Article pubs.acs.org/JAFC
Polyphenols in Strawberry (Fragaria × ananassa) Leaves Induced by Plant Activators Anna Kårlund,† Juha-Pekka Salminen,‡ Piia Koskinen,‡ Jeffrey R. Ahern,‡ Maarit Karonen,‡ Kari Tiilikkala,§ and Reijo O. Karjalainen*,† †
Department of Biology, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland Department of Chemistry, University of Turku, FI-20014 Turku, Finland § Plant Production Research, MTT AgriFood Research Finland, FI-31600 Jokioinen, Finland ‡
S Supporting Information *
ABSTRACT: Strawberry leaves contain high amounts of diverse phenolic compounds potentially possessing defensive activities against microbial pathogens and beneficial properties for human health. In this work, young strawberry plants were treated with two plant activators, S-methylbenzo-1,2,3-thaidiazole-7-carbothiate (BTH) and birch wood distillate. Phenolic compounds from activator-treated and control leaves were subjected to quantitative analyses by HPLC-DAD, HPLC-ESI-MS, and microQTOF ESI-MS. Thirty-two different phenolic compounds were detected and characterized, and 21 different ellagitannins constituted the largest group of compounds in the strawberry leaves (37.88−45.82 mg/g dry weight, 47.0−54.3% of total phenolics). Treatment with BTH resulted in higher levels of individual ellagitannins, whereas treatment with birch wood distillate strongly increased the levels of chlorogenic acid in strawberry leaves compared with the control. The results suggest that different plant activators may be useful tools for the activation of different branches in the phenylpropanoid biosynthesis in strawberry. KEYWORDS: polyphenols, strawberry (Fragaria × ananassa), leaves, BTH, activators
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INTRODUCTION During the evolutionary history of terrestrial plants, plant surfaces have been continuously exposed to abiotic and biotic stresses caused by environmental factors such as ultraviolet-B (UV-B) radiation, ozone, and numerous plant pathogens and insects. In agriculture, pesticides are extensively used to control diseases, insects, and weeds to protect and improve yields and might be considered a necessity of modern farming.1−3 Despite the benefits of pesticides to crop yields, these chemicals can additionally cause some health hazards to pesticide-manufacturing workers and farmers, and also consumers and workers in the food industry are exposed daily to plant pesticides and pesticide residues via plants or food of plant origin.1−3 In the future, changing climatic variables, such as altered wind patterns and rising temperatures, may further increase pest outbreaks and the need for pesticide application and directly or indirectly alter the behavior of pesticides, for example, fungicides, in agricultural lands and plant surfaces.4−6 Altogether, application of pesticides can also cause some external, delayed costs from damage to the environment, human health, and other production sectors.3,5 Apart from better awareness and safety practices, there is likely a growing need and demand for inexpensive and effective but at the same time more natural pesticides. Phenolic compounds, a large class of plant secondary metabolites, are an important component of plants’ natural defense against pathogens. Elicitor molecules are exogenous or endogenous compounds that can activate defense compounds, secondary metabolites, or their synthesis in an organism. Elicitors that induce pathways enhancing the accumulation of phenolic compounds can be utilized to protect plants from infections.7 © 2014 American Chemical Society
Polyphenolic compounds have been, and still are, important mediators in the adaptation and survival responses of plants in acute and chronic challenges. Polyphenols, such as ellagitannins, appear to play an important role in plant defense against insect pests8 and plant pathogens.9 Also, plants growing naturally in high latitudes,10 high temperature, and high light conditions11 or under low nitrogen and phosphorus availability12,13 may contain relatively high concentrations of phenolic compounds. UV-B radiation14 and ozone-induced stress15,16 are capable of causing variable changes in polyphenol profiles. Polyphenols also act in plants regulating cell growth, differentiation, pollen fertility, and nodulation and thus may contribute some vital features of plant fitness.17,18 The concentration of polyphenols in plants varies widely during plant development and in different plant organs.19 The concentration of antimicrobial polyphenols can be increased by exposing plants to mild microbial infection or infection mimicking treatment, that is, by application of elicitor-active compounds capable of activation of defensive pathways. For example, S-methylbenzo-1,2,3-thaidiazole-7-carbothiate (BTH) is successfully used in numerous plants to activate plant defense systems, increasing concentrations of different polyphenols.20 Recently it has been found that leaves of berry plants are rich in bioactive polyphenols. High concentrations of polyphenols have been measured from black currant (Ribes nigrum),21 raspberry (Rubus ideaus),22 bilberry (Vaccinium myrtillus),10 Received: Revised: Accepted: Published: 4592
December 16, 2013 April 16, 2014 April 16, 2014 April 16, 2014 dx.doi.org/10.1021/jf405589f | J. Agric. Food Chem. 2014, 62, 4592−4600
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Article
saskatoon (Amelanchier alnifolia),23 and strawberry (Fragaria × ananassa)24 leaves. Strawberries are extensively grown horticultural crop plants possessing a wide range of bioactive compounds. In strawberry, antioxidant capacity in leaves has been found higher than in berries,19 and the extract made from wild strawberry leaves has demonstrated a direct, endotheliumdependent vasodilator activity.25 Potentially beneficial health effects of strawberry polyphenols against, for example, cancer,26 cardiovascular diseases,27 inflammation-related diseases,28 and Alzheimer’s disease,29 have been reported. These observations suggest that strawberry leaves may be a promising bioresource for diverse health-related applications; this could increase the level of utilization and the value of the plant material. Although polyphenols from strawberry fruits have been extensively analyzed from different cultivars during the past few years, little is known about the individual phenolic compounds occurring in strawberry leaves.30,31 We previously analyzed some of the phenolic compounds in greenhouse-grown strawberry leaves.32 We also detected improved resistance of strawberry leaves to powdery mildew (Sphaerotheca macularis Wall. ex. Fr) after BTH exposure.32 Byproducts of a slow thermal conversion process, also known as pyrolysis of wood material, have been used in Asian countries for centuries as growth stimulants, fertilizers, fungicides, herbivore repellents, and herbicides, and today they are marketed globally as biological control agents.33,34 Birch wood distillate is a byproduct of birch wood processing formed during pyrolysis,33 and its tar-free fraction contains mainly acetic acid, methanol, hydroxypropanone, furfural, and acetone.34 Fractions of birch wood distillate may act as plantbased biological control agents that can possibly be used as a commercial plant-protecting agent of low environmental risk.34,35 In the present study, extensive analysis using powerful liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry (HPLC-DAD, HPLC-ESIMS, and microQTOF ESI-MS) was conducted to further identify and characterize the induced polyphenols from BTHand birch wood distillate-treated strawberry leaves to clarify the potential of strawberry leaves as a source of health-beneficial compounds and BTH and birch wood distillate as plantprotecting agents.
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treatment/sampling date combination, we randomly selected 10 plants and destructively harvested leaves for chemical analyses. From these 10 plants, leaves from 2−3 plants were pooled, creating four unique sample replicates per treatment/sampling date combination. Samples were immediately frozen with liquid nitrogen and stored at −80 °C until they were freeze-dried. Sample Preparation, Extraction, and Fractionation. Freezedried samples were homogenized into a fine powder using a ball grinder (Retsch MM200, Haan, Germany) and stored in a −20 °C freezer until the chemical analyses were conducted. Our analytic workflow was as follows: (1) initial sample screening; (2) preparative scale extraction, fractionation, and MRM method development; (3) quantitative analysis of phenolic compounds by UPLC-DAD-MS/MS; and (4) qualitative analysis of phenolics by HPLC-ESI-QTOF-MS. Initial Sample Screening. For initial screening and subsequent quantitative analysis, analytical scale extractions were used: samples of 10 mg were extracted with 2 × 1400 μL of acetone/water (80:20, v/v). The extracts were concentrated into water phase with a vacuum concentrator (Eppendorf concentrator 5301, Hamburg, Germany), freeze-dried (Christ Alpha 2-4, Osterode, Germany), redissolved into 1 mL of water, and filtered through 0.20 μm PTFE filters (VWR International, Darmstadt, Germany). We first conducted preliminary analyses to identify the major phenolic compounds present in all samples using UPLC-MS/MS (see experimental methods below). Preparative Scale Extraction, Fractionation, and MRM Method Development. For preparative scale extractions, 8 g of ground leaf material was extracted with 5 × 200 mL acetone/water (80:20, v/v). Acetone was evaporated from the pooled extracts by rotary evaporation and the water phase freeze-dried. Two grams of the freeze-dried extract was dissolved into 30 mL of water and fractionated with Sephadex LH-20 (Pharmacia, Uppsala, Sweden) gel chromatography as described by Salminen and Karonen.36 The Sephadex fractions containing the polyphenols were further fractionated by semipreparative HPLC using a Waters Delta 600 (Waters, Milford, MA, USA) system equipped with Fraction Collector III. The column used was Phenomenex Gemini C18 (10 μm, 150 × 21.2 mm, Phenomenex, Torrance, CA, USA) with 0.1% formic acid and acetonitrile as eluents. These purified compounds were used by direct infusion with a Waters XEVO triple-quadrupole mass spectrometer to develop compound-specific multiple reaction monitoring (MRM) methods and quantitative calibration curves for the specific measurement of each major metabolite in our samples (Table 1).37 Isomeric compound pairs with the same molecular masses were quantified using the same calibration curve for both of the compounds. Quantitative Analysis of Phenolic Compounds by UPLC-DADMS/MS. For quantitative analyses, phenolic extracts from each sample (after 30× dilution with water) were analyzed using ultraperformance liquid chromatography with an electrospray triple-quadrupole mass spectrometry detector (UPLC-MS/MS) and compound-specific MRM methods. The Waters Acquity Xevo UPLC-MS/MS (Waters, Milford, MA, USA) system utilized a Waters Acquity UPLC BEH Phenyl column (1.7 μm, 2.1 × 100 mm, Waters, Dublin, Ireland) column with acetonitrile (A) and 0.1% formic acid (B) as eluents. The gradient was as follows: 0−0.5 min, 0.1% A (isocratic); 0.5−5 min, 0.1−30% A in B (linear gradient); 5−5.1 min, 30−90% A in B (linear gradient); 5.1− 7.1 min, 90% A (isocratic); 7.1−7.2 min, 90−0.1% A in B (linear gradient); 7.2−8.5 min, 0.1% A (isocratic). The flow rate was 0.500 mL/min. Five microliters of the sample extract was injected into the UPLC column. Phenolic concentrations were calculated as milligrams per gram dry weight. Qualitative Analysis of Phenolics by HPLC-ESI-QTOF-MS. All fractions and extracts were analyzed by the HPLC-DAD-ESI-QTOFMS system consisting of an Agilent 1200 series HPLC equipped with a diode array detector (Agilent Technologies, Waldbronn, Germany) and micrOTOF-Q ESI-mass spectrometer (Bruker Daltonics, Bremen, Germany). Chromatographic separations were performed using a Waters Acquity UPLC BEH C18 column (1.7 μm, 50 mm × 2.1 mm, Waters, Dublin, Ireland). The binary mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B). The elution profile was as follows: 0−0.1 min, 0% A (isocratic); 0.1−5 min, 0−30% A in B
MATERIALS AND METHODS
Plant Material. Strawberries (cv. Polka) were grown at the MTT AgriFood Research Finland (Jokioinen, Finland) greenhouses in 7 cm pots in a peat−sand mixture (3:1) and fertilized weekly with Superflex9 fertilizer (19% N, 5% P, 20% K). The following growth conditions were used: relative humidity, 50−70% (night−day); daylight, 16 h; and temperature, 19−22 °C (night−day). Plants were grown until they had at least four fully developed leaves before the treatments were begun. Two hundred strawberry plants were randomly divided into three groups of 40−80 plants. One group was sprayed with 0.5 g/L active BTH (Bion 50 WG, Syngenta, ca. 5 mL per plant) according to our previous work.32 One group was sprayed with 10% birch wood distillate (ca. 5 mL per plant) containing, for example, acids, ethersolubles, ether-insolubles (“wood syrup”), and polycyclic aromatic hydrocarbons.34 Further composition of the distillate is described in Fagernäs et al.34 The control group was sprayed with water until runoff (ca. 5 mL per plant). Samples for chemical analysis were collected from control plants immediately after treatment (day 0) and from control and BTH- and birch wood distillate-treated plants 7 days after treatment (day 7).32 All samples were collected at the same time of day (at 12−2 p.m.) under uniform light conditions. For each 4593
dx.doi.org/10.1021/jf405589f | J. Agric. Food Chem. 2014, 62, 4592−4600
4594
E11
F1 F2
23
24 25
E7c
16
E10
E7b
15
22
E7a
14
E9b
E6c
13
21
E6b
12
E9a
E6a
11
20
E4 E5
9 10
E8b
E3c
8
19
E3b
7
E8a
E2 E3a
5 6
18
G3 E1
3 4
E7d
G1 G2
1 2
17
code
no.
trimeric ellagitannin 2502 Da trimeric ellagitannin 2804 Da catechin quercetin diglycoside 610 Da
1-O-galloylglucose monogalloyl quinic acid trigalloyl glucose 2,3-(S)-HHDPglucose pedunculagin galloyl-HHDPglucose a galloyl-HHDPglucose b galloyl-HHDPglucose c tellimagrandin I dimeric ellagitannin 1416 Da dimeric ellagitannin 1718 Da a dimeric ellagitannin 1718 Da b dimeric ellagitannin 1718 Da c dimeric ellagitannin 2020 Da a dimeric ellagitannin 2020 Da b dimeric ellagitannin 2020 Da c dimeric ellagitannin 2020 Da d dimeric ellagitannin 2038 Da a dimeric ellagitannin 2038 Da b dimeric ellagitannin 1870 Da agrimoniin
compd identity
2804.2293 290.0790 610.1170
C15H14O6 C26H26O17
2502.2231
1870.1581
C123H80O78
C109H74O70
C82H54O52
1870.1581
2038.1640
C89H58O57
C82H54O52
2038.1640
2020.1534
2020.1534
2020.1534
2020.1534
C89H58O57
C89H56O56
C89H56O56
C89H56O56
C89H56O56
1718.1472
1718.1472
C75H50O48
C75H50O48
1718.1472
786.0916 1416.1409
634.0806
634.0806
784.0759 634.0806
C75H50O48
C34H26O22 C61H44O40
C27H22O18
C27H22O18
C34H24O22 C27H22O18
636.0963 482.0697
332.0743 344.0743
C13H16O10 C14H16O10
C27H24O18 C20H18O14
calcd
mol formula
290.0797 610.1164
2804.2282
2502.2216
1870.1538
1870.1616
2038.1676
2038.1623
2020.1523
2020.1556
2020.1583
2020.1615
1718.1545
1718.1494
1718.1596
786.0916 1416.141
634.0809
634.0802
784.0760 634.0802
636.0980 482.0669
332.072 344.0729
measd
2GLUC + G + 4HHDP 2GLUC + G + 4HHDP 2GLUC + G + 4HHDP 2GLUC + 2G + 4HHDP + GA − H2O 2GLUC + 2G + 4HHDP + GA − H2O 2GLUC + 2G + 4HHDP + GA − H2O 2GLUC + 2G + 4HHDP + GA − H2O 2GLUC + 2G + 4HHDP + GA 2GLUC + 2G + 4HHDP + GA 2GLUC + 2G + 4HHDP 2GLUC + 2G + 4HHDP 3GLUC + 3G + 5HHDP
−7.2 −1.3 −4.2 −4.0
4.31 2.86 3.75
−2.4 1.0
4.01
4.15
3.95
3.90
3.84 (3.90)
4.63
4.44
4.16
3.77
3.74
3.65
3.26
3.08, 3.31 3.18
3.08
2.71
2.49, 2.82 2.52
46 55 32
632.97 → 300.92 784.97 → 300.97 707.16 → 300.97
45 32
1250.40 → 301.00 933.90 → 300.99
32 45
38
934.10 → 300.99
289.03 → 245.07 609.03 → 301.00
38
934.10 → 300.99
42
42
996.16 → 300.96 996.16 → 300.96
40
40
40
40
36
36
1009.16 → 300.99
1009.16 → 300.99
1009.16 → 300.99
1009.16 → 300.99
858.10 → 300.96
858.10 → 300.96
36
46
632.97 → 300.92
858.10 → 300.96
56 46
32 26
782.97 → 301.04 632.97 → 300.92
634.97 → 464.97 481.03 → 301.00
34 38
331.03 → 168.92 343.03 → 191.00
1.25 (1.47, 2.00, 2.44) 1.73 3.35 1.02, 1.10
cone voltage (V)
quantitative MRM method: m/z of the mother ion → m/z of the daughter ion
retention time in UPLC-MS/MS analysis (min)
0.4
0.6
2.3
−1.9
−1.8
0.8
0.5
−1.1
−2.4
GLUC + 2G + HHDP 2GLUC + G + 3HHDP
GLUC + G + HHDP
0.0 −0.1
0.6
GLUC + G + HHDP
GLUC + 2HHDP GLUC + G + HHDP
−0.1 −0.1 0.6
GLUC + 3G GLUC + HHDP
GLUC + G GLUC + Q
composition
−2.7 5.8
6.9 4.1
error (ppm)
16 30
44
65
46
46
55
55
54
54
54
54
42
42
42
40 40
32
32
44 32
24 26
22 20
collision energy (eV)
Table 1. Phenolic Compounds Quantified from Strawberry Leaves, Their Molecular Formulas, High-Resolution Mass Spectral Data, Composition, Retention Times in the UPLC-MS/MS Analysis, and Multiple Reaction Monitoring (MRM) Methods Used for Their Selective Quantificationa
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6.5 354.0928 354.0951 C16H18O9
578.1424 C30H26O12
F5
P1
P2a
P3b
CH
28
29
30
31
32
(linear gradient); 5−6 min, 30−70% A in B (linear gradient); 6−8 min, 70% A in B (isocratic). The flow rate was 0.45 mL/min, and the injection volume was 5 μL. Chromatograms were recorded at 280 nm. The HPLC system was controlled by Hystar software (version 3.2., Bruker BioSpin, Rheinstetten, Germany). The mass spectrometer was controlled by Compass micrOTOF control software (Bruker Daltonics) and operated in negative ion mode. The capillary voltage was maintained at +4000 V with the end plate offset at −500 V. The pressure for the nebulizer gas (N2) was set at 1.6 bar, and the drying gas (N2) flow rate was 12.0 L/min and temperature, 200 °C. The full scan mass ranged from m/z 100 to 3000. Calibration with 5 mM sodium formate injected via a six-port valve was used at the end of the LC-MS experiment to provide high-accuracy mass measurements. The data were handled by Compass DataAnalysis software (version 4.0, Bruker Daltonics). Data Handling. The phenolic chemical data set contained both quantitative variation (in the total concentrations of phenolics in a sample) and qualitative variation (relative amounts of different chemical in comparison to the total concentration). To distinguish between these two effects, for each sample we divided the concentration of each individual compound by the total concentration of phenolics in the sample (the sum of all compounds combined). These values were used in further analysis as measures of qualitative variation in the phenolic profile of each sample. In addition to this, we included the total concentration of phenolics as a measure of quantitative variation between samples. Statistical Analysis. Our data set contained a large number of response variables (concentrations of individual chemicals), with strong positive and negative correlations between individual traits. This correlated data structure precluded the use of multiple univariate analysis of variance (ANOVA) analyses. Similarly, because the number of response variables (P = 33) exceeded our sample size (N = 16), we did not have the statistical power to utilize standard statistical methods such as MANOVA. Thus, we opted to utilize principal component analysis (PCA) to explain observed variation between samples in phenolic profiles. PCA uses orthogonal transformation to generate linearly uncorrelated principal components.38 We used the PRINCOMP procedure in SAS to perform a PCA with all of our treatment levels together. We selected the optimum number of principal component axes using a scree plot,38 which resulted in two axes (Supporting Information Table S1). To test whether groups of samples separated in the PCA were significantly different, from one another we used the GLM procedure in SAS to perform ANOVA. We used our treatment levels as predictor variables and the two PC axes as response variables. We used the LSMEANS statement to test for differences between treatments, correcting for multiple comparisons using the Tukey adjustment.
Isomeric compounds with the same molecular mass that were quantified with the same calibration curves are denoted in their identifying code (e.g. E6a, E6b). Compounds with multiple reported retention times are known epimers (e.g., tellimagrandin I) or other isomers that were detected in samples but below the limits of quantification (in parentheses, e.g., 1-O-galloylglucose). Ellagitannin and galloyl derivative characterization based on Moilanen, Sinkkonen, and Salminen, 2013. GA, gallic acid; G, galloyl; Q, quinic acid; GLUC, glucose; HHDP, hexahydroxydiphenoyl.
18 24 353.03 → 191.00
3.44 −1.0 578.1430
2.90 (3.28)
2.89 −2.6 578.1439 578.1424 C30H26O12
866.2058 C45H38O18
446.1577 C23H26O9
478.0747 C21H18O13 F4 27
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RESULTS AND DISCUSSION Identification and Characterization of Strawberry Leaf Phenolics. The phenolic compounds from strawberry leaves were characterized and tentatively identified according to their characteristic UV spectra, molecular masses, and MS fragmentation patterns. In total, 32 phenolic compounds were quantified using sensitive and compound-specific MRM methods that were separately developed for each compound (Figure 1; Table 1). Isomeric compounds with the same molecular mass were treated as unique compounds, but separate analyses pooling these compounds resulted in qualitatively similar results (results not shown). Strawberry leaves contained a diverse suite of polyphenols, including gallic acid derivatives (6), ellagitannins (21), flavonoids (5), proanthocyanidins (3), and chlorogenic acids (2). The total amounts and percentages of different groups of phenolic compounds in different treatment groups are presented in Table 2. Twenty-one different ellagitannins constituted the largest group of the compounds in the leaves,
a
16 30 577.09 → 425.10
16 30
2.18 −0.3 866.2061
577.09 → 425.10
36 40
4.99 7.4 446.1544
865.07 → 289.05
28 40 445.10 → 144.98
22 32 2.9 478.0733
4.08
477.03 → 301.01
25 34 593.03 → 284.99 4.4 594.1195 594.1221 C26H26O16 F3 26
kaempferol diglycoside 594 Da quercetin glycoside 478 Da flavonoid glycoside 446 Da trimeric proanthocyanidin dimeric proanthocyanidin a dimeric proanthocyanidin b chlorogenic acid a6
calcd mol formula compd identity code no.
Table 1. continued
composition measd
4.06
cone voltage (V) error (ppm)
retention time in UPLC-MS/MS analysis (min)
quantitative MRM method: m/z of the mother ion → m/z of the daughter ion
collision energy (eV)
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Figure 1. Representative UV and compound-specific MRM chromatograms. The use of MRM allowed the highly sensitive and selective quantification of a number of phenolic compounds simultaneously. Pictures are selected MRM chomtograms, with individual measured compounds identified.
quercetin-3-O-glucuronide, and these leaves contain also many other quercetin drivatives and other flavonols.40 The saskatoon leaves consisted mainly of quercetin- and kaempferol-derived glycosides (41% of the phenols), hydroxycinnamic acids (36%), and minor amounts of catechins and neolignans.23 In general, we observed strong positive correlations between the concentrations of gallic acid derivatives and ellagitannins (Supporting Information Table S2). This was expected, because these compounds are formed along the same hydrolyzable tannin biosynthetic pathway that uses the metabolic flux of one branch of the shikimate pathway.36 In contrast, all of the hydrolyzable tannins showed negative correlations with the concentrations of flavonoids, proanthocyanidins, and chlorogenic acids. This finding is in agreement with flavonoids, proanthocyanidins, and chlorogenic acids needing the metabolic flux of the shikimate pathway that is not directed toward hydrolyzable tannins. Similarly, we found strong positive correlations between the relative concentrations of flavonoids, proanthocyanidins, and chlorogenic acids as they all need the shikimate pathway for their biosynthesis. However, flavonoids and proanthocyanidins were more strongly correlated with each other than with chlorogenic acids, because they utilize also the acetate/malonate pathway that is not needed for the biosynthesis of chlorogenic acids. Among the correlations between individual compounds there were three exceptions that did not fit well with the general pattern: HHDP-glucose (E1), ellagitannin 2038 Da (E13), and flavonoid glycoside 594 Da (F3). Of these, only the pattern observed with HHDP-glucose and other HTs is explained by HHDP-glucose being the catabolic product of ellagitannins.41 To explain the patterns observed with E13 and F3 would need further investigation of the specific structures. The PCA and subsequent ANOVA were able to detect significant differences in the polyphenolic profiles of different treatments (Figure 2). The first two PCA axes were able to
representing 47.0−54.3% of the total phenolic compounds in differently treated strawberry leaves. Ellagitannins occur in high levels also in strawberry fruits (9.7−22.9 mg/100 g).30 Sanguiin H-6, lambertianin C, galloyl bis-hexahydroxydiphenic acid (HHDP) glucose,30 agrimoniin, and five other ellagitannins31 were recently detected in fruits of different strawberry cultivars. In this work, agrimoniin (Table 2, code E9b) was the most abundant compound in the strawberry leaves, supporting the previous findings32 and agrimoniin was also recently found to be the main ellagitannin in both wild and cultivated strawberry fruits.31,39 In the present study, flavonoids were found to be the second largest group of polyphenols in strawberry leaves, and their percentage of the total phenolic compounds in different treatment groups was 36.4−41.4%. However, Oszmiański et al.22 found quercetin derivatives as the most abundant group of phenolic compounds in the strawberry leaves. Proanthocyanidins are the most abundant phenolic compounds in strawberry fruits; the concentrations vary among cultivars between 53.9 and 163.2 mg/100 g.30 Proanthocyanidins have also been detected in strawberry leaves32 and in the leaves of white strawberry.24 In the present study, proanthocyanidins represented the third largest group of phenolic compounds in strawberry leaves (4.7−8.0% of total, Table 2). Galloyl derivatives and chlorogenic acids were the other phenolic compounds identified from the strawberry leaf samples, and their percentages of total phenolic compounds altered between 0.9 and 3.1%. When compared to the recent information about polyphenols analyzed from the leaves of other berries, strawberry appears to be a rich source of different ellagitannins. For example, leaves of bilberry contain mainly quercetin-3-Oglucuronide, trans-chlorogenic acid, cis-chlorogenic acid, cinchonain, and some other minor phenolics, whereas the main compound in the lingonberry leaves seems to be 4596
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Table 2. Concentrations of Phenolic Compounds Measured in Strawberry Leaves in Different Treatmentsa concentrations (mg/g dw) no.
code
compound
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
G1 G2 G3 E1 E2 E3a E3b E3c E4 E5 E6a E6b E6c E7a E7b E7c E7d E8a E8b E9a E9b E10 E11 F1 F2 F3 F4 F5 P1 P2a P2b CH TOTAL
1-O-galloylglucose monogalloyl quinic acid trigalloyl glucose 2,3-(S)-HHDP-glucose pedunculagin galloyl-HHDP-glucose a galloyl-HHDP-glucose b galloyl-HHDP-glucose c tellimagrandin I dimeric ellagitannin 1416 Da dimeric ellagitannin 1718 Da dimeric ellagitannin 1718 Da dimeric ellagitannin 1718 Da dimeric ellagitannin 2020 Da a dimeric ellagitannin 2020 Da b dimeric ellagitannin 2020 Da c dimeric ellagitannin 2020 Da d dimeric ellagitannin 2038 Da a dimeric ellagitannin 2038 Da b dimeric ellagitannin 1870 Da agrimoniin trimeric ellagitannin 2502 Da a trimeric ellagitannin 2804 Da catechin quercetin diglycoside 610 Da kaempferol diglycoside 594 Da quercetin glycoside 478 Da flavonoid glycoside 446 Da trimeric proanthocyanidin dimeric proanthocyanidin a dimeric proanthocyanidin b chlorogenic acid total phenolics
control day 0 0.84 1.38 0.31 1.14 2.83 0.07 0.10 0.26 0.20 0.89 1.24 0.84 0.71 0.15 0.8 1.00 0.21 1.39 0.21 1.94 27.85 1.65 0.63 6.89 10.51 8.56 3.79 0.22 0.28 3.23 0.29 0.74 81.15
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.01 (1.0) 0.08 (1.7) 0.02 (0.4) 0.07 (1.4) 0.14 (3.5) 0.01 (0.1) 0.01 (0.1) 0.02 (0.3) 0.01 (0.2) 0.04 (1.1) 0.08 (1.5) 0.02 (1.0) 0.02 (0.9) 0.02 (0.2) 0.05 (1.0) 0.07 (1.2) 0.01 (0.3) 0.14 (1.7) 0.03 (0.3) 0.06 (2.4) 0.2 (34.3) 0.08 (2.0) 0.06 (0.8) 0.28 (8.5) 0.28 (12.9) 0.23 (10.6) 0.18 (4.7) 0.04 (0.3) 0.07 (0.3) 0.13 (4.0) 0.03 (0.4) 0.02 (0.9) 0.64
control day 7 0.38 1.25 0.19 1.43 2.72 0.05 0.08 0.18 0.16 0.93 1.14 0.57 0.45 0.13 0.72 0.80 0.19 1.49 0.29 1.17 23.33 1.64 0.41 9.68 12.36 5.25 5.86 0.30 0.50 5.51 0.49 1.07 80.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.04 0.01 0.01 0.04 0.01 0.01 0.01 0.01 0.04 0.02 0.04 0.03 0.01 0.04 0.06 0.01 0.04 0.03 0.11 0.58 0.07 0.02 0.27 0.17 0.27 0.17 0.06 0.09 0.47 0.05 0.03 0.32
(0.5) (1.5) (0.2) (1.8) (3.4) (0.1) (0.1) (0.2) (0.2) (1.2) (1.4) (0.7) (0.6) (0.2) (0.9) (1.0) (0.2) (1.8) (0.4) (1.4) (28.9) (2.0) (0.5) (12.0) (15.2) (6.5) (7.3) (0.4) (0.6) (6.8) (0.6) (1.3)
BTH day 7 0.78 1.50 0.31 1.41 2.87 0.06 0.10 0.33 0.17 0.95 1.47 0.70 0.50 0.10 0.84 1.12 0.21 1.64 0.25 2.46 28.02 2.03 0.59 8.35 11.09 7.60 3.86 0.25 0.30 3.87 0.36 1.26 85.36
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.03 0.04 0.02 0.03 0.08 0.01 0.01 0.02 0.02 0.08 0.06 0.05 0.02 0.03 0.03 0.08 0.01 0.13 0.03 0.13 0.50 0.02 0.08 0.23 0.29 0.35 0.12 0.03 0.07 0.24 0.02 0.08 1.75
(0.9) (1.8) (0.4) (1.7) (3.4) (0.1) (0.1) (0.4) (0.2) (1.1) (1.7) (0.8) (0.6) (0.1) (1.0) (1.3) (0.2) (1.9) (0.3) (2.9) (32.8) (2.4) (0.7) (9.8) (12.9) (8.9) (4.5) (0.3) (0.4) (4.5) (0.4) (1.5)
birch day 7 0.35 1.14 0.21 1.33 2.85 0.05 0.06 0.13 0.17 0.90 1.06 0.59 0.46 0.13 0.75 0.88 0.20 1.53 0.25 0.92 23.61 1.66 0.44 8.92 11.88 5.54 5.99 0.36 0.44 5.11 0.36 2.26 80.57
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.02 0.03 0.01 0.03 0.06 0.01 0.01 0.01 0.01 0.05 0.03 0.04 0.03 0.02 0.05 0.05 0.01 0.14 0.05 0.06 0.87 0.06 0.05 0.40 1.00 0.49 0.21 0.07 0.07 0.19 0.04 0.04 2.40
(0.4) (1.4) (0.3) (1.7) (3.5) (0.1) (0.1) (0.2) (0.2) (1.1) (1.3) (0.7) (0.6) (0.2) (0.9) (1.1) (0.2) (1.9) (0.3) (1.1) (29.3) (2.1) (0.5) (11.1) (14.7) (6.9) (7.4) (0.4) (0.5) (6.3) (0.4) (2.8)
Values are presented as the mean ± standard deviation with percent concentration in parentheses. Sample size for each treatment was 4. BTH, BTH-treated leaves; Birch, birch wood distillate-treated leaves. a
plants was found to be reduced to the same level as in control plants within 7 days even if the concentration was higher in treated than in untreated plants after 2 days of exposure. This is in line with the recent data demonstrating that majority of the phenolic compounds accumulated in plants in a genotypedependent manner42 because a different cultivar (cv. ‘Polka’) was used in the present study. The total amount of ellagitannins in BTH-treated strawberry leaves was found to increase within the 7 days after BTH exposure, but the total content of galloyl derivatives was not much altered in BTH-treated plants over the test period. In control plants, the total contents of both ellagitannins and galloyl derivatives were somewhat decreased during the 7 day experiment. Instead, the total amount of proanthocyanidins was increased in all leaf samples within the 7 day test period, but the increase was more pronounced in control samples than in BTH-exposed samples. In our previous work32 we found that the most significant early accumulation of strawberry leaf phenolic compounds after BTH application occurred in the ellagitannin group but that the increased total ellagitannin content of BTH-treated plants did not persist over 7 days. However, the total amounts of tannin groups do not indicate any specific biological activities in a
summarize 77% of the variance in the data set (Supporting Information Table S1). These principal components were able to separate samples into unique clusters corresponding to different treatments (Figure 2A). ANOVA and Tukey’s pairwise comparisons revealed these clusters formed significantly different groups. The first principal component (PC1) separated samples into three clusters, corresponding to different treatments. All treatments were differentiated in PC1 except for control day 7 and birch wood distillate-treated day 7 (Figure 2A, x-axis). The second principal component (PC2) further differentiated groups on the basis of their combined phenolic profiles. Here, control day 7 and birch wood distillatetreated day 7 were differentiated (Figure 2A, y-axis). The individual traits with the highest and lowest loading values contributed most to these differences (Figure 2B; Supporting Information Table S1). Impact of BTH Treatment on Strawberry Leaf Phenolics. The total amount of phenolic compounds in untreated control leaves was not much altered in 7 days, but in the BTH-treated leaves the concentration of total phenolic compounds was increased 7 days after the BTH exposure (Table 2). Interestingly, in a previous work32 using strawberry cultivar ‘Jonsok’, the concentration of phenolics in BTH-treated 4597
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dependent manner, even though the total amount of phenolic compounds is not necessarily altered.41 Salicylic acid (SA), a 2-hydroxyl derivative of benzoic acid, is an important player not only in plant innate immunity but also in systemic-acquired resistance (SAR), and both SA and its analogue BTH as well as incompatible microbial interaction are known to induce rapid oxidative burst (OB) in plants.43,44 OB is a plant defense mechanism causing rapid and extensive production of reactive oxygen species, mainly superoxide radical (•O2−), hydrogen peroxide (H2O2), and hydroxyl radical (•OH), which destroy invading pathogens and act as signal molecules in further stress-related reactions in plant cells; however, they can also damage plant tissues and organelles.45,46 Proanthocyanidins, ellagitannins, and galloyl-glucose esters are strong radical scavengers.43 For example, in sugar cane (Saccharum spp.), exposure to H2O2 has been shown to cause up-regulation of phenylpropanoid pathways and, hence, increase the production of phenolic metabolites in the plants 24 h after the H2O2 treatment.46 Ellagitannins generally play protective roles in plants against microbial pathogens and insects pests.8,9 It was recently found that a penta-esterified ellagitannin, identified as 1-O-galloyl-2,3;4,6-bis-hexahydroxydiphenoyl-β-D-glucopyranose, is strongly accumulated in strawberry leaves as a result of infection by nonvirulent Colletotrichum fragariae;47 this ellagitannin may have properties of common plant defense molecule and capability of activating defense cascades perhaps via the transcription factor WRKY1 following elicitor treatment or pathogen infection.48 In addition, (+)-catechin previously isolated from strawberry leaves was shown to contribute the induced resistance against fungal diseases.49 Proanthocyanidins are oligomers and polymers of flavan-3-ol units, such as (+)-catechin, and flavan-3-ols are products of the flavonoid biosynthesis pathway.50 Like total proanthocyanidins, total flavonoid compounds in the control and BTH-treated plants were increased 7 days after the exposure, but the flavonoid level in the BTH-exposed samples stayed lower than in the control plants. In a previous study,32 the amount of catechin was not altered upon BTH application. Stress factors, such as competition, herbivory, and diseases, can cause changes in allocation patterns in plants and plant leaves, and these changes are often dependent on both plant genotype and resource availability.51 Effect of Birch Wood Distillate on Leaf Phenolics. To examine if plant-derived natural elicitors are capable of activation of the phenolic defense compound production in strawberry, leaves were treated with birch wood distillate. Interestingly, our data clearly indicate that birch wood distillate exposure increased the concentrations of chlorogenic acids in strawberry leaves. Chlorogenic acids were increased in control plants as well, but the increase in distillate-treated plants was much more pronounced (Table 2). The other phenolic compounds exhibited no changes in response to birch wood distillate treatment. In plants, chlorogenic acid has been demonstrated to play an important role in plant resistance to fungal diseases.52 The strong antibacterial activity of chlorogenic acid appears to be based on its ability to modulate outer and plasma membrane permeability, resulting in the loss of the important barrier function.53 In peach (Prunus persica), another member of the Rosaceae family, neochlorogenic acid, an isomer of chlorogenic acid, has been found to inhibit brown rot (Monilinia laxa) infections, and also chlorogenic acid has a reducing effect on melanin production and, hence, on the growth of the fungus.54
Figure 2. Results of principal component analysis. Panel A shows the sample scores on PC1 and PC2 from PCA. Bars and letters on each PC axis show the results of ANOVA analyses, groups with different letters were significantly different at α = 0.05. BTH, BTH-treated strawberry leaves; Birch, birch wood distillate-treated strawberry leaves. Panel B shows the loading values of response values (phenolic compound concentrations and total phenolics) on the same PCA axes.
plant sample because the activity of an individual tannin compound is affected by its chemical structure and the contents of different active and nonactive compound forms may vary.41 It is known that the contents of individual phenolics in tree leaves can change during leaf development and in a season4598
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internalization, reduced pesticide application rates, and climate change. Proc. Environ. Sci. 2011, 6, 153−161. (6) Steffens, K.; Larsbo, M.; Moeys, J.; Jarvis, N.; Lewan, E. Predicting pesticide leaching under climate change: Importance of model structure and parameter uncertainty. Agric. Ecosyst. Environ. 2013, 172, 24−34. (7) Krishna, V. V.; Kumar, K. G.; Pradeepa, K.; Kumar, S. R. S.; Kumar, R. S. Biochemical markers assisted screening of Fusarium wilt resistant Musa paradisiacal (L.) cv. Puttabale micropropagated clones. Indian J. Exp. Biol. 2013, 51, 531−542. (8) Ruuhola, T.; Salminen, P.; Salminen, J.-P.; Ossipov, V. Ellagitannins: defences of Betula nana against Epirrita autumnata folivory? Agric. For. Entomol. 2013, 15, 187−196. (9) Daglia, M. Polyphenols as antimicrobial agents. Curr. Opin. Biotechnol. 2012, 23, 174−181. (10) Martz, F.; Jaakola, L.; Julkunen-Tiitto, R.; Stark, S. Phenolic composition and antioxidant capacity of bilberry (Vaccinium myrtillus) leaves in Northern Europe following foliar development and along environmental gradients. J. Chem. Ecol. 2010, 36, 1017−1028. (11) Wang, S. Y.; Chen, C.-T.; Wang, C. Y. The influence of light and maturity on fruit quality and flavonoid content of red raspberries. Food Chem. 2009, 112, 676−684. (12) Anttonen, M.; Hoppula, K.; Nestby, R.; Verheul, M.; Karjalainen, R. Influence of fertilization, mulch color, early forcing, fruit order, planting date, shading, growing environment, and genotype on the contents of selected phenolics in strawberry (Fragaria × ananassa Duch.) fruits. J. Agric. Food Chem. 2006, 54, 2614−2620. (13) Lillo, C.; Lea, U.; Ruoff, P. Nutrient depletion as a key factor for manipulating gene expression and product formation in different branches of the flavonoid pathway. Plant Cell Environ. 2008, 31, 587− 601. (14) Jansen, M. A. K.; Hectors, K.; O’Brien, N. M.; Guisez, Y.; Potters, G. Plant stress and human health: do human consumers benefit from UV-B acclimated crops? Plant Sci. 2008, 175, 449−458. (15) Kangasjärvi, J.; Talvinen, J.; Utriainen, M.; Karjalainen, R. Plant defense systems induced by ozone: review. Plant Cell Environ. 1994, 17, 783−794. (16) Agati, G.; Azzarello, E.; Pollastri, S.; Tattini, M. Flavonoids as antioxidants in plants: location and functional significance. Plant Sci. 2012, 196, 67−76. (17) Boudet, A.-M. Evolution and current status of research in phenolic compounds. Phytochemistry 2007, 68, 2722−2735. (18) Pourcel, L.; Routaboul, J.-M.; Cheynier, V.; Lepiniec, L.; Debeaujon, I. Flavonoid oxidation in plants: from biochemical properties to physiological functions. Trends Plant Sci. 2007, 12, 29− 36. (19) Wang, Y.; Lin, H. H. Antioxidant activity in fruits and leaves of blackberry, raspberry, and strawberry varies with cultivar and developmental stage. J. Agric. Food Chem. 2000, 48, 140−146. (20) Walters, D. R.; Fountaine, J. M. Practical application of induced resistance to plant diseases: an appraisal of effectiveness under field conditions. J. Agric. Sci. 2009, 147, 523−535. (21) Tabart, J.; Franck, T.; Kevers, C.; Pincemail, J.; Serteyn, D.; Defraigne, J.-O.; Dommes, J. Antioxidant and anti-inflammatory activities of Ribes nigrum extracts. Food Chem. 2006, 131, 1116−1122. (22) Oszmiański, J.; Wojdyło, A.; Gorzelany, J.; Kapusta, I. Identification and characterization of low molecular weight polyphenols in berry leaf extracts by HPLC-DAD and LC-ESI/MS. J. Agric. Food Chem. 2011, 59, 12830−12835. (23) Lavola, A.; Karjalainen, R.; Julkunen-Tiitto, R. Bioactive polyphenols in leaves, stems and berries of Saskatoon (Amelancier alnifolia Nutt) cultivars. J. Agric. Food Chem. 2012, 60, 1020−1027. (24) Simirgiotis, M. J.; Schmeda-Hirschmann, G. Determination of phenolic composition and antioxidant activity in fruits, rhizomes and leaves of the white strawberry (Fragaria chiloensis spp. chiloensis form chiloensis) using HPLC-DAD-ESI-MS and free radical quenching techniques. J. Food Compos. Anal. 2010, 23, 545−553. (25) Mudnic, I.; Modun, D.; Brizic, I.; Vukovic, J.; Generalic, I.; Katalinic, V.; Bilusic, T.; Ljubenkov, I.; Boban, M. Cardiovascular
Collectively, our data demonstrate that strawberry leaves possess a substantial amount of ellagitannins and that the accumulation of specific ellagitannins in young strawberry leaves can be further increased by elicitor treatments such as BTH application. Our data also suggest that plant-derived activators such as birch wood distillate can activate different branches in the strawberry phenylpropanoid biosynthetic pathway from synthetic ones (such as BTH); treatments of young plants in greenhouses with different types of plant activators may, hence, hold great potential for enhancing specific compounds in plants for the production of valuable biomaterial for industrial applications, for example, cosmetics, dietary supplements, pharmaceuticals, and animal feed. Consequently, young greenhouse-grown strawberry plants provide a homogeneous and inexpensive material for the production of specific ellagitannins. BTH treatments have also been found to improve resistance in grape plants to gray mold and in strawberry to powdery mildew.32,55 In addition, some studies support the idea of using pyrolysis byproducts as environmentally and economically sustainable plant-protecting agents.33 Hence, more extensive study on the application and mechanisms of action of BTH and wood-derived biological activators in protecting plants from fungal diseases could also be worthwhile.
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ASSOCIATED CONTENT
S Supporting Information *
Tables S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(R.O.K.) E-mail: reijo.karjalainen@uef.fi. Funding
This study was financially supported by the Academy of Finland (Grant 258992 to J.P.S. and Grant 251388 to M.K.). We thank the Maiju and Yrjö Rikala Horticultural Foundation and the Northern Savo ELY-center for financial support. Chemical analyses on the ultraperformance liquid chromatography−mass spectrometry system were made possible by a Strategic Research Grant of University of Turku (Ecological Interactions) to J.P.S. Notes
The authors declare no competing financial interest.
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REFERENCES
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