American Journal of Analytical Chemistry, 2013, 4, 104-110 http://dx.doi.org/10.4236/ajac.2013.42014 Published Online February 2013 (http://www.scirp.org/journal/ajac) Development of a Performant Method for Glucocapparin Determination in Boscia senegalensis Lam Ex. Poir.: A Study of the Variability Momar Talla Gueye1,2,3*, Dogo Seck4, Abdoulaye Diallo1, Danny Trisman2, Christophe Fischer3, Jean-Paul Barthelemy3, Jean-Paul Wathelet2, Georges Lognay3 1Institute of Food Technology (ITA), Hann-Dakar, Senegal 2University of Liege, Gembloux Agro-Bio Tech, Unit of General and Organic Chemistry, Gembloux, Belgique 3Gembloux Agro-Bio Tech, Unit Analysis, Quality, Risks—Laboratory of Analytical Chemistry, University of Liege, Gembloux, Belgium 4Regional Centre for Research in Ecotoxicology and Environmental Safety (CERES/Locustox), Dakar, Senegal Email: *[email protected] Received December 12, 2012; revised January 12, 2013; accepted January 18, 2013 ABSTRACT This study describes a glucocapparin determination method. Based on rapeseed determination of glucosinolate (GSL), the equation of the average straight regression line is Y = 100.42X − 0.03 (R2 = 0.9998). Enzymatic hydrolysis of glu- cocapparin extracted from leaves and fruits of B. senegalensis, analyzed by SPME-GC-MS confirmed the presence of methylisothiocyanate as the main hydrolysis glucocapparin product. Monitoring glucocapparin contents in B. sene- galensis leaves and fruits collected in 4 localities in Senegal showed differences between organs according localities and periods of harvest. Glucocapparin content was very high in dry season particularly in January and the lowest rates were recorded during the rainy period between August and November. Keywords: Boscia Senegalensis; Validation Method; Glucocapparin; Monitoring; Senegal 1. Introduction are well known. The allylisothiocyanates are used as conservatives in food industry. Although they do not The glucosinolates (GSLs) are particularly abundant neutralize the free radicals before they can harm cells, as sulphur-containing compounds in the families of the Or- antioxidants and carotenoids would do, GSLs and their der Capparales - Brassicaceae, Tovariaceae, Resedaceae, hydrolysis products are considered as indirect antioxi- Capparaceae and Moringaceae [1]. At least, 120 GSLs were identified in sixteen angiosperm families including dants by modulating the activity of xenobiotic metabo- a large number of edible species. The type of major GSL lizing enzymes [3]. They have a protective effect by in- present varies largely between different plant species and hibiting the proliferation of cancer cells [4]. The hydro- the organ considered [2]. GSLs are associated in plants lytic products of alkyl and aryl GSL are cytotoxic to with enzymes called myrosinases (thioglucoside gluco- Salmonella thyphimurium (Tiedink et al., 1991 cited by hydrolase, EC.3.2.3.1). Their hydrolysis into D-glucose [3]. Aurinia sinuate showed a powerful antimicrobial and a variety of breakdown products is responsible for effect on Gram-positive and Gram-negative bacteria and the pungent bitter taste of many cruciferous and spice fungi probably by the GSL degradation products such as plants. As a result of mechanical wound or pathogen at- isothiocyanates and nitriles taking into account their im- tack, the GSLs and hydrolyzing enzymes come into con- portance in the volatile products [5]. Many other works tact and produce a number of hydrolytic products, in situ highlighted the biocide activity of GSL hydrolytic prod- [3]. According to the substrate and the reaction condi- ucts. Given their occurrence, the use as substitutes to tions, the secondary products are mainly isothiocyanates, pesticides in seed and stored product treatments is de- oxazolidines thiones, thiocyanates, nitriles and glucose sired in IPM. In this fact, the Capparaceae Boscia sene- (Figure 1). galensis was successfully tested in cowpea storage and Many properties of GSLs and their hydrolysis products peanut [6-8]. Its efficacy is due to the occurrence of glu- cocapparin in its organs. Indeed methylisothiocyanate *Corresponding author. (MITC) is released by hydro l ysis of glucocapparin and Copyright © 2013 SciRes. AJAC M. T. GUEYE ET AL. 105 OH O HO HS R HO S R MYROSINASE C OH C + D-Glucose H2O N N - - O3SO Glucosinolate O3SO Thiohydroxamate-O-sulphonate - HSO4 S R' R' CH2 R-C N + S R-N=C=S R-S-C N CH2-CH-CH-(CH2)n-C N C N-H - Nitriles R'' S-C N O C Isothiocyanates Cyano-epithioalkanes S Oxazolidine-2-thiones Thiocyanates Figure 1. Myrosinase degradation pattern of glucosinolate. responsible for the insecticidal activity on the bruchids 2. Experimental Callosobruchus maculatus and C. serratus. Glucocap- 2.1. Plant Material parin was identified in leaves of Cleome spinosa and C. chelidonii [9,10]. However, glucocapparin is less docu- Leaves and fruits of B. senegalensis were collected in mented than other glucosinolates, undoubtedly due to the four localities of Senegal: Dakar (1442'00"N, 1728'00"W), lack of commercial interest in it. It has been reported that Thies (1656'W, 1448'N), Louga (1542'00"N, 1612'00"W) in Western Africa, B. senegalensis holds a considerable and Richard Toll (1628'00"N, 1541'00"W) between April nutritive value. The fresh and immature seeds are used 2010 and November 2011. Samples were collected from like lentils after a process of cooking and complex plants with the ripest fruits. Different phenological states scrubbing. In Senegal, these preparations are sold in the leaves were collected at various levels of the plants. The markets. Dried, they are transformed into flour and pre- plant identification was done at University of Dakar pared into a type of couscous which is often used during (IFAN) in the botanical section, maintaining the Integrity the hardest scarth. A terrible year of dryness (1958) was of the Specifications. baptized “the year of Boscia”, because it was with this species and its seeds or “peas” that many people survived 2.2. Preparation of Samples [11]. In the laboratory, samples were dried at ambient tem- Due to the fact that specific biological effects of dif- perature (25˚C ± 5˚C) to avoid the premature attack of ferent glucosinolates vary considerably, it is appropriate the myrosinase. For the GSLs extraction, a fine crushing to develop sensitive and reliable method for efficient with crusher IKA ®A11basic was carried out. identification and quantification for the particular glu- cosinolate under investigation [12]. 2.3. Determination of Glucocapparin Content The object of this work is to develop a reliable and ef- fective method to analyze glucocapparin in leaves and 2.3.1. Preparation of Sulfatase and of Sinigrine fruits of B. senegalensis and to analyze the effect of its Solution localization on main secondary metabolite concentration. Sulfatase: 3 ml of water and 2 ml of ethanol were added Therefore, we adapted the standard ISO 9167-1 method to 75 mg sulfatase of Helix Pomatia (Sigma EC3161). [13] described to analyze the glucosinolates in rapeseeds The tube was placed in a vortex for 3 min before being (Brassica napus). After validation of the analytical me- centrifuged at 3000 rpm for 10 minutes. The deposit was thod, leaves and fruits of B. senegalensis from four lo- discarded and 8 ml of ethanol was added to the super- calities in Senegal and different seasons are monitored in natant. This mixture was agitated again with a vortex order to evaluate the variability of glucocapparin. In ad- machine for 45 minutes after which the supernatant was dition, an identification of the glucocapparin breakdown collected. A last round of centrifugation at 3000 rpm was products is carried out by gas chromatography coupled done after which the residue was transferred in 5 ml of with the mass spectrometry. distilled water. Copyright © 2013 SciRes. AJAC 106 M. T. GUEYE ET AL. Sinigrine: Sinigrine was taken as internal standard. A dase from Sinapis alba, Sigma) were added to facilitate solution (20 µmol/ml) was prepared from the pure prod- hydrolysis. The flask was placed at 25˚C during two uct of Sigma Aldrich (Ref.: 51647) which was dissolved hours. The flask was placed at 25˚C during two hours. in methanol/water 70:30 v/v solution. The volatile derivatives were sampled by SPME (Car- boxen PDMS 75 µm fiber Ref 57344Supelco). GC-MS 2.3.2. Glucocapparin Extraction was realized on a HP-5MS ((5%-phenylmethyl)-siloxane, 50 mg of powdered B. senegalensis dry leaves or fruits Agilent) column (length 30 m; diameter 250 µm; df = were placed in 12 ml glass tubes. In a water-bath at 74˚C 0.25 µm. The oven temperature program was initiated at ± 1˚C, it was added in the test tubes (10 ml) 4.5 ml of a 40˚C, held for 5 min then raised first at 5˚C/min to 230˚C, mixture methanol/water (70/30) beforehand heated to raised in a second ramp at 30˚C/min to 280˚C with a final boiling then 1 ml of sinigrine 20 μM. 4.5 ml of the hot hold at this temperature for 5 min. The carrier gas is he- solvent mixture were added again in the glass. The solu- lium (pressure: 49.9 KPa, flow: 1 mL/min) and the in- tion is agitated during 15 min by means of a magnet. The jector split/splitless (in splitless mode) is maintained at whole was then centrifuged at 3500 rpm during 10 min. 250˚C. The source of the mass spectrometer was fixed at The supernatant was recovered thereafter. a temperature of 230˚C and the scanned mass range was 50 to 350. 2.3.3. Desulfatation and Purification of Extracted Glucocapparin 2.4. Validation of the Glucocapparin 30 ± 5 mg of DEAE Séphadex A-25 are placed in a Quantification Method minicolonne (1 ml syringe). The column is washed with The procedure of validation is in conformity with the distilled water (2 fold 1 ml). pH is adjusted by elution of Quality System of Good Laboratory Practice (Mono- an acetate buffer (pH 5.8, 0.5 M, 2 ml).