Lipopeptide-containing fat dissolving product as well as preparation method and application thereof

Abstract

The invention provides a lipopeptide-containing fat dissolving product as well as a preparation method and application thereof, and relates to the field of medical cosmetology. According to the invention, the lipopeptide is applied to the preparation of the fat-dissolving medicine for the first time, and the lipopeptide can be selected from one or more of surfactin, iturin, interleukin, abrumogen, daptomycin, polymyxin, bacitramycin L, phospholipid rhzomorph and bacitramycin D. According to the invention, the lipopeptide is directly injected into the subcutaneous adipose tissue for the first time, so that the accumulation of the local adipose tissue is reduced, and a new active ingredient is provided for the fat dissolving needle; the invention also proves that compared with sodium deoxycholate, the lipopeptide has higher fat-dissolving activity and lower effective dose when being used as a fat-dissolving component; lipopeptide is adopted as a new effective component of the fat-dissolving needle, so that the fat-dissolving needle is safer and more effective, the rupture of an injection part can be reduced, the fat-dissolving needle better fits the pH value of a human body, and the pain in the injection process is reduced.

Description

Technical Field

[0001] The present application relates to the field of medical cosmetology, and in particular to a lipopeptide-containing lipolysis product and a preparation method and application thereof. Background Art

[0002] Weight loss and slimming have always been key areas of concern. Among the methods of weight loss and slimming, surgical liposuction has the most obvious effect, but its disadvantages are large surgical trauma and long recovery time. Non-surgical lipolysis can solve the pain points of high risk and long recovery period of surgical liposuction, and improve customer comfort and safety. At present, the mainstream technologies of non-surgical lipolysis include cryolipolysis, radiofrequency lipolysis, ultrasonic lipolysis and lipolysis needles. Among them, cryolipolysis, radiofrequency lipolysis and ultrasonic lipolysis all require the assistance of special equipment, while lipolysis needles do not require the use of special equipment. It is a technology that directly injects drugs into the subcutaneous fat layer of the human body to dissolve the fat tissue. At present, lipolysis needles are still scarce products.

[0003] The active ingredient of Kybella lipolysis injection is 1% sodium deoxycholate. The solution also contains 0.9% benzyl alcohol, 0.44% sodium chloride, and inorganic salt components such as sodium hydroxide and phosphate, with a pH value of 8.3. Sodium deoxycholate is a chemically synthesized small molecule compound with the same structure as endogenous deoxycholic acid. Some researchers believe that when the pH value of the solution of sodium deoxycholate is lower than 8.2, it will form a precipitate after being placed for a long time for one month. Precipitation will not occur only when the pH value is greater than 8.2. Therefore, the pH value of Kybella lipolysis injection preparation is 8.3. However, the normal range of human physiological pH is usually between 7.35-7.45, and when the pH value and osmotic pressure of the injection preparation are different from the normal physiological values ​​of the human body, it will cause pain.

[0004] In response to the above problems, patent CN112955125A relates to a dry powder preparation for injection of deoxycholic acid component, which has excellent storage stability and a pH of 8.2 or lower. When it is dissolved in water for injection, it can reduce the pain during injection by forming a pH environment closer to the human body than conventional injections, and allow sodium deoxycholate to dissolve well in water for injection without forming precipitation.

[0005] Biological cell membranes are composed of lipid bilayer structures. The hydrophobic chains of amphiphilic surfactants can be embedded in the lipid bilayer membrane, making the cell membrane unstable and decomposing, causing cell lysis, and the cell membrane lipids or cell lysates are mixed with surfactants to form micelles, which then induce the surrounding macrophages to aggregate and phagocytose and trigger tissue inflammatory response. Sodium deoxycholate, as an ionic surfactant, exerts a lipolysis effect by dissolving fat cells at the injection site. Although the clinical efficacy of Kybella lipolysis injection has been verified, according to clinical applications and market feedback, the existing lipolysis injection treatment effect is not obvious and has a certain degree of side effects. Common side effects of sodium deoxycholate lipolysis injection include inflammatory reactions after injection, manifested as erythema and edema. Repeated inflammatory reactions caused by multiple injections may lead to fibrosis, and hard nodules occasionally appear at the injection site, which disappear within weeks to months. Therefore, consumers will not choose lipolysis injections because they are dissatisfied with the existing treatment effects or are afraid of side effects. If safer and more effective lipolysis injections can be developed, it will greatly increase consumers' willingness to use them and expand new unmet body shaping markets, officially leading body shaping into the era of medical aesthetics and micro-surgery.

[0006] Lipopeptides are composed of fatty acids connected to short linear or circular peptide chains. The differences between this family of structural homologues can be the type, number (7-25) and configuration of amino acids in the peptide fragment, the length (12-17 carbons) and composition of the fatty acid chain, and the different connecting groups between the two parts. Lipopeptides are generally produced by the metabolism of Gram-positive bacteria and exhibit a variety of biological activities. In current research, the biological activities exhibited by lipopeptides mainly include: surfactants, emulsifiers, antibacterial, antiviral, antitumor, hemolytic, anti-inflammatory, etc., which can be used in medicine, cosmetics, oil production, agriculture, washing, textiles and other fields.

[0007] At present, the use of lipopeptides is becoming more and more extensive. Patent CN103570809B relates to an anti-inflammatory lipopeptide, which binds to the TLR2 receptor, induces the nuclear entry of β-catenin, and β-catenin and p65 competitively bind to PPAR-γ, thereby playing an anti-inflammatory role. Similarly, patent CN111643656A proposes that lipopeptides have broad-spectrum coronavirus membrane fusion inhibitors and their use against HIV. The lipopeptide is a compound obtained by modifying a polypeptide with antiviral activity with a lipophilic compound. Patent CN112679576A relates to a method for preparing an antibacterial lipopeptide hydrogel and its use in antibacterial and tissue damage repair. The antibacterial lipopeptide is obtained by coupling the amino acid sequence of KKAAA(K)nAAAKK with an aliphatic hydrocarbon acid, where n is an integer of 1-4. Patent CN113683673A discloses a lipopeptide based on bee venom peptide, which is obtained by coupling C10-18 fatty acid to the N-terminus of bee venom peptide and amidation modification of the C-terminus. Compared with bee venom peptide, it has better inhibition of Gram-positive and Gram-negative bacteria and lower hemolysis. Patent CN113908252A mentions that surfactin can significantly inhibit Propionibacterium acnes and / or Malassezia, and involves the use of surfactin in the preparation of antimicrobial drugs or cosmetics and its preparation method. It can be seen that the application of lipopeptides has gradually expanded, especially in the field of medicine, which is attracting more and more interest from researchers. However, at present, no studies have shown that lipopeptides are related to lipolysis.

[0008] In summary, there is an urgent need to develop a lipolysis injection that is both safe and effective in lipolysis. Summary of the invention

[0009] The present invention adopts lipopeptide as a lipolytic active substance for the first time and applies it to lipolytic drugs and lipolytic processes. The present invention proves that lipopeptide can replace the existing sodium deoxycholate component as an effective ingredient for lipolysis, effectively reducing local fat accumulation, and at the same time, the side effects are less than those of lipolytic preparations on the market.

[0010] On the one hand, the present application provides the use of lipopeptides in the preparation of lipolysis products.

[0011] Lipopeptide substances include, but are not limited to, the following substances, such as surfactant, iturin, fennel, fengycin, daptomycin, polymyxin, bacillus antibiotic L, lecithin, bacillus antibiotic D, etc.

[0012] Further, the lipopeptide is a cyclic lipopeptide; preferably, the lipopeptide is selected from one or more of surfactin, iturin, fennel, fengycin, daptomycin, polymyxin, bacillary antibiotic L, phospholipid inhibitor, and bacillary antibiotic D; more preferably, the lipopeptide is surfactin and / or daptomycin; more preferably, the lipopeptide is surfactin; more preferably, the surfactin is surfactin-C14 and / or surfactin-C15, that is, a β-hydroxy fatty acid whose acyl structure is C14 and / or C15.

[0013] Among them, surfactin (general molecular formula: C 53 H 93 N 7 O 13 , CAS No.: 24730-31-2) is an anionic biosurfactant produced by fermentation of Bacillus subtilis, soluble in water and polar organic solvents. The surfactin family is composed of a hydrophilic group of a cyclic heptapeptide and a β-hydroxy fatty acid chain. Its peptide ring contains 7 amino acids, and the acyl structure is C 12 -C 16 The beta-hydroxy fatty acid of the peptide chain, the carboxyl group of the amino acid at the end of the peptide chain and the beta-hydroxyl group on the fatty acid chain are connected to form a ring through an ester bond. It has biodegradability, good biocompatibility, and ultra-low irritation. It is one of the most efficient biosurfactants, and its physicochemical properties are relatively stable. It is a surfactant and emulsifier with excellent performance. Since surfactin is formed by connecting fatty acids and cyclic heptapeptides, its degradation products are fatty acids and amino acids, which are harmless to the human body. The present invention has proved through in vitro cell experiments and in vivo animal experiments that surfactin is an ideal ingredient for injection to eliminate local fat, which has a lipolysis effect comparable to that of sodium deoxycholate and has fewer side effects.

[0014] Although both lipopeptides and sodium deoxycholate are amphiphilic substances with hydrophilic and hydrophobic groups, they are very different. First of all, the molecular structure and molecular weight are different. Lipopeptides are composed of fatty acids and peptides, while sodium deoxycholate is a C 7 Sodium cholate lacks hydroxyl groups, and the molecular weights of the two are about 1036Da and 414Da respectively. Sodium deoxycholate has a smaller molecular weight and acts on the cell membrane of fat cells in a short time, and the reaction is more violent, causing a stronger inflammatory reaction. In addition, the former can be decomposed and metabolized in the body, while the latter does not decompose in the body. Therefore, the use of lipopeptides as the main active ingredient for fat dissolution in this application is safer and has fewer side effects than the currently commonly used sodium deoxycholate fat dissolving injection.

[0015] In addition, the applicant found that sodium deoxycholate is not easy to dissolve in water and needs to be heated to completely dissolve into a transparent and clear fluid state. When the temperature drops to room temperature or below, the aqueous solution of sodium deoxycholate turns into a gel state, and the pH value needs to be adjusted to above 8.2 to avoid the gel state. Therefore, the pH of the sodium deoxycholate fat-dissolving injection is greater than 8.2. However, the normal range of the physiological pH value of the human body is usually between 7.35-7.45, so it will cause pain during injection. Surfactant is soluble in water, and its aqueous solution is in a transparent and clear fluid state. Therefore, the pH value of the fat-dissolving injection containing surfactant can be close to the physiological pH value of the human body and will not cause pain.

[0016] In a preferred embodiment, the surfactin (HXRZ) is a laboratory-made sample, and the preparation method can refer to patents CN114214252A and CN105695543B.

[0017] A person skilled in the art can also prepare or purchase surfactin (surfactin-C13, surfactin-C14 and surfactin-C15) to complete the technical solution of the present application. The source of surfactin is not further limited herein.

[0018] In a preferred embodiment, surfactin-C13, surfactin-C14 and surfactin-C15 are surfactins with different acyl structures obtained by liquid phase separation and purification of surfactin (HXRZ). Those skilled in the art can complete the separation and purification process according to actual conditions and prior art, so it is not specifically limited in this application.

[0019] Furthermore, the lipolysis includes dissolving fat cells and / or degrading adipose tissue.

[0020] Furthermore, the lipolysis drug does not break down during the lipolysis process.

[0021] On the other hand, the present application also provides a lipopeptide-containing lipolysis product.

[0022] Further, the lipopeptide is a cyclic lipopeptide; preferably, the lipopeptide is selected from one or more of surfactin, iturin, fennel, fengycin, daptomycin, polymyxin, bacillary antibiotic L, phospholipid inhibitor, and bacillary antibiotic D; more preferably, the lipopeptide is surfactin and / or daptomycin; more preferably, the lipopeptide is surfactin; more preferably, the surfactin is surfactin-C14 and / or surfactin-C15, that is, a β-hydroxy fatty acid whose acyl structure is C14 and / or C15.

[0023] Furthermore, in terms of mass percentage, the content of lipopeptide in the fat-dissolving product is 0.05%-3%; preferably, the content of lipopeptide is 0.1%-1%.

[0024] Preferably, the content of lipopeptides in the fat-dissolving product is 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3% and any percentage in between.

[0025] It can be understood by those skilled in the art that the lipolysis product also includes a pharmaceutically acceptable solvent, carrier and / or excipient, and the solvent can be selected from one or more of water, physiological saline, and PBS. In addition, the lipolysis product can also include pH regulators, osmotic pressure regulators, antibacterial agents, analgesics, anti-inflammatory agents and other known functional ingredients to enrich the use effect of the lipolysis product.

[0026] As mentioned above, it is known that sodium deoxycholate easily forms precipitation under physiological pH conditions of 7.35-7.45. In order to avoid this disadvantage, the pH value must be adjusted to 8.3, which causes pain during injection. Surfactin does not have this disadvantage. Therefore, those skilled in the art can adjust the pH of the fat-reducing product described in this application as needed, and no specific limitation on the pH range is made herein.

[0027] In a preferred embodiment, the pH value of the fat-dissolving product is in the range of 7.35-7.45.

[0028] Preferably, the dosage form of the product is selected from injection and / or topical preparation; preferably, the dosage form is injection.

[0029] On the other hand, the present application also provides a method for preparing the above-mentioned liposoluble product, the method comprising: uniformly dissolving the lipopeptide in a solvent.

[0030] Further, the lipopeptide is a cyclic lipopeptide; preferably, the lipopeptide is selected from one or more of surfactin, iturin, fennel, fengycin, daptomycin, polymyxin, bacillary antibiotic L, phospholipid inhibitor, and bacillary antibiotic D; more preferably, the lipopeptide is surfactin and / or daptomycin; more preferably, the lipopeptide is surfactin; more preferably, the surfactin is surfactin-C14 and / or surfactin-C15, that is, a β-hydroxy fatty acid whose acyl structure is C14 and / or C15.

[0031] More preferably, in terms of mass percentage, the content of the lipopeptide is 0.05%-3%; more preferably, the content of the lipopeptide is 0.1%-1%.

[0032] More preferably, the content of the lipopeptide is 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3% and any percentage in between.

[0033] Furthermore, the solvent is selected from one or more of water, physiological saline, and PBS.

[0034] The present invention has the following beneficial effects:

[0035] 1. The present invention first discovered that lipopeptides can be directly injected into subcutaneous adipose tissue to destroy the fat cell membrane, induce fat cell rupture, cause local dissolution of adipose tissue, and achieve the purpose of fat dissolution;

[0036] 2. The present invention proves that, compared with sodium deoxycholate, lipopeptide as a new active ingredient for lipolysis injection has stronger surface activity and lipolysis activity, and its effective dose is lower. It can also be seen from the onset time related experiments that the lipolysis mechanism of surfactin is different from that of sodium deoxycholate;

[0037] 3. The present invention uses lipopeptide as a new active ingredient for lipolysis injection, which can overcome the problem that the currently available sodium deoxycholate lipolysis injection is prone to adverse reactions and obvious pain during use, making the lipolysis injection safer and less prone to side effects, and can reduce ulceration at the injection site. It is more in line with the pH of the human body and reduces pain during the injection process. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0039] Figure 1 It is a statistical graph of relative proliferation rate of surfactin cells;

[0040] Figure 2 is the statistical graph of relative proliferation rate of daptomycin cells;

[0041] Figure 3 It is a statistical graph of relative proliferation rate of sodium deoxycholate cells;

[0042] Figure 4 This is the effect of surfactin and sodium deoxycholate on mouse adipocytes (observation results of adipocytes under an inverted microscope), with a magnification of 200 times;

[0043] Figure 5 This is the result of hematoxylin-eosin staining of the fat tissue of the left hip of the mouse, with a magnification of 5 times;

[0044] Figure 6 This is the lipolysis of adipose tissue of transgenic mice by 0.5% surfactin (A) and 1% surfactin (B), with a magnification of 5 times;

[0045] Figure 7 This is a picture of the ulceration of the administration site of each mouse in the 2% sodium deoxycholate group and the 2% surfactin group, where the circle marks the administration site;

[0046] Figure 8 The diagram is a picture of the ulceration of the administration site of each mouse in the 1% sodium deoxycholate group and the 1% surfactin group after two administrations, where the circle marks the administration site;

[0047] Fig. 9 This is a diagram of inguinal fat degradation in mice in the 0.5% surfactin group, where the circle marks the administration site;

[0048] Fig.10 It is the statistical graph of the left inguinal adipose tissue weight (A) and fat coefficient (B) of each group of mice;

[0049] Fig.11 The following are typical images of hematoxylin-eosin staining of adipose tissue of mice in each group (A, negative control; B, 1% sodium deoxycholate group; C, 1% surfactin group) and the statistical analysis results of average adipocyte density (D), where the magnification is 20 times;

[0050] Fig.12 This is a diagram showing the lysis effect of HXRZ and HXRZ-A, HXRZ-B and HXRZ-C on red blood cells;

[0051] Fig.13 These are the HE staining images of the pathological sections of adipose tissue in each group (each picture was taken at 200 times magnification, A. blank control group; B. positive control group; C. HXRZ-B high-dose administration group; D. HXRZ-B medium-dose administration group; E. HXRZ-B low-dose administration group; F. HXRZ-C high-dose administration group; G. HXRZ-C medium-dose administration group; H. HXRZ-C low-dose administration group). DETAILED DESCRIPTION

[0052] Technical terms:

[0053] Adipose tissue: is loose connective tissue composed mainly of fat cells. In humans, adipose tissue is located: beneath the skin (subcutaneous fat), around internal organs (visceral fat), in the bone marrow (yellow marrow), between muscles (muscular system), and in the breasts (breast tissue).

[0054] Ulceration: usually refers to skin ulceration, which often occurs when skin problems such as blisters, eczema, boils, etc. occur.

[0055] Topical preparations: Liquid, semisolid or solid preparations that act locally.

[0056] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0057] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0058] Among them, surfactin (HXRZ) is a laboratory-made sample, and the preparation method refers to patents CN114214252A and CN105695543B, with a purity of >98.5%; surfactin HXRZ-A is surfactin-C13, which is a laboratory-made sample and is obtained by purifying surfactin HXRZ through liquid phase separation; surfactin HXRZ-B is surfactin-C14, which is a laboratory-made sample and is obtained by purifying surfactin HXRZ through liquid phase separation; surfactin HXRZ-C is surfactin-C15, which is a laboratory-made sample and is obtained by purifying surfactin HXRZ through liquid phase separation.

[0059] Daptomycin was purchased from Hangzhou Sino-US Huadong Pharmaceutical Jiangdong Co., Ltd.; sodium deoxycholate was purchased from Solebao Biotechnology Co., Ltd.; and basal culture medium was purchased from Nanjing Keygene Biotechnology Development Co., Ltd.

[0060] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0061] Example 1 Comparison of the Surface Activity of Surfactin and Sodium Deoxycholate

[0062] Purified water was used to prepare 0.1% surfactin HXRZ solution and sodium deoxycholate solution, and then diluted to different gradient concentrations by stepwise dilution method, and the surface tension at different concentrations was measured by JZ-200A automatic interfacial tension meter. The results are shown in Table 1.

[0063] Table 1 CMC value determination results

[0064]

[0065] As shown in Table 1, 0.05% concentration of surfactin HXRZ can reduce the surface tension of water from 80mN / m to 33mN / m, while 0.1% concentration of sodium deoxycholate can only reduce the surface tension of water from 80mN / m to 53mN / m, which shows that the surface activity of surfactin HXRZ is stronger than that of sodium deoxycholate, that is, a lower concentration of surfactin HXRZ in water can form micelles, thereby reducing the surface tension of water. It can be inferred that when surfactin HXRZ is injected into adipose tissue, it can act on the cell membrane of adipocytes at a lower concentration to form micelles, thereby exerting a lipolysis effect.

[0066] Example 2 Comparison of half inhibitory concentration (IC50) of surfactin HXRZ, daptomycin and sodium deoxycholate on adipocytes

[0067] According to GB / T 16886.5 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test (GB / T16886.5-2017 / ISO 10993-5:2009, IDT), the test was conducted with reference to the method therein. The specific experimental process is as follows:

[0068] 1. Paving

[0069] Mouse adipocyte cell line 3T3-F442A in logarithmic growth phase was used as the 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate. Each well was cultured with 100 μL of basal culture medium supplemented with 10% fetal bovine serum. The inoculated cells were placed in a carbon dioxide incubator at 37°C and 5% CO 2 Routine culture for 24 hours.

[0070] 2. Sample solution preparation

[0071] Surfactin HXRZ, daptomycin or sodium deoxycholate was first prepared into a 0.5% solution using PBS, filtered through a 0.22 μm filter membrane for sterilization, and then diluted to the final concentration using serum-free cell culture medium for immediate use.

[0072] 3. Adding medicine

[0073] After 24 h of routine culture, the old culture medium was discarded and replaced with 100 μL sample solution in the experimental group and an equal amount of serum-free culture medium in the normal control group (control). At least 3 parallel wells were added for each level.

[0074] 4. Detection

[0075] After 24 hours of continuous culture, CCK-8 was used to detect the relative proliferation rate of cells. The culture medium was discarded, and 100 μL of CCK-8 diluted 10 times with serum-free culture medium was added to each well, and the cells were placed in a cell culture incubator for another 2 hours. The absorbance was measured at a wavelength of 450 nm using an enzyme marker. The relative proliferation rate (RGR) is the ratio of the absorbance of the experimental group to that of the normal control group.

[0076] 5. Results

[0077] The results of the relative proliferation rate determination of 3T3-F442A cells treated with surfactin HXRZ, daptomycin or sodium deoxycholate are shown in Table 2.

[0078] Table 2 Relative cell proliferation rate (%)

[0079]

[0080] According to the data in Table 2, the concentration is plotted as the horizontal axis and the relative cell proliferation rate is plotted as the vertical axis. Figure 1 , Figure 2 and Figure 3 The half-inhibitory concentrations IC50 of surfactin HXRZ, daptomycin and sodium deoxycholate were 0.0271 mg / mL, 0.1444 mg / mL and 0.2605 mg / mL, respectively. This shows that at the cellular level, surfactin HXRZ and daptomycin both have certain lipolysis activity, and the lipolysis activity of surfactin HXRZ is better than daptomycin and sodium deoxycholate.

[0081] Example 3 Comparison of the onset time of surfactin HXRZ and sodium deoxycholate on adipocytes

[0082] The experimental steps of Example 3 are the same as those of Example 2, except that the drug addition time is 0.5h, 4h and 24h. The relative proliferation rate determination results of 3T3-F442A cells treated with surfactin HXRZ and sodium deoxycholate for different time periods are shown in Table 3.

[0083] Table 3 Relative cell proliferation rate (%)

[0084]

[0085] As shown in Table 3, 0.5 mg / mL sodium deoxycholate can completely dissolve adipocytes after being treated for 0.5 h, while 0.08 mg / mL surfactin HXRZ needs to act on adipocytes for 24 h to completely dissolve them.

[0086] Figure 4 These are inverted microscope images taken after 0.08 mg / mL surfactin HXRZ and 0.5 mg / mL sodium deoxycholate acted on adipocytes for 4 hours and 24 hours, respectively. Figure 4It can be clearly seen that compared with the negative control, after 24 hours of treatment with surfactin HXRZ and 4 and 24 hours of treatment with sodium deoxycholate, there was almost no complete cell morphology and almost all the cells died.

[0087] It can be seen that compared with sodium deoxycholate, the effective concentration of surfactin HXRZ on adipocytes is lower, but the onset time is longer. It is speculated that the mechanism of action of surfactin HXRZ and sodium deoxycholate in dissolving adipocytes may be different.

[0088] Example 4 Experiment on the degradation of buttocks adipose tissue of transgenic obese mice by surfactin HXRZ and sodium deoxycholate

[0089] According to the results of Example 2, the half-inhibitory concentrations IC50 of surfactin HXRZ and sodium deoxycholate are 0.0271 mg / mL and 0.2605 mg / mL, respectively. Since the active ingredient of Kybella lipolysis injection is 1% sodium deoxycholate, the concentrations of surfactin HXRZ and sodium deoxycholate in the animal experiment were designed to be 0.1% and 1%, respectively.

[0090] Six-week-old transgenic obese mice (ob / ob) were used as the model. Different drugs were injected into the left buttock fat pad and divided into three groups, with 7-10 mice in each group. Specifically:

[0091] 1) Negative control group: injection of 200 μL of sterile saline;

[0092] 2) 1% sodium deoxycholate group: injection of 200 μL of 1% sodium deoxycholate sterile saline solution;

[0093] 3) 0.1% Surfactant HXRZ group: 200 μL of 0.1% Surfactant HXRZ sterile saline solution was injected.

[0094] The first administration was considered as the first day of the experiment, followed by administration on the third and fifth days. The mice were regularly observed for their condition and the site of administration during the experiment. The mice were killed on the 21st day of the experiment, and the adipose tissue on the left hip of the mice was removed by dissection and quickly fixed in adipose tissue fixative, followed by paraffin embedding. The adipose tissue was pathologically stained with hematoxylin-eosin staining, followed by optical microscopy analysis. The results are shown in Table 1. Figure 5 shown.

[0095] like Figure 5As shown, through the observation of adipose tissue sections stained with hematoxylin-eosin, it can be seen that compared with the negative control group, sodium deoxycholate and surfactin HXRZ both caused the degradation of adipose tissue at the administration site, which was manifested by the irregular morphology of the originally tightly arranged fat cells, swelling or rupture of the cells, inflammatory cell infiltration between cells, and the formation of new fibrous tissue in the area where the fat cells were degraded and cleared.

[0096] In addition, in the 1% sodium deoxycholate group, three mice had obvious lumps on the injection site, and one mouse had ulceration on the injection site. However, the above phenomenon did not occur in the surfactin HXRZ group and the negative control group, indicating that no adverse reactions occurred in the two groups.

[0097] Example 5 Experiment on the degradation of adipose tissue in transgenic mice with different concentrations of surfactin HXRZ

[0098] In this example, the degradation of adipose tissue in the buttocks of transgenic mice by surfactin HXRZ at different concentrations was further investigated.

[0099] Specifically, 6-week-old transgenic obese mice (ob / ob) were used as a model, with 1 mouse in each group, and 200 μL of 0.5% and 1% surfactin HXRZ saline solution (test sample) were injected into the left buttock fat pad. The first administration was regarded as the first day of the experiment, followed by administration once on the 3rd and 5th days. The condition of the mice and the site of administration were observed regularly during the experiment. On the 21st day of the experiment, the mice were killed, and the adipose tissue on the left buttock of the mice was dissected and removed, quickly fixed in adipose tissue fixative, and then embedded in paraffin. The adipose tissue was pathologically stained with hematoxylin-eosin staining, and then observed and analyzed under an optical microscope. The results are as follows: Figure 6 shown.

[0100] like Figure 6 As shown, both 0.5% and 1% surfactin HXRZ caused the degradation of mouse adipose tissue. The degradation range of adipose tissue in the 1% surfactin HXRZ group was greater than that in the 0.5% surfactin HXRZ group, indicating that the lipolysis activity of surfactin HXRZ is dose-dependent, and the greater the dose, the better the lipolysis effect.

[0101] Example 6 Preliminary study on the degradation effect of surfactin HXRZ and sodium deoxycholate on inguinal adipose tissue in mice

[0102] Six-week-old Kunming mice (20-30 g) were used as the model. 200 μL of different drugs were injected subcutaneously at multiple points in the left groin. The specific groups were as follows:

[0103] 1) Negative control group (3 rats): 200 μL sterile saline;

[0104] 2) 1% sodium deoxycholate group (3 mice): 200 μL 1% sodium deoxycholate saline solution;

[0105] 3) 2% sodium deoxycholate group (3 rats): 200 μL 2% sodium deoxycholate saline solution;

[0106] 4) 0.5% surfactin HXRZ group (3 mice): 200 μL 0.5% surfactin HXRZ saline solution;

[0107] 5) 1% surfactin HXRZ group (3 rats): 200 μL 1% surfactin HXRZ saline solution;

[0108] 6) 2% surfactin HXRZ group (3 rats): 200 μL 2% surfactin HXRZ saline solution;

[0109] The drug was administered every 7 days, and the ulceration of the administration site of each mouse was observed during the administration period. The mice were killed on the 21st day of the experiment, and the degradation of the left inguinal fat tissue of the mice was observed. Figure 7-9 shown.

[0110] Depend on Figure 7 It can be seen that after two administrations, the administration sites of mice in the 2% sodium deoxycholate group and the 2% surfactin HXRZ group all ulcerated, and they still could not heal after 7 days, so the administration was terminated. The degree of ulceration at the administration site of mice in the 2% surfactin HXRZ group was less than that in the 2% sodium deoxycholate group.

[0111] Depend on Figure 8 It can be seen that after two administrations, the administration sites of mice in the 1% surfactin HXRZ group and the 1% sodium deoxycholate group also had ulceration. It can be seen with the naked eye that the ulceration of the administration site of mice in the 1% surfactin HXRZ group was lighter than that in the 1% sodium deoxycholate group. However, after a 7-day administration interval, the ulceration was completely healed, and the administration was continued for 4 times thereafter, and no ulceration occurred in the mice in each group during this period.

[0112] Depend on Fig. 9 It can be seen that during the entire dosing period, no ulceration occurred at the dosing site of each mouse in the 0.5% surfactin HXRZ group. After the experiment, the inguinal fat tissue of the mice at the dosing site was dissected, and it was found that the edges of the inguinal tissue on the dosing side were blurred compared with the tissue on the non-dosing side, indicating that a certain degree of degradation had occurred. Example 7 Study on the efficacy and side effects of surfactin HXRZ and sodium deoxycholate on the degradation of inguinal fat tissue in mice

[0113] In this example, 6-week-old Kunming mice (20-30 g) were used as the model, and 200 μL of different drugs were injected subcutaneously at multiple points in the left groin. The specific groups are as follows:

[0114] 1) Negative control group (9 rats): 200 μL sterile saline;

[0115] 2) Sodium deoxycholate group (9 rats): 200 μL 1% sodium deoxycholate sterile saline solution;

[0116] 3) Surfactin HXRZ group (9 mice): 200 μL of 1% Surfactin HXRZ sterile saline solution.

[0117] Compared with Example 6, this example changed the dosing interval and administered the drug once every 14 days. On the 42nd day of the experiment, the mice were killed, and the left inguinal fat tissue of the mice was dissected and removed, and quickly fixed in fat tissue fixative, and then embedded in paraffin. The specific fat tissue weight and fat coefficient are shown in Table 4 and Fig.10 The adipose tissue was stained with hematoxylin-eosin staining, followed by optical microscopy analysis, and the adipocyte density was calculated. The results are shown in Table 5 and Fig.11 shown.

[0118] Table 4 Left inguinal adipose tissue weight and fat coefficient of each group of mice

[0119]

[0120] Note: Fat coefficient = (weight of inguinal fat tissue on the administration side / body weight) × 100%

[0121] *Compared with negative control group, p<0.05; **Compared with negative control group, p<0.01

[0122] As shown in Table 4 and Fig.10 As shown, compared with the negative control group, subcutaneous injection of 1% surfactant HXRZ and 1% sodium deoxycholate both caused a significant reduction in the inguinal fat tissue of mice, and the effects of the two were comparable without significant difference.

[0123] Table 5 Fat cell density of mice in each group

[0124]

[0125] Note: Adipocyte density = number of adipocytes in each image / adipose tissue area;

[0126] *Compared with negative control group, p<0.05

[0127] From Table 5 and Fig.11The results showed that hematoxylin-eosin staining of paraffin sections of adipose tissue, panoramic scanning of the sections using 3DHISTECH (Hungary), and analysis by Media Cybemetics (USA) showed that both 1% sodium deoxycholate and 1% surfactin HXRZ degraded the cells of the inguinal adipose tissue of mice, thereby reducing the average cell density of adipose tissue, such as Fig.11 As shown, the average adipocyte density in the 1% sodium deoxycholate group and the 1% surfactin HXRZ group was 727.84±56.64 ( The same below) and 676.94±51.326 were both lower than 868.2±57.02 of the negative control group, and there was a significant difference between the 1% surfactin HXRZ group and the negative control group. It can be seen that surfactin HXRZ and sodium deoxycholate both have a degradative effect on the inguinal adipose tissue of mice and there is no significant difference between the two. Surfactant can be used as a new active ingredient for lipolysis.

[0128] In addition, in this application, relevant experiments were conducted on the local inflammatory response of the administration site caused by surfactin HXRZ and sodium deoxycholate. After each administration, mice in the surfactin HXRZ group and the sodium deoxycholate group developed local inflammation, manifested as ulceration and subcutaneous nodules and lumps. Ulceration was used as a marker of severe local inflammation, and subcutaneous nodules and lumps were used as markers of mild local inflammation for statistics. The macrophages of the pathological sections were stained by F480 immunohistochemical staining, and then optical microscopy was performed for analysis. The specific situation is shown in Table 6.

[0129] Table 6 Local inflammation in mice in each group

[0130]

[0131] As can be seen from Table 6, except for the 35th day, the local inflammation of mice in the 1% surfactin HXRZ group was not higher than that in the 1% sodium deoxycholate group, and no ulceration of the administration site occurred in the mice in the 1% surfactin HXRZ group during the entire administration cycle, while some mice in the 1% sodium deoxycholate group had ulceration of the administration site after the first administration. It can be seen that compared with sodium deoxycholate, the present application proves that the use of surfactin HXRZ as a lipolytic component has the advantages of being safer and having fewer side effects.

[0132] Example 8 Comparison of the efficacy and safety of surfactin-C13, surfactin-C14 and surfactin-C15 - toxic effects on adipocytes

[0133] In this example, the effect of the length of the fatty acid chain and the effect of surfactin on the efficacy of adipocytes was further studied, and HXRZ and HXRZ-A, HXRZ-B and HXRZ-C were used as samples for the experiment. Specifically, surfactin HXRZ-A was surfactin-C13, surfactin HXRZ-B was surfactin-C14 and surfactin HXRZ-C was surfactin-C15.

[0134] 3T3-F442A mouse adipocytes in the logarithmic growth phase were collected and 1×10 5 The cells were inoculated in a 96-well plate at a density of 100 μL per well. After culturing for 24 h, 100 μL of culture medium containing samples of different concentrations was added, and the blank control group was added with an equal amount of serum-free culture medium and continued to be cultured for 24 h. The specific concentrations are shown in Table 7. CCK-8 was used to detect the relative cell proliferation rate. The blank control was set to 100%, and the relative proliferation rate and half inhibition concentration of each group were calculated using the same method as in Example 2.

[0135] As shown in Table 7, the IC values ​​of HXRZ and HXRZ-A, HXRZ-B, and HXRZ-C on adipocytes 50 Gradually decreasing, among which HXRZ-C IC 50 The lowest is about 1 / 2 of HXRZ-A. In other words, the killing effect on fat cells will increase with the extension of fatty acid chains, and HXRZ-C has the strongest toxicity to fat cells.

[0136] Table 7 Toxic concentrations of HXRZ and HXRZ-A, HXRZ-B, and HXRZ-C on adipocytes of 3T3-F442A mice

[0137]

[0138]

[0139] Note: The values ​​in the cells are the relative proliferation rates of each group when the proliferation rate of the blank control group is 100%. * indicates a significant difference compared with Control (p<0.05); # indicates a significant difference compared with HXRZ group at the same concentration (p<0.05); a indicates a significant difference compared with HXRZ-A group at the same concentration (p<0.05); b indicates a significant difference compared with HXRZ-B group at the same concentration (p<0.05); c indicates a significant difference compared with HXRZ-C group at the same concentration (p<0.05)

[0140] Example 9 Comparison of the efficacy and safety of surfactin-C13, surfactin-C14 and surfactin-C15 - Lysis of red blood cells

[0141] Take the finished sheep red blood cells and add physiological saline to make a red blood cell suspension. Prepare different concentrations of fermented surfactant HXRZ and HXRZ-A, HXRZ-B and HXRZ-C sample solutions with physiological saline, take 1mL of sample solution and add 1mL of red blood cell suspension to mix, and add an equal amount of physiological saline to the control group. Incubate in a 37℃ water bath for 10min, centrifuge and take the supernatant, and measure OD 575nm The values, determination results and specific concentrations are shown in Table 8 and Fig.12 shown.

[0142] Table 8 Lysis of red blood cells by HXRZ and HXRZ-A, HXRZ-B, and HXRZ-C

[0143] Concentration (mg / mL) HkDJ HXRZ-A HXRZ-B HXRZ-C 0.0078125mg / mL 0.268 0.2727 0.2657 0.2644 0.015625mg / mL 0.2834 0.2848 0.2746 0.2731 0.03125mg / mL 0.2841 0.2814 0.286 0.2777 0.0625mg / mL 0.4866 0.3333 0.4726 0.3761 0.125mg / mL 0.9445 1.0014 0.9256 0.7537 0.25mg / mL 0.9439 1.0377 1.0187 0.914

[0144] As shown in Table 8 and Fig.12 As shown in the figure, HXRZ and HXRZ-A, HXRZ-B and HXRZ-C began to lyse red blood cells at concentrations above 0.03125 mg / mL, and red blood cells were almost completely lysed at 0.25 mg / mL. There was no significant difference in the hemolytic effect of HXRZ-A, HXRZ-B and HXRZ-C.

[0145] Example 10 Comparison of the efficacy and safety of surfactin-C13, surfactin-C14 and surfactin-C15 - Animal experiment

[0146] (1) Experimental plan

[0147] Female Kunming mice (7-8 weeks old, weighing 25.55-33.76g) were selected and sorted by weight from small to large, and divided into 7-8 mice / group according to the Latin method. Each cage was marked with numbers on the tail of the mice with an oil-based marker pen. The specific groups were as follows: 1) blank control group; 2) positive control group (10 mg / mL sodium deoxycholate); 3) HXRZ-B high-dose group (10 mg / mL); 4) HXRZ-B medium-dose group (5 mg / mL); 5) HXRZ-B low-dose group (1 mg / mL); 6) HXRZ-C high-dose group (10 mg / mL); 7) HXRZ-C medium-dose group (5 mg / mL); 8) HXRZ-C low-dose group (1 mg / mL).

[0148] According to the positive drug injection dosage reported in the literature, the subcutaneous injection dose of this test was determined to be 10 mg / mL, 200 μL. A 4-needle water light needle (34G, 1.5 mm long, 0.6 mm inner diameter) was used for subcutaneous injection. The drugs were administered from top to bottom along the right groin. The drug was administered once every 14 days, for a total of 4 times.

[0149] During the administration period, the experimental animals in each group were kept under the same conditions and environment, and their eating, drinking and activities were observed every day. The administration site of each mouse was observed every day for ulceration, lumps, etc. Two mice in each group were killed and dissected 14 days after the second administration, and the remaining mice were all killed and dissected 14 days after the fourth administration.

[0150] (2) Experimental results

[0151] 2.1 Local adverse reactions

[0152] In this embodiment, statistics were conducted on the local adverse reactions at the administration site of experimental mice. The statistical results are shown in Table 9. Except for the abnormal adverse reaction of the first administration in the HXRZ-B high-dose group, the incidence of adverse reactions in the positive control group was higher than 60%, and the incidence of adverse reactions in the sample group was lower than 60%.

[0153] Table 9 Incidence of local adverse reactions in mice in each group after administration

[0154]

[0155] Note: The value in each grid is the percentage (%) of mice with abnormal inoculation site; abnormal inoculation site refers to redness, swelling, dark red scabs, and skin ulceration at the inoculation site; before the third administration, two mice were taken from each group for dissection to observe the interim results of the administration, so the number of animals in each group was reduced.

[0156] 2.2 Changes in adipose tissue at the administration site

[0157] In this embodiment, the changes in the fat tissue at the administration site of the experimental mice were statistically analyzed. Specifically, the inguinal fat tissue on the inoculated side (right side) was removed and weighed and recorded, and then the percentage of fat tissue on the inoculated side of each mouse - body weight was calculated [(weight of fat tissue on the inoculated side / mouse weight) × 100%]. The experimental results are shown in Table 10. The average wet fat weight and wet fat weight - body weight percentage of the positive drug and each administration group of mice were lower than those of the blank control group. The higher the drug concentration, the more obvious the effect, while there was no significant difference between the high-concentration and medium-concentration drugs and the positive drug group.

[0158] Table 10 Changes in adipose tissue weight at the administration site

[0159]

[0160] Note: * indicates comparison between the test group and the blank control group, *p<0.05, **p<0.01

[0161] The adipose tissue at the administration site was prepared into pathological sections and stained with HE. Fig.13As shown, each administration group resulted in the degradation of adipocytes, the formation of lipid lakes, the infiltration of immune cells and the generation of new connective tissue.

[0162] The density and of fat cells were further counted. The inoculated side fat was immersed in adipose tissue fixative and sent for hematoxylin-eosin (HE) staining. After imaging, the area of ​​fat tissue in each picture was measured in millimeters as the standard unit; the number of fat cells in each picture was counted; and the area of ​​a single fat cell was calculated = adipose tissue area / number of fat cells; fat cell density = number of fat cells / area of ​​fat tissue. The statistical results are shown in Table 11. After the drug acted, the fat tissue cells were destroyed, the cells enlarged, and the cell density decreased. After 4 doses, the fat tissue at the administration site had been reconstructed to form new connective tissue; the higher the drug concentration, the more obvious the effect. The effect of HXRZ-C was slightly better than that of HXRZ-B, which was similar to the results of wet fat weight.

[0163] Table 11 Changes in the average adipocyte density and average adipocyte area in each group of pathological sections

[0164]

[0165] Note: * indicates comparison between the test group and the blank control group, *p<0.05, **p<0.01

[0166] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Application of lipopeptides in the preparation of lipolysis products.

2. The use according to claim 1, characterized in that: The lipopeptide is a cyclic lipopeptide; preferably, the lipopeptide is selected from one or more of surfactin, iturin, feninterferon, fengyuansu, daptomycin, polymyxin, bacillary antibiotic L, phospholipid inhibitor, and bacillary antibiotic D; more preferably, the lipopeptide is surfactin and / or daptomycin; more preferably, the lipopeptide is surfactin; more preferably, the surfactin is surfactin-C14 and / or surfactin-C15.

3. The use according to claim 1, characterized in that: The lipolysis includes dissolving fat cells and / or degrading adipose tissue.

4. A fat-dissolving product containing lipopeptides.

5. The fat dissolving product according to claim 4, characterized in that: The lipopeptide is a cyclic lipopeptide; preferably, the lipopeptide is selected from one or more of surfactin, iturin, feninterferon, fengyuansu, daptomycin, polymyxin, bacillary antibiotic L, phospholipid inhibitor, and bacillary antibiotic D; more preferably, the lipopeptide is surfactin and / or daptomycin; more preferably, the lipopeptide is surfactin; more preferably, the surfactin is surfactin-C14 and / or surfactin-C15.

6. The fat dissolving product according to claim 4, characterized in that: In terms of mass percentage, the content of lipopeptide in the fat-dissolving product is 0.05%-3%; preferably, the content of lipopeptide is 0.1%-1%.

7. A method for preparing a fat dissolving product as claimed in any one of claims 4 to 6, characterized in that: The method comprises: uniformly dissolving the lipopeptide in a solvent.

8. The method according to claim 7, characterized in that The lipopeptide is a cyclic lipopeptide; preferably, the lipopeptide is selected from one or more of surfactin, iturin, feninterferon, fengyuansu, daptomycin, polymyxin, bacillary antibiotic L, phospholipid inhibitor, and bacillary antibiotic D; more preferably, the lipopeptide is surfactin and / or daptomycin; more preferably, the lipopeptide is surfactin; more preferably, the surfactin is surfactin-C14 and / or surfactin-C15.

9. The method according to claim 7, characterized in that: In terms of mass percentage, the content of the lipopeptide is 0.05%-3%; more preferably, the content of the lipopeptide is 0.1%-1%.

10. The method according to claim 7, characterized in that The solvent is selected from one or more of water, physiological saline and PBS.

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