Food Chemistry 166 (2015) 215–222
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Genistein isoflavone glycoconjugates in sour cherry (Prunus cerasus L.) cultivars }s b László Abrankó a,⇑, Ádám Nagy a, Blanka Szilvássy a, Éva Stefanovits-Bányai a, Attila Hegedu a b
Department of Applied Chemistry, Faculty of Food Science, Corvinus University of Budapest, 29-33 Villányi, 1118 Budapest, Hungary Department of Genetics and Plant Breeding, Faculty of Horticultural Science, Corvinus University of Budapest, Ménesi út 44, 1118 Budapest, Hungary
a r t i c l e
i n f o
Article history: Received 30 June 2013 Received in revised form 19 May 2014 Accepted 3 June 2014 Available online 12 June 2014 Keywords: Prunus cerasus L. Sour cherry Genistin Genistein Isoflavone qTOFMS
a b s t r a c t The isoflavone genistein on the contrary to its well-established health-beneficial effects is not a major component of the Western diet, since soy consumption, considered as the main dietary source of genistein, in these populations is low. Genistein compounds in twelve commercial sour cherry (Prunus cerasus L.) cultivars grown in Hungary were studied. High performance liquid chromatography coupled to electrospray ionisation quadrupole/time-of-flight mass spectrometry (HPLC-ESI-qToF-MS) was used for screening and confirmatory analyses. Genistin and genistein were found in ‘Pipacs1’, ‘Kántorjánosi’, } termo } ’ and ‘Éva’, which are native cultivars to Hungary. Genistein content of the latter three ‘Debreceni bo were in the range of 0.4–0.6, while in ‘Pipacs1’ in total 4.4 mg genistein compounds were measured expressed as aglycone equivalents per 100 g of fresh fruit flesh. These cultivars may play important role as complementary genistein sources in the Western diet. Especially ‘Pipacs 1’, may be best utilised in functional food products. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Polyphenols are plant secondary metabolites, which have drawn interest in food science, since an impressive list of health benefits are associated to these compounds when they are consumed with diet in various forms of polyphenol-rich plant foods (Crozier, Jaganath, & Clifford, 2009; Del Rio et al., 2013). Genistein – a polyphenol compound belonging to the subclass of isoflavones – also showed various health-beneficial effects in numerous experiments due to its multiple mechanisms of action. For instance, genistein can improve lipid profile and lower blood pressure and hence exert cardiovascular protection (Schwab, Stein, Scheler, & Theuring, 2012). Genistein was also proved to be a promising therapeutic agent at least for ameliorating diabetes and obesity states (Behloul & Wu, 2013). Most of the studies relating to the health beneficial effects of genistein however, have focussed on its cancer preventive properties. It has been shown that genistein can induce apoptosis in haematological tumour cells through multiple mechanisms, while protecting normal cells from toxicity. Genistein was also shown to be a potent growth inhibitor of breast, prostate, pancreatic, melanoma, and kidney cancer cells in vitro (Li, Frame, Hirsch, & Cobos, 2010). In a recent study,
⇑ Corresponding author. Tel.: +36 1482 6163; fax: +36 1466 4272. E-mail address: [email protected] (L. Abrankó). http://dx.doi.org/10.1016/j.foodchem.2014.06.007 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
daidzein and genistein were effectively induced apoptosis in HT29 colon cancer cells (Kim, Song, Kim, Choi, & Jang, 2012). In substantial amounts, genistein has been found almost exclusively in leguminous plants so far with highest concentrations occurring in soybean (Glycine max). Therefore, soybean is considered as the main dietary source of genistein (Liggins et al., 2000a,b). Concentrations of genistein in soybean are inherently heterogeneous depending on type, climate, crop year, and location of growth (Chan et al., 2009). According to a comprehensive database for the isoflavone content of selected foods, published by the US Department of Agriculture, genistein was varied in the range of 5.6–276 mg/100 g in raw mature soybeans (Bhagwat, Haytowitz, & Holden, 2008). Soy is still the staple food in Asia, but it is a relative newcomer at the dinner table in other parts of the world, which means that human exposure to genistein varies widely because of cultural differences in diet (Li et al., 2010). This might explain the conclusion achieved in a number of human studies i.e., risk of those cancers, where genistein was proved to have preventive actions, is lower among populations in Japan and China compared to the populations of the USA and Europe (Kurahashi, Iwasaki, Inoue, Sasazuki, & Tsugane, 2008; Lampe et al., 2007; Yang et al., 2012). The isoflavone genistein on the contrary to its well-established health beneficial effects not a major component of the Western diet, since soy food intake in these populations is typically low (Crozier, Del Rio, & Clifford, 2010; Crozier et al., 2009; Lampe
216
L. Abrankó et al. / Food Chemistry 166 (2015) 215–222
et al., 2007). However, in addition to soybean, genistein is present in small quantities in a number of other edible plants. According to the studies of Liggins et al., legumes was the next richest group of foods following ones derived from soybean. Legumes contained between 0.2 and 0.6 mg genistein and daidzein combined per 100 g of wet weight of food (Liggins et al., 2000b). With respect to fruits, they found that currants and raisins were the richest sources of the isoflavones, containing around 0.2 mg of the genistein and daidzein combined per 100 g of wet weight of food. (Liggins et al., 2000a). In this context, peanut should also be mentioned containing genistein and daidzein around 0.03 mg/100 g (Chukwumah, Walker, Vogler, & Verghese, 2012). In a recent study it was shown that in groundnut, genistein was also present in the form of genistein-7-O-genitiobioside, which glycoconjugate was not observed before (Nara, Nihei, Ogasawara, Koga, & Kato, 2011). Considering the small isoflavone concentrations typical in plant foods other than soy, it is evident that the inclusion of a soy product in a diet will expose the consumer to significant concentrations of daidzein and genistein. Nevertheless, in fruits, nuts, and vegetables the concentration of isoflavones varies significantly therefore those ones containing higher concentrations can contribute to the daily dietary genistein intake as well (Liggins et al., 2000a). Sour or tart cherries (Prunus cerasus L.) are commercially important and consumed in a variety of ways, including fresh, frozen and canned, or as juice, brined or dried. Anthocyanins and other flavonoids, as well as melatonin, in various cultivars were analysed and it has been known that sour cherries contain substantial amounts of anthocyanins and phenolic acids (Ficzek et al., 2011; Kirakosyan, Seymour, Llanes, Kaufman, & Bolling, 2009). So far, studies focus on the health-beneficiary effects of sour cherry consumption are scarce, however a few interesting papers investigating the biological effects of sour cherry constituents present in fruit (Hevesi et al., 2012; Khoo, Clausen, Pedersen, & Larsen, 2011; Kim, Heo, Kim, Yang, & Lee, 2005) and seed kernel are available (Bak et al., 2010). With respect to genistein content of sour cherries, published data are very limited (Wang, Nair, Strasburg, Booren, & Gray, 1999). One of the explanations for that might be that based on the available data in this context, it has been considered that sour cherries in general do not contain genistein in substantial amounts. However, natural variation among sour cherry cultivars could be considerable, which means, as is known to occur in soya (Bhagwat et al., 2008; Chan et al., 2009), certain sour cherry cultivars contain above-average amounts of polyphenols also including some unexpected ones such as genistein. In this study, twelve sour cherry (Prunus cerasus L.) cultivars grown in Hungary were screened for genistein compounds in a
non-target manner. High performance liquid chromatography coupled to quadrupole/time-of-flight mass spectrometry, equipped with electrospray ion source (HPLC-ESI-qTOFMS) was used for screening and confirmatory analyses of indicated genistein glycoconjugates. In addition, UV spectra of indicated compounds were also applied for confirmatory purposes. Quantitative determination of genistein compounds was also performed. 2. Materials and methods 2.1. Plant material Sour cherries (Prunus cerasus L.) grown in Hungary were studied. Samples were harvested in 2009 and 2010. Twelve Hungarian sour cherry (Prunus cerasus L.) genotypes were tested in the present study, which are commercial cultivars of Carpathian Basin origin (Hungary or Serbia). Studied cultivars are listed in Table 2. All cultivars were cultivated at the same germplasm collection in the Research and Extension Centre for Fruit Growing (Újfehétró, Eastern Hungary, 47 °N latitude, 21 °E longitude and 122 m altitude). Fruits were harvested in June–July 2009 and 2010 at consumption maturity stage. Pictures of the fruits are presented in Fig. 1. 2.2. Chemicals and standards Acetonitrile and methanol (Prolabo HiPerSolv, VWR International, Radnor, PA, USA) used were super gradient grade. Formic acid (98% for mass spectrometry) was obtained from Fluka (Sigma–Aldrich, St. Luis, MO, USA). Crystalline reference substances of genistein aglycone (purity P 95% based on HPLC-UV) and genistein-7-O-b-D-glucoside (genistin, purity P 99% based on HPLC-UV) and daidzein (purity P 95% based on HPLC-UV) were obtained from Extrasynthese (Genay, France). A Milli-Q ultrapure water system (Merck Millipore, Billerica, MA, USA) was used throughout the study to obtain high purity water. 2.3. Sample preparation Sour cherry fruits were halved and pitted before lyophilisation. Lyophilised samples were pulverised and an amount of 200 mg was extracted for 40 min with 10 ml 60/39/1 methanol/water/formic acid solution using an ultrasonic bath. Extracts were centrifuged and 4 ml supernatant was evaporated approximately to 0.5–0.7 ml in a vacuum centrifuge. Afterwards 100 ll acetonitrile and 10 ll 1:1 diluted formic acid was added and the samples were reconstituted to 1 ml with water and were thoroughly vortexed. Daidzein aglycone was added to all samples as surrogate standard.
Table 1 Suspected genistein compounds found in some sour cherry samples. Formulae of observed diagnostic ions and their identification errors obtained as a result of in-source fragmentations using 210 V fragmentor voltage. Retention time, min
Compound
Found diagnostic ions
Ion formula
Exact mass
18.33
Genistein-dihexoside
Gen (Y+0) Gen-H Gen-H-H Gen-H-H-Na
C15H11O5 C21H21O10 C27H31O15 C27H30O15Na
271.0601 433.1129 595.1658 617.1483
Error, ppm 0.37 1.85 1.18 1.78
21.03
Genistin (genistein-7-O-b-D-glucoside)
Gen (Y+0) Gen-H Gen-H-Na
C15H11O5 C21H21O10 C21H20O10Na
271.0601 433.1129 455.0954
0.00 0.69 1.10
21.60
Genistein-hexoside (a)
Gen (Y+0) Gen-H-Na
C15H11O5 C21H20O10Na
271.0601 455.0954
0.37 1.98
22.73
Genistein-hexoside (b)
Gen (Y+0) Gen-H Gen-H-Na
C15H11O5 C21H21O10 C21H20O10Na
271.0601 433.1129 455.0954
0.00 0.69 1.32
31.33
Genistein aglycone
Gen (Y+0)
C15H11O5
271.0601
0.00
L. Abrankó et al. / Food Chemistry 166 (2015) 215–222 Table 2 Measured concentrations of genistin, genistein and a tentatively identified Gen-H in sour cherry samples grown in Hungary in 2009 and 2010. Values are the average of results obtained in two individual sample preparations and are given in mg per 100 g wet weight. (The ± values are estimations for the deviation of the mean values expressed as the corrected standard deviation.) Genistin and genistein was quantified based on MS/MS data, while Gen-H was quantified based on the calibration equation of genistin using the UV signal at 260 nm. MS/MS
a
UV
Variety
Year
Genistin
Genistein
Gen-H (tR = 22.73)
Pipacs1 Kántorjánosi }termo } Debreceni bo Éva Oblachiskha VN-7 }termo } Érdi bo } di Csengo Cigány404 Korai pipacs VN-4 Sárdy SF
2009 2009 2009 2010 2009 2009 2009 2009 2009 2009 2009 2010
4.49 ± 0.62 0.54 ± 0.01 0.52 ± 0.01 0.79 ± 0.01
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Genistein isoflavone glycoconjugates in sour cherry (Prunus cerasus L.) cultivars }s b László Abrankó a,⇑, Ádám Nagy a, Blanka Szilvássy a, Éva Stefanovits-Bányai a, Attila Hegedu a b
Department of Applied Chemistry, Faculty of Food Science, Corvinus University of Budapest, 29-33 Villányi, 1118 Budapest, Hungary Department of Genetics and Plant Breeding, Faculty of Horticultural Science, Corvinus University of Budapest, Ménesi út 44, 1118 Budapest, Hungary
a r t i c l e
i n f o
Article history: Received 30 June 2013 Received in revised form 19 May 2014 Accepted 3 June 2014 Available online 12 June 2014 Keywords: Prunus cerasus L. Sour cherry Genistin Genistein Isoflavone qTOFMS
a b s t r a c t The isoflavone genistein on the contrary to its well-established health-beneficial effects is not a major component of the Western diet, since soy consumption, considered as the main dietary source of genistein, in these populations is low. Genistein compounds in twelve commercial sour cherry (Prunus cerasus L.) cultivars grown in Hungary were studied. High performance liquid chromatography coupled to electrospray ionisation quadrupole/time-of-flight mass spectrometry (HPLC-ESI-qToF-MS) was used for screening and confirmatory analyses. Genistin and genistein were found in ‘Pipacs1’, ‘Kántorjánosi’, } termo } ’ and ‘Éva’, which are native cultivars to Hungary. Genistein content of the latter three ‘Debreceni bo were in the range of 0.4–0.6, while in ‘Pipacs1’ in total 4.4 mg genistein compounds were measured expressed as aglycone equivalents per 100 g of fresh fruit flesh. These cultivars may play important role as complementary genistein sources in the Western diet. Especially ‘Pipacs 1’, may be best utilised in functional food products. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Polyphenols are plant secondary metabolites, which have drawn interest in food science, since an impressive list of health benefits are associated to these compounds when they are consumed with diet in various forms of polyphenol-rich plant foods (Crozier, Jaganath, & Clifford, 2009; Del Rio et al., 2013). Genistein – a polyphenol compound belonging to the subclass of isoflavones – also showed various health-beneficial effects in numerous experiments due to its multiple mechanisms of action. For instance, genistein can improve lipid profile and lower blood pressure and hence exert cardiovascular protection (Schwab, Stein, Scheler, & Theuring, 2012). Genistein was also proved to be a promising therapeutic agent at least for ameliorating diabetes and obesity states (Behloul & Wu, 2013). Most of the studies relating to the health beneficial effects of genistein however, have focussed on its cancer preventive properties. It has been shown that genistein can induce apoptosis in haematological tumour cells through multiple mechanisms, while protecting normal cells from toxicity. Genistein was also shown to be a potent growth inhibitor of breast, prostate, pancreatic, melanoma, and kidney cancer cells in vitro (Li, Frame, Hirsch, & Cobos, 2010). In a recent study,
⇑ Corresponding author. Tel.: +36 1482 6163; fax: +36 1466 4272. E-mail address: [email protected] (L. Abrankó). http://dx.doi.org/10.1016/j.foodchem.2014.06.007 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
daidzein and genistein were effectively induced apoptosis in HT29 colon cancer cells (Kim, Song, Kim, Choi, & Jang, 2012). In substantial amounts, genistein has been found almost exclusively in leguminous plants so far with highest concentrations occurring in soybean (Glycine max). Therefore, soybean is considered as the main dietary source of genistein (Liggins et al., 2000a,b). Concentrations of genistein in soybean are inherently heterogeneous depending on type, climate, crop year, and location of growth (Chan et al., 2009). According to a comprehensive database for the isoflavone content of selected foods, published by the US Department of Agriculture, genistein was varied in the range of 5.6–276 mg/100 g in raw mature soybeans (Bhagwat, Haytowitz, & Holden, 2008). Soy is still the staple food in Asia, but it is a relative newcomer at the dinner table in other parts of the world, which means that human exposure to genistein varies widely because of cultural differences in diet (Li et al., 2010). This might explain the conclusion achieved in a number of human studies i.e., risk of those cancers, where genistein was proved to have preventive actions, is lower among populations in Japan and China compared to the populations of the USA and Europe (Kurahashi, Iwasaki, Inoue, Sasazuki, & Tsugane, 2008; Lampe et al., 2007; Yang et al., 2012). The isoflavone genistein on the contrary to its well-established health beneficial effects not a major component of the Western diet, since soy food intake in these populations is typically low (Crozier, Del Rio, & Clifford, 2010; Crozier et al., 2009; Lampe
216
L. Abrankó et al. / Food Chemistry 166 (2015) 215–222
et al., 2007). However, in addition to soybean, genistein is present in small quantities in a number of other edible plants. According to the studies of Liggins et al., legumes was the next richest group of foods following ones derived from soybean. Legumes contained between 0.2 and 0.6 mg genistein and daidzein combined per 100 g of wet weight of food (Liggins et al., 2000b). With respect to fruits, they found that currants and raisins were the richest sources of the isoflavones, containing around 0.2 mg of the genistein and daidzein combined per 100 g of wet weight of food. (Liggins et al., 2000a). In this context, peanut should also be mentioned containing genistein and daidzein around 0.03 mg/100 g (Chukwumah, Walker, Vogler, & Verghese, 2012). In a recent study it was shown that in groundnut, genistein was also present in the form of genistein-7-O-genitiobioside, which glycoconjugate was not observed before (Nara, Nihei, Ogasawara, Koga, & Kato, 2011). Considering the small isoflavone concentrations typical in plant foods other than soy, it is evident that the inclusion of a soy product in a diet will expose the consumer to significant concentrations of daidzein and genistein. Nevertheless, in fruits, nuts, and vegetables the concentration of isoflavones varies significantly therefore those ones containing higher concentrations can contribute to the daily dietary genistein intake as well (Liggins et al., 2000a). Sour or tart cherries (Prunus cerasus L.) are commercially important and consumed in a variety of ways, including fresh, frozen and canned, or as juice, brined or dried. Anthocyanins and other flavonoids, as well as melatonin, in various cultivars were analysed and it has been known that sour cherries contain substantial amounts of anthocyanins and phenolic acids (Ficzek et al., 2011; Kirakosyan, Seymour, Llanes, Kaufman, & Bolling, 2009). So far, studies focus on the health-beneficiary effects of sour cherry consumption are scarce, however a few interesting papers investigating the biological effects of sour cherry constituents present in fruit (Hevesi et al., 2012; Khoo, Clausen, Pedersen, & Larsen, 2011; Kim, Heo, Kim, Yang, & Lee, 2005) and seed kernel are available (Bak et al., 2010). With respect to genistein content of sour cherries, published data are very limited (Wang, Nair, Strasburg, Booren, & Gray, 1999). One of the explanations for that might be that based on the available data in this context, it has been considered that sour cherries in general do not contain genistein in substantial amounts. However, natural variation among sour cherry cultivars could be considerable, which means, as is known to occur in soya (Bhagwat et al., 2008; Chan et al., 2009), certain sour cherry cultivars contain above-average amounts of polyphenols also including some unexpected ones such as genistein. In this study, twelve sour cherry (Prunus cerasus L.) cultivars grown in Hungary were screened for genistein compounds in a
non-target manner. High performance liquid chromatography coupled to quadrupole/time-of-flight mass spectrometry, equipped with electrospray ion source (HPLC-ESI-qTOFMS) was used for screening and confirmatory analyses of indicated genistein glycoconjugates. In addition, UV spectra of indicated compounds were also applied for confirmatory purposes. Quantitative determination of genistein compounds was also performed. 2. Materials and methods 2.1. Plant material Sour cherries (Prunus cerasus L.) grown in Hungary were studied. Samples were harvested in 2009 and 2010. Twelve Hungarian sour cherry (Prunus cerasus L.) genotypes were tested in the present study, which are commercial cultivars of Carpathian Basin origin (Hungary or Serbia). Studied cultivars are listed in Table 2. All cultivars were cultivated at the same germplasm collection in the Research and Extension Centre for Fruit Growing (Újfehétró, Eastern Hungary, 47 °N latitude, 21 °E longitude and 122 m altitude). Fruits were harvested in June–July 2009 and 2010 at consumption maturity stage. Pictures of the fruits are presented in Fig. 1. 2.2. Chemicals and standards Acetonitrile and methanol (Prolabo HiPerSolv, VWR International, Radnor, PA, USA) used were super gradient grade. Formic acid (98% for mass spectrometry) was obtained from Fluka (Sigma–Aldrich, St. Luis, MO, USA). Crystalline reference substances of genistein aglycone (purity P 95% based on HPLC-UV) and genistein-7-O-b-D-glucoside (genistin, purity P 99% based on HPLC-UV) and daidzein (purity P 95% based on HPLC-UV) were obtained from Extrasynthese (Genay, France). A Milli-Q ultrapure water system (Merck Millipore, Billerica, MA, USA) was used throughout the study to obtain high purity water. 2.3. Sample preparation Sour cherry fruits were halved and pitted before lyophilisation. Lyophilised samples were pulverised and an amount of 200 mg was extracted for 40 min with 10 ml 60/39/1 methanol/water/formic acid solution using an ultrasonic bath. Extracts were centrifuged and 4 ml supernatant was evaporated approximately to 0.5–0.7 ml in a vacuum centrifuge. Afterwards 100 ll acetonitrile and 10 ll 1:1 diluted formic acid was added and the samples were reconstituted to 1 ml with water and were thoroughly vortexed. Daidzein aglycone was added to all samples as surrogate standard.
Table 1 Suspected genistein compounds found in some sour cherry samples. Formulae of observed diagnostic ions and their identification errors obtained as a result of in-source fragmentations using 210 V fragmentor voltage. Retention time, min
Compound
Found diagnostic ions
Ion formula
Exact mass
18.33
Genistein-dihexoside
Gen (Y+0) Gen-H Gen-H-H Gen-H-H-Na
C15H11O5 C21H21O10 C27H31O15 C27H30O15Na
271.0601 433.1129 595.1658 617.1483
Error, ppm 0.37 1.85 1.18 1.78
21.03
Genistin (genistein-7-O-b-D-glucoside)
Gen (Y+0) Gen-H Gen-H-Na
C15H11O5 C21H21O10 C21H20O10Na
271.0601 433.1129 455.0954
0.00 0.69 1.10
21.60
Genistein-hexoside (a)
Gen (Y+0) Gen-H-Na
C15H11O5 C21H20O10Na
271.0601 455.0954
0.37 1.98
22.73
Genistein-hexoside (b)
Gen (Y+0) Gen-H Gen-H-Na
C15H11O5 C21H21O10 C21H20O10Na
271.0601 433.1129 455.0954
0.00 0.69 1.32
31.33
Genistein aglycone
Gen (Y+0)
C15H11O5
271.0601
0.00
L. Abrankó et al. / Food Chemistry 166 (2015) 215–222 Table 2 Measured concentrations of genistin, genistein and a tentatively identified Gen-H in sour cherry samples grown in Hungary in 2009 and 2010. Values are the average of results obtained in two individual sample preparations and are given in mg per 100 g wet weight. (The ± values are estimations for the deviation of the mean values expressed as the corrected standard deviation.) Genistin and genistein was quantified based on MS/MS data, while Gen-H was quantified based on the calibration equation of genistin using the UV signal at 260 nm. MS/MS
a
UV
Variety
Year
Genistin
Genistein
Gen-H (tR = 22.73)
Pipacs1 Kántorjánosi }termo } Debreceni bo Éva Oblachiskha VN-7 }termo } Érdi bo } di Csengo Cigány404 Korai pipacs VN-4 Sárdy SF
2009 2009 2009 2010 2009 2009 2009 2009 2009 2009 2009 2010
4.49 ± 0.62 0.54 ± 0.01 0.52 ± 0.01 0.79 ± 0.01
