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WO2023197248A1 - 一种生物制剂及促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法 - Google Patents

一种生物制剂及促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法 Download PDF

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WO2023197248A1
WO2023197248A1 PCT/CN2022/086841 CN2022086841W WO2023197248A1 WO 2023197248 A1 WO2023197248 A1 WO 2023197248A1 CN 2022086841 W CN2022086841 W CN 2022086841W WO 2023197248 A1 WO2023197248 A1 WO 2023197248A1
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mesenchymal stem
compound
stem cells
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biological
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汤熙翔
孙坪
黄文�
徐广鑫
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Xiamen Qianfu Marine Biotechnology Co Ltd
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Xiamen Qianfu Marine Biotechnology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention belongs to the technical field of cell biology, and specifically relates to the application of a biological agent and QF-1 compound in the preparation of biological agents that promote the adipogenic differentiation of mesenchymal stem cells and adipogenesis of adipose precursor cells and the promotion of mesenchymal stem cell differentiation.
  • Methods for adipogenesis and adipogenesis in adipogenic precursor cells are also known.
  • Wnt/ ⁇ -catenin signaling inhibits adipogenesis, either through the expression of Wnt1 or Wnt10b, or through the activation of Wnt/ ⁇ -catenin signaling by chemicals that stabilize free intracellular ⁇ -catenin, preventing adipogenesis.
  • Wnt/ ⁇ -catenin signaling prevents the induction of peroxisome proliferator-activated receptor- ⁇ (PPAR ⁇ ) and CCAAT/enhancer-binding protein ⁇ (C/EBP ⁇ ), two major adipogenic transcription factors that coordinately control Preadipocyte differentiation (adipogenesis).
  • PPAR ⁇ peroxisome proliferator-activated receptor- ⁇
  • C/EBP ⁇ CCAAT/enhancer-binding protein ⁇
  • ⁇ -catenin inhibits the transcriptional activity of PPAR ⁇ .
  • the purpose of the present invention is to study the pharmacological effects of QF-1 compounds in mesenchymal stem cells and adipocyte precursor cells, and find that QF-1 compounds can promote the adipogenic differentiation ability of mesenchymal stem cells and promote the adipogenic differentiation of adipocyte precursor cells.
  • Adipose synthesis ability thereby providing a biological preparation containing a QF-1 compound, the application of the QF-1 compound in the preparation of a biological preparation that promotes adipogenic differentiation of mesenchymal stem cells and adipogenesis of adipose precursor cells, and the use of QF-1 Methods for compounds to promote adipogenic differentiation of mesenchymal stem cells and adipogenesis of adipogenic precursor cells.
  • the first aspect of the present invention provides a biological preparation, wherein the biological preparation uses a QF-1 compound having a structure represented by formula (1) as an active ingredient:
  • the biological preparation is a cosmetic, health product, medical device, medicine or external dressing.
  • the biological preparation in addition to the QF-1 compound, also includes various pharmaceutically acceptable excipients.
  • the auxiliary material is at least one of a coating material, a solvent, a solubilizer, a binder, a stabilizer, an antioxidant, a pH adjuster and a flavoring agent.
  • the biological preparation is administered by injection, oral administration or transdermal absorption.
  • the dosage form of the biological preparation is a tablet, capsule, oral liquid, injection, powder injection, dressing or transdermal preparation.
  • a second aspect of the present invention provides the use of a QF-1 compound represented by formula (1) in the preparation of biological preparations that promote adipogenic differentiation of mesenchymal stem cells and adipogenesis of adipose precursor cells.
  • the QF-1 compound is the active ingredient in the biological preparation.
  • the biological preparation in addition to the QF-1 compound, also includes various pharmaceutically acceptable excipients.
  • the auxiliary material is at least one of a coating material, a solvent, a solubilizer, a binder, a stabilizer, an antioxidant, a pH adjuster and a flavoring agent.
  • the biological preparation is administered by injection, oral administration or transdermal absorption.
  • the dosage form of the biological preparation is a tablet, capsule, oral liquid, injection, powder injection, dressing or transdermal preparation.
  • a third aspect of the present invention provides a method for promoting adipogenic differentiation of mesenchymal stem cells and adipogenesis of adipocyte precursor cells, by administering a QF-1 compound having a structure represented by formula (1) to a subject:
  • the methods include therapeutic methods and non-therapeutic methods.
  • the non-therapeutic methods include health care methods and scientific research methods.
  • the subject is a human or other mammal.
  • the single application dose of the QF-1 compound is 10 -3 ⁇ 10 3 mg/kg.
  • the applicable scenarios of the method include body beauty shaping, breast enlargement, and buttock enlargement.
  • the QF-1 compound provided by the present invention is used in mesenchymal stem cells and adipocyte precursor cells. Through research, it is found that the QF-1 compound can activate the PPAR ⁇ signaling pathway of cells and promote the formation of fat. Therefore, the QF-1 compound can promote the adipogenic differentiation ability of mesenchymal stem cells and the fat synthesis ability of adipocyte precursor cells, and can therefore be used in the technical fields of body beauty shaping, breast enlargement, buttock augmentation and other fat increase technologies.
  • Figure 1 shows the absorbance values at 500nm after adding different final concentrations of 0 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, 40 ⁇ M and 60 ⁇ M QF-1 compounds after 14d and 28d adipogenic induction;
  • Figure 2 shows the microscopic observation of umbilical cord mesenchymal stem cells after adding 0 ⁇ M and 10 ⁇ M QF-1 compounds for 28 days;
  • Figure 3 shows the relative luciferase activity of adipose precursor cells induced by adding QF-1 compounds at different final concentrations of 0 ⁇ M, 5 ⁇ M, 10 ⁇ M, and 20 ⁇ M. ****P ⁇ 0.0001 indicates comparison with the control group.
  • the QF-1 compound can be purchased commercially or prepared according to various existing methods.
  • the QF-1 compound is prepared according to the following method:
  • step S2 Extract the Aspergillus terreus fermentation product obtained in step S1 with an ester solvent, and obtain an ester-soluble Aspergillus terreus crude extract after concentration of the obtained extract;
  • step S3 Separate the ester-soluble Aspergillus terreus crude extract obtained in step S2 by silica gel chromatography, and then remove the solvent in the obtained QF-1 enriched solution to obtain the QF-1 crude extract;
  • step S4 Heat the QF-1 crude extract obtained in step S3 to prepare a methanol-water supersaturated solution, slowly cool down, add distilled water to the obtained supernatant and then centrifuge.
  • the solid product obtained by the centrifugal separation is the QF-1 compound.
  • the purity of the QF-1 compound obtained by using the above preferred methods is higher, and the effect is better when it is used to promote adipogenic differentiation of mesenchymal stem cells and adipogenesis of adipose precursor cells.
  • the key to using the above preferred method to prepare QF-1 compounds is to use Aspergillus terreus as a strain for fermentation and culture, and at the same time, use extraction, silica gel chromatography, and hot alcohol water supersaturated impurity removal phase in combination.
  • the difference in solubility of QF-1 and related impurities in the methanol-water supersaturated solution is used to heat the mixture to form a methanol-water supersaturated solution. After slowly cooling the temperature, the impurities are removed. In this way, QF-1 is easy to enrich. Collect, the purity of the obtained QF-1 compound can reach more than 95% by HPLC analysis, and the total yield can reach more than 23.3%.
  • step S1 Aspergillus terreus is fermented in a fungus culture medium to obtain an Aspergillus terreus fermentation product.
  • the fermentation conditions are not particularly limited as long as they can produce the QF-1 compound.
  • the fermentation temperature is preferably 25 to 30°C, such as 25°C, 26°C, 27°C, 28°C, 29°C, 30°C. ° C, etc.;
  • the fermentation time is preferably 7 to 60 days.
  • the culture time of liquid fungal fermentation medium is preferably 7 to 15 days, such as 7, 8, 9, 10, 11, 12, 13, 14, 15 days, etc.
  • the culture time of solid fungal fermentation medium is preferably 20 to 60 days, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 days wait.
  • the Aspergillus terreus can be obtained in various existing ways, usually through commercial purchase.
  • the type of the fungal culture medium is not particularly limited and can be conventionally selected in the art.
  • it can be based on cereal starch (corn, potato, tapioca starch), glucose, molasses, ammonia, ammonium salt or nitrate, At least one of commonly used nitrogen sources and carbon sources such as urea, corn steep liquor, soybean cake powder, peanut cake powder, fish meal, yeast extract, etc. is used as the culture medium for carbon and nitrogen sources.
  • cereal starch corn, potato, tapioca starch
  • glucose molasses
  • ammonia ammonium salt or nitrate
  • At least one of commonly used nitrogen sources and carbon sources such as urea, corn steep liquor, soybean cake powder, peanut cake powder, fish meal, yeast extract, etc. is used as the culture medium for carbon and nitrogen sources.
  • step S2 the Aspergillus terreus fermentation product obtained in step S1 is extracted with an ester solvent, and then the solvent in the obtained QF-1 enriched liquid is removed to obtain a QF-1 crude extract.
  • the extraction method only needs to be able to separate the QF-1 compound from most impurities, with QF-1 entering the extraction liquid and the impurities entering the raffinate.
  • the extraction method is to use an equal volume of ester organic solvent to ultrasonically extract the Aspergillus terreus fermentation product 2 to 4 times (such as 2, 3, 4 times), and extract 20 times each time independently.
  • ester organic solvent include, but are not limited to: at least one of methyl acetate, ethyl acetate, butyl acetate and phenyl acetate.
  • step S3 the ester-soluble Aspergillus terreus crude extract obtained in step S2 is separated by silica gel chromatography, and then the solvent in the obtained QF-1 enriched solution is removed to obtain the QF-1 crude extract.
  • the particle size of the silica gel filler packed in the silica gel chromatography column used for the silica gel chromatography separation can be 70-230 mesh, 230-400 mesh, 60-100 mesh, 100-200 mesh or 200-300 mesh, preferably 200 ⁇ 300 mesh.
  • the elution solvent used can be used as long as it can separate QF-1 from impurities, preferably dichloromethane and acetone in a volume ratio of 100: (1 to 10) (such as 100:1, 100:2, 100:3, 100 :4, 100:5, 100:6, 100:7, 100:8, 100:9, 100:10) mixed solvents.
  • the method of removing the solvent may be, for example, rotary evaporation, freeze-drying, etc., and is not particularly limited.
  • step S4 the QF-1 crude extract obtained in step S3 is heated to form a methanol-water supersaturated solution, the temperature is slowly cooled, the obtained supernatant is added with distilled water and centrifuged.
  • the solid product obtained by the centrifugal separation is QF-1. compound.
  • the QF-1 crude extract is dissolved in methanol, and then the resulting QF-1 methanol solution is heated while adding water to form a methanol-water supersaturated solution, and then slowly cooled to 0 to 5°C and absorbed.
  • the supernatant is added with an equal volume of distilled water and then centrifuged.
  • the solid product obtained by the centrifugal separation is the QF-1 compound.
  • This step utilizes the principle of different solubilities of QF-1 compounds and related impurities in good solvent methanol and poor solvent water.
  • the QF-1 crude extract is heated to make a methanol-water supersaturated solution. After slowly cooling down, the solubility of the impurities decreases. Precipitate out to further separate QF-1 from impurities, thereby improving the purity of QF-1 product.
  • the heating conditions are preferably such that the temperature of the obtained supersaturated solution is 50°C to 55°C.
  • the amount of methanol used is preferably just enough to completely dissolve the QF-1 crude extract, so that the purity of the QF-1 product obtained is better.
  • the slow cooling is performed in a low temperature environment, for example, at 0 to 5°C.
  • the QF-1 compound is preferably the active ingredient in the biological preparation.
  • the biological preparations in addition to the QF-1 compound, also include various acceptable excipients.
  • the auxiliary materials may be at least one of coating materials, solvents, solubilizers, adhesives, stabilizers, antioxidants, pH adjusters and flavoring agents.
  • the administration mode of the biological preparation is injection, oral administration or transdermal absorption.
  • the non-therapeutic methods include but are not limited to health care methods and scientific research methods, and the implementation objects can be humans or other mammals, and the other mammals can be mice, rabbits, and dogs.
  • the total yield of the product actual yield of the target product/theoretical yield of the target product ⁇ 100%, where the theoretical yield of the target product is derived from the content ratio of the HPLC spectrum.
  • Ferment Aspergillus terreus purchased from Shanghai Preservation Biotechnology Center, ATCC 34571 in rice culture medium (composed of rice and water according to a mass ratio of 1:1.5) at 25°C for 45 days to obtain Aspergillus terreus fermentation product.
  • step S3 Separate the ester-soluble Aspergillus terreus crude extract obtained in step S2 through 200-300 mesh silica gel chromatography, use a dichloromethane-acetone mixed solution with a volume ratio of 100:3 as the eluent, and collect QF-1 The enriched liquid was evaporated to dryness using a rotary evaporator to obtain a crude extract of QF-1. After HPLC analysis, the QF-1 crude extract contained QF-1 compound with a purity of 70%.
  • step S4 Dissolve 1g of QF-1 crude extract obtained in step S3 in 1mL of methanol, add 1.6mL of distilled water while heating to form a methanol-water supersaturated solution with a temperature of 50°C, and then place it in a 4°C refrigerator to cool slowly for 30 minutes. Then suck out the supernatant; continue to add 200 ⁇ L of methanol to the lower layer precipitate, and add 400 ⁇ L of distilled water while heating to form a methanol-water supersaturated solution with a temperature of 50°C. Place it in a 4°C refrigerator to slowly cool down. After 30 minutes, suck out the supernatant and precipitate the lower layer.
  • Example 1 QF-1 promotes adipogenic differentiation of mesenchymal stem cells
  • step S2 Resuspend the cells that were completely digested in step S1 in DMEM/F2 culture medium with 10% FBS and 1% 100X streptomycin-penicillin double antibody solution, and use 10% FBS and 1% 100X streptomycin-penicillin double antibody solution. Dilute the cell concentration to 4 ⁇ 10 5 cells/mL in anti-solution DMEM/F2 culture medium, and transfer 1 mL per well into a 24-well cell culture plate for overnight culture;
  • step S3 Cultivate the cells in step S2 until they adhere to the wall, aspirate the culture medium, and add the QF-1 compound obtained from the preparation example (the same below) containing different final concentrations of 0, 1, 5, 10, 20, 40, and 60 ⁇ M.
  • the adipogenesis induction medium is cultured, and the adipogenesis induction medium is 10% FBS, 1% 100X streptomycin-penicillin double antibody solution, 1 ⁇ M dexamethasone, 0.5mM 3-isobutyl-1-methyl yellow Purine, 0.2mM indomethacin, and 10mg/L insulin in DMEM medium; and leave 3 wells for culture in DMEM/F2 medium containing 10% FBS and 1% 100X streptomycin-penicillin double antibody solution as a control;
  • Oil Red O staining steps include: (1) Aspirate the adipogenic induction medium and add PBS buffer for washing twice, then add 4% paraformaldehyde for cell fixation for 20 minutes, rinse twice with double-distilled water, then add 60% isoenzyme Infiltrate with the propanol solution for 5 minutes; (2) Remove the isopropyl alcohol and dry the cells naturally; (3) Add 0.5 mL of Oil Red O staining working solution (composed of 0.35% Oil Red O isopropyl alcohol solution and double-distilled water) to each well Mix according to the volume ratio of 3:2 and let it stand for 20 minutes, then filter it through a 0.22 ⁇ m pore size filter membrane to obtain) and stain it for 10 minutes; (4) Aspirate the staining solution and immediately rinse it 4 times with
  • the detection steps of OD 500nm absorbance include: (1) Add 250 ⁇ L of 100% isopropyl alcohol to each well, shake gently for 20 minutes, then pipet 150 ⁇ L into a 96-well plate, use 100% isopropyl alcohol as a blank control, and use enzyme Use a standard instrument to measure the absorbance of OD 500nm in different well plates respectively; (2) Use the measured value of OD 500nm of each well and subtract the background value of the absorbance of OD 500nm of mesenchymal stem cells (MSC) to calculate adipogenic differentiation. The Oil Red O absorption value was compared with the obtained results. The obtained results are shown in Table 1 and Figure 1.
  • Example 2 QF-1 promotes adipogenesis of adipocyte precursor cells
  • PPAR ⁇ response element (5'-taggtcaaaggtcaa-3') to synthesize DNA with BmtI restriction site at 5', BglII restriction site at 3', and 4 repeated PPAR ⁇ response elements inside.
  • the sequence is as follows: 5'-gccGCTAGC taggtcaaaggtca taggtcaaaggtca taggtcaaaggtca taggtcaaaggtca taggtcaaaggtcaAGATCTccg-3'.
  • the synthesized DNA and pGL6-TA plasmid were subjected to restriction enzyme digestion with BmtI enzyme and BglII enzyme and then gel recovery. After overnight ligation with T4 DNA ligase, they were introduced into the DH5 ⁇ strain to construct the pGL6-PPAR ⁇ -Luc plasmid with PPAR ⁇ transcription activity detection function. .
  • step S3 Resuspend the cells that were completely digested in step S2 in DMEM/F2 culture medium with 10% FBS and 1% 100X streptomycin-penicillin double antibody solution, count them, and seed them in a 96-well plate at 1 ⁇ 10 4 cells/well. , the final total volume of culture medium per well is 100 ⁇ L.
  • adipogenesis induction medium is 10% FBS, 1% 100X streptomycin- Penicillin double antibody solution, 1 ⁇ M dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine, 0.2mM indomethacin, 10mg/L insulin (DMEM medium) for adipogenic culture, and add the final concentration QF-1 compounds of 0 ⁇ M, 5 ⁇ M, 10 ⁇ M, and 20 ⁇ M were continuously cultured for 24 h, and three replicate wells were made for each concentration.
  • the adipogenesis induction medium is 10% FBS, 1% 100X streptomycin- Penicillin double antibody solution, 1 ⁇ M dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine, 0.2mM indomethacin, 10mg/L insulin (DMEM medium) for adipogenic culture, and add the final concentration QF-1 compounds of 0 ⁇ M, 5 ⁇ M, 10 ⁇ M, and 20 ⁇

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Abstract

本发明属于细胞生物学技术领域，公开了一种生物制剂、QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用及促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法，所述QF-1化合物具有式(1)所示的结构。将QF-1化合物应用于培养充质干细胞和脂肪前体细胞后可以激活细胞的PPARγ信号通路，促进脂肪的生成。因此，QF-1化合物能够促进间充质干细胞的成脂分化能力以及促进脂肪前体细胞的脂肪合成能力，从而可应用于机体美容塑形、丰胸、丰臀等脂肪增多技术领域。

Description

一种生物制剂及促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法 技术领域
本发明属于细胞生物学技术领域，具体涉及一种生物制剂、QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用及促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法。
背景技术
在医学美容领域，促进特定部位的脂肪组织生长具有重要的应用。目前丰胸、丰臀等医学美容项目普遍采用可植入假体或采用自体脂肪进行填充。然而，以上方式均为手术项目，存在一定的风险，且自体脂肪填充由于植入的自体脂肪无法在植入部位获得有效的营养供给导致其在较短的时间内为机体所吸收，从而降低了实际效果。
研究结果表明，Wnt/β-catenin信号抑制脂肪的生成，通过Wnt1或Wnt10b的表达，或通过稳定细胞内游离β-连环蛋白的化学物质激活Wnt/β-连环蛋白信号，阻止脂肪生成。Wnt/β-catenin信号可阻止过氧化物酶体增殖物激活受体-γ(PPARγ)和CCAAT/增强子结合蛋白α(C/EBPα)的诱导，这两种主要的脂肪生成转录因子协同控制前脂肪细胞分化(脂肪生成)。此外，β-连环蛋白抑制PPARγ的转录活性。
为了使得医学美容项目顺利推进，亟需依据皮肤屏障的结构特点，寻找一种可以透皮吸收的、促进局部脂肪增生的化合物，从而促进间充质干细胞的成脂分化能力、促进脂肪前体细胞的脂肪合成能力。
发明内容
本发明的目的在于通过对QF-1化合物在间充质干细胞和脂肪前体细胞中的药理作用研究，发现QF-1化合物能够促进间充质干细胞的成脂分化能力以 及促进脂肪前体细胞的脂肪合成能力，从而提供了一种含有QF-1化合物的生物制剂、QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用以及通过QF-1化合物促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法。
具体地，本发明的第一方面提供了一种生物制剂，其中，所述生物制剂中以具有式(1)所示结构的QF-1化合物作为活性成分：
Figure PCTCN2022086841-appb-000001
在一种优选实施方式中，所述生物制剂为化妆品、保健品、医疗器械、药品或外敷料。
在一种优选实施方式中，所述生物制剂中，除QF-1化合物外，还包括药学上可接受的各种辅料。
在一种优选实施方式中，所述辅料为包衣材料、溶剂、增溶剂、粘合剂、稳定剂、抗氧化剂、pH调节剂和矫味剂中的至少一种。
在一种优选实施方式中，所述生物制剂的使用方式为注射、口服或者透皮吸收。
在一种优选实施方式中，所述生物制剂的剂型为片剂、胶囊剂、口服液、针剂、粉针剂、敷剂或透皮给药制剂。
本发明的第二方面提供了QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用，所述QF-1化合物具有式(1)所示的结构：
Figure PCTCN2022086841-appb-000002
在一种优选实施方式中，所述生物制剂中QF-1化合物为活性成分。
在一种优选实施方式中，所述生物制剂中，除QF-1化合物外，还包括药 学上可接受的各种辅料。
在一种优选实施方式中，所述辅料为包衣材料、溶剂、增溶剂、粘合剂、稳定剂、抗氧化剂、pH调节剂和矫味剂中的至少一种。
在一种优选实施方式中，所述生物制剂的给药方式为注射、口服或者透皮吸收。
在一种优选实施方式中，所述生物制剂的剂型为片剂、胶囊剂、口服液、针剂、粉针剂、敷剂或透皮给药制剂。
本发明的第三方面提供了一种促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法，向对象施用具有式(1)所示结构的QF-1化合物：
Figure PCTCN2022086841-appb-000003
在一种优选实施方式中，所述方法包括治疗方法和非治疗方法。
在一种优选实施方式中，所述非治疗方法包括保健方法和科学研究方法。
在一种优选实施方式中，所述对象为人或或其他哺乳动物。
在一种优选实施方式中，所述QF-1化合物的单次应用剂量为10 -3～10 3mg/kg。
在一种优选实施方式中，所述方法的适用场景包括机体美容塑形、丰胸、丰臀。
本发明的有益技术效果：
本发明所提供的QF-1化合物用于间充质干细胞和脂肪前体细胞中，通过研究发现，QF-1化合物可以激活细胞的PPARγ信号通路，促进脂肪的生成。因此，QF-1化合物能够促进间充质干细胞的成脂分化能力以及促进脂肪前体细胞的脂肪合成能力，从而可应用于机体美容塑形、丰胸、丰臀等脂肪增多技术领域。
附图说明
图1为添加不同终浓度为0μM、5μM、10μM、20μM、40μM、60μM QF-1 化合物在进行14d和28d成脂诱导后于500nm下的吸光度值；
图2为添加0μM与10μM QF-1化合物经28d诱导后的脐带间充质干细胞显微镜观察图；
图3为添加不同终浓度为0μM、5μM、10μM、20μM QF-1化合物诱导脂肪前体细胞的相对荧光素酶活性，****P＜0.0001表示与对照组的比较。
具体实施方式
在本发明中，所述QF-1化合物可以通过商购获得，也可以按照现有的各种方法制备得到。
在一种优选实施方式中，所述QF-1化合物按照以下方法制备得到：
S1、将土曲霉在真菌培养基中进行发酵，得到土曲霉发酵物；
S2、将步骤S1所得土曲霉发酵物采用酯类溶剂进行萃取，所得萃取液经浓缩后得到酯溶性土曲霉粗提物；
S3、将步骤S2所得酯溶性土曲霉粗提物进行硅胶色谱分离，再将所得QF-1富集液中的溶剂去除，得到QF-1粗提物；
S4、将步骤S3所得QF-1粗提物加热制成甲醇-水过饱和溶液，缓慢降温，将所得上清液加蒸馏水后离心分离，所述离心分离所得固体产物即为QF-1化合物。
较采用现有方式而言，采用以上优选方式所得QF-1化合物的纯度更高，将其应用于促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成时效果更佳。采用以上优选方式制备QF-1化合物的关键在于以土曲霉作为菌株进行发酵培养，同时将萃取、硅胶色谱分离以及热醇水过饱和除杂相配合使用，其中，在热醇水过饱和除杂过程中，利用QF-1及相关杂质在甲醇-水过饱和溶液中溶解度的不同，将混合物加热制成甲醇-水过饱和溶液，缓慢降温之后再将杂质去除，如此操作，QF-1易于富集，所得QF-1化合物经HPLC分析纯度可达95％以上，总收率可达到23.3％以上。
步骤S1中，将土曲霉在真菌培养基中进行发酵，得到土曲霉发酵物。其中，所述发酵的条件只要能够生成QF-1化合物即可，没有特别的限定，例如， 发酵温度优选为25～30℃，如25℃、26℃、27℃、28℃、29℃、30℃等；发酵时间优选为7～60天，其中，选用液体真菌发酵培养基的培养时间优选为7～15天，如7、8、9、10、11、12、13、14、15天等；选用固体真菌发酵培养基的培养时间优选为20～60天，如20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60天等。此外，所述土曲霉可以采用现有的各种方式获得，通常通过商购获得。此外，所述真菌培养基的种类没有特别的限定，可以为本领域的常规选择，例如，可以为以谷物淀粉(玉米、马铃薯、木薯淀粉)、葡萄糖、糖蜜、氨水、铵盐或硝酸盐、尿素、玉米浆、豆饼粉、花生饼粉、鱼粉、酵母浸出膏等常用氮源、碳源中的至少一种作为碳氮源的培养基。
步骤S2中，将步骤S1所得土曲霉发酵物采用酯类溶剂进行萃取，再将所得QF-1富集液中的溶剂去除，得到QF-1粗提物。其中，所述萃取的方式只要能够将QF-1化合物与大部分杂质分离，QF-1进入萃取液，杂质进入萃余液即可。在一种优选实施方式中，所述萃取的方式为将土曲霉发酵物采用等体积的酯类有机溶剂超声萃取2～4次(如2、3、4次)，每次各自独立地萃取20～40min(如20、22、25、28、30、32、35、38、40min)，之后合并萃取液并蒸干，得到酯溶性土曲霉粗提物。此外，所述酯类有机溶剂的具体实例包括但不限于：乙酸甲酯、乙酸乙酯、乙酸丁酯和乙酸苯酯中的至少一种。
步骤S3中，将步骤S2所得酯溶性土曲霉粗提物进行硅胶色谱分离，再将所得QF-1富集液中的溶剂去除，得到QF-1粗提物。其中，所述硅胶色谱分离所采用的硅胶色谱柱中装填的硅胶填料的粒径可以为70～230目、230～400目、60～100目、100～200目或200～300目，优选为200～300目。所采用的洗脱溶剂只要能够使得QF-1与杂质分离即可，优选为二氯甲烷和丙酮按照体积比100:(1～10)(如100:1、100:2、100:3、100:4、100:5、100:6、100:7、100:8、100:9、100:10)的混合溶剂。此外，将溶剂去除的方式例如可以为旋蒸、冻干等，没有特别的限定。
步骤S4中，将步骤S3所得QF-1粗提物加热制成甲醇-水过饱和溶液，缓 慢降温，将所得上清液加蒸馏水后离心分离，所述离心分离所得固体产物即为QF-1化合物。具体地，将所述QF-1粗提物溶于甲醇中，再将所得QF-1甲醇溶液边加热边加入水形成甲醇-水过饱和溶液，之后放入缓慢冷却至0～5℃，吸取上清液加等体积蒸馏水后离心分离，所述离心分离所得固体产物即得QF-1化合物。此步骤利用QF-1化合物及相关杂质在良溶剂甲醇和不良溶剂水中溶解度的不同的原理，将QF-1粗提物加热制成甲醇-水过饱和溶液，缓慢降温后，由于杂质溶解度降低而析出沉淀，以实现QF-1与杂质的进一步分离，从而提高QF-1产物的纯度。其中，所述加热的条件优选使所得过饱和溶液的温度为50～55℃。此外，所述甲醇的用量优选恰好使得QF-1粗提物完全溶解，如此所得QF-1产物的纯度更佳。所述缓慢冷却在低温环境中进行，例如，在0～5℃下进行。
在本发明中，所述生物制剂中QF-1化合物优选为活性成分。此外，所述生物制剂中，除QF-1化合物外，还包括可接受的各种辅料。所述辅料可以为包衣材料、溶剂、增溶剂、粘合剂、稳定剂、抗氧化剂、pH调节剂和矫味剂中的至少一种。所述生物制剂的给药方式为注射、口服或者透皮吸收。
在本发明中，当通过QF-1化合物促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成时，包括治疗方法和非治疗方法。其中，所述非治疗方法包括但不限于保健方法以及科学研究方法，实施对象可以为人或其他哺乳动物，所述其他哺乳动物可以为鼠、兔、犬。
以下将通过实施例对本发明进行详细描述。
以下制备例中，产物的总收率＝目标产物实际产量/目标产物理论产量×100％，其中，目标产物理论产量由HPLC谱图含量比例得出。
制备例
S1、将土曲霉(购自上海保藏生物技术中心，ATCC 34571)在大米培养基(由大米和水按照质量比1:1.5组成)中于25℃下发酵45天，得到土曲霉发酵物。
S2、将步骤S1所得土曲霉发酵物采用等体积的乙酸乙酯超声萃取三次， 每次30min，所得萃取液合并后用旋转蒸发仪蒸干，得到酯溶性土曲霉粗提物。
S3、将步骤S2所得酯溶性土曲霉粗提物经200～300目的硅胶色谱分离，以体积配比为100:3的二氯甲烷-丙酮混合溶液作为洗脱剂进行洗脱，收集QF-1富集液，将其用旋转蒸发仪蒸干，得到QF-1粗提物。经HPLC分析，该QF-1粗提物中含有纯度为70％的QF-1化合物。
S4、将步骤S3所得QF-1粗提物1g溶于1mL甲醇中，边加热边加入1.6mL蒸馏水形成温度为50℃的甲醇-水过饱和溶液，之后放入4℃冰箱中缓慢冷却，30min后吸出上清液；下层沉淀继续加入200μL甲醇，边加热边加入400μL蒸馏水形成温度为50℃的甲醇-水过饱和溶液，放入4℃冰箱缓慢降温，30min后吸出上清液，将下层沉淀采用该步骤重复操作三次；将所有上清液合并，加等体积蒸馏水后离心分离，得到240mg浅黄色的沉淀物，即为QF-1固体，经HPLC检测，其纯度为95.53％，收率为34.29％。从 1H NMR图谱和 13C NMR图谱可以看出，QF-1化合物具有式(1)所示的结构。
实施例1 QF-1促进间充质干细胞的成脂分化
(1)实验过程
S1、将第三代的脐带间充质干细胞采用含10％FBS、1％100X链霉素-青霉素双抗溶液的DMEM/F2培养基培养至80％融合后，加入0.25％胰酶消化至细胞悬浮后再加入3倍体系的10％FBS、1％100X链霉素-青霉素双抗溶液的DMEM/F2培养基终止消化，300g离心5min后用PBS缓冲液同等条件洗涤2次，得到消化完全的细胞；
S2、将步骤S1中消化完全的细胞用10％FBS、1％100X链霉素-青霉素双抗溶液的DMEM/F2培养基进行重悬，并用10％FBS、1％100X链霉素-青霉素双抗溶液的DMEM/F2培养基稀释细胞浓度至4×10 5cells/mL，每孔1mL转入24孔细胞培养板中进行过夜培养；
S3、将步骤S2中的细胞培养至贴壁后吸去培养基，分别加入含0、1、5、10、20、40、60μM不同终浓度的由制备例所得QF-1化合物(下同)成脂诱导培养基进行培养，所述成脂诱导培养基为10％FBS、1％100X链霉素-青霉 素双抗溶液、1μM地塞米松、0.5mM 3-异丁基-1-甲基黄嘌呤、0.2mM吲哚美辛、10mg/L胰岛素的DMEM培养基；并留3孔采用含10％FBS、1％100X链霉素-青霉素双抗溶液的DMEM/F2培养基进行培养作为对照；
S4、将步骤S3中的细胞进行每3d换液，分别培养至14d和28d后终止培养并进行油红O染色，检测500nm下的吸光度值。其中，油红O染色步骤包括：(1)吸取成脂诱导培养基并加入PBS缓冲液进行冲洗2遍后加入4％多聚甲醛进行细胞固定20min，双蒸水冲洗2遍后加入60％异丙醇溶液浸润5min；(2)去除异丙醇后对细胞进行自然干燥；(3)每孔加入0.5mL的油红O染色工作液(由0.35％油红O异丙醇溶液与双蒸水按体积比3:2混合后静置20min，而后经0.22μm孔径滤膜过滤得到)进行染色10min；(4)吸去染色液，立即用1mL的双蒸水冲洗4遍；(5)于显微镜下观察细胞的染色情况并拍照后让培养孔自然干燥，留待500nm下的吸光度值(OD 500nm)的检测。其中，OD 500nm吸光度的检测步骤包括：(1)于每孔中加入250μL的100％异丙醇，轻晃20min后吸取150μL至96孔板中，以100％异丙醇为空白对照，用酶标仪分别测量不同孔板中的OD 500nm的吸光度；(2)以每孔的OD 500nm测得值，减去以间充质干细胞(MSC)的OD 500nm吸光度的背景值，计算成脂诱导分化的油红O吸收值，将所得结果进行对比，所得结果如表1和图1所示。
表1
Figure PCTCN2022086841-appb-000004
从实验结果可知，分别采用QF-1进行14d与28d的脐带间充质干细胞成 脂诱导，通过油红O染色后检测发现于间充质干细胞中添加10～60μM浓度的QF-1化合物可有效促进间充质干细胞的成脂分化。特别是于间充质干细胞中添加浓度为10μM的QF-1化合物效果最佳，经过28d的成脂诱导后生成的脂肪量为对照组(QF-1化合物的添加量为0μM)的2.17倍。实验结果证明QF-1化合物具有优异的促进间充质干细胞的成脂分化能力。
(2)成脂诱导后的染色显微观察
将上述诱导后的脐带间充质干细胞进行显微镜观察，如图2所示，对比添加0μM与10μM QF-1化合物经28d诱导的显微照片，可以明显地发现添加了QF-1化合物诱导后的脐带间充质干细胞经油红O染色后出现更多的红色沉淀，证明QF-1化合物有效促进了间充质干细胞的成脂分化。
实施例2 QF-1促进脂肪前体细胞的脂肪生成
S1、将PPARγ反应元件(5‘-taggtcaaaggtcaa-3’)通过DNA合成方式合成5‘带有BmtI酶切位点、3’带有BglII酶切位点、内部含有4个重复PPARγ反应元件的DNA序列，序列如下：5‘-gccGCTAGC taggtcaaaggtca taggtcaaaggtca taggtcaaaggtca taggtcaaaggtcaAGATCTccg-3’。将合成的DNA与pGL6-TA质粒采用BmtI酶与BglII酶进行限制性酶切后胶回收，T4DNA连接酶过夜连接后导入DH5α菌株，构建成带有PPARγ转录活性检测功能的pGL6-PPARγ-Luc质粒。
S2、将3T3-L1细胞用10％FBS、1％100X链霉素-青霉素双抗溶液的DMEM/F2培养基培养至60％融合后，采用脂质体Lipo2000将步骤S1中的pGL6-PPARγ-Luc质粒与内参质粒PRL-SV40-C质粒导入3T3-L1细胞。经过夜培养后，加入0.25％胰酶消化至细胞悬浮后加入3倍体系的10％FBS、1％100X链霉素-青霉素双抗溶液的DMEM/F2培养基终止消化，300g离心5min后用PBS缓冲液同等条件洗涤2次，得到消化完全的细胞。
S3、将步骤S2中消化完全的细胞用10％FBS、1％100X链霉素-青霉素双抗溶液的DMEM/F2培养基重悬并计数，以1×10 4细胞/孔接种于96孔板中，每孔最终的培养基总量为100μL。
S4、待7h后，待步骤S3中的3T3-L1细胞贴壁后吸去培养基，更换成成脂诱导培养基(所述成脂诱导培养基为10％FBS、1％100X链霉素-青霉素双抗溶液、1μM地塞米松、0.5mM 3-异丁基-1-甲基黄嘌呤、0.2mM吲哚美辛、10mg/L胰岛素的DMEM培养基)进行成脂培养，并添加终浓度为0μM、5μM、10μM、20μM的QF-1化合物持续培养24h，每个浓度做3个重复孔。
S5、采用双萤光素酶报告基因检测试剂盒(碧云天RG027)按照使用说明书的检测方法进行荧光检测。以萤火虫荧光素酶活性(Firefly luciferase)与海肾荧光素酶(Renilla lucieferase)活性的比值F/R反应PPARγ的转录活性水平，所得结果如表2和图3所示。
表2
Figure PCTCN2022086841-appb-000005
需要说明的是，图3中，****P＜0.0001表示与对照组的比较。
从实验结果可知，与不添加QF-1化合物(0μM)的对照组相比，于3T3-L1细胞中添加了5μM、10μM和20μM的QF-1化合物显著增加了相对荧光素酶活性，差异具有统计学意义，由此证明QF-1化合物提升了PPARγ转录水平，促进了脂肪前体细胞的脂肪生成。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在不脱离本发明的原理和宗旨的情况下在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (17)

  1. 一种生物制剂，其特征在于，所述生物制剂中以具有式(1)所示结构的QF-1化合物作为活性成分：
    Figure PCTCN2022086841-appb-100001
  2. 根据权利要求1所述的生物制剂，其特征在于，所述生物制剂为化妆品、保健品、医疗器械、药品或外敷料。
  3. 根据权利要求1所述的生物制剂，其特征在于，所述生物制剂中，除QF-1化合物外，还包括可接受的各种辅料。
  4. 根据权利要求3所述的生物制剂，其特征在于，所述辅料为包衣材料、溶剂、增溶剂、粘合剂、稳定剂、抗氧化剂、pH调节剂和矫味剂中的至少一种。
  5. 根据权利要求1所述的生物制剂，其特征在于，所述生物制剂的使用方式为注射、口服或者透皮吸收。
  6. 根据权利要求1所述的生物制剂，其特征在于，所述生物制剂的剂型为片剂、胶囊剂、口服液、针剂、粉针剂、敷剂或透皮给药制剂。
  7. QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用，其特征在于，所述QF-1化合物具有式(1)所示的结构：
    Figure PCTCN2022086841-appb-100002
  8. 根据权利要求7所述的QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用，其特征在于，所述生物制剂中QF-1化合物为活性成分。
  9. 根据权利要求7所述的QF-1化合物在制备促进间充质干细胞成脂分 化和脂肪前体细胞脂肪生成的生物制剂中的应用，其特征在于，所述生物制剂中，除QF-1化合物外，还包括可接受的各种辅料。
  10. 根据权利要求9所述的QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用，其特征在于，所述辅料为包衣材料、溶剂、增溶剂、粘合剂、稳定剂、抗氧化剂、pH调节剂和矫味剂中的至少一种。
  11. 根据权利要求7所述的QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用，其特征在于，所述生物制剂的给药方式为注射、口服或者透皮吸收。
  12. 根据权利要求7所述的QF-1化合物在制备促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的生物制剂中的应用，其特征在于，所述生物制剂的剂型为片剂、胶囊剂、口服液、针剂、粉针剂、敷剂或透皮给药制剂。
  13. 一种促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法，其特征在于，向对象施用具有式(1)所示结构的QF-1化合物：
    Figure PCTCN2022086841-appb-100003
  14. 根据权利要求13所述的促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法，其特征在于，所述方法包括治疗方法和非治疗方法；所述非治疗方法包括保健方法和科学研究方法。
  15. 根据权利要求13所述的促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法，其特征在于，所述对象为人或其他哺乳动物。
  16. 根据权利要求13所述的促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法，其特征在于，所述QF-1化合物的单次应用剂量为10 -3～10 3mg/kg。
  17. 根据权利要求13所述的促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法，其特征在于，所述方法的适用场景包括机体美容塑形、丰胸、丰臀。
PCT/CN2022/086841 2022-04-14 2022-04-14 一种生物制剂及促进间充质干细胞成脂分化和脂肪前体细胞脂肪生成的方法 Ceased WO2023197248A1 (zh)

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