CN114199840A - Heart comforting tablet quality control method based on biological effect - Google Patents
Heart comforting tablet quality control method based on biological effect Download PDFInfo
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- CN114199840A CN114199840A CN202111478096.9A CN202111478096A CN114199840A CN 114199840 A CN114199840 A CN 114199840A CN 202111478096 A CN202111478096 A CN 202111478096A CN 114199840 A CN114199840 A CN 114199840A
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- BBFYUPYFXSSMNV-UHFFFAOYSA-N quercetin-7-o-galactoside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=CC=3)OC2=C1 BBFYUPYFXSSMNV-UHFFFAOYSA-N 0.000 description 1
- 235000005412 red sage Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- ZMMKVDBZTXUHFO-DDWIOCJRSA-M sodium;(2r)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate Chemical compound [Na+].[O-]C(=O)[C@H](O)CC1=CC=C(O)C(O)=C1 ZMMKVDBZTXUHFO-DDWIOCJRSA-M 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000013513 substance screening Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
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- 229930189533 tanshinol Natural products 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
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Abstract
The invention provides a quality control method of a Xinkeshu tablet based on biological effect, which specifically comprises the following steps: preparing a methanol extract of a Xinkeshu tablet sample; adopting a zebra fish internode angiogenesis obstacle model to carry out biological effect detection on the Xinkeshu tablet sample methanol extract, wherein the drug concentration is based on the concentration of the Xinkeshu tablet sample of 85 microgram/ml; performing biological effect detection on the methanol extract of the KESHU tablet sample by adopting a zebra fish hyperlipidemia model, wherein the drug concentration is based on the concentration of the KESHU tablet sample of 680 microgrammes/ml; the length of the zebra fish internode blood vessels in the zebra fish internode angiogenesis obstacle model reaches over 2184 mu m, the lipid-lowering rate in the zebra fish hyperlipidemia model reaches over 43 percent, and the comfort tablet sample is judged to be a qualified sample when the two index values are reached simultaneously.
Description
Technical Field
The invention particularly relates to a quality control method of Xinkeshu tablets based on biological effect, belonging to the technical field of quality control of Chinese patent medicines.
Background
'Xinkeshu tablet' is composed of salvia miltiorrhiza, radix puerariae, pseudo-ginseng, hawthorn and costustoot, has the effects of promoting blood circulation to remove blood stasis, promoting qi circulation and relieving pain, and is commonly used for chest distress, palpitation, dizziness, headache and neck pain caused by qi stagnation and blood stasis; coronary heart disease, angina pectoris, hyperlipemia, hypertension, arrhythmia with the above syndromes. The current quality standard of the Chinese pharmacopoeia (2020 edition department) (hereinafter referred to as pharmacopoeia) includes items such as characters, identification, inspection, characteristic spectrum, content measurement and the like. The identification item mainly comprises a pseudo-ginseng and hyperin identification method. The characteristic map comprises 8 characteristic peaks such as sodium danshensu, protocatechualdehyde, 3 '-hydroxypuerarin, puerarin, 3' -methoxy puerarin, puerarin-7-xyloside, daidzin, salvianolic acid B, etc. The content measurement takes the contents of salvianolic acid B, tanshinol, protocatechualdehyde and salvianolic acid B, costunolide and dehydrocostus lactone as indexes. As can be seen from the above, the pharmacopeia quality control system basically covers the control of all the medicinal flavors of the prescription of the xinkeshu tablet, but the system involves a large and complex number of experiments, the correlation between the quality control index and the clinical efficacy is poor, and only a few compounds are detected, so that the overall quality of the xinkeshu tablet cannot be accurately evaluated. In recent years, quality control methods based on biological effects are receiving wide attention, and a new idea is provided for quality control of traditional Chinese medicines. The biological effect detection is a method for determining the effectiveness and safety of a medicament by using the biological effect of the medicament on a test system, taking biological statistics as a tool and applying specific experimental design from the viewpoint of correlation with clinical functional indications of the medicament. The quality control method based on biological effect can realize the quality control of Chinese patent medicine at the level of biological effect related to clinical efficacy without component theory. Especially for Chinese patent medicines with complex medicinal taste and undefined active ingredients, the integrity and superiority of the Chinese patent medicines can be highlighted.
The zebra fish has clear genetic background, has 87 percent homology with human genes, has highly similar physiological structures and functions of organs such as heart, liver, kidney and the like as well as wide P450 enzyme, has a plurality of drug metabolism approaches similar to human, is an internationally recognized model organism, and has continuous important attention to the application in early-stage drug activity and safety evaluation by international magazine, drug evaluation management department, famous toxicologists, large pharmaceutical enterprises and the like. The experiment using zebra fish as a model has the advantages of quick and efficient in vitro experiment, low component and small dosage, and also has the advantages of strong predictability of the experiment of mammals, high contrast, capability of observing multiple organs and the like. Research shows that zebra fish juvenile fish can predict cardiotoxicity more reliably than a HiPSC cardiomyocyte system, and the zebra fish is similar to dog prediction, so that tens of innovative drugs based on zebra fish phenotype screening are already in clinical research. At present, zebra fish models are widely applied to the aspects of traditional Chinese medicine activity evaluation, drug effect substance screening, action mechanism research and the like.
Chinese literature, "application research of zebra fish hyperlipidemia model in quality control of Shanqing tablets" (Guo palaine, Zhou Juan, etc., Chinese medicine standard 2013, 14 th volume, 4 th phase), discloses biological activity evaluation and quality stability detection of the blood lipid reducing effect of the Shanqing tablets by using the zebra fish hyperlipidemia model. Chinese literature, "Shanqing tablet auxiliary blood lipid lowering action clinical observation" (Von Feiyu, Tang Gift, Zhejiang traditional Chinese and western medicine combined impurities 2009, 19 th volume 9) discloses that the Shanqing tablet has blood lipid lowering effect, mainly contains red peony root extract and dangshen extract, and both red peony root and dangshen have certain blood lipid lowering effect. Because the sheet of the liriodendron purpureum has single function and relatively simple composition, the quality stability can be detected by only using a single biological effect function index; however, the Chinese patent medicine has the characteristics and advantages of multiple components, multiple targets and multiple ways for treating diseases, most Chinese patent medicines have the characteristics of multiple compatible medicines, complex component compositions, wide functional indications and the like, can take effect synergistically through multiple different pharmacological actions, is not feasible to control the quality by only utilizing a single biological effect function index, and is easy to have wrong assessment.
The XINKESHU tablet is prepared from Saviae Miltiorrhizae radix, radix Puerariae, radix aucklandiae, Notoginseng radix, and fructus crataegi; the Xinkeshu tablet has various biological activities of expanding coronary artery, improving myocardial ischemia, promoting blood circulation to remove blood stasis, improving cardiac microcirculation, increasing coronary blood flow, enhancing activities of mitochondria of myocardial cells and Adenosine Triphosphate (ATP) enzyme and the like. In the prior art, the quality control of the traditional Chinese medicine with complex prescription and multiple functions and main indications by utilizing biological effect is not reported.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a quality control method of a Xinkeshu tablet based on biological effect.
According to experimental research, the single-function biological effect of the Xinkeshu tablet is easy to cause wrong evaluation, and even some functional biological effects have poor stability and cannot be used as the evaluation index of the biological effect of the Xinkeshu tablet. According to the invention, a large number of experimental researches show that the quality control of the Xinkeshu tablet based on biological effect can be realized by combining a zebra fish internode angiogenesis obstacle model and a hyperlipidemia model, a quality control method of the Xinkeshu tablet based on biological effect is established, and methodology investigation is carried out. The invention provides a new idea and method for the quality control research based on biological effect for the Chinese patent medicine with complex prescription and multiple functions and main indications.
Description of terms:
(1) internodal vessels (ISV).
(2) Tail region intensity of lipid staining (IOD).
(3) hpf (hours post fertilization): biological specific term, refers to the time after fertilization. For example 24hpf refers to an embryo 24 hours after fertilization.
(4) dpf (days post fertilization)): biological specific term, refers to the number of days after fertilization. For example, 1dpf refers to an embryo at 1 day after fertilization.
The technical scheme of the invention is as follows:
a quality control method of Xinkeshu tablet based on biological effect comprises the following steps:
(1) preparing a methanol extract of a Xinkeshu tablet sample;
(2) performing biological effect detection on the methanol extract of the Xinkeshu tablet sample in the step (1) by adopting a zebra fish internode angiogenesis obstacle model, wherein the medicine concentration is based on that the concentration of the Xinkeshu tablet sample is 85 microgram/ml; performing biological effect detection on the methanol extract of the Xinkeshu tablet sample in the step (1) by adopting a zebra fish hyperlipidemia model, wherein the medicine concentration is based on the concentration of the Xinkeshu tablet sample of 680 mu g/ml;
(3) step (2), detecting results of the central comfort tablet sample: the length of the zebra fish internode blood vessel in the zebra fish internode angiogenesis obstacle model reaches over 2180 mu m, the lipid-lowering rate in the zebra fish hyperlipidemia model reaches over 40 percent, and the sample of the Chinese medicinal comfort piece is judged to be a qualified sample when the two index values are reached.
Preferably, according to the present invention, methanol is used in step (1) to extract the sample of the perhexiline tablet.
Further preferably, the preparation method of the sample extract of the Xinkeshu tablet by using methanol comprises the following steps:
taking a Xinkeshu tablet sample, grinding into powder, weighing, adding 10 times of methanol, soaking at normal temperature for 30min, ultrasonically extracting for 30min, carrying out solid-liquid separation, retaining an extracting solution, repeatedly extracting the residual medicine residues for 2 times, retaining the extracting solution, then combining the extracting solutions, concentrating to obtain an extract, and drying the extract to obtain the Xinkeshu sample extract.
Preferably, the Xinkeshu tablet sample is ground into powder, weighed, added with 10 times of methanol, soaked for 30min at normal temperature, ultrasonically extracted for 30min, subjected to suction filtration, retained in the extracting solution, the dregs of a decoction are repeatedly extracted for 2 times, the extracting solution is retained, then the extracting solutions are combined, the extracting solution is concentrated under reduced pressure to 1/10 volume, transferred to water bath and steamed to obtain an extract, and then the extract is dried in vacuum to obtain the Xinkeshu tablet sample extract.
According to the invention, in the step (1), the extract of the sample of the cardiopulmonary bypass tablet is prepared into stock solution containing 100mg/ml of the extract of the cardiopulmonary bypass tablet sample by using 10% of dimethyl sulfoxide (DMSO) by volume fraction, and the concentration of the cardiopulmonary bypass tablet sample is calculated according to the yield of the extract of the cardiopulmonary bypass tablet sample.
Preferably, the length of the zebra fish internode blood vessels in the zebra fish internode angiogenesis disorder model in the step (3) reaches 2180 μm-2382 μm, and the lipid-lowering rate in the zebra fish hyperlipidemia model reaches 43% -56%.
Preferably, according to the invention, in step (2) the internodal vessels are measured using Imagepro Plus 5.1.
Preferably, in step (2), Imagepro Plus 5.1 is used to measure the staining intensity of the cardiopulmonary bypass treatment group and calculate the fat reduction rate (%) (model group IOD-cardiopulmonary bypass treatment group IOD)/model group IOD × 100%; the IOD is the intensity of the tail region lipid staining.
Preferably, in step (2), the method for detecting the biological effect of the extract of the central comfort tablet sample in step (1) by using the zebra fish internode angiogenesis disorder model comprises the following steps:
selecting 23hpf Tg (flk1: EGFP) blood vessel green fluorescence labeling transgenic zebra fish, demoulding by 1mg/ml streptokinase E, randomly dividing the health embryos after demoulding into a blank control group, a model group and a cardiopalmus treatment group, randomly distributing the embryos, wherein each group is provided with more than 2 parallel embryos, and each group is provided with 10-12 parallel embryos. Blank control group administration: the fish culture water contains DMSO with the volume fraction of 0.1%; model group administration: fish water containing 0.1% DMSO by volume + PTK787 at a final concentration of 0.175. mu.g/ml; the cardiotonic tablet treatment group was given: the fish culture water contains 0.1 percent of DMSO by volume fraction, PTK787 with the final concentration of 0.175 mu g/ml and Xinkeshu tablets with the final concentration of 85 mu g/ml; incubating at the temperature of 28 ℃ until the incubation temperature reaches 47hpf, and observing the blood vessel morphology of the zebra fish embryo under a fluorescence microscope;
quantitative analysis: internodal blood vessels (ISVs) were measured using Imagepro Plus 5.1.
Preferably, in step (2), the method for detecting the biological effect of the extract of the sample of the central comfort tablet in step (1) by using the zebra fish hyperlipidemia model comprises the following steps:
selecting 5dpf (days post fertilization) wild AB line zebra fish, randomly dividing the zebra fish into a blank control group, a model group and a Xinkeshu piece treatment group, wherein 28-32 zebra fish are selected from each group, the blank control group is given with fish rearing water containing 0.015mg/ml Phenylthiourea (PTU), the model group and the Xinkeshu piece treatment group are fed with egg yolk solution with the mass fraction of 0.1%, treating for 48h, removing yolk solution, feeding fish culture water containing 0.015mg/ml PTU + 0.1% DMSO to a blank control group and a model group, feeding fish culture water containing 0.015mg/ml PTU + 0.1% DMSO +680 mu g/ml Xinkeshu tablet to a Xinkeshu tablet treatment group, incubating at 28 ℃, treating for 24h for liquid change, continuously treating for 24h, the staining intensity of the cardiopulmonary bypass treatment group was measured using Imagepro Plus 5.1 and the lipid reduction rate (%) was calculated as (model group IOD-cardiopulmonary bypass treatment group IOD)/model group IOD × 100%.
The quality control method is applied to quality inspection of the cardiotocolic tablet sample.
According to the invention, the method is preferably applied to detection of a Xinkeshu tablet lacking sample and a Xinkeshu tablet feeding halved sample.
Drawings
FIG. 1 is a graph showing the result of the determination of the angiogenesis promoting activity of a qualified sample of a KEKESHU tablet;
in the figure: a is a surface pattern diagram of cardiotonic tablet sample for promoting angiogenesis; B. c, D are respectively a bar chart of the results of different 3-day repeated experiments of the angiogenesis promoting activity of qualified samples of the Xinkeshu tablet;
FIG. 2 is a graph showing the results of measurement of the angiogenesis promoting activity of each sample;
in the figure: a is a phenotype graph of angiogenesis promotion of qualified Xinkeshu tablet samples, and B is a column graph of angiogenesis promotion activity of qualified Xinkeshu tablet samples;
c is a phenotype diagram of angiogenesis promotion of the deficient negative sample and the feeding reduced half negative sample, and D is a column diagram of angiogenesis promotion activity of the deficient negative sample and the feeding reduced half negative sample.
FIG. 3 is a result chart of the measurement of hypolipidemic activity of a qualified Xinkeshu tablet sample;
in the figure: a is a blood fat reducing activity phenotype diagram of a qualified Xinkeshu tablet sample, and B, C, D is a bar chart of different 3-day repeated experiment IOD results of the qualified Xinkeshu tablet sample respectively; F. e, G are respectively column charts of blood lipid reducing rate results of repeated experiments for 3 days on qualified Xinkeshu tablets.
FIG. 4 is a diagram of the evaluation result of the hypolipidemic activity of qualified samples of each lot of perhexiline tablets;
in the figure: a is a blood fat reducing activity phenotype diagram of qualified samples of each batch of the Xinkeshu tablets; b is a column diagram of the hypolipidemic active IOD of qualified samples of each batch of the Xinkeshu tablets; and C is a column diagram of the blood fat reducing activity and the blood fat reducing rate of qualified samples of each batch of the Xinkeshu tablets.
FIG. 5 is a diagram of the evaluation result of the hypolipidemic activity of the negative deficient sample and the negative half-reduced sample;
in the figure: a is a phenotype diagram of the hypolipidemic activity of the negative sample of the lacking side and the negative sample of the feeding material halving; b is a column diagram of the blood fat reducing activity IOD of the deficient negative sample and the feeding reduced half negative sample; c is a column diagram of the blood fat reducing activity and the blood fat reducing rate of the deficient negative sample and the feeding reduced half negative sample.
FIG. 6 is a graph showing the result of determination of antithrombotic activity of the perhexiline tablets;
in the figure: a is the surface pattern of antithrombotic activity of the Xinkeshu tablet; B. c, D is a graph of the results of the Area repeated experiments for different 3 days; E. f, G graphs showing IOD results of experiments repeated for different 3 days.
Advantageous effects
1. According to the invention, a large number of experimental researches show that the quality control of the Xinkeshu tablet based on biological effect can be realized by combining a zebra fish internode angiogenesis obstacle model and a hyperlipidemia model, a quality control method of the Xinkeshu tablet based on biological effect is established, and methodology investigation is carried out. The invention provides a new idea and method for the quality control research based on biological effect for the Chinese patent medicine with complex prescription and multiple functions and main indications.
2. The invention establishes the integral quality control method of the Xinkeshu tablets based on the biological effect of the zebra fish, and provides important supplement for the current standard of the Xinkeshu tablets with chemical components as detection indexes.
3. The invention establishes the quality control method of the biological effect of the Xinkeshu tablet by taking the angiogenesis promotion and the blood fat reduction activity as indexes, the method is closely related to the clinical efficacy of the Xinkeshu tablet, and the quality of the Xinkeshu tablet can be more accurately controlled regardless of the theory.
4. The method established by the invention has demonstration effect on the application of the zebra fish in the quality control method of the biological effect of the Chinese patent medicine with complex prescription and multiple functional indications.
Detailed Description
The invention is further illustrated with reference to the following examples, without limiting the scope of the invention.
The details not described in the examples are according to the state of the art.
Sources of materials
Fish farming water: containing 5.0mM NaCl, 0.17mM KCl, 0.4mM CaCl2，0.16mM MgSO4。
0.1% methylene blue: 100mg of methylene blue powder is weighed and dissolved in 100ml of purified water, and the mixture is stored at 4 ℃ for standby.
1mg/ml PTU: weighing 1g of phenylthiourea powder, dissolving in 1L of purified water, performing ultrasonic treatment for 2h to fully dissolve, and storing at 4 ℃ for later use.
10% dimethylsulfoxide: dimethyl sulfoxide and purified water in a volume ratio of 1: 9 mixing and preparing.
1mg/ml pronase E: 1mg of the pronase E is dissolved in 1ml of purified water and is prepared as it is.
0.1% egg yolk solution: 1g of egg yolk powder was weighed out and dissolved in 1000ml of fish farming water containing 0.015mg/ml of PTU, and sufficiently stirred uniformly.
25% propylene glycol solution: PBS and propylene glycol in a volume ratio of 3: 1, mixing and preparing.
50% propylene glycol solution: PBS and propylene glycol were mixed in a volume ratio of 1: 1, mixing and preparing.
75% propylene glycol solution: PBS and propylene glycol were mixed in a volume ratio of 1: 3, mixing and preparing.
0.3% oil red O staining solution: taking 0.25g of oil red O powder, adding 50ml of isopropanol solution, performing ultrasonic treatment for 90min to fully dissolve, and storing at 4 ℃ in dark for a long time. When in use, a proper amount of the water is mixed with purified water according to the volume ratio of 3: 2, mixing evenly, and filtering by a 0.22 mu m microporous membrane.
PBT: 100 μ l Tween 20 was added to 100ml PBS.
Xinkeshu tablets are available from Shandong Wahua pharmaceutical science and technology Co., Ltd under the batch numbers 0100714, 0110277, 0110278, 0110279, 0110280 and 0110281.
The zebra fish is provided by a zebra fish drug screening platform (a zebra fish model of human diseases and a drug screening engineering research center) of biological research institute of academy of sciences of Shandong province, and can also be purchased by a national zebra fish resource center.
PBS:1×PBS:GENVIEW,Cat#:GS3101-500mL。
Example 1
Preparation of sample methanol extract
The preparation method of the qualified Xinkeshu tablet sample, the deficient negative sample and the half-feeding reduced negative sample methanol extract comprises the following steps:
grinding the sample into powder, weighing, transferring into a beaker, adding 10 times of methanol, soaking at normal temperature for 30min, ultrasonically extracting (40Khz) for 30min, filtering, and repeatedly extracting the residue for 2 times. Mixing the filtrates, concentrating under reduced pressure to 1/10 volume, transferring to evaporating dish, steaming in water bath to obtain extract, vacuum drying to obtain methanol extract, and calculating the yield. Preparing stock solution containing 100mg/ml of the ethanol extract of the Xinkeshu tablets by using 10% of dimethyl sulfoxide (DMSO), and calculating the concentration of the Xinkeshu tablets according to the yield when in use.
The extraction yield of the methanol extract of each sample is shown in table 1:
TABLE 1
Example 2
A detection method for evaluating the activity of different samples on internode angiogenesis by adopting a zebra fish internode angiogenesis obstacle model comprises the following steps:
(1) 23hpf Tg (flk1: EGFP) was selected as a vascular green fluorescent marker for transgenic zebra fish, which was then decapsulated with 1mg/ml of pronase E. The health embryos after stripping are randomly divided into a blank control group, a model group and a Xinkeshu tablet treatment group, the embryos are randomly placed in 24-hole plates, each group is provided with 3 parallel holes, and each hole contains 10 embryos. Blank control group: the fish culture water contains DMSO with the volume fraction of 0.1%; model group: fish water containing 0.1% DMSO by volume + PTK787 at a final concentration of 0.175. mu.g/ml; the Xinkeshu tablet treatment group: the fish culture water contains 0.1% DMSO by volume fraction, PTK787 with a final concentration of 0.175 μ g/ml, and cardiopalmus tablets with final concentrations of 21.25, 42.5 and 85 μ g/ml respectively. And (3) when the culture temperature is 28 ℃ and the culture is incubated to 47hpf, observing the result after 24h of administration (14 h of illumination and 10h of dark place), observing the blood vessel morphology of the zebra fish embryo under a fluorescence microscope, and taking a picture at a side view angle.
(2) Quantitative analysis: the ISV was measured using Imagepro Plus 5.1 and statistically analyzed, and the results of the measurements are shown in FIG. 1.
The ISV of the zebra fish treated by the Xinkeshu tablets is larger than that of a model group, and reaches a statistical significance level (p-value is less than 0.05), which indicates that the Xinkeshu tablets at the concentration have a promoting effect on angiogenesis of the zebra fish.
The zebra fish is anesthetized by soaking 0.3 per mill of tricaine for 40-90 s. Collecting an image, namely fixing the side body position of the zebra fish embryo, and observing and taking a picture under a fluorescence microscope to obtain a zebra fish embryo side position image; the acquired images were obtained at the same magnification.
ISV is an evaluation index of zebra fish angiogenesis, and the influence of a drug on angiogenesis can be estimated. When the medicine has the function of inhibiting angiogenesis, the ISV has the phenomena of quantity loss, length shortening, developmental deformity and the like; when the medicine has the effect of promoting angiogenesis, the phenomena of ISV number loss, length shortening, developmental deformity and the like can be reversed. As shown in figure 1, 85 mug/ml Xinkeshu tablet has remarkable angiogenesis promoting activity.
The detection method is examined for daytime precision.
The measurement was carried out in three days according to the methods in steps (1) and (2), and the reproducibility of the method was evaluated based on the Relative Standard Deviation (RSD) of the results of 3 activity measurements, with a final concentration of 85. mu.g/ml in the COKE tablet-treated group, and the results of the measurements are shown in Table 2.
TABLE 2 precision investigation of the cardiovascular angiogenesis promoting Activity experiments
As shown in figure 1, 85 microgram/ml of the cardiotonic tablet has remarkable angiogenesis promoting activity, and the daytime precision RSD is 4.52% (see table 2), which indicates that the method can be used for measuring the angiogenesis promoting activity of the cardiotonic tablet. The concentration of the Xinkeshu tablets is 85 mug/ml as a standard, the corresponding concentration of each sample is calculated according to the yield, the experiment is carried out under the same condition, and the influence of different samples on the angiogenesis of the internodes of the zebra fish is evaluated by adopting a zebra fish internode angiogenesis model.
Example 3
A method for evaluating the activity of different samples on internode angiogenesis by adopting a zebra fish internode angiogenesis obstacle model comprises the following steps:
(1) 23hpf Tg (flk1: EGFP) was selected as a vascular green fluorescent marker for transgenic zebra fish, which was then decapsulated with 1mg/ml of pronase E. Dividing the stripped healthy embryos into a blank control group, a model group and each sample treatment group at random, placing the embryos in 24-hole plates at random, wherein each group is provided with 3 parallel holes, and each hole contains 10 embryos. Blank control group: the fish culture water contains DMSO with the volume fraction of 0.1%; model group: fish water containing 0.1% DMSO by volume + PTK787 at a final concentration of 0.175. mu.g/ml; each sample treatment group: the fish water contained a volume fraction of 0.1% DMSO + PTK787+ at a final concentration of 0.175. mu.g/ml + respective samples at a final concentration of 85. mu.g/ml. And (3) when the culture temperature is 28 ℃ and the culture is incubated to 47hpf, observing the result after 24h of administration (14 h of illumination and 10h of dark place), observing the blood vessel morphology of the zebra fish embryo under a fluorescence microscope, and taking a picture at a side view angle.
(2) Quantitative analysis: the ISV was measured using Imagepro Plus 5.1 and statistically analyzed, and the results are shown in FIG. 2 and Table 3.
The ISV of the zebra fish treated by the Xinkeshu tablets is larger than that of a model group, and reaches a statistical significance level (p-value is less than 0.05), which indicates that the Xinkeshu tablets at the concentration have a promoting effect on angiogenesis of the zebra fish.
The zebra fish is anesthetized by soaking 0.3 per mill of tricaine for 40-90 s. Collecting an image, namely fixing the side body position of the zebra fish embryo, and observing and taking a picture under a fluorescence microscope to obtain a zebra fish embryo side position image; the acquired images are obtained at the same magnification,
as shown in fig. 2AB, each qualified lot of cardiocosable tablets had significant pro-angiogenic activity. As shown in fig. 2CD, only the 1/2DS (red sage root halved) sample among the deficient or minus negative samples had pro-angiogenic activity. As shown in Table 3, the lengths of the blood vessels between the zebra fish internodes in the group treated by the 6 batches of qualified Xinkeshu tablets are obviously increased compared with the model group, and the lengths are 2184.20-2381.64 μm. The internode blood vessel length of the feeding halved negative sample treatment group is 1721.03-2070.51 mu m. The internode blood vessels of the deletion negative sample treatment groups were 1628.74-1739.60 μm. The angiogenesis promoting activity of the negative sample is lower than that of the qualified Xinkeshu tablet sample; the experimental result shows that the length of the zebra fish internode blood vessel of the qualified sample reaches 2184.20-2381.64 mu m.
However, as shown in fig. 2D, the ISV 2070.51 of the 1/2DS (salvia miltiorrhiza minus square negative sample) treated group was significantly different from that of the PTK787 treated group, indicating that the 1/2DS negative sample has pro-angiogenic activity and is very close to that of the qualified sample, and the sample cannot be determined as a non-qualified sample only from the experimental results of the pro-angiogenic; the detection result of the sample is easy to be misjudged.
TABLE 3 internodal vessel length for each sample treatment group
Example 4
A detection method for evaluating the blood fat reducing activity of different samples by adopting a zebra fish hyperlipidemia model comprises the following steps:
(1) selecting 5dpf (days post fertilization) wild AB line zebra fishes, randomly dividing the zebra fishes into a blank control group, a model group and a cardioplegia treatment group, wherein each group comprises 30 strips, the blank control group is fed with fish culture water containing 0.015mg/ml PTU, the model group and the cardioplegia treatment group are fed with egg yolk solution with the mass fraction of 0.1%, after 48 hours of treatment, the egg yolk solution is removed, the blank control group and the model group are fed with fish culture water containing 0.015mg/ml PTU + 0.1% DMSO, the cardioplegia treatment group is fed with fish culture water containing 0.015mg/ml PTU + 0.1% DMSO + the final concentration of each of cardioplegia tablets is 170, 340 and 680 μ g/ml, the incubation temperature is 28 ℃, the treatment is carried out for 24 hours for liquid change (14 hours of illumination and 10 hours of dark place), and after 24 hours of continuous treatment (14 hours of illumination and 10 hours of dark place), oil red O dyeing is carried out.
(2) Dyeing with oil red O:
firstly, fixing: removing the treatment liquid of zebra fish, adding 4% by mass of Paraformaldehyde (PFA), and fixing at room temperature for 4h or overnight at 4 ℃.
And (2) dehydrating: dehydration with 25%, 50%, 75% and 100% propylene glycol in sequence for 10 min.
③ dyeing: 0.3% oil red O staining solution is used for staining for 4h at 28 ℃.
Fourthly, rehydration: decolorized with 100%, 75%, 50%, 25% propylene glycol and PBS in that order for 10 minutes.
Fifth, elution: eluting with PBT for 15min and 4 times.
(3) Quantitative analysis
According to the prior art, the zebra fish dyed with the oil red O in the step (2) is observed under a microscope, whether the hyperlipidemia model is successfully established or not is judged qualitatively by observing the blood fat dyeing intensity of the zebra fish, after the zebra fish hyperlipidemia model is successfully modeled, Imagepro Plus 5.1 is used for measuring the blank control group IOD and the model group IOD, Imagepro Plus 5.1 is used for measuring the dyeing intensity of the cardiopalmus treating group, and the fat reduction rate is calculated. Fat reduction rate (%) (model group IOD-xinkeshu tablet treatment group IOD)/model group IOD × 100%.
IOD of the Dangxing tablet treatment group is lower than that of the model group and reaches a statistical significance level (p-value is less than 0.05), which indicates that the Xinkeshu tablets at the concentration have a treatment effect on the zebra fish hyperlipidemia.
Hyperlipidemia refers to abnormal metabolism of lipid components in human body, and is mainly manifested by various dyslipidemia components with high cholesterol (TC) and/or high Triglyceride (TG), high low-density lipoprotein cholesterol (LDL-C) or low high-density lipoprotein cholesterol (HDL-C) in plasma. Hyperlipidemia is a major pathogenic factor of atherosclerosis, and often causes serious consequences due to invasion of important organs, such as diabetes, cardiovascular and cerebrovascular diseases, intractable hypertension and nephrotic syndrome, pancreatitis, lithiasis, fatty liver, etc. In the invention, the yolk solution with the mass fraction of 0.1% is fed to the zebra fish to form a zebra fish hyperlipidemia disease model. As shown in figure 3, the fat reducing rate of the Xinkeshu tablet treatment group is increased in a dose-dependent manner, and has a significant difference with that of the model group, and the final concentration of the Xinkeshu tablet is selected to be 680 mu g/ml for subsequent experiments.
The detection method is examined for daytime precision.
The measurement was carried out in three days according to the methods in steps (1) and (2), and the reproducibility of the method was evaluated based on the Relative Standard Deviation (RSD) of the results of 3 activity measurements, with a final concentration of the COKE tablet in the COKE tablet treatment group of 680. mu.g/ml, and the results of the measurements are shown in Table 4.
TABLE 4 precision investigation of the hypolipidemic Activity of XINKESHU tablet
As shown in figure 3, the fat reducing rate of the Xinkeshu tablet treatment group is increased in a dose-dependent manner, and has a significant difference with that of the model group. The blood fat reducing daytime precision RSD of the 680 mu g/ml Xinkeshu tablet treatment group is 7.89% (see table 4), which indicates that the method can be used for the blood fat reducing activity determination of the Xinkeshu tablets. The concentration of the Xinkeshu tablets is 680 mu g/ml as a standard, the corresponding concentration of each sample is calculated according to the yield, the experiment is carried out under the same condition, and the influence of different samples on the hyperlipidemia of the zebra fish is evaluated by adopting a zebra fish blood lipid reducing model.
Example 5
A detection method for evaluating the blood fat reducing activity of different samples by adopting a zebra fish hyperlipidemia model comprises the following steps:
(1) selecting 5dpf (days post fertilization) wild AB line zebra fishes, randomly dividing the zebra fishes into a blank control group, a model group and each sample treatment group, wherein each group comprises 30 strips, feeding the blank control group with 0.015mg/ml PTU fish culture water, feeding the model group and each sample treatment group with an egg yolk solution with the mass fraction of 0.1%, removing the egg yolk solution after 48h of treatment, feeding the blank control group and each sample treatment group with fish culture water containing 0.015mg/ml PTU + 0.1% DMSO, feeding each sample treatment group with fish culture water containing 0.015mg/ml PTU + 0.1% DMSO + the final concentration of 680 mu g/ml, incubating at the temperature of 28 ℃, treating 24h of liquid change (illumination 14h is protected from light for 10h), continuously treating for 24h (illumination 14h is protected from light for 10h), and then carrying out oil red O dyeing.
(2) Dyeing with oil red O:
firstly, fixing: removing the treatment liquid of zebra fish, adding 4% by mass of Paraformaldehyde (PFA), and fixing at room temperature for 4h or overnight at 4 ℃.
And (2) dehydrating: dehydration with 25%, 50%, 75% and 100% propylene glycol in sequence for 10 min.
③ dyeing: 0.3% oil red O staining solution is used for staining for 4h at 28 ℃.
Fourthly, rehydration: decolorized with 100%, 75%, 50%, 25% propylene glycol and PBS in that order for 10 minutes.
Fifth, elution: eluting with PBT for 15min and 4 times.
(3) Quantitative analysis
According to the prior art, the zebra fish dyed with the oil red O in the step (2) is observed under a microscope, whether the hyperlipidemia model is successfully established or not is judged qualitatively by observing the blood fat dyeing intensity of the zebra fish, after the zebra fish hyperlipidemia model is successfully modeled, Imagepro Plus 5.1 is used for measuring the blank control group IOD and the model group IOD, Imagepro Plus 5.1 is used for measuring the dyeing intensity of the cardiopalmus treating group, and the fat reduction rate is calculated. Fat reduction rate (%) (model group IOD-xinkeshu tablet treatment group IOD)/model group IOD × 100%.
As shown in fig. 4 and table 5, each qualified perhexiline tablet has significant hypolipidemic activity, with a lipid-lowering rate of 43.95% -55.20%. As shown in fig. 5 and table 5, except for the lack of the salvia miltiorrhiza negative sample (QDS), each negative sample had a certain lipid-lowering effect, but the activity was significantly weakened compared to the qualified sample. The lipid-lowering rate of the deficient sample is only 7.49% -13.43%, and the lipid-lowering rate of the reduced negative sample is 17.22% -31.64%.
The lipid-lowering rate of the qualified sample is 43.95-55.20% according to the experimental result. However, the lipid-lowering rate of the negative sample 1/2SZ reaches 31.64%, compared with the lipid-lowering activity of a model group, the lipid-lowering activity effect is obvious, the misjudgment is easy to occur when the negative sample 1/2SZ is evaluated by only using the lipid-lowering activity, and the angiogenesis promoting activity of the negative sample 1/2SZ in the embodiment 3 is not obvious because the blood vessel length is only 1853.66 μm.
TABLE 5 lipid lowering Rate for each sample treatment group
Comparative example 1
A detection method for evaluating the antithrombotic activity of the Xinkeshu tablets by adopting a zebra fish antithrombotic model comprises the following steps:
(1) the embryos of AB strain zebrafish with 72hpf are taken, normal-developing embryos are selected under a stereomicroscope, and transferred into 6-well culture plates with 30 embryos per well. Set up 6 experimental groups. The blank group was given PTU fish water, the model group was given PTU fish water, and after placing in an incubator at 28 ℃ for 6 hours, the final concentration was changed to 80 μ M AA solution. The positive control group was administered ASP solution to a final concentration of 22.5. mu.g/mL, and placed in an incubator at 28 ℃ for 6 hours, and then changed to AA solution at a final concentration of 80. mu.M. The administration group is administered with ethanol extract of KEKESHU tablet of 10.625, 21.25, 42.5, 85, 170, 340, and 680 μ g/ml, and placed in 28 deg.C incubator for 6 hr, and then changed into AA solution of 80 μ M.
(2) The drug treatment is carried out for 6h, AA molding is carried out for 1h, then the product is taken out and dyed with 1mg/mL o-dianisidine dyeing solution for 10min in a dark place. The fish is washed 3 times, and each group of zebra fish is placed under a microscope to observe the venous thrombosis and take a picture of the heart (the magnification is 80 times). The stained areas and optical densities of the erythrocytes of the heart were quantitatively analyzed with image pro plus 5.1 image processing software.
FIG. 6A is a table of antithrombotic activity of XINKESHU tablet, FIG. 6B, C, D is a bar chart of Area of 3-day repeat experiment, and FIG. 6E, F, G is a bar chart of IOD of 3-day repeat experiment. As shown, after AA molding, the zebrafish heart area was significantly reduced and the IOD was significantly reduced compared to the blank control group. And the cardioplegia tablet 170-680 mu g/mL can obviously reduce the thrombus at the heart of the zebra fish caused by AA. However, through methodology examination, the invention discovers that the blank control group dyeing area RSD reaches 19.36%, the IOD value RSD reaches 16.63%, the model group dyeing area RSD reaches 14.40%, and the cardiopalmus tablet treatment group IOD value RSD reaches 11.30%, which indicates that the stability of the experiment is poorer than that of the interjoint angiogenesis activity model and the blood fat reducing model (tables 6 and 7), and the experiment is difficult to be used as a biological effect index for quality control of the cardiopalmus tablet.
TABLE 6 antithrombotic Activity test day precision investigation (Area)
TABLE 7 precision Investigation (IOD) of the antithrombotic Activity of Xinkeshu tablets
In summary, as shown in fig. 2D, the ISV 2070.51 of the 1/2DS (salvia miltiorrhiza minus square negative sample) treated group is significantly different from that of the PTK787 treated group, which indicates that the 1/2DS negative sample has the angiogenesis promoting activity and is very close to the angiogenesis promoting activity of the qualified sample, and the sample cannot be determined to be a non-qualified sample only from the angiogenesis promoting experimental result; the detection result of the sample is easy to be misjudged. As shown in fig. 5C and table 5, the lipid-lowering rate of the 1/2DS negative sample treatment group was only 28.97%, which was significantly lower than the lipid-lowering rate of the qualified sample (43.95% -55.20%), and the sample was judged to be failed.
The lipid-lowering rate of the subtractive negative sample 1/2SZ reaches 31.64%, compared with the lipid-lowering activity of a model group, the hypolipidemic activity is remarkable, the hypolipidemic activity is only used for evaluating the subtractive negative sample 1/2SZ, misjudgment is easy to occur, the blood vessel length of the subtractive negative sample 1/2SZ in the embodiment 3 is only 1853.66 μm, the angiogenesis promoting activity is not remarkable, and the sample can be judged to be unqualified.
The Xinkeshu tablet has the treatment characteristics of multiple components, multiple targets and multiple ways, adopts a single biological activity model, and is difficult to comprehensively evaluate the quality; the invention finds that the method for evaluating the antithrombotic activity of the Xinkeshu tablets by adopting a zebra fish antithrombotic model has poor stability and is difficult to be used as a biological effect index for controlling the quality of the Xinkeshu tablets; according to the invention, a large number of experiments show that the length of the zebra fish internode blood vessel in the zebra fish internode angiogenesis disorder model reaches over 2184 mu m, the lipid-lowering rate in the zebra fish hyperlipidemia model reaches over 43 percent, and the cardiovascular relaxation sample can be judged to be a qualified sample when the two index values are reached, preferably, the length of the zebra fish internode blood vessel in the zebra fish internode angiogenesis disorder model reaches 2180 mu m-2382 mu m, and the lipid-lowering rate in the zebra fish hyperlipidemia model reaches 43 percent-56 percent. The method can effectively reduce the phenomenon of misjudgment of the bioactivity detection of the Xinkeshu tablet sample, and realizes the quality control of the Xinkeshu tablet based on biological effect.
Claims (10)
1. A quality control method of a Xinkeshu tablet based on biological effect is characterized by comprising the following steps:
(1) preparing a methanol extract of a Xinkeshu tablet sample;
(2) performing biological effect detection on the methanol extract of the Xinkeshu tablet sample in the step (1) by adopting a zebra fish internode angiogenesis obstacle model, wherein the medicine concentration is based on that the concentration of the Xinkeshu tablet sample is 85 microgram/ml; performing biological effect detection on the methanol extract of the Xinkeshu tablet sample in the step (1) by adopting a zebra fish hyperlipidemia model, wherein the medicine concentration is based on the concentration of the Xinkeshu tablet sample of 680 mu g/ml;
(3) step (2), detecting results of the central comfort tablet sample: the length of the zebra fish internode blood vessel in the zebra fish internode angiogenesis obstacle model reaches over 2180 mu m, the lipid-lowering rate in the zebra fish hyperlipidemia model reaches over 40 percent, and the sample of the Chinese medicinal comfort piece is judged to be a qualified sample when the two index values are reached.
2. The method according to claim 1, wherein methanol is used for extracting the sample extract of the KESHU tablet in step (1);
preferably, the preparation method of the sample extract of the Xinkeshu tablet by using methanol comprises the following steps:
taking a Xinkeshu tablet sample, grinding the sample into powder, weighing, adding 10 times of methanol, soaking at normal temperature for 30min, ultrasonically extracting for 30min, carrying out solid-liquid separation, retaining an extracting solution, repeatedly extracting the residual medicine residues for 2 times, retaining the extracting solution, then combining the extracting solutions, concentrating to obtain an extract, and drying the extract to obtain a Xinkeshu sample extract;
preferably, the Xinkeshu tablet sample is ground into powder, weighed, added with 10 times of methanol, soaked for 30min at normal temperature, ultrasonically extracted for 30min, subjected to suction filtration, the extracting solution is reserved, the medicine residue is repeatedly extracted for 2 times, the extracting solution is reserved, then the extracting solutions are combined, the extracting solution is concentrated under reduced pressure to 1/10 volume, transferred to water bath and steamed to an extract, and then the extract is subjected to vacuum drying to obtain the Xinkeshu tablet sample extract.
3. The method according to claim 1, wherein in the step (1), the extract of the sample of the KECOSHU tablet is prepared into a stock solution containing 100mg/ml of the extract of the sample of the KECOSHU tablet by using 10% by volume of dimethyl sulfoxide, and the concentration of the sample of the KECOSHU tablet is calculated according to the yield of the extract of the sample of the KECOSHU tablet.
4. The method of claim 1, wherein in step (2) internodal blood vessels are measured using Imagepro Plus 5.1.
5. The method according to claim 1, wherein in step (2), Imagepro Plus 5.1 is used to measure the staining intensity of the cardiopulmonary bypass treatment group and to calculate the fat reduction rate (%) (model IOD-cardiopulmonary bypass treatment group IOD)/model IOD x 100%; the IOD is the intensity of the tail region lipid staining.
6. The method of claim 1, wherein the step (2) of detecting the biological effect of the extract from the sample of the central comfort tablet of step (1) using the zebrafish internode angiogenesis disorder model comprises the steps of:
selecting 23hpf Tg (flk1: EGFP) blood vessel green fluorescence labeled transgenic zebra fish, demoulding by 1mg/ml streptokinase E, randomly dividing the health embryos after demoulding into a blank control group, a model group and a cardiopalmus treatment group, randomly distributing the embryos, wherein each group is provided with more than 2 parallel embryos, and each group is provided with 10-12 parallel embryos; blank control group administration: the fish culture water contains DMSO with the volume fraction of 0.1%; model group administration: fish water containing 0.1% DMSO by volume + PTK787 at a final concentration of 0.175. mu.g/ml; the cardiotonic tablet treatment group was given: the fish culture water contains 0.1 percent of DMSO by volume fraction, PTK787 with the final concentration of 0.175 mu g/ml and Xinkeshu tablets with the final concentration of 85 mu g/ml; incubating at the temperature of 28 ℃ until the incubation temperature reaches 47hpf, and observing the blood vessel morphology of the zebra fish embryo under a fluorescence microscope;
quantitative analysis: internodal blood vessels (ISVs) were measured using Imagepro Plus 5.1.
7. The method according to claim 1, wherein the step (2) of detecting the biological effect of the extract from the sample of the central comfort piece of step (1) by using the zebrafish hyperlipidemia model comprises the following steps:
selecting 5dpf (days post fertilization) wild AB line zebra fish, randomly dividing the zebra fish into a blank control group, a model group and a Xinkeshu piece treatment group, wherein 28-32 zebra fish are selected from each group, the blank control group is given with fish rearing water containing 0.015mg/ml Phenylthiourea (PTU), the model group and the Xinkeshu piece treatment group are fed with egg yolk solution with the mass fraction of 0.1%, treating for 48h, removing yolk solution, feeding fish culture water containing 0.015mg/ml PTU + 0.1% DMSO to a blank control group and a model group, feeding fish culture water containing 0.015mg/ml PTU + 0.1% DMSO +680 mu g/ml Xinkeshu tablet to a Xinkeshu tablet treatment group, incubating at 28 ℃, treating for 24h for liquid change, continuously treating for 24h, the staining intensity of the cardiopulmonary bypass treatment group was measured using Imagepro Plus 5.1 and the lipid reduction rate (%) was calculated as (model group IOD-cardiopulmonary bypass treatment group IOD)/model group IOD × 100%.
8. The method of claim 1, wherein the length of the zebrafish internode blood vessels in the zebrafish internode angiogenesis disorder model in step (3) is 2180 μm-2382 μm, and the lipid-lowering rate in the zebrafish hyperlipidemia model is 43% -56%.
9. Use of the method of any one of claims 1 to 8 for quality inspection of a sample of a cardiopalmus sheet.
10. Use according to claim 9, wherein the method is used in the detection of a lack of square sample of a cardiotocol tablet, a half-reduced sample of the feeding of a cardiotocol tablet.
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