Journal of Functional Foods 27 (2016) 503–516

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Comprehensive characterization and antioxidant

activities of the main biflavonoids of Garcinia
madruno: A novel tropical species for developing
functional products

Luis Carrillo-Hormaza a,*, Ana M. Ramírez a, Camilo Quintero-Ortiz a,

Marlon Cossio a, Sonia Medina b, Federico Ferreres b,
Angel Gil-Izquierdo b, Edison Osorio a,*
a
Grupo de Investigación en Sustancias Bioactivas, Facultad de Ciencias Farmacéuticas y Alimentarias,
Universidad de Antioquia UdeA, Calle 70 No. 52-21, 050010 Medellín, Colombia
b
Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology,
CEBAS (CSIC), P.O. Box 164, Campus University Espinardo, 30100 Murcia, Spain

A R T I C L E I N F O A B S T R A C T

Article history: The aim of the study was to determine the qualitative and quantitative plant secondary
Received 28 April 2016 metabolite profile of Garcinia madruno by LC-MSn and to evaluate the structure–activity re-
Received in revised form 20 lationship (SAR) with its antioxidant properties. A total of 21 biflavonoids and 3 organic acids
September 2016 were identified. The leaves were the most promising source of biflavonoids, and the epicarp
Accepted 4 October 2016 was the richest source of morelloflavone type biflavonoids (>10%). Morelloflavone and
Available online fukugiside were the major biflavonoids found in all samples as well as the compounds re-
sponsible for antioxidant activity. The inter-flavonoid bond did not increase the antioxidant
Keywords: activity by single electron transfer (SET) mechanism but increased the activity by single proton-
Garcinia madruno loss electron transfer (SPLET) mechanism. The results suggest that G. madruno contains
Biflavonoids potentially useful amounts of bioactive biflavonoids; in particular, the leaves and epicarp
Metabolic profiling may be a potential source of functional products.
Structure–antioxidant activity © 2016 Elsevier Ltd. All rights reserved.
relationship

from one of the mentioned groups. For instance, the benzo-

1. Introduction
Garcinia is considered the most diverse and bountiful genus
of the Clusiaceae family (Jamila, Khairuddean, Khan, & Khan,
2014). Phytochemical studies have reported the isolation and
identification of biflavonoids, flavonoids, benzophenones,
xanthones, and organic acids. As a chemotaxonomic particu-
larity, some species of Garcinia overexpress specific metabolites
phenones are the most representative secondary metabolites
from Garcinia xanthochymus, G. mannii, G. staudtii, and
G. subelliptica (Acuña, Dastmalchi, Basile, & Kennelly, 2012;
Hemshekhar et al., 2011); xanthones are found in the fruits of
G. mangostana (Wittenauer, Falk, Schweiggert-Weisz, & Carle,
2012); organic acids (specially hydroxycitric acid) are found in
the fruits of G. cambogia (Semwal, Semwal, Vermaak, & Viljoen,
2015); and biflavonoids are found in the seeds of G. kola (Ayepola,

* Corresponding authors. Grupo de Investigación en Sustancias Bioactivas, Sede de Investigación Universitaria, Universidad de Antioquia,
Carrera 53 # 61-30 Lab 229. 050010 Medellin, Colombia. Fax: +57 (4) 2196590.
E-mail addresses: luis.carrillo@udea.edu.co (L. Carrillo-Hormaza); edison.osorio@udea.edu.co (E. Osorio)
http://dx.doi.org/10.1016/j.jff.2016.10.001
1756-4646/© 2016 Elsevier Ltd. All rights reserved.
504 Journal of Functional Foods 27 (2016) 503–516
Cerf, Brooks, & Oguntibeju, 2014), the fruits of G. brasiliensis
(Gontijo et al., 2012) and the cortex of G. hombroniana (Jamila
et al., 2014). A compilation of scientific data shows that many
of those species have been exploited in pharmaceutical and
food fields. In fact, G. mangostana, G. cambogia and G. kola are
the most recognized Garcinia species used in nutraceutical,
dietary supplements or functional foods products. The use of
these species have been reported for the prevention or treat-
ment of multiple symptoms and diseases such as ulcers,
diarrhoea, hypertension, obesity, inflammatory disorders,
hepatic damage, among others, and it has been related mainly
to the content of biflavonoids, hydroxycitric acid, xanthones
and benzophenones (Adegbehingbe et al., 2008; Farombi, 2011;
Hemshekhar et al., 2011; Saiyed et al., 2015; Tang et al., 2009;
Udani, Singh, Barrett, & Singh, 2009; Vasques et al., 2014). Mean-
while, G. madruno (Kunt) Hammel (known as “madroño”) is a
tropical tree of Central and South America characterized by its
exotic yellow fruit with edible pulp of mangosteen-like bitter-
sweet flavour (Cury, Abela, Bravo, Peñarrieta, & Rendón, 2012).
Previously, we isolated and identified from the leaves
some known biflavonoids: amentoflavone, morelloflavone,
volkensiflavone, fukugiside and espicataside, as well as
madrunoudeaside, a novel acetylglucoside of morelloflavone
(Osorio, Londoño, & Bastida, 2013; Osorio, Montoya, & Bastida,
2009).
Biflavonoids are characterized as the covalent union of two
monomeric units of flavonoids through C—C or C—O—C bonds
between flavanone–flavone, flavone–flavone, flavanone–
flavanone or flavone–flavanonol (Ferreira, De Carvalho, & Da
Silva, 2012). At the same time, those monomers may present
several substitutions, giving rise to glycosylated, methylated,
sulphated or isoprenylated biflavonoids, among others (Acuña
et al., 2010; Ito et al., 2013; Yang et al., 2010). Therefore, a theo-
retical number of biflavonoids could exist. However, unlike some
of their monomeric constituents, biflavonoids are character-
ized by presenting a distribution among nature restricted to
some species, highlighting the Ginkgo biloba and some species
from the genera Garcinia and Selaginella (Ferreira et al., 2012;
Kim, Park, Son, Chang, & Kang, 2008). The Garcinia biflavonoids
generally lead to 3→8″ biflavanones or 3→8″ flavanone–
flavone and carry at least one stereogenic centre but also show
atropisomeric behaviour due to restricted rotation about the
central axis (Li et al., 2002; Osorio et al., 2013). Nonetheless,
the majority of the reports regarding these biflavonoids involve
laborious pre-treatment and methodological approaches based
on chromatographic isolations and identification by spectro-
scopic techniques. Thereupon, the results obtained from these
methodologies, while confirming the presence of a series of
metabolites, their limitations and the lack of quantitative strat-
egies have not allowed determination of the metabolic profiles
in relation to the expression and content of biflavonoids,
preventing us from performing qualitative–quantitative com-
parisons intra- and inter-species, as well as establishing the
main natural sources of these compounds.
A number of studies have shown that biflavonoids gener-
ally possess a high level of antioxidant activity and relevant
pharmacological activities such as being antimicrobial, anti-
allergenic, anti-inflammatory, hepatoprotective and antiviral
(Ferreira et al., 2012). In relation to the G. madruno biflavonoids,
previous studies have shown that these compounds have the
ability to inhibit the lipid peroxidation of the human LDL and
to stabilize the DPPH• radical (Osorio et al., 2009, 2013).
Morelloflavone has also been associated with hypocholes-
terolemic (Tuansulong, Hutadilok-Towatana, Mahabusarakam,
Pinkaew, & Fujise, 2011), anti-inflammatory (Otuki et al., 2011)
and atheroprotective (Decha-Dier & Hutadilok-Towatana, 2008;
Pinkaew et al., 2009; Pinkaew, Hutadilok-Towatana, Teng,
Mahabusarakam, & Fujise, 2012) activities. The chemical and
antioxidant profiles of G. madruno biflavonoids may point to a
better understanding of the role of these substances in physi-
ology processes. However, although the functionality of
biflavonoids is highly related to their antioxidant capacity, no
clear comparison has been made to understand the structural
differences among biflavonoids with the antioxidant activity
or that compares the activity between biflavonoids and their
respective flavonoids monomers. Therefore, the aim of the
present study was to provide a comprehensive antioxidant ac-
tivity and a qualitative and quantitative characterization of the
G. madruno biflavonoids using a metabolomics approach based
on HPLC–DAD–MSn and a SAR analysis.
2. Material and methods
2.1. Chemical and reagents
All solvents used were analytical or HPLC grade (Merck Chemi-
cals, Darmstadt, Germany). Deionized water was obtained with
a Mill-Q water purification system (Millipore, Bedford, MA, USA).
Standard compounds (±)-6-hydroxy-2,5,7,8-tetramethylchromane-
2-carboxylicacid (Trolox), naringenin, apigenin, luteolin, luteolin-
7-O-glucoside, quercetin and (+)-catechin were obtained
from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). The
reference standard amentoflavone and biapigenin were pur-
chased from TCI Chemical Co. (Tokyo, Japan) and Phytolab
(Vestenbergsgreuth, Germany), respectively; whereas morello-
flavone, fukugiside and volkensiflavone were isolated from
a biflavonoid fraction previously obtained (Osorio et al., 2013)
using an HPTLC methodology (Camag-Linomat 5, Switzer-
land). The chromatography purity of the isolated standard
compounds was >97% for each compound. The reagents 2,4,6-
tris (2-pyridyl)-s-triazine (TPTZ), fluorescein and 2,2′-azobis (2-
methylpropionamidine) dihydrochloride (AAPH) were obtained
from Sigma Chemical Co. (St. Louis, MO, USA); Na2HPO4 and
KH2PO4 were purchased from Carlo Erba reagents (Milan, Italy);
and sodium acetate and FeCl3 were obtained from J.T. Baker
(Xalostoc, México).
2.2. Plant material
Five different G. madruno samples were used: (1) leaves, (2)
stems, (3) epicarp, (4) mesocarp and (5) seeds. All samples were
collected in February of 2014 from trees located on the Antioquia
University campus. Samples 1 and 2 were dried at 40 °C for five
days in a drying oven, and samples 3, 4 and 5 were frozen with
liquid nitrogen and freeze-drying (EYELA Freeze Dried FDU-
1200). All dried samples were mechanically blended until
obtaining a homogeneous particle size. Finally, all the pow-
dered samples were stored at room temperature, protected from
light and moisture.
 Journal of Functional Foods 27 (2016) 503–516
2.3. Extraction procedure
For the qualitative, quantitative and antioxidant activity, the
powdered G. madruno samples (100 mg) were extracted in a
temperature-controlled sonication bath (Elma P60H, Singer,
Germany) at a fixed power of 180 W, a frequency of 37 kHz, and
an acoustic energy density (AED) of 36 W L−1. All extractions
were carried out at 30 °C using 1.3 mL of an ethanol/water (74:26)
mixture for 60 minutes. After extraction, the mixture was cen-
trifuged for 5 min at 13.000 rpm at 4 °C. The supernatant was
decanted and transferred to a volumetric flask, and the pellet
was washed with an additional 500 µL of extraction solution.
Samples were centrifuged, decanted and transferred to the same
volumetric flask. The volume was finally adjusted with water
to 2.0 mL. This extract was later diluted, filtered through a 0.45-
µm nylon membrane and stored in the dark at −20 °C until
further analysis.
2.4. HPLC–DAD–ESI–MSn qualitative analysis of
G. madruno samples
Chromatographic analyses were carried out on an Agilent
Zorbax SB RRHT ® (Rapid Resolution High Throughput) C18
(50 mm × 4.6 mm, with a 1.8 µm particle size) column. The
mobile phase consisted of two solvents: water with 0.1% formic
acid (A) and acetonitrile (B). The following linear gradient was
used: 0 min, 10% B; 9 min, 28% B; 19 min, 38% B; 22 min, 70%
B; 30 min, 90% B; 35 min, 90% B and finally a reconditioning
cycle of the column over 5 min with the initial settings for the
next analysis. The flow rate was 800 µL min−1, and the injec-
tion volume was 5 µL. Spectral data from all peaks were
accumulated in the range of 240–400 nm, and chromato-
grams were recorded at 280 and 335 nm. The HPLC–DAD–ESI/
MSn analyses were carried out in an Agilent HPLC 1200 series
equipped with a diode array detector and mass detector in
series (Agilent Technologies, Palo Alto, CA, USA). The HPLC con-
sisted of a binary pump (model G1376A), an autosampler (model
G1377A) refrigerated at 4 °C (G1330B), a degasser (model G1379B),
and a diode array detector (model G1315D). The HPLC system
was controlled by ChemStation software (Agilent, v. B.01.03-
SR2). The mass detector was a Bruker ion trap spectrometer
(model HCT Ultra) equipped with an electro-spray ionization
interface and was controlled by LCMSD software (Agilent, v.
6.1). The ionization conditions were adjusted at 350 °C and 4.0 kV
for capillary temperature and voltage, respectively. The nebu-
lizer pressure and flow rate of nitrogen were 65.0 psi and
11 L min−1, respectively. The full scan mass covered the range
from m/z 150 up to m/z 1100. Collision-induced fragmenta-
tion experiments were performed in the ion trap using helium
as the collision gas, with voltage ramping cycles from 0.3 up
to 2 V. Mass spectrometry data were acquired in the negative
ionization mode. MSn was carried out in the automatic mode
on the more abundant fragment ion.
2.5. HPLC–DAD quantitative analysis of biflavonoids
For the quantification of G. madruno biflavonoids, each extract
was analyzed on an analytical HPLC Agilent 1200 Series LC
system (Agilent Technologies, Palo Alto, CA, USA) equipped with
a vacuum degasser, an autosampler, a quaternary pump and
505
a Diode Array Detector (DAD). Separation of the compounds
was performed using an Agilent Zorbax SB RRHT (Rapid Reso-
lution High Throughput) ® C18 (50 mm × 4.6 mm, with a 1.8 µm
particle size) column, with a flow rate of 1.0 mL min−1 at 35 °C.
The mobile phase consisted of water with 0.1% acetic acid (A)
and acetonitrile (B). The following linear gradient was used:
0 min, 22.5% B; 3.5 min, 22.5% B; 12 min, 50% B; 14 min, 90%
B; 18 min, 90% B, and finally a reconditioning cycle of the
column during 3 min with the initial settings for the next analy-
sis. The injection volume was 2 µL. The different compounds
were monitored using the DAD set at 280 and 335 nm, and the
spectra were recorded between 200 and 400 nm.
An external standard method was used for the quantifica-
tion of fukugiside, morelloflavone, volkensiflavone and
amentoflavone. A stock solution of 1 mg mL−1 in methanol was
prepared for each analyte. Consecutive dilutions of the stock
solutions in the mobile phase (80% A and 20% B) were ob-
tained to provide a linear dynamic range of seven concentration
levels for each compound. The content of fukugiside,
morelloflavone, volkensiflavone and amentoflavone was ex-
pressed as milligrams of compound by gram of dried sample
(mg g−1 sample). The limit of quantitation (LOQ) was calcu-
lated experimentally according to the values of precision and
accuracy but was not necessarily fixed as the lower limit of
the dynamic range. For the intra-day precision determina-
tion, each matrix was analyzed six times on the same day (n = 6)
and the corresponding RSD were calculated. To verify the ac-
curacy of the analyses, the method was tested against three
calibration levels using the standard references of fukugiside,
morelloflavone, volkensiflavone and amentoflavone (n = 9). The
relative bias was determined for each level and each compound.
2.6. Antioxidant activity
2.6.1. Oxygen radical absorbance capacity (ORAC) value
The ORAC assay was adapted from previously described pro-
cedures (Jiménez et al., 2015). AAPH was used as a peroxyl
radical generator, and Trolox was used as a standard. More-
over, fluorescein was used as the fluorescent probe. Fluorescein,
AAPH and the samples were prepared in a 75 mM phosphate
buffer at pH 7.4. The samples were mixed with 1.6 µM fluo-
rescein (1:4) in black 96-well microplates for 30 min at 37 °C.
Subsequently, 125 mM AAPH in PBS was added at a 1:5 ratio.
Fluorescein intensity was measured every two minutes for
120 min at excitation and emission wavelengths of 485 and
520 nm, respectively, in a Spectra Max Gemini EM – Molecu-
lar Devices – (Orleans, USA). The relative ORAC values were
calculated using the differences of areas under the decay curves,
and the results are expressed as µmol equivalents per gram
of sample (µmol TE g−1) based on a calibration curve with Trolox
(12.5–100 µmol).
2.6.2. Ferric reducing antioxidant power (FRAP) value
The ability of the samples to reduce the TPTZ–Fe (III) complex
was measured according to the method reported by Jiménez
et al. (2015). The FRAP working solution contained 300 mM of
acetate buffer (pH 3.6), 40 mM of TPTZ and 20 mM of FeCl3.6H2O
in water at a 10:1:1 ratio (v:v:v). Samples and the working FRAP
solution were mixed at a 1:25 ratio for 10 min at 37 °C in the
506 Journal of Functional Foods 27 (2016) 503–516
dark. The absorbance was read at 593 nm using an UV/Vis
PowerWave TM XS2 spectrophotometer, and it was interpo-
lated in a calibration curve with Trolox (31.25–1000 µM). The
results are expressed in µmol Trolox equivalents per gram of
sample (µmol TE g−1).
2.6.3. Total phenol content (TPC)
Total phenol content was determined by the Folin–Ciocalteu
method with some modifications (Jiménez et al., 2015). The re-
action mixture contained sample or standard 0.2 N of Folin–
Ciocalteu reagent and 7.5% sodium carbonate at a ratio of
25:125:100 (v:v:v). After 60 min at room temperature and with
protection from light, the absorbance was recorded at 764 nm
using an UV/Vis PowerWave TM XS2 spectrophotometer (BioTek
USA). The results are expressed in mg gallic acid equivalents
per gram of sample (mg EAG g−1) based on a calibration curve
with gallic acid (10–100 g mL−1).
2.6.4. Structure antioxidant–activity relationship of
biflavonoids and related flavonoids
For the SAR study of G. madruno biflavonoids and structurally
related flavonoids, a total of 11 compounds (Table 4) were evalu-
ated through FRAP and ORAC assay at 100 and 10 µM,
respectively. The flavonoids used were as follows: (i) G. madruno
biflavonoids (morelloflavone, volkensiflavone, amentoflavone
and fukugiside; (ii) other biflavonoids (biapigenin [3→8″
biapigenina]); (iii) flavonoid monomers of G. madruno
biflavonoids (apigenin, naringenin, luteolin and luteolin-7-O-
glucoside); and (iv) reference flavonoids (catechin and quercetin).
2.7. Statistical analysis
All the experiments were carried out at least in triplicate, and
the results are expressed as the mean ± standard deviations.
Statistical analyses were performed by using the Graph Pad
Prism® version 5.00 for Windows (Graph Pad Software, Inc.-
San Diego CA2007) software. Analysis of variance (ANOVA) was
carried out for each variable to test the statistical signifi-
cance using a p-value at 5% level.
3. Results and discussion
3.1. Metabolic profile of G. madruno By
HPLC–DAD–ESI–MSn
Several classes of compounds have been identified in the
Garcinia genus, including biflavonoids, flavonoids, benzophe-
nones, xanthones, and organic acids. However, only
morelloflavone and amentoflavone type biflavonoids have been
isolated and identified in G. madruno (Li et al., 2002; Osorio et al.,
2013). The identification by LC–DAD–ESI–MSn of the main sec-
ondary metabolites of G. madruno matrices (leaves, stems,
epicarp, mesocarp and seeds) is shown in the Table 1. The com-
pound number is based on the elution order shown in Fig. 1.
The compounds were tentatively identified based on the in-
terpretation of their mass spectral behaviour obtained from
MSn experiments and by comparison with the literature. Thus,
a total of 26 peaks were identified. The proposed structures of
the identified compounds are shown in Fig. 2. Overall, 3 organic
acids and 21 biflavonoids were identified and more than 15
peaks structurally associated with known benzophenones of
Garcinia species were detected.
3.1.1. Organic acids
Compound 1 was identified as hydroxycitric acid (HCA) based
on its m/z 207 and MS2 fragments. Compound 2 was identi-
fied by MS as the hydroxycitric acid lactone (HCAL)
(Rodríguez-Medina et al., 2009). Citric acid (compound 3) was
found in all matrices and seems to be the major organic acid
of G. madruno, and HCA and HCAL were not found in the seeds.
HCA and HCAL have not been previously reported in G. madruno,
and this fruit may have been overlooked as an important source
of hypocholesterolemic and anti-obesity compounds
(Hemshekhar et al., 2011).
3.1.2. Polyisoprenylated benzophenones
The peaks observed in the last part of the chromatographic
profile were associated with polyisoprenylated benzophe-
nones (PIBs) (Fig. 1). About twenty peaks associated with PIBs
from their molecular ion and fragmentation patterns were
found, although more than eighty PIBs have been reported from
Garcinia genus (Kumar, Sharma, & Chattopadhyay, 2013). We
were not able to identify PIBs of G. madruno due to the low re-
sponse and similarities of the molecular ions and MS n
fragments. The main PIBs observed in all G. madruno matri-
ces were the compounds 25 and 26 eluting at 27.66 and 31.79
minutes, respectively (Fig. 1). According to the Msn spectrum,
both compounds had the same molecular ion at m/z 601 and
similar MS2 and MS3 fragments, whereas the UV spectrum and
the retention time were pretty different (Table 1). Thus, for the
molecular ion of compounds 25 and 26, there are 13 possible
structures identified in the Garcinia species so far (camboginol,
cambogin, eugeniaphenone, (−) cycloxanthochymol, garci-
nialiptone D, garcinol, guttiferone A, E, F, H o M, isogarcinol or
isoxanthochymol) (Kumar et al., 2013). Therefore, further studies
using high resolution tandem mass spectrometry and some
reference standards are required, taking to account the bio-
logical relevance of these compounds (Kumar et al., 2013).
3.1.3. Biflavonoids
The biflavonoids were the most abundant secondary metabo-
lites found in G. madruno. These compounds were located in
the middle part of each UV chromatogram (Fig. 1) between 7
and 23 minutes. In total, 21 different biflavonoids were iden-
tified, ten of them as glycosylated and three as sulphate
derivatives. Additionally, eight biflavonoid aglycones were found,
one of them methylated and another prenylated. According to
the classification of biflavonoids, morelloflavone type biflavonoids
were the most abundant group with a total of 10 different com-
pounds, followed by GB-a type biflavonoids (8 compounds),
amentoflavone type biflavonoids (2 compounds) and GB-1 type
biflavonoids (1 compound).
3.1.3.1. Morelloflavone type biflavonoids. Compounds 4, 8, 9,
11, 15, 16, 17, 19, 21 and 22 were identified as morelloflavone
type biflavonoids (flavanone-(3→8″)-flavone). Compound 19
(TR = 14.89 min) with a [M-H]− ion at m/z 555 was identified as
morelloflavone (naringenin-(3→8″)-luteolin) according to its MSn
Table 1 – Metabolic profile of Garcinia madruno samples by HPLC–DAD–ESI–MSn.
Compound Sample Rt (min) UV (nm) [M-H]− (m/z) LC-ESI/MSn (% base peak)
1 Hydroxycitric acid Leaves, stems, epicarp and mesocarp. 0.79 – 207 MS2 [207]: 188.9 (100), 126.9 (10.3)
2 Hydroxycitric acid lactone Leaves, stems, epicarp and mesocarp. 0.84 – 189 MS2 [189]: 126.8 (100), 110.8 (82.9)
3 Citric acid Leaves, stems, epicarp, mesocarp and seeds. 0.89 – 191 MS2 [191]: 126.8 (77.1), 110.8 (100)
4 Morelloflavone-4‴, 7″-di-O-glucoside Leaves, stems, epicarp and seeds 7.16 290; 335 879 MS2 [879]: 717.0 (100), 428.9 (9.9)
MS3 [879→717]: 591.0 (30.86), 565.0 (100), 428.9 (46.3)
5 GB-2a-di-O-hexoside Leaves, stems, epicarp and seeds 7.33 288; 324sh 881 MS2 [881]: 719.0 (25.5), 593.0 (46.11), 557.0 (22.9), 431.0 (100)
MS3 [881→431]: 412.9 (16.6), 320.9 (15.52), 294.8 (100)
6 GB-1a-di-O-hexoside Leaves 7.80 – 865 MS2 [865]: 703.0 (96.8), 597 (26.8), 540.9 (25), 415.0 (100)
MS3 [865→415]: 320.9 (51.6), 309.7 (11.9), 294.8 (100), 268.9 (8.3)
7 GB 2a-O-hexoside-sulphate Leaves 8.61 284; 330sh 799 MS2 [799]: 719.0 (100), 593.0 (98), 431.0 (36.25)
MS3 [799→719]: 592.9 (26), 557.0 (12.1), 430.9 (100), 294.7 (6.7)
8 Morelloflavone-7″-O-sulphate-4‴- Stems, epicarp and seeds 8.80 274sh, 290, 797 MS2 [797]: 717.0 (100), 590.9 (21.9), 555.0 (21.2), 428.9 (24.7)

Journal of Functional Foods 27 (2016) 503–516

O-glucoside 335 MS3 [797→717]: 591.0 (33.1), 554.9 (24.7), 428.9 (100)
9 Fukugiside Leaves, stems, epicarp, mesocarp and seeds. 9.92 255sh, 274sh, 717 MS2 [717]: 591.0 (65.6), 565.0 (100), 428.9 (64.4) 402.9 (20.9)
288, 346 MS3 [717→565]: 402.9 (100), 444.9 (13.6)
10 GB-2a-O-hexoside Leaves, stems, epicarp, mesocarp and seeds. 10.81 292; 320sh 719 MS2 [719]: 593.0 (84.0), 557.0 (5.5), 430.9 (100)
MS3 [719→431]: 320.9 (5.8), 294.8 (100)
11 Morelloflavone-4‴-O-glucoside Leaves, stems, epicarp, mesocarp and seeds. 11.02 255sh, 275sh, 717 MS2 [717]: 590.9 (16.8), 555.0 (53.3) 428.9 (100)
287, 334 MS3 [717→429]: 400.9 (100), 386.9 (46.4), 294.8 (36.4), 249.0 (42.9)
12 GB-2 Mesocarp and seeds 11.26 286, 320sh 573 MS2 [573]: 446.9 (100), 428.9 (16.8), 418.9 (22.5)
MS3 [573→447]: 429.0 (36.8), 418.9 (100), 402.8 (7.4)
13 GB-2a-sulphate Leaves, stems, epicarp, mesocarp and seeds. 11.47 286, 320sh 637 MS2 [637]: 557.0 (30.3), 430.9 (100), 298.8 (2.8)
MS3 [637→431]: 412.9 (15.2), 320.9 (8.1), 294.8 (100)
14 GB-1a-O-hexoside Leaves and epicarp 11.70 Co-eluted 703 MS2 [703]: 540.9 (96.7), 414.9 (100)
MS3 [703→414.9]: 320.8 (8.1), 294.8 (100)
15 Morelloflavone-7″-O-sulphate Leaves, stems, epicarp, mesocarp and seeds. 11.80 Co-eluted 635 MS2 [635]: 554.9 (100), 428.9 (91.36)
MS3 [635→555]: 448.9 (5.7), 428.9 (100), 402.8 (8.2)
16 Espicataside Leaves, stems and seeds. 11.90 Co-eluted 701 MS2 [701]: 575.0 (15.4), 538.8 (100), 444.8 (19.1), 412.9 (30.4)
MS3 [701→539]: 445.0 (18.3), 432.9 (19.3), 412.9 (100)
17 Volkenisflavone-7″-O-sulphate Leaves, stems, epicarp, mesocarp and seeds. 14.13 270sh, 288, 619 MS2 [619]: 538.9 (100), 444.9 (3.6), 413.0 (17.7)
335 MS3 [619→539]:433.0 (11.6), 412.9 (100)
18 GB-2a Leaves, stems, epicarp, mesocarp and seeds. 14.49 292, 320sh 557 MS2 [557]: 450.9 (13.7), 430.9 (100)
MS3 [557→431]: 320.9 (8.4), 294.8 (100)
19 Morelloflavone Leaves, stems, epicarp, mesocarp and seeds. 14.89 254sh, 274sh, 555 MS2 [555]: 403.0 (6.1), 428.9 (100)
288, 345 MS3 [555→429]: 400.9 (100), 356.9 (15.2), 294.8 (8.4)
20 GB-1a Leaves, stems, epicarp, mesocarp and seeds. 17.30 – 541 MS2 [541]: 446.9 (15.2), 414.9 (100), 295.8 (7.2)
MS3 [541→415]:320.8 (19.0), 294.8 (100)
21 Volkensiflavone Leaves, stems, epicarp, mesocarp and seeds. 17.55 274sh, 288, 539 MS2 [539]: 444.9 (20.9), 433.0 (11.6), 412.9 (100)
330sh MS3 [539→413]: 384.9 (100), 294.9 (2.8), 264.8 (4.0), 184.8 (3.5)
22 3‴-O-methylmorelloflavone Stems 18.00 – 569 MS2 [569]: 462.8 (10.5), 442.9 (100)
MS3 [569→443]: 427.9 (100), 410.9 (4.8)
23 Amentoflavone Leaves and stems 18.74 338, 268 537 MS2 [537]: 442.9 (14.0), 416.9 (20.21), 398.9 (13.2), 374.9 (100)
MS3 [537→375]: 330.8 (100), 306.9 (11.7), 244.8 (6.2), 188.8 (4.5)
24 Prenylated-amentoflavone Leaves and stems 23.20 334, 270 605 MS2 [605]: 510.9 (11.1), 485.0 (17.6), 467.0 (13.2), 442.9 (100)
MS3 [605→443]: 399.9 (44.2), 387.8 (100), 374.9 (72.9)
25 Polyisoprenylated benzophenone Leaves, stems, epicarp, mesocarp and seeds. 27.66 278, 310sh 601 MS2 [601]: 557.1 (12), 465.0 (100), 447.1 (28), 409.0 (37), 355.0 (52)
MS3 [601→465]: 327.0 (25), 272.9 (100)
26 Polyisoprenylated benzophenone Leaves, stems, epicarp, mesocarp and seeds. 31.79 250, 354sh 601 MS2 [601]: 557.1 (12), 465.2 (100), 409.0 (19), 327.0 (26.4)

507
MS3 [601→465]: 327.0 (11), 272.9 (100)
508 Journal of Functional Foods 27 (2016) 503–516
Fig. 1 – HPLC fingerprint of Garcinia madruno (280 nm). Peak: 1–26 (see Table 1).
fragmentation pattern (Fig. 3a) and the retention time of the
respective reference standard. Regarding the fragmentation
route of morelloflavone, the MS2 product at m/z 429 [(M-H)-
126]− was the most abundant fragment found and corresponded
to the loss of -C6H6O3 by the cleavage of the C-ring in the
naringenin monomer at position “a” (Fig. 3a). Meanwhile, the
neutral loss of CO in the C-ring of the naringenin monomer
at position “c”, following the cleavage of the C-ring in flavo-
noid II at position “d”, were the most representative MS2
cleavage sites in the fragmentation pathway of morelloflavone
and almost all morelloflavone type biflavonoids (Fig. 3a and
Table 1). In contrast, compound 9 (TR = 9.92 min) showed a dif-
ferent MS cleavage site conducted mainly by position “b”
(Fig. 3a and Table 1). According to its MSn spectra and the ref-
erence standards available, this compound was identified as
fukugiside, the 7″-O-glucoside of morelloflavone. However, com-
pound 11 with the same molecular ion [M-H]− and UV spectra
of the fukugiside was found at 11.02 min, suggesting the pres-
ence of another morelloflavone–hexoside. In fact, the MS2
spectra of both compounds differ significantly, whereas
fukugiside showed a fragmentation pathway dependent on “b”
site cleavage, compound 11 showed the MS2 product ion at m/z
555 [(M-H)-162]−, resulting from the loss of a hexose and the
representative fragmentation pathway of morelloflavone (Fig. 3a
and Table 1). Thus, based upon the reports of biflavonoids
in Garcinia genus, compound 11 could correspond to
morelloflavone-4″’-O-glucoside (Ferreira et al., 2012). The ESI–
MS spectrum of compound 4 (TR = 7.16 min) showed a
molecular ion at m/z 879, the MS2 spectrum had a peak at m/z
717 [(M-H)-162]−, showing the loss of a hexose, and the MS3
spectrum corresponded to the MS spectrum of fukugiside
(Table 1). Thus, this compound was tentatively identified as
morelloflavone-4‴,7″-di-O-glucoside.
Compound 15 (TR = 11.8) with a [M-H]− ion at m/z 635 was
identified as morelloflavone-7″-O-sulphate, a compound previ-
ously isolated and identified in G. madruno (Li et al., 2002). The
MS2 spectrum had a peak at m/z 555 [(M-H)-80]−, showing the
loss of a sulphate (SO3), and the MS3 spectrum corresponded to
the MS2 spectrum of morelloflavone. Whereas compound 8
(TR = 8.80 min) with a molecular ion at m/z 797 had a MS2 spec-
trum peak at m/z 717 [(M-H)-80]−, showing the loss of a sulphate,
and the MS3 spectrum [797 →717] corresponded to the MS2 spec-
trum of morelloflavone-4‴-O-glucoside (compound 11). Thus,
this compound tentatively was identified as morelloflavone-
7″-O-sulphate-4‴-O-glucoside. Additionally, compound 22
(TR = 18.0 min) with a [M-H]− ion at m/z 569 was identified as
O-methylated morelloflavone derivate. The MS2 spectrum found
was in accordance with the morelloflavone type biflavonoids frag-
mentation pathway (Fig. 3a), showing the major MS2 fragment
at m/z 443 [(M-H)-126]−, and the MS3 spectrum had a peak at m/z
428 [(M-H-126)-15]−, showing the loss of a methyl (-CH3). There-
fore, taking into account the biflavonoids found in Garcinia genus,
compound 22 was tentatively identified as 3‴-O-methyl-
morelloflavone (Ferreira et al., 2012). Following the same
fragmentation pathway of morelloflavone, compound 21
(TR = 17.55) was identified as volkensiflavone (naringenin-(3→8″)-
apigenin), a morelloflavone type biflavonoid previously
identified in this species (Li et al., 2002; Osorio et al., 2013).
In the same way, two known G. madruno volkensiflavone
derivates were found. Compounds 16 and 17 (RT = 11.90
and 17.55 min, respectively) were identified as espica-
taside (volkensiflavone-7″-O-glucoside) and volkenisflavone-
7″-O-sulphate, respectively (Li et al., 2002; Osorio et al.,
2013).
3.1.3.2. GB-1a type biflavonoids. Compounds 5, 6, 7, 10, 13, 14,
18 and 20 were identified as GB-1a type biflavonoids (flavanone-
(3→8″)-flavanone). According to the biflavonoids found in Garcinia
genus, compound 18 (RT = 14.49 min) with a molecular ion at
m/z 557 could be the biflavonoid GB-2a (naringenin-(3→8″)-
eriodictyol) or GB-1 (naringenin-(3→8″)-dihydrokaempferol).
Following the proposed fragmentation pathway of morelloflavone
type biflavonoids (Fig. 3a), the MS and the MS2 spectrum of GB-
2a and GB-1 theoretically will not present significant differences,
 Journal of Functional Foods 27 (2016) 503–516 509

Fig. 2 – Chemical structures of the main compounds detected in Garcinia madruno. *The structure showed is tentative. **The
garcinol structure was associated to the compounds 25 and 26.

as well as the UV spectrum. However, the MS3 spectrum of this
peak showed a characteristic ion fragment at m/z 321 that was
associated only with the fragmentation of GB-2a and could be
explained by the loss of the B ring of the flavonoid II (eriodictyol)
[(M-H-126)-110]− (cleavage site “c” Fig. 3b). While this cleavage
site is commonly reported for the fragmentation of eriodictyol
and dihydrokaempferol (Tsimogiannis, Samiotaki, Panayotou,
& Oreopoulou, 2007), applying the GB-2a fragmentation pathway
to GB-1, the MS3 fragment formed will be at m/z 337, 16 m/z units
more than the ion observed. Therefore, based upon GB-2a iden-
tification, the fragmentation pathway of GB-1a type biflavonoids
is shown in Fig. 3b. Overall, there are few significant differ-
ences in comparison with morelloflavone type biflavonoids. The
MS2 products of the aglycones are formed mainly by the same
cleavage site of morelloflavone type biflavonoids, correspond-
ing to the loss of -C6H6O3 by the cleavage of the C-ring in
510 Journal of Functional Foods 27 (2016) 503–516

Fig. 3 – MSn fragmentation pathway proposed for morelloflavone (A), GB-1a (B) and amentoflavone (C) type biflavonoids.
 Journal of Functional Foods 27 (2016) 503–516 511

Fig. 4 – Chromatographic separation with a Rapid Resolution High Throughput column by HPLC–DAD for the quantitative
analysis. (A) Chromatogram of biflavonoid standard compounds. (B) Comparison of the chromatographic profiles of different
G. madruno matrices. Compounds 9, 19, 21 and 23 correspond to fukugiside, morelloflavone, volkensiflavone and
amentoflavone, respectively.

naringenin monomer at position “a” (Fig. 3a and b). Nonethe-
less, position “d” was the most representative MS2 cleavage site
in the fragmentation pathway of GB-a biflavonoids instead of
position “c” of the morelloflavone type biflavonoids (Fig. 3a). Fol-
lowing the GB-1a fragmentation pathway, compound 20 was
identified as GB-1a (naringenin-(3→8″)-naringenin). On the other
hand, six derivates of those compounds were found. Com-
pounds 5, 6, 10 and 14 were identified as GB-2a-di-O-hexoside,
GB-1a-di-O-hexoside, GB-2a-O-hexoside and GB-1a-O-hexoside,
respectively. The main MS2 fragments ions of these com-
pounds were obtained by the loss of -C6H6O3 [(M-H)-126]−, the
loss of one or two hexoside moiety [(M-H-162) or (M-H)-162-
162]− and the successive loss of -C6H6O3 and one hexoside moiety
[(M-H)-126-162]−. Whereas compounds 13 and 7 were identi-
fied as GB-2a-sulphate and GB-2a-O-hexoside-sulphate,
respectively, and both showed a MS2 major fragment loss of
80 m/z (SO3) in regard to their molecular ion.
3.1.3.3. Other type of biflavonoids. Compounds 23 and 24 were
identified as amentoflavone type biflavonoids (flavone-(3′→8″)-
flavone). Compound 23 (TR = 18.74 min) with a [M-H]− ion at
m/z 537 was identified as amentoflavone (apigenin-(3′→8″)-
apigenin) according to its MSn fragmentation pattern (Fig. 3c),
the retention time and the UV spectrum of the respective ref-
erence standard. The MS2 spectrum of this compound showed
ions at m/z 443, 417, 399 and 375 (Table 1). These fragments have
already been found in other LC–ESI–MS/MS analysis of
amentoflavone (Liao et al., 2015; Wang et al., 2014). In all these
cases, the main MS2 fragment was found at m/z 375, corre-
sponding to the loss of —C9H6O3 [(M-H)-162]− by cleavage at
position “a” of the C-ring in one of the apigenin monomers
(Fig. 3c). On the other hand, compound 24 (TR = 23.20) with a
[M-H]− ion at m/z 605 had the UV spectrum characteristic for
bi-flavones, as well as for amentoflavone (Table 1). In addi-
tion, according to its MSn spectrum, it was tentatively identified
as prenylated-amentoflavone because the fragmentation pattern
of this compound was the same as the amentoflavone, adding
a prenylated group in the main fragment found (68 m/z units
more) (Table 1). Finally, compound 12 (TR = 11.26 min) with
a [M-H] − ion at m/z 573 was tentatively identified as
GB-2 (naringenin-(3→8″)-taxifolin), a GB-1 type biflavonoid
(flavanone-(3→8″)-flavanonol).
3.2. Qualitative and quantitative analysis of
G. madruno biflavonoids
For the quantitative analysis of G. madruno biflavonoids, a
shorter RP–HPLC–DAD method was developed using the avail-
able reference standards. Under optimized conditions, the best
separation of fukugiside (9), morelloflavone (19), volkensiflavone
(21), and amentoflavone (23) was obtained in 10 minutes
(Fig. 4a), assuring an adequate separation for a quantitative ap-
proach of all target biflavonoids in each of the G. madruno
samples evaluated (Fig. 4b). The analytical performance of the
method is shown in the Table 2. The range selected was fixed
according to the chromatographic response for each biflavonoid
with the aim to quantify all analytes in each sample using only
one dilution. Good linearity was achieved over the concentra-
tion range presented; the four calibration curves exhibited linear
regressions of at least r2 > 0.992 and met the homoscedasticity
criteria. The LOQ was determined experimentally according to
the parameters of precision and accuracy fixed in a coeffi-
cient of variation and relative bias (less than 10%). For all
analytes, the LOQ were 0.625 µg mL−1 (Table 2). In regard to the
repeatability and accuracy of the method, the results ob-
tained were satisfactory. The RSD were lower than 10% for each
512 Journal of Functional Foods 27 (2016) 503–516

analyte in each G. madruno matrix evaluated, and the relative

Highd
Accuracy (%RE, n = 9)
bias was lower than 10% in all levels and compounds evaluated.

1.75
2.19
5.47
1.74
According to the results, there are significant differences
in the chromatographic profiles and content of biflavonoids

Halfc
4.28
5.81
8.92
7.02
between the different G. madruno samples (Fig. 1, Tables 1 and
3). As a whole, the leaves followed by the stems had a higher
diversity of biflavonoids, whereas the mesocarp was the sample
Lowb
5.84
4.77
6.72
6.74
with the lowest expression of these compounds. On the other
hand, amentoflavone type biflavonoids were not detected in
Seeds

any of the G. madruno fruit samples, and compound 12 was

3.38
0.76
1.28
ND
found only in the mesocarp and the seeds. Thus, the pres-
ence and absence of those compounds could be identity
Mesocarp

markers of the G. madruno fruits. Nevertheless, further robust

chemometric approaches are required to establish the iden-
4.95
8.76
5.69

tity and quality markers between the different G. madruno

ND
Intra-day precision (RSD, n = 6)

matrices.
Table 2 – Analytical performance of the developed HPLC–DAD method for the quantification of Garcinia madruno biflavonoids.

Quantitatively (Table 3), morelloflavone and fukugiside were

Epicarp

found in higher concentrations than volkensiflavone and

5.05
5.32
5.31
ND

amentoflavone in G. madruno samples. Indeed, morelloflavone

was the main biflavonoid found in the epicarp, stems and me-
Stems

socarp, whereas fukugiside was the main biflavonoid found in

4.31
3.17
4.73
4.18

the leaves and stems. The morelloflavone content ranged

between 94.0 ± 4.99 and 1.67 ± 0.14 mg g−1 of the dried samples,
decreasing in the order of epicarp > stems > leaves > seeds > me-
Leaves
1.37
3.73
3.77
6.34

socarp. In comparison with morelloflavone, the content

of fukugiside was lower, ranging from 35.05 ± 0.81 to
6.73 ± 0.29 mg g−1, decreasing in the order of leaves > epicarp >
LOQ (µg mL−1)

seeds > stems. In the mesocarp, this compound was detect-

able, but the response was lower than the LOQ (Table 2).
200.00 µg/mL to fukugiside and morelloflavone, 35.00 µg mL−1 to volkensiflavone and amentoflavone.
50.00 µg/mL to fukugiside and morelloflavone, 10.00 µg mL−1 to volkensiflavone and amentoflavone.
10.00 µg/mL to fukugiside and morelloflavone, 1.25 µg mL−1 to volkensiflavone and amentoflavone.

Volkensiflavone was found in all matrices, and its content ranged

0.625
0.625
0.625
0.625

from 5.83 ± 0.31 to 0.12 ± 0.01 mg g−1, with the epicarp and leaves
being the main sources of this compound. Finally, amentoflavone
Range (µg mL−1)

was detected and quantified only in the leaves and stems, and
its content was the lowest of the biflavonoids evaluated. The
sum of the content of biflavonoids was expressed as total percent
5–200
5–200
0.625–35
1.250–35

of biflavonoids (Table 3). According to this result, the samples

ranged from 11.4 to 0.18% (w/w, dry wt.), in which the epicarp
was the matrix with the highest content of biflavonoids, fol-
lowed by the leaves, stems, seeds and mesocarp. In all cases,
G expa

0.337
0.444
0.349
0.441

the content of morelloflavone plus fukugiside reached values

above 90% of the total biflavonoid reported for each matrix.
In comparison with the biflavonoid content reported in other
0.9992
0.9984
0.9927
0.9961

species, the quantitative results obtained were highly rel-

r2

evant. Within the genus Garcinia, Acuña et al. (2012) determined

the content of morelloflavone, volkensiflavone, amentoflavone
Calibration curve

Cochran’s homoscedasticity test, Gtables = 0.561.

Y = 9.691x − 8.254
Y = 6.635x − 7.987
Y = 3.671x − 0.479
Y = 19.90x − 9.850

and fukugiside in fruits of eight different Garcinia species by RP-

HPLC-DAD. In general, these authors have reported lower content
of biflavonoids. In fact, between the eight species analyzed,
G. xanthochymus reached the highest value of biflavonoids (2.5%
w/w, dry wt.). Moreover, Anu Aravind et al., using HPTLC, found
significant fukugiside content in the leaves of G. travancorica (7%
w/w, dry wt.); however, other compounds were not quantified
λ (nm)

in the study (Anu Aravind, Asha, & Rameshkumar, 2015). In other

290
290
290
335

species of the genus, the information found did not provide

quantitative data to be able to make a comparison. Mean-
Volkensiflavone
Amentoflavone
Morelloflavone

while, in another genus, the scenario was similar. For instance,

Compound

Fukugiside

in Ginkgo biloba, the maximum content of biflavonoids reached

was only 1.68% in optimized extracts (Van Beek, 2002); similar
behaviour was found in some Selaginella species (Aguilar et al.,
d
b
a

2013) and Hypericum perforatum (Hosni, Msaâda, Taârit,

 Journal of Functional Foods 27 (2016) 503–516
Table 3 – Biflavonoids content, total phenolic content and antioxidant activity of the samples of Garcinia madruno.
Sample Biflavonoids (BF) amount (mg g−1)
Mo Fu Vo Am
Leaves 26.0 ± 1.05c 35.00 ± 0.81d 3.63 ± 0.17d 2.26 ± 0.10b
Stems 33.5 ± 1.06d 6.73 ± 0.29a 2.83 ± 0.13c 0.88 ± 0.04a
Epicarp 94.0 ± 4.99e 13.90 ± 0.69c 5.83 ± 0.31e ND
Mesocarp 1.7 ± 0.14a <LOQ 0.12 ± 0.01a ND
Seeds 6.6 ± 0.05b 8.40 ± 0.40b 1.26 ± 0.01b ND
Data are presented as mean ± SD (n = 3). Values within a column followed by the same superscript letters are not significantly different at the
0.05 level according to ANOVA.
ND: Not detected.
a
Expressed as mg Galic acid equivalents g−1 of sample.
b
Expressed as µmol Trolox equivalents g−1 of sample.
c
Expressed as µmol Trolox equivalents 100 g−1 of sample.
Hammami, & Marzouk, 2010). The comparison of these results
with those of previous researchers is difficult due to the differ-
ences in the nature of the sample and pre-concentration
technologies, extraction systems, and assay methodologies.
However, taking to account that the total biflavonoid content
found in G. madruno is underestimated by the compounds iden-
tified as biflavonoids but not quantified (Fig. 1 and Table 1), our
results propose that G. madruno is one of the sources with a high
biflavonoid content in nature; specifically, the leaves and the
epicarp will reach a biflavonoid content comparable with some
species recognized by their high concentrations of flavonoid, such
as the content of total catechins reported in some varieties of
tea (more of 20% of total catechins, w/w, dry wt.).
3.3. Antioxidant activity of G. madruno samples
A single antioxidant method can provide basic information
about antioxidant properties, but a combination of methods
can describe the antioxidant properties of the samples in
more detail (Jiménez et al., 2015). In this study, a set of anti-
oxidant assays was performed to fully characterize the
antioxidant potential of G. madruno. Table 3 shows the TPC
content and the ORAC and FRAP value of G. madruno matri-
ces. The decreasing order obtained was the same for the three
assays (epicarp > leaves > stems > seeds > mesocarp), as well as
the total percent of biflavonoids, indicating a high correla-
tion between antioxidant activity and biflavonoid content
(Table 3). As a whole, epicarp and leaves were the matrices with
the highest antioxidant activity. The results obtained in the
ORAC assay were higher in comparison with the values re-
ported in the database of fruits recognized by its scavenger
potential, such as blackberries, blueberries, pomegranate and
apples (Speisky, López-Alarcón, Gómez, Fuentes, &
Sandoval-Acuña, 2012).
3.3.1. Structure antioxidant–activity relationship (SAR) of
biflavonoids and structurally related flavonoids
Table 4 shows the results obtained for the SAR study of
G. madruno biflavonoids and structurally related flavonoids. The
single electron transfer (SET) and the single proton-loss elec-
tron transfer (SPLET) were the antioxidant mechanism evaluated
through FRAP and ORAC assay, respectively (Firuzi, Lacanna,
Petrucci, Marrosu, & Saso, 2005; Zhang et al., 2013). Regarding
513
TPCa FRAPb ORACc
Total BF (%)
66.96 (6.70)d 44.930 ± 1.41d 215.40 ± 6.13d 109,505 ± 2569d
43.93 (4.39)c 35.076 ± 0.48c 147.28 ± 3.30c 102,373 ± 1254c
113.80 (11.40)e 70.770 ± 0.62e 327.61 ± 15.20e 175,834 ± 6609e
1.79 (0.18)a 11.660 ± 0.13a 130.74 ± 1.35b 21,670 ± 1285a
16.29 (1.63)b 19.190 ± 1.24b 94.053 ± 5.82a 48,588 ± 373b
the SET mechanism, the FRAP value obtained increased in
the following order: naringenin < apigenin < amentoflavone
< biapigenin < volkensiflavone < catechin < morelloflavone
< luteolin-7-O-glucosyde < fukugiside < luteolin and querce-
tin. The compounds naringenin, apigenin, amentoflavone,
biapigenin and volkensiflavone had a ferric reducing poten-
tial comparable to the reference compound used (Trolox).
Meanwhile, catechin, morelloflavone, luteolin-7-O-glucoside,
fukugiside, luteolin and quercetin had at least twice the ac-
tivity of Trolox. Regarding the flavonoid monomers, quercetin,
followed by luteolin and catechin, were the compounds with
the highest activity, whereas naringenin had the lowest re-
ducing capability. These results are in agreement with other
reports and confirm that the combination of the catechol group
in the B ring, the 3-hydroxy group, the 2,3-double bond and
the 4-oxo function in the C ring are the functional groups that
give the major contribution to effectively reducing the complex
[Fe-(TPTZ)2)]3+ (Firuzi et al., 2005). On the other hand, the results
showed that the activity of the biflavonoids is conducted mainly
for just one of its monomers. According to the ANOVA analy-
sis (Table 4), the flavonoid–flavonoid binding of biapigenin,
amentoflavone and volkensiflavone had a negligible synergis-
tic effect in comparison with their respective flavonoid
monomers (apigenin and naringenin; p-value > 0.05). Indeed,
an antagonistic effect was observed for the naringenin–
luteolin binding of morelloflavone, significantly decreasing the
activity of this compound in regard to the most active monomer
(luteolin, p-value < 0.05). This could be explained by a steric im-
pediment promoted by the naringenin monomer, which
decreases the effective reducing capability of luteolin against
the complex [Fe-(TPTZ)2)]3+.
In the SPLET mechanism, the results obtained were
significantly different (Table 4). The ORAC value obtained
for all compounds were at least 3-fold more than the
activity found by Trolox, in which luteolin-7-O-glucoside
was the flavonoid with the lowest activity, followed
by volkensiflavone < amentoflavone < apigenin < querce-
tin < luteolin < naringenin < morelloflavone < catechin < fukugiside
and biapigenin. According to these results, the main struc-
tural requirements for an effective reducing capability previously
observed does not seem to be the functional groups that give
the major contribution and explain the peroxyl radical-
scavenging activity of flavonoids and biflavonoids. In fact, under
514 Journal of Functional Foods 27 (2016) 503–516

Table 4 – Structure–antioxidant activity relationship (SAR) of G. madruno biflavonoids and related flavonoids by SPLET
and SET mechanism.
Compound Functional groups Antioxidant activity
R1 R2 R3 R4 R5 R6 R7 SPLET ORAC value* SET FRAP value*
Naringenin (Na) H H CO OH OH H OH 6.408 ± 0.206d
0.942 ± 0.014a
Apigenin (Ap) DB H CO OH OH H OH 5.198 ± 0.359b 0.939 ± 0.039a
Amentoflavone (Am) [Ap-(3′→8″)-Ap] DB H CO OH OH AP (C8) OH 5.063 ± 0.056b 0.953 ± 0.017a
Biapigenin (Bia) [Ap-(3→8″)-Ap] DB Ap (C8) CO OH OH H OH 11.070 ± 0.158g 1.027 ± 0.026ab
Volkenisflavone (Vo) [Na-(3→8″)-Ap] H Ap (C8) CO OH OH H OH 4.948 ± 0.302b 1.112 ± 0.021b
(+)-Catechin (Ca) H OH C OH OH OH OH 8.226 ± 0.293e 2.181 ± 0.087c
Morelloflavone (Mo) [Na-(3→8″)-Lu] H Lu (C8) CO OH OH H OH 7.898 ± 0.088e 2.373 ± 0.093d
Luteolin 7-O glycoside (Lu 7G) DB H CO OH O-Gly OH OH 2.863 ± 0.056a 2.661 ± 0.085e
Fukugiside (Fu) [Na-(3→8″)-Lu 7G] H Lu 7G (C8) CO OH OH H OH 9.511 ± 0.134f 2.914 ± 0.055f
Luteolin (Lu) DB H CO OH OH OH OH 5.819 ± 0.222c 3.160 ± 0.131g
Quercetin (Q) DB OH CO OH OH OH OH 5.358 ± 0.224b 3.766 ± 0.013h

Data are presented as mean ± SD (n = 3). Values within a column followed by the same superscript letters are not significantly different at the
0.05 level according to ANOVA. DB = double bond.
* Expressed as µmol Trolox equivalents/µmol compound.

this mechanism, naringenin instead of luteolin was the flavo-

noid monomer of G. madruno biflavonoids with the highest
activity found, whereas the biflavonoids were more active than
the flavonoid monomers, specifically the flavonoid–flavonoid
binding of biapigenin, morelloflavone and fukugiside, which sig-
nificantly enhances the activity in regard to its respective
monomers, promoting a synergistic effect that was not ob-
served by SET mechanism (Table 4). Nevertheless, a structure–
activity relationship based just in the functional groups applicable
to biflavonoids and related flavonoids could not be performed
with our results. Thus, a quantum chemical analysis focused
on structural and kinetic parameters of flavonoids and
biflavonoids is required for a better understanding of the peroxyl
radical-scavenging activity of these compounds by the SPLET
mechanism (Zhang et al., 2013).
4. Conclusion
This study allowed to characterize for the first time the chemi-
cal profile of G. madruno by HPLC–DAD–ESI/MSn. The results
obtained are the basis for future quality control and identity
chemical marker analysis for this species. Undoubtedly, our
results propose G. madruno as a prominent species of the Garcinia
genus on the expression of highly valuated metabolites. The
high content of biflavonoids, antioxidant activity, and the pres-
ence of hydroxycitric acid found mainly in the leaves and
epicarp could be the starting point for developing functional
ingredients for the food and pharmaceutical industries.
Acknowledgements
The authors thank the Comité para el Desarrollo de la
Investigación (CODI) of the University of Antioquia for the fi-
nancial support (Cod: CPT-1219). Luis Carrillo-Hormaza is
grateful to the National Doctoral Program of COLCIENCIAS (Cod:
647) for provision of a doctoral fellowship.
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