1065Original Article
Pharmacogn J. 2021; 13(5): 1065-1071
A Multifaceted Journal in the Theld of Natural Products and Pharmacognosy
www.phcogj.com
Cite this article: Muchtaromah B, Wahyudi D, Ahmad M, Ansori ANM, Annisa R, Hanifah
L. Chitosan-Tripolyphosphate Nanoparticles of Mango Ginger (Curcuma mangga) Extract:
Phytochemical Screening, Formulation, Characterization, and Antioxidant Activity. Pharmacogn
J. 2021;13(5): 1065-1071.Phcogj.com
Pharmacognosy Journal, Vol 13, Issue 5, Sep-Oct, 2021
Chitosan-Tripolyphosphate Nanoparticles of Mango Ginger
(Curcuma mangga) Extract: Phytochemical Screening,
Formulation, Characterization, and Antioxidant Activity
Bayyinatul Muchtaromah
1,
*, Didik Wahyudi
1
, Mujahidin Ahmad
1
, Arif Nur Muhammad Ansori
2
, Rahmi
Annisa
3
, Lil Hanifah
1
INTRODUCTION
The use of plants for medicinal purposes is
common in Indonesia.
1,2
A medicinal plant widely
used in various communities is the mango ginger
(Curcuma mangga).
3
This is used as a lust booster
and detoxification drug. Preliminary studies in
identifying the group of compounds in its extracts
showed some pharmacological effects, making it
important in pharmaceutical industries.
4
The separation of secondary metabolite compounds
such as alkaloids, flavonoids, triterpenoids,
steroids, saponins, and tannins from this plant is
carried out through TLC methods. This research
is a preliminary screening to determine the profile
of mango ginger extracts as a basis for making the
right drug delivery system. Compounds with low
solubility in water also have low bioavailability in
the body. This could be prevented by developing
mango ginger extract nanoparticles using
ionic gelation methods by chitosan polycation.
Nanoparticles are materials with particle size
on the nanometer scale, and the application of
nanotechnology is common in the health and
pharmaceutical world, used in drug delivery and is
not harmful to the human body.
5
Chitosan is a non-toxic natural polysaccharide,
which is biodegradable. It has a cellulose-like
structure with the ability to form a gel in an
acidic atmosphere, and matrix-like in drug delivery
systems.
6
One of the methods used for synthesizing
chitosan nanoparticles is the ionic gelation method.
7

Most researchers make use of this method due to
its simple process, and the particles released are
easily controlled. The principle of nanoparticles
formation in this method is based on the occurrence
of electrostatic interactions between amine groups
in positively charged chitosan with negatively
charged polyanion TPP to form a three-dimensional
intramolecular structure.
8
This study involves the
phytochemical identification of chitosan-TPP
nanoparticles synthesized from mango ginger
extract, determination of its characteristics, as well
as its antioxidant activity.
MATERIALS AND METHODS
Materials
Mango ginger revealed from Balai Materia Medika,
Batu, Indonesia. 70% ethanol (Bratachem), chitosan
(Himedia), tripolyphosphate (Sigma Aldrich), and
acetic acid (Merck).
Extraction of mango ginger extract
Mango ginger was macerated using 70% ethanol
solvent. The mixture was soaked for 24 hours, then
filtered. Maceration was repeated three times to
obtain a clear colored filtrate. The filtrate obtained
was concentrated with a rotary evaporator at 50 °C.
ABSTRACT
Introduction: Mango ginger (Curcuma mangga) is one of Indonesia's medicinal plants widely used in most
communities as a lust booster and for detoxifying purposes. Therefore, the purpose of this study is to
synthesize chitosan-tripolyphosphate nanoparticles from mango ginger extract, determine their chemical
contents, the nano chitosan characteristics, and its antioxidant activity. Methods: In this study, we
macerated mango ginger using 70% ethanol solvent, then performed phytochemical test and formulation
of chitosan nanoparticles of mango ginger extract. The group of secondary metabolites that showed
positive results with the reagent test was further identified through TLC. Results: The results showed
that the extract contained flavonoids and triterpenoids. Also, characterization of chitosan nanoparticles
from the extract was conducted with FTIR test, PSA, XRD, and SEM. Based on the results, the nano
chitosan particle size was 993 nm and examination with FTIR showed the presence of N-H and P=O
groups, indicating ammonium ion interaction from chitosan with the polyanion from TPP and Mango
ginger. Additionally, the XRD results showed that the crystals formed were in an amorphous form, which
was supported by particle morphology images from SEM. Furthermore, the nanoparticles showed very
strong antioxidant activity based on the reaction with DPPH. Conclusion: Based on these results, the
phytochemical identification of mango ginger extract showed positive results in flavonoid and triterpenoid
compounds. In addition, based on the characterization of the nanoparticles, the mango ginger extract
showed positive results, illustrating that the nano chitosan synthesis was successful. Furthermore, the
nano chitosan has a very strong antioxidant activity with an IC
50
value of 18.08 µg/mL.
Key words: Chemical identification, Nanoparticles, Chitosan, Mango ginger, TPP.
Bayyinatul Muchtaromah
1,
*, Didik
Wahyudi
1
, Mujahidin Ahmad
1
,
Arif Nur Muhammad Ansori
2
,
Rahmi Annisa
3
, Lil Hanifah
1
1
Department of Biology, Faculty of Science
and Technology, Maulana Malik Ibrahim
State Islamic University, Malang, INDONESIA.
2
Doctoral Program in Veterinary Science,
Faculty of Veterinary Medicine, Universitas
Airlangga, Surabaya, INDONESIA.
3
Department of Pharmacy, Faculty of Medical
and Health Sciences, Maulana Malik Ibrahim
State Islamic University, Malang, INDONESIA.
Correspondence
Bayyinatul Muchtaromah
Department of Biology, Faculty of
Science and Technology, Maulana Malik
Ibrahim State Islamic University, Malang,
INDONESIA.
E-mail: [email protected]
History
• Submission Date: 18-04-2021;
• Review completed: 12-06-2021;
• Accepted Date: 18-06-2021.
DOI : 10.5530/pj.2021.13.138
Article Available online
http://www.phcogj.com/v13/i5
Copyright
© 2021 Phcogj.Com. This is an open-
access article distributed under the terms
of the Creative Commons Attribution 4.0
International license.

1066Muchtaromah B, et al .: Chitosan-Tripolyphosphate Nanoparticles of Mango Ginger (Curcuma mangga ) Extract: Phytochemical Screening, Formulation,
Characterization, and Antioxidant Activity
Pharmacognosy Journal, Vol 13, Issue 5, Sep-Oct, 2021
Phytochemical test with reagent test
Phytochemical tests were carried out on the active compounds of the
ethanol extract dissolved in a little solvent. Compounds tested with the reagent
include; alkaloid, flavonoid, triterpenoid, steroid, saponin, and tannin.
Separation of active compounds with TLC
The group of secondary metabolites that showed positive results with
the reagent test was further identified through TLC. The GF
254
silica
gel plate was used in the chromatography, and the Spots formed on
it were at a wavelength between 254 and 366 nm. These were further
sprayed with Spot’s viewer and then viewed under the same UV light.
Characteristics observed in the Spot include; the number, color, and
distance of migration from its original place, i.e., the Rf value.
Formulation of chitosan nanoparticles from mango
ginger extract
The ionic gelation method was used with sonification time of 90
minutes. This involved dissolving 0.1 gram of mango ginger extract in
5 ml of 70% ethanol. Then, 0.5, 0.75, and 1% of chitosan solution were
separately put into 100 ml of acetic acid and stirred until it dissolved.
Also, 0.1, 0.15, and 0.2 gram of TPP were dissolved in 20, 30, and 40
ml of distilled water, respectively. Then, 1 ml of tween 80 was added to
each solution concentration and stirred using a homogenizer at speed
of 1000 rpm for 10 minutes. This mixture of chitosan, TPP, and mango
ginger extract was turned into homogeneous using a disperser at speed
of 3000 rpm for 30 minutes. The mixture was then left for 24 hours
and lyophilized (freeze-drying) to obtain nanoparticles in the form of
powder samples.
9
Characterization of chitosan nanoparticles in mango
ginger extract
The particle size measurement and distribution were carried out using
the Nanotrac Wave II Q by Microtrac MRB. Results were calculated
from the average fluctuations in the light scattering intensity.
Functional group examination
This was conducted using FTIR. The mixture was kept in a vacuum
freeze dryer for one day, and the resulting powder was irradiated with
infrared light at a wavelength of 4000-400 cm
-1
.
9
Crystal formation examination
This was conducted using XRD, and the level of crystallinity was
determined using wavelength source of 1.5406 °A.
9
Particle morphology examination
The particle morphology examination was carried out with a SEM at
magnification of 500×.
5,9
RESULTS AND DISCUSSION
The extraction process with 101.300 grams of mango ginger powder
produced a thick extract of 19.657 grams (yield of 19.405%) (Table
1). The phytochemical test provided an overview of the secondary
metabolite compounds contained in each extract. This was performed
on alkaloids, flavonoids, triterpenoids, steroids, saponins, and
tannins groups (Table 2). The results of phytochemical testing were
strengthened with the separation of active compounds, flavonoids,
and triterpenoids, through TLC. The separation results of flavonoid
from mango ginger ethanol extract in 5 eluent variations are presented
below (Table 3). The best eluent for flavonoid separation in the mango
ginger extract was methanol:chloroform (1:9), which produced orange
Spots and turned green after being evaporated with ammonia, and
two other green Spots before and after being steamed with ammonia
with Rf of 0.813, 0.9, and 0.95 respectively. Similarly, the results of
triterpenoid separation from mango ginger extract using seven
eluent variations are shown below (Table 4). The best eluent for
triterpenoid separation in mango ginger extract is n-hexane: ethyl
acetate (6:4), which produced 6 Spots; purple (0.188), orange
(0.356), green (0.275), (0.325), (0.85) and yellow (0.906). The Spots
were clear, and the separation was good.
Extract
Sample Weight +
Solvent
Filtrate Color
Change
Solid Extract ColorExtract Weight Yield
Mango Ginger 101.3 g + 1000 mLSolid black to pale blackBlack 19.657 g 19.405 %
Table 1: The Result of Mango Ginger Extract through the Maceration Method.
Compound group Reagents Test Results
Alkaloids
Dragendorff -
Mayer -
Flavonoids Wilstater +
Triterpenoid Lieberman-Burchard +
Steroids Lieberman-Burchard -
Saponin Forth -
Tannin FeCl
3
-
Table 2: Results of Phytochemical Test of Mango Ginger Extract.
Note:
+: positive towards compounds/color is formed.
-: negative towards compounds/no color.
Eluent
Rf Value
Spot 1 Spot 2 Spot 3
Butanol:Acetic Acid:Water (3:1:1) - 0.938 -
Butanol:Acetic Acid:Water (3:1:1) 0.125 0.225 -
Methanol:Chloroform (1:39) 0.125 0.225 -
Chloroform:Methanol:Water (9.7:0.2:0.1) 0.088 0.125 0.375
Methanol:Chloroform (1:9) 0.813 0.900 0.950
Table 3: Analytical TLC Rf Value of Flavonoid in Mango Ginger Extract.

1067Pharmacognosy Journal, Vol 13, Issue 5, Sep-Oct, 2021
Muchtaromah B, et al .: Chitosan-Tripolyphosphate Nanoparticles of Mango Ginger (Curcuma mangga ) Extract: Phytochemical Screening, Formulation,
Characterization, and Antioxidant Activity
Eluent
Rf value
Spot 1 Spot 2 Spot 3 Spot 4 Spot 5 Spot 6
n-Hexane:Ethyl acetate (2:8)0.775 0.831 0.8875 0.925 - -
n-Hexane:Ethyl acetate (1:1)0.438 0.481 0.550 0.875 0.925 -
n-Hexane:Ethyl acetate (8:2)0.056 0.081 - - - -
n-Hexane:Ethyl acetate (6:4)0.188 0.356 0.275 0.325 0.850 0.906
Benzene:Chloroform (3:7) 0.050 0.450 0.737 0.8125 - -
Chloroform:Methanol (3:7) 0.812 0.850 0.900 - - -
n-Hexane:Acetone (80:20) 0.012 0.937 - - - -
Table 4: Analytical TLC Rf Value of Triterpenoid in Mango Ginger Extract.
Antioxidant Testing IC
50
(µg/mL)
Mango Ginger Extract Nanoparticles 18.08
Mango Ginger Extract 16.18
Ascorbic Acid 14.81
Table 5: The IC
50
value on the Mango Ginger Extract Nanoparticles, Mango Ginger Extract, and Ascorbic Acid.
This was carried out using the ionic gelation method, which involved
a reaction between the mixture of chitosan and TPP which was used
as a stabilizer. Also, the TPP added acted as a crosslinking agent,
strengthening the chitosan nanoparticle matrix, hence, producing
stable chitosan nanoparticles. Additionally, surfactant (tween 80) was
added, which also functioned as a stabilizer. The surfactant, in addition,
helped the chitosan particles in the solution to come together and
stabilize with one another, thus, forming effective nanoparticles that
are smaller in size. Mainly, the use of TPP was to avoid the formation of
aggregates and as a stabilizer of the formed nanoparticles.
10,11
The success in transforming a sample into nanoparticles is known by
measuring the sample size. Results obtained showed an average particle
size of 993 nm, indicating a nanometer particle size. Therefore, chitosan
nanoparticles can increase the bioavailability of active compounds in
the body. Particle measurement results are presented below (Figure 1).
The FTIR instrument was used to identify the complex groups in the
compounds but was unable to determine their constituent elements.
With FTIR, infrared radiation passes through the sample, and while
some of the radiation is absorbed, some are transmitted by the sample.
In general, the molecule absorbs the radiation if the frequency of its
vibration is equal to the infrared radiation frequency, which is directed
towards it. The results of the functional group's examination on
chitosan nanoparticles from mango ginger extract are shown below
(Figure 2).
Figure 2 shows its chemical profile in the form of different spectrum
patterns with distinctive characteristics. Chitosan has specific groups,
such as -NH
2
and -OH.
12
The FTIR results showed the presence of
hydroxyl groups at wavelength of 3425.56 cm
-1
due to the vibrational
strain interaction between the hydroxyl group and the amide group
on chitosan. However, the chitosan amide function group was at a
wavelength of 1640.56 cm
-1
. The FTIR results showed the functional
groups in chitosan from mango ginger extract nanoparticle within 90
minutes of sonication period. Determination of chitosan presence is
needed to determine the extract coating ability, and a method used to
determine its presence is through FTIR. The infrared spectrum has the
ability to detect functional groups and identify compounds in a polymer
sample.
13
The FTIR in this study used intermediate level wavenumbers,
in the range of 4000-400 cm
-1
and the determination of the wavelength
value was in accordance with the functional groups in organic
compounds.
14
The working principle of FTIR is based on the absorption
or transmission of infrared light by the molecules of compounds in the
sample. The molecule absorbs the light if the frequency of its functional
group’s vibration is the same as the frequency of the infrared radiation.
However, not all of the infrared light is absorbed by the molecule.
15

The result obtained from FTIR is in the form of a transmittance graph.
Analysis of the crystal structure of the catalyst was conducted using
XRD. It is one of the oldest and most commonly used methods in
material characterization to date. This XRD technique is used to
identify a material based on its crystalline state by determining the
lattice parameters and its particle size. The method is based on the
fact that XRD patterns for each crystalline material have different
characteristics. The result of the crystal formation examination is
shown below (Figure 3).
The XRD analysis was used to determine the physical structure of the
sample. The characterization results of the nanoparticles showed an
amorphous property, indicating that the constituent particles were
irregularly arranged and less compact. This irregular arrangement
allows the easy insertion of other molecules. In addition, the more
amorphous a molecule is, the easier it is to insert other molecules into
it.
16
Normally, the amorphous shape of a particle is marked by a valley
peak at diffraction angle of 20°.
The SEM is a technique widely used for material characterization,
with the capacity of viewing particle surface morphology up to 1 nm
in size.
17
It is also a method used to examine the shape and surface
microstructure of an object which cannot be seen by the eye or
with an optical microscope.
18
Its large magnification range and the
3-dimensional image makes SEM results easier to observe and analyze.
The results of this study showed that chitosan nanoparticles were
mostly spherical. The result of the morphological examination of
chitosan nanoparticles is presented below (Figure 4).
Analysis of antioxidant activity was carried out on mango ginger
extract nanoparticles, mango ginger extract, and ascorbic acid used as
control. Each of the samples were allowed to react with free radicals
in the form of DPPH, and their absorbance was measured using a
spectrophotometer. The absorbance measurement was carried out with
test solution concentrations at 0, 2, 5, 10, and 25 ppm, measured from
low to high concentration. The IC
50
results are shown below (Table 5).
The table shows that the nanoparticles of mango ginger extract have an
IC
50
value of 18.08 µg/mL, mango ginger extract has 16.18 µg/mL while
ascorbic acid has 14.81 µg/mL. These values in the three samples are
indication that the antioxidant activities are very strong. The substance
with an IC
50
value of ≤50 µg/mL, has a very strong antioxidant activity.
However, values within 50-100 indicate a strong antioxidant activity,
within 100-150 is classified as a weak, 150-250 is classified as weak,
while ≥250 is considered as inactive, but still has potential as an
antioxidant substance.

1068Muchtaromah B, et al .: Chitosan-Tripolyphosphate Nanoparticles of Mango Ginger (Curcuma mangga ) Extract: Phytochemical Screening, Formulation,
Characterization, and Antioxidant Activity
Pharmacognosy Journal, Vol 13, Issue 5, Sep-Oct, 2021
Figure 3: The results of the examination on chitosan nanoparticles crystals formation using XRD with sonication time of 90
minutes.
Figure 1: The size distribution of chitosan nanoparticles-mango ginger extract with sonication of 90 minutes using particle
size analyzer.
Figure 2: The results of functional groups examination on chitosan nanoparticles-mango ginger extract with sonication time
of 90 minutes using FTIR.

1069Pharmacognosy Journal, Vol 13, Issue 5, Sep-Oct, 2021
Muchtaromah B, et al .: Chitosan-Tripolyphosphate Nanoparticles of Mango Ginger (Curcuma mangga ) Extract: Phytochemical Screening, Formulation,
Characterization, and Antioxidant Activity
CONCLUSION
Based on these results, the phytochemical identification of mango
ginger extract showed positive results in flavonoid and triterpenoid
compounds. In addition, based on the characterization of the
nanoparticles, the mango ginger extract showed positive results,
illustrating that the nano chitosan synthesis was successful.
Furthermore, the nano chitosan has a very strong antioxidant activity
with an IC
50
value of 18.08 µg/mL.
ACKNOWLEDGMENT
We thank EJA Team, Indonesia for editing the manuscript.
DISCLOSURE STATEMENT
The authors have no conflicts of interest to declare.
ABBREVIATIONS
DPPH: 2,2-diphenyl-1-picrylhydrazyl; FTIR: Fourier transform
infrared spectroscopy; IC
50
: 50% antiviral concentration; PSA:
Particle size analyzer; Rf: Retardation factor; SEM: Scanning electron
microscope; TLC: Thin layer chromatography; TPP: Tripolyphosphate;
XRD: X-ray diffraction.
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1070Muchtaromah B, et al .: Chitosan-Tripolyphosphate Nanoparticles of Mango Ginger (Curcuma mangga ) Extract: Phytochemical Screening, Formulation,
Characterization, and Antioxidant Activity
Pharmacognosy Journal, Vol 13, Issue 5, Sep-Oct, 2021
GRAPHICAL ABSTRACT
Bayyinatul Muchtaromah received her doctoral degree at Brawijaya University, Indonesia
(2007). She is currently a lecturer at Biology Department of Universitas Islam Negeri (UIN)
Maulana Malik Ibrahim Malang, Indonesia. Her research interest includes animal physiology
and reproduction.
Didik Wahyudi received his master degree in Brawijaya University, Indonesia. He is a
lecturer in Biology Department of Universitas Islam Negeri Maulana Malik Ibrahim Malang,
Indonesia. His research interest includes botany, plant systematic and bioinformatics.
Mujahidin Ahmad received his master degree in Brawijaya University, Indonesia and
King Mongkut’s University of Technology Thonburi Bangkok. He is a lecturer in Biology
department of Universitas Islam Negeri Maulana Malik Ibrahim Malang, Indonesia. His
research interest includes biotechnology and animal systematic.
Arif Nur Muhammad Ansori is a Ph.D. candidate in Veterinary Science at Universitas
Airlangga. He completed his B.Sc. in Biology and M.Sc. in Vaccinology and
Immunotherapeutics at Universitas Airlangga. Currently, he is an awardee of PMDSU
Scholarship (Batch III) at Universitas Airlangga. His research projects related to virology,
bioinformatics, and molecular biology. His actual research focus is the application of
molecular biology to the development of a novel bivalent vaccine against COVID-19.
ABOUT AUTHORS

1071Pharmacognosy Journal, Vol 13, Issue 5, Sep-Oct, 2021
Muchtaromah B, et al .: Chitosan-Tripolyphosphate Nanoparticles of Mango Ginger (Curcuma mangga ) Extract: Phytochemical Screening, Formulation,
Characterization, and Antioxidant Activity
Cite this article: Muchtaromah B, Wahyudi D, Ahmad M, Ansori ANM, Annisa R, Hanifah L. Chitosan-Tripolyphosphate Nanoparticles
of Mango Ginger (Curcuma mangga) Extract: Phytochemical Screening, Formulation, Characterization, and Antioxidant Activity.
Pharmacogn J. 2021;13(5): 1065-1071.
Rahmi Annisa received her master degree in Universitas Airlangga, Indonesia. She is
currently a lecture in Pharmacy Department of Universitas Islam Negeri Maulana Malik
Ibrahim Malang, Indonesia. Her research focused in drug delivery system.