PRELIMINARY PHYTOCHEMICAL ANALYSIS AND IN VITRO ANTIOXIDANT ACTIVITY OF ARAUCARIA COLUMNARIS BARK PEEL AND COSMOS SULPHUREUS FLOWERS

Abstract

Objective: Four different extracts of Araucaria columnaris (bark peel) and Cosmos sulphureus (flowers) were screened for their phytochemical composition, and free radical scavenging activities.Methods: DPPH method was used to test the antioxidant activity for extracts.Results: Among the different extracts tested, the methanol extract of both the plant species showed significant radical scavenging activities. Phytochemical analysis of the extracts revealed that the radical scavenging activities might be due to the presence of flavonoids, tannins and phenolic compounds.Conclusion: The results obtained suggest that Araucaria columnaris (bark peel) and Cosmos sulphureus(flowers) could be exploited in the treatment of various diseases like cancer, cardiovascular diseases and infection diseases.

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Original Article
PRELIMINARY PHYTOCHEMICAL ANALYSIS AND IN VITRO ANTIOXIDANT ACTIVITY OF
ARAUCARIA COLUMNARIS BARK PEEL AND COSMOS SULPHUREUS FLOWERS
KRISHMA M. JADAV
1*
, K. N. NINGE GOWDA
2
1,2
Department of Apparel Technology and Management, Bangalore University, Bangalore 560001, India
Email: krishma.jadav@gmail.com
Received: 28 Jan 2017, Revised and Accepted: 20 Apr 2017
ABSTRACT
Objective: Four different extracts of Araucaria columnaris (bark peel) and Cosmos sulphureus (flowers) were screened for their phytochemical
composition, and free radical scavenging activities.
Methods: DPPH method was used to test the antioxidant activity for extracts.
Results: Among the different extracts tested, the methanol extract of both the plant species showed significant radical scavenging activities.
Phytochemical analysis of the extracts revealed that the radical scavenging activities might be due to the presence of flavonoids, tannins and
phenolic compounds.
Conclusion: The results obtained suggest that Araucaria columnaris (bark peel) and Cosmos sulphureus (flowers) could be exploited in the
treatment of various diseases like cancer, cardiovascular diseases and infection diseases.
Keywords: Cosmos sulphureus, Araucaria columnaris, Antioxidant activity, DPPH, Phytochemicals, Radical scavenging
© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijcpr.2017v 9i4.20967
INTRODUCTION
Medicinal plants are resources of new drugs and many of the
modern medicines are produced indirectly from plants [1]. In the
last years, interest in medicinal plants as an alternative to synthetic
drugs is more and more increasing because of safety concerns,
particularly against oxidative stress [2, 3]. Oxidative stress is
potential when there is an imbalance between ROS (Reactive Oxygen
Species) production and cellular antioxidant activity [4]. Oxidative
stress is implicated in over hundred human disease conditions, such
as cancer, cardiovascular diseases, aging and neurological disorders.
This free radical induced oxidative stress can be prevented by the
intake of sufficient amount of antioxidants [5]. Antioxidants are
molecules that are capable of neutralizing the harmful effects of the
ROS through the endogenous enzymatic defence system such as the
superoxide dismutase (SOD), glutathione peroxidase (GPX) and
catalase (CAT) in human system [6, 4]. Several studies have shown
that the mechanism underlying polar antioxidant involves reactions
with the hydroxyl (OH) group present in phenolics. Indeed, phenolics
are composed of one or more aromatic rings bearing one or more
hydroxyl groups and are therefore potentially able to quench free
radicals by forming stabilized phenoxyl radicals and most of the
current antioxidants isolated so far from flowering plants are simple
phenolic compounds which owe their properties to the mere fact that
their aromatic hydroxyl moieties react with free radicals [7].
Research has quite convincingly shown that foods and herbs rich in
antioxidants reap health benefits. Foods may possibly enhance
antioxidant levels because foods contain a lot of antioxidant
substances [8]. Potential sources of antioxidant compounds have
been found in several types of plant materials such as vegetables,
fruits, leaves, oilseeds, cereal crops, barks and roots, spices and
herbs, and crude plant drugs [9]. Natural products, mainly obtained
from dietary sources provide a large number of antioxidants.
Phytoconstituents are also an important source of antioxidant and
capable to terminate the free radical chain reactions [10, 11].
Araucaria columnaris belongs to the family Araucariaceae and genus
Araucaria. It is distributed throughout the New Caledonia and
Peshawar. It is commonly used as an ornamental plant all around the
world [12, 13].
Cosmos sulphureus Cav. is a member of the family Asteraceae [14]. It is
cultivated as an ornamental plant or it has been found as growing wild
weeds along the roadsides and other undisturbed areas. Cosmos
flowers are available in three different colours, orange, yellow and red.
Flower capitulum composed of the disc and ray florets (7-8 petals
like), straight and long-stalked. The height of the plant is 90-120 cm
and has compound leaves. Flowering season is October to December
[15]. The present study was undertaken to investigate the
phytochemical and antioxidants properties of bark peel extract of
Araucaria columnaris and flower extracts of Cosmos sulphurous with a
view to assess the potentials of both the plants as a source for phenolic
antioxidants.
MATERIALS AND METHODS
Plant material
The bark peel of Araucaria columnaris was collected from Lalbagh
Botanical Garden, Bangalore and Cosmos sulphurous flowers were
collected from different Botanical gardens situated in and around
Bangalore. Both the plant materials collected were air-dried and
ground to fine powder.
Extraction
Successive solvent extraction [16, 17] procedure was adopted for
the preparation of various extracts Araucaria columnaris and Cosmos
sulphureus. The powdered plant materials were subjected to
successive extraction using Soxhlet apparatus with solvents in their
ascending order of polarity. The solvents used were petroleum ether
(60-80 °C), chloroform, methanol and distilled water. The extracts
were filtered using Whatman no. 1 and further dried by using rotary
evaporator until semi-solid is obtained.
Preliminary phytochemical screening
The phytochemical screening was carried out using standard
procedures [18]. The tests for phytochemical screening include:
A. Tests for Carbohydrates
i. Benedict’s test: Equal volumes of Benedict’s reagent and test
solution were mixed in a test tube and heated in boiling water bath
International Journal of Current Pharmaceutical Research
ISSN- 0975-7066 Vol 9, Issue 4, 2017

Jadav et al.
Int J Curr Pharm Res, Vol 9, Issue 4, 96-99
97
for 5 min. Appearance of green, yellow or red indicated the presence
of carbohydrates depending on the amount of reducing sugar
present in test solution.
B. Test for Proteins
i. Million’s test: 3 ml of test solution was mixed with 5 ml of
Million’s reagent. White ppt. forms; when made warm turns brick
red or ppt. dissolves giving red coloured solution indicating the
presence of proteins.
C. Tests for Amino acids
i. Tyrosine test: 3 ml of test solution was heated with 3 drops of Million’s
reagent. Dark red colour formed shows the presence of Amino acids.
D. Tests for Fats and Oils
i. Filter paper test: Filter paper was treated with the test solution
and dried. Permanent oil stain on the filter paper indicated the
presence of Fats and Oils.
E. Tests for Steroid
i. Liebermann’s reaction: 3 ml of test solution was mixed with 3 ml
acetic anhydride. Heated and cooled. On addition of few drops of H
2
SO
4
,
the appearance of blue colour indicated the presence of steroids.
F. Tests for Volatile oils
i. Hydro distillation method: The test solution was
hydrodistillated. Volatile oil was separated from the distillate. The
filter paper was treated with volatile oil and dried. The filter paper is
not permanently stained with volatile oil.
G. Tests for Glycosides
i. Liebermann’s test: 3 ml of test solution was mixed with 3 ml acetic
anhydride. Heated and cooled. On addition of few drops of H
2
SO
4
,
appearance of blue colour indicated the presence of Glycosides
H. Tests for Saponins
i. Foam test: Persistent foam was observed when test solution was
shaken vigorously with water indicating the presence of saponins.
I. Test for Coumarins
i. Odour test: Aromatic odour indicated presence of coumarin glycosides
ii. Alkalinity test: Alcoholic extract when made alkaline showed
blue or fluorescence indicating the presence of coumarins.
J. Tests for Flavonoids
i. Sulphuric acid test: On addition of sulphuric acid (66% or 80%)
flavones and flavonols dissolve into it giving a deep yellow solution.
Chalcones and aurones gave red or red-bluish solutions. And orange
to red colours indicated the presence of Flavanes.
ii. Lead acetate solution test: to a small quantity of residue, lead
acetate solution was added. Yellow coloured ppt. formed indicated
the presence of flavonoids.
K. Tests for Alkaloids
i. Tannic acid test: Test solution treated with tannic acid solution
gave buff coloured ppt. indicating the presence of alkaloids.
L. Tests for Tannins and Phenolic compounds
i. 5% FeCl
3
test: To 2-3 ml of aqueous or alcoholic extract, few
drops FeCl
3
(5%) was added. Deep blue-black colour observed
indicated the presence of Tannins and Phenolic compounds.
Determination of antioxidant activity (DPPH assay)
Radical scavenging activity of the extracts was determined by
measuring the decrease in absorbance of 2, 2-Diphenyl-1-
picrylhydrazyl radical (DPPH•) at 517 nm [19]. The DPPH assay is
a widely used method to evaluate the ability of antioxidants [20] to
scavenge free radicals which are known to be a major factor in
biological damages caused by oxidative stress. This assay is known
to give reliable information concerning the antioxidant ability of
the tested compounds [21-23]. This method is based on the ability
of DPPH radical to react with hydrogen donor species such as
phenolics and flavonoids present in the extracted material. Upon
receiving a proton from the donor species it loses its color and
becomes yellow. As the concentration of phenolic compounds
increases, their DPPH radical scavenging activity also increases
[24]. The crude extracts were weighed and dissolved in DMSO (10
mg/ml). This was considered as pure sample extract for testing the
antioxidant property. 0.3 mmol solution of DPPH was prepared in
100% methanol. To 1 ml of this solution, three different
concentrations 100 µl, 300 µl and 500 µl of sample extract and
standard solution (Ascorbic acid) were added separately. The final
volume was made up to 4 ml by adding 100% methanol to each
sample mixture and also for a standard solution (Ascorbic acid).
The same reaction mixture without the extracted sample but with
an equivalent amount of standard phosphate buffer was taken as
control. All the sample mixtures and control were shaken
thoroughly and kept in dark at room temperature for 30 min. The
absorbance of the reaction mixtures was measured at 517 nm [25].
The radical scavenging activities were expressed as a percentage
of inhibition and calculated according to the following equation.
DPPH radical scavenging activity (%) = [ (Abs
control
–Abs
sample)
/(Abs
control)
] x 100
Where Abs
control
is absorbance control and Abs
sample
is absorbance
test sample.
RESULTS AND DISCUSSION
Preliminary phytochemical screening
The phytochemical analysis conducted on Araucaria columnaris and
Cosmos sulphureus extracts revealed the presence of proteins,
coumarins, saponins, amino acids, flavonoids, tannins, and phenolic
compounds (table 1 and table 2). These phytochemical compounds are
known to support bioactive activities in medicinal plants and thus
responsible for the antioxidant activities of this plant extract used in
this study.
Table 1: Results of the qualitative test for preliminary phytochemical analysis of Araucaria columnaris (bark peel) extracts
Phytochemical
constituents
Tests
Blank
Control
Petroleum ether
Chloroform
Methanol
Aqueous
Carbohydrates
Benedict’s test
-
+
-
-
-
-
Proteins
Million’s test
-
+
-
-
+
-
Amino acids
Tyrosine test
-
+
-
-
-
-
Fats
and
oils
Filter paper test
-
+
-
-
+
+
Steroids
Liebermann
-
Burchard Test
-
+
-
-
-
-
Glycosides
Liebermann’s test
-
+
-
-
-
-
Saponins
Foam test
-
+
-
-
-
+
Coumarins
Odour test
-
+
-
-
-
+
A
lkalinity test
-
+
-
-
-
-
Flavonoids
H
2
SO
4
test
-
+
-
-
++
+
Lead acetate solution test
-
+
-
-
++
+
Alkaloids
Tannic acid test
-
+
-
-
-
-
Tannins
and
phenolic compounds
5% FeCl
3
test
-
+
-
-
++
+
+++(High),++(Moderate)+(low) and-(Nil)

Jadav et al.
Int J Curr Pharm Res, Vol 9, Issue 4, 96-99
98
Table 2: Results of the qualitative test for preliminary phytochemical analysis of Cosmos sulphureus (flower) extracts
Phytochemical
constituents
Tests
Blank
Control
Petroleum ether
Chloroform
Methanol
Aqueous
Carbohydrates
Benedict’s test
-
+
-
-
-
-
Proteins
Mil
lion’s test
-
+
-
-
-
-
Amino acids
Tyrosine test
-
+
-
-
-
+
Fats
and
oils
Filter paper test
-
+
-
-
+
+
Steroids
Liebermann
-
Burchard Test
-
+
-
-
-
-
Glycosides
Liebermann’s test
-
+
-
-
-
-
Saponins
Foam test
-
+
-
-
-
+
Coumarins
Odour test
-
+
-
-
-
-
Alkalinity test
-
+
-
-
-
-
Flavonoids
H
2
SO
4
test
-
+
-
-
+++
++
Lead acetate solution test
-
+
-
-
+++
-
Alkaloids
Tannic acid test
-
+
-
-
-
-
Tannins
and
phenolic compounds
5% FeCl
3
test
-
+
-
-
++
+
+++(High),++(Moderate)+(low) and-(Nil)
Determination of antioxidant activity (DPPH assay)
The in vitro antioxidant assay of both the plant extracts (fig. 1 and
fig. 2) reveals significant antioxidant potential compared with
standard Ascorbic acid. The methanol extracts of both the plants
Araucaria columnaris and Cosmos sulphurous showed highest
antioxidant activity. The percentage inhibition of Araucaria
columnaris and Cosmos sulphureus at the concentration of 500 µl
were 90.37 % and 89.87 % respectively, compared to ascorbic acid
(94.05%). The extracts showed increased antioxidant activity with
the increase in concentration (µl) of the extracts.
Fig. 1: Antioxidant properties of Araucaria columnaris plant extract in comparison with standard ascorbic acid as determined with DPPH method
Fig. 2: Antioxidant properties of Cosmos sulphureus plant extract in comparison with standard ascorbic acid as determined with DPPH method
CONCLUSION
As antioxidants play a vital role in fighting against many diseases,
especially those that are due to oxidative stress, it is important to
identify which natural plants are effective in fighting against the free
radicals. Based on the results obtained, the radical scavenging
activity is concentration-dependent, as it increases when the
concentration changes from 100 µl to 300 µl to 500 µl. The
investigation confirms the in vitro antioxidant potential of solvent
extracts of Araucaria columnaris and Cosmos sulphureus, with results

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Int J Curr Pharm Res, Vol 9, Issue 4, 96-99
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comparable to those of the standard compound ascorbic acid and
can, therefore, be proposed as new potential sources of natural
additives for the food and/or pharmaceutical industries. The
phytochemical screening revealed the presence of flavonoids,
tannins and phenolic compounds in methanol and aqueous extracts
of both the plant species. However, the components responsible for
the antioxidant activity of the extracts were not identified and
further work should be conducted to isolate and identify these
bioactive compounds.
CONFLICTS OF INTERESTS
All authors have none to declare
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How to cite this article
• Krishma M Jadav, KN Ninge Gowda. Preliminary phytochemical
analysis and in vitro antioxidant activity of Araucaria columnaris
bark peel and Cosmos sulphureus flowers. Int J Curr Pharm Res
2017;9(4):96-99.