Herbal bioactivation: The good, the bad and the ugly

Pulegone toxicity

Pulegone is a monoterpene ketone present in essential oils from many mint species (e.g. Hedeoma pulegoides and Mentha pulegium, both called pennyroyal) Budavari, 1996, Grundschober, 1979. Pennyroyal oil has been used as a flavoring agent in foods and beverages, a fragrant constituent and a flea repellent (Hall and Oser, 1965). Pennyroyal has also been used to induce menstruation and abortion. However, pennyroyal oil, especially at high doses, has been reported to cause hepatic failure, central nervous system toxicity, gastritis, renal and pulmonary toxicity, and death (Anderson et al., 1996). Pulegone, constituting >80% of the terpenes in pennyroyal oils, was found to be hepatotoxic and pneumotoxic in mice (Gordon et al., 1982).

The metabolites of pulegone have been considered to be responsible for its toxicity (Gordon et al., 1987). Both menthofuran and p-cresol were potential candidates. Menthofuran is a naturally occurring hepatotoxin (Thomassen et al., 1992) and p-cresol is a known glutathione depletory and toxin (Thompson et al., 1994). In vitro studies have demonstrated CYP-mediated conversion of pulegone to menthofuran which was then bioactivated to reactive γ-ketoenal Gordon et al., 1987, Thomassen et al., 1991. γ-Ketoenal was capable of covalently binding to cellular proteins and caused hepatic injury (Thomassen et al., 1992). The γ-ketoenal can be converted to mintlactones, as well as other reactive intermediates in CYP-mediated oxidation of menthofuran.

The in vivo metabolism of pulegone is very complicated, with about 14 Phase I and 10 Phase II metabolites being identified and characterized in rats Chen et al., 2001, Madyastha and Raj, 1993, McClanahan et al., 1989, Moorthy et al., 1989, Thomassen et al., 1991. These metabolites accounted for only 3% of total radioactivity excreted in bile, and their structures could not be established solely by mass spectral analysis. The metabolism of pulegone in rats involves three major metabolic pathways: a) conjugation by glucuronic acid following C-5 or methyl group hydroxylation or by GSH; with the conjugates being further metabolized; b) the formation of menthofuran; and c) the formation of piperitenone beginning with 5-hydroxylation followed by dehydration (Fig. 2) Chen et al., 2001, Madyastha and Gaikwad, 1999, Madyastha and Raj, 1992, Madyastha and Raj, 1993. The CYP system plays an important role in the metabolism of R-(+)-pulegone Madyastha and Gaikwad, 1999, Madyastha and Raj, 1992. Most of the metabolites of pulegone are derived from menthofuran and piperitenone Madyastha and Gaikwad, 1999, Madyastha and Raj, 1992, Madyastha and Raj, 1993. Studies carried out in vivo have shown 4-methyl-2-cyclohexenone as one of the metabolites of R-(+)-pulegone (Madyastha and Raj, 1993), and conversion of 4-methyl-2-cyclohexenone to p-cresol has been demonstrated in vitro Madyastha and Raj, 1990, Madyastha and Raj, 1993, Madyastha and Raj, 2002.

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Fig. 2

Proposed bioactivation of pulegone.

Capsaicin

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a major pungent phenolic constituent present in a variety of capsicum fruits such as hot peppers, and thus represents an important ingredient of the majority of spicy foods Park et al., 1998, Surh and Lee, 1995. It has shown a wide range of pharmacological properties, including pain-killing, antigenotoxic, antimutagenic, and anticarcinogenic effects Surh et al., 1998, Surh and Lee, 1995. Capsaicin has been used to treat various peripheral painful conditions such as rheumatoid arthritis and diabetic neuropathy (Surh and Lee, 1995).However, capsaicin has been reported to be a tumor promoter, carcinogen and potential mutagen Agarwal et al., 1986, Lopez-Carillo et al., 1994, Toth and Gannett, 1992, Ward and Lopez-Carrillo, 1999. It caused strand scission in calf thymus and plasmid DNA in the presence of Cu(II). This breakage is mediated by reactive oxygen species arising from capsaicin, especially the hydroxyl radical Singh et al., 2001a, Singh et al., 2001b.

Capsaicin undergoes bioactivation by CYP2E1 to reactive species (Gannett et al., 1990). The major bioactivation pathways include: a) epoxidation of the vanillyl ring moiety to produce an arene oxide; b) one-electron oxidation of the ring hydroxyl group to a phenoxy radical; and c) O-demethylation at the aromatic ring and subsequent oxidation of the resulting catechol metabolite to semiquinone and quinone derivatives (Fig. 4) . The resultant reactive species are capable of binding covalently to the active site of CYP2E1 as well as DNA (Surh and Lee, 1995). The interaction with target cell DNA would trigger mutagenicity and malignant transformation. However, metabolism of capsaicinoids by CYPs may also represent a detoxification process (in contrast to bioactivation), resulting in a reduction in cytotoxicity (Reilly et al., 2003). For example, a novel dehydrogenation metabolic pathway of capsaicin yields macrocyclic, diene and imide metabolites (Reilly et al., 2003).

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Fig. 4

Proposed bioactivation of capsaicin.

Furanocoumarins (bergamottin and 8-methoxypsoralen)

Bergamottin is a major furanocoumarin in grapefruit juice. It reversibly inhibited the activities of CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4 in human liver microsomes (He et al., 1998). It also inactivated CYP3A4 following metabolic activation in a time- and concentration-dependent manner, with k_inact and K_I values of 0.3 min⁻¹ and 7.7 μM, respectively (He et al., 1998). The loss of catalytic activity exhibited pseudo-first-order kinetics. During bergamottin-induced inactivation, CYP3A4 retained more than 90% of the heme, but 50% of the apoprotein in the inactivated CYP3A4 could not be recovered. This suggests that the inactivation may involve apoprotein modification in the active site of the enzyme instead of heme adduct formation or heme fragmentation (He et al., 1998). The reactive metabolites of bergamottin remain unknown, but it may undergo oxidation to form a reactive furanoepoxide that covalently binds to CYP3A4 (Fig. 5) .

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Fig. 5

Proposed bioactivation of bergamottin.

8-Methoxypsoralen is a natural furanocoumarin rich in many foodstuffs such as parsnips and parsley. It is used in combination with long wavelength ultraviolet light to treat psoriasis, vitiligo and T cell lymphoma Anderson and Voorhees, 1980, Edelson et al., 1987, Parrish et al., 1974. The usefulness of 8-methoxypsoralen in treating these diseases resides in its ability to be photoactivated to a species capable of binding covalently to nucleic acids and lymphocytes, and thus inhibiting DNA synthesis and cellular proliferation. Early studies indicated that 8-methoxypsoralen was a potent mechanism-based inhibitor of CYPs in rodents Fouin-Fortunet et al., 1986, Labbe et al., 1989, Letteron et al., 1986, Mays et al., 1990 and humans (Tinel et al., 1987). Further studies found that 8-methoxypsoralen was a potent mechanism-based inhibitor of CYP2A6, with K_I of 0.8–1.9 μM and k_inact of 1–2 min⁻¹ in a reconstituted and native liver microsomes Koenigs et al., 1997, Koenigs and Trager, 1998a, Koenigs and Trager, 1998b, Maenpaa et al., 1993. 8-Methoxypsoralen was also a potent inactivator of CYP2B1, with a K_I of 2.9 μM and k_inact of 0.34 min⁻¹ Koenigs and Trager, 1998a, Koenigs and Trager, 1998b. However, the reactive species of 8-methoxypsoralen remains unknown, but it is suggested that 8-methoxypsoralen as a furanocoumarin undergoes CYP2A6/2B1-mediated oxidation to form a furanoepoxide (Fig. 6) . The resultant epoxide is then converted to dihydrofuranocoumarin products following hydrolytic attack or direct attack by exogenous nucleophiles Koenigs and Trager, 1998a, Koenigs and Trager, 1998b.

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Fig. 6

Proposed bioactivation of 8-methoxypsoralen.

Glabridin

The dried roots of the licorice (Glycyrrhiza glabra) have been consumed for the past 6000 years and are used as flavoring and sweating agents, as demulcents and expectorants in the Western world and as antiallergic and anti inflammatory agents in Asian countries including Japan and China (Chandler, 1985). Licorice contains glycyrrhizin (glycyrrhizic acid, a glycoside which is 50 times sweeter than sugar), oleane triterpenoids, glucose, ammonia, polyphenols, flavonoids, and sucrose Hatano et al., 1991a, Hatano et al., 1991b. Flavanoid components of licorice root have been shown to have antitumorigenic, antimicrobial, antiviral, anti inflammatory, estrogen-like, and antioxidative activity Fujisawa et al., 2000, Fukai et al., 2002, Shibata, 2000, Tamir et al., 2001. Licorice root extract, as well as its major flavanoid, the isoflavan glabridin, are potent antioxidants against LDL oxidation in mice and humans (Rosenblat et al., 1999). Glabridin was also shown to inhibit the activity of macrophage NADPH-oxidase presumably by inhibiting protein kinase C and serotonin re-uptake Ofir et al., 2003, Rosenblat et al., 1999. In addition, glycyrrhizin was recently found to be a potent inhibitor of the SARS-associated coronavirus in vitro (Cinatl et al., 2003). However, licorice may cause hypermineralocorticoidism (Nobata et al., 2001), arrhythmia (Bocker and Breithardt, 1991), pseudoaldosteronism (Ferrari et al., 2001), and hypertension (Astrup, 2001). These toxicities have been ascribed to the inhibitory activity of glycyrrhizin and glycyrrhetic acid from licorice on 11-hydroxy-steroid dehydrogenase (Ferrari et al., 2001).

An in vitro study indicated that glabridin inactivated CYP3A4 and 2B6 in a time- and concentration-dependent manner, but CYP2C9 was competitively inhibited (Kent et al., 2002). Metabolism of glabridin by CYP3A4 resulted in the destruction of the heme moiety, as the loss in activity with different concentrations of glabridin correlated well with the loss in the CYP3A4-reduced carbon oxide spectrum and detectable heme at 405 nm (Kent et al., 2002). No other peaks indicative of glabridin-alkylated heme eluting after the native heme were detected at 405 nm. Presumably glabridin undergoes metabolism, forming reactive intermediates causing heme fragmentation. Incubations with 2,4-dimethylglabridin did not lead to a loss in the enzymatic activity of CYP3A4. Incubation of glabridin with CYP3A4 and NADPH resulted in a metabolites, suggesting that the two hydroxyl groups on the 2′ and 4′ position of the flavonoid B ring of glabridin are required for CYP3A4 inactivation (Fig. 7) (Kent et al., 2002). Indeed, these two hydroxyl groups are also believed to be essential for its antioxidative activity (Belinky et al., 1998).

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Fig. 7

Chemical structure of glabridin.

Isothiocyanates

Isothiocyanates are released upon chewing or maceration of cruciferous vegetables, such as cabbage, cauliflower, and broccoli where they occur as thioglucoside conjugates called glucosinolates (Fenwick et al., 1983). The enzyme myrosinase is released at the same time and hydrolyzes the glucosinolate with rearrangement of intermediates producing the isothiocyanates, hydrogen sulfate, and glucose as the major products. Typical isothiocyanates include benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC). These compounds have been shown to be potent modulators of CYPs in vitro and in vivo.

Isothiocyanates inhibited CYP1A1, 1A2, 2A1, 2B1, and 2E1 which are involved in carcinogen activation (Yang et al., 1994). BITC and PEITC have been shown to inhibit N-nitrosodimethylamine demethylation activity. BITC is a potent mechanism-based inactivator of rat CYP1A1, 1A2, 2B1, and 2E1, as well as human CYP2B6 and 2D6 Goosen et al., 2000, Moreno et al., 1999. The K_I for CYP1A1, 1A2, 2B1, and 2E1 were 35, 28, 16, and 18 μM, respectively; and the corresponding k_inact values were 0.26, 0.09, 0.18, and 0.05 min⁻¹, respectively Goosen et al., 2001, Moreno et al., 1999. Human CYP2C9 and rat CYP3A2 were not inactivated by BITC. BITC was also a potent inactivator of CYP2B1, as indicated by a partition ratio of approximately 11:1 (Goosen et al., 2001).

The mechanism of CYP inhibition by isothiocyanates involves both competitive and mechanims-based inhibition. The competitive inhibition involves competition with substrates at the active site of CYP Ishizaki et al., 1990, Smith et al., 1993, while mechanism-based inhibition would involve the metabolic activation of isothiocyanates Goosen et al., 2000, Ishizaki et al., 1990, Lee, 1996, Moreno et al., 1999, Smith et al., 1996. The metabolic activation of isothiocyanates results in reactive intermediates that could ultimately bind to the heme moiety or apoprotein, thereby inactivating the enzyme. For example, BITC was metabolized to a reactive benzyl isocyanate intermediate that is capable of binding covalently to CYP apoprotein or hydrolyzed to form benzylamine (Fig. 8) (Goosen et al., 2001). BITC may be oxidized at the α-carbon to benzaldehyde, instead of being desulfurated to benzyl isocyanate. Alternatively, benzylamine could be deaminated to give benzaldehyde and ammonia. The subsequent oxidation of benzaldehyde would yield benzoic acid. The formation of benzoic acid was also observed in dogs, where administration of BITC resulted in the excretion of hippuric acid, the glycine conjugate of benzoic acid (Brüsewitz et al., 1997). In humans and rats, BITC is metabolized through conjugation with GSH and finally excreted as mercapturic acid. In addition, oxidative desulfuration of the isothiocyanate could also release elemental sulfur, which could further contribute to enzymatic modification and possibly inactivation, as in the case of parathion (Butler and Murray, 1993).

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Fig. 8

Proposed bioactivation of benzyl isothiocyanate (BITC).

The in vivo administration of isothiocyanates to rodents also modulate CYP content and activity Guo et al., 1992, Smith et al., 1993. The effects appeared to depend on the experimental conditions, the isothiocyanate used and treatment regimen, the target tissue, and the specific CYP evaluated (Zhang and Talalay, 1994). For example, acute administration of PEITC to rats decreased liver CYP2E1 activity, whereas CYP2B1 activity and content increased approximately 10-fold, but without any significant effect on lung CYP1A2 and 2B1 activities (Guo et al., 1992). However, chronic administration of PEITC induced both CYP2B1 and 2E1 with related increases in carcinogenesis (Smith et al., 1993).

Kavalactones (methysticin and dihydromethysticin)

Kava (Piper methysticum, found in Polynesia, Melanesia, and Micronesia) is an effective herbal medicine for the therapy of anxiety and insomnia Pittler and Ernst, 2000, Rouse, 1998, Singh and Singh, 2002, Volz and Kieser, 1997. Clinical studies have shown that kava and kavalactones are effective in the treatment of anxiety at subclinical and clinical levels, anxiety associated with menopause and various other medical conditions Bilia et al., 2002, Pittler and Ernst, 2000, Stevinson et al., 2002, Volz and Kieser, 1997, Wheatley, 2001 The major constituents of kava are pharmacologically active kavalactones which are responsible for about 95% of the total activity of kava (Singh and Singh, 2002). Yangonin, desmethoxyyangonin, methysticin, 7,8-dihydromethysticin, kawain, and 7,8-dihydrokawain are present in the highest levels of kavalactones, accounting for approximately 96% of lipidic extracts (Lebot and Lévesque, 1989).

Both methysticin and dihydromethysticin (Fig. 9) were mechanism-based inhibitors of CYPs, as indicated by the unidentified metabolic intermediate complexes at 455 nm when incubation with human liver microsomes (Mathews et al., 2002). This 455-nm absorbing complex was both NADPH- and time-dependent, with absorption increasing with increasing incubation time. Methysticin analogs in kava extract have been shown to inhibit multiple CYPs after metabolic activation through the formation of metabolic intermediate complexes Murray and Reidy, 1989, Murray et al., 1983. Preincubation of whole kava extract with human liver microsomes inhibited CYP1A2, 2C9, 2C19, 2D6, 3A4, and 4A9/11; whereas CYP2A6, 2C8, and 2E1 were unaffected (Mathews et al., 2002). Various kavalactones inhibited different CYPs. Kava extract and kavalactones inhibited CYP3A4 in cDNA-expressed microsomes (Unger et al., 2002).

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Fig. 9

Chemical structures of methysticin and dihydromethysticin from kava.

Oleuropein

The Mediterranean diet has been associated with longevity and low rates of coronary artery disease and cancers despite its high dietary fat content Braga et al., 1998, Owen et al., 2000, Roche et al., 1998. The Mediterranean diet includes the consumption of large amounts of olive oil. Olive oil is a source of at least 30 phenolic compounds (total: 60–1000 mg/kg) (Tuck and Hayball, 2002). The beneficial effect of olive oil has been mainly attributable to oleuropein and its two major metabolites, hydroxytyrosol and tyrosol. Oleuropein is the major phenolic compound in olive fruit, which can be as much as 14% in dried fruit. It is an ester consisting of hydroxytyrosol and elenolic acid which are potent antioxidant agents (Briante et al., 2001).

The phenolic components in olive oil are believed to play a vital role in the prevention of coronary artery disease and atherosclerosis. These compounds, in particular hydroxytyrosol, are potent antioxidants and radical scavengers Tuck and Hayball, 2002, Visiol and Galli, 2002, Visioli and Galli, 1994, Visioli et al., 2002. They exert protective effects on cardiovascular system mainly by inhibiting platelet aggregation and endothelial activation (Carluccio et al., 2003), modulating arachidonic acid metabolism (Kohyama et al., 1997) and preventing the oxidation of low density lipids Visiol and Galli, 2002, Visioli et al., 1995, Visioli et al., 2002. It is presumed that hydroxytyrosol as water and lipid soluble molecule can penetrate in cell membranes and thus inhibit the production of leukotriene B4 effectively from endogenous arachidonic acid (Kohyama et al., 1997).

In vitro studies indicated that oleuropein inactivated androstenedione 6β-hydroxylase (CYP3A4) activity in human liver microsomes, with k_inact and K_I of 0.09 min⁻¹ and 22.2 μM, respectively Stupans et al., 2001, Stupans et al., 2000. The reactive metabolites of oleuropein remain undetermined, but oleuropein might undergo CYP3A-mediated oxidation to unidentified metabolite(s) capable of binding and inactivating CYP3A4 (Fig. 10) . In addition, hydroxytyrosol and tyrosol and/or their metabolites may be involved. Metabolism studies have indicated that hydroxytyosol is excreted into the urine unchanged and as its glucuronide and sulfate conjugates (Fig. 10) Miro-Casas et al., 2003, Tsarbopoulos et al., 2003, Tuck and Hayball, 2002. Hydroxytyrosol can also be sequentially oxidized to 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylacetaldehyde by alcohol and aldehyde dehydrogenase, or methylated by catechol methyltransferase to homovanillic acid and then oxidized to homovanillic alcohol (D'Angelo et al., 2001).

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Fig. 10

Metabolism of oleuropein.

Organosulfur compounds

Garlic is frequently used as a flavor-enhancing ingredient in food preparation and healthy supplement in the practice of folk medicine. Organosulfur compounds are the major active components of garlic. Garlic also contains numerous flavonoids/isoflavonoids (such as nobiletin, quercetin, rutin, and tangeretin), polysaccharides, prostaglandins, saponins, and terpenes (such as citral, geraniol, linalool, α- and β-phellandrene) Dausch and Nixon, 1990, Singh et al., 2001a, Singh et al., 2001b. Alliin (S-allylcysteine sulfoxide) is one major component of garlic, which is converted by alliinase to allicin. Allicin is further transformed to other garlic compounds, including diallyl sulfide. Organosulfur compounds have been reported to have hypolipidemic, antiplatelet, immune-enhancing, anticancer, chemopreventive, hepatoprotective, antihypertension, and procirculatory effects Rahman, 2001, Spigelski and Jones, 2001.

Organosulfar compounds modulated CYPs in vitro and in vivo (Brady et al., 1991). For example, extracts of fresh garlic, and samples of garlic oil, freeze dried garlic, and aged garlic inhibited human CYP2C9, 2C19, 3A4, 3A5 and 3A7 activity, whereas the CYP2D6 activity was unaltered (Foster et al., 2001). The organosulfur compounds 4,4′-dipyridyl disulfide, di-n-propyl disulfide and diallyl sulfide were also potent competitive inhibitors of coumarin 7-hydroxylase (CYP2A6), with a K _i value of 0.06, 1.7 and 2.1 μM respectively (Fujita and Kamataki, 2001). In vivo studies in the mouse indicated that garlic administration increased CYP2E1 and 1A2 levels (Kishimoto et al., 1999), whereas the administration of garlic constituents in the rat decreased the CYP2E1 activity and/or protein level, but increased or did not alter the CYP1A, CYP2B and CYP3A activities Dalvi, 1992, Haber et al., 1995, Haber et al., 1994. A single dose of garlic oil in the rat resulted in a significant inhibition of hepatic CYP-catalyzed reactions including aminopyrine N-demethylase (CYP2C) and aniline hydroxylase (CYP2E1) activity, but administration of garlic for 5 days led to a significant increase in these hepatic CYP activities (Fitzsimmons and Collins, 1997). In addition, administration of garlic oil to healthy subjects for 28 days reduced CYP2E1 activity by 39% when chlorzoxazone was used as a probe compound (Gurley et al., 2002).

Diallyl sulfide is converted by CYP2E1 to diallyl sulfoxide and sequentially to diallyl sulfone (Fig. 11) (Teyssier et al., 1999). All these compounds are competitive inhibitors of CYP2E1 (Brady et al., 1991). A single oral dose of diallyl sulfone inactivated CYP2E1- and 1A-dependent activities in mouse liver microsomes (Lin et al., 1996). Diallyl sulfone is also a mechanism-based inhibitor of CYP2E1, involving the formation of a reactive metabolite which forms a CYP complex leading to autocatalytic destruction Brady et al., 1991, Jin and Baillie, 1997, Premdas et al., 2000. A recent study has demonstrated that diallyl sulfone was oxidized at one of the terminal double bonds, yielding the monoallyleopoxide that inactivated CYP2E1 (Fig. 11) (Premdas et al., 2000). This monoallyleopoxide was conjugated by GSH. It appeared that the reactive intermediate from diallyl sulfone might initially affect the heme and/or apoprotein.

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Fig. 11

Metabolism of diallyl sulfide by CYP2E1.

The authors appreciate the support by the National University of Singapore Academic Research Funds.