Fast and efficient identification of hyaluronidase specific inhibitors from Chrysanthemum morifolium Ramat. using UF-LC-MS technique and their anti-inflammation effect in macrophages

Abstract

The purpose of the study was to establish a rapid analytical strategy to screen potential anti-inflammatory compounds from Flos Chrysanthemum flower. The enzyme assay was conducted to prescreen botanical extracts, in which Chrysanthemum morifolium aqueous extract (CME) displayed hyaluronidase (HAase) inhibitory activity in a dose-dependent manner with the values of 8.31, 24.25, and 66.51% at concentrations of 1.00, 2.00, and 4 0.00 mg/mL, respectively. Eight potential compounds targeting HAase (compounds 9, 10, 11, 13, 15, 17, 20 and 21) from CME were screened using ultrafiltration affinity liquid chromatography coupled with mass spectrometry (UF-LC-MS) technology. The well-known inhibitor, dipotassium glycyrrhizinate (DG), was used as a positive control and competitive ligand to eliminate false positives. Then, four of these potential components (compounds 9, 10, 17, and 21), namely eriodictyol-7-O-glucoside, luteoloside, apigenin-7-O-glucoside and diosmetin-7-O-glucoside, were distinguished as potent HAase specific inhibitor candidates with high BD and CBD values. The enzyme inhibitory activities of candidate compounds were verified using enzyme inhibition assay. At a concentration of 1000 μM, compounds 9, 10, 17, and 21 showed 40.15, 44.85, 18.04, and 24.15% inhibition of HAase, respectively. Furthermore, all the four compounds significantly decreased the production of nitric oxide (NO) and IL-6, and significantly suppressed the mRNA expression of inducible NO synthase (iNOS) and IL-1β in both murine and human macrophages.

Keywords: Anti-inflammation; Chrysanthemum morifolium; Hyaluronidase inhibitors; Macrophages; UF-UPLC-Q-TOF-MS.

Fig. 1

The influence of different enzyme concentration (a), different temperature (b), and culture time (c) on the reaction.

Fig. 2

Scheme of UF-UPLC-PDA-MS assay for screening for HAase inhibitors.

Fig. 3

UF-UPLC-MS analysis of CME for identifying the ligands bound to HAase. A. Chromatogram of the blank group. B. Chromatogram of the sample group. C. Chromatogram of the DG-blocked HAase group.

Fig. 4

EIC of screened compounds (9, 10, 11, 13, 15, 17, 20, and 21) from CME for identifying the ligands bound to HAase. Group A. EIC of the blank group; Group B. EIC of the sample group; Group C. EIC of the DG-blocked HAase group.

Fig. 5

Chemical structure and fragmentation pathway of (a) eriodictyol-7-O-glucoside, (b) luteoloside，(c) apigenin-7-O-glucoside, and (d) diosmetin 7-O-glucoside.

Fig. 6

Anti-inflammatory activity of four compounds in macrophages. The level of NO (A), IL-6 (B), TNF-α (C) was determined using an assay kit. The mRNA expressions of IL-1β (D), iNOS (E) and MyD88 (F) were measured by RT-qPCR. Values are presented as the x ± s, n = 3. Analyses were performed using a one-way ANOVA. ####p < 0.001 vs. the con group; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs. the LPS group. Compounds 9, 10, 17, and 21 were identified as eriodictyol-7-O-glucoside, luteoloside, apigenin 7-O-glucoside, and diosmetin 7-O-glucoside, respectively.