Crocin Bleaching Assay Using Purified Di-gentiobiosyl Crocin (a-crocin) from Iranian Saffron

Document Type: Original Article

Authors

1 Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

2 Department of Biochemistry, Payame Noor University, Tehran, Iran

3 Children Medical Center, Tehran University of Medical Sciences, Tehran, Iran

Abstract

Objective(s)
Crocin bleaching assay (CBA) is a new method for determination of antioxidant capacity. In CBA, addition of hydrogen to the conjugated double bonds of crocin results in reduction of crocin and increase in the absorbance at 440 nm, which is considered as a measure of antioxidant potential. Here CBA method was set up using di-gentiobiosyl crocin or a-crocin from Iranian saffron. Then, the antioxidant activity of some known antioxidants i.e. L-ascorbic acid, bilirubin, Trolox, uric acid, and some plasma samples of infants, were tested. The results were compared to that obtained by ferric reducing antioxidant power" (FRAP) as a standard method.
Materials and Methods
Di-gentiobiosyl crocin was extracted and purified from Iranian saffron as previously described by us. Then, the CBA procedure using a-crocin was done in 2 different aquatic conditions, >50% or >90% of water. Results were analyzed by both the Bors method (calculating the relative rate constants= Krel) and the Tsimidou method (calculating the percent of a-crocin bleaching inhibition=% Inh).
Results
Our results indicated the following order of antioxidant potential for the above mentioned agents: ascorbic acid > uric acid > Trolox. However, these results are very similar to the data reported by others, but they are strongly related to the aqueous condition. In addition, uric acid showed different properties at different concentrations; so that it showed the antioxidant activity at low concentrations but it acted as a prooxidant at higher concentrations. Bilirubin interfered with this test, possibly because its maximum absorbance is close to the crocin. The obtained data for the antioxidant capacity of the serums was comparable with FRAP assay, except for the sample that contains high bilirubin concentration.
Conclusion
In conclusion, it seems that CBA using the main fraction of crocin (a-crocin) is a simple and useful method for determination of antioxidant potential of aqueous samples. In addition, the CBA ability to distinguish the samples that contain bilirubin or high uric acid content is helpful in clinical laboratories.

Keywords


1.Somogyi A, Rosta K, Pusztai P, Tulassay Z, Nagy G. Topical Review: Antioxidant measurements. Physiol Meas 2007; 28:R41-R55.

2.Manzocco L, Calligaris S, Nicoli MC. Assessment of Pro-oxidant activity of foods by kinetic analysis of crocin bleaching. J Agric Food Chem 2002; 50:2767-2771.

3.Cao G, Alessio HM, Cutler RG. Oxygen-radical absorbance capacity assay for antioxidants. Free Radic Biol Med 1993; 14:303-311.

4.Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. J Anal Biochem 1996; 239:70-76.

5.Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evanse C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol 1999; 26:1231-1237.

6.Wayner D, Burton G, Ingold K, Locke S. Quantitative measurement of the total peroxyl radical-trapping antioxidant capability of human blood plasma by controlled lipid peroxidation. FEBS Lett 1985; 187:33-37.

7.Tubaro F, Ghiselli A, Rapuzzi P, Maiorino M, Ursini F. Analysis of plasma antioxidant capacity by competition kinetics. Free Radic Biol Med 1998; 24:1228-1234.

8.Bors W, Michel C, Saran M. Inhibition of the bleaching of the carotenoid crocin a rapid test for quantifying antioxidant activity. J Biochem Biophys 1985; 796:312-319.

9.Ordoudi SA, Tsimidou MZ. Crocin bleaching assay step by step: observations and suggestions for an alternative validated protocol. J Agric Food Chem 2006; 54:1663-1671.

10.Bolhasani A, Bathaie SZ. Sepration and purification of some components of Irainan saffron.Chem Asian J 2005; 17:725-729.

11.Ashrafi M. Study on the effect of saffron carotenoids and monoterpene aldehydes on H1 structure and H1-DNA interaction. MSc Thesis, Tarbiat Modares University, Tehran, Iran, 1382.

12.Ashrafi M, Bathaie SZ, Taghikhani M, Moosavi-Movahedi AA. The Effect of carotenoids obtained from saffron on histone H1 structure and H1-DNA interaction. Int J Biol Macromol 2005; 36:246-252.

13.Bathaie SZ, Bolhasani A, Hoshyar R, Ranjbar B, Sabouni F, Moosavi-Movahedi AA. Interaction of saffron carotenoids as anticancer compounds with ctDNA, Oligo (dG.dC)15, and Oligo (dA.dT)15. DNA Cell Biol 2007; 26:533-540.

14.Standard Operating Procedure for FRAP Assay on Plasma and Faecal extracts. Robert Gordon University School of Life Sciences. Availble at: http://www4.rgu.ac.uk/files/sop 11 FRAP on faecal extracts plasma.pdf

15.Lussignoli S, Fraccaroli M, Andrioli G, Brocco G, Bellavite P. A microplate-based colorimetric assay of the total peroxyl radical trapping capability of human plasma. J Anal Biochem 1999; 269:38-44.

16.Sendra JM, Sentandreu E, Navarro JL. Kinetic model for the antiradical activity of the isolated P- catechol group in flavanone type structures using the free stable radical 2,2 -diphenyl-1-picrylhydrazyl as the Antiradical Probe. J Agri Food Chem 2007; 55:5512-5522.

17.Majoa DD, Giammancoa M, Guardiaa ML, Tripolia E, Giammancoa S, Finot E. Flavanones in citrus fruit: structure-antioxidant activity relationships. J Food Res Int 2005; 38:1161-1166.

18.Howard D, Haskins MD. The Uric acid solvent power of normal urine. J Biol Chem 1916; 321:205-215.

19.Nalsen C. Measurement and evaluation of antioxidant status and relation to oxidative stress in humans. Swedish Thesis English Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 2006.