International Congress BIOLOGICAL PRODUCTS - Gruppo di ...

Supelco Supelclean LC-18 SPE tubes (3ml). The other phenolic compoundswere quercetin, rutin (quercetin-3-Orutinoside),
kaempferol, kaempferol-3-glucoside, kaempferol-3-rutinoside, rhamnetin, isorhamnetin, and were
also supplied by Sigma.
Wine Analysed
The analyses were carried out on 23 Italian wine samples (red and white), coming from three different Italian
vineyards: one located in Tuscany, one in Campania and one in Sicily. In particular, wines from Tuscany were
produced from 30 years old plants (vines were grafted with Sangiovese variety, Morellino clone) and each sample
was vinified red. Wines from Campania were produced from 20 years old plants (vines were grafted with Fiano
d’Avellino variety) and each sample was vinified white. Sicilian wines were produced from 15-20 years old
plants (vines were grafted with Inzolia and Carricante variety in ratio 1:1) and each sample was vinified white.
The vineyard was subdivided in thesis; each thesis consisted of four rows of vines and the remotest was located
with the function to prevent drift effect. The fungicides treatments were carried out at the end of blossoming and
maturation. The scheme of experimental work provided the treatment of each thesis with different antioidics and
antiperonosporics: sulphur, azoxystrobin, dinocap, penconazolo, fenarimol, quinoxyfen (tab.1).
Analysis of cis- and trans-resveratrolo
cis- and trans-resveratrol were extracted from wines using the SPE cartridges prior to the HPLC analysis. For the
analysis of total wine resveratrol was used an enzymatic hydrolysis method, improved by our working group (25).
The wine resveratrol O-glycosides (trans- and cis-piceid) were totally hydrolysed in ~ 9 hours after incubation
with β-glucosidase at 50°C; then the trans- and cis-aglycones were measured by HPLC after solid phase
extraction (SPE). An ODS Hypersil 5µm (250x4,6 mm i.d.) column was used as the stationary phase and was
preceded by a precolumn of the same material. The mobile phase consisted of phase (A) water/acetic acid (pH =
3) and phase (B) acetonitrile/acetic acid (pH = 3). cis- and trans-resveratrol were eluted with a gradient time
program and the eluted was monitored at 285 and 307 nm, where cis- and trans- isomers have absorbance
maxima, respectively. All chromatographic experiments were performed at room temperature with a flow rate
1ml/min. The chromatograms were recorded according to the retention time. Before the HPLC analysis the
extract were filtered through a 0,45µm glass-microfiber GMF Whatman chromatographic filter.
HPLC analysis methodology for flavonoids
The analysis were carried out on aliquot of wine filtered through a 0,45 µm nylon Supelco chromatographic filter,
using an HPLC system operating at room temperature with a flow–rate of 0,8 ml/min.. The spectrophotometric
detector was a photodiode array operating at wavelengths of 265 nm for aglycon-flavonoids and 254 for
flavonoid-glucoside. The analytical column was a Alltech Alltime C-18, 5 µm ( 250 x 4,6 mm i.d.). A SUPELCO
guard column packed with the same stationary phase was also used. The mobile phase consisted of
H2O/CH3COOH at pH= 3 (phase A) and CH3CN/CH3COOH at pH = 3 (phase B). Elution was carried out in
gradient.
RESULTS AND DISCUSSION
Analytical data related to the free isomeric forms of resveratrol (cis- and trans-) and to the total resveratrol (free
and glucosides isomers) are reported in fig. 1,2 and 3. This investigation, once again, clearly shows that red wines
have a greater resveratrol content than white wines, regardless of the pesticide treatments of the vines. Analysing
the data related to wines from Campania, it can be noticed that in the samples Thesis 3, 4, 5 and 6 the amount of
cis-resveratrol is between 0.04 and 0.05 mg/L, while the samples Thesis 1, Thesis 2 and Thesis 7 have an amount
of cis-resveratrol impossible to verify with the adopted analytic method. The concentration of trans-resveratrol
results highest in the sample Thesis 5 (0.13 mg/L), and a lowest in the sample Thesis 3 (0.07 mg/L). The total
concentration of cis- and trans-resveratrol deserve a particular attention because, generally, it is similar in every
sample while, this balance does not occur in free form of cis- and trans-resveratrol. In fact, the concentration of
free trans-resveratrol is a little lower than that of total trans-resveratrol, but in sample Thesis 2 it is even almost
identical; the same situation does not occur in cis-resveratrol. Examining the concentration of free cis-resveratrol
to that of total, it results that the amount of cis-piceid is much higher than that of trans-piceid, regardless of the
pesticides treatment. A completely different situation is shown for wines produced and vinified in Sicily. In this
case, the concentration of total trans-resveratrol (between 0.17 and 0.34 mg/L) is always higher than that of total
cis-resveratrol (between 0.09 and 0.20 mg/L). Moreover, differently from what was noticed in wines produced
and vinified in Campania, the cis- and trans-piceid concentration in each sample is comparable, and is between a
minimum value of 0.03 mg/L and a maximum of 0.18 mg/L. The samples Thesis 4 and Thesis 5 differ from this
general trend, as they are characterized by an amount of cis-piceid clearly higher than that of trans-isomer. As
shown in fig.3, the wines obtained from cv Sangiovese have very different quantities of free trans-resveratrol and
they range from 0.22 to 1.76 mg/L. The same trend can be noticed with free cis-resveratrol, but in this case the
concentrations are more homogeneous. Comparing total cis- and trans-resveratrol concentrations to those of the
equivalent free isomers, it emerges that the levels of trans-piceid are approximately twice as high as those of the
corresponding cis-piceid, except for samples Thesis 4 and Thesis 5 that present comparable levels. The
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