# NMR Analysis of Pulegone in Food Products
## 1 Introduction  
Pulegone, (_R_)-2-isopropylidene-5-methyl-cyclohexanone, is a monoterpene ketone that is a major component of mint flavor. It occurs naturally in the essential oils of various mint species, such as peppermint (_Mentha x pipertia_ L) and pennyroyal (_Mentha pulegium_ L) [1, 2, 3]. In addition to mint, pulegone is a component of the essential oils of buchu (_Agathosma betulina_) and muna (_Mithustachys mollis_ Griseb.) [4, 5, 6]. At 59.1%-77.9%, the pulegone content in the essential oil of pennyroyal is the highest among the mint species [1, 7, 8]. In peppermint, a proportion of pulegone in the oil of 1.2%-12.3% can be found [2, 9]. In spearmint (_Mentha spicata_ L.), the proportion of pulegone is in the range of 0.30%-29.59% [3]. In corn mint (_Mentha arrensis_ L.) oil, the pulegone content is 0.855-2.27% [10]. Horse mint (_Mentha longifolia_ L.) has a pulegone content of 0.17%-69.49% in the essential oil, lesser calamint (_Calaminthu nepeta_ L.) has a pulegone content of 2.4%-64.6%, apple mint (_Mentha suaveolens_ Ehrh.) has a pulegone content of 1.15-2.5%, and water mint has a pulegone content of 0.45% [8, 11, 12, 13]. In _Mentha haplocalyx_ Briq., the proportion of pulegone is 28.7%-80.2% [14]. In Germany, peppermint is one of the most popular types of infusion. In 2018, sales of peppermint tea in Germany totaled 5511 t [15]. Peppermint, corn mint, and spearmint are often used as flavoring in foods such as sweets and chewing gum [16]. In the kitchen,mint leaves are also used fresh or dried. In Great Britain and the USA, mint is used as a sauce and is served with savory dishes. In addition, spearmint is often used to prepare lamb dishes. Pennyroyal is also used in aromatherapy and as a spice [16]. [PERSON] et al. [10] examined various sweets, pastilles, and chewing gums with mint flavor for their pulegone content and found 0.45-29.91 mg/kg in confectionery, 1.23-14.29 mg/kg in pastilles, and 1.63-138.15 mg/kg in chewing gum. A pulegone content of 6.2 mg/kg was quantified for a mint-flavored chocolate brand [17]. A carcinogenicity study was conducted by the National Toxicology Program (NTP) in 2011. They classified the activity of pulegone in male rats as \"no evidence of carcinogenic activity\". Increased formation of urinary bladder papillomas was observed in female rats. This evidence was acknowledged by the NTP as \"clear evidence of carcinogenic activity\". An increased incidence of liver neoplasia was observed in male and female mice, with the formation of hepatoblasts in males and adenomas in both males and females [18]. The occurrence of this liver tumor was classified as \"clear evidence of carcinogenic activity\". Considering this evidence, the International Agency for Research on Cancer (IARC) has classified pulegone as possibly carcinogenic to humans (Group 2B). Furthermore, the use of pulegone as such as a flavoring agent in food is prohibited in the European Union in accordance with Regulation (EC) No. 1334/2008 Annex III [19]. Nevertheless, the possibility of pulegone occurrence in flavored foods cannot be entirely discounted, given that the use of mint flavoring is permitted. For this reason, Regulation (EC) No. 1334/2008 sets maximum levels for confectionery (250 mg/kg), very small breath-freshening confectionery (2000 mg/kg), chewing gum (350 mg/kg), nonalcoholic beverages (20 mg/kg), and alcoholic beverages (100 mg/kg) with mint or peppermint flavoring. The Joint FAO/WHO Expert Committee on Food Additives (JEFCFA) estimates the average intake of pulegone to be 2 ug/day/person or 0.04 ug/kg body weight (bw) in Europe [20]. The European Union recommends limiting peppermint oil consumption to a maximum of 1.2 mL per day. Given a maximum content of 32.97 mg of pulegone in 1.2 mL of peppermint oil, the daily intake of pulegone for a 60 kg individual is 0.55 mg/kg bw. The Committee of Experts on Flavouring Substances has established a tolerable daily intake (TDI) of 0.1 mg/kg bw for pulegone [21].