CN113016844B - 一种具有咖啡风味及保肝的酸枣仁提取物及其制备方法和应用 - Google Patents
一种具有咖啡风味及保肝的酸枣仁提取物及其制备方法和应用 Download PDFInfo
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- CN113016844B CN113016844B CN202110276775.1A CN202110276775A CN113016844B CN 113016844 B CN113016844 B CN 113016844B CN 202110276775 A CN202110276775 A CN 202110276775A CN 113016844 B CN113016844 B CN 113016844B
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- wild jujube
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Abstract
本发明属于酸枣仁提取技术领域,提供了一种具有咖啡风味及保肝的酸枣仁提取物及其制备方法和应用。采用炒制的方式制备具有咖啡风味的酸枣仁提取物,制备方法包括以下步骤:炒制,粉碎,浸泡,提取,过滤,干燥。采用GC‑MS方法分析炒制酸枣仁成分,表明炒制后酸枣仁具有8种与咖啡一致成分,采用感官评价的方式评价炒制酸枣仁及其提取物,结果提示其具有明显的咖啡风味。本发明所涉及的酸枣仁提取物的制备方法能使保肝功效物质最大化的保留富集,有效的改善急性肝损伤,增强酸枣仁提取物的保肝作用,显著提高酸枣仁提取物的咖啡风味。本发明也可在制备食品、保健品及制药领域中应用。
Description
技术领域
本发明属于酸枣仁提取技术领域,具体涉及一种具有咖啡风味及保肝的酸枣仁提取物及其制备方法和应用;该酸枣仁提取物在食品,保健品及医药领域中的应用。
背景技术
肝脏是人体重要的脏器,是人体内重要的代谢通路。肝脏疾病一直是医学上研究的重点对象之一,临床上常见肝脏疾病有慢性肝炎,乙肝,肝癌,酒精肝等。每年患肝脏疾病的人数不断攀升,对人类的健康造成一定威胁。肝损伤是多种肝疾病的共同病理状态,导致病变的因素主要有药物、酒精、病毒等,肝损伤造成肝排毒不畅,机体代谢失调等病理表现。临床治疗肝损伤的大多数药物存在有效性差、不良反应多等问题,故研发疗效确切、安全性高的药物尤为重要。
酸枣为鼠李科植物,是一种野生资源,广泛分布于我国北方地区,其成熟种子为酸枣仁。酸枣仁味苷、酸、平,归肝、心、胆,具有补肝,宁心,敛汗、生津等功效。酸枣仁主要成分为皂苷类、黄酮类,生物碱类成分。酸枣仁作为临床常用药,因其具有镇静催眠的作用,常用来治疗失眠症,也有研究表明酸枣仁具有保肝作用。现代研究发现黄酮类成分和皂苷类成分可能为保肝作用的主要成分(李国栋等.野菊花中萜类和黄酮类化合物保肝作用研究[J]. 中草药,2013,44(24):3510-3514.)、(张华锋,刘炯,张杰,杨云,卫冰,王启帅.基于聚类分析和典型相关分析的北柴胡保肝作用谱效关系研究[J]. 中草药,2013,44(19):2696-2702.)。
研究发现烘焙咖啡的主要成分同样具有大量挥发性成分、脂肪酸类成分,包括油酸,亚油酸,硬脂酸、棕榈酸、肉豆蔻酸,β-谷甾醇、胆甾醇、花生酸等(邱明华,张枝润,李忠荣,周琳,储瑞,刘接卿,王卫华.咖啡化学成分与健康[J]. 植物科学学报,2014,32(05):540-550.)。酸枣仁中同样具有有大量的脂肪酸和挥发性成分,与咖啡中化学成分一定程度类似。
发明内容
本发明提供了一种具有咖啡风味及保肝的酸枣仁提取物及其制备方法和应用。
本发明由如下技术方案实现:即一种具有咖啡风味及保肝的酸枣仁提取物的制备方法,具体步骤如下:
(1)干燥酸枣仁:酸枣仁30-60℃加热干燥15-60 min,取出摊放于温度为20-25℃、湿度为44%-75%的环境内,酸枣仁冷却至18-25℃,得到水分含量为7%-15%的干燥酸枣仁;
(2)炒制酸枣仁:干燥酸枣仁平铺于电热恒温油浴锅或多功能炒制锅内,控制炒制锅的温度为130-300℃,锅内温度为100-250℃,锅内温度升温后稳定1-10min,进行炒制酸枣仁,每隔10-40s翻炒一次,炒制时间为10-40min;炒至酸枣仁鼓起,表皮颜色由微黄色变至焦黑色,有咖啡香气外溢,停止炒制,取出自然晾凉;
(3)酸枣仁水提液的制备:步骤(2)中炒制的酸枣仁粉碎至10-50目,加入酸枣仁粉重量的1-16倍的蒸馏水,浸泡0.5-5h,然后加热回流提取或超声提取或浸提1-2h,过滤,滤液备用,残渣中继续加入残渣重量的1-16倍的蒸馏水,继续浸泡、提取,提取1-4次后,滤液合并,即为酸枣仁水提液,酸枣仁水提液的浓度为0.01-4 g/mL;
(4)酸枣仁提取物干燥:酸枣仁水提物-20至-60℃、0-20pa冷冻干燥0.5-48h,即为酸枣仁提取物。
本发明还提供了利用上述方法制备的一种具有咖啡风味及保肝的酸枣仁提取物。
另外,本发明还提供了所述具有咖啡风味及保肝的酸枣仁提取物在制备食品中的应用,所述食品包含咖啡、饮料,饼干,面包。所述具有咖啡风味及保肝的酸枣仁提取物在制备保肝保健品中的应用,所述保健品包括保肝茶,保肝凝胶软糖。所述具有咖啡风味及保肝的酸枣仁提取物在制备保肝药物中的应用,所述保肝药物包括,颗粒剂,丸剂,片剂,胶囊。
本发明所制备的酸枣仁提取物经检测评价,色泽为颜色深浅,为棕黄-焦黑;所述酸枣仁溶液为咖啡味香程度,为微香-焦香;口感为苦涩程度、是否有酸枣仁特殊风味、口感优劣为微苦涩味,口感较好;溶液颜色为浅棕—深褐;溶液澄清程度为澄清-浑浊分层。
本发明采用GC-MS的方法分析炒酸枣仁成分,表明有8种化学成分与咖啡的成分共有,分别为棕榈酸,亚油酸,油酸,硬脂酸,肉豆蔻酸,豆甾醇,二十烷酸,β-谷甾醇等,提示炒酸枣仁可能具有咖啡的风味。
采用HPLC-DAD-ELSD的测定方法对生、炒后酸枣仁提取物的潜在药效成分进行定量分析,分别为乌药碱、木兰花碱、斯皮诺素、维采宁、、酸枣仁皂苷A、酸枣仁皂苷B,炒制后成分发生显著变化,含量显著升高,提示主要有效成分含量升高可能显著增强酸枣仁的保肝作用。
采用肝损伤小鼠动物模型评价具有咖啡风味的酸枣仁提取物的保肝作用。评价指标为血清中ALT和肝脏中MDA的含量、SOD活力及肝组织病理切片。给药14天后,小鼠空白组,模型组,低剂量酸枣仁提取物给药组,高剂量酸枣仁提取物给药组中ALT、SOD、MDA的含量分别为82.45±18.56,185.13±25.23,158.78±23.45,154.46±26.12;120.34±27.46,90.22±36.18,128.34±25.79,130.18±36.34;3.46±1.12;9.39±1.51,4.45±1.87,4.16±1.59;与空白组相比,模型组SOD活力显著降低,ALT、MDA含量显著升高,表明肝损伤模型成立。给药组相比模型组SOD活力显著升高;ALT、MDA含量显著降低。肝组织病理切片的结果为空白组颜色正常,红润且有光泽,显微镜下可见肝组织结构完整,肝细胞形态正常,组织未见硬化或变性、坏死状况,基本无炎性细胞浸润;模型组肝组织出现点状坏死,肝细胞胞质疏松,并伴有大量的炎性细胞浸润;给药组肝组织颜色红润,肝细胞病变明显减轻,仅少量肝细胞胞质疏松或呈空泡状,无明显肝细胞坏死、炎性细胞浸润和淤血。三个检测指标的变化及肝组织病理切片的结果说明酸枣仁提取物能有效改善小鼠急性肝损伤,具有明显的保肝作用。
本发明所述的酸枣仁提取物可以被制成咖啡,饮料,饼干,面包等;保肝茶、保肝凝胶糖果等;颗粒剂,丸剂,片剂,胶囊等。
本发明制备的酸枣仁提取物具有以下优点和效果:本发明制备工艺可使保肝功效物质最大化的保留富集,增强酸枣仁提取物的保肝作用。对炒制后的酸枣仁提取物进行感官评价,得到色泽均匀,口感较好,稳定性较好。本发明同时对酸枣仁提取物的保肝作用进行评价,采用小鼠急性肝损伤模型进行评价,以血清中ALT含量和肝脏中MDA含量、SOD活力及肝组织病理切片结果为评价指标。结果均显示炒制酸枣仁提取物,能显著改善急性肝损伤,丰富了酸枣仁提取物的应用范围,有利于促进酸枣仁相关产业发展。为市场提供具有咖啡风味兼具有保肝作用的酸枣仁提取物,满足消费者日益差异化的消费需求。
本发明采用酸枣仁作为开发对象,开发一种既具有咖啡风味又能增强保肝作用的酸枣仁新产品,扩大酸枣仁的应用范围,不仅在临床上治疗肝损伤有更好的选择,在功能食品市场上也具有独特优势。
附图说明
图1为保肝生化指标和酸枣仁主要成分的相关性分析;图中:V维采宁,S斯皮诺素,F, JA酸枣仁皂苷A, JB酸枣仁皂苷B,M木兰花碱,C乌药碱;
图2为实验例3中各组肝组织病理切片结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明的一部分实施例,而不是全部的实施例;基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法;下述实施例中所用的试剂、材料等,如无特殊说明,均可从商业途径得到。
实施例1:一种具有咖啡风味及保肝的酸枣仁提取物的制备方法,具体步骤如下:
取酸枣仁,50℃加热干燥,干燥20 min,取出放凉至20℃,控制室温为25℃,湿度为55%,测得酸枣仁的水分含量为18%;将酸枣仁平铺于锅底,将酸枣仁140℃炒制10 min,隔30s翻炒一次,得到的酸枣仁棕黄色,有淡淡的咖啡香气。然后进行粉碎,过10目筛,用14倍量蒸馏水浸泡0.5 h,加热回流提取1 h,提取2次,过滤,得到浓度为0.5g/ml的酸枣仁水提物;将酸枣仁水提物于-60℃,20 pa下进行冷冻干燥24 h,得炒酸枣仁提取物并于4℃保存。
对得到的炒制后酸枣仁提取物进行感官评价,此条件下的酸枣仁提取物色泽均匀呈浅棕色;咖啡香较淡,略有酸枣仁特有烘焙香;其溶液形态呈多层分层现象,浑浊;色泽成浅棕色;风味单一,无咖啡焦苦味;口感较差,余味短;所得酸枣仁提取物感官评分为60分。此制备条件下得到的酸枣仁提取物,测得的成分含量见表3,结果为炒制后的成分含量显著升高。
实施例2:一种具有咖啡风味及保肝的酸枣仁提取物的制备方法,具体步骤如下:
取酸枣仁,60℃加热干燥,干燥15min,取出放凉至18℃,控制室温为25℃,湿度为75%,测得酸枣仁的水分含量为7%;将酸枣仁平铺于锅底,酸枣仁250℃炒制25min ,隔10s翻炒一次,得到的酸枣仁棕褐色,有较浓咖啡香。然后进行粉碎,过20目筛,用1倍量蒸馏水浸泡1 h,加热回流提取2 h,提取4次,过滤,得到浓度为4g/ml的酸枣仁水提物;将酸枣仁水提物于-40℃,10 pa下进行冷冻干燥0.5 h,得酸枣仁提取物并于4℃保存。
对得到的炒制后的酸枣仁提取物进行感官评价,此条件下的酸枣仁提取物色泽均匀呈棕褐色;咖啡香较香,有酸枣仁特有烘焙香;其溶液形态稳定,底部无沉淀;色泽成棕褐色;风味协调,焦苦味适宜;口感较好,余味悠长;所得酸枣仁提取物感官评分为90分。此制备条件下得到的酸枣仁提取物,测得的成分含量见表3,结果为炒制后的成分含量显著升高。
实施例3:一种具有咖啡风味及保肝的酸枣仁提取物的制备方法,具体步骤如下:
取酸枣仁,30℃加热干燥,干燥60 min,取出放凉至25℃,控制室温为25℃,湿度为44%,测得酸枣仁的水分含量为20%;将酸枣仁平铺于锅底,酸枣仁100℃炒制40 min,隔40s翻炒一次,得到的酸枣仁棕褐色,有较浓咖啡香。然后进行粉碎,过50目筛,用16倍量蒸馏水浸泡5 h,加热回流提取1.5 h,提取1次,过滤,得到浓度为0.01g/ml的酸枣仁水提物;将酸枣仁水提物于-20℃,0 pa下进行冷冻干燥48 h,得酸枣仁提取物。
对得到的炒制后酸枣仁提取物进行感官评价,此条件下的酸枣仁提取物色泽均匀呈棕褐色;有较浓咖啡香味,有酸枣仁特有烘焙香;其溶液形态稳定,色泽呈棕褐色;风味协调,咖啡焦苦味较淡;口感较好,余味长;所得酸枣仁水提物冻干粉感官评分为80分。此制备条件下得到的酸枣仁提取物,测得的成分含量见表3,结果为炒制后的成分含量显著升高。
实施例4:取实施例1所得酸枣仁提取物,加入南瓜粉,山药粉,鸡蛋,植物油,淡奶粉,蛋白糖等辅料,均与酸枣仁提取物等重量配比混合,加入适量的纯净水,220℃烘烤1-2h,即得饼干。
实施例5:取实施例2所得酸枣仁提取物10 g,分别取小麦粉,大麦粉,莜麦粉,燕麦片,荞麦粉,小米面,玉米面,大豆面,绿豆面各5g混合为辅料;所述辅料由糖与酸枣仁提取物质量比为1:0.2-0.3,奶油与酸枣仁提取物的质量比为1:0.1-0.2,鸡蛋与酸枣仁的提取物的质量比为1:0.3-0.4,果料与酸枣仁的提取物的质量比为1:0.2-0.3。水,啤酒花发酵液和辅料经混合后加热发酵3-4 h,220℃烘烤1-2 h,即得面包。
实施例6:取实施例3所得酸枣仁提取物10 g,加蒸馏水稀释,取体积比为30%的稀释液,加入白砂糖量为加入酸枣仁提取物质量的0.5%、柠檬酸量为加入酸枣仁提取物质量的0.1%、膳食纤维量为加入酸枣仁提取物质量的0.1%、精制卡拉胶为加入酸枣仁提取物质量的0.2%等混匀即得饮料。
实施例7:取实施例1所得酸枣仁提取物0.5-2.0 g,咖啡4-15 g,甜味剂4-9 g,奶粉2-6 g,混合即得咖啡。
实施例8:取实施例1酸枣仁提取物20 g,另取酸枣叶干燥,粉碎,两者配比为2:1,混匀,装袋封口即得保肝茶。
实施例9:取酸枣仁提取物,加入凝胶,白糖,柠檬酸等,加入量分别为酸枣仁提取物质量的5倍,3倍,0.1倍,加热混匀,放凉,即得保肝凝胶糖果。
实施例10:取酸枣仁提取物,加2倍量糊精,混合均匀,制粒,过筛,干燥,即得酸枣仁提取物颗粒剂。
实施例11:取酸枣仁提取物,加2倍量糊精,混合均匀,制粒,过筛,干燥,将制得的颗粒经进一步压片,干燥,即得酸枣仁水提物冻干粉片剂。
实施例12:取酸枣仁提取物10 g,加入纤维素-水羟硅钠石复合气凝胶粉末,占酸枣仁提取物质量的0.1%,搅拌均匀,干燥,加入聚乙烯吡咯烷酮醇液制备成母核,占酸枣仁提取物质量的0.2%;再加入糊精、聚乙烯吡咯烷酮醇液进行包衣,分别占酸枣仁提取物质量的10%、0.2%,制备成丸剂,即得酸枣仁提取物丸剂。
实施例13:取酸枣仁提取物10 g,加入相当于2倍量的酸枣仁提取物的微粉硅胶,混合均匀,采用胶囊制备工艺制备,即得酸枣仁提取物胶囊。
实验例1:采用GC-MS代谢组学技术分析炒酸枣仁成分,成分分析说明炒制后酸枣仁成分与咖啡风味物质一致,从而也体现出本发明所制备的酸枣仁水提物的咖啡风味。
1、实验仪器、材料:GC-MS联用仪(Agilent 5977 B气质联用仪,G4513A自动进样器,四级杆质量分析器);HP-5MS毛细管柱(30 m×0.25 mm×0.25 μm);电子天平(FA3204B,上海精科天美科学仪器有限公司);恒温振荡金属浴(型号:Mixing Block MB-102);旋转蒸发仪(RE-52,上海亚荣生化仪器厂)。
2、试剂:TMCS(双(三甲基甲硅烷基)三氟乙酰胺+ 1%三甲基氯硅烷)购自RegisTechnologies(美国莫顿格罗夫),石油醚、正己烷为分析纯。
3、实验方法
(1)样品制备:精密称定炒酸枣仁粉末2g,取6份,置于索氏提取器中,加石油醚50mL浸泡过夜,再加入40mL,90℃加热回流4 h,石油醚提取液加无水硫酸镁吸水,过滤,减压回收石油醚,得酸枣仁脂肪油。取50 μL脂肪油加入TMCS(三甲基氯硅烷)衍生化试剂150μL,放入恒温振荡金属浴中以温度为90℃,转速300 r/min,衍生1.5 h,过0.22 µm微孔滤膜,得GC-MS供试品溶液。从每份供式品溶液中取50 μL,制备QC样品。
(2)分析条件:气相色谱条件:HP-5毛细管柱(30 m×0.25 mm×0.25 μm),载气为高纯氦气,流速为1.0 mL/min,分流比为100:1,进样量为0.2 μL,进样口温度250℃,传输线温度280℃,程序升温:起始温度150℃,保持1 min,然后以5℃ /min升至215℃,保持18min,之后以5℃/min升至240℃,保持6 min,然后以10℃/min升至270℃保持13 min。
质谱条件:EI电离70 eV,离子源温度230℃,四级杆温度150℃,扫描范围50-500m/ z,溶剂延迟3 min。
(3)统计方法:GC-MS采集的质谱数据以XML的文件格式输出,然后采用R软件xcms程序包进行去卷积,峰匹配和峰积分等。
4、实验结果
表1为GC-MS代谢组学技术分析炒酸枣仁成分。共鉴定出13种成分,其中有8种成分是与咖啡的共有成分,分别为棕榈酸,亚油酸,油酸,硬脂酸,肉豆蔻酸,豆甾醇、二十烷酸、β-谷甾醇等,提示可能炒酸枣仁中具有咖啡的风味。
表1 GC-MS测定炒酸枣仁成分
(注:其中2,3,5,6,9,11,12,13为炒酸枣仁与咖啡的共有成分。)
实验例2:有研究表明(闫艳等. 酸枣仁与理枣仁的研究进展及质量标志物预测分析 [J]. 中草药, 2019.)酸枣仁皂苷A、酸枣仁皂苷B、斯皮诺素、、维采宁、乌药碱、木兰花碱可作为酸枣仁潜在质量标志物,且以上几种成分在酸枣仁中的含量较高,故选取该7个主要成分进行含量测定,分析生、炒后酸枣仁提取物的7种成分含量差异,并以此为基础说明炒制后酸枣仁提取物对保肝作用的影响。
1、实验材料仪器:对照品:乌药碱(批号HC225036198)、木兰花碱(批号20160710)、维采宁Ⅱ(批号HV187847198)、斯皮诺素(批号20160314)、(批号20160303)、酸枣仁皂苷A(批号20160315)均购于宝鸡市辰光生物科技有限公司,酸枣仁皂苷B(批号20170210)购于春秋生物工程有限公司(南京)。所有对照品经 HPLC 测定质量分数均大于98%;色谱级甲醇、乙腈购自美国 Fisher 公司;其他试剂均为分析纯。
Waters e 2695高效液相色谱仪,配自动进样器、四元梯度泵、柱温箱、UV检测器以及Empower色谱工作站(美国Waters公司);ELSD 6000蒸发光散射检测器(美国奥泰公司);电子天平(FA3204B,上海精科天美科学仪器有限公司)。
2、实验方法
(1)对照品溶液的制备:精密称取乌药碱、维采宁II、、酸枣仁皂苷A和酸枣仁皂苷B对照品适量,各自加70%甲醇水制成浓度为1 mg/mL的母液;分别称取木兰花碱和斯皮诺素,加70%甲醇水制成2 mg/mL对照品母液。精密吸取各成分对照品母液,加70%甲醇水配制为含乌药碱80 μg/mL、木兰花碱320 μg/mL、维采宁Ⅱ 20 μg/mL、斯皮诺素200 μg/mL、/>180 μg/mL、酸枣仁皂苷A 200 μg/mL和酸枣仁皂苷B100 μg/mL的混合对照品工作溶液。精密量取上述混合对照品工作溶液适量,用70%甲醇水逐级稀释成7个浓度梯度的标准曲线工作溶液。
(2)供试品溶液的制备:精密称定不同制备条件下生、炒后酸枣仁提取物粉末2 g,生酸枣仁提取物按照实施例1-3所对应步骤制备(去掉炒制步骤);置于索氏提取器中,加石油醚50 mL浸泡过夜,再加入40 mL,90℃加热回流4 h,药渣挥去溶剂转移至圆底烧瓶,加50mL70%乙醇回流提取2 h,过滤,药渣用70%乙醇5 mL洗涤,合并滤液并挥干,甲醇复溶并定容至10 mL,过0.22微孔滤膜即得HPLC-DAD-ELSD供试品溶液。
(3)分析条件:色谱柱为Apollo C18柱(250 mm × 4.6 mm,5 μm);流动相为0.1%甲酸水(A)和乙腈(B),梯度洗脱:0~26 min,10%~20% B;26~30 min,20%~23% B;30~43min,23%~26% B;43~45 min,26%~37% B;45~47 min,37% B;47~54 min,37%~39% B;54~63 min,39%~100% B,流速为1.0 mL/min;柱温25 ℃,进样量10 μl;紫外检测波长为227 nm和335 nm;ELSD参数:漂移管温度105 ℃,空气体积流量为2.5 L/min。采用227、335nm和ELSD检测器同时进行测定。
3.实验结果:乌药碱、木兰花碱、维采宁II、斯皮诺素、、酸枣仁皂苷A和酸枣仁皂苷B分别按照分析条件进行分析,建立标准曲线,结果见表2。各标准曲线的相关系数(R 2)值均大于0.99。
表2:7种成分的回归方程、相关系数、线性范围
含量测定结果见表3,由表3可知炒制后以上几种成分含量显著升高,结果说明炒制酸枣仁主要成分含量发生显著变化,提示可能对保肝作用有一定影响。
表3:实施例1-3中在不同制备条件下生炒后酸枣仁提取物中7种成分的含量测定
(注:Z为实施例1酸枣仁提取物,F为实施例1炒制后酸枣仁提取物;2Z为实施例2酸枣仁提取物,2F为实施例2;3Z为实施例3酸枣仁提取物,3F为实施例3炒制后酸枣仁提取物。炒制后酸枣仁提取物与生酸枣仁相比,**P<0.01 ***P<0.001)
实验例3:酸枣仁提取物动物实验
1、实验材料:生理盐水,四氯化碳,大豆调和油,ALT(谷丙转氨酶),超氧化物歧化酶(SOD)、丙二醛(MDA)试剂盒;
健康ICR小鼠(18-22 g)84只,雌雄各半,购买回来先饲养7天,昼夜交替,室温25~28℃,相对湿度55%~65%,环境安静,通风,自由饮水饮食。随机分为4组,每组12只。
2.实验方法
A、造模及给药:小鼠随机分为4组,每组12只,分别为空白组,模型组(CCl4),低剂量给药组,高剂量给药组。给药组灌胃给药,低剂量组按10 g/kg给药,高剂量组按20 g/kg给药。每天1次,按0.2ml/10g连续灌胃14天,空白组与模型组给等体积的蒸馏水,末次给药后禁食不禁水,1h后除空白组腹腔注射等体积的植物油(共需3 mL)外,其余各组均腹腔注射0.1%的CCl4植物油溶液,注射量为0.1 ml/10g,造模时间为16 h。
B、检查指标:ALT,SOD,MDA等指标的含量测定。
C、肝组织病理切片:将肝组织用4%多聚甲醛固定,用石蜡进行包埋,切片,HE染色,光镜下观察肝组织切片的病理形态学变化。
D、统计学处理:该实验采用统计软件进行数据处理,结果以均值加减标准差表示,P<0.05表示有统计学意义。
E、实验结果:实施例1-3所提取得到酸枣仁提取物灌胃小鼠14天测得血清中ALT和肝脏中MDA含量、SOD活力结果见表4,肝脏病理切片结果见图2。
表4 实施例1-3血清ALT和肝脏MDA含量、SOD活力
与模型组相比,*P<0.05,**P<0.1,***P<0.001;模型与空白组相比,# P<0.05,## P<0.1,###P<0.001。
结果显示:模型组与空白组相比ALT、MDA显著升高,SOD显著降低,模型成立;与模型组相比给药组显著降ALT、MDA含量,与模型组相比给药组SOD活力降低,但无差异。肝脏病理切片结果见图2,模型组与空白组相比,肝组织出现严重病变及坏死,与模型组相比给药组明显减轻肝组织损伤程度。
实验例4:将实验例2所测7种成分与实验例3保肝生化指标ALT、MDA、SOD进行相关性分析。生化指标MDA和ALT与保肝作用呈负相关,MDA和 ALT含量越低,保肝作用越强;SOD与保肝作用呈正相关,SOD含量越高,保肝作用越强。
所得结果如图1所示,结合相关系数分析MDA与斯皮诺素(0.9)、(0.9)、酸枣仁皂苷A(0.9)相关性较强,呈负相关,说明黄酮类和皂苷成分可能具有保肝作用;ALT与斯皮诺素(0.8)、/>(0.9)、酸枣仁皂苷A(0.9)相关性较强,呈负相关,说明黄酮类和皂苷成分可能具有保肝作用。SOD与斯皮诺素(0.4)、/>(0.4)、酸枣仁皂苷A(0.4)相较于其他成分相关性强,呈正相关,说明黄酮类和皂苷成分可能具有保肝作用。综上所述,保肝生化指标与斯皮诺素、/>和皂苷A相关性较强,提示酸枣仁中黄酮类成分和皂苷成分是发挥保肝作用的有效成分。
结合实验例2的结果,炒制后的酸枣仁提取物黄酮类及皂苷类成分含量升高,可能会使酸枣仁提取物保肝作用增强。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (6)
1.一种具有咖啡风味及保肝的酸枣仁提取物的制备方法,其特征在于:具体步骤如下:
(1)干燥酸枣仁:酸枣仁30-60℃加热干燥15-60min,取出摊放于温度为20-25℃、湿度为44%-75%的环境内,酸枣仁冷却至18-25℃,得到水分含量为7%-20%的干燥酸枣仁;
(2)炒制酸枣仁:干燥酸枣仁平铺于电热恒温油浴锅或多功能炒制锅内,控制炒制锅的温度为130-300℃,锅内温度为100-250℃,锅内温度升温后稳定1-10min,进行炒制酸枣仁,每隔10-40s翻炒一次,炒制时间为10-40min;炒至酸枣仁鼓起,色由微黄变为焦黑,有咖啡香气外溢,停止炒制,取出自然晾凉;
采用GC-MS的方法分析炒酸枣仁成分,表明有8种化学成分与咖啡的成分共有,分别为棕榈酸,亚油酸,油酸,硬脂酸,肉豆蔻酸,豆甾醇,二十烷酸,β-谷甾醇;
(3)酸枣仁水提液的制备:步骤(2)中炒制的酸枣仁粉碎至10-50目,加入酸枣仁粉重量的1-16倍的蒸馏水,浸泡0.5-5h,然后加热回流提取或超声提取1-2h,过滤,滤液备用,残渣中继续加入残渣重量的1-16倍的蒸馏水,继续浸泡、提取,提取1-4次后,滤液合并,即为酸枣仁水提物,酸枣仁水提物的浓度为0.01-4g/mL;
采用HPLC-DAD-ELSD的测定方法对炒后酸枣仁提取物的潜在药效成分进行定量分析,分别为乌药碱、木兰花碱、斯皮诺素、维采宁、、酸枣仁皂苷A、酸枣仁皂苷B,炒制后含量显著升高,显著增强酸枣仁的保肝作用;
(4)酸枣仁提取物干燥:酸枣仁水提物-20至-60℃、0-20pa冷冻干燥0.5-48h,即为酸枣仁提取物。
2.利用权利要求1所述方法制备的一种具有咖啡风味及保肝的酸枣仁提取物。
3.一种如权利要求2所述的酸枣仁提取物在制备辅助保肝食品中的应用,其特征在于:所述食品包含饮料,饼干或面包。
4.根据权利要求3所述的应用,其特征在于:所述食品包含咖啡。
5.一种如权利要求2所述酸枣仁提取物在制备辅助保肝保健品中的应用,其特征在于:所述保健品包括保肝茶或保肝凝胶糖果。
6.一种如权利要求2所述酸枣仁提取物在制备辅助保肝药物中的应用,其特征在于:所述药物包括颗粒剂,片剂,丸剂或胶囊。
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