CN110156855A - 糖基化黄酮类化合物及其制备方法和应用 - Google Patents
糖基化黄酮类化合物及其制备方法和应用 Download PDFInfo
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- CN110156855A CN110156855A CN201910433218.9A CN201910433218A CN110156855A CN 110156855 A CN110156855 A CN 110156855A CN 201910433218 A CN201910433218 A CN 201910433218A CN 110156855 A CN110156855 A CN 110156855A
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- glycosylated
- diosmin
- flavanoid
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Abstract
本发明提供了一种糖基化黄酮类化合物及其制备方法和应用。及其在治疗痔疮/冻疮等先关的静脉曲张功能不全的相关症状以,及在活血化瘀及抗炎方面的应用。本发明用糖苷酶和糖基转移酶与黄酮类化合物在缓冲液中进行糖基化反应,可以制备出高溶解度的地奥司明或其衍生物。本发明制备糖基化黄酮类化合物的方法具有转化率高、溶解度高、条件温和、使用无毒性试剂等特点,本发明制备的糖基化黄酮类化合物在治疗痔疮、促进血液循环及抗炎的疗效均优于地奥司明。
Description
技术领域
本发明涉及有机化学技术领域。
技术背景
黄酮类化合物因其独特的化学结构而对哺乳动物和其它类型的细胞具有许多重要的生理、生化作用。随着国内外化学家对其构效关系的深入研究,发现了黄酮类化合物部分药理作用的作用机制,为其在医药、食品领域的应用提供了理论依据,加快了黄酮类化合物的开发利用。如:将橙皮苷脱氢,即可得到地奥司明;将地奥司明糖基化,可以提高溶解度,增加地奥司明的疗效,提高了地奥司明的经济价值。
地奥司明是一种黄酮类药物,如:商品名爱脉朗(施维雅(天津)制药有限公司),地奥司明片(马应龙药业集团股份有限公司),主治:1、静脉淋巴功能不全相关的各种症状(腿部沉重、疼痛、晨起时肿胀不适等);2、急性痔疮发作相关的各种症状。但目前制备地奥司明的方法存在诸多缺点。
①中国专利CN 201110027247.9公开的生产方法包括将橙皮苷、碘和吡啶进行主反应,然后再经清洗、纯化、干燥、粉碎与混合,最后回收碘。其工艺线路长,反应复杂,相关杂质难于控制,收率低,实现工业化生产难度高。
②中国专利CN 201310255497.7公开了将橙皮甙加入吡啶和二甲亚砜的混合溶剂中,再加入碘化钾与硫酸进行脱氢反应,再加入含氢氧化钠的甲醇溶液,然后加酸调pH值,静置析晶。但是该方法在生产过程中加入固体碱,反应速度降低,同时造成产品相关杂质难于控制,会降低产品纯度。
③中国专利CN 201610474671.0公开的制备方法包括:(a)将四季青粉碎,用65~75%乙醇热回流提取,合并提取液,浓缩至无醇味,依次用石油醚、乙酸乙酯和水饱和的正丁醇萃取,分别得到石油醚萃取物、乙酸乙酯萃取物和正丁醇萃取物;(b)步骤(a)中正丁醇取物用大孔树脂除杂,先用20%乙醇洗脱9个柱体积,再用65%乙醇洗脱10个柱体积,收集65%洗脱液,减压浓缩得65%乙醇洗脱浓缩物;(c)步骤(b)中65%乙醇洗脱浓缩物用正相硅胶分离,依次用体积比为65:1、35:1、15:1和7:1的二氯甲烷-甲醇梯度洗脱得到4个组分;(d)步骤(c)中组分4用正相硅胶进一步分离,依次用体积比为12:1、7:1和1:1的二氯甲烷-甲醇梯度洗脱得到3个组分;(e)步骤(d)中组分2用十八烷基硅烷键合的反相硅胶分离,用体积百分浓度为58%的甲醇水溶液等度洗脱,收集11~15个柱体积洗脱液,洗脱液减压浓缩。该制备方法中主要辅料碘的用量较大,由于碘的价格较高,造成地奥司明生产成本高,没有成本优势,而且后期碘回收操作危险,环境污染严重。
上述合成方法在技术上虽然已经成熟,但是会用到哌啶等有机碱刺激性气味的,对操作工人的刺激性很大,而且卤素的价格昂贵,回收难度大,造成地奥司明的成本高居不下和环境污染大等因素。
另一方面,地奥司明在临床应用中,具有明显缺陷,如:溶解性差,生物利用低,疗效局限等,严重限制了地奥司明的应用。
发明内容
本发明通过对黄酮化合物进行生物酶转化,通过糖基化、去糖基等生物合成的方法,得到高纯度、高质量、高生物活性的糖基化地奥司明及其糖基化衍生物,满足了市场的需求、提高了地奥司明的疗效。
本发明提供的糖基化黄酮类化合物结构如式(Ⅰ)所示
其中:R1为H、F、Cl、Br、CH3、OCH3或OCH2CH3;
R2为H、F、Cl、Br、CH3、OCH3或OCH2CH3;
R3为Glu,Rha,Xyl或Gal,n=1~9。
优选地,所述R1为H、CH3或OCH3;R2为H、CH3或OCH3;R3为Glu或Rha,n=1~5。
上述糖基化黄酮类化合物是通过生物酶制备方法实现的,具体为:将原料、糖基供体、反应缓冲液、糖苷酶和糖基转移酶混合均匀,所述原料与反应缓冲液的比例为1:(10~50),反应的时间为10~24h,反应温度为35~75℃,所述原料包括但不限于地奥司明和香叶木素。
其中,所述糖基供体包括葡萄糖、UDP-葡萄糖、木糖、UDP-木糖、半乳糖、UDP-半乳糖、赤藓糖、α-环糊精、β-环糊精、淀粉、木聚糖、蔗糖、麦芽糊精,所述糖基供体的浓度为1~10g/L。所述反应缓冲液是质量分数为0.05~1%的磷酸氢二钠-柠檬酸钠缓冲液或质量分数为0.9%的NaCl缓冲液,所述反应缓冲液的pH值为2~10。所述糖苷酶为鼠李糖苷酶,所述糖基转移酶为葡萄糖基转移酶、木糖基转移酶或半乳糖基转移酶中的一种或两种组合。所述糖苷酶与糖基转移酶的比例为(5:95)~(95:5);所述糖苷酶和糖基转移酶总量的质量分数为原料的5%~50%,进一步优选为10%~30%。
所述的糖基化黄酮类化合物可通过与药学上可接受的盐、载体、赋形剂、稀释剂、媒介物或它们的组合相结合,进而制作成药物组合物。
上述糖基化黄酮类化合物或药物组合物可应用于痔疮、冻疮、活血化瘀、抗炎及静脉淋巴功能不全方面;亦可应用于护肤品、保健品、食品和药品。
通过对地奥司明和糖基化地奥司明及其衍生物的研究,在动物细胞模型条件下,糖基化地奥司明及其衍生物具有良好的抗炎效果,糖基化地奥司明的抗炎效果和地塞米松的效果相当;在溶解度实验中发现糖基化地奥司明的溶解度要比地奥司明的溶解度高出约150倍,有利于提高地奥司明的疗效;在动物试验中糖基化地奥司明比地奥司明的生物利用度高了约45.3%;糖基化地奥司明在增加血液流动,增加血管弹性方面的疗效优于地奥司明,且剂量也小于地奥司明的用量。
附图说明
图1是实施例4所述糖基化地奥司明HPLC液相图;
图2是实施例5所述葡萄糖基橙皮苷拟合曲线图;
图3是实施例5所述糖基化地奥司明与常规地奥司明的溶解度对比图。
具体实施方式
以下各步骤仅用以说明本公开的技术方案,而非对其限制;尽管参照前述各步骤对本公开进行了详细的说明,但本领域的普通技术人员应当理解:其依然可以对前述各步骤所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本公开各步骤技术方案的范围。
实施例1
以香叶木素和鼠李糖、葡萄糖为原料,反应缓冲液为质量分数为0.5%的磷酸氢二钠-柠檬酸缓冲液(pH=6);在反应体系中按1:1的量加入鼠李糖苷酶和葡萄糖基转移酶,两种酶的质量分数为投入香叶木素的20%;该反应体系中葡萄糖浓度为30g/L,鼠李糖浓度为10g/L,反应温度52℃,反应时间控制6h。在该反应体系中,得到糖基化地奥司明(n=1~9),产率97.6%。
实施例2
以香叶木素和鼠李糖、葡萄糖为原料,反应缓冲液为质量分数为0.9%的磷酸氢二钠-柠檬酸缓冲液(pH=5);在反应体系中先加入葡萄糖基转移酶,3h后再加入鼠李糖苷酶,再持续3h后终止反应。所述鼠李糖苷酶和葡萄糖基转移酶的量为1:1,两种酶的质量分数为投入香叶木素的15%;该反应体系中葡萄糖浓度为10g/L,鼠李糖浓度为30g/L,反应温度65℃。在该反应体系中,得到糖基化地奥司明(n=1~9),产率96.6%。
实施例3
以香叶木素和UDP-葡萄糖、UDP-鼠李糖为原料,反应缓冲液为质量分数为0.9%的磷酸氢二钠-柠檬酸缓冲液(pH=5);在反应体系中先加入葡萄糖基转移酶,3h后再加入鼠李糖苷酶,再持续3h后终止反应。所述鼠李糖苷酶和葡萄糖基转移酶的量为1:1,两种酶的质量分数为投入香叶木素的15%;该反应体系中UDP-葡萄糖浓度为10g/L,UDP-鼠李糖浓度为30g/L,反应温度65℃。在该反应体系中,得到糖基化地奥司明(n=1~9),产率96.4%。
实施例4
以地奥司明和鼠李糖、木糖为原料,反应缓冲液为质量分数为0.5%的磷酸氢二钠-柠檬酸缓冲液(pH=6);在反应体系中同时加入木糖基转移酶和鼠李糖苷酶,两种酶的质量分数为投入地奥司明的20%;该反应体系中木糖浓度为20g/L,鼠李糖浓度为20g/L,反应温度52℃,反应时间控制6h。在该反应体系中,得到糖基化地奥司明(n=1~9),产率95.7%。结果如图1,据图可知,该反应体系可以有效的糖基化地奥司明,由于多糖基化橙皮素链接的糖基的数量不同,在HPLC的图中出峰时间会出现一次减小的峰。
实施例5,糖基化地奥司明(n=1~9)的溶解度测定
化合物溶解能力的大小直接影响到药物在溶液体系和细胞体系的应用。因为糖基化地奥司明在水溶液中稳定,所以我们利用紫外分光光度法对饱和状态的糖基化地奥司明的水溶液进行溶解度值的测定。
分别精密量取10mg、50mg、100mg、200mg、1000mg的葡萄糖基橙皮苷,置于100mL容量瓶中,加DMSO稀释至刻度,摇匀,得到一系列的浓度分别为0.1mg/mL,0.5mg/mL,1mg/mL,2mg/mL,10mg/mL的葡萄糖基橙皮苷标准溶液,用HPLC分析,对283nm区间的特征峰进行积分,记录峰面积。以葡萄糖基橙皮苷的浓度为纵坐标,峰面积为横坐标作图,并进行线性回归。标准曲线图如图2。
标准曲线方程1:y=0.00151A+0.00966,R2=0.9999为葡萄糖基橙皮苷拟合曲线。
然后将过量的糖基化地奥司明和地奥司明分别加入到2mL水相中,置于25±1℃恒温振荡器上连续振荡72h,取出后转移到离心管中,8000r/min离心15min,取上清液用0.45μm微孔滤膜过滤,并用甲醇稀释至线性范围之内,采用紫外分光光度法测定糖基化地奥司明和地奥司明在水中的溶解度。结果见图3,糖基化的地奥司明的溶解度是地奥司明的45倍左右。
实施例6,糖基化地奥司明(n=1~9)急毒性测定
在28±1℃的温度,70±5%的湿度条件下,选取7~8周龄,健康的清洁级NIH小鼠20只,雌雄各半,分2组雌雄各5只,体重在20~22g。将饲料和水消毒,试验前和试验的观察期内,均按正常饲料条件饲养。
将分别将糖基化地奥司明溶解在0.5%Tween80中,浓度为500mg/ml,分别将该液体经口给药小鼠,按小鼠体重0.02ml/g为给药剂量。给药后观察1,4,8,12h,以后每12h观察一次。观察死亡情况,每天记录小鼠体重变化以及其他的症状。第10天,以断颈的方式处死小鼠,取各器官进行病理检查。
在第10天,全部小鼠存活,0.02ml/g剂量的糖基化地奥司明未见毒性反应。小鼠各器官病理检查正常,没有发现病变,10天内小鼠体重未见减轻。因此,说明本发明的糖基化地奥司明在口服给药动物时未见毒性。
实施例7,糖基化地奥司明(n=1~9)耳肿涨实验
1、实验材料
实验动物来自广东省实验动物中心的SPF级的5-6周雌性Balb/c小鼠。
糖基化地奥司明,广东金骏康生物技术有限公司;TPA-99.99%,中山大学药学院;丙酮,广州试剂厂;二氯甲烷,广州试剂厂;DMSO,广州试剂厂,布洛芬,美国阿拉丁工业公司。
2、实验方法
1)、将40只Balb/c小鼠随机分成五组,分别是丙酮空白组、TPA对照组、姜黄素组、厚朴酚以及化合物组,每组1只小鼠,重复3次。
2)、丙酮空白组和TPA对照组分别用15μL自配的溶液(二氯甲烷:丙酮=80:20)均匀涂于小鼠两只耳朵,而糖基化地奥司明均匀涂于小鼠两只耳朵。
3)、6min后,丙酮空白组用丙酮均匀涂于小鼠两只耳朵:而TPA对照组、糖基化地奥司明组则分别用15μL TPA均匀涂于小鼠两只耳朵两面致炎。
4)、6h后,将小鼠处死,随后及时剪下小鼠双耳,并用直径6mm的打孔器分别在同一部位打下圆耳片。
5)、称重,以丙酮空白组为阴性,将实验组耳片重量与空白组耳片重量的差异作为肿胀度。
6)、抑制率=(1-给药组肿胀度/对照组的肿胀度)×100%。
3、实验结果
3.1糖基化地奥司明对TPA诱导的小鼠耳廓肿胀的抑制作用
为了研究糖基化地奥司明对TPA诱导的小鼠耳廓肿胀的抑制作用,选定地糖基化奥司明的工作浓度为0.75μM。通过对糖基化地奥司明对小鼠耳廓肿胀的抑制实验研究发现,单纯涂抹TPA诱导小鼠耳廓肿胀导致小鼠耳廓的平均重量由6.1mg增重至12.1mg,而且糖基化地奥司明对小鼠耳廓肿胀都显示不同程度的抑制作用,实施例1和4制得的糖基化地奥司明的抑制率范围为94.6%、90.1%。
糖基化地奥司明对TPA诱导的小鼠耳廓肿胀起抑制作用很明显。由动物实验结果表明,实施例1和4制得的糖基化地奥司明的抑制率分别为94.6%和90.1%,布洛芬和NSAIDs的抑制率为96.1%和83.4%。
实施例8,糖基化地奥司明(n=1~9)凝胶对巴豆油致大鼠急性痔疮的影响
Wistar大鼠,雄性。设空白对照组、阳性药马痔膏对照组和糖基化地奥司明凝胶组低、中、高三个浓度组。造模后将动物按肛周肿胀程度分到各组,每组10只。
羟丙甲基纤维素2.5%,卡波姆0.75%,三乙胺0.75%,甘油5%,丙二醇5%,氮酮2%,分别取实施例1制得的糖基化地奥司明3%、5%、8%,其余为蒸馏水,充分混合均匀,灭菌,即为低、中、高三个浓度组的糖基化地奥司明凝胶。
按照1份蒸馏水、4份吡啶、5份乙醚和10份6%的巴豆油乙醚溶液配成致炎剂。然后把浸吸0.16mL巴豆油混合液的棉球插入6周龄大鼠肛门内10s,建立大鼠肛门巴豆油肿胀模型。次日按肿胀程度分组,空白基质对照组、阳性药马痔膏对照组和受试药地糖基化奥司明凝胶、糖基化新地奥司明凝胶组分别涂抹空白基质、马痔膏、七叶皂苷钠凝胶,每日涂一次,涂皮量均为1.0g/kg.bw(0.2g/只),连续涂7天,于末次给药后30min,处死各组大鼠,切取自肛门皮毛缘起15mm的直肠组织,以冷生理盐水清洗后,用滤纸吸干水分。每组随机选取5只动物,其组织以10%甲醛固定,石蜡包埋,切片,HE染色,行病理组织学观察;剩余组织用以测定各组大鼠的肛门肿胀情况。以病检结果及直肠肛门肿胀系数为指标,观察七叶皂苷钠凝胶对巴豆油致大鼠肛门肿胀的治疗作用。
结果见表1,本发明药物中、高浓度组均能明显改善巴豆油所致大鼠肛门肿胀及炎症反应。与空白对照组比较,差异有显著性意义(P<0.05)。
表1本发明药物对巴豆油致大鼠肛门肿胀的影响(n=10)
注:与空白对照组比较,*P<0.05。
实施例9,糖基化地奥司明(n=1~9)血管扩张的影响
SD大鼠60只,雄性,体重150-170g,由中山大学实验动物中心提供,合格证:粤医动字20090043号。采用分层随机法,将动物分成3组,每组20只。先在实验的环境下给A普通饲料、B含有地奥司明1g/kg饲料、C含有多糖基化1g/kg饲料,饲养一个月,然后再用普通饲料喂养A、B、C组SD大鼠一周。
从SD大鼠中取出胸主动脉,将其横切为血管环,每节长度1.0mm左右。置于37℃恒温的血管张力仪中,持续通入5%CO2和95%O2的混合气。安置好动脉环后,给予2mN负荷,20min换一次溶液,持续稳定1.0h。然后用K+-Kreb’s液(K+60mM)检验动脉环活性,两次收缩幅度差<10%可进行后续实验。先用1.0μM苯肾上腺素预收缩,待张力上升并稳定后,加入干扰线粒体功能的药物FCCP(1.0μM),Valinomycin(1.0μM),Nigericin(1.0μM)和Monesin(1.0μM),检测大鼠血管张力的变化情,发现给予多糖基化地奥司明后30min腹主动脉收缩最为明显,在60min后收缩开始减弱,而在给药后360min时恢复正常;给予地奥司明后30min腹主动脉收缩最为明显,在50min后收缩开始减弱,而在给药后240min时恢复正常;给予葡萄糖后30min腹主动脉收缩最为明显,在40min后收缩开始减弱,而在给药后180min时恢复正常。
从实验中可以看出,糖基化地奥司明在促进血液循环上明显好于地奥司明,可用于急性痔疮/冻疮的治疗、活血化瘀的缓解剂。
Claims (10)
1.一种糖基化黄酮类化合物,其特征在于,结构如式(Ⅰ)所示
其中:R1为H、F、Cl、Br、CH3、OCH3或OCH2CH3;
R2为H、F、Cl、Br、CH3、OCH3或OCH2CH3;
R3为Glu,Rha,Xyl或Gal,n=1~9。
2.根据权利要求1所述的糖基化黄酮类化合物,其特征在于,R1为H、CH3或OCH3;R2为H、CH3或OCH3;R3为Glu或Rha,n=1~5。
3.糖基化黄酮类化合物的制备方法,其特征在于,将原料、糖基供体、反应缓冲液、糖苷酶和糖基转移酶混合均匀,所述原料与反应缓冲液的比例为1:(10~50),反应的时间为10~24h,反应温度为35~75℃,所述原料包括但不限于地奥司明和香叶木素。
4.根据权利要求3所述的制备方法,其特征在于,所述糖基供体包括葡萄糖、UDP-葡萄糖、木糖、UDP-木糖、半乳糖、UDP-半乳糖、赤藓糖、α-环糊精、β-环糊精、淀粉、木聚糖、蔗糖、麦芽糊精,所述糖基供体的浓度为1~10g/L。
5.根据权利要求3所述的制备方法,其特征在于,所述反应缓冲液是质量分数为0.05~1%的磷酸氢二钠-柠檬酸钠缓冲液或质量分数为0.9%的NaCl缓冲液,所述反应缓冲液的pH值为2~10。
6.根据权利要求3所述的制备方法,其特征在于,所述糖苷酶为鼠李糖苷酶,所述糖基转移酶为葡萄糖基转移酶、木糖基转移酶或半乳糖基转移酶中的一种或两种组合。
7.根据权利要求3~6任一项所述的制备方法,其特征在于,所述糖苷酶与糖基转移酶的比例为(5:95)~(95:5);所述糖苷酶和糖基转移酶总量的质量分数为原料的5%~50%,进一步优选为10%~30%。
8.一种药物组合物,其特征在于,包含权利要求1中所述的糖基化黄酮类化合物及药学上可接受的盐、载体、赋形剂、稀释剂、媒介物或它们的组合。
9.权利要求1所述糖基化黄酮类化合物在痔疮、冻疮、活血化瘀、抗炎及静脉淋巴功能不全方面的应用。
10.权利要求1所述糖基化黄酮类化合物在护肤品、保健品、食品和药品上的应用。
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