A composition for treating non-alcoholic fatty liver, a pharmaceutical preparation, and a preparation method and use thereof

Abstract

The application belongs to the technical field of traditional Chinese medicines, and particularly relates to a composition for treating non-alcoholic fatty liver, a pharmaceutical preparation and a preparation method and application thereof. The application provides a composition DJZ-x for treating non-alcoholic fatty liver, which is composed of ginsenoside Rg1, 6-gingerol and / or hydroxyl-alpha-piperine in a specific proportion, and the components have a synergistic effect. The composition has clear components, stable and controllable quality, and a lower effective dose, and can replace traditional large Jianzhong decoction, and has a good application prospect in treating non-alcoholic fatty liver disease (NAFLD).

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine, and particularly relates to a composition for treating non-alcoholic fatty liver, a pharmaceutical preparation and a preparation method and use thereof. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disease with a global incidence of up to 25%. NAFLD has become the first chronic liver disease in China and is the primary cause of abnormal liver biochemical indicators in health checkups. NAFLD often manifests as a multi-system disease, and the main feature is the accumulation of a large amount of triglyceride (TG) in liver tissue. According to the occurrence stage of the disease, NAFLD can be divided into simple hepatic steatosis (NAFL), fatty liver hepatitis (NASH), liver fibrosis, liver cirrhosis and hepatocellular carcinoma. NAFLD is closely related to various chronic diseases such as obesity, hyperlipidemia and diabetes; in addition, the occurrence of NAFLD can lead to the occurrence of various diseases and intrahepatic and extrahepatic tumors such as cardiovascular system diseases, breast cancer and colon cancer. Although the current diagnosis level of NAFLD has been significantly improved, there are few choices for the clinical treatment of NAFLD, and the main treatment strategy still relies on diet, exercise therapy and weight loss surgery. There are relatively few drugs specifically used for the treatment of NAFLD in the clinic, and the existing drugs are mainly weight loss and lipid-lowering drugs such as orlistat, metformin, betaine and statins; liver-protecting drugs such as vitamin E, obeticholic acid and silymarin. However, these drugs currently have no definite therapeutic effect on diseases at the stages of NASH and liver fibrosis. Therefore, the development of NAFLD drugs is imminent, and the market prospect of drug research and development is huge.

[0003] In recent years, more and more studies have reported that traditional Chinese medicine and its compound have potential positive effects on the prevention and treatment of NAFLD. The pathogenesis of NAFLD is complex, and it is related to the dysfunction of multiple organs in the body. Therefore, the treatment of NAFLD by relying on single or several target chemical drugs often has no obvious advantage. The significant feature of traditional Chinese medicine and its compound is to rely on the synergistic mechanism of multi-component, multi-target and multi-pathway, which has a natural advantage in the treatment of NAFLD. In traditional Chinese medicine theory, NAFLD is attributed to 'liver stagnation', 'accumulation', 'accumulation disease', 'phlegm turbidity', 'fat', etc. According to the 'Difficult Classic', 'liver accumulation' is named 'fat', which is located in the left hypochondrium, like a covered cup, with head and foot. Although the disease is located in the liver, the key to the disease is the spleen. The spleen is earth, and the liver is wood. If the earth is empty, the wood will be dry. If the earth is full, the wood will be lush. The spleen and the liver are complementary and mutually restrictive. The medical sage Zhang Zhongjing said, 'When you see liver disease, you know that the liver transmits to the spleen. You should first nourish the spleen.' Ancient and modern related literature believes that the main pathogenesis of NAFLD is improper diet, internal injury of the spleen and stomach, spleen dysfunction, and phlegm turbidity. As the 'Discussion of Bi' in 'Plain Questions' says, 'Excessive diet and injury of the stomach and intestines.' 'Materia Medica' says, 'Excessive diet, good food, oil, and lard, thick and solid, leading to spleen dysfunction, causing problems, all caused by phlegm.' The 'Nineteen Pathogenesis of Diseases' in 'Plain Questions' says, 'All wet swelling and fullness are related to the spleen.' Spleen dysfunction and phlegm turbidity are the main pathogenesis of non-alcoholic fatty liver disease. The spleen is responsible for transportation and transformation, likes dryness and hates dampness. Spleen deficiency leads to phlegm and dampness, and water and food cannot be normally transported and distributed, leading to the accumulation of phlegm turbidity,'swelling' or 'fullness', etc. As 'Standard for Treating Syndromes' says, 'Spleen deficiency cannot separate clear and turbid, and phlegm is generated.' Therefore, traditional Chinese medicine believes that the pathogenesis of NAFLD is spleen deficiency and phlegm obstruction, and the spleen deficiency is the root and the phlegm obstruction is the branch. Spleen deficiency leads to dysfunction, internal phlegm turbidity, and obstruction of liver qi, and finally the disease occurs. Therefore, 'warming spleen yang to transform phlegm turbidity' and 'treating from the spleen' are one of the main strategies for treating NAFLD in traditional Chinese medicine.

[0004] Dajianzhong Decoction (DJZ) is derived from 'Abdominal Distension, Cold Hernia, and Food Retention Disease, Pulse, Syndrome, and Treatment' in 'Synopsis of Golden Chamber' by Zhang Zhongjing of Eastern Han Dynasty. It is a traditional Chinese medicine classic prescription for treating middle jiao deficiency, spleen and stomach yang deficiency, and is composed of four Chinese medicines: Zanthoxylum bungeanum, Zingiber officinale, Panax ginseng, and Glycine. In DJZ, 'Zanthoxylum bungeanum is the main ingredient, Zingiber officinale is the auxiliary ingredient, and Panax ginseng is added. Zanthoxylum bungeanum is hot and pungent, which can dispel cold and reverse adverse flow in the lung, spleen, stomach, and kidney. Zingiber officinale is hot and pungent, which can warm the heart and dispel cold. Panax ginseng is sweet and warm, which can tonify the spleen and lung. Glycine is sweet, which can tonify the spleen and stomach, and can also balance the side effects of Zanthoxylum bungeanum and Zingiber officinale. The whole prescription has the functions of warming and tonifying, which can warm without drying, and can tonify deficiency, ascend yang, transform turbidity, and relieve pain. According to traditional Chinese medicine theory, the efficacy of Dajianzhong Decoction meets the requirements of 'warming spleen yang to transform phlegm turbidity' and 'treating from the spleen' in the treatment of NAFLD.

[0005] Traditional Chinese medicine and its compound have the characteristics of multi-target, multi-pathway and integrated mechanism; however, the effective substance basis and the clinical treatment mechanism of traditional Chinese medicine are unknown, and the treatment of traditional Chinese medicine and its compound by what component and mechanism is still a "black box", which leads to unclear regularity of traditional Chinese medicine and its compound in treating diseases, affects the stability of clinical curative effect of traditional Chinese medicine, and seriously limits the quality stability and further improvement of traditional Chinese medicine, and becomes the bottleneck of traditional Chinese medicine and its compound to the mainstream market of international medicine.

[0006] Therefore, developing a medicine based on the compatibility of traditional Chinese medicine and having clear components and stable and controllable quality for treating non-alcoholic fatty liver disease (NAFLD) is a problem faced by the field. SUMMARY

[0007] In view of the defects of the prior art, the present application provides a composition for treating non-alcoholic fatty liver, a pharmaceutical preparation and a preparation method and use thereof.

[0008] The present application provides a composition for treating non-alcoholic fatty liver, which consists of ginsenoside Rg1 and 6-gingerol, and the molar ratio of the two is 6.25-100:6.25-100.

[0009] Preferably, the weight ratio of ginsenoside Rg1 to 6-gingerol is 20:7.

[0010] The present application provides a composition for treating non-alcoholic fatty liver, which consists of ginsenoside Rg1, hydroxy-α-shogaol and 6-gingerol, and the weight ratio of the three is 18-22:5-9:2-6.

[0011] Preferably, the weight ratio of ginsenoside Rg1, 6-gingerol and hydroxy-α-shogaol is 20:7:3.

[0012] The present application provides a pharmaceutical preparation for treating non-alcoholic fatty liver, which is prepared by adding a pharmaceutically acceptable excipient to the composition for treating non-alcoholic fatty liver described in any of the above.

[0013] Preferably, the preparation is an oral preparation.

[0014] Preferably, the oral preparation is a decoction, an oral liquid, a granule or a pill.

[0015] The present application provides a preparation method of the composition for treating non-alcoholic fatty liver described in any of the above, which comprises:

[0016] The composition for treating non-alcoholic fatty liver consists of ginsenoside Rg1 and 6-gingerol, and / or the composition for treating non-alcoholic fatty liver consists of ginsenoside Rg1, 6-gingerol and hydroxy-α-shogaol.

[0017] wherein the molar ratio of ginsenoside Rg1 to 6-shogaol is 6.25-100:6.25-100.

[0018] The application provides use of the composition for treating non-alcoholic fatty liver or the pharmaceutical preparation for treating non-alcoholic fatty liver described in any one of the above for preparing a medicine for treating non-alcoholic fatty liver.

[0019] The molecular formula of ginsenoside Rg1 is C 42 H 72 O 14 The structural formula is

[0020] ;

[0021] The molecular formula of 6-shogaol is C 17 H 26 O4, and the structural formula is ;

[0022] The molecular formula of hydroxyl-alpha-homosaligenin is C 16 H 25 NO2, and the structural formula is .

[0023] The application provides a composition DJZ-x for treating non-alcoholic fatty liver, which is composed of ginsenoside Rg1, 6-shogaol and / or hydroxyl-alpha-homosaligenin in specific proportions, and has a synergistic effect among the components. The composition has clear components, stable and controllable quality, and a lower effective dose, and can replace traditional Dajianzhong Decoction, and has a good application prospect in treating non-alcoholic fatty liver disease (NAFLD).

[0024] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above technical idea of the application.

[0025] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the above subject matter of the application is limited to the following examples. Any technology achieved based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The figure shows the influence of ginsenoside Rg1, hydroxyl-alpha-homosaligenin and 6-shogaol on the size of lipid droplets in AML12 cells, and the figure shows the results of oil red O and hematoxylin staining. Figure 1 A is the oil red O and hematoxylin staining result figure,Figure 1 B is the statistical result diagram of lipid droplet size in the dyeing experiment, the experimental data is in the form of mean ± standard deviation, n = 3, the concentration of DJZ-x is 30 μg / ml.

[0027] Figure 2 B is the statistical result diagram of lipid droplet size in the dyeing experiment, the experimental data is in the form of mean ± standard deviation, n = 3, the concentration of DJZ-x is 30 μg / ml. Figure 2 A is the oil red O and hematoxylin staining result diagram, Figure 2 B is the statistical result diagram of lipid droplet size in the dyeing experiment, the experimental data is in the form of mean ± standard deviation, n = 3, ** represents P < 0.01, *** represents P < 0.001, the P value of each group is calculated by comparison with the model (model group), the concentration of DJZ extract and DJZ-x is 200 μg / ml and 30 μg / ml respectively.

[0028] Figure 3 B is the statistical result diagram of lipid droplet size in the dyeing experiment, the experimental data is in the form of mean ± standard deviation, n = 3, ** represents P < 0.01, *** represents P < 0.001, the P value of each group is calculated by comparison with the model (model group), the concentration of DJZ extract and DJZ-x is 200 μg / ml and 30 μg / ml respectively. Figure 3 A is the ZIP synergistic score three-dimensional diagram of ginsenoside Rg1 and hydroxy-α-hydroxy-α-momordicae, the ZIP synergistic score is 16.7, Figure 3 B is the ZIP synergistic score three-dimensional diagram of ginsenoside Rg1 and 6-gingerol, the ZIP synergistic score is 18.31, Figure 3 C is the ZIP synergistic score three-dimensional diagram of hydroxy-α-momordicae and 6-gingerol, the ZIP synergistic score is 7.98. DETAILED DESCRIPTION

[0029] In the following examples and experimental examples, the reagents and materials not specifically stated are commercially available.

[0030] The preparation method of DJZ extract is as follows:

[0031] The Dajianzhong Decoction is composed of pepper, dried ginger, ginseng and sugar, and the preparation method is as follows: accurately weigh 6 g of Sichuan pepper, 12 g of dried ginger and 6 g of ginseng, soak them in 500 mL of pure water for 30 min, then decoct for 30 min, remove the drug residue, concentrate to 1 g / mL of medicinal liquid, place it at 4℃ overnight, collect the supernatant layer, freeze-dry, and get it.

[0032] Example 1 A composition for treating non-alcoholic fatty liver and its anti-NAFLD effect

[0033] I. A composition for treating non-alcoholic fatty liver

[0034] The formula of the composition for treating non-alcoholic fatty liver of the present embodiment is as follows:

[0035] Ginsenoside Rg1: 20 μg / ml (25 μM), 6-shogaol: 7 μg / ml (12.5 μM), Hydroxy-α-sanshool 3 μg / ml (12.5 μM).

[0036] II. Application to Anti-NAFLD

[0037] 1. Establishment of NAFLD model in vitro and drug administration

[0038] Aml12 cells were seeded in 6-well plates in vitro, and a high-fat acid environment was simulated by incubating with oleic acid (OA) and palmitic acid (PA) at a concentration of 2:1 for 24 h to establish a NAFLD model in vitro. After modeling, the cells were treated with the composition DJZ-x (final concentration in the well 30 μg / ml) of the present example, DJZ extract (final concentration in the well 200 μg / ml), ginsenoside Rg1 (final concentration in the well 20 μg / ml), hydroxy-α-sanshool (final concentration in the well 3 μg / ml), and 6-shogaol (final concentration in the well 7 μg / ml) for 24 h.

[0039] After the experiment, the cells were fixed in 4% paraformaldehyde for 30 min. After washing with PBS for three times, 60% isopropanol was added for washing, and then oil red O working solution was added for staining for 30 min. Then, hematoxylin was added for staining for 2 min, and the PBS was washed. The size of lipid droplets was observed under a microscope. Lipid droplets are organelles for storing triglycerides and other lipids, and the changes in their size and number reflect the lipid level in the cells.

[0040] 2. Anti-NAFLD effect

[0041] (1) Comparison of the intervention effects of monomer compounds and DJZ-x on the NAFLD model

[0042] The results are shown in Table 1. Figure 1 As shown in Table 1, in the normal group, the lipid droplets were small, and in the model group, the number of lipid droplets became dense, and the volume of lipid droplets increased, indicating that the model group was successfully established. Compared with the model group (set as 100%), the number of lipid droplets in the ginsenoside Rg1: 20.0 μg / ml (25 μM), hydroxy-α-sanshool 3.0 μg / ml (12.5 μM), and 6-shogaol 7 μg / ml (12.5 μM) treatment groups was 90%, 87%, and 91%, respectively. The total number of lipid droplets in the three compounds alone was reduced by 32%, while DJZ-x reduced by 46%. This proves that the anti-NAFLD effect of DJZ-x is not simply the sum of the effects of the three compounds, but has a significant synergistic effect. Therefore, the combination of the three is considered to be a potential combination drug for anti-NAFLD.

[0043] (2) Comparison of the intervention effects of DJZ and DJZ-x on the NAFLD model

[0044] Results as shown in Figure 2 The results showed that compared with the model group, after the administration of DJZ-x, the lipid droplets became smaller, and part of the cells returned to normal without fatty degeneration. The staining area had a significant difference (P<0.001) after statistics. Compared with DJZ extract (200 μg / ml), after the administration of DJZ-x (30 μg / ml), the oil red area was smaller, with a significant difference (P<0.01). It showed that DJZ-x (30 μg / ml) significantly reduced the size of lipid droplets, and its anti-NAFLD effect was better than that of DJZ extract (200 μg / ml).

[0045] Therefore, the composition DJZ-x of the present embodiment has a synergistic anti-NAFLD effect, which is better than the sum of the effects of each component and also better than the effect of a larger dose of DJZ extract.

[0046] Example 2 A composition for treating non-alcoholic fatty liver and its anti-NAFLD effect

[0047] I. A composition for treating non-alcoholic fatty liver

[0048] The formula of the composition for treating non-alcoholic fatty liver of the present embodiment is as follows:

[0049] Ginsenoside Rg1: 20 μg / ml (25 μM), 6-gingerol: 7 μg / ml (12.5 μM).

[0050] II. Application in anti-NAFLD

[0051] 1. Establishment of in vitro NAFLD model and administration

[0052] The in vitro NAFLD model was established according to the method of Example 1.

[0053] After the modeling was completed, ginsenoside Rg1, 6-gingerol, and hydroxy-α-shogaol were respectively combined in pairs at concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, 75 μM, and 100 μM to intervene in the cells for 24 h.

[0054] After the experiment, oil red O staining was performed: first, the cells were washed with PBS for 2-3 times to remove the residual culture medium. The cells were fixed with 4% paraformaldehyde for 20 minutes. After fixation, they were washed with PBS for three times, then 60% isopropanol was added for washing, and oil red staining was performed for 30 min. After staining, 60% isopropanol was added for washing, and then the sample was dissolved in 100% isopropanol. The absorbance value at 490 nm wavelength was read by a spectrophotometer, which was proportional to the lipid content in the sample. The ZIP synergy score was calculated by Synergy Finder software to quantify the synergistic effect. The ZIP synergy score is the overall synergistic effect score of two drugs in different concentration gradient combinations. ZIP synergy score < 0 indicates no synergy, ZIP synergy score > 0 indicates additivity, and ZIP synergy score > 10 indicates synergy.

[0055] 2. Anti-NAFLD effect

[0056] The three-dimensional graph of the ZIP synergy score is shown in Figure 3 The ZIP synergy score of ginsenoside Rg1 and hydroxy-α-sanshool is 16.7, the ZIP synergy score of ginsenoside Rg1 and 6-gingerol is 18.31, and the ZIP synergy score of hydroxy-α-sanshool and 6-gingerol is 7.98. Therefore, ginsenoside Rg1 and 6-gingerol, and ginsenoside Rg1 and hydroxy-α-sanshool have significant synergistic anti-NAFLD effects when combined in the concentration range of 6.25 μM-100 μM, and the combination of ginsenoside Rg1 and 6-gingerol has the strongest synergistic effect at a concentration of 20 μg / ml: 7 μg / ml.

[0057] As can be seen from the above examples, the present application provides a composition DJZ-x for treating non-alcoholic fatty liver, which is composed of ginsenoside Rg1, 6-gingerol and / or hydroxy-α-sanshool in a specific ratio, and the components have synergistic effects. The composition has clear components, stable and controllable quality, and lower effective dose, and can replace traditional Dajianzhong Decoction, and has good application prospect in treating non-alcoholic fatty liver disease (NAFLD).

Claims

1. A composition for treating non-alcoholic fatty liver, characterized by comprising: It is composed of ginsenoside Rg1 and 6-shogaol, and the weight ratio of the two is 20:

7.

2. A composition for treating non-alcoholic fatty liver, characterized by: It is composed of ginsenoside Rg1, hydroxyl-α-sanshool and 6-shogaol, and the weight ratio of the three is 18-22:5-9:2-6.

3. The composition for treating non-alcoholic fatty liver according to claim 2, wherein: The weight ratio of ginsenoside Rg1, 6-shogaol and hydroxyl-α-sanshool is 20:7:

3.

4. A pharmaceutical preparation for treating non-alcoholic fatty liver, characterized by: It is a preparation prepared from the composition for treating non-alcoholic fatty liver disease according to any one of claims 1-3, plus pharmaceutically acceptable adjuvants.

5. The pharmaceutical preparation for treating non-alcoholic fatty liver according to claim 4, characterized by: The preparation is an oral preparation.

6. The pharmaceutical preparation for treating non-alcoholic fatty liver according to claim 5, characterized by: The oral preparation is a decoction, an oral liquid, granules or pills.

7. The method of preparing the composition for treating non-alcoholic fatty liver according to any one of claims 1 to 3, wherein, It comprises: The composition for treating non-alcoholic fatty liver disease is composed of ginsenoside Rg1 and 6-shogaol, and / or the composition for treating non-alcoholic fatty liver disease is composed of ginsenoside Rg1, 6-shogaol and hydroxyl-α-sanshool.

8. Use of the composition for treating non-alcoholic fatty liver disease according to any one of claims 1-3, or the pharmaceutical preparation for treating non-alcoholic fatty liver disease according to any one of claims 5-7, for the preparation of a medicament for treating non-alcoholic fatty liver disease.

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