CN107233511A - A kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and purposes - Google Patents

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 the method that method determines dispelling wind detoxicating capsule active component content is switched using RP HPLC dual wavelengths simultaneously, while there is provided the new pharmaceutical use of dispelling wind detoxicating capsule.

Description

A kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and purposes

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 of 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 are constituted.With wind and heat dispersing, the work(for relieving sore-throat of detoxifying belongs to wind-heat syndrome, symptoms include heating for treating acute upper respiratory infection, Foul wind, pharyngalgia is had a headache, nasal obstruction, flows turbid tears, cough etc., clinical practice for many years, determined curative effect.This product is listed in the Ministry of Public Health《A type H1N1 influenza diagnosis and treatment schemes》(second edition, the third edition, version in 2010 in 2009) 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 the national medical insurance catalogue of version in 2009.

This product is the key product of applicant Anhui Ji people's medicine company, and annual value of production, annual sales amount are crossed in hundred million The big kind of medicine.Obtain within 2011 National modern Chinese medicine development for hi-tech industry special project to support, successively win that " state key is newly produced 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), has preferable curative effect 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 of this product development.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 this product is 8 kinds of Chinese medicine flavour of a drug raw materials composition, 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, therefore, and 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 is 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 is respectively：2014105823185th, 201410583418X, patent publication No. is respectively：104316613B、 104330489B, two pieces patent proposes 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, Propose and establish simultaneously 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 Very prominent, unexpected therapeutic effect.

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 background technology exist technical problem, 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, 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 is the flavour of a drug raw material system 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, adopts and prepares with the following method：

S1：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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 merges, reclaim ethanol be simultaneously concentrated under reduced pressure at 60 DEG C it is relative The thick paste of density 1.35~1.40, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby； The dregs of a decoction and decoction coarse filtration, filtrate are centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract the dregs of a decoction after volatile oil and closed And, 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 extract 1h, coarse filtration, filtrate from The heart is separated, and the supernatant that radix bupleuri that the supernatant extracted twice is obtained with S2 steps, the capsule of weeping forsythia extract after volatile oil merges, and decompression is 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, sieves, and mixes, and loads capsule, produces.

A kind of detection method of dispelling wind detoxicating capsule, the dispelling wind detoxicating capsule is the flavour of a drug raw material system 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 adopted to be prepared with the following method：

S1：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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 merges, reclaim ethanol be simultaneously concentrated under reduced pressure at 60 DEG C it is relative The thick paste of density 1.35~1.40, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby； The dregs of a decoction and decoction coarse filtration, filtrate are centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract the dregs of a decoction after volatile oil and closed And, 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 extract 1h, coarse filtration, filtrate from The heart is separated, and the supernatant that radix bupleuri that the supernatant extracted twice is obtained with S2 steps, the capsule of weeping forsythia extract after volatile oil merges, and decompression is 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, sieves, and mixes, and loads capsule, produces；

L-Epicatechin gallate, Cassia in dispelling wind detoxicating capsule are determined using RP-HPLC dual wavelengths switching method simultaneously The step of content of pine -8-O- glucosides and malonyl saikosaponin D, its detection method, is as follows：

(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 is：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%, from 11 to 20min, and 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 With malonyl saikosaponin D reference substance, it is accurately weighed, put in same measuring bottle, with 0.2mol/L sodium dihydrogen phosphates dissolve 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 is let cool, then weighed quality, is supplied with 0.2mol/L sodium dihydrogen phosphates The quality of institute's less loss, centrifugation takes supernatant, after filter paper filtering, takes subsequent filtrate, then is filtered with 0.45 μm of miillpore filter, takes continuous filter Liquid, produces need testing solution；

(4) determine：Mixed reference substance solution, need testing solution injection high performance liquid chromatograph are taken, is determined.

Above-mentioned detection method, switches method using RP-HPLC dual wavelengths and does not have while determining epicatechin in dispelling wind detoxicating capsule 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 is：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%, from 11 to 20min, and 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 With malonyl saikosaponin D reference substance, it is accurately weighed, put in same 100mL brown measuring bottles, use 0.2mol/L sodium dihydrogen phosphates Solution dissolves and is diluted to scale, shakes up, and mass concentration respectively 300.0mgL 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 is added 0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, is let cool, then Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifugation, is taken supernatant, after filter paper filtering, is taken Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, need testing solution is produced；

(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, high performance liquid chromatograph is injected, determined.

A kind of application of dispelling wind detoxicating capsule in treatment NASH medicine is prepared, the dispelling wind detoxicating capsule is 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：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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 merges, reclaim ethanol be simultaneously concentrated under reduced pressure at 60 DEG C it is relative The thick paste of density 1.35~1.40, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby； The dregs of a decoction and decoction coarse filtration, filtrate are centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract the dregs of a decoction after volatile oil and closed And, 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 extract 1h, coarse filtration, filtrate from The heart is separated, and the supernatant that radix bupleuri that the supernatant extracted twice is obtained with S2 steps, the capsule of weeping forsythia extract after volatile oil merges, and decompression is 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, sieves, and mixes, and loads capsule, produces；

L-Epicatechin gallate, Cassia in dispelling wind detoxicating capsule are determined using RP-HPLC dual wavelengths switching method simultaneously The step of content of pine -8-O- glucosides and malonyl saikosaponin D, its detection method, is as follows：

(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 is：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%, from 11 to 20min, and 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 With malonyl saikosaponin D reference substance, it is accurately weighed, put in same 100mL brown measuring bottles, use 0.2mol/L sodium dihydrogen phosphates Solution dissolves and is diluted to scale, shakes up, and mass concentration respectively 300.0mgL 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 is added 0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, is let cool, then Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifugation, is taken supernatant, after filter paper filtering, is taken Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, need testing solution is produced；

(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, high performance liquid chromatograph is injected, determined.

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, by Shanghai, big broadcasting and TV supersonic generator is public Department's production；HC-2516 supercentrifuges, are manufactured by Shanghai Li Xinjian centrifuges Co., Ltd.

L-Epicatechin gallate reference substance (purity Coriolis mass fraction is 98.5%), breathes out clever biotechnology limited 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%), by Chengdu, Rui Fensi biotechnologies are limited Company produces.Acetonitrile (chromatographically pure), by the production of Town in Shanghai spectrum experiment Science and Technology Co., Ltd.；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：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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 merges, reclaim ethanol be simultaneously concentrated under reduced pressure at 60 DEG C it is relative The thick paste of density 1.35~1.40, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby； The dregs of a decoction and decoction coarse filtration, filtrate are centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract the dregs of a decoction after volatile oil and closed And, 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 extract 1h, coarse filtration, filtrate from The heart is separated, and the supernatant that radix bupleuri that the supernatant extracted twice is obtained with S2 steps, the capsule of weeping forsythia extract after volatile oil merges, and decompression is 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, sieves, and mixes, and loads capsule, is made 1000, produces.

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, use 0.2mol/L phosphoric acid Dihydro sodium solution dissolves and is diluted to scale, shakes up, and mass concentration respectively 300.0mgL 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, is let cool, Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates again, centrifugation, takes 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 is：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%, from 11 to 20min, and 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 is calculated, 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 is flat 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 is repeated 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, is analyzed by " 2.2 " bar chromatographic condition sample introduction, and 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, L-Epicatechin gallate, every gram of content of Cassia pine -8-O- glucosides and malonyl saikosaponin D exist respectively In 1.20~1.45mg, 0.90~1.55mg and 2.40~3.60mg.

The content assaying method that this research institute sets up, 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 for the determination method that improves dispelling wind detoxicating capsule.

Experiment two：Therapeutic action of the dispelling wind detoxicating capsule to mouse NASH

Nonalcoholic fatty liver model is set up in this research 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：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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 merges, reclaim ethanol be simultaneously concentrated under reduced pressure at 60 DEG C it is relative The thick paste of density 1.35~1.40, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby； The dregs of a decoction and decoction coarse filtration, filtrate are centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract the dregs of a decoction after volatile oil and closed And, 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 extract 1h, coarse filtration, filtrate from The heart is separated, and the supernatant that radix bupleuri that the supernatant extracted twice is obtained with S2 steps, the capsule of weeping forsythia extract after volatile oil merges, and decompression is 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, sieves, and mixes, and loads capsule, is made 1000, produces.

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, is manufactured, lot number by the Lai Bo Science and Technology Ltd.s of Beijing hundred：20151223-9；It is super Superoxide dismutase (SOD) detection kit, is manufactured, lot number by the Lai Bo Science and Technology Ltd.s of Beijing hundred：20160120；Automatically Biochemical Analyzer, is manufactured, lot number by the permanent Science and Technology Ltd.s of Beijing Hua Aozhi：20151227；Three light microscopes, by Guilin Matt optical instrument Co., Ltd of city manufactures.

1.3 animal

Kunming mouse, SPF grades, ♂, weight (20 ± 2) g, by Anhui University of Chinese Medecine, Experimental Animal Center is provided, and is moved Thing production licence number：SCXK (Anhui) 2012-2013.Standard chow is given, free water keeps illumination and lucifuge circulation to raise (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, (34) feedings of remaining mouse 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, compares the serum and liver biochemical indexes of 2 groups of mouse, and liver organization pathological section, judges 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, respectively every group 10, 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.All mouse are after last dose, and 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.Take and mouse is put to death after blood, take out liver, a part uses 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 is made, wherein TG, TC, MDA, SOD content or activity is determined.

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, the visible hepatic cell fattydegeneration of hepatic tissue pathology sections observation, be the results are shown in Table.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, promotes 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 TG, TC content and ALT in reduction NASH mice serum, TG, TC, MDA content, the effect (P of increased SOD vigor in the effect of AST activity, reduction NASH mouse liver< 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 the TC contents in reduction 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

Be administered after 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, and the almost deposition without fat drips in liver cell, liver cell is bad Dead degree reduction.

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 are substantially reduced, and the amount of the Serum ALT of NASH mouse, AST levels and liver MDA is substantially reduced, SOD activity significantly rise, hepatic cell fattydegeneration is 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 conventional reasoning from logic, 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 has 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：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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 merges, reclaim ethanol be simultaneously concentrated under reduced pressure at 60 DEG C it is relative The thick paste of density 1.35~1.40, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby； The dregs of a decoction and decoction coarse filtration, filtrate are centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract the dregs of a decoction after volatile oil and closed And, 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 extract 1h, coarse filtration, filtrate from The heart is separated, and the supernatant that radix bupleuri that the supernatant extracted twice is obtained with S2 steps, the capsule of weeping forsythia extract after volatile oil merges, and decompression is 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, sieves, and mixes, and loads capsule, is made 1000, produces.

Positive drug：Bifendate, is produced, authentication code by Hebei east wind pharmaceutcal corporation, Ltd：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 is solid Alcohol (TC) kit, Shanghai Foxing Changzheng medical science Co., Ltd.

1.2 experimental animal ICR mouse, male, 18~22g.By Anhui University of Chinese Medecine, Experimental Animal Center is provided, animal Production licence number：SCXK (Anhui) 2012-2013.

1.3 laboratory apparatus SELECTRA-E full automatic biochemical apparatus, are manufactured by the permanent Science and Technology Ltd.s of Beijing Hua Aozhi；Low speed Large Copacity Multi-pipe centrifugal machine, is manufactured by Shanghai Jia Peng Science and Technology Ltd.s；Electronic balance, is had by the flat instrument and equipment in Changzhou day Limit company manufactures；J-50 type fluidized bed air flow crushers, are 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, are manufactured by the flat experimental instruments and equipment limited in Changzhou day.

2. experimental method

2.1 animal packet

Male ICR mouse, 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

Daily gastric infusion 1 time, 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 being administered 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.

2.3 index determining last gavages, which give fasting after alcohol and can't help plucking eyeball after water, 12h, takes blood, 4000r/min centrifugations 15min, takes 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, is 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, it is unobvious to the improvement result 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：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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 merges, reclaim ethanol be simultaneously concentrated under reduced pressure at 60 DEG C it is relative The thick paste of density 1.35~1.40, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby； The dregs of a decoction and decoction coarse filtration, filtrate are centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae and S2 steps to obtain extract the dregs of a decoction after volatile oil and closed And, 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 extract 1h, coarse filtration, filtrate from The heart is separated, and the supernatant that radix bupleuri that the supernatant extracted twice is obtained with S2 steps, the capsule of weeping forsythia extract after volatile oil merges, and decompression is 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, sieves, and mixes, and loads capsule, is made 1000, produces.

L-Epicatechin gallate, Cassia in dispelling wind detoxicating capsule are determined using RP-HPLC dual wavelengths switching method simultaneously The step of content of pine -8-O- glucosides and malonyl saikosaponin D, its detection method, is as follows：

(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 is：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%, from 11 to 20min, and 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 With malonyl saikosaponin D reference substance, it is accurately weighed, put in same 100mL brown measuring bottles, use 0.2mol/L sodium dihydrogen phosphates Solution dissolves and is diluted to scale, shakes up, and mass concentration respectively 300.0mgL 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 is added 0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, is let cool, then Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifugation, is taken supernatant, after filter paper filtering, is taken Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, need testing solution is produced；

(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, high performance liquid chromatograph is injected, determined；

(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 (6)

1. a kind of dispelling wind detoxicating capsule, 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, 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.

2. a kind of preparation method of dispelling wind detoxicating capsule, 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 weight of Verbena officinalis 4 Part, the parts by weight of reed rhizome 3, the parts by weight of radix glycyrrhizae 2, it is characterised in that the dispelling wind detoxicating capsule is adopted to be prepared with the following method：

S1：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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, filtration, filtrate merging, reclaim ethanol and are simultaneously concentrated under reduced pressure into relative density at 60 DEG C again 1.35~1.40 thick paste, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby；The dregs of a decoction With decoction coarse filtration, filtrate is centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae to be obtained with S2 steps extract the dregs of a decoction after volatile oil and merged, Add water to cook and extract secondary, first time plus 18 times of amount water extract 2h, add 12 times of amount water to extract 1h, coarse filtration, filtrate centrifugation for the second time Supernatant after separation, radix bupleuri that the supernatant extracted twice is obtained with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and is 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 was 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 are obtained Oil, sieves, and mixes, and loads capsule, produces.

3. a kind of detection method of dispelling wind detoxicating capsule, 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 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 is adopted to be prepared with the following method：

S1：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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, filtration, filtrate merging, reclaim ethanol and are simultaneously concentrated under reduced pressure into relative density at 60 DEG C again 1.35~1.40 thick paste, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby；The dregs of a decoction With decoction coarse filtration, filtrate is centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae to be obtained with S2 steps extract the dregs of a decoction after volatile oil and merged, Add water to cook and extract secondary, first time plus 18 times of amount water extract 2h, add 12 times of amount water to extract 1h, coarse filtration, filtrate centrifugation for the second time Supernatant after separation, radix bupleuri that the supernatant extracted twice is obtained with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and is 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 was 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 are obtained Oil, sieves, and mixes, and loads capsule, produces；

Characterized in that, switching method using RP-HPLC dual wavelengths determines epicatechin gallate in dispelling wind detoxicating capsule simultaneously The step of content of ester, Cassia pine -8-O- glucosides and malonyl saikosaponin D, its detection method, is as follows：

(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 is：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%, from 11 to 20min, and 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：0.5~2.0mLmin^-1；Detection wavelength：0 to 10min detection 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 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) 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 is let cool, then weighed quality, is supplied and is subtracted with 0.2mol/L sodium dihydrogen phosphates The quality of mistake, centrifugation takes supernatant, after filter paper filtering, takes subsequent filtrate, then is filtered with 0.45 μm of miillpore filter, takes subsequent filtrate, i.e., Obtain need testing solution；

(4) determine：Mixed reference substance solution, need testing solution injection high performance liquid chromatograph are taken, is determined.

4. the detection method of dispelling wind detoxicating capsule as claimed in claim 3, it is characterised in that cut using RP-HPLC dual wavelengths Method is changed while determining L-Epicatechin gallate, Cassia pine -8-O- glucosides and malonyl bavin in dispelling wind detoxicating capsule The step of content of Hu saponin D, its detection method, is as follows：

(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 is：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%, from 11 to 20min, and 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；

(2) 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, use 0.2mol/L sodium dihydrogen phosphates Dissolve and be diluted to scale, shake up, mass concentration respectively 300.0mgL is made^-1、350.0mg·L^-1And 450.0mgL^-1 Mixed 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 is added 0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, is let cool, then Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifugation, is taken supernatant, after filter paper filtering, is taken Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, need testing solution is produced；

(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, high performance liquid chromatograph is injected, determined.

5. a kind of application of dispelling wind detoxicating capsule in treatment NASH medicine is prepared, 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, the dispelling wind detoxicating capsule recklessly Adopt and prepare with the following method：

S1：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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, filtration, filtrate merging, reclaim ethanol and are simultaneously concentrated under reduced pressure into relative density at 60 DEG C again 1.35~1.40 thick paste, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby；The dregs of a decoction With decoction coarse filtration, filtrate is centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae to be obtained with S2 steps extract the dregs of a decoction after volatile oil and merged, Add water to cook and extract secondary, first time plus 18 times of amount water extract 2h, add 12 times of amount water to extract 1h, coarse filtration, filtrate centrifugation for the second time Supernatant after separation, radix bupleuri that the supernatant extracted twice is obtained with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and is 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 was 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 are obtained Oil, sieves, and mixes, and loads capsule, produces；

L-Epicatechin gallate, Cassia pine -8- in dispelling wind detoxicating capsule are determined using RP-HPLC dual wavelengths switching method simultaneously The step of content of O- glucosides and malonyl saikosaponin D, its detection method, is as follows：

(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 is：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%, from 11 to 20min, and 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；

(2) 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, use 0.2mol/L sodium dihydrogen phosphates Dissolve and be diluted to scale, shake up, mass concentration respectively 300.0mgL is made^-1、350.0mg·L^-1And 450.0mgL^-1 Mixed 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 is added 0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, is let cool, then Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifugation, is taken supernatant, after filter paper filtering, is taken Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, need testing solution is produced；

(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, high performance liquid chromatograph is injected, determined.

6. a kind of application of dispelling wind detoxicating capsule in treatment alcoholic liver injury medicine is prepared, it is characterised in that the dispelling wind solution Toxic 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, radix bupleuri 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, the dispelling wind detoxicating capsule is Adopt what is prepared with the following method：

S1：Giant knotweed, Radix Isatidis is taken to be ground into coarse granule, plus 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, filtration, filtrate merging, reclaim ethanol and are simultaneously concentrated under reduced pressure into relative density at 60 DEG C again 1.35~1.40 thick paste, it is standby；

S2：The water of the capsule of weeping forsythia, radix bupleuri plus 7 times of amounts is taken, heating and refluxing extraction volatile oil 4h divides the volatile oil for taking layering, standby；The dregs of a decoction With decoction coarse filtration, filtrate is centrifuged, and supernatant and the dregs of a decoction are saved backup respectively；

S3：Radix bupleuri, the capsule of weeping forsythia for taking field pennycress, Verbena officinalis, reed rhizome, radix glycyrrhizae to be obtained with S2 steps extract the dregs of a decoction after volatile oil and merged, Add water to cook and extract secondary, first time plus 18 times of amount water extract 2h, add 12 times of amount water to extract 1h, coarse filtration, filtrate centrifugation for the second time Supernatant after separation, radix bupleuri that the supernatant extracted twice is obtained with S2 steps, capsule of weeping forsythia extraction volatile oil merges, and is 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 was 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 are obtained Oil, sieves, and mixes, and loads capsule, produces；

L-Epicatechin gallate, Cassia pine -8- in dispelling wind detoxicating capsule are determined using RP-HPLC dual wavelengths switching method simultaneously The step of content of O- glucosides and malonyl saikosaponin D, its detection method, is as follows：

(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 is：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%, from 11 to 20min, and 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；

(2) 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, use 0.2mol/L sodium dihydrogen phosphates Dissolve and be diluted to scale, shake up, mass concentration respectively 300.0mgL is made^-1、350.0mg·L^-1And 450.0mgL^-1 Mixed 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 is added 0.2mol/L sodium dihydrogen phosphates 4mL, weighed quality, with power 300W, frequency 60kHz ultrasonic extraction 40min, is let cool, then Weighed quality, the quality of institute's less loss is supplied with 0.2mol/L sodium dihydrogen phosphates, centrifugation, is taken supernatant, after filter paper filtering, is taken Subsequent filtrate, then filtered with 0.45 μm of miillpore filter, subsequent filtrate is taken, need testing solution is produced；

(4) determine：Each 20 μ L of mixed reference substance solution, need testing solution are taken, high performance liquid chromatograph is injected, determined.