CN113786423A - 一种金丝皇菊挥发油及其组合物在制备治疗糖脂代谢综合紊乱的产品方面的应用 - Google Patents
一种金丝皇菊挥发油及其组合物在制备治疗糖脂代谢综合紊乱的产品方面的应用 Download PDFInfo
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- CN113786423A CN113786423A CN202111111541.8A CN202111111541A CN113786423A CN 113786423 A CN113786423 A CN 113786423A CN 202111111541 A CN202111111541 A CN 202111111541A CN 113786423 A CN113786423 A CN 113786423A
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- volatile oil
- chrysanthemum
- silk
- extract
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Abstract
本发明公开了一种金丝皇菊挥发油及其组合物在制备治疗糖脂代谢综合紊乱的产品方面中的应用。本发明首次发现金丝皇菊花和/或叶中的挥发油具有显著的降血糖和/或降血脂活性,扩大了农产品金丝皇菊本身以及下游高附加值产品的应用领域,金丝皇菊挥发油以及挥发油和提取物组合物可以应用在制备治疗糖脂代谢综合紊乱以及相关疾病的产品方面中。
Description
技术领域
本发明属于医药和功能食品技术领域,涉及天然植物分子及其组合物的新应用,尤其是一种金丝皇菊挥发油及其组合物在制备治疗糖脂代谢综合紊乱的产品方面的应用。
背景技术
金丝皇菊,因南宋诗人黄庭坚将修水药菊进贡朝廷,被皇上赐名金丝皇菊而得名。金丝皇菊不仅具有很好的观赏价值,而且气味芬芳,味甘苦,性微寒;属药、茶两用佳品,更因其个大形美、口感清香甘甜被誉为菊花中的上品。现代医学研究表明,金丝皇菊含有的黄酮素成分极高,富含多种氨基酸、维生素和微量元素,具有“香、甜、润”三大特点,还有散风热、明目等功效,具有抗疲劳抗衰老的神奇功效,深受消费者青睐。
金丝皇菊作用较多,有文献报道,其中一些黄酮类化合物可通过抑制磷酸烯醇式丙酮酸羧化酶和葡萄糖-6-磷酸酶的活性而减少肝葡萄糖异生,绿原酸能够有效降低糖尿病大鼠的血糖水平,奎宁酸能够改善糖尿病大鼠的胰腺损伤程度。在所了解到的现有技术中并没有将金丝皇菊花和叶中挥发油及其与提取物组合物兼具降血糖和降血脂功效的报道与应用实例。
糖脂代谢紊乱相关疾病是当前全球健康关注的重点,是全球主要的公共卫生问题之一。糖脂代谢紊乱会引起糖脂代谢相关疾病,包括2型糖尿病(T2DM)、高脂血症、非酒精性脂肪肝(NAFLD)、肥胖、高血压和动脉粥样硬化等,这些疾病如T2DM,作为慢性疾病,对个人和社会都造成了极大的影响和经济负担。目前临床上对于高血糖合并高血脂的患者主要以降血糖和降血脂的联合用药的方式进行治疗,但存在药物相互作用,体内代谢方式不统一等问题,因此,开发兼具降血糖和降血脂活性的药物或功能性食品,具有良好的应用前景。
植物挥发油是植物体内的次生代谢物质,由分子量相对较小的简单化合物组成,具有一定芳香气味,在常温下能挥发的油状液体物质。该物质所含化学成分比较复杂,按其化学结构的不同可分为脂肪族、芳香族和萜类三大类化合物以及它们的含氧衍生物如醇、醛、酮、酸、醚、酯等,此外还有含氮和含硫的化合物,这些化合物均有沸点低且易挥发的特点。植物挥发油几乎没有毒副作用,因此被广泛地应用在医药、食品、化妆品等行业。近年来,国内外学者对植物挥发油的研究主要集中在医药保健、食品工业、抗菌、害虫防治及果蔬保鲜等方面。因此,随着医药、食品、化妆品等行业的飞速发展,研究植物挥发油有着重要的现实意义。
通过检索,发现如下几篇与本发明专利申请相关的专利公开文献:
1、一种具有美白祛斑功效的金丝皇菊挥发油制备方法和应用(CN112057369A),从金丝皇菊花、茎、叶中提取得到挥发油,提取分离方法中加入减压和循环冷凝装置,和/或用乳木果油替代有机溶剂,用气质联用方法分析和鉴定了挥发油的成分,并对不同部分挥发油以及乳木果油和金丝皇菊挥发油的混合物进行了美白祛斑活性评价,结果表明金丝皇菊花、金丝皇菊茎、金丝皇菊叶的挥发油,以及金丝皇菊挥发油和乳木果油混合物均具有抑制酪氨酸酶的活性,以及抑制黑色素瘤细胞黑色素生成活性。该专利公开文献未涉及降血糖和降血脂活性以及在治疗糖脂代谢综合紊乱方面产品的应用。
2、一种金丝皇菊纳米技术护肤品及其制备方法和应用(CN111481481A),护肤品的原料成分为:金丝皇菊花瓣精油,金丝皇菊水提物,金丝皇菊醇提物,金丝皇菊茎叶水提物,金丝皇菊茎叶醇提物,从金丝皇菊中分离出的香叶木素黄酮及金合欢素黄酮;二棕榈酰磷脂酰胆碱,卵磷脂,聚乙二醇400,透明质酸,海藻糖,甘油硬脂酸酯,羟苯甲酯钠,聚山梨醇酯-20,柠檬酸,柠檬酸钠,碳酸钠。该专利公开文献未涉及降血糖和降血脂活性以及在治疗糖脂代谢综合紊乱方面产品的应用。
3、香叶基柑橘查尔酮及组合物在制备治疗脂肪肝的产品方面中的应用(CN111388457A),公开了3'-香叶基柑橘查尔酮及其与金丝皇菊提取物的组合物用于制备治疗脂肪肝方面的应用,该专利主要活性成分为3'-香叶基柑橘查尔酮,金丝皇菊提取物只是协同增效的作用,不涉及金丝皇菊挥发油,且不涉及降血糖和降血脂双重功效。
4、期刊文献:胡文杰等,皇菊不同部位挥发油化学成分比较分析,《天然产物研究与开发》,2015,27:1187-1193。该论文分析了采自井冈山大学生物园的皇菊,采用普通的水蒸汽蒸馏法提取其挥发油,用气质联用技术鉴定了皇菊叶、茎、根和花四部分中挥发油的结构,但未对成分进行功效检测。
通过对比,本发明专利申请与上述公开文献存在本质的不同。
发明内容
本发明的目的在于克服现有技术上存在的问题,提供一种金丝皇菊挥发油及其组合物在制备治疗糖脂代谢综合紊乱的产品方面的应用。
本发明解决技术问题所采用的技术方案是:
一种金丝皇菊挥发油及其组合物在制备治疗糖脂代谢综合紊乱的产品方面中的应用。
进一步地,所述金丝皇菊挥发油为金丝皇菊花挥发油、或金丝皇菊叶挥发油、或金丝皇菊花挥发油和金丝皇菊叶挥发油的混合物;或者,所述组合物为金丝皇菊花和/或叶的挥发油与金丝皇菊提取物的组合物,其中,金丝皇菊提取物为金丝皇菊醇提物或水提物。
进一步地,所述金丝皇菊挥发油、金丝皇菊挥发油和提取物的组合物均具有抑制α-葡萄糖苷酶、果糖-1,6-二磷酸酯酶(FBPase)和胰脂肪酶的活性;金丝皇菊花挥发油的0.25mg/mL浓度下对α-葡萄糖苷酶抑制率82.25%,金丝皇菊叶挥发油的0.25mg/mL浓度下对α-葡萄糖苷酶抑制率88.81%,两种挥发油对α-葡萄糖苷酶的抑制活性高于相同浓度的阿卡波糖,阿卡波糖的0.25mg/mL浓度下抑制率66.53%,同时也均高于同浓度下金丝皇菊乙醇提取物、金丝皇菊水浸提提取物;
金丝皇菊花挥发油的0.25mg/mL浓度下对FBPase酶抑制率62.14%,金丝皇菊叶挥发油0.25mg/mL浓度下对FBPase酶抑制率58.02%,两种挥发油对FBPase酶的抑制活性均高于同浓度下金丝皇菊水提物,金丝皇菊水提物的0.25mg/mL浓度下抑制率25.48%;金丝皇菊乙醇提取物的0.5mg/mL浓度下对胰脂肪酶抑制率90.08%,其对胰脂肪酶的抑制活性均高于相同浓度下金丝皇菊花挥发油和金丝皇菊叶挥发油,金丝皇菊花挥发油的0.5mg/mL浓度下抑制率68.02%,金丝皇菊叶挥发油的0.5mg/mL浓度下抑制率89.99%;
细胞水平上,金丝皇菊花和/或叶的挥发油以及金丝皇菊挥发油与提取物的组合物均具有细胞水平降血糖和降血脂的活性:在1μg/mL浓度下,金丝皇菊花和/或叶挥发油以及金丝皇菊花和/或叶挥发油与提取物组合物能够促进细胞的葡萄糖消耗量,比相同浓度下的临床用药二甲双胍活性强;在0.2μg/mL浓度下,金丝皇菊叶挥发油以及金丝皇菊叶挥发油与水提物组合物均能够降低细胞脂肪含量,比临床用药洛伐他汀活性高。
进一步地,所述金丝皇菊花挥发油、金丝皇菊叶挥发油的成分如下:
一种金丝皇菊挥发油及其组合物在制备降血糖、降血脂活性方向的食品和/或保健食品和/或药品方面中的应用。
进一步地,所述金丝皇菊挥发油为金丝皇菊花挥发油、或金丝皇菊叶挥发油、或金丝皇菊花挥发油和金丝皇菊叶挥发油的混合物;或者,所述组合物为金丝皇菊花和/或叶的挥发油与金丝皇菊提取物的组合物,其中,金丝皇菊提取物为金丝皇菊醇提物或水提物。
一种金丝皇菊挥发油的提取方法,步骤如下:
S1:采用水蒸气蒸馏提取法,称取金丝皇菊花和/或叶进行粉碎,转移至容器中,加入料液比1:2~20的水,连接挥发油提取器,并在挥发油提取器中加入乙醚或正己烷或环己烷或二氯甲烷中的一种或多种,在回流冷凝管上部连接减压装置,回流冷凝管连接低温恒温循环冷凝器用于冷凝;
S2:减压情况下,加热水至沸腾,回流提取1~10小时,收集有机相,水层用有机相萃取,合并有机相,干燥,蒸馏除去有机相,即得金丝皇菊花和/或叶的挥发油。
进一步地,所述步骤S1中,采用金丝皇菊花、金丝皇菊叶粉末中一种或两种混合进行提取;
或者,所述步骤S1中,回流冷凝管冷凝所采用的仪器是低温恒温循环冷凝器,冷媒为异丙醇、乙二醇,循环冷凝温度为0℃至-80℃;
或者,所述步骤S2中,所述减压的压力范围为0.01至0.1MPa。
一种金丝皇菊提取物的提取方法,步骤如下:
称量金丝皇菊花和/或叶,进行粉碎,将粉末加入5~50倍体积的提取溶剂,回流提取1~5次,每次0.1~12h,抽滤,合并提取液,除去提取液,干燥,即得金丝皇菊提取物。
进一步地,所述提取溶剂为水和/或乙醇。
本发明取得的有益效果是:
1、本发明首次发现金丝皇菊花和叶中的挥发油具有显著的降血糖和/或降血脂活性,扩大了农产品金丝皇菊本身以及下游高附加值产品的应用领域,金丝皇菊挥发油以及挥发油和提取物组合物可以应用在制备治疗糖脂代谢综合紊乱以及相关疾病的产品方面中。
2、本发明将金丝皇菊花和/或叶的挥发油与金丝皇菊水提物或醇提物以一定的比例混合,可以制成一种具有降血糖和/或降血脂功效的金丝皇菊新型组合物。该组合物在保持相似活性前提下可以减少挥发油添加量,利于保存,且成本更低。
3、本发明研究的不仅是常规食品中金丝皇菊花的挥发油,且首次对金丝皇菊叶的挥发油叶进行了活性研究,发现了其良好的降血糖和降血脂活性,这对于金丝皇菊茶整个植株的充分利用,变废为宝,增加农户的收入,具有积极的作用。
4、本发明对金丝皇菊花和金丝皇菊叶的挥发油分别用气相色谱一质谱检测(GC-MS)进行了成分分析:经搜索谱库以及标准样品比对结合人工解析质谱图,鉴定了金丝皇菊挥发油中的76个化合物,其中含量比较高的有石竹烯、环氧芳烃、(-)-芦丁烯醇、冰片、桃金娘烯醇、石竹烯氧化物等。
5、本发明分别进行了金丝皇菊花挥发油和金丝皇菊叶挥发油、金丝皇菊水提物和醇提物,以及金丝皇菊挥发油与提取物组合物的酶活水平与细胞水平的降血糖和降血脂活性评价。
酶活水平上,评价了不同浓度的金丝皇菊挥发油、提取物以及挥发油和提取物组合物对α-葡萄糖苷酶、FBPase和胰脂肪酶的抑制活性,分别以阿卡波糖、5’腺嘌呤核苷酸(AMP)和奥利司他为阳性对照,结果表明金丝皇菊花、金丝皇菊叶的挥发油,以及金丝皇菊提取物和金丝皇菊挥发油和提取物的组合物均具有良好的抑制α-葡萄糖苷酶、FBPase酶和胰脂肪酶的活性。金丝皇菊花挥发油(0.25mg/mL浓度下抑制率82.25%)和金丝皇菊叶挥发油(0.25mg/mL浓度下抑制率88.81%)对α-葡萄糖苷酶的抑制活性高于相同浓度的阿卡波糖(0.25mg/mL浓度下抑制率66.53%),同时也均高于同浓度下金丝皇菊乙醇提取物、金丝皇菊水浸提提取物。金丝皇菊花挥发油(0.25mg/mL浓度下抑制率62.14%)和金丝皇菊叶挥发油(0.25mg/mL浓度下抑制率58.02%)对FBPase酶的抑制活性均高于同浓度下金丝皇菊水提物(0.25mg/mL浓度下抑制率25.48%)。金丝皇菊乙醇提取物(0.5mg/mL浓度下抑制率90.08%)对胰脂肪酶的抑制活性高于相同浓度下金丝皇菊花挥发油(0.5mg/mL浓度下抑制率68.02%)和金丝皇菊叶挥发油(0.5mg/mL浓度下抑制率89.99%)。
细胞水平上,用葡萄糖检测试剂盒和油红O染色实验来检测金丝皇菊挥发油、金丝皇菊提取物和金丝皇菊挥发油与提取物的组合物对细胞葡萄糖消耗量和脂肪含量的影响。分别以二甲双胍和洛伐他汀为阳性对照。结果表明金丝皇菊花、叶的挥发油以及金丝皇菊挥发油与提取物的混合物均具有良好的细胞水平降血糖和降血脂的活性。
附图说明
图1为本发明中金丝皇菊挥发油、提取物、挥发油和提取物组合物的胰脂肪酶抑制率柱状图。
具体实施方式
为更好理解本发明,下面结合实施例对本发明做进一步地详细说明,但是本发明要求保护的范围并不局限于实施例所表示的范围。
本发明中所使用的的原料,如无特殊说明,均为常规市售产品,本发明中所使用的方法,如无特殊说明,均为本领域常规方法,本发明所使用的各物质质量均为常规使用质量。
一种金丝皇菊挥发油及其组合物在制备治疗糖脂代谢综合紊乱的产品方面中的应用。
较优地,所述金丝皇菊挥发油为金丝皇菊花挥发油、或金丝皇菊叶挥发油、或金丝皇菊花挥发油和金丝皇菊叶挥发油的混合物;或者,所述组合物为金丝皇菊花和/或叶的挥发油与金丝皇菊提取物的组合物,其中,金丝皇菊提取物为金丝皇菊醇提物或水提物。
较优地,所述金丝皇菊挥发油、金丝皇菊挥发油和提取物的组合物均具有抑制α-葡萄糖苷酶、FBPase和胰脂肪酶的活性;金丝皇菊花挥发油的0.25mg/mL浓度下对α-葡萄糖苷酶抑制率82.25%,金丝皇菊叶挥发油的0.25mg/mL浓度下对α-葡萄糖苷酶抑制率88.81%,两种挥发油对α-葡萄糖苷酶的抑制活性高于相同浓度的阿卡波糖,阿卡波糖的0.25mg/mL浓度下抑制率66.53%,同时也均高于同浓度下金丝皇菊乙醇提取物、金丝皇菊水浸提提取物;
金丝皇菊花挥发油的0.25mg/mL浓度下对FBPase抑制率62.14%,金丝皇菊叶挥发油0.25mg/mL浓度下对FBPase抑制率58.02%,两种挥发油对FBPase酶的抑制活性均高于同浓度下金丝皇菊水提物,金丝皇菊水提物的0.25mg/mL浓度下抑制率25.48%;金丝皇菊乙醇提取物的0.5mg/mL浓度下对胰脂肪酶抑制率90.08%,其对胰脂肪酶的抑制活性均高于相同浓度下金丝皇菊花挥发油和金丝皇菊叶挥发油,金丝皇菊花挥发油的0.5mg/mL浓度下抑制率68.02%,金丝皇菊叶挥发油的0.5mg/mL浓度下抑制率89.99%;
细胞水平上,金丝皇菊花和/或叶的挥发油以及金丝皇菊挥发油与提取物的组合物均具有细胞水平降血糖和降血脂的活性:在1μg/mL浓度下,金丝皇菊花和/或叶挥发油以及金丝皇菊花和/或叶挥发油与提取物组合物能够促进细胞的葡萄糖消耗量,比相同浓度下的临床用药二甲双胍活性强;在0.2μg/mL浓度下,金丝皇菊叶挥发油以及金丝皇菊叶挥发油与水提物组合物均能够降低细胞脂肪含量,比临床用药洛伐他汀活性高。
较优地,所述金丝皇菊花挥发油、金丝皇菊叶挥发油的成分如下:
一种金丝皇菊挥发油及其组合物在制备降血糖、降血脂活性方向的食品和/或保健食品和/或药品方面中的应用。
较优地,所述金丝皇菊挥发油为金丝皇菊花挥发油、或金丝皇菊叶挥发油、或金丝皇菊花挥发油和金丝皇菊叶挥发油的混合物;或者,所述组合物为金丝皇菊花和/或叶的挥发油与金丝皇菊提取物的组合物,其中,金丝皇菊提取物为金丝皇菊醇提物或水提物。
一种金丝皇菊挥发油的提取方法,步骤如下:
S1:采用水蒸气蒸馏提取法,称取金丝皇菊花和/或叶进行粉碎,转移至容器中,加入料液比1:2~20的水,连接挥发油提取器,并在挥发油提取器中加入乙醚或正己烷或环己烷或二氯甲烷中的一种或多种,在回流冷凝管上部连接减压装置,回流冷凝管连接低温恒温循环冷凝器用于冷凝;
S2:减压情况下,加热水至沸腾,回流提取1~10小时,收集有机相,水层用有机相萃取,合并有机相,干燥,蒸馏除去有机相,即得金丝皇菊花和/或叶的挥发油。
进一步地,所述步骤S1中,采用金丝皇菊花、金丝皇菊叶粉末中一种或两种混合进行提取;
或者,所述步骤S1中,回流冷凝管冷凝所采用的仪器是低温恒温循环冷凝器,冷媒为异丙醇、乙二醇或其他制冷剂,循环冷凝温度为0℃至-80℃;
或者,所述步骤S2中,减压情况下利于不同沸点的挥发油的提取,所述减压的压力范围为0.01至0.1MPa。
一种金丝皇菊提取物的提取方法,步骤如下:
称量金丝皇菊花和/或叶,进行粉碎,将粉末加入5~50倍体积的提取溶剂,回流提取1~5次,每次0.1~12h,抽滤,合并提取液,除去提取液,干燥,即得金丝皇菊提取物。
较优地,所述提取溶剂为水和/或乙醇。
具体地,相关的制备及检测如下:
实施例1
一种金丝皇菊挥发油提取方法优化:
表1不同条件下金丝皇菊花挥发油提取率
由表1得知,挥发油提取器中提取溶剂和后处理中萃取溶剂为乙醚时,提取率较高,当外浴加热温度为150℃、提取时间9h时,提取率最高。
实施例2
一种金丝皇菊挥发油提取流程:
S1采用水蒸气蒸馏提取法,分别称取一定量的金丝皇菊花、叶进行超微粉碎,转移至圆底烧瓶中,加入料液比1:10的去离子水,再加入沸石,连接挥发油提取器,并在挥发油提取器中加入乙醚,在回流冷凝管上部连接减压装置,回流冷凝管连接低温恒温循环冷凝器用于冷凝。
S2减压情况下,加热水至沸腾,回流提取3-9小时,收集乙醚层,水层用乙醚萃取两次,合并乙醚层,无水硫酸钠干燥,蒸馏除去乙醚,即得淡黄色金丝皇菊花、叶的挥发油,放入冰箱保存。
较优地,所述步骤S1中,回流冷凝管冷凝所采用的仪器是低温恒温循环冷凝器,冷媒为异丙醇、乙二醇,循环冷凝温度为-10至-40℃。
较优地,所述步骤S2中,减压情况下利于不同沸点的挥发油的提取,所述减压的压力范围为0.04-0.09MPa。
实施例3
提取的金丝皇菊挥发油的液质联用分析:
按照《中华人民共和国国家药典2010版》挥发油测定方法进行化学成分分析采用气相色谱-质谱检测(GC-MS)分析,分析条件如下:
检测仪器:美国安捷伦5975C+7890A气相色谱-质谱联用仪。
色谱条件:HP-5MS弹性石英毛细管柱(0.25μm×30m×0.25mm),程序升温的初始温度为40℃,保留3min,以4℃/min升至160℃,保留3min,以6℃/min升至280℃,保留10min,以高纯氦气(99.99%)为载气,体积流量为1.0mL/min,分流比为50:1,进样量为1.0μL。
质谱条件:电子轰击(EI)离子源,电子能量70eV,溶剂延迟3min,离子源温度230℃,四极杆的温度150℃,全扫描采集模式,质量范围m/z 50~500amu,标准谱库为NIST库。
金丝皇菊花、叶分别提取的挥发油用GC-MS进行了分析,经搜索谱库以及标准样品比对结合人工解析质谱图,首次鉴定了金丝皇菊挥发油中的119个化合物,具体如表2所示:
表2提取制备的金丝皇菊花、金丝皇菊叶挥发油的成分分析结果
实施例4
金丝皇菊提取物的提取方法:
称量一定量的金丝皇菊粉末于圆底烧瓶中,加入5-50倍水或乙醇提取溶剂,回流提取2次,每次0.5~5h,抽滤,合并提取液,除去提取液,干燥,称重。
实施例5
金丝皇菊挥发油及其组合物的α-葡萄糖苷酶活性评价:
微孔板法检测不同浓度下金丝皇菊挥发油、金丝皇菊提取物和金丝皇菊挥发油与提取物的组合物(8:2,v/v)对α-葡萄糖苷酶的抑制活性,以阿卡波糖为阳性对照,方法如下:
化合物测试组(A):30μL底物溶液+10μL待测溶液+20μL酶溶液+140μL 1×磷酸缓冲液(PBS);化合物空白组(B):30μL底物溶液+10μL待测溶液+160μL 1×PBS;对照组(C):30μL底物溶液+20μL酶溶液+10μLDMSO+140μL 1×PBS;空白组(D):30μL底物溶液+170μL 1×PBS。
依次加入1×PBS缓冲液、不同浓度的待测溶液和0.04U/mL的酶溶液,将其置于96孔酶标板中,37℃培育5min后,加入0.5mM 4-硝基苯基-α-D-吡喃葡萄糖苷的底物溶液,37℃恒温箱中反应30min,随后立即于405nm波长下用酶标仪测定吸光值(OD)。磷酸盐缓冲溶液pH值6.8,实验结果为3次独立实验,每次为2个平行。
按下式计算出α-葡萄糖苷酶活性的抑制率:抑制率/%=1-(ODA-ODB)/(ODC-ODD)]×100%。
表3金丝皇菊挥发油、提取物和挥发油与提取物组合物的α-葡萄糖苷酶抑制活性
如表3所示,金丝皇菊花挥发油和金丝皇菊叶挥发油都具有良好的抑制α-葡萄糖苷酶的活性,相同浓度下活性高于阳性对照阿卡波糖。而金丝皇菊乙醇和水的提取物在浓度达到1mg/mL时也没有明显的抑制活性,但是当把挥发油和提取物组合使用时,α-葡萄糖苷酶的活性比单独使用活性有所提高。
实施例6
金丝皇菊挥发油及其组合物的果糖-1,6-二磷酸酯酶的活性评价:
微孔板法检测不同浓度下金丝皇菊挥发油、金丝皇菊提取物和金丝皇菊挥发油与提取物的组合物(8:2,v/v)对果糖-1,6-二磷酸酯酶(FBPase)酶的抑制活性,以单磷酸腺苷(AMP)为阳性对照,方法如下:
化合物测试组(A):30μL底物溶液+10μL待测溶液+10μLNADP++10μL GPI+10μLG-6-PD+10μL FBPase+120μL 1×PBS;化合物空白组(B):10μL待测溶液+10μLNADP++180μL 1×PBS;对照组(C):30μL底物溶液+10μL NADP++10μL GPI+10μLG-6-PD+10μLFBPase+10μLDMSO+120μL 1×PBS;空白组(D):10μLNADP++190μL1×PBS。
依次加入1×PBS缓冲液、不同浓度的待测溶液、NADP+、GPI、G-6-PD和FBPase酶溶液,最后加入底物FBP,将其置于96孔酶标板中,30℃培育30min后立即于340nm波长下用酶标仪测定吸光值(OD)。磷酸盐缓冲溶液pH值7.5,实验结果为3次独立实验,每次为2个平行。
按下式计算出FBPase酶活性的抑制率:抑制率/%=1-(ODA-ODB)/(ODC-ODD)]×100%。
表4金丝皇菊挥发油、提取物和挥发油与提取物组合物的FBPase酶抑制活性
如表4所示,金丝皇菊花挥发油,金丝皇菊叶挥发油都具有抑制FBPase酶的活性,而水或乙醇提取物在测试浓度下没有明显的抑制活性,但是当挥发油和提取物以一定比例组合后,具有一定的抑制活性,这说明通过加入一定比例的提取物可以减少挥发油的添加量并保持相应的抑制活性。由于挥发油比提取物含量少且制备工艺复杂,因此该组合物也有应用前景。
实施例7
金丝皇菊挥发油及其组合物的胰脂肪酶的活性评价:
微孔板法检测不同浓度下金丝皇菊挥发油、金丝皇菊提取物和金丝皇菊挥发油与提取物的组合物(8:2,v/v)对胰脂肪酶的抑制活性,以奥利司他为阳性对照,方法如下:
化合物测试组(A):50μL底物溶液+10μL待测溶液+25μL酶溶液+100μL柠檬酸钠+15μLTris-HCl;化合物空白组(B):50μL底物溶液+10μL待测溶液+100μL柠檬酸钠+40μL Tris-HCl;对照组(C):50μL底物溶液+25μL酶溶液+100μL柠檬酸钠+25μLTris-HCl;空白组(D):50μL底物溶液+100μL柠檬酸钠+50μLTris-HCl。
依次加入Tris-HCl缓冲液、底物溶液和不同浓度的待测溶液,将其置于96孔酶标板中充分混合后加入酶溶液引发反应,25℃培育30min后,每孔加入100μL柠檬酸钠终止反应,随后立即于340nm和460nm发射光波波长下用酶标仪测定吸光值(OD)。Tris-HCl缓冲液pH值8.0,实验结果为3次独立实验,每次为2个平行。
按下式计算出胰脂肪酶活性的抑制率:抑制率/%=1-(ODA-ODB)/(ODC-ODD)]×100%。
表5金丝皇菊挥发油、提取物和挥发油与提取物组合物的胰脂肪酶抑制活性
如图1、表5所示,金丝皇菊花和叶挥发油,以及金丝皇菊提取物在测试浓度下均具有明显的抑制胰脂肪酶的活性,其中金丝皇菊叶挥发油和金丝皇菊乙醇提取物的活性最好,添加20%的金丝皇菊乙醇提取物至挥发油中,可以提高抑制活性,因此,添加金丝皇菊乙醇提取物的组合物,在保持活性前提下可以减少挥发油用量。由于挥发油比提取物含量少且制备工艺复杂,因此该组合物也有应用前景。
实施例8
金丝皇菊挥发油及其组合物的葡萄糖消耗促进活性的评价:
用葡萄糖消耗实验来评价不同浓度的挥发油、提取物和挥发油与提取物组合物的促进葡萄糖消耗的量,从而反应细胞水平降血糖活性。以二甲双胍为阳性对照,具体方法如下:
取对数生长期的HepG2细胞常规消化,用DMEM低糖培养基培养,接种细胞密度为5×104个/mL于96孔培养板中,每孔100μL,置于培养箱中培养过夜,细胞贴壁后弃上清液,用1×PBS洗1次,用无血清的DMEM高糖培养基饥饿24h,用1×PBS洗1次,换用DMEM高糖培养基培养并设立药物对照组(培养基+待测物+细胞),阴性对照组(培养基+DMSO+细胞)和空白对照组(单纯培养基+待测物)+阳性对照组(培养基+二甲双胍+细胞)。每一浓度均设置3个复孔,每孔加入0.5μL含有不同浓度受试物的稀释液,将细胞置于二氧化碳培养箱中继续培养。24h后用葡萄糖测定试剂盒检测相对葡萄糖消耗量。
表6金丝皇菊挥发油、提取物和金丝皇菊挥发油与提取物组合物的葡萄糖消耗活性
由表6得知,金丝皇菊花和叶挥发油、金丝皇菊乙醇提取物和金丝皇菊挥发油与提取的混合物均具有一定的促进葡萄糖消耗的活性,也就是具有一定的降血糖活性,其中金丝皇菊叶挥发油的活性最好,在1μg/mL浓度下就具有显著的活性,比相同浓度下的临床用药二甲双胍活性还强。在5μg/mL和1μg/mL浓度下,金丝皇菊花挥发油、金丝皇菊叶挥发油、金丝皇菊花挥发油与醇提物组合物的促进葡萄糖消耗的活性均比二甲双胍强。
实施例9
金丝皇菊挥发油及其组合物的细胞水平降血脂活性研究:
用油红O染色实验评价不同浓度的挥发油、提取物和挥发油与提取物混合物的细胞水平降低脂肪含量的活性。以洛伐他汀为阳性对照,具体方法如下:
取对数生长期的HepG2细胞,用DMEM低糖培养基以细胞密度为1×105个/mL接种于6孔板中,每孔2mL,置于37℃培养箱中培养过夜,细胞贴壁后弃上清液,用1×PBS洗1次,用无血清的DMEM高糖培养基饥饿24h,用1×PBS洗1次,用配制好的长链脂肪酸(FFAS)诱导剂诱导HepG2细胞建立脂肪蓄积模型24h,并设立药物对照组(诱导剂+待测物+细胞),阴性对照组(诱导剂+DMSO+细胞)和空白对照组(仅含1%BSA的DMEM低糖培养基溶液)+阳性对照组(诱导剂+洛伐他汀+细胞)。每一浓度均设置3个复孔,每孔加入10μL含有不同浓度受试物的稀释液,将细胞置于二氧化碳培养箱中继续培养24h后用1×PBS清洗3次,用4%多聚甲醛每孔2mL固定细胞30min,用1×PBS清洗3次,60%异丙醇每孔2mL作用细胞5min增加细胞的通透性,在黑暗室温条件下用油红O每孔2mL染色1h,蒸馏水清洗细胞4次后每孔加入1mL异丙醇结合10min,震荡洗出,用酶标仪在492nm处测定吸光度。
表7金丝皇菊挥发油、提取物和挥发油与提取物组合物的降低脂肪含量的活性
由表7得知,金丝皇菊挥发油、金丝皇菊提取物和金丝皇菊挥发油与提取的混合物均具有一定的降低细胞脂肪含量的活性。其中,金丝皇菊叶挥发油的活性最高,在0.2μg/mL浓度下仍具有明显的活性,比临床用药洛伐他汀活性还高,相似活性下,金丝皇菊叶挥发油的浓度比洛伐他汀浓度低25倍。
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
Claims (10)
1.一种金丝皇菊挥发油及其组合物在制备治疗糖脂代谢综合紊乱的产品方面中的应用。
2.根据权利要求1所述的应用,其特征在于:所述金丝皇菊挥发油为金丝皇菊花挥发油、或金丝皇菊叶挥发油、或金丝皇菊花挥发油和金丝皇菊叶挥发油的混合物;或者,所述组合物为金丝皇菊花和/或叶的挥发油与金丝皇菊提取物的组合物,其中,金丝皇菊提取物为金丝皇菊醇提物或水提物。
3.根据权利要求2所述的应用,其特征在于:所述金丝皇菊挥发油、金丝皇菊挥发油和提取物的组合物均具有抑制α-葡萄糖苷酶、果糖-1,6-二磷酸酯酶和胰脂肪酶的活性;金丝皇菊花挥发油的0.25mg/mL浓度下对α-葡萄糖苷酶抑制率82.25%,金丝皇菊叶挥发油的0.25mg/mL浓度下α-葡萄糖苷酶抑制率88.81%,两种挥发油对α-葡萄糖苷酶的抑制活性高于相同浓度的阿卡波糖,阿卡波糖的0.25mg/mL浓度下抑制率66.53%,同时也均高于同浓度下金丝皇菊乙醇提取物、金丝皇菊水浸提提取物;
金丝皇菊花挥发油的0.25mg/mL浓度下对果糖-1,6-二磷酸酯酶抑制率62.14%,金丝皇菊叶挥发油0.25mg/mL浓度下对果糖-1,6-二磷酸酯酶抑制率58.02%,两种挥发油对果糖-1,6-二磷酸酯酶的抑制活性均高于同浓度下金丝皇菊水提物,金丝皇菊水提物的0.25mg/mL浓度下抑制率25.48%;金丝皇菊乙醇提取物的0.5mg/mL浓度下对胰脂肪酶抑制率90.08%,其对胰脂肪酶的抑制活性均高于相同浓度下金丝皇菊花挥发油和金丝皇菊叶挥发油,金丝皇菊花挥发油的0.5mg/mL浓度下抑制率68.02%,金丝皇菊叶挥发油的0.5mg/mL浓度下抑制率89.99%;
细胞水平上,金丝皇菊花和/或叶的挥发油以及金丝皇菊挥发油与提取物的组合物均具有细胞水平降血糖和降血脂的活性:在1μg/mL浓度下,金丝皇菊花和/或叶挥发油以及金丝皇菊花和/或叶挥发油与提取物组合物能够促进细胞的葡萄糖消耗量,比相同浓度下的临床用药二甲双胍活性强;在0.2μg/mL浓度下,金丝皇菊叶挥发油以及金丝皇菊叶挥发油与水提物组合物均能够降低细胞脂肪含量,比临床用药洛伐他汀活性高。
5.一种金丝皇菊挥发油及其组合物在制备降血糖、降血脂活性方向的食品和/或保健食品和/或药品方面中的应用。
6.根据权利要求5所述的应用,其特征在于:所述金丝皇菊挥发油为金丝皇菊花挥发油、或金丝皇菊叶挥发油、或金丝皇菊花挥发油和金丝皇菊叶挥发油的混合物;或者,所述组合物为金丝皇菊花和/或叶的挥发油与金丝皇菊提取物的组合物,其中,金丝皇菊提取物为金丝皇菊醇提物或水提物。
7.一种金丝皇菊挥发油的提取方法,其特征在于:步骤如下:
S1:采用水蒸气蒸馏提取法,称取金丝皇菊花和/或叶进行粉碎,转移至容器中,加入料液比1:2~20的水,连接挥发油提取器,并在挥发油提取器中加入乙醚或正己烷或环己烷或二氯甲烷中的一种或多种,在回流冷凝管上部连接减压装置,回流冷凝管连接低温恒温循环冷凝器用于冷凝;
S2:减压情况下,加热水至沸腾,回流提取1~10小时,收集有机相,水层用有机相萃取,合并有机相,干燥,蒸馏除去有机相,即得金丝皇菊花和/或叶的挥发油。
8.根据权利要求7所述的金丝皇菊挥发油的提取方法,其特征在于:所述步骤S1中,采用金丝皇菊花、金丝皇菊叶粉末中一种或两种混合进行提取;
或者,所述步骤S1中,回流冷凝管冷凝所采用的仪器是低温恒温循环冷凝器,冷媒为异丙醇、乙二醇,循环冷凝温度为0℃至-80℃;
或者,所述步骤S2中,所述减压的压力范围为0.01至0.1MPa。
9.一种金丝皇菊提取物的提取方法,其特征在于:步骤如下:
称量金丝皇菊花和/或叶,进行粉碎,将粉末加入5~50倍体积的提取溶剂,回流提取1~5次,每次0.1~12h,抽滤,合并提取液,除去提取液,干燥,即得金丝皇菊提取物。
10.根据权利要求9所述的金丝皇菊提取物的提取方法,其特征在于:所述提取溶剂为水和/或乙醇。
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