278 Eur. J. Lipid Sci. Technol.

104 (2002) 278–281

Dora Enith García Torresa, Antioxidant activity of macambo

Dalva Assunção
Portari Mancinib, (Theobroma bicolor L.) extracts
Rosângela Pavan Torresa,
Macambo is an edible fruit species from the Amazon Region with a high content of
Jorge Mancini-Filhoa
lipids (33%). Macambo samples were obtained from the Amazon Region, dried at
60 °C, milled (32 mesh sieve) and submitted to sequential extraction using ether,
a ethanol and water as solvents. The antioxidant activity of the ether, ethanol and aque-
Food Science and
Experimental Nutrition ous extracts was 47%, 68% and 67%, respectively, and the antioxidant activity of
Department, Faculty of the control butylated hydroxytoluene (BHT) was 64%. Three phenolic fractions were
Pharmaceutical Sciences, obtained with tetrahydrofuran: free phenolic acids, soluble and insoluble phenolic
University of São Paulo, esters. The antioxidant activity of these fractions was 83%, 85% and 93% respective-
São Paulo, Brazil ly, and the antioxidant activity of the control BHT was 64%. The phenolic acids were
b Scientific Development identified on the basis of relative retention times as compared with standards. The
Division, Butantan Institute, fractions obtained showed the presence of the following phenolic acids: salicylic, trans-
São Paulo, Brazil cinnamic, sinapinic, chlorogenic, protocatechinic, gallic, quinic, and p-hydroxibenzoic
acid. The presence of phenolic compounds in macambo fruit extracts may be respon-
sible for its rather high antioxidant activity and the results obtained suggest that
macambo extracts could be used as food antioxidants. Further studies are in progress
to analyze the use of its extracts and phenolic fractions in edible oil shelf life.

Keywords: Macambo, antioxidants, phenolic compounds, Theobroma bicolor L.

1 Introduction in food products, leading to their deterioration and rejec-

tion by consumers. Products of lipid oxidation influence
Macambo (Theobroma bicolor L.) is a member of the
other food constituents and can also cause pathological
Sterculiacea family originating from the Amazon Region
changes in the mucous membrane of the alimentary tract.
in South America. Its popular names are macambo in Pe-
They can inhibit the activity of enzymes and increase the
ru, cacau from Peru in Brazil, macambo and bacau in
content of peroxides in the blood serum [2]. Fatty acid
Colombia, and pataste in the other Latin-American coun-
peroxidation can be prevented by adding antioxidants to
tries. The macambo fruit is voluminous, ellipsoidal, mea-
Research Paper

the food. Antioxidants are regarded as compounds which

suring up to 35 cm in length and 15 cm in width and
are able to delay, retard or prevent oxidation processes.
weighing 3 kg. It is yellowish-brown in color when ripe. It
They can interfere with oxidation by reacting with free
has numerous seeds, an average of 40 per fruit which are
radicals, chelating catalytic metals and also by acting as
arranged in series and surrounded by a yellowish pulp. It
oxygen scavengers. Synthetic antioxidants such as butylat-
is fibrous and has an uncommon taste and aroma. The
ed hydroxyanisole (BHA), butylated hydroxytoluene (BHT)
fruit pulp is edible, and consumed in the natural state, but
and tert-butylhydroquinone (TBHQ) have been used to
it can also be used in the preparation of juices and ice
control lipid oxidation. Nowadays there is a keen interest
creams. The seeds are consumed boiled or roasted, and
in replacing the synthetic antioxidants by natural alterna-
are also used in cooking just as nuts and for the prepara-
tives found in plants because of the worldwide trend to-
tion of chocolate. Macambo fruit contains a high amount
wards the use of natural additives in foods [3, 4]. Natural
of lipids (33%), 42% being unsaturated fatty acids that
antioxidants can occur in all higher plants and in all of
can be easily oxidized [1]. The oxidation of lipids, which
their parts. They are usually phenolic or polyphenolic
occurs during raw material storage, processing and heat
compounds. Typical antioxidant molecules are deriva-
treatment, is one of the basic processes causing rancidity
tives or isomeric forms of polyphenols, flavones, iso-
flavones, flavonols, catechins, coumarin, tocopherols,
cinnamic acid, phosphatides, polyfunctional organic
acids, and others [5].
Correspondence: Jorge Mancini-Filho, Food Science and
Experimental Nutrition Department, Faculty of Pharmaceutical
Sciences, University of São Paulo, Av. Prof. Lineu Prestes, 580 –
The aim of the present study was to identify the phenolic
Bloco 14, São Paulo, 05508-900, Brazil. Phone: +5511-38183674, compounds in macambo fruit extracts and to evaluate
Fax: +5511-38154410; e-mail: [email protected] their antioxidant activity.

© WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 0931-5985/2002/0505-0278 $17.50+.50/0

Eur. J. Lipid Sci. Technol. 104 (2002) 278–281 Antioxidant activity of macambo extracts 279

2 Material and methods tion containing 100 ppm of pure BHT solely were added
to a series of tubes containing 5 ml of an emulsion of
Macambo fruits were obtained from Iquitos City, Peru, linoleic acid and β-carotene stabilized with Tween 60.
sufficiently dried and milled in order to permit their pas- Immediately after the addition of the extracts or BHT the
sage through a 32 mesh sieve, and then submitted to ex- zero-time absorbance at 470 nm was recorded. Samples
traction with ether, ethanol and water for 60 min under were kept in stoppered tubes placed in a water bath at
shaking at room temperature (approximately 23 °C). 50 °C and the absorbance was measured at 15 minute
Ether, alcohol and aqueous extracts were sequentially intervals for two hours with a Spectronic 20D apparatus
obtained from 20 g of fruit following the increasing polari- (Milton Roy Company).
ty of the solvent. Each extract was made up to 100 ml
with the corresponding solvent. The amount of dry mate- The antioxidant activity of the extracts, of BHT and of the
rial in each extract was determined gravimetrically [6]. extracts combined with BHT was calculated as percent-
age in comparison with the oxidation of linoleic acid and
The content of water, ash, protein as well as of insoluble
β-carotene without antioxidant (Blank). This system was
and soluble fiber was determined according to the official
also used for the evaluation of the antioxidant activity of
Methods of Analysis of the Association of Official Analyti-
the phenolic compound fractions.
cal Chemists (AOAC) [7]. By subtracting the percentage
value of water, protein, fat and ash from 100, the percent- The relationship between antioxidant activity and an ex-
age value of carbohydrate of the components was ob- tract/substrate quantity of 50 or 100 ppm was determined
tained [8] with insoluble and soluble fibers being excluded. with different concentrations of each extract using the
previously described methodology. The association of
Total lipids were extracted according to the method of
macambo extracts with a synthetic antioxidant (BHT) was
Folch et al. and determined gravimetrically after solvent
performed to evaluate the antioxidant activity.
evaporation [9]. Aliquots of the final lipid extracts were
used to prepare the fatty acid methyl esters (FAME) ac-
cording to the method of Hartman and Lago [10]. Analysis 3 Results and discussion
was performed with a SHIMADZU gas chromatograph
model 17 A equipped with a flame ionization detector and The percent composition of macambo fruit is shown in
a 30 m × 0.25 mm ID capillary column (Carbowax 20). Tab. 1; the amounts of lipids and protein were 32.95%
The temperature for operation was set initially at 150 °C, and 13.30%, respectively.
and programmed to increase to 210 °C at a rate of
3 °C/min. Standard mixtures of FAME were used to ob- The lipids at hand in macambo fruit were composed of
tain relative retention times and to identify the fatty acids the following fatty acids: palmitic (6.1%), heptadecenoic
in the macambo fruit. The helium carrier gas flow rate (0.2%), stearic (49.6%), arachidic (1.9%), oleic (39.9%),
was 1 ml/min. linoleic (2.1%) and linolenic acid (0.2%). The total amount
of unsaturated fatty acids was 42.2%. This elevated
According to the method of Krygier [11] three fractions amount of unsaturated fatty acids in macambo fruit sug-
were obtained with tetrahydrofuran in the fractionation of gested the existence of some protection mechanism
phenolic compounds: free phenolic acids, soluble pheno- against oxidation in the fruit itself (Tab. 2).
lic esters and insoluble phenolic esters, The analysis of
phenolic compounds was performed with a SHIMADZU Fractions with specific solubility in apolar and polar sol-
gas chromatograph model 17 A equipped with a flame vents were obtained through a sequential extraction
ionization detector and a 25 m × 0.25 mm ID DB-5 capil-
lary column (J&W). The temperature for operation was Tab. 1. Centesimal determination of macambo fruits (pulp
initially set at 112 °C, and programmed to increase to and seed).
290 °C in steps of 10 °C/min. Standard mixtures of phe-
Determination Content [%]
nolic compounds were obtained from SIGMA® and used
to obtain the relative retention times and to identify the Water 35.58 ± 0.18*
phenolic acids in the macambo extracts. The helium car- Lipids 32.95 ± 0.23
rier gas flow rate was 2 ml/min. Ash 0.84 ± 0.02
Protein 13.30 ± 0.06
The antioxidant activity of macambo fruit extracts was Insoluble fiber 9.90 ± 0.12
evaluated using the β-carotene plus linoleic acid model Soluble fiber 2.30 ± 0.00
system as described by Marco [12]. The extract solution Carbohydrate** 5.13 ± 0.37
(0.2 ml) containing 100 ppm or 50 ppm of dry matter as- ** Standard deviation.
sociated with 50 ppm of pure BHT, and 0.2 ml of a solu- ** By difference.
280 Torres et al. Eur. J. Lipid Sci. Technol. 104 (2002) 278–281

Tab. 2. Fatty acid composition of fat from macambo fruit. ity. Moure et al. worked with Genuina avellana, a native
Chilean oilseed, using solvents with different polarity to
Fatty acid Content [%]
extract total phenolics [14]. The authors observed that the
C16:0 6.1 ± 0.04* alcohol extract was more active in the β-carotene assay,
C17:0 0.2 ± 0.01 while the ethanol and methanol extracts were the most
C18:0 49.6 ± 0.08 active substances in terms of hydrogen radical scaveng-
C20:0 1.9 ± 0.01 ing activity. The aqueous and methanol extracts inhibited
Σ Saturated fatty acids 57.8±
±
the oxidation of soybean oil more efficiently at 70 °C and
C18:1 39.9 ± 0.30 80 °C, respectively.
C18:2 2,1 ± 0.00
C18:3 0.2 ± 0.01 As shown in Tab. 3, the combination of the extracts
Σ Unsaturated fatty acids 42.2
± (50 ppm) with BHT (50 ppm) showed a higher antioxidant
* Standard deviation. activity than the extracts (100 ppm) or BHT (100 ppm)
alone. These results demonstrate the potential effect of
processes. Extraction with ethyl ether released apolar the antioxidant when BHT is combined with the extracts.
compounds, ethanol extraction yielded compounds with
intermediate polarity, and water extraction yielded com- Phenolic compound fractionation was performed with
pounds of high polarity [6]. The final volume of each ex- tetrahydrofuran and resulted in three fractions. The dry
tract was 100 ml. The total dry matter was 54.2 mg/ml, matter of free phenolic acids (FPA), soluble phenolic es-
3.2 mg/ml and 5.3 mg/ml for the ether, alcohol and aque- ters (SPE) and insoluble phenolic esters (IPE) contents
ous extracts, respectively (Tab. 3). Ya-Lun et al., working were 2.8 mg/ml, 5.0 mg/ml and 1.0 mg/ml, respectively,
with Scutellaria rehderiana, obtained extracts with hexa- as shown in Tab. 4. The antioxidant activity of these frac-
ne, acetone and methanol by sequential extraction [13] tions (100 ppm) is shown in Tab. 4. FPA, SPE and IPE
and observed higher antioxidant activity in the acetone showed 82.58%, 85.02% and 93.10% inhibition, respec-
extract than in the hexane and methanol extracts. tively, while the synthetic antioxidant BHT (100 ppm)
showed 70.46% inhibition. When associated with BHT
The antioxidant activity was evaluated with the β-carotene/ (50 ppm), each phenolic fraction (50 ppm) showed syner-
linoleic acid system. This spectrophotometric method is gistic antioxidant effects.
based on the ability of the different extracts to decrease
oxidative losses of β-carotene in an emulsion [6]. The chromatographic analysis of phenolic acids in the
fractions of free phenolic acids, soluble phenolic acids
The antioxidant activities of the extracts are shown and insoluble phenolic esters showed the following phe-
in Tab. 3. The ether, alcohol and aqueous extracts nolic acids: salicylic, trans-cinnamic, p-hydroxybenzoic,
(100 ppm of dry matter) showed 47.10%, 68.21%, and protocatequinin, gallic, synaptic, catequinic, and chloro-
67.02% antioxidant activity, respectively, while pure BHT genic acid. Tab. 5 shows that the amount of salicylic acid
at the same concentration showed 64% antioxidant activ- (94%) was the highest of all phenolic acids in all three

Tab. 3. Antioxidant activity of the ether, alcohol and aqueous macambo extracts and of the extract combined with BHT.
Extract 100 ppm BHT (100 ppm) 50 ppm BHT + 50 ppm Extract Dry matter [mg/ml]
Ether 47.10 64.00 72.41 54.2 ± 0.04**
Alcohol 68.20 64.00 85.96 3.2 ± 0.01**
Aqueous 67.02 64.00 81.85 5.3 ± 0.01**
** Inhibition oxidation [%].
** Standard deviation.

Tab. 4. Antioxidant activity* of phenolic fractions from macambo and of the fraction combined with BHT and dry matter.
Fraction** 100 ppm BHT 100 ppm Fraction 50 ppm + BHT 50 ppm Dry matter [mg/ml]
FPA 82.58 70.46 89.40 2.8 ± 0.03
SPE 85.02 70.46 85.23 5.0 ± 0.05
IPE 93.10 70.46 91.71 1.0 ± 0.00
** Inhibition oxidation [%].
** FPA – free phenolic acids, PE – soluble phenolic esters, IPE – insoluble phenolic esters.
Eur. J. Lipid Sci. Technol. 104 (2002) 278–281 Antioxidant activity of macambo extracts 281

Tab. 5. Phenolic acids in fractions from macambo fruits*. Acknowledgements

Phenolic acids Composition [%] We acknowledge FAPESP for financial support and
CAPES – PEC/PG and CNPq for fellowships.
FPA SPE IPE
Salicylic 94.88 94.72 93.76
References
Trans-cinnamic 0.35 0.18 ND***
Pi-hydroxybenzoic 0.14 0.23 0.17 [1] S. Flores Paytan: Cultivo de frutales nativos amazónicos:
Protocatequinic ND 3.56 ND manual para el extensionista. Secretaría Pro Tempore,
Gallic ND 0.17 0.39 Tratado de Cooperación Amazónica, Lima (Peru) 1997.
Synaptic 1.46 0.18 0.7 [2] A. Koresh, A. M. Youdim, A. J. James: Incorporation of el-
Catequinic 0.18 ND ND derberry anthocyanins by endothelial cells increases pro-
Chlorogenic 0.25 0.30 ND tection against oxidative stress. Free Radical Biol. Med. 9
NI** 2.74 0.66 4.45 (2000) 51–60.
[3] R. Amarowicz, M. Naczk, F. Shahidi: Antioxidant activity of
*** FPA – free phenolic acids, SPS – soluble phenolic esters,
crude tannins of canola and rapeseed hulls. J. Am. Oil
IPE – insoluble phenolic esters.
Chem. Soc. 77 (2000) 957–961.
*** NI – not identified.
*** ND – not detected. [4] C. Sanchez-Moreno, M. T. Satué-Gracia, E. N. Frankel:
Antioxidant activity of selected Spanish wine in corn oil
emulsions. J. Agric. Food Chem. 48 (2000) 5581–5587.
fractions. The presence of phenolic compounds in [5] E. M. Marinova, N .V. Yanisaleva: Effect of lipid unsaturation
macambo fruit is related to its antioxidant activity. on the antioxidative activity of some phenolic acids. J. Am.
Oil Chem. Soc. 71 (1994) 427–447.
The global effect of antioxidants on the decrease in ab- [6] M. O. S. O. M. Melo, J. Mancini-Filho: Antioxidantes natu-
sorbance at 470 nm of the ether, alcohol and aqueous rais da castanha do Brasil. Ciênc. Tecnol. Aliment. 11
macambo extracts was used to evaluate the antioxidant (1991) 263–271.
activity. The alcohol and aqueous extracts inhibited the [7] Association of Official Analytical Chemists (AOAC): Official
oxidative process up to 68% and 67%, respectively, while Methods of Analysis. Washington (USA) 1998.
the control sample, BHT, inhibited it by 64%. [8] D. A. T. Sonthgate, H. Greenfield: Food composition data –
Production, Management and Use. Chapman & Hall, Wein-
The antioxidant activity of the extracts depends on the heim (Germany) 1992.
solvent, the concentration of the extract and the antioxi- [9] J. Folch, M. Lees, G. H. S. Stanley: A simple method for the
dant assay. Acute differences in activity are due to differ- isolation and purification of total lipids. J. Biol. Chem. 226
ences in the relative partition between phases in different (1957) 497–509.
polar systems. Assaying the antioxidant activity by the [10] L. Hartman, B. C. A. Lago: Rapid preparation of fatty acid
β-carotene method showed, as the main result, that the methyl esters from lipids. Lab. Pract. 22 (1973) 475–477.
higher the extract concentration, the higher the antioxi- [11] K. Krygier, F. W. Sosulski, L. Hogge: Free, esterified and in-
soluble-bound phenolic acids. I. Extraction and purification
dant activity coefficient [14]. This property was observed
procedure. J. Agric. Food Chem. 30 (1982) 330–334.
for several antioxidants by other authors [15–17].
[12] G. J. A. Marco: Rapid method for evaluation of antioxidants.
Further investigation of antioxidant activity of macambo J. Am. Oil. Chem. Soc. 45 (1968) 594–598.
fruit in food systems is currently underway. [13] S. Ya-Lun, K. L. Lai, B. Yu-Rong, Y. Huang, C. Zhen-Yu:
Antioxidant activity of flavonoids isolated from Scutellaria
rehderiana. J. Am. Oil Chem. Soc. 77 (2000) 807–812.
4 Conclusions [14] A. Moure, D. Franco, J. Sineiro, H. Dominguez, M. J. Nunez,
J. M. Lema: Evaluation of extracts from Gevuina avellana
● The reported data indicated that ether, alcohol and hulls as antioxidants. J. Agric. Food Chem. 48 (2000) 3890–
aqueous macambo extracts possess antioxidant activ- 3897.
ity that can be measured by the β-carotene/linoleic [15] O. I. Aruoma, J. P. E. Spencer, D. Warren, P. Jenner, J. But-
acid system. ler, B. Halliwell: Characterization of food antioxidants, illus-
● The fractionation of phenolic compounds from macam- trated using commercial garlic and ginger preparation.
Food Chem. 60 (1997) 149–156.
bo fruit yielded three fractions with antioxidant activity.
The combination of these with BHT showed a higher [16] M. Karamac, R. Amarowicz: Actividad antioxidante de BHA,
BHT y TBHQ examinada con el test de Miller. Grasas y
effect in terms of antioxidant activity. aceites 48(2) (1997) 83–86.
● The three phenolic fractions contained several pheno-
[17] A. Von Gadow, E. Joubert, C. F. Hansmann: Comparison of
lic acids, and salicylic acid was present in all three frac- the antioxidant activity of aspalathin with that of other plant
tions in larger quantities than the other compounds. phenols of rooibos (Aspalathus linearis), α-tocopherol,
The phenolic acids are related to the antioxidant activ- BHT, and BHA. J. Agric. Food Chem. 45 (1997) 632–638.
ity of macambo extracts. [Received: August 23, 2001; accepted: February 20, 2002]