Comprehensive Comparisons between Grafted Kynam Agarwood and Normal Agarwood on Traits, Composition, and In Vitro Activation of AMPK

Abstract

Agarwood, a highly valuable resin/wood combination with diverse pharmacological activities but scarce supply, has a long history of being used as a medicine in several medical systems. Grafted Kynam agarwood (GKA) has been cultivated successfully recently and has the qualities meeting the definition of premium Kynam agarwood. However, there are few comprehensive comparisons between GKA and normal agarwood in terms of traits, global composition, and activity, and some key issues for GKA to be adopted into the traditional Chinese medical (TCM) system have not been elaborated. The two types of agarwood samples were evaluated in terms of trait characteristics, physicochemical indicators, key component groups, and global compositional profile. Furthermore, a molecular docking was performed to investigate the active ingredients. In vitro activity assays were performed to evaluate the activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) by GKA and normal agarwood. The results revealed that, overall, the traits, microscopic characteristics, chemical composition types, and bioactivity between GKA and normal agarwood were similar. The main differences were the content of resin (ethanolic extract content), the content of key component groups, and the composition of the different parent structural groups of 2-(2-phenethyl) chromones (PECs). The contents of total PEC and ethanol extract content of GKA were significantly higher than those of normal agarwood. The MS-based high-throughput analysis revealed that GKA has higher concentrations of sesquiterpenes and flindersia-type 2-(2-phenylethyl) chromones (FTPECs) (m/z 250-312) than normal agarwood. Molecular docking revealed that parent structural groups of FTPECs activated multiple signaling pathways, including the AMPK pathway, suggesting that FTPECs are major active components in GKA. The aim of this paper is to describe the intrinsic reasons for GKA as a high-quality agarwood and a potential source for novel drug development. We combined high-throughput mass spectrometry and multivariate statistical analysis to infer the different components of the two types of agarwood. Then we combined virtual screening and in vitro activity to construct a component/pharmacodynamic relationship to explore the causes of the activity differences between agarwood with different levels of quality and to identify potentially valuable lead compounds. This strategy can also be used for the comprehensive study of other TCMs with different qualities.

Keywords: AMPK; Aquilaria sinensis; Flindersia-type 2-(2-phenylethyl) chromones; Grafted Kynam agarwood; MS-based high-throughput analysis; Molecular docking.

Figure 1

Morphology of the samples of two representative types of agarwood as well as their transverse section microscopic features (magnification of 100×) and wood flake morphology.

Figure 2

Content of agarwood ethanol extracts (a) and total PECs (b). The ethanol-soluble extract content was determined according to the procedures of the Chinese Pharmacopoeia. The total PEC content was measured at 254 nm with agarotetrol as the standard.

Figure 3

BPI plots of positive ion mode (a) and negative ion mode (b) of the ethanol extracts of two representative samples of agarwood.

Figure 4

Overview of the results of untargeted GC-MS analysis of the metabolic profiles of all samples by XCMS-Online. The overlap of total ion chromatograms (TIC) before (a) and after (c) retention time (RT) alignment, the retention time alignment curve (b), multidimensional scaling (d), and differential feature plot (“mirror plot”) (e). The red indicated the features that is more highly expressed in the GKA, and the green represented the opposite. p value is represented by how dark or light the color is. Fold change is represented by the radius of each feature.

Figure 5

Multivariate statistical analysis revealed metabolic differences between the two types of agarwood. Score plot of the PCA model (a), score plot (b), S-line plot (c), and permutation analysis line plots (d) of the OPLS model. The numbers in the S-line plot are the codes for the ion features. The left, middle, and right columns are the multivariate statistical results obtained by GC-MS and UPLC/Q-TOF-MS positive and negative ion modes, respectively.

Figure 6

Differential analysis of PECs biosynthesis pathway between GKA and normal agarwood. The biosynthetic pathway of PECs (a), relative quantitative analysis of structural diversification in secondary metabolite FTPECs biosynthesis (b), relative quantitative analysis of structural diversification in secondary metabolite THPECs biosynthesis (c).

Figure 7

Molecular docking revealed the potential signaling pathways regulated by agarwood related to diseases. The predicted bioavailability radar of 2-(2-phenylethyl) chromone and agarotetrol (a), Simulation of a potential target-compound-pathway docking network visualized by Cytoscape (b). The depth of the red connecting line between the compound and the target gene represents the result of the free energy of molecular docking. Protein-protein interactions (PPI) of potential targets (c), the bubble plot of KEGG pathway enrichment analysis (d), and GO analysis of potential targets with top 10 terms displayed in Bar graph (e).

Figure 8

In vitro activity and validation study of the agarwood ethanol extracts activates AMPK in 293T cells. The cell lysates were subjected to SDS-PAGE and Western blot analysis using antibodies specific for phospho-AMPK or total AMPK. The results are presented relative to the vehicle control (DMSO) and as the mean ± SD (n = 3). ** p < 0.01; *** p < 0.001 (a). The binding mode of some representative compounds with AMPK; the codes for the compounds in the figure correspond to those in Supplementary Materials Table S2 (b).