Impaginato 85 1. Introduction Sunflower (Helianthus annuus L.) is one of the most important oilseed crops. Besides soy and rape- seed oil, sunflower oil is ranking third with a world- wide production of about 44 million tons each year f r o m 2 0 1 2 t o 2 0 1 6 , 2 8 m i l l i o n t o n s i n t h e E U ( C o m m i t t e e f o r t h e C o m m o n O r g a n i z a t i o n o f Agricultural Markets, 2016). Sunflowers prove to be a protein source of great interest for human nutrition; moreover, the residues originating from oil extrac- tion are rich in phenolic antioxidants, which account for 1-4% of the total mass with chlorogenic acid being the predominant component (Weisz et al., 2009). Polyphenols in sunflower seeds were identi- fied and quantified after HPLC analysis (Aramendia et al., 2000; Pedrosa et al., 2000). The main phenolic compounds present in both the kernel and hull besides chlorogenic acid are caffeic acid and caf- feoylquinic derivatives. Phenolic compounds in sun- flower seeds have been shown to exert a high antiox- idative potential, which might be beneficial from a biofunctional point of view and may be used as effec- tive antioxidants for sunflower oils (De Leonardis et al., 2003, 2005; Anjum et al., 2012). Organic agriculture is most widely used and its benefits concern overall the environment and the health of food, which is not contaminated with pesti- cides and synthetic fertilizers. However organic fertil- izer of biological origin may lead in the long term to t h e “ c o n v e n t i o n a l i z a t i o n o f o r g a n i c f a r m i n g ” (Darnhofer et al., 2010). In order to go deeper in the organic management, the Montepaldi Long Term Experiment (MOLTE) trial in central Italy has been set up to compare three agro-ecosystems with different management: two organic (old organic since 1992 and young organic since 2001) and one conventional (Migliorini, 2014). The aim of this research is to fol- low the fate of polyphenols in teguments, kernels and oil from sunflower seeds grown under the above-mentioned three different conditions (one conventional and two organic) in order to assess whether such condition may affect the polyphenols profile and which compounds may be regarded as innovative parameters in order to deeper investigate the relation among soil condition, agricultural man- agement and seed characteristics. 2. Materials and Methods The Montepaldi Long Term Experiment (MOLTE) h a s b e e n a c t i v e s i n c e 1 9 9 1 i n t h e f a r m o f t h e University of Florence (location Montepaldi, San Adv. Hort. Sci., 2017 31(2): 85-88 DOI: 10.13128/ahs-20608 Seeds and oil polyphenol content of sunflower (Helianthus annuus L.) grown with different agricultural management A. Romani 1, P. Pinelli 1, V. Moschini 2, D. Heimler 2 (*) 1 Dipartimento di Statistica, Informatica, Applicazioni “G. Parenti”, Università degli Studi di Firenze, Viale Morgagni, 40, 50134 Firenze, Italy. 2 Dipartimento di Scienze delle Produzioni Agroalimentari e dell’Ambiente, Università degli Studi di Firenze, Piazzale delle Cascine, 18, 50144 Firenze, Italy. Key words: organic management, polyphenol content, sunflower. Abstract: Using a long term experiment (20 and 11 years of organic cultivation on the same soil), sunflower was cultivat- ed under organic management and in a different part of the same farm under conventional management. Kernels, tegu- ments and oils were analyzed for their polyphenols content. Five caffeoylquinic acids were identified. No qualitative dif- ferences were found in the three cases, while quantitative differences have been pointed out and discussed. (*) Corresponding author: daniela.heimler@unifi.it Received for publication 2 May 2017 Accepted for publication 15 May 2017 Copyright: © 2017 Author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2017 31(2): 85-88 86 C a s c i a n o , V a l d i P e s a , L o n g . 1 1 ° 0 9 ’ 0 8 ’ ’ E , L a t . 43°40’16’’ N) covering a slightly sloping surface of about 15 ha at 90 m a.s.l. The MOLTE experiment has a system approach and includes the following three different micro agro-ecosystem managements: a) “Old Organic” (OldO) with an area of 5.2 ha, divid- ed into 4 fields under organic since 1992; b) “YoungOrganic” (YngO) with an area of 5.2 ha, divided into 4 fields (integrated farming) from 1992 to 2000 and converted into organic manage- ment since 2001; c) “Conventional” (Conv) area of 2.6 ha divided into 2 conventional fields, where farming techniques used were those normally used in the territory of the study area for conventional management. On October 28, 2013, after harvesting the whole sunflower plant, seeds were put in an oven at 60°C for 48 hours and then preserved in paper bags. Seeds were manually dehulled in order to obtain kernel and tegument. Phenolic compounds were extracted for 30 min from kernels (about 2 g) and from teguments (about 1 g) with 70:30 ethanol/water (25 and 15 ml, respectively). The extracts were evaporated under vacuum at room temperature and finally dissolved in 10 ml ethanol/water (70:30). Cold-pressed seeds oils ( 2 5 m l ) w e r e e x t r a c t e d w i t h 3 x 2 5 m l o f 7 0 : 3 0 ethanol/water, adjusted to pH 2.0 with formic acid; each step involved an extraction for 30 min at room temperature. The extracts were combined and defat- ted with 3x50 ml hexane. The defatted extracts were evaporated to dryness under vacuum at room tem- perature and finally dissolved in ethanol/water (70:30) to a final volume of 4 ml. Solvents and reagents All the solvents (HPLC grade) and formic acid (ACS reagent) were purchased from Aldrich Chemical C o m p a n y I n c . ( M i l w a u k e e , W i s c o n s i n , U S A ) . Chlorogenic acid was obtained from Extrasynthese S.A. (Lyon, Nord-Genay, France). The HPLC-grade water was obtained via double-distillation and purifi- cation with a Labconco Water Pro PS polishing sta- tion (Labconco Corporation, Kansas City, USA). HPLC analysis HPLC/DAD Analysis. The HPLC/DAD analyses were performed with an HP 1100L liquid chromatograph equipped with HP DAD (Agilent Technologies, Palo Alto, CA, USA). A Kinetex C18 column 100×2.1 mm, 5 μm (Phenomenex) operating at 30°C was used. The eluents were H2O adjusted to pH 3.2 by formic acid and acetonitrile. A four-step linear solvent gradient was performed starting from 100% water up to 100% acetonitrile, with a flow rate of 0.2 ml/min for a 30- min period (Table 1). HPLC/ESI-MS Analysis. The HPLC-MS analyses were performed using an HP 1100L liquid chromato- graph equipped with a DAD and 1100 MS detectors. The interface was an HP 1100 MSD API-electrospray (Agilent Technologies). Mass spectrometer operating conditions were the following: gas temperature 350°C at a flow rate of 10.0 l/min, nebulizer pressure 30 psi, quadrupole temperature 30°C and capillary voltage 3500 V. The mass spectrometer operated in positive and negative ionization mode at 80-120 eV, for both ionization modes. 3. Results and Discussion In figure 1, the chromatographic profile of organic sunflower kernel, registered at 330 nm, maximum wavelength of absorbance of caffeic acid and its derivatives, is reported. Eight compounds have been identified: 3-O-caffeoylquinic acid, chlorogenic acid, 4-O-caffeoylquinic acid, three derivatives of caffeic acid, 3,5-O-dicaffeoylquinic acid, and 4,5-O-dicaf- Table 1 - Linear solvent gradient system used in HPLC-DAD and HPLC-MS analysis Time (min) A B 0.10 100.0 0.0 5.00 80.0 20.0 7.00 80.0 20.0 13.00 70.0 30.0 18.00 70.0 30.0 22.00 30.0 70.0 26.00 30.0 70.0 30.00 0.0 100.0 Fig. 1 - HPLC chromatogram of sunflower kernel extract record- ed at 330 nm. Peaks= 1. 3-O-caffeoylquinic acid; 2. chlorogenic acid; 3. 4-O-caffeoylquinic acid; 4-6. caffeic acid derivatives; 7. 3,5- O-dicaffeoylquinic acid; 8. Ac. 4,5- O-dicaffeoylquinic acid. Solvent A= H2O adjusted to pH 3.2 by HCOOH. Solvent B= CH3CN. Romani et al. - Seeds and oil polyphenol content of sunflower 87 feoylquinic acid. In the shells the same compounds with the exception of 4-O-caffeoylquinic acid and the two dicaffeoylquinic acids were identified. The same caffeoyl and dicaffeoylquinic acid derivatives were already found by Aramendia et al. (2000), Pedrosa et al. (2000), and Weisz et al. (2009). The qualitative pattern does not change along with the agricultural management. As concerns quantitative data, Table 2 reports the quantitative data of kernel and tegu- ments. Total polyphenols amount is the lowest, in the case of kernel, when the plant grows on the old organic field, while it is almost the same with the other two managements; in the case of teguments, on the contrary, the lowest amount is observed in the case of conventional management. It should however be noted that in the case of teguments, polyphenols content is very low changing from about 1 0 % t o 2 % w i t h r e s p e c t t o k e r n e l c o n t e n t . C h l o r o g en i c a c i d i s t h e m a i n c o m p o u n d a n d i t accounts for about 90% in tegument. Chlorogenic acid in our samples, both kernel and tegument, is about ten times more abundant with respect to the quantitative data reported by Aramedia et al. (2000) and Pedrosa et al. (2000). The comparison of quanti- tative data is however difficult since different tech- niques and sample treatments are involved (Weisz et al., 2009). Polyphenols content along with agriculture management has recently reviewed and in most veg- etables grown under organic conditions, a higher content of polyphenols has been found; on the other hand a higher soil nitrogen availability decreases polyphenols content (Heimler et al., 2017). Our data support previous findings that indicate how old man- aged organic soil was the most efficient in term of C and N storage (Migliorini et al., 2014). Furthermore, when the individual compounds are taken into account the relative percentages of compounds from new organic and conventional managed soils are sim- ilar, while in the case of the old managed organic soil, notwithstanding the lowest total polyphenol content, a higher dicaffeoylquinic acids content was found (17% with respect to 12%). Chlorogenic acid and d i c a f f eo yl q u i n i c a c i d s d eri ve f ro m t h e p h en yl - propanoid pathway. Generally, this pathway is induced by biotic and abiotic stress such as wound- ing, UV irradiation, or pathogen attack (Moglia et al., 2008). No information is available on the regulation of dicaffeoylquinic acids in any plant species, even if the same regulation of chlorogenic acid synthesis could be foreseen (Moglia et al., 2008). In Table 3, the data of sunflower oil are reported. Oil has been obtained by means of a mechanical OldO= old organic; YoungO= young organic; CO= conventional (see experimental section). ND= not determined. Standard deviation within brackets. Table 2 - Polyphenols (mg/g, fresh weight) in sunflower kernel and tegument Compound Agricultural management Kernel Tegument OldO YoungO CO OldO YoungO CO 3-O-caffeoylquinic acid 2.51(0.37) 4.57(0.82) 4.74(0.76) 0.08(0.01) 0.08(0.01) 0.05(0.01) chlorogenic acid 6.25(1.12) 11.39(1.94) 11.82(1.42) 0.95(0.18) 0.96(0.18) 0.41(0.07) 4-O-caffeoylquinic acid 0.44(0.08) 0.8(0.11) 0.83(0.13) 0.03(0.005) traces traces caffeic acid derivatives 0.21(0.04) 0.38(0.04) 0.39(0.06) traces n.d. traces 3,5-O-dicaffeoylquinic acid 1.3(0.24) n.d. n.d. n.d. n.d. n.d. 4,5-O-dicaffeoylquinic acid 0.77(0.11) 2.36(0.34) 2.45(0.27) n.d. n.d. n.d. Total polyphenols 11.48 19.5 20.23 1.06 1.04 0.46 OldO= old organic; YoungO= young organic; CO= conventional (see experimental section). ND= not determined. Standard deviation within brackets. Table 3 - Polyphenols (mg/l) in sunflower oil Compound Agricultural management OldO YoungO CO 3-O-caffeoylquinic acid 0.232(0.192) 0.35(0.09) 0.176(0.057) chlorogenic acid 1.96(0.393) 2.43(0.11) 1.936(0.12) p-coumaroylquinic acid 2.36(0.318) 0.112(0.022) 0.592(0.079) 4-O-caffeoylquinic acid 0.048(0.005) 0.104(0.011) 0.08(0.02) caffeic acid derivatives 2.28(0.644) 0.328(0.079) 3.216(0.415) not identified polar compounds 0.504(0.011) 1.2(0.045) 1.456(0.429) Total polyphenols 7.384 4.368 7.456 Adv. Hort. Sci., 2017 31(2): 85-88 88 press, which is the only technology that allows the maintenance of polyphenols high content (Bendini et al., 2011). 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