**1. Introduction**

Additive colorants are found in large quantities in food because consumers often associate them with the flavor, safety, and nutritional value of the foodstuff, thus making it more attractive [1]. The food industry uses synthetic food colorants to a large extent, mainly due to their low cost and high stability [2]; however, most of them contain azo functional groups and aromatic ring structures, which can be harmful to human health [3], with additional environmentally harmful effects during food processing [4]. Thus, an acceptable daily intake (ADI) of authorized food additives is continuously evaluated by regulatory bodies, such as the European Food Safety Authority (EFSA) in Europe and the

Food and Drug Administration (FDA) in the United States (US), to adequately protect consumers [5]. Even so, consumers remain cautious with regard to the safety of synthetic dyes, whereas they are also aware that many natural colorants provide health benefits; thus, the replacement of artificial food colorants with natural ones is a current market demand [2].

Naturally occurring color additives from vegetable and mineral sources were used to color foods, drugs, and cosmetics in ancient times. Paprika, turmeric, and saffron are some examples obtained from vegetables. From the second half of the 19th century onward, artificial colors rapidly replaced natural colorants. Artificial colorants are used in a wide variety of foods, mainly to make them more attractive to consumers; in fact, their intake by consumers has increased since 1950 [2]. Currently, consumers prefer natural colorants in foodstuff or for cookery since almost all of them are hypoallergenic and nontoxic, while displaying salutary properties in humans, which is a significant advantage over many artificial dyes. In addition, their production, use, and elimination is environmentally friendly and can contribute to sustainable development [6]. The current market trend involves the replacement of artificial dyes with natural ones [2].

The most important use of saffron (*Crocus sativus* L.) is in cookery; its dried stigmas constitute the saffron spice. This spice is greatly valued for its coloring, flavoring, and aromatizing properties in some traditional dishes, as well as in modern cuisine. In addition to its use as a spice, saffron has long been considered a medicinal plant for its therapeutic properties [7]. However, for years, the use of this spice as a food colorant, due to its high price, has been replaced by low-cost synthetic dyes (e.g., tartrazine (TTZ)) [8]. The consumption of TTZ can produce adverse metabolic effects [3,9–11]. Indeed, TTZ has been banned in some countries including Norway and Austria [12]. Turmeric (whose main coloring component is curcumin) has excellent heat stability, and it is often used as a replacement for TTZ; however, this pigment is unstable when exposed to light and it is susceptible to oxidation [2]. In contrast, saffron pigments are quite light- and heat-resistant [13].

Saffron contains several bioactive compounds, of which crocins (crocetin esters), a group of water-soluble carotenoids derived from crocetin (CCT, the aglycon of crocin), are responsible for the intense color that saffron provides to aqueous solutions [14]. Saffron displays numerous functional and bioactive properties. Therefore, research into the effects of saffron and its components is necessary to achieve a more widespread use of the spice. Today, along with the current trend of using natural colorants, there is a growing interest in therapeutic diets, which include culinary herbs or spices to support therapies for chronic diseases, including obesity [14]. Obesity is a global health problem that is acquiring an enormous epidemiological relevance due to its increasing prevalence rate [15]. The World Health Organization defines obesity as an abnormal or excessive accumulation of fat (adiposity) that can be harmful to health, and it is a risk factor for diabetes, cardiovascular disease, and cancer [16].

Recently, the most important therapeutic effects of saffron were attributed to CCT in its free-acid form [17]. Typical carotenoids contain 40 carbon atoms (C40); however, CCT is a C20 apocarotenoid (C20H24O4; molecular weight 328.4 g/mol), and it is generated via the hydrolysis of crocin glycosides. Crocin is crocetin digentiobiase ester, whereas CCT is 8,8-diapo-ψ,ψ-carotenoic acid. CCT contains a carboxyl group at each end of the polyene chain; when ionized, it can function as an acid (anionic) dye for biological staining [18]. On the other hand, CCT has high antioxidant power and possesses a wide range of beneficial properties for humans including anti-inflammatory, antiatherosclerotic, antihypertensive, and anticancer activities [19–22].

Adipose tissue function is essential for health; it is pivotal in the synthesis and storage of triacylglycerol in lipid droplets (lipogenesis) and the release of fatty acids into systemic circulation during periods of scarcity. In addition, adipocytes are a source of numerous proteins and hormones with actions relevant in practically every aspect of human physiology, including cardiovascular physiology. A crucial process for the homeostatic maintenance of lipid metabolism is the generation of new adipocytes from preadipocytes, a process known as adipogenesis. The process of adipogenesis involves growth arrest, mitotic clonal expansion, early differentiation, and terminal differentiation [23]. In vitro, adipogenesis takes place in two sequential stages: (1) the early stage, dependent on the

activation of early transcription factors: CCAAT/enhancer-binding protein (*C*/*EBP*)β and *C*/*EBP*δ, which in turn activate the transcription factors of the (2) late stage, dependent on the activation of late genes: *C*/*EBP*α and peroxisome proliferator-activated receptor γ (*PPAR*γ) [24–26]. In this way, preadipocytes differentiate into an adipocytic phenotype, causing morphological changes in the cell, including lipogenesis [27]. An exquisitely accurate adipogenesis process preserves lipid health [28], with increased lipid accumulation caused by an altered adipogenic process being a key factor in obesity. Thus, intervention in the regulation of adipogenesis, in terms of reducing fat mass, has been proposed as a possible therapy to prevent adipose tissue development and obesity [29]. In this sense, several studies have shown that CCT could play a preventive or even therapeutic role in some aspects related to the comorbidities that accompany obesity. Indeed, CCT has been shown to prevent visceral fat accumulation and insulin resistance induced by a hypercaloric diet in rats [30]. In addition, CCT regulates the expression of adiponectin in the adipose tissue of fructose-fed rats [31].

We observed in previous studies that different components of saffron, such as CCT and crocins, on some occasions, have opposite vasoactive properties [21]. Therefore, during this trend of a change toward healthier and more sustainable natural products, coinciding with the rapid advance of obesity in the world, we aimed to study CCT isolated from saffron (*C. sativus* L.) to broaden the existing knowledge on its beneficial properties and to promote its use as a healthy natural food colorant in the future. Specifically, our aim was to test the ability of CCT to reduce adipocytic lipid accumulation. We examined the ability of the CCT to induce differentiation in cultured murine 3T3-L1 preadipocytes by studying the amount of intracellular fat, the number and size of lipid droplets, and the viability and expression of the main early (*C*/*EBP*β and *C*/*EBP*δ) and late (*C*/*EBP*α and *PPAR*γ) genes involved in differentiation from preadipocytes to adipocytes. CCT decreased intracellular fat in mature adipocytes, showing potential antiadipogenic properties. Additionally, CCT did not affect lipid droplet generation or cellular viability. On the other hand, we report here that CCT diminished the messenger RNA (mRNA) levels of the transcription factor *C*/*EBP*<sup>α</sup>, which is implicated in lipid accumulation. Therefore, we propose that CCT reduces intracellular fat by decreasing *C*/*EBP*α mRNA levels.
