WO2009079860A1 - Médicament destiné au traitement de l'obésité ou de la stéatose hépatique - Google Patents

Médicament destiné au traitement de l'obésité ou de la stéatose hépatique Download PDF

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WO2009079860A1
WO2009079860A1 PCT/CN2007/003772 CN2007003772W WO2009079860A1 WO 2009079860 A1 WO2009079860 A1 WO 2009079860A1 CN 2007003772 W CN2007003772 W CN 2007003772W WO 2009079860 A1 WO2009079860 A1 WO 2009079860A1
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icariin
mice
liver
fatty
fat
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PCT/CN2007/003772
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English (en)
French (fr)
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Nan Zhang
Rong Zhong
Stuart K. Calderwood
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Nan Zhang
Rong Zhong
Calderwood Stuart K
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Priority to PCT/CN2007/003772 priority Critical patent/WO2009079860A1/zh
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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

Definitions

  • the invention relates to a medicament for treating obesity or fatty liver. Background technique
  • Epimedium is a spicy ornamental herb commonly found in Asia and the Mediterranean. The Chinese call it Epimedium, which roughly means "a lame goat grass.” Epimedium is a genus, including a variety of related plants, some of which have been used as medicinal, including the leaves of Epimedium, the genus Epimedium, the scorpion, and the Korean Epimedium. Epimedium is an important traditional Chinese herbal medicine widely used as a tonic and antirheumatic drug in China, Japan and Korea, and has been proven to be effective in the treatment of osteoporosis, cardiovascular disease and cancer.
  • Icariin (molecular weight 676), a flavonol glycoside, is a major component of Epimedium.
  • NMU2R Neuromedin U2 Receptor
  • Icariin administered food-induced obesity (DI0) mice can inhibit eating and reduce body weight.
  • DI0 food-induced obesity mice
  • agonists are through the adrenocorticotropic hormone releasing hormone (CRH) signaling pathway. It has been shown that intracerebroventricular administration of CRH also inhibits rat feeding.
  • mice that were knocked out by (CRH) In mice that were knocked out by (CRH), the effect of neuromedin U (NemU) on inhibition of food intake, increased oxygen consumption, and body weight regulation completely disappeared. Neurons containing CRH were found in the paraventricular Nucleus (PVN) region of the hypothalamus. In addition, NmU can stimulate the release of CRH from the hypothalamus.
  • PVN paraventricular Nucleus
  • NmU can stimulate the release of CRH from the hypothalamus.
  • Fatty Acid Synthase is a potential therapeutic target for obesity and tumors.
  • Fatty Acid Synthase FAS
  • flavonoids inhibit FAS in a reversible and irreversible manner.
  • EGCG green tea bioactive flavonoid epigallocate
  • the FAS inhibitor light cyanobacterin and a synthetic compound C75 treated the mice systemically and intraventricularly, inhibiting eating and significantly reducing body weight.
  • C75 inhibits the expression of neuropeptide Y in the hypothalamic signal, which has been mediated by malonyl-CoA since its role in Putin (anti-obesity)-dependent manner. Therefore, FAS may be an important link in dietary regulation and a potential therapeutic target.
  • the present invention provides an obesity or fatty liver treatment drug using icariin as an active ingredient.
  • a therapeutically effective amount of icariin and a pharmaceutically acceptable carrier and/or excipient may also be included in the obesity or fatty liver treatment.
  • icariin is a very effective FAS antagonist, and oral administration of icariin to DIO mice strongly inhibits eating and significantly reduces body weight.
  • Mouse experiments have shown that administration of icariin reduces liver steatosis and has a reduced degree of hepatic ischemia/reperfusion injury. Icariin has great potential to be developed into an obesity or fatty liver treatment drug.
  • Icariin has an activity of inhibiting cellular fatty acid production
  • prostate cancer cells LnCAP were treated with different concentrations of icariin for 5 hours to quantitatively determine the amount of 2- 14 C-labeled acetate in cell lipids.
  • 0.5 ⁇ of icariin inhibited lipogenesis by more than 50%.
  • Higher concentrations of icariin reduced fatty acid production in a dose-dependent manner. After 24 hours of treatment, it is further reduced.
  • Icariin reduces fatty acid production in cells by inhibiting FAS activity
  • icariin is a FAS inhibitor
  • pretreatment of LnCAP cell protein extracts with different concentrations of icariin with different concentrations of icariin, and quantitative determination of the incorporation of 2- 14 C-labeled malonyl-CoA in fatty acids the amount.
  • icariin reduced FAS enzyme activity in vitro with an enzyme activity of less than 50%.
  • Western blot analysis showed that icariin did not affect FAS protein levels. It was therefore confirmed that inhibition of fatty acid production was a direct result of the FAS enzyme activity, rather than by reducing FAS expression.
  • Icariin can strongly inhibit DIO mice from eating and significantly reducing body weight
  • the purpose of analyzing the body composition of DIO mice was to determine whether the reduction in body weight caused by repeated administration of icariin was mainly due to fat loss. As shown in Table 1, during the 14-day treatment period,
  • DIO mice treated with 100 mg/kg and 50 mg/kg icariin had significant differences in body fat weight loss (-6.1 g and -3.7 g). These differences in body fat content are the main reason for the difference in body weight between icariin-treated DIO mice and control mice.
  • icariin in protecting the steatosis liver from ischemia/reperfusion injury was further confirmed by histological analysis. Hematoxylin and eosin stained samples were analyzed using the standard for assessing necrosis (Fig. 5), and 5 high power fields were observed for each slice, ranging from 0 (no apoptosis) to 4
  • the mice in the oral icariin-treated group received ischemia/reperfusion injury, and then measured serum alanine aminotransferase levels. To determine whether icariin reduces liver cell damage. Serum alanine aminotransferase levels are shown in Figure 6.
  • Icariin can reduce the ischemic/reperfusion injury in the steatotic liver.
  • the liver administered by icariin changed from macrovesicular steatosis to vesicular steatosis.
  • the fatty acid composition of the liver, palmitic acid and linoleic acid content can be determined by gas chromatography hydrogen flame ionization assay.
  • Figures 8 and 9 show under the area under the standard curve. These two fatty acids are abundantly present in the steatotic liver, and their levels are significantly decreased after administration of icariin.
  • the area under the standard curve of the fatty acid components of palmitic acid and linoleic acid in the control group before ischemia/reperfusion Each was 6856 and 1823, while the treatment group was 2116.6 and 689 each. Tests have shown that icariin can reduce the amount of fatty acids present in the liver, which significantly reduces the fatty degeneration of the liver.
  • Icariin can strongly inhibit DIO mice from eating and significantly reducing body weight, and weight loss is dose- and time-dependent; Icariin has the effect of reducing hepatic ischemia/reperfusion injury Using ob/ob mouse model to determine the serum alanine aminotransferase of epimedium against ischemia/reperfusion injury markers. The results of the testFig. 6 show that the serum alanine aminotransferase level of the treatment group is higher than that of the control group. Significantly reduced; Icariin can reduce the amount of fatty acids present in the liver, which can significantly reduce the fatty degeneration of the liver.
  • icariin is an effective new drug for obesity/fatty liver treatment with great development prospects.
  • the therapeutic drug may also contain an effective amount of icariin and a commonly known pharmaceutically acceptable carrier and/or Or an excipient.
  • a commonly known pharmaceutically acceptable carrier and/or Or an excipient for example Captisol, crop oil, sodium carboxymethylcellulose.
  • the effective dose for oral administration is recommended to be 750-1000 mg/body surface area M 2 per day for a course of two weeks. Specific cases can be adjusted by the physician according to the condition. Table 1 Effect of Icariin Treatment on Body Composition of DI0 Mice
  • mice 100 mg/k Icariin 39.0 (1.8) 37.2 (1.3) 14.1 (0.5) 10.5 (0.2) 20.4 (1.2) 6.1 g Note: The standard error average is shown in parentheses. Table 2 Pharmacokinetic parameters of icariin in mice
  • Figure 1 is a graph showing the concentration of lipogenesis-icarisin in LnCAP cells treated with icariin.
  • Figure 1 is a graph showing the concentration of fatty acid synthase-icarisin in LnCAP cells treated with icariin.
  • Figure 3 is a graph showing the ingestion of icariin in DIO mice.
  • Figure 4 is a graph showing the weight loss of DIO mice induced by Icariin treatment.
  • Figure 5 is a graph showing the necrosis index of the control group and the treatment group.
  • Figure 6 is a graph showing the levels of serum alanine aminotransferase in the control and treatment groups.
  • Figure 7 is a graph showing the percentage of steatosis in the control and treatment groups.
  • Fig. 8 is a graph showing the contents of fatty acids (palmitic acid) in the liver of the control group and the treatment group.
  • Figure 9 is a graph showing the content of fatty acids (linoleic acid) in the liver of the control group and the treatment group.
  • Epimedhasin can then be prepared by hydrolyzing the precursors as described briefly below.
  • Solution A 80 mg precursor was sonicated in 18 ml of methanol solution;
  • solution B 500 U (0. 433 g) cellulase dissolved in 180 ml (0.1 mol/L), pH 5. 0 acetate buffer In the liquid.
  • Solution A was slowly added to Solution B while stirring, and then the resulting mixture was incubated overnight in a shaking water bath at 37 ° C to sufficiently hydrolyze glucose and rhamnose attached to the precursor.
  • the next day three equal volumes (about 200 ml) of ethyl acetate were extracted, and the organic layer was vacuum dried on a rotary evaporator to obtain icariin (150 mg).
  • Lipids were extracted using the Bligh Dyer method (Swinnen JV et al., Endocrinology 1996; 137: 4468-4474), and the amount of (2- 14 C) acetate incorporation of cell lipids was quantified by scintillation counting. The results obtained were normalized to the sample protein content.
  • the lipogenicity-icarisin concentration curve of icariin after treatment of LnCAP cells is shown in Figure 1. ' Example 3
  • LnCAP cell protein extract fatty acid synthase activity by icariin
  • the FAS enzyme activity was determined using LnCAP cell protein extract (Brussdmans K. et al., J Biol Chem. 2005 Feb 18;280(7):5636- 5645).
  • LnCAP cells were collected by centrifugation, resuspended in hypotonic buffer (1 mM EDTA, 20 mM Tris-HCl, pH 7.5), and then the protein content of the samples was determined by BCA method (Pierce).
  • Equal amounts of protein 50 ⁇ ⁇ ) were placed in 2.5 ml phosphate buffer (100 ⁇ , ⁇ 7.0) with different concentrations of icariin (0.03-30 ⁇ ) and pre-incubated for 30 minutes at 37 °C.
  • DIO mice were prepared on a high-fat diet (D12492) or a low-fat diet (D12450B) (Research Diets, New Brunswick, N: C57BL6/J mice for 7 weeks. 60% of the calories in a high-fat diet are fat, to lard Mainly, only 10% of calories in low-fat diets are derived from fat. Weigh twice a week. After 7 weeks, mice fed a low-fat diet gained 34% 4%, while mice fed a high-fat diet gained weight. >70%, defined as DIO mice.
  • mice were randomly divided into 3 groups: The first group was the control group, and the vehicle was intraperitoneally injected with 1% sodium carboxymethylated cellulose per day for 10 days.
  • the second and third groups were treated with icariin (prepared with 1% sodium carboxymethylcellulose) and administered orally daily at 50 mg/kg and 100 mg/kg for 10 days. Monitor food intake several times a day, a total of 5 day. Body weight was measured once a day for 2 weeks. The inhibition of eating and weight loss results are shown in Figures 3 and 4.
  • Body composition analysis (see Table 1): Animals were sacrificed and carcasses were stored at -80 °C. When the analysis was taken out, the gastrointestinal tract was removed, and the remaining corpses were dried at 60 Torr. The difference between wet weight and dry weight is the weight of the body. The fat mass of a thousand corpses was determined using a Soxlet extractor and petroleum ether extract lipid (Chen RZ et al. 5 Eur J Pharmacol. 2004 Jul 8; 495(l): 63-66). The dry weight after extraction is the fat-free residual weight. The weight of the viscera removed minus the fat weight is fat-free.
  • mice 6-8 weeks old adult ob/ob mice (25-28 g) were purchased from the Jackson Laboratory (Bar Harbor, ME:). 4 per cage, served in a room with temperature and light control, 12 hours of light and dark, free diet.
  • Icariin is formulated with 1% sodium carboxymethylcellulose powder.
  • the treatment group was orally administered at 100 mg/kg for 5 days.
  • the control group was orally administered with 1% sodium carboxymethylcellulose for 5 days.
  • mice Hepatic ischemia/reperfusion according to the literature (Chavin KD et al., J Biol Chem, 1999; 274: 5692-5700; Chavin KD et al, Am J Transplant, 2004; 4: 1440-1447) perfusion. Briefly, mice were anesthetized with pentobarbital (50 mg/kg), and a small incision was placed in the abdominal cavity to expose the liver. Ischemia was caused by a non-invasive vascular clamp closing the hilar. After 15 minutes of ischemia, the vessel clamp was released, the abdominal cavity was closed, and the incision was sutured. The mice were transferred to a temperature-controlled cage and returned to a free diet.
  • Serum alanine aminotransferase assay Open the chest under anesthesia, collect whole blood from the right atrium, leave it at room temperature for 15 minutes to be coagulated, centrifuge at room temperature 3500 g for 5 minutes, and collect serum. Serum alanine aminotransferase activity was then determined and expressed in IU/ml. The results of the measurement are shown in Figure 6.
  • Red oil O staining test Red oil 0 staining as described in the literature (Luna LG, editor, Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York, NY: McGraw-Hill Blakiston Division; 1968).
  • a quantitative grading score using red oil O staining Figure 7 is a graph showing the percentage of fat degeneration in the control and treatment groups.
  • GC/FID gas chromatography hydrogen flame ionization detection

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Description

一种肥胖症或脂肪肝治疗药物 技术领域
本发明涉及一种肥胖症或脂肪肝治疗药物。 背景技术
淫羊藿常见于亚洲和地中海地区， 是一种辛辣性观赏草本植物。 中 国人称之为淫羊藿， 大致的意思为 "放肆的山羊草" 。 淫羊藿是一个 属， 包括多种相关植物， 其中一些已作为药用， 包括箭叶淫羊藿、 小檗 科淫羊藿、 大花淫羊藿和朝鲜淫羊藿。 箭叶淫羊藿是一种重要的传统中 草药， 在中国、 日本和韩国被广泛用作滋补药和抗风湿药， 并被证明能 有效治疗骨质疏松、 心血管疾病和肿瘤。
淫羊藿苷 （ICA) (分子量为 676) ， 一种黄酮醇糖苷， 是箭叶淫 羊藿的主要成分。 近来有文献报道， 分离于淫羊藿的淫羊藿苷是一个神 经介素 U2受体（Neuromedin U2 Receptor, NMU2R) 的强力激动剂， 能选择性激活 NMU2R。 淫羊藿苷给予食物诱导的肥胖 （DI0) 小鼠可 以抑制进食， 并降低体重。 NMU2R激动剂调节进食行为和能量平衡的 分子机制和信号途径尚不清楚。 激动剂的一个可能效应是通过促肾上腺 皮质释放激素 (CRH)信号途径。 已经表明， 经脑室内给予 CRH也能抑 制大鼠进食。 在 （CRH)敲除的小鼠， 神经介素 U (Neuromedin U, NmU)对抑制摄食、 增加耗氧量、 调节体重的作用完全消失。 在下丘脑 室旁核 （Paraventricular Nucleus, PVN)区发现含有 CRH的神经元。 另 夕卜， NmU能剌激下丘脑释放 CRH。 这些资料强烈提示， 激活 NMU2R 可能增加 CRH释放， 调节进食行为以及能量内平衡。 进一步的研究， 如 NMU2R敲除小鼠或 NMU-2R/CRH双基因敲除小鼠的表型分析， 可 能有助于完全理解该受体的分子基础和生理学功能。
近来文献报道， 动物脂肪酸合成酶 (Fatty Acid Synthase, FAS)是肥 胖和肿瘤的一个潜在治疗靶标。 鉴定新的 FAS抑制剂一直引起人们很 大的兴趣。 现已发现， 黄酮类以可逆和不可逆方式抑制 FAS。 并且证 实， 一种绿茶生物活性黄酮类化合物表没食子儿 (EGCG)， 通过减少能 量吸收， 增加脂肪氧化， 可以使 DIO小鼠变痩。 而且还发现， FAS抑 制剂 （浅蓝菌素和一个合成化合物 C75 ) 经全身和脑室内处理小鼠， 可 抑制进食， 显著减轻体重。 C75抑制下丘脑前期信号神经肽 Y的表达， 以来普汀 （抗肥胖药） 依赖性方式发挥作用， 似乎由丙二酰辅酶 A介 导。 因此， FAS可能是饮食调节方面的一个重要链环， 是一个潜在的治 疗靶标。
有文献报道（Liu J and Lou YJ, J Pharrn Biomed Anal. 2004 Oct 29;36(2):365-370; Liu J et al., Pharmazie, 2005, 60 (2), 120-125), 淫羊藿 苷已被生物转化， 至少被转化成 3种代谢产物： 淫羊藿次甙 II (分子量 为 514 ) ， 淫羊藿素 （分子量为 368 ) 以及去甲淫羊藿素 （分子量为 354) 。 但对淫羊藿素通过抑制 FAS , 从而抑制小鼠进食及减肥的研究 未见报道。 发明内容 本发明提供一种以淫羊藿素作为有效成分的肥胖症或脂肪肝治疗药 物。 肥胖症或脂肪肝治疗药物中也可含有治疗有效量的淫羊藿素及可药 用载体和 /或赋形剂。
本发明人通过试验证明淫羊藿素是一个非常有效的 FAS拮抗物， 给 DIO小鼠口服淫羊藿素可强烈抑制进食， 显著减轻体重。 小鼠试验 显示， 淫羊着素给药可降低肝脏的脂肪变性， 并具有减轻脂肪变性肝脏 缺血 /再灌注损伤的程度。 淫羊藿素具有被开发成一种肥胖症或脂肪肝 治疗药物的巨大潜力。
淫羊藿素的制备
参考文献报道（叶海涌等，浙江大学学报（医学版)， 2005, 34 (2) : 131- 136 ) 简述如下： 将箭叶淫羊藿磨成粉末， 用溶剂萃取， 萃取物通过硅 胶柱层析， 用溶剂洗脱， 可制得淫羊藿素前体淫羊藿苷。 再按照下列示 意的反应路线， 将淫羊藿苷通过水解反应制得淫羊藿素。
Figure imgf000004_0001
淫羊藿苷 淫羊藿素 淫羊鼈素的药理试验
1.体外试验
a)淫羊藿素具有抑制细胞脂肪酸生成的活性 为了证实淫羊藿素抑制脂肪酸生成的活性， 用不同浓度的淫羊藿素 处理前列腺癌细胞 LnCAP 5小时， 定量测定 2-14C标记的醋酸盐在细胞 脂质中的惨入量。 如图 1 所示， 0.5 μΜ淫羊藿素抑制脂肪生成大于 50%。 更高浓度淫羊藿素以剂量依赖性方式减少脂肪酸生成。 处理 24 小时后则进一步降低。
b)淫羊藿素通过抑制 FAS活性以降低细胞中脂肪酸生成
为了证实淫羊藿素是一个 FAS抑制剂， 用不同浓度的淫羊藿素预 处理 LnCAP细胞蛋白质提取物， 然后定量测定 2-14C标记的丙二酸单酰 辅酶 A在脂肪酸中的掺入量。 如图 2所示， 0.5 μΜ淫羊藿素在体外降 低 FAS酶活性，酶活性小于 50%。 Western blot分析表明， 淫羊藿素不影 响 FAS蛋白水平。 因此证实抑制脂肪酸生成是 制 FAS酶活性的直接 结果， 而不是通过降低 FAS表达实现的。
2.淫羊藿素能使 DIO小鼠强烈抑制进食和显著减轻体重
本试验应用 DIO模型评价淫羊藿素对来普汀 （抗肥胖药） 抵抗的 肥胖模型的作用。 通过喂养 C57BL6/J小鼠高脂肪饮食诱导肥胖症， 高 脂肪饮食中 60%总热量来自于脂肪。 对照小鼠喂食低脂肪饮食， 其中 10%总热量来源于脂肪。 7周后， 喂养高脂肪饮食的小鼠体重增加 76士 7%, 而对照小鼠体重仅增加 34 ±4%。 如图 3 所示， 给予剂量为 50 mg/kg或 100 mg/kg淫羊藿素， 第一天可使 DIO小鼠摄食减少 25%或 50%。 淫羊藿素处理小鼠的摄食抑制表现为剂量依赖性效应。 但是， 在 整个试验过程中淫羊藿素处理小鼠的这种摄食抑制并未呈现时间依赖 性。 尽管如此， 在整个试验过程中经淫羊藿素处理的 DIO小鼠的体重 不断减轻 （见图 4) 。 淫羊藿素处理动物的这种体重减轻具有剂量依赖 性和时间依赖性。
分析 DIO小鼠身体组成的目的是确定淫羊藿素反复给药引起的体 重减轻是否主要源于脂肪减少。 如表 1所示， 在 14天治疗期间内， 用
100 mg/kg和 50 mg/kg淫羊藿素分别处理的 DIO小鼠， 其身体脂肪重量 失重差别明显 (-6.1 g和- 3.7 g)。 这些身体脂肪含量的差别正是淫羊藿 素处理的 DIO小鼠与对照小鼠之间体重差别的主要原因。
3.淫羊藿素具有减轻脂肪变性肝脏缺血 /再灌注损伤的程度
通过组织学分析进一步证实淫羊藿素在保护脂肪变性肝脏免于缺血 /再灌注损伤方面的作用。 采用评定坏死的标准分析苏木素和伊红染色 样本 (图 5)， 每张切片观察 5个高倍视野， 评分范围从 0 (无凋亡） 到 4
(融合病灶） 。 结果显示， 治疗组动物的肝坏死指数为 0.80 (n=10) ， 较对照组的 2.57 ( n=10 ) 有显著下降， 给药后肝坏死指数平均下降 68.9%。 为进一步鉴定淫羊藿素保护脂肪变性肝脏减轻缺血 /再灌注损伤 的能力， 对口服淫羊藿素治疗组小鼠接受缺血 /再灌注损伤， 然后测定 血清丙氨酸氨基转移酶水平， 以确定淫羊藿素是否减轻了肝细胞损伤。 血清丙氨酸氨基转移酶水平见图 6， 口服淫羊藿素小鼠治疗组的血清丙 氨酸氨基转移酶水平明显降低， 为 92.3 IU/ml (n=10),而口服赋形剂对照 组为 1170 IU/ml(n=10)o 结果证实， 与对照组相比， 口服淫羊藿素的治 疗组可显著保护肝脏， 减轻缺血 /再灌注的损伤。 与痩鼠相比， 脂肪变 性肝脏的小鼠对伴有肠充血的全肝缺血 /再灌注更敏感。 实验评估再灌 注后 24小时动物生存的情况： 治疗组的生存率为 100%， 而对照组为 65%， 显示生存率有显著增加。 4.淫羊藿素能降低肝脏的脂肪变性
淫羊藿素能使脂肪变性的肝脏降低缺血 /再灌注损伤， 为了阐明其 作用机制， 确定淫羊藿素是否减少 ob/ob鼠的肝脏的脂肪变性， 使用油 红 0染色的定量分级评分。 试验结果显示， 缺血 /再灌注前， 未操作的 对照组鼠样本的脂肪变性百分比为 50%， 而淫羊藿素给药的治疗组鼠样 本的脂肪变性百分比明显下降 （18.5% ) (图 7)(n=6)。 此外， 淫羊藿素 给药的肝脏从大泡性脂肪变改变为小泡性脂肪变。 考虑到脂肪中可能有 某些特别的成分对较高等级的脂肪变移植器官的衰竭的重要性， 使用气 相色谱氢焰离子化检测法测定肝脏的脂肪酸成分， 软脂酸和亚油酸含量 如图 8和图 9 (以标准曲线下面积显示） 所示。 这两种脂肪酸大量存在 于脂肪变性的肝脏， 他们的含量在淫羊藿素给药后均明显下降， 缺血 / 再灌注前对照组的脂肪酸成分软脂酸和亚油酸的标准曲线下面积各为 6856和 1823， 而治疗组各为 2116.6和 689。 试验证实淫羊藿素能降低 肝脏中存在的脂肪酸量， 即显著降低肝脏的脂肪变性。
此外还进行了下列试验：
淫羊藿素在小鼠体内的药代动力学试验
口饲给药的淫羊藿素在 4个小鼠体内的药代动力学参数见表 2, 这 些实验数据说明淫羊藿素能在体内保持一定的药物浓度， 有利于治疗肥 胖症或脂肪肝的效果。
给药后淫羊藿素对小鼠的急性毒性试验
受试小鼠处死后进行病理分析， 结果显示， 以单次剂量 1000mg/kg 和多次剂量 200mg/kg (Q2D X 15)给予淫羊藿素， 均未观察到毒性， 所有 受试小鼠生长良好， 无一死亡。 由以上药理试验的效果证明， 来源于天然物质中草药箭叶淫羊藿的 淫羊藿素具有抑制脂肪酸生成的活性； 淫羊藿素是一个有效的 FAS抑 制剂， 能通过抑制 FAS活性以降低细胞中脂肪酸生成； 淫羊藿素能使 DIO小鼠强烈抑制进食和显著减轻体重， 且体重减轻具有剂量依赖性和 时间依赖性； 淫羊藿素具有减轻脂肪变性肝脏缺血 /再灌注损伤的程 度， 应用 ob/ob小鼠模型测定了淫羊藿素对缺血 /再灌注损伤标记物血清 丙氨酸氨基转移酶， 试验结果图 6显示治疗组的血清丙氨酸氨基转移酶 水平较对照组明显降低； 淫羊藿素能降低肝脏中存在的脂肪酸量， 即能 显著降低肝脏的脂肪变性。 此外， 淫羊藿素对小鼠的急性毒性试验结果 也显示未观察到毒性， 所有受试小鼠生长良好， 无一死亡。 因此淫羊藿 素是一种有效的具有很大开发前景的肥胖症 /或脂肪肝治疗新药， 治疗 药物中也可含有有效量的淫羊藿素及通常已知常规的可药用载体和 /或 赋形剂。 例如 Captisol, 作物油， 羧甲基纤维素钠。
口服的有效剂量推荐为每天 750-1000 mg/体表面积 M2， 持续两周 为一疗程。 具体病例可由医师根据病情调整。 表 1淫羊藿素处理对 DI0小鼠身体组成的影响
体重 无脂肪重量 脂肪 处理 水 (g)
活体 (g) 去除内脏 fe) (g) 总量 (g) 失重 赋形剂 48.0(1.4) 45.2(0.8) 17.5(0.6) 13.2(0.5) 26.5(0.9) 一
50mg/k 淫羊藿素 42.5(2.0) 40.0(1.5) 17.2(0.8) 11.4(0.6) 22.8(1.7) 3-7g
100mg/k 淫羊藿素 39.0(1.8) 37.2(1.3) 14.1(0.5) 10.5(0.2) 20.4(1.2) 6.1g 注： 括号内为标准误差平均值。 表 2 淫羊藿素在小鼠体内的药代动力学参数
Figure imgf000009_0001
附图说明
图 1是 LnCAP细胞经淫羊藿素处理后的脂肪生成-淫羊藿素浓度曲 线图。
图 1是 LnCAP细胞经淫羊藿素处理后的脂肪酸合成酶 -淫羊藿素浓 度曲线图。
图 3是淫羊藿素抑制 DIO小鼠摄食示图。
图 4是淫羊藿素治疗导致 DIO小鼠体重减轻示图。
图 5是对照组和治疗组的坏死指数示图。
图 6是对照组和治疗组的血清丙氨酸氨基转移酶水平示图。
图 7是对照组和治疗组的脂肪变性百分比示图。
图 8是对照组和治疗组的肝脏中脂肪酸 （软脂酸） 的含量示图。 图 9是对照组和治疗组的肝脏中脂肪酸 （亚油酸） 的含量示图。 具体实施方式
实施例 1 淫羊藿素的制备 （参见叶海涌等， 浙江大学学报（医学 版）， 2005， 34 (2) : 131-136 )
取在中国南部山区采集获得的箭叶淫羊藿 2 kg磨成粉末， 然后用 95%乙醇 (10 L)萃取。 获得 250 g浓缩原料， 然后用 2. 5 L氯仿、 乙酸 乙酯和 n -丁醇进一步萃取。 将 8 g乙酸乙酯萃取物和 10 g n-丁醇萃取 物装入硅胶层析柱， 用 CHC13- MeOH- HC00H (15 : 1 : 0. 5)和 CHC13- Me0H(8 : 2) 分别洗脱。 通过这一过程， 可制得 300 mg淫羊藿素和去甲淫羊藿素前 体淫羊藿苷。 然后按照以下简要描述的方法将前体水解， 即可制得淫羊 藿素。 溶液 A: 将 80 mg前体超声溶解于 18 ml 甲醇溶液中； 溶液 B: 500 U (0. 433 g)纤维素酶溶于 180 ml (0. 1 mol/L) ， pH 5. 0 乙酸缓冲 液中。 一边搅拌一边将溶液 A缓慢加入溶液 B中， 然后将所得混合物在 37°C的振荡水浴中孵育过夜， 以充分水解连接在前体上的葡萄糖和鼠李 糖。 第二天用三份等体积（约 200 ml)的乙酸乙酯萃取， 并将有机物层 在旋转蒸发仪上真空干燥得淫羊藿素（150 mg)。
实施例 2
用 （2-14C)醋酸盐掺入法测定淫羊藿素对细胞脂肪酸生成的抑制活性 用不同浓度的淫羊藿素处理 LnCAP细胞 5小时或 24小时后， 2-14C标 记的醋酸盐 (57 mCi/mmol; 2 μα/dish; Amersham Biosciences)加入 LnCAP 细胞培养基中。 孵育 4小时后， 收集细胞和培养基， 重悬离心收集的细 胞于 0.8 ml PBS。 应用 Bligh Dyer法 (Swinnen J. V. et al.， Endocrinology 1996; 137： 4468-4474)抽提脂类， 通过闪烁计数法定量测定细胞脂类 的 （2-14C)醋酸盐掺入量。 获得的结果被标准化为样品蛋白质含量。 淫 羊藿素处理 LnCAP细胞后脂肪生成-淫羊藿素浓度曲线图见图 1。 ' 实施例 3
淫羊藿素对 LnCAP细胞提取物脂肪酸合成酶活性抑制的试验 利用 LnCAP细胞蛋白抽提物测定 FAS酶活性 （Brussdmans K. et al.， J Biol Chem. 2005 Feb 18;280(7):5636-5645 ) 。 通过离心收集 LnCAP细 胞， 重悬于低渗缓冲液中（1 mM EDTA,20 mM Tris-HCl,pH7.5), 然后应 用 BCA法 (Pierce)测定样品蛋白质含量。 等量的蛋白 （50 μ§) 与不同浓 度的淫羊藿素 （0.03-30 μπι ) 置于 2.5 ml磷酸盐缓冲液 (100 πιΜ,ρΗ 7.0), 在 37°C预孵育 30分钟。 之后加入 20 μΐ反应液 [2.5 mM NADPH,1.25 mM乙酰 -辅酶 A, 1.25 mM丙二酰 -辅酶 A,0.02 mM[2-14C]丙二酰 -辅酶 A(60 mCi/mmol;PerkinElmer Life Sciences)], 37°C再孵育 15分钟， 加入 3 ml冰 冷的 1 M盐酸 /甲醇混合液 （6 : 4， V/V) 终止反应， 并用石油醚抽提脂 肪酸， 通过闪烁计数分析掺入 2-14C的丙二酰辅酶 A。 结果如图 2所示。
实施例 4 ' 用淫羊藿素治疗 DIO小鼠抑制进食和减轻体重的试验
用高脂肪饮食 (D12492)或低脂肪饮食 (D12450B)(Research Diets, New Brunswick, N： 喂养C57BL6/J小鼠7周， 制备 DIO小鼠。 高脂肪饮 食中 60%热量是脂肪， 以猪油为主， 而低脂肪饮食只有 10%热量来源于 脂肪。 每周 2次称重。 7周后， 喂养低脂肪饮食的小鼠体重增加 34士 4%， 而喂养高脂肪饮食的小鼠体重增加 >70%， 被定义为 DIO小鼠。
适应 7天后， 小鼠被随机分成 3个组： 第一组为对照组， 每天腹腔 内注射赋形剂 1%羧甲基化纤维素钠， 共 10天。 第二组和第三组为治疗 组， 用淫羊藿素 （以 1%羧甲基化纤维素钠配制） 治疗， 每天口服， 分 别为 50 mg/kg和 100 mg/kg, 共 10天。 每天数次监视摄食情况， 共 5 天。 每天测定体重 1次， 共 2周。 抑制进食和减轻体重结果见图 3和图 4。
身体组成分析（见表 1 ) ： 处死动物， 动物尸体贮存在 -80°C。 分析 时取出， 除去胃肠道， 剩余尸体在 60Ό干燥。 湿重与干重的差即为尸 体水重。 应用索克斯雷特萃取器和石油醚萃取脂质测定千尸的脂肪量 (Chen RZ et al.5 Eur J Pharmacol. 2004 Jul 8;495(l):63-66) 。 萃取后干重 即为无脂肪余重。 去除内脏的尸重减去脂肪重即为无脂肪重。
实施例 5
用淫羊藿素治疗 ob/ob小鼠 （肥胖小鼠， II型糖尿病动物模型） 脂 肪变性肝脏缺血 /再灌注损伤的试验
动物喂养： 6-8周成年雄性 ob/ob小鼠 (25-28 g) 购自 Jackson实 验室 (Bar Harbor, ME：)。 每笼 4只， 在温度、 光线均可控的房间内伺养， 明暗周期为 12小时， 自由饮食。
淫羊藿素给药： 淫羊藿素用 1%羧甲基纤维素钠粉剂配制。 治疗组 按 100 mg/kg口服 5天。 对照组用 1%羧甲基纤维素钠口服 5天。
缺血 /再灌注： 按照文献 （Chavin KD et al., J Biol Chem,1999; 274: 5692-5700; Chavin KD et al, Am J Transplant, 2004; 4: 1440-1447)进行肝 脏缺血 /再灌注。 简述如下， 戊巴比妥麻醉小鼠 （50 mg/kg) ， 小切口打 幵腹腔， 暴露肝脏。 以无创伤血管夹钳闭肝门造成缺血。 局部缺血 15 分钟后解除血管夹， 关闭腹腔， 缝合切口。 将小鼠转至温控的笼子中恢 复， 自由饮食。
组织学染色试验： 苏木素和伊红染色如文献所述 （preece A., A Manual for Histologic Technicians. 3rd ed. Boston, Mass: Carolina Press; 1985 ) ， 坏死的分级依照一个改良的评分标准 （0-4) (Neil DA et al., Transplantation, 2001; 71: 1566-1572) 。 每张切片分析与中央静脉有关的 5个高倍视野。 图 5是对照组和治疗组的肝坏死指数示图。
血清丙氨酸氨基转移酶测定： 在麻醉状态下开胸， 从右心房收集全 血， 室温放置 15分钟待凝固， 室温 3500 g离心 5分钟， 收集血清。 然 后测定血清丙氨酸氨基转移酶活性， 以 IU/ml表示。 测定结果见图 6。
红油 O染色试验： 红油 0染色如文献所述 （Luna LG , editor, Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York, NY: McGraw-Hill Blakiston Division; 1968) 。 使用红油 O染色的定量分级评分， 图 7是对照组和治疗组的脂 肪变性百分比示图。
实施例 6
脂肪变性肝脏中的脂肪酸含量测定
肝脏的脂肪酸含量测定使用气相色谱氢焰离子化检测法 (GC/FID) (参见 Fiorini RN et al., Hepatology, 2003; 38: 562A) 简述如 下： 将肝脏样本制成匀浆， 氯仿 -甲醇 （2 : 1 ) 混合物提取脂质。 匀浆 经有机分离滤器分离， 去除水分和肝脏匀浆。 加入 BF3-10%甲醇进行催 化反应， 将脂肪分解成自身成分脂肪酸甲酯。 然后将试管置于蒸气浴 45分钟， 促使反应完成。 样本经氮蒸气干燥后， 盖封， 冷冻 (-80°C)备 用。 分析前将其重新溶解于甲醇 (500 μΐ), 使用 Agilent 7683自动进样器 进样， 进样口选择脉冲不分流模式， Agilent6890气相色谱仪， DB-5(内 径 30 mmx0.25 mm,薄层厚度 0.25)毛细管柱。 柱温保持在 70°C 2分钟， 而后温度以每分钟 15°C的梯度增至 300°C， 保持 12分钟。 氢焰离子化 检测法检测。 色谱峰通过对比 FAME标准鉴定， 肝脏的脂肪酸成分软 脂酸和亚油酸含量数据显示为曲线下面积， 以总蛋白校正。 图 8和图 9 分别是对照组和治疗组肝脏中脂肪酸软脂酸和亚油酸的标准曲线下面积 示图。

Claims

权利要求
1.一种肥胖症或脂肪肝治疗药物， 其特征在于该药物是以淫羊藿素 作为有效成份。
2.如权利要求 1所述的肥胖症或脂肪肝治疗药物， 其特征在于该药 物中含有治疗肥胖症或脂肪肝的有效量的淫羊藿素及可药用载体和 /或 赋形剂。
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WO2022065550A1 (ko) * 2020-09-25 2022-03-31 대구한의대학교산학협력단 이카리틴 및 퀘르세틴을 포함하는 간질환의 예방 또는 치료용 조성물
CN112451515A (zh) * 2020-12-04 2021-03-09 中国科学院广州生物医药与健康研究院 一种人胰高血糖素样肽-1受体激活剂及其应用

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