technologies

Article
Proximate Composition, Extraction, and Purification
of Theobromine from Cacao Pod Husk
(Theobroma Cacao L.)
Van Tang Nguyen 1,2, * and Nghia Huu Nguyen 3
1 School of Environmental and Life Sciences, Faculty of Science and Information Technology,
University of Newcastle, Ourimbah, NSW 2258, Australia
2 Department of Food Technology, Faculty of Food Technology, Nha Trang University,
No. 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa 8458, Vietnam
3 Department of Thermal and Refrigeration Technology, Faculty of Mechanical Engineering,
Nha Trang University, No. 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa 8458, Vietnam;
huunghiaktl@gmail.com
* Correspondence: vantang.nguyen@uon.edu.au; Tel.: +61-4-3423-8842

Academic Editor: Manoj Gupta

Received: 4 March 2017; Accepted: 30 March 2017; Published: 2 April 2017

Abstract: The aims of this study were to determine the proximate composition of cacao pod husk as
well as the optimal conditions for extraction and purification of theobromine from cacao pod husk.
The results indicated that cacao pod husk had high contents of moisture and carbohydrate (87.06% and
11.03% by fresh weight, respectively), but low contents of crude protein, crude lipid, and ash (0.31%,
0.12%, and 1.48% by fresh weight, respectively). The optimal conditions for extraction of theobromine
from cacao pod husk were of 70% ethanol, with an extraction time of 90 min, and 1 as the number of
extractions. A concentration of 10% by volume of 10% lead acetate solution was the best selection for
purification of the crude extracts containing theobromine from cacao pod husk. Under these optimal
conditions, theobromine content obtained from cacao pod husk was 6.79 mg/100 g dry weight.
The finding from this study is a valuable contribution for obtaining theobromine from an abundant,
inexpensive, renewable, and sustainable source for potential application in the nutraceutical, medical,
and pharmaceutical industries.

Keywords: cacao pod husk; theobromine; proximate composition; extraction; purification

1. Introduction
Theobroma cacao L. has been known as “the food of the gods”. Cacao tree is wildly grown in tropical
and subtropical areas around the world including in Africa, such as Côte d'Ivoire, Ghana, Nigeria,
and Cameroon, in American countries, like Brazil, Ecuador, Columbia, and Mexico, in Caribbean
and Southwestern Pacific countries, such as the Dominican Republic and Papua New Guinea, and in
Southeast Asian countries, like Indonesia, Malaysia, and Vietnam. Total production of cacao bean is
estimated worldwide to be approximately 4.0 million tonnes in 2013, with a value about $12 billion [1].
Residues or wastes from the cacao processing industry consist of cacao pod shell, husk, pulp/mucilage,
and hull, which account for a high proportion, approximately 85% by fresh weight of total cacao
pod mass [2], in which the annual worldwide amount of cacao pod husk is estimated to be about 55
million tonnes, which is equal to 13 times the total amount of cacao bean. Therefore, cacao pod husk
needs to be exploited to produce high-value-added products and this waste has been considered as an
abundant, inexpensive, and renewable source of theobromine, a valuable compound, which exhibits
stimulatory effects on the central nervous, gastrointestinal, cardiovascular, renal, and respiratory
systems [3]. The chemical structure of theobromine is shown in Figure 1.

Technologies 2017, 5, 14; doi:10.3390/technologies5020014 www.mdpi.com/journal/technologies

Technologies 2017, 14, 14 2 of 10

cardiovascular, renal, and respiratory systems [3]. The chemical structure of theobromine is shown
Technologies 1. 5, 14
in Figure 2017, 2 of 10

Figure 1. Chemical structure of theobromine [4].

Figure 1. Chemical structure of theobromine [4].

As of late, few studies on cacao residues have been performed. For example, example, Arlorio
Arlorio et al. [5]
reported that cacao bean hull, principalprincipal by-product
by-product from
from cacao
cacao industry, was commonly used as a
secondary source of theobromine (1.29 g/100 g/100 gg byby dry
dry weight),
weight), while
while Hammerstone
Hammerstone and Chimel [6]
extracted theobromine
theobrominefrom fromcacao
cacaosolids
solidsusing
usingvarious
varioussolvents,
solvents,including
includingisopropanol,
isopropanol,methanol, and
methanol,
ethanol
and at different
ethanol temperatures
at different temperatures andand
found thatthat
found the the
highest theobromine
highest theobromine content waswas
content obtained by
obtained
ethanol at 50 ◦ C. Peralta-Jiménez and Cañizares-Macías [7] also used an ultrasound-assisted method
by ethanol at 50 °C. Peralta-Jiménez and Cañizares-Macías [7] also used an ultrasound-assisted
with
method heated
with water 80 ◦ Catfor
heatedatwater 80 the extraction
°C for of theobromine
the extraction of theobromineand caffeine from from
and caffeine cacaocacao
seedsseeds
and
chocolate
and products,
chocolate and and
products, indicated that that
indicated the extraction efficiency
the extraction of theobromine
efficiency of theobromine fromfrom
cacao seeds
cacao and
seeds
chocolate
and products
chocolate increased
products 43.6%43.6%
increased and 23%,
andrespectively, when compared
23%, respectively, when comparedto conventional stirring
to conventional
extraction
stirring methods.
extraction MunierMunier
methods. and Hartadi [8] reported
and Hartadi that fresh
[8] reported thatcacao
fresh pod
cacao husk
podcontained 0.40%
husk contained
theobromine
0.40% and it was
theobromine andgreatly
it was reduced during fermentation
greatly reduced by A. oryzae,
during fermentation by and Adamafio
A. oryzae, and [9] mentioned
Adamafio [9]
theobromine
mentioned toxicity, theobromine
theobromine catabolism, and
toxicity, theobromine de-theobromine
catabolism, methods from
and de-theobromine cacao by-products.
methods from cacao
With regard to extraction and purification processes of bioactive compounds, water or organic
by-products.
solvent,
Withorregard
a mixture of water and
to extraction andorganic solvent,
purification has been
processes of used to extract
bioactive xanthine
compounds, alkaloids,
water such
or organic
as caffeine
solvent, or aand theobromine
mixture of water[3,10], while bleaching
and organic solvent, hasearth
been and
usedactivated
to extractcarbon are usually
xanthine used
alkaloids, suchto
remove
as foreign
caffeine matter in water
and theobromine andwhile
[3,10], oils [11,12].
bleachingHowever,
earth andthe activated
study on thecarbonproximate composition
are usually used to
of cacao foreign
remove pod husk and optimal
matter in waterconditions for extraction
and oils [11,12]. However, andthepurification
study on the of theobromine from cacao
proximate composition
pod
of huskpod
cacao hashusk
not been sufficiently
and optimal reported.
conditions forTherefore,
extractionthis
andstudy aimed to
purification of determine
theobromine thefrom
proximate
cacao
composition
pod husk has of notcacao
been pod husk, as
sufficiently well asTherefore,
reported. optimal conditions
this study foraimedextraction and purification
to determine the proximate of
theobromine of
composition from cacao
cacao pod pod huskasaswell
husk, an abundant,
as optimal inexpensive,
conditions renewable,
for extraction andand
sustainable source
purification of
using a non-toxic,
theobromine fromaccessible,
cacao podand huskcheap
as ansolvent for potential
abundant, application
inexpensive, in theand
renewable, nutraceutical,
sustainablemedical,
source
and pharmaceutical
using industries.
a non-toxic, accessible, and cheap solvent for potential application in the nutraceutical, medical,
and pharmaceutical industries.
2. Materials and Methods
2. Materials and Methods
2.1. Plant Material
2.1. Plant Material
Twenty kilograms of cacao pods (Theobroma cacao trinitario) were purchased from the Western
Highlands
Twenty kilogramsand
Agriculture Forestry
of cacao Science
pods Institute,
(Theobroma Buon
cacao Ma Thuot
trinitario) werecity, Dak Lak province,
purchased Vietnam
from the Western
(latitude 12.6667 ◦ N, longtitude ◦
108.0500Science
E). After collection, cacao
Highlands Agriculture and Forestry Institute, Buon Mapods
Thuotwere packed
city, DakinLak
juteprovince,
bags and
immediately taken to the laboratory at Nha Trang University for further treatments.
Vietnam (latitude 12.6667° N, longtitude 108.0500° E). After collection, cacao pods were packed The fresh samples
in
were then stored in the freezer at − 20 ◦ C until used for further experiments. The overall experimental
jute bags and immediately taken to the laboratory at Nha Trang University for further treatments.
procedure
The is described
fresh samples wereinthen
Figure 2. in the freezer at –20 °C until used for further experiments. The
stored
overall experimental procedure is described in Figure 2.
Technologies 2017, 5, 14 3 of 10
Technologies 2017, 14, 14 3 of 10

Figure2.
Figure Overallexperimental
2.Overall experimentalprocedure
procedure of
of the
the study.
study.

2.2. Chemicals
2.2. Chemicals
All chemicals
All chemicals used
usedininthis
thisresearch were
research of analytical
were grade.
of analytical Standard
grade. theobromine
Standard (purity(purity
theobromine 98.5%)
was purchased
98.5%) from Sigma-Aldrich
was purchased Pte Ltd. (Nucleos,
from Sigma-Aldrich Pte Ltd. Singapore). Absolute ethanol,
(Nucleos, Singapore). Absolutechloroform,
ethanol,
acetonitrile, acetonitrile,
chloroform, phosphoric acid, lead acetate,
phosphoric and sodium
acid, lead acetate, sulfate were purchased
and sodium from
sulfate were the Chemical
purchased from and
the
Scientific Technological
Chemical Join Stock Company
and Scientific Technological (Ho Company
Join Stock Chi Minh (HoCity,Chi
Vietnam).
Minh City, Vietnam).

2.3. Preparation
2.3. Preparation of
of Dried
Dried Samples
Samples
Fresh cacao
Fresh cacao pod
pod husk
husk waswas separated
separated from
from cacao
cacao pods,
pods, then
then cut
cut into
into small
small pieces,
pieces, and
and dried
dried at
at
◦C
45 °C to
to constant
constant weight
weight using
using an
an infrared
infrared drying
drying cabinet
cabinet (SHN-L,
(SHN-L, Nha
Nha Trang
Trang University,
University, Vietnam)
Vietnam)
45
based on the formerly reported method of Nguyen et al. [13]. The dried samples were
based on the formerly reported method of Nguyen et al. [13]. The dried samples were packed in packed insealed
sealed
plastic bags
plastic bags and
and stored
stored in
in aa dry
dry place
place until
until required.
required. Prior
Prior to
touse
usefor
forall
allexperiments,
experiments, the
thedried
driedsamples
samples
were ground to fine particles using a hammer mill
were ground to fine particles using a hammer mill [14]. [14].

2.4. Analysis of Proximate Composition of Cacao Pod Husk

2.4. Analysis of Proximate Composition of Cacao Pod Husk
The moisture, crude protein, crude lipid and ash contents of cacao pod husk were analyzed
The moisture, crude protein, crude lipid and ash contents of cacao pod husk were analyzed
according to the Association of Official Analytical Chemists (AOAC) official method (1998) and based
according to the Association of Official Analytical Chemists (AOAC) official method (1998) and based
on the previously-described method by Nguyen [2]. Moisture content was determined using a hot-air
on the previously-described method by Nguyen [2]. Moisture content was determined using a hot-
oven at 100 ◦ C overnight. Nitrogen content was measured using the Kjeldahl method and the crude
air oven at 100 °C overnight. Nitrogen content was measured using the Kjeldahl method and the
protein content was then calculated by multiplying nitrogen content by a factor of 6.25. Crude lipids
crude protein content was then calculated by multiplying nitrogen content by a factor of 6.25. Crude
were extracted using the Soxhlet method and the crude lipid content was determined after oven-drying
lipids were extracted using the Soxhlet method and the crude lipid content was determined after
the extract at 100 ◦ C for 30 min. Ash content was measured by heating the cacao pod shells in a furnace
oven-drying the extract at 100 °C for 30 min. Ash content was measured by heating the cacao pod
at 600 ◦ C for 5 h. Carbohydrates (include crude fiber) was calculated from Equations (1) and (2) [15]
shells in a furnace at 600 °C for 5 h. Carbohydrates (include crude fiber) was calculated from
as follows:
Equations (1) and (2) [15] as follows:
Carbohydrate (% by(%fresh
Carbohydrate weight)
by fresh = 100
weight) − −moisture
= 100 moisture − crudeprotein
− crude protein − crude
− crude − ash− ash (1)(1)
lipidlipid

Carbohydrate (% by
Carbohydrate (%dry weight)
by dry weight) = 100−−crude
= 100 protein−−
crude protein crude
crude lipid
lipid − ash
− ash (2)(2)

2.5. Extraction of Theobromine from Cacao Pod Husk

2.5. Extraction of Theobromine from Cacao Pod Husk
The extracts from cacao pod husk were prepared according to the prior methods of Nguyen [2]
withThe
someextracts
minorfrom cacao pod Briefly,
modifications. husk were prepared
to select according
the most to the
effective prior 10
solvent, methods of Nguyen
g of dried [2]
cacao pod
with
husksome minor modifications.
was extracted with 270 mL Briefly, to select
of various the most
solvents effective
for 30 min atsolvent, 10 g of of
temperatures dried
100 cacao
◦ C, 80pod
◦ C,
husk was
◦ extracted with 270 mL of various solvents for 30 min at temperatures of 100
and 60 C for water, 70% ethanol and chloroform, respectively, using a Memmert WB29 thermostatic °C, 80 °C, and
60 °C(Memmert
bath for water, 70%
GmbHethanol and chloroform,
+ Co.KG, Schwabach,respectively, using was
Germany), which a Memmert WB29 thermostatic
closely covered bath
by a lid to avoid
(Memmert GmbH +during
water evaporation Co.KG,extraction.
Schwabach, Germany),
Different which was
extraction timesclosely covered
and the by aoflid
number to avoid water
extractions were
evaporation during extraction. Different extraction times and the number of extractions
then applied with selected solvent to identify optimal extraction time and number of extractions. were then
applied with selected solvent to identify optimal extraction time and number of extractions. After
extraction, the extracts were immediately cooled to room temperature using an ice water bath, and
Technologies 2017, 5, 14 4 of 10
Technologies 2017, 14, 14 4 of 10

then filtered
After extraction, through a Whatman
the extracts were No. 1 filter paper
immediately cooledto to
obtain
roomthe crude extracts
temperature using foranfurther
ice wateranalysis
bath,
and then
and experiments.
filtered through a Whatman No. 1 filter paper to obtain the crude extracts for further analysis
and experiments.
2.6. Purification of Crude Extracts from Cacao Pod Husk
2.6. Purification of Crude Extracts from Cacao Pod Husk
Purification of the crude extracts from cacao pod husk containing theobromine is to remove
foreign matters (protein,
Purification lipid, extracts
of the crude pectin, tannins,
from cacao pigments, etc.) containing
pod husk within the crude extracts is
theobromine fortoobtaining
remove
the pure
foreign extracts
matters or theobromine-enriched
(protein, lipid, pectin, tannins, extracts.
pigments,Briefly, 10% lead
etc.) within theacetate solutionforatobtaining
crude extracts different
concentrations
the pure extracts of or
2.5%, 5.0%, 7.5%, 10.0%, and
theobromine-enriched 12.5%Briefly,
extracts. by volume10% was leadadded
acetatetosolution
40 mL of at the crude
different
extracts. The mixture
concentrations of 2.5%, was then7.5%,
5.0%, left at10.0%,
room and temperature
12.5% by(32 ± 2 °C)was
volume for added
15 min.toAfter
40 mL treatment with
of the crude
10% leadThe
extracts. acetate solution,
mixture the residual
was then left at roomlead temperature
acetate in the(32 ± 2 ◦ C)
extracts wasforcompletely
15 min. After removed
treatmentby using
with
10% lead
10% sodium sulfate
acetate solution
solution, the(based
residual on lead
formation
acetate ofin
white precipitate
the extracts wasnamed as lead
completely sulfate) by
removed andusing
then
filtered
10% sodiumthrough a Whatman
sulfate No. 1 filter
solution (based paper toofobtain
on formation whitethe pure extracts.
precipitate namedTheseas lead pure extracts
sulfate) and were
then
then measured
filtered through viscosity
a Whatman usingNo.a1viscometer
filter paperLVDV-Eto obtain(Brookfield, NY, USA)
the pure extracts. Theseatpure
a speed of 60were
extracts rpmthen and
spindle S03,
measured and absorbance
viscosity at 436 nm
using a viscometer LVDV-Eusing(Brookfield,
a Cary 50 NY, UV-VISUSA)spectrophotometer
at a speed of 60 rpm(Varian, and spindleInc.,
Manasquan,
S03, and absorbanceNJ, USA). at The
436 nmresults
using of viscosity
a Cary 50and absorbance
UV-VIS at 436 nm were
spectrophotometer expressed
(Varian, Inc., as centipoise
Manasquan,
(cP)USA).
NJ, and absorbance
The resultsunit, respectively.
of viscosity The best pure
and absorbance at 436extract
nm were obtained
expressedafteraspurification
centipoise was also
(cP) and
analyzed theobromine
absorbance content to
unit, respectively. Theevaluate
best pure the extract
effectiveness
obtained of the purification
after purification process.
was also analyzed
theobromine content to evaluate the effectiveness of the purification process.
2.7. Analysis of Theobromine Content
2.7. Analysis of Theobromine Content
Theobromine content in the extracts from cacao pod husk was analyzed using HPLC system
Theobromine
(Shimadzu, Kyoto, content in theon
Japan) based extracts from cacao
the previously pod huskmethod
developed was analyzed
of Nguyen using HPLC
et al. [13] system
Briefly,
(Shimadzu,
the extracts Kyoto, Japan) theobromine
and standard based on thesolutionpreviously (100developed
µg/mL in method
methanol) of were
Nguyen et al.through
filtered [13] Briefly,
0.45
the extracts and standard theobromine solution (100 µg/mL in methanol)
µm nylon membranes, and 20 µL were then individually injected by an auto injector (Shimadzu, were filtered through 0.45 µm
nylon
Kyoto,membranes,
Japan) onto aand 20 µLCwere
column 18 (250 then
× 4.6individually injected by
mm 5 µm; Shimadzu, an auto
Kyoto, injector
Japan), which (Shimadzu, Kyoto,
was maintained
Japan) onto a column C (250 × 4.6 mm 5 µm; Shimadzu, Kyoto, Japan),
at 35 °C by a column18 oven (Shimadzu, Kyoto, Japan). The isocratic elution consisted of 0.05% which was maintained at 35 ◦ C
by a column acid
phosphoric ovenin(Shimadzu,
distilled waterKyoto, (A)Japan).
and 100% The acetonitrile
isocratic elution consisted
(B) (85:15). Flow of rate
0.05% was phosphoric acid in
set at 1 mL/min.
distilled water (A) compound
The theobromine and 100% acetonitrile
was detected (B) (85:15).
at 272 nm Flow rate was
using set at 1detector
a UV-VIS mL/min. The theobromine
(Shimadzu, Kyoto,
compound was detected at 272 nm using a UV-VIS detector (Shimadzu,
Japan). Theobromine content in the extracts from cacao pod husk was quantified based on the Kyoto, Japan). Theobromine
content
calibration in the extracts
curve from cacao
of standard pod husk was
theobromine quantified based
by comparing retentionon thetimecalibration
and peakcurve area of standard
theobromine by comparing
with those in retention
the extracts timefromand peak
cacaoareapodofhusk.
standardThe theobromine
calibration curve with those in the
of standard
extracts from cacao pod husk. The calibration curve of standard theobromine
theobromine (concentration ranged from 0 to 5 mg/L) is indicated in Figure 3. Limit of detection (concentration ranged
from
(LOD) 0 to
and 5 mg/L)
limit ofis quantification
indicated in Figure (LOQ) 3. of
Limit of detection
standard (LOD) and
theobromine were limit of quantification
found to be 0.10 and (LOQ)
0.31
of standard theobromine
mg/L, respectively. were found to be 0.10 and 0.31 mg/L, respectively.

Figure 3.
Figure 3. Calibration
Calibration curve
curve of
of standard
standard theobromine.
theobromine.

2.8. Statistical Analysis

All experiments were run in triplicate. The data were analyzed using SAS software (version 9.2,
SAS Inst., Inc., Cary, NC, USA) and expressed as the mean ± standard deviation (n = 3). Statistical
Technologies 2017, 5, 14 5 of 10

2.8. Statistical Analysis

All experiments were run in triplicate. The data were analyzed using SAS software (version 9.2,
SAS Inst., Inc., Cary, NC, USA) and expressed as the mean ± standard deviation (n = 3). Statistical
comparisons were made using analysis of variance (ANOVA) and t-tests (LSD). Differences were
considered significantly when p-values were below 0.05 (p < 0.05).

3. Results and Discussion

3.1. Proximate Composition of Cacao Pod Husk

The proximate composition of cacao pod husk was calculated based on fresh weight and dry
weight (Table 1) to use for different purposes. The results showed that cacao pod husk had high
contents of moisture and carbohydrates (87.06% and 11.03% by fresh weight, respectively), but low
contents of crude proteins, crude lipids, and ash (0.31%, 0.12%, and 1.48% by fresh weight, respectively).
Based on dry weight, the contents of crude protein, crude lipid, ash, and carbohydrate were of 2.42%,
0.93%, 11.44%, and 85.21% by dry weight, respectively, indicating that cacao pod husk contains high
carbohydrate content which forms a firm structure for its husk. The moisture content in cacao pod
husk found in this study was similar to that in cacao pod shell (87.0%), but the contents of crude
protein, crude lipid, and ash in cacao pod husk were lower than those in cacao pod shell (1.26%, 0.16%,
2.30% by fresh weight and 9.69%, 1.23%, and 17.69% by dry weight, respectively), and carbohydrate
content in cacao pod husk was higher than that in cacao pod shell (9.28% by fresh weight and 71.39%
by dry weight, respectively) [2]. Marcel et al. [16] indicated that crude protein content in cacao pod
shell, cacao pod husk, and cacao bean hull (16.0%, 9.14%, and 17.9% by dry weight, respectively) was
much higher than that in cacao pod husk found in this study, whereas ash content in cacao pod shell,
cacao pod husk and cacao bean hull (7.5%, 9.07%, and 9.3% by dry weight, respectively) was lower
than that in cacao pod husk found in this study. Arlorio et al. [5] reported that moisture, the contents
of crude lipid, crude protein and ash in pre-roasted cacao bean hull were of 10.12%, 6.81%, 18.12%, and
8.1% by dry weight, respectively. These results showed that proximate composition in different parts
of cacao pod was greatly different that directly relates to structural characteristics of cacao pod, in that
cacao pod husk is a main part against insects and harmful effects from the environment in terms of
physical, chemical, and microbial agents.

Table 1. Proximate composition of cacao pod husk.

Proximate Composition Fresh Weight (%) Dry Weight (%)

Moisture 87.06 ± 0.58 * -
Crude protein (protein factor: 6.25) 0.31 ± 0.21 2.42 ± 0.37
Crude lipid 0.12 ± 0.12 0.93 ± 0.34
Ash 1.48 ± 0.31 11.44 ± 0.41
Carbohydrate (include crude fiber) 11.03 ± 0.21 85.21 ± 0.29
* Means and standard deviations were of triplicate.

3.2. Effect of Extraction Conditions on Theobromine Content from Cacao Pod Husk

3.2.1. Effect of Various Solvents on Theobromine Content

Theobromine has been known as the most valuable compound in cacao and cacao products.
Figure 4 illustrates HPLC chromatograms of standard theobromine (Figure 4A) and theobromine in the
crude extract from cacao pod husk (Figure 4B). Theobromine content from cacao pod husk extracted
by various solvents is indicated in Figure 5.
Among three solvents tested, 70% ethanol obtained the greatest level of theobromine
(2.23 mg/100 g dry weight) as compared to those extracted by water and chloroform (0.09 and
0.35 mg/100 g dry weight), revealing that theobromine had a limited dissolvability in water, which
Technologies 2017, 5, 14 6 of 10

is considered as a “green” solvent. This greatly related to the intermediate polarity of 70% ethanol,
which allows it to solvate theobromine with low molecular weight containing functional groups (–C=O
and –CH3 ) [17]. Hu et al. [10] reported that 95% theobromine and 99% caffeine could be recovered by
using a chloroform-water system, while Kasabe and Badhe [4] recovered theobromine in tea samples
Technologies 2017, 14, 14 6 of 10
usingTechnologies
various 2017,
organic
14, 14 solvents and aqueous mixtures and found that an increase of recovery in10order
6 of
by n-hexane,
recovery ethyl
in orderacetate, methylene
by n-hexane, dichloride,
ethyl acetate, chloroform,
methylene methanol,
dichloride, water,
chloroform, 5% sulphuric
methanol, water, 5% acid in
water,recovery
and 5% indiethyl
order byaminen-hexane,
in ethyl acetate,
water, with a methylene
maximum dichloride,
level of chloroform, methanol,
theobromine was
sulphuric acid in water, and 5% diethyl amine in water, with a maximum level of theobromine was foundwater,
to 5%2.31%
be
sulphuric acid in water, and 5% diethyl amine in water, with a maximum level of theobromine was
by dry weight.
found to beThis
2.31% data by illustrated
dry weight.that
Thisthe extractability
data of theobromine
illustrated that wasofgreatly
the extractability affected
theobromine was by the
found to be 2.31% by dry weight. This data illustrated that the extractability of theobromine was
greatly
character of affected by the character of the solvent.
the solvent.
greatly affected by the character of the solvent.

Figure 4. HPLC
4. HPLC chromatograms
chromatograms ofstandard
ofof standard theobromine (A) and theobromine in the crude extract
Figure
Figure 4. HPLC chromatograms standard theobromine (A)and
theobromine (A) andtheobromine
theobromine in the
in the crude
crude extract
extract
from cacao pod husk (B).
from from
cacaocacao
podpod
husk (B).(B).
husk

Figure 5. Effect of solvents on theobromine content from cacao pod husk. Different letters (a, b, c)
Figure 5. Effect
5. Effect of solvents
of solvents onon theobromine content
theobromine content from cacao
cacaopod husk. Different letters (a, b,(a,
c) b, c)
Figure
indicated significant differences between treatments (pfrom
< 0.05). pod husk. Different letters
indicated
indicated significant
significant differences
differences betweentreatments
between treatments (p
(p <<0.05).
0.05).
Of these, 70% ethanol was selected for further extraction of theobromine from cacao pod husk,
Of these, 70% ethanol was selected for further extraction of theobromine from cacao pod husk,
which is inexpensive, accessible, and friendly to humans and the environment.
which is inexpensive, accessible, and friendly to humans and the environment.
Technologies 2017, 5, 14 7 of 10

Of these, 70% ethanol was selected for further extraction of theobromine from cacao pod husk,
Technologies 2017, 14, 14 7 of 10
which is inexpensive, accessible, and friendly to humans and the environment.

3.2.2. Effect
3.2.2. Effect of
of Extraction
Extraction Time
Time and
and Number
Number of
of Extractions
Extractions on
on Theobromine
TheobromineContent
Content
Figure 66 indicates
Figure indicates the the effect
effect of
of different
different extraction
extraction timestimes and
and number
number of of extractions
extractions onon theobromine
theobromine
content from
content from cacao
cacaopodpodhusk.
husk.Of Ofwhich,
which, theobromine
theobromine content
content was significantly
was significantlyhigher when
higher cacao
when pod
cacao
husk was extracted by 70% ethanol at longer times (2.23%, 3.29%, and 5.98
pod husk was extracted by 70% ethanol at longer times (2.23%, 3.29%, and 5.98 mg/100 g dry weight mg/100 g dry weight for 30,
60, 30,
for and60,90and
min,90respectively). However,
min, respectively). theobromine
However, contentcontent
theobromine was notwas significantly different
not significantly when
different
number of extractions increased from 1 time to 2 times (5.98 and 6.06 mg/100
when number of extractions increased from 1 time to 2 times (5.98 and 6.06 mg/100 g dry weight, g dry weight, respectively).
This finding isThis
respectively). supported
findingbyisNguyen
supported andby Pham
Nguyen [14] and
and Pham
Nguyen [2]and
[14] whoNguyen
reported [2]that
whothe extract
reported
concentration
that the extractfrom different parts
concentration fromofdifferent
artichokeparts
plantof(leaf, stalk, root,
artichoke plantand flower)
(leaf, stalk,and
root,pigment yield
and flower)
from cacao pod shell attained an equilibrium at extraction times of 60 and
and pigment yield from cacao pod shell attained an equilibrium at extraction times of 60 and 80 min, 80 min, respectively, and
Tan et al. [18] also found that and the phenolic extraction efficiency from bitter
respectively, and Tan et al. [18] also found that and the phenolic extraction efficiency from bitter melon melon was not
significantly
was differentdifferent
not significantly after an extraction
after time time
an extraction of 5ofmin5 min andandone
oneextraction.
extraction. Considering
Considering the the
production costs, longer extraction times and more number of extractions
production costs, longer extraction times and more number of extractions consume more energy for consume more energy for
extraction and
extraction andremove
remove solvent
solvent afterafter extraction,
extraction, leading leading to higher
to higher production
production costs,
costs, this is an this is an
important
important factor and needs to be properly considered when selecting extraction
factor and needs to be properly considered when selecting extraction time and number of extractions. time and number of
extractions. Therefore, an extraction time of 90 min and one as the number of extractions
Therefore, an extraction time of 90 min and one as the number of extractions were suitable for extraction were suitable
fortheobromine
of extraction offrom
theobromine
cacao podfrom husk.cacao pod husk.

Figure 6. Effect
Figure 6. Effect of
of different
different extraction
extraction times
times and
and number
number of of extractions
extractions on
on theobromine
theobromine content
content from
from
cacao
cacao pod
pod husk.
husk. Different letters (a, b, c) indicated significant differences between treatments (p < 0.05).

3.3. Effect of Purification

3.3. Purification on
on Physicochemical
Physicochemical Properties
Properties of
of Extracts
Extracts and
and Theobromine
TheobromineContent
Contentfrom
fromCacao
Cacao
Pod
Pod Husk
Husk
In
In this
thisstudy,
study,lead
leadacetate waswas
acetate used as an
used asagent to remove
an agent foreignforeign
to remove matter matter
within the crude
within theextracts
crude
from cacao pod husk including protein, lipid, pectin, tannins, pigments, and so on
extracts from cacao pod husk including protein, lipid, pectin, tannins, pigments, and so on due to due to bleaching
earth and earth
bleaching activated
and carbon
activatedcould absorb
carbon couldgreatly
absorbtheobromine in the extracts,
greatly theobromine leading leading
in the extracts, to the loss of
to the
theobromine. The effectThe
loss of theobromine. of 10% lead
effect of acetate
10% leadsolution concentration
acetate on the viscosity
solution concentration on and
the absorbance
viscosity andof
the extracts of
absorbance from
thecacao pod
extracts husk
from is shown
cacao in Figure
pod husk 7. Ininthis
is shown instance,
Figure the instance,
7. In this viscosity the
of the extracts
viscosity of
was significantly
the extracts reduced at a reduced
was significantly concentration of 2.5% by volume
at a concentration of 2.5%of by
10% lead acetate
volume of 10%solution (4.77
lead acetate
to 3.90 cP),
solution but
(4.77 tono significant
3.90 cP), but nodifference
significantindifference
the absorbance of the extracts
in the absorbance was
of the observed
extracts was (1.87 and
observed
1.83
(1.87absorbance units). However,
and 1.83 absorbance units).when the concentration
However, of 10% lead acetate
when the concentration of 10% solution increased
lead acetate from
solution
increased from 2.5% to 5.0% by volume, both the viscosity and absorbance of the extracts were
significantly decreased (3.90 to 3.63 cP and 1.83 to 0.37 absorbance unit, respectively), and then they
were significantly reduced with an increase of the concentration of 10% lead acetate solution from
5.0% to 7.5% by volume (3.63 to 3.54 cP and 0.37 to 0.30 absorbance units, respectively). The
Technologies 2017, 5, 14 8 of 10

2.5% to 5.0% by volume, both the viscosity and absorbance of the extracts were significantly decreased
(3.90 to 3.63 cP and 1.83 to 0.37 absorbance unit, respectively), and then they were significantly reduced
Technologies 2017, 14, 14
with an increase of the concentration of 10% lead acetate solution from 5.0% to 7.5% by volume 8 of 10
(3.63 to 3.54 cP and 0.37 to 0.30 absorbance units, respectively). The absorbance of the extracts was
absorbance
continuouslyofreduced
the extracts
whenwas continuously of
the concentration reduced
10% lead when the solution
acetate concentration of 10%
rose from 7.5%lead acetate
to 10.0% by
solution rose from 7.5% to 10.0% by volume, whereas the viscosity of the extracts
volume, whereas the viscosity of the extracts was not significantly different. These results indicated was not significantly
different. These resultsofindicated
that the concentration 10.0% bythat the concentration
volume of 10.0%
of 10% lead acetate by volume
solution was the of best
10% selection
lead acetate for
solution was the best selection for purification of the extracts from cacao
purification of the extracts from cacao pod husk. Under the extraction conditions of 70% ethanol, onepod husk. Under the
extraction
extraction conditions
for 90 min,of 70%
and ethanol, one
purification extraction
with for 90 min,
a concentration of and
10.0% purification
by volumewith a concentration
of 10% lead acetate
of
solution, theobromine content achieved 6.79 mg/100 g dry weight, which was higher thanmg/100
10.0% by volume of 10% lead acetate solution, theobromine content achieved 6.79 g dry
that without
weight,
purifying which
by 10% was higher
lead than
acetate that without
solution purifying
(5.58 mg/100 byweight).
g dry 10% leadThis acetate
is duesolution (5.58 of
to the effect mg/100
foreign g
dry weight). This is due to the effect of foreign matter present in the extracts, leading
matter present in the extracts, leading to a decrease in the detectability of theobromine in the extracts to a decrease in
the detectability
by HPLC analysis.of theobromine
Howlader etinal.the extracts
[19] by HPLC
and Reddy and analysis.
Lakshmi Howlader et al.10%
[20] also used [19]lead
and acetate
Reddy
and Lakshmi
solution as an[20]
agentalsotoused
detect10%thelead acetate
presence of solution
tannins in as the
an agent to detect
methanolic the presence
extract of tannins
from Diospyros in
blancoi
the methanolic extract from Diospyros blancoi leaf and phenolic compounds in
leaf and phenolic compounds in the extract from the whole plant of Oxalis corniculata L, based on the the extract from the
whole plant
formation ofof Oxalis
white corniculata
precipitate inL, based
the on the formation of white precipitate in the end-products.
end-products.

Figure 7. Effect of concentration of 10% lead acetate solution on viscosity and absorbance of the extracts
from cacao
cacao pod
pod husk.
husk. Different
Differentletters
letters(a,
(a,b,
b, c,
c, d, A, B,
B, BC,
BC, C)
C)indicated
indicatedsignificant
significant differences
differences between
treatments(p
treatments (p<<0.05).
0.05).

By
By applying
applying thethe conventional
conventional extraction
extraction technique
technique inin this
this study,
study, theobromine
theobromine content
content obtained
obtained
from cacao pod
from cacao pod husk
huskwas waslower
lowerthan
thanthat
thatfound
foundinincacao
cacaobean
bean hull
hull (1.29
(1.29 g/100
g/100 gg dry
dry weight)
weight) [5][5]
andand
in
in tea samples (2.31 g/100 g dry weight) [4]. This outcome revealed that although theobromine
tea samples (2.31 g/100 g dry weight) [4]. This outcome revealed that although theobromine content content
in
in cacao pod husk
cacao pod husk isis lower
lower than
thanthat
thatin
insome
someother
othermaterials,
materials,a avery
verylarge
largeamount
amountofof cacao
cacao pod
pod husk
husk is
is discarded from the cacao processing industry, approximately 55 million tonnes
discarded from the cacao processing industry, approximately 55 million tonnes per year. Therefore,per year. Therefore,
cacao
cacao podpod husk
husk isis an
an inexpensive,
inexpensive, renewable,
renewable, andand sustainable
sustainable source
source for
for extraction
extraction ofof theobromine,
theobromine,
with
with an estimation about 457.1 tonnes per year. To increase the extraction efficiency of
an estimation about 457.1 tonnes per year. To increase the extraction efficiency of theobromine
theobromine
and
and reduce extraction time, further research can be applied some advanced extraction techniques,
reduce extraction time, further research can be applied some advanced extraction techniques,
such
such asas ultrasound-assisted
ultrasound-assistedextraction
extraction [17],
[17], microwave-assisted
microwave-assisted extraction
extraction [21],[21], and supercritical
and supercritical fluid
fluid extraction
extraction [22]. [22].

4. Conclusions
This study was an exploratory and valuable assessment for further studies. The findings from
this study indicated that cacao pod husk had high contents of moisture and carbohydrates, but low
contents of crude protein, crude lipid, and ash. The extraction and purification processes greatly
influenced on theobromine content from cacao pod husk, particularly solvent, extraction time, and
concentration of 10% lead acetate solution. Therefore, the use of these extraction and purification
conditions for effective exploitation of theobromine from cacao pod husk is a promising and
Technologies 2017, 5, 14 9 of 10

4. Conclusions
This study was an exploratory and valuable assessment for further studies. The findings from this
study indicated that cacao pod husk had high contents of moisture and carbohydrates, but low contents
of crude protein, crude lipid, and ash. The extraction and purification processes greatly influenced on
theobromine content from cacao pod husk, particularly solvent, extraction time, and concentration
of 10% lead acetate solution. Therefore, the use of these extraction and purification conditions for
effective exploitation of theobromine from cacao pod husk is a promising and sustainable trend.
In further studies, it is necessary to determine the biological variability of theobromine in cacao pod
husk samples, as well as produce theobromine-enriched powder and evaluate its biological activity in
various models for potential application in the medical and pharmaceutical industries.

Acknowledgments: We sincerely acknowledge Nha Trang University, Vietnam for financial and mechanical
support. The authors also would like to kindly thank Huu Tuan Nguyen and Thi Be Phan for their
experimental support.
Author Contributions: Van Tang Nguyen conceived and designed the experiments, performed the experiments,
analyzed the data, and wrote the paper. Nghia Huu Nguyen reviewed and suggested the paper.
Conflicts of Interest: The authors declare no conflict of interest.

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