CN116898880B - 一种人工熊胆粉的制备方法 - Google Patents
一种人工熊胆粉的制备方法 Download PDFInfo
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- CN116898880B CN116898880B CN202311176419.8A CN202311176419A CN116898880B CN 116898880 B CN116898880 B CN 116898880B CN 202311176419 A CN202311176419 A CN 202311176419A CN 116898880 B CN116898880 B CN 116898880B
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- Prior art keywords
- hydroxysteroid dehydrogenase
- chicken
- bile
- ldh
- chicken bile
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Classifications
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
本发明公开了一种人工熊胆粉的制备方法,这种制备方法使用包含7α‑羟基类固醇脱氢酶、7β‑羟基类固醇脱氢酶及胆绿素还原酶的混合酶及辅酶,以鸡胆汁为底物通过一锅法反应,从而得到人工熊胆粉,本发明得到的人工熊胆粉色泽金黄,牛磺熊去氧胆酸含量超过40%,牛磺熊去氧胆酸与牛磺鹅去氧胆酸的含量总和超过70%。
Description
技术领域
本发明属于中医药领域,具体涉及一种人工熊胆粉的制备方法。
背景技术
关于熊胆粉在治疗急慢性肝炎以及胆石疾病等领域,已经得到临床认证,具有较好的治疗的效果。但由于全国养熊企业较少,无管引流产量不高,无法满足日益增长的市场需求。
熊胆粉中主要组分包括胆汁酸类及非胆汁酸类,其中主要有效成分在胆汁酸类中,包括牛磺熊去氧胆酸(TUDCA)、牛磺鹅去氧胆酸(TCDCA)、非结合性牛磺鹅去氧胆酸(CDCA)、胆红素、去氧胆酸(DCA)及胆酸(CA),其中牛磺熊去氧胆酸(TUDCA)用于区别其他动物胆汁的主要组分。
专利申请号CN201210162695.4中公开了一种人工熊胆,该人工熊胆通过牛磺熊去氧胆酸钠与混合胆素及氨基酸、微量元素混合得到。
专利申请号CN201510996540.4中公开了以过氧化钠为氧化剂使牛磺鹅去氧胆酸(TCDCA)被氧化成3α-羟基-7-羰基-5β-胆烷酰-N-牛磺酸(T7-ketoLCA),以金属钠为还原剂将氧化物T7-ketoLCA被还原为牛磺熊去氧胆酸(TUDCA)和牛磺鹅去氧胆酸(TCDCA),反应完成后胆汁酸成分变化为TUDCA、TCDCA和TCA,三者比例接近47:36:17,该产物与天然熊胆胆汁酸成分相似,上述方法为化学合成方法。
专利申请号CN201410588581.5中公开了通过7α-羟基类固醇脱氢酶、7β-羟基类固醇脱氢酶将禽畜类胆粉中牛磺鹅去氧胆酸(TCDCA)转化为牛磺熊去氧胆酸(TUDCA),从而得到人工熊胆粉, TUDCA占人工熊胆粉的质量百分比为23~33%,TCDCA占人工熊胆粉的质量百分比为12~30%, 其总胆酸含量:40~65%,上述专利中辅酶采用辅酶Ⅰ(NAD+)或辅酶Ⅱ(NADPH),而上述方法得到的一种人工熊胆粉未考虑到胆红素。在现有技术中记载了鸡胆粉中TCDCA含量为48.5%,大于38%,其鸡胆粉中非胆汁酸成分与熊胆粉的相似度高于其他禽胆粉,鸡胆粉为“体外模拟熊胆粉”的最佳转化基质,但是鸡胆中不含有胆红素,确定含有胆绿素。
国内外大量研究表明,胆红素具有良好的抗炎、抗氧化和免疫调节作用,而在现有技术中有记载胆红素纳米颗粒在多种胃肠道疾病、肝肾疾病、自身免疫性疾病等具有巨大的应用潜力。
在现有技术中还记载了熊胆中含有0.4%的胆红素,胆红素同样为熊胆中重要的有效组分。
综上所述,将鸡胆中胆绿素转化为胆红素,对于制备人工熊胆粉显得尤为重要。
发明内容
本发明的目的在于提供一种人工熊胆粉的制备方法,这种制备方法采用鸡胆作为原料,通过酶对鸡胆汁催化,采用一锅法反应,得到牛磺熊去氧胆酸含量高,色泽金黄,并含有胆红素的人工熊胆粉。
为了实现上述目的,本发明提供以下技术方案:
一种人工熊胆粉的制备方法,包括以下步骤:
1)准备底物:准备鸡胆汁精制液,检测鸡胆汁精制液中TCDCA的含量,鸡胆汁精制液中TCDCA的含量不少于20%;
2)准备混合酶及辅酶:所述混合酶包括LDH-7α-羟基类固醇脱氢酶、GDH-7β-羟基类固醇脱氢酶及胆绿素还原酶,辅酶包括NAD+及NADP+,LDH-7α-羟基类固醇脱氢酶的酶活为200-220U/mL,GDH-7β-羟基类固醇脱氢酶为30-40U/mL,胆绿素还原酶的酶活为0.2U/g;
3)底物酶转化:将鸡胆汁精制液与混合酶及辅酶混合反应0.5-1.5h,LDH-7α-羟基类固醇脱氢酶的添加量为反应体系容积的10%;GDH-7β-羟基类固醇脱氢酶的添加量为反应体系容积的40%;胆绿素还原酶的添加量为0.1mg/mL,鸡胆汁精制液为反应体系容积的5.6%。
准备底物具体为:
选取鸡胆,破胆取胆汁,得到鸡胆汁;对上述鸡胆汁进行浓缩,浓缩至原容积50%,得到浓缩鸡胆汁,浓缩温度为70℃;
对浓缩鸡胆汁进行醇提,将浓缩鸡胆汁降温至30℃,向浓缩鸡胆汁加入3倍容积的95%乙醇,然后搅拌200min;将完成搅拌后浓缩鸡胆汁温度维持在4℃,以3500rpm的转速,离心25min,每次离心5min,分5次离心;完成离心后取上清液,对上清液进行二次浓缩,浓缩时间40min,浓缩温度为45℃,得到鸡胆汁精制液。
优选地、所述LDH-7α-羟基类固醇脱氢酶中,7α-羟基类固醇脱氢酶来源于大肠杆菌,乳酸脱氢酶来源于乳酸杆菌;GDH-7β-羟基类固醇脱氢酶中7β-羟基类固醇脱氢酶来源于活泼瘤胃球菌,葡萄糖脱氢酶来源于芽孢杆菌,胆绿素还原酶由源自集胞藻(Synechocystis sp.PCC6803)。
底物酶催化(转化)具体为:
取鸡胆汁精制液调解pH至7,然后加入丙酮酸钠、一水合葡萄糖,混合酶、辅酶、正己醇及水构成反应体系,反应体系pH调节至7;丙酮酸钠与鸡胆汁精制液中TCDCA的摩尔比为1.7:1,一水合葡萄糖与鸡胆汁精制液中TCDCA的摩尔比为2.4:1,正己醇占反应体系容积的8%,水余量。
熊胆中含有多种胆色素,其中胆红素相较于其他胆色素是重要的活性成分,现有文献中指出,在熊胆粉中胆红素含量对熊胆外观并无影响,而外观是评价熊胆粉品质的重要条件。
鸡胆中胆汁酸组分,包括牛磺鹅去氧胆酸(TCDCA)、牛磺酸(TCA)、胆酸(CA)、去氧胆酸(DCA)及甘氨胆酸(GCA),不含有牛磺熊去氧胆酸(TUDCA),非胆汁酸中胆色素主要为胆绿素,不含胆红素。
附图说明
图1为本申请具体实施例中TUDCA标品液相色谱图;
图2为本申请具体实施例中TCDCA标品液相色谱图;
图3为本申请具体实施例中实施例1产品液相色谱图;
图4为本申请具体实施例中实施例1重复产品液相色谱图;
图5为本申请具体实施例中实施例2产品液相色谱图;
图6为本申请具体实施例中实施例2重复产品液相色谱图;
图7为本申请具体实施例中对比例1产品液相色谱图;
图8为本申请具体实施例中对比例1重复产品液相色谱图;
图9为本申请具体实施例中对比例2产品液相色谱图;
图10为本申请具体实施例中对比例2重复产品液相色谱图;
图11为本申请具体实施例中对比例3产品液相色谱图;
图12为本申请具体实施例中对比例3重复产品液相色谱图。
有益效果:1、本发明中,LDH-7α-羟基类固醇脱氢酶、GDH-7β-羟基类固醇脱氢酶的功能为将牛磺鹅去氧胆酸转化为牛磺熊去氧胆酸,胆绿素还原酶的功能为将鸡胆汁中部分胆绿素还原为胆红素,本发明得到的人工熊胆粉色泽金黄,牛磺熊去氧胆酸含量超过40%,牛磺熊去氧胆酸与牛磺鹅去氧胆酸的含量总和超过70%;
2、本发明中通过鸡胆自制鸡胆汁,通过破胆、浓缩、醇提、离心,得到浓缩鸡胆汁;使用鸡胆制作鸡胆汁作为原料较于直接购买鸡胆粉作为原料,一方面大幅降低其生产成本,另一方面牛磺鹅去氧胆酸含量及非胆汁酸类含量更加稳定;
3、本发明中采用一锅法反应,节约反应时间,降低生产成本。
具体实施方式
为使本发明实施例的目的、技术方案及优点更加清楚,下面将结合本发明实施例,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作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。
制备鸡胆汁精制液
选取市售鸡胆,常温解冻,破胆取胆汁,得到鸡胆汁;
对上述鸡胆汁进行浓缩,浓缩至原容积50%,得到浓缩鸡胆汁,浓缩温度为70℃;
对浓缩鸡胆汁进行醇提,将浓缩鸡胆汁降温至30℃,向浓缩鸡胆汁加入3倍容积的95%乙醇,然后搅拌200min;将完成搅拌后浓缩鸡胆汁温度维持在4℃,以3500rpm的转速,离心25min,每次离心5min,分5次离心;完成离心后取上清液,对上清液进行二次浓缩,浓缩时间40min,浓缩温度为45℃,得到鸡胆汁精制液。
通过NaOH调节pH至7,备用;经检测,每100g鸡胆汁精制液中含有20.35gTCDCA。
LDH-7α-羟基类固醇脱氢酶、GDH-7β-羟基类固醇脱氢酶及酶构建可见专利申请号CN202111304130.0中公开的LDH-7α-羟基类固醇脱氢酶、GDH-7β-羟基类固醇脱氢酶及其构建方法。
酶的制备
pRSFDuet-1载体的获得:-80°C保存的Top10菌株(含有pRSFDuet-1载体)三线划区加了50μg/ml卡那霉素(Kan)的LB固体平板培养基,37°C过夜培养后挑取单克隆接种5mlLB液体培养基(Kan=50μg/ml),37°C、220rpm过夜培养后用天根质粒小提试剂盒提取质粒。
载体pRSF-7α重组载体的构建:由于LDH基因片段上含有NdeI酶切位点,7α上游的酶切位点也是NdeI,故构建双表达载体时先将7α片段连接上去;pRSFDuet-1及7α片段用NdeI和XhoI双酶20μl体系:pRSFDuet-1质粒或7α片段各5μl,10x buffer 2μl,NdeI和XhoI各0.5μl,双蒸水12μl,37℃水浴酶切1h,80℃灭活15min后加入10x loading buffer 2μl,用1%的琼脂糖凝胶电泳切胶回收酶切产物,胶回收试剂盒用的是全式金的DNA纯化试剂盒。回收纯化的质粒大片段和7α连接反应:7α片段和质粒片段各3μl,10x T4 buffer 1μl,T4连接酶0.5μl,双蒸水3.5μl,22℃连接1h。3μl连接产物转化Top10感受态细胞,复苏1h涂布含有50μg/ml卡那霉素的LB固体平板,37℃过夜培养。用7α-F/R进行转化子的菌落PCR验证,验证无误的转化子转接LB液体培养基(Kan=50μg/ml),37℃培养过夜提取质粒pRSF-7α待用。
载体pRSF-LDH-7α重组载体的构建:pRSF-7α及LDH片段用NcoI和HindIII双酶20μl体系:pRSF-7α及LDH片段各5μl,10x buffer 2μl,NcoI和HindIII各0.5μl,双蒸水12μl,37℃水浴酶切1h,80℃灭活15min后加入10x loading buffer2μl,用1%的琼脂糖凝胶电泳切胶回收酶切产物。回收纯化的质粒大片段和LDH连接反应:LDH片段和质粒片段各3μl,10xT4 buffer 1μl,T4连接酶0.5μl,双蒸水3.5μl,22℃连接1h。3μl连接产物转化Top10感受态细胞,复苏1h涂布含有50μg/ml卡那霉素的LB固体平板,37℃过夜培养。用LDH-F/R进行转化子的菌落PCR验证,验证无误的转化子转接LB液体培养基(Kan=50μg/ml),37℃培养过夜提取质粒pRSF-LDH-7α待用。
pRSF-LDH-7α质粒转化50μl BL21(DE3)感受态细胞,37℃复苏1h涂布含有50μg/ml卡那霉素的LB固体平板,37℃过夜培养。
挑取单克隆转接5ml LB液体培养基(Kan=50μg/ml),37℃过夜培养,1:100转接50ml LB液体培养基(Kan=50μg/ml),37℃、220rpm培养2~3h,OD600为0.5~0.8,加入0.5mMIPTG,30℃诱导4h,8000rpm离心10min收集菌体。
菌体加入50ml的PBS缓冲液(100mM,pH8.0),30%功率超声10min,10000rpm离心收集上清液,即为LDH-7α酶液,LDH-7α酶液酶活为200U/mL。
载体pRSF-7β重组载体的构建:pRSFDuet-1及7β片段用NdeI和XhoI双酶切20μl体系:pRSFDuet-1质粒或7β片段各5μl,10x buffer 2μl,NdeI和XhoI各0.5μl,双蒸水12μl,37℃水浴酶切1h,80℃灭活15min后加入10x loading buffer2μl,用1%的琼脂糖凝胶电泳切胶回收酶切产物,胶回收试剂盒用的是全式金的DNA纯化试剂盒。回收纯化的质粒大片段和7β连接反应:7β片段和质粒片段各3μl,10x T4 buffer 1μl,T4连接酶0.5μl,双蒸水3.5μl,22℃连接1h。3μl连接产物转化Top10感受态细胞,复苏1h涂布含有50μg/ml卡那霉素的LB固体平板,37℃过夜培养。用7β_F/R进行转化子的菌落PCR验证,验证无误的转化子转接LB液体培养基(Kan=50μg/ml),37℃培养过夜提取质粒pRSF-7β待用。
载体pRSF-GDH-7β重组载体的构建:pRSF-7β及GDH片段用NcoI和HindIII双酶切20μl体系:pRSF-7β及GDH片段各5μl,10x buffer 2μl,NcoI和HindIII各0.5μl,双蒸水12μl,37℃水浴酶切1h,80℃灭活15min后加入10x loading buffer2μl,用1%的琼脂糖凝胶电泳切胶回收酶切产物。回收纯化的质粒大片段和GDH连接反应:GDH片段和质粒片段各3μl,10xT4 buffer 1μl,T4连接酶0.5μl,双蒸水3.5μl,22℃连接1h。3μl连接产物转化Top10感受态细胞,复苏1h涂布含有50μg/ml卡那霉素的LB固体平板,37℃过夜培养。用GDH-F/R进行转化子的菌落PCR验证,验证无误的转化子转接LB液体培养基(Kan=50μg/ml),37℃培养过夜提取质粒pRSF-GDH-7β待用。
pRSF-GDH-7β质粒转化50μl BL21(DE3)感受态细胞,37℃复苏1h涂布含有50μg/ml卡那霉素的LB固体平板,37℃过夜培养。
挑取单克隆转接5ml LB液体培养基(Kan=50μg/ml),37℃过夜培养,1:100转接50ml LB液体培养基(Kan=50μg/ml),37℃、220rpm培养2~3h,OD600=0.5~0.8,加入0.5mM IPTG,30℃诱导4h,8000rpm离心10min收集菌体。
菌体加入50ml的PBS缓冲液(100mM,pH8.0),30%功率超声10min,10000rpm离心收集上清液,即为GDH-7β酶液,酶液酶活为40U/mL。
胆绿素还原酶及构建可见专利申请号CN201210395683.6的专利。
将含胆绿素还原酶基因的质粒pRSET-BR转化感受态细菌细胞E.coli HB101,在Luria broth (LB)平板(含100mg/L卡那霉素)上37°C培养24小时。接种单个克隆于5毫升LB液体培养基(含100mg/L卡那霉素)中于30°C培养20-24小时。离心收集菌体,并悬浮于1毫升100mM Tris盐酸缓冲液(pH7.5)中。然后用超声波裂解细菌细胞。离心(10°C, 17, 800g,10分钟)并收集上清液,即为粗提蛋白(或称粗提物)。
取胆绿素还原酶粗提蛋白,用洗酶缓冲液(0.02M Tris-HCl/0.001MEDTA,pH7.0溶液)稀释至蛋白含量5-10mg/ml。将酶稀释液与PB溶液(2.0mol/L磷酸二氢钾,pH7.5)等体积混合,加入环氧型固定化酶载体LX-3000 (10毫克酶/克载体),于摇床(转速100rpm)中25V反应20小时。反应完成后用滤袋过滤,用洗酶缓冲液清洗5-6次,得到胆绿素还原酶。
胆绿素还原酶的固定化载体可选用传统的无机载体材料二氧化硅、活性炭、玻璃珠等,再如有机高分子载体大孔型聚N-氨乙基丙烯酰胺-聚乙烯等固定化载体,本申请具体实施方式中使用的为环氧型载体LX-3000。
丙酮酸钠、一水合葡萄糖、NAD+、NADP+、正己醇及NaOH等均为市售产品,丙酮酸钠分子量110.04,一水合葡萄糖 CAS号为:14431-43-7,分子量为240.2509。
其中NAD+为辅酶Ⅰ,全称为烟酰胺腺嘌呤二核苷酸,又称二磷酸烟苷,存在每一个细胞中参与上千项反应。NAD+是三羧酸循环的重要辅酶,促进糖、脂肪、氨基酸的代谢,参与能量的合成;NAD+又是辅酶I消耗酶的唯一底物(DNA修复酶PARP的唯一底物、长寿蛋白Sirtuins的唯一底物、环ADP核糖合成酶CD38/157的唯一底物)。
NADP+为辅酶Ⅱ,NADP+的中文名称是烟酰胺腺嘌呤二核苷磷酸,是还原型辅酶II(NADPH)的氧化形式,表示失去了一个电子而带上一个正电荷。
NAD+和NADP+作为脱氢酶的辅酶,在酶促反应中起递氢体的作用,为单递氢体。
实施例1:人工熊胆粉的制备,80ml小试反应:
取20g备用鸡胆汁精制液(TCDCA含量约4.07g),与丙酮酸钠1.5g、一水合葡萄糖4.7g,胆绿素还原酶8 mg(0.1mg/mL),NAD+0.96g(10mg/mL),NADP+0.8g(10mg/mL),并添加8mL的LDH-7α-羟基类固醇脱氢酶,32mL的GDH-7β-羟基类固醇脱氢酶,6.4ml正己醇,一同加入搅拌器中,添加适量的水构成80ml反应体系,反应体系pH调节至7,LDH-7α-羟基类固醇脱氢酶的酶活为200U/mL,GDH-7β-羟基类固醇脱氢酶的酶活为40U/mL,胆绿素还原酶的酶活为0.2U/g,反应90min。
反应后减压浓缩正己醇,然后醇提,过树脂柱后得到人工熊胆粉。
实施例1为实验室小试反应,目的在于验证通过LDH-7α-羟基类固醇脱氢酶,GDH-7β-羟基类固醇脱氢酶与胆绿素还原酶的催化结果,其中反应中pH控制在7,环境温度维持在25-28℃,经检测TCDCA含量为54.51%。
实施例2:人工熊胆粉的制备,50L反应:取12.5kg备用鸡胆汁精制液(TCDCA含量约2.543kg),加入丙酮酸钠0.9375kg,胆绿素还原酶5g(0.1mg/mL),一水合葡萄糖(工业级)2.93kg,NAD+0.6kg(10mg/mL),NADP+0.5kg(10mg/mL),4L正己醇,5L LDH-7α-羟基类固醇脱氢酶,20L GDH-7β-羟基类固醇脱氢酶,添加适量的水构成50L反应体系,反应90min。
反应后减压浓缩正己醇,然后醇提得到人工熊胆粉。
实施例2为中试放大实验,其目的在于确定放大生产的稳定性。
对比例1:对比例1与实施例1的区别仅在于未添加胆绿素还原酶;
取20g备用鸡胆汁精制液(TCDCA含量约4.07g),与丙酮酸钠1.5g、一水合葡萄糖4.7g, NAD+0.96g(10mg/mL),NADP+0.8g(10mg/mL),并添加8mL的LDH-7α-羟基类固醇脱氢酶,32mL的GDH-7β-羟基类固醇脱氢酶,6.4ml正己醇,一同加入搅拌器中,添加适量的水构成80ml反应体系,反应体系pH调节至7,LDH-7α-羟基类固醇脱氢酶的酶活为200U/mL,GDH-7β-羟基类固醇脱氢酶及40U/mL,反应90min。
反应后减压浓缩正己醇,然后醇提得到人工熊胆粉。
对比例1的目的在于验证胆绿素还原酶对最终产品的影响。
对比例2:对比例2与实施例1的区别仅在于添加胆绿素还原酶时机不同;
取20g备用鸡胆汁精制液(TCDCA含量约4.07g),与丙酮酸钠1.5g、一水合葡萄糖4.7g,NAD+0.96g(10mg/mL),NADP+0.8g(10mg/mL),并添加8mL的LDH-7α-羟基类固醇脱氢酶,32mL的GDH-7β-羟基类固醇脱氢酶,6.4ml正己醇,一同加入搅拌器中,添加适量的水构成80ml反应体系,反应体系pH调节至7,LDH-7α-羟基类固醇脱氢酶的酶活为200U/mL,GDH-7β-羟基类固醇脱氢酶为40U/mL,待反应60min后加入胆绿素还原酶8 mg(0.1mg/mL),再反应30℃。
反应后减压浓缩正己醇,然后醇提得到人工熊胆粉。
对比例2的目的在于区别一锅法及分开反应对最终产品的影响。
对比例3:对比例3与实施例1的区别仅在于未添加胆绿素还原酶,直接添加胆红素;
取20g备用鸡胆汁精制液(TCDCA含量约4.07g),与丙酮酸钠1.5g、一水合葡萄糖4.7g,NAD+0.96g(10mg/mL),NADP+0.8g(10mg/mL),并添加8mL的LDH-7α-羟基类固醇脱氢酶,32mL的GDH-7β-羟基类固醇脱氢酶,6.4ml正己醇,一同加入搅拌器中,添加适量的水构成80ml反应体系,反应体系pH调节至7,LDH-7α-羟基类固醇脱氢酶的酶活为200U/mL,GDH-7β-羟基类固醇脱氢酶酶活为40U/mL,反应90min。
反应后加入3mg胆红素,减压浓缩正己醇,然后醇提得到人工熊胆粉。
对比例3的目的在于证明直接添加胆红素和将鸡胆中胆绿素转化为胆红素对最终产品的影响。
实施例1-实施例2,对比例1-对比例3中所述的醇提具体为:1)加入乙醇搅拌沉淀,在30-35℃的条件下搅拌1h,乙醇为95%乙醇;2)离心过滤,得到上清液;3)在65-70℃下浓缩上清液,浓缩90min,完成浓缩后降至室温,放入冻干机冻干,冻干温度为-15℃,真空度为20pa;4)完成冻干后取出,粉碎得到人工熊胆粉。
对实施例1-实施例2、对比例1-对比例2得到的人工熊胆粉进行检测,观测成品的颜色,称重,TUDCA标品、TCDCA标品及实施例1-实施例2和对比例1-对比例3 使用甲醇定容,10mL,浓度约2mg/mL,然后通过液相质谱仪进行检测,实施例1-实施例2和对比例1-对比例3液相测定均重复一次,通过比峰法测算TUDCA及TCDCA的含量。
TUDCA检测结果见下表1,TCDCA检测结果见下表2, TUDCA标品液相色谱图见图1,TCDCA标品液相色谱图见图2,实施例1-实施例2和对比例1-对比例3液相色谱图见图3-图12。
由图1可见TUDCA出峰时间为6min,由图2可见TCDCA出峰时间为12min。
TCDCA及TUDCA的百分含量计算方式为:
标品浓度/标品峰面积=百分含量×样品浓度/样品峰面积
表1
表2
表3
熊胆粉有三个等级,分别是金胆、铁胆、菜胆,金胆(又称铜胆或琥珀胆)金黄色,有光泽,半透明如琥珀,质松脆。墨胆(又称铁胆)黑色,质坚而脆或呈稠膏状,品质次于金胆。菜花胆(又称青茶胆)黄绿色,光亮较差,质较脆。
由表3可见熊胆粉收率不低于109%,其中TUDCA的含量不低于54%,由实施例1的数据可见,使用LDH-7α-羟基类固醇脱氢酶,GDH-7β-羟基类固醇脱氢酶,及NAD+、NADP+、一水合葡萄糖的酶再生系统,然后控制反应体系pH为7,最终使得TUDCA含量为54.51%,TCDCA含量为28.12%,明显高于现有公开的人工熊胆粉资料中的TUDCA含量;而胆绿素还原酶将鸡胆汁中胆绿素转化为胆红素,成品颜色为金黄色,其中NADP+不仅为LDH-7α-羟基类固醇脱氢酶,GDH-7β-羟基类固醇脱氢酶的辅酶,同时也为胆绿素还原酶的辅酶,并且胆绿素还原酶能够在pH为7,环境在25-28℃的条件下保证酶的活性稳定;
由对比例1与实施例1对比可见,未添加胆绿素还原酶,对收率影响较小,对比例1收率为103.43%,对TUDCA含量有一定的影响,含量为42.11%,最终得到的成品颜色为黄绿色,即添加胆绿素还原酶将鸡胆汁中胆绿素转化为胆红素可以提高人工熊胆粉的品质。
由对比例2与实施例1对比可见,先对TCDCA进行催化,再对胆绿素进行催化,在同样的反应时间内,最终得到的成品颜色为黄绿色,即中途添加胆绿素还原酶在同样的反应时间内,无法完全反应,而增加反应时间,一方面可能会造成TCDCA含量过低,另一方面生产成本会大幅增加。
由对比例3与实施例1对比可见,在完全反应后再掺入胆红素,最终得到成品颜色为红棕色,人工熊胆粉品质明显不如实施例1,猜测胆绿素的含量过高会导致人工熊胆粉的品相不佳。
Claims (3)
1.一种人工熊胆粉的制备方法,其特征在于,所述制备方法包括以下步骤:
1)准备底物:准备鸡胆汁精制液,检测鸡胆汁精制液中TCDCA 的含量,鸡胆汁精制液中TCDCA 的含量不少于20%;
2)准备混合酶及辅酶:所述混合酶包括LDH-7α-羟基类固醇脱氢酶、GDH-7β-羟基类固醇脱氢酶及胆绿素还原酶,辅酶包括NAD+及NADP+,LDH-7α-羟基类固醇脱氢酶的酶活为200-220U/mL,GDH-7β-羟基类固醇脱氢酶为30-40U/mL,胆绿素还原酶的酶活为0.2U/g;
3)底物酶转化:将鸡胆汁精制液与混合酶及辅酶混合反应0.5-1.5h,LDH-7α-羟基类固醇脱氢酶的添加量为反应体系容积的10%;GDH-7β-羟基类固醇脱氢酶的添加量为反应体系容积的40%;胆绿素还原酶的添加量为0.1mg/mL,鸡胆汁精制液与反应体系的质量体积比为25g/mL;
所述步骤3)具体为:取鸡胆汁精制液20g、丙酮酸钠1.5g、一水合葡萄糖4.7g、胆绿素还原酶8 mg、NAD+0.96g、NADP+0.8g,添加8mL的LDH-7α-羟基类固醇脱氢酶、32mL的GDH-7β-羟基类固醇脱氢酶、6.4ml正己醇,鸡胆汁精制液含4.07gTCDCA,一同加入搅拌器中,添加适量的水构成80ml反应体系,反应体系pH调节至7,反应90min。
2. 根据权利要求1 所述的制备方法,其特征在于,所述步骤1)具体为:选取鸡胆,破胆取胆汁,得到鸡胆汁;对上述鸡胆汁进行浓缩,浓缩至原容积50%,得到浓缩鸡胆汁,浓缩温度为70°C;对浓缩鸡胆汁进行醇提,将浓缩鸡胆汁降温至30°C,向浓缩鸡胆汁加入3 倍容积的95%乙醇,然后搅拌200min;将完成搅拌后浓缩鸡胆汁温度维持在4°C,以3500rpm 的转速,离心25min,每次离心5min,分5 次离心;完成离心后取上清液,对上清液进行二次浓缩,浓缩时间40min,浓缩温度为45°C,得到鸡胆汁精制液。
3. 根据权利要求1 所述的制备方法,其特征在于,步骤2)中,所述LDH-7α-羟基类固醇脱氢酶中,7α-羟基类固醇脱氢酶来源于大肠杆菌,乳酸脱氢酶来源于乳酸杆菌;GDH-7β-羟基类固醇脱氢酶中7β-羟基类固醇脱氢酶来源于活泼瘤胃球菌,葡萄糖脱氢酶来源于芽孢杆菌,胆绿素还原酶源自Synechocystis sp.PCC6803。
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