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Investigating the Photochemical Changes of Chlorogenic Acids Induced by UV Light in Model Systems and in Agricultural Practice with Stevia rebaudiana Cultivation as an Example Hande Karaköse, Rakesh Jaiswal, Sagar Deshpande, and Nikolai Kuhnert J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00838 • Publication Date (Web): 20 Feb 2015 Downloaded from http://pubs.acs.org on March 3, 2015
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Journal of Agricultural and Food Chemistry
An Investigation of the Photochemical Changes of Chlorogenic Acids Induced by UV Light in Model Systems and in Agricultural Practice with Stevia rebaudiana Cultivation as an Example
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Hande Karaköse, Rakesh Jaiswal, Sagar Deshpande and Nikolai Kuhnert*
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Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen, Germany
*Author to whom correspondence should be addressed Tel: 49 421 200 3120; Fax: 49 421 200 3229; E-mail:
[email protected] 1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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ABSTRACT
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Mono and di acyl chlorogenic acids undergo photochemical trans-cis isomerization under UV
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irradiation. The photochemical equilibrium composition was established for eight selected
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derivatives. In contrast to all other dicaffeoylquinic acid derivatives cynarin (1,3-
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dicaffeoylquinic acid) undergoes a [2+2] photochemical cycloaddition reaction, constituting a
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first example of Schmidt’s law in a natural product family. The relevance of photochemical
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isomerization in agricultural practice was investigated using 120 samples of Stevia
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rebaudiana leave samples grown under defined cultivation conditions. Ratios of cis to trans
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chlorogenic acids were determined in leaf samples and correlated with climatic and harvesting
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conditions.
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derivatives and sunshine hours prior to harvesting and illustrate the relevance of UV exposure
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to plant material affecting its phytochemical composition.
The data indicate a clear correlation between the formation of cis-caffeoyl
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Keywords:
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photocycloaddition; tandem mass spectrometry
Chlorogenic
acids;
Stevia
rebaudiana;
cis-trans
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isomerization;
[2+2]
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Journal of Agricultural and Food Chemistry
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INTRODUCTION
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All dietary plants are exposed to significant amounts of UV light. The upper part of the plant,
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in particular leaves and fruits are exposed to UV irradiation from the sun during their growth
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period. If photochemically active compounds are present in the irradiated plant organs,
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dramatic photochemical changes can be expected and new products, which could be
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considered as natural products, are formed. Vitamin D must be considered as the prime
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example of such a photochemically produced natural product.1 Additionally human
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intervention uses UV irradiation on many occasions to control microbial growth and therefore
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extend shelf-life times in dietary materials of plant origin, again resulting in novel
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photoproducts being produced.2
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The photoproducts of polyphenols have been investigated on numerous occasions.3-5 In
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general phenolics are considered to be photostable, linked to their proposed role in UV
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protection of plant tissue. Chlorogenic acids (CGAs), which we define as hydroxycinnamoyl
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esters of quinic acid, are ubiquitous plant secondary metabolites associated with UV B light
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protection of plant tissue due to their cinnamoyl moiety maximum of absorption around 320-
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330 nm.6-8 Recently we reported the presence of photoisomerisation products of CGAs in
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plant leaf material exposed to natural UV light. In particular in leaf samples significant
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amounts of cis-cinamoylquinic acids could be detected in a variety of dietary relevant plant
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species including coffee leaves,7 Stevia rebaudiana,9 lettuce (Lactuca sativa L.),10 Gallium
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odoratum,11 Rudbeckia hirta,12 Ilex paraguariensis13 and others.
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In this study we investigated the photochemistry of CGAs in detail. Important factors to be
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established include the position of the photochemical equilibrium of CGA derivatives, the
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question of whether trans-cis isomerization is the only photochemical reaction pathway
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possible, and finally the relevance of photochemical reactions for agricultural practice. The
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question to be answered is whether growth and harvesting conditions actually influence the 3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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phytochemical profile of a crop and whether these photoproducts can be used as quality
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markers for a certain crop. Since currently no data exist on the biological activity or biological
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role of CGA photoproducts any impact assessment and evaluation is a task for the future.
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MATERIALS AND METHODS
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All the chemicals (Analytical grade) and the authentic standards of CGAs, 3-caffeoylquinic
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acid (3-CQA),1, 4-caffeoylquinic acid (4-CQA),2, 5-caffeoylquinic acid (5-CQA),3, 1,3-
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dicaffeoylquinic acid (1,3-diCQA) (cynarin),4, 3,4-dicaffeoylquinic acid (3,4-diCQA),5, 3,5-
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dicaffeoylquinic acid (3,5-diCQA),6 and 4,5-dicaffeoylquinic acid (4,5-diCQA),7 were
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purchased from Sigma-Aldrich and Phytolab (Germany).
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Methanolic Extract of S. rebaudiana. S. rebaudiana leaves (2 g) was immersed in liquid
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nitrogen, ground in a hammer mill, and extracted first with 150 mL of chloroform in a Soxhlet
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apparatus (Büchi B-811 Soxhlet extraction system, Büchi, Essen, Germany) for 2 h and then
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with 150 mL of methanol for another 2 h. Solvents were removed from the methanolic extract
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in vacuo, and extracts were stored at - 20 oC until required.
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LC-MSn. The LC equipment (Agilent, Karlsruhe, Germany) comprised a binary pump, an
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auto sampler with a 100 μL loop, and a DAD detector with a light-pipe flow cell, recording at
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254, 280 and 320 nm, and scanning from 200 to 600 nm. This was interfaced with an ion-trap
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mass spectrometer fitted with an ESI source (Bruker Daltonics, Bremen, Germany) operating
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in full scan, Auto MSn mode to obtain fragment ion m/z. Tandem mass spectra were acquired
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in the Auto-MSn mode (smart fragmentation) using a ramping of the collision energy.
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Maximum fragmentation amplitude was set to 1 Volt, starting at 30% and ending at 200%.
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The MS operating conditions (negative mode) had been optimized using 5-CQA, 3 with a
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capillary temperature of 365 oC, a dry gas flow rate of 10 L/min, and a nebulizer pressure of
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10 psi. High resolution LC-MS was carried out using the same HPLC equipped with a 4 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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MicrOTOF Focus mass spectrometer (Bruker Daltonics, Bremen, Germany) fitted with an
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ESI source and internal calibration was achieved with 10 mL of a 0.1 M sodium formate
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solution injected through a six port valve prior to each chromatographic run. Calibration was
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carried out using the enhanced quadratic mode and the mass error was below 5 ppm.
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HPLC. Separation was achieved on a 150 mm x 3 mm i.d., 5 m, diphenyl column, with a 5
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mm x 3 mm i.d. guard column of the same material (Varian, Darmstadt, Germany). Solvent A
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was water/formic acid (1000:0.005 v/v) and solvent B was methanol. Solvents were delivered
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at a total flow rate of 500 μL/min. The gradient profile was from 10% B to 70% B linearly in
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60 min followed by 10 min isocratic, and a return to 10% B at 90 min and 10 min isocratic to
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re-equilibrate.14
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NMR. 1H NMR spectra were acquired on a JEOL ECX-400 spectrometer operating at 400
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MHz at room temperature in methanol-d4 using a 5 mm probe.
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UV Irradiation. The prepared samples of CGAs (0.5 mg/mL of methanol) were placed in a
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photoreactor a LZC-4 V photoreactor (Luzchem, Ottawa, Canada) under a shortwave UV
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lamp and irradiated at 254 nm.
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1
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Products). CD3OD, δ (ppm) 1.69 (1H, dd, J=11.45, 10.99), 2.08 (1H, dd, J=15.45, 1.85), 2.39
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(1H, m), 2.93 (1H, m), 3.59 (1H, dd, J=9.62, 3.21), 3.88 (2H, d, J=6.87), 4.0 (2H, d, J=7.33),
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4.15 (1H, dd, J=8.24, 3.2), 5.06 (1H, m), 6.30 (1H, dd, J=8.24, 1.83), 6.34 (1H, dd, J=8.24,
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1.83), 6.39 (2H, dd, J=8.70, 1.83), 6.51 (1H, dd, J=8.24, 4.1).
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Statistical Analysis. Statistical analyses of the data were performed with software package of
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IBM SPSS Statistics 20.0. The distribution of the data set was analyzed by descriptive
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statistics and Kolmogorov-Smirnov test. The correlation studies were performed by Pearson's
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correlation for Gaussian (normal) distributed data set (5-CQA), 3 and Spearman correlation
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coefficients test was performed for non-Gaussian data set (e.g. cis-5-CQA, 8). A value of p
