CN116726070A - 一种具有抗肿瘤作用的夏枯草籽油自乳化软胶囊及其制备方法与应用 - Google Patents
一种具有抗肿瘤作用的夏枯草籽油自乳化软胶囊及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种具有抗肿瘤作用的夏枯草籽油自乳化软胶囊及其制备方法与应用,属于医药技术领域。本发明以夏枯草籽油、表面活性剂、助表面活性剂和抗氧剂一起制成一种具有抗肿瘤作用的夏枯草籽油自乳化软胶囊;本发明还首次公开了一种夏枯草籽油中富含以熊果酸为代表的三萜类及以迷迭香酸为代表的苯丙素类成分,并通过其自乳化软胶囊的制备增强其夏枯草籽油的抗肿瘤作用。本发明所提供的方法工艺流程简单,操作方便,适合于工业生产;产品的质量稳定,质量控制方便,可用于药物和食品。
Description
技术领域
本发明属于医药技术领域,特别涉及一种具有抗肿瘤作用的夏枯草籽油自乳化软胶囊及其制备方法与应用。
背景技术
夏枯草为唇形科(Lamiaceae)植物夏枯草Prunella vulgaris L.的干燥果穗,始载于《神农本草经》,因“此草夏至后即枯”而得名,是一种药食同源的多年生草本植物,至今已有几千年的药用历史,现收载于2015年版之《中国药典》。夏枯草味苦、辛,性寒,归肝、胆经,可清肝散火、明目、散结消肿,对目赤肿痛、畏光流泪、目珠夜痛、头晕目眩、瘰疬、瘿瘤、乳腺癌、高血压、淋巴结核、浸润性肺结核、单纯性甲状腺肿、腮腺炎、急性黄疸型传染性肝炎等多种疾病均有良好的临床治疗效果。现代研究表明,夏枯草中含有多种化学成分:包括三萜、甾醇、黄酮、有机酸、香豆素等类型化合物,具有抗肿瘤、抗炎、抗菌、抗病毒、免疫调节、降血压、降血糖、降血脂等药理作用。夏枯草籽为唇形科(Lamiaceae)植物夏枯草Prunella vulgaris L.的干燥成熟种子,为夏枯草的生殖种子,应具有夏枯草的遗传活性物质与作用,但其在食用、保健或药物中的应用价值还未受到重视,还未见到其相关报导,特别是夏枯草籽油的研究与应用更是空白。
熊果酸是一种五环三萜类化合物,以游离体和配糖体的形式存在于许多植物中,熊果酸(Ursolic acid)又名乌索酸、乌苏酸,存在于山茱萸、夏枯草、山楂等植物中,为该中药的有效成份之一。熊果酸具有镇静、抗炎、抗菌、抗糖尿病、抗溃疡、降低血糖等多种生物学效应。近年来研究人员发现熊果酸具有抗致癌、抗促癌、诱导F9畸胎瘤细胞分化和抗血管生成作用,对肾癌、黑色素瘤、结肠癌、卵巢癌、中枢神经系统癌、非小细胞肺癌、表皮样瘤、肝癌、卵巢癌、急性早幼粒细胞白血病及畸胎瘤等癌细胞有一定的细胞毒效应。迷迭香酸(rosmarinic acid,RA)是一种多酚酸,化学名为:R(+)2-[3-(3,4-二羟基苯基)-1-氧代-2-丙烯基]氧基-3,4-二羟基苯丙酸,广泛分布于唇形科、紫草科、葫芦科等植物中,是夏枯草的主要有效成分之一;现代药理研究表明:迷迭香酸具有抗氧化、抗菌抗炎、抗肿瘤、抗抑郁和抗焦虑、改善肾脏疾病、保肝等多方面药理学活性。其作用及作用机理主要为:⑴抗氧化作用:迷迭香酸具有较强的抗氧化和清除体内自由基的作用,该作用与其邻二酚羟基的结构有关;⑵抗菌抗炎作用:迷迭香酸对金黄色葡萄球菌、藤黄细球菌、大肠杆菌、枯草杆菌等细菌均有明显的抑制作用,对牛皮癣、皮肤炎、呼吸道炎症、急性肺损伤等多种炎症均有一定的抑制作用;⑶抗肿瘤作用:迷迭香酸对结直肠癌、乳腺癌、宫颈癌、白血病、肺癌、肝癌等多种肿瘤有明显的抑制作用,其抗肿瘤机制可能与提高机体免疫功能和有效地抑制肿瘤细胞的增殖和侵袭并诱导凋亡有关;⑷抗抑郁和抗焦虑作用:迷迭香酸的抗抑郁作用,体外实验发现迷迭香酸可促进新生大鼠星形胶质细胞的增殖,体内实验发现迷迭香酸可改善慢性不可预见应激抑郁模型大鼠的抑郁样行为,低剂量的迷迭香酸能够发挥抗焦虑作用;⑸保肝作用:迷迭香酸能够抑制肝星状细胞HSCs的增殖分化,通过抑制TGF-β1和CTGF的表达对抗四氯化碳诱导的抗纤维化;⑹改善肾脏疾病作用:迷迭香酸有改善肾脏相关疾病的作用,主要表现为减少尿酸生成,抑制肾小球肾炎以及延缓慢性肾功能不全。熊果酸、迷迭香酸都是夏枯草中药中的重要活性成分,但对夏枯草的生殖种子--夏枯草籽却未见其成分的相关报道与开发研究。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足,提供一种具有抗肿瘤作用的夏枯草籽油自乳化软胶囊。该自乳化软胶囊包括富含以熊果酸为代表的三萜类及以迷迭香酸为代表的苯丙素类成分的夏枯草籽油,且具有生物利用度高,使用安全、方便的能显著增夏枯草籽油抗肿瘤作用的特点。
本发明的另一目的在于提供通过上述具有抗肿瘤作用的夏枯草籽油自乳化软胶囊的制备方法。
本发明的再一目的在于提供上述具有抗肿瘤作用的夏枯草籽油自乳化软胶囊的应用。
本发明的目的通过下述技术方案实现:一种具有抗肿瘤作用的夏枯草籽油自乳化软胶囊,包括芯材和包裹芯材的囊材;其中,芯材为夏枯草籽油自乳化系统,由夏枯草籽油、表面活性剂、或助表面活性剂、或和抗氧剂或和防腐剂制备得到;优选由以下按质量百分比计的成分的成分组成:夏枯草籽油40~75%、表面活性剂20~50%、助表面活性剂0~25%、抗氧剂0.1~1.0%、防腐剂0~0.3%。
所述的夏枯草籽油优选通过如下步骤制备得到:
1)夏枯草籽的收集:采摘完全成熟的干燥的夏枯草果穗,将干燥的夏枯草果穗中的夏枯草种子抖落,收集并获得夏枯草籽粗品;
2)夏枯草籽的净选:将夏枯草籽粗品中的杂质去除,得到纯净的夏枯草籽;
3)夏枯草籽的破壁:将步骤2)得到的纯净的夏枯草籽置于粉碎破壁机中进行破壁,得到破壁后的夏枯草籽粉;其中粉碎破壁机筛网的孔径小于或等于40目,粉碎破壁机在15℃以下的环境工作;
4)与夹带剂混匀:
①加夹带剂混合:将破壁后的夏枯草籽粉和极性夹带剂混合均匀;
②装填:将步骤①得到的混匀的含极性夹带剂的粉末均匀地填充于超临界CO2萃取釜中;
5)超临界萃取:
①静态浸泡萃取:将步骤4)得到的夏枯草籽粉颗粒置于超临界CO2萃取釜升温至30-60℃的温度,通以CO2超临界流体升压至10-25MPa的压力,停泵,静态浸泡提取20-60min;
②动态萃取:静态浸泡提取后,当分离器的温度及压力达到设定值时开始动态萃取并计时,萃取时间为100-180min;
③分离:采用二级分离,分离的工艺条件是:一级解析器压力为9~13MPa,分离温度37~43℃;二级解析器压力为4~6Mpa,分离温度27~33℃;获得黄绿色的夏枯草籽油;
6)过滤除杂:取步骤5)得到的夏枯草籽油,置于孔径等于或小350目的过滤装置中减压抽滤,弃去不能滤过的杂质,得清澈的夏枯草籽油。
步骤2)中所述的净选的具体步骤优选如下:
A、将收集的夏枯草籽粗品置于15~20目的筛网中,让粒径小于筛网网孔的夏枯草籽通过,除去粒径大于筛网网孔的较粗大的杂质,得到初筛的夏枯草籽;
B、将初筛的夏枯草籽置于30~40目的筛网中,让粒径小于筛网网孔的杂质通过,除去粒径小于筛网网孔的较细小的杂质,收集不能通过筛网网孔的夏枯草籽,获得纯净的夏枯草籽。
步骤A中所述的筛网优选为20目。
步骤B中所述的筛网优选为30目。
步骤3)中所述的粉碎破壁机筛网优选孔径为40~50目的粉碎破壁机筛网。
步骤3)中所述的破壁后的夏枯草籽粉优选为能过小于或等于40目的筛的夏枯草籽粉。
步骤3)中所述的夏枯草籽需经破壁处理至破壁率≥96%,得到破壁夏枯草籽粉。
步骤3)中所述的环境的温度优选为0~15℃;更优选为5~12℃。
所述的环境是整个粉碎破壁机完全放置的环境;或是在粉碎破壁机的发热部位外部加装制冷循环水装置得到的环境。
步骤4)中所述的极性夹带剂优选为甲醇、乙醇、乙醇水溶液和丙酮中的至少一种。
所述的乙醇水溶液的浓度优选为体积百分比95%。
步骤4)中所述的极性夹带剂的用量优选按夏枯草籽粉:极性夹带剂=1kg:300-1500mL计算;更优选按夏枯草籽粉:极性夹带剂=1kg:700-800mL计算。
步骤5)中所述的静态浸泡萃取的条件优选为:温度为43-50℃,压力为18-20MPa,静态浸泡提取的时间为30-50min。
步骤5)中所述的动态萃取的萃取时间优选为120-150min。
步骤5)中所述的分离的工艺条件优选为:一级解析器压力为9~11MPa,分离温度37~42℃;二级解析器压力为4~5Mpa,分离温度27~32℃。
所述的夏枯草籽油富含以熊果酸为代表的三萜类及以迷迭香酸为代表的苯丙素类成分;并通过其自乳化系统的制备增强其夏枯草籽油的抗肿瘤作用。
所述的夏枯草籽油在所述的夏枯草籽油自乳化系统中的含量优选为质量百分比45~75%;更优选为质量百分比50~65%。
所述的表面活性剂优选为大豆磷脂、蛋黄卵磷脂、液态卵磷脂和脑磷脂中的至少一种。
所述的表面活性剂在所述的夏枯草籽油自乳化系统中的含量优选为质量百分比20~50%;更优选为质量百分比20~26%。
所述的助表面活性剂优选为甘油、乙醇和聚乙二醇400中至少一种。
所述的助表面活性剂在所述的夏枯草籽油自乳化系统中的含量优选为质量百分比13.5~23%。
所述的抗氧剂优选为维生素E和维生素C中的至少一种。
所述的抗氧剂在所述的夏枯草籽油自乳化系统中的含量优选为质量百分比0.5~1%。
所述的防腐剂优选为尼泊金酯类防腐剂和山梨酸钾中的至少一种。
所述的防腐剂在所述的夏枯草籽油自乳化系统中的含量优选为质量百分比0~0.05%。
所述的囊材的组成成分包括食用胶、增塑剂和水;或再选择性添加防腐剂、香精、调味剂和色素中的至少一种;优选由如下按质量百分比计的成分组成:食用胶30~45%、增塑剂15~25%、防腐剂0~适量、调味剂0~适量、香精0~适量、遮光剂0~适量、着色剂0~适量,余量为水;更优选由如下按质量百分比计的成分组成:食用胶35~40%、增塑剂18~19%、防腐剂0.1%,余量为水。
所述的食用胶优选为明胶。
所述的增塑剂优选为甘油。
所述的适量是依据实际需求制定,且符合国家或行业的相关规定。
所述的防腐剂优选为尼泊金酯类防腐剂和山梨酸钾中的至少一种。
所述的调味剂优选为甜菊糖苷、阿斯巴甜、葡萄糖和果糖中的至少一种。
所述的香精优选为薄荷香精、柠檬香精、玫瑰香精、牛奶香精和薄荷香精中的至少一种。
所述的水优选为蒸馏水和纯化水中的至少一种。
所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊的制备方法,包括如下步骤:
(1)夏枯草籽油自乳化系统的制备:取夏枯草籽油,在20-80℃条件温度下,加入表面活性剂、助表面活性剂和抗氧剂,必要时加入防腐剂溶解,混合均匀,滤过,得到均一的夏枯草籽油自乳化系统;
(2)软胶囊的制备:将软胶囊囊材液和步骤(1)制备的夏枯草籽油自乳化系统通过软胶囊机,采用压制法制备软胶囊,经干燥,洗涤,再干燥,分检,包装,得到软胶囊成品。
步骤(1)更优选为:先将表面活性剂、助表面活性剂和防腐剂混合均匀,得到混合液A;将混合液A与夏枯草籽油、抗氧化剂混匀,滤过,得到均一的夏枯草籽油自乳化系统。
所述的混合均匀优选于6000~10000rpm的转速进行剪切2~20min;更优选为于7000~8000rpm的转速进行剪切5~15min。
步骤(1)优选为于60~80℃的条件下进行。
步骤(2)中所述的软胶囊囊材液优选通过如下步骤制备得到:将食用胶和水混合,必要时加入防腐剂、调味剂、香精、遮光剂、着色剂,密闭同时加热,搅拌至融化溶胀完全,加入增塑剂,密闭配胶罐,关闭搅拌,开启真空泵,抽真空,待气泡抽净,即得软胶囊囊材液,于50~70℃保温。
所述的水是温度为60~80℃的水。
所述的加热的温度优选为70~95℃。
上述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊在食品、保健食品或药物领域中的应用。
夏枯草是我国药典收载的一种药食同源的常见药味,其野生与种植资源十分丰富,夏枯草种子(夏枯草籽)资源同样也十分丰富,但由于夏枯草籽还未被开发利用,致使每年成千上万吨的夏枯草籽未被采集,在采收夏枯草时被散落在荒地野外,造成夏枯草籽资源浪费;本发明的利用有如下的有益效果:
1、本发明首次发现了一种富含以熊果酸为代表的三萜类及以迷迭香酸为代表的苯丙素类成分的夏枯草籽油,并利用其脂肪油进行开发研究,为夏枯草籽油的保健与药用提供了依据;
2、本发明首次提供了一种夏枯草籽油自乳化系统及其制备方法,为夏枯草籽及夏枯草籽油开发利用提供了方法和依据,将减少夏枯草籽的资源浪费;
3、本发明首次夏枯草籽油优化的自乳化系统的制备条件制备成一种稳定的夏枯草籽油自乳化软胶囊制剂,不仅可提高夏枯草籽油的生物利用度,而且起效快,克服了夏枯草籽油普通制剂生物利用度相对较低的缺点,显著增强了夏枯草籽油抗肿瘤的作用。
附图说明
图1是夏枯草籽油中三萜成分熊果酸的TLC鉴别结果在可见光下的照片图;其中,1、2为实施例1夏枯草籽油供试品,3、5为实施例2夏枯草籽油供试品,4为熊果酸对照品,6、7为实施例3夏枯草籽油供试品。
图2是夏枯草籽油中三萜成分熊果酸的TLC鉴别结果在紫外光下的照片图;其中,1、2为实施例1夏枯草籽油供试品,3、5为实施例2夏枯草籽油供试品,4为熊果酸对照品,6、7为实施例3夏枯草籽油供试品。
图3是夏枯草籽油中迷迭香酸成分的TLC鉴别结果在254nm下的照片图;其中,1、2为实施例1夏枯草籽油供试品,3、5为实施例2夏枯草籽油供试品,4为迷迭香酸对照品,6、7为实施例3夏枯草籽油供试品。
图4是夏枯草籽油中迷迭香酸成分的TLC鉴别结果在365nm下的照片图;其中,1、2为实施例1夏枯草籽油供试品,3、5为实施例2夏枯草籽油供试品,4为迷迭香酸对照品,6、7为实施例3夏枯草籽油供试品。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
实施例1夏枯草籽油使用极性夹带剂的超临界CO2萃取的制备方法一
(1)夏枯草籽的收集:采摘完全成熟的干燥的夏枯草果穗,置于干净的收集装置中,将干燥的夏枯草果穗中的夏枯草种子抖落于收集装置中,收集获得的夏枯草籽,获得夏枯草籽粗品5.94kg。
(2)夏枯草籽的净选
A、除去粗大杂质:选用孔径20目的筛网,将上述收集到的夏枯草籽粗品5.94kg置于筛网中,振摇过筛,让粒径小于筛网网孔的草籽通过,除去粒径大于筛网网孔的较粗大的杂质,获得除去粗大杂质的夏枯草籽粗品5.63kg;
B、除去细小杂质:选用孔径30目的筛网,将上述除去粗大杂质的夏枯草籽粗品5.63kg置于筛网中,振摇过筛,让粒径小于筛网网孔的杂质通过,除去粒径小于筛网网孔的较细小的杂质,收集不能通过筛网网孔的夏枯草籽,获得纯净的夏枯草籽5.09kg。
(3)夏枯草籽的破壁:
A、将上述纯净的夏枯草籽置于粉碎破壁机中,选用40目孔径的粉碎破壁机筛网进行粉碎破壁;
B、将粉碎破壁机置于10±2℃的环境下;或是在粉碎破壁机的发热部位外部加装制冷循环水装置,并保持粉碎温度在15℃以下;
C、开启机粉碎,粉碎后的夏枯草籽粉置于孔径为40目的筛网中,振摇过筛,让粒径小于筛网网孔的草籽粉通过,不能通过的草籽粉加入粉碎机中继续粉碎破壁,直至全部通过为止,获得破壁的夏枯草籽粉4.88kg;破壁率为99%以上。
(4)与夹带剂混匀:
①加夹带剂混合:往上述破壁夏枯草籽粉原料中加入极性夹带剂甲醇,夏枯草籽粉与甲醇夹带剂的比例为1千克夏枯草籽粉加入800毫升甲醇夹带剂,混合均匀;
②装填:将上述夏枯草籽粉与甲醇夹带剂混合均匀的粉体均匀地填充于超临界CO2萃取釜中。
(5)超临界萃取:
①静态浸泡萃取:将上述填充好夏枯草籽混合粉体的超临界CO2萃取釜,升温至45℃的温度,通以CO2超临界流体升压至20MPa的压力,停泵,静态浸泡提取40min;
②动态萃取:静态浸泡提取40min后,当分离器的温度及压力达到设定值(即下面“③分离”中的条件)时开始动态萃取并计时,萃取时间120分钟;
③分离:采用二级分离,分离的工艺条件是:一级解析器压力为10MPa,分离温度37℃;二级解析器压力为4Mpa,分离温度27℃;获得黄绿色的夏枯草籽油0.95kg,以净选后夏枯草籽重量计,得油率为18.6%。
(6)过滤除杂:取上述夏枯草籽油,置于孔径为350目的过滤装置中减压抽滤,弃去不能滤过的杂质,得清澈的夏枯草籽油0.93kg。
(7)质检及灌装:抽取上述夏枯草籽油样品进行质量检测,达标后充氮气灌装。
实施例2夏枯草籽油使用极性夹带剂的超临界CO2萃取的制备方法二
(1)夏枯草籽的收集:采摘完全成熟的干燥的夏枯草果穗,置于干净的收集装置中,将干燥的夏枯草果穗中的夏枯草种子抖落于收集装置中,收集获得的夏枯草籽,获得夏枯草籽粗品6.02kg。
(2)夏枯草籽的净选
A、除去粗大杂质:选用孔径20目的筛网,将上述收集到的夏枯草籽粗品6.02kg置于筛网中,振摇过筛,让粒径小于筛网网孔的草籽通过,除去粒径大于筛网网孔的较粗大的杂质,获得除去粗大杂质的夏枯草籽粗品5.72kg;
B、除去细小杂质:选用孔径30目的筛网,将上述除去粗大杂质的夏枯草籽粗品5.72kg置于筛网中,振摇过筛,让粒径小于筛网网孔的杂质通过,除去粒径小于筛网网孔的较细小的杂质,收集不能通过筛网网孔的夏枯草籽,获得纯净的夏枯草籽5.16kg。
(3)夏枯草籽的破壁:
A、将上述纯净的夏枯草籽置粉碎机中,选用40目孔径的粉碎机筛网进行粉碎破壁;
B、将粉碎破壁机置于10±2℃的环境下;或是在粉碎破壁机的发热部位外部加装制冷循环水装置,并保持粉碎温度在15℃以下;
C、开启机粉碎,粉碎后的夏枯草籽粉置于孔径为40目的筛网中,振摇过筛,让粒径小于筛网网孔的草籽粉通过,不能通过的草籽粉加入粉碎机中继续粉碎破壁,直至全部通过为止,获得破壁的夏枯草籽粉4.95kg;破壁率为99%以上。
(4)与夹带剂混匀:
①加夹带剂混合:往上述破壁后的夏枯草籽粉中加入极性夹带剂乙醇(95%vol.的乙醇水溶液),夏枯草籽粉与乙醇水溶液夹带剂的比例为1千克夏枯草籽粉加入700毫升乙醇水溶液夹带剂,混合均匀;
②装填:将上述夏枯草籽粉与乙醇水溶液夹带剂混合均匀的粉体均匀地填充于超临界CO2萃取釜中。
(5)超临界萃取:
①静态浸泡萃取:将上述填充好夏枯草籽混合粉体的超临界CO2萃取釜,升温至50℃的温度,通以CO2超临界流体升压至20MPa的压力,停泵,静态浸泡提取30min;
②动态萃取:静态浸泡提取30min后,当分离器的温度及压力达到设定值时开始动态萃取并计时,萃取时间150分钟;
③分离:采用二级分离,分离的工艺条件是:一级解析器压力为11MPa,分离温度42℃;二级解析器压力为5Mpa,分离温度32℃;获得黄绿色的夏枯草籽油0.95kg,以净选后夏枯草籽重量计,得油率为18.3%。
(6)过滤除杂:取上述夏枯草籽油,置于孔径为350目的过滤装置中减压抽滤,弃去不能滤过的杂质,得清澈的夏枯草籽油0.93kg。
(7)质检及灌装:抽取上述夏枯草籽油样品进行质量检测,达标后充氮气灌装。
实施例3夏枯草籽油使用极性夹带剂的超临界CO2萃取的制备方法三
(1)夏枯草籽的收集:采摘完全成熟的干燥的夏枯草果穗,置于干净的收集装置中,将干燥的夏枯草果穗中的夏枯草种子抖落于收集装置中,收集获得的夏枯草籽,获得夏枯草籽粗品6.32kg。
(2)夏枯草籽的净选
A、除去粗大杂质:选用孔径20目的筛网,将上述收集到的夏枯草籽粗品6.32kg置于筛网中,振摇过筛,让粒径小于筛网网孔的草籽通过,除去粒径大于筛网网孔的较粗大的杂质,获得除去粗大杂质的夏枯草籽粗品6.13kg;
B、除去细小杂质:选用孔径30目的筛网,将上述除去粗大杂质的夏枯草籽粗品6.13kg置于筛网中,振摇过筛,让粒径小于筛网网孔的杂质通过,除去粒径小于筛网网孔的较细小的杂质,收集不能通过筛网网孔的夏枯草籽,获得纯净的夏枯草籽5.42kg。
(3)夏枯草籽的破壁:
A、将上述纯净的夏枯草籽5.42kg置粉碎机中,选用40目孔径的粉碎机筛网进行粉碎破壁;
B、将粉碎破壁机置于10±2℃的环境下;或是在粉碎破壁机的发热部位外部加装制冷循环水装置,并保持粉碎温度在15℃以下;
C、开启机粉碎,粉碎后的夏枯草籽粉置于孔径为40目的筛网中,振摇过筛,让粒径小于筛网网孔的草籽粉通过,不能通过的草籽粉加入粉碎机中继续粉碎破壁,直至全部通过为止,获得破壁的夏枯草籽粉5.25kg;破壁率为99%以上。
(4)与夹带剂混匀:
①加夹带剂混合:往上述破壁后的夏枯草籽粉中加入极性夹带剂丙酮,夏枯草籽粉与丙酮夹带剂的比例为1千克夏枯草籽粉加入800毫升丙酮夹带剂,混合均匀;
②装填:将上述夏枯草籽粉与丙酮夹带剂混合均匀的粉体均匀地填充于超临界CO2萃取釜中。
(5)超临界萃取:
①静态浸泡萃取:将上述填充好夏枯草籽混合粉体的超临界CO2萃取釜,升温至43℃的温度,通以CO2超临界流体升压至18MPa的压力,停泵,静态浸泡提取50min;
②动态萃取:静态浸泡提取50min后,当分离器的温度及压力达到设定值时开始动态萃取并计时,萃取时间130分钟;
③分离:采用二级分离,分离的工艺条件是:一级解析器压力为9MPa,分离温度40℃;二级解析器压力为5Mpa,分离温度28℃;获得黄绿色的夏枯草籽油0.98kg,以净选后夏枯草籽重量计,得油率为18.1%。
(6)过滤除杂:取上述夏枯草籽油,置于孔径为350目的过滤装置中减压抽滤,弃去不能滤过的杂质,得清澈的夏枯草籽油0.96kg。
(7)质检及灌装:抽取上述夏枯草籽油样品进行质量检测,达标后充氮气灌装。
实施例4夏枯草籽油中熊果酸与迷迭香酸的TLC检测
夏枯草籽油主要组成为脂肪油成分,其组成脂肪油的成分还有待进一步研究。本研究所用夏枯草籽油为使用极性夹带剂的超临界CO2萃取方法制备的夏枯草籽油,富含熊果酸与迷迭香酸:
1、夏枯草籽油中熊果酸的薄层色谱(TLC)检测
(1)供试品溶液的制备:取夏枯草籽油样品6份(取实施例1、2、3中制备的夏枯草籽油各2份),分别各称取约0.8g;分别加入30mL石油醚(60-90℃)溶解后,再分别转移至分液漏斗中,分别加入含80%(v/v)甲醇的水溶液30mL,振摇萃取二次,每次振摇1分钟,静置分层,分取下层的甲醇的水溶液,分别置蒸发皿中,水浴加热蒸发至干,残渣分别加甲醇2mL使溶解,分别得夏枯草籽油的1、2、3、5、6、7号供试品溶液,备用。
(2)对照品溶液的制备:称取熊果酸对照品2.01mg,加甲醇2mL使溶解,成每lmL约含l.01mg熊果酸的溶液,作为对照品溶液,编号4号,备用。
(3)薄层色谱(TLC)检测:
按照《中华人民共和国药典》一部2015年版一部中薄层色谱法(通则0502)试验,吸取上述两种溶液各5μL,分别点于同一高效硅胶薄层板上,以甲苯-乙酸乙酯-甲酸(体积比8.2:1.6:0.2)为展开剂,展开,取出,晾干,喷以10%硫酸乙醇溶液,在105℃加热至斑点显色淸晰,分别置可见光灯与紫外光灯下检识。
可见光灯下检识:供试品色谱中,在与对照品熊果酸色谱相应的位置上,显相同的紫红色斑点;见图1。
置紫外光灯(365nm)下检视:供试品色谱中,在与对照品熊果酸色谱相应的位置上,显相同的橙黄色荧光斑点;见图2。
(4)薄层色谱(TLC)检测结果与结论:
从可见光灯下检识:供试品色谱中,在与对照品熊果酸色谱相应的位置上,显相同的紫红色斑点及置紫外光灯(365nm)下检视:供试品色谱中,在与对照品熊果酸色谱相应的位置上,显相同的橙黄色荧光斑点的结果证明:6份夏枯草籽油样品中均含有三萜类成分熊果酸,且6份供试品色谱中的主要三萜类成分色谱基本相同。
2、夏枯草籽油中迷迭香酸的薄层色谱(TLC)检测
(1)供试品溶液的制备:取夏枯草籽油样品6份(取实施例1、2、3中制备的夏枯草籽油各2份),分别各称取约0.8g;分别加入30mL石油醚(60-90℃)溶解后,再分别转移至分液漏斗中,分别加入含80%(v/v)甲醇的水溶液30mL,振摇萃取二次,每次振摇1分钟,静置分层,分取下层的甲醇的水溶液,分别置蒸发皿中,水浴加热蒸发至干,残渣分别加甲醇2mL使溶解,分别得夏枯草籽油的1、2、3、5、6、7号供试品溶液,备用。
(2)对照品溶液的制备:称取迷迭香酸对照品1.81mg,加甲醇2mL使溶解,制成每lmL约含0.91mg迷迭香酸的溶液,作为对照品溶液,编号4号,备用。
(3)薄层色谱(TLC)检测:
按照《中华人民共和国药典》一部2015年版一部中薄层色谱法(通则0502)试验,吸取上述两种溶液各4μL,分别点于同一高效硅胶GF254薄层板上,以环已烷-乙酸乙酯-异丙醇-甲酸(体积比6.7:1.4:1.6:0.3)为展开剂,展开,取出,晾干;分别置紫外光灯254nm与紫外光灯365nm下检识。
紫外光灯254nm下检识:供试品色谱中,在与对照品迷迭香酸色谱相应的位置上,显相同的荧光萃灭斑点;见图3。
置紫外光灯(365nm)下检视:供试品色谱中,在与对照品迷迭香酸色谱相应的位置上,显相同的颜色荧光斑点;见图4。
(4)薄层色谱(TLC)检测结果与结论:
从紫外光灯254nm下检识:供试品色谱中,在与对照品迷迭香酸色谱相应的位置上,显相同的荧光淬灭斑点及置紫外光灯(365nm)下检视:供试品色谱中,在与对照品迷迭香酸色谱相应的位置上,显相同的颜色荧光斑点的结果证明:6份夏枯草籽油样品中均含有迷迭香酸成分。且6份供试品色谱中的迷迭香酸成分色谱基本相同。
可见,本发明所用的夏枯草籽油为使用极性夹带剂的超临界CO2萃取方法制备的夏枯草籽油,主要为脂肪油成分,其中除富含以熊果酸为代表的三萜类成分外,还富含以迷迭香酸为代表的苯丙素类成分,为夏枯草籽油的开发利用提供了物质基础。
实施例5.夏枯草籽油自乳化软胶囊的制备方法之一
夏枯草籽油(实施例1制备得到)的自乳化软胶囊的制备一。
1、自乳化软胶囊成分组成如表1所示:
表1夏枯草籽油自乳化软胶囊成分组成
2、夏枯草籽油的自乳化软胶囊的制备:
(1)夏枯草籽油的自乳化系统制备:取蛋黄卵磷脂200g、甘油190g混合,加热至60℃,用剪切机(8000转/分)搅拌,使其分散均匀,约需搅拌10分钟,加入加热至60℃的夏枯草籽油600g、维生素E10g,搅拌3次,每次5分钟,滤过,形成均一的液体即夏枯草籽油的自乳化系统。
(2)软胶囊囊材液的制备:将明胶200g加入溶胶罐中,加入240g温度为60-80℃的水,加入防腐剂尼泊金乙酯0.2g,必要时加入遮光剂和着色剂,密闭同时加热,搅拌至融化溶胀完全,加入100g甘油,密闭配胶罐,关闭搅拌,开启真空泵,抽真空,待气泡抽净,即得软胶囊囊材液。50-70℃保温,备用。
(3)自乳化软胶囊成品的制备:取软胶囊囊材液、夏枯草籽油的自乳化系统上软胶囊机,采用压制法制备软胶囊,经干燥,洗涤,再干燥,分检,包装,得成品。
(4)自乳化效果检测:取该夏枯草籽油的自乳化系统1.0mL,加至20mL人工胃液中,用玻璃棒缓和搅拌,即可迅速形成带有蓝色乳光的乳剂。对乳剂的粒径用TSM超细颗粒粒径分析仪进行测定,测得结果为乳滴的平均粒径在0.28μm左右。
实施例6夏枯草籽油自乳化软胶囊的制备方法之二
1、自乳化软胶囊成分组成如表2所示:
表2夏枯草籽油自乳化软胶囊成分组成
2、夏枯草籽油的自乳化软胶囊的制备:
(1)夏枯草籽油的自乳化系统制备:取大豆磷脂260g、甘油230g混合,加热至70℃,用剪切机(8000转/分)搅拌,使其分散均匀,约需搅拌15分钟,加入加热至70℃的夏枯草籽油500g、维生素E10g,搅拌3次,每次5分钟,滤过,形成均一的液体即夏枯草籽油的自乳化系统。
(2)软胶囊囊材液的制备:将明胶200g加入溶胶罐中,加入240g温度为60-80℃的水,加入防腐剂尼泊金乙酯0.2g,必要时加入遮光剂和着色剂,密闭同时加热,搅拌至融化溶胀完全,加入100g甘油,密闭配胶罐,关闭搅拌,开启真空泵,抽真空,待气泡抽净,即得软胶囊囊材液。50-70℃保温,备用。
(3)自乳化软胶囊成品的制备:取软胶囊囊材液、夏枯草籽油的自乳化系统上软胶囊机,采用压制法制备软胶囊,经干燥,洗涤,再干燥,分检,包装,得成品。
(4)自乳化效果检测:取该夏枯草籽油的自乳化系统1.0mL,加至20mL人工胃液中,用玻璃棒缓和搅拌,即可迅速形成带有蓝色乳光的乳剂。对乳剂的粒径用TSM超细颗粒粒径分析仪进行测定,测得结果为乳滴的平均粒径在0.31μm左右。
实施例7夏枯草籽油自乳化软胶囊的制备方法之三
1、自乳化软胶囊成分组成如表3所示:
表3夏枯草籽油自乳化软胶囊成分组成
2、夏枯草籽油的自乳化软胶囊的制备:
(1)夏枯草籽油的自乳化系统制备:取液态卵磷脂210g、甘油135g混合、山梨酸钾0.5g,加热至75℃,用剪切机(8000转/分)搅拌,使其分散均匀,约需搅拌5分钟,加入加热至75℃的夏枯草籽油650g、维生素C 5g,搅拌3次,每次5分钟,滤过,形成均一的液体即夏枯草籽油的自乳化系统。
(2)软胶囊囊材液的制备:将明胶200g加入溶胶罐中,加入240g温度为60-80℃的水,加入防腐剂尼泊金乙酯0.2g,必要时加入遮光剂和着色剂,密闭同时加热,搅拌至融化溶胀完全,加入100g甘油,密闭配胶罐,关闭搅拌,开启真空泵,抽真空,待气泡抽净,即得软胶囊囊材液。50-70℃保温,备用。
(3)自乳化软胶囊成品的制备:取软胶囊囊材液、夏枯草籽油的自乳化系统上软胶囊机,采用压制法制备软胶囊,经干燥,洗涤,再干燥,分检,包装,得成品。
(4)自乳化效果检测:取该夏枯草籽油的自乳化系统1.0mL,加至20mL人工胃液中,用玻璃棒缓和搅拌,即可迅速形成带有蓝色乳光的乳剂。对乳剂的粒径用TSM超细颗粒粒径分析仪进行测定,测得结果为乳滴的平均粒径在0.33μm左右。
实施例8夏枯草籽油自乳化系统与纯夏枯草籽油抑瘤率比较实验
下面通过药效试验比较本发明所提供的夏枯草籽油与夏枯草籽油自乳化系统抗肿瘤作用的效果。
1、供试样品
夏枯草籽油及其自乳化系统的制备:取用来制备夏枯草籽油自乳化系统的夏枯草籽油(实施例1制备得到)及实施例6制备的夏枯草籽油自乳化系统作为夏枯草籽油及其自乳化系统的供试品。
2、实验动物分组及实验条件
雄性封闭群昆明种小鼠(KM小鼠,南方医科大学实验动物管理中心)90只,体重18~22g,制成EAC模型鼠,随机分成A、B、C三组,每组30只。分别为A组:生理盐水对照组;B组:纯夏枯草籽油组(实施例1制备的夏枯草籽油);治疗组;C:夏枯草籽油自乳化系统(实施例6制备)治疗组。实验温度23℃~25℃,湿度58%~60%。
3、S180腹水瘤模型小鼠制作
S180腹水瘤细胞(ATCC)经体内传代培养后,在无菌条件下取对数生长期的S180腹水瘤细胞,用PBS制备成2.5×107个细胞/mL密度的细胞悬液,接种于90只小鼠右侧腋部皮下,每只小鼠0.2mL,共计5×106细胞。
4、实验方法
待肿瘤长至约100mm3后,将动物随机分为A组生理盐水对照组、B组纯夏枯草籽油治疗组、C组夏枯草籽油自乳化系统治疗组,30只/组,治疗组给药剂量以含相同的纯夏枯草籽油净含量为基准。
4.1A组生理盐水对照组:每只小鼠灌胃给予0.9%生理盐水0.2mL,每天灌胃一次,连续灌胃30天。
4.2B组纯夏枯草籽油治疗组:每只小鼠灌胃给予纯夏枯草籽油0.1mL(96mg,相当于C组夏枯草籽油自乳化系统0.2mL中所含的纯夏枯草籽油的量),每天灌胃一次,连续灌胃30天。
4.3C组夏枯草籽油自乳化系统治疗组:每只小鼠灌胃给予夏枯草籽油自乳化系统治疗组0.2mL,每天灌胃一次,连续灌胃给予30天。
4.4动物饲养与处置
在给药周期内小鼠饲喂常备饲料,正常饮水。30天末次给药24h后,断锥处死小鼠,剖取肿瘤,分离出腹水瘤体,去除脂肪、结缔组织,精确称重,并计算抑瘤率。
抑瘤率=(A-B或C)/A×100%
(A为生理盐水对照组的平均瘤重,B、C为治疗组的平均瘤重)
比较生理盐水对照组与纯夏枯草籽油治疗组之间的抑瘤率,以及纯夏枯草籽油治疗组与夏枯草籽油自乳化系统治疗组之间的抑瘤率是否存在显著性差异。
5、实验结果
连续灌胃给予30天后,A组(生理盐水对照组)平均瘤重为4.31克,B组(纯夏枯草籽油治疗组)平均瘤重为2.04克,抑制率为52.67%;C组(夏枯草籽油自乳化系统治疗组)平均瘤重为1.47克,抑制率为65.89%。与生理盐水对照组比较,纯夏枯草籽油治疗组和夏枯草籽油自乳化系统治疗组的抑瘤率均有显著性差异(P<0.01);纯夏枯草籽油治疗组和夏枯草籽油自乳化系统治疗组的抑瘤率之间比较亦存在显著性差异(P<0.05),夏枯草籽油自乳化系统治疗组抑瘤率约是纯夏枯草籽油抑瘤率的1.25倍。
6、结论与分析
6.1纯夏枯草籽油治疗组与对照组比较,抑瘤率有非常显著差异,说明纯夏枯草籽油对肿瘤有较好的抑制作用。
6.2同样,夏枯草籽油自乳化系统治疗组与对照组比较,抑瘤率有非常显著差异,说明夏枯草籽油自乳化系统对肿瘤有非常好的抑制作用。由于夏枯草籽油被自乳化,在胃里由于胃粘膜的作用,夏枯草籽油自乳化系统自乳化为纳米乳,其活性及药效均有较大提高。
6.3夏枯草籽油自乳化系统治疗组与纯夏枯草籽油治疗组比较,抑瘤率又有显著差异,说明夏枯草籽油自乳化系统对肿瘤有非常好的抑制作用。
6.4由于纯夏枯草籽油中含有具有传统抗肿瘤作用的三萜类和苯丙素类成分,所以夏枯草籽油具有较好的抑制肿瘤作用;而夏枯草籽油自乳化系统,由于自乳化作用使夏枯草籽油的作用速度加快、吸收度增加,所以夏枯草籽油的活性及药效有较大的提高,其抑瘤率增加了0.25倍。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种具有抗肿瘤作用的夏枯草籽油自乳化软胶囊,包括芯材和包裹芯材的囊材;其特征在于:芯材为夏枯草籽油自乳化系统,由夏枯草籽油、表面活性剂、或助表面活性剂、或和抗氧剂或和防腐剂制备得到。
2.根据权利要求1所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊,其特征在于:所述的夏枯草籽油自乳化系统由以下按质量百分比计的成分的成分组成:夏枯草籽油40~75%、表面活性剂20~50%、助表面活性剂0~25%、抗氧剂0.1~1.0%、防腐剂0~0.3%。
3.根据权利要求2所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊,其特征在于:
所述的夏枯草籽油在所述的夏枯草籽油自乳化系统中的含量为质量百分比45~75%;
所述的表面活性剂在所述的夏枯草籽油自乳化系统中的含量为质量百分比20~50%;
所述的助表面活性剂在所述的夏枯草籽油自乳化系统中的含量为质量百分比13.5~23%;
所述的抗氧剂在所述的夏枯草籽油自乳化系统中的含量为质量百分比0.5~1%;
所述的防腐剂在所述的夏枯草籽油自乳化系统中的含量为质量百分比0~0.05%。
4.根据权利要求1~3任一项所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊,其特征在于:
所述的夏枯草籽油通过如下步骤制备得到:
1)夏枯草籽的收集:采摘完全成熟的干燥的夏枯草果穗,将干燥的夏枯草果穗中的夏枯草种子抖落,收集并获得夏枯草籽粗品;
2)夏枯草籽的净选:将夏枯草籽粗品中的杂质去除,得到纯净的夏枯草籽;
3)夏枯草籽的破壁:将步骤2)得到的纯净的夏枯草籽置于粉碎破壁机中进行破壁,得到破壁后的夏枯草籽粉;其中粉碎破壁机筛网的孔径小于或等于40目,粉碎破壁机在15℃以下的环境工作;
4)与夹带剂混匀:
①加夹带剂混合:将破壁后的夏枯草籽粉和极性夹带剂混合均匀;
②装填:将步骤①得到的混匀的含极性夹带剂的粉末均匀地填充于超临界CO2萃取釜中;
5)超临界萃取:
①静态浸泡萃取:将步骤4)得到的夏枯草籽粉颗粒置于超临界CO2萃取釜升温至30-60℃的温度,通以CO2超临界流体升压至10-25MPa的压力,停泵,静态浸泡提取20-60min;
②动态萃取:静态浸泡提取后,当分离器的温度及压力达到设定值时开始动态萃取并计时,萃取时间为100-180min;
③分离:采用二级分离,分离的工艺条件是:一级解析器压力为9~13MPa,分离温度37~43℃;二级解析器压力为4~6Mpa,分离温度27~33℃;获得黄绿色的夏枯草籽油;
6)过滤除杂:取步骤5)得到的夏枯草籽油,置于孔径等于或小350目的过滤装置中减压抽滤,弃去不能滤过的杂质,得清澈的夏枯草籽油。
5.根据权利要求4所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊,其特征在于:
步骤4)中所述的极性夹带剂为甲醇、乙醇、乙醇水溶液和丙酮中的至少一种;
步骤4)中所述的极性夹带剂的用量按夏枯草籽粉:极性夹带剂=1kg:300-1500mL计算;
步骤5)中所述的静态浸泡萃取的条件为:温度为43-50℃,压力为18-20MPa,静态浸泡提取的时间为30-50min;
步骤5)中所述的动态萃取的萃取时间为120-150min;
步骤5)中所述的分离的工艺条件为:一级解析器压力为9~11MPa,分离温度37~42℃;二级解析器压力为4~5Mpa,分离温度27~32℃。
6.根据权利要求1~3任一项所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊,其特征在于:
所述的表面活性剂为大豆磷脂、蛋黄卵磷脂、液态卵磷脂和脑磷脂中的至少一种;
所述的助表面活性剂为甘油、乙醇和聚乙二醇400中至少一种;
所述的抗氧剂为维生素E和维生素C中的至少一种;
所述的防腐剂为尼泊金酯类防腐剂和山梨酸钾中的至少一种。
7.根据权利要求1~3任一项所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊,其特征在于:
所述的囊材由如下按质量百分比计的成分组成:食用胶30~45%、增塑剂15~25%、防腐剂0~适量、调味剂0~适量、香精0~适量、遮光剂0~适量、着色剂0~适量,余量为水。
8.权利要求1~7任一项所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊的制备方法,其特征在于包括如下步骤:
(1)夏枯草籽油自乳化系统的制备:取夏枯草籽油,在20-80℃条件温度下,加入表面活性剂、助表面活性剂和抗氧剂,必要时加入防腐剂溶解,混合均匀,滤过,得到均一的夏枯草籽油自乳化系统;
(2)软胶囊的制备:将软胶囊囊材液和步骤(1)制备的夏枯草籽油自乳化系统通过软胶囊机,采用压制法制备软胶囊,经干燥,洗涤,再干燥,分检,包装,得到软胶囊成品。
9.根据权利要求8所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊的制备方法,其特征在于:
步骤(1)为:先将表面活性剂、助表面活性剂和防腐剂混合均匀,得到混合液A;将混合液A与夏枯草籽油、抗氧化剂混匀,滤过,得到均一的夏枯草籽油自乳化系统;
步骤(2)中所述的软胶囊囊材液通过如下步骤制备得到:将食用胶和水混合,必要时加入防腐剂、调味剂、香精、遮光剂、着色剂,密闭同时加热,搅拌至融化溶胀完全,加入增塑剂,密闭配胶罐,关闭搅拌,开启真空泵,抽真空,待气泡抽净,即得软胶囊囊材液,于50~70℃保温。
10.权利要求1~7任一项所述的具有抗肿瘤作用的夏枯草籽油自乳化软胶囊在食品、保健食品或药物领域中的应用。
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