CN110201130A - A kind of preparation of Xiaochaihutang extract and mass spectrum fingerprint pattern construction method - Google Patents

A kind of preparation of Xiaochaihutang extract and mass spectrum fingerprint pattern construction method Download PDF

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Publication number
CN110201130A
CN110201130A CN201910195785.5A CN201910195785A CN110201130A CN 110201130 A CN110201130 A CN 110201130A CN 201910195785 A CN201910195785 A CN 201910195785A CN 110201130 A CN110201130 A CN 110201130A
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xiaochaihutang
extract
xiao chaihu
chaihu tang
mass spectrum
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杜婷
孙荣进
杜士明
郜嵩
胡明
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Shiyan Taihe Hospital
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Shiyan Taihe Hospital
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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
    • 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)
    • A61K36/25—Araliaceae (Ginseng family), e.g. ivy, aralia, schefflera or tetrapanax
    • A61K36/258—Panax (ginseng)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/484—Glycyrrhiza (licorice)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A61K36/539—Scutellaria (skullcap)
    • 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)
    • A61K36/72—Rhamnaceae (Buckthorn family), e.g. buckthorn, chewstick or umbrella-tree
    • A61K36/725—Ziziphus, e.g. jujube
    • 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)
    • A61K36/888—Araceae (Arum family), e.g. caladium, calla lily or skunk cabbage
    • A61K36/8888—Pinellia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9068—Zingiber, e.g. garden ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

Abstract

The invention belongs to Chinese materia medica preparation atlas analysis technical field, preparation and the mass spectrum fingerprint pattern construction method of a kind of Xiaochaihutang extract are disclosed；Minor decoction of Bupleurum formulation is prepared using the preparation method of Xiaochaihutang extract；According to the mass spectrum fingerprint pattern construction method, test sample and reference substance solution are prepared；LC-ESI-QTOF MS/MS measurement is injected, chromatogram is recorded；The foundation in Xiao Chaihu Tang chemical composition data library；Gained map is handled with software, obtains the substance group compound information of Xiaochaihutang extract.Xiaochaihutang extract provided by the invention meets the dosage form of clinical use；The present invention is by LC-MS finger-print, the material base for having detected all medicinal materials in Xiao Chaihu Tang of comprehensive decorum, while this method can also be used for the analysis detection of vivo biodistribution sample.It can be widely applied to the quality control and effective substance research of Xiao Chaihu Tang and its preparation.

Description

A kind of preparation of Xiaochaihutang extract and mass spectrum fingerprint pattern construction method
Technical field
The invention belongs to the preparation of Chinese materia medica preparation atlas analysis technical field more particularly to a kind of Xiaochaihutang extract and Mass spectrum fingerprint pattern construction method.
Background technique
Currently, the prior art commonly used in the trade is such that
Xiao Chaihu Tang is a kind of warp formed with bupleurum Chinese, ginseng, Radix Glycyrrhizae, ginger, radix scutellariae, jujube and tuber of pinellia seven flavor medicine Allusion quotation prescription.In ancient Chinese medicine book treatise on Febrile Diseases before being recorded in 2000 earliest.(Xiao Chaihu Tang is in day before about 1500 This is referred to as Chinese prescription So saiko to) as a kind of east allusion drug intake sheet from China, it is originally manufactured into mouth The drug of modern industry scale.It is estimated that the millions of patients of Treated by Minor Decoction of Bupleurum, do not report that any serious poison is secondary and make With.Currently, XCHT is also used as dietary supplements to sell in the U.S..
Xiao Chaihu Tang is recorded by existing Chinese Pharmacopoeia and Pharmacopeia of Japan, and clinic is widely used in the various inflammation for the treatment of (bronchitis, common cold, pneumonia), liver and gallbladder (chronic liver disease, liver fibrosis, liver cancer), respiratory tract and enterogastric diseases.Recently Clinical verification it have antidepressant effect.Although in China, Japan and South Korea etc., Asias sale is up to billions of every year First minor decoction of Bupleurum formulation.But the chemical feature and its absorption characteristic of Xiao Chaihu Tang prescription obtain comprehensive and systematic test not yet Card.2010 editions " Chinese Pharmacopoeia " quality with scutelloside, saikosaponin a and d control Xiao Chaihu Tang, Pharmacopeia of Japan are basic herein Upper add increases enoxolone.Xiao Chaihu Tang compound ingredient is numerous, complicated, and existing quality control method and quality control index cannot The inherent quality of reflection drug comprehensively.The present invention has quantified 17 main components in Xiao Chaihu Tang in the research of early period, this A little ingredients account for the 16.06% of Xiaochaihutang extract total amount, but remaining 80% ingredient is still unclear.In addition, in these components In, body absorption metabolic characteristics is insufficient, therefore the quality of Xiao Chaihu Tang controls and its Absorption Characteristics is far from satisfying clinic Demand.
Mass spectrum (MS) is one of most steady and sensitive method of current Chinese herbal medicine secondary metabolite Structural Identification, Because it can not only provide the quality information of molecule, the information of its molecule fragment can also be provided, is especially advantageous for illustrating chemical combination Object structure.In addition, the combination of MS and LC liquid chromatogram can be with separating mixture, this makes identification is simpler to be easy.
For the effective substance for illustrating Xiao Chaihu Tang, base of the present invention in building each medicinal material chemical component information bank of prescription On plinth, using high performance liquid chromatography and flight time mass spectrum joint technology (HPLC-ESI-TOF/MS) to the main color of Xiao Chaihu Tang The source of spectral peak is belonged to, its chemical structure is inferred and identified.Its inventive result can be the matter of Xiao Chaihu Tang Amount control and further Pharmacological Analysis lay the foundation.The main component of Xiao Chaihu Tang is saponins, flavonoids and gingerol The bioavilability of class compound, flavonoids and saponins is excessively poor.Therefore the present invention copies FDA in compound medicine experiment to push away The external Caco2 model recommended summarizes to mechanism of absorption of the Xiao Chaihu Tang prescription in small intestine, evaluates main in Xiao Chaihu Tang The bioavilability of ingredient；The intracorporal metabolism group and pharmacokinetics of Xiao Chaihu Tang and its main component.
In conclusion problem of the existing technology is: being come in the prior art using high-efficient liquid phase technique DAD UV detector The content of saikosaponin a, saikoside d, baicalein and enoxolone in Xiao Chaihu Tang is controlled, some scholars attempt to use Chinese medicine Finger-print is to control its quality, but each chromatographic peak fails to carry out making ownership, poor reproducibility clear in its finger-print；Due to small Bupleurum root decoction database and mass spectrometric analysis method it is not perfect, the chemical feature and its absorption characteristic of Xiao Chaihu Tang prescription is not yet To comprehensive and systematic verifying.
Solve the difficulty and meaning of above-mentioned technical problem: the present invention combines each single medicinal material in Xiao Chaihu Tang compound first Material base and mainstream mass spectrometry database establish the compound database of Xiao Chaihu Tang, enumerate 862 comprehensively to obtain small bavin The information profile of compound in Hu soup applies positive and negative two kinds from scan pattern respectively.The results show that under negative ion mode The detection sensitivity of most standard substance is five times of positive ion mode, sees Fig. 2, and has higher signal-to-noise ratio, partization Closing object can only just detect in the negative ion mode.In addition, present invention discover that some compounds are shown not in different modes Same fracture mode.Therefore, this experiment analyzes and identifies the chemical component in Xiao Chaihu Tang using both of which, and complementation obtains completeer Whole structural information.To obtain more fragment ions, positive and negative ion mode uses different collision voltage: 10,20,40V, That is collision energy gradient acquisition mode.This test to compound use different level collision energy, evaluation collision generate from Subtype.When collision energy increases, generates complicated fragment and increase, generate more fragment ions.All precursor ions Valuable ionic type will be split into, generation is most had into significant fragment ion.The present invention explores collision energy to change The influence of object is closed, and observes the ionic type generated as collision energy function.Induction and conclusion is acquired in collision energy gradient Generating under mode most has significant fragment ion and cracking mode (table 7).Than the high-resolution matter that compound is used alone at present The analysis method that spectrum information (both first mass spectrometrics) is identified is more acurrate.Secondly it utilizes and standard control and same class compound The comparison of second level high resolution mass spectrum information, 122 in precise Identification Xiao Chaihu Tang in compound, and comprehensive and systematic tracking These material composition groups are from vitro to intracorporal Absorption And Metabolism process.
The present invention improves the method for quality control of this product and discloses its effective substance, develops a kind of simple easy Row, accuracy are high, avoid matrix interference, the reproducible and stable fingerprint map construction method for compound, for small bavin The control of the quality of Hu soup compound and its preparation and effective substance evaluation are of great significance.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of preparation of Xiaochaihutang extract and mass spectrum fingerprints Map construction method.
The invention is realized in this way a kind of preparation method of Xiaochaihutang extract, specifically includes the following steps:
Step 1: choosing medicinal material radix bupleuri, radix scutellariae, Radix Glycyrrhizae, the tuber of pinellia, rhizoma zingiberis, ginseng and the jujube of good Xiao Chaihu Tang, does It is dry to smash it through pharmacopeia 2 sieves；
Step 2: radix bupleuri 150g, radix scutellariae 56g, pinellia 56g, ginseng 56g, ginger 56g, Radix Glycyrrhizae are weighed in prescription ratio 56g, jujube 56g, add 8 times of amount water, cold soaking 2h, slightly boiled reflux 1h, pour out medical fluid, then plus 6 times of amount water, slightly boiled reflux 30min incline Medical fluid out；
Step 3: merging medical fluid twice, and filter paper filtering is concentrated under reduced pressure, and Xiaochaihutang extract is made in freeze-drying, It is spare.
Another object of the present invention is to provide a kind of mass spectrum fingerprint pattern construction methods of Xiaochaihutang extract, specifically The following steps are included:
Step 1: it prepares test solution: weighing the freeze-dried powder of the above-mentioned full side of 2g, add water 16mL, 80 DEG C of heating water baths 30min, centrifugation, take supernatant to get；
Step 2: preparation reference substance solution: take the stock solution of 22 kinds of standard items compounds use respectively ethyl alcohol/DMSO (4: 1) it dissolves, the final concentration of 10mM of mother liquor；Then every kind of stock solution is subjected to LC-ESI- to 1 μM with 50% methanol dilution QTOF MS/MS analysis；
Step 3: measurement: accurate respectively to measure Xiaochaihutang extract test solution, reference substance solution and blank control Solution, injection LC-ESI-QTOF MS/MS measurement, records chromatogram；
Step 4: the foundation in Xiao Chaihu Tang chemical composition data library: according to the chemical component of Xiao Chaihu Tang each group recipe material, By retrieving in the database, saponins, the flavonoids, Diphenylheptane class, gingerol class, alkaloid in Xiao Chaihu Tang are arranged With organic acid chemical component totally 862, establish corresponding chemical component formula database；
Step 5: with Agilent Qualitative Analysis, B.06.00 software handles gained map, Respectively obtain the substance group compound information of Xiaochaihutang extract.
Further, it in step 1, is centrifuged as 14000rpm × 30min.
Further, in step 2,22 kinds of standard items compounds be caffeic acid, liquiritin, rutin, isoliquiritigenin, scutelloside, Scutelloside, baicalein, ginsenoside Rg1, Rd, Re, Rf, Rb1, wogonin, gingerol, glycyrrhizic acid, saikosaponin a, radix bupleuri Saponin(e b2, saikoside d, Quercetin, enoxolone, Saponin A, zingiberone.
Further, in step 3, chromatographic condition is chromatographic column: ACQUITY UPLC BEH C18 (50mm × 2.1mm ID, 1.7 μm)；ACQUITY UPLC BEH C18 (100mm × 2.5mm ID, 1.8 μm)； Agilent Poroshell 120 PFP (150mm × 2.5mm, 2.7 μm) mobile phase A: 0.1% aqueous formic acid mobile phase；B: acetonitrile, 40 DEG C of column temperature；Sample temperature 4℃；Sampling volume is 10 μ L.
Further, UHPLC-TOF/MS Mass Spectrometry Conditions use the source ESI, under positive ion mode: capillary voltage 4000V, mist Change atmospheric pressure 30psi, dry gas stream speed 8L/min, dry 350 DEG C of temperature degree, fragment voltage 160eV；Under negative ion mode: hair Tubule voltage 3500V, nebulizer pressure 30psi, dry gas stream speed 8L/min, dry 350 DEG C of temperature degree, fragment voltage 160V； Mass number scanning range m/z 100~3200.
Further, it in step 4, retrieves in the database, database includes Scifinder, PubMed, TCM data The library Taiwan@version (TCM Database@Taiwan), China Tsinghua University State Intellectual information bank (Chinese National Knowledge Infrastructure of Tsinghua University) and KNApSAcK.
Further, in step 4, using Agilent PCDL software create compound database, database include molecular formula, Accurate molecular quality, compound name and plant origin.
In conclusion advantages of the present invention and good effect are as follows:
(1) Xiaochaihutang extract provided by the invention meets the dosage form of clinical use；
(2) present invention passes through LC-MS finger-print, the substance base for having detected all medicinal materials in Xiao Chaihu Tang of comprehensive decorum Plinth, while this method can also be used for the analysis detection of vivo biodistribution sample.It can be widely applied to the matter of Xiao Chaihu Tang and its preparation Amount control and effective substance research.
(3) present invention combines the material base of each single medicinal material and mainstream mass spectrometry database in Xiao Chaihu Tang compound to build first The PCDL compound database for having found Xiao Chaihu Tang enumerates 862 kinds of compounds in Xiao Chaihu Tang, and is currently known most total It is 415 according to library.
It (4) is the comprehensive information profile for obtaining compound in Xiao Chaihu Tang, this experiment is analyzed using positive and negative both of which Identify the chemical component in Xiao Chaihu Tang, complementation obtains more complete structural information.It is broken to obtain more second order ms ions Piece information, positive and negative ion mode use different collision voltage: 10,20,40V, i.e. collision energy gradient acquisition mode.This examination Test the collision energy that different level is used to compound, the ionic type that evaluation collision generates.When collision energy increases, generate Complicated fragment increases, and generates more fragment ions.Induction and conclusion, which generates under collision energy gradient acquisition mode, most to be had Significant fragment ion and cracking mode (table 7).Than high resolution mass spectrum information (the both level-one matter that compound is used alone at present Spectrum) analysis method identified is more acurrate.
(4) for the first time with the Absorption Characteristics of 122 kinds of compounds in vivo in Caco2 modeling Xiao Chaihu Tang.
(5) 122 kinds of compounds have been tracked in Xiao Chaihu Tang from vitro to internal blood, urine, gallbladder using this method system Metabolic profile in juice, and 17 kinds of metabolites are identified, it lays a good foundation to disclose its material composition for playing drug effect.
Detailed description of the invention
Fig. 1 is the preparation method flow chart of Xiaochaihutang extract provided in an embodiment of the present invention.
Fig. 2 is the mass spectrum fingerprint pattern construction method flow chart of Xiaochaihutang extract provided in an embodiment of the present invention.
Fig. 3 is chromatography schematic diagram of the 22 kinds of significant substances provided in an embodiment of the present invention under both positive and negative mode.
Fig. 4 is that the base peak ion stream of the Xiao Chaihu Tang of 25mg/ml provided in an embodiment of the present invention in the positive-ion mode is shown It is intended to.
Fig. 5 is that the base peak ion stream of the Xiao Chaihu Tang of 25mg/ml provided in an embodiment of the present invention in the negative ion mode is shown It is intended to.
Fig. 6 is the chemical structure of organic acid, flavones and ginger phenolic compounds in Xiao Chaihu Tang provided in an embodiment of the present invention Schematic diagram.
Fig. 7 is the compounds such as saikoside, ginsenoside and glycyrrhizin in Xiao Chaihu Tang provided in an embodiment of the present invention Chemical structure schematic diagram.
Fig. 8 is Absorption Characteristics schematic diagram of the Xiao Chaihu Tang provided in an embodiment of the present invention on Caco-2 cell model.
Fig. 9 is total ion current of the Xiao Chaihu Tang in FVB mice plasma under positive ion mode provided in an embodiment of the present invention Schematic diagram.
Figure 10 is total ion current of the Xiao Chaihu Tang in FVB mice plasma under negative ion mode provided in an embodiment of the present invention
Figure 11 is total ion current of the Xiao Chaihu Tang in FVB mouse urine under positive ion mode provided in an embodiment of the present invention Schematic diagram.
Figure 12 is total ion current of the Xiao Chaihu Tang in FVB mouse urine under negative ion mode provided in an embodiment of the present invention Schematic diagram.
Figure 13 is total ion current of the Xiao Chaihu Tang in FVB mouse bile under positive ion mode provided in an embodiment of the present invention Schematic diagram.
Figure 14 is total ion current of the Xiao Chaihu Tang in FVB mouse bile under negative ion mode provided in an embodiment of the present invention Schematic diagram.
Figure 15 is the noval chemical compound schematic diagram that Xiao Chaihu Tang provided in an embodiment of the present invention is generated in Mice Body intracellular metabolite.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.
Application principle of the invention is described in detail with reference to the accompanying drawing.
As shown in Figure 1, the preparation method of Xiaochaihutang extract provided in an embodiment of the present invention, specifically includes following step It is rapid:
S101: choosing medicinal material radix bupleuri, radix scutellariae, Radix Glycyrrhizae, the tuber of pinellia, rhizoma zingiberis, ginseng and the jujube of good Xiao Chaihu Tang, dry Smash it through pharmacopeia 2 sieves；
S102: radix bupleuri 150g, radix scutellariae 56g, pinellia 56g, ginseng 56g, ginger 56g, Radix Glycyrrhizae are weighed in prescription ratio 56g, jujube 56g, add 8 times of amount water, cold soaking 2h, slightly boiled reflux 1h, pour out medical fluid, then plus 6 times of amount water, slightly boiled reflux 30min incline Medical fluid out；
S103: merging medical fluid twice, and filter paper filtering is concentrated under reduced pressure, freeze-drying, and Xiaochaihutang extract is made, standby With.
As shown in Fig. 2, the mass spectrum fingerprint pattern construction method of Xiaochaihutang extract provided in an embodiment of the present invention, specifically The following steps are included:
S201: it prepares test solution: weighing the freeze-dried powder of the above-mentioned full side of 2g, add water 16mL, 80 DEG C of heating water baths 30min, centrifugation, take supernatant to get；
S202: preparation reference substance solution: the stock solution of 22 kinds of standard items compounds is taken to use ethyl alcohol/DMSO (4: 1) respectively Dissolution, the final concentration of 10mM of mother liquor；Then every kind of stock solution is subjected to LC-ESI-QTOF to 1 μM with 50% methanol dilution MS/MS analysis；
S203: measurement: accurate measurement Xiaochaihutang extract test solution, reference substance solution and blank control are molten respectively Liquid, injection LC-ESI-QTOF MS/MS measurement, records chromatogram；
S204: the foundation in Xiao Chaihu Tang chemical composition data library: according to the chemical component of Xiao Chaihu Tang each group recipe material, lead to Cross and retrieve in the database, arrange Xiao Chaihu Tang in saponins, flavonoids, Diphenylheptane class, gingerol class, alkaloid and Organic acid chemical component totally 862, establish corresponding chemical component formula database；
S205: with Agilent Qualitative Analysis, B.06.00 software handles gained map, i.e., Respectively obtain the substance group compound information of Xiaochaihutang extract.
In step S201, centrifugation provided in an embodiment of the present invention is 14000rpm × 30min.
In step S202,22 kinds of standard items compounds provided in an embodiment of the present invention are caffeic acid, liquiritin, rutin, different Glycyrrhizin, scutelloside, scutelloside, baicalein, ginsenoside Rg1, Rd, Re, Rf, Rb1, wogonin, gingerol, glycyrrhizic acid, Saikosaponin a, saikoside b2, saikoside d, Quercetin, enoxolone, Saponin A, zingiberone.
In step S203, chromatographic condition provided in an embodiment of the present invention is chromatographic column: ACQUITY UPLC BEH C18 (50mm × 2.1mm ID, 1.7 μm)；ACQUITY UPLC BEH C18 (100mm × 2.5mm ID, 1.8 μm)；Agilent 120 PFP of Poroshell (150mm × 2.5mm, 2.7 μm) mobile phase A: 0.1% aqueous formic acid mobile phase；B: acetonitrile, column 40 DEG C of temperature；4 DEG C of sample temperature；Sampling volume is 10 μ L.
In step S203, UHPLC-TOF/MS Mass Spectrometry Conditions provided in an embodiment of the present invention use the source ESI, positive ion mode Under: capillary voltage 4000V, atomization gas pressure 30psi, dry gas stream speed 8L/min, dry 350 DEG C of temperature degree, fragment voltage 160eV；Under negative ion mode: capillary voltage 3500V, nebulizer pressure 30psi, dry gas stream speed 8L/min, dry temperature 350 DEG C of degree, fragment voltage 160V；Mass number scanning range m/z 100~3200.
It is provided in an embodiment of the present invention to retrieve in the database in step S204, database include Scifinder, PubMed, the TCM data library Taiwan@version (TCM Database@Taiwan), Chinese Tsinghua University State Intellectual information bank (Chinese National Knowledge Infrastructure of Tsinghua University) and KNApSAcK。
It is provided in an embodiment of the present invention to create compound database, data using Agilent PCDL software in step S204 Library includes molecular formula, accurate molecular quality, compound name and plant origin.
Application principle of the invention is further elaborated combined with specific embodiments below；
The object of the present invention is to provide a kind of preparation methods of Xiao Chaihu Tang compound extract, and use LC-MS fingerprint image The method of spectrum carries out quality control to it and illustrates its effective substance.In order to achieve this, the Xiao Chaihu Tang initially set up Extract LC-MS finger-print identifies 122 kinds of effective components altogether with the mass spectrographic cracking rule of reference substance and compound, wraps Flavone compound, saponins compound, ginger phenolic compounds etc. are included, and carry out the comprehensive matter of multicomponent using above-mentioned technology Amount detects and controls；Secondly the method for using metabolism of Chinese medicine group identifies Xiaochaihutang extract in human colon carcinoma Caco2 cell Absorption and Xiaochaihutang extract on model make the effective substance base of Xiao Chaihu Tang in metabolic characteristics in the mammalian body Plinth is definitely.
Embodiment 1；
To achieve the above object, the present invention adopts the following technical solutions:
1, the preparation method of Xiaochaihutang extract provided in an embodiment of the present invention, specifically includes the following steps:
S101: choosing medicinal material radix bupleuri, radix scutellariae, Radix Glycyrrhizae, the tuber of pinellia, rhizoma zingiberis, ginseng and the jujube of good Xiao Chaihu Tang, dry Smash it through pharmacopeia 2 sieves；
S102: radix bupleuri 150g, radix scutellariae 56g, pinellia 56g, ginseng 56g, ginger 56g, Radix Glycyrrhizae are weighed in prescription ratio 56g, jujube 56g, add 8 times of amount water, cold soaking 2h, slightly boiled reflux 1h, pour out medical fluid, then plus 6 times of amount water, slightly boiled reflux 30min incline Medical fluid out；
S103: merging medical fluid twice, and filter paper filtering is concentrated under reduced pressure, freeze-drying, and Xiaochaihutang extract is made, standby With.
It 2, the embodiment of the invention provides a kind of Xiaochaihutang extract, is extracted basically according to this 1 method It arrives.
3, the mass spectrum fingerprint pattern construction method of Xiaochaihutang extract provided in an embodiment of the present invention, specifically includes following Step:
(1) preparation of test solution: weighing the freeze-dried powder of the above-mentioned full side of 2g, adds water 16mL, 80 DEG C of heating water baths 30min, be centrifuged (14000rpm × 30min), take supernatant to get；
(2) preparation of reference substance solution: caffeic acid, liquiritin, rutin, isoliquiritigenin, scutelloside, scutelloside, baicalein, Ginsenoside Rg1, Rd, Re, Rf, Rb1, wogonin, gingerol, glycyrrhizic acid, saikosaponin a, saikoside b2, saikoside D, the stock solution of 22 kinds of standard items compounds such as Quercetin, enoxolone, Saponin A, zingiberone uses ethyl alcohol/DMSO respectively (4: 1) it dissolves, the final concentration of 10mM of mother liquor.Then every kind of stock solution is subjected to LC-ESI- to 1 μM with 50% methanol dilution QTOF MS/MS analysis；
(3) measure: accurate measurement Xiaochaihutang extract test solution, reference substance solution and blank control are molten respectively Liquid, injection LC-ESI-QTOF MS/MS measurement, records chromatogram；
A. chromatographic condition is chromatographic column: ACQUITY UPLC BEH C18 (50mm × 2.1mm ID, 1.7 μm)； ACQUITY UPLC BEH C18 (100mm × 2.5mm ID, 1.8 μm)；Agilent Poroshell 120 PFP(150mm× 2.5mm, 2.7 μm) mobile phase A: 0.1% aqueous formic acid mobile phase；B: acetonitrile, 40 DEG C of column temperature.4 DEG C of sample temperature；Sample introduction body Product is 10 μ L.Mobile phase elution program is shown in Table 3
3 gradient elution program of table
B.UHPLC-TOF/MS Mass Spectrometry Conditions use the source ESI, under positive ion mode: capillary voltage 4000V, atomization gas Pressure 30psi, dry gas stream speed 8L/min, dry 350 DEG C of temperature degree, fragment voltage 160eV；Under negative ion mode: capillary Voltage 3500V, nebulizer pressure 30psi, dry gas stream 8 L/min of speed, dry 350 DEG C of temperature degree, fragment voltage 160V.Quality Number scanning range m/z 100~3200.
(4) foundation in Xiao Chaihu Tang chemical composition data library
According to the chemical component of Xiao Chaihu Tang each group recipe material, by Scifinder, PubMed, TCM data library@ Taiwan version (TCM Database@Taiwan), China Tsinghua University State Intellectual information bank (Chinese National Knowledge Infrastructure of Tsinghua University) and the databases such as KNApSAcK in retrieve, it is whole Saponins, flavonoids, Diphenylheptane class, gingerol class, alkaloid and the organic acid chemical component in Xiao Chaihu Tang are managed Totally 862, establish corresponding chemical component formula database.Compound database is created using Agilent PCDL software.Number It include molecular formula, accurate molecular quality, compound name and plant origin according to library；
(5) with Agilent Qualitative Analysis, B.06.00 software handles gained map, that is, divides The substance group compound information of Xiaochaihutang extract is not obtained.
According to step (5) mass spectrum fingerprint pattern in above scheme have altogether in obtain 122 compound in Xiao Chaihu Tang.
4, provided in an embodiment of the present invention that ingredient and/or Metabolite are absorbed as the Xiaochaihutang extract of index using prototype Mass spectrum fingerprint pattern construction method, specifically comprise the following steps:
(I) preparation of test solution: weighing the freeze-dried powder of the above-mentioned full side of 2g, adds water 16mL, 80 DEG C of heating water baths 30min, be centrifuged (14000rpm × 30min), take supernatant to get；
(II) preparation of reference substance solution: caffeic acid, liquiritin, rutin, isoliquiritigenin, scutelloside, scutelloside, radix scutellariae Element, ginsenoside Rg1, Rd, Re, Rf, Rb1, wogonin, gingerol, glycyrrhizic acid, saikosaponin a, saikoside b2, radix bupleuri The stock solution of 22 kinds of standard items compounds such as saponin(e d, Quercetin, enoxolone, Saponin A, zingiberone use respectively ethyl alcohol/ DMSO (4: 1) dissolution, the final concentration of 10mM of mother liquor.Then every kind of stock solution is subjected to LC- to 1 μM with 50% methanol dilution ESI-QTOF MS/MS analysis；
(III) measure: accurate measurement Xiaochaihutang extract test solution, reference substance solution and blank control are molten respectively Liquid, injection LC-ESI-QTOF MS/MS measurement, records chromatogram；
A. chromatographic condition is chromatographic column: ACQUITY UPLC BEH C18 (50mm × 2.1mm ID, 1.7 μm)； ACQUITY UPLC BEH C18 (100mm × 2.5mm ID, 1.8 μm)；Agilent Poroshell 120 PFP(150mm× 2.5mm 2.7 μm) mobile phase A: 0.1% aqueous formic acid mobile phase；B: acetonitrile, 40 DEG C of column temperature.4 DEG C of sample temperature；Sample introduction body Product is 10 μ L.Mobile phase elution program is shown in Table 4.
4 gradient elution program of table
B.UHPLC-TOF/MS Mass Spectrometry Conditions use the source ESI, under positive ion mode: capillary voltage 4000V, atomization gas Pressure 30psi, dry gas stream speed 8L/min, dry 350 DEG C of temperature degree, fragment voltage 160eV；Under negative ion mode: capillary Voltage 3500V, nebulizer pressure 30psi, dry gas stream 8 L/min of speed, dry 350 DEG C of temperature degree, fragment voltage 160V.Quality Number scanning range m/z 100~3200.
(IV) foundation in Xiao Chaihu Tang chemical composition data library
According to the chemical component of Xiao Chaihu Tang each group recipe material, by Scifinder, PubMed, TCM data library@ Taiwan version (TCM Database@Taiwan), China Tsinghua University State Intellectual information bank (Chinese National Knowledge Infrastructure of Tsinghua University) and the databases such as KNApSAcK in retrieve, it is whole Saponins, flavonoids, Diphenylheptane class, gingerol class, alkaloid and the organic acid chemical component in Xiao Chaihu Tang are managed Totally 862, establish corresponding chemical component formula database.Compound database is created using Agilent PCDL software.Number It include molecular formula, accurate molecular quality, compound name and plant origin according to library；
(V) Absorption And Metabolism finger-print of the Xiaochaihutang extract on Caco-2 cell
Up-to-standard kind is had to the transwell plate of Caco-2 cell, 2mL HBSS buffering is added in the two sides in every hole Liquid (pH=7.4), 37 DEG C are placed on air constant-temperature table and are incubated for 1h.After 1h, HBSS liquid is siphoned away.Xiao Chaihu Tang is from mucous membrane side (AP) to serosa side (BL) transport process (AP-BL): from the side AP be added the 5mg/ml of Xiao Chaihu Tang containing target HBSS liquid 2mL, Isometric blank HBSS liquid (pH=7.4) is added in the side BL simultaneously.It is 37 DEG C that temperature, which is arranged, in air table, revolving speed 50rpm. 0 after dosing, 0.5,1,1.5,2h, 0.5mL is sampled respectively at two sides, while isometric drug containing is added in supplement in the side AP again HBSS solution, and the side BL also supplements isometric blank HBSS liquid, every group of sample is 3 parts parallel.Drug turns from the side BL to the side AP It is identical as the transport experiment operation of the side AP-BL to transport (BL-AP), but the side BL is added in Xiao Chaihu Tang, blank is then added in the side AP HBSS liquid.It is separately added into the inner mark solution (daidzin is dissolved in acetonitrile solution) of 100 μ L in sample, is vortexed and mixes 3min, After 14000rpm is centrifuged 20min, supernatant is taken to analyze using LC-ESI-QTOF MS/MS.
(VI) mammal is made to take orally the Xiaochaihutang extract of claim 1 respectively, this experimental design Xiao Chaihu Tang Metering becomes radix bupleuri 24g, radix scutellariae 9g, tuber of pinellia 9g, ginger 9g, ginseng 9g, Radix Glycyrrhizae 9g with reference to clinical metering, the group of Xiao Chaihu Tang, Jujube 9g.With reference to formula rate in treatise on Febrile Diseases, 2010 " Chinese Pharmacopoeia " Xiao Chai Hu granules, radix bupleuri and radix scutellariae ratio are 8: 3.People Weight according to 70kg, so the dosage of people is 78g/70kg-1.1g/kg (small Radix Bupleuri extractum recovery rate is 18.3%), It is converted into extract 0.20g/kg.It is 3.6g/kg so being converted into mouse multiplied by coefficient 9.3；Rat is 1.2g/ multiplied by coefficient 6 Kg, other animals and so on.0~the biological sample that takes for 24 hours after continuous gavage is administered five days.It extracts biological sample and obtains biology Sample test solution is analyzed with LC-ESI-QTOF MS/MS.
(VIII) with Agilent Qualitative Analysis, B.06.00 software handles gained map, i.e., Respectively obtain the compound information of the metabolism group of Xiaochaihutang extract.
It is provided by the invention to refer to using prototype absorption ingredient and/or Metabolite as the mass spectrum of the Xiaochaihutang extract of index Line map construction method can verify small radix bupleuri by step (III) and (IV) two ways with external sample and vivo sample The quality or the true and false of decoction extract, while step (V) and (VI) complementally absorb ingredient and/or Metabolite with prototype to test Demonstrate,prove the true and false or quality of drug or extract.
It is provided by the invention to refer to using prototype absorption ingredient and/or Metabolite as the mass spectrum of the Xiaochaihutang extract of index Line map construction method, wherein mammal described in step (VI) is selected from people, rat, mouse, dog, monkey etc..Implement at one In scheme, the mammal is mouse.
It is provided by the invention to refer to using prototype absorption ingredient and/or Metabolite as the mass spectrum of the Xiaochaihutang extract of index Line map construction method, wherein biological sample described in step (VI) includes but is not limited to blood sample, urine sample and bile sample.
It is provided by the invention to refer to using prototype absorption ingredient and/or Metabolite as the mass spectrum of the Xiaochaihutang extract of index Line map construction method wherein obtains 23 prototype compounds and 17 metabolins in step (VIII) biological sample.
Embodiment 2；
A kind of preparation of Xiaochaihutang extract provided in an embodiment of the present invention and its mass spectrum fingerprint pattern construction method
(1) radix bupleuri, radix scutellariae, Radix Glycyrrhizae, the tuber of pinellia, rhizoma zingiberis, ginseng and jujube are purchased from Shiyan City, Hubei Province legendary god of farming book on Chinese herbal medicine Chinese medicine Medicine materical crude slice Co., Ltd identifies, specifying information is shown in Table 5 through Hubei Medicine and pharmacy college pharmaceutical college professor Chen Jiyan.
5 Chinese medicine compound prescription Xiao Chaihu Tang of table forms crude drug source information table
(2) preparation of full formula extraction
Radix bupleuri 150g, radix scutellariae 56g, pinellia 56g, ginseng 56g, ginger 56g, Radix Glycyrrhizae 56g, jujube are weighed in prescription ratio 56g, adds 8 times of amount water, cold soaking 2h, slightly boiled reflux 1h, pours out medical fluid, then plus 6 times of amount water, slightly boiled reflux 30min pour out medical fluid, Merge medical fluid twice, filter paper filtering is concentrated under reduced pressure, and freeze-drying is spare.
(3) preparation of test solution: weighing the freeze-dried powder of the above-mentioned full side of 2g, adds water 16mL, 80 DEG C of heating water baths 30min, be centrifuged (14000rpm × 30min), take supernatant to get；
(4) preparation of reference substance solution: caffeic acid, liquiritin, rutin, isoliquiritigenin, scutelloside, scutelloside, baicalein, Ginsenoside Rg1, Rd, Re, Rf, Rb1, wogonin, gingerol, glycyrrhizic acid, saikosaponin a, saikoside b2, saikoside D, the stock solution of 22 kinds of standard items compounds such as Quercetin, enoxolone, Saponin A, zingiberone uses ethyl alcohol/DMSO respectively (4: 1) it dissolves, the final concentration of 10mM of mother liquor.Then every kind of stock solution is subjected to LC- to 1 μM with 50% methanol dilution ESI-QTOF MS/MS analysis；
(5) measure: accurate measurement Xiaochaihutang extract test solution, reference substance solution and blank control are molten respectively Liquid, injection LC-ESI-QTOF MS/MS measurement, records chromatogram；
A. chromatographic condition is chromatographic column: ACQUITY UPLC BEH C18 (50mm × 2.1mm ID, 1.7 μm)； ACQUITY UPLC BEH C18 (100mm × 2.5mm ID, 1.8 μm)；Agilent Poroshell 120 PFP(150mm× 2.5mm, 2.7 μm) mobile phase A: 0.1% aqueous formic acid mobile phase；B: acetonitrile, 40 DEG C of column temperature.4 DEG C of sample temperature；Sample introduction body Product is 10 μ L.Mobile phase elution program is shown in Table 6.
6 gradient elution program of table
B.UHPLC-TOF/MS Mass Spectrometry Conditions use the source ESI, under positive ion mode: capillary voltage 4000V, atomization gas Pressure 30psi, dry gas stream speed 8L/min, dry 350 DEG C of temperature degree, fragment voltage 160eV；Under negative ion mode: capillary Voltage 3500V, nebulizer pressure 30psi, dry gas stream 8 L/min of speed, dry 350 DEG C of temperature degree, fragment voltage 160V.Quality Number scanning range m/z 100~3200.
(6) foundation in Xiao Chaihu Tang chemical composition data library
According to the chemical component of Xiao Chaihu Tang each group recipe material, by Scifmder, PubMed, TCM data library@ Taiwan version (TCM Database@Taiwan), China Tsinghua University State Intellectual information bank (Chinese National Knowledge Infrastructure of Tsinghua University) and the databases such as KNApSAcK in retrieve, it is whole Saponins, flavonoids, Diphenylheptane class, gingerol class, alkaloid and the organic acid chemical component in Xiao Chaihu Tang are managed Totally 862, establish corresponding chemical component formula database.Compound database is created using Agilent PCDL software.Number It include molecular formula, accurate molecular quality, compound name and plant origin according to library.
(7) with Agilent Qualitative Analysis, B.06.00 software handles gained map, that is, divides The substance group compound information of Xiaochaihutang extract is not obtained.
(8) result:
A.UHPLC and MS condition optimizing
Optimize the UHPLC analysis condition such as different types of chromatographic column, mobile phase, column temperature and flow velocity, to main in Xiao Chaihu Tang Want the separating effect of ingredient.Zorbax SB-C18 column is compared with 120 SB-AQ Column column of proshell, is used The chromatographic peak peak shape of ACQUITY UHPLC BEH C18 post separation is best.To obtain more preferable chromatographic peak and stronger total ion current, Several flow phase systems, including organic phase methanol, acetonitrile are compared, water phase includes water, 0.1% formic acid and 2.5mM ammonium acetate.Most Afterwards, acetonitrile and 0.1% formic acid as mobile phase, separating effect is best, and ionising effect is best.The results show that Xiao Chaihu Tang In most of ingredients separated, for next step Structural Identification provide greatly ensure (table 7).
B. the identification strategy of chemical component
For the information for obtaining compound in maximum Xiao Chaihu Tang, both positive and negative ion scan mode is applied respectively.Knot Fruit shows, bearing the detection sensitivities from mark substances most of under mode is five times (Fig. 3) just from mode, and have compared with High signal-to-noise ratio, part of compounds can only be detected just in the negative ion mode.In addition, some compounds table in different modes Reveal different fracture modes.Therefore, this experiment analyzes and identifies the chemical component in Xiao Chaihu Tang using both of which, complementary to obtain To more complete structural information.To obtain more fragment ions, positive and negative ion mode is using different collision voltages: 10,20 And 40V, both collision energy gradient acquisition modes.This test uses the collision energy of different level to compound, and evaluation collision produces Raw ionic type.When collision energy increases, generates complicated fragment and increase, generate more fragment ions.Before all Body ion can all split into valuable ionic type, and generation is most had significant fragment ion.It explores and touches in the present invention Influence of the energy to compound is hit, and observes the ionic type generated as collision energy function.With the increase of collision energy, The abundance for the ion that fragment generates increases.Ideally, all precursor ions will be broken down into available ionic type (but Will not excessive fragmentation), to generate the highest possible signal of fragment ion.
C. reference standards identify chemical structure characteristic
22 compound standard product of this experimental applications, including organic acid, flavones, saikoside, ginsenoside, Radix Glycyrrhizae soap Glycosides and ginger.These compounds use 50% methanol dilution to take 1 μM of sample, LC-ESI Q-TOF MS/ to a certain concentration respectively It is analyzed under MS both positive and negative mode, as a result sees Fig. 3.
Chromatogram of Fig. 3: 22 kinds of significant substances under both positive and negative mode；
As shown in figure 3,2 caffeic acids, 12 liquiritins, 14 rutins, 25 glycyrrhizins, 26 isoliquiritins, 29 scutellosides, 40 Chinese Scutelloside, 41, ginsenoside Rg1,43 ginsenoside Rds/Re, 45 baicaleins, 50Rf, 52 wogonins, 65 gingerols, wild jujube Benevolence saponin A, 71 Rb1,78 glycyrrhizic acids, 86ssa, 89 ssb2,94 ssd, 107 Quercetins, enoxolone.
As shown in figure 3, by comparing in the UHPLC retention time, accurate molecular weight and second order ms of compound standard product Fragment ion, authenticating compound structure.
D. in Xiao Chaihu Tang compound identification
The cracking mode of reference standard product of the present invention is analyzed using identical structure and its derivative parent nucleus having the same Identification.Therefore, these compounds are identified by two-stage identification.The first order is referred to as " identification ", wherein by comparing MS/ Retention time in MS, accurate molecular masses (mass deficit filters < 5ppm) and fragmentation pathway (mass deficit filters < 10ppm) It is compared with reference standard product.The second level is known as " tentative identification ", wherein by comparing accurate point in the MS/MS with correlation Son measures (mass deficit filters < 5ppm) and fragmentation pathway (mass deficit filters < 10ppm) to identify with identical basic framework Compound.For the isomers with identical molecular weight, by the fragmentation pathway in MS/MS come authenticating compound.
Such as table 7 it is found that 122 compounds have been determined in total in Xiao Chaihu Tang.Wherein, 29 compounds come from radix bupleuri, 42 compounds come from radix scutellariae, and 22 come from Radix Glycyrrhizae, and 1 comes from the tuber of pinellia, and 19 come from ginseng, and 5 come from ginger, and 4 come from Jujube.
As shown in Figure 4 and Figure 5, the chromatography peak figure under the negative ions mode of Xiao Chaihu Tang.
As shown in fig. 6, the compound structure figure of flavonoids.
As shown in fig. 7, saikoside, ginsenoside, glycyrrhizin, gingerol class compound structure figure.
Embodiment 3；
A kind of Absorption And Metabolism finger-print of the Xiaochaihutang extract provided in an embodiment of the present invention on Caco-2 cell.
1, the foundation of Caco-2 cell traffic model
1) the freezing of Caco-2 cell, recover and secondary culture
(1) Caco-2 cell is frozen to be donated by houston, U.S.A university Ming professor Hu.The purpose of Caco-2 cell cryopreservation It is to keep cell algebra close in experimentation and experiment reproducibility.Cell is in logarithmic growth phase, degrees of fusion reaches When 80% or so, cell cryopreservation can be carried out, operating procedure is as follows: by cell from 5%CO2It is taken out in incubator, falling, it is micro- to set When the growthform and degrees of fusion of microscopic observation cell reach 80% or so.It is cleaned twice with 5mlPBS.2ml 0.25% is added Trypsin digestion and cell 3-5min is rapidly added the DMEM culture medium containing 10%FBS and terminates digestion, and 10ml pipettor is blown repeatedly The cell being adhered on culture bottle is beaten, and so that cell is dispersed into individual cells as far as possible.Collect cell suspension, 800rpm centrifugation 5 Min is discarded supernatant, and configured frozen stock solution is added, and (DMEM culture medium: FBS: DMSO=6: 3: 1) being resuspended cell, soft to blow and beat Disperse cell, makes cell density control 5 × 106/ mL or so.Label is sub-packed in cryopreservation tube, and every pipe dispenses 1mL.Cell It is put into gradient in freezing storing box to freeze, is finally transferred in liquid nitrogen container and saves for a long time.
(2) recovery of cell
The cell frozen is quickly removed from liquid nitrogen, is immediately placed in 37 DEG C of waters bath with thermostatic control, weak vibrations melt it rapidly Change, is then transferred quickly in the 15ml centrifuge tube for being pre-loaded with the DMEM culture medium that 10mL is heated to 37 DEG C.Pipettor is slight After piping and druming, 800rpm is centrifuged 3min, and 5mL DMEM culture medium is added, and gently blows and beats number down toward being visible by naked eyes cell mass, fast Speed moves to cell suspension in the Tissue Culture Flask of 25cm2, is put into incubator and cultivates.
(3) secondary culture of cell
After cell recovery, next day changes liquid, changes weekly liquid 3~4 times.Cell fusion degree reaches 80%-90% and is passed on, It is passed on according to 1: 3-1: 5 ratio.
2, Caco-2 cell kind plate
Logarithmic growth phase Caco-2 cell is pressed 2.5 × 105A/cm2Density be seeded in six holes for being coated with Collagen type-I On Transwell culture plate, as shown in Figure 4.2nd day replacement culture solution after kind plate then every other day changes once, and culture is extremely At 19-21 days, chrotoplast is stepped up with the measurement of cell resistance instrument.Before measuring resistance, first with the HBSS (pH=7.4,37 DEG C) prepared Buffer, which is gently swung, to be washed cell 3 times, as 420 Ω of its resistance value >/cm2When, illustrate to have formed complete and close cell monolayer. Transport plates meet the requirements, and can be used for drug transport experiment.
3, Caco-2 cell traffic is tested
1) two-way transport experiment process on Caco-2 cell
After having surveyed resistance, 2mL HBSS buffer (pH=7.4) is added in the two sides in every hole, 37 DEG C are placed in air constant temperature and shake 1h is incubated on bed.After 1h, HBSS liquid is siphoned away.Xiao Chaihu Tang is from mucous membrane side (AP) to the transport process (AP- of serosa side (BL) BL): the HBSS liquid 2mL of the 5mg/ml of Xiao Chaihu Tang containing target being added from the side AP, while isometric blank HBSS being added in the side BL Liquid (pH=7.4).It is 37 DEG C that temperature, which is arranged, in air table, revolving speed 50rpm.0 after dosing, 0.5,1 .1.5,2h, respectively at Two sides sample 0.5mL, while in the side AP, isometric drug containing HBSS solution is added in supplement again, and the side BL also supplements isometric sky White HBSS liquid, every group of sample are 3 parts parallel.The transport experiment of transhipment (BL-AP) of the drug from the side BL to the side AP and the side AP-BL operates It is identical, but the side BL is added in Xiao Chaihu Tang, the HBSS liquid of blank is then added in the side AP.The internal standard of 100 μ L is separately added into toward sample Solution (daidzin is dissolved in acetonitrile solution) is vortexed and mixes 3min, after 14000rpm is centrifuged 20min, supernatant is taken to use LC-ESI Q-TOF MS/MS analysis.
4, result: absorption and Metabolic fingerprinting of the Xiaochaihutang extract on Caco-2 cell.
Fig. 8: Absorption Characteristics schematic diagram of the Xiao Chaihu Tang on Caco-2 cell model.
: "+" represents positive ion mode, and "-" represents negative ion mode.
As shown in figure 8, being can detecte in the substrate liquid of Caco-2 cell model to 33 compounds, Absorption Characteristics figure. When Xiao Chaihu Tang is attached to top surface, 19 flavone compounds are detected in substrate side, 6 glycyrrhizins, 7 saikosides, 1 When a gingerol.
Embodiment 4；
It is provided in an embodiment of the present invention a kind of using prototype absorption ingredient and/or Metabolite as the extraction of the Xiao Chaihu Tang of index The mass spectrum fingerprint pattern construction method of object.
1, Xiaochaihutang extract gastric infusion
Due to Xiao Chaihu Tang prescription clinic be Long-term taking medicine at least one it is more than week, the present invention designs small bavin thus The metering of Hu soup becomes radix bupleuri 24g, radix scutellariae 9g, tuber of pinellia 9g, ginger 9g, ginseng 9g with reference to clinical metering, the group of Xiao Chaihu Tang, Radix Glycyrrhizae 9g, jujube 9g.With reference to formula rate, radix bupleuri and radix scutellariae ratio in treatise on Febrile Diseases, 2010 " Chinese Pharmacopoeia " Xiao Chai Hu granules It is 8: 3.The weight of people is according to 70kg, so the dosage of people is 78g/70kg=1.1g/kg (Xiao Chaihu Tang medicinal extract recovery rate 18.3%), to be converted into extract 0.20g/kg.It is 3.6g/kg so being converted into mouse multiplied by coefficient 9.3.FVB male is small Mouse 5, continuous gavage is administered five days, and stomach-filling volume is 0.1ml/10g.
2, the collection of the biological samples such as mice plasma, bile and urine
FVB mouse the 5th day administration after 5,10,30min eye socket take blood in be pre-coated with 1% heparin sodium physiological saline EP manage Middle 50ul is centrifuged 3min (8000rpm/min), takes supernatant, obtain plasma sample.Bile duct is then carried out after anesthesia and inserts art, is received Collect 1h bile.Before experiment, mouse elder generation fasting 8-12 hours, but free water can be carried out.Biliary drainage pipe (No. 10) is before surgery It first uses physiological saline rinse 3 times, dehydrated alcohol rinse 2 times, is dried up with dry disposable sterilized insulin syringe.Mouse claims The preparatory gastric infusion Xiao Chaihu Tang suspension of dosage of 3.6g/kg is pressed after weight.After half an hour is administered in Xiao Chaihu Tang, intraperitoneal injection 50% urethane is anaesthetized, and anaesthesia dosage is (0.05ml/10g).The mouse tongue that turns up after anesthesia ensures respiration unobstructed, sets Normal body temperature is maintained at 37 DEG C of small animal surgical heating cushion, fixed, shaving opens abdominal cavity about 3cm extremely along mouse ventrimeson Diaphragm lower end.Find mouse gall-bladder, surgical thread is ligatured along duodenum end, wait gall-bladder completely it is full heave after penetrate No. 10 biliary drainage pipes.Bile collection terminates, and mouse plucks after eyeball takes blood, dislocation to put to death and excises one's gallbladder, and takes out liver, kidney, Physiological saline blots water after rinsing 2 times
3, the preparation of mouse biological sample test liquid
20 μ L of plasma containing drug and blank plasma is taken, 10 times of amount acetonitrile precipitation albumen are added, high speed, which is vortexed, mixes 4min, from Heart 30min (14 000rpm/min) is taken 80% supernatant in being dried with nitrogen, is redissolved with 50% acetonitrile to 100 μ L, be centrifuged 30min (14000rpm/min) takes 4 μ L of supernatant to analyze for UHPLC-Q-TOF/MS.The system of mouse urine, bile sample test liquid It is standby: to take urine or bile to dilute 10 times, then prepare the method preparation urine of sample according to blood plasma, bile is used for mass spectral analysis.
4, UHPLC testing conditions and mass spectrum acquisition parameter
5, result；
1) plasma containing drug UHPLC-Q-TOF/MS is analyzed
Using the accurate molecular weight (mass deficit filters < 10ppm) of UHPLC-Q-TOF/MS analysis control compounds, knot The second level MS/MS fragment ion information (being included under negative ions mode) of polymerisable compounds gradually confirms biological sample blood plasma, gallbladder The prototype compound confirmed in juice and urine.The metabolism that Agilent Metabolite ID software calculates each prototype compound produces Object carries out data analysis retrieval so as to form new Xiao Chaihu Tang metabolite data library again.In mice plasma, urine and bile One isolates and identifies totally 49 kinds of chemical constituents (including 32 prototype compounds and 17 metabolins have determined that or tentative true Recognize).Wherein 20 kinds of compound prototypes and n-compound are composed by comparing retention time and mass spectrometric data data, and with drug The Absorption Characteristics schemed on the Xiao Chaihu Tang Caco2 model of sum are matched, and the results are shown in Table 8-10, various compounds further into The matching gone on chromatogram, as shown in Fig. 9 to Figure 15.
As shown in figure 9, under positive ion mode provided in an embodiment of the present invention Xiao Chaihu Tang in FVB mice plasma always from Subflow schematic diagram.
As shown in Figure 10, Xiao Chaihu Tang is total in FVB mice plasma under negative ion mode provided in an embodiment of the present invention Ion stream
As shown in figure 11, Xiao Chaihu Tang is total in FVB mouse urine under positive ion mode provided in an embodiment of the present invention Ion flow diagram.
As shown in figure 12, Xiao Chaihu Tang is total in FVB mouse urine under negative ion mode provided in an embodiment of the present invention Ion flow diagram.
As shown in figure 13, Xiao Chaihu Tang is total in FVB mouse bile under positive ion mode provided in an embodiment of the present invention Ion flow diagram.
As shown in figure 14, Xiao Chaihu Tang is total in FVB mouse bile under negative ion mode provided in an embodiment of the present invention Ion flow diagram.
As shown in figure 15, the noval chemical compound signal that Xiao Chaihu Tang provided in an embodiment of the present invention is generated in Mice Body intracellular metabolite Figure.
Due to the interference of fecal specimens mesostroma effect and Xiao Chaihu Tang, non-analysis detection fecal specimens, according to bile sample Product speculate.
In blood plasma, bile and urine, there is the differences of conspicuousness for the metabolism distribution of various compounds.Three kinds of biological samples There are also a large amount of flavones and its metabolites for product, but saponin component is nearly no detectable in urine, with this speculate class at The main metabolic pathway divided passes through bile and drains.There are 23 kinds of prototype ingredients and 12 kinds of metabolins, compound letter in bile sample It ceases most abundant.For urine sample, there are 19 kinds of prototype ingredients and 5 kinds of metabolins.There are also blood samples 23 prototype ingredients and 7 Metabolin.The summary of all compounds and structural information are listed in table 8 and Fig. 9 and Figure 10.The cracking of various compounds is advised Rule can refer to the cracking rule of the compound second level in chapter 2 annex.
The metabolic response that chemical component in Xiao Chaihu Tang mainly occurs is the redox reactions and two such as hydrolysis, hydroxylating The reaction of phase glucuronidation.This is similar with the experimental result of other scholars, and most important metabolite is mainly its prototype Close the hydrolysate of object, such as metabolite the 24-Hydroxy glycyrrhetic acid and 18 β-of liquiritin Glycyrrhetinic acid；The metabolite prosaikogenin G of saikoside.But inventor is in vivo It detects it could be theoretically argued that being difficult the macromolecular saikoside 2 '-O-xylosyl the saikosaponin b2 or b1 that absorb. Speculate the absorption that can promote wherein many compounds in Xiao Chaihu Tang.
In short, the metabolic profile of Chinese herbal medicine has very important effect the research on pharmacodynamic substances basis.Due to it Significant meaning and complexity, a science and advanced analysis system demand are extremely urgent.In the present invention, with Xiao Chaihu Tang For the foreign bodies basis of understanding Xiao Chaihu Tang of system, Absorption in vitro model in evaluation and final prototype and metabolin in vivo Verifying.Based on UHPLC-ESI-Q-TOFMS metabonomic analysis technical tactic, the external substance base of Xiao Chaihu Tang can be clarified Plinth and internal metabolic profiling analysis.Temporarily or identify 122 kinds of prototype compounds and 17 kinds of cylinder metabolism-ures altogether.Small radix bupleuri Main component in soup in vivo is flavone compound saponin component, and flavones ingredient mainly passes through urine and bile row It lets out, but based on saponin component drained with bile.
Xiao Chaihu Tang ingredient in table 10:FVB mouse bile
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.

Claims (10)

1. a kind of preparation method of Xiaochaihutang extract, which is characterized in that the preparation method of the Xiaochaihutang extract has Body the following steps are included:
Step 1: medicinal material radix bupleuri, radix scutellariae, Radix Glycyrrhizae, the tuber of pinellia, rhizoma zingiberis, ginseng and the jujube of Xiao Chaihu Tang, mistake after drying and crushing are chosen Pharmacopeia 2 sieves；
Step 2: in prescription ratio weigh radix bupleuri 150g, radix scutellariae 56g, pinellia 56g, ginseng 56g, ginger 56g, Radix Glycyrrhizae 56g, Jujube 56g, adds 8 times of amount water, cold soaking 2h, slightly boiled reflux 1h, pours out medical fluid, then plus 6 times of amount water, slightly boiled reflux 30min pour out medicine Liquid；
Step 3: merging medical fluid twice, and filter paper filtering is concentrated under reduced pressure, freeze-drying, and Xiaochaihutang extract is made, spare.
2. Xiaochaihutang extract prepared by a kind of preparation method of Xiaochaihutang extract as described in claim 1.
3. a kind of mass spectrum fingerprint pattern construction method of Xiaochaihutang extract as claimed in claim 2, which is characterized in that described The mass spectrum fingerprint pattern construction method of Xiaochaihutang extract the following steps are included:
Step 1: it prepares test solution: weighing the freeze-dried powder of the full side 2g, add water 16mL, 80 DEG C of heating water bath 30min, be centrifuged, Take supernatant to get；
Step 2: preparation reference substance solution: the stock solution of 22 kinds of standard items compounds is taken to use ethyl alcohol/DMSO=4: 1 molten respectively Solution, the final concentration of 10mM of mother liquor；Then every kind of stock solution is subjected to LC-ESI-QTOF to 1 μM with 50% methanol dilution MS/MS analysis；
Step 3: measurement: accurate measurement Xiaochaihutang extract test solution, reference substance solution and blank control are molten respectively Liquid, injection LC-ESI-QTOF MS/MS measurement, records chromatogram；
Step 4: the foundation in Xiao Chaihu Tang chemical composition data library: according to the chemical component of Xiao Chaihu Tang each group recipe material, pass through It retrieves, arrange the saponins in Xiao Chaihu Tang, flavonoids, Diphenylheptane class, gingerol class, alkaloid and has in the database Machine acids chemical component totally 862, establish corresponding chemical component formula database；
Step 5: with Agilent Qualitative Analysis, B.06.00 software handles gained map, that is, divides The substance group compound information of Xiaochaihutang extract is not obtained.
4. the mass spectrum fingerprint pattern construction method of Xiaochaihutang extract as claimed in claim 3, which is characterized in that the step In rapid one, it is centrifuged as 14000rpm × 30min.
5. the mass spectrum fingerprint pattern construction method of Xiaochaihutang extract as claimed in claim 3, which is characterized in that the step In rapid two, 22 kinds of standard items compounds are caffeic acid, liquiritin, rutin, isoliquiritigenin, scutelloside, scutelloside, baicalein, people Join saponin(e Rg1, Rd, Re, Rf, Rb1, wogonin, gingerol, glycyrrhizic acid, saikosaponin a, saikoside b2, saikoside d, Quercetin, enoxolone, Saponin A, zingiberone.
6. the mass spectrum fingerprint pattern construction method of Xiaochaihutang extract as claimed in claim 3, which is characterized in that the step In rapid three, chromatographic condition is chromatographic column: ACQUITY UPLC BEH C 18 (50mm × 2.1mm ID, 1.7 μm)；ACQUITY UPLC BEH C18 (100mm × 2.5mm ID, 1.8 μm)；120 PFP of Agilent Poroshell (150mm × 2.5mm, 2.7 μm) mobile phase A: 0.1% aqueous formic acid mobile phase；B: acetonitrile, 40 DEG C of column temperature；4 DEG C of sample temperature；Sampling volume is 10 μ L。
7. the mass spectrum fingerprint pattern construction method of Xiaochaihutang extract as claimed in claim 3, which is characterized in that described UHPLC-TOF/MS Mass Spectrometry Conditions use the source ESI, under positive ion mode: capillary voltage 4000V, atomization gas pressure 30psi are done Pathogenic dryness flow velocity 8L/min, dry 350 DEG C of temperature degree, fragment voltage 160eV；Under negative ion mode: capillary voltage 3500V, mist Change device pressure 30psi, dry gas stream speed 8L/min, dry 350 DEG C of temperature degree, fragment voltage 160V；Mass number scanning range m/z 100~3200.
8. the mass spectrum fingerprint pattern construction method of Xiaochaihutang extract as claimed in claim 3, which is characterized in that the step In rapid four, compound database is created using Agilent PCDL software, database includes molecular formula, accurate molecular quality, chemical combination Name claims and plant origin.
9. the original in a kind of Xiao Chaihu Tang of the construction method of the mass spectrum fingerprint pattern using Xiao Chaihu Tang described in claim 3-8 The characteristic spectrum of type compound and metabolic compounds.
10. a kind of construction method of the mass spectrum fingerprint pattern using Xiao Chaihu Tang described in claim 3-8 is extracted in Xiao Chaihu Tang Application in object and its quality of the pharmaceutical preparations evaluation.
CN201910195785.5A 2019-03-06 2019-03-06 A kind of preparation of Xiaochaihutang extract and mass spectrum fingerprint pattern construction method Pending CN110201130A (en)

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CN113030340A (en) * 2021-04-09 2021-06-25 北京市药品检验所 Method for measuring content of characteristic components of radix bupleuri in radix bupleuri and preparation thereof by liquid phase coupling high-resolution mass spectrometry
CN113138239A (en) * 2021-04-19 2021-07-20 华润三九医药股份有限公司 Small radix bupleuri compound preparation fingerprint spectrum and construction method thereof and content determination method of small radix bupleuri compound preparation
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CN114563510B (en) * 2022-03-01 2022-10-14 山东大学 Establishing method of fingerprint spectrum of minor radix bupleuri decoction formula based on shaoyang disease formula
CN114577941A (en) * 2022-03-07 2022-06-03 陕西步长制药有限公司 Fingerprint detection method for cough-relieving and phlegm-reducing traditional Chinese medicine
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Application publication date: 20190906