CN109115933A - A method of screening Protective effects of Sini decoction myocardial ischemia effective substance - Google Patents
A method of screening Protective effects of Sini decoction myocardial ischemia effective substance Download PDFInfo
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Abstract
The invention discloses a kind of methods for screening Protective effects of Sini decoction myocardial ischemia effective substance, comprising the following steps: one, the measurement of the determination of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia and peak area；Two, the determination of effective constituents and the measurement of peak area in Sini Tang body；Three, the correlation analysis of the peak area of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia and the peak area of effective constituents in Sini Tang body, filters out Protective effects of Sini decoction myocardial ischemia effective substance.The present invention is based on isoprels to cause myocardial ischemia in rats model, metabonomic technology is targeted using the non-targeted metabonomic technology of ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry and based on ultra performance liquid chromatography-triple quadrupole bar mass spectrum, filter out 12 kinds of Protective effects of Sini decoction myocardial ischemia effective substances, improve the accuracy of Protective effects of Sini decoction myocardial ischemia effective substance, the screening period is shortened, screening Expenses Cost is reduced.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of screening Protective effects of Sini decoction myocardial ischemia effective substance
Method.
Background technique
Sini Tang is made of monkshood, rhizoma zingiberis, Radix Glycyrrhizae, is the classics recipe in treatise on Febrile Diseases written by Zhang Zhongjing, has Hui Yang
Inverse effect is rescued, the modern times are usually used in the cardiovascular diseases such as myocardial ischemia, myocardial infarction and heart failure.Sini Tang is high because of the efficacy of a drug
Suddenly, the distinguished attention for being constantly subjected to ancient Chinese medicine doctor of curative effect, research find the aconite alkaloids in monkshood, the gingerol in rhizoma zingiberis
Class compound and flavones in total volatile oil, Radix Glycyrrhizae and saponins compound are the main chemical compositions in Sini Tang.However, four
Does is it which that inverse soup, which plays the real effective component of cardiac vascular activity,? interaction between these effective components and bioactivity is closed
How is system? these are the bases for further researching and developing safe and effective, quality controllable Sini Tang modern Chinese herbal medicine.
Chinese medicine has the characteristics that multicomponent, mostly by way of, multiple target point, and playing a role is that " system (drug system)-system is (raw
Object system) " binding mode.Excavating herbal medicine efficacy ingredient is the premise for disclosing Chinese medicine scientific meaning, instructing clinical rational drug use.
It is still that bioactive components study most common method that the system separation of biological effect guiding, which combines effect experiment,.But this
The kind method period is long, flux is low, and decomposes and ignore the essence of Chinese medicine " multicomponent-multiple target point " mass action.And Chinese medicine
Multicomponent " spectrum-effect " research is confined to grinding for " system-point " between " multicomponent spectrum " and Chinese medicine " single effect " index mostly
Study carefully, is still not able to reflection Chinese medicine and passes through multiple ingredients and the coefficient feature of multiple targets.In addition, due to Chinese medicine effect
Diversity, there are also problems with reasonability for the science for causing pharmacodynamics index to select.It is therefore desirable to establish and Chinese medicine " system-
The adaptable active ingredient group New Research Method of system " feature provides support for bioactive components research.
Metabolism group is emerging " group is learned " as one in systems biology, and core concept is Study of Exogenous substance pair
The group effect of organism metabolism network regulation, and using organism intracellular metabolite small molecule as object, drug is studied to body generation
Thank to the systemic effect of group.Since metabolism group is all genes of organism, the movable terminal of protein function, thus metabolism group is regarded
Small molecule marker group energy for " the biochemical phenotype " of organism allomeric function state, metabolism group discovery is enough instant, sensitive, true
Reflect the response and adjusting of organism metabolism network under disease or drug effect on the spot, it has also become the potential target spot of drug therapy,
It can be used as the Substitute Indexes of evaluating drug effect.Serum pharmaceutical chemistry is from separation is identified in the Contained Serum after oral prescription
The thinking and researching and designing of medicine effective substance make result of study embody the interior therapeutic state of traditional Chinese medicine ingredients, are embodied
The internal direct working substance of prescription response to treatment, theory and method meet Chinese medicine mass action mode.Medicine serum drug
Chemical research method provides methodology support for discovery bioactive components.Chinese medicine enters between blood component and herbal medicine efficacy
Relevance is certainly existed, can use the dynamic that correlation analysis will enter peak area (content) and drug action intensity of blood component spectrum
Variation connects, and real effective component in numerous compositions is determined by statistical analysis and by biological experiment.
Summary of the invention
Technical problem to be solved by the present invention lies in view of the above shortcomings of the prior art, providing, a kind of screening four is inverse
Soup resists myocardial ischemia the method for drug effect material base.This method is based on isoprel and causes myocardial ischemia in rats model, uses
The non-targeted metabonomic technology of ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry (UHPLC/Q-TOFMS) and based on super
High performance liquid chromatography-triple quadrupole bar mass spectrum (UHPL/QQQ-MS) targets metabonomic technology, filters out 12 kinds of Protective effects of Sini decoction hearts
Myocardial ischemia effective substance improves the accuracy of Protective effects of Sini decoction myocardial ischemia effective substance, shortens the screening period,
Reduce screening Expenses Cost.
In order to solve the above technical problems, the technical solution adopted by the present invention is that: a kind of screening Sini Tang myocardial ischemic antagonist
The method for imitating material base, which is characterized in that method includes the following steps:
Step 1: the determination of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia and the measurement of peak area
Step 101, the endogenous metabolism object for determining Protective effects of Sini decoction myocardial ischemia:
The preparation of step 1011, Sini Tang: 60g monkshood, 40 rhizoma zingiberis and 60g Radix Glycyrrhizae are weighed respectively, 1600mL is then added
Pure water decocts 2h after impregnating 1h, while hot with four layers of filtered through gauze, obtains first-time filtrate and a residue, adds into a residue
Enter 1200mL pure water and decoct 1h, obtain secondary filtrate and secondary residual with four layers of filtered through gauze while hot again, merges primary filter
Liquid and secondary filtrate, obtain the Sini Tang that concentration is 1g/mL after being concentrated under reduced pressure, i.e. every milliliter of Sini Tang is equivalent to monkshood
0.375g, rhizoma zingiberis 0.25g, Radix Glycyrrhizae 0.375g；
The acquisition and preparation of step 1012, sample: taking male SD rat to be divided into three groups, respectively blank group, model group and
Model administration group, wherein model administration group is oral gives Sini Tang, and Sini Tang dosage is 10mL solution/1000g rat body weight, empty
White group and model group are oral to give same volume physiological saline, the daily gastric infusion of each group 1 time, continuous 10d, in 9d and 10d
Prepared by myocardial infarction and ischemia model to model administration group and model group intraperitoneal injection isoprel, isoprel dosage is
85mg/1000g rat body weight, to the physiological saline of blank group intraperitoneal injection same volume, each group is injected 1 time daily, last time
When 2h and 4h after injection, the equal rat eye socket of each group takes blood, is then centrifuged 10min under conditions of 4 DEG C, 3000 × g, obtains
Supernatant is blood serum sample, and each group blood serum sample is saved at -70 DEG C；
Step 1013, sample analysis: will respectively take 100 μ L after each group blood serum sample freeze thawing obtained in step 1012, respectively
The methanol vortex 1min that 400 μ L contain 12.5 μ g/mL internal standard compound L-2- chlorine propyl benzene propylhomoserins is added, is then placed in ice-water bath and surpasses
Sound extracts 10min, then is centrifuged 15min under conditions of 4 DEG C, 14000 × g, obtained supernatant using ultra performance liquid chromatography/
Quadrupole time-of-flight mass spec-trometry is tested and analyzed, the data of each group blood serum sample are obtained, wherein superelevation liquid-phase chromatographic analysis
Condition are as follows: superelevation liquid chromatogram uses Agilent1290Infinity liquid chromatographic system, and chromatographic column filler uses ACQUITY
UPLC HSS T3C18, specification are 2.1mm × 100mm, and 1.7 μm, column temperature is 40 DEG C, and the A phase in mobile phase is that formic acid quality contains
The aqueous formic acid of amount 0.1%, B phase are the formic acid acetonitrile solution of formic acid mass content 0.1%, and flow velocity is 400 μ L/min, sample introduction
Amount is 4 μ L, wherein Mass Spectrometry Conditions are as follows: mass spectrum uses Agilent 6530Accurate-Mass Quadrupole Time-of-
Flight (Q-TOF) mass spectrograph, is detected using positive ion mode, and the capillary voltage in detection parameters is 3500V, dry gas stream
Speed is 11L/min, and dry temperature degree is 350 DEG C, spray pressure 45psig, and being crushed voltage is 120V, orifice potential 60V, number
It is 50~1000 according to acquisition range mass-to-charge ratio m/z；
Step 1014, Protective effects of Sini decoction myocardial ischemia endogenous metabolism object determination: first according to each group in step 1013
The data of blood serum sample compare the variation of the endogenous metabolism object of blank group and model group, using orthogonal Partial Least Squares point
It analyses and determines the endogenous metabolism object of model group anomalous variation, be then potential with the endogenous metabolism object of model group anomalous variation
Pharmacodynamics index, the endogenous metabolism object of the anomalous variation of contrast model group and model administration group analyzed using method of analysis of variance
The metabolin obviously reversed in the endogenous metabolism object of anomalous variation after Sini Tang intervention, it is determined that Protective effects of Sini decoction myocardial ischemia
Endogenous metabolism object；P < 0.05 in the results of analysis of variance of the metabolin obviously reversed；
Step 102, Protective effects of Sini decoction myocardial ischemia endogenous metabolism object peak area measurement:
Step 1021, using ultra-high performance liquid chromatography/triplquadrupole quadrupole mass spectrum, by multiple-reaction monitoring pattern to step
The characteristic ion of the endogenous metabolism object of the 13 kinds of Protective effects of Sini decoction myocardial ischemias determined in 1014 is scanned, and is composed accordingly
Figure；
Step 1022 passes through quantitative analysis software MassHunter Quantitative Analysis in step 1021
The obtained chromatographic peak area in corresponding spectrogram is integrated, and containing for the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia is obtained
Measure information；
Step 2: in Sini Tang body the determination of effective constituents and peak area measurement
The determination of effective constituents in step 201, Sini Tang body: according to the data of each group blood serum sample in step 1013, divide
It does not establish between the orthogonal Partial Least Squares discriminant analysis model between blank group and model group and blank group and model administration group
Orthogonal Partial Least Squares discriminant analysis model, then with the orthogonal Partial Least Squares discriminant analysis between blank group and model group
Model Weight load factor is abscissa, with blank group Partial Least Squares discriminant analysis model power orthogonal with model administration group
Repeated factor is ordinate, shares unique texture figure using Simca-p statistical software (version 14.1) building SUS-plot,
It screens Sini Tang and the intracorporal effective constituents of rat is administered in model, determine that the variable fallen in axis of ordinates positive direction is Sini Tang
The intracorporal effective constituents of rat are administered in model；
The measurement of the peak area of effective constituents in step 202, Sini Tang body:
Step 2021, using ultra-high performance liquid chromatography/triplquadrupole quadrupole mass spectrum, by multiple-reaction monitoring pattern to step
The characteristic ion of effective constituents is scanned in the Sini Tang body determined in 201, obtains corresponding spectrogram；
Step 2022 passes through quantitative analysis software MassHunter Quantitative Analysis in step 2021
The obtained chromatographic peak area in corresponding spectrogram is integrated, and the content information of effective constituents in Sini Tang body is obtained；
Step 3: the peak of the peak area of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia and effective constituents in Sini Tang body
The correlation analysis of area: by the content information of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia obtained in step 1022 with
The content information of effective constituents carries out Pearson correlation analysis in Sini Tang body obtained in step 2022, with correlation coefficient r table
Show effective constituents in Sini Tang body to the effect degree of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia, wherein | r | >=0.6 table
Show significant correlation, to filter out Protective effects of Sini decoction myocardial ischemia effective substance.
Compared with the prior art, the present invention has the following advantages:
1, the present invention is based on isoprels to cause myocardial ischemia in rats model, screens Sini Tang using metabolism group method
In resist myocardial ischemia drug effect substance, first using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry to each group blood serum sample
It is tested and analyzed, passes through the variation of the endogenous metabolism object of comparison blank group and model group, model group and model administration group
The endogenous metabolism object of anomalous variation determines the endogenous metabolism object of 13 kinds of Protective effects of Sini decoction myocardial ischemias, using ultra high efficiency liquid phase
Chromatography/triple quadrupole bar mass spectrum determines the spectrogram of endogenous metabolism object characteristic ion by multiple-reaction monitoring pattern and obtains peak
Area, then according to the analysis of each group blood serum sample as a result, determine model administration group rat body in 19 kinds of Sini Tang effective constituents,
Using ultra-high performance liquid chromatography/triplquadrupole quadrupole mass spectrum, the spectrogram of effective constituents is determined by multiple-reaction monitoring pattern and is obtained
Peak area, then the peak area to effective constituents in the peak area and Sini Tang body of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia
Correlation analyzed, to filter out 12 kinds of Protective effects of Sini decoction myocardial ischemia effective substances, be respectively as follows: isotalatisidine,
Songorine, Fuziline, neoline, Talatisamine, 14- acetyl Talatisamine, glycyrrhizin, Benzoylmesaconitine,
Isoliquiritin, benzoyl aconine, benzoyl Hypaconitine, 6-gingerol improve Protective effects of Sini decoction myocardial ischemia effective substance
The accuracy on basis shortens the screening period, reduces screening Expenses Cost.
2, the present invention analyzes each group blood serum sample using ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry
Detection finally determines effective constituents in the endogenous metabolism object and Sini Tang body of Protective effects of Sini decoction myocardial ischemia, due to ultra high efficiency liquid
Phase chromatography-quadrupole time-of-flight mass spec-trometry chromatogram separating capacity with higher and mass spectrum accuracy, to improve each group blood
In final proof product in endogenous metabolism object and Sini Tang body effective constituents detection accuracy, be conducive to endogenous metabolism object and Sini Tang
The Structural Identification of internal effective constituents, and expand the detection range of effective constituents in endogenous metabolism object and Sini Tang body.
3, the present invention is using ultra performance liquid chromatography-triple quadrupole bar mass spectrum to the endogenous generation of Protective effects of Sini decoction myocardial ischemia
It thanks to effective constituents in object and Sini Tang body and carries out quantitative analysis, obtain the content information and dynamic law of the two, simplify quantitative
The process of analysis improves the accuracy and repeatability of data analysis result, is conducive to subsequent Protective effects of Sini decoction myocardial ischemia
Endogenous metabolism object peak area and effective constituents in Sini Tang body peak area correlation analysis, finishing screen select with it is interior
The exogenous traditional Chinese medicine ingredients of source property metabolin highlights correlations are substantially increased as Protective effects of Sini decoction myocardial ischemia effective substance
The accuracy of the selection result.
4, the present invention provides a kind of Chinese medicines to treat " system-system " new method that myocardial ischemia effective component screens, by
Extremely complex in traditional Chinese medicine ingredients, most mechanism of action are unclear, one or several single pharmacodynamics indexs are difficult to embody Chinese medicine " multicomponent
The action characteristic of " system-system " between system " and body " biosystem ", to be difficult to determine effective component in Chinese medicine, this hair
Bright Innovation Integration is based on the non-targeted metabonomic technology of ultra performance liquid chromatography-quadrupole time-of-flight mass spec-trometry and based on super
High performance liquid chromatography-triple quadrupole bar mass spectrum targets metabonomic technology, the determining and quantitative endogenous metabolism object with curative effect phase
Group, and in this, as drug effect Substitute Indexes, carry out effective constituents group and the endogenous metabolism object faciation of curative effect phase in Chinese medicine body and closes
Journal of Sex Research discloses bioactive components, provides effective means for the screening of bioactive components.
Technical solution of the present invention is described in further detail below by embodiment.
Detailed description of the invention
Fig. 1 is the base peak chromatograms that the Sini Tang of the embodiment of the present invention 1 is analyzed through UHPLC-Q-TOFMS.
Fig. 2 a is the SUS-plot for the blood serum sample that 2h is acquired after 1 model administration group last time of the embodiment of the present invention is injected
Figure.
Fig. 2 b is the SUS-plot for the blood serum sample that 4h is acquired after 1 model administration group last time of the embodiment of the present invention is injected
Figure.
Fig. 3 is that 1 model administration group blood serum sample of the embodiment of the present invention is more through ultra-high performance liquid chromatography/triplquadrupole quadrupole mass spectrum
The characteristic ion spectrogram of effective constituents in 19 kinds of Sini Tang bodies that reaction monitoring obtains.
Fig. 4 is the Sini Tang in the blood serum sample that 2h is acquired after 1 model administration group last time of the embodiment of the present invention is injected
The related coefficient figure of effective constituents in the endogenous metabolism object and Sini Tang body to resist myocardial ischemia.
Fig. 5 is the Sini Tang in the blood serum sample that 4h is acquired after 1 model administration group last time of the embodiment of the present invention is injected
The related coefficient figure of effective constituents in the endogenous metabolism object and Sini Tang body to resist myocardial ischemia.
Fig. 6 is 8 kinds of ingredients in the Sini Tang body of the embodiment of the present invention 1 in effective constituents to anoxia-induced apoptosis cell viability
Influence diagram.
Specific embodiment
Embodiment 1
The present embodiment screening Protective effects of Sini decoction myocardial ischemia effective substance method the following steps are included:
Step 1: the determination of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia and the measurement of peak area
Step 101, the endogenous metabolism object for determining Protective effects of Sini decoction myocardial ischemia:
The preparation of step 1011, Sini Tang: 60g monkshood, 40 rhizoma zingiberis and 60g Radix Glycyrrhizae are weighed respectively, 1600mL is then added
Pure water decocts 2h after impregnating 1h, while hot with four layers of filtered through gauze, obtains first-time filtrate and a residue, adds into a residue
Enter 1200mL pure water and decoct 1h, obtain secondary filtrate and secondary residual with four layers of filtered through gauze while hot again, merges primary filter
Liquid and secondary filtrate, obtain the Sini Tang that concentration is 1g/mL after being concentrated under reduced pressure, i.e. every milliliter of Sini Tang is equivalent to monkshood
0.375g, rhizoma zingiberis 0.25g, Radix Glycyrrhizae 0.375g；
Fig. 1 is the base peak chromatograms that the Sini Tang of the present embodiment is analyzed through UHPLC-Q-TOFMS, the number in Fig. 1
Label respectively represents: the attached spirit in the river 1-, 2- karacoline, the new aconine of 3-, 4- isotalatisidine, 5- songorine, 6-
Fuziline, 7- neoline, 8- Talatisamine, 9- Cha Simanlin, 10-14- acetyl Talatisamine, 11- glycyrrhizin, 12- benzoyl
Middle aconine, 13- isoliquiritin, 14- benzoyl aconine, 15- benzoyl Hypaconitine, 16- benzoyl deoxygenate the rhizome of Chinese monkshood
Alkali, 17-6- gingerol, 18-6- salad oil, 19- enoxolone；
The acquisition and preparation of step 1012, sample: 35 male SD rats are taken to be divided into three groups, respectively blank group (10
Only), model group (10) and model administration group (15), wherein model administration group is oral gives Sini Tang, and Sini Tang dosage is
10mL solution/1000g rat body weight, blank group and model group are oral to give same volume physiological saline, the daily gastric infusion 1 of each group
Secondary, continuous 10d prepares myocardial ischemia to model administration group and model group intraperitoneal injection isoprel in 9d and 10d
Model, isoprel dosage are 85mg/1000g rat body weight, to the physiological saline of blank group intraperitoneal injection same volume, respectively
The daily injection of group 1 time, last time inject after 2h and 4h when, the equal rat eye socket of each group takes blood, then in 4 DEG C, 3000 × g
Under the conditions of be centrifuged 10min, obtained supernatant be blood serum sample, each group blood serum sample is saved at -70 DEG C；
Step 1013, sample analysis: will respectively take 100 μ L after each group blood serum sample freeze thawing obtained in step 1012, respectively
The methanol vortex 1min that 400 μ L contain 12.5 μ g/mL internal standard compound L-2- chlorine propyl benzene propylhomoserins is added, is then placed in ice-water bath and surpasses
Sound extracts 10min, then is centrifuged 15min under conditions of 4 DEG C, 14000 × g, obtained supernatant using ultra performance liquid chromatography/
Quadrupole time-of-flight mass spec-trometry is tested and analyzed, the data of each group blood serum sample are obtained, wherein superelevation liquid-phase chromatographic analysis
Condition are as follows: superelevation liquid chromatogram uses Agilent1290Infinity liquid chromatographic system, and chromatographic column filler uses ACQUITY
UPLC HSS T3C18, specification are 2.1mm × 100mm, and 1.7 μm, column temperature is 40 DEG C, and the A phase in mobile phase is that formic acid quality contains
The aqueous formic acid of amount 0.1%, B phase are the formic acid acetonitrile solution of formic acid mass content 0.1%, and flow velocity is 400 μ L/min, sample introduction
Amount is 4 μ L, wherein Mass Spectrometry Conditions are as follows: mass spectrum uses Agilent 6530Accurate-Mass Quadrupole Time-of-
Flight (Q-TOF) mass spectrograph, is detected using positive ion mode, and the capillary voltage in detection parameters is 3500V, dry gas stream
Speed is 11L/min, and dry temperature degree is 350 DEG C, spray pressure 45psig, and being crushed voltage is 120V, orifice potential 60V, number
It is 50~1000 according to acquisition range mass-to-charge ratio m/z；
Step 1014, Protective effects of Sini decoction myocardial ischemia endogenous metabolism object determination: according to each group serum in step 1013
The data of sample compare the variation of the endogenous metabolism object of blank group and model group, then using orthogonal Partial Least Squares point
The endogenous metabolism object for analysing and determining 21 kinds of model group anomalous variations is respectively as follows: valine, hydroxybutyric acid fatty acyl carnitine, junket ammonia
Acid, isoleucine, heteroauxin, tryptophan, hippuric acid, toluric acid, 3- indolepopionic acid, sphingosine 1-phosphate, haemolysis phosphorus
Phosphatidylcholine (20:5), lysophosphatidyl choline (15:0), lysophosphatidyl choline (18:2), tetradecane fatty acyl carnitine, haemolysis
Phosphatidyl-ethanolamine (20:1 (11Z)/0:0), lysophosphatidyl choline (22:5), linseed oil fatty acyl carnitine, hemolytic phosphatidyl gallbladder
Alkali (20:2), palmitoyl carnitine, octadecene acylcarnitine, stearoyl carnitine, then with the endogenous generation of model group anomalous variation
Thanking to object is potential pharmacodynamics index, and the endogenous metabolism object of contrast model group and 21 kinds of anomalous variations of model administration group uses
The substance obviously reversed in the endogenous metabolism object of anomalous variation after method of analysis of variance analysis Sini Tang intervention, it is determined that 13 kind four
The endogenous metabolism object that inverse soup resists myocardial ischemia is respectively as follows: valine, isoleucine, heteroauxin, tryptophan, 3- indoles third
Acid, sphingosine 1-phosphate, lysophosphatidyl choline (20:5), tetradecane fatty acyl carnitine, lysophosphatidyl choline (22:5), flax
Oleoyl carnitine, lysophosphatidyl choline (20:2), palmitoyl carnitine, stearoyl carnitine；The metabolin obviously reversed
P < 0.05 in the results of analysis of variance；
Step 102, Protective effects of Sini decoction myocardial ischemia endogenous metabolism object peak area measurement:
Step 1021, using ultra-high performance liquid chromatography/triplquadrupole quadrupole mass spectrum, by multiple-reaction monitoring pattern to step
The characteristic ion of the endogenous metabolism object of the 13 kinds of Protective effects of Sini decoction myocardial ischemias determined in 1014 is scanned, and is composed accordingly
Figure；The multiple-reaction monitoring pattern of the endogenous metabolism object of 13 kinds of Protective effects of Sini decoction myocardial ischemias is respectively as follows: valine m/z
118.0 → 72.0, isoleucine m/z 132.1 → 86.0, heteroauxin m/z 176.0 → 130.0, tryptophan m/z 205.1
→ 118.1,3- indolepopionic acid m/z 190.1 → 130.0, sphingosine 1-phosphate m/z 380.2 → 264.2, hemolytic phosphatidyl gallbladder
Alkali (20:5) m/z 542.3 → 104.1, tetradecane fatty acyl carnitine m/z 372.3 → 85.0, lysophosphatidyl choline (22:5) m/
Z 570.3 → 184.1, linseed oil fatty acyl carnitine m/z 424.3 → 85.0, lysophosphatidyl choline (20:2) m/z 548.3 →
104.1, palmitoyl carnitine m/z 400.3 → 85.0, stearoyl carnitine m/z 428.3 → 85.0；
Step 1022 passes through quantitative analysis software MassHunter Quantitative Analysis in step 1021
The obtained chromatographic peak area in corresponding spectrogram is integrated, and the endogenous metabolism object of 13 kinds of Protective effects of Sini decoction myocardial ischemias is obtained
Content information；
Step 2: in Sini Tang body the determination of effective constituents and peak area measurement
The determination of effective constituents in step 201, Sini Tang body: according to the data of each group blood serum sample in step 1013, divide
It does not establish between the orthogonal Partial Least Squares discriminant analysis model between blank group and model group and blank group and model administration group
Orthogonal Partial Least Squares discriminant analysis model, then with the orthogonal Partial Least Squares discriminant analysis between blank group and model group
Model Weight load factor is abscissa, with blank group Partial Least Squares discriminant analysis model power orthogonal with model administration group
Repeated factor is ordinate, shares unique texture figure using Simca-p statistical software (version 14.1) building SUS-plot,
It screens Sini Tang and the intracorporal effective constituents of rat is administered in model, determine that the variable fallen in axis of ordinates positive direction is Sini Tang
The intracorporal effective constituents of rat are administered in model；
Fig. 2 a, Fig. 2 b are the blood serum sample of 2h and 4h acquisition after the present embodiment model administration group last time is injected respectively
SUS-plot figure, wherein the variation in the small frame in SUS-plot figure axis of ordinates positive direction is Sini Tang in model administration rat
Intracorporal effective constituents determine 19 kinds of Sini Tangs in model administration rat body from the figure software initial data of building of Fig. 2 a and Fig. 2 b
Effective constituents, be respectively as follows: the attached spirit in river, karacoline, new aconine, isotalatisidine, songorine, Fuziline,
Neoline, Talatisamine, Cha Simanlin, 14- acetyl Talatisamine, glycyrrhizin, Benzoylmesaconitine, isoliquiritin, benzene
Formyl aconine, benzoyl Hypaconitine, benzoyl deoxyaconitine, 6-gingerol, (E)-1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one, enoxolone；
The measurement of the peak area of effective constituents in step 202, Sini Tang body:
Step 2021, using ultra-high performance liquid chromatography/triplquadrupole quadrupole mass spectrum, by multiple-reaction monitoring pattern to step
The characteristic ion of effective constituents is scanned in the 19 kinds of Sini Tang bodies determined in 201, obtains spectrogram as shown in Figure 3；It is described
The multiple-reaction monitoring pattern of effective constituents is respectively as follows: the attached spirit m/z 394.3 → 376.1 in river, the more rhizome of Chinese monkshoods in 19 kinds of Sini Tang bodies
Alkali m/z 378.2 → 360.1, new aconine m/z 486.2 → 436.1, isotalatisidine m/z 408.3 → 390.1, quasi- Ge Er
Aconitine m/z 358.2 → 342.1, Fuziline m/z 454.3 → 436.2, neoline m/z 438.2 → 370.3, Talatisamine
M/z 422.3 → 390.1, Cha Simanlin m/z452.3 → 420.1,14- acetyl Talatisamine m/z 464.3 → 414.1, Radix Glycyrrhizae
Plain m/z 257.1 → 137.3, Benzoylmesaconitine m/z 590.3 → 540.2, isoliquiritin m/z 419.1 → 257.1,
Benzoyl aconine m/z 604.3 → 554.2, benzoyl Hypaconitine m/z574.3 → 542.2, benzoyl deoxyaconitine
M/z 588.3 → 556.2,6-gingerol m/z 295.2 → 177.3, (E)-1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one m/z 277.2 → 137.3, enoxolone m/z
471.3→453.2；
Step 2022 passes through quantitative analysis software MassHunter Quantitative Analysis in step 2021
The obtained chromatographic peak area in corresponding spectrogram is integrated, and the content information of effective constituents in Sini Tang body is obtained；
Step 3: the peak of the peak area of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia and effective constituents in Sini Tang body
The correlation analysis of area: by the content information of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia obtained in step 1022 with
The content information of effective constituents carries out Pearson correlation analysis in Sini Tang body obtained in step 2022, with correlation coefficient r table
Show effective constituents in Sini Tang body to the effect degree of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia, wherein | r | >=0.6 table
Show significant correlation, to filter out Protective effects of Sini decoction myocardial ischemia effective substance.
Fig. 4 and Fig. 5 is in the blood serum sample that 2h and 4h is acquired after the present embodiment model administration group last time is injected respectively
Protective effects of Sini decoction myocardial ischemia endogenous metabolism object and effective constituents in Sini Tang body related coefficient figure, whereinTable
Show high-positive correlation (r >=0.6),It indicates high negative correlation (r≤- 0.6), expression low correlation (| r | < 0.6), from Fig. 4
With Fig. 5 as can be seen that 12 kinds of effective constituents in 19 kinds of Sini Tang bodies in effective constituents and Protective effects of Sini decoction myocardial ischemia it is endogenous
Property the significant correlation of metabolin, i.e. 12 kinds of effective constituents are Protective effects of Sini decoction myocardial ischemia effective substances, are respectively as follows: different tower and draw
The rhizome of Chinese monkshood is former in fixed, songorine, Fuziline, neoline, Talatisamine, 14- acetyl Talatisamine, glycyrrhizin, benzoyl
Alkali, isoliquiritin, benzoyl aconine, benzoyl Hypaconitine, 6-gingerol.
Isotalatisidine, songorine, Fuziline, neoline, Talatisamine, 14- acetyl Thalassa in Sini Tang
Quick, glycyrrhizin, Benzoylmesaconitine, isoliquiritin, benzoyl aconine, benzoyl Hypaconitine, 6-gingerol etc. 12
Kind of effective constituents are the ingredient of Protective effects of Sini decoction myocardial ischemia drug effect, select isotalatisidine therein, Fuziline, Talatisamine, sweet
8 kinds of ingredients such as careless element, Benzoylmesaconitine, isoliquiritin, benzoyl aconine, 6-gingerol, it is former using rat suckling mouse
It is divided into normal group, model group (H/R) and effect group for Myocytes Anoxia/reoxygenation (H/R) damage model, and by cardiac muscle cell
(above-mentioned 8 kinds of ingredients is taken to act on respectively) it is multiple to Myocytes Anoxia to verify each ingredient using cardiomyocyte viability as evaluation index
The protective effect of oxygen injury, as a result as shown in Figure 6.
Fig. 6 is influence of 8 kinds of ingredients to anoxia-induced apoptosis cell viability in the Sini Tang body of the present embodiment in effective constituents
Figure, from fig. 6, it can be seen that model group (H/R) p < 0.01 compared with normal group, effect group p < 0.05 compared with model group (H/R),
Illustrate isotalatisidine, Fuziline, Talatisamine, glycyrrhizin, Benzoylmesaconitine, isoliquiritin, benzoyl aconine,
6-gingerol can obviously increase the vigor of cardiac muscle cell.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way.It is all according to invention skill
Art any simple modification, change and equivalence change substantially to the above embodiments, still fall within technical solution of the present invention
Protection scope in.
Claims (1)
1. a kind of method for screening Protective effects of Sini decoction myocardial ischemia effective substance, which is characterized in that this method includes following step
It is rapid:
Step 1: the determination of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia and the measurement of peak area
Step 101, the endogenous metabolism object for determining Protective effects of Sini decoction myocardial ischemia:
The preparation of step 1011, Sini Tang: weighing 60g monkshood, 40 rhizoma zingiberis and 60g Radix Glycyrrhizae respectively, and it is pure that 1600mL is then added
Water decocts 2h after impregnating 1h, while hot with four layers of filtered through gauze, obtains first-time filtrate and a residue, is added into a residue
1200mL pure water decocts 1h, obtains secondary filtrate and secondary residual with four layers of filtered through gauze while hot again, merges first-time filtrate
With secondary filtrate, the Sini Tang that concentration is 1g/mL is obtained after being concentrated under reduced pressure, i.e. every milliliter of Sini Tang is equivalent to monkshood
0.375g, rhizoma zingiberis 0.25g, Radix Glycyrrhizae 0.375g；
The acquisition and preparation of step 1012, sample: male SD rat is taken to be divided into three groups, respectively blank group, model group and model
Administration group, wherein model administration group is oral gives Sini Tang, and Sini Tang dosage is 10mL solution/1000g rat body weight, blank group
It is taken orally with model group and gives same volume physiological saline, the daily gastric infusion of each group 1 time, continuous 10d, in 9d and 10d to mould
Type administration group and model group intraperitoneal injection isoprel prepare myocardial infarction and ischemia model, and isoprel dosage is 85mg/
1000g rat body weight, to the physiological saline of blank group intraperitoneal injection same volume, each group is injected 1 time daily, after last time is injected
2h and 4h when, the equal rat eye socket of each group takes blood, is then centrifuged 10min under conditions of 4 DEG C, 3000 × g, obtained supernatant
For blood serum sample, each group blood serum sample is saved at -70 DEG C；
Step 1013, sample analysis: 100 μ L will be respectively taken after each group blood serum sample freeze thawing obtained in step 1012, be separately added into
400 μ L contain the methanol vortex 1min of 12.5 μ g/mL internal standard compound L-2- chlorine propyl benzene propylhomoserins, are then placed in ultrasound in ice-water bath and mention
10min is taken, then is centrifuged 15min under conditions of 4 DEG C, 14000 × g, obtained supernatant uses ultra performance liquid chromatography/quadrupole
Bar-flight time mass spectrum is tested and analyzed, and the data of each group blood serum sample are obtained, wherein the condition of superelevation liquid-phase chromatographic analysis
Are as follows: superelevation liquid chromatogram uses Agilent1290 Infinity liquid chromatographic system, and chromatographic column filler uses ACQUITY
UPLC HSS T3 C18, specification are 2.1mm × 100mm, and 1.7 μm, column temperature is 40 DEG C, and the A phase in mobile phase is that formic acid quality contains
The aqueous formic acid of amount 0.1%, B phase are the formic acid acetonitrile solution of formic acid mass content 0.1%, and flow velocity is 400 μ L/min, sample introduction
Amount is 4 μ L, wherein Mass Spectrometry Conditions are as follows: mass spectrum uses 6530 Accurate-Mass Quadrupole Time-of- of Agilent
Flight (Q-TOF) mass spectrograph, is detected using positive ion mode, and the capillary voltage in detection parameters is 3500V, dry gas stream
Speed is 11L/min, and dry temperature degree is 350 DEG C, spray pressure 45psig, and being crushed voltage is 120V, orifice potential 60V, number
It is 50~1000 according to acquisition range mass-to-charge ratio m/z；
Step 1014, Protective effects of Sini decoction myocardial ischemia endogenous metabolism object determination: first according to each group serum in step 1013
The data of sample compare the variation of the endogenous metabolism object of blank group and model group, simultaneously using orthogonal Partial Least Squares Method
The endogenous metabolism object of model group anomalous variation is determined, then with the endogenous metabolism object of model group anomalous variation for potential medicine
Index is imitated, the endogenous metabolism object of contrast model group and the anomalous variation of model administration group is inverse using method of analysis of variance analysis four
The metabolin obviously reversed in the endogenous metabolism object of anomalous variation after soup intervention, it is determined that Protective effects of Sini decoction myocardial ischemia it is endogenous
Property metabolin；P < 0.05 in the results of analysis of variance of the metabolin obviously reversed；
Step 102, Protective effects of Sini decoction myocardial ischemia endogenous metabolism object peak area measurement:
Step 1021, using ultra-high performance liquid chromatography/triplquadrupole quadrupole mass spectrum, by multiple-reaction monitoring pattern in step 1014
The characteristic ion of the endogenous metabolism object of 13 kinds of determining Protective effects of Sini decoction myocardial ischemias is scanned, and obtains corresponding spectrogram；
Step 1022 is obtained by quantitative analysis software MassHunter Quantitative Analysis in step 1021
Corresponding spectrogram in chromatographic peak area integrated, obtain the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia content letter
Breath；
Step 2: in Sini Tang body the determination of effective constituents and peak area measurement
The determination of effective constituents in step 201, Sini Tang body: it according to the data of each group blood serum sample in step 1013, builds respectively
It is orthogonal between vertical blank group Partial Least Squares discriminant analysis model orthogonal between model group and blank group and model administration group
Partial Least Squares discriminant analysis model, then with the orthogonal Partial Least Squares discriminant analysis model between blank group and model group
Weight load factor be abscissa, with blank group Partial Least Squares discriminant analysis Model Weight orthogonal with model administration group because
Son is ordinate, using the i.e. shared unique texture figure of Simca-p statistical software (version 14.1) building SUS-plot, screening
The intracorporal effective constituents of rat are administered in model in Sini Tang, determine that the variable fallen in axis of ordinates positive direction is Sini Tang in mould
The intracorporal effective constituents of rat are administered in type；
The measurement of the peak area of effective constituents in step 202, Sini Tang body:
Step 2021, using ultra-high performance liquid chromatography/triplquadrupole quadrupole mass spectrum, by multiple-reaction monitoring pattern in step 201
The characteristic ion of effective constituents is scanned in determining Sini Tang body, obtains corresponding spectrogram；
Step 2022 is obtained by quantitative analysis software MassHunter Quantitative Analysis in step 2021
Corresponding spectrogram in chromatographic peak area integrated, obtain the content information of effective constituents in Sini Tang body；
Step 3: the peak area of the peak area of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia and effective constituents in Sini Tang body
Correlation analysis: by the content information and step of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia obtained in step 1022
The content information of effective constituents carries out Pearson correlation analysis in Sini Tang body obtained in 2022, indicates four with correlation coefficient r
Effective constituents are to the effect degree of the endogenous metabolism object of Protective effects of Sini decoction myocardial ischemia in inverse soup body, wherein | r | >=0.6 indicates aobvious
It is related, to filter out Protective effects of Sini decoction myocardial ischemia effective substance.
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