Phytosphingosine and application of phytosphingosine supplement in metabolic disorder and human body constitution direction

Abstract

The invention belongs to the technical field of application of phytosphingosine, and particularly relates to application of phytosphingosine and supplements thereof in metabolic disorder and human body constitution direction, and application of phytosphingosine in metabolic disorder early screening and human body constitution recognition methods. Phytosphingosine is used as a metabolic marker to be applied to screening in the early stage of metabolic disorders and before the metabolic disorders are attacked, the metabolic disorders comprise obesity, hyperlipidemia, hypertension, hyperuricemia and non-alcoholic fatty liver, and the phytosphingosine is used as a metabolic marker to be applied to recognition of the phlegm-dampness constitution of the human body. According to the content change of the phytosphingosine, metabolic disorder early screening and human body constitution recognition are carried out, and the supplement containing the phytosphingosine can be applied to metabolic disorder treatment. Effectiveness and reliability of phytosphingosine application are verified through a verification method.

Description

Technical Field

[0001] This application belongs to the technical field of the application of phytosphingosine, and particularly relates to the application of phytosphingosine and its supplements in metabolic disorders and human physique. Background Art

[0002] The statements in this part only provide background technical information related to this application, and do not necessarily constitute prior art.

[0003] Metabolic disorders (MDs) are a group of diseases aggregated by a variety of interrelated pathological states. They have a high incidence, are difficult to prevent and treat, have serious consequences, and have a great impact. Many of these metabolic disorders have a common pathological basis, such as insulin resistance, chronic low-grade inflammation, etc. Therefore, they often occur simultaneously or successively, bringing great difficulties to treatment.

[0004] Phlegm-damp constitution is a common constitution type in traditional Chinese medicine constitution theory. Due to the failure of fluid transportation and transformation in phlegm-damp constitution, and the long-term condensation of phlegm and dampness, Meta-analyses of epidemiological and clinical studies have confirmed that phlegm-damp constitution is a high-risk population for metabolic disorders.

[0005] At present, the prevention and control of metabolic disorders mainly adopt measures such as health check-ups and health education, and certain results have been achieved, but far from the expected effect. Traditional Chinese medicine constitution theory believes that phlegm-damp constitution is a high-risk population for metabolic disorders. Through the identification of phlegm-damp constitution, "early screening" and "early warning" of metabolic disorders can be achieved. Through the intervention of phlegm-damp constitution, it is possible to achieve the prevention from "one disease" to "a group of diseases", providing theoretical guidance and technical support for the early prevention and control of metabolic disorders.

[0006] In recent years, more and more epidemiological studies have used metabolomics to find metabolites that reflect specific exposures, are early markers of diseases, represent pathways that promote disease development, or help predict disease risk.

[0007] The correlation between phlegm-damp constitution and metabolic diseases has been supported by clinical and experimental studies. Modern biomarker research shows that the contents of metabolites such as short-chain fatty acids, bile acids, and trimethylamine N-oxide in individuals with phlegm-damp constitution are closely related to their disease risks in the body.

[0008] Phytosphingosine, also known as sphingosine, is an 18-carbon amino alcohol containing an unsaturated hydrocarbon chain. It exists in specific tissues, including the epidermis and small intestine of mammals, and is widely present in cell membranes, participating in the regulation of various physiological processes such as cell signal transduction, cell growth, differentiation, and apoptosis. In addition to intracellular signal transduction, phytosphingosine also has biological effects when added externally, such as being used for moisturizing in cosmetics.

[0009] Disease screening methods mostly rely on imaging examinations, traditional blood indicators, etc. However, these methods often have problems such as single disease screening and high screening costs. The early screening method based on the change of phytosphingosine level is expected to overcome these disadvantages, provide a new means for the early detection of diseases in people with metabolic disorders, and achieve early prevention of diseases.

[0010] Currently, the screening of physical constitution mostly relies on the traditional four diagnostic methods of "inspection, auscultation and olfaction, interrogation, and palpation", or on industry-standard physical scales, which are greatly affected by subjective factors, have poor stability, and rely on the doctor group. The early screening method based on the change of phytosphingosine level is expected to overcome these disadvantages, provide a new means for physical constitution identification, and achieve early prevention of diseases.

[0011] In addition, the exogenous addition of phytosphingosine may also produce biological effects, and the application of phytosphingosine may have important application potential in the prevention and treatment of metabolic disorders and the improvement of physical constitution. Summary of the Invention

[0012] To solve the above problems, the present application provides the application of phytosphingosine and its supplements in the directions of metabolic disorders and human physical constitution.

[0013] The first object of the present application is to provide the application of phytosphingosine in the methods of early screening of metabolic disorders and human physical constitution identification.

[0014] To achieve the first object of the present application, the technical solution of the present application is:

[0015] The application of phytosphingosine in the methods of early screening of metabolic disorders and human physical constitution identification, where phytosphingosine is used as a metabolic marker in the early screening of metabolic disorders before the onset of the disease. Metabolic disorders include obesity, hyperlipidemia, hypertension, hyperuricemia, non-alcoholic fatty liver. Human physical constitution includes balanced constitution, phlegm-dampness constitution, yang-deficiency constitution, yin-deficiency constitution, qi-deficiency constitution, damp-heat constitution, blood stasis constitution, special endowment constitution, qi stagnation constitution. Phytosphingosine is used as a metabolic marker in the identification of human phlegm-dampness constitution, and the early screening of metabolic disorders and the identification of human physical constitution are carried out according to the change of the content of phytosphingosine.

[0016] The second object of the present application is to provide the application of supplements in the intervention of phlegm-dampness constitution and the treatment of metabolic disorders.

[0017] To achieve the second object of the present application, the technical solution of the present application is:

[0018] The supplement contains phytosphingosine. Metabolic disorders include obesity, diabetes, hyperlipidemia, hyperuricemia, and non-alcoholic fatty liver. The phytosphingosine in the supplement can improve the insulin sensitivity of patients with metabolic disorders and reduce blood glucose, blood lipid, and blood uric acid levels by regulating glucose metabolism, lipid metabolism, and uric acid metabolism. Specifically, the phytosphingosine in the supplement can activate the PPAR signaling pathway, enhance the PPAR-related insulin signaling pathway, promote the activation of insulin receptors, increase the uptake and utilization of glucose by cells, thereby reducing fasting blood glucose and postprandial blood glucose levels and improving insulin resistance. The phytosphingosine in the supplement can activate the PPAR signaling pathway, reduce the synthesis of cholesterol in the liver, and at the same time increase the production and utilization of high-density lipoproteins in the liver and tissues, reducing the levels of low-density lipoproteins, triglycerides, and total cholesterol, thereby improving blood lipid disorders. The phytosphingosine in the supplement can improve abnormal liver function caused by metabolic disorders by reducing fat deposition in the liver and adipose tissue and reducing the content of triglycerides and total cholesterol. The phytosphingosine in the supplement can reduce the weight and number of adipose tissues in the body, reduce lipid deposition in liver fat cells, reduce liver fat deposition, and reduce the risk of obesity and related metabolic complications. The phytosphingosine in the supplement can significantly reduce the serum uric acid level and reduce the deposition of uric acid in the kidneys and joints, thereby preventing and treating hyperuricemia and gout.

[0019] Furthermore, the number of methylene groups in phytosphingosine is 16 - 20.

[0020] Furthermore, the supplement is a pharmaceutically acceptable dosage form, including tablets, capsules, dripping pills, granules, injections, and oral liquid dosage forms.

[0021] The third object of the present application is to provide a verification method for verifying the application of phytosphingosine in the methods of early screening of metabolic disorders and identification of human constitutions.

[0022] To achieve the third object of the present application, the technical solution of the present application is as follows:

[0023] In this verification method, the subjects are judged for peaceful constitution and phlegm-dampness constitution, and the subjects are divided into a group of those with peaceful constitution and no disease, a group of those with peaceful constitution and disease, a group of those with phlegm-dampness constitution and no disease, and a group of those with phlegm-dampness constitution and disease. Blood samples are collected from the four groups of people, and then serum untargeted metabolomics detection and data analysis are carried out, and statistical analysis methods are used for application verification.

[0024] When collecting blood samples, peripheral venous blood is drawn on an empty stomach, left standing at room temperature for 30 minutes, then centrifuged at 3000 rpm for 5 minutes. The serum is transferred to a centrifuge tube to form a serum sample. The serum sample is tested, and the test indicators include blood routine, four lipid items, ApoA1, ApoB, Lpα, fasting blood glucose, and uric acid. The remaining serum sample is quickly frozen to -80 °C in liquid nitrogen for storage. After the first blood draw, the subject eats, and the time is counted from the first bite of food. Two hours later, blood is drawn again to test the two-hour postprandial blood glucose;

[0025] When performing the detection of serum untargeted metabolome, the serum sample in liquid nitrogen is first processed. The serum sample is thawed on ice, 100 μL of the serum sample and 100 μL of the internal standard solution are mixed. The internal standard solution is L-2-chlorophenylalanine prepared with methanol at 0.3 mg / mL. 300 μL of the protein precipitant is added. The volume ratio of methanol to acetonitrile in the protein precipitant is 2:1. Vortex for 1 min, stand at -20 °C for 30 min, then centrifuge at 13000 rpm and 4 °C for 10 min. Take 300 μL of the supernatant and transfer it to an LC-MS injection vial to evaporate to dryness. Then redissolve it with 300 μL of methanol solution under the conditions of vortexing for 30 s and ultrasonic treatment for 3 min. The volume ratio of methanol to water in the methanol solution is 1:4. After redissolution, stand at -20 °C for 2 hours, centrifuge at 13000 rpm and 4 °C for 10 min. Aspirate 150 μL of the supernatant with a syringe, filter it through a 0.22 μm organic phase needle filter, transfer it to an LC-MS injection vial, and store it at -80 °C until LC-MS analysis. Each sample is mixed in equal amounts to prepare a quality control sample;

[0026] Before data analysis, the content of phytosphingosine is analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain liquid chromatography-tandem mass spectrometry data;

[0027] When analyzing data, the liquid chromatography-tandem mass spectrometry data is processed using Progenesis software, including baseline filtering, peak identification, integration and retention time correction, peak alignment and normalization. Then the extracted data is processed to generate a data matrix, which is composed of the data obtained in the positive ion and negative ion modes. Unsupervised principal component analysis is applied to observe the overall distribution among samples and the stability of the entire analysis process. Then orthogonal partial least squares discriminant analysis is applied to discriminate different groups. Metabolites with differences between patients and the control group are determined according to the following criteria: variable importance in the projection: VIP > 1.0, two-sided student t-test: p < 0.05, and fold change, Fold Change < 1 or > 1. To understand the mechanism of metabolic pathway changes in differential samples, metabolic pathway enrichment analysis is performed on differential metabolites based on the KEGG database. Differential metabolites include up-regulated differential metabolites and down-regulated differential metabolites;

[0028] In the statistical analysis method, the statistical analysis of all clinical data was performed using IBM SPSS statistical software. Continuous data were presented as mean ± standard deviation. The normal distribution hypothesis of continuous variables was tested using the Shapiro-Wilk test and Q-Q plot. For variables with a normal distribution, an independent samples t-test was used for comparison between two groups. For non-normally distributed data, the Mann-Whitney U rank sum test was used. For comparison among multiple groups, ANOVA analysis was performed. For those with homogeneous variances, the Turkey and R-E-G-WQ methods were used, and for those with inhomogeneous variances, the Dunnetts T3 method was used. For non-normally distributed variables, the Mann-Whitney U rank sum test was used for comparison between two groups, and the Kruskal-Wallis test was used for comparison among multiple groups. Categorical variables were analyzed using the chi-square test, and the Spearman correlation analysis method was used for correlation analysis.

[0029] The fourth object of the present application is to provide a verification method for verifying the application of a supplement containing phytosphingosine in the treatment of metabolic disorders.

[0030] To achieve the fourth object of the present application, the technical solution of the present application is as follows:

[0031] In this verification method, animal model experiments and cell intervention model experiments are carried out. In the animal model experiments, biochemical index detection and histological analysis are carried out. In the cell intervention model experiments, cell culture is carried out, and the cultured cells are intervened with phytosphingosine.

[0032] Animal model experiments are carried out. Based on the human microbiota of phlegm-dampness constitution and the high-fat diet animal model, mice are randomly divided into a model group, a high-dose phytosphingosine group, and a low-dose phytosphingosine group. Phytosphingosine is vortexed and mixed evenly with sterile PBS buffer. The dosing doses are as follows: the low-dose phytosphingosine group is 25 mg / kg, and the high-dose phytosphingosine group is 50 mg / kg. The dosing method is as follows: after microbiota transplantation, the high-dose phytosphingosine group and the low-dose phytosphingosine group are respectively given phytosphingosine by gavage at the corresponding dosing doses, and the model group is given an equal amount of PBS buffer by gavage once a day for 5 consecutive weeks;

[0033] During the whole animal experiment process, the reactivity, hair color, diet, eyes, excreta, and mental state of the mice are closely observed. After one week of adaptive feeding of the mice, the body weight change data are recorded starting from the following week, and the food intake of the mice is randomly selected and recorded for 10 days during the experimental period;

[0034] All experimental mice were fasted for 8 hours before sacrifice. Subsequently, they were anesthetized with sodium pentobarbital, and blood was collected by eye socket enucleation. The plasma was allowed to stand for 3 - 4 hours, and then centrifuged at a speed of 3000 rpm, a temperature of 4°C, and for 10 minutes in a refrigerated centrifuge to obtain the supernatant. Before sacrifice, the feces of each experimental mouse were collected into a sterile 1.5 ml centrifuge tube using a sterile acrylic box, quickly frozen in a liquid nitrogen tank, and then transferred to an -80°C freezer for storage;

[0035] After the mouse died from blood collection, the abdominal cavity of the mouse was opened, and the liver tissue was carefully dissected on an ice box and rinsed thoroughly with pre-cooled PBS buffer. A 0.5×0.5 cm large liver lobe was cut with a blade and placed in 4% paraformaldehyde for fixation and standby. Subsequently, the liver tissue was cut into small pieces, sub-packed into cryotubes, frozen quickly in liquid nitrogen, and then transferred to an -80°C freezer for storage. The adipose tissue was separated on an ice box, weighed and recorded. A 0.5×0.5 cm adipose tissue from the same site was cut off and placed in adipose tissue fixative, and the remaining adipose tissue was sub-packed and frozen;

[0036] When performing biochemical index detection, a fully automatic biochemical analyzer was used to detect the levels of four lipid items, blood glucose, and uric acid in the serum samples. According to the requirements for animal tissue homogenate in the reagent kit used for the detection index, 100 mg of liver tissue was accurately weighed, and 900 μL of PBS buffer was added according to the ratio of weight:volume = 1:9 (weight unit is gram, volume unit is milliliter). The homogenizer was held and the sample was ground and homogenized under the conditions of a 4°C ice-water bath, a rotation speed of 2500 rpm, and centrifugation for 10 minutes. The supernatant was taken, the liver homogenate was diluted to different multiples, BCA protein quantification was performed, the best concentration was selected to dilute each sample, and the final protein concentration of each sample was detected and calculated. The contents of TG, TC, ALT, and AST in the liver homogenate samples were detected and calculated using the reagent kit;

[0037] When performing histological analysis, the liver and adipose tissues were fixed in 4% paraformaldehyde solution for 24 hours, then embedded in paraffin, sectioned with hematoxylin-eosin for the liver and adipose tissues. The section thickness was about 3 μm, and staining was performed. The stained samples were observed under a 400-fold magnifying glass, and ImageJ software was used to analyze the lipid levels of the liver and adipose tissues.

[0038] Cell culture was carried out. HepG2 cells were cultured in a high-glucose medium containing 10% fetal bovine serum and 1% penicillin or streptomycin. The culture temperature was 37°C, and the culture environment had 5% CO 2, Observe the cell growth status every day, change the fresh culture medium every 2 - 3 days. When the cells grow to 80% of the bottom area of the culture flask, perform cell modeling and drug administration intervention. Divide the 96-well plate into 10 groups, and each group uses the model culture medium with 6 replicates in each group. The first group is the cell-free blank control group, and the second group is the model group. The remaining 8 groups corresponding to phytosphingosine are: Phy-0.01, Phy-0.05, Phy-0.1, Phy-0.25, Phy-0.5, Phy-1, Phy-2.5, Phy-5, where Phy is phytosphingosine and the numbers are concentrations with the concentration unit of %. Seed the plate with HepG2 cells that have been cultured for 48 h. Calculate according to 10,000 cells and 100 μL of model culture medium per well of the 96-well plate. Add the model culture medium and the corresponding concentration of phytosphingosine to make the cell concentration reach 100,000 cells / mL. After seeding and culturing in the incubator for 48 h, add 10 μL of CCK-8 solution to each well, and then incubate for another 2 h. Measure the absorbance at 450 nm with an enzyme-labeling instrument;

[0039] In the phytosphingosine intervention insulin resistance cell model, first culture HepG2 cells with insulin at a dose of 1 μM for 48 hours, then change to serum-free medium and culture for another 12 hours. Finally, add 1 μM insulin medium to HepG2 cells and add high-dose phytosphingosine at 2.5 μM, low-dose phytosphingosine at 1 μM, and no phytosphingosine and culture in dimethyl sulfoxide for 48 hours;

[0040] In the phytosphingosine intervention high-fat cell model, use palmitic acid reagent and oleic acid reagent to intervene and model HepG2 cells for 48 hours according to a concentration ratio of 1:2, and then culture with 1 μM and 2.5 μM phytosphingosine culture solution for 48 hours;

[0041] Freshly prepare the Oil Red O working solution, stain HepG2 cells for 30 min, then wash the cells with 60% isopropanol to remove the excess dye, then rinse with PBS buffer, then stain with Mayer hematoxylin for 2 min, then rinse with PBS buffer, and observe the stained HepG2 cells under a microscope to verify the effect of phytosphingosine on cells in the cell intervention model experiment.

[0042] Compared with the prior art, the beneficial effects of this application are:

[0043] 1. Apply phytosphingosine to the human body. According to the change in the content of phytosphingosine, it is possible to identify the physical constitution of the human body, screen before metabolic disorders are found in the human body, and utilize the change in the content of phytosphingosine to improve the accuracy of screening. By combining the detection of various clinical indicators, "early screening" and "early warning" of metabolic disorders can be achieved. After the physical constitution is identified, through the intervention of phlegm-dampness constitution, it is possible to achieve the prevention from "one disease" to "a class of diseases".

[0044] 2. This application uses a supplement containing phytosphingosine as an exogenous supplement substance, which can be applied in the treatment of phlegm-dampness constitution intervention. The phytosphingosine in the supplement intervenes in the phlegm-dampness constitution by alleviating typical characteristics of phlegm-dampness constitution such as laziness.

[0045] 3. This application uses a supplement containing phytosphingosine as an exogenous supplement substance, which can be applied in the treatment of phlegm-dampness constitution intervention and metabolic disorders. The phytosphingosine in the supplement intervenes in the phlegm-dampness constitution by alleviating typical characteristics of phlegm-dampness constitution such as laziness. The phytosphingosine in the supplement improves the insulin sensitivity of patients with metabolic disorders by regulating glucose metabolism, lipid metabolism and uric acid metabolism, and reduces blood glucose, blood lipid and blood uric acid levels, thereby achieving the overall treatment of a class of diseases such as obesity, diabetes, hyperlipidemia, hyperuricemia, etc. in metabolic disorders, and can also reduce the risk of metabolic syndrome and its complications.

[0046] 4. This application uses a supplement containing phytosphingosine as an exogenous supplement substance, which can be applied in the treatment of the disease of hyperuricemia. This supplement can significantly reduce the serum uric acid level and reduce the deposition of uric acid in the kidneys and joints, thereby preventing and treating hyperuricemia and gout.

[0047] 5. Through the verification method, this application verifies that according to the change in the content of phytosphingosine, it can be applied in the early screening of metabolic disorders and the identification of human physical constitution, and verifies that a supplement containing phytosphingosine can be applied in the treatment of phlegm-dampness constitution intervention and metabolic disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The specification drawings forming a part of this application are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation of this application.

[0049] Figure 1Spearman correlation between phytosphingosine and clinical obesity indicators. A is the Spearman correlation between phytosphingosine and body weight among clinical obesity indicators, B is the Spearman correlation between phytosphingosine and BMI among clinical obesity indicators, C is the Spearman correlation between phytosphingosine and waist circumference among clinical obesity indicators, D is the Spearman correlation between phytosphingosine and hip circumference among clinical obesity indicators, and E is the Spearman correlation between phytosphingosine and waist-to-hip ratio among clinical obesity indicators.

[0050] Figure 2 Spearman correlation between phytosphingosine and clinical lipid metabolism indicators. A is the Spearman correlation between phytosphingosine and TC in clinical lipid metabolism indicators, where TC is total cholesterol, B is the Spearman correlation between phytosphingosine and TG in clinical lipid metabolism indicators, where TG is triglyceride, C is the Spearman correlation between phytosphingosine and LDLC in clinical lipid metabolism indicators, where LDLC is low-density lipoprotein, D is the Spearman correlation between phytosphingosine and HDLC in clinical lipid metabolism indicators, where HDLC is high-density lipoprotein, E is the Spearman correlation between phytosphingosine and ApoA1 in clinical lipid metabolism indicators, where ApoA1 is apolipoprotein A1, F is the Spearman correlation between phytosphingosine and ApoB in clinical lipid metabolism indicators, where ApoB is apolipoprotein B, and G is the Spearman correlation between phytosphingosine and Lpα in clinical lipid metabolism indicators, where Lpα is lipoprotein α.

[0051] Figure 3 Spearman correlation between phytosphingosine and clinical glucose metabolism indicators. A is the Spearman correlation between phytosphingosine and FBG in clinical glucose metabolism indicators, where FBG is fasting blood glucose, B is the Spearman correlation between phytosphingosine and 2hPG in clinical glucose metabolism indicators, where 2hPG is two-hour postprandial blood glucose, C is the Spearman correlation between phytosphingosine and HBA1C in clinical glucose metabolism indicators, where HBA1C is glycated hemoglobin, D is the Spearman correlation between phytosphingosine and Fasting insulin in clinical glucose metabolism indicators, where Fasting insulin is fasting insulin, and E is the Spearman correlation between phytosphingosine and HOMA-IR in clinical glucose metabolism indicators, where HOMA-IR is insulin resistance index.

[0052] Figure 4Spearman correlation between phytosphingosine and blood uric acid and blood pressure. A is the Spearman correlation between phytosphingosine and Serumuric acid, where Serum uric acid is blood uric acid. B is the Spearman correlation between phytosphingosine and SBP of blood pressure, where SBP is systolic blood pressure. C is the Spearman correlation between phytosphingosine and DBP of blood pressure, where CBP is diastolic blood pressure.

[0053] Figure 5 Comparison of the content of phytosphingosine in four groups of subjects: BCN, BCD, PDN, and PDD. The BCN group is the group of subjects with a peaceful constitution and no disease. The BCD group is the group of subjects with a peaceful constitution and existing disease. The PDN group is the group of subjects with a phlegm-dampness constitution and no disease. The PDD group is the group of subjects with a phlegm-dampness constitution and existing disease.

[0054] Figure 6 Constructing an ROC curve with phytosphingosine as the explanatory variable. A is the ROC curve of the BCN group and the PDN group. B is the ROC curve of the PDN group and the PDD group.

[0055] Figure 7 Box plot comparing the model group, the high-dose phytosphingosine group, and the low-dose phytosphingosine group when using the phlegm-dampness non-diseased human gut microbiota transplantation model. A is the comparison of the skin greasiness of mice. B is the comparison of the lethargy state of mice. PBS is the model group. PhyH is the high-dose phytosphingosine group. PhyL is the low-dose phytosphingosine group.

[0056] Figure 8 Graph comparing the body weights and food intakes of the model group, the high-dose phytosphingosine group, and the low-dose phytosphingosine group. A is the change in the body weight of mice over weeks. B is the box plot comparing the body weights of the three groups. C is the box plot comparing the food intakes of the three groups.

[0057] Figure 9 Graph comparing the fat weights and morphologies. A is the box plot comparing the weight changes of the three groups in white adipocytes, beige adipocytes, and brown adipocytes. Among them, Inguinal is beige fat, Epididymal is white fat, and perirenal is brown fat. B is the box plot comparing the fat cell sizes of the three groups. C is the morphological comparison diagram of the volume of white adipocytes.

[0058] Figure 10 Graph comparing the three groups in four blood lipid parameters, blood glucose, and blood uric acid. A is the graph comparing the four blood lipid parameters. Among them, TC is total cholesterol, TG is triglyceride, HDLC is high-density lipoprotein, and LDLC is low-density lipoprotein. B is the graph comparing blood glucose, where serum GLU is blood glucose. C is the graph comparing blood uric acid, where serum UA is blood uric acid.

[0059] Figure 11 These are three groups of comparison charts regarding fat deposition in mouse livers. Chart A shows the comparison of lipid droplet sizes in liver tissues, Chart B shows the comparison of the proportion of lipid droplet areas calculated using ImageJ, Chart C shows the comparison of total cholesterol detected in liver tissue homogenates, Chart D shows the comparison of triglycerides detected in liver tissue homogenates, Chart E shows the comparison of AST detected in liver tissue homogenates, and Chart F shows the comparison of ALT detected in liver tissue homogenates.

[0060] Figure 12 These are comparison charts of the cell viability of HepG2 cells at various concentrations under a high-fat cell model. "cell viability" refers to cell viability.

[0061] Figure 13 These show the effects of phytosphingosine treatment at 1 μM and 2.5 μM on lipid deposition in a high-fat cell model. Chart A shows the comparison of TC in the high-fat cell model, Chart B shows the comparison of TG in the high-fat cell model. "Oleic acid" refers to Oil Red O staining, and Chart C shows the number of HepG2 cells observed under a microscope after staining.

[0062] Figure 14 These are comparison charts of the cell viability of HepG2 cells at various concentrations under an insulin resistance cell model. "cellviability" refers to cell viability.

[0063] Figure 15 These show the effects of phytosphingosine treatment at 1 μM and 2.5 μM on glucose consumption in an insulin resistance cell model. Specific Embodiments

[0064] The present application will be further described below in conjunction with the accompanying drawings and embodiments.

[0065] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. When using standard units, it can be expressed in Chinese characters, such as "hour", or represented by "h".

[0066] Example 1

[0067] In this embodiment, the application of phytosphingosine in the early screening of metabolic disorders and the identification of human constitutions. Phytosphingosine is used as a metabolic marker in the screening before the onset of early-stage metabolic disorders. Metabolic disorders include obesity, hyperlipidemia, hypertension, hyperuricemia, and non-alcoholic fatty liver. Human constitutions include balanced constitution and phlegm-dampness constitution. Phytosphingosine is used as a marker in the identification of human phlegm-dampness constitution, and the early screening of metabolic disorders and the identification of human constitutions are carried out according to the content change of phytosphingosine.

[0068] In addition, human constitutions also include seven constitutions such as yang-deficiency constitution, yin-deficiency constitution, qi-deficiency constitution, damp-heat constitution, blood stasis constitution, special endowment constitution, and qi-stagnation constitution. There are a total of 9 constitutions including balanced constitution and phlegm-dampness constitution. And the phytosphingosine in this embodiment is used as a marker in the identification of human constitutions, and the phlegm-dampness constitution is screened out from the 9 human constitutions.

[0069] Specifically, when using phytosphingosine for the early screening of metabolic disorders and the identification of human constitutions, it cannot achieve the risk assessment of suffering from metabolic disorders, nor can it predict the treatment effect of metabolic disorders. Instead, through the content change of phytosphingosine, intermediate information in the early screening of metabolic disorders and the identification of human constitutions is obtained from a living human body. For example, the intermediate information for the early screening of metabolic disorders is that the content change is correlated with multiple clinical indicators such as obesity, blood lipid, uric acid, and blood pressure, but the above clinical indicators cannot be clearly obtained. The intermediate information for the identification of human constitutions is the classification of constitutions. This provides a direction for the judgment of metabolic disorders. Using the content change of phytosphingosine can improve the accuracy of screening, and combined with the detection of various clinical indicators, "early screening" and "early warning" of metabolic disorders can be achieved. After the constitution is identified, through the intervention of the phlegm-dampness constitution, it is possible to achieve the prevention from "one disease" to "a class of diseases".

[0070] The number of methylene groups in phytosphingosine is 16 - 20. For example, as an embodiment, the molecular formula of phytosphingosine is C 18 H 39 NO 3 。

[0071] As the basic process of the verification method in this embodiment, the subjects are determined for their balanced constitution and phlegm-dampness constitution, and the subjects are divided into a balanced constitution group, a phlegm-dampness constitution pre-disease group, and a phlegm-dampness constitution disease group. Among them, the balanced constitution group is further divided into a balanced constitution pre-disease group and a balanced constitution disease group. Blood samples are collected from the four groups of people, and then serum untargeted metabolomics detection and data analysis are carried out, and statistical analysis methods are used for application verification. Among them, the balanced constitution group is abbreviated as BC or BC group (Balanced constitution), the BCN group is the balanced constitution pre-disease group, the BCD group is the balanced constitution disease group, the phlegm-dampness constitution pre-disease group is abbreviated as PDN or PDN group, and the phlegm-dampness constitution disease group is abbreviated as PDD or PDD group (Phlegm dampness constitution, metabolic disorders).

[0072] In the stage of differentiating the constitution of the subjects, in this embodiment, according to the "Classification and Judgment of Traditional Chinese Medicine Constitution" promulgated by the Chinese Association of Chinese Medicine, the standard number is ZZYXH / T157-2009. The sub-scales of balanced constitution and phlegm-dampness constitution are shown in Table 1 - Table 2, and the scoring methods and judgment criteria are shown in Table 3 and Table 4 respectively. The volunteers first fill in the "Traditional Chinese Medicine Constitution Scale" to determine the constitution type, and then it is confirmed by manual identification by professional doctors of traditional Chinese medicine constitution science.

[0073] Table 1: Sub-scale of Balanced Constitution in "Classification and Judgment of Traditional Chinese Medicine Constitution"

[0074]

[0075]

[0076] Table 2: Sub-scale of Phlegm-dampness Constitution in "Classification and Judgment of Traditional Chinese Medicine Constitution"

[0077]

[0078]

[0079] Table 3: Scoring Methods for Balanced Constitution and Phlegm-dampness Constitution

[0080] Score Score range Calculation method Original score 8 - 40 points Original score = sum of scores of each item Converted score 0 - 100 points Converted score = (Original score - 8) / 32 × 100

[0081] Note: The items marked with * need to be reverse-integrated, that is: 1 is recorded as 5, 2 is recorded as 4, 3 is recorded as 3, 4 is recorded as 2, 5 is recorded as 1, and then the conversion score is calculated.

[0082] Table 4: Judgment Criteria for Balanced Constitution and Phlegm-dampness Constitution

[0083]

[0084] As a diagnostic criterion for metabolic disorders, at least one of the following obesity, diabetes, hyperlipidemia, hypertension, and hyperuricemia can be diagnosed as metabolic disorders (MDs).

[0085] The diagnostic criteria for obesity follow "Epidemiology and Determinants of Obesity in China" published in The Lancet. See Table 5 for details.

[0086] Table 5: Body Mass Index for Overweight and Obesity in Chinese Adults

[0087] Item <![CDATA[Body Mass Index (Kg / m 2 )]]> Underweight ＜18.5 Normal weight 18.5-23.9 Overweight 24.0-27.9 Obese ≥28.0

[0088] The diagnostic criteria for dyslipidemia follow "Chinese Guidelines for the Prevention and Treatment of Dyslipidemia in Adults (2016 Revised Edition)". See Table 6 for details.

[0089] Table 6: Appropriate Blood Lipid Levels and Abnormal Stratification Criteria for Primary Prevention of ASCVD [mmol / L (mg / dl)]

[0090]

[0091] Note: The meanings of the letters in Table 6 are as follows: ASCVD: Atherosclerotic Cardiovascular Disease; TC: Total Cholesterol; LDL-C: Low-Density Lipoprotein Cholesterol; HDL-C: High-Density Lipoprotein Cholesterol; non-HDL-C: Non-High-Density Lipoprotein Cholesterol; TG: Triglyceride. LDL-C can also be written as LDLC, and HDL-C can also be written as HDLC.

[0092] The diagnostic criteria for prediabetes follow "Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes (2017 Edition)". See Table 7 for details.

[0093] Table 7: Classification of Glucose Metabolism Status in "Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes (2017 Edition)" (WHO 1999 Standard)

[0094]

[0095] Note: The meanings of the letters in Table 7 are as follows: IFG is impaired fasting glucose, IGT is impaired glucose tolerance, and IFG and IGT are collectively referred to as impaired glucose regulation.

[0096] The diagnostic criteria for hyperuricemia follow "Chinese Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout (2019)". See Table 8 for details.

[0097] Table 8: Diagnostic Criteria for Hyperuricemia in "Chinese Guidelines for the Diagnosis and Treatment of Hyperuricemia and Gout (2019)"

[0098] Concept Diagnostic criteria Hyperuricemia Fasting blood uric acid > 420 umol / L (for adults, regardless of gender)

[0099] The diagnostic criteria for hypertension follow the "2018 Revised Edition of the Chinese Guidelines for the Prevention and Control of Hypertension", as shown in Table 9 specifically.

[0100] Table 9: Classification and Definition of Blood Pressure Levels in the "2018 Revised Edition of the Chinese Guidelines for the Prevention and Control of Hypertension"

[0101]

[0102]

[0103] Note: The meanings of the letters in Table 9 are as follows: SBP: Systolic blood pressure; DBP: Diastolic blood pressure.

[0104] The following are the specific inclusion criteria for the subjects:

[0105] (1) According to the research diagnostic criteria, the physical constitution is determined as a balanced constitution or a phlegm-dampness constitution;

[0106] (2) Age range: 18 years ≤ age ≤ 50 years, and the age is calculated based on the difference between the screening survey date and the date of birth;

[0107] (3) Gender is not restricted;

[0108] (4) Having lived stably in the survey area for at least 1 year and having a fixed residence;

[0109] (5) Having good memory and activity ability, being able to take care of oneself in life, and having mental health;

[0110] (6) The subjects give informed consent and sign the informed consent form;

[0111] The following are the specific exclusion criteria for the subjects:

[0112] (1) Having used antibiotics within the past 3 months; having used other foods and drugs that affect the intestinal flora status, such as gastrointestinal motility drugs and microecological regulators within the past 3 months;

[0113] (2) Those who have lost weight by any drug method or surgical method within the past 3 months. Drug methods include appetite suppressant drugs, thyroid hormones, progesterone, laxatives, etc. and various traditional Chinese medicine ingredient weight loss drugs, etc.

[0114] (3) Those suffering from gastrointestinal diseases or having undergone gastrointestinal surgery;

[0115] (4) Those suffering from severe organic diseases or infectious diseases.

[0116] When collecting clinical data in this embodiment, trained professional doctors collected detailed clinical information of eligible participants in a face-to-face manner. The collected clinical data included relevant information such as the demographic information, anthropometric characteristics, health status, disease history, and medication use of the participants. The body mass index (BMI) of the participants was calculated by dividing the weight (in kilograms) by the square of the height (in square meters). The waist circumference was measured by going around the abdomen horizontally at 5 cm above the umbilicus, and the hip circumference was measured at the most prominent part of the buttocks. The vital signs (heart rate, blood pressure) were measured at rest, and the blood pressure was taken on the right side in a sitting position.

[0117] Among the numerous participants, subjects were selected and repeatedly verified according to the method of this embodiment.

[0118] When collecting blood samples, peripheral venous blood was drawn in a fasting state, left to stand at room temperature for 30 minutes, and then centrifuged at 3000 rpm for 5 minutes. The serum was transferred to a centrifuge tube to form a serum sample. The serum sample was detected, and the detection indicators included blood routine, four lipid items, ApoA1, ApoB, Lpα, fasting blood glucose, and uric acid. The remaining serum sample was quickly frozen to -80°C in liquid nitrogen for storage. After the first blood draw, the participant ate, and the time was counted from the first bite of food. Two hours later, blood was drawn again to detect the blood glucose two hours after a meal.

[0119] When performing the detection of serum untargeted metabolomics, the serum samples in liquid nitrogen were first processed. The serum samples were thawed on ice, 100 μL of the serum sample and 100 μL of the internal standard solution were mixed. The internal standard solution was L-2-chlorophenylalanine configured with methanol at 0.3 mg / mL. 300 μL of the protein precipitant was added. The volume ratio of methanol to acetonitrile in the protein precipitant was 2:1. It was vortexed for 1 min and left to stand at -20°C for 30 min. Then it was centrifuged at 13000 rpm and 4°C for 10 min. 300 μL of the supernatant was taken and put into an LC-MS injection vial to evaporate to dryness. Then it was redissolved with 300 μL of methanol solution under the conditions of vortexing for 30 s and ultrasonic treatment for 3 min. The volume ratio of methanol to water in the methanol solution was 1:4. After redissolution, it was left to stand at -20°C for 2 hours, centrifuged at 13000 rpm and 4°C for 10 min. 150 μL of the supernatant was aspirated with a syringe, filtered through a 0.22 μm organic phase needle filter, and transferred to an LC-MS injection vial, and stored at -80°C until LC-MS analysis. And each sample was mixed in equal amounts to prepare a quality control sample.

[0120] Before data analysis, the content of phytosphingosine was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry to obtain liquid chromatography-tandem mass spectrometry data. An UPLC BEH C18 column (1.7 μm, 2.1 mm × 100 mm) and a QExactive HF-X mass spectrometer were used, and positive and negative ion modes were adopted. Table 10 shows the mass spectrometry conditions of this method:

[0121] Table 10: Mass Spectrometry Conditions

[0122] Parameter Positive ion Negative ion Electrospray capillary voltage (capillary voltages, KV) 2.5 2.5 Injection voltage (DP, V) 40 40 Collision voltage (CE, eV) 4 4 Ion source temperature (source temperature, ℃) 115 115 Desolvation temperature (desolvation temperature, m) 450 450 Carrier gas flow rate (desolvation gas flow, L / h) 900 900 Mass spectrometry scan range (mass range, amu) 50-1000 50-1000 Scan time (Scan time, s) 0.2 0.2 Interval time (interscan delay, s) 0.02 0.02

[0123] During data analysis, the original liquid chromatography-tandem mass spectrometry data was processed using Progenesis software (Nonlinear Dynamics, Newcastle, UK), including baseline filtering, peak identification, integration and retention time correction, peak alignment and normalization. Then, the extracted data was processed to generate a data matrix, which was composed of the data obtained in positive and negative ion modes. Unsupervised principal component analysis (PCA) was applied to observe the overall distribution among samples and the stability of the entire analysis process. Then, orthogonal partial least squares discriminant analysis (OPLS-DA) was applied to discriminate different groups. Metabolites with differences between patients and the control group were determined according to the following criteria: variable importance in the projection: VIP > 1.0, two-sided student t-test: p < 0.05, and fold change: Fold Change < 1 or > 1. To understand the mechanism of metabolic pathway changes in differential samples, metabolic pathway enrichment analysis was performed on differential metabolites based on the KEGG database. Differential metabolites include up-regulated differential metabolites and down-regulated differential metabolites.

[0124] In the statistical analysis method, all statistical analyses of clinical data were performed using IBM SPSS statistical software. Continuous data were presented as mean ± standard deviation. The normal distribution hypothesis of continuous variables was tested using the Shapiro-Wilk test and Q-Q plot. For variables with a normal distribution, an independent samples t-test was used for comparison between two groups, the Mann-Whitney U rank sum test was used for non-normally distributed data, ANOVA analysis was used for comparison among multiple groups, the Turkey and R-E-G-WQ methods were used for those with homogeneous variances, and the Dunnetts T3 method was used for those with heterogeneous variances. For non-normally distributed variables, the Mann-Whitney U rank sum test was used for comparison between two groups, and the Kruskal-Wallis test was used for comparison among multiple groups. Categorical variables were analyzed using the chi-square test, and Spearman correlation analysis method was used for correlation analysis.

[0125] From the analysis, whether it is all phlegm-dampness constitution individuals, or the phlegm-dampness constitution is divided into two subgroups, PDN and PDD, according to whether there is metabolic disorder, compared with the peaceful constitution, phytosphingosine shows significant changes, and it has the strongest Spearman correlation with the phlegm-dampness constitution transformation score PDC score. Therefore, we believe that phytosphingosine has the potential to become a characteristic metabolite of the phlegm-dampness constitution. Next, we will conduct a correlation analysis between phytosphingosine and clinical indicators and use sphingosine to construct a classifier to determine the efficacy of phytosphingosine.

[0126] Specifically, in the relationship between phytosphingosine and obesity, as Figure 1 shown, Spearman correlation analysis shows that phytosphingosine has a significant correlation with obesity-related indicators BMI (r = -0.242, p < 0.001), body weight (r = -2.202, p = 0.003), waist circumference (r = -0.280, p < 0.001), hip circumference (r = -0.209, p = 0.002), waist-hip ratio WHR (r = -0.222, p = 0.001).

[0127] Specifically, in the relationship between phytosphingosine and lipid metabolism indicators, as Figure 2 shown, Spearman correlation analysis shows that phytosphingosine has a significant correlation with lipid metabolism-related indicators total cholesterol (r = -0.205, p = 0.003), triglyceride (r = -0.183, p = 0.008), low-density lipoprotein (r = -0.232, p = 0.001), apolipoprotein B (r = -0.197, p = 0.004); no significant correlation was observed between phytosphingosine and high-density lipoprotein (r = 0.089, p = 0.200), apolipoprotein A1 (r = 0.039, p = 0.576), lipoprotein α (Lpα) (r = 0.016, p = 0.816).

[0128] Specifically, in the relationship between phytosphingosine and glucose metabolism indicators, as Figure 3 shown, Spearman correlation analysis shows that there is no significant correlation between phytosphingosine and glucose metabolism-related indicators fasting blood glucose FBG (r = 0.001, p = 0.991), two-hour postprandial blood glucose 2hPG (r = -0.061, p = 0.381), glycated hemoglobin (r = -0.082, p = 0.238), fasting insulin (r = -0.083, p = 0.233), insulin resistance index IR (r = -0.079, p = 0.255).

[0129] Specifically, in the relationship between phytosphingosine and other metabolic indicators, as Figure 4As shown, Spearman correlation analysis showed that phytosphingosine was significantly correlated with uric acid UA (r = -0.230, p < 0.001); it was also significantly correlated with systolic blood pressure SBP (r = -0.147, p = 0.033) and diastolic blood pressure DBP (r = -0.199, p = 0.004).

[0130] Based on the above analysis, combined with Figure 5 , we observed that phytosphingosine was significantly decreased in both the PDN group and the PDD group, and was closely related to multiple clinical indicators such as obesity, blood lipids, uric acid, and blood pressure.

[0131] To explore the potential of phytosphingosine in the identification of phlegm-dampness constitution, especially for the determination of those with latent phlegm-dampness disease, we used phytosphingosine as an explanatory variable to draw an ROC curve. The ROC curve is the Receiver Operating Characteristic curve, and its English name is The Receiver Operating Characteristic curve, which is used to classify BCN or PDN from all subjects. As Figure 6 shown, phytosphingosine had extremely strong ability to identify PDN, and the area under the ROC curve AUC (the area under the ROC curve, AUC) was as high as 0.89, which could distinguish the phlegm-dampness constitution population from the healthy population with peaceful constitution. However, the ability of phytosphingosine to distinguish PDN and PDD was poor, with AUC = 0.53. An AUC less than 0.6 indicates no diagnostic significance and cannot distinguish the disease status of the population with phlegm-dampness constitution. These results suggest that phytosphingosine is a good predictor for distinguishing phlegm-dampness constitution and peaceful constitution, and further indicates that phytosphingosine is a biomarker of phlegm-dampness constitution.

[0132] From the above analysis, phytosphingosine, as a biomarker, has a high correlation with phlegm-dampness constitution and can significantly improve the specificity of screening.

[0133] This embodiment can be used to prepare phytosphingosine into a supplement and apply it in the treatment of metabolic disorders. The supplement contains phytosphingosine. The metabolic disorders include obesity, diabetes, hyperlipidemia, hyperuricemia, and non-alcoholic fatty liver. The phytosphingosine in the supplement can improve the insulin sensitivity of patients with metabolic disorders, and reduce blood glucose, blood lipid, and blood uric acid levels by regulating glucose metabolism, lipid metabolism, and uric acid metabolism. Specifically, the phytosphingosine in the supplement can enhance the PPAR-related insulin signaling pathway, promote the activation of insulin receptors, increase the uptake and utilization of glucose by cells, thereby reducing fasting blood glucose and postprandial blood glucose levels, and improving insulin resistance. The phytosphingosine in the supplement can reduce the synthesis of cholesterol in the liver by activating the PPAR signaling pathway, while increasing the production and utilization of high-density lipoproteins in the liver and tissues, and reducing the levels of low-density lipoproteins, triglycerides, and total cholesterol, thereby improving blood lipid disorders. The phytosphingosine in the supplement can improve the abnormal liver function caused by metabolic disorders by reducing fat deposition in the liver and adipose tissue, and reducing the content of triglycerides and total cholesterol. The phytosphingosine in the supplement can reduce the weight and number of adipose tissues in the body, reduce lipid deposition in liver fat cells, reduce liver fat deposition, and reduce the risk of obesity and related metabolic complications. The phytosphingosine in the supplement can significantly reduce the serum uric acid level and reduce the deposition of uric acid in the kidneys and joints, thereby preventing and treating hyperuricemia and gout.

[0134] The supplement is a pharmaceutically acceptable dosage form, including tablets, capsules, dripping pills, granules, injections, and oral liquid dosage forms, and can be used as a health food or a drug.

[0135] This embodiment provides a verification method for verifying the application of a supplement containing phytosphingosine in the treatment of metabolic disorders.

[0136] In this verification method, animal model experiments and cell intervention model experiments are carried out. In the animal model experiments, biochemical index detection and histological analysis are carried out. In the cell intervention model experiments, cell culture is carried out, and the cultured cells are intervened with phytosphingosine.

[0137] Specifically, animal model experiments are carried out. Based on the human flora with phlegm-damp constitution and the high-fat diet animal model, mice are randomly divided into a model group, a high-dose phytosphingosine group, and a low-dose phytosphingosine group. The phytosphingosine is vortexed and mixed evenly with sterile PBS buffer. The dosing doses are as follows: the low-dose phytosphingosine group is 25 mg / kg, and the high-dose phytosphingosine group is 50 mg / kg. The dosing method is as follows: after flora transplantation, the high-dose phytosphingosine group and the low-dose phytosphingosine group are respectively given phytosphingosine by gavage at the corresponding dosing doses, and the model group is given an equal amount of PBS buffer by gavage, once a day for 5 consecutive weeks;

[0138] During the entire animal experiment process, closely observe the reactivity, hair color, diet, eyes, excreta, and mental state of the mice. After one week of adaptive feeding, record the body weight change data starting from the following week, and randomly select 10 days during the experimental period to record the food intake of the mice;

[0139] During the entire experiment process, closely observe the reactivity, hair color, diet, eyes, excreta, and mental state of the animals. Conduct phlegm-dampness state scoring one day before euthanasia. The scoring criteria are 0 - 3 points, divided into the following two categories: (1) Obvious lethargy: motionless (unless stimulated) 3 points, mild reduction in movement 2 points, moderate reduction in movement 2 points, normal 0 points; (2) Greasy hair: severely greasy and poorly groomed 3 points, mildly greasy 2 points, moderately greasy 1 point, normal 0 points. Obvious lethargy and greasy fur are respectively similar to human body heaviness and skin greasiness.

[0140] All experimental mice are fasted for 8 hours before euthanasia, then anesthetized with sodium pentobarbital, blood is taken by eye enucleation, the plasma is allowed to stand for 3 - 4 h, and the supernatant is centrifuged at a rotation speed of 3000 rpm, a temperature of 4 °C, and a time of 10 min in a refrigerated centrifuge. All experimental mice are collected with sterile acrylic boxes for the feces of each mouse into sterile 1.5 ml centrifuge tubes before euthanasia, and quickly placed in a liquid nitrogen bucket for quick freezing, and then transferred to an -80 °C refrigerator for storage;

[0141] After the mice die from blood collection, open the abdominal cavity of the mice, carefully dissect the liver tissue on an ice box, and rinse it clean with pre-cooled PBS buffer. Cut a 0.5×0.5 cm large liver lobe with a blade and place it in 4% paraformaldehyde for fixation and standby. Then cut the liver tissue into small pieces, sub-pack it into cryopreservation tubes, quickly freeze it in liquid nitrogen, and then transfer it to -80 °C for storage. Separate the adipose tissue on the ice box, weigh and record it, cut off a 0.5×0.5 cm adipose tissue at the same site and place it in adipose tissue fixative, and sub-pack and store the remaining adipose tissue;

[0142] When conducting biochemical index detection, use an automatic biochemical analyzer to detect the levels of four lipid items, blood glucose, and uric acid in the serum samples. According to the requirements for animal tissue homogenate in the reagent kits used for the detection indicators, accurately weigh 100 mg of liver tissue, and add 900 μL of PBS buffer according to the ratio of weight:volume = 1:9. The weight unit is grams and the volume unit is milliliters. Hold the homogenizer and grind and homogenize it under the conditions of an ice-water bath at a temperature of 4 °C, a rotation speed of 2500 rpm, and centrifugation for 10 min. Take the supernatant, dilute the liver homogenate into different multiples, perform BCA protein quantification, select the best concentration to dilute each sample and detect and calculate the final protein concentration of each sample, and use the reagent kit to detect and calculate the contents of TG, TC, ALT, and AST in the liver homogenate samples.

[0143] For histological analysis, the liver and adipose tissues were fixed in 4% paraformaldehyde solution for 24 hours, then embedded in paraffin. The liver and adipose tissue sections were stained with hematoxylin-eosin, with a section thickness of approximately 3 μm, and observed under a 400-fold magnifying glass. The lipid levels in the liver and adipose tissue were analyzed using ImageJ software, and the website of ImageJ software is https: / / imagej.nih.gov / ij / .

[0144] When targeting the detection of phytosphingosine, the content of phytosphingosine was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The samples were extracted with an extraction solution, which was a liquid with a chloroform:methanol ratio of 9:1. First, vortex for 5 minutes, then centrifuge at 12000 r / min for 10 minutes, and then concentrate 400 μL of the supernatant to complete dryness at 20 °C, and then re-dissolve with 100 μL of methanol. In this example, water and methanol were used as the mobile phase, and phytosphingosine was separated on an ACQUITY HSS T3 chromatographic column (2.1×100 mm, 1.8 μm), and MRM detection was performed on a QTRAP 6500 mass spectrometer (Sciex, Concord, Canada). Quantitative analysis was carried out using internal and external standard curves. The chromatographic conditions were as follows: water contained 5 mM ammonium acetate and 0.1% formic acid, methanol contained 0.1% formic acid, the injection volume was 2 μL, and the column temperature was 40 °C.

[0145] The mass spectrometry conditions and corresponding parameters are shown in Table 11.

[0146] Table 11: Mass spectrometry conditions and parameters

[0147] IonMode ESI CurtainGas 20 IonSprayVoltage 5500 Temperature 500 IonSourceGas1 40 IonSourceGas2 50 CollisionGas Medium Scan type MRM EntrancePotential 10 CollisionCellExitPotential 10

[0148] For the statistical analysis method used in this example, physiological and biochemical data were statistically analyzed, and GraphPad Prism (version 9.5) was used for data statistical analysis and graphing. For data that conform to a normal distribution, an independent samples t-test was used for comparison between two groups, and ANOVA analysis was used for comparison between multiple groups. For those with homogeneous variances, the Turkey and R-E-G-WQ methods were used for analysis, and for those with heterogeneous variances, the Dunnetts T3 method was used for analysis; for variables that do not conform to a normal distribution, the Mann-Whitney U rank sum test was used for comparison between two groups, and the Kruskal-Wallis test was used for comparison between multiple groups. Correlation analysis was performed using the Spearman's rho statistic, and p < 0.05 indicates statistical significance. Among them, in the comparative analysis, *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance.

[0149] Cell culture was carried out. HepG2 cells were cultured in high-glucose medium containing 10% fetal bovine serum and 1% penicillin or streptomycin. The culture temperature was 37 °C, and the culture environment had 5% CO 2 , and the cell growth state was observed every day. The medium was replaced every 2 - 3 days. When the cells grew to 80% of the bottom area of the culture flask, cell modeling and drug administration intervention were carried out. A 96-well plate was divided into 10 groups, and each group used the model medium with 6 replicates per group. The first group was the cell-free blank control group, and the second group was the model group. The remaining 8 groups corresponding to phytosphingosine were: Phy-0.01, Phy-0.05, Phy-0.1, Phy-0.25, Phy-0.5, Phy-1, Phy-2.5, Phy-5. Phy is the abbreviated expression of phytosphingosine, and the numbers are concentrations with the concentration unit of %. The HepG2 cells that had been cultured for 48 h were counted and seeded. For each well of the 96-well plate, according to 10,000 cells and 100 μL of the model medium, the model medium and the corresponding concentration of phytosphingosine were added to make the cell concentration reach 100,000 cells / mL. After seeding, the cells were cultured in an incubator for 48 h, then 10 μL of CCK-8 solution was added to each well, and after incubation for another 2 h, the absorbance value at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader;

[0150] In the phytosphingosine intervention insulin resistance cell model, HepG2 cells were first cultured with 1 μM insulin for 48 hours, then changed to serum-free medium and cultured for another 12 hours. Finally, 1 μM insulin medium was given to HepG2 cells, and 2.5 μM of high-dose phytosphingosine, 1 μM of low-dose phytosphingosine, and no phytosphingosine were added and cultured in dimethyl sulfoxide for 48 hours;

[0151] In the phytosphingosine intervention high-fat cell model, palmitic acid reagent and oleic acid reagent were used to intervene and model HepG2 cells for 48 hours according to a concentration ratio of 1:2, and then the cells were cultured with 1 μM and 2.5 μM phytosphingosine culture solutions for 48 hours;

[0152] Freshly prepare the Oil Red O working solution, stain HepG2 cells for 30 min, then wash the cells with 60% isopropanol to remove the excess dye, then rinse with PBS buffer, stain with Mayer hematoxylin for 2 min, then rinse with PBS buffer, and observe the stained HepG2 cells under a microscope to verify the effect of phytosphingosine on cells in the cell intervention model experiment.

[0153] Judging from the results, as Figure 7As shown, under the condition of high-fat feeding, compared with the model group intragastrically administered with PBS, supplementation with phytosphingosine can improve the phlegm-dampness phenotype in mice. Although there was no significant difference between the mice intragastrically administered with low-dose phytosphingosine and the PBS group, supplementation with high-dose phytosphingosine alleviated the skin greasiness and lethargy in mice in a dose-dependent manner.

[0154] As Figure 8 shown, compared with the model group of the phlegm-dampness non-disease human gut microbiota transplantation model intervened with PBS, treatment with high-dose phytosphingosine for 5 weeks can significantly reduce the body weight of mice, while there was no significant change in the low-dose phytosphingosine group. There was no significant difference in food intake among the model group, the high-dose phytosphingosine group, and the low-dose phytosphingosine group, indicating that high-dose phytosphingosine can effectively reduce obesity, and this effect is not due to reduced food consumption or energy acquisition.

[0155] As Figure 9 shown, compared with the PBS group, the weights of white fat (epididymal fat) and beige fat (inguinal fat) in the PhyH group were significantly reduced, while the weight of brown fat (perirenal fat) showed no obvious change, and there was no significant difference in the weights of the three types of fat in the PhyL group.

[0156] Using HE staining to observe the morphology of white adipocytes, compared with the PBS group, the white adipocytes in the PhyH and PhyL groups were reduced to varying degrees, and the number of cells in the field of view increased significantly, which was more obvious in the PhyH group. Using ImageJ to calculate the volume of white adipocytes, it was found that the adipocyte volumes in the PhyH and PhyL groups were significantly reduced, and it was more significant in the PhyH group, indicating that phytosphingosine reduces lipid deposition in adipocytes in a dose-dependent manner.

[0157] As Figure 10 shown, after treatment with phytosphingosine for 5 weeks, compared with the model group intervened with PBS, the total cholesterol (TC), low-density lipoprotein (LDLC), and high-density lipoprotein (HDLC) in the PhyH and PhyL groups were significantly reduced. Compared with the PhyL group, the lipid-lowering effect in the PhyH group was more significant. After treatment with phytosphingosine for 5 weeks, compared with the PBS group, the fasting blood glucose levels in the PhyH and PhyL groups were significantly reduced. After treatment with phytosphingosine for 5 weeks, compared with the PBS group, the fasting blood uric acid levels in the PhyH and PhyL groups were significantly reduced. The above results indicate that phytosphingosine intervention can effectively alleviate the metabolic disorders of blood lipid, blood glucose, and blood uric acid caused by phlegm-dampness non-disease.

[0158] As Figure 11As shown, histological analysis showed that compared with the PBS group, the lipid droplet size in the liver tissues of the PhyH and PhyL groups was significantly reduced. Using ImageJ to calculate the proportion of the lipid droplet area, compared with the PBS group, both the PhyH and PhyL groups were significantly decreased. The results of detecting TC and TG in the liver tissue homogenate showed that TG in the PhyH and PhyL groups was significantly reduced, and TC in the PhyH group was significantly reduced. Compared with the PBS group, there were no significant changes in AST and ALT in the livers of the PhyH and PhyL groups.

[0159] As Figure 12 shown, through an oleic acid-induced hyperlipidemic cell model, to explore the effect of phytosphingosine in vitro intervention on lipid metabolism disorders. In this example, different concentration gradients of phytosphingosine (Phy-0.01, Phy-0.05, Phy-0.1, Phy-0.25, Phy-0.5, Phy-1, Phy-2.5, Phy-5) were set to observe cell viability in order to screen for safe and effective drug doses. After incubating HepG2 cells in each group for 48 h, compared with the model group, the cell viability of the Phy-5 group was significantly decreased (p < 0.001). In subsequent experiments, the phytosphingosine concentrations used were the maximum concentrations of 2.5 μM and 1 μM that had no effect on cell viability.

[0160] As Figure 13 shown, consistent with the animal studies, after incubating HepG2 cells in each group for 48 h, TG and TC in the model group cells were significantly higher than those in the blank group, indicating that the lipid deposition model was successfully established. And under the concentration conditions of Phy-2.5 and Phy-1, both could significantly reduce TG and TC in the hyperlipidemic model cells. Oil red O staining showed that after treatment with high doses of Phy-2.5 and Phy-1 concentrations, compared with the model group, the number of red cells in the visual field decreased, indicating that exogenous supplementation of phytosphingosine effectively reduced lipid deposition.

[0161] As Figure 14 shown, in this example, through an insulin-induced HepG2 cell insulin resistance model, to explore the effect of phytosphingosine in vitro intervention on glycolipid metabolism disorders. In this example, different concentration gradients of phytosphingosine (Phy-0.01, Phy-0.05, Phy-0.1, Phy-0.25, Phy-0.5, Phy-1, Phy-2.5, Phy-5) were set to observe cell viability in order to screen for safe and effective drug doses. After incubating HepG2 cells in each group for 48 h, compared with the model group, the cell viability of the Phy-5 group was significantly decreased (p < 0.01). In subsequent experiments, the phytosphingosine concentrations used were the maximum concentrations of 2.5 μM and 1 μM that had no effect on cell viability.

[0162] As Figure 15As shown, after incubation for 48 hours, the glucose consumption of HepG2 cells in each group was significantly lower in the model group than in the blank group, indicating successful establishment of the insulin resistance model. Under the concentration conditions of Phy-2.5 and Phy-1, the glucose consumption of model cells was significantly increased, indicating that exogenous supplementation of phytosphingosine effectively alleviated insulin resistance.

[0163] Based on the above data analysis, phytosphingosine in the supplement improves insulin sensitivity in patients with metabolic disorders and reduces blood glucose, blood lipid, and blood uric acid levels by regulating glucose metabolism, lipid metabolism, and uric acid metabolism.

[0164] The above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

[0165] Although the specific implementation manners of the present application have been described above in conjunction with the accompanying drawings, it is not a limitation on the protection scope of the present application. Those skilled in the art should understand that various modifications or deformations that can be made without creative efforts on the basis of the technical solution of the present application are still within the protection scope of the present application.

Claims

1. Application of phytosphingosine in a method for early screening of metabolic disorders and identification of human constitution, characterized in that: Phytosphingosine is used as a metabolic marker in the early and pre-onset screening of metabolic disorders, including obesity, hyperlipidemia, hypertension, hyperuricemia, and non-alcoholic fatty liver disease. Human body constitutions include balanced constitution, phlegm-damp constitution, yang deficiency constitution, yin deficiency constitution, qi deficiency constitution, damp-heat constitution, blood stasis constitution, special constitution, and qi stagnation constitution. Phytosphingosine is used as a metabolic marker in the identification of human phlegm-damp constitution.

2. The use of supplements in the intervention of phlegm-damp constitution and the treatment of metabolic disorders, characterized by: The supplement contains phytosphingosine. Phlegm-dampness constitution is one of the nine body constitutions in the human body. Metabolic disorders include obesity, diabetes, hyperlipidemia, hyperuricemia, and non-alcoholic fatty liver disease.

3. The use according to claim 1 or 2, characterized in that: The number of methylene groups of the phytosphingosine is 16-20.

4. The use according to claim 2, characterized in that: The phytosphingosine in the supplement improves the insulin sensitivity of patients with metabolic disorders and reduces the levels of blood sugar, blood lipids and blood uric acid by regulating sugar metabolism, lipid metabolism and uric acid metabolism.

5. The use according to claim 4, characterized in that: The supplement is in pharmaceutically acceptable dosage forms, including tablets, capsules, pills, granules, injections, and oral liquid dosage forms.

6. The use according to claim 4, characterized in that: The phytosphingosine in the supplement activates the PPAR signaling pathway, increasing the uptake and utilization of glucose by cells, thereby reducing fasting blood sugar and postprandial blood sugar levels and improving insulin resistance.

7. The use according to claim 4, characterized in that: The phytosphingosine in the supplement activates the PPAR signaling pathway, reduces the synthesis of cholesterol in the liver, and increases the production and utilization of high-density lipoprotein by the liver and tissues, thereby reducing the levels of low-density lipoprotein, triglycerides and total cholesterol, thereby improving blood lipid disorders.

8. The use according to claim 4, characterized in that: The phytosphingosine in the supplement improves liver function abnormalities caused by metabolic disorders by reducing fat deposits in the liver and adipose tissue, lowering triglyceride and total cholesterol levels; the phytosphingosine in the supplement can reduce the weight and amount of adipose tissue in the body, reduce lipid deposition in liver fat cells, reduce liver fat deposition, and reduce the risk of obesity and related metabolic complications.

9. The use according to claim 4, characterized in that: The phytosphingosine in the supplement can significantly reduce serum uric acid levels and reduce the deposition of uric acid in the kidneys and joints, thereby preventing and treating hyperuricemia and gout.

10. The use according to claim 4, characterized in that: The supplemental dose of phytosphingosine is 5mg / kg-50mg / kg.

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