WO2022183616A1 - 天门冬氨酸在预防或治疗肥胖症中的应用 - Google Patents

天门冬氨酸在预防或治疗肥胖症中的应用 Download PDF

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WO2022183616A1
WO2022183616A1 PCT/CN2021/098044 CN2021098044W WO2022183616A1 WO 2022183616 A1 WO2022183616 A1 WO 2022183616A1 CN 2021098044 W CN2021098044 W CN 2021098044W WO 2022183616 A1 WO2022183616 A1 WO 2022183616A1
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aspartic acid
obesity
pharmaceutical composition
aspartate
derivatives
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French (fr)
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黄志纾
饶勇
陆勇军
郭诗瑶
邝芷琪
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中山大学
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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents

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  • the invention belongs to the field of biomedicine, and particularly relates to the application of aspartic acid in preventing or treating obesity.
  • obesity is due to the imbalance between the body's energy intake and energy consumption, resulting in excessive fat accumulation and abnormal accumulation.
  • obesity has become a major health problem worldwide.
  • About 2.1 billion people in the world are overweight or obese, with an adult overweight incidence rate of 28.1% and an obesity incidence rate of 5.2%.
  • Related technical studies have shown that obesity may cause a variety of complications, such as non-alcoholic fatty liver disease (NAFLD), cardiovascular and cerebrovascular diseases, hypertension, type 2 diabetes (T2D), Cancer, etc., seriously endanger human health.
  • NAFLD non-alcoholic fatty liver disease
  • T2D type 2 diabetes
  • Cancer etc.
  • obesity treatment methods are very limited. At present, the treatment of obesity is mostly lifestyle intervention based on diet control and exercise intervention, but the curative effect is mediocre.
  • Clinical bariatric surgery (such as liposuction, gastrectomy) is generally suitable for severely obese patients. This method may cause surgical complications and has a high risk.
  • commercially available obesity treatment drugs are divided into three categories: drugs that inhibit intestinal lipid absorption, drugs that regulate nerve center and suppress appetite, and drugs that promote energy metabolism.
  • Drugs that inhibit intestinal lipid absorption can reduce fat absorption by about 30%, but easily affect the absorption of fat-soluble vitamins in patients, have gastrointestinal adverse reactions, and may cause serious Liver damage; appetite-suppressing drugs that regulate nerve centers, such as central appetite suppressants phentermine/topiramate, naltrexone/bupropion hydrochloride, all act on the hypothalamus appetite-regulating pathway to reduce appetite, but there is a risk of addiction and central side effects; drugs that promote energy metabolism, such as peripheral appetite suppressant liraglutide, are relatively safe in vivo, but are expensive and require injection.
  • the present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art.
  • the present invention proposes the application of aspartic acid in preventing or treating obesity.
  • the inventors have verified through research that aspartic acid can inhibit adipogenesis, reduce the level of triglycerides in adipocytes, and effectively control glucose, Insulin and free fatty acid levels reduce the content of total adipose tissue and adipose tissue in various parts of mice, and inhibit the maturation of adipocytes, thereby effectively improving or treating obesity.
  • the first aspect of the present invention provides a pharmaceutical composition for obesity treatment, which contains 40-200 ⁇ M of aspartic acid or a derivative thereof.
  • L-aspartate also known as aspartic acid, aminosuccinic acid, etc.
  • Its chemical structural formula is as follows Formula (I).
  • L-aspartate was originally found in aspartate plants. It is a non-essential amino acid belonging to the arginine family and one of the basic amino acids that make up proteins in living organisms. In vivo, L-aspartate can be generated by the transfer of amino groups in glutamic acid to oxaloacetate under the action of aspartate aminotransferase, and it can also directly participate in the urea cycle as an amino donor. body. Since aspartic acid is one of the endogenous basic amino acids in the body, it has high safety. , Improve chronic fatigue syndrome, protect cardiomyocytes, prevent and treat non-alcoholic fatty liver disease and improve bacterial sensitivity to antibiotics.
  • L-aspartate also inhibits adipogenesis, reduces the level of triglyceride in adipocytes, and effectively controls the levels of glucose, insulin and free fatty acids in the body, reduces the content of total adipose tissue and adipose tissue in various parts, and inhibits fat.
  • the effect of cell maturation thus has potential medicinal value as an effective improvement or treatment of obesity.
  • the aspartic acid or its derivatives include L-aspartic acid and its derivatives or salts, D-aspartic acid and its derivatives Derivatives or salts, DL-aspartic acid and derivatives or salts thereof.
  • the aspartic acid or a derivative thereof is L-type aspartic acid.
  • the pharmaceutical composition further contains pharmaceutically acceptable excipients.
  • the auxiliary materials include, but are not limited to, one or more of diluents, absorbents, wetting agents, binders, disintegrants, lubricants, flavoring agents or transdermal absorption enhancers.
  • diluents include, but are not limited to, one or more of diluents, absorbents, wetting agents, binders, disintegrants, lubricants, flavoring agents or transdermal absorption enhancers.
  • diluents include, but are not limited to, one or more of diluents, absorbents, wetting agents, binders, disintegrants, lubricants, flavoring agents or transdermal absorption enhancers.
  • those skilled in the art can also reasonably add other auxiliary materials or carrier materials according to actual use requirements.
  • the dosage form of the pharmaceutical composition is selected from oral preparations, injection preparations or transdermal preparations.
  • the dosage form of the pharmaceutical composition is an oral formulation.
  • the second aspect of the present invention provides the use of aspartic acid or a derivative thereof in the preparation of a medicine for preventing obesity.
  • L-aspartate also inhibits adipogenesis, reduces the level of triglyceride in adipocytes, and effectively controls the levels of glucose, insulin and free fatty acids in the body, reduces the content of total adipose tissue and adipose tissue in various parts, and inhibits fat.
  • the role of cell maturation thus has potential medicinal value as an effective prevention of obesity.
  • the aspartic acid or its derivative is L-aspartic acid, and the effective amount of the L-aspartic acid is 40- 200 ⁇ M.
  • the third aspect of the present invention provides the use of aspartic acid or a derivative thereof in the preparation of a drug for treating obesity.
  • L-aspartate also inhibits adipogenesis, reduces the level of triglyceride in adipocytes, and effectively controls the levels of glucose, insulin and free fatty acids in the body, reduces the content of total adipose tissue and adipose tissue in various parts, and inhibits fat. cell maturation, and thus has potential medicinal value as an effective treatment for obesity.
  • the aspartic acid or its derivative is L-aspartic acid, and the effective amount of the L-aspartic acid is 40- 200 ⁇ M.
  • the fourth aspect of the present invention provides the use of aspartic acid or a derivative thereof in the preparation of an adipogenic precursor cell differentiation inhibitor.
  • L-aspartate can significantly inhibit the maturation process of adipocytes and reduce their differentiation level.
  • the aspartic acid or its derivative is L-aspartic acid, and the effective amount of the L-aspartic acid is 40- 200 ⁇ M.
  • the present invention provides a medicinal composition containing L-aspartate, which has the functions of inhibiting adipogenesis, reducing triglyceride levels in adipocytes, effectively controlling the levels of glucose, insulin and free fatty acids in vivo, reducing total adipose tissue and various parts
  • the content of adipose tissue and the effect of inhibiting the maturation of adipocytes have potential medicinal value as effective prevention or treatment of obesity.
  • Figure 1 is a photo (B) of oil red O-stained cells under the level of triglyceride (TG) in adipocytes (A) and a representative concentration (40 ⁇ M) in the example of the present invention
  • Fig. 2 is a line graph (A) of the body weight change of the mouse model in the embodiment of the present invention, a comparison chart of the body weight of the mouse model after 7 weeks of administration (B), and the fat mass of the mouse model after 7 weeks of administration (C) ;
  • Fig. 3 is the content level of glucose (A), insulin (B) and free fatty acid (FFA) (C) in the blood of the mouse model in the embodiment of the present invention, and the glucose tolerance (D) and insulin of the mouse model Sensitivity (E) comparison chart;
  • Figure 4 shows the mouse model total adipose tissue (tWAT) and adipose tissue of various parts (abdominal adipose tissue (sWAT), subcutaneous adipose tissue (eWAT), inguinal adipose tissue (iWAT), perirenal adipose tissue ( pWAT)) content comparison chart, wherein, A is the fat mass comparison chart, and B is the pathological staining comparison chart.
  • sWAT randominal adipose tissue
  • eWAT subcutaneous adipose tissue
  • iWAT inguinal adipose tissue
  • pWAT perirenal adipose tissue
  • the experimental materials and reagents used are conventional consumables and reagents that can be obtained from commercial sources.
  • the formula of differentiation induction solution I is: add 500 ⁇ M 3-isobutyl-1-methyl-xanthine, 100 ng/mL dexamethasone and 2 ⁇ g/mL insulin to the DMEM complete culture medium.
  • the formula of the differentiation induction solution II is: add insulin with a final concentration of 2 ⁇ M to the DMEM complete culture solution.
  • the L-configuration (left-handed) (L-aspartate, L-aspartate) of aspartic acid is used as the test object to further verify the effect of aspartic acid on the content of triglycerides in adipocytes.
  • the 3T3-L1 adipogenic precursor cells in logarithmic growth phase were taken, and 5.0 ⁇ 10 4 cells/well were evenly seeded into a 48-well plate, and cultured in a cell culture incubator.
  • the culture medium was replaced every two days. When the cells grew close to 80% confluence, the culture medium was replaced, and the culture was continued for 2 days until the cells reached 100% confluence (day 0).
  • the medium was replaced with DMEM complete medium containing differentiation inducer I (DMEM containing 10% FBS and 1% double antibody), and different concentrations (10, 40) diluted with DMEM complete medium containing differentiation inducer I were added.
  • L-aspartic acid statically cultured at 37° C., 5% CO 2 for 3 days (days 1 to 3). After culturing for 3 days, the medium was replaced with DMEM complete medium containing differentiation inducer II, and L-aspartum of the same concentration (10, 40, 100, 200) diluted with DMEM complete medium containing differentiation inducer II was added. acid, and continue to culture for 3 days (4th to 6th days).
  • the blank control group and the differentiation control group were set up, and the same volume of DMSO solution as the experimental group L-aspartic acid was added on the 0th and 3rd days when the medium was changed (the same volume of DMEM with differentiation induction solution I and II, respectively, was used to complete the cells). culture medium as a diluent). After culturing on the 6th day, oil red O staining and triglyceride content analysis were performed on each group.
  • Oil Red O fat staining method is as follows:
  • the cells to be stained were washed once with pre-cooled PBS and fixed with 4% frozen paraformaldehyde fixative solution at room temperature for 60 min. Add 0.3% Oil Red O staining solution for 30 min at room temperature. After staining, rinse with deionized water for 2-3 times at room temperature, and take pictures with an inverted microscope (40 times).
  • the cells to be tested were rinsed twice with pre-cooled PBS. Remove PBS, add deionized solution containing 0.2% Triton X-100, let stand for 1 h at room temperature, collect cell suspension, sonicate for 10 min to fully lyse the cells, and collect supernatant by centrifugation. ) instructions to determine the triglyceride content.
  • the experimental results are the average of three independent experiments, and the results are statistically analyzed according to "mean ⁇ standard deviation”.
  • L-aspartate can effectively inhibit adipogenesis at concentrations of 40-200 ⁇ M.
  • Quantitative detection of triglycerides indicated that the use of L-aspartate reduced triglyceride levels in adipocytes (1A).
  • Oil red O staining further demonstrated the lipid-lowering activity of L-aspartate.
  • a large amount of lipids could be clearly seen in the cells of the control group, while the lipid content in the cells of the L-aspartate-treated group was significantly reduced (1B).
  • the L-configuration (L-rotation) (L-aspartate, L-aspartate) of aspartic acid is used as the test object, and an in vivo verification experiment is carried out on a high-fat and high-cholesterol diet-induced obesity mouse model.
  • HFC high-fat and high-cholesterol diet
  • normal diet by provided by the Experimental Animal Center of the East Campus of Sun Yat-sen University
  • HFC-fed mice were randomly divided into two groups (HFC control group and L-aspartate-administered group) with 10 mice in each group.
  • mice with normal diet continued to be fed with normal diet, and mice in HFC group and L-aspartate group continued to be given HFC diet.
  • the L-aspartate group was given the L-aspartate solution prepared in step (2) by gavage at a dose of 200 mg/kg, and the control group was given normal saline.
  • the mice were treated every two days, and the food intake and body weight of the mice were observed and recorded. After 3 weeks of dosing, mice were tested for glucose tolerance. After 5 weeks of administration, the mice were tested for insulin resistance. All mice were anesthetized for blood collection 7 weeks after administration, sacrificed by cervical dislocation and dissected, and the body weight and adipose tissue weight of mice in each group were recorded.
  • mice to be tested were fasted for 6 hours, intraperitoneally injected with glucose solution (2 g/L), and the blood glucose changes of the mice were detected at 0, 15, 30, 60, 90, and 120 minutes after injection.
  • the specific methods of the insulin resistance test are as follows:
  • mice to be tested were fasted for 6 hours, and insulin solution (0.6 U/kg) was injected intraperitoneally.
  • insulin solution 0.6 U/kg
  • the changes in blood glucose of the mice were detected at 0, 15, 30, 60, 90, and 120 minutes after injection.
  • the mouse blood sample collected in step (3) was centrifuged at 3000 rpm for 10 min, and the supernatant was taken. Serum glucose, fatty acids, triglycerides and other indicators were detected.
  • mice adipose tissue samples collected in step (3) were subjected to histopathological analysis to evaluate the effect of L-aspartate treatment on the maturation process of adipocytes.
  • L-aspartate significantly reduced the total adipose tissue (tWAT) and the content of adipose tissue in various parts of the mice ( Figure 4A), such as abdominal adipose tissue (sWAT). ), subcutaneous adipose tissue (eWAT), inguinal adipose tissue (iWAT), perirenal adipose tissue (pWAT) and other adipose tissues were significantly reduced. Pathological staining results showed that L-aspartate could significantly inhibit the maturation process of adipocytes and reduce the cell surface area size (Fig. 4B).
  • both in vivo and in vitro experiments can effectively demonstrate that aspartate can inhibit adipogenesis, reduce triglyceride levels in adipocytes, and effectively control the levels of glucose, insulin and free fatty acids in vivo, and reduce the amount of adipogenesis in mice.
  • the content of total adipose tissue and adipose tissue in each part of the body can inhibit the maturation of adipocytes, thereby effectively improving or treating obesity.

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Abstract

一种治疗肥胖症的药用组合物，所述药用组合物中含有40-200μm的天门冬氨酸或其衍生物。天门冬氨酸或其衍生物在制备预防或治疗肥胖症药物中的应用。

Description

天门冬氨酸在预防或治疗肥胖症中的应用 技术领域
本发明属于生物医药领域，具体涉及天门冬氨酸在预防或治疗肥胖症中的应用。
背景技术
肥胖的发生是由于机体能量摄入与能量消耗失衡，导致脂肪过度增生而产生异常累积。近年来，肥胖症已经成为了世界范围内的主要健康问题，世界上约有21亿人处于超重或肥胖状态，成人超重发生率达到28.1％，肥胖发生率为5.2％。相关技术研究表明肥胖可能会引发多种并发症，如非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)、心脑血管疾病、高血压、Ⅱ型糖尿病(type 2 diabetes,T2D)、癌症等，严重危害人体健康。
相关技术中，肥胖治疗方法十分有限。目前，肥胖的治疗多以饮食控制和运动干预为主的生活方式干预，但疗效一般。临床减肥手术(如抽脂、胃切除术)一般适用于严重肥胖患者，该方法可能会产生手术并发症，风险较大。在药物治疗方面，市售的肥胖症治疗药物分为三类：抑制肠道脂质吸收类药物、调控神经中枢抑制食欲类药物和促进能量代谢类药物。抑制肠道脂质吸收类药物，如肠道脂肪酶抑制剂奥利司他可以减少约30％的脂肪吸收，但容易影响患者脂溶性维生素的吸收，存在胃肠道不良反应，并且可能导致严重肝损伤；调控神经中枢抑制食欲类药物，如中枢食欲抑制剂苯丁胺/托吡酯，纳曲酮/盐酸安非他酮均是作用于下丘脑食欲调节通路，降低食欲，但存在成瘾性风险和中枢副作用；促进能量代谢类药物，如外周食欲抑制剂利拉鲁肽体内安全性相对较高，但价格昂贵，且需注射给药。除上市药物外，其他广受关注的肥胖症药物治疗策略还包括激活白色脂肪组织棕色化、下调甾醇调节元件结合蛋白(sterol regulatory-element binding proteins,SREBPs)、过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ,PPAR-γ)开发生长激素释放肽(Ghrelin)反向激动剂、Bombesin受体亚型3(bombesin receptor subtype-3,BRS-3)激动剂、黑色素浓缩激素(melanin concentrating hormone,MCH)拮抗剂等，但其安全性和长期疗效仍待进一步验证。
因此，开发一种高效低毒的、甚至无毒害的抗肥胖药物对解决目前的肥胖问题具有极为重要的临床意义。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出天门冬氨 酸在预防或治疗肥胖症中的应用，发明人通过研究验证天门冬氨酸能够抑制脂肪生成，降低脂肪细胞内甘油三酯水平，并在体内有效控制葡萄糖、胰岛素与游离脂肪酸水平，减少了小鼠体内总脂肪组织与各部位脂肪组织含量，抑制脂肪细胞成熟，从而有效改善或治疗肥胖症。
本发明的第一个方面，提供一种肥胖症治疗药用组合物，该药用组合物中含有40-200μM的天门冬氨酸或其衍生物。
L-天冬氨酸(L-aspartate)，又称门冬氨酸、氨基丁二酸等，分子式为C 4H 7NO 4，分子量133.1，CAS号为6899-03-2，其化学结构式如式(Ⅰ)所示。
Figure PCTCN2021098044-appb-000001
L-aspartate最初在天门冬植物中被发现，是一种非必需氨基酸，属于精氨酸家族，也是生物体内组成蛋白质的基本氨基酸之一。在体内，L-aspartate可在谷草转氨酶的作用下由谷氨酸中的氨基转移至草酰乙酸生成，也可直接作为氨基供体参与尿素循环，同时，L-aspartate也是嘌呤和嘧啶的合成前体。由于天冬氨酸是机体内源性基础氨基酸之一，安全性较高，相关技术中，常被用作改善神经系统疾病、提高运动能力等方面的药物或补充剂，具有增强肌肉质量和力量、改善慢性疲劳综合征、保护心肌细胞、防治非酒精性脂肪肝和提高细菌对抗生素敏感性的作用。发明人通过研究发现，L-aspartate还具有抑制脂肪生成，降低脂肪细胞内甘油三酯水平，并在体内有效控制葡萄糖、胰岛素与游离脂肪酸水平，减少总脂肪组织与各部位脂肪组织含量，抑制脂肪细胞成熟的作用，从而具有作为有效改善或治疗肥胖症的潜在药用价值。
根据本发明的第一个方面，在本发明的一些实施方式中，所述天门冬氨酸或其衍生物包括L型天门冬氨酸及其衍生物或盐、D型天门冬氨酸及其衍生物或盐、DL型天门冬氨酸及其衍生物或盐。
在本发明的一些优选的实施方式中，所述天门冬氨酸或其衍生物为L型天门冬氨酸。
在本发明的一些优选的实施方式中，所述药用组合物中还含有药学上可接受的辅料。
其中，所述辅料包括但不限于稀释剂、吸收剂、润湿剂、粘合剂、崩解剂、润滑剂、矫味剂或透皮吸收促进剂中的一种或多种。当然，本领域技术人员也可以根据实际使用需求，合理添加其他辅料或载体材料。
在本发明的一些优选的实施方式中，所述药用组合物的剂型选自口服制剂、注射制剂或透皮制剂。
在本发明的一些更优选的实施方式中，所述药用组合物的剂型为口服制剂。
本发明的第二个方面，提供天门冬氨酸或其衍生物在制备预防肥胖症药物中的应用。
发明人通过研究发现，L-aspartate还具有抑制脂肪生成，降低脂肪细胞内甘油三酯水平，并在体内有效控制葡萄糖、胰岛素与游离脂肪酸水平，减少总脂肪组织与各部位脂肪组织含量，抑制脂肪细胞成熟的作用，从而具有作为有效预防肥胖症的潜在药用价值。
根据本发明的第二个方面，在本发明的一些实施方式中，所述天门冬氨酸或其衍生物为L型天门冬氨酸，所述L型天门冬氨酸的有效量为40-200μM。
本发明的第三个方面，提供天门冬氨酸或其衍生物在制备治疗肥胖症药物中的应用。
发明人通过研究发现，L-aspartate还具有抑制脂肪生成，降低脂肪细胞内甘油三酯水平，并在体内有效控制葡萄糖、胰岛素与游离脂肪酸水平，减少总脂肪组织与各部位脂肪组织含量，抑制脂肪细胞成熟的作用，从而具有作为有效治疗肥胖症的潜在药用价值。
根据本发明的第三个方面，在本发明的一些实施方式中，所述天门冬氨酸或其衍生物为L型天门冬氨酸，所述L型天门冬氨酸的有效量为40-200μM。
本发明的第四个方面，提供天门冬氨酸或其衍生物在制备脂肪前体细胞分化抑制剂中的应用。
发明人通过病理学染色发现，L-aspartate可显著抑制脂肪细胞成熟过程，降低其分化水平。
根据本发明的第三个方面，在本发明的一些实施方式中，所述天门冬氨酸或其衍生物为L型天门冬氨酸，所述L型天门冬氨酸的有效量为40-200μM。
本发明的有益效果是：
本发明提供了一种含有L-aspartate的药用组合物，具有抑制脂肪生成，降低脂肪细胞内甘油三酯水平，并在体内有效控制葡萄糖、胰岛素与游离脂肪酸水平，减少总脂肪组织与各部位脂肪组织含量，抑制脂肪细胞成熟的作用，从而具有作为有效预防或治疗肥胖症的潜在药用价值。
附图说明
图1为本发明实施例中的脂肪细胞内甘油三酯(TG)水平(A)和代表浓度(40μM)下油红O染色细胞照片(B)；
图2为本发明实施例中的小鼠模型体重变化折线图(A)、给药7周后的小鼠模型体重对 比图(B)和给药7周后的小鼠模型脂肪质量(C)；
图3为本发明实施例中的小鼠模型血液中的葡萄糖(A)、胰岛素(B)与游离脂肪酸(FFA)(C)的含量水平，以及小鼠模型的葡萄糖耐受(D)与胰岛素敏感性(E)对比图；
图4为本发明实施例中的小鼠模型总脂肪组织(tWAT)与各部位脂肪组织(腹部脂肪组织(sWAT)、皮下脂肪组织(eWAT)与腹股沟脂肪组织(iWAT)、肾周脂肪组织(pWAT))含量对比图，其中，A为脂肪质量对比图，B为病理学染色对比图。
具体实施方式
为了使本发明的发明目的、技术方案及其技术效果更加清晰，以下结合具体实施方式，对本发明进行进一步详细说明。应当理解的是，本说明书中描述的具体实施方式仅仅是为了解释本发明，并非为了限定本发明。
所使用的实验材料和试剂，若无特别说明，均为常规可从商业途径所获得的耗材和试剂。
实验材料
分化诱导液的配制：
(1)分化诱导液Ⅰ的配方为：在DMEM完全培养液中加入终浓度为500μM的3-异丁基-1-甲基-黄嘌呤、100ng/mL的地塞米松和2μg/mL胰岛素。
(2)分化诱导液ⅠI的配方为：在DMEM完全培养液中加入终浓度为2μM的胰岛素。
天门冬氨酸对脂肪细胞中甘油三酯含量的影响
本实施例中以天门冬氨酸的L构型(左旋)(L-天冬氨酸，L-aspartate)为测试对象，以进一步验证天门冬氨酸对脂肪细胞中甘油三酯含量的影响。
具体试验步骤如下：
取对数生长期的3T3-L1脂肪前体细胞，按照5.0×10 4个细胞/孔，均匀接种至48孔板，在细胞培养箱静置培养，每两天更换一次培养液。待细胞生长接近至80％汇合度时，更换培养液，继续培养2天至细胞达到100％汇合度(第0天)。将培养基更换含有分化诱导液Ⅰ的DMEM完全培养液(含10％FBS及1％双抗的DMEM培养液)，加入使用含有分化诱导液Ⅰ的DMEM完全培养液稀释的不同浓度(10、40、100、200μM)的L-天冬氨酸，37℃、5％CO 2静置培养3天(第1～3天)。培养3天后，将培养基更换为含有分化诱导液Ⅱ的DMEM完全培养液，加入使用含有分化诱导液ⅠI的DMEM完全培养液稀释的同样浓度(10、40、100、200)的L-天冬氨酸，继续培养3天(第4～6天)。设置空白对照组与分化对照组，在第0天和第3天更换培养基时分别加入与实验组L-天冬氨酸等体积的DMSO溶液(同样分别以分化诱导液I和Ⅱ的DMEM完全培养液作为稀释液)。在第6天培养结束后，对各组进行油 红O染色拍照以及甘油三酯含量分析。
油红O脂肪染色法的具体操作为：
将待染色细胞经预冷PBS润洗1次，4％冰冻多聚甲醛固定液室温固定60min。加入0.3％油红O染色工作液室温染色30min。染色完成后，室温条件下，用去离子水漂洗2-3次，倒置显微镜拍照(40倍)。
拍照完成后，每孔分别加入300uL异丙醇溶液，使用摇床平缓摇动，室温萃取油红O染料30min。分别吸取100μM萃取后的染液在510nm处进行吸光度检测。
甘油三酯含量分析实验的具体步骤为：
将待检测细胞经预冷PBS润洗2次。除去PBS，加入含0.2％Triton X-100的去离子溶液，室温静置1h，收集细胞悬液，超声破碎10min，使细胞充分裂解，离心收集上清液，按照甘油三酯检测试剂盒(罗氏)说明书测定甘油三酯含量。
其中，甘油三酯含量分析以分化对照组作为对照(Ctrl)，倍数关系为：
Figure PCTCN2021098044-appb-000002
实验结果为三次独立实验的平均值，结果按照“平均值±标准差”进行统计学分析。
结果如图1所示。
如图1所示，L-aspartate在40-200μM浓度下均能有效的抑制脂肪生成。甘油三酯定量检测结果表明使用L-aspartate可以降低脂肪细胞内甘油三酯水平(1A)。油红O染色进一步证明L-aspartate的降脂活性。对照组的细胞内能清晰可见大量的脂质，而L-aspartate处理组细胞内的脂质含明显减少(1B)。
天门冬氨酸体内实验验证天门冬氨酸对肥胖症的影响
本实施例以天门冬氨酸的L构型(左旋)(L-天冬氨酸，L-aspartate)为测试对象，通过在高脂高胆固醇饮食诱导的肥胖小鼠模型上进行体内验证实验。
具体步骤如下：
(1)构建肥胖小鼠模型：
以8周龄雄性C57BL/6小鼠(体重18-20克)为对象，喂食高脂肪高胆固醇饲料(HFC，含60％脂肪和1.2％胆固醇，购自美国Research Diet公司)或正常饲料(由中山大学东校区实验动物中心提供)，饲养10周。当HFC饮食小鼠体重为正常饮食小鼠体重1.2倍时，则认为肥胖小鼠模型建立成功。
(2)配制天门冬氨酸溶液：
使用超纯水配制浓度为20g/L的L-aspartate溶液，并使用10mol/L氢氧化钠溶液调节pH至7.4，充分混匀。
(3)生理盐水或L-aspartate干预处理：
将HFC饮食小鼠随机分成两组(HFC对照组和L-aspartate给药组)，每组10只。
正常饲料小鼠继续给予普通饲料喂养，HFC组和L-aspartate组小鼠继续给予HFC饲料。其中，L-aspartate组按200mg/kg剂量经灌胃给予步骤(2)配制的L-aspartate溶液，对照组给与生理盐水处理。每两天处理一次，观察并记录小鼠摄食量和体重。给药3周后，对小鼠进行葡萄糖耐受检测。给药5周后，对小鼠进行胰岛素抵抗检测。所有小鼠于给药7周后麻醉采血，并颈椎脱臼处死并解剖，记录各组小鼠体重和脂肪组织重量。
其中，葡萄糖耐受检测实验的具体方法为：
将待测小鼠禁食6小时，腹腔注射葡萄糖溶液(2g/L)，于注射后第0、15、30、60、90、120分钟分别检测小鼠血糖变化。
胰岛素抵抗检测实验的具体方法为：
将待测小鼠禁食6小时，腹腔注射胰岛素溶液(0.6U/kg)，于注射后第0、15、30、60、90、120分钟分别检测小鼠血糖变化。
(4)小鼠血液样本分析：
将步骤(3)中收集的小鼠血液样本以3000转离心10min，取上清。检测血清中葡萄糖、脂肪酸、甘油三酯等指标。
(5)小鼠脂肪组织样本分析：
将步骤(3)中收集的小鼠脂肪组织样本进行组织病理学分析，评估L-aspartate治疗对脂肪细胞成熟过程影响。
结果如图2-4所示。
如图2所示，小鼠口服L-aspartate可有效抑制长期HFC饮食诱导的小鼠肥胖症发生。在L-aspartate给药7周后，与HFC对照组小鼠相比，L-aspartate组小鼠体重减轻约15.6％，说明L-aspartate显著改善了小鼠肥胖症。如图3所示，与HFC对照组小鼠相比，L-aspartate组小鼠血液中的葡萄糖(图3A)、胰岛素(图3B)与游离脂肪酸(图3C)水平显著降低，而且，L-aspartate组小鼠葡萄糖耐受(图3D)与胰岛素敏感性(图3E)也发生了显著性增强。此外，如图4所示，与HFC对照组小鼠相比，L-aspartate显著减少了小鼠体内总脂肪组织(tWAT)与各部位脂肪组织含量(图4A)，具体如腹部脂肪组织(sWAT)、皮下脂肪组织(eWAT)与腹股沟脂肪组织(iWAT)、肾周脂肪组织(pWAT)等脂肪组织中的脂肪含量都发生了显著 降低。病理学染色结果表明L-aspartate可显著抑制脂肪细胞成熟过程，降低细胞表面积大小(图4B)。
综上所述，通过体内和体外实验，均可以有效说明天门冬氨酸能抑制脂肪生成，降低脂肪细胞内甘油三酯水平，并在体内有效控制葡萄糖、胰岛素与游离脂肪酸水平，减少了小鼠体内总脂肪组织与各部位脂肪组织含量，抑制脂肪细胞成熟，从而有效改善或治疗肥胖症。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种肥胖症治疗药用组合物，其特征在于，所述药用组合物中含有40-200μM的天门冬氨酸或其衍生物。
  2. 根据权利要求1所述的肥胖症治疗药用组合物，其特征在于，所述天门冬氨酸或其衍生物包括L型天门冬氨酸及其衍生物或盐、D型天门冬氨酸及其衍生物或盐、DL型天门冬氨酸及其衍生物或盐。
  3. 根据权利要求2所述的肥胖症治疗药用组合物，其特征在于，所述天门冬氨酸或其衍生物为L型天门冬氨酸。
  4. 根据权利要求1～3任一项所述的肥胖症治疗药用组合物，其特征在于，所述药用组合物中还含有药学上可接受的辅料。
  5. 根据权利要求1～3任一项所述的肥胖症治疗药用组合物，其特征在于，所述药用组合物的剂型选自口服制剂、注射制剂或透皮制剂。
  6. 天门冬氨酸或其衍生物在制备预防肥胖症药物中的应用。
  7. 天门冬氨酸或其衍生物在制备治疗肥胖症药物中的应用。
  8. 根据权利要求6或7所述的应用，其特征在于，所述天门冬氨酸或其衍生物为L型天门冬氨酸，所述L型天门冬氨酸的有效量为40-200μM。
  9. 天门冬氨酸或其衍生物在制备脂肪前体细胞分化抑制剂中的应用。
  10. 根据权利要求9所述的应用，其特征在于，所述天门冬氨酸或其衍生物为L型天门冬氨酸，所述L型天门冬氨酸的有效量为40-200μM。
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