CN105434807B - The pharmaceutical composition for the treatment of diabetes mellitus encephalopathy and its preparation method and detection method and purposes - Google Patents

Abstract

The invention belongs to the field of Chinese medicines, there is provided a kind of pharmaceutical composition treating diabetes mellitus encephalopathy, the good wine decoction thing of invention entitled treatment diabetes mellitus encephalopathy and its preparation method and detection method and purposes, this pharmaceutical composition is to be made up of the raw material of following weight portion：Rhizoma Dioscoreae 30 weight portion, the Radix Astragali 15 weight portion, the Rhizoma Anemarrhenae 15 weight portion, Radix Puerariae 15 weight portion, Fructus Schisandrae Chinensis 15 weight portion, Radix Trichosanthis 15 weight portion, Endothelium Corneum Gigeriae Galli 10 weight portion.This pharmaceutical composition is adopted and is prepared with the following method：Pharmaceutical decocting piece is weighed by prescribed dose, with 5～15 times amount, 40～60% alcohol reflux 3 times, each extraction time 0.5～1.5h, united extraction liquid, reclaims ethanol, concentrates, dry, pulverize, obtain final product.The invention provides the method that assay is carried out to diosgenin using high performance liquid chromatography, additionally provide the method that assay is carried out to cuparene, thujopsene using gas chromatography.

Description

The pharmaceutical composition for the treatment of diabetes mellitus encephalopathy and its preparation method and detection method and purposes

Technical field

The invention belongs to the field of Chinese medicines is and in particular to a kind of pharmaceutical composition treating diabetes mellitus encephalopathy.

Background technology

First, diabetes（Diabetes mellitus, abbreviation DM）

Diabetes（Diabetes mellitus, abbreviation DM）Be due to insulin secretion and（Or）Insulin action is not enough The Metabolic Syndrome with hyperglycemia as principal character causing, epidemiological study shows, with social development and life The change of mode, the sickness rate of DM is in rising trend in the world.At present, the whole world has 1.5 hundred million DM patients, to 2025 Year, this numeral will rise to 3.3 hundred million.The current number of patients of China about 40,000,000, accounts for the whole world about 1/5.Diabetes are a kind of Systemic metabolic disease, can cause Multisystem damage to lead to cardiovascular and cerebrovascular disease, retinopathy, nephropathy, diabetic foot, surrounding The complication such as neuropathy, diabetic animals are all confirmed with clinical research in a large number at present, and persistent high blood sugar can also result in maincenter god Through system（Central nervous system, CNS）Cognitive dysfunction（Mild cognitive impairment, MCI）Diabetes mellitus encephalopathy, that is, central nervous system cause the infringement of cerebral tissue 26S Proteasome Structure and Function, MCI cognitive dysfunction is again Claim cognitive function decline, Cognitive or cognitive disabled, refer to various degrees of cognitive impairment, from MCI until Dull-witted.Multinomial research all shows, type Ⅱdiabetes mellitus cognitive dysfunction and sex, age, schooling, life style, blood Sugar level, metabolism disorder, insulin resistant, hypothalamic-pituitary-adrenal（hypothalamic-pituitary- Adrenal, HPA）S function imbalance, inflammatory mediator etc. are relevant, and the research to diabetes cognitive dysfunction is current research heat One of point.

2nd, central cholinergic system

Central cholinergic system is considered as the important path constituting learning and memory, acetylcholine（Acetylcholine, ACh）It is wherein important neurotransmitter, choline acetyltransterase（Choline acetyl-transferase, ChAT）As The rate-limiting enzyme of Ach synthesis, the mark frequently as research cholinergic neuron or estimation Ach content（Burst size）Indirect indicator. Intracerebral cholinergic neurotransmitter Ach content and the change of ChAT activity, all can reflect the activity of cholinergic nerve, as current One of old and feeble leading indicator of research mankind's brain function.With the proposition of " cholinergic damage theory ", cholinergic system is in glycosuria Effect in sick encephalopathy morbidity is increasingly subject to people and payes attention to.The striatum of diabetes rat and sea caused by multinomial research confirmation STZ Acetylcholine Developmental and Metabolic Disorder in horse area, is also found diabetes rat cerebral tissue（Estrangement, corpus amygdaloideum, Hippocampus etc.）Cholinergic nerve System enzyme activity decrease, cholinergic nerve conduction reduces.Scholar is therefore had to think, diabetes mellitus encephalopathy early stage is that cognitive function is relevant Basal forebrain, the cholinergic neuron degeneration such as Hippocampus and cortex.

Research shows, glucose occurs non-enzymatic glycation and oxidation reaction institute shape with the free radical of protein or lipid The product becoming, i.e. Advanced glycation endproducts（AGEs）, Advanced glycation endproducts（AGEs）In-vivo tissue one can be caused Serial pathophysiological change, is the important paathogenic factor leading to chronic complicating diseases of diabetes.AGE receptor（RAGE）It is present in very Many cells surface, its combination with AGE may result in Cellular Oxidation stress and NF- κ B path activation.After 30 years old, people In intracerebral Hippocampus and neurogliocyte, AGEs starts to gather.AGEs is co-deposited with beta-amyloyd speckle in extracellular, in the cell Then accumulate in neuron and astrocyte.AGEs is combined with its receptor RAGE, can activate oxidative stress, causes a series of Damage, and the finally transcription factor sensitive to redox reaction such as activation such as NF- κ B, lead to the rise of cytokine, such as IL- 1 β and TNF-α.The protective effect to the cognitive impairment that AGE induces for the good wine soup of this experimental evaluation, including Behavior test and latent Mechanism probe into.

3rd, the related introduction of good wine soup

TCM Treatment of Diabetes is with a history of thousands of years, LIUWEI DIHUANG WAN, BUZHONG YIQI TANG, XIAOYAO POWDER, good wine soup, The Chinese medicine compound such as compound Salviae Miltiorrhizae, Herba Dendrobii mixture play the role of prevention, treatment diabetes or mitigate chronic complicating diseases.Wherein good wine soup Come from Zhang Xichun《Records of Tradition Chinese and Western Medicine in Combination》, it is that Chinese traditional treatment is quenched one's thirst recipe, record in former book and " control and quench one's thirst.Quench one's thirst, i.e. west Cure so-called diabetes, the sweet thing of diet.”

The present invention is on the basis of good wine soup, there is provided a kind of pharmaceutical composition and preparation method thereof, and this medicine is in preventing and treating T2DM and improve cognitive function aspect, determined curative effect, the medicine of the present invention not only can improve the symptom of diabetes, also can improve pancreas Insulin resistance, and have the cognitive function effect that has some improvement.

Content of the invention

It is an object of the invention to provide a kind of pharmaceutical composition treating diabetes mellitus encephalopathy.

It is a further object of the present invention to provide the preparation method of this pharmaceutical composition.

The purpose of the present invention is achieved in the following ways.

A kind of pharmaceutical composition treating diabetes mellitus encephalopathy is to be made up of the raw material of following weight portion：Rhizoma Dioscoreae 30 weight Part, in the Radix Astragali 15 weight portion, the Rhizoma Anemarrhenae 15 weight portion, Radix Puerariae 15 weight portion, Fructus Schisandrae Chinensis 15 weight portion, Radix Trichosanthis 15 weight portion, chicken Golden 10 weight portions.

This pharmaceutical composition is adopted and is prepared with the following method：Pharmaceutical decocting piece is weighed by prescribed dose, with 5～15 times amount 40～ 60% alcohol reflux 3 times, each extraction time 0.5～1.5h, united extraction liquid, reclaim ethanol, concentrate, dry, pulverize, Obtain final product.

This pharmaceutical composition preferably employs following method preparation：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount, 50% alcohol reflux 3 times, each extraction time 1h, united extraction liquid, reclaims ethanol, concentrates, be dried, powder Broken, obtain final product.

This pharmaceutical composition makes hard capsule, tablet, granule according to conventional preparation method in pharmacy.

Described medicine, adopts and detects with the following method：Using high performance liquid chromatography carry out diosgenin containing measurement Fixed：

（1）Chromatographic condition：Using HypersilDs chromatographic column；Mobile phase：Ratio is 20～40：60～80 acetonitrile-water； Detection wavelength：190～210nm；Column temperature：15～25 DEG C；Flow velocity：0.5～1.5mL min^-1；Sample size：5～20 μ L；

（2）Prepared by reference substance solution：Precision weighs the dry diosgenin reference substance to constant weight in right amount, plus methanol dissolving Make reference substance solution；

（3）The preparation of need testing solution：Precision weighs medicine of the present invention, plus methanol, is heated to reflux, extracting solution reflux solvent And be concentrated to dryness, residue is dissolved in water, and is shaken with water saturated n-butyl alcohol and extracts, and merges n-butanol extracting liquid, is washed with ammonia solution Wash, to doing, residue adds methanol dissolving to n-butanol extracting liquid recycling design, shakes up, and filtration takes filtrate, obtains final product need testing solution；

（4）Measure：Precision measures above-mentioned need testing solution, each 5～20 μ L of reference substance solution respectively, injects high-efficient liquid phase color Spectrometer, is detected.

The above-mentioned detection method that using high performance liquid chromatography, diosgenin is carried out with assay, preferred method is such as Under：

（1）Chromatographic condition：Using HypersilDs chromatographic column；Mobile phase：Ratio is 30：70 acetonitrile-water；Detection ripple Long：200nm；Column temperature：20℃；Flow velocity：1.0mL·min^-1；Sample size：10μL；

（2）Prepared by reference substance solution：It is appropriate to the diosgenin reference substance of constant weight that precision weighs 80 DEG C of dryings, plus methanol The reference substance solution that every 1g contains 0.2mg is made in dissolving；

（3）The preparation of need testing solution：Precision weighs medicine 10mL of the present invention, plus methanol 40mL, is heated to reflux 4h, extracts Liquid reflux solvent is simultaneously concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, close And n-butanol extracting liquid, washed with ammonia solution 3 times, each 15mL, to doing, it is molten that residue adds methanol to n-butanol extracting liquid recycling design Solve and be transferred in 10mL volumetric flask, plus methanol, to scale, shakes up, filtration, take filtrate, obtain final product need testing solution；

（4）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected.

Described medicine carries out assay using gas chromatography to cuparene, thujopsene：

（1）Chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary column；Carrier gas：N₂, 0.5～1.5mL mL^-1；Hydrogen Gas, 30～50mL mL^-1；Air, 300～500mL min^-1；Split ratio：5～15：1；Injector temperature：240～260 DEG C, Detector temperature：290～310 DEG C；Temperature programming：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；

（2）The preparation of inner mark solution：Take Ketohexamethylene appropriate, plus anhydrous alcohol solution dilute and make solution, shake up, as Inner mark solution；

（3）The preparation of need testing solution：Precision measures this product, puts in volumetric flask, plus dehydrated alcohol is diluted to scale, then essence Close measure, put in volumetric flask, accurate add inner mark solution, plus dehydrated alcohol is diluted to scale, shakes up；

（4）The preparation of reference substance solution：Precision weighs cuparene reference substance, thujopsene reference substance in right amount, plus anhydrous Ethanol dissolves and dilutes the reference substance stock solution made containing cuparene and thujopsene, standby；

（5）Measure：Precision measures above-mentioned need testing solution, each 5～20 μ L of reference substance solution respectively, injects gas chromatogram Instrument, is detected.

Above-mentioned cuparene is carried out using gas chromatography, thujopsene carries out assay, it is preferred to use following method：

（1）Chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary column；Carrier gas：N₂, 1.0mL mL^-1；Hydrogen, 40mL·mL^-1；Air, 400mL min^-1；Split ratio：10：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Program Heat up：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；

（2）The preparation of inner mark solution：Take Ketohexamethylene appropriate, plus anhydrous alcohol solution dilute and make every 1mL and contain 12.5mg Solution, shake up, as inner mark solution；

（3）The preparation of need testing solution：Precision measures this product 1.0mL, puts in 10mL volumetric flask, plus dehydrated alcohol is diluted to Scale, then precision measures 1.0mL, puts in 10mL volumetric flask, accurate add inner mark solution 1.0mL, plus dehydrated alcohol is diluted to quarter Degree, shakes up；

（4）The preparation of reference substance solution：Precision weighs cuparene reference substance, thujopsene reference substance in right amount, plus anhydrous Ethanol dissolves and dilutes makes the mL of 0.301mg containing cuparene^-1And thujopsene 0.901mg mL^-1Reference substance deposit molten Liquid, standby；

（5）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects gas chromatograph, enters Row detection.

Application in preparation treatment diabetes mellitus encephalopathy medicine for the described pharmaceutical composition.

Application in preparation treatment diabetic foot and asthmatic medicament for the described pharmaceutical composition.

In pharmaceutical composition of the present invention, the Ji Yuan of crude drug is as follows：

Rhizoma Dioscoreae：For Dioscoreaceae yam Rhizoma DioscoreaeDioscorea opposita Thunb. [D.batatas Decne.]Tuber.

The Radix Astragali：For pulse family Astragalus Radix AstagaliAstragalus membranaceus Bunge var. Mongholius(Bunge)P.K.HsiaoAnd Radix AstragaliA. Membranaceus (Fisch.) BungeRoot.

The Rhizoma Anemarrhenae：For the Liliaceae Rhizoma Anemarrhenae platymiscium Rhizoma AnemarrhenaeAne-marrhena asphodeloides BurgeRhizome.

Radix Puerariae：For pulse family Pueraria Herba Gelsemii ElegantisPueraria lobata (Willd.) Ohwi [P.thunbergiana (Sieb. et Zucc.) Benth.;P.hirsuta (Thunb.) Schneid.]Or Radix Puerariae rattanP. Thomsonii Benth.Tuber.

Fructus Schisandrae Chinensis：For Schisandraceae schisandra plant Fructus Schisandrae ChinensisSchisandra chinensis (Turca.) Baill.[Kadsura chinensis Turca.]Or schisandra chinensisS.sphenanthera Rehd. et Wils.Fruit Real.

Radix Trichosanthis：For Cucurbitaceae Trichosanthes Fructus TrichosanthissTrichosanthes kiriowii MaximAnd China's Fructus Trichosanthiss Root.

Endothelium Corneum Gigeriae Galli：For Phasianidae Phasianus animal family chickenGallus gallus domesticus BrissonGizzard tunica intima.

Above-mentioned medical material is all purchased from medical material and is purchased from Xianyang continuous feast medicine supermarket.

Verify the technique effect of the present invention by following experimentation：

Experiment one：Pharmaceutical composition of the present invention is to diabetes mellitus encephalopathy rat model neuroprotective and Mechanism Study

1. material

1.1 medicines and reagent

（1）Medicine

Medicine of the present invention：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 g, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, is returned with 10 times amount, 8 times amount, 6 times amount 50% ethanol respectively Stream extracts 3 times, each extraction time 1h, united extraction liquid, reclaims ethanol, concentrates, dry, pulverize, obtain final product.

Drugs compared A：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount water reflux, extract, 3 times, each extraction time 1h, united extraction liquid, concentrate, dry, pulverize, obtain final product.

Drugs compared B：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 30% alcohol reflux Extract 3 times, each extraction time 1h, united extraction liquid, reclaim ethanol, concentrate, dry, pulverize, obtain final product.

Drugs compared C：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 70% alcohol reflux Extract 3 times, each extraction time 1h, united extraction liquid, reclaim ethanol, concentrate, dry, pulverize, obtain final product.

Drugs compared D：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：(1) Radix Astragali of described weight, Radix Puerariae, Fructus Schisandrae Chinensis Chinese medicine coarse powder are taken, with 70% ethanol of 5 times of weight Heating and refluxing extraction 2 times, 1 hour every time, united extraction liquid, filtration, filtrate recycling ethanol is to no alcohol taste, standby；(2) described in taking The Rhizoma Dioscoreae of weight, the Rhizoma Anemarrhenae, Radix Trichosanthis, plus 7 times of weight water decoct 2 times, 1 hour every time, united extraction liquid, filtration, filtrate decompression Being concentrated into the clear paste that relative density is 1.10-1.20 (room temperature), cooling, plus ethanol makes alcohol content reach 70%, stirs evenly, standing 12 is little When, filtration, filtrate recycling ethanol is to no alcohol taste, standby；(3) take (1) and (2) gained filtrate to merge, stir evenly, powder after vacuum drying It is broken into 80～100 mesh fine powders, mix with the Endothelium Corneum Gigeriae Galli fine powder being ground into 100 mesh, obtain final product.

Drugs compared E：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：First, prepare medical material：Weigh the 15 g Radixs Astragali, the 15 g Rhizoma Anemarrhenaes, 30g Rhizoma Dioscoreae, 10g Endothelium Corneum Gigeriae Galli, 15g first Radix Puerariae, 15g Fructus Schisandrae Chinensis and 15g Radix Trichosanthis, that is, obtain treating decocting herbs；2nd, decoct：1. first to treat in decocting herbs add Deionized water, and decoct 1～2h, after then adopting filter method to decoct first time decocting liquid with first time, medical material separates；2. to Step 2 1. in first time of being isolated to decoct after add deionized water in medical material, and decoct 1～2h, then using filtering After second decocting liquid is decocted by method with second, medical material separates；3. to step 2 2. in be isolated to second decoction Add deionized water in medical material afterwards, and decoct 1～2h, then adopt filter method to remove medical material after decoction, and pan-fried by obtain Liquid is merged with first time decocting liquid and second decocting liquid, obtains pending decocting liquid；3rd, finished product：By pending decocting liquid temperature be 70 DEG C～90 DEG C under conditions of be concentrated into relative density 1.04g/cm³～1.12g/cm³, obtain just clear paste, by first clear paste in centrifugation speed Spend for being centrifuged 8min～12min under 3500r/min～4500r/min, be then 70 DEG C by separating the supernatant obtaining in temperature It is concentrated into relative density 1.02g/cm under conditions of～90 DEG C³～1.10g/cm³, obtain clear paste, finally adopt lyophilization skill Art by clear paste sublimation drying 35h～45h altogether, that is, obtains good wine soup lyophilized oral formulations.

Streptozotocin（STZ）, purchased from Sigma company；

Piracetam（Piracetam）, Hua Yuan Anhui, Shanghai Ren Ji pharmaceutical Co. Ltd, Chinese medicines quasi-word H34020741；

Huperzine-A Tablets, Tests for Uniformity, Tailong Pharmaceutical Co., Ltd., Henan, Chinese medicines quasi-word H10940156；

Metformin Hydrochloride Tablets, one one-tenth of Liaoning pharmaceutcal corporation, Ltd, Chinese medicines quasi-word H21024375；

（2）Reagent

AchE（ELISA）Test kit, AGEs（ELISA）Test kit and ChAT（ELISA）Test kit, above test kit is all purchased In Ai Laisa biotechnology（Shanghai）Company limited；RAGE and NF- κ B immunohistochemical kit, purchased from Wuhan doctor's moral biology work Journey company limited.

1.2 instrument

Morris water maze, Jiliang Software Sci-Tech Co., Ltd., Shanghai；BT224S electronic balance, Sai Duolisi scientific instrument Company limited；BI-2000 medical image analysis system, Chengdu TME Technology Co., Ltd.；Eon all-wave length microplate reader, the U.S. BioTek；Thermo-86 degree cryogenic refrigerator；Micropipettor, Qiujing Bio-Chemical Reagent Instrument Co., Ltd., Shanghai, TGL-16M platform Formula High speed refrigerated centrifuge, Hunan Xiang Yi Laboratory Instruments development corporation, Ltd.；The stable blood glucose meter of three promises, three promise bio-sensing stocks Part company limited.

1.3 animal

Male SD rat, cleaning grade, body weight 250～300g, provided by The Fourth Military Medical University's Experimental Animal Center, the quality certification Number：SCXK（Army）2012-006；Feeding environment：ShanXi Chinese Medicine Academy herbal pharmacology laboratory, animal sub-cage rearing, keep 12h Circadian rhythm, room temperature（22±1）DEG C, free water is ingested.

2. method

2.1 modelings and packet

Male SD rat adaptability is fed 1 week, and in addition to blank group gives normal feedstuff, remaining rat gives to make high fat height by oneself Sugared feedstuff（Normal feedstuff 59%, Adeps Sus domestica 18%, sucrose 20%, egg yolk 3%）After feeding 50 days, disposable celiac injects STZ 30mg/ kg（It is dissolved in the concentration being configured to 1% in the citrate buffer solution of 0.1mol/L, pH4.2 before use）, after 72 hours, tail vein is adopted Blood blood glucose meter surveys blood glucose, and with blood sugar concentration >=16.7mmol/L for modeling success, after model success, rat is randomly divided into model Group, piracetam group（0.48g/kg）, huperzine A group（40μg/kg）, metformin hydrochloride group（0.15g/kg）, medicine of the present invention Group（11.5g/kg）, drugs compared A group（11.5g/kg）, drugs compared B group（11.5g/kg）, drugs compared C group（11.5g/ kg）, drugs compared D group（11.5g/kg）, drugs compared E group（11.5g/kg）, latter second day beginning gastric infusion of model packet, The capacity normal saline such as blank group and the equal gavage of model group, 1 time/d, is administered 30d, animal ad lib water inlet during administration, not Using insulin and other hypoglycemic medicine.

2.2 Morris water maze laboratories

Carry out the mensure of Rats With Memory function using Morris water maze：1. orientation navigation experiment：Fill in last After stomach, 1h starts.Every group takes 10 rats at random, every natural gift upper and lower two time periods of noon, and each time period trains 4 times, every time Respectively from 4 different labelling points（4 quadrants are evenly distributed）, rat is put in water towards pool wall, seeks in record 90s Levelling required time（Escape latency）And detection range.At least it is spaced 30min every time, training lasts 4d altogether.Position of platform Keep constant in the training of a collection of rat and test process.If rat finds platform in 90s not yet, it is replaced in Platform, removes labyrinth after 20s, is designated as 90s its incubation period.2. space exploration experiment：Remove platform within 5th day, optional 1 place of entry Rat is put in water towards pool wall, record rat in 90s across the traversing times of virtual platform and rat in target quadrant （Former platform quadrant）Swimming time.

2.3 draw materials and sample disposal

After water maze laboratory terminates, after water 12h is can't help in fasting, every group in addition to 4 rats of random residual remaining rat claim Weight, tail vein blood is put to death after surveying blood glucose, takes brain rapidly, is placed on rinsing in ice-cold normal saline, filter paper suck dry moisture, separates sea Horse is organized, and weighs, and is homogenized with homogenizer, makes the tissue homogenate that w/v is 10% under ice bath, centrifugation（3500r/ min）10 min, take supernatant strictly to illustrate to measure AchE, AGES and ChAT indices by test kit.Every group by remaining 4 Rat, after anesthesia, 4% paraformaldehyde 0.1mol L^-1Phosphate buffer（pH 7.4）Intracardiac perfusion is fixed, to be fixed fully after, Open cranium and take brain, Coronal takes optic chiasma caudad 3-4mm piece of tissue, after the identical fixative of input fixes 1 week in 4 DEG C, conventional stone Wax embeds, and coronal section carries out RAGE and NF- κ B immunohistochemical staining；Each group is chosen Hippocampus position and is not repeated the visual field for totally 8, With BI-2000 medical image analysis system, SABC slice, thin piece is analyzed, statistical average gray value.

2.4 statistical procedures

All experimental datas are used± s represents, compare between group all even variance of normal distribution neat when adopt single factor test variance Analysis, normal distribution is uneven or non parametric testss during heterogeneity of variance, with SPSS 13.0 software statistics.

3. result

The impact of 3.1 pairs of diabetes mellitus encephalopathy rat model water maze laboratory achievements

Between the 1st day each group of water maze laboratory, incubation period, exploration, apart from there was no significant difference substantially, are compared with blank group, from From 2nd day, model group escape latency, the notable prolongation of exploration distance（P<0.01 or P<0.05）, target quadrant（II quadrant）Trip The swimming time substantially shortens（p<0.01）, platform traversing times significantly reduce（p<0.01）；Compared with model group, the 3rd day, piracetam Organize incubation period, explore the obvious shortening of distance（P<0.05）, medicine group exploration distance of the present invention is obvious to be shortened（P<0.05）, the 4th day, Piracetam group, huperzine A group, metformin hydrochloride group, medicine group of the present invention substantially shorten incubation period（P<0.05 or P< 0.01）, huperzine A group, metformin hydrochloride group, medicine group of the present invention explore the obvious shortening of distance（P<0.05）, hydrochloride Biguanide group, medicine group target quadrant of the present invention（II quadrant）Swimming time be obviously prolonged（P<0.05）, piracetam group, huperzine First group, metformin hydrochloride group, medicine group of the present invention, drugs compared A group, drugs compared B group, drugs compared C group, drugs compared D group, drugs compared E group platform traversing times increase（P<0.05 or P<0.01）（It is shown in Table 1-1 to table 1-3）

Table 1-1 preclinical impact on diabetes mellitus encephalopathy rat model water maze test（ ±s,n=10）

Note：Compare with blank group,^△P<0.05,^△△P<0.01；Compare with model group,^▲P<0.05,^▲▲P<0.01

Table 1-2 explores the impact of distance to diabetic model rats water maze test（ ±s,n=10）

Note：Compare with blank group,^△P<0.05,^△△P<0.01；Compare with model group,^▲P<0.05

The impact to diabetes mellitus encephalopathy rat water maze test target quadrant time and traversing times for the table 1-3（ ±s,n= 10）

Note：Compare with blank group,^△P<0.05,^△△P<0.01；Compare with model group,^▲P<0.05,^▲▲P<0.01

3.2 pairs of diabetes mellitus encephalopathy rat model blood glucose, body weight, the impacts of AchE, ChAT and AGEs

Compare with blank group, model group blood glucose, AchE, AGEs level dramatically increase（P<0.01）, body weight, ChAT level show Write and reduce（P<0.01）；Compare with model group, metformin hydrochloride group, medicine group blood glucose value of the present invention significantly reduce（P<0.05 Or P<0.01）, metformin hydrochloride group, medicine group rat body weight value significance of the present invention raise（P<0.05）, piracetam group, stone China fir alkali group, medicine group AchE, AGEs level of the present invention significantly reduce（P<0.05 or P<0.01）, piracetam group, huperzine A Group, metformin hydrochloride group, medicine group ChAT level of the present invention dramatically increase（P<0.05 or P<0.01）, it is shown in Table 1-4 and table 1- 5.

Table 1-4 is to diabetes mellitus encephalopathy rat model blood glucose, Body Mass Index test result（ , n=10）

Note：Compare with blank group,^△△P<0.01；Compare with model group,^▲P<0.05,^▲▲P<0.01

Table 1-5 is to diabetes mellitus encephalopathy rat model AchE, ChAT and AGEs index test result（ , n=10）

Note：Compare with blank group,^△△P<0.01；Compare with model group,^▲P<0.05,^▲▲P<0.01

The impact of 3.3 pairs of each group Rat hippocampus pathological changes

Mainly in endoglin expression, immunohistochemical staining is positive to be in brown color to RAGE, and immunohistochemical staining shows, with blank Group compares, model group rats Hippocampus DG area RAGE immuning positive cell number showed increased, and coloring is substantially deepened, and average gray value is bright Aobvious reduction（P<0.01）；Compare with model group, piracetam group, huperzine A group, metformin hydrochloride group, medicine group of the present invention are big Mus Hippocampus DG area RAGE immuning positive cell number substantially reduces, and coloring is shallower, and average gray value is significantly raised（P<0.01）, it is shown in Table 1-6.Nuclear factor NF- κ B expresses in neuron plasma, and immunohistochemical staining shows, compares with blank group, model group rats Hippocampus DG area's granular cell layer and cortical neuron NF- κ B immunoreation positive cell many and color depth, average gray value is obvious Reduce（P<0.01）；Compare with model group, piracetam group, huperzine A group, metformin hydrochloride group, medicine group rat of the present invention Hippocampus DG area's granular cell layer and cortical neuron NF- κ B immunoreation positive cell are less, and average gray value is significantly raised（P< 0.05 or P<0.01）It is shown in Table 1-6.

Table 1-6 expresses the impact of average gray value to RAGE, NF- κ B in diabetes mellitus encephalopathy rat model hippocampal tissue（）

Note：Compare with sham operated rats,^△△P<0.01；Compare with model group,^▲P<0.05,^▲▲P<0.01

4. conclusion and discussion

4.1 conclusion

This experimental studies results shows, medicine of the present invention can obviously improve the cognitive disorder of diabetes mellitus encephalopathy rat model, There is certain cerebral protection, its mechanism of action is by blood sugar lowering, improves cholinergic nerve function, improves diabetes cerebral tissue The approach such as middle AGEs- RAGE-NF- κ B path are improving diabetic model rats hippocampal tissue pathological changes.

This experiment carries out the checking of rat cognitive model and the assessment of nerve understanding treatment feasibility by water maze, Rat escape the time, grope distance shorter, traversing times are more, the target quadrant activity time is longer, and learning and memory in rats is described Ability is stronger.This research water maze laboratory data display, medicine of the present invention can reduce rat escape latency and grope distance, increase Plus traversing times and target quadrant activity time, show that medicine of the present invention can obviously improve the study of diabetes mellitus encephalopathy rat model Memory ability, its effect is substantially better than drugs compared A, drugs compared B, drugs compared C, drugs compared D, drugs compared E.

Hyperglycemia leads to the deposition of AGEs in cerebral tissue, and the focusing energy of AGEs causes a lot of toxic reactions, and it is to cell Toxic action be by with its specific receptor AGER（RAGE）Be implemented in combination in.RAGE is cell A member in surface molecular immunoglobulin superfamily, is initially that the bind receptor as AGEs is found, RAGE and glycosuria Sick complication has substantial connection.AGEs is combined rear interaction with its cell surface receptor RAGE, can activate NF- κ B mediation Path, Hofmann etc. confirms the activation of NF- κ B first in diabeticss, and the NF- κ B of activation can stimulate numerous target bases Because of expression, such as tissue factor（TF）, vascular cell slit attached molecule-l（VCAM-l）, the expression of RAGE etc., promote diabetes to enter one Step deteriorates, and increases the further degradation degeneration of cerebral cortex, leads to neurodegeneration.

Test result indicate that, RAGE-NF- κ B path can be activated in rat hippocampus DG area injection AGE, infringement rat is in water fan The cognitive performance of palace experiment.After Drug therapy of the present invention, the cognitive competence of rat model is improved.Blood glucose, AchE, AGEs Level significantly reduces, and this experimental result illustrates that medicine of the present invention can improve the cognitive impairment of AGEs induction, and its effect is substantially better than Drugs compared A, drugs compared B, drugs compared C, drugs compared D, drugs compared E.

Experiment two：Medicine high performance liquid chromatography quality determining method research of the present invention

1 instrument and reagent

1.1 instrument

High performance liquid chromatograph（Agilent110 high performance liquid chromatograph and work station, G1311Aquat pump, G1314 is purple External detector）.

1.2 reagent

Diosgenin reference substance（Chinese pharmaceutical biological product examines and determine academy）；Medicine of the present invention；Chinese crude drug（Xianyang sky Its happy medicine supermarket provides）；Methanol（Chromatograph alcohol, Shanghai biochemistry work auxiliary reagent factory）；Other reagent are that analysis is pure.

2 methods and result

2.1 prescription

Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 g, Endothelium Corneum Gigeriae Galli 10 g.

2.2 preparation

Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount, 50% alcohol reflux 3 times, often Secondary extraction time 1h, united extraction liquid, reclaims ethanol, concentrates, dry, pulverize, obtain final product.

The assay of 2.3 diosgenins

2.3.1 HPLC chromatogram condition

Using HypersilDs（4.0mm × 125mm, 5 μm）Chromatographic column；Mobile phase：Ratio is 30：70 acetonitrile-water；Inspection Survey wavelength：200nm；Column temperature：20℃；Flow velocity：1.0mL·min^-1；Sample size：10μL；Under this chromatographic condition, reference substance and sample Product chromatographic peak is good, and no Rhizoma Dioscoreae negative control is noiseless to measuring.

2.3.2 the preparation of reference substance solution

Precision weighs that 80 DEG C of dryings are appropriate to the diosgenin reference substance of constant weight, plus methanol is made every 1mL and contained 0.2mg's Solution.

2.3.3 the preparation of need testing solution and negative controls

Precision weighs medicine 10g of the present invention, plus methanol 40mL, is heated to reflux 4h, and extracting solution reflux solvent is simultaneously concentrated to dryness, Residue add water 10mL dissolving, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, merge n-butanol extracting liquid, tried with ammonia Liquid washs 3 times, each 15mL, and to doing, residue adds methanol and dissolves and be transferred to 10mL volumetric flask n-butanol extracting liquid recycling design In, plus methanol, to scale, shakes up, filtration, takes filtrate to obtain final product；The another negative controls in the preparation of prescription ratio without Rhizoma Dioscoreae, with Method makes negative controls.

2.3.4 the drafting of standard curve

It is appropriate to the diosgenin reference substance of constant weight that precision weighs 80 DEG C of dryings, makes 10.4,20.8 with methanol, 41.6,83.2,166.4 μ gmL^-1The solution of series concentration, respectively precision measure each 10 μ L of above-mentioned 5 kinds of strength solution, injection is efficiently Chromatograph of liquid is measured.

Linear regression is carried out with peak area ratio and concentration, obtaining regression equation is：A=21.2763C-0.1391, r= 0.9999.Show diosgenin in 10.4～166.4 μ gmL^-1In good linear relationship in concentration range.

2.3.5 stability test

Accurate absorption need testing solution 10 μ L, respectively at 0,1,2,4,8h sample introduction, and calculates Determination of Diosgenin.Result In 8h, RSD is 0.45%（n=5）.Show that sample solution is stable in 8h.

2.3.6 replica test

By 5 parts of need testing solution preparation method parallel processing sample, measure Determination of Diosgenin in accordance with the law and calculate.Result Recording diosgenin average content is 0.12mg mL^-1, RSD is 1.3%.

2.3.7 Precision Experiment

Accurate absorption diosgenin reference substance solution, repeats sample introduction 5 times, measures peak area in accordance with the law.Result RSD is 0.23%（n=5）.Show that precision is preferable.

2.3.8 response rate experiment

Precision weighs 6 parts of the sample of the same lot number of known Determination of Diosgenin, accurate respectively by high, medium and low concentration Add appropriate diosgenin reference substance solution, operate by under sample determination item, measure in accordance with the law, calculate the response rate.Result is put down All the response rate is 0.45% for 100.3%, RSD（n=5）.

2.3.9 sample size measures

Measure reference substance solution and appropriate need testing solution respectively, with microporous filter membrane filtration, each sample introduction 10 μ L, by above-mentioned color Spectral condition measures 3 batch samples, parallel assay 5 times.By external standard method with the content of calculated by peak area need testing solution diosgenin. This product should be containing diosgenin and indicates the 95%～105% of content, is contained in terms of diosgenin by every 1g this product, must not be less than 0.12mg.3 batch sample contents are respectively 100.8%（RSD=1.2%）, 101.7%（RSD=1.3%）, 99.2%（RSD=1.1%）.

Experiment three：Medicine gas chromatography Simultaneous Determination cuparene of the present invention, the content of thujopsene

1 instrument and reagent

Agilent7890N gas chromatograph：Fid detector, A.01.12.1 chromatographic work station；SGH-300 high-purity hydrogen Generator（Beijing Orient elite science and technology garden Science and Technology Ltd.）；Chromatographic column fused-silica capillary column（30m × 0.25mm, 0.25μm）；Prunus mume (sieb.) sieb.et zucc. Teller-support benefit 100,000/electronic analytical balance；Cuparene reference substance（Content 99.9%, China's food Product drug assay academy）；Thujopsene reference substance（Content 99.9%, National Institute for Food and Drugs Control）；Medicine of the present invention （Prepare with reference to the embodiment 1 in description of the invention specific embodiment）, reagent：Ketohexamethylene, dehydrated alcohol is chromatographically pure.

2 chromatographic conditions

Chromatographic column：ZB-WAX fused-silica capillary column（30m × 0.25mm, 0.25 μm）；Carrier gas：N₂, 1.0mL mL^-1； Hydrogen, 40mL mL^-1；Air, 400mL min^-1；Split ratio：10：1；Injector temperature：250 DEG C, detector temperature 300 ℃；Temperature programming：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard Method.

3 test methods and result

The preparation of 3.1 inner mark solutions

Take Ketohexamethylene appropriate, plus anhydrous alcohol solution dilute and make the solution that every 1g contains 12.5mg, shake up, as internal standard Solution.

The preparation of 3.2 need testing solutions

Precision measures this product 1.0g, puts in 10mL volumetric flask, plus dehydrated alcohol is diluted to scale, then precision measures 1.0mL, Put in 10mL volumetric flask, precision adds inner mark solution 1.0mL, plus dehydrated alcohol is diluted to scale, shakes up.

The preparation of 3.3 reference substance stock solutions

It is appropriate that precision weighs cuparene reference substance, thujopsene reference substance, plus anhydrous alcohol solution dilute to make and contain Cuparene 0.301mg mL^-1And thujopsene 0.901mg mL^-1Reference substance stock solution, standby；

The preparation of 3.4 negative control solutions

Take by the blank solution not adding cuparene and thujopsene in prescription, by preparation method under " 3.2 " item, make the moon Property contrast solution.

The investigation of 3.5 linear relationships

Respectively precision pipette 0.2,0.5,1.0,1.5,3.5mL reference substance storing solution in 10mL volumetric flask, plus internal standard is molten Liquid 1.0mL, plus dehydrated alcohol is diluted to scale, shakes up, and as reference substance solution, takes 1 μ L sample introduction respectively, records chromatogram, with Cuparene, thujopsene and interior target peak area ratio are vertical coordinate（Y）, concentration（C）For abscissa（X）, draw mark respectively Directrix curve, obtains regression equation and is respectively：Y（Cuparene）=1.1148X-0.0016, R²=0.9999, cuparene concentration exists 0.146～2.555mg mL^-1In the range of, linear relationship is good；Y（Thujopsene）=1.1347X+0.0035, R²=0.9998, Thujopsene concentration is in 0.198～3.465mg mL^-1In the range of, linear relationship is good.

3.6 precision test

Cuparene concentration is taken to be 0.230mg mL^-1It is 0.290mg mL with thujopsene concentration^-1Reference substance molten Liquid, repeats sample introduction 6 times, records peak area, calculates 2 kinds of compositions and interior target peak area ratio respectively（A/A internal standard）, flower Cacumen Platycladi Alkene, the RSD of thujopsene are respectively 0.24% and 1.2%（n=6）.

3.7 replica test

Take same batch sample, by the method replication under sample determination item 6 times, result cuparene, thujopsene RSD is respectively 0.36%, 1.8%（n=6）.

3.8 stability test

Take same batch sample solution, place 0 respectively at room temperature, 2,4,6,8, measure after 12h, result press cuparene, The RSD of thujopsene is respectively 0.38%, 1.7%, illustrates that sample solution measures in 12h, result is stable.

3.9 average recovery tests

Take 9 parts of the sample solution of known content, and add suitable basic, normal, high reference substance solution, survey by sample determination method Determine cuparene, thujopsene content, calculate the response rate respectively, the results are shown in Table 3-1.

Table 3-1 determination of recovery rates result（N=9, %）

Result shows, the response rate of this method preferably, the response rate of cuparene respectively 99.4%～100.2%, arhat Between 98.1%～100.7%, relative standard deviation is respectively 0.28% and 0.86% to the response rate of cupressene, and this assay method can be full The assay of cuparene and thujopsene in sufficient medicine of the present invention.

3.10 quantitative limit and test limit

To determine quantitative limit and the test limit of this research using " signal to noise ratio method ", line taking standard solution is appropriate, to adopt and add Progressively dilution method is diluted dehydrated alcohol, when sample introduction concentration is 6.27,9.90 μ g mL^-1When, take 1 μ L sample introduction, continuous sample introduction 3 Secondary, obtain cuparene, internal standard, thujopsene signal to noise ratio meansigma methodss respectively close to 10.0, can be with this concentration as quantitative limit；Continue Continuous dilution sample introduction, when sample introduction concentration is 1.044,1.65 μ g mL^-1, continuous sample introduction 3 times, obtain cuparene, internal standard, Thujopsis dolabrata Alkene signal to noise ratio meansigma methodss, can be with this concentration as test limit close to 3.0.

3.11 serviceability test

Investigate through different chromatographic columns and stability of solution is investigated, and column temperature, injector temperature and detector temperature investigation, Show this method good tolerance it is adaptable in medicine of the present invention two components assay.

3.11.1 the impact of chromatographic column

From the chromatographic column of 3 different commercial specifications, measure the content of same batch sample, calculate the RSD% of content value respectively For 1.3,1.7,1.6.Result shows, sample measures content, cuparene, thujopsene and internal standard with different PEG chromatographic columns All can efficiently separate, illustration method good tolerance.

3.11.2 the impact of column temperature

Column temperature is mainly on detached impact affects the appearance time of main peak, and temperature is higher, and main peak appearance time is shorter, When first stage is 80 DEG C, cuparene main peak cuparene and impurity peaks can guarantee that baseline separation, sieve during 120 DEG C of second stage Chinese cupressene main peak and impurity peaks can guarantee that baseline separation, and the RSD of content at each temperature is less than 2.0%.

3.11.3 the impact of injector temperature

When injector temperature is higher than column temperature, cuparene and impurity peaks ensure that baseline separation, thujopsene and impurity Separate good, and the RSD of content at each temperature is less than 2.0%.

3.11.4 the impact of detector temperature

When detector temperature is higher than injector temperature, cuparene and impurity peaks ensure that baseline separation, thujopsene Separate well with impurity, and at each temperature content RSD be less than 2.0%.

3.12 sample size measurement result

Through Method validation, this content assaying method is easy and simple to handle, accuracy is high, favorable reproducibility, can more effectively control Product quality processed.Therefore application the method, to 10 batch samples, carries out assay according to preceding method using internal standard method, result is shown in Table 3-2.

Table 3-2 sample size measurement result

4 discussion

4.1 system suitability test

Under this test gas chromatography system, respectively pipette samples measure with mixed reference substance solution, need testing solution with The each 1 μ L of negative control solution, records chromatogram.2 kinds of components all can preferably be separated with internal standard substance, negative noiseless.This is System adaptability the results are shown in Table 3-3.

Table 3-3 system suitability test

The selection of 4.2 internal standard substances

Once Ketohexamethylene, naphthalene, biphenyl, methyl salicylate etc. were tried out, because sample volatile ingredient is many, result is with Ketohexamethylene Retention time and separating effect are most suitable.

The selection of 4.3 column temperatures

The boiling point difference ratio of cuparene, Ketohexamethylene and thujopsene is larger, when column temperature is low, the retention time of thujopsene Long, when column temperature is high, cuparene can not efficiently separate with impurity, through meeting two kinds of compositions using two sections of temperature-programmed modes While analysis.

The content limit of 4.4 this product

By 10 batch products measurement results, the content limit of tentative this product is：The every 1g of this product must not be less than containing cuparene 0.200mg, must not be less than 0.200mg containing thujopsene.

This method carries out separating and detection to 2 kinds of compositions simultaneously, and method is quick, sensitive, and separating degree is good, specificity is good, energy Efficiently control drug quality.

Implement four：The present invention treats the pharmacodynamic study of asthma

1st, experiment material：

Medicine of the present invention：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 g, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, is returned with 10 times amount, 8 times amount, 6 times amount 50% ethanol respectively Stream extracts 3 times, each extraction time 1h, united extraction liquid, reclaims ethanol, concentrates, dry, pulverize, obtain final product.

Drugs compared A：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount water reflux, extract, 3 times, each extraction time 1h, united extraction liquid, concentrate, dry, pulverize, obtain final product.

Drugs compared B：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 30% alcohol reflux Extract 3 times, each extraction time 1h, united extraction liquid, reclaim ethanol, concentrate, dry, pulverize, obtain final product.

Drugs compared C：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 70% alcohol reflux Extract 3 times, each extraction time 1h, united extraction liquid, reclaim ethanol, concentrate, dry, pulverize, obtain final product.

Drugs compared D：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：(1) Radix Astragali of described weight, Radix Puerariae, Fructus Schisandrae Chinensis Chinese medicine coarse powder are taken, with 70% ethanol of 5 times of weight Heating and refluxing extraction 2 times, 1 hour every time, united extraction liquid, filtration, filtrate recycling ethanol is to no alcohol taste, standby；(2) described in taking The Rhizoma Dioscoreae of weight, the Rhizoma Anemarrhenae, Radix Trichosanthis, plus 7 times of weight water decoct 2 times, 1 hour every time, united extraction liquid, filtration, filtrate decompression Being concentrated into the clear paste that relative density is 1.10-1.20 (room temperature), cooling, plus ethanol makes alcohol content reach 70%, stirs evenly, standing 12 is little When, filtration, filtrate recycling ethanol is to no alcohol taste, standby；(3) take (1) and (2) gained filtrate to merge, stir evenly, powder after vacuum drying It is broken into 80～100 mesh fine powders, mix with the Endothelium Corneum Gigeriae Galli fine powder being ground into 100 mesh, obtain final product.

Drugs compared E：Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 G, Endothelium Corneum Gigeriae Galli 10 g；Preparation method：First, prepare medical material：Weigh the 15 g Radixs Astragali, the 15 g Rhizoma Anemarrhenaes, 30g Rhizoma Dioscoreae, 10g Endothelium Corneum Gigeriae Galli, 15g first Radix Puerariae, 15g Fructus Schisandrae Chinensis and 15g Radix Trichosanthis, that is, obtain treating decocting herbs；2nd, decoct：1. first to treat in decocting herbs add Deionized water, and decoct 1～2h, after then adopting filter method to decoct first time decocting liquid with first time, medical material separates；2. to Step 2 1. in first time of being isolated to decoct after add deionized water in medical material, and decoct 1～2h, then using filtering After second decocting liquid is decocted by method with second, medical material separates；3. to step 2 2. in be isolated to second decoction Add deionized water in medical material afterwards, and decoct 1～2h, then adopt filter method to remove medical material after decoction, and pan-fried by obtain Liquid is merged with first time decocting liquid and second decocting liquid, obtains pending decocting liquid；3rd, finished product：By pending decocting liquid temperature be 70 DEG C～90 DEG C under conditions of be concentrated into relative density 1.04g/cm³～1.12g/cm³, obtain just clear paste, by first clear paste in centrifugation speed Spend for being centrifuged 8min～12min under 3500r/min～4500r/min, be then 70 DEG C by separating the supernatant obtaining in temperature It is concentrated into relative density 1.02g/cm under conditions of～90 DEG C³～1.10g/cm³, obtain clear paste, finally adopt lyophilization skill Art by clear paste sublimation drying 35h～45h altogether, that is, obtains good wine soup lyophilized oral formulations.

Animal：SD healthy rat, male, cleaning grade, body weight（130±10）g.

Primary drug, reagent and instrument：Asmeton capsule（Japanese Sankyo Co., Ltd）OVA（Sigma product）；Chlorination second Phatidylcholine（Shanghai San'aisi Reagent Co., Ltd.）；Histamine phosphate（Shanghai Li Zhu east wind Bioisystech Co., Ltd）；402A surpasses Sound nebulizer（Yuyue Medical Apparatus Co., Ltd., Jiangsu）.

2nd, experimental technique：

Animal model replication：SD rat：99, it is divided into 9 groups immediately, i.e. Normal group, model control group, medicine of the present invention Thing group, drugs compared A group, drugs compared B group, drugs compared C group, drugs compared D group, drugs compared E, Asmeton group.Yu Shi Test the 0th, 7 days, except Normal group gives 1% aluminum hydroxide solution, each group animal is all with freshly prepared sensitization liquid（1mg/mL OVA, is dissolved in 1% aluminum hydroxide solution）Make subcutaneous injection, every Mus take 10 altogether in the both sides metapedes sole of the foot, groin, waist, the back of the body, cervical region Point, every injection 0.05mL, lumbar injection 0.5mL simultaneously, 1mL altogether.In experiment the 14th day, rat is placed in self-control mist Change case（40×30×20cm³) in, with ultrasound atomizer with atomization quantity 0.8mL/min, it is atomized 1%OVA normal saline solution 15min（Normal group is atomized with normal saline）, one time a day, to excite asthma.After continuous agitation 3w, stop exciting 6d, so After excite 1 time, after 24h put to death.

The preparation of medicine gavage solution of the present invention：Take medicine 15g of the present invention to add in 1500mL water, be made into suspension water-soluble Liquid.

The preparation of drugs compared A gavage solution：Take drugs compared A15g to add in 1500mL water, be made into suspension aqueous solution.

The preparation of drugs compared B gavage solution：Take drugs compared B15g to add in 1500mL water, be made into suspension aqueous solution.

The preparation of drugs compared C gavage solution：Take drugs compared C15g to add in 1500mL water, be made into suspension aqueous solution.

The preparation of drugs compared D gavage solution：Take drugs compared D15g to add in 1500mL water, be made into suspension aqueous solution.

The preparation of drugs compared E gavage solution：Take drugs compared D15g to add in 1500mL water, be made into suspension aqueous solution.

Medication：In experiment, the 14th day starts gastric infusion, and one time a day, until putting to death first 1 day.Medicine group of the present invention, Drugs compared A group, drugs compared B group, drugs compared C group, drugs compared D group, drugs compared E group, press 10mL/kg body weight respectively Gavage give train decocting liquid 26g crude drug/（kg·d）.Asmeton group：Give Asmeton aqueous solution by 10mL/kg body weight gavage 25mg/（kg·d）.Normal group and model control group：Give normal saline.

Non-specific brinchial provocation test：Carry out within 27th, 41 days respectively at experiment.2% acecoline that will prepare With 0.2% histamine phosphate mixed in equal amounts, as exciting liquid, with nebulizer（When atomization quantity excites with OVA）After atomization exciting liquid 60s Stop, observing the reaction of animal in 10min, the asthmatic latent period of record animal.Asthmatic latent period, more than 10min person, is just designated as 10min.

Specificity brinchial provocation test：Carry out within 41st day in experiment, with 1%OVA exciting liquid Neulized inhalation 15min, root Drawing of severity of symptom record animal according to animal performance breathes heavily result, is divided into 4 grades：No send out author（-）：It is designated as 0 point；Gently Degree（+）：Only scratch where it itches, sneeze, cough person, be designated as 1 point；Moderate（++）：Rapid breathing, ventral breathing, dyspnea occur Person, is designated as 2 points；Severe（+++）：Breathing is the devil, and motionless, the bradykinesia person that lies prostrate, and is designated as 3 points.

Statistical analysis：Data adopts mean ± standard deviation（±s）Represent, compare between group using homogeneity test of variance and One factor analysis of variance, selects LSD according to homogeneity test of variance result（Variance is neat）Or Tamhane ' s T2（Heterogeneity of variance）, adopt Completed with SPSS 11. 0 for Windows software kit；Sxemiquantitative data adopts Ridit to analyze.

3rd, experimental result：

Ordinary circumstance is observed：Normal group animal excites before and after's general state substantially not change, and breathing steadily, is lived Jump.

Model control group rat excites the rear typical performance to be：Scratch nose, sneeze or cough, rapid breathing, severe patient audible And obvious wheezing or dyspnea with rapid and short breath sound, respiratory rhythm is whole, and sometimes fast and sometimes slow, amplitude increases and uneven, ventral breathing substantially, mouth and nose, Extremity and the slight cyanosis of tail point, activity significantly reduces or prostrate motionless.With the growth of firing time, above-mentioned symptom gradually adds Weight, and body weight starts mitigation, hair is matt, bradykinesia, and respiratory rhythm and amplitude are disorderly.

Medicine group of the present invention has obvious improvement to above-mentioned symptom.

Drugs compared A group, drugs compared B group, drugs compared C group, drugs compared D group, drugs compared E group are to above-mentioned symptom There is improvement, but inconspicuous.

The comparison of each group rat non-specificity bronchus excitation experiment asthmatic latent period：Compare with Normal group, model 2 asthmatic latent period of matched group all substantially shorten（P < 0.01）；Compare with model control group, medicine group of the present invention is drawn for 1st time Breathe heavily and no substantially change incubation period；2nd time asthmatic latent period is all obviously prolonged（P <0.05 or P<0.01）.Drugs compared A group, right No substantially change than medicine B group, drugs compared C group, drugs compared D group, the 1st asthmatic latent period of drugs compared E group, the 2nd time Asthmatic latent period all has prolongation, but effect inconspicuous.

The results are shown in Table 4-1：

The comparison of table 4-1 each group rat non-specificity brinchial provocation test asthmatic latent period（）

In table 4-1, compare with Normal group,^△△P< 0.01；Compare with model control group, * P<0.05, * * P< 0.01.

Each group rat specificity brinchial provocation test draws the comparison breathing heavily classification：Model control group OVA bronchus excite reality Test to draw to breathe heavily classification results and compare with Normal group and have notable difference.Compare with model control group, medicine group of the present invention and A Si Beautiful group is drawn to the 3rd time and breathes heavily result and have remarkable effect.

The results are shown in Table 4-2：

The 41st day specificity brinchial provocation test of table 4-2 each group rat draws the comparison breathing heavily classification（）

From table 4-2, compare with Normal group,^△P< 0.05；Compare with model control group, * P< 0.05.

4th, experiment conclusion

Medicine of the present invention can significantly reduce experimental rat model of asthma sneeze and grab nose number of times, alleviate asthma attack degree, and increase is exhaled Gas peak flow, can treat asthma and allergic asthma.Particularly, the action effect of medicine of the present invention be substantially better than drugs compared A, Drugs compared B, drugs compared C, drugs compared D and drugs compared E, illustrate that the compatibility between medicine taste of Chinese medicine of the present invention is superior, Indispensable, the combination between flavour of a drug creates obvious synergistic function, creates the technique effect that one-plus-one is more than two.

Specific embodiment：

Embodiment 1：

Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 g, Endothelium Corneum Gigeriae Galli 10 g.

Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 50% alcohol reflux 3 Secondary, each extraction time 1h, united extraction liquid, reclaims ethanol, concentrates, dry, pulverize, obtain final product.

Detection method：

1st, assay is carried out to diosgenin using high-efficient liquid spectrum method：

（1）Chromatographic condition：Using HypersilDs chromatographic column；Mobile phase：Ratio is 30：70 acetonitrile-water；Detection ripple Long：200nm；Column temperature：20℃；Flow velocity：1.0mL·min^-1；Sample size：10μL；

（2）Prepared by reference substance solution：It is appropriate to the diosgenin reference substance of constant weight that precision weighs 80 DEG C of dryings, plus methanol The reference substance solution that every 1mL contains 0.2mg is made in dissolving；

（3）The preparation of need testing solution：Precision weighs medicine 10g of the present invention, plus methanol 40mL, is heated to reflux 4h, extracts Liquid reflux solvent is simultaneously concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, close And n-butanol extracting liquid, washed with ammonia solution 3 times, each 15mL, to doing, it is molten that residue adds methanol to n-butanol extracting liquid recycling design Solve and be transferred in 10mL volumetric flask, plus methanol, to scale, shakes up, filtration, take filtrate, obtain final product need testing solution；

（4）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected；

（5）Measurement result：Contained in terms of diosgenin by every 1g this product, 0.12mg must not be less than.

2nd, assay is carried out to cuparene, thujopsene using gas chromatography：

（1）Chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary column；Carrier gas：N₂, 1.0mL mL^-1；Hydrogen, 40mL·mL^-1；Air, 400mL min^-1；Split ratio：10：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Program Heat up：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；

（2）The preparation of inner mark solution：Take Ketohexamethylene appropriate, plus anhydrous alcohol solution dilute and make every 1mL and contain 12.5mg Solution, shake up, as inner mark solution；

（3）The preparation of need testing solution：Precision measures this product 1.0g, puts in 10mL volumetric flask, plus dehydrated alcohol is diluted to Scale, then precision measures 1.0mL, puts in 10mL volumetric flask, accurate add inner mark solution 1.0mL, plus dehydrated alcohol is diluted to quarter Degree, shakes up；

（4）The preparation of reference substance solution：Precision weighs cuparene reference substance, thujopsene reference substance in right amount, plus anhydrous Ethanol dissolves and dilutes makes the mL of 0.301mg containing cuparene^-1And thujopsene 0.901mg mL^-1Reference substance deposit molten Liquid, standby；

（5）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects gas chromatograph, enters Row detection；

（6）Measurement result：The every 1g of this product must not be less than 0.200mg containing cuparene, must not be less than containing thujopsene 0.200mg.

Embodiment 2：

Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 g, Endothelium Corneum Gigeriae Galli 10 g.

Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 15 times amount, 10 times amount, 5 times amount 40% alcohol reflux 3 Secondary, each extraction time 1.5h, united extraction liquid, reclaim ethanol, concentrate, dry, pulverize, add suitable dressing, such as starch or Cellulose, mixes, and makes hard capsule using conventional pharmaceutical methods in pharmacy, obtains final product.

Detection method：

1st, assay is carried out to diosgenin using high-efficient liquid spectrum method：

（1）Chromatographic condition：Using HypersilDs chromatographic column；Mobile phase：Ratio is 30：70 acetonitrile-water；Detection ripple Long：200nm；Column temperature：20℃；Flow velocity：1.0mL·min^-1；Sample size：10μL；

（2）Prepared by reference substance solution：It is appropriate to the diosgenin reference substance of constant weight that precision weighs 80 DEG C of dryings, plus methanol The reference substance solution that every 1mL contains 0.2mg is made in dissolving；

（3）The preparation of need testing solution：Precision weighs medicine 10g of the present invention, plus methanol 40mL, is heated to reflux 4h, extracts Liquid reflux solvent is simultaneously concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, close And n-butanol extracting liquid, washed with ammonia solution 3 times, each 15mL, to doing, it is molten that residue adds methanol to n-butanol extracting liquid recycling design Solve and be transferred in 10mL volumetric flask, plus methanol, to scale, shakes up, filtration, take filtrate, obtain final product need testing solution；

（4）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected；

（5）Measurement result：Contained in terms of diosgenin by every 1g this product, 0.12mg must not be less than.

2nd, assay is carried out to cuparene, thujopsene using gas chromatography：

（1）Chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary column；Carrier gas：N₂, 1.0mL mL^-1；Hydrogen, 40mL·mL^-1；Air, 400mL min^-1；Split ratio：10：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Program Heat up：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；

（2）The preparation of inner mark solution：Take Ketohexamethylene appropriate, plus anhydrous alcohol solution dilute and make every 1mL and contain 12.5mg Solution, shake up, as inner mark solution；

（3）The preparation of need testing solution：Precision measures this product 1.0g, puts in 10mL volumetric flask, plus dehydrated alcohol is diluted to Scale, then precision measures 1.0mL, puts in 10mL volumetric flask, accurate add inner mark solution 1.0mL, plus dehydrated alcohol is diluted to quarter Degree, shakes up；

（4）The preparation of reference substance solution：Precision weighs cuparene reference substance, thujopsene reference substance in right amount, plus anhydrous Ethanol dissolves and dilutes makes the mL of 0.301mg containing cuparene^-1And thujopsene 0.901mg mL^-1Reference substance deposit molten Liquid, standby；

（5）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects gas chromatograph, enters Row detection；

（6）Measurement result：The every 1g of this product must not be less than 0.200mg containing cuparene, must not be less than containing thujopsene 0.200mg.

Embodiment 3：

Prescription：Rhizoma Dioscoreae 30g, the Radix Astragali 15 g, the Rhizoma Anemarrhenae 15 g, Radix Puerariae 15 g, Fructus Schisandrae Chinensis 15 g, Radix Trichosanthis 15 g, Endothelium Corneum Gigeriae Galli 10 g.

Preparation method：Pharmaceutical decocting piece is weighed by prescribed dose, respectively with 12 times amount, 10 times amount, 6 times amount 60% alcohol reflux 3 Secondary, each extraction time 0.5h, united extraction liquid, reclaim ethanol, concentrate, dry, pulverize, add suitable dressing, such as starch or Cellulose, mixes, and makes tablet using conventional pharmaceutical methods in pharmacy, obtains final product.

Detection method：

1st, assay is carried out to diosgenin using high-efficient liquid spectrum method：

（1）Chromatographic condition：Using HypersilDs chromatographic column；Mobile phase：Ratio is 30：70 acetonitrile-water；Detection ripple Long：200nm；Column temperature：20℃；Flow velocity：1.0mL·min^-1；Sample size：10μL；

（2）Prepared by reference substance solution：It is appropriate to the diosgenin reference substance of constant weight that precision weighs 80 DEG C of dryings, plus methanol The reference substance solution that every 1mL contains 0.2mg is made in dissolving；

（3）The preparation of need testing solution：Precision weighs medicine 10g of the present invention, plus methanol 40mL, is heated to reflux 4h, extracts Liquid reflux solvent is simultaneously concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, close And n-butanol extracting liquid, washed with ammonia solution 3 times, each 15mL, to doing, it is molten that residue adds methanol to n-butanol extracting liquid recycling design Solve and be transferred in 10mL volumetric flask, plus methanol, to scale, shakes up, filtration, take filtrate, obtain final product need testing solution；

（4）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected；

（5）Measurement result：Contained in terms of diosgenin by every 1g this product, 0.12mg must not be less than.

2nd, assay is carried out to cuparene, thujopsene using gas chromatography：

（1）Chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary column；Carrier gas：N₂, 1.0mL mL^-1；Hydrogen, 40mL·mL^-1；Air, 400mL min^-1；Split ratio：10：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Program Heat up：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；

（2）The preparation of inner mark solution：Take Ketohexamethylene appropriate, plus anhydrous alcohol solution dilute and make every 1mL and contain 12.5mg Solution, shake up, as inner mark solution；

（3）The preparation of need testing solution：Precision measures this product 1.0g, puts in 10mL volumetric flask, plus dehydrated alcohol is diluted to Scale, then precision measures 1.0mL, puts in 10mL volumetric flask, accurate add inner mark solution 1.0mL, plus dehydrated alcohol is diluted to quarter Degree, shakes up；

（4）The preparation of reference substance solution：Precision weighs cuparene reference substance, thujopsene reference substance in right amount, plus anhydrous Ethanol dissolves and dilutes makes the mL of 0.301mg containing cuparene^-1And thujopsene 0.901mg mL^-1Reference substance deposit molten Liquid, standby；

（5）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects gas chromatograph, enters Row detection；

（6）Measurement result：The every 1g of this product must not be less than 0.200mg containing cuparene, must not be less than containing thujopsene 0.200mg.

Claims (8)

1. a kind of pharmaceutical composition treating diabetes mellitus encephalopathy, this pharmaceutical composition is to be made up of the raw material of following weight portion： Rhizoma Dioscoreae 30 weight portion, the Radix Astragali 15 weight portion, the Rhizoma Anemarrhenae 15 weight portion, Radix Puerariae 15 weight portion, Fructus Schisandrae Chinensis 15 weight portion, Radix Trichosanthis 15 weight Amount part, Endothelium Corneum Gigeriae Galli 10 weight portion are it is characterised in that this pharmaceutical composition is adopted and prepared with the following method：Pharmaceutical decocting piece presses place's prescription Amount weighs, respectively with 10 times amount, 8 times amount, 6 times amount, 50% alcohol reflux 3 times, each extraction time 1h, and united extraction liquid, Reclaim ethanol, concentrate, dry, pulverize, obtain final product.

2. pharmaceutical composition as claimed in claim 1 is it is characterised in that this pharmaceutical composition is according to conventional preparation in pharmacy Method makes hard capsule, tablet, granule.

3. pharmaceutical composition as claimed in claim 1 is it is characterised in that entered to diosgenin using high performance liquid chromatography Row assay：

（1）Chromatographic condition：Using HypersilDs chromatographic column；Mobile phase：Ratio is 20～40：60～80 acetonitrile-water；Detection Wavelength：190～210nm；Column temperature：15～25 DEG C；Flow velocity：0.5～1.5mL min^-1；Sample size：5～20 μ L；

（2）Prepared by reference substance solution：Precision weighs the dry diosgenin reference substance to constant weight in right amount, plus methanol dissolving is made Reference substance solution；

（3）The preparation of need testing solution：Precision weighs described pharmaceutical composition, plus methanol, is heated to reflux, and extracting solution backflow is molten Agent is simultaneously concentrated to dryness, and residue is dissolved in water, and is shaken with water saturated n-butyl alcohol and extracts, and merges n-butanol extracting liquid, uses ammonia solution Washing, to doing, residue adds methanol dissolving to n-butanol extracting liquid recycling design, shakes up, and filtration takes filtrate, obtains final product need testing solution；

（4）Measure：Precision measures above-mentioned need testing solution, each 5～20 μ L of reference substance solution respectively, injects high performance liquid chromatography Instrument, is detected.

4. pharmaceutical composition as claimed in claim 3 is it is characterised in that contained to diosgenin using high-efficient liquid spectrum method It is fixed to measure：

（1）Chromatographic condition：Using HypersilDs chromatographic column；Mobile phase：Ratio is 30：70 acetonitrile-water；Detection wavelength： 200nm；Column temperature：20℃；Flow velocity：1.0mL·min^-1；Sample size：10μL；

（2）Prepared by reference substance solution：It is appropriate to the diosgenin reference substance of constant weight that precision weighs 80 DEG C of dryings, plus methanol dissolving Make the reference substance solution that every 1mL contains 0.2mg；

（3）The preparation of need testing solution：Precision weighs described pharmaceutical composition 10mL, plus methanol 40mL, is heated to reflux 4h, carries Take liquid reflux solvent and be concentrated to dryness, residue add water 10mL dissolving, with water saturated n-butyl alcohol shaking extract 5 times, each 20mL, Merge n-butanol extracting liquid, washed with ammonia solution 3 times, each 15mL, to doing, residue adds methanol to n-butanol extracting liquid recycling design Dissolve and be transferred in 10mL volumetric flask, plus methanol, to scale, shakes up, filtration, take filtrate, obtain final product need testing solution；

（4）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects high performance liquid chromatograph, enters Row detection.

5. pharmaceutical composition as claimed in claim 1 is it is characterised in that adopt gas chromatography to cuparene, Thujopsis dolabrata Alkene carries out assay：

（1）Chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary column；Carrier gas：N₂, 0.5～1.5mL mL^-1；Hydrogen, 30 ～50mL mL^-1；Air, 300～500mL min^-1；Split ratio：5～15：1；Injector temperature：240～260 DEG C, detection Device temperature：290～310 DEG C；Temperature programming：Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, protect Hold 3.5min；Internal standard method；

（2）The preparation of inner mark solution：Take Ketohexamethylene appropriate, plus anhydrous alcohol solution dilute and make solution, shake up, as internal standard Solution；

（3）The preparation of need testing solution：Precision measures described pharmaceutical composition, puts in volumetric flask, plus dehydrated alcohol is diluted to Scale, then precision measures, and puts in volumetric flask, accurate add inner mark solution, plus dehydrated alcohol is diluted to scale, shakes up；

（4）The preparation of reference substance solution：Precision weighs cuparene reference substance, thujopsene reference substance in right amount, plus dehydrated alcohol Dissolve and dilute the reference substance stock solution made containing cuparene and thujopsene, standby；

（5）Measure：Precision measures above-mentioned need testing solution, each 5～20 μ L of reference substance solution respectively, injects gas chromatograph, enters Row detection.

6. pharmaceutical composition as claimed in claim 5 is it is characterised in that adopt gas chromatography to cuparene, Thujopsis dolabrata Alkene carries out assay：

（1）Chromatographic condition：Chromatographic column：ZB-WAX fused-silica capillary column；Carrier gas：N₂, 1.0mL mL^-1；Hydrogen, 40mL mL^-1；Air, 400mL min^-1；Split ratio：10：1；Injector temperature：250 DEG C, 300 DEG C of detector temperature；Temperature programming： Initial 80 DEG C, 5 DEG C per minute rise to 120 DEG C, and 10 DEG C per minute rise to 180 DEG C, keep 3.5min；Internal standard method；

（2）The preparation of inner mark solution：Take Ketohexamethylene appropriate, plus anhydrous alcohol solution dilute and make every 1mL and contain the molten of 12.5mg Liquid, shakes up, as inner mark solution；

（3）The preparation of need testing solution：Precision measures described pharmaceutical composition 1.0mL, puts in 10mL volumetric flask, plus anhydrous second Alcohol is diluted to scale, then precision measures 1.0mL, puts in 10mL volumetric flask, accurate addition inner mark solution 1.0mL, plus dehydrated alcohol It is diluted to scale, shake up；

（4）The preparation of reference substance solution：Precision weighs cuparene reference substance, thujopsene reference substance in right amount, plus dehydrated alcohol Dissolve and dilute and make the mL of 0.301mg containing cuparene^-1And thujopsene 0.901mg mL^-1Reference substance stock solution, Standby；

（5）Measure：Precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, injects gas chromatograph, is examined Survey.

7. application in preparation treatment diabetes mellitus encephalopathy medicine for the pharmaceutical composition as claimed in claim 1.

8. application in preparation treatment asthmatic medicament for the pharmaceutical composition as claimed in claim 1.