Dammarane type tetracyclic triterpenoid saponin composition and application thereof

Abstract

The invention discloses a dammarane type tetracyclic triterpenoid saponin composition and application thereof, and belongs to the technical field of biological medicine. According to the invention, three saponins (notoginsenoside Ft1, 20 (R)-ginsenoside Rh2 and 20 (R)-ginsenoside Rg3) are combined to prepare the composition, and the composition can effectively inhibit the increase of triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), aspartate transaminase (AST) and alanine transaminase (ALT) of mouse livers in a high-dose (40mg / kg) group and a medium-dose (20mg / kg) group; the synthesis of glutathione (GSH) is effectively promoted, and the reduction of mouse body weight and the weight gain of liver are restrained; the high-dose composition effectively inhibits the generation of liver fat particles; the traditional Chinese medicine composition has a good treatment effect on alcoholic fatty liver.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a dammarane-type tetracyclic triterpenoid saponin composition and application thereof. Background Art

[0002] Alcoholic fatty liver disease (AFLD) accounts for approximately 5% of all fatty liver disease cases. The primary cause of liver disease is chronic excessive alcohol consumption. Symptoms include fat accumulation and inflammation in the liver. In severe cases, it can progress to alcoholic hepatitis, liver fibrosis, cirrhosis, and even liver cancer. Reportedly, approximately 70% of patients with AFLD will develop liver cancer, and the incidence is increasing annually.

[0003] The current treatment for alcoholic fatty liver disease mainly focuses on quitting drinking, supplemented by surgical treatment and drug support. However, there are still challenges such as high relapse rate, late diagnosis, and drug limitations. To date, there is still a lack of effective therapeutic drugs. Because many patients have psychological dependence and physiological reactions, quitting drinking faces the problem of high relapse rate; although surgical treatment can remove diseased tissue, the operation itself has risks and requires time for postoperative recovery. Current drug treatments mainly include anti-inflammatory, liver-protecting and anti-fibrosis drugs. The most commonly used drugs include polyene phosphatidylcholine, Fuzheng Huayu capsules, silybin capsules, etc. These drugs can relieve symptoms to a certain extent, but long-term use of such drugs is prone to drug resistance and side effects.

[0004] Therefore, there is an urgent need for an effective and side-effect-free saponin drug for the treatment of alcoholic fatty liver disease, which can provide research ideas for the treatment of fatty liver disease. Summary of the Invention

[0005] In view of this, the present invention provides a dammarane-type tetracyclic triterpenoid saponin composition, which has a protective effect on alcoholic fatty liver, especially improving the blood biochemical indicators and lipid accumulation in the liver tissue of mice with alcoholic fatty liver disease, and effectively inhibiting liver weight gain and the formation of liver fat particles.

[0006] On the one hand, the present application provides a dammarane-type tetracyclic triterpenoid saponin composition, wherein the dammarane-type tetracyclic triterpenoid saponin composition is notoginsenoside Ft1, 20(R)-ginsenoside Rh2 and 20(R)-ginsenoside Rg3.

[0007] Furthermore, the mass ratio of the notoginsenoside Ft1, 20(R)-ginsenoside Rh2 and 20(R)-ginsenoside Rg3 is 1:2:3.

[0008] Furthermore, the composition can be prepared into one of tablets, capsules, granules, injections, tinctures, suppositories, patches, pills, syrups, mixtures, powders, films, and dripping pills.

[0009] On the other hand, the present application also provides a use of the above-mentioned dammarane-type tetracyclic triterpenoid saponin composition in the preparation of a drug for treating alcoholic fatty liver disease.

[0010] Furthermore, the concentration of the dammarane-type tetracyclic triterpenoid saponin composition is 20 mg / kg or 40 mg / kg.

[0011] On the other hand, the present application also provides a use of the above-mentioned dammarane-type tetracyclic triterpenoid saponin composition in the preparation of alcohol-relieving medicines, foods or health products.

[0012] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application.

[0013] Beneficial effects 1. The dammarane-type tetracyclic triterpenoid saponin composition provided by the present invention has a protective effect on alcoholic fatty liver and can be used to prepare a drug for preventing or treating alcoholic fatty liver.

[0014] 2. This application used a mouse model of alcoholic liver disease (NIAAA model) and normal mice (control group) as research subjects. Serum biochemical index testing results showed that compared with the control group, the serum biochemical indicators aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the model group were significantly elevated. After treatment with the composition (20 mg / kg and 40 mg / kg), ALT and AST were significantly reduced. Hepatic tissue lipid metabolism indicators total cholesterol (TC), triglycerides (TG), and malondialdehyde (MDA) were also measured, showing that intracellular TC, TG, and MDA levels decreased after treatment with the composition (20 mg / kg and 40 mg / kg). Liver anatomy results showed that the composition effectively curbed liver weight gain in mice. HE staining results showed that the composition (40 mg / kg) significantly reduced the formation of fat particles in the liver. Therefore, the composition of the present invention can effectively improve alcoholic fatty liver disease in mice. In addition, compared with the model group, glutathione (GSH) synthesis in mice treated with the composition increased, indicating that the composition enhances the anti-fatigue effect of mice.

[0015] 3. When the oral administration of the present application was performed on mice, the oral composition (20 mg / kg and 40 mg / kg) in C57BL / 6 mice had a significant protective effect against alcoholic fatty liver disease. Based on conventional inferences, the daily dosage of the composition for an adult in clinical application can be inferred. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of exemplary embodiments of the present application in conjunction with the accompanying drawings.

[0017] Figure 1 This is an anatomical diagram of mouse liver tissue in Example 1 provided by the present invention.

[0018] Figure 2 This is the test result of the effect of serum biochemical indexes aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels on mice in Example 1 provided by the present invention.

[0019] Figure 3 This is the test result of the influence of total cholesterol (TC) and triglyceride (TG) content on lipid metabolism indicators of mouse liver tissue in Example 1 provided by the present invention.

[0020] Figure 4 These are the test results of the effects of malondialdehyde (MDA) and glutathione (GSH) content on lipid peroxidation indicators in mouse liver tissue in Example 1 provided by the present invention.

[0021] Figure 5 This is the H&E test result of the mouse liver tissue in Example 1 provided by the present invention.

[0022] Figure 6 Schematic diagram of the molecular structure of notoginseng saponin Ft1.

[0023] Figure 7 Schematic diagram of the molecular structure of 20(R)-ginsenoside Rh2.

[0024] Figure 8 Schematic diagram of the molecular structure of 20(R)-ginsenoside Rg3. DETAILED DESCRIPTION

[0025] The following describes embodiments of the present application in more detail with reference to the accompanying drawings. Although the accompanying drawings illustrate embodiments of the present application, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. Rather, these embodiments are provided to make the present application more thorough and complete, and to fully convey the scope of the present application to those skilled in the art.

[0026] Saponins are a class of natural compounds found widely in plants, exhibiting diverse biological and pharmacological effects. While total saponins from Panax ginseng leaves and Panax notoginseng have demonstrated promising effects on non-alcoholic fatty liver disease (NAFLD), their effectiveness in treating AFLD remains unclear, hindering the development of clinical drugs.

[0027] Unless otherwise specified, all reagents and materials used in the following examples were purchased from the market.

[0028] Example 1 Preparation method of a dammarane-type tetracyclic triterpene composition Ginseng leaf saponins and Panax notoginseng saponins have beneficial effects on non-alcoholic fatty liver disease. Ginseng leaf saponins primarily include tetracyclic triterpenoid saponins (dammarane and octilon types) and oleanolic acid-type pentacyclic triterpenoid saponins, while Panax notoginseng saponins primarily belong to the tetracyclic triterpenoid saponins (dammarane type). 20(R)-ginsenoside Rg3, a dammarane-type tetracyclic triterpenoid saponin within ginseng saponins, has been shown to alleviate and treat alcoholic liver disease (AFLD). Ginsenoside Rh2, also a dammarane-type tetracyclic triterpenoid saponin within ginseng saponins, has been reported to significantly shorten the duration of intoxication when combined with other compounds, namely, a 10:10:1:1 ratio of reduced β-nicotinamide mononucleotide (NADH): (NMNH): ginsenoside Rh2: ginsenoside Rg3. Ginsenoside Rh2 has two configurations, S-type and R-type. The ginsenoside Rh2 in this invention does not clearly state whether it is S-type or R-type; Panax notoginsenoside Ft1 is a dammarane-type tetracyclic triterpenoid saponin in Panax notoginseng total saponins. Literature studies have shown that it can increase serum BA levels and then activate TGR5 in adipose tissue to increase energy consumption, thereby producing beneficial metabolic effects on obese mice (Ding L, Yang Q, Zhang E, et al. Notoginsenoside Ft1 acts as a TGR5 agonist but FXR antagonist to alleviate high fat diet-induced obesity and insulin resistance in mice[J]. Journal Of Medicinal Chemistry, 2021(6). DOI:10.1016 / j.apsb.2021.03.038.). Therefore, the applicant speculates that the dammarane-type tetracyclic triterpenoid saponin composition has the effect of treating alcoholic fatty liver. Therefore, the present application combines notoginsenoside Ft1, 20(R)-ginsenoside Rh2 and 20(R)-ginsenoside Rg3 to determine whether the resulting composition has an inhibitory effect on alcoholic fatty liver.

[0029] Cocktail therapy was originally a drug combination therapy used to treat AIDS. It uses three or more drugs in combination to reduce the side effects of a single drug through multi-party coordination. It now plays an important role in the field of pain management. Among them, TBC cocktail therapy (Thalidomide Based Cocktail) provides a "low-toxic and high-efficiency" solution for advanced liver cancer. It mainly includes three common drugs: thalidomide, carmofur and compound banchelidon. The combination of these three drugs is superior to single-drug or double-drug regimens in terms of efficacy and cost through multi-target synergy, drug resistance risk and survival rate improvement. Therefore, this application uses the above-mentioned three saponin combinations to prevent and treat alcoholic fatty liver disease and test their efficacy.

[0030] Notoginsenoside Ft1, 20(R)-ginsenoside Rh2, and 20(R)-ginsenoside Rg3 were thoroughly mixed in a mass ratio of 1:2:3 to prepare a dammarane-type tetracyclic triterpene composition. All three compounds were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0031] (1) CAS number of notoginsenoside Ft1: 155683-00-4; molecular formula: C 47 H 80 O 17 , the structural formula is Figure 6 shown.

[0032] (2) CAS number of 20(R)-ginsenoside Rh2: 112246-15-8; molecular formula: C 36 H 62 O8, structural formula Figure 7 shown.

[0033] (3) CAS number of 20(R)-ginsenoside Rg3: 38243-03-7; molecular formula: C 42 H 72 O 13 , the structural formula is Figure 8 shown.

[0034] Example 2 Application of a dammarane-type tetracyclic triterpene composition in the preparation of a drug for treating alcoholic fatty liver disease Step 1: Establishment of an alcoholic fatty liver disease mouse model Male C57BL / 6 mice weighing 20-22 g were used in the experiment. They were first acclimated for 7 days in an SPF-grade barrier environment (temperature: 25 ± 1°C, humidity: 50 ± 5%). The mice were then randomly divided into five groups, each consisting of 12 mice: a control group (Control), a model group (Model), a positive drug group (Silybin), a low-dose drug group (L-ZLB03), a medium-dose drug group (M-ZLB03), and a high-dose drug group (H-ZLB03). Then, mice in the model, positive drug, low-dose drug, medium-dose drug, and high-dose drug groups were fed a diet containing increasing amounts of alcohol (1%-5% vol / vol) from day 1 to day 7. The alcohol concentration was 1% on day 1, 2% on day 2, 3% on day 3, 4% on day 4, and so on, reaching 5% on day 5 and thereafter. After the mice adapted to the alcohol concentration, they were fed a 5% (vol / vol) alcohol liquid diet for another five days. The control group was fed an alcohol control liquid diet (without alcohol). Both the alcohol liquid diet and the alcohol control diet were purchased from Xiaoshu Youtai Biotechnology Co., Ltd. (Beijing, China).

[0035] Table 1 1%-5% (vol / vol) alcohol liquid feed (1000 mL) Step 2: Administer medication Liquid feed; the control group continued to be fed an alcohol control feed. The above-mentioned composition refers to the dammarane type prepared in Example 1. On the sixth day, the positive drug group was treated with a silybin solution (50 mg / kg) by gavage; the low-dose drug group was treated with a 10 mg / kg solution of the composition (the composition was mixed with the liquid feed in Table 1 to form a solution) (based on the mouse's body weight; a 1 kg mouse was fed 10 mg of the composition) by gavage; the medium-dose drug group was treated with a 20 mg / kg solution of the composition by gavage; and the high-dose drug group was treated with a 40 mg / kg solution of the composition by gavage; the model group continued to be fed 5% (vol / vol) alcohol.

[0036] Step 3: Sampling and weighing a) On the eleventh day of the experiment, all mice were gavaged with 95% ethanol (5 g / kg). Mice in each group were killed suddenly 9 hours after gavage.

[0037] b) After weighing the mice, blood was collected from the eyeballs and allowed to stand for at least 1 hour. The blood was then centrifuged at 3000 rpm for 10 minutes and the supernatant was collected.

[0038] c) After removing the eyeballs and collecting blood, the entire liver was immediately removed and rinsed several times in pre-chilled saline to remove any residual blood. The total mass of the liver was determined, and the middle portion of the left lobe was immersed in 4% paraformaldehyde for tissue section preparation.

[0039] d) Separately, accurately weigh 200 mg of liver tissue and accurately measure 9 times its mass of pre-chilled saline (approximately 1.8 mL). Mince the tissue and place it in a homogenizer along with the saline. Place the homogenizer on ice and repeatedly grind until thoroughly broken down to obtain a liver homogenate with a mass concentration of 100 mg / mL. Centrifuge the prepared liver homogenate at 3500 r / min at 4°C for 15 min. Remove the supernatant and place it in a 1.5 mL centrifuge tube. Store the sample frozen at -20°C for later use.

[0040] See the results Figure 1 The results showed that compared with the model group (Model), the liver morphology and weight of the positive drug group (Silybin), the medium-dose drug group (M-ZLB03) and the high-dose drug group (H-ZLB03) were improved.

[0041] Step 4: Serum biochemical index testing The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of each group of mice were detected for the samples in step 3 b) according to the instructions of the AST and ALT kits, respectively.

[0042] See the results Figure 2 The results showed that the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum of the model group mice were significantly increased. After treatment with the composition (20 mg / kg and 40 mg / kg), various serum biochemical indicators were significantly reduced, indicating that the composition prepared by Example 1 can inhibit the serum biochemical indicators of alcoholic fatty liver, thereby exerting a protective effect on alcoholic fatty liver.

[0043] Step 5: Detection of liver tissue lipid metabolism indicators According to the instructions of the TC and TG kits, the contents of total cholesterol (TC) and triglyceride (TG) in the 100 mg / mL liver homogenate of each group of mice were detected for the sample in step 3c).

[0044] See the results Figure 3 The results showed that the total cholesterol (TC) and triglyceride (TG) of the model group mice increased significantly. After treatment with the composition (20 mg / kg and 40 mg / kg), various liver tissue lipid metabolism indicators were significantly reduced, indicating that the composition of the present invention can play a protective effect on alcoholic fatty liver by inhibiting the liver tissue lipid metabolism indicators of alcoholic fatty liver.

[0045] Step 6: Liver tissue lipid peroxidation indicators The contents of malondialdehyde (MDA) and glutathione (GSH) in 100 mg / mL liver homogenate of each group of mice were detected for the sample c) in step 3 according to the instructions of the MDA and GSH kits.

[0046] See the results Figure 4 The results showed that the malondialdehyde (MDA) content of the model group mice increased significantly, while the glutathione (GSH) content decreased significantly. After treatment with the composition (20 mg / kg and 40 mg / kg), the malondialdehyde (MDA) content decreased significantly, indicating that the composition of the present invention can exert a protective effect against alcoholic fatty liver by inhibiting the production of malondialdehyde (MDA). In addition, the results showed that compared with the model group, the glutathione (GSH) content increased after treatment with 40 mg / kg of the composition, indicating that the composition enhanced the anti-fatigue effect of mice.

[0047] Step 7: Liver tissue pathology examination Conventional HE staining: The liver was fixed with 4% paraformaldehyde in step 3 (c), embedded in paraffin, and then sectioned, dewaxed, hydrated, and stained with hematoxylin-eosin. Finally, the pathological changes of the liver tissue were observed using an optical microscope and imaging system.

[0048] See the results Figure 4 The results showed that after treatment with 40 mg / kg of the composition, the fat particles in the mouse liver were significantly reduced, indicating that the composition can also protect the liver by inhibiting the formation of fat particles in the liver.

[0049] The experimental methods and 11-day observation period of the mouse NIAAA model in this invention are based on extensive literature research and pharmacological theoretical foundations to ensure scientificity and reliability. In combination with the literature: Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc. 2013, 8(3):627-37. https: / / doi.org / 10.1038 / nprot.2013.032. and the literature: You Y, Li WZ, Zhang S, Hu B, Li YX, Li HD, Tang HH, LiQW, Guan YY, Liu LX, Bao WL, Shen X. SNX10 mediates alcohol-induced liver injury and steatosis by regulating the activation of chaperone-mediated autophagy. J Hepatol. 2018, 69(1):129-141. https: / / doi.org / 10.1016 / j.jhep.2018.01.038. This indicates that the model is reliable, and that both symptoms and indicators were alleviated after administration, demonstrating the efficacy of the drug of the present invention. Furthermore, the experimental period of 11 days is sufficient to cover the five half-lives of the compound (t½ = 53 hours) and achieve steady-state concentrations, meeting the requirements of OECD document 407 for subacute exposure assessment.

[0050] The embodiments of the present application have been described above. The above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or improvements to the technology in the market, or to enable other persons skilled in the art to understand the embodiments disclosed herein.

Claims

1. A dammarane-type tetracyclic triterpenoid saponin composition, characterized in that: The dammarane-type tetracyclic triterpenoid saponin composition comprises notoginsenoside Ft1, 20(R)-ginsenoside Rh2 and 20(R)-ginsenoside Rg3.

2. The dammarane-type tetracyclic triterpenoid saponin composition according to claim 1, characterized in that The mass ratio of the notoginsenoside Ft1, 20(R)-ginsenoside Rh2 and 20(R)-ginsenoside Rg3 is 1:2:

3.

3. The dammarane-type tetracyclic triterpenoid saponin composition according to claim 1, characterized in that The composition can be prepared into tablets, capsules, granules, injections, tinctures, suppositories, patches, pills, syrups, mixtures, powders, films and dripping pills.

4. Use of the dammarane-type tetracyclic triterpenoid saponin composition according to claim 1 in the preparation of a medicament for treating alcoholic fatty liver disease.

5. The use according to claim 4, characterized in that The concentration of the dammarane-type tetracyclic triterpenoid saponin composition is 20 mg / kg or 40 mg / kg.

Images