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Industrial Crops and Products 95 (2017) 227–234

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Industrial Crops and Products


journal homepage: www.elsevier.com/locate/indcrop

Elderberry (Sambucus nigra L.) by-products a source of anthocyanins


and antioxidant polyphenols
Pedro Silva 1 , Sandrine Ferreira 1 , Fernando M. Nunes ∗
Chemistry Research Centre – Vila Real (CQ-VR), Chemistry Department, University of Trás-os-Montes and Alto Douro, School of Life Sciences and
Environment, Vila Real, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: Elderberry industrial by-products can be a potential low cost source of some unique bioactive polyphe-
Received 21 May 2016 nols. Hence, the main objective of this work was to perform a comparative study of the polyphenol
Received in revised form 3 October 2016 profile of elderberry branches, the scavenging activity towards 2,2 -azino-bis(3-ethylbenzothiazoline)-
Accepted 9 October 2016
6-sulfonic acid, hydroxyl and nitric oxide radicals and to compare with those of the elderberry berries.
Available online 28 October 2016
Total phenolic compounds and anthocyanins were significantly higher in the berries when compared to
the branches (1.68 and 3.21 times, respectively), nevertheless the 2,2 -azino-bis(3-ethylbenzothiazoline-
Keywords:
6-sulfonic acid) and hydroxyl radicals scavenging activities were similar between the two materials, and
Elderberry
By-products
branches extract presented a higher nitric oxide radical scavenging activity. A total of 23 polyphenolic
Branches compounds were detected in all samples, of which thirteen compounds were present both in berries
Anthocyanins and branches, including cyanidin 3,5-diglucoside, cyanidin 3-sambubioside-5-glucoside, cyanidin 3-
Polyphenols glucoside and cyanidin 3-sambubioside. As elderberry production is increasing, as well the generation of
Antioxidant activity associated by products, the polyphenolic composition of the elderberry branches opens the possibility of
its valorization, using simple and relatively easy extraction procedures yielding extracts rich in polyphe-
nols possessing a significant antioxidant activity, useful to other emerging industrial and agricultural
applications.
© 2016 Elsevier B.V. All rights reserved.

1. Introduction complements in the form of concentrates, juices and infusions


(Duymuş et al., 2014), this last one being traditionally used for the
European elderberry (Sambucus nigra L.) is a deciduous treatment of constipation, diuretic and respiratory tract infections.
shrub native from northern hemisphere, being nowadays present Several studies have demonstrated that elderberry extracts besides
throughout the temperate and subtropical regions of Asia, North having antioxidant activity, present anti-inflammatory, arthero-
Africa and North America (Fazio et al., 2013; Veberic et al., 2009). protective, immune-stimulating and chemopreventive potential
Its high commercial value is due to its fruits, the elderberry berries, effect. So elderberry phytochemicals may have an important action
which contain large amounts of anthocyanins and other polyphe- in the prevention of several degenerative diseases, such as cardio-
nols (Dawidowicz et al., 2006; Veberic et al., 2009), being used vascular and inflammatory disease, cancer and diabetes (Duymuş
as food colorants in jams and jellies, pies, yoghurts, syrups, and et al., 2014; Fazio et al., 2013; Ozgen et al., 2010; Schmitzer
alcoholic beverages (Cernusca et al., 2012; Lee and Finn, 2007; et al., 2010). In the last two decades the elderberry plantation has
Lima-Brito et al., 2011; Schmitzer et al., 2010). Due to the large increased significantly in Portugal, being mainly cultivated in the
amounts of phytochemicals present in the berries and to the sig- Varosa Valley located in the northern part, where it has excellent
nificant antioxidant properties, berries are also used as dietary edaphoclimatic conditions, being produced annually between 1500
and 2000 ton of elderberry berries (Neto, 2007; Seabra et al., 2010).
In recent years, the elderberry production and commercializa-
tion has changed significantly, whereas traditionally elderberries
∗ Corresponding author.
were commercialized dried, today the fresh refrigerated elderber-
E-mail address: fnunes@utad.pt (F.M. Nunes).
1
These authors contributed equally to this project. ries are the main product exported to other European countries

http://dx.doi.org/10.1016/j.indcrop.2016.10.018
0926-6690/© 2016 Elsevier B.V. All rights reserved.
228 P. Silva et al. / Industrial Crops and Products 95 (2017) 227–234

for further industrial processing. The production of fresh refriger- 2013 in Varosa Valley, Portugal. Umbels were harvested from
ated elderberries generates a large amount of by-products, with four different points of tree, and the branches were removed
branches being the most abundant, accounting for more than 10% manually. Elderberry berries and branches were freeze-dried and
of the total elderberry production, being at present composted or milled in an electric mill, and then stored at −80 ◦ C until further
through away. During ripening of elderberry berries, especially in analysis. Representative voucher samples of elderberry branches
the last ripening stages, the branches acquire a distinct reddish and berries were deposited at the herbarium of the Univer-
color, suggesting the presence of anthocyanins, and probably other sity of Trás-os-Montes e Alto Douro with the voucher number
polyphenols, that might be related or identical to those present HVR21095.
in the berries. So elderberry wastes resultant from the elderberry
industry could be a potential low cost and alternative source of 2.3. Preparation of the elderberry berries and branches extracts
some unique natural bioactive polyphenols for other industries, like
pharmaceutical and cosmetic industries, with many applications For the exhaustive extraction of polyphenols from elder-
related to the health benefits like scavenging activity of free rad- berry berries and branches, 250 mg of freeze-dried material were
icals and anti-inflammatory properties (Murthy and Naidu, 2012; extracted with 5 mL methanol containing 1% HCl in an orbital
Nandakumar et al., 2008; Peralbo-Molina and de Castro, 2013; Ping shaker (Orbital Shaker GFL 3005 series, Hanover, Germany) for
et al., 2012; Seabra et al., 2010; Terra et al., 2007). Also polyphenols 20 min at 200 rpm. After extraction the suspension was centrifuged
may have potential uses as biobased phytosanitary products, able to (5 min at 50,000 rpm; Sigma Centrifuges 3–30 K, St. Louis, MO, USA)
control the incidence of crop diseases (Benouaret et al., 2014). The and the supernatant was removed. This process was repeated 8
use of this by-product for the extraction of valuable polyphenols times (number of exactions needed for removing all the color from
can reduce the environmental impact of some of these substances elderberry berries), and supernatants were added up and diluted to
on the cultivated fields and on the irrigation water (Kuppusamy a final volume of 50 mL with the extraction solution. For the devel-
et al., 2015; Seabra et al., 2010). Although is possible to find in the opment of a simpler and environmental friendly extraction method
literature some research on the polyphenolic content and antioxi- for elderberry branches, water and ethanol (95%) were used as sol-
dant capacity of elderberry berries and elderflowers, when we look vents. To 25 g of branches (as is basis), 1 L of solvent was added and
for information regarding to elderberry branches extracts no stud- boiled during 15 min. After cooling, the water or ethanol extracts,
ies have been done, hindering valorization. Thus, in this study we depending of the solvent used for extraction, were filtered and con-
evaluate the potential of elderberry branches, by-products of elder- centrated by rotary evaporation (Stuart RE300, Staffordshire, UK)
berries berries production, as alternative source of anthocyanins to 100 mL.
and other polyphenols. For this purpose, the polyphenolic pro-
file, total phenols, anthocyanin contents, as well the antioxidant 2.4. Determination of the total phenolic content of elderberry
activity of extracts from branches were analyzed and compared berries and branches
with those of the elderberry berries. This paper represents the first
attempt to assess the polyphenolic content and the antioxidant pro- The total phenolic content of elderberries and branches were
file of the elderberry branches from the Varosa Valley in northern determined using the Folin-Ciocalteu method according to Cheok
Portugal. et al. (2013) with some modifications. Briefly, 1 mL of properly
diluted extract was mixed with 1 mL of Folin-Ciocalteu reagent,
2 mL of sodium carbonate (7.5% w/w) and 6.5 mL of milli-Q water
2. Material and methods
and the mixture was incubated during 30 min at 70 ◦ C. After cool-
ing to room temperature, the absorbance was measured at 750 nm
2.1. Reagents
(PerkinElmer-Lambda 25, PerkinElmer, London, United Kingdom).
The total phenolic content was expressed in gallic acid equiv-
Folin–Ciocalteu’s reagent, 2,2-azino-bis(3-
alents (mg/100 g fresh weight), using a gallic acid calibration
ethylbenzothiazoline)6 sulfonic acid (ABTS),
curve.
(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
(Trolox), methanol, sodium carbonate, gallic acid, hydrogen
2.5. Determination of the total monomeric anthocyanins of
peroxide solution, 2-deoxy-d-ribose, potassium persulfate, thio-
elderberry berries and branches
barbituric acid (TBA) and all organic solvents were purchased
from Sigma-Aldrich (Spain). Sodium nitroprusside, Sulfanil-
Total monomeric anthocyanins of elderberry berries and
amide, Naphthylethylenediamine dihydrochloride, ascorbic acid,
branches were determined using the UV–vis spectroscopy pH dif-
trichloroacetic acid were purchased from Merck (Germany).
ferential method according to Giusti and Wrolstad (2001). Two
Ethylenediamine tetraacetic acid (EDTA), Ferric(II) chloride
dilutions of elderberries and branches extracts were prepared, one
were purchased from Panreac (Spain). For HPLC analysis, ultra-
containing hydrogen chloride (pH 1.0) and the second containing
pure water was used (Milli-Q system, Millipore, Bedford, MA).
sodium acetate buffer (pH 4.5). The absorbance of both dilu-
Quercetin 3-glucoside; quercetin 3-rutinoside; isorhamnetin
tions was measured at 520 and 700 nm (PerkinElmer-Lambda 25,
3-rutinoside; isorhamnetin 3-glucoside; cryptochlorogenic
PerkinElmer, London, United Kingdom) and the total monomeric
acid; chlorogenic acid; cyanidin 3,5-diglucoside; cyanidin
anthocyanins were expressed as cyanidin-3-glucoside equivalents
3-sambubioside-5-glucoside; cyanidin 3-glucoside; cyanidin
(mg/100 g of fresh weight) using the molar extinction coefficient
3-sambubioside and quercetin were from Extrasynthese (France).
of 26,900 L cm−1 mol−1 and molecular weight of 449.2 g/mol of
Stock solutions of standard phenolic acids were prepared in
cyanidin-3-glucoside.
methanol (1 mg/mL), stored at −80 ◦ C, and used within a 1 week
period.
2.6. Determination of the antioxidant activity of elderberry
berries and branches extracts
2.2. Elderberry berries and branches samples
2.6.1. Trolox equivalent antioxidant capacity assay
The samples of elderberry berries and branches were picked in The antioxidant activity against ABTS radical was determined
quadruplicate at theoptimum fruit maturity at the end of August according to Barros et al. (2011). Briefly, the ABTS radical was
P. Silva et al. / Industrial Crops and Products 95 (2017) 227–234 229

prepared after addiction of 7 mM ABTS solution and 2.45 mM potas-


sium persulfate, and incubated in the dark at room temperature
for 12–16 h. The ABTS radical formed was diluted with 20 mM
sodium acetate buffer (pH 4.5) until an absorbance at 0.70 ± 0.02
at 734 nm was obtained. The reaction started by addition of prop-
erly diluted extracts with 2 mL of the diluted ABTS radical solution.
After 15 min at room temperature, the absorbance was measured
at 734 nm (PerkinElmer-Lambda 25, PerkinElmer, London, United
Kingdom). The antioxidant activity of the extracts was expressed
in Trolox equivalents (mmol Trolox/100 g fresh weight), using a
Trolox calibration curve.

Fig. 1. Inhibition of the formation of malondialdehyde by the OH radical induced


2.6.2. Hydroxyl radical scavenging activity of elderberry berries oxidation of 2-deoxyribose. Determination of the malondialdehyde-thiobarbituric
acid adduct by HPLC to avoid interference from the anthocyanins present in the
and branches extracts extracts.
The hydroxyl radical scavenging activity of elderberry berries
and branches extracts was performed according to Genaro-Mattos
et al. (2009) with some modifications. The proper diluted extracts
of elderberries and branches were added to a solution containing
idant activity of the samples was calculated in Trolox equivalents
28 mM of 2-deoxy-ribose; EDTA 1 mM, FeCl2 1 mM, ascorbic acid
(mmol Trolox/100 g fresh weight), using a Trolox calibration curve.
1.0 mM; 10 mM H2 O2 and 20 mM phosphate buffer (pH 7.4). The
reaction was incubated during 1 h at 37 ◦ C. After the incubation,
0.5 mL of a TBA solution (0.5%w/v in 5% trichloroacetic acid solu- 2.7. Polyphenols profile of elderberry berries and branches
tion) was added and the mixture boiled during 15 min. After cooling extracts by reversed phase (RP) high performance liquid
to room temperature the malondialdehyde –TBA complex formed chromatography (HPLC) and quantification
was analyzed by RP-HPLC (Dionex Ultimate 3000 pump, PDA-100
photodiode array detector, a WPS-3000TSL Analyt auto sampler The polyphenols profile of elderberry berries and branches
and Ultimate 3000 column compartment, USA), after extraction extracts was determined by RP-HPLC using an Ultimate 3000 HPLC
with 1 mL of butanol. For the HPLC analysis an Ace UltraCore5 (Dionex, USA) equipped with an Ultimate 3000 pump, PDA-100
Super C18 (150 × 4.6 mm id) column was used. The mobile phases photodiode array detector, a WPS-3000TSL Analyt auto sampler
were composed by: (B) methanol and (A) 50 mM phosphate buffer and Ultimate 3000 column compartment. For the determination
solution at pH = 6.8. The analysis conditions were carried out using of the polyphenol profile of the exhaustive methanol-HCl extrac-
a flow rate of 1 mL/min with initial conditions of 15% (B) during tion, samples prepared according to 2.3 were concentrated 5
10 min, changed to 60% (B) until 25 min and changed again to the times by rotary evaporation (Stuart RE300, Staffordshire, United
initial conditions and maintained during 5 min. The column tem- Kingdom) and re-suspended in aqueous 5% formic acid. For the
perature was maintained at 30 ◦ C during analysis. The UV detection extracts obtained using water or ethanol the samples prepared
was performed at 532 nm and the injection volume was 100 ␮L. For according to 2.3 were analyzed directly. For the separation a
equipment control and data acquisition, the software Chromeleon Kinetex C18 column (100 mm × 4.6 mm, 2.6 ␮m diameter parti-
version 7.1 (Dionex, USA) was used. The hydroxyl radical scaveng- cles; Model TCC-3200) from Phenomenex (Torrance, CA, USA)
ing activity was expressed as percentage of inhibition of formation was used. The eluents used were: (A) aqueous 5% formic acid,
of malondialdehyde in relation to control (with addition of water and (B) methanol. The eluents were prepared daily, filtered
instead of the extracts). through a 0.45 ␮m membrane filter Millipore and on-line degassed.
The analysis was performed using a flow rate of 1 mL/min and
with initial conditions of 98% (A) during 35 min, changed to
2.6.3. NO scavenging activity of elderberry berries and branches 35% (A) until 55 min and maintained during 10 min. Afterwards
extracts the eluent was changed to 98% (A) at 65.1 min, and kept dur-
The NO radical scavenging activity of elderberry berries and ing more 10 min. The column temperature was maintained at
branches extracts was determined according to the protocol of 30 ◦ C during analysis. The UV detection was performed at wave-
Awah and Verla (2010). A sodium nitroprusside solution (5 mM) lengths between 200 and 650 nm and the injection volume was
in 20 mM phosphate buffer (pH 7.3), was incubated with or 25 ␮L. For equipment control and data acquisition, the software
without elderberries and branches extracts at 25 ◦ C for 180 min Chromeleon version 7.1 (Dionex, USA) was used. Peak identifica-
under of a 25W tungsten lamp. The nitric oxide radical produced tion was confirmed by retention time and UV-spectra comparison
was measured every 30 min at 546 nm (PerkinElmer-Lambda 25, with those of commercial standards (quercetin 3-glucoside;
PerkinElmer, London, United Kingdom) after addition of 1.0 mL of quercetin 3-rutinoside; isorhamnetin 3-rutinoside; isorhamnetin
Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% 3-glucoside; cryptochlorogenic acid; chlorogenic acid; cyanidin
naphthyl ethylenediamine dihydrochloride). The nitrite generated 3,5-diglucoside; cyanidin 3-sambubioside-5-glucoside; cyanidin
was estimated by a standard curve of sodium nitrite and the antiox- 3-glucoside; cyanidin 3-sambubioside and quercetin). Cyanidin

Table 1
Percentage of humidity, total phenol content, total monomeric anthocyanins and antioxidant activity (fresh weight basis) of the branches and berries.

Humidity g/100 g Total Phenols nmg/100 g Anthocyanins mg/100 g TEACx mmol Trolox/100 g OH• scavenging % NO• scavenging mmol Trolox/100 g

Branches 37.0 ± 0.8a


708 ± 22 a
253 ± 47 a
10.7 ± 0.6a
89 ± 5.2a
220 ± 17a
Berries 75.2 ± 1.4b 1191 ± 85b 813 ± 156b 10.9 ± 2.3a 93 ± 0.9a 190 ± 7b

Values are expressed as mean ± standard deviation (n = 4). Means within a column for each sample followed by the same letter are not significantly different (t-Student test,
p < 0.05).
x
Trolox equivalent antioxidant capacity.
230 P. Silva et al. / Industrial Crops and Products 95 (2017) 227–234

Fig. 2. Chromatograms at 280 nm, 325 nm and 525 nm of extracts from berries and branches of elderberry obtained by exhaustive methanol-1% HCl extraction. Peak
identification: 6 – quercetin 3-glucoside; 7 – quercetin 3-rutinoside; 9 – isorhamnetin 3-glucoside; 11 – cryptochlorogenic acid; −12 – chlorogenic acid; 14 – cyanidin
3,5-diglucoside; 15 – cyanidin 3-sambubioside-5-glucoside; 16 – cyanidin 3-glucoside; 17 – cyanidin 3-sambubioside; 22 – quercetin; 1, 2, 3, 4, 8, 10, 13, 18–21 and 23 –
unknown compounds.

3-sambubioside-5-glucoside was identified by comparison of its glucoside – 0.2 mg/L of extract and 1.1 mg/100 g fresh weight;
retention time in relation to the others anthocyanins described quercetin 3-rutinoside – 0.5 mg/L of extract and 2.4 mg/100 g
in the literature (Duymuş et al., 2014). For quantification of the fresh weight; isorhamnetin 3-glucoside – 1.5 mg/L of extract and
polyphenolic compounds a calibration curve was constructed in 7.5 mg/100 g fresh weight; cryptochlorogenic acid – 0.3 mg/L of
the range of 0–250 mg/mL for cyanidin 3-glucoside; cyanidin extract and 1.2 mg/100 g fresh weight; chlorogenic acid – 0.2 mg/L
3-sambubioside, and between 0 and 75 mg/L for cyanidin 3,5- of extract and 1.0 mg/100 g fresh weight; cyanidin 3,5-diglucoside
diglucoside and 0–100 mg/L for the other polyphenolic compounds – 0.05 mg/L of extract and 0.8 mg/100 g fresh weight; cyanidin
(quercetin 3-glucoside; quercetin 3-rutinoside; isorhamnetin 3- 3-glucoside – 0.2 mg/L of extract and 0.8 mg/100 g fresh weight;
rutinoside; isorhamnetin 3-glucoside; cryptochlorogenic acid; cyanidin 3-sambubioside – 0.9 mg/L of extract and 4.3 mg/100 g
chlorogenic acid; and quercetin). Cyanidin 3-sambubioside-5- fresh weight; quercetin – 1.9 mg/L of extract and 9.5 mg/100 g fresh
glucoside was quantified using the cyanidin 3-sambubioside weight. For those compounds which no standard was available cal-
calibration curve. Each calibration curve was checked for linearity ibration curves of gallic acid, chlorogenic acid and quercetin were
using the method described by (Eurachem/CITAC, 2012; Thompson used for benzoic acid derivatives, cinnamic acid derivatives and
et al., 2002), and the limits of detection and quantification were flavonoid derivatives, respectively. Recovery experiments were
determined using the residual standard deviation of the cali- performed for chlorogenic acid and the recovery obtained was
bration curve using the method described by (Eurachem/CITAC, 98.1 ± 2.7%.
2012; Thompson et al., 2002). Limits of detection: quercetin 3-
P. Silva et al. / Industrial Crops and Products 95 (2017) 227–234 231

2.8. Statistical analysis 450


mAU 9
11 12 6
Experimental results obtained were compared using the t- 7
300
student test to indicate significant differences between means, 22
p < 0.05. All statistical treatments were performed on the Statistica
200 1617
7 (StatSoft, Inc., Tulsa, Oklahoma, USA) statistical software.
100 14

3. Results and discussion min


-50
0 10 20 30 40 50 60 70
3.1. Total phenols, total anthocyanins and antioxidant activity of
elderberry berries and branches Fig. 3. Chromatogram showing the retention time of commercial standards (see
also Table 2): (6) quercetin 3-glucoside, (7) quercetin 3-rutinoside, and (9) isorham-
netin 3-glucoside (11) cryptochlorogenic acid, (12) chlorogenic acid, (14) cyanidin
In order to evaluate the potential of elderberry branches as alter-
3,5-diglucoside, (16) cyanidin 3-glucoside; (17) cyanidin 3-sambubioside and (22)
native source of anthocyanins and other bioactive polyphenols and quercetin.
compare its composition with that of elderberry berries, an exhaus-
tive extraction method using methanol containing 1% of HCl was
used. The total phenol content, total anthocyanins, polyphenols tion of the polyphenols present, so the polyphenolic profile of the
profile by HPLC, and the antioxidant activity against different rad- branches was analyzed and compared with that of the berries.
icals (ABTS+ , OH and NO radicals) were evaluated. The results are
presented in Table 1. As can be observed, on a fresh basis, the total 3.2. Polyphenols profile of elderberry berries and branches
phenolic compounds and anthocyanins are significantly higher in
the berries when compared to the branches (1.68 and 3.21 times, The polyphenolic profiles of elderberry berries and branches are
respectively). Despite the different total phenolic compounds con- presented in Fig. 2. A total of 23 polyphenolic compounds were
tent between berries and branches, the antioxidant activity was detected in all samples, of which thirteen compounds (2, 3, 6, 7, 9,
similar between the two samples (11 mmol Trolox per 100 g FW) 10–17) were present in both in berries and branches, three (1, 4 and
and the OH radical scavenging activities (Table 1 and Fig. 1) was 8) were detected only in berries and six (18–23) only in branches
also similar in the branches and berries (89% and 93% of malondi- (Fig. 2, Table 2). Of the detected polyphenolic compounds, 9 of them
aldehyde formation inhibition, respectively). Furthermore, for the were unequivocally identified by comparison of the retention time
NO radical scavenging activities, the branches possessed a higher and UV-spectra with that of commercial standards (Fig. 3), includ-
antioxidant activity than the berries (220 mmol Trolox per 100 g ing: (6) quercetin 3-glucoside, (7) quercetin 3-rutinoside, and (9)
FW and 190 mmol Trolox per 100 g FW, respectively). These results isorhamnetin 3-glucoside (11) cryptochlorogenic, (12) chlorogenic
show that branches are a potential source of bioactive polyphenols, acid, (14) cyanidin 3,5-diglucoside, (16) cyanidin 3-glucoside; (17)
and the differences observed can be related to a different composi- cyanidin 3-sambubioside and (22) quercetin. The polyphenol pro-

Table 2
Amount of the polyphenolic compounds in elderberry berries and branches and concentration of extracts obtained with hot water and hot ethanol from elderberry branches
determined by HPLC.

Peak no. Rtw (min) Identification ␭máx


v
Berries Branches Water Ethanol

g/100 g dry weight mg/L

1 2.71 ± 0.01 Unkx 275 0.094 ± 0.023a ndb 0.25 ± 0.01a 0.27 ± 0.02a
2 3.56 ± 0.01 Unk 285 0.25 ± 0.03a 0.30 ± 0.03a nd nd
3 8.22 ± 0.02 Unk 279; 288shy 0.039 ± 0.007a 1.1 ± 0.2b 0.33 ± 0.02a 0.34 ± 0.02a
4 9.16 ± 0.03 Unk 282 0.009 ± 0.002a ndb 0.18 ± 0.01a 0.21 ± 0.01b
5 43.11 ± 0.01 Quercetin-derivative 351; 258 ndz nd 1.39 ± 0.08a 1.47 ± 0.08a
6 44.21 ± 0.01 Quercetin 3-glucoside 354; 256 0.13 ± 0.02a 0.029 ± 0.006b 2.40 ± 0.15a 2.78 ± 0.17b
7 44.70 ± 0.01 Quercetin 3-rutinoside 354; 256 1.38 ± 0.19a 0.010 ± 0.001b 30.3 ± 6.7a 40.7 ± 3.5b
8 47,16 ± 0.14 Unk 281 0.006 ± 0.001a ndb nd nd
9 48.45 ± 0.02 Isorhamnetin 3-glucoside 373; 255 0.21 ± 0.03a 1.1 ± 0.1b 0.58 ± 0.44a 2.15 ± 0.58b
10 63.68 ± 0.02 Unk. 277 0.031 ± 0.001a 0.007 ± 0.002b 1.71 ± 0.03a 1.69 ± 0.02a
11 10.56 ± 0.02 Cryptochlorogenic acid 325; 297sh 0.064 ± 0.009a 0.017 ± 0.004a 0.62 ± 0.03a 0.29 ± 0.01b
12 11.61 ± 0.04 Chlorogenic acid 325; 297sh 0.006 ± 0.002a 0.012 ± 0.005b 3.77 ± 0.20a 4.56 ± 0.22b
13 39.11 ± 0.03 Unk 326; 297sh 0.21 ± 0.04a 0.34 ± 0.01b nd nd
14 20.56 ± 0.07 Cyanidin 3,5-diglucoside 512; 277 0.61 ± 0.17a 0.081 ± 0.029b 0.22 ± 0.03a 0.32 ± 0.03b
15 22.73 ± 0.13 Cyanidin 3-sambubioside-5-glucoside 513; 277 1.79 ± 0.45a 0.032 ± 0.010b 1.45 ± 0.13a 2.16 ± 0.11b
16 32.40 ± 0.19 Cyanidin 3-glucoside 513; 280 4.27 ± 0.52a 0.43 ± 0.04b 0.29 ± 0.06a 0.72 ± 0.05b
17 35.79 ± 0.20 Cyanidin 3-sambubioside 515; 280 5.59 ± 0.63a 0.12 ± 0.01b 0.97 ± 0.17a 2.33 ± 0.13b
18 18.57 ± 0.07 Unk. 294; 260 nda 0.073 ± 0.004b 0.09 ± 0.03a 0.09 ± 0.01a
19 51.43 ± 0.11 Unk 270 nda 0.012 ± 0.001b nd nd
20 36.38 ± 0.08 Unk 323; 295sh nda 2.11 ± 0.07b nd nd
21 45.34 ± 0.02 Unk. 311; 300sh nda 0.30 ± 0.02b nd nd
22 47.12 ± 0.03 Quercetin 354; 256 nda 0.021 ± 0.002b 1.39 ± 0.34a 1.87 ± 0.19b
23 47.31 ± 0.02 Unk. 324; 297sh nda 0.23 ± 0.01b nd nd

Values are expressed as mean ± standard deviation (n = 4); Means between two column for each polyphenol followed by the same letter are not significantly different
(t-Student test, p < 0.05).
w
Rt – Retention time.
v
␭máx – maximum wavelength.
x
Unk – unknown.
y
sh – shoulder.
z
n.d. – not detected.
232 P. Silva et al. / Industrial Crops and Products 95 (2017) 227–234

file of berries is in accordance with previous studies of elderberry


berries from European cultivars (Dawidowicz et al., 2006; Kaack
et al., 2008; Veberic et al., 2009). By inspection of the UV–vis spec-
tra of the unidentified compounds (Table 2), compounds 1–4, 8, 10
and 19 presented a spectra characteristic of benzoic acids deriva-
tives, peaks 13, 20, 21 and 23 presented a spectra characteristic of
cinnamic acid derivatives, and peak 5 a spectra characteristics of
flavonol (Lin et al., 2012). The amount of each polyphenol in elder-
berry berries and branches is presented in Table 2, and the total
amount of anthocyanins, flavonols and cinnamic acid derivatives
content in shown in Fig. 4. As can be observed the total amount of
anthocyanins and flavonols were significantly higher in the berries
when compared to the branches. Nevertheless the amount of cin-
namic acid derivatives was significantly higher in the branches
when compared to the berries (Fig. 4). The highest NO radical scav-
Fig. 4. Content of anthocyanins, flavonols and cinnamic acid derivatives in elder-
enging activity of the branches when compared to berries, and the berry branches (open bars) and berries (filled bars) exhaustive methanol-1% HCl
similar TEAC values and OH radical scavenging activities obtained extraction. Significant differences by t-Student test **, p < 0.01; ***p < 0.001.
in the branches and berries, are certainly due to the higher amount

Fig. 5. Chromatograms at 280 nm, 325 nm and 525 nm of elderberry branches extracts obtained with water and ethanol. Peak identification: 6 – quercetin 3-glucoside; 7 –
quercetin 3-rutinoside; 9 – isorhamnetin 3-glucoside; 11 – cryptochlorogenic acid; 12 – chlorogenic acid; 14 – cyanidin 3,5-diglucoside; 15 – cyanidin 3-sambubioside-5-
glucoside; 16 – cyanidin 3-glucoside; 17 – cyanidin 3-sambubioside; 22 – quercetin; 1, 3, 4, 10, 12, 13 and 18 – unknown compounds.
P. Silva et al. / Industrial Crops and Products 95 (2017) 227–234 233

in the cinnamic acid derivatives (Table 2 and Fig. 4), especially the flavonols and cinnamic acids esters, presenting similar or even bet-
presence of compounds 20, 21 and 23. ter antioxidant activities when compared to berries, showing the
potential of elderberry branches as a low cost alternative source of
natural antioxidants.
3.3. Evaluation of a simpler and environmentally friendly
extraction method for elderberry branches
Acknowledgments
Methanol is usually used as good extraction solvent for
hydrophilic phenolic compounds and for the extraction of antho- The authors want to acknowledge QREN, ADI, Programa Opera-
cyanins methanol it is normally acidified (Dai and Mumper, 2010), cional do Norte and FEDER for the financial support of the Project
nevertheless the use of water or ethanol as extraction solvents are SambucusFresh n◦ 23109 and to the financial support provided to
more safe (Dai and Mumper, 2010; Shi et al., 2005) and environ- the Research Unit in Vila Real (PEst-OE/QUI/UI0616/2014) by FCT
mentally friendly (Corrales et al., 2009). Especially ethanol due and COMPETE.
to its physicochemical characteristics (low boiling point) make
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