CN107164436A - β‑葡萄糖苷酶在转化淫羊藿总黄酮制备宝霍苷I中的应用 - Google Patents
β‑葡萄糖苷酶在转化淫羊藿总黄酮制备宝霍苷I中的应用 Download PDFInfo
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- CN107164436A CN107164436A CN201710333064.7A CN201710333064A CN107164436A CN 107164436 A CN107164436 A CN 107164436A CN 201710333064 A CN201710333064 A CN 201710333064A CN 107164436 A CN107164436 A CN 107164436A
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Abstract
β‑葡萄糖苷酶在转化淫羊藿总黄酮制备宝霍苷I中的应用。该方法是利用一种特异的β‑葡萄糖苷酶定向转化淫羊藿总黄酮,使淫羊藿总黄酮中含量丰富的多个组分都转化为宝霍苷I。本发明筛选获得的一种β‑葡萄糖苷酶,不仅能高效地酶解淫羊藿总黄酮中富含的淫羊藿苷、朝霍定A、箭藿苷A制得宝霍苷I,而且能特异地将朝霍定B和箭藿苷B制得宝霍苷I。本发明酶使用成本低,只需一种酶;总黄酮的转化效率高,摩尔转化率大于95%。而且所制备的宝霍苷I对乳腺癌、肝癌、结肠癌、肺癌等肿瘤增殖的抑制作用等方面的药理学活性显著高于淫羊藿总黄酮中其它的主要淫羊藿苷。
Description
技术领域
本发明属于生物医药及保健品领域,具体涉及β-葡萄糖苷酶在转化淫羊藿总黄酮制备宝霍苷I中的应用及宝霍苷I在制备治疗乳腺癌、肺癌、结肠癌、肝癌药物中的应用。
背景技术
黄酮具有广泛的药理活性,体外、体内实验及临床试验都表明,部分黄酮类化合物对于癌症的预防与治疗具有重要的意义。它们的抗氧化特性能够减少细胞DNA的养化损伤、从而降低DNA的突变,还可以抑制各种致癌物的体内激活,并提高机体清除致癌物质,对于癌细胞黄酮类化合物还可以抑制其增殖型号的转导,阻滞癌细胞周期诱发癌细胞凋亡从而控制癌细胞的增殖。因此,黄酮类化合物对癌症的治疗与防治具有重要的意义。此外部分研究表明,某些肿瘤细胞表面存在鼠李糖凝集素,能够特异性地结合鼠李糖。抗肿瘤药物的糖基化修饰会增强药物的靶向性、减少对正常细胞的损害、减少药物给剂量以及提高疗效,而且还可以用来研究不同肿瘤细胞的药物敏感性。
淫羊藿属于小檗科植物,具有增加心脑血管血流量、促进造血功能、免疫功能及骨代谢,具有补肾壮阳、抗衰老、抗肿瘤等功效。而淫羊藿富含特有的鼠李糖苷黄酮类化合物,同时在我国多地区实现规模化人工种植,其资源丰富,有利于获取大量的淫羊藿提取物。通过对淫羊藿提取物成分的分析,已发现并确认了其中几种含量较高的富含鼠李糖苷黄酮类化合物,包括朝霍定A、朝霍定B、朝霍定C、淫羊藿苷。但研究表明,在淫羊藿中含量较低的组分宝霍苷I药理活性独特,对癌细胞具有显著的抑制作用并可诱导癌细胞凋亡,在肿瘤治疗方面存在广阔的应用前景。然而,由于其在淫羊藿植株中含量太低,无法高效大量地提取分离供以实际应用。根据对淫羊藿黄酮类化合物的结构研究发现,霍定A、朝霍定B、朝霍定C、淫羊藿苷的黄酮母核与宝霍苷I完全一致,仅在糖基个数及种类上存在差异。
综合考虑上述问题,通过选择性地去除与宝霍苷I有差异的糖基,即可获得目的产物宝霍苷I,根据已公布文献可见,目前制备宝霍苷I生物转化方法较为单一,几乎都是以多组分淫羊藿苷为底物,未对淫羊藿总黄酮达到高效利用,如CN103160553通过葡聚糖酶水解淫羊藿苷制备宝霍苷I;CN103305572通过酵母菌厌氧发酵仅将淫羊藿中的淫羊藿苷转化生成宝霍苷I。从而在制备过程中,首先要获得淫羊藿提取物,然后分离得到淫羊藿苷,然后通过酶法或微生物法转化,不仅大大增加了成本,而且没有充分高效利用朝霍定A、朝霍定B、朝霍定C。另一方面,使得大量获取淫羊藿苷资源问题非常突出,无法保障规模化制备宝霍苷I。并且,现有的酶法或微生物法转化淫羊藿苷的效率还有待提高。而本发明提供了一种酶法转化淫羊藿总黄酮制备宝霍苷I的方法,仅仅通过一种具有高效水解葡糖糖苷酶和木糖苷酶的双功能酶特性的β-葡萄糖苷酶,不仅能高效地酶解淫羊藿总黄酮中富含的淫羊藿苷、朝霍定A、箭藿苷A制得宝霍苷I,而且能特异地将朝霍定B和箭藿苷B制得宝霍苷Ⅰ。
发明内容
解决的技术问题:本发明提供一种β-葡萄糖苷酶在转化淫羊藿总黄酮制备宝霍苷I中的应用,仅利用一种酶不仅能高效地酶解淫羊藿总黄酮中富含的淫羊藿苷,而且能高效地酶解富含的朝霍定A、箭藿苷A制得宝霍苷I,并且能特异地将朝霍定B和箭藿苷B都制得宝霍苷I的方法。
技术方案:β-葡萄糖苷酶在转化淫羊藿总黄酮制备宝霍苷I中的应用,利用β-葡萄糖苷酶酶解淫羊藿总黄酮中富含的淫羊藿苷、朝霍定A、箭藿苷A制得宝霍苷I,且特异地将朝霍定B和箭藿苷B制得宝霍苷I。
所述β-葡萄糖苷酶来源于细菌或真菌。
所述β-葡萄糖苷酶优选来源于Dictyoglomus thermophilum DSM 3960的GH3家族β-葡萄糖苷酶的大肠杆菌重组菌。
酶转化体系为:淫羊藿总黄酮浓度5g/L,pH 5.0 25mM柠檬酸-磷酸氢二钠缓冲液,再加入β-葡萄糖苷酶Dth3至终浓度25U/mL,在85℃下反应1h后生成宝霍苷I。
来源于Dictyoglomus thermophilum DSM 3960的GH3家族β-葡萄糖苷酶及其基因重组菌在转化淫羊藿总黄酮制备宝霍苷I中的应用。
制备的富含宝霍苷I的产品在制备治疗乳腺癌、肺癌、结肠癌、肝癌药物中的应用。
该方法筛选了多种不同来源不同家族的β-葡萄糖苷酶,包括有来源于Thermotogathermarum DSM 5069GH1家族、Thermotoga thermarum DSM 5069GH3家族、Thermotogapetrophila DSM 13995GH1家族、Thermotoga petrophila DSM 13995GH3家族、Dictyoglomus thermophilum DSM3960等,并确定来源于Dictyoglomus thermophilumDSM3960的β-葡萄糖苷酶Dth3具有将多组分淫羊藿黄酮淫羊藿苷、朝霍定A,朝霍定B、箭藿苷A及箭藿苷B转化生成宝霍苷I。
有益效果:本发明利用成分确定的重组酶催化淫羊藿总黄酮制备宝霍苷I,酶法转化的摩尔转化率大于95%。转化效率高。本发明首次筛选到不仅能高效地水解淫羊藿苷、箭藿苷A上的葡萄糖苷,能够高效水解朝霍定B、箭藿苷B上的葡萄糖苷和木糖苷的β-葡萄糖苷酶,该酶的最适反应温度为90℃,温度稳定性优良。在优选条件下可高效表达目的蛋白。而且,淫羊藿总黄酮经本发明提供的β-葡萄糖苷酶转化后得到以宝霍苷I为主要黄酮成分的产品,宝霍苷I在抑制人肝癌HepG2细胞、肺癌A549细胞、小鼠结肠癌CT26细胞和乳腺癌4T1细胞增殖方面的活性,显著强于淫羊藿总黄酮中富含的其他黄酮单体。因此,本发明所述的β-葡萄糖苷酶可应用于高抗肿瘤活性宝霍苷I的制备。
附图说明
图1为实施例2纯化的β-葡萄糖苷酶Dth3的纯度鉴定结果图;其中泳道M为蛋白Marker(购自Thermo scientific公司,货号2661),泳道1为纯酶蛋白;泳道2为热处理后的粗酶液;泳道3为粗酶液;泳道4为PET-20b转化宿主菌空白对照的全细胞裂解液;泳道5为诱导表达后全细胞裂解液。
图2为实施例3所述β-葡萄糖苷酶的定性测定结果图;其中A图为最适反应温度的测定结果图,横坐标为温度,单位摄氏度(℃),纵坐标为相对酶活力,单位%;B图为最适反应pH的测定结果图,横坐标为pH,纵坐标为相对酶活力,单位%;C图为pH稳定性的测定结果图,横坐标为pH,纵坐标为相对酶活力,单位%;D图为温度稳定性的测定结果图,横坐标为保温时间,单位分钟(min),纵坐标为相对酶活力,单位%。
图3为实施例4所述β-葡萄糖苷酶诱导表达条件优化结果图;其中A图为不同诱导剂IPTG浓度下重组菌产酶结果图,纵坐标为酶活力,单位为U/mL;横坐标为诱导剂IPTG的浓度,B为不同诱导温度下重组菌产酶结果图,纵坐标为酶活力,单位为U/mL;横坐标为诱导温度。
图4为实施例5所述5种不同来源不同家族β-葡萄糖苷酶转化淫羊藿黄酮效果比较图。其图A为5种不同来源不同家族β-葡萄糖苷酶对朝霍定B转化生成宝霍苷Ⅰ的效果比较;图B是5种不同来源不同家族β-葡萄糖苷酶对淫羊藿提取物的转化生成宝霍苷Ⅰ的效果比较,其中转化率以目标产物宝霍苷Ⅰ含量计。注:TtBGL1表示来源于Thermotoga thermarumDSM5069GH1家族;TpBGL1表示来源于Thermotoga petrophila DSM 13995GH1家族的β-葡萄糖苷酶;TtBGL3表示来源于Thermotoga thermarum DSM 5069GH3家族;TpBGL3表示来源于Thermotoga petrophila DSM 13995GH3家族的β-葡萄糖苷酶;DthBGL3表示Dictyoglomusthermophilum DSM3960的β-葡萄糖苷酶。
图5为实施例5所述β-葡萄糖苷酶Dth3对淫羊藿提取物转化前后样品的HPLC分析图谱,其中1为其中1.朝霍定A;2.朝霍定B;3.朝霍定C;4.淫羊藿苷;6.淫羊藿苷元及目标产物5(宝霍苷Ⅰ),显示目前的HPLC方法可有效地对这几种组分进行分析,且方法高效稳定。
图6为淫羊藿总黄酮中各黄酮单体的抗肿瘤活性比较图。其中HepG2为人肝癌细胞,A549为人肺癌细胞,CT26为小鼠结肠癌细胞,4T1为小鼠乳腺癌细胞。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步的详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定发明。
下面结合附图及具体实施例对本发明的应用原理作进一步描述。
实施例1:本发明所述β-葡萄糖苷酶基因的获得及重组质粒pET-DthBGL3的构建
1.1Dictyoglomus thermophilum DSM 3960的培养
Dictyoglomus thermophilum DSM 3960购于DSMZ菌种保藏中心(www.dsmz.de)编号为13995,其培养基配方为:磷酸二氢钾1.5g/L,十二水合磷酸氢二钠4.2g/L,氯化铵0.5g/L,六水合氯化镁0.38g/L,二水合氯化钙0.06g/L,六水合硫酸铁铵0.04g/L,六水氯化钴2.9mg/L,二水合钼酸钠2.4mg/L,五水合硒酸钠1.7mg/L,四水合氯化锰2mg/L,硫酸锌2.8mg/L,可溶性淀粉5g/L、蛋白胨2g/L、酵母提取物2g/L、碳酸钠1g/L、盐酸半胱氨酸1g/L、刃天青钠1g/L,在氮气环境下脱氧,调整pH为7.2。用注射器按照0.5wt.%接种量接种,85℃静止培养24h,收集细胞。
1.2基因组DNA的提取
(1)静置培养Dictyoglomus thermophilum DSM 3960 24小时,取30mL菌液4,000g离心10min收集细胞。
(2)用9.5mL TE缓冲液重悬菌体,加入0.5mL 10wt.%十二烷基硫酸钠(SDS)和50μL蛋白酶K(20mg/mL),混合均匀,37℃保温1h。
(3)加入1.8mL 5mol/L NaCl,1.5mL十六烷基三乙基溴化铵(CTAB)/NaCl,混匀,65℃温育20min。
(4)加入等体积(13mL)氯仿/异戊醇(体积比42:1),混匀,6,000g离心10min。
(5)为防止剪切力造成基因组DNA断裂,用粗口吸管将上清转入另一离心管中,加入等体积(13mL)酚/氯仿/异戊醇(体积比25:24:1)混匀,6,000g离心10min。
(6)上清转移至新离心管,加入0.6倍体积异丙醇,轻轻晃动至白色丝状DNA沉淀清晰可见。
(7)用吸管将DNA缠绕其上,在70%酒精中清洗。
(8)用无菌牙签将DNA从吸管上刮下,转入1.5mL离心管中。
(9)室温下风干,加500μL TE缓冲液溶解。
(10)取50μL用核酸蛋白检测仪检测DNA浓度。
1.3重组质粒pET-DthBGL3的构建
按照已知的Dictyoglomus thermophilum DSM 3960β-葡萄糖苷酶基因(登录号:WP_041723615.1)设计引物,引物由上海生物工程有限公司合成。以提取的Dictyoglomusthermophilum DSM 3960的基因组DNA为模板,用合成的引物进行PCR扩增,扩增的条件是94℃,3min;30次循环(94℃,10s;58℃,30s;72℃,2.7min);72℃,5min;反应停止,4℃保温。通过凝胶回收试剂盒对PCR扩增产物进行纯化。得到β-葡萄糖苷酶DthBgl3的DNA分子。
将得到的β-葡萄糖苷酶DthBgl3的DNA分子和pET-28a分别用Nco I和XhoI进行双酶切,并分别割胶回收,浓缩后16℃连接过夜,将连接产物转化大肠杆菌JM109感受态细胞,筛选阳性克隆,进行序列分析;挑选序列正确的克隆提取质粒,获得含有β-葡萄糖苷酶DNA分子的重组质粒pET-DthBGL3。
实施例2:本发明所述β-葡萄糖苷酶Dth3的制备
将重组质粒pET-DthBGL3转化大肠杆菌JM109(DE3)宿主菌(购自Novagen公司),在含有卡那霉素(50μg/mL)的LB平板(LB培养基:胰蛋白胨10g/L,酵母提取物5g/L,NaCl 5g/L,琼脂15g/L)上经过37℃培养过夜,挑转化子到200mL的LB培养基中(50μg/mL卡那霉素)37℃,200rpm振荡培养至OD600为0.6时,加入终浓度为0.005-0.01mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂,30℃培养6h,用高速冷冻离心机将培养液在4℃下,以13,000rpm离心15min,收集菌体。
由于重组质粒pET-DthBGL3中含有His-tag标签,通过His·Bind PurificationKit(购自Novagen公司)进行纯化,得到纯化的重组酶。具体操作过程:
A.样品的处理
(1)将洗涤过的菌体,用1×Binding Buffer 8mL重悬,超声波破壁。
(2)破壁后,13,000g离心30min,取上清即为样品。
B.处理柱子
(1)取1mL填料装柱。
(2)用3mL的无菌水洗柱子。
(3)用5mL的1×Charge Buffer洗柱子。
(4)用3mL的1×Binding Buffer洗柱子。
C.上样
(1)将样品加入柱子,控制流速约每分钟6滴。
(2)用3mL 1×Binding Buffer洗柱子,除去未结合的蛋白质。
(3)用8mL含有20mM咪唑的洗脱液洗柱子,除去杂蛋白。
(4)用200mmol/L咪唑的洗脱液洗柱子,将目的蛋白洗脱下来。
(5)用4mL 1×Strip Buffer洗柱子。
通过此过程得到纯化的β-葡萄糖苷酶,通过SDS-PAGE电泳后染色鉴定β-葡萄糖苷酶的纯度,结果如图1所示。
Dth3基因在宿主菌JM109(DE3)中表达量较高,目的蛋白通过HisTag标签纯化后,其洗脱液中β-葡萄糖苷酶Dth3纯度较高,在80kDa处有单一的条带,达到电泳纯级别。
实施例3:本发明所述β-葡萄糖苷酶的定性测定
1、酶活的测定方法
反应体系100μL,5μL 20mmol/L对硝基苯-β-L-葡萄糖苷(pNPG)中加入85μL100mmol/L柠檬酸-磷酸氢二钠缓冲液(pH 5.0),先在90℃孵育2min,再加入10μL稀释到合适倍数的酶液反应10min,显色后再加入1mol/L的碳酸钠溶液600μL终止反应。在405nm下测定吸光值。酶活力单位(U)定义为:在测定条件下,每分钟产生1μmol p-硝基苯酚所需要的酶量为1个酶活力单位。
2、最适反应温度的测定
在60-100℃范围内,每隔5℃,分别测定酶活。缓冲为100mmol/L柠檬酸-磷酸氢二钠缓冲液,pH 5.0,结果如图2-A所示。
由图2-A结果可见,本发明所述β-葡萄糖苷酶的最适反应温度为90℃。
3、最适反应pH的测定
在不同的pH(3.5-7.5,100mmol/L柠檬酸-磷酸氢二钠缓冲液)条件下,90℃分别测定酶活,结果如图2-B所示。
由图2-B结果可见,本发明所述β-葡萄糖苷酶的最适反应pH为5.0。
4、pH稳定性的测定
将纯化的重组酶DthBgl3在不同的pH(3.5-7.5,100mmol/L柠檬酸-磷酸氢二钠缓冲液)下70℃处理1h,与不保温酶的酶相比,结果如图2-C所示。
由图2-C结果可见,本发明所述β-葡萄糖苷酶在pH4.5-7.5条件下75℃保温1h后仍能具有80%以上的残余酶活力。
5、温度稳定性的测定
在pH 5.0下,使酶在75℃,85℃,90℃温度下分别保温不同的时间(0,10,30,60,90,120min),再测定相对酶活,以未保温(4℃保存)的酶活性为100%,结果如图2-D所示:三角形表示75℃;正方形表示85℃;圆形表示90℃。
由图2-D结果可见,本发明所述β-葡萄糖苷酶在75℃下保温2h残余酶活力高于85%。
实施例4:本发明所述β-葡萄糖苷酶优选制备方法
将重组质粒pET-DthBGL3转化大肠杆菌JM109(DE3)宿主菌(购自Novagen公司),在含有卡那霉素(50μg/mL)的LB平板(LB培养基:胰蛋白胨10g/L,酵母提取物5g/L,NaCl 5g/L,琼脂15g/L)上经过37℃培养过夜,挑转化子到200mL的LB培养基中(50μg/mL卡那霉素)37℃,200rpm振荡培养至OD600为0.6时,加入终浓度分别为0mM,0.005mM,0.01mM,0.05mM,0.1,0.5mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂,37℃培养7h,用高速冷冻离心机分别将2mL培养液在4℃下,以13,000rpm离心15min,收集菌体。在菌体中加入缓冲溶液,重悬后,超声波破碎细胞,获得全细胞裂解液,取全细胞裂解液以13,000rpm离心15min,获得上清液可溶性蛋白溶液,沉淀即为不溶性蛋白—包涵体。我们通过测定上清液中β-葡萄糖苷酶活力来评价不同表达条件的效果,结果如图3-A所示。
由图3-A可见,在30℃诱导下,加入微量的IPTG至于终浓度0.005-0.01mM时,重组酶即可获得最高表达;但是随着IPTG添加浓度越大,酶产率越低,可能是高浓度的诱导剂对菌体生长造成影响。由此可见,本发明所述表达β-葡萄糖苷酶的基因重组菌在37℃下加入0.005mM诱导剂IPTG即可达到高效表达。
挑取含有表达质粒PET-DthBGL3转化子到200mL的LB培养基中(50μg/mL卡那霉素)37℃,200rpm振荡培养至OD600为0.6时,加入终浓度0.005mM异丙基β-D-硫代吡喃半乳糖苷(IPTG)诱导剂,分别在22℃、28℃、32℃、37℃、43℃诱导培养7h,用高速冷冻离心机分别将2mL培养液在4℃下,以13,000rpm离心15min,收集菌体。在菌体中加入缓冲溶液,重悬后,超声波破碎细胞,获得全细胞裂解液,取全细胞裂解液以13,000rpm离心15min,获得上清液可溶性蛋白溶液,沉淀即为不溶性蛋白—包涵体。我们通过测定上清液中β-葡萄糖苷酶活力来评价不同表达条件的效果,结果如图3-B。
由图3-B可见,在37-32℃诱导下,加入微量的IPTG至终浓度0.005M时,重组酶可达到最高表达;但是随着诱导温度的降低,酶产量也相应降低,而当培养温度升高时,酶表达量也有一定的降低,这可能是由于低温或者高温影响了重组菌的生长速度。
实施例5:本发明所示5中多种不同来源不同家族的β-葡萄糖苷酶对淫羊藿提取物中的多组分黄酮糖苷转化效果的比较。
分别选取不同来源不同家族的β-葡萄糖苷酶,包括Thermotoga thermarum DSM5069GH1家族、Thermotoga thermarum DSM 5069GH3家族、Thermotoga petrophila DSM13995GH1家族、Thermotoga petrophila DSM 13995GH3家族、Dictyoglomus thermophilumDSM3960共5种β-葡萄糖苷酶,在相同底物浓度及酶添加量及5种β-葡萄糖苷酶各自最适合反应条件下对淫羊藿多组分黄酮朝藿定A、朝藿定B以及淫羊藿苷进行转化,其转化反应体系均100μL,其中朝藿定A、朝霍定、淫羊藿苷的含量均为1g/L,酶添加量均为25U/mL,其中Thermotoga thermarum DSM 5069GH1家族、Thermotoga petrophila DSM 13995GH1家族的β-葡萄糖苷酶转化条件为pH 6.0,反应温度90℃;Thermotoga thermarum DSM 5069GH3家族、Thermotoga petrophila DSM 13995GH3家族以及Dictyoglomus thermophilumDSM3960的β-葡萄糖苷酶转化条件为pH 5.0,反应温度90℃。反应时间为30min。
高效液相检测条件如下,色谱柱:安捷伦C18XDB;流动相流速:1mL/min;检测波长(UV):203nm;柱温:30℃;流动相比例如表1。
表1淫羊藿总黄酮提取物的高效液相色谱流动相条件
时间(min) | 水(V/V,%) | 乙腈(V/V,%) |
0 | 80 | 20 |
35 | 72 | 28 |
45 | 63 | 37 |
50 | 10 | 90 |
50.1 | 80 | 20 |
55 | 80 | 20 |
结果如图4所示5种不同来源不同家族的β-葡萄糖苷酶均能高效地将朝定霍A和淫羊藿苷转化为目标产物宝霍苷I,然而仅有来源于Dictyoglomus thermophilum DSM3960的β-葡萄糖苷酶DthBGL3具有β-葡萄糖苷酶和β-木糖苷酶活力,因此仅有DthBGL3能够将朝霍定B高效地转化为目标产物宝霍苷I,其转化率达到99.2%。
实施例6:本发明所述β-葡萄糖苷酶转化淫羊藿提取物反应前后HPLC检测图谱。
将实施例2中所述,纯化后的β-葡萄糖苷酶DthBGL3应用于淫羊藿提取物的催化转化。其中淫羊藿总黄酮提取物由江苏康源药业股份有限公司提供,其主要成分的含量如下,朝藿定A:1.44%、朝藿定B:2.76%、朝藿定C:5.96%、淫羊藿苷:9.05%、宝霍苷I:0.95%。
反应体系为:淫羊藿提取物5g/L,酶添加量为25U/L,反应pH5.0,反应温度90℃,反应时间1小时。产物加入甲醇,用于HPLC分析。其液相检测方法同实施例5。
结果如图5所示,β-葡萄糖苷酶DthBGL3对淫羊藿提取物具有显著的转化能力,反应1小时后,几乎完全转化为宝霍苷I。
实施例7:本发明所述的淫羊藿提取物经β-葡萄糖苷酶DthBGL3转化后抗肿瘤将有所提高。
在96孔板中每孔接种3000个细胞,放入37℃培养,待细胞贴壁6h后,每孔加入不同浓度的淫羊藿黄酮培养72h,于实验终点前4h向每孔加入20μL MTT(4mg/mL),实验终点时取出96孔板,于1000rcf离心,然后吸去上清,加入200μL DMSO,570nm处测吸光值。根据以下公式计算待测样品对肿瘤细胞体外增殖的抑制率:
抑制率=[100-(OD570(实验孔)-OD570(空白对照))/(OD(不加药对照空)-OD570(空白对照)×100)%
根据实施例5中淫羊藿总黄酮经β-葡萄糖苷酶DthBGL3转化前后的HPLC图谱可见,淫羊藿总黄酮本来主要由淫羊藿苷、朝藿定A、B、C和宝霍苷I等黄酮类化合物组成,其中,宝霍苷I含量不高,而经转化后主要由宝霍苷I组成。如图6所示的体外抗肿瘤活性研究表明,在100μM的浓度下,宝霍苷I对人肝癌HepG2、肺癌A549、小鼠结肠癌CT26、乳腺癌4T1的增殖抑制率分别高达97%、90%、92%和96%,显著高于淫羊藿总黄酮中的其他单体成分。因此,淫羊藿总黄酮经本发明提供的β-葡萄糖苷酶转化为宝霍苷I后,抗肿瘤活性得到显著提高。
淫羊藿总黄酮结构如下所示:
Claims (6)
1.β-葡萄糖苷酶在转化淫羊藿总黄酮制备宝霍苷I中的应用,其特征在于利用β-葡萄糖苷酶酶解淫羊藿总黄酮中富含的淫羊藿苷、朝霍定A、箭藿苷A制得宝霍苷I,且特异地将朝霍定B和箭藿苷B制得宝霍苷I。
2.根据权利要求1所述的应用,其特征在于所述β-葡萄糖苷酶来源于细菌或真菌。
3.根据权利要求2所述的应用,其特征在于所述β-葡萄糖苷酶优选来源于Dictyoglomus thermophilum DSM 3960的 GH3家族β-葡萄糖苷酶的大肠杆菌重组菌。
4.根据权利要求1所述的应用,其特征在于酶转化体系为:淫羊藿总黄酮浓度5 g/L,pH 5.0 25 mM柠檬酸-磷酸氢二钠缓冲液,再加入β-葡萄糖苷酶Dth3至终浓度25 U/mL,在85℃下反应1 h后生成宝霍苷I。
5.来源于Dictyoglomus thermophilum DSM 3960的 GH3家族β-葡萄糖苷酶及其基因重组菌在转化淫羊藿总黄酮制备宝霍苷I中的应用。
6.权利要求1所述应用制备的富含宝霍苷I的产品在制备治疗乳腺癌、肺癌、结肠癌、肝癌药物中的应用。
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