Traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition and a preparation method and application thereof. The traditional Chinese medicine composition provided by the invention comprises astragalus membranaceus, cinnamon, semen cassiae, lotus leaves, poria cocos, lophatherum gracile, malt, hawthorn, rose, pogostemon cablin, sophora flower, semen torreya and emblic leafflower fruit. The formula takes the effects of tonifying spleen and promoting diuresis, soothing liver and regulating qi, and removing food retention and dissolving turbidity as the core, and has multiple effects of promoting urination and relieving stasis. The traditional Chinese medicine composition provided by the invention can regulate and control abnormal signal pathways of fatty liver and atherosclerosis; the compound has no obvious inhibition effect on a hepatic cell line HepG2 and has an inhibition effect on a hepatic stellate cell line LX2; the obvious lipid-lowering effect is achieved on a HepG2 high-lipid model; liver fat accumulation and liver injury in a TAA-induced zebrafish fatty liver model can be remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, and particularly to traditional Chinese medicine compositions, their preparation methods, and applications. Background Technology

[0002] Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of conditions ranging from simple fatty liver to metabolic-associated steatohepatitis, and further to liver fibrosis, cirrhosis, and even liver cancer. Simple fatty liver is defined as when the fat content in the liver exceeds 5%-10% of the liver's wet weight (normally, the fat content in the liver accounts for 3%-5% of the liver's wet weight), but there is no significant inflammation or hepatocellular damage. Clinicians and the general public have poor adherence to fatty liver guidelines, and their understanding of the dangers of fatty liver is far from sufficient. Treatment for fatty liver remains at the level of 30 years ago; there are no drugs specifically effective against it.

[0003] Traditional Chinese medicine (TCM) treatment emphasizes holistic regulation and syndrome differentiation, enabling simultaneous treatment of fatty liver from multiple aspects, such as regulating lipid metabolism, improving liver function, and anti-inflammatory and liver-protective effects. Developing a TCM compound formula with core functions of "strengthening the spleen and eliminating dampness, soothing the liver and regulating qi, and eliminating stagnation and turbidity," while also possessing multiple effects such as "promoting urination and relieving stagnation," to reduce liver fat accumulation is both highly feasible and necessary. Summary of the Invention

[0004] In view of this, the present invention provides a traditional Chinese medicine composition, its preparation method and application.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a traditional Chinese medicine composition comprising Astragalus membranaceus, Cinnamomum cassia, Cassia tora, Nelumbo nucifera leaf, Poria cocos, Lophatherum gracile, Malt, Hawthorn, Rose, Patchouli, Sophora japonica, Torreya grandis, and Phyllanthus emblica.

[0007] In some specific embodiments of the present invention, the traditional Chinese medicine composition comprises the following components in parts by weight:

[0008] Astragalus membranaceus 9-30 parts;

[0009] 1-10 parts cinnamon;

[0010] Cassia seeds 9-15 parts;

[0011] 3-10 lotus leaves;

[0012] Poria cocos 3-15 parts;

[0013] 6-10 parts of Lophatherum gracile leaves;

[0014] 9-15 parts malt;

[0015] 9-12 servings of hawthorn;

[0016] 3-6 portions of roses;

[0017] Patchouli 3-9 parts;

[0018] 5-10 portions of locust flowers;

[0019] 3-15 portions of Torreya nuts;

[0020] Phyllanthus emblica 4-9 parts.

[0021] In some specific embodiments of the present invention, the traditional Chinese medicine composition comprises the following components in parts by weight:

[0022] 30 portions of Astragalus membranaceus;

[0023] 10 parts cinnamon;

[0024] 12 portions of cassia seeds;

[0025] 10 lotus leaves;

[0026] 15 parts of Poria cocos;

[0027] 6 parts of Lophatherum gracile;

[0028] 15 parts malt;

[0029] 12 servings of hawthorn;

[0030] Six portions of roses;

[0031] Patchouli (9 parts);

[0032] 10 portions of locust flowers;

[0033] 3 portions of torreya nuts;

[0034] Nine portions of Phyllanthus emblica;

[0035] or

[0036] 15 portions of Astragalus membranaceus;

[0037] 1 part cinnamon;

[0038] 9 parts of cassia seeds;

[0039] 3 lotus leaves;

[0040] 12 parts of Poria cocos;

[0041] 8 parts of Lophatherum gracile;

[0042] 12 parts malt;

[0043] 9 portions of hawthorn;

[0044] Four portions of roses;

[0045] Patchouli (3 parts);

[0046] 5 portions of locust flowers;

[0047] 15 portions of torreya nuts;

[0048] 4 portions of Phyllanthus emblica;

[0049] or

[0050] Astragalus membranaceus 9 parts;

[0051] 5 parts cinnamon;

[0052] 15 portions of cassia seeds;

[0053] 6 portions of lotus leaves;

[0054] 3 parts of Poria cocos;

[0055] 10 parts of Lophatherum gracile;

[0056] 9 parts malt;

[0057] 10 servings of hawthorn;

[0058] Three portions of roses;

[0059] Patchouli 6 parts;

[0060] 8 portions of locust flowers;

[0061] 12 portions of torreya nuts;

[0062] Phyllanthus emblica, 6 portions.

[0063] Secondly, the present invention also provides a method for preparing the traditional Chinese medicine composition, comprising the following steps:

[0064] Step 1: Weigh each herb according to the formula, crush it, mix it with water at a material-to-liquid ratio of 1:10, perform a first heating reflux extraction, collect the reflux liquid, filter it, and obtain the first extract.

[0065] Step 2: Take the residue from the first heating and reflux extraction, mix it with water at a material-to-liquid ratio of 1:8, and perform a second heating and reflux extraction. Collect the reflux liquid, filter it, and obtain the second extract.

[0066] Step 3: Mix the first extract and the second extract, concentrate them first, collect the supernatant after centrifugation, and then concentrate them second time to obtain the traditional Chinese medicine composition.

[0067] In some specific embodiments of the present invention, in step 1, the first heating reflux extraction is boiling followed by maintaining a gentle boil for 90 minutes; the filtration is filtration through 200-mesh gauze; and / or

[0068] In step 2, the second heating reflux extraction involves boiling and then maintaining a gentle boil for 60 minutes; the filtration is done through 200-mesh gauze.

[0069] In some specific embodiments of the present invention, in step 3, the first concentration is carried out at 60°C and -0.06 to -0.09 MPa to a relative density of 1.1 to 1.2; and / or

[0070] The centrifugation speed is 4000 rpm, and the centrifugation time is 10 min; and / or

[0071] The second concentration is carried out at 60°C and -0.06 to -0.09 MPa until the relative density is 1.25 to 1.3.

[0072] In some specific embodiments of the present invention, the second concentration further includes the steps of vacuum drying, pulverization and / or sieving;

[0073] Preferably, the vacuum drying conditions are 70~75℃ and -0.08~0.09Mpa;

[0074] Preferably, the pulverized material has a mesh size of 100.

[0075] Thirdly, the present invention also provides a traditional Chinese medicine composition prepared by the aforementioned preparation method.

[0076] Fourthly, the present invention also provides the use of the traditional Chinese medicine composition in the preparation of remedies for any of the following diseases;

[0077] (I) Prevention and / or treatment of fatty liver;

[0078] (II) Prevention and / or treatment of liver fibrosis;

[0079] (III) Prevention and / or treatment of atherosclerosis;

[0080] (IV) Lowering lipids;

[0081] Preferably, the traditional Chinese medicine composition has no significant inhibitory effect on the HepG2 hepatocyte cell line;

[0082] Preferably, the traditional Chinese medicine composition inhibits the activation and proliferation of hepatic stellate cells (LX2 cells);

[0083] Preferably, the traditional Chinese medicine composition regulates abnormal signaling pathways in fatty liver and / or atherosclerosis;

[0084] Preferably, the traditional Chinese medicine composition has a lipid-lowering effect on the HepG2 hyperlipidemia model;

[0085] Preferably, the traditional Chinese medicine composition improves fat accumulation in a zebrafish fatty liver model.

[0086] Fifthly, the present invention also provides a medicine comprising the aforementioned traditional Chinese medicine composition.

[0087] In some specific embodiments of the present invention, the dosage form of the drug includes powders, granules, etc. All dosage forms commonly used in the art are within the scope of protection of the present invention, and the present invention does not limit them.

[0088] This invention relates to a traditional Chinese medicine formula with the core functions of "strengthening the spleen and eliminating dampness, soothing the liver and regulating qi, and eliminating stagnation and turbidity," while also possessing multiple effects such as "promoting urination and relieving depression." Most of the components are food-grade medicinal materials with mild properties, and it is used for the prevention and treatment of fatty liver. The traditional Chinese medicine composition provided by this invention includes Astragalus membranaceus, Cinnamomum cassia, Cassia tora, Nelumbo nucifera leaf, Poria cocos, Lophatherum gracile, Hordeum vulgare, Crataegus pinnatifida, Rosa rugosa, Pogostemon cablin, Sophora japonica, Torreya grandis, and Phyllanthus emblica.

[0089] (1) The traditional Chinese medicine composition provided by the present invention can regulate the abnormal signaling pathways of fatty liver and atherosclerosis;

[0090] (2) The traditional Chinese medicine composition provided by the present invention has no significant inhibitory effect on the HepG2 hepatocyte cell line;

[0091] (3) The traditional Chinese medicine composition provided by the present invention has an inhibitory effect on the hepatic stellate cell line LX2;

[0092] (4) The traditional Chinese medicine composition provided by the present invention has a significant lipid-lowering effect in the HepG2 hyperlipidemia model;

[0093] (5) The traditional Chinese medicine composition provided by the present invention can significantly improve fat accumulation and liver damage in zebrafish fatty liver model. Attached Figure Description

[0094] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0095] Figure 1 This invention demonstrates the regulation of abnormal signaling pathways in fatty liver and atherosclerotic plaques in Example 1 and Comparative Examples 1-3 of the formulation of this application on HepG2 and ox-LDL-treated THP1 cell lines.

[0096] Figure 2 This invention demonstrates the effect of the formulation on the proliferation of HepG2 and LX2 cells;

[0097] Figure 3The hepatotoxicity test results of different formulations are shown.

[0098] Figure 4 This shows the quantitative results of BODIPY 493 / 503 fluorescence staining of the traditional Chinese medicine composition;

[0099] Figure 5 The results of fluorescent staining and photography of the traditional Chinese medicine composition using BODIPY 493 / 503 are shown.

[0100] Figure 6 This shows the quantitative results of liver fat accumulation in zebrafish liver using Red O detection method.

[0101] Figure 7 The image shows the results of oil red O staining of zebrafish liver.

[0102] Figure 8 The results of fluorescence detection in zebrafish livers are shown.

[0103] Figure 9 Results showing the fluorescence area in the liver region of zebrafish. Detailed Implementation

[0104] This invention discloses a traditional Chinese medicine composition, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0105] Currently, there is no specific drug for fatty liver disease; treatment mainly targets the underlying causes and complications. Traditional Chinese medicine (TCM) is characterized by its multi-target, multi-pathway, and holistic regulatory approach, enabling it to treat fatty liver simultaneously from multiple angles, such as regulating lipid metabolism, improving liver function, and providing anti-inflammatory and liver-protective effects. This invention relates to a TCM formula with the effects of strengthening the spleen and kidneys, clearing heat and removing dampness, and harmonizing the stomach and reducing turbidity. Most of its components are food-grade medicinal herbs with mild properties, used for the prevention and treatment of fatty liver disease. Related TCM herbs include Astragalus membranaceus, Cinnamomum cassia, Cassia tora, Nelumbo nucifera leaf, Poria cocos, Lophatherum gracile, Hordeum vulgare, Crataegus pinnatifida, Rosa rugosa, Pogostemon cablin, Sophora japonica, Torreya grandis, and Phyllanthus emblica.

[0106] The composition and dosage of the prescription are as follows:

[0107] Drug Name Dosage

[0108] Astragalus 9–30g

[0109] 1–10g of cinnamon

[0110] Cassia seeds 9–15g

[0111] 3–10g of lotus leaves

[0112] Poria cocos 3–15g

[0113] Lophatherum gracile 6–10g

[0114] 9–15g of malt

[0115] Hawthorn 9–12g

[0116] 3–6g of rose petals

[0117] Patchouli 3–9g

[0118] 5–10g of Sophora japonica flowers

[0119] Torreya nuts 3–15g

[0120] Phyllanthus emblica 4–9g.

[0121] Astragalus is derived from the dried root of *Astragalus mongholicus* or *Astragalus membranaceus*, both belonging to the legume family. It is sweet and warm in nature. It has the effects of tonifying qi and raising yang, strengthening the defensive qi and consolidating the exterior, promoting tissue regeneration and detoxification, and promoting diuresis and reducing swelling. It is commonly used for symptoms such as qi deficiency and fatigue, poor appetite and loose stools, qi sinking, chronic diarrhea and rectal prolapse, hematochezia and metrorrhagia, spontaneous sweating due to exterior deficiency, and edema due to qi deficiency.

[0122] Cinnamon is derived from the dried bark of the cinnamon tree, a plant in the Lauraceae family. It is pungent, sweet, and very hot in nature. It has the effects of tonifying fire and assisting yang, guiding fire back to its source, dispelling cold and relieving pain, and warming the meridians and promoting blood circulation. It is commonly used for impotence, cold uterus, lower back and knee pain, kidney deficiency causing asthma, upward floating of deficient yang, dizziness and red eyes, cold pain in the heart and abdomen, vomiting and diarrhea due to deficiency and cold, and abdominal pain due to cold hernia.

[0123] Phyllanthus emblica comes from the fruit of the Phyllanthus emblica plant, belonging to the Euphorbiaceae family. It has a sour, astringent, and cool nature. It possesses properties of clearing heat and detoxifying, strengthening the stomach and aiding digestion, and promoting the production of body fluids and relieving cough. It is commonly used for sore throat, cough with loss of voice, and abdominal distension due to food stagnation.

[0124] Sophora japonica flowers are derived from the dried flowers and buds of the Sophora japonica plant (a legume). They are bitter and slightly cold in nature. They have the effects of cooling the blood and stopping bleeding, clearing liver heat and purging fire. They are commonly used for hematochezia, hemorrhoidal bleeding, bloody dysentery, metrorrhagia, hematemesis, epistaxis, red eyes due to liver heat, headache, and dizziness.

[0125] Lophatherum gracile is derived from the dried stems and leaves of the Lophatherum gracile plant, a member of the Poaceae family. It has a sweet, bland, and cold nature. It possesses the effects of clearing heat and purging fire, relieving irritability, and promoting urination. It is commonly used for feverish thirst, mouth and tongue sores, and scanty dark urine.

[0126] Lotus leaves are derived from the leaves of the lotus plant (Nelumbo nucifera), a member of the Nymphaeaceae family. They are bitter and neutral in nature. They have the effects of clearing heat and dampness, promoting the upward movement of clear yang, cooling the blood and stopping bleeding. They are commonly used for summer heat and thirst, damp-heat diarrhea, and blood-heat vomiting and nosebleeds.

[0127] Malt is derived from the mature fruit of barley, a plant belonging to the Poaceae family, through germination and drying. It has a sweet and neutral flavor. It is used to promote digestion, strengthen the spleen and stomach, reduce lactation, and relieve bloating. It is commonly used for indigestion, abdominal distension and pain, and poor appetite due to spleen deficiency.

[0128] Hawthorn is the dried, ripe fruit of the hawthorn plant, belonging to the Rosaceae family. It has a sour and sweet taste, and is slightly warm in nature. It has the effects of aiding digestion, strengthening the stomach, promoting qi circulation, dispersing blood stasis, and reducing turbidity and lipids. It is commonly used for indigestion, abdominal distension, diarrhea, and abdominal pain caused by excessive meat consumption.

[0129] Patchouli is derived from the aerial parts of the patchouli plant, belonging to the Lamiaceae family. It has a pungent and slightly warm nature. It possesses the functions of resolving dampness, stopping vomiting, releasing exterior pathogens, and relieving summer heat. It is commonly used for dampness obstructing the middle burner, vomiting, and summer-heat colds.

[0130] Torreya grandis Fort., a plant in the Taxaceae family, is a dried, mature seed. It is sweet and neutral in nature, and enters the lung, stomach, and large intestine meridians. Its main functions and indications include killing parasites and eliminating food stagnation, moistening dryness and promoting bowel movements, and moistening the lungs and relieving coughs.

[0131] Roses are derived from the dried flower buds or newly opened flowers of the Rosa rugosa Thunb., a plant in the Rosaceae family. They have the effects of soothing the liver and relieving depression, promoting blood circulation and relieving pain, regulating menstruation and blood, and invigorating the spleen and promoting qi circulation.

[0132] Cassia seeds are the dried, mature seeds of the legumes Cassia obtusifolia L. or Cassia tora L. Their main benefits include clearing the liver and improving eyesight, and moistening the intestines and relieving constipation.

[0133] Poria cocos is the dried sclerotium of the fungus Poria cocos (Schw.) Wolf, belonging to the Polyporaceae family. Its effects include promoting diuresis and eliminating dampness, strengthening the spleen and replenishing the middle jiao (digestive system), and calming the mind and soothing the nerves.

[0134] This invention rationally combines the aforementioned 13 traditional Chinese medicines to achieve synergistic regulation among the components, thereby modulating fatty liver-related signaling pathways on hepatocytes and hepatic stellate cells. The composition of this invention has "strengthening the spleen and eliminating dampness, soothing the liver and regulating qi, and eliminating stagnation and turbidity" as its core functions, while also possessing multiple effects such as "promoting urination and relieving stagnation." It can be used for the treatment or adjuvant treatment of fatty liver.

[0135] The traditional Chinese medicine composition, its preparation method, and the raw materials and reagents used in its application provided by this invention can all be purchased from the market.

[0136] The present invention will be further illustrated below with reference to the embodiments:

[0137] The prepared formula improved the signaling pathways of fatty liver and atherosclerotic plaques.

[0138] 1. Acquisition of disease signaling pathways

[0139] For fatty liver, publicly published data were retrieved from the NCBI GEO database. Transcriptome sequencing data (GSE135251) for fatty liver were retrieved from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). KEGG pathway gene set enrichment analysis (GSEA) was performed on the sequencing data to select abnormal signaling pathways associated with fatty liver.

[0140] For atherosclerotic plaques, based on the transcriptome sequencing data of atherosclerotic plaques in the GEO database (GSE226790), the sequencing data were subjected to KEGG pathway gene set enrichment analysis (GSEA) to select abnormal signaling pathways associated with atherosclerotic plaques.

[0141] 2. Preparation method of traditional Chinese medicine formula: The weight proportions of each component are shown in Table 1:

[0142] Table 1. Examples and comparative weight fractions of traditional Chinese medicine compositions

[0143]

[0144] Preparation method: Weigh the powder formula according to the dosage. For the first extraction, add 10 times the volume of purified water at a material-to-liquid ratio of 1:10, heat and reflux for extraction, boil, and maintain a gentle boil for 90 minutes. Filter the liquid through 200-mesh gauze, store it separately, and record the volume of the liquid. For the second extraction, add 8 times the volume of purified water to the residue after the first extraction and reflux at a material-to-liquid ratio of 1:8, boil, and maintain a gentle boil for 60 minutes. Filter the liquid through 200-mesh gauze, store it separately, and record the volume of the liquid. The two solutions were mixed and concentrated at 60℃ (-0.06~-0.09MPa) to a relative density of 1.1-1.2. The mixture was then centrifuged at 4000rpm for 10min to remove impurities. The supernatant was collected and further concentrated at 60℃ (-0.06~-0.09MPa) to a relative density of 1.25-1.3 to form a thick paste. The paste was then dried in a vacuum drying oven (70~75℃, -0.08~0.09MPa), pulverized, and passed through a 100-mesh pharmacopoeia sieve to obtain the dry paste powder.

[0145] 3. Transcriptome sequencing of THP1 stimulated by HepG2 and ox-LDL with traditional Chinese medicine

[0146] 1) Extraction of traditional Chinese medicine:

[0147] Extraction was performed on Examples 1 and Comparative Examples 1-3. An appropriate amount of lyophilized powder was weighed and dissolved in phosphate buffered saline (PBS) to prepare the extract stock solution with a concentration of 50 mg / mL.

[0148] 2) Cell plating and drug administration

[0149] Human HepG2 hepatocytes were cultured in MEM medium (Gibco) containing 1% penicillin-streptomycin solution (Hyclone) and 10% fetal bovine serum (Excell). During the logarithmic growth phase of HepG2 cells, they were seeded at a density of 3000 cells / well in 384-well plates. After overnight adhesion, the cells were treated with 200 μg / mL of the drug for 24 h.

[0150] THP1 cell lines were seeded in 96-well plates with RPMI 1640 complete medium and PMA induction. After directional induction for 48 h, the cells were treated with oxidized low-density lipoprotein (ox-LDL) and drug extract for 48 h.

[0151] 4. Transcriptome sequencing and analysis

[0152] Cell lysis, reverse transcription, and library construction for drug screening were performed according to the instructions of the relevant kits. After high-throughput sequencing of the samples, the data were analyzed. Gene set enrichment analysis (GSEA) was used to enrich signaling pathways (FDR < 0.25 was considered a significantly regulated pathway).

[0153] 5. Regulation of abnormal signaling pathways in fatty liver by traditional Chinese medicine and its combinations.

[0154] Depend on Figure 1 The results show that Example 1 and Comparative Examples 1-3 modulate the abnormal signaling pathways associated with fatty liver and atherosclerotic plaques. The results demonstrate that Example 1 completely modulates the pathways associated with fatty liver and atherosclerotic plaques in HepG2 and ox-LDL-stimulated THP1 cells.

[0155] Example 1: Extracts from a combination of traditional Chinese medicines inhibited the proliferation of hepatic stellate cell line LX2.

[0156] The formula of the present invention was extracted and prepared using the method described in the preparation example.

[0157] 1) Cell plating and drug administration:

[0158] Human HepG2 hepatocytes and LX2 hepatic stellate cells were cultured in MEM medium (Gibco) containing 1% penicillin-streptomycin solution (Hyclone) and DMEM medium (Hyclone) containing 10% fetal bovine serum (Excell), respectively. HepG2 and LX2 cells were seeded in 384-well plates at a seeding density of 3000 cells / well. After overnight adhesion, each cell was treated with the extract from Example 1 for 48 hours.

[0159] 2) Cell viability detection

[0160] Remove the cell culture plate from the incubator and slowly discard the culture medium. Add the prepared CCK8 complete medium. After incubating for 3 hours, measure the cell culture using a microplate reader (wavelength 450 nm).

[0161] 3) Experimental Results

[0162] Experiments showed that the extract from Example 1 provided by this invention, at concentrations of 50 μg / mL to 400 μg / mL, had no significant inhibitory effect on the HepG2 hepatocyte cell line, but significantly inhibited the activation and proliferation of hepatic stellate cells (LX2 cells). The activation and proliferation of hepatic stellate cells is a central link in liver fibrosis. Inhibiting the proliferation of hepatic stellate cells can reduce their ability to transform into a pro-fibrotic cell phenotype. This may reduce the secretion of collagen and other ECM components, thereby alleviating liver fibrosis, such as... Figure 2 As shown.

[0163] Example 2: Hepatotoxicity test of the formula

[0164] The formula of the present invention was extracted and prepared using the methods described in Examples 1-3 of the preparation examples.

[0165] 1) Cell plating and drug administration:

[0166] Human HepG2 hepatocytes were cultured in MEM (Gibco) and DMEM (Hyclone) medium containing 1% penicillin-streptomycin solution (Hyclone) and 10% fetal bovine serum (Excell). They were seeded in 384-well plates at a seeding density of 3000 cells / well, and after overnight adhesion, the cells were treated with the drug for 48 hours.

[0167] 2) Cell viability detection

[0168] Remove the cell culture plate from the incubator and slowly discard the culture medium. Add the prepared CCK8 complete medium. After incubating for 3 hours, measure the cell culture using a microplate reader (wavelength 450 nm).

[0169] 3) Experimental Results

[0170] Experiments showed that the extracts of this invention, at concentrations ranging from 100 μg / mL to 400 μg / mL, had no significant inhibitory effect on the HepG2 hepatocyte cell line. Figure 3 As shown.

[0171] Example 3: Validation of lipid-lowering efficacy based on a HepG2 cell hyperlipidemia model

[0172] (1) Cell plating, modeling and drug administration

[0173] Human HepG2 hepatocytes were cultured in MEM (Gibco) and DMEM (Hyclone) media containing 1% penicillin-streptomycin solution (Hyclone) and 10% fetal bovine serum (Excell). Cells were seeded at a density of 3000 cells / well in 96-well plates and allowed to adhere overnight. In the high-fat model group, sodium oleate was added to a final concentration of 60 μg / mL, while in the normal control group, only an equal volume of PBS was added. The traditional Chinese medicine composition provided in Example 1 of this invention was added to a final concentration of 400 μg / mL. After treatment, the cells were cultured for another 48 hours.

[0174] (2) Lipid-lowering efficacy test

[0175] The lipid content of cells in each group was detected using the BODIPY 493 / 503 staining method. BODIPY 493 / 503 is a bright green fluorescent dye that can be used as a staining agent for neutral lipids and a tracer for oils and other nonpolar lipids. 1 mg of BODIPY 493 / 503 was weighed and dissolved in 382 μL of DMSO to obtain a 10 mM stock solution. The stock solution was diluted with serum-free cell culture medium to obtain a 5 μM working solution. After 48 h of cell culture, the culture medium in the wells was discarded, and the cells were gently and thoroughly washed twice with PBS buffer. 100 μL of BODIPY 493 / 503 working solution was added to each well, and the cells were gently shaken to completely cover them. The cells were incubated at room temperature in the dark for 30 minutes. The cells were washed twice with serum-free culture medium for 5 minutes each time to remove unbound dye. The fluorescence signal intensity was measured using a microplate reader with an excitation wavelength of 493 nm and an emission wavelength of 511 nm.

[0176] (3) Experimental results

[0177] The test results are as follows Figure 4 , Figure 5 As shown, the traditional Chinese medicine composition provided in Example 1 of this invention has a significant lipid-lowering effect at a concentration of 400 μg / mL.

[0178] Example 4: Efficacy Validation Based on Zebrafish Fatty Liver Animal Model

[0179] Normally developed zebrafish individuals were selected at 3dpf and placed in 6-well plates for induction treatment with thioacetamide (TAA) for 48 hours. After induction, the four groups of juvenile fish were divided into a control group, a model group (10 mMTAA), a milk thistle group (100 mg / L), and a group treated with the traditional Chinese medicine composition provided in Example 1 of this invention (1000 mg / L) for 72 hours. During the treatment, the survival status of the juvenile fish was observed and recorded every 24 hours, and the corresponding fresh culture medium was replaced.

[0180] Oil Red O staining: Dissolve 0.5 g of Oil Red O powder completely in 100 mL of isopropanol and store at 4°C in the dark. Before use, dilute the stock solution with distilled water at a ratio of 3:2 (v / v) and filter thoroughly through a 0.22 μm microporous membrane to obtain a working solution without precipitate. Fix zebrafish samples treated with the drug in 4% PFA at room temperature for 60 min, and then dehydrate them in a sequential gradient of 25%, 50%, 75%, and 100% propylene glycol / phosphate-buffered saline (PBS) for 10 min each. Stain the samples overnight with Oil Red O working solution at room temperature and rehydrate them in 100%, 75%, 50%, and 25% propylene glycol / PBS. Observe pathological changes using a stereomicroscope. Perform grayscale quantitative analysis of the Oil Red O staining in the liver samples to assess the amount of fat accumulation in the liver.

[0181] The results showed that the extract of Example 1 of the present invention could significantly reduce fat accumulation in the liver of a zebrafish fatty liver model. Figure 6 , Figure 7 ).

[0182] Example 5: Effect of the formula of this invention on liver fluorescence morphology in a TAA-induced zebrafish model.

[0183] Zebrafish of the Tg[-1.7apoa2:eGFP] liver fluorescent strain, selected at 3dpf, were placed in 6-well plates and induced with thioacetamide (TAA) for 48 hours. After induction, the four groups of juvenile fish were treated accordingly: control group, model group (10 mM TAA), milk thistle group (100 mg / L), and the traditional Chinese medicine composition provided in Example 1 of this invention (1000 mg / L), for 72 hours.

[0184] After anesthetizing zebrafish samples of the Tg[-1.7apoa2:eGFP] liver fluorescent strain, the zebrafish livers were imaged under a fluorescence stereomicroscope. The fluorescent area of ​​the zebrafish liver in the images was measured using ImageJ, and the changes in liver morphology were assessed by analyzing the area values ​​of the fluorescent regions in each group of samples. The results showed that the formula of this invention can effectively reverse TAA liver toxicity, restore the fluorescent area of ​​the liver region, and has hepatoprotective effects. Figure 8, Figure 9 ).

[0185] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, It includes astragalus, cinnamon, cassia seed, lotus leaf, poria cocos, lophatherum gracile, malt, hawthorn, rose, patchouli, sophora japonica, torreya grandis, and emblica.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, Based on parts by weight, it comprises the following components: Astragalus membranaceus 9-30 parts; 1-10 parts cinnamon; Cassia seeds 9-15 parts; 3-10 lotus leaves; Poria cocos 3-15 parts; 6-10 parts of Lophatherum gracile leaves; 9-15 parts malt; 9-12 servings of hawthorn; 3-6 portions of roses; Patchouli 3-9 parts; 5-10 portions of locust flowers; 3-15 portions of Torreya nuts; Phyllanthus emblica 4-9 parts.

3. The traditional Chinese medicine composition as described in claim 1 or 2, characterized in that, Based on parts by weight, it comprises the following components: 30 portions of Astragalus membranaceus; 10 parts cinnamon; 12 portions of cassia seeds; 10 lotus leaves; 15 parts of Poria cocos; 6 parts of Lophatherum gracile; 15 parts malt; 12 servings of hawthorn; Six portions of roses; Patchouli (9 parts); 10 portions of locust flowers; 3 portions of torreya nuts; Nine portions of Phyllanthus emblica; or 15 portions of Astragalus membranaceus; 1 part cinnamon; 9 parts of cassia seeds; 3 lotus leaves; 12 parts of Poria cocos; 8 parts of Lophatherum gracile; 12 parts malt; 9 portions of hawthorn; Four portions of roses; Patchouli (3 parts); 5 portions of locust flowers; 15 portions of torreya nuts; 4 portions of Phyllanthus emblica; or Astragalus membranaceus 9 parts; 5 parts cinnamon; 15 portions of cassia seeds; 6 portions of lotus leaves; 3 parts of Poria cocos; 10 parts of Lophatherum gracile; 9 parts malt; 10 servings of hawthorn; Three roses; Patchouli 6 parts; 8 portions of locust flowers; 12 portions of torreya nuts; Phyllanthus emblica, 6 portions.

4. The method for preparing the traditional Chinese medicine composition according to any one of claims 1 to 3, characterized in that, Includes the following steps: Step 1: Weigh each herb according to the formula, crush it, mix it with water at a material-to-liquid ratio of 1:10, perform a first heating reflux extraction, collect the reflux liquid, filter it, and obtain the first extract. Step 2: Take the residue from the first heating and reflux extraction, mix it with water at a material-to-liquid ratio of 1:8, and perform a second heating and reflux extraction. Collect the reflux liquid, filter it, and obtain the second extract. Step 3: Mix the first extract and the second extract, concentrate them first, collect the supernatant after centrifugation, and then concentrate them second time to obtain the traditional Chinese medicine composition.

5. The preparation method according to claim 4, characterized in that, In step 1, the first heating reflux extraction involves boiling followed by maintaining a gentle boil for 90 minutes; the filtration is performed using 200-mesh gauze; and / or In step 2, the second heating reflux extraction involves boiling and then maintaining a gentle boil for 60 minutes; the filtration is done through 200-mesh gauze.

6. The preparation method according to claim 4 or 5, characterized in that, In step 3, the first concentration is carried out at 60°C and -0.06 to -0.09 MPa until the relative density is 1.1 to 1.2; and / or The centrifugation speed is 4000 rpm, and the centrifugation time is 10 min; and / or The second concentration is carried out at 60°C and -0.06 to -0.09 MPa until the relative density is 1.25 to 1.

3.

7. The preparation method according to any one of claims 4 to 6, characterized in that, The second concentration process also includes vacuum drying, pulverization, and / or sieving steps; Preferably, the vacuum drying conditions are 70~75℃ and -0.08~0.09Mpa; Preferably, the pulverized material has a mesh size of 100.

8. The traditional Chinese medicine composition prepared by the preparation method according to any one of claims 4 to 7.

9. The use of the traditional Chinese medicine composition according to any one of claims 1 to 3 or the traditional Chinese medicine composition according to claim 8 in the preparation of a medicament for any of the following diseases; (I) Prevention and / or treatment of fatty liver; (II) Prevention and / or treatment of liver fibrosis; (III) Prevention and / or treatment of atherosclerosis; (IV) Lowering lipids; Preferably, the traditional Chinese medicine composition has no significant inhibitory effect on the HepG2 hepatocyte cell line; Preferably, the traditional Chinese medicine composition inhibits the activation and proliferation of hepatic stellate cells (LX2 cells); Preferably, the traditional Chinese medicine composition regulates abnormal signaling pathways in fatty liver and / or atherosclerosis; Preferably, the traditional Chinese medicine composition has a lipid-lowering effect on the HepG2 hyperlipidemia model; Preferably, the traditional Chinese medicine composition improves fat accumulation and liver damage in a zebrafish fatty liver model.

10. A drug, characterized in that, This includes the traditional Chinese medicine composition as described in any one of claims 1 to 3 or the traditional Chinese medicine composition as described in claim 8.

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