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Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine and Tufts-New England Medical Center, Boston, MA
4 To whom correspondence should be addressed: E-mail: dj.greenblatt{at}tufts.edu.
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • aged garlic extract • Cytochromes P450 • in vitro metabolism • drug interaction
The increasingly extensive utilization of complementary or alternative medical therapies over the last decade is now well documented (1–7). This incorporates the use of botanical medicines either alone or in combination with prescription medications. With the increased prevalence of botanical use comes the need for clinical and scientific data on the pharmacologic properties, mechanisms of action, drug interactions, and adverse effects of these agents such that consumers and health care providers will have the information available to maximize therapeutic benefits of botanicals while minimizing the likelihood of unwanted effects.
Commercial promotion of botanicals usually emphasizes that they are "safe," "natural," and contain "no chemicals." In fact, plant systems have their own metabolic processes, and human evolution has created processes to biotransform and eliminate ingested plants. As such, plants can induce, inhibit, or be toxic to human metabolic systems, and seemingly safe and natural plant products may have predictable influences on human drug metabolism. Of particular concern are the increasing numbers of clinical and scientific reports of drug interactions involving botanical products (8–19). Some of these interactions, for example, those involving St. John's wort, are of major clinical importance. Mechanisms investigated to date include the possibility that botanical medicines may induce or inhibit the activity of human Cytochrome P450 enzymes or the activity of transport proteins such as P-glycoprotein (20). Because the number of botanical medicines in clinical use is very large, it is simply not feasible to conduct clinical studies for all possible drug interactions that need to be studied and understood. Accordingly, there are now large gaps in knowledge, and recommendations regarding which drug combinations with botanicals are safe or unsafe are often based on incomplete data.
An extensive literature supports the existence of the therapeutically beneficial effects of garlic preparations in the prevention of atherogenesis and neoplastic disease (21–31). A number of components in garlic are postulated to act synergistically to provide these health benefits (32–39). Due to the complex chemistry of garlic, variations in processing yield quite different preparations. Highly unstable thiosulfinates, such as allicin, disappear during processing and are quickly and extensively transformed (34). Efficacy and safety are also contingent upon processing methods. The process of extraction has been assumed to increase the potency and bioavailability of various crude herbs and eliminate their harsh and toxic characteristics. The irritating, acidic, and oxidizing compounds in raw garlic can be eliminated and modified through the extraction process. In fact, in some cultures, garlic is soaked or extracted with alcohol, wine, milk, or vinegar before being used as a therapeutic agent. Many adverse reactions to garlic ingestion can be attributed to the oil-soluble constituents derived from allicin. The lipid-lowering effect attributed to oil-soluble sulfur compounds in hepatocytes may be due to cytotoxicity, as revealed by increased lactate dehydrogenase from cells exposed to various oil-soluble components. Acetone has been detected in the breath of subjects consuming allicin-derived oil-soluble compounds, further suggesting the cytotoxicity of such compounds. On the other hand, water-soluble sulfur compounds, though effective at reducing cholesterol, are not cytotoxic. Aged garlic extract contains a number of the water-soluble constituents, such as S-allyl-cysteine, that significantly reduce its toxicity, as confirmed by various toxicological studies together with (32).
Clinical studies evaluating drug interactions with garlic preparations are limited (40–42). This is not surprising, since design and execution of pharmacokinetic drug interaction studies in humans are costly and time consuming. Recently, in vitro systems, using human liver microsomal preparations, have been increasingly utilized as approaches to screening for drug interactions that may be probable, possible, or unlikely (43–47). Data from these in vitro models can be utilized as a guide for targeting of clinical resources such that the most important research priorities are addressed.
The present study utilized the in vitro model to screen for potential inhibitory metabolic effects of a number of components of aged garlic extract.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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Microsomes were prepared by ultracentrifugation; microsomal pellets were suspended in 0.1 mmol/L potassium phosphate buffer containing 20% glycerol and stored at –80°C until use.
Incubation mixtures contained 50 mmol/L phosphate buffer, 5 mmol/L Mg++, 0.5 mmol/L NADP+, and an isocitrate/isocitric dehydrogenase regenerating system. Appropriate substrates (Table 1), with and without an inhibitor in methanol solution, were added to a series of incubation tubes. The solvent was evaporated to dryness at 40°C under conditions of mild vacuum. Reactions were initiated by addition of microsomal protein. After an appropriate incubation duration at 37°C, reactions were stopped by cooling on ice and addition of 100 µL of acetonitrile. Internal standard was added, the incubation mixture centrifuged, and the supernatant transferred to an autosampling vial for HPLC analysis.
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-OH-triazolam and 4-OH-triazolam. Pure samples of water-soluble components of aged garlic extract (Table 2) were donated by Dr. Harunobu Amagase of Wakunaga of America. Solutions were prepared in methanol. Inhibitory effects of 100 µmol/L concentrations of each component were evaluated in each of the in vitro systems. For studies of CYP3A activity using triazolam as the substrate, incubations were performed both without and with preincubation of inhibitor with triazolam. This is done to evaluate the possibility of irreversible or "mechanism-based" inhibition (58–60).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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In only 2 instances, inhibition of CYP3A exceeded 50%. S-allyl-L-cysteine, an important component in terms of biologic effects of aged garlic extract, reduced CYP3A activity to 40% of the control (Fig. 1). An evaluation of the concentration dependence of the inhibitory action did not demonstrate clear evidence of classical concentration effect. Furthermore, inhibition of the 2 parallel pathways of triazolam hydroxylation (-OH- and 4-OH-triazolam formation) revealed differential inhibition of the 2 pathways (Fig. 2). There was no evidence that the character of inhibition was "mechanism based."
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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The present study indicates that water-soluble garlic components are highly unlikely to inhibit activity of the 6 human cytochrome P450 isoforms responsible for the majority of drug metabolism reactions. The in vitro screen did reveal the possibility of modest inhibition of CYP3A by S-methyl-L-cysteine and S-allyl-L-cysteine. Although available data (41) provides no evidence that garlic inhibits CYP3A in vivo, the possibility could be confirmed or ruled out through a straightforward clinical drug-interaction study involving a suitable CYP3A substrate such as midazolam (67).
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FOOTNOTES |
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2 Author disclosure: No relationships to disclose.
3 This work was supported in part by grants AT-01381, AI-55412, MH-58435, DA-13209, DK/AI-58496, DA-13834, AG-17880, AI-58784, and RR-00054 from the U.S. Department of Health and Human Services.
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LITERATURE CITED |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONLITERATURE CITED |
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