Vol 4, Issue 1, 2022 (208-218)
http://journal.unpad.ac.id/idjp
*Corresponding author,
e-mail : norisca@unpad.ac.id (N. A. Putriana)
https://doi.org/10.24198/idjp.v4i1.40477
© 2022 N. A. Putriana et al
Formulation and Evaluation of Black Garlic (Allium Sativum L.) Lozenges As An
Antioxidant Supplement
Norisca Aliza Putriana1, Efri Mardawati2, Yoga Windu Wardhana1, Ismiatun1, Anting
Wulandari2, Dwi Wahyudha Wira3, Nanang Masruchin4
1Department of Pharmaceutical and Formulation Technology, Faculty of Pharmacy, Universitas
Padjadjaran, Jalan Raya Bandung-Sumedang KM 21, Jatinangor 45363, Indonesia
2Faculty of Agricultural Industrial Technology, Universitas Padjadjaran, Jalan Raya
Bandung-Sumedang KM 21, Jatinangor 45363, Indonesia
3Faculty of Veterinary, Universitas Padjadjaran, Jalan Raya Bandung-Sumedang KM 21,
Jatinangor 45363, Indonesia
4Biomaterial Research Center, Indonesian Science Institutions, Jalan Raya Bogor Km 46,
Cibinong Bogor, 16319, Indonesia
Submitted : 04/07/ 2022, Revised : 16/07/ 2022,, Accepted : 01/08/ 2022, Published : 19/08/2022
Abstract
Free radicals are species of reactive chemical compounds that can cause
degenerative diseases. Antioxidants work to deactivate free radicals by binding to
these radicals to stabilize the radicals. One of the plants that contain antioxidants is
black garlic. This study aimed to obtain the best formula from the preparation of
lozenges of black garlic extract using the wet granulation method as an antioxidant
supplement. The formula optimization was carried out using a statistical approach
with a two-level factorial method using expert design software. The lozenges
formula was made using wet granulation method. The granules and tablets were
evaluated for its characteristic. Besides, the levels of polyphenols and antioxidant
activity of the extracts and lozenges were also determined. The best formula is F2
obtained from a ratio of gum arabic and starch pregelatin of 3: 5 with a desirability
value of 0.922. The Evaluation of black garlic extract granules is eligible, including
the moisture content of 2.04%, an excellent flow rate is 18.3 g/s, and good
compressibility of 9%. The uniformity of lozenge weight obtained is eligible, which
is 3%. The hardness and disintegration time of 9.1 kg and 11.28 minutes,
respectively. Meanwhile, the antioxidant activity of the extract and lozenges was
263 µg/mL and 323.9 µg/mL, respectively. The best formula obtained is with 3%
gum arabic and 5% starch pregelatin.
Keywords: Antioxidant, Black Garlic, Design expert, Lozenges
1. Introduction
Free radicals are species of reactive
chemical compounds, which have an
unpaired free electron in their outermost
orbital [1]. Excessive exposure of free
radicals can cause degenerative diseases,
such as cancer, and cardiovascular [2]. So it
is needed antioxidants that work to
N. A. Putriana et al / Indo J Pharm 4 (2022) 208-218
209
deactivate free radicals by binding to these
radicals, through electron donation so that
radical species become more stable. It is
will be neutral and unreactive [1]. One of
the plants that contain antioxidants is black
garlic [3].
Figure 1. Black Garlic
Black garlic is a product of garlic
that undergoes an aging process (heating),
at 65-80oC for several days. The heating
process uses an electric rice cooker so the
color changes due to the Millard reaction.
This change also increases its bioactive
compounds. These compounds are s-allyl
cysteine, amino acids, polyphenols, and
flavonoids [4]. In another study, it was
proven that black garlic fermented for 15
days had a strong antioxidant, which is 2.41
g/ml [5].
Black garlic also has activity as
antibacterial [6], antifungal [7],
hepatoprotective [8], antidiabetic [9]. Black
garlic has great potential to be developed
into supplements and pharmaceutical
preparations. However, black garlic has an
unattractive shape, black color, sweet and
sour taste, and a short shelf life [6]. To
improve the taste and make it easier to use,
lozenges can be made. In addition, by
making lozenges, it can increase shelf life
compared to the extract and can be made
with the right dose. So in this study,
lozenges were made from black garlic.
2. Methods
2.1 Materials
The materials to be used were
aquadest, gallic acid, black garlic thick
extract, 70% ethanol (Merck), folin-
ciocalteau, methanol (Merck), NaOH, black
garlic simplicia, DPPH reagent (2,2-
Diphenyl-1-pikrilhidrazi). For the
formulation materials, Avicel (CV.
Quadrant), Gum Arabic (CV. Quadrant),
starch pregelatin (CV. Quadrant), sucralos
(CV. Quadrant) and talcum (CV. Quadrant)
were used.
1.1 Extraction of Black Garlic
Garlic was heated using an electric
rice cooker with several variations of time,
which are 7, 14 and 21 days so that black
garlic was obtained. Then black garlic
simplicia was chopped and extracted by
maceration method using 70% ethanol. It
was carried out for 4x24 h while stirring
occasionally. The extract obtained was
filtered, then the filtrate was evaporated
using a rotary evaporator at 30 rpm and a
temperature of 50-60oC to obtain a
concentrated extract.
1.2 Total Polyphenol Determination
of extract and tablets of black
garlic
The standard solution (gallic acid)
was prepared with various concentrations
of 20, 30, 40, 50, and 60 g/mL. Meanwhile,
the test solution was prepared by weighing
0.2 g of extract and adding 25 ml of
methanol and then stirring for 30 min with
a magnetic stirrer. Then it was filtered and
put into a 25 ml volumetric flask and it was
added methanol up to the mark. Each 1 ml
of the test solution and the comparison
solution were put in a test tube and 5 ml of
folin-ciocalteu (7.5% in water) was added.
It was allowed to stand for 8 minutes, then
added 4 ml of 1% NaOH and incubated for
1 hour. The polyphenol content of the
extract was measured using a
spectrophotometer at 765 nm [14].
1.3 Antioxidant activity test of
extract and tablets of black garlic
Antioxidant activity test of black
garlic extract and tablets is using DPPH.
The antioxidant activity was quantitatively
measured using a UV-Vis
spectrophotometer. The concentration
variations of the samples used were 100,
200, 300, 400, 500 ppm. Samples were
N. A. Putriana et al / Indo J Pharm 4 (2022) 208-218
210
added with DPPH reagent and incubated for
30 minutes in a dark room and then measure
the absorbance at the optimal wavelength.
Determination of the optimum wavelength
by measuring the stock solution DPPH (2,2-
diphenyl-1-picrylhydrazyl) at a wavelength
of 400-800 nm with uv vis
spectrophotometry [16].
2.2 Optimization of formulas and
formulations of lozenges
Optimization using design expert
software, with two factorial methods, to get
the best formula from several variations.
Optimization was carried out to get the best
formula composition to be used. The
formulation was carried out using the wet
granulation method by mixing the internal
phase and adding binders to form lumps and
granulation. Oven-dried at 400C and printed
using a tablet printer.
2.3 Evaluation of granules and
lozenges
2.3.1 Granule flow time test
This was carried out by flowing 10
grams of granules in a diverter with a time
of not more than 1 second [10].
2.3.2 Granule compressibility test
Determination was using a Tapped
density tester. Compressibility was
calculated from the density of granules by
entering 100 ml of granules into a
measuring cup. initial volume was recorded
and the device was turned on. The final
volume was observed and calculate the
compressibility used this equation.
2.3.3 Tablet visual test
It was carried out by observing the
general appearance of the tablet such as:
size, shape, color, presence or absence of
odor, taste, surface shape and physical
defects [10].
2.3.4 Tablet weight uniformity test
It was carried out by weighing 20
tablets and calculating the average weight
of each tablet. The uniform requirement is
that no more than 2 tablets deviate by more
than 5%, and not one tablet deviates more
than 10% [10].
2.3.5 Tablet size uniformity test
It used a caliper, by taking 20 or 10
tablets, then measuring the diameter and
thickness of the tablets one by one. The
requirements for a good tablet are to have a
diameter of not more than 3 times and not
less than one-third of the tablet [10].
2.3.6 Tablet hardness test
It used a hardness tester, by
applying pressure to the tablet until the
tablet cracks or breaks, the minimum tablet
strength is 4 kg/cm3 [10].
2.3.7 Tablet friability test
The examination was carried out
by weighing the weight of 20 large tablets.
It was inserted into the friability instrument
and the instrument was operated for 4
minutes at a speed of 25 rpm, about 100
rotation. Then the tablets were cleaned and
weighed again and the percent friability
was calculated using the formula below.
The requirement for a good tablet is the
losses of weight less than 1% [10].
*
*F = Percent of friability ; Wo = initial weight ; W1
= final weight
2.3.8 Disintegration time test of tablets
It used a disintegration testertester,
the disintegration time test was carried out
on 6 tablets with the condition that all
tablets disintegrated in no more than 30
minutes [11].
Data analysis
Determination of the best formula was
using the two-level factorial method using
the Design Expert software, by entering the
responses generated from the evaluation of
the tablet preparations in it. Furthermore,
the optimal value will be obtained by
N. A. Putriana et al / Indo J Pharm 4 (2022) 208-218
211
setting goals and limits for each response.
The Design Experts software perform analysis of variance (ANOVA) on the
responses used.
3. Results
3.1 Black Garlic Extract
The heated black garlic was oval
in shape and soft in texture, sweet and sour
in taste, and the odor produced was not as
strong as garlic. The longer heating will
result in a darker black garlic color. This
occurs because of the Millard reaction [15].
During the heating process, the substances
contained in garlic will not be damaged
because during the process the onions are
wrapped in aluminum foil [20]. The results
of the extraction of black garlic simplicia
from each heating time obtained black
garlic extract which was concentrated like
caramel, brownish-black in color and has a
distinctive smell.
3.2 Total Polyphenol Determination
of extract and tablets of black
garlic
3.2.1 Extract polyphenol testing
The principle of this method is that
the phenolic compounds contained in the
extract will be oxidized by Folin Ciocalteu
reagent to form a blue complex solution.
The highest total polyphenol value was
found in the 14-day heating period, which
was 15.18 mg GAE/g. x
Table 1. Polyphenol’s total of black garlic extract
Heating time (days)
Polyphenol’s total (mg GAE/g)
7
4.77 ± 0.0321
14
15.18 ± 0.0215
21
14.59 ± 0,057
3.2.2 Tablet polyphenol testing
From the results of the total
polyphenol test, the extract which has the
largest polyphenol was carried out for tablet
formulations. The total polyphenol values
obtained from black garlic extract tablets by
heating for 14 days is shown in Table 2.
Table 2. Polyphenol content of tablets
Absorbance
Average
Polyphenol content
(mg GAE/g)
n = 6
0.4495
0.4453± 0.65
11.998 ± 0.00415
0.4452
0.4412
The total polyphenol content of the
extract decreased after it was made into
tablets, the decrease was not significant.
This happens because polyphenols are
compounds that are less stable to the effects
of light, oxygen, temperature, and chemical
changes [21].
3.3 Antioxidant activity test of
extract and tablets of black garlic
3.3.1 Antioxidant activity of black
garlic extract
The test was carried out using the
DPPH (1,1-diphenyl-2-picrylhydrazyl)
method. This method is a simple and fast
method [11]. The initial determination of
the maximum wavelength of the DPPH was
carried out so that the measurements
N. A. Putriana et al / Indo J Pharm 4 (2022) 208-218
212
obtained have high sensitivity to changes
that occur. The maximum wavelength of
DPPH in this test was 516 nm. The
measurement results obtained that the
highest antioxidant activity was on heating
for 14 days, which can be seen from the
IC50 value. The smaller the IC50 value, the
antioxidant activity is the greater.
Table 3. Antioxidant Activity of Black Garlic Extract
3.3.2 Tablet antioxidant activity test
The principle of reaction change
used is colorimetry, namely the change
from purple color (DPPH radical) to yellow
in the presence of antioxidants
(polyphenols). The color change is
proportional to the added antioxidant
content [12].
The result of the antioxidant activity of
black garlic extract tablet with heating for
14 days was 323.9 µg/mL. This is in
accordance with the decrease in the levels
of polyphenols in the preparation so that the
antioxidant activity also decreases. This is
because compounds that have a role as
antioxidants are polyphenols contained in
the extract.
Table 4. Antioxidant activity of black garlic tablet
Repetition
IC50
Antioxidant
activity (µg/mL)
n = 6
1
325.5
323.9 ± 4.04
2
319.3
3
326.9
3.4 Optimization and formulation of
lozenges
In the design expert optimization
mode, the factors used were Arabic gum as
a binder with a value of 1% (low) - 3%
(High) and starch pregelatin as a
disintegrant with a value of 5% (low) - 10%
(High). The responses used were flow rate
(g/s) and disintegration time (s). It was
obtained 4 formula designs with varying
concentrations of Gum Arab and Starch
pregelatin.
Table 5. Optimization of lozenges preparation formula
Materials
F1 (%)
F2 (%)
F3 (%)
F4 (%)
Function
Black garlic
extract
46.86%
46.86%
46.86%
46,86%
Bioactive
component
Pregelatin strach
5%
5%
10%
10%
Disintegrant
Gummi arabicum
1%
3%
1%
3%
Binder
Heating time (days)
IC50 (µg/mL)
n = 6
7
720.5 ± 0.843
14
263 ± 0.432
21
32.8 ± 0.581
N. A. Putriana et al / Indo J Pharm 4 (2022) 208-218
213
Talcum
2%
2%
2%
2%
Lubricants
Sucralose
0.1%
0.1%
0.1%
0.1%
Sweetener
Avicel
Ad.100%
Ad.100%
Ad.100%
Ad.100%
Diluent
The tablet formulation was carried
out using the wet granulation method. The
black garlic extract to be formulated has a
poor flow rate, so this method is used to
improve the flow rate and compressibility
in order to form tablets that are
homogeneous in content.
3.5 Evaluation of granules and
tablets of black garlic extract
3.5.1 Granule flow rate evaluation
From the results of the tests carried out, the
flow rate of formula 1 (F1) to formula 3
(F3) had a "very good" flow rate because
they had a value of >10 g/s. While the
formula 4 (F4) had a "good" flow rate [10].
2.5.2 Moisture evaluation
Black garlic extract granules have
good moisture content except for formula 1,
this may be due to the content of excipients
added in formulas is different, such as gum
arabic and starch pregelatin which have
water absorption.
3.5.3 Evaluation of granule
compressibility This test is carried out to
determine the nature of the material if given
Figure 2. Granule flow rate
11
18.3
13.2
9.6
0
5
10
15
20
F1 F2 F3 F4
Flow rate (g/s)
Figure 3. Humidity content of granule
8.50%
2.04%
2.94%
1.76%
0%
2%
4%
6%
8%
10%
12%
F1 F2 F3 F4
Humidity content (%)
N. A. Putriana et al / Indo J Pharm 4 (2022) 208-218
214
pressure it will form a stable and compact
mass [13]. In this work, the granules of F1,
F2, and F4 had "very good" %
compressibility and F3 had "good"
compressibility.
3.5.4 Tablet visual evaluation
The visual form of each formula
(F1, F2, F3 and F4) is not much different.
Where the tablet was round flat, dark brown
in color, sweet and sour in taste and
characteristic in odor.
3.5.5 Evaluation of weight uniformity
All formulas (F1, F2, F3, and F4)
met the requirements of good tablet. That
was, no more than 2 tablets had a storage
percentage of more than 5% and there was
not a single tablet that deviates more than
10%.
Figure 5. Tablet weight uniformity
3.5.6 Evaluate tablet hardness
The hardness of the tablet is
strongly influenced by the compression
pressure provided by the tool. The higher
the pressure applied, the hardness will
increase. In this study, all formulas (F1, F2,
F3, and F4) met the requirements of good
tablet which was more than 4 kg/cm3.
Figure 6. Hardness of tablet
2.4
3.0
1.9
1.4
F1 F2 F3 F4
Deviation (%)
3.5
3.0
2.5
2.0
1,5
1.0
0.5
0.0
10.4
9.1 10.1 10.4
0
2
4
6
8
10
12
F1 F2 F3 F4
Hardness (kg/cm3)
Figure 4. Compressibility of granule
6.7
9
13.3
6.7
0
2
4
6
8
10
12
14
F1 F2 F3 F4
Compressibility (%)
N. A. Putriana et al / Indo J Pharm 4 (2022) 208-218
215
3.5.7 Evaluation of tablet friability
The formulas (F1, F2 and F4) meet the
requirements as good tablet. While F3 was
not eligible. The eligible conditions were
the tablet friability of less than 1%. This
may be due to the influence of the binder
and disintegrant used. F3 used a binder with
a low concentration of 1% and a high
concentration of disintegrant, which was
10%, so that the tablet formed had a high
friability.
Figure 7. Friability of tablet
3.5.8 Evaluation of tablet disintegration
time Disintegration time testing was
carried out to determine the tablet's ability
to dissolve in the body. Where a good
disintegration time requirement for
lozenges is less than 30 minutes. The test
results show that all formulas (F1, F2, F3,
and F4) were eligible which were less than
30 minutes of disintegration time.
Figure 8. disintegration time of tablet
3.6 Data analysis
Response data included in the
design expert were flow rate and
disintegration time. Where the flow rate
category used is "maximize", because a
large flow rate value is directly proportional
to good flowability. And the disintegration
time category entered is “minimize”
because the disintegration time of lozenges
must be under 30 minutes. So the smaller of
disintergartion time, the tablet is better.
The P-value of gum arabic and
starch pregelatin on the response to flow
rate and disintegration time showed a value
of <0.05. So there was a significant
difference between gum arabic and starch
pregelatin on flow rate and disintegration
time. So it shows that H0 is rejected and H1
is accepted. However, the value of gum
arabic in response to disintegration had a
value of more than 0.05 so that the effect of
disintegration time on gum arabic was not
significantly different. To determine the
best formula, it can be seen from the
desirability value of each formula.
0.064 0.006
1.3
0.22
F1 F2 F3 F4
Friability (%)
12.34 11.28
13.26 13.77
0
2
4
6
8
10
12
14
16
F1 F2 F3 F4
Disintegration time (min)
1.0
1.5
0.0
-0.5
0.5
N. A. Putriana et al / Indo J Pharm 4 (2022) 208-218
216
Table 6. The desirability value of each formula
Formula
% Composition
(Arabic gum :
primojel )
Flow rate
response
(g/s)
Disintegration
time response
(s)
Desirability
value
1
1:5
10.31
721
0.345
10.53
735
12.05
766
2
3:5
18.8
691
0.922
18.5
664
17.5
676
3
1:10
13.3
774
0.104
13.5
789
12.8
825
4
3:10
9.3
807
0.376
9.4
810
10
861
The desirability value is the value
of the program's ability to meet the good
characteristic, the best desirability value is
close to 1. In this work the best desirability
value from the above formula was formula
2 with a gum arabic value of 3% and a
starch gelatin value of 5%, with a
desirability value of 0.922.
4. Discussion
Antioxidants work to deactivate free
radicals by binding to these radicals,
through electron donation so that radical
species become more stable [1]. The use of
antioxidants is widely used from several
plants such as turmeric, green tea, and black
garlic [17,18]. Black garlic is a product of
garlic that undergoes an aging process
(heating), at a temperature of 65-80oC for
several days using an electric ricecooker so
that changes occur due to the Millard
reaction, this change also increases the
bioactive compounds it contains. These
compounds are s-allyl cysteine, amino
acids, polyphenols and flavonoids [19]. So
that black garlic is one of the plants that has
high antioxidant activity and contains
active compounds, namely phenols,
flavonoids, pyruvate, thiosulfate, s-
alilcysteine, and s-allymercaotocysteine.[3]
From some of these activities,
according to Zhafira's research (2018),
black garlic has a high antioxidant activity
of 3,475 µg/g [18], and in Agustina et al's
study (2020), proved that black onions
fermented for 15 days have strong
antioxidants, namely 2, 41 µg/ml.[5] In this
study, it was found that the antioxidant
activity of the extract was 263 µg/mL, and
the antioxidant activity of lozenges was
323.9 ± 4.04 µg/mL. The result can be seen
in Table 4. This shows that the antioxidant
activity of black garlic in this study is
smaller than that of other studies. This can
occur due to several factors such as the
heating process in the manufacture of black
garlic which in this study showed that too
long the heating process can reduce
antioxidant activity but lack of heating also
causes low antioxidant activity. This result
can be seen in Table 3. Therefore,
sufficient heating time is required to
produce large antioxidant activity. In
addition, the storage process can affect
because storage for too long can reduce
antioxidant activity. Then, the addition of
excipients in the manufacture of these
lozenges can also affect the content and
antioxidant activity in them. In addition, the
antioxidant activity is influenced by its
N. A. Putriana et al / Indo J Pharm 4 (2022) 60-70
217
polyphenol content. The antioxidant
activity of extract decreases, along with the
decreasing levels of polyphenols in the
preparation. We can conclude that it can be
seen from tables 1,2, and 4 in result.
Then, to facilitate the consumption of
antioxidants, lozenges can be made. In this
manufacture of lozenges from this study,
the best formula was obtained, namely F2
with a ratio of gum arabic and starch
pregelatin (3:5) with a desirability value of
0.922. The evaluation of the black garlic
extract granules met the requirements,
including the moisture content of 2.04%.
The flow rate is very good, namely 18.3 g/s.
Good compressibility is 9%. The evaluation
results of lozenges for the size uniformity
test met the requirements, the weight
uniformity test met the requirements,
namely 3%. hardness of 9.1 kg. The
disintegration time was 11.28 minutes. This
shows that lozenges are suitable and good
for making preparations from black garlic.
5. Conclusion
The results show that the best formula is F2
with a ratio of gum arabic and starch
pregelatin (3:5) with a desirability value of
0.922. The antioxidant activity of the
extract and tablet of black garlic is 263
µg/mL and 323.9 ± 4,04 µg/mL,
respectively.
Acknowledgement
The authors are grateful to the Rector of
Universitas Padjadjaran for support this
study. The authors are also thankful to
Faculty of Pharmacy, Universitas
Padjadjaran, for providing the support
and their facilities.
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