CN116637141B - 一种基于微生物发酵得栀子降尿酸代谢产物发酵浸膏的方法 - Google Patents
一种基于微生物发酵得栀子降尿酸代谢产物发酵浸膏的方法 Download PDFInfo
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- CN116637141B CN116637141B CN202310453575.8A CN202310453575A CN116637141B CN 116637141 B CN116637141 B CN 116637141B CN 202310453575 A CN202310453575 A CN 202310453575A CN 116637141 B CN116637141 B CN 116637141B
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Abstract
本发明属于生物技术领域，具体涉及一种基于微生物发酵得栀子降尿酸代谢产物发酵浸膏的方法。该方法通过以下步骤实现：（1）复苏菌株后于灭菌后的MRS培养基中培养得种子液；（2）将栀子粉和蒸馏水混合后灭菌，然后将混合浆料接种种子液，接种后进行厌氧发酵，将发酵液过滤浓缩得栀子降尿酸代谢产物发酵浸膏。本发明利用微生物发酵转化技术和质谱分子网络，开展以抑制XOD酶活性为导向的天然产物发现研究，为天然来源治疗高尿酸血症药物先导化合物的发现探索新途径，对本学科的发展具有重要的科学价值和现实意义。
Description
技术领域
本发明属于生物技术领域，具体涉及一种基于微生物发酵得栀子降尿酸代谢产物发酵浸膏的方法。
背景技术
高尿酸血症（Hyperuricemia）主要是由于人体嘌呤类物质代谢发生紊乱而引起的一种代谢类疾病，主要病理表现为体内尿酸生成增多或尿酸排泄明显减少而引起人体血清中尿酸浓度异常增高的现象。近年来，随着我国社会经济的发展，居民的饮食结构发生了较大改变，由于高嘌呤食物摄入过多，使得高尿酸血症发病率呈逐年上升趋势。流行病学调查显示，近10年我国高尿酸血症患病率为5.5%-19.3%，痛风患病率为0.8%-2.2%。高尿酸血症不仅发病率高，还可引起一系列的并发症。肾功能损伤是其常见的并发症，这是由于尿酸盐结晶在肾小管部位不断沉积而引发的肾脏组织炎症反应，表现为血液中尿酸、肌酐、尿素氮等指标的异常升高。此外，高尿酸血症患者还往往伴有冠心病、心血管疾病、糖尿病等，严重者甚至引起尿毒症、肾衰竭而导致死亡。高尿酸血症严重威胁人类健康，已成为不容忽视的全民健康问题。
基于对高尿酸血症发病机制的研究，目前临床上治疗高尿酸血症的主要策略为减少尿酸生成和促进尿酸排泄。黄嘌呤氧化酶（XOD）作为一种广泛表达于肝脏、肾脏等的羟化酶，为尿酸生成的限速酶，催化次黄嘌呤转化为黄嘌呤，再进一步氧化为尿酸。大量证据表明，抑制XOD活性进而减少尿酸生成是当前临床上控制高尿酸血症的一线治疗手段。
目前经FDA批准上市的黄嘌呤氧化酶抑制剂类药物包括别嘌醇和非布索坦，但是临床数据报告显示，长期服用别嘌醇容易引起皮肤不良反应、白细胞减少等血液系统疾病，严重时可引起继发性感染或内脏器官功能衰竭。非布索坦虽具有较好的治疗效果，但其易引起头痛、肝功能和肾功能损伤。高尿酸血症治疗药物多具有明显的副作用，临床使用并不理想。因此，研究开发新型低毒高效的治疗高尿酸血症药物具有重要的现实意义。
发明内容
针对现有技术中存在的天然来源创新药物研发过程中效率低、针对性差等问题，本发明提供了一种基于微生物发酵得栀子降尿酸代谢产物的方法。
本发明为了实现上述目的所采用的技术方案为：
本发明提供了一种基于微生物发酵得栀子降尿酸代谢产物发酵浸膏的方法，包括以下步骤：
（1）复苏菌株后于灭菌后的MRS培养基中培养得种子液；
（2）将栀子粉和蒸馏水混合后灭菌，然后将混合浆料接种种子液，接种后进行厌氧发酵，将发酵液过滤浓缩得栀子降尿酸代谢产物发酵浸膏。
本发明发酵过程中，所使用的菌株为植物乳杆菌（LPLC）、嗜酸乳杆菌（LACC）和长双岐杆菌（BLOC）；所述培养为37 ℃培养1 d。
进一步的，步骤（2）中，所述栀子粉和蒸馏水的比例为6 g:80 mL；所述灭菌为在121 ℃下，灭菌25 min；所述种子液接种的浓度为108CFU/mL；所述厌氧发酵为37 ℃厌氧培养3 d。
本发明通过发酵得到的栀子降尿酸代谢产物发酵浸膏中，栀子降尿酸代谢产物的分离方法为：将发酵液过滤浓缩得栀子降尿酸代谢产物发酵浸膏，将发酵浸膏分散在10倍体积的水中，经石油醚、乙酸乙酯、正丁醇依次萃取得石油醚部位、乙酸乙酯部位、正丁醇部位和水部位四个组分，经过XOD酶活性导向，对乙酸乙酯部位通过正反硅胶柱色谱和HPLC半制备柱分离纯化，得栀子降尿酸代谢产物。
上述栀子降尿酸代谢产物的具体分离纯化过程为：
（a）将乙酸乙酯部位通过200-300目的硅胶柱分离，并用CH2Cl2/MeOH逐步洗脱产生六个组分Fr1-Fr6；
（b）Fr2通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和SephadexLH-20分离后，再用半制备型HPLC制备得到芹菜素（apigenin）、木犀草素（luteolin）、异鼠李素（isorhamnetin）和甲基麦冬黄酮A（methylophiopogonone A）；
（c）Fr3通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和SephadexLH-20分离后，由半制备型HPLC制备得到吐叶醇（blumenol A）和阿魏酸（ferulic acid）；
（d）Fr4通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和SephadexLH-20后，由半制备型HPLC制备得到南烛木树脂酚（lyoniresinol）和柳叶柴胡酚（salicifoliol）。
进一步的，步骤（a）中，所述CH2Cl2/MeOH逐步洗脱的比例为20:1、10:1、5:1、2:1、1:1、1:5、0:1。
进一步的，步骤（b）中，所述石油醚/乙酸乙酯的洗脱比例为2:1；所述SephadexLH-20为MeOH/CH2Cl2= 1/1；所述半制备型HPLC的参数为：MeOH/H2O = 60/40，2.0mL/min。
进一步的，步骤（c）中，所述石油醚/乙酸乙酯的洗脱比例为3:2；所述SephadexLH-20为MeOH/CH2Cl2= 1/1；所述半制备型HPLC的参数为：MeOH/H2O = 50/50，2.0mL/min。
进一步的，步骤（d）中，所述石油醚/乙酸乙酯的洗脱比例为1:1；所述SephadexLH-20为MeOH/CH2Cl2= 1/1；所述半制备型HPLC的参数为：MeOH/H2O = 40/60，2.0mL/min。
本发明通过发酵后分离纯化得到的栀子苷尿酸代谢产物为：
。
本发明通过微生物发酵对栀子粉进行转化，通过微生物细胞将复杂的底物进行结构修饰和改造，以微生物为载体，利用其代谢过程中产生的酶对外源化合物进行的水解、氧化还原、脱水、缩合、羟基化等催化反应，具有较高的区域和立体选择性更易得到结构新颖的化合物，为新药开发提供更有价值的先导化合物。
本发明的有益效果为：
（1）本发明通过肠道微生物发酵栀子，发酵之后的栀子浸膏对XOD酶活性的抑制作用显著增强（提高了4.2倍），生成了黄酮类、环烯醚萜类、生物碱类等差异代谢物；对发酵前后栀子的浸膏进行UPLC-QTOF-HRMS实验，对数据结果进行代谢组学分析，结果显示发酵组较未发酵组（GFE）的化学成分出现了较显著的差异，以长双岐杆菌（BLOC）发酵组/GFE组为例，一是化学成分的相对含量发生了变化，如栀子苷发生水解反应生成了入血苷元京尼平（含量增加了33.89倍）；二是新产生了栀子中原来没有的化合物，如原有化学成分发生氧化、酯化等反应新产生了harpagide、eugenol acetate、isorhapontigenin等化合物。
（2）本发明利用微生物发酵转化技术和质谱分子网络，开展以抑制XOD酶活性为导向的天然产物发现研究，为天然来源治疗高尿酸血症药物先导化合物的发现探索新途径，对本学科的发展具有重要的科学价值和现实意义。
附图说明
图1为栀子发酵浸膏对高尿酸血症小鼠的影响；血浆中尿酸（A）、尿素氮（B）、肌酐（C）含量；肝脏XOD酶活性（D）；小鼠肾脏组织病理形态图（E）、病理切片评分（F）；与正常组相比，#P<0.05，##P<0.01；与模型组相比，*P<0.05，**P<0.01；
图2为微生物发酵栀子浸膏的酶促反应活性对比图；
图3为栀子发酵浸膏对小鼠血浆中尿酸（A）、尿素氮（B）、肌酐（C）和小鼠肾脏（D、E）的影响；与正常组相比，##P<0.01；与模型组相比，*P<0.05；
图4为栀子发酵前后对XOD酶活性的影响；与正常组相比，#P<0.05；与模型组相比，*P<0.05；
图5为栀子发酵前后对肾脏中OAT1（A：肾脏切片免疫组化染色；B：免疫组化评分）和URAT1（C：肾脏切片免疫组化染色；D：免疫组化评分）表达的影响；
图6 栀子发酵浸膏的化学成分分析。（A）总离子流图（TIC图）；（B）正负离子条件下的PCA分析图；（C）以BLOC/GFE代表的差异代谢物火山图；
图7为BLOC发酵栀子发生的代表性反应和产生的代表性差异代谢物；
图8为代谢产物apigenin的氢谱（左）和碳谱（右）；
图9为代谢产物luteolin的氢谱（左）和碳谱（右）；
图10为代谢产物ferulic acid的氢谱（左）和碳谱（右）；
图11为代谢产物lyoniresinol的氢谱（左）和碳谱（右）。
具体实施方式
下面通过具体的实施例对本发明的技术方案作进一步的解释和说明。
实施例1
（1）目标菌株筛选确定和发酵条件优化
目标菌株筛选确定：选取8种肠道微生物包括长双歧杆菌（Bifidobacteriumlongum, BLOC）、短双歧杆菌（B.breve, BBRC）、嗜酸乳杆菌（Lactobacillus acidophilus, LACC）、副干酪乳杆菌（L.paracasei, LPAC）、干酪乳杆菌（L.casei, LCAC）、鼠李糖乳杆菌（L.rhamnosus, LRHC）、植物乳杆菌（L.plantarum, LPLC）和保加利亚乳杆菌（L.bulgaricus, LBUC）作为研究对象，复苏菌株后于灭菌后的MRS培养基中培养1 d得种子液，250 mL锥形瓶中加入6 g栀子粉（过80目筛）和80 mL蒸馏水于灭菌锅中121 ℃，灭菌25 min。接种2 mL种子液（浓度为108CFU/mL），厌氧培养3 d对栀子进行发酵转化，将发酵液进行过滤浓缩，得发酵浸膏。
效果实施例1
综合利用体外XOD酶活实验，HepG2细胞模型和氧嗪酸钾/腺嘌呤建立的高尿酸血症小鼠模型对发酵浸膏的降尿酸活性进行评价。分别采用磷钨酸还原法、肌氨酸氧化酶法、脲酶法评价各组发酵浸膏对尿酸、肌酐和尿素氮水平的影响。
通过对发酵浸膏进行UPLC-QTOF-HRMS检测，整合化合物信息，通过代谢组学的数据处理软件进行处理，如主成分分析（PCA），正交偏最小二乘判别分析（OPLS-DA）为菌株中化合物的信息提供可视化的分析结果，以此筛选出具有产生独特化学成分的菌株。结合体内外降尿酸活性测试结果和发酵浸膏表现出的化学特征，筛选活性较强、代谢产物具有特异性和多样性的目标菌株。最终确定长双歧杆菌（BLOC）、植物乳杆菌（LPLC）和嗜酸乳杆菌（LACC）为后续的研究对象。
（一）XOD酶抑制活性化合物的筛选
化合物抑制XOD酶的体外活性筛选：利用体外酶促反应检测化合物对XOD酶的抑制作用，用磷酸缓冲液（PBS，10 mM，pH = 7.4）作为溶解液，将50 μL（100 U/L）的黄嘌呤氧化酶和100 μL单体化合物（100 μM）混合，同时别嘌醇（allopurinol，100 μM）为阳性对照组，PBS组为空白对照组。37 ℃恒温箱孵育5 min，然后加入50 μL的黄嘌呤（0.5 mM）引发反应，30 min后通过酶标仪测量每个孔的OD值，分析各组的抑制率。
化合物抑制XOD酶的体内活性筛选：对量大且体外活性较好的化合物利用建立的采用氧嗪酸钾/腺嘌呤建立的高尿酸血症小鼠模型，对活性化合物的降尿酸活性进行验证和作用机制研究。小鼠造模成功后随机分为高尿酸血症模型组、阳性对照组（allopurinol，10 mg/kg）、化合物给药组（10 mg/kg）等。连续治疗给药14天后，分别采用磷钨酸还原法、肌氨酸氧化酶法、脲酶法评价各化合物对尿酸、肌酐和尿素氮水平的影响。利用XOD酶催化次黄嘌呤生成黄嘌呤时产生的超氧阴离子自由基与电子受体及显色剂生成紫红色结合物，于530 nm处测吸光度，计算XOD酶的活力。通过Western-Blot等实验检测与尿酸生成相关蛋白的表达情况；病理切片HE染色分析肝脏和肾脏组织病变情况。
（二）栀子具有降尿酸活性和肾脏保护作用
利用高尿酸血症模型对栀子的降尿酸活性进行了评价，阳性对照组分别为别嘌醇组（Allopurinol，给药量10 mg/kg）和苯溴马隆组（Benzbromarone，给药量5 mg/kg），栀子给药组分为低、中、高三个剂量组（GFL、GFM、GFH，栀子生药给药量分别为0.5、1、2 g/kg）。结果表明，与模型组相比，栀子给药组小鼠血浆中的尿酸（图1A）、尿素氮（图1B）、肌酐（图1C）的含量明显降低。HE病理切片和病理切片评分表明（图1E和1F），栀子能够缓解氧嗪酸钾/腺嘌呤联合给药造成的小鼠肾小管萎缩、扩张，细胞坏死以及炎性细胞浸润。XOD酶作为尿酸生成的重要靶点，虽然栀子也能降低小鼠肝脏中XOD酶的活性（图1D），但与模型组和阳性药组相比栀子给药组对小鼠XOD酶的抑制活性不强，这一发现提示我们可以通过提高栀子对XOD酶的抑制作用来增强栀子的降尿酸活性。
（三）体内外实验证明微生物发酵能提高栀子的降尿酸活性
（1）栀子发酵浸膏对XOD酶活性的影响
发明人选取8种肠道微生物对栀子进行发酵转化，对得到的发酵浸膏（未发酵栀子和不同菌种发酵栀子组给药浓度均为200 μg/mL）进行体外抑制XOD酶活性的研究。结果表明，在同等给药剂量条件下，未发酵的栀子组（GFE）不能显著的降低XOD酶活性，而植物乳杆菌（LPLC）、嗜酸乳杆菌（LACC）和长双岐杆菌（BLOC）发酵栀子组显示出较强的抑制XOD酶活性（图2）。
利用建立的高尿酸血症小鼠模型对体外降尿酸活性较强的LPAC、LACC和BLOC发酵栀子组进行体内降尿酸活性验证。结果表明，与模型组和GFE组相比，BLOC发酵栀子组小鼠血浆中的尿酸（图3A）、尿素氮（图3B）、肌酐（图3C）的含量明显降低。HE病理切片和病理评分表明，栀子发酵浸膏能够缓解肾小管萎缩、扩张，细胞坏死以及炎性细胞浸润，特别是LPLC发酵栀子组显示较强的肾脏保护活性（图3D和E）。以上结果表明，栀子经肠道微生物发酵后，其降尿酸活性和肾脏保护作用进一步提高。
（2）栀子发酵浸膏降尿酸的作用机制研究
对小鼠肝脏中尿酸生成关键酶XOD的活性进行评价，结果显示，与模型组和未发酵栀子组相比，发酵后的栀子能够显著的抑制肝脏XOD酶的活性（图4）。特别是BLOC发酵栀子组的活性比阳性药allopurinol组强。同时利用免疫组化方法检测了尿酸分泌排泄、重吸收的关键蛋白OAT1和URAT1的表达，结果表明相较于高尿酸血症模型组和未发酵栀子组，栀子发酵产物特别是LACC组和BLOC组能够提高尿酸分泌蛋白OAT1的表达（图5A和B），降低尿酸重吸收蛋白URAT1的表达（图5C和D）。
综合以上药理学研究结果表明，经微生物发酵后的栀子显示了较强的降尿酸和肾脏保护作用，特别是发酵栀子较未发酵栀子对尿酸生成的关键酶XOD具有更显著的抑制活性。
效果实施例2微生物发酵能够提高栀子化学成分的多样性
（1）栀子发酵浸膏的代谢组学分析
对发酵前后栀子的浸膏进行UPLC-QTOF-HRMS实验，对数据结果进行代谢组学分析。利用中药色谱指纹图谱相似度评价系统比较发酵前后的共有峰和非共有峰，分析发酵浸膏的整体化学成分变化（图6A）。根据相似度计算结果显示，LACC和BLOC组与未发酵GFE组相比相似度最低，分别为0.584和0.611，说明LACC和BLOC组与未发酵GFE组相比化学成分差异较大。主成分分析（PCA）显示各组发酵浸膏样本之间的总体代谢产物与GFE存在差异（6B），火山图（6C）显示了各组与GFE组比较的差异代谢物，如BLOC/GFE有266个差异代谢物（相对含量差异倍数大于2或小于0.5），主要包括黄酮类、环烯醚萜类、生物碱类等。经过微生物的发酵栀子浸膏产生了差异代谢物，一是化学成分的相对含量发生了变化，以栀子的活性成分环烯醚萜类化合物为例，BLOC发酵栀子后栀子苷（geniposide）发生水解反应，生成入血成分苷元京尼平（genipin）使其含量增加了33.89倍。p-coumaric acid经过乙酰化、氧化、甲基化反应生成ethyl ferulate，使后者的含量提高了219.74倍（图7）；二是新产生了栀子中原来没有的化合物，如BLOC发酵栀子浸膏中新产生了harpagide、eugenolacetate、isorhapontigenin等化合物。
综合以上药理学和化学分析结果，微生物发酵栀子后栀子浸膏对尿酸生成关键酶XOD活性的抑制作用增强，原有化学成分发生了变化。
（2）栀子发酵浸膏中代谢产物的初步分离
对BLOC发酵栀子的发酵浸膏进行过滤浓缩，浓缩液分散在水中，经过石油醚、乙酸乙酯、正丁醇依次萃取得石油醚、乙酸乙酯、正丁醇和水部位，经过XOD酶活性导向、正反硅胶柱色谱和HPLC半制备柱色谱分离纯化，目前从乙酸乙酯部位共分离鉴定了8个化合物，包括4个黄酮类化合物，其中的4个黄酮类化合物和blumenol A、lyoniresinol为首次从栀子中分离得到。
将发酵液过滤浓缩得栀子降尿酸代谢产物发酵浸膏，将浓缩液分散在水中，经石油醚、乙酸乙酯、正丁醇依次萃取得石油醚部位、乙酸乙酯部位、正丁醇部位和水部位四个组分，经过XOD酶活性导向、正反硅胶柱色谱和HPLC半制备柱分离纯化，得栀子降尿酸代谢产物。
XOD酶活性导向：利用体外酶促反应检测化合物对XOD酶的抑制作用，用磷酸缓冲液（PBS，10 mM，pH = 7.4）作为溶解液，将50 μL（100 U/L）的黄嘌呤氧化酶分别与待测的四个组分（100 μL，100 μg/mL）混合，同时别嘌醇（allopurinol，100 μM）为阳性对照组，PBS组为空白对照组。37 ℃恒温箱孵育5 min，然后加入50 μL的黄嘌呤（0.5 mM）引发反应，30min后通过酶标仪测量每个孔的OD值，分析各组的抑制率。石油醚部位、乙酸乙酯部位、正丁醇部位和水部位四个组分的抑制率分别为15%、53%、40%和20%，乙酸乙酯部位的XOD酶抑制活性较高，接下来采用色谱分离技术对乙酸乙酯部位进行分离纯化。
乙酸乙酯提取物通过200-300目的硅胶柱分离，并用CH2Cl2/MeOH逐步洗脱产生六个组分Fr1-Fr6；Fr2通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和SephadexLH-20（MeOH/CH2Cl2= 1/1）分离后，再用半制备型HPLC（MeOH/H2O= 60/40，2.0mL/min）制备得到apigenin（5.3 mg）、luteolin（8.2 mg）、isorhamnetin（10.5 mg）和methylophiopogononeA（15.2 mg）；Fr3通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和SephadexLH-20（MeOH/CH2Cl2= 1/1）分离后，由半制备型HPLC（MeOH/H2O = 50/50，2.0mL/min）制备得到blumenolA（5.3 mg）和ferulic acid（25.8 mg）；Fr4通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和Sephadex LH-20后，由半制备型HPLC（MeOH/H2O = 40/60，2.0mL/min）制备得到lyoniresinol（4.5 mg）和salicifoliol（6.6 mg）。
apigenin：黄色固体，易溶于二氯甲烷。ESI-MSm/z: 269.05 [M-H]-，结合核磁碳信号，分子式确定为C15H10O5，不饱和度为11；氢谱碳谱图如图8所示。
luteolin：黄色固体，易溶于二氯甲烷。ESI-MSm/z: 287.05 [M+H]+，结合核磁碳信号，分子式确定为C16H10O6，不饱和度为12；氢谱碳谱图如图9所示。
isorhamnetin：黄色固体，易溶于二氯甲烷。ESI-MSm/z: 315.05 [M-H]-，结合核磁碳信号，分子式确定为C16H12O7，不饱和度为11。
methylophiopogonone A：黄色固体，易溶于二氯甲烷。ESI-MSm/z: 339.09 [M-H]-，结合核磁碳信号，分子式确定为C19H16O6，不饱和度为12。
blumenol A：固体，易溶于丙酮。ESI-MSm/z: 225.14 [M+H]+，结合核磁碳信号，分子式确定为C13H20O3，不饱和度为4。
ferulic acid：固体，易溶于二氯甲烷。ESI-MSm/z: 195.06 [M+H]+，结合核磁碳信号，分子式确定为C10H10O4，不饱和度为6；氢谱碳谱图如图10所示。
lyoniresinol：固体，易溶于甲醇。ESI-MSm/z: 421.18 [M+H]+，结合核磁碳信号，分子式确定为C22H28O8，不饱和度为9；氢谱碳谱图如图11所示。
salicifoliol：固体，易溶于二氯甲烷。ESI-MSm/z: 251.09 [M+H]+，结合核磁碳信号，分子式确定为C13H14O5，不饱和度为7。
筛选得到的化合物结构式如下所示：
。
利用肠道微生物发酵栀子后，栀子浸膏对XOD酶的抑制活性显著增强，并生成了黄酮类、环烯醚萜类、生物碱类等差异代谢物。栀子是临床在用治疗高尿酸血症的药食同源植物，但栀子提取物或单体化合物对尿酸生成的关键酶XOD的抑制活性不强。本项目以提高栀子XOD酶的抑制活性，高效获得具有XOD酶抑制活性单体化合物为出发点，利用8种肠道微生物对栀子进行发酵，提高栀子的降尿酸活性，结合活性追踪和质谱分子网络技术从发酵栀子中高效获得抑制XOD酶活性的化合物。在活性方面，植物乳杆菌（LPLC）、嗜酸乳杆菌（LACC）和长双岐杆菌（BLOC）发酵栀子后栀子浸膏抑制XOD酶的活性显著提高，与发酵前相比提高了4.2倍；在化学成分方面，利用UPLC-QTOF-HRMS分析发现发酵后栀子的化学成分更加丰富，生成了黄酮类、环烯醚萜类、生物碱类等差异代谢物（266个）。如栀子苷发生水解反应生成入血苷元京尼平，并使其相对含量增加了33.89倍。另外，栀子中原有化学成分发生氧化、酯化、水解等反应新产生了harpagide、eugenol acetate、isorhapontigenin等化合物。
Claims (6)
1.一种基于微生物发酵得栀子降尿酸代谢产物发酵浸膏的方法，其特征在于，包括以下步骤：
（1）复苏菌株后于灭菌后的MRS培养基中培养得种子液；
所述菌株为植物乳杆菌、嗜酸乳杆菌和长双岐杆菌；所述培养为37 ℃培养1 d；
（2）将栀子粉和蒸馏水混合后灭菌得混合浆料，然后将混合浆料接种种子液，接种后进行厌氧发酵得发酵液，将发酵液过滤浓缩得栀子降尿酸代谢产物发酵浸膏；
所述栀子降尿酸代谢产物发酵浸膏中，栀子降尿酸代谢产物的分离方法为：将发酵液过滤浓缩得栀子降尿酸代谢产物发酵浸膏，将发酵浸膏分散在10倍体积的水中，经石油醚、乙酸乙酯、正丁醇依次萃取得石油醚部位、乙酸乙酯部位、正丁醇部位和水部位四个组分，经过XOD酶活性导向，对乙酸乙酯部位通过正反硅胶柱色谱和HPLC半制备柱分离纯化，得栀子降尿酸代谢产物；
所述栀子降尿酸代谢产物，乙酸乙酯部位具体分离纯化过程为：
（a）将乙酸乙酯部位通过200-300目的硅胶柱分离，并用CH2Cl2/MeOH逐步洗脱产生六个组分Fr1-Fr6；
所述CH2Cl2/MeOH逐步洗脱的比例为20:1、10:1、5:1、2:1、1:1、1:5、0:1；
（b）Fr2通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和SephadexLH-20分离后，再用半制备型HPLC制备得到apigenin、luteolin、isorhamnetin和methylophiopogonone A；
所述石油醚/乙酸乙酯的洗脱比例为2:1；所述SephadexLH-20为MeOH/CH2Cl2 = 1/1；所述半制备型HPLC的参数为：MeOH/H2O = 60/40；
（c）Fr3通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和SephadexLH-20分离后，由半制备型HPLC制备得到blumenol A和ferulic acid；
所述石油醚/乙酸乙酯的洗脱比例为3:2；所述SephadexLH-20为MeOH/CH2Cl2 = 1/1；所述半制备型HPLC的参数为：MeOH/H2O = 50/50；
（d）Fr4通过200-300目硅胶柱经石油醚/乙酸乙酯洗脱和Sephadex LH-20后，由半制备型HPLC制备得到lyoniresinol和salicifoliol；
所述石油醚/乙酸乙酯的洗脱比例为1:1；所述SephadexLH-20为MeOH/CH2Cl2 = 1/1；所述半制备型HPLC的参数为：MeOH/H2O = 40/60。
2.根据权利要求1所述的方法，其特征在于，步骤（2）中，所述栀子粉和蒸馏水的比例为6g:80mL；所述灭菌为在121 ℃下，灭菌25 min；所述种子液接种的浓度为108 CFU/mL；所述厌氧发酵为37 ℃厌氧培养3 d。
3.根据权利要求1所述的方法，其特征在于，步骤（b）中，所述半制备型HPLC的参数为：2.0mL/min。
4.根据权利要求1所述的方法，其特征在于，步骤（c）中，所述半制备型HPLC的参数为：2.0mL/min。
5.根据权利要求1所述的方法，其特征在于，步骤（d）中，所述半制备型HPLC的参数为：2.0mL/min。
6.根据权利要求1-5任一项所述的方法，其特征在于，所述栀子降尿酸代谢产物为：
。
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