Adi A.C. et al. Foods and Raw Materials.

2023;11(2):206–214 E-ISSN 2310-9599

Foods and Raw Materials, 2023, vol. 11, no. 2 ISSN 2308-4057

Research Article [Link]

Open Access Available online at [Link]
[Link]

Effect of cocoa husk Criollo tea

on hypercholesterolemia in animal model
Annis C. Adi1,* , Ali I. Tawakal2 , Mohammad F. Rasyidi2 ,
Wizara Salisa2 , Farapti Farapti1 , Heni Rachmawati3
Airlangga University , Surabaya, Indonesia
1

Rumah Inovasi Natura, Surabaya, Indonesia

2

3
Bandung Institute of Technology , Bandung, Indonesia

* e-mail: annis_catur@[Link]

Received 27.08.2022; Revised 03.12.2022; Accepted 10.01.2023; Published online 04.04.2023

Abstract:
Organic waste is a problem the cocoa industry has to handle. The industry produces a lot of cocoa bean husk, also called criollo
cocoa husk. Cocoa bean husk is an underutilized cocoa waste that contains bioactive components in the form of phenols and
flavonoids. Processed cocoa bean husk can be brewed as a functional beverage.
The research objective was to test cocoa husk tea for sensory properties, bioactive components, and impact on blood cholesterol.
This study used a randomized experimental design with six repetitions. Sensory data were processed using the Friedman and
Wilcoxon signed-rank tests (α = 0.05) to determine the difference in sensory properties between each formulation of cocoa husk
tea.
The sensory evaluation involved 30 untrained panelists who gave the highest score to the formulation with 62.5% cocoa bean
husk, 25% lemongrass, and 12.5% aromatic ginger, which could also reduce 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals
(IC50 = 264.8675). The animal test showed that the cocoa husk formulation produced no significant difference (p > 0.05) in pre-
and post-treatment, but was able to keep cholesterol within normal limits.
Cocoa bean husk showed health benefits by its antioxidant properties and ability to control blood cholesterol.

Keywords: Сriollo cocoa husk, sensory characteristic, bioactive value, blood cholesterol, food waste

Funding: This research was supported by the Public Health Faculty of Airlangga University , grant no. 2059/UN3.1.10/
PT/2020.

Please cite this article in press as: Adi AC, Tawakal AI, Rasyidi MF, Salisa W, Farapti F, Rachmawati H. Effect of cocoa
husk Criollo tea on hypercholesterolemia in animal model. Foods and Raw Materials. 2023;11(2):206–214. [Link]
10.21603/2308-4057-2023-2-567

INTRODUCTION weight of cocoa beans [3]. A proper utilization of this

Organic waste produced by the food or beverage by-product could prevent many environmental problems
industry covers a wide range of substances that cannot that arise from it being dumped as waste.
be consumed for nutrition purposes [1]. Dried cocoa Cocoa husk has a good biofunctional potential for
beans and fermented cocoa beans are the main raw human health. It contains a lot of polyphenols that
materials for all types of cocoa products, while cocoa possess numerous biofunctional properties and health
bean husk is a by-product of the cocoa industry. The benefits. Flavonoids are the main polyphenol class in
three most popular types of cocoa are Criollo, Forastero, cocoa and their by-products [4]. According to scientific
and Trinitario, which together make up 95% of the total evidence, polyphenols are good for human health
cocoa produced in the world. Criollo cocoa husk is a due to their antioxidant properties: they act as free
by-product with highly valuable bioactive components. radical scavengers and reduce oxidative stress. The
However, it is discarded without recycling [2]. The bioactive substances contained in cocoa husk have
weight of cocoa husk ranges from 10–17% of the total antibacterial, antiviral, anticarcinogenic, antidiabetic,

Copyright © 2023, Adi et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International
License ([Link] allowing third parties to copy and redistribute the material in any medium or format and to remix,
transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.

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 Adi A.C. et al. Foods and Raw Materials. 2023;11(2):206–214

Table 1 Cocoa bean husk beverage formulations

Ingredients Formulation
Control 1 2 3 4
g % g % g % g % g %
Cocoa bean husk 8.0 100.0 5.0 62.5 5.0 62.5 5.0 62.5 5.0 62.5
Lemongrass – – 2.0 25.0 1.0 12.5 2.0 25.0 1.0 12.5
Ginger – – 1.0 12.5 1.0 12.5 – – – –
Turmeric – – – – 1.0 12.5 – – 1.0 12.5
Aromatic ginger – – – – – – 1 12.5 1.0 12.5
Mineral water 200.0 96.2 200 96.2 200.0 96.2 200.0 96.2 200.0 96.2

and neuroprotective properties, not to mention their the Health Research Ethics Committee, Department
beneficial effect on the cardiovascular system [5]. The of Dental Medicine, Universitas Airlangga (Number:
total polyphenol content in one cocoa pod is slightly 379/[Link]/ VIII/2020).
higher than in cocoa husk, but the total flavonoid con- Cocoa husk tea development. We designed four
tent in cocoa husk is almost twice higher than in cocoa cocoa husk tea formulations (Table 1). Formulations 1
pods [6]. and 2 contained such spices as lemongrass, ginger, and
Cocoa polyphenols can affect the lipid profile and turmeric. Formulations 3 and 4 included lemongrass,
enhance the antiatherogenic effect. Saad tested cocoa turmeric, and aromatic ginger. The spices were intended
polyphenols in vitro and in cell culture to demonstrate to improve the taste. Ginger (Zingiber officinale) was
the inhibition of low-density lipoprotein oxidation chosen because it contains active compounds with
and reduction of low-density lipoprotein oxidative anti-inflammatory and antioxidant properties [9, 10].
susceptibility [7]. Experimental rats after a month Lemongrass (Cymbopogon citratus) is known to prevent
on a cocoa powder diet improved their lipid profile several diseases because it has antibacterial, antifungal,
and demonstrated a low cardiovascular risk. Polyphe- antioxidant, antiseptic, anti-inflammatory, analgesic,
nols have a protective effect against atherosclerosis: and antipyretic properties [11, 12]. Turmeric (Curcuma
they alter the hepatic cholesterol homeostasis by redu- longa) is useful as an anti-inflammatory, anti-oxidant,
cing cholesterol absorption. Polyphenols were also anti-microbial, cancer-prevention, and anti-tumor agent.
reported to lower blood pressure and the activity of It can reduce fat and cholesterol in the blood and acts as
enzymes in the renin-angiotensin-aldosterone system, a blood purifier [13]. It also lowers blood pressure and
which are involved in the renin-angiotensin-aldosterone improves rheumatism [14]. Likewise, aromatic ginger
system [8]. (Kaempferia galanga L) also contains antioxidants, like
A functional cocoa-husk beverage is a solution to other spices [15]. The production process required an
the cocoa waste problem, not to mention its potential oven, a baking sheet, a tray, a measuring spoon, a digital
for human health. Cocoa bean husk can be processed scale, and a grinding machine, as well as tea brewing
equipment in the form of hollow tea bags.
into a functional beverage with a good antiradical
CHC phytochemical screening. Dry cocoa beans
and cardioprotective potential because it contains
(Theobroma cacao L.) served as the main raw material.
antioxidants in the form of polyphenols (flavonoids).
The list of reagents included Dragendorff’s reagent,
Cocoa bean husk is processed into functional beverage
Mayer’s reagent, and Stiasny’s reagent. Dragendorff’s
starting with sorting and sterilization, followed by
reagent was prepared by mixing 0.8 g of bismuth nitrate
packaging into brewed bags. The present research
and 20 mL of HNO3 (p) with 27.2 grams of Kl dissolved
objective was to find out the sensory profile of cocoa
in 50 mL of water. The solutions were allowed to stand
husk functional beverages and their effect on blood
for 24 h, filtered, and brought up to 100 mL with Aqua
cholesterol.
Dest. Mayer’s reagent was made by mixing 1.36 g of
HgCl2 and 60 mL of Aqua Dest with 5 g of Kl dissolved
STUDY OBJECTS AND METHODS in 10 mL of water. The two solutions were mixed and
Research design. We used a complete randomized brought up to 100 mL with Aqua Dest. Stiasny’s reagent
experimental design with six repetitions. We develo- was prepared by mixing two parts of 30% formalde-
ped several tea formulations based on criollo cocoa hyde with one part of concentrated hydrochloric acid.
husk. The research included a qualitative analysis of The method used in this research was a laboratory
bioactive and sensory components based on such experimental method. The tests covered flavonoids,
parameters as color, smell, taste, and concentration, as tannins, quinones, saponins, steroids/triterpenoids, and
well as a pre-clinical animal test. The panelists gave alkaloids.
an informed consent before the sensory evaluation. The flavonoid examination referred to Farnsworth:
The evaluation procedure had no adverse effect the sample was heated with hot water and mixed with
on the panelists. The research was approved by magnesium powder, hydrochloric acid, and amyl

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alcohol solution [16]. Yellow, orange, and red staining very much, 2 = disliked, 3 = disliked a little, 4 = neutral,
indicated the presence of flavonoid compounds. This 5 = liked a little, 6 = liked, and 7 = liked very much. The
test as described by Stefova et al. and Nugroho used samples were tea from the cocoa husk with mineral water
the standard High-Performance Liquid Chromatography and spices. The panelists were given mineral water and
(HPLC) method [17, 18]. advised to drink it before moving to the next tea sample.
The method for examining tannins was approved Blood cholesterol test in experimental animals.
by WHO and the Association of Official Agricultural The experimental animals used in this study were male
Chemists [19]. The tannin testing followed the pro- Wistar strains rats (Rattus norvegicus) aged 2–3 months
cedure described by Farnsworth: the heated sample with a weight of 160–240 g. Before entering the treat-
solution was mixed with three different reagents, namely ment stage, all experimental animals were adapted
iron (III) chloride, gelatin, and Stiasny’s reagent [16]. for 7 days. Each rat was placed in a different cage ac-
The positive test for tannins was indicated by a change cording to the treatment group. The rats were treated
in color for each reagent: blue-black, white, and pink, and controlled in a fixed environment to make them
respectively. able to adapt to the new conditions. The conditions pre-
The quinone test was based on the method developed supposed room temperature and sufficient light. Food
by Farnsworth [16]. The sample solution was processed and drinks were given ad libitum. Induction of hyper-
through the initial stages of heating. The boiling solution cholesterolemia was performed by testing cholesterol
was then mixed with sodium hydroxide. Red staining tolerance by giving extra egg yolk.
indicated a positive quinone test. The same initial The experimental animals were grouped into three
preparation process was also valid for the saponin tests: phases: the adaptation experiment phase, the induction
after adding a reagent of hydrochloric acid, the vile was phase, and the intervention phase. During the adaptation
shaken vertically for 10 s. stage, all the rats were treated normally using pellet feed
The test for steroids/triterpenoids involved adding for 7 days. During the induction stage, they were divi-
20 mL of ether to 1 g of sample followed by grinding ded into five groups: positive control, negative control,
and filtering. The filtrate was put into a vaporizer cup Treatment 1, Treatment 2, and Treatment 3. During the
and allowed evaporating. Then few drops of Lieber- induction stage, all the groups were induced using egg
mann-Burchard reagent were added. Green-blue or red- yolk, except for the negative control group (–), where the
purple staining indicated the presence of steroids/tri- pellet induction lasted two weeks.
terpenoids. For the alkaloid test, a solution of 10 mL The intervention stage involved three types of treat-
HCl was mixed with 2 g of sample, crushed in a mortar, ment using simvastatin at a dose of 1 mg/kg BW, as well
and then filtered. After that, 5 mL of 25% ammonia was as 200 and 400 mg cocoa bean husk extract. Figure 1
added to the filtrate and extracted with 20 mL of chlo- summarizes the details.
roform. The chloroform layer was removed, and part of Statistical analysis. The sensory data were first
it was dropped on filter paper, where it reacted with tested for normality and homogeneity by the Saphiro-
Dragendorff’s reagent. Orange staining indicated a Wilk test. If the data were distributed normally, they
positive alkaloid test. were processed by the analysis of variance (ANOVA)
Cocoa husk antioxidant test. The antioxidant acti- to determine the difference between parameters. The
vity test relied on the procedure described by Filbert parameters that were found to be different underwent
with several modifications [20]. It involved a sample so- Fisher’s test (p ≤ 0.05). If the assumption of normality
lution with a concentration of 1000 µg/mL and a 0.4 mM was not met, the data were subjected to the Friedman
DPPH solution. The sample stock solution was diluted test and the Wilcoxon test (α = 0.05).
to various concentrations with a total volume of 1.6 mL. The statistical data analysis of the cholesterol test
The solution was then put into a test tube as a test was conducted by using the normality test according
solution, followed by producing a blank solution. At to the Saphiro-Wilk test. If the data were distributed
the next stage, the test solution was mixed with 0.4 mL normally (p > 0.05), it was followed by the paired
of DPPH in the test tube and underwent an incubation sample t-test analysis. If the data were not distributed
process for 30 min in the dark. After that, the blank normally, then the Wilcoxon test was used. The test was
and the test solution were measured for absorbance conducted to determine the differences in cholesterol
value using UV-Vis spectrophotometry at a maximal levels in the pre- and post-test through the post hoc test.
wavelength of 516 nm. Each sample was tested for If the significance value obtained was < 0.05, it meant
absorbance value of the inhibition percentage (%) and that there was a difference between cholesterol levels in
IC50 value [21]. the pre-test and post-test in all treatments. The statistical
Sensory evaluation. The sensory evaluation revea- analysis was tested using the IBM Statistics SPSS 22
led the consumer appeal and feasibility [22]. It included software.
such parameters as color, smell, taste, and concentration
of cocoa bean husk beverages. Five trained panelists RESULTS AND DISCUSSION
and 30 untrained panelists filled in a questionnaire. The Cocoa husk tea development. The cocoa bean
panelists were asked to assess the samples based on husk tea included several types of spices (Table 1).
their level of preference on a 7-point scale: 1 = disliked The spices were intended to improve the taste. Ginger

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Wistar rat

Sample criteria

Wistar rat

2–3 months old, 160–240 g

Experimental
stages (7 days
adaptation)
Pre induction
(pellet)

Induction stage (2 weeks)

Control (–) Control (+) Treatment 1 Treatment 2 Treatment 3

– pellet – pellet – pellet – pellet – pellet

– egg yolk – egg yolk – egg yolk – egg yolk

Intervention Stage (2 weeks)

Treatment 1 Treatment 2 Treatment 3

– pellet – pellet – pellet

– egg yolk – egg yolk – egg yolk
– simvastatin – cocoa husk – cocoa husk
extract 200 mg extract 400 mg

Figure 1 Cholesterol tests in experimental animals

Phytochemical screening results. The phytochemi-
Table 2 Phytochemical screening of cocoa bean husk cal screening employed laboratory experimental me-
thods. It included tests for flavonoids, tannins, quinones,
Compounds Result saponins, steroids/triterpenoids, and alkaloids (Table 2).
Alkaloids – The results of the phytochemical screening showed
Flavonoids + that cocoa bean husk contained flavonoids, condensed
Saponins – tannins, and steroids. The fact that it also contained fla-
Quinones – vonoid compounds, condensed tannins, and steroids/
Hydrolyzable tannins – triterpenoids was in line with Yumas, who subjected
Condensed tannins + cocoa bean husk to phytochemical screening and
Steroids/triterpenoids + reported flavonoids, tannins, and triterpenoids [23].
The abovementioned substances belong to secondary
+: secondary metabolite detected
metabolites. The screening detected no alkaloid com-
–: secondary metabolite undetected
pounds, saponins, quinones, or hydrolyzable tannins.
Flavonoids are secondary metabolite compounds that
(Zingiber officinale) was chosen for its bioactive compo- are commonly found in plant tissues. Flavonoids belong
to phenolic compounds with a chemical structure of C6-
unds that have anti-inflammatory and antioxidant pro-
C3-C6 [24]. They are powerful antioxidants as they are
perties [9, 10]. Lemongrass (Cymbopogon citratus) is
able to release hydrogen atoms [25].
known to prevent several diseases because it possesses Tannins are another type of secondary metabolites
antibacterial, antifungal, antioxidant, antiseptic, anti- found in plants. Tannins are antioxidants: the larger their
inflammatory, analgesic, and antipyretic properties [11, content, the greater their antioxidant activity. Tannins
12]. Turmeric (Curcuma longa) is an anti-coagulant and owe their antioxidant activity to the fact that they con-
anti-oxidant. Aromatic ginger (Kaempferia galanga L) tain polyphenolic compounds that are able to capture
also contains antioxidants [15]. free radicals [26].

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Steroids are natural compounds with a carbon ske- Table 3 Average sensory test score of cocoa bean husk
leton. They belong to the type of secondary metabo- beverage
lite compounds. Steroids are found in nature and deri-
Formulation
ved from triterpene. Steroids of plant origin are derived
Indicators Control 1 2 3 4
from cycloartenol triterpenes. Early in their formation,
Color 3.0 5.0 6.5 6.0 6.0
acetic acid converts into cycloartenol through meva-
Smell 6.0 4.5 5.0 6.0 5.0
lonic acid and squalene [27]. Steroids are also classified
Taste 2.0 5.0 3.8 5.0 4.0
as secondary metabolites and are known to possess anti-
Concentration 1.5 5.5 4.5 5.5 5.5
oxidant and antibacterial properties [28].
Total 12.5 20.0 19.8 22.5 20.5
Antioxidant test results on cocoa bean husk Average 3.1 5 4.9 5.6 5.1
simplicial. Simplicia is a natural material that has not
undergone any changes or processing, e.g., it has not
been dried [29]. We tested the antioxidant activity of F4
F4
cocoa bean husk simplicia to measure the effect of F3
F3
phytochemical substances in the initial raw material, i.e., F2
F2
before processing. F1
F1
The simplicia calibration showed that the percentage Fcontrol
Fcontrol
of inhibition increased together with concentration, 0%
0% 50%
50% 100%
100%
which was raised gradually. The resulting value of
R² = 0.9956 demonstrated that these two variables had a Liked
Liked Disliked
Disliked
strong effect.
The maximal wavelength was 516 nm. The control Figure 2 Color assessment of cocoa bean husk beverage, %
absorbance values were 0.973, 0.905, and 0.934 with
a mean value of 0.937. The values of IC50 and IAA for
cocoa bean husk simplicia were very weak – 1302.414 elucidation was not conducted). Therefore, the
and 0.034551, respectively. Thus, the antioxidant activity antioxidant effect could be provided only by regular and
was weak: IC50 < 50 ppm = very strong antioxidant; continuous consumption of cocoa bean husk infusion.
50–100 ppm = strong; 101–150 ppm = moderate; 150– Sensory profile. Five formulations of cocoa bean
200 ppm = weak [30]. husk beverages (Table 1) where bean husk was sub-
Antioxidant activity of cocoa bean husk extract. stituted with various spices were tested for consumer
The next antioxidant test was carried out on the cocoa appeal. Table 3 shows the results of the sensory test for
bean husk extract to determine the effect of processing each parameter. Among the five samples, Formulation 3
on the antioxidant activity of cocoa bean husk. The (62.5% cocoa bean husk with 25% lemongrass and
antioxidant test preceded both the sensory test and the 12.5% aromatic ginger) attained the highest preference
experimental animal test. value based on the mean value of all parameters, i.e.,
The results of the extract calibration showed that color, smell, taste, and concentration.
the percentage of inhibition increased together with Color is an important aspect that can affect food and
concentration. The obtained value of R² = 0.9949 beverage preferences [31]. The sensory assessment of
demonstrated that these two variables had a strong effect. color showed that the range of color acceptance scores
The maximal wavelength was 516 nm. The control was 3.0–6.5, which means that it ranged from “disliked
absorbance values were 0.977, 0.951, and 0.954 with a a little” to “liked”. The highest color score was obtained
mean value of 0.961. The IC50 and IAA values of cocoa by Formulation 2 (62.5% cocoa bean husk, 12.5%
bean husk extract were found weak – 264.8675 and lemongrass, 12.5% ginger, 12.5% turmeric, 12.5%
0.169896, respectively. Thus, the antioxidant activity of aromatic ginger), while the lowest preference score was
cocoa bean extract was still weak. Compared with the obtained by the control formulation with 100% cocoa
results for simplicia, the extract showed even lower IC50 bean husk.
values. The panelists preferred a lighter shade to the dark
The antioxidant activity test was based on the 2,2- one: the lighter samples got a higher score than the dark-
diphenyl-1-picrylhydrazyl (DPPH) method. Cocoa bean colored ones. The more cocoa bean husk was added,
husk simplicia demonstrated antioxidant properties. the darker the color became [22]. When a part of cocoa
However, when compared to the antioxidant standards, husk was substituted with spices, it produced a yellowish
the antioxidant activity of cocoa bean husk fell into color or affected the brightness. Extra spices made the
the weak category, as indicated by its IC50 value. This drink slightly yellow or bright in color. Turmeric was
antioxidant ability was in line with the phytochemical responsible for the yellow color as it is known to contain
screening results for flavonoid content (Table 2). The three pigments: curcumin, dimethoxy-curcumin, and
weak antioxidant power might have been due to the low bis dimethoxy-curcumin [32]. Ginger powder alone
content of flavonoid compounds (not yet quantified) produces a yellowish-white color when dissolved in
or the type of flavonoid compound that had a chemical water [33]. Pure lemongrass powder dissolved in water
structure with a weak electron transfer ability (structural produces a yellowish color [34].

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F4
F4 F4
F4
F3
F3 F3
F3
F2
F2 F2
F2
F1
F1 F1
F1
Fcontrol
Fcontrol Fcontrol
Fcontrol
0%
0% 50%
50% 100%
100% 0%
0% 50%
50% 100%
100%
Liked
Liked Neutral
Neutral Disliked
Disliked Liked
Liked Neutral
Neutral Disliked
Disliked

Figure 3 Smell assessment of cocoa bean husk beverage, % Figure 4 Taste assessment of cocoa bean husk beverage, %

F4 Color
F4
7
F3
F3 6
F2 5
F2 4
F1
F1 3
Fcontrol 2
Fcontrol 1
0% 50% 100% Concentration 0 Smell
0% 50% 100%
Liked
Liked Neutral
Neutral Disliked
Disliked

Figure 5 Concentration assessment of cocoa bean husk

beverage, % Taste
F0 F0F0 F1F1 F1F1
Fcontrol
F0 F1 F2 F2 F2
F2 F3 F3 F3F3F4 F4 F4F4
F2
F0F0 F1F1 F2F2 F3
F3F3 F4
F4
F4
The percentage of panelists who claimed to like the
color was as follows: Formulation 0 – 40%, Formulation
Figure 6 Hedonic test results
1 – 60%, Formulation 2 – 100%, Formulation 3 – 60%,
and Formulation 4 – 80%. Formulation 2 had the best
score for color: all 30 panelists appreciated it (Fig. 2). a lower proportion of cocoa bean husk. Polyphenols that
Figure 3 demonstrates smell evaluation of the give cocoa bean husk its antioxidant properties produce
cocoa bean husk beverages. The highest score for smell a slightly bitter and sour taste [23].
went to the control sample and Formulation 2 (62.5% The score for the concentration ranged from 1.5
cocoa bean husk, 12,5% lemongrass, 12.5% ginger, to 5.5, which means that it ranged from “disliked very
12,5% turmeric). The lowest smell score belonged to much” to “liked a little” (Fig. 5). The highest concentra-
Formulation 1 (62.5% cocoa bean husk, 25% lemongrass, tion score belonged to Formulation 1 (62.5% cocoa bean
12.5% ginger). The panelists preferred the beverage with husk, 25% lemongrass, 12.5% ginger), Formulation 3
a distinctive cocoa flavor. The characteristic cocoa smell (62.5% cocoa bean husk, 25% lemongrass, 12.5%
appears as a result of the reaction between cocoa smell aromatic ginger), and Formulation 4 (62.5% cocoa bean
precursors (free amino acids and peptides) and sugar husk, 12.5% lemongrass, 12.5% turmeric, 12.5% aro-
that enter the Maillard reaction to produce such smell matic ginger). The control sample received the lowest
components as alcohol, ether, furan, thiazole, pyrone, concentration score.
acid, ester, aldehyde, amine, amine, oxazole, pyrazine, The acceptability value of cocoa bean husk beve-
and pyrrole. One of the compounds responsible for the rages was based on the sensory test which included color,
characteristic smell of cocoa is 2,3-butanediol [35]. smell, taste, and concentration (Fig. 6).
The highest taste score belonged to Formulation 3 Formulation 3 (62.5% cocoa bean husk, 25% lemon-
(62.5% cocoa bean husk, 25% lemongrass, 12.5% aro- grass, 12.5% aromatic ginger) received the highest
matic ginger), while the lowest score belonged to the preference value based on all the sensory indicators,
control (Fig. 4). The panelists preferred the samples with i.e., color, smell, taste, and concentration.

Table 7 Paired sample t-test: pre- and post-intervention cholesterol levels

Treatment groups Cholesterol levels, mg/dL A p-value of paired t-test results Conclusion
Pre-test Post-test
Negative control 29.8 ± 4.38 38.0 ± 8.64 0.902 No difference (normal cholesterol level)
Positive control 33.8 ± 5.54 86.5 ± 8.87 0.002 Difference detected (high cholesterol level)
Treatment 1 34.8 ± 6.18 58.0 ± 4.55 0.606 No difference (high cholesterol level)
Treatment 2 44.0 ± 3.39 42.0 ± 14.16 0.841 No difference (normal cholesterol level)
Treatment 3 33.0 ± 2.86 33.8 ± 8.32 0.942 No difference (normal cholesterol level)

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86.5 Treatment 2 and Treatment 3, which involved cocoa

100 58.0
44.0 42.0 33.0 33.8 bean husk extract, maintained cholesterol levels in
33.8 86.5 34.8
Cholesrol Levels

100 58.0 the normal range but provided no significant changes.

33.8 34.8 44.0 42.0 33.0 33.8 Treatment 1, which involved simvastatin, demonstra-
10 ted a significant difference in blood cholesterol levels.
10 The positive control also showed a significant difference,
which was inversely proportional to the negative
1 control. According to Ahmad & Amy, cocoa injections
C+ T1 T2 T3
1 in rats reduced their low-density lipoprotein (LDL)
C+ T1Groups
Pre Test Post Test
T2 T3 levels. Darand et al. also mentioned the effect of cocoa
consumption which could significantly reduce blood
Pre Test Post Test
cholesterol levels in humans [37, 38]. Sweety also stated
that cocoa products had a significant anti-cholesterol
Figure 7 Effect of cocoa bean husk extract on cholesterol impact [39].
in rats The mechanism of reducing LDL cholesterol and
total cholesterol in experimental animals could be
explained by the high flavonoid content in cocoa
Blood cholesterol value in experimental animals.
bean husk. Flavonoids with their antioxidant and anti-
The data collected for blood cholesterol tests were the
inflammation properties increased the function of
initial data before the intervention and the final data
high-density lipoprotein (HDL) cholesterol, which
collected two weeks after the intervention period. The
decreased the total cholesterol levels. HDL choles-
pre-test and post-test data were compared to reveal
terol removed reverse cholesterol transport in the
differences in the results of the intervention activities
blood [40]. For instance, Martinez et al. also reported
(Table 4).
that an increase in the consumption of cocoa flavonoids
Normal blood cholesterol level in rats is 10–
improved the level of HDL cholesterol [41]. Flavonoid-
54 mg/dL [36]. The paired sample t-test showed that the
rich foods could improve the endothelial function in
negative control treatment with pellets increased cho-
cells [42]. Out of the five types of intervention trials,
lesterol from 29.8 mg/dL in pre-intervention to 38 mg/dL
Treatments 2 and 3, which involved cocoa bean extract,
in post-intervention (Table 7). The negative control de-
managed to keep cholesterol levels in rats within the
monstrated no significant difference in cholesterol levels
normal limits, although they provided no significant
(p-value = 0.902 (> 0.05)). Thus, the negative control
change.
treatment had no significant effect on cholesterol.
The positive control group received duck egg yolk.
CONCLUSION
As a result, the pre-cholesterol level of 33.8 mg/Dl (nor-
mal cholesterol) increased to 86.5 mg/dL (high chole- Cocoa bean husk extract, a by-product of the cocoa
sterol). The difference between pre-test and post-test industry, showed health benefits through its antioxidant
was significant with a p-value of 0.002 (< 0.05). Hence, activity. It was able to reduce DPPH free radicals
the positive control treatment had a significant effect on (IC50 = 264.8675) better than cocoa bean husk simplicia
the changes in cholesterol (Fig. 7). (IC50 = 1302.414). The blood cholesterol animal tests
Treatment 1 included pellets, duck egg yolk, and registered no significant difference, but the extract was
simvastatin. As a result, the pre-cholesterol level of able to maintain cholesterol within normal limits. The
34.8 mg/dL reached 58 mg/dL. No significant difference lighter-colored samples with little cocoa husk received
in cholesterol levels was detected (p-value = 0.841 the best score for color and concentration, while the
(> 0.05)). Thus, Treatment 1 had no significant effect on samples with a higher proportion of cocoa bean husk
cholesterol. received a higher score for the characteristic cocoa
Treatment 2 involved pellets, duck egg yolks, and smell. As for the taste, the panelists preferred the sample
200 mg/kg BW of cocoa bean husk extract. From the with less cocoa bean husk. The highest mean score for
precholesterol level of 44 mg/dL, it fell down to sensory properties belonged to the sample with 62.5%
42 mg/dL. Therefore, we detected no significant dif- cocoa bean husk, 25% lemongrass, and 12.5% aromatic
ference in cholesterol levels between pre-test and post- ginger.
test (p-value = 0.841). Treatment 2 produced no sig-
nificant effect on cholesterol but was able to maintain it CONTRIBUTION
within normal limits. Conceptualization: A.C. Adi and H. Rachmawati;
Treatment 3 involved pellets, duck egg yolks, and methodology: H. Rachmawati and F. Farapti; software:
400 mg/kg BW of cocoa bean husk extract. The pre- W. Salisa; validation: A.I. Tawakal; formal analysis:
cholesterol level of 33.3 mg/dL remained almost the W. Salisa and M.F. Rasyidi; investigation: W. Salisa
same: 33.8 mg/dL. The difference between pre-test and and A.I. Tawakal; resources: F. Farapti; data curation:
post-test was not significant (p-value = 0.942). Thus, W. Salisa; original draft: M. Rasyidi; review and editing:
Treatment 3 had no significant effect on cholesterol but H. Rachmawati, F. Farapti, M.F. Rasyidi; visualization:
was able to maintain it within normal limits. W. Salisa; supervision: A.C. Adi and H. Rachmawati;

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 Adi A.C. et al. Foods and Raw Materials. 2023;11(2):206–214

project administration: A.C. Adi and H. Rachmawati. CONFLICTS OF INTEREST

All the authors have read and agreed to the published The authors declared no conflict of interests
version of the manuscript. regarding the publication of this article.

REFERENCES
1. Barišić V, Jozinović A, Flanjak I, Šubarić D, Babić J, Milicević B, et al. Difficulties with use of cocoa bean shell in food
production and high voltage electrical discharge as a possible solution. Sustainability. 2020;12(10). [Link]
10.3390/SU12103981
2. Panak Balentić J, Ačkar Đ, Jokić S, Jozinović A, Babić J, Miličević B, et al. Cocoa shell: a by-product with great
potential for wide application. Molecules. 2018;23(6). [Link]
3. Hashimoto JC, Lima JC, Celeghini RMS, Nogueira AB, Efraim P, Poppi RJ, et al. Quality control of commercial
cocoa beans (Theobroma cacao L.) by near-infrared spectroscopy. Food Analytical Methods. 2018;11(5):1510–1517.
[Link]
4. Socci V, Tempesta D, Desideri G, de Gennaro L, Ferrara M. Enhancing human cognition with cocoa flavonoids.
Frontiers in Nutrition. 2017;4. [Link]
5. Rojo-Poveda O, Barbosa-Pereira L, Zeppa G, Stévigny C. Cocoa bean shell – A by-product with nutritional properties
and biofunctional potential. Nutrients. 2020;12(4). [Link]
6. Karim AA, Azlan A, Ismail A, Hashim P, Abdullah NA. Antioxidant properties of cocoa pods and shells. Malaysian
Cocoa Journal. 2014;8:49–56.
7. Saad S. Biochemical and nutritional studies on cocoa powder and its effect on blood lipids in experimental rats.
African Journal of Biological Sciences. 2018;14(1):121–130.
8. Murillo AG, Fernandez ML. The relevance of dietary polyphenols in cardiovascular protection. Current Pharmaceutical
Design. 2017;23(17):2444–2452. [Link]
9. Aryanta IW. Benefits of ginger for health. Widya Kesehatan; 2019.
10. Ginger: Health benefits and dietary tips [Internet]. [cited 2022 Jul 7]. Available from: [Link]
com/articles/[Link]
11. Minasari, Nasution DL. The effectivity of lemongrass (Cymbopogon citratus) extract against porphyromonas gingi-
valis ATCC® 33277™ (in-vitro). Advances in Health Sciences Research; 2018;8:169–172. [Link]
idcsu-17.2018.45
12. Oladeji OS, Adelowo FE, Ayodele DT, Odelade KA. Phytochemistry and pharmacological activities of Cymbopogon
citratus: A review. Scientific African. 2019;6. [Link]
13. Bavappa KV, Leon J, Withers LA. Turmeric (Curcuma domestica Val). In: Leon J, Withers LA, editors. Guidelines for
seed exchange and plant introduction in tropical crops. Rome: FAO; 1986. pp. 162–163.
14. Prabawati T, Pujimulyani D. Effect of addition of kencur extract on color, antioxidant activity, and preference level of
white turmeric instant drink. Local Food Innovation to Support Food Security. Yogyakarta: Mercu Buana University
Yogyakarta; 2018.
15. Farnsworth NR. Biological and phytochemical screening of plants. Journal of Pharmaceutical Sciences. 1966;55(3):225–
276. [Link]
16. Stefova M, Stafilov T, Kulevanova S. HPLC analysis of flavonoids. In: Cazes J, editor. Encyclopedia of chromatography.
New York: Marcel Dekker; 2003. pp. 113–119.
17. Nugroho A. Identification and HPLC quantification of flavonoid compounds in Chrysanthemum boreale. Prosiding
Seminar Nasional. 2015:299–302. (In Indonesian).
18. The International Pharmacopoeia, Ninth Edition. World Health Organization; 2019. 1865 p.
19. Filbert, Koleangana HSJ, Runtuwenea MRJ, Kamu VS. Determination of antioxidant activity based on IC50 value
of methanol extract and partition result fraction on yaki betel nut (Areca vestiaria giseke) peel. MIPA Unsrat Online.
2014;3(2):149–154. (In Indonesian).
20. Lisi AKF, Runtuwene MRJ, Wewengkang DS. Phytochemical test and antioxidant activity of soyogic flower methanol
extract (Saurauia bracteosa DC). Pharmacon. 2017;6(1). (In Indonesian). [Link]
21. Langkong J, Mahendradatta M, Tahir MM, Rahman ANF, Abdullah N, Marina N. Utilization of cocoa bean husk
extract (Theobroma cacao L) on the product chocolate cookies. Canrea Journal: Food Technology, Nutritions, and
Culinary Journal. 2020;3(1):42–48. [Link]
22. Rastogi S, Pandey MM, Rawat AKS. Spices: Therapeutic potential in cardiovascular health. Current Pharmaceutical
Design. 2016;23(7):989–998. [Link]

213
 Adi A.C. et al. Foods and Raw Materials. 2023;11(2):206–214

23. Yumas M. Utilization of cocoa beans epidermis waste (Theobroma cacao L) as antibacterial streptococcus mutans.
Jurnal Industri Hasil Perkebunan. 2017;12(2):7–20. [Link] (In Indonesian).
24. Zaynab M, Fatima M, Abbas S, Sharif Y, Umair M, Zafar MH, et al. Role of secondary metabolites in plant defense
against pathogens. Microbial Pathogenesis. 2018;124:198–202. [Link]
25. Vo QV, Nam PC, Thong NM, Trung NT, Phan C-TD, Mechler A. Antioxidant motifs in flavonoids: O−H versus C−H
bond dissociation. ACS Omega. 2019;4(5):8935–8942. [Link]
26. Sawunggaling F. Identification of tannin compounds and antioxidant activity on parasite mango leaves (Dendropthoe
pentandra L) from tegal and brebes regions. Harapan Bersama Polytechnic City of Tegal; 2020.
27. Salempa PM, Muharram H. Steroid compounds in bayur plants. Badan Penerbit UNM; 2016. 71 p. (In Malaysian).
28. Lin M, Han P, Li Y, Wang W, Lai D, Zhou L. Quinoa secondary metabolites and their biological activities
or functions. Molecules. 2019;24(13). [Link]
29. Indonesian herbal pharmacopoeia Edition II. Jakarta: Kementerian Kesehatan Republik Indonesia; 2017. 561 p.
(In Indonesian).
30. Widyasanti A, Rohdiana D, Ekatama N. Antioxidant activity of white tea extract (Camellia sinensis) by DPPH method
(2,2 diphenyl-1-pikrylhydrazil). Fortech. 2016;1(1).
31. Krishna A, Cian L, Aydınoğlu NZ. Sensory aspects of package design. Journal of Retailing. 2017;93(1):43–54. https://
[Link]/10.1016/[Link].2016.12.002
32. Dabas D. Polyphenols as colorants. Advances in Food Technology and Nutritional Sciences. 2018;SE(2):S1–S6.
[Link]
33. Singha P, Muthukumarappan K. Quality changes and freezing time prediction during freezing and thawing of ginger.
Food Science and Nutrition. 2016;4(4):521–533. [Link]
34. Utomo D, Ariska SB. The quality of drinks powders instant lemongrass (Cymbopogon citratus) with method foam mat
drying. Teknologi Pangan. 2020;11(1):42–51. (In Indonesian). [Link]
35. Moreira IMDV, de Figueiredo Vilela L, Santos C, Lima N, Schwan RF. Volatile compounds and protein profiles
analyses of fermented cocoa beans and chocolates from different hybrids cultivated in Brazil. Food Research
International. 2018;109:196–203. [Link]
36. Harini M, Astirin OP. Blood cholesterol level of hypercholesterolemia rat (Rattus norvegicus) after VCO treatment.
Nusantara Bioscience. 2009;1(2):53–58. [Link]
37. Ahmad MN, Amr AM. The effect of defatted cocoa powder on cholesterol-induced changes of serum lipids in rats.
Nutrición Hospitalaria. 2017;34(3):680–687. [Link]
38. Darand M, Hajizadeh Oghaz M, Hadi A, Atefi M, Amani R. The effect of cocoa/dark chocolate consumption on lipid
profile, glycemia, and blood pressure in diabetic patients: A meta-analysis of observational studies. Phytotherapy
Research. 2021;35(10):5487–5501. [Link]
39. Sweety GJ. Assess the effectiveness of cocoa powder in reducing cholesterol levels among hypertensive clients in the
rural area, of Medavakkam, Chennai. Asian Journal of Nursing Education and Research. 2020;10(3):260–264. https://
[Link]/10.5958/2349-2996.2020.00055.5
40. Millar CL, Duclos Q, Blesso CN. Effects of dietary flavonoids on reverse cholesterol transport, HDL metabolism, and
HDL function. Advances in Nutrition. 2017;8(2):226–239. [Link]
41. Munguia L, Rubio-Gayosso I, Ramirez-Sanchez I, Ortiz A, Hidalgo I, Gonzalez C, et al. High flavonoid cocoa
supplement ameliorates plasma oxidative stress and inflammation levels while improving mobility and quality of life
in older subjects: A double-blind randomized clinical trial. The Journals of Gerontology: Series A. 2019;74(10):1620–
1627. [Link]
42. Bondonno NP, Bondonno CP, Blekkenhorst LC, Considine MJ, Maghzal G, Stocker R, et al. Flavonoid-rich apple
improves endothelial function in individuals at risk for cardiovascular disease: A randomized controlled clinical trial.
Molecular Nutrition and Food Research. 2018;62(3). [Link]

ORCID IDs
Annis C. Adi [Link]
Ali I. Tawakal [Link]
Mohammad F. Rasyidi [Link]
Wizara Salisa [Link]
Farapti Farapti [Link]
Heni Rachmawati [Link]

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