CN109222099A - 青刺果果皮和果肉提取物的用途 - Google Patents
青刺果果皮和果肉提取物的用途 Download PDFInfo
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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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)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- 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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- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Obesity (AREA)
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- Organic Chemistry (AREA)
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- Child & Adolescent Psychology (AREA)
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- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
本发明公开了青刺果果皮和果肉提取物在作为胰脂肪酶抑制剂中的应用,属于食品或医药技术领域;本发明以青刺果果皮和果肉为原料,利用有机试剂或水与有机试剂混合液提取,然后经过大孔树脂、凝胶及C18小柱进行纯化和分离,得到青刺果果皮和果肉的不同提取物,本发明经体外胰脂肪酶活性实验显示,所有提取物均能够有效地抑制胰脂肪酶的活性,从而可以有效地抑制胃肠道中脂肪的水解和吸收。因此,本发明可作为脂肪酶抑制剂用以制备降低脂肪吸收的保健食品和药品。
Description
技术领域
本发明涉及青刺果提取物在作为胰脂肪酶抑制剂中的应用,属于食品或医药技术领域。。
背景技术
随着人们物质生活的不断提高,不健康的高脂饮食和不良生活习惯诱发多种慢性疾病的发生。脂质的过多摄入和运动的严重缺乏,导致脂肪沉积在身体各个部位,逐渐演变成肥胖症。目前肥胖症以惊人的速率增长,危及着全世界特别是具有高脂饮食习惯国家人口的健康,已成为一个世界性的健康话题。肥胖与许多慢性疾病有关,如糖尿病、脂肪肝和心血管疾病等,据世界卫生组织数据显示,肥胖的死亡人数远远高于体重轻的人,而发达国家中约有65%的人属于肥胖人群。食物中的脂肪需要经过胃肠道中相应的消化酶的水解后才能够被身体吸收,饮食中70%左右脂肪是由胰脂肪酶进行水解消化。因此,通过抑制胰脂肪酶的活性,降低和延缓餐后脂肪水解和吸收,从而可以有效缓解肥胖和高血脂等健康问题。所以,积极寻找和开发新的药物和保健食品以控制高脂饮食带来的不健康影响至关重要。
目前,市场针对胰脂肪酶的抑制剂主要为奥利司他,这一抑制剂作为单一化合物虽然具有良好的作用效果,但是通常伴随着一定的副作用。目前全世界都在积极寻找新型的胰脂肪酶抑制剂,从而应对日益严峻的高脂饮食所引发的肥胖和高血脂等相关疾病问题。因此,新型胰脂肪酶抑制剂的探寻具有极其重要的意义。
青刺果(PrinsepiautilisRoyle)又名阿纳斯果,属于蔷薇科扁核木属的一种多年生落叶灌木,主要分布在中国的西南部和印度北部高海拔地区。在中国,青刺果主要生长在云南丽江海拔2300米到3200米的高寒冷凉山区,是一种具有药食两用的特色经济作物。目前国内外对青刺果果皮和果肉的研究报道很少,尤其是青刺果果皮和果肉的酚类物质提取物作为胰脂肪酶抑制剂用以降低肠道脂肪吸收的研究尚未有专利文献报道。
发明内容
本发明的目的是提供青刺果果皮和果肉提取物的新用途,用于作为胰脂肪酶的天然抑制剂,并进一步用于制备降低肠道脂肪消化和吸收的药品或保健食品;本发明将为开发安全有效纯天然的胰脂肪酶抑制剂的保健食品或药品提供一定的科学依据。
经实验证实青刺果果皮和果肉不同提取物富含有植物酚类物质,均具有确切的抑制胰脂肪酶活性,能够非常显著地抑制胰脂肪酶的活性,因此可以用于制备和开发为降低肠道脂肪消化吸收的新药或保健食品。青刺果果皮和果肉的酚类物质提取物用于制备胰脂肪酶抑制剂用以降低肠道脂肪消化吸收的药物或保健食品具有以下这些优势:青刺果树对土壤要求较低,能够在较为恶劣的自然环境中生长,不占用粮田,对其附加值的开发可以使农民进一步创收;另外,在云南和印度北部,青刺果常被用作食材,且其种子更是被作为高档植物油的榨油原材料,是一种传统药食两用的果实,安全性高。其酚类物质提取物的提取和制备工艺流程简单、但市场前景可观、蕴含着广阔的市场前景。
本发明中青刺果果皮和果肉提取物的制备工艺如下:
将新鲜青刺果去除核后,将果肉和果皮匀浆;按料液比为1:4-5,在匀浆液中加入甲酸-甲醇混合液(为含有1-2%甲酸的质量浓度75-85%的甲醇溶液),在30-40℃水浴下超声提取25-35min,超声波功率200-300W,过滤得到提取液,滤渣重复提取2-3次,合并提取液,减压浓缩去除溶剂;浓缩液上Amberlite XAD-7HP柱子,用质量浓度1-2%的甲酸水溶液冲洗柱子去除非多酚物质,用甲醇洗柱子,收集甲醇洗脱液浓缩后,冷冻干燥得到总酚组分,为提取物1;取部分总酚组分用pH 7的去离子水溶解样品,上Sephadex LH-20柱子,先用pH 7的去离子水冲洗,然后用甲酸-甲醇混合液(为含5-10%甲酸的质量浓度70-85%甲醇溶液)洗脱样品,收集洗脱液浓缩后,冷冻干燥得到黄酮和花色苷的混合物,将该混合物组分用质量浓度5-10%甲酸水溶液溶解,上样到Sepax Extraction 小柱(SPE)上,首先用质量浓度5-10%甲酸水溶液淋洗,接下来用乙酸乙酯淋洗,收集乙酸乙酯洗脱液得到富含黄酮的组分,为黄酮组分,为提取物2;最后用甲酸-甲醇混合液(为含8-10%甲酸的质量浓度70-85%甲醇溶液)洗脱,得到花色苷组分,为提取物3;分别将黄酮组分和花色苷组分的样品浓缩冻干,放置在-20℃待用。
与现有技术相比,本发明具有如下优点:
1、本发明为青刺果发掘了新的保健食品或医疗用途,开拓了一个新的研究领域;
2、本发明通过实验发现青刺果果皮和果肉的提取物在较低浓度下均可以有效地抑制胰脂肪酶活性;且青刺果是可以用作食物原料的植物果实,安全性高。
附图说明
图1是青刺果果皮和果肉不同提取物对胰脂肪酶的抑制活性。
具体实施方式
下面结合具体实例来进一步地阐述本发明。这些实验的事例仅用于说明本发明,不用于限制本发明的应用范围。在阅读了本发明的记载以后,本领域的科技人员对其等效的各种改动、修改和修饰,都属于本发明权利要求所限定的范围。
实施例1
将新鲜的青刺果(P. utilisRoyle)去除种子后,将果肉和果皮匀浆;破碎后的匀浆液(50g)用甲酸-甲醇混合液(含1%甲酸的质量浓度80%甲醇),料液比g:mL为1:5,水浴超声提取30min,超声波功率210W,超声温度35℃,过滤得到提取液,滤渣重复提取两次,合并提取液,然后在40℃下减压浓缩去除溶剂;浓缩液上Amberlite XAD-7HP柱子,用质量浓度1%的甲酸水溶液冲洗柱子去除非多酚物质,用纯甲醇将样品洗脱下来,收集甲醇洗脱液浓缩后,冷冻干燥得到总酚组分,样品1。取部分总酚组分用pH 7的去离子水溶解样品,上SephadexLH-20柱子,先用pH 7的去离子水冲洗,然后用甲酸-甲醇混合液(含10%甲酸的质量浓度70%甲醇)洗脱样品,收集流出液浓缩后,冷冻干燥得到黄酮和花色苷的混合物;将该混合物组分用质量浓度5%甲酸水溶液溶解,上样到Sepax Extraction 小柱(SPE)上,首先用质量浓度5%甲酸水溶液淋洗,接下来用乙酸乙酯淋洗得到富含黄酮的组分,为黄酮组分样品,样品2。最后,用甲酸-甲醇混合液(10%甲酸的质量浓度70%甲醇)洗脱,得到花色苷组分样品,样品3。分别将黄酮组分和花色苷组分的样品浓缩冻干,放置在-20℃待用。
实施例2
青刺果各组分样品中总酚含量的测定:用质量浓度80 %甲醇溶液溶解冻干的样品,制成1mg/mL的样液,取1 mL加入10 mL离心管后,加入6mL蒸馏水以及0.5mL福林酚,混匀后加入1.5 mL 20% Na2CO3溶液,最后用蒸馏水将整个反应体系定容至10 mL; 70℃水浴(10min)后恢复至室温,用酶标仪测量765 nm处的吸光值。以没食子酸为当量作标准曲线,样品的总酚含量表示为:mg没食子酸/g提取物干重,结果如表1所示;
青刺果各组分样品中总黄酮含量:用70 %的乙醇溶解冻干的提取物,取1 mL样液于离心管,并加入2.5 mL 70 %乙醇,再依次向离心管中加入0.15 mL 5% NaNO2、0.15 mL 10 %Al(NO3)3,摇匀;再加入1mol/L NaOH 1 mL,然后用70 %乙醇定容至5 mL,混匀后室温避光静置30 min;用酶标仪测定510 nm处的吸光值,标准曲线以芦丁为当量,样品的TFC表示为:mg芦丁/ g提取物干重,结果如表1所示。
采用pH示差法测定花色苷含量:首先配制氯化钾缓冲液(0.03 mol/L),用1mol/L的HCl调节pH至1.0;配制乙酸钠缓冲液(0.4 mol/L),用乙酸调节pH至4.5,用80%甲醇溶解样品,制成1.0 mg/mL,取1 mL样液分别加入9 mL上述两种缓冲液,用紫外分光光度计,分别在520 nm、700 nm处测定吸光值,以1 mL水分和9 mL缓冲液为空白,做三个平行。花色苷的含量 (mg/g干重)=浓度C(mg/L)= (A×Mw×DF×103)/(ε×l),其中A为吸光值等于(A520 -A700) pH1.0 - (A520 - A700) pH4.5,花青素-3-葡萄糖为当量,分子量Mw为449.2 g/mol,DF为稀释系数即10,ε为消光系数26900,l为路径长度1 cm,结果如表1所示;
表1青刺果各组分中总酚、总黄酮、花色苷含量(mg/g)
。
实施例3
采用UHPLC-ESI- MS对青刺果各组分中化合物组成分析,具体操作如下:液相的色谱条件为Reprospher 100 C18分析柱(100 × 2 mm,1.8 μm,),流动相为2.0 %甲酸水(A)和质谱级乙腈(B),较为适宜的流动相梯度为:0.01–5 min, 10% B; 5–8 min, 10%–15% B, 8–15 min, 15%–25% B, 15–20 min, 25%–10% B, 20–23 min, 10% B。流速为0.2 mL/min,进样量为2.0 µL,柱温设为35℃。质谱条件为:为全扫描(150~1000 m/z);毛细管温度为320℃;电离电压为3.3 kV;辅助气流流速为8 L/min;鞘气流速为32 L/min;加热器温度为350℃。总酚、黄酮类在负离子模式下检出,花色苷在正离子模式下检出,主要酚类物质的检测结果如表2中所示;
表2 青刺果果皮和果肉中主要酚类物质组成的鉴定
。
实施例4:青刺果果实各提取物对胰脂肪酶的抑制活性测定
猪胰脂肪酶用蒸馏水配成15mg/mL,10000 rpm离心5分钟后,取上清液;在5mmoL/L的乙酸钠缓冲溶液中加入1%的曲拉X-100和1%的对硝基苯月桂酸酯配成反应底物;棕榈果各提取物用DMSO溶解,用Tris缓冲溶液稀释成适当浓度(见图1);样品组加入100μL提取物溶液,400μL Tris缓冲溶液,350μL底物反应液和150μL酶液,空白对照组则加入100μL的样品溶剂;各组混匀后于37℃孵育2小时,孵育结束,10000 rpm离心5分钟后取上清液,于400nm处测定各组吸光值。胰脂肪酶抑制率(%)=(ODcontrol-ODsample)/ODcontrol×100,其中ODsample为样品组的吸光值,ODcontrol为空白对照组的吸光值,结果如图1所示;青刺果果实各提取物对胰脂肪酶的抑制作用的检测结果表明,总酚组分、黄酮组分和花色苷组分对胰脂肪酶的抑制活性IC50值分别为:423.15 ± 11.11 μg/mL、439.99 ± 1.08 μg/mL和407.43 ± 33.89 μg/mL。本结果表明各提取组分对胰脂肪酶均有良好的抑制活性。不同组分提取物随着浓度的增加,对胰脂肪酶的抑制率逐渐增强。
综上所述,可以确定青刺果果皮和果肉的不同酚类物质提取物富含有植物多酚,均可以有效地抑制胰脂肪酶的活性。从而可以确定青刺果果皮和果肉的不同酚类物质提取物可以用于制备降低肠道脂肪消化吸收的药品或保健食品。
Claims (5)
1.青刺果果皮和果肉提取物在作为胰脂肪酶抑制剂中的应用。
2.根据权利要求1所述的应用,其特征在于:青刺果果皮和果肉提取物是将新鲜青刺果去除核后,将果肉和果皮匀浆;按料液比为1:4-5的比例,在匀浆液中加入甲酸-甲醇混合液,在30-40℃水浴下超声提取25-35min,超声波功率200-300W,过滤得到提取液,滤渣重复提取2-3次,合并提取液,减压浓缩去除溶剂;浓缩液上Amberlite XAD-7HP柱子,用质量浓度1-2%的甲酸水溶液冲洗柱子去除非多酚物质,用甲醇洗柱子,收集甲醇洗脱液浓缩后,冷冻干燥得到总酚组分,为提取物1;取部分总酚组分用pH 7的去离子水溶解样品,上Sephadex LH-20柱子,先用pH 7的去离子水冲洗,然后用甲酸-甲醇混合液洗脱样品,收集洗脱液浓缩后,冷冻干燥得到黄酮和花色苷的混合物,将该混合物组分用质量浓度5-10%甲酸水溶液溶解,上样到Sepax Extraction 小柱上,首先用质量浓度5-10%甲酸水溶液淋洗,接下来用乙酸乙酯淋洗,收集乙酸乙酯洗脱液得到富含黄酮的组分,为黄酮组分,作为提取物2;最后用甲酸-甲醇混合液洗脱,得到花色苷组分,为提取物3;浓缩冻干待用。
3.根据权利要求2所述的应用,其特征在于:超声提取时用的甲酸-甲醇混合液为含有1-2%甲酸的质量浓度75-85%的甲醇溶液。
4.根据权利要求2所述的应用,其特征在于:洗脱Sephadex LH-20柱时用的甲酸-甲醇混合液为含5-10%甲酸的质量浓度70-85%甲醇溶液。
5.根据权利要求2所述的应用,其特征在于:洗脱花色苷组分时用的甲酸-甲醇混合液为含8-10%甲酸的质量浓度70-85%甲醇溶液。
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