CN116574797A - Construction method and application of anti-osteoporosis zebra fish biomarker and anti-osteoporosis zebra fish model of American ginseng - Google Patents
Construction method and application of anti-osteoporosis zebra fish biomarker and anti-osteoporosis zebra fish model of American ginseng Download PDFInfo
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Abstract
The invention provides a zebra fish biomarker for resisting osteoporosis of American ginseng, a construction method and application of a zebra fish anti-osteoporosis model, and belongs to the technical field of traditional Chinese medicine research, in particular to application of the zebra fish biomarker in evaluation of anti-osteoporosis activity of American ginseng or quality control of American ginseng, wherein the zebra fish biomarker comprises the following components: gene markers and/or metabolic markers; the gene markers are vdrb, col1a2, spark, mmp9, trap and ctsk genes; the metabolic substance markers are: gluconic acid, D-sedoheptulose-7-phosphate, D-ribulose-5-phosphate, uric acid, citric acid, guanine, hypoxanthine, creatinine, guanosine, and phosphopyruvic acid; the American ginseng anti-osteoporosis biomarker provided by the invention provides a theoretical basis for revealing an action mechanism of an American ginseng extract for exerting anti-osteoporosis activity through multiple targets and multiple paths.
Description
Technical Field
The invention belongs to the technical field of Chinese medicine research, and particularly relates to a method for constructing an anti-osteoporosis biomarker of American ginseng and an anti-osteoporosis model of zebra fish and application of the anti-osteoporosis biomarker.
Background
Osteoporosis is a common skeletal disease characterized by reduced bone mass and damaged bone tissue microstructure, resulting in increased bone fragility and susceptibility to fracture.
American ginseng (Panax quiquefolium L.) is a perennial herb of the genus Panax of the family Araliaceae. American ginseng is used as a root medicament, mainly enters heart, lung and kidney channels, and has the effects of strengthening and nourishing, nourishing yin and moisturizing lung, nourishing stomach and promoting fluid production and the like. The American ginseng has rich chemical component types and contains saponin, polysaccharide, polyacetylene, flavonoid and the like, wherein the saponin component is one of main active components of the American ginseng, and has various biological activities such as neuroprotection, cardiovascular protection, metabolism regulation, immunoregulation, anti-tumor, antioxidation and the like. In addition, research reports are provided, american ginseng can be clinically used for treating osteoporosis, american ginseng powder can effectively suppress and improve osteoporosis symptoms of female castrated guinea pigs, however, no systematic research reports on the anti-osteoporosis activity and the action mechanism of the anti-osteoporosis activity are provided at present.
In theory, the action mechanism of the American ginseng for resisting osteoporosis is related to the characteristics of multiple components, multiple targets and multiple functions, but the American ginseng is exotic for introduction and cultivation, and the change of the growing environment tends to cause the American ginseng to be different in components and biological functions to a certain extent, so that the common biological quality marker for resisting osteoporosis is found from the American ginseng in different places by adopting effective methods and technical means, and the method has great significance for researching the mechanism of resisting osteoporosis of the American ginseng and screening osteoporosis therapeutic drugs.
Metabonomics is to perform qualitative and quantitative analysis on metabolites in biological samples by modern detection means, so as to further analyze the change rule of endogenous metabolites and the influenced metabolic pathways of the organisms after being stimulated, and search the relative relation between the metabolites and physiological and pathological changes. Therefore, metabonomics technology is an effective means for quality marker and mechanism of action research.
Hard bone animals and mammals share many common genetic characteristics in terms of skeletal elements, ossification mechanisms, and bone matrix components. The zebra fish is used as a teleosts, the skeletal development of the zebra fish is very similar to that of mammals, the zebra fish has a complete system of bone formation and bone absorption activities, genes and signal paths in the skeletal development process are highly homologous with human beings, and compared with other animal models, the zebra fish has the characteristics of small individuals, suitability for high-throughput screening, transparency of young fish bodies and easiness in observing skeletal development. In recent years, a zebra fish bone osteoporosis model has been widely applied to pharmaceutical activity screening, and in basic and preclinical studies, zebra fish has become an important model organism for studying skeletal development and related diseases. Therefore, the chemical induced osteoporosis zebra fish model is adopted, so that the high-throughput screening and action mechanism research of the anti-osteoporosis active medicaments are realized.
The prior research reports on American ginseng in different producing areas are mainly focused on chemical components, and the research discovers that the American ginseng in different producing areas has no obvious difference in total saponins, reducing sugars, total sugars, amino acids and trace element types and is mainly represented by different degrees of difference in component content (Gao Lijiang, zhao Fangjie, zhang Jiguang and the like; the main active component detection and quality evaluation of American ginseng in different producing areas [ J ]. North-northwest agriculture report, 2021,30 (09): 1402-1409; zhang Cuicui, ai, guo Ruiji and the like; 12 ginsenoside contents in American ginseng in different producing areas are measured based on HPLC and fingerprint spectrum research [ J ]. Liaoning J. Traditional Chinese medicine, 2022,49 (09): 144-147+223.DOI: 10.13192/j.isn.1000-1719.2022.09.041). The existing technology for evaluating the quality of the American ginseng mainly surrounds chemical component detection technology, comprises a High Performance Liquid Chromatography (HPLC) method, an inductively coupled plasma mass spectrometry (ICP-MS) method, a spectrophotometry method and the like for measuring components such as saponins, microelements and polysaccharides in the American ginseng, an American ginseng fingerprint spectrum measuring method based on the HPLC chromatography technology is disclosed in an invention patent CN201010215325.3, the quality control of the American ginseng can be carried out according to characteristic chromatographic peaks, and an American ginseng origin detection method based on the terahertz spectrum technology is disclosed in an invention patent CN202111397557.X, and the American ginseng origin can be predicted according to the thickness and extinction coefficient of a sample of the American ginseng. However, the existing technology for the types or contents of the components is not sufficient to effectively distinguish the American ginseng from the ginseng in different places of production, and even is easy to be confused with the ginseng. In addition, the prior art also fails to disclose quality control methods related to the efficacy of American ginseng.
The existing model animals used for osteoporosis research mainly comprise mice, rats and zebra fish. Zebra fish has similar skeleton development with human, transparent embryo, short experiment period, less medicine consumption and low experiment cost, and has the features of long molding period and high cost, and is used widely in screening osteoporosis resisting medicine in recent years (Jiang Ruixue, jiang Xinquan, wen Jin. Osteoporosis animal model research state and progress [ J ]. Chinese osteoporosis journal 2022,28 (07): 1039-1044). The existing zebra fish anti-osteoporosis model mainly adopts a mode of combining chemical induction and dyeing, as reported by a published technology, the activity of the osteoporosis zebra fish is improved based on the combination of glucocorticoid induction and alizarin red dyeing, the model takes 3dpf zebra fish larvae as experimental animals, the zebra fish larvae are collected through 6 days of continuous modeling administration, and the zebra fish vertebrae dyeing conditions (Zheng Huili, hua Yongqing, liu Xinhui and the like) are counted through the operation of fixing, bleaching, dyeing and the like in the near 2 days; zhan Yang et al disclose an evaluation of the anti-osteoporosis activity of prednisolone induction combined with calcein staining by continuously molding zebra fish developing 3dpf for 4 days (Zhan Yang, li Yingmeng, yuting, etc.. A model of zebra fish bone mass loosening based on two diets evaluates the anti-osteoporosis effect of bone peptides [ J ]. Chinese food additives, 2022,33 (12): 134-138.). However, the staining method generally has the problems of relatively complicated operation steps, easiness in damaging tissue samples, relatively long experimental period, uneven dye staining, sensitivity to illumination, poor experimental repeatability and the like (Peng Wei, zhang Wenjuan, xue. The zebra fish is used as a bone disease model to be researched and developed [ J ]. The Chinese laboratory animal school, 2019,27 (02): 248-253).
In the prior art, the experimental period of the osteoporosis model induced by dexamethasone based on the transgenic zebra fish is 8d, and the zebra fish can eat food through an opening when developing 7dpf, so that the phenomenon is easy to have great influence on experimental results.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for constructing an anti-osteoporosis biomarker of American ginseng and an anti-osteoporosis model of zebra fish and application thereof.
According to the technology disclosed by the invention, the prednisolone-induced osteoporosis zebra fish model with the experimental period controlled within 5 days is constructed by examining the glucocorticoid modeling medicine concentration and the modeling time, so that the defects of the prior art are overcome. In addition, as related technical reports of the anti-osteoporosis activity evaluation of American ginseng based on the zebra fish model are not found at present, and research technology and reports of the anti-osteoporosis zebra fish biomarker of American ginseng are not found, the technology fills the blank of the anti-osteoporosis research field of American ginseng.
The invention adopts metabonomics technology and combines with an osteoporosis zebra fish model, takes American ginseng saponin extracts of different producing areas as research objects, develops the anti-osteoporosis activity research of the American ginseng saponin extracts, explores the anti-osteoporosis action mechanism of the American ginseng saponin extracts, and combines with a bioinformatics method to clarify the change rule of endogenous metabolites, so as to find potential biomarkers and metabolic pathways, thereby providing references and bases for preventing and treating the osteoporosis.
The invention also provides a construction method of the zebra fish anti-osteoporosis model.
The technical scheme of the invention is as follows:
an application of zebra fish biomarkers in the evaluation of anti-osteoporosis activity of American ginseng or the quality control of American ginseng, wherein the zebra fish biomarkers comprise: gene markers and/or metabolic markers;
the gene markers are vdrb, col1a2, spark, mmp9, trap and ctsk genes;
the metabolic substance markers are: gluconic acid, D-sedoheptulose-7-phosphate, D-ribulose-5-phosphate, uric acid, citric acid, guanine, hypoxanthine, creatinine, guanosine, and phosphopyruvic acid.
According to a preferred embodiment of the invention, the primer sequences for the vdrb, col1a2, spark, mmp9, trap and ctsk genes are as follows:
the primer sequences of the vdrb gene are: SEQ ID NO.7 and SEQ ID NO.8;
the primer sequences of the col1a2 gene are: SEQ ID NO.9 and SEQ ID NO.10;
the primer sequences of the spark gene are: SEQ ID NO.13 and SEQ ID NO.14;
the primer sequences of the mmp9 gene are as follows: SEQ ID NO.11 and SEQ ID NO.12;
the primer sequences of the trap gene are: SEQ ID NO.5 and SEQ ID NO.6;
the primer sequences of the ctsk gene are as follows: SEQ ID NO.3 and SEQ ID NO.4.
According to the invention, preferably, the zebra fish is a transgenic zebra fish with green fluorescence markers on bones.
A method for evaluating the anti-osteoporosis activity of American ginseng or the quality control of American ginseng by using zebra fish biomarkers, which comprises the following steps:
the American ginseng extract acts on the zebra fish bone osteoporosis model to serve as an experimental group, and a corresponding blank group and a zebra fish bone osteoporosis model group are arranged; compared with a blank group, in the zebra fish osteoporosis model group, the beta-actin is taken as an internal reference gene, the expression of vdrb, col1a2 and spark genes in the zebra fish body is obviously reduced, and the expression of mmp9, trap and ctsk genes is obviously increased; compared with a model group, the experimental group can obviously up-regulate the expression of vdrb, col1a2 and spark genes in zebra fish bodies by taking beta-actin as an internal reference gene; the expression of the mmp9, trap and ctsk genes in the zebra fish body can be obviously reduced; proved by the demonstration that the American ginseng extract has the anti-osteoporosis activity; or proving that the American ginseng is a genuine product;
or, the American ginseng extract is acted on the zebra fish bone osteoporosis model to be used as an experimental group, and a corresponding blank group and a zebra fish bone osteoporosis model group are arranged; the zebra fish bone loosening model group significantly increases the expression levels of gluconic acid, D-sedoheptulose-7-phosphate, D-ribulose-5-phosphate, uric acid, citric acid, guanine, hypoxanthine, creatinine, guanosine and phosphopyruvate in zebra fish compared to the blank group (P < 0.001), the experimental group significantly decreases the expression levels of the biological standard in the zebra fish compared to the blank group; proved by the demonstration that the American ginseng extract has the anti-osteoporosis activity; or prove that the American ginseng is a genuine product.
According to a preferred embodiment of the present invention, the blank group is a zebra fish water treatment group.
Further preferably, the zebra fish water for breeding fish contains: 5.0mM NaCl,0.17mM KCl,0.4mM CaCl2,0.16mM MgSO4.
According to a preferred embodiment of the present invention, the American ginseng extract is a 50% ethanol extract of American ginseng.
Further preferably, in the method, the preparation method of the American ginseng extract comprises the following steps:
pulverizing American ginseng, adding 5-20 times of 45-55% ethanol water solution, ultrasonic-treating and extracting for 2-4 times, 1-3 h each time, collecting extract, concentrating under reduced pressure to obtain extract, and freeze-drying to obtain powder.
More preferably, american ginseng is prepared into powder, sieved by a No. 3 sieve, added with 10 times of 50% ethanol water solution, extracted for 2 times by ultrasonic treatment for 2 hours each time, filtered and combined with filtrate, decompressed and concentrated into extract, and then freeze-dried into powder.
According to the invention, preferably, the zebra fish is a transgenic zebra fish with green fluorescence markers on bones.
According to the method, the construction method of the zebra fish bone loss model comprises the following steps:
taking Tg (-2.2col10a1a: GFP) transgenic zebra fish embryo of 1dpf, removing egg membrane, selecting normal zebra fish juvenile fish, randomly grouping, and dividing into a blank group and a model group, wherein the blank group is treated by zebra fish water; adding prednisolone with the final concentration of 5-11 mu M into zebra fish culture water of a model group, continuously culturing the juvenile zebra fish to 5dpf, then observing the condition of zebra fish fluorescent cells, photographing, calculating the bone fluorescent area and the fluorescent density of the zebra fish head, and obtaining that the model group has the bone fluorescent area and the fluorescent density which are remarkably reduced (P is less than 0.05) compared with the blank group data, thus obtaining the successful modeling.
Further preferably, prednisolone with a final concentration of 5-10 mu M is added into the zebra fish culture water of the model group.
Further preferably, 8-15 zebra fish are used in each group, and 2-3 zebra fish are used in each group in parallel.
Further preferably, 1.0 mg/mL is used -1 The pronase E solution was stripped of egg membranes.
Further preferably, the culture condition of the juvenile zebra fish is 28 ℃.
According to the invention, the experimental groups are in particular: compared with the model group, the American ginseng extract is added while the same modeling agent is added.
Further preferably, the molding agent is prednisolone and the American ginseng extract is added at a final concentration of 2.5, 5 or 10 μg/mL.
According to a preferred embodiment of the present invention, the primer sequences of vdrb, col1a2, spark, mmp9, trap and ctsk genes are as follows:
the primer sequences of the vdrb gene are: SEQ ID NO.7 and SEQ ID NO.8;
the primer sequences of the col1a2 gene are: SEQ ID NO.9 and SEQ ID NO.10;
the primer sequences of the spark gene are: SEQ ID NO.13 and SEQ ID NO.14;
the primer sequences of the mmp9 gene are as follows: SEQ ID NO.11 and SEQ ID NO.12;
the primer sequences of the trap gene are: SEQ ID NO.5 and SEQ ID NO.6;
the primer sequences of the ctsk gene are as follows: SEQ ID NO.3 and SEQ ID NO.4.
A construction method of a zebra fish bone osteoporosis model comprises the following steps:
taking Tg (-2.2col10a1a: GFP) transgenic zebra fish embryo of 1dpf, removing egg membrane, selecting normal zebra fish juvenile fish, randomly grouping, and dividing into a blank group and a model group, wherein the blank group is treated by zebra fish water; adding prednisolone with the final concentration of 5-11 mu M into zebra fish culture water of a model group, continuously culturing the juvenile zebra fish to 5dpf, then observing the condition of zebra fish fluorescent cells, photographing, calculating the bone fluorescent area and the fluorescent density of the zebra fish head, and obtaining that the model group has the bone fluorescent area and the fluorescent density which are remarkably reduced (P is less than 0.05) compared with the blank group data, thus obtaining the successful modeling.
Further preferably, prednisolone with a final concentration of 5-10 mu M is added into the zebra fish culture water of the model group.
Further preferably, 8-15 zebra fish are used in each group, and 2-3 zebra fish are used in each group in parallel.
Further preferably, 1.0 mg/mL is used -1 The pronase E solution was stripped of egg membranes.
Further preferably, the culture condition of the juvenile zebra fish is 28 ℃.
The application of American ginseng as an active ingredient in preparing an anti-osteoporosis medicament.
According to a preferred aspect of the present invention, in the application, the American ginseng is an American ginseng extract.
Further preferably, the American ginseng extract is an American ginseng 50% ethanol extract.
More preferably, the American ginseng extract is prepared by the preparation method of the American ginseng extract.
Advantageous effects
1. The invention discloses an anti-osteoporosis biomarker for American ginseng based on metabonomics technology and application thereof for the first time; the invention is based on a prednisolone-induced zebra fish osteoporosis model, takes a bone fluorescence area and a fluorescence density as evaluation indexes, confirms the anti-osteoporosis of the American ginseng extract, and confirms the expression level of the related genes vdrb, col1a2 and sparc of the osteoblast for the first time through real-time fluorescence quantitative PCR detection, and obviously reduces the expression levels of the related genes mmp9, trap and ctsk of the osteoblast. Based on the metabonomics technical means, 24 key differential metabolites of the American ginseng for resisting osteoporosis are defined for the first time, and the path analysis shows that the American ginseng can call back 10 key biological quality marker contents by participating in purine metabolism, tricarboxylic acid circulation and pentose phosphate metabolism, thereby improving the osteoporosis state of the zebra fish.
2. The American ginseng anti-osteoporosis biomarker based on the metabonomics technology provides a theoretical basis for revealing an action mechanism of an American ginseng extract for exerting anti-osteoporosis activity through multiple targets and multiple paths.
3. The invention provides data support for research and development of the anti-osteoporosis high-added-value product of American ginseng.
4. The invention also provides a construction method of the zebra fish osteoporosis model, which adopts the transgenic zebra fish, can visually observe the improvement effect of the medicine on bone development within 5dpf, and improves the problems of uneven dyeing, long experimental period, poor repeatability and the like caused by the traditional dyeing method.
5. The invention also discovers for the first time that the American ginseng has the activity of resisting osteoporosis and can be used as an effective component for treating osteoporosis symptoms.
Drawings
FIG. 1 is a diagram of the bone fluorescent phenotype of the head of transgenic zebra fish at different developmental stages.
FIG. 2 effect of prednisolone at different concentrations on bone fluorescence of transgenic zebra fish heads.
Figure 3 phenotypes of prednisolone treated zebra fish were observed by different staining methods.
FIG. 4 is a graph showing the effect of drug treatment on the fluorescence area and optical density of osteoporotic zebra fish skull;
in the figure: ctl is a blank control group; PD is a prednisolone model group; ED is etidronate disodium positive control group; WD, US, CAN and JL are American ginseng extracts produced in the Chinese Wendent, the United states, canada and China Jilin, respectively.
FIG. 5 is a bar graph of statistical results of the effect of drug treatment on the fluorescence area of osteoporotic zebra fish cranium;
in the figure: ## P＜0.01vs Ctl； * P＜0.05, ** P＜0.01, *** P＜0.001, **** P＜0.0001vs PD。
FIG. 6 is a bar graph of statistical results of the effect of drug treatment on the fluorescence optical density of osteoporotic zebra fish cranium;
in the figure: ### P＜0.001vs Ctl； * P＜0.05, ** P＜0.01, *** P＜0.001, **** P＜0.0001vs PD。
fig. 7 is a graph showing the effect of different traditional Chinese medicine extracts on bone development of the head of the osteoporosis zebra fish.
FIG. 8 is a bar graph of statistical results of the effect of drug treatment of American ginseng at different producing regions on the level of gene expression of osteoporotic zebra fish;
in the figure: ctl is a blank control group; PD is a prednisolone model group; WD, US, CAN and JL are extracts of American ginseng produced in chinese text dence, united states, canada, and chinese Jilin, respectively;
## P＜0.01vs control； *** P＜0.001, **** P＜0.0001vs PD。
FIG. 9 is a bar graph of statistical results of the effect of Jilin produced American ginseng on the level of expression of osteoporotic zebra fish genes at mass concentrations of 2.5, 5 and 10 μg/mL;
in the figure: * P＜0.05, *** P＜0.001, **** P＜0.0001vs PD。
FIG. 10 is a graph of metabonomic profile analysis results;
in the figure: PCA score plot (A), PLS-DA score plot (B), PLS-DA validation plot (C) and S-plot (D) in OPLS-DA mode for each experimental group in positive ion mode; PCA score plot (E), PLS-DA score plot (F), PLS-DA validation plot (G) and S-plot (H) in OPLS-DA mode for each experimental group in negative ion mode.
FIG. 11 is a bar graph of the relative content statistics of 50 different metabolites in each experimental group;
in the figure: c is blank group, M is model group, JL is Jilin American ginseng treatment group, CAN is Canadian American ginseng treatment group, US is American ginseng treatment group, WD is Wendenty American ginseng treatment group;
#### P＜0.0001vs C； * P＜0.05, ** P＜0.001, *** P＜0.0001, **** P＜0.0001vs M。
FIG. 12 is a bar graph of relative content statistics of 50 different metabolites in each experimental group;
in the figure: c is blank group, M is model group, JL is Jilin American ginseng treatment group, CAN is Canadian American ginseng treatment group, US is American ginseng treatment group, WD is Wendenty American ginseng treatment group;
#### P＜0.0001vs C； * P＜0.05, ** P＜0.001, *** P＜0.0001, **** P＜0.0001vs M。
FIG. 13 is a graph of metabolic channel dredging analysis results of key differential metabolites.
Detailed Description
The technical scheme of the invention is further described below with reference to the attached drawings of the specification so as to enable the person skilled in the art to implement the technical scheme according to the description.
The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.
The main material sources are as follows:
american ginseng decoction pieces in different producing areas are prepared from the following raw materials: american ginseng decoction pieces are produced in Jilin, the United states, canada and the different places of production of Wendelia respectively and purchased from Wendelia mountain area and Wendelia development Limited.
Zebra fish water for culturing fish: containing 5.0 mmol.L -1 NaCl，0.17mmol·L -1 KCl，0.4mmol·L -1 CaCl 2 ，0.16mmol·L -1 MgSO 4 。
Tg (-2.2col10a1a:gfp) transgenic zebra fish embryo: the adult zebra fish is in darkness for 10 h/illumination for 14h,
feeding at the temperature of (28+/-0.5) DEG C, and feeding the brine shrimp at regular time and quantity every day. Mating and spawning healthy zebra fish in a spawning jar according to the proportion of 2:2, collecting fertilized eggs, sterilizing and washing the fertilized eggs, transferring the fertilized eggs into zebra fish raising water (containing 5.0mM NaCl,0.17mM KCl,0.4mM CaCl2,0.16mM MgSO4), and culturing under controlled light at 28 ℃ until the fertilized eggs develop to 1dpf for experiment; the culture method belongs to the conventional culture method in the field.
Preparation of American ginseng extract: based on the saponin components in the American ginseng as main active components, a 50% ethanol extraction method is selected to prepare an American ginseng extract sample.
The specific method comprises the steps of preparing American ginseng decoction pieces by Jilin, american, canada and Wendengden, grinding into powder by a grinder, and sieving by a No. 3 sieve of pharmacopoeia. Precisely weighing 10g of American ginseng powder in different producing areas in a conical flask with a plug, adding a ten-fold amount of 50% ethanol water solution, sealing, performing ultrasonic-assisted extraction at room temperature for 2 times each time for 2 hours, filtering, combining the filtrates, concentrating under reduced pressure to obtain an extract, freeze-drying to obtain powder, and preserving at 4 ℃ for later use.
Construction method of anti-osteoporosis zebra fish model
Skeletal green fluorescently labeled transgenic zebra fish Tg (-2.2col10a1a: GFP) was purchased from the national zebra fish resource center. The experiment constructs a glucocorticoid-induced osteoporosis evaluation model based on transgenic zebra fish Tg (-2.2col10a1a: GFP) marked by bone green fluorescence. And the modeling concentration and the model experiment period of the prednisolone are determined by dynamically observing the development condition of osteoblasts.
The bone structure and morphology of the head bones of transgenic zebra fish within days 4-7 after fertilization were examined by feeding only zebra fish water (figure 1). The head skeleton of zebra fish is mainly formed by cartilage ossification, the head ossification generally starts from 3d, the head skeleton of zebra fish juvenile fish generally forms substantially from 5d, and consists of three parts of cartilage, cranium and pharyngeal skeleton (Peng Wei, zhang Wenjuan, xue. The research progress of zebra fish as a bone disease model [ J ]. Chinese laboratory animal journal, 2019,27 (02): 248-253.). From the results, it was found that the fluorescence area and fluorescence density of the bone of zebra fish head, in which the Michael cartilage, the gill strip bone, the gill cover bone and the spoon bone were substantially formed, were used as the evaluation index of the anti-osteoporosis activity within 4 to 7 days. The zebra fish gill strips developed for 4d are relatively weak in ossification, head bones do not form a stable state, and the individual weight is small due to short development time, so that the zebra fish gill strips are not beneficial to sample collection in q-PCR and metabonomic analysis. The ossification degree of the branchia bones of the zebra fish growing for more than 6 days is deepened, 4-6 branchia bones can be observed, however, at the moment, the zebra fish can eat food through openings, the medicine absorption mode is changed from skin permeation to oral and skin permeation, and experimental data are easy to be scattered due to individual difference of medicine intake of the zebra fish, and the model is unstable. Thus, the experimental period selected for the present patent was 5dpf.
And (3) setting a blank group and a prednisolone molding module, and examining the influence of the molding drug concentration in the concentration range of 2.5-12.5 mu M on the bone development of the head of the transgenic zebra fish (figure 2). The concentration of the prednisolone is within the concentration ranges of 2.5, 5, 7.5, 10 and 12.5 mu M, the prednisolone has positive correlation with the modeling effect to a certain extent, the prednisolone can form a stable osteoporosis state in the range of 5-10 mu M, the modeling concentration is too low (2.5 mu M), the development of the skull of a model group is not obviously inhibited (P is more than 0.05 compared with a blank group), the modeling concentration is too high (12.5 mu M), the bone development inhibition effect is obvious, the toxicity is certain, and the swimming bladder of the young zebra fish can be caused. In conclusion, prednisolone concentration in the range of 5-10 μm can be used as molding concentration.
The modeling key factors are integrated, and the constructed zebra fish osteoporosis resistant model is as follows: when the zebra fish embryo grows to 1dpf, the egg membrane is removed by using 1.0mg/mL pronase E solution, normal zebra fish juvenile fish are selected under a stereoscopic microscope and transferred into a 24-hole culture plate, a blank group, a model group and 3 groups of American ginseng experiment groups with gradient concentration are arranged, 10 zebra fish are arranged in each group, 3 compound holes are arranged in each group, each group is cultivated by zebra fish cultivation water, and the final volume of each hole is 2.0mL. Except that the blank group is treated by zebra fish culture water, the other groups are added with a prednisolone solution, so that the concentration of the prednisolone in each hole is 5-10 mu M, the model group is not added with other medicines for treatment, the experimental groups are added with American ginseng extract, the final concentrations are 2.5, 5 and 10 mu g/mL respectively, the cover is added, the fish is placed in an illumination incubator (28 ℃) to enable the juvenile fish to continue to develop to 5dpf, the zebra fish of each experimental group is placed in anesthetic for anesthesia, the condition of zebra fish fluorescent cells is observed under a microscope and photographed, and the Image-Pro Plus software is utilized to calculate the bone fluorescent area and the fluorescent density of the head of the zebra fish. Compared with blank data, the bone fluorescence area and fluorescence density of the model group are obviously reduced (P is less than 0.05), and the model is successfully manufactured.
Comparison of the constructed osteoporosis model with the traditional osteoporosis model:
the method is characterized in that the method comprises the steps of adopting alizarin red staining method (Zheng Huili, hua Yongqing, liu Xinhui and the like, screening the active site of the osteoporosis based on the wolfberry fruit of the zebra fish model, and the mechanism of the method is initially discovered [ J ]. Pharmaceutical journal, 2023,58 (01): 127-138), double staining method (Pu Shiya, pei win the influence of phthalate exposure on the growth and development of young zebra fish [ C ]// Chongqing market environmental science (Chongqing Society for Environmental Sciences), chongqing market ecological environment science institute (Chongqing Academy of Eco-environmental Sciences), chongqing market second-day ecological environment technical society and the society of Chongqing environmental science of 2019, the influence of resveratrol on the osteoporosis of the zebra fish induced by prednisone [ J ], [ Guo Donggui, kangqin, li Li and the like ] and the method for constructing the osteoporosis-resistant model of the zebra fish by using the method is researched by the invention (2019,30).
The traditional osteoporosis resistance activity evaluation method adopts a chemical staining method of AB wild zebra fish induced by prednisolone, and dyes comprise alizarin red, calcein, alizarin red, and Ab Li Xinlan double staining, but have the advantages of complex operation (multiple chemical reagents are needed), long experiment period (repeated operations such as bleaching are needed by the alizarin red and double staining method, the experiment period is about 9 days), unstable dye marking (the chemical dyes have the defects of light sensitivity or poor absorptivity, the experimental repeatability cannot be ensured), large dye fluorescence background interference, multiple times of cleaning with a large amount of water are needed, and zebra fish tissue breakage is easy to cause (calcein method). Therefore, the green fluorescence transgenic zebra fish osteoporosis model induced by the glucocorticoid provided by the invention has the advantages of simple operation, short experimental period and good reproducibility.
(III) evaluation of anti-osteoporosis Activity of American ginseng extracts in different producing areas
Selecting healthy skeleton green fluorescence marked transgenic zebra fish as an experimental object, removing egg membranes by using 1.0mg/mL pronase E solution when embryos develop to 1dpf, selecting normal zebra fish juvenile fish under a stereoscopic microscope, transferring the zebra fish juvenile fish into a 24-hole culture plate, and setting a blank control group, a model group, a positive control group and a low-medium-high three-concentration American ginseng medicine treatment group, wherein the total volume of each group is 2.0mL. The blank control group (Ctl) is treated by adding only zebra fish culture water. Zebra fish water (5.0 mmol.L) -1 NaCl，0.17mmol·L -1 KCl，0.4mmol·L -1 CaCl 2 ，0.16mmol·L - 1 MgSO 4 ). ModelGroup (PD) was added with prednisolone modeling agent in addition to zebra fish water, and the modeling agent concentration in the wells was 10. Mu.M. The positive control group (ED) was added with the modeling agent at the same concentration as the model group, and then with etidronate disodium, so that the concentration of etidronate disodium in the wells was 25. Mu.g/mL. The American ginseng medicine treatment group is added with the prednisolone with the same concentration as the model group, and simultaneously added with the American ginseng extract with gradient concentration, and the final concentration of the American ginseng extract in the low, medium and high concentration groups is 2.5, 5 and 10 mug/mL respectively. 3 compound holes are arranged in each group, the cover is covered, and the young fish is placed in an illumination incubator (28 ℃) to continue to develop to 5dpf.
And (3) putting the zebra fish of each experimental group into an anesthetic for anesthesia, observing the condition of fluorescent cells of the zebra fish under a microscope, photographing, and calculating the bone fluorescence area and the fluorescence density of the head of the zebra fish by using Image-Pro Plus software. The skeletal development of zebra fish in each experimental group is shown in figure 4. The statistics of the fluorescence area of the zebra fish bones of each experimental group are shown in fig. 5, and the statistics of the fluorescence density of the bones are shown in fig. 6. Compared with a blank group, the model group has obviously reduced bone fluorescence area and fluorescence density (P is less than 0.01), which indicates that the osteoporosis model induced by prednisolone is successful; compared with the model group, the positive control group can obviously improve the fluorescence area (P is less than 0.05) of the zebra fish bones, which indicates that the model result is reliable. The 50% ethanol extracts of the American ginseng in 4 producing areas can improve the reduction of the fluorescence area and the fluorescence density of the zebra fish bones caused by prednisolone to a certain extent. Wherein, the Jilin American ginseng extract (JL) CAN obviously improve the fluorescence area and the fluorescence density of the zebra fish bones (P < 0.0001) in the concentration range of 2.5-10 mug/mL, the Canadian American ginseng extract (CAN) has inferior osteoporosis improvement activity, the Wedelin American ginseng extract (WD) CAN obviously improve the fluorescence area and the fluorescence density of the zebra fish bones (P < 0.01) only at the concentration of 10 mug/mL, and the American ginseng extract (US) CAN only obviously improve the fluorescence area of the zebra fish bones (P < 0.05) at the concentration of 10 mug/mL. The results show that the American ginseng extract in each producing area has the anti-osteoporosis activity.
Evaluation of anti-osteoporosis activity of other traditional Chinese medicine extracts based on zebra fish model
The extracts of the epimedium herb, the nutgrass galingale rhizome, the szechuan lovage rhizome and the tree peony bark are prepared by adopting a 50% ethanol ultrasonic auxiliary extraction method, and the anti-osteoporosis activity of the extracts is evaluated according to the anti-osteoporosis activity evaluation method based on the zebra fish (shown in figure 7), so that the result shows that the traditional anti-osteoporosis traditional Chinese medicine epimedium herb can remarkably improve the zebra fish osteoporosis state induced by prednisolone, and the traditional Chinese medicines with the effects of soothing liver, relieving depression, activating blood circulation, removing blood stasis and the like such as the nutgrass galingale rhizome, the szechuan lovage rhizome and the tree peony bark have no obvious effect of improving the osteoporosis activity. The results show that the technology disclosed by the invention can be used for evaluating the osteoporosis activity of the medicine.
(IV) qRT-PCR analysis of influence of American ginseng extracts with different concentrations on expression level of related genes of zebra fish osteoporosis
1) Zebra fish grouping and processing
Selecting healthy skeleton green fluorescence marked transgenic zebra fish as an experimental object, removing egg membranes by using 1.0mg/mL pronase E solution when embryos develop to 1dpf, selecting normal zebra fish juvenile fish under a stereoscopic microscope, transferring the zebra fish juvenile fish into a 24-hole culture plate, and setting a blank control group, a model group, a positive control group and a low-medium-high three-concentration American ginseng medicine treatment group, wherein the total volume of each group is 2.0mL. The blank control group (Ctl) is treated by adding only zebra fish culture water. Zebra fish water (5.0 mmol.L) -1 NaCl，0.17mmol·L -1 KCl，0.4mmol·L -1 CaCl 2 ，0.16mmol·L - 1 MgSO 4 ). In addition to zebra fish water, prednisolone molding agent is added into the model group (PD), and the concentration of molding agent in the holes is 10 mu M. The positive control group (ED) was added with the modeling agent at the same concentration as the model group, and then with etidronate disodium, so that the concentration of etidronate disodium in the wells was 25. Mu.g/mL. The American ginseng medicine treatment group is added with the prednisolone with the same concentration as the model group, and simultaneously added with the American ginseng extract with gradient concentration, and the final concentration of the American ginseng extract in the low, medium and high concentration groups is 2.5, 5 and 10 mug/mL respectively. 3 compound holes are arranged in each group, the cover is covered, and the young fish is placed in an illumination incubator (28 ℃) to continue to develop to 5dpf.
2) Effect of American ginseng extract on osteoporosis-related Gene expression level
30 5dpf zebra fish strips are respectively collected from a blank control group, a prednisolone model group and an American ginseng drug treatment experimental group, and frozen at-80 ℃ for standby. Each group of zebra fish total RNA was extracted according to the FastPure cell// Tissue Total RNA Isolation kit V2 kit (Vazyme) instructions, reverse transcribed into cDNA using the HiScript// Q RT Supermix kit (Vazyme), and real-time fluorescent quantitative PCR (Quantitative Real-time PCR (qRT-PCR)) was performed using ChamQ Universal SYBR Qpcr master mix (Vazyme). The amplification conditions were: pre-denaturation at 95℃for 10min, annealing at 60℃for 30s, extension at 72℃for 15s,40 cycles. Detecting the expression of each gene, and taking beta-actin as a reference gene. The primer synthesis sequences are shown in Table 1.
TABLE 1 primer sequences
The effect of four production place American ginseng extract experimental groups with mass concentration of 10 mug/mL on the osteoporosis related Gene expression level of zebra fish is shown in figure 8, compared with the blank group, the expression of the vitamin D receptor transcription factor (vdrb, gene ID: 564511), alpha 1-II collagen Gene (col 1a2, gene ID: 336471), cysteine-rich acid secretion protein Gene (sparc, gene ID: 321357) is obviously reduced (P < 0.0001), the expression of the matrix metalloproteinase 9 (mmp 9, gene ID: 406397), the tartrate-resistant acid phosphatase (trap, gene ID: 100187907) and the expression of the cathepsin K (ctsk, gene ID: 550475) are obviously increased (P < 0.01) after the prednisolone treatment,
the dry prognosis of American ginseng can obviously improve the influence of prednisolone on the expression level of the genes (P <0.001 compared with a model group), and the improvement effect of American ginseng at each producing area on the expression level of zebra fish genes does not show obvious difference (P > 0.05) under the condition of the same experimental concentration, so that the influence of American ginseng at different producing areas on the key genes of osteoblast generation and osteoclast absorption is the same.
The effect of improving the above genes at concentrations of 2.5, 5 and 10 μg/mL is shown in figure 9, and compared with the model group, the treatment of the extract of American ginseng in Jilin production can significantly improve the abnormal expression (P <0.05, P <0.001, P < 0.0001) of the above genes in the zebra fish organism induced by prednisolone.
(V) metabonomics-based American ginseng anti-osteoporosis biomarker and action mechanism research
(1) Sample collection and processing
Selecting healthy skeleton green fluorescence marked transgenic zebra fish as an experimental object, removing egg membranes by using 1.0mg/mL pronase E solution when embryos develop to 1dpf, selecting normal zebra fish juvenile fish under a stereoscopic microscope, transferring the zebra fish juvenile fish into a 6-hole culture plate, setting a blank control group (Ctl), a prednisolone model group (PD), a Jilin American ginseng medicine treatment group (JL), a Canada American ginseng medicine treatment group (CAN), a American ginseng medicine treatment group (US) and a Wendengden American ginseng medicine treatment group (WD) at a final volume of 5.0mL per hole, and carrying out three-dimensional culture on the zebra fish. The blank control group (Ctl) is treated by adding only zebra fish culture water. Zebra fish water (5.0 mmol.L) -1 NaCl，0.17mmol·L -1 KCl，0.4mmol·L -1 CaCl 2 ，0.16mmol·L -1 MgSO 4 ). In addition to zebra fish water, prednisolone molding agent is added into the model group (PD), and the concentration of molding agent in the holes is 10 mu M. Adding prednisolone with the same concentration as that of the model group into the American ginseng medicine treatment group, adding American ginseng extracts in different producing areas, wherein the final concentration of each group of medicine is 10 mug/mL, setting 3 composite holes in each group, collecting young fish with each hole when the young fish grows to 5dpf, grinding the young fish in liquid nitrogen in an EP pipe, adding 500 mu L of 80% methanol water, vortex oscillating for 1min, standing in an ice bath for 5min, centrifuging for 20min at 4 ℃ at 15000g, adding mass spectrum grade ultrapure water into the supernatant to dilute the supernatant to 53%, centrifuging for 2 times according to the conditions, and taking the supernatant for LC-MS analysis.
(2) Chromatographic conditions
Thermo Fisher Hypesil Gold column column (100×2.1mm,1.9 μm), positive ion mode: mobile phase a (0.1% formic acid) and mobile phase B (methanol), flow rate 0.2mL/min, column temperature 40 ℃, gradient elution: 2% B,0-1.5min;2-85% B,1.5-3min;85-100% B for 3-10min;100-2% B,10-10.1min;100-2% B,10-10.1min;2% B,10.1-12min.
(3) Mass spectrometry conditions
The scanning range is selected from m/z 100-1500; the ESI source settings were as follows: spray Voltage (Spray Voltage): 3.5kV; sheath air flow rate (Sheath gas flow rate): 35psi; auxiliary gas flow rate (Aux Gas flow rate): 10L/min; ion transport tube temperature (calilla Temp): 320 ℃; ion introduction ring projection frequency amplitude (S-lens RF level): 60 percent; auxiliary gas heater temperature (Aux gas heater temp): 350 ℃; polarity (Polarity): positive, negative; the MS/MS secondary scan is a data-dependent scan (data-dependent scans).
(4) Metabonomics profile analysis
And the UPLC-MS is adopted to collect data of non-targeted metabonomics of zebra fish samples, equal volume mixing of each group of test samples is adopted as a quality control sample (QC), and total ion flow diagrams of QC samples are basically overlapped in positive and negative ion modes, so that the stability of a detection system is good. The analysis results of metabonomics profile of each experimental group are shown in fig. 10, the clustering condition of each group of data is analyzed by adopting a principal component analysis method (PCA) and a partial least squares discriminant analysis method (PLS-DA), PCA score diagrams in positive and negative ion modes are shown in fig. 10A and 10E, a model group (PD) is obviously separated from a blank group (Ctl), each producing area American ginseng medicament group, particularly a Jilin American ginseng medicament group (JL) and a Canadian American ginseng medicament group (CAN) are close to the blank group, and further analysis of each experimental group of data by adopting PLS-DA also shows that the metabolite level in the zebra fish body CAN be improved by adding American ginseng protection, so that the zebra fish body is close to the blank group (fig. 10B and 10F). The model is verified through substitution test, as shown in fig. 10C and 10G, in the positive and negative ion modes, R2 and Q2 are lower than the rightmost original value from left to right, and Q2 and Y axis intersect at the negative half axis, which indicates that the model has no overfitting and good prediction capability.
(5) Screening of potential biomarkers for osteoporosis resistance
Differential metabolites of prednisolone-induced zebra fish osteoporosis were screened based on variable importance Values (VIP), significance analysis P values, and components at both ends of S-plot of the blank and model groups (fig. 10D and 10H). Compared with a blank group (Ctl group), the VIP value of 70 metabolites in the model group (M group) is larger than 1, the P value is obviously analyzed by combining the Ctl group and the model group, the P value is less than 0.05 as a further screening condition, 50 differential metabolites are determined to be related to prednisolone-induced zebra fish bone osteoporosis, and after the intervention treatment of the osteoporosis zebra fish by American ginseng extracts in different producing places, the differential metabolite level can be obviously changed compared with that of the model group (figure 11 and figure 12). The method is characterized in that VIP & gt 1 and P & lt 0.05 are used as indexes, in-vivo biomarkers of zebra fish after American ginseng drug intervention are studied, compared with a model group, JL groups are changed in 72 metabolites, CAN groups are changed in 82 metabolites, US groups are changed in 81 metabolites, WD groups are changed in 82 metabolites, 33 intersection difference metabolites are obtained through Wen graph analysis and co-screening, and the content of the intersection difference metabolites is further compared, so that 24 metabolites in the zebra fish body treated by prednisolone CAN be obviously recalled into a blank group through 50% alcohol extract of American ginseng (tables 2-1 and 2-2). The results indicate that the 24 key differential metabolites may be key biological quality markers for the American ginseng to play the anti-osteoporosis activity.
Table 2-1 relative levels ∈and ∈of the differential metabolites of each experimental group represent the decrease and increase in relative levels
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Table 2-2 relative levels of differential metabolites
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(6) Analysis of related metabolic pathways
Through metabolic pathway enrichment analysis of the 24 different metabolites, and taking-LogP > 1.5 and Impact > 0.02 as standards, the American ginseng is found to improve osteoporosis caused by prednisolone mainly through 3 metabolic pathways including purine metabolism, tricarboxylic acid cycle and pentose phosphate metabolism (figure 13). The 3 differential metabolic pathways described above involved 10 key biomarkers in total, and the identification information is shown in table 3. The results show that compared with a blank group, the prednisolone treatment can obviously raise the level of the above-mentioned marker in the zebra fish body (P < 0.001), compared with a model group, the intervention of the 50% ethanol extract of American ginseng can obviously lower the expression level of the above-mentioned biomarker in the zebra fish body (P < 0.001) by adding the 50% ethanol extract of American ginseng, so that the expression level of the biomarker in the zebra fish body is adjusted back to be close to the level of the blank group.
Table 3 biomarker identification information
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The invention discloses a zebra fish biomarker for resisting osteoporosis of American ginseng and application thereof for the first time; the invention makes clear for the first time that American ginseng can obviously up-regulate the expression level of osteoblast related genes vdrb, col1a2 and spark and obviously down-regulate the expression level of osteoclast related genes mmp9, trap and ctsk. Based on the metabonomics technical means, 24 key differential metabolites of the American ginseng for resisting osteoporosis are defined for the first time, and the path analysis shows that the American ginseng can call back 10 key biological quality marker contents by participating in purine metabolism, tricarboxylic acid circulation and pentose phosphate metabolism, thereby improving the osteoporosis state of the zebra fish.
The American ginseng anti-osteoporosis biomarker based on the metabonomics technology provides a theoretical basis for revealing an action mechanism of an American ginseng extract for exerting anti-osteoporosis activity through multiple targets and multiple paths.
The invention also provides a construction method of the zebra fish osteoporosis model, which adopts the transgenic zebra fish, can visually observe the improvement effect of the medicine on bone development within 5dpf, and improves the problems of uneven dyeing, long experimental period, poor repeatability and the like caused by the traditional dyeing method.
The invention also discovers for the first time that the American ginseng has the activity of resisting osteoporosis and can be used as an effective component for treating osteoporosis symptoms.
Claims (11)
1. An application of zebra fish biomarkers in the evaluation of anti-osteoporosis activity of American ginseng or the quality control of American ginseng, wherein the zebra fish biomarkers comprise: gene markers and/or metabolic markers;
the gene markers are vdrb, col1a2, spark, mmp9, trap and ctsk genes;
the metabolic substance markers are: gluconic acid, D-sedoheptulose-7-phosphate, D-ribulose-5-phosphate, uric acid, citric acid, guanine, hypoxanthine, creatinine, guanosine, and phosphopyruvic acid.
2. The use according to claim 1, wherein in the use, the primer sequences of vdrb, col1a2, spark, mmp9, trap and ctsk genes are as follows:
the primer sequences of the vdrb gene are: SEQ ID NO.7 and SEQ ID NO.8;
the primer sequences of the col1a2 gene are: SEQ ID NO.9 and SEQ ID NO.10;
the primer sequences of the spark gene are: SEQ ID NO.13 and SEQ ID NO.14;
the primer sequences of the mmp9 gene are as follows: SEQ ID NO.11 and SEQ ID NO.12;
the primer sequences of the trap gene are: SEQ ID NO.5 and SEQ ID NO.6;
The primer sequences of the ctsk gene are as follows: SEQ ID NO.3 and SEQ ID NO.4.
3. The use according to claim 1, wherein the zebra fish is a transgenic zebra fish that is fluorescently labeled for bone green.
4. The method for evaluating the anti-osteoporosis activity or the quality control of the American ginseng by using the zebra fish biomarker is characterized by comprising the following steps of:
the American ginseng extract acts on the zebra fish bone osteoporosis model to serve as an experimental group, and a corresponding blank group and a zebra fish bone osteoporosis model group are arranged; compared with a blank group, in the zebra fish osteoporosis model group, the beta-actin is taken as an internal reference gene, the expression of vdrb, col1a2 and spark genes in the zebra fish body is obviously reduced, and the expression of mmp9, trap and ctsk genes is obviously increased; compared with a model group, the experimental group can obviously up-regulate the expression of vdrb, col1a2 and spark genes in zebra fish bodies by taking beta-actin as an internal reference gene; the expression of the mmp9, trap and ctsk genes in the zebra fish body can be obviously reduced; proved by the demonstration that the American ginseng extract has the anti-osteoporosis activity; or proving that the American ginseng is a genuine product;
or, the American ginseng extract is acted on the zebra fish bone osteoporosis model to be used as an experimental group, and a corresponding blank group and a zebra fish bone osteoporosis model group are arranged; the zebra fish bone loosening model group has the advantages that the expression level of gluconic acid, D-sedoheptulose-7-phosphate, D-ribulose-5-phosphate, uric acid, citric acid, guanine, hypoxanthine, creatinine, guanosine and phosphopyruvic acid in zebra fish body is obviously increased compared with a blank group, the experimental group has the advantages that the expression level of the biological standard substance in the zebra fish body is obviously reduced compared with the model group, and compared with the blank group, the experimental group has no obvious difference; proved by the demonstration that the American ginseng extract has the anti-osteoporosis activity; or prove that the American ginseng is a genuine product.
5. The method of claim 4, wherein the blank group is a zebra fish aquaria treatment group;
preferably, the zebra fish water contains: 5.0mM NaCl,0.17mM KCl,0.4mM CaCl2,0.16mM MgSO4;
preferably, the zebra fish is a transgenic zebra fish with green fluorescence markers on bones.
6. The method of claim 4, wherein in the method, the American ginseng extract is a 50% ethanol extract of American ginseng;
preferably, in the method, the preparation method of the American ginseng extract comprises the following steps:
pulverizing radix Panacis Quinquefolii into powder, adding 5-20 times of 45-55% ethanol water solution, ultrasonic extracting for 2-4 times each for 1-3 hr, collecting extractive solution, concentrating under reduced pressure to obtain extract, and lyophilizing to obtain powder;
preferably, american ginseng is prepared into powder, sieved by a No. 3 sieve, added with 10 times of 50% ethanol water solution, extracted for 2 times by ultrasonic treatment for 2 hours each time, filtered and combined with filtrate, decompressed and concentrated into extract, and then freeze-dried into powder.
7. The method of claim 4, wherein the method for constructing the zebra fish bone loss model comprises the following steps:
Taking Tg (-2.2col10a1a: GFP) transgenic zebra fish embryo of 1dpf, removing egg membrane, selecting normal zebra fish juvenile fish, randomly grouping, and dividing into a blank group and a model group, wherein the blank group is treated by zebra fish water; adding prednisolone with the final concentration of 5-11 mu M into zebra fish culture water of a model group, continuously culturing juvenile zebra fish to 5dpf, observing the condition of zebra fish fluorescent cells, photographing, and calculating the bone fluorescent area and the fluorescent density of the zebra fish head, wherein compared with blank group data, the bone fluorescent area and the fluorescent density of the model group are obviously reduced, namely the modeling is successful;
preferably, prednisolone with the final concentration of 5-10 mu M is added into zebra fish culture water of a model group;
preferably, 8-15 zebra fish in each group, and 2-3 zebra fish in each group are parallel;
preferably, 1.0 mg.multidot.mL is used -1 Removing egg membranes from the pronase E solution;
preferably, the culture condition of the young zebra fish is 28 ℃.
8. The method according to claim 4, wherein the experimental group is specifically: compared with the model group, the American ginseng extract is added while the same modeling agent is added;
preferably, the molding agent is prednisolone, and the American ginseng extract is added to a final concentration of 2.5, 5 or 10 mug/mL.
9. The method of claim 4, wherein the primer sequences for vdrb, col1a2, spark, mmp9, trap, and ctsk genes are as follows:
the primer sequences of the vdrb gene are: SEQ ID NO.7 and SEQ ID NO.8;
the primer sequences of the col1a2 gene are: SEQ ID NO.9 and SEQ ID NO.10;
the primer sequences of the spark gene are: SEQ ID NO.13 and SEQ ID NO.14;
the primer sequences of the mmp9 gene are as follows: SEQ ID NO.11 and SEQ ID NO.12;
the primer sequences of the trap gene are: SEQ ID NO.5 and SEQ ID NO.6;
the primer sequences of the ctsk gene are as follows: SEQ ID NO.3 and SEQ ID NO.4.
10. The construction method of the zebra fish bone osteoporosis model is characterized by comprising the following steps of:
taking Tg (-2.2col10a1a: GFP) transgenic zebra fish embryo of 1dpf, removing egg membrane, selecting normal zebra fish juvenile fish, randomly grouping, and dividing into a blank group and a model group, wherein the blank group is treated by zebra fish water; adding prednisolone with the final concentration of 5-11 mu M into zebra fish culture water of a model group, continuously culturing juvenile zebra fish to 5dpf, observing the condition of zebra fish fluorescent cells, photographing, and calculating the bone fluorescent area and the fluorescent density of the zebra fish head, wherein compared with blank group data, the bone fluorescent area and the fluorescent density of the model group are obviously reduced, namely the modeling is successful;
Preferably, prednisolone with the final concentration of 5-10 mu M is added into zebra fish culture water of a model group;
preferably, 8-15 zebra fish in each group, and 2-3 zebra fish in each group are parallel;
preferably, 1.0 mg.multidot.mL is used -1 Removing egg membranes from the pronase E solution;
preferably, the culture condition of the young zebra fish is 28 ℃.
11. The application of American ginseng as an active ingredient in preparing an anti-osteoporosis medicament;
preferably, in the application, the American ginseng is an American ginseng extract;
preferably, the American ginseng extract is an American ginseng 50% ethanol extract;
preferably, the American ginseng extract is prepared by the preparation method of the American ginseng extract in claim 6.
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