Traditional Chinese medicine compound and application of active monomer components of traditional Chinese medicine compound in improvement of heat loss

Abstract

The invention provides a traditional Chinese medicine compound for improving heat loss on the first aspect, the traditional Chinese medicine compound is prepared from cinnamon, ginseng, bighead atractylodes rhizome, honey-fried licorice root and dried ginger, the invention discloses a preparation method of the traditional Chinese medicine compound, and four potential compounds with the heat loss improving effect are screened out from blood entering components in the compound through molecular docking; experiments prove that the traditional Chinese medicine compound and the application of main active ingredients entering blood of the traditional Chinese medicine compound in the aspects of promoting body temperature rising and lipid metabolism, and the traditional Chinese medicine compound can be used for preparing high-efficiency and low-toxicity medicines for promoting body rewarming and can be applied to treatment of metabolic diseases at the same time. The invention provides a traditional Chinese medicine compound and active monomer component for promoting body rewarming and improving body cold resistance for workers in cold regions, and is expected to be developed into a high-efficiency and low-toxicity medicine for preventing and treating body heat loss.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals and relates to the application of a traditional Chinese medicine compound with the effect of improving hypothermia of the body. Background Art

[0002] China's land border stretches for thousands of miles, and alpine regions are widely distributed. The decline in production and living capabilities caused by extreme low-temperature environments has constituted a major public health problem, seriously restricting the living quality of residents in cold regions and social development. Under low-temperature conditions, the hypothermia situation of residents in cold regions is serious and has been considered the key to causing multi-system complications such as cardiovascular, skin, and endocrine abnormalities. Therefore, developing specific drugs that can promote body temperature recovery, effectively reduce the incidence of hypothermia, and enhance the body's heat production level has become a core proposition that urgently needs to be overcome in the field of environmental medicine in cold regions. Summary of the Invention

[0003] The purpose of the present invention is to provide the uses of a traditional Chinese medicine compound and its main blood-active components in improving hypothermia and regulating fat metabolism. Four potential compounds with the effect of improving hypothermia are screened out from the blood components of the compound through molecular docking; and the uses of the traditional Chinese medicine compound and its main blood-active components in promoting body temperature recovery and lipid metabolism are proved through experiments.

[0004] To achieve the above purpose, the present invention takes the following technical solutions: In the first aspect of the present invention, a traditional Chinese medicine compound for improving hypothermia is proposed, which is made from cinnamon, ginseng, atractylodes macrocephala, roasted licorice root, and dried ginger.

[0005] Preferably, the weight parts of each component in the compound are as follows: cinnamon 9 - 12 parts; roasted licorice root 9 - 12 parts; atractylodes macrocephala 7 - 9 parts; ginseng 7 - 9 parts; dried ginger 7 - 9 parts.

[0006] The present invention also proposes a preparation method of the above traditional Chinese medicine compound, and the specific steps are as follows: Step 1: Take peeled cinnamon, ginseng, atractylodes macrocephala, roasted licorice root, and dried ginger, and add ethanol with a volume 8 times that of the raw materials to soak at room temperature; Step 2: Heat the soaked medicinal materials together with ethanol, boil, reflux and extract, and then recover the filtrate; add ethanol with a volume 8 times that of the raw materials to the medicinal residues again, heat to boil, reflux and extract, and recover the filtrate again; after several times of reflux extraction, combine the filtrates recovered from each reflux extraction to obtain a medicinal liquid; Step 3: Concentrate the medicinal liquid into an extract to obtain the traditional Chinese medicine compound.

[0007] Preferably, the ethanol in Step 1 and Step 2 is an absolute ethanol solution, the number of times of reflux extraction in Step 2 is 3 times, and the time of each reflux extraction is 2 hours.

[0008] In the second aspect of the present invention, a method for analyzing the active monomer components of a traditional Chinese medicine compound entering the blood is proposed. Four compounds that bind to the key proteins of brown adipose tissue thermogenesis screened by HPLC-Q / TOF-MS and molecular docking are potential compounds for improving hypothermia in the body, namely: 3-Methylcinnamic acid (3-Methyl CA), 8-Gingerol, Cinnamic acid, and Ginsenoside Rd; The structural formulas are respectively: (1): 3-Methylcinnamic acid (3-Methyl CA) (2): 8-Gingerol (3): Cinnamic acid (4): Ginsenoside Rd The specific analysis process is as follows: Step 1: Analyze the components of the traditional Chinese medicine compound entering the blood by HPLC-Q / TOF-MS to obtain the total ion current chromatogram of the full scan of the positive and negative ion time-of-flight mass spectra of the traditional Chinese medicine compound; Step 2: Upload the targets related to hypothermia obtained from the previous transcriptomics (i.e., the gene transcriptomics of brown adipose tissue, specifically the composition and transcriptional regulation changes of RNA in brown adipose tissue) to the STRING11.5 database, construct a PPI network, and use Cytoscape to analyze the PPI network to obtain the top five Hubba nodes; Step 3: Perform molecular docking of the monomer components obtained by HPLC-Q / TOF-MS analysis with the top five targets; Step 4: According to the content of each monomer compound in the monomer components obtained by HPLC-Q / TOF-MS analysis in Step 1, obtain the main active monomer components of the traditional Chinese medicine compound entering the blood.

[0009] In the third aspect of the present invention, the above traditional Chinese medicine compound and its active monomer components are proposed for use in the preparation of drugs for regulating fat metabolism, improving hypothermia, promoting body temperature recovery, improving energy metabolism, preventing and treating metabolic syndrome, and the effects of changing lipid droplet consumption, mitochondrial membrane potential level and reactive oxygen species generation, and enhancing the expression of key thermogenic proteins in brown adipocytes and the thermogenic efficiency of induced brown adipocytes are verified by experiments; Among them, hypothermia refers to the imbalance between heat production and heat dissipation; common inducing factors include exposure to low-temperature environments, wet and cold clothing, physical exhaustion, etc.; the application of regulating fat metabolism includes promoting cholesterol and triglyceride metabolism, and activating brown adipose tissue in the scapular region; the metabolic syndrome includes hyperlipidemia, obesity, and type II diabetes caused by metabolic problems.

[0010] Beneficial effects: The present invention provides a traditional Chinese medicine compound and its active ingredients with the effects of improving hypothermia and regulating fat metabolism, which can be developed into a new type of drug for better promoting body rewarming and increasing lipid metabolism, alleviating the problems of the attenuation of work ability and severe hypothermia of personnel in cold regions under low-temperature conditions. Description of the drawings

[0011] Figure 1 shows the effect of traditional Chinese medicine compounds at different concentrations on lipid droplet consumption in brown adipocytes; Figure 2 shows the effect of traditional Chinese medicine compounds at different concentrations on the change of mitochondrial membrane potential in brown adipocytes; Figure 3 shows the effect of traditional Chinese medicine compounds at different concentrations on the ROS level in brown adipocytes; Figure 4 shows the effect of traditional Chinese medicine compounds at different concentrations on the expression of UCP1 protein in brown adipocytes; Figure 5 shows the effect of traditional Chinese medicine compounds at different concentrations on the expression of CPT1 protein in brown adipocytes; Figure 6 shows the effect of traditional Chinese medicine compounds at different concentrations on the expression of PGC1 protein in brown adipocytes; Figure 7 shows the effect of traditional Chinese medicine compounds at different doses on the rectal temperature of C57BL / 6J mice in a -20°C cold environment; Figure 8 shows the effect of traditional Chinese medicine compounds at different doses on the body surface temperature of C57BL / 6J mice in a -20°C cold environment; Figure 9 shows the effect of traditional Chinese medicine compounds at different doses on the protein heat production of C57BL / 6J mice in a -20°C cold environment (the left figure is the protein consumption; the right figure is the heat production); Figure 10 shows the effect of traditional Chinese medicine compounds at different doses on the respiratory level and energy metabolism of C57BL / 6J mice in a 4°C cold environment (from top to bottom are the relevant indicators of VO2, VCO2, and EE); Figure 11 shows the effect of traditional Chinese medicine compounds at different doses on the activation of brown adipose tissue in the scapular region of C57BL / 6J mice; Figure 12 shows the effect of traditional Chinese medicine compounds at different doses on the morphology of brown adipose tissue in the scapular region of C57BL / 6J mice; Figure 13 shows the total ion current chromatogram of the positive and negative ion TOF MS full scan of the traditional Chinese medicine compound (the upper figure is the positive ion chromatogram, and the lower figure is the negative ion chromatogram); Figure 14 is the chemical structure diagram of the main active monomer components in the traditional Chinese medicine compound; Figure 15 shows the effects of active components of traditional Chinese medicine compounds at different doses on the lipid droplet consumption of brown adipocytes; Figure 16 shows the effects of 3-Methyl CA and 8-Gingerol on the lipid levels in brown adipocytes (the left and right figures are the TC and TG levels in brown adipocytes respectively); Figure 17 shows the effects of 3-Methyl CA and 8-Gingerol on the changes in mitochondrial membrane potential of brown adipocytes; Figure 18 shows the effects of 3-Methyl CA and 8-Gingerol on the ROS levels in brown adipocytes; Figure 19 shows the effects of 3-Methyl CA and 8-Gingerol on the mitochondrial respiration levels of brown adipocytes. Specific implementation manners

[0012] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0013] The present invention provides the effect of a traditional Chinese medicine compound in improving the hypothermia of the body in a cold environment. Through HPLC-Q / TOF-MS and molecular docking screening, potential compounds (active monomer components) for improving the hypothermia of the body are selected, aiming to develop the traditional Chinese medicine compound and its active monomer components into novel drugs for better promoting body rewarming and increasing lipid metabolism.

[0014] In the present invention, primary pre-brown adipocytes are used to prove through experiments such as a mitochondrial membrane potential assay kit, a lipid detection kit, a mitochondrial oxidative respiration seahorse experiment, and Western Blot that the traditional Chinese medicine compound and its active components may promote the conversion of energy into heat by promoting lipid consumption, and improve the thermogenic capacity of BAT by increasing the expression levels of related thermogenic proteins.

[0015] Example 1: Traditional Chinese medicine compound and its preparation method.

[0016] The traditional Chinese medicine compound is made of the following components in parts by mass: 12 parts of cinnamon; 12 parts of roasted licorice; 9 parts of atractylodes macrocephala; 9 parts of ginseng; 9 parts of dried ginger.

[0017] Preparation method of traditional Chinese medicine compound: Take peeled cinnamon, ginseng, atractylodes macrocephala, roasted licorice, and dried ginger, add ethanol with a volume 8 times that of the medicinal materials, and soak at room temperature for 4 h. The soaked medicinal materials and ethanol are heated together, boiled, and reflux-extracted for 2 h, and then the filtrate is recovered. The medicinal residues are added with ethanol with a volume 8 times that of the medicinal materials again, heated to boiling, reflux-extracted for 2 h, and the filtrate is recovered again. The reflux extraction is carried out three times in total. The filtrates recovered three times are combined, and an appropriate amount of the filtrate is concentrated to a thick and flowing state to obtain the traditional Chinese medicine compound extract.

[0018] Furthermore, the present invention verified through experiments that the above traditional Chinese medicine compound promotes the conversion of energy into heat by promoting lipid consumption, and improves the thermogenic capacity of BAT by increasing the expression level of related thermogenic proteins.

[0019] The specific experimental process is as follows: 1) Instruments and reagents: DMEM medium (Hyclone); fetal bovine serum (Hyclone); Oil Red O staining solution (Shanghai Beyotime Biotechnology Co., Ltd.); mitochondrial membrane potential detection kit (Shanghai Beyotime Biotechnology Co., Ltd.); BCA protein concentration determination kit (Shanghai Beyotime Biotechnology Co., Ltd.); reactive oxygen species (ROS) detection kit (Wuhan Ealrite Biotech Co., Ltd.); CKX31 inverted microscope (Olympus, Philippines); constant temperature CO2 incubator (Thermo, USA); 5810 R bench-top high-speed low-temperature centrifuge (Eppendorf, Germany).

[0020] 2) Cell culture: The preadipocytes obtained by primary culture are grown in DMEM complete medium containing 10% FBS. When approaching confluence (confluence greater than 90%), it is changed to induction differentiation medium I (DMEM complete medium added with 0.5 mmol / L IBMX, 1 μmol / L dexamethasone, 10 μg / mL insulin), which is recorded as day 0 of induction differentiation, and continue to culture. On the 2nd day of induction differentiation, that is, after 48 h, the culture medium is changed to induction differentiation medium II (DMEM complete medium added with 10 μg / mL insulin), and continue to culture. Thereafter, the culture medium is changed to DMEM culture medium once every 2 days, and the cell morphology changes are observed under a microscope. Culture in a 37°C incubator (5% CO2, relative humidity 90%). After the cells are induced to differentiate and mature, subsequent experiments are carried out.

[0021] 3) Oil Red O staining: After the primary pre-brown adipocytes were induced to differentiate and mature, the corresponding concentrations of the Chinese medicine compound-containing serum and CL-316243 were added respectively. After 48 h, the original culture medium was discarded, and 1 mL of 10% neutral formaldehyde solution was added to each well to fix the cells at room temperature for about 1 h. Then, Oil Red O application solution was added to the fixed cells for staining. 1 mL of isopropanol was added to each well to extract Oil Red O, and it was oscillated on a micro-oscillator for 10 min. The OD value (recorded as the absorbance A value) was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay detector. Calculate the lipid droplet consumption rate (lipid droplet consumption rate = 1 - A sample / A control), and the experiment was repeated 3 times. The results showed that compared with the control group, the Chinese medicine compound-containing serum could increase the lipid droplet consumption rate of primary brown adipocytes. The results indicated that the Chinese medicine compound might increase energy consumption by consuming lipid droplets in brown adipose tissue. The specific results are as Figure 1 shown.

[0022] 4) Mitochondrial membrane potential measurement: After the primary pre-brown adipocytes were induced to differentiate and mature, the corresponding concentrations of the Chinese medicine compound-containing serum and CL-316243 were added respectively and incubated for 48 h before the experiment. The specific experimental steps were carried out according to the instructions of the "Mitochondrial Membrane Potential Detection Kit (JC-1)" from Beyotime Biotechnology Co., Ltd. Results: Compared with the control group, the positive drug group of CL-316243 could significantly reduce the mitochondrial membrane potential in primary brown adipocytes, and the Chinese medicine compound decreased the mitochondrial membrane potential in cells in a concentration-dependent manner. The results indicated that the Chinese medicine compound might change the electron transport across the mitochondrial membrane. The specific results are as Figure 2 shown.

[0023] 5) Reactive oxygen species (ROS) level measurement: After the primary pre-brown adipocytes were induced to differentiate and mature, the corresponding concentrations of the Chinese medicine compound-containing serum and CL-316243 were added respectively and incubated for 48 h before the experiment. The specific experimental steps were carried out according to the instructions of the "Reactive Oxygen Species Detection Kit (ROS)" from Wuhan Ealite Biotechnology Co., Ltd. Results: Compared with the control group, the positive drug group of CL-316243 could significantly increase the ROS level in primary brown adipocytes, and the Chinese medicine compound increased the production of ROS in cells in a concentration-dependent manner. The results indicated that the Chinese medicine compound might promote the modification of UCP1 by ROS (such as sulfinylation modification) to activate its thermogenic activity. The specific results are as Figure 3 shown.

[0024] 6) Determination of the contents of thermogenic proteins UCP1, CPT1, and PGC1: After the primary adipocytes were induced to differentiate and mature, the positive drug CL-316243 and the serum containing the traditional Chinese medicine compound at the corresponding concentrations were added. After 48 hours of treatment, the cellular proteins were extracted, and protein quantification was performed using the "BCA Protein Concentration Assay Kit" from Shanghai Beyotime Biotechnology Co., Ltd. The protein concentration was determined according to the instructions. The expression levels of UCP1, PGC1, and CPT1 proteins in the brown adipocytes after drug administration were determined by Western Blot. The results showed that the traditional Chinese medicine compound could increase the protein expression of UCP1, PGC1, and CPT1 in brown adipocytes. The results indicated that the traditional Chinese medicine compound might improve the thermogenic capacity of BAT by increasing the expression levels of the key thermogenic proteins in BAT. The specific results are as Figures 4 - 6 shown.

[0025] The following is a brief description of the experimental phenomena and results in combination with the attached drawings: From Figure 1 the results of Oil Red O staining, it can be seen that compared with the control group, the number of lipid droplets in the serum containing the traditional Chinese medicine compound at different concentrations was significantly less than that in the control group. After 48 h of treatment, the traditional Chinese medicine compound could consume the lipid droplets generated by brown adipocytes in a concentration-dependent manner. The serum containing the traditional Chinese medicine compound at 10% and 20% concentrations (blood was taken 30 min and 60 min after administration) could significantly promote the consumption of lipid droplets, and it was statistically significant.

[0026] From Figure 2 the results, it can be known that the traditional Chinese medicine compound could significantly reduce the mitochondrial membrane potential level of brown adipocytes in a concentration-dependent manner. When UCP1 was activated, it would mediate a large amount of H + to flow from the mitochondrial intermembrane space into the matrix, directly consuming the proton gradient on which the mitochondrial membrane potential depends. Therefore, the higher the activity of UCP1, the more H + flows in, and the lower the mitochondrial membrane potential. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form polymers, emitting strong red fluorescence; when the mitochondrial membrane potential is low, JC-1 cannot aggregate in the mitochondrial matrix, producing green fluorescence. Thus, the change in mitochondrial membrane potential can be detected by the change in fluorescence color. The change in electron transport across the inner mitochondrial membrane is measured by the ratio of red to green fluorescence.

[0027] From Figure 3 the results, it can be known that after 48 hours of administration of the serum containing the traditional Chinese medicine compound to brown adipocytes, the mitochondrial reactive oxygen species level decreased in a concentration-dependent manner, suggesting that the compound activates its thermogenic activity by promoting the modification of UCP1 by ROS (such as sulfinylation modification).

[0028] From Figures 4 - 6It can be seen that, compared with the control group, the traditional Chinese medicine compound can dose-dependently increase the protein levels of UCP1, PGC1, and CPT1 in brown adipocytes, and the serum containing the traditional Chinese medicine compound at a concentration of 20% (blood was collected 60 min after administration) can significantly increase the expression of related proteins. The results indicate that the traditional Chinese medicine compound can increase the thermogenic protein levels in brown adipocytes, such as the expression of UCP1, PGC1, and CPT1. *p < 0.05, **p < 0.01; p < 0.05 indicates a statistically significant difference.

[0029] Furthermore, the present invention verified the improvement of the thermogenic capacity of the above traditional Chinese medicine compound in C57BL / 6J mice under cold exposure conditions, and the specific process is as follows: 1) Instruments and reagents: Mouse rectal thermometer (Beijing Zhongshi Dichuang Technology Development Co., Ltd.); Infrared thermal imager (E5, FLIR, USA); Paraffin slicer (RM2145, Leica, Germany); Constant temperature experimental animal refrigerator (RXZ-0250, Haier Co.); Small animal in vivo optical two-dimensional imaging system (PerkinElmer, USA); Constant temperature and humidity respiratory metabolism system (Ningbo Jiangnan Instrument Co., Ltd., China); Energy metabolism acquisition system (Sable system, USA).

[0030] 2) Grouping of experimental animals and administration method: Forty C57BL / 6J mice were randomly divided into 5 groups according to body weight: blank control group, model control group, traditional Chinese medicine compound groups at doses of 0.3 g / kg, 0.6 g / kg, and 1.2 g / kg (calculated based on the crude drug amount), with 8 mice in each group. The mice were administered by gavage at a volume of 0.1 mL / 10 g, and the blank control group and the model control group were respectively administered an equal volume of 0.5% CMC-Na by gavage for 14 consecutive days.

[0031] 3) Cold exposure experiment: After the mice in each experimental group were continuously administered for 14 d, the mice in each administration group were placed in a constant temperature experimental refrigerator at -20°C. After 60 min, the rectal temperature and body surface temperature of the mice were measured after they were taken out (0 min, 15 min, 30 min, 60 min, 90 min). The results indicate that the traditional Chinese medicine compound can significantly increase the rectal temperature and body surface temperature of mice under cold exposure (-20°C). The specific results are as Figures 7 - 8 shown. # p < 0.05, ## p < 0.005 vs. Model.

[0032] 4) Indirect calorimetry: After continuously administering drugs to the mice in each experimental group for 14 days, the mice after cold exposure (-20 °C) were placed in a metabolic cage, one mouse per cage, with free access to food and water. Urine was continuously collected from the mice for 24 h. After collection, the urine was immediately stored at low temperature until measurement. The urinary nitrogen content in the mouse urine was measured, and the calorific value of protein oxidation was calculated. Protein consumption (g) = urinary nitrogen excretion × 6.25; protein calorific value (KJ) = 18 × protein consumption. The results showed that the traditional Chinese medicine compound could significantly increase the protein calorific value of mice under cold exposure (-20 °C). The specific results are as Figure 9 shown.

[0033] 5) Measurement of respiratory and energy metabolism levels: After continuously administering drugs to the mice in each experimental group for 14 days, the changes in the respiratory and energy metabolism levels of the mice in the cold exposure environment were deeply investigated using a thermostatic and humidostatic metabolism system. During the experiment, the mice were individually placed in a metabolic cage and cold-exposed at 4 °C for 24 h. During this period, the mice could freely ingest food and water. At the same time, an energy metabolism acquisition system was used to continuously monitor and record related indicators such as the mice's VO2, VCO2, and EE at 5-min intervals. The results showed that the traditional Chinese medicine compound could significantly increase the measurement of the respiratory and energy metabolism levels of mice under cold exposure (4 °C). The specific results are as Figure 10 shown.

[0034] 6) Near-infrared imaging of mouse iBAT: After continuously administering drugs to the mice in each experimental group for 14 days, CyHF-8 (0.5 mg / Kg, dissolved in a mixed solution of 45% DMSO and 55% propylene glycol) was administered via tail vein injection. Using a small animal in vivo optical two-dimensional imaging system, fluorescence images (excitation wavelength: 780 nm, emission wavelength: 845 nm) were collected before intravenous injection of CyHF-8 and at 10 min and 30 min after injection. The fluorescence intensity of iBAT was analyzed in Living image software to evaluate its activation state. The results showed that the traditional Chinese medicine compound could significantly activate the brown adipose tissue in the scapular region of mice. The specific results are as Figure 11 shown.

[0035] 7) Morphological changes in adipose tissue of mice: The HE staining method was used to examine the morphological changes in adipose tissue: Tissue samples were fixed, dehydrated, cleared, infiltrated with wax, embedded, sectioned, dewaxed, and stained to obtain HE staining results, and the changes in lipid droplets in adipose tissue were observed. The results showed that the traditional Chinese medicine compound could increase the consumption of lipid droplets in brown adipose tissue and promote lipolysis. The specific results are as Figure 13 shown.

[0036] The experimental phenomena and results are briefly described below in combination with the attached drawings: From Figures 7 - 8, compared with the model group, after 60 minutes of cold exposure at -20°C, the mice in each administration group of the traditional Chinese medicine compound had higher anal temperature and body surface temperature. The results showed that the traditional Chinese medicine compound could maintain the anal temperature and body surface temperature of mice in a cold exposure environment and improve the body temperature recovery ability of mice.

[0037] It can be seen from Figures 9 - 10 that, compared with the model group, the traditional Chinese medicine compound group could increase the body's respiratory level, promote the body's energy metabolism, and increase the body's heat production under cold exposure.

[0038] It can be seen from Figures 11 - 12 that, compared with the model group, the traditional Chinese medicine compound group could promote the activation of brown adipose tissue, and by increasing the consumption of lipid droplets in brown adipose tissue, promote lipolysis, thereby activating heat production and improving hypothermia.

[0039] Example 2: Traditional Chinese medicine compound and its preparation method.

[0040] The traditional Chinese medicine compound is made of the following components by mass: 9 parts of cinnamon; 9 parts of roasted licorice; 7 parts of atractylodes macrocephala; 7 parts of ginseng; 7 parts of dried ginger. In this example, the preparation method of the compound and the experimental verification method are the same as those in Example 1, and will not be elaborated here.

[0041] Example 3: Analysis of the active ingredients of the traditional Chinese medicine compound and the effect of this ingredient on the heat production of brown adipocytes.

[0042] The specific process is as follows: 1) Instruments and reagents: CL - 316243 (Sigma Company, USA, purity > 98%); Cell Mitochondrial Oxidative Respiration Assay Kit (Agilent Seahorse XFP Cell Mito Stress Test Kit, USA); Total Cholesterol and Triglyceride Content Detection Kit (Nanjing Jiancheng Bioengineering Institute); Waters Time-of-Flight Mass Spectrometer (Waters Company, USA).

[0043] 2) Analysis and screening of the components of the traditional Chinese medicine compound entering the blood Analysis was performed using high performance liquid chromatography - tandem high resolution mass spectrometry (HPLC-Q / TOF-MS) to obtain the total ion chromatogram (TIC) of the positive and negative ion full scans of time-of-flight mass spectrometry (TOF MS). In order to predict the interactions between targets and screen the main targets of Shengui Quhan Granules against cold injury, the cold injury-related targets obtained from previous omics were uploaded to the STRING 11.5 database to construct a PPI network. The PPI network was analyzed to obtain the top five Hubba nodes. The compounds obtained from the analysis were docked with the top five targets, and combined with the contents of each monomer compound determined by HPLC-Q / TOF-MS, and finally the main active ingredients were obtained. The specific results are as Figures 13 - 14 shown.

[0044] 2) Determination of lipid metabolism level: Total cholesterol (TC) and triglyceride (TG) are used as substrates for adipose tissue. When brown adipose tissue is activated and UCP1 is upregulated, TC and TG are degraded into free fatty acids as fuels, thereby playing a role in heat production in the body. The levels of TC and TG in brown adipocytes were measured using the "Total Cholesterol Assay Kit (enzymatic method)" and "Triglyceride Assay Kit (enzymatic method)" from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. Results: The main active ingredients in the traditional Chinese medicine compound can dose-dependently reduce the levels of TC and TG in serum. The results indicate that: The main active ingredients in the traditional Chinese medicine compound can reduce the lipid level in brown adipocytes, suggesting that it can play a role in preventing cold and generating heat by increasing lipolysis. The specific results are as Figure 16 shown.

[0045] 3) Determination of mitochondrial oxidative respiration level: The "Agilent Seahorse XFp Cell MitoStress Test Kit" was used to measure the mitochondrial oxidative respiration level of brown adipocytes. Primary pre-brown adipocytes were seeded in the culture plates provided in the kit. After induction of differentiation and maturation, the main monomer components in the traditional Chinese medicine compound and CL-316243 at each corresponding concentration were added, and the experiment was carried out according to the kit instructions after 48 hours. Results: Compared with the control group, the basal and maximal oxidative respiration capacities of the cells in the main monomer component group of the traditional Chinese medicine compound and the positive drug group of CL-316243 were significantly increased. The results indicate that: The main monomer components in the traditional Chinese medicine compound can significantly upregulate the oxygen consumption rate at the basal and maximal levels of the cells, suggesting that it promotes mitochondrial respiration and thus increases energy metabolism. The specific results are as Figure 19 shown.

[0046] The methods for the remaining activity experiments are the same as those for the traditional Chinese medicine compound.

[0047] The experimental phenomena and results are briefly described below in conjunction with the accompanying drawings: From Figure 14 it can be seen that the main active monomer components in the traditional Chinese medicine compound are 3-Methylcinnamic acid (3-MethylCA), 8-Gingerol, Cinnamicacid, and Ginsenoside Rd; From Figure 15 it can be seen that the active monomers in the traditional Chinese medicine compound can significantly promote the lipid droplet consumption of brown adipocytes, and 3-Methyl CA and 8-Gingerol have the best effects; From Figure 16 it can be seen that 3-Methyl CA and 8-Gingerol can significantly reduce the levels of TC and TG in brown adipocytes; From Figures 17 - 18 it can be seen that 3-Methyl CA and 8-Gingerol can significantly reduce the mitochondrial membrane potential and ROS levels; From Figure 19 it can be seen that 3-Methyl CA and 8-Gingerol can significantly increase the mitochondrial respiration level.

[0048] Summary: The traditional Chinese medicine compound can promote the activation of brown adipocytes, increase the expression of thermogenic proteins, and at the same time reduce the mitochondrial membrane potential, uncoupling ATP generation from mitochondrial oxidative respiration, thereby playing a role in improving the body's hypothermia. The traditional Chinese medicine compound can significantly increase the rectal temperature and body surface temperature of mice after cold exposure (-20°C), and significantly improve the respiratory and energy metabolism levels of mice in a cold exposure environment. In addition, the traditional Chinese medicine compound can activate brown adipose tissue, increase the consumption of lipid droplets in brown adipose tissue, promote lipolysis, thereby activating thermogenesis and improving hypothermia. Furthermore, through molecular docking and a series of activity experiments, there are 4 compounds in the traditional Chinese medicine compound that may improve hypothermia, mainly 3-Methylcinnamic acid (3-Methyl CA), 8-Gingerol, Cinnamicacid, and Ginsenoside Rd, and 3-Methyl CA and 8-Gingerol have the best effects.

[0049] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A Chinese medicinal compound for improving hypothermia, characterized in that: The compound is made of cinnamon, ginseng, atractylodes, roasted licorice and dried ginger.

2. A Chinese medicinal compound for improving hypothermia according to claim 1, characterized in that: The weight proportions of the components in the compound are: 9-12 parts of cinnamon bark; 9-12 parts of roasted licorice root; 7-9 parts of atractylodes macrocephala; 7-9 parts of ginseng; and 7-9 parts of dried ginger.

3. A method for preparing a Chinese medicinal compound for improving hypothermia as claimed in claim 1 or 2, characterized in that: The following steps are involved: Step 1: Take peeled cinnamon, ginseng, white atractylodes, licorice, and dried ginger, add 8 times the volume of ethanol and soak at room temperature; Step 2: The soaked medicinal materials are heated with ethanol, boiled, and refluxed for extraction, and then the filtrate is recovered; 8 times the volume of ethanol is added to the medicinal residue again, heated and boiled, refluxed for extraction, and the filtrate is recovered again; after several reflux extractions, the filtrate recovered from each reflux extraction is combined to obtain a medicinal solution; Step 3: Concentrate the medicinal liquid into an extract to obtain a Chinese medicine compound.

4. The preparation method according to claim 3, characterized in that: The ethanol in step 1 and step 2 adopts anhydrous ethanol solution, the number of reflux extraction in step 2 is 3 times, and the time of each reflux extraction is 2 hours.

5. A method for analyzing blood-active monomer components of a traditional Chinese medicine compound as claimed in claim 1, characterized in that: The following steps are involved: Step 1: Analyze the blood components of the Chinese medicine compound by using HPLC-Q / TOF-MS to obtain a total ion flow chromatogram of the full scan of the positive and negative ion time-of-flight mass spectrometry of the Chinese medicine compound; Step 2: Upload the hypothermia-related targets obtained from the previous transcriptomics to the STRING11.5 database, construct a PPI network, and use Cytoscape to analyze the PPI network to obtain the top five Hubba nodes; the previous transcriptomics is the gene transcriptomics of brown adipose tissue, specifically the composition and transcriptional regulation changes of RNA in brown adipose tissue; Step 3: Molecular docking of the monomer components obtained by HPLC-Q / TOF-MS analysis with the top five targets; Step 4: Based on the content of each monomer compound in the monomer component obtained by HPLC-Q / TOF-MS analysis in step 1, the main blood-entering active monomer component of the traditional Chinese medicine compound is obtained.

6. An active monomer obtained by the method of claim 5, characterized in that: The active monomers are composed of the following compounds: （1）： （2） （3） （4） 。 7. Use of the active monomer as claimed in claim 6 in the preparation of drugs for regulating fat metabolism, improving hypothermia, promoting body temperature recovery, improving energy metabolism, and preventing and treating metabolic syndrome.

8. Use of the Chinese medicinal compound according to claim 1 or 2 in the preparation of drugs for regulating fat metabolism, improving hypothermia, promoting body temperature recovery, improving energy metabolism, and preventing and treating metabolic syndrome.

Images