Changes in the Triterpenoid Content of Cuticular Waxes during Fruit

Article pubs.acs.org/JAFC

Changes in the Triterpenoid Content of Cuticular Waxes during Fruit Ripening of Eight Grape (Vitis vinifera) Cultivars Grown in the Upper Rhine Valley Flora Pensec,† Cezary Pączkowski,‡ Marta Grabarczyk,‡ Agnieszka Woźniak,‡ Mélanie Bénard-Gellon,† Christophe Bertsch,† Julie Chong,† and Anna Szakiel*,‡ †

Laboratoire Vigne Biotechnologies et Environnement EA 3391, Université de Haute Alsace, 33 rue de Herrlisheim, 68000 Colmar, France ‡ Department of Plant Biochemistry, Faculty of Biology, University of Warsaw, ul. Miecznikowa 1, 02-096 Warszawa, Poland S Supporting Information *

ABSTRACT: Triterpenoids present in grape cuticular waxes are of interest due to their potential role in protection against biotic stresses, their impact on the mechanical toughness of the fruit surface, and the potential industrial application of these biologically active compounds from grape pomace. The determination of the triterpenoid profile of cuticular waxes reported here supplements existing knowledge of the chemical diversity of grape, with some compounds reported in this species for the first time. Common compounds identified in eight examined cultivars grown in the Upper Rhine Valley include oleanolic acid, oleanolic and ursolic acid methyl esters, oleanolic aldehyde, α-amyrin, α-amyrenone, β-amyrin, cycloartanol, 24methylenecycloartanol, erythrodiol, germanicol, lupeol accompanied by lupeol acetate, campesterol, cholesterol, sitosterol, stigmasterol, and stigmasta-3,5-dien-7-one, whereas 3,12-oleandione was specific for the Muscat d’Alsace cultivar. Changes in the triterpenoid content of cuticular waxes were determined at three different phenological stages: young grapes, grapes at véraison (the onset of ripening), and mature grapes. The results reveal a characteristic evolution of triterpenoid content during fruit development, with a high level of total triterpenoids in young grapes that gradually decreases with a slight increase in the level of neutral triterpenoids. This phenomenon may partially explain changes in the mechanical properties of the cuticle and possible modulations in the susceptibility to pathogens of mature grapes. KEYWORDS: triterpenoids, grape (Vitis vinifera L.), grape berry, cuticular waxes, fruit ripening, GC-MS

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from internal tissues. However, the cuticle also acts as the first protective barrier against abiotic and biotic environmental stresses and provides the external mechanical support to maintain fruit integrity, which is particularly important for fruits that lack hard internal tissue.7 Cuticles are formed as layers that are primarily composed of two classes of lipids: lipids derived from very long chain fatty acids and lipids derived from isoprene. Lipids derived from isoprene, which primarily include triterpenoids (pentacyclic triterpenes and steroids), are present in cuticular waxes.8 Triterpenoids represent a large group of natural products synthesized from isopentenyl diphosphate via the C30 precursor squalene. The diversity of enzymes capable of cyclizing 2,3-oxidosqualene and the large number of possibilities to establish different internal linkages during cyclization yields more than 100 different natural tetra- or pentacyclic triterpene skeletons.9,10 Triterpenoids have been shown to exhibit numerous biological activities and demonstrate various pharmacological effects.11−13 Therefore, the presence of triterpenoids in fruit cuticular waxes is of interest due to their potential role in protection against biotic stresses and health benefits ascribed to whole fruit consumption, in

INTRODUCTION Grapes (Vitis vinifera L.) are one of the most widely cultivated fruit crops of great economic importance. Regular consumption of grape products (fresh berries, raisins, juice, and wine) has been associated with a reduction in the incidence of chronic illnesses, such as cancer, cardiovascular diseases, ischemic stroke, and neurodegenerative disorders.1−3 In the European Union, the majority of the total crop is used in the winemaking industry and generates large quantities of pomace. Grape seeds and “skins” (which is a commonly used term to denote the outer fruit layers that primarily include the cuticle and epidermis) represent the majority of the volume of grape pomace and are thus considered large-scale waste that can be utilized in sustainable recycling.4,5 The global characterization of compounds present in the grape cuticle can therefore serve as a foundation for an integrated exploitation of this inexpensive and readily available source of products that are potentially suitable for the pharmaceutical, cosmetics, and food supplement industries.6 However, in addition to a winemaking byproduct, the grape cuticle may play a potential role in pathogen susceptibility, fruit quality, storage ability, and postharvest shelf life. The primary functions attributed to the fruit cuticle, which forms the hydrophobic coating on the epidermis, include the prevention of desiccation by minimizing water loss and the restriction of leaching of organic and inorganic compounds © 2014 American Chemical Society

Received: Revised: Accepted: Published: 7998

April 29, 2014 July 23, 2014 July 24, 2014 July 24, 2014 dx.doi.org/10.1021/jf502033s | J. Agric. Food Chem. 2014, 62, 7998−8007

Journal of Agricultural and Food Chemistry

Article

and ketones); (ii) triterpene acids; and (iii) triterpenoid (sterol and triterpene) low-polarity esters. The individual fractions were localized on plates by comparison with standards of oleanolic acid, sitosterol, αamyrin, and sitosterol palmitate and were visualized by spraying the appropriate area on the plate with 50% H2SO4, followed by heating with a hot-air stream. Fractions were eluted from the gel in at least 10 volumes of diethyl ether relative to the volume of the isolated gel, with the average recovery of individual triterpenoids of 96−98%.22 The fractions containing free neutral triterpenes and steroids (RF 0.3−0.9) were directly analyzed using GC-MS, whereas fractions containing triterpene acids (RF 0.2−0.3) were first methylated with diazomethane, and fractions containing triterpenoid (triterpene and sterol) lowpolarity esters (RF 0.9−1) were subjected to alkaline hydrolysis. Methylation of Triterpene Acids. Nitrosomethylurea (2.06 g) was added to a mixture of 20 mL of diethyl ether and 6 mL of 25% aqueous KOH, and the organic layer was washed with water (3 × 50 mL) and separated from the aqueous layer. Samples containing triterpene acids (not more than 10 mg) were dissolved in 2 mL of the obtained solution of diazomethane in diethyl ether and incubated at 2 °C for 24 h. Alkaline Hydrolysis. The ester fraction was subjected to alkaline hydrolysis using 10% NaOH in 80% MeOH at 80 °C for 3 h. Subsequently, 5 volumes of water was added to each hydrolysate, adjusted to neutral pH with 5% CH3COOH, and the obtained mixtures were extracted with diethyl ether (3 × 10 mL). These extracts were fractionated using preparative TLC as previously described. Subsequently, fractions containing free triterpene alcohols and sterols were directly analyzed using GC-MS, whereas triterpene acid fractions were methylated prior to this analysis. Identification and Quantitation of Triterpenoids using GCMS/Flame Ionization Detector (FID). An Agilent Technologies 7890A gas chromatograph (Perlan Technologies, Warsaw, Poland) equipped with a 5975C mass spectrometric detector was used for qualitative and quantitative analyses. Samples dissolved in diethyl ether/methanol (5:1, v/v) were applied (in a volume of 1−4 μL) using 1:10 split injection. All samples were analyzed in triplicate. The column used was a 30 m × 0.25 mm i.d., 0.25 μm, HP-5MS (Agilent Technologies, Santa Clara, CA, USA). Helium was used as the carrier gas at a flow rate of 1 mL/min. The following parameters were employed: column temperature, 280 °C; inlet and FID temperature, 290 °C; MS transfer line temperature, 275 °C; quadrupole temperature, 150 °C; ion source temperature, 230 °C; EI, 70 eV; m/z range, 33−500; FID gas (H2) flow, 30 mL/min (hydrogen generator); and air flow, 400 mL/min. Individual compounds were identified by comparing their mass spectra with library data from Wiley 9th ed. and NIST 2008 Lib. SW version 2010 or previously reported data and by comparison of their retention times and corresponding mass spectra with those of authentic standards, when available. Quantitation was performed using an external standard method based on calibration curves determined for the following representative triterpenoid classes: α-amyrin, oleanolic acid methyl ester, and sitosterol. Separation by High-Performance Liquid Chromatography (HPLC). Chromatographic analysis of samples containing a mixture of α-amyrin and lupeol was performed using a chromatographic system (Shimadzu, Shim-pol, Izabelin, Poland) equipped with two LC-10AT pumps, a CTO-10AS oven, and an SPD-10A spectrophotometer set at 200 and 254 nm. A 250 mm × 4.6 mm i.d., 4 μm, Synergi Max-RP 80Å column (Phenomenex, Torrance, CA, USA) was used. All data were acquired and processed using Shimadzu CLASS-VP version 5.032 chromatography data system software. The mobile phase (flow rate of 0.6 mL/min) was 100% acetonitrile (isocratic system), and separation was performed at 30 °C. Quantitative analysis of α-amyrin and lupeol was based on the relative ratios of their peak areas.22 Statistical Analysis. Data are presented as the means ± standard deviations of three independent samples analyzed in triplicate. The data were subjected to one-way analysis of variance (ANOVA), and the differences between means were evaluated using Duncan’s multiple-range test. Statistical significance was considered at p < 0.05.

which fruits are regarded as a functional mixture of watersoluble compounds from the fruit flesh and lipophilic constituents of the cuticle.14 Nevertheless, current knowledge of the triterpenoid composition and content in grape cuticular waxes remains limited.15 Cuticle biosynthesis during fruit development has been primarily investigated from a morphological or quantitative perspective.7 However, a limited number of studies have reported the compositional alterations in individual cuticular components during fruit development. These reports revealed substantial differences in time-dependent changes, in particular, constituents that are dependent on the type of fruit, indicating that these studies require case-by-case investigation. Moreover, alterations in a limited number of compounds, for example, proanthocyanidins,16 polysaccharides,17 and sterols,18,19 that are present in the cuticle and the whole “skin” during grape berry development have been investigated. The presence of several triterpenoids in grape berry cuticular waxes has been reported in several studies.15,19 However, a complete identification of the triterpenoids in grape berry cuticular waxes has not been reported, although the number of identified compounds continues to increase due to the chemical diversity of grapevines. Therefore, the aim of this study was to determine the composition and content of triterpenoids in cuticular waxes of grape berries of the following eight cultivars that are characteristic of the Upper Rhine Valley: the white varieties Chasselas, Gewurztraminer, Muscat d’Alsace, Riesling, Sylvaner, Pinot auxerrois, and Pinot gris; and the red variety Pinot noir. Moreover, to enhance the understanding of the compositional and functional evolution of cuticular waxes during fruit development, alterations in the triterpenoid content were examined at three different phenological stages: young grapes, grapes at véraison (the onset of ripening), and mature grapes.

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MATERIALS AND METHODS

Plant Material. Randomly selected bunches of eight grape cultivars (Chasselas, Gewurztraminer, Muscat d’Alsace, Pinot auxerrois, Pinot gris, Pinot noir, Riesling, and Sylvaner) were collected in vineyards in Ingersheim, Ammerschwihr, and Katzenthal in the Upper Rhine Valley in the summer of 2011 at three different stages corresponding to young grapes (denoted T1, collected on June 27), grapes at véraison (denoted T2, collected on July 25), and mature grapes (denoted T3, collected on August 22). T1 corresponded to stage 75 (berries peasized, bunches hang) of the BBCH scale,20 T2 to stage 79 (berry touch complete), and T3 to stage 89 (berries ripe for harvest). The replicate samples of fruits (4−5 g for T1, 6−7 g for T2, and 11−13 g for T3) were prepared from different pooled sample sets of 20−25 g. Chemicals and Standards. All solvents used for extraction and analysis were of analytical grade. Authentic standards of α-amyrin and ursolic acid methyl ester were purchased from Roth (Karlsruhe, Germany); β-amyrin, lupeol, oleanolic acid, campesterol, cholesterol, sitosterol, and stigmasterol were purchased from Sigma-Aldrich (Steinheim, Germany). α-Amyrenone and β-amyrenone were obtained by oxidation of the α-amyrin and β-amyrin standards with chromium trioxide-pyridine in dichloromethane.21 Extraction and Fractionation of Cuticular Waxes. Each sample of entire fruit was extracted by incubation in 40 mL of chloroform with gentle stirring for 30 s at room temperature. The extracts were decanted and evaporated to dryness. After weighing, the obtained wax extracts (masses of approximately 3.7 mg for T1 samples, 5.0 mg for T2, and 8.8 mg for T3) were separated by preparative thin layer chromatography on 20 cm × 20 cm glass plates coated with a 0.25 mm layer of silica gel 60G (Merck, Darmstadt, Germany) in the solvent system CHCl3/MeOH (97:3, v/v) into three fractions: (i) free (nonesterified) steroids and neutral triterpenes (alcohols, aldehydes, 7999

dx.doi.org/10.1021/jf502033s | J. Agric. Food Chem. 2014, 62, 7998−8007


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Table 1. Masses of Cuticular Wax Extracts Obtained from Grapes at Three Phenological Stagesa mg/g dry wt ± SDb

average water content (%) cultivar

T1

T2

T3

Chasselas Gewurztraminer Muscat d’Alsace Pinot auxerrois Pinot gris Pinot noir Riesling Sylvaner

82 86 76 82 75 78 79 72

91 89 92 91 88 90 89 91

85 83 86 85 78 81 81 84

T1 6.0 5.7 3.5 5.4 4.6 4.1 4.9 3.4

± ± ± ± ± ± ± ±

0.6 0.3 0.4 0.5 0.4 0.6 0.4 0.5

T2 a a a a a a a a

7.6 8.6 8.3 6.5 7.9 7.7 7.6 7.8

± ± ± ± ± ± ± ±

0.9 0.3 1.3 0.3 2.3 1.9 0.9 0.8

T3 a b b b b b b b

3.5 5.5 3.8 4.4 4.7 5.0 3.9 3.9

± ± ± ± ± ± ± ±

0.2 0.9 0.4 0.3 0.9 0.9 0.4 0.4

b a a c a a c a

a T1, young grapes; T2, grapes at véraison; T3, mature grapes. bValues are expressed as the means of three independent samples. Values in a row not sharing a common letter are significantly different (p < 0.05).

Figure 1. Chemical structures of triterpenoids identified in grape cuticular waxes: α-amyrenone, 1; α-amyrin, 2; ursolic acid methyl ester, 3; βamyrin, 4; erythrodiol, 5; oleanolic aldehyde, 6; oleanolic acid, 7; oleanolic acid methyl ester, 8; lupeol, 9; lupeol acetate, 10; germanicol, 11; 3,12oleandione, 12; campesterol, 13; cholesterol, 14; cycloartanol, 15; 24-methylenocycloartanol, 16; sitosterol, 17; stigmasterol, 18; stigmasta-3,5-dien7-one, 19.

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epidermis, collenchyma, or parenchyma.15,22 The masses of the obtained extracts are summarized in Table 1. The development of the grape berry involves several phases, and each growth period is characterized by distinct physiological processes, which may be reflected in different chemical compositions.16,24 In this study, the amount of chloroform-soluble cuticular waxes, which is expressed per 1 g of berry dry weight, was found to be highest at the véraison

RESULTS AND DISCUSSION

Cuticular wax extracts were obtained by incubation of fresh entire grapes in chloroform with gentle stirring for 30 s. Despite potential incomplete recovery (up to 73% of the cuticular wax, depending on the plant species23), this brief, gentle method of extraction is commonly applied because it precludes solvent penetration across the cuticle and prevents contamination by compounds originating from deeper tissues such as the 8000



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Figure 2. GC-FID chromatograms of the fraction containing methyl esters of triterpene acids obtained from cuticular waxes of Riesling young grapes (A) and the fractions containing sterols and neutral triterpenes (alcohols, aldehydes, and ketones) from cuticular waxes of Gewurztraminer and Sylvaner mature grapes (B, C). Peaks are numbered according to Figure 1.

stage (T2). The increase in the amount of wax extract from young grapes (T1) to grapes at véraison (T2) can be >2-fold, as observed for Muscat d’Alsace and Sylvaner. With berry ripening (T3), the amount of chloroform-soluble waxes sharply decreases for cultivars such as Chasselas, Gewurztraminer, Pinot auxerrois, and Riesling to a level below that of young grapes (T1). However, this decrease does not necessarily reflect a dramatic decrease in the amount of these waxes on the berry surface during ripening. The lower levels of extracted wax per 1 g of T3 berry dry weight are caused by the increase in the dry weight of the berry due to chemical changes that are characteristic for maturing and ripening fruit, that is, the accumulation of anthocyanin pigments, other phenolic compounds, and, primarily, substantial levels of sugars. The different stages of berry development can also be reflected in changes in the water content. The young grapes (T1) contained the lowest amount of water, which ranged from 72

to 86%. The grapes at véraison stage (T2), which are entering the period of rapid cell expansion and tissue softening, contained the highest amount of water (88−92%). In the mature grapes (T3), the amount of water decreased to 78−85% due to the accumulation of sugars and other compounds and the growing density of mesocarp cells; however, these phenomena may occur from the evaporative loss of water. Indeed, the observed water content in grapes at different developmental stages may depend on external conditions that influence water availability; for example, periods of dryness or high humidity can considerably modify the water content in grape berries at various stages of development.24 Identification of Triterpenoids in Grape Cuticular Waxes. The obtained chloroform extracts of grape cuticular waxes were separated into three fractions using preparative TLC. The fractions containing free (nonesterified) steroids and neutral triterpenes, triterpene acids after methylation, and 8001



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Figure 3. Triterpenoid content in the grape berry waxes of Chasselas, Gewurztraminer, Muscat d’Alsace, and Riesling at three phenological stages (black bars, young grapes; dark gray bars, grapes at véraison; light gray bars, mature grapes). The results are expressed as the means of three independent samples. The results for each triterpenoid not sharing a common letter are significantly different (p < 0.05).

NIST library and with previously reported data.25 Additional significant peaks, with relative tR of 1.44 and 1.55, were identified as sitosterol, 17, and β-amyrin, 4, respectively. Certain smaller peaks present in all chromatograms were associated with other steroids, that is, cholesterol, 14, campesterol, 13, stigmasterol, 18, and stigmasta-3,5-dien-7one, 19, as well as triterpene alcohols, that is, germanicol (olean-18-en-3-ol), 11, α-amyrin, 2, and lupeol, 9, forming one common peak with a relative tR of 1.68, cycloartanol, 15, and its derivative 24-methylenecycloartanol, 16, erythrodiol, 5, and the triterpene ketone α-amyrenone, 1. In addition to free triterpenoids, three triterpenoid esters, that is, lupeol acetate, 10, and the naturally occurring methyl esters of oleanolic acid, 8, and ursolic acid, 3, which cofractionated with free neutral triterpenoids due to a similar chromatographic mobility, were also identified in the chromatograms of all cultivars. Moreover, the triterpene diketone 3,12-oleandione (relative tR of 2.1), 12, was exclusively detected in the fraction obtained from the wax

triterpenoids released from their hydrolyzed esters were individually subjected to GC-MS/FID analysis. The chemical structures of the triterpenoid compounds identified in grape cuticular waxes are shown in Figure 1. The only significant peak with a relative retention time of 2.29 (relative retention time values are relative to cholesterol, for which the retention time is set to 1) detected in the fractions containing triterpene acids after methylation obtained from cuticular waxes of grapes of all evaluated cultivars was identified as oleanolic acid methyl ester. A representative GCFID chromatogram is shown in Figure 2A. By contrast, the chromatograms of the fractions containing steroids and neutral triterpenes (triterpene alcohols, aldehydes, and ketones) were much more complex, containing several peaks of various tR and relative intensity. The representative chromatograms are shown in Figure 2B,C. The principal peak with a relative tR of 2.35 was associated with oleanolic aldehyde, 6, and its identification was confirmed by comparison with mass spectra from the Wiley8002



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Figure 4. Triterpenoid content in the grape berry waxes of Sylvaner, Pinot noir, Pinot gris, and Pinot auxerrois at three phenological stages (black bars, young grapes; dark gray bars, grapes at véraison; light gray bars, mature grapes). The results are expressed as the means of three independent samples. The results for each triterpenoid not sharing a common letter are significantly different (p < 0.05).

and (viii) five steroids, campesterol, cholesterol, sitosterol, stigmasterol, and stigmasta-3,5-dien-7-one. The presence of oleanolic acid (which is a triterpene acid that contains an oleanane-type carbon skeleton that is very common in higher plants) in grape berry wax was known as early as 1938.26 Notably, the triterpene acid ursolic acid, an isomer of oleanolic acid that differs in the position of methyl groups at C29 and C30, which contains an ursane-type skeleton, was found in grape waxes only as methyl esters rather than as free forms. Thus, it can be concluded that triterpenoids derived from the oleanane skeleton are predominant in V. vinifera species, comprising sequential oxidation products of the precursor β-amyrin that contain additional hydroxymethyl and carbonyl groups (i.e., erythrodiol and oleanolic aldehyde) and can proceed to the final product that contains a carboxylic group (oleanolic acid), which can undergo additional esterification. In contrast to the presence of different oleanane triterpenoids, the series of ursane compounds is unexpectedly

extract of T1 Muscat d’Alsace samples. Other peaks that have not been numbered were in most cases identified as hydrocarbons typically occurring in plant cuticular waxes. Chromatograms of the fractions obtained following alkaline hydrolysis of low-polar esters revealed only the presence of some aliphatic compounds and did not contain detectable peaks of any triterpenoids. Thus, according to GC-MS analysis, the major triterpenoid profile of cuticular waxes of grapes from the eight examined cultivars exhibits the following composition: (i) one triterpene acid occurring in a free (nonesterified) form, oleanolic acid; (ii) two esters of triterpene acids, oleanolic and ursolic methyl esters; (iii) six monohydroxy alcohols, α-amyrin, β-amyrin, cycloartanol, 24-methylenecycloartanol, germanicol, and lupeol; (iv) one acetyl ester of a triterpene monohydroxy alcohol, lupeol acetate; (v) one dihydroxy alcohol, erythrodiol; (vi) one aldehyde, oleanolic aldehyde; (vii) one ketone, α-amyrenone; 8003



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incomplete, beginning with the alcohol and the ketone αamyrin and α-amyrenone, respectively, and ending with ursolic acid (which is immediately esterified), whereas the intermediate compounds uvaol and ursolic aldehyde were not detected. In the cultivars studied herein, triterpenoids that contain a lupanetype carbon skeleton, which is the third most commonly found triterpene skeleton in plants, were represented only by the precursor of the series, lupeol, which was present as a free form and as an acetyl ester. Neither betulin (lupane dihydroxy alcohol) nor betulinic acid was found in grape waxes except for the samples of young (T1) Muscat d’Alsace grapes, in which a negligible peak of betulinic acid methyl ester was detected. However, the presence of betulin and betulinic acid has been previously reported in grapes of the California raisin cultivar Thompson seedless.27 According to our results and those previously reported, the presence of oleanolic acid, oleanolic aldehyde, erythrodiol, and lupeol, as well as sterols, that is, campesterol, cholesterol, sitosterol, and stigmasterol, in grape cuticular wax appears to be a common feature in all V. vinifera cultivars studied to date.15 Our current results also revealed the presence of α- and βamyrins, α-amyrenone, cycloartanol, 24-methylenecycloartanol, and germanicol, an oleanane monohydroxy alcohol with a double bond between C18 and C19, which can be synthesized in addition to β-amyrin by a multifunctional triterpenoid synthase.28 These compounds have not been previously described in grape cuticular wax, perhaps due to the genuine absence of these compounds in other cultivars, but most probably because of their presence at low levels that are difficult to detect. The presence of certain other triterpenoids, for example, betulin and betulinic acid, is rather restricted to specific cultivars, for example, Thompson seedless grapes.27 Additionally, the presence of the diketone 3,12-oleandione reported in this study appears to be characteristic for Muscat d’Alsace. Triterpenoid Content in Grape Cuticular Waxes and Its Evolution during Fruit Ripening. Although the utilized method of wax extraction does not allow the 100% yield that is necessary for absolute quantitation, this study enables the evaluation of the relative changes in the content of pentacyclic triterpenes and steroids at three stages of fruit development: young grapes (T1), grapes at véraison (T2), and mature grapes (T3). The quantitative determination of individual triterpenoids identified in cuticular wax extracts of grapes of the eight evaluated cultivars are presented in Figures 3 and 4. The total triterpenoid content in 1 mg of cuticular wax extract was the highest in young grapes (T1 samples) and significantly differed among the evaluated cultivars, ranging from the lowest levels of 42 and 48% of the wax extract mass in Muscat d’Alsace and Riesling, respectively, to 62% in Gewurztraminer and nearly 80% in Sylvaner, which exhibited the highest level of triterpenoids of all cultivars evaluated in this study. All Pinot cultivars exhibited relatively similar levels of triterpenoids in T1 samples (53−59%), which were comparable to those in Chasselas (58%). During subsequent phases of fruit development, the total triterpenoid content decreased, and in mature grapes (T3 samples), the total triterpenoid content ranged from 34% in Muscat d’Alsace to 45 and 49% of the wax extract mass in Gewurztraminer and Sylvaner, respectively. This decrease in triterpenoid content was particularly sharp in Pinot gris, Sylvaner, and Pinot noir (the level in respective T3 samples decreased by 41, 39, and 38% compared with the level in T1 samples). In Muscat d’Alsace and Riesling, the initial

triterpenoid content decreased by only 18 and 15%, respectively, during fruit ripening. The most abundant triterpenoid present in grape cuticular waxes of all cultivars was oleanolic acid, as previously reported in numerous studies.15 A molecular model of the threedimensional arrangement of oleanolic acid and aliphatic constituents of cuticular wax (e.g., n-hexacosanol) was proposed to explain the organization of the grape berry cuticle characterized by such a high abundance of a single triterpene acid.29 In our study, oleanolic acid constituted on average 92% of the total triterpenoids in all cultivars. The level of oleanolic acid was highest in young grapes (T1) and ranged from 406 ± 22 μg/mg of wax extract in Muscat d’Alsace to 782 ± 58 μg/mg in Sylvaner; this level subsequently decreased during fruit maturation to 309 ± 12 μg/mg in Muscat d’Alsace and 440 ± 30 μg/mg in Sylvaner (Figures 3 and 4). The total neutral triterpenoid (pentacyclic triterpenes and steroids) content in 1 mg of cuticular wax extract was low in comparison to oleanolic acid and ranged from 5.25 μg/mg of wax extract in T1 Riesling samples to 53.6 μg/mg in T3 Gewurztraminer samples. In contrast to oleanolic acid, the levels of neutral triterpenoids tended to increase during fruit development, although not in an identical manner in all cultivars. In Chasselas, Muscat d’Alsace, Riesling, and Sylvaner, this increase was constant from T1 to T3, with a final neutral triterpenoid content that was 4- or 5-fold higher in the waxes of mature grapes compared with young fruits (a particularly substantial increase was observed in Muscat d’Alsace, Riesling, and Sylvaner). For all Pinot cultivars, the highest total neutral triterpenoid content was observed at véraison (T2), and this level subsequently slightly decreased during the last stage of ripening. Unexpectedly, in Gewurztraminer, the total level of neutral triterpenoids was lowest in grapes of the T2 samples. Among neutral triterpenoids, oleanolic aldehyde was the most abundant triterpenoid, except for Gewurztraminer, in which this compound was second to sitosterol. Indeed, in Gewurztraminer, oleanolic aldehyde constituted only 3% of all triterpenoids, whereas in other cultivars, the level of oleanolic aldehyde was higher, up to 6% of all triterpenoids in T2 samples of Pinot auxerrois and Pinot noir. The oleanolic aldehyde content increased during fruit development in grape waxes of all cultivars except for the three Pinot cultivars in which the highest concentration was observed at véraison (Figure 4). Considerable levels (>1 μg/mg of wax extract) of βamyrin, erythrodiol (except for Chasselas), and germanicol were detected in all evaluated cultivars. The levels of β-amyrin and its derivative erythrodiol, which contains an additional hydroxyl group, were altered in a similar manner in all cultivars. The highest levels of erythrodiol were observed in T3 samples of Chasselas, Gewurztraminer, Pinot auxerrois, Pinot gris, Riesling, and Sylvaner and at véraison in Pinot noir and Muscat d’Alsace (Figures 3 and 4). Moreover, the level of germanicol increased uniformly in grape wax extracts of all cultivars during fruit ripening. Additional pentacyclic triterpenoids and their esters, that is, α-amyrin, α-amyrenone, lupeol, lupeol acetate, and ursolic acid methyl ester, were found at only low levels (up to 0.2% of total triterpenoids) in all cultivars. The total steroid content generally increased during fruit development, reaching the highest level in the cuticular wax extract of mature grapes (5% of all triterpenoids in T3 samples of Gewurztraminer). As previously reported,15,18,19 sitosterol was the most abundant phytosterol in the grape waxes of all cultivars, following campesterol and stigmasterol. Among the 8004



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necessary to confirm the possible role of biologically active triterpenoids, which are known for their antibacterial, antifungal, and antiparasitic properties15 in grape resistance to pathogens. The initially very high level of triterpenoids in cuticular waxes followed by a gradual decrease in this level during fruit development has also been demonstrated for other fruits, for example, sweet cherry (Prunus avium) fruit;34 however, it is not a general rule in fruit ontogeny. In contrast, triterpenoids were shown to be progressively accumulated during olive fruit development, and the profile of these compounds was altered according to the different phases in its long maturation.35 The evolution of triterpenoid content in grape berry is also of interest in terms of the metabolic pathways of these compounds. In young grapes, the rapid biosynthesis of high levels of oleanolic acid occurs, encompassing the entire sequence of oxidation of the precursor, β-amyrin. Intriguingly, in T2 and T3 samples, the final reaction appears to be terminated, and intermediates, including oleanolic aldehyde and erythrodiol, begin to be accumulated in larger quantities. The metabolic behavior of phytosterols appears to be more readily accounted for. At fruit developmental phases involving growth by cell division and consecutive synthesis of membranes, the level of these compounds “wasted” for accumulation in surface waxes is relatively low. During the stages of water and sugar accumulation, the excess steroids can be exported into cuticular waxes and accumulate there at more substantial levels. The results presented in this study were obtained from samples collected during only one vegetative season. However, the similar patterns of changes in triterpenoid content observed in eight grape cultivars might suggest that this phenomenon is general, although the relative levels of individual compounds can vary in consecutive years due to the influence of external abiotic and biotic stimuli (e.g., meterological conditions, pathogen infections). In recent years, studies that have investigated the constituents in grape skins have been of increasing importance due to the possible utilization of winemaking byproducts, as well as unripe green grapes collected during “green harvesting” to reduce the yield of vineyards.36 The high level of oleanolic acid in grape pomace and green grapes makes these two agroindustrial waste residues an economically advantageous alternative source of this compound. One of the first reported methods was extraction of a freeze-dried grape pomace macerate of red grape Nerello Mascalese with ethyl acetate for obtaining oleanolic acid with a yield of 0.16% of pomace dry weight.37 However, other triterpenoid constituents present in grape cuticular waxes, for example, triterpene alcohols or phytosterols, may also be worthwhile to obtain from pomace.15,19,37 Oleanolic acid is known to have numerous pharmacological properties including anticancer, antidiabetogenic, antimicrobial, and hepatoprotective effects; therefore, the high abundance of this compound in grape berry cuticular wax can contribute to health benefits ascribed to fresh grape consumption, for example, the reduction of the incidence of type 2 diabetes and the prevention of liver disorders.15,38 Both oleanolic acid and oleanolic aldehyde obtained from Thompson seedless raisins were demonstrated to display significant antimicrobial activity against oral pathogens associated with human dental carries and periodontal diseases,27 which makes the raisins a healthy alternative to widely consumed sugary snack foods.39

evaluated cultivars, Gewurztraminer grape wax contained the highest level of sitosterol, constituting 4% of the total triterpenoids in T3 samples. Interestingly, the level of sitosterol was lowest at véraison in Gewurztraminer, in contrast to the other cultivars in which it increased relatively uniformly during grape maturation. In Chasselas and all Pinot cultivars, the sitosterol content linearly increased during fruit development; in Muscat d’Alsace and Sylvaner the level of sitosterol nearly doubled after véraison, and in Riesling, the level of sitosterol exhibited a sharp, 8-fold increase during fruit maturation (Figures 3 and 4). In addition to typical phytosterols (i.e., sitosterol, campesterol, and stigmasterol), grape cuticular wax extracts also contained small amounts of cholesterol, which is not considered a typical plant sterol, although it is commonly found in plant cuticular waxes.30 The steroid ketone stigmasta3,5-dien-7-one, also commonly found in plant cuticular waxes,23,31 was the most abundant in Chasselas, exceeding the level of sitosterol and constituting 1% of the total triterpenoids in T3 samples. The level of stigmasta-3,5-dien7-one decreased during fruit ripening in all cultivars except Sylvaner and Pinot noir. Finally, in all evaluated cultivars, the level of cycloartanol, which is the precursor of sterols, decreased during fruit maturation in all cultivars, whereas the amount of its derivative, 24-methylenecycloartanol, increased. Previous studies on the evolution of phytosterol content in grape skins reported a decrease in the level of phytosterols either during the last stage of maturity18 or during the transition from pre-véraison to véraison.19 However, in these studies, phytosterol content was expressed per dry weight of fruits rather than per mass of the wax extract. Therefore, according to chemical changes that occur during fruit development, the relative content of compounds can be lowered due to an increase in the dry weight caused by the accumulation of sugars and other metabolites associated with grape maturation. In this study, a considerable decrease in the level of oleanolic acid was observed during fruit development. It is unlikely that oleanolic acid, once accumulated in cuticular waxes, is either transported back to the cell or undergoes additional chemical transformations. Hence, this decrease could be explained by an increase in the level of aliphatic constituents of cuticular waxes and, consequently, by the dilution of oleanolic acid in the grape wax extract mass. It was previously demonstrated that the composition of grape cuticular waxes was altered during fruit development, with an increase in waxy deposits and a simultaneous decrease in the thickness of the cuticle.32 It can be assumed that with the growing size of the berry and the increase in water content during fruit ripening, some aliphatic hydrophobic compounds (primarily hydrocarbons32) are accumulated in surface layers of the cuticle to prevent the uncontrolled loss of water. It is commonly believed that triterpenoid constituents do not participate in the protection against water loss, and a high level of triterpenoids in cuticular wax is not positively correlated with water impermeability.15 However, as recently demonstrated for Fuyu persimmon fruit,33 triterpenoids affect the mechanical properties of the fruit cuticle and endow its toughness by functioning as a nanofiller, that is, filling the gaps in the cutin matrix with low-ordered crystals. Moreover, a decrease in the oleanolic acid content could explain the growing susceptibility of berries to fungal diseases during maturation. The compositional changes in cuticular components has been suggested to influence the susceptibility to Botrytis cinerea infection in grapes.32 Additional studies are 8005



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(6) González-Centeno, M. R.; Jourdes, M.; Femenia, A.; Simal, S.; Roselló, C.; Teissedre, P. L. Characterization of polyphenols and antioxidant potential of white grape pomace byproducts (Vitis vinifera L.). J. Agric. Food Chem. 2013, 61, 11579−11587. (7) Lara, I.; Belge, B.; Goulao, L. F. The fruit cuticle as a modulator of postharvest quality. Postharvest Biol. Technol. 2014, 87, 103−112. (8) Buschhaus, C.; Jetter, R. Composition differences between epicuticular and intracuticular wax substructures: how do plants seal their epidermal surfaces? J. Exp. Bot. 2011, 62, 841−853. (9) Sawai, S.; Saito, K. Triterpenoid biosynthesis and engineering in plants. Front. Plant Biotechnol. 2011, 2, 25. (10) Xu, R.; Fazio, G. C.; Matsuda, S. P. T. On the origins of triterpenoid skeletal diversity. Phytochemistry 2004, 65, 261−291. (11) Patočka, J. Biologically active pentacyclic triterpenes and their current medicine signification. J. Appl. Biomed. 2003, 1, 7−12. (12) Petronelli, A.; Pannitteri, G.; Testa, U. Triterpenoids as new promising anticancer drugs. Anti-Cancer Drugs 2009, 20, 880−892. (13) Yadav, V. R.; Prasad, S.; Sung, B.; Kannappan, R.; Aggarwal, B. B. Targeting inflammatory pathways by triterpenoids for prevention and treatment of cancer. Toxins 2010, 2, 2428−2466. (14) Liu, R. H. Health benefits of fruits and vegetables are from additive and synergistic combination of phytochemicals. Am. J. Clin. Nutr. 2003, 78, 517S−520S. (15) Szakiel, A.; Pączkowski, C.; Pensec, F.; Bertsch, C. Fruit cuticular waxes as a source of biologically active triterpenoids. Phytochem. Rev. 2012, 263−284. (16) Kennedy, J. A.; Hayasaka, Y.; Vidal, S.; Waters, E. J.; Jones, G. P. Composition of grape skin proanthocyanidins at different stages of berry development. J. Agric. Food Chem. 2001, 49, 5348−5355. (17) Vicens, A.; Founard, D.; Williams, P.; Sidhoum, L.; Moutounet, M.; Doco, T. Changes in polysaccharide and protein composition of cell walls in grape berry skin (cv. Shiraz) during ripening and overripening. J. Agric. Food Chem. 2009, 57, 2955−2960. (18) Le Fur, Y.; Hory, C.; Bard, M. H.; Olsson, A. Evolution of phytosterols in Chardonnay grape berry skins during last stages of ripening. Vitis 1994, 33, 127−131. (19) Ruggiero, A.; Vitalini, S.; Burlini, N.; Bernasconi, S.; Iriti, M. Phytosterols in grapes and wine, and effects of agrochemicals on their levels. Food Chem. 2013, 141, 3473−3479. (20) Lorenz, D. H.; Eichhorn, K. W.; Bleiholder, H.; Klose, R.; Meier, U.; Weber, E. Phenological growth stages of the grapevine (Vitis vinifera L. ssp. vinifera) − codes and descriptions according to the extended BBCH scale. Aust. J. Grape Wine Res. 1995, 1, 100−110. (21) Tojo, G.; Fernández, M. Oxidation of Alcohols to Aldehydes and Ketones; Springer: Berlin, Germany, 2006; pp 1−97. (22) Szakiel, A.; Pączkowski, C.; Huttunen, S. Triterpenoid content of berries and leaves of bilberry Vaccinium myrtillus from Finland and Poland. J. Agric. Food Chem. 2012, 60, 11839−11849. (23) Santier, S.; Chamel, A. Reassessment of the role of cuticular waxes in the transfer of organic molecules through plant cuticles. Plant Physiol. Biochem. 1998, 36, 225−231. (24) Conde, C.; Silva, P.; Fontes, N.; Dias, A. C. P.; Tavares, R. M.; Sousa, M. J.; Agasse, A.; Delrot, S.; Gerós, H. Biochemical changes throughout grape berry development and fruit and wine quality. Food 2007, 1, 1−22. (25) Dagna, L.; Gasparini, G.; Icardi, M. L.; Sessia, E. Study of some components of the unsaponifiable fraction in the skin of grapes. Am. J. Enol. Vitic. 1982, 33, 201−206. (26) Radler, F.; Horn, D. H. S. The composition of grape cuticle wax. Aust. J. Chem. 1965, 18, 1059−1069. (27) Rivero-Cruz, J. F.; Zhu, M.; Kinghorn, A. D.; Wu, C. D. Antimicrobial constituents of Thompson seedless raisins (Vitis vinifera) against selected oral pathogens. Phytochem. Lett. 2008, 1, 151−154. (28) Basyuni, M.; Oku, H.; Tsujimoto, E.; Kinjo, K.; Baba, S.; Takara, K. Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae. FEBS J. 2007, 274, 5028−5042.

In conclusion, this study examining the triterpenoid profile of fruit cuticular waxes of eight V. vinifera cultivars enhances the knowledge of the chemical diversity of grapevines. The profile of identified triterpenoids comprises compounds commonly present at significant levels in all grape cultivars studied to date, that is, oleanolic acid, oleanolic aldehyde, erythrodiol, lupeol, and phytosterols (campesterol, sitosterol, and stigmasterol). We also identified compounds that have been rarely or not previously reported in grape berry waxes, for example, α- and βamyrins, α-amyrenone, cycloartanol, 24-methylenecycloartanol, germanicol, and 3,12-oleandione. The triterpenoid content in cuticular waxes exhibits characteristic alterations during fruit development, with early deposition of high levels of oleanolic acid and its gradual decrease, which is not entirely compensated by the concomitant slight increase in the level of neutral triterpenoids. Termination of oleanolic acid biosynthesis may result in the modulation of the mechanical toughness of the cuticle and a decrease in disease resistance, features that typically appear in numerous fruits during ripening and storage.

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ASSOCIATED CONTENT

S Supporting Information *

GC-MS data of triterpenoids identified in grape berry cuticular waxes (Table 1S), calibration data for quantitative determination of identified triterpenoids (Table 2S), triterpenoid content in grape berry waxes at three phenological stages (Table 3S), and identification of 3,12-oleandione based on comparison of the mass spectra (Figure 1S). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(A.S.) Phone: +48 22 55 43 316. Fax: +48 22 55 43 221. Email: [email protected]. Funding

Research subject carried out with the use of CePT infractructure financed by the European Union, the European Regional Development Fund within the Operational Programme “Innovative economy” for 2007−2013. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Jean-Marc Bertsch and Hubert Straub for their kind permission for fruit collection in their vineyards. REFERENCES

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Changes in the Triterpenoid Content of Cuticular Waxes during Fruit

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