CN105434807A - Yuye decoction medicine for treating diabetic encephalopathy, preparation method thereof, detection method and application - Google Patents

Yuye decoction medicine for treating diabetic encephalopathy, preparation method thereof, detection method and application Download PDF

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CN105434807A
CN105434807A CN201610050742.4A CN201610050742A CN105434807A CN 105434807 A CN105434807 A CN 105434807A CN 201610050742 A CN201610050742 A CN 201610050742A CN 105434807 A CN105434807 A CN 105434807A
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solution
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medicine
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CN105434807B (en
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刘继平
程玥
张晓双
史永恒
胡锐
邓翀
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Shaanxi University of Chinese Medicine
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刘继平
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/894—Dioscoreaceae (Yam family)
    • A61K36/8945—Dioscorea, e.g. yam, Chinese yam or water yam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/57—Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • A61K36/428—Trichosanthes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/481—Astragalus (milkvetch)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/488—Pueraria (kudzu)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/57—Magnoliaceae (Magnolia family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/79—Schisandraceae (Schisandra family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8964—Anemarrhena
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2059—Starch, including chemically or physically modified derivatives; Amylose; Amylopectin; Dextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/025—Gas chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography

Abstract

The invention belongs to the field of traditional Chinese medicines and provides a medicine composition for treating diabetic encephalopathy and particularly provides a Yuye decoction medicine for treating the diabetic encephalopathy, a preparation method thereof, a detection method and application. The medicine composition is prepared from, by weight, 30 parts of yams, 15 parts of astragali radix, 15 parts of rhizoma anemarrhenae, 15 parts of radix puerariae, 15 parts of schisandra chinensis, 15 parts of radix trichosanthis and 10 parts of endothelium corneum gigeriae galli. The medicine composition is prepared by the following method: medicinal material slices are weighed according to prescription doses, reflux extraction is performed by using 40-60% ethanol 5-15 times of the amount of the slices for 3 times, the extraction time each time is 0.5-1.5 hours, extraction solutions are mixed, and ethyl alcohol recovery, concentration, drying and smashing are performed to obtain the Yuye decoction medicine. The invention further provides a method for adopting a high-performance liquid chromatography to determine the diosgenin content and a method for adopting a gas chromatographic method to determine the contents of cuparene and thujopsene.

Description

The treatment good wine decoction thing of diabetes mellitus encephalopathy and method for making thereof and detection method and purposes
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to a kind of pharmaceutical composition for the treatment of diabetes mellitus encephalopathy.
Background technology
One, diabetes (diabetesmellitus is called for short DM)
Diabetes (diabetesmellitus, be called for short DM) be that what to cause due to insulin secretion and (or) insulin action deficiency take hyperglycemia as the Metabolic Syndrome of principal character, epidemiological study shows, along with development and the living-pattern preservation of society, the sickness rate of DM is in rising trend in the world.At present, the whole world has 1.5 hundred million DM patients, and by 2025, this numeral will rise to 3.3 hundred million.The current number of patients of China about 4,000 ten thousand, accounts for the whole world about 1/5.Diabetes are a kind of systemic metabolic diseases, Multisystem damage can be caused to cause cardiovascular and cerebrovascular disease, retinopathy, nephropathy, diabetic foot, the complication such as peripheral neuropathy, a large amount of diabetic animal and clinical research at present all confirms, persistent high blood sugar also can cause central nervous system (Centralnervoussystem, CNS) cognitive dysfunction (mildcognitiveimpairment, MCI)---diabetes mellitus encephalopathy, namely central nervous system causes the infringement of cerebral tissue 26S Proteasome Structure and Function, MCI cognitive dysfunction fails also known as cognitive function, Cognitive or cognitive disabled, make a general reference the cognitive impairment of various degree, from MCI until dementia.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, are one of current study hotspot to the research of diabetes cognitive dysfunction.
Two, central cholinergic system
Central cholinergic system is considered to the important path forming learning and memory, acetylcholine (acetylcholine, ACh) be wherein important neurotransmitter, choline acetyltransterase (Cholineacetyl-transferase, ChAT) as the rate-limiting enzyme of Ach synthesis, Chang Zuowei studies the mark of cholinergic neuron or estimates the indirect indicator of Ach content (burst size).The change of brain Cholinergic neurotransmitter Ach content and ChAT activity, all can reflect the activity of cholinergic nerve, as one of the leading indicator of current research mankind brain function aging.Along with the proposition of " cholinergic damage theory ", the effect of cholinergic system in diabetes mellitus encephalopathy morbidity is day by day subject to people and payes attention to.Multinomial research confirms acetylcholine Developmental and Metabolic Disorder in the striatum of diabetes rat caused by STZ and hippocampus, and be also found diabetes rat cerebral tissue (estrangement, corpus amygdaloideum, Hippocampus etc.) cholinergic nerve system enzymatic activity and decline, cholinergic nerve conduction reduces.Therefore have scholar to think, diabetes mellitus encephalopathy is relevant basal forebrain, the cholinergic neuron degeneration such as Hippocampus and cortex of cognitive function in early days.
Research shows, the product that the free radical generation non-enzymatic glycation of glucose and protein or lipid and oxidation reaction are formed, i.e. Advanced glycation endproducts (AGEs), Advanced glycation endproducts (AGEs) can cause a series of pathophysiological change of in-vivo tissue, is the important paathogenic factor causing chronic complicating diseases of diabetes.AGE receptor (RAGE) is present in a lot of cell surface, the combination of itself and AGE can cause Cellular Oxidation stress with the activation of NF-κ B path.After 30 years old, in human brain, in Hippocampus and neurogliocyte, AGEs starts to gather.AGEs, at extracellular and beta-amyloyd speckle codeposition, then accumulates in neuron and astrocyte in cell.AGEs is combined with its receptor RAGE, can activate oxidative stress, causes a series of damage, and final activates as the transcription factor to redox reaction sensitivity such as NF-κ B, causes the rise of cytokine, as IL-1 β and TNF-α.The protective effect of good wine soup to the cognitive impairment that AGE induces of this experimental evaluation, comprises Behavior test and potential mechanism is probed into.
Three, the related introduction of good wine soup
TCM Treatment of Diabetes has the history of several thousand, and the Chinese medicine compound such as LIUWEI DIHUANG WAN, BUZHONG YIQI TANG, XIAOYAO POWDER, good wine soup, compound Salviae Miltiorrhizae, Herba Dendrobii mixture have prevention, treat diabetes or alleviate the effect of chronic complicating diseases.Wherein good wine soup comes from " Records of Tradition Chinese and Western Medicine in Combination " of Zhang Xichun, for Chinese traditional treatment is quenched one's thirst recipe, namely records and " control and quench one's thirst in former book.Quench one's thirst, i.e. the so-called diabetes of doctor trained in Western medicine, the sweet thing of diet.”
The present invention is on the basis of good wine soup, provide a kind of pharmaceutical composition and preparation method thereof, this medicine is at control T2DM and improve in cognitive function, determined curative effect, medicine of the present invention not only can improve the symptom of diabetes, also insulin resistant can be improved, the cognitive function effect and tool has some improvement.
Summary of the invention
The object of this invention is to provide a kind of pharmaceutical composition for the treatment of diabetes mellitus encephalopathy.
Another object of the present invention is to provide the preparation method of this pharmaceutical composition.
The object of the invention is to realize in the following manner.
The pharmaceutical composition for the treatment of diabetes mellitus encephalopathy is 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 takes by prescribed dose, and with 5 ~ 15 times amount 40 ~ 60% alcohol reflux 3 times, each extraction time 0.5 ~ 1.5h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
This pharmaceutical composition is preferably adopted and is prepared with the following method: pharmaceutical decocting piece takes by prescribed dose, and respectively with 10 times amount, 8 times amount, 6 times amount 50% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
This pharmaceutical composition makes hard capsule, tablet, granule according to preparation method conventional in pharmacy.
Described medicine, adopt and detect with the following method: adopt high performance liquid chromatography to carry out the assay of diosgenin:
(1) chromatographic condition: adopt HypersilDs chromatographic column; Mobile phase: ratio is the acetonitrile-water of 20 ~ 40:60 ~ 80; Determined wavelength: 190 ~ 210nm; Column temperature: 15 ~ 25 DEG C; Flow velocity: 0.5 ~ 1.5mLmin -1; Sample size: 5 ~ 20 μ L;
(2) reference substance solution preparation: it is appropriate that precision takes the diosgenin reference substance being dried to constant weight, adds dissolve with methanol and make reference substance solution;
(3) preparation of need testing solution: precision takes medicine of the present invention, adds methanol, reflux, extracting solution reflux solvent is also concentrated into dry, and residue is dissolved in water, and extracts with water saturated n-butyl alcohol jolting, merge n-butanol extracting liquid, with ammonia solution washing, n-butanol extracting liquid recycling design is to dry, and residue adds dissolve with methanol, shake up, filter, get filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 ~ 20 μ L of reference substance solution respectively, inject high performance liquid chromatograph, detect.
Above-mentioned employing high performance liquid chromatography carries out the detection method of assay to diosgenin, preferred method is as follows:
(1) chromatographic condition: adopt HypersilDs chromatographic column; Mobile phase: ratio is the acetonitrile-water of 30:70; Determined wavelength: 200nm; Column temperature: 20 DEG C; Flow velocity: 1.0mLmin -1; Sample size: 10 μ L;
(2) reference substance solution preparation: it is appropriate that precision takes 80 DEG C of diosgenin reference substances being dried to constant weight, adds dissolve with methanol and makes the reference substance solution of every 1g containing 0.2mg;
(3) preparation of need testing solution: precision takes medicine 10mL of the present invention, adds methanol 40mL, reflux 4h, extracting solution reflux solvent is also concentrated into dry, residue add water 10mL dissolve, extract 5 times with water saturated n-butyl alcohol jolting, each 20mL, merges n-butanol extracting liquid, washs 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and residue adds dissolve with methanol and is transferred in 10mL volumetric flask, add methanol to scale, shake up, filter, get filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, inject high performance liquid chromatograph, detect.
Described medicine adopts gas chromatography to carry out assay to cuparene, thujopsene:
(1) chromatographic condition: chromatographic column: ZB-WAX fused-silica capillary column; Carrier gas: N 2, 0.5 ~ 1.5mLmL -1; Hydrogen, 30 ~ 50mLmL -1; Air, 300 ~ 500mLmin -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) preparation of inner mark solution: get Ketohexamethylene appropriate, adds anhydrous alcohol solution and solution is made in dilution, shakes up, as inner mark solution;
(3) preparation of need testing solution: precision measures this product, puts in volumetric flask, add dehydrated alcohol and be diluted to scale, then precision measures, and put in volumetric flask, precision adds inner mark solution, adds dehydrated alcohol and is diluted to scale, shake up;
(4) preparation of reference substance solution: precision takes cuparene reference substance, thujopsene reference substance is appropriate, adds anhydrous alcohol solution and also dilutes the reference substance stock solution made containing cuparene and thujopsene, for subsequent use;
(5) measure: precision measures above-mentioned need testing solution, each 5 ~ 20 μ L of reference substance solution respectively, and inject gas chromatograph, detects.
Above-mentioned employing gas chromatography carries out cuparene, thujopsene carries out assay, preferably adopts with the following method:
(1) chromatographic condition: chromatographic column: ZB-WAX fused-silica capillary column; Carrier gas: N 2, 1.0mLmL -1; Hydrogen, 40mLmL -1; Air, 400mLmin -1; Split ratio: 10:1; Injector temperature: 250 DEG C, detector temperature 300 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) preparation of inner mark solution: get Ketohexamethylene appropriate, adds anhydrous alcohol solution and the solution of every 1mL containing 12.5mg is made in dilution, shakes up, as inner mark solution;
(3) preparation of need testing solution: precision measures this product 1.0mL, puts in 10mL volumetric flask, add dehydrated alcohol and be diluted to scale, then precision measures 1.0mL, and put in 10mL volumetric flask, precision adds inner mark solution 1.0mL, adds dehydrated alcohol and is diluted to scale, shake up;
(4) preparation of reference substance solution: precision takes cuparene reference substance, thujopsene reference substance is appropriate, adds anhydrous alcohol solution and dilution is made containing cuparene 0.301mgmL -1and thujopsene 0.901mgmL -1reference substance stock solution, for subsequent use;
(5) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, and inject gas chromatograph, detects.
The application of described pharmaceutical composition in preparation treatment diabetes mellitus encephalopathy medicine.
The application of described pharmaceutical composition in preparation treatment diabetic foot and asthmatic medicament.
The Ji Yuan of pharmaceutical composition Raw medicine of the present invention is as follows:
Rhizoma Dioscoreae: be the tuber of Dioscoreaceae yam Rhizoma Dioscoreae DioscoreaoppositaThunb. [D.batatasDecne.].
The Radix Astragali: be the root of pulse family Astragalus Radix Astagali AstragalusmembranaceusBungevar.Mongholius (Bunge) P.K.Hsiao and Radix Astragali A.Membranaceus (Fisch.) Bunge.
The Rhizoma Anemarrhenae: be the rhizome of Liliaceae Anemarrhena plant Rhizoma Anemarrhenae Ane-marrhenaasphodeloidesBurge.
Radix Puerariae: be pulse family Pueraria Herba Gelsemii Elegantis Puerarialobata (Willd.) Ohwi [P.thunbergiana (Sieb.etZucc.) Benth.; P.hirsuta (Thunb.) Schneid.] or the tuber of Radix Puerariae rattan P.ThomsoniiBenth..
Fructus Schisandrae Chinensis: be the fruit of Schisandraceae schisandra plant Fructus Schisandrae Chinensis Schisandrachinensis (Turca.) Baill. [KadsurachinensisTurca.] or schisandra chinensis S.sphenantheraRehd.etWils..
Radix Trichosanthis: be the root of Cucurbitaceae Trichosanthes Fructus Trichosanthis TrichosantheskiriowiiMaxim and Chinese Fructus Trichosanthis.
Endothelium Corneum Gigeriae Galli: be the gizzard tunica intima of Phasianidae Phasianus animal man chicken GallusgallusdomesticusBrisson.
Above-mentioned medical material be all purchased from medical material all purchased from Xianyang continuous feast medicine supermarket.
Technique effect of the present invention is verified 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, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 50% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
Drugs compared A: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with the water reflux, extract, 3 times of 10 times amount, 8 times amount, 6 times amount, each extraction time 1h, merge extractive liquid, concentrated, dry, pulverize, to obtain final product.
Drugs compared B: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 30% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
Drugs compared C: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 70% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
Drugs compared D: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: (1) gets the Radix Astragali, Radix Puerariae, the Fructus Schisandrae Chinensis Chinese medicine coarse powder of described weight, extracts 2 times, each 1 hour, merge extractive liquid, filtration with 70% alcohol heating reflux of 5 times of weight, filtrate recycling ethanol is to without alcohol taste, for subsequent use; (2) Rhizoma Dioscoreae of described weight, the Rhizoma Anemarrhenae, Radix Trichosanthis is got, add 7 times of weight water and decoct 2 times, each 1 hour, merge extractive liquid, filtration, filtrate reduced in volume to relative density is the clear paste of 1.10-1.20 (room temperature), cooling, adds ethanol and makes alcohol content reach 70%, stir evenly, leave standstill 12 hours, filter, filtrate recycling ethanol is to without alcohol taste, for subsequent use; (3) get the merging of (1) and (2) gained filtrate, stir evenly, after vacuum drying, be ground into 80 ~ 100 order fine powders, and be ground into 100 object Endothelium Corneum Gigeriae Galli fine powders and mix, to obtain final product.
Drugs compared E: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: one, prepare medical material: first take the 15g Radix Astragali, the 15g Rhizoma Anemarrhenae, 30g Rhizoma Dioscoreae, 10g Endothelium Corneum Gigeriae Galli, 15g Radix Puerariae, 15g Fructus Schisandrae Chinensis and 15g Radix Trichosanthis, namely obtain treating decocting herbs; Two, decoction: 1. first to treating to add deionized water in decocting herbs, and decoct 1 ~ 2h, then employing filter method will decocting liquid and the rear medical material of first time decoction be separated for the first time; 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 adopt filter method that second time decocting liquid and second time are decocted rear medical material and be separated; 3. to step 2 2. in the second time that is isolated to decoct after add deionized water in medical material, and decoct 1 ~ 2h, medical material after then adopting filter method to remove to decoct, and by the decocting liquid obtained with first time decocting liquid and second time decocting liquid merge, obtain pending decocting liquid; Three, finished product: be concentrated into relative density 1.04g/cm under the condition of 70 DEG C ~ 90 DEG C in temperature by pending decocting liquid 3~ 1.12g/cm 3, obtain just clear paste, by first clear paste centrifugal 8min ~ 12min under centrifugal speed is 3500r/min ~ 4500r/min, the supernatant then separation obtained is be concentrated into relative density 1.02g/cm under the condition of 70 DEG C ~ 90 DEG C in temperature 3~ 1.10g/cm 3, obtain clear paste, finally adopt Freeze Drying Technique by clear paste sublimation drying 35h ~ 45h altogether, namely obtain good wine soup lyophilized oral formulations.
Streptozotocin (STZ), available from Sigma;
Piracetam (piracetam), Hua Yuan Anhui, Shanghai Ren Ji pharmaceutical Co. Ltd, the accurate word H34020741 of traditional Chinese medicines;
Huperzine-A Tablets, Tests for Uniformity, Tailong Pharmaceutical Co., Ltd., Henan, the accurate word H10940156 of traditional Chinese medicines;
Metformin hydrochloride tablet, one one-tenth, Liaoning pharmaceutcal corporation, Ltd, the accurate word H21024375 of traditional Chinese medicines;
(2) reagent
AchE(ELISA) test kit, AGEs(ELISA) test kit and ChAT(ELISA) test kit, above test kit is all purchased from Ai Laisa biotechnology (Shanghai) Co., Ltd.; RAGE and NF-κ B immunohistochemical kit, purchased from Wuhan Boster Biological Technology Co., Ltd..
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.; The long microplate reader of Eon all-wave, U.S. BioTek; Thermo-86 degree cryogenic refrigerator; Micropipettor, Qiujing Bio-Chemical Reagent Instrument Co., Ltd., Shanghai, TGL-16M table-type high-speed refrigerated centrifuge, Hunan Xiang Yi Laboratory Instruments development corporation, Ltd.; The stable blood glucose meter of three promises, Sinocare Biosensing Co., Ltd.
1.3 animal
Male SD rat, cleaning grade, body weight 250 ~ 300g, is 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, freely drinks water and ingests.
2. method
2.1 modelings and grouping
Male SD rat adaptability is fed 1 week, and except blank group gives except normal feedstuff, all the other rats give self-control high-sugar-fat-diet (normal feedstuff 59%, Adeps Sus domestica 18%, sucrose 20%, egg yolk 3%) feed after 50 days, disposable celiac injection STZ30mg/kg(is dissolved in 0.1mol/L before use, the concentration of 1% is configured in the citrate buffer solution of pH4.2), after 72 hours, tail vein blood blood glucose meter surveys blood glucose, and with blood sugar concentration >=16.7mmol/L for modeling success, after model success, rat is divided into model group at random, piracetam group (0.48g/kg), huperzine A group (40 μ g/kg), metformin hydrochloride group (0.15g/kg), medicine group (11.5g/kg) of the present invention, 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), model grouping starts gastric infusion in latter second day, and the capacity normal saline such as blank group and the equal gavage of model group, 1 time/d, administration 30d, animal ad lib water inlet during administration, does not use insulin and other hypoglycemic medicine.
2.2Morris water maze laboratory
Morris water maze is adopted to carry out the mensuration of Rats With Memory function: 1. orientation navigation experiment: in 1h after last gavage.Often organize and get 10 rats at random, two time periods of every natural gift upper and lower noon, each time period trains 4 times, each respectively from 4 different gauge points (being evenly distributed 4 quadrants), rat is put into water towards pool wall, in record 90s, finds platform required time (escape latency) and detection range.Each at least interval 30min, training lasts 4d altogether.Position of platform remains unchanged in a collection of rat training and test process.If rat finds platform not yet in 90s, it is placed in platform again, shifts out labyrinth after 20s, its incubation period is designated as 90s.2. space exploration experiment: remove platform on the 5th day, rat is dropped in water towards pool wall by optional 1 place of entry, and record rat strides across the traversing times of virtual platform and the rat swimming time at target quadrant (former platform quadrant) in 90s.
2.3 draw materials and sample disposal
After water maze laboratory terminates, after water 12h is can't help in fasting, often group all the other rat weight except 4 rats of random residual, tail vein blood is put to death after surveying blood glucose, get brain rapidly, be placed on rinsing in ice-cold normal saline, filter paper suck dry moisture, be separated hippocampal tissue, weigh, under ice bath, use homogenizer homogenate, make the tissue homogenate that w/v is 10%, centrifugal (3500r/min) 10min, gets supernatant and strictly by test kit, mensuration AchE, AGES and ChAT indices is described.Often group is by residue 4 rats, after anesthesia, and 4% paraformaldehyde 0.1molL -1phosphate buffer (pH7.4) intracardiac perfusion is fixed, to be fixed fully after, open cranium and get brain, Coronal gets optic chiasma caudad 3-4mm piece of tissue, and drop into after identical fixative fixes 1 week in 4 DEG C, routine paraffin wax embeds, coronal section, carries out RAGE and NF-κ B immunohistochemical staining; Each group choose Hippocampus position totally 8 do not repeat the visual field, use BI-2000 medical image analysis system to analyze SABC slice, thin piece, 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 time adopt one factor analysis of variance, non parametric tests during normal distribution uneven or heterogeneity of variance, uses SPSS13.0 software statistics.
3. result
3.1 impacts on diabetes mellitus encephalopathy rat model water maze laboratory achievement
Incubation period between each group of water maze laboratory the 1st day, explore apart from basic there was no significant difference, compare with blank group, from the 2nd day, model group escape latency, exploration distance significant prolongation (P<0.01 or P<0.05), target quadrant (II quadrant) swimming time obviously shortens (p<0.01), and platform traversing times obviously reduces (p<0.01), compared with model group, the 3rd day, piracetam group incubation period, explore distance and obviously shorten (P<0.05), medicine group of the present invention is explored distance and is obviously shortened (P<0.05), the 4th day, piracetam group, huperzine A group, metformin hydrochloride group, medicine group of the present invention obviously shortens (P<0.05 or P<0.01) incubation period, huperzine A group, metformin hydrochloride group, medicine group of the present invention is explored distance and is obviously shortened (P<0.05), metformin hydrochloride group, the swimming time of medicine group target quadrant (II quadrant) of the present invention obviously extends (P<0.05), piracetam group, huperzine A 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 increases (P<0.05 or P<0.01) (see table 1-1 to showing 1-3)
Table 1-1 on the preclinical impact of 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 on diabetic model rats water maze test explore distance impact ( ± s, n=10)
Note: compare with blank group, △p<0.05, △ △p<0.01; Compare with model group, ▲p<0.05
Table 1-3 on the impact of diabetes mellitus encephalopathy rat water maze test target quadrant time and traversing times ( ± 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 on the impact of diabetes mellitus encephalopathy rat model blood glucose, body weight, AchE, ChAT and AGEs
Compare with blank group, model group blood glucose, AchE, AGEs level significantly increase (P<0.01), and body weight, ChAT level significantly reduce (P<0.01), compare with model group, metformin hydrochloride group, medicine group blood glucose value of the present invention significantly reduces (P<0.05 or P<0.01), metformin hydrochloride group, medicine group rat body weight value significance of the present invention raises (P<0.05), piracetam group, huperzine A group, medicine group AchE of the present invention, AGEs level significantly reduces (P<0.05 or P<0.01), piracetam group, huperzine A group, metformin hydrochloride group, medicine group ChAT level of the present invention significantly increases (P<0.05 or P<0.01), in Table 1-4 and table 1-5.
Table 1-4 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 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
3.3 on each impact organizing Rat hippocampus pathological change
RAGE is mainly at endoglin expression, and the immunohistochemical staining positive is in brown color, and immunohistochemical staining shows, compare with blank group, model group rats Hippocampus DG district RAGE immuning positive cell number showed increased, painted obvious intensification, average gray value obviously reduces (P<0.01); Compare with model group, piracetam group, huperzine A group, metformin hydrochloride group, medicine group rat hippocampus DG district RAGE immuning positive cell number of the present invention obviously reduce, painted more shallow, average gray value obviously raises (P<0.01), in Table 1-6.Nuclear factor NF-κ B expresses at neuron plasma, immunohistochemical staining shows, compare with blank group, model group rats Hippocampus DG district's granular cell layer and cortical neuron NF-κ B immunoreation positive cell many and color depth, average gray value obviously reduces (P<0.01); Compare with model group, piracetam group, huperzine A group, metformin hydrochloride group, medicine group rat hippocampus DG district's granular cell layer of the present invention and cortical neuron NF-κ B immunoreation positive cell are less, and average gray value obviously raises (P<0.05 or P<0.01) in Table 1-6.
Table 1-6 in diabetes mellitus encephalopathy rat model hippocampal tissue RAGE, NF-κ B express average gray value impact ( )
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 obviously can improve the cognitive disorder of diabetes mellitus encephalopathy rat model; there is certain cerebral protection, its mechanism of action is by blood sugar lowering, improve cholinergic nerve function, improve the approach such as AGEs-RAGE-NF-κ B path in diabetes cerebral tissue and improve diabetic model rats hippocampal tissue pathological changes.
The checking of rat cognitive model and the assessment of neural understanding treatment feasibility are carried out in this experiment by water maze, rat escapes the time, gropes apart from shorter, and traversing times is more, the target quadrant activity time is longer, illustrates that learning and memory in rats ability is stronger.This research water maze laboratory data show, medicine of the present invention can reduce rat escape latency and grope distance, increase traversing times and target quadrant activity time, show that medicine of the present invention obviously can improve the ability of learning and memory of diabetes mellitus encephalopathy rat model, its successful is better than drugs compared A, drugs compared B, drugs compared C, drugs compared D, drugs compared E.
Hyperglycemia causes the deposition of AGEs in cerebral tissue, and the focusing energy of AGEs causes a lot of toxic reaction, and it is by realizing with the combination of its specific receptor AGER (RAGE) to the toxic action of cell.RAGE is a member in cell surface molecule immunoglobulin superfamily, is at first to be found as the bind receptor of AGEs, and RAGE and diabetic complication have substantial connection.Interact after AGEs is combined with its cell surface receptor RAGE, the path that NF-κ B mediates can be activated, Hofmann etc. confirm the activation of NF-κ B first in diabetics, the NF-κ B of activation can stimulate numerous expression of target gene, as tissue factor (TF), vascular cell slit attached molecule-l(VCAM-l), the expression of RAGE etc., promote that diabetes worsen further, increase the weight of the further degradation degeneration of cerebral cortex, cause neurodegeneration.
Experimental result shows, in rat hippocampus DG district, injection AGE can activate RAGE-NF-κ B path, and infringement rat is in the cognitive performance of water maze laboratory.After Drug therapy of the present invention, the cognitive competence of rat model is improved.Blood glucose, AchE, AGEs level significantly reduce, and this experimental result illustrates the cognitive impairment that medicine of the present invention can improve AGEs and brings out, and its successful is 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 UV-detector).
1.2 reagent
Diosgenin reference substance (Chinese pharmaceutical biological product calibrating academy); Medicine of the present invention; Chinese crude drug (Xianyang continuous feast medicine supermarket provides); Methanol (chromatograph alcohol, biochemical work auxiliary reagent factory, Shanghai); Other reagent are analytical pure.
2 methods and result
2.1 prescription
Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g.
2.2 preparation
Pharmaceutical decocting piece takes by prescribed dose, and respectively with 10 times amount, 8 times amount, 6 times amount 50% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
The assay of 2.3 diosgenins
2.3.1HPLC chromatographic condition
Adopt HypersilDs(4.0mm × 125mm, 5 μm) chromatographic column; Mobile phase: ratio is the acetonitrile-water of 30:70; Determined wavelength: 200nm; Column temperature: 20 DEG C; Flow velocity: 1.0mLmin -1; Sample size: 10 μ L; Under this chromatographic condition, reference substance and sample chromatogram peak are well, noiseless to mensuration without Rhizoma Dioscoreae negative control.
2.3.2 the preparation of reference substance solution
It is appropriate that precision takes 80 DEG C of diosgenin reference substances being dried to constant weight, adds methanol and make the solution of every 1mL containing 0.2mg.
2.3.3 the preparation of need testing solution and negative controls
Precision takes medicine 10g of the present invention, adds methanol 40mL, reflux 4h, extracting solution reflux solvent is also concentrated into dry, residue add water 10mL dissolve, extract 5 times with water saturated n-butyl alcohol jolting, each 20mL, merges n-butanol extracting liquid, washs 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and residue adds dissolve with methanol and is transferred in 10mL volumetric flask, add methanol to scale, shake up, filter, get filtrate and get final product; Separately do not contain the negative controls of Rhizoma Dioscoreae in the preparation of prescription ratio, be made in the same way of negative controls.
2.3.4 the drafting of standard curve
It is appropriate that precision takes 80 DEG C of diosgenin reference substances being dried to constant weight, makes 10.4,20.8,41.6,83.2,166.4 μ gmL with methanol -1the solution of series concentration, precision measures each 10 μ L of above-mentioned 5 kinds of strength solution respectively, injects high performance liquid chromatograph and measures.
Carry out linear regression with peak area ratio and concentration, obtaining regression equation is: A=21.2763C-0.1391, r=0.9999.Show that diosgenin is at 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.In result 8h, RSD is 0.45%(n=5).Show that sample solution is stable in 8h.
2.3.6 replica test
By 5 parts, need testing solution preparation method parallel processing sample, measure Determination of Diosgenin in accordance with the law and calculate.It is 0.12mgmL that result records diosgenin average content -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 better.
2.3.8 response rate experiment
Precision takes 6 parts, the sample of the same lot number of known Determination of Diosgenin, adds appropriate diosgenin reference substance solution, operate, measure in accordance with the law, calculate the response rate by under sample determination item by high, medium and low concentration is accurate respectively.Result average recovery rate is 100.3%, RSD is 0.45%(n=5).
2.3.9 sample size measures
Measure reference substance solution and need testing solution respectively appropriate, filter with microporous filter membrane, each sample introduction 10 μ L, measures 3 batch samples by above-mentioned chromatographic condition, 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 95% ~ 105% of content, in every 1g this product containing diosgenin, must not be less than 0.12mg.3 batch sample content are respectively 100.8%(RSD=1.2%), 101.7%(RSD=1.3%), 99.2%(RSD=1.1%).
Experiment three: the content of medicine air phase chromatography Simultaneous Determination cuparene of the present invention, 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, 0.25 μm); Prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit 100,000/electronic analytical balance; Cuparene reference substance (content 99.9%, National Institute for Food and Drugs Control); Thujopsene reference substance (content 99.9%, National Institute for Food and Drugs Control); Medicine of the present invention (preparing with reference to the embodiment 1 in description detailed description of the invention of the present invention), 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: N 2, 1.0mLmL -1; Hydrogen, 40mLmL -1; Air, 400mLmin -1; Split ratio: 10:1; Injector temperature: 250 DEG C, detector temperature 300 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.
3 test methods and result
The preparation of 3.1 inner mark solutions
Get Ketohexamethylene appropriate, add anhydrous alcohol solution and dilute and make the solution of every 1g containing 12.5mg, shake up, as inner mark solution.
The preparation of 3.2 need testing solutions
Precision measures this product 1.0g, puts in 10mL volumetric flask, add dehydrated alcohol and be diluted to scale, then precision measures 1.0mL, and put in 10mL volumetric flask, precision adds inner mark solution 1.0mL, adds dehydrated alcohol and is diluted to scale, shake up.
The preparation of 3.3 reference substance stock solutions
Precision takes cuparene reference substance, thujopsene reference substance is appropriate, adds anhydrous alcohol solution and dilute to make containing cuparene 0.301mgmL -1and thujopsene 0.901mgmL -1reference substance stock solution, for subsequent use;
The preparation of 3.4 negative control solutions
Getting the blank solution by not adding cuparene and thujopsene in prescription, by preparation method under " 3.2 " item, making negative control 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 is in 10mL volumetric flask, add inner mark solution 1.0mL, add dehydrated alcohol and be diluted to scale, shake up, product solution in contrast, get 1 μ L sample introduction respectively, record chromatogram, with cuparene, thujopsene and interior target peak area ratio for vertical coordinate (Y), concentration (C) is abscissa (X), drawing standard curve, obtains regression equation and is respectively: Y(cuparene respectively)=1.1148X-0.0016, R 2=0.9999, cuparene concentration is at 0.146 ~ 2.555mgmL -1in scope, linear relationship is good; Y(thujopsene)=1.1347X+0.0035, R 2=0.9998, thujopsene concentration is at 0.198 ~ 3.465mgmL -1in scope, linear relationship is good.
3.6 precision test
Getting cuparene concentration is 0.230mgmL -1be 0.290mgmL with thujopsene concentration -1reference substance solution, repeat sample introduction 6 times, record peak area, calculate 2 kinds of compositions and interior target peak area ratio (A/A is interior to be marked) respectively, the RSD of cuparene, thujopsene be respectively 0.24% and 1.2%(n=6).
3.7 replica test
Get same batch sample, by the method replication under sample determination item 6 times, the RSD of result cuparene, thujopsene be respectively 0.36%, 1.8%(n=6).
3.8 stability test
Get same batch sample solution, at room temperature place 0 respectively, 2,4,6,8, measure after 12h, result is respectively 0.38%, 1.7% by the RSD of cuparene, thujopsene, and interpret sample solution measures in 12h, and result is stablized.
3.9 average recovery tests
Get the sample solution 9 parts of known content, and add suitable basic, normal, high reference substance solution, measure cuparene, thujopsene content by sample determination method, 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 is better, the response rate of cuparene is respectively 99.4% ~ 100.2%, the response rate of thujopsene is between 98.1% ~ 100.7%, relative standard deviation is respectively 0.28% and 0.86%, and this assay method can meet the assay of cuparene and thujopsene in medicine of the present invention.
3.10 quantitative limit and detectability
Adopt " signal to noise ratio method " to determine quantitative limit and the detectability of this research, line taking standard solution is appropriate, employing add dehydrated alcohol progressively dilution method dilute, when sample introduction concentration is 6.27,9.90 μ gmL -1time, get 1 μ L sample introduction, continuous sample introduction 3 times, obtain cuparene, interior mark, thujopsene signal to noise ratio meansigma methods respectively close to 10.0, can with this concentration for quantitative limit; Continue dilution sample introduction, when sample introduction concentration is 1.044,1.65 μ gmL -1, continuous sample introduction 3 times, obtains cuparene, interior mark, thujopsene signal to noise ratio meansigma methods close to 3.0, can with this concentration for detectability.
3.11 serviceability test
Investigate and stability of solution investigation through different chromatographic column, and column temperature, injector temperature and detector temperature are investigated, and show this method good tolerance, are applicable to the assay of two components in medicine of the present invention.
3.11.1 the impact of chromatographic column
Select the chromatographic column of 3 different commercial specifications, measure the content of same batch sample, the RSD% calculating content value is respectively 1.3,1.7,1.6.Result shows, the different PEG chromatographic column of sample measures content, and cuparene, thujopsene all can effectively be separated with interior mark, illustration method good tolerance.
3.11.2 the impact of column temperature
Column temperature is mainly on the impact be separated the appearance time affecting main peak, temperature is higher, main peak appearance time is shorter, when the first stage is 80 DEG C, cuparene main peak cuparene and impurity peak energy ensure baseline separation, during second stage 120 DEG C, thujopsene main peak and impurity peak energy ensure baseline separation, and the RSD of content is less than 2.0% at each temperature.
3.11.3 the impact of injector temperature
When injector temperature is higher than column temperature, cuparene and impurity peaks can ensure baseline separation, and thujopsene and magazins' layout are good, and the RSD of content is less than 2.0% at each temperature.
3.11.4 the impact of detector temperature
When detector temperature is higher than injector temperature, cuparene and impurity peaks can ensure baseline separation, and thujopsene and magazins' layout are good, and the RSD of content is less than 2.0% at each temperature.
3.12 sample size measurement result
Through Method validation, this content assaying method is easy and simple to handle, accuracy is high, favorable reproducibility, more effectively can control product quality.Therefore apply the method to 10 batch samples, adopt internal standard method to carry out assay according to preceding method, the results are shown in Table 3-2.
Table 3-2 sample size measurement result
4 discuss
4.1 system suitability test
Under this test gas chromatography system, each 1 μ L of pipette samples mensuration mixing reference substance solution, need testing solution and negative control solution respectively, record chromatogram.2 kinds of components all can be separated preferably with internal standard substance, negative noiseless.This system suitability the results are shown in Table 3-3.
Table 3-3 system suitability test
The selection of 4.2 internal standard substances
Once tried out Ketohexamethylene, naphthalene, biphenyl, methyl salicylate etc., because sample volatile ingredient is many, result with the retention time of Ketohexamethylene and separating effect most suitable.
The selection of 4.3 column temperatures
The boiling point difference of cuparene, Ketohexamethylene and thujopsene is larger, and when column temperature is low, the retention time of thujopsene is long, and during column temperature height, cuparene can not effectively be separated with impurity, analyzes while meeting two kinds of compositions through adopting two sections of temperature-programmed modes.
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 0.200mg containing cuparene, must not be less than 0.200mg containing thujopsene.
This method is carried out being separated to 2 kinds of compositions simultaneously and is detected, and method is quick, sensitive, and separating degree is good, specificity is good, effectively can control drug quality.
Implement four: the present invention treats the pharmacodynamic study of asthma
1, experiment material:
Medicine of the present invention: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 50% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
Drugs compared A: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with the water reflux, extract, 3 times of 10 times amount, 8 times amount, 6 times amount, each extraction time 1h, merge extractive liquid, concentrated, dry, pulverize, to obtain final product.
Drugs compared B: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 30% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
Drugs compared C: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 70% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
Drugs compared D: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: (1) gets the Radix Astragali, Radix Puerariae, the Fructus Schisandrae Chinensis Chinese medicine coarse powder of described weight, extracts 2 times, each 1 hour, merge extractive liquid, filtration with 70% alcohol heating reflux of 5 times of weight, filtrate recycling ethanol is to without alcohol taste, for subsequent use; (2) Rhizoma Dioscoreae of described weight, the Rhizoma Anemarrhenae, Radix Trichosanthis is got, add 7 times of weight water and decoct 2 times, each 1 hour, merge extractive liquid, filtration, filtrate reduced in volume to relative density is the clear paste of 1.10-1.20 (room temperature), cooling, adds ethanol and makes alcohol content reach 70%, stir evenly, leave standstill 12 hours, filter, filtrate recycling ethanol is to without alcohol taste, for subsequent use; (3) get the merging of (1) and (2) gained filtrate, stir evenly, after vacuum drying, be ground into 80 ~ 100 order fine powders, and be ground into 100 object Endothelium Corneum Gigeriae Galli fine powders and mix, to obtain final product.
Drugs compared E: prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g; Method for making: one, prepare medical material: first take the 15g Radix Astragali, the 15g Rhizoma Anemarrhenae, 30g Rhizoma Dioscoreae, 10g Endothelium Corneum Gigeriae Galli, 15g Radix Puerariae, 15g Fructus Schisandrae Chinensis and 15g Radix Trichosanthis, namely obtain treating decocting herbs; Two, decoction: 1. first to treating to add deionized water in decocting herbs, and decoct 1 ~ 2h, then employing filter method will decocting liquid and the rear medical material of first time decoction be separated for the first time; 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 adopt filter method that second time decocting liquid and second time are decocted rear medical material and be separated; 3. to step 2 2. in the second time that is isolated to decoct after add deionized water in medical material, and decoct 1 ~ 2h, medical material after then adopting filter method to remove to decoct, and by the decocting liquid obtained with first time decocting liquid and second time decocting liquid merge, obtain pending decocting liquid; Three, finished product: be concentrated into relative density 1.04g/cm under the condition of 70 DEG C ~ 90 DEG C in temperature by pending decocting liquid 3~ 1.12g/cm 3, obtain just clear paste, by first clear paste centrifugal 8min ~ 12min under centrifugal speed is 3500r/min ~ 4500r/min, the supernatant then separation obtained is be concentrated into relative density 1.02g/cm under the condition of 70 DEG C ~ 90 DEG C in temperature 3~ 1.10g/cm 3, obtain clear paste, finally adopt Freeze Drying Technique by clear paste sublimation drying 35h ~ 45h altogether, namely obtain 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); Acecoline (Shanghai San'aisi Reagent Co., Ltd.); Histamine phosphate (Shanghai Li Zhu east wind Bioisystech Co., Ltd); 402A ultrasound atomizer (Yuyue Medical Apparatus Co., Ltd., Jiangsu).
2, experimental technique:
Animal model replication: SD rat: 99, is divided into 9 groups immediately, i.e. Normal group, model control 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, Asmeton group.In experiment the 0th, 7 days, except Normal group gives 1% aluminum hydroxide solution, each treated animal is all with freshly prepared sensitization liquid (1mg/mLOVA, be dissolved in 1% aluminum hydroxide solution) make subcutaneous injection, every Mus gets 10 points altogether at the both sides metapedes sole of the foot, groin, waist, the back of the body, cervical region, often some injection 0.05mL, simultaneously lumbar injection 0.5mL, amount to 1mL.In experiment the 14th day, rat is placed in self-control atomization box (40 × 30 × 20cm 3) in, with ultrasound atomizer with atomization quantity 0.8mL/min, the atomization of atomization 1%OVA normal saline solution 15min(Normal group is with normal saline), every day 1 time, to excite asthma.After continuous agitation 3w, stop exciting 6d, then excite 1 time, put to death after 24h.
The preparation of medicine gavage solution of the present invention: get medicine 15g of the present invention and add in 1500mL water, be made into suspendible aqueous solution.
The preparation of drugs compared A gavage solution: get drugs compared A15g and add in 1500mL water, be made into suspendible aqueous solution.
The preparation of drugs compared B gavage solution: get drugs compared B15g and add in 1500mL water, be made into suspendible aqueous solution.
The preparation of drugs compared C gavage solution: get drugs compared C15g and add in 1500mL water, be made into suspendible aqueous solution.
The preparation of drugs compared D gavage solution: get drugs compared D15g and add in 1500mL water, be made into suspendible aqueous solution.
The preparation of drugs compared E gavage solution: get drugs compared D15g and add in 1500mL water, be made into suspendible aqueous solution.
Medication: start gastric infusion on the 14th day in experiment, every day 1 time, until put 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, give training decocting liquid 26g crude drug/(kgd) by 10mL/kg body weight gavage respectively.Asmeton group: give Asmeton aqueous solution 25mg/(kgd by 10mL/kg body weight gavage).Normal group and model control group: give normal saline.
Non-specific brinchial provocation test: carry out for the 27th, 41 days respectively at experiment.By 2% acecoline of preparation and 0.2% histamine phosphate mixed in equal amounts, as exciting liquid, stop after being atomized exciting liquid 60s with nebulizer (when atomization quantity excites with OVA), observe 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 for the 41st day in experiment, with 1%OVA exciting liquid Neulized inhalation 15min, the drawing of severity of symptom record animal according to Animal performance breathes heavily result, is divided into 4 grades: acomia author (-): be designated as 0 point; Slightly (+): only scratch where it itches, sneeze, cough person, be designated as 1 point; Moderate (++): occur rapid breathing, ventral breathing, dyspnea person, be designated as 2 points; Severe (+++): breathe and be the devil, and occur lying prostrate motionless, bradykinesia person, be designated as 3 points.
Statistical analysis: data acquisition mean ± standard deviation ( ± s) represent, compare between group and adopt homogeneity test of variance and one factor analysis of variance, neat according to homogeneity test of variance result selection LSD(variance) or Tamhane ' sT2(heterogeneity of variance), adopt SPSS11.0forWindows software kit to complete; Sxemiquantitative data adopts Ridit to analyze.
3, experimental result:
Ordinary circumstance is observed: normal control treated animal excites front and back general state obviously not change, and breathes steadily, active.
Model control group rat excites rear typical performance to be: scratch nose, sneeze or cough, rapid breathing, severe patient audible and significantly wheezing or dyspnea with rapid and short breath sound, respiratory rhythm is not whole, sometimes fast and sometimes slow, amplitude strengthen and uneven, obviously, mouth and nose, extremity and tail point slightly cyanosis, activity is minimizing or prostrate motionless obviously in ventral breathing.Along with the growth of firing time, above-mentioned symptom increases the weight of gradually, and body weight starts to alleviate, and hair is matt, bradykinesia, respiratory rhythm and amplitude disorder.
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 improved above-mentioned symptom, but not obvious.
The comparison of each group of rat non-specific bronchus excitation experiment asthmatic latent period: compare with Normal group, model control group 2 asthmatic latent period all obviously shorten (P<0.01); Compare with model control group, medicine group of the present invention 1st asthmatic latent period is without obvious change; 2nd time asthmatic latent period all obviously extends (P<0.05 or P<0.01).Drugs compared A group, drugs compared B group, drugs compared C group, drugs compared D group, drugs compared E group the 1st asthmatic latent period are without obvious change, and the 2nd time asthmatic latent period all has prolongation, but effect not obvious.
The results are shown in Table 4-1:
Table 4-1 respectively organize the non-specific brinchial provocation test asthmatic latent period of rat comparison ( )
In table 4-1, compare with Normal group, △ △p<0.01; Compare with model control group, * P<0.05, * * P<0.01.
Each group of rat specificity brinchial provocation test draws the comparison of breathing heavily classification: model control group OVA bronchus excitation experiment is drawn and breathed heavily classification results and compare with Normal group and have notable difference.Compare with model control group, medicine group of the present invention and Asmeton group are breathed heavily result have remarkable effect to be drawn for the 3rd time.
The results are shown in Table 4-2:
Table 4-2 respectively organize rat the 41st day specificity brinchial provocation test draw breathe heavily classification comparison ( )
From table 4-2, compare with Normal group, △p<0.05; Compare with model control group, * P<0.05.
4, experiment conclusion
Medicine of the present invention obviously can reduce experimental rat model of asthma sneeze and grab nose number of times, relieving asthma attack degree, increases peak expiratory flow rate, can treat asthma and allergic asthma.Particularly, the action effect of medicine of the present invention is obviously 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, combination between flavour of a drug creates obvious synergistic function, creates the technique effect that one-plus-one is greater than two.
Detailed description of the invention:
Embodiment 1:
Prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g.
Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 50% alcohol reflux 3 times, each extraction time 1h, merge extractive liquid, reclaims ethanol, concentrated, dry, pulverize, to obtain final product.
Detection method:
1, high-efficient liquid spectrometry is adopted to carry out assay to diosgenin:
(1) chromatographic condition: adopt HypersilDs chromatographic column; Mobile phase: ratio is the acetonitrile-water of 30:70; Determined wavelength: 200nm; Column temperature: 20 DEG C; Flow velocity: 1.0mLmin -1; Sample size: 10 μ L;
(2) reference substance solution preparation: it is appropriate that precision takes 80 DEG C of diosgenin reference substances being dried to constant weight, adds dissolve with methanol and makes the reference substance solution of every 1mL containing 0.2mg;
(3) preparation of need testing solution: precision takes medicine 10g of the present invention, adds methanol 40mL, reflux 4h, extracting solution reflux solvent is also concentrated into dry, residue add water 10mL dissolve, extract 5 times with water saturated n-butyl alcohol jolting, each 20mL, merges n-butanol extracting liquid, washs 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and residue adds dissolve with methanol and is transferred in 10mL volumetric flask, add methanol to scale, shake up, filter, get filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, inject high performance liquid chromatograph, detect;
(5) measurement result: in every 1g this product containing diosgenin, must not 0.12mg be less than.
2, gas chromatography is adopted to carry out assay to cuparene, thujopsene:
(1) chromatographic condition: chromatographic column: ZB-WAX fused-silica capillary column; Carrier gas: N 2, 1.0mLmL -1; Hydrogen, 40mLmL -1; Air, 400mLmin -1; Split ratio: 10:1; Injector temperature: 250 DEG C, detector temperature 300 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) preparation of inner mark solution: get Ketohexamethylene appropriate, adds anhydrous alcohol solution and the solution of every 1mL containing 12.5mg is made in dilution, shakes up, as inner mark solution;
(3) preparation of need testing solution: precision measures this product 1.0g, puts in 10mL volumetric flask, add dehydrated alcohol and be diluted to scale, then precision measures 1.0mL, and put in 10mL volumetric flask, precision adds inner mark solution 1.0mL, adds dehydrated alcohol and is diluted to scale, shake up;
(4) preparation of reference substance solution: precision takes cuparene reference substance, thujopsene reference substance is appropriate, adds anhydrous alcohol solution and dilution is made containing cuparene 0.301mgmL -1and thujopsene 0.901mgmL -1reference substance stock solution, for subsequent use;
(5) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, and inject gas chromatograph, detects;
(6) measurement result: the every 1g of this product must not be less than 0.200mg containing cuparene, must not be less than 0.200mg containing thujopsene.
Embodiment 2:
Prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g.
Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 15 times amount, 10 times amount, 5 times amount 40% alcohol reflux 3 times, each extraction time 1.5h, merge extractive liquid, reclaim ethanol, concentrated, dry, pulverize, add suitable dressing, as starch or cellulose, mixing, adopt pharmaceutical methods conventional in pharmacy to make hard capsule, to obtain final product.
Detection method:
1, high-efficient liquid spectrometry is adopted to carry out assay to diosgenin:
(1) chromatographic condition: adopt HypersilDs chromatographic column; Mobile phase: ratio is the acetonitrile-water of 30:70; Determined wavelength: 200nm; Column temperature: 20 DEG C; Flow velocity: 1.0mLmin -1; Sample size: 10 μ L;
(2) reference substance solution preparation: it is appropriate that precision takes 80 DEG C of diosgenin reference substances being dried to constant weight, adds dissolve with methanol and makes the reference substance solution of every 1mL containing 0.2mg;
(3) preparation of need testing solution: precision takes medicine 10g of the present invention, adds methanol 40mL, reflux 4h, extracting solution reflux solvent is also concentrated into dry, residue add water 10mL dissolve, extract 5 times with water saturated n-butyl alcohol jolting, each 20mL, merges n-butanol extracting liquid, washs 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and residue adds dissolve with methanol and is transferred in 10mL volumetric flask, add methanol to scale, shake up, filter, get filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, inject high performance liquid chromatograph, detect;
(5) measurement result: in every 1g this product containing diosgenin, must not 0.12mg be less than.
2, gas chromatography is adopted to carry out assay to cuparene, thujopsene:
(1) chromatographic condition: chromatographic column: ZB-WAX fused-silica capillary column; Carrier gas: N 2, 1.0mLmL -1; Hydrogen, 40mLmL -1; Air, 400mLmin -1; Split ratio: 10:1; Injector temperature: 250 DEG C, detector temperature 300 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) preparation of inner mark solution: get Ketohexamethylene appropriate, adds anhydrous alcohol solution and the solution of every 1mL containing 12.5mg is made in dilution, shakes up, as inner mark solution;
(3) preparation of need testing solution: precision measures this product 1.0g, puts in 10mL volumetric flask, add dehydrated alcohol and be diluted to scale, then precision measures 1.0mL, and put in 10mL volumetric flask, precision adds inner mark solution 1.0mL, adds dehydrated alcohol and is diluted to scale, shake up;
(4) preparation of reference substance solution: precision takes cuparene reference substance, thujopsene reference substance is appropriate, adds anhydrous alcohol solution and dilution is made containing cuparene 0.301mgmL -1and thujopsene 0.901mgmL -1reference substance stock solution, for subsequent use;
(5) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, and inject gas chromatograph, detects;
(6) measurement result: the every 1g of this product must not be less than 0.200mg containing cuparene, must not be less than 0.200mg containing thujopsene.
Embodiment 3:
Prescription: Rhizoma Dioscoreae 30g, Radix Astragali 15g, Rhizoma Anemarrhenae 15g, Radix Puerariae 15g, Fructus Schisandrae Chinensis 15g, Radix Trichosanthis 15g, Endothelium Corneum Gigeriae Galli 10g.
Method for making: pharmaceutical decocting piece takes by prescribed dose, respectively with 12 times amount, 10 times amount, 6 times amount 60% alcohol reflux 3 times, each extraction time 0.5h, merge extractive liquid, reclaim ethanol, concentrated, dry, pulverize, add suitable dressing, as starch or cellulose, mixing, adopt pharmaceutical methods conventional in pharmacy to make tablet, to obtain final product.
Detection method:
1, high-efficient liquid spectrometry is adopted to carry out assay to diosgenin:
(1) chromatographic condition: adopt HypersilDs chromatographic column; Mobile phase: ratio is the acetonitrile-water of 30:70; Determined wavelength: 200nm; Column temperature: 20 DEG C; Flow velocity: 1.0mLmin -1; Sample size: 10 μ L;
(2) reference substance solution preparation: it is appropriate that precision takes 80 DEG C of diosgenin reference substances being dried to constant weight, adds dissolve with methanol and makes the reference substance solution of every 1mL containing 0.2mg;
(3) preparation of need testing solution: precision takes medicine 10g of the present invention, adds methanol 40mL, reflux 4h, extracting solution reflux solvent is also concentrated into dry, residue add water 10mL dissolve, extract 5 times with water saturated n-butyl alcohol jolting, each 20mL, merges n-butanol extracting liquid, washs 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and residue adds dissolve with methanol and is transferred in 10mL volumetric flask, add methanol to scale, shake up, filter, get filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, inject high performance liquid chromatograph, detect;
(5) measurement result: in every 1g this product containing diosgenin, must not 0.12mg be less than.
2, gas chromatography is adopted to carry out assay to cuparene, thujopsene:
(1) chromatographic condition: chromatographic column: ZB-WAX fused-silica capillary column; Carrier gas: N 2, 1.0mLmL -1; Hydrogen, 40mLmL -1; Air, 400mLmin -1; Split ratio: 10:1; Injector temperature: 250 DEG C, detector temperature 300 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) preparation of inner mark solution: get Ketohexamethylene appropriate, adds anhydrous alcohol solution and the solution of every 1mL containing 12.5mg is made in dilution, shakes up, as inner mark solution;
(3) preparation of need testing solution: precision measures this product 1.0g, puts in 10mL volumetric flask, add dehydrated alcohol and be diluted to scale, then precision measures 1.0mL, and put in 10mL volumetric flask, precision adds inner mark solution 1.0mL, adds dehydrated alcohol and is diluted to scale, shake up;
(4) preparation of reference substance solution: precision takes cuparene reference substance, thujopsene reference substance is appropriate, adds anhydrous alcohol solution and dilution is made containing cuparene 0.301mgmL -1and thujopsene 0.901mgmL -1reference substance stock solution, for subsequent use;
(5) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, and inject gas chromatograph, detects;
(6) measurement result: the every 1g of this product must not be less than 0.200mg containing cuparene, must not be less than 0.200mg containing thujopsene.

Claims (10)

1. treat the pharmaceutical composition of diabetes mellitus encephalopathy for one kind, it is characterized in that, this pharmaceutical composition is 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.
2. pharmaceutical composition as claimed in claim 1, it is characterized in that, this pharmaceutical composition is adopted and is prepared with the following method: pharmaceutical decocting piece takes by prescribed dose, with 5 ~ 15 times amount 40 ~ 60% alcohol reflux 3 times, and each extraction time 0.5 ~ 1.5h, merge extractive liquid, reclaim ethanol, concentrated, dry, pulverize, to obtain final product.
3. pharmaceutical composition as claimed in claim 2, it is characterized in that, this pharmaceutical composition is adopted and is prepared with the following method: pharmaceutical decocting piece takes by prescribed dose, respectively with 10 times amount, 8 times amount, 6 times amount 50% alcohol reflux 3 times, and each extraction time 1h, merge extractive liquid, reclaim ethanol, concentrated, dry, pulverize, to obtain final product.
4. the pharmaceutical composition as described in claims 1 to 3 any one, is characterized in that, this pharmaceutical composition makes hard capsule, tablet, granule according to preparation method conventional in pharmacy.
5. as the medicine in claims 1 to 3 as described in any one, it is characterized in that, adopt high performance liquid chromatography to carry out assay to diosgenin:
(1) chromatographic condition: adopt HypersilDs chromatographic column; Mobile phase: ratio is the acetonitrile-water of 20 ~ 40:60 ~ 80; Determined wavelength: 190 ~ 210nm; Column temperature: 15 ~ 25 DEG C; Flow velocity: 0.5 ~ 1.5mLmin -1; Sample size: 5 ~ 20 μ L;
(2) reference substance solution preparation: it is appropriate that precision takes the diosgenin reference substance being dried to constant weight, adds dissolve with methanol and make reference substance solution;
(3) preparation of need testing solution: precision takes medicine of the present invention, adds methanol, reflux, extracting solution reflux solvent is also concentrated into dry, and residue is dissolved in water, and extracts with water saturated n-butyl alcohol jolting, merge n-butanol extracting liquid, with ammonia solution washing, n-butanol extracting liquid recycling design is to dry, and residue adds dissolve with methanol, shake up, filter, get filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 5 ~ 20 μ L of reference substance solution respectively, inject high performance liquid chromatograph, detect.
6. medicine as claimed in claim 5, is characterized in that, adopts high-efficient liquid spectrometry to carry out assay to diosgenin:
(1) chromatographic condition: adopt HypersilDs chromatographic column; Mobile phase: ratio is the acetonitrile-water of 30:70; Determined wavelength: 200nm; Column temperature: 20 DEG C; Flow velocity: 1.0mLmin -1; Sample size: 10 μ L;
(2) reference substance solution preparation: it is appropriate that precision takes 80 DEG C of diosgenin reference substances being dried to constant weight, adds dissolve with methanol and makes the reference substance solution of every 1mL containing 0.2mg;
(3) preparation of need testing solution: precision takes medicine 10mL of the present invention, adds methanol 40mL, reflux 4h, extracting solution reflux solvent is also concentrated into dry, residue add water 10mL dissolve, extract 5 times with water saturated n-butyl alcohol jolting, each 20mL, merges n-butanol extracting liquid, washs 3 times with ammonia solution, each 15mL, n-butanol extracting liquid recycling design is to dry, and residue adds dissolve with methanol and is transferred in 10mL volumetric flask, add methanol to scale, shake up, filter, get filtrate, obtain need testing solution;
(4) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, inject high performance liquid chromatograph, detect.
7. as the medicine in claims 1 to 3 as described in any one, it is characterized in that, adopt gas chromatography to carry out assay to cuparene, thujopsene:
(1) chromatographic condition: chromatographic column: ZB-WAX fused-silica capillary column; Carrier gas: N 2, 0.5 ~ 1.5mLmL -1; Hydrogen, 30 ~ 50mLmL -1; Air, 300 ~ 500mLmin -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) preparation of inner mark solution: get Ketohexamethylene appropriate, adds anhydrous alcohol solution and solution is made in dilution, shakes up, as inner mark solution;
(3) preparation of need testing solution: precision measures this product, puts in volumetric flask, add dehydrated alcohol and be diluted to scale, then precision measures, and put in volumetric flask, precision adds inner mark solution, adds dehydrated alcohol and is diluted to scale, shake up;
(4) preparation of reference substance solution: precision takes cuparene reference substance, thujopsene reference substance is appropriate, adds anhydrous alcohol solution and also dilutes the reference substance stock solution made containing cuparene and thujopsene, for subsequent use;
(5) measure: precision measures above-mentioned need testing solution, each 5 ~ 20 μ L of reference substance solution respectively, and inject gas chromatograph, detects.
8. medicine as claimed in claim 7, is characterized in that, adopts gas chromatography to carry out assay to cuparene, thujopsene:
(1) chromatographic condition: chromatographic column: ZB-WAX fused-silica capillary column; Carrier gas: N 2, 1.0mLmL -1; Hydrogen, 40mLmL -1; Air, 400mLmin -1; Split ratio: 10:1; Injector temperature: 250 DEG C, detector temperature 300 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) preparation of inner mark solution: get Ketohexamethylene appropriate, adds anhydrous alcohol solution and the solution of every 1mL containing 12.5mg is made in dilution, shakes up, as inner mark solution;
(3) preparation of need testing solution: precision measures this product 1.0mL, puts in 10mL volumetric flask, add dehydrated alcohol and be diluted to scale, then precision measures 1.0mL, and put in 10mL volumetric flask, precision adds inner mark solution 1.0mL, adds dehydrated alcohol and is diluted to scale, shake up;
(4) preparation of reference substance solution: precision takes cuparene reference substance, thujopsene reference substance is appropriate, adds anhydrous alcohol solution and dilution is made containing cuparene 0.301mgmL -1and thujopsene 0.901mgmL -1reference substance stock solution, for subsequent use;
(5) measure: precision measures above-mentioned need testing solution, each 10 μ L of reference substance solution respectively, and inject gas chromatograph, detects.
9. the application of the pharmaceutical composition as described in claims 1 to 3 any one in preparation treatment diabetes mellitus encephalopathy medicine.
10. the application of the pharmaceutical composition as described in claims 1 to 3 any one in preparation treatment diabetic foot and asthmatic medicament.
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