CN109100442A - The detection method of internal directly action component in Heiguteng exract medicinal material - Google Patents
The detection method of internal directly action component in Heiguteng exract medicinal material Download PDFInfo
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Abstract
The invention discloses the detection methods of directly action component internal in Heiguteng exract medicinal material, (UHPLC-Q-TOF-MS) technology is combined using ultra performance liquid chromatography-level four bars flight time tandem mass spectrum, by comparing serum HPLC fingerprint and blank rat serum HPLC fingerprint after black bone boisiana extract, Oral Administration in Rats black bone boisiana extract, determine rat constituents absorbed into blood after taking orally black bone boisiana extract, the internal direct working substance of Heiguteng exract medicinal material is specified, to determine that Heiguteng exract plays the Effective Compounds of pharmacological action and quality control provides reference.
Description
Technical field
The present invention relates to Pharmaceutical Analysis field, in particular to the directly detection of action component in vivo in a kind of Heiguteng exract medicinal material
Method.
Technical background
Traditional Chinese medicine ingredients are complicated, and oral medication is the main means of traditional Chinese medicine treatment, modern study proves that, extract oral administration
Afterwards, ingredient (i.e. effective constituents, the irritability generated including original shape ingredient, metabolite and body of blood are only absorbed into
Ingredient), curative effect can be generated when being distributed to each histoorgan or target spot with systemic circulatory system, and reaching certain blood concentration.Therefore,
Be administered orally, the ingredient for really entering blood be possible to be Chinese medicine internal direct working substance basis, analysis and find mouth
The prototype ingredient and metabolite of blood can be entered after clothes, certain foundation can be provided to illustrate its effective substance.
The dry root or complete that Heiguteng exract is the black keel Periploca forrestii Schltr. of Asclepiadaceae Periploca Plants
Strain has promoting menstruation, and promoting blood circulation and wind-dispelling and other effects are used for rheumatic arthritis, and the diseases such as traumatic injury are recorded in " Guizhou Province's Chinese medicine, the people
Race's quality of medicinal material standard " 2003 years versions；It is Guizhou minority nationality medication, there is the work such as relaxing tendons and activating collaterals, dispelling wind and eliminating dampness, clearing heat and detoxicating
With equally there is stronger swelling and pain relieving effect for the soft tissue aseptic inflammation as caused by rheumatism, rheumatoid.Modern times grind
Study carefully and shows in Heiguteng exract plant containing there are many chemical components, mainly cardiac glycosides, C21 steroid, triterpenes, Anthraquinones, flavones
Class, phenylpropanoids etc..Although the document for studying Heiguteng exract chemical component is more, the effective substance about Heiguteng exract
The internal direct agent Quality Research of basis, especially Heiguteng exract still belongs to blank, affects the reasonable development of Heiguteng exract and makes
With.Therefore, using serum drug chemical research method and Modern Mass Spectrometry joint technology, study Heiguteng exract be absorbed into it is intracorporal at
Divide and its existence form, analysis metabolite structure type specify the chemical component directly acted in vivo in Heiguteng exract medicinal material, it can
It lays the foundation for its innovation drug research and quality control.
Summary of the invention
In view of the above-mentioned problems, the object of the present invention is to provide directly make in vivo in a kind of accurate, easy Heiguteng exract medicinal material
With the detection method of ingredient.
To achieve the above object, the technical scheme adopted by the invention is that: using the flight of ultra performance liquid chromatography-level four bars
Time tandem mass spectrum is combined (UHPLC-Q-TOF-MS) technology, extracts by comparing black bone boisiana extract, Oral Administration in Rats Heiguteng exract
Serum HPLC fingerprint and blank rat serum HPLC fingerprint after object are determined and are migrated into rat serum after taking orally black bone boisiana extract
Point, specify the internal direct working substance of Heiguteng exract medicinal material, for determine Heiguteng exract play pharmacological action Effective Compounds and
Quality control provides reference.
Specifically, the detection method of direct action component, including following step in vivo in a kind of Heiguteng exract medicinal material of the invention
Rapid: by cleaning grade SD rat, half male and half female is divided into blank group and two groups of administration group, and every group 6, stomach-filling black bone boisiana extract, often
Day 2 times, successive administration 3 days, femoral artery was taken a blood sample after last dose 45min, prepared blood serum sample, was used for UHPLC-Q-TOFMS
It tests and analyzes.
According to pharmacodynamics test early period it is preferable to determine the preparation process of black bone boisiana extract is as follows, Heiguteng exract medicinal material is taken
6kg is mixed well, and is taken the ethyl alcohol that 8 times of amount volume fractions are 0.7, is extracted 3 times, first time 1.5h, after each 1h twice, merge 3 times
Filtrate is concentrated under reduced pressure, and recycles ethyl alcohol, obtains 6L concentrate.1 times of amount extracting n-butyl alcohol 3 times of above-mentioned concentrate saturation, merge 3
Secondary extract liquor recycles n-butanol, is evaporated, obtains medicinal extract, micro-wave vacuum to obtain the final product.
Preferably, the Oral Administration in Rats medication of the black bone boisiana extract is the following steps are included: take cleaning grade SD rat,
Half male and half female.It tests and is divided into blank group and administration group, every group 6.Fasting 12 hours, free water.Administration group is according to 116g
Kg-1 crude drug amount stomach-filling black bone boisiana extract, blank group give the 1%CMC-Na aqueous solution of stomach-filling equal volume.2 times a day, even
Continuous administration 3d, femoral artery is taken a blood sample after last dose 45min, and taking whole blood to be placed in 37 DEG C of water-baths and keep the temperature to upper layer has weak yellow liquid
It is precipitated, 3000rmin-1 is centrifuged 10min in 4 DEG C of centrifuges after taking-up.Upper serum is taken, same group of each serum is mixed,
To eliminate individual difference, -20 DEG C of preservations are placed in, it is spare.
Preferably, the blood serum sample preparation method is the following steps are included: take Contained Serum and each 600 μ of blank serum
L sequentially adds aqueous formic acid, 2400 μ L methanol, whirlpool that 120 μ L volume fractions are 0.2 and mixes 2min, ultrasonic machine ultrasound 5min,
12000r·min-1Low-temperature centrifugation 10min.Then supernatant is taken to set in nitrogen evaporator, is dried up at 37 DEG C.Volume point is added in residue
Number is that 0.5 methanol-water, 150 μ L redissolves, vortex 1min, ultrasonic 10min, in high-speed refrigerated centrifuge 12000rmin-1Low temperature from
Heart 10min takes Contained Serum sample, blank serum samples for detection.
Preferably, the chromatographic condition that the UHPLC-Q-TOF-MS is tested and analyzed are as follows: chromatographic column: Agilent Eclipse
Plus Cl8RRHD (100mm × 2.1mm, 1.8 μm)；Mobile phase: -0.1% formic acid acetonitrile (B) of 0.1% formic acid water (A), flow velocity:
0.250ml·min-1, column temperature: 40.0 DEG C, sampling volume: 1 μ L, program gradient elution, condition of gradient elution: 0~1min, 5%
~10% (B)；1~2min, 8%~10% (B)；2~8min, 10%~15% (B)；8~14min, 15%~28% (B)；
14~16min, 28%~90% (B)；16~17min, 90%~90% (B)；17~18min, 90%~5% (B).
Preferably, the Mass Spectrometry Conditions that the UHPLC-Q-TOF-MS is tested and analyzed are as follows: electric spray ion source, scanning mode are
Anion scans (ESI-, m/z100~1000)；Capillary voltage: 4.5KV；Orifice potential: 150V；Ion source temperature: 110
℃；Atomization gas (N2) pressure: 0.12MPa；200 DEG C of atomization gas temperature；Flow velocity: 8.0mLmin-1。
By adopting the above technical scheme, detect that 17 enter blood component, including 10 prototype ingredients: 5- from Heiguteng exract serum
O- caffeoyl quinic acid, 4-O- caffeoyl quinic acid, 3-O- caffeoyl quinic acid, 3,4-O- two-caffeoyl quinic acid
3,5-O- two-caffeoyl quinic acid, 4,5-O- two-caffeoyl quinic acid, Glucoperiplocymarin and 7 metabolites, including original
The methylation of type ingredient, desugar, de-hydroxylated metabolite.
In the present invention, if without specified otherwise, mass percent that percentage % is referred both to.
The invention has the following advantages: the UHPLC-Q-TOF/MS that the technical program is established is to Heiguteng exract Contained Serum
The powerful separating capacity of ultra performance liquid chromatography and mass spectrographic high-resolution feature are reasonably used in herbal mixture by detection method
In system, has many advantages, such as that ion transmission efficiency height, high sensitivity, reproducibility are high, identified from Heiguteng exract medicinal material Contained Serum
7 prototype components ingested into the blood out, it is most of for single chemistry such as caffeoyl quinic acid class and two-caffeoyl quinic acid class at
Point, pertinent literature report, caffeoyl quinic acid class chemical component has apparent antibacterial, antiviral, anti-inflammatory, removing freely
The effects of base, hypoglycemic, reducing blood lipid.And another ingredient Glucoperiplocymarin is with prototype absorbed into serum, to treatment gastric cancer, colon cancer,
Human breast carcinoma, liver cancer etc. all have effect.7 metabolic peaks are also found in Heiguteng exract Contained Serum, thus it is speculated that be prototype ingredient
Methylation, desugar, de-hydroxylated metabolite.Above-mentioned Heiguteng exract absorbs directly into blood component and metabolite, is Heiguteng exract
The active component group of drug effect is played in vivo, and above-mentioned chemical component is for carrying out the further pharmacology activity research of Heiguteng exract and innovation
Drug development research provides reference.
Detailed description of the invention
Fig. 1 is the ESI- total ion chromatogram of black bone boisiana extract sample；
Fig. 2 is the ESI- total ion chromatogram of Contained Serum sample after last stomach-filling black bone boisiana extract 45min；
Fig. 3 is the ESI- total ion chromatogram of rat blank serum samples；
Fig. 4 is Contained Serum and blank serum ESI- difference chromatogram；
Fig. 5 is the ESI- total ion chromatogram for mixing reference substance.
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.It is attached
To observe the wave crest tendencies of lines as main points, for some texts for not seeing Chu of Software Create in figure, this field skill in figure
Art personnel can ignore, and not influence the understanding to technical solution of the present invention.
1. material
1.1 reagent
5-O- caffeoyl quinic acid, 4-O- caffeoyl quinic acid, 3,4-O- two-caffeoyl quinic acid 3,5-O- bis-
Caffeoyl quinic acid, 4,5-O- two-caffeoyl quinic acid reference substance (lot number: X20-20141012, Y58-20141012,
1384-101215,1384-101215) it is purchased from solid preparation of traditional Chinese medicine manufacturing technology National Engineering Research Centre；Glucoperiplocymarin and
3-O- caffeoyl quinic acid reference substance (lot number: 111793-200901,110753-201415) is purchased from the inspection of Chinese food drug
Determine research institute.Black bone boisiana extract (self-control).Chromatographic Pure Methanol (Tianjin Kermel Chemical Reagent Co., Ltd.), acetonitrile (moral
State Merck)；Remaining reagent is that analysis is pure.
1.2 instrument
Agilent 1290Infinity UHPLC-DAD-Bruker MicrOTOF-Q II (U.S. Agilent, Germany-
Bruker)；Allegra64R low-temperature and high-speed centrifuge (U.S. Beckman)；DK-98-11 thermostat water bath (Tianjin Stettlen
Instrument Ltd.)；MTN-2800D nitrogen blows enrichment facility (Ao Te Sainz instrument company)；WP-UP-II-20 ultrapure water machine
(Sichuan water that Water Management Equipment Ltd.)；METTLER electronic analytical balance (EL204, plum Teller-support benefit instrument (on
Sea) Co., Ltd)；CQ250A-TS supersonic wave cleaning machine (Shanghai leap medical optical instrument factory).
1.3 animals and plants
Healthy SD rat, weight 200g~240g, half male and half female, quarantine is qualified, is purchased from Guizhou medical university animal house, perhaps
It can the number of card: SCXK (Guizhou Province) 2018-0001.Rat feeding is in SPF grades of animal housing.Heiguteng exract medicinal material is purchased from Guizhou, by Guizhou medical courses in general
Pharmacognosy teaching and research room of University Medical institute associate professor Long Qingde is accredited as the black keel (Periploca of Asclepiadaceae Periploca Plants
Forrestii Schltr.) drying root diameter.
2. method
The preparation of 2.1 serum
Take cleaning grade SD rat, half male and half female.It is randomly divided into blank group and two groups of administration group, every group 6.Fasting 12 is small
When, but free water.According to 116gkg-1Crude drug amount stomach-filling black bone boisiana extract, 2 times a day, successive administration 3d gives in last
Femoral artery is taken a blood sample after medicine 45min, and taking whole blood to be placed in 37 DEG C of water-baths and keep the temperature to upper layer has weak yellow liquid precipitation, in 4 DEG C after taking-up
3000rmin in centrifuge-1It is centrifuged 10min.Upper serum is taken, same group of each serum is mixed, to eliminate individual difference, is set
It is saved in -20 DEG C, it is spare.
The processing of 2.2 blood serum samples
Take Contained Serum and each 600 μ L of blank serum, sequentially add 120 μ L volume fractions be 0.2 aqueous formic acid,
2min, ultrasonic machine ultrasound 5min, 12000rmin are mixed in 2400 μ L methanol, whirlpool-1Low-temperature centrifugation 10min.Then supernatant is taken to set nitrogen
It blows in instrument, is dried up at 37 DEG C.Residue is added volume fraction and redissolves for 0.5 methanol-water, 150 μ L, vortex 1min, ultrasonic 10min,
In high-speed refrigerated centrifuge 12000rmin-1Low-temperature centrifugation 10min takes Contained Serum sample, blank serum samples, for serum
Pharmaceutical chemistry research.
The preparation of 2.3 standard solution
Precision weighs the reference substances such as Glucoperiplocymarin, in 10ml volumetric flask, adds methanol ultrasonic dissolution, constant volume.Obtain 3-O- coffee
Coffee acyl group quininic acid (1.11gL-1), 5-O- caffeoyl quinic acid (1.032gL-1), 4-O- caffeoyl quinic acid
(1.026g·L-1), 3,4- two-caffeoyl quinic acid (1.032gL-1), 3,5- two-caffeoyl quinic acid (1.038gL-1), 4,5- two-caffeoyl quinic acid (1.204gL-1), Glucoperiplocymarin (1.058gL- 1).(- 20 DEG C) of refrigerator preservations are set,
It is spare.
2.4 chromatographic condition
Chromatographic column: Agilent Eclipse Plus Cl8RRHD (100mm × 2.1mm, 1.8 μm)；Mobile phase: 0.1%
- 0.1% formic acid acetonitrile (B) of formic acid water (A), flow velocity: 0.250mlmin-1, column temperature: 40.0 DEG C, sampling volume: 1 μ L, program ladder
Degree elution, condition of gradient elution: 0~1min, 5%~10% (B)；1~2min, 8%~10% (B)；2~8min, 10%~
15% (B)；8~14min, 15%~28% (B)；14~16min, 28%~90% (B)；16~17min, 90%~90%
(B)；17~18min, 90%~5% (B).
2.5 Mass Spectrometry Conditions
Electric spray ion source, scanning mode are negative ion scan (ESI-, m/z 100~1000)；Capillary voltage:
4.5KV；Orifice potential: 150V；Ion source temperature: 110 DEG C；Atomization gas (N2) pressure: 0.12MPa；Atomization gas temperature 200
℃；Flow velocity: 8.0mLmin-1.Exact mass measurement uses sodium formate calibration standard liquid；Data analysis: Data Analysis is soft
Part, Metabolite Detect software.
3. result
The acquisition of 3.1 maps
According to established Heiguteng exract constituents absorbed into blood analysis method, using negative ion mode to black bone boisiana extract,
The samples such as Contained Serum and blank serum carry out data acquisition.
3.2 Heiguteng exracts enter blood component analysis and identification
According to the analysis method under " 2.4 and 2.5 " item, with Metabolite Detect comparison black bone boisiana extract
Sample liquid, black bone boisiana extract Contained Serum and blank serum difference chromatography graph discovery occur 17 in Heiguteng exract Contained Serum
Enter blood component, in conjunction with reference substance comparison and document report, wherein 10 are prototype ingredient, 7 are metabolite, and identify 7
A prototype components ingested into the blood, the results are shown in Table 1.
1 black bone boisiana extract total ionic chromatographic peak retention time of table and ownership
3.2.1 Heiguteng exract enters the identification of blood component
3.2.1.1 monosubstituted caffeoyl quinic acid class Compound Compound 1,5,6 generates quasi-molecular ion peak m/z
353.09, it is known that its molecular formula is C16H17O9.It is compared by retention time and reference substance, compound 1,5,6 is accurately reflected respectively
It is set to 5-O- caffeoyl quinic acid, 3-O- caffeoyl quinic acid and 4-O- caffeoyl quinic acid.
3.2.1.2 disubstituted caffeoyl quinic acid class Compound Compound 14,15,16 generates quasi-molecular ion peak
m/z 515.12[M-H]-.It is C according to its molecular formula of high resolution mass spectrum accurate molecular quality deducibility obtained25H23O12.It loses
1 molecule caffeic acid is gone to obtain single 353 [M-H-C of caffeoyl quinic acid deprotonation fragment m/z16H17O9]-.Thus infer this 3
A compound is double caffeoylquinic acids.By with the chromatographic retention of reference substance, mass spectrum quasi-molecular ion peak and compare
Compound 14,15,16 precise Identifications can be Isochlorogenic acid B, 3,5-, bis- caffeoyl quinine compared to pair by product information
Acid and 4,5- dicaffeoylquinic acid.
3.2.1.3 caffeoyl quinic acid class compounds methyl product Compound 12 is in TRThe quasi-molecule of 8.6min from
Son is m/z 367.1086 [M-H]–, predict that its chemical formula is C by Smart Formula17H19O9, former with monosubstituted quinine acids
Type compound m/z 353.09 [M-H]–Compared to 14Da is reduced, to lose 1 methylene, and the more monosubstituted Kui of its retention time
Peaceful acids is long, and hydrophily reduces, it may be possible to monosubstituted quinine acids methylation as a result, may be disubstituted quinine acids
Compound loses 1 molecule caffeic acid and is transformed into monosubstituted quinine acids to methylate again.Therefore speculate TRThe compound of 8.6min is single
Replace quinine acids methylation metabolite, but the data of this experiment can not illustrate the position specifically to methylate, need into one
Step research.
3.2.1.4 caffeoyl quinic acid Compound Compound 13 carries out the compound with Data Analysis software
Prediction, in TRWhen 8.9min, obtaining quasi-molecular ion peak is [M-H]-M/z 559.1539 is easy to lose a molecule sinapic acid
Obtaining fragment ion is [M-H-C11H12O5+H2O]-M/z 353, therefore speculate compound 13
(1S,3R,4-R,5R)-3-{[(2E)-3-(3,4-Dihydroxyphenyl)-2-propenoyl]oxy}-1,5-
dihydr oxy-4-{[(2E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-2-propenoyl]oxy}
cyclohexanecarb oxyl-icacid。
3.2.1.5 cardiac glycoside compounds compound 17 is in TRThe quasi-molecular ion peak of 15.8min chromatographic peak is m/z
741.3836[M+HCOO]-, main fragment ion is m/z 695 [M-H]-.It is identical as Glucoperiplocymarin standard items, thus identificationization
Conjunction object 17 is Glucoperiplocymarin.
3.2.2 Heiguteng exract enters the possible metabolic pathway analysis of blood component
Heiguteng exract enters the methylation and Glucoperiplocymarin that the possible metabolic pathway of blood component is monosubstituted quinine acid compounds
The glucose for sloughing two molecules is de-hydroxylated again.Monosubstituted quinine acid compounds speculate by taking 5-O- caffeoyl quinic acid as an example
Possible metabolic pathway is methylation；The possible biotransformation pathway of Glucoperiplocymarin includes desugar, oxygen bond rupture and removes hydroxyl.
This experiment establishes UHPLC-Q-TOF/MS to the detection method of Heiguteng exract Contained Serum, reasonably by ultra high efficiency liquid
The powerful separating capacity of phase chromatography and mass spectrographic high-resolution feature are used in herbal mixture system, have ion transmission efficiency
The advantages that height, high sensitivity, high reproducibility.After rat oral gavage black bone boisiana extract, 10,30,45,60,120min are investigated respectively
The Information in Mass Spectra of serum by chromatogram peak out intensity to different samples, peak number mesh and enters the results such as blood peak area and compares,
It is final to determine that the best blood sampling time point after stomach-filling is 45min.And positive and negative ion scanning mode is respectively adopted to black in testing
Bone boisiana extract sample liquid carries out full scan, as a result, it has been found that the compound in black bone boisiana extract sample liquid in the negative ion mode
It responds relatively high, and realizes preferable separation between each chromatographic peak, therefore experimental selection negative ion mode is test
Mode.
This research is used by finger-prints such as comparison black bone boisiana extract, Contained Serum, blank serum and reference substances
Data Analysis, Metabolite Detect software carry out blank serum and the difference spectrum of Contained Serum is analyzed, from blood
Detect that 17 enter blood component in clear, wherein 10 are prototype ingredient, 7 are metabolite.Retained according to provided by mass spectrum
The control of time, accurate relative molecular mass and standard substance identify 7 prototype components ingested into the blood, are 5-O- respectively
Caffeoyl quinic acid, 4-O- caffeoyl quinic acid, 3-O- caffeoyl quinic acid, 3,4-O- two-caffeoyl quinic acid 3,
5-O- two-caffeoyl quinic acid, 4,5-O- two-caffeoyl quinic acid, Glucoperiplocymarin etc..Heiguteng exract constituents absorbed into blood is most
Number is single chemical components such as caffeoyl quinic acid class and two-caffeoyl quinic acid class, pertinent literature report, coffee acyl Kui
Peaceful acids chemical component has the effects that apparent antibacterial, antiviral, anti-inflammatory, removing free radical, hypoglycemic, reducing blood lipid.And it is another
One ingredient Glucoperiplocymarin all has effect with prototype absorbed into serum, to treatment gastric cancer, colon cancer, human breast carcinoma, liver cancer etc..?
7 metabolic peaks are also found in Contained Serum, thus it is speculated that be methylation, the desugar, de-hydroxylated metabolite of prototype ingredient.Speculate
Above-mentioned Heiguteng exract absorbs directly into blood component and metabolite, and the active component group of drug effect may be played in vivo for Heiguteng exract,
Above-mentioned chemical component provides foundation for carrying out the further pharmacological activity of Heiguteng exract and pharmacokinetic, also black to illustrate
Bone rattan effective substance and mechanism of action provide reference.
The above is only preferable examples of the present invention, not to making any data and formal limitation, it is all according to
According to technical spirit of the invention to any data modification, equivalent variations made by example is applied above, the technology of the present invention reality is still fallen within
Within the scope of matter.
Claims (6)
1. the detection method of internal directly action component in a kind of Heiguteng exract medicinal material, it is characterised in that: use ultra high efficiency liquid phase color
Spectrum-level four bars flight time tandem mass spectrum is combined (UHPLC-Q-TOF-MS) technology, by comparing black bone boisiana extract, rat mouthful
Serum HPLC fingerprint and blank rat serum HPLC fingerprint after black bone boisiana extract are taken, determines rat after oral black bone boisiana extract
Constituents absorbed into blood specifies the internal direct working substance of Heiguteng exract medicinal material.
2. the detection method of internal directly action component in Heiguteng exract medicinal material according to claim 1, it is characterised in that packet
Include following steps: by cleaning grade SD rat, half male and half female is divided into blank group and two groups of administration group, and every group 6, stomach-filling Heiguteng exract
Extract, 2 times a day, successive administration 3 days, femoral artery was taken a blood sample after last dose 45min, is prepared blood serum sample, is used for
UHPLC-Q-TOF MS test and analyze, detect that 17 enter blood component from Heiguteng exract blood serum sample, wherein 10 for prototype at
Point, 7 are metabolite.
3. the detection method of internal directly action component in Heiguteng exract medicinal material according to claim 2, it is characterised in that: institute
The Oral Administration in Rats medication of black bone boisiana extract is stated the following steps are included: taking cleaning grade SD rat, half male and half female；Experiment is divided into
Blank group and administration group, every group 6；Fasting 12 hours, free water.Administration group is according to 116gkg-1 crude drug amount stomach-filling black bone
Boisiana extract, blank group give the 1%CMC-Na aqueous solution of stomach-filling equal volume；2 times a day, successive administration 3d gives in last
Femoral artery is taken a blood sample after medicine 45min, and taking whole blood to be placed in 37 DEG C of water-baths and keep the temperature to upper layer has weak yellow liquid precipitation, in 4 DEG C after taking-up
3000rmin-1 is centrifuged 10min in centrifuge；Upper serum is taken, same group of each serum is mixed, to eliminate individual difference, is set
It is saved in -20 DEG C, it is spare.
4. the detection method of internal directly action component in Heiguteng exract medicinal material according to claim 2, it is characterised in that: institute
The blood serum sample preparation method stated sequentially adds 120 μ L bodies the following steps are included: take Contained Serum and each 600 μ L of blank serum
The aqueous formic acid, 2400 μ L methanol, whirlpool that fraction is 0.2 mix 2min, ultrasonic machine ultrasound 5min, 12000rmin-1Low temperature from
Heart 10min；Then supernatant is taken to set in nitrogen evaporator, is dried up at 37 DEG C；It is 0.5 methanol-water, 150 μ L that volume fraction, which is added, in residue
It redissolves, vortex 1min, ultrasonic 10min, in high-speed refrigerated centrifuge 12000rmin-1Low-temperature centrifugation 10min, takes Contained Serum
Sample, blank serum samples are for detection.
5. the detection method of internal directly action component in Heiguteng exract medicinal material according to claim 2, it is characterised in that: institute
State the chromatographic condition of UHPLC-Q-TOF-MS detection and analysis are as follows: chromatographic column: AgilentEclipse Plus Cl8RRHD(100mm
× 2.1mm, 1.8 μm)；Mobile phase: -0.1% formic acid acetonitrile (B) of 0.1% formic acid water (A), flow velocity: 0.250mlmin-1, column
Temperature: 40.0 DEG C, sampling volume: 1 μ L, program gradient elution, condition of gradient elution: 0~1min, 5%~10% (B)；1~
2min, 8%~10% (B)；2~8min, 10%~15% (B)；8~14min, 15%~28% (B)；14~16min, 28%
~90% (B)；16~17min, 90%~90% (B)；17~18min, 90%~5% (B)；Mass Spectrometry Conditions are as follows: electron spray from
Component, scanning mode are negative ion scan (ESI-, m/z 100~1000)；Capillary voltage: 4.5KV；Orifice potential: 150V；
Ion source temperature: 110 DEG C；Atomization gas (N2) pressure: 0.12MPa；200 DEG C of atomization gas temperature；Flow velocity: 8.0mLmin-1。
6. the detection method of internal directly action component in Heiguteng exract medicinal material according to claim 2, it is characterised in that: from
Detect that 17 enter blood component, including 10 prototype ingredients: 5-O- caffeoyl quinic acid, 4-O- caffeoyl in Heiguteng exract serum
Base quininic acid, 3-O- caffeoyl quinic acid, 3,4-O- two-caffeoyl quinic acid 3,5-O- two-caffeoyl quinic acid, 4,5-
Hydroxyl is removed in O- two-caffeoyl quinic acid, Glucoperiplocymarin and 7 metabolites, methylation, desugar including prototype ingredient
Change metabolite.
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