CN111965315A - Method for screening radix astragali total saponin extraction process based on zebra fish vascular injury model and application thereof - Google Patents
Method for screening radix astragali total saponin extraction process based on zebra fish vascular injury model and application thereof Download PDFInfo
- Publication number
- CN111965315A CN111965315A CN202010813955.4A CN202010813955A CN111965315A CN 111965315 A CN111965315 A CN 111965315A CN 202010813955 A CN202010813955 A CN 202010813955A CN 111965315 A CN111965315 A CN 111965315A
- Authority
- CN
- China
- Prior art keywords
- zebra fish
- astragalus
- vascular injury
- blood vessels
- extraction process
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Steroid Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a method for screening an extraction process of total saponins of astragalus based on a zebra fish vascular injury model and application thereof, belonging to the technical field of detection of active ingredients of traditional Chinese medicines. The invention simulates myocardial cell injury or necrosis symptoms of a patient by using the zebra fish internode vascular injury, inspects the regeneration effect of the total astragalosides on the zebra fish blood vessels, and measures the regeneration rate of the blood vessels, thereby establishing a sensitive, feasible and good-repeatability test method for promoting the zebra fish ISV generation by the total astragalosides, and optimizing the optimal extraction process of the total astragalosides by promoting the relative growth rate of the zebra fish internode blood vessels through the total astragalosides, so that the invention has good practical application value.
Description
Technical Field
The invention belongs to the technical field of detection of active ingredients of traditional Chinese medicines, and particularly relates to a method for screening an extraction process of total saponins of astragalus based on a zebra fish vascular injury model and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Astragalus root, used as a commonly used qi-tonifying drug, mainly contains saponins (such as astragaloside IV), polysaccharides (such as calycosin glucoside), flavonoids (astragalus flavone) and other effective components. The plant source of the astragalus is generally dry roots and rhizomes of leguminous plant astragalus membranaceus or astragalus mongholicus, is listed as a top-grade medicine, is sweet in nature, has the effect of supporting sores and promoting tissue regeneration, and is a Chinese clinical medicine which is highly advocated by China all the time. The main active ingredients in the astragalus root comprise total saponins of the astragalus root (such as astragaloside IV) and astragalus polysaccharide (such as calycosin glucoside), and researches show that the total saponins of the astragalus root and the astragalus polysaccharide have important pharmacological effects. At present, various researches find that astragalus has two kinds of medicinal materials: separating more than 40 triterpene saponin compounds from Astragalus membranaceus and Mongolian milkvetch root.
Zebra fish is used as a new generation of model organism applied to drug activity detection, and the pharmacological model of zebra fish is basically mature and widely applied to pharmaceutical and pharmacological research at present. Zebrafish are widely used in drug screening as a biological model and become a model for studying cardiovascular growth promotion and inhibition. In the process of pharmacological research of medicines, zebra fish can be used for researching organ development and disease models, is one of common model organisms, the embryo of the zebra fish is transparent under the visual observation, and the zebra fish can observe organs in vivo through a microscope under the condition of a stain or a fluorescent marker. When the zebra fish is used as a biological model for detecting the pharmacological effect of the active ingredients, the drug dosage of the zebra fish is small. When the zebra fish is used for screening the drug activity, the growth cycle is short, the time consumption is low, and the efficiency is high. In the activity detection experiment, the experimental operation of the zebra fish is simple and convenient. After 48 hours post fertilization, a simple circulatory network was formed between the dorsal artery and the axial vein of zebrafish. After 72 hours after fertilization, the vascular system was formed in zebrafish bodies, and the sub-intestinal venous vessels (SIVs) were primarily formed.
Zebrafish are widely used for drug screening as a biological model and become one of the main models for studying cardiovascular growth promotion and inhibition. The method has short propagation and growth period and large number of sub-generations, and can simultaneously detect different samples on a large scale. The method applies the large-scale rapid comparison of the drug content of the zebra fish, embodies the screening effect of model organisms, and can provide a certain reference for researching the activity evaluation of the cardiovascular and cerebrovascular drugs. The zebra fish is used as a mode for rapidly comparing the high and low drug content on a large scale to screen the pharmacological action of the drugs, so that the detection efficiency is greatly improved. However, the inventor finds that no research report exists so far about the application of a zebra fish vascular injury model in the activity evaluation of the total saponins of astragalus membranaceus and further the optimization of the extraction process of the total saponins of astragalus membranaceus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for screening an extraction process of total saponins of astragalus based on a zebra fish vascular injury model and application thereof. The invention uses zebra fish internode vascular injury to simulate myocardial cell injury or necrosis symptoms of a patient, inspects the regeneration effect of the total saponins of astragalus to zebra fish blood vessels, and determines the rate of the regeneration of the blood vessels, thereby establishing a sensitive, feasible and good-repeatability test method for promoting the generation of zebra fish ISV by the total saponins of astragalus, and optimizing the extraction process of the total saponins of astragalus for promoting the relative growth rate of the zebra fish internode blood vessels and optimizing the total saponins of astragalus, thereby having good value of practical application.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided a method for evaluating the activity of total saponins of astragalus based on a zebra fish vascular injury model, the method comprising: establishing a zebra fish vascular injury model, and applying the astragalus total saponin extract to the zebra fish vascular injury model.
The method further comprises the following steps: and (3) inspecting the effect of the total saponins extract of astragalus on the regeneration of the zebra fish blood vessels, and determining the regeneration rate of the zebra fish blood vessels.
The method for establishing the zebra fish vascular injury model specifically comprises the following steps: endothelial cytokine receptor tyrosine kinase inhibitor (VRI) is administered to zebrafish to induce vascular injury.
In a second aspect of the invention, the application of the method in the extraction process of screening the total saponins of astragalus is provided.
The application specifically comprises: the best extraction process of the corresponding astragalus total saponin extract is screened by investigating the promotion effect of the astragalus total saponin extract on the relative growth rate of the blood vessels between the zebra fish joints.
The third aspect of the invention provides a method for screening an extraction process of total saponins of astragalus membranaceus based on a zebra fish vascular injury model, which comprises the step of utilizing the method for evaluating the activity of the total saponins of astragalus membranaceus based on the zebra fish vascular injury model to further screen the optimal extraction process of the total saponins of astragalus membranaceus.
The beneficial technical effects of one or more technical schemes are as follows:
according to the technical scheme, the activity of the total saponins of astragalus is evaluated through a zebra fish vascular injury model, the optimization of the extraction process of the total saponins of astragalus is realized, the method is simple and rapid, the sensitivity is high, the specificity is good, the repeatability is good, and a reference basis can be provided for the optimization of the extraction process of the total saponins of astragalus.
Meanwhile, the activity evaluation method adopting the zebra fish vascular injury model in the technical scheme has the advantages of accuracy, reliability, high sensitivity, good specificity and the like, and has more effectiveness on the activity analysis of medicinal material components, so that the method has good value of practical application.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 shows the ISV effect of 9 HQIs on zebrafish in example 2 of the present invention; wherein A is a normal control group; b is a PTK787 model group, and 1-9 respectively correspond to 1-9 of an additive group;
FIG. 2 is a bar graph showing the ISV generation rate of zebra fish promoted by 9 groups of HQIs in example 2 of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
As is known in the art, the total saponins of astragalus have complex components and large content difference of different saponins, and the optimal extraction process of the total saponins of astragalus obtained by comparing the content of single component by an instrumental analysis method cannot reflect the activity and comprehensiveness of the total saponins of astragalus. In the process optimization method, a zebra fish vascular injury model method is provided for optimizing the extraction process of the total saponins of astragalus membranaceus. The astragaloside extract is currently used as a raw material substance of a traditional Chinese medicine injection for treating cardiovascular diseases, and has the effects of promoting the function of heart and lung and the like. Therefore, the best extraction process of the astragalus is researched from the aspect of drug effect, and the verification of the effect influence of the total saponins of the astragalus on the corresponding pathological model is pointed and extremely important.
In view of the above, in one embodiment of the present invention, there is provided a method for evaluating total saponins of astragalus activity based on a zebra fish vascular injury model, the method comprising: establishing a zebra fish vascular injury model, and applying the astragalus total saponin extract to the zebra fish vascular injury model.
In another embodiment of the present invention, the method further comprises: and (3) inspecting the effect of the total saponins extract of astragalus on the regeneration of the zebra fish blood vessels, and determining the regeneration rate of the zebra fish blood vessels.
In another embodiment of the present invention, the method for establishing a zebra fish vascular injury model specifically includes: endothelial cytokine receptor tyrosine kinase inhibitor (VRI) is administered to zebrafish to induce vascular injury.
In yet another embodiment of the present invention, the zebrafish is a fluorescently labeled zebrafish embryo, preferably an eggshell removed zebrafish embryo.
In yet another embodiment of the present invention, the endothelial cytokine receptor tyrosine kinase inhibitor is a small molecule inhibitor of Vatalanib (Vatalanib, PTK 787). PTK787 can block the phosphorylation process in the VEGF receptor signal transduction process, can effectively inhibit VEGF, effectively reduce the expression of vascular VEGF biological effect to inhibit the growth of human tumor cells planted in nude mice and the formation of microvessels in tumor tissues, and has good effect on the treatment of liver metastasis of colorectal cancer.
The preparation method of the PTK787 solution is as follows: 10mg of PTK787 powder was dissolved in 1mL of DMSO solvent to prepare a 10mg/mL PTK787 mother solution, and the solution was diluted with the mother solution to obtain 0.2 to 0.4. mu.g/mL PTK787 solution.
In another specific embodiment of the present invention, the method for investigating the effect of the total saponins of astragalus extract on the regeneration of zebra fish blood vessels and determining the regeneration rate of zebra fish blood vessels comprises: observing the conditions of zebra fish internodal blood vessels (ISVs), and measuring and counting the zebra fish blood vessels.
The preparation method of the astragalus total saponin extract (HQI) solution comprises the following steps: taking 5mL of astragalus saponin extract to an EP tube, evaporating ethanol in a water bath kettle at 60 ℃, weighing 10mg of extract, and adding 1mL of DMSO to prepare 10mg/mL of HQI solution. Adding 2-15 μ L HQI solution into zebra fish feeding solution.
In another specific embodiment of the present invention, an application of the method in screening the total saponins of astragalus is provided.
The application specifically comprises: the best extraction process of the corresponding astragalus total saponin extract is screened by investigating the promotion effect of the astragalus total saponin extract on the relative growth rate of the blood vessels between the zebra fish joints.
In another embodiment of the present invention, the extraction process of the total saponins of astragalus membranaceus comprises:
extracting radix astragali with ethanol as extraction solvent, and purifying the extractive solution.
In another embodiment of the invention, the feed-liquid ratio of the astragalus root to the ethanol is 1: 4-10, such as 1:4, 1:8 or 1: 10; preferably 1: 10;
in another embodiment of the present invention, the number of extraction times is 1 to 3, preferably 1;
in another embodiment of the invention, the extraction time is 1-3 h, preferably 3 h;
in another embodiment of the present invention, the specific purification method is: concentrating, removing impurities, eluting, recovering solvent under reduced pressure, and drying to obtain radix astragali total saponin extract; more specifically, the extract is passed through D101 macroporous adsorption resin at the speed of 1mL/min (the mass ratio of the medicinal materials to the resin is 3.6: 1), water-soluble impurities are washed by 8 times of column volume of water, the extract is eluted by 80% ethanol at the speed of 1mL/min, the dosage of an eluent is 4 times of column volume, the solvent is recovered from the eluent by decompression, and the extract is dried to obtain the compound.
In another embodiment of the present invention, a method for screening an extraction process of total saponins of astragalus based on a zebra fish vascular injury model is provided, and the method comprises the step of using the method for evaluating the activity of total saponins of astragalus based on the zebra fish vascular injury model to further screen an optimal extraction process of total saponins of astragalus.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
(1) Preparation of total saponins of astragalus
Pulverizing radix astragali, sieving with No. 3 sieve, precisely weighing 5g radix astragali coarse powder, placing in 100mL round bottom flask, adding 50mL 80% ethanol, heating and reflux-extracting in electric heating jacket for 2 hr, extracting once, hot filtering, adding 50mL water into medicinal material residue, reflux-extracting for 2 hr, extracting twice, and hot filtering. Regulating water extractive solution to 85% alcohol content, precipitating polysaccharide, mixing the filtered clear solution with the ethanol extractive solution, separating the extract into saponin part and polysaccharide part, and further separating and purifying the two parts to obtain saponin and polysaccharide.
The purification process of the alcohol extract comprises the following steps: taking the astragalus ethanol extract (0.6g crude drug/mL), passing through D101 macroporous adsorption resin (the mass ratio of the drug to the resin is 3.6: 1) at the speed of 1mL/min, washing with water soluble impurities by 8 times of the column volume, eluting with 80% ethanol at the speed of 1mL/min, wherein the eluent is 4 times of the column volume, decompressing the eluent, recovering the solvent, and drying to obtain the sample.
(2) Zebra fish embryo collection
Zebra fish was bred by circulating water according to the Westerfield method with reference to Zebraphis book, the breeding temperature was maintained at (28.5 + -1) ° C, the feeding was performed alternately in 14h light treatment and 10h dark treatment every day, and shrimp and fish food were fed 2 times a day. When eggs are collected, the adult zebra fish with the fluorescent marker is placed in the same culture tank for mating and breeding according to the sex 1/1 or 1/2. Fertilized eggs are obtained after 20 h.
(3) Zebra fish embryo treatment
Selecting zebra fish embryo with good state, fluorescence color development and development for 21h under electric fluorescence zoom biological stereomicroscope, sterilizing and cleaning fertilized egg with 0.1% methylene blue reagent, treating with mold release agent (protease), and washing with small amount of culture solution to remove egg shell. Transferring the zebra fish embryo into a culture solution, and culturing at 28 ℃ by using a light control incubator under the condition of light control and temperature control. (4) Endothelial cytokine receptor tyrosine kinase inhibitor (VRI) induced vascular injury
Taking out zebra fish embryos from a light culture box, selecting embryos with egg shells removed, putting not less than 10 embryos in each hole into a 24-hole plate of a feeding liquid, adding a Vatalanib small molecule inhibitor (PTK787) in advance, treating for 3 hours in the feeding box, and then washing for 3-4 times by using the feeding liquid.
PTK787 can block the phosphorylation process in the VEGF receptor signal transduction process, can effectively inhibit VEGF, effectively reduce the expression of vascular VEGF biological effect to inhibit the growth of human tumor cells planted in nude mice and the formation of microvessels in tumor tissues, and has good effect on the treatment of liver metastasis of colorectal cancer.
(5) Adding radix astragali total saponin extract (HQI) into zebra fish feeding liquid
Collecting fertilized eggs of zebra fish of the same parent, putting the fertilized eggs into a 28 ℃ illumination incubator for 21h of development, randomly grouping the fertilized eggs after embryo demoulding treatment, putting the fertilized eggs into 24-hole plates (8 eggs/hole), adding 2mL of a feeding liquid into each hole, sterilizing, cleaning and demoulding the fertilized eggs after the zebra fish embryos develop for 21h, transferring the fertilized eggs into the feeding liquid containing corresponding PTK787 solution, treating the fertilized eggs in the feeding incubator for 3h, cleaning the fertilized eggs for 3-4 times by using the feeding liquid, transferring the fertilized eggs into the feeding liquid containing corresponding astragaloside extracts, and putting the zebra fish eggs into the 28 ℃ illumination incubator for light-controlled temperature-controlled culture for 24 h. And taking a picture after 48h, and statistically processing the analysis data.
The preparation method of the PTK787 solution in the step (4) is as follows: 10mg of PTK787 powder was dissolved in 1mL of DMSO solvent to prepare a 10mg/mL PTK787 mother solution, which was diluted to give a 0.4. mu.g/mL PTK787 solution.
The preparation method of the HQI solution in the step (5) is as follows: taking 5mL of astragalus saponin extract to an EP tube, evaporating methanol to dryness in a water bath kettle at 60 ℃, weighing 10mg of extract, and adding 1mL of DMSO to prepare 10mg/mL of HQI solution. To the zebrafish feeding solution was added 10. mu.L of HQI solution.
(6) Taking fertilized eggs of zebra fish, adding 1mg/mL of a mold release agent Pronase E, keeping for 2-3min, carrying out embryo demolding, placing 10 zebra fish in 24 pore plates respectively per pore, arranging a blank control group, adding a 0.2-0.4 mu g/mL PTK787 model control group and a PTK787 treated HQI adding medicine group (sample liquid 5-15 mu L), adding the same amount of DMSO solution into the blank control group, enabling each group to be parallel twice, adding 2mL of a feeding liquid into each pore, after an embryo develops for 21h, sterilizing, cleaning and demolding, transferring into a feeding liquid containing a corresponding PTK787 solution, treating for 3h in a feeding box, then washing for 3-4 times by using the feeding liquid, transferring into the feeding liquid containing a corresponding astragaloside extract, and placing in a light-irradiation culture box for light-controlled temperature culture for 24 h.
The experimental result of the zebra fish is observed by using an electric fluorescence stereomicroscope. When the length of the zebra fish ISVs is observed and measured, in order to enable the zebra fish to stably stay on a glass slide to be convenient for observation and photographing, 0.6% Ethyl 3-aminobenzoate methane salt saiL is needed to be used for anesthetizing the zebra fish body, measurement software Image Pro PLus 5.0 is adopted for calculating the length of the zebra fish ISVs, the experimental result of variance analysis has statistical significance by taking the significance of being less than 0.05, the length data of the zebra fish ISVs is recorded, and the blood vessel growth rate of the zebra fish is calculated.
SV blood vessel growth rate calculation formula: the increase rate (%) was (HQI dosing group-PTK 787 model control)/(normal control-PTK 787 model control) × 100%.
Table 1 is the results of the effect of HQI on zebrafish.
TABLE 1 Effect of HQI on Zebra Fish
Note: in comparison with the normal group,*p is less than 0.05; in comparison with the PTK787 model group,△P＜0.05。
(7) observation method
Sucking out the culture solution and the liquid medicine after the zebra fish is cultured in the culture solution, washing for 3-5 times by using the culture solution, transferring the zebra fish onto a glass slide, and observing the conditions of the zebra fish internode blood vessels (ISVs) by using a fluorescence microscope. Blood vessels of 5 zebrafish were measured manually and counted separately.
(8) Statistical method
The variance and range analysis function of SPSS16.0 statistical software is adopted for analysis, the mean value is expressed above and below the standard deviation when experimental data accord with normal distribution, the data comparison among groups adopts one-factor variance analysis, and the comparison among groups adopts the minimum significant difference method for inspection. Statistical differences were indicated by P < 0.05.
Example 2
(1) Preparation of nine groups of total saponins of astragalus under different extraction process conditions
Firstly, ethanol is taken as an extraction solvent, the ethanol dosage (A), the extraction times (B) and the extraction time (C) of the extraction solvent which influence the extraction effect of the medicinal materials are determined as investigation factors, and the three investigation factors are respectively examined in 3 levels, which is shown in Table 2. Mixing extractive solutions, concentrating by rotary evaporation to 2 column volumes, passing through D101 macroporous adsorbent resin at a speed of 1mL/min (mass ratio of medicinal material to resin is 3.6: 1), eluting with 8 times of column volume of water to remove water-soluble impurities, eluting with 80% ethanol at a speed of 1mL/min, eluting with 4 times of column volume, recovering solvent from eluate under reduced pressure, and drying to obtain sample.
TABLE 2 experiment factor level table
(2) Zebra fish embryo collection
The zebra fish is fed by circulating water, the feeding temperature is kept at (28.5 +/-1) DEG C, the zebra fish is alternately fed by 14h of light treatment and 10h of dark treatment every day, and the zebra fish is fed with shrimp and fish food 2 times every day. When eggs are collected, the adult zebra fish with the fluorescent marker is placed in the same culture tank for mating and breeding according to the sex 1/1 or 1/2. Fertilized eggs are obtained after 20 h.
(3) Zebra fish embryo treatment
Selecting zebra fish embryo with good state, fluorescence color development and development for 21h under electric fluorescence zoom biological stereomicroscope, sterilizing and cleaning fertilized egg with 0.1% methylene blue reagent, treating with mold release agent (protease), and washing with small amount of culture solution to remove egg shell. Transferring the zebra fish embryo into a culture solution, and culturing at 28 ℃ by using a light control incubator under the condition of light control and temperature control.
(4) VRI-induced vascular injury
Taking out zebra fish embryos from the illumination incubator, selecting embryos with no less than 10 eggs removed, putting the embryos in a 24-hole plate of the feeding liquid per hole, adding 3 mu L of VRI (PTK787)0.1mg/mL in advance, treating for 3h in the feeding incubator, and then washing for 3-4 times by using the feeding liquid.
(5) Nine groups of astragalus total saponin extracts tested by zebra fish ISV injury model
Collecting fertilized eggs of zebra fish of the same parent, placing in a 28 ℃ illumination incubator for development for 21h, randomly grouping the fertilized eggs after embryo demoulding treatment, and placing in a 24-hole plate (8 eggs/hole). Transferring the embryos to a feeding solution containing a corresponding PTK787 solution, treating for 3h in a feeding box, then cleaning for 3-4 times by using the feeding solution, and transferring to the feeding solution containing a corresponding astragalus saponin extract, wherein the feeding solution containing the astragalus saponin extract is a DMSO solution which is prepared by 9 groups of astragalus total saponins in advance and has the same optimal dose of the searched astragalus total saponins and the optimal dose. Setting a normal control group, a PTK787 model group (added with 0.3 mu g/mL inhibitor) and No. 1-9 dosing treatment groups (added with a sample solution of corresponding total saponins of astragalus after being treated with 0.3 mu g/mL PTK787 inhibitor). Placing in a light incubator at 28 ℃ for light-controlled culture for 24 h. And taking a picture after 48h, and statistically processing the analysis data.
(6) Observation method
Sucking out the culture solution and the liquid medicine after the zebra fish is cultured in the culture solution, washing for 3-5 times by using the culture solution, transferring the zebra fish onto a glass slide, and observing the conditions of the zebra fish internode blood vessels (ISVs) by using a fluorescence microscope. The blood vessels of each group of 5 zebra fish were manually measured and counted.
(7) Statistical method
The variance and range analysis function of SPSS16.0 statistical software is adopted for analysis, the mean value is expressed above and below the standard deviation when experimental data accord with normal distribution, the data comparison among groups adopts one-factor variance analysis, and the comparison among groups adopts the minimum significant difference method for inspection. Statistical differences were indicated by P < 0.05.
The ISV growth status of 9 groups of zebrafish is shown in table 3. The result of the relative increase rate of the blood vessels of the zebra fish ISVs shows that the blood vessels of the zebra fish in the treatment group are remarkably recovered, and the ISV of the zebra fish in the PTK787 model group is remarkably cracked. The angiogenesis promoting rate of the 9 groups of samples is between 15 and 60 percent, and the drug-added treatment group has significant difference (P is less than 0.05) compared with the PTK787 model group.
Table 39 results of using HQI to promote ISV (ISV-producing rate) of zebra fish
Note: p <0.05 compared to the PTK787 model group
As shown in FIG. 1, photographs 1 to 9 show the growth of the internode blood vessels of zebra fish after nine groups of astragaloside extracts were added to the zebra fish culture solution, respectively. Compared with the PTK787 model group, the normal control group has significant difference (P is less than 0.05), and compared with the PTK787 model group and the HQI dosing treatment groups of 1-9 groups, the PTK787 model group has significant difference (P is less than 0.05); as can be seen from the data in table 3 and fig. 1 and 2, the relative ISV production rates of the groups 7, 8 and 9 are significantly higher than those of the other experimental groups, the ISV production rate of the zebra fish corresponding to the group 7 is 56.18%, the ISV production rate of the zebra fish corresponding to the group 8 is 46.17%, and the ISV production rate of the zebra fish corresponding to the group 9 is 54.16%. The astragaloside extract of group 7 had the most significant effect on promoting the internode blood vessels of zebra fish.
And (3) according to the relative growth rate data of the zebra fish ISV obtained by the experiment, making an intuitive analysis table and an analysis of variance table, wherein the factor D is an error item, and the table is shown in tables 4 and 5.
TABLE 4 ISV relative growth Rate review L9(34) Orthogonal test table
TABLE 5 ISV relative growth Rate investigation ANOVA Table
From the results of the orthogonal test, the influence of each factor on the ISV relative growth rate of the zebra fish is in the following order: a (ethanol dosage) > C (extraction time) > B (extraction times); the trend among the levels of each factor 3 is A3> A1> A2, B1> B3> B2, C3> C2> C1, and the optimal extraction process is A3B1C3 through visual analysis, namely 10 times of alcohol is added, and the extraction is carried out for 1 time and 3 hours each time. The results of the anova analysis of tables 4-5 show that: the effect of factor a has statistical differences (P <0.05), i.e. ethanol usage has a significant effect on the relative rate of increase of zebrafish ISVs.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. A method for evaluating the activity of total saponins of astragalus based on a zebra fish vascular injury model is characterized by comprising the following steps: establishing a zebra fish vascular injury model, and applying the astragalus total saponin extract to the zebra fish vascular injury model.
2. The method of claim 1, wherein the method further comprises: and (3) inspecting the effect of the total saponins extract of astragalus on the regeneration of the zebra fish blood vessels, and determining the regeneration rate of the zebra fish blood vessels.
3. The method of claim 1, wherein the method for establishing the zebra fish vascular injury model specifically comprises: an endothelial cytokine receptor tyrosine kinase inhibitor is administered to zebrafish to induce vascular injury.
4. The method of claim 3, wherein the zebrafish is a fluorescently labeled zebrafish embryo, preferably an eggshell removed zebrafish embryo; or the like, or, alternatively,
the endothelial cell factor receptor tyrosine kinase inhibitor is a small molecule inhibitor PTK787 of vardanib.
5. The method as claimed in claim 2, wherein the method for investigating the effect of the astragalus total saponin extract on the regeneration of the zebra fish blood vessels and determining the regeneration rate of the zebra fish blood vessels comprises the following steps: observing the conditions of zebra fish internodal blood vessels (ISVs), and measuring and counting the zebra fish blood vessels.
6. Use of the method of any one of claims 1-5 in an extraction process for screening total saponins of astragalus membranaceus.
7. The application of claim 6, wherein the application specifically comprises: the best extraction process of the corresponding astragalus total saponin extract is screened by investigating the promotion effect of the astragalus total saponin extract on the relative growth rate of the blood vessels between the zebra fish joints.
8. The use as claimed in claim 7, wherein the extraction process of the total saponins of astragalus membranaceus comprises the following steps:
extracting radix astragali with ethanol as extraction solvent, and purifying the extractive solution.
9. The use according to claim 8, wherein the feed-to-liquid ratio of the astragalus to the ethanol is 1:4 to 10, such as 1:4, 1:8 or 1: 10; preferably 1: 10;
the extraction times are 1-3 times, preferably 1 time;
the extraction time is 1-3 h, preferably 3 h;
the purification method comprises the following steps: concentrating, removing impurities, eluting, recovering solvent under reduced pressure, and drying to obtain radix astragali total saponin extract; more specifically, the extract is passed through D101 macroporous adsorption resin at the speed of 1mL/min (the mass ratio of the medicinal materials to the resin is 3.6: 1), water-soluble impurities are washed by 8 times of column volume of water, the extract is eluted by 80% ethanol at the speed of 1mL/min, the dosage of an eluent is 4 times of column volume, the solvent is recovered from the eluent by decompression, and the extract is dried to obtain the compound.
10. A method for screening an extraction process of total saponins of astragalus membranaceus based on a zebra fish vascular injury model, which is characterized by comprising the step of utilizing the method for evaluating the activity of the total saponins of astragalus membranaceus based on the zebra fish vascular injury model in any one of claims 1 to 5, and further screening the optimal extraction process of the total saponins of astragalus membranaceus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010813955.4A CN111965315A (en) | 2020-08-13 | 2020-08-13 | Method for screening radix astragali total saponin extraction process based on zebra fish vascular injury model and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010813955.4A CN111965315A (en) | 2020-08-13 | 2020-08-13 | Method for screening radix astragali total saponin extraction process based on zebra fish vascular injury model and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111965315A true CN111965315A (en) | 2020-11-20 |
Family
ID=73365583
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010813955.4A Pending CN111965315A (en) | 2020-08-13 | 2020-08-13 | Method for screening radix astragali total saponin extraction process based on zebra fish vascular injury model and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111965315A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114577763A (en) * | 2022-02-14 | 2022-06-03 | 山东省科学院生物研究所 | Kunxian capsule quality control method based on biological effect and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425674A (en) * | 2002-12-19 | 2003-06-25 | 上海博泰医药科技有限公司 | Process for preparing astraglus base total saponin |
| CN1981800A (en) * | 2005-12-12 | 2007-06-20 | 北京奇源益德药物研究所 | Chinese-medicinal preparation for treating cardiovascular diseases, its production and use |
| CN101084979A (en) * | 2006-06-08 | 2007-12-12 | 天津天士力之骄药业有限公司 | Medicinal preparation for cancer auxiliary treatment and its preparation method |
| CN102499135A (en) * | 2011-10-25 | 2012-06-20 | 澳门大学 | Zebra fish vascular injury model for screening vascular injury resisting medicament as well as building method and application thereof |
| CN104152491A (en) * | 2014-08-18 | 2014-11-19 | 中国农业科学院兰州畜牧与兽药研究所 | Fermented Radix Astragali preparation method, and method for extracting total saponins of fermented Radix Astragali |
-
2020
- 2020-08-13 CN CN202010813955.4A patent/CN111965315A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425674A (en) * | 2002-12-19 | 2003-06-25 | 上海博泰医药科技有限公司 | Process for preparing astraglus base total saponin |
| CN1981800A (en) * | 2005-12-12 | 2007-06-20 | 北京奇源益德药物研究所 | Chinese-medicinal preparation for treating cardiovascular diseases, its production and use |
| CN101084979A (en) * | 2006-06-08 | 2007-12-12 | 天津天士力之骄药业有限公司 | Medicinal preparation for cancer auxiliary treatment and its preparation method |
| CN102499135A (en) * | 2011-10-25 | 2012-06-20 | 澳门大学 | Zebra fish vascular injury model for screening vascular injury resisting medicament as well as building method and application thereof |
| CN104152491A (en) * | 2014-08-18 | 2014-11-19 | 中国农业科学院兰州畜牧与兽药研究所 | Fermented Radix Astragali preparation method, and method for extracting total saponins of fermented Radix Astragali |
Non-Patent Citations (4)
| Title |
|---|
| 侯美如 等: "不同提取工艺对黄芪总皂苷提取效果的研究", 《中国兽药杂志》 * |
| 先红 等: "黄芪活性成分(黄芪多糖和黄芪总皂甙)的促血管生成作用", 《岭南心血管病杂志》 * |
| 张哲睿 等: "三七及三七花总皂苷对斑马鱼模型促血管新生作用的研究", 《上海中医药大学学报》 * |
| 王鑫 等: "黄芪提取工艺研究", 《中国实验方剂学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114577763A (en) * | 2022-02-14 | 2022-06-03 | 山东省科学院生物研究所 | Kunxian capsule quality control method based on biological effect and application |
| CN114577763B (en) * | 2022-02-14 | 2023-08-15 | 山东省科学院生物研究所 | Kunxian capsule quality control method based on biological effect and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101744869B (en) | Compound traditional Chinese medicine extract composition for preventing and treating diabetes | |
| CN111965315A (en) | Method for screening radix astragali total saponin extraction process based on zebra fish vascular injury model and application thereof | |
| US20050287230A1 (en) | Method of producing ginsenoside 20 (R)-Rh2 and composition of matter thereof | |
| CN114199840B (en) | Quality control method of Xinkeshu tablet based on biological effect | |
| CN102048770B (en) | Artificially-cultured cordceps militaris sporocarp extract and preparation process thereof | |
| CN104274727B (en) | The quality determining method of clear battalion's oral liquid | |
| CN102688264B (en) | New use of Primula sikkmensis Hook or extract thereof | |
| CN102488722A (en) | Preparation method and quality detection method of bupleurum oral solution | |
| CN112794923B (en) | Ligusticum wallichii polysaccharide and preparation method, identification method and application thereof | |
| CN1057210C (en) | Radix sophorae flavescentis, large-flowered skullcap root and coptis root tablet | |
| CN106928376B (en) | The separation method of skunk bush polysaccharide and its application | |
| CN100354625C (en) | Quality control method for Chinese medicinal formula for treating urination difficulty and urination disturbance | |
| CN100574781C (en) | A kind of veterinary drug for the treatment of gout and its production and use | |
| CN106942737A (en) | Hippophate flavone and its application | |
| CN107513092B (en) | Malonyl ginsenoside Rb1Preparation method and medical application thereof | |
| CN107050010A (en) | Application of the Cichoric acid in the medicine of anti respiratory syncytial virus is prepared | |
| CN110041386A (en) | Preparation method, Specnuezhenide and its application of Specnuezhenide | |
| CN102028698B (en) | Medicine for treating colon cancer and preparation method thereof | |
| CN110075164A (en) | Preparation method, fragrans seed extract and its application of fragrans seed extract | |
| CN105044301B (en) | A kind of detection method of pseudo-ginseng blood-circulation-invigovating blood stasis dispelling activity | |
| CN1323676C (en) | Method for preparing traditional Chinese medicine capsule for treating weakness of spleen and stomach, and its quality control method | |
| CN115414429B (en) | Active extractive of wolfberry fruit for resisting osteoporosis, preparation method and application | |
| CN1150204C (en) | Cyclobornane triterpenoid saponin compound and its application in immunosuppresion and treating tumor | |
| CN117448445B (en) | Application of PTEN, TSC1 and TSC2 genes in aristolochic acid sensitivity detection | |
| CN112759661B (en) | Cherokee rose fruit polysaccharide preparation method, identification method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |