基于Py-GC-MS和UHPLC-TOF-MS研究不同靛蓝提取物的化学组成差异

The Chemical Composition Variance of Different Indigo Extracts Based on Py-GC-MS and UHPLC-TOF-MS

天然靛蓝来源于可再生含靛植物资源,具有杀菌消炎、清热解毒等药理作用,为探究不同提取工艺得到的靛蓝提取物的化学组成的差异,本研究以马蓝鲜叶自然发酵和生石灰打靛产生的靛蓝膏(粗靛)为原料,通过预处理去杂、盐酸酸化、葡萄糖还原和乙酸乙酯溶剂萃取等工艺,制备靛蓝粗提物(ICE)、酸化-靛蓝提取物(AIE)、葡萄糖还原-靛蓝提取物(GRIE)及乙酸乙酯萃取-靛蓝提取物(EAEI),并采用高效液相色谱(HPLC)、热裂解-气相色谱/质谱联用(Py-GC-MS)和超高效液相色谱-四级杆飞行时间串联质谱(UHPLC-TOF-MS)对提取物进行分析。研究结果表明:相比于靛蓝膏,4种提取工艺制备的靛蓝提取物中靛蓝质量分数均显著提高,灰分含量均大幅减少。其中,还原-靛蓝提取物中靛蓝质量分数由靛蓝膏的2.09%提高到85%。4种靛蓝提取物的裂解产物组分中含12~20个碳的大分子化合物较多,相应的GC含量分别是7.91%、57.09%、54.4%和62.7%。4种提取工艺中,盐酸酸化和乙酸乙酯溶剂萃取提取的靛蓝提取物的裂解产物组分数量远高于预处理去杂和葡萄糖还原工艺。AIE和EAEI分别鉴定出61和184种化合物,主要包括酸类、多糖类、黄酮类、生物碱类、苷类、多酚类、醛类、蛋白类、酯类9类物质,且EAEI中酸类、多酚类和黄酮类的相对峰面积分别是48.72%、4.56%和1.71%,均较AIE的有大幅度提高即乙酸乙酯萃取工艺实现了对靛蓝提取物中酸类、多酚类、黄酮类等有效富集。

Natural indigo came from renewable indigo plant resources and had pharmacological effects such as sterilization,anti-inflammatory,heat clearing,and detoxification.In order to investigate the differences in the chemical composition of indigo extracts obtained by different extraction processes,the raw material of indigo extract(crude indigo)used in this study was produced by natural fermentation and quick lime indigo beating of fresh leaves of Baphicacanthus cusia(Nees)Bremek.The indigo crude extract,acidification indigo extract,glucose reduction indigo extract and ethyl acetate extraction indigo extract were prepared through pretreatment,impurity removal,hydrochloric acid acidification,glucose reduction,and ethyl acetate solvent extraction,respectively.The samples were analyzed by high performance liquid chromatography(HPLC),pyrolysis gas chromatography/mass spectrometry(Py-GC-MS)and ultra performance liquid chromatography quadrupole time of flight mass spectrometry(UHPLC-TOF-MS).The results indicated that compared to indigo paste,the mass fraction of indigo in the indigo extract prepared by the four extraction processes had been significantly increased,and the ash content had been significantly reduced.Among them,the mass fraction of indigo in the reduced indigo extract increased from 2.09%of indigo paste to 85%.The cracking product components of four indigo extracts contain more macromolecular compounds with 12 to 20 carbon atoms,with corresponding GC contents of 7.91%,57.09%,54.4%and 62.7%,respectively.Among the four extraction processes,the hydrochloric acid acidification and ethyl acetate solvent extraction significantly increased the variety and quantity of cracking products in indigo extract compared to those of pre-treatment impurity removal and glucose reduction processes.Acidification indigo extract and ethyl acetate extraction indigo extract identified 61 and 184 compounds respectively,mainly including acids,polysaccharides,flavonoids,alkaloids,glycosides,polyphenols,aldehydes,proteins,and esters.Compared to acidification indigo extract,the relative peak areas of acids,polyphenols,and flavonoids in ethyl acetate extraction indigo extract were 48.72%,4.56%,and 1.71%,respectively.This demonstrated that the ethyl acetate extraction process had achieved effective enrichment of acids,polyphenols,flavonoids,and other compounds in indigo extract.