藤椒油粕黄酮类成分的提取纯化工艺及成分鉴定和抑菌作用研究

Extraction-purification procedure of flavonoids in Zanthoxylum armatum Residue with chemical identification and antibacterial activity

目的建立藤椒Zanthoxylum armatum油粕中黄酮类成分的提取纯化工艺,鉴定提取纯化物中的化学成分,并评价其抗细菌作用。方法以总黄酮提取率为考察指标,通过单因素实验和Box-Behnken设计-响应面试验优化提取工艺。以总黄酮转移率、提取纯化物得率和总黄酮质量分数为考察指标,通过大孔吸附树脂类型、上样溶液pH值和乙醇体积分数、洗脱剂种类和用量等因素考察优化纯化工艺。对整合的提取纯化工艺进行6次重复性试验。利用UPLC-Q-TOF-MS/MS技术鉴定提取纯化物中的化学成分。采用抑菌圈法及微量稀释法检测提取纯化物的抑菌效果。结果藤椒油粕先用石油醚回流提取3次,料液比1∶10,每次提取60 min;再用65%乙醇回流提取130 min,料液比1∶20。提取液浓缩后,调节乙醇体积分数为10%~15%,调节pH值至3.0。将酸性乙醇溶液上样至HPD500树脂柱,先用5倍柱体积水洗脱,再用60%乙醇洗脱。乙醇洗脱液经浓缩干燥,得总黄酮提取纯化物。从该提取纯化物中鉴定了32种成分,包括23种黄酮、6种酚酸和3种酰胺。该提取纯化物对金黄色葡萄球菌Staphylococcus aureus和表皮葡萄球菌S.epidermidis的抑菌圈直径分别为(15.43±0.25)mm和(15.16±0.09)mm,最小杀菌质量浓度分别为5 mg/mL和>5 mg/mL;对大肠杆菌Escherichia coli和铜绿假单胞菌Pseudomonas aeruginosa未形成明显的抑菌圈,最小抑菌质量浓度分别156.25μg/mL和312.50μg/mL。结论建立的藤椒油粕中黄酮类成分的提取纯化工艺稳定,提取效率高,纯化效果好。该提取纯化物主要成分为黄酮类成分,对金黄色葡萄球菌和表皮葡萄球菌具有抑制作用。

Objective To develop the extraction-purification procedure of flavonoids from Zanthoxylum armatum Residue(ZAR),identify chemical components in the extracted-purified extract and evaluate its antibacterial activity.Methods The extraction rate of total flavonoid was used as indicator to evaluate extraction procedure of total flavonoids from ZAR.This procedure was optimized by single factor experiments and Box-Behnken design-response surface methodology.Then,according to the transfer rate of total flavonoids,the yield of extracts and the mass fraction of total flavonoids,the purification procedure was optimized by the macroporous adsorption resin type,pH value of sample solution,ethanol volume fraction,eluent type and dosage.Repeatability tests of the integrated extraction and purification procedure were carried out for six times.Meanwhile,the UPLC-Q-TOF-MS/MS technique was employed to identify chemical constituents from the extracted-purified extract.The antibacterial activity of extracted-purified extract was detected by the inhibition circle and microdilution method.Results ZAR was extracted in petroleum ether for three times by reflux,the solid-liquid ratio was 1:10,and each extraction was 60 min.Then,the defatted ZAR was extracted in 65%ethanol for 130 min by reflux,with a solid-liquid ratio of 1:20.The ethanol extraction solution was concentrated,and adjusting the pH value to 3.0,volume fraction of ethanol to 10%—15%.The acidic ethanol solution was loaded to the HPD500 resin column.This column was firstly washed with 5-folds column volume of water,and then with 60%ethanol.Then ethanol eluent was concentrated and dried to obtain total flavonoid extracts.A total of 32 compounds were identified in the final extracted-purified extract including 23 flavonoids,six phenolic acids and three amides.The diameter of circle for flavonoid extracts inhibiting Staphylococcus aureus and S.epidermidis were(15.43±0.25)mm and(15.16±0.09)mm,respectively.The minimum bactericidal concentration for the flavonoid extracts on S.aureus and S.epidermidis were 5 mg/mL and more than 5 mg/mL,respectively.The flavonoid extracts did not produce an obvious antibacterial circle against Escherichia coli and Pseudomonas aeruginosa,with the minimum inhibitory concentration of 156.25μg/mL and 312.50μg/mL,respectively.Conclusion The established extraction-purification procedure of flavonoids from ZAR is stable with high extraction efficiency and purification.This extracted-purified extract mainly contains flavonoids and has an inhibitory effect on S.aureus and S.epidermidis.