Isolation and quantitative analysis of phenolic antioxidants, free sugars, and polyols from mango (Mangifera indica L.) stem bark aqueous decoction used in Cuba as a nutritional supplement.

Abstract

An aqueous decoction of mango (Mangifera indica L.) stem bark has been developed in Cuba on an industrial scale to be used as a nutritional supplement, cosmetic, and phytomedicine. Previously we reported its antioxidant activity, and we concluded that the product could be useful to prevent the production of reactive oxygen species and oxidative tissue damage in vivo. A phytochemical investigation of mango stem bark extract has led to the isolation of seven phenolic constituents: gallic acid, 3,4-dihydroxy benzoic acid, gallic acid methyl ester, gallic acid propyl ester, mangiferin, (+)-catechin, (-)-epicatechin, and benzoic acid and benzoic acid propyl ester. All structures were elucidated by ES-MS and NMR spectroscopic methods. Quantitative analysis of the compounds has been performed by HPLC, and mangiferin was found to be the predominant component. Total polyphenols were assayed also by the Folin-Ciocalteu method. The free sugars and polyols content was also determined by GC-MS.

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Isolation and Quantitative Analysis of Phenolic Antioxidants,
Free Sugars, and Polyols fromMango (Mangifera indica L.)
StemBark Aqueous Decoction Used in Cuba as a Nutritional
Supplement
ALBERTO J. NU Ä N ˜EZ SELLE ÄS,†HERMAN T. VE ÄLEZ CASTRO,†
JUAN AGU ¨ERO-AGU ¨ERO,†JOHANES GONZA Ä LEZ-GONZA Ä LEZ,†FABIO NADDEO,‡
FRANCESCO DE SIMONE,‡AND LUCA RASTRELLI*,‡
Centro de Quı ´mica Farmace ´utica, Calle 200 y 21, Atabey, Apdo. 16042, CP 11600, Ciudad de La
Habana, Cuba, and Dipartimento di Scienze Farmaceutiche, Universita ` degli Studi di Salerno,
Via Ponte don Melillo, 84084, Fisciano (SA), Italy
An aqueous decoction of mango (Mangifera indica L.) stem bark has been developed in Cuba on an
industrial scale to be used as a nutritional supplement, cosmetic, and phytomedicine. Previously we
reported its antioxidant activity, and we concluded that the product could be useful to prevent the
production of reactive oxygen species and oxidative tissue damage in vivo. A phytochemical
investigation of mango stem bark extract has led to the isolation of seven phenolic constituents:
gallic acid, 3,4-dihydroxy benzoic acid, gallic acid methyl ester, gallic acid propyl ester, mangiferin,
(+)-catechin, (-)-epicatechin, and benzoic acid and benzoic acid propyl ester. All structures were
elucidated by ES-MS and NMR spectroscopic methods. Quantitative analysis of the compounds
has been performed by HPLC, and mangiferin was found to be the predominant component. Total
polyphenols were assayed also by the Folin-Ciocalteu method. The free sugars and polyols content
was also determined by GC-MS.
KEYWORDS:
sugars; polyols; antioxidant activity; 1D and 2D NMR; HPLC; GC-MS
Mangifera indica L.; nutritional supplement; phytomedicine; phenolic constituents; free
INTRODUCTION
Mango (Mangifera indica L.), which belongs to the family
Anacardiaceae, is widely found in Cuba and in many other
tropical and sub-tropical regions, and is one of the most popular
edible fruits in the world. Mango stem bark has been tradition-
ally used for the treatment of menorrhagia, scabies, diarrhea,
syphilis, diabetes, cutaneous infections, and anemia, using an
aqueous extract obtained by decoction as reported in the
Napralert Data Base (1). An extract of this plant has been
developed in Cuba on an industrial scale to be used as a
nutritional supplement, cosmetic, and phytomedicine (2). The
industrial extract obtained by decoction and drying of mango
stem bark is a homogeneous brown powder which melts with
decomposition from 215 to 218 °C and has a particle size of
30-60 µm. Previously, we compared the protective abilities of
the following at the indicated concentrations: extract, 50-250
mg/kg; mangiferin, 50 mg/kg; vitamin C, 100 mg/kg; vitamin
E, 100 mg/kg; and vitamin C + E, 100 mg/kg each; and
?-carotene, 50 mg/kg body weight, respectively, in mice, against
the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxida-
tive damage in serum, liver, and brain, as well as in the hyper-
production of reactive oxygen species (ROS) by peritoneal
macrophages. We concluded that mango stem bark extract could
be useful to prevent the production of ROS and oxidative tissue
damage in vivo, and it is more active than vitamin C, vitamin
E, mangiferin, and ?-carotene (3).
In this study, we report the isolation, structure characteriza-
tion, and HPLC quantitative determination of phenolic con-
stituents from the n-BuOH soluble portion of Mangifera indica
stem bark. In the past few years there has been an increased
interest in the study of mango phenolics, due to their antioxidant
activity and health-promoting properties that make consumption
of fruits and derived processed products from fruits, seed kernel,
and stem bark a very healthy habit. Previously, alkylgallates
were isolated from the blossoms of M. indica (4), whereas
galloyl, hydroxybenzoyl esters, and epicatechin were isolated
from the leaves (5). Saleh et al. (6) reported the polyphenolic
composition of leaves, twigs, fruits, and seeds. The phenolic
composition of mango stem bark is reported here for the first
time. Quantitative determination of phenolics was essential for
estimating their content in the extract and for quality control
* To whom correspondence should be addressed. Phone: 39 89 964356.
Fax: 39 89 964356. E-mail: rastrelli@unisa.it
†Centro de Quı ´mica Farmace ´utica.
‡Universita ` degli Studi di Salerno.
762 J. Agric. Food Chem . 2002, 50, 762−766
10.1021/jf011064b CCC: $22.00©2002 Am erican Chem ical Society
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purposes. Quantitative evaluation of free sugars and polyols from
the water-soluble fraction was also performed by GC-MS.
MATERIAL AND METHODS
Chemicals. All of the pure standards were purchased from Sigma
Chemicals (Milan, Italy). All organic solvents were products of Carlo
Erba, Milano (Italy). Water was purified by a Milli-Qplussystem from
Millipore (Milford, MA).
Plant Material. The stem bark of Mangifera indica L. (Anacardi-
aceae) was collected from plants grown in a fruit farm (Alquizar,
Havana, January, 1998) without affecting the ecosystem. Bark was
collected free of microbial contamination and subsequently dried and
milled to obtain particles of around 5 cm. The industrial extract obtained
by decoction and drying of the same plant material, hereafter coded as
Vimang, was a homogeneous brown powder, which melted from 215
to 218 °C with decomposition, with a particle size of 30-60 µm, and
was provided by the Center of Pharmaceutical Chemistry, Havana,
Cuba.
Preparative Reversed-Phase (RP) HPLC Condition. Separations
were performed on a Waters 590 series pumping system equipped with
a Waters R401 refractive index detector, a U6K injector, and a Waters
µ-Bondapak C-18 column (30 cm × 7.8 mm i.d., 4 mm) using MeOH-
H2O (30:70 v/v, flow rate 2.5 mL/min) to yield compounds 1 (8.5 mg,
Rt) 10.1 min), 2 (9.0 mg, Rt) 11.8 min), 3 (8.1 mg, Rt) 12.7 min),
4 (18.4 mg, Rt) 20.6 min), 5 (21.5 mg, Rt) 28.4 min), 6 (323.1 mg,
Rt ) 33.3 min), 7 (66.1 mg, Rt ) 39.4 min), 8 (36.4 mg, Rt ) 40.2
min), and 9 (20.1 mg, Rt) 41.2 min).
Sample Preparation for Analytical Determination. For sample
preparation, 500 mg of pulverized plant material was partitioned twice
with 80 mL of n-BuOH/H2O (1:1, v/v) at room temperature. The
aqueous extract contained only free sugars and polyols that were
analyzed by GC-MS. A portion (3 mg) of the dried BuOH-soluble
material (87 mg) was diluted to a volume of 3 mL (1 mg/mL) in a
volumetric flask. Standard solutions containing 1 mg/mL of gallic acid,
(-)-epicatechin, and benzoic acid in methanol were also prepared.
Quantitative HPLC Conditions. HPLC analyses were performed
on a Waters 600 E-Multisolvent Delivery System liquid chromatograph,
equipped with a U6-K injector (fitted with a 20-µL loop), a Waters
486 tunable UV-Vis spectrophotometric detector, and a Waters 746-
data module integrator. Following are the operating conditions used.
The column was a C18 µ-Bondapak (300 × 4.0 mm i.d.). Chromato-
graphic separation was carried out using isocratic elution with two
solvents [A ) acetonitrile; B ) water/2% acetic acid (98:2 v/v)] in the
ratio 15:85 (v/v). Detection wavelength was 278 nm. Flow rate was
0.7 mL/min, and the injection volume was 10 µL. The tR values for
gallic acid (1), 3,4-dihydroxy benzoic acid (2), benzoic acid (3), methyl
gallate (4), propyl gallate (5), mangiferin (6), (+)-catechin (7), (-)-
epicatechin (8), and propyl benzoate (9) were 3.40, 3.98, 4.30, 6.95,
9.61, 11.25, 13.65, 16.17, and 22.02 min, respectively. Gallic acid, 3,4-
dihydroxy benzoic acid, benzoic acid, methyl gallate, (+)-catechin, and
(-)-epicatechin, for which standards were available, were identified
by chromatograms according to their retention times. Propyl gallate,
mangiferin, and propyl benzoate were identified by comparing the
retention times of the peaks in the extracts with those of the same
compounds previously isolated and characterized by NMR analysis.
Quantitation. Quantitation was performed by reporting the measured
integration area in the calibration equation of the corresponding
standard. Gallic acid, 3,4-dihydroxy benzoic acid, methyl gallate, and
propyl gallate were assayed as gallic acid equivalent. Mangiferin, (+)-
catechin, and (-)-epicatechin were assayed as (-)-epicatechin equiva-
lent. Benzoic acid and propyl benzoate were assayed as benzoic acid
equivalent.
Linearity. Linearity was determined for standards and for the
constituents 1-9 in Vimang. Linearity of responses was determined
on six levels of concentration with three injections for each level. A
linear relationship between peak area and concentration (5-25 µg/mL)
was observed for each standard with a correlation coefficient r )
0.9997. The minimum detection limit was 0.2 ng, which resulted in a
signal-to-noise ratio of 3:1.
Reproducibility. The reproducibility of the injection integration
procedure was determined for standards and for the Vimang constituents
1-9. The solutions were injected 10 times and the relative standard
deviations were calculated (gallic acid, 1.01%; 3,4-dihydroxy benzoic
acid, 0.98%; benzoic acid, 1.04%; methyl gallate, 2.5%; propyl gallate,
1.7%; mangiferin, 1.9%; (+)-catechin, 3.2%; (-)-epicatechin, 4.9%;
and propyl benzoate, 2.6%). Relative standard deviation for retention
times was less than 1%.
Repeatability. To evaluate the repeatability of the method, three
solutions at different concentrations (0.7 mg/mL, 1.0 mg/mL, and 1.3
mg/mL of dry extract in methanol) of Vimang BuOH extract were
prepared; each solution was injected three times. The contents of
constituents 1-9 were calculated in order to estimate the RSD (gallic
acid, 1.12%; 3,4-dihydroxy benzoic acid, 1.93%; benzoic acid, 2.43%;
methyl gallate, 2.01%; propyl gallate, 1.64%; mangiferin, 3.11%; (+)-
catechin, 1.87%; (-)-epicatechin, 1.23%; and propyl benzoate, 2.32%).
Total Polyphenols Assay. Estimation of the global polyphenol
content in the extracts was performed by the Folin-Ciocalteu method.
Vimang (1.15 mg) was dissolved in MeOH (2 mL), and the extract
was diluted 10-fold with water. Folin-Ciocalteu reagent (0.5 mL;
Merck) was added to the diluted solutions (0.5 mL), then 0.5 mL of a
100 g/L solution of Na2CO3was added. The absorbance was measured
at 720 nm (Shimadzu UV-2101 spectrophotometer) with a blank sample
(water plus reagents) in the reference cell. Quantification was obtained
by reporting the absorbances in the calibration curve of gallic acid used
as standard phenol.
GC-MS Analysis of Free Sugars and Polyols. The aqueous
extract, obtained as described above, was dried, and 10 mg of residue
was diluted with 40 µL of dry pyridine. The diluted residue was then
directly silylated at 80 °C for 30 min with 50 µL of N,O-bis-
(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethyl-
chlorosilane as catalyst. The TMS derivatives were kept in isooctane
and analyzed by GC-MS as follows: Shimadzu MS-QP5050 system;
column, DB1 J&W, 25m × 0.2 mm i.d., 0.33 µm; injection, 1 µL;
split ratio, 2; oven temperature, 100° for 1 min, to 180 °C at 4 °C/min,
then to 290 °C at 10 °C/min, and held for 20 min.; injector temperature,
290 °C; detector EMV 1.35-1.5 kV; carrier gas, helium; flow, 1 mL/
min. Individual sugars and polyols were identified by comparison of
MS spectra and retention times of peaks with those of TMS derivatives
of authentic compounds. The results represents the mean ( SD of 10
determinations. The concentrations of each compound in the extract
were calculated from the experimental peak areas by interpolation to
standard calibration curves.
Spectroscopic Apparatus. The ES-MS spectra were determined
on a Fisons Platform spectrometer both in the positive (90 V) and
negative (100 V) modes. The sample was dissolved in MeOH and
injected directly.
UV spectra were measured with a HP 8472-A spectrometer in
MeOH, (c ) 1) and IR spectra with a Nicolet Impact 400, in KBr.
A Bruker DRX-600 spectrometer, operating at 599.19 MHz for1H
and 150.858 for13C, with the UXNMR software package, was used
for NMR experiments measured in CD3OD. The DEPT experiments
were performed using a transfer pulse of 135° to obtain positive signals
for CH and CH3and negative ones for CH2. Polarization transfer delays
were adjusted to an average CH coupling of 135 Hz.1H-1H DQF-
COSY (7),
obtained using the conventional pulse sequences as described in the
literature, and 1D TOCSY (10) was acquired using waveform generator-
based GAUSS shaped pulse, mixing time ranging from 80 to 100 ms,
and a MLEV-17 spin-lock field of 10 kHz was preceded by a 2 ms
trim pulse. CD measurement was performed on a Jasco J-7140
spectropolarimeter.
Gallic Acid (1). White amorphous solid; ES-MS, m/z (rel int.) (100
V, negative mode): 169 [M-H]-(100).
3,4-Dihydroxy Benzoic Acid (2). White amorphous solid; ES-MS,
m/z (rel int.) (100 V, negative mode): 153 [M-H]-(100).
Benzoic Acid (3). White amorphous solid; ES-MS, m/z (rel int.)
(100 V, negative mode): 121 [M-H]-(100).
Methyl Gallate (4). White amorphous solid; ES-MS, m/z (rel int.)
(100 V, negative mode): 183 [M-H]-(100).
1H-13C HSQC, and HMBC (8, 9) experiments were
Constituents of Mango StemBark Nutritional Supplem entJ. Agric. Food Chem ., Vol. 50, No. 4, 2002 763
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Propyl Gallate (5). White amorphous solid; ES-MS, m/z (rel int.)
(100 V, negative mode): 211 [M-H]-(100).
Mangiferin (6). Colorless amorphous solid; UV λ max (MeOH):
237, 254; 268, 312, 364; +KOH: 235, 268, 298, 341; +AlCl3: 235,
268 321sh; 352, 392; +AlCl3+ HCl: 226, 258, 276sh, 316, 330 395;
+ NaOAc: 237, 263, 301sh. ES-MS, m/z (rel int.) (100 V, negative
mode): 421 [M-H]-(100).
(+)-Catechin (7). Colorless amorphous solid; ES-MS, m/z (rel int.)
(100 V, negative mode): 289 [M-H]-(100).
(-)-Epicatechin (8). Colorless amorphous solid; ES-MS, m/z (rel
int.) (100 V, negative mode): 289 [M-H]-(100).
Benzoic Acid Propyl Ester (9). White amorphous solid; EI-MS,
m/z (rel int.) (100 V, negative mode): 163 [M-H]-(100).
RESULTS AND DISCUSSION
Vimang obtained by decoction and drying of Mangifera
indica L. stem bark was extracted at room temperature with
MeOH. The MeOH extract (5 g) was partitioned between
n-BuOH and H2O to afford an n-BuOH soluble portion (0.87
g) which was submitted to preparative reversed-phase HPLC
to yield phenolic compounds gallic acid (1), 3,4-dihydroxy
benzoic acid (2), benzoic acid (3), methyl gallate (4), propyl
gallate (5), mangiferin (6), (+)-catechin (7), (-)-epicatechin (8),
and propyl benzoate (9) (Figure 1). The structures and molecular
formulas of compounds 1-9 were determined from their ES-
MS spectra, as well as from 1D and 2D1H and13C NMR data.
Compound 1 was identified as gallic acid from the1H NMR
spectrum and further confirmed by comparison of its chromato-
graphic behavior with that of an authentic sample. Compounds
2 and 3 showed spectroscopic data identical with those of 3,4-
dihydroxy benzoic acid and benzoic acid, respectively. The
NMR spectra of compounds 4 and 5 also showed the presence
of a gallic acid moiety, but in addition there was a methyl ester
signal in 4, and resonances for a propyl ester in 5. Compounds
4 was methyl gallate and 5 was propyl gallate. This assignment
was also corroborated by comparison with published data (3).
Compound 6 was the most abundant phenolic and the13C
NMR spectrum showed 19 carbon atom signals. The presence
of a glycosyl moiety was clearly suggested from the analysis
of the 2D COSY, HSQC, and HMBC experiments. The sugar
was identified as a ?-D-glucopyranose on the basis of its1H
and13C NMR data (11). The mass spectra did not show the
usual fragmentation pattern for O-glycoside derivatives, and
these data together with the chemical shifts of H-1 (δ 4.86)
and C-1 (δ 73.4) unambiguously showed the C-linkage of the
sugar.1H and13C NMR chemical shifts of the aglycon were in
accordance with those reported in the literature for tetrahydroxy-
xanthones (12). The glycosidic linkage of 6 was determined to
be at the C-4 position based on the cross-peaks due to3J long-
range coupling between the anomeric-H (δ 4.86, H-1) and C-2
(δ 107.9), C-1 (162.1), and C-3 (164.1) in the HMBC spectrum.
From these considerations the structure of mangiferin (13) was
assigned to 6.
Compounds 7 and 8 were identified as catechin and epicat-
echin by HPLC comparison with authentic samples, and their
identities were also apparent from their13C NMR spectra which
showed similar carbon signals for the phloroglucinol A-ring and
catechol B-ring, but slight difference in signals for the pyran
ring. The absolute configuration of C-2 and C-3 for (+)-catechin
and (-)-epicatechin were deduced as 2S from the CD spec-
troscopy measurement in comparison with the data reported in
the literature (14).
Compound 9 was identified easily as propyl benzoate from
its NMR and also by comparison with compound 5.
The quantitative analysis of the phenolic compounds from
mango stem bark was performed by HPLC. The concentrations
of each compound in the extract, calculated from the experi-
mental peak areas by interpolation to standard calibration curves,
Figure1. Com pounds isolatedfromMangiferaindicaL. stembarkextract: gallic acid(1), 3,4-dihydroxybenzoic acid(2), benzoic acid(3), m ethylgallate
(4), propyl gallate (5), m angiferin (6), (+)-catechin (7), (−)-epicatechin (8), and propyl benzoate (9). Glc ) ?-D-glucopyranosyl.
764 J. Agric. Food Chem ., Vol. 50, No. 4, 2002Nu ´ n ˜ ez Selle ´ s et al.
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were 1.12% for compound 1, 1.30% for 2, 1.14% for compound
3, 2.56% for 4, 2.74% for 5, 41.06% for 6, 7.52% for 7, 4.64%
for 8, and 2.29% for 9, corresponding to 208.0 mg/100 g dry
weight for compound 1, 226.2 mg for 2, 198.6 mg for 3, 445.2
mg for 4, 476.2 mg for 5, 7140.1 mg for 6, 1308.0 mg for 7,
807.4 mg for 8, and 398.7 mg for 9. The sum of all phenolic
compound concentrations obtained by quantitative HPLC (10.61
g/100 g dry weight) was compared with the results obtained
from the Folin-Ciocalteu assays, generally considered as the
method of choice to estimate total phenol contents in plant
extracts (15). No significant difference was found between the
two methods, as the total polyphenols in Vimang determined
by the Folin-Ciocalteu method, and expressed as gallic acid
equivalents, was 9.4 g/100 g dry weight.
The aqueous extract, containing free sugars and polyols, was
also analyzed by GC-MS. The major sugars were galactose,
glucose, and arabinose (839.7, 495.6, and 483.7 mg/100 g dry
weight, respectively); fructose was detected at the low concen-
tration of 155.9 mg/100 g. Additionally, Vimang contained three
polyols: sorbitol, present in appreciable quantities (685.2 mg),
myoinositol (303.2 mg), and xylitol (52.5 mg/100 g dry weight).
Vimang contained a variety of polyphenols, which included
phenolic acids (gallic acid, 3,4-dihydroxy benzoic acid, benzoic
acid), phenolic esters (methyl gallate, propyl gallate, propyl
benzoate), flavan-3-ols (catechin and epicatechin), and a xan-
thone (mangiferin). These phenolics made up approximately
11% of Vimang. The phenolic composition of mango stem bark
is reported here for the first time.
There is now much interest in polyphenolic products of the
plant phenylpropanoid pathway as they have considerable
antioxidant activity in vitro and are ubiquitous in our diet. Rich
sources include tea, wine, fruits and vegetables (although levels
are affected by species), light, degree of ripeness, processing,
and storage. There is little convincing epidemiological evidence
that intakes of polyphenols are inversely related to the incidence
of cancer. In contrast, numerous cell culture and animal models
indicate potent anticarcinogenic activity by certain polyphenols
mediated through a range of mechanisms including antioxidant
activity, enzyme modulation, gene expression, apoptosis, up-
regulation of gap junction communication and P-glycoprotein
activation (16).
In a previous work, we tested mango bark extract against
vitamins (C, E, and C + E) and ?-carotene in vivo, in terms of
lipid peroxidation inhibition (LPI) and protection to oxidative
damage (POD). LPI values for Vimang were similar to those
of vitamins in plasma, significantly higher than vitamin C and
?-carotene in liver, and similar to the combination of vitamins
C + E in brain. POD values for Vimang were significantly
higher than those for all products tested in plasma, macrophages,
liver, and brain, being the most significant difference in brain
(3).
We also evaluated the protective abilities of Vimang against
the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxida-
tive damage in blood serum, liver, and brain, as well as in the
hyper-production of reactive oxygen species (ROS) by peritoneal
macrophages (3).
Mangiferin, the predominant component (7%), has been
reported to have multiple biological effects such as hypolipi-
demic activity in cholesterol-fed mice (17), antidiabetic activity
(18), inhibitory effects in bowel carcinogenesis of male F344
rats (19), and antitumor, immunomodulatory, and anti-HIV
effect (20). Its mechanism as an anti-oxidant has been described
elsewhere (21). The metabolism of mangiferin yields norathyriol
after cleavage of the C-C linkage of the glucose moiety, which
exhibits a potent iron chelating effect, and an inhibitory effect
of the induced-respiratory burst in rat neutrophils (22). In our
experiments, mangiferin did not show higher biological activity
than the whole extract; and we therefore hypothesized that the
strong antioxidant activity of the mango stem bark extract may
be due to the presence of a combination of polyphenolic
compounds such as catechins, and galloyl esters derivatives,
known for their antioxidant activities (23, 24), together with
important microelements (copper, zinc, and selenium) deter-
mined by ICP spectrometric techniques in a previous study (25).
Those elements play important roles in the activity of enzymes
involved in the endogenous antioxidant defense mechanism in
the human organism, such as superoxide dismutase (SOD) and
gluthation peroxidase (GPx). The mineral content of mango stem
bark extract was 40 mg/100 g dry weight.
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