Preparative isolation and purification of 12 main antioxidants from the roots of Polygonum multiflorum Thunb. using high-speed countercurrent chromatography and preparative HPLC guided by 1,1'-diphenyl-2-picrylhydrazyl-HPLC.

A hyphenated strategy by off-line coupling of 1,1'-diphenyl-2-picrylhydrazyl-high-performance liquid chromatography, high-speed countercurrent chromatography, and preparative high-performance liquid chromatography was established to screen and separate antioxidants from ethyl acetate fraction of the roots of Polygonum multiflorum. Under the targeted guidance of 1,1'-diphenyl-2-picrylhydrazyl-high-performance liquid chromatography experiment, 12 compounds were identified as potential antioxidants and readily isolated by high-speed counter-current chromatography and preparative high-performance liquid chromatography. Ultraviolet spectroscopy, mass spectrometry, and 1 H NMR spectroscopy were employed to identify their structures, which were assigned as gallic acid (1, 6.2 mg, 98.28%), catechin (2, 8.8 mg, 90.69%), epicatechin (3, 4.1 mg, 96.71%), polydatin (4, 5.3 mg, 94.91%), 2,3,5,4'-tetrahydroxy stilbene-2-Ο-ß-D-glucoside (5, 20.2 mg, 95.23%), piceatannol (6, 5.3 mg, 96.85%), rutin (7, 5.4 mg, 97.92%), resveratrol (8, 5.2 mg, 96.94%), isorhapontigenin (9, 11.4 mg, 94.81%), hyperoside (10, 9.7 mg, 98.52%), rhein (11, 4.9 mg, 97.46%), and emodin (12, 8.2 mg, 95.74%). Notably, compounds 6 and 9 were isolated from Polygonum multiflorum for the first time. In addition, antioxidant activity of compounds 1-12 were evaluated, and compounds 1-8 and 10 exhibited stronger antioxidant activity than ascorbic acid (positive control). These results indicated that the proposed method is a highly efficient strategy to screen and isolate antioxidants from complex natural products.

4-Mercaptophenylboronic acid-modified spirally-curved mesoporous silica nanofibers coupled with ultra performance liquid chromatography-mass spectrometry for determination of brassinosteroids in plants.

Herein, for the first time, thiol-functionalized mesoporous silica (mSiO2-SH) nanofibers with a spirally-curved twisted hexagonal morphology were synthesized via a simple one-pot protocol. 4-Mercaptophenylboronic acids (4-MPBA) were attached onto the mSiO2-SH nanofibers via disulfide bond, serving as boronate affinity sorbent to selectively capture brassinosteroids (BRs) from plant extract. The resulting BRs-MPBA derivatives were easily eluted from the sorbent by cleaving the disulfide bond, which was subsequently subjected to ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Thus, the in situ extraction/derivatization/desorption method coupled with UPLC-MS was established for the fast, sensitive and selective detection of BRs in plant tissues. Finally, based on the developed method, endogenous BRs were successfully detected in leaf of H. lupulus L., silique of A. thaliana, and panicle of O. sativa L.

Rapid screening and identification of antioxidants in the leaves of Malus hupehensis using off-line two-dimensional HPLC-UV-MS/MS coupled with a 1,1'-diphenyl-2-picrylhydrazyl assay.

The leaves of Malus hupehensis have a strong antioxidant activity and are commonly consumed as a healthy tea. However, detailed information about its antioxidants is incomplete. Herein, we developed an effective strategy based on combining off-line two-dimensional high-performance liquid chromatography with ultraviolet and tandem mass spectrometry detection with a 1,1'-diphenyl-2-picrylhydrazyl assay to rapidly screen and identify the antioxidants from the leaves of M. hupehensis. In the orthogonal two-dimensional liquid chromatography system, a Venusil HILIC column was used for the first dimension, while a Universil XB-C18 column was installed in the second dimension. As a result, 32 antioxidants, including ten dihydrochalcones, two flavanones, nine flavonols, four flavones, and seven phenolic acids were tentatively identified, out of which 23 compounds, as far as we know, were isolated and characterized from the leaves of M. hupehensis for the first time. To the best of our knowledge, this is the first systematic investigation of the antioxidants from the leaves of M. hupehensis. The results indicated that the proposed method is an efficient technique to rapidly investigate antioxidants, especially for coeluted and minor compounds in a complex system.