POTENTIALITY OF INCORPORATING COCOA LIQUOR IN SKIN CARE COSMETICS PJAEE, 17 (10) (2020)

POTENTIALITY OF INCORPORATING COCOA LIQUOR IN SKIN CARE

COSMETICS
Alyaa Nurathirah Abd Halim1, Siti Salwa Abd Gani2*, Uswatun Hasanah Zaidan3, Mohd Izuan
Effendi Halmi 4, Norliza Abdul Wahab 5, Arief Huzaimi Md Yusof 6
1
Department of Agriculture Technology, Faculty of Agriculture.
2
Halal Products Research Institute, Putra Infoport.
3
Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences.
4
Department of Land Management, Faculty of Agriculture.
5,6
Malaysia Cocoa Board, Cocoa Innovative and Technology Centre, Lot 12621 Nilai
Industrial Area, 71800 Nilai, Negeri Sembilan, Malaysia.
* Corresponding author: 2ssalwaag@upm.edu.my or ssalwa.abdgani@gmail.com
1
alyaanurathirah@gmail.com , 3uswatun@upm.edu.my , 4m_izuaneffendi@upm.edu.my ,
5
naw@koko.gov.my , 6ariefhuzaimi@koko.gov.my

Alyaa Nurathirah Abd Halim , Siti Salwa Abd Gani , Uswatun Hasanah Zaidan , Mohd
Izuan Effendi Halmi Norliza Abdul Wahab Arief Huzaimi Md Yusof. Potentiality Of
Incorporating Cocoa Liquor In Skin Care Cosmetics--- Palarch’s Journal Of Archaeology
Of Egypt/Egyptology 17(10), 1039-1046. ISSN 1567-214x

Keywords: Antioxidants, Skin Care Cosmetics, Cocoa Liquor, Phytochemicals,

Phenolic Content, Flavonoid Content.

ABSTRACT
Halal cosmetics, generally derived from plant-based materials, are receiving much interest
amongst health-conscious consumers and cosmetic industry. Cocoa liquor, a paste produced
from ground cocoa (Theobrama cacao L.) beans, is a natural source of antioxidants with
potential health benefits. The present study was conducted to determine the prospect of
incorporating cocoa liquor in skin care cosmetics complementing its ability in protecting the
skin by warding off free radicals from the environment. The amount of total phenolic contents
(TPC) in cocoa liquor was determined by Folin-Ciocalteau colorimetric method using gallic
acid as a standard, and various concentrations of extract sample were measured at 765 nm.
Total flavonoid contents (TFC) were measured by using aluminium chloride colorimetric
assay. Rutin was used as a standard and the absorbance was measured at 405 nm. The 1,1-
diphenyl-1-picylhydrazyl (DPPH) free radical scavenging activity was estimated and

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absorbance was measured at 517 nm. At the highest concentration of 1000 ppm, both TPC and
TFC recorded values of 131.97 mg GAE/g and 4.10 mg RE/g dried weight of sample
respectively. DPPH free radical scavenging activity was recorded at the highest concentration
of 87.99% with EC50 value of 30.33 mg/mL. The various concentrations implied different
levels of antioxidant properties. The results suggest that cocoa liquor is a potential source of
phytochemicals. The study presented scientific validation on phytochemical contents of cocoa
liquor showing presence of bioactive compounds with nutritional and therapeutic values which
have positive impact on skin health and suggesting its prospective use in value-added products
such as skin care cosmetics.

INTRODUCTION
The term “halal” is, by and large, associated with food. In recent years, the term
has become increasingly affixed to other items such as skin care cosmetics, a
preparation used to clean, improve or change skin’s appearance. The demand
towards halal cosmetic products has increased among younger generations of
conscious consumers. “Halal” certification rests not only on the ingredients
that go into the making of the product, but also the packaging, manufacturing,
and distribution methods. For a product to be considered halal, it must be free
of alcohol, not tested on animals, and contains no animal fat or other harmful
chemicals [11].

Empirical studies have documented various beneficial effects of cocoa liquor,

also called chocolate liquor, unsweetened chocolate, cocoa mass or simply
liquor [15]. The thick paste texture of cocoa liquor contains about 55% fats from
cocoa butter and cocoa cake [2]. The chemical composition of cocoa liquors
depends on cocoa varieties, fermentation and roasting conditions in processing
the cocoa beans [14]. Cocoa liquor does not contain any alcohol liquids, halal
and safe to consume or for use as topical applications for the skin. Its phenolic
and flavonoid contents have been documented to be higher than any of other
phytochemical-rich foods [20]. The abundance of polyphenols has also been
reported by Porter et al. (1991) to be specifically in the form of flavonoids.

Previous study by Nichols and Katiyar (2010) reported that topical applications
of polyphenols can protect the skin against ultraviolet radiation effectively.
Flavanol-rich cocoa has been reported to enhance skin elasticity while
improving skin thickness and structure [22]. Long-term effect of ultraviolet
radiation causes photo-damage such as wrinkles, dermal connective alteration
and accumulation of collagen and can be prevented by similar topical
application [10].

Against this background, the present study was conducted to establish the
antioxidant contents in cocoa liquor with a view of determining its potentiality
in skin care cosmetics or incorporating with other halal materials giving the skin
an added protection in fighting free radicals from the environment.

MATERIALS AND METHODS

Cocoa Liquor

General

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POTENTIALITY OF INCORPORATING COCOA LIQUOR IN SKIN CARE COSMETICS PJAEE, 17 (10) (2020)

Cocoa liquor was directly purchased from Pusat Inovatif dan Teknologi Koko
(Cocoa Innovative and Technology Centre) Nilai, Negeri Sembilan, Malaysia.

Preparation of extracts

Cocoa liquor blocks were melted in an oven at 40℃. Extraction procedures

followed the method of Radojčić et al. (2009) with some modifications. Melted
cocoa liquor was subsequently defatted with hexane (C6H14), an organic alkane
compound. Cocoa liquor was extracted with 10 mL ethanol in an ultrasonic
bath. The supernatant was centrifuged for 10 minutes at 5000 rpm and
condensed in a rotary evaporator until dry. The prepared extracts were subjected
to total phenolic content (TPC), total flavonoid content (TFC) and antioxidant
capacity determinations of DPPH. All extracts were prepared and analysed in
triplicates.

Total phenolic content (TPC) determination

Total phenolic content (TPC) was determined following method described by

Kaur and Kapoor (2002) with some modifications using Folin-Ciocalteau (FC)
reagent. An extracted sample and a standard were dissolved in ethanol (serial
dilution) and 100 μL was pipetted into vials. An amount 500 μL of FC reagent
was added to a sample and mixed for about 8 seconds. Sodium carbonate
solution (20% w/v) was added at an amount of 1500 μL to the sample mixed
and allowed to stand in the dark at 40℃ for an hour. Measurement of phenolic
acid content was carried out using an UV-Vis microplate reader at wavelength
of 765 nm against gallic acid standard. Results obtained were expressed in mg
gallic acid equivalent (GAE)/g sample.

Total flavonoid content (TFC) determination

Total flavonoid content (TFC) was determined following method by Chang et

al. (2002). An amount 2 g of aluminium chloride was dissolved in 100 mL
ethanol resulting in a 2% (w/v) aluminium chloride solution. An extracted
sample and standard (serial dilution) of 100 μL each were pipetted into a 96-
well microplate and 100 μL aluminium chloride solution was added. The
mixtures were incubated for 10 minutes and the absorbance was recorded at
wavelength of 405 nm using an UV-Vis microplate reader. Readings of samples
were expressed in mg Rutin Equivalent (RE)/g sample.

Determination of DPPH scavenging assay

The DPPH free radical scavenging activity was carried out following method
described by Blois (1958) with minor modifications. An extracted sample and
a standard were weighed and dissolved in ethanol (serial dilution).
Subsequently, 10 mg of 1,1-diphenyl-2-picryhdrazyl (DPPH) was weighed and
dissolved in ethanol and the solution was made up to 100 mL in a volumetric
flask. An amount 50 µL of the extracted samples and standard were mixed with
150 µL DPPH solution in a 96-well microliter plate and incubated in the dark
for about 30 minutes for reaction to occur. Absorbance was measured at
wavelength of 517 nm using an UV-Vis microplate reader. A sample control

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was prepared by omitting sample extract from DPPH working solutions. All
analyses were performed in triplicates. The radical inhibition activity of the
extracts was calculated by the reduction of DPPH radical using the following
equation (Eq. 1):

DPPH Scavenging effect (%) = [(A0 – (1)

A1)/A0 x 100]
Where,
A0 = Absorbance of control reaction
A1 = Absorbance in the presence of sample

The plotted graph of DPPH scavenging

effect against concentration of plant extract
determined the EC50 value, described as the
reduction of initial DPPH radical
concentration by 50% as affected by the
total antioxidant in need.

Statistical analysis

All data were expressed as mean ± standard deviation and independent analyses
were performed in triplicates. Data were evaluated using one-way (unstacked)
analysis of variance (ANOVA) using Tukey test by Minitab Software version
14.

RESULTS AND DISCUSSION

Total phenolic content

The amount of TPC in extracted cocoa liquor was based on the absorbance of
extracted sample and Folin-Ciocalteau reagent mixture at wavelength of 765
nm. The phenolic compounds presented in each of sample was recorded as mg
GAE per gram sample. A standard curve was plotted to quantify the phenolic
compound in samples as mg GAE per gram samples. The total amount of TPC
in sample is presented as mean mg GAE per gram sample (Figure 1).
Total phenolic content (mg

160
140
GAE/g extract)

120
100
80
60
40
20
0
31.25 62.5 125 250 500 1000
Concentration (mg/mL)

Figure 1. Total phenolic content (TPC) of cocoa liquor extracts. Values are
means ± SD (n=3); Means with different letters are significantly different at
level of (p<0.05) by Turkey test.

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POTENTIALITY OF INCORPORATING COCOA LIQUOR IN SKIN CARE COSMETICS PJAEE, 17 (10) (2020)

Previous studies reported that different extracting solvents influenced

antioxidant activity and yield of phenolic content [21]. Among several studies,
only one concentration was used to show TPC values. In addition, ethanol and
water were safer solvents than methanol and other organic solvents [18]. In the
present study, an extraction medium was used for preparing cocoa liquor
extracts. Total phenolic contents were recorded in each concentration of
extracted cocoa liquor in the order of 1000 > 500 > 250 > 125 > 62.5 > 31.25.

The study recorded significant differences between concentrations (p<0.005)

using Tukey’s test. Higher concentration of cocoa liquor extract showed higher
level of phenol in the serial dilution methods which started from highly
concentrated to less concentrated suggesting less amount of TPC in the diluted
extract compared to concentrated samples. The results of TPC were similar as
reported by Brabo de Sousa et al. (2018) on fruit of palm species, Oenocarpus
distichus.

TPC of extracted cocoa liquor recorded in the present study was highest when
compared with studies. Radojčić et al. (2009) documented that the order of TPC
yield from highest to lowest level were Madagascar > Mexico > Ecuador >
Venezuela > Sao Tome > Ghana. A report by Natsume et al. (2000) reaffirmed
that phenolic content in cocoa liquor varied with countries where the study was
conducted.

Total flavonoid content

The amount of TFC in cocoa liquor extract was based on the absorbance of
sample using aluminium chloride colorimetric assay at wavelength of 405 nm.
Flavonoid compounds present in each concentration of samples was reported as
mg RE per gram sample. One standard curve was plotted to quantify the
flavonoid compound in the samples as mg Rutin Equivalent per gram sample
(mg RE/g).

Compounds in flavanols known as catechin are usually found in cocoa or

chocolate [9]. In extracted cocoa liquor, the total flavonoids contents detected
in each concentration followed the order of 1000 > 500 > 250 > 125. There were
significant differences (p< 0.05) between the concentrations using Tukey’s test.
The highest flavonoid content was in 1000 (mg/L) concentration giving a value
of 4.10 mg RE/g sample (Table 1). The results in TFC were in agreement with
a study by Zhang et al. (2019) in petioles of sweet potato (Ipomoea batatas L.).
The amount of flavonoid in the extracted cocoa liquor depicted lower yield due
to several factors such as quality of cocoa beans producing the cocoa liquor [3],
as well as fermentation process and roasting conditions [20]. These conditions
caused phenol levels to be reduced thus affecting flavonoids content.

Table 1: Total flavonoid content from cocoa liquor

Concentration (mg/mL) (TFC) (mg RE/g)

1000.0 4.10 ± 0.000

500.0 -5.65 ± 0.001

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250.0 -8.21 ± 0.002

125.0 -12.33 ± 0.001
DPPH scavenging assays

DPPH scavenging assay is the simplest and most widely reported method for
screening antioxidant activity in food and many other plant-derived drugs [1].
The change of colour from deep purple fading to light yellow indicated that
antioxidant compounds in the extracted cocoa liquor reacted with DPPH
radicals by donating hydrogen radicals resulting in bleaching of DPPH solution.
Antioxidants assay determines scavenging of stable radical species of DPPH by
antioxidants.

In the present study, serial dilution methods were used to determine scavenging
activity. At 1000 mg/mL concentration, the highest activity was affected
followed by others at lower concentrations. Scavenging activity increased as the
concentration of sample extract was increased until a plateau was reached at 500
mg/mL as shown in Figure 2. There were significant differences (p<0.005)
between the concentrations with the highest recorded at 87.99%. The EC50 value
was determined from the plotted graph of scavenging activity against the
concentration of the extract, which was the amount of antioxidant necessary to
decrease the initial DPPH radical concentration by 50% [20]. From the study,
the EC50 of the sample of extracted cocoa liquor was at 30.33 mg/mL suggesting
that reactions occurred to fight free radicals in maintaining skin health.

120

100
Ascorbic Acid
Scavenging Activity (%)

80

60

40
Extracted Cocoa
20
Liquor
0
0 200 400 600 800 1000 1200
Concentration (mg/mL)

Figure 2. DPPH of cocoa liquor extracts. Values are means SD (n=3); Means with different
letters are significantly different at the level of (p<0.05) with Turkey test.

CONCLUSION
In the present study, polyphenols level was dependent on fermentation, roasting
and quality of cocoa beans resulting in a variation of results. The total phenolic
was highest at 1000 mg/mL giving a value of 131.97 mg GAE/g dried weight
of sample while the total flavonoid content was 4.10 mg RE/g dried weight of
sample. The antioxidant activity’s highest percentage was at 1000 mg/mL with
87.99% and EC50 of 30.33 mg/mL. The data suggest presence of high
antioxidants in cocoa liquor indicating its potentiality in the development of
skin care cosmetics.

ACKNOWLEDGEMENT

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The authors would like to express their gratitude to Malaysia Cocoa Board,
Halal Products Research Institute and Faculty of Agriculture, Universiti Putra
Malaysia (UPM) for their support in the study.

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