天然酯型儿茶素EGCG的分离纯化及药学研究
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摘要
本文建立了等梯度高效液相色谱(HPLC)法定性、定量分析儿茶素,并对儿茶素中主要成分EGCG含量测定方法进行方法学考察。最佳色谱条件为:ODS (5μm,4.6×250 mm)柱,乙腈∶乙酸乙酯∶水(0.05% H3PO4)=86∶12∶2(V/V)为流动相(pH=3.0),流速:0.8 ml/min,紫外检测波长:280 nm。此分析方法简便易行,准确可靠。
文中以茶多酚粗品为原料,进行提取富集得到酯型儿茶素(EGCG,GCG,ECG)并将其作为色谱分离纯化样品。采用单因素试验考察了EGCG在分配色谱柱上的分离及纯化条件。确定最佳分离纯化条件:柱床高度为40 cm,流速为5 ml/min,每4 min收集一个馏分,基本实现了儿茶素单体EGCG的有效分离。本实验方法简便,成本低廉,产品纯度均较高,并可实现EGCG一次性克量级以上的制备。
工艺放大中,以含EGCG≥60%的茶多酚粗品为原料,在实验室小试的基础上,采用均匀放大的方法,最终实现了EGCG的成功分离纯化。分离纯化条件为:分别以蒸馏水为固定相,乙醚为移动相,洗脱流速为10 ml/min;径高比1∶1 0;每隔30 min收集一个洗脱液。另外,初步探讨了酯型儿茶素的稳定性。本研究经过一次柱层析,即可得到高纯的EGCG,所采用的分离设备经济,方法简便,易于工业化。
药学部分重点研究了EGCG的处方和冻干工艺。以外观、含水量和复溶性为指标,经处方筛选和工艺优化后,以25 mg EGCG,200 mg乳糖为赋形剂,规格为2 ml/支,于-40.0℃预冻5 h,-25.0℃升华干燥32 h,再升温0℃解析干燥5 h。对制得的注射用EGCG进行质量研究,内容包括:一般检查项目(澄明度、pH值、水分)、含量测定研究。室温留样3个月和加速试验3个月的初步稳定性考察表明本品稳定。
A kind of analytical method by high performance liquid chromatography (HPLC) for tea catechins was established. In the meantime, the method was investigated in the methodological aspect through determining the main catechins EGCG of tea polyphenols. The optimum chromatographic conditions were as follows: ODS column (5μm,4.6×250 mm),distilled water (0.05% orthophosphoric)/ acetonitrile/ ethyl acetate is 86: 12: 2 (V/V) as mobile phase (pH=3.0), UV wavelength is 280 nm, the flow rate is 0.8 ml/min. The analytical method is simple, precise and easy to operate.
The ester catechins was enriched containing EGCG, GCG, ECG from the raw TP in the pretreatment process and employed as raw stuff in purification process. Single factor experiment method was used to systematically study the separation and purification conditions of EGCG by liquid-liquid partition chromatography. Took silica gel as inert stuffing with the height of 40 cm and ether as elution agent with the elution speed of 5 ml/min in the separation process, and collect an elution every 4 minutes, we effectively obtained EGCG with high-purity. Besides, this method needed low cost, produced EGCG with high purity and achieved gram magnitude for once time.
Tea polyphenols with the EGCG content about 60% were used as the raw materials, used the liquid-liquid partition column chromatography and choosed silica gel as the inert supporter to amplify the result uniformly based on the experiment condition, which was carried out in a small scale. As a result, we got a good method of separation and purification to obtain EGCG from the partition column: the deionized water was used as stationary phase, and ether was used as mobile phase, the elution speed was 10 ml/min, the ratio diameter to height was 1:10, collect the eluted liquid every 30 minutes. In addition, based on the experimental data, the stability of ester catechins was researched to provide scientific proof for storage of the finished product. In this work, more economic equipments had been used and EGCG of high purity can be obtained by using single partition column chromatography. The developed technology was simple and easy to control with the advantages of low cost. The separation technique was easy to be industrialized.
The injection formulation and lyophilizing process was studied in the pharmic research on EGCG. The formulation and lyophilizing process are optimized by the criterion of appearance, water content and resolubility. The study showed that the formulation was 25 mg EGCG, 300 mg lactose and the specification was 2 ml each bottle. The optimized technological process was as followed: the drug was pre-freezed for 5 h under -40℃and dried for 32 h in the lyophilizer under–25.0℃. After that, the temperature was raised to 0℃to continue drying for 5 hours. The quality evaluation of final product was researched including the general examination items (clarity, pH, water content), the content and the stability experiment. Three months tests under room temperature or under accelerated circumstance indicate that the preparation was stable.
文中以茶多酚粗品为原料,进行提取富集得到酯型儿茶素(EGCG,GCG,ECG)并将其作为色谱分离纯化样品。采用单因素试验考察了EGCG在分配色谱柱上的分离及纯化条件。确定最佳分离纯化条件:柱床高度为40 cm,流速为5 ml/min,每4 min收集一个馏分,基本实现了儿茶素单体EGCG的有效分离。本实验方法简便,成本低廉,产品纯度均较高,并可实现EGCG一次性克量级以上的制备。
工艺放大中,以含EGCG≥60%的茶多酚粗品为原料,在实验室小试的基础上,采用均匀放大的方法,最终实现了EGCG的成功分离纯化。分离纯化条件为:分别以蒸馏水为固定相,乙醚为移动相,洗脱流速为10 ml/min;径高比1∶1 0;每隔30 min收集一个洗脱液。另外,初步探讨了酯型儿茶素的稳定性。本研究经过一次柱层析,即可得到高纯的EGCG,所采用的分离设备经济,方法简便,易于工业化。
药学部分重点研究了EGCG的处方和冻干工艺。以外观、含水量和复溶性为指标,经处方筛选和工艺优化后,以25 mg EGCG,200 mg乳糖为赋形剂,规格为2 ml/支,于-40.0℃预冻5 h,-25.0℃升华干燥32 h,再升温0℃解析干燥5 h。对制得的注射用EGCG进行质量研究,内容包括:一般检查项目(澄明度、pH值、水分)、含量测定研究。室温留样3个月和加速试验3个月的初步稳定性考察表明本品稳定。
A kind of analytical method by high performance liquid chromatography (HPLC) for tea catechins was established. In the meantime, the method was investigated in the methodological aspect through determining the main catechins EGCG of tea polyphenols. The optimum chromatographic conditions were as follows: ODS column (5μm,4.6×250 mm),distilled water (0.05% orthophosphoric)/ acetonitrile/ ethyl acetate is 86: 12: 2 (V/V) as mobile phase (pH=3.0), UV wavelength is 280 nm, the flow rate is 0.8 ml/min. The analytical method is simple, precise and easy to operate.
The ester catechins was enriched containing EGCG, GCG, ECG from the raw TP in the pretreatment process and employed as raw stuff in purification process. Single factor experiment method was used to systematically study the separation and purification conditions of EGCG by liquid-liquid partition chromatography. Took silica gel as inert stuffing with the height of 40 cm and ether as elution agent with the elution speed of 5 ml/min in the separation process, and collect an elution every 4 minutes, we effectively obtained EGCG with high-purity. Besides, this method needed low cost, produced EGCG with high purity and achieved gram magnitude for once time.
Tea polyphenols with the EGCG content about 60% were used as the raw materials, used the liquid-liquid partition column chromatography and choosed silica gel as the inert supporter to amplify the result uniformly based on the experiment condition, which was carried out in a small scale. As a result, we got a good method of separation and purification to obtain EGCG from the partition column: the deionized water was used as stationary phase, and ether was used as mobile phase, the elution speed was 10 ml/min, the ratio diameter to height was 1:10, collect the eluted liquid every 30 minutes. In addition, based on the experimental data, the stability of ester catechins was researched to provide scientific proof for storage of the finished product. In this work, more economic equipments had been used and EGCG of high purity can be obtained by using single partition column chromatography. The developed technology was simple and easy to control with the advantages of low cost. The separation technique was easy to be industrialized.
The injection formulation and lyophilizing process was studied in the pharmic research on EGCG. The formulation and lyophilizing process are optimized by the criterion of appearance, water content and resolubility. The study showed that the formulation was 25 mg EGCG, 300 mg lactose and the specification was 2 ml each bottle. The optimized technological process was as followed: the drug was pre-freezed for 5 h under -40℃and dried for 32 h in the lyophilizer under–25.0℃. After that, the temperature was raised to 0℃to continue drying for 5 hours. The quality evaluation of final product was researched including the general examination items (clarity, pH, water content), the content and the stability experiment. Three months tests under room temperature or under accelerated circumstance indicate that the preparation was stable.
引文
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[3]吴琼洁,蔡碧琼.茶多酚的研究进展及应用前景[J].武夷科学, 2005, 1(12): 145-149.
[4]宛晓春.茶叶生物化学[M].北京:中国农业出版社, 2003.26-27.
[5]郑裕国,王远山,薛亚平等.抗氧化剂的生产及应用[M].北京:化学工业出版社, 2003, 334-336.
[6]李小洁,晏良军,杨贤强等.茶多酚清除自由基和抗氧化作的研究[J].环境化学, 1992, 4(12): 13-18.
[7] Zhang S, Liang G, Lei Y X. Separate monomer of tea polyphenols from Guangxi green tea[J]. J Guangxi Med, 2003, 20(6): 891-892.
[8]罗晓明,蒋雪薇,徐协文等.聚酰胺色谱法分离制备茶多酚的研究[J].食品添加剂, 2002, 5(1): 34-37.
[9]范小兵,殷梦龙,林涛等.一种没食子儿茶素没食子酸酯单体的纯化方法[P]. CN: 1733753A, 2006, 02, 15.
[10]戚向阳等.高纯度表没食子儿茶素没食子酸(EGCG)的分离与制备[J].精细化工, 1994, 4(11): 40-46.
[11]张星海.儿茶素富集和EGCG单体纯化新工艺研究[J].茶叶, 2002, 28(3): 136-137.
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