Rapid isolation and characterization of crocins, picrocrocin, and crocetin from saffron using centrifugal partition chromatography and LC–MS

Abstract

This study demonstrates a simple method for one-step isolation of the main secondary metabolites of a hydroalcoholic extract of Crocus sativus stigmas (saffron) using step-gradient centrifugal partition chromatography. The analysis was performed in dual and elution-extrusion mode, using five biphasic systems of the solvents heptane/ethyl acetate/butanol/ethanol/water in ratios of 4:10:0:4:10, 1:13:0:4:10, 1:12:1:4:10, 1:10:3:4:10, and 1:7:6:4:10. Five major crocins, picrocrocin, and crocetin were directly isolated in one step. Scaling up to preparative level, allowed the recovery of significantly high quantities of pure compounds, especially trans-crocin-4, saffron's principal crocin. Comparing dual-mode and elution-extrusion, in dual-mode, the trans-crocin-4 containing fractions were co-eluted with a high amount of free β-d-glucose. In contrast, absence of free β-d-glucose was observed in the corresponding trans-crocin-4 fractions obtained by the second method denoting its superiority against dual-mode. Initiating analysis with the 4 solvent-system afforded selective isolation of trans-crocin-4, with reduction in experimental time and solvent consumption. Structure elucidation was performed by nuclear magnetic resonance spectroscopy, liquid chromatography with mass spectrometry, and high-resolution tandem mass spectrometry. The proposed methodology comprises an integrated approach for the purification and characterization of biologically active saffron components in a fast, selective, and environmentally friendly manner.

Preparation, chemical characterization and determination of crocetin's pharmacokinetics after oral and intravenous administration of saffron (Crocus sativus L.) aqueous extract to C57/BL6J mice

“…Safranal, crocetin, curcumin (used as internal standard, IS) and β‐glucuronidase (Type H‐1 from Helix pomatia , 338 000 units/g) were purchased from Sigma Aldrich. Standard crocin‐4 (TC4) was kindly donated from the Laboratory of Pharmacognosy & Chemistry of Natural products, NKUA (LPCNP‐NKUA), purity >90% . The plant material, Saffron stigmas, was kindly donated by Kozani Saffron Producers Cooperative (Kozani, Greece) and a voucher specimen has been deposited at the Herbarium of the LPCNP‐NKUA, under the code number ATHU Valsami & Skaltsa 01.…”

“…Standard crocin-4 (TC4) was kindly donated from the Laboratory of Pharmacognosy & Chemistry of Natural products, NKUA (LPCNP-NKUA), purity >90%. [24] The plant material, Saffron stigmas, was kindly donated by Kozani Saffron Producers Cooperative (Kozani, Greece) and a voucher specimen has been deposited at the Herbarium of the LPCNP-NKUA, under the code number ATHU Valsami & Skaltsa 01. HPLC grade solvents, reagents and Methanol-D4 (99.8% for NMR, Art.…”

Separation and isolation of major polyphenols from maritime pine (Pinus pinaster) knots by two‐step centrifugal partition chromatography monitored by LC‐MS and NMR spectroscopy

“…Moreover, the absence of a solid support provides a wide range of advantages such as no irreversible adsorption of compounds onto the column, a minimal risk of sample denaturation, a maximal recovery of the injected sample, and a considerable potential for scale‐up . In addition, CPC is considered a “green” technique due to the low consumption of solvent and short analysis time . Thus, several recent studies have shown a successful isolation of complex mixtures of polyphenolic compounds in different matrices using CPC .…”

Crocins, the Bioactive Components of Crocus sativus L., Counteract the Disrupting Effects of Anesthetic Ketamine on Memory in Rats

“…The major component is a digentiobiosyl ester of crocetin (approximately 60% of the total crocins) [46]. The chemical profile of crocins has been well documented in previous studies [14,[47][48][49], and we evaluated the quality of the fresh prepared extract used in this study. The purity of the crocins was 85% (according to HPLC., Figure 4).…”