CN114832022A - 一种桑黄子实体酚类活性物质的制备及其在调节肠道菌群、尿酸代谢中的应用 - Google Patents
一种桑黄子实体酚类活性物质的制备及其在调节肠道菌群、尿酸代谢中的应用 Download PDFInfo
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- CN114832022A CN114832022A CN202210679329.XA CN202210679329A CN114832022A CN 114832022 A CN114832022 A CN 114832022A CN 202210679329 A CN202210679329 A CN 202210679329A CN 114832022 A CN114832022 A CN 114832022A
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- active substance
- phenolic active
- phellinus igniarius
- uric acid
- phellinus
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Abstract
本发明提供了一种桑黄子实体酚类活性物质的制备及其应用,该方法以桑黄子实体粉末作为原料,先用水浸泡后,再加入丙酮乙醇混合液多频超声提取;获得粗品后,采用乙酸乙酯正丁醇混合液分级萃取;获得萃取物后利用膜分离技术除去样品中多糖、蛋白等大分子物质;旋蒸冻干后得到桑黄酚类活性物质。本发明提取的桑黄子实体酚类活性物质纯度较高,活性较强,不仅能有效提高肠道菌群多样性,促进肠道有益菌群的生长,还能增加尿液中尿酸、肌酐和尿素氮的***以及有效降低血清中尿酸、肌酐和尿素氮的含量,可作为天然、无副作用的尿酸代谢稳态维持及肠道菌群调节的辅助食品。
Description
技术领域
本发明属于食用菌深加工技术领域,具体而言,涉及一种桑黄子实体活性物质的制备及其在尿酸代谢调控中的应用。
背景技术
桑黄又称桑耳、桑臣和胡孙眼,在分类学上隶属于担子菌门(Basidiomycota)伞菌纲(Agaricomycetes)锈革菌目(Hymenochaetales)锈革菌科(Hymenochaetaceae)桑黄属(Sanghuangporus),是一类珍贵的药用真菌。现代研究发现,桑黄中含有多糖、黄酮、三萜、多酚类、吡喃酮类和呋喃类等多种活性物质,因此具有抗氧化、免疫调节、降血糖、抗肿瘤和抗癌等多种功效。近年来,随着桑黄药理作用研究的不断深入及其天然抗癌产品属性的深入人心,桑黄已经为国内外医药制剂和保健品行业研究开发的热点。与欧盟、日韩等一些国家相比,我国对于桑黄产品的开发与利用技术仍存在较大差距。目前,杨树桑黄(Sanghuangporus vaninii)人工驯化栽培已经获得成功,因此可以从桑黄子实体中大量提取生物活性物质。
目前,关于桑黄活性物质提取技术方面申请了许多的专利,主要包括:CN101032532A公开了从桑黄子实体中提取多糖PT1和蛋白多糖PT2的方法及其在抗癌药物或免疫调节药物中的应用。CN112656820A公开了利用氯化胆碱和苹果酸组成的深共熔溶剂提取桑黄深层发酵菌丝中桑黄多酚的方法。CN112791108A公开了利用有机溶剂从桑黄菌株发酵液中萃取活性物质的方法,发现萃取物具有一定降血糖活性;CN101474211A和CN111096983A分别公开了一种从桑黄子实体中制备乙醇提取物的方法及其具有降血糖活性。CN112138030A公开了乙醇提取、大孔吸附树脂纯化的桑黄黄酮提取方法及其在防治痛风方面的作用;CN108379305A和CN112189505A分别公开了桑黄水提物和醇提物具有降尿酸效果;CN110693921B公开了桑黄子实体醇提物在制备痛风和高尿酸血症的预防和治疗的药物中的应用。上述专利从不同角度公开了桑黄粗提物的制备方法,虽然其最终提供得到“多酚、黄酮”,但由于多酚和黄酮是一类物质的统称,因此究竟降血糖、降尿酸活性到底是哪种、哪些或者是哪一类主要成分起作用,我们都无从知晓。此外,在降尿酸活性(痛风)研究中,所提到的都是醇提物,并没有考虑不同活性物质在不同溶剂中的溶解性。因此亟需开发一种高效、廉价制备桑黄多酚类活性物质的方法,能明确其主要成分并能增强其生物活性。
发明内容
针对目前桑黄子实体中多酚类活性物质的提取率较低、提取不充分、功能成分混杂等问题,本发明的第一个目的在于提供一种多酚含量高达60%以上的高纯度桑黄子实体酚类活性物质的制备方法。
为了实现上述技术目的,发明人结合多年来的提取分离经验,并通过大量试验研究并不懈探索,最终获得了如下技术方案:一种桑黄子实体酚类活性物质的制备方法,该方法包括如下步骤:
(1)取栽培年限1~3年的杨树桑黄子实体,烘干后粉碎,将得到的子实体粉末加入2~10倍量的水浸泡过夜,加入丙酮乙醇混合液,搅拌均匀后,采用多频超声装置提取1~3次,每次20~40min,所述多频超声装置的提取条件为:超声频率40KHz和80KHz交替使用,每次使用4~6min,共计20~40min,超声功率为450~600W,提取温度为25~35℃;提取结束后离心,收集上清液,旋转蒸发浓缩后得到丙酮乙醇粗提物干品;
(2)将步骤(1)得到的丙酮乙醇粗提物干品与蒸馏水混合制成悬浮液,使用乙酸乙酯正丁醇混合液进行萃取1~3次,每次静置萃取1h~6h,分离有机层,旋蒸后挥发有机溶剂,得到乙酸乙酯正丁醇萃取物;
(3)将步骤(2)得到的乙酸乙酯正丁醇萃取物用所述乙酸乙酯正丁醇混合液溶解后,将溶解液与蒸馏水按照1:(6~15)的体积比震荡混匀,过超滤膜,除去蛋白质、多糖等大分子物质;收集滤液,旋蒸冻干后得到桑黄子实体酚类提取物纯品。
进一步优选地,如上所述桑黄子实体酚类活性物质的制备方法,其步骤(1)中桑黄子实体烘干后粉碎后过20-80目筛。
进一步优选地,如上所述桑黄子实体酚类活性物质的制备方法,其步骤(1)加入丙酮乙醇混合液后,所得料液中V丙酮:V乙醇:V水=(0.5~3):(6~8):1。
再进一步优选地,如上所述桑黄子实体酚类活性物质的制备方法,其步骤(1)加入丙酮乙醇混合液后,所得料液中V丙酮:V乙醇:V水=(0.8~1.5):(7~8):1。
进一步优选地,如上所述桑黄子实体酚类活性物质的制备方法,其步骤(2)中所述的乙酸乙酯正丁醇混合液中V乙酸乙酯:V正丁醇=(5~30):1。
再进一步优选地,如上所述桑黄子实体酚类活性物质的制备方法,其步骤(2)中所述的乙酸乙酯正丁醇混合液中V乙酸乙酯:V正丁醇=(5~10):1。
进一步优选地,如上所述桑黄子实体酚类活性物质的制备方法,其步骤(2)中萃取时每次静置1h~2h。
进一步优选地,如上所述桑黄子实体酚类活性物质的制备方法,其步骤(3)中超滤膜规格为10~20kDa,压力为0.2~0.4MPa,温度为25~30℃。
另外,本发明的第二个目的在于提供上述制备方法得到的桑黄子实体酚类活性物质在制备调节肠道菌群结构或/和调节尿酸代谢的药品、食品或保健品中的应用,所述调节肠道菌群结构为选择性刺激肠道Muri菌(Muribaculaceae)、拟杆菌属(Bacteroides)及毛螺菌科(Lachnospiraceae)益生菌属的生长,降低乳酸杆菌属(Lactobacillus)菌群的丰度;所述调节尿酸代谢为降低血清中肌酐、尿素氮和尿酸浓度,增加尿液中肌酐、尿素氮和尿酸的排放。
与现有技术相比,本发明制备的桑黄子实体酚类活性物质中多酚含量高达60%以上,总黄酮含量超过10%,并具有如下的有益效果:
(1)体外黄嘌呤氧化酶抑制实验显示,本发明制备的桑黄子实体酚类活性物质具有显著的黄嘌呤氧化酶抑制活性,且其IC50值较低,为0.45mg/mL。
(2)小鼠模型试验显示,喂食本发明制备的桑黄子实体酚类活性物质能显著刺激肠道Muri菌(Muribaculaceae),拟杆菌属(Bacteroides),毛螺菌科(Lachnospiraceae)等益生菌属的生长,降低乳酸杆菌属(Lactobacillus)菌群的丰度,有效调节肠道菌群结构并改善模型小鼠肠道屏障的保护作用。
(3)小鼠模型试验显示,喂食本发明制备的桑黄子实体酚类活性物质降低了模型小鼠血清中肌酐、尿素氮和尿酸浓度,增加尿液中肌酐、尿素氮和尿酸的排放。
(4)本发明制备的桑黄子实体酚类活性物质还增加了小鼠肠道内总SCFAs及乙酸、丙酸、丁酸等小分子有机酸的含量,改善了SCFAs介导的能量供应调控情况。
综上所述,本发明制备方法简便高效,制备了高纯度桑黄子实体酚类活性物质,该活性物质具有增加肠道有益菌群、降低血清尿酸和增强尿酸***的特性。
附图说明
图1为各实验组小鼠肠道菌群在门和属水平上的差异性分析图;
图2为各实验组小鼠的血清尿酸、肌酐和尿素氮含量图
图3为各实验组小鼠的尿液中尿酸、肌酐和尿素氮含量图。
具体实施方式
通过以下实施例对本发明进行详细描述,以使本发明的优点和特征能更易于被本领域技术人员理解,但并不以任何方式限制本发明。在不背离本发明的技术解决方案前提下,凡是利用本发明说明书内容所作的、本领域普通技术人员易于实现的任何改动或改变都将包括在本发明的专利保护范围内。
实施例1
取栽培年限3年的杨树桑黄(Sanghuangporus vaninii)子实体100g,烘干后粉碎并过40目筛,将得到的子实体粉末加入5倍量的水浸泡过夜,加入丙酮乙醇混合液,使得丙酮:乙醇:水的体积比为10:80:10,搅拌均匀后,采用多频超声装置提取,其中多频超声装置设置为:超声功率500W,提取温度为30℃;超声频率40KHz和80KHz交替使用,每次使用5min,共计20min。5000r/min离心,沉淀中再加入丙酮-乙醇-水混合液(V丙酮:V乙醇:V水=10:80:10),搅拌均匀后,采用多频超声装置重复提取一次,合并两次的上清液,旋转蒸发浓缩后得到丙酮乙醇粗提物6.98g(提取率为6.98%)。将所得粗提物与一定量蒸馏水混合制成悬浮液,使用乙酸乙酯正丁醇混合液(体积比为85:15)进行分级萃取2次,每次静置萃取2h,旋蒸后挥发有机溶剂,得到乙酸乙酯正丁醇萃取物。溶解后,过20kDa超滤膜,压力为0.2MPa,温度为25℃,除去蛋白质、多糖等大分子物质,收集滤液,旋蒸冻干后得到桑黄酚类活性物质纯品(该活性物质记为S1)。
对比例1:单频超声提取工艺
取栽培年限3年的杨树桑黄(Sanghuangporus vaninii)子实体100g,烘干后粉碎并过40目筛,将得到的子实体粉末加入5倍量的水浸泡过夜,加入丙酮乙醇混合液,使得丙酮:乙醇:水的体积比为10:80:10,搅拌均匀后,采用超声装置提取,其中超声装置设置为:超声功率500W,提取温度为30℃;超声频率80KHz,超声时间20min。5000r/min过滤,沉淀中再加入丙酮-乙醇-水混合液(V丙酮:V乙醇:V水=10:80:10),搅拌均匀后,采用超声装置重复提取一次,合并两次上清液,旋转蒸发浓缩后得到丙酮乙醇粗提物6.05g(提取率为6.05%)。
对比例2:使用乙醇作为溶剂直接从桑黄中提取桑黄酚类活性物质
取栽培年限3年的杨树桑黄子实体100g,烘干后粉碎并过40目筛,将得到的子实体粉末加入5倍量的水浸泡过夜,加入乙醇,使得料液中乙醇:水的体积比为90:10,搅拌均匀后,采用多频超声装置提取,其中多频超声装置设置为:超声功率500W,提取温度为30℃;超声频率40KHz和80KHz交替使用,每次使用5min,共计20min。5000r/min离心,沉淀中再加入乙醇-水混合液(V乙醇:V水=90:10),搅拌均匀后,采用多频超声装置重复提取一次,合并两次上清液,旋转蒸发浓缩后得到乙醇粗提物6.51g(提取率为6.51%,该活性物质记为D1)。
对比例3:乙醇粗提物纯化处理
将对比例2制备的D1粗提物与一定量蒸馏水混合制成悬浮液,使用乙酸乙酯正丁醇混合液(体积比为85:15)进行分级萃取2次,每次静置萃取2h,旋蒸后挥发有机溶剂,得到乙酸乙酯正丁醇萃取物。溶解后,过20kDa超滤膜,压力为0.2MPa,温度为25℃,收集滤液,旋蒸冻干后得到桑黄酚类活性物质纯品(该活性物质记为D2)。
实施例2:桑黄子实体酚类活性物质的效果实验
一、实验步骤
(1)以没食子酸为标准品,采用福林-酚法测定各组样品中的总酚含量;以芦丁为标准品,采用亚硝酸钠-硝酸铝法测定各组样品中的黄酮含量。
(2)对黄嘌呤氧化酶抑制活性测定:取磷酸缓冲液(0.1mol/L,pH=8.5)0.6mL,黄嘌呤溶液(2mmol/L)0.2mL以及不同样液(3.0mg/mL,DMSO配制)0.1mL于1.5mL离心管中涡旋混匀;然后加入0.2mL黄嘌呤氧化酶液(0.1U/mL)并在25℃水浴反应30min;完成后加入0.2mL的HCl溶液(1.0mol/L)终止反应。以别嘌呤醇作为阳性对照,于波长290nm处测定吸光值。黄嘌呤氧化酶抑制率的计算公式如下:
抑制率(%)={[(A-B)-(C-D)]/(A-B)}×100
其中,A为加酶不加样品管吸光值;B为不加酶不加样品管吸光值;C为加酶加样品管吸光值;D为不加酶加样品管吸光值。
(3)动物实验:准备50只昆明小鼠,雄性,体重为20~22g,并随机分为5组,每组10只。1为空白对照组,2为模型组,3为实施例1中S1样品(100mg/kg),4为对比例1中D1样品(100mg/kg),5为对比例2中D2样品(100mg/kg)。除空白对照组,其余各组每天灌胃200mg/kg氧嗪酸钾和200mg/kg次黄嘌呤制备高尿酸血症小鼠模型,连续造模7天,空白对照组灌胃等量0.5%CMC-Na。第8天开始,各组在灌胃200mg/kg氧嗪酸钾和200mg/kg次黄嘌呤的基础上,并分别灌胃相应药物,空白对照组和模型组灌胃等量0.5%CMC-Na,持续灌胃28天。灌胃体积为0.1mL/10g。喂养最后1天,收集小鼠肠道粪便用于测序和短链脂肪酸含量测定。
(4)盲肠短链脂肪酸含量测定:称取约100mg粪便样本于2mL研磨管中,加入一颗钢珠,加入900μL甲醇和100uL内标(1000μg/mL的2-乙基丁酸,甲醇配制)于50HZ冷冻研磨仪中研磨3min,重复研磨2次。然后置于冰水浴超声30min后,于-20℃静置30min,4℃、13000r/min条件下离心15min。将上清转移至1.5mL离心管中,加入50mg无水硫酸钠,涡旋,4℃、13000r/min条件下继续离心15min,取上清。使用0.22μm有机滤膜过滤,进行GC-MS检测分析。
(5)微生物菌群结构及多样性分析:小鼠直肠粪便委托公司进行肠道微生物测序(n=6)。基于OTU可以进行多种多样性指数分析,基于OTU聚类分析结果,可以对OTU进行多种多样性指数分析以及对测序深度的检测;基于分类学信息,可以在各个分类水平上进行群落结构的统计分析。
二、结果分析
(1)不同样品的多酚黄酮含量、黄嘌呤氧化酶抑制率及其对小鼠肠道中SCFA含量的影响。
表1不同样品化学组成及不同实验组中短链脂肪酸含量
实施例1、对比例1和对比例2获得的桑黄酚类活性物质常规指标如表1所示。实施例1中S1所含总酚含量和黄酮含量显著高于对比例1和对比例2。实施例1中S1对黄嘌呤氧化酶酶抑制活性显著高于对比例1和对比例2,这表明经过混合溶剂的提取及纯化,多酚类物质降尿酸活性显著升高。喂养4周后,实施例1中酚类活性物质喂养的小鼠盲肠中SCFA总量、乙酸含量、丙酸含量和丁酸含量显著高于模型组、对比例1和对比例2。另外,对比例2中SCFA总量、乙酸含量、丙酸含量和丁酸含量显著高于对比例1。
(2)不同样品对小鼠肠道菌群组成和含量的影响
各实验组小鼠的肠道菌群在门分类水平上的组成如图1和表2所示。由图1a可知:小鼠的肠道菌群主要由5个门组成,即拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、疣微菌门(Verrucomicrobia)、放线细菌门(Actinobacteriota)和弯曲杆菌门(Campilobacterota),其中的优势菌群是拟杆菌门和厚壁菌门。与模型组相比,实施例1、对比例1和对比例2中厚壁菌门含量大大减少,拟杆菌门含量显著增加。各实验组小鼠的肠道菌群在属水平上的差异性分析如图1b所示。与模型组相比,实施例1中Lactobacillus菌大量减少,Muribaculaceae菌、Bacteroides菌和Lachnospiraceae菌显著增加。与对比例1和对比例2相比,实施例1中Muribaculaceae菌和Bacteroides菌显著增加。
表2不同实验组中肠道菌群组成、血清及尿液中相关指标含量
(3)不同样品对小鼠血清中尿酸指标的影响
各实验组小鼠血清中尿酸、肌酐和尿素氮含量如图2和表2所示。所有实验组中,模型组中尿酸、肌酐和尿素氮含量最高。相对于对比例1和对比例2,实施例1中血清中尿酸、肌酐和尿素氮含量相对较低;另外,对比例2中各项指标显著低于对比例1。
(4)不同样品对小鼠尿液中尿酸指标的影响
各实验组小鼠尿液中尿酸、肌酐和尿素氮含量如图3和表2所示。实施例1尿液中尿酸、肌酐和尿素氮含量最高,这表明实施例1中S1活性物质促进了小鼠体内尿酸、肌酐和尿素氮等的***。
以上的研究结果显示,本发明制备的桑黄酚类活性物质能够增加Muribaculaceae菌、Bacteroides菌和Lachnospiraceae菌等有益菌群含量,减少Lactobacillus菌含量,有效调节肠道菌群结构。本发明制备的桑黄酚类活性物质可能通过促进了小鼠体内尿酸、肌酐和尿素氮等的***来降低血清中尿酸、肌酐和尿素氮等含量,维持了小鼠体内尿酸代谢的稳定性。除此之外,本发明制备的桑黄酚类活性物质还增加了小鼠肠道内总SCFAs及乙酸、丙酸和丁酸等小分子有机酸的含量,改善了SCFAs介导的能量供应调控情况。本发明的研究结果表明,桑黄酚类活性物质可以作为潜在的健康补充剂或尿酸代谢调节剂。
Claims (10)
1.一种桑黄子实体酚类活性物质的制备方法,其特征还在于,该方法包括如下步骤:
(1)取栽培年限1~3年的杨树桑黄子实体,烘干后粉碎,将得到的子实体粉末加入2~10倍量的水浸泡过夜,加入丙酮乙醇混合液,搅拌均匀后,采用多频超声装置提取1~3次,每次20~40min,所述多频超声装置的提取条件为:超声频率40KHz和80KHz交替使用,每次使用4~6min,共计20~40min,超声功率为450~600W,提取温度为25~35℃;提取结束后离心,收集上清液,旋转蒸发浓缩后得到丙酮乙醇粗提物干品;
(2)将步骤(1)得到的丙酮乙醇粗提物干品与蒸馏水混合制成悬浮液,使用乙酸乙酯正丁醇混合液进行萃取1~3次,每次静置萃取1~6h,分离有机层,旋蒸后挥发有机溶剂,得到乙酸乙酯正丁醇萃取物;
(3)将步骤(2)得到的乙酸乙酯正丁醇萃取物用所述乙酸乙酯正丁醇混合液溶解后,将溶解液与蒸馏水按照1:(6~15)的体积比震荡混匀,过超滤膜,除去大分子物质;收集滤液,旋蒸冻干后得到桑黄子实体酚类提取物纯品。
2.根据权利要求1所述桑黄子实体酚类活性物质的制备方法,其特征在于,步骤(1)中桑黄子实体烘干后粉碎后过20-80目筛。
3.根据权利要求1所述桑黄子实体酚类活性物质的制备方法,其特征在于,步骤(1)加入丙酮乙醇混合液后,所得料液中V丙酮:V乙醇:V水=(0.5~3):(6~8):1。
4.根据权利要求3所述桑黄子实体酚类活性物质的制备方法,其特征在于,步骤(1)加入丙酮乙醇混合液后,所得料液中V丙酮:V乙醇:V水=(0.8~1.5):(7~8):1。
5.根据权利要求1所述桑黄子实体酚类活性物质的制备方法,其特征在于,步骤(2)中所述的乙酸乙酯正丁醇混合液中V乙酸乙酯:V正丁醇=(5~30):1。
6.根据权利要求5所述桑黄子实体酚类活性物质的制备方法,其特征在于,步骤(2)中所述的乙酸乙酯正丁醇混合液中V乙酸乙酯:V正丁醇=(5~10):1。
7.根据权利要求1所述桑黄子实体酚类活性物质的制备方法,其特征在于,步骤(2)中萃取时每次静置1h~2h。
8.根据权利要求1所述桑黄子实体酚类活性物质的制备方法,其特征在于,步骤(3)中超滤膜规格为10~20kDa,压力为0.2~0.4MPa,温度为25~30℃。
9.根据权利要求1所述制备方法得到的桑黄子实体酚类活性物质在制备调节肠道菌群结构或/和调节尿酸代谢的药品、食品或保健品中的应用,所述调节肠道菌群结构为选择性刺激肠道Muri菌(Muribaculaceae)、拟杆菌属(Bacteroides)及毛螺菌科(Lachnospiraceae)益生菌属的生长,降低乳酸杆菌属(Lactobacillus)菌群的丰度。
10.根据权利要求9所述的应用,其特征在于,所述调节尿酸代谢为降低血清中肌酐、尿素氮和尿酸的浓度,增加尿液中肌酐、尿素氮和尿酸的排放。
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