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B I O D I V E R S IT A S
ISSN: 1412-033X
Volume 20, Number 2, February 2019 E-ISSN: 2085-4722
Pages: 576-580 DOI: 10.13057/biodiv/d200238
Antioxidant activity, total phenolic and flavonoid content of several
indigenous species of ferns in East Kalimantan, Indonesia
HENNY NURHASNAWATI1,♥, REKSI SUNDU1, SAPRI1, RISA SUPRININGRUM1, HARLINDA KUSPRADINI2,
ENOS TANGKE ARUNG2,♥♥
1Akademi Farmasi Samarinda. Jl. A. Wahab Syahrani 226, Samarinda 75124, East Kalimantan, Indonesia.
Tel./fax. +62-541-7777363, ♥email: henny.nurhasnawati@gmail.com
2Department of Forest Product Technology, Faculty of Forestry, Universitas Mulawarman. Jl. Ki Hajar Dewantara, Kampus Gunung Kelua, Samarinda
75123, East Kalimantan, Indonesia. Tel./fax.: +62-541-735379, ♥♥email: tangkearung@yahoo.com
Manuscript received: 7 December 2018. Revision accepted: 30 January 2019.
Abstract. Nurhasnawati H, Sundu R, Sapri, Supriningrum R, Kuspradini H, Arung ET. 2019. Antioxidant activity, total phenolic and
flavonoid content of several indigenous species of ferns in East Kalimantan, Indonesia. Biodiversitas 20: 576-580. This study aimed to
determine the total phenolic and flavonoids content and antioxidant activity of ethanol extract of several indigenous species of ferns in
East Kalimantan. Total phenolic content was determined by Folin-Ciocalteau method and flavonoid content was measured by
colorimetric method. Antioxidant activity was evaluated by DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The result of photochemical
screening indicated that the leaves of Plagiogyria pycnophylla, Plagiogyria glauca, and Stenochlaena palustris contained alkaloid,
flavonoid, tannin, saponin, and steroid while Acrostichum aureum contained flavonoid, tannin, saponin and steroid. The result showed
extract Acrostichum aureum had the highest total phenolic content (366.4573 ± 2.2117 mg GAE.g-1), flavonoid content (228.6087 ±
2.2548 mg QE.g-1), and very strong antioxidant activity with IC50 value 29.5303 ppm. There is positive correlation between total
phenolic content, flavonoid with antioxidant activity.
Keywords: ferns, antioxidant, DPPH, total phenolic, flavonoid
INTRODUCTION
Ferns are vascular plants that are widespread
throughout Indonesia. There are approximately 12,000
species of ferns around the world and 1,300 of them are
found in Indonesia (Wang 2017; Imaniar 2017). Local
people use ferns for food, as ornamental plants and
traditional medicine (Ridianingsih et al. 2017). Plagiogyria
pycnophylla (Kunze) Mett. and Plagiogyria glauca
(Blume) Mett. are species of ferns that can be found in
West Kutai District in East Kalimantan. Empirically, the
Dayak Benuaq ethnic group uses the leaves and rhizomes
of those plants as anti-cancer, especially breast cancer and
antitoxin. The rhizome of Acrostichum aureum L. is used
to treat snake bites, wounds and pustules while the leaves
are used to stop bleeding (Khan et al. 2013). Empirically,
the Dayak people use the leaves of Stenochlaena palustris
(Burm. F.) Bedd. for face powder.
A number of diseases are caused by excessive oxidation
in the body which increases the amount of free radicals.
Free radical is a substance or molecule having one or more
unpaired electrons in its outer orbit (Phaniendra 2015).
Free radicals are continuously produced endogenously by
human body through normal metabolic processes,
inflammation and malnutrition. Apart from those, free
radical can also be derived exogenously through pollution,
ultraviolet radiation and cigarette smoke.
Antioxidant is a substance that provides protection from
oxidation reaction (Cross et al. 1994). The use of synthetic
antioxidants as the source of exogenous antioxidants might
have side effects, so natural antioxidants are a necessary
alternative. The choice and availability of natural antioxidant
are still limited so that research on natural antioxidant becomes
a trend among researchers (Sayuti and Yenrina 2015).
In this study, we determine the total phenolic, flavonoid
content and the evaluation of antioxidant activity of several
indigenous species of ferns in East Kalimantan as
preliminary study considering the potential of ferns as
source of medicine and cosmetics.
MATERIALS AND METHODS
Collection and ferns identification
Several species of ferns that have been used empirically
for medicines and cosmetics were collected from West
Kutai District and Samarinda City, East Kalimantan,
Indonesia during October-December 2017. Identification of
ferns was carried out at the Plant Anatomy and Systematics
Laboratory of the Faculty of Mathematics and Natural
Sciences in Mulawarman University, Samarinda,
Indonesia. Species of ferns used in this study were:
Plagiogyria pycnophylla (Kunze) Mett.; Plagiogyria
glauca (Blume) Mett.; Acrostichum aureum L. and
Stenochlaena palustris (Burm. F.) Bedd.
Chemical reagents
The reagents used were distilled water, acetic
anhydride, gallic acid, aluminum chloride, hydrochloric
NURHASNAWATI et al. – Phytochemical characteristics of several indigenous ferns
577
acid, sulfuric acid, amyl alcohol, iron (III) chloride,
dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picrylhydrazyl
(DPPH), 70% ethanol, ethyl acetate, Folin-Ciocalteau,
chloroform, quercetin, sodium hydroxide, sodium nitrite, n-
hexane, Bouchard reagent, Dragendorf reagent, Mayer
reagent, magnesium powder.
Extraction and phytochemical screening
Ferns were sorted, washed, drained, air-dried in the
open air and protected from direct sunlight exposure due to
in the humid condition, fungi will easily grow. Dried
samples were ground using a blender and sieved with a 60
mesh sieve. Weighed as much as 250 g of simplicia soaked
in ethanol solvent by maceration extraction method.
Phytochemical screening was conducted using procedures
described by Marjoni (2016) and Harborne (1987) to
determine the presence of alkaloid, flavonoid, tannin,
saponin, and steroid; that are described below.
Alkaloid. The sample was weighed as much as 0.5
grams, 1 ml of 2 N hydrochloric acid and 9 ml of distilled
water were added, heated over water bath for 2 minutes,
cooled and then filtered. The filtrate was used for the
following test: 3 drops of filtrate were taken to add 2 drops
of Mayer reagent (to produce white/yellow precipitate),
Bouchardat reagent (dark brown precipitate), Dragendorf
reagent (red brick precipitate). If two of the three tests
above give a positive result, then the extract contains an
alkaloid.
Flavonoids. A total of 0.5 grams of sample was
extracted using 10 mL of distilled water, filtered, taken 5
ml then added 0.1 g of Mg powder, 1 mL of concentrated
HCl and 2 mL of amyl alcohol, shaken and allowed to
separate. Flavonoids are positive if there is red, yellow,
orange in the amyl alcohol layer.
Tanin. A total of 0.5 grams of sample was extracted
using 10 mL of distilled water. The extraction results are
filtered then the filtrate obtained is diluted with distilled
water until it is colorless. Taken as much as 2 mL, then
added with 1-2 drops of FeCl3 1%. Blue or blackish green
occurs indicating the presence of tannins.
Saponin. A total of 0.5 grams of sample is put into a
test tube and added 10 mL of hot distilled water, cooled
then shaken vigorously for 10 seconds, foam or foam is
formed which for no less than 10 minutes is 1-10 cm high.
1 HCl 2 N was added, if the froth did not disappear
indicating saponins.
Steroids/terpenoids. A sample of 1 gram is macerated
with 20 mL n-hexane for 2 hours, then filtered. The filtrate
is evaporated over water. At the residue, 2 drops of
anhydrous acetic acid were added and 1 drop of
concentrated sulfuric acid. A purple or red color indicates a
terpenoid, then turns blue-green indicating steroids.
Determination of total phenolic
Total phenolic content in each extract was evaluated
using Folin-Ciocalteau (FC) method, according to the
method by Bajalan et al. (2017) and Adesegun et al. (2007)
research with slight modifications. This method based on
the inhibition power of phenolic hydroxyl group. Phenolic
compounds react with Folin-Ciocalteau reagent. The
principle of Folin-Ciocalteau method was the formation of
blue-color complex compound (Alfian and Susanti 2012).
Making a calibration curve
Gallic acid solution with a concentration of 200 mg/L
(5 mg gallic acid was made in 25 ml of distilled water) was
then made into a series of solutions with a concentration of
12.5; 25; 50; 100 and 200 mg/L (or ppm) of 10 ml each.
Then 0.1 ml of each of the standard gallic acid series
solution was taken and added 2 ml of Na2CO3, left for 5
minutes. Next, 1 ml of Folin Ciocalteau solution was
diluted with distilled water (1:10) and incubated for 30
minutes. The absorbance is measured at a wavelength of
780 nm against reagent blank for each series of standard
solutions so that a curve with a linear regression equation is
obtained (y = bx + a).
Measurement of absorbance of the sample
A sample solution of 1000 mg/L was made (10 mg
extract in 1 ml DMSO, the volume was sufficient to 10 ml
with distilled water), then 0.1 ml was taken and added
Na2CO3, left for 5 minutes. Then added 1 ml of Folin
Ciocalteau solution and incubate for 30 minutes. The
absorbance is measured at a wavelength of 780 nm against
reagent blank.
Calculation of total phenolic levels
The sample absorbance value is entered as the y value
in the linear regression equation, so the value of x can be
determined. Total phenolic levels are calculated using the
following formula: TPC = (C . V)/W ; where TPC = total
phenolic content (mg.g-1 extract); C = concentration of
sample established from the calibration curve (mg/L); V=
volume of sample solution (L); W = weight of ethanolic
plant extract (g). Total phenolic content was expressed as
mg gallic acid equivalent per gram extract (mg GAE.g-1).
Determination of flavonoid content
The determination of flavonoid content in extract was
done using colorimetric method, according to method by
Chandra et al. (2014) and Bajalan et al. (2017).
Making a calibration curve
Quercetin solution with a concentration of 200 mg/L (5
mg in 1 ml DMSO, volume up to 25 ml with ethanol) was
then made into a series of solutions with a concentration of
12.5; 25; 50; 100 and 200 mg/L (or ppm) of 10 ml each.
Then 0.5 ml were taken from the standard quercetin series
solution, added 0.15 ml 15% NaNO2, left for 6 minutes.
Then added 0.15 ml of AlCl3 10% and incubated for 60
minutes. Then added 2 ml of 4% NaOH, 2 ml of distilled
water and left for 15 minutes. The absorbance is measured
at a wavelength of 420 nm against reagent blank for each
standard solution series to obtain a curve with a linear
regression equation is obtained (y = bx + a).
Measurement of absorbance of the sample
A sample solution of 1000 mg/L was made (10 mg of
extract in 1 ml of DMSO, sufficient volume of 10 ml with
aquades), 0.5 ml was taken added 0.15 ml of 15% NaNO2,
B I O D I V E R S I T A S
20 (2): 576-580, February 2019
578
left for 6 minutes. Then added 0.15 ml of AlCl3 10% and
incubated for 60 minutes. Then added 2 ml of 4% NaOH, 2
ml of distilled water and left for 15 minutes. The
absorbance is measured at a wavelength of 420 nm against
reagent blank.
Calculation of flavonoid levels
The sample absorbance value is entered as the y value
in the straight line equation, so the value of x can be
determined. Total phenolic levels are calculated using the
formula: FC = (C . V)/W ; where FC = flavonoid content
(mg/g extract); C = concentration of sample established
from the calibration curve (mg/L); V= volume of sample
solution (L); M = weight of ethanolic plant extract (g).
Total flavonoid content was expressed as mg quercetin
equivalent per gram extract (mg QE.g-1).
Antioxidant assay
Antioxidant activity assay refers to Arung et al. (2006)
with slight modifications. Antioxidant activity was
determined by decolorization of DPPH solution and carried
out using a UV/Vis spectrophotometer at room
temperature. The range of concentration of the samples
were 12,5; 25; 50; 100 and 200 mg/L (or ppm). One ml of
the sample was added with 2 ml of DPPH 60 mg/L, and
incubated at room temperature in the dark for 30 minutes.
Absorbance was measured at 523 nm. Quercetin is used as
a positive control with concentrations of 2, 4, 6, 8 and 10
mg/L. The percentage of inhibition was calculated using
the following equation: % DPPH inhibition = [(Ab –
As)/Ab]. 100 % ; where Ab = absorbance of blank sample,
As = absorbance of sample.
The absorbance of each sample was measured and
calculated to determine the value of inhibition percentage
(reduction). Linear regression equation obtained from the
scatter plot of extract concentration and inhibition
percentage was used to calculate IC50 value. The IC50 value
is the concentration of extract required to inhibit 50% of
DPPH free radical. According to Miryanti et al. (2011)
states that antioxidant activity is considered extremely high
if the value of IC50 is less than 50 ppm, high if the value of
IC50 is between 50 – 100 ppm, moderate if the value is
between 100 – 150 ppm and low if the value is between
151 – 200 ppm.
Data analysis
The qualitative and quantitative data obtained were
analyzed descriptively. Antioxidant activity was grouped
according to IC50 criteria, while total phenolic and
flavonoids were determined as mean ± SD. All
measurements were done in triplicate.
RESULTS AND DISCUSSION
Phytochemical screening
Phytochemical screening was carried out to determine
the secondary metabolites in the sample. The result of the
phytochemical screening assay can be seen in Table 1.
Phytochemical screening was carried out due to its
simplicity, speed, minimum use of equipment and
selectivity (Nohong 2009). The result of phytochemical
screening shown in Table 1 indicated that P. pycnophylla,
P. glauca, and S. palustris contain alkaloid, flavonoid,
tannin, saponin, and steroid group, but alkaloid was absent
in A. Aureum. The principle of the alkaloid test is the
formation of sediment due to ligand replacement. Nitrogen
atoms which are alkaloid lone pairs replace iodine ions in
Dragendorf and Mayer reagents (Sangi et al. 2008).
All samples containing flavonoids were characterized
by the formation of yellow/orange layers in the amyl
alcohol layer. Flavonoids have the potential as antioxidants
which can prevent the formation of free radicals.
Saponin contains glycosyltransferase acting as polar
groups, while steroid and triterpene to function as nonpolar
groups. All samples contain saponin. According to Saxena
et al. (2013), many saponins are known to be antimicrobial,
to inhibit mold, and to protect plants from insect attack.
Based on the results in Table 1, all samples contain
condensed tannins. The addition of FeCl3 causes the
hydrolyzed tannin to turn into blue while the condensed
tannin turns into green. Decolorization occurs because
FeCl3 reacts to one of the hydroxyl groups in the tannin
(Sangi et al. 2008).
Steroid analysis was based on the ability of the
compound to change color with concentrated H2SO4 in
acetic anhydride (Sangi et al. 2008). The results show the
formation of a greenish blue ring which means the extract
contains steroids.
As far as we know, there is no report about
phytochemical screening of P. glauca and P. pycnophylla.
Study of Khan et al. (2013) showed that ethanol extract A.
aureum contained alkaloids, glycosides, tannins,
flavonoids, and terpenoids. The results of the study differ
from Khan (2013) alleged because according to Salim et al.
(2016) differences in the place of growth of a species affect
the precursors of biosynthesis of secondary metabolites.
While methanol extract contained proteins and amino
acids, glycosides, steroids, triterpenes, saponins, and
flavonoids (Raja and Ravindranadh 2014). Study of Chai
(2012) showed that S. palustris contains polyphenols,
flavonoids, cinnamic acid, and anthocyanins.
Table 1. Phytochemical detected in fern extracts collected from East Kalimantan, Indonesia
Species
Local name
Part
Alkaloid
Flavonoid
Tanin
Saponin
Steroid/Terpenoid
Plagiogyria pycnophylla
Paku atai merah
Leaf
+
+
+
+
+
Plagiogyria pycnophylla
Paku atai merah
Rhizome
+
+
+
+
+
Plagiogyria glauca
Paku atai putih
Leaf
+
+
+
+
+
Acrostichum aureum
Paku laut
Leaf
-
+
+
+
+
Stenochlaena palustris
Kelakai
Leaf
+
+
+
+
+
Note : (+) = presence; (-) = absence
NURHASNAWATI et al. – Phytochemical characteristics of several indigenous ferns
579
Table 3. Total phenolic and flavonoid content in ferns ethanolic extract
Sample
Part
Total Phenolic Content
(mg GAE.g-1 extract)
Flavonoid Content
(mg QE.g-1 extract)
P. pycnophylla
Leaf
264.0565 ± 4.0365
114.8439 ± 1.6012
P. pycnophylla
Rhizome
286.9202 ± 9.4492
127.1995 ± 0.4558
P. glauca
Leaf
83.8114 ± 8.7162
81.1347 ± 0.8025
A. aureum
Leaf
366.4573 ± 2.2117
228.6087 ± 2.2548
S. palustris
Leaf
245.3011 ± 3.2382
166.1779 ± 4.1420
Note: The data are averaged ± SD, n=3
Table 2. IC50 value and antioxidant activity of fern extracts
collected from East Kalimantan
Sample
Part
IC50 (ppm)
Category
P. pycnophylla
Leaf
124.6806
Moderate
P. pycnophylla
Rhizome
106.5234
Moderate
P. glauca
Leaf
995.0497
No activity
A. aureum
Leaf
29.5303
Very strong
S. palustris
Leaf
140.7528
Moderate
Total phenolic
Phenolics, which are secondary metabolites of plants,
exhibit remarkable bioactivities. A large body of
epidemiological studies has proven the bioactivities of
phenolics in both standard compounds and natural extracts:
namely, anticancer, anti-inflammatory, and antibacterial
activities as well as reducing diabetes, cardiovascular
disease, and neurodegenerative disease. Phenolics also
display anti-analgesic, anti-allergic, and anti-Alzheimer’s
properties (Shahidi and Yeo 2018). The results of total
phenolic and flavonoid content were presented in Table 3.
Total phenolic has been known to have biological
activity as free radical scavengers and antioxidants. Its
antioxidant activity is mainly caused by redox properties
which act as reducing agents, hydrogen donors and singlet
oxygen quenchers (Chandra et al. 2014). Ethanol extract of
A. aureum leaves showed the highest phenolic content
(366.4573 ± 2.2117 mg GAE.g-1) followed by P.
pycnophylla rhizome (286.9202 ± 9.4492 mg GAE.g-1), P.
pycnophylla leaves (264.0565 ± 4,0365 mg GAE.g-1), S.
palustris (245.3011 ± 3.2382 mg GAE.g-1) and P. glauca
(83.8114 ± 8.7162 mg GAE.g-1). The results of this study
showed that total phenolic content was positively
correlated with antioxidant activity. Based on Tables 2 and
3 it can be concluded that the higher the total content of
phenol, the antioxidant activity is also getting stronger.
Kumar et al. (2014) reported that phenolic compounds as
the main contributors to antioxidant activity.
Flavonoid
Flavonoids are one of the largest natural phenolic
compounds and are found in all plants, therefore it is
certain that flavonoids are found in every plant extract
(Markham 1988). The results in Table 3 showed that A.
aureum had the highest flavonoid content (228.6087 ±
2.2548 mg QE.g-1), followed by S. palustris (166.1779 ±
4.1420 mg QE.g-1), P. pycnophylla rhizome (127.1995 ±
0.4558 mg QE.g-1), P. pycnophylla leaves (114.8439 ±
1.6012 mg QE.g-1), and P. glauca (81.1347 ± 0.8025 mg
QE.g-1). Generally, phenolics and flavonoids constitute a
major group of compounds, which act as main antioxidant
(Adesegun et al. 2007). Differences in flavonoid structure
and group substitution affect the stability of phenoxyl
radicals, thus affecting the antioxidant properties of
flavonoids (Wojdyło et al. 2007).
Yao et al. (2010) showed a correlation between
antioxidant activity and total flavonoid content and total
phenolics in celery. This is in accordance with the results
of this study on ferns where A. aureum extract contains the
highest total phenolic, and flavonoids and antioxidant
activity.
Antioxidant activity
Antioxidant activity of ethanol extract of several
species of ferns was carried out using the DPPH method,
and measured using a UV-Vis spectrophotometer. DPPH
free radical scavenger method was chosen due to its
simplicity, ease, speed, sensitivity and the little amount of
samples needed (Marzuki et al. 2012). DPPH, is a stable
free radical which possesses purple color will turn to
yellow compound when it reacts with antioxidant
compounds. In this reaction, antioxidants release electrons
to DPPH.
The results in Table 2 show the IC50 value of A. aureum
extract is 29.5303 ppm which is categorized as a very
strong antioxidant. According to Miryanti et al. (2011) IC50
values < 50 ppm for antioxidant was very strong. The IC50
value of quercetin as a positive control is 5.9866 ppm
which was also categorized as very strong antioxidant
activity. Based on the IC50 value, the extracts of P.
pycnophylla and S. palustris are categorized as moderate
antioxidant activity, with IC50 values ranging from
106.5234 - 140.7528, while P. glauca extract does not have
antioxidant activity based on its IC50 value (995.0497 ppm)
Antioxidant activity is influenced by total phenol levels
and flavonoids. Phenol and flavonoid compounds have a
linear contribution to antioxidant activity, so the higher the
level the better the antioxidants (Ghasemzadeh and
Ghasemzadeh 2011). High levels of total phenolic in the
ethanol extract of A. aureum are thought to have an
important role as antioxidants. Besides phenol and
flavonoids, other phenolic components such as tannins,
alkaloids and terpenoids (Saxena et al. 2013) also
contribute as antioxidants.
Based on research, ethanol extract of A. aureum has the
highest levels of total phenolic, flavonoids with very strong
B I O D I V E R S I T A S
20 (2): 576-580, February 2019
580
antioxidant activity. There is a positive correlation between
levels of total phenolic, flavonoids with antioxidant
activity. Further research needs to be done to isolate active
compounds of A. aureum as antioxidants.
ACKNOWLEDGEMENTS
The authors were grateful to Institute for Research and
Community Services of Akademi Farmasi Samarinda and
the Directorate General of Strengthening Research and
Development, Ministry of Research, Technology, and
Higher Education of Republic of Indonesia for the
cooperation provided. This research was funded through
the Collaborative Research Inter-University Scheme with
Contract No.: 560/KONTRAK-PENELITIAN/ K11/KM/2018.
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