Original research articles

Stilbenes in the different organs of Vitis vinifera cv. Merlot grafted on Teleki Kober 5BB rootstock

Vol 51, No 3 (2017): OENO One

DOI: http://dx.doi.org/10.20870/oeno-one.2016.50.4.1068

Abstract

Aim: To determine which of the grapevine organ is the richest in stilbene, like the t-piceid, t-resveratrol and ε-viniferin and would it be useful as by-product of viticulture for processing stilbene concentrate.

Methods and results: Nine organs, such as cane, buds, shoot tips, inflorescences, clusters at veraison, matured berry skins, seeds and cluster stems, of Merlot grafted on Teleki Kober 5BB rootstock were collected during a year and its ethanolic extractions were analyzed via HPLC-FLD. Stilbene content of the different organs occurred in a wide range, the lowest was 3.15 mg/kg dry weight (dw) measured in the seeds and the highest 2265 mg/kg dw in the buds.

Conclusion: The present research demonstrated that different grapevine organs contain different amount of stilbene in the vineyard. The winter-buds, the roots and the matured cane internodes of Merlot are significantly richer in t-resveratrol and t-ε-viniferin than the green vegetative and generative parts we examined in one growing year.

Significance and impact of the study: According to our knowledge there was no stilbenes measuring in several plant organs of a vine cultivar in field study, in vineyard. Our research pointed out the pruned cane could be primary source of stilbene of the health-industry. Beside that grape root is a by-product of grapevine nursery is also rich in stilbene.

Introduction

Stilbenes are natural phenolic compounds occurring in a number of plant families including Vitaceae. Resveratrol is a low molecular weight stilbene molecule, its 3-O-glucoside is piceid and one of the resveratrol oligomers is ε-viniferin, a dehydrodimer. The putative medical benefits of stilbenes were intensively studied during the last decade. Resveratrol is an antioxidant agent that is thought to fend off cardiovascular diseases and cancer; influence Alzheimer’s disease and ageing; and prevent bone loss and diabetes (Tosun and Inkaya, 2009). The biological activities of piceid have not been thoroughly studied, but it seems it may also have beneficial effects on health (Chong et al., 2009). ε-Viniferin has promising actions in vitro, like decreasing glucose absorption (Guschlbauer et al., 2013) and inhibiting vascular muscle cell proliferation (Zghonda et al., 2011) and amyloid-beta peptide aggregation (Rivière et al., 2010). Resveratrol and its derivatives are naturally produced by plants as self-defence agents either in response to biotic or abiotic stress or constitutively without elicitors (Jeandet et al., 2010), and thus can be considered as phytoalexins and phytoanticipins. Stilbenes were detected or quantified in different vine organs, such as berries, seeds, berry skin, cluster stems, leaves, canes, roots (Waffo-Teguo et al., 2008), flowers (Keller et al., 2003; Timperio et al., 2012) and buds (Wang et al., 2010; Qsaib et al., 2014). To our knowledge, no measurement of stilbene content in several plant organs of a vine cultivar in a field study has been reported, and only limited data are available on grape root resveratrol and ε-viniferin content.

Materials and methods

1. Plant material and field conditions

Vitis vinifera cv. Merlot red wine grape cultivar was established on Teleki Kober 5BB (TK5BB) rootstock in 2004 in Szekszárd, Hungary (46°19’30’’N/18°41’09’’E, 130 m altitude). The effective temperature was 3538°C and rainfall was 798 mm in 2013.

2. Sample collection

Visually healthy plant organs were collected from 10 randomly chosen vines from the vineyard. Samples were pooled replicates and we made three technical repeats. The shoot tips and inflorescences were collected at full bloom in 2013. Clusters were collected at veraison and at harvest in 2013. The internodes of the cane, the bud and the root samples were collected in the dormant season in February 2014 (Figure 1).

Figure 1. investigated organs of the grape plant. Winter-buds (1), cane internodes (2), surface roots of the rootstock (3), shoot tip with a young leaf and a tendril at the beginning of the leaf expansion (4), inflorescence at full bloom (5), cluster at veraison (6), and cluster stem (7a), berry skins (7b) and seeds (7c) of a ripe grape cluster.

3. Sample processing

Sample preparation and extraction was performed with minor modifications according to the method of Rayne et al. (2008) within a day after collection. The ripe cluster was destemmed, and the skin and seeds were separated from the berries. The samples of plant material were treated with liquid nitrogen, then ground into powder (Moulinex AR100) and kept at -70°C in the dark until further processing. 1 g of fresh sample powder (particle size <1 mm) was extracted in 80% ethanol with 10 minutes ultrasound treatment at 38 kHz (Realsonic RS-26), and 10 minutes mixing (VWR-IKA VMS-C4) at room temperature. The extraction was performed three times with the samples. The mixture was centrifuged at 5500 g at 2°C for 10 minutes and filtered. The combined supernatants were evaporated under a nitrogen stream at 35°C (IKA-WERKE RV06-ML, HB4 basic). The dried residue was re-dissolved in 1 mL of extracting solution and kept at -20 °C in the dark. The solution was filtered (pore size 0.45 µm cellulose filter) prior to HPLC injection.

4. Chemicals and reagents

All chemicals and t-resveratrol (>99% purity), t-piceid (>95% purity) and ε-viniferin (>95% purity) standards were obtained from VWR International.

5. Chromatographic methods

HPLC-FLD analyses were conducted on Perkin Elmer Flexar chromatograph system and Chromera-Flexar FL data collection software. The analytical column Zorbax Eclipse XDB-C8 4.6×150 mm was thermostatically controlled at 30 °C. Mobile phase “A” was water with 0.5% formic acid and mobile phase “B” was acetonitrile with 10% water and 0.5% formic acid. The flow rate was 1 mL/min and the injection volume was 20 μL. Gradient elution was performed from 10% to 55% eluent “B” in 45 minutes with the following gradient program: 0 -> 1.0 min 10% B; 1.0 -> 45.0 min 10% -> 55% B; 46.0 -> 51.0 min 80% B; 51.0 -> 53.0 min 80% B; 53.0 -> 58.0 min 80% -> 10% B; 58.0 -> 61.0 min 10% B. The fluorimetric detector was set at 330 nm excitation and 374 nm emission wavelength for stilbenes according to Pezet et al. (1994) and Timperio et al. (2012).

Quantification was performed using external 5-point calibration curve (0.15, 0.3, 3.0, 9.0, 30.0 mg/L for t-piceid and ε-viniferin; 0.075, 0.15, 1.5, 4.5, 15.0 mg/L for t-resveratrol). The assay for stilbene standards was linear with high correlation coefficient (r=0.996 for t-piceid, 0.998 for t-resveratrol and 0.991 for ε-viniferin). The slope of the calibration curve was 4x10-6 (t-piceid and ε-viniferin) and 2x10-6 ppm/mFLUs (t-resveratrol). The limit of detection was 0.03 mg/L for t-piceid and ε-viniferin and 0.015 mg/L for t-resveratrol. The limit of quantification was the lowest concentrations of the calibration curves.

6. Statistical analysis

Data were analysed by two-way analysis of variance (ANOVA), and Duncan Multiple Range Post Hoc test was performed with SPSS 15. P<0.05 was considered statistically significant.

Results and discussion

In this study, biologically valuable stilbene molecules of viticultural by-products were measured from different organs of the plant. Mature canes with winter-buds are waste products of pruning and grafting; roots are available from the nurseries or sell off vineyards; shoot tips, inflorescences and immature clusters are removed from the vines during summer pruning; and cluster stems and berry skin and seeds of mature clusters are by-products of the wine making process. These plant organs are available each year from vine cultivation. The examined organs contained a wide range of stilbene contents. The sum of the three examined molecules was lowest in seeds (3.15 mg/kg dry weight (dw)) and highest in buds (2265 mg/kg dw) (Table 1). In the inflorescences of Merlot at full bloom, stilbenes were not detectable. Timperio et al. (2012) reported that Merlot flowers without Botrytis cinerea inoculation showed low concentrations of t-resveratrol (pg/µL magnitude in samples) and no δ-viniferin, which is a different resveratrol dimer than what we have investigated. Keller et al. (2003) in healthy Gamay flowers detected no ε-viniferin and resveratrol but 3.69 mg/kg fresh weight (fw) piceid. The winter-pruned canes contain nodes with buds and internodes. Most studies on cane stilbene content do not specify whether the canes were measured with or without buds. Lambert et al. (2013) reported 1181 mg/kg dw t-resveratrol and 2263 mg/kg dw ε-viniferin in Merlot canes. Our results are higher (2081 ± 170 mg/kg dw and 385.1 ± 33.4 mg/kg dw ε-viniferin in buds and internodes, respectively) but in line with Qsaib et al. (2014), who measured 90 mg/kg dw ε-viniferin in buds and 60 mg/kg dw ε-viniferin in internodes of Merlot. Buds were significantly richer in ε-viniferin than internodes and other grape organs. Cane internodes were significantly the richest organs in t-resveratrol and t-piceid (Table 1).

Table 1. Stilbene content of Merlot plant organs


Plant organs

t-piceid

t-resveratrol

ε-viniferin

bud

14.65 ± 1.57 c

168.3 ± 10.4 b

2081 ± 170 a

cane internode

125.9 ± 10.5 a

344.5 ± 32.2 a

385.1 ± 33.4 c

root

26.14 ± 1.26 b

113.0 ± 6.2 c

1476 ± 181 b

shoot tip

4.97 ± 0.08 d

2.96 ± 0.29 e

6.48 ± 0.29 d

inflorescence

nd*

nd*

nd*

cluster at veraison

16.08 ± 5.64 c

2.26 ± 0.16 e

18.46 ± 4.81 d

cluster stem

14.50 ± 2.26 c

17.43 ± 2.34 de

98.51 ± 6.13 d

berry skin

nd*

31.24 ± 1.37 d

121.54 ± 3.82 d

seed

nd*

0.78 ± 0.02 e

2.37 ± 0.38 d

Results are expressed as mg/kg dry weight and represent average values of triplicate determinations (n=3) ± standard deviation. *not detectable.

Values in the same column with different letters are statistically different (p<0.05) based on Duncan Multiple Range Post-Hoc test.

The rootstock of the investigated grafting was Vitis berlandieri x Vitis riparia TK5BB. The t-resveratrol content in roots was remarkable (113.0 ± 6.2 mg/kg dw) and that of ε-viniferin was the second highest among all organs (1476 ± 181 mg/kg dw). Wang et al. (2010) in roots of young Vitis vinifera cv. Cabernet sauvignon raised in pot measured approx. 4.8 mg/kg fw t-resveratrol, and Ji et al. (2014) in roots of wild Vitis amurensis reported 61.2-123.4 mg/kg dw over a year. Bavaresco et al. (2003) measured 15.5 mg/kg fw t-resveratrol and 32.3 mg/kg fw t-piceid in roots of Chardonnay TK5BB grafts. Bavaresco et al. (2000) reported 640 mg/kg fw t-resveratrol and 980 mg/kg fw ε-viniferin in roots of ungrafted TK5BB rootstocks.

Berries of other varieties were investigated by Bavaresco et al. (1997) at different developmental stages. They measured a few mg/kg fw stilbenes in berries at veraison. Our results contain berry and cluster stem stilbenes together at veraison, which are similarly low, as previously mentioned.

Bavaresco et al. (1997) measured 38 mg/kg fw t-resveratrol and 54 mg/kg fw ε-viniferin in cluster stems of Merlot and an average of 113 mg/kg dw and 288 mg/kg dw in other cultivars, respectively (Anastasiadi et al., 2012). Our data (17.43 ± 2.43 mg/kg dw and 98.51 ± 6.13 mg/kg dw, respectively) are comparable with these obtained from the literature.

The skin of mature grapes contains t-piceid besides the cis isomer (Romero-Pérez et al., 2001; Liu et al., 2013; Vincenzi et al., 2013), although it was not measured in this work. Our results for t-resveratrol contents in mature Merlot grape skin (31.24 ± 1.37 mg/kg dw) are similar to the values measured by Romero-Pérez et al. (2001) (38.26 mg/kg dw). The ε-viniferin values measured in Merlot grape skin (121.54 ± 3.82 mg/kg dw) were higher than in other blue grape cultivars measured by Anastasiadi et al. (2010) (5.1-65.3 mg/kg dw).

In Merlot grape seeds, t-piceid was not detectable and a low concentration of t-resveratrol (0.78 ± 0.02 mg/kg dw) was measured, as reported by Sun et al. (2006). In grape seeds, ε-viniferin was detected and low amount was measured (2.37 ± 0.38 mg/kg dw), contrary to Anastasiadi et al. (2010).

The variation of stilbene concentrations measured in grape organs could be high between studies. Ji et al. (2014) showed that the t-resveratrol concentration in V. amurensis organs varied significantly between seasons. The time between pruning and the analysis of the cane samples has an effect on the measurement results (Vergara et al., 2012). t-Resveratrol concentration in berries is significantly affected by variety and elevation (Bavaresco et al., 2007) and stilbene concentration by genotype, ripening (Gatto et al., 2008) and viticultural factors (Bavaresco, 2003). The sample preparation and the extraction method may cause differences as well (Romero-Pérez et al., 2001; Aaviksaar et al., 2003; Liu et al., 2013; Soural et al., 2015). Studies including concentrations expressed in fresh weight of plant material are difficult to compare with others using dry weight.

Conclusion

As a consequence of increasing interest in natural bioactive compounds to meet the demands of the health industry, the present research provides an overall characterization of stilbene content in grapevine organs originating from the vineyard. The winter-buds, the roots and the mature cane internodes of Merlot are significantly richer in t-resveratrol and ε-viniferin than the green vegetative and generative parts we examined over one year. Canes may be an adequate, stable (Zhang et al., 2011) and economic (Rayne et al., 2008) source of resveratrol. In addition, roots and buds may be good source of ε-viniferin-rich ethanolic extracts. Our research pointed out that roots could be an easily accessible source of stilbenes, as they are by-products of the grafting preparation for storage or planting.

Acknowledgements

We are grateful to László Wágner and Károly Dublecz for the opportunity to work with the laboratory of the Department of Animal Sciences and Animal Husbandry. We thank Takler Winery, with special thanks to Ferenc Takler Jr. for the plant material, and the OMSz for the meteorological data.

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Authors


Gyöngyi Németh

Affiliation : University of Pannonia, Georgikon Faculty, Department of Horticulture, Deák F. street 16, 8360 Keszthely, Hungary


Orsolya Hegyi

Affiliation : University of Pannonia, Georgikon Faculty, Department of Animal Sciences and Animal Husbandry, Deák F. street 16, 8360 Keszthely, Hungary


Attila Dunai

Affiliation : University of Pannonia, Georgikon Faculty, Department of Crop Production and Soil Science, Deák F. street 16, 8360 Keszthely, Hungary


Laszlo Kocsis

http://georgikon.hu

Affiliation : University of Pannonia, Georgikon Faculty, Department of Horticulture, Deák F. street 16, 8360 Keszthely, Hungary

kocsis-l@georgikon.hu

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