Vol 3, Issue 3, 2021 (116-127)
http://journal.unpad.ac.id/idjp
Formulation and Evaluation of instant granules from Ketapang Badak fruit (Ficus lyrata
Warb) using wet granulation method as an antioxidant supplement
Norisca Aliza Putriana*.1, Efri Mardawati2, Yoga Windu Wardhana1, Nurul Afifah1, 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
e-mail: norisca@unpad.ac.id
Abstract
Submitted : 09/12/2021, Revised : 25/01/2022, Accepted : 12/02/2022, Published : 13/02/ 2022
Free radicals are naturally produced from the body's metabolic processes, but the
excessive amount of free radicals can interfere with human health because they cause
oxidative stress. Therefore, our bodies need antioxidants that can protect against free
radicals. Ficus lyrata W. is one of the antioxidant sources. This study aim to formulate
instant granules from the Ethanol extract of Ficus lyrata W. using the wet granulation
method. The formula was optimized using the Design Expert with the two-level factorial
method. The optimized factors were xanthan gum 0.8-1.5% and polyvinylpyrrolidone
(PVP) 0.5-5%. Granules were evaluated and analyzed using the Design Expert. As a
result, all the four formulas obtained showed that Formula 4 with a combination of
xanthan gum 0.8% and PVP 5% is the best formula, which the evaluation result is Loss
On Drying (LOD) 3.28%, Flowability 16.043 ± 0.221 (g/s), Angle of Repose 21.77 ±
0.862, no precipitate for 15 minutes, pH = 4.7, dispersed in 31 seconds and sedimentation
time is 52.213 ± 1.7878 minutes, the results of the antioxidant activity test of the ethanol
extract of Ficus lyrata W. is 38.27 µg/ml, and instant granules is 145.02 µg/ml.
Keywords: Antioxidant, Ficus lyrata W., Instant granules, Design expert
1. Introduction
Free radicals naturally are produced by
the cellular metabolism process. It plays an
important role in our body's physiology which
the excessive amount of free radicals will cause
health problems (1). Free radicals are defined
as a molecule that contains one or more unpair
electrons in the outside orbital, which is
relatively unstable and reactive, so it can
protect against another molecule to reach the
stable state (2).
Oxidative stress is a condition where
there’s unbalance condition between the
production and accumulation of Reactive
Oxidant Species (ROS) and its ability to
balance it back (3). Free radicals that induced
oxidative stress have been reported to be
involved in the development of several
degenerative diseases such as cancer, diabetes
*Corresponding author,
e-mail : norisca@unpad.ac.id (N. A. Putriana)
https://doi.org/10.24198/idjp.v3i3.347062
2021 N. A. Putriana et al
N. A. Putriana et al / Indo J Pharm 3 (2021) 116-127
118
mellitus, rheumatoid arthritis, respiratory
diseases, cardiovascular and neurodegenerative
diseases (2).
Ketapang Badak (Ficus lyrata W.) is an
annual plant that is usually used as an
ornamental and shade plant because of its long
stems and thick leaves (4). Ketapang Badak
(Ficus lyrata W.) is one of the plants of the
Ficus genus whose utilization is still lacking.
According to the research, from the six types of
Ficus genus plants, Ficus lyrata w. has the
highest antioxidant activity with an IC50 value
is 38,37 ppm (5). The antioxidant activity of
Ketapang Badak (Ficus lyrata W.) is because
there are phenolic compounds, which
according to research (6).
Ketapang Badak (Ficus lyrata W.) has
the potential to be developed as an antioxidant
source. So far, there have been no reports that
Plant material used was collected from
Padjadjaran university surroundings. The plant
was determined at the Taxonomy Laboratory,
Biology Department, Faculty of Mathematics
and Natural Sciences, Padjadjaran University.
2.3 The Extraction of Ketapang Badak (
Ficus lyrata W.)
Every 100 grams of simplicia powder
were extracted using three different solvents
(ethanol, methanol, and ethyl acetate) with
maceration method for 3x24 hours, which
every 24 hours, the filtrate was collected and
changed with a new solvent. It is concentrated
using the rotary evaporator with the maximum
temperature is 40℃ (7)
Rendement (%) =
have developed the fruit of Ketapang Badak
(Ficus lyrata W.) to be formulated into a dosage
𝑇𝑜𝑡𝑎𝑙 𝑤𝑒𝑖𝑔ℎ 𝑜𝑓 𝑒𝑥𝑡𝑟𝑎𝑐𝑡
𝑇𝑜𝑡𝑎𝑙 𝑤𝑒𝑖𝑔ℎ 𝑜𝑓 𝑠𝑖𝑚𝑝𝑙𝑖𝑐𝑖𝑎
𝑥 100% … (Eq. 1)
form as an antioxidant supplement, including
instant granules. Granules preparations have
better stability, flowability, more practical, and
suitable for preparations.
So, this study aim to formulate the
Ketapang Badak (Ficus lyrata W.) extract to
become instant granules as an antioxidant
supplement.
2. Methods
2.1 Materials
Spectrophotometer UV-Vis (TECAN
M200 pro), analytical measurement (Mettler
Toledo). Granulator, powder density tester,
flowability and angle of repose tester
(ERWEKA GT), oven (Memmert), macerator,
rotary evaporator (BUCHI Rotavapor R-300),
water bath (Memmert), ethanolic extract of
Ficus lyrata W, DPPH (1,1-diphenyl-2-
picyhydrazyl) (Sigma Aldrich), Vitamin C
(Bratachem), ethanol 70%, ethanol 95%, n-
hexane, ethyl acetate, Citric acid (Quadrant),
Sodium Citrate (Quadrant)l, Xanthan Gum
(Fufeng), Sucrose (ROFA), and Lactose
(Dwilab Mandiri).
2.2 Material collection and plant
determination
2.4 Antioxidant Activity Test
2.4.1 Sample preparation
Ethanol extracts were prepared with a
stock solution of 100 ppm. 100 ppm stock
solution was diluted to concentrations 10, 20,
30, 40, and 50 ppm.
2.4.2 Preparations of Comparative solutio
ns
Ascorbic acid was prepared with a
solution of 100 ppm and diluted the stock
solution standard to concentrations 0, 1, 2, 3,
and 4 ppm.
2.4.3 Preparation of DPPH solution
DPPH was weighed and dissolved in
ethanol p.a at a concentration of 0.1 M for
immediate use and maintained in low
temperatures, and protected from light
exposure.
2.4.4 Maximum Wavelength Determination
DPPH 8 mg was dissolved with 50 mL
ethanol. 0.6 mL of DPPH solution was put to
the cuvette and added methanol until its volume
was 3 mL. The solution was incubated for 30
minutes. It was measured at a wavelength from
400-800 nm (8)
N. A. Putriana et al / Indo J Pharm 3 (2021) 116-127
119
2.4.5 Determination of percent inhibition
The sample solutions of each
concentration were put to the cuvette and then
added 0.6 mL of DPPH solution. It was added
methanol until the volume is 3 mL. The mixture
solution was incubated for 30 minutes,
protected from light exposure, and measured at
its maximum wavelength (517 nm)
Inhibition (%) =
𝐵𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒−𝑆𝑎𝑚𝑝𝑙𝑒 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
𝑥 100%
…
NaOH 1% was added and incubated for an
hour. The solutions were measured at 765
wavelengths (9)
2.5.2. Determination of Total Phenolic
Content
200 mg extract was dissolved at 25 mL
methanol. 1 mL of extract solution was pipetted
to 10 mL of a volumetric flask with methanol.
1 mL sample solution was added to the test tube
with 5 mL of Folin-Ciocalteau and incubated
(Eq.
𝐵𝑙𝑎𝑛𝑘 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
for 8 minutes. The solution was added with 4
mL of NaOH 1% and incubated for an hour (9).
2.4.6 Determination of IC50 with DPPH
method
Substitute the sample concentration and
percent inhibition on the x and y axes in the
linear regression equation, respectively. The
equation was used to determine the IC50 value
of each sample, their value is 50, and the x
values will be obtained as IC50 (8)
2.5 Determination of Total Phenolic
Content of Extract
2.5.1 Determination of standard curve
using Gallic Acid
Gallic acid was prepared with a solution
of 400 ppm. Diluted the stock solution standard
to concentrations 30, 40, 60, and 80 ppm with
methanol p.a. 1 mL of each solution was added
5 mL of Folin-Ciocalteau reagent and
incubated for 8 minutes. After that, 4 mL of
The total phenolic content can be calculated by
the equation:
Total Phenolic Content (TPC) 𝑐 𝑥 𝑣 𝑥 𝑓𝑝
𝑔
Notes:
c = Phenolic concentration (x value)
v = volume used (mL)
fp = dilution factor
g = sample weigh (g)
2.6 The Formulation of Instant
Granules
The formulating was optimized by a
Design Expert, where there are four formulas
that contain two factors that have been varied:
Suspending agent and the binder
Tabel 1. Instant granules formulas
F1 (%)
F2 (%)
F3 (%)
F4 (%)
Function
Ficus lyrata
extract
3.6
3.6
3.6
3.6
Active
substance
PVP
5
0.5
5
0.5
Binder
Xanthan
Gum
1.5
0.8
1.5
0.8
Suspending
agent
Citric acid
0.3
0.3
0.3
0.3
Acid
flavoring
N. A. Putriana et al / Indo J Pharm 3 (2021) 74-81
121
Sodium
citrate
0.3
0.3
0.3
0.3
pH balancing
Sucrose
40
40
40
40
Sweetening
agent
Lactose
Ad 100
Ad 100
Ad 100
Ad 100
Filler
2.7 Formula Optimization using
ANOVA Design Expert
Determination of the best formula is
done using Design-Expert software by entering
the results of the Evaluation as a response and
determining the optimum value of each
response used. Furthermore, the Design Expert
will perform an analysis of variance (ANOVA)
on the responses used. The results of the
analysis of the response can be seen from
several results, including the value of the
contribution of factors to the response, the P-
Value to see the significance of each response,
the mathematical equation of the response used,
the interaction graph of each formula design on
the response and the desirability value (10).
2.8 Evaluation of Granules
2.8.1 Loss on Drying (LOD)
The granules were weighed at 10 grams and
placed on a moisture content analyzer. Then
click start and after 10 minutes, the instrument
showed the LOD results in percentage (11).
2.8.2 Flowability and The Angle of Repose
The granules were weighed at 20 g and put into
the funnel with a closed bottom hole. When
measuring, the bottom hole was opened and the
time needed by the granules to flow down was
calculated using a stopwatch. Flow rate was
determined by the following equation.
Flowability = 𝑤 … (Eq. 3)
𝑡
Description:
w = granul weight (g)
t = the time needed by the granules to flow (s)
The granules that were formed like cones on a
flat plane and measured for their angle of
repose, with the following equation:
α = arc tan ℎ … (Eq. 4)
𝑟
Description:
α = angle of repose(°)
h = heap height (cm)
r = heap radius(cm)
2.8.3 Dispersion Time
5 grams of granules were dispersed into 100
mL of aquadest in a beaker. The dispersion time
was recorded by measuring the time from the
start until all the granules were homogeneously
dispersed in aquadest (Husni et al., 2020).
2.8.4 pH Solution
5 grams of instant granules were dissolved into
100 mL aquadest. pH solution was measured by
pH meter that was previously calibrated using
pH 4 and 7 buffers. The pH meter was inserted
into the instant granule solution and the
measured pH was recorded.
2.8.5 Sedimentation Volume
5 grams of instant granules were dissolved into
100 mL aquadest. The solution was stirred until
it was dispersed. The amount of sediment that
occurs was observed in a period of 1-15
minutes (12).
2.8.6 Dispersibility
5 grams of instant granules were dissolved into
100 mL aquadest. The solution was stirred until
it was dispersed. The time until the formation
of a precipitate is observed and recorded.
2.8.7 Redispersibility
The redispersion test was carried out after the
evaluation of the sedimentation volume was
completed. The test tube containing the
dispersed preparation was rotated 180o and
inverted to its original position. Good
N. A. Putriana et al / Indo J Pharm 3 (2021) 74-81
121
redispersibility if the sediment in the solution
can be completely dispersed (13).
2.8.8 Homogeneity Test
Homogeneity test was done by measuring the
total phenolic content of instant granule
preparations. A number of granules were
weighed equivalent to 0.2 grams of extract (5
times) and then dissolved in 25 ml of methanol.
The absorbance of the sample solution was
measured and the polyphenol content was
calculated.
1. Result
3.1 Determination
The result of plant determination show
Ficus lyrata W. fruit belongs to the
Family Moraceae, Genus: Ficus,
Species: Ficus lyrata W., which
Indonesian name is Ketapang or Biola
cantik.
3.2 Extraction
Extraction using three kinds of solvents with
the maceration method shows the result as in
the Table 2.
Tabel 2. Rendement result of Ficus lyrata extract
Solvent
Rendement
Methanol
8.39%
Ethanol
11.49%
Ethyl acetate
1.39%
3.3 Antioxidant activity
Antioxidant activity of the ethanol extract of
Ketapang Badak fruit (Ficus lyrata W.)
decreased after being made in the form of
instant granules. It can be caused by the
influence of pH on the granule preparation. In
a study conducted by (14), pH significantly
affected the ability of polyphenols to reduce
lipid oxidation where the caffeic acid content
increased at pH 6 compared to pH 2 and 4.
Tabel 3. IC50 values
Concentration
Absorbance
average
Inhibition (%)
IC50
(ppm)
Ascorbic acid
1
0.629
29.2463
1.7
2
0.222
75.0281
3
0.088
90.1012
4
0.082
90.7762
Ethanol
extract
10
0.7743
14.0902
38.1575
20
0.6240
30.7692
30
0.5150
42.8624
40
0.3967
55.9911
50
0.3710
58.8388
Methanol
extract
10
0.763
17.5432
37.056
20
0.622
32.7810
N. A. Putriana et al / Indo J Pharm 3 (2021) 74-81
127
30
0.50767
45.1369
40
0.40567
56.1599
50
0.37067
59.9424
Ethyl acetate
extract
50
0.730
17.8853
116.62
100
0.524
41.0574
150
0.246
72.3285
200
0.157
82.3397
Instant
granules
25
0.732
9.1346
145.020
50
0.662
17.8329
75
0.588
27.0170
100
0.55867
30.6578
125
0.48733
39.5118
150
0.375
53.4547
175
0.29867
62.9293
200
0.251
68.8457
3.4 Total phenolic content
Tabel 4. Total phenolic content results
Sample
Average of
absorbance
Test
solution
volume
(mL)
Weight
(g)
Linear
equation
R2
Total
polyphenol
content (µg
GAE/g)
Extract
0.5513
25
0.2053
Y = 0.0126x
+ 0.0064
0.9966
52665.218
Instant
granules
0.4367
25
0.2
y = 0.0085x
+ 0.1841
0.9957
37066.46
3.5 Evaluation of Instant Granules
Several evaluations were completed,
including LOD, flowability and the angle of
repose, dispersion time, the pH solution,
sedimentation volume, dispersibility,
redispersibility, and homogeneity test.
LOD (Loss on Drying) test is to
measure the moisture balance in the granules
that can affect the stability of the granules,
which is the lower the water content is, the
higher the stability of the granules. The
requirement of the good LOD value is in the
range of 2-4% (12).
Flowability and the angle of repose
N. A. Putriana et al / Indo J Pharm 3 (2021) 74-81
127
Flow rate is defined as the time it takes for a
number of grams of granules to flow through a
funnel. Good granules are granules that can
flow easily. Flow properties can be influenced
by particle shape, particle size and
cohesiveness between particles, while he angle
of repose is the maximum angle that the granule
surface can form in the horizontal plane. The
size of the angle of repose is influenced by the
size of the particles, the magnitude of the
attractive force and the friction between the pa
rticles (12).
Dispersion time aims to determine the
time required for the granules to be dispersed in
water. The dispersed time of granules is
influenced by the size distribution of the
granules, where smaller granules are dispersed
faster than larger granules. The uniform particle
size also causes the water medium to more
easily penetrate into the granule particles
thereby increasing the granule dispersal
time. The dispersed time requirement is less th
an 5 minutes (15).
pH solution was evaluated to know the
exact pH of the granules after being dispersed
in water, which all the four formulas of the gra
nules showed the range pH is about 4.
Sedimentation volume test was carried
out to determine the deposition ratio (F) that
occurred during a certain time storage. A good
F value is close to 1, meaning that the particles
in the suspension are evenly dispersed in the
carrier liquid (16). Meanwhile the
sedimentation evaluation was carried out to
measure the time needed to form a
sedimentation of the granules after being
dispersed.
The homogeneity test was carried out to
determine the uniformity of the extract content
in the granule preparation. This test was carried
out by measuring the total polyphenol content
several times in instant granule preparations.
Tabel 5. Evaluation of instant granules
Evaluation
Formula
1
(n=3)
2
(n=3)
3
(n=3)
4
(n=3)
LOD (%)
2.85 ± 0.00
2.30 ± 0.00
4.08 ± 0.00
3.28 ± 0.00
Flowability (s)
15.593 ± 2.114
16.053 ± 0.488
15.063 ±
0.685
16.043 ± 0.221
Angle of repose (o)
21.204±1.355
19.057±1.363
21.933±0.368
21.770±0.862
Dispersion time
3’ 23”
2’ 21”
1’ 26”
31”
pH
4.645
4.535
4.845
4.735
Sedimentation
volume (mL)
15 min
0.91
0.92
-
-
60 min
0.9
0.87
0.6
0.87
Sedimentation time (s)
7.590 ± 0.829
6.227 ± 0.935
26.890 ± 1.64
52.213 ± 1.788
Redispersibility
+
+
+
+
N. A. Putriana et al / Indo J Pharm 3 (2021) 74-81
127
Homogeneity
Tested by looking at the total phenolic content of granules for five
times replication
+: Dispersed granules can be redisperse
-: Dispersed granules cannot be redisperse
4. Discussion
Simplicia was extracted using three
different solvents to obtain which one is the
best solvent to get the better rendement and
IC50 value. As presented in the Table 3., the
ethanol extract is the one that has the highest
rendement and the IC50 value categorized as the
strong antioxidant. As it mention in the
research conducted by Do, Ethanol has been
known as a good solvent for polyphenol
extraction and is safe for human
consumption(17). So, for the formulation of
instant granules, the chosen solvent is ethanol.
The antioxidant activity test was carried
out using a UV-Vis spectrophotometer with
DPPH-method. The DPPH method has
advantages such as being simple, easy, fast and
requiring a small number of samples. The
working principle of the DPPH method is that
the hydrogen atom (H+) in the antioxidant
compound will bind to free electrons in the
DPPH radical compound so that it causes a
change from free radicals to non-radical
compounds (diphenylpicrylhydrazine). This is
indicated by a change in color from purple to
yellow (free radical compounds are reduced by
the presence of antioxidants) (Setiawan et al.,
2018). The ability of a sample to capture DPPH
radicals is an indication that the sample has
antioxidant activity. The parameter used for the
DPPH radical scavenging test is the IC50 value,
which is the concentration of a test sample
required to reduce DPPH radicals by 50%. The
IC50 value is obtained from a linear regression
equation which states the relationship between
the concentration of the test sample and the
percent inhibition of the DPPH radical.
A compound is said to have very strong
antioxidant activity if the IC50 value is less than
50 ppm, strong if the IC50 value is 50-100 ppm,
moderate if it is 100-150 ppm, and weak if the
IC50 value is 150-200 ppm (18). Based on the
results of the study of IC50 ethanol extract
Ficus lyrata W. is 38.27 ppm categorized as a
very strong antioxidant, and the IC50 of instant
granules is 145.02 ppm is categorized as a
moderate antioxidant.
Determination of total polyphenol
content was carried out on extracts and granules
using Folin-Ciocalteau reagent. Folin-
Ciocalteau reagent is used because this reagent
can react with phenolic compounds to form a
colored solution which can then be measured
absorbance (9).
The principle of this method is the
formation of a complex blue compound. When
phenolic compounds are reacted with the Folin-
Ciocalteau reagent, a change from yellow to
blue will occur (19). Phenolic compounds will
only react with Folin-Ciocalteau in an alkaline
environment so that proton dissociation occurs
in phenolic compounds into phenolic ions, so
the addition of a base is required (9). The
intensity of the blue color is determined by the
amount of phenol content in the test compound.
The greater the concentration of phenolic
compounds in the sample, the darker the blue
color seen (19).
From the results of testing the total
polyphenol content, it can be seen that the
instant granule compound decreased the total
polyphenol content. This is in line with the
decrease in antioxidant activity (IC50), which
may be due to the influence of pH on the
granule preparation. In a study conducted by
(Kim and Choe, 2018), pH significantly
affected the ability of polyphenols to reduce
lipid oxidation where the caffeic acid content
increased at pH six compared to pH 2 and 4.
4.1 Formula optimization using Design
Expert
The response data for the optimization
of the formula entered into the Design Expert is
the Evaluation of the flowability and
sedimentation time by determining the
category criteria for the response of flowability
and sedimentation time, namely "maximize"
N. A. Putriana et al / Indo J Pharm 3 (2021) 74-81
127
because the higher the flowability value, the
better the flow properties of the granules. The
longer the sedimentation time, the better the
quality of the granule preparation. The effect of
using Xanthan Gum and PVP on the flowability
and sedimentation time is shown in Table 6.
Tabel 6. Contribution percentage of excipients
No
Excipient
%
Contribution to
Flowability
%
Contribution to
Sedimentation time
1
Xanthan
Gum
9.22257
10.2091
2
PVP
0.118008
76.5825
3
Xanthan
Gum - PVP
0.106408
12.6747
Tabel 7. Desirability values
Formula
Xanthan Gum
PVP
Desirability value
1
1.5
5
0.467
2
0.8
0.5
0.210
3
1.5
0.5
0.229
4
0.8
5
0.723
Figure 1. Contour plot dan 3D surface formula to sedimentation time
Figure 2. Contour plot dan 3D surface formula to flowability
N. A. Putriana et al / Indo J Pharm 3 (2021) 74-81
127
Figure 3. Contour plot dan 3D surface formula to desirability value
5. Conclusion
The best formula for instant granule
ethanol extract of the Ketapang Badak fruit
(Ficus lyrata W.) was Formula 4 with a
concentration of 0.8% xanthan gum and 5%
PVP, which met the requirements for good
granule quality. The results of the Evaluation of
Formula 4 showed a LOD value of 3.28%,
flowability 16.043 ± 0.221 (g/s), angle of
repose 21.77 ± 0.862, did not form a precipitate
for 15 minutes, pH = 4.7, dispersed within 31
seconds and a sedimentation time of 52.213 ±
1.7878 (minute).
The antioxidant activity of extracts and instant
granules was obtained, where the IC50 value of
the ethanol extract was 38.27 g/ml, which was
a very good antioxidant group, while the IC50
value of the granule preparations was 145.02
g/ml, which was included in the moderate
antioxidant group.
Acknowledgements
The authors are grateful to the Rector of
Universitas Padjadjaran for funding this study
and the Covid Research Grant from the
Ministry of Education the Directorate Career
and Competency of Human Resources,
Ministry of Research, Technology and Higher
Education, Indonesia.
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