CN106596432A - Method for determining activity contribution degrees of compounds in traditional Chinese medicine for promoting blood circulation to remove blood stasis - Google Patents
Method for determining activity contribution degrees of compounds in traditional Chinese medicine for promoting blood circulation to remove blood stasis Download PDFInfo
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Abstract
The embodiment of the invention discloses a method for determining the activity contribution degrees of compounds in a traditional Chinese medicine for promoting blood circulation to remove blood stasis. The method comprises the following steps: (1) obtaining the traditional Chinese medicine for promoting blood circulation to remove blood stasis, and determining at least two compounds contained in the medicine; (2) determining an antioxidant activity parameter a of each compound in the step (1); (3) determining an anti-platelet aggregation parameter b of each compound in the step (1); (4) determining a content parameter c of each compound in the traditional Chinese medicine for promoting blood circulation to remove blood stasis, which is obtained in the step (1); (5) determining a high-temperature stability parameter d corresponding to each compound; (6) determining a high-light stability parameter e corresponding to each compound (6); and (7) taking the determined values a, b, c, d and e corresponding to each compound as five-element network parameters of each compound to determine a five-element network regression area A of each compound.
Description
Technical field
The present invention relates to the activity of compound contributes journey in Chinese medicine quality control field, more particularly to blood-activating stasis-removing kind Chinese medicine
The determination method of degree.
Background technology
Traditional Chinese medical theory thinks that blood-activating stasis-removing kind Chinese medicine has functions that smooth blood, dissipation blood stasis, promoting blood circulation to remove obstruction in the collateral；
Modern pharmacological research shows, blood-activating stasis-removing kind Chinese medicine have very strong antioxidation, antiplatelet aggregation, blood vessel endothelium protection and
Improve the effect such as lectin from hemolymph.Wherein, antioxidation and antiplatelet aggregation are that blood-activating stasis-removing kind Chinese medicine can play disease treatment work
One of important mechanisms.
The antioxidation of blood-activating stasis-removing kind Chinese medicine and the size of antiplatelet aggregation ability typically by its antioxidant activity and
Platelet aggregation inhibitory activity is embodying.With the development of the Modern Analytical Instruments such as high performance liquid chromatography, by the analysis for modernizing
Instrument determines that the classes of compounds and content included in Chinese medicine has not been problem, to chemical combination in each blood-activating stasis-removing kind Chinese medicine
The report of species and content is also to emerge in an endless stream.For example, for DANHONG ZHUSHEYE, it has been disclosed and has contained compound pellet
Ginseng element, L-phenylalanine, uridnine, cytidine, protocatechualdehyde, protocatechuic acid, rosmarinic acid, salvianolic acid B, salvianolic acid A, salvianolic acid C,
Caffeic acid, alkannic acid, adenosine, syringoside and 5 hydroxymethyl furfural etc..
At present, to the research of blood-activating stasis-removing kind Antioxidant Capacity of Chinese Herb and platelet aggregation inhibitory activity via whole with Chinese medicine
Body develops into contained compound with Chinese medicine as object of study as object of study, by antioxidation and antiplatelet aggregation
Experiment screening goes out the compound with antioxidant activity and platelet aggregation inhibitory activity, and with the antioxidant activity of this compound
And/or the height of platelet aggregation inhibitory activity is determining its size to blood-activating stasis-removing kind active Chinese drug component percentage contribution.
However, in the practical clinical of blood-activating stasis-removing kind Chinese medicine, compound is whole to blood-activating stasis-removing kind Chinese medicine in Chinese medicine
The comprehensive contribution degree of body not only has with the antioxidant activity of the compound itself and/or the height of platelet aggregation inhibitory activity
Close, the content, storage stability (including high light stability and high-temperature stability) also with it in Chinese medicine is relevant, if in Chinese medicine
The antioxidant activity and/or platelet aggregation inhibitory activity of certain compound itself is very high, but its storage stability is very poor, it is likely that
The compound has just been degraded in its storing process, so as in practical clinical, the compound has been out antioxidant activity
And/or platelet aggregation inhibitory activity, then the actual active percentage contribution to Chinese medicine of the compound is very low.In the same manner, if
Content of the content of the compound much smaller than other compounds, then in practical clinical, its activity contribution to Chinese medicine
Degree is likely to the active percentage contribution less than the compound that other activity are lower slightly but content is very high in Chinese medicine to Chinese medicine.So
It is only right to determine its according to the antioxidant activity of compound in blood-activating stasis-removing kind Chinese medicine and/or the height of platelet aggregation inhibitory activity
The active percentage contribution of Chinese medicine is irrational.
The content of the invention
In order to solve only with the antioxidant activity and/or antiplatelet aggregation of the compound in blood-activating stasis-removing kind Chinese medicine
The height of activity determining the irrationality of its active percentage contribution to Chinese medicine, the invention provides a kind of blood circulation promoting and blood stasis dispelling apoplexy due to endogenous wind
The determination method of the active percentage contribution of compound in medicine.Technical scheme is as follows：
The determination method of the active percentage contribution of compound in blood-activating stasis-removing kind Chinese medicine, including：
(1) blood-activating stasis-removing kind Chinese medicine is obtained, and determines at least two compounds for wherein containing；
(2) antioxidant activity parameter a of each compound in step (1) is determined；Wherein,
Antioxidant activity parameter a of each compound is determined by following methods：
The free radical scavenging activity for determining the compound is tested using free radical scavenging activity, if the compound is specified first
Free radical scavenging activity under concentration is more than or equal to 50%, it is determined that the IC corresponding to the compound free radical scavenging activity50Value, and will
The IC50Data normalization process is carried out divided by maximum therein after value is inverted, the antioxidant activity ginseng of the compound is obtained
Number a；If free radical scavenging activity of the compound under the first prescribed concentration is less than 50%, the antioxygen of the compound is directly determined
It is zero to change reactivity parameter a；
(3) antiplatelet aggregation parameter b of each compound in step (1) is determined；Wherein,
Antiplatelet aggregation parameter b of each compound is determined by following methods：
The platelet aggregation inhibition rate for determining the compound is tested using platelet aggregation inhibitory activity, if the compound is the
Platelet aggregation inhibition rate under two prescribed concentrations is more than or equal to 50%, it is determined that the compound platelet aggregation inhibition rate correspondence
IC50Value, and by the IC50Data normalization process is carried out divided by maximum therein after value is inverted, the compound is obtained
Antiplatelet aggregation parameter b；If platelet aggregation inhibition rate of the compound under the second prescribed concentration is less than 50%, directly
Determine that compounds for resisting platelet aggregation parameter b is zero；
(4) content of each compound in the blood-activating stasis-removing kind Chinese medicine obtained in step (1) is determined, and by each compound
Content carries out data normalization process divided by maximum therein, obtains content parameter c of each compound；
(5) the blood-activating stasis-removing kind Chinese medicine obtained in step (1) is carried out into high-temperature process, the high-temperature process is at 60 DEG C
Under the conditions of avoid light place Preset Time, after high-temperature process, determine each compound in blood circulation promoting and blood stasis dispelling apoplexy due to endogenous wind using the method for step (4)
Content in medicine；And corresponding high-temperature stability parameter d of each compound is determined according to formula d=1- ︱ CR-100% ︱, wherein, CR
Represent the ratio of content of the same compound after high-temperature process and the content before high-temperature process；
(6) the blood-activating stasis-removing kind Chinese medicine obtained in step (1) is carried out into strong illumination process, the strong illumination is processed
It is that Preset Time is irradiated under conditions of intensity of illumination is 4500lx ± 500lx, using the side of step (4) after strong illumination process
Method determines content of each compound in blood-activating stasis-removing kind Chinese medicine；And each compound is determined according to the ︱ of formula e=1- ︱ CR ' -100%
Corresponding high light stability parameter e, wherein, CR ' is at content and strong illumination of the same compound after strong illumination process
The ratio of the content before reason；
(7) the corresponding a of each compound using determined by, b, c, d, e-value as each compound five metanetwork parameters, according to
Formula
Five metanetworks for determining each compound return area A, and return the size of area A come really according to five metanetwork
The five metanetworks recurrence area A of fixed active percentage contribution of each compound in blood-activating stasis-removing kind Chinese medicine, i.e. compound is bigger, its
Active percentage contribution in blood-activating stasis-removing kind Chinese medicine is bigger.
In this application, described Chinese medicine may refer to Chinese medicine compound, it is also possible to refer to single medicinal material, need further exist for
Bright, the concrete dosage form of Chinese medicine has no effect on the enforcement of technical solution of the present invention, and specifically, the dosage form of Chinese medicine can
To include：Tablet, capsule, soft capsule, pill, granule, injection etc..And due to the chemical composition of Chinese medicine it is more multiple
It is miscellaneous, multiple compounds can be included.How in Chinese medicine compound is determined in step (1), and here of the present invention is not defined, both can be with
Determined by the compound contained in the Chinese medicine disclosed by the data of document of the prior art or other forms, it is also possible to existing
The compound contained in having the means such as extraction usual in technology, chromatographic isolation to determine Chinese medicine.
In step (2), 50% whether is more than come really by free radical scavenging activity of the compound under the first prescribed concentration
The antioxidant activity of the fixed compound is strong and weak, and the first prescribed concentration described in step (2) is to be used for the solution that compound is prepared
After the experimental group of test free radical scavenging activity, concentration of the compound in experimental group reaction system, because receiving drug administration dosage
Restriction and drug metabolism in vivo impact, compound is extremely difficult in vivo very high concentration, so in the present invention, it is preferred to will
First prescribed concentration is set to 50 μ g/mL.The prescribed concentration is very high, if still cannot be real in this high concentration compound
Existing free radical scavenging activity is more than or equal to 50%, then illustrates that the antioxidant activity of the compound is very weak, can ignore, i.e., directly really
Antioxidant activity parameter a of the fixed compound is zero.
In the present invention, the corresponding IC of compound free radical scavenging activity50When value refers to free radical scavenging activity arrival 50%
The concentration of compound in reaction system.IC50The determination method of value is techniques known, and here of the present invention is not limited
It is fixed.Determine generally by S type dose-effect curves (abbreviation amount effect curve).That is first determine dense known to difference
The corresponding free radical scavenging activity of certain compound of degree.Then the concentration (value of taking the logarithm) with the compound is as abscissa, with freedom
Base clearance rate is vertical coordinate, draws the S type dose-effect curves of the compound, determines that free radical scavenging activity is from curve
When 50% in reaction system compound concentration, the as corresponding IC of the compound50Value.
In the preferred embodiment of the present invention, determinization is tested using free radical scavenging activity in step (2)
The method of the free radical scavenging activity of compound, including testing by DPPH free radical scavenging activities, determines compound known to difference
Free radical scavenging activity under concentration.
In the preferred embodiment of the present invention, compound difference is determined by the experiment of DPPH free radical scavenging activities
The method of the free radical scavenging activity under different concentration knowns includes：
The reference substance of compound is obtained, and compound control product are configured to into a series of samples with different concentration knowns
Solution；
Each sample solution is added separately to be configured to experimental group solution in the microwell plate containing DPPH methanol solutions；And arrange
Blank control group solution and DPPH matched group solution, the blank control group solution includes sample solution and methanol, the DPPH
Matched group solution includes preparing the solvent and DPPH methanol solutions of sample solution；Wherein, sample solution in experimental group solution
Solvent volume in the volume and DPPH matched group solution of the sample solution in volume, blank control group solution is identical；Experimental group
DPPH in volume, the blank control group solution of the DPPH methanol solutions in solution in the volume and DPPH matched group solution of methanol
The volume of methanol solution is identical.
The absorbance OD of each experimental group solution is determined by plate reading machineSample, blank control group solution absorbance ODIt is blank
And the absorbance OD of DPPH matched group solutionControl, and according to formula
SR=[1-(ODSample–ODIt is blank)/ODControl] × 100%
Determine free radical scavenging activity SR of the compound under different concentration knowns.
In a kind of preferred implementation of the present invention, the condition of plate reading machine mensuration absorbance value includes：Test temperature：20～
50 DEG C, preferred 30～40 DEG C, more preferably 37 DEG C；Read plate speed：45s/ time；Acquisition time：45 minutes；Detection wavelength：
517nm。
In step (3), whether 50% is more than by platelet aggregation inhibition rate of the compound under the second prescribed concentration
Platelet aggregation inhibitory activity to determine the compound is strong and weak.In the present invention, the second prescribed concentration described in step (3) is to change
Polymer solution is configured to for after the experimental group for testing platelet aggregation inhibition rate, compound to be dense in experimental group reaction system
Degree because by drug administration dosage restriction and drug metabolism in vivo affected, in the present invention, it is preferred to by this specify it is dense
Degree is set to 100 μM.Second prescribed concentration is very high, if still cannot realize platelet aggregation in this high concentration compound
Collection suppression ratio is more than or equal to 50%, then illustrates that the platelet aggregation inhibitory activity of the compound is very weak, can ignore, i.e., directly really
Fixed compounds for resisting platelet aggregation parameter b is zero.
In the present invention, the IC corresponding to compound platelet aggregation inhibition rate50Value refers to platelet aggregation inhibition rate
When reaching 50% in reaction system compound concentration.The IC50The determination method of value is referred to aforesaid free radical scavenging
The corresponding IC of rate50The method of value.The platelet aggregation inhibition rate corresponding to the compound of different concentration knowns is first determined, so
Afterwards the concentration (value of taking the logarithm) with the compound is as abscissa, with platelet aggregation inhibition rate as vertical coordinate, draw S type dosage-
Effect curve, then determines the concentration of platelet aggregation inhibition rate compound in reaction system when being 50%, i.e., from curve
For the corresponding IC of the compound50Value.
In a kind of preferred implementation in the present invention, step (3) tests determinization using platelet aggregation inhibitory activity
The platelet aggregation inhibition rate of compound is comprised the following steps：
The reference substance of compound is obtained, and compound control product are configured to into the need testing solution of concentration known；
Experimental group is configured to by the need testing solution, agonist and thrombocyte suspension, by thrombocyte suspension and slow
Rush solution and be configured to blank control group；Negative control group is configured to by buffer solution, agonist and thrombocyte suspension；By described
Need testing solution and buffer preparation are into background color group；The cumulative volume of each group is identical.And blank control group, negative control group
And in experimental group thrombocyte suspension volume it is identical；Agonist volume in negative control group and experimental group is identical.Background face
Colour cell, the volume of the need testing solution of experimental group are identical.
Determination experiment group, blank control group, the platelet aggregation rate maximum of negative control group；According to formula
[(negative control group maximum platelet aggregation rate-blank control group maximum platelet aggregation rate)-(experimental group blood is little
Plate maximum agglutination rate-blank control group maximum platelet aggregation rate)]/(negative control group maximum platelet aggregation rate-blank is right
According to a group maximum platelet aggregation rate) × 100%
Determine the corresponding platelet aggregation inhibition rate of compound.
In the preferred embodiment of the present invention, determination experiment group, blank control group, negative control group, blood it is little
The method of plate aggregation rate maximum includes：
By plate reading machine determination experiment group, blank control group, negative control group and background color group t absorbance
Value, and the absorbance of experimental group, blank control group, negative control group t is individually subtracted into the suction of background color group t
After shading value, experimental group absorbance, blank control group absorbance as t, negative control group absorbance；
And according to formula：Platelet aggregation ratet=(ODT=0-ODt)/ODT=0× 100%
Determine the time dependent kinetic curve of platelet aggregation rate, wherein, platelet aggregation ratetExpression experimental group,
Blank control group or negative control group t platelet aggregation rate, ODT=0Represent initial experimental group absorbance, blank
Matched group absorbance or negative control group absorbance；ODtRepresent that the experimental group absorbance of t, blank control group are inhaled
Shading value or negative control group absorbance；
The kinetic curve according to determined by determines the platelet aggregation rate of experimental group, blank control group, negative control group
Maximum.
Wherein, the agonist contains thromboxane A2 analog, preferably comprises thromboxane A2 analog U46619；Positive drug
For Ticagrelor (platelet aggregation inhibitor).
For the assay method of compounds content in Chinese medicine, in prior art to have been reported that, these known methods are same more
The blood-activating stasis-removing kind Chinese medicine in the present invention is can apply to, in the preferred embodiment of the present invention, in step (4),
High performance liquid chromatography can be passed through or Ultra Performance Liquid Chromatography determines the content of each compound.
In step (5), according to 2010 editions《Pharmacopoeia of People's Republic of China》With regard to " crude drug and pharmaceutical preparation stability
Test direction principle " specifies：Hot test selects 60 DEG C, and described Preset Time can be by those skilled in the art according to " former
Material medicine and pharmaceutical preparation stability test guideline " is typically chosen 10 days determining.In addition, the one kind in step (5) is preferred
In embodiment, blood-activating stasis-removing kind Chinese medicine can be placed in the climatic chamber without lamp and be tested.So can exclude wet
The interference of the other factorses such as degree so that assay result is more accurate.
In step (6), according to 2010 editions《Pharmacopoeia of People's Republic of China》With regard to " crude drug and pharmaceutical preparation stability
Test direction principle " specifies：Intensity of illumination selects 4500lx ± 500lx, and described Preset Time can be by people in the art
Member is typically chosen 10 days according to " crude drug and pharmaceutical preparation stability test guideline " determination.In addition, in step (6)
In a kind of preferred implementation, blood-activating stasis-removing kind Chinese medicine can be placed in the climatic chamber equipped with daylight lamp and be irradiated.
The interference of the other factorses such as humidity, temperature can so be excluded so that assay result is more accurate.
In a kind of specific embodiment of the present invention, aforementioned blood-activating stasis-removing kind Chinese medicine is DANHONG ZHUSHEYE.
The compound contained in DANHONG ZHUSHEYE includes：Danshensu, S-A Hydroxysafflor yellow A, L-phenylalanine, uridnine,
Cytidine, protocatechualdehyde, protocatechuic acid, rosmarinic acid, salvianolic acid B, salvianolic acid A, salvianolic acid C, caffeic acid, alkannic acid, adenosine, purple
Ligustrin and 5 hydroxymethyl furfural.
By such scheme, the present invention determines that compound with oxidation resistance in blood-activating stasis-removing kind Chinese medicine is active, anti-blood is little
Plate aggregation activity, content, high-temperature stability, the relevant parameter of high light stability this five aspects, and by the parameter of this five aspect
Integrated by formula, five metanetworks for obtaining compound return area, and return the big of area according to five metanetwork
It is little determining active percentage contribution of each compound in blood-activating stasis-removing kind Chinese medicine.Because the present invention is it is determined that pharmaceutically active contribution
During degree, not only allow for antioxidant activity, the platelet aggregation inhibitory activity of compound itself, it is also contemplated that should in actual clinical
With the middle content that can affect compound activity percentage contribution, high-temperature stability, high light stability, therefore, the promoting blood circulation of the present invention
The determination method of the active percentage contribution in stasis of blood class Chinese medicine is more reasonable, is more beneficial for the quality control of Chinese medicine.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described, it should be apparent that, drawings in the following description are only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, can be with
Other accompanying drawings are obtained according to these accompanying drawings.
Figure 1A is the free radical scavenging activity and dose-effect curve of salvianolic acid B；Figure 1B is clear for the free radical of protocatechuic acid
Except activity and dose-effect curve；Fig. 1 C are the free radical scavenging activity and dose-effect curve of protocatechualdehyde；Fig. 1 D are pellet
The free radical scavenging activity and dose-effect curve of phenolic acid C；Fig. 1 E are caffeinic free radical scavenging activity and docs-effect
Curve；Fig. 1 F are the free radical scavenging activity and dose-effect curve of salvianolic acid A；Fig. 1 G are the free radical scavenging of rosmarinic acid
Activity and dose-effect curve；Fig. 1 H are the free radical scavenging activity and dose-effect curve of alkannic acid；Fig. 1 I are danshensu
Free radical scavenging activity and dose-effect curve；
Fig. 2A is the platelet aggregation inhibition rate and amount effect curve of salvianolic acid A, and Fig. 2 B press down for the platelet aggregation of salvianolic acid B
Rate processed and amount effect curve；
Fig. 3 is five metanetwork figures of 16 compounds in DANHONG ZHUSHEYE；
Fig. 4 is that five metanetworks of 16 compounds in DANHONG ZHUSHEYE return area block diagram.
Specific embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than the embodiment of whole.It is based on
Embodiment in the present invention, it is every other that those of ordinary skill in the art are obtained under the premise of creative work is not made
Embodiment, belongs to the scope of protection of the invention.
DANHONG ZHUSHEYE is a kind of by Radix Salviae Miltiorrhizae, the taste of Flos Carthami two tradition blood-activating and stasis-removing Jing scientific compatibility and using modern system
Complex injection of Chinese materia medica made by agent technique, the effect of with blood circulation promoting and blood stasis dispelling, TONGMAI SHULUO.Just with DANHONG ZHUSHEYE it is below
Example, is described to technical scheme.It should be noted that, although the technical scheme is that with DANHONG ZHUSHEYE
As a example by illustrate, but other Chinese medicines can also equally realize the purpose of the present invention using technical scheme,
Simply as space is limited, the citing that differs is illustrated the present invention.
DANHONG ZHUSHEYE used by following examples is provided by Heze Buchang Pharma Co., Ltd., and required key instrument is such as
Shown in table 1.
The key instrument that the embodiment of table 1 is adopted
It is reported that mainly containing 16 compounds in DANHONG ZHUSHEYE, respectively danshensu, hydroxyl is red in prior art
Anthoxanthin A, L-phenylalanine, uridnine, cytidine, protocatechualdehyde, protocatechuic acid, rosmarinic acid, salvianolic acid B, salvianolic acid A, pellet
Phenolic acid C, caffeic acid, alkannic acid, adenosine, syringoside and 5 hydroxymethyl furfural.
Reference substance：Danshensu (lot number：D-014-150318), S-A Hydroxysafflor yellow A (HSYA) (lot number：Q-008-
141216), L-phenylalanine (B074-150727), uridnine (lot number：N-025-150730), cytidine (lot number：B-109-
150715) it is purchased from Chengdu Rui Fensi bio tech ltd；Protocatechualdehyde (lot number：W06-2-5), protocatechuic acid (lot number：
W06-3-4), rosmarinic acid (lot number：W10-5-3), salvianolic acid B (lot number：W17-7-1), salvianolic acid A (lot number：W1324 it is), red
Phenolic acid C (lot numbers：W17-7-4), caffeic acid (lot number：W05-5-6), alkannic acid (lot number：W16-5-6), adenosine (lot number：W17-
0-9), syringoside (lot number：W12-2-7) it is purchased from Zhongxin Innova Laboratories；5 hydroxymethyl furfural (5-HMF) is purchased from U.S.
Sigma-Aldrich companies of state.Its purity is all higher than 98%.
1st, antioxidant activity parameter a of each compound in DANHONG ZHUSHEYE is determined；
The preparation of 1.1 sample stock solutions
Salvianolic acid B, protocatechuic acid, protocatechualdehyde, salvianolic acid C, caffeic acid, salvianolic acid A, rosmarinic acid, Radix Arnebiae (Radix Lithospermi) are taken respectively
Acid, danshensu reference substance, accurately weighed 5.02mg, 5.01mg, 5.03mg, 5.00mg, 5.01mg, 5.03mg, 5.01mg,
5.02mg, 5.01mg, in being respectively placed in 5mL brown volumetric flasks, add methanol (chromatographically pure, the limited public affairs of Tianjin Concord science and technology
Department) to groove, shake up, as 1.004mg/mL, 1.002mg/mL, 1.006mg/mL, 1.000mg/mL, 1.002mg/mL,
1.006mg/mL, 1.002mg/mL, 1.004mg/mL, 1.002mg/mL sample stock solution.
HSYA, L-phenylalanine, syringoside, adenosine, cytidine, uridnine, 5-HMF reference substances are taken respectively, it is accurately weighed
5.01mg, 5.03mg, 5.00mg, 5.02mg, 5.02mg, 5.02mg, 5.01mg, in being respectively placed in 2mL brown volumetric flasks, methanol
Dissolving and constant volume to groove, shake up, as 2.505mg/mL, 2.515mg/mL, 2.500mg/mL, 2.510mg/mL,
2.510mg/mL, 2.510mg/mL, 2.505mg/mL sample stock solution.
1.2 the preparation of sample solution
Salvianolic acid B, alkannic acid sample stock solution are taken respectively appropriate, use methanol stepwise dilution, be configured to 200.80 μ g/
mL、133.87μg/mL、89.24μg/mL、59.50μg/mL、39.66μg/mL、26.44μg/mL、17.63μg/mL、11.75μ
The sample solution of g/mL, 7.83 μ g/mL, 5.22 μ g/mL, 3.48 μ g/mL and 2.32 μ g/mL12 concentration, it is standby.
Protocatechuic acid, caffeic acid, rosmarinic acid, danshensu sample stock solution are taken respectively appropriate, use methanol stepwise dilution,
Be configured to 200.40 μ g/mL, 133.60 μ g/mL, 89.07 μ g/mL, 59.38 μ g/mL, 39.59 μ g/mL, 26.39 μ g/mL,
The sample of 17.59 μ g/mL, 11.73 μ g/mL, 7.82 μ g/mL, 5.21 μ g/mL, 3.48 μ g/mL and 2.32 12 concentration of μ g/mL
Solution, it is standby.
Take salvianolic acid A sample stock solution appropriate, use methanol stepwise dilution, be configured to 201.20 μ g/mL, 134.13 μ g/
mL、89.42μg/mL、59.61μg/mL、39.74μg/mL、26.50μg/mL、17.66μg/mL、11.78μg/mL、7.85μg/
The sample solution of mL, 5.23 μ g/mL, 3.49 μ g/mL and 2.33 12 concentration of μ g/mL, it is standby.
Take protocatechualdehyde sample stock solution appropriate, use methanol stepwise dilution, be configured to 59.61 μ g/mL, 39.74 μ g/
mL、26.50μg/mL、17.66μg/mL、11.78μg/mL、7.85μg/mL、5.23μg/mL、3.49μg/mL、2.33μg/mL、
The sample solution of 1.55 μ g/mL, 1.03 μ g/mL and 0.69 12 concentration of μ g/mL, it is standby.
Take salvianolic acid C sample stock solution appropriate, use methanol stepwise dilution, be configured to 333.33 μ g/mL, 200.00 μ g/
mL、133.33μg/mL、88.89μg/mL、59.26μg/mL、39.51μg/mL、26.34μg/mL、17.56μg/mL、11.71μ
The sample solution of g/mL, 7.80 μ g/mL, 5.20 μ g/mL and 3.47 12 concentration of μ g/mL, it is standby.
Take HSYA samples stock solution appropriate, use methanol stepwise dilution, be configured to 2505.00 μ g/mL, 1670.00 μ g/
mL、1113.33μg/mL、742.22μg/mL、494.81μg/mL、329.88μg/mL、219.92μg/mL、146.61μg/mL、
97.74 μ g/mL, 65.16 μ g/mL, 43.44 μ g/mL, the sample solution of 28.96 12 concentration of μ g/mL, it is standby.
It is (molten as sample that L-phenylalanine, syringoside, adenosine, cytidine, uridnine, 5-HMF sample stock solutions are taken respectively
Liquid), it is standby.
The assay method (DPPH micropore quantitative determination process) of 1.3DPPH free radical scavenging activities
Using3 type desk type multifunctional plate reading machines 37 DEG C at the 517nm at determine DANHONG ZHUSHEYE 16
The DPPH scavenging capacities of compound.Experimental group：Sequentially add the μ L of sample solution 50 and 150 μ L of each 12 variable concentrations of composition
DPPH methanol solutions (DPPH concentration in reaction system：0.1875mM, DPPH (2,2- diphenyl -1- hardship diazanyls), analyze pure,
Sigma-Aldrich companies), parallel three multiple holes；Blank control group：50 μ L samples solution and 150 μ L methanol are sequentially added, with
Deduct the impact of medicine background；DPPH matched groups：50 μ L methanol and 150 μ L DPPH methanol solutions are sequentially added (in reaction system
DPPH concentration：0.1875mM), parallel three multiple holes.Each group solution is immediately placed in multi-functional plate reading machine, the record one per 45s
Secondary absorbance, continuously records 45min, all test parallel testings 3 times.
DPPH radical scavenging activities are represented with free radical scavenging activity SR, are calculated by formula 1：
SR=[1-(ODSample–ODIt is blank)/ODControlThe formula of] × 100% 1
Wherein, ODSampleRepresent the absorbance of experimental group solution, ODIt is blankThe absorbance of blank control group solution is represented,
ODControlRepresent the absorbance of DPPH matched group solution；SR values are bigger, and oxidation resistance is stronger.
The test result of 1.4DPPH free radical scavenging activities
Each compound is obtained under " 1.3 " item after the free radical scavenging activity under variable concentrations, is determined 50 μ g/mL's
Free radical scavenging activity is more than or equal to 50% under concentration (concentration refers to concentration of the compound in experimental group reaction system)
Compound；The corresponding free radical scavenging activity of each compound of different concentration knowns is further determined, then with the compound
Concentration (concentration of the compound in experimental group reaction system) is abscissa, with free radical scavenging activity as vertical coordinate, and is adopted
" Origin Pro 8.5.1 SR2 " fits the S type dose-effect curves of compound, and draws each compound by amount effect curve
Corresponding IC50Value.It should be noted that drawing in S type dose-effect curves, it is compound in reality that compound concentration is referred to
The concentration tested in group reaction system.
Found after test, salvianolic acid B, protocatechuic acid, protocatechualdehyde, salvianolic acid C, caffeic acid, salvianolic acid A, fan change
Fragrant acid, alkannic acid, danshensu this 9 compounds free radical scavenging activity under the concentration of 50 μ g/mL are more than or equal to 50%；Further
The corresponding free radical scavenging activity of each compound of different concentration knowns is determined, corresponding S types dose-effect curve is drawn, is determined
Its IC50Value, the free radical scavenging activity and amount effect curve of each compound are shown in Figure 1A～Fig. 1 I.
HSYA determines its DPPH free radical scavenging activity, as a result shows that HSYA has a free radical scavenging activity, but activity compared with
Weak, it is impossible to complete the drafting of S amount effect curves, SR values concrete outcome is shown in Table 2 under each concentration.
The free radical scavenging activity result of the variable concentrations HSYA of table 2 (concentration in reaction system)
During final concentration about 0.625mg/mL in experimental group reaction system, determine cytidine, adenosine, uridnine, syringoside,
The DPPH free radical scavenging activities of 6 compositions of 5-HMF and L-phenylalanine, as a result show, syringoside has weaker activity, SR
It is worth for 32.53%；Other 5 chemical compositions can consider do not have DPPH free radical scavenging activities.Concrete outcome is shown in Table 3.
The free radical scavenging of (concentration 0.625mg/mL in experimental group reaction system) 6 compounds is lived under the finite concentration of table 3
Property
By the IC of 9 compounds such as salvianolic acid B50Carry out at data normalization divided by maximum therein after value is inverted
Reason, obtains antioxidant activity parameter a of 9 compounds, the results are shown in Table 4；Remaining 7 compounds (HSYA, cytidine, adenosine, urine
Glycosides, syringoside, 5-HMF and L-phenylalanine) it is less than due to the free radical scavenging activity under prescribed concentration (50 μ g/mL)
50%, then antioxidant activity parameter a for directly determining them is zero；
4 DANHONG ZHUSHEYE of table, 16 compound with oxidation resistance activity IC50Value and antioxidant activity parameter a
2nd, antiplatelet aggregation parameter b of each compound in DANHONG ZHUSHEYE is determined；
The preparation of 2.1 experimental solutions
HEPES Modified Tyrode's solution are prepared：Sodium Chloride (NaCl), potassium chloride (KCl), magnesium chloride
(MgCl2), sodium bicarbonate (NaHCO3), sodium dihydrogen phosphate (NaH2PO4), 4- hydroxyethyl piperazine ethanesulfonic acid (HEPES), glucose
(D-Glucose), bovine serum albumin (Bovine serum albumin, BSA) is dissolved in above-mentioned medicine by certain proportioning double
In boiling off ionized water, its proportioning is as shown in table 5 below, adjusts PH=7.4, is settled to 40mL, matching while using.
The formula table of the HEPES Modified Tyrode's solution of table 5
Agonist is prepared：8 μM of U46619 concentration, Ca2+Solution concentration 8mM, with containing Fibrinogen (concentration 1mg/mL)
HEPES Modified Tyrode's solution prepare.Final concentration in resuspended platelet experimental system is preparation
The 1/4 of concentration.
PGE1 (PGE1)：Storing solution DMSO is formulated as concentration 10mM, HEPES Modified Tyrode's
Solution is diluted to desired concn, final concentration of 1.25 μM in resuspended platelet experimental system.
The preparation of test sample storing solution：Cytidine, uridnine, adenosine, Phenylalanine, 5-HMF, danshensu, protocatechuic acid, former youngster
Tea aldehyde, HYSA, syringoside, caffeic acid, rosmarinic acid, alkannic acid, salvianolic acid B, salvianolic acid A and salvianolic acid C reference substance are appropriate,
Storing solution is prepared with DMSO, and HEPES Modified Tyrode's solution are diluted to concentration for 400 μM.
The preparation of need testing solution：Using HEPES Modified Tyrode's solution by salvianolic acid A, salvianolic acid
B storing solutions be diluted to concentration be 400 μM, 360 μM, 320 μM, 280 μM, 240 μM, 200 μM, 160 μM, 120 μM of test sample it is molten
Liquid.
The preparation of positive drug solns：Using Ticagrelor as positive drug, storing solution is prepared with DMSO, HEPES Modified
Tyrode's solution are diluted to concentration for 4 μM.
2.2 platelet aggregation inhibitory activity experimental techniques
The platelet aggregation inhibitory activity of each compound is tested using microwell plate quantitative method, experiment packet be set to experimental group,
Blank control group (for monitoring spontaneous platelet aggregation extent), negative control group are (for detecting platelet the stress function and medicine
The calculating of thing suppression ratio), positive controls (for determining activation and the antagonism of specific receptor), background color group is (for examining
Survey and deduct medicine background color), the concrete table 6 below of each group.
The preparation program of each group of table 6
2.3 determine blood of each compound under prescribed concentration (100 μM, the concentration in reaction system) in DANHONG ZHUSHEYE
Platelet assembles suppression ratio
Using under " 2.1 " item concentration be the test sample storing solution of 400 μM of 16 kinds of compounds as need testing solution, by table 6
Difference preparation experiment group, blank control group, negative control group, positive controls and background color group.In experimental group and background face
In colour cell, the concentration of 16 kinds of compounds in reaction system is 100 μM；Then tested as follows：
Experimental group：100 μ L human blood platelets suspensions (OD values are that absorbance is about 0.65) are added to into respectively the hole of standard 96
Tissue Culture Plate, adds the need testing solution that 50 μ L concentration are 400 μM, 10min and sustained oscillation is incubated in 37 DEG C, at 405nm
Detection dynamic process, reading time interval 45s makes medicine fully mix with platelet；50 μ L agonist are added, is detected at 405nm
Dynamic process, intermittent sustained oscillation 15min finally gives the time dependent kinetics of OD values bent per 45s readings once
Line.
Blank group：100 μ L human blood platelets suspensions (OD values are that absorbance is about 0.65) are added to into respectively the hole of standard 96
Tissue Culture Plate, adds 100 μ L buffer, and in 37 DEG C 10min and sustained oscillation are incubated, and dynamic process is detected at 405nm, reads
Number interval 45s, makes fully mixing；Intermittent sustained oscillation 15min, detects dynamic process at 405nm, per 45s readings once,
Finally give the time dependent kinetic curve of OD values.
Negative control group：100 μ L human blood platelets suspensions (OD values are that absorbance is about 0.65) are added to into respectively standard
96 porocyte culture plates, add 50 μ L buffer, and in 37 DEG C 10min and sustained oscillation are incubated, and kinetics mistake is detected at 405nm
Journey, reading time interval 45s makes fully mixing；50 μ L agonist are added, dynamic process is detected at 405nm, per 45s readings once,
Intermittent sustained oscillation 15min, finally gives the time dependent kinetic curve of OD values.
Positive controls：100 μ L human blood platelets suspensions (OD values are that absorbance is about 0.65) are added to into respectively standard
96 porocyte culture plates, add the positive drug solns that 50 μ L concentration are 4 μM, and in 37 DEG C 10min and sustained oscillation, 405nm are incubated
Place's detection dynamic process, reading time interval 45s makes medicine fully mix with platelet；50 μ L agonist are added, is examined at 405nm
Dynamic process is surveyed, intermittent sustained oscillation 15min finally gives the time dependent kinetics of OD values per 45s readings once
Curve.
Background color group：150 μ L buffer are added to into respectively the porocyte culture plates of standard 96, adds 50 μ L test samples molten
Liquid, in 37 DEG C 10min and sustained oscillation are incubated, and dynamic process, reading time interval 45s are detected at 405nm；Continue intermittent continuing
Concussion 15min, detects dynamic process at 405nm, finally give the time dependent kinetics of OD values per 45s readings once
Curve.
Using the OD Value Datas of each each hole Each point in time of group of Office Excel software analysis, background color is cut
After the corresponding numerical value of group, the platelet aggregation rate of the Each point in time that converted using formula 2 finally gives platelet aggregation rate at any time
Between change kinetic curve.
Platelet aggregation ratet=(ODT=0-ODt)/ODT=0× 100% formula 2
After platelet aggregation reaction is complete or reaches default reaction duration, platelet maximum (final) aggregation rate is obtained.
Using the corresponding platelet aggregation inhibition rate of the 16 kinds of compounds of calculating of formula 3, concrete outcome is as shown in table 7.
[(negative control group maximum agglutination rate-blank control group maximum agglutination rate)-(experimental group maximum agglutination rate-blank group
Maximum agglutination rate)]/(negative control group maximum agglutination rate-blank control group maximum agglutination rate) × 100% formula 3
The corresponding platelet aggregation inhibition rate of Ticagrelor is calculated using formula 4, concrete outcome is as shown in table 7.
[(negative control group maximum agglutination rate-blank control group maximum agglutination rate)-(positive controls maximum agglutination rate-sky
White matched group maximum agglutination rate)]/(negative control group maximum agglutination rate-blank control group maximum agglutination rate) × 100% formula 4
Platelet aggregation inhibition rate of the 7 16 kinds of compounds of table in prescribed concentration 100 μM (concentration in reaction system)
Compare with negative control group, * p ＜ 0.05, * * p ＜ 0.01, * * * p ＜ 0.001
As can be seen from Table 7, in addition to salvianolic acid A and salvianolic acid B, the platelet suppression ratio of other each compounds does not have
Have and reach 50%, illustrate that their platelet aggregation inhibitory activity is very weak, even without platelet aggregation inhibitory activity, therefore, directly
Determine that these compounds for resisting platelet aggregation parameters b are zero；
2.4 antiplatelet aggregation parameters b for determining salvianolic acid A and salvianolic acid B
According to the experimental technique in " 2.3 ", it is determined that the salvianolic acid A of the variable concentrations prepared under " 2.1 " item, salvianolic acid B
The platelet aggregation inhibition rate of need testing solution.Then salvianolic acid A, the corresponding S types dose-effect curve of salvianolic acid B are drawn, and is surveyed
Fixed its corresponding IC50Value, salvianolic acid A, the corresponding IC of platelet aggregation inhibition rate of salvianolic acid B50Value is respectively 77.3 μM
(38.19μg/mL)、54.4μM(39.06μg/mL).The platelet aggregation inhibition rate and amount effect curve of two compounds is shown in figure
2A-2B.By salvianolic acid A, the IC of salvianolic acid B50Enter divided by maximum therein after the μ g/mL of value 38.19,39.06 μ g/mL are inverted
Row data standardization, obtains salvianolic acid A, antiplatelet aggregation parameter b of salvianolic acid B is respectively 1.00 and 0.98.
3rd, content parameter c of each compound in DANHONG ZHUSHEYE is determined；
3.1 determine content of each compound in DANHONG ZHUSHEYE using Ultra Performance Liquid Chromatography instrument；
3.1.1 chromatographic condition
Using Waters Acquity Ultra Performance Liquid Chromatography systems (on-line degassing machine, binary solvent pump, sample managing
Device, column oven, TUV detectors), control software is Masslynx4.1.Chromatographic column Agilent Zorbax RRHT SB-C18Post
(4.6mm × 100mm, 1.8 μm), with 0.1% aqueous formic acid (A)～acetonitrile (B) as mobile phase, gradient elution program such as table 8
It is shown；40 DEG C of column temperature；Flow velocity is 0.5mL/min；Initial Gradient balances chromatographic column 5min.
The gradient elution program of table 8
Detection wavelength arranges as follows：Passage 1：0.00～5.00min, 260nm determine cytidine, uridnine, adenosine；5.01～
13.70min, 286nm determine 5-HMF, danshensu, protocatechuic acid, protocatechualdehyde；13.71～14.50min, 403nm are determined
HYSA；14.50～45.50min, 286nm determine caffeic acid, rosmarinic acid, alkannic acid, salvianolic acid B, salvianolic acid A, salvianolic acid C.
Passage 2：0.00～45.50min, 257nm determine Phenylalanine and syringoside.Sample manager temperature is 4 DEG C.
3.1.2 the preparation of reference substance solution and need testing solution
The preparation of reference substance solution：Take cytidine, uridnine, adenosine, Phenylalanine, 5-HMF, danshensu, protocatechuic acid, former youngster
Tea aldehyde, HYSA, syringoside, caffeic acid, rosmarinic acid, alkannic acid, salvianolic acid B, salvianolic acid A and salvianolic acid C reference substance are appropriate,
Accurately weighed, in being respectively placed in 5mL brown measuring bottles, in addition to Phenylalanine is dissolved in 50% methanol aqueous solution, remaining is dissolved in methanol
Scale is dissolved and be settled to, concentration is obtained and is respectively：Cytidine (2.004mg/mL), uridnine (1.136mg/mL), adenosine
(2.006mg/mL), Phenylalanine (2.016mg/mL), 5-HMF (3.486mg/mL), danshensu (3.930mg/mL), former catechu
Sour (2.012mg/mL), protocatechualdehyde (2.010mg/mL), HYSA (2.006mg/mL), syringoside (2.002mg/mL), coffee
Coffee acid (2.006mg/mL), rosmarinic acid (2.014mg/mL), alkannic acid (2.012mg/mL), salvianolic acid B (2.004mg/mL),
The reference substance stock solution of salvianolic acid A (1.114mg/mL) and salvianolic acid C (0.382mg/mL), cold preservation is standby in 4 DEG C of refrigerators.
Precision measures above-mentioned 16 reference substance stock solutions in right amount, is configured to mixed reference substance solution, wherein cytidine 20.04
μ g/mL, the μ g/mL of uridnine 68.16, the μ g/mL of adenosine 14.04, the μ g/mL of Phenylalanine 120.96,5-HMF 55.78 μ g/mL, Radix Salviae Miltiorrhizae
746.70 μ g/mL of element, the μ g/mL of protocatechuic acid 20.12, the μ g/mL of protocatechualdehyde 120.60, HYSA 16.05 μ g/mL, syringoside
16.02 μ g/mL, the μ g/mL of caffeic acid 16.05, the μ g/mL of rosmarinic acid 241.68, the μ g/mL of alkannic acid 80.48, salvianolic acid B
340.68 μ g/mL, the μ g/mL of salvianolic acid A 155.96, the μ g/mL of salvianolic acid C 6.11, cold preservation is standby in 4 DEG C of refrigerators.Carry out
Linear relationship study when, by this mixed reference substance solution stepwise dilution into a series of concentration mixed reference substance solution.
The preparation of need testing solution：Accurate measuring 1.00mL DANHONG ZHUSHEYE uses 50% methanol-water in 5mL brown volumetric flasks
Solution is settled to scale, obtains final product.
3.1.3 Method validation
Ultra-performance liquid chromatography to being set up under " 3.1.1 " item carries out Method validation, and Method validation includes line
Sexual intercourse (r >=0.999), quantitative limit (0.038～0.756 μ g/mL), test limit (0.011～0.227 μ g/mL), precision
(withinday precision, day to day precision RSD≤2.3%), repeated (RSD≤2.0%), stability (8h stablizes at 4 DEG C) and plus
The experiment of the sample response rate (91.47%～108.46%, RSD≤3.1%).Checking concrete grammar and result have been described in the literature
(Liu HT,Wang YF,Olaleye Olajide,et al.Characterization of in vivo antioxidant
constituents and dual-standard quality assessment of Danhong injection[J]
.Biomedical chromatogr,DOI 10.1002/bmc.2842).The document is incorporated by herein by quoting it
In, here of the present invention is not repeated.By aforesaid Method validation, it may be determined that the superelevation set up under " 3.1.1 " item
Effect liquid phase chromatogram method disclosure satisfy that the requirement that compounds content is determined.
3.1.4 assay result of the compound in DANHONG ZHUSHEYE
16 chemical compositions in 10 batches of DANHONG ZHUSHEYE are detected using the chromatographic condition under " 3.1.1 " item, and is calculated
Cytidine, uridnine, adenosine, Phenylalanine, 5-HMF, danshensu, protocatechuic acid, protocatechualdehyde, HYSA, purple fourth in DANHONG ZHUSHEYE
The content of fragrant glycosides, caffeic acid, rosmarinic acid, alkannic acid, salvianolic acid B, salvianolic acid A and salvianolic acid C, by the content results of each measure
Average, to reflect that content height of each composition in DANHONG ZHUSHEYE the results are shown in Table 9；
The assay result of 16 compounds in the DANHONG ZHUSHEYE of table 9
The calculating of 3.2 each compounds content parameters c
The content meansigma methodss of each compound in table 9 are carried out into data normalization process divided by maximum therein, obtains each
Content parameter c of compound, the results are shown in Table 10；Wherein, the content of HSYA is less than quantitative limit, it is believed that its content parameter c is zero.
10 DANHONG ZHUSHEYE of table, 16 chemical composition assays and content parameter c
4th, high-temperature stability parameter d of each compound in DANHONG ZHUSHEYE is determined
The DANHONG ZHUSHEYE with batch with sequence number 10 in table 9 is randomly selected, being put in climatic chamber carries out high-temperature process,
High-temperature process condition is：Avoid light place 10 days at 60 DEG C；Using ultra-performance liquid chromatography, by the chromatostrip under " 3.1.1 " item
The preparation method of reference substance solution and need testing solution under part and " 3.1.2 " item, to high-temperature process before (do not carry out intense light irradiation yet
Penetrate process, it is understood that into high-temperature process 0 day) and high-temperature process after DANHONG ZHUSHEYE detected, it is therein to determine
The content of 16 kinds of compounds, concrete outcome is shown in Table 11.The contrast of the compounds content by determining twice, determines DANHONG ZHUSHEYE
Compound each compounds content after high-temperature process situation of change.
Specifically, corresponding high-temperature stability parameter d of each compound is determined according to formula d=1- ︱ CR-100% ︱, specifically
12 are the results are shown in Table, wherein, CR represents the ratio of content of the same compound after high-temperature process and the content before high-temperature process；CR
The degree of the volume of compounds is represented, changes of contents is bigger, represents its stability poorer；Subtract the absolute value with 1 again, make calculating
As a result with stability positive correlation, so as to obtain high-temperature stability parameter d.Because HSYA content parameters c are zero, its high-temperature stability
Parameter d ought to also be designated as zero.
The changes of contents situation of 16 chemical compositions of DANHONG ZHUSHEYE under the high temperature of table 11, strong illumination
Changes of contents ratio and high-temperature stability parameter d after the placement of table 12 DANHONG ZHUSHEYE 16,60 DEG C of compound high temperature
5th, high light stability parameter e of each compound in DANHONG ZHUSHEYE is determined
Randomly select and be put in the climatic chamber equipped with daylight lamp with the DANHONG ZHUSHEYE of batch with sequence number 10 in table 9
Strong illumination process is carried out, strong illumination treatment conditions are：10 are irradiated under conditions of intensity of illumination is 4500lx ± 500lx
My god；Using ultra-performance liquid chromatography, by the reference substance solution under the chromatographic condition under " 3.1.1 " item and " 3.1.2 " item and confession
The preparation method of test sample solution, detects, to determine 16 kinds of chemical combination therein to the DANHONG ZHUSHEYE after strong illumination process
The content of thing, concrete outcome is shown in Table 11.With the content of each compound in the DANHONG ZHUSHEYE of the same lot number determined under aforementioned " 4 " item
As the content (it also will be understood that processing 0 day into strong illumination) of the compound of strong illumination before processing, by what is determined twice
The contrast of compounds content is determining each compounds content of the compound of DANHONG ZHUSHEYE after processing through strong illumination
Change.
Specifically, corresponding high light stability parameter e of each compound is determined according to the ︱ of formula e=1- ︱ CR ' -100%, specifically
13 are the results are shown in Table, wherein, CR ' represents the content of content of the same compound after strong illumination process and strong illumination before processing
Ratio；CR ' represents the degree of the volume of compounds, and changes of contents is bigger, represents its stability poorer；Subtract this with 1 again exhausted
To value, result of calculation and stability positive correlation are made, so as to obtain high light stability parameter e.Because HSYA content parameters c are zero,
Its high light stability parameter e is also denoted as zero.
Changes of contents ratio and high light stability parameter e after table 13 DANHONG ZHUSHEYE, 16 chemical composition strong illuminations
6th, five metanetworks of each compound return the determination of area
According to antioxidant activity parameter a of hereinbefore fixed each compound, antiplatelet aggregation parameter b, content ginseng
Number c, high-temperature stability parameter d, high light stability parameter e, as five metanetwork parameters of each compound, according to formulaFive metanetworks for determining each compound return area A, calculate knot
Fruit is as shown in table 14, Fig. 3, Fig. 4.
Five metanetworks of table 14 DANHONG ZHUSHEYE, 16 chemical compositions return area
The size of area is returned according to five metanetworks of each compound, can be with 16 kinds of chemical combination in overall merit DANHONG ZHUSHEYE
Active percentage contribution of the thing to DANHONG ZHUSHEYE.Fig. 4 is that five metanetworks of 16 kinds of compounds in DANHONG ZHUSHEYE return area post
Shape figure, can intuitively find out the active percentage contribution of each compound very much from figure.As figure 4, it is seen that activity contribution
What degree came first six digits is successively danshensu, protocatechualdehyde, salvianolic acid B, salvianolic acid A, protocatechuic acid and rosmarinic acid, wherein
Danshensu shows maximum contribution, and it is 1.03 that it returns area.Comprehensive tribute of the mentioned component in terms of activity, content, stability
Offer larger.Can be used as the selection indicators of DANHONG ZHUSHEYE quality control.
The determination method of the active percentage contribution of compound in blood-activating stasis-removing kind Chinese medicine provided by the present invention is entered above
Go and be discussed in detail.Specific embodiment used herein is set forth to the principle and embodiment of the present invention, above reality
The explanation for applying example is only intended to help and understands the method for the present invention and its central idea.It should be pointed out that common for this area
For technical staff, under the premise without departing from the principles of the invention, some improvement and modification can also be carried out to the present invention, these
Improve and modify the protection for also falling into the claims in the present invention.
Claims (10)
1. in blood-activating stasis-removing kind Chinese medicine the active percentage contribution of compound determination method, it is characterised in that include：
(1) blood-activating stasis-removing kind Chinese medicine is obtained, and determines at least two compounds for wherein containing；
(2) antioxidant activity parameter a of each compound in step (1) is determined；Wherein,
Antioxidant activity parameter a of each compound is determined by following methods：
The free radical scavenging activity for determining the compound is tested using free radical scavenging activity, if the compound is in the first prescribed concentration
Under free radical scavenging activity be more than or equal to 50%, it is determined that the corresponding IC of the compound free radical scavenging activity50Value, and by the IC50
Data normalization process is carried out divided by maximum therein after value is inverted, antioxidant activity parameter a of the compound is obtained；If
Free radical scavenging activity of the compound under the first prescribed concentration is less than 50%, then directly determine the antioxidant activity of the compound
Parameter a is zero；
(3) antiplatelet aggregation parameter b of each compound in rapid (1) is determined；Wherein,
Antiplatelet aggregation parameter b of each compound is determined by following methods：
The platelet aggregation inhibition rate for determining the compound is tested using platelet aggregation inhibitory activity, if the compound refers to second
The platelet aggregation inhibition rate determined under concentration is more than or equal to 50%, it is determined that the compound platelet aggregation inhibition rate is corresponding
IC50Value, and by the IC50Data normalization process is carried out divided by maximum therein after value is inverted, the anti-of the compound is obtained
Platelet aggregation parameter b；If platelet aggregation inhibition rate of the compound under the second prescribed concentration is less than 50%, directly really
Fixed compounds for resisting platelet aggregation parameter b is zero；
(4) content of each compound in the blood-activating stasis-removing kind Chinese medicine obtained in step (1) is determined, and by the content of each compound
Data normalization process is carried out divided by maximum therein, content parameter c of each compound is obtained；
(5) the blood-activating stasis-removing kind Chinese medicine obtained in step (1) is carried out into high-temperature process, the high-temperature process is in 60 DEG C of conditions
Lower avoid light place Preset Time, after high-temperature process, determines each compound in blood-activating stasis-removing kind Chinese medicine using the method for step (4)
Content；And corresponding high-temperature stability parameter d of each compound is determined according to formula d=1- ︱ CR-100% ︱, wherein, CR is represented
The ratio of the content before content of the same compound after high-temperature process and high-temperature process；
(6) the blood-activating stasis-removing kind Chinese medicine obtained in step (1) is carried out into strong illumination process, the strong illumination is processed as
Intensity of illumination is that Preset Time is irradiated under conditions of 4500lx ± 500lx, true using the method for step (4) after strong illumination process
Fixed content of each compound in blood-activating stasis-removing kind Chinese medicine；And each compound correspondence is determined according to the ︱ of formula e=1- ︱ CR ' -100%
High light stability parameter e, wherein, CR ' is content and strong illumination before processing of the same compound after strong illumination process
Content ratio；
(7) the corresponding a of each compound using determined by, b, c, d, e-value as each compound five metanetwork parameters, according to formula
Five metanetworks for determining each compound return area A, and it is each to determine that the size of area A is returned according to five metanetwork
The five metanetworks recurrence area A of active percentage contribution of the compound in blood-activating stasis-removing kind Chinese medicine, i.e. compound is bigger, and it is being lived
Active percentage contribution in blood blood stasis dispelling class Chinese medicine is bigger.
2. the method for claim 1, it is characterised in that the freedom for determining compound is tested using free radical scavenging activity
The method of base clearance rate, including being tested by DPPH free radical scavenging activities, determine compound under different concentration knowns from
By base clearance rate.
3. method as claimed in claim 2, it is characterised in that compound point is determined by the experiment of DPPH free radical scavenging activities
The method of the free radical scavenging activity not under different concentration knowns includes：
The reference substance of compound is obtained, and it is molten that compound control product are configured to into a series of samples with different concentration knowns
Liquid；
Each sample solution is added separately to into preparation experiment group solution in the microwell plate containing DPPH methanol solutions；And arrange blank right
According to group solution and DPPH matched group solution, the blank control group solution includes sample solution and methanol, the DPPH matched groups
Solution includes preparing the solvent and DPPH methanol solutions of sample solution；
The absorbance OD of each experimental group solution is determined by plate reading machineSample, blank control group solution absorbance ODIt is blankAnd
The absorbance OD of DPPH matched group solutionControl, and according to formula
SR=[1-(ODSample–ODIt is blank)/ODControl] × 100%
Determine free radical scavenging activity SR of the compound under different concentration knowns.
4. method as claimed in claim 3, it is characterised in that the condition of plate reading machine mensuration absorbance value includes：Test temperature：
20～50 DEG C, preferred 30～40 DEG C, more preferably 37 DEG C；Read plate speed：45s/ time；Acquisition time：45 minutes；Detection wavelength：
517nm。
5. the method for claim 1, it is characterised in that the blood for determining compound is tested using platelet aggregation inhibitory activity
Platelet aggregation suppression ratio is comprised the following steps：
The reference substance of compound is obtained, and compound control product are configured to into the need testing solution of concentration known；
Experimental group is configured to by the need testing solution, agonist and thrombocyte suspension, it is molten by thrombocyte suspension and buffering
Liquid is configured to blank control group；Negative control group is configured to by buffer solution, agonist and thrombocyte suspension；By described for trying
Product solution and buffer preparation are into background color group；
Determination experiment group, blank control group, the platelet aggregation rate maximum of negative control group；According to formula
[(negative control group maximum platelet aggregation rate-blank control group maximum platelet aggregation rate)-(experimental group platelet is most
Big aggregation rate-blank control group maximum platelet aggregation rate)]/(negative control group maximum platelet aggregation rate-blank control group
Maximum platelet aggregation rate) × 100%
Determine the platelet aggregation inhibition rate of compound.
6. method as claimed in claim 5, it is characterised in that determination experiment group, blank control group, negative control group blood it is little
The method of plate aggregation rate maximum includes：
By plate reading machine determination experiment group, blank control group, negative control group and background color group t absorbance, and
The absorbance of experimental group, blank control group, negative control group t is individually subtracted into the absorbance of background color group t
After value, experimental group absorbance, blank control group absorbance as t, negative control group absorbance；
And according to formula：Platelet aggregation ratet=(ODT=0-ODt)/ODT=0× 100%
Determine the time dependent kinetic curve of platelet aggregation rate, wherein, platelet aggregation ratetRepresent that experimental group, blank are right
According to group or negative control group t platelet aggregation rate, ODT=0Represent initial experimental group absorbance, blank control group
Absorbance or negative control group absorbance；ODtRepresent experimental group absorbance, the blank control group absorbance of t
Or negative control group absorbance；
The kinetic curve according to determined by determines the platelet aggregation rate maximum of experimental group, blank control group, negative control group
Value.
7. method as claimed in claim 5, it is characterised in that the agonist contains thromboxane A2 analog, preferably comprises
Thromboxane A2 analog U46619；Positive drug is Ticagrelor.
8. the method for claim 1, it is characterised in that in step (4), by high performance liquid chromatography or ultra high efficiency liquid
The content of each compound in phase chromatographic determination blood-activating stasis-removing kind Chinese medicine.
9. the method as any one of claim 1-8, it is characterised in that the blood-activating stasis-removing kind Chinese medicine is DANHONG injection
Liquid.
10. method as claimed in claim 9, it is characterised in that the compound contained in DANHONG ZHUSHEYE includes：Danshensu,
S-A Hydroxysafflor yellow A, L-phenylalanine, uridnine, cytidine, protocatechualdehyde, protocatechuic acid, rosmarinic acid, salvianolic acid B, red phenol
Sour A, salvianolic acid C, caffeic acid, alkannic acid, adenosine, syringoside and 5 hydroxymethyl furfural.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108918437A (en) * | 2018-05-16 | 2018-11-30 | 天津中医药大学 | A kind of the antioxidant activity detection method and application of blood-activating and stasis-removing preparation |
| CN110441456A (en) * | 2019-06-06 | 2019-11-12 | 天津宏仁堂药业有限公司 | A kind of 'Xue Fu Zhu Yu ' capsule Quality Control marker discovery method based on the steady multidimensional network mode living of amount |
| WO2021233239A1 (en) | 2020-05-18 | 2021-11-25 | 南方医科大学 | Application of active ingredient of root of ligulilobe sage or pharmaceutically acceptable salt thereof in preparing antiviral drug |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108918437A (en) * | 2018-05-16 | 2018-11-30 | 天津中医药大学 | A kind of the antioxidant activity detection method and application of blood-activating and stasis-removing preparation |
| CN110441456A (en) * | 2019-06-06 | 2019-11-12 | 天津宏仁堂药业有限公司 | A kind of 'Xue Fu Zhu Yu ' capsule Quality Control marker discovery method based on the steady multidimensional network mode living of amount |
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| WO2021233239A1 (en) | 2020-05-18 | 2021-11-25 | 南方医科大学 | Application of active ingredient of root of ligulilobe sage or pharmaceutically acceptable salt thereof in preparing antiviral drug |
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