CN110680840A - 小黑药提取物在制备降血脂药物中的应用 - Google Patents
小黑药提取物在制备降血脂药物中的应用 Download PDFInfo
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Abstract
本发明公开了小黑药提取物在制备降血脂药物中的应用,属于降血脂药物技术领域。所述小黑药提取物为小黑药醇提物、小黑药石油醚部位和小黑药乙酸乙酯部位中的一种或多种;所述小黑药醇提物的制备方法为:将小黑药干燥根茎采用粉碎机粉碎,过筛,加入体积百分比20‑100%的C1‑C4醇溶液提取0.5‑2小时,离心取上清液、旋转蒸发、浓缩得到小黑药醇提取物。与现有技术相比,本发明提供的小黑药醇提物具有安全性高、降脂作用好的性能,可应用于预防和辅助治疗肥胖、酒精性脂肪肝、非酒精性脂肪肝、2型糖尿病、代谢综合征、冠心病、动脉粥样硬化疾病的药品、食品或功能食品。
Description
技术领域
本发明涉及小黑药提取物在制备降血脂药物中的应用,属于降血脂药物技术领域。
背景技术
小黑药是旋覆花属植物显脉旋覆花(Inula nervosa wall)的根茎,又称云威灵,民间常用抗炎中草药,能通经络,祛风湿,健胃消食,止咳祛痰,2010年经卫生部批准为新食品原料,是一种药食两用资源。有研究表明,显脉旋覆花富含酚类和挥发油成分,其中麝香草酚和异丁酸百里香酚酯为挥发油中的主要成分,由其挥发油研制的云威灵油软胶囊用于治疗风湿痹病(李柯,史蕾喆,陈丹,胡薏冰,卢茂芳,李若存,李顺祥.HPLC测定不同药用部位、产地及采收期显脉旋覆花中麝香草酚和异丁酸百里香酯的含量[J].湖南中医药大学学报,2013,33(07):41-44)。另外,小黑药总挥发油还被报道具有抑制哮喘、祛痰、抑菌、抗炎作用(舒晔,王懋德,江滨,贾向云,林桦.显脉旋复花总挥发油的药理研究[J].云南中医学院学报,1990(01):22-24)。但目前尚未有小黑药醇提物、低极性成分降血脂活性的报道。
高脂血症是由于脂肪转运或代谢异常所致的血浆或血清中一种或者多种脂质高于正常范围内的全身性疾病,具体表现为总胆固醇、甘油三酯、低密度脂蛋白胆固醇异常升高,是一种常见的慢性疾病。过量脂肪在多个组织、脏器异位沉积,诱发氧化应激、炎症反应,损伤细胞形态和功能,是脂代谢异常慢性疾病,如肥胖、酒精性脂肪肝、非酒精性脂肪肝、2型糖尿病、代谢综合征、冠心病、动脉粥样硬化疾病发生、发展的重要因素。现阶段的生活方式干预及针对疾病症状的对抗治疗,远期疗效不佳,难以逆转疾病的发展,凸显开发新型干预策略及防治药物的重要性。从传统药食两用资源中寻找安全、降脂作用好的组分,是发展脂代谢异常慢性疾病干预策略的重要方向之一。我们首先研究发现小黑药醇提取物,特别是低极性部位提取物具有良好的降血脂效果。
发明内容
为了解决上述问题,本发明提供了一种小黑药醇提物在制备降血脂药物中的应用,具有安全性高、降脂作用好的性能,可应用于预防和辅助治疗肥胖、酒精性脂肪肝、非酒精性脂肪肝、2型糖尿病、代谢综合征、冠心病、动脉粥样硬化疾病的药品、食品或功能食品,具有广阔的经济效益和社会效益。
本发明的第一个目的是提供一种小黑药提取物,所述小黑药提取物为小黑药醇提物、小黑药石油醚部位和小黑药乙酸乙酯部位中的一种或多种;
所述小黑药醇提物的制备方法为:将小黑药干燥根茎采用粉碎机粉碎,过筛,加入体积百分比20-100%的C1-C4醇溶液提取0.5-2小时,离心取上清液、旋转蒸发、浓缩得到小黑药醇提取物;
所述小黑药石油醚部位和小黑药乙酸乙酯部位的制备方法为:取制备得到的小黑药醇提取物分散于蒸馏水中,配制成浓度为5~20%的小黑药醇提取溶液,分别加入石油醚、乙酸乙酯进行分配萃取,旋转蒸发、浓缩得到小黑药石油醚部位和小黑药乙酸乙酯部位。
在本发明一种实施方式中,所述小黑药与醇溶液的质量体积比为1:(5-50);所述小黑药醇提取溶液和石油醚、乙酸乙酯的体积比为(1-5):1。
本发明的第二个目的是提供一种小黑药提取物在制备降血脂药物中的应用,所述小黑药提取物为小黑药醇提物;所述是小黑药醇提物采用体积百分比20-100%的C1-C4醇溶液进行提取得到的。
在本发明一种实施方式中,所述小黑药提取物还包括小黑药石油醚部位、小黑药乙酸乙酯部位和小黑药水溶性部位。
在本发明一种实施方式中,所述小黑药石油醚部位、小黑药乙酸乙酯部位和小黑药水溶性部位是分别采用石油醚、乙酸乙酯和水对上述制备得到的小黑药醇提物进一步进行分配萃取得到的。
在本发明一种实施方式中,所述小黑药提取物的制备方法如下:
(1)制备小黑药醇提物:将小黑药干燥根茎采用粉碎机粉碎,过10-200目筛,加入80%-95%乙醇,置超声清洗机中进行超声提取0.5-2小时,离心取上清液进行旋转蒸发仪减压浓缩至干,得小黑药醇提取物;小黑药与醇溶液的质量体积比为1:(5-50)。
(2)制备小黑药石油醚部位、小黑药乙酸乙酯部位和小黑药水溶性部位:取小黑药乙醇提取物分散于蒸馏水中,配制成浓度为5~20%的小黑药乙醇提取溶液,分别加入石油醚、乙酸乙酯进行分配萃取,得到中低极性的小黑药石油醚部位、小黑药乙酸乙酯部位;所述小黑药乙醇提取溶液和石油醚、乙酸乙酯的体积比为(1-5):1。
在本发明一种实施方式中,所述应用包括可应用于预防和辅助治疗肥胖、酒精性脂肪肝、非酒精性脂肪肝、2型糖尿病、代谢综合征、冠心病、动脉粥样硬化疾病。
本发明的第三个目的是提供一种降血脂药物组合物,所述药物组合物包含小黑药醇提物、小黑药石油醚部位、小黑药乙酸乙酯部位和小黑药水溶性部位中的一种或多种。
在本发明一种实施方式中,所述的药物组合物还包括药物载体和/或药用辅料。
在本发明一种实施方式中,所述的药物组合物的剂型为医学上认可的任意一种剂型。
本发明的第四个目的是提供一种降血脂药物制剂,所述药物制剂包含小黑药醇提物、小黑药石油醚部位、小黑药乙酸乙酯部位和小黑药水溶性部位中的一种或多种。
在本发明一种实施方式中,所述剂型包括粉剂、注射液、胶囊、片剂、口服液。
在本发明一种实施方式中,小黑药提取物的剂量至少为0.5μg/mL。
本发明的第五个目的是提供一种在降血脂食品或饲料,所述食品或饲料的配方包含小黑药醇提物、小黑药石油醚部位、小黑药乙酸乙酯部位和小黑药水溶性部位中的一种或多种。
本发明的第六个目的是提供一种降血脂的膳食补充剂,所述膳食补充剂的配方包含小黑药醇提物、小黑药石油醚部位、小黑药乙酸乙酯部位和小黑药水溶性部位中的一种或多种。
本发明的第七个目的是提供一种降血脂的保健品,所述保健品的配方包含小黑药醇提物、小黑药石油醚部位、小黑药乙酸乙酯部位和小黑药水溶性部位中的一种或多种。
本发明的有益效果:
与现有技术相比,本发明提供了一种具有降脂作用的小黑药醇提物,给予2μg/mL小黑药醇提物时能有效降低细胞内脂质累积36.87%,给予2μg/mL小黑药醇提物乙酸乙酯部位时降低细胞内脂质累积54.31%。小黑药醇提物在动物体内具有较好的降血脂效果,甘油三酯水平由4.83±0.24mmol/mg prot显著降低至3.35±0.46mmol/mg prot。小黑药醇提物具有安全性高、降脂作用好的性能,可应用于预防和辅助治疗肥胖、酒精性脂肪肝、非酒精性脂肪肝、2型糖尿病、代谢综合征、冠心病、动脉粥样硬化疾病的药品、食品或功能食品。
附图说明
图1小黑药乙酸乙酯部位对游离脂肪酸诱导的HepG2细胞内脂质累积量(油红O染色)和活性氧生成量(DCFH-DA荧光探针)的影响。
具体实施方式
以下对本发明的优选实施例进行说明,应当理解实施例是为了更好地解释本发明,不用于限制本发明。
实施例1:小黑药醇提物、中低极性部位的制备
按照下述方法制备得到:
(1)制备小黑药醇提物:100g小黑药干燥根茎采用药材粉碎机(黄城HC-2500Y304,武义海纳电器有限公司)粉碎,过100目筛,按1:20质量体积比加入95%乙醇(分析纯,常州恒光化学试剂有限公司),置超声清洗机(东森DS-100S,深圳市品凰科技有限公司)中进行超声提取1小时,离心取上清液进行旋转蒸发仪(上海亚荣RE-52A,上海亚荣生化仪器厂)减压浓缩至干,得9.5g小黑药乙醇提取物。
(2)提取中低极性部位:取9.0g小黑药乙醇提取物分散于90mL蒸馏水中,按3:1体积比分别加入石油醚、乙酸乙酯(分析纯,常州恒光化学试剂有限公司)进行分配萃取,旋转蒸发、浓缩得到中低极性的小黑药石油醚部位0.68g、乙酸乙酯部位1.25g以及水溶性部位6.80g。
实施例2:小黑药醇提物、各极性部位的降脂作用(体外实验)
将摩尔比1:2的油酸钠、棕榈酸钠(Sigma-Aldrich)分别用无脂肪酸牛血清白蛋白(上海翊圣生物科技有限公司)偶联后混合,制得混合游离脂肪酸。将1.2mM混合游离脂肪酸加入到96孔板中预先贴壁生长的人肝癌HepG2细胞(中国科学院典型培养物保藏委员会细胞库)中,在37℃的5%CO2细胞培养箱(Forma,Thermo Scientific)中培养24h,建立游离脂肪酸诱导的脂质累积模型。通过考察实施例1中制备的小黑药醇提物(XHY-Q)、石油醚部位(PE-Q)、乙酸乙酯部位(AE-Q)、水部位(W-Q)对HepG2细胞增殖的影响后,确定适宜的给药浓度为0.1-2ug/mL。因此在0.1-2ug/mL浓度下,分别通过尼罗红(上海阿拉丁生化科技股份有限公司,纯度>95%)、DCFH-DA荧光探针(碧云天生物技术有限公司)测定了小黑药醇提物(XHY-Q)、石油醚部位(PE-Q)、乙酸乙酯部位(AE-Q)、水部位(W-Q)的降低脂质累积的效果和维持氧化还原稳态的作用,结果见表1。实验结果显示,XHY-Q、PE-Q、AE-Q、W-Q浓度依赖性地降低细胞的脂质累积和活性氧产生,其中,给予2μg/mL XHY-Q时降低游离脂肪酸诱导的细胞内脂质累积和活性氧产生水平效果最好,细胞内脂质累积水平模型组的640.8±50.7(%)显著降低至393.03±58.78(%),降低比例为38.67%,细胞内氧化还原水平由模型组的146.30±9.19(%)显著降低至104.54±9.60(%),接近正常组水平。比较不同极性部位的降低脂质累积和活性氧生成效果,在0.5-2μg/mL浓度下极显著地降低细胞内脂质累积和ROS生成(p<0.05),发现中低极性的乙酸乙酯部位降低细胞内脂质累积和ROS生成效果最好。分别给予0.5、1、2ug/mL AE-Q后,细胞内脂质累积水平由模型组的640.8±50.7(%)分别显著降低至489.37±38.62(%)、408.68±37.89(%)、292.78±28.12(%),ROS由模型组的146.30±9.19(%)分别显著降低至116.06±4.83(%)、103.83±5.45(%)、90.00±6.61(%)。
分别使用油红O染色(北京索莱宝科技有限公司)、甘油三酯试剂盒(TG,南京建成生物工程研究所)、总胆固醇试剂盒(T-CHO,南京建成生物工程研究所)测定小黑药乙酸乙酯部位(AE-Q)的脂代谢调控作用;采用试剂盒测定丙二醛(MDA,南京建成生物工程研究所)、总抗氧化能力(T-AOC,南京建成生物工程研究所)、谷胱甘肽过氧化物酶(GSH-px,南京建成生物工程研究所)、超氧化物歧化酶(SOD,南京建成生物工程研究所)、过氧化氢酶(CAT,南京建成生物工程研究所)、还原型谷胱甘肽与氧化型谷胱甘肽的比值(南京建成生物工程研究所)的水平,深入评价小黑药乙酸乙酯部位的调控脂代谢和氧化应激的效果,结果见表2和表3。实验结果表明,小黑药乙酸乙酯部位主要以降低甘油三酯含量的方式降低细胞内脂质累积水平,给予2μg/mL AE-Q时甘油三酯下降最明显,含量由0.158±0.011mmol/g prot显著降低至0.087±0.007mmol/g prot,而降低胆固醇含量的作用并不明显;小黑药乙酸乙酯部位通过提高细胞抗氧化酶水平,降低脂质氧化终产物MDA水平,给予2μg/mL AE-Q时作用最明显,MDA含量由9.90±1.31ng/mg prot显著降低至5.30±0.78ng/mg prot,总抗氧化能力T-AOC由1.40±0.15U/mg prot显著升高至2.92±0.22U/mgprot,SOD含量由1.53±0.36ng/mg prot显著升高至3.34±0.36ng/mg prot,CAT含量由14.19±0.98ng/mg prot显著降低至22.86±2.36ng/mg prot,GSH-px含量由15.34±1.71ng/mg prot显著降低至20.06±1.86ng/mg prot,GSH/GSSG由0.80±0.15显著降低至1.17±0.12。因此结合脂质积累量与活性氧生成量的实验结果,说明小黑药醇提物中的具有中低极性的乙酸乙酯部位具有良好的降脂、抗氧化效果。
表1小黑药醇提物及各极性部位对游离脂肪酸诱导的HepG2细胞内脂质累积量和活性氧生成量的影响
表2小黑药乙酸乙酯部位对游离脂肪酸诱导的HepG2细胞氧化还原稳态的影响
表3小黑药乙酸乙酯部位对游离脂肪酸诱导的HepG2细胞脂代谢的影响
实施例3:小黑药醇提物的降脂作用(体内实验)
40只初始体重30g的4周龄ICR雄性小鼠(上海斯莱克实验动物有限公司)适应性饲喂1周后,随机分为4组,每组10只,分别为正常组CON,高脂组HFD,高脂小黑药醇提物HFD+XHY-Q。CON组给予正常日粮,并每日灌胃0.2mL生理盐水;HFD给予高脂日粮(江苏省协同医药生物工程有限责任公司),并每日灌胃0.2mL生理盐水;HFD+XHY-Q给予高脂日粮(江苏省协同医药生物工程有限责任公司),并每日按20mg/kg体重灌胃小黑药醇提物。第5周开始,HFD组小鼠平均体重超过CON组平均体重的130%,表明高脂日粮诱导小鼠肥胖造模成功。于第6周处死各组小鼠,处死前将小鼠称重(表4),取肝脏测定肝重(表5)和肝脏内氧化应激水平(表6),取血浆测定小鼠血脂(表7)。实验结果表明,给予20mg/kg小黑药提取物能有效降低肥胖小鼠体重和肝重,在第6周时体重由48.91±0.38g显著降低至44.78±0.28g,肝重由2.19±0.08g显著降低至1.97±0.05g。小黑药醇提物能显著改善甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平,但对胆固醇(TC)水平无明显影响,甘油三酯水平由4.83±0.24mmol/mg prot显著降低至3.35±0.46mmol/mg prot,高密度脂蛋白胆固醇由1.43±0.17mmol/mg prot显著升高至2.43±0.38mmol/mg prot,低密度脂蛋白胆固醇由1.84±0.33mmol/mg prot显著降低至1.33±0.33mmol/mg prot,总胆固醇由7.67±0.82mmol/mg prot降低至7.09±0.54mmol/mg prot。小黑药醇提物能显著降低肝脏脂质氧化终产物丙二醛(MDA)水平,升高总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-px)、过氧化氢酶(CAT)、GSH/GSSG的水平,丙二醛(MDA)水平由12.59±1.28nmol/mg prot显著降低至9.58±0.99nmol/mg prot,总抗氧化能力由6.36±0.24U/mg prot显著升高至7.08±0.24U/mg prot,超氧化物歧化酶由0.50±0.03U/mgprot显著升高至0.57±0.05U/mg prot,谷胱甘肽过氧化物酶由443.55±60.03U/mg prot显著升高至556.84±40.44U/mg prot,GSH/GSSG由1.53±0.40显著升高至3.29±0.36。实验表明,小黑药醇提物能有效控制肥胖体重,降低血脂,改善肝脏氧化应激。
表4小黑药醇提物对高脂饮食小鼠体重的影响
表5小黑药醇提物对高脂饮食小鼠肝脏重量的影响
表6小黑药醇提物对高脂饮食小鼠氧化还原稳态的影响
表7小黑药醇提物对高脂饮食小鼠脂代谢的影响
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一种小黑药提取物,其特征在于,所述小黑药提取物为小黑药醇提物、小黑药石油醚部位和小黑药乙酸乙酯部位中的一种或多种;
所述小黑药醇提物的制备方法为:将小黑药根茎粉碎,加入C1-C4醇溶液提取0.5-2小时,得到小黑药醇提取物;
所述小黑药石油醚部位和小黑药乙酸乙酯部位的制备方法为:取制备得到的小黑药醇提取物溶解得到小黑药醇提取溶液,分别加入石油醚、乙酸乙酯进行分配萃取,得到小黑药石油醚部位和小黑药乙酸乙酯部位。
2.根据权利要求1所述的小黑药提取物,其特征在于,所述小黑药与醇溶液的质量体积比为1:(5-50);所述小黑药醇提取溶液浓度为5~20%;所述小黑药醇提取溶液和石油醚、乙酸乙酯的体积比为(1-5):1。
3.一种在辅助调节血脂的食品或饲料或膳食补充剂或保健品,其特征在于,所述食品或饲料或膳食补充剂或保健品的配方包含权利要求1或2所述的小黑药提取物。
4.权利要求1或2所述的小黑药提取物在制备降血脂药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述应用包括可应用于预防和辅助治疗肥胖、酒精性脂肪肝、非酒精性脂肪肝、2型糖尿病、代谢综合征、冠心病、动脉粥样硬化疾病。
6.一种降血脂药物组合物,其特征在于,所述药物组合物包含权利要求1或2所述的小黑药提取物。
7.根据权利要求6所述的药物组合物,其特征在于,所述的药物组合物还包括药物载体和/或药用辅料。
8.根据权利要求6所述的药物组合物,其特征在于,所述的药物组合物的剂型为医学上认可的任意一种剂型。
9.根据权利要求8所述的药物组合物,其特征在于,所述剂型包括粉剂、注射液、胶囊、片剂、口服液。
10.根据权利要求6所述的药物组合物,其特征在于,小黑药提取物的剂量至少为0.5μg/mL。
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