Application of surfactin family lipopeptide in preparation of medicine for preventing and / or treating non-alcoholic fatty liver disease

Abstract

The invention discloses application of surfactin family lipopeptide in preparation of a medicine for preventing and / or treating non-alcoholic fatty liver disease, and belongs to the field of biological medicine. Experiments prove that the surfactin family lipopeptide can significantly reduce weight gain and liver weight of a model mouse, reduce the ratio of liver weight to weight, relieve HFD-induced hepatic distension and lipid droplet accumulation, improve hepatic lesion, and reduce the liver weight of the model mouse. In addition, according to verification of the invention, single surfactin A, surfactin B or surfactin C can also play a role in improving NAFLD (non-alcoholic fatty liver disease) of mice induced by high fat diet. The significant effect and transformation potential of the surfactin family lipopeptide in prevention and treatment of the non-alcoholic fatty liver are verified, so that the surfactin family lipopeptide can be applied to preparation of drugs for prevention and treatment of the non-alcoholic fatty liver, and a new thought is provided for clinical treatment of prevention and treatment of the non-alcoholic fatty liver.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the use of surfactin family lipopeptides in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide, especially in Western countries, and is becoming the most common indication for liver transplantation. NAFLD includes a series of histological features, ranging from simple fatty degeneration characterized by excessive lipid accumulation in hepatocytes to nonalcoholic fatty liver hepatitis associated with inflammation or fibrosis, and finally developing into cirrhosis and liver malignancies. Currently, NAFLD has become the number one chronic liver disease in my country. The prevalence of NAFLD in adults has reached 29.2%, and it is gradually showing a trend of younger age. In addition, a large number of clinical studies have shown that NAFLD is not only closely related to type 2 diabetes, chronic kidney disease and cardiovascular disease, but is also a major risk factor for liver malignancies. It can be seen that NAFLD is a serious hazard and needs further attention. At present, some treatments for NAFLD have been proven to have good efficacy in clinical trials, such as pioglitazone, vitamin E, polyene phosphatidylcholine and other drugs commonly used in clinical treatment. However, the above drugs have some adverse reactions in clinical use. For example, taking pioglitazone can increase the patient's body weight by 3-5 kg, and also increase the risk of bladder cancer; vitamin E can increase the risk of prostate cancer and hemorrhagic stroke; vomiting, rash, diarrhea and other adverse reactions often occur during the use of polyene phosphatidylcholine, which seriously limits the promotion of the above drugs in clinical practice. Therefore, it is imperative to find safer and more effective drugs for the treatment of NAFLD.

[0003] Bioactive peptides are widely used active substances that can regulate the functions of animal bodies and protect the body from a variety of diseases and infections. The study of active peptides has been widely conducted in vitro (molecular and cellular levels) or in vivo (through animal experiments). Surfactin is a compound with special biological surface activity isolated from the extracellular secondary metabolites of Bacillus subtilis by Arima et al. in 1968 and identified as a lipopeptide structure. The carbon chain length of the fatty acid in the Surfactin group is 13-16. The chiral heptapeptide with LLDLLDL is connected to the β-hydroxyl of the carbon atom of the fatty acid chain through a lactone bond. The molecule is in a "saddle-shaped" conformation in aqueous solution. The family members include surfactin produced by Bacillus subtilis, lichenyishin produced by Bacillus licheniformis, Pumilacidin and Esperin of Bacillus pumilus. Among them, Lihenyishin, Pumilacidin and Esperin are mainly used in industry and environmental management. Surfactin is a lipopeptide antibacterial substance, which is composed of β-hydroxy fatty acid and a small peptide of 7 amino acid residues. The carboxyl group on the 7th amino acid of the peptide chain and the β-hydroxyl of the fatty acid are condensed to form a ring structure. There are two main types of Surfactin, that is, the amino acid at the 7th position is Leu or Val. The typical amino acid sequence in the peptide chain is (L-)Glu-(L-)Leu-(D-)Leu-(L-)Val-(L-)Asp-(D-)Leu-(L-)Leu. However, due to the differences in the amino acids at position 2 (Leu / Ile / Val), position 4 (Val / Leu / Ile / Ala), and position 7 (Leu / Val / Ile) and the length of the fatty acid chain (C13-C15), there are many analogs. In 1991, Baumgart used two-dimensional H-NMR to prove that Surfactin extracted from the culture medium of Bacillus subtilis ATCC21332 and Bacillus subtilis OKB105 has three structural analogs, named Surfactin A, Surfactin B, and Surfactin C; among them, Surfactin A is the main component, and the 7th amino acid on its cyclic peptide chain is Leu, the 7th amino acid of Surfactin B is Val, and the 7th amino acid of Surfactin C is Ile. In 1992, Oka et al. used optimized high-performance liquid chromatography (HPLC) to successfully separate six surfactants produced by Bacillus subtilis for the first time, and discovered two new surfactants with different fatty acid substitutions.In 1995, Russian scientists obtained five structural analogs of Surfactin from fermentation broth and determined their structures through mass spectrometry, chemical modification and two-dimensional nuclear magnetic resonance technology. Subsequent studies have more accurately determined more than ten homologues of Surfactin. There are no reports in the prior art on the use of surfactin family lipopeptides for the prevention and / or treatment of fatty liver or non-alcoholic fatty liver. Summary of the invention

[0004] The purpose of the present invention is to provide the use of surfactin family lipopeptides in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease, so as to solve the problems existing in the above-mentioned prior art. The present invention verifies the significant effect and transformation potential of surfactin family lipopeptides in preventing and treating non-alcoholic fatty liver disease, and provides a new idea for the clinical treatment of non-alcoholic fatty liver disease.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of surfactin family lipopeptides in preparing a drug for preventing and / or treating non-alcoholic fatty liver disease.

[0007] Furthermore, the surfactin family lipopeptide comprises one or more of surfactin A, surfactin B and surfactin C.

[0008] The present invention also provides a medicine for preventing and / or treating non-alcoholic fatty liver disease, which comprises surfactin family lipopeptides and pharmaceutically acceptable excipients.

[0009] Furthermore, the surfactin family lipopeptide comprises one or more of surfactin A, surfactin B and surfactin C.

[0010] Furthermore, the auxiliary materials include one or more of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

[0011] Furthermore, the dosage form of the drug includes injection, infusion, drops, powder, tablet, pill, granule, capsule, nasal drops or spray.

[0012] Furthermore, the drug is taken orally or parenterally.

[0013] The present invention discloses the following technical effects:

[0014] The present invention discloses a new application of surfactin family lipopeptides, namely, application in the prevention or treatment of non-alcoholic fatty liver disease. It has been verified by a non-alcoholic fatty liver mouse model that surfactin family lipopeptides can significantly reduce the weight gain and liver weight of model mice, reduce their liver-to-body weight ratio, alleviate HFD-induced liver expansion and lipid droplet accumulation, and improve liver lesions. The present invention has also been verified to find that a single surfactin A, surfactin B or surfactin C can also play a role in improving high-fat diet-induced NAFLD in mice. The present invention verifies the significant effect and conversion potential of surfactin family lipopeptides in the prevention and treatment of non-alcoholic fatty liver disease. Therefore, surfactin family lipopeptides can be used in the preparation of drugs for the prevention and treatment of non-alcoholic fatty liver disease, providing a new idea for the clinical treatment of non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1 The figure is a validation diagram of the protective effect of surfactin on the liver; A: grouping diagram; B: statistical graph of weight gain of mice in each group; C: statistical graph of weight of mice in each group; D: statistical graph of liver weight of mice in each group; E: statistical graph of the ratio of liver to body weight of mice in each group; F: statistical graph of the proportion of vacuolar areas in H&E staining of each group; G: statistical graph of the proportion of Oil Red O positive areas in each group; H: H&E and Oil Red O staining; NCD: blank control group; HFD: control group; HFD+Surf: surfactin group; **: p<0.01, ***: p<0.001;

[0017] Figure 2 Effects of surfactin on HFD-induced non-alcoholic fatty liver mice; A: Schematic diagram of grouping; B: Statistical graph of the ratio of liver to body weight of mice in each group; C: Statistical graph of the proportion of vacuolar areas in H&E staining of each group; D: Statistical graph of the proportion of Oil Red O positive areas in each group; E: H&E and Oil Red O staining; NCD: blank control group; HFD: control group; HFD+Surf: surfactin group; **: p < 0.01, ***: p < 0.001;

[0018] Figure 3The effects of various components of surfactin on HFD-induced non-alcoholic fatty liver mice; A: statistical graph of weight gain of mice in each group; B: statistical graph of the proportion of vacuolar areas in H&E staining of each group; C: statistical graph of the proportion of Oil Red O staining positive areas in each group; NCD: blank control group; HFD: control group; HFD1-HFD12 are groups that orally take compounds 1-12 in Example 1; *: p < 0.05, **: p < 0.01, ***: p < 0.001. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0022] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0023] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0024] Example 1 Preparation of Surfactant

[0025] (1) Bacillus subtilis BSD-2 (with a deposit number of CGMCC No. 8440, disclosed in patents CN114717165B-a composite bacterial agent and a biological control seed coating agent containing the composite bacterial agent, CN103651774B-a biological storage agent for edible fungi, a preparation method thereof and edible fungi fresh-keeping paper) is expanded and cultured to obtain a fermentation liquid;

[0026] (2) taking the fermentation broth and centrifuging it to remove the bacterial cells to obtain a sterile supernatant;

[0027] (3) Add Amberlity XAD-7HP macroporous resin to the sterile supernatant for adsorption and shake at 4°C and 30 rpm for 12 h;

[0028] (4) After the resin is collected by filtration, it is washed three times with distilled water and then washed with 60% ethanol (volume fraction), and the eluate is concentrated to obtain a crude extract;

[0029] (5) Further purification by high performance liquid chromatography: mobile phase A is acetonitrile containing 0.1% (volume ratio) trifluoroacetic acid, and mobile phase B is ultrapure water containing 0.1% (volume ratio) trifluoroacetic acid. The above sample was loaded into a WondaSil C18 (4.6mm×150mm) column with a particle size of 5μm, and eluted with a linear gradient of 10% to 90% acetonitrile, a flow rate of 1mL / min, a detection wavelength of 214nm, and a column temperature of 30°C. The components corresponding to 15-20min were collected and detected by mass spectrometry. The components of surfactin obtained are shown in Table 1. The mixed solution of the 15-20min components is a surfactin aqueous solution.

[0030] Table 1 Mass spectrometry results of purified products

[0031]

[0032]

[0033] Example 2 Preventive Effect of Surfactin on Nonalcoholic Fatty Liver Disease in Mice

[0034] (1) 8-week-old C57BL / 6JGpt mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The mice were individually placed in ventilated cages, with 4 mice in each cage, provided with sufficient food and water, maintained at a temperature of 24.5±0.5℃, and followed a 12 / 12-hour light / dark cycle. After one week of adaptation, the mice were randomly divided into a normal mouse chow control group (NCD, fed with normal mouse chow and drinking normal water), a 60% high-fat diet control group (HFD, fed with 60% high-fat diet and drinking normal water) and a 60% high-fat diet treatment group (HFD+Surf, fed with 60% high-fat diet and drinking 150 mg / L surfactant aqueous solution) (see Figure 1 Middle A), 8 mice per group for 11 weeks.

[0035] (2) During the experiment, the clinical manifestations of mice in each group were closely observed, and the coat color and mental state of the mice were observed and recorded daily; the weight of the mice was weighed and recorded daily. At 11 weeks of the experiment, the mice were killed by dislocation of the neck and dissected to observe whether there was any damage or inflammation in the chest and abdominal cavities of the mice. The livers of the mice were removed, weighed, and stored in 4% paraformaldehyde tissue fixative. Preparation of paraffin sections of liver tissue: Prepare the corresponding tissue sections according to the SOP of experiments such as pathological tissue sampling, fixation, embedding, paraffin sectioning, frozen sectioning, dehydration, and staining. Preparation of oil red sections of liver tissue: Prepare the corresponding tissue sections according to the SOP of experiments such as pathological tissue sampling, fixation, OCT embedding, frozen sectioning, and staining.

[0036] (3) The results showed that in the HFD-fed mouse cohort, the weight gain of mice in the surfactin aqueous solution treatment group (HFD+Surf) was significantly slower than that of mice in the control group (HFD) drinking normal water ( Figure 1 Middle B), mice lost weight ( Figure 1 In addition, surfactin also reduced liver weight ( Figure 1 D), decreased the ratio of liver weight to body weight ( Figure 1 Middle E). Histological analysis using H&E and Oil Red O staining showed that mice receiving surfactin solution showed significant liver protection ( Figure 1 FH). Compared with the HFD group, the number of vacuoles in liver tissue of mice in the HFD+Surf group was significantly reduced, the hepatocytes were evenly arranged, the amount of fat vacuoles was significantly reduced, and fat accumulation was significantly reduced. Histological analysis of Oil Red O staining showed that the NCD+Surf group had less fat accumulation. Collectively, these findings suggest that surfactin has a hepatoprotective effect and can play a potential role in preventing and alleviating NAFLD.

[0037] Example 3 The therapeutic effect of surfactin on non-alcoholic fatty liver disease in mice

[0038] (1) 6-week-old C57BL / 6JGpt mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The mice were individually placed in ventilated cages, with 4 mice per cage, provided with sufficient food and water, maintained at a temperature of 24.5±0.5℃, and followed a 12-h light / 12-h dark cycle. After one week of adaptation, they were treated with a 60% high-fat diet for 14 weeks to establish a NAFLD model.

[0039] (2) After successful modeling, the mice were randomly divided into three groups (see Figure 2Middle A): control group (HFD, NAFLD model mice were orally administered with 0.9% saline (SA)), surfactin group (HFD+Surf, NAFLD model mice were orally administered with 40 mg / kg surfactin) and blank control group (NCD, normal mice were orally administered with 0.9% saline). Each group included at least 8 mice for 4 weeks.

[0040] (3) The results showed that in the HFD-fed mouse cohort, the surfactin group had a reduced liver-to-body weight ratio ( Figure 2 Middle B). Histological analysis using H&E and Oil Red O staining showed that surfactin attenuated HFD-induced liver swelling and lipid droplet accumulation, indicating that surfactin has the potential to improve high-fat diet-induced NAFLD in mice ( Figure 2 (CE).

[0041] Example 4 Therapeutic effects of different components of surfactin on non-alcoholic fatty liver disease in mice

[0042] Six-week-old C57BL / 6JGpt mice were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. After C57 mice were adapted to the constant temperature animal room for one week, they were treated with a 60% high-fat diet for 14 weeks to establish NAFLD model mice.

[0043] After successful modeling, the mice were randomly divided into the following 14 groups:

[0044] NCD (blank control group): normal mice were orally administered 0.9% saline;

[0045] HFD (control group): NAFLD model mice were orally administered 0.9% saline;

[0046] HFD1: NAFLD model mice were orally administered 40 mg / kg of the compound 1 in Example 1;

[0047] HFD2: NAFLD model mice were orally administered 40 mg / kg of compound 2 in Example 1;

[0048] HFD3: NAFLD model mice were orally administered 40 mg / kg of compound 3 in Example 1;

[0049] HFD4: NAFLD model mice were orally administered 40 mg / kg of compound 4 in Example 1;

[0050] HFD5: NAFLD model mice were orally administered 40 mg / kg of compound 5 in Example 1;

[0051] HFD6: NAFLD model mice were orally administered 40 mg / kg of compound 6 in Example 1;

[0052] HFD7: NAFLD model mice were orally administered 40 mg / kg of compound 7 in Example 1;

[0053] HFD8: NAFLD model mice were orally administered 40 mg / kg of compound 8 in Example 1;

[0054] HFD9: NAFLD model mice were orally administered 40 mg / kg of compound 9 in Example 1;

[0055] HFD10: NAFLD model mice were orally administered 40 mg / kg of compound 10 in Example 1;

[0056] HFD11: NAFLD model mice were orally administered 40 mg / kg of compound 11 in Example 1;

[0057] HFD12: NAFLD model mice were orally administered 40 mg / kg of the compound 12 in Example 1.

[0058] The treatment lasted for 4 weeks, with each group including at least 8 mice.

[0059] Results showed that among the HFD-fed groups, the HFD1, HFD2, HFD3, HFD4, HFD5, HFD6, HFD7, HFD8, HFD9, HFD10, HFD11, and HFD12 groups all significantly reduced weight gain compared to the saline-treated HFD mice ( Figure 3 Middle A). Histological analysis using H&E and Oil Red O staining showed that HFD1, HFD2, HFD3, HFD4, HFD5, HFD6, HFD7, HFD8, HFD9, HFD10, HFD11, and HFD12 groups all attenuated HFD-induced liver expansion and lipid droplet accumulation ( Figure 3 BC), indicating that each component of surfactin can improve NAFLD in mice induced by high-fat diet.

[0060] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of surfactin family lipopeptides in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that The surfactin family lipopeptides include one or more of surfactin A, surfactin B and surfactin C.

3. A drug for preventing and / or treating non-alcoholic fatty liver disease, characterized in that: The medicine comprises surfactin family lipopeptide and pharmaceutically acceptable excipients.

4. The drug according to claim 3, characterized in that The surfactin family lipopeptides include one or more of surfactin A, surfactin B and surfactin C.

5. The drug according to claim 3, characterized in that The auxiliary materials include one or more of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

6. The drug according to claim 3, characterized in that The dosage form of the drug includes injection, infusion, drops, powder, tablet, pill, granule, capsule, nasal drops or spray.

7. The drug according to claim 3, characterized in that The drug can be taken orally or parenterally.

Images