Application of tea tree root extract and camellia glycoside A in preparation of medicine for treating metabolism-related fatty liver disease

Abstract

The invention belongs to the technical field of biological pharmacy, and provides application of a tea tree root extract and camellia glycoside A in preparation of a medicine for treating metabolism-related fatty liver diseases. According to the application, a method for establishing a fatty liver model through induction of HFD high-fat, high-fructose and high-cholesterol feed is adopted; the treatment effect of the tea tree root extract and the camellia glycoside A in the tea tree root extract on the MASLD is evaluated, and the result shows that the tea tree root extract and the camellia glycoside A can achieve the purpose of treating the MASLD by reducing the content of TC and TG in serum, adjusting the content of ALT and AST in liver tissue, improving liver inflammation and adiposis, adjusting hepatocyte ferroptosis and exerting the anti-lipid peroxidation effect.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceutical technology and relates to a tea tree root extract, and in particular to an application of a tea tree root extract and camelliaside A in the preparation of a medicine for treating metabolism-related fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury that affects multiple systemic systems. Its spectrum includes simple non-alcoholic fatty liver disease, non-alcoholic steatohepatitis (NASH), and associated liver fibrosis and cirrhosis, ultimately progressing to hepatocellular carcinoma. Following two name changes in consensus statements jointly developed by global multidisciplinary expert groups in 2020 and 2022, NAFLD has been redefined as the more representative metabolic dysfunction-associated steatotic liver disease (MASLD).

[0003] Epidemiological studies have shown that the prevalence of MASLD has been increasing year by year and has become the leading cause of chronic liver disease worldwide, significantly impacting human health and placing a significant economic burden on public health systems. Notably, the age of MASLD patients is increasing, suggesting that an increasing number of patients may be affected by MASLD and its related complications over the long term. Furthermore, recent studies have shown that even mild fatty liver disease can significantly increase mortality from various factors, and the severity of this risk is closely related to the course of the disease.

[0004] Currently available therapeutic agents fail to provide satisfactory intervention strategies for addressing the significant challenges posed by this major health problem. In recent years, progress has been made in the development of several drugs, including GLP-1 receptor agonists (e.g., semaglutide), farnesoid X receptor agonists (e.g., obeticholic acid), and PPARα / δ dual agonists (e.g., elafibranor, resmetirom, cenicriviroc, and selonsertib). However, in clinical practice, these drugs exhibit varying degrees of limitations, including significant adverse reactions and a single therapeutic approach that fails to fully address the systemic metabolic problems of MASLD. Therefore, exploring innovative treatment options and shifting from single-cause control to multi-mechanism intervention strategies may provide further assistance in the development of therapeutics for MASLD.

[0005] In this context, Traditional Chinese Medicine (TCM), thanks to its systemic and low-toxicity treatment advantages, is gaining increasing attention in the prevention and treatment of MASLD. Some natural herbal medicines and monomeric compounds derived from natural medicines have demonstrated systemic, multi-target therapeutic potential.

[0006] Camellia japonica radix (CJR) is the root of Camellia japonica, also known as oil tea or single-seed oil tea, and is an evergreen shrub or tree in the Theaceae family. The roots, leaves, flowers, and seeds of Camellia japonica can all be used as medicine, with the root having the effects of clearing away heat and detoxifying, promoting blood circulation, and regulating menstruation. Currently, the use of Camellia japonica root is primarily limited to treating arrhythmias, mouth sores, and psoriasis. Research on its use in metabolic diseases such as fatty liver is limited, and the selected drug sources are mostly ordinary tea roots, while the roots of Camellia japonica or Camellia japonica are less commonly used. Therefore, one of the objectives of the present invention is to explore Camellia japonica-derived tea root extracts for their application in the preparation of drugs for metabolic diseases such as fatty liver.

[0007] Camellianoside A (CA) is a natural compound extracted from the Camellia genus and belongs to the flavonoid glycoside class. Currently, the commonly used methods for preparing CA primarily source it from the leaves of Camellia sinensis and Camellia chinensis, resulting in relatively high costs. However, CA is also found in the flowers of Camellia sinensis and the roots and seeds of Camellia oleifera. Therefore, another object of the present invention is to develop a novel method for preparing CA.

[0008] In addition, current studies have shown that camelliaside A can be used to treat acute chemical liver damage induced by carbon tetrachloride. However, research on its effect in metabolic diseases such as fatty liver is also limited. Therefore, another purpose of the present invention is to explore the efficacy of camelliaside A and apply it to the preparation of drugs for metabolic diseases such as fatty liver. Summary of the Invention

[0009] The purpose of the present invention is to provide a use of a tea tree root extract and camelliaside A in the preparation of a drug for treating metabolic-related fatty liver disease, and to use the tea tree root extract and camelliaside A in the preparation of a drug for treating MASLD, thereby providing a new approach and means for treating MASLD.

[0010] In order to achieve the above object, the technical solution of the present invention is as follows:

[0011] The present invention provides an application of a tea tree root extract in preparing a medicine for treating metabolism-related fatty liver disease.

[0012] Preferably, the active ingredients of the tea tree root extract include but are not limited to camelliaside A.

[0013] Preferably, the preparation method of the tea tree root extract is as follows: the roots of Camellia oleifera are used as raw materials, which are crushed and extracted with 60-80% ethanol solution in three extraction devices connected in series at a temperature of 50-60°C, the extraction time of each extraction device is 3-4 hours, the mass volume ratio of Camellia oleifera roots to 60-80% ethanol is 3-5kg:15-25L, the tea tree root ethanol extract is collected, concentrated and dried to constant weight, which is the tea tree root extract.

[0014] The present invention also provides a use of camelliaside A in the preparation of a medicine for treating metabolism-related fatty liver disease.

[0015] Preferably, the preparation method of camelliaside A is as follows: the above-mentioned tea tree root ethanol extract is microfiltered and ultrafiltered in sequence to obtain a permeate, which is passed through an HPD100 macroporous adsorption resin column, first eluted with water until colorless, then eluted with 40% to 60% ethanol, the ethanol eluate is collected, and then the obtained crude product is separated and purified by high-speed countercurrent chromatography to obtain camelliaside A.

[0016] Preferably, the volume ratio of the tea tree root ethanol extract to the column volume of the macroporous adsorption resin column is 24-28:1, and the amount of the 40%-60% ethanol elution is equivalent to 3-5 times the column volume of the macroporous adsorption resin column.

[0017] Preferably, the solvent of the high-speed countercurrent chromatography is: n-hexane-n-butanol-methanol-0.5% acetic acid aqueous solution solvent system, and the ratio of n-hexane, n-butanol, methanol and 0.5% acetic acid aqueous solution is 1-3:3-5:1-3:4-6.

[0018] The present invention also provides a drug for treating metabolism-related fatty liver disease prepared from the above-mentioned tea tree root extract or the above-mentioned camellia glycoside A. The tea tree root extract or camellia glycoside A is used as an active ingredient or in combination with other drugs to prepare the drug for treating metabolism-related fatty liver disease.

[0019] Preferably, the drug is used to reduce the levels of TC and TG in serum to lower blood lipids; the drug is used to reduce the levels of ALT and AST in liver tissue to promote liver function recovery; the drug is used to improve liver inflammation and fatty degeneration; the drug is used to regulate liver cell ferroptosis and exert anti-lipid peroxidation effects.

[0020] Preferably, the drug comprises tea tree root extract or camelliaside A with or without medically permitted excipients, and is prepared into various acceptable dosage forms, such as injection, pill, capsule, granule, tablet or oral solution.

[0021] Beneficial effects of the present invention:

[0022] The present invention adopts a method of inducing a fatty liver model with a high-fat, high-fructose, high-cholesterol diet (HFD) to explore the weight-reducing, lipid-lowering, and anti-inflammatory effects of tea tree root extract and camelliaside A, and evaluates the therapeutic effects of tea tree root extract and camelliaside A on metabolism-related fatty liver disease. The results show that tea tree root extract and camelliaside A can reduce the levels of TC and TG in serum, regulate the levels of ALT and AST in liver tissue, improve liver inflammation and steatosis, regulate hepatocyte ferroptosis, and exert anti-lipid peroxidation effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the base peak ion chromatogram (BPC) of the UPLC-HRMS sample of the tea tree root single prescription in the present invention (A is the negative ion mode, B is the positive ion mode);

[0024] Figure 2 The UPLC ultraviolet chromatogram of the tea tree root single prescription sample in the present invention is UV (300nm);

[0025] Figure 3 Graphs showing the effects of tea root extract on body weight and liver weight in MASLD mice induced by HFD (A is a graph showing changes in body weight of HFD-induced MASLD mice induced by tea root extract, and B is a graph showing changes in liver weight of HFD-induced MASLD mice induced by tea root extract; CON is a normal group, HFD is a model group, LCJR is a low-dose tea root extract treatment group, MCJR is a medium-dose tea root extract treatment group, HCJR is a high-dose tea root extract treatment group, and PPC is a polyene phosphatidylcholine positive control group); *** P < 0.001 compared with the CON group; ** P < 0.01, *** P < 0.001, compared with the HFD group);

[0026] Figure 4 : This is a graph showing the effect of the tea tree root extract on the liver function of HFD-induced MASLD mice (A is a graph showing the effect of the tea tree root extract on the TG content of blood lipids in HFD-induced MASLD mice, and B is a graph showing the effect of the tea tree root extract on the TC content of blood lipids in HFD-induced MASLD mice; wherein CON is a normal group, HFD is a model group, LCJR is a low-dose treatment group of the tea tree root extract, MCJR is a medium-dose treatment group of the tea tree root extract, HCJR is a high-dose treatment group of the tea tree root extract, and PPC is a polyene phosphatidylcholine positive control group; *** P < 0.001, compared with the CON group; * P<0.05, ** P < 0.01, *** P < 0.001, compared with the HFD group);

[0027] Figure 5 Graphs showing the effects of the tea tree root extract on liver function in HFD-induced MASLD mice (A is a graph showing the effects of the tea tree root extract on ALT content in liver tissue of HFD-induced MASLD mice, and B is a graph showing the effects of the tea tree root extract on AST content in liver tissue of HFD-induced MASLD mice; CON is a normal group, HFD is a model group, LCJR is a low-dose tea tree root extract treatment group, MCJR is a medium-dose tea tree root extract treatment group, HCJR is a high-dose tea tree root extract treatment group, and PPC is a polyene phosphatidylcholine positive control group); *** P < 0.001, compared with the CON group; * P<0.05, ** P < 0.01, *** P < 0.001, compared with the HFD group);

[0028] Figure 6 Graph showing the effect of the tea tree root extract of the present invention on liver tissue pathology in HFD-induced MASLD mice (the graph shows HE cell staining results and Oil Red O staining results (200×); wherein, CON is the normal group, HFD is the model group, LCJR is the low-dose tea tree root extract treatment group, MCJR is the medium-dose tea tree root extract treatment group, HCJR is the high-dose tea tree root extract treatment group, and PPC is the polyene phosphatidylcholine positive control group);

[0029] Figure 7 The tea tree root extract of the present invention has an effect on the iron death markers in the liver tissue of HFD-induced MASLD mice. (A is a graph showing the effect of tea tree root extract on the malondialdehyde content in the liver tissue of HFD-induced MASLD mice, and B is a graph showing the effect of tea tree root extract on the Fe 2+ Content diagram; CON is the normal group, HFD is the model group, LCJR is the low-dose treatment group of tea tree root extract, MCJR is the medium-dose treatment group of tea tree root extract, and HCJR is the high-dose treatment group of tea tree root extract; *** P < 0.001, compared with the CON group; * P<0.05, ** P < 0.01, *** P < 0.001, compared with the HFD group);

[0030] Figure 8: This is a graph showing the effect of the tea tree root extract on the lipid peroxidation signaling pathway in the liver tissue of HFD-induced MASLD mice (A is a graph showing the detection results of the tea tree root extract on the NOX1 and GPX4 proteins in HFD-induced MASLD mice, B is a graph showing the relative expression of the GPX4 protein, and C is a graph showing the relative expression of the NOX1 protein; wherein CON is the normal group, HFD is the model group, LCJR is the low-dose treatment group of the tea tree root extract, MCJR is the medium-dose treatment group of the tea tree root extract, and HCJR is the high-dose treatment group of the tea tree root extract; *** P < 0.001, compared with the CON group; ** P < 0.01, *** P < 0.001, compared with the HFD group);

[0031] Figure 9 The present invention is a graph showing the effect of camelliaside A on the body weight and liver weight of HFD-induced MASLD mice (A is a graph showing the changes in body weight of HFD-induced MASLD mice by camelliaside A, and B is a graph showing the changes in liver weight of HFD-induced MASLD mice by camelliaside A; wherein CON is a normal group, HFD is a model group, LCA is a low-dose camelliaside A treatment group, MCA is a medium-dose camelliaside A treatment group, HCA is a high-dose camelliaside A treatment group, PPC is a polyene phosphatidylcholine positive control group, and PPC is a polyene phosphatidylcholine positive control group; *** P < 0.001, compared with the CON group; * P < 0.01, ** P < 0.01, compared with the HFD group);

[0032] Figure 10 The present invention is a graph showing the effect of camelliaside A on blood lipids in HFD-induced MASLD mice (A is a graph showing the effect of camelliaside A on TG content in liver tissue of HFD-induced MASLD mice, and B is a graph showing the effect of camelliaside A on TC content in liver tissue of HFD-induced MASLD mice; wherein CON is a normal group, HFD is a model group, LCA is a low-dose camelliaside A treatment group, MCA is a medium-dose camelliaside A treatment group, HCA is a high-dose camelliaside A treatment group, and PPC is a polyene phosphatidylcholine positive control group; *** P < 0.001, compared with the CON group; * P<0.05, ** P < 0.01, *** P < 0.001, compared with the HFD group);

[0033] Figure 11The present invention is a graph showing the effect of camelliaside A on liver function in HFD-induced MASLD mice (A is a graph showing the effect of camelliaside A on ALT content in liver tissue of HFD-induced MASLD mice, and B is a graph showing the effect of camelliaside A on AST content in liver tissue of HFD-induced MASLD mice; wherein CON is a normal group, HFD is a model group, LCA is a low-dose camelliaside A treatment group, MCA is a medium-dose camelliaside A treatment group, HCA is a high-dose camelliaside A treatment group, and PPC is a polyene phosphatidylcholine positive control group; *** P < 0.001, compared with the CON group; * P<0.05, ** P < 0.01, *** P < 0.001, compared with the HFD group);

[0034] Figure 12 This is a graph showing the effect of camelliaside A on liver tissue pathology in HFD-induced MASLD mice of the present invention (the graph shows HE cell staining results and Oil Red O staining results (200×); wherein CON is the normal group, HFD is the model group, LCA is the camelliaside A low-dose treatment group, MCA is the camelliaside A medium-dose treatment group, HCA is the camelliaside A high-dose treatment group, and PPC is the polyene phosphatidylcholine positive control group);

[0035] Figure 13 The present invention is the effect of camelliaside A on the iron death markers in the liver tissue of HFD-induced MASLD mice (A is the malondialdehyde content in the liver tissue of camelliaside A-induced MASLD mice, B is the Fe 2+ Content diagram; CON is the normal group, HFD is the model group, LCA is the camelliaside A low-dose treatment group, MCA is the camelliaside A medium-dose treatment group, and HCA is the camelliaside A high-dose treatment group; *** P < 0.001, compared with the CON group; * P<0.05, ** P < 0.01, *** P < 0.001, compared with the HFD group);

[0036] Figure 14 The present invention is a graph showing the effect of camelliaside A on the lipid peroxidation signaling pathway in the liver tissue of HFD-induced MASLD mice (A is a graph showing the detection results of camelliaside A on NOX1 and GPX4 proteins in HFD-induced MASLD mice, B is a graph showing the relative expression of GPX4 protein, and C is a graph showing the relative expression of NOX1 protein; wherein CON is the normal group, HFD is the model group, LCA is the low-dose camelliaside A treatment group, MCA is the medium-dose camelliaside A treatment group, and HCA is the high-dose camelliaside A treatment group;*** P < 0.001, compared with the CON group; * P<0.05, ** P < 0.01, *** P < 0.001, compared with the HFD group). DETAILED DESCRIPTION

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0040] It should be noted that the extraction rate calculation formula of the embodiment of the present invention is: (mass of dry paste / mass of raw materials)×100%.

[0041] Example 1 Preparation method of tea tree root extract

[0042] 3 kg of Camellia oleifera root was crushed and placed in three series-connected dynamic continuous countercurrent extraction devices. 15 L of 60% (food-grade) ethanol solution was added. Extraction was performed at 50°C for 3 hours in each device, yielding 10.2 L of ethanol extract. The extract was dried in a vacuum oven to constant weight, yielding 762.9 g of dry extract with an extraction yield of 25.43%.

[0043] Example 2 Preparation method of tea tree root extract 2

[0044] 4 kg of Camellia oleifera root was crushed and placed in three series-connected dynamic continuous countercurrent extraction devices. 20 L of 70% ethanol (food-grade) was added. Extraction was performed at 55°C for 3.5 hours in each device, yielding 13.8 L of ethanol extract. The extract was dried in a vacuum oven to constant weight, yielding 1030.8 g of dry extract, representing an extraction yield of 25.77%.

[0045] Example 3 Preparation method of tea tree root extract

[0046] 5 kg of Camellia oleifera root was crushed and placed in three series-connected dynamic continuous countercurrent extraction devices. 25 L of 80% ethanol (food-grade) was added. Extraction was performed at 60°C for 4 hours in each device, yielding 16.1 L of ethanolic extract. The extract was dried in a vacuum oven to constant weight, yielding 1290.5 g of dry extract, representing an extraction yield of 25.81%.

[0047] Example 4 Preparation method of camelliaside A 1

[0048] 12.6 L of tea tree root ethanol extract (taken from the tea tree root ethanol extract preparation method in Example 2) was taken and microfiltered and ultrafiltered in a spiral membrane separation device in sequence to obtain a permeate, which was adsorbed on an HPD100 macroporous adsorption resin column with a column volume of 0.5 L. The product was first eluted with water until colorless and then eluted with 2 L of 40% ethanol. The ethanol eluate was collected and separated and purified by high-speed countercurrent chromatography. A solvent system of n-hexane-n-butanol-methanol-0.5% acetic acid aqueous solution was prepared in a volume ratio of 1:3:1:4. The main engine speed was set to 800 r / min. After dynamic equilibrium, the mobile phase flow rate was set to 1.5 ml / min. The obtained sample was dissolved in the lower phase and injected through the injection valve. HPLC detection was performed (Shanghai Shidande Biotechnology Co., Ltd.). The target component was collected and the dried fraction was concentrated under reduced pressure to obtain 4.58 g of camelliaside A.

[0049] Example 5 Preparation method 2 of Camelliaside A

[0050] 13.3 L of tea tree root ethanol extract (taken from the tea tree root ethanol extract preparation method in Example 2) was taken and microfiltered and ultrafiltered in a spiral membrane separation device in sequence to obtain a permeate, which was adsorbed on an HPD100 macroporous adsorption resin column with a column volume of 0.5 L. The solution was first eluted with water until colorless and then eluted with 1.5 L of 50% ethanol. The ethanol eluate was collected and separated and purified by high-speed countercurrent chromatography. A solvent system of n-hexane-n-butanol-methanol-0.5% acetic acid aqueous solution was prepared in a volume ratio of 2:5:2:4. The main engine speed was set to 850 r / min. After dynamic equilibrium, the mobile phase flow rate was set to 2.0 ml / min. The obtained sample was dissolved in the lower phase and injected through the injection valve. HPLC detection was performed, the target component was collected, and the dried fraction was concentrated under reduced pressure to obtain 4.72 g of camelliaside A.

[0051] Example 6 Preparation Method 3 of Camelliaside A

[0052] 13.5 L of tea tree root ethanol extract (taken from the tea tree root ethanol extract preparation method in Example 2) was taken and microfiltered and ultrafiltered in a spiral membrane separation device in sequence to obtain a permeate, which was adsorbed on an HPD100 macroporous adsorption resin column with a column volume of 0.5 L. The product was first eluted with water until colorless and then eluted with 2.5 L of 60% ethanol. The ethanol eluate was collected and separated and purified by high-speed countercurrent chromatography. A solvent system of n-hexane-n-butanol-methanol-0.5% acetic acid aqueous solution was prepared in a volume ratio of 3:5:3:6. The main engine speed was set to 900 r / min. After dynamic equilibrium, the mobile phase flow rate was set to 2.5 ml / min. The obtained sample was dissolved in the lower phase and injected through the injection valve. HPLC detection was performed, the target component was collected, and the dried fraction was concentrated under reduced pressure to obtain 5.04 g of camelliaside A.

[0053] The following method was used to detect the components of tea tree root extract:

[0054] The tea tree root ethanol extracts obtained in the previous Examples 1-3 were mixed, and the tea tree root extract samples were analyzed using ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS).

[0055] The analysis results of tea tree root extract components are shown in Table 1 and Figure 1 、 Figure 2 .

[0056] Table 1 Identification of the main components of tea tree root extract

[0057]

[0058]

[0059]

[0060] Based on the sample multi-level mass spectrometry information combined with the natural product high-resolution mass spectrometry database and related literature, 33 compounds were identified from the tea tree root extract sample.

[0061] Application Example 1 Effect of Tea Tree Root Extract on Liver Tissue of HFD-Induced MASLD Mice

[0062] A fatty liver model was established by inducing a high-fat, high-fructose, high-cholesterol diet using an HFD to verify the therapeutic effect of the tea tree root ethanol extract mixture prepared in Examples 1-3 on MASLD.

[0063] 1 Experimental methods

[0064] 1.1 Modeling and grouping

[0065] Male C57BL / 6 mice, 6-8 weeks old, were housed in a SPF-adapted environment for one week. Mice were weighed, their toes clipped, and numbered according to a random number table. The mice were divided into six groups, each consisting of eight mice. The control group (CON) was fed a standard control diet, while the model group (HFD) and all treatment groups were fed a high-fat, high-fructose, high-cholesterol diet (Synergy Biotech, XT310). The mice were housed for 16 weeks with free access to the diet. The treatment groups were given a low-, medium-, and high-dose (LCJR, MCJR, and HCJR) tea root extract (0.975, 1.95, and 3.9 g / kg / day, calculated based on the human-animal body surface area conversion ratio, according to the Methodology of Pharmacological Research in Traditional Chinese Medicine) by oral gavage from the start of modeling. Polyene phosphatidylcholine (PPC, 178 mg / kg / day, human equivalent) was also administered from the beginning of modeling.

[0066] 1.2TG and TC testing

[0067] Detected by automatic biochemical analyzer.

[0068] 1.3ALT and AST testing

[0069] Detected by automatic biochemical analyzer.

[0070] 1.4 Malondialdehyde detection

[0071] Mouse liver tissue was homogenized and lysed in PBS buffer at a tissue-to-reagent ratio of 1:9 (g / mL). After homogenization, the mixture was centrifuged at 10,000 g for 10-15 min at 4°C. The supernatant was used for subsequent MDA assays. 5 mL of ultrapure water was added to the MDA assay probe vial and thoroughly dissolved under boiling conditions. While still hot, 5 mL of glacial acetic acid was added and mixed to prepare the MDA probe working solution. A 200 μM MDA standard was serially diluted in PBS or ultrapure water for standard curve analysis. 20 μL of the MDA standard / PBS / test sample solution and 800 μL of the MDA working solution were thoroughly mixed and incubated at 95°C for 40 min. After the incubation period, the mixture was immediately placed on ice for 5 min. Following the ice bath, the mixture was centrifuged at 10,000 g for 10 min. 200 μL of the supernatant was transferred to a clear 96-well plate and the absorbance at 532 nm was measured using a microplate reader.

[0072] 1.5 Ferrous ion detection

[0073] Mouse liver tissue was homogenized in PBS buffer at a tissue-to-reagent ratio of 1:9 (g / mL). After homogenization, the solution was centrifuged at 10,000 g for 10-15 min at 4°C, and the supernatant was collected for analysis. Accurately weigh 27.8 mg of the standard solution and dissolve it in 10 mL of ultrapure or double-distilled water to obtain a 10 mM standard solution. This solution was serially diluted to concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 μmol / L for standard curve analysis. 50 μL of the water / standard / test sample solution was mixed with 150 μL of assay buffer and 100 μL of colorimetric solution. The solution was thoroughly mixed and incubated at 37°C for 40 min. After centrifugation at 10,000 g for 5 min, 200 μL of the supernatant was transferred to a clear 96-well plate and the absorbance at 593 nm was measured using a microplate reader.

[0074] 1.6 UPLC-Q-TOF / MS detection

[0075] Chromatographic separation was performed using a Waters ACQUITY UPLC HSS T3 column at 30°C at a flow rate of 0.3 mL / min. The mobile phase consisted of acetonitrile (a) and 0.1% formic acid in water (B) with the following gradient program: 0-4 min, 0-100% B; 4-8 min, 0-6% A, 100-94% B; 8-40 min, 6-28% A, 94-72% B; 40-50 min, 28-50% A, 72-50% B; 50-55 min, 50-95% A, 50-5% B; 55-58 min, 95% A, 5% B; 58.1-61 min, 0% A, 100% B. The injection volume was 2 μL, and the detection wavelength range was 190 to 400 nm, with a dominant wavelength of 254 nm. Analyzers were run on an AB Sciex Triple Mass spectrometry analysis was performed on a 4600 instrument in positive and negative ion modes. Instrument parameters included: TOF mass range 50–1700 m / z, MS / MS mass range 50–1250 m / z, nebulizer gas pressure 50 psi, auxiliary heater gas pressure 50 psi, curtain gas pressure 35 psi, ionization voltage -4500 / 5000 V, source temperature 500°C, declustering voltage 100 V, collision energy ±40 eV, energy spread 20 eV, ion release delay 30 msec, and beam width 15 msec. Compound identification was based on comparison with literature data and the Sundia Natural Products High-Resolution Mass Spectral Database.

[0076] 1.7 Protein content determination

[0077] Mouse livers were assayed by Western blot to determine the relative expression of NOX1 and GPX4. Beta-actin was used as an internal control. NOX1 (DF8684, Affinity), GPX4 (DF6701, Affinity), and beta-actin (abs171598, Absin) were purchased from commercial suppliers.

[0078] 1.8HE staining

[0079] Liver tissue samples were removed from the formalin fixative and rinsed continuously with running water for 4 hours to remove residual fixative and impurities. The tissue samples were then dehydrated in an automatic dehydrator. After dehydration, they were embedded in paraffin in an embedding machine to create wax blocks. The wax blocks were sliced ​​into 5-μm sections using a microtome and mounted on glass slides. The dried sections were immersed in xylene for 10 minutes, repeated once, to completely remove the paraffin. Next, they were immersed in anhydrous ethanol for 5 minutes, then again for 2 minutes, followed by immersion in 95%, 90%, 80%, and 70% ethanol for 2 minutes each. Finally, they were rinsed with distilled water and then washed three times with PBS for 5 minutes each to ensure full hydration and remove impurities. The sections were stained with hematoxylin and eosin according to conventional methods. The stained sections were immersed in 95% ethanol for 5 minutes, then in anhydrous ethanol for 5 minutes, and finally in xylene for 5 minutes to completely dehydrate the sections. After the slices were dried, neutral resin was added to seal the slices and photographed.

[0080] 1.9 Oil Red O staining

[0081] Pre-cool the cryostat to ensure the machine temperature remains stable at an appropriate low temperature. Remove the frozen tissue sample and, after the sample temperature equilibrates, thaw it on ice in preparation for subsequent sectioning. Adhere the thawed tissue sample to the cryostat with OCT glue. Place the sample-attached slide holder on the pre-cooled cryostat. Once the OCT glue has solidified, section the sample at an 8μm thickness. Gently adhere the section to a pre-labeled slide. Remove the frozen section and rinse it in a container of 60% isopropyl alcohol for 20 seconds to remove surface impurities and residual OCT glue. Mix Oil Red O dye and diluent in a 5:2 ratio and filter twice through filter paper to ensure the stain is uniform and free of impurities. Stain the sections with Oil Red O according to conventional methods. Then, rinse the sections with double-distilled water preheated to 37°C to ensure a clean surface. Counterstain the sections with hematoxylin (counter-stain) for 4 minutes to stain the cell nuclei. The slides were mounted with an aqueous mounting medium preheated to a liquid state at 60°C and photographed.

[0082] 1.10 Data Analysis

[0083] GraphPad Prism 7.0 software was used to process the data, and the data are expressed as mean ± standard deviation. One-way analysis of variance was used to compare the indicators between groups, and repeated measurement data were compared using repeated measurement analysis of variance. P < 0.05 indicated a significant difference.

[0084] 2 Experimental results

[0085] 2.1 Identification of ingredients in tea tree root extract

[0086] The tea tree root extract samples were analyzed by ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-Q-TOF / MS). According to the multi-level mass spectrometry information of the samples combined with the natural product high-resolution mass spectrometry database and related literature, 33 compounds ( Figure 1 、 Figure 2 , Table 1).

[0087] Effects of tea tree root extract on body weight and liver weight in HFD-induced MASLD mice

[0088] During the modeling period, the mice were in good spirits, flexible in movement, and ate and drank as usual. The weight test of the mice showed that at the beginning of the experiment, there was no significant difference in the weight of the mice in each group (P>0.05). During the modeling treatment, the weight of the mice in each group fed with a high-fat, high-fructose, and high-cholesterol diet increased rapidly, among which the weight of the mice in the HFD group increased the fastest. By the end of the experiment, the weight of the mice in the CON group was basically stable, while the weight of the mice in the HFD group continued to increase, and the difference was statistically significant compared with the CON group (P<0.05). The weight of the mice in each treatment group also continued to increase, but the increase was significantly lower than that in the HFD group. Among them, the specific treatment effect was HCJR>MCJR>LCJR>PPC( Figure 3 A). At the end of the experiment, the livers of mice were weighed. Compared with the CON group, the liver weight of the HFD group was significantly increased (P < 0.05); compared with the HFD group, the liver weight of mice in each treatment group was significantly decreased, and the differences were statistically significant (P < 0.05). Among them, the specific treatment effect was HCJR > MCJR > PPC > LCJR ( Figure 3 B) These results indicate that tea tree root extract can inhibit body weight gain and reduce liver weight in HFD mice.

[0089] Effects of tea tree root extract on blood lipids in HFD-induced MASLD mice

[0090] The detection of TG and TC levels in the serum of mice showed that compared with the CON group, the serum TC and TG levels of mice in the HFD group were significantly increased (P < 0.05); compared with the HFD group, the TC and TG levels of mice in each treatment group were significantly decreased, and the differences were statistically significant (P < 0.05) ( Figure 4 ). Among them, the specific treatment effect of TG is HCJR>MCJR>PPC>LCJR( Figure 4 A), the specific treatment effect of TC HCJR>MCJR>PPC>LCJR( Figure 4 B) This result indicates that tea tree root extract can reduce the serum TG and TC levels in HFD-induced MASLD mice and improve the serum lipid profile of mice.

[0091] Effects of tea tree root extract on liver function in HFD-induced MASLD mice

[0092] ALT and AST are sensitive indicators of liver damage and are called liver tests or liver blood tests. The results of the mouse serum test showed that compared with the CON group, the serum ALT and AST levels of the mice in the HFD group were significantly increased (P < 0.05); and compared with the HFD group, the ALT and AST levels of the mice in each treatment group were significantly decreased, and the differences were statistically significant (P < 0.05). Figure 5 Among them, the specific treatment effect of ALT is HCJR>MCJR>PPC>LCJR( Figure 5 A), the specific treatment effect of AST HCJR>MCJR>PPC>LCJR( Figure 5 B) This result indicates that tea tree root extract can reduce serum ALT and AST levels and repair liver damage.

[0093] Effects of tea tree root extract on liver histopathology in HFD-induced MASLD mice

[0094] The results of HE and Oil Red O staining of the mouse liver cells showed that compared with the CON group, the HFD group had obvious fatty degeneration, fat vacuoles and inflammatory cell infiltration in the liver tissue; compared with the HFD group, the liver inflammation and fatty degeneration of the mice in each treatment group were improved ( Figure 6 Among them, the most obvious improvement was in the HCJR group ( Figure 6 ). This result suggests that tea tree root extract can improve liver inflammation and fatty degeneration.

[0095] Effects of tea tree root extract on liver ferroptosis markers in HFD-induced MASLD mice

[0096] The detection of malondialdehyde and ferrous ion contents in the liver of mice showed that compared with the CON group, the malondialdehyde and ferrous ion contents in the liver tissue of mice in the HFD group were significantly increased (P < 0.05); compared with the HFD group, the malondialdehyde and ferrous ion contents in the mice in each treatment group were significantly decreased, and the differences were statistically significant (P < 0.05) ( Figure 7 This result indicates that tea tree root extract can reduce the levels of malondialdehyde and ferrous ions in liver tissue, regulate the levels of liver tissue ferroptosis markers, and thus prevent liver tissue ferroptosis.

[0097] Effects of tea tree root extract on lipid peroxidation signaling pathways in liver tissue of HFD-induced MASLD mice

[0098] Protein detection of mouse liver tissue showed that all groups expressed two proteins, NOX1 and GPX4 ( Figure 8 A). Compared with the CON group, the GPX4 content in the liver tissue of mice in the HFD group was significantly decreased (P < 0.05), and the NOX1 content was significantly increased (P < 0.05); compared with the HFD group, the GPX4 and NOX1 contents of mice in each treatment group were restored, and the differences were statistically significant (P < 0.05) ( Figure 8 This result suggests that tea tree root extract can regulate liver tissue peroxidation and play an anti-lipid peroxidation role.

[0099] Application Example 2 Effect of Camelliaside A on Liver Tissue of HFD-Induced MASLD Mice

[0100] A fatty liver model was established by inducing a high-fat diet using HFD to verify the therapeutic effect of the camelliaside A prepared in Examples 4-6 on MASLD.

[0101] 1 Experimental methods

[0102] 1.1 Modeling and grouping

[0103] Male C57BL / 6 mice, 6-8 weeks old, were selected and housed in a SPF-compliant environment for one week. Mice were weighed, their toes clipped, and then randomly assigned to six groups of eight mice each. The control group (CON) was fed a standard control diet, while the model group (HFD) and all treatment groups were fed a high-fat, high-fructose, high-cholesterol diet (Synergy Bio, XT310). Mice were maintained with free access to the diet for 16 weeks.

[0104] The treatment groups were given camelliaside A (10, 20, 40 mg / kg / d, refer to the previous pilot experiment data) by gavage starting from week 12 of modeling as the low, medium, and high dose (LCA, MCA, HCA) treatment groups, and polyene phosphatidylcholine (PPC, 178 mg / kg / d, human equivalent dose) as a positive control.

[0105] 1.2TG and TC testing

[0106] Same as Application Example 1.

[0107] 1.3ALT and AST testing

[0108] Same as Application Example 1.

[0109] 1.4 Malondialdehyde detection

[0110] Same as Application Example 1.

[0111] 1.5 Ferrous ion detection

[0112] Same as Application Example 1.

[0113] 1.6 UPLC-Q-TOF / MS detection

[0114] Same as Application Example 1.

[0115] 1.7 Protein content determination

[0116] Same as Application Example 1.

[0117] 1.8HE staining

[0118] Same as Application Example 1.

[0119] 1.9 Oil Red O staining

[0120] Same as Application Example 1.

[0121] 1.10 Data Analysis

[0122] Same as Application Example 1.

[0123] 2 Experimental results

[0124] 2.1 Effects of Camelliaside A on Body Weight and Liver Weight in HFD-Induced MASLD Mice

[0125] During the modeling period, the mice were in good spirits, flexible in movement, and ate and drank as usual. At the beginning of the experiment, there was no significant difference in the weight of mice in each group (P>0.05). During the modeling treatment, the weight of mice in each group fed with high-fat, high-fructose, and high-cholesterol diets increased rapidly, among which the weight of mice in the HFD group increased the fastest; by the end of the experiment, the weight of CON mice was basically stable, while the weight of mice in the HFD group continued to increase, and the difference was statistically significant compared with CON (P<0.05); the weight of mice in each treatment group also continued to increase, but the increase was significantly lower than that in the HFD group. Among them, the specific treatment effect was HCA>MCA>PPC>LCA( Figure 9A). At the end of the experiment, the livers of mice were weighed. Compared with CON, the liver weight of HFD group was significantly increased (P < 0.05); and compared with HFD group, the liver weight of mice in each treatment group was significantly decreased, and the difference was statistically significant (P < 0.05). Among them, the specific treatment effect was HCA > PPC > MCA > LCA ( Figure 9 B) This result indicates that camelliaside A can inhibit the body weight gain and reduce the liver weight of HFD mice.

[0126] 2.2 Effects of camelliaside A on blood lipids in HFD-induced MASLD mice

[0127] The results of the detection of TG and TC levels in the serum of mice showed that compared with CON, the serum TC and TG levels of mice in the HFD group were significantly increased (P < 0.05); compared with the HFD group, the TC and TG levels of mice in each treatment group were significantly decreased, and the differences were statistically significant (P < 0.05); the specific treatment effect was HCA > PPC > MCA > LCA ( Figure 10 This result indicates that camelliaside A can reduce the levels of TG and TC in the serum of HFD-induced MASLD mice and improve the serum lipid profile of mice.

[0128] Effects of camelliaside A on liver function in HFD-induced MASLD mice

[0129] The results of the mouse serum test showed that compared with the CON group, the ALT and AST levels in the HFD group were significantly increased (P < 0.05); and compared with the HFD group, the ALT and AST levels in the mice in each treatment group were significantly decreased, and the differences were statistically significant (P < 0.05) ( Figure 11 ). Among them, the specific treatment effect is HCA>PPC>MCA>LCA( Figure 11 This result shows that camelliaside A can reduce the levels of ALT and AST in serum and repair liver damage.

[0130] 2.4 Effect of Camelliaside A on Liver Histopathology in HFD-Induced MASLD Mice

[0131] The results of HE and Oil Red O staining of the mouse liver cells showed that compared with the CON group, the HFD group had obvious fatty degeneration, fat vacuoles and inflammatory cell infiltration in the liver tissue; compared with the HFD group, the liver inflammation and fatty degeneration of the mice in each treatment group were improved ( Figure 12 Among them, the most obvious improvement effect was in the HCA group ( Figure 12 ).

[0132] Effects of Camelliaside A on Ferroptosis Markers in Liver Tissue of HFD-Induced MASLD Mice

[0133] The detection of malondialdehyde and ferrous ion contents in the liver of mice showed that compared with the CON group, the malondialdehyde and ferrous ion contents in the liver tissue of mice in the HFD group were significantly increased (P < 0.05); compared with the HFD group, the malondialdehyde and ferrous ion contents in the mice in each treatment group were significantly decreased, and the differences were statistically significant (P < 0.05) ( Figure 13 This result indicates that camelliaside A can reduce the levels of malondialdehyde and ferrous ions in liver tissue, regulate the levels of liver tissue ferroptosis markers, and thus prevent liver tissue ferroptosis.

[0134] Effects of Camelliaside A on Lipid Peroxidation Signaling Pathways in the Liver of HFD-Induced MASLD Mice

[0135] Protein detection of mouse liver tissue showed that all groups expressed two proteins, NOX1 and GPX4 ( Figure 14 A). Compared with the CON group, the GPX4 content in the liver tissue of mice in the HFD group was significantly decreased (P < 0.05), and the NOX1 content was significantly increased (P < 0.05); compared with the HFD group, the GPX4 and NOX1 contents of mice in each treatment group were restored, and the differences were statistically significant (P < 0.05) ( Figure 14 This result indicates that camelliaside A can regulate liver tissue peroxidation and play an anti-lipid peroxidation role.

[0136] In summary, tea tree root extract and camelliaside A can reduce serum TC and TG levels, regulate ALT and AST levels in liver tissue, improve liver inflammation and steatosis, modulate hepatocyte ferroptosis, and exert anti-lipid peroxidation effects. Tea tree root extract or camelliaside A can be used as an active ingredient or in combination with other drugs to prepare drugs for the treatment of metabolic-related fatty liver disease.

[0137] The above-described embodiments merely represent preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Use of a tea tree root extract in the preparation of a medicament for treating metabolism-related fatty liver disease.

2. The use according to claim 1, characterized in that The effective components of the tea tree root extract include but are not limited to camelliaside A.

3. The use according to claim 1, characterized in that The preparation method of the tea tree root extract is as follows: using the roots of Camellia oleifera as raw materials, crushing them and extracting them with a 60-80% ethanol solution in three extraction devices connected in series at a temperature of 50-60°C, the extraction time of each extraction device being 3-4 hours, the mass volume ratio of Camellia oleifera roots to 60-80% ethanol being 3-5 kg:15-25 L, collecting the tea tree root ethanol extract, concentrating and drying to a constant weight, thereby obtaining the tea tree root extract.

4. A use of camelliaside A in the preparation of a drug for treating metabolism-related fatty liver disease.

5. The use according to claim 4, characterized in that The preparation method of camelliaside A is as follows: the tea tree root ethanol extract described in claim 3 is sequentially microfiltered and ultrafiltered to obtain a permeate, which is passed through an HPD100 macroporous adsorption resin column, first eluted with water until colorless, then eluted with 40% to 60% ethanol, the ethanol eluate is collected, and then the obtained crude product is separated and purified by high-speed countercurrent chromatography to obtain camelliaside A.

6. The use according to claim 5, characterized in that The volume ratio of the tea tree root ethanol extract to the column volume of the macroporous adsorption resin column is 24-28:1, and the amount of the 40%-60% ethanol elution is equivalent to 3-5 times the column volume of the macroporous adsorption resin column.

7. The use according to claim 5, characterized in that The solvent of the high-speed countercurrent chromatography is: n-hexane-n-butanol-methanol-0.5% acetic acid aqueous solution solvent system, and the ratio of n-hexane, n-butanol, methanol and 0.5% acetic acid aqueous solution is 1-3:3-5:1-3:4-6.

8. A medicament for treating metabolic-related fatty liver disease prepared from the tea tree root extract or camelliaside A according to claim 1 or claim 4, characterized in that: The tea tree root extract or camelliaside A is used as an active ingredient or in combination with other drugs to prepare a drug for treating metabolism-related fatty liver disease.

9. The drug according to claim 8, characterized in that The drug is used to reduce the TC and TG content in serum to lower blood lipids; the drug is used to reduce the ALT and AST content in liver tissue to promote liver function recovery; the drug is used to improve liver inflammation and fatty degeneration symptoms; the drug is used to regulate liver cell ferroptosis and exert an anti-lipid peroxidation effect.

10. The drug according to claim 8, characterized in that The medicine comprises tea tree root extract or camelliaside A with or without medically permitted excipients, and is prepared into various acceptable dosage forms, including injection, pill, capsule, granule, tablet or oral solution.

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