CN107233511B - A kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and purposes - Google Patents
A kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and purposes Download PDFInfo
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- CN107233511B CN107233511B CN201710449157.6A CN201710449157A CN107233511B CN 107233511 B CN107233511 B CN 107233511B CN 201710449157 A CN201710449157 A CN 201710449157A CN 107233511 B CN107233511 B CN 107233511B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/19—Acanthaceae (Acanthus family)
- A61K36/195—Strobilanthes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
- A61K36/233—Bupleurum
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
- A61K36/315—Isatis, e.g. Dyer's woad
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
- A61K36/484—Glycyrrhiza (licorice)
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- 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)
- A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
- A61K36/634—Forsythia
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- 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)
- A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
- A61K36/704—Polygonum, e.g. knotweed
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- 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)
- A61K36/84—Valerianaceae (Valerian family), e.g. valerian
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- 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)
- A61K36/85—Verbenaceae (Verbena family)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/4841—Filling excipients; Inactive ingredients
- A61K9/4866—Organic macromolecular compounds
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- 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
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- 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
Abstract
The present invention relates to the field of Chinese medicines, and in particular to a kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and pharmaceutical applications.The dispelling wind detoxicating capsule is made up of the flavour of a drug raw material of following parts by weight：The parts by weight of giant knotweed 5, the parts by weight of the capsule of weeping forsythia 4, the parts by weight of Radix Isatidis 4, the parts by weight of radix bupleuri 4, the parts by weight of field pennycress 4, the parts by weight of Verbena officinalis 4, the parts by weight of reed rhizome 3, the parts by weight of radix glycyrrhizae 2.The invention provides use RP HPLC dual wavelengths switching method to determine the method for dispelling wind detoxicating capsule active component content simultaneously, while provides the new pharmaceutical use of dispelling wind detoxicating capsule.
Description
Technical field
The present invention relates to the field of Chinese medicines, and in particular to a kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and system
Medicinal way.
Background technology
Dispelling wind detoxicating capsule, is the exclusive product of applicant Anhui Jiren Pharmacy Co., Ltd., and authentication code is：
Chinese medicines quasi-word Z2009004, specification：Every dress 0.52g.The kind is the hereditary recipe to Chu Xian according to the famous old docter of TCM in China,
Original name " toxic removing dissipates ", the new Chinese medicine of development, by giant knotweed, the capsule of weeping forsythia, field pennycress, radix bupleuri, Verbena officinalis, Radix Isatidis, reed rhizome, radix glycyrrhizae etc.
Flavour of a drug form.With wind and heat dispersing, the work(for relieving sore-throat of detoxifying, for treating acute upper respiratory infection category wind-heat syndrome, symptoms include generate heat,
Foul wind, pharyngalgia, have a headache, nasal obstruction, flow turbid tears, cough etc., clinical practice is for many years, curative for effect.This product is listed in the Ministry of Public Health《A type
H1N1 influenza diagnosis and treatment schemes》(second edition in 2009, the third edition, version in 2010) treatment wind-heat violate defend preferred Chinese patent drug,《It is popular
Flu Clinics and Practices guide (version in 2011)》, State Administration of Traditional Chinese Medicine《Fever caused by exogenous pathogens (infection of the upper respiratory tract) diagnosis and treatment side
Case》With《Influenza (Influenza A H1N1) diagnosis and treatment scheme》Recommended drug, and include version country medical insurance catalogue in 2009.
This product is the key product of applicant Anhui Ji people's medicine company, is that annual value of production, annual sales amount are crossed in hundred million
The big kind of medicine.Obtain within 2011 that National modern Chinese medicine development for hi-tech industry is special to be supported, successively win that " state key newly produces
A series of honorary titles such as product ", " Anhui Province's autonomous innovation product ", " respiratory disease class Chinese medicine top ten ".This product has
Independent intellectual property right (patent No. ZL200510117119.8), there are the effect of preferable and city in treatment acute upper respiratory infection
, how to ensure the stable uniformity and its safety and effectiveness of product, be the urgent problem to be solved that this product develops.Existing dredges
Wind detoxicating capsule detection method only defines the capsule of weeping forsythia, radix bupleuri, the TLC Identification and tlc scanning determination of Verbena officinalis
The method of emodin content, and to be 8 kinds of Chinese medicine flavour of a drug raw materials form this product, complicated component, existing detection method be still in compared with
The simple primary stage, it is clear that be unfavorable for the guarantee of product quality and curative effect.
The raising of product quality be unable to do without the raising of detection method, and therefore, applicant of the present invention Anhui Ji people's medicine company has
Limit company is significantly lifted to dispelling wind detoxicating capsule detection method, has once applied for two pieces invention on October 27th, 2014
Patent, denomination of invention are respectively：A kind of method for building up of dispelling wind detoxicating capsule finger-print, a kind of detection of dispelling wind detoxicating capsule
Method, application number are respectively：2014105823185th, 201410583418X, patent publication No. are respectively：104316613B、
104330489B, two pieces patent propose the detection method of the finger-print to dispelling wind detoxicating capsule respectively, and to its horsewhip
Several compositions such as careless glycosides, verbenalin, polygonin, forsythiaside A, acteoside, mono-ammonium glycyrrhizinate, rheum emodin are examined
The method of survey, these detection methods serve important impetus for the quality of lifting dispelling wind detoxicating capsule.But, exist
In production practices and scientific experiment, it has been found that L-Epicatechin gallate, Cassia pine -8-O- glucosides and the third two
Acyl group saikosaponin D is dispelling wind detoxicating capsule important activity composition, so, applicant has carried out experimental study repeatedly to this,
It is proposed and establish while to L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl saikosaponin D
The method that content is detected.
In addition, guiding theory of the applicant according to national Chinese medicine big variety development strategy, to the new of dispelling wind detoxicating capsule
Therapeutical uses have carried out systematic research, have been surprisingly found that under study for action, and dispelling wind detoxicating capsule has to NASH
Therapeutic effect protrude very much, unexpected.
This project has obtained the emphasis support of national science technology department small medium S＆T enterprises innovation fund, entry name
Claim：Dispelling wind detoxicating capsule, item types：Key project, code of setting up the project：10C26213404286, authentication code：Section of state hair meter word
[2010] No. 543.
The content of the invention
Based on technical problem existing for background technology, the present invention propose a kind of dispelling wind detoxicating capsule and preparation method thereof with
Detection method and pharmaceutical applications.
It is an object of the invention to provide a kind of dispelling wind detoxicating capsule medicine and preparation method thereof.
It is a further object of the present invention to provide the drug test method of dispelling wind detoxicating capsule.
Present invention also offers the new pharmaceutical applications of dispelling wind detoxicating capsule.
The purpose of the present invention is achieved in the following ways：
A kind of dispelling wind detoxicating capsule, it is made up of the flavour of a drug raw material of following parts by weight：The parts by weight of giant knotweed 5, the weight of the capsule of weeping forsythia 4
Part, the parts by weight of Radix Isatidis 4, the parts by weight of radix bupleuri 4, the parts by weight of field pennycress 4, the parts by weight of Verbena officinalis 4, the parts by weight of reed rhizome 3, the weight of radix glycyrrhizae 2
Measure part.
A kind of preparation method of dispelling wind detoxicating capsule, the dispelling wind detoxicating capsule are the flavour of a drug raw material systems by following parts by weight
Into：The parts by weight of giant knotweed 5, the parts by weight of the capsule of weeping forsythia 4, the parts by weight of Radix Isatidis 4, the parts by weight of radix bupleuri 4, the parts by weight of field pennycress 4, the weight of Verbena officinalis 4
Part, the parts by weight of reed rhizome 3, the parts by weight of radix glycyrrhizae 2 are measured, are prepared with the following method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction
Again plus 3 times of 70% ethanol heating and refluxing extraction 1h of amount, filtration, filtrate merge, and reclaim ethanol and are concentrated under reduced pressure at 60 DEG C relative
The thick paste of density 1.35~1.40, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；
The dregs of a decoction and decoction coarse filtration, filtrate centrifuge, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction close after radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract volatile oil
And it is secondary to add water to cook extraction, for the first time plus 18 times of amount water extraction 2h, second plus 12 times of amount water extraction 1h, coarse filtration, filtrate from
The heart separates, and the supernatant after radix bupleuri that the supernatant extracted twice obtains with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and depressurizes dense
The thick paste of relative density 1.35~1.40 at 60 DEG C is reduced to, it is standby；
S4：Dextrin, superfine silica gel powder are taken, is mixed, the water extraction concentration thick paste that above-mentioned S1 steps obtain is added to and is obtained with S3 steps
To alcohol extracting concentration thick paste in, stir, laying, be dried in vacuo, crush, add dextrin, spray into S2 steps and obtain
Volatile oil, sieve, mix, load capsule, produce.
A kind of detection method of dispelling wind detoxicating capsule, the dispelling wind detoxicating capsule are the flavour of a drug raw material systems by following parts by weight
Into：The parts by weight of giant knotweed 5, the parts by weight of the capsule of weeping forsythia 4, the parts by weight of Radix Isatidis 4, the parts by weight of radix bupleuri 4, the parts by weight of field pennycress 4, the weight of Verbena officinalis 4
Part, the parts by weight of reed rhizome 3, the parts by weight of radix glycyrrhizae 2 are measured, the dispelling wind detoxicating capsule is prepared with the following method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction
Again plus 3 times of 70% ethanol heating and refluxing extraction 1h of amount, filtration, filtrate merge, and reclaim ethanol and are concentrated under reduced pressure at 60 DEG C relative
The thick paste of density 1.35~1.40, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；
The dregs of a decoction and decoction coarse filtration, filtrate centrifuge, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction close after radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract volatile oil
And it is secondary to add water to cook extraction, for the first time plus 18 times of amount water extraction 2h, second plus 12 times of amount water extraction 1h, coarse filtration, filtrate from
The heart separates, and the supernatant after radix bupleuri that the supernatant extracted twice obtains with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and depressurizes dense
The thick paste of relative density 1.35~1.40 at 60 DEG C is reduced to, it is standby；
S4：Dextrin, superfine silica gel powder are taken, is mixed, the water extraction concentration thick paste that above-mentioned S1 steps obtain is added to and is obtained with S3 steps
To alcohol extracting concentration thick paste in, stir, laying, be dried in vacuo, crush, add dextrin, spray into S2 steps and obtain
Volatile oil, sieve, mix, load capsule, produce；
L-Epicatechin gallate, Cassia in dispelling wind detoxicating capsule are determined using RP-HPLC dual wavelengths switching method simultaneously
The content of pine -8-O- glucosides and malonyl saikosaponin D is as follows the step of its detection method：
(1) chromatographic condition：Chromatographic column：C18Post, specification：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.4mol/L phosphorus
Acid dihydride sodium solution, gradient elution, elution program are：0 to 10min, acetonitrile is from 5% linear rise to 15%, 0.4mol/L phosphorus
Acid dihydride sodium solution is from 95% linear decline to 85%, and from 11 to 20min, acetonitrile is from 15% linear rise to 35%, 0.4mol/
L sodium dihydrogen phosphates are from 85% linear decline to 65%；Flow velocity：0.5~2.0mLmin-1；Detection wavelength：0 to 10min inspection
Survey wavelength 230~240nm, 11 to 330~340nm of 20min Detection wavelengths；Column temperature：30~40 DEG C；Sample size：5~20 μ L；
(2) preparation of mixed reference substance solution：L-Epicatechin gallate, Cassia pine -8-O- glucosides are taken respectively
It is accurately weighed with malonyl saikosaponin D reference substance, put in same measuring bottle, dissolved with 0.2mol/L sodium dihydrogen phosphates
And scale is diluted to, shake up, mixed reference substance solution is made；
(3) preparation of need testing solution：This product content is taken, it is accurately weighed, put in centrifuge tube, precision adds 0.2mol/L
Sodium dihydrogen phosphate, weighed quality, ultrasonic extraction, lets cool, then weighed quality, is supplied with 0.2mol/L sodium dihydrogen phosphates
The quality of institute's less loss, centrifugation, supernatant is taken, after filter paper filtering, take subsequent filtrate, then filtered with 0.45 μm of miillpore filter, take continuous filter
Liquid, produce need testing solution；
(4) determine：Take mixed reference substance solution, need testing solution injection high performance liquid chromatograph, measure.
Above-mentioned detection method, epicatechin in dispelling wind detoxicating capsule is determined simultaneously using RP-HPLC dual wavelengths switching method and not had
The content of infanticide acid esters, Cassia pine -8-O- glucosides and malonyl saikosaponin D, the preferred steps of its detection method are such as
Under：
(1) chromatographic condition：Chromatographic column：C18Post, specification：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.4mol/L phosphorus
Acid dihydride sodium solution, gradient elution, elution program are：0 to 10min, acetonitrile is from 5% linear rise to 15%, 0.4mol/L phosphorus
Acid dihydride sodium solution is from 95% linear decline to 85%, and from 11 to 20min, acetonitrile is from 15% linear rise to 35%, 0.4mol/
L sodium dihydrogen phosphates are from 85% linear decline to 65%；Flow velocity：1.5mL·min-1；Detection wavelength：0 to 10min detection ripple
Long 235nm, 11 to 20min Detection wavelengths 336nm；Column temperature：36℃；Sample size：20μL；
(2) preparation of mixed reference substance solution：L-Epicatechin gallate, Cassia pine -8-O- glucosides are taken respectively
It is accurately weighed with malonyl saikosaponin D reference substance, put in same 100mL brown measuring bottles, with 0.2mol/L sodium dihydrogen phosphates
Solution dissolves and is diluted to scale, shakes up, and it is respectively 300.0mgL that mass concentration, which is made,-1、350.0mg·L-1With
450.0mg·L-1Mixed reference substance solution；
(3) preparation of need testing solution：This product content 0.1g is taken, it is accurately weighed, put in 5mL centrifuge tubes, precision adds
0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, let cool, then
Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifuged, taken supernatant, after filter paper filtering, take
Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, produces need testing solution；
(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, inject high performance liquid chromatograph, measure.
A kind of application of dispelling wind detoxicating capsule in treatment NASH medicine is prepared, the dispelling wind detoxicating capsule are
It is made up of the flavour of a drug raw material of following parts by weight：The parts by weight of giant knotweed 5, the parts by weight of the capsule of weeping forsythia 4, the parts by weight of Radix Isatidis 4, the parts by weight of radix bupleuri 4,
The parts by weight of field pennycress 4, the parts by weight of Verbena officinalis 4, the parts by weight of reed rhizome 3, the parts by weight of radix glycyrrhizae 2, the dispelling wind detoxicating capsule are using as follows
Prepared by method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction
Again plus 3 times of 70% ethanol heating and refluxing extraction 1h of amount, filtration, filtrate merge, and reclaim ethanol and are concentrated under reduced pressure at 60 DEG C relative
The thick paste of density 1.35~1.40, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；
The dregs of a decoction and decoction coarse filtration, filtrate centrifuge, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction close after radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract volatile oil
And it is secondary to add water to cook extraction, for the first time plus 18 times of amount water extraction 2h, second plus 12 times of amount water extraction 1h, coarse filtration, filtrate from
The heart separates, and the supernatant after radix bupleuri that the supernatant extracted twice obtains with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and depressurizes dense
The thick paste of relative density 1.35~1.40 at 60 DEG C is reduced to, it is standby；
S4：Dextrin, superfine silica gel powder are taken, is mixed, the water extraction concentration thick paste that above-mentioned S1 steps obtain is added to and is obtained with S3 steps
To alcohol extracting concentration thick paste in, stir, laying, be dried in vacuo, crush, add dextrin, spray into S2 steps and obtain
Volatile oil, sieve, mix, load capsule, produce；
L-Epicatechin gallate, Cassia in dispelling wind detoxicating capsule are determined using RP-HPLC dual wavelengths switching method simultaneously
The content of pine -8-O- glucosides and malonyl saikosaponin D is as follows the step of its detection method：
(1) chromatographic condition：Chromatographic column：C18Post, specification：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.4mol/L phosphorus
Acid dihydride sodium solution, gradient elution, elution program are：0 to 10min, acetonitrile is from 5% linear rise to 15%, 0.4mol/L phosphorus
Acid dihydride sodium solution is from 95% linear decline to 85%, and from 11 to 20min, acetonitrile is from 15% linear rise to 35%, 0.4mol/
L sodium dihydrogen phosphates are from 85% linear decline to 65%；Flow velocity：1.5mL·min-1；Detection wavelength：0 to 10min detection ripple
Long 235nm, 11 to 20min Detection wavelengths 336nm；Column temperature：36℃；Sample size：20μL；
(2) preparation of mixed reference substance solution：L-Epicatechin gallate, Cassia pine -8-O- glucosides are taken respectively
It is accurately weighed with malonyl saikosaponin D reference substance, put in same 100mL brown measuring bottles, with 0.2mol/L sodium dihydrogen phosphates
Solution dissolves and is diluted to scale, shakes up, and it is respectively 300.0mgL that mass concentration, which is made,-1、350.0mg·L-1With
450.0mg·L-1Mixed reference substance solution；
(3) preparation of need testing solution：This product content 0.1g is taken, it is accurately weighed, put in 5mL centrifuge tubes, precision adds
0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, let cool, then
Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifuged, taken supernatant, after filter paper filtering, take
Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, produces need testing solution；
(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, inject high performance liquid chromatograph, measure.
Following experimental study is used for the technology contents and technique effect for verifying technical scheme, but is not intended to limit this
The protection domain of invention.
Experiment one：RP-HPLC dual wavelengths switching method determines L-Epicatechin gallate in dispelling wind detoxicating capsule, determined simultaneously
The content of Ming Song -8-O- glucosides and malonyl saikosaponin D
1 instrument and material
HITACHIL2130 high performance liquid chromatographs, including HI-TACHIL-2400 UV-detectors, D-2000Ellte colors
Work station is composed, is manufactured by Te Saiensi Instrument Ltd.；Kromasil C18Chromatographic column, specification：250mm × 4.6mm, 5 μm,
Manufactured by Agilent Technologies (China) Co., Ltd；SK250H ultrasonic generators, it is public by the big broadcasting and TV supersonic generator in Shanghai
Department's production；HC-2516 supercentrifuges, manufactured by Shanghai Li Xinjian centrifuges Co., Ltd.
L-Epicatechin gallate reference substance (purity Coriolis mass fraction is 98.5%), it is limited to breathe out clever biotechnology by Shanghai
Company produces；Cassia pine -8-O- glucosides reference substance (purity Coriolis mass fraction is 99.0%), by Chengdu De Site biotechnologys
Co., Ltd produces；Malonyl saikosaponin D (purity Coriolis mass fraction is 99.0%), it is limited by Chengdu Rui Fensi biotechnologies
Company produces.Acetonitrile (chromatographically pure), the production of experiment Science and Technology Co., Ltd. is composed by Town in Shanghai；Other reagents (analysis is pure), by
The production of Town in Shanghai spectrum experiment Science and Technology Co., Ltd.；Water is double distilled water, by Anhui Jiren Pharmacy Co., Ltd. laboratory certainly
System.
Dispelling wind detoxicating capsule：Prescription：Giant knotweed 450g, capsule of weeping forsythia 360g, Radix Isatidis 360g, radix bupleuri 360g, field pennycress 360g, horse
Whip grass 360g, reed rhizome 270g, radix glycyrrhizae 180g；
Preparation method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction
Again plus 3 times of 70% ethanol heating and refluxing extraction 1h of amount, filtration, filtrate merge, and reclaim ethanol and are concentrated under reduced pressure at 60 DEG C relative
The thick paste of density 1.35~1.40, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；
The dregs of a decoction and decoction coarse filtration, filtrate centrifuge, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction close after radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract volatile oil
And it is secondary to add water to cook extraction, for the first time plus 18 times of amount water extraction 2h, second plus 12 times of amount water extraction 1h, coarse filtration, filtrate from
The heart separates, and the supernatant after radix bupleuri that the supernatant extracted twice obtains with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and depressurizes dense
The thick paste of relative density 1.35~1.40 at 60 DEG C is reduced to, it is standby；
S4：Dextrin, superfine silica gel powder are taken, is mixed, the water extraction concentration thick paste that above-mentioned S1 steps obtain is added to and is obtained with S3 steps
To alcohol extracting concentration thick paste in, stir, laying, be dried in vacuo, crush, add dextrin, spray into S2 steps and obtain
Volatile oil, sieve, mix, load capsule, be made 1000, produce.
2 methods and result
The preparation of 2.1 solution
2.1.1 the preparation of mixed reference substance solution：L-Epicatechin gallate, Cassia pine -8-O- glucose are taken respectively
Glycosides and malonyl saikosaponin D reference substance are appropriate, accurately weighed, put in same 100mL brown measuring bottles, with 0.2mol/L phosphoric acid
Dihydro sodium solution dissolves and is diluted to scale, shakes up, and it is respectively 300.0mgL that mass concentration, which is made,-1、350.0mg·L-1With
450.0mg·L-1Mixed reference substance solution.
2.1.2 the preparation of need testing solution：Take this product content 0.1g, it is accurately weighed, put in 5mL centrifuge tubes, precision plus
Enter 0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, let cool,
Weighed quality again, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifuged, take supernatant, after filter paper filtering,
Subsequent filtrate is taken, then is filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, produces need testing solution.
2.1.3 the preparation of negative control solution
With the prescription ratio of capsules preparation technique, the feminine gender of scarce giant knotweed and scarce radix bupleuri is prepared respectively by " 2.1.2 " bar method
Contrast solution.
2.2 chromatographic conditions and system suitability
Chromatographic column：Kromasil C18Post, specification：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.4mol/L di(2-ethylhexyl)phosphates
Hydrogen sodium solution, gradient elution, elution program are：0 to 10min, acetonitrile is from 5% linear rise to 15%, 0.4mol/L di(2-ethylhexyl)phosphate
Hydrogen sodium solution is from 95% linear decline to 85%, and from 11 to 20min, acetonitrile is from 15% linear rise to 35%, 0.4mol/L phosphorus
Acid dihydride sodium solution is from 85% linear decline to 65%；Flow velocity：1.5mL·min-1；Detection wavelength：0 to 10min Detection wavelengths
235nm, 11 to 20min Detection wavelengths 336nm；Column temperature：36℃；Sample size：20μL.
Take L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl respectively under above-mentioned chromatographic condition
Mixed reference substance solution, negative control solution and each 20 μ L sample introductions analysis of need testing solution of base saikosaponin D, each tested composition
The separating degree of chromatographic peak and adjacent chromatographic peak be more than 1.5, tailing factor meets the requirements, and theoretical cam curve presses malonyl radix bupleuri
Saponin D, which calculates, is not less than 5000, and chromatogram is shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4.
2.3 Method validation
2.3.1 the investigation of linear relationship
Respectively precision measure mixed reference substance solution 1.0,2.0,4.0,6.0,8.0mL put in 10mL measuring bottles, methanol dilution
To scale, the mixing reference standard serial solution of different quality concentration is made into.Analyzed by " 2.2 " bar chromatographic condition sample introduction, with each
From peak area (Y) be ordinate, with mass concentration (X, mgL-1) it is abscissa, draw standard curve.Gained epicatechin
Gallate, Cassia pine -8-O- glucosides and malonyl saikosaponin D regression equation, the results are shown in Table 1.
The linear relationship of table 1
2.3.2 precision test
Take L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl saikosaponin D mass concentration point
Not Wei 190.0,220.5,290.5mgL-1Same mixed reference substance solution, by " 2.2 " bar chromatographic condition continuous sample introduction 6 times,
Measure the RSD difference of L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl saikosaponin D peak area
For 1.6%, 1.2%, 1.1%.Show that the precision of instrument is good.
2.3.3 replica test
Precision weighs same lot number dispelling wind detoxicating capsule (lot number：160128) content 0.1g, put down by " 2.1.2 " bar method
Row prepare 6 parts of need testing solutions, by " 2.2 " bar chromatographic condition sample introduction analyze, measure L-Epicatechin gallate, Cassia pine-
8-O- glucosides and the RSD of malonyl saikosaponin D content are respectively 1.5%, 1.3%, 1.4%.Show that method repeats
Property is good
2.3.4 stability test
Take same need testing solution, room temperature is placed, respectively at 0,2,4,6,8,10h, by " 2.2 " bar chromatographic condition sample introduction point
Analysis, try to achieve L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl saikosaponin D peak area RSD points
Wei 2.3%, 1.9%, 2.8%.As a result show, need testing solution is stable in 10h at room temperature.
2.3.5 average recovery is tested
Weigh the dispelling wind detoxicating capsule (lot number of known content：160128) 9 parts of content, 3 parts are one group, and every part about
50mg, it is accurately weighed.The accurate contrast solution that adds is appropriate respectively, and basic, normal, high 3 mass concentrations are made by " 2.1.2 " bar method
Need testing solution, by " 2.2 " bar chromatographic condition sample introduction analyze, the results are shown in Table 2.
The rate of recovery of the L-Epicatechin gallate of table 2, Cassia pine -8-O- glucosides and malonyl saikosaponin D
Test (n=9)
The assay of 2.4 test samples
Each lot number dispelling wind detoxicating capsule 10 is weighed respectively, it is accurately weighed.It is molten that test sample is prepared by " 2.1.2 " bar method
Liquid, analyzed by " 2.2 " bar chromatographic condition sample introduction, L-Epicatechin gallate, Cassia pine -8-O- grapes are calculated using external standard method
The content of glucosides and malonyl saikosaponin D, the results are shown in Table 3.
L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl radix bupleuri in the dispelling wind detoxicating capsule of table 3
Content (n=3) (mgg of saponin D-1)
3 conclusions
Using this research method 10 batches of dispelling wind detoxicating capsule have been carried out with the assay of index composition.As a result
Show, every gram of L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl saikosaponin D content exist respectively
In 1.20~1.45mg, 0.90~1.55mg and 2.40~3.60mg.
The content assaying method that this research institute establishes, while 3 index compositions are determined, it is as a result accurately, easy to operate, point
The analysis time is quick, and foundation is provided to improve the determination method of dispelling wind detoxicating capsule.
Experiment two：Therapeutic action of the dispelling wind detoxicating capsule to mouse NASH
This research establishes nonalcoholic fatty liver model with high glucose and high fat forage feed mouse, studies dispelling wind detoxicating capsule pair
The therapeutic action of NASH.
1 instrument and reagent
1.1 reagent
Dispelling wind detoxicating capsule：Prescription：Giant knotweed 450g, capsule of weeping forsythia 360g, Radix Isatidis 360g, radix bupleuri 360g, field pennycress 360g, horse
Whip grass 360g, reed rhizome 270g, radix glycyrrhizae 180g；
Preparation method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction
Again plus 3 times of 70% ethanol heating and refluxing extraction 1h of amount, filtration, filtrate merge, and reclaim ethanol and are concentrated under reduced pressure at 60 DEG C relative
The thick paste of density 1.35~1.40, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；
The dregs of a decoction and decoction coarse filtration, filtrate centrifuge, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction close after radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract volatile oil
And it is secondary to add water to cook extraction, for the first time plus 18 times of amount water extraction 2h, second plus 12 times of amount water extraction 1h, coarse filtration, filtrate from
The heart separates, and the supernatant after radix bupleuri that the supernatant extracted twice obtains with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and depressurizes dense
The thick paste of relative density 1.35~1.40 at 60 DEG C is reduced to, it is standby；
S4：Dextrin, superfine silica gel powder are taken, is mixed, the water extraction concentration thick paste that above-mentioned S1 steps obtain is added to and is obtained with S3 steps
To alcohol extracting concentration thick paste in, stir, laying, be dried in vacuo, crush, add dextrin, spray into S2 steps and obtain
Volatile oil, sieve, mix, load capsule, be made 1000, produce.
Kezhi capsule (trade name：Sweet demutation), produced by Inner Mongolia Furui Medical Science Co., Ltd., approval text
Number：Chinese medicines quasi-word Z20050665, specification：0.25g*30s, lot number：20160125-8.
1.2 reagents and instrument
MDA (MDA) detection kit, manufactured by the Lai Bo Science and Technology Ltd.s of Beijing hundred, lot number：20151223-9；It is super
Superoxide dismutase (SOD) detection kit, manufactured by the Lai Bo Science and Technology Ltd.s of Beijing hundred, lot number：20160120；Automatically
Biochemical Analyzer, manufactured by Beijing Hua Aozhi perseverances Science and Technology Ltd., lot number：20151227；Three light microscopes, by Guilin
Matt optical instrument Co., Ltd of city manufactures.
1.3 animal
Kunming mouse, SPF levels, ♂, weight (20 ± 2) g, provided, moved by Anhui University of Chinese Medecine's Experimental Animal Center
Thing production licence number：SCXK (Anhui) 2012-2013.Standard chow is given, free water, keeps illumination and lucifuge circulation raising
(12/12h)；Experiment keeps indoor feeding 1 week before starting.
2 methods
The foundation of 2.1 NASH mouse models
Mouse is randomly divided into 2 groups, (14) feeding normal diets of Normal group mouse, remaining mouse (34) is fed with
High lipid food (40% normal diet powder, 20% lard, 15% yolk powder, 10% milk powder, 10% sucrose, 2.5% cholesterol,
2.5% sodium taurocholate), freely ingest, drink water.After continuous nursing 5 weeks, from Normal group and Models of Fatty Liver component other places dead 4
Mouse, compare the serum and liver biochemical indexes of 2 groups of mouse, and liver organization pathological section, judge non-alcoholic fatty
Whether liver model mice is successfully established.
2.2 packets and administration
Modeling success mouse 30 is only randomly divided into 3 groups, every group 10, respectively model control group, positive drug group (shell
Fat capsule 100mgkg-1, 1 d-1), dispelling wind detoxicating capsule group (100mgkg-1, 1 d-1).Successive administration 6 weeks, just
Normal control group (10) and model control group give equivalent distilled water.During administration, in addition to Normal group standard mouse material is raised,
Remaining each group continues to give high fat diet, until experiment terminates.For all mouse after last dose, water 12h, mouse are can't help in fasting
Eyeball takes blood, separates serum, and serum paddy T-CHOL (TC), triglyceride (TG) content are detected using automatic clinical chemistry analyzer
With pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST) activity.Mouse is put to death after taking blood, takes out liver, it is a part of with 4% formal
Woods is fixed, and HE dyeing, section, send Anhui University of Chinese Medecine attached First Hospital pathology department, for histopathological examination；Another portion
Point, 10% liver tissue homogenate, measure wherein TG, TC, MDA, SOD content or activity is made.
2.3 data processing
Statistical description and analysis are carried out to data using SPSS12.0 softwares, as a result so that (x ± s is represented, using single factor test
Variance analysis carries out multigroup mean and compared.
3 results
The foundation of 3.1 nonalcoholic fatty liver models
Compared with Normal group, TG, TC content significantly rise in model mice serum and liver that high lipid food is fed
Height (P<0.05 or P<0.01), MDA contents significantly raise (P in serum alt, AST activity and liver<0.01), SOD vigor drops
Low (P<0.05) 1, is the results are shown in Table, the visible hepatic cell fattydegeneration of hepatic tissue pathology sections observation.Show NASH
Establishment of mouse model success.
The formula of high lipid food is that cholesterol, lard, sodium taurocholate etc. are added on the basis of basal feed, and sodium taurocholate can be with
Chyle fat, promote to absorb.This experiment feeds mouse by high lipid food and has been successfully established NASH animal model.
3.2 mice serums and the detection of hepatic tissue biochemical indicator
At the end of experiment, every biochemical indicator of positive drug group mouse has improvement (P<0.05 or P<0.01).With mould
Type control group is compared, dispelling wind detoxicating capsule group show to reduce TG, TC content and ALT in NASH mice serum,
The effect of AST activity, reduces TG, TC, MDA content in NASH mouse liver, the effect (P of increased SOD vigor<
0.05 or P<0.01).And from the comparison of every biochemical indicator, the therapeutic effect and positive drug group of dispelling wind detoxicating capsule group
Quite (P>0.05), for reducing the TC contents in serum, dispelling wind detoxicating capsule group is also advantageous over positive drug group (P<0.05).Knot
Fruit is shown in Table 4.
Table 4 to NASH mouse biochemical indicator influence (n=10,)
Note：Compared with model control group, * P<0.05, #P<0.01
3.3 dispelling wind detoxicating capsules are on the pathological influence of NASH murine liver tissue
After being administered 6 weeks, positive drug group and dispelling wind detoxicating capsule group mouse liver color close to normal liver kermesinus,
And the liver of model control group mouse is milky.Tissue pathological slice is shown in Fig. 5, Fig. 6, Fig. 7, Fig. 8, and experimental result is shown, dredges
The mouse liver cell fat lesion degree of wind detoxicating capsule group mitigates, almost bad without the deposition of fat drips, liver cell in liver cell
Dead degree reduces.
4 conclusions
This experimental result shows that dispelling wind detoxicating capsule group is treated 6 weeks, in NASH mice serum and liver
TG, TC content substantially reduce, and the Serum ALT of NASH mouse, AST are horizontal and the amount of liver MDA substantially reduces,
SOD activity significantly rise, hepatic cell fattydegeneration are repaired, and dispelling wind detoxicating capsule high dose group mouse liver tissue morphology connects
Nearly normal condition.Research shows that dispelling wind detoxicating capsule has therapeutic action for NASH.
Experiment three：Experimental study of the dispelling wind detoxicating capsule to murine chronic alcoholic liver injury model
Dispelling wind detoxicating capsule has therapeutic action for NASH, according to the reasoning from logic of routine, dispelling wind removing toxic substances
Capsule should also have certain therapeutic action, the experimental study that inventor is also soundd out this to alcoholic liver injury.
1 experiment material
1.1 medicines and reagent
Modeling agent：Red Star Erguotou wine (fen-flavor type white spirit, alcoholic strength 56%vol), 200ml/ bottles, Beijing Red Star share have
Limit company, after unpacking 4 DEG C it is stored refrigerated.
By reagent：
Dispelling wind detoxicating capsule：Prescription：Giant knotweed 450g, capsule of weeping forsythia 360g, Radix Isatidis 360g, radix bupleuri 360g, field pennycress 360g, horse
Whip grass 360g, reed rhizome 270g, radix glycyrrhizae 180g；
Preparation method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction
Again plus 3 times of 70% ethanol heating and refluxing extraction 1h of amount, filtration, filtrate merge, and reclaim ethanol and are concentrated under reduced pressure at 60 DEG C relative
The thick paste of density 1.35~1.40, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；
The dregs of a decoction and decoction coarse filtration, filtrate centrifuge, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction close after radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract volatile oil
And it is secondary to add water to cook extraction, for the first time plus 18 times of amount water extraction 2h, second plus 12 times of amount water extraction 1h, coarse filtration, filtrate from
The heart separates, and the supernatant after radix bupleuri that the supernatant extracted twice obtains with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and depressurizes dense
The thick paste of relative density 1.35~1.40 at 60 DEG C is reduced to, it is standby；
S4：Dextrin, superfine silica gel powder are taken, is mixed, the water extraction concentration thick paste that above-mentioned S1 steps obtain is added to and is obtained with S3 steps
To alcohol extracting concentration thick paste in, stir, laying, be dried in vacuo, crush, add dextrin, spray into S2 steps and obtain
Volatile oil, sieve, mix, load capsule, be made 1000, produce.
Positive drug：Bifendate, produced by Hebei east wind pharmaceutcal corporation, Ltd, authentication code：Chinese medicines quasi-word
H13023295, rule：25mg, lot number：20160221.The decoction that concentration is 0.40mg/mL, 4 DEG C of refrigerations are configured to distilled water
Preserve.
Reagent：ALT (ALT) kit, aspartate amino transferase (AST) kit, courage are solid
Alcohol (TC) kit, Shanghai Foxing Changzheng medical science Co., Ltd.
1.2 experimental animal ICR mouse, male, 18~22g.There is provided by Anhui University of Chinese Medecine's Experimental Animal Center, animal
Production licence number：SCXK (Anhui) 2012-2013.
1.3 laboratory apparatus SELECTRA-E full automatic biochemical apparatus, manufactured by Beijing Hua Aozhi perseverances Science and Technology Ltd.；Low speed
Large Copacity Multi-pipe centrifugal machine, manufactured by Shanghai Jia Peng Science and Technology Ltd.s；Electronic balance, had by the flat instrument and equipment in Changzhou day
Limit company manufactures；J-50 type fluidized bed air flow crushers, produced by Shanghai Sai Shan powder machineries Manufacturing Co., Ltd；
The dry-wet integrated laser particle instrument of the intelligent gamuts of Winner2308, is manufactured by Jinan Winner Particle Instrument Co., Ltd.；
BP211D type electronic analytical balances, manufactured by the flat experimental instruments and equipment limited in Changzhou day.
2. experimental method
2.1 animal packet
Male ICR mouse, it is divided into dispelling wind detoxicating capsule, Normal group, model control group, positive control (DDB
Dripping pill) group, every group 10, totally 10 groups.
2.2 administrations and modeling
Gastric infusion 1 time daily, gavage volume is 0.1mL/10g body weight.Normal group and model control group are given
Water；Dispelling wind detoxicating capsule gavage gives 0.5g/mL dispelling wind detoxicating capsule.2h after administration every time, in addition to Normal group, remaining
Group gives 56 ° of white wine by 10mL/kg body weight gavages, causes chronic alcohol liver injury model within continuous 60 days.
Water is can't help in fasting after 2.3 index determining last gavages give alcohol, and eyeball is plucked after 12h and takes blood, 4000r/min centrifugations
15min, take serum is standby to survey ALT, AST, TC.
2.4 statistical procedures：Experimental data represents that group difference is examined using t with x ± s.
3. result
Influence to alcoholic liver injury mice serum ALT, AST, TC
The result of table 5 is shown, compared with Normal group, the significantly raised (P of model control group mice serum ALT, AST, TC<
0.05, P<0.01).Compared with model control group, dispelling wind detoxicating capsule can not reduce Serum ALT (P ＞ 0.05), serum AST (P
＞ 0.05) and serum TC (P ＞ 0.05).Illustrate dispelling wind detoxicating capsule to chronic alcohol liver injury mice serum ALT, AST, TC
Do not improve significantly, to the improvement result unobvious of alcoholic liver injury mouse.
Influence of the table 5 to alcoholic liver injury mice serum ALT, ASTN=10
Compared with normal group:△P<0.05,△△P<0.01；Compared with model group:*P<0.05
Brief description of the drawings：
Fig. 1 is the high-efficient liquid phase chromatogram of mixed reference substance solution
Fig. 2 is the high-efficient liquid phase chromatogram for lacking radix bupleuri negative control solution
Fig. 3 is the high-efficient liquid phase chromatogram for lacking giant knotweed negative control solution
Fig. 4 is the high-efficient liquid phase chromatogram of dispelling wind detoxicating capsule sample
Fig. 5 is Normal group mouse liver tissue pathological slice figure (200 ×)
Fig. 6 is model control group mouse liver tissue pathological slice figure (200 ×)
Fig. 7 is positive controls mouse liver tissue pathological slice figure (200 ×)
Fig. 8 is dispelling wind detoxicating capsule group mouse liver tissue pathological slice figure (200 ×)
Embodiment
The present invention is made with reference to specific embodiment further to explain.
Embodiment 1：
Dispelling wind detoxicating capsule：
Prescription：Giant knotweed 450g, capsule of weeping forsythia 360g, Radix Isatidis 360g, radix bupleuri 360g, field pennycress 360g, Verbena officinalis 360g, reed rhizome
270g, radix glycyrrhizae 180g；
Preparation method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction
Again plus 3 times of 70% ethanol heating and refluxing extraction 1h of amount, filtration, filtrate merge, and reclaim ethanol and are concentrated under reduced pressure at 60 DEG C relative
The thick paste of density 1.35~1.40, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；
The dregs of a decoction and decoction coarse filtration, filtrate centrifuge, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction close after radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract volatile oil
And it is secondary to add water to cook extraction, for the first time plus 18 times of amount water extraction 2h, second plus 12 times of amount water extraction 1h, coarse filtration, filtrate from
The heart separates, and the supernatant after radix bupleuri that the supernatant extracted twice obtains with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and depressurizes dense
The thick paste of relative density 1.35~1.40 at 60 DEG C is reduced to, it is standby；
S4：Dextrin, superfine silica gel powder are taken, is mixed, the water extraction concentration thick paste that above-mentioned S1 steps obtain is added to and is obtained with S3 steps
To alcohol extracting concentration thick paste in, stir, laying, be dried in vacuo, crush, add dextrin, spray into S2 steps and obtain
Volatile oil, sieve, mix, load capsule, be made 1000, produce.
L-Epicatechin gallate, Cassia in dispelling wind detoxicating capsule are determined using RP-HPLC dual wavelengths switching method simultaneously
The content of pine -8-O- glucosides and malonyl saikosaponin D is as follows the step of its detection method：
(1) chromatographic condition：Chromatographic column：C18Post, specification：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.4mol/L phosphorus
Acid dihydride sodium solution, gradient elution, elution program are：0 to 10min, acetonitrile is from 5% linear rise to 15%, 0.4mol/L phosphorus
Acid dihydride sodium solution is from 95% linear decline to 85%, and from 11 to 20min, acetonitrile is from 15% linear rise to 35%, 0.4mol/
L sodium dihydrogen phosphates are from 85% linear decline to 65%；Flow velocity：1.5mL·min-1；Detection wavelength：0 to 10min detection ripple
Long 235nm, 11 to 20min Detection wavelengths 336nm；Column temperature：36℃；Sample size：20μL；
(2) preparation of mixed reference substance solution：L-Epicatechin gallate, Cassia pine -8-O- glucosides are taken respectively
It is accurately weighed with malonyl saikosaponin D reference substance, put in same 100mL brown measuring bottles, with 0.2mol/L sodium dihydrogen phosphates
Solution dissolves and is diluted to scale, shakes up, and it is respectively 300.0mgL that mass concentration, which is made,-1、350.0mg·L-1With
450.0mg·L-1Mixed reference substance solution；
(3) preparation of need testing solution：This product content 0.1g is taken, it is accurately weighed, put in 5mL centrifuge tubes, precision adds
0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, let cool, then
Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifuged, taken supernatant, after filter paper filtering, take
Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, produces need testing solution；
(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, inject high performance liquid chromatograph, measure；
(5) measurement result：Every gram of this product contains L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl
Base saikosaponin D is respectively 1.35mg, 1.26mg, 2.88mg.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
Claims (3)
1. a kind of detection method of dispelling wind detoxicating capsule, the dispelling wind detoxicating capsule are made up of the flavour of a drug raw material of following parts by weight：
The parts by weight of giant knotweed 5, the parts by weight of the capsule of weeping forsythia 4, the parts by weight of Radix Isatidis 4, the parts by weight of radix bupleuri 4, the parts by weight of field pennycress 4, the weight of Verbena officinalis 4
Part, the parts by weight of reed rhizome 3, the parts by weight of radix glycyrrhizae 2, the dispelling wind detoxicating capsule are prepared with the following method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction add 3 again
70% ethanol heating and refluxing extraction 1h of amount again, filtration, filtrate merge, and reclaim ethanol and are simultaneously concentrated under reduced pressure into relative density at 60 DEG C
1.35~1.40 thick paste, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；The dregs of a decoction
With decoction coarse filtration, filtrate centrifuges, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction merge after taking radix bupleuri, the capsule of weeping forsythia extraction volatile oil that field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae obtain with S2 steps,
It is secondary to add water to cook extraction, for the first time plus 18 times of amount water extract 2h, and second plus 12 times of amount water extraction 1h, coarse filtration, filtrate centrifuge
Separation, radix bupleuri that the supernatant extracted twice obtains with S2 steps, the capsule of weeping forsythia extract the supernatant after volatile oil and merged, be concentrated under reduced pressure
The thick paste of relative density 1.35~1.40, standby to 60 DEG C；
S4：Dextrin, superfine silica gel powder are taken, is mixed, is added to what the water extraction concentration thick paste that above-mentioned S1 steps obtain obtained with S3 steps
In alcohol extracting concentration thick paste, stir, laying, be dried in vacuo, crush, add dextrin, spray into the volatilization that S2 steps obtain
Oil, sieve, mix, load capsule, produce；
Characterized in that, switch method using RP-HPLC dual wavelengths determines epicatechin gallate in dispelling wind detoxicating capsule simultaneously
The content of ester, Cassia pine -8-O- glucosides and malonyl saikosaponin D is as follows the step of its detection method：
（1）Chromatographic condition：Chromatographic column：C18Post, specification：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.4mol/L di(2-ethylhexyl)phosphates
Hydrogen sodium solution, gradient elution, elution program are：0 to 10min, acetonitrile is from 5% linear rise to 15%, 0.4mol/L biphosphate
Sodium solution is from 95% linear decline to 85%, and from 11 to 20min, acetonitrile is from 15% linear rise to 35%, 0.4mol/L biphosphate
Sodium solution is from 85% linear decline to 65%；Flow velocity：0.5~2.0mLmin-1；Detection wavelength：0 to 10min Detection wavelengths 230~
240nm, 11 to 330~340nm of 20min Detection wavelengths；Column temperature：30~40 DEG C；Sample size：5~20 μ L；
（2）The preparation of mixed reference substance solution：L-Epicatechin gallate, Cassia pine -8-O- glucosides and third are taken respectively
Diacyl saikosaponin D reference substance, it is accurately weighed, put in same measuring bottle, dissolved with 0.2mol/L sodium dihydrogen phosphates and dilute
Release to scale, shake up, mixed reference substance solution is made；
（3）The preparation of need testing solution：This product content is taken, it is accurately weighed, put in centrifuge tube, precision adds 0.2mol/L phosphoric acid
Dihydro sodium solution, weighed quality, ultrasonic extraction, lets cool, then weighed quality, is supplied and is subtracted with 0.2mol/L sodium dihydrogen phosphates
The quality of mistake, centrifugation, supernatant is taken, after filter paper filtering, take subsequent filtrate, then filtered with 0.45 μm of miillpore filter, take subsequent filtrate, i.e.,
Obtain need testing solution；
（4）Measure：Take mixed reference substance solution, need testing solution injection high performance liquid chromatograph, measure.
2. the detection method of dispelling wind detoxicating capsule as claimed in claim 1, it is characterised in that cut using RP-HPLC dual wavelengths
Change method while determine L-Epicatechin gallate in dispelling wind detoxicating capsule, Cassia pine -8-O- glucosides and malonyl bavin
The content of Hu saponin D is as follows the step of its detection method：
（1）Chromatographic condition：Chromatographic column：C18Post, specification：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.4mol/L di(2-ethylhexyl)phosphates
Hydrogen sodium solution, gradient elution, elution program are：0 to 10min, acetonitrile is from 5% linear rise to 15%, 0.4mol/L biphosphate
Sodium solution is from 95% linear decline to 85%, and from 11 to 20min, acetonitrile is from 15% linear rise to 35%, 0.4mol/L biphosphate
Sodium solution is from 85% linear decline to 65%；Flow velocity：1.5mL·min-1；Detection wavelength：0 to 10min Detection wavelengths 235nm, 11 to
20min Detection wavelengths 336nm；Column temperature：36℃；Sample size：20μL；
（2）The preparation of mixed reference substance solution：L-Epicatechin gallate, Cassia pine -8-O- glucosides and third are taken respectively
Diacyl saikosaponin D reference substance, it is accurately weighed, put in same 100mL brown measuring bottles, with 0.2mol/L sodium dihydrogen phosphates
Dissolve and be diluted to scale, shake up, it is respectively 300.0mgL that mass concentration, which is made,-1、350.0mg·L-1And 450.0mgL-1
Mixed reference substance solution；
（3）The preparation of need testing solution：This product content 0.1g is taken, it is accurately weighed, put in 5mL centrifuge tubes, precision adds
0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, let cool, then
Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifuged, taken supernatant, after filter paper filtering, take
Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, produces need testing solution；
（4）Measure：Each 20 μ L of mixed reference substance solution, need testing solution are taken, inject high performance liquid chromatograph, measure.
3. a kind of dispelling wind detoxicating capsule is preparing the application in treating NASH medicine, it is characterised in that the dispelling wind
Detoxicating capsule is made up of the flavour of a drug raw material of following parts by weight：The parts by weight of giant knotweed 5, the parts by weight of the capsule of weeping forsythia 4, the parts by weight of Radix Isatidis 4, bavin
4 parts by weight, the parts by weight of field pennycress 4, the parts by weight of Verbena officinalis 4, the parts by weight of reed rhizome 3, the parts by weight of radix glycyrrhizae 2 recklessly, the dispelling wind detoxicating capsule
Prepare with the following method：
S1：Take giant knotweed, Radix Isatidis to be ground into coarse granule, add 5 times of 70% ethanol heating and refluxing extraction 2h of amount, filtration；The dregs of a decoction add 3 again
70% ethanol heating and refluxing extraction 1h of amount again, filtration, filtrate merge, and reclaim ethanol and are simultaneously concentrated under reduced pressure into relative density at 60 DEG C
1.35~1.40 thick paste, it is standby；
S2：Take the capsule of weeping forsythia, radix bupleuri to add the water of 7 times of amounts, heating and refluxing extraction volatile oil 4h, divide the volatile oil for taking layering, it is standby；The dregs of a decoction
With decoction coarse filtration, filtrate centrifuges, and supernatant and the dregs of a decoction save backup respectively；
S3：The dregs of a decoction merge after taking radix bupleuri, the capsule of weeping forsythia extraction volatile oil that field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae obtain with S2 steps,
It is secondary to add water to cook extraction, for the first time plus 18 times of amount water extract 2h, and second plus 12 times of amount water extraction 1h, coarse filtration, filtrate centrifuge
Separation, radix bupleuri that the supernatant extracted twice obtains with S2 steps, the capsule of weeping forsythia extract the supernatant after volatile oil and merged, be concentrated under reduced pressure
The thick paste of relative density 1.35~1.40, standby to 60 DEG C；
S4：Dextrin, superfine silica gel powder are taken, is mixed, is added to what the water extraction concentration thick paste that above-mentioned S1 steps obtain obtained with S3 steps
In alcohol extracting concentration thick paste, stir, laying, be dried in vacuo, crush, add dextrin, spray into the volatilization that S2 steps obtain
Oil, sieve, mix, load capsule, produce；
L-Epicatechin gallate in dispelling wind detoxicating capsule, Cassia pine -8- are determined using RP-HPLC dual wavelengths switching method simultaneously
The content of O- glucosides and malonyl saikosaponin D is as follows the step of its detection method：
（1）Chromatographic condition：Chromatographic column：C18Post, specification：250mm × 4.6mm, 5 μm；Mobile phase：Acetonitrile -0.4mol/L di(2-ethylhexyl)phosphates
Hydrogen sodium solution, gradient elution, elution program are：0 to 10min, acetonitrile is from 5% linear rise to 15%, 0.4mol/L biphosphate
Sodium solution is from 95% linear decline to 85%, and from 11 to 20min, acetonitrile is from 15% linear rise to 35%, 0.4mol/L biphosphate
Sodium solution is from 85% linear decline to 65%；Flow velocity：1.5mL·min-1；Detection wavelength：0 to 10min Detection wavelengths 235nm, 11 to
20min Detection wavelengths 336nm；Column temperature：36℃；Sample size：20μL；
（2）The preparation of mixed reference substance solution：L-Epicatechin gallate, Cassia pine -8-O- glucosides and third are taken respectively
Diacyl saikosaponin D reference substance, it is accurately weighed, put in same 100mL brown measuring bottles, with 0.2mol/L sodium dihydrogen phosphates
Dissolve and be diluted to scale, shake up, it is respectively 300.0mgL that mass concentration, which is made,-1、350.0mg·L-1And 450.0mgL-1
Mixed reference substance solution；
（3）The preparation of need testing solution：This product content 0.1g is taken, it is accurately weighed, put in 5mL centrifuge tubes, precision adds
0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, let cool, then
Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifuged, taken supernatant, after filter paper filtering, take
Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, produces need testing solution；
（4）Measure：Each 20 μ L of mixed reference substance solution, need testing solution are taken, inject high performance liquid chromatograph, measure.
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