PHYTOCHEMICAL SCREENING AND ANTIOXIDANT ACTIVITY OF Epipremnum
pinnatum L. LEAF, Crescentia cujete LEAF, AND Lantana camara FLOWER EXTRACTS
ELLAINE S. BULAWIT, ABIGAIL L. DELA CRUZ, CHERRIE MAE J. MARTINEZ,
CHRISTINE JOY ANN V. PADIERNOS, AND SYDNEY S. SANTIAGO
Department of Chemistry, Central Luzon State University
ABSTRACT
Background: Some natural products possess antioxidant activity which protects cells from
destructive effects of reactive oxygen species (ROS). Consequently, these antioxidant
compounds such as tannins, alkaloids, terpenoids, steroids, and flavonoids are being isolated
and developed as neutraceutics and/or phytomedicines. This study aimed to assess for total
phenolic content and antioxidant activity of the extracts of Epipremnum pinnatum L. leaves,
Crescentia cujete leaves, and Lantana camara flowers. Methods: The bioactive components
were extracted by ethanol solution. The phytochemical constituents were determined using
qualitative test tube method. The total phenolic content (TPC) of the extracts was measured
using the Folin-Ciocalteu method, while the antioxidant assay was done by DPPH free radical
scavenging activity. Ascorbic acid was used as standard. Results: All phytochemical
constituents tested for L. camara flower and C. cujete leaf extracts was positive except for
ketones and phlobatannins, and carbohydrates and flavonoids, respectively. Alkaloids,
carbohydrates, and saponins were found present on E. pinnatum L. leaf extract. Measurements
of their antioxidant activity by DPPH gave a percent scavenging activity of 96.94±1.81%,
94.82±0.74%, and 36.76±5.75% for L. Camara, C. cujete, and E. pinnatum L. The TPC of the
extracts was found to be 204.26±0.108565 mg GAE/g crude extract for L. camara flower
extract, 143.26±0.032517 mg GAE/g crude extract for C. cujete leaf extract and 130.88
±0.033382 mg GAE/g crude extract for E. pinnatum L. leaf extract. Conclusion: The higher
antioxidant property observed in L. Camara flower extract was due to its high phenolic content.
Further study is necessary for the isolation and characterization of active antioxidant agents that
can be possibly used to treat oxidative stress-related diseases.
Keywords: Epipremnum pinnatum L., Crescentia cujete, Lantana camara, phytochemicals,
phenolic, antioxidant
INTRODUCTION
Plants, as extracts and in various forms,
have historically been the most productive
source of bioactive compounds and chemical
lead structures for the discovery and
development of traditional and modern
medicine. Interestingly, plants are currently
considered as a rapidly growing health
system worldwide together with the
developing science and technology where in
the recent decades, many plants led to
1|Page
approved products (Abdallah, 2011). Many
of them possess antioxidant activity which
protects cells from destructive effects of
reactive oxygen species (ROS) (Edoga et. Al,
2005). Consequently, these antioxidant
compounds such as tannins, alkaloids,
terpenoids, steroids, and flavonoids are being
isolated and developed as neutraceutics
and/or phytomedicines (Johnson, 2013).
Noteworthy, the Philippines abounds in
medicinal and indigenous plants which
present potential sources of new compounds
with interesting bioactivities. Though many
plants are known to be a source of medically
useful compounds, there are species that
remain unexplored for its nutritional and
antioxidant value (Gurib-fakim, 2006). As
humans, it is our role to discover the aesthetic
value of nature.
Epipremnum pinnatum L. commonly
known as “Dragon Tail Plant” or “centipede
togavine” is a large root-climber that is
widely found in Malaysia and Singapore and
has had a reputation as a traditional
anticancer preparation as well as a remedy for
skin diseases. This plant is native to
Southeastern Asia and New Guinea. (Huxley
A, Griffiths and Grifiths, 1994)
This contain the broad spectrum of
phytochemicals including alkaloids, steroidal
terpenoids, flavonoids, tannins, and cardiac
glycosides which has been investigated by
preliminary analysis of the ethanolic plant
extract (leaves and root) (Lalitha, Arathi,
Sripathi, Hemalatha & Jayanthi, 2010).
Moreover, various solvent extracts of E.
pinnatum leaves and aerial roots revealed
antibacterial
activity
against
microorganisms.
Crescentia cujete, also known as Calabash
is considered as a miracle tree where all parts
of the calabash tree was found to be useful in
many aspects (Ejelonu, Lasisi, Olaremu, and
Ejelonu, 2011). It has been used by different
local communities because of its chemical
composition and medicinal prospects.
A study showed that Calabash fruit and
leaf extracts contain flavonoids such as
quercetin and anthraquinone. These
compounds have the ability to prevent blood
vessel growth and development. Because of
the presence of these phytochemicals it was
also believed that calabash tree could be a
natural resource for anti-cancer treatments.
The leaves and barks also shown potential
as anti-inflammatory, antibacterial activities,
2|Page
and potential cure to disease caused by the
damage of biological membranes (Parvin,
Das, Jahan, Akhter & Nahar, 2015).
On a study conducted by Parente, De
Oliveira, Rodrigues, De Oliveira Jr., Paulo,
Nunes, Delange, and Almeida (2016),
Calabash was found to contain significant
amount of phenolic compounds that makes it
possible to contain antioxidant activity.
Lantana camara is a species of flowering
plant
within
the
verbena
family
(Verbenaceae). They are toxic ornamental
shrubs that are originally from tropical
America, but have spread through many of
the tropical and subtropical locations of the
world.
Two of the primary hepatotoxic and
allelopathic
secondary
metabolites,
pentacyclic triterpenoids, lantadene A and
lantadene B are present in the roots, leaves,
stems, and unripe fruit of L. camara.
On a study conducted by Ahmed, Shoaib,
Wassel, and El–Sayyad (1972), the presence
of myristic, palmitic, arachidic, oleic, and
linoleic acids as well as a-amyrin, p-sitosterol
and 1-triacontanol revealed on L. Camara
extract. Also, the presence of glucose,
maltose, and rhamnose before hydrolysis of
L. Camara was found, while glucose and
rhamnose revelaved after hydrolysis.
The aim of this study was to screen plant
material extracts of E. pinnatum L., C. cujete,
and L. camara with respect to their total
phenolic content and antioxidant activity in
order to find new potential sources of natural
antioxidants.
MATERIALS AND METHODS
Plant Collection
Plant materials were harvested along
Central Luzon State University, Science City
of Muñoz, Nueva Ecija, Philippines on April,
2019. It was rinsed with clean tap water then,
dried under room conditions until it becomes
crispy. Dried samples were pulverized into
coarse powder and stored in different air tight
glass containers prior to subsequent analysis.
Preparation of Plant Extract
The powdered samples were extracted
with ethanol. The mixtures were filtered and
the filtrates were concentrated by evaporating
it in a water bath at 60°. Subsequently, the
crude
extracts
were
subjected
to
phytochemical screening and antioxidant
activity tests.
Phytochemical Screening
Phytochemical screening was performed
using test tube method from the standard
procedure (Farnsworth, 1966).
Antioxidant activity
i. Diphenyl-1-picrylhydrazyl (DPPH) Free
Radical Scavenging Assay
A 2.5-mL of 6.0 mg/100-mL DPPH in
ethanol was added to 1-mL of 1000, 300, 100,
10, 1 and 0.1 ppm of the crude extracts. The
mixtures were shaken vigorously and left to
stand in the dark for 30 minutes at room
temperature. Ascorbic acid was used as
standard. Absorbance of the mixtures was
read
at
517 nm
using UV-Vis
spectrophotometer.
The
inhibition
percentage for scavenging DPPH radical was
calculated according to the equation:
absorbance of control − absorbance of sample
%scavenging = (
) x 100.
absorbance of control
A software named Graphpad Prism was
used to identify the EC50 value of the extracts.
Determination of Total Phenolic Content
The total phenolic content of the extracts
was determined according to the FolinCiocalteu spectrophotometric method used
by Mahdi-pour, Jothy, Latha, Chen, &
Sasidharan (2012) with some modifications.
A 120, 100, 80, 60, 40, and 20 ppm of
gallic acid-ethanol solution was prepared for
3|Page
calibration curve and 3000, 1000, 800, 500
and 300 ppm of the crude extract-ethanol
solution were also prepared. From each
solution, 400-μL were diluted with 800
microliters of distilled water then 1-mL of
Folin-Ciocalteu reagent was added. The
mixtures were allowed to stand for 5 minutes
at room temperature. Then, 2-mL of 7.5%
Sodium carbonate solution was added. The
mixtures were shaken vigorously and left to
stand in the absence of light for one and a half
hour. Absorbance was obtained at 765 nm
using UV spectrophotometer. The test was
done in triplicate for each extract. A
calibration curve was plotted to determine the
level of phenolics in the samples. Results
were expressed as milligrams of Gallic acid
equivalent (GAE) per gram dried sample.
RESULTS
Phytochemical screening
The result of phytochemical screening of
alkaloids, carbohydrates, flavonoids, cardiac
glycosides, ketones, phlobatannins, saponins,
phenols, and tannins for L. Camara flower
extract, C. cujete and E. pinnatum L. leaves
was summarized in Table 1-3.
Table 1. Phytochemical analysis of L.
Camara flower extract
L. camara
CONSTITUENTS
FLOWER
EXTRACT
Alkaloids
+
Carbohydrates
+
Flavonoids
+
Cardiac glycosides
+
Ketones
Phlobatannins
Saponins
+
Phenols
+
Tannins
+
Table 2. Phytochemical analysis of E.
pinnatum L. leaf extract
E. pinnatum L.
CONSTITUENTS
LEAF EXTRACT
Alkaloids
+
Carbohydrates
+
Flavonoids
Cardiac glycosides
Ketones
Phlobatannins
Saponins
+
Phenols
Tannins
DPPH assay
The results were reported in terms of ppm
equivalence of ascorbic acid from the extract
of L. Camara flower, C. cujete and E.
pinnatum L. leaves. The value obtained were
calculated and plotted in terms of %
scavenging against the concentration the
extracts as shown in Figure 1.
At 100 ppm, ascorbic acid exhibited a
maximum
scavenging
activity
of
98.12±0.04% followed by L. Camara of
96.94±1.81%, C. cujete of 94.82±0.74%, and
E. pinnatum L. of 36.76±5.75%. There is no
significant difference among the extracts and
standard.
EC50 was obtained using graphpad
application (Table 4). EC50 of ascorbic acid as
4|Page
a standard was found to be 2.363 while L.
Camara, C. cujete, and E. pinnatum L. were
found to be 19.37, 16.95, and 183.6,
respectively.
Total phenolic content
The total phenolic content was reported as
gallic acid equivalents by reference to
standard curve (y = 0.0072x + 0.1029 and
R²=0.9945) as shown in Figure 2.
Gallic acid
1.5
Absorbance
Table 2. Phytochemical analysis of C. cujete
leaf extract
C. cujete LEAF
CONSTITUENTS
EXTRACT
Alkaloids
+
Carbohydrates
Flavonoids
Cardiac glycosides
+
Ketones
+
Phlobatannins
+
Saponins
+
Phenols
+
Tannins
+
1
0.241
0.5
0.406 0.513
0.699
0.85 0.952
0
0
50
100
150
Concentration
Figure 2. Standard curve calibration of gallic
acid
At 3000 ppm, L. Camara had the highest
phenolic content of 204.26±0.108565 mg
GAE/g crude extract. This is followed by C.
cujete 143.26±0.032517 mg GAE/g crude
extract and E. pinnatum L. of 130.88
±0.033382 mg GAE/g crude extract (Table
3).
Table 3. Total phenolic content of C. cujete,
E. pinnatum L. and L. Camara at 3000 ppm.
Phenolic concentration
SAMPLE
(mg GAE/ g crude
extract)
C. cujete
143.26±0.032517
E. pinnatum L.
130.88 ±0.033382
L. Camara
204.26±0.108565
*readings from the average of (3) replicate
average phenolic concentration ± SD.
CONCLUSION
This study determined the presence of
phytochemical constituents and evaluated the
use of E. pinnatum L. leaf, C. cujete leaf, and
L. camara flower extracts as antioxidants.
Figure 1. %free radical scavenging activity against log concentration of sample
Phytochemical screening of the extracts
was performed for the determination of the
presence of reducing sugars, alkaloids,
saponins,
flavonoids,
and
phenolic
compounds. Total phenolic content and
antioxidant activity was also carried out.
Measurements of their antioxidant activity
by DPPH gave a percent scavenging activity
of 96.94±1.81%, 94.82±0.74%, and
36.76±5.75% for L. Camara, C. cujete, and
E. pinnatum L. The TPC of the extracts was
found to be 204.26±0.108565 mg GAE/g
crude extract for L. camara flower extract,
143.26±0.032517 mg GAE/g crude extract
for C. cujete leaf extract and 130.88
±0.033382 mg GAE/g crude extract for E.
pinnatum L. leaf extract.
5|Page
The results calculated showed that the
three extracts contain varied concentration of
phenolic compounds and L. camara flowers
having the largest concentration. Based on
the antioxidant activity via DPPH
scavenging, L. camara showed the highest
scavenging comparable to vitamin C among
the three plant species. The high total
phenolic content of L. camara flower may
have contributed to its high antioxidant
activity. The results showed that among the
three collected plant species, L. camara
flower extract has the highest potentiality in
using as a plant based natural product
antioxidant.
REFERENCES
6|Page