Food ChemistryFood Chemistry 100 (2007) 1126–1131 www.elsevier.com/locate/foodchem HPLC quantification of major active components from 11 different saffron (Crocus sativus L.) sources Heriberto Caballero-Ortega a, Rogelio Pereda-Miranda b, Fikrat I. Abdullaev a a,* ´a Experimental, Instituto Nacional de Pediatrı ´a, Avenida del Ima ´ n # 1, Torre de Investigacio ´ n, 6° piso, 04530 Laboratorio de Oncologı DF, Mexico City, Mexico b ´mica, Universidad Nacional Auto ´ noma de Me ´ xico, 04510 DF, Mexico City, Mexico Departamento de Farmacia, Facultad de Quı Received 28 July 2005; received in revised form 17 November 2005; accepted 17 November 2005 Abstract Eleven certified saffron samples (Crocus sativus L.), one each from Azerbaijan, China, Greece, France, India, Iran, Italy, New Zealand, Spain, Turkey and the Sigma Chemical Company, were analyzed by using an HPLC photodiode array detection method. This analysis quantified the 10 major saffron compounds in each sample and their concentration was analyzed at three different wavelengths. Results indicated that the Greek, Indian, New Zealand, and Spanish saffron extracts possessed the highest concentrations of water-soluble glycosidic carotenoids (P8.0%) suggesting that they could be a good source of this type of metabolites for further biological evaluation. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Saffron; Crocus sativus L.; Quantitative analysis; HPLC; Glycosidic carotenoids; Crocins 1. Introduction Saffron is the dried stigmas of a flower scientifically identified as Crocus sativus L. Although the source of saffron is unknown, it apparently originated in the area of Iran, Turkey and Greece, but now it is also successfully cultivated in such European countries as Spain, Italy, France, and Switzerland, as well as in Morocco, Egypt, Israel, Azerbaijan, Pakistan, India, New Zealand, Australia and Japan. While the world’s total annual saffron production is estimated to be 190 tons, Iran produces about 90% of the total (Fernandez, 2004; Negbi, 1999; Xuabin, 1992). Since ancient times, saffron has been used as a medicinal plant and a culinary spice (Basker & Negbi, 1983; Rios, Recio, Giner, & Man ˜ ez, 1996; Zhou, Sun, & Zhang, 1978). The stigmas contain carbohydrates, minerals, vita* Corresponding author. Tel.: +52 55 10 84 09 00x1474; fax: +52 55 10 84 38 83. E-mail addresses: fi
[email protected], fi
[email protected] (F.I. Abdullaev). mins and such pigments as carotenes, and flavonoids (Abdullaev, 1993; Winterhalter & Straubinger, 2000). The bitter-taste is produced by the picrocrocin (C16H26O7), a monoterpene glycoside precursor of safranal (C10H14O), the main volatile oil responsible for the aroma. b-Glucosidase action on picrocrocin liberates the aglycone, 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde (HTCC, C10H16O2; MW 168), which is then transformed to safranal by dehydration during the drying process of the ´ mez, Rubio, & plant material (Lozano, Delgado, Go Iborra, 2000; Sujata, Ravishankar, & Venkataraman, 1992). The colour of saffron comes from the water-soluble glycosidic cis- and trans-carotenoids crocins, glucosyl esters of crocetin (8,8 0 -diapocarotene-8,8 0 -dioic acid; C20H24O4). For commercial purposes the quality of the colouring power of a saffron sample depends on the quantification of the crocin analogues by colorimetric (Li, Lin, Kwan, & Min, 1999) and chromatographic techniques, such as TLC, GC-MS, and HPLC (Zareena, Variyar, Gholap, & Bongirwar, 2001). 0308-8146/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2005.11.020 USA) equipped with a pump (Model 600E). & Polissiou. 2-Nitroaniline (Sigma) was used as the internal standard for HPLC analysis. MA. Extraction Saffron stigmas (50 mg) were suspended in 10 ml of methanol–water (50:50. After extraction. 2001). Sujata et al. 25 cm length. 1992. Tarantilis et al. Tarantilis. Tarantilis & Polissiou. a commercial sample was purchased from Sigma (Product S8381. USA). China (Tibetan saffron). Moosavi-Movahedi. 1999. Simancas. The material was stored in our laboratory in the dark at 4 °C until processed. and 1. HPLC equipment HPLC analysis was performed on a multisolvent delivery system (Waters. Therefore.S. 1999) and they are expressed in milligrams per gram of saffron stigmas. 1 ml of 2-nitroaniline (0. Montijano. 0. 2. MA. Germany (type Electus pulvis) (Straubinger. Up-scaling the same protocol permits preparative level isolation of the major compounds for biological activity investigation.5. Germany). Milford. 0.H.. Greece (Kanakis.4. & Iborra. 4. v/v) and extracted as described above.0 mg/ml in triplicate (Lindsay.031. 10 lm parti˚ ) was used for all analcle size with a pore diameter of 80 A yses.e.. ´n. Barnstead Thermolyne. linked to a computer system (Optiflex GX280. Iran (Abbaszadeh.3. picrocrocin. Tsoupras. Dubuque. Materials and methods 2. Picrocrocin. samples were centrifuged at 30. Bla ´ z1999. Methanol and acetonitrile were of HPLC grade (Merck KGaA. China (Li & Wu. Darmstadt. Zalacaı quez. 1999). ´ n.25. a multiple UV wavelength photo-diode array detector (Model 996). MO. Water was distilled. and Iran (Bolhasani. A linear gradient of methanol (10–100%) in water (15% of acetonitrile) was used as a mobile phase with a flow-rate of 1. 0. & Winterhalter. Eckstein. 25 cm length. & Manfait.).. & safranal and crocins (Castellar.D. i. Apsheron). Spain (La Mancha) and Turkey (No brand name). & Winterhalter. 1995). Reg... Jezussek. 1999). & Ghaffari. Polissiou.000g for 20 min to eliminate plant residues and then the supernatant was collected and filtered through a nylon membrane (Acrodisc 13. no reports analysing and comparing the chemical composition of four or more saffron samples originating from different locations (Li et al. Carmona. v/v) and magnetically stirred during 24 h at 4 °C in the dark. 2004. 1992. 1999. Castellar. Iborra. a calibration curve was prepared using final concentrations of 0. Bau. Sigma– Aldrich Corp. Milford. Manjo ´ n. All samples were directly provided by local expert saffron growers in order to avoid any adulteration with foreign plant material. 1994. & Manjo 1992. 1994. The sample size was 50 ll of the test solution (Abdullaev et al. Plant materials and chemicals Certified dried saffron stigma samples were obtained from plants harvested in 2003 from each of the following countries: Azerbaijan (Belgya.5.015. For preparative HPLC. Lozano et al. Empower Software 2002 (Waters) was used for equipment control. Quantitative determination A Spherisorb RP C18 column (Waters. & Polissiou. There are however.. 1999).1. Milford. 1992). 2004). 2002. 1997). Tarantilis et al..9833 to 0. 20 mm internal diameter. The chemical composition of saffron samples from Spain (Iborra et al. USA). Rangiora). Dell Computer Corp. 2.. India (Baby Brand). Zareena et al. Fink. Castellar. extraction. For the internal standard..5 mg/ml) was added as an internal standard to 1 ml of each tested sample (Caballero-Ortega et al. 0.062. Tarantilis. 1993. whereas the internal standard was detected at the above three mentioned wavelengths (Lozano et al. Dı ´az-Plaza. 70070). Waters.6 mm internal diameter. the aim of the present investigation was to develop an HPLC analytical protocol useful for the identification and quantification of the major components in 11 commercial samples of saffron from different countries for quality control comparison. 0. & Alonso. Yavari. The R2 range was from 0. Greece (Krokos Kozanis).125. MA. St. Louis. 1998. 2.45 lm pore size · 13 mm diameter. Straubinger. In addition. Li et al. separation and quantification. and processing of the chromatographic information. 2005) indicated that the estimated amounts of constituents strongly depended on the methods employed for drying. IA. USA. safranal at 310 nm and all crocins at 440 nm. Quantitative determinations were made taking into account the molecular coefficient absorbance of each peak obtained at the wavelength of maximum absorbance of the respective ingredient as previously reported (Lozano et al. Daferera. No. Lozano et al. values between injected samples were lower than 5%. Lozano. Preparative analysis and spectrometric identification of pure compounds A Spherisorb RP C18 column (Waters.. 0. 2. Tarantilis. 2000). 1997. / Food Chemistry 100 (2007) 1126–1131 1127 Several methodologies have been described using different analytical tools to establish purification protocols for the bioactive constituents of saffron. Bathaie. Before quantitative chromatographic analysis. 20 mg of the Greek sample were pulverized and suspended in 1 ml of methanol–water (50:50. 10 lm particle size with a pore .2. 2002). Iborra. Italy (Cooperativa Altopiano di Navelli).. HTCC and kaempferol were detected at 250 nm. 2004). France (Poitou). Canovas.. New Zealand (Eight Moon Saffron. Waibel. The analyses were triplicated for each sample... USA) (Lozano et al. 1995). 2. data acquisition. Caballero-Ortega et al.0 ml/min for a maximum elution time of 60 min at room temperature. India (Sujata et al..9990 and the slope R. deionized and further purified through an ultrafiltration system (Easypure RF. 1992.. 2005. 2 and 3 detected at 250 nm were picrocrocin. 2005). 3-gentiobiosyl-kaempferol (peak 3). HTCC and 3gentiobiosyl-kaempferol. trans-crocin 4 (peak 4). transcrocin 3 (peak 5). / Food Chemistry 100 (2007) 1126–1131 ˚ ) was used for this analysis. trans-crocin 2 (peak 9). HTCC (peak 2). MO. 3.1 (trans-crocin 2). The chromatographic conditions employed allowed identification of 10 major components in each sample and a well-resolved baseline separation was obtained. 1999. cis-crocin 4 (peak 8). Tarantilis et al. 1 shows three representative chromatograms. recorded at three wavelengths. 38. 17. Caballero-Ortega et al.7 (trans-crocin 3). the second as an example for the Sigma product (1B) and a third one showing the profile for the Tibetan sample (1C). IS: internal standard. 25. and 8 to 10 (440 nm) were trans-crocin 4.4 (cis-crocin 2) were collected by the technique of heart cutting (Hostettmann. Positive-ion LRFAB-MS was registered using a glycerol matrix on a SX102A spectrometer (JEOL. Eluates across the peaks with tR of 8.1. Lozano et al. Mass spectra were determined in duplicate and the pseudomolecular ion [M + Na]+ was detected for each peak. tran-crocin 2.0 (HTCC). Tarantilis et al. Each component was identified by comparison of its retention time as previously described in the literature (Li et al. transcrocin 2 0 .8 (3-gentiobiosyl-kaempferol). a linear gradient of methanol (10–100%) in water (15% of acetonitrile). Japan).9 (trans-crocin 2 0 ). The amount of sample injected was 500 ll of the test solution.1128 H. and cis-crocin 2. and 44. Fig. A Waters Spherisorb C18 column. 31. The observed fragmentation pattern was used for crocin identification by comparison with previously reported spectra (Carmona et al. HPLC fingerprint chromatograms of saffron samples from Greece (cultivated sample) [A]. Tokyo. and the resolving power was 3000. Saffron components: picrocrocin (peak 1). and China (noncultivated sample) [C]. 1998).4 (trans-crocin 4). The above-mendiameter of 80 A tioned linear gradient was used with a flow-rate of 10 ml/min during an elution time of 60 min. 1995). . one with the highest concentration of analysed glycosidic carotenoids (1A). 1999.0 ml/min were used for quantitative determinations.9 (safranal). The accelerating voltage was 10 keV. 1. cis-crocin 2 (peak 10). The peak identification is as follows: number 1. respectively. and a flow rate of 1. Quantitative analysis Table 1 shows the concentration of each detected compound in the 11 tested samples. Each collected peak was rapidly evaporated under vacuum using a centrifugal solvent evaporator system (CentriVap. 28. Xenon was used as collision gas. The results indicate Fig. but did differ in the concentration of each component. Labconco.0 (cis-crocin 4).2. trans-crocin 3. trans-crocin 2 0 (peak 6).. According to our analysis different saffron samples did not differ in their chemical composition.3 (picrocrocin)... 3. cis-crocin 4.. 35.. 2-nitroaniline. & Hostettmann. peak number 7 (310 nm) was safranal and peak numbers 4 to 6. 35. 16. USA) and further submitted to low resolution fast atom bombardment mass spectrometry (LRFAB-MS) analysis. Qualitative analysis The chemical composition of saffron samples from 11 different sources was determined using reverse phase C18 HPLC. 1995) as well as by LRFAB-MS analysis through the detection (m/z) of its corresponding pseudomolecular ion (M + H)+ (Carmona et al. respectively. safranal (peak 7). Marston.. Results and discussion 3. Sigma Product S8381 [B]. gas chromatography (GC) (Kanakis et al.053 0..15 ± 0.001 nd 0.14 ± 0. In case of Tibetan saffron.28 ± 0. 1999.43 ± 0.119 0..001 1. 1994.26 ± 0.001 0.27 ± 0. as precursor of the saffron aroma components (safranal and HTCC).001 5.04 5. vaccum head space method (VHS).07 85.02 ± 0.13 ± 0.33 ± 0.001 >0. cis-crocin 2 was present in large amounts in the Indian sample.43 mg/g of stigmas) as well as the Tibetan variety from China (9.031 Table 1 HPLC quantitative analyzes of 10 saffron metabolites from 11 different saffron sources 8.003 1129 that the differences might be due to the origin of the sample.09 ± 0.001 0.86 mg/g of stigmas) showed the lowest. 2000)..74 ± 0.001 France 78. Caballero-Ortega et al.006 7.001 2.21 12.001 0.34 ± 0.21 ± 0. respectively).69 ± 0.. 1995).001 2.027 0. while the sample purchased from Sigma (12.53 ± 0.9 and 40.001 0.001 Greece 1.001 2. the lower quantities of components could be due to the shorter and non-protruding stigmas found in this non-cultivated sample collected in the field (Tibetan Medicine Encyclopaedia). 1992).04 ± 0.31 ± 0.001 2. respectively. 2004) and high-performance liquid chromatography (HPLC) have been employed (Iborra et al.004 Italy 3.25 ± 0.17 ± 0. Spanish.04 ± 0.43 ± 0.47 ± 0..04 ± 0.12 82. to the dissimilar drying processes possibly involving varied time periods.064 0.64 ± 0. Tarantilis et al.89 ± 0.001 1.01 China .98 ± 0. Kanakis and collaborators (2004) reported that the amount of safranal obtained by MSDE and USE were in the range of 288. was present in large amounts in both the Spanish and New Zealand samples (8.001 0.311 0.001 4.1–687.45 ± 0.. Results are expressed as means ± standard deviations.001 >0.08 ± 1.001 0.697 27.067 41.25 ± 0. different strategies as microsimultaneous hydrodistillation-extraction (MSDE). all of which could affect the concentration of glycosidic carotenoids as they are thermally labile and photochemically sensitive components (Tarantilis et al.001 3.001 9.001 1. steam distillation (SD).32 ± 0.76 ± 0.H.02 ± 0.65 ± 0.578 0.73 ± 0.25 ± 2.010 nd Sample (mg/g of stigmas) 5.29 ± 0.83 ± 0. Quality control for crocins has been obtained by HPLC assays (Li et al.062 28. Total 94. it had low concentration of components in comparison to the sample analysed from La Mancha..41 ± 0.29 ± 0.049 38.72 ± 0.001 0. 3Gentiobiosyl-kaempferol was not detected in either the Spanish or the New Zealand sample.001 nd 0.025 0.11 ± 0.14 and 7.22 ± 0.85 ± 0.06 mg/g of stigmas) followed in order by the Indian.160 25.132 0.32 ± 0.457 1. Table 1 also shows that picrocrocin. and Iranian saffron extracts.20 ± 0.001 22.14 ± 0.001 0. 2 and cis-crocin 4.32 ± 0. 1.87 ± 0.53 ± 0.29 ± 0.58 ± 1.188 27.41 ± 0.001 0.13 ± 0.42 mg/g of stigmas). French.65 ± 0.19 ± 0. 1992.86 ± 0. Previously obtained results from Spanish saffron also reported a similar picrocrocin and HTCC concentration (Iborra et al.44 ± 0.53 ± 0.001 2.25 ± 0.35 ± 0.001 0. The high concentration of safranal in the tested Greek sample agrees with a previous report describing it as a spice rich in safranal.43 ± 0. Azerbaijanian.14 ± 0.084 37. Greek saffron had the highest concentrations of transcrocin 2 0 .00 ± 0.28 ± 0.80 ± 0.001 10.002 0. New Zealand saffron had the highest concentration of trans-crocin 4 and 3. 1995).108 0.001 3.58 ± 0.21 ± 0.54 ± 0.07 ± 0.001 0.97 ± 0.001 5.13 83.07 ± 0.01 ± 0.53 ± 0.001 >0.35 74. nd: not detected. Although.77 ± 0.16 ± 0.03 ± 0.049 38.004 0. Sigma described their product as authentic Spanish saffron. Italian.005 Picrocrocin HTCC 3-Gentiobiosylkaempferol Safranal trans-crocin 4 trans-crocin 3 trans-crocin 2 0 cis-crocin 4 trans-crocin 2 cis-crocin 2 Compound 1.371 1.059 39. or the time and conditions under which the plant product was packed and stored in each country.580 24. Turkish. Greek saffron had the highest total concentration of components (94.67 ± 0.35 ± 2.24 ± 0.13 ± 0.61 ± 0.001 0. Tarantilis et al..36 ± 0. show similar data.0 ± 0. Our results in Table 1 show that trans-crocin 4 is the 0.050 6. To obtain and quantify HTCC and safranal.90 ± 0.001 1. / Food Chemistry 100 (2007) 1126–1131 0.008 0.003 0.093 36.407 0. This variation could be the result of different drying processes used.23 ± 0.001 2.068 0.007 Iran 5.20 Instrumental conditions as described in Fig.16 ± 0.223 0.12 ± 0.88 ± 0.001 2.12 ± 0.00 ± 0.05 ± 0.28 76.007 0.0 ± 0.10 ± 0.1997).28 ± 0.091 4.001 New Zealand 0.001 7.004 0.46 ± 0.84 ± 0.001 1.42 ± 0.16 ± 0.125 38.120 2.420 30.010 nd 7.32 ± 1.7 mg/100 g. Our results (Table 1) on 100 g basis for each tested samples.004 Turkey 5.001 0.95 ± 0.84 ± 0.72 ± 0.001 Spain 0. New Zealand. ultrasound-assisted extraction (USE).001 India 0.096 0.001 1. as well as to storage conditions.003 Azerbaijan 81.003 0. 1995.054 6.7–647. These components are present in very small amounts in the fresh stigma and its degree of concentration strongly depends on drying and storage conditions (Lozano et al. Spain.100 0.03 ± 0.95 ± 0.051 40.09 ± 0.92 ± 0. 1999.03 ± 0. Lozano et al.81 ± 0.008 0.001 1.21 ± 0.69 ± 0. while the highest concentration of HTCC was found in the Turkish sample (0.21 ± 0.355 0..003 0.49 ± 0.001 5.02 Sigma 9.04 ± 0.31 ± 0.02 83.197 31.06 ± 0.31 ± 0.9 mg/g of stigmas.685 23.001 0.27 ± 0.001 0.064 38. A. 849. Kanakis. Bolhasani. Negbi. Z. Rios. Bathaie. L.. & Man ˜ ez. Spain: International Society for Horticultural Science.. Crocins.g. J. as other carotenoids. 229–250). In J.. Giner. Alvarruiz. present and future prospects. Simancas. J. A. 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