Flavonol Glycosides in Currant Leaves and Variation with Growth

Article pubs.acs.org/JAFC

Flavonol Glycosides in Currant Leaves and Variation with Growth Season, Growth Location, and Leaf Position Wei Yang,† Aino-Liisa Alanne,§ Pengzhan Liu,† Heikki Kallio,†,# and Baoru Yang*,† †

Food Chemistry and Food Development, Department of Biochemistry, §Instrument Centre, Department of Chemistry, and #The Kevo Subarctic Research Institute, University of Turku, FI-20014 Turku, Finland S Supporting Information *

ABSTRACT: Flavonol glycosides (FG) were analyzed in the leaves of six currant cultivars (Ribes spp.) with HPLC-DAD, HPLC-MS/MS, and NMR. The average amounts of the 12 major, identified FG constituted 86−93% (9.6−14.1 mg/g DW) of the total of 27 FG found. Quercetin and kaempferol were the major aglycones with trace amounts of myricetin. Quercetin-3-O(2,6-α-dirhamnopyranosyl-β-glucopyranoside), quercetin-3-O-(2-β-xylopyranosyl-6-α-rhamnopyranosyl-β-glucopyranoside), and kaempferol-3-O-(3,6-α-dirhamnopyranosyl-β-glucopyranoside) were identified for the first time in currant leaves and existed in a white currant cultivar ‘White Dutch’ only. Kaempferol-3-O-β-(6′-malonyl)glucopyranoside was also a new compound existing in abundance in five cultivars but not in the white one. The results show the primary importance of the genetic background of the cultivars. The content of malonylated FG of special importance in cardiovascular health decreased regularly during summer. Time of collection and leaf position were more prominent factors affecting the composition than were the year of harvest or the growth latitude. Randomly collected leaves differed in their FG profiles from those collected from the middle position of new branches. KEYWORDS: flavonol glycosides, growth latitude, 1H and 13C NMR, HPLC-MS/MS, leaves, Ribes nigrum, Ribes rubrum, seasonal variation

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rich in flavonols such as rutin, hyperoside, and isoquercitrin.16,17 The compounds have potential in reducing the risk of heart disease and also show biological activities against inflammation, oxidation, cancer, viruses, and bacteria.13,18,19 Moreover, quercetin glycosides also inhibit oxidative modification of human low-density lipoprotein (LDL).20 Thus, currant leaves are potential new raw materials for the production of nutraceuticals and functional ingredients of food. Factors such as cultivar, harvesting time, and leaf position are known to influence significantly the content and composition of black currant leaf flavonol glycosides16,17,21−23 This study was focused on the major flavonol glycosides in leaves of six currant (Ribes spp. L.) cultivars commonly used in Finland. Quantitative analysis of the compounds was carried out with high-performance liquid chromatography combined with a diode array detector (HPLC-DAD) and electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). Nuclear magnetic resonance (1H and 13C NMR) spectrometry was used to identify the earlier unknown major flavonol glycosides after isolation with preparative HPLC. Variation in the content and profile of flavonol glycosides in currant leaves was investigated as a function of cultivar, growth location, growth season, harvesting date, and leaf position. The results provide new insights into the accumulation of these compounds and guidance for identifying optimal conditions for leaf collection.

INTRODUCTION Ribes nigrum L. (black currants and green currants) and Ribes rubrum L. (red currants and white currants) are widely cultivated species both for commercial use and in home gardens in European countries and in North America.1−3 Currant berries are generally rich sources of flavonoids and other polyphenols,3,4 which may possess potential health-promoting properties due to their evident antiallergenic, antihemostatic, anti-inflammatory, antiviral, antioxidative, and anticarcinogenic properties.5 In addition, convincing evidence has suggested positive effects of black currants on chronic diseases, such as cardiovascular disease and stroke.6−10 In Europe, the annual harvest of currants adds up to >1 million tons mostly cultivated in Poland, Germany, Great Britain, and the Nordic and Baltic regions. As a side stream of berry production, large quantities of leaves are produced every year. Whereas currant berries are important food for household consumption and raw materials for the food industry, only a small fraction of black currant leaves are processed to produce tea-type products and preserved food.11 The majority of the leaves are left unused as agricultural waste. Improved utilization of currant leaves is essential for sustainable agriculture. Black currant leaves contain considerable amounts of bioactive compounds.11 In ancient times, the leaves were used as herbal medicine for treatment of chronic rheumatic and inflammatory disorders due to the diaphoretic and diuretic properties of the products.12 Many investigations have demonstrated the extracts of currant leaves to possess significant anti-inflammatory and antioxidative activity both in vitro and in vivo models.13,14 Tabart et al. showed that black currant leaves had a higher content of total phenolics than the fully ripened berries.15 Currant leaves are © XXXX American Chemical Society

Received: July 31, 2015 Revised: October 2, 2015 Accepted: October 8, 2015

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DOI: 10.1021/acs.jafc.5b04171 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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kV; cone voltage, 60 V; extractor voltage, 6 V; source temperature, 120 °C; and desolvation temperature, 300 °C. Mass spectra were obtained by scanning ions between m/z 100 and 1000. Isolation of Flavonol Glycosides with Semipreparative HPLC. A Phenomenex Aeris PEPTIDE XB-C18 column (5 μm, 250 mm × 10 mm) combined with a Phenomenex Prodigy guard column was used for isolation of FG for NMR analyses. The HPLC separation was carried out by a gradient elution of formic acid/water (0.1:99.9, v/v) as solvent A and formic acid/acetonitrile (0.1:99.9, v/v) as solvent B at the mobile phase flow rate of 5 mL/min. For compound 25 to be isolated from ‘Mikael’, the gradient program of solvent B in A (v/v) was 0−15 min with 15−30% B, 15−20 min with 30−60% B, 20−25 min with 60−15% B, and 25−30 min with 15% B. For the other compounds, 3, 4, and 9 of ‘White Dutch’, the gradient program of solvent B in A (v/v) was 0−17 min with 15% B, 17−25 min with 15−60% B, 25−30 min with 60−15% B, and 30−35 min with 15% B. For both samples, the injection volumes were 300 μL. The peaks were monitored at the wavelength of 360 nm for the FG. The HPLC-DAD system consisted of a DGU-20A5 vacuum degasser, an LC-20AB pump, an SIL 20AC automatic injector, a CTO10AC column oven, an SPD-20A UV/vis detector, an FRC-10A fraction collector, and a CBM-20A system controller (Shimadzu, Kyoto, Japan). The chromatograph was operated using Labsolution Workstation software (Shimadzu). To verify the quality of the isolation, the collected samples were reanalyzed with the analytical HPLC-MS. Each of the collected four compounds was divided in two parts, one for NMR analysis and one to be used as external standard in the quantitative analysis. NMR Experiments. The unknown compounds collected by HPLC were analyzed by 1H and 13C NMR on a Bruker Avance 500 spectrometer (Billerica, MA, USA) operating at 500.13 and 125.77 MHz, respectively. The spectra were recorded at 25 °C using CD3OD as a solvent with a nonspinning sample in a 5 mm NMR tube. Concentrations of the samples varied between 2 and 15 mg/mL. The spectra were calibrated on the solvent residual signal of CD3OD at 3.31 ppm for 1H and 49.15 for 13C and processed by TopSpin 3.2 software. Quantitative HPLC-DAD Analysis of Flavonol Glycosides. The quantitative analysis of 12 major FGs in the leaf extracts was carried out with the HPLC-DAD method using external standards of the 10 major FG of quercetin and kaempferol identified in the leaves. Six reference compounds were commercially purchased, and the remaining four were isolated from leaf extracts by semipreparative HPLC and identified with NMR analysis. Standard solutions in methanol were analyzed in the concentration range of 0.01−0.15 mg/mL. The injection volume was 10 μL for each solution. Calibration curves were constructed by plotting the peak areas against the concentrations. The combined amounts of the other tentatively identified FG were calculated on each chromatogram on the basis of the chromatographic peak areas on a dry weight (DW) basis at 360 nm. The concentrations were expressed on DW basis.

MATERIALS AND METHODS

Plant Materials. The leaves of three black currant (R. nigrum L.) cultivars, ‘Mortti’, ‘Mikael’, and ‘Jaloste n:o 15’, a green currant (R. nigrum) ‘Vertti’, a red currant (R. rubrum) ‘Red Dutch’, and a white currant (R. rubrum) ‘White Dutch’ were collected from the test fields of MTT Agrifood Research Finland in both Piikkiö (southern Finland, 60°23′15.7″ N) and Apukka (northern Finland, 66°34′41.2″ N) in 2012 and 2013. The leaf samples were collected at four time points in 2012 (July 3 and 24, August 14, September 4) and in 2013 (July 3 and 23rd, August 14, September 3). Leaves from the middle position of new, berry-bearing branches (referred to as “middle leaves”) were collected from four blocks of each cultivar, every block consisting of three to four bushes. Five branches from each bush were selected. The leaves from different bushes within each block were well mixed as one sample. Changes in the content and composition of flavonol glycosides (FG) were followed during the seasons 2012 and 2013 in the two growth locations. A separate set of samples was randomly collected from different positions of the bushes in Piikkiö in 2012 (July 3 and 24, August 14, September 4) (referred to as “random leaves”). The samples from each block were randomly collected from different sites of the three or four bushes and mixed well after collection. All of the samples were frozen immediately after picking and stored at −18 °C until analysis. For dry weight measurement ca. 5 g of fresh leaves was weighed accurately, milled into fine powder in a mortar in liquid nitrogen, dried to a constant weight at 103−105 °C, cooled in a desiccator, and weighed. Chemicals and Reagents. Methanol (HPLC grade) and formic acid (MS grade) were purchased from LGC Standards GmbH (Wesel, Germany) and acetone (HPLC grade) and acetonitrile (HPLC grade) from VWR International Oy (Espoo, Finland). Reference compounds myricetin-3-O-glucoside, kaempferol-3-O-rutinoside, kaempferol-3-Oglucoside, rutin (quercetin-3-O-rutinoside), and hyperoside (quercetin3-O-galactoside) were purchased for identification and quantitative analyses from Extrasynthese (Genay, France). Quercetin-3-O-(6″malonyl)-glucoside and isoquercitrin (quercetin-3-O-glucoside) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Sample Preparation. A sample of 5 g of frozen leaves of each cultivar was milled into a fine powder in liquid nitrogen. An aliquot of 1 g of leaf powder was extracted three times with 20 mL of 70% aqueous acetone by sonicating for 20 min during each extraction, followed by centrifugation (4420g) for 10 min. The supernatants were combined. The extracts were dried by a vacuum rotary evaporator, redissolved in 1 mL of methanol, and filtered through a polytetrafluoroethylene (PTFE) filter (13 mm i.d., 0.22 μm, VWR International, West Chester, PA, USA) before analysis. For preparative chromatography, samples of 10 g of frozen leaves of the cultivars ‘Mikael’ and ‘White Dutch’ were milled into fine powder in liquid nitrogen and extracted according to the same method as described above. Analysis by HPLC-DAD-ESI-MS/MS. The samples were analyzed with a Waters Acquity ultrahigh-performance LC system (Waters Corp., Milford, MA, USA) with a sample manager, binary solvent delivery system, Waters 2996 PDA detector, and Waters Quattro Premier Tandem Quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) source. The instrument was operated using MassLynx 4.1 software. A Phenomenex Aeris peptide XB-C18 (3.6 μm, 150 × 4.60 mm) column combined with a Phenomenex Security Guard Cartridge Kit (Torrance, CA, USA) was used for the analysis of the samples. The analyses were carried out by a gradient elution with formic acid/water (0.1:99.9, v/v) as solvent A and formic acid/acetonitrile (0.1:99.9, v/v) as solvent B. The gradient program of solvent B in A (v/v) was 0−15 min with 15−20% B, 15−20 min with 20−25% B, 20−25 min with 25% B, 25−30 min with 25−60% B, 30−35 min with 60−15% B, and 35−40 min with 15% B. The flow rate of the mobile phase was 0.5 mL/min. The injection volume was 10 μL. The peaks were monitored at 360 nm for the flavonol glycosides. The whole flow of 0.5 mL/min was directed into the mass spectrometer after the UV detector. The mass spectrometer was operated in a positive ion mode. The ESI inlet conditions were as follows: capillary voltage, 3.2

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RESULTS AND DISCUSSION

Identification of Flavonol Glycosides in Currant Leaves. Analysis by HPLC-DAD-ESI-MS. The leaf extracts of all the samples of black, green, red, and white currants were analyzed by HPLC-DAD-ESI/MS. Results of the qualitative analyses of favonol glycosides are shown in Figure 1 and in Supporting Information Supplementary Table 1. Figure 1 was composed of example chromatograms (360 nm) of the leaf extracts from three black currant cultivars (Figure 1A− C), the green currant ‘Vertti’ (Figure 1D), the red currant ‘Red Dutch’ (Figure 1E), and the white currant ‘White Dutch’ (Figure 1F) collected in Piikkiö on August 14, 2013. As summarized in Supplementary Table 1, identifications of the compounds were based on retention characteristics, UV and mass spectra, NMR analyses, and reference compounds as well as literature comparisons.16,17,24 Most of the FG have typical absorption maxima around 250 and 360 nm, but small shifts in the maxima may be caused by B


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was found in the cultivar ‘Mikael’ only, whereas all others were common in all of the varieties studied. In addition to kaempferol-3-O-rutinoside (17), compound (14) was preliminarily identified as a kaempferol-rhamnosylhexoside due to a mass spectrum similar to that of [M + H]+ at m/z 595 (287 + 162 + 146) and a fragment ion at m/z 449 (287 + 162). Each of the compounds 23−27 had [M + H]+ at m/z 535 (287 + 248) and a fragment ion at m/z 287. They also showed an [M + H + Na]+ at m/z 557 in the full scan mode. The five compounds were thus tentatively identified by MS analysis as kaempferol-malonylhexosides I, II, III, IV, and V, respectively. Compound 25 was typically a major component in the berries except ‘White Dutch’ and was chosen to be analyzed later by NMR. Compounds 9 and 10 existed in ‘White Dutch’ only. The more abundant compound 9 had the [M + H]+ ion at m/z 741 (287 + 146 + 146 + 162) and provided fragment ions at m/z 595 (287 + 162 + 132) and m/z 449 (287 + 162). The compound was worth selecting for NMR analysis. Identification of compound 9 as a kaempferol-dihexoside was confirmed on the basis of UV and MS analyses. Compound 10 was preliminarily defined as a kaempferol-rhamnosylhexoside due to a mass spectrum similar to that of kaempferol-3-O-rutinoside (17) with [M + H]+ at m/z 595 (287 + 162 + 146) and a fragment ion at m/z 449 (287 + 162). Two major glycosides of quercetin (3 and 4) and one of kaempferol (9) were identified in white currants only. Compound 3 with [M + H]+ at m/z 757 (303 + 146 + 146 + 146) and fragment ions at m/z 611 (303 + 162 + 146) and m/z 465 (303 + 162) was preliminarily identified as quercetinrhamnosylglucoside-rhamnoside (Supplementary Table 1). Compound 4 displayed [M + H]+ at m/z 743 (303 + 162 + 146 + 132) and fragment ions at m/z 611 (303 + 162 + 146) and m/z 465 (303 + 162). It was evidently a quercetinrhamnosylglucoside-pentoside. The exact structures were selected for isolation and analysis by NMR. A trace compound, 12, had UV and mass spectra similar to those of compound 13 (Supplementary Table 1), and it was tentatively identified as quercetin-rhamnosylhexoside, an isomer of rutin. Analogously, compound 21 had [M + H]+ at m/z 551 (303 + 248), a fragment ion at m/z 287, and UV and mass spectra similar to those of quercetin-3-O-(6″-malonyl)-glucoside (compound 19) (Supplementary Table 1). Compound 21 was preliminarily identified as a quercetin-malonylhexoside. Four myricetin glycosides were found in trace quantities only. Compound 5 was identified as myricetin-3-O-glucoside on the basis of the retention time and UV and mass spectra identical to those of the reference compound. Compound 6 was tentatively identified as myricetin-rhamnosylhexoside by diagnostic ions at m/z 481 (319 + 162) and 627 (319 + 162 + 146). Compound 7 with [M + H]+ at m/z 481 (319 + 162) was likely a myricetinhexoside. Compound 11 showed [M + H]+ at m/z 567 (319 + 248) and was tentatively identified as myricetin-malonylhexoside. The major hexose among the compounds identified was glucose as indicated also in earlier literature.16,17,22 Galactose was confirmed in hyperoside and only in the cultivar ‘Mikael’. In 14 of the minor compounds not completely identified, a hexose was one of the sugar moieties (Figure 1, compounds 1, 5−8, 10−12, 14, 18, 21, 23, 24, and 27). However, the structure of glucose could not be confirmed even though the common literature supported its preference over galactose. Of these, compound 23 (a kaempferol-malonylhexoside) was the only more abundant

Figure 1. HPLC-DAD chromatograms (360 nm) of leaf extracts of currant cultivars, collected on August 14, 2103: (A) ‘Mikael’; (B) ‘Mortti’; (C) ‘Jaloste no. 15’; (D), ‘Vertti’; (E) ‘Red Dutch’; (F) ‘White Dutch’.

detailed structural characteristics of the aglycones. The glycosides of quercetin have common maxima at 256 and 353 nm and a shoulder at 265 nm, glycosides of kaempferol at 265 and 247 nm, and those of myricetin at 268 and 353 nm. Full scan function of ESI-MS and parent ion scan function of ESI-MS/MS (MS2) were used to reveal the more detailed structures. The MS2 analysis proved the existence of kaempferol (m/z 287), quercetin (m/z 303), and myricetin (m/z 319). In all, 27 FG were found. Kaempferol (14 compounds) and quercetin (9 compounds) were found to be the predominant aglycones in FG, but also some myricetin glycosides (4 compounds) existed in trace quantities. Exact structures of 12 major compounds were in the end determined. Structures of six major FG, rutin (13), hyperoside (15), isoquercitrin (16), kaempferol-3-O-rutinoside (17), quercetin-3O-(6″-malonyl)-glucoside (19), and kaempferol-3-O-glucoside (20), were confirmed directly with the aid of reference compounds (Supplementary Table 1). The retention times (HPLC-DAD), UV spectra, and mass spectral data ([M + H]+ and other mass spectral ions of MS2) were identical in the analytes and the respective reference compounds. Hyperoside C


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Figure 2. Chemical structures of 3, 4, 9, and 25 and their 1H and 13C NMR chemical shifts in CD3OD (coupling constants shown as hertz in parentheses).

Identification of Compounds 3, 4, 9, and 25 by NMR. Structures of several major FG (13, 15−17, 19, and 20) were confirmed with the aid of commercial reference compounds by UV and MS results. Structures of four remaining major compounds (3, 4, 9, and 25) were first inferred from the MS

kaempferol FG with an evident galactose moiety and existed in ‘Mikael’ only. Likewise, the deoxyhexose in compounds 2, 6, 10, 12, and 14 may be assumed to be rhamnose on the basis of previous literature.16 D


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Figure 3. Changes in the content of the identified FG in middle leaves of ‘Mikael’, ‘Mortti’, ‘Jaloste no. 15’, ‘Vertti’, ‘Red Dutch’, and ‘White Dutch’ from (A) Piikkiö 2012, (B) Piikkiö 2013, (C) Apukka 2012, and (D) Apukka 2013.

carbon at δ 169.1 to the H6 protons of the glucose moiety as well as the correlation from the second CO carbon (δ 171.2) to the malonyl CH2 protons at δ 3.15. These compounds were identified as glycosides of quercetin and kaempferol, quercetin-3-O-(2,6-α-dirhamnopyranosyl-βglucopyranoside) (3), quercetin-3-O-(2-β-xylopyranosyl-6-αrhamnopyranosyl-β-glucopyranoside) (4), kaempferol-3-O(3,6-α-dirhamnopyranosyl-β-glucopyranoside) (9), and kaempferol-3-O-β-(6′-malonyl)glucopyranoside (25). The purified compounds were used as external standards in quantitative analyses. All four of theses compounds have been previously characterized by NMR in other materials but not in currants. The NMR data obtained in our study were consistent with the data published earlier.25−27 To our best knowledge, this is the first time that the four compounds have been reported in currant leaves. Retention Time Comparison. Retention time comparison gives additional information and confirmation for identifications (Supplementary Table 1; Figure 1). Analogous glycoside pairs of quercetin and kaempferol, such as 3 versus 9, 13 versus 17, 16 versus 20, 19 versus 25, and 21 versus 27, were obtained showing attachments of the same sugar moieties commonly in both aglycones. The only apparent discrepancy was compound 15, which included the only galactose proved to exist in the FG. However, it is evident that also compound 23 identified as “kaempferol-malonylhexoside I” contained galactose and was a sugar anomer of compound 25, but we did not have a proper reference compound for confirmation. Furthermore, it is also possible that compound 18 would be a kaempferol-galactoside.

and MS/MS data, after which the exact structures were further elucidated by NMR. The compounds were isolated by semipreparative HPLC from the extracts of ‘Mikael’ (25) and ‘White Dutch’ (3, 4, and 9) and obtained as light yellow amorphous powders. The purified compounds were analyzed by 1 H and 13C NMR using 1D-TOCSY, DQF-COSY, HSQC, and HMBC measurements. The structures and the 1H and 13C NMR chemical shifts of the characterized compounds are presented in Figure 2. Compounds 3 and 4 could not be separated and were identified from the mixture, which caused overlapping of the signals, especially in the area of the signals arising from the sugar moieties (δ 3.2−4.1). However, in the sample, compound 3 accounted for about one-third of the total concentrations of the compounds and compound 4 around two-thirds. The clear difference in the integrals of the signals made the analysis easier. Despite the overlapping, the HMBC correlation from the flavonoid part (carbon 3, at δ 134.6 and 135.1) to the glucose moiety could be detected in either of the compounds. In addition, the HMBC correlations from the second and sixth carbons of the glucose to the anomeric proton signals of xylose and rhamnose moieties, respectively, were clearly recognized. The sugar moieties of all compounds were identified using selective 1D-TOCSY experiment revealing the protons that belong to the same spin system with the chosen anomeric proton signal. Due to overlapping and second-order effects, some of the coupling constants of the sugars could not be accurately determined despite the 1D-TOCSY experiments and thus are marked as multiplets in Figure 2. The malonyl part of compound 25 was identified by the clear HMBC correlation of CO E


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Figure 4. Ratios between of quercetin-3-O-(6″-malonyl)-glucoside and isoquercitrin (A) and between kaempferol-3-O-β-(6′-malonyl)glucopyranoside and kaempferol-3-O-glucoside (B) in the leaves of black, green, and red currants.

had the lowest content (9.6 ± 1.5 mg/g DW) among all of the cultivars studied. Results of the quantitative analyses of the 12 compounds identified are given in Supplementary Table 2. Changes in Flavonol Glycosides during the Growing Season. Leaves were collected from the middle position of the bushes at four different time points during the growing seasons of 2012 and 2013. The considerably fluctuating trends of the FG are shown in Figure 3. In general, their contents reached the highest levels around late July to mid-August, followed often by a decrease. In the samples from Apukka (northern Finland), the peaks were typically postponed by some weeks but not regularly. Also, sampling year and cultivars affected the trends, which shall be considered when currant leaves are harvested as raw materials for high contents of FG. The major compound, quercetin-3-O-(6″-malonyl)-glucoside (19), defined the common trends in all of the samples, except in ‘White Dutch’. The nonmalonylated isoquercitrin (quercetin-3O-glucoside) (16) was typically less abundant than compound 19. During summer the ratio of compound 19 to compound 16 decreased regularly each year in both growth places in all five cultivars in which the compounds existed (Figure 4A). The same trend applied also between compounds 25 (kaempferol-3-O-β(6′-malonyl)-glucoside) and 20 (kaempferol-3-O-glucoside) (Figure 4B). This phenomenon seems to be part of the common metabolism in currant leaves, and by the end of the season the ratios of 19 to 16 and of 25 to 20 had typically leveled to about 1. It is worth remembering that quercetin-3-O-(6″-malonyl)glucoside, the main component of FG in currant leaves, reduces oxidation of LDL and oxidative stress in the liver, lowers glycemic response, and attenuates atherosclerotic lesion.28−30 Information concerning bioactivity of the other malonylated flavonol glycosides in currant leaves may not be available. This should be taken into account when planning a strategy for exploitation of currant leaves for food and supplement purposes. Flavonoid glycosides with malonyl moieties are resistant to lactase phlorizin hydrolase relative to their simple glucoside counterparts, potentially limiting their bioavailability.31 Malonylglucosides of isoflavones are relatively less bioavailable than their respective nonconjugated β-glucosides in vivo.32 Variation between Growth Locations. The flavonol glycosides were compared between the leaves from Piikkiö (southern

Only the cultivar ‘Mikael’ was proved to contain galactose (compound 15, hyperoside), and compound 18 was found only in ‘Mikael’. Myricetin FG appeared in such low quantities that comparisons of these compounds were not possible. Comparison of the Cultivars. The four R. nigrum cultivars and two R. rubrum cultivars were analyzed. The differences and similarities in the flavonol profiles among the six cultivars did not follow the taxonomy. The FG in leaves of only one of the R. nigrum cultivar (‘Mikael’) contained galactose and practically only ‘White Dutch’ of R. rubrum contained rhamnose, and it was at the same time very low in glucose. Quercetin and kaempferol were the major aglycones in the FG of the leaves of all the currant cultivars studied, and myricetin existed in trace quantities only. Amounts and Development of Flavonol Glycosides in Currant Leaves. The contents of the 12 most abundant compounds (3, 4, 9, 13, 15−20, 23, and 25) were determined on the basis of external standard curves of reference compounds. Compound 18, kaempferol-hexoside I, was tentatively identified as a kaempferol-galactoside according to the UV, MS, and retention data, and the quantitative analysis was based on the standard curve of kaempferol-3-O-glucoside. Likewise, for compound 23, kaempferol-malonylhexoside I, which is evidently a malonylated kaempferol-galactoside, kaempferol-3-O-β-(6′malonyl)-glucopyranoside was used as reference. The other minor 15 FG compounds were not fully identified, but their total peak area was analyzed at 360 nm. Of the 27 FG defined, the peak areas of the 12 identified compounds varied between 86 and 93% of the total FG, indicating their approximate proportion of 90% in the leaves. In quantitative comparisons, six compounds (13, 16, 17, 19, 20, and 25) were taken into account in cultivars ‘Mortti’, ‘Jaloste no. 15’, ‘Vertti’, and ‘Red Dutch’, nine compounds (13, 15−20, 23, and 25) in ‘Mikael’, and seven (3, 4, 9, 13, 16, 17, and 20) in ‘White Dutch’. The average contents of flavonol glycosides in each of the six cultivars were calculated on the basis of measurements in each year, each location, and at each time point in the middle leaves. Leaves of ‘Red Dutch’ had the highest average content of FG (14.1 ± 2.1 mg/g DW), followed by ‘Mortti’ (13.1 ± 1.1 mg/g DW), ‘Vertti’ (12.9 ± 0.9 mg/g DW), ‘Jaloste no. 15’ (12.3 ± 1.7 mg/g DW), and ‘Mikael’ (11.9 ± 3.0 mg/g DW). “White Dutch” F


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declining. Compounds 3, 4, and 9 in white currants showed analogous patterns. An earlier study has reported that young leaves of blackberry, raspberry, and strawberry contain higher contents of phenolic compounds than mature leaves.33 In addition, UV radiation, sunlight, latitudes, altitudes, and genetic background affect the content of flavonoids and flavonoid synthesis in leaves.34−37 A recent study compared flavonol glycosides in leaves from different positions in new shoots of black currant cultivar ‘BRi 9508-3B’. The content of total phenolic compounds in basal leaves was lower than the levels in apical and middle leaves during the growing season. The content of isoquercitrin in leaves from basal position was higher than in apical and middle positions, but the quercetin-3-O-(6″-malonyl)-glucoside level was lower in the basal position.22 The results were in agreement with the findings of the current study suggesting that the position of leaves may have an influence on the content and profile of FG. In conclusion, 27 flavonol glycosides were found, and 12 of them were identified on the basis of UV spectra, mass spectra, reference compounds, and NMR analysis in the leaf extracts of three black currant cultivars ‘Mortti’, ‘Mikael’, and ‘Jaloste no. 15’, a green currant cultivar ‘Vertti’, a red currant cultivar ‘Red Dutch’, and a white currant cultivar ‘White Dutch’. Four major flavonol glycosides were identified by NMR for the first time in currant leaves. The flavonol profiles were highly dependent on the varieties and, for example, ‘Red Dutch’ and ‘White Dutch’ differed clearly from each other even though both of them are R. rubrum species. Also, there were major differences in black currant ‘Mikael’ compared with ‘Mortti’ and ‘Jaloste no. 15’. This is the first systematic report of FG in currant leaves and variation among cultivars, locations, growing seasons. and leaf positions. The results suggest that, for medicinal or industrial purpose, currant leaves should preferentially be collected from the end of July to early September, when they contain the highest level of FG. Our findings relate to the accumulation of FG compounds and provide guidance for the cultivation and utilization of currant leaves as raw material of nutraceuticals and functional foods.

Finland) and Apukka (northern Finland). The average total contents at the four time points in both years were compared. In Piikkiö, the FG contents of ‘Jaloste no. 15’, ‘Mortti’, ‘Mikael’, ‘Vertti’, ‘Red Dutch’, and ‘White Dutch’ were 13.0 ± 4.1, 10.5 ± 3.9, 10.1 ± 3.8, 13.2 ± 5.4, 14.0 ± 4.3, and 9.1 ± 1.9 mg/g DW, respectively. The corresponding values from Apukka were 10.9 ± 1.8, 13.8 ± 3.3, 12.7 ± 1.6, 12.5 ± 1.3, 13.7 ± 2.0, and 9.9 ± 1.8 mg/g DW, respectively. Independent t test showed no statistically significant difference in FG contents in the leaves collected from the two growth locations (P > 0.05). Contents of the major FG in samples from Piikkiö reached the highest levels during late July to mid-August and had an obvious decrease in early September. In the samples from Apukka, the corresponding peak of the analytes was postponed to mid-August or early September but the descending trend was less clear than in Piikkiö. The ratios of compounds 19 to 16 and 25 to 20 had commonly a decreasing trend during the season (Figure 4). In all black currant varieties, in both years in early July, both of the ratios were higher in leaves from Apukka compared with Piikkiö, revealing higher malonylation level in the north. In green and red currants this difference was not evident. The latitude difference between Piikkiö and Apukka is close to 700 km. However, due to the extreme deviation between individual samples, no effect of the latitude difference could be found. Variation between Years. Samples of the middle leaves picked in Piikkiö and Apukka were compared in 2012 and 2013 (Figure 3). The trends of FG in ‘White Dutch’ were equal in both years, unlike most of the other samples. In ‘Jaloste no. 15’ and ‘Mikael’, the changing patterns were quite analogous between the two years. The contents of total FG increased from early July and reached the highest levels toward the end of July, followed by a decrease to early September. The FG content of green currant leaves picked on July 23, 2013, was as high as 24.5 mg/g DW (Figure 3B). The small number of collection points and different weather conditions in the two years may have caused these clear irregularities. Previous studies reported that the total phenolic content in black currant leaves was high during June, followed by a decrease until early August.15,23 Variation between Leaf Positions. The leaves of green, red, and white currants picked in Piikkiö (southern Finland) in 2012 were compared between the random and middle positions (Supplementary Figure 1). Between the two leaf collections, differences were noted in individual compounds in all of the varieties investigated. In all three cultivars, the contents of total FG reached the highest level in mid-August in middle leaves, but was postponed to early September in random leaves. The increasing trend in the total FG contents of the random leaves was recognized in the beginning of September unlike the middle-position leaves. This is well in agreement with our earlier investigations related to black currants.16 The trend is due to the special increase of compounds 13, 16, 17, and 20 toward the end of summer. Isoquercitrin composed almost half of the total FG in the random September samples. In white currant, kaempferol-3-O-glucoside was the most abundant FG in random leaves, whereas it had the lowest content in middle position. During the whole summer the contents of the malonylated FG species were higher in the middle-position leaves of green and red currants, even though the concentrations were regularly

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04171. Supplementary Tables 1 and 2 and Supplementary Figure 1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(B.Y.) Phone: +358 2 333 68 44. E-mail: baoru.yang@utu.fi. Funding

This work was supported by the China Scholarship Council (CSC) and TEKESthe Finnish Funding Agency for Technology and Innovation within the project “Blackcurrant as Unique Source of Functional Ingredients of Food: Novel Processes and Innovations”, cofounded by Finnish and European food companies. Notes

The authors declare no competing financial interest.

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REFERENCES

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Flavonol Glycosides in Currant Leaves and Variation with Growth

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