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. 2021 Nov 18:12:779801.
doi: 10.3389/fphar.2021.779801. eCollection 2021.

Drug-Drug Interaction Potential, Cytotoxicity, and Reactive Oxygen Species Production of Salix Cortex Extracts Using Human Hepatocyte-Like HepaRG Cells

João Victor Dutra Gomes  1 , Corinna Herz  1 , Simone Helmig  2 , Nadja Förster  3 , Inga Mewis  3 , Evelyn Lamy  1
Affiliations

Drug-Drug Interaction Potential, Cytotoxicity, and Reactive Oxygen Species Production of Salix Cortex Extracts Using Human Hepatocyte-Like HepaRG Cells

João Victor Dutra Gomes et al. Front Pharmacol. .

Abstract

Herbal preparations of willow bark (Salix cortex) are available in many countries as non-prescription medicines for pain and inflammation, and also as dietary supplements. Currently only little information on toxicity and drug interaction potential of the extracts is available. This study now evaluated the effects of two Salix cortex extracts on human hepatocyte-like HepaRG cells, in view of clinically relevant CYP450 enzyme activity modulation, cytotoxicity and production of reactive oxygen species (ROS). Drug metabolism via the CYP450 enzyme system is considered an important parameter for the occurrence of drug-drug interactions, which can lead to toxicity, decreased pharmacological activity, and adverse drug reactions. We evaluated two different bark extracts standardized to 10 mg/ml phenolic content. Herein, extract S6 (S. pentandra, containing 8.15 mg/ml total salicylates and 0.08 mg/ml salicin) and extract B (industrial reference, containing 5.35 mg/ml total salicylates and 2.26 mg/ml salicin) were tested. Both Salix cortex extracts showed no relevant reduction in cell viability or increase in ROS production in hepatocyte-like HepaRG cells. However, they reduced CYP1A2 and CYP3A4 enzyme activity after 48 h at ≥25 μg/ml, this was statistically significant only for S6. CYP2C19 activity inhibition (0.5 h) was also observed at ≥25 μg/ml, mRNA expression inhibition by 48 h treatment with S6 at 25 μg/ml. In conclusion, at higher concentrations, the tested Salix cortex extracts showed a drug interaction potential, but with different potency. Given the high prevalence of polypharmacy, particularly in the elderly with chronic pain, further systematic studies of Salix species of medical interest should be conducted in the future to more accurately determine the risk of potential drug interactions.

Keywords: CYP450 enzymes; Salix species; drug interaction; herb-drug interaction; willow bark.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
CYP450 enzyme activity quantification after treatment with Salix cortex extracts using a luminescent method. (A–D) Differentiated HepaRG cells were exposed to extracts for 1 or 48 h before analysis. (E) CYP2C19 enzyme activity was analysed in a cell-free assay after 0.5 h incubation of extracts with a human recombinant CYP2C19 enzyme, followed by analysis. Positive control (PC): 320 μM naringenin (CYP1A2 inhibition, PC1), 50 μM omeprazole (CYP1A2 induction, PC2), 10 μM rifampicin (CYP3A4 inhibition, PC3), 10 μM ketoconazole, (CYP3A4 induction, PC4), and 22.6 μM troglitazone (CYP2C19 inhibition, PC5). ASA, acetylsalicylic acid. The values are presented as means + SD (CYP1A2 1 and 48 h, n = 3; CYP3A4 1 h, n = 3, 48 h n = 4; CYP2C19 n = 3). Ordinary one-way ANOVA was used for statistical analysis, followed by a Dunnett test. Significance was evaluated between extracts and solvent control (a. d.) as well as between extract S6 and B. *p > 0.05, **p > 0.01.
FIGURE 2
FIGURE 2
mRNA levels of CYP2C19 in differentiated HepaRG cells after Salix cortex extracts treatment. mRNA levels were quantified by qRT-PCR. Differentiated HepaRG cells were treated with Salix cortex extracts (extract B or S6) by 6 h (A) or 48 h (B). CYP2C19 expression levels were expressed as mean + SD; (n = 3; S6, 48 h n = 2). Ordinary one-way ANOVA was used for statistical analysis, followed by a Dunnett test versus solvent control (SC: 0.5% destilled water) group. **p < 0.001.
FIGURE 3
FIGURE 3
Cytotoxicity and ROS production of Salix cortex extracts in differentiated HepaRG cells. ATP levels were analysed after (A) 24 h (B) 48 h of extract treatment. n = 3 (C) LDH cell release was measured after 24 h extract treatment. 0.2% Triton-X was used as positive control (PC). n = 4 (D, E) ROS production was measured by EPR spectroscopy after (D) 1 h or (E) 24 h of extract treatment. 200 μM menadione for 30 min. was used as positive control (PC). n = 3. The values are presented as means + SD. Ordinary one-way ANOVA was used for statistical analysis, followed by a Dunnett test versus solvent control (SC: 0.5% destilled water) group. **p < 0.01.
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