Walnut Compound Juglone Blocks Key Drug-Metabolizing Enzyme CYP2C9 in Lab Study

A redox-active compound found in walnut green husks has emerged as a surprisingly potent inhibitor of one of the liver’s most important drug-metabolizing enzymes. Juglone, a naphthoquinone long studied for its antimicrobial, anti-inflammatory, and anticancer properties, was put through a systematic battery of enzyme assays by researchers at East China Normal University, and the results, published in Frontiers in Pharmacology, show that it interferes most strongly with CYP2C9, the cytochrome P450 enzyme responsible for clearing warfarin, sulfonylureas, phenytoin, and many common anti-inflammatory drugs. The work does not demonstrate a clinical interaction, but it provides the kind of kinetic baseline that regulators and pharmacologists rely on when deciding whether a natural product deserves closer scrutiny.

The team, led by Luping Zhao, Yan Shen, Yuanjin Zhang, and Xin Wang, evaluated juglone against six major CYP activities in both pooled human liver microsomes and microsomes prepared from rat liver. Using a cocktail of probe substrates—phenacetin for CYP1A2, bupropion for CYP2B6, tolbutamide for CYP2C9, dextromethorphan for CYP2D6, chlorzoxazone for CYP2E1, and midazolam for CYP3A4—they quantified metabolite formation by liquid chromatography–tandem mass spectrometry after incubating the microsomes with juglone concentrations ranging from 1 to 100 micromolar. The screening revealed a clear hierarchy of susceptibility in the human preparations, with CYP2C9 and CYP2E1 standing out well ahead of the rest.

In human liver microsomes, CYP2C9 was the most vulnerable activity, with a half-maximal inhibitory concentration of 8.12 micromolar. Kinetic experiments that varied both substrate and inhibitor concentrations pointed to competitive inhibition, with an apparent inhibition constant of 4.97 micromolar—the lowest Ki recorded among the human activities examined. CYP2E1 was the next most susceptible, showing an IC50 of 12.67 micromolar and a mixed inhibition pattern, with apparent Ki and alpha-Ki values of 19.59 and 8.74 micromolar respectively. By contrast, CYP1A2 and CYP2B6 were only weakly inhibited, with IC50 values near 50 micromolar, and CYP2D6 and CYP3A4 were not inhibited by half even at the highest concentration tested.

The rat microsomes told a different story. There, CYP1A2 was the most strongly inhibited activity, with an IC50 of 12.03 micromolar, followed by CYP2E1 at 18.50 micromolar. Follow-up kinetic analysis identified mixed inhibition of rat CYP1A2, with an apparent Ki of 12.31 micromolar, and competitive inhibition of rat CYP2E1, with an apparent Ki of 19.89 micromolar. Notably, rat CYP2C11—the nominal counterpart of human CYP2C9—was considerably less sensitive, with an IC50 of 33.39 micromolar. The authors are careful to note that CYP2C11 is not a one-to-one equivalent of human CYP2C9, and that CYP abundance differs between the two species, so the rat profile should be used to design animal studies rather than as a quantitative surrogate for human risk.

One of the more consequential findings concerns what the study did not detect. Time-dependent inhibition experiments, in which microsomes were preincubated with juglone for up to twenty minutes before the probe reaction was started, showed no consistent progressive loss of enzyme activity for any of the selected CYPs. That argues against mechanism-based inactivation under the tested conditions, though the authors acknowledge important caveats: the preincubation window was short, the concentration range limited, and—critically—no dedicated positive control for time-dependent inhibition was included. Definitive conclusions about reversibility, and any measurement of the inactivation parameters kinact and KI, will require follow-up experiments with appropriate controls and longer preincubation periods.

The species comparison around CYP2E1 is particularly instructive. The apparent Ki values in human and rat microsomes were numerically almost identical—19.59 versus 19.89 micromolar—yet the fitted inhibition models differed, with mixed inhibition in the human system and competitive inhibition in the rat system. The authors resist the temptation to read this as a genuine species difference. The two assays used different microsomal protein concentrations and buffer compositions, factors known to alter apparent inhibitory potency and catalytic activity. Without matched conditions and verified reaction linearity, they write, similar Ki values cannot be taken as evidence of equivalent intrinsic susceptibility, and the observed model differences cannot be attributed solely to species.

To add structural context, the team docked juglone into crystal structures of human CYP2C9 and CYP2E1. The docking produced GlideScores of −6.758 and −7.502 kilocalories per mole respectively, and the top-ranked CYP2C9 pose placed the naphthoquinone ring close to the heme catalytic center, flanked by hydrophobic residues including Leu102, Ala103, Leu208, and Val237, with a predicted hydrogen bond between a juglone carbonyl and Arg108. Interestingly, the docking scores did not reproduce the experimental potency ranking—CYP2E1 scored better yet was inhibited more weakly in human microsomes—a discrepancy the authors attribute to the limits of empirical scoring functions, which cannot be translated directly into inhibition constants and do not model substrate competition or the enzyme–substrate–inhibitor states relevant to mixed inhibition.

The translational question—could juglone-containing products actually perturb the metabolism of prescription drugs?—remains open, and the paper is unusually candid about why. Reliable human plasma data for unchanged juglone under relevant use conditions are simply unavailable. Preclinical evidence suggests partial absorption in rats, formulation-dependent half-lives, and more than 99 percent binding to serum albumin, but none of this establishes the unbound systemic concentration that would determine whether the micromolar inhibition seen in vitro is ever approached in vivo. The authors convert their key numbers for perspective: the CYP2C9 IC50 of 8.12 micromolar corresponds to roughly 1.41 micrograms per milliliter, and the Ki of 4.97 micromolar to about 0.866 micrograms per milliliter—nominal in vitro concentrations, not clinical thresholds. Measuring the unbound, unchanged compound in plasma is flagged as the essential next step.

The study also fits into a broader research program. The same laboratory previously showed that the related naphthoquinones plumbagin, shikonin, and deoxyshikonin inhibited all six tested human CYP activities, whereas juglone’s effects concentrated on CYP2C9 and CYP2E1. That contrast underscores a point the authors emphasize repeatedly: structural relatedness alone does not define a shared inhibition profile, and each compound must be assessed on its own. It also matters clinically, because CYP2C9 clears drugs with narrow therapeutic windows, and inhibitors of this enzyme have well-documented potential to raise exposure to anticoagulants and anticonvulsants.

The authors are equally transparent about the study’s statistical and analytical limits. All replicates were technical incubation replicates within a single pooled microsomal preparation, so the reported variability says nothing about inter-individual differences in enzyme activity. The apparent Ki and alpha-Ki values were derived from Lineweaver–Burk plots and secondary replots, now accompanied by 95 percent confidence intervals, and were cross-checked with global nonlinear regression and the corrected Akaike information criterion. For human CYP2C9, the lowest-AICc competitive model agreed with the graphical analysis, yielding a Ki of 5.11 micromolar versus 4.97 micromolar from the replots. For human CYP2E1, the mixed model was only modestly favored over a pure noncompetitive alternative, and some alpha-Ki estimates—particularly for the rat enzymes—were poorly constrained. Comprehensive analytical validation of the LC-MS/MS method under the study’s exact conditions was not performed, leaving quantitative bias incompletely characterized.

Where does this leave juglone? The compound, enriched in Pericarpium Juglandis—the green walnut husk used in Chinese medicine as Qinglongyi—has attracted attention as a scaffold for derivatives targeting cancer, inflammation, and bacterial quorum sensing. This study does not sound an alarm for consumers, but it does something arguably more useful: it converts a vague concern about natural product–drug interactions into a concrete, testable hypothesis centered on CYP2C9. The recommended roadmap includes recombinant human CYP2C9 experiments, measurements of plasma and microsomal protein binding, metabolite identification to check whether juglone’s breakdown products also inhibit CYPs, and in vivo pharmacokinetic studies that quantify unchanged juglone under defined dosing and formulation conditions. Only with that exposure data, combined with binding-adjusted inhibition estimates and knowledge of how much of a co-administered drug’s clearance depends on CYP2C9, can a defensible prediction of interaction risk be made. For now, the message is measured: juglone is a potent CYP2C9 inhibitor in a test tube, and that fact alone makes it a compound worth watching.

Subject of Research: In vitro inhibition of cytochrome P450 enzymes by the natural naphthoquinone juglone in human and rat liver microsomes

Article Title: Inhibition of cytochrome P450 activities by juglone in human and rat liver microsomes: kinetic characterization and implications for natural product–drug interaction assessment

Article References: Zhao, L., Shen, Y., Xu, X., Liu, L., Huang, J., Liang, C., Zhang, Y., & Wang, X. (2026). Inhibition of cytochrome P450 activities by juglone in human and rat liver microsomes: kinetic characterization and implications for natural product–drug interaction assessment. Frontiers in Pharmacology, 17, Article 1968844. https://doi.org/10.3389/fphar.2026.1968844

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1968844

Keywords: juglone, cytochrome P450, CYP2C9, CYP2E1, liver microsomes, drug metabolism, enzyme kinetics, natural product-drug interactions, naphthoquinone, pharmacokinetics, molecular docking, time-dependent inhibition