CN112451546A - 仿刺参海参花脂类提取物及其制备方法和应用 - Google Patents
仿刺参海参花脂类提取物及其制备方法和应用 Download PDFInfo
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- CN112451546A CN112451546A CN202011349839.8A CN202011349839A CN112451546A CN 112451546 A CN112451546 A CN 112451546A CN 202011349839 A CN202011349839 A CN 202011349839A CN 112451546 A CN112451546 A CN 112451546A
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Abstract
本发明提供仿刺参海参花脂类提取物及其制备方法和应用,海参花脂类提取物主要含有二十碳五烯酸和二十二碳六烯酸等20种脂肪酸,其中不饱和脂肪酸13种,相对百分含量不低于70%。经提取纯化后的海参花脂类提取物经口途径给药可显著降低高脂血症大鼠总胆固醇和低密度脂蛋白,该作用与阳性对照药阿托伐他汀钙片效果接近。并且有显著降低高脂血症大鼠血清甘油三脂的作用,降低甘油三脂的作用优于阳性对照药阿托伐他汀钙片,同时对高脂血症大鼠的高密度脂蛋白有明显升高作用。海参花脂类提取物比海参花具有更显著的调节血脂活性,其效果远远好于海参花,提示海参花脂类提取物可应用于调节高血脂症的药物或保健功能食品的研究开发。
Description
技术领域
本发明涉及医药技术领域,尤其涉及仿刺参海参花脂类提取物及其制备方法和应用。
背景技术
仿刺参(Apostichopus japonicus Selenka)又称辽参,属海参纲刺参科仿刺参属动物。主产于我国山东、辽宁和河北、江苏北部等沿海地区。海参在《本草纲目拾遗》列为补益药物,称其“补肾经、益精髓、消痰涎、摄小便,壮阳,治疗溃疡生疽。”《本草从新》载:“海参辽海产者良,有刺者名刺参,无刺者名光参”。海参花为海参的肠卵部分。海参在捕捞后,一般都要被立即解剖去脏,用长形小刀在海参腹部近肛门处插刀,向前方纵割一小口,长约参体的三分之一,从中取出海参肠后,就可以看见金黄色的海参卵了。海参籽外形像是连在一起的一串串细长、绵软的微型麦穗。如果把海参卵在水中摊开,则其连绵不断,随水漂动如同一朵绽放在水中的菊花,因此,海参卵才得名海参花。海参花营养极为丰富,被称为海黄金,海参花含有对人体有特殊功效的酸性粘多糖、脂质、内脏色素(β胡萝卜素、海胆紫酮、虾青素、玉米黄素、角黄素等),以及比海参体壁含量更高的钒、精氨酸等活性物质,还含有多种海参皂甙、硒、铁、钙等成分。
仿刺参海参花脂是一种从仿刺参海参花中提取的脂肪酸脂类提取物,对其研究鲜有报道。
发明内容
本发明的第一个目的在于,提供一种仿刺参海参花脂类提取物。
本发明第二个目的在于,提供一种从生长于我国渤海海域的仿刺参海参花中提取海参花脂类提取物的制备方法。
本发明第三个目的在于,提供仿刺参海参花脂类提取物的应用。
为了实现上述第一个目的,本发明提供了一种仿刺参海参花脂类提取物,所述脂类提取物含有十五烷酸、十六烯酸、十六烷酸、十七烷酸、十八烯酸、硬脂酸、十八烷酸、5,8,11,14-二十碳四烯酸、二十碳五烯酸(EPA)、二十烯酸、异二十烯酸、二十三烯酸、二十烷酸、二十一烷酸、4,7,10,13,16,19-二十二碳六烯酸(DHA)、二十二稀酸、异二十二稀酸、二十二烷酸、二十三稀酸和15-二十四稀酸。
作为一个优选方案,所述脂类提取物含有不饱和脂肪酸13种,相对质量百分含量不低于70%。
为了实现上述第二个目的,本发明提供了仿刺参海参花脂类提取物的制备方法,通过溶剂提取法提取,将海参花经水洗和冷冻干燥,得干燥的海参花粉,取海参花粉加入60%~95%(V/V)的乙醇搅拌提取,离心过滤,合并提取液,减压回收乙醇得流浸膏,将流浸膏溶解于50%~85%(V/V)乙醇中,加入活性炭,加热回流脱色,过滤,滤液减压回收,有机溶剂萃取,减压回收至干,得棕色油脂状物海参花脂类提取物。
作为一个优选方案,所述萃取用的有机溶剂为氯仿、二氯甲烷、正丁醇、醋酸乙酯、乙醚和石油醚中的一种。
为了实现上述第三个目的,本发明提供了仿刺参海参花脂类提取物在制备调节高血脂的药物或保健食品中的应用。
优选地,本发明提供了仿刺参海参花脂类提取物在制备降低高脂血症总胆固醇、低密度脂蛋白和甘油三脂药物或保健食品中的应用。
优选地,本发明提供了仿刺参海参花脂类提取物在制备升高高脂血症的高密度脂蛋白药物或保健食品中的应用。
本发明的优点在于,本发明提取的海参花脂类提取物活性好,得率高,杂质含量少。经提取纯化后的海参花脂类提取物比海参花具有更显著的调节血脂活性,其效果远远好于海参花,提示海参花脂可应用于调节高血脂症的药物或保健功能食品的研究开发。经口途径给药可显著降低高脂血症大鼠总胆固醇和低密度脂蛋白,该作用与阳性对照药阿托伐他汀钙片效果接近。并且有显著降低高脂血症大鼠血清甘油三脂的作用,降低甘油三脂的作用优于阳性对照药阿托伐他汀钙片,同时对高脂血症大鼠的高密度脂蛋白有明显升高作用。
附图说明
图1海参花脂类提取物脂肪酸甲酯的总离子流图。
具体实施方式
以下,结合具体实施方式对本发明的技术进行详细描述。应当知道的是,以下具体实施方式仅用于帮助本领域技术人员理解本发明,而非对本发明的限制。
实施例1.
新鲜采集的海参花经水洗和冷冻干燥,得干燥的海参花粉末。取海参花粉1000克,每次加入5倍量95%乙醇(V/V)搅拌提取3次,每次1小时,离心过滤,合并提取液,减压回收乙醇得流浸膏,溶解于80%乙醇(V/V),加入活性炭,加热回流1小时脱色,过滤,滤液减压回收,二氯甲烷萃取,萃取液水洗4次,减压回收至干,得棕色油脂状物海参花脂类提取物72克,得率约7%左右。
海参花脂类提取物(下称海参花脂)的鉴定:
本研究对海参花脂中的脂肪酸先进行甲酯化处理,再用气相色谱一质谱联用仪对其进行分析测定,分离鉴定了其中的脂肪酸成分和各组分相对含量。
实验方法:称取海参花脂100克,甲醇300毫升超声提取6次,旋转蒸发回收溶剂,得海参花脂甲醇提取浸膏16.7克,将浸膏均匀分散于水中,正丁醇提取3次,旋转蒸发回收溶剂,得海参花脂正丁醇层浸膏11.8克,正丁醇层浸膏反复水洗得到黄色油状物质7.9克。称取上述试样500mg至具塞试管中,用移液管移取5mL正庚烷溶解试样,加入2mL氢氧化钾甲醇溶液,盖上玻璃盖猛烈振摇1min后,加蒸馏水至试管颈部,置于离心机以3000r/min离心5min,移取2mL上清液至具塞试管,定容置10mL,取经处理过的样品0.4mL,用GC/MS进行分析鉴定.通过G170LBA化学工作站数据处理系统,检索NIST98谱图库,确定样品中各个化学成分。选取峰面积占比前20的化合物,按峰面积归一化法进行定量分析,分别求得20个主要化学成分的相对含量。
气相色谱条件:HP 5890II气相色谱仪(附电子轰击质谱检测器),甲醇、正丁醇、氢氧化钾为国药集团化学试剂有限公司,均为分析纯。海参花由烟台东宇海珍品有限公司提供。色谱条件:毛细管色谱柱Alltech EC.1 000,30m×O.25mm;色谱柱温度:198℃恒温3min,以10℃/min升温至248℃,维持12min;进样口温度:280℃;EI-MS检测器温度:280℃;进样方式:分流,分流比30:1;进样量:1;载气:氮气。质谱条件:离子源为电子轰击源;离子源温度230℃;电离电压70eV;电子倍增器电压1988V;发射电流34.6mA;接口温度230℃;扫描范围m/z 20—500。定性分析:取经上述处理过的样品0.4nl,用GC/MS进行分析鉴定.通过G170LBA化学工作站数据处理系统,检索NIST98谱图库,确定样品中各个化学成分。定量分析:通过G170I BA化学工作站数据处理系统,按峰面积归一化法进行定量分析,分别求得各化学成分的相对含量。
结果:样品经过GC/MS分析鉴定,得到海参花脂脂肪酸甲酯的总离子流图(图1)。
表1海参花脂脂肪酸甲酯GC/MS分析结果
实验结果表明,海参花脂中主要的20种脂肪酸中,饱和脂肪酸7种,相对百分含量为19.50%;不饱和脂肪酸13种,相对百分含量为80.50%,其中单不饱和脂肪酸9种,相对百分含量为47.91%;多不饱和脂肪酸4种,相对百分含量为32.59%。二十碳五烯酸(EPA)的相对百分含量为15.08%,二十二碳六烯酸(DHA)的相对百分含量为7.61%。
实施例2.海参花脂调节血脂实验:
1.实验目的
了解供试品海参花脂是否具有预防大鼠高脂血症的作用。
2.实验材料
2.1.受试药物
名称或代码:海参花脂(代号:HSZ-1)
来源:由易杨华教授提供;
性状:棕色油脂状物;
规格:50g;
批号:20200418
保存条件:4℃;
有效期:2年
配制方法:匀浆器研磨后加0.5%CMC-Na混悬。
2.2.阳性对照品
名称或代码:阿托伐他汀钙片;
来源:兴安药业有限公司;
性状:白色片状;
规格:10mg;
批号:042004071;
保存条件:密封,在阴凉处保存;
有效期:2年。
2.3.其它试剂
大鼠高脂饲料:常州鼠一鼠二生物科技有限公司生产,型号:D12109T,产品批号:18072702。
羧甲基纤维素钠盐:国药集团化学试剂有限公司生产,批号:F20050302。
总胆固醇(总胆固醇)测试盒:南京建成生物工程研究所生产,批号:20200730。
甘油三酯(甘油三酯)测试盒:南京建成生物工程研究所生产,批号:20200730。
低密度脂蛋白胆固醇(低密度脂蛋白)测试盒:南京建成生物工程研究所生产,批号:20200731。
高密度脂蛋白胆固醇(高密度脂蛋白)测试盒:南京建成生物工程研究所生产,批号:20200730。
2.4.实验动物
品系:SD大鼠;
级别:SPF级;
性别:雄性;
数量:48只;
体重:200g;
动物生产许可证号:SCXK(沪)2018-0006;
动物质量合格证编号:20180006016463;
来源:上海西普尔-必凯实验动物有限公司;
动物购回后在本单位实验室分笼适应性饲养3天,用普通大鼠饲料喂养,自由饮水,光线12小时明暗自动转换,室内温度20±2℃。
2.5.实验仪器
电子天平:梅特勒-托利多仪器(上海)有限公司,型号:AL204。
电热恒温鼓风干燥箱:上海精宏实验设备有限公司,型号:DHG-9123A
离心机:湖南湘仪仪器厂,型号:L-500型。
酶标仪:赛默飞世尔(上海)仪器有限公司,型号:Multiskan Mk3型。
3.实验方法
3.1.动物分组
实验分为7组,如下:
正常对照组:普通饲料+0.5%CMC-Na
模型对照组:高脂饲料+0.5%CMC-Na;
阳性对照组:高脂饲料+阿托伐他汀钙片2mg/kg;
海参花对照组:高脂饲料+600mg/kg;
海参花脂低剂量组:高脂饲料+150mg/kg;
海参花脂中剂量组:高脂饲料+300mg/kg;
海参花脂高剂量组:高脂饲料+600mg/kg;
共7组,每组动物数量为8只。
3.2.给药途径及给药时间
给药途径:灌胃;
给药容积:5ml/kg;
给药时间:每天1次,连续2周。
3.3.模型复制
大鼠购置后在实验室动物房适应性饲养1周,然后将大鼠随机分为7组,分组后除正常对照组用普通大鼠饲料喂养以外,模型对照组和药物治疗组用高脂饲料喂养,同时给予相应的受试药物灌胃,每天1次,连续2周。
末次给药1h后,各组大鼠眼眶取血1ml,3000r/min离心10min取上清,-20℃冻存。然后用血脂试剂盒检测大鼠的血脂水平,包括:血清总胆固醇(总胆固醇),甘油三脂(甘油三酯),低密度脂蛋白(低密度脂蛋白)和高密度脂蛋白(高密度脂蛋白)。
4.观察指标
血清总胆固醇、甘油三酯、低密度脂蛋白和高密度脂蛋白的水平。
5.统计方法
实验结果以均数±标准差(x_±s)表示,用SPSS 18.0软件对组间数据采用单因素ANOVA分析、Scheffe`s检验测定两组间的差异显著性,以P<0.05定义为具有显著的统计学差异。
6.实验结果
大鼠经过2周的高脂饲料喂养并同时灌胃给予各组受试药物,检测血脂结果提示,海参花脂能够有效地调节大鼠血脂水平,主要体现在降低总胆固醇、甘油三酯和低密度脂蛋白水平,并有升高高密度脂蛋白的作用。
以总胆固醇为例,正常对照组为1.93±0.26mmol/L,模型对照组大鼠在高脂饲料喂养2周后升高到5.04±0.89mmol/L;在高脂饮食的同时,其它各组每天分别给予受试药物灌胃发现,阿托伐他汀给药2周后,总胆固醇降低非常显著,为3.52±0.47mmol/L(P<0.001),降低百分率达30.3%;海参花对照组没有明显降低;供试品海参花脂也有较好的降低总胆固醇作用,海参花脂低中高剂量组总胆固醇水平分别可降低到3.86±0.41mmol/L(P<0.01)、3.72±0.39mmol/L(P<0.01)和3.68±0.35mmol/L(P<0.01),与模型对照组比较分别降低了23.6%、26.3%和27.1%,并随着剂量增加,海参花脂降低总胆固醇的作用也逐渐增强,有显著的量效关系存在。
海参花脂还具有降低甘油三酯、低密度脂蛋白和增加高密度脂蛋白的作用,并且作用效果均强于阳性对照组药阿托伐他汀钙片。以甘油三酯为例,阳性对照组甘油三酯水平为1.16±0.11mmol/L,相较于模型对照组1.48±0.40mmol/L降低了21.8%;海参花对照组没有明显的降低;而海参花脂从低剂量开始降低甘油三酯的作用即强于阳性对照药,其低中高剂量组甘油三酯水平分别为1.04±0.18mmol/L、1.01±0.20mmol/L、1.03±0.14mmol/L,分别降低了29.6%、31.4%和30.7%。
表1.海参花脂对大鼠高脂血症的预防作用血脂水平(x_±s;n=8;单位:mmol/L)
*p<0.05,**p<0.01,***p<0.001与模型对照组比较
7.实验结论
7.1.供试品海参花脂经口途径给药有显著降低高脂血症大鼠血清中总胆固醇和低密度脂蛋白的水平,该作用与阳性对照药阿托伐他汀钙片效果接近。而海参花不能显著降低高脂血症大鼠血清中总胆固醇和低密度脂蛋白的水平。
7.2.供试品海参花脂经口服途径给药有显著降低高脂血症大鼠血清中甘油三酯的水平,降低甘油三酯的作用优于阳性对照药阿托伐他汀钙片。而海参花不能显著降低高脂血症大鼠血清中甘油三酯的水平。
7.3.供试品海参花脂经口服途径给药对高脂血症大鼠血清中的高密度脂蛋白有明显升高作用。而海参花对高脂血症大鼠血清中的高密度脂蛋白没有明显升高作用。
上述实验显示,经提取纯化后的海参花脂较海参花更具有显著的调节血脂活性,海参花脂可应用于防治高血脂症的药物或保健功能食品的研究开发。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (7)
1.一种仿刺参海参花脂类提取物,其特征在于,所述提取物含有十五烷酸、十六烯酸、十六烷酸、十七烷酸、十八烯酸、硬脂酸、十八烷酸、5,8,11,14-二十碳四烯酸、二十碳五烯酸、二十烯酸、异二十烯酸、二十三烯酸、二十烷酸、二十一烷酸、4,7,10,13,16,19-二十二碳六烯酸、二十二稀酸、异二十二稀酸、二十二烷酸、二十三稀酸和15-二十四稀酸。
2.根据权利要求1所述的一种仿刺参海参花脂类提取物,其特征在于,所述脂类提取物含不饱和脂肪酸13种,不饱和脂肪酸相对质量百分含量不低于70%。
3.权利要求1所述仿刺参海参花脂类提取物的制备方法,其特征在于,通过溶剂提取法提取,将海参花经水洗和冷冻干燥,得干燥的海参花粉,取海参花粉加入60%~95%(V/V)的乙醇搅拌提取,离心过滤,合并提取液,减压回收乙醇得流浸膏,将流浸膏溶解于50%~85%(V/V)乙醇中,加入活性炭,加热回流脱色,过滤,滤液减压回收,有机溶剂萃取,减压回收至干,得棕色油脂状海参花脂类提取物。
4.根据权利要求3所述仿刺参海参花脂类提取物的制备方法,其特征在于,所述萃取用的有机溶剂为氯仿、二氯甲烷、正丁醇、醋酸乙酯、乙醚和石油醚中的一种。
5.权利要求1所述仿刺参海参花脂类提取物在制备调节高血脂的药物或保健食品中的应用。
6.权利要求1所述仿刺参海参花脂类提取物在制备降低高脂血症总胆固醇、低密度脂蛋白和甘油三脂药物或保健食品中的应用。
7.权利要求1所述仿刺参海参花脂类提取物在制备升高高脂血症的高密度脂蛋白药物或保健食品中的应用。
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