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Phenolic compounds, which are classified as secondary metabolites, are produced in the shikimic acid of plants and pentose phosphate through phenylpropanoid metabolization (1). These compounds have various health benefits, assisting with inflammation and cardiovascular protection while being full of antioxidants. They can be found in many objects, including the flowers of Chrysanthemum morifolium cv. Fubaiju. In a study published in the Journal of Separation Science, 12 phenolic compounds were successfully separated from the flowers; namely, five flavonoid glycosides and seven quinic acid derivatives were separated (2).
This separation was accomplished via a combination of high-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). HSCCC is a two-phase solvent system with a liquid stationary phase, meant to “resolve target compounds relying on the different partitioning of solutes between two immiscible solvents” (3). Preparative HPLC, meanwhile, isolates and purifies valuable products (4).
The solvent system consisted of ethyl acetate-n-butanol–acetonitrile–water–acetic acid was selected as a solvent system, with the aim of removing six fractions from the flowers. To purify the fractions, 20% aqueous acetonitrile (containing 0.1% formic acid) was used as the eluent solvent. The twelve phenolic compounds that were isolated include luteolin-7-O-β-D-glucoside, luteolin-7-O-β-D-glucuronide, apigenin-7-O-β-D-glucoside, luteolin-7-O-β-D-rutinoside, diosmetin-7-O-β-D-glucoside, chlorogenic acid, 1,5-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, 3,4-dicaffeoyl-epi-quinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid, all of which had purities above 95%.
Once identified, the isolates were later evaluated for how much they prevented H2O2-induced oxidative damage in adult retinal pigment epithelial cells. A 2019 study, published in the Journal of Biochemical and Molecular Toxicology set the precedent for this research, centered on monitoring cymaroside, a different flavonoid compound, and its protective effects against these effects. The results revealed that cymaroside “effectively attenuated the decrease of cell activity induced by H2O2”, so that effect could possibly be replicated with the phenolic compounds in Chrysanthemum morifolium (5).
(1) Lin, D.; Xiao, M.; Zhao, J.; Li, Z.; Xing, B.; Li, X.; Kong, M.; Li, L.; Zhang, Q.; Liu, Y.; Chen, H.; Qin, W.; Wu, H.; Chen, S. An Overview of Plant Phenolic Compounds and Their Importance in Human Nutrition and Management of Type 2 Diabetes. Molecules 2016, 21 (10), 1374. DOI: https://doi.org/10.3390/molecules21101374
(2) Dong, X.; Huang, H.; Wang, R.; Luo, S.; Mi, Y.; Pan, Y.; Shen, W.; Cui, J.; Hu, X.; Cheng, X.; Shi, X.; Wang, H. High-speed counter-current chromatography assisted preparative isolation of phenolic compounds from the flowers of Chrysanthemum morifolium cv. Fubaiju. J. Sep. Sci. 2023. DOI: https://doi.org/10.1002/jssc.202300172
(3) Goldhaber-Pasillas, G. D.; Choi, Y. H.; Verpoorte, R. New Light on Alkaloid Biosynthesis and Future Prospects. Adv. Bot. Res. 2013, 68, 232–272. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/countercurrent-chromatography (accessed 2023-08-29)
(4) Huber, U.; Majors, R. E. Introduction into preparative HPLC. In Principles in preparative HPLC: A Primer. Agilent Technologies, Inc. 2007, 2. https://www.agilent.com/cag/EMEA/HPLC-LCMS_Literature/Primer/5989-6639EN.pdf (accessed 2023-08-29)
(5) Yu, H.; Li, J.; Hu, X.; Feng, J.; Wang, H.; Xiong, F. Protective effects of cynaroside on oxidative stress in retinal pigment epithelial cells. J. Biochem. Mol. Toxicol. 2019, 33 (8). DOI: https://doi.org/10.1002/jbt.22352