CN114949017A - 一种刺山柑醇提物、制备方法及其在制备抗氧化产品和/或降血脂产品中的应用 - Google Patents
一种刺山柑醇提物、制备方法及其在制备抗氧化产品和/或降血脂产品中的应用 Download PDFInfo
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Abstract
本发明提供了一种刺山柑醇提物、制备方法及其在制备抗氧化产品和/或降血脂产品中的应用,属于植物活性物质提取技术领域。本发明提供的刺山柑醇提方法克服了现有技术中刺山柑醇提物步骤繁琐、涉及溶剂种类较多、提取率低以及提取物活性成分损失等问题。本发明提供的刺山柑醇提物制备方法提取率高,产物中黄酮得率高,且所得刺山柑醇提物具有显著的体外抗氧化活性以及体外降血脂活性,能够有效改善高血脂机体糖脂代谢,抑制脂质累积,修复氧化应激损伤,逆转脂肪组织和各脏器损伤,具有显著的降血脂作用。
Description
技术领域
本发明涉及植物活性物质提取技术领域,尤其涉及一种刺山柑醇提物、制备方法及其在制备抗氧化产品和/或降血脂产品中的应用。
背景技术
刺山柑(Capparis spinosa L.)属山柑科或白花菜科,山柑属植物,又名马槟榔、野西瓜,是一种药用植物,在我国主要分布在新疆、甘肃、西藏等地区。研究表明,刺山柑果实、叶、根皮均可入药,主要有祛风除湿、止痛、消肿、消炎、降血脂、降血糖等功效,可用于治疗关节炎和肩周炎等疾病,在抗炎、抗高脂血症药物上具有极大的开发前景。
中国专利CN101406497A中公开了一种刺山柑提取物制备方法。该方法包括将药材粉碎后用水煎煮或用不同浓度的乙醇加热回流提取、提取液浓缩后再加入乙醇沉淀杂质、上清液或浓度高于80%的乙醇提取液减压浓缩,回收乙醇后经干燥后得提取物1。提取物1用水分散溶解后石油醚脱酯,然后用乙酸乙酯萃取,乙酸乙酯萃取部位和水溶性部位分别经减压浓缩和干燥后,分别制成提取物2和3。经药效学实验显示提取物1、2和3具有镇痛、抗炎活性,可用于制备治疗风湿性关节炎、类风湿性炎或肩周炎的药物。
中国专利CN101185663A中公开了一种从刺山柑中获得提取物的方法,包括将刺山柑药用部位粉碎,加热回流,冷浸或渗漉提取,减压浓缩,得到粗提物,将粗提物先用石油醚脱脂,其特征在于,脱脂后的残余物用氯仿萃取,得到提取物。该提取物可用于治疗风湿性关节炎、类风湿性关节炎、肩周炎。
杨涛等的研究公开了如下方法:刺山柑果实粉碎,加入乙醇回流提取3次,合并提取液,减压浓缩,冷冻干燥成乙醇总提取物,用水溶解后,先后分别用石油醚和乙酸乙酯萃取3次,所得萃取液合并后,减压浓缩并干燥,得石油醚部位和乙酸乙酯部位。剩余的水溶液部分减压浓缩后冷冻干燥得水溶性部位。并记载刺山柑的抗炎活性部位主要集中在乙酸乙醋部位和水溶性部位(杨涛,于富生,王长虹,等.刺山柑果实醇提物及不同萃取部位的抗炎与镇痛活性研究[J].上海中医药大学学报,2009,23(1):4.)。
然而上述提取方法均存在步骤繁琐、涉及溶剂种类较多、提取率低以及提取物活性成分损失等问题,如何对刺山柑果实的醇提方法进行改进,尚不得而知。
发明内容
本发明的目的在于提供一种刺山柑醇提物、制备方法及其在制备抗氧化产品和/或降血脂产品中的应用,以解决现有技术中刺山柑醇提物步骤繁琐、涉及溶剂种类较多、提取率低以及提取物活性成分损失等问题。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种刺山柑醇提物的制备方法,包含如下步骤:
将刺山柑果实粉末与乙醇水溶液混合,进行超声处理,得到超声提取产物;
将超声提取产物进行第一醇提处理,得到一次提取液以及一次醇提渣滓;
将所得一次醇提渣滓与乙醇水溶液混合进行第二醇提处理,得到二次提取液以及二次醇提渣滓;
将所得二次醇提渣滓与乙醇水溶液混合进行第三醇提处理,得到三次提取液以及三次醇提渣滓;
将所得一次提取液、二次提取液和三次提取液合并,得到混合醇提液;
将混合醇提液依次进行浓缩、冻干,即得刺山柑醇提物;
所述第一醇提处理、第二醇提处理和第三醇提处理的温度为60~70℃。
优选的,所述刺山柑果实粉末与乙醇水溶液的料液比为1g:7~11mL;
所述乙醇水溶液的体积浓度为60~80%。
优选的,所述超声处理的功率为600~1000W,所述超声梳理的频率为20~60kHz,所述超声处理的时间为10~20min。
优选的,所述第一醇提处理、第二醇提处理和第三醇提处理的时间分别为1~3h;
所述乙醇水溶液的体积浓度为60~80%。
优选的,所述浓缩采用旋转蒸发;
所述旋转蒸发的温度为30~50℃,所述旋转蒸发的转速为20~50rpm。
优选的,所述冻干采用真空冻干;
所述真空冻干的温度为-50~-60℃;
所述真空冻干的压强为0.1~0.3mbar。
优选的,所述刺山柑果实粉末与乙醇水溶液混合之前还包括进行预处理,所述预处理包含如下步骤:
将刺山柑果实粉末依次进行脱脂、离心、干燥;
所述脱脂采用石油醚;
所述刺山柑果实粉末与石油醚的料液比为1g:8~12mL。
优选的,所述脱脂的时间为12~36h;
所述离心的转速为3000~4000rpm,所述离心的时间为5~15min。
本发明还提供了一种上述刺山柑醇提物的制备方法得到的刺山柑醇提物。
本发明还提供了一种上述刺山柑醇提物在制备抗氧化产品和/或降血脂产品中的应用。
本发明的技术效果和优点:
本发明提供的刺山柑醇提物制备方法提取率高,产物中黄酮得率高,且所得刺山柑醇提物具有显著的体外抗氧化活性以及体外降血脂活性,能够有效改善高血脂机体糖脂代谢,抑制脂质累积,修复氧化应激损伤,逆转脂肪组织和各脏器损伤,具有显著的降血脂作用。
附图说明
图1为总抗氧化能力结果;
图2为DPPH自由基相对清除活性结果;
图3为DPPH自由基相对清除率结果;
图4为超氧阴离子相对清除率结果;
图5为细胞内TG水平检测结果;
图6为脂质沉积检测结果;
图7为ROS荧光染色流式检测结果;
图8为ROS检测分析结果;
图9为SOD活性检测结果;
图10为MDA含量检测结果;
图11为线粒体膜电位检测结果;
图12为空腹血糖含量测定结果;
图13为脏器指数统计结果;
图14为肝肾损伤指标检测结果;
图15为脂质代谢指标检测结果;
图16为高血脂小鼠动脉粥样硬化指数(AI),抗动脉粥样硬化指数(AAI),冠心指数(R-CHR)检测结果;
图17为小鼠血清和肝脏组织氧化应激反应指标检测结果;
图18为脂肪组织形态HE染色结果;
图19为脏器病理学形态观察结果。
具体实施方式
本发明提供了一种刺山柑醇提物的制备方法,包含如下步骤:
将刺山柑果实粉末与乙醇水溶液混合,进行超声处理,得到超声提取产物;
将超声提取产物进行第一醇提处理,得到一次提取液以及一次醇提渣滓;
将所得一次醇提渣滓与乙醇水溶液混合进行第二醇提处理,得到二次提取液以及二次醇提渣滓;
将所得二次醇提渣滓与乙醇水溶液混合进行第三醇提处理,得到三次提取液以及三次醇提渣滓;
将所得一次提取液、二次提取液和三次提取液合并,得到混合醇提液;
将混合醇提液依次进行浓缩、冻干,即得刺山柑醇提物;
所述第一醇提处理、第二醇提处理和第三醇提处理的温度为60~70℃。
在本发明中,所述刺山柑果实粉末优选由刺山柑果实通过干燥、粉碎过筛制得,所述干燥优选为自然干燥,所述过筛的目数优选为50~100目,进一步优选为60~90目;所述刺山柑果实粉末与乙醇水溶液混合之前还优选进行预处理,所述预处理优选为:将刺山柑果实粉末依次进行脱脂、离心、干燥,所述脱脂优选采用石油醚进行,所述刺山柑果实粉末与石油醚的料液比优选为1g:8~12mL,进一步优选为1g:9~11mL;所述脱脂的时间优选为12~36h,进一步优选为18~30h,所述脱脂优选为静置脱脂;所述脱脂后进行离心,所述离心的转速优选为3000~4000rpm,进一步优选为3300~3700rpm,所述离心的时间优选为5~15min,进一步优选为8~12min;所述离心后进行干燥,所述干燥优选采用风干。
在本发明中,所述刺山柑果实粉末与乙醇水溶液的料液比优选为1g:7~11mL,进一步优选为1g:8~10mL,所述乙醇水溶液的体积浓度优选为60~80%,进一步优选为65~75%;所述刺山柑果实粉末与乙醇水溶液混合后进行超声处理,所述超声处理的功率优选为600~1000W,进一步优选为700~900W,所述超声梳理的频率优选为20~60kHz,进一步优选为30~50kHz,所述超声处理的时间优选为10~20min,进一步优选为13~17min,所述超声处理的温度优选为24~26℃。
在本发明中,所述超声处理结束后将超声提取产物进行第一醇提处理,所述第一醇提处理优选在水浴条件下进行,所述水浴的温度优选为60~70℃,进一步优选为64~66℃,所述第一醇提处理的时间优选为1~3h,进一步优选为1.5~2.5h;所述第一醇提处理结束后优选进行抽滤,所述抽滤为真空抽滤,所述真空抽滤的压力优选为0.05~0.1MPa,抽滤后添加新的乙醇水溶液进行第二醇提处理,所述乙醇水溶液的体积浓度优选为60~80%,进一步优选为65~75%;所述第二醇提处理优选在水浴条件下进行,所述水浴的温度优选为60~70℃,进一步优选为64~66℃,所述第二醇提处理的时间优选为1~3h,进一步优选为1.8~2.2h;所述第二醇提处理结束后同样优选进行抽滤,所述抽滤的条件同上述抽滤,抽滤结束后添加新的乙醇水溶液进行第三醇提处理,所述乙醇水溶液的体积浓度优选为60~80%,进一步优选为65~75%;所述第三醇提处理优选在水浴条件下进行,所述水浴的温度优选为60~70℃,进一步优选为64~66℃,所述第三醇提处理的时间优选为1~3h,进一步优选为1.8~2.2h。
在本发明中,将混合醇提液依次进行浓缩、冻干,即得刺山柑醇提物,所述浓缩优选采用旋转蒸发,所述旋转蒸发的温度优选为30~50℃,进一步优选为35~45℃,所述旋转蒸发的转速优选为20~50rpm,进一步优选为30~40rpm,所述旋转蒸发得到的产品状态优选为浸膏状;所述浓缩后进行冻干,所述冻干优选采用真空冻干,所述真空冻干的温度优选为-50~-60℃,进一步优选为-53~-57℃,所述真空冻干的压强优选为0.1~0.3mbar,进一步优选为0.1~0.3mbar;本发明刺山柑醇提物优选在-10~-30℃条件下进行保存。
本发明还提供了一种上述刺山柑醇提物在制备抗氧化产品和/或降血脂产品中的应用,所述抗氧化产品和/或降血脂产品优选为保健品和药物;所述产品优选的以本发明刺山柑醇提物为唯一活性成分;所述产品优选的还包括辅料,本发明对所述辅料的种类没有特殊限定,采用本领域常规的产品辅料即可;本发明所述的产品剂型优选为散剂、片剂、颗粒剂、胶囊剂、溶液剂、乳剂、混悬剂、注射剂、喷雾剂、气雾剂或粉雾剂等。在本发明中,所述产品包括载体,所述载体优选为口服制剂用的粘合剂、润滑剂、崩解剂、助溶剂、稀释剂、稳定剂、悬浮剂、色素、矫味剂等;可注射制剂用的防腐剂、加溶剂、稳定剂等;局部制剂用的基质、稀释剂、润滑剂、防腐剂等。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
挑选质量、形态优质的自然干燥的刺山柑果实,粉碎并过80目筛。精密称取500.00g刺山柑果实粉末,以1:10(g/mL)料液比加入石油醚溶液进行脱脂处理,静置24h,3500rpm离心10min,收集滤渣,风干,得到预处理的刺山柑果实粉末;将上述获得的刺山柑脱脂粉末精密称取100.00g,以1:9(g/mL)料液比加入70%的乙醇溶液,室温25℃下超声功率800W,超声频率40KHZ超声14min后,水浴锅66℃醇提2h,0.085MPa真空抽滤,加入新的70%乙醇溶液重复共提取3次;收集并合并滤液;40℃,30rpm旋蒸浓缩至浸膏状,-55℃,0.22mbar真空冷冻干燥,即得刺山柑醇提物,称量并-20℃保存备用。
实施例2
挑选质量、形态优质的自然干燥的刺山柑果实,粉碎并过80目筛。精密称取500.00g刺山柑果实粉末,以1:8(g/mL)料液比加入石油醚溶液进行脱脂处理,静置12h,3000rpm离心5min,收集滤渣,风干,得到预处理的刺山柑果实粉末;将上述获得的刺山柑脱脂粉末精密称取100.00g,以1:7(g/mL)料液比加入65%的乙醇溶液,室温25℃下超声功率800W,超声频率40kHz超声14min后,水浴锅60℃醇提1.5h,0.05MPa真空抽滤,加入新的65%乙醇溶液再次水浴锅60℃醇提1.5h,0.05MPa真空抽滤,加入新的65%乙醇溶液再次水浴锅60℃醇提1.5h,共提取3次;收集并合并滤液;40℃,30rpm旋蒸浓缩至浸膏状,-55℃,0.22mbar真空冷冻干燥,即得到刺山柑醇提物,并-20℃保存备用。
实施例3
挑选质量、形态优质的自然干燥的刺山柑果实,粉碎并过80目筛。精密称取500.00g刺山柑果实粉末,以1:12(g/mL)料液比加入石油醚溶液进行脱脂处理,静置36h,4000rpm离心15min,收集滤渣,风干,得到预处理的刺山柑果实粉末;将上述获得的刺山柑脱脂粉末精密称取100.00g,以1:11(g/mL)料液比加入80%的乙醇溶液,室温25℃下超声功率1000W,超声频率50kHz超声15min后,水浴锅68℃醇提3h,0.085MPa真空抽滤,加入新的80%乙醇溶液再次水浴锅68℃醇提2h,0.085MPa真空抽滤,加入新的80%乙醇溶液再次水浴锅68℃醇提3h,共提取3次;收集并合并滤液;50℃,35rpm旋蒸浓缩至浸膏状,-60℃,0.25mbar真空冷冻干燥,即得到刺山柑醇提物,并-20℃保存备用。
对比例1
挑选质量、形态优质的自然干燥的刺山柑果实,粉碎并过80目筛。精密称取500.00g刺山柑果实粉末,以1:10(g/mL)料液比加入石油醚溶液进行脱脂处理,静置24h,3500rpm离心10min,收集滤渣,风干,得到预处理的刺山柑果实粉末;将上述获得的刺山柑脱脂粉末精密称取100.00g,以1:10(g/mL)料液比加入70%的乙醇溶液,室温25℃下超声功率800W,超声频率40kHz超声20min后,60℃热水浸提2h,共提取3次;收集并合并滤液;40℃,30rpm旋蒸浓缩至浸膏状,-55℃,0.22mbar真空冷冻干燥,即得刺山柑醇提物,-20℃保存备用。
实验例1得率及黄酮得率计算
分别计算实施例1与对比例1的刺山柑醇提物提取率以及黄酮得率。
提取率按照如下公式计算:
提取率=(刺山柑醇提物冻干粉质量/刺山柑果实粉末质量)×100%
黄酮得率按照如下方法计算:
准确称取6.00mg芦丁标准品,无水乙醇定容得到0.60mg/mL母液,再用无水乙醇将其稀释配置成0、0.06、0.12、0.18、0.24、0.36、0.48、0.60mg/mL的标准溶液,依次取50μL加入到96孔板中,加入30μL 0.1mol/LAlCl3溶液和50μL 1mol/LKAc溶液,以加入80μL蒸馏水替代AlCl3和KAc溶液作为对照,混匀,415nm处测定吸光度值(OD)。
将上述配置好的刺山柑醇提物进行稀释,按一定体积加到96孔板中,按照与测定标准曲线同样的方法(AlCl3-KAc法),每个浓度设置三个复孔,415nm处测定OD值,将其代入标准曲线得到待测液质量浓度。带入标准曲线回归方程后计算黄酮浓度,再换算成黄酮提取量以及提取率:
结果显示:
实施例1:刺山柑醇提物的提取率为41.52%,黄酮得率为2.37%;
对比例1:刺山柑醇提物的提取率为24.6%,黄酮得率为0.67%。
实验例2体外抗氧化活性研究
取实施例1获得的刺山柑醇提物,采用乙醇分别调节浓度至0.25mg/mL、0.5mg/mL、1mg/mL、2mg/mL、4mg/mL,以抗坏血酸(Vc)作为对照组,利用“铁离子还原/抗氧化能力法”通过酶标仪检测反应体系吸光度的变化,探究了刺山柑醇提物的总抗氧化能力,总抗氧化能力结果如图1所示,DPPH自由基相对清除活性结果如图2所示,DPPH自由基相对清除率结果如图3所示,超氧阴离子相对清除率结果如图4所示(数据进行单因素方差分析,Control组与刺山柑醇提物处理组比较:*p<0.05,**p<0.01,***p<0.001)。
由结果可知,当刺山柑醇提物浓度从0.25mg/mL升高到4mg/mL时,总抗氧化能力逐渐增强,表现出良好的剂量依赖性;随着样品浓度的增大,刺山柑醇提物呈剂量依赖性降低DPPH自由基活性,最高浓度4mg/mL时,相对清除活性与Vc相当。同时刺山柑醇提物也具有呈现剂量依赖性清除超氧阴离子自由基的能力。
实验例3体外降血脂活性研究
肝脂质变性细胞模型的主要表现是TG含量显著增加。通过油酸(OA)诱导HepG2细胞(人源肝癌HepG2细胞株,购买于中国科学院干细胞库)建立肝脂质变性细胞模型。首先以8×104/mL密度将HepG2细胞接种于12孔板中,待细胞继续长至80%-90%后,更换含有1%BSA的培养液,并在培养液中加入0.4mM OA孵育24h,用预冷的PBS洗涤2次,加入100μL RIPA溶液裂解细胞,根据试剂盒说明书测定细胞内TG含量,同时油红O染色观察脂滴形成情况。与阴性对照Control组相比,OA组脂滴显著增多,且TG含量显著升高,即模型诱导成功。
采用不同剂量100、200、400μg/mL刺山柑醇提物处理OA-HepG2细胞24h,未作处理的模型组作为对照(Control),检测细胞内TG水平,结果如图5所示。由结果可知,与OA组相比,刺山柑醇提物呈剂量依赖性降低细胞内TG水平,表明刺山柑醇提物可以有效抑制OA-HepG2细胞脂质水平。
脂滴是甘油三酯在细胞内的一种储存形式,当细胞发生脂质变性,脂滴的数量也相对增加。利用4%多聚甲醛室温固定细胞,油红O染液染色,倒置荧光显微镜下拍照观察,结果如图6所示。从图6可以看出,OA组相比于Control组,HepG2细胞内脂滴明显增多,说明模型组诱导成功;给予不同浓度刺山柑醇提物处理后,与模型组相比,刺山柑醇提物处理组细胞红色脂滴呈剂量依赖性减少,说明刺山柑醇提物可以降低OA-HepG2细胞内脂质沉积。
游离脂肪酸过多摄入,会产生过量活性氧(ROS),进而使得氧化与抗氧化系统间的平衡被破坏,造成氧化损伤。因此检测了刺山柑醇提物对OA-HepG2细胞ROS产生的影响。荧光探针DCFH-PA染液按1:1000的比例稀释后进行细胞染色,流式上机检测并分析,结果如图7~8所示。可知,与OA组相比,200μg/mL、400μg/mL刺山柑醇提物处理组ROS水平分别为34.8%±0.59,43.8%±8.48,表现出一定的下降趋势;表明刺山柑醇提物可降低OA-HepG2细胞ROS水平。
称取1.0g肝脏组织,按照每10mg肝组织加入100μL SOD溶液的比例4℃匀浆,12,000g离心5min,取上清,WST-8法测定SOD活性;按试剂盒比色法测定MDA含量,结果如图9~10所示;荧光探针JC-1染液按1:200的比例稀释后进行细胞染色,流式上机检测并分析线粒体膜电位,如图11所示(数据进行单因素方差分析,#p<0.05,##p<0.01,###p<0.001,OA组与Control组比较;*p<0.05,**p<0.01,***p<0.001,刺山柑醇提物处理组与OA组比较),与OA组相比,200μg/mL、400μg/mL刺山柑醇提物可一定程度提高OA-HepG2细胞SOD活性,降低MDA含量,减轻脂质氧化。此外,与OA组相比,刺山柑醇提物线粒体膜电位呈现下降趋势,推测刺山柑醇提物可能可以修复线粒体功能。以上结果表明,刺山柑醇提物通过降低脂质积累和逆转氧化应激损伤,从而发挥抑制OA-HepG2脂肪变性细胞脂质生成和代谢的作用。
实验例4对高血脂小鼠脂质代谢和氧化应激损伤的干预作用
雄性C57BL/6J小鼠(6~8周龄,购于北京维通利华实验动物技术有限公司),适应性饲养7天后,按体重随机分出6只,饲喂高脂饲料,在造模14周后(禁食不禁水12h后),测定其空腹血清中TC、TG、LDL-C、HDL-C的含量,建立高血脂小鼠模型,待造模期间血清中TC、TG或LDL-C含量均明显高于正常组,HDL-C低于正常组,高脂动物模型建立成功。剔除建模失败的小鼠,将模型组小鼠根据体重随机分为7组,分别为NFD组(普通饲料)、HFD组(60%kcal高脂饲料)、T-80组(HFD+0.1%吐温80)、刺山柑醇提物药物治疗组(HFD+400mg/kg/d、HFD+800mg/kg/d、HFD+1200mg/kg/d),阳性对照辛伐他汀治疗组10mg/kg/d,每组小鼠6只。灌胃给药治疗5周,每周测定小鼠体重,结束后测定空腹血糖含量,结果如表1及图12所示。
表1小鼠体重变化结果(g)
由结果表明,刺山柑醇提物能够减轻模型小鼠体重和空腹血糖含量。
观察各脏器组织形态,记录脏器指数,如图13所示。由结果可知,与NFD组相比,HFD组脏器指数有下降趋势;1200mg/kg刺山柑醇提物给药治疗后,HFD小鼠肝脏、心脏和脾脏指数均有所增加;同时发现HFD组小鼠肺部组织受到一定程度的损伤,而刺山柑醇提物可有效修复脏器损伤。
谷丙转氨酶(ALT)和谷草转氨酶(AST)是反映肝功能的指标,肌酐(Cr)和尿素氮(BUN)是反映肾功能的指标。利用南京建成公司试剂盒检测小鼠相关指标变化,通过酶标仪检测吸光度,结果如图14所示。与NFD组相比,HFD组小鼠血清AST水平显著升高,表明高脂饮食导致小鼠肝脏受损;刺山柑醇提物治疗后,呈剂量依赖性降低HFD小鼠AST含量,且800mg/kg和1200mg/kg刺山柑醇提物也可降低Cr水平,表明刺山柑醇提物对HFD小鼠肝肾损伤具有较强的保护作用。
进一步探究刺山柑醇提物对高血脂小鼠脂质代谢的作用,对小鼠肝脏组织进行匀浆,对血液进行离心取上清,利用试剂盒及酶标仪检测相关指标,如图15~16所示。发现在肝脏组织中刺山柑醇提物显著降低模型小鼠TG、LDL-C水平,提高HDL-C水平。动脉粥样硬化指数(AI),抗动脉粥样硬化指数(AAI)是可以衡量动脉硬化程度的关键指标,AAI降低,机体抗动脉粥样硬化作用水平低。由图16可以看出,刺山柑醇提物可以随着作用剂量的增大,降低HFD小鼠AI值,提升AAI值。LDL-C的升高,会促进脂肪滴在血管内皮的沉积,进而促进动脉粥样硬化的发展,冠心指数(R-CHR)是反映冠心病的重要参数,我们发现刺山柑醇提物组小鼠R-CHR有降低趋势。
高脂饮食引起的肥胖,导致脂解作用增加,释放大量FFAs进入血液并在周围组织异位沉积,产生脂毒性,诱导线粒体功能障碍以及氧化应激损伤。因此收集血清,准确称取肝脏组织并加入9倍体积的生理盐水,冰水浴条件下机械匀浆、离心收集上清,试剂盒法分别检测小鼠血清和肝脏组织氧化应激反应指标变化,结果如图17所示。结果发现,与HFD组相比,高剂量刺山柑醇提物可一定程度提高HFD小鼠总抗氧化能力(T-AOC)及超氧化物歧化酶(SOD)水平;同时,脂质过氧化反应严重,脂质会进一步分解为其他复杂化合物,终产物为丙二醛(MDA)。1200mg/kg刺山柑醇提物能够显著降低HFD小鼠血清MDA含量。因此,刺山柑醇提物对HFD小鼠氧化应激损伤具有一定的保护作用。
甘油三酯以脂滴的形式存储于脂肪组织中。对HFD小鼠各脂肪组织形态进行HE染色观察,包括皮下脂肪组织(scWAT)、内脏脂肪组织(vWAT)、附睾白色脂肪组织(eWAT)、肾周白色脂肪组织(pWAT)及棕色脂肪组织(BAT),结果如图18所示。结果表明,与NFD组相比,HFD组scWAT和vWAT脂肪细胞体积增大,刺山柑醇提物组脂肪细胞体积明显变小,在相同视野下细胞数量增多。在BAT中,HFD出现单个、较大的空泡脂肪室,刺山柑醇提物组空泡数呈剂量依赖性减少,且细胞核明显,细胞间毛细血管丰富。表明刺山柑醇提物显著改善脂肪组织肥大和病变。
给药五周后处死小鼠,收集各脏器,进一步对小鼠肝脏、肾脏、心脏进行病理学形态观察,结果如图19所示,NFD组小鼠肝细胞排列紧密,未出现空泡,胞质丰富,无脂滴;而HFD组肝细胞排列紊乱,出现大量空泡,细胞膜不完整,细胞核分布不均匀,并伴随有大量脂滴产生,肝脏组织发生脂肪变性;刺山柑醇提物高剂量组和阳性对照辛伐他汀组肝脏细胞基本排列整齐,空泡减少,脂滴基本消失,明显改善了肝脏受损情况。观察小鼠肾脏形态结构发现,NFD组肾小球呈球形,肾小囊包绕肾小球,肾小管上皮细胞排列整齐;而HFD组肾小球囊腔部分消失,肾小球变形,上皮细胞排列紊乱;刺山柑醇提物高剂量组肾小球囊腔重新明显,肾小球基本呈球形,改善了小鼠肾脏受损情况。且NFD组心肌细胞排列紧密,细胞结构完整,细胞质均一,心肌纤维横纹完整;而HFD组心肌细胞排列紊乱,心肌纤维断裂,刺山柑醇提物高剂量治疗后细胞结构基本完整,心肌纤维横纹有所改善,改善了小鼠心脏受损情况。表明刺山柑醇提物对小鼠肝脏、肾脏和心脏损伤有显著的修复作用。
综上可知,刺山柑醇提物能够有效改善高血脂机体糖脂代谢,抑制脂质累积,修复氧化应激损伤,逆转脂肪组织和各脏器损伤,具有显著的降血脂作用。
由以上实施例可知,本发明提供的刺山柑醇提物制备方法提取率高,产物中黄酮得率高,且所得刺山柑醇提物具有显著的体外抗氧化活性以及体外降血脂活性,能够有效改善高血脂机体糖脂代谢,抑制脂质累积,修复氧化应激损伤,逆转脂肪组织和各脏器损伤,具有显著的降血脂作用。
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来说,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 (10)
1.一种刺山柑醇提物的制备方法,其特征在于,包含如下步骤:
将刺山柑果实粉末与乙醇水溶液混合,进行超声处理,得到超声提取产物;
将超声提取产物进行第一醇提处理,得到一次提取液以及一次醇提渣滓;
将所得一次醇提渣滓与乙醇水溶液混合进行第二醇提处理,得到二次提取液以及二次醇提渣滓;
将所得二次醇提渣滓与乙醇水溶液混合进行第三醇提处理,得到三次提取液以及三次醇提渣滓;
将所得一次提取液、二次提取液和三次提取液合并,得到混合醇提液;
将混合醇提液依次进行浓缩、冻干,即得刺山柑醇提物;
所述第一醇提处理、第二醇提处理和第三醇提处理的温度为60~70℃。
2.根据权利要求1所述的刺山柑醇提物的制备方法,其特征在于,所述刺山柑果实粉末与乙醇水溶液的料液比为1g:7~11mL;
所述乙醇水溶液的体积浓度为60~80%。
3.根据权利要求2所述的刺山柑醇提物的制备方法,其特征在于,所述超声处理的功率为600~1000W,所述超声梳理的频率为20~60kHz,所述超声处理的时间为10~20min。
4.根据权利要求3所述的刺山柑醇提物的制备方法,其特征在于,所述第一醇提处理、第二醇提处理和第三醇提处理的时间分别为1~3h;
所述乙醇水溶液的体积浓度为60~80%。
5.根据权利要求4所述的刺山柑醇提物的制备方法,其特征在于,所述浓缩采用旋转蒸发;
所述旋转蒸发的温度为30~50℃,所述旋转蒸发的转速为20~50rpm。
6.根据权利要求5所述的刺山柑醇提物的制备方法,其特征在于,所述冻干采用真空冻干;
所述真空冻干的温度为-50~-60℃;
所述真空冻干的压强为0.1~0.3mbar。
7.根据权利要求6所述的刺山柑醇提物的制备方法,其特征在于,所述刺山柑果实粉末与乙醇水溶液混合之前还进行预处理,所述预处理包含如下步骤:
将刺山柑果实粉末依次进行脱脂、离心、干燥;
所述脱脂采用石油醚;
所述刺山柑果实粉末与石油醚的料液比为1g:8~12mL。
8.根据权利要求7所述的刺山柑醇提物的制备方法,其特征在于,所述脱脂的时间为12~36h;
所述离心的转速为3000~4000rpm,所述离心的时间为5~15min。
9.权利要求1~8任一项所述的刺山柑醇提物的制备方法得到的刺山柑醇提物。
10.权利要求9所述的刺山柑醇提物在制备抗氧化产品和/或降血脂产品中的应用。
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CN114099558A (zh) * | 2021-12-06 | 2022-03-01 | 新疆农业大学 | 一种刺山柑果提取物的制备方法及其应用 |
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