Effects of short-term saffron (Crocus sativus L.) intake on the in vivo activities of xenobiotic metabolizing enzymes in healthy volunteers
Introduction
Crocus sativus L. (C. sativus) is a perennial plant of the Iridaceae family grown in regions around the Mediterranean, Iran, India and China. Saffron is a spice derived from the dried red style branches, stigmas, of C. sativus flower. It is used as food additive for flavoring and coloring and is one of the most expensive spices reflected by the labor intensiveness and costs associated with its production. Although the world's total annual production of saffron originates from Iran, the largest European saffron production is Greece (Schmidt et al., 2007) where its cultivation dates back as early as 16th century B.C. as documented by Minoan frescos brought to light in Santorini and Crete. Saffron consists of a complex mixture of volatile and non-volatile compounds. The main bioactive compounds of saffron, derived from the oxidative cleavage of the carotenoid zeaxanthine, are crocin and its aglycone crocetin, which are responsible for the colouring properties of saffron, and picrocrocin and its aglycone safranal which give saffron its bitter flavour and characteristic aroma, respectively (Fig. 1). Other constituents such as mineral agents, anthocyanins, flavonoids and kaempferol have also been reported (Tarantilis et al., 1995; Papandreou et al., 2006; Sánchez et al., 2008; Sobolev et al., 2014; Karkoula et al., 2018). Apart from its value as a food additive and dye material, saffron has been also used as an effective herbal medicine. Its constituents exhibit a spectrum of beneficial pharmacological effects (Alavizadeh and Hosseinzadeh, 2014) such as anti-nociceptive, anti-inflammatory and antioxidative effects (Assimopoulou et al., 2005; Papandreou et al., 2006, 2011), neuroprotective (Linardaki et al., 2013) and memory improving effects (Pitsikas and Sakellaridis, 2006; Pitsikas et al., 2007; Pitsikas, 2015) and has been evaluated as a potential agent for depression (Wang et al., 2010; Ghasemi et al., 2015) and cancer prevention (Tavakkol-Afshari et al., 2008; Mousavi et al., 2009; Zhang et al., 2013; Hoshyar and Mollaei, 2017; Khorasanchi et al., 2018).
The Cytochrome P450 (CYP) enzyme superfamily, present in the smooth endoplasmic reticulum of the liver and other extrahepatic tissues, is involved in the metabolism of endogenous compounds and xenobiotics such as drugs or environmental chemicals. Within the CYP superfamily, only isozymes belonging to the subfamily of CYP1, 2, and 3 are responsible for the biotransformation of most foreign substances including approximately 70% of all drugs in clinical practice (Pelkonen et al., 2008). CYP1A2, is the only hepatic member of the CYP1 family and it accounts for the 13% of the total hepatic content of cytochrome isoenzymes (Shimada et al., 1994). It is involved in the metabolism of drugs such as clozapine, olanzapine and theophylline (Pelkonen et al., 2008) as well as the metabolic activation of several procarcinogens, including aromatic and heterocyclic amines, nitroaromatic compounds, and mycotoxins (Pelkonen et al., 2008). The wide inter-subject variability observed in CYP1A2 activity has been attributed to factors such as gender, race and exposure to inducers or inhibitors, while the role of the genetic polymorphisms identified to date is questionable (Jiang et al., 2006; Pelkonen et al., 2008). Inter-individual variations in CYP1A2 activity may influence the therapeutic efficacy of some drugs and even the susceptibility to cancer risk (Le Marchand et al., 1997; Zhou et al., 2010).
CYP2A6 has a predominant role in the overall metabolism of nicotine and its metabolite cotinine as well as compounds that are of toxicological significance such as nitrosamines and aflatoxin B1 (Pelkonen et al., 2008; Di et al., 2009). Due to its low levels in human liver (∼4%) (Shimada et al., 1994) its contribution to overall drug metabolism is expected to be limited (Rendic and Di Carlo, 1997). CYP2A6 exhibits relatively large variability among individuals owed to genetic polymorphisms (López-Flores et al., 2017).
Xanthine oxidase (XO) is an interconvertible form of the enzyme xanthine oxidoreductase and it is present in the cytoplasm and on the outer surface of the cell membrane (Harrison, 2002). It is the rate-limiting enzyme in purine catabolism and can oxidize a variety of endogenous substrates such as aldehydes, purines, pyrimidines and pteridines; in addition, it catabolizes aminopurines, such as 2-aminopurine, heterocyclic compounds, such as 4-hydroxypyrimidine, retinol (Krenitsky et al., 1972; Battelli et al., 2014) and different xenobiotics, such as antiviral and anticancer agents, thus, contributing to liver detoxification (Pritsos, 2000; Battelli et al., 2014).
N-acetyltransferase-2 (NAT2) is one of the two closely related isoforms of the xenobiotic-metabolizing enzymes arylamine N-acetyltransferases involved in the N-acetylation of arylamines and the O-acetylation of N-hydroxylated heterocyclic amines (Dupret and Rodrigues-Lima, 2005). It is a cytosolic conjugating enzyme expressed mainly in the liver and gut (Sim et al., 2012) and it participates in the acetylation of several prescribed drugs, such as sulfamethazine, sulfapyridine, procainamide, dapsone, nitrazepam, hydralazine, clonazepam, and isoniazid (Evans, 1989) and in the metabolism of environmental carcinogens including aromatic and heterocyclic amines (Dupret and Rodrigues-Lima, 2005). It is a polymorphic enzyme with 107 alleles arising from the combination of 43 point mutations; these polymorphisms separate individuals into slow or rapid acetylators (Boukouvala and Fakis, 2005; Hein et al., 2018).
Caffeine is an almost universal component of the human diet either as a natural constituent or as a common additive in various food products. It is a drug with large consumption among humans, it is easily accessible, well-tolerated and is characterized by excellent oral bioavailability (Nehlig, 2018). More than 80% of caffeine is metabolized by CYP1A2 to 1,7-dimethylxanthine (17X) which is bio-transformed to 1,7-dimethyluric acid (17U) by CYP2A6 and to 1-methylxanthine (1X) by CYP1A2. 1X is subsequently converted to 1-methyluric acid (1U) by XO. A small part of 1,7-dimethylxanthine is metabolized to 5-acetylamino-6-formylamino-3-methyluracil (AFMU) by NAT2 (Fig. 2) (Gu et al., 1992; Kot and Daniel, 2008). Therefore, the overall metabolism of caffeine is mediated mostly by CYP1A2, CYP2A6, XO and NAT2. Subsequently, caffeine metabolic ratios determined in saliva and urine have long been used as safe and non-invasive methods for the simultaneous assessment of the in vivo activity of CYP1A2, CYP2A6, XO and NAT2 (Asprodini et al., 1998; Begas et al., 2007; Hakooz, 2009).
While drugs remain the cornerstone of medicine, the use of herbal products and supplements as a source of healthcare, either as alternatives or as complementary to approved medications, has increased tremendously in the Western world over the past few years (Bodeker et al., 2005; Bandaranayake, 2006; Ekor, 2014). Unlike prescription drugs, however, over-the-counter herbal products are often used without prior proof of safety or efficacy introducing considerable risk for adverse herbal-drug interactions, especially when administered with drugs with narrow therapeutic index (Zhou et al., 2003; Bent, 2008; Zhang et al., 2015). Herbal-drug interactions may involve altered pharmacokinetic properties of administered drugs through inhibition or induction of metabolic enzymes mediated by various natural phytochemicals contained in herbs (Zhou et al., 2003; Wanwimolruk and Prachayasittikul, 2014). A common example is hyperforin, the active ingredient in St. John's wort, which promotes the expression of CYP3A4 in the small intestine and liver through interaction with pregnane xenobiotic receptor (PXR) (Moore et al., 2000) ultimately leading to altered pharmacokinetics of many drugs (Awortwe et al., 2019) and subsequent herb-drug interactions (Hammerness et al., 2003). Knowledge of possible effects of herbs on xenobiotic metabolizing enzymes is, therefore, necessary for assessing and minimizing clinical risks related to drug-herb interactions.
Despite the wealth of clinical data on the effects of C. sativus on depression, anxiety and other mental disorders (Lopresti and Drummond, 2014; Christodoulou et al., 2015; Shafiee et al., 2018; Tóth et al., 2019), diabetes and cardiovascular risk factors (Hausenblas et al., 2015; Pourmasoumi et al., 2019) or sexual dysfunction (Leone et al., 2018) in the literature, no study has to date addressed possible interactions of saffron with xenobiotic metabolism enzymes in humans. In fact, data of the effect of saffron constituents on enzyme activity is limited to an experimental animal study showing significant modulation of the activity of CYP3A, CYP2C11, CYP2B, and CYP2A in rat liver microsomes (Dovrtělová et al., 2015). Therefore, the purpose of the present study was to examine the effect of C. sativus on the in vivo activity of the xenobiotic metabolizing enzymes CYP1A2, CYP2A6, XO, and NAT2 in healthy volunteers using caffeine as a probe-drug.
Section snippets
Subjects
Thirty-four non-smoking volunteers (twenty females and fourteen males) with mean age 38.79 ± 14.58 years (range 19–62), mean weight 70.63 ± 16.34 kg (range 52–130), mean height 1.70 ± 0.10m (range 1.57–1.90) and mean BMI 24.09 ± 3.66 (range 18.65–37.98) participated in the present study. Their health status was based on medical history, physical examination and recent routine blood tests. Volunteers did not have any history of medical illnesses (such as heart disease, inflammatory of autoimmune
Results
All 34 subjects completed the treatment period with no report of any adverse effects suggesting good tolerability of the C. sativus infusion intake. The metabolite ratios calculated for all volunteers reflecting the activity of CYP1A2, CYP2A6, XO, and NAT2 at baseline (free diet) and before and after C. sativus infusion intake under restricted diet, are presented in Table 1 and Fig. 4, Fig. 5.
CYP1A2 metabolite ratios in saliva exhibited a statistically significant 13.7% reduction (range −36.0
Discussion
Herbal products may alter the activity of xenobiotic metabolizing enzymes leading to potential pharmacokinetic herb-drug interactions and, subsequently, to important clinical consequences. Given the growing interest in the medicinal uses of C. sativus, its potential influence upon drug metabolizing enzymes becomes an important issue. The present study is the first to demonstrate that six-day consumption of C. sativus infusion decreases CYP1A2, but not CYP2A6, XO or NAT2, metabolic ratios in
Conclusions
C. sativus is increasingly used for its medicinal properties and has gained the interest of researchers and the public for its multiple health benefits. One important implication of the growing medicinal use of C. sativus is the possible interactions between its phytochemical content and the activity of enzymes involved in the biotransformation of clinically administered drugs. The present study is the first to address the effects of consumption of C. sativus infusion prepared from saffron of
Conflicts of interest
The authors declare that they have no conflict of interests.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was funded by the Research Committee of the University of Thessaly (Code No. 4822). The funding source had no involvement in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication. The authors wish to thank all volunteers who participated in the study. They are also grateful to M. Daviti and N. Govaris for their excellent assistance in the experimentation.
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2020, Environmental ResearchCitation Excerpt :CMRs were used as phenotypic indices for the assessment of the in vivo activity of the enzymes examined. CYP1A2, CYP2A6, XO and NAT2 activities were estimated by the ratios (AFMU+1U+1X)/17U (Campbell et al., 1987; Begas et al., 2007), 17U/(17U + 17X) (Grant et al., 1983; Begas et al., 2017), 1U/(1X+1U) (Kalow and Tang, 1991) and AFMU/(AFMU+1U+1X) (Rostami-Hodjegan et al., 1996; Begas et al., 2007, 2019; Asprodini et al., 2019), respectively. Caffeine metabolites 17X, 17U, and (1U) were purchased from Sigma (Steinheim, Germany); 1-methylxanthine (1X) was purchased from TCI-Europe (Zwijndrecht, Belgium); 5-Acetylamino-6-formylamino-3-methyluracil (AFMU) was kindly provided by Wolfgang Pfleiderer (University of Konstanz, Konstanz, Germany).
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