CN109557203A - The detection method of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body - Google Patents

The detection method of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body Download PDF

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CN109557203A
CN109557203A CN201811437131.0A CN201811437131A CN109557203A CN 109557203 A CN109557203 A CN 109557203A CN 201811437131 A CN201811437131 A CN 201811437131A CN 109557203 A CN109557203 A CN 109557203A
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bletillae
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rhizoma bletillae
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CN109557203B (en
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黄勇
李勇军
郑林
潘洁
巩仔鹏
李月婷
孙慧园
杨畅
陆苑
刘亭
马雪
刘春花
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Guizhou Medical University
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    • 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
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    • 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
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Abstract

The invention discloses the detection methods of prototype ingredient and Metabolite in a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract body, this method comprises: after Oral Administration in Rats Pseudobulbus Bletillae (Rhizoma Bletillae) extract, collect serum, urine, excrement, bile biological sample, using ultra performance liquid chromatography-level four bars flight time tandem mass spectrum combination (UHPLC-Q-TOF-MS) technology detection and metabolism software analysis, determine the intracorporal prototype ingredient of rat and Metabolite after taking orally Pseudobulbus Bletillae (Rhizoma Bletillae) extract, specify chemical component in Pseudobulbus Bletillae (Rhizoma Bletillae) extract in vivo be primarily present form, prepareation and quality control for bletilla original new drug provides reference.

Description

The detection method of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body
Technical field
The present invention relates to Pharmaceutical Analysis field, in particular to prototype ingredient and Metabolite in a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract body Detection method.
Background technique
Oral medication is the main means of traditional Chinese medicine treatment, and modern study confirms that oral drugs are thin through the epithelium of gastrointestinal tract Born of the same parents enter blood circulation system and are distributed to each histoorgan or target spot, and while reaching certain blood concentration can generate curative effect.In reality Common many Chinese medicines and its compound do not detect proto-drug ingredient in blood in the research work of border, but drug effect is still very significant, Supposition may be that the Metabolite that generates after vivo biodistribution converts of drug has played therapeutic effect.It can be seen that the body of Chinese medicine Intracellular metabolite research is absorbed into the existence form and its metabolic pathway and mechanism of internal ingredient to clear Chinese medicine, and then really illustrates The working substance basis that Chinese medicine works plays a significant role.
Bletilla is the dry tuber of orchid bletilla Bletilla striata (Thunb.) Reichb.F, has convergence The effect of hemostasis, clearing heat and promoting diuresis, detumescence and promoting granulation, is recorded going through version " Chinese Pharmacopoeia ", and main product is in Guizhou, Sichuan, Hunan, lake North etc. saves, and is widely used in treating traumatic hemorrhage, tuberculosis haematemesis, hemorrhage of digestive tract, sore swollen toxin, chapped skin etc., curative effect is aobvious It writes.Bletilla mainly contains bibenzyl, dihydro phenanthrene class, joins the multiple compounds such as luxuriant and rich with fragrance class, glycoside and bletilla polysaccharide, has hemostasis, promotees Into wound healing, antiulcer, prevention intestinal adhesion, antibacterial, antitumor and other effects.But existing research has been proved in addition to bletilla polysaccharide Outside anastalsis, the other working substances and mechanism of action of bletilla hemostasis are unclear, the especially internal direct effect of bletilla Object Quality Research still belongs to blank, seriously constrains the deep level development of bletilla product.Therefore, research Pseudobulbus Bletillae (Rhizoma Bletillae) extract is absorbed into Intracorporal ingredient and its existence form analyze metabolite structure type, specify the chemistry directly acted in vivo in Bletilla striata medicinal materials Ingredient can lay the foundation for its innovation drug research and quality control.
Summary of the invention
The purpose of the present invention is to overcome prior art defect, provide prototype ingredient and metabolism in a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract body The detection method of ingredient,
Technical scheme is as follows:
The detection method of prototype ingredient and Metabolite in a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract body, after Oral Administration in Rats Pseudobulbus Bletillae (Rhizoma Bletillae) extract, One of serum, urine, excrement, bile biological sample or combination are prepared and collected, is flown using ultra performance liquid chromatography-level four bars Time tandem mass spectrum is combined the detection of (UHPLC-Q-TOF-MS) technology and metabolism software is analyzed, big after determining oral Pseudobulbus Bletillae (Rhizoma Bletillae) extract The intracorporal prototype ingredient of mouse and Metabolite.
Wherein, the rat blood serum sample preparation methods after taking orally Pseudobulbus Bletillae (Rhizoma Bletillae) extract are the following steps are included: in after the last administration Abdominal aorta blood sampling, taking whole blood to be placed in 37 DEG C of water bath with thermostatic control to upper layers has yellow liquid precipitation, and desk centrifuge centrifugation takes upper layer Serum；It will be mixed with each serum is organized, and to eliminate individual difference, be placed in -20 DEG C of preservations, it is spare；Rat blood serum is taken, centrifuge tube is placed in In, methanol is added, after the mixed concussion in whirlpool, successively ultrasonic treatment and centrifugal treating, take supernatant to be dried with nitrogen in 37 DEG C, and methanol is added In the sample of drying, methanol secondary protein precipitation is added by above-mentioned processing method, 50% methanol aqueous solution dissolution residual is added Object, after the mixed concussion in whirlpool, successively ultrasonic treatment and centrifugal treating, taking supernatant UHPLC-Q-TOF MS, sample introduction is analyzed.
Wherein, take orally Pseudobulbus Bletillae (Rhizoma Bletillae) extract after rat urine sample preparation methods the following steps are included: respectively collect 12, 24, the 48, urine of 72h period, and the volume of urine of each period is recorded, -20 DEG C of preservations are placed in, it is spare；Before sample treatment, Each time point urine of mixed in equal amounts；Rat urine is taken, is placed in centrifuge tube, methanol is added and is successively ultrasonically treated after the mixed concussion in whirlpool And centrifugal treating, it takes supernatant to be dried with nitrogen in 37 DEG C, methanol is added in the sample of drying, first is added by above-mentioned processing method 50% methanol aqueous solution dissolution residual substance is added in alcohol secondary protein precipitation, after the mixed concussion in whirlpool, successively at ultrasonic treatment and centrifugation Reason, taking supernatant UHPLC-Q-TOF MS, sample introduction is analyzed.
Wherein, take orally Pseudobulbus Bletillae (Rhizoma Bletillae) extract after rat fecal specimens preparation method the following steps are included: respectively collect 12, 24, the 48, excrement of 72h period, and the stool weight after each period drying is recorded, -20 DEG C of preservations are placed in, it is spare；Sample Before processing, each time point excrement of mixed in equal amounts；Rat excrement after drying is taken, is made into 25% homogenate with physiological saline, it is successively ultrasonic Processing and centrifugal treating, separation upper liquid take homogenate, add methanol, and whirlpool is mixed, successively ultrasonic treatment and centrifugal treating, supernatant It is dried with nitrogen in 37 DEG C, methanol is added in the sample of drying, by above-mentioned processing method secondary protein precipitation, residue is with 50% Methanol aqueous solution dissolution, after the mixed concussion in whirlpool, successively ultrasonic treatment and centrifugal treating, take supernatant UHPLC-Q-TOF MS sample introduction point Analysis.
Wherein, rat bile sample preparation methods after taking orally Pseudobulbus Bletillae (Rhizoma Bletillae) extract the following steps are included: Fractional Collections 0~ 4h, 4~12h, 12~bile for 24 hours, after 24~48h period；Above-mentioned sample is placed in -20 DEG C of refrigerators and saves, spare；At sample Before reason, each time point bile of mixed in equal amounts；Rat bile is taken, 1% formic acid water is added, ethyl acetate is then added and extracts 3 times, closes And extract liquor is dried with nitrogen in 37 DEG C, and 50% methanol aqueous solution dissolution residual substance is added, after the mixed concussion in whirlpool, successively ultrasonic treatment and Centrifugal treating, taking supernatant UHPLC-Q-TOF MS, sample introduction is analyzed.
Further, the chromatographic condition that UHPLC-Q-TOF-MS is tested and analyzed are as follows: chromatographic column is Waters BEH C18 (2.1mm × 50mm, 1.7 μm) column；Mobile phase: -0.01% formic acid acetonitrile (B) of 0.01% aqueous formic acid (A), gradient elution: 0 ~2min, 5%B, Curve:initial；2~5min, 5%~15%B, Curve:6；5~8min, 15%~15%B, Curve:1；8~10min, 15%~45%B, Curve:6；10~14min, 45%~95%B, Curve:6；14~15min, 95%~5%B；Flow velocity: 0.5mL/min；Column temperature: 35 DEG C；Sampling volume is 1 μ L；Mass Spectrometry Conditions are as follows: electric spray ion source is swept The mode of retouching is negative ion scan (ESI-, m/z 50~1200)；Capillary voltage: 1.5KV；Orifice potential: 30V；Ion source temperature Degree: 100 DEG C；300 DEG C of desolvation temperature；Taper hole throughput 50L/h；Collision energy 20-30V；Desolventizing gas flow 10L/ min；Data acquisition scheme: MSE Continuum；Sodium formate correction；Correction mode: sensitivity；Mass spectrometric data acquisition and Processing software are as follows: Masslynx V4.1 work station；Scanning mode is MS Centroid mode.
According to the method described above, from after oral Pseudobulbus Bletillae (Rhizoma Bletillae) extract rat blood serum, urine, excrement, examine in bile biological sample 10 ingredients in Pseudobulbus Bletillae (Rhizoma Bletillae) extract, including 5 prototype ingredients: gastrodin, α-isobutylmalic acid are measured, Gymnoside I, dactylorhin A and militarine and 5 Metabolite, it is hydrolysis including prototype ingredient, de- Sugar, glucuronidation, sulphation metabolite.
It is passed the invention has the following advantages: technical solution of the present invention is used with high duplication, high throughput, ion The time of-flight mass spectrometer of the defeated advantages such as high-efficient carries out the detection of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract rat body, It specifies it and is absorbed into intracorporal ingredient and its existence form, analyze metabolite structure type, illustrate its directly effect in vivo Substance.Above-mentioned bletilla absorbs directly into blood component and metabolite, plays the active principle of drug effect in vivo for Pseudobulbus Bletillae (Rhizoma Bletillae) extract Group, above-mentioned chemical component provide reference for further carrying out the control of bletilla quality, pharmacokinetic studies and original new drug exploitation.
Detailed description of the invention
Fig. 1 is reference substance (A) and Pseudobulbus Bletillae (Rhizoma Bletillae) extract (B) UPLC chromatogram；
Seven main component chemical structures in Fig. 2 Pseudobulbus Bletillae (Rhizoma Bletillae) extract；
Fig. 3 is blank serum samples (A) and blood serum sample (B) ESI- total ion chromatogram is administered in Pseudobulbus Bletillae (Rhizoma Bletillae) extract；
Fig. 4 is blank diaper sample (C) and urine sample (D) ESI- total ion chromatogram is administered in Pseudobulbus Bletillae (Rhizoma Bletillae) extract；
Fig. 5 is blank fecal specimens (E) and blood serum sample (F) ESI- total ion chromatogram is administered in Pseudobulbus Bletillae (Rhizoma Bletillae) extract；
Fig. 6 is blank bile sample (G) and bile sample (H) ESI- total ion chromatogram is administered in Pseudobulbus Bletillae (Rhizoma Bletillae) extract.
The main metabolites of Fig. 7 Pseudobulbus Bletillae (Rhizoma Bletillae) extract and possible metabolic pathway (1: desugar；2: hydrolysis；3: water twice Solution；4: desugar sulphation；S: serum；U: urine；F: excrement；B: bile).
Specific embodiment
Specific embodiments of the present invention will be further explained with reference to the accompanying drawing.It should be noted that for The explanation of these embodiments is used to help understand the present invention, but and does not constitute a limitation of the invention.In addition, disclosed below The each embodiment of the present invention involved in technical characteristic can be combined with each other as long as they do not conflict with each other.With Numerical value or number ratios involved in lower content refer both to mass figures or mass ratio such as without especially mark.
1. material
1.1 reagent
Militarine reference substance (lot number: PS180413-03, content >=98%) is purchased from the general think of biotechnology share in Chengdu Co., Ltd；Gastrodin reference substance (lot number: 110807-201608, content 97.6%) is ground purchased from Chinese food drug assay Study carefully institute；α-isobutylmalic, gymnoside I reference substance are laboratory self-control (purity >=95%) its structure and purity It is confirmed through IR, 1H NMR, MS and HPLC.Pseudobulbus Bletillae (Rhizoma Bletillae) extract (self-control), acetonitrile is chromatographically pure (German Merck company), formic acid is Chromatographically pure, water are pure water, other reagents are that analysis is pure.
1.2 instrument
UPLC-Q-TOF (water generation company, the U.S. Xevo G2-XS, electron spray level four bars time-of-flight mass spectrometry instrument, MassLynx V4.1 mass spectrum work station, UNIFI database), KQ-300DE numerical control ultrasonic cleaner (Sichuan water that science and technology Development Co., Ltd), NA-5L nitrogen sky all-in-one machine (Beijing Zhong Xinghui benefit development in science and technology Co., Ltd), EL204 electronic balance (plum Teller-support benefit Instrument Ltd.), GILSON pipettor (Denmark is triumphant rich), metabolic cage is (by Italian Tecniplast company Import).
1.3 experimental animal
Healthy SD rat, male and female dual-purpose, weight are (220 ± 20) g, are provided by Chongqing Teng Xin Bioisystech Co., Ltd [quality certification number: SCXK (Chongqing) 2015-0001].After rat is introduced into laboratory, by each rearging cage packing 8, female mice and hero Mouse is separately raised, and illumination is sufficient in animal house, and air-conditioning and ventilation equipment system are good, and temperature is controlled at 18~25 DEG C, relatively wet Degree is 50%~60%.Laboratory periodically carries out disinfection and sterilizes to animal house, and rat is raised 1 week in animal house, moves wait adapt to Zoopery is used further to after object room environment.
2. method
The preparation of 2.1 Pseudobulbus Bletillae (Rhizoma Bletillae) extracts
Bletilla striata medicinal materials are weighed with appropriate, with 4 times amount 95% ethanol solution refluxing extraction 3 times, 2h/ time, filtration, merging is filtered Liquid, is concentrated into medicinal extract, medicinal extract upper D101 large pore resin absorption column chromatography after being dissolved with water, after water elution, with 80% ethanol solution Eluent is collected in elution, is concentrated under reduced pressure, and residue micro-wave vacuum obtains bletilla active component, recovery rate 6.82%.
2.2 chromatographic condition
Guard column is Waters VanGuard BEH C18 (2.1mm × 5mm, 1.71.7 μm)；Chromatographic column is Waters BEH C18 (2.1mm × 50mm, 1.7 μm) column；Mobile phase: -0.01% formic acid acetonitrile (B) of 0.01% aqueous formic acid (A), ladder Degree elution: 0~2min, 5%B, Curve:initial；2~5min, 5%~15%B, Curve:6；5~8min, 15%~ 15%B, Curve:1；8~10min, 15%~45%B, Curve:6；10~14min, 45%~95%B, Curve:6；14~ 15min, 95%~5%B, Curve:1；Flow velocity: 0.5mL/min；Column temperature: 35 DEG C；Sampling volume is 1 μ L.
2.3 Mass Spectrometry Conditions
Electric spray ion source；Scanning mode is negative ion scan (ESI-, m/z 50~1200)；Capillary voltage 1.5kV； 100 DEG C of ion source temperature；Orifice potential 30V；300 DEG C of desolvation temperature；Taper hole throughput 50L/h；Collision energy 20-30V； Desolventizing gas flow 10L/min；Data acquisition scheme: MSE Continuum；Sodium formate correction；Correction mode: sensitivity；Mass spectrometric data acquisition and processing software are as follows: Masslynx V4.1 work station.Scanning mode is MS Centroid mode.
The collection of 2.4 biological samples
2.4.1 the collection of serum
Selection upgrowth situation is healthy SD rat 12 similar, and half male and half female, weight (220 ± 20) g normally raises one Week, to adapt to laboratory environment.It is randomly divided into 2 groups (administration group and blank groups), 6/group, raises in metabolic cage, before administration Fasting 12h, free water.Bletilla active component and 1%CMC-Na solution are given respectively.By each 44gKg-1 (crude drug amount) Dosage continuous gavage administration 3 days, 2 times/d, the 1%CMC-Na solution of blank group stomach-filling equal volume.Respectively at after the last administration The blood sampling of 30min abdominal aorta, taking whole blood to be placed in 37 DEG C of water bath with thermostatic control to upper layers has yellow liquid precipitation, desk centrifuge 5000rmin-1 (2683g) is centrifuged 10min, takes upper serum.It will be mixed with each serum is organized, to eliminate individual difference, be placed in- 20 DEG C of preservations, it is spare.
2.4.2 the collection of urine and excrement
It is healthy SD rat 6 similar to choose upgrowth situation, half male and half female, weight (220 ± 20) g, normal raising one week, It to adapt to laboratory environment, raises in metabolic cage, fasting 12h, free water before being administered.Urine for 24 hours and excrement are collected respectively, Then bletilla active component and 1%CMC-Na solution are given respectively.It is given by each 44gKg-1 (crude drug amount) dosage continuous gavage Medicine 3 days, 2 times/d, the 1%CMC-Na solution of blank group stomach-filling equal volume.12,24,48, the urine of 72h period are collected respectively Liquid and excrement, and record the weight of excrement after the volume of urine and drying of each period.- 20 DEG C of preservations are placed in, it is spare.Sample Before processing, each time point urine of mixed in equal amounts and excrement.
2.4.3 the collection of bile
Healthy SD rat 12 are taken, is randomly divided into 2 groups (administration group and blank groups), operation consent fasting 12h, urethane fiber crops Implement cystic duct cannula operation under liquor-saturated state, the silicone rubber tube (1.5mm) for selecting internal diameter small finds bile duct, cuts off an osculum and inserts Enter cystic duct cannula, is sutured after fixed cystic duct cannula.Rat four limbs are fixed on metabolic cage, while keeping bile outflow smooth. By 44gKg-1Dosage gives rat oral gavage bletilla active component, blank group stomach-filling equal volume 1%CMC-Na solution.Fractional Collections 0~4h, 4~12h, 12~bile for 24 hours, after 24~48h period.Above-mentioned sample is placed in -20 DEG C of refrigerators and saves, spare.Sample Before product processing, each time point bile of mixed in equal amounts.
The processing of 2.5 biological samples
2.5.1 serum, urine sample processing method
Rat blood serum or urine 1mL are taken, is placed in 5mL Entry plastic centrifuge tube, 4mL methanol, the mixed concussion 2min in whirlpool are added Afterwards, ultrasonic 5min, 15000rpm are centrifuged 10min, supernatant are taken to be dried with nitrogen in 37 DEG C, and 1mL methanol is added in the sample of drying In, 1mL methanol secondary protein precipitation is added by above-mentioned processing method, 200 μ L, 50% methanol aqueous solution dissolution residual substance is added, After the mixed concussion 2min in whirlpool, ultrasonic 5min, 15000rpm are centrifuged 10min, and taking supernatant UHPLC-Q-TOF MS, sample introduction is analyzed.
2.5.2 bile sample processing method
Rat bile 1ml is taken, 1% formic acid water of 1mL is added, 2mL ethyl acetate is then added and extracts 3 times, combining extraction liquid It is dried with nitrogen in 37 DEG C, 200 μ L, 50% methanol aqueous solution dissolution residual substance is added, after concussion 2min is mixed in whirlpool, ultrasonic 5min, 15000rpm is centrifuged 10min, and taking supernatant UHPLC-Q-TOF MS, sample introduction is analyzed.
2.5.3 fecal specimens processing method
Rat excrement 1g after drying is taken, is made into 25% homogenate with physiological saline, after ultrasonic 5min, 5000rpm (centrifugation 10min, separation upper liquid take 1mL homogenate, add methanol 4ml, and 1min mix in whirlpool, ultrasonic 5min, 15000rpm (it is centrifuged 10min, Supernatant is dried with nitrogen in 37 DEG C, and 1ml methanol is added in the sample of drying, residual by above-mentioned processing method secondary protein precipitation Object 200 μ L, 50% methanol aqueous solution is stayed to dissolve, after the mixed concussion 2min in whirlpool, ultrasonic 5min, 15000rpm are centrifuged 10min, take Sample introduction is analyzed by supernatant UHPLC-Q-TOF MS.
3. Pseudobulbus Bletillae (Rhizoma Bletillae) extract is in the intracorporal metabolism of rat
The analysis of 3.1 serum, urine, excrement and bile sample
Using Pseudobulbus Bletillae (Rhizoma Bletillae) extract as experimental subjects, by serum, urine, excrement and the bile after collection rat administration, utilize High-resolution quadrupole rod-flight time tandem mass spectrometer detects each biological sample, obtains blank serum, Contained Serum figure Spectrum；Blank diaper, drug containing urine map；Blank excrement, drug containing excrement map；Blank bile, drug containing bile map.Each ingredient exists Preferable response signal is obtained under ESI- mode.
3.2 serum, urine, excrement, the metabolite identification analysis in bile
In conjunction with MassLynx V4.1 mass spectrum work station, UNIFI database, to after rat oral gavage bletilla active component Metabolite is quickly analyzed, and research head bletilla active component is in the intracorporal bioconversion rule of rat, thus it is speculated that living in drug Property or the possible metabolic pathway of potential active constituent, provide line to illustrate material base and the mechanism of action of bletilla pharmacological action Rope.
3.2.1 prototype Components identification
C1: there are m/z 331.1018 [M+HCOO] at 0.55min-The compound at peak, display m/z 285.0990, 123.0443 main fragment ion peak is divided into C by the chemical formula that Single Mass Analysis is predicted14H19O9, with Gastrodin reference substance is identical, thereby determines that TRThe C1 of 0.55min is gastrodin.
C2: there are m/z 189.0756 [M-H] at 4.23min-The compound at peak, display m/z's 129.0556 is main Fragment ion peak is divided into C by the chemical formula that Single Mass Analysis is predicted8H13O5, with α-isobutylmalic Acid reference substance is identical, thereby determines that TRThe C2 of 4.23min is α-isobutylmalic acid.
C4: there are m/z 457.1710 [M-H] at 8.72min-The compound at peak, display m/z's 285.1025 is main Fragment ion peak is divided into C by the chemical formula that Single Mass Analysis is predicted21H29O11, with gymnoside I reference substance It is identical, thereby determine that TRThe C4 of 8.72min is gymnoside I.
C5: there are m/z 887.3223 [M-H] at 9.32min-The compound at peak, display 725.2758,457.1754, 189.0765 main fragment ion peak is divided into C by the chemical formula that Single Mass Analysis is predicted40H55O22, with Dactylorhin A reference substance is identical, thereby determines that TRThe C5 of 9.32min is dactylorhin A.
C6: it is found that there are m/z in 10.06min in the difference spectrum obtained by Masslynx V4.1 software The compound at 725.2648 [M-H]-peaks shows the main fragment ion peak of m/z 457.1797, by Single Mass The chemical formula of Analysis prediction is divided into C34H45O17, it is identical as militarine reference substance, thereby determine that TR 10.06min's C6 is militarine.
3.2.2 ester linkage hydrolyzing product is identified
M2: there are m/z 351.1286 [M-H] at 3.31min-The compound at peak, display m/z 189.0765, 129.0556 main fragment ion peak is divided into C by the chemical formula that Single Mass Analysis is predicted14H23O10, thus it is speculated that M2 For the metabolite of dactylorhin A C-1 and the C-4 ester linkage hydrolyzings of metabolite or M4 of C-4 ester linkage hydrolyzings.
M4: there are m/z 619.2284 [M-H]-peak compound at 6.32min, show m/z 439.1647, 189.0793 main fragment ion peak loses a molecule glucose by the chemical formula M4 of Single Mass Analysis prediction Show the fragment ion peak of m/z 439.1647.Consulting literatures are it is found that such C-4 bit esterified compound can just generate loss one The ion of molecule glucose, and such C-1 bit esterified compound then basically will not produce the ion [1], therefore speculate that M4 is The metabolite of C-1 ester linkage hydrolyzings of dactylorhin A.
C4:C3 is accredited as gymnoside I.Analyze militarine chemical structure known to militarine C-1 with C-4 are ester bond；Analyzing gymnoside I C-1 known to the chemical structure of gymnoside I is ester bond.Speculate that C4 is The metabolite of C-4 ester linkage hydrolyzings of militarine.
C2:C2 is accredited as α-isobutylmalic acid.Speculate that C2 is the generation of C-1 ester linkage hydrolyzings of gymnoside I Thank to the metabolite of product or militarine C-1 and C-4 ester linkage hydrolyzings.
C1:C1 is accredited as gastrodin.Speculate C1 be C-1 ester linkage hydrolyzings of gymnoside I metabolite or The metabolite of C-4 ester linkage hydrolyzings of militarine ester linkage hydrolyzing or M4 or dactylorhin A ester linkage hydrolyzing.
3.2.3 desugar product is identified
C6:C6 is accredited as militarine.Molecular weight few 162 compared with dactylorhin A militarine, analysis Dactylorhin A C-2 have glucosyl group substitution known to the chemical structure of dactylorhin A, therefore speculate that C6 is The metabolite of a dehydrated glucose is lost in C-2 glycosidic bond fractures of dactylorhin A.
C2:C2 is accredited as α-isobutylmalic acid.Molecular weight few 162 compared with M2, thus it is speculated that C2 is M2C-2 sugar Glycosidic bonds lose the metabolite of a dehydrated glucose.
3.2.4 desugar sulfating product is identified
C6: there are m/z 203.0023 [M-H] at 0.58min-The compound at peak, display m/z's 123.0443 is main Fragment ion peak is divided into C by the chemical formula that Single Mass Analysis is predicted7H7O5S, thus it is speculated that M1 is gastrodin desugar The metabolite of sulphation afterwards.Since specifying information is not comprehensive enough, the site that metabolism and conversion occurs not can determine that.
3.2.5 sulfating product is identified
M5: there are m/z 321.0447 [M-H] at 7.93min-The compound at peak, mass number ratio Dihydrophenanthrene more than 5 80 shows the main fragment ion peak of m/z 241.0866, by Single Mass The chemical formula of Analysis prediction is divided into C15H13O6S, thus it is speculated that M5 is that the metabolism of 5 sulphation of dihydrophenanthrene produces Object.Since specifying information is not comprehensive enough, the site that metabolism and conversion occurs not can determine that.
3.2.6 glucuronidation product is identified
M3: there are m/z 417.1174 [M-H] at 6.22min-The compound at peak, mass number ratio Dihydrophenanthrene more than 5 176 shows the main fragment ion peak of m/z 241.0834, by Single Mass The chemical formula of Analysis prediction is divided into C21H21O9, thus it is speculated that M3 is the generation of 5 glucuronidation of dihydrophenanthrene Thank to product.Since specifying information is not comprehensive enough, the site that metabolism and conversion occurs not can determine that.
Main metabolites information in 3.3 serum, urine, excrement and bile summarizes
After handling with Masslynx V4.1 software, detect Pseudobulbus Bletillae (Rhizoma Bletillae) extract in rat blood serum, urine, excrement and gallbladder Main metabolites information in juice is shown in Table 1.Including 5 prototype ingredients: gastrodin (C1), α-isobutylmalic Acid (C2), gymnoside I (C4), dactylorhin A (C5) and militarine (C6) and 5 Metabolites, Hydrolysis, desugar, glucuronidation including prototype ingredient, sulphation metabolite.
Main metabolites (S: serum after 1 Oral Administration in Rats head Pseudobulbus Bletillae (Rhizoma Bletillae) extract of table in serum, urine, excrement, bile U: urine F: excrement B: bile；*: with standard control)
The analysis of 3.3 metabolic pathways
This research gives rat Pseudobulbus Bletillae (Rhizoma Bletillae) extract by oral, from Pseudobulbus Bletillae (Rhizoma Bletillae) extract serum, urine, excrement, bile biology 10 ingredients, including 5 prototype ingredients: gastrodin (C1), α-isobutylmalic acid (C2) are detected in sample, gymnoside I(C4),dactylorhinA(C5),and militarine(C6)；And 5 Metabolites, show bletilla Bioconversion can occur in vivo for active component, metabolite to hydrolyze, desugar, glucuronidation, based on sulphation.At this Research identifies 5 prototype ingredients in metabolite, analyzes known to the chemical structure of these ingredients gymnoside I (C4), Dactylorhin A (C5), and militarine (C6) are benzyl succinate glycosides compound, are by 2- isobutyl group malic acid The monoesters or dibasic acid esters analog derivative formed with 4- glucosyl group benzylalcohol；α-isobutylmalic acid (C2) is 2- isobutyl group apple Tartaric acid；gastrodin(C1).The fracture of glycosidic bond and grape glycosyloxy benzyl mainly occurs for benzyl succinate glycosides compound.Institute With there may be metabolic conversion relationships between benzyl succinate glycosides compound.Content of the Militarine in bletilla active component Up to 26%, it Militarine can be observed disappears substantially from the chromatogram of Contained Serum, urine, excrement, bile.Prompt bletilla Extract is absorbed into and violent biotransformation has occurred in vivo, therefore it plays the form of drug effect in addition to prototype in vivo Outside ingredient, metabolite is also likely to be its active constituent.
In conjunction with attached drawing, the embodiments of the present invention are described in detail above, but the present invention is not limited to described implementations Mode.For a person skilled in the art, in the case where not departing from the principle of the invention and spirit, to these embodiments A variety of change, modification, replacement and modification are carried out, are still fallen in protection scope of the present invention.

Claims (7)

1. the detection method of prototype ingredient and Metabolite in a kind of Pseudobulbus Bletillae (Rhizoma Bletillae) extract body, it is characterised in that: Oral Administration in Rats bletilla After extract, one of serum, urine, excrement, bile biological sample or combination are prepared and collect, using ultra performance liquid chromatography- Level four bars flight time tandem mass spectrum is combined the detection of (UHPLC-Q-TOF-MS) technology and metabolism software analysis, determines oral bletilla The intracorporal prototype ingredient of rat and Metabolite after extract.
2. the detection method of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body according to claim 1, feature exist In: the rat blood serum sample preparation methods after oral Pseudobulbus Bletillae (Rhizoma Bletillae) extract in abdominal aorta after the last administration the following steps are included: adopt Blood, taking whole blood to be placed in 37 DEG C of water bath with thermostatic control to upper layers has yellow liquid precipitation, and desk centrifuge centrifugation takes upper serum；It will be same Each serum mixing of group, to eliminate individual difference, is placed in -20 DEG C of preservations, spare；Rat blood serum is taken, is placed in centrifuge tube, first is added Alcohol, after the mixed concussion in whirlpool, successively ultrasonic treatment and centrifugal treating, take supernatant to be dried with nitrogen in 37 DEG C, and methanol is added in drying In sample, methanol secondary protein precipitation is added by above-mentioned processing method, 50% methanol aqueous solution dissolution residual substance, the mixed shake in whirlpool is added After swinging, successively ultrasonic treatment and centrifugal treating, taking supernatant UHPLC-Q-TOF MS, sample introduction is analyzed.
3. the detection method of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body according to claim 1, feature exist In: the rat urine sample preparation methods after oral Pseudobulbus Bletillae (Rhizoma Bletillae) extract are the following steps are included: when collecting 12,24,48,72h respectively Between section urine, and record the volume of urine of each period, be placed in -20 DEG C of preservations, it is spare；Before sample treatment, mixed in equal amounts is each Time point urine；Rat urine is taken, is placed in centrifuge tube, adds methanol, after the mixed concussion in whirlpool, successively at ultrasonic treatment and centrifugation Reason, takes supernatant to be dried with nitrogen in 37 DEG C, and methanol is added in the sample of drying, and it is secondary heavy that methanol is added by above-mentioned processing method 50% methanol aqueous solution dissolution residual substance is added in shallow lake albumen, and after the mixed concussion in whirlpool, successively ultrasonic treatment and centrifugal treating, take supernatant Sample introduction is analyzed by liquid UHPLC-Q-TOF MS.
4. the detection method of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body according to claim 1, feature exist In: the rat fecal specimens preparation method after oral Pseudobulbus Bletillae (Rhizoma Bletillae) extract is the following steps are included: when collecting 12,24,48,72h respectively Between section excrement, and record the stool weight after the drying of each period, be placed in -20 DEG C of preservations, it is spare；Before sample treatment, equivalent Mix each time point excrement；Take drying after rat excrement, be made into 25% homogenate with physiological saline, successively ultrasonic treatment and centrifugation at Reason, separation upper liquid take homogenate, add methanol, and whirlpool is mixed, and successively ultrasonic treatment and centrifugal treating, supernatant are blown in 37 DEG C of nitrogen It is dry, methanol is added in the sample of drying, by above-mentioned processing method secondary protein precipitation, residue is molten with 50% methanol aqueous solution Solution, after the mixed concussion in whirlpool, successively ultrasonic treatment and centrifugal treating, taking supernatant UHPLC-Q-TOF MS, sample introduction is analyzed.
5. the detection method of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body according to claim 1, feature exist In: the rat bile sample preparation methods after oral Pseudobulbus Bletillae (Rhizoma Bletillae) extract are the following steps are included: Fractional Collections 0~4h, 4~12h, 12 ~bile for 24 hours, after 24~48h period；Above-mentioned sample is placed in -20 DEG C of refrigerators and saves, spare；Before sample treatment, equivalent is mixed Close each time point bile；Take rat bile, 1% formic acid water be added, ethyl acetate is then added and extracts 3 times, combining extraction liquid in 37 DEG C are dried with nitrogen, and 50% methanol aqueous solution dissolution residual substance is added, after the mixed concussion in whirlpool, successively ultrasonic treatment and centrifugal treating, Taking supernatant UHPLC-Q-TOF MS, sample introduction is analyzed.
6. the detection method of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body according to claim 1, feature exist In chromatographic condition that the UHPLC-Q-TOF-MS is tested and analyzed are as follows: chromatographic column be Waters BEH C18 (2.1mm × 50mm, 1.7 μm) column；Mobile phase: -0.01% formic acid acetonitrile (B) of 0.01% aqueous formic acid (A), gradient elution: 0~2min, 5%B, Curve:initial；2~5min, 5%~15%B, Curve:6；5~8min, 15%~15%B, Curve:1；8~ 10min, 15%~45%B, Curve:6；10~14min, 45%~95%B, Curve:6；14~15min, 95%~5%B； Flow velocity: 0.5mL/min；Column temperature: 35 DEG C；Sampling volume is 1 μ L；Mass Spectrometry Conditions are as follows: electric spray ion source, scanning mode be negative from Son scanning (ESI-, m/z 50~1200)；Capillary voltage: 1.5KV；Orifice potential: 30V；Ion source temperature: 100 DEG C；Precipitation 300 DEG C of agent temperature degree；Taper hole throughput 50L/h；Collision energy 20-30V；Desolventizing gas flow 10L/min；Data acquisition module Formula: MSE Continuum；Sodium formate correction；Correction mode: sensitivity；Mass spectrometric data acquisition and processing software are as follows: Masslynx V4.1 work station；Scanning mode is MS Centroid mode.
7. the detection side of prototype ingredient and Metabolite in Pseudobulbus Bletillae (Rhizoma Bletillae) extract body described in any one of -6 according to claim 1 Method, it is characterised in that: from after oral Pseudobulbus Bletillae (Rhizoma Bletillae) extract rat blood serum, urine, excrement, detect bletilla in bile biological sample 10 ingredients in extract, including 5 prototype ingredients: gastrodin, α-isobutylmalic acid, gymnoside I, dactylorhin A and militarine and 5 Metabolite, hydrolysis, desugar, grape alditol including prototype ingredient Acidification, sulphation metabolite.
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