Corresponding Author: Agung
Ari Chandra Wibawa
E-mail: [email protected]
INTRODUCTION
Cocoa can
be regarded as an important crop in the Province of Bali, Indonesia. Jembrana
Regency currently is the main contributor to total cocoa production in Bali,
producing approximately 2942 tons of cacao beans in 2019 (BPS, 2021). The cocoa industry in Indonesia faces
many issues, one of which is a relatively lower quality of the beans than that
of most other areas (Fibrianto
et al., 2021). The COVID-19 pandemic poses additional
problems by lowering the price of cocoa beans, at least in Jembrana, Bali (Darmada,
2020). COVID-19 eats away at Balinese cocoa
farmers' income, further adding detrimental effects to the regional economy of
Bali, which relies heavily on the tourism sector. To support the government of
Indonesia in tackling the issues above in the cocoa industry, it is crucial to
explore additional values of Balinese cocoa beans in Jembrana through
scientific study.
Extraction,
using various solvents, is one of the most widely deployed methods to
investigate the phytochemical and pharmacological profile of natural products. Methanol
compound is relatively polar and possess a role as universal solvent capable of
extracting non-polar components (Riyadi et al., 2023). Based on a study using spirulina
extract, using methanol solvent can increase the antioxidant activity of the
extract compared to other solvents with a lower polarity level. A group of
phytoconstituents named as flavonoids are regarded as endogenous antioxidants. Flavonoids
protect plants from a myriad of biotic and abiotic stressors, and act as unique
UV filters (Panche et
al., 2016). Flavonoids
are also recognized to exert medicinal benefits to human health. Catechins are classified as flavonoids, also known as
flavanols or flavan-3-ols. Catechin,
gallocatechin, epicathecin (EC), epigallocatechin (EGC) are non-esterified cathecins, whereas the esterified catechins are epigallocathechin
gallate (EGCG), epicatechin gallate (ECG), gallocatechin gallate (GCG), and
catechin gallate. Catechins are
distributed in a variety of natural products such
as cocoa (Rauf et
al., 2019). Other
important phytoconstituents that contribute to human health are tannins. Tannins
are proteins-precipitating polyphenols reported to undergo biotransformation by
human gut microbiota, which determine their bioavailability and pharmacological
effects. Most of the health-promoting properties of natural products can be
attributed to the presence of tannins (Sallam et al., 2021), thus it is
important to reveal the availability of tannins in studied samples of natural
product.
To harvest
antioxidants (i.e, flavonoids and tannins) from plants, maceration and
partition extraction are considered as relatively simple methods (Slamet et
al., 2022). These phytoconstituents are soluble in
polar solvent such as methanol (Ng et al.,
2020) and n-butanol (Larit et
al., 2019). The higher the polarity of the solvent,
the higher the extract's antioxidant content (Guo et
al., 2020). Up to date, scientific data regarding
total flavonoids, tannins and antioxidant activity of Balinese cocoa beans
obtained from Jembrana regency is non-existent. Therefore, we conducted a phytochemical
profiling study on cacao bean extract using methanol solvent and its n-butanol
fraction as a preliminary step in developing this bioproduct as an additional
source of therapeutic compounds (i.e., as a pharma food). The results of this
study are expected to partially contribute to the added value of Balinese cocoa
beans from Jembrana in the future.
In a
phytochemical profile study on Bali cocoa bean extract from Jembrana Regency
using methanol solvent and its n-butanol fraction, the aim of the study was to
evaluate the difference in phytochemical profile and antioxidant activity
between cocoa bean methanol extract and its n-butanol fraction obtained from
Jembrana, Bali. This research aims to contribute to the development of
bioproducts as an additional source of therapeutic compounds, so as to increase
the added value of Bali cocoa beans from Jembrana in the future. The benefit of
this research is to provide a deeper understanding of the potential
phytochemical compounds contained in cocoa beans from Jembrana, as well as
provide useful information for the cocoa industry related to the development of
products with high added value, such as pharmaceutical foods containing
therapeutic compounds. Thus, this research can make a real contribution in
increasing the added value of Bali cocoa beans from Jembrana and supporting the
economic growth of cocoa farmers in the region.
METHOD
Sample Preparation
Cocoa beans
were obtained from Gumbrih Village, Jembrana Regency, Bali Province, Republic
of Indonesia. The cocoa beans separated from the fruit were washed using clean
water to remove impurities. Cleansed cocoa beans were then air-dried at room
temperature. After the cocoa beans were dry, they were peeled to obtain them
without the epidermis (pallets). Next, the pallets were blended until a fine
powder of cocoa beans was obtained.
Extraction
����������� Two
hundred grams of cocoa bean powder was put into a glass bottle, mixed with
methanol solution, covered and left for 1 x 24 hours, protected from light, and
stirred every 2 hours. After 24 hours, the dregs and filtrate were filtered.
Subsequently, the dregs were re-macerated using methanol for 24 hours. All the
macerates were combined and evaporated using a rotary vacuum evaporator until a
thick methanol extract was obtained.�
Partitioning
����������� The
thick methanol extract was partitioned based on its polarity to determine the
fraction that has the potential to be a bioactive compound. The concentrated
extract was dissolved in ethanol: water (3: 7) and then evaporated to produce
cocoa bean extract in the water phase. The extract was then partitioned using
n-butanol. The n-butanol fraction was quantitatively tested for flavonoid,
tannin and antioxidant activity using UV-Vis spectrophotometry.
Total
Flavonoid Content Analysis
Standard
Solutions
Total
flavonoid analysis was conducted according to the protocol published by (Cong-Hau et al., 2021). Standard quercetin solutions with
concentrations of 2, 4, 8, 12, 16 and 20 �g/mL were pipetted with a volume of 1.00
mL. The next step was adding 0.30 mL of 5% w/v NaNo2 (wait for 5
minutes). Then add 0.30 mL of 10% w/v AlCl3, 2.00 mL NaOH 1 mol, and
distilled water to 10.00 mL into the pipetted standard quercetin solutions.
These mixtures were incubated for 30 minutes at room temperature, and the
absorbances were measured using UV-Vis spectrophotometry with a wavelength of 510
nm.
Test Samples
One
hundred milligrams of extract were weighed and dissolved in 10 ethanol. Pipette
1 mL of the extract, then mixed with 0.30 mL of 5% w/v NaNo2 (wait
for 5 minutes). Then add 0.30 mL of 10% w/v AlCl3, 2.00 mL NaOH 1
mol, and distilled water to 10.00 mL into the pipetted standard quercetin
solutions. These mixtures were incubated for 30 minutes at room temperature,
and the absorbances were measured using UV-Vis spectrophotometry with a
wavelength of 510 nm. The sample solution was made in three replications so
that the flavonoid levels obtained were equivalent to quercetin.
Total
Tannin Analysis
Standard Solutions
Analysis of total
tannin content was conducted according to a method disseminated by (Ci, 2016). For each concentration of tannic acid
(20, 40, 60, 80 and 10 �g/mL), 0.1 mL was pipetted, then 0.5 mL of Folin
Ciocalteau reagent was added, shaken and left for 3 minutes. 1mL of 35% Na2CO3
solution was added, shaken until homogeneous, and left for 60 minutes at room
temperature. The absorbances were measured at a wavelength of 765 nm. The
results were plotted into a calibration curve.
Test Samples
Weigh
10 mg of methanol extract and n-butanol fraction samples each, put them in a
beaker, and dissolve them in 10 mL of 70% ethanol: distilled water (1:1). A
volume of 0.1 mL of the extract solution was mixed with 0.5 mL of
Folin-Ciocalteau reagent, homogenized then left for 3 minutes. Add 1.00 mL of 35%
sodium carbonate (Na2CO3) solution to the mixture and let
stand for 60 minutes at room temperature. The absorbance of the solution was
measured with a UV-Vis spectrophotometer at the maximum wavelength.
RESULTS AND DISCUSSION
Taxonomically,
Balinese cocoa is identified as Theobroma
cacao L. (Letter Number: B-491/IPH.7/AP/VI/2019). Detailed taxonomic
information on Balinese cacao is written as follows:
Table 1. Balinese
cocoa taxonomy
|
Kingdom:
|
Plantae
|
|
Subkingdom:
|
Tracheobionta
|
|
Division:
|
Magnoliophyta
|
|
Superdivision:
|
Spermatophyta
|
|
Class:
|
Magnoliopsida
|
|
Subclass:
|
Rosidae
|
|
Order:
|
Malvales
|
|
Family:
|
Malvaceae
|
|
Genus:
|
Theobroma
|
|
Species:
|
Theobroma cacao L.
|
Comparative
selected chemical analyses have been conducted on the methanolic extract of
Balinese cocoa and its n-butanol fraction. Cocoa is globally known as the raw
material for chocolate and derivative products such as chocolate beverages,
bakeries and pastries. It is also developed into cosmetic products. In the
current study, Balinese cocoa beans from Jembrana, Bali, Indonesia, were
revealed to contain secondary metabolites and demonstrate potent antioxidant
activity. These findings further validate the extended spectrum of Balinese
cocoa beans utilization, at least in the form of methanol extract and its
n-butanol fraction, as a pharmafood and potentially as cosmeceutical products.
Pharmafood is endowed with medicinal benefits due to the existence of specific
constituents, such as those with antioxidant activity (Mahendra
& Pramartha, 2021).
Flavonoid
Content Analysis
Total flavonoid levels were analysed using the UV-Vis
spectrophotometric method with quercetin as a standard based on the study conducted by (Yahya et al., 2021). The measurement analysis results
obtained on the quercetin standard showed the line equation y=0.0297x+0.0066
with an R2 of 0.999. Through the linear equation values, the total flavonoid
content in the methanol extract of cocoa beans was 0.3358 � 0.002 g QE/100g.
The analysis results of total flavonoid levels in the n-butanol fraction were
higher than the methanol extract at 0.372 � 0.011 g QE/100g (Table 1).
A study measured the total levels of
flavonoids in the methanol extract of C. Decapetala wood at 3.93 � 0.005
mg QE/g (Pawar
& Surana, 2010). Other research on total flavonoid
levels using Enna singing plant extract revealed a total flavonoid level
of 7.37 mg QE/g (Mwamatope
et al., 2020). The ability of flavonoid compounds as
antioxidants can be seen from the total flavonoid content. Through the
mechanism of action of flavonoids, they can act as ROS scavengers by donating
electrons to free radicals (Dias et
al., 2021). Flavonoid compounds can play a role in
preventing lipid peroxidation caused by ROS in vivo (Ebuehi et
al., 2019). They may act as an organ protector
(i.e. nephroprotective) against a constellation of pathophysiological events
triggered by damage-associated molecular patterns (DAMP) such as uric acid (Mahendra
et al., 2023; Mahendra & Dewi, 2020).
Figure 1. Calibration Curve of Total Flavonoid Content.
Tannin Content Analysis
Total tannin content was analysed using the UV-vis spectrophotometric
method with tannic acid as a standard. The measurement analysis results
obtained on the tannic acid standard showed the line equation y=0.013x + 0.0099
with R2 of 0.998 (figure 2). The line equation values revealed that the total
tannin content in the methanol extract was 15.3446 � 1.34 g TAE/100g. Total
flavonoid levels in the n-butanol fraction were higher compared to the methanol
extract, amounting to 20.1565 � 0.82 g TAE/100g. Complete data on total
flavonoid and total tannin levels can be seen in Table 1.
Tannin compounds are secondary metabolites that have roles as
antioxidants, UV and antibacterial agents (Guo et
al., 2020). An example of a condensed tannin compound is proanthocyanidin (Rauf et
al., 2019b). In another study using methanol extract of Indigofera
tincoria, tannin levels were 229 � 0.001 mg TAE/g (Guo et al., 2020). This finding shows that cocoa beans are superior to this test
bioproduct in terms of tannin content.
Figure
2. Calibration Curve of Tannin Total Content
Table 2. Flavonoid and Tannin Content of Cocoa Beans Methanol
Extract vs. Its N-butanol Fraction
|
Sample
|
Replication
|
Flavonoid
(g QE/100g)
|
Tannin (g
TAE/100g)
|
|
Methanol
Extract
|
I
|
0,33405
|
14,8871
|
|
II
|
0,33491
|
16,8544
|
|
III
|
0,33851
|
14,2923
|
|
N-butanol
Fraction
|
I
|
0,38534
|
19,3696
|
|
II
|
0,36846
|
20,0854
|
|
III
|
0,36273
|
21,0147
|
Antioxidant Activity Index (AAI)
The DPPH method, with a concentration of 0.1 mM, was used to
investigate the antioxidant activity of the test substance in this study. The
results of antioxidant testing of the methanol extract of cocoa beans obtained
an IC50 value of 29.66 �g/mL and the n-butanol fraction with an IC50
value of 15.23 �g. /mL. The antioxidant activity value can be seen from how big
the IC50 (Inhibitor Concentration) value is obtained from the linear
regression equation of extract concentration on the free radical scavenger
activity value as in Figures 3 and 4. The y coefficient in this regression
equation is IC50.
In contrast, the x coefficient in the equation is the fraction's
concentration whose value will be sought, where the value of x obtained is the
concentration required to reduce 50% of DPPH radical activity. From the
equation, it can also be seen that the R2 value shows the correlation between
the percentage (%) of free radical scavenger activity and the sample
concentration and sample immersion. The results provide a positive correlation,
as seen from the R2 value of the methanol extract of cocoa beans of 0.958. At
the same time, the n-butanol fraction is 0.9898, and both R2 values
of the fractions are close to 1, illustrating that increasing
extract concentration increases the free radical scavenger activity (Scherer
& Godoy, 2009).
The IC50 value is obtained from the regression equation, which is a
reference for high and low antioxidant activity. IC50 is a parameter
for interpreting DPPH test results. The IC50 value is the concentration of the
test compound that can absorb free radicals by 50%, or IC50 is a
number that shows the concentration of the extract that can inhibit the
oxidation process by 50%. The classification of antioxidants is divided into 5,
namely < 50 ppm (very strong), 50-100 ppm (strong), 100-150 ppm (medium),
150-200 ppm (weak), and > 200 ppm is very weak (Scherer & Godoy, 2009).
From the test results obtained, the cocoa bean fraction has a strong potential
as an antioxidant. Based on research conducted with the results of the ethyl
acetate fraction, an IC50 value of 8.66 �g/mL was obtained, and the
n-butanol fraction had an IC50 value of 15.41 �g/mL. So, the ethyl
acetate fraction and n-butanol fraction are classified as very strong
antioxidants because they are at <50 ppm.
The AAI value serves to classify the
extract's antioxidant properties, where the AAI is obtained by calculating the
final concentration of the DPPH solution divided by the IC50 value
of the antioxidant compound. Based on the AAI value, if the AAI value is less
than 0.5, then it is said to have "weak" antioxidant activity; it is
said to be "medium" if the AAI value is between 0.5 - 1, it is said
to be "strong" if the AAI value is between 1 - 2. It is "very
strong" if the AAI value exceeds 2 (Scherer
& Godoy, 2009). Based on research that has been carried
out, it was found that the AAI value for the methanol extract of cocoa beans
was 1.35, and the n-butanol fraction was 2.63. Referring to the AAI values,
cocoa beans' methanol extract and n-butanol fraction have very strong activity.
Figure 3. IC50 of Balinese cocoa beans methanol extract
Based on the curve results obtained, the
line equation y = 20.8157x + 25.799 with an R2 of 0.958 was obtained, so the
IC50 value of the ethanol extract of cocoa beans was 29.66 �g/mL. Another study
that carried out tests carried out by (Mzid et
al., 2017) found that the reduction of free
radicals in Urtica urens ethanol extract was 245.65 � 10.2 l �g/mL.�
Figure 4. IC50 Value of n-butanol fraction
Table 3. IC50 and AAI values of ethanol extract and
n-butanol fraction
|
Sample
|
IC50
|
AAI
|
|
Methanol
extract
|
29,66 �g.ml-1
|
1,35
|
|
N-butanol
fraction
|
15,23 �g.ml-1
|
2,63
|
Medicinal
properties of bioactive compounds in cocoa beans and cocoa-derived products are
subjects of scientific pursuit. Specific bacterial-fungal alliance and the
resulting metabolites associated with cocoa bean fermentation may also
contribute to the medicinal effect of cocoa beans, which exert potential
medicinal benefits on human health. The integrality of these microbiological
interactions and the resulting metabolites is called cocobiota, a novel term
coined by (Petyaev
& Bashmakov, 2016). The microbial diversity of cocobiota of
cocoa beans harvested from southern Brazil revealed the presence of Hanseniaspora
spp., Saccharomyces spp., and Pichia spp. as the native yeast
population. Lactococcus spp., Staphylococcus spp., Acetobacter
spp. and Lactobacilli strains were demonstrated as the bacterial
components of this cocobiota (Bastos et
al., 2018). Cocobiota, thus, is an important factor
that shall be considered in future studies to elucidate the medicinal
properties of Balinese cocoa beans.
It is
essential to examine further the bioactive compound armamentarium contained in
Balinese cocoa beans using various solvents and pre-extraction treatments.
Safety issues (i.e., acute and chronic toxicity profiling) and other relevant
issue, such as cocobiota, shall be addressed. These studies will provide a
sound basis for further investigations in developing Balinese cocoa beans as a
source of bioactive compounds with medicinal properties.
CONCLUSION
The n-butanol fraction of Balinese cocoa
(Theobroma cacao L.) beans exhibits higher total flavonoid and tannin content
than the methanolic extract counterpart. The AAI value of the methanol extract
is lower than its n-butanol fraction, but both test agents display very strong
antioxidant activity. Further studies should be conducted to elucidate the
complete picture of Balinese cocoa methanol extract's and its n-butanol
fraction�s phytochemical profile and antioxidant properties.
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