3.1.3. Metabolism of Elemicin

Elemicin is the natural continuation of methyleugenol, bearing two *meta*- and one *para*methoxy group relative to the allyl side chain. For this compound, the *O*-demethylation pathway becomes more prominent, which leads to some divergent metabolites, compared to methyleugenol. In 1980, Solheim and Scheline revealed that the two major metabolic pathways of elemicin in rats follow the cinnamoyl pathway and the epoxide-diol pathway [6]. The former route gives 3,4,5-trimethoxyphenyl-propionic acid and its glycine conjugate as major urinary metabolites, whereas 2- ,3- -dihydroxy-elemicin is the most prominent metabolite of the latter route. In addition, elemicin can also be 1- -hydroxylated at the allylic side chain. When comparing the kinetic constants for conversion of elemicin and 1- -hydroxy-elemicin by male rat liver and mixed gender pooled human liver fractions, van den Berg et al. concluded that glucuronidation of 1- -hydroxy-elemicin, representing a detoxification pathway, is the most important pathway in rats and in humans. In contrast, bioactivation of 1- -hydroxy-elemicin by sulfonation was suggested to be only a minor pathway in both rat and human liver [76].

In 2019, Wang et al. confirmed and extended these studies. They found a total of 22 metabolites for elemicin in mice, e.g., in urine, feces, and plasma [88]. In vivo, elemicin and most of its metabolites were mainly excreted in urine collected from 0 to 24 h post-procedure in metabolic cages of male C57BL/6 mice that were orally administered

100 mg/kg elemicin. The obtained results indicate that phase I metabolic reactions of elemicin included demethylation, hydroxylation, hydration, allyl rearrangement, reduction, hydroformylation, and carboxylation. Phase II metabolism of elemicin yielded several conjugates, e.g., with cysteine, *N*-acetyl cysteine, glucuronic acid, glycine, or taurine [88]. In addition, the 4-demethoxylated forms of elemicin and of 2- ,3- -dihydroxy-elemicin could be detected in human urine after nutmeg abuse [8].

#### 3.1.4. Metabolism of Safrole

From a toxicological point of view, safrole bioactivation by sequential 1- -hydroxylation and sulfonation, resulting in reactive sulfate esters capable of forming adducts with cellular nucleophiles such as DNA, is of high relevance [71,89]. In 1983, Boberg et al. identified 1- -sulfoxy-safrole as an ultimate electrophilic metabolite of safrole and as an initiator of hepatic carcinogenicity in vivo. The toxicological relevance of this pathway was demonstrated in mice co-treated with the hepatic SULT inhibitor pentachlorophenol (0.05% added to the diet of mice) in vivo and in mice being genetically defective with respect to the hepatic synthesis of PAPS [75].

However, work in rats, mice, and guinea pigs elucidated multiple metabolic pathways of safrole far beyond 1- -hydroxylation and sulfo conjugation. Upon intraperitoneal (i.p.) injection safrole is metabolized in rat and guinea pig by the epoxide-diol pathway and by cleavage of the methylenedioxy ring to form a catechol [98,99]. Since an allylic double bond is still present in the catechol and 1- -hydroxy-safrole, both metabolites can be further metabolized via epoxides to the corresponding dihydrodiols. A small amount of a triol 1- ,2- ,3- -trihydroxy-safrole was found in rat urine by Stillwell and colleagues [70]. Interestingly, 2- ,3- -epoxy-safrole apparently has sufficient stability in vivo to be absorbed from the peritoneal cavity to the circulatory system, and to persist even in urine. The major urinary metabolites identified by GC-MS were 4-allylcatechol, 1- -hydroxy-safrole, 2- ,3- -dihydroxy-safrole, 2- ,3- -dihydroxy-4-propyl-catechol, 2- -hydroxy-1- - (3,4-methylenedioxy-phenyl)-propanoic acid, and 3,4-methylenedioxy-benzoyl glycine [70] (Figure 2).

Urinary metabolites of safrole in the rat were also identified via GC-MS in a further study performed in 1982. Metabolite excretion was 93% within 72 h, and most of this material (86%) consisted of metabolites formed via demethylenation of the methylenedioxy moiety. The other metabolic routes observed were allylic hydroxylation and the epoxidediol pathway [79].

#### 3.1.5. Metabolism of Myristicin

Myristicin is well absorbed following oral exposure and is metabolized extensively.

Metabolism of the volatile alkenylbenzene myristicin results in the formation of less volatile metabolites, predominantly remaining in the aqueous phase on extraction with ether [99].

Early experiments highlighted the cleavage of the methylenedioxyphenyl moiety concomitant with CO2 release from myristicin as an important metabolic pathway. Within 48 h after oral administration of radiolabeled myristicin to male albino mice, 73% of the radiocarbon was set free as 14CO2 [98], which was potentially formed from the hydroxylation of the methylene group of myristicin and subsequent release and degradation of formate-14C. This demethylenation reaction was found to be catalyzed by microsomal CYPs and would yield the corresponding catechol derivative.

Later analytical studies in rat and human urine indeed revealed further water-soluble metabolites of myristicin, including the catecholic derivatives. In male Wistar rats that were administered myristicin once by oral gavage (100 mg/kg), different metabolites were identified in urine, including 1- -hydroxy-myristicin, 5-allyl-2,3-dihydroxy-1-methoxybenzene, 5-allyl-2-hydroxy-1,3-methoxy-benzene, 5-allyl-1-hydroxy-2,3-methylendioxybenzene, 5-(2- ,3- -dihydroxypropyl)-1-hydroxy-2,3-methylendioxy-benzene [8].

Incubation of myristicin in rat liver microsomes formed two major metabolites, 1- -hydroxy-myristicin and 5-allyl-1-methoxy-2,3-dihydroxy-benzene, bearing a catechol moiety [7]. Those metabolites were also identified in the above mentioned study by Beyer et al. in 2006 [8].

Isolation of metabolites from male Sprague–Dawley rat urine after a single oral administration of 100 mg/kg myristicin, and comparison before and after glucuronidase treatment, suggests that the catecholic metabolites 5-allyl-1-methoxy-2,3-dihydroxy-benzene and 1- -hydroxy-myristicin are also excreted in their respective conjugated forms [7].

Currently, no comprehensive studies with respect to quantitative metabolism and excretion of myristicin in humans are available. However, one study examined metabolites present in the urine of a patient who ingested five nutmeg seeds, resulting in an intoxication [8].
