CN110389179A - A kind of detection method of Chinese medicine composition - Google Patents
A kind of detection method of Chinese medicine composition Download PDFInfo
- Publication number
- CN110389179A CN110389179A CN201810353717.2A CN201810353717A CN110389179A CN 110389179 A CN110389179 A CN 110389179A CN 201810353717 A CN201810353717 A CN 201810353717A CN 110389179 A CN110389179 A CN 110389179A
- Authority
- CN
- China
- Prior art keywords
- solution
- group
- preparation
- reference substance
- test solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
- A61K36/238—Saposhnikovia
-
- 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)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
- A61K36/284—Atractylodes
-
- 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
- A61K36/484—Glycyrrhiza (licorice)
-
- 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)
- A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
-
- 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)
- A61K36/57—Magnoliaceae (Magnolia family)
- A61K36/575—Magnolia
-
- 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)
- A61K36/65—Paeoniaceae (Peony family), e.g. Chinese peony
-
- 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)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
-
- 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)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
-
- 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
- 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)
- A61K36/906—Zingiberaceae (Ginger family)
- A61K36/9068—Zingiber, e.g. garden ginger
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
- A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/02—Nasal agents, e.g. decongestants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- 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
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- 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
-
- 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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Medical Informatics (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Pulmonology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Otolaryngology (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention provides a kind of detection method of Chinese medicinal composition preparation, which comprises determination condition: using octadecylsilane key and silica gel as filler;With acetonitrile-water (25~35): (65~75) are mobile phase, evaporative light scattering detector detection;Number of theoretical plate is calculated by Astragaloside IV peak should be not less than 4000;The preparation of reference substance solution: taking Astragaloside IV reference substance, and organic solvent dissolution obtains reference substance solution;The preparation of test solution: taking Chinese medicinal composition preparation, and organic solvent extracts, and obtains test solution;Measuring method: drawing reference substance solution and test solution respectively, injects liquid chromatograph, measurement.
Description
Technical field
The present invention relates to a kind of detection methods of Chinese medicine composition, belong to field of medicaments.
Background technique
Allergic rhinitis (allergic rhinitis, AR) i.e. allergic rhinitis refer to that atopic individuals contact allergen
It is mainly discharged afterwards by the medium (mainly histamine) that IgE is mediated, and there are many immunocompetent cell and cell factors etc. to participate in
Schneiderian membrance non-infectious inflammatory disease.AR is one of most common anaphylactia, and disease incidence rises in obvious in recent years
Trend about influences the crowd of global 10%-25%, especially higher in its national illness rate of industry prosperity.Conservative estimation
Global AR patient is more than 500,000,000.11, China key city telephone questionnaire investigation display AR illness rate is about 11%.AR can lead to very
More diseases and disability, have become major global public health problem.
Western medical treatment AR is based on immunotherapy and drug therapy, and there is deficiencies: the more difficult determination of immunotherapy allergen
And standard is difficult consistent, works slow, the course for the treatment of is long, should not adhere to, it is difficult to cure;Drug therapy mainly has decongestant, antihistamine
Medicine, glucocorticoid, anticholinergic drug, mast cell stabilizers, leukotriene receptor antagonists etc. with symptomatic treatment, alleviate disease
Based on shape, although alleviation symptom is very fast, ideal radical cure method there is no at present.
Chinese traditional treatment AR, therapy concentrate on sensible, dampness removing stagnation resolvation of inducing diaphoresis etc., also slightly take into account righting.But mainly to dispel table
It is not comprehensive to human righteousness's solid protection based on card;Globality and systematicness to interpretation of the cause, onset and process of an illness essence understand that deficiency, the level of understanding need to be mentioned
It is high;Therapeutic effect is still weak, and is not able to satisfy clinical demand.
Summary of the invention
In order to solve the problems existing in the prior art, the present invention provide a kind of Chinese medicine composition for treating allergic rhinitis and its
Preparation method.
As one aspect of the present invention, the present invention provides a kind of Chinese medicine composition for treating allergic rhinitis, the Chinese medicine
Composition is made of the following raw material medicine: Radix Astragali, the flower bud of lily magnolia, radix saposhnikoviae, Rhizoma Atractylodis Macrocephalae, rhizoma zingiberis, cortex cinnamomi, Radix Paeoniae Alba, dark plum, Schisandra chinensis, is known ramulus cinnamomi
Female, Radix Glycyrrhizae.
In a particular embodiment, the Chinese medicine composition is made of the following raw material medicine: Radix Astragali 5-50 parts by weight, ramulus cinnamomi
3-27 parts by weight, flower bud of lily magnolia 2-20 parts by weight, radix saposhnikoviae 3-24 parts by weight, Rhizoma Atractylodis Macrocephalae 3-27 parts by weight, rhizoma zingiberis 2-20 parts by weight, cortex cinnamomi 1-
8 parts by weight, Radix Paeoniae Alba 2-20 parts by weight, dark plum 4-30 parts by weight, Schisandra chinensis 2-20 parts by weight, rhizoma anemarrhenae 2-20 parts by weight, Radix Glycyrrhizae 2-
20 parts by weight.
In a preferred embodiment, the Chinese medicine composition is made of the following raw material medicine: Radix Astragali 8-40 parts by weight, ramulus cinnamomi
4-22 parts by weight, flower bud of lily magnolia 3-16 parts by weight, radix saposhnikoviae 4-18 parts by weight, Rhizoma Atractylodis Macrocephalae 4-22 parts by weight, rhizoma zingiberis 3-16 parts by weight, cortex cinnamomi 1-
6 parts by weight, Radix Paeoniae Alba 3-16 parts by weight, dark plum 6-24 parts by weight, Schisandra chinensis 3-16 parts by weight, rhizoma anemarrhenae 3-16 parts by weight, Radix Glycyrrhizae 3-
16 parts by weight.
In further preferred embodiment, the Chinese medicine composition is made of the following raw material medicine: Radix Astragali 10-30 weight
Part, ramulus cinnamomi 5-18 parts by weight, flower bud of lily magnolia 4-12 parts by weight, radix saposhnikoviae 5-14 parts by weight, Rhizoma Atractylodis Macrocephalae 5-18 parts by weight, rhizoma zingiberis 4-12 weight
Part, cortex cinnamomi 2-5 parts by weight, Radix Paeoniae Alba 4-12 parts by weight, dark plum 8-20 parts by weight, Schisandra chinensis 4-12 parts by weight, rhizoma anemarrhenae 4-12 weight
Part, Radix Glycyrrhizae 4-12 parts by weight.
In further preferred embodiment, the Chinese medicine composition is made of the following raw material medicine: Radix Astragali 15-25 weight
Part, ramulus cinnamomi 7-12 parts by weight, flower bud of lily magnolia 5-8 parts by weight, radix saposhnikoviae 6-9 parts by weight, Rhizoma Atractylodis Macrocephalae 7-12 parts by weight, rhizoma zingiberis 5-8 parts by weight, meat
Osmanthus 2-4 parts by weight, Radix Paeoniae Alba 5-8 parts by weight, dark plum 10-15 parts by weight, Schisandra chinensis 5-8 parts by weight, rhizoma anemarrhenae 5-8 parts by weight, Radix Glycyrrhizae
5-8 parts by weight.
In most preferred embodiments, the Chinese medicine composition is made of the following raw material medicine: 21 parts by weight of Radix Astragali, ramulus cinnamomi
9 parts by weight, 6 parts by weight of the flower bud of lily magnolia, windproof 7 parts by weight, 9 parts by weight of Rhizoma Atractylodis Macrocephalae, 6 parts by weight of rhizoma zingiberis, 3 parts by weight of cortex cinnamomi, 6 weight of Radix Paeoniae Alba
Part, 12 parts by weight of dark plum, 6 parts by weight of Schisandra chinensis, 6 parts by weight of rhizoma anemarrhenae, 6 parts by weight of Radix Glycyrrhizae;
Or, the Chinese medicine composition is made of the following raw material medicine: 16 parts by weight of Radix Astragali, 11 parts by weight of ramulus cinnamomi, 5 weight of the flower bud of lily magnolia
Part, windproof 8 parts by weight, 8 parts by weight of Rhizoma Atractylodis Macrocephalae, 7 parts by weight of rhizoma zingiberis, 2 parts by weight of cortex cinnamomi, 7 parts by weight of Radix Paeoniae Alba, 11 parts by weight of dark plum,
7 parts by weight of Schisandra chinensis, 5 parts by weight of rhizoma anemarrhenae, 7 parts by weight of Radix Glycyrrhizae;
Or, the Chinese medicine composition is made of the following raw material medicine: 24 parts by weight of Radix Astragali, 8 parts by weight of ramulus cinnamomi, 7 weight of the flower bud of lily magnolia
Part, windproof 6 parts by weight, 11 parts by weight of Rhizoma Atractylodis Macrocephalae, 5 parts by weight of rhizoma zingiberis, 4 parts by weight of cortex cinnamomi, 5 parts by weight of Radix Paeoniae Alba, 14 parts by weight of dark plum,
5 parts by weight of Schisandra chinensis, 7 parts by weight of rhizoma anemarrhenae, 5 parts by weight of Radix Glycyrrhizae.
As preferred embodiment, the Radix Astragali is made a living Radix Astragali;The Rhizoma Atractylodis Macrocephalae is rhizoma atractylodis macrocephalae;The Radix Glycyrrhizae is made a living sweet
Grass.
Chinese medicine composition of the present invention can be the composition mixed after each bulk pharmaceutical chemicals crush, can also mix or
The active component that the extract or the further polishing purification of extract obtained after individually extracting obtains, can also be addition
Regular dosage form made of pharmaceutically acceptable auxiliary material.
Wherein, the extracting method includes decocting extraction, refluxing extraction, Soakage extraction, ultrasonic extraction, seepage pressure effects, micro-
Wave extraction etc.;The purification process includes water extract-alcohol precipitation, alkali soluble acid sinks and various column chromatography purification process, such as macroreticular resin
Column, silicagel column, gel column, reversed-phase column etc.;The regular dosage form include but is not limited to injection, capsule, tablet, granule,
Gelling agent, sustained release agent, oral solution, pill or nanometer formulation;The pharmaceutically acceptable auxiliary material include: filler, disintegrating agent,
Lubricant, suspending agent, adhesive, sweetener, corrigent, preservative, matrix etc..Filler include: starch, pregelatinized starch,
Lactose, mannitol, chitin, microcrystalline cellulose, sucrose etc.;Disintegrating agent includes: starch, pregelatinized starch, microcrystalline cellulose, carboxylic
Methyl starch sodium, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, croscarmellose sodium etc.;Lubricant packet
It includes: magnesium stearate, lauryl sodium sulfate, talcum powder, silica etc.;Suspending agent includes: polyvinylpyrrolidone, crystallite fibre
Tie up element, sucrose, agar, hydroxypropyl methyl cellulose etc.;Adhesive includes starch slurry, polyvinylpyrrolidone, hydroxypropyl methyl
Cellulose etc..
As the second aspect of the invention, the present invention provides the Chinese medicine composition in preparation treatment allergic rhinitis
Application in drug.
As the third aspect of the invention, the present invention provides the medicine that the Chinese medicine composition has anti-inflammatory effect in preparation
Application in object.
As the fourth aspect of the invention, the present invention, which provides the Chinese medicine composition, has anti-allergic effects in preparation
Application in drug.
As the fifth aspect of the invention, the present invention provides the preparation method of the Chinese medicinal composition granules, described
Method includes:
Composition extract powder is taken, with excipient in 3:(1~2) ratio mix, it is dry using wet granulation, obtain particle
Agent;
Wherein, the composition extract powder is the extract obtained after each bulk pharmaceutical chemicals extract according to a conventional method, or is mentioned
The active component for taking the further polishing purification of object to obtain.The extracting method include decoct extraction, refluxing extraction, Soakage extraction,
Ultrasonic extraction, seepage pressure effects, Microwave Extraction etc.;The purification process includes water extract-alcohol precipitation, alkali soluble acid sinks and various column chromatographys
Purification process, such as macroporous resin column, silicagel column, gel column, reversed-phase column.
In a particular embodiment, the excipient in dextrin, lactose, soluble starch any one or it is several
Kind, preferably dextrin;The ethanol solution that the wetting agent is 90%~95%, preferably 94% ethanol solution.The combination
Object extract powder and excipient ratios are preferably 3: 2.
In a particular embodiment, in the pelletization envionmental humidity below 74%.
In a preferred embodiment, the composition extract powder is prepared as follows:
Step a takes ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, four taste of the flower bud of lily magnolia to obtain volatile oil using extraction by steam distillation volatile oil
A, the aqueous B and dregs of a decoction C after distillation;
Step b will volatilize oily A and be included with betadex;Dregs of a decoction C extracting in water, obtains extracting solution D;
Step c, takes Radix Astragali, radix saposhnikoviae, Rhizoma Atractylodis Macrocephalae, Radix Paeoniae Alba, dark plum, Schisandra chinensis, rhizoma anemarrhenae, eight taste of Radix Glycyrrhizae, and extracting in water must extract
Liquid E;
Step d merges the aqueous B after extracting solution D, E and step a distillation, and adding ethyl alcohol to alcohol content is 50%~70%,
Filtrate recycling ethanol is taken, dry, pulverize, is mixed with betadex inclusion compound to get composition extract powder.
Wherein, the ratio of step b volatile oil and betadex is 1: (5~10);It is preferred that 1: (6~9);Most preferably 1: 8;
Step d adds ethyl alcohol to alcohol content to be 60%.
As the sixth aspect of the invention, the present invention provides the detection method of the Chinese medicinal composition granules, described
Method includes:
Assay A:
Determination condition: using octadecylsilane key and silica gel as filler;With acetonitrile-water (25~35): (65~75) are
Mobile phase;
The preparation of reference substance solution: taking Astragaloside IV reference substance, and organic solvent dissolution obtains reference substance solution;
The preparation of test solution: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Measuring method: drawing reference substance solution and test solution respectively, injects liquid chromatograph, measurement.
Assay B:
Determination condition: using octadecylsilane key and silica gel as filler;With methanol-water (10~20): (80~90) are
Mobile phase;
The preparation of reference substance solution: taking Paeoniflorin reference substance, and organic solvent dissolution obtains reference substance solution;
The preparation of test solution: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Measuring method: drawing reference substance solution and test solution respectively, injects liquid chromatograph, measurement.
In a particular embodiment, the organic solvent be selected from methanol, acetone, n-butanol, ethyl acetate, petroleum ether,
Any one in chloroform;The extracting method is selected from ultrasonic extraction, refluxing extraction, cold soaking extraction, seepage pressure effects, Microwave Extraction
The methods of.
In a preferred embodiment, it in the preparation of assay A test solution, is extracted using methanol eddy;More into one
Step, test solution the preparation method is as follows: take Chinese medicinal composition granules, add methanol cold soaking to stay overnight, refluxing extraction, filtrate is returned
It receives solvent and is concentrated to dryness, residue is dissolved in water, and is saturated with water n-butanol shaking and extracts, n-butanol extracting liquid liquid is washed with ammonia solution
Wash, ammonia solution discards, and the n-butanol liquid after washing is evaporated, residue add methanol make dissolution to get.
In a preferred embodiment, mobile phase is acetonitrile-water (31: 69) in assay A;It is flowed in assay B
Mutually it is methanol-water (15: 85).
In a particular embodiment, the detection method further includes one or more of following identification:
Identify A:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Control medicinal material solution preparation: taking Radix Astragali control medicinal material, and organic solvent extracts, and obtains control medicinal material solution;
Detection: drawing test solution and control medicinal material solution respectively, and point is on same silica gel plate, with chloroform-first
Alcohol-water (10~15): (5~9): (1~3) is solvent, and expansion is heated to the colour developing of 10% ethanol solution of sulfuric acid at 105 DEG C
Spot development is clear;
Identify B:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Reference substance solution preparation: taking Paeoniflorin reference substance, and organic solvent dissolution obtains reference substance solution;
Detection: drawing test solution and control solution respectively, and point is on same silica gel plate, with chloroform-acetic acid
Ethyl ester-methyl alcohol-formic acid (5~10): (1~3): (2~7): (0.1~0.3) is solvent, and expansion is molten with 5% vanillin-sulfuric acid
Liquid colour developing, it is clear to be heated to spot development at 105 DEG C;
Identify C:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Control medicinal material solution preparation: taking windproof control medicinal material, and organic solvent extracts, and obtains control medicinal material solution;
Reference substance solution preparation: taking 5-O- methyl visamminol glycosides reference substance, and it is molten to obtain reference substance for organic solvent dissolution
Liquid;
Detection: drawing test solution, control medicinal material solution and control medicinal material solution respectively, point on same silica gel plate,
With chloroform-methanol (3~8): (0.5~2) is solvent, and expansion is set and inspected under ultraviolet lamp (254nm);
Identify D:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Control medicinal material solution preparation: taking Schisandra chinensis control medicinal material, and organic solvent extracts, and obtains control medicinal material solution;
Reference substance solution preparation: taking schizandrin reference substance, and organic solvent dissolution obtains reference substance solution;
Detection: drawing test solution, control medicinal material solution and control medicinal material solution respectively, point on same silica gel plate,
With petroleum ether-Ethyl formate-formic acid (12~18): (4~8): (0.5~2) is solvent, and expansion is set under ultraviolet lamp (254nm)
It inspects;
Identify E:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Control medicinal material solution preparation: extracting liquorice control medicinal material, organic solvent extract, and obtain control medicinal material solution;
Detection: drawing test solution and control medicinal material solution respectively, and point is on same silica gel plate, with ethyl acetate-first
Acid-glacial acetic acid-water (12~18): (0.5~2): (0.5~2): (1~3) is solvent, and expansion is molten with 10% sulfuric acid ethyl alcohol
It is clear to be heated to spot development at 105 DEG C for liquid;
In a particular embodiment, the organic solvent be selected from methanol, acetone, n-butanol, ethyl acetate, petroleum ether,
Any one in chloroform;The extracting method is selected from ultrasonic extraction, refluxing extraction, cold soaking extraction, seepage pressure effects, Microwave Extraction
The methods of.
In a preferred embodiment, identify A sample solution preparation method are as follows: take Chinese medicinal composition granules, add first
Alcohol ultrasonic extraction, filtrate recycling design are simultaneously concentrated to dryness, and residue is dissolved in water, and are saturated with water n-butanol shaking and are extracted, n-butanol
Extracting solution liquid is washed with ammonia solution, and ammonia solution discards, and the n-butanol liquid after washing is evaporated, residue add methanol make dissolution to get.
Each bulk pharmaceutical chemicals of the present invention are recorded in " Chinese Pharmacopoeia " 2015 editions, and concocting method is " pharmacopeia " record method.
Radix Astragali in prescription of the present invention, sweet, warm, return lung, the spleen channel, invigorating qi for strengthening superficies, strengthening spleen and tonifying lung;Ramulus cinnamomi, pungent, sweet, warm is returned
The heart, lung, bladder meridian, warming meridian, helps yang transforming qi;Two medicines are total to the function of figure warming Yang, enlivening spleen benefit lung, with for monarch drug in a prescription.Ministerial drug one is (pungent
It is smooth), it is pungent, warm, it attaches to the lung and stomach meridians, function is apt to expelling cold, eliminating, clearing the nasal passage, nasal obstruction, the key medicine of nasosinusitis;Ministerial drug two (radix saposhnikoviae), it is pungent, sweet, micro-
Temperature returns bladder, liver, the spleen channel, expels pathogenic wind from the body surface, and tool rises the property of clear eliminating dampness, can be used for the insufficiency of the spleen with overabundance of dampness, lucid yang failing to raise;Ministerial drug three is (white
Art), bitter, sweet, warm, returns spleen, stomach meridian, strengthening the spleen and replenishing qi;Ministerial drug four (rhizoma zingiberis), pungent, hot, returns spleen, stomach, kidney, the heart, lung channel, middle benefit gas dissipate
It is cold, recover Yang and smooth venation, eliminating dampness dissolving phlegm;Ministerial drug five (cortex cinnamomi), pungent, sweet, big heat.Return kidney, spleen, the heart, Liver Channel, mends fire supporing yang, cold dispelling stops
Bitterly, warming meridians and promoting circulation of qi, let the fire back to its origin;Five medicines or walk to express key or warming meridians and promoting circulation of qi or strengthening the spleen and replenishing qi to help lung be ministerial drug altogether.Adjutant one
(Radix Paeoniae Alba), it is bitter, sour, slightly cold, return liver, the spleen channel, yin, the analgesic of soft liver, suppressing liver-YANG are held back in blood-nourishing, and astringing lung-QI yin consolidates lung body, smooth mechanism of qi benefit
Qi and blood;Adjutant two (dark plum), it is sour, puckery, it puts down, returns liver, spleen, lung, large intestine channel, astringe the lung to stop cough, relieving diarrhea with astringents promotes the production of body fluid to quench thirst, adjutant
Three (Schisandra chinensis), acid, sweet, warm, return lung, the heart, kidney channel, astringency inducing, supplementing qi and promoting the production of body fluid, tonifying kidney and calming nerves are kind to control long-last cough and virtual asthma, saliva wound
It is thirsty, feeble pulse of losing heart, Heat Diabetes, palpitation and insomnia;Adjutant four (rhizoma anemarrhenae), it is bitter, sweet, it trembles with fear, return lung, stomach, kidney channel, clearing heat-fire,
Fluid dryness, the anti-hot and suffocating interior life of deficiency of both lung and spleen;Above-mentioned four medicine, convergence gas yin, facilitates strengthening spleen and tonifying lung, reaches the same goal by different routes and mutually shares as assistant
Medicine.Radix Glycyrrhizae, sweet and neutral, return heart, lung, spleen, stomach meridian are invigorated the spleen and benefited qi, clearing heat and detoxicating, expelling phlegm and arresting coughing, coordinating the drug actions of a prescription, with to make medicine.Entirely
" gentle pungent Jin Peiben " principle is closed by side, and acridness and sweetness activating yang, sour-sweet herbs nourishing yin, cold temperature, which dissipate to receive, to be used in combination, and is searched for the primary cause of disease in treatment, play altogether strengthening spleen and tonifying lung,
The function of warm tonneau key.
Clinical research show 100 AR patients after the traditional chinese medicine composition of the invention is treated, effective 76, effective 15, nothing
Effect 9, effective percentage 91%.Symptom and sign integral, VAS scale integral and the former total IgE of serum anaphylaxis are aobvious before relatively treating after treatment
Writing reduces (P < 0.01).Show that the traditional chinese medicine composition of the invention can improve the clinical symptoms and sign of AR patient, reduces patient's blood
The clear total IgE of anaphylactogen is horizontal, significant in efficacy.
Detailed description of the invention
Fig. 1 is Astragaloside IV reference substance solution chromatogram
Fig. 2 is Chinese medicine composition test solution chromatogram
Fig. 3 is Radix Astragali feminine gender test solution chromatogram
Fig. 4 is Paeoniflorin reference substance solution chromatogram
Fig. 5 is Chinese medicine composition test solution chromatogram
Fig. 6 is Radix Paeoniae Alba feminine gender test solution chromatogram
Specific embodiment
1 Chinese medicine composition decoction of embodiment
Prescription: Radix Astragali 21g, ramulus cinnamomi 9g, flower bud of lily magnolia 6g, radix saposhnikoviae 7g, Rhizoma Atractylodis Macrocephalae 9g, rhizoma zingiberis 6g, cortex cinnamomi 3g, Radix Paeoniae Alba 6g, dark plum
12g, Schisandra chinensis 6g, rhizoma anemarrhenae 6g, Radix Glycyrrhizae 6g;
Preparation method: taking each bulk pharmaceutical chemicals in proportion, and water boiling and extraction obtains Chinese medicine composition decoction.
2 Chinese medicine composition decoction of embodiment
Prescription: Radix Astragali 16g, ramulus cinnamomi 11g, flower bud of lily magnolia 5g, radix saposhnikoviae 8g, Rhizoma Atractylodis Macrocephalae 8g, rhizoma zingiberis 7g, cortex cinnamomi 2g, Radix Paeoniae Alba 7g, dark plum
11g, Schisandra chinensis 7g, rhizoma anemarrhenae 5g, Radix Glycyrrhizae 7g;
Preparation method: taking each bulk pharmaceutical chemicals in proportion, and water boiling and extraction obtains Chinese medicine composition decoction.
3 Chinese medicine composition decoction of embodiment
Prescription: Radix Astragali 24g, ramulus cinnamomi 8g, flower bud of lily magnolia 7g, radix saposhnikoviae 6g, Rhizoma Atractylodis Macrocephalae 11g, rhizoma zingiberis 5g, cortex cinnamomi 4g, Radix Paeoniae Alba 5g, dark plum
14g, Schisandra chinensis 5g, rhizoma anemarrhenae 7g, Radix Glycyrrhizae 5g;
Preparation method: taking each bulk pharmaceutical chemicals in proportion, and water boiling and extraction obtains Chinese medicine composition decoction.
4 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 21g, ramulus cinnamomi 9g, flower bud of lily magnolia 6g, radix saposhnikoviae 7g, Rhizoma Atractylodis Macrocephalae 9g, rhizoma zingiberis 6g, cortex cinnamomi 3g, Radix Paeoniae Alba 6g, dark plum
12g, Schisandra chinensis 6g, rhizoma anemarrhenae 6g, Radix Glycyrrhizae 6g;
Preparation method:
Ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, the flower bud of lily magnolia extract volatile oil 10 hours, and volatile oil is included with betadex, the water after distillation
Liquid is spare, and the dregs of a decoction add 8 times of amount water to extract 1 hour, and filtration, the another device of filtrate stores for future use;
Eight taste such as remaining Radix Astragali, Radix Paeoniae Alba adds water to cook three times, 1.5 hours every time, collecting decoction, filtration, filtrate with it is above-mentioned
Two liquid merge, and are concentrated into the clear cream that relative density is 1.15~1.20 (60 DEG C), add ethyl alcohol to make alcohol content up to 60%, stirring is quiet
It sets overnight, filtrate recycling ethanol, filtrate is concentrated into right amount, is dried under reduced pressure, and fine powder is ground into, with above-mentioned betadex inclusion compound
It mixes, obtains composition extract powder;
Composition extract powder is uniformly mixed with dextrin by 3: the 2 auxiliary ratio of medicine, wet granulation, is wetting with 94% ethyl alcohol
Agent, 74% hereinafter, preparing particle, dry, packaging gets product particle for envionmental humidity control.
The research of the invention finds that wanting strictly controlled environment relative humidity 74% hereinafter, if relatively wet in pelletization
Degree is higher than 74%, and it will cause the material moisture absorption, granulation is difficult.
5 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 16g, ramulus cinnamomi 11g, flower bud of lily magnolia 5g, radix saposhnikoviae 8g, Rhizoma Atractylodis Macrocephalae 8g, rhizoma zingiberis 7g, cortex cinnamomi 2g, Radix Paeoniae Alba 7g, dark plum
11g, Schisandra chinensis 7g, rhizoma anemarrhenae 5g, Radix Glycyrrhizae 7g;
Preparation method is the same as embodiment 4.
6 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 24g, ramulus cinnamomi 8g, flower bud of lily magnolia 7g, radix saposhnikoviae 6g, Rhizoma Atractylodis Macrocephalae 11g, rhizoma zingiberis 5g, cortex cinnamomi 4g, Radix Paeoniae Alba 5g, dark plum
14g, Schisandra chinensis 5g, rhizoma anemarrhenae 7g, Radix Glycyrrhizae 5g;
Preparation method is the same as embodiment 4.
7 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 18g, ramulus cinnamomi 10g, flower bud of lily magnolia 5g, radix saposhnikoviae 9g, Rhizoma Atractylodis Macrocephalae 7g, rhizoma zingiberis 8g, cortex cinnamomi 3g, Radix Paeoniae Alba 5g, dark plum
12g, Schisandra chinensis 5g, rhizoma anemarrhenae 8g, Radix Glycyrrhizae 5g;
Preparation method:
Ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, the flower bud of lily magnolia extract volatile oil 12 hours, and volatile oil is included with betadex, the water after distillation
Liquid is spare, and the dregs of a decoction add 10 times of amount water to extract 1 hour, and filtration, the another device of filtrate stores for future use;
Eight taste such as remaining Radix Astragali, Radix Paeoniae Alba adds water to cook three times, 1.5 hours every time, collecting decoction, filtration, filtrate with it is above-mentioned
Two liquid merge, and are concentrated into the clear cream that relative density is 1.15~1.20 (60 DEG C), add ethyl alcohol to make alcohol content up to 70%, stirring is quiet
It sets overnight, filtrate recycling ethanol, filtrate is concentrated into right amount, is dried under reduced pressure, and fine powder is ground into, with above-mentioned betadex inclusion compound
It mixes, obtains composition extract powder;
Composition extract powder is uniformly mixed with dextrin by 3: the 1 auxiliary ratio of medicine, wet granulation, is wetting with 94% ethyl alcohol
Agent, 74% hereinafter, preparing particle, dry, packaging gets product particle for envionmental humidity control.
8 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 23g, ramulus cinnamomi 7g, flower bud of lily magnolia 8g, radix saposhnikoviae 6g, Rhizoma Atractylodis Macrocephalae 10g, rhizoma zingiberis 5g, cortex cinnamomi 3g, Radix Paeoniae Alba 8g, dark plum
10g, Schisandra chinensis 8g, rhizoma anemarrhenae 5g, Radix Glycyrrhizae 8g;
Preparation method is the same as embodiment 7.
9 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 12g, ramulus cinnamomi 15g, flower bud of lily magnolia 4g, radix saposhnikoviae 12g, Rhizoma Atractylodis Macrocephalae 6g, rhizoma zingiberis 10g, cortex cinnamomi 2g, Radix Paeoniae Alba 10g, crow
Plum 9g, Schisandra chinensis 10g, rhizoma anemarrhenae 4g, Radix Glycyrrhizae 10g;
Preparation method is the same as embodiment 7.
10 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 27g, ramulus cinnamomi 6g, flower bud of lily magnolia 10g, radix saposhnikoviae 5g, Rhizoma Atractylodis Macrocephalae 15g, rhizoma zingiberis 4g, cortex cinnamomi 5g, Radix Paeoniae Alba 4g, dark plum
18g, Schisandra chinensis 4g, rhizoma anemarrhenae 10g, Radix Glycyrrhizae 4g;
Preparation method:
Ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, the flower bud of lily magnolia extract volatile oil 8 hours, and volatile oil is included with betadex, the water after distillation
Liquid is spare, and the dregs of a decoction add 15 times of amount water to extract 1 hour, and filtration, the another device of filtrate stores for future use;
Eight taste such as remaining Radix Astragali, Radix Paeoniae Alba adds water to cook three times, 1.5 hours every time, collecting decoction, filtration, filtrate with it is above-mentioned
Two liquid merge, and are concentrated into the clear cream that relative density is 1.15~1.20 (60 DEG C), add ethyl alcohol to make alcohol content up to 50%, stirring is quiet
It sets overnight, filtrate recycling ethanol, filtrate is concentrated into right amount, is dried under reduced pressure, and fine powder is ground into, with above-mentioned betadex inclusion compound
It mixes, obtains composition extract powder;
Composition extract powder is uniformly mixed with dextrin by 3: the 2 auxiliary ratio of medicine, wet granulation, is wetting with 95% ethyl alcohol
Agent, 74% hereinafter, preparing particle, dry, packaging gets product particle for envionmental humidity control.
11 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 9g, ramulus cinnamomi 20g, flower bud of lily magnolia 3g, radix saposhnikoviae 16g, Rhizoma Atractylodis Macrocephalae 4g, rhizoma zingiberis 14g, cortex cinnamomi 1g, Radix Paeoniae Alba 14g, dark plum
7g, Schisandra chinensis 14g, rhizoma anemarrhenae 3g, Radix Glycyrrhizae 14g;
The preparation method is the same as that of Example 10.
12 Chinese medicinal composition granules of embodiment
Prescription: Radix Astragali 35g, ramulus cinnamomi 4g, flower bud of lily magnolia 14g, radix saposhnikoviae 4g, Rhizoma Atractylodis Macrocephalae 20g, rhizoma zingiberis 3g, cortex cinnamomi 6g, Radix Paeoniae Alba 3g, dark plum
22g, Schisandra chinensis 3g, rhizoma anemarrhenae 14g, Radix Glycyrrhizae 3g;
The preparation method is the same as that of Example 10.
13 Chinese medicine composition decoction of embodiment
Prescription: Radix Astragali 7g, ramulus cinnamomi 25g, flower bud of lily magnolia 2g, radix saposhnikoviae 22g, Rhizoma Atractylodis Macrocephalae 3g, rhizoma zingiberis 18g, cortex cinnamomi 1g, Radix Paeoniae Alba 18g, dark plum
5g, Schisandra chinensis 18g, rhizoma anemarrhenae 2g, Radix Glycyrrhizae 18g;
Preparation method: taking each bulk pharmaceutical chemicals in proportion, and water boiling and extraction obtains Chinese medicine composition decoction.
14 Chinese medicine composition decoction of embodiment
Prescription: Radix Astragali 45g, ramulus cinnamomi 3g, flower bud of lily magnolia 18g, radix saposhnikoviae 3g, Rhizoma Atractylodis Macrocephalae 25g, rhizoma zingiberis 2g, cortex cinnamomi 7g, Radix Paeoniae Alba 3g, dark plum
26g, Schisandra chinensis 2g, rhizoma anemarrhenae 18g, Radix Glycyrrhizae 2g;
Preparation method: taking each bulk pharmaceutical chemicals in proportion, and water boiling and extraction obtains Chinese medicine composition decoction.
15 Chinese medicine composition decoction of embodiment
Prescription: Radix Astragali 15g, ramulus cinnamomi 12g, flower bud of lily magnolia 5g, radix saposhnikoviae 9g, Rhizoma Atractylodis Macrocephalae 7g, rhizoma zingiberis 8g, cortex cinnamomi 2g, Radix Paeoniae Alba 8g, dark plum
10g, Schisandra chinensis 8g, rhizoma anemarrhenae 5g, Radix Glycyrrhizae 8g;
Preparation method: taking each bulk pharmaceutical chemicals in proportion, and water boiling and extraction obtains Chinese medicine composition decoction.
16 Chinese medicine composition tablet of embodiment
Prescription: Radix Astragali 25g, ramulus cinnamomi 7g, flower bud of lily magnolia 8g, radix saposhnikoviae 6g, Rhizoma Atractylodis Macrocephalae 12g, rhizoma zingiberis 5g, cortex cinnamomi 4g, Radix Paeoniae Alba 5g, dark plum
15g, Schisandra chinensis 5g, rhizoma anemarrhenae 8g, Radix Glycyrrhizae 5g;
Preparation method:
Ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, the flower bud of lily magnolia extract volatile oil 10 hours, and volatile oil is included with betadex, the water after distillation
Liquid is spare, and the dregs of a decoction add 8 times of amount water to extract 1 hour, and filtration, the another device of filtrate stores for future use;
Eight taste such as remaining Radix Astragali, Radix Paeoniae Alba adds water to cook three times, 1.5 hours every time, collecting decoction, filtration, filtrate with it is above-mentioned
Two liquid merge, and are concentrated into the clear cream that relative density is 1.15~1.20 (60 DEG C), add ethyl alcohol to make alcohol content up to 60%, stirring is quiet
It sets overnight, filtrate recycling ethanol, filtrate is concentrated into right amount, is dried under reduced pressure, and fine powder is ground into, with above-mentioned betadex inclusion compound
It mixes, obtains composition extract powder;
Tablet is made in composition extract powder addition conventional tablet auxiliary material.
17 Chinese medicine composition tablet of embodiment
Prescription: Radix Astragali 30g, ramulus cinnamomi 5g, flower bud of lily magnolia 12g, radix saposhnikoviae 5g, Rhizoma Atractylodis Macrocephalae 18g, rhizoma zingiberis 4g, cortex cinnamomi 5g, Radix Paeoniae Alba 4g, dark plum
20g, Schisandra chinensis 4g, rhizoma anemarrhenae 12g, Radix Glycyrrhizae 4g;
The preparation method is the same as that of Example 16.
18 Chinese medicine composition tablet of embodiment
Prescription: Radix Astragali 10g, ramulus cinnamomi 18g, flower bud of lily magnolia 4g, radix saposhnikoviae 14g, Rhizoma Atractylodis Macrocephalae 5g, rhizoma zingiberis 12g, cortex cinnamomi 2g, Radix Paeoniae Alba 12g, crow
Plum 8g, Schisandra chinensis 12g, rhizoma anemarrhenae 4g, Radix Glycyrrhizae 12g;
The preparation method is the same as that of Example 16.
Embodiment 19
Prescription: Astragalus Root P.E 21g, Ramulus Cinnamomi extract 9g, Flos Magnoliae extract 6g, Radix Saposhnikoviae extract 7g, Rhizoma Atractylodis Macrocephalae extract
9g, Rhizoma Zingiberis extract 6g, cinnamomum cassia extract 3g, radix paeoniae alba extraction 6g, smoked plum extractive 12g, Schisandra chinens P.E 6g, rhizoma anemarrhenae mention
Take object 6g, licorice 6g.
Embodiment 20
Prescription: Astragalus Root P.E 16g, Ramulus Cinnamomi extract 11g, Flos Magnoliae extract 5g, Radix Saposhnikoviae extract 8g, Rhizoma Atractylodis Macrocephalae extract
8g, Rhizoma Zingiberis extract 7g, cinnamomum cassia extract 2g, radix paeoniae alba extraction 7g, smoked plum extractive 11g, Schisandra chinens P.E 7g, rhizoma anemarrhenae mention
Take object 5g, licorice 7g.
Embodiment 21
Prescription: Astragalus Root P.E 24g, Ramulus Cinnamomi extract 8g, Flos Magnoliae extract 7g, Radix Saposhnikoviae extract 6g, Rhizoma Atractylodis Macrocephalae extract
11g, Rhizoma Zingiberis extract 5g, cinnamomum cassia extract 4g, radix paeoniae alba extraction 5g, smoked plum extractive 14g, Schisandra chinens P.E 5g, rhizoma anemarrhenae
Extract 7g, licorice 5g.
Embodiment 22
Prescription: Astragalus Root P.E 18g, Ramulus Cinnamomi extract 10g, Flos Magnoliae extract 5g, Radix Saposhnikoviae extract 9g, Rhizoma Atractylodis Macrocephalae extract
7g, Rhizoma Zingiberis extract 8g, cinnamomum cassia extract 3g, radix paeoniae alba extraction 5g, smoked plum extractive 12g, Schisandra chinens P.E 5g, rhizoma anemarrhenae mention
Take object 8g, licorice 5g.
Embodiment 23
Prescription: Astragalus Root P.E 23g, Ramulus Cinnamomi extract 7g, Flos Magnoliae extract 8g, Radix Saposhnikoviae extract 6g, Rhizoma Atractylodis Macrocephalae extract
10g, Rhizoma Zingiberis extract 5g, cinnamomum cassia extract 3g, radix paeoniae alba extraction 8g, smoked plum extractive 10g, Schisandra chinens P.E 8g, rhizoma anemarrhenae
Extract 5g, licorice 8g.
Ramulus Cinnamomi extract described in above embodiments 19-23, Rhizoma Zingiberis extract, cinnamomum cassia extract, Flos Magnoliae extract are respectively
Ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, the flower bud of lily magnolia extractive of volatile oil;The Astragalus Root P.E, Radix Saposhnikoviae extract, Rhizoma Atractylodis Macrocephalae extract, Radix Paeoniae Alba mention
Take object, smoked plum extractive, Schisandra chinens P.E, anemarrhena asphodefoides extract, licorice be respectively Radix Astragali, radix saposhnikoviae, Rhizoma Atractylodis Macrocephalae, Radix Paeoniae Alba,
The extract that dark plum, Schisandra chinensis, rhizoma anemarrhenae, Radix Glycyrrhizae prepare through aqueous extraction-alcohol precipitation technology, the alcohol precipitation concentration are 60%.
The experiment of 24 clinical effectiveness of embodiment
1 data and method
1.1 general information
Control group: it goes to a doctor in October, 2009 in July, 2010 in China-Japan Friendship Hospital, meet syndrome of qi deficiency (deficiency of both lung and spleen)
AR patient cases data 20, the age (42.1 ± 12.3) year, wherein male 12, female 8.
Experimental group: it goes to a doctor in October, 2010 in October, 2014 in China-Japan Friendship Hospital, meet syndrome of qi deficiency (deficiency of both lung and spleen)
AR patient cases data 100, the age (44.2 ± 14.1) year, wherein male 57, female 43.
1.2 diagnostic criteria
The diagnostic criteria of AR is referring to Chinese Journal of Otorhinolaryngology Head And Neck Surgery editorial board, Chinese Medical Association's otolaryngology-head and neck
" diagnosis of allergic rhinitis and the treatment guidelines (2009) " that surgery branch nasology group is formulated are drafted.Chinese medical discrimination diagnostic criteria
It combines and faces referring to National Standard of the People's Republic of China GB/T16751.2-1997 " tcm clinical practice diagnosis and treatment term syndrome part "
Bed is studied and accumulates Consensus of experts triplicity formulation, specific manifestation are as follows: nasal obstruction caused by deficiency of both lung and spleen, the failure of lung key, sneeze frequency
Make, runny nose is like water;Or with fatigue and weakness, or with cold, anemophobia, or with skin wheal or Na Gu be not fragrant or abdominal distention after meal, or
Companion defecates, and uncomfortable, half congealed, discharge is powerless, or sleep not firm or dreaminess or dry or dry hardship, or white glutinous with cough, or with coughing up
Phlegm;Tongue is fat to have an indentation greatly, and tongue fur is white or white micro- greasy or Bai Weihuang;Arteries and veins is thin and delicate or heavy thin.
It is included in standard
1. being diagnosed as AR and meeting the card type person of Chinese medicine syndrome of qi deficiency (deficiency of both lung and spleen);2. the age between 18-70 years old, gender
It is unlimited;3. the course of disease was at 1 year or more.
Exclusion criteria
1. gestation or breast feeding women;2. with other serious disease patients;3. merging mental disease or serious neurological official
It can disease patient;4. subjective malaise symptoms person cannot be expressed;5. receiving to suck corticosteroid treatment simultaneously, or there is nose other are different
The often person that may interfere with curative effect evaluation.
1.3 curative effect index
1.3.1 curative effect index
Doctor records patient symptom integral joint observation patient sign integral (the two integral is added, to treat total mark).
1.3.1.1 symptom integral standard: being shown in Table 1.
1 symptom integral standard of table
Classification is scored | Rhiocnesmus | Sneeze * | Runny nose * * | Nasal obstruction |
0 point | Nothing | Nothing | Nothing | Nothing |
1 point | Interruption | 3-5 | < 4 | Feel when conscious air-breathing |
2 points | Ant row sense, but can endure | 6-10 | 5-9 | Intermittent or interactivity |
3 points | Ant row sense, unbearably | > 11 | > 10 | Almost whole day mouth breathing |
Note: * 1 continuous sneeze number;* blows nose number daily.
1.3.1.2 somatic feature score standard: being shown in Table 2.
2 somatic feature score standard of table
1.3.2 secondary efficacy index
1.3.2.1 patient voluntarily assesses the visual analogue scale scale (visual analog scale, VAS) of its symptom
Score: advising patient according to nearest 1 week Symptoms, mark the resulting score of the symptom on scale, detailed programs be rhiocnesmus,
Nasal obstruction, sneeze, runny nose, eye symptom include that eye itches, is envious and swells, shed tears, ophthalmodynia, feel suffocated, wheeze, coughing, pressure in chest.
Slide calliper rule shape is made in VAS, and respectively there is the lateral scale of 10cm long on two sides, and slide calliper rule one side has smile and pain at the both ends of horizontal line
Bitter two kinds of different cartoon expressions, slide calliper rule another side horizontal line one end mark 0 indicate asymptomatic;The other end is 10, indicates that symptom is extremely tight
Weight;Sandwich digit indicates different degrees of.When questionnaire survey, slide calliper rule card expression one is facing patient by researcher, allow patient self
Rhinitis and related symptoms severity, the shifting mark on mobile slide calliper rule are assessed, researcher records the corresponding numerical value in the slide calliper rule other side.Meter
Calculate the sum of each symptom VAS (Multi-VAS) scoring.(Uni-VAS, total symptom VAS are that patient exists to the another record total symptom VAS of patient
Its overall sense of discomfort of self-assessment is marked on the slide calliper rule of 0-10, patient's " your disease of your common sensation is generally inquired by researcher
Sick distress level is how " and allow patient that the number on slide calliper rule is specified to assess).
1.3.2.2 the former total IgE measurement of serum anaphylaxis:
The former total IgE of pretherapy and post-treatment serum anaphylaxis is detected by China-Japan Friendship Hospital's division of respiratory disease control laboratory, < 60kU/L
It is judged as negative;>=60kU/L is judged as positive.
1.4 curative effect determinate standard
Referring to the curative effect determinate standard of 2009 " diagnosis of allergic rhinitis and treatment guidelines (2009) ".According to pretherapy and post-treatment
The summation scored, curative effect are evaluated by following equation: the preceding total mark of (total mark after total mark-treatment before treating)/treatment ×
100%.>=51% is effective, 50%-21% be it is effective ,≤20% is invalid.
1.5 method
1.5.1 drug
Control drug: raw Radix Astragali 21g, ramulus cinnamomi 9g, windproof 6g, flower bud of lily magnolia 6g, rhizoma atractylodis macrocephalae 9g, rhizoma zingiberis 6g, Radix Paeoniae Alba 6g, licorice
6g;
Experimental drug: Radix Astragali 21g, ramulus cinnamomi 9g, flower bud of lily magnolia 6g, radix saposhnikoviae 7g, Rhizoma Atractylodis Macrocephalae 9g, rhizoma zingiberis 6g, cortex cinnamomi 3g, Radix Paeoniae Alba 6g, crow
Plum 12g, Schisandra chinensis 6g, rhizoma anemarrhenae 6g, Radix Glycyrrhizae 6g.
1.5.2 instructions of taking
Daily 1 dose, 2 clothes of decocting point, 7d is 1 course for the treatment of, even served 2 courses for the treatment of, total 14d.
1.5.3 statistical method
It counted, analyzed using 17.0 pairs of SPSS every data, parameters are indicated with x- ± s, using pairing
Wilcoxon signed rank sum test is that difference is statistically significant with P < 0.05.
2 experimental results
2.1 efficient results
It the results are shown in Table 1.
3 clinical efficacy result of table
Sample size | Obvious effective rate | It is efficient | |
Control group | 20 | 4 (20%) | 15 (75%) |
Experimental group | 100 | 76 (76%) | 15 (15%) |
The result shows that obvious effective rate 20% in the experiment of 20 people of control group, effective percentage 75%;Experimental group expands sample size,
Prescription is optimized simultaneously, more obvious improvement and alleviation of the improvement prescription to patient clinical symptom, wherein 100 patients
Middle obvious effective rate is 76%, total effective rate 91%.
The pretherapy and post-treatment symptom integral result of 2.2 experimental groups
It the results are shown in Table 4.
The pretherapy and post-treatment shape somatic feature score of 4 experimental group of table, 100 patients compares (x- ± s divides)
Project | Before treatment | After treatment |
Rhiocnesmus | 2.58±0.50 | 1.20±0.55** |
Sneeze | 1.97±0.86 | 0.08±0.27** |
Runny nose | 2.72±0.53 | 0.06±0.23** |
Nasal obstruction | 2.44±0.77 | 0.76±0.71** |
Sign score | 2.04±0.27 | 0.89±0.72** |
Note: compared with before treatment, * * P < 0.01.Following table is same.
The pretherapy and post-treatment VAS total mark of 2.3 experimental groups, total symptom VAS compare
It the results are shown in Table 5.Before VAS total mark and total symptom VAS are substantially less than treatment after treatment, difference is statistically significant
(P < 0.01).
5 100 patients of table pretherapy and post-treatment VAS and the total IgE of anaphylactogen compare (x- ± s divides)
Project | Before treatment | After treatment |
VAS total mark | 16.43±2.63 | 5.21±2.57** |
Total symptom VAS | 8.24±1.90 | 3.17±3.20** |
IgE | 77.56±11.16 | 59.63±9.63* |
The total IgE value of the pretherapy and post-treatment anaphylactogen of 2.4 experimental groups compares.
It is significantly reduced after the total IgE treatment of anaphylactogen, or even normal level (< 60kU/L) can be down to, difference has statistics meaning
Adopted (P < 0.01).
Actual situation cold-heat jumble is presented in AR interpretation of the cause, onset and process of an illness essence, and Characteristics of Syndromes is related to the function of multiple internal organs based on deficiency of both lung and spleen
It is deficient, so, only progress " using lung spleen as the internal organs integral syndrome differentiation of core ", with the treatment principle of " gentle pungent Jin Peiben ",
AR could thoroughly be cured.This research is by illustrating Chinese medicine of the present invention to 100 AR patient itself cross-reference clinical research observations
Composition treatment AR clinical efficacy is preferable, and before primary and secondary wants curative effect index to be superior to treatment after treatment, total effective rate reaches 91%;
Organic conception and the syndrome differentiation of zang-fu viscera for sufficiently inheriting Chinese medicine are theoretical, reflect AR patient and lose by the viscera function of core of lung spleen
It adjusts, the essential problem of physical function state entire lowering;It compensates for current Chinese patent drug mostly to set about from real example, is to dispel exterior syndrome
It is main, righting is slightly taken into account, it is overall to the incomplete defect of human righteousness's solid protection.
25 pharmacodynamic study of embodiment
This research be based on the traditional chinese medicine composition of the invention effect and AR pathogenesis, establish cavy AR model caused by TDI,
The passive skin mistake of AR model caused by egg protein, xylene-induced ear swelling in mice model, swollen hyperplasia of rat granuloma model, allotransplantation in rats
Quick reaction model and mouse ear xenogenesis passive cutaneous anaphylaxis model, observation the traditional chinese medicine composition of the invention make the treatment of AR
With to provide experimental basis for its further clinical application.
The influence of cavy allergic rhinitis caused by 1 couple of TDI
AR guinea pig model is established using 10%TDI olive oil solution nasal drip, after model success, after successive administration 14d, is seen
It examines each group behaviouristics and scores, enzyme linked immunosorbent assay (ELISA) detects SERUM IgE, IFN-γ, IL-12, IL-4, IL-
6, nasal mucosa IL-6, TNF-α, IFN-γ detect in TNF-α and the content of schneiderian membrance HIS, immunohistochemistry staining method
Content.Optical microphotograph microscopic observation nasal mucosa Pathologic changes and pathological score, and carry out eosinophil (EOS) meter
Number.
1.1 materials and methods
1.1.1 material
1.1.1.1 animal and grouping
SPF grades of cavys, 70 (Beijing Vital River Experimental Animals Technology Co., Ltd.), animal credit number is SCXK
(capital) 2012-0001,220~240g of weight, be randomly divided into normal group, model group, rhinitis group (0.41g/kg), chlorine thunder he
Determine group (0.91mg/kg), Chinese medicine composition high dose group (3.04g/kg), middle dose group (1.52g/kg), low dose group
(O.76g/kg), every group 10, male and female each 5.
1.1.1.2 drug and reagent
Loratadine tablet (Xi'an Yang Sen, batch number: 050218967);
Rhinitis piece (Chinese medicines group, batch number: 14272);
The traditional chinese medicine composition of the invention granule (is prepared, 1g is equivalent to 5.7577g crude drug amount, distilled water by 4 method of embodiment
It is spare to be configured to 0.304g/mL);
2,4- toluene di-isocyanate(TDI)s (TDI, sigma company, bw-d0007);
Olive oil (Chinese medicines group, batch number: 20140725);
Cavy IgE, IL-4, IL-6, IL-12, TNF-α, IFN-γ, HIS kit (Lan Ji Biotechnology Co., Ltd,
Batch number: 20150724);
Ethyl alcohol (Beijing chemical plant, batch number: 20140607);
Formalin (Tianjin good fortune morning chemical reagent factory, batch number: 20141020);
Dimethylbenzene (Tong Guang fine chemistry industry company, Beijing, batch number: 20140719);
Breathe out Rui Shi haematoxylin dye liquor (Beijing Shiji Heli Biological Technology Co., Ltd., batch number: 20141203);
Breathe out Rui Shi eosin stain (Beijing Shiji Heli Biological Technology Co., Ltd., product batch: 20141203);
DAB (Dako, batch number: K5007);
Anti-TNF alpha antibody (abcam, batch number: ab6671);
IL-6Polyclonal Antibody (proteintech, batch number: 21865-1-AP);
Interferon gamma Polyclonal Antibody (proteintech, batch number: 15365-1-
AP)。
1.1.1.3 laboratory apparatus
Microscope (Japanese OLYMPUS company), image analysis software (MIAS);
Adopt figure software Nikon NIS-Elements D 3.2 (Japanese Nikon company);
ASP300 takes off full-automatic multi-functional microplate reader: MULTISKAN MK3 (Thermo company);
GL-21M high speed freezing centrifuge (Shanghai Lu Xiangyi centrifuge Instrument Ltd.);
BX51 water dispenser (German LEICA company);
EG1150H embedding machine (German LEICA company);
RM2235 slicer (German LEICA company);
H I1 210 spreads out piece machine (German LEICA company).
1.2 method
1.2.1 model foundation
Using 10%TDI olive oil solution to cavy bilateral nostril collunarium (every 5 μ L of side), one time a day, it is changed to after continuous 7d
The next day 1 time.After the completion of daily collunarium, score according to following standards of grading: 1. rhiocnesmus: slight: dab nose several times, 1 point;Severe:
It is more than to scratch nasal surface, 2 points.2. sneeze: 1~3 1 point, 4~10 2 points, 11 or more 3 points.3. runny nose: it flow to prenaris, 1
Point;Beyond prenaris, 2 points;Runny nose is had one's face covered with 3 points.The allergic conditions of each group cavy in 30min are observed, each symptom integral is superimposed,
Total score is greater than 5 points of persons for modeling success.Normal group substitutes 10%TDI olive oil solution with olive oil, and method and steps is the same as cause
Quick group.
1.2.2 grouping and administration
Modeling the 8th day, according to neurological deficit score as a result, being grouped gastric infusion again, successive administration 14d, one time a day, TDI
The next day of being changed to collunarium with maintain allergic symptom until experiment terminate, continue to observe the symptoms after collunarium and score.Model group and normal
Group gives the distilled water stomach-filling of equivalent.
1.2.3 Testing index and method
1.2.3.1 animal behavioral study
The allergic conditions of each group cavy, and the record that scores are observed after administration 14d.
1.2.3.2 biochemical and amynologic index detection
After last excitation for 24 hours, 10% chloraldurate (300mg/kg) intraperitoneal injection of anesthesia, abdominal aortic blood, 3500r/
Min is centrifuged 10min.Serum is separated, is placed under -80 DEG C of refrigerators and saves.Serum is detected according to ELISA kit specification step
IgE, IFN-γ, IL-4, IL-6, IL-12, TNF-α are horizontal.Take cavy schneiderian membrance physiological saline be homogenized, ELISA method detection its
HIS content.
1.2.3.3 nasal mucosa pathological observation
5 cavys of every group of selection, open rapidly bridge of the nose, peel off upper jaw osseous part skin, the vestibular wall on the outside of the nasal septum of right side
It cuts, opens exposed nasal septum and bilateral nasal cavity upwards, take bilateral mucous membrane of nasal septum immediately later, be fixed on 10% neutral formalin
It is fixed in solution, paraffin embedding, slice, HE dyeing, observation schneiderian membrance pathological change, and carry out EOS counting.
HE staining procedure: the fixed sample of 10% neutral formalin, conventional alcohol dehydration, dimethylbenzene processing, stone
Wax embedding, 3 μm of serial section are dried spare.Dimethylbenzene I, II dewaxing 15min.Dehydrated alcohol I, dehydrated alcohol II, 95% second
Each 2min of aquation, flowing water rinse step by step for alcohol, 80% ethyl alcohol, and haematoxylin dyeing 10min washes 3min, color separation liquid 10s, washing 3
Secondary, 0.25% returns blue liquid 40s, washes 3 times, 1% eosin stains 1min, washes 3 times.95% ethyl alcohol I, 95% ethyl alcohol II take off step by step
Water 1min, dehydrated alcohol I, II are dehydrated 2min, dimethylbenzene I2min, the transparent 5min of dimethylbenzene II, III, neutral gum envelope step by step
Gu.
Pathological score standard: 0 grade, cilium is completely neat, and columnar epithelium is normal, a small amount of goblet cell, remembers 0 point;I grades, cup
Shape cytosis, the inflammatory cell that minority is dispersed in, the glandular hyperplasia of film lamina propria accounts for the 1/3 of mucosa lamina propria hereinafter, 1 point of note;
II grades, goblet cell gathers, and cilium lodging is irregular, accidental to fall off, and more acidophic cell is swum out of, and the body of gland of film lamina propria increases
It is raw to account for the 1/3~2/3 of mucosa lamina propria, remember 2 points;III level, goblet cell gather, and cilium largely falls off, epithelium damage layer or group
Texture disorder is knitted, a large amount of acidophic cells, which are swum out of, accounts for 2/3 or more of whole epithelial inflammation cells, and the glandular hyperplasia of film lamina propria accounts for
2/3 or more of mucosa lamina propria remembers 3 points.
1.2.3.4 immunohistochemical staining
Immunohistochemical staining (IHC) method detects nasal mucosa IFN-γ, IL-6, TNF-α expression.
Operating procedure: 2 μm of schneiderian membrance paraffin section are used to water with distillation washing 2 times through conventional dewaxing (dyeing with HE)
PBS (phosphate buffer) is rinsed 1 time, each 5min, pressure cooker antigen retrieval, and PBS is rinsed 2~3 times, each 5min;3%
H2O2 is stored at room temperature 10min;It is rinsed 2~3 times with PBS, each 5min.37 DEG C of primary antibody incubation 2h, PBS rinsing 3 times is added dropwise, every time
5min;Secondary antibody is added dropwise and is incubated at room temperature 50min, DAB (diaminobenzidine) 3~5min of colour developing grasps dyeing journey under the microscope
Degree, tap water rinse, haematoxylin redyeing 2min, and hydrochloride alcohol differentiation, dehydration, dimethylbenzene is transparent, neutral gum mounting.
Every picture randomly selects 5 visuals field under high power lens, and brown particle is positive mark in endochylema, using Image-
Pro Plus Version 6.0 analyzes software, analyzes image area according to formula (IOD/area) and it is close to acquire positive expression light
Angle value.
1.2.4 statistical method
All data are with mean ± standard deviationIt indicates, handled with 9.3 statistical software of SAS, data obey normal state
Distribution uses variance analysis, compares examined using t two-by-two, and data are disobeyed normal distribution or heterogeneity of variance and examined using nonparametric
It tests, wherein guinea-pig nasal non-cancer lesion degree is analyzed using Ridit method, is that difference has statistics meaning with P < 0.05 or P < 0.01
Justice.
1.3 experimental result
1.3.1 on the ethological influence of AR cavy
Occur rhiocnesmus, sneeze symptom after sensitized guinea pig 1d collunarium, in 30min, performance degree weight is different, but without bright
Aobvious runny nose symptom, since 4d, with the increase of collunarium number, there is clear nasal discharge in each group, and behaviouristics symptom is also more and more brighter
Aobvious, then without evident act symptom, hints model replicates successfully Normal group.It is administered since the 8th day, after 14d is administered, removes
Outside low dose group, each administration group symptom compared with model group is substantially reduced (P < 0.01 or P < 0.05).It the results are shown in Table 6.
6 each group cavy nose behaviouristics symptom score of table (N=10)
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01。
1.3.2 to the influence of AR guinea pig serum biochemical immunity index
1.3.2.1 to the influence of AR guinea pig serum IgE
Compared with normal group, model group serum IgE level is significantly raised (P < 0.05).Compared with model group, each administration group
Serum IgE level is substantially reduced (P < 0.01).It the results are shown in Table 7.
The variation of 7 ELISA method detection SERUM IgE of table
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01。
1.3.2.2 influence of the traditional chinese medicine composition of the invention to AR guinea pig serum IFN-γ
Compared with normal group, model group guinea pig serum IFN-γ level is remarkably decreased (P < 0.05).Compared with model group,
High, middle dose group serum I FN- γ level has significant raising (P < 0.05 or 0.01), the results are shown in Table 8.
8 ELTSA method of table detection serum I FN- γ variation (N=10)
Group | Dosage (g/kg) | IFN-γ(pg/mL) |
Normal group | - | 119.03±41.32 |
Model group | - | 82.99±21.87▲ |
Loratadine group | 0.00091 | 116.02±41.35* |
Rhinitis group | 0.405 | 123.79±22.87** |
Chinese medicine composition high dose group | 3.04 | 107.80±13.36** |
Chinese medicine composition middle dose group | 1.52 | 107.93±22.03* |
Chinese medicine composition low dose group | 0.76 | 89.04±30.11 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
1.3.2.3 influence of the traditional chinese medicine composition of the invention to AR guinea pig serum IL-12
Compared with normal group, model group guinea pig serum IL-12 level is remarkably decreased (P < 0.05).Compared with model group, this
The horizontal significant raising (P < 0.01) of invention Chinese medicine composition high dose group guinea pig serum IL-12, the results are shown in Table 9.
The variation of 9 ELTSA method of table detection serum IL -12 (N=10)
Group | Dosage (g/kg) | IL-12(pg/mL) |
Normal group | - | 120.85±45.36 |
Model group | - | 77.32±23.15▲ |
Loratadine group | 0.00091 | 123.37±47.77* |
Rhinitis group | 0.405 | 123.97±25.43** |
Chinese medicine composition high dose group | 3.04 | 111.25±19.09** |
Chinese medicine composition middle dose group | 1.52 | 99.83±32.28 |
Chinese medicine composition low dose group | 0.76 | 86.00±34.71 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
1.3.2.4 to the influence of AR guinea pig serum IL-4
Compared with Normal group, model group guinea pig serum IL-4 level has raising trend, but without significant difference (P >
0.05).Compared with model group, each dosage group levels of IL-4 is substantially reduced (P < 0.01 or P < 0.05).It the results are shown in Table 10.
The variation of 10 ELISA method detection serum IL -4 of table
Group | Dosage (g/kg) | n | IL-4(pg/mL) |
Normal group | _ | 9 | 673.71±74.31 |
Model group | _ | 10 | 683.91±77.51 |
Loratadine group | 0.00091 | 10 | 631.83±85.98 |
Rhinitis group | 0.405 | 10 | 603.26±82.55* |
Chinese medicine composition high dose group | 3.04 | 10 | 502.14±1116.91** |
Chinese medicine composition middle dose group | 1.52 | 10 | 555.37±121.31* |
Chinese medicine composition low dose group | 0.76 | 10 | 507.89±90.96** |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
1.3.2.5 to the influence of AR guinea pig serum IL-6
Compared with normal group, the horizontal significant raising (P < 0.01) of model group guinea pig serum IL-6.Compared with model group, respectively
Administration group Serum IL-6 levels are substantially reduced (P < 0.01 or P < 0.05).It the results are shown in Table 11.
The variation of 11 ELISA method detection blood serum IL-6 of table
Group | Dosage (g/kg) | n | IL-6(pg/mL) |
Normal group | - | 9 | 326.10±96.20 |
Model group | - | 10 | 449.09±57.58▲▲ |
Loratadine group | 0.00091 | 10 | 342.84±52.10** |
Rhinitis group | 0.405 | 10 | 336.00±103.38** |
Chinese medicine composition high dose group | 3.04 | 10 | 350.48±70.70** |
Chinese medicine composition middle dose group | 1.52 | 10 | 364.32±94.72* |
Chinese medicine composition low dose group | 0.76 | 10 | 292.24±54.60** |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
1.3.2.6 to the influence of AR guinea pig serum TNF-α
Compared with normal group, model group serum TNF-cc level is significantly raised (P < 0.01).Compared with model group, the present invention
Each administration group serum TNF-cc level of Chinese medicine composition is substantially reduced (P < 0.01 or P < 0.05).It the results are shown in Table 12.
The variation of 12 ELISA method detection Serum TNF-α of table
Group | Dosage (g/kg) | n | TNF-α(pg/mL) |
Normal group | _ | 9 | 281.20±71.23 |
Model group | _ | 10 | 364.32±70.09▲ |
Loratadine group | 0.00091 | 10 | 319.42±83.85 |
Rhinitis group | 0.405 | 10 | 302.96±67.95 |
Chinese medicine composition high dose group | 3.04 | 10 | 302.99±61.55* |
Chinese medicine composition middle dose group | 1.52 | 10 | 277.03±83.57** |
Chinese medicine composition low dose group | 0.76 | 10 | 266.06±42.30** |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
1.3.2.7 to the influence of AR cavy schneiderian membrance HIS
Compared with normal group, model group schneiderian membrance HIS level has raising trend, but difference is not significant (P > 0.05);With
Model group compares, and the traditional chinese medicine composition of the invention group schneiderian membrance HIS level has certain decreasing trend, but unobvious (the P > of difference
0.05).It the results are shown in Table 13.
13 ELISA method detection schneiderian membrance HIS of table variation (N=5)
Group | Dosage (g/kg) | Schneiderian membrance HIS (ng/mL) |
Normal group | _ | 5.23±1.42 |
Model group | _ | 7.03±1.98 |
Loratadine group | 0.00091 | 6.59±0.99 |
Rhinitis group | 0.405 | 6.02±0.77 |
Chinese medicine composition high dose group | 3.04 | 5.74±1.06 |
Chinese medicine composition middle dose group | 1.52 | 4.97±0.72 |
Chinese medicine composition low dose group | 0.76 | 5.97±2.03 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
1.3.3 to the influence of AR guinea-pig nasal Mucosal inflammatory index
1.3.3.1 to the influence of AR cavy schneiderian membrance IFN-γ expression
Light microscopic observation guinea-pig nasal mucosa-immune histochemical staining slice, the region for being dyed to brown granular is IFN-γ
Positive expression region.The results show that there are a large amount of brown granulars between normal rats nasal epithelial cells, IFN-γ is prompted
Largely it is present between normal nasal epithelial cells, and cavy schneiderian membrance IFN-γ positive expression amount is apparently higher than model group (P <
0.05);Compared with model group, each administration group cavy schneiderian membrance IFN-γ expression quantity significantly increases (P < 0.05);It the results are shown in Table
14。
Influence that 14 the traditional chinese medicine composition of the invention of table expresses AR cavy schneiderian membrance IFN-γ (N=5)
Group | Dosage (g/kg) | OD value |
Normal group | - | 0.156±0.080 |
Model group | - | 0.046±0.041▲ |
Loratadine group | 0.00091 | 0.151±0.016** |
Rhinitis group | 0.405 | 0.198±0.080** |
Chinese medicine composition high dose group | 3.040 | 0.113±0.043* |
Chinese medicine composition middle dose group | 1.520 | 0.159±0.048* |
Chinese medicine composition low dose group | 0.760 | 0.115±0.029* |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
1.3.3.2 to the influence of AR cavy schneiderian membrance IL-6 expression
Light microscopic observation guinea-pig nasal mucosa-immune histochemical staining slice, the region for being dyed to brown granular is IL-6 sun
Property expression region.Compared with normal group, model group cavy schneiderian membrance IL-6 OD value significantly increases (P < 0.05);With model
Group compares, and (P < 0.05, the results are shown in Table 15 for each administration group schneiderian membrance IL-6 positive expression OD value significant decrease.
Influence that 15 the traditional chinese medicine composition of the invention of table expresses AR cavy schneiderian membrance IL-6 (N=5)
Group | Dosage (g/kg) | OD value |
Normal group | - | 0.048±0.034 |
Model group | - | 0.149±0.064▲ |
Loratadine group | 0.00091 | 0.067±0.027* |
Rhinitis group | 0.405 | 0.020±0.008** |
Chinese medicine composition high dose group | 3.040 | 0.061±0.018* |
Chinese medicine composition middle dose group | 1.520 | 0.063±0.029* |
Chinese medicine composition low dose group | 0.760 | 0.050±0.014* |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P < 0.01
1.3.3.3 to the influence of AR cavy schneiderian membrance TNF-α expression
Light microscopic observation guinea-pig nasal mucosa-immune histochemical staining slice, the region for being dyed to brown granular is TNF-α
Positive expression region.Compared with normal group, the visible apparent positive expression product of model group cavy schneiderian membrance TNF-α, optical density
Value is significant to increase (P < 0.05);Compared with model group, each administration group schneiderian membrance TNF-α positive expression OD value is significantly reduced
(P < 0.05) shows that the traditional chinese medicine composition of the invention can reduce positive expression of the TNF-α in schneiderian membrance, the results are shown in Table 16.
Influence that 16 the traditional chinese medicine composition of the invention of table expresses AR cavy schneiderian membrance TNF-α (N=5)
Group | Dosage (g/kg) | OD value |
Normal group | - | 0.042±0.038 |
Model group | - | 0.312±0.210▲ |
Loratadine group | 0.00091 | 0.032±0.029* |
Rhinitis group | 0.405 | 0.033±0.022* |
Chinese medicine composition high dose group | 3.040 | 0.027±0.019* |
Chinese medicine composition middle dose group | 1.520 | 0.210±0.099* |
Chinese medicine composition low dose group | 0.760 | 0.041±0.038* |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P < 0.01
1.3.4 on the pathological influence of AR cavy nasal mucosa
1.3.4.1 schneiderian membrance EOS is counted
Every slice randomly selects 5 high power fields (× 400), carries out EOS counting.Compared with normal group, model group EOS
Infiltrate obvious (P < 0.01);Compared with model group, in addition to the traditional chinese medicine composition of the invention low dose group, other each groups EOS infiltrates feelings
Condition mitigates (P < 0.01 or P < 0.05).Show that the traditional chinese medicine composition of the invention can reduce AR cavy schneiderian membrance EOS infiltration, subtracts
Light schneiderian membrance inflammatory reaction, the results are shown in Table 17.
17 schneiderian membrance EOS weighted mean value of table (N=5)
Group | Dosage (g/kg) | EOS counts (a) |
Normal group | - | 2.28±1.28 |
Model group | - | 17.04±7.20▲▲ |
Loratadine group | 0.00091 | 2.32±0.84** |
Rhinitis group | 0.405 | 3.64±2.17** |
Chinese medicine composition high dose group | 3.04 | 5.48±2.30** |
Chinese medicine composition middle dose group | 1.52 | 7.12±4.39* |
Chinese medicine composition low dose group | 0.76 | 9.80±5.24 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
1.3.4.2 schneiderian membrance pathological observation and scoring
Normal group nasal mucosa is thin by pseudostratified ciliated columnar epithelium cell, goblet cell, sertoli cell and substrate
The composition such as born of the same parents, the dense fibrous connective tissue of submucosa is very thin, mucosal epithelium structural integrity, marshalling, cilium thickness one
It causes, has no obvious inflammatory cell infiltration;Model group nasal mucosa epithelium is imperfect, there is obscission, and glandular hyperplasia is obvious, sticks
Film lamina propria is shown in a large amount of EOS;Loratadine group mucosal epithelium is substantially intact, a small amount of inflammatory cell infiltration, on rhinitis group mucous membrane
Skin portion falls off, and part glandular hyperplasia has no inflammatory cell infiltration;The traditional chinese medicine composition of the invention high dose group mucosal epithelium is completeer
It is good, it is seen that part inflammatory cell infiltration;Middle dose group mucous membrane is substantially intact, there is a small amount of inflammatory cell infiltration;Low dose group mucous membrane
Partial exfoliation, glandular hyperplasia is obvious, and inflammatory cell infiltration is more apparent.
Compared with normal group, model group pathological score is significantly raised (P < 0.01).Compared with model group, Loratadine group,
The scoring of rhinitis group is substantially reduced (P < 0.05), and after the traditional chinese medicine composition of the invention treats 14d, high dose group schneiderian membrance pathology is commented
Divide and is substantially reduced (P < 0.05).It the results are shown in Table 18.
The scoring of 18 cavy schneiderian membrance pathological grading of table (N=5)
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P < 0.01
The influence of allergic rhinitis rat caused by 2 pairs of egg proteins
AR rat model is prepared using egg protein injection and schneiderian membrance stimulus method, after model is successfully established, observes each group row
To learn and scoring, ELISA method detection SERUM IgE, IFN-γ, IL-4, IL-6, TNF-α, the content of IL-17, immuning tissue
Chemical dyeing method detects nasal mucosa IL-6, TNF-α, IFN-γ expression, carries out nasal secretion EOS meter under optical microscopy
Number observes nasal mucosa Pathologic changes and scores.
2.1 materials and methods
2.1.1 material
2.1.1.1 animal and grouping
SPF grades SD rat 70, half male and half female, 200~300g of weight is purchased from Beijing dimension tonneau China experimental animal technology
Co., Ltd, animal credit number: SCXK (capital) 2012-0001.Animal is divided by normal group, model group, chlorine using randomized
Lei Tading group (1.17mg/kg), rhinitis group (0.6g/kg), the traditional chinese medicine composition of the invention high dose group (4.6g/kg), this hair
Bright Chinese medicine composition middle dose group (2.3g/kg), the traditional chinese medicine composition of the invention low dose group (1.15g/kg), every group 10.
2.1.1.2 drug and reagent
Egg protein (Ovalbumin, OVA, lot number: SLBK6455V, sigma company, the U.S.);
The traditional chinese medicine composition of the invention granule (is prepared into, 1g is equivalent to 5.7577g crude drug amount, distillation by 4 method of embodiment
It is spare that water is configured to 0.304g/mL);
Loratadine tablet (Xi'an Yang Sen, batch number: 050218967);
Rhinitis piece (Foshan Dezhong Pharmaceutical Co., Ltd., batch number: 14272);
Gel aluminum hydroxide (Thermo company, batch number: pc 197962);
Sodium chloride injection (Shijiazhuang Siyao Co., Ltd, batch number: 1511023204);
Rat IgE ELISA kit (Xin Bosheng, batch number: R160322-117b);
Rat IL-4 ELISA kit (Xin Bosheng, batch number: R160415-002a);
Rat IL-17A ELISA kit (Xin Bosheng, batch number: R160415-17a);
Rat TNF-α ELISA kit (Xin Bosheng, batch number: R160415-102a);
Rat IFN-γ ElISA kit (Xin Bosheng, batch number: R160415-101a);
Rat IL-6 kit (CSB, batch number: D10033395);
Anti-TNF alpha antibody (abcam, batch number: ab6671);
IL-6 Polyclonal Antibody (proteintech, batch number: 21865-1-AP);
Interferon gamma Polyclonal Antibody (proteintech, batch number: 15365-1-
AP);
Ethyl alcohol (Beijing chemical plant, batch number: 20140607);
Formalin (Tianjin good fortune morning chemical reagent factory, batch number: 20141020);
Dimethylbenzene (Tong Guang fine chemistry industry company, Beijing, batch number: 20140719);
Breathe out Rui Shi haematoxylin dye liquor (Beijing Shiji Heli Biological Technology Co., Ltd., batch number: 20141203);
Breathe out Rui Shi eosin stain (Beijing Shiji Heli Biological Technology Co., Ltd., batch number: 20141203);
DAB (Dako, batch number: K5007).
2.1.1.3 laboratory apparatus
Microscope (Japanese OLYMPUS company), image analysis software (MIAS);
Adopt figure software Nikon NIS-Elements D 3.2 (Japanese Nikon company);
ASP300 takes off full-automatic multi-functional microplate reader: MULTISKAN MK3 (Thermo company);
GL-21M high speed freezing centrifuge (Shanghai Lu Xiangyi centrifuge Instrument Ltd.);
BX51 water dispenser (German LEICA company);
EG1150H embedding machine (German LEICA company);
RM2235 slicer (German LEICA company);
H I1 210 spreads out piece machine (German LEICA company).
2.2 method
2.2.1 model copy
2.2.1.1 basic sensitization
Experimental group: being dissolved in 1mL physiological saline for 1mg OVA and 30mg Al (OH) 3 Gel Adjuvant and be made into suspension, in
0.2mL OVA adjuvant suspension is injected in rat two sides groin, toes behind two sides, abdominal cavity by 1d, 5d, 10d respectively.
Normal group substitutes sensitization adjuvant, the same experimental group of method and steps with same amount of normal saline.
2.2.1.2 local sensitization
Except for the normal group, in 15d, with 10%OVA normal saline solution bilateral collunarium, every side 50uL, collunarium 7d.Just
Normal group replaces 10%OVA suspension, the same experimental group of method and steps with same amount of normal saline.
2.2.2 administration
After model success, 1 collunarium maintains the stimulation to schneiderian membrance the next day model group, and administration group 1h before daily collunarium is filled
Stomach administration.14d is administered in continuous gavage, and every 7d observation is primary, records symptom and scores.
2.2.3 observation index
2.2.3.1 animal behavioral study
Sniffle is observed after local excitation in 30min, calculating behaviouristics total score by standards of grading, (being greater than 5 points is animal
Model success).Standards of grading: 1. 0 point: no sneeze scratches nose and nasal discharge;2. 1 point: sneeze < 4, slightly scratching nose, prenasal
There is nasal discharge in hole;3. 2 points: sneeze 4~10, frequently scratching nose, nasal discharge is more than prenaris;4. 3 points: sneeze > 11,
Nose, friction are persistently scratched, face is covered with nasal discharge.
2.2.3.2 biochemical and immune index detection
After last excitation for 24 hours, 10% chloraldurate (350mg/kg) intraperitoneal injection of anesthesia, abdominal aortic blood, 3500r/
Min is centrifuged 10min.Serum is separated, is placed under -80 DEG C of refrigerators and saves.According to ELISA kit specification step detect IgE,
IFN-γ, IL-4, IL-6, TNF-α, IL-17 are horizontal.
2.2.3.3 nasal secretion EOS is counted
Bridge of the nose is opened rapidly, peels off upper jaw osseous part skin, and vestibular wall is cut on the outside of the nasal septum of right side, opens exposure upwards
Nasal septum and bilateral nasal cavity, first dip secretion with cotton swab, are applied on glass slide in the same direction, and row HE dyes (every group of selection 5
Only), (400) randomly select 5 visuals field (size is 677.3mm × 508mm) under optical microscopy, carry out EOS counting.
2.2.3.4 observing nasal mucosa Pathologic changes
Rats with bilateral mucous membrane of nasal septum is taken, is fixed in 10% neutral formalin solution, paraffin embedding, HE dyeing observation nose is glutinous
Film pathological change simultaneously scores (the same first part of operating procedure).
Pathological score standard: 0 grade, cilium is completely neat, and columnar epithelium is normal, a small amount of goblet cell, remembers 0 point;I grades, cup
Shape cytosis, the inflammatory cell that minority is dispersed in, the glandular hyperplasia of film lamina propria accounts for the 1/3 of mucosa lamina propria hereinafter, 1 point of note;
II grades, goblet cell gathers, and cilium lodging is irregular, accidental to fall off, and more acidophic cell is swum out of, and the body of gland of film lamina propria increases
It is raw to account for the 1/3~2/3 of mucosa lamina propria, remember 2 points;III level, goblet cell gather, and cilium largely falls off, epithelium damage layer or group
Texture disorder is knitted, a large amount of acidophic cells, which are swum out of, accounts for 2/3 or more of whole epithelial inflammation cells, and the glandular hyperplasia of film lamina propria accounts for
2/3 or more of mucosa lamina propria remembers 3 points.
2.2.3.5 immunohistochemical staining
Immunohistochemical staining (IHC) method detects nasal mucosa IFN-γ, IL-6, TNF-α expression.Every figure under high power lens
Piece randomly selects 5 visuals field, analyzes software using Image-Pro Plus Version 6.0, image area is analyzed, according to public affairs
Formula (IOD/area), acquires positive expression OD value.The same first part of operating procedure.
2.2.4 statistical analysis
Experimental data is with mean ± standard deviationIt indicates, is handled using 9.3 statistical software of SAS.If each group is obeyed
Normal distribution and variance is neat, using one-way analysis of variance, two groups are compared and are examined using t;If each group disobey normal distribution or
Heterogeneity of variance, using non-parametric test.
2.3 experimental result
2.3.1 influence of the traditional chinese medicine composition of the invention to AR rat behavior
Before administration, experimental group rat is compared with normal group, and sneeze, scratching nose, runny nose etc., behaviouristics symptom score obviously increases
(P < 0.01), hints model replicates successfully.After drug treatment 14d, compared with normal group, neurological deficit score obviously increases model group
Add (P < 0.01);For each dosage group of the traditional chinese medicine composition of the invention compared with model group, neurological deficit score is substantially reduced (P < 0.01),
Prompt the traditional chinese medicine composition of the invention can obviously improve AR rat sniffle.It the results are shown in Table 19.
Table 19 be administered after neurological deficit score record sheet (N=10)
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P < 0.01
2.3.2 influence of the traditional chinese medicine composition of the invention to AR rat biochemistry and amynologic index
2.3.2.1 influence of the traditional chinese medicine composition of the invention to AR rat blood serum IgE
After drug treatment 14d, model group is compared with normal group, the horizontal obvious rising (p < 0.01) of rat blood serum IgE;This
Compared with model group, rat blood serum IgE level is substantially reduced (p < 0.01 or 0.05) invention Chinese medicine composition high dose group, knot
Fruit is shown in Table 20.
The variation of 20 ELISA method detection SERUM IgE of table
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P < 0.01
2.3.2.2 influence of the traditional chinese medicine composition of the invention to AR rat blood serum IFN-γ
After drug treatment 14d, for model group compared with normal group, rat blood serum IFN-γ level is decreased obviously (P < 0.01);
The traditional chinese medicine composition of the invention high and low dose group compared with model group, rat blood serum IFN-γ it is horizontal it is significantly raised (P < 0.01 or
0.05) 21, be the results are shown in Table.
21 ELISA method detection serum I FN- γ of table variation
Group | Dosage (g/kg) | n | IFN-γ(pg/mL) |
Normal group | - | 10 | 12.99±5.79 |
Model group | - | 10 | 7.92±1.53▲▲ |
Loratadine group | 0.00091 | 9 | 9.35±0.76** |
Rhinitis group | 0.405 | 10 | 11.06±3.63* |
Chinese medicine composition high dose group | 3.04 | 9 | 10.40±2.05** |
Chinese medicine composition middle dose group | 1.52 | 10 | 8.65±1.50 |
Chinese medicine composition low dose group | 0.76 | 10 | 10.88±2.24** |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.2.3 influence of the traditional chinese medicine composition of the invention to AR rat blood serum IL-4
After drug treatment 14d, model group is compared with normal group, the horizontal obvious rising (P < 0.01) of rat blood serum IL-4;This
For invention Chinese medicine composition middle dose group compared with model group, rat blood serum IL-4 level is decreased obviously (P < 0.05), the results are shown in Table
22。
The variation of 22 ELISA method detection serum IL -4 of table
Group | Dosage (g/kg) | n | IL-4(pg/mL) |
Normal group | _ | 10 | 5.33±1.97 |
Model group | _ | 10 | 10.33±5.78▲▲ |
Loratadine group | 0.00091 | 10 | 5.86±2.10** |
Rhinitis group | 0.405 | 10 | 8.00±2.30 |
Chinese medicine composition high dose group | 3.04 | 10 | 6.74±1.49 |
Chinese medicine composition middle dose group | 1.52 | 10 | 5.91±1.97* |
Chinese medicine composition low dose group | 0.76 | 10 | 9.60±4.42 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.2.4 influence of the traditional chinese medicine composition of the invention to AR rat blood serum IL-6
After drug treatment 14d, model group is compared with normal group, and rat blood serum IL-6 level is in rising trend, but without significant
Sex differernce (P > 0.05);Each dosage group of the traditional chinese medicine composition of the invention is compared with model group, and rat blood serum IL-6 level is without conspicuousness
Change (P > 0.05), the results are shown in Table 23.
The variation of 23 ELISA method detection blood serum IL-6 of table
Group | Dosage (g/kg) | n | IL-6(pg/mL) |
Normal group | _ | 10 | 17.78±3.94 |
Model group | _ | 10 | 21.15±6.21 |
Loratadine group | 0.00091 | 10 | 18.91±2.88 |
Rhinitis group | 0.405 | 10 | 20.58±7.87 |
Chinese medicine composition high dose group | 3.04 | 10 | 20.59±4.47 |
Chinese medicine composition middle dose group | 1.52 | 10 | 22.63±6.58 |
Chinese medicine composition low dose group | 0.76 | 10 | 25.22±8.18 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.2.5 influence of the traditional chinese medicine composition of the invention to AR rat blood serum TNF-α
After drug treatment 14d, model group is compared with normal group, and ascendant trend is presented in rat blood serum TNF-α level, but without aobvious
It writes difference (P > 0.05);Compared with model group, rat blood serum TNF-α is horizontal obvious for the traditional chinese medicine composition of the invention height, middle dose group
Decline (P < 0.05 or 0.01), the results are shown in Table 24.
The variation of 24 ELISA method detection Serum TNF-α of table
Group | Dosage (g/kg) | n | TNF-α(pg/mL) |
Normal group | _ | 10 | 74.80±14.13 |
Model group | _ | 10 | 86.68±9.64 |
Loratadine group | 0.00091 | 9 | 76.49±9.28* |
Rhinitis group | 0.405 | 10 | 65.49±16.54** |
Chinese medicine composition high dose group | 3.04 | 9 | 74.29±12.92* |
Chinese medicine composition middle dose group | 1.52 | 10 | 72.25±9.26** |
Chinese medicine composition low dose group | 0.76 | 10 | 77.18±17.32 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.2.6 influence of the traditional chinese medicine composition of the invention to AR rat blood serum IL-17
After drug treatment 14d, model group is compared with normal group, the horizontal obvious rising (P < 0.01) of rat blood serum IL-17;
The traditional chinese medicine composition of the invention low dose group is compared with model group, and rat blood serum IL-17 level is decreased obviously (P < 0.05), as a result
It is shown in Table 25.
The variation of 25 ELISA method detection serum IL -17 of table
Group | Dosage (g/kg) | n | IL-17(pg/mL) |
Normal group | _ | 10 | 8.45±3.17 |
Model group | _ | 10 | 14.91±7.58▲ |
Loratadine group | 0.00091 | 9 | 9.03±3.55* |
Rhinitis group | 0.405 | 10 | 11.76±2.55 |
Chinese medicine composition high dose group | 3.04 | 9 | 14.33±4.89 |
Chinese medicine composition middle dose group | 1.52 | 10 | 11.29±3.16 |
Chinese medicine composition low dose group | 0.76 | 10 | 9.20±3.88* |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.3 the influence that the traditional chinese medicine composition of the invention expresses AR rat schneiderian membrance IFN-γ, IL-6, TNF-α
2.3.3.1 the influence that the traditional chinese medicine composition of the invention expresses AR rat schneiderian membrance IFN-γ
Light microscopic observation rat schneiderian membrance immunohistochemical staining slice, the results show that compared with normal group, model group rats
Schneiderian membrance IFN-γ positive expression OD value is substantially reduced (P < 0.05);Compared with model group, Chinese medicine high and low dose group is big
Mouse schneiderian membrance OD value significantly increases (P < 0.05 or 0.01);It the results are shown in Table 26.
Influence that 26 the traditional chinese medicine composition of the invention of table expresses AR rat schneiderian membrance IFN-γ (N=5)
Group | Dosage (g/kg) | OD value |
Normal group | - | 0.126±0.058 |
Model group | - | 0.044±031▲ |
Loratadine group | 0.0017 | 0.056±0.02* |
Rhinitis group | 0.60 | 0.168±0.014** |
Chinese medicine composition high dose group | 4.60 | 0.161±0.026** |
Chinese medicine composition middle dose group | 2.30 | 0.128±0.062 |
Chinese medicine composition low dose group | 1.15 | 0.160±0.028** |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.3.2 the influence that the traditional chinese medicine composition of the invention expresses AR rat schneiderian membrance IL-6
Light microscopic observation rat schneiderian membrance immunohistochemical staining slice, the results show that compared with normal group, model group rats
Schneiderian membrance IL-6 positive expression OD value significantly increases (P < 0.01);Compared with model group, Chinese medicine is high, middle dose group nose is glutinous
Film IL-6 OD value significantly reduces (P < 0.01), the results are shown in Table 27.
Influence that 27 the traditional chinese medicine composition of the invention of table expresses AR rat schneiderian membrance IL-6 (N=5)
Group | Dosage (g/kg) | Average optical density value |
Normal group | - | 0.052±0.031 |
Model group | - | 0.142±0.029▲▲ |
Loratadine group | 0.0017 | 0.057±0.013** |
Rhinitis group | 0.60 | 0.039±0.033** |
Chinese medicine composition high dose group | 4.60 | 0.050±0.014** |
Chinese medicine composition middle dose group | 2.30 | 0.042±0.022** |
Chinese medicine composition low dose group | 1.15 | 0.086±0.042 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.3.3 the influence that the traditional chinese medicine composition of the invention expresses AR rat schneiderian membrance TNF-α
Light microscopic observation rat schneiderian membrance immunohistochemical staining slice, the results showed that, compared with normal group, model group rats
Schneiderian membrance TNF-α positive expression OD value significantly increases (P < 0.01);Compared with model group, Chinese medicine high dose group schneiderian membrance
TNF-α positive expression OD value significantly reduces (P < 0.05 or 0.01), shows that the traditional chinese medicine composition of the invention can reduce TNF-
Positive expression of the α in schneiderian membrance, the results are shown in Table 28.
Influence that 28 the traditional chinese medicine composition of the invention of table expresses AR rat schneiderian membrance TNF-α (N=5)
Group | Dosage (g/kg) | OD value |
Normal group | - | 0.013±0.009 |
Model group | - | 0.084±0.029▲▲ |
Loratadine group | 0.0017 | 0.010±0.014** |
Rhinitis group | 0.60 | 0.027±0.021** |
Chinese medicine composition high dose group | 4.60 | 0.037±0.016** |
Chinese medicine composition middle dose group | 2.30 | 0.051±0.023 |
Chinese medicine composition low dose group | 1.15 | 0.049±0.025 |
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.4 the traditional chinese medicine composition of the invention is on the pathological influence of AR rat nasal mucosa
2.3.4.1 influence of the traditional chinese medicine composition of the invention to AR rat nasal secretion EOS
After 14d is administered, for model group compared with normal group, nasal secretion EOS quantity is significantly raised (P < 0.01).This hair
For bright each dosage group of Chinese medicine composition compared with model group, EOS quantity is substantially reduced (P < 0.05 or 0.01).Loratadine group, nose
Scorching Kang Zuyu model group compares, and EOS infiltration is decreased obviously (P < 0.05 or 0.01), the results are shown in Table 29.
29 rat nasal secretion EOS of table counting (N=5)
Note: compared with normal group, ▲ P < 0.05, ▲ ▲ P < 0.01;Compared with model group, * P < 0.05, * * P <
0.01.
2.3.4.2 the traditional chinese medicine composition of the invention is on the pathological influence of AR rat tissue
After HE dyeing it is in the form of a column arrangement, has no the congestion of blood vessel and tissue as it can be seen that normally group schneiderian membrance epithelium is intact under light microscopic
Oedema, inflammatory cell infiltration, glandular hyperplasia;Mucosal epithelium damage in model group part falls off, and goblet cell is more, lamina propria blood vessel
Dilatation and congestion, with more eosinophil and other inflammatory cell infiltrations, glandular hyperplasia under mucous membrane, compared with normal group, disease
Reason scoring has significant difference (P < 0.01);Loratadine group, rhinitis group mucosal epithelium are substantially intact, and a small amount of inflammation is thin
Born of the same parents infiltrate, and body of gland has slight hyperplasia under mucous membrane, and compared with model group, pathological score is decreased obviously (P < 0.05 or 0.01);This hair
Bright each dosage group mucosal epithelium of Chinese medicine composition is substantially intact, partially visible glandular hyperplasia, blood vessel mild hyperaemia, a small amount of acidophilia
Granulocyte infiltration, compared with model group, pathological score is decreased obviously (P < 0.05 or 0.01), the results are shown in Table 30.
The scoring of 30 rat schneiderian membrance pathological grading of table
Note: compared with normal group,▲P < 0.05,▲▲P < 0.01;Compared with model group,*P < 0.05,**P < 0.01
3 the traditional chinese medicine composition of the invention are anti-inflammatory, anti-allergic effects are studied
Method is formed using xylene-induced ear swelling in mice method and swollen hyperplasia of rat granuloma, studies the traditional chinese medicine composition of the invention
Anti-inflammatory effect;This hair is observed using allotransplantation in rats passive cutaneous anaphylaxis method and mouse ear xenogenesis passive cutaneous anaphylaxis method
The anti-allergic effects of bright Chinese medicine composition.
3.1 materials and methods
3.1.1 experimental material
3.1.1.1 animal
Selection SPF grades kunming mice 100, half male and half female, 18~22g of weight.SPF grades SD rat 110, male and female are each
Half, 180~200g of weight.The above animal is purchased from Beijing Vital River Experimental Animals Technology Co., Ltd., animal credit number:
SCXK (capital) 2012-0001.
3.1.1.2 drug and reagent
The traditional chinese medicine composition of the invention granule (is prepared, 1g is equivalent to 4.8804g crude drug amount) by 4 method of embodiment;
Loratadine (Xian-Janssen Pharmaceutical Ltd., lot number: 150923784);
Dexamethasone acetate tablets (Tianjin Lisheng Pharmaceutical Co., Ltd., lot number: 1501006);
Dimethylbenzene (Beijing Chemical Plant, lot number: 20150506);
Dual anti-(CORNING, the suspension of mycillin, lot number: 30-002-CI);
Egg protein (sigma company, the U.S., lot number: SLBK6455V);
It adsorbs DPT vaccine (Changchun Changsheng Biological Technology Co., Ltd, lot number: 20150918-02);
Evans blue (Sinopharm Chemical Reagent Co., Ltd., lot number: wc20070607);
Acetone (Beijing Chemical Plant, lot number: 20150316);
Potassium hydroxide powder (Beijing Chemical Plant, lot number: 20141118);
Phosphoric acid (Sinopharm Chemical Reagent Co., Ltd., lot number: 20160127).
3.1.1.3 instrument
Labofuge refrigerated centrifuge (German Heraeus company);
Punch (diameter 6.5mm);
Electronic analytical balance (Ohaus company, the U.S.);
WFJ2000 type ultraviolet-uisible spectrophotometer (Shanghai You Nike Instrument Ltd.);
QL-901 vortex oscillator (its woods Bell's instrument manufacturing Co., Ltd, Haimen City);
Electric heating constant-temperature blowing drying box (Hengfeng Medical Instruments Co., Ltd., Huangsih City);
Vertical pressure steam sterilization pan (Medical Equipment Plant, Shanghai Boxun Industrial Co., Ltd.).
3.2 experimental method
3.2.1 xylene-induced ear swelling in mice is tested
Animal is divided into control group, Dexamethasone group (5mg/kg), the traditional chinese medicine composition of the invention high dose group at random
(7.86g/kg), middle dose group (3.93g/kg), low dose group (1.96g/kg), every group 10, half male and half female.By above-mentioned grouping
Mode, successive administration 5d, 1 times/day.After the 5th day administration 45min, 50 μ L caused by dimethylbenzene xylene are uniformly smeared on mouse right ear two sides
Inflammation, after smearing 20min, cervical dislocation is lethal, and two ears of left and right, the left and right ear of same area is beaten with punch under auricle edge scissor
Piece is weighed immediately after.Using the difference of two auricle weight as swelling, the inhibition percentage of drug is calculated.Inhibiting rate (%)=
(control group swelling-medication group swelling)/control group swelling × 100%.Control group gives the distilled water of equivalent, method and
The same experimental group of step.
3.2.2 swollen hyperplasia of rat granuloma is tested
Be randomly divided into control group, the traditional chinese medicine composition of the invention agent high dose group (4.6g/kg), middle dose group (2.3g/kg),
Low dose group (1.15g/kg), Dexamethasone group (0.79mg/kg), every group 10, half male and half female.Rat chloraldurate
An opening is made in (350mg/kg) anesthesia, the disinfection of 75% ethyl alcohol abdomen among abdomen, removes skin to groin with haemostatic clamp
Place, at preparatory high pressure sterilization and 50mg cotton balls (dual anti-100 μ L is added) implantation rat groin, skin suture is clear to animal
Start to be administered after waking up.In addition to distilled water is given in control group stomach-filling, relative medicine is given in other each administration group stomach-fillings, one time a day,
1mL/100g, continuous 7d.1h after the last administration, cervical dislocation put to death rat, open skin of abdomen, carefully remove cotton balls, be placed in electricity
It weighs on sub- balance, as cotton balls weight in wet base.Cotton balls is placed in 60 DEG C of baking ovens, dry 12h takes out and weighs as dry weight.
3.2.3 models of passive skin irritability of rats reaction experiment
3.2.3.1 the preparation of rat antisera
SD rat male and female each 5, every 5% egg protein normal saline solution 0.2mL of mouse hind leg intramuscular injection are chosen in experiment, simultaneously
DPT vaccine 0.2mL is injected intraperitoneally.After normal raising 13d, anesthesia, abdominal aortic blood, 3500r/min is centrifuged 15min, quiet
After setting 2h, rat blood serum is separated, -20 DEG C of refrigerators is placed in and saves.Prepare anti-egg protein rat blood serum.
3.2.3.2 passive cutaneous anaphylaxis
SD rat is divided into control group, the traditional chinese medicine composition of the invention high dose group (4.6g/kg), middle dose group (2.3g/kg),
Low dose group (1.15g/kg), Loratadine group (1.16mg/kg), every group 10, male and female each 5.By above-mentioned group technology, chlorine
7d is administered in Lei Tading group and Chinese medicine each group continuous gavage, one time a day, antigen attack is given after not secondary administration 45min.Control group fills
Stomach distilled water.It is administered the 6th day, dorsal line two sides is cut off under the anesthesia of chloraldurate away from hair (every side at 1.5~2cm of backbone
3cm × 3cm), and by above-mentioned antiserum normal saline dilution at 1: 2 and 1: 6, intracutaneous injection is in rat spine two sides shaving portion
Point, each dilution injects two o'clock, every 0.1mL.It is administered the 7th day, tail vein injection ovalbumin (2%) after last dose 1h
With Evans blue (0.5%) physiological saline mixed solution 1mL.
3.2.3.3 collection of specimens and Indexs measure
Sacrificed by decapitation after 30min, rat dorsum skin is overturn, and is cut back locus coeruleus skin, is shredded, with 5mL acetone-life
It manages salt water (7: 3) to impregnate, is placed in 37 DEG C of water-baths and heats 48h, be centrifuged, supernatant taken, with ultraviolet-uisible spectrophotometer in wave
Long 620nm measures OD value.
3.2.4 mouse ear xenogenesis passive cutaneous anaphylaxis
Mouse is randomly divided into control group, the traditional chinese medicine composition of the invention agent high dose group (7.86g/kg), middle dose group
(3.93g/kg), low dose group (1.96g/kg), Loratadine group (2mg/kg), every group 10, half male and half female.Continuous gavage is given
Medicine 7d, administered volume 0.2mL/10g, give antigen attack after last stomach-filling 45min by 1 times/day.It is administered the 6th day, mouse two
Auricle respectively injects 25 μ L of rat antisera.Antigen attack after for 24 hours, 2% ovalbumin of tail vein injection and 0.5% Evans blue are molten
Liquid 0.25mL.After 30min, mouse cervical dislocation is put to death, cuts Mice Auricle (indigo plant dye position).Cut auricle is placed in test tube
In, the potassium hydroxide solution (1mol/L) that 0.75mL is added impregnates, and 37 DEG C are digested overnight.Next day adds 3.75mL phosphoric acid
(0.6mol/L) and acetone mixture (5: 13, v/v), shakes through vortex oscillator, then is centrifuged 15min with 2500r/min, will be upper
Clear liquid takes out, and ultraviolet-uisible spectrophotometer measures OD value at wavelength 640nm, calculates the inhibiting rate of drug.Inhibiting rate (%)
=(control group OD value-medication group OD value)/control group OD value × 100%.
3.2.5 statistical method
Experimental data usesIt indicates, is handled using 9.3 statistical software of SAS.If each group Normal Distribution and variance
Qi Ze uses variance test, compares examined using LSD two-by-two, use non-parametric test if each group disobeys normal distribution.With P
< 0.05 is that difference is statistically significant.
3.3 experimental result
3.3.1 the traditional chinese medicine composition of the invention paraxylene causes the influence of mouse ear swelling
Compared with the control group, Dexamethasone group and the high, medium and low dosage group of the traditional chinese medicine composition of the invention can significantly reduce diformazan
Benzene causes mice auricle swelling (P < 0.05 or 0.01), shows that the traditional chinese medicine composition of the invention can inhibit dimethylbenzene induced mice auricle
Inflammatory reaction.It the results are shown in Table 31.
31 the traditional chinese medicine composition of the invention paraxylene of table cause mouse ear swelling influence (N=10)
Note: compared with the control group, * P < 0.05, * * P < 0.01.
3.3.2 the influence that the traditional chinese medicine composition of the invention agent forms swollen hyperplasia of rat granuloma
Compared with the control group, Dexamethasone group can significantly lower granuloma induced by implantation of cotton pellets weight (P < 0.01), Chinese medicine group of the present invention
Each dosage group rat cotton balls dry weight of object, weight in wet base are closed without significant difference (P > 0.05), prompts the traditional chinese medicine composition of the invention to cotton balls institute
It causes rat granulation tissue hyperplasia without apparent inhibiting effect, the results are shown in Table 32.
32 the traditional chinese medicine composition of the invention of table to swollen hyperplasia of rat granuloma influence (N=10)
Group | Dosage | Cotton balls weight in wet base/mg | Cotton balls dry weight/mg |
Control group | - | 1416.0±907.3 | 215.9±25.4 |
Dexamethasone group | 0.79mg/kg | 1128.0±575.2** | 173.1±12.7** |
Chinese medicine composition high dose group | 4.60g/kg | 1363.8±177.1 | 212.8±29.7 |
Chinese medicine composition middle dose group | 2.30g/kg | 1395.6±794.4 | 216.5±20.3 |
Chinese medicine composition low dose group | 1.15g/kg | 1398.7±103.3 | 212.9±16.2 |
Note: compared with the control group, * * P < 0.01.
3.3.3 influence of the traditional chinese medicine composition of the invention to allotransplantation in rats passive cutaneous anaphylaxis
Compared with the control group, Loratadine group, the traditional chinese medicine composition of the invention each group can obviously reduce Locus Coeruleus In The Rat OD value
(P < 0.05 or 0.01) shows that the traditional chinese medicine composition of the invention has allotransplantation in rats passive cutaneous anaphylaxis caused by egg protein
Certain inhibiting effect.It the results are shown in Table 33.
Influence that 33 the traditional chinese medicine composition of the invention of table reacts models of passive skin irritability of rats (N=10)
Group | Dosage | OD | Inhibiting rate |
Control group | - | 0.134±0.081 | - |
Loratadine group | 1.16mg/kg | 0.059±0.039* | 55.97% |
Chinese medicine composition high dose group | 4.6g/kg | 0.036±0.018* | 73.13% |
Chinese medicine composition middle dose group | 2.3g/kg | 0.048±0.045* | 64.18% |
Chinese medicine composition low dose group | 1.15g/kg | 0.024±0.020** | 82.09% |
Note: compared with the control group, * P < 0.05, * * P < 0.01.
3.3.4 influence of the traditional chinese medicine composition of the invention to mouse ear xenogenesis passive cutaneous anaphylaxis
Compared with the control group, Loratadine group can substantially reduced Mice Auricle locus coeruleus OD value (P < 0.01), Chinese medicine of the present invention
Composition high dose group can significantly reduce Mice Auricle indigo plant dye degree (P < 0.05), prompt the traditional chinese medicine composition of the invention can be obvious
Inhibit the xenogenesis passive cutaneous anaphylaxis of egg protein induced mice ear, the results are shown in Table 34.
34 the traditional chinese medicine composition of the invention of table to mouse ear xenogenesis passive cutaneous anaphylaxis influence (N=10)
Note: compared with the control group, * P < 0.05, * P < 0.01.
26 process for producing granula of embodiment is investigated
1 dry granulation
The big advantage of the one of dry granulation is to reduce the dosage of auxiliary material, and then reduce patient's dose, therefore take appropriate combination
Object extract powder adds dextrin, lactose respectively, is uniformly mixed with 3: 1 ratio, carries out dry-pressing granulation with GZL20 type dry granulating machine,
Dry granulation the results are shown in Table 35.
Composition extract powder is prepared as follows:
Ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, the flower bud of lily magnolia extract volatile oil 10 hours, and volatile oil is included with betadex, the water after distillation
Liquid is spare, and the dregs of a decoction add 8 times of amount water to extract 1 hour, and filtration, the another device of filtrate stores for future use;
Eight taste such as remaining Radix Astragali, Radix Paeoniae Alba adds water to cook three times, 1.5 hours every time, collecting decoction, filtration, filtrate with it is above-mentioned
Two liquid merge, and are concentrated into the clear cream that relative density is 1.15~1.20 (60 DEG C), add ethyl alcohol to make alcohol content up to 60%, stirring is quiet
It sets overnight, filtrate recycling ethanol, filtrate is concentrated into right amount, is dried under reduced pressure, and fine powder is ground into, with above-mentioned betadex inclusion compound
It mixes, obtains composition extract powder.
35 dry granulation experimental result of table
Excipient | Roll wheel speed (HZ) | Roll wheel pressure (MPa) | One-pass molding rate (%) |
Extract powder: dextrin | 15 | +4 | 44.85 |
Extract powder: lactose | 15 | +4 | 36.00 |
The experimental results showed that roll wheel pressure be 4MPa when, granule fines are more, and one-pass molding rate is lower, respectively less than 50, and
Extract powder viscosity is stronger, occurs gluing in pelletization and rolls wheel phenomenon, pelletizes more difficult, be unable to meet production requirement, therefore use instead wet
Method granulation.
2 wet granulations
The selection of 2.1 wetting agents
The common wetting agent of wet granulation is ethyl alcohol, therefore selects ethyl alcohol for wetting agent, and investigate different ethanol concentration to system
The influence of grain situation.
2.2 supplementary product kinds are investigated
Extract powder and dextrin, lactose, soluble starch are pressed into 3: 1 mixings respectively, 95% alcohol granulation is sprayed into, with softwood
Shape, the ratio of briquetting of particle, granulation complexity and particle color, fine powder, bonding situation etc. be evaluation index, the results are shown in Table
36。
36 supplementary product kind of table is investigated
Excipient | Softwood character | Granulation complexity | Ratio of briquetting (%) |
Extract powder: dextrin | Loosely, no agglomerate | Sieve can be passed through completely | 60.08 |
Extract powder: lactose | Loosely, no agglomerate | Sieve can be passed through completely | 47.20 |
Extract powder: soluble starch | Loosely, no agglomerate | Sieve can be passed through completely | 53.74 |
As can be seen from the results, three kinds of auxiliary materials can make grain forming, and granulation is easy, but when dextrin is added particle ratio of briquetting most
Height, and dextrin is cheap compared to lactose and soluble starch, stability is good, good fluidity, therefore select dextrin for auxiliary material.
The dosage of 2.3 auxiliary materials and the investigation of wetting agent concentration of alcohol
Appropriate extract powder and dextrin are uniformly mixed in 3: 1,3: 2,1: 1 ratio respectively, spraying appropriate concentration respectively is
90%, 94%, 95% alcohol granulation, granulation situation the results are shown in Table 37.
37 granule moulding process optimization test result of table
As can be seen from the above results, extract powder is to wet more sensitive, and when to select 90% ethyl alcohol be wetting agent, agglomerate is more,
Granulation is difficult;Selecting 95% ethyl alcohol is wetting agent, particle is made loosely, fine powder is more, and ratio of briquetting is lower;Select 94% ethyl alcohol
When for wetting agent, it is easy granulation, grain graininess is uniform, and ratio of briquetting is higher.When the auxiliary ratio of medicine is 3: 1,3: 2, particle can be made,
Therefore selecting hydroscopicity is index, further determines that the auxiliary ratio of best medicine.
The determination of the auxiliary ratio of 2.4 medicines
Using the hygroscopicity of particle as evaluation index, the auxiliary ratio of optimal medicine of particle is screened, extract powder and appropriate dextrin are weighed,
It mixes according to 3: 1 and 3: 2, pelletizes using 94% ethyl alcohol as wetting agent respectively, measure its Moisture percentage, it is bent to draw moisture absorption
Line.
The hygroscopicity that can be seen that particle after pelletizing from the hygroscopicity result of particle is improved compared to extract powder,
Compare medicine it is auxiliary than be 3: 1 and 3: 2 particle, medicine it is auxiliary than 3: 1 made from particle hydroscopicity it is higher, and 3: 1 granulation more difficult, agglomerates
More, when the auxiliary ratio of medicine is 3: 2, granulation is easy, and compared with medicine is auxiliary than 3: 1, dose difference is little, therefore the auxiliary ratio of final choice medicine is
3: 2,94% ethyl alcohol is wetting agent, and granulation is easy, and grain forming rate is high.
The thin layer of 27 Chinese medicinal composition granules of embodiment identifies
Identify A:
The preparation of test solution: Chinese medicinal composition granules 4g prepared by Example 4, it is finely ground, add methanol 60mL, surpasses
Sonication 30 minutes, filtration, filtrate was set and is evaporated on water-bath, and residue adds water 50mL to dissolve, and is saturated with water n-butanol shaking and extracts 4
Secondary, each 50mL merges n-butanol liquid, is washed twice with ammonia solution, each 50mL, ammonia solution discards, and n-butanol liquid is evaporated, residual
Slag adds methanol 5mL to make to dissolve, as test solution.
The preparation of control medicinal material solution: taking Radix Astragali control medicinal material 4g, and water 200mL is added to decoct 1 hour, filtration, and filtrate is appropriate
It is concentrated (about 20mL), is saturated with water n-butanol shaking and extracts 4 times, each 20mL, merge n-butanol liquid, wash two with ammonia solution
Secondary, each 20mL, ammonia solution discards, and n-butanol liquid is evaporated, and residue adds methanol 5mL to dissolve, as control medicinal material solution.
The preparation of negative control solution: in prescription ratio, autogamy is free of group's medicine of Radix Astragali, and negative sky is made by preparation process
Negative control solution is made in white preparation, the preparation method with test solution.
Indentification by TLC: drawing above-mentioned each 10 μ L of three kinds of solution, is put respectively on same silica gel g thin-layer plate, with trichlorine
Lower layer's solution of methane-methanol-water (13: 7: 2) is solvent, is unfolded, and takes out, dries, and is sprayed with 10% ethanol solution of sulfuric acid, In
105 DEG C to be heated to spot development clear, sets and inspects under ultraviolet lamp (365nm).
Test result: in sample chromatogram, on position corresponding with reference medicine chromatography, show identical brown under daylight
Color spot point negative sample does not interfere with.
Identify B:
The preparation of test sample solution: Chinese medicinal composition granules 1g prepared by Example 4, it is finely ground, add methanol 20mL, ultrasound
Processing 20 minutes, filtration, filtrate are set and are evaporated on water-bath, and residue adds methanol 1mL to make to dissolve, as test solution.
The preparation of reference substance solution: taking Paeoniflorin reference substance, adds methanol that solution of every 1mL containing 0.6mg is made, as control
Product solution.
The preparation of negative control solution: in prescription ratio, autogamy is free of group's medicine of Radix Paeoniae Alba, and negative sky is made by preparation process
Negative control solution is made in white preparation, the preparation method with test solution.
Indentification by TLC: draw 4 μ L of reference substance solution, each 10 μ L of test solution, negative control solution, put respectively in
On same silica gel g thin-layer plate, with chloroform-acetate-methanol-formic acid (8: 2: 5: 0.2) for solvent, it is unfolded, takes out,
It dries, sprays with 5% vanillin-sulfuric acid solution, it is clear to be heated to spot development at 105 DEG C.
Test result: in sample chromatogram, at the position corresponding to the chromatogram of the reference substance, the spot of same color is shown;Yin
Property sample does not interfere with.
Identify C
The preparation of test solution: Chinese medicinal composition granules 4g prepared by Example 4, it is finely ground, add acetone 20mL, surpasses
Sonication 20 minutes, filtration;Filtrate is set and is evaporated on water-bath, and residue adds methanol 1mL to make to dissolve, as test solution.
The preparation of reference substance solution: taking 5-O- methyl visamminol glycosides reference substance, adds methanol that every 1mL is made containing 0.5mg
Solution, as reference substance solution.
The preparation of control medicinal material solution: taking windproof control medicinal material 2g, adds acetone 20mL, is ultrasonically treated 20 minutes, filters, filter
Liquid is set and is evaporated on water-bath, and residue adds methanol 1mL to make to dissolve, as control medicinal material solution.
The preparation of negative control solution: in prescription ratio, negative sky is made by preparation process in group's medicine of the autogamy without radix saposhnikoviae
Negative control solution is made in white preparation, the preparation method with test solution.
Indentification by TLC: 4 μ L of reference substance solution is drawn, control medicinal material solution, test solution, negative control solution are each
10 μ L put respectively on same silica GF254 lamellae, with chloroform-methanol (5: 1) for solvent, are unfolded, take out, dry in the air
It is dry, it sets and is inspected under ultraviolet lamp (254nm).
Test result: in sample chromatogram, on position corresponding with reference substance and reference medicine chromatography, same color is shown
Spot;Negative sample does not interfere with.
Identify D
The preparation of test solution: Chinese medicinal composition granules 2g prepared by Example 4, it is finely ground, add chloroform
25mL is ultrasonically treated 30 minutes, and filtration, filtrate sets and be evaporated on water-bath, and the residue 1mL that adds methylene chloride makes to dissolve, as examination
Product solution.
The preparation of reference substance solution: taking schizandrin reference substance, adds methanol that solution of every 1mL containing 0.5mg is made, as
Reference substance solution.
The preparation of control medicinal material solution: taking Schisandra chinensis control medicinal material 1g, adds chloroform 25mL, is ultrasonically treated 30 minutes,
Filtration, filtrate sets and is evaporated on water-bath, and the residue 1mL that adds methylene chloride makes to dissolve, as control medicinal material solution.
The preparation of negative control solution: in prescription ratio, autogamy is free of group's medicine of Schisandra chinensis, feminine gender is made by preparation process
Negative control solution is made in blank formulation, the preparation method with test solution.
Indentification by TLC: drawing above-mentioned each 10 μ L of three kinds of solution, puts respectively on same silica GF254 lamellae, with
Petroleum ether (30~60 DEG C)-Ethyl formate-formic acid (15: 6: 1) upper solution is solvent, is unfolded, and takes out, hangs ultraviolet lamp
It is inspected under (254nm).
Test result: in sample chromatogram, on position corresponding with reference substance and reference medicine chromatography, same color is shown
Spot;Negative sample does not interfere with.
Identify E
The preparation of test solution: Chinese medicinal composition granules 3g prepared by Example 4, it is finely ground, add methanol 30mL, surpasses
Sonication 30 minutes, filtration, filtrate was set and is evaporated on water-bath, and residue adds water 20mL to dissolve, and was extracted 2 times with ethyl acetate shaking,
Each 20mL, combined ethyl acetate liquid, is evaporated, and residue adds methanol 10mL to make to dissolve, as test solution.
The preparation of control medicinal material solution: extracting liquorice control medicinal material 2g adds water 200mL to decoct 1 hour, filtration, and filtrate is appropriate
It is concentrated (about 20mL), is extracted 2 times, each 20mL with ethyl acetate shaking, combined ethyl acetate liquid is evaporated, and residue adds methanol
10mL makes to dissolve, as control medicinal material solution.
The preparation of negative control solution: in prescription ratio, taking the extract of scarce Radix Glycyrrhizae to mix, negative blank formulation be made,
Negative control solution is made in preparation method with test solution.
Indentification by TLC: 5 μ L of control medicinal material solution, test solution and each 10 μ L of negative control solution are drawn, respectively
Point with acetic ether-methanoic acid-glacial acetic acid-water (15: 1: 1: 2) for solvent, is unfolded, takes out on same silica gel g thin-layer plate,
It dries, sprays with 10% ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C.
Test result: in sample chromatogram, on position corresponding with reference medicine chromatography, the fluorescent spot of same color is shown
Point;Negative sample does not interfere with.
The present invention is attempted using cinnaldehydrum as Index Establishment detection method, but ramulus cinnamomi and the principle active component of cortex cinnamomi are equal
For cinnaldehydrum, specificity is not strong, therefore does not set up the detection method;Refer in addition, the present invention has also investigated camphor in the flower bud of lily magnolia and has been used as
Ingredient is marked, but discovery has negative interference, specificity is not strong, due to the more difficult determination of index components or it is difficult to obtain reference substance,
The present invention does not set up corresponding detection method;Ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, four taste of the flower bud of lily magnolia, the spot of aobvious same color in test sample
Point, the principle active component atractylone of Rhizoma Atractylodis Macrocephalae do not show spot, and corresponding other ingredients have negative interference, therefore above-mentioned a few taste medicines are not made
For the index for detecting the traditional chinese medicine composition of the invention.
The assay of 28 Chinese medicinal composition granules of embodiment
The foundation of 1 Astragaloside IV chromatographic condition
The foundation of 1.1 chromatographic conditions
Chromatographic condition: Agilent Zorbax SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), acetonitrile-water (31: 69)
For mobile phase, flow velocity 1.0mLmin-1, 30 DEG C of column temperature, ELSD parameter: 100 DEG C of drift tube temperature, carrier gas (compressed air) flow velocity
2.7L·min-1, 10 μ L of sample volume.
The preparation and measurement of 1.2 solution
(1) preparation of reference substance solution
Take Astragaloside IV reference substance appropriate, it is accurately weighed, add methanol that the molten of every 1mL 0.30225mg containing Astragaloside IV is made
Liquid to get.
(2) preparation of test solution
(2.1) investigation of extracting method
It was found that, being easy when index components Astragaloside IV is directly dissolved with water in test sample with when extracting n-butyl alcohol
There is emulsion, and common breaking method cannot be such that emulsion layer eliminates well, present invention trial is first extracted with methanol
To remove a part of impurity, then when with extracting n-butyl alcohol, i.e., without emulsion.
The influence that circumfluence method and ultrasonic method extract Astragaloside IV is investigated, concrete operations are as follows:
Circumfluence method: taking Chinese medicinal composition granules 4g, accurately weighed, sets in stuffed conical flask, adds methanol 60mL, cold soaking mistake
Night refluxing extraction 1 hour, lets cool, and filtering, filtrate is concentrated to dryness, and residue adds water 40mL to make to dissolve, and is saturated with water n-butanol shaking
It extracts 4 times, each 50mL, merges n-butanol liquid, washed 2 times, each 50mL with ammonia solution, n-butanol liquid is evaporated, and residue adds first
Alcohol makes to dissolve, and is settled to 5mL volumetric flask, shake up to get.
Ultrasonic method: taking Chinese medicinal composition granules 4g, accurately weighed, sets in stuffed conical flask, adds methanol 60mL, ultrasound mentions
30 minutes, 45 minutes are taken, are let cool, are filtered, remaining operates same circumfluence method.
Each 10 μ L of above two test solution is taken respectively, is measured in accordance with the law, is calculated the content of Astragaloside IV, the results are shown in Table
38。
38 extracting method of table is investigated
Method | Astragaloside IV mg/g |
Reflux 1 hour | 0.44 |
Ultrasound 30 minutes | 0.39 |
Ultrasound 45 minutes | 0.34 |
As a result it is found that circumfluence method extract Astragaloside IV content be higher than ultrasonic method, and ultrasound 30 minutes when be higher than 45 minutes,
The content of the prompt too long Astragaloside IV of ultrasonic time declines instead, therefore determines that the extracting method of Astragaloside IV is circumfluence method.
(2.2) investigation of extraction time
To extract the Astragaloside IV in Chinese medicinal composition granules to greatest extent, return time is further investigated to Radix Astragali
Reflux 1 hour, 2 hours, 3 hours have been investigated in the influence of glucoside content, experiment, remaining operates same circumfluence method, it is molten to prepare test sample
Liquid, 10 μ L of sample introduction, measures in accordance with the law, calculates the content of Astragaloside IV, the results are shown in Table 39.
39 extraction time of table is investigated
Extraction time (hour) | Astragaloside IV mg/g |
Reflux 1 hour | 0.44 |
Reflux 1 hour | 0.45 |
Reflux 3 hours | 0.47 |
As a result: refluxing extraction 3 hours, the content highest of Astragaloside IV determined the preparation side that refluxing extraction 3 hours are sample
Method.
The content of Astragaloside IV, measurement result are aobvious in precipitating after also having investigated reflux, ultrasound in above-mentioned experimentation
Showing may be due to the too low chromatographic peak for failing to detect Astragaloside IV of content in precipitating.
To sum up, determine test solution solution the preparation method is as follows: takes this product, finely ground, takes about 4g, accurately weighed, sets
In stuffed conical flask, add methanol 60mL, cold soaking is overnight, refluxing extraction 3 hours, lets cool, and filters, with a small amount of methanol (about 30mL) point
Secondary washing container and residue, merge washing lotion and filtrate, recycling design are simultaneously concentrated to dryness, and residue adds water 40mL to make to dissolve, full with water
It is extracted 4 times, each 50mL with n-butanol shaking, merges n-butanol liquid, sufficiently washed 2 times with ammonia solution, each 50mL, ammonia solution
It discards, merges n-butanol liquid, be evaporated, residue adds methanol to make to dissolve and be transferred in 5mL measuring bottle, with methanol constant volume to scale, shakes
It is even, filtration, take subsequent filtrate to get.
The preparation of negative test solution: self-control is free of the blank sample of Radix Astragali, is prepared into negative test solution.
Measuring method: accurate respectively to draw 10 μ L of reference substance solution, 20 μ L, 10 μ L of test solution injects liquid chromatograph,
It measures in accordance with the law.
1.3 methodological study
(1) specificity is tested
Accurate absorption 10 μ L of reference substance solution, 20 μ L respectively, test solution 10 μ L, negative 10 μ L of test solution,
HPLC measurement.The result is shown in Figure 1-3.
The result shows that negative test solution has no chromatographic peak at retention time identical with Astragaloside IV reference substance,
Feminine gender does not interfere with.
(2) linear relationship is investigated
The preparation of reference substance solution: taking Astragaloside IV reference substance appropriate, accurately weighed, adds methanol that every 1mL is made containing Radix Astragali
The solution of first glycosides 1.209mg is to get reference substance stock solution.Precision absorption Astragaloside IV stock solution is appropriate, sets 10mL volumetric flask, adds
Methanol dilution at concentration be respectively 0.0755625mg/mL, 0.151125mg/mL, 0.30225mg/mL, 0.43524mg/mL,
The solution of 0.6045mg/mL, 0.7254mg/mL.It is accurate respectively to draw 20 μ L, high performance liquid chromatograph is injected, peak area is recorded,
With Astragaloside IV peak area logarithm (Y) for ordinate, Astragaloside IV sample volume logarithm (X) is that abscissa does figure, obtains Astragaloside IV
Regression equation be y=1.6908x+11.362 (R2=0.9993).The result shows that Astragaloside IV 0.0755625mg/mL~
Linear relationship is good in 0.7254mg/mL.
(3) precision test
Precision draws 20 μ L of Astragaloside IV reference substance solution, and continuous sample introduction measures 6 times, measures the peak area result of reference substance
It is shown in Table 40.
40 Precision test result of table (n=6)
The result shows that: relative standard deviation RSD < 3% shows that precision is good.
(4) stability test
Chinese medicinal composition granules (finely ground) is taken, about 4g is taken, it is accurately weighed, Chinese medicinal composition granules are prepared in accordance with the law for examination
Product solution, 0,2,4,6,8,12,24 after preparation hour measures in accordance with the law respectively, and Astragaloside content is averaged in 24 hours
Value is 0.4518mg/g, RSD=2.76%, it was demonstrated that Chinese medicinal composition granules sample is good in 24 hours internal stabilities, as a result
It is shown in Table 41.
41 stability test result of table
(5) reappearance test
It takes with a collection of Chinese medicinal composition granules sample, according to Chinese medicinal composition granules sample solution preparation method,
Test solution is prepared, 6 parts of operation repetitive, is analyzed into HPLC, corresponding peak area is recorded, calculates Astragaloside content, as a result see
Table 42.
42 reproducible test results of table (n=6)
The result shows that: the content average value for measuring Astragaloside IV in test sample is 0.4865mg/g, and RSD < 3% shows
Reproducibility is good.
(6) sample recovery rate is tested
The Chinese medicinal composition granules sample of 6 parts of known contents is taken, finely ground, every part of about 2g is accurately weighed, is separately added into dense
Degree is the Astragaloside IV reference substance solution 1.0mL of 0.976mg/mL, prepares test solution, measures content, calculates sample-adding recycling
Rate the results are shown in Table 43.
43 sample recovery rate test result (n=6) of table
The result shows that: the average recovery rate of Astragaloside IV is 98.05%, RSD=2.93%, n=6, sample recovery rate symbol
It closes and requires.
Conclusion: to sum up, the precision of Astragaloside IV method, linear, reproducibility, stability, accuracy meet the requirements,
It is feasible to illustrate that the content assaying method is stablized.
The foundation of 2 Paeoniflorin chromatographic conditions
The foundation of 2.1 chromatographic conditions
Chromatographic condition: Waters symmetryshieldTMRP18 chromatographic column (4.6mm × 150mm, 5 μm);Methanol-water
It (15: 85) is mobile phase, flow velocity 1.0mLmin-1, 30 DEG C of column temperature, sample volume is 10 μ L, Detection wavelength: 230nm.
The preparation and measurement of 2.2 solution
The preparation of reference substance solution: taking Paeoniflorin reference substance appropriate, accurately weighed, adds methanol that every 1mL first containing Radix Astragali is made
The solution of glycosides 0.04112mg to obtain the final product.
The preparation of test solution solution: taking this product, finely ground, takes about 1g, accurately weighed, sets in stuffed conical flask, accurate
Methanol 50mL, close plug is added, weighed weight is ultrasonically treated (power 100W, frequency 40KHZ) 30 minutes, lets cool, then weighed heavy
Amount, the weight of less loss is supplied with methanol, is shaken up, filter, take subsequent filtrate to get.
The preparation of negative test solution: in prescription taste of traditional Chinese medicine ratio, autogamy is free of group's medicine of Radix Paeoniae Alba, by preparation process system
It is standby to prepare at blank formulation, then by sample solution preparation method to get negative test solution.
Measuring method: it is accurate respectively to draw reference substance solution, test solution and each 10 μ L of negative test solution, inject liquid
Chromatography, measurement.
Paeoniflorin content measuring method is found in establishing, and uses the lower content assaying method of 2015 editions " Chinese Pharmacopoeia " Radix Paeoniae Albas
When measuring Paeoniflorin, the hangover of Paeoniflorin peak;Different mobile phase condition (1. Wondasil TM C18 chromatographies have been attempted afterwards and respectively
Column (250mm × 4.6mm, 5 μm), -0.1% phosphoric acid solution of acetonitrile are mobile phase, gradient elution, Detection wavelength is respectively 230,
286nm, flow velocity 1.0mlmin-1;2. Eclipse XDB-C18 chromatographic column (250mm × 4.6mm, 5 μm), -0.1% phosphorus of acetonitrile
Aqueous acid (14: 86) is mobile phase, flow velocity: 1.0mlmin-1, Detection wavelength: 230nm;3. Agilent ZORBAXSBC18
(4.6mm × 250mm, 5 μm) chromatographic column, -0.1% phosphoric acid water of acetonitrile are mobile phase, gradient elution, flow velocity 1.0mlmin-1,
25 DEG C of column temperature, Detection wavelength: 230nm), different test samples prepare impurity-removing method, as a result, it has been found that the hangover of Paeoniflorin peak also without
To improvement;Finally discovery uses Waters symmetryshieldTMWhen RP18 chromatographic column (4.6mm × 150mm, 5 μm), flowing
Mutually acid adding can also not improve the tailing factor at Paeoniflorin peak, therefore final choice Waters symmetryshieldTMRP18 is surveyed
Determine Paeoniflorin.
2.3 methodological study
(1) specificity is investigated
It is accurate respectively to draw reference substance solution, negative test solution and 10 μ L of test solution, liquid chromatograph is injected,
Measurement.As a result see Fig. 4-6.
The result shows that negative sample is in retention time identical with Paeoniflorin reference substance, no chromatographic peak, therefore think negative
It does not interfere with.
(2) the relational investigation of line
The preparation of reference substance solution: taking Paeoniflorin reference substance appropriate, accurately weighed, adds methanol that every 1mL is made containing Paeoniflorin
The solution of 0.1028mg is to get reference substance stock solution.Precision absorption Paeoniflorin stock solution is appropriate, sets 10mL volumetric flask, adds methanol
Be diluted to concentration be respectively 0.01028mg/mL, 0.02056mg/mL, 0.04112mg/mL, 0.049344mg/mL,
The standard solution of 008224mg/mL, 0.1028mg/mL.It is accurate respectively to draw 10 μ L, high performance liquid chromatograph is injected, is measured,
Paeoniflorin standard curve is drawn, the regression equation for obtaining Paeoniflorin is y=107x-19813(R2=0.9999).The result shows that Chinese herbaceous peony
Glycosides linear relationship in 0.01028mg/mL~0.1028mg/mL is good.
(3) precision test
Precision draws 10 μ L of Paeoniflorin reference substance solution, continuous sample introduction 6 times, measures the peak area of reference substance, the results are shown in Table
44。
44 Precision test result of table (n=6)
The result shows that: relative standard deviation RSD < 3% shows that precision is good.
(4) stability test
Prepare Chinese medicinal composition granules test solution, respectively after preparation 2,4,6,8,10,12,14,16,18,
Sample introduction is analyzed within 24 hours, and the average value of paeoniflorin content is 1.86mg/g, RSD=2.55% in 24 hours, shows sample 24
Hour internal stability is good, the results are shown in Table 45.
45 stability test result of table
(5) reappearance test
It takes and prepares 6 parts of test solution according to preparation method of test article with a collection of Chinese medicinal composition granules sample, measure
Peak area calculates paeoniflorin content, the results are shown in Table 46.
46 reproducible test results of table (n=6)
The result shows that: the content average value for measuring Paeoniflorin in test sample is 1.7184mg/g, RSD < 3%, shows weight
Existing property is good.
(6) sample recovery rate is tested
The Chinese medicinal composition granules sample of 6 parts of known contents is taken, finely ground, every part of about 0.5g is accurately weighed, is separately added into
Paeoniflorin reference substance 0.879mg is prepared into test solution, measures content, calculates sample recovery rate, the results are shown in Table 47.
47 sample recovery rate test result (n=6) of table
The result shows that: the mean sample recovery rate of Paeoniflorin is 102.27, RSD=2.28%, n=6, shows that the method is loaded
The rate of recovery meets the requirements.
Conclusion: to sum up, the methodology of Paeoniflorin meets the requirements, and it is feasible to illustrate that the content assaying method is stablized.
Three batches of sample assays
Three batches of traditional Chinese medicine particle samples are prepared, content is measured by the content assaying method of above-mentioned foundation, calculates Radix Astragali
The content of first glycosides and Paeoniflorin.It the results are shown in Table 48.
48 3 batches of sample assay results of table
Batch | Astragaloside IV mg/g | Paeoniflorin mg/g |
20150528 | 0.54 | 1.68 |
20150628 | 0.49 | 1.88 |
20150728 | 0.50 | 1.81 |
Average value | 0.51 | 1.79 |
As a result: in present composition particle, the average content of Astragaloside IV is 0.51mg/g, the average content of Paeoniflorin
For 1.79mg/g.
It draws up and determines content limit are as follows: the every 1g of this product is containing Radix Astragali with Astragaloside IV (C31H68O14) meter, 0.41mg must not be less than.
The every 1g of this product is containing Radix Paeoniae Alba with Paeoniflorin (C23H28O11) meter, 1.43mg must not be less than.
Claims (10)
1. a kind of detection method of Chinese medicinal composition preparation, which comprises
Assay A:
Determination condition: using octadecylsilane key and silica gel as filler;With acetonitrile-water (25~35): (65~75) are flowing
Phase, evaporative light scattering detector detection;Number of theoretical plate is calculated by Astragaloside IV peak should be not less than 4000;
The preparation of reference substance solution: taking Astragaloside IV reference substance, and organic solvent dissolution obtains reference substance solution;
The preparation of test solution: taking Chinese medicinal composition preparation, and organic solvent extracts, and obtains test solution;
Measuring method: drawing reference substance solution and test solution respectively, injects liquid chromatograph, measurement;
Assay B:
Determination condition: using octadecylsilane key and silica gel as filler;With methanol-water (10~20): (80~90) are flowing
Phase, Detection wavelength 230nm.Number of theoretical plate is calculated by Paeoniflorin peak should be not less than 2000;
The preparation of reference substance solution: taking Paeoniflorin reference substance, and organic solvent dissolution obtains reference substance solution;
The preparation of test solution: taking Chinese medicinal composition preparation, and organic solvent extracts, and obtains test solution;
Measuring method: drawing reference substance solution and test solution respectively, injects liquid chromatograph, measurement;
Wherein, the bulk pharmaceutical chemicals composition of the Chinese medicine composition are as follows: Radix Astragali 8-40 parts by weight, ramulus cinnamomi 4-22 parts by weight, flower bud of lily magnolia 3-16
Parts by weight, radix saposhnikoviae 4-18 parts by weight, Rhizoma Atractylodis Macrocephalae 4-22 parts by weight, rhizoma zingiberis 3-16 parts by weight, cortex cinnamomi 1-6 parts by weight, Radix Paeoniae Alba 3-16 weight
Measure part, dark plum 6-24 parts by weight, Schisandra chinensis 3-16 parts by weight, rhizoma anemarrhenae 3-16 parts by weight, Radix Glycyrrhizae 3-16 parts by weight.
2. detection method as described in claim 1, which is characterized in that the organic solvent be selected from methanol, acetone, n-butanol,
Ethyl acetate, petroleum ether, any one in chloroform;The extracting method be selected from ultrasonic extraction, refluxing extraction, cold soaking extract,
Seepage pressure effects, Microwave Extraction.
3. detection method as described in claim 1, which is characterized in that in the preparation of assay A test solution, using methanol
Refluxing extraction.
4. detection method as claimed in claim 3, which is characterized in that assay A test solution is prepared as follows:
Chinese medicinal composition preparation is taken, methanol cold soaking is added to stay overnight, refluxing extraction, filtrate recycling design is simultaneously concentrated to dryness, and residue is dissolved in water,
It is saturated with water n-butanol shaking to extract, n-butanol extracting liquid liquid is washed with ammonia solution, and ammonia solution discards, the n-butanol liquid after washing
Be evaporated, residue add methanol make dissolution to get.
5. detection method as described in claim 1, which is characterized in that mobile phase is acetonitrile-water (31: 69) in assay A.
6. detection method as described in claim 1, which is characterized in that mobile phase is methanol-water (15: 85) in assay B.
7. detection method as described in claim 1, which is characterized in that the detection method includes following one identified in A-E
Kind is several:
Identify A:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Control medicinal material solution preparation: taking Radix Astragali control medicinal material, and organic solvent extracts, and obtains control medicinal material solution;
Detection: drawing test solution and control medicinal material solution respectively, and point is on same silica gel plate, with chloroform-methanol-water
(10~15): (5~9): (1~3) is solvent, and expansion is heated to spot at 105 DEG C with the colour developing of 10% ethanol solution of sulfuric acid
Colour developing is clear;
Identify B:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Reference substance solution preparation: taking Paeoniflorin reference substance, and organic solvent dissolution obtains reference substance solution;
Detection: drawing test solution and control solution respectively, and point is on same silica gel plate, with chloroform-ethyl acetate-
Methyl alcohol-formic acid (5~10): (1~3): (2~7): (0.1~0.3) is solvent, and expansion is aobvious with 5% vanillin-sulfuric acid solution
It is clear to be heated to spot development at 105 DEG C for color;
Identify C:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Control medicinal material solution preparation: taking windproof control medicinal material, and organic solvent extracts, and obtains control medicinal material solution;
Reference substance solution preparation: taking 5-O- methyl visamminol glycosides reference substance, and organic solvent dissolution obtains reference substance solution;
Detection: drawing test solution, control medicinal material solution and control medicinal material solution respectively, and point is on same silica gel plate, with three
Chloromethanes-methanol (3~8): (0.5~2) is solvent, and expansion is set and inspected under ultraviolet lamp (254nm);
Identify D:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Control medicinal material solution preparation: taking Schisandra chinensis control medicinal material, and organic solvent extracts, and obtains control medicinal material solution;
Reference substance solution preparation: taking schizandrin reference substance, and organic solvent dissolution obtains reference substance solution;
Detection: drawing test solution, control medicinal material solution and control medicinal material solution respectively, and point is on same silica gel plate, with stone
Oily ether-Ethyl formate-formic acid (12~18): (4~8): (0.5~2) is solvent, and expansion is set and examined under ultraviolet lamp (254nm)
Depending on;
Identify E:
Test solution preparation: taking Chinese medicinal composition granules, and organic solvent extracts, and obtains test solution;
Control medicinal material solution preparation: extracting liquorice control medicinal material, organic solvent extract, and obtain control medicinal material solution;
Detection: drawing test solution and control medicinal material solution respectively, and point is on same silica gel plate, with acetic ether-methanoic acid-ice
Acetic Acid-Water (12~18): (0.5~2): (0.5~2): (1~3) is solvent, expansion, with 10% ethanol solution of sulfuric acid, In
105 DEG C to be heated to spot development clear.
8. detection method as claimed in claim 7, which is characterized in that the organic solvent be selected from methanol, acetone, n-butanol,
Ethyl acetate, petroleum ether, any one in chloroform;The extracting method be selected from ultrasonic extraction, refluxing extraction, cold soaking extract,
Seepage pressure effects, Microwave Extraction.
9. detection method as claimed in claim 7, which is characterized in that the identification A sample solution preparation method are as follows: in taking
Drug composition granule adds methanol ultrasonic extraction, and filtrate recycling design is simultaneously concentrated to dryness, and residue is dissolved in water, and is saturated with water just
Butanol shaking is extracted, and n-butanol extracting liquid liquid is washed with ammonia solution, and ammonia solution discards, and the n-butanol liquid after washing is evaporated, residue
Add methanol make dissolution to get.
10. such as the described in any item detection methods of claim 1-9, which is characterized in that the Chinese medicinal composition preparation is by as follows
Method preparation:
Ramulus cinnamomi, rhizoma zingiberis, cortex cinnamomi, the flower bud of lily magnolia extract volatile oil, and volatile oil is included with betadex, and the aqueous after distillation is spare, medicine
Pulp water extracts 1 hour, and filtration, the another device of filtrate stores for future use;
Eight taste such as remaining Radix Astragali, Radix Paeoniae Alba adds water to cook, filtrate with distillation after aqueous and dregs of a decoction extracting solution merge, add ethyl alcohol to make to contain
Up to 60%, filtering, filtrate recycling ethanol is dried under reduced pressure alcohol amount, is mixed with above-mentioned betadex inclusion compound, is obtained composition medicinal extract
Powder;
Composition extract powder is uniformly mixed with dextrin by 3: the 2 auxiliary ratio of medicine, wet granulation, using 94% ethyl alcohol as wetting agent, system
Standby particle, obtains granule.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810353717.2A CN110389179A (en) | 2018-04-19 | 2018-04-19 | A kind of detection method of Chinese medicine composition |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810353717.2A CN110389179A (en) | 2018-04-19 | 2018-04-19 | A kind of detection method of Chinese medicine composition |
Publications (1)
Publication Number | Publication Date |
---|---|
CN110389179A true CN110389179A (en) | 2019-10-29 |
Family
ID=68283319
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810353717.2A Pending CN110389179A (en) | 2018-04-19 | 2018-04-19 | A kind of detection method of Chinese medicine composition |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110389179A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115792017A (en) * | 2022-12-06 | 2023-03-14 | 中国中医科学院眼科医院 | Quality detection method of traditional Chinese medicine composition for reducing recurrence of herpes simplex viral keratitis |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1768854A (en) * | 2005-10-26 | 2006-05-10 | 三九医药股份有限公司 | Chinese medicinal capsule with spleen-supplementing, intestine-benefiting function |
CN101158670A (en) * | 2005-09-22 | 2008-04-09 | 北京奇源益德药物研究所 | Quality control method of pharmaceutical composition |
-
2018
- 2018-04-19 CN CN201810353717.2A patent/CN110389179A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101158670A (en) * | 2005-09-22 | 2008-04-09 | 北京奇源益德药物研究所 | Quality control method of pharmaceutical composition |
CN1768854A (en) * | 2005-10-26 | 2006-05-10 | 三九医药股份有限公司 | Chinese medicinal capsule with spleen-supplementing, intestine-benefiting function |
Non-Patent Citations (10)
Title |
---|
周滢 等: "RP-HPLC测定芍石护睛胶囊中芍药苷的含量", 《华西药学杂志》 * |
国家药典委员会: "《中华人民共和国药典 2015年版 一部》", 30 June 2015, 中国医药科技出版社 * |
国家食品药品监督管理总局执业药师资格认证中心: "《中药学专业知识(一) 第七版》", 31 January 2017, 中国医药科技出版社 * |
孙雪妹 等: "市售五味子与南五味子的鉴别研究和质量分析", 《内蒙古中医药》 * |
李四维 等: "芪防鼻敏汤治疗过敏性鼻炎100例临床观察", 《中华中医药杂志》 * |
毕映燕 等: "HPLC测定复方赤芍颗粒中芍药苷含量", 《中国中医药信息杂志》 * |
续艳 等: "芪防鼻敏颗粒对变应性鼻炎大鼠行为学与组织病理学的影响", 《中国药师》 * |
续艳 等: "芪防鼻敏颗粒抗炎、抗过敏作用研究", 《中国药师》 * |
蓝鸣生 等: "五淋散胶囊中栀子苷和芍药苷的HPLC含量测定", 《中国新药杂志》 * |
薛丹 等: "多指标正交设计优选芪防鼻敏颗粒水提醇沉工艺", 《天津中医药》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115792017A (en) * | 2022-12-06 | 2023-03-14 | 中国中医科学院眼科医院 | Quality detection method of traditional Chinese medicine composition for reducing recurrence of herpes simplex viral keratitis |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN1239187C (en) | Chinese medicine composition for curing chronic atrophic sastritis and its preparing method and quality control method | |
CN103830577A (en) | Medicine composition for treating liver and gall calculus and kidney stone and application thereof | |
CN101991785B (en) | Lonicerae and Forsythiae detoxication soft capsule medicine and preparation method and quality detection method thereof | |
CN114246918B (en) | Traditional Chinese medicine composition for treating hashimoto thyroiditis and preparation method thereof | |
CN110384787A (en) | A kind of Chinese medicine composition and preparation method thereof for treating allergic rhinitis | |
CN101002929A (en) | Medicine for treating functional hypopepsia or complicating Helicobacter pylori infection, and its preparing method | |
CN109718273A (en) | Perilla leaf extract is preventing or is treating the application in Osteoarthritis | |
CN103341092B (en) | Preparation method of powder for treating atrophic vaginitis | |
CN105056128B (en) | A kind of Chinese medicine composition and preparation method and application for treating atrophic gastritis and intestines | |
CN105343260B (en) | The drug for treating chronic ulcerative colitis | |
CN110389179A (en) | A kind of detection method of Chinese medicine composition | |
CN110384788A (en) | A kind of preparation method for treating allergic rhinitis Chinese medicine composition | |
CN101850091B (en) | Chinese medicament for treating colorectal polypus and preparation method thereof | |
CN103446500B (en) | Traditional Chinese medicine composition for treating cyclomastopathy, mammary pain and postpartum milk obstruction | |
CN105194460A (en) | Traditional Chinese medicine preparation for treating acute episode stage of wind-damp-heat type gout | |
CN102309705B (en) | Medicine for reducing serum uric acid, preparation method thereof and purpose thereof | |
WO2021169682A1 (en) | Traditional chinese medicine composition, and preparation method therefor and application thereof | |
CN114939150A (en) | Traditional Chinese medicine emulsifiable paste for treating hyperplasia of mammary glands with anxiety and depression and preparation method thereof | |
CN105327115B (en) | A kind of prevention and treatment type II diabetes Phellinus is logical to rush down formula and preparation process | |
CN101569682B (en) | Medical composition for treating rheumatoid arthritis and preparation method | |
CN107029084A (en) | A kind of Chinese medicine composition and its pharmaceutical preparation for treating Colon and rectum precancerous lesion and application | |
CN103191399A (en) | Traditional Chinese medicine composition for treating bronchial asthma in acute-outbreak period | |
CN108434287B (en) | Traditional Chinese medicine composition and preparation method and application thereof | |
CN108452008A (en) | A kind of application including Common Leafflower Herb, rainbow conk, Radix Salviae Miltiorrhizae and the Chinese medicine composition of Asian puccoon in preparing the drug for inhibiting recurrence of PHC | |
CN110064016A (en) | A kind of Chinese medicine composition and preparation method thereof adjusting chronic kidney disease immune state |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191029 |