Berberine and magnolol exert cooperative effects on ulcerative colitis in mice by self-assembling into carrier-free nanostructures

Abstract

The risk of ulcerative colitis (UC) is increasing worldwide with limited success using classical drugs, which has underscored the development of novel agents. Recently, carrier-free molecular assembly has been proven to be an effective drug delivery system, but it has yet to be examined for UC drug development using phytochemicals. Based on traditional Chinese medicine compatibility and potential medicinal uses, a pair of natural compounds, berberine (BBR) and magnolol (MAG), were found to self-assemble into nanostructures in aqueous solutions. Spectral analysis revealed that the assembly mechanisms of BBR and MAG were mediated through charge interactions and π-π stacking. Pharmacokinetic studies and animal imaging showed that BBR-MAG self-assembly (BM) effectively promoted the oral bioavailability and biodistribution of BBR in the colon. BM exhibited superior effects in regulating inflammatory factors, maintaining colon barrier integrity, and regulating gut microbiota in a dextran sulfate sodium salt-induced colitis mouse model. Additionally, no apparent signs of toxicity were observed, suggesting that BM has a favorable safety profile. This study presents a new strategy for UC management and highlights the cooperative effects of combined phytochemicals.

Keywords: Berberine; Gut microbiota; Magnolol; Self-assembly; Ulcerative colitis.

Fig. 1

BBR and MAG formed stable nanoassembly in aqueous solution: (A) Preparation process of BM; (B) BM morphology under transmission electron microscopy (TEM) (C) Size distribution of BM; (D) Zeta potential of BM and Tindall effect of BM solution; (E) Fully atomistic simulation of BM at different simulation time (0–150 ns); (F) Radial distribution functions of BM; (G) DLS results regarding the size and PDI of BM for seven days; (H) Drug release of BM and BBR in weakly alkaline (pH 7.4) and (I) acidic solutions (pH 2.4)

Fig. 2

BM was formed by electrostatic attraction and π–π stacking. (A) Self-assembling mode between BBR and MAG in BM; (B) UV-vis spectra of BBR, MAG, and BM; (C) XRD spectra of BBR, MAG, and BM; (D) FT-IR spectra of BBR, MAG, and BM; (E) ¹H-NMR spectra of BBR, MAG, and BM; (F) ROESY 2D NMR spectrum correlations of BM and signal annotation in spatial structure

Fig. 3

BM improved the bioavailability and colon accumulation compared with free BBR: (A) In vivo fluorescence images of oral deliverance of DiR@BM to mice; (B) Fluorescence images of mice colon isolated after 24 h drug administration; (C) Living Image 4.5 software measurements of the region of interest (ROI) fluorescence intensity of colons (n = 3); (D) Pharmacokinetic analysis process of BM and free BBR in SD rats; (E) BBR concentration of BBR in colon tissue after administration for 3 h (n = 7); (F) BBR concentration in plasma (n = 7). Significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.001; values are denoted by mean ± SD

Fig. 4

BM showed therapeutic efficacy advantages against DSS-induced colitis: (A) Drug intervention plan; (B) Body-weight changing trend, (C) DAI changing trend, and (D) Colon length of various groups (n = 6); (E) Digital colon photos isolated from mice; Representative images of (F) H&E-stained histological section (scale bar: 100 µm), (G) PAS-stained histological section scans, and (H) PSR-stained histological sections (scale bar: 200 µm); (I) TNF-α, IL-6, IL-1β and INF-γ levels in colon tissues isolated from various groups (n = 5). Significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.001; values are denoted as mean ± SD

Fig. 5

BM protected the epithelial barrier and reduces cell apoptosis: (A) IF images of β-catenin and apo-BrdU in colon tissues (scale bar = 100 µm); (B) IF images of ZO-1 and occludin in colon tissues (scale bar = 100 µm); (C) Histogram of semiquantitative analysis of β-catenin, ZO-1, apo-BrdU, and occludin in mouse colons. Significance is denoted as *p < 0.05, **p < 0.01, and ***p < 0.001; values are denoted as mean ± SD (n = 3)

Fig. 6

Intestinal microbiota regulated by BM in colitis induced by DSS: (A) Alpha diversity index for Ace, observed species, and Chao; (B) Venn diagram of five groups of species; (C) Area plot of cross groups showing phylum abundance; (D) NMDS and PCoA analysis of the level of operational taxonomic units; (E) Family-level LEfSe analysis with top 10 features according to significance; (F) Genus-level LEfSe analysis; (G and H) individual phylum abundance; (I and L) individual family and genus abundance. Significance level: *p < 0.05, **p < 0.01, and ***p < 0.001; values expressed as mean ± SD (n = 5)

Fig. 7

BM showed good safety in mice: (A) Scanning images of the mice heart, liver, spleen, lungs, and kidneys after H&E staining; (B) Individual blood biochemistry analysis. Scale bar = 100 µm; values expressed as mean ± SD (n = 5)