Two in vitro studies on cell-free enzymes [8, 9] demonstrated that garlic constituents competitively inhibit several recombinant CYP enzymes, namely CYP2C9, CYP2C19 and CYP3A4. Aqueous extracts from odorless garlic products inhibited CYP3A4 (range of inhibition by the six separate products, 30.8–53.8%) to a slightly greater extent than CYP2C9 (range of inhibition: 4.8–33.5%). Steam-distilled garlic oil inhibited CYP2C9 and CYP3A4 to a comparable extent on average, while freeze-dried garlic tablets did not inhibit CYP2C9 activity [8]. Allicin, a major organosulfur constituent of some garlic supplements, was a more potent inhibitor of cDNA-expressed CYP2C9 than CYP3A4 (IC₅₀ 5.4 vs. 43.7 μM, respectively) [8]. On the other hand, a human microsomal P450 activity study suggested that only two garlic constituents, S-allyl- or S-methyl-L-cysteine at 0.1 mg/mL significantly inhibited CYP3A4 with no impact on CYP2C9, CYP2C19 or CYP1A2 [10].

We also determined the effects of garlic on RNA transcripts of CYP2C9 and CYP3A4 in Fa2N-4 cells. The RNA levels were determined on day four using a validated, quantitative RT-PCR assay for each of the target gene transcripts. As shown in Table 2, exposure of these cells to rifampin induces both CYP2C9 and CYP3A4 mRNA transcripts by about four-fold compared to control medium. When the same cells were exposed to garlic, CYP2C9 showed a garlic concentration-dependent reduction in transcript levels. In contrast, exposure to even up to 100 μg/mL of garlic extract did not appear to significantly alter the CYP3A4 transcript concentration in hepatocytes. These results suggest that garlic extract had a selective suppressive effect on the mRNA expression of CYP2C9 in Fa2N-4 hepatocytes. Taken together, the enzymatic activity of CYP2C9 and CYP3A4 in these hepatocytes appeared to have tracked the effects of garlic on RNA transcript levels.

Although it has been suggested that garlic supplements may induce CYP activity in vivo, there is little direct evidence that garlic extract up-regulates human CYP2C9 or CYP3A4 expression. Using a novel immortalized hepatocyte Fa2N-4 cell line that exhibits similar characteristics as those of primary human hepatocytes, we have systematically studied the modulating effects of garlic extract on CYP 2C9 and CYP3A4 mRNA expression and catalytic activity. Our results show that garlic suppresses CYP2C9 expression and activity in a remarkable, concentration-dependent manner, but that garlic has no discernable effect on CYP3A4 within the same cells. The changes in CYP2C9 activity paralleled those of RNA transcript levels. The decrease in transcript concentration is not due to any detrimental effects of garlic extract on cell viability, since expression and activity of CYP3A4 were maintained in the same cells. Also, the degree of induction observed with rifampin in our Fa2N-4 cultures (Table 2) is comparable to that reported by others [11]. In regards to mechanism, the decrease in CYP2C9 transcript could reflect either reduction in transcription or increased RNA degradation, or both. As CYP3A4 transcription is not affected by garlic exposure, it appears that the putative effects of garlic on intracellular transcription or message destabilization are a target-gene specific process. The exact mechanism awaits further investigation. It should be noted that garlic extract could also directly inhibit CYP2C9, but not CYP3A4 activity. Using cDNA expressed recombinant CYP3A4 enzyme, in microsome (cell-free) preparations, Foster et al. have shown that various preparations of garlic extract at a fixed 25 mg/mL concentration inhibited CYP3A4 and CYP2C9 activity [9]. It is not known whether garlic extract at the much lower concentrations employed in our experiments (between 5–200 μg/mL) can directly inhibit CYP2C9 activity (Tables 1 and ​and2).2). Nonetheless, it is clear that the activity of CYP3A4 in Fa2N-4 was not affected by the chosen medium concentrations of garlic extract (Table 2).

It has been proposed that garlic supplements’ effects on CYP3A4 in humans is likely to be biphasic in nature; that is, inhibition during the early phase of treatment [8, 9], and simultaneous inhibition and induction following chronic treatment. This hypothesis is supported by the minimal effects of short term exposure (four to five days) to garlic on modifying the area under the concentration-time curve (AUC) of a single dose of ritonavir, a substrate for CYP3A4 [12]. Chronic (i.e., 21-days) administration of dehydrated garlic (Natrol's GarliPure™ caplets BID) significantly reduced the AUC and Cmax of the HIV protease inhibitor saquinavir (a CYP3A4 and P glycoprotein substrate) in nine healthy volunteers [6]. It was proposed that garlic constituents lowered the saquinavir plasma concentrations by inducing the drug-metabolizing enzyme CYP3A4 in the intestine and the liver, and/or the membrane efflux transporter P-glycoprotein at the intestinal mucosa. CYP3A4 activity was not affected by 14-day administration of a different dehydrated garlic product (Kwai garlic supplement, three tablets BID) [13]. Only one study to date has examined the effects of steam-distilled garlic oil on CYP3A4; midazolam hydroxylation, a marker of CYP3A4, was not affected by steam-distilled garlic oil (500 mg TID for 28 days) [14]. It appears that garlic has an equivocal effect on hepatic CYP3A4 that may be substrate-dependent.