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P-ISSN 2586-9000
E-ISSN 2586-9027
Homepage : https://tci-thaijo.org/index.php/SciTechAsia
Science & Technology Asia
Vol.28 No.2 April - June 2023
Page: [183-195]
Original research article
*Corresponding author: nokluksamee@hotmail.com
In vitro Screening of Phytochemicals,
Antioxidant and Anticancer Activities of
Derris indica Extracts
Luksamee Vittaya1,*, Juntra Ui-eng1, Nararak Leesakul2
1Faculty of Science and Fisheries Technology, Rajamangala University of Technology Srivijaya,
Trang 92150, Thailand
2Division of Physical Science and Center of Excellence for Innovation in Chemistry, Faculty of Science,
Prince of Songkla University, Songkhla 90000, Thailand
Received 11 September 2021; Received in revised form 30 September 2022
Accepted 1 December 2022; Available online 14 June 2023
ABSTRACT
Derris indica ( Leguminosae) has been used in folk medicine to treat ulcers, bronchitis,
coughing, whooping cough, and diabetes and has also been used pharmacologically for its anti-
inflammatory, antimicrobial, antidiabetic, and anticancer activities. Fifteen extracts from leaf,
flower, fruit, twig, and bark of D. indica were investigated by standard phytochemical screening
tests, and the activity was confirmed by quantitative analysis based on the determination of total
flavonoid contents. Free radical scavenging activities were evaluated in vitro by two methods:
DPPH and ABTS, and the anticancer activity was measured by cell growth inhibition
determined using a resazurin microplate assay (REMA). Phytochemical screening revealed the
phenolics, flavonoids, terpenoids, alkaloids, saponins, and anthraquinones, and quantitative
analysis showed that hexane and ethyl acetate extracts showed significantly higher TFC than
methanol extracts. A positive correlation was established by the Pearson correlation test
between DPPH and ABTS measurements (r = 0.831). Interestingly, the free radical scavenging
activity depended on the structure-function relationship of flavonoids, with a negative
correlation (r = 0.758, 0.846, 0.972, 0.601) for DPPH and (r = 0.946, 0.480, 0.965, 0.686) for
ABTS which was observed in leaf, flower, fruit, and twig methanolic extracts, respectively.
Anticancer activity was tested against the MCF7 breast cancer cell line and the NCI-H187 small
cell lung cancer line. Hexane extract of the fruit, and ethyl acetate extract of the twig and bark
were active against NCI-H187 cancer cells.
Keywords: ABTS; Anticancer; Antioxidant; Derris indica; DPPH; Phytochemical
1. Introduction
Cancer kills more people worldwide
than any other illness and the number of
deaths from cancer increases every year. The
World Health Organization reported that in
2018 there were 18.1 million new cases of
doi: 10.14456/scitechasia.2023.37
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
184
cancer and 9.6 million deaths from cancer.
The five most common cancers in the world
are lung cancer, breast cancer, colon cancer,
prostate cancer, and stomach cancer. The risk
factors of cancer may be environmental,
dietary, or genetic. In Thailand, cancer has
been the number one cause of death for over
20 years. According to statistics, liver cancer
is the most serious type of cancer, lung and
breast cancer are the second and third most
serious, respectively. Even though it is a
terrible disease, it is possible to prevent and
reduce the risk of cancer. At present, there is
growing interest in the preventive and
therapeutic use of natural products.
Phytochemicals from leaf, flower, fruit, seed,
stem, and bark of plants [1] contain active
components that may help to reduce chronic
disease risks, including the risk of cancer [2-
4]. Active components may be compounds
such as terpenoids, glycosides, alkaloids,
tannins, and flavonoids, which scavenge free
radicals that are important mediators of
several diseases. These compounds have
been reported to possess antioxidant [5],
antibacterial [6, 7], and anticancer activities
[8].
Derris indica (Lam.) Bennet
[synonyms: Derris pinnata (Lour),
Pongamia pinnata (L.) Pierre, Pongamia
pinnata (L.) Merr., Pongamia glabra Vent.,
and Cytisus pinnaus (L.)] is a medicinal plant
belonging to the Leguminosae family. Its
phytochemicals are produced under the
extreme environmental conditions of strong
winds, high temperatures, and strong salinity
prevalent in mangrove areas. Various parts of
D.indica have been used in folk medicine as
antimicrobials, antiseptics, as liniment for
rheumatism and diabetes, or even to cure
tumors, skin ailments, bronchitis, and
whooping cough [9]. In traditional Thai
medicine, the leaf, stem, seed, root, bark, and
fruit are often used for their anti-
inflammatory, anti-plasmodial, antioxidant,
and anti-diarrheal activities [9, 10]. An
ethanolic extract of the flower has been used
for its anti-hyperglycemic and anti-lipid
peroxidation effects and to enhance the
antioxidant defense system in alloxan-
induced diabetic rats [11]. An ethanolic bark
extract exhibited anti-inflammatory activity
in a rat model [12] and the isolated pure
compounds showed antioxidant activity [13].
In addition, compounds from a hexane
extract of the fruit of this plant showed
cytotoxicity against cholangiocarcinoma and
HepG2 cell lines [14]. Previous studies have
established that plants containing these and
similar bioactive compounds have been used
in traditional medicine to treat cancer.
However, the growth of plants is
affected by location, climate, rainfall, and
altitude. Variations in these conditions
produce major variations in bioactive ingre-
dients, even in plants grown in the same
country [15]. Therefore, qualitative phyto-
chemical screening must be the first step in
identifying interesting bioactive compounds
in medicinal plants for extraction, isolation,
purification, and further investigation of
pharmacological activities. Some data have
been obtained from the phytochemical
screening of various parts of D. indica. An
aqueous bark extract showed the presence of
alkaloids, terpenoids, and saponins [16]. The
leaf, root bark, and root heart-wood extracted
with various solvents (petrol, dichlorome-
thane, ethyl acetate, butanol, and methanol)
contains alkaloids, steroids, triterpenoids,
flavonoids, saponins, and tannins, and
exhibits antimicrobial activity, especially as
a methanolic extract of leaf and root heart-
wood. Petrol, butanol, and methanol extracts
of the root bark showed good antibacterial
activity [17]. The results of these studies
suggest that the solvent systems selected for
extraction play a significant role in the
recovery of desired biomolecules.
The chemical investigation of various
parts of this plant has identified several
bioactive compounds (flavonoids, flavone,
chalcone derivatives, and furanoflavonoid
glycosides) that exhibit different biological
activities. For instance, candidone, a flavone
derivative isolated from a hexane extract of
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
185
D. indica fruit, had an antitumor effect on a
model cell line and could be a treatment for
cholangiocarcinoma [8]. The compounds
derrivanone and derrischalcone showed
strong activity against human hepatoma
HepG2 cells [14]. Karanjin, pongamol,
pongagalabrone, pongapin, pinnatin,
kanjone, glabrachalcone, and isopongachro-
mene were isolated from the seeds of D.
indica [9]. The leaf and stem of the plant
yielded several flavone and chalcone
derivatives, such as galbone, pongone,
pongalabol and pongagallone A and B [18]
and flavonoids were isolated from the root
bark [19]. D. indica fruit yielded the new
compounds, furanoflavonoid glycoside, and
pongamoside A-C, as well as the new
flavonols glycoside, and pongamoside D
[20]. Scientists have continued to study the
pharmacological activity of extracts of the
leaf, fruit, and flower of this plant but very
little information exists about the activity of
the twig and bark.
Therefore, the main objective of this
work was to study the different
phytochemical constituents of various parts
of D. indica such as leaf, flower, fruit, twig,
and bark, to investigate the correlation
between total flavonoid content and free
radical scavenging activity by DPPH and
ABTS methods, and to study the anticancer
activities of these extracts.
2. Materials and Methods
2.1 Collection and preparation of extracts
Fresh leaf, flower, fruit, twig, and bark
samples of D. indica were collected in April
2017 from Rajamangala Mangrove Forest,
Rajamangala University of Technology
Srivijaya, Trang Province and identified by
Assistant Professor Sittichoke Junyong. The
specimen voucher CMUB. 39893 was
deposited in the Department of Biology,
Faculty of Science, Chiang Mai University.
The dried powder of each part was extracted
for a week with sequentially polar organic
solvents hexane, ethyl acetate, and methanol
in the ratio of 1:5 (w/v). The resulting
extracts were evaporated to dry residue using
a rotary evaporator at 45oC and refrigerated
until further use. The yields of leaf, flower,
fruit, twig, and bark extracts were recorded
and the dried extracts were screened for
phytochemicals and biological activities.
Fig. 1. Derris indica (A) flower, (B) leaf, (C) fruit
and (D) twig and bark.
2.2 Phytochemical screening test
The analysis of the phytochemical
constituents of the various extracts of D.
indica followed established standard
techniques [5]. The detection of phenolics,
flavonoids, terpenoids, alkaloids, saponins,
and anthraquinone was carried out as
previously described [21]. In the qualitative
analysis, absence was expressed as a
negative (-) and presence as a positive (+),
and the intensity of characteristic color or
solidity was expressed as (+), (++), or (+++).
2.3 Determination of total flavonoid
contents
The flavonoid contents (mg/mL) of D.
indica extracts were determined as
previously described [22]. For each extract, a
reaction mixture was composed consisting of
0.1 mL of the extract at a concentration of 1
mg/mL and 0.5 mL of 5% NaNO2. The
mixture was left to stand for 6 minutes at
room temperature before the addition of 0.2
ml of 10% AlCl3. The mixture was then
vortexed for 5 min, after which 0.5 mL of 1
M NaOH was added and the volume was
A
B
C
D
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
186
made up to 1.5 mL with distilled water. The
solution was mixed well again and
absorbance was measured against a blank at
510 nm using a UV-Visible spectrophoto-
meter. The total flavonoid content of each
sample was calibrated alongside the standard
curve of rutin at concentrations of 50-500
µg/mL and is expressed in terms of rutin
equivalents per gram of dried crude extract
(mg RU/g CE).
2.4 Free radical scavenging activity
2.4.1 DPPH scavenging assay
The free radical scavenging effect on
DPPH radicals was determined by slightly
modifying the method of Vittaya et al. [5].
Briefly, 0.5 mL of 0.15 mM methanolic
DPPH solution was mixed with 0.5 mL of
each sample (1 mg/mL) and standard. The
reaction mixture was shaken vigorously and
allowed to stand at room temperature in
darkness for 30 min. The control was
prepared as above without the addition of the
extract sample. Ascorbic acid and butylated
hydroxytoluene (BHT) were used as positive
controls. The absorbance of the solution was
measured at 517 nm against the blank. The
scavenging ability of each plant extract was
calculated using the following equation:
DPPH Scavenging activity
where is the absorbance of the test
sample with DPPH solution, is
the absorbance of the test sample only, and
is the absorbance of DPPH
solution. All measurements were performed
in triplicate and are expressed as average
values.
2.4.2 ABTS scavenging assay
The 2,2/-azino-bis-3-ethylbenzothi
azoline-6-sulphonate radical cation (ABTS+)
decoloration assay was performed as
described by Vittaya et al. [21]. The ABTS+
working solution was prepared by mixing 5
mL of 7 mM ABTS with 880 µL of 140 mM
K2S2O8 (potassium persulfate). The mixture
was allowed to stand in darkness for 16 h at
room temperature and then diluted with
methanol to give an absorbance of 0.700 ±
0.25 units using the spectrophotometer at an
excitation wavelength of 734 nm. A sample
extract aliquot of 0.1 mL was added to 0.9
mL of diluted ABTS+ solution. The reaction
mixture was shaken and left to stand for 6
min in darkness. After incubation,
absorbance was measured at 734 nm. The
percentage of scavenging inhibition of
ABTS+ was determined in triplicate and was
calculated using the following equation:
where is the absorbance of the
extract without ABTS+ solution and
is the absorbance of the extract with ABTS+
solution.
2.5 Anticancer screening
The anticancer activity of each extract
was evaluated from cell growth inhibition
determined using a resazurin microplate
assay (REMA) in a 2-cell line panel
consisting of MCF7 breast cancer cells and
NCI-178 small cell lung cancer cells. Results
were compared with positive controls of
Ellipticin, Doxorubicin, and Tamoxifen. The
extracts were tested in a range of
concentrations (0.21-50.00 µg/mL) and the
positive controls were dissolved in DMSO.
The primary anticancer assay was performed
at the National Center for Genetic
Engineering and Biotechnology (BIOTEC)
of the National Science and Technology
Development Agency (NSTDA). Anticancer
activity was determined by % cytotoxicity: <
50% was considered non-cytotoxic and >
50% was considered cytotoxic (IC50
included) [3].
( )
(%) 1 100,
sample sample blank
control
Abs Abs
Abs
æö
-
ç÷
=- ´
ç÷
èø
sample
Abs
sample blank
Abs
control
Abs
( )
% inhibition 100,
control sample
control
Abs Abs
Abs
-
=´
control
Abs
sample
Abs
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
187
2.6 Statistical analysis
The results are expressed as mean ±
standard error of triplicate determination.
Statistical analysis was carried out using a
one-way analysis of variance (ANOVA)
followed by Duncan multiple comparison. A
value of p < 0.05 was considered significant.
Two-way ANOVA was used to determine
the interaction between plant parts and the
three organic solvents. Correlation between
the flavonoid contents and free radical
scavenging activity was carried out using
Person correlation coefficients.
3. Results and Discussion
3.1 Phytochemical screening
Various parts of D. indica were
analyzed to determine their phytochemical
composition, which showed the presence of
several secondary metabolites such as
anthraquinones, saponins, alkaloids,
phenolics, flavonoids, and terpenoids. The
results are summarized in Table 1. All
studied parts contained flavonoids and
phenolic compounds, which are among the
most important groups of plant metabolites
[23]. Phenolics were found in the fractions
extracted with ethyl acetate and methanol
(medium to high polarity solvents). In
contrast to phenolics, flavonoids were found
in the fractions extracted with hexane and
ethyl acetate. Both metabolites have been
reported to possess biological properties such
as anticandidal activity [24] and antioxidant
activity [25-29]. Terpenoids were found in
leaf, flower, and twig extracted with hexane,
ethyl acetate, and methanol but only in fruit
and bark extracted with hexane. The
therapeutic properties of terpenoids have
generally been reported in use against
bacteria, fungi, and cancers [24, 30, 31].
Alkaloids and saponins were detected only in
methanolic extracts and several works have
reported their anti-inflammatory [33],
antimicrobial [33], and antimalarial [34]
activities. Saponins were found to exercise
antimicrobial activity against a wide range of
microorganisms in vitro [35]. However,
anthraquinone was found only in leaf
samples extracted with ethyl acetate and
methanol.
Table 1. Qualitative phytochemical screening of extracts of D. indica.
Note: + means present and - means absent; H=hexane; E=ethyl acetate; M=methanol.
3.2 Determination of flavonoid contents
Total flavonoid contents (TFC) were
analyzed using the equation:
as rutin equivalents (mg rutin equivalents/g
crude extract), and are presented in Table 2.
The TFC was in the range 0.12 to 1.76 mg
RU/g CE as shown in Table 2. Two-way
ANOVA revealed that the main effects (plant
parts and solvents) have a significant effect
on the TFC (F = 63.745, p < 0.001 for plant
parts; F = 339.194, p < 0.001 for solvents).
Interaction between plant parts and solvents
also had significant effects on the TFC (F =
( )
2
0.0016 0.0033 0.9995 ,yx R=-=
Plant
constituent
Procedure
Observations
D. indica extracts
Leaf
Flower
Fruit
Twig
Bark
H
E
M
H
E
M
H
E
M
H
E
M
H
E
M
Phenolics
Extract+FeCl3
Dark green
precipitate
-
+
+
-
+
+
-
-
+
-
-
+
-
-
+
Flavonoids
Extract+Mg+HCl
Formation of a cherry
color
+
+
-
+
+
-
+
+
+
+
+
-
+
+
-
Terpenoids
Extract+chloroform+
conc.H2SO4
Reddish brown ring
+
+
+
+
+
+
+
-
-
+
+
+
+
-
-
Alkaloids
Extract+Dragendroff’s
reagent
Orange-yellow
precipitate
-
-
+
-
-
+
-
-
-
-
-
+
-
-
+
Saponins
Extract+H2O+shaking
Formation of a stable
form
-
-
+
-
-
-
-
-
+
-
-
+
-
-
+
Anthraquinones
Extract+dil. H2SO4+
chloroform+dil.NH3
Reddish color
-
+
+
-
-
-
-
-
-
-
-
-
-
-
-
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
188
45.214; p < 0.001). Based on a two-way
ANOVA, TFC was affected by plant parts
and solvents (p <0.001). Considering the
effects of solvents ( averaged across plant
parts), TFC was found highest in hexane
(1.30±0.31 mg RU/g CE) and ethyl acetate
(1.24±0.52 mg RU/g CE), as shown in Fig.
2. When the effect of plant parts was
analyzed, the TFC was found to be highest in
the leaf (1.31±0.60 mg Ru/g CE) followed by
bark (1.20±0.87 mg RU/g CE). Fruit
(1.00±0.27 mg Ru/g CE) and twig
(0.97±0.37 mg RU/g CE) were similar and
the lowest was found in flower (0.64±0.20
mg RU/g CE).
Fig. 2. Interaction effect of plant parts and
solvents on the quantity of TFC in Derris indica
extracts, where different lowercase letters
indicate significant (p < 0.05) differences among
means and error bars represent ± SD.
Table 2. Percentage of extract yield, concentration of total flavonoid contents and free radical
scavenging activity of D. indica extracts.
Note: Data shown as mean ± SD values from triplicate analysis. Different lower case letters (a-k) in each sample denote significant
differences (p < 0.05). H=hexane; E=ethyl acetate; M=methanol.
3.3 Free radical scavenging activity
Due to the complex nature of
phytochemical substances, the antioxidant
activity of a plant extract cannot be evaluated
by any single method. It is also important to
use generally accepted assays to determine
antioxidant activity. DPPH and ABTS were
selected to determine the free radical
scavenging activity. The DPPH method was
used to test the oxidation reaction of crude
Part
used
Sol
Extract yield
(%)
Total flavonoid
Content (mg RU/g CE)
Free radical scavenging percentage (%)
DPPH assay
ABTS assay
Leaf
H
1
.
11
1
.
76 ± 0
.
17 b
55
.
52 ± 1
.
45 d
22
.
44 ± 0
.
72 i
E
1.33
1.43 ± 0.02 c
92.56 ±0.17 a
48.31 ± 0.84 fg
M
10.56
0.70 ± 0.06 i
94.12 ± 0.89 a
67
.
82 ± 1
.
61 b
Flower
H
3
.
00
0
.
87 ± 0
.
14 gh
11
.
99 ± 7
.
31 g
9
.
70 ± 0
.
23 j
E
5.40
0.64 ± 0.03 i
95.11 ± 0.61 a
99
.
55 ± 0
.
78 a
M
54.60
0.43 ± 0.02 j
95.02 ± 1.19 a
53
.
81 ± 1
.
96 e
Fruit
H
2.70
1.31 ± 0.14 cd
65.95 ± 2.39 c
50.76 ± 3.12 f
E
7.40
0.98 ± 0.07 fg
82.38 ± 3.71 b
70
.
46 ± 2
.
50 b
M
23.20
0.72 ± 0.01 hi
94.25 ± 1.29 a
99.09 ± 0.69 a
Twig
H
0.62
1.21 ± 0.02 de
50.31 ± 3.16 e
32
.
34 ± 0
.
12 h
E
2
.
31
1
.
08 ± 0
.
22 ef
92
.
39 ± 1
.
72 a
63
.
92 ± 0
.
08 c
M
4.93
0.48 ± 0.02 j
91.33 ± 1.21 a
68.38 ± 1.26 b
Bark
H
1.10
1.37 ± 0.03 cd
28.23 ± 3.01 f
22
.
55 ± 2
.
22 i
E
5.75
2.10 ± 0.10 a
65.55 ± 1.94 c
47.69 ± 0.49 g
M
10.50
0.12 ± 0.01 k
70.05 ± 3.75 c
57
.
13 ± 3
.
87 d
BHT
94.12 ± 0.06 a
98
.
90 ± 0
.
57 a
Ascorbic
acid
96.22 ± 0.17 a
99.74 ± 0.18 a
Two-way ANOVA
Variable
df
F
P
F
P
F
P
Plant Parts (PP)
4
63
.
745
< 0.001
143.973
< 0.001
415.528
< 0
.
001
Solvents (S)
2
339.194
< 0.001
1258.003
< 0.001
2601.350
< 0.001
PP x S
8
45
.
214
< 0.001
66.459
< 0.001
277.032
< 0
.
001
Error
30
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
189
extracts with stable, purple DPPH at room
temperature. Taking all extracts of all parts
of D. indica into consideration, scavenging
of the DPPH free radical ranged from 11% to
95% (Table 2). The ABTS+ method was also
chosen as it is effective in determining
antioxidant activity. The decolorization of
the ABTS+ radical indicates the capacity of
an antioxidant species to inactivate radical
species by donating electrons or hydrogen
atoms. The observed reductions in
absorbance of the samples indicated
moderate scavenging activity that ranged
from 9% to 99% when all fifteen D. indica
extracts were taken into consideration. It was
found that the ethyl acetate and methanol
extracts from all studied parts of Derris
indica possessed higher free radical
scavenging activity than the hexane extracts
by both DPPH and ABTS assays, despite
their lower flavonoid contents. This is caused
by the presence of an active flavonoid
structure obtained through high polar solvent
extraction. Although the flavonoid contents
from methanol crude extract were lower than
other solvents, there might exist some
specific flavonoid derivatives which are
potentially active to free-radical scavengers,
that provided activity against the DPPH and
ABTS radicals. In this work, we did not
isolate each derivative of flavonoid.
However, based on a previous literature
review, it is possible that the presence of a
diphenylpropane (C6-C3-C6) skeleton,
substitution on the flavonoid skeleton,
gallate and galactouronate moieties [36]
could give rise to free radical scavenging
activity (the structure-function relationship).
Such compounds acting as antioxidants due
to their conjugated π-electron systems allow
donation of electrons or hydrogen atoms
from the hydroxyl moieties to free radicals
[37]. Two-way ANOVA revealed the main
effects (plant parts and solvents) showing
significant effect on antioxidant capacity.
The highly significant effect, plant parts ×
solvents, on DPPH (F = 66.459, p < 0.001),
and on ABTS (F = 277.032, p < 0.001) are
shown in Table 3.
Fig. 3 and Fig. 4 show that antioxidant
activity increases as the concentration of
sample extracts increases. Extracts of fruit
and twig, especially, showed potent
antioxidant activities. The antioxidant
activity of fruit and twig extracts was
influenced by secondary metabolites in the
TFC (Table 2). This detected compound
could be responsible for antioxidant activity.
Flavonoids are oxidized by free radicals of
DPPH and the ABTS ion, resulting in a more
stable and less reactive radical [33]. On the
other hand, hydroxyl groups of flavonoids
stabilize the reactive oxygen species by
reacting with the radicals. Both these
reactions produce inactive radicals. TFC was
higher in the hexane and ethyl acetate
extracts than in the methanolic extracts
which correlated with the results of free
radical scavenging with various
concentrations of each extract. This negative
correlation of flavonoids with antioxidant
capacity was observed in previous research
[38], that reported that flavonoids could be
related to other antioxidant compounds
contained in extracts. The results of the
DPPH and ABTS assays were also well in
agreement, with a correlation coefficient of
0. 831 at a statistically significant level (p <
0.01) (Table 3).
Table 3. Correlation coefficient between variables of total flavonoid and free radical scavenging
activity in organic extract of D. indica.
Variables
TFC
DPPH
ABTS
TFC
1
DPPH
-0.345*
1
ABTS
-0.455**
0
.
831
**
1
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
190
Note: TFC = total flavonoid content; DPPH = 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity; ABTS = 2,2/-azinobis-3
ethylbenzothiazoline-6-sulfonic acid; * Correlation is significant at the 0.05 level; ** Correlation is significant at the 0.01 level.
3.3 Correlation between TFC and the free
radical scavenging in all parts extracts
Since the strength of free radical
scavenging activity of extracts depended on
the presence of considerable quantities of
flavonoids (Table 1), we investigated the
correlation between TFC in D. indica
extracts and free radical scavenging activity
as measured by the DPPH and ABTS assays.
The results are presented in Table 4. In this
work, the correlation between TFC and the
DPPH/ABTS assay results was satisfactory
for all five parts of the plant. Good
correlations were found between TFC and
ABTS scavenging for leaf, fruit, and twig
extracts and between TFC and DPPH
scavenging for leaf, flower, and fruit
extracts. In the case of fruit extracts, TFC
correlated significantly with the results of the
DPPH and ABTS assays. Bark extracts
showed a poor correlation and no
significance. Table 4 shows the negative
correlation between each sample of D. indica
and free radical scavenging, indicating that
the activity did not depend on TFC but rather
on the structure-function relationship of
flavonoids. This result was used to explain
the results of all extracts except bark. In
addition, ethyl acetate and methanol are more
effective organic solvents for extracting
bioactive flavonoid compounds than hexane.
These correlations confirm that the
flavonoids are the main active component
contributing to the antioxidant activities in
extracts of leaf, flower, fruit, and twig but not
bark. It is possible that other secondary
metabolic compounds such as phenolics,
terpenoids, alkaloids, and saponins have an
effect on activity in the bark. These
compounds are certain to be phenolics and
alkaloids [39] which were only found in
methanolic extracts of D. indica. This result
supports the use of this plant in traditional
medicines. Its bioactive compounds can
scavenge the free radicals implicated in
several disease conditions.
Table 4. Correlation coefficient (r) between variables of total flavonoid followed by each of
parts and free radical activity analyzed by different methods.
Methods
TFC
Leaf
Flower
Fruit
Twig
Bark
DPPH
-0.758
(
p
=
0
.
018
)
-0.846
(
p
=
0
.
004
)
-0.972
(
p
=
0
.
000
)
-0.601
(
p
=
0
.
087
)
-0.246
(
p
=
0
.
523
)
ABTS
-0.946
(
p
=
0
.
000
)
-0.480
(p = 0.191)
-0.965
(p = 0.000)
-0.686
(
p
=
0
.
041
)
-0.399
(p = 0.288)
Note: TFC = total flavonoid content; DPPH = 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity; ABTS = 2,2/-azinobis-3
ethylbenzothiazoline-6-sulfonic acid; * Correlation is significant at the 0.05 level; ** Correlation is significant at the 0.01 level.
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
191
3.4 Anticancer activity
Cancer is one of the most serious
diseases to affect humans. Plants are
bioactive sources of drugs for the treatment
of cancer and can help the development of
novel anticancer agents. This research aimed
to find new potential anticancer agents from
a natural source. In this work, the anticancer
activity of D. indica extracts is shown in
Table 5. The hexane extract of fruit, and the
ethyl acetate extracts of twig and bark
showed anticancer activity against the NCI-
H187 small cell lung cancer cells. The IC50
values were 46.23±3.37 for the hexane
extract of fruit, 47.77±1.13 the ethyl acetate
extract of twig, and 46.87±0.57 µg/mL for
the ethyl acetate extract of bark. The
anticancer activity of these extracts could be
attributed to the presence of flavonoids and
terpenoids in the hexane and ethyl acetate
extracts. Previous studies have shown that
flavonoids exhibit cytotoxic activity against
human cholangiocarcinoma cells [8, 20, 40,
41]. Reports also mentioned that hexane
extract of D. indica fruit contained flavone
derivatives [8], derrivanone and derrischal-
cone compounds [13], furanoflavonoid
glycoside, pongamoside, and flavonol
glycoside [19]. Other extracts appeared to be
inactive against this cell line (IC50 > 50
µg/mL). None of the extracts inhibited the
growth of MCF7 breast cancer cells. These
results support the potential benefits of the
reported use of this plant for the treatment of
cancer in traditional medicine [8, 9, 19, 38].
Fig. 3. The DPPH free radical scavenging activity (%) of D. indica leaf, flower, fruit, twig, and bark
extracts at different concentrations, where H, E, and M are hexane, ethyl acetate, and methanol,
respectively, and error bars indicate ± SD from triplicate analysis.
Fig. 4. The ABTS free radical scavenging activity (%) of D. indica leaf, flower, fruit, twig, and bark
extracts at different concentrations, where H, E, and M are hexane, ethyl acetate, and methanol,
respectively, and error bars indicate ± SD from analysis of triplicate analysis.
L. Vittaya et al. | Science & Technology Asia | Vol.28 No.2 April – June 2023
192
Table 5. IC50 values of the D. indicia extracts against cancer cell lines.
Part used
Solvent
Cell lines IC50 (
µ
g/mL)
MCF7
NCI-H187
Leaf
H
> 50
> 50
*E
> 50
> 50
M
> 50
> 50
Flower
H
> 50
> 50
E
> 50
> 50
M
> 50
> 50
Fruit
H
> 50
46.23 ± 3.37
E
> 50
> 50
M
> 50
> 50
Twig
*H
> 50
> 50
*E
> 50
47.77 ± 1.13
M
> 50
> 50
Bark
H
> 50
> 50
E
> 50
46.87 ± 0.57
M
> 50
> 50
Ellipticine
-
2.02 ± 0.57
Doxorubicin
-
0.10 ± 0.03
Tamoxifen
7.92 ± 0.84
-
Note: The extracts with an IC50 value > 50 µg/ mL were considered inactive. Data shown as mean ± SD values from analysis of
triplicate analysis. H= hexane, E=ethyl acetate, M=methanol; * = partially soluble 100 % DMSO. H=hexane; E=ethyl acetate;
M=methanol.
4. Conclusion
The bioactive compounds in leaf,
flower, fruit, and twig of D. indica showed
good free radical scavenging activity
arising from the structure-function
relationship of the flavonoids, rather than
TFC. In addition, ethyl acetate and
methanol solvents were good organic
solvents for extracting bioactive
flavonoids. The activities of hexane and
ethyl acetate extracts of fruit and twig
against small cell lung cancer cells were
comparable with the ethyl acetate bark
extract. The results of this study showed
that extracts of D. indica could be
promising sources of antioxidants for
pharmaceutical applications. To fully
understand the structure-function
relationship of flavonoids, further study is
needed to purify, characterize, and identify
bioactive compounds from the plant
extracts.
Acknowledgements
The authors acknowledge the
Faculty of Science and Fisheries
Technology, Rajamangala University of
Technology Srivijaya, Trang Campus for
providing laboratory facilities and the
Department of Biology, Faculty of Science,
Chiang Mai University for the specimen
voucher of D. indica. We thank Mr.
Thomas Duncan Coyne for editing a draft
of this manuscript and the Division of
Physical Science and the Center of
Excellence for Innovation in Chemistry,
Faculty of Science, Prince of Songkla
University for supplying some materials
used for experiments.
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