**1. Introduction**

Mustard has been known as one of the oldest condiments ever and has been considered as one of the most widely grown and multifunctional plant in the world for thousands of years. The first known cultivars and use of mustard plants dating back to 3000 B.C. [1]. The following section gives a short overview about mustard plants, their taxonomic classification and the economic relevance of mustard seeds.

Mustard plants belong to the commonly known mustard family Brassicaceae of the order Brassicales (previously denoted as Capparales) including over 330 genera and over 3700 species that are distributed worldwide [2]. Notable characteristics of this family are the four sepals in median position of the flowers followed by four alternating petals that are arranged in a crossform referring to the old family name Cruciferae. The presence of organosulphur compounds is also a unique characteristic of this plant family.

The most cultivated varieties within this family almost all belong to the six species *Brassica rapa*, *Brassica juncea*, *Brassica nigra*, *Brassica carinata*, *Brassica oleracea* and *Brassica napus* in which also two of three of the most important mustard species are included (*B. juncea* and *B. nigra*). Based on cytogenetic and hybridization experiments of the Korean botanist U Nagaharu (1935), a well-known model named "U's triangle" have demonstrated the genetic relationship among these different species. The triangle is based on the theory that the genomes of three ancestral diploid species of Brassica combined to give rise to three common tetraploid vegetables and oilseed crop species. Figure 1 illustrates the triangle and shows the main forms *B. rapa*, *B. nigra* and *B. oleracea* with their own elementary genome A, B and C. The three amphidiploid species *B. juncea* (AB), *B. napus* (AC) and *B. carinata* (BC) are natural hybrids of the diploids. A genetic relationship exists also between *B. nigra* and the genera *Sinapis* L. in which the species *Sinapis alba,* belonging to one of the important mustard species*,* is included [1,3–5].

**Figure 1.** Taxa, genomes, chromosome number (n) and genetic relationship among Brassica species represented by U's triangle. The most cultivated mustard species that are subject of this article are framed in black.

Amongst the approximately 40 species of mustard plants in the Brassicaceae family, three types, derived from the genera *Sinapis* and *Brassica*, are mainly commercially cultivated throughout the world and used to make the mustard condiment.

The genus *Sinapis* L. contains several different types of mustard species, including *Sinapis alba* (L.) (white mustard, syn. *B. hirta* Moench or *B. alba* Linn) or *Sinapis arvensis* (charlock mustard). As mentioned above the cultivated form *S. alba* L. is phylogenetically close to Brassica species and occasionally referred to as *Brassica alba* or *B. hirta.* The seeds of *S. alba* are light straw yellow-colored and are slightly larger than the seeds of *B. nigra* and *juncea*. White mustard), believed to be native to the Mediterranean region, is the most used mustard species in Europe [1,6]. In warm regions, the mustard plant is primarily cultivated for its seeds, as a spice and for its high oil content, whilst in cold regions it is grown as catch crop, green fodder crop or as green manure. Varieties of mustard blends made from white mustard are available on the market. For example the American ballpark-style mustard is made from the white seeds, blended with vinegar and spices and usually coloured with turmeric [7]. Characteristic of the white mustard seeds is the glucosinolate sinalbin, which is enzymatically hydrolysed to 4-hydroxybenzyl isothiocyanate contributing significantly to the irritating odour and the sharp flavour of the mustard. However, white mustard is milder in taste than other mustard species. The seeds are often used for pickling gherkins or mixed pickles or making sausages [7]. Several varieties of yellow mustard exist with different qualities such as earlier maturing, nematode-resistance or altered content of glucosinolates or erucic acid. According to the catalogue of varieties of agricultural plant species more than 100 varieties of *S. alba* L. are listed [8].

*Brassica nigra* (L.) W.D.J. Koch (black mustard, syn.: *S. nigra, B. sinapioides*) is widely cultivated for its blackish brown-red seeds which are slightly bitter and more pungent than the seeds of the white (*S. alba)* or brown (*B. juncea*) mustard. Sinigrin is the major glucosinolate in the seeds of black mustard and can be hydrolysed to allyl-isothiocyanate (AITC) giving the characteristic of a pungent irritating odour [9]. Black mustard is commonly used as spice, potherb and as source of oil, however in the Unites States (U.S.) and Europe difficulties in harvesting reduce its popularity [7]. Likewise, the plant plays an important role in traditional medicine since eternal times, either used internally or externally. Black mustard oil is also utilized for production of soap and for medicinal remedies [10]. When

black mustard seeds are grounded to meal and mixed with vinegar, a pungent condiment occurs. Among the different mustard varieties, the traditional Dijon mustard originated in the city of Dijon in France, is very popular and made from black or brown mustard seeds blended with wine (vinegar) and/or verjuice and seasonings. If vinegar is added to a ground mixture of seeds of black and white mustard a milder blend is produced (e.g., German mustard) [7].

Plants of *Brassica juncea* (L.) Czern. and Coss. (brown mustard, syn. *B. integrifolia*) are grown worldwide, in North America and Europe mainly for the seeds to be used as condiment, in the Indian subcontinent for oil production, and in Asiatic countries like China and Japan the mustard plant is used as a root and leaf vegetable [1,6]. *B. juncea* plants are shorter and have larger seeds than its relative *B. nigra. B. juncea* is more suitable for its agricultural cultivation and mechanical harvesting than *B. nigra* due to its higher growth and its pods, which are less prone to bursting. Therefore, brown mustard enables less labour-intensive and cost-effective production and increasingly replaces cultivation of black mustard [9]. *B. juncea* is a hybrid form derived from interspecific crosses between *B. nigra* and *B. rapa* giving it the characteristics of rapid growth from *B. rapa* and the mustard oil of *B. nigra*. Several subspecies of *B. juncea* exist with different morphologies and characteristics. Besides the oilseed types, vegetable and root type forms are cultivated for its edible leaves, inflorescences, stems and roots [1,11]. Due to its close relative to *B. napus* (oilseed rape, canola) a canola variety known as *Brassica juncea var. juncea canola* was developed from *B. juncea* resulting in low erucic acid and glucosinolate content and comparable oil and meal quality as canola (rapeseed) species [12,13]. The most important constituent in brown mustard is the natural glucosinolate sinigrin which on hydrolysis yields up to 1.4% allyl-isothiocyanate, known as volatile oil responsible for the pungent taste of brown mustard. *B. juncea* is also used as fodder and in traditional medicine, e.g., as diuretic or stimulant [10].

The world's largest producer of mustard seeds in the last years was Nepal, accounting for more than 32% of the global production in 2019, followed by Russia with 25% and Canada with 21%. In the recent past, the largest importers of mustard seeds across the globe were the U.S. and Germany, followed by France, however the import values strongly vary depending on the different mustard species intended to be market. In this case, North America is the major market for yellow mustard used for the condiment industry, whereas Europe is important for brown mustard for use as a condiment, often in the form of specialty mustards. The Asia–Pacific region is the major market for brown mustard used as spicy cooking oil and condiments [14]. According to data provided by FAOSTAT (Food and Agriculture Organization of the United Nations), the countries UK (221 K tonnes), Germany (202 K tonnes) and Italy (190 K tonnes) had the highest volumes of prepared mustard consumption in 2019, together comprising 44% of total consumption [15]. In 2019, the average consumption of mustard per capita in Germany was around 900 g [16]. So far, the average daily consumption of mustard might be in the range from 1 to 2 g and it is very common to consume around 10–20 g mustard (as condiment) during a meal.

Mustard plants, especially the seeds, are used as or in food in many different forms and due to different functional properties. Processed foods are often mixed with natural mustard seeds, such as in pickled gherkins or small white onions. Mustard is also used as an ingredient in many ready-cooked dishes like crackers, appetizers, various flours and dehydrated products for soups. Moreover, various spicy sauces, vinaigrettes and mayonnaises often contain mustard condiment. Mustard may even be present in baby food. The risk of contamination is high, e.g., in fast food restaurants or snack stands where several products with mustard are handled [9,10].

To put it briefly, the seeds of mustard plants are characterized by various nutritional and functional properties and the use in the food and beverage industry is constantly growing. As mustard plants contain several bioactive compounds, the purpose of this review is to present a comprehensive overview about potentially harmful substances in the seeds from a toxicological perspective and to evaluate possible risks to human health.

#### **2. Biologically Active Compounds in Mustard Seeds**

Long ago, mustard was considered as medicinal plant rather than as culinary one pointing out the existence of biologically active compounds in the plants and their seeds. In general, these so-called "bioactive compounds" are typically present in various naturalbased sources characterized by specific functionalities. In recent years, the research interest in these phytochemicals increased exponentially in various commercial industries. Moreover, scientific and (bio)technologic developments have led to immense nutritional discoveries and product developments resulting in increasing numbers of food products with potential medical and health benefits. Due to its broad use as nutraceutical or functional constituents for several health purposes, biologically active compounds have entered the market in many different forms (e.g., as food supplements), partially without reliable scientific basis with respect to efficacy or safety [17,18].

The growing consumer interest towards different taste preferences and healthy eating habits increases the use of mustard seeds and products made thereof. Due to its different functional and technological properties, mustard is gradually utilized in a wide-range by the food and beverage industry as well as in the cosmetic and pharmaceutical industry Moreover, the use of mustard plants has gained increasing interest for several non-food uses. For instance, seed meal of yellow mustard (*S. alba*) was shown to be efficient in controlling weeds, and oriental mustard (*B. juncea*) seed meal has been used as a broad-spectrum pesticide to control nematodes, insects, and fungi [19,20]. Current and future studies will undoubtedly focus on the health benefits of mustard plants and seeds whereas information on undesirable and antinutritional compounds in these products intended for human consumption will tend to fade into the background. Therefore, this article underscores and evaluates those compounds in mustard seeds potentially exerting undesired effects in humans.
