CN112370459A - 红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用 - Google Patents
红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用 Download PDFInfo
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- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明属于海洋药物领域,具体涉及一种红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用。以含有D‑/L‑半乳糖的红藻多糖为原料,经物理法、化学法、生物酶法或上述方法的任意组合进行可控降解,进而定向氧化或还原获得不同聚合度的红藻半乳寡糖及其衍生物,其分子骨架中含有D‑半乳糖和L‑半乳糖及其衍生物。本发明产品的原料来源于海洋红藻,具有资源丰富、制备工艺简单、安全性高,易于产业化等优点,作为功能因子和益生元,在改善肝损伤患者肠道菌群紊乱,调节胆汁酸代谢平衡,防治急性肝脏组织损伤及在护肝和特医食品的开发领域,具有广阔的应用前景。
Description
技术领域
本发明属于海洋生物资源利用及药物开发领域,具体涉及一种红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用。
背景技术
临床上造成急性肝损伤的原因较多,但主要有病毒感染、口服用药物不当、乙醇摄入过量、放射线损伤及误服有毒食物等。尤其是,随着临床用药种类的迅速增加及患者自行口服药或随意加大药物剂量概率的增加,因药物诱发的急性肝损伤的发生率显著增加。外源化学物质及毒性代谢物可在肝脏中转化及储存,进而在富含磷脂的膜状组织(如线粒体和内质网)中引起脂质过氧化和其他连锁反应。当肝细胞发生钙稳态紊乱、抗氧化系统失衡以及氧自由基与核酸或蛋白质间形成共价键等一系列变化发时,细胞生物膜的通透性会被破坏,诱发急性肝损伤。炎症反应会激活中性粒细胞呼吸爆发产生大量氧自由基,从而加重氧化应激和肝损伤。这些启动因子可以进一步激活Toll样受体、丝裂原活化蛋白激酶和其他相关信号通路,诱发肝细胞凋亡和坏死,甚至可能发展为肝癌。目前,对急性肝损伤的治疗方法很多并有一定的疗效,但也存在着明显的不足。现有临床药物对急性肝损伤的治疗效果并不十分突出,且具有较大的毒副作用。还有一些疗法虽然对急性肝损伤有效,但因其价格昂贵,使患者经济负担较重。近年来,天然产物(如中药)的保肝作用受到极大关注,其作用机制可能与自由基的清除和抗氧化作用有关。基于肝损伤发生、发展机制的复杂性,天然产物的多作用途径和多靶点的特点在对抗急性肝损伤方面具有一定的优势。因此,开发新型的价格低廉、副作用小且药效较好的抗急性肝损伤药物制剂、临床特医食品迫在眉睫,且有重要的临床应用开发价值。
肠道菌群是一组与宿主协同作用的共生微生物群。肠道菌群中有1000多种不同的细菌种类,细菌总数超过1014个,基因组数量多达300万个,是人类基因组数量的150倍,被称为“人体第二基因组”。人体内的肠道菌群并非固定不变,它很容易受到遗传、饮食、药物及卫生环境等多种因素的影响。Gong等人研究表明,对乙酰氨基酚诱导的小鼠急性肝损伤伴随着盲肠菌群及肠道菌群代谢产物(如胆汁酸)的显著改变,且通过粪菌移植进一步证明急性肝损伤小鼠粪便可显著加重无菌小鼠的肝损伤程度,提示肠道菌群可能在急性肝损伤的发病中起重要作用。由于肝脏和肠道通过门静脉相连,使肝脏更容易暴露于易位的细菌,细菌产物,脂多糖和炎症介质中。在某些病理条件下,肠道屏障的破坏可导致细菌及其代谢产物的易位和免疫系统的异常活化,引发肝脏炎症和损伤。肠-肝轴是指肠道与肝脏之间的相互作用,其作为连接肠道与肝脏的重要途径,在急性肝损伤的发病机制中起关键作用。
益生菌是指对宿主健康有益的活性微生物。在小鼠模型中,用Saccharomycescerevisiae菌株干预后,对乙酰氨基酚诱导的肝脏损伤在生物化学和组织学水平均改善,揭示了肠道益生菌在化学物质诱导急性肝损伤中的保护作用。肠道菌群可通过影响肝脏氧化应激状态、缓解肝脏炎症以及调节胆汁酸代谢等多种机制促进急性肝损伤的发病,由此可见肠道菌群在急性肝损伤发病机制中发挥重要作用。益生元是指一组非消化性食物成分,可选择性地改变结肠中某些细菌的生长或活性,对人体健康产生益处。动物研究显示,补充膳食菊粉可显著缓解亚硝胺前体诱导的急性肝损伤,并调节菌群的稳态。这表明益生元可通过调节肠道菌群来缓解急性肝损伤,为急性肝损伤的临床治疗治疗提供了新的方向和途径。
本团队研究表明,酸性多糖和寡糖,如硫酸软骨素及其寡糖、硫酸角质素、岩藻聚糖硫酸酯和浒苔多糖(公开号CN108440681A)等,可作为益生元改善肠道菌群紊乱,进而发挥降血糖、降血脂、抗炎和改善代谢综合征作用。本团队研究还发现,琼胶寡糖(公开号CN105168232A)具有降血脂等活性,岩藻聚糖硫酸酯具有抑制α-糖苷酶活性(公开号CN103288978A),褐藻胶寡糖及其衍生物具有改善胰岛素抵抗、降血糖活性(公开号CN101649004A,公开号CN101691410A)等,但迄今还没有发现含有红藻来源半乳寡糖及其衍生物可改善急性肝损伤并改善小鼠肠道菌群紊乱的报道。红藻来源的半乳聚糖主要有三个结构系列,即卡拉胶系列、琼胶系列和紫菜胶系列,其中,卡拉胶系列的多糖与寡糖均由D-半乳糖及其硫酸酯衍生物组成(公开号CN1513880A,公开号CN101012249A,公开号CN101279991A),而琼胶和紫菜胶系列的多糖与寡糖则是由D-半乳糖和L-半乳糖二种糖残基及硫酸酯衍生物组成。琼胶与紫菜胶的不同在于琼胶含有较多的L-AnG,而紫菜胶含有较多6-硫酸-L-Gal(Gal6S),单糖种类不同则理化性质和生物学功能也不同。例如,虽然本课题组发现各种结构的卡拉胶(含有4-硫酸-D-半乳糖糖残基)及其低聚糖或寡糖具有抑制PTP1B活性(公开号CN111875714A)和作为喷剂具有抗流感病毒活性(公开号CN102516323A,公开号CN104546895A)以及抗新冠病毒活性(公开号CN111773240A),但我们发现该类硫酸多糖口服却有一定的致肠炎安全隐患(ShangQ.,ToxicolLet.,2017,279:87-95);琼胶和紫菜胶及其寡糖衍生物口服则安全性很高,我们也证明了琼胶类多糖和寡糖及其衍生物,具有防治非酒精性脂肪肝(公开号CN111388488A)、可以作为SGLTs抑制剂降低血糖(公开号CN110812364A)、具有防治神经退行性疾病(公布号CN111004296A)、改善线粒体功能并防治胰岛素抵抗(公开号CN110669149A)等作用,是海洋药物及功能食品开发的优质原料。虽然,琼胶和紫菜胶及其寡糖衍生物发现了以上应用,也有学者发现琼胶低聚糖对LPS+D-GalN急性肝损伤小鼠有清除自由基作用(薛长湖等,水产学报,2003,3:283),但在化学有毒试剂所导致的急性肝损伤的防治方面以及对肠道微生物影响没有研究。此外,在质量控制方面,由于半乳聚糖溶解性较差,结构序列不明确,致使其质量难控制,所以寡糖的制备与应用是近年来研究开发的重点。琼胶寡糖制备技术主要有酸法降解和酶法降解,不同方法能获得结构与活性不同的寡糖,例如酸法降解可以得到奇数琼胶寡糖(公开号CN1513860A),酶法降解得到偶数新琼胶寡糖(公开号CN102827899A;公开号CN109576328A),还原酸降解得到偶数糖醇(公开号CN100999537A),自由基降解则得到混合的琼胶寡糖(公开号CN109400756A);紫菜胶采用酸法降解(LiuY.,etal,MarDrugs.2018,16(3).pii:E82)或者酶法降解(ZhangY.,etal,J.Agr.FoodChem.2019,67,9307-9313)均能获得紫菜胶寡糖等。本发明在已有的降解技术基础上,进一步对所制备的各种寡糖进行定向还原和氧化反应,得到了结构与序列不同且还原端含有糖醇或糖酸结构的新寡糖衍生物,并通过实验证明了这些新型结构的半乳寡糖及衍生物能作为益生元,具有治疗急性肝损伤活性,可用做制备防治急性肝损伤、肝脏纤维化以及作为保肝、护肝药物及其功能制品。
发明内容
本发明的目的是提供一种半乳寡糖及衍生物在作为防治急性肝损伤药物和特医食品中的应用,从海洋红藻多糖中获得系列红藻半乳寡糖及其衍生物,并证明其具有改善肠道微生物紊乱和胆汁酸代谢,在防治急性肝损伤等相关疾病中具有广阔应用前景。
为实现上述发明目的,本发明采用下述技术方案:
一种红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,红藻半乳寡糖及其衍生物的结构通式如下:
式中,R=-H或-SO3Na,n=0~30;
所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,以富含D-/L-半乳糖及其衍生物的红藻多糖为原料,经物理降解、化学降解、酶法降解之一种或两种以上降解方法的组合,制备不同聚合度的寡糖及其衍生物,所制备的化合物结构中同时含有β-1,3-D-半乳糖(D-Gal)残基和α-1,4-L-半乳糖(L-Gal)残基,或同时含有D-Gal与α-1,4-L-3,6-内醚半乳糖(L-AnG)残基;在D-Gal与L-Gal糖残基的C6位羟基含有不同程度的硫酸酯基(Gal6S);所制备寡糖的非还原端是Gal、Gal6S或AnG,还原端是Gal或糖醇(Gal-OH)与糖酸(Gal-OOH),或AnG糖醇(AnG-OH),或Gal6S及其糖醇(Gal6S-OH)与糖酸(Gal6S-OOH)。
所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物中的应用,该红藻半乳寡糖及其衍生物采用下述制备工艺:
将红藻来源的琼脂糖(Agarose)溶于60℃热水,用缓冲液配成10mg/mL溶液,放置于30℃水浴锅内添加β-琼胶酶(CAS#37288-57-6)并搅拌降解4小时,冷却后离心,收集上清液,加3倍体积95%医用乙醇于4℃过夜,离心,收集沉淀并用水将其溶解,用200Da透析袋透析脱盐,将内液旋蒸浓缩并冷冻干燥,得到新琼寡糖混合物,并进一步采用硼氢化钠还原得到新琼胶寡糖醇,或者用本尼迪克试剂氧化得到新琼胶寡糖酸;或者,将琼脂糖用60℃热水溶解,采用摩尔浓度0.1M稀盐酸配成10mg/mL溶液,于80℃搅拌降解0.5小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,然后加2倍体积95%医用乙醇于4℃过夜,离心收集沉淀,将沉淀用水溶解后,用200Da透析袋透析脱盐,旋蒸浓缩并冷冻干燥得到寡糖混合物,进一步采用硼氢化钠还原得到琼胶寡糖醇,或者采用本尼迪克试剂氧化得到琼胶寡糖酸;或者,将硫琼胶(Agaropectin)用摩尔浓度0.1M稀硫酸配成10mg/mL水溶液,加热到60℃后搅拌降解1.5小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,然后加入3倍体积95%医用乙醇于4℃过夜,离心收集沉淀,将沉淀溶水溶解后,用200Da透析袋透析脱盐,之后旋蒸浓缩并冷冻干燥得到硫琼胶寡糖混合物,然后进一步经硼氢化钠还原得到硫琼胶寡糖醇,或者采用本尼迪克试剂氧化得到硫琼胶寡糖酸;或者,将紫菜胶(Porphyran)用摩尔浓度0.1M稀硫酸配成10mg/mL水溶液,加热到80℃后搅拌降解2小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,然后加入3倍体积95%医用乙醇于4℃过夜,离心收集沉淀,将沉淀用水溶解后,用200Da透析袋透析脱盐,之后旋蒸浓缩并冷冻干燥得到寡糖混合物,进一步采用硼氢化钠还原得到紫菜胶寡糖醇,或者采用本尼迪克试剂氧化得到紫菜胶寡糖酸。
所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,半乳寡糖及衍生物可作为功能因子和益生元发挥防治急性肝损伤作用,具有这些结构特征的红藻半乳寡糖及其衍生物有效改善急性肝损伤患者肠道的紊乱,调节肠道菌群稳态,进而调节胆汁酸代谢(包括初级胆汁酸和次级胆汁酸),起到缓解肝脏损伤的作用。此外,红藻半乳寡糖及其衍生物可显著上调肝脏中CYP2E1的表达量,降低Bax、细胞核内NF-κBp65表达,激活Nrf2/HO-1信号通路以及FXR胆汁酸,发挥解毒作用,从而显著缓解CCl4造成的急性肝损伤,作为防治急性肝损伤及相关疾病的药物或保健品。
所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,在门水平上,该红藻半乳寡糖及其衍生物显著降低急性肝损伤小鼠结肠Bacteroidetes的相对丰度,同时提高Proteobacteria的相对丰度;在属水平上,该红藻半乳寡糖及其衍生物显著增加NAFLD小鼠结肠Bacteroides spp.、Helicobacter spp.和Anaerobiospirillumspp的相对丰度,并降低Prevotella spp.的相对丰度,使肠道菌群恢复正常水平。
所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物中的应用,红藻半乳寡糖及其衍生物可显著上调肝脏中CYP2E1的表达量,降低Bax、细胞核内NF-κB p65表达,激活Nrf2/HO-1信号通路以及FXR胆汁酸,该红藻半乳寡糖及其衍生物能显著降低氧化应激,发挥解毒作用,从而显著缓解CCl4造成的急性肝损伤,用做制备抗急性肝损伤、保肝、胰岛素抵抗、抗代谢综合征、抗高脂血症或降血脂的药物。
所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,该红藻半乳寡糖及其衍生物用于抗脂肪肝、保护肝脏或降脂的保健品;或者用于饮料、啤酒、饮食补充剂,或者与其它保肝的药物联用,或者与降血脂的药物联用;或者包含该红藻半乳寡糖及其衍生物的复配制剂;或以该红藻半乳寡糖及其衍生物为母核制备的衍生物用于抗肝损伤、抗胰岛素抵抗、抗代谢综合征的药物、功能食品或者生物制品中。
所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,该红藻半乳寡糖及其衍生物与联苯双酯、甘草酸二铵、双环醇、还原型谷胱甘肽或相关临床药物形成复配制剂。
本发明的优点及有益效果是:
1、本发明含D-和L-半乳糖残基的寡糖及其衍生物能改善急性肝损伤小鼠肠道菌群的紊乱和胆汁酸代谢失衡。
2、本发明含D-和L-半乳糖残基的寡糖及其衍生物具有显著的降低肝脏氧化应激和炎症以及肝脏纤维化效果,可用于肝损伤、脂肪肝、非酒精脂肪性肝炎等相关肝脏疾病的防治。
3、本发明产品的原料来源于海洋红藻多糖,具有资源丰富、制备工艺简单、产品稳定性好,易于产业化,安全性高、效果独特等优点,用于改善急性肝损伤所致肠道菌群的紊乱,在防治急性肝损伤及在保肝、降脂和代谢综合征等新药和特医食品的开发领域,具有广阔的开发应用前景。
4、本发明采用CCl4诱导构建的急性肝损伤动物模型对所制备的系列半乳寡糖进行缓解急性肝损伤等相关功能评价。研究结果表明,含D-和L-半乳糖残基及其衍生物的寡糖能显著改善CCl4诱导的急性肝损伤小鼠肠道菌群紊乱,进而调节胆汁酸代谢并显著降低肝脏氧化应激、炎症和肝脏损伤,进而对肝脏起到很好的保护作用,具有治疗急性肝损伤和保肝的作用。
附图说明
图1A图、B图、C图和D图分别为紫菜胶寡糖(PYOs)三糖、五糖、七糖和九糖的高分辨质谱图及结构式。图中,横坐标m/z代表质荷比,纵坐标Relative Abundance代表相对丰度。
图2A图、B图、C图分别为新琼四糖及其糖醇、糖酸的高分辨质谱图及结构式。图中,横坐标m/z代表质荷比,纵坐标Relative Abundance代表相对丰度。
图3为PYOs对CCl4诱导的急性肝损伤小鼠肝脏生理状态的改善效果图。Control代表腹腔注射生理盐水组;Model代表腹腔注射2mg/kg CCl4;DDB代表腹腔注射2mg/kg CCl4及200mg/kg/d联苯双酯;DGL代表腹腔注射2mg/kg CCl4及25mg/kg/d甘草酸二铵;PYOs-L代表腹腔注射2mg/kg CCl4及1mg/kg/d PYOs;PYOs-H代表腹腔注射2mg/kg CCl4及16mg/kg/dPYOs。
图4为PYOs对CCl4诱导的急性肝损伤小鼠肝脏功能的改善结果图。Control代表腹腔注射生理盐水组;Model代表腹腔注射2mg/kg CCl4;DDB代表腹腔注射2mg/kg CCl4及200mg/kg/d联苯双酯;DGL代表腹腔注射2mg/kg CCl4及25mg/kg/d甘草酸二铵;PYOs-L代表腹腔注射2mg/kg CCl4及1mg/kg/d PYOs;PYOs-H代表腹腔注射2mg/kg CCl4及16mg/kg/dPYOs。*P<0.05,与Control组相比;#P<0.05,与Model组相比;&P<0.05,PYOs-H与PYOs-L组相比。其中,A图为不同处理组小鼠血清中ALT(alanine aminotransferase,谷丙转氨酶)的酶活图;B图为不同处理组小鼠血清中AST(aspartate aminotransferase,谷草转氨酶)的酶活图;C图为不同处理组小鼠血清中TBA(total bile acid,总胆汁酸)的含量图。
图5为门水平PYOs对CCl4诱导的急性肝损伤小鼠结肠菌群结构的调节作用图。Control代表腹腔注射生理盐水组;Model代表腹腔注射2mg/kg CCl4;DDB代表腹腔注射2mg/kg CCl4及200mg/kg/d联苯双酯;DGL代表腹腔注射2mg/kg CCl4及25mg/kg/d甘草酸二铵;PYOs-L代表腹腔注射2mg/kg CCl4及1mg/kg/d PYOs;PYOs-H代表腹腔注射2mg/kg CCl4及16mg/kg/d PYOs。*P<0.05,与Control组相比;#P<0.05,与Model组相比;&P<0.05,PYOs-H与PYOs-L组相比。其中,A图为不同处理组小鼠结肠菌群的PCoA图;B图为不同处理组小鼠结肠菌群门水平的总体变化图,纵坐标Relative Abundance代表相对丰度(%);C图和D图分别代表小鼠肠道菌群在门水平Bacteroidetes和Proteobacteria的相对丰度变化,纵坐标Relative Abundance代表相对丰度(%)。图中,Bacteroidetes代表拟杆菌门,Firmicutes代表厚壁菌门,Proteobacteria代表变形菌门,Deferribacteres代表脱铁杆菌门,Candidatus Saccharibacteria代表暂定螺旋体门,Actinobacteria代表放线菌门,Tenericutes代表软壁菌门,Elusimicrobia代表迷踪菌门,Cyanobacteria/Chloroplast代表蓝藻菌门,Verrucomicrobia代表疣微菌门,Other代表其他菌门,Phylum Level Barplot代表菌门水平柱状图。
图6为属水平PYOs对CCl4诱导的急性肝损伤小鼠结肠菌群结构的调节作用图。Control代表腹腔注射生理盐水组;Model代表腹腔注射2mg/kg CCl4;DDB代表腹腔注射2mg/kg CCl4及200mg/kg/d联苯双酯;DGL代表腹腔注射2mg/kg CCl4及25mg/kg/d甘草酸二铵;PYOs-L代表腹腔注射2mg/kg CCl4及1mg/kg/d PYOs;PYOs-H代表腹腔注射2mg/kg CCl4及16mg/kg/d PYOs。*P<0.05,与Control组相比;#P<0.05,与Model组相比;&P<0.05,PYOs-H与PYOs-L组相比。其中,A图为不同处理组小鼠结肠菌群在属水平的总体变化图;B图、C图、D图和E图分别为Bacteroides spp、Anaerobiospirillum spp、Prevotella spp.和Helicobacter spp的相对丰度图,其中横坐标代表不同组别,纵坐标代表Relativeabundance(%)代表相对分度。图中,Bacteroidetes代表拟杆菌属,Phascolarctobacterium代表考拉杆菌属,Prevotella代表普雷沃菌属,Helicobacter代表螺杆菌属,Paraprevotella代表帕拉普氏菌属,Anaerobiospirillum代表厌氧螺菌属,Clostridium XlVa代表芽胞杆菌XlVa,Alloprevotella代表拟普雷沃菌属,Blautia代表布劳特氏菌属,Megamonas代表巨单胞菌属,Lactobacillus代表乳杆菌属,Mucispirillum代表粘液菌属,Parabacteroides代表狄氏副拟杆菌属,Barnesiella代表巴恩斯氏菌属,Escherichia/Shigella代表大肠杆菌/志贺氏菌属,Parasutterella代表副沙门氏菌属,Alistipes代表另枝菌属,Oscillibacter代表颤杆菌属,Roseburia代表罗斯氏菌属,Saccharibacteria_genera_incertae_sedis代表糖杆菌属,Other代表其他菌属。
图7为LEfSe分析PYOs对CCl4诱导的急性肝损伤小鼠结肠菌群关键系统发育类型的影响结果图。Control代表腹腔注射生理盐水组;Model代表腹腔注射2mg/kg CCl4;DDB代表腹腔注射2mg/kg CCl4及200mg/kg/d联苯双酯;DGL代表腹腔注射2mg/kg CCl4及25mg/kg/d甘草酸二铵;PYOs-L代表腹腔注射2mg/kg CCl4及1mg/kg/d PYOs;PYOs-H代表腹腔注射2mg/kg CCl4及16mg/kg/d PYOs。*P<0.05,与Control组相比;#P<0.05,与Model组相比;&P<0.05,PYOs-H与PYOs-L组相比。LDA:Latent Dirichlet Allocation。其中,A图为16mg/kg/dPYOs处理前后,急性肝损伤小鼠的肠菌改变LEfSe图;B图为16mg/kg/d PYOs处理前后,急性肝损伤小鼠的肠菌LDAscore差异分析结果图,选取了log10>2的进行分析。
图8为属水平与代谢相关参数之间的Spearman’s相关性热图分析揭示PYOs缓解CCl4诱导的急性肝损伤。Control代表腹腔注射生理盐水组;Model代表腹腔注射2mg/kgCCl4;DDB代表腹腔注射2mg/kg CCl4及200mg/kg/d联苯双酯;DGL代表腹腔注射2mg/kg CCl4及25mg/kg/d甘草酸二铵;PYOs-L代表腹腔注射2mg/kg CCl4及1mg/kg/d PYOs;PYOs-H代表腹腔注射2mg/kg CCl4及16mg/kg/d PYOs。*P<0.05,与Control组相比;#P<0.05,与Model组相比;&P<0.05,PYOs-H与PYOs-L组相比。其中,A图、B图、C图分别为与肝损伤(TBA、ALT和AST指标)、氧化应激水平(ROS、MDA、CAT和SOD水平)以及炎症因子(LBP、MCP-1、IL-1β、TNF-α水平)与肠道菌群之间的相关性分析。
图9为PYOs对CCl4诱导的急性肝损伤小鼠血清胆汁酸谱的结果图。Control代表腹腔注射生理盐水组;Model代表腹腔注射2mg/kg CCl4;DDB代表腹腔注射2mg/kg CCl4及200mg/kg/d联苯双酯;DGL代表腹腔注射2mg/kg CCl4及25mg/kg/d甘草酸二铵;PYOs-L代表腹腔注射2mg/kg CCl4及1mg/kg/d PYOs;PYOs-H代表腹腔注射2mg/kg CCl4及16mg/kg/dPYOs。*P<0.05,与Control组相比;#P<0.05,与Model组相比;&P<0.05,PYOs-H与PYOs-L组相比。其中,A图、B图、C图、D图、E图、F图分别为不同处理组βMCA(β-鼠胆酸)、ωMCA(ω-鼠胆酸)、TβMCA(牛磺-β-鼠胆酸)、TCA(牛磺胆酸)、GCA(甘氨胆酸)和TωMCA(牛磺-ω-鼠胆酸)的UPLC-MS/MS分析绝对定量结果图。
图10为PYOs对CCl4诱导的急性肝损伤小鼠肝脏中肝损伤相关蛋白表达水平westernblot结果图。Control代表腹腔注射生理盐水组;Model代表腹腔注射2mg/kg CCl4;DDB代表腹腔注射2mg/kg CCl4及200mg/kg/d联苯双酯;DGL代表腹腔注射2mg/kg CCl4及25mg/kg/d甘草酸二铵;PYOs-L代表腹腔注射2mg/kg CCl4及1mg/kg/d PYOs;PYOs-H代表腹腔注射2mg/kg CCl4及16mg/kg/d PYOs。其中,A图、B图、C图、D图、E图和F图分别为与CYP2E1(细胞色素P4502E1A)、Bax(Bcl2相关X蛋白)、Nuc NF-κB p65(核内的核因子κB p65)、NucNrf-2(核内核因子E2相关因子蛋白)、HO-1(Ⅰ型血红素氧合酶)和FXR1(法尼醇X受体)蛋白表达水平。
具体实施方式
在具体实施过程中,本发明以含有D-/L-半乳糖的红藻多糖为原料,经物理法、化学法、生物酶法或上述方法的任意组合进行可控降解,制备获得不同聚合度的红藻半乳寡糖及其衍生物,其分子骨架中含有D-半乳糖和L-半乳糖及其衍生物。本发明产品的原料来源于红藻多糖,具有资源丰富、制备工艺简单、安全性高,易于产业化等优点,作为益生元和功能因子,在改善急性肝损伤患者肠道菌紊乱,防治化学因素引起的急性肝损伤,及在护肝、防治肝脏纤维化及特医食品的开发领域,具有广阔的应用前景。
下面,结合具体实施例对本发明的技术方案作进一步的说明。
实施例1:含有6-O-硫酸-β-1,3-D-半乳糖(Gal6S)和α-1,4-L-3,6-内醚半乳糖(AnG)硫琼胶寡糖(SAOs)、硫琼胶寡糖醇(SAOs-OH)及硫琼胶寡糖酸(SAOs-OOH)的制备。
将1g硫琼胶多糖用摩尔浓度0.1M的稀硫酸配成10mg/mL水溶液,加热到60℃,搅拌降解1.5小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,然后加入3倍体积95%医用乙醇(体积浓度)于4℃过夜,离心收集沉淀,用少量水溶解后,用200Da透析袋透析脱盐,内液经旋蒸浓缩后冷冻干燥,得到SAOs。取100mg SAOs,将其溶于10mL摩尔浓度100mM的NaBH4水溶液(含摩尔浓度100mM的NaOH)于4℃过夜反应,加入醋酸调pH至7.0,经透析脱盐,冷冻干燥得寡糖醇SAOs-OH。再取200mg SAOs,将其溶于5mL新配制的本尼迪克试剂中,55℃加热反应至无砖红色沉淀产生,离心取上清液,经阳离子交换树脂去除残余铜离子,调pH至中性,经透析脱盐,冷冻干燥,得到寡糖酸SAOs-OOH。
所制得的SAOs系列硫琼胶寡糖醇、寡糖酸和寡糖的结构式如下:
式中,R=-SO3Na;n=0~30;
实施例2:含有β-1,3-D-半乳糖(Gal)和6-O-硫酸-α-1,4-半乳糖(Gal6S)的紫菜胶寡糖(PYOs)、寡糖醇(PYOs-OH)和寡糖酸(PYOs-OOH)的制备。
将紫菜胶用摩尔浓度0.1M的稀硫酸配成10mg/mL水溶液,加热到80℃,搅拌降解2小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,加入4倍体积95%医用乙醇于4℃过夜,离心收集沉淀并用少量水溶解后用200Da透析袋脱盐,经旋蒸浓缩、冷冻干燥得到紫菜胶寡糖PYOs(如图1A-D所示)。取PYOs寡糖150mg,将其溶于15mL摩尔浓度为150mM的NaBH4水溶液(含摩尔浓度150mM的NaOH)于4℃过夜反应,加入醋酸调pH至7.0,经透析脱盐,冷冻干燥,得紫菜胶寡糖醇PYOs-OH。再取100mg的PYOs,将其溶于3mL新配制的本尼迪克试剂中,55℃加热搅拌反应,至无转红色沉淀产生后,离心取上清液,经阳离子交换树脂去除残余铜离子,调pH至中性,经透析脱盐,冷冻干燥,得到紫菜胶寡糖酸PYOs-OOH。
所制备的紫菜胶PYOs寡糖醇、寡糖酸及其寡糖的结构式如下:
式中,R=-H,或-SO3Na;n=0~30;
实施例3:含有β-1,3-D-半乳糖(Gal)和α-1,4-L-3,6-内醚半乳糖(AnG)的琼胶寡糖及其寡糖醇和寡糖酸的制备。
将琼脂糖用热水溶解,采用摩尔浓度0.1M的稀盐酸配成10mg/mL溶液,于80℃搅拌降解0.5小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,然后加3.5倍体积95%医用乙醇于4℃过夜,离心收集沉淀,水溶解后用200Da透析袋透析脱盐,旋蒸浓缩并冷冻干燥得到琼寡糖AOs,进一步采用硼氢化钠还原得到琼胶寡糖醇AOs-OH,或者采用本尼迪克试剂氧化得到琼胶寡糖酸AOs-OOH。琼胶寡糖醇、寡糖酸及其寡糖的化学结构式如下:
式中,n=0~30;
实施例4:含有α-1,4-L-3,6-内醚半乳糖(AnG)和β-1,3-D-半乳糖(Gal)新琼胶寡糖及其糖醇和寡糖酸的制备。
将琼脂糖用60℃热水溶解,配成10mg/mL水溶液,放置35℃水浴锅内搅拌降温后,加入β-琼胶酶,恒温搅拌酶解3小时,立即置于95℃水浴锅使酶变性10分钟后冷却到室温,离心收集上清液,然后加3倍体积95%医用乙醇于4℃过夜,离心收集沉淀,少量水溶解后,用200Da透析袋透析脱盐,旋蒸浓缩并冷冻干燥得到新琼寡糖NAOs,进一步采用硼氢化钠还原得到新琼胶寡糖醇NAOs-OH,或者采用本尼迪克试剂氧化得到新琼胶寡糖酸NAOs-OOH。所制备的新琼胶寡糖醇、寡糖酸及寡糖的化学结构式如下:
式中,n=0~30;
为了验证所得寡糖醇的序列结构,可将酶解得到的新琼胶寡糖用Superdex 30柱分离纯化,得到新琼四糖纯品(图2A),采用碱性硼氢化钠还原方法制得新琼四糖糖醇,所得产品的高分辨质谱(ESI-MS)分析结果如图2B所示。同样,将所得新琼四糖采用本尼迪克定向氧化方法获得新琼四糖酸产品,其高分辨质谱(ESI-MS)分析结果如图2C所示。
实施例5:PYOs对CCl4诱导的急性肝损伤小鼠肝脏生理状态的影响。
昆明小鼠(雄性,18~22g)在SPF级动物房通风的笼子里饲养,饲养条件为在23℃~25℃下每天12h光照,自由饮水和进食。经过1周的适应期后,36只小鼠随机平均分为以下6组:Control组(腹腔注射等体积生理盐水);Model组(腹腔注射等体积生理盐水);DDB组(腹腔注射200mg/kg溶于生理盐水的联苯双酯);DGL组(腹腔注射25mg/kg溶于生理盐水的甘草酸二铵);PYOs-L组(腹腔注射1mg/kg溶于生理盐水的PYOs);PYOs-H组(腹腔注射16mg/kg溶于生理盐水的PYOs)。连续给药7天后,在最后一次给药后2h腹腔注射2mg/kg0.5%溶于橄榄油的CCl4,Contol组给予等量橄榄油。注射CCl4后48h处死小鼠,采集血液、肝脏和结肠内容物。一部分肝脏组织立即固定在4%多聚甲醛中用于组织病理学观察,另一部分肝脏组织则冻存于-80℃冰箱。H&E染色(苏木精—伊红染色法)结果表明(如图3所示),Control组肝细胞以中央静脉为中心呈放射状排列,结构正常;Model组能观察到严重的病理组织变化,肝细胞损伤和广泛坏死,细胞膜不清楚,炎性细胞浸润;DDB、DGL和PYOs组均能明显改善由CCl4造成的肝损伤,修复坏死细胞。其中,PYOs使肝脏组织形态趋于正常且组织坏死面积显著减少,呈现一定的剂量依赖性。
实施例6:PYOs对CCl4诱导的急性肝损伤小鼠肝脏功能的影响。
肝脏功能相关酶主要存在于肝脏组织内,但在肝细胞膜受损时可大量进入血液,血清中这些酶的活性可以反映肝损伤的程度。如图4A和4B所示,急性肝损伤小鼠血清中ALT和AST活性明显高于Control组(P<0.05),而PYOs处理组则显著逆转这种情况(P<0.05)。此外,PYOs显著降低急性肝损伤小鼠血清TBA水平(P<0.05)(图4C),TBA血清含量显著增加是肝损伤的重要指标。以上结果表明,16mg/kg/d的PYOs补充对ALI小鼠肝脏功能有显著的保护作用,且效果优于200mg/kg/d的DDB和25mg/kg/d的DGL。以上结果表明,PYOs显著改善CCl4诱导的急性肝损伤小鼠肝脏功能且呈现一定的剂量依赖性。
实施例7:PYOs对CCl4诱导的急性肝损伤小鼠结肠菌群门水平的影响。
研究表明,肝脏损伤的发生和发展与肠道菌群的紊乱密切相关。通过PCoA分析可知(图5A),与Control组相比,急性肝损伤小鼠结肠菌群的组成结构发生显著改变,但是补充PYOs显著改变了急性肝损伤小鼠结肠菌群的结构且更趋向于Control组。PYOs-L组和PYOs-H组的结肠菌群结构几乎完全分离,表明PYOs对结肠菌群的调控呈剂量依赖性(图5A)。PYOs处理会使急性肝损伤小鼠结肠菌群结构发生显著的变化,这表明PYOs能有效地重塑急性肝损伤小鼠结肠菌群。在门水平对不同处理组小鼠结肠菌群进行了比较分析(图5B),与Control组相比,急性肝损伤小鼠结肠的Bacteroidetes相对丰度升高(P<0.05)(图5C),而补充PYOs能够显著逆转这种情况(P<0.05);PYOs还以剂量依赖性的方式显著增加了急性肝损伤小鼠体内Proteobacteria的丰度(P<0.05)(图5D),这些变化对能改善小鼠急性肝损伤有积极作用。上述结果表明,PYOs可以在门水平改善急性肝损伤小鼠的结肠菌群紊乱,对急性肝损伤的缓解发挥积极的作用。
实施例8:PYOs对NAFLD小鼠肝脏氧化应激和炎症状态的影响。
在属水平评价PYOs对急性肝损伤小鼠结肠菌群的调节作用(图6A-E),与Model组相比,PYOs显著降低了急性肝损伤小鼠结肠中Prevotella spp.的相对丰度(P<0.05),同时升高了Bacteroides spp.、Helicobacter spp.和Anaerobiospirillum spp.的相对丰度(P<0.05),使它们恢复到相对正常的水平。这些结果表明,PYOs可以在属水平缓解急性肝损伤小鼠的结肠微生物群失调,进而缓解急性肝损伤。
实施例9:PYOs对CCl4诱导的急性肝损伤小鼠结肠菌群关键系统发育类型的影响。
采用LEfSe分析比较Model组和PYOs-H组结肠微生物群的变化(图7A-B),与Model组相比,PYOs可显著增加Proteobacteria和Deferribacteres的相对丰度(P<0.05),降低Bacteroidetes的相对丰度(P<0.05),它们的相对丰度与急性肝损伤的发生和发展密切相关。在科水平,PYOs-H显著提高了急性肝损伤小鼠的Deferribacteraceae和Ruminococcaceae的相对丰度(P<0.05),同时显著降低了急性肝损伤小鼠Eubacteriaceae和Eubacteriaceae的相对丰度(P<0.05)。在属水平,PYOs-H处理可显著增加Odoribacterspp.、Flavonifractor spp.和Mucispinillum spp.的相对丰度(P<0.05),同时减少了Eubacterium spp.、Corynebacterium spp.、Staphylococcus spp.和Parabacteroidesspp.的相对丰度(P<0.05)。上述结果表明,PYOs改善了急性肝损伤小鼠结肠微生物群的紊乱,包括有益菌的增加和有害菌的减少,这有利于宿主健康。
实施例10:PYOs调控的结肠菌群与急性肝损伤小鼠生理指标的相关性分析。
通过Spearman相关性分析阐明结肠菌群与急性肝损伤小鼠生理指标之间的关系(图8A-C),Butyricimonas spp.丰度与血清ALT、ALP和TBA水平呈正相关;Alloprevotellaspp.、Bacteroides spp和Butyricimonas spp.丰度与CAT、SOD呈正相关,与ROS、MDA、IL-1β、TNFα、MCP-1、LBP含量呈负相关。综上所述,PYOs可通过调节上述菌属的丰度改善急性肝损伤小鼠体内脂质积累、氧化应激水平、炎症反应和肝功损伤,从而缓解CCl4诱导的急性肝损伤。
实施例11:PYOs对CCl4诱导的急性肝损伤小鼠血清胆汁酸谱的影响。
由图9A-F可知,急性肝损伤小鼠血清中总胆汁酸水平显著较Control组小鼠显著增加(P<0.05)。PYOs补充可显著改善上述不利状况,具体来讲:PYOs降低ALI小鼠血清初级胆汁酸,如βMCA、UDCA、TβMCA、TUDCA、TωMCA和GCA的含量;PYOs降低急性肝损伤小鼠次级胆汁酸,如TCA和ωMCA的含量。这表明,PYOs可能通过调节急性肝损伤小鼠肠道微生物进而调节肠道中胆汁酸的代谢,并通过肝门静脉循环入肝脏内发挥其调节作用,从而改善CCl4诱导的急性肝损伤。
实施例12:PYOs对CCl4诱导的急性肝损伤小鼠肝损伤相关蛋白表达水平的影响。
由图10A-F可知,与正常组小鼠相比,急性肝损伤小鼠肝脏中CYP2E1、Nuc Nrf-2和FXR1的表达量显著降低(图10A、D和F),Bax、Nuc NF-κB p65和HO-1(图10B、C和E)的表达量显著提高。然而,PYOs可显著上调肝脏中CYP2E1的表达量,降低Bax、细胞核内NF-κB p65表达,激活Nrf2/HO-1信号通路以及FXR胆汁酸,发挥解毒作用,从而显著缓解CCl4造成的急性肝损伤。
利用CCl4诱导的急性肝损伤小鼠模型评价了PYOs的抗急性肝损伤功效。结果表明,PYOs减少CCl4诱导的急性肝损伤小鼠肝脏生理学改变,缓解肝脏损伤和改善肝脏功能。PYOs缓解CCl4诱导的ALI小鼠结肠菌群紊乱。在门水平,PYOs显著降低Bacteroidetes相对丰度,增加Proteobacteria的相对丰度;在属水平,PYOs显著增加Bacteroides spp.、Helicobacter spp.和Anaerobiospirillum spp.的相对丰度,同时降低Prevotella spp.的相对丰度。这些结果表明PYOs可以在门水平和属水平缓解急性肝损伤小鼠的结肠微生物群紊乱。通过Spearman相关性分析阐明结肠菌群与急性肝损伤小鼠生理指标之间的关系。结果表明,PYOs在减少急性肝损伤小鼠体内有害微生物含量的同时,增加了益生菌的相对丰度,它们中特定菌属与改善肝损伤、提高肝脏功能、降低氧化应激水平和炎症反应密切相关。PYOs通过改变急性肝损伤小鼠血清胆汁酸谱,降低血清总胆汁酸含量以及在ALI中发挥重要作用的胆汁酸水平,发挥缓解急性肝损伤的作用。此外,PYOs可显著上调肝脏中CYP2E1的表达量,降低Bax、细胞核内NF-Kb p65表达,激活Nrf2/HO-1信号通路以及FXR胆汁酸,发挥解毒作用,从而显著缓解CCl4造成的急性肝损伤。综上所述,与阳性药DDB和DGL相比,在较低剂量时PYOs具有良好的益生元效果,可能通过改善急性肝损伤小鼠结肠菌群紊乱,发挥调节血清胆汁酸水平的作用,进而激活gut-liver axis发挥缓解肝脏损伤和改善肝脏功能的效果,这也为治疗急性及其相关疾病提供了新的思路。
综上,本发明的寡糖通过改善肠道菌群稳态及相关胆汁酸代谢水平进而起到抗急性肝损伤的效果,宜于作为抗急性肝损伤、保肝护肝、改善非酒精性脂肪性肝炎或者与其他保肝药物(如联苯双酯、甘草酸二铵、双环醇、还原型谷胱甘肽)形成复配制剂的应用。实施例结果表明,本发明的紫菜胶寡糖(PYOs)可作为益生元,调节急性肝损伤相关肠道菌群紊乱,从而发挥抗急性肝损伤作用,可作为防治急性肝损伤相关疾病的药物或保健品。PYOs能够显著减少肝组织损伤、缓解肝脏氧化应激和炎症,从而实现保护肝脏和缓解急性肝损伤的作用。本发明产品来源于海洋红藻寡糖,具有资源丰富、易于产业化,安全有效等诸多优点,在防治急性肝损伤、肝脏保护、肝脏纤维化以及作为保肝护肝药物等方面具有广阔的开发应用前景。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例中所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (8)
2.按照权利要求1所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,其特征在于,以富含D-/L-半乳糖及其衍生物的红藻多糖为原料,经物理降解、化学降解、酶法降解之一种或两种以上降解方法的组合,制备不同聚合度的寡糖及其衍生物,所制备的化合物结构中同时含有β-1,3-D-半乳糖(D-Gal)残基和α-1,4-L-半乳糖(L-Gal)残基,或同时含有D-Gal与α-1,4-L-3,6-内醚半乳糖(L-AnG)残基;在D-Gal与L-Gal糖残基的C6位羟基含有不同程度的硫酸酯基(Gal6S);所制备寡糖的非还原端是Gal、Gal6S或AnG,还原端是Gal或糖醇(Gal-OH)与糖酸(Gal-OOH),或AnG糖醇(AnG-OH),或Gal6S及其糖醇(Gal6S-OH)与糖酸(Gal6S-OOH)。
3.按照权利要求2所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,其特征在于,该红藻半乳寡糖及其衍生物采用下述制备工艺:
将红藻来源的琼脂糖(Agarose)溶于60℃热水,用缓冲液配成10mg/mL溶液,放置于30℃水浴锅内添加β-琼胶酶(CAS#37288-57-6)并搅拌酶解4小时,冷却后离心,收集上清液,加3倍体积95%医用乙醇于4℃过夜,离心,收集沉淀并用水将其溶解,用200Da透析袋透析脱盐,将内液旋蒸浓缩并冷冻干燥,得到偶数新琼寡糖,并进一步采用硼氢化钠还原得到偶数新琼寡糖醇,或者用本尼迪克试剂氧化得到偶数新琼胶寡糖酸;或者,将琼脂糖用60℃热水溶解,采用摩尔浓度0.1M稀盐酸配成10mg/mL溶液,于80℃搅拌降解0.5小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,然后加3倍体积95%医用乙醇于4℃过夜,离心收集沉淀,将沉淀用水溶解后,用200Da透析袋透析脱盐,旋蒸浓缩并冷冻干燥得到奇数琼胶寡糖,进一步采用硼氢化钠还原得到奇数琼胶寡糖醇,或者采用本尼迪克试剂氧化得到奇数琼胶寡糖酸;或者,将硫琼胶(Agaropectin)用摩尔浓度0.1M稀硫酸配成10mg/mL水溶液,加热到60℃后搅拌降解1.5小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,然后加入3倍体积95%医用乙醇于4℃过夜,离心收集沉淀,将沉淀溶水溶解后,用200Da透析袋透析脱盐,之后旋蒸浓缩并冷冻干燥得到硫琼胶寡糖,然后进一步经硼氢化钠还原得到硫琼胶寡糖醇,或者采用本尼迪克试剂氧化得到硫琼胶寡糖酸;或者,将紫菜胶(Porphyran)用摩尔浓度0.1M稀硫酸配成10mg/mL水溶液,加热到80℃后搅拌降解2小时,冷却后用摩尔浓度2M的NaOH水溶液中和,离心收集上清液,然后加入3倍体积95%医用乙醇于4℃过夜,离心收集沉淀,将沉淀用水溶解后,用200Da透析袋透析脱盐,之后旋蒸浓缩并冷冻干燥得到紫菜胶寡糖,进一步采用硼氢化钠还原得到紫菜胶寡糖醇,或者采用本尼迪克试剂氧化得到紫菜胶寡糖酸。
4.按照权利要求1至3之一所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,其特征在于,半乳寡糖及衍生物作为益生元和功能因子发挥防治急性肝损伤作用,具有这些结构特征的红藻半乳寡糖及其衍生物有效改善急性肝损伤患者肠道的紊乱,通过改善肠道菌群和胆汁酸代谢紊乱,发挥肝脏抗氧化应激、抗炎、减少肝细胞损伤,作为防治急性肝损伤及相关疾病的药物或保健品。
5.按照权利要求4所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,其特征在于,在门水平上该红藻半乳寡糖及其衍生物显著增加急性肝损伤小鼠结肠内Proteobacteria的相对丰度,同时降低Bacteroidetes的相对丰度;在属水平上,该红藻半乳寡糖及其衍生物显著增加急性肝损伤小鼠结肠内Bacteroides spp.、Helicobacter spp.和Anaerobiospirillum spp.的相对丰度,并降低Prevotella spp.的相对丰度,显著改善急性肝损伤小鼠肠道菌群紊乱。
6.按照权利要求4所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,其特征在于,该红藻半乳寡糖及其衍生物显著上调急性肝损伤个体肝脏中CYP2E1的表达量,降低Bax、细胞核内NF-κB p65表达,激活Nrf2/HO-1信号通路以及FXR胆汁酸,发挥解毒作用,从而显著缓解CCl4造成的氧化应激、炎症、肝脏细胞损伤,作为防治急性肝损伤及相关疾病的药物或保健品。
7.按照权利要求4所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,其特征在于,该红藻半乳寡糖及其衍生物用于抗急性肝损伤或保护肝脏的保健品;或者用于饮料、啤酒、饮食补充剂,或者与其它保肝的药物联用;或者包含该红藻半乳寡糖及其衍生物的复配制剂;或以该红藻半乳寡糖及其衍生物为母核制备的衍生物用于抗急性肝损伤的药物、功能食品或者生物制品中。
8.按照权利要求4所述的红藻半乳寡糖及其衍生物在作为防治急性肝损伤药物和特医食品中的应用,其特征在于,该红藻半乳寡糖及其衍生物与联苯双酯、甘草酸二铵、双环醇、还原型谷胱甘肽或相关临床口服药物形成复配口服制剂。
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