CN111281863A - 用于减脂的化合物及其应用 - Google Patents
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Abstract
本发明涉及医药领域,尤其是用于减脂的化合物及其应用。本发明提供3‑(4‑羟基‑3‑甲氧苯基)丙酸用于减少脂肪累积的医药组合物。本发明亦提供一种包含3‑(4‑羟基‑3‑甲氧苯基)丙酸的组合物用于制备减少脂肪累积的医药组合物的用途。该组合物可进一步包含6,7,8,3’,4’‑五甲氧基黄酮、3,5,7,8,3’,4’‑六甲氧基黄酮、或其组合。
Description
技术领域
本发明涉及医药领域,尤其是用于减脂的化合物及其应用。
背景技术
高油脂与高糖分的饮食方式及运动量不足使多数现代人面临肥胖问题,并且因为肥胖有较高机率罹患代谢相关疾病,例如糖尿病、高血脂症、高血压、心血管疾病、脂肪肝等,最终令身体健康受到严重威胁。亦有研究显示肥胖是重要的致癌因子。此外,肥胖者较容易出现心理问题与遭遇社交障碍。因此,近年来许多医学研究皆着重于寻求抑制肥胖的方法,试图以此方式促进身心健康。
抑制肥胖的方法包括饮食控制、运动、改变生活型态、药物治疗、及手术。除了严重肥胖的患者需要手术,一般大众多采饮食控制与运动的方式减脂,因为现代人出于工作繁忙不易改变生活型态,又出于崇尚自然疗法而不愿意使用非必要的药物。然而,饮食控制严格要求个人的饮食均衡与热量摄取,在实行上颇有困难;运动方式若不适当可能会造成身体损伤。此外,这二种方法对减脂的效果有限,因为它们并非直接针对脂肪细胞,特别是内脏中的脂肪组织。
有鉴于此,开发一种便于大众使用又能有效减少脂肪细胞内脂肪含量的组合物,以预防肥胖及降低前述多种代谢疾病发生的可能性,实有其必要。
发明内容
缘此,本发明的一目的在提供一种3-(4-羟基-3-甲氧苯基)丙酸用于制备减少脂肪累积的医药组合物的用途。
在本发明的一实施例中,该3-(4-羟基-3-甲氧苯基)丙酸减少一脂肪细胞的脂肪含量。
本发明的另一目的在提供一种组合物用于制备减少脂肪累积的医药组合物的用途,其中该组合物包含3-(4-羟基-3-甲氧苯基)丙酸及一药学上可接受的载体。
在本发明的一实施例中,前述组合物进一步包含6,7,8,3’,4’-五甲氧基黄酮、3,5,7,8,3’,4’-六甲氧基黄酮、或其组合。
本发明揭露经特殊发酵过程而获得的橘皮发酵物中可分离出数种具有减少脂肪细胞的脂肪含量的化合物,包括3-(4-羟基-3-甲氧苯基)丙酸、6,7,8,3’,4’-五甲氧基黄酮、及3,5,7,8,3’,4’-六甲氧基黄酮。因此,本发明提供3-(4-羟基-3-甲氧苯基)丙酸或包含该化合物的组合物用于制备减少脂肪累积的医药组合物的用途。该医药组合物可具有粉末、颗粒、溶液、胶体、或膏体的剂型,其可藉由口服方式给予一个体。
以下将配合图式进一步说明本发明的实施方式,下述所列举的实施例是用以阐明本发明的发明特点及应用,而非以限定本发明的范围,任何熟习此技艺者,在不脱离本发明的精神和范围内,当可做些许更动与润饰,因此本发明的保护范围当视后附的申请专利范围所界定者为准。
附图说明
图1是脂肪细胞以化合物1、2或3处理后的相对脂肪含量柱形图。
具体实施方式
本发明提供一种3-(4-羟基-3-甲氧苯基)丙酸用于制备减少脂肪累积的医药组合物的用途,以及一种包含3-(4-羟基-3-甲氧苯基)丙酸的组合物用于制备减少脂肪累积的医药组合物的用途。以下实施例说明经特殊发酵过程而获得的橘皮发酵物中可分离出3-(4-羟基-3-甲氧苯基)丙酸、6,7,8,3’,4’-五甲氧基黄酮、及3,5,7,8,3’,4’-六甲氧基黄酮等三种化合物,各该化合物显著减少脂肪细胞的脂肪含量。
定义
本文中所使用数值为近似值,所有实验数据皆表示在20%的范围内,较佳为在10%的范围内,最佳为在5%的范围内。
本文中所谓「药学上可接受的载体」是指该载体必须与组合物的活性成分兼容(较佳为能够稳定该活性成分),并且对使用者个体无害。药学上可接受的载体包括水、缓冲溶液、赋形剂、稳定剂、抗氧化剂、多肽、胺基酸、碳水化合物。
材料与方法
材料
MEM-α培养基(Minimum Essential Medium α medium)、胎牛血清(fetal bovineserum,FBS)、青霉素/链霉素(penicillin/streptomycin)、及磷酸缓冲盐溶液(phosphatebuffered saline,PBS)购自Thermo Fisher Scientific公司。油红O(Oil red O)购自Sigma公司。甲醛(formaldehyde)及异丙醇(isopropanol)购自Echo chemical公司。正己烷(n-hexane)、乙酸乙酯(ethyl acetate)、丙酮(acetone)、甲醇(methanol)、乙醇(ethanol)、正丁醇(n-butanol)、乙腈(acetonitrile)、氯仿-d1(氘化程度99.5%)、甲醇-d6(氘化程度99.5%)、重水(deuterium oxide,氘化程度>99.8%)、及二甲基亚砜-d6(dimethyl sulfoxide-d6,氘化程度>99.9%)购自中国台湾默克公司。
微生物
自食品工业发展研究所生物资源保存及研究中心(Bioresource Collection andResearch Center,BCRC)购买酿酒酵母(Saccharomyces cerevisiae;BCRC 20271)。
化学分析仪器
化合物的分离是利用管柱层析法(column chromatography)及薄层层析法(thinlayer chromatography,TLC)。中压液相层析(medium pressure liquid chromatography,MPLC)系统是Rf+(Teledyne ISCO);管柱是选用自Sephadex LH-20(Amersham Biosciences)、Diaion HP-20(Mitsubishi Chemical)、Merck Kieselgel 60(40-63μm,Art.9385)、及MerckRP-18(40-63μm,Art.0250)。高效液相层析(HPLC)系统装配Hitachi L-2310系列泵、Hitachi L-2420UV-VIS侦测器(侦测波长为200nm至380nm)、及D-2000Elite资料处理软件;管柱是选用自分析级HS C18(250×4.6mm,5μm;SUPELCO)与Mightysil RP-18GP 250(250×4.6mm,5μm;Kanto Chemical),以及半制备级HS C18(250×10.0mm,5μm;SUPELCO)与制备级HS C18(250×21.0mm,5μm;SUPELCO)。层析系统配备紫外灯UVP UVGL-25(波长为254nm及365nm)。薄层层析片是涂覆硅胶60F254(0.25mm;Merck)或RP-18F254S(0.25mm;Merck)的铝片。
化合物的化学结构是以质谱法(mass spectrometry,MS)及核磁共振光谱法(nuclear magnetic resonance spectrometry,NMR)确定。具体而言,使用二维离子阱串联傅立叶变换质谱(Bruker amaZon SL system)及电喷洒离子化串联质谱(ESI-MS/MS;Thermo Scientific Orbitrap Elite system);并使用400MHz Varian 400FT-NMR取得一维与二维NMR光谱,以四甲基硅烷(tetramethylsilane,TMS)作为内部标准品。
减脂功效试验
以下实施例使用购自美国典型培养物保存中心(American Type CultureCollection,ATCC)的小鼠间质细胞株OP9(ATCC CRL-2749)分化而得的脂肪细胞。OP9细胞依8×104个细胞/孔接种于含有500μL前脂肪细胞扩充培养基(90%MEM-α培养基,20%FBS,1%青霉素/链霉素)的24孔培养盘,在37℃下培养7天。细胞培养期间每隔3天更换培养基为脂肪细胞分化培养基(90%MEM-α培养基,20%FBS,1%青霉素/链霉素)。7天后,以显微镜(ZEISS;放大倍率400x)观察细胞内油滴形成以确认其完全分化为脂肪细胞。其后,将待测样品添加至细胞,于37℃下培养7至10天,期间每隔3天更换脂肪细胞分化培养基。最终,移除培养基并以PBS溶液清洗细胞,以便进行油红O染色及测定各组细胞的相对脂肪含量。该相对脂肪含量是实验组细胞相对于控制组细胞的脂肪含量比值(以百分比表示)。控制组细胞是依类似方式以不含待测样品的脂肪细胞分化培养基处理。
油红O染液的制备
将染剂油红O彻底溶解于100%异丙醇以配制3mg/mL的油红O储备溶液。为获得可供使用的油红O染液,于使用前实时将该储备溶液以二次水稀释至浓度1.8mg/mL并以0.22μm滤膜过滤。
油红O染色
细胞中的脂肪含量是经过油红O染色测定。染色前,使用PBS溶液清洗细胞,再以10%甲醛固定细胞30分钟。该固定的细胞经PBS溶液清洗1次后,以60%异丙醇润洗1分钟。其后,以油红O染液对细胞染色1小时,移除染液后再以60%异丙醇退染5秒。染色后细胞经PBS溶液清洗即以显微镜(ZEISS Axio Vert.A1)观察,或以100%异丙醇溶解细胞内染剂10分钟以进行定量。定量时,将100μL前述溶染剂-异丙醇溶液移入一96孔盘,使用ELISA(enzyme-linked immunosorbent assay,酵素结合免疫吸附法)读盘机(BioTek)测量在510nm的吸光值。定量结果以Excel软件的学生t检定进行统计分析。
实施例1
橘皮发酵物的制备
首先,将橘子(Citrus reticulata)果皮洗净、干燥、及切碎(边长约0.5至1公分)。另外,配制一酵母培养液,其是包含1%至5%(w/v)酵母蛋白水解物(yeast peptone)及10%至15%(w/v)葡萄糖的水溶液。该酵母培养液可在50℃至100℃下加热0.5至2小时,再降温至室温。将0.01%至0.5%(w/v)酿酒酵母(Saccharomyces cerevisiae;BCRC 20271)添加至该酵母培养液,在25℃至35℃静置培养3至5天可获得一酵母培养物(yeastculture)。其后,依重量比1:15至3:10混合前述橘皮与该酵母培养物,并且在25℃至35℃进行静置发酵3至10天,即可获得一橘皮发酵物。
实施例2
橘皮发酵物的减脂活性成分分析
为确认依实施例1的方法所制得的橘皮发酵物中的减脂活性成分,依实施例1所述方法制备10公升橘皮发酵物,并依下列步骤进行分析。首先,藉由乙酸乙酯(ethylacetate)与水等比例液相分配的方式萃取该橘皮发酵物三次。将所得乙酸乙酯层萃取液合并,再经减压浓缩干燥可得到乙酸乙酯层萃取物合计约3.6g。其后,余下水层萃取液以正丁醇与水等比例液相分配的方式萃取三次。将所得正丁醇层萃取液及水层萃取液分别合并,再经减压浓缩干燥可得到正丁醇层萃取物合计约10.8g及水层萃取物合计约199g。
橘皮发酵物及前述三种橘皮发酵物的萃取物(乙酸乙酯层萃取物、正丁醇层萃取物与水层萃取物)分别进行减脂功效试验,其结果显示橘皮发酵物及该三种萃取物皆可显著减少脂肪细胞中的脂肪,特别是乙酸乙酯层萃取物表现出抑制脂肪累积的最佳效果。因此,进一步自乙酸乙酯层萃取物中分离出具有减脂活性的成分。
依据生物活性导引分离方法(bioassay guided fractionation),使用SephadexLH-20管柱及甲醇冲提液,对乙酸乙酯层萃取物(约3.6g)进行管柱层析,分离得到5个划分层(分别标记为F1至F5)。其后,使用硅胶管柱Merck Kieselgel 60(40-63μm,Art.9385)及二氯甲烷与甲醇依体积比19:1混合的冲提液,对F2划分层进行管柱层析,所得流出液再经薄层层析片分离,可得到11个划分层(分别标记为F2-1至F2-11)。接着,使用硅胶管柱及正己烷与乙酸乙酯依体积比2:3混合的冲提液,对F2-4划分层进行管柱层析,所得流出液再经薄层层析片分离,可得到13个划分层(分别标记为F2-4-1至F2-4-13)。
F2-4-11及F2-4-13划分层进一步以高效液相层析(HPLC)分离。移动相为甲醇与水依体积比3:7的混合液时,可自F2-4-11划分层获得化合物1(约16.2mg),并自F2-4-13划分层获得化合物2(约9.3mg)。
此外,使用硅胶管柱及正己烷与乙酸乙酯依体积比1:1混合的冲提液,对F3划分层进行管柱层析,所得流出液再经薄层层析片分离,可得到8个划分层(分别标记为F3-1至F3-8)。其后,使用硅胶管柱及二氯甲烷与甲醇依体积比9:1混合的冲提液,对F3-5划分层进行管柱层析,所得流出经薄层层析片分离,再以HPLC分离(移动相为甲醇与水依体积比3:7的混合液),可得到化合物3(约4.7mg)。
藉由质谱仪与核磁共振光谱仪分析确定化合物1、2、及3的化学结构,其名称及结构式如下表1所示。
表1
实施例3
化合物1-3的减脂效果评估
本实施例利用减脂功效试验评估化合物1、2、及3(浓度为4μg/mL)的减脂效果,其结果如图1及下表2所示;图中*及**分别表示p<0.05及p<0.01。依据表2,相比控制组,化合物1、2、及3皆显著抑制脂肪细胞的脂肪累积,其分别降低脂肪含量达约20%、17%、及24%。
表2
处理 | 相对脂肪含量(%) |
控制组 | 100±9.52 |
化合物1 | 79.89±13.58 |
化合物2 | 82.88±3.91 |
化合物3 | 75.75±1.33 |
综上所述,上述实验显示本发明的橘皮发酵过程可产出数种可降低脂肪累积的化合物。因此,本发明提供一种3-(4-羟基-3-甲氧苯基)丙酸(即化合物3)或包含该化合物的组合物用于制备减少脂肪累积的医药组合物的用途。该医药组合物可具有粉末、颗粒、溶液、胶体、或膏体的剂型,其可藉由口服方式给予一个体。
Claims (6)
1.一种3-(4-羟基-3-甲氧苯基)丙酸用于制备减少脂肪累积的医药组合物的用途。
2.根据权利要求1所述的用途,其特征在于,所述3-(4-羟基-3-甲氧苯基)丙酸减少脂肪细胞的脂肪含量。
3.一种组合物用于制备减少脂肪累积的医药组合物的用途,其特征在于,所述组合物包含3-(4-羟基-3-甲氧苯基)丙酸及一药学上可接受的载体。
4.根据权利要求3所述的用途,其特征在于,所述组合物进一步包含6,7,8,3’,4’-五甲氧基黄酮、3,5,7,8,3’,4’-六甲氧基黄酮、或其组合。
5.根据权利要求3项所述的用途,其特征在于,所述医药组合物是以口服方式给予。
6.根据权利要求3项所述的用途,其特征在于,所述医药组合物具有粉末、颗粒、溶液、胶体、或膏体的剂型。
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