Phytochemical Screening Chromolaena odorata L.

Ethanolic Leaf
Extract
(Ungab, Reymar S.)

Abstract: This activity with objective of identifying and confirming the

presence of five phytochemical constituents namely: alkaloids, flavonoids,
phenols, saponins and tannins from sthe ethanolic leaf extract of the locally
known medicinal plant Hagonoy (Chromolaena odorata L.). Plant samples
were collected at the back of the Mahogany Residence Hall, Central
Mindanao University, Musuan, Maramag, Bukidnon. Result showed negative
result indicating that there are no presence of the five phytochemicals in the
ethanolic leaf extract of the plant sample. This result does not coincide with
the data provided by literatures that could be due to certain factors such as
location where the plant samples were collected, concentration of sample
solution and choice of extracting solvent. Due to the negative result obtained
in this activity using different extracting solvent in the next conduct of the
same activity is recommended.

Introduction (secondary protective tissue),

besides retarding water loss,
Plants are essential to the
provide passive barriers to
balance of nature and in people's
bacterial and fungal entry.
lives. They are expected to be fully
A diverse group of plant
utilized because they have been
compounds, commonly referred to
created with a purpose. In natural
as secondary metabolites, also
habitats, plants are surrounded by
defends plants against a variety of
an enormous number of potential
herbivores and pathogenic
enemies. Nearly all ecosystems
microbes. These secondary
contain a wide variety of bacteria,
metabolites are phytochemicals
viruses, fungi, nematodes, mites,
which are of great interest to
insects, mammals, and other
pharmaceutical companies for the
herbivorous animals. By their
production of new drugs to combat
nature, plants cannot avoid these
various illnesses.
herbivores and pathogens simply
Medicinal plants are proven
by moving away; they must protect
to be useful for curing human
themselves in other ways. Their
diseases because of the presence
first line of defence involves the
of phytochemical constituents that
plant surface. The cuticle (a waxy
produce definite physiological
outer layer) and the periderm
action on the human body behind using this plant came from
(Akinmoladun et al., 2007). the ethno medicinal use of this
Phytochemicals are naturally found plant among the traditional and
in the Plant kingdom with various tribal people.
chemical structure and defence
mechanisms. Methodology
Phytochemicals are plant
based natural constituent can be Materials and Apparatuses:
derived from the various parts of The following materials and
the plants that may contain active apparatuses were used in order of
components. The systematic utilization; sampling cellophane
screening of plant species with the bags, GPS, labelling tape, stainless
purpose of discovering new knife, drying net, aluminium plates,
bioactive compounds is a routine mortar and pestle, 250-mL
activity in many laboratories beakers, 1000-mL flask, pipette
nowadays. Plants with medicinal and aspirator, rotary evaporator,
values are collected either glass slides, stirring rod, glass
randomly or by following leads slides, micropipette, volumetric
supplied by local healers in flask 50-mL, sprayer and lead
geographical areas where the pencil.
plants are abundantly found (Kumar Procedure:
et al., 2004). Fresh or dried plant
materials can be used as a source A. Sample Collection &
for the extraction of secondary Preparation of Herbarium
plant components. Specimen
In this activity, Hagonoy
(Chromolaena odorata L.) a plant
used locally in treating and
disinfecting fresh wounds was
subjected to phytochemical
screening. The initial steps include
sample collection and preparation
of herbarium specimen while the
final steps include Thin Layer
Chromatography and visualization
Figure 1 Sampling Site
via spray method. This activity
aimed to confirm the presence of The plant samples,
some phytochemicals namely; Chromolaena odorata L.,
alkaloids, flavonoids, phenols, were collected at the back of
saponins and tannins. The logic Mahogany Residence Hall,
Central Mindanao University,
Musuan, Maramag, Bukidnon
having site coordinates of
75150.7 N 1250253.2 E
asshown in the figure 1.
Figure 2 Herbarium Specimen
The collected fresh
mature leaves were
thoroughly cleaned through
washing with tap water and
rinsing with distilled water.
Five plants with its flower
were meticulously picked for
herbarium specimen and
were sent to the CMU
museum for drying. The
herbarium specimen is shown
in the figure 2 above.
Cleaned plant leaf
samples were air and oven-
dried at 40 C. Dried samples
were then cut into pieces
using scissors and were
ground using mortar and
pestle.
B. Extraction & Concentration of
Plant Extract
Fifty grams of the
ground plant samples were
subjected to solvent
extraction and concentration
of the extract following the
method used by Aguinaldo et
al in order to extract the
secondary metabolites from
the collected plant sample.
This extraction method
includes soaking the ground
plant sample with 95%
ethanol for 24 hours.
Ethanolic extracts obtained
were then concentrated
through distillation via rotary
evaporator. The concentrated
plant extracts were stored at
0 to 5 oC in the refrigerator
until it were used.
C. Phytochemical Screening via
Thin Layer Chromatography
To qualitatively identify
and confirm the type and
presence of the natural
products in the plant sample
collected, thin layer
chromatography was used
following the method of Pavia
et, al., (1988). In preparing
the TLC plates, silica gel was
dissolved in water (3:1) in a
wide mouth screw cap jar.
The mixture was swirled to
obtain the slurry. The TLC
plates were made by using a
 thoroughly cleaned glass Solvent for Development: To
slides. The glass slides were qualitatively test the presence
washed with soap and water of different kinds of natural
then it was rinsed with tap products several solvent
water and 50% aqueous system were used. A 250-mL
methanol. It was then air beaker was used as a
developing chamber.
dried on top of white paper
towels. The slides were then a. Test for Alkaloids: One
coated with the previously TLC plate were placed
prepared slurry by in the developing
sandwiching the two glass chamber. The solvent
slide and then dipped in the system used for the
slurry for 2 seconds. It was determination of
Alkaloids was
then slowly withdrew and
chloroform: methanol
separated and placed in an
(5:1). The developed
oven at 105 oC. Prepared TLC chromatograms were
plates are shown in the figure air-dried and visualized
3 below. using Dragendorrfs
reagent by spraying.
Orange spots would
indicate the presence
of alkaloids.

Preparation of
Dragendorrfs reagent:
Solution A: A mass of
0.85 g of bismuth (III)
nitrate was dissolved in
a mixture of 10.00 mL
Figure 3 TLC Plates acetic acid and 40.00
Spotting the Plates: In mL water.
spotting the plates a
Solution B: A mass of
micropipette was used and
8.00 g potassium
2000ppm of the concentrated
iodide (KI) was
sample was prepared from the
dissolved in 20.00 mL
concentrated plant extract.
of water. Stock
Approximately 5 L prepared
solution: Equal parts of
extract solution was liberally
solutions A & B were
placed in the spot plate. The
mixed.
solvent was then allowed to
evaporate.
 Dragendorrfs reagent:
One mL of stock d. Test for Saponins: One
solution, 2.00 mL of TLC plate were placed
acetic acid and 10.00 in the developing
mL of water were chamber. The solvent
mixed. system used for the
determination saponins
b. Test for Flavonoids: was chloroform:
One TLC plate were methanol (30:5). The
placed in the developed
developing chamber. chromatograms were
The solvent system oven-dried and sprayed
used for the with vanillin sulfuric
determination of acid solution. Violet
flavonoids was ethyl spots would indicate
acetate: glacial acetic the presence of
acid: water (90:10:10). saponins.
The developed
chromatograms were e. Test for Tannins: One
air-dried and sprayed TLC plate were placed
with 5% ferric chloride in the developing
solution. Gray spots chamber. The solvent
would indicate the system used for the
presence of flavonoids. determination of
tannins was
c. Test for Phenols: One chloroform: methanol:
TLC plate were placed water (65:35:10). The
in the developing developed
chamber. The solvent chromatograms were
system used for the oven-dried and sprayed
determination of with 0.5% (w/v) vanillin
phenols was ethyl solution prepared in 4%
acetate: formic acid: (w/v) HCl. Blue spots
acetic acid: water would indicate the
(100:11:11:26). The presence of tannins.
developed
Results & Discussion
chromatograms were
air-dried and sprayed Phytochemical screening is of
with 2% ferric chloride great interest and importance
in ethanol. Bright blue especially for the pharmaceuticals
spots would indicate in discovering bio-active
the presence of compounds. Together with this,
phenols. the systematic screening of plant
species with the purpose of preliminary phytochemical
discovering new bioactive screening of the Chromolaena
compounds is a common activity odorata L. ethanolic extract. The
in many laboratories nowadays qualitative phytochemical analysis
with the objective of identifying revealed no positive result as
the phytochemical constituents shown in the figure 4 below in any
present. of the five phytochemical
constituents tested. These results
Preliminary data that should
disagree with the study of
be obtained this activity was the
Vijayaraghavan et al. (2013) in
moisture content of the leaf
which presence of flavonoids,
samples from Hagonoy
phenols, saponins and tannins
(Chromolaena odorata L.) plant.
were confirmed from the
According to the study of Igboh et
Chromolaena odorata L. ethanolic
al. (2009), moisture content of
extracts. Using methanol as
hagonoy leaf is 60% but
extracting solvent on the other
experimentally it was not
hand as with the study of Harini et
determined due to loss of some of
al. (2014), all of the five
the dried samples because of
phytochemical constituents tested
heavy winds. The percent yield
in this activity which are alkaloids,
was not also calculated as
flavonoids, phenols, saponins and
samples was not totally dried.
tannins were present.
Table 1. Preliminary
phytochemical screening of
the ethanolic extract of
Chromolaena
Plant Observation
Constitu
ent
Alkaloids (-) no visible orange
spots
Flavonoi (-) visible gray spots Figure 4 Developed TLC Plate
ds
Phenols (-) no visible bright The discrepancies of the
blue spots result of this activity from the data
Saponins (-) visible violet spots provided by literatures can be
Tannins (-) no visible blue accounted primarily to the
spots different preferences on sampling
Legend: + = positive; - = sites, which consequently would
negative mean variation on environmental
factors such as climate, altitude
The table 1 above shows the and rainfall that would eventually
summary of the results of the produce major variations in the
bioactive compounds present in all the five phytochemicals. But
the plants (GEETHA, 2014). this disagreement could be
Another major factors that could attributed to several factors
be the reason of the negative namely: location, concentration of
results are the concentration of sample solution and choice of
the sample solution, the choice of extracting solvent.
extracting solvent and premature
Considering the results
visualization. Concentration of the
obtained given the certain
sample solution used was 2000
conditions, it is recommended that
ppm, this concentration might be
extraction must be done using
a little bit low to be able observe
different solvents, more
the presence of the tested
concentrated sample solution and
phytochemicals qualitatively. The
using plant sample exposed to
choice of extracting solvent is also
different environmental stresses.
a factor that cannot be ignored.
Type of solvent plays an important Reference
role in the extraction of
phytochemical composition since AKINMOLADUN, A.C., E.O. IBUKUN,
different type of different solvent E. AFOR, E.M. OBUOTOR AND E.O
have different extracting impact FAROMBI, 2007. Pytochemical
(Ngo, 2017). With this reason, the constituents and antioxidant
capacity of ethanol might not be activity of extract from the leaves
enough to extract enough amount of the Ocimum graticcimum. Sci.
of phytochemical constituents Res. Essay, 2:163_166
from the Chromolaena odorata L. GEETHA, T. & GEETHA, N. 2014.
leaf. Lastly, during visualization Phytochemical Screening,
the plate may not be dried enough Quantitative Analysis of Primary
and could therefore causen pre- and Secondary Metabolites of
mature visualization and will
Cymbopogan citratus(DC) stapf.
eventually give a negative result.
leaves from Kodaikanal hills,
Conclusions & Tamilnadu
Recommendations
HARINI, K., JERLIN SHOWMYA J. AND
The result showed negative GEETHA N. (2014). Phytochemical
presence of the five Constituents of Different Extracts
phytochemical constituents. from the Leaves of Chromolaena
Therefore it was concluded that
odorata L. King and Robinson.
Chromolaena odorata L. is not a
International Journal of
good candidate to a source of
useful drugs. Although literatures Pharmaceutical Sciences and
disagree with this result stating Business Management, Vol.2 Issue.
that the plant sample 12, December- 2014, pg. 13-20
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THANH VAN NGO, CHRISTOPHER

JAMES SCARLETT, MICHAEL
CHRISTIAN BOWYER, PHUONG DUC