Production Practices and Quality Assessment of Food Crops. Vol. 1

222 S. A. Ordoudi and M. Z. Tsimidou
Table 2. Recommended protocols for crocin isolation.
Step Action
Weber and Grosch (1976)
1 Removal of fat in a Soxhlet apparatus
saffron (2.5 g) + Et 2O
2 Extraction with MeOH (2 × 100 ml)
3 Crude crocin + celite (2 g) purification on column chromatography
(silic acid, Mallinckrodt 100 mesh, in Et 2O (1.8 cm × 30 cm)
a. removal of impurities (100 ml Et 2O + 100 ml Et 2O:MeOH, 70:30,v/v)
b. crocin elution with 20% methanolic Et 2O
4 Evaporation in vacuo
Adjustment of pH with 5 ml 0.05 M Na 3PO 4 (pH 6.5)
5 Further purification with column chromatography (Sephadex G-10 column, 22 cm × 1.8 cm)
Equilibration of column with the same buffer as above.
6 Collection of fractions containing pure crocin
Speranza et al., 1984
1 saffron (10 g)
Successive extraction with n-hexane (100 ml), Et 2O (100 ml), Et 2O satur. with H 2O (200 ml),
AcOEt (150 ml), aqueous EtOH 70% (300 ml)
2 The ethanolic extract a) evaporation of ethanol, b) lyophilisation
(residue: 5.1 g)
3 Purification on column chromatography: silica gel 60 (230–400 mesh, 1300 g)
Eluent: AcOEt-EtOH-H 2O, 6:3:1, v/v/v
4 Collection of fractions containing crocins – Evaporation to dryness
(residue: 0.7 g)
5 Further separation on preparative HPLC (Lichrosorb RP-18, 250 × 10 mm, 7 µ
Eluent: ACN-H 2O, 20–45% ACN in 15 min, flow rate 5 ml/min, 440 nm
[crocin 1: UV(H 2O) λ max = 462, 443, 410 nm]
[13-cis crocin: UV (H 2O) λ max = 462, 437, 410, 328 nm]
Straubinger et al. (1997)
1 saffron (8.8 g)
successive extraction in a Soxhlet apparatus with petroleum ether (250 ml), Et 2O (250 ml),
MeOH (3 × 250 ml)
evaporation under reduced pressure to dryness
2 Fractionation with multilayer coil countercurrent chromatography (75 m × 2.6 mm)
Eluent: CHCl 3: MeOH:H 2O, 7:13:8, v/v/v
3 Combination of 70 fractions (× 10 ml each)
I: 1–12; II: 13–29; III: 30–34; IV: 35–64; V: 65–70 (study of the first three)
1987; Koyama et al., 1988; Isa et al., 1990; Sarma et al., 1990, 1991; Isa and
Ogasawara, 1991; Dufresne et al., 1997; Bhagyalakshmi, 1999; Zhao et al., 2001).
The in vitro synthesis of crocin (and also picrocrocin and safranal) in stigma-like
structures developed by young stigmas and ovaries of C. sativus was first reported
by Himeno and Sano (1987). The stigma-like structures were formed and grown
for 20 weeks on a Linsmaier and Skoog medium supplemented with napthaleneacetic