Characterization of tannins from two wild

blackberries (Rubus spp) by LC–ESI–MS/

MS, NMR and antioxidant capacity

Oscar Abel Sánchez-Velázquez, Julio

Montes-Ávila, Jorge Milán-Carrillo,
Cuauhtémoc Reyes-Moreno, Saraid
Mora-Rochin, et al.
Journal of Food Measurement and
Characterization

ISSN 2193-4126
Volume 13
Number 3

Food Measure (2019) 13:2265-2274

DOI 10.1007/s11694-019-00146-z

1 23
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 Author's personal copy
Journal of Food Measurement and Characterization (2019) 13:2265–2274
https://doi.org/10.1007/s11694-019-00146-z

ORIGINAL PAPER

Characterization of tannins from two wild blackberries (Rubus spp)

by LC–ESI–MS/MS, NMR and antioxidant capacity
Oscar Abel Sánchez‑Velázquez1 · Julio Montes‑Ávila1 · Jorge Milán‑Carrillo1,2 · Cuauhtémoc Reyes‑Moreno1,2 ·
Saraid Mora‑Rochin1 · Edith‑Oliva Cuevas‑Rodríguez1,2 

Received: 8 January 2019 / Accepted: 30 April 2019 / Published online: 13 May 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019

Abstract
Tannins are phenolics found in fruits such as wild Rubus berries. The aim of this study was to determine the profile by LC–
ESI–MS/MS and NMR, antioxidant capacity (AOX) of the tannins in two Mexican wild blackberries. Fractions rich in tannins
(FRT) were isolated using different solvents and chromatographic resins. The LC–ESI–MS/MS and NMR results showed
that catechin and epicatechin (m/z ­289−1), which are precursors of proanthocyanidins, as well as other six oligomeric forms
of ellagitannins (m/z ­301−1) were found in R. Liebmannii, whereas in R. Palmeri, only ellagitannins were found, highlight-
ing oligomeric forms of lambertianins and sanguiins (m/z ­783−1 to ­1869−1). The purification pathway made it possible to
increase the AOX 19.7 times from fruits to FRT showing strong positive correlations (R2 > 0.9) with total phenolic content.
This study provided a good assessment of the tannin composition of wild blackberries from the Northwest of Mexico and
their bioactivity as a potential edible source.

Keywords  Wild blackberries · Tannins · LC–ESI–MS/MS · NMR · Antioxidant capacity · Rubus

Introduction
Rubus fruits (family Rosaceae) are field berries with more
than 300 accepted species worldwide, including grown and
wild blackberries. In Mexico, almost 40 wild Rubus species
have been recorded, among them R. Liebmannii Focke (an
endemic species) and R. Palmeri Rydb. (rare native bram-
ble) [1–3]. These two species, like other Mexican Rubus,
are agriculturally underused and are consumed only as a
seasonal fruit and/or an ethnomedical resource [2, 4].
Electronic supplementary material  The online version of this
article (https​://doi.org/10.1007/s1169​4-019-00146​-z) contains
supplementary material, which is available to authorized users.
* Edith‑Oliva Cuevas‑Rodríguez
edith.cuevas.r@uas.edu.mx
1 have been reported in wild Mexican Rubus berries collected
Programa Regional de Posgrado en Biotecnología,
Universidad Autónoma de Sinaloa, Av. Universitarios s/n,
Col. Universitarios, AP 1354, CP 80000 Culiacán Rosales,
Sinaloa, Mexico
2
Posgrado en Ciencia Y Tecnología de Alimentos, Facultad
de Ciencias Químico‑Biológicas, Universidad Autónoma de
Sinaloa, Av. Universitarios s/n, Col. Universitarios, AP 1354,
CP 80000 Culiacán Rosales, Sinaloa, Mexico
Wild Rubus fruits are reach in polyphenols like tannins [2,
5–10]. Tannins are hydroxylated polymers (up to 20,000 Da)
with a large diversity of chemical structures reported fre-
quently in many Rubus berries, as one of the main second-
ary metabolites in Rubus fruits (Fig. 1) [9, 10]. Rubus tan-
nins are classified in two major groups: (1) hydrolyzable
tannins or ellagitannins (ETs) are esterified polymers of
hexahydroxydiphenic acid (HHDP), gallic acid, and polyol
units (i.e., d-glucopyranose) and are the main phenolic com-
pound in seeds, but are also present in low concentrations
in the flesh and peel [11]; (2) condensed tannins or proan-
thocyanidins (PAs) are oligomers of flavan-3-ol units, such
as (−)-catechin and (+)-epicatechin, and their galloylated
forms [i.e. (epi)gallocatechin (epi)gallocatechin-gallate,
etc.]. PAs occur as an important phytochemical constituent
of the peel and seeds in berries [11, 12]. Bioactive effects
(anti-inflammatory, antiparasitic, antioxidant and genotoxic)
in the Central and Southern of the country, attributing them
to phenolic diversity (including phenolic acids, anthocyanins
and tannins) [1, 2, 4–8].
To study Rubus berries tannins, multiple solvent
extraction protocols has been improved. In some cases,
they are followed by purification stages with commercial

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Fig. 1  Common tannin precursors (a, b, c) and oligomeric ellagitan-
nins found in Rubus berries (d, e, f): gallic acid (a), ellagic acid (b)
(epi)catechin (c), casuarictin (a), sanguiin H-6/lambertianin A (b)
adsorption and/or ion-exchange resins [8]. Liquid chro-
matography (LC) and mass spectrophotometry (MS) are
the major qualitative and quantitative analyses to suppose
the tannin profile in red fruits [13–20], but researchers
have been suggested, that is necessary to complement the
LC–MS/MS analysis with nuclear magnetic resonance
(NMR) to guarantee the chemical structure of the com-
pounds present in the samples. NMR is a technique that
details angles, distances, and chiral forms of atoms [15,
16, 18, 20].
13
O. Abel Sánchez‑Velázquez et al.
and lambertianin C (c). Structures adapted from Mertz et al. (2007),
Kool et  al. (2010), Lee et  al. (2012), Melone et  al. (2015), and Li
et al. (2016)
On the other hand, many assays are used to determine
the total phenolic content (TPC) and antioxidant capac-
ity (AOX) of Rubus fruits, but there is no real consensus
about the right mechanisms of phenolic-antioxidant action.
However, tests such as Folin–Ciocalteu reaction (for TPC)
and oxygen radical absorbance capacity assay (ORAC) (for
AOX), have been extensively used in studies on Rubus ber-
ries showing good results [8, 17, 18]. Despite the above,
the phenolic profile of Northern-Mexican wild Rubus ber-
ries, including R. Liebmannii and R. Palmeri, has not been
 Author's personal copy
Characterization of tannins from two wild blackberries (Rubus spp) by LC–ESI–MS/MS, NMR and…
studied. Thus, a purification pathway it is necessary to evalu-
ate the profile of tannins, TPC and AOX of fractions of both
berry species. The aim of this study was to determine the
profile by LC–ESI–MS/MS and NMR and AOX of tannins
in two unstudied Northern Mexican wild blackberries.
Material and methods
Materials and reagents
The following chemicals were used: standards of gallic acid,
ellagic acid (+)-catechin, and (±)-6-hydroxy-2,5,7,8-tetra-
methylchromane-2-carboxylic acid (Trolox); Folin–Ciocal-
teu reagent; dichlorofluorescein (DCF); 2,2-azo-bis-(2-ami-
dinopropane) dihydrochloride (AAPH); trichloroacetic acid
(TCA); methanol (MeOH); acetone (­ CH3COCH3); acetoni-
trile ­(CH3CN); formic acid ­(HCO2H); deuterated methanol
­(CD3OD); and deuterated dimethyl sulfoxide-d6 (DMSO-d6).
The chemicals were of different degrees of purity. Standards
and reagents were supplied by Sigma-Aldrich (St. Louis,
MO, USA), unless otherwise specified.
Plant samples
Ripened fruits of two wild blackberry species were col-
lected in July 2014 in the Chara Pinta Preserve in El
Palmito, Concordia, Sinaloa, Mexico (23°35′22.2″N,
105°52′11.8″W, 2170  m a.s.l.), identified and recorded
as Rubus liebmannii and R. Palmeri under folio numbers
IBUNAM:MEXU:1417228 and IBUNAM:MEXU:1417230,
respectively, at Herbarium of Biology Institute of National
Autonomous University of Mexico. The fruit samples
were washed, freeze-dried, and stored at − 80 °C for future
analysis.
Isolation and purification of the fraction rich
in tannins
The techniques used were those reported previously by
Cuevas-Rodríguez et al. [7], with some modifications, as
illustrated in Online Source 1. Freeze-dried fruits (100 g)
(FreeZone Dryer System; Labconco, Kansas City, MO,
USA) were blended with 250 mL of acidified 80% metha-
nol (MeOH:water:TCA, 80:19.7:0.3, v/v/w) for 24 h under
cold agitation (100 rpm at 4 °C) into the incubator LOM-
150-Series (MRC, St. Haifa, Israel) to obtain a crude extract
(CE), which was then filtered through Whatman #4 and #1
filter paper (Florham Park, NJ, USA) layers to remove solids.
The above extraction process was done two times, and the
filtered CEs came together. The pooled CE (500 mL) was
concentrated by rotary evaporation ( < 40 °C) to eliminate
alcohol (Rotavapor R-100; BÜCHI, Flawil, Switzerland).
2267
The aqueous CE was mixed with ethyl acetate (1:1, v/v)
to remove non-polar compounds (two times). The defatted
CE was evaporated again. The CE was mixed with 100 g of
Amberlite XAD-7 resin (Sigma Life Science, St. Louis, MO,
USA) and then packed in a glass column (300 × 25 mm).
Polyphenols were adsorbed by the resin, since impurities
were eluted with acidified water (water:TCA, 99.7:0.3, v/w).
After that, polyphenols were eluted with 1 L of acidified
80% MeOH (0.3% TCA) to obtain a fraction rich in polyphe-
nols (FRP). Alcohol was removed, and the aqueous FRP was
freeze-dried. The dry FRP (2.0 g) was diluted in 100 mL of
acidified MeOH and loaded into a glass column packed with
Sephadex LH-20 resin (Sigma Life Science, St. Louis, MO,
USA) previously activated with acidified MeOH. First, frac-
tions rich in anthocyanins (FRAs) were eluted with acidi-
fied 80% MeOH (0.3% TCA), and then fractions rich in tan-
nins (FRTs) were eluted with 70% acetone (acetone:water,
70:30, v/v). Ketonic solvent was evaporated ( < 40 °C), and
the aqueous FRTs were immediately freeze-dried ( − 80 °C)
for later use.
Identification and quantification
Liquid chromatography–electrospray ionization–tandem
mass spectrometry (LC–ESI–MS/MS) analysis of tan-
nins began with an LCQ DECA XP mass spectrometer
(Thermo Finnigan Corp., San Jose, CA, USA) version 1.3
SRI, electrospray ionization (ESI) in positive-ion mode (m/z
100–2000), along with a photodiode array (PDA) detector
(200–600 nm) version 1.2, an autosampler, and Xcalibur
2.2 software (Thermo Fisher Scientific Inc., Austin, TX,
USA). The capillary temperature was 250 °C. The spray
voltage was 10 kV. Liquid chromatography (LC) elution was
carried out using acidified water (high-performance liquid
chromatography [HPLC]-grade water:formic acid, 95:5, v/v)
as mobile phase A and acidified acetonitrile (HPLC-grade
acetonitrile:formic acid, 99.9:0.1, v/v) as mobile phase B.
The flow was constant (1 mL/min) at 25 °C with a step gra-
dient of 0%, 5%, 30%, 60%, 90%, 90%, and 0% solvent B at
0, 40, 45, 50, 55, 60, and 70 min, respectively, and reading
at 280 nm. Catechin and ellagic acid were taken as reference
standards. The FRTs (5 mg/mL) and standards (0.25 mg/mL)
were diluted in 100% HPLC-grade MeOH, filtered through a
0.22–µm nylon filter (Millipore, Darmstadt, Germany), and
placed in 2.0–mL amber vials. All samples were analyzed
in triplicate.
Nuclear magnetic resonance (NMR) of proton (1H-NMR)
and carbon (13C-NMR) spectra were obtained using a Bruker
Spectrophotometer (Bruker Biospin GmbH, Rheinstetten,
BW, Germany) at 400 and 500 Hz. Samples were diluted in
­CD3OD and DMSO-d6. Tetramethyl saline was used as an
internal standard. The values of chemical displacement (δ)
were expressed in parts per million (ppm). The values of the
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coupling constant (J ) were expressed in hertz in all cases.
1
H-NMR analysis was run at 200 and 400 Hz, and 13C-NMR
analysis was run at 50 and 100 Hz.
Total phenolic content
The TPCs of the extracts and fractions were measured by
the method of Nurmi et al. [21] with some modifications.
Briefly, 0.5 mL of 1 N Folin–Ciocalteu reagent was added
to 0.5 mL of diluted CE, FRP, and FRT (50 µg/mL). This
mixture was allowed to stand for 2 to 5 min before the addi-
tion of 1 mL of 20% ­Na2CO3. The solution was then allowed
to stand for an additional 10 min before reading was per-
formed at 765 nm in a microplate reader spectrophotometer
(SpectraMax® Plus 384, Molecular Devices Corp., Sunny-
vale, CA, USA). The measurement was compared against
the standard curve of gallic acid, and the TPC results were
expressed as mg of gallic acid equivalent (GAE) per g of
extract or fraction (freeze-dried weight). All samples were
analyzed by triplicate.
Antioxidant activity
The AOX of the samples were determined using the ORAC
assay [22]. Peroxyl radicals were generated by AAPH, and
fluorescence loss was monitored in a microplate reader (Syn-
ergy HT multi-detection microplate reader; BioTek Instru-
ments, Inc., Winooski, VT, USA). The CE, FRP, and FRT
were evaluated against the Trolox curve. The absorbances of
excitation and emission were set at 485 and 538 nm, respec-
tively. The antioxidant capacities were expressed as micro-
moles of Trolox equivalent (TE) per 100 g of dry weight
(dw). All samples were analyzed in triplicate.
Statistical analysis
Data were reported as means ± standard deviation in trip-
licate and analyzed using one-way analysis of variance
(ANOVA) under Tukey’s test. Pearson correlation (R2) was
determined between TPC and AOX. Statistical analysis was
performed using Minitab software version 2013 (Minitab
Inc., USA).
Results
Isolation and purification
Acidic hydroalcoholic extraction was performed to obtain a
crude fraction rich in soluble compounds, among them sug-
ars, lipids, and phenolics. Ethyl acetate was used to remove
non-polar chemicals that could interfere the next steps of
purification. Amberlite XAD-7 resin allowed to separate the
13
O. Abel Sánchez‑Velázquez et al.
FRPs from other organic compounds, such as free sugars,
pectins, and peptides. Afterwards, with Sephadex LH-20
ion-exchange resin, FRAs and FRTs were obtained [10].
Tannin profile
A total of 11 signals corresponding to tannins (PAs and ETs)
were identified and quantified by LC–ESI–MS/MS (Table 1)
and confirmed by 1H-NMR and 13C-NMR in R. Liebmannii
(RL) and R. Palmeri (RP) berries (Table 2).
Compound  1 (retention time ­[RT] = 8.28  min) was
recorded only in RL berries and had a molecular ion
([M−H]−) at a mass-to-charge ratio(m/z) 289 and a maxi-
mum absorption (λmax) of 276 and 278 nm, which indicate
the presence of (−)-catechin and its isomer (+)-epicatechin
based on a pure standard. 1H-NMR spectrum exhibited sig-
nals such as a multiplet (m) at 6.99 ppm, associated with
the C6 and C8 protons of PAs [19]; the singlet (s) signal
at 7.25 δ indicates the presence of the (−)-catechin form,
whereas the s signal at 7.68 δ was due to the isomeric form
(+)-epicatechin; and 13C-NMR signals at 64.3 and 69.2 δ
were associated with chemical shift of C1′ in catechin and
epicatechin, respectively [13].
Compound 2 ­(RT = 8.57 min) was found in both Rubus
berries. Based on the standards, it corresponds to ellagic
acid, since this compound had a [M−H]− at m/z 301 and a
λmax of 254 nm. 1H-NMR spectrum showed a chemical shift
of two doublets at 7.63 δ (J = 4.0 Hz) and 7.23 δ (J = 4.2 Hz),
corresponding to C3 and C3′, respectively, in ellagic acid
and/or HHDP, and was found to have an s at 7.99 δ due the
presence of a COOH group in those structures; the signal at
157.4 δ in 13C-NMR spectrum corresponds to the C3 and
C3′ of the galloyl groups (gal), since the signal at 211.6 is
associated with the C = O group present in gallotannins and
ETs.
Compound 3 ­(RT = 14.83) was determined to be a pedun-
culagin (ET) isomer with a [M−H]− at m/z 783, a MS/MS
fragment pattern at m/z 481 (loss of HHDP) and 301 (loss of
glu), and a λmax of 258 nm. Pedunculagin has been reported
in other Rubus fruits, such as Apache blackberry, red rasp-
berries, and cloudberries [11, 23]. 1H-NMR spectrum
revealed the presence of a carbohydrate (glu) s at 6.21, cor-
responding to β-d-glucose, since it is bonded to two HHDP
molecules by C–O links; the H-1 glu signal was reported in
13
C-NMR spectrum at 29.6 δ as a high signal that appeared
in the following galloylated ET.
Compound 4 ­(RT = 18.58 min) was a casuarictin/potentil-
lin isomer and showed [M−H]− at m/z 935, MS/MS frag-
ments at m/z 783 (loss of gal), 633 (loss of HHDP), and
301 (loss of gal-glu), and a λmax of 259 nm. 1H-NMR spec-
trum of compound 4 presented signals similar to those for
pedunculagin, but the presence of a doublet (d) at 5.83 δ
(J = 3.5 Hz) indicates an additional gal group bonded to
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Characterization of tannins from two wild blackberries (Rubus spp) by LC–ESI–MS/MS, NMR and… 2269

Table 1  Profile of tannins from R.Liebmannii (RL) and R.Palmeri (RP) by LC–ESI–MS/MS

Signal RT (min) Compound (CID) M+ (m/z) Fragment ions (m/z) λmax (nm) References

1 8.28 Epicatechin/catechin (9064/72276) 290 289 ([M−H]−) 276, 278 [7, 13, 14]
2 8.57 Ellagic acid (5281855) 302 301 ([M−H]−) 254 [11, 12, 20]
3 14.83 Pedunculagin isomer (442688) 784 783 ([M−H]−), 481 ([M−H]− - HHDP), 258 [11, 16, 15]
301 ([M−H]− - HHDP-glu)
4 18.58 Casuarictin/Potentillin isomer 936 935 ([M−H]−), 783 ([M−H]− - gal), 633 259 [11, 15]
(73644/5315734) ([M−H]− - HHDP), 301 ([M−H]− - gal-
HHDP-glu)
5 18.75 UC [952−]2 951 ([M−H]−), 907 ([M−H]− -CO2), 783 250 [11]
[(M−H)]− - ­CO2-gal), 633 ([M−H]− -
gal-glu), 301 ([M−H]− -gal-glu-HHDP)
6 18.89 Sanguiin H-2 isomer (74079453) 1104 1103 ([M−H]−), 1058 ([M−H]- ­CO2), 248 [11, 15]
935 ([M−H]−), 783 ([M−H]− - gal),
301 ([M−H]− - gal-HHDP-HHDP-glu)
7 19.45 Lambertianin B isomer (N/A) 1251 1250 ([M−H]−), 783 ([M−H]− -HHDP- 250 [15]
gal), 301 ([M−H]− -HHDP-gal-HHDP-
glu)
8 19.49 Lambertianin C isomer (101196665) [1402−]2 [1401−]2 ([M−H]−/2), 1250 {([M−H]−/2) 250 [11, 12, 15, 16]
- gal}, 1103 {([M−H]−/2) - HHDP},
934 {[M−H]−/2) - gal-HHDP}, 633
{([M−H]−/2) - gal-HHDP-HHDP},
301 {[M−H]−/2) - gal-HHDP-HHDP-
glu-gal}
9 19.82 Sanguiin H-10 isomer (131752591) 1568 1567 ([M−H]-), 1265 ([M−H]− - 250 [15, 16]
HHDP), 1103 ([M−H]− - HHDP-glu),
934 ([M−H]− - HHDP-glu-gal), 632
([M−H]− - HHDP-glu-gal-HHDP),
301 ([M−H]− - HHDP-glu-gal-HHDP-
HHDP)
10 24.36 Nobotanin A/ Malabathrin B isomer 1719 1718 ([M−H]−), 1567 ([M−H]− - gal), 250 [11, 16]
(103616133/N/A) 1250 ([M−H]− - gal-gal), 1015
([M−H]− - gal-HHDP-glu), 951
([M−H]− - gal-gal-HHDP), 859
([M−H]− - gal-HHDP-glu-galloyl)
11 30.78 Sanguiin H-6/ Lambertianin A isomer 1870 1869 ([M−H]−), 1567 ([M−H]− - 250 [11, 12, 15, 16]
(16181831/101632066) HHDP), 1265 ([M−H]− - HHDP-gal-
glu), 1250 ([M−H]− - HHDP-gal-gal),
935 ([M−H]− -HHDP-gal-glu-HHDP),
633 ([M−H]− -HHDP-gal-glu-HHDP-
HHDP)

RT retention time, M± molecular ion, PubChem CID PubChem Compound ID, RL R. Liebmannii, RP R. Palmeri, UC unknown compound,
HHDP hexahydroxydiphenoyl group, gal galloyl group; glu glucosyl group, N/A non-available

C2 of the structural H-1 glu group, which structurally cor-
responds to a casuarictin/pedunculagin isomer; 13C NMR
spectrum showed a gal signal at 11.4 δ, associated with the
bond between l-COOH gal group and the C2 of the H-1
glu. The absence of another signal related to the COOH end
of the gal group indicates that a casuarictin isomer is not
present in these fruits.
Compound 5 ­(RT = 18.75 min) was determined to be an
“unknown” compound with a [M−H]− at m/z 951, a MS/
MS fragmentation pattern typical for ET at m/z 907 (loss of
­CO2), 783 (loss of glu), 633 (loss of gal), and 301 (loss of
HHDP), and a λmax at 250 nm, which is typical for an ET
such as a nobotanin A/malabathrin B isomer. A d was shown
in 1H-NMR spectrum at 6.14 δ (J = 4.0 Hz) and in 13C-NMR
spectrum at 14.4 δ, which indicate a non-lactonized gal
group bonded to C2 of the H-1′ glu, and it could be associ-
ated with the monolactonized moieties of a nobotanin A/
malabathrin B isomer [11].
Compound 6 ­(RT = 18.89 min) was identified in RP ber-
ries as a sanguiin H-2 isomer with a [M−H]− at m/z 1103,
a MS/MS fragment pattern at m/z 1058 (loss of ­CO2), 935
(loss of gal), 783 (loss of gal), and 301 (loss of HHDP), and
a λmax at 248 nm. 1H-NMR spectrum showed a d at 6.51 δ
(J = 2.4 Hz), indicating the galloylated form of HHDP from
gal-2-bis-HHDP-glu; this signal corresponds to 13C-NMR
spectrum at 70.6 δ.

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2270 O. Abel Sánchez‑Velázquez et al.

Table 2  Chemical shift of Compound 1

H (δ/ppm) Multipl. (J/Hz) Signal (Fig. 2) 13
C (δ/ppm) Signal (Fig. 3) Species
1
H and 13C NMR of isolated
tannins from wild blackberries (-)-Catechin 6.99 m (2.0) f 64.3 i RL
7.25 s (2.2) d
(+)-Epicatechin 6.99 m (2.0) f 69.2 h RL
7.68 s (2.0) b
Ellagic acid 7.63 dd (4.0) c 157.4 b RL, RP
7.99 s (4.0) a 211.6 a
HHDP 7.23 dd (4.2) e 157.4 b RL, RP
7.99 s (4.0) a 211.6 a
Pedunculagin 6.21 s (4.5) k 29.6 k RL, RP
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
Casuarictin 5.83 d (3.5) n 11.4 n RL, RP
6.21 s (4.5) k 29.6 k
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
UC 5.83 d (3.5) h 11.4 n RP
6.14 d (3.5) l 14.4 m
6.21 s (4.5) k 29.6 k
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
Sanguiin H-2 5.83 d (3.5) n 11.4 n RP
6.51 d (2.4) h 70.6 g
6.21 s (4.5) k 29.6 k
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
Lambertianin B 5.83 d (3.5) h 11.4 n RL, RP
6.47 s (1.2) i 29.6 k
6.21 s (4.5) k 30.5 j
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
Lambertianin C 5.83 d (3.5) n 11.4 n RL, RP
6.21 s (4.5) k 29.6 k
6.72 t (8.0) g 125.0 f
129.0 d
132.0 c
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
Sanguiin H-10 5.83 d (3.5) n 11.4 n RP
5.98 d (4.8) m 126.3 e
6.21 s (4.5) k 29.6 k
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
Nobotanin A/ 5.83 d (3.5) n 11.4 n RL, RP
Malabathrin B 6.14 d (3.5) l 14.4 m
6.21 s (4.5) k 29.6 k
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
Sanguiin H-6 5.83 s (3.5) n 11.4 n RL, RP
6.21 s (4.5) k 29.6 k
6.38 s (4.5) j 24.0 i
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a
Lambertianin A 5.83 s (3.5) n 11.4 n RL, RP
6.21 s (4.5) k 29.6 k
125.0 f
7.23 dd (4.2) e 157.4 b
7.99 s (4.0) a 211.6 a

s singles, d doublet, dd double doublet, t triplet, m multiplet, RL R.Liebmannii, RP R. Palmeri

13
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Characterization of tannins from two wild blackberries (Rubus spp) by LC–ESI–MS/MS, NMR and… 2271

Fig. 2.  1H NMR spectra of tannins: a ellagitannins found in R. Liebmannii and R.Palmeri fruits; b signals corresponding to flavan-3-ols from R.
Liebmannii. Individual letters corresponding to Table 2. Ppm, parts per million

Fig. 3.  13C NMR spectra of tannins: a ellagitannins found in R. Lieb- DMSO-d6, deuterated dimethyl sulfoxide; ­CD3OD, deuterated metha-
mannii and R.Palmeri fruits; b signals corresponding to flavan-3-ols nol; ppm, parts per million
from R. Liebmannii. Individual letters corresponding to Table  2.

Compound 7 ­(RT = 19.45 min) was present in both Rubus of HHDP-gal) and 301 (loss of HHDP-glu), and a λmax at
fruits and was identified as a lambertianin B isomer with a 250 nm. A s on 1H–NMR spectrum at 6.47 δ is due to the
[M−H]− at m/z 1250, MS/MS fragments at m/z 783 (loss additional lactonized HHDP linked to gal-2-bis-HHDP-glu.

13
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2272 O. Abel Sánchez‑Velázquez et al.

Compound 8 ­(RT = 19.49 min) was found to be associ-
ated with an isomeric form of lambertianin C precursor,
coeluting with a lambertianin C moiety by its ([M−H] − )2
doubly charged ion at m/z 1401, and had MS/MS fragments
at m/z 1250 (loss of gal), 1103 (loss of HHDP), 934 (loss
of gal), 633 (loss of HHDP), and 301 (loss of glu-gal) and
a λmax of 250 nm. In fact, the molecular mass (Mr) of this
compound is 2805, and it was determined indirectly by the
doubly charged ion at m/z 1401, since the mass spectrometer
could not detect m/z higher than 2000. 1H-NMR spectrum
indicates the presence of a triplet (t) at 6.72 δ (J = 8.0 Hz)
related to the oligomeric structure of lambertianin C, since
13
C-NMR spectrum showed signals at 132, 129, and 125 δ
related to gal–glu (C–O–COOH) bonds present in the
macromolecule.
Compound 9 ­(RT = 19.82 min) is related to a sanguiin
H-10 isomer with a [M−H]− at m/z 1869, MS/MS ionic frag-
ments at m/z 1265 (loss of HHDP), 1103 (loss of glu), 934
(loss of gal), 632 (loss of HHDP), and 301 (loss of HHDP),
and a λmax of 250 nm. Sanguiin H-10 1H-NMR spectrum
showed signals similar to those for compound 6, but also
had a d signal at 5.98 δ (J = 4.8 Hz), indicating a glu-HHDP
group linked to gal of the sanguiin H-2. 13C-NMR spectrum
showed a signal at 126 δ related to the H1′-glu core.
Compound 10 ­(RT = 24.36 min) was identified as an iso-
mer of nobotanin A/malabathrin B with a [M−H]− at m/z
1718, its MS/MS showed fragments at m/z 1567 (loss of
gal), 1250 (loss of gal), 1015 (loss of gal-glu), 951 (loss of
gal), and 859 (loss of gal), and it had a λmax of 250 nm. 1H-
NMR and 13C-NMR spectra were similar to those described
for compound 5, except that criteria to distinguish among
isomers were not found in the spectra for compound 5.
Compound 11 ­(RT = 30.78 min), a sanguiin H-6/lamber-
tianin A isomer, was detected with a [M−H]− at m/z 1869
and a MS/MS fragmentation pattern at m/z 1567 (loss of
HHDP), 1265 (loss of gal-glu), 1250 (M-1250, loss of gal-
gal), 935 (loss of HHDP), and 633 (loss of HHDP), with a
λmax of 250 nm. 1H-NMR spectrum showed a s associated
with isomeric forms of gal at 6.21 and 6.38 δ, which cor-
respond to lambertianin A and sanguiin H-6, respectively;
the signals of these isomers appear at 24.0 and 125.0 δ in the
13
C-NMR spectrum for sanguiin H-6 and lambertianin A,
respectively. Sanguiin H-6/lambertianin A is the dimer form
of casuarictin/potentillin, and their respective l- or d- posi-
tion of the COOH of gallic acid bonded to the H-1 and H-1′
glu group, is the key to distinguishing between them. Thus,
l-gal in H-1 glu is casuarictin, and for the dimeric form, l-
gal in H-1′ is lambertianin A, but with a d-gal in H-1 glu it is
known as potentillin, and the dimeric form appears as l-gal
in H-1 glu and d-gal in H-1′ glu for sanguiin H-6 [11, 23],
Total phenolic content and antioxidant activity
TPC results are shown in Table 3. TPC values in the CEs
were 15.2 mg GAE/g dw for the RL fruits and 17.3 mg
GAE/g dw for the RP fruits, with significant differences
(p < 0.05) between them. Previous studies reported TPC
values indicated ranges from 0.59 to 22.4 mg GAE/g dw
for cultivated Rubus brambles and from 18.4 to 25.5 mg
GAE/g dw for wild fruits [7, 24]. TPC values after defatting
and purification with Amberlite XAD-7 resin were 201.0
and 184.4 mg GAE/g dw for RL and RP fruits, respectively,
with significant differences (p < 0.05) between them. FRTs
obtained after purification with Sephadex LH-20 resin
showed concentrations of 713.1 mg GAE/g dw for RL fruits
and 637.5 mg GAE/g dw for RP fruits, with significant dif-
ferences (p < 0.05) between them.
AOX results are summarized in Table 3. ORAC values
of RL and RP fruits were 277.4 and 249.2 µM TE/g dw,
respectively, with significant differences (p < 0.05) between
them. AOX of FRP was 2921.2 µM TE/g dw for RL fruits
and 2280.2 µM TE/g dw for RP fruits, with significant dif-
ferences (p < 0.05) between them. AOX values of the FRPs
increased 10.5 and 9.2 times from those in the CEs of RL
and RP fruits, respectively. Post-Sephadex resin, AOX of
FRTs were 5243.8 and 4908.1 µM TE/g dw for RL and RP
fruits, respectively, with significant differences (p < 0.05).
Discussion
The use of Amberlite XAD-7 and Sephadex LH-20 resins
polymer with appropriate solvent mixtures facilities the
separation and isolation of tannins from Rubus aqueous

Table 3  Total phenolic content SP Total phenolic content (mg GAE/g dw) Antioxidant capacity (μM TE/g dw) R2
(TPC), antioxidant capacity and
analysis of correlation of TPC CE FRP FRT CE FRP FRT
vs antioxidant capacity
RL 15.2 ± 0.6b 201.0 ± 6.9a 713.1 ± 58.1a 277.4 ± 9.5a 2921.2 ± 97.4a 5243.8 ± 124.9a 0.95
RP 17.3 ± 0.7a 184.4 ± 13.1b 637.5 ± 31.9b 249.2 ± 8.9b 2280.2 ± 126.2b 4908.1 ± 54.3b 0.98

No common letters in different columns indicates significant difference (mean ± SD), n = 3

SP species, CE crude extract, FRP fraction rich in polyphenols, FRT fraction rich in tannins, mg GAE/
gdw milligrams gallic acid equivalents/g of dry weight, μM TE/gdw micromole Trolox equivalents/g of dry
weight, R2 coefficient of correlation among TPC and AC, RL R. Liebmannii, RP R. Palmeri

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Characterization of tannins from two wild blackberries (Rubus spp) by LC–ESI–MS/MS, NMR and…
extracts [7, 18, 25]. Methanolic (0.3% TCA) extraction
showed a good carryover of intact tannins from freeze-
dried berries, and subsequent purification steps increased
their concentration.
The detection of catechin and epicatechin monomers
(Compound 1) may indicate the presence of macromolecular
PAs in these fruits, but are present more frequently in seeds,
which remain nearly intact after acid-hydromethanolic
extraction, whereas ETs are easily carried out from more
soft fruits structures as peel and pulp. Other works showed
high contents of PAs obtained from crushed fruits [7, 12].
These polyhydroxyflavans (flavan-3-ols) contributes to the
berries’ high AOX and multiple health benefits, including
anti-inflammatory, antimicrobial, metallic-ion-chelating,
antiprotozoal, anti-tumoral anti-proliferation, and among
others [5, 7, 8, 14, 23, 26, 27].
Ellagic acid (Compound 2) and its lactonized form,
HHDP (hexahydroxydiphenyl) and its moieties (gal), occur
in Rubus species in low concentrations ( ~ 5% of TPC) but
are commonly esterified to β-d-glucose (glucosyl [glu]) to
build macromolecular oligomeric ETs [10, 11]. Polymeric
forms of ellagic acid with low molecular weight ( < 950 g/
mol) as pedunculagin (compound 3) and casuarictin/poten-
tillin (compound 4), are often in Rubus fruits and have been
shown as potential secondary metabolites with free-radical
scavenging, lipoperoxidative, and anti-inflammatory activi-
ties [7, 28]. Other macromolecular ETs, as sanguiin H-2,
H-10 and H-6/lambertianin A (compounds 6, 9 and 11,
respectively), were reported previously in other Rubus spe-
cies, but this is the first report of sanguiin H-10 in Rubus
fruits, since only has been recorded in other aerial parts of
these plants [15, 28, 29]. Lambertianin B (compound 7), was
detected easily in both Rubus species, but lambertianin C is
a molecule relatively heavy, and its presence was inferred
thought its molecular moiety. Lambertianin C in its natural
state (m/z 3470) is the trimeric form of casuarictin and has
been reported in several grown and wild Rubus berries with
highly potent beneficial effects on human health, also, is
one of the ETs most studied in Rubus berries. Few works
have achieved evaluate the bioactivity of these compounds
(sanguiins and lambertianins), including antioxidant, DNA-
preventive (on a human lymphocyte model), antifungal,
anti-inflammatory, and lipoperoxidative effects [14, 25, 28,
30]. An unknown molecule (compound 5) was reported as
the moiety of nobotanin A/malabathrin B (compound 10).
Some studies indicated antioxidant, anti-inflammatory, and
lipoperoxidative effects as the main bioactivity of nobota-
nin A/malabathrin B [7, 28]. LC–ESI–MS/MS analysis is
a tool to identify the compounds of low molecular weight,
but its main desadventage is the needed to use standards,
databases and bibliography to suggest the presence of some
compounds. In this study, we isolated and characterized
rigorously the tannin profile in both Rubus fruits by NMR,
2273
which allowed us to obtain additional information to under-
stand the complexity of their isomeric forms and oligomeric
structures.
Additionally, cleaning-up methodologies improve the
concentration of phenolic compounds. These values were
increased around 3.5 times higher than the contents before
clean-up with Sephadex LH-20 and 46.9 and 36.8 times
higher than the contents in the CEs of RL and RP fruits,
respectively. In another study, TPC values in fractions puri-
fied with Sephadex LH-20 resin revealed a greater increase
in this Rubus species in comparison with CEs from other
wild Rubus fruits, whose values were less than 31 times
higher [7]. The double exposure of dry fruits to acidified
80% MeOH (0.3% TCA) and to the purification steps could
increase the extraction of tannins from Rubus fruits and con-
serve chemical structures until the subsequent procedures
are carried out [18, 24]. Fruit phytochemistry is not stable
for plants, even in grown with same conditions, and TPC
is variable according to genotype and environmental and
seasonal conditions, including within established crops [18].
AOX of post-Sephadex fractions were 1.8 and 18.9 times
higher than FRPs and CEs, respectively, in RL samples and
2.2 and 19.7 times higher than FRPs and CEs, respectively,
in RP products. Srivastava et al. [31] studied blackberry cul-
tivars and reported increases in AOX from 1.1 to 1.7 times
in post-Sephadex LH-20 fractions, that had been purified
previously with Amberlite XAD-16 resin, while, using the
same procedures, Cuevas-Rodríguez et  al. [7] observed
increases in AOX from 7.0 to 19.8 times from a CE to an
FRP, of 1.0 to 1.7 times from an FRP to an FRT, and of 10.8
to 18.0 times from a CE to an FRT in Mexican wild black-
berries. In Rubus fruits, tannins have been reported as main
responsible of AOX, inclusive, showing higher values than
anthocyanins, due to the high presence of hydroxyl groups
in their structures [7, 25, 30, 31].
CEs, FRPs, and FRTs extracts from both wild Rubus
species, showed a strong positive correlation (Rubus RL
R2 = 0.95 and RP R2 = 0.98) among TPC vs AOX accord-
ing to the degree of purification. These results indicate that
the AOX of these Rubus fruits is influenced significantly by
purification tannin degree (Table 3).
Conclusion
Purification methodologies made it possible to determine
the tannin profile of Rubusliebmannii and R. Palmeri fruits
by LC–ESI–MS/MS and NMR (1H- and 13C-NMR). Two
flavan-3-ol (catechin and epicatechin) precursors of proan-
thocyanidins as well as six other oligomeric forms of ellagic
acid, including pedunculagin, casuarictin, nobotanin  A/
malabathrin B, lambertianins and sanguiins, were found in
R. Liebmannii, whereas in R. Palmeri fruits, 10 forms of
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2274
the above ellagitannins were found. TPC and AOX were
significantly corelated (R2 ≥ 0.95) during following purifi-
cation steps. This is the first study of tannin profile of wild
blackberries from Sinaloa, Mexico and their bioactivity as
a potential new healthy edible source.
Acknowledgements  This research was supported by Universidad
Autónoma de Sinaloa (Grants PROFAPI2014 and PROFAPI2015).
Oscar Abel Sánchez-Velázquez’s scholarship was provided by the
Consejo Nacional de Ciencia y Tecnología (CONACYT). The authors
would also like to thank Dr. Alfredo Leal Orduño for his invaluable
support of this research.
Compliance with ethical standards  jfca.2015.01.015
Conflicts of interest  The authors declare that there are no conflicts of
interest.
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