Multicomponent therapeutics of berberine alkaloids

Abstract

Although berberine alkaloids (BAs) are reported to be with broad-spectrum antibacterial and antiviral activities, the interactions among BAs have not been elucidated. In the present study, methicillin-resistant Staphylococcus aureus (MRSA) was chosen as a model organism, and modified broth microdilution was applied for the determination of the fluorescence absorption values to calculate the anti-MRSA activity of BAs. We have initiated four steps to seek the optimal combination of BAs that are (1) determining the anti-MRSA activity of single BA, (2) investigating the two-component combination to clarify the interactions among BAs by checkerboard assay, (3) investigating the multicomponent combination to determine the optimal ratio by quadratic rotation-orthogonal combination design, and (4) in vivo and in vitro validation of the optimal combination. The results showed that the interactions among BAs are related to their concentrations. The synergetic combinations included "berberine and epiberberine," "jatrorrhizine and palmatine" and "jatrorrhizine and coptisine"; the antagonistic combinations included "coptisine and epiberberine". The optimal combination was berberine : coptisine : jatrorrhizine : palmatine : epiberberine = 0.702 : 0.863 : 1 : 0.491 : 0.526, and the potency of the optimal combination on cyclophosphamide-immunocompromised mouse model was better than the natural combinations of herbs containing BAs.

Figure 1

Molecular structures of berberine alkaloids.

Figure 2

Schematic diagram of 36-point dose matrices for two-component combination assay. The five test concentrations for each compound were chosen by first determining the EC₅₀ of each compound as a single agent in this assay, and then selecting 4-fold and 16-fold higher and lower concentrations.

Figure 3

Inhibiting ratios of different concentrations of cefoxitin and five berberine alkaloids on MRSA.

Figure 4

Fluorescent determination using resazurin. (a) 96-well fluorescent plates with 2-fold dilutions of Ber; (b) fluorescent image of MRSA suspension without any chemical; (c) the microphotograph of MRSA at 0 h without any chemical (stained with gram stain); (d) the microphotograph of MRSA at 24 h without any chemical; (e) 96-well translucent plates with 2-fold dilutions of Ber; (f) fluorescent image of MRSA suspension with 64 μg/mL cefoxitin; (g) the microphotograph of MRSA at 24 h with 64 μg/mL cefoxitin; (h) the microphotograph of MRSA at 24 h with 128 μg/mL Ber.

Figure 5

The excess over the highest single agent model, including 10 combinations of the 5 berberine alkaloids. Purple squares indicate strong antagonistic effect, orange squares indicate slight antagonistic effect, dark blue squares indicate additive effect, light blue squares indicate slight synergistic effect, brilliant yellow squares indicate strong slight synergistic effect, and green squares indicate strong synergistic effect.

Figure 6

The excess over Bliss additivism model, including 10 combinations of the 5 berberine alkaloids.

Figure 7

Comparison of the antimicrobial activities of different combinations of berberine alkaloids on five laboratory strains, including E. coli, S. aureus, S. dysenteriae, S. pneumonia, and C. albicans.

Figure 8

In vivo anti-MRSA effects of BAs combinations. (a) Body weight of ICR mice from day −3 to 16; (b) percent survival of ICR mice during the therapeutic process. (a: cefoxitin group; b: combination in Rhizoma Coptidis group; c: combination in Phellodendri Chinensis Cortex group; d: combination in Berberidis Radix group; e: optimal combination in this study group; f: geometric proportion of alkaloids group; g: negative group (noninfection mice without being given drugs); h: placebo control).

Figure 9

Bacterial density in kidney, lung, and brain tissues. (a) Pathogen-free female ICR mice; (b) MRSA-infected ICR mice; (c) brain tissue of died or killed mice; (d) lung tissue of died or killed mice; (e) kidney tissue of died or killed mice; (f) micrograph; (g) colony counts on day 12 (log cfu). (a: cefoxitin group; b: optimal combination in this study group; c: combination in Rhizoma Coptidis group; d: combination in Phellodendri Chinensis Cortex group; e: combination in Berberidis Radix group; f: geometric proportion of alkaloids group; g: placebo group).