Comparison of the Blood-Brain Barrier Penetration Ability and Anti-Neuroinflammatory Activity of Chromones in Two Types of Agarwood

Abstract

Background/Objectives: Agarwood has a good neuroprotective effect and is often used to relieve anxiety and treat insomnia. This study compared the similarities and differences in the chromone components of two types of agarwood. It further investigated the absorption and brain distribution characteristics of these components in rats and their neuroprotective effects mediated through anti-neuroinflammatory pathways. Methods: This study confirmed, through ITS2 barcoding and chloroplast genome analysis, that both the ordinary and Qi-Nan agarwood are derived from Aquilaria sinensis. A comparative analysis of chromones in ethanol extracts derived from ordinary and Qi-Nan agarwood, as well as those capable of penetrating the blood-brain barrier in vivo, was conducted using UPLC-Q-TOF-MS. Subsequently, an in vitro neuroinflammatory model was established via lipopolysaccharide (LPS)-stimulated BV-2 cells to evaluate the anti-neuroinflammatory activity of differential chromones. Results: UPLC-Q-TOF-MS characterization revealed the chromone components in the two types of agarwood: A total of 81 chromone compounds were identified in the ethanol extracts of ordinary agarwood (OAE) (20 THPECs, 42 FTPECs, and 19 BI), while 41 were identified in the ethanol extracts of Qi-Nan agarwood (QNE) (11 THPECs and 30 FTPECs). Pharmacokinetic analysis in rats showed that 14 components from OAE (eight THPECs and six FTPECs) penetrated the rat serum, and 10 of these 14 components penetrated the blood-brain barrier (BBB). Twelve FTPECs from QNE penetrated the rat serum, all of which penetrated the BBB. The total peak area of the total ion current (TIC) was calculated for the samples, and the TIC of the serum was compared to that of the brain tissue from the same rat to roughly estimate the ratio. The results demonstrated that the capability of FTPECs to traverse the blood-brain barrier is substantially superior to that of THPECs. Correspondingly, only FTPECs were detected using DESI-MS imaging; no THPECs were detected in rat brain tissue, and DESI-MS imaging localized FTPECs to neuroanatomic regions (cerebral cortex, thalamus, and hippocampus). In vitro neuroinflammatory assays revealed the superior anti-inflammatory efficacy of QNE over OAE (IL-6/TNF-α suppression, p < 0.05), correlating with its FTPEC-rich composition. Conclusions: Structure-activity relationships identified FTPECs as potent inhibitors of pro-inflammatory cytokines, exhibiting enhanced BBB penetration (blood-brain relative abundance > 1). These findings establish FTPECs as prioritized candidates for CNS-targeted therapeutics, with QNE's pharmacological superiority attributed to its FTPEC dominance and optimized BBB transit capacity.

Keywords: Qi-Nan agarwood; blood–brain partitioning; chromones; neuroinflammation; ordinary agarwood.

Figure 1

Skeleton structure of THPECs and FTPECs.

Figure 2

Species identification results using the Herb-Q method: 10 bp base information before and after the 7464 locus in the chloroplast genome of Aquilaria Species (A). The forward and reverse Sanger sequencing results of Sample A01 showed the expected results at the 7464 site (B). The forward and reverse Sanger sequencing results of Sample B01 showed the expected results at the 7464 site (C). Different colors of peaks correspond to different bases below. Red represents base T; green represents base A; black represents base G; blue represents base C.

Figure 3

Results of the chromone components in ethanol extracts of two types of agarwood. The number of different chromone types in the ethanol extracts of two types of agarwood (A). Venn diagram of the chromone components in OAE (a) and QNE (b) (B). The relative peak area ratio of different types of chromones in the ethanol extracts of ordinary agarwood (OAE) (C). The relative peak area ratio of different types of chromones in the ethanol extracts of Qi-Nan agarwood (QNE) (D). Proportion of the relative peak area of each component of two types of agarwood (E).

Figure 4

Results of the blood-penetrating components in two types of agarwood. The relative peak area ratio of each detected blood-penetrating component in two types of agarwood ethanol extracts and two types of agarwood serum: OAE and QNE (A) and OAS and QNS (B), respectively. The production pathway of each metabolite (C).

Figure 5

The results of the blood- and brain-penetrating components of two types of agarwood. Relative peak area ratio of the brain-penetrating components of two types of agarwood in ethanol extract, serum, and brain tissue (A). Transfer process of the brain-penetrating components of two types of agarwood in ethanol extract, serum, and brain tissue (B). The number of chromone components in the ethanol extract, serum, and brain tissue of two types of agarwood (C).

Figure 6

Blood–brain relative abundance of each component in two types of agarwood: ordinary agarwood (A) and Qi-Nan agarwood (B). Yellow represents THPECs; Green represents FTPECs; Blue represents the FTPECs with the highest blood–brain relative abundance.

Figure 7

The distribution of the brain components in each group detected using DESI-MS.

Figure 8

Cell experiment results. The CCK-8 results of BV-2 cells treated with ordinary agarwood (5 μg/mL) and Qi-Nan agarwood (5 μg/mL) (A). The CCK-8 results of BV-2 cells based on ten types of compounds (10 μM each) (B). Inhibitory effects of dexamethasone (Dex, 10 μM), ordinary agarwood (5 μg/mL), and Qi-Nan agarwood (5 μg/mL) on LPS-induced IL-6 production in BV-2 cells (C). Inhibitory effects of dexamethasone (Dex, 10 μM), ordinary agarwood (5 μg/mL), and Qi-Nan agarwood (5 μg/mL) on TNF-α production in BV-2 cells induced by LPS (D). Inhibitory effects of dexamethasone (Dex, 10 μM) and 10 compounds (10 μM) on LPS-induced IL-6 production in BV-2 cells (E). Inhibitory effects of dexamethasone (10 μM) and 10 compounds (10 μM) on TNF-α production in BV-2 cells induced by LPS (F). * Compared to the model group LPS, there are significant differences between the administration group and the model group, * p < 0.05, ** p < 0.002, and *** p < 0.001; # significant differences between the Qi-nan agarwood (5 μg/mL) group and the ordinary agarwood (5 μg/mL) group, ## p < 0.002, and ### p < 0.001; $ among the four FTPEC components, compared to compound FT-4, the tree compounds are significantly different from the FT-4 components, namely, FT-1, FT-2, and FT-3, and $$$ p < 0.001.