Determination of Total Phenolic Content, Total Flavonoid Content and Antioxidant Activity of Various Organic Crude Extracts of Licuala Spinosa Leaves from Sabah, Malaysia

Abstract

In this study, the leaves of Licuala spinosa were used to determine the total phenolic and flavonoid content as well as antioxidant activity of different crude extracts. The samples were extracted successively with organic solvents such as hexane, chloroform and ethyl acetate respectively. The total phenolic content was determined by Folin-Ciocalteu's assay. Chloroform crude extract showed the highest total phenolic content (9.42± 0.06 mg GAE/g), followed by ethyl acetate crude extract (8.91± 0.06 mg GAE/g) and hexane crude extract (6.78±0.26 mg GAE/g). The total flavonoid content was determined by Aluminium chloride colometric assay and expressed as QE equivalent. Chloroform crude extract showed the highest total flavonoid content (8.96 ± 0.21 mg QE/g), followed by ethyl acetate crude extract (7.04 ± 0.02 mg QE/g) and hexane crude extract (3.05 ± 0.09 mg QE/g). The antioxidant activity of extracts were evaluated by 2,2-diphenyl-1-picyhydrazyl (DPPH) assay. In DPPH assay, IC50 values were used to determine the antioxidant potential of the sample. The lower the IC50 value, the higher the antioxidative property. Among all the extracts, chloroform extracts exhibited higher DPPH radical scavenging activity with IC50 value of 0.032 mg/mL. BHT used as the positive control showed IC50 value of 0.089 mg/mL

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*Corresponding author’s e-mail: shaheenchem@gmail.com
ASM Sc. J., 11, Special Issue 3, 2018 for SANREM, 53-58
Determination of Total Phenolic Content, Total
Flavonoid Content and Antioxidant Activity of Various
Organic Crude Extracts of Licuala Spinosa Leaves from
Sabah, Malaysia
Mohammad Shaheen Khan1,*, Samina Khan Yusufzai2, Mohd Rafatullah2, Mohd. Sani Sarjadi1 and
Mohd Razlan2
1Industrial Chemistry Programme, Faculty of Science and Natural Resources, Universiti Malaysia Sabah,
88400 Kota Kinabalu, Sabah, Malaysia
2School of Industrial Technology, Universiti Sains Malaysia, 11800 Minden, Pulau Penang
In this study, the leaves of Licuala spinosa were used to determine the total phenolic and
flavonoid content as well as antioxidant activity of different crude extracts. The samples were
extracted successively with organic solvents such as hexane, chloroform and ethyl acetate
respectively. The total phenolic content was determined by Folin-Ciocalteu’s assay. Chloroform
crude extract showed the highest total phenolic content (9.42± 0.06 mg GAE/g), followed by ethyl
acetate crude extract (8.91± 0.06 mg GAE/g) and hexane crude extract (6.78±0.26 mg GAE/g).
The total flavonoid content was determined by Aluminium chloride colometric assay and expressed
as QE equivalent. Chloroform crude extract showed the highest total flavonoid content (8.96 ± 0.21
mg QE/g), followed by ethyl acetate crude extract (7.04 ± 0.02 mg QE/g) and hexane crude extract
(3.05 ± 0.09 mg QE/g). The antioxidant activity of extracts were evaluated by 2,2-diphenyl-1-
picyhydrazyl (DPPH) assay. In DPPH assay, IC50 values were used to determine the antioxidant
potential of the sample. The lower the IC50 value, the higher the antioxidative property. Among all
the extracts, chloroform extracts exhibited higher DPPH radical scavenging activity with IC50 value
of 0.032 mg/mL. BHT used as the positive control showed IC50 value of 0.089 mg/mL
Keywords:
Licuala spinosa, Total phenolics, Total flavonoids, DPPH activity, BHT
I.
INTRODUCTION
Medicinal plant usage dates back practically to the
existence of human civilization. From ancient
records, the uses of medicinal plants by human have
been traced such as the cinchona bark in
Mesoamerica, opium poppy in Egypt and snakeroot
plant in India (Tiwari et al. 2011). According to
WHO (World Health Organization), 80% of people
worldwide were estimated rely on herbal medicines
for some aspect of their main health care needs
(Ekor, 2014). Moreover, around 21,000 plants
species have the possibility or potentials for being
used as medicinal plants (Joy et al. 2001). Due to
their pain-relieving and healing abilities, medicinal
plants have been prized and relied on in about 75%
of our medicines until today (Chevallier, 2012).
Despite the advances and advantages of modern
medicines,

ASM Science Journal, Volume 11, Special Issue 3, 2018 for SANREM
54
medicinal plants offer the benefits that
pharmaceutical drugs often lack, aiding to support
the body’s effort to regain good health. In Malaysia
around 2,000 plants species have therapeutic
characteristics and can be used in traditional
treatments (Rukayah et al. 2006). These medicinal
plants are considered to contain rich resources of
ingredients, which can be used in drug development
and synthesis (Hassan, 2012)). The medicinal value
of these plants lies in some chemical active
substances that produce a definite psychological
action on the human body and the most important
bioactive constituents of plants are alkaloids,
tannins, flavonoid and phenolic compounds
(Rajendra et al. 2011). The L. spinosa belongs to
family aracaceae (palmae) native to vast area of the
Asiatic south-east, which includes Cambodia, India
(Andaman and Nicobar Island), Indonesia (Java,
Borneo and Sumatra), Malaysia, Myanmar,
Philippines, Thailand and Vietnam. It grows mainly
along the coasts and the banks of the river. The
name of the species “spinosa” refers to the thorns
present on the margins of the petiole (Henderson,
2009). L. spinosa is a densely clumping palm of
medium height, with slender stems and heads of
circular, divided fan leaves. The upright stems to 4
m tall and 8-10 cm in diameter with remnant fibers.
The leaf is circular shaped with squared-off ends.
The fruit and flowers are inflorescences (1-2.5 m
long) are branched to two orders in long drooping
spikes.The colour of the fruit is bright red with 0.5
inch in diameter. The seed is small, round and it can
germinate in 6–8 weeks. L. spinosa grows well in
shade or filtered light in acidic or neutral condition
of pH soil (Riffle et al. 2003)). The present study
was designed to investigate the total phenolic and
flavonoid content of L. spinosa leaves by using some
of significant methods and evaluate the antioxidant
activity using DPPH scavenging assays.
Figure 1. L. spinosa plants and fruits in natural
habit
II.
MATERIALS AND METHOD
A.
Plant Material and Sample Collection
The plant L. spinosa was used in this experiment.
The fresh samples (leaves) were taken around
Universiti Malaysia Sabah (UMS). The samples were
washed with distilled water to remove dust and were
dried under shade at room temperature for one
week. About 1kg leaves were pulverized in a grinder
for 3 min and stored in dark bags to protect from
humidity and light, prior to analysis.
B. Preparation of Crude Extract
The dried leaves powder (200 g) was extracted
with methanol using soxhlet extraction method at
room temperature. The methanolic extract was
recovered by evaporating the solvent by vacuum
rotatory evaporator. This crude extract was further
diluted with water and subsequently extracted with
hexane, chloroform and ethyl acetate to get their
respective residual fractions. These fractions were
filtered using Whatmanns filter paper, and then
evaporated under reduced pressure using rotary

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55
evaporator in order to obtain the crude extracts. The
extraction processes was repeated in triplicate. After
solvent evaporation, all the crude extracts were
weighed and kept for further usage in caped vials at
4 °C.
C. Determination of total phenolic contents
The total phenolic content was determined using
spectroscopic method as described by Ainsworth et
al. (2007). The reaction mixture was prepared by
mixing 1 mL plant extracts (1mg/mL), 1 mL of 10%
Folin-Ciocalteu’s reagent dissolved in 13 mL of
deionized water followed by the addition of 5 mL of
7% Na2CO3 solution. The mixture was mixed
thoroughly and kept in the dark at room
temperature for 2 h. The blank solution was also
prepared. The absorbance was recorded using
spectrometer at 760 nm. All the analysis was
repeated three times and the mean value of
absorbance was obtained. Total phenolic content
was determined by extrapolating calibration line
which was construed by gallic acid solution. The
TPC was expressed as gallic acid equivalent (mg
GAE) per gram of the dried sample.
D. Determination of total flavonoid contents
The total flavonoids content of the L. spinosa was
determined by using aluminium chloride
calorimetric method based on the methodology
reported by Afify et al. (2012) with some
modifications. 0.5 mL of sample (1mg/mL) was
mixed with 1mL of 10% aluminium chloride, 1mL of
potassium acetate (1M) and 2.5 mL of distilled
water. Quercetin was used to make the calibration
curve. The absorbance of the mixtures was
measured at 415
nm by using UV-spectrophotometer. The total
flavonoid content was expressed in terms of
quercetin equivalent (mg QE/g of sample). All the
analyses were repeated three times and the mean
value of absorbance was obtained.
E. DPPH radical scavenging activity
The antioxidant activity of the extracts were
quantitatively assessed on the basis of free radical
scavenging activity of stable1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical according to the
reported method of Brand-Williams et al. with slight
modification. 1mL of plant extract solution of
various concentrations, ranging from 0.05-0.20
mg/mL was mixed with 1 mL of 0.5 mM DPPH
solution in methanol. Incubation of the resulting
solution was carried out for 30 min in dark room at
37 °C. BHT was used as positive control under the
same assay condition. The absorbance was
measured calorimetrically at 517 nm. The
experiments were carried out in triplicate. The
percentage inhibition was calculated using the
following formula.
DPPH Scavenging Activity (%) = [(Ao-As)/Ao)]
×100
Here, Ao is the absorbance of the control (no
sample, DPPH solution only) and As is the
absorbance in the presence of the sample.
III.
RESULTS AND DISCUSSION
In this study, the leaves of L. spinosa were extracted

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56
with hexane, chloroform and ethyl acetate. Initial
weight of the sample used was 300 g for leaves. The
highest percentage yield of solid residue was
obtained for hexane extract (Table 1).
Table 1. The percentage yield of extracts obtained
from solid residue of plant material.
Solvents
Percentage Yield (%)
Hexane
1.057
Chloroform
0.677
Ethylacetate
0.397
The total phenolic content was determined by
using Folin-Ciocalteu method (Annisworth et al.
2007). Gallic acid was used as standard calibration
and total phenolic content in mg gallic acid
equivalence (mg GAE/g). The total phenolic content
of the crude extracts was solvent-dependent. The
higher the polarity of solvent the more will be the
total phenolic and flavonoid content in the extract.
In this present study, the total phenolic content in
fractions of different polarities varied, ranging from
6.78 ± 0.26 to 9.42 ± 0.06 mg of GAE/100 g powder
weight. Chloroform extract exhibited the highest
total phenolic content (9.42 ± 0.06 mg GAE/g),
followed by ethyl acetate (8.912 ± 0.06 mg GAE/g)
and hexane (6.778 ± 0.257 mg GAE/g) (Table 2).
Table 2. Total phenolic and total flavonoid
contents in various plant extracts of L. spinosa red
leaves
Solvents
Total phenolic
contents
(mg GA/gm)
Total flavonoid
contents
(mg QE/gm)
Hexane
6.78±0.26
3.05 ± 0.09
Chloroform
9.42± 0.06
8.96 ± 0.21
Ethylacetate
8.91± 0.06
7.04 ± 0.02
The total flavonoid content was determined by
aluminium chloride colorimetric method as describe
by Chang et al. (2002). Quercetin was used as
standard calibration and total flavonoid content in
mg quercetin equivalence (mg QE/g). The total
content of flavonoids in different extracts of L.
spinosa was determined from the regression
equation of the calibration curve (y= 0.0072x +
0.0026, R2= 0.999) and expressed as milligrams of
quercetin equivalents (QE). In this study, the
chloroform extract exhibited the highest total
flavonoid content (8.96 ± 0.21 mg QE/g), followed
by ethyl acetate (7.04 ± 0.02 mg QE/g) and hexane
(3.05 ± 0.09 mg QE/g).
Antioxidants are compound that having the
ability to either delay or inhibit the oxidation
processes (Pisoschi et al. 2011). This reaction takes
place under the presence of atmospheric oxygen or
reactive oxygen species (ROS). Antioxidant involved
as a defensive mechanism of the organism from an
attack of free radicals.
In this present study, the antioxidant activity of
crude extracts from L. spinosa was determined by
using 2, 2-diphenyl-1-picrylhydrazyl (DPPH)
antioxidant capacity assay. The delocalization of the
spare electron on the molecule makes DPPH as a
stable free radical and does not dimerize like the
other free radical. The appearance of a violet colour
with an absorption band around 520 nm indicates
the delocalization of DPPH molecule (Pisoschi et al.
2009). The leaves of L. spinosa in different organic
solvents were tested for their free radical scavenging
activity using DPPH assay. The IC50 value was
calculated and found 1.181 mg/mL for hexane

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57
extract, 0.088 mg/mL for chloroform extract and
0.121 mg/mL for ethyl acetate extract whereas IC50
value of BHT was found to be 0.032 mg/mL.
IV.
SUMMARY
In conclusion, this study shows that higher total
flavonoid content (8.96 ± 0.21 mg QE/g) and total
phenolic content (9.42± 0.06 mg GA/g) is higher in
chloroform extract followed by ethyl acetate. The
lowest phenolic and flavonoid concentration was
reported in the hexane extract i.e. 6.78±0.26 mg
GA/g and 3.05 ± 0.09 mg QE/g, respectively.
V.
ACKNOWLEDGMENT
The authors thank Faculty of Science and Natural
Resources, Universiti Malaysia Sabah and School of
Industrial Technology, Universiti Sains Malaysia for
providing necessary research facilities to carry out
the research work. Thanks are due to Malaysian
Government and UMS for the providing the grant
SBK0329-2017 to conduct this work.
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