CN105920114A - 具有降血糖功能的黑苦荞米提取物及其制备方法 - Google Patents
具有降血糖功能的黑苦荞米提取物及其制备方法 Download PDFInfo
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- CN105920114A CN105920114A CN201610246915.XA CN201610246915A CN105920114A CN 105920114 A CN105920114 A CN 105920114A CN 201610246915 A CN201610246915 A CN 201610246915A CN 105920114 A CN105920114 A CN 105920114A
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- rhizoma fagopyri
- fagopyri tatarici
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- black radix
- tartary buckwheat
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/70—Polygonaceae (Buckwheat family), e.g. spineflower or dock
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/50—Methods involving additional extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
本发明公开了一种具有降血糖功能的黑苦荞米提取物及其制备方法,包括以下步骤:(1)将黑苦荞米粉碎、过20目筛处理后,得到黑苦荞米原料;(2)取得到的黑苦荞米原料,加入料液比为1:20‑1:25、体积分数为50‑60%的乙醇溶液;在150~250W频率下超声波处理10~30min后,继续在40‑75℃条件下,搅拌提取30‑75min后,冷却至室温,将提取液在4℃、3000~6000rpm下冷冻离心10‑30min,收集上清液;(3)取上清液,减压浓缩,回收乙醇,浓缩得到浓缩液;(4)将得到的浓缩液进行液相色谱分离,收集分离液,分离液经真空冷冻干燥得到黑苦荞米提取物。采用本发明的方法制备得到的黑苦荞米提取物主要成分为芦丁,纯度为87‑95%。同时,本发明的黑苦荞米提取物具有较好的降血糖功效。
Description
技术领域
本发明涉及功能食品领域,特别涉及一种具有降血糖功效的黑苦荞米提取物及其制备方法。
背景技术
荞麦属于蓼科(Polygonaceae)荞麦属(Fagopyrium),一年生草本,双子叶植物。荞麦又名三角麦、乌麦,生育期短、耐冷冻瘠薄,是粮食作物中比较理想的填闲补种作物。到目前为止,荞麦属中研究较多的化学成分是黄酮类化合物,该类化合物是荞麦中最重要的生物活性物质之一,广泛存在于荞麦特别是苦荞的花、茎、也和种子中,具有抗氧化、防治冠心病、降低胆固醇等多种保健功能(B,MrázováZ.Prophylacticcomponents of buckwheat[J].Food Research International,2005,38(5):561-568.)。荞麦分为苦荞麦和甜荞麦,苦荞种子和植株中黄酮类化合物的含量均高于甜荞。荞麦黄酮类化合物主要包括芦丁、槲皮素、山奈酚、桑色素、金丝桃苷等化合物,其中芦丁又称芸香苷,维生素P,占苦荞中黄酮总量的75%以上(中国苦荞[M].科学出版社,2009.;李丹.苦荞麦加工与利用的研究:博士学位论文[D].,2001.)。
糖尿病是由多种病因所引起的以高血糖为特征的代谢紊乱性疾病。高血糖主要是由胰岛素分泌不足或其生物功能障碍引起,进而会导致蛋白质、脂肪等代谢紊乱,造成各种组织,特别是眼、心脏、肾、血管、神经的慢性损害及功能衰竭。随着社会经济的快速发展,生活水平的逐渐提高和生活模式的改变,全球糖尿病患者数逐年成上升趋势。糖尿病目前已经成为威胁人类健康的重大疾病,是世界上最普遍和最具挑战性的重大公共卫生问题之一。相关研究表明,黄酮具有体外和体内的降血糖效果(李胜华,伍贤进,曾军英,等.蛇含委陵菜总黄酮的体外和体内降血糖效果研究[J].食品科学,2014,11:049.)。肝脏是糖代谢作用的地点之一,参与糖原的合成,HepG2细胞来源于人体肝组织,具有肝细胞的功能和形态,因而可以采用HepG2细胞模型,来观察黑苦荞米提取物的降糖作用。
发明内容
本发明要解决的技术问题是提供一种具有黑苦荞米提取物的制备方法,采用超声处理、乙醇提取和液相色谱分离制备方法获得高纯度黑苦荞米提取物,经该法制备所得的黑苦荞米提取物具有降血糖活性。
本发明通过以下技术方案实现,提取步骤如下:
(1)将黑苦荞米粉碎、过20目筛处理后,得到黑苦荞米原料;
(2)取步骤(1)得到的黑苦荞米原料,加入料液比为1:20-1:25、体积分数为50-60%的乙醇溶液;在150~250W频率下超声波处理10~30min后,继续在40-75℃条件下,搅拌提取30-75min后,冷却至室温,将提取液在4℃、3000~6000rpm下冷冻离心10-30min,收集上清液;
(3)取步骤(2)的上清液,在40℃条件下减压浓缩,回收乙醇,浓缩至原体积的20-25%,得到浓缩液;
(4)将步骤(3)得到的浓缩液进行液相色谱分离,收集分离液,分离液经真空冷冻干燥得到黑苦荞米提取物。
作为本发明的黑苦荞米黄酮提取方法的改进,步骤(4)所述的液相色谱分离条件为:
色谱柱为Promosil C18(4.6×250mm,5μm),即色谱柱孔内径4.6mm,长度250mm,色谱柱填料颗粒直径5μm,流速为1mL/min,柱温控制为30℃;
流动相由流动相A和流动相B组成,流动相A为0.1%甲酸水溶液,流动相B为乙腈;
采用梯度洗脱方式,洗脱流程为:
0-5min时,流动相B的体积浓度为5%;
5-25min时,流动相B的体积浓度为5-16%;
25-33min时,流动相B的体积浓度为16-30%;
33-35min时,流动相B的体积浓度为30-90%;
35-40min时,流动相B的体积浓度为90%;
40-45min时,流动相B的体积浓度为90-5%;
45-50min时,流动相B的体积浓度为5%;
收集20-30min时间段内的洗脱液,冷冻干燥至恒重后,得到黑苦荞米提取物。
本发明中,所得到的黑苦荞米提取物中有效成分为芦丁,芦丁质量百分比含量为87-95%。
本发明的黑苦荞米提取物具有降血糖功效。可用于制备以高血糖为特征的医学病症的治疗药物或用于制备具有降血糖功能的功能性食品。
作为优选,黑苦荞米提取物的有效降血糖剂量为50μg/ml。
本发明具有以下优点及效果:
1、本发明主要采用超声波辅助乙醇提取,具有环保、方便和高效的优点
2、通过液相色谱制备的方法,可以得到高纯度的苦荞米提取物
3、所得的苦荞米提取物具有较好的降血糖生物活性
附图说明
图1黑苦荞米提取物液相图;
图2为实施例1制备的黑苦荞米提取物提高HepG2细胞对葡萄糖的消耗作用;
图3为实施例2制备的黑苦荞米提取物对α-淀粉酶活性的抑制作用。
具体实施方式
下面结合具体实施例对本发明作进一步描述,以下列举的仅是本发明的具体实施例,但本发明的保护范围并不仅限于此:
本发明的实施例以黑苦荞米为原料。
实施例1
(1)将黑苦荞米粉碎、过20目筛处理后,得到黑苦荞米原料;
(2)取5g步骤(1)得到的黑苦荞米原料,加入125mL食品级50%的乙醇溶液,在150W频率下超声波处理20min;然后在65℃条件下,搅拌提取70min后,冷却至室温,将提取液在4℃、5000rpm下冷冻离心10min,收集上清液;
(3)取步骤(2)的上清液,在40℃条件下减压浓缩,回收乙醇,浓缩至30ml,得到浓缩液;
(4)将步骤(3)得到的浓缩液进行液相色谱分离,收集分离液,分离液经真空冷冻干燥得到黑苦荞米提取物,具体液相色谱分离条件为:
液相色谱仪器为Dionex ultimate 3000,检测器为UV检测器,检测波长为260nm,色谱柱为Promosil C18(4.6×250mm,5μm),进样量为10μl,流速为1mL/min,柱温控制为30℃;
流动相由流动相A和流动相B组成,流动相A为0.1%甲酸水溶液,流动相B为乙腈;
采用梯度洗脱方式,洗脱流程为:
0-5min时,流动相B的体积浓度为5%;
5-25min时,流动相B的体积浓度为5-16%;
25-33min时,流动相B的体积浓度为16-30%;
33-35min时,流动相B的体积浓度为30-90%;
35-40min时,流动相B的体积浓度为90%;
40-45min时,流动相B的体积浓度为90-5%;
45-50min时,流动相B的体积浓度为5%;
收集20-30min时间段内的洗脱液,-60℃冷冻干燥至恒重后,得到黑苦荞米提取物103mg。
经HPLC检测,所得到的黑苦荞米提取物中有效成分为芦丁,芦丁质量百分比含量为95%,见图1。
实施例2
将实施例1步骤(2)中的乙醇浓度由50%改为55%,超声频率由150W改为200W,超声波处理时间由20min改为10min,搅拌提取时间由70min改为75min,离心条件由5000rpm改为3000rpm,离心时间由10min改为30min,其余同实施例1.
最终得到黑苦荞米提取物97mg。
经HPLC检测,所得到的黑苦荞米提取物中有效成分为芦丁,芦丁质量百分比含量为90%。
实施例3
将实施例1步骤(2)中提取温度由65℃改为40℃,超声频率由150W改为250W,超声波处理时间由20min改为30min,搅拌提取时间由70min改为30min,离心条件由5000rpm改为4000rpm,离心时间由10min改为20min,步骤(3)中浓缩体积由500ml改为625ml,其余同实施例1.
最终得到黑苦荞米提取物101mg。
经HPLC检测,所得到的黑苦荞米提取物中有效成分为芦丁,芦丁质量百分比含量93%。
实施例4
将实施例1步骤(2)中乙醇浓度由50%改为60%,超声频率由150W改为250W,离心条件由5000rpm改为6000rpm,离心时间由10min改为15min,,其余同实施例1.
最终得到黑苦荞米提取物118mg。
经HPLC检测,所得到的黑苦荞米提取物中有效成分为芦丁,芦丁质量百分比含量为89%。
实施例5
将实施例1步骤(2)中的乙醇浓度由50%改为60%,超声波处理时间由20min改为15min,提取温度由65℃改为75℃,搅拌提取时间由70min改为60min,其余同实施例1.
最终得到黑苦荞米提取物96mg。
经HPLC检测,所得到的黑苦荞米提取物中有效成分为芦丁,芦丁质量百分比含量为91%。
实施例6
将实施例1步骤(2)中乙醇溶液体积由125mL改为100mL,乙醇浓度由50%改为55%,搅拌提取时间由70min改为65min,步骤(3)中浓缩体积由500ml改为600ml,其余同实施例1.
最终得到黑苦荞米提取物107mg。
经HPLC检测,所得到的黑苦荞米提取物中有效成分为芦丁,芦丁质量百分比含量为87%。
实施例7
将实施例1步骤(2)中中乙醇溶液体积由125mL改为110mL,乙醇浓度由50%改为55%,提取温度由65℃改为60℃,搅拌提取时间由70min改为65min,步骤(3)中浓缩体积由500ml改为450ml,其余同实施例1.
最终得到黑苦荞米提取物109mg。
经HPLC检测,所得到的黑苦荞米提取物中有效成分为芦丁,芦丁质量百分比含量为92%。
性能测试
以实施例1制备的黑苦荞米提取物为样品,测试其降血糖活性
1、HepG2细胞葡萄糖消耗实验
收集对数期的HepG2细胞,将细胞接种到96孔细胞培养板中(浓度为5×103个细胞/孔),孵育24h后;用黑苦荞米提取物处理HepG2细胞24h,然后收集细胞培养液,使用葡萄糖试剂盒测定培养液中的葡萄糖消耗量。按照参考文献的方法,采用MTT法检测细胞的存活率用以矫正数据。将移除培养基后的细胞用MTT(0.5mg/mL)孵育4h。生成的沉淀物溶解于150μL的DMSO中,用酶标仪检测费490nm处的吸光度。实验结果表明(见图2),50μg/ml黑苦荞米提取物可以提升HepG2细胞50%的葡萄糖消耗能力,具有较好的降血糖效果。
以实施例2制备的黑苦荞米提取物为样品,测试其降血糖活性
2、α-淀粉酶活性抑制作用实验
a)500μl黑苦荞米提取物(1mg/ml)和500μl 0.02mol/l磷酸钠缓冲液(含有0.5mg/mlα-淀粉酶和6mmol/l氯化钠,pH6.9)混合,在25℃下反应30分钟;
b)加入500μl的0.02M磷酸钠缓冲液(含0.5%淀粉和6mmol/l氯化钠,pH6.9);
c)加入1ml水杨酸(DNS试剂,含1%3,5-二硝基水杨酸和12%酒石酸钾钠溶于0.4mol/l NaOH).)以终止反应;
d)在沸水条件下水浴10分钟,然后冷却至室温;
e)稀释至15ml,在540nm波长下测定吸光值;
f)以二甲双胍作为阳性对照;
g)抑制率计算公式:抑制率=(1-Asample 540/Acontrol 540)×100%
实验结果表明(见图3)1mg/ml的黑苦荞米提取物对α-淀粉酶的抑制效果与4mg/ml二甲双胍相当。
Claims (6)
1.一种黑苦荞米提取物的制备方法,其特征在于,提取步骤如下:
(1)将黑苦荞米粉碎、过20目筛处理后,得到黑苦荞米原料;
(2)取步骤(1)得到的黑苦荞米原料,加入料液比为1:20-1:25、体积分数为50-60%的乙醇溶液;在150~250W频率下超声波处理10~30min后,继续在40-75℃条件下,搅拌提取30-75min后,冷却至室温,将提取液在4℃、3000~6000rpm下冷冻离心10-30min,收集上清液;
(3)取步骤(2)的上清液,在40℃条件下减压浓缩,回收乙醇,浓缩至原体积的20-25%,得到浓缩液;
(4)将步骤(3)得到的浓缩液进行液相色谱分离,收集分离液,分离液经真空冷冻干燥得到黑苦荞米提取物。
2.根据权利要求1所述的黑苦荞米提取物,其特征在于,步骤(4)所述的液相色谱分离条件为:
色谱柱为Promosil C18(4.6×250mm,5μm),流速为1mL/min,柱温控制为30℃;
流动相由流动相A和流动相B组成,流动相A为0.1%甲酸水溶液,流动相B为乙腈;
采用梯度洗脱方式,洗脱流程为:
0-5min时,流动相B的体积浓度为5%;
5-25min时,流动相B的体积浓度为5-16%;
25-33min时,流动相B的体积浓度为16-30%;
33-35min时,流动相B的体积浓度为30-90%;
35-40min时,流动相B的体积浓度为90%;
40-45min时,流动相B的体积浓度为90-5%;
45-50min时,流动相B的体积浓度为5%;
收集20-30min时间段内的洗脱液,冷冻干燥至恒重后,得到黑苦荞米提取物。
3.一种如权利要求1所述方法制备的黑苦荞米提取物。
4.根据权利要求3所述的黑苦荞米提起物,其特征在于所述的黑苦荞米提取物中的芦丁质量百分比含量为87-95%。
5.一种根据权利要求3所述的黑苦荞米提取物的应用,其特征在于用于制备以高血糖为特征的医学病症的治疗药物或用于制备具有降血糖功能的功能性食品。
6.根据权利要求5所述应用,其特征在于,所述的黑苦荞米提取物的有效降血糖剂量为50μg/ml。
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