Optimization of Preparative Separation and Purification of Total Flavonoids from Radix Puerariae by Macroporous Resin Method

Aim To screen the optimum macroporous resin and conditions for the isolation and purification of flavonoids from Radix Puerariae. Methods The static and dynamic adsorption/desorption methods were used, and the separation and purification process was evaluated by measuring the concentration of total flavonoid in the fractions with UV spectrophotometer. Results The SP70 macroporous resin was the most effective compared with other macroporous resins. The optimum conditions were screened, which were 0.5 g· mL^- 1 corresponding to crude drug for concentration of extract, pH 5 - 6, and appended 60 times the volume of the resin bed （BV） with the adsorption speed 2 BV·h^-1, and the volume of aq. 70% （V/V） ethanol as eluant was 5 BV with desorption speed 2 BV·h^-1. By this method, the final contents of total flavonoids exceeded 80%. Conclusion The SP70 macroporous resin is the most effective one for large-scale isolation and purification of flavonoids from Radix Pueraria, which meets industrial needs.

Kinetics of Leaching Flavonoids from Pueraria Lobata with Ethanol

The kinetics of leaching flavonoids from Pueraria Lobata with ethanol was investigated. The effects of leaching temperature, mechanical agitation rate, concentration of ethanol and feed particle size on leaching kinetics were examined. It is found that the smaller the feed particle size or the higher the leaching temperature, the higher the leaching rate. The leaching process can be described by the shrinking-core model. The apparent activation energy is 10.8kJ·mol^-1, suggesting that the leaching process is controlled by the inner diffusion. An empirical equation relating the flavonoids leaching rate constant to the feed particle size and leaching temperature was expected.

Kinetics of Leaching Flavonoids from Pueraria Lobata with Ethanol

The kinetics of leaching flavonoids from Pueraria Lobata with ethanol was investigated. The effects of leaching temperature, mechanical agitation rate,concentration of ethanol and feed particle size on leaching kinetics were examined.It is found that the smaller the feed particle size or the higher the leaching temperature,the higher the leaching rate. The leaching process can be described by the shrinking-core model. The apparent activation en- ergy is 10.8kJ·mol-1, suggesting that the leaching process is controlled by the inner diffusion. An empirical equa- tion relating the flavonoids leaching rate constant to the feed particle size and leaching temperature was expected.

Extraction and separation of total flavonoids from Flos Puerariae and its antioxidant activity

Objective To study the optimum extraction and separation process of total flavonoids in the flowers of Flos Puerariae and its antioxidative activity.Methods The total flavonoids were extracted with assistance of ultrasonic wave and the content was determined at 265 nm wavelength by Spectrophotometric method.Orthogonal experiment L9(34)was carried out to investigate the effects of concentration of solvent,the ratio of material to liquid,time length of extraction,and frequency of extraction on extraction results of total flavonoids from Flos Puerariae.The extracted solution was purified by petroleum ether and ethanol sequently.Under these conditions,the total flavonoids was eluted gradiently with mixed mobile phase of methanol-chloroform solution in the silica gel column system,and then determined by UV scanning and HPLC.Fenton reaction was used to produce and detect hydroxyl radicals(·OH),and pyrogallol system was used to produce and detect the superoxide radical anion(O2-·).Results The optimum conditions were as follows;using 40%(V/V)methanol as extractor with the ratio of material to liquid at 1∶30,and extracting for 2.5 h a time for 3 times.The extraction yield of total flavonoids was 13.6%.Six isoflavone compounds were isolated from the flowers of Flos Puerariae by the method of silica gel column chromatography.Antioxidative test results showed good performance of flavonoid scavenging capacity in both hydroxyl radical system and superoxide radical system and its IC50 was 7.65 μg·mL-1 and 0.18 mg·mL-1,respectively.Conclusions This study provided scientific basis for further development and utilization of Flos Puerariae and make preparation for later pharmacological research.

Extraction Technology of Total Flavonoids from Pueraria lobata (Willd.) Ohwi and Content Determination of Pueraria lobata (Willd.) Ohwi at Different Altitudes

[Objectives]To optimize the extraction technology of total flavonoids from Pueraria lobata( Willd.) Ohwi. [Methods]The ultraviolet-visible spectrophotometry was used to determine the content of total flavonoids in Pueraria lobata( Willd.) Ohwi. With the puerarin as index,the reflux extraction and single factor test were employed to investigate the effects of temperature,time,ethanol concentration and solid-liquid ratio on the content of total flavonoids in Pueraria lobata( Willd.) Ohwi,respectively. Under the optimal extraction technology,the content of total flavonoids in Pueraria lobata( Willd.) Ohwi at different altitudes was determined.[Results] The optimum extraction process was as follows: 70%ethanol; solid-liquid ratio of 1∶ 30; 1 h reflux extraction. Under these conditions,the extraction rate of flavonoids in Pueraria lobata( Willd.) Ohwi was 11. 48%,the total flavonoids content of different kudzu parts was in the order of roots > stems > leaves,and the total flavonoids content of the sample at about an altitude of 1000 m was significantly higher than at the altitudes of 1400 m and 1700 m.[Conclusions]It was suggested that the Pueraria lobata( Willd.) Ohwi should not be cultivated as medicinal plant in too high mountains,and the stems and leaves of Pueraria lobata( Willd.) Ohwi could be used as raw materials for extracting total flavonoids.

Effects of Ethanol Condensation Reflux Extraction and Ultrasound-assisted Extraction on the Content of Total Flavonoids of Pueraria edulis

With Pueraria edulis from different habitats in Yunnan Province,Guangdong Province and Guangxi Zhuang autonomous region as materials,the medicinal materials of P.edulis were extracted by ethanol condensation reflux extraction and ultrasound-assisted extraction,and the effects of different extraction methods the contents of total flavonoids in P.edulis from different areas were compared.The results showed that the best extraction time for P.edulis in Yunnan Province was 20 min,and that for Guangdong and Guangxi Zhuang Autonomous Region was 30 min.The yield of total flavonoids from P.edulis extracted by the condensation reflux extraction method was relatively better.The content of total flavonoids in P.edulis in Yunnan was the highest with an average content of 0.2557%,while the average content of total flavonoids in P.edulis in Guangdong was the lowest with an average content of 0.2108%.

葛根中总黄酮含量测定方法比较研究

目的:紫外-可见分光光度测定葛根总黄酮含量的两种方法(显色法、直接测定)结果具有显著差异,因此将两种测定方法与HPLC法进行对比,找出导致其差异的原因和解决方案。方法:以芦丁为对照品,采用亚硝酸钠-硝酸铝-氢氧化钠显色,通过UV法测定葛根总黄酮含量;以葛根素为对照品,直接于250 nm处测定吸光度计算葛根总黄酮含量;采用高效液相色谱法测定葛根素的含量,对比三种方法的含量差异,评价两种紫外测定法所得结果的准确性。结果:显色法测定23批野葛总黄酮含量为0.93%~8.02%,23批粉葛总黄酮含量为0.04%~1.12%,直接测定法测得23批野葛总黄酮含量为3.31%~15.74%,23批粉葛总黄酮含量为0.80%~7.59%;显色法测得葛根总黄酮含量均显著低于非显色法。HPLC测定23批葛根的葛根素含量为1.98%~5.85%,23批粉葛的葛根素含量为0.17%~2.37%。结论:显色法测定的总黄酮含量大部分低于单一成分葛根素的含量,不显色法测得总黄酮含量均高于单一成分葛根素的量。在葛根总黄酮的测定中,以葛根素为对照品的直接测定法更加合理准确。

葛根黄酮浸取工艺优化研究

通过六组单因素试验和四因素正交试验探讨了葛根黄酮的最佳浸取工艺。结果表明：在温度８５℃、ｐＨ值为８的条件下，用１０倍于葛根量的浓度为７０％的乙醇，浸取２次，每次３ｈ，葛根黄酮浸取效果最好，得率在９％以上、浸出物黄酮含量大于４０％。

葛根黄酮降血糖防治糖尿病并发症的实验研究

通过尾静脉注射四氧嘧啶(50mg/kg体重)建立糖尿病小鼠模型,实验分为正常组、糖尿病组、糖脉康药物对照组和葛根黄酮组,实验40d后检测各组动物体重、饮水量、血糖、糖耐量、果糖胺、山梨醇、醛糖还原酶、山梨醇脱氢酶和糖基化终产物(AGEs)等实验指标.结果表明,葛根黄酮能明显改善糖尿病小鼠的病理症状,降低血糖水平,降低血液中果糖胺、山梨醇、醛糖还原酶和AGEs的含量,升高血液中山梨醇脱氢酶的含量,同时还能降低肝脏、肾脏、大脑、心脏和皮肤组织中AGEs和山梨醇的含量,与糖尿病组小鼠比较差异显著(P<0.01).由此得出结论,葛根黄酮除具有明显降血糖作用外,还对糖尿病并发症有一定的预防和治疗效果.

超声波预处理微波萃取葛根总黄酮的工艺优化

以湘西野葛根为原料,优化葛根总黄酮超声微波双辅助萃取工艺。研究超声提取温度、超声作用时间、微波功率、微波作用时间、固液比5个影响因素对葛根总黄酮提取量的影响,采用紫外分光光度计测定葛粉中总黄酮的含量,建立葛根总黄酮的最佳提取工艺条件。结果表明,在以80%的乙醇为提取溶剂、微波辐射功率300W、微波辐射时间9min、固液比1:25(g/mL)、超声提取温度50℃、超声提取时间30min的条件下,总黄酮的提取量可达14.685mg/g。

葛根多糖和黄酮综合提取工艺优化

研究热水浸提葛根多糖和黄酮综合提取的工艺路线。在单因素试验的基础上,选定料液比、提取时间及提取温度3个因素的3个水平进行中心组合试验,建立多糖和黄酮总含量的二次回归方程,进一步优化提取工艺条件,验证模型的有效性,通过响应面分析得到优化组合条件。结果表明,提取温度、提取时间对多糖得率均有显著影响,而料液比无显著影响。当提取工艺条件为提取时间2.2h、料液比1:25.2(g/mL)和提取温度98.4℃时,水提葛根多糖黄酮提取量达到极大值。此条件下多糖和黄酮总提取量预测值为29.0591%,验证值为28.9472%。

葛根黄酮提取物诱导HL-60细胞凋亡调控研究

用RT-PCR技术和蛋白质免疫印迹技术,检测caspase-8和caspase-3基因转录和蛋白质翻译水平的变化,以探讨葛根黄酮提取物诱导HL-60细胞凋亡的调控途径。结果表明,50μg/mL葛根总黄酮提取物可以上调caspase-8和caspase-3基因转录和蛋白质翻译水平,且其能力分别强于葛根素和大豆甙元单体;说明葛根黄酮提取物诱导HL-60细胞凋亡可能通过经F as死亡受体和/或线粒体途径共同介导执行,葛根黄酮提取物抑制诱导凋亡能力强于葛根素和大豆甙元单体。

葛根提取方法的比较研究及其工艺条件优化

采用水提法、回流法、渗漉法、索氏提取法、微波辅助萃取法、超声提取法等不同的方法提取葛根药材,以干浸膏得率、总黄酮得率及葛根素粗品得率为考察指标,对6种提取方法进行比较,并对其中一种最优方法进行工艺优化。结果表明,传统水提法提取葛根药材的效果最好。通过正交试验获得最佳提取工艺条件为8倍量溶剂,提取3次,每次1.5 h,其中溶剂倍量和提取次数为显著影响因素。水提法提取葛根药材,方法简便,有效成分的提取率高。

葛根黄酮类物质美白作用的研究

通过研究葛根黄酮类物质对酪氨酸酶的抑制作用和抗氧化作用探索葛根黄酮类物质的美白作用机理。实验结果表明,葛根黄酮类物质对酪氨酸酶的抑制作用明显强于熊果苷,其半抑制浓度(IC50)为1.72 g·L-1。葛根黄酮类物质的总还原能力较强,对超氧阴离子自由基、.OH和DPPH.均有一定的清除作用,对超氧阴离子自由基和DPPH.的IC50分别为0.98和0.81 g·L-1。

CO_2超临界萃取葛根总黄酮的研究

以葛根为原料,采用CO2超临界提取法提取葛根中的总黄酮。用L9(34)正交试验考查了提取温度、提取时间、料液比、萃取压力四因素对总黄酮提取率的影响。实验结果表明,在温度为50℃,料液比为280:330(g/mL),萃取压力35MPa条件下萃取2h,总黄酮的提取率为1.4572%。

大孔树脂分离葛根总黄酮工艺优化

目的 :筛选分离葛根总黄酮的最佳树脂 ,并对影响分离的各种因素进行系统的研究 ,使分离工艺达到最优化。方法 :采用静态与动态的吸附 解吸两种方法 ,用紫外分光光度法测定葛根总黄酮的含量对工艺进行评价。结果 :SP70分离效果最好 ,其最佳工艺为药液浓度 0 .5 g·mL-1 (相当于原生药 )、pH为 5～ 6、上样量为 6 0BV(树脂床体积 ) ,以 2BV/h吸附速率进行吸附 ,4BV的 70 %乙醇、2BV/h的流速进行洗脱效果最佳。经SP70处理后的葛根总黄酮可达 80 %以上。结论 :该法简单可行 ,分离效果好 ,能满足于大生产的要求。

正交实验优选葛根复方的半仿生提取工艺

目的 :为葛根复方 (葛根 ,川芎 ,山楂 ,槐花米 ,麦冬、甘草等 )研究半仿生提取工艺。方法 :以葛根黄酮得率为指标 ,在粒度、提取次数、提取工艺、浸渍剂浓度等条件相同时 ,考虑乙醇溶液 pH值、提取时间、提取温度、配伍比例 ,选用L16(45)正交进行实验 ,确定最佳提取工艺。结果 :第 1煎提取液 pH为 7,第 2煎提取液pH为 11,提取温度为85℃ ,提取时间为 4h ,配伍比例为 4∶3∶3∶2∶2∶2 (葛根 :川芎 :山楂 :槐花米 :麦冬 :甘草 )。结论 :用正交实验优选半仿生提取工艺 ,是可行的。

不同干燥方法对葛根黄酮含量的影响研究

目的:研究不同干燥方法对葛根总黄酮及葛根素、大豆苷、大豆素、染料木苷、染料木素含量的影响。方法:采用分光光度法和高效液相色谱法测定热风干燥、真空干燥、冷冻干燥法干制葛根粉中葛根总黄酮、葛根素、大豆苷、大豆素、染料木苷、染料木素含量,研究干燥方法对葛根黄酮含量变化的影响规律,探寻最佳葛根干燥方法。结果:冷冻干燥葛根总黄酮含量最高,热风干燥葛根总黄酮含量最低,冷冻干燥、真空干燥和低温热风干燥葛根总黄酮含量无显著差异;热风干燥中,随着干燥温度升高,葛根总黄酮、葛根素、染料木苷、大豆素含量降低,染料木素损失殆尽,大豆苷含量增加;冷冻干燥、真空干燥对葛根素、大豆苷、染料木素含量影响差异显著,对大豆素、染料木苷含量影响差异不显著。结论:葛根干燥时,考虑干燥成本宜采用低温热风干燥;考虑葛根内在品质宜采用真空干燥,冷冻干燥对葛根内在品质提高有限。

响应面法优化葛根总黄酮的超声辅助提取工艺

为提高葛根总黄酮的得率,采用响应面试验设计优化超声辅助提取葛根总黄酮的工艺条件。在单因素试验基础上确定以超声时间、料液比及超声温度为3个主要因素,以总黄酮得率为响应值,根据Box-Behnken原理采用三因素三水平响应面试验设计进行优化。结果表明超声辅助法提取葛根总黄酮的最佳工艺条件为:以蒸馏水作为超声提取溶剂,超声时间43min、料液比1:10(g/mL)、超声温度68℃,总黄酮得率为5.28%,与预测得率5.36%相比,相对误差仅为1.4 9%。说明该模型可靠,与实际情况拟合较好。

6种晋产野生葛根中葛根素、大豆苷和总黄酮含量比较

采用高效液相色谱法对山西省5地6处野生葛根中主要有效成分葛根素、大豆苷及总黄酮含量进行了分析,并对HPLC法的测定条件（流动相和洗脱程序）进行了优化。结果表明,6种晋产野生葛根中主要有效成分含量差异显著,葛根素的含量为1.46%~4.48%,大豆苷的含量为0.11%~0.62%,总黄酮的含量为11.19%~26.14%。葛根素、总黄酮含量与纬度高低呈正相关。晋南绛县么里镇小北山与夏县泗交镇太宽河自然保护区的野生葛根中葛根素含量低于《中国药典》规定的2.4%,而晋北灵丘县的野生葛根中葛根素含量则高达4.48%。