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Effect of some growth regulators on polyphenols content and antioxidant

activity of grapes and wines of mavrud, cabernet sauvignon and chardonnay
grape varieties

Article  in  Oxidation Communications · January 2012

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 Oxidation Communications 35, No 1, 120–127 (2012)

Antioxidants in biological systems

Effect of Some Growth Regulators on Polyphenols

Content and Antioxidant Activity of Grapes
and Wines of Mavrud, Cabernet Sauvignon and
Chardonnay Grape Varieties

N. Gougouliasa , L. Mashevab*
a
Department of Plant Production, Technological Educational Institute of Larissa,
41 110 Larissa, Greece
E-mail: ngougoulias@teilar.gr
b
Agricultural University, 12 D. Mendeleev Blvd., 4000 Plovdiv, Bulgaria
E-mail: nimash@abv.bg

ABSTRACT
The effect of phytohormone preparation CP (combined preparation) and Ethrel-48
alone on the contents of total phenols (ТР), anthocyanins (АС), antiradical activity
(DPPH•) and ferric reducing power (FRAP) on the grape and wine of three grape
varieties: Mavrud, Cabernet Sauvignon and Chardonnay has been assayed. It was
found that treatment of grapevines after florescence with CP and Ethrel-48 affects
favourably the content of ТР, АС, DPPH• and FRAP activity of the grape and wine of
the three grape varieties. Of the two preparations the Ethrel-48 preparation was found
to affect stronger the polyphenol complex and the antioxidant activity depending on
the variety type. The Mavrud and Chardonnay grape varieties react more sensitively
to the Ethrel-48 treatment in comparison with the Cabernet Sauvignon, inducing
higher increase of the polyphenols content and antioxidant activity of the grape and
the wine as well.
Keywords: grape, wine, phytohormones, total phenols, anthocyanins, DPPH• activity,
ferric reducing antioxidant power (FRAP).

AIMS AND BACKGROUND

The endogeneous phytohormones auxins, gibberellins, cytokinins, abscisic acid and
ethylene act as major modulators in regulation of basic plant biological processes1,2.
Grapevine, similar to other plants, is subjected also to the effect of endogeneous
phytohormones3–5 in its annual cycle of development. Their study, both in qualitative
*
For correspondence.

120
and quantitative respect, in the separate organs and phases of the grapevine leads to
a more complete understanding of the role in the producing of higher quality grape
products as well as the need of their application in grapevine agrotechnique. They are
used for eliminating different ‘defects’ caused by unfavourable climatic conditions as
well as for the increase in the berries size of the seedless table varieties. It has been
established that the treatment of the grapevine with ecologically safe phytohormone
preparations leads to improvement and regulation of the biosynthesis of some grape
components, including polyphenol profile and anthocyanins that are of particular
importance for grape products quality6–8. Particular attention is paid to the key role of
the grape compounds related to the human health, which is attributed to their strong
antioxidant properties and their capability to defend the human organism against the
harmful action of the free radicals in the cells1,2. The Mavrud grape variety is one of
the most popular local varieties, grown from ancient times in the region of the Plov-
div town, and on which basis were and are produced famous high-quality red wines.
There are studies which show that the treatment with phytohormones can stimulate
its ripening phase and improve the polyphenol and the antioxidant composition of the
grape9. The 2 grape varieties selected for comparison purposes of the Mavrud grape and
wine are Cabernet Sauvignon and Chardonnay, which are widely distributed European
grapevine varieties used for producing of high-grade red and white wines10.
The purpose of the present research is to study the effect of the treatment of
grapevines with ethylene-releasing preparation Ethrel-48 and combined preparation
CP, containing auxins, gibberellins and cytokinines on the content of total phenols
(ТР) and anthocyanins (АС), and their correlation with antiradical activity (DPPH•)
and ferric reducing antioxidant power (FRAP).

experimental
Materials. The grapevines of the 3 grape varieties were grown at the experimental
field of the Department of Viticulture at AU–Plovdiv, near the village Brestnik. The
grapevines were cultivated using a conventional for the region technology. The Mavrud
and Chardonnay varieties were grown according to the Moser modified formulation,
and Cabernet Sauvignon – Ombrella. The test variants were the following: V1 – un-
treated grapevine (control); V2 – treated with the combined preparation (CP), and
V3 – treated with 0.1% Ethrel-48.
The combined preparation (CP) contains equal molar concentrations of gibberel-
lic acid (GA3), indol-3-acetic acid (IAA) and 6-furfarylaminopurpurine (kinetin),
supplied by Merck. Ethrel-48 is an ethylene-releasing compound, a product of the
American Corporation Rhone-Poulenc and contains 48% Etafon. The solutions were
prepared afresh before use in a concentration of 0.1%. The spraying with CP was
2-fold – after fertilisation, and the second one – after 10 days. The treatment with
Ethrel-48 was done once at the beginning of veraison. The grape was collected in the

121
phase of technological maturity from which were randomly selected mean samples
for chemical analysis and vinification.
Vinification of the grape. 25 l wine were produced from the grape of the studied grape
variants according to the methods applied in the wine cellar at the experimental field
in the village Brestnik.
Extracts preparation. The grape extracts were obtained after 2-fold treatment of 20 g
sample with 60%-methanol solution after 1-hour storage at dark and room tempera-
ture. The collected extracts after centrifugation (filtration) were brought to 50 ml with
aqueous methanol and further used for chemical analysis.
Determination of total polyphenols content. Total polyphenols (TP) contents were
determined with the Folin–Ciocalteu reagent according to the method of Singleton
and Rossi11, modified by Baderschnieder et al.12and were expressed as gallic acid
equivalent (GAE).
Determination of anthocyanins. The anthocyanins (AC) were determined according
to the method of Rivas-Gonzalo et al.13, slightly modified by Burns et al.14, by fol-
lowing the change in colour at different pH. They were presented as µmol malvidin-
3-glicoside/g fresh mass.
Determination of antioxidant activity. The antioxidant activity was assayed by 2
methods. The antiradical activity of the methanol extracts was determined according
to the method of Brand-Williams et al.15 applying the stable radical 2,2′-diphenyl-1-
picrylhydrazyl (DPPH). The activity was evaluated in µmol DPPH/g grape as well
as in mg% fresh mass, equivalent to Trolox (synthetic vitamin E).
The ferric reducing antioxidant power (FRAP) was evaluated according to the
method of Benzie et al.16 and was expressed as µmol FRAP reagent/g grape. The ac-
tivity was also presented as ascorbic acid equivalent (AAE) in mg% per fresh mass.
The results are means of 3 years (2003–2005) and the chemical analyses were
the means of 3 parallel samples. The statistic treatment of the results was performed
according to well-accepted methods using a program making possible the calculation
of t of the Student test at a confidence level p<0.05, 0.01 and 0.001.

RESULTS AND DISCUSSSION

The polyphenols content depending on the grape variety increases in the grapevine
treated with CP, containing the 3 phytohormones (indol-3-acetic acid, gibberellic acid
and cytokinin) and Ethrel-48 preparation, which releases the phytohormone ethylene
by 8–12% with respect to the non-treated grapevines (Table 1). It should be noted
that the 3 grape varieties under study react in different way to the treatment with the
preparations used. The treatment of the 3 grape varieties with Ethrel-48 induces an
increase in TP content in comparison with the treatment with CP. The tendency of
increase in the amount of total anthocyanins depending on the treatment of the red

122
varieties with ethylene-releasing preparation Ethrel-48 is strongly expressed, particu-
larly for the Mavrud variety. In our previous work we have established also a higher
AC amount in the Mavrud grape after treatment with Ethrel-48 preparation.

Table 1. Polyphenols content of the grape of the studied grape varieties

Grape variety Variant ТР (GAE ) Anthocyanins
ррm mM µmol malv.- mg/100 g
3-glu
Mavrud V0 – control 1347±26 7.16 132±2.3   6.98
V1 – 0.1% CP 1473±31 7.84 141±2.5   7.46
V2 – 0.1% Ethrel-48 1469±28 7.81 163±3.2   8.63
Cabernet V0 – control 1509±34 8.03 204±2.8 10.79
Sauvignon V1 – 0.1% CP 1650±37 8.78 239±1.8 12.64
V2 – 0.1% Ethrel 1696±31 9.02 252±2.1 13.33
Chardonnay V0 – control   610±11 3.24 – –
V1 – 0.1% CP   620±8 3.30 – –
V2 – 0.1% Ethrel   670±13 3.56 – –

The differences in the increase of TP and AC, depending on the treatment with the
investigated preparations and grape varieties, are statistically proved (р < 0.05 ).
The wine produced from the grape of the 3 varieties treated with the phytohormo-
nal preparations is also characterised by higher content of TP and AC, in dependence on
the grape variety and the preparations used (Table 2). The treatment with the combined
preparation (CP) affects more slightly the increase in the amount of TP and AC in the
wine of the red grape varieties Mavrud and Cabernet Sauvignon in comparison with
the treatment with Ethrel-48 preparation. Particularly strong is the effect of Ethrel-48
treatment on the amount of anthocyanins in the wine from the Mavrud grape, which
is increased by 33% with respect to that of the control (р < 0.001), and with 11% in
the wine from the grape variety Cabernet Sauvignon (р < 0.05 ). The specific effect
of ethylene on the polyphenols biosynthesis in the grape of the Chardonnay variety
affects more favourably their amount in the wine which increases with 17.5% with
respect to the control variant (р < 0.001). It is known that the polyphenols content of
the wine depends not only on their amount in the grape but on the microbiological
and chemical processes during its vinification17–19. Although in some recent studies is
underlined the multifunctional role of phytohormone ethylene in the different stages of
plants development and its interaction with phytohormones, its biochemical function
in fruit ripening, including grape, remains still an object of immense research2,20 .

123
Table 2. Polyphenols content in the wine
Grape variety Variant ТР ( GAE ) Anthocyanins
ррm mM µmol malv.- mg /100 g
3-glu
Mavrud V0 – control 2083±39 11.08 116±2.2 6.14
V1 – 0.1% CP 2342±42 12.46 124±2.8 6.56
V2 – 0.1% Ethrel 2384±48 12.68 174±3.1 9.20
Cabernet V0 – control 2246±36 11.95 180±3.4 9.52
Suavignon V1 – 0.1% CP 2393±43 12.73 197±3.9 10.42
V2 – 0.1% Ethrel 2526±32 13.44 202±32 10.69
Chardonnay V0 – control 315±4.2 1.68 – –
V1 – 0.1% CP 325±3.9 1.73 – –
V2 – 0.1% Ethrel 370±3.3 1.97 – –

The grape antiradical activity in the case of the 3 grapevine varieties treated
with CP and Ethrel-48 was determined using the stable free radical 2,2′-diphenyl-1-
picrylhydrazyl (DPPH•), which is widely used in biochemical investigations for as-
say of in vitro antioxidant activity of various bioactive chemical natural or synthetic
substances and foods21.
Depending on the treatment the antiradical activity of the Mavrud grape varies
from 1.14 to 1.28 µmol DPPH•/g, of the Cabernet Sauvignon – from 1.35 to 1.57 µmol
DPPH•/g, and of the Chardonnay variety – from 0.66 to 0.79 µmol DPPH•/g (Table 3).
The antiradical capacity of the grape increases after the treatment with the 2 prepara-
tions (р < 0.05), but the treatment with Ethrel-48 is more efficient (р < 0.01). The
grape of the Chardonnay variety treated with the ethylene-containing preparations is
characterised by the highest antiradical activity (р < 0.001). Concerning the antiradi-
cal activity the grape varieties are arranged in the following order: Chardonnay >
Cabernet Sauvignon > Mavrud.

Table 3. Grape antioxidant activity

Grape variety Vraiant Antiradical activity Ferric-reducing antioxidant
(DPPH) power (FRAP)
µmolDPPH/g µmolTrolox/g µmol/ml µmol AAE/g
Mavrud V0 – control 1.14±0.021 4.15 6.09±0.092 2.77
V1 – 01% CР 1.26±0.012 4.59 6.46±0.112 2.93
V2 – 0.1% Ethrel 1.28±0.015 4.66 6.96±0.118 3.16
Cabernet V0 – control 1.35±0.018 4.91 6.90±0.131 3.13
Sauvignon V1 – 0.1% CР 1.46±0.026 5.31 7.20±0.128 3.27
V2 – 0.1% Ethrel 1.57±0.031 5.71 7.41±0.137 3.36
Chardonnay V0 – control 0.66±0.006 2.40 1.14±0.012 0.52
V1 – 0.1% CР 0.68±0.008 2.48 1.20±0.023 0.54
V2 – 0.1% Ethrel 0.79±0.011 2.88 1.42±0.018 0.64

124
 The determination of the ferric reducing antioxidant power of different products
is widely used in the current biochemical studies for evaluation of the in vitro anti-
oxidant activity22. The results obtained of the grape varieties under consideration vary
from 1.14 to 7.41 µmol FRAP/g, depending on the preparations used and the variety
nature. The treatment of the grapevines of the 3 varieties with CP leads to a slight
increase in the ferric reducing power of the grape (р < 0.05), whereas the treatment
with Ethrel-49 causes a higher increase, which for the Chardonnay variety reaches
25% with respect to the control variant (р < 0.001).
The antiradical activity of the wine was determined 6 months after fermentation,
applying the both methods, which have been used in the case of the grape (Table 4).
The antiradical activity (DPPH) of the wine produced from the grape of the treated
with phytohormones (CP and Ethrel-48) grapevines increases (р < 0.05), but depends
on the type of the preparation and the variety nature. The treatment with Ethrel-48
promotes stronger increase of DPPH-activity of the wine produced from the 3 varie-
ties in comparison with the control variant. It should be underlined that for the Char-
donnay variety it reaches 30% as compared to the control. Taking into consideration
the antiradical activity of the investigated grape varieties treated with Ethrel-48, the
wines have the following sequence: Chardonnay > Mavrud > Cabernet Sauvignon.
The ferric reducing antioxidant power (FRAP) of the investigated wines ranges from
2.4 6 to 17.66 µmol FRAP/g (Table 4). The activity of the wines produced from
the grape of all grape varieties treated with the combined preparation (CР) is lower
than the activity of the wines after treatment with Ethrel-48 preparation. The ferric
reducing antioxidant power of the wine from the Mavrud grape is increased most
significantly – 17.02 µmol FRAP/g, and amounts to 18% with respect to the non-
treated grapevines. Related to the ferric reducing antioxidant power, the wines from
the investigated grape varieties treated with Ethrel-48 can be arranged as follows:
Mavrud > Chardonnay > Cabernet Sauvignon.

Table 4. Wine antioxidant activity

Grape variety Variant Antiradical activity Ferric-reducing antioxidant
(DPPH) power (FRAP)
µmol µmol µmol µmol
DPPH/ml Trolox/ml FRAP/ml AAE/ml
Mavrud V0 – control 4.66±0.11 16.96 14.48±0.12 6.57
V1 – 0.1% CР 5.05±0.09 18.38 16.67±0.18 7.57
V2 – 0.1% Ethrel 5.20±0.12 18.93 17.09±023 7.76
Cabernet Sauvi- V0 – control 5.03±0.08 18.31 16.57±0.31 7.52
gnon V1 – 0.1% CР 5.35±0.13 19.47 17.44±0.28 7.92
V2 – 0.1% Ethrel 5.41±0.10 19.69 17.66±0.34 8.02
Chardonnay V0 – control   0.52±0.005  1.88 2.46±0.091 1.12
V1 – 0.1% CР   0.61±0.008   2.21 2.68±0.083 1.22
V2 – 0.1% Ethrel   0.69±0.006   2.51 2.83±0.074 1.28

125
 The nutritive polyphenols constitute a large group of secondary metabolites in
the plants, which are widely distributed in fruits, vegetables, seeds, berries, kernels,
tea, cacao, essential oils and other foods and exert strong antioxidant properties. The
results of many clinical and epidemiological studies support their defending action
on the human organism against the oxidative stress and degenerative diseases23,24. The
grape and wine are among the richest sources of polyphenols with high antioxidant
potential25,26. Our results show that the endogenous growth regulators used exogenously
at the different grapevines can to a certain extent induce the growth and metabolism
of the grape plant substances, the grape and wine quality, the polyphenols content
and increase their nutritive and healthy characteristics.

conclusions
The comparative study on the effect of the treatment with phytohormones preparations
CP and Ethrel-48 on the content of total phenols (ТР) and anthocyanins from the one
side and, from the other, on the antiradical activity and ferric reducing antioxidant
power of the grape and the wine, supports the assumption that the endogenous phy-
tohormones can be applied exogenously for the grape varieties Mavrud, Chardonnay
and Cabernet Sauvignon for promoting the nutritive and healthy properties of the grape
and the wine produced from them. The application of the 2 preparations alone on the
local variety Mavrud and the widely distributed Cabernet Sauvignon, as well as on
the white grape variety Chardonnay leads to improvement of the biosynthesis of total
phenols (ТР) in the 3 varieties and anthocyanins (АС) in the red grape varieties. The
Ethrel-48 preparation has a greater effect than CP regarding the content of TP and AC,
particularly for the grape and wine of the Mavrud and Chardonnay grape varieties.

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