Saussurea involucrata cell culture and use thereof

Abstract

Provided are a saussurea involucrata cell culture and use thereof. The saussurea involucrata cell culture comprises the following components: a) a flavonoid compound with a content of 7 wt% to 20 wt%; b) a phenylpropanoid derivative with a content of 2.1 wt% to 14.1 wt%; c) a carboxylic acid and a derivative thereof, wherein the carboxylic acid and the derivative thereof comprise dicaffeoylquinic acid with a content of 1.6 wt% to 35 wt%; and d) other components.

Description

Snow lotus cell culture and application thereof Cross-references

[0001] This application claims priority to Chinese application No. 202410209155.X filed on February 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and in particular to a snow lotus cell culture and its application. Background Art

[0003] Saussurea involucrata Kar.et Kir., also known as "snow lotus," is a perennial herbaceous plant of the genus Saussurea in the Asteraceae family. It is a rare and precious Chinese herbal medicine unique to Xinjiang. Of the 12 species and one variant of Saussurea commonly used as a crude drug, only Saussurea involucrata is currently included in the 2005 edition of the Chinese Pharmacopoeia. Traditional Chinese Medicine believes that Saussurea involucrata dispels cold and dampness, promotes blood circulation and menstruation, and balances yin and yang. Modern scientific research shows that Saussurea involucrata primarily contains flavonoids and lignans, as well as bioactive compounds such as coumarins, sesquiterpenes, phenolic aldehydes, phenylpropanols, sterols, polysaccharides, and ceramides. Consequently, it possesses therapeutic effects in inhibiting tumors, leukemia, various cancers, and inflammation, and holds great medicinal value. However, Saussurea involucrata grows slowly in its specific habitat, making it difficult to cultivate artificially. Long-term predatory harvesting has put the wild Saussurea involucrata at risk of extinction.

[0004] Plant cell culture technology, which cultivates and propagates plant cells, tissues, or organs in an organism-free environment, is an effective means of addressing the scarcity of medicinal plant resources. Using plant cell culture technology to address the issue of natural snow lotus resources is an important path to sustainable development. Therefore, Tianshan Snow Lotus plant cell culture technology and snow lotus cell culture hold enormous potential for application.

[0005] Atherosclerosis (AS) is a serious threat to human health and is the most common cause of coronary thrombosis and ischemic stroke. Its characteristic lesion begins in the intima of the affected artery, gradually leading to lipid accumulation, fibrosis, and calcification, and even calcification of the arterial media. This can lead to intraplaque hemorrhage, plaque rupture, and localized thrombosis, which are the primary causes of most cardiovascular diseases. Therefore, preventing and treating atherosclerosis is a key step in the prevention of cardiovascular and cerebrovascular diseases. Research on lipid-lowering and anti-inflammatory drugs is crucial for the prevention and treatment of atherosclerosis.

[0006] Long-term chronic liver damage is a chronic inflammatory liver disease caused by various factors, including drug-induced hepatitis, viral infection, chronic alcohol consumption, fatty liver disease, and immune responses. Drug-induced and viral damage are the primary causes. Long-term chronic liver damage is often accompanied by an inflammatory response, which can lead to liver cell damage and apoptosis, as well as excessive release of transaminases into the bloodstream, thus impairing normal liver function. Researching and using appropriate medications is crucial for the prevention and treatment of long-term chronic liver damage.

[0007] Therefore, it is necessary to provide a snow lotus cell culture that can replace the plant-derived Tianshan snow lotus and can be prepared by plant cell culture technology, as well as to provide the use of the snow lotus cell culture in the preparation of drugs for specific diseases / indications. Summary of the Invention

[0008] Some embodiments of the present specification provide a snow lotus cell culture, comprising the following components: a) flavonoids, with a content of 7-20 wt%; b) phenylpropanoid derivatives, with a content of 2.1-14.1 wt%; c) carboxylic acids and their derivatives, wherein the carboxylic acids and their derivatives contain dicaffeoylquinic acid, with a content of 1.6-35 wt%; and d) other components.

[0009] In some embodiments, the phenylpropanoid derivative comprises 0.3 wt%-5 wt% of syringin and 0.3 wt%-1.3 wt% of chlorogenic acid.

[0010] In some embodiments, the dicaffeoylquinic acid comprises 1,5-dicaffeoylquinic acid, the content of the 1,5-dicaffeoylquinic acid is 1.5 wt %-7.7 wt %, and the proportion of the 1,5-dicaffeoylquinic acid in the dicaffeoylquinic acid is 22%-95%.

[0011] In some embodiments, the HPLC characteristic spectrum of the snow lotus cell culture at 265 nm includes 9 characteristic peaks; among them, peak 1 is syringin, peak 2 is chlorogenic acid, peak 4 is 1,4-dicaffeoylquinic acid, peak 5 is 1,5-dicaffeoylquinic acid, peak 6 is 3,5-dicaffeoylquinic acid, peak 7 is dicaffeoylquinic acid malate, peak 8 is 1,3,5-tricaffeoylquinic acid malate, and peak 9 is 1,3,5-tricaffeoylquinic acid apple methyl ester.

[0012] In some embodiments, in the HPLC characteristic spectrum of the snow lotus cell culture at 265 nm, with peak No. 5 as the reference peak, the relative retention time of each characteristic peak is within ±5% of the following specified values: the specified value of peak No. 1 is 0.369, the specified value of peak No. 2 is 0.450, the specified value of peak No. 3 is 0.668, the specified value of peak No. 4 is 0.918, the specified value of peak No. 5 is 1.000, the specified value of peak No. 6 is 1.057, the specified value of peak No. 7 is 1.140, the specified value of peak No. 8 is 1.622, and the specified value of peak No. 9 is 1.645.

[0013] Some embodiments of this specification also provide a method for preparing a snow lotus cell culture. The preparation method comprises: obtaining a population of differentiated embryonic pluripotent stem cells derived from wild snow lotus; performing amplification culture using the differentiated embryonic pluripotent stem cell population, and harvesting a culture fluid containing the snow lotus cell culture; wherein the differentiated embryonic pluripotent stem cell population is obtained through differentiation control, wherein the differentiation control causes the differentiated embryonic pluripotent stem cell population to have the following characteristics: compared with in vitro cells from wild snow lotus as a control, the differentiated embryonic pluripotent stem cell population has low expression of differentiation-related genes, including WOX4, LBD18, WOX5, and PLT4.

[0014] In some embodiments, the snow lotus cell culture comprises the following components: a) flavonoids, with a content of 7wt%-20wt%; b) phenylpropanoid derivatives, with a content of 2.1wt%-14.1wt%; c) carboxylic acids and their derivatives, wherein the carboxylic acids and their derivatives contain dicaffeoylquinic acid with a content of 6.8wt%-8.1wt%; and d) other components.

[0015] In some embodiments, the dicaffeoylquinic acid comprises 1,5-dicaffeoylquinic acid, the content of the 1,5-dicaffeoylquinic acid is 1.5 wt %-7.7 wt %, and the proportion of the 1,5-dicaffeoylquinic acid in the dicaffeoylquinic acid is 22%-95%.

[0016] In some embodiments, the expression levels of WOX4 and LBD18 in the differentiated embryonic pluripotent stem cell population are less than 1 / 10 of the expression levels of the corresponding genes in the control group; the expression levels of WOX5 and PLT4 in the differentiated embryonic pluripotent stem cell population are less than 1 / 2 of the expression levels of the corresponding genes in the control group.

[0017] In some embodiments, the preparation method further comprises, during the expansion culture process, performing metabolic regulation on the differentiated embryonic pluripotent stem cell population based on enhanced flavonoid metabolic pathways.

[0018] In some embodiments, the metabolic regulation enables the differentiated embryonic pluripotent stem cell population to have the following characteristics: expression of genes related to the flavonoid metabolic pathway in the differentiated embryonic pluripotent stem cell population, including PAL, C4H, 4CL, CHI, C3H, F5H and CAD; and, using in vitro cells of wild snow lotus as a control, the expression of C3H, F5H and CAD in the differentiated embryonic pluripotent stem cell population is increased.

[0019] In some embodiments, the method also includes separating the snow lotus cell culture from the culture medium and preparing an extract using the snow lotus cell culture; the preparation of the extract includes: repeatedly freezing and thawing the snow lotus cell culture to obtain a molten mixture; extracting the molten mixture using an ethanol aqueous solution to obtain an extract, wherein the extraction treatment is at least one of ultrasonic extraction and stirring extraction; separating the solid part and the liquid part of the extract; and drying the liquid part to obtain the extract.

[0020] Some embodiments of this specification also provide the use of the aforementioned snow lotus cell culture in the preparation of a drug for atherosclerosis.

[0021] In some embodiments, the formation of atherosclerotic plaques in blood vessels is reduced in the treatment group administered with the saussurea cell culture compared to the atherosclerosis model group.

[0022] In some embodiments, compared with the atherosclerosis model group, in the treatment group administered with the Saussurea cell culture, the distribution of intestinal flora is regulated, and the regulation reduces the activation and migration of macrophages and the apoptosis of endothelial cells, thereby reducing the formation of atherosclerotic plaques.

[0023] In some embodiments, the snow lotus cell culture is obtained by amplifying and culturing a differentiated embryonic pluripotent stem cell population derived from wild snow lotus, and the differentiated embryonic pluripotent stem cell population is obtained by differentiation control, and the differentiation control enables the differentiated embryonic pluripotent stem cell population to have the following characteristics: using in vitro cells of wild snow lotus as a control, the differentiation-related genes of the differentiated embryonic pluripotent stem cell population are lowly expressed, and the differentiation-related genes include WOX4, LBD18, WOX5 and PLT4.

[0024] Some embodiments of this specification also provide the use of the aforementioned snow lotus cell culture in the preparation of a medicine for long-term chronic liver damage.

[0025] In some embodiments, compared with the model group of long-term chronic liver injury, in the treatment group administered with snow lotus cell culture, liver function injury-related indicators are reduced, including ALT, AST, GGT and AKP; and, in the treatment group, the generation of liver tissue fibrosis is reduced, and the formation and accumulation of adipose tissue are reduced, thereby reducing the formation of alcoholic fatty liver.

[0026] In some embodiments, compared with the model group of long-term chronic liver injury, in the treatment group administered with snow lotus cell culture, the intestinal flora distribution is regulated, and the regulation increases the number of beneficial bacteria and reduces the number of harmful bacteria, thereby reducing the difference in intestinal flora distribution between the treatment group and the normal group.

[0027] In some embodiments, the snow lotus cell culture is obtained by amplifying and culturing a differentiated embryonic pluripotent stem cell population derived from wild snow lotus, and the differentiated embryonic pluripotent stem cell population is obtained by differentiation control, and the differentiation control enables the differentiated embryonic pluripotent stem cell population to have the following characteristics: using in vitro cells of wild snow lotus as a control, the differentiation-related genes of the differentiated embryonic pluripotent stem cell population are lowly expressed, and the differentiation-related genes include WOX4, LBD18, WOX5 and PLT4. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] This specification will be further described in the form of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, and in these embodiments, like numbers represent like structures, wherein:

[0029] FIG1 is a bar graph showing the relative expression levels of differentiation-related genes in embryonic pluripotent stem cell populations at the mRNA level according to some embodiments of the present specification;

[0030] FIG2 is a bar chart showing the relative expression levels of key enzymes in the flavonoid metabolic pathway at the mRNA level according to some embodiments of the present specification;

[0031] FIG3 is a chromatogram comparison of characteristic peaks of Saussurea involucrata cell culture and Saussurea involucrata at 265 nm according to some embodiments of this specification;

[0032] FIG4A is a UV spectrum of 1,5-dicaffeoylquinic acid according to some embodiments of the present specification;

[0033] FIG4B is a UV spectrum of characteristic peaks of snow lotus cell culture No. 5 according to some embodiments of this specification;

[0034] FIG5A is a UV spectrum of rutin according to some embodiments of the present specification;

[0035] FIG5B is a UV spectrum of characteristic peak No. 6 of snow lotus cell culture according to some embodiments of this specification;

[0036] FIG5C is a UV spectrum of characteristic peaks of Tianshan Snow Lotus No. 6 according to some embodiments of this specification;

[0037] FIG6A is a bar graph showing the cell activity of HUVEC cells in different groups according to some embodiments of the present specification;

[0038] FIG6B is a bar graph showing the range of cell apoptosis according to some embodiments of the present specification;

[0039] FIG6C is a bar chart showing intervals of response cell necrosis according to some embodiments of the present specification;

[0040] FIG6D is a bar graph showing intervals of response to cell mechanical damage according to some embodiments of the present specification;

[0041] FIG6E is a bar graph showing the fluorescence intensity of HUVEC cells in different groups based on TUNEL staining according to some embodiments of the present specification;

[0042] FIG6F is a fluorescence image of HUVEC cells in different groups obtained by laser confocal microscopy based on TUNEL / DAPI double staining according to some embodiments of the present specification;

[0043] FIG6G is a bar graph showing the fluorescence intensity of the quantitative analysis of ROS production by HUVEC cells in different groups by flow cytometry according to some embodiments of the present specification;

[0044] FIG6H is a fluorescence image of HUVEC cells in different groups obtained by laser confocal microscopy based on DCFH-DA / DAPI double staining according to some embodiments of the present specification;

[0045] FIG6I is a bar graph showing the fluorescence intensity of HUVEC cells in different groups stained with DCFH-DA according to some embodiments of the present specification;

[0046] FIG7A is a bar graph showing the gene expression levels of TNF-α in different groups of RAW264.7 cells according to some embodiments of the present specification;

[0047] FIG7B is a bar graph showing the gene expression levels of IL-6 in different groups of RAW264.7 cells according to some embodiments of the present specification;

[0048] FIG7C is a bar graph showing the gene expression levels of IL-10 in different groups of RAW264.7 cells according to some embodiments of the present specification;

[0049] FIG7D is a bar graph showing the gene expression levels of IL-1β in different groups of RAW264.7 cells according to some embodiments of the present specification;

[0050] 7E-7G are images of different groups of RAW264.7 cells stained with Oil Red according to some embodiments of the present specification;

[0051] FIG7H is a bar graph showing the relative areas of lipid droplets in different groups of RAW264.7 cells according to some embodiments of the present specification;

[0052] FIG8A is a bar graph showing the concentration of the key factor GLU in the blood of different groups of mice according to some embodiments of the present specification;

[0053] FIG8B is a bar graph showing the concentration of the key factor CHOI in the blood of different groups of mice according to some embodiments of the present specification;

[0054] FIG8C is a bar graph showing the concentrations of key factors TG in the blood of different groups of mice according to some embodiments of the present specification;

[0055] FIG8D is a bar graph showing the concentration of HDL-C, a key factor in the blood of different groups of mice according to some embodiments of the present specification;

[0056] FIG8E is a bar graph showing the concentration of LDL-C, a key factor in the blood of different groups of mice according to some embodiments of the present specification;

[0057] FIG8F is an oil red staining image of the thoracic aorta of mice in different groups according to some embodiments of the present specification;

[0058] FIG8G is a staining diagram of tissue sections of thoracic aorta of mice from different groups according to some embodiments of the present specification;

[0059] FIG8H is a quantitative analysis bar graph of Oil Red staining according to some embodiments of the present specification;

[0060] FIG8I is a bar graph showing quantitative analysis of Masson staining according to some embodiments of the present specification;

[0061] FIG8J is a bar graph showing quantitative analysis of TUNEL cell staining according to some embodiments of the present specification;

[0062] FIG8K is an IHC staining image of tissue sections of arteries of mice from different groups according to some embodiments of the present specification;

[0063] FIG9A is a diagram of species richness of the intestinal flora evaluated using the Chao1 richness estimator, Shannon diversity index, and Simpson index according to some embodiments of the present specification;

[0064] FIG9B is a principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) diagram of the intestinal flora according to some embodiments of the present specification;

[0065] FIG9C is a Venn diagram of the intestinal flora of each group of samples according to some embodiments of the present specification;

[0066] 9D-9E are cluster histograms of intestinal flora according to some embodiments of the present specification;

[0067] 9F-9G are graphs showing the relative abundance of families and genera of different microorganisms among various groups according to some embodiments of the present specification;

[0068] 9H-9I are PCA analysis diagrams of the families and genera of different microorganisms in each group according to some embodiments of the present specification;

[0069] FIG10A is a HE staining image of liver sections of mice from different groups according to some embodiments of the present specification;

[0070] FIG10B is an oil red staining image of liver sections of mice from different groups according to some embodiments of the present specification;

[0071] FIG10C is an immunohistochemical staining image of liver sections of mice from different groups according to some embodiments of the present specification. DETAILED DESCRIPTION

[0072] It should be understood that although the terms "first," "second," "third," etc., may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first product may be referred to as a second product, and similarly, a second product may be referred to as a first product without departing from the scope of the exemplary embodiments of this specification.

[0073] As used in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not refer to the singular but also include the plural. Generally speaking, the terms "comprises" and "include" only indicate the inclusion of the steps and elements specifically identified, and these steps and elements do not constitute an exclusive list. A method or apparatus may also include other steps or elements.

[0074] 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 disclosure belongs.

[0075] Some embodiments of the present specification provide a snow lotus cell culture, which comprises the following components: a) flavonoids; b) phenylpropanoid derivatives; c) carboxylic acids and their derivatives; and d) other components.

[0076] As used herein, the term "flavonoids" refers to a class of compounds containing an aromatic ring and two benzene rings with a ketone group in the middle.

[0077] In some embodiments, rutin is a flavonoid commonly found in the plant source Saussurea involucrata.

[0078] In some embodiments, the flavonoid component of the snow lotus cell culture comprises one or more of quinic acid, protocatechuic aldehyde, protocatechuic acid, eriodictyol, ferulic acid, syringin, chlorogenic acid, caffeic acid, coumaroylquinic acid, luteolin, eriodictyol-7-O-glucoside, 1,5-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, caffeic acid methyl ester, luteolin, and apigenin.

[0079] In some embodiments, the content of the flavonoid component in the snow lotus cell culture is 7wt%-20wt%. For example, the content of the flavonoid component in the snow lotus cell culture can be 7wt%, 8wt%, 9wt%, 10wt%, 12wt%, 14wt%, 16wt%, 18wt% or 20wt%, or a range composed of any two end values. "Content" as used herein refers to mass content or mass percentage, based on dry weight. In some preferred embodiments, the content of the flavonoid component in the snow lotus cell culture is 14wt%-20wt%.

[0080] The term "phenylpropanoid derivatives" refers to a class of compounds containing one or more C6-C3 units.

[0081] In some embodiments, the content of the phenylpropanoid derivative component in the snow lotus cell culture is 2.1wt%-14.1wt%. For example, the content of the phenylpropanoid derivative component in the snow lotus cell culture can be 2.1wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 12wt%, 14wt% or 14.1wt%, or a range formed by combining any two of these end values. In some preferred embodiments, the content of the phenylpropanoid derivative component in the snow lotus cell culture is 6wt%-10wt%.

[0082] In some embodiments, the phenylpropanoid derivative component of the snow lotus cell culture comprises syringin and chlorogenic acid.

[0083] In some embodiments, the content of syringin in the saussurea cell culture is 0.3-5 wt%. For example, the content of syringin in the saussurea cell culture can be 0.3 wt%, 0.6 wt%, 1.0 wt%, 2.0 wt%, 3.0 wt%, 4.0 wt%, or 5.0 wt%, or a range formed by combining any two of these values. In some preferred embodiments, the content of syringin in the saussurea cell culture is 0.5 wt%-0.9 wt%.

[0084] In some embodiments, the content of chlorogenic acid in the saussurea cell culture is 0.3 wt%-1.3 wt%. For example, the content of chlorogenic acid in the saussurea cell culture can be 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1.0 wt%, 1.1 wt%, 1.2 wt%, or 1.3 wt%, or a range formed by combining any two of these values. In some preferred embodiments, the content of chlorogenic acid in the saussurea cell culture is 0.6 wt%-1.0 wt%.

[0085] The term "carboxylic acid and its derivatives" refers to a class of compounds containing a carboxyl (-COOH) functional group, as well as compounds in which the hydroxyl group on the carboxyl group of such compounds is replaced by other atoms or atomic groups.

[0086] In some embodiments, the carboxylic acid and its derivative components of the snow lotus cell culture include dicaffeoylquinic acid. In some embodiments, the content of dicaffeoylquinic acid in the snow lotus cell culture is 1.6wt%-35wt%. For example, the content of dicaffeoylquinic acid in the snow lotus cell culture can be 1.6wt%, 2wt%, 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%, 31wt%, 32wt%, 33wt%, 34wt% or 35wt%, or a range composed of any two of the end values. In some preferred embodiments, the content of dicaffeoylquinic acid in the snow lotus cell culture is 3.2 wt%-28 wt%.

[0087] In some embodiments, the dicaffeoylquinic acid in the saussurea cell culture comprises 1,5-dicaffeoylquinic acid. In some embodiments, the content of 1,5-dicaffeoylquinic acid in the saussurea cell culture is 1.5 wt % to 7.7 wt %. For example, the content of dicaffeoylquinic acid in the snow lotus cell culture can be 1.5wt%, 1.7wt%, 1.9wt%, 2.1wt%, 2.3wt%, 2.5wt%, 2.7wt%, 2.9wt%, 3.1wt%, 3.3wt%, 3.5wt%, 3.7wt%, 3.9wt%, 4.1wt%, 4.3wt%, 4.5wt%, 4.7wt%, 4.9wt%, 5.1wt%, 5.3wt%, 5.5wt%, 5.7wt%, 5.9wt%, 6.1wt%, 6.3wt%, 6.5wt%, 6.7wt%, 6.9wt%, 7.1wt%, 7.3wt%, 7.5wt% or 7.7wt%, or a range formed by combining any two end values ​​therein. In some preferred embodiments, the content of 1,5-dicaffeoylquinic acid in the snow lotus cell culture is 3.0 wt%-6.2 wt%.

[0088] In some embodiments, the proportion of 1,5-dicaffeoylquinic acid in the dicaffeoylquinic acid in the saussurea cell culture is 22%-95%. For example, the proportion of 1,5-dicaffeoylquinic acid in the dicaffeoylquinic acid in the saussurea cell culture can be 22%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or a range consisting of any two of these values. In some preferred embodiments, the proportion of 1,5-dicaffeoylquinic acid in the dicaffeoylquinic acid in the saussurea cell culture is 60% or 70%.

[0089] In some embodiments, the dicaffeoylquinic acid in the saussurea cell culture comprises 3,5-dicaffeoylquinic acid and 1,4-dicaffeoylquinic acid.

[0090] In some embodiments, the carboxylic acid and its derivative components in the snow lotus cell culture further comprise dicaffeoylquinic acid malate, 1,3,5-tricaffeoylquinic acid malate and 1,3,5-tricaffeoylquinic acid malate methyl ester.

[0091] In some embodiments, the other components in the saussurea cell culture include protein. In some embodiments, the protein content in the saussurea cell culture is 20%-35%. For example, the protein content in the saussurea cell culture can be 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, or 35%, or a range formed by combining any two of these values.

[0092] In some embodiments, other components in the snow lotus cell culture further include organic oxides, lipid compounds, and prenol compounds.

[0093] In some embodiments, the HPLC characteristic spectrum of the snow lotus cell culture at 265 nm includes 9 characteristic peaks, wherein Peak 1 is syringin, Peak 2 is chlorogenic acid, Peak 4 is 1,4-dicaffeoylquinic acid, Peak 5 is 1,5-dicaffeoylquinic acid, Peak 6 is 3,5-dicaffeoylquinic acid, Peak 7 is dicaffeoylquinic acid malate, Peak 8 is 1,3,5-tricaffeoylquinic acid malate, and Peak 9 is 1,3,5-tricaffeoylquinic acid malate methyl ester.

[0094] In some embodiments, in the HPLC characteristic spectrum of snow lotus cell culture at 265 nm, with peak No. 5 as the reference peak, the relative retention time of each characteristic peak is within ±5% of the following specified values: the specified value of peak No. 1 is 0.369, the specified value of peak No. 2 is 0.450, the specified value of peak No. 3 is 0.668, the specified value of peak No. 4 is 0.918, the specified value of peak No. 5 is 1.000, the specified value of peak No. 6 is 1.057, the specified value of peak No. 7 is 1.140, the specified value of peak No. 8 is 1.622, and the specified value of peak No. 9 is 1.645.

[0095] Some embodiments of this specification also provide a method for detecting the HPLC characteristic spectrum of snow lotus cell culture. In some embodiments, the detection of the HPLC characteristic spectrum of the analyte includes step a1 and step a2.

[0096] Step a1, preparing a test solution of snow lotus cell culture and a reference solution.

[0097] In some embodiments, the test solution is a methanol extract of saussurea cell culture. In some embodiments, the test solution is prepared by adding saussurea cell culture powder to 60% methanol, sonicating, cooling to room temperature, adding 60% methanol to the mixture until the weight loss is reached, and filtering through a 0.45 μm microporous filter to obtain the test solution. In some embodiments, the saussurea cell culture powder weighs approximately 0.2 g and the volume of methanol is 25 mL.

[0098] In some embodiments, the reference solution is a methanol solution of 1,5-dicaffeoylquinic acid. In some embodiments, the reference solution is prepared using 60% methanol and a 1,5-dicaffeoylquinic acid reference substance, with each mL of the reference solution containing 0.3 mg of the 1,5-dicaffeoylquinic acid reference substance.

[0099] Step a2: Under predetermined chromatographic conditions, a predetermined amount of the test solution and the reference solution are measured using a liquid chromatograph to generate an HPLC characteristic spectrum.

[0100] In some embodiments, the predetermined chromatographic conditions include: octadecylsilane bonded silica gel as the filler; methanol as the mobile phase A, 0.3% formic acid aqueous solution as the mobile phase B; a flow rate of 0.9 mL / min, a detection wavelength of 265 nm; and gradient elution conditions of:

[0101] 0.01-15min, phase A: 20%, phase B: 80%;

[0102] 15-25 min, phase A: 20% → 36%, phase B: 80% → 64%;

[0103] 25-60 min, phase A: 36% → 37%, phase B: 64% → 63%;

[0104] 60-85 min, phase A: 37% → 70%, phase B: 63% → 30%;

[0105] 85-90 min, phase A: 70%→100%, phase B: 30%→0%;

[0106] 90-100 min, phase A: 100%, phase B: 0%;

[0107] 100-100.01 min, phase A: 100%→20%, phase B: 0%→80%;

[0108] 115 minutes, stop.

[0109] In some embodiments, the injection volume of the test solution and the reference solution is 5 μL.

[0110] Some embodiments of this specification also provide a method for quality control of a snow lotus cell culture, which comprises comparing the HPLC characteristic spectrum of the test substance with the HPLC characteristic spectrum of the snow lotus cell culture.

[0111] It can be understood that the detection method of the HPLC characteristic spectrum of the analyte is the same as the detection method of the aforementioned HPLC characteristic spectrum of the snow lotus cell culture. The spectral characteristics of the HPLC characteristic spectrum of the snow lotus cell culture are the same as the spectral characteristics of the HPLC characteristic spectrum of the snow lotus cell culture at 265nm mentioned above, and will not be repeated here.

[0112] Some embodiments of this specification also provide a method for preparing a snow lotus cell culture, which includes steps b1 and b2.

[0113] Step b1, obtaining a population of differentiated embryonic pluripotent stem cells derived from wild snow lotus.

[0114] In some embodiments, a population of differentiated embryonic pluripotent stem cells can be obtained by differentiation control, and the differentiation control enables the population of differentiated embryonic pluripotent stem cells to have the following characteristics: using wild snow lotus in vitro cells as a control, the population of differentiated embryonic pluripotent stem cells has low expression of differentiation-related genes, including WOX4 (NCBI Gene ID: 841113), LBD18 (NCBI Gene ID: 819150), WOX5 (NCBI Gene ID: 820297) and PLT4 (NCBI Gene ID: 59281483), as shown in Table 1. Table 1

[0115] The term "differentiation-related gene" refers to a gene that is associated with the regulation of directed differentiation of a population of embryonic pluripotent stem cells after differentiation.

[0116] In some embodiments, the above-mentioned differentiation-related genes consist of WOX4, LBD18, WOX5 and PLT4.

[0117] In some embodiments, the expression levels of WOX4 and LBD18 in the differentiated embryonic pluripotent stem cell population are less than 1 / 10 of the expression levels of the corresponding genes in the control group. For example, the expression levels of WOX4 and LBD18 in the differentiated embryonic pluripotent stem cell population can be about 1 / 11, 1 / 12, 1 / 13, 1 / 14, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 50, or 1 / 100 of the expression levels of the corresponding genes in the control group.

[0118] In some embodiments, the expression levels of WOX5 and PLT4 in the differentiated embryonic pluripotent stem cell population are less than 1 / 2 of the expression levels of the corresponding genes in the control group. For example, the expression levels of WOX5 and PLT4 in the differentiated embryonic pluripotent stem cell population can be 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 30, 1 / 40, or 1 / 50 of the expression levels of the corresponding genes in the control group.

[0119] In some embodiments, differentiation control can be performed based on cell feature screening. For example, cell features can include morphology, microscopic features, compound composition, and color.

[0120] In some embodiments, differentiation control can be used to direct the differentiation of a population of embryonic pluripotent stem cells into cells, tissues, or organs that are conducive to the synthesis of target metabolites. For enzymes involved in the metabolic pathways of target metabolites, the aforementioned cells, tissues, or organs can be conducive to maintaining the expression levels of these enzymes and / or can be conducive to increasing the expression levels of one or more of these enzymes. In some embodiments, the target metabolite can include at least one of flavonoids, phenylpropanoid derivatives, carboxylic acids, and their derivatives.

[0121] Step b2: using the differentiated embryonic pluripotent stem cell population for expansion culture, and harvesting the culture fluid containing the snow lotus cell culture.

[0122] In some embodiments, the preparation method further comprises performing metabolic regulation based on the enhanced flavonoid metabolic pathway on the differentiated embryonic pluripotent stem cell population during the expansion culture process.

[0123] The term "enhanced flavonoid metabolic pathway" refers to an increase in the metabolic synthesis level of flavonoids compared to that without metabolic regulation.

[0124] In some embodiments, metabolic regulation enables the differentiated embryonic pluripotent stem cell population to have the following characteristics: the expression of genes related to the flavonoid metabolic pathway in the differentiated embryonic pluripotent stem cell population, the flavonoid metabolic pathway-related genes include PAL (NCBI Gene ID: 818280), C4H (NCBI Gene ID: 817599), 4CL (NCBI Gene ID: 841593), CHI (NCBI Gene ID: 824678), C3H (NCBI Gene ID: 817599), F5H (NCBI Gene ID: 829779) and CAD (NCBI Gene ID: 829572), see Table 2. In some embodiments, the flavonoid metabolic pathway-related genes consist of PAL, C4H, 4CL, CHI, C3H, F5H and CAD. Table 2

[0125] In some embodiments, genes associated with the flavonoid metabolic pathway are genes encoding enzymes associated with the flavonoid metabolic pathway and genes regulating the expression of key enzymes in flavonoid anabolism. Metabolic regulation of the flavonoid metabolic pathway can maintain a certain expression level of enzymes at various processes / nodes in the flavonoid metabolic pathway, thereby achieving a relatively uniform flux distribution in the metabolic pathway.

[0126] In some embodiments, using wild snow lotus in vitro cells as a control, the expression of C3H, F5H, and CAD in the differentiated embryonic pluripotent stem cell population was increased. Fluorescence quantitative PCR (qPCR) was used to detect the expression of key enzymes in the flavonoid metabolic pathway at the mRNA level. In the embryonic pluripotent stem cell population (purple), cinnamic acid 3-hydroxylase (C3H), flavonoid 3',5'-hydroxylase (F5H), and cinnamyl alcohol dehydrogenase (CAD) showed significant upregulation of expression, which were 1.3 times, 2.7 times, and 7.2 times that of the control group, respectively.

[0127] Increased expression levels of genes related to specific flavonoid metabolic pathways can strengthen the flavonoid metabolic pathway and increase the synthesis of metabolites.

[0128] In some embodiments, the snow lotus cell culture in the culture medium is dark purple in a living state and grows in clusters. The diameter of the cell clusters of the snow lotus cell culture is distributed between 0.02 mm and 2 mm.

[0129] In some embodiments, the preparation method further comprises separating the saussurea cell culture from the culture medium and preparing an extract using the saussurea cell culture. The preparation of the extract comprises: repeatedly freezing and thawing the saussurea cell culture to obtain a molten mixture; extracting the molten mixture with an ethanol-water solution to obtain an extract, wherein the extraction process is at least one of ultrasonic extraction and stirring extraction; separating a solid portion and a liquid portion of the extract; and drying the liquid portion to obtain the extract.

[0130] In some embodiments, the number of freeze-thaw cycles is greater than or equal to 3 times.

[0131] In some embodiments, freeze-thawing comprises freezing the saussurea cell culture by cooling to -20°C or placing in liquid nitrogen for 1.5-2.5 hours, and then thawing the saussurea cell culture to 4°C in a water bath.

[0132] In some embodiments, separation of the solid portion and the liquid portion is performed by centrifugation and / or filtration.

[0133] In some embodiments, the drying process is freeze drying or oven drying.

[0134] In some embodiments, the obtained extract has the following characteristics: the extract is a powder with uniform tissue state, gray-purple and uniform color; it has a slight fragrance and a slight glycoside taste; moisture / % ≤ 10, ash / % ≤ 10, acid-insoluble ash / % ≤ 2.0, and extract / % ≥ 30.0.

[0135] The preparation method of the extract can freeze the water in the cells into ice crystals and increase the volume through repeated freezing and thawing, thereby achieving cell wall rupture of the snow lotus cell culture while ensuring the activity of the bioactive substances, thereby efficiently obtaining high-quality extracts.

[0136] Some embodiments of this specification also provide the use of the aforementioned snow lotus cell culture in the preparation of a drug for atherosclerosis.

[0137] In some embodiments, the formation of atherosclerotic plaques in blood vessels is reduced in the treatment group administered with the saussurea cell culture compared to the atherosclerosis model group.

[0138] In some embodiments, compared with the atherosclerosis model group, in the treatment group administered with the Saussurea cell culture, the distribution of intestinal flora is regulated, and the regulation reduces the activation and migration of macrophages and the apoptosis of endothelial cells, thereby reducing the formation of atherosclerotic plaques.

[0139] In some embodiments, the above-mentioned drug is used to prevent, treat or alleviate atherosclerosis.

[0140] In some embodiments, the above-mentioned drug can be formulated into one of injections, tablets, capsules, oral liquids, granules, decoctions, powders, pills, ointments, pills, and suppositories.

[0141] Some embodiments of this specification also provide the use of snow lotus cell culture in preparing medicine for long-term chronic liver damage.

[0142] In some embodiments, compared with the model group of long-term chronic liver injury, in the treatment group administered with snow lotus cell culture, liver function injury-related indicators are reduced, including ALT, AST, GGT and AKP; and, in the treatment group, the generation of liver tissue fibrosis is reduced, and the formation and accumulation of adipose tissue are reduced, thereby reducing the formation of alcoholic fatty liver.

[0143] In some embodiments, compared with the model group of long-term chronic liver injury, in the treatment group administered with snow lotus cell culture, the intestinal flora distribution is regulated, which increases the number of beneficial bacteria and reduces the number of harmful bacteria, thereby reducing the difference in intestinal flora distribution between the treatment group and the normal group.

[0144] In some embodiments, the above-mentioned drug is used to prevent, treat or alleviate long-term chronic liver damage.

[0145] In some embodiments, the above-mentioned drug can be formulated into one of injections, tablets, capsules, oral liquids, granules, decoctions, powders, pills, ointments, pills, and suppositories.

[0146] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative experiments in the following examples are all repeated three times and the results are averaged. Example 1: Cell differentiation control and metabolic regulation

[0147] Plant stem cells exist in a unique microenvironment and are regulated by complex endogenous and exogenous signals. The positive and negative feedback loops formed by differentiation-related genes are one of the key mechanisms for maintaining stem cell stability.

[0148] Through controlled differentiation, embryonic pluripotent stem cell populations (purple) suitable for snow lotus cell culture were obtained. Quantitative PCR (qPCR) was used to examine the expression levels of differentiation-related genes, including WOX4, LBD18, WOX5, and PLT4. WOX4 is a key gene involved in cell division in the cambium and plays a pivotal role in regulating cambium activity. WOX4 responds to various hormones, including auxin, as well as small peptide signals, thereby participating in the maintenance of cambium activity. The LBD18 transcription factor is a key factor in controlling callus formation during in vitro plant regeneration. LBD18 controls callus formation by ectopically activating signals for lateral root development, thereby mimicking auxin-induced callus formation. WOX5 plays a crucial role in determining the fate of the quiescent center (QC). PLT4, also known as BABY BOOM (BBM), is a plant-specific transcription factor of the AP2 / ERF (APETALA2 / ethylene-responsive factor) family. It plays a crucial regulatory role in somatic embryogenesis and is widely used as a star factor to improve the efficiency of transgenic transformation. The expression levels of differentiation-related genes were quantitatively determined by Quant Studio™ 6Flex (ABI). The primer information is shown in Table 3. Table 3. PCR primer sequence information for RT-qPCR

[0149] Figure 1 is a bar graph showing the relative expression of differentiation-related genes at the mRNA level in embryonic pluripotent stem cell populations according to some embodiments of this specification. The experimental group (P) is a sample of the aforementioned embryonic pluripotent stem cell population, the control group (W) is sample 1 of in vitro cells of Saussurea tianshanica without differentiation control, and (G) is sample 2 of in vitro cells of Saussurea tianshanica without differentiation control. As shown in Figure 1, compared with the W group, the expression of WOX4 and LBD18 in the P group was downregulated by 5.56-fold and 11.04-fold, respectively, compared with the control group. The relative expression of WOX5 and PLT4 in the P group remained essentially at the same level.

[0150] For the metabolically regulated embryonic pluripotent stem cell population, qPCR technology was used to detect the expression levels of key enzymes of secondary metabolism, including phenylalanine ammonia-lyase 1 (PAL1), chalcone isomerase (CHI), flavonoid 3'-hydroxylase-1 (F3H-1), flavonoid 3'-hydroxylase-2 (F3H-2), dihydroflavonol 4-reductase (DFR1), anthocyanidin synthase (ANS), cinnamate-4-hydroxylase (C4H), 4-coumarate CoA ligase (4-coumarate hydroxylase), and 4-hydroxylase-1 (F3H-1). The expression levels of genes related to key enzymes in flavonoid metabolism were quantified using Quant Studio™ 6Flex (ABI). Primer information is shown in Table 4. Table 4. PCR primer sequence information for RT-qPCR. Note: ELF5A is an internal reference gene

[0151] Figure 2 is a bar graph showing the relative expression levels of key enzymes in the flavonoid metabolic pathway at the mRNA level according to some embodiments of this specification. The experimental group (P) is a sample of the aforementioned metabolically regulated embryonic pluripotent stem cell group, the control group (W) is sample 1 of Tianshan Saussurea involucrata cells that have not been differentiated, and (G) is sample 2 of Tianshan Saussurea involucrata cells that have not been differentiated. As shown in Figure 2, compared with the P group, cinnamic acid-3-hydroxylase (C3H), flavonoid 3', 5'-hydroxylase (F5H) and cinnamyl alcohol dehydrogenase (CAD) in the W group showed significant up-regulation of expression, which were 1.3 times, 2.7 times and 7.2 times that of the control group, respectively. The high expression of C3H, F5H and CAD made the metabolism of phenolic compounds (small molecule phenolic acids, lignans), flavonoids and isoflavone compounds in the phenylpropionic acid PAL pathway in the Saussurea cell culture higher than that of ordinary wild plants. Example 2: Detection of HPLC characteristic spectrum of Saussurea cell culture Establishment of HPLC characteristic spectrum of Saussurea cell culture

[0152] Chromatographic conditions: octadecylsilane bonded silica gel as filler; methanol as mobile phase A, 0.3% formic acid aqueous solution as mobile phase B; flow rate 0.9 mL / min, detection wavelength 265 nm; gradient elution conditions:

[0153] 0.01-15min, phase A: 20%, phase B: 80%;

[0154] 15-25 min, phase A: 20% → 36%, phase B: 80% → 64%;

[0155] 25-60 min, phase A: 36% → 37%, phase B: 64% → 63%;

[0156] 60-85 min, phase A: 37% → 70%, phase B: 63% → 30%;

[0157] 85-90 min, phase A: 70%→100%, phase B: 30%→0%;

[0158] 90-100 min, phase A: 100%, phase B: 0%;

[0159] 100-100.01 min, phase A: 100%→20%, phase B: 0%→80%;

[0160] 115 minutes, stop.

[0161] Preparation of reference solution: Accurately weigh appropriate amounts of syringin reference, chlorogenic acid reference, and 1,5-dicaffeoylquinic acid reference, and add 60% methanol to prepare a solution containing 0.05 mg of syringin, 0.08 mg of chlorogenic acid, and 0.3 mg of 1,5-dicaffeoylquinic acid reference per mL. Prepare the reference solution for positioning by adding an appropriate amount of rutin reference to 60% methanol.

[0162] Preparation of test solution: Take about 0.2 g of the product powder (passed through No. 4 sieve) (the Tianshan Saussurea control medicinal material in this example was purchased from the China Food and Drug Administration, and the sample weight was 0.5 g), accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 60% methanol, weigh the weight, and ultrasonically treat it for 10 minutes (power 250 W, frequency 40 kHz), take it out, let it cool to room temperature, make up the loss with 60% methanol, shake well, and filter through a 0.45 μm microporous filter membrane to obtain the product.

[0163] Determination: Prepare test solutions from multiple batches of Saussurea cell culture samples. Accurately pipette 5 μL of each of the reference solution and multiple batches of test solution into a liquid chromatograph (Shimadzu LC-2030C 3D high performance liquid chromatograph, Phenomenex Gemini 5 μm C18 250×4.6 column) and measure and record the chromatogram.

[0164] Characteristic peak identification: Based on the principle of stable relative retention time and the fact that peaks can be detected in all batches of samples and are relatively high, a total of 9 peaks with good reproducibility were selected as characteristic peaks. The results showed that when Peak 5 was used as the S peak, the RSDs of the relative retention times of the remaining characteristic peaks were within an acceptable range. Identification of characteristic peaks in HPLC characteristic spectrum

[0165] Figure 3 is a 265 nm chromatogram comparison of characteristic peaks from saussurea cell culture and Saussurea involucrata, according to some embodiments of this specification. Comparison of the HPLC chromatograms of saussurea cell culture and plant-derived Saussurea involucrata reveals that: saussurea cell culture contains more components than Saussurea involucrata, and at higher concentrations; of the nine characteristic peaks from saussurea cell culture, peaks 1, 2, 4, and 5 are shared by both saussurea cell culture and plant-derived Saussurea involucrata; and compared to saussurea cell culture, a peak at a similar position to Peak 6 in the plant-derived Saussurea involucrata exhibits a retention time (RT) shift of 0.5.

[0166] The UV spectrum of the compound corresponding to the characteristic peak of snow lotus cell culture was measured by UV spectroscopy. By comparing it with the UV spectrum of the standard substance reference substance, the following identification can be made.

[0167] The compound corresponding to peak No. 1 in snow lotus cell culture is syringin.

[0168] The compound corresponding to peak No. 2 in snow lotus cell culture is chlorogenic acid.

[0169] The compound corresponding to peak No. 4 in snow lotus cell culture is 1,4-dicaffeoylquinic acid.

[0170] The compound corresponding to peak No. 5 in the snow lotus cell culture was 1,5-dicaffeoylquinic acid (see Figures 4A and 4B).

[0171] The maximum UV absorbance of Peak 6 (252,330) in Saussurea involucrata cell culture is inconsistent with that of the rutin reference substance (see Figures 5A and 5B). The compound corresponding to Peak 6 in Saussurea involucrata cell culture is not rutin. However, the maximum UV absorbance of a peak at a position similar to Peak 6 in Saussurea involucrata is consistent with that of the rutin reference substance (see Figures 5A and 5C). The peak at a position similar to Peak 6 in Saussurea involucrata is rutin. Comparison of the UV spectrum with that of a 3,5-dicaffeoylquinic acid reference substance indicates that the compound corresponding to Peak 6 is 3,5-dicaffeoylquinic acid.

[0172] The compound corresponding to peak 7 in snow lotus cell culture is dicaffeoylquinic acid apple ester.

[0173] The compound corresponding to peak No. 8 in snow lotus cell culture is 1,3,5-tricaffeoylquinic acid apple ester.

[0174] The compound corresponding to peak No. 9 in snow lotus cell culture is 1,3,5-tricaffeoylquinic acid apple methyl ester.

[0175] Therefore, Tianshan Saussurea contains syringin, chlorogenic acid, 1,5-dicaffeoylquinic acid, and rutin; and the amount and content of the components in the Saussurea cell culture are higher than those in Tianshan Saussurea. Example 3: Determination of the content of components in Saussurea cell culture

[0176] The above HPLC method was used to determine the content of each component in multiple batches of snow lotus cell culture samples. The results are shown in Table 5. Table 5 Example 4: Evaluation of the Antioxidant Properties of Snow Lotus Cell Culture Biomaterials

[0177] Human umbilical vein endothelial cells (HUVEC): HUVEC cells obtained from the Atcc Biobank were cultured in ECM medium (Sciencell) supplemented with 10% fetal bovine serum (Sangon Biotech) and 1% penicillin / streptomycin solution (Gibco, Thermo Fisher). HUVEC cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0178] This example uses HUVEC cells and H2O2 to simulate the initial damage to endothelial function during the development of atherosclerosis, and evaluates the antioxidant properties of saussurea cell culture by analyzing its improvement on the aforementioned damage.

[0179] Control group (Con): received ECM complete culture medium.

[0180] Model group (H2O2): oxidative stress was induced with 650 μM H2O2 for 4 h.

[0181] Protective group (SI): Pre-treated with saussurea cell culture (10 μg / mL) for 4 hours, and then induced with 650 μM H2O2 for 4 hours. Cell viability assay (CCK-8)

[0182] HUVEC cells were plated at a density of 10,000 cells per well in a 96-well plate and assessed for cell viability according to the experimental grouping design described in this example. Briefly, after 4 hours of H₂O₂ treatment, CCK8 reagent (DOJINDO LABORATORIES) was added to each well at a 1:100 volume ratio and treated for 2 hours. Absorbance was then measured at 450 nm using a SpectraMax i3X Microplate Reader.

[0183] FIG6A is a bar graph showing the cell activity of HUVEC cells in different groups according to some embodiments of the present disclosure. As shown in FIG6A , compared to the HUVEC cells in the H2O2 group that were subjected to oxidative stress, the cell activity of HUVEC cells in the SI group was improved due to the addition of snow lotus cell culture. Cell apoptosis detection - Annexin V-APC / PI double staining

[0184] HUVEC cells were loaded into six-well plates at a density of 200,000 cells per well and incubated for 48 h. The cells were then grouped according to the aforementioned experimental design of the present embodiment and apoptosis detection was performed using a flow cytometer. In brief, after 4 hours of H2O2 treatment, cells were digested and collected using trypsin and washed 2-3 times with PBS. The cells were resuspended in 100 μL of binding buffer, and 5 μL of Annexin V-APC and 5 μL of PI were added to the cell suspension for staining. The cell suspension was placed in the dark and incubated at room temperature for 30 min. The incubated cell suspension was diluted to 500 μL with binding buffer, and the fluorescence signal was then detected using a flow cytometer (BD FACSCelesta). The detection data were analyzed using Flowjo software.

[0185] Figures 6B-6D are bar graphs of the range of cell apoptosis responses shown in some embodiments of this specification, which are based on flow cytometry measurements of different groups of HUVEC cells stained with Annexin V-APC / PI double staining. As shown in Figures 6B-6D, the addition of snow lotus cell culture significantly reduced H2O2-induced cell apoptosis, necrosis, and mechanical damage in the SI group compared to the H2O2 group. Cell apoptosis detection - TUNEL method

[0186] HUVEC cells were loaded into eight-well plates (μ-Slide 8Well high, ibidi) at a density of 20,000 cells per well and incubated for 24 hours. The TUNEL method was then used to detect apoptosis according to the aforementioned experimental grouping design of the present embodiment. In brief, after H2O2 treatment for 4 hours, the cells of each group were stained using TUNEL BrightRed Apoptosis Detection Kit (Vazyme), and the fluorescence signal was observed at a wavelength of 620 nm using a laser confocal microscope (ZEISS LSM 980). In addition, laser confocal microscopy was used to perform fluorescence imaging of the cells of each group whose nuclei were stained with DAPI, and a fusion fluorescence image of TUNEL and DAPI double staining was obtained by Merge technology.

[0187] Figure 6E is a bar graph of the reaction fluorescence intensity according to some embodiments of this specification, which is measured by flow cytometry based on different groups of HUVEC cells stained with TUNEL. Figure 6F is a fluorescence image according to some embodiments of this specification, which is obtained by laser confocal microscopy based on different groups of HUVEC cells stained with TUNEL / DAPI double staining. As shown in Figures 6E and 6F, compared with the H2O2 group, the addition of snow lotus cell culture significantly reduced H2O2-induced cell apoptosis, necrosis and mechanical damage in the SI group. Intracellular reactive oxygen species detection-reactive oxygen species (ROS) fluorescent probe detection method

[0188] HUVEC cells were loaded into six-well plates at a density of 200,000 cells per well. When the cell density reached 80%, the production of intracellular reactive oxygen species was detected using a ROS fluorescent probe detection method according to the aforementioned experimental grouping design of this embodiment. Briefly, after 4 hours of H2O2 treatment, the cells were rinsed 2-3 times with serum-free medium. The cells were stained with the ROS-specific probe DCFH-DA (GlpBio) at a concentration of 1:1000 and incubated in the dark at 37°C for 30 minutes. Subsequently, flow cytometry was used to detect green fluorescence at 525nm after cell collection. In addition, a laser confocal microscope was used to perform fluorescence imaging of the cells in each group whose nuclei were stained with DAPI, and a fused fluorescence image of DCFH-DA / DAPI double staining was obtained by Merge technology.

[0189] Figure 6G is a bar graph of the fluorescence intensity of the quantitative analysis of ROS production by flow cytometry in different groups of HUVEC cells according to some embodiments of this specification. Figure 6H is a fluorescence image according to some embodiments of this specification, which is obtained by laser confocal microscopy based on different groups of HUVEC cells stained with DCFH-DA / DAPI double staining. Figure 6I is a bar graph of the fluorescence intensity of different groups of HUVEC cells stained with DCFH-DA according to some embodiments of this specification. As shown in Figures 6G-6I, compared with the H2O2 group, the intracellular reactive oxygen species level of the SI group was significantly reduced, and was similar to the intracellular reactive oxygen species level of the Con group. Snow lotus cell culture exhibits a strong ability to inhibit the production and release of reactive oxygen species in HUVEC cells.

[0190] In summary, under the model of H2O2-induced oxidative stress, the snow lotus cell culture showed good antioxidant properties. Example 5: Evaluation of the anti-inflammatory properties of snow lotus cell culture Biomaterials

[0191] Macrophages: RAW264.7 cells transferred to 25 cm 2 Cells were grown in DMEM medium containing 10% FBS and 1% penicillin / streptomycin mixture (100 U / mL penicillin; 100 μg / mL streptomycin) at 37° C. in a 5% CO 2 atmosphere in culture flasks.

[0192] Oxidized low-density lipoprotein (ox-LDL): ox-LDL (1 mg / mL) was incubated with 10 μmol / L CuSO4 at 37°C for 18 h for deep oxidation to obtain oxidized ox-LDL. Methods Effect of snow lotus cell culture on the expression levels of pro-inflammatory / anti-inflammatory factors

[0193] In this example, LPS (Sigma) was used as an inflammatory stimulus to activate the mouse macrophage RAW264.7 cell line to construct an inflammatory response cell model. This inflammatory response cell model was used to evaluate the anti-inflammatory properties of saussurea cell culture.

[0194] RAW264.7 cells were cultured at 1×10 5 Cells were plated at a density of 10 cells / well into six-well plates and incubated for 24 hours. Anti-inflammatory properties were then evaluated according to the following experimental group design.

[0195] Control group (Con): RAW264.7 cells were cultured normally without LPS stimulation.

[0196] Model group (LPS): RAW264.7 cells were stimulated with LPS (0.5 μg / mL) for 24 h.

[0197] Protection group (SI): RAW264.7 cells were pretreated with different concentrations of saussurea cell culture (1 μg / mL; 10 μg / mL) for 4 h, and then stimulated with LPS (0.5 μg / mL) for 24 h.

[0198] After LPS treatment, total RNA from each group of samples was extracted using TRIzol reagent (Sigma), and the quantity and purity of RNA were measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). The Abs 260 / 280 nm ratio of all samples was in the range of 1.8-2.0. Then, cDNA was synthesized from the extracted total RNA using the Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2AG11728 (AG). RT-qPCR analysis was performed using the SYBR Green Premix Pro Taq HS qPCR Kit AG11701 (AG). Primer information is shown in Table 6. TM 6Flex (ABI) quantitatively measured the expression levels of pro-inflammatory factors TNF-α, IL-6, and IL-1β, as well as the anti-inflammatory factor IL-10. Table 6. PCR primer sequence information for RT-qPCR Note: GAPDH is an internal reference gene

[0199] Figures 7A-7D are bar charts of the gene expression levels of pro-inflammatory factors and anti-inflammatory factors in different groups of RAW264.7 cells according to some embodiments of this specification. As shown in Figures 7A-7D, compared with the LPS group, the gene expression of pro-inflammatory factors TNF-α, IL-6 and IL-1β in the SI group was downregulated, while the gene expression of the anti-inflammatory factor IL-10 was upregulated. This shows that the snow lotus cell culture has a certain degree of anti-inflammatory ability. Effect of snow lotus cell culture on foam cell formation

[0200] Foam cells are a key contributor to the development of atherosclerotic plaques. Their formation is induced by macrophage phagocytosis of ox-LDL. This example used RAW264.7 cells and ox-LDL to simulate foam cell formation during the development of atherosclerosis and evaluated the effect of saussurea cell culture on foam cell formation. Briefly, the evaluation was performed using the following experimental design.

[0201] Normal group (Con): RAW264.7 cells were cultured normally, and the medium was changed every two days.

[0202] Model group (ox-LDL): RAW264.7 cells and oxidized ox-LDL, the medium was changed every two days.

[0203] Treatment group (SI): RAW264.7 cells + oxidized ox-LDL + Saussurea cell culture (0.01 μg / mL), the medium was changed every two days.

[0204] After 14 days of culture, cells from each group were stained with Oil Red and observed under a microscope.

[0205] Figures 7E-7G are images of different groups of RAW264.7 cells stained with Oil Red according to some embodiments of this specification. Figure 7H is a bar chart of the relative areas of lipid droplets in different groups of RAW264.7 cells according to some embodiments of this specification. As shown in Figures 7E-7G, compared with the Con group, positive Oil Red-stained cells appeared in the images of the ox-LDL group, indicating that the addition of ox-LDL promoted the formation of foam cells in RAW 264.7. Compared with the ox-LDL group, the number of positively stained cells in the SI group to which the Saussurea cell culture was added decreased, indicating that the Saussurea cell culture has potential in inhibiting the formation of foam cells. The quantitative analysis results shown in Figure 7H further confirmed that the Saussurea cell culture has a significant inhibitory effect on the formation of foam cells.

[0206] In summary, in the inflammatory response cell model and the model simulating the formation of foam cells in the process of atherosclerosis development, the snow lotus cell culture showed good anti-inflammatory properties. Example 6: Effect of snow lotus cell culture on the formation of atherosclerotic plaques Biomaterials

[0207] Atherosclerosis model mice (ApoE) and C57BL / 6J mice: 8-week-old, male, ear-tagged, weighing between 18-22 g. These mice were housed at the Animal Experimental Center of Zhengzhou University under conditions including a 12:12 h light cycle, a normal diet, and access to purified water.

[0208] This example uses high-fat fed ApoE mice to evaluate the therapeutic effect of snow lotus cell culture on atherosclerosis.

[0209] Blank control group (Bla): Age- and sex-matched wild-type C57BL / 6J mice (n=5) were fed a normal diet (wheat flour 5%, oatmeal 25%, cornmeal 35%, soybean meal 19%, fish meal 8%, sesame 1%, bone meal 4%, vitamins and yeast powder 2%, and refined salt 1%).

[0210] Control group (Con): Age- and sex-matched wild-type C57BL / 6J mice (n=4) were fed a high-fat diet (49.9% normal feed, 10% protein powder, 22% lard, 2% cholesterol, 0.1% bile salt, and 16% sugar).

[0211] Model group (Dis): ApoE mice (n=4) were fed a high-fat diet and gavaged with 500 mg / kg of hydroxymethylcellulose sodium once a day for 16 weeks.

[0212] Experimental group (Saussurea involucrate SI): ApoE mice (n=5) were fed a high-fat diet and gavaged with Saussurea involucrate cell culture at a dose of 500 mg / kg once a day for 16 weeks. Blood sample analysis

[0213] Blood was drawn from mice in each group on the first and seventh days, and the concentrations of key factors in the blood samples were used, including glucose (GLU), cholesterol (CHOl), triglycerides (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL).

[0214] Figures 8A-8E are bar graphs showing the concentrations of key factors in the blood of different groups of mice according to some embodiments of this specification. As shown in Figures 8A-8E, compared with the Dis group, the growth rate of CHO1 concentration in the blood of mice in the SI group was significantly slower between the two time points, indicating that Saussurea cell culture has therapeutic potential for some diseases, such as lipid disorders, hyperlipidemia and / or hypercholesterolemia. Tissue / Cell Staining Analysis

[0215] At the end of the experiment, the arteries and thoracic aorta of mice in each group were obtained for tissue / cell staining and analysis: (1) Oil red staining was used to observe and analyze the formation of plaques on the thoracic aorta; (2) Masson staining was used to identify the fibrin-positive areas in the plaques on the thoracic aorta; (3) TUNEL cell staining was used to detect and analyze cell apoptosis; (4) IHC staining of IL-1β, IL-6, and TNF-α was used to observe and analyze the inflammatory response around the arterial wall.

[0216] Figure 8F is an Oil Red staining image of the thoracic aorta of mice in different groups according to some embodiments of the present specification. Figure 8F shows that compared with the Dis group, the accumulation of plaques on the arterial wall of mice in the SI group was reduced.

[0217] Figure 8G is a staining image of tissue sections of the thoracic aorta of mice in different groups according to some embodiments of the present specification. Figure 8G shows that although fibrous tissue was observed in the Masson staining of the thoracic aorta of mice in all three groups, the area of ​​fibrous tissue in the SI group was smaller.

[0218] Figures 8H-8J are quantitative analyses of Oil red staining, Masson staining, and TUNEL cell staining according to some embodiments of this specification. Figures 8H-8J show quantitative analysis bar graphs of Oil red staining, Masson staining, and TUNEL staining, demonstrating that Snow Lotus has statistically significant effects in reducing plaque formation, reducing fibrous tissue area, and reducing cell apoptosis.

[0219] Figure 8K is an IHC staining image of tissue sections of arteries of mice in different groups according to some embodiments of this specification. Figure 8K shows that compared with the Dis group, local inflammation around the arterial wall of mice in the SI group was suppressed, indicating that the snow lotus cell culture has certain anti-inflammatory properties.

[0220] Sample collection: The fecal samples of mice in each group were collected and stored at -80°C for further analysis.

[0221] DNA Extraction, Amplification, and Sequencing: Total genomic DNA was extracted from stool samples. DNA quantity and quality were measured using a spectrophotometer and agarose gel electrophoresis. The V3-V4 region of the bacterial 16S rRNA gene was amplified by PCR using primers containing a sample-specific 7-base barcode for multiplex sequencing. Paired-end sequencing was performed at 2 × 250 bp using the Illumina NovaSeq platform.

[0222] Sequence and bioinformatics analysis:

[0223] The raw data are processed by DADA2 or deblur to obtain the initial Amplicon Sequence Variants (ASVs).

[0224] ASVs with an abundance greater than 95% were retained. Species annotation was performed using QIIME2 software.

[0225] Intergroup calculations were performed using QIIME2 software to explore the phylogenetic relationships among ASVs and identify dominant species differences between samples.

[0226] Subsequently, α-diversity and β-diversity analyses were performed based on the output ASVs data.

[0227] Alpha diversity was estimated by calculating the Chao1 richness estimator, observed species, Shannon diversity index, Simpson index, Faith's PD, Pielou evenness, and Good's coverage, focusing on the abundance and evenness of the microbial community.

[0228] β-diversity was estimated using the Bray-Curtis distance and the Anosim method.

[0229] To compare different species between individual samples, principal coordinates analysis (PCoA) and nonmetric multidimensional scaling (NMDS) were performed using the vegan R package.

[0230] The dominance of bacterial communities among the groups was analyzed using linear discriminant analysis (LDA) effect size (LEfSe) (LDA score = 2 as the cutoff value).

[0231] The molecular ecology R package was used to draw a taxonomic cladogram showing the relationships among the major taxonomic levels from phylum to genus in different sample communities.

[0232] Statistical Analysis: All data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism (version 8.0.2) using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. A P value < 0.05 was considered statistically significant.

[0233] Figure 9A is a community species richness diagram evaluated by the Chao1 richness estimator, Shannon diversity index and Simpson index of the intestinal flora shown in some embodiments of this specification. As can be seen from the figure, treatment with snow lotus cell culture reduced the richness and diversity of the intestinal flora. Figure 9B is a principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS) diagram of the intestinal flora shown in some embodiments of this specification. As can be seen from the figure, there are significant differences in the microbial composition between the groups. The Anosim method showed that there were significant differences in the intestinal microbiota between the model group and the experimental group (p = 0.012), and there were also significant differences in the intestinal microbial community structure between the control group and the experimental group (p = 0.012), which shows that snow lotus cell culture treatment changed the heterogeneous community structure. Figure 9C is a Venn diagram of the intestinal flora of each group of samples shown in some embodiments of this specification. As can be seen from the figure, there are differences in species distribution between the control group, model group and experimental group. Figures 9D-9E are cluster histograms of the intestinal flora shown in some embodiments of this specification. As can be seen from the figures, the specific changes in the bacterial community, the species composition of each sample, and the changes in the intestinal flora at the family and genus level are highlighted. Figures 9F-9G are relative abundance diagrams of the families and genera of different microorganisms between the groups shown in some embodiments of this specification. As can be seen from the figure, at the family level, compared with the control group, the F16, Clostridiaceae, and Peptostreptococcaceae in the model group decreased, while the Bacillaceae, Erysipelotrichaceae, and Nocardiaceae increased. In contrast, the snow lotus cell culture experimental group showed the opposite trend, with an increase in the relative abundance of F16, Clostridiaceae, and Lactobacillaceae, and a decrease in the relative abundance of Bacillaceae, Erysipelotrichaceae, and Nocardiaceae. At the genus level, relative to the control group, the abundance of Lactococcus, Melissococcus, Desulfovibrio, AF12, SMB53, 02d06, Clostridium, and Anaerostipes decreased in the model group, while Allobaculum, Olsenella, Rhodococcus, Gemella, and Barnesiella increased in abundance. Snow lotus cell culture treatment reduced the relative abundance of SMB53 and Anaerostipes and significantly increased the relative abundance of Lactobacillus, while the relative abundance of Lactobacillus remained unchanged in the other groups. Figures 9H-9I are PCA analysis diagrams of the families and genera of different microorganisms in each group shown in some embodiments of this specification. It can be seen that there are significant differences between the experimental groups.

[0234] In summary, administration of snow lotus cell culture can effectively regulate the number of intestinal flora associated with cardiovascular diseases (Banesiellaceae, Enterococcaceae), metabolic disorders (AF12) and inflammation (Pseudomonadacea) in atherosclerosis model mice. Example 7: Effect of snow lotus cell culture on long-term chronic liver injury Materials Animals

[0235] Blank mice: C57BL / 6 mice, male (4-5 weeks).

[0236] Alcoholic liver disease model mice: C57BL / 6 mice, male (4-5 weeks), continuously fed with alcohol.

[0237] Dissolving solution: 0.5% methylcellulose in saline solution;

[0238] High-dose solution of snow lotus cell culture: 1.1 g of snow lotus cell culture was dissolved in 22 mL of dissolution solution;

[0239] Low-dose solution of snow lotus cell culture: 2 mL of high-dose solution of snow lotus cell culture is diluted to 20 mL with 18 mL of dissolving solution.

[0240] The following experimental design was used to evaluate the effect of snow lotus cell culture on improving / treating long-term chronic liver injury. Specifically:

[0241] Normal group: blank mice were given distilled water by oral gavage every day at a dose of 10 mL / kg.

[0242] Model group: Alcoholic liver model mice were given distilled water by oral gavage every day at a dose of 10 mL / kg.

[0243] Low-dose group: Alcoholic liver model mice were orally gavaged with a low-dose solution at a dose of 50 mg / kg daily.

[0244] High-dose group: Alcoholic liver model mice were orally gavaged with a high-dose solution daily at a dose of 500 mg / kg.

[0245] The animals were weighed twice a week, and the dose of the test samples was adjusted according to their body weight. The following indicators were measured once a month: ALT, alkaline phosphatase (ALP), AST, GGT, albumin (Albumin), malondialdehyde (MDA), glutathione (GSH), triglycerides (TG) and fecal sampling. The mice were fasted but not deprived of water for 12 hours and blood was collected from the eye sockets. At the end of two months of administration of the test samples, the animals were killed by fasting for 16 hours. After anesthesia with sodium pentobarbital, a midline incision was made into the abdomen, and the inferior vena cava was punctured to draw blood samples, separate liver tissue, and collect feces, colon, ileum, and jejunum for testing of various indicators and pathological histological examination. Detection and analysis of liver function-related indicators in the first month

[0246] The liver and blood of mice in each group were collected for analysis one month after administration.

[0247] Table 7.1 shows the changes in AKP, ALT, AST, MDA, TG, and GSH in the livers of mice in each group one month after the experiment. Compared with the normal group, the AKP, ALT, AST, MDA, and TG levels in the livers of the model group were significantly increased, and the GSH content was significantly decreased, indicating that the liver injury model was successfully established, and the differences were statistically significant (P < 0.05). Compared with the model group, the AKP, ALT, AST, MDA, and TG levels in the livers of the high-dose and low-dose groups were reduced to varying degrees, and the GSH content was increased, and the differences were statistically significant (P < 0.05). Table 7.1 - Changes in AKP, ALT, AST, MDA, TG, and GSH in the liver (x ± s) Note: Compared with the normal group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, ** P<0.01.

[0248] Table 7.2 shows the changes in GGT, ALB, and GSH in the plasma of each group of mice one month after the experiment. Compared with the normal group, the GGT content in the plasma of the model group increased significantly, the ALB content did not change significantly, and the GSH content decreased significantly, indicating that the liver injury model was successfully established, and the differences were statistically significant (P < 0.05). Compared with the model group, the GGT content in the plasma of the high-dose group and the low-dose group decreased significantly, and the GSH content increased, and the differences were statistically significant (P < 0.05). Table 7.2 - Changes in GGT, ALB, and GSH in Plasma (x ± s) Note: Compared with the normal group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, **P<0.01. Analysis of liver function related indicators in the second month

[0249] The liver and blood of mice in each group were collected for analysis 2 months after administration.

[0250] Table 7.3 shows the changes in AKP, ALT, AST, MDA, TG, and GSH in the livers of mice in each group two months after the experiment. Compared with the normal group, the AKP, ALT, AST, MDA, and TG levels in the livers of the model group were significantly increased, and the GSH level was significantly decreased, indicating that the long-term chronic liver injury model was successfully established, and the differences were statistically significant (P < 0.05). Compared with the model group, the AKP, ALT, AST, MDA, and TG levels in the livers of the high-dose and low-dose groups were reduced to varying degrees, and the GSH level was increased, and the differences were statistically significant (P < 0.05). Table 7.3 - Changes in AKP, ALT, AST, MDA, TG, and GSH in the liver (x ± s) Note: Compared with the normal group: # P<0.05, ## P<0.01; compared with the model group: * P<0.05, ** P<0.01.

[0251] Table 7.4 shows the changes in GGT, ALB, and GSH in the plasma of mice in each group two months after the experiment. Compared with the normal group, the GGT content in the plasma of the model group increased significantly, the ALB content did not change significantly, and the GSH content decreased significantly, indicating that the long-term chronic liver injury model was successfully established, and the difference was statistically significant (P < 0.05). Compared with the model group, the GGT content in the plasma of the high-dose group and the low-dose group decreased significantly, and the GSH content increased, and the difference was statistically significant (P < 0.05). Table 7.4 - Changes in GGT, ALB, ALT, AST, and GSH in plasma (x ± s) Note: Compared with the normal group: # P<0.05, ## P < 0.01; compared with the model group: *P < 0.05, **P < 0.01. Liver tissue section analysis

[0252] Two months later, transverse sections were taken from the middle of the left lobe of the liver of mice in each group, frozen sections were made, and HE staining, oil red staining, immunohistochemical staining and microscopic examination were performed.

[0253] Figure 10A is a HE-stained image of liver sections from different groups of mice, as described in some examples of this specification. Compared with the normal group, the model group showed severe liver tissue cell damage, with hepatocyte swelling and disorganization around the central vein and extensive lipid vacuolar deposition. Compared with the model group, the high-dose and low-dose groups showed improved liver structural disorder, mild hepatocyte steatosis, inflammatory infiltration, and significantly reduced liver damage, with the low-dose group showing greater improvement in liver damage.

[0254] Figure 10B shows oil red staining of liver sections from different groups of mice, as described in some examples of this specification. Compared to the normal group, the liver tissue of the model group showed significant fatty vacuolar changes and diffuse granular lipid droplets, indicating the histopathological changes of alcoholic liver injury. Furthermore, compared to the model group, the fatty vacuolar changes in the liver tissue of the high-dose and low-dose groups were significantly reduced, indicating significantly less liver damage.

[0255] Figure 10C is an immunohistochemical staining of liver sections from different groups of mice as shown in some embodiments of this specification. Compared with the normal group, the expression of proinflammatory cytokines (CD68, COX2, iNOS, TNF-α, and NGAL) in the liver of the model group was significantly increased, and the number and size of vacuoles were significantly increased. Compared with the model group, the expression of proinflammatory cytokines in the liver of the high-dose group and the low-dose group was significantly reduced, alcohol-induced lipid metabolism disorders and hepatic steatosis were reduced, and liver damage was significantly reduced. Liver damage was significantly reduced.

[0256] In summary, snow lotus cell culture has a certain degree of protective effect on the liver of mice with long-term chronic liver injury model, has the effect of significantly reducing liver damage, and the effect is better when administered at a low dose.

[0257] Those skilled in the art will appreciate that the above embodiments are intended only to illustrate the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and variations made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

[0258] This specification also uses specific terms to describe the embodiments of this specification. For example, "one embodiment," "an embodiment," and / or "some embodiments" refer to a feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "one embodiment," "an embodiment," or "an alternative embodiment" two or more times in different locations in this specification do not necessarily refer to the same embodiment. Furthermore, certain features, structures, or characteristics of one or more embodiments of this specification may be appropriately combined.

[0259] In some embodiments, numbers are used to describe the quantity of components and attributes. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise stated, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the description and claims are approximate values, which may change according to the required characteristics of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the settings of such numerical values ​​are as accurate as possible within the feasible range.

[0260] Each patent, patent application, patent application publication, and other materials, such as articles, books, specifications, publications, and documents, cited in this specification is hereby incorporated by reference in its entirety. This includes application history documents that are inconsistent with or conflict with the content of this specification, as well as documents (currently or subsequently attached to this specification) that limit the broadest scope of the claims of this specification. It should be noted that if the descriptions, definitions, and / or terminology used in the accompanying materials are inconsistent or conflicting with the content of this specification, the descriptions, definitions, and / or terminology used in this specification will control.

[0261] Finally, it should be understood that the embodiments described in this specification are intended only to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, by way of example and not limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly described and illustrated in this specification.

Claims

1. A snow lotus cell culture, characterized in that The snow lotus cell culture comprises the following components: a) flavonoids, with a content of 7wt%-20wt%; b) phenylpropanoid derivatives, with a content of 2.1wt%-14.1wt%; c) carboxylic acids and their derivatives, wherein the carboxylic acids and their derivatives contain dicaffeoylquinic acid with a content of 1.6wt%-35wt%; and d) other components.

2. The snow lotus cell culture according to claim 1, wherein The phenylpropanoid derivatives contain syringin in an amount of 0.3 wt% to 5 wt% and chlorogenic acid in an amount of 0.3 wt% to 1.3 wt%.

3. The snow lotus cell culture according to claim 1, wherein The dicaffeoylquinic acid contains 1,5-dicaffeoylquinic acid, the content of the 1,5-dicaffeoylquinic acid is 1.5wt%-7.7wt%, and the proportion of the 1,5-dicaffeoylquinic acid in the dicaffeoylquinic acid is 22%-95%.

4. The snow lotus cell culture according to claim 1, wherein The HPLC characteristic spectrum of the snow lotus cell culture at 265 nm includes 9 characteristic peaks; among them, peak 1 is syringin, peak 2 is chlorogenic acid, peak 4 is 1,4-dicaffeoylquinic acid, peak 5 is 1,5-dicaffeoylquinic acid, peak 6 is 3,5-dicaffeoylquinic acid, peak 7 is dicaffeoylquinic acid malate, peak 8 is 1,3,5-tricaffeoylquinic acid malate, and peak 9 is 1,3,5-tricaffeoylquinic acid apple methyl ester.

5. The snow lotus cell culture according to claim 4, wherein In the HPLC characteristic spectrum of the snow lotus cell culture at 265 nm, with peak No. 5 as the reference peak, the relative retention time of each characteristic peak is within ±5% of the following specified values: the specified value of peak No. 1 is 0.369, the specified value of peak No. 2 is 0.450, the specified value of peak No. 3 is 0.668, the specified value of peak No. 4 is 0.918, the specified value of peak No. 5 is 1.000, the specified value of peak No. 6 is 1.057, the specified value of peak No. 7 is 1.140, the specified value of peak No. 8 is 1.622, and the specified value of peak No. 9 is 1.

645.

6. A method for preparing a snow lotus cell culture, characterized in that: The preparation method comprises: Obtaining differentiated embryonic pluripotent stem cell populations from wild snow lotus; and using the differentiated embryonic pluripotent stem cell population for expansion culture, and harvesting the culture fluid containing the snow lotus cell culture; Among them, the differentiated embryonic pluripotent stem cell population is obtained through differentiation control, and the differentiation control enables the differentiated embryonic pluripotent stem cell population to have the following characteristics: using the in vitro cells of wild snow lotus as a control, the differentiation-related genes of the differentiated embryonic pluripotent stem cell population are lowly expressed, and the differentiation-related genes include WOX4, LBD18, WOX5 and PLT4.

7. The preparation method according to claim 6, wherein The snow lotus cell culture comprises the following components: a) flavonoids, with a content of 7wt%-20wt%; b) phenylpropanoid derivatives, with a content of 2.1wt%-14.1wt%; c) carboxylic acids and their derivatives, wherein the carboxylic acids and their derivatives contain dicaffeoylquinic acid with a content of 6.8wt%-8.1wt%; and d) other components.

8. The preparation method according to claim 7, wherein The dicaffeoylquinic acid contains 1,5-dicaffeoylquinic acid, the content of the 1,5-dicaffeoylquinic acid is 1.5wt%-7.7wt%, and the proportion of the 1,5-dicaffeoylquinic acid in the dicaffeoylquinic acid is 22%-95%.

9. The preparation method according to claim 6, wherein The expression levels of WOX4 and LBD18 in the differentiated embryonic pluripotent stem cell population are less than 1 / 10 of the corresponding gene expression levels in the control group; the expression levels of WOX5 and PLT4 in the differentiated embryonic pluripotent stem cell population are less than 1 / 2 of the corresponding gene expression levels in the control group.

10. The preparation method according to claim 6, wherein The preparation method further comprises, during the expansion culture process, performing metabolic regulation on the differentiated embryonic pluripotent stem cell population based on the enhancement of the flavonoid metabolic pathway.

11. The preparation method according to claim 10, characterized in that The metabolic regulation enables the differentiated embryonic pluripotent stem cell population to have the following characteristics: expression of genes related to the flavonoid metabolic pathway in the differentiated embryonic pluripotent stem cell population, and the flavonoid metabolic pathway related genes include PAL, C4H, 4CL, CHI, C3H, F5H and CAD; and, using in vitro cells of wild snow lotus as a control, the expression of C3H, F5H and CAD in the differentiated embryonic pluripotent stem cell population is increased.

12. The preparation method according to claim 6, wherein The method further comprises separating the snow lotus cell culture from the culture fluid, and using the snow lotus cell culture to prepare an extract; the preparation of the extract comprises: Repeatedly freezing and thawing the snow lotus cell culture to obtain a molten mixture; extracting the molten mixture using an ethanol aqueous solution to obtain an extract, wherein the extraction process is at least one of ultrasonic extraction and stirring extraction; separating a solid portion and a liquid portion of the extract; and The liquid portion is dried to obtain the extract.

13. Use of the snow lotus cell culture according to claim 1 in preparing a medicine for treating atherosclerosis.

14. The use according to claim 13, characterized in that Compared with the model group of atherosclerosis, the formation of atherosclerotic plaques in blood vessels was reduced in the treatment group administered with the saussurea cell culture.

15. The use according to claim 13, characterized in that Compared with the atherosclerosis model group, in the treatment group administered with snow lotus cell culture, the distribution of intestinal flora was regulated, which reduced the activation and migration of macrophages and the apoptosis of endothelial cells, thereby reducing the formation of atherosclerotic plaques.

16. The use according to claim 13, characterized in that The snow lotus cell culture is obtained by amplifying and culturing a differentiated embryonic pluripotent stem cell population derived from wild snow lotus. The differentiated embryonic pluripotent stem cell population is obtained by differentiation control, and the differentiation control enables the differentiated embryonic pluripotent stem cell population to have the following characteristics: using in vitro cells of wild snow lotus as a control, the differentiation-related genes of the differentiated embryonic pluripotent stem cell population are lowly expressed, and the differentiation-related genes include WOX4, LBD18, WOX5 and PLT4.

17. Use of the snow lotus cell culture according to claim 1 in the preparation of a medicament for long-term chronic liver damage.

18. The use according to claim 17, characterized in that Compared with the model group of long-term chronic liver damage, in the treatment group administered with snow lotus cell culture, liver function damage-related indicators were reduced, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT) and alkaline phosphatase (AKP); and, in the treatment group, the generation of liver tissue fibrosis was reduced, as well as the formation and accumulation of adipose tissue was reduced, thereby reducing the formation of alcoholic fatty liver.

19. The use according to claim 17, characterized in that Compared with the model group of long-term chronic liver injury, the intestinal flora distribution was regulated in the treatment group administered with snow lotus cell culture, which increased the number of beneficial bacteria and reduced the number of harmful bacteria, thereby reducing the difference in intestinal flora distribution between the treatment group and the normal group.

20. The use according to claim 17, wherein The snow lotus cell culture is obtained by amplifying and culturing a differentiated embryonic pluripotent stem cell population derived from wild snow lotus. The differentiated embryonic pluripotent stem cell population is obtained by differentiation control, and the differentiation control enables the differentiated embryonic pluripotent stem cell population to have the following characteristics: using in vitro cells of wild snow lotus as a control, the differentiation-related genes of the differentiated embryonic pluripotent stem cell population are lowly expressed, and the differentiation-related genes include WOX4, LBD18, WOX5 and PLT4.

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