschin-Beck disease arthralgia, dyspepsia, malaria, traumatic injury, carbuncle malignant boil." " Shandong Chinese medicine " then think that Rhizoma Dioscoreae Nipponicae " can control anemofrigid-damp arthralgia." " Chinese medicine voluminous dictionary " middle record Rhizoma Dioscoreae Nipponicae function " easypro warp of invigorating blood circulation, help digestion diuretic, eliminates the phlegm, preventing the attack (or recurrence) of malaria ".

In recent years, because Rhizoma Dioscoreae Nipponicae is used widely at Chinese medicine preparation, after Chinese Pharmacopoeia version in 1977 has recorded this kind, Chinese Pharmacopoeia version one in 2010 records this kind again, and effect of clear stipulaties Rhizoma Dioscoreae Nipponicae medical material is wind-damp dispelling, pain relieving, relaxing muscles and tendons and activating QI and blood in the collateral, relieving cough and asthma eliminates the phlegm.For rheumatic arthritis, lumbago and skelalgia, numbness, Kaschin-Beck disease, injury from falling down, lumbar sprain and QI divergeny, chronic bronchitis, the diseases such as cough and asthma.

(4) Radix Salviae Miltiorrhizae: come from Shennong's Herbal.Another name Arisaema balansae Engl..For the root of Labiatae herbaceos perennial Radix Salviae Miltiorrhizae Salviamiltiorrhiza Bge., also known as Radix Salviae Miltiorrhizae.Nature and flavor are bitter, be slightly cold.GUIXIN, pericardium, Liver Channel.Effect blood circulation promoting and blood stasis dispelling, removing heat from blood eliminating carbuncle, nourishing blood to tranquillize the mind." herbal classic " is recorded " main trusted subordinate's pathogen ... cold and heat gather ， Po Disorder except abdominal mass, only stuffy sensation with restlessness, QI invigorating." " record is not " " nourish blood, remove trusted subordinate's obstinate disease stagnation of QI, back is strong, and foot numbness, exorcising stays heat for meaning." " Japan hanako materia medica " claim Radix Salviae Miltiorrhizae to have " sedate of reposing, concerted effort GUAN-pulse.Control cold and hot labor, arthralgia, extremity are unsuccessful.Evacuation of pus pain relieving, granulation promoting is put on flesh.Under hemostasis metrorrhagia and leukorrhagia, adjust married woman's passages through which vital energy circulates irregular.Blood evil thought is tired of, malignant boil scabies, and wart is gone to live in the household of one's in-laws on getting married toxic swelling, erysipelas." Compendium of Material Medica says: " invigorating blood circulation, logical percardium ".Comprehensively above-mentioned, Radix Salviae Miltiorrhizae can be used for menoxenia, amenorrhea dysmenorrhea ， lumps in the chest and abdomen, breast ventral spine pain, pyretic arthralgia pain, and skin infection swells and ache, dysphoria and insomnia, and blood void waits disease [7] less.

(5) Herba Clinopodii: Herba Clinopodii is the dry aerial parts of labiate Clinopodium Polycephalum Herba Clinopodii (Herba Clinopodii Polycephali) Cliropodoium polycephalum (vaniot) C.Y.Wu etHsuanex Hsuan or Herba Clinopodii Polycephali Cliropodoium chinense (Benth) O.Kuntze.Property mildly bitter flavor, pungent, cool.Return Liver Channel.Effect is stopped blooding.Be mainly used in metrorrhagia, hysteromyoma hemorrhage, hematuria, epistaxis, gingival hemorrhage, traumatic hemorrhage etc.

(6) Radix Rehmanniae Preparata: Radix Rehmanniae Preparata is sweet, tepor.Return liver, kidney channel.Enter blood system.Have enrich blood moist; Benefit essence fills out the effect of marrow; Radix Rehmanniae Preparata can be enriched blood YIN nourishing and liver-nourishing kidney-boosting, and all blood deficiency the moon loses, dizzy caused by deficiency of the liver and kindey, all can apply.Enrich blood often to be equal to Radix Angelicae Sinensis, the Radix Paeoniae Alba and use; " book on Chinese herbal medicine is just ": Radix Rehmanniae Preparata is put down, and abnormal smells from the patient is pure quiet, therefore can tonifying five ZANG-organs Kidney-Yin, and in many blood dirty be will, obtain non-taste through medicine that and why madam has survivor, gas and Tremella samguinea Peng.The main sun of gas and moving, main the moon of blood and quiet, QI invigorating is based on Radix Ginseng, and stilbene, art but auxiliary can be helped for it foretell blood based on Radix Rehmanniae Preparata, and Radix Ginseng, Radix Rehmanniae Preparata, then QI and blood must nothing, therefore the deficiency of YANG QI person of all warps, Radix Ginseng, all through it bright blood deficiency person, Radix Rehmanniae Preparata.All all Kidney-Yin loss persons, generate heat effectively, are headache, for terribly thirsty, being sore throat, is expectorant of coughing, for breathing, or spleen renal cold is inverse for vomitting, or asthenic fire carries blood in mouth and nose, or water is general in skin, or the deficiency of YIN and let out profit, or sun is floating and manic, or prolapse of the uterus and ground of falling forward, the deficiency of YIN and the loose person of god, the keeping of non-Radix Rehmanniae Preparata be not enough to poly-it; The deficiency of YIN and the fiery person of liter, non-Radix Rehmanniae Preparata be heavily not enough to fall it; The deficiency of YIN and restless person, non-Radix Rehmanniae Preparata quiet be not enough to town it; The deficiency of YIN and firm anxious person, non-Radix Rehmanniae Preparata sweet be not enough to slow it; The deficiency of YIN and the pathogenic water person of spreading unchecked, house Radix Rehmanniae Preparata is how made by oneself; The deficiency of YIN and true ' gas person of scattering and disappearing, house Radix Rehmanniae Preparata how Gui Yuan; The deficiency of YIN and essence and blood all damages, the residual thin person of fat, house Radix Rehmanniae Preparata how thick the intestines and stomach.The merit of the nourishing YIN and supplementing blood of Radix Rehmanniae Preparata is got in this side.

Pharmacodynamic test of active extract data:

1, experiment purpose:

ITP mouse model is set up by immune molding method, observe the impact of stilbene Chinese herbaceous peony Hematogenesis capsule on its peripheral blood platelet number, Significance of Megakaryocyte Number and classification, immune organ, evaluate this medicine therapeutic effect, for new drug development provides pharmacological data and clinical application foundation.

2, experiment material

2.1 Experimental agents

(1) the former cream of stilbene Chinese herbaceous peony Hematogenesis capsule: clinical adult's consumption is 33 grams of crude drug/60kg/d.This test adopts the unguentum of fixing preparation technology's scale up test, is provided by Shaanxi Hao Qijun Pharmaceutical Co., Ltd.; Every gram of coated granule is containing crude drug 6.63 grams, and lot number is 20140501, and capsule 's content physiological saline solution is made into debita spissitudo, and Refrigerator store is for subsequent use.

(2) XUEKANG KOUFUYE: produced by Jingang Pharmaceutical Co., Ltd., Dalian, lot number 140121; During use with normal saline dilution to desired concn.3. prednisolone acetate: produced by Tianjin Lik-Sang pharmaceutical Co. Ltd, lot number 140203; During use with normal saline dilution to desired concn.

2.2 experiment reagents:

(1) bSA: be purchased from Beijing with positive chemical reagents corporation, produced by sigma company, lot number: 2508-19-2.

(2) PBS: be purchased from doctor's moral Bioisystech Co., Ltd.

(3) Wright's staining liquid and Rui Shi buffer: be purchased from sky, Beijing and make a concerted effort grace chemical reagents corporation.

(4) EDTA-Na2 anticoagulant: be purchased from doctor's moral Bioisystech Co., Ltd.

(5) anti-mouse platelet serum (APS): prepared by Xiyuan Hospital, Chinese Medicine Academy of China's hematopathy laboratory ,-20 degree Refrigerator stores, dilute with physiological saline solution during use.

(6) other reagent: platelet diluent, platelet buffer, platelet cleanout fluid: be all purchased from Beijing Lan Qiao chemical reagents corporation.

2.3 experimental apparatus:

(1) automatic blood analyzer: produced by China blue bridge Instruments Medicaux G.B. Inc., model: M-2100.

(2) fluorescence microscope: Japan produces, model: OLYMPUS-CHB type.

(3) cytospin: Britain SHANDON produces, model: Cytospin2 type.

(4) other instruments: microbalance, digital camera and operating equipment etc. are all domestic manufacture.

2.4 laboratory animals:

Select BALB/c mouse, body weight is 18-22g, 8 week age, and male and female half and half, are provided by Beijing Vital River Experimental Animals Technology Co., Ltd. totally by 84, and licence is numbered: SCXK (capital) 2013-0007.Mouse feeder and experiment are all carried out in cleaning grade zoopery indoor, and the quality certification number is: capital is dynamic is permitted numbers (2010) No. 039, and doctor moves No. 01-2094th, word (2010).Mouse feed for sterilization feedstuff, by Beijing Australia of section pull together feed corporation,Ltd supply.Mice bedding and padding are sterilization bedding and padding.Cage tool peracetic acid soln is sterilized.Drinking water is for purifying waste water.

3, experimental technique:

3.1 anti-mouse platelet serum preparations:

With reference to pertinent literature ^[1], APS is by following preparation method: 1. get BALB/C mice, after etherization, with EDTA-Na ₂anticoagulant gets whole blood from heart, is separated platelet and washs, with normal saline dilution; 2. get the platelet be separated to be mixed into Water-In-Oil shape with equivalent Split completely and incomplete freund adjuvant respectively and to perform antigen, to be injected in Cavia porcellus vola, the back of the body and subcutaneous 4 points containing Split completely antigen in 0 week; With containing incomplete freund adjuvant antigen respectively at 1, within 2,4 weeks, be injected in Cavia porcellus vola, the back of the body and subcutaneous abdomen 4 point.Within 5th week, get not anticoagulated whole blood from guinea pig heart, get supernatant after 560g × 10 are minute centrifugal, obtain Cavia porcellus anti-mouse platelet antiserum (GP-APS), and it is stand-by to be stored in-20 DEG C of refrigerators; 3. improved with reference to ELISA method, replace alkali phosphatase-protein A enzyme labelled antibody to detect antiserum titre by domestic lyophilizing Enzyme-linked SPA sterling (Shanghai Rong Sheng preparation factory); 4. APS is taken out from-20 DEG C, put 56 DEG C of water-baths 30 minutes, with equivalent BALB/C mice hemadsorption at least twice, become 1: 4 concentration APS stand-by with normal saline dilution.

3.2 modeling methods:

List of references improves slightly ^[2], 84 BALB/C mice are divided into normally at random by peripheral blood platelet number, model, XUEKANG KOUFUYE, prednisolone acetate, stilbene Chinese herbaceous peony Hematogenesis capsule heavy dose, middle dosage, low dose totally 7 groups, often organize 12.Except Normal group is to the normal saline of equal volume after grouping, refuse outside special handling, all the other respectively organize the APS all injecting 1: 1 dilution by 100 μ l/20g dosage in mouse peritoneal, a duplicate injection in every 2 days, modeling the 8th day ITP mouse model is stablized, and peripheral blood platelet maintains reduced levels.Within 9th day, start except continuing by above method to except each group of injected in mice APS, model control group gavages the normal saline of equal volume by 0.2ml/10g; XUEKANG KOUFUYE group gavages XUEKANG KOUFUYE by 0.2ml/20g; Prednisolone acetate group gavages lysate by 1ml/mg concentration (20 times of quantity); Stilbene Chinese herbaceous peony Hematogenesis capsule is respectively organized respectively by gavage the former cream diluent of stilbene Chinese herbaceous peony Hematogenesis capsule containing crude drug 0.3179g/20g (heavy dose), 0.1589g/20g (middle dosage), 0.0795g/20g (low dose).Every day 1 time, after continuous 8 days, put to death mice (i.e. final injection APS48 hour), get blood and taking internal organ carries out index of correlation inspection.

3.3: statistical method:

Experimental data mean ± standard deviation represent, carry out t inspection between One-Way variance analysis and group with SPSS software kit, P < 0.05 indicates statistical significance.Date processing carries out on SPSS software kit, and ranked data compare with the inspection of Fisher precise probabilistic method or the analysis of Kendall method of rank correlation.Compare between group with t inspection process.

4, observation index:

4.1 Pharmacodynamics indexs:

(1) lab index: 6 hours, 24 hours, 48 hours three different time sections after injection APS respectively, randomly draw 12 from modeling mice, tail venous blood sampling surveys peripheral blood platelet number, and preliminary judgement APS tires.Experiment terminates rear Testing index to be had: 1. get each group of Mouse whole blood, divides two parts, a detection platelet, leukocyte, red blood cell count(RBC) and content of hemoglobin; Another part detects clotting time.2. get mouse bone marrow cells smear, observe megalokaryocyte quantity, form and classification.3. get each group of mouse spleen, thymus, weigh and calculate organ coefficient, make spleen and thymus pathological section, carry out PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM.

(2) extent of hemorrhage: with reference to " new Chinese medicine guideline of clinical investigations " second volume " guideline of clinical investigations of new Chinese medicine treatment idiopathic thrombocytopenic purpura ^[3]" with Society of Internal Medicine of China Association of Traditional Chinese Medicine hematopathy special interest group about extent of hemorrhage grade scale, in conjunction with zoopery feature, improved, drafted extent of hemorrhage grade scale.And observe extent of hemorrhage change before and after treatment.Bleeding grading standard: 1. I level: injection site hyporrhea, other positions are dispersed in petechia.2. II level: injection site is obviously hemorrhage, and see there are other position ecchymosis, petechias.3. III level: injection site is obviously hemorrhage, a large amount of ecchymosis of skin, petechia, visible dermis blackout or ulceration.

4.2 TCM Syndrome Theories indexs:

For the tcm syndrome change in objective reproduction mankind ITP pathogenic process, experimentation adopts and does not add any artificial intervention factor, the similar TCM Syndrome Theories index change of the dynamic observation modeling mice from macroscopic view to microcosmic." new Chinese medicine treatment syndrome of deficiency of QI guideline of clinical investigations in syndrome of blood stasis due to qi deficiency CM syndrome differentiation criterion Primary Reference " new Chinese medicine guideline of clinical investigations " second volume ^[4]", the syndrome of blood stasis diagnostic criteria of formulating of " new Chinese medicine treatment syndrome of blood stasis guideline of clinical investigations " and syndrome of blood stasis Professional Committee of CAIM (Chinese Association Of Integrative Medicine); combine dynamic mouse model feature correct, draft the dialectical and diagnostic criteria of modeling mice blood stasis due to qi deficiency.

(1) CM syndrome differentiation criterion: 1. subjective observation foundation: A. experiment mice short of breath; B. movable minimizing; C. be slow in action; D. bradykinesia; E. tired sleeping drowsiness; F. inappetence; G soft stool; H. hair color is withered; I. the dermal ecchymosis, or petechia.2. objective determination foundation: A. loses weight; B. body temperature is normal or reduce; C. swimming time shortens.3. pathologic finding foundation: A. organ weights alleviates; B. internal organs are dissected or pathological observation finds blood stasis, hemorrhage or thrombosis etc.

(2) diagnostic criteria: 1. possess any three+I items of A, B, C, D, E, F, G, H of subjective observation foundation and diagnosable; 2. any binomial+I item+objective basis any one of A, B, C, D, E, F, G, H of subjective observation foundation and diagnosable is possessed; 3. any one+I the item of A, B, C, D, E, F, G, H of subjective observation foundation+pathologic finding is possessed according to holding any one and diagnosable.

5, experimental result:

5.1 Pharmacodynamics experimental results:

Main detection model control group and Drug therapy respectively organize that mouse peripheral blood resembles, bone marrow smear, clotting time and organize sick learning to change.Main research is as follows:

(1) platelet count: 6 hours, 24 hours, 48 hours 3 time period rating model control group mice peripheral blood platelet number after injection APS, it the results are shown in Table 1.After treatment terminates, Normal group and model control group mouse peripheral blood platelet number change in table 2.

Table 1, different time sections platelet count change

Compare with Normal group, ^*p < 0.05

Upper table illustrates, model control group all has in different time sections platelet count and declines in various degree, compares, have statistical significance (P < 0.05) with Normal group.Can prove: 1. test APS used and tire normally.2. adopt seroimmunity molding method that mouse peripheral blood platelet can be made to decline rapidly, show the success of ITP model manufacturing.

Table 2, peripheral blood platelet number compare

Compare with Normal group, ^*p < 0.01; Compare with model control group, ^△p < 0.05, ^{△ △}p < 0.01; Compare with XUEKANG KOUFUYE, ^#p < 0.05

Table 2 result shows, and except XUEKANG KOUFUYE group, treating each group of platelet count all has and rise in various degree.Wherein, each dosage group of stilbene Chinese herbaceous peony Hematogenesis capsule compares with model control group, XUEKANG KOUFUYE group, has statistical significance (P < 0.01 and P < 0.05).And though prednisolone acetate group mouse peripheral blood platelet number has necessarily, amplitude is little, compares, no significant difference (P > 0.05) with model control group.Illustrate that stilbene Chinese herbaceous peony Hematogenesis capsule has obvious therapeutic effect to the minimizing of ITP mouse platelets, obvious with heavy dose group.

(2) numeration of leukocyte: after experiment terminates, numeration of leukocyte testing result is in table 3.

Table 3, numeration of leukocyte compare

Compare with Normal group, ^*p < 0.05; ^*p < 0.01, compares with model control group, ^△p < 0.05

Table 3 is pointed out, and each group numeration of leukocyte all has and raises in various degree, compares, have statistical significance (P < 0.05 and 0.01) with Normal group.Except model control group, other treatment is respectively organized also all within normal range.Mouse peripheral blood leukocyte raises may have certain relation with injection APS irritant reaction.

(3) red blood cell count(RBC): after experiment terminates, red blood cell count testing result is in table 4.

Table 4, red blood cell count(RBC) compare

Compare with Normal group, * P < 0.05; * P < 0.01

As can be seen from Table 4, after experiment terminates, each group red blood cell count(RBC) all has and declines in various degree, compares, have statistical significance (P < 0.01) with Normal group.But still in normal range.Analyze that its reason may hemorrhage that erythrocyte is lost be relevant with modeling mice.

(4) Hemoglobin Value: to a certain degree decline though each group Mouse Blood Lactoferrin has, also within normal range, the results are shown in Table 5.

Table 5, experiment terminate rear each group of mice Hemoglobin Value and compare

Compare with Normal group, ^*p < 0.01; Compare with model control group, ^△p < 0.05

Table 5 proves, testing each group of Hemoglobin Value all has different minimizing, compares, have statistical significance (P < 0.01) with Normal group; Prednisolone acetate group compares with model control group, has statistical significance (P < 0.05).But except model control group, treat each group all also within normal range.Analysing haemoglobin dampening factor may be relevant with hemorrhage loss.

(5) clotting time: after experiment terminates, coagulation time test value is in table 6.

Table 6, clotting time compare

Compare with Normal group, * P < 0.01, compares with model control group, ^△p < 0.01

Table 6 shows, and model control group clotting time obviously extends, and compares with Normal group, has statistical significance (P < 0.01).Clotting time is respectively organized in administration all to be had and shortens in various degree, and compare with model control group, difference all has significance (P < 0.01).Show that above various medicine all effectively can improve hypocoagulability, utilize clinical hemostasis.More obvious with stilbene Chinese herbaceous peony Hematogenesis capsule heavy dose group.

(6) megalokaryocyte number: after experiment terminates, each group mouse bone marrow cells megalokaryocyte counting all has certain change, the results are shown in Table 7.

Table 7, bone marrow megakaryocyte count ratio are comparatively

Compare with model control group, ^*p < 0.01; ^*p < 0.05

Table 7 proves, model control group bone marrow megakaryocyte counting obviously increases, and compares, have significant difference (P < 0.05) with Normal group, illustrates that this model meets the change of chronic ITP bone marrow.After treatment except XUEKANG KOUFUYE group, other respectively organize bone marrow megakaryocyte counting all has and to a certain degree increases, and has statistical significance (P < 0.05).Wherein, the big or middle dosage of stilbene Chinese herbaceous peony Hematogenesis capsule and prednisone group bone marrow megakaryocyte counting recovery amplitude more obvious.Show that stilbene Chinese herbaceous peony Hematogenesis capsule and prednisolone acetate are improving therapeutic equivalence in bone marrow macronucleus DH.

(7) megalokaryocyte classification: after experiment terminates, the classification of each group mice megalokaryocyte also has significant change, the results are shown in Table 8.

The classification of table 8, bone marrow megakaryocyte is compared

Compare with model control group, ^*p < 0.05; Compare with blood health group, ^△p < 0.05; Compare with prednisone group, ^#p < 0.05; Compare with small dose group, ^▲p < 0.05

As can be seen from Table 8, model control group mouse bone marrow cells is produced plate megalokaryocyte number and is obviously reduced, and compares, have statistical significance (P < 0.05) with Normal group; Treat each group of bone marrow product plate megalokaryocyte number and all have showed increased, wherein, more obvious with stilbene Chinese herbaceous peony Hematogenesis capsule big or middle dosage group curative effect, compare with model control group, have statistical significance (P < 0.05).Illustrate that stilbene Chinese herbaceous peony Hematogenesis capsule can promote ITP mouse bone marrow cells megakaryocyte differentiation, and promote peripheral blood platelet numerical value by increasing product plate megalokaryocyte number.

(7) thymus and spleen weight: test after terminating, each group mouse thymus and spleen all have to be increased in various degree, the results are shown in Table 9.

Table 9, thymus and index of spleen compare (g/20g)

Compare with Normal group, ^△p < 0.01; Compare with model control group, * P < 0.01

Table 9 shows, model and treatment each group of mouse spleen is all shown in obvious increase, compare with Normal group, there is statistical significance (P < 0.01), and prednisolone acetate group spleen increases not obvious, compare with model control group, have statistical significance (P < 0.01).The pathological change that spleen increases is similar to mankind ITP clinical sign.Treat each group of mouse thymus size to compare with Normal group, no significant difference (P > 0.05).

(8) histopathology: thymic tissue form does not all find obvious exception with light microscopic substantially.Spleen tissue morphology gross examination of skeletal muscle volume obviously increases, and is about 3 times of normal mouse.Om observation Normal group mouse spleen organizational structure is clear, and tunicle, red pulp, white pulp are clear, and under high power lens, in splenic red pulp, in medullary cord, medullary sinus, each cellular morphology shows no obvious abnormalities.Model control group white pulp becomes large, and red pulp reduces, and lymphocyte significantly increases, and distribution volume megalokaryocyte in medullary cord, medullary sinus in splenic red pulp, show that extramedullary hemopoiesis function is enlivened, under high power lens, megalokaryocyte Assessment of Nuclear Volume is large, and endochylema enriches.Blood health treatment group spleen organizes the megalokaryocyte quantity in red pulp to reduce to some extent; Prednisolone acetate and stilbene Chinese herbaceous peony Hematogenesis capsule treatment group spleen organize the megalokaryocyte quantity in red pulp obviously to reduce.

5.2 hemostatic healing efficacies:

Press out blood system grade standard, observe each group respectively and inject bleeding after APS24 hour, and by hemorrhage grading criterion evaluation hemostatic healing efficacy after treatment.The results are shown in Table 10,11.

Table 10, the dermal ecchymosis compare

The every data of upper table are through inspection, and stilbene Chinese herbaceous peony Hematogenesis capsule group and Normal group and model control group the dermal ecchymosis all have statistical significance (P < 0.05); Stilbene Chinese herbaceous peony Hematogenesis capsule group and XUEKANG KOUFUYE group and prednisolone acetate group not statistically significant (P > 0.05).Respectively organize mice by after observation injection APS 3 days and all have obvious bleeding tendency, show as ecchymosis, petechia.Attach most importance to injection site (abdominal part), afterbody, extremity.Bleeding tendency continues beginning in about 1 week and alleviates gradually.It is also hemorrhage based on III, IV level that experiment terminates rear model control group; All the other are respectively organized bleeding tendency and are all improved in varying degrees.Wherein, with prednisolone acetate group, stilbene Chinese herbaceous peony Hematogenesis capsule heavy dose group haemostatic effect the most obvious.

Table 11, hemostatic healing efficacy are evaluated

Above result is through inspection, and stilbene Chinese herbaceous peony Hematogenesis capsule group and Normal group, model control group hemostatic healing efficacy have statistical significance (P < 0.05); Stilbene Chinese herbaceous peony Hematogenesis capsule group and XUEKANG KOUFUYE group and prednisolone acetate group not statistically significant (P > 0.05).Illustrate that the overall haemostatic effect of stilbene Chinese herbaceous peony Hematogenesis capsule is better than other each group, wherein, more obvious with stilbene Chinese herbaceous peony Hematogenesis capsule heavy dose group hemostatic healing efficacy.

5.3 impacts on traditional Chinese medical science syndrome of blood stasis due to qi deficiency:

(1) subjective observation index: after experiment terminates, each group traditional Chinese medical science subjective symptom the results are shown in Table 12.

Table 12, blood stasis due to qi deficiency observation index compare

Experiment terminates rear stilbene Chinese herbaceous peony Hematogenesis capsule group and each group of all blood stasis due to qi deficiency observation index assay is as follows:

(2) objective determination index: inspection method and result as follows:

1. body weight inspection: in experiment pre-test once, surveys once to testing end for after this every 3 days.For ease of statistical procedures, three body weight testing results are in table 13.

Table 13, weight ratio are comparatively

Compare with model control group, ^△p < 0.05; Compare with blood health group, ^#p < 0.05; Compare with prednisolone acetate group, ^▲p < 0.05

As can be seen from Table 13, the big or middle dosage group of stilbene Chinese herbaceous peony Hematogenesis capsule has obviously prevention ITP Mouse Weight to alleviate effect.Compare with model control group, prednisolone acetate group, XUEKANG KOUFUYE group, have statistical significance (P < 0.05).Illustrate that stilbene Chinese herbaceous peony Hematogenesis capsule has good tonify deficiency effect.

2. feed and amount of drinking water: measure once before testing, surveys and measures once to testing end for after this every 3 days.For ease of statistical procedures, three measurement results are in table 14,15.

Table 14, daily inleting appetite compare

Compare with model control group, * P < 0.05; Compare with blood health group, ^△p < 0.05

Table 14 proves, after modeling type, each group food-intake obviously reduces, and compares, have statistical significance (P < 0.05) with Normal group.Illustrate that model mice has deficiency-weakness of spleen-QI symptom.Experiment terminates rear model control group, blood health group, stilbene Chinese herbaceous peony Hematogenesis capsule small dose group food-intake can not be recovered, and compares, have statistical significance (P < 0.05) with Normal group.Other are respectively organized food-intake and all can recover, and compare, have statistical significance (P < 0.05) with model control group, blood health group.Illustrate that the big or middle dosage group of stilbene Chinese herbaceous peony Hematogenesis capsule has invigorating the spleen and benefiting QI, appetite stimulator effect.

Table 15, day enter amount of drinking water and compare

Table 15 proves, after modeling type, each group amount of drinking water obviously reduces, and compares, have statistical significance (P < 0.05) with Normal group.Illustrate that model mice has deficiency-weakness of spleen-QI symptom.Experiment terminates rear model control group, blood health group, stilbene Chinese herbaceous peony Hematogenesis capsule small dose group amount of drinking water can not recover, and compares, have statistical significance (P < 0.05) with Normal group.Other are respectively organized amount of drinking water and all can recover, and compare, have statistical significance (P < 0.05) with model control group, blood health group.Illustrate that the big or middle dosage group of stilbene Chinese herbaceous peony Hematogenesis capsule has invigorating the spleen and benefiting QI effect.

3. body temperature measurement: adopt WMz-03 digital temperature indicator, respectively surveys limb temperature (left fore), anus temperature once and after modeling for 2-3 hour before experiment, after modeling, experiment terminates from each group, to randomly draw 5 mice take temperatures respectively afterwards.The results are shown in 16.

As can be seen from Table 16, mice body temperature appearance rising in 2-3 hour after first time injection APS is respectively organized in modeling, compares, have statistical significance (P < 0.05) with Normal group.After this APS body temperature is injected normal.Illustrate that injecting APS first may cause irritative response and occur the temporary rising of body temperature.

Table 16, different time Temperature changing

Compare with Normal group, * P < 0.05

4. swimming time: add water in glass jar, depth of water 20cm, water temperature remains on 0.5 DEG C, 20 soil.And at the plasticine of afterbody bundle one 2g.Mice is put into respectively glass jar to swim, timing, note observing, when mouse head submerged 3 can not the person of emerging be muscle power exhaustion, timing at once second, calculate mice swimming time.Before experiment, after modeling, test and terminate rear each survey once.Experimental result is in table 17.

Table 17, swimming time compare

Compare with before modeling, * P < 0.01, * * P < 0.001; Compare with before administration, ^△p < 0.05, ^{△ △}p < 0.01; Compare with model control group, ^▲p < 0.05

Table 17 result shows, test each group after modeling (before administration) mice swimming time all see obvious shortening, compare with Normal group, have statistical significance (P < 0.05).Illustrate that ITP mice has positive QI-insufficiency, the deficiency of vital energy performance of not resistance to labour.Experiment terminates rear model control group mice swimming time and continues to shorten; After treatment, prednisolone acetate group, blood health group mice swimming time can not recover normal, and each dosage group swimming time of stilbene Chinese herbaceous peony Hematogenesis capsule extends as seen, with before administration, model control group compares, and has statistical significance (P < 0.05).Illustrate that stilbene Chinese herbaceous peony Hematogenesis capsule has good QI invigorating, antifatigue effect.

(3) pathology detection index: 1. experiment terminates rear observations discovery, model control group mice is subcutaneous have obviously hemorrhage.After dissecting, gross examination of skeletal muscle also finds, model control group mouse core, lung, liver, brain, kidney all can see obvious petechia and blood stasis.Medication is respectively organized without obvious above-mentioned performance.2. pathology section examination result shows, above-mentioned internal organs model control group has hemorrhage and blood stasis pathological change, but without obvious internal organs atrophy phenomenon.And administration is respectively organized and is not found above-mentioned pathological phenomenon.

6, discuss:

6.1 immunizations manufacture ITP Model Practical:

ITP is with hemorrhage and peripheral blood thrombocytopenia, and Significance of Megakaryocyte Number is normal or increase, and is the common hemorrhage of main manifestations with dysmaturity.Think now, ITP belongs to the one of autoimmune disease, and its pathogenesis may be that humoral immunization and cellular immunization, platelet function and endocrine etc. cause extremely.The pathogenesis comparatively affirmed and the disorder of autoimmune regulatory function closely related.

Adopted Busulfan, cytosine arabinoside, cyclophosphamide or radiation exposure legal system to make animal model more in the past, and for pharmacodynamic study.But this modeling method can damage marrow hemopoietic stem cells, bone marrow and peripheral blood three is caused to be that cell all reduces.To carrying out aplastic anemia, the pharmacodynamic study of chemicotherapy bone marrow injury medicine has important value, and is not suitable for the pharmacodynamic study of ITP.Because though this kind of model has periphery thrombocytopenia, the immunologic derangement mechanism of its genesis mechanism and ITP is inconsistent, therefore seldom adopts now.The foundation of the chronic ITP animal model of immune-mediated method is initiated in 1975.First Bentield establishes SD rat chronic ITP model, repeats 5-6 injection APS in 10 days, and platelet count low-level during modeling can be made to maintain.Nineteen ninety, Stenberg improved on this basis, and research finds, to stop after injection APS mouse peripheral blood platelet number and megalokaryocyte form and the existing dynamic change of polyploid distribution in 5 days, proved this kind of model and mankind ITP pathogenic process basic simlarity ^[5].Yang Yufei etc. ^[1]first set up at home BALB/c mouse ITP model, injected the method for APS totally 6 times at 0,1,2,4,6,7 day, observe peripheral hemogram, bone marrow smear and respectively organize internal organs pathological change.Result of study with abroad report basically identical ^[1].Illustrate that to set up ITP Animal models by the exogenous antiplatelet antibody of infusion ripe.Its principle is simple, and interference factor is little.Thus, there is obvious superiority in pharmacodynamic study ^[6-7].

The feasibility that 6.2 disease combination models are set up:

At present, for the unification still not of ITP traditional Chinese medical science pathogenesis understanding, substantially there is deficiency-weakness of spleen-QI, QI failing to control blood, yin asthenia generating intrinsic heat, if forcing blood is capable, obstruction of collaterals by blood stasis, escape of blood from meridian three kinds of pathogenesis changes.Clinical commonly have the deficiency of vital energy, the deficiency of YIN, deficiency of both QI and YIN, deficiency of both YIN and YANG syndrome.But most scholar thinks in close relations with " deficiency-weakness of spleen-QI, QI failing to control blood ".Thus, when carrying out pharmacodynamic study, also with " invigorating the spleen and benefiting QI is enriched blood and is taken the photograph blood " for base therapy rule.But in clinical practice, with the passing of time ITP patient mostly often is the course of disease, controls for a long time and is difficult to produce effects, clinical commonly have lusterless complexion, fatigue and weak, deficiency of QI with disinclination to talk, asthenia spontaneous perspiration, inappetence, the dermal ecchymosis, or petechia, and body of the tongue is dull, and pulse condition is thin and delicate waits performance.Feel frustrated and the theory of Chinese medical science of pathogen usually intruding into collateral in protracted disease according to prolonged illness, when with " blood stasis due to qi deficiency " for main syndromes.

The ITP animal mould previously set up is also many to be associated with insufficiency of the spleen.And to set up on disease model basis when pharmacodynamic study, by under applying hunger, rushing down, the method establishment disease combination model such as tired is for pharmacodynamic study ^[8].In fact this modeling mode has been run counter to disease self and has been occurred and law of development.Any disease has its most basic pathological change in generation with evolution, and Chinese and western medicine is basically identical.Therefore, setting up in ITP animal model, artificial applying intervention factor still can not be observed disease and the essential phenomenon with evolution occurs.This experiment is while setting up ITP disease model, according to disease development rules, not applying any artificial intervention factor, with reference to evaluating human diseases tcm symptom (syndrome) standard, by macroscopic view, dynamically observing method evaluation ITP animal model Characteristics of traditional Chinese syndromes.Through experimental studies have found that, the ITP model that immunity molding method is set up both had had periphery thrombocytopenia, hemorrhage (ecchymosis, or petechia), bone marrow megakaryocyte increases and the disease model characteristic of producing plate megalokaryocyte and reducing, there is again short of breath, movable to reduce, be slow in action, bradykinesia, tired sleeping drowsiness, inappetence, soft stool, hair color withered, the dermal ecchymosis, or petechia, lose weight, swimming time shortening, organ weights alleviate and to dissect with main organs and pathological observation finds blood stasis, " blood stasis due to qi deficiency syndrome " feature such as hemorrhage.Can think, not add any artificial intervention factor and set up ITP disease combination model there is feasibility, can be used for the research of new Chinese medicine pharmacy.On the other hand, also reproduce and demonstrate chronic ITP with " blood stasis due to qi deficiency " for main pathogenesis and Characteristics of Syndromes.

6.3 experimental results:

Successfully replicate ITP mouse model by the immune molding method injecting APS, and traditional Chinese medical science pathogenic process and the clinical manifestation of mankind ITP can be reproduced.And obvious positive findings is obtained after the treatment of stilbene Chinese herbaceous peony Hematogenesis capsule, Pharmacodynamics conclusion is as follows:

(1) platelet count is promoted: stilbene Chinese herbaceous peony Hematogenesis capsule obviously can promote ITP mouse peripheral blood platelet number, compares with model control group, has statistical significance (P < 0.05 and 0.01).Wherein, the most obvious with the effect of heavy dose group.

(2) shorten clotting time: ITP mice not only has the minimizing of periphery platelet counts, also there is clotting mechanism abnormal.Experimental result shows, and after treatment, stilbene Chinese herbaceous peony Hematogenesis capsule respectively organizes clotting time all has and shorten in various degree, compares, have statistical significance (P < 0.01) with model control group.

(3) bone marrow megakaryocyte differentiation is promoted: bone marrow smear megalokaryocyte can normally or increase, and is the most important feature of ITP with dysmaturity.After the treatment of stilbene Chinese herbaceous peony hemostasis capsule, Significance of Megakaryocyte Number can recover normal, produces plate macronucleus showed increased, compares, have statistical significance (P < 0.05 and 0.01) with model control group.Wherein, be excellent with the big or middle dosage group curative effect of stilbene Chinese herbaceous peony Hematogenesis capsule.

(4) immune organ protective effect: found by pathological research, model control group mouse spleen enlarges markedly, under light microscopic, white pulp becomes large, red pulp reduces, number and the area of lymph follicle area, germinal center significantly increase, lymphocyte obviously increases, distribution volume megalokaryocyte in medullary cord, medullary sinus in red pulp; Under high power lens, megalokaryocyte Assessment of Nuclear Volume is large, and endochylema enriches.These pathological changes illustrate that the outer hemopoietic function of model control group Murine Bone Marrow is enlivened.After the treatment of stilbene Chinese herbaceous peony Hematogenesis capsule, the enlargement of ITP mouse spleen all has to be reduced in various degree, shows that stilbene Chinese herbaceous peony Hematogenesis capsule has protective effect to ITP mouse immune organ.

(5) improve tcm syndrome: through stilbene Chinese herbaceous peony Hematogenesis capsule treatment after, the traditional Chinese medical science " blood stasis due to qi deficiency " syndrome can be improved significantly.Meanwhile, stilbene Chinese herbaceous peony Hematogenesis capsule can increase ITP mice swimming time, stops Mouse Weight to decline and obvious haemostatic effect.This result of study illustrates that stilbene Chinese herbaceous peony Hematogenesis capsule not only has obvious benefiting QI for activating blood circulation hemostatic effect, also demonstrate that ITP is with " syndrome of blood stasis due to qi deficiency " and the traditional Chinese medical science " gas hemopoietic " most important theories with " promoting tissue regeneration by removing blood stasis ".

7, conclusion:

Stilbene Chinese herbaceous peony Hematogenesis capsule of the present invention has good therapeutical effect to the ITP mice modeling that injection APS immunity manufactures.Its action character can promote peripheral blood platelet number, promotes bone marrow macronucleus differentiation and maturation, improve blood stasis due to qi deficiency syndrome for this medicine.Thus, this medicine may be used for the treatment of human chronic ITP.

Acute toxicity testing result shows: stilbene Chinese herbaceous peony Hematogenesis capsule is by the concentration of 57.1% and the administration capacity of 0.3ml/10g, within 24 hours, give ICR mouse stomach 1 time, mice is without 1 death, also without obvious toxic reaction, recording the maximum test medicine amount of stilbene Chinese herbaceous peony Hematogenesis capsule is 17.13g/kgbw, for clinical people intends 174.8 times of consumption.Stilbene Chinese herbaceous peony Hematogenesis capsule does not measure LD50 to ICR Strains of Mouse gavage, is administered once in 24 hours, and observe 7 days, mice, without death, does not observe the toxic reaction of stilbene Chinese herbaceous peony Hematogenesis capsule under this dosage, shows that stilbene Chinese herbaceous peony Hematogenesis capsule is without acute toxic reaction.

Long term toxicity test result shows: stilbene Chinese herbaceous peony Hematogenesis capsule is by 35.90g crude drug/kgbw, 17.95g crude drug/kgbw, 8.976g crude drug/kgbw tri-dosage to rat oral gavage three months, and quite clinical people intends 67.12,33.56,16.78 times of consumption respectively.Large, medium and small dosage group rat body weight increases, and compares without significant difference with matched group, and normally, administration is after three months, and often group is lived and killed 10, ♂ ♀ half and half, and electrocardiogram, routine urinalysis detect normally for diet, hair color, activity.Common carotid artery gets blood, carries out hematological examination and blood biochemistry checking, and result compares without significant difference with matched group, and separate taking internal organ tissue, organ index compares without significant difference with matched group.Histopathologic examination shows, each organs and tissues compares without obvious pathological change with matched group.Drug withdrawal after 2 weeks again work kill residue rat, often organize 10, ♂ ♀ half and half, observation project the same, except platelet obviously raises, each test item is all normal.Visible, stilbene Chinese herbaceous peony Hematogenesis capsule non-toxic reaction, long-term prescription is safe and reliable.

Detailed description of the invention:

Embodiment 1:

Prescription:

Preparation method is:

Embodiment 2:

Prescription:

Radix Astragali 720g Radix Paeoniae Alba 470g Rhizoma Dioscoreae Nipponicae 720g

Radix Salviae Miltiorrhizae 470g Herba Clinopodii 470g Radix Rehmanniae Preparata 220g.

Preparation method is:

Embodiment 3:

Prescription:

Radix Astragali 780g Radix Paeoniae Alba 530g Rhizoma Dioscoreae Nipponicae 780g

Radix Salviae Miltiorrhizae 530g Herba Clinopodii 530g Radix Rehmanniae Preparata 280g.

Preparation method is:

Embodiment 4:

Prescription:

Preparation method is:

Above Six-element, Rhizoma Dioscoreae Nipponicae, Radix Salviae Miltiorrhizae, Herba Clinopodii are by 60% soak with ethanol after 12 hours, and heating and refluxing extraction secondary, 2 hours first times, adds alcohol 10 times amount, and second time 1.5 hours, adds alcohol 8 times amount, filters, merging filtrate; Medicinal residues add the Radix Paeoniae Alba, the Radix Astragali, Radix Rehmanniae Preparata decoct with water secondary, and first time adds water 8 times amount, decocts 2 hours, add water 6 times amount for the second time, decoct 1.5 hours, collecting decoction, filter, filtrate record when being concentrated into 60 DEG C relative density be 1.10 ~ 1.20 thick paste, let cool, add ethanol and make alcohol content reach 60%, leave standstill 48 hours, get supernatant, filter; Merge above-mentioned filtrate, reclaim ethanol, record the thick paste that relative density is 1.20 ~ 1.30 when being concentrated into 60 DEG C, dry, pulverize into fine powder, granulate, dry, coating, encapsulated, make 1000, obtain capsule.

Embodiment 5:

Prescription:

Preparation method is:

Above Six-element, Rhizoma Dioscoreae Nipponicae, Radix Salviae Miltiorrhizae, Herba Clinopodii are by 80% soak with ethanol after 12 hours, and heating and refluxing extraction secondary, 2 hours first times, adds alcohol 10 times amount, and second time 1.5 hours, adds alcohol 8 times amount, filters, merging filtrate; Medicinal residues add the Radix Paeoniae Alba, the Radix Astragali, Radix Rehmanniae Preparata decoct with water secondary, and first time adds water 8 times amount, decocts 2 hours, add water 6 times amount for the second time, decoct 1.5 hours, collecting decoction, filter, filtrate record when concentrating 60 DEG C relative density be 1.10 ~ 1.20 thick paste, let cool, add ethanol and make alcohol content reach 80%, leave standstill 48 hours, get supernatant, filter; Merge above-mentioned filtrate, reclaim ethanol, record the thick paste that relative density is 1.20 ~ 1.30 when being concentrated into 60 DEG C, dry, pulverize into fine powder, granulate, dry, coating, encapsulated, make 1000, obtain capsule.

Embodiment 6:

Prescription:

Preparation method is:

Embodiment 7:

Prescription:

Preparation method is:

Claims

1. treat a Chinese medicine composition for immunologic thrombocytopenic purpura, it is characterized in that described traditional Chinese medicinal composition raw materials weight proportion is:

2. Chinese medicine composition according to claim 1, is characterized in that described Traditional Chinese medicine composition formula consists of:

3. the preparation method of Chinese medicine composition according to claim 1 or 2, is characterized in that:

4. the preparation method of Chinese medicine composition according to claim 1 or 2 or 3, is characterized in that:

5. the preparation method of Chinese medicine composition according to claim 1 or 2 or 3, is characterized in that:

6. the preparation method of Chinese medicine composition according to claim 1 or 2 or 3, is characterized in that: