Trad. Med. J., January-April 2022
Vol. 27(1), p 51-59
ISSN-p : 1410-5918 ISSN-e : 2406-9086
Traditional Medicine Journal, 27(1), 2022 | DOI: 10.22146/mot.72210 51
Submitted : 13-01-2022
Revised : 23-03-2022
Accepted : 26-03-2022
Evaluation of Total Flavonoid, Total Phenolic, and Antioxidant Activity
of Etlingera elatior (Jack) R.M.Sm Flower, Fruit, and Leaf
Ulya Safrina
*
, Wardiyah, Harpolia Cartika
Department of Pharmacy, Politeknik Kesehatan Kemenkes Jakarta II, Jakarta Pusat, DKI Jakarta, Indonesia
ABSTRACT
Etlingera elatior (E. elatior) plant has long been used as a kitchen spice and daily food. E. elatior has
potential as an antioxidant because it contains polyphenol and flavonoid compounds widely found in the
leaves, fruit, and flowers. This study measures the total flavonoid content, total phenol content, and
antioxidant activity using the ABTS method. Total phenol content was determined by the Follin-Ciocalteu
method and total flavonoid content was determined by the Aluminium Chloride method. The results
showed that the total flavonoid content from the highest to the lowest value from E. elatior was fruit extract,
leaf extract, and flower extract, respectively. The value of total flavonoid content was 8.38 ± 0.15; 4.86 ±
0.10; and 2.60 ± 0.04 % w/w Catechin Equivalent (CE). The total phenol content from the highest to the
lowest value from E. elatior was fruit extract, leaf extract, and flower extract, respectively. The total phenolic
content values were 54.48 ± 1.89, 46.20 ± 0.83, and 4.80 ± 0.53 % w/w Gallic Acid Equivalent (GAE). The
highest to lowest antioxidant activity values (IC50) were leaf extract at 58.82 ppm (strong activity), fruit
extract at 103.05 ppm (moderate activity), and flower extract at 251.40 (weak activity), respectively.
Keywords: Etlingera elatior; total phenol; total flavonoid; antioxidant activity
INTRODUCTION
Antioxidants are natural compounds that
can prevent oxidation reactions. These compounds
can protect the body's cells from damage caused by
free radicals. Herbal plants are sources of natural
antioxidants. Therefore, they can fulfill the body's
antioxidant needs (Ulewicz-Magulska and
Wesolowski, 2019). In its performance, these
natural compounds inhibit oxidation reactions by
binding to free radical molecules and maintaining
the genetic structure in normal conditions (Lingga,
2012).
Antioxidant compounds commonly found in
foodstuffs are vitamin E, vitamin C, beta-carotene,
selenium, superoxide dismutase (SOD), and
flavonoids. Natural antioxidants are dominated by
plants and generally contain phenolic compounds
such as phenolic acids, flavonoids, lignin, stilbenes,
and tannins, which are spread throughout the plant
(Silvia et al., 2016; Ulewicz-Magulska and
Wesolowski, 2019).
Extracts are liquid, viscous, or dry
preparations resulting from the extraction process
of a matrix or simplicial using an appropriate
method (Endang, 2019). Antioxidant compounds
from plants can be obtained by extraction using a
solvent. In this case, the type of solvent influences
the antioxidant activity obtained.
One of the plants that are efficacious as
antioxidants are E. elatior. Traditionally, E. elatior
has long been used and utilized by the community
*Corresponding author : Ulya Safrina
Email : [email protected]
as medicine and food flavoring. The compound
content of E. elatior leaves consists of saponins,
flavonoids, and chlorogenic acid. E. elatior has
various kinds of pharmacological activities such as
antioxidant, antibacterial, larvicidal, and repellent
(Farida and Maruzy, 2016).
In a previous study, antioxidant activity was
tested in the aqueous extract of E. elatior leaves
using the DPPH method (2,2 -diphenyl-1-
picrylhydrazil), which gave an IC50 value of 24.39
mg/L, which was classified as very strong
(Ningtyas, 2011). Meanwhile, the antioxidant
activity of 96% ethanol extract of E. elatior leaves
obtained an IC50 value of 4.7645 ppm, classified as
having very strong antioxidant activity with total
phenolic content of 48.223 mg GAE/gram
(Handayani et al., 2014; Pramiastuti et al., 2018). In
addition, the antioxidant activity of the leaf, flower,
and stem fraction of E. elatior was tested using the
ferric thiocyanate method. The method is based on
the formation of peroxide, which results from the
oxidation of linoleic acid. The study results on E.
elatior leaves showed that the type of fraction had
a significant effect on total phenol and antioxidant
activity. The ethyl acetate fraction gave the total
phenol and antioxidant values, respectively,
ranging from 522.08 to 1776.08 mg/100 g and
62.30 to 73.87%. Meanwhile, the ethanol fraction
ranged from 854.10 to 4851.30 mg/100 g and
47.47 to 75.07% (Naufalin and Rukmini, 2011).
One of the antioxidant testing methods is
ABTS (2,2-Azinobis 3-Ethyl Benzothiazoline 6
Sulfonic Acid). ABTS is a method used to test
Ulya Safrina
52 Traditional Medicine Journal, 27(1), 2022
antioxidants in plants. The advantages of the ABTS
method are reacting quickly with antioxidants, can
be used at different pH levels, and being soluble in
water and organic solvents.
Based on previous studies, the potential of
the E. elatior plant as an antioxidant has been
proven in previous studies using E. elatior leaf
extract using the DPPH method. In addition, testing
was carried out on the E. elatior leaf fraction using
the ferric thiocyanate method. Thus, the ABTS
method encouraged the authors to research
different antioxidant activities by determining the
total phenolic and flavonoid levels in flower, fruit,
and leaf extracts. A compound is proven to function
as an antioxidant by testing at least 3 test methods.
METHODOLOGY
Source of plant material
The samples used in this research were the
leaf, fruit, and flowers of E. elatior obtained from
the gardens of local farmers in the Samadua region,
South Aceh Regency, Aceh, on April 22, 2021. The
plant parts were then inspected at the Laboratory
of the Biological Research Center, LIPI, Indonesia,
to prove the correctness of the plants used in this
study. The results showed that the sample used
was the correct E. elatior plant, with the certificate
number for the determination result being B-
147/V/DI.05.07/10/2021.
Source of chemical material
The chemicals used in this study were ABTS,
potassium persulfate, Catechin, Folin-Ciocalteu
(Sigma Aldrich), Gallic Acid (MP Biomedicals),
AlCl3, Ethanol, Acetic acid, Ferric chloride (Merck),
and Aquadest.
Extraction of E. elatior leaf, fruit, and flower
extract
Fresh E. elatior leaf, flowers, and fruit were
cut into pieces, dried, then powdered. Extraction
was carried out by the maceration method using
ethanol as a solvent. E. elatior leaf, flowers, and
fruit powder (100 g) were weighed and put into a
maceration chamber with the addition of 70%
ethanol as much as 750 mL. The powder was
soaked for 6 hours while stirring every 30 minutes,
then held for 3 days at room temperature
(Maserate I). After three days, the preparations
were separated and added with 250 ml of 70%
ethanol. After being dissolved, 70% ethanol was
allowed to be held for 2 days at room temperature
(Maserate II). Maserates I and II were mixed, then
the extraction solution was filtered using the
Büchner funnel. The filter results were then
evaporated in a rotary evaporator until it became a
thick extract (Sivanandham, 2015; Wardiyah et al.,
2021). The thick extract obtained was then
measured for its water content using a moisture
analyzer.
Phytochemical screening of E. elatior leaf, fruit,
and flower extract
E. elatior leaf, flower, and fruit extracts were
screened qualitatively for phenolic compounds,
flavonoids, saponins, and alkaloids groups.
Identification of flavonoids was carried out by
adding 2 mg of magnesium powder and 2 mL of
concentrated hydrochloric acid into the extract
and then shaking it with 10 mL of amyl alcohol. A
positive reaction is indicated by the orange, yellow,
or red color on the amyl alcohol layer (Harborne,
2012). Identification of alkaloids was carried out
with 1 gram of extract with three drops of 10%
ammonia and 1.5 mL of chloroform, then shaken.
The chloroform layer was taken and then dissolved
in 1 mL of 2 N sulfuric acid, then shaken. After that,
the mixture was added with Meyer's reagent. The
mixture contains alkaloids if there is a white
precipitate (Departemen Kesehatan Republik
Indonesia, 1989). Saponin identification was
carried out by placing 1 gram of extract into a test
tube, then adding 20 mL of hot water, cooling it,
then shaking vigorously vertically for 10 seconds.
If the foam is formed as high as 1 to 10 cm, which
is stable for no less than 10 minutes and does not
disappear with one drop of 2 N hydrochloric acid,
it indicates the presence of saponins (Departemen
Kesehatan Republik Indonesia, 1989) .
Identification of phenol by placing the extract into
a test tube and then add two drops of 5% FeCl3. If a
greenish, red-purple, blue, or black color is formed
in the mixture, indicating the presence of phenolic
compounds (Harborne, 2012).
Determination of total phenolic content
Sample solutions (250 and 500 µL) and
standard solutions of gallic acid (25,50, 100, 150,
and 200 µL) were pipetted into a test tube, and 4
mL of distilled water were added. Then, 250 µL of
Follin-Ciocalteu reagent was added and shaken.
After being allowed to stand for 8 minutes at room
temperature, 750 µL of 20% Sodium Carbonate
was added and shaken homogeneously. Next, the
mixture was allowed to stand for 2 hours at room
temperature and the absorbance was measured at
a wavelength of 765 nm (Singleton et al., 1999).
Determination of total flavonoid content
E. elatior leaf, fruit, and flower extracts and
standard solutions of quercetin were made in
various concentrations of 205.2 to 680 ppm. Each
concentration was taken 5 mL and put in a 10.0 mL
volumetric flask. Then, 0.3 mL of 5% NaNO2 and 0.3
Evaluation of Total Flavonoid, Total Phenolic, and Antioxidant Activity
Traditional Medicine Journal, 27(1), 2022 53
mL of 10% AlCl3 were added to the sample
solution. Then, the sample solution was incubated
at room temperature for 5 minutes. Next, 2 ml of 1
M NaOH was added and distilled water up to 10.0
mL. The sample solution was measured using a
UV/Vis Spectrophotometer at a wavelength of 415
nm.
Evaluation of antioxidant activity using ABTS
method
The testing procedure was carried out based
on previous research (Arnao, 2000; Wardiyah et
al., 2021). E. elatior leaf, fruit, and flower extract
sample solutions were made with 25 to 400 ppm
varying concentrations. Vitamin C standard
solution was made with a concentration variation
of 0.625 to 5 ppm. The ABTS solution and the
sample were pipetted in a 1:1 ratio into a 96-well
microplate, then homogenized. The absorbance of
the sample solutions and standard solutions was
then measured with a microplate reader at a
wavelength of 516 nm.
RESULT AND DISCUSSION
Phytochemical Screening of E. elatior Leaf, Fruit
and Flower Extracts
Phytochemical screening was carried out
simply using color reagents using qualitative
analysis methods. The results of phytochemical
screening of E. elatior flower, leaf, and stem
extracts are in Table I.
Phytochemical screening aims to determine
the class of compounds in the extracts of leaves,
fruits, and flowers of E. elatior. Phytochemical
screening tests were carried out on four main
compounds: flavonoids, phenols, alkaloids, and
saponins. The results of phytochemical screening
on E. elatior leaf and flower extracts showed
positive results for flavonoid, alkaloid, saponin,
and phenolic compounds. This is in line with other
studies showing the same results: E. elatior leaves
contain many flavonoid and saponin compounds
(Nisrina Effendi et al., 2019; Roslim and Umam,
2021). In addition, the phytochemical screening of
E. elatior fruit extract showed positive results
containing flavonoids, alkaloids, and phenolics.
This is in line with other studies that show the
phytochemical content of E. elatior fruit from
Nagan Raya, Aceh, which contains alkaloids,
phenols, flavonoids, tannins, and terpenoids. E.
elatior flower parts also contain alkaloids, phenols,
flavonoids, saponins, tannins, and terpenoids
(Ernilasari et al., 2021). The difference between
the results of this study and previous studies is the
intensity of the color produced during testing. The
more intense the color produced, the more
secondary metabolites in the extract (Das and
Gezici, 2018). Differences in secondary metabolites
in plants are influenced by variations in plant
growth height, light, climate, temperature,
groundwater, soil fertility, and salinity (Giweli
et al., 2013; Liu et al., 2016).
(a) (b) (c) (d)
Figure 1. Results of phytochemical screening of E.elatior leaf, fruit, and flower extracts sequentially. (a)
Saponin test with aquadest, (b) Flavonoid test with Mg powder and concentrated HCl, (c) Phenolic test
with FeCl3 reagent, (d) Alkaloid test with Dragendorf and Mayer reagents.
Table I. Phytochemical Screening of E. elatior Leaf, Fruit, and Flower Extracts Qualitatively
Sample Flavonoid Phenol Alkaloid Saponin
E. elatior Leaf Extract +++ +++ ++ +++
E. elatior Fruit Extract +++ +++ +++ -
E. elatior Flower Extract +++ +++ +++ +++
Notes :
+
Weak; ++Strong; +++Very strong, -Negative/none
Ulya Safrina
54 Traditional Medicine Journal, 27(1), 2022
Total Flavonoid and Total Phenol Content of E.
elatior Leaf, Fruit, and Flower Extract
Measurement of total phenolic in extracts of
leaves, fruit, and flowers of E. elatior using gallic
acid standard. The results of total phenolic
measurements can be seen in Table II.
The total phenolic content of E. elatior leaf,
fruit, and flower extract was tested using the
Follin-Ciocalteu method, with gallic acid as the
standard. The test results are expressed in % mg/g
gallic acid equivalent. The total phenolic content
was obtained from the linear regression equation y
= 0.0517x + 0.2155, R
2
= 0.9918 (Appendix 8).
From table 5, it can be seen that the highest to
lowest total phenolic content were fruit extract,
leaf extract, and E. elatior flower extract with
values of 54.48 ± 1.89, 46.20 ± 0.83, and 4.80 ± 0,
53. This result is different from several other
studies on several etlingera species. The ethanol
extract of E. elatior leaves showed a higher total
phenolic content than the fruit ethanol extract
(Ahmad et al., 2015; Isyanti et al., 2019; Shahid-Ud-
Daula et al., 2019). This can happen because the
fruit and leaves are parts of the plant that are easier
to get light and sunlight, so the concentration of
phenolic acids and flavonoids is greater in the fruit
and leaves than the flower parts (Shahid-Ud-Daula
et al., 2019). Another study also showed the same
results regarding the total phenolic content of E.
elatior flower extract, where the total phenolic
content was lower than that of the leaves and
stems. This is because the leaves contain many
polar compounds and chlorophyll (Nuryanti et al.,
2021). The high temperature during simplicial
drying can also affect the total phenolic content in
the E. elatior flower extract. The flower parts
contain many volatile essential oils, which can
evaporate when drying simplicial. This is in line
with other studies, which showed the total
phenolic content of fresh E. elatior flower
simplicial was higher than that of dried E. elatior
flower simplicial (Nuryanti et al., 2021). In this
study, dry simplicial was used.
Measurement of total flavonoids in the
extract of leaves, fruit, and flowers of E. elatior was
using catechin standards. The measurement of
total flavonoids can be seen in Table III.
Testing the total flavonoid content of E.
elatior leaf, fruit, and flower extracts were carried
out using catechins as a standard. The test is
expressed in % mg/g Catechin equivalent (CE).
From table 6, the total flavonoid content from the
highest to the lowest is E. elatior fruit extract, leaf
extract, and flower extract with a value of 8.38 ±
0.15; 4.86 ± 0.10; and 2.60 ± 0.04. This is following
previous research, which states that the E. elatior
plant's total flavonoid and phenolic content are
primarily contained in the leaves, flowers, stems,
and rhizomes (Mai, 2009). The results of the total
flavonoid content were linear with the results of
the total phenolic content. Extracts that have a high
total phenolic content also contain high total
flavonoids. Plants that contain lots of flavonoids
can function as a source of antioxidants that can
increase the antioxidant capacity of organisms and
fight lipid peroxidation (Khorasani Esmaeili et al.,
2015).
The difference in the amount of total
phenolic content and total flavonoid in each part of
the plant is influenced by various environmental
factors, such as light and ultraviolet radiation,
temperature, lack of water in the soil, salt content
in the soil, soil composition, differences in plant
age, metal content, and other chemical factors. (Li
et al., 2020). The different types of solvents used in
the extraction process also affect the content of
metabolite compounds (Herni et al., 2018).
Extraction of phenolic compounds using water as a
solvent resulted in a total phenolic content of
15.9% more than ethanol (Ghasemzadeh et al.,
Table II. Comparison of Total Phenolic Content in E. elatior Leaf, Fruit, and Flower Extracts (n = 3
replication)
No Part of Plants Average Total Phenolic Content in Extract (% w/w Gallic Acid Equivalent)
1 E. elatior Fruit 54.48 ± 1.89
2 E. elatior Leaf 46.20 ± 0.83
3 E. elatior Flower 4.80 ± 0.53
Table III. Comparison of Total Flavonoid Content in E. elatior Leaf, Fruit, and Flower Extracts (n = 3
replication)
No Part of Plants Average Total Flavonoid Content in Extract (% w/w Catechin Equivalent)
1 E. elatior Fruit 8.38 ± 0.15
2 E. elatior Leaf 4.86 ± 0.10
3 E. elatior Flower 2.60 ± 0.04
Evaluation of Total Flavonoid, Total Phenolic, and Antioxidant Activity
Traditional Medicine Journal, 27(1), 2022 55
2015). The solubility of polyphenolic compounds is
better in ethanol solvents than in water solvents.
The simplicial drying process can also damage
some phenolic compounds because, in dry
conditions, phenolic compounds are trapped in
plant cells and cannot be extracted (Nuryanti et al.,
2021). However, the high temperature during the
extraction process can increase the solubility of
phenolic compounds. Since high temperatures
can cause phenolic compounds to come out of
plant cell walls, more phenolic compounds are
extracted (Wazir et al., 2011). Thus, the drying
time and temperature need to be validated during
the drying process so that the bioactive
components contained in the extract are not
damaged.
Phenolic compounds and flavonoids are
metabolites in many plants and have been
reported to have effective antioxidant activity due
to their specific redox characteristics. Flavonoids
belong to the phenolic group that can effectively
reduce Reactive Oxygen Species (ROS). Differences
in the amount of flavonoid content in plants can be
caused by different types of flavonoid compounds
contained in these plants. Less polar flavonoid
compounds (isoflavones, flavanones, flavones, and
flavonols) are better extracted with non-polar
solvents, and more polar flavonoid compounds
(glycosides and aglycones) are better extracted
using polar solvents (alcohol, water-alcohol)
(Aryal et al., 2019; Muflihah et al., 2021).
Antioxidant Activity of E. elatior Leaf, Fruit, and
Flower Extracts
Measure antioxidant activity in extracts of
leaves, fruit, and flowers of E. elatior using the
ABTS method with Vitamin C as a standard. The
percentage value inhibition and IC 50 of
antioxidants were obtained from the linear
regression equation of the standard curve of the
standard and the sample. Antioxidant activity
testing was conducted to measure E. elatior leaf,
fruit, and flower extracts' relative antioxidant
ability to reduce free radicals in the reagents.
Antioxidant testing using ABTS reagents is the
direct production mechanism of ABTS•+, which is
blue or green due to the oxidation reaction of ABTS
with potassium persulfate, followed by a reduction
reaction due to the presence of hydrogen donor
antioxidants (Al-Mansoub et al., 2021). The ABTS
method is used because free radicals are more
stable when receiving hydrogen ion donors from
antioxidants. Thus, the blue color of ABTS
disappears. The ABTS method is more sensitive to
determining antioxidant activity because the
kinetic reaction is faster and detects antioxidants
better than the DPPH method. ABTS reagent is also
soluble in water and organic solvents. Therefore, it
can determine both hydrophilic and lipophilic
antioxidants (Lee et al., 2015; Shah and Modi,
2015).
The IC50 value was obtained from the
calibration curve of the vitamin C standard
Figure 2. Vitamin C Standard Solution Calibration Curve
y = -0,0816x + 0,6187
R² = 0,99
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,0 1,0 2,0 3,0 4,0 5,0 6,0
Absorbance
Concentration(ppm)
Ulya Safrina
56 Traditional Medicine Journal, 27(1), 2022
solution. The calibration curve was obtained from
the concentration vs. absorbance plot of the
sample so that the linear regression equation y = -
0.0816x + 0.6187 with R
2
= 0.9900 (Figure 2) was
obtained.
After obtaining the calibration curve, the
percentage of ABTS radical scavenging activity was
calculated using the formula. The results of
percentage ABTS inhibition vs. concentration are
in Figure 3. The percentage ABTS radical
scavenging activity is sorted from highest to
lowest: E. elatior leaf extract, fruit extract, and
flower extract, respectively. The difference in the
results of free radical reduction is due to the
difference in the total phenol and flavonoid content
in the plant parts. The highest total phenol and
flavonoid content was in the E. elatior fruit extract,
but the highest % ABTS reduction was in the E.
elatior leaf extract. This can happen because the
leaves of E. elatior contain other non-polar
compounds that have antioxidant activity, such as
steroids and terpenoids (Wardiyah et al., 2021).
Further research needs to be done to see other
compounds responsible for antioxidants besides
phenols and flavonoids in the E. elatior.
The results of antioxidant activity were
obtained in IC50 values, as shown in Table 4. The
highest antioxidant activity was found in E. elatior
leaf extract with an IC50 value of 58.82 ppm and
classified as strong activity. This is in line with
previous studies, which showed that the ethanolic
extract of E. elatior leaves has strong antioxidant
activity with an IC50 value of 23.45 ug/mL
(Wardiyah et al., 2021). This difference in the value
of antioxidant activity may occur due to differences
in the number of secondary metabolite compounds
contained in the plant parts since the E. elatior
plants used in this study, and previous studies
were harvested in different areas. In addition,
variations in plant age, growing location, soil
composition, and weather also affect the levels of
secondary metabolites (Li et al., 2020).
The positive control used in this study was
vitamin C. Vitamin C is a hydrophilic compound
and has very strong antioxidant activity. The IC50
value is 1.51 ppm. The use of positive control aims
Figure 3. ABTS radical scavenging activity
Table IV. Value of IC50 E. elatior Leaf, Fruit and Flower Extract (n = 3 replication)
No Part of Plants Value of IC50 (ppm) Activities
1 E. elatior Fruit 58.82 Strong
2 E. elatior Leaf 103.05 Medium
3 E. elatior Flower 251.40 Weak
4 Vitamin C 1.51 Very Strong
0
10
20
30
40
50
60
70
80
90
100
0 100 200 300 400 500
ABTS Inhibition (%)
Concentration (ppm)
ABTS Radical Scavenging Activity
Vit. C
E. elatior leaves extract
E. elatior flower extract
E. elatior fruit extract
Evaluation of Total Flavonoid, Total Phenolic, and Antioxidant Activity
Traditional Medicine Journal, 27(1), 2022 57
to compare the antioxidant activity of E. elatior leaf,
fruit, and flower extracts with positive controls.
In previous studies, the antioxidant activity
of E. elatior leaf was tested using the DPPH and
ABTS methods. The results of previous studies
showed that the antioxidant activity of E. elatior
leaf was in the very strong range in both methods.
This is different from the current study, where E.
elatior leaf was only in the strong range. This can
occur due to differences in the location of plant
growth used as samples in two studies (Li et al.,
2020). In this study, the highest antioxidant
activity was shown by the fruit extract E. elatior.
The advantage of this study is to use several parts
of the E. elatior plant to compare the value of the
antioxidant activity. ABTS method can measure
hydrophilic and lipophilic antioxidant compounds
in E. elatior. However, this study has several
limitations, such as antioxidant testing using the
ABTS method, which is an in vitro test model so it
cannot describe all antioxidant activities in E.
elatior. In addition, antioxidant activity is also
influenced by the solvent used during extraction
(Floegel et al., 2011). It needs to be considered for
further research in the selection of appropriate
methods and solvents.
CONCLUSION
E. elatior fruit extract has the highest total
phenol and flavonoid content compared to other
plant parts, but the highest antioxidant activity is
obtained from E. elatior leaf extract. This can
happen because the leaves of E. elatior contain
other non-polar compounds that have antioxidant
activity, such as steroids and terpenoids.
ACKNOWLEDGEMENT
The authors would like to thank the
Politeknik Kesehatan Kemenkes Jakarta II,
Indonesia, for funding this research. The authors
also thank all parties involved in this research.
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