Chemistry of Saffron
Saffron contains more than 150 volatile and aroma-yielding compounds. It also has many nonvolatile active components, many of which are carotenoids, including zeaxanthin, lycopene, and various α- and β-carotenes. However, saffron's golden yellow-orange colour is primarily the result of α-crocin. This crocin is trans-crocetin di-(β-D-gentiobiosyl) ester; it bears the systematic (IUPAC) name 8,8-diapo-8,8-carotenoic acid. This means that the crocin underlying saffron's aroma is a digentiobiose ester of the carotenoid crocetin. Crocins themselves are a series of hydrophilic carotenoids that are either monoglycosyl or diglycosyl polyene esters of crocetin. Crocetin is a conjugated polyene dicarboxylic acid that is hydrophobic, and thus oil-soluble. When crocetin is esterified with two water-soluble gentiobioses, which are sugars, a product results that is itself water-soluble. The resultant α-crocin is a carotenoid pigment that may comprise more than 10% of dry saffron's mass. The two esterified gentiobioses make α-crocin ideal for colouring water-based and non-fatty foods such as rice dishes.
The bitter glucoside picrocrocin is responsible for saffron'sflavour. Picrocrocin (chemical formula: C
7; systematic name: 4-(β-D-glucopyranosyloxy)-2,6,6-trimethylcyclohex-1-ene-1-carboxaldehyde) is a union of an aldehyde sub-molecule known as safranal (systematic name: 2,6,6-trimethylcyclohexa-1,3-diene-1-carboxaldehyde) and a carbohydrate. It has insecticidal and pesticidal properties, and may comprise up to 4% of dry saffron. Picrocrocin is a truncated version of the carotenoid zeaxanthin that is produced via oxidativecleavage, and is the glycoside of the terpene aldehyde safranal.
When saffron is dried after its harvest, the heat, combined with enzymatic action, splits picrocrocin to yield D–glucose and a free safranal molecule. Safranal, a volatile oil, gives saffron much of its distinctive aroma. Safranal is less bitter than picrocrocin and may comprise up to 70% of dry saffron's volatile fraction in some samples. A second molecule underlying saffron's aroma is 2-hydroxy-4,4,6-trimethyl-2,5-cyclohexadien-1-one, which produces a scent described as saffron, dried hay-like.Chemists find this is the most powerful contributor to saffron's fragrance, despite its presence in a lesser quantity than safranal. Dry saffron is highly sensitive to fluctuating pH levels, and rapidly breaks down chemically in the presence of light and oxidising agents. It must, therefore, be stored away in air-tight containers to minimise contact with atmospheric oxygen. Saffron is somewhat more resistant to heat.
Picrocrocin is a monoterpene glycoside precursor of safranal. It is found in the spice saffron, which comes from the crocus flower. Picrocrocin has a bitter taste, and is the chemical most responsible for the taste of saffron.
During the drying process, picrocrocin liberates the aglycone (HTCC, C10H16O2) due to the action of the enzyme glucosidase. The aglycone is then transformed to safranal by dehydration. Picrocrocin is a degradation product of the carotenoid zeaxanthin
The best authenticity biomarker in saffron is picrocrocin. So far, saffron is the only plant in which it has been found and this is why it is an excellent marker of saffron authenticity and purity even against the most sophisticated adulteration with Gardenia jasminoides. This plant contains members from the same crocetin ester family, but in different proportions. It is a fraudulent practice to increase the colour of saffron. Recently, a rapid method for picrocrocin routine control has been proposed using UV–vis technique (Sánchez et al., 2009) which could be very useful in the future for saffron authentication. If the picrocrocin content is below the usual levels (Del campo et al., 2010), the sample can be suspected of tampering and should be analysed in detail with specific antifraud methodologies.
In addition to picrocrocin, several major and minor constituents can be used as biomarkers for origin differentiation. The crocetin esters profile is determined using several techniques: Fourier transform-near infrared (FT-NIR) spectroscopy, Fourier transform-mid infrared (FT-MIR) spectroscopy, liquid chromatography or 1H nuclear magnetic resonance (NMR) (Zalacain et al. 2005a,b; Yilmaz et al., 2010), have all proven useful for origin discrimination purposes among Iranian, Greek and Spanish saffron disputes. Gas chromatography (GC) offers the possibility of establishing the fingerprint of saffron volatiles which can permit geographical differentiation. Several authors have established it for different countries, such as Iran (Jalali-Heravi et al., 2010), Greece (Kanakis et al., 2004) and Spain (Alonso et al., 2007; Maggi et al., 2009). The contents of three compounds, 3, 5, 5-trimethyl-2-cyclohexenone, 2, 6, 6-trimethylcyclohexane-1, 4-dione and acetic acid, are capable of differentiating saffron according to its country of origin. Iranian and Moroccan samples show high contents of acetic acid while low or undetectable content is observed in Greek and Spanish samples. These differences are attributed to the different post-harvest processes that saffron undergoes depending on its origin (Carmona et al., 2006a). GC can be also applied to train an electronic nose based on metal oxides to perform the differentiation (Carmona et al., 2006a).
There are also significant differences in the content of minor constituents such as the kaempferol-3-O-sophoroside in saffron from Spain, Greece, Iran and Morocco (Carmona et al., 2007a), as well as free amino acids and ammonium ion content (Del Campo et al., 2009). Finally, a recent study (Maggi et al., 2011) has proved that analysis of the stable isotopes of the bio-elements H, C and N of saffron from the production areas of Western Macedonia in Greece, Khorasan in Iran, Sardinia in Italy and Castilla-La Mancha in Spain, combined with multivariate analysis, is a reliable tool in determining the geographical origin of saffron.
Cell cultures of C. sativus are expected to lead to the development of a biotechnological process for in vitro saffron production. Callus cultures were obtained from floral buds on Murashige and Skoog's medium supplemented with 3% sucrose, 2,4-dichlorophenoxy acetic acid (2 mg/liter), and kinetin (0.5 mg/liter). The cultures could be induced to produce red globular callus (RGC) and red filamentous structures (RFS) which produced crocin, crocetin, picrocrocin, and safranal. The quantity of picrocrocin in both RGC and RFS was higher than in stigmata. The safranal content of RGC was comparable to that of stigmata, whereas the crocin content in RGC and RFS was less. The biosynthetic capability of callus cultures of saffron to produce crocin, crocetin, picrocrocin, and safranal for biotechnological application is established.
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