CN116715647A - 一种从陈皮中提取槲皮素的方法和应用 - Google Patents
一种从陈皮中提取槲皮素的方法和应用 Download PDFInfo
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- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 title claims abstract description 35
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- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
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- C07D311/28—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only
- C07D311/30—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3 with aromatic rings attached in position 2 only not hydrogenated in the hetero ring, e.g. flavones
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- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
- C07D311/26—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with aromatic rings attached in position 2 or 3
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Abstract
本发明公开了一种从陈皮中提取槲皮素的方法和应用,该方法包括步骤1、将陈皮用乙酸乙酯浸泡并用超声加速浸提,重复浸提3次,合并浸提液,旋转蒸发仪抽干溶剂,得到乙酸乙酯层提取物AE;将乙酸乙酯层提取物AE用50%甲醇水溶解,用中低压反相C18柱分离纯化,流速设置30mL/min,40%甲醇水溶液洗脱,收集第64~93min组分,为组分AE‑4;将组分AE‑4用甲醇溶解,用Sephadex LH‑20分离纯化,流速设置为0.25mL/min,甲醇做洗脱剂,收集第1395~1500min组分,为组分AE‑4‑9;经核磁鉴定,为槲皮素;该方法提高了槲皮素的提取率,产品纯度高,并且槲皮素具有抑制黄嘌呤氧化酶活性,可制成降尿酸血症的药物。
Description
技术领域
本发明涉及生物医学技术领域,具体涉及一种从陈皮中提取槲皮素的方法和应用。
背景技术
黄嘌呤氧化酶是一种多功能的钼黄素蛋白,分布广泛,存在于牛奶、肾脏、肺、心脏和血管内皮中。该酶参与嘌呤的代谢,催化次黄嘌呤和黄嘌呤转化为尿酸,在高尿酸血症和痛风中发挥重要作用。黄嘌呤氧化酶在正常生理条件下以黄嘌呤脱氢酶的形式存在,通过氧化巯基残基或有限的蛋白水解作用转化为黄嘌呤氧化酶。在嘌呤底物的催化氧化羟化过程中,黄嘌呤氧化酶产生超氧阴离子和H2O2,这些超氧阴离子在螯合铁的存在下转化为高反应性的羟自由基。别嘌呤醇是最常用的临床XOD嘌呤类抑制剂,但其因史蒂文斯—约翰逊综合征、肾毒性等毒副作用而备受争议。非布索坦作为非嘌呤XOD抑制剂也会导致心血管并发症,食品和药物管理局(FDA)也要补充该药物的警示性声明。
陈皮为芸香科植物橘或其栽培品种的干燥成熟果皮,是药食同源的宝贵原料之一,具有极高的药用价值,并广泛用于治疗消化不良和一些炎症综合征,如支气管炎和哮喘。植物化学研究表明,陈皮含有丰富的黄酮类、生物碱类、酚酸类和挥发油类化合物,其中黄酮类化合物被认为是中草药的主要生物活性分。药理学研究表明,陈皮具有抗氧化、抗炎、抗肿瘤、促消化、降血压和心脏保护等多种作用。
相关技术中记载陈皮的水提物和陈皮的醇提物可抑制XOD活性,降低血清中的尿酸。也记载通过水浴提取柑橘皮中的槲皮素,然而其槲皮素的提取率较低,并且槲皮素的纯度较低,不适合大规模工业生产、制备。
发明内容
为解决上述问题,本发明的实施例在第一方面提出了一种从陈皮中提取槲皮素的方法,其包括以下步骤:
步骤1、将陈皮用乙酸乙酯浸泡并用超声加速浸提,重复浸提3次,合并浸提液,旋转蒸发仪抽干溶剂,得到乙酸乙酯层提取物AE;
步骤2、将乙酸乙酯层提取物AE用50%甲醇水溶解,用中低压反相C18柱分离纯化,流速设置30mL/min,40%甲醇水溶液洗脱,收集第64~93min组分,为组分AE-4;
步骤3、将组分AE-4用甲醇溶解,用Sephadex LH-20分离纯化,流速设置为0.25mL/min,甲醇做洗脱剂,收集第1395~1500min组分,为组分AE-4-9;经核磁鉴定,为槲皮素。
根据本发明实施例的一种从陈皮中提取槲皮素的方法,该方法以陈皮为原料进行分离,原料易得,能够对陈皮进行充分回收利用,避免浪费;制备过程简单易操作;所得的槲皮素从陈皮中提取、分离纯化得到,产品纯度高,并发现具有抑制黄嘌呤氧化酶活性,可制成降尿酸血症的药物,为降高尿酸血症的防治提供了新的来源。
可选地,步骤1中,陈皮与乙酸乙酯的料液比为1:10g/mL浸提1h。
本发明的实施例在第二方面提出了上述从陈皮中提取槲皮素的方法制得的槲皮素在制备降尿酸血症的药物中的应用。
根据本发明的实施例,上述提取的槲皮素可以有效地抑制黄嘌呤氧化酶的活性,可应用于制备防治高尿酸血症的药物中。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
图1为根据本发明实施例的化合物AE-4-9的HPLC分析图;
图2为根据本发明实施例的化合物AE-4-9的1H-NMR图;
图3为根据本发明实施例的化合物AE-4-9的13C-NMR图。
图4为根据本发明实施例的化合物浓度与XOD酶抑制率关系图。
具体实施方式
以下通过特定的具体实例说明本发明的技术方案。应理解,本发明提到的一个或多个方法步骤并不排斥在所述组合步骤前后还存在其他方法步骤或在这些明确提到的步骤之间还可以插入其他方法步骤;还应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。而且,除非另有说明,各方法步骤的编号仅为鉴别各方法步骤的便利工具,而非为限制各方法步骤的排列次序或限定本发明可实施的范围,其相对关系的改变或调整,在无实质变更技术内容的情况下,当亦视为本发明可实施的范畴。
为了更好的理解上述技术方案,下面更详细地描述本发明的示例性实施例。虽然显示了本发明的示例性实施例,然而应当理解,可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反,提供这些实施例是为了能够更透彻地理解本发明,并且能够将本发明的范围完整的传达给本领域的技术人员。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面参考具体实施例,对本发明进行描述,需要说明的是,这些实施例仅仅是描述性的,而不以任何方式限制本发明。
实施例1
一种从陈皮中提取槲皮素的制备方法,包括以下步骤:
步骤1、取100g的陈皮,于粉碎机粉碎,置于2L锥形瓶中,以10倍体积的乙酸乙酯浸泡并用超声加速浸提1h,重复浸提3次,合并浸提液,以旋转蒸发仪抽干溶剂,得到乙酸乙酯层提取物34.65g。
步骤2、取步骤1得到的乙酸乙酯层提取物(AE)34.65g,用50mL 50%甲醇水溶解,用中低压反相C18柱(填料为:YMC-GEL ODS-AQ-HG,s-50μm;柱长为4.5×35cm),流速设置为30mL/min,用40%甲醇水溶液洗脱,洗脱组分进行抑制XOD酶活性追踪,选取活性较高部分进行后续试验,即收集第64~93min组分(AE-4)。
步骤3、取步骤2得到的(AE-4)1.34g用20mL 100%甲醇溶解,用Sephadex LH-20(2.5×170cm),100%甲醇做洗脱剂,过柱流速为0.25mL/min,洗脱组分进行抑制XOD酶活性追踪,选取活性较高部分进行后续试验,即收集第1395~1500min组分(AE-4-9),浓缩得到化合物约0.291g,提取率为10.233%,纯度为98.0%。
纯度的测定方式:精密称取槲皮素标准品250μg,加甲醇少量超声溶解,并定容于5mL量瓶中,分别制成浓度为0、0.5、1、5、10、25、50μg·mL-1的对照品溶液,再用微孔滤膜过滤,得待测标准品溶液。设定HPLC的检测条件:检测时间:15min;流速:1mL/min,柱温:35℃,进样量:20.0μL;检测波长:254nm。以浓度为横坐标,峰面积为纵坐标,得到回归曲线Y=5.32x102X-0.2009。同时配置一定浓度的提取物样品,以同样的方法进行检测,计算出样品中槲皮素纯度。
化合物AE-4-9为黄色粉末状,对其进行HPLC分析,HPLC参数如下:HPLC型号:Agilent 1260Infinity II;检测器:1260DAD WR;色谱柱:H&E-SP ODS-TA柱,4.6×250mm,s-5μm;液相条件:流动相:60%甲醇-40%水混合液,再加入0.1%的三氟乙酸;检测时间:15min;流速:1mL/min,柱温:35℃,进样量:20.0μL;检测波长:254nm。
结果如图1所示,图1为化合物AE-4-9在254nm检测波长下的高效液相色谱图,保留时间为5.432min。
对化合物AE-4-9进行核磁测定:用Bruker AvanceII-500核磁共振仪(Bruker,瑞士)记录了1H和13C核磁共振谱。1H和13C NMR的工作频率分别为500MHz和125MHz。样品用600μL CD3OD溶解,试验温度为300K。耦合常数(J)以Hz表示,化学位移以δ(ppm)表示,并以四甲基硅烷(TMS)作为内标物。结果如图2和图3。
1H-NMR(500MHz,CD3OD)数据如下:δ7.73(1H,d,J=2.0Hz,H-2'),7.63(1H,dd,J=8.5,1.9Hz,H-6'),6.88(1H,d,J=8.5Hz,H-5'),6.38(1H,d,J=1.9Hz,H-8),6.18(1H,d,J=2.0Hz,H-6)。
13C-NMR(125MHz,CD3OD)数据如下:δ177.5(C-4),165.7(C-7),162.6(C-5),158.3(C-9),148.9(C-2),148.1(C-4'),146.3(C-3'),137.3(C-3),124.3(C-1′),121.8(C-6'),116.4(C-5'),116.1(C-2'),104.7(C-10),99.4(C-6),94.6(C-8)。
综合化合物AE-4-9的核磁数据及液相数据并与文献进行比对,推测化合物AE-4-9为槲皮素;英文名:Quercetin;分子式:C15H10O7;相对分子量:302.2357;结构如下:
对比例1
取100g的陈皮,于粉碎机粉碎,按照料液比1:50(g/mL)加入浓度为80%乙醇,用塑料薄膜将锥形瓶口封紧,将其在80℃的恒温水浴锅中水浴3.5h,待浸提液晾凉后,取上清液,测定槲皮素的得率,此条件下槲皮素的提取率为9.685%,纯度为90.3%。
对比例2
步骤1、取100g的陈皮,于粉碎机粉碎后加入锥形瓶中,并加入2L乙醇,将混合物在60℃下水浴提取2h后使用400目滤网过滤。提取实验重复三次,将所得滤液合并后减压蒸馏浓缩得到陈皮乙醇提取物,此条件下槲皮素的提取率为9.731%,纯度为89.6%。
步骤2、在85℃的水浴中用2L水作为溶剂萃取步骤1醇提后的陈皮残渣2h,后减压过滤分离滤渣和滤液,提取实验重复三次,将所得滤液合并后减压蒸馏浓缩并冻干得到水提取物27.03g,产率为13.52%,槲皮素的提取率为9.586%,纯度为83.6%。
实施例2
抑制黄嘌呤氧化酶活性的测定:
PBS缓冲液(0.1M,pH=7.0):精密称取磷酸氢二钠2.0660g,磷酸二氢钠0.6590g,NaCl 0.9000g,用超纯水溶解,并定容至100mL。
0.1mM NaOH溶液:精密称取NaOH 0.0200g,超纯水溶解,定容于5mL容量瓶备用。
1mM黄嘌呤反应液:精密称取黄嘌呤粉末0.0075g,加入0.5mL的0.1mM NaOH溶液进行助溶,用PBS缓冲液定容至5mL容量瓶,置于4℃避光冷藏备用。
0.1U/mL黄嘌呤氧化酶溶液:将10U/mL黄嘌呤氧化酶的原液用PBS缓冲液稀释至0.1U/mL,置于4℃避光冷藏备用。
80μM别嘌呤醇溶液:精密称取0.0065g的别嘌呤醇,用PBS缓冲液定容至5mL容量瓶,得到10mM的别嘌呤醇溶液,加PBS缓冲液稀释至80μM别嘌呤醇溶液,置于4℃避光冷藏备用。
样品溶液:精密称取实施例1制得的化合物AE-4-9 0.0050g,用DMSO溶解,并用PBS缓冲液进行稀释,控制DMSO浓度在3%以内,得到浓度1mg/mL的样品溶液。
黄嘌呤氧化酶活抑制试验方法:将100μL PBS缓冲液,25μL样品溶液,25μL 0.1U/mL黄嘌呤氧化酶依次加入96孔板中,震荡混匀,在37℃下孵育20min。孵育结束后加入50μL的1mM黄嘌呤反应液,震荡混匀,在37℃下孵育5min。在290nm下检测样品孔的吸光度。空白对照组由PBS缓冲液代替样品和黄嘌呤反应液。在完全反应组中,用PBS缓冲液代替样品。样品对照组用PBS缓冲液代替黄嘌呤氧化酶。别嘌呤醇作为阳性对照。样品组黄嘌呤氧化酶的抑制率计算公式如下(式1)。
式中,C1为空白对照组;C2为完全反应组;C3为样品反应组;C4为样品对照组。
在酶活性测定中,槲皮素有效的降低了黄嘌呤氧化酶的活性,其IC50值为6.52±0.56μM,阳性对照别嘌呤醇IC50值为22.3±0.22μM,这说明申请制备的槲皮素可以有效地抑制黄嘌呤氧化酶的活性,可应用于防治高尿酸血症的药物中。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不应理解为必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外,本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (3)
1.一种从陈皮中提取槲皮素的方法,其特征在于,包括以下步骤:
步骤1、将陈皮用乙酸乙酯浸泡并用超声加速浸提,重复浸提3次,合并浸提液,旋转蒸发仪抽干溶剂,得到乙酸乙酯层提取物AE;
步骤2、将乙酸乙酯层提取物AE用50%甲醇水溶解,用中低压反相C18柱分离纯化,流速设置30mL/min,40%甲醇水溶液洗脱,收集第64~93min组分,为组分AE-4;
步骤3、将组分AE-4用甲醇溶解,用Sephadex LH-20分离纯化,流速设置为0.25mL/min,甲醇做洗脱剂,收集第1395~1500min组分,为组分AE-4-9;经核磁鉴定,为槲皮素。
2.如权利要求1所述的方法,其特征在于,步骤1中,陈皮与乙酸乙酯的料液比为1:10g/mL浸提1h。
3.如权利要求1或2所述的从陈皮中提取槲皮素的方法制得的槲皮素在制备降尿酸血症的药物中的应用。
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