CN114588175A - 黑木耳膳食纤维在酒精性肝损伤中的应用 - Google Patents
黑木耳膳食纤维在酒精性肝损伤中的应用 Download PDFInfo
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Abstract
本发明公开了黑木耳膳食纤维在酒精性肝损伤中的应用，涉及生物医药技术领域。本发明要求保护黑木耳膳食纤维在制备治疗酒精性肝损伤的药物中的应用。本发明证实了黑木耳可溶性膳食纤维素可通过抗氧化及炎症调节2个途径发挥保护酒精性肝损伤作用。本发明揭示了黑木耳可溶性膳食纤维对酒精性肝损伤具有良好的防治作用，本发明为研制以黑木耳可溶性膳食纤维活性组分为基础的解酒保肝药物及保健品提供科学依据，具有重大的应用价值。
Description
技术领域
本发明涉及生物医药技术领域，特别是涉及黑木耳膳食纤维在酒精性肝损伤中的应用。
背景技术
酒精性肝病是长期大量饮酒所致的慢性疾病，随着饮酒量的增加及饮酒史的延长而逐渐表现为脂肪肝、酒精性肝炎、酒精性肝纤维化、酒精性肝硬化等疾病。目前酒精性肝病患者数量逐年增加，已成为仅次于病毒性肝炎的第二肝脏疾病。与非酒精性肝损伤患者相比，酒精性肝损伤疾病发展更快，发生肝衰竭、肝癌等恶性疾病的风险更高，而目前还没有酒精性肝损伤的特效药。
黑木耳隶属于担子菌门，层菌纲，木耳目，木耳科，木耳属，是重要的胶质食药用菌，产量位居食用菌产量的前三名，约占世界黑木耳产量的95％。
目前，对于黑木耳膳食纤维的研究集中于提取方法的工艺条件优化及产品开发，对于功能活性的研究较少，更未有其对酒精性肝损伤的作用的报道。
发明内容
本发明的目的是提供黑木耳膳食纤维在酒精性肝损伤中的应用，以解决现有技术中研制以黑木耳可溶性膳食纤维活性组分为基础的解酒保肝药物及保健品提供了科学依据。
为实现上述目的，本发明提供了如下方案：
技术方案一：黑木耳膳食纤维在制备治疗酒精性肝损伤的药物中的应用。
进一步地，所述黑木耳膳食纤维为可溶性膳食纤维。
进一步地，所述可溶性膳食纤维的主要组成成分为β-吡喃糖。
进一步地，所述可溶性膳食纤维素的分子量范围为230kDa-1260kDa。
进一步地，所述治疗酒精性肝损伤是通过调节抗氧化和炎症来达到保护酒精性肝损伤的作用。
进一步地，所述调节抗氧化和炎症具体的为：降低血清谷丙转氨酶和谷草转氨酶的酶活力及甘油三酯的含量；提高谷胱甘肽含量；提高谷胱甘肽过氧化物酶和超氧化物歧化酶活性；降低丙二醛和一氧化氮生成量；抑制IL-1β、IL-6和TNF-α的表达水平。
技术方案二：一种黑木耳膳食纤维的制备方法，包括以下步骤：
(1)黑木耳干燥后过20目筛，按体积比为1：80料液比加入纯净水，破壁，得到匀浆；
(2)向所述匀浆加入Na2CO3溶液，超声提取后，离心，得上清液1和下层沉淀；
(3)将所述下层沉淀按体积比为1：30料液比加入蒸馏水，加入纤维素酶和果胶酶酶解3.5h，灭酶、离心，得上清液2，调pH值至7.0；
(4)将所述上清液1和上清液2混合，浓缩后冻干。
技术方案三：一种治疗酒精性肝损伤的药物，所述药物的成分包括所述的黑木耳膳食纤维。
技术方案四：一种食品或保健品，所述食品或保健品的成分包括所述的黑木耳膳食纤维。
本发明公开了以下技术效果：
本发明通过小鼠体内实验证实，黑木耳可溶性膳食纤维可降低慢性酒精性肝损伤小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)酶活力及甘油三酯(TG)含量，提高谷胱甘肽(GSH)含量及谷胱甘肽过氧化物酶(GSH-Px)酶活性，提高超氧化物歧化酶(SOD)活性，降低氧化产物丙二醛(MDA)及一氧化氮生成量，抑制免疫因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)表达水平。本发明证实了黑木耳可溶性膳食纤维素可通过抗氧化及炎症调节2个途径发挥保护酒精性肝损伤作用。本发明揭示了黑木耳可溶性膳食纤维对酒精性肝损伤具有良好的防治作用，本发明为研制以黑木耳可溶性膳食纤维活性组分为基础的解酒保肝药物及保健品提供了科学依据，具有重大的应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为黑木耳可溶性膳食纤维的高效凝胶渗透色谱图；
图2为黑木耳可溶性膳食纤维单糖组成HPLC图；
图3为黑木耳可溶性膳食纤维红外光谱图；
图4为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠血清谷丙转氨酶活性的影响；
图5为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠谷草转氨酶活性的影响；
图6为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠总甘油三酯含量的影响；
图7为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠总胆固醇含量的影响；
图8为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠谷胱甘肽含量的影响；
图9为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠超氧化物歧化酶活性的影响；
图10为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠谷胱甘肽过氧化物酶活性的影响；
图11为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠丙二醛生成量的影响；
图12为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠一氧化氮生成量的影响；
图13为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠白介素-1β水平的影响；
图14为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠白介素-6水平的影响；
图15为黑木耳可溶性膳食纤维对酒精性肝损伤小鼠肿瘤坏死因子-α水平的影响。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值，以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
实施例1
1.黑木耳可溶性膳食纤维提取及组成分析
黑木耳干燥子实体粉碎，20目过筛，料液比按体积比为1:80加入纯净水，至于破壁机中破壁2min，重复4次，得黑木耳匀浆。将黑木耳匀浆置于烧杯中，加入Na2CO3使黑木耳匀浆中Na2CO3浓度为0.5％，超声提取30min，7000r/min下离心15min，分离得上清液1；下层沉淀用蒸馏水洗至中性，烘干，按体积比为1:30料液比加入蒸馏水，HCl调节pH至5.0，按照百分比计，加入0.9％纤维素酶及果胶酶，其中按质量体积比算，纤维素酶：果胶酶＝1：2，50℃酶解3.5h，100℃加热20min灭活酶活性，7000r/min下离心15min，分离得上清液2并调节pH值至7.0，合并上清液1、上清液2，浓缩，冻干即为黑木耳可溶性膳食纤维。黑木耳可溶性膳食纤维提取率85.63％，膳食纤维含量72.45％。
1.2黑木耳可溶性膳食纤维结构分析
1.2.1黑木耳可溶性膳食纤维分子量测定
采用高效凝胶渗透色谱法对黑木耳可溶性膳食纤维进行分子量测定，详见图1，根据样品的保留时间，确定样品的分子量大小，保留时间越小，分子量越大。色谱条件为RID-10A视差折光检测器，TSK-gel G-3000PWXL色谱柱(7.8×300nm)，流动相为蒸馏水，流速为0.6mL/min，柱温为35℃，进样量为10μL。得到分子量标准曲线方程y＝0.1588x+7.3239，R2＝0.9954。根据保留时间和标准曲线，计算黑木耳可溶性膳食纤维分子量范围为230kDa-1260kDa，平均分子量为501kDa。
1.2.2黑木耳可溶性膳食纤维单糖组成分析
取干净色谱瓶，称量黑木耳可溶性膳食纤维样品5mg，加入配制好的TFA酸溶液，121℃加热2小时。通氮气吹干，加入甲醇清洗再吹干，重复3次。加入无菌水溶解，转入色谱瓶中待测。流动相；A相：ddH2O；B相：200mM NaOH；C相：200mM NaOH/500mM NaAC。流速：0.5mL/min。黑木耳可溶性膳食纤维组成为甘露糖：葡萄糖醛酸：葡萄糖：半乳糖：木糖：岩藻糖＝52.03：8.51：14.42：1.90：10.09：1.00。黑木耳可溶性膳食纤维单糖组成HPLC图详见图2。
1.2.3黑木耳可溶性膳食纤维红外光谱
黑木耳可溶性膳食纤维粉和溴化钾(KBr)放入烘干机中，烘干4小时；将样品与溴化钾以质量比1：100的比例充分研磨，混合均匀，用压片机将混合物压成透明片，在4000-500cm-1范围内扫描，扫描次数为64，分辨率为4cm-1。经红外光谱分析，确定黑木耳可溶性膳食纤维为β-吡喃糖。黑木耳可溶性膳食纤维红外光谱图详见图3。
2.黑木耳可溶性膳食纤维抗酒精性肝损伤活性的研究
2.1动物实验设计及处理
2.1.1动物分组
将90只Balb/c小鼠随机分为6组，分别为空白对照组、模型组、水飞蓟宾组、黑木耳可溶性膳食纤维高剂量组、黑木耳可溶性膳食纤维中剂量组、黑木耳可溶性膳食纤维低剂量组，每组15只。
2.1.2动物模型建立及给药
除空白组外的各组小鼠，于造模开始的第1-15天灌胃40％乙醇，空白组注射生理盐水；实验第15-36天，除空白组外，各组小鼠灌胃40％乙醇后的第2小时灌胃各组对应给药药物，建立小鼠酒精性肝损伤模型。
2.2血清生化指标的测定
小鼠眼眶取血后，血浆放置30min，在6000r/min离心10min，分离血清，4℃冰箱保存，3d内按照试剂盒说明书方法检测血清中AST、ALT酶活力及TC含量，详见图4-6。结果显示，灌胃乙醇后，模型组、水飞蓟宾组、黑木耳可溶性膳食纤维高、中、低剂量组小鼠血清AST、ALT酶活力及TG含量升高，给予水飞蓟宾和黑木耳可溶性膳食纤维后，水飞蓟宾组、黑木耳可溶性膳食纤维高、中、低剂量组血清AST、ALT酶活力及TG含量下降，水飞蓟宾组、黑木耳可溶性膳食纤维高低剂量组接近正常组水平。
2.3肝组织脂质过氧化指标的测定
取其余肝组织，用4℃预冷生理盐水冲洗并制备10％肝脏组织匀浆(生理盐水制备)，4℃，6000r/min离心10min，收集上清液，测定肝指标，按照试剂盒说明书方法检测GSH含量、GSH-Px活性、SOD酶活性、MDA及NO含量，详见图8-12。结果显示，灌胃乙醇后，小鼠肝脏GSH含量、GSH-Px、SOD酶活性下降，给予水飞蓟宾和黑木耳可溶性膳食纤维后，水飞蓟宾组、黑木耳可溶性膳食纤维高、中、低剂量组肝脏GSH含量、GSH-Px、SOD酶活性升高，水飞蓟宾组、黑木耳可溶性膳食纤维高、中剂量组接近正常组水平。灌胃乙醇后，小鼠肝脏MDA及NO生成量上升，给予水飞蓟宾和黑木耳可溶性膳食纤维后，水飞蓟宾组、黑木耳可溶性膳食纤维高、中、低剂量组肝脏MDA及NO生成量下降，水飞蓟宾组、黑木耳可溶性膳食纤维高剂量组接近正常组水平。
2.4肝组织免疫因子的测定
取其余肝组织，用4℃预冷生理盐水冲洗并制备10％肝脏组织匀浆(生理盐水制备)，4℃，6000r/min离心10min，收集上清液，测定肝指标，按照试剂盒说明书方法检测IL-1β、IL-6、TNF-α含量。结果显示，灌胃乙醇后，小鼠肝脏IL-1β、IL-6、TNF-α含量上升，详见图13-15，给予水飞蓟宾和黑木耳可溶性膳食纤维后，水飞蓟宾组、黑木耳可溶性膳食纤维高、中、低剂量组肝脏IL-1β、IL-6、TNF-α含量相对于MOD组的含量，显著下降，水飞蓟宾组、黑木耳可溶性膳食纤维高剂量组接近正常组(CON)水平。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (9)
1.黑木耳膳食纤维在制备治疗酒精性肝损伤的药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述黑木耳膳食纤维为可溶性膳食纤维。
3.根据权利要求2所述的应用，其特征在于，所述可溶性膳食纤维的主要组成成分为β-吡喃糖。
4.根据权利要求2所述的应用，其特征在于，所述可溶性膳食纤维素的分子量范围为230kDa-1260kDa。
5.根据权利要求1所述的应用，其特征在于，所述治疗酒精性肝损伤是通过调节抗氧化和炎症来达到保护酒精性肝损伤的作用。
6.根据权利要求5所述的应用，其特征在于，所述调节抗氧化和炎症具体的为：降低血清谷丙转氨酶和谷草转氨酶的酶活力及甘油三酯的含量；提高谷胱甘肽含量；提高谷胱甘肽过氧化物酶和超氧化物歧化酶活性；降低丙二醛和一氧化氮生成量；抑制IL-1β、IL-6和TNF-α的表达水平。
7.一种黑木耳膳食纤维的制备方法，其特征在于，包括以下步骤：
(1)黑木耳干燥后过20目筛，按体积比为1：80料液比加入纯净水，破壁，得到匀浆；
(2)向所述匀浆加入Na2CO3溶液，超声提取后，离心，得上清液1和下层沉淀；
(3)将所述下层沉淀按体积比为1：30料液比加入蒸馏水，加入0.9％纤维素酶和果胶酶酶解3.5h，灭酶、离心，得上清液2，调pH值至7.0；
(4)将所述上清液1和上清液2混合，浓缩后冻干。
8.一种治疗酒精性肝损伤的药物，其特征在于，所述药物包括权利要求1所述的黑木耳膳食纤维。
9.一种食品或保健品，其特征在于，所述食品或保健品包括黑木耳膳食纤维。
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