Flavonoid Determination in the Quality Control of Floral Bioresidues from Crocus sativus L. Jéssica Serrano-Díaz,†,§ Ana M. Sánchez,#,⊥ Magdalena Martínez-Tomé,§ Peter Winterhalter,# and Gonzalo L. Alonso*,† †
Cátedra de Quı ́mica Agrı ́cola, ETSI Agrónomos, Universidad de Castilla-La Mancha, Campus Universitario, 02071 Albacete, Spain Food Technology, Nutrition and Food Science Department, Regional Campus of International Excellence “Campus Mare Nostrum”, Murcia University, 30100 Murcia, Spain # Institut für Lebensmittelchemie, Technische Universität Braunschweig, 38106 Braunschweig, Schleinitzstrasse 20, Germany ⊥ Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), C/Nicolás Cabrera 9, Campus de Cantoblanco, 28049 Madrid, Spain §
ABSTRACT: A high-performance liquid chromatography with photodiode array detection method (HPLC-DAD) was validated for the analysis of ﬂoral bioresidues obtained in saﬀron spice production by using three diﬀerent solvent mixtures [water/ hydrochloric acid (HCl) (100:1, v/v), water/acetonitrile/triﬂuoroacetic acid (47:50:3, v/v/v), and water/acetonitrile/HCl (50:50:1, v/v/v)]. Fifteen phenolic compounds were tentatively identiﬁed, kaempferol 3-O-sophoroside and delphinidin 3,5-diO-glucoside being the main ones. The extracts showed very diﬀerent phenolic proﬁles obtained by HPLC-DAD coupled with electrospray ionization mass spectrometry (ESI-MSn), and several experiments were carried out to explain this. The use of acetonitrile as solvent causes the chromatographic splitting of the peak of the delphinidin 3,5-di-O-glucoside into two peaks. Results obtained in this paper show that the extract prepared with water/HCl (100:1, v/v) would be the best suited for determining phenolic compounds in the quality control of the ﬂoral bioresidues from Crocus sativus L. KEYWORDS: Crocus sativus L., ﬂower waste, validation, quality control, kaempferol 3-O-sophoroside, delphinidin 3,5-di-O-glucoside
Flowers of saﬀron, along with the ﬂoral bioresidues from saﬀron spice production, are valuable natural sources of antioxidants,1 and they have an adequate nutritional composition to be employed as food ingredients.6 Saﬀron stamen and perianth have shown signiﬁcant antifungal, cytotoxic, and antioxidant activities, prompting us to expand the possible uses of this valuable plant.11 Bergoin characterized fresh ﬂowers, leaves and corms of saﬀron by analysis of their plant matter, volatile compounds, and liposoluble colorants to utilize them in the cosmetic, perfume, and fragrance industries.12 The high content in ﬂavonoids of the ﬂoral bioresidues from C. sativus L. has increased their interest to be exploited as neutraceuticals.13−15 Flavonoids are a class of secondary plant phenolics that include ﬂavonols, ﬂavones, ﬂavanones, catechins, anthocyanins, isoﬂavones, dihydroﬂavonols, and chalcones. Because ﬂavonoids have a wide range of diﬀerent biological properties, the contribution of these health-promoting compounds is being investigated for many purposes. Diﬀerent solvent systems (water, aqueous mixtures of ethanol, methanol, acetone, or acetonitrile, among others) have been used to extract ﬂavonoids from plant materials such as fruits, vegetables, and other foodstuﬀs.13,16−18 Many studies show that the extractive ability of plant ﬂavonoids considerably depends on the type of solvent used.17,18 Their conclusions associated the
INTRODUCTION Flowers of Crocus sativus L. are composed of six violet-colored tepals, three yellow stamens, and a ﬁliform white style, which culminates in a red stigma divided into three threads representing only 7.4% (w/w) of total weight of the fresh ﬂowers.1 The ﬂower part currently used is the stigma, which is converted into saﬀron spice, the most valued spice worldwide due to its color, taste, and aromatic properties and its numerous bioactive properties in humans.2−5 About 173,250 ﬂowers of saﬀron, which weighed over 68 kg, were required to obtain 1 kg of saﬀron spice.6 Once the stigma is separated from the ﬂowers, large quantities of tepals, stamens, and styles are discarded as agricultural bioresidues. These ﬂoral bioresidues generated in the production of saﬀron spice accounted for 92.6 g per 100 g of C. sativus ﬂowers.1 Currently, the largest saﬀron spice producer and exporter is Iran, followed by India, Greece, Spain, Morocco, and Italy. Spain was a major producer in the past and continues to lead the saﬀron spice trade due to the excellent quality of its production. Almost all manipulations to produce saﬀron spice require great manual labor, which increments the cost of the production process. However, in recent years, mechanized and forced production under greenhouse and controlled microclimatic conditions are being implemented in saﬀron cultivation by public institutions and several associations of producers and traders.7−10 Mechanization in C. sativus cultivation is a revolution that increases the productivity and, in turn, the generation of ﬂoral bioresidues. Hence, new options for their exploitation are required. © 2014 American Chemical Society
Received: Revised: Accepted: Published: 3125
December March 14, March 20, March 20,
19, 2013 2014 2014 2014
dx.doi.org/10.1021/jf4057023 | J. Agric. Food Chem. 2014, 62, 3125−3133
Journal of Agricultural and Food Chemistry
Thin-Layer Chromatography (TLC). The screening of the composition of the volumes collected by HSCCC was performed by TLC using water/methanol/dichloromethane (3:27:75, v/v/v) in silica gel 60 F254 aluminum plates and water/ACN/TFA (90:9.5:0.5, v/v/v) in C18 plates. Anthocyanins were visualized under a UV lamp at 254 and 366 nm, and then TLC plates were sprayed with anisaldehyde/sulfuric acid/glacial acetic acid spray reagent and heated at 100 °C. Tubes with a similar anthocyanin composition were reunited. After the evaporation of organic solvents, the fractions were frozen and freeze-dried. The HSCCC fractions with their corresponding retention times (tR) are shown in Table 1.
type of solvent used in the extraction with the yield and eﬃciency achieved in the ﬂavonoid extraction. Most of the papers are aimed at ﬁnding the solvents with the best extraction power,16,19 but very few authors have considered the eﬀect that solvents can have on the analysis of the compounds.20 Therefore, the aim of this work was to propose a quality control method of the ﬂoral bioresidues from C. sativus L. by the analysis of phenolic compounds after extraction with diﬀerent solvents.
MATERIALS AND METHODS
Table 1. Isolated Amounts and Retention Times (tR) of the Fractions Obtained by HSCCC
Chemicals. All reagents and solvents employed were of HPLC purity or analytical grade. Standards of kaempferol 3-O-glucoside (purity > 99%), kaempferol aglycone (purity > 99%), and delphinidin 3,5-di-O-glucoside were purchased from Extrasynthese (Genay, France). Kaempferol 3-O-sophoroside was isolated as described Sánchez et al.21 Ultrahigh-purity water was produced using a Milli-Q System from Millipore (Bedford, MA, USA). Acetonitrile (ACN), ethanol, acetone, tert-butyl methyl ether (TBME), n-butanol, dichloromethane, methanol, hydrochloridric acid (HCl), triﬂuoroacetic acid (TFA), anisaldehyde, sulfuric acid, glacial acetic acid, and formic acid were supplied from Scharlau (Barcelona, Spain). Plant Material. The ﬂoral bioresidues from the production of saﬀron spice were obtained from the company Agrı ́cola Técnica de Manipulación y Comercialización S.L. (Minaya, Spain) after separation of the stigma from the ﬂower of saﬀron during the 2010−2011 harvest. Stigma separation was performed using traditional procedures for the Protected Designation of Origin “Azafrán de La Mancha” according to DOCM.22 Floral bioresidues were formed by all parts of the ﬂowers except the stigmas. Fresh ﬂoral bioresidues from C. sativus L. were freeze-dried in a LyoAlfa 6-50 freeze-dryer (Telstar, Terrassa, Spain) for 48 h to constant weight.14 The conditions used for freeze-drying the samples were as follows: mass load, 100 g; initial temperature, −50 ± 2 °C; and pressure, 10−3 mbar. They were crushed, sieved through a 500 μm mesh size, and then stored at −20 °C until their further analysis. Isolation of Anthocyanins. Sample Preparation for High-Speed Counter-Current Chromatography (HSCCC). Floral bioresidues (835 g) were extracted with 25 L of water/HCl (100:1, v/v) at 40 ± 5 °C by stirring for 1 h and then decanted and ﬁltered through a fabric ﬁlter. The supernatant was ﬁltered through a Buchner funnel (sintered disk of porosity grade 2) and concentrated with a rotary evaporator under vacuum and freeze-dried. This crude extract was redissolved in water, centrifuged at 3000 rpm for 5 min, and ﬁltered with a paper ﬁlter prior to the application onto an Amberlite XAD-7 column (Fluka, 100 × 7 cm), conditioned with 2 L of methanol and then with 2 L of water. The column was rinsed with 3 L of water, and anthocyanins were eluted with 2 L of a mixture of water/ACN/TFA (49.5:50:0.5, v/v/v). The eluate was concentrated, dissolved in water, and freeze-dried. An amount of 3.9 g of puriﬁed XAD-7 extract was obtained from 74.1 g of crude extract. High-Speed Counter-Current Chromatography. The XAD-7 extract was fractioned with a CCC-1000 high-speed countercurrent chromatograph manufactured by Pharma-Tech Research Corp. (Baltimore, MD, USA) equipped with three preparative coils and connected in series (tubing diameter of 2.6 mm and total volume of 850 mL). TBME/n-butanol/water/ACN (1:3:5:1, v/v/v/v) acidiﬁed with 0.5% TFA was used as solvent system. The elution mode was head to tail with the lighter (organic) phase acting as the stationary phase and the aqueous phase acting as the mobile phase. The ﬂow rate was set at 3 mL min−1 and delivered by a BT 3020 HPLC pump (Jasco, GrossUmstadt, Germany). The separation was run at a speed of 1000 rpm. One gram of XAD-7 extract was dissolved in 25 mL of a mixture of the upper (organic) and lower (aqueous) phases (50:50, v/v) and injected into the system by a loop injection. Volumes of 12 mL were collected with a fraction collector. Elution was monitored with a K-2501 UV−vis absorbance detector (Knauer, Berlin, Germany) at 280 nm to detect anthocyanins and also ﬂavonols.
isolated amount (mg)
F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11
204−212 212−220 220−232 232−252 252−308 308−324 324−336 336−364 364−380 coil residue cleaning in methanol
301.93 77.54 46.24 35.20 39.43