Isolation Of Plant Polyphenols By High-Speed Counter-Current Chromatography And Their Characterization

Polyphenols are secondary metabolites in plants, which possess special chemical and physiological activities. Separation and purification of phenolic compounds from plants for molecule structure identification are key steps of their pharmacological activities, rational combination and high-value utilization of renewable resources. This paper first described the qualitative determination of phenolic compounds in Cassia tora L. and sugarcane by HPLC- ESI-MS. And then high-speed counter-current chromatography (HSCCC) was used to establish the rapid and high efficiency preparation platforms of phenolic compounds from Cassia tora L. and sugarcane. The characterizations of main phenolic compounds were also investigated. The main contents and results are as follows:(1) The extraction of anthraquinones in Cassia tora L. with microwave-assisted method was studied. The optimal condition was 60% ethanol (V:V), the ratio of solid to liquid (1:4), extraction 40 minutes at 60°C by orthogonal experiments.(2) The chemical components were analyzed by HPLC-ESI-MS in Cassia tora L., and 22 molecule structures were elucidated according to mass-to-charge (m/z) of quasi-molecule ion peak and reference. Five anthraquinones including aurantio-obtusin, 1-desmethylaurantio- obtusin, chryso-obtusin, obtusin, and 1-desmethylchryso-obtusin were isolated and purified by HSCCC and semi-HPLC from Cassia tora L. The purity of aurantio-obtusin, 1-desmethylaurantio-obtusin, chryso-obtusin, obtusin, and 1-desmethylchryso-obtusin was over 97% as determined by HPLC. The structures of five anthraquinones were identified by UV, MS, ¹H NMR.(3) HPLC-ESI-MS was utilized for the identification of nine flavonoid glycosides from sugarcane juice. Three flavonoid glycosides including swertisin, isoorientin-7, 3'-dimethyl ether, and tricin-7-O-glycoside were isolated and purified from sugarcane juice by HSCCC with a two-phase solvent system composed of ethyl acetate-n-butanol-water (9:1:10, v/v/v). The isolation produced a total of 19.7 mg of swertisin, 38.3 mg of isoorientin-7, 3'-dimethyl ether, and 8.5 mg of tricin-7-O-glycoside from 250 mg of crude extract in one-step elution. The purity of each compound was over 97% as determine by HPLC. The chemical identities of these components were confirmed by UV, MS and ¹H NMR. (4) The muti-stage tandem collision-induced dissociation mass spectrum (MS⁵-MS⁹) of four anthraquinones (aurantio-obtusin, obtusin, chryso-obtusin, 1-desmethylaurantio-obtusin) and two flavonoid glycosides (isoorientin-7, 3'-dimethyl ether, tricin-7-O-glycoside) were investigated in negative mode by electrospray ionization-ion trap mass spectrometry (ESI-ITMS). The fragmentation mechanisms of these compounds were explored according to some interesting losses. We also summarized the relationship between structural features of similar compounds and corresponding fragmentation behavior.(5) The crystals of five phenolic compounds were analyzed by single-crystal X-ray diffraction. The results showed that chryso-obtusin and caffeic acid crystallized in the monoclinic space group P2₁/n, and alizarin-1-methyl ether, 1,2-dimethoxy-3-hydroxy- anthraquinone and protocatechuic acid (PCA) crystallized in the triclinic space group P -1. It was one important feature of these crystals that their molecules were interconnected by themselves through hydrogen bonds,π···πstacking, and weak intermolecular interactions to form 3D supramolecular network. Single-crystal X-ray analysis revealed that, in the complexes K-protocatechuic acid (K-PCA) and Ca-protocatechuic acid (Ca-PCA), the central metallic ions were coordinated to eight oxygen atoms from 3-OH and 4-OH of four PCA ligands, crystallizing in the triclinic space group P -1 and the orthorhombic space group P 2₁2₁2 respectively. The main chemical stoichiometry of complexes K-PCA and Ca-PCA were K(C₇H₅O₄)(C₇H₆O₄) and Ca(C₇H₅O₄)₂(C₇H₆O₄)₂.(6) The antioxidant activity of PCA, complexes K-PCA and Ca-PCA were investigated. The results demonstrated that the ligand and its complexes showed good ability for scavenging superoxide and hydroxyl free radicals, and the order of antioxidant activity was Ca-PCA > K-PCA > PCA. At the molecular level, we deduced that the formation of K-O and Ca-O coordination bonds reduced the electron density of 3-OH and 4-OH, and increased the departure ability of H. On the other hand, PCA and its complexs also were tested in vitro to evaluate their antibacterial activity against bacteria Escherichia coli and Staphylococcus aureus, but it was not found that Ca-PCA and K-PCA showed higher activity than PCA.