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International Journal of Pharmacognosy and Phytochemical Research 2017; 9(6); 814-819
ISSN: 0975-4873
Research Article
*Author for Correspondence:sowjypulipati@gmail.com
Determination of Total Phenolic, Tannin, Flavonoid Contents and
Evaluation of Antioxidant Property of Amaranthus tricolor (L)
Sowjanya Pulipati*, P Srinivasa Babu, U Naveena, S K Rafeeka Parveen, S K Sumaya
Nausheen, M Tanmai Naga Sai
Department of Biotechnology, Vignan Pharmacy College, Vadlamudi-522213, Andhra Pradesh, India
Received: 24th April, 17; Revised 8th June, 17, Accepted: 15th June, 17; Available Online:25th June, 2017
ABSTRACT
Free radicals or reactive oxygen species are involved in various pharmacological conditions. As synthetic antioxidants
possess numerous adverse health effects, the medicinal plants possessing antioxidant components can be used to prevent
harmful effects of reactive oxygen species. In the present study leaves of Amaranthus tricolor Linn were used to prepare
chloroform (CEAT), methanolic (MEAT) and aqueous (AEAT) extracts, analyze the presence of phytochemicals and
evaluation of in-vitro antioxidant property. Quantitative determination of phenols, tannins and flavonoids in leaves
A.tricolor was carried out using spectrophotometric methods. The antioxidant activity was performed by DPPH, p-NDA
radical scavenging methods for different extracts of the plant. The plant species showed that methanolic extract (MEAT)
on higher concentration possess better antioxidant potential when compared with reference standard ascorbic acid. The
plant extracts exhibited strong antioxidant DPPH radical scavenging activity with the IC50 values 290, 657, 830 & 130μg/ml
of MEAT, CEAT, AEAT & ASA respectively. In scavenging hydroxyl radical by p-NDA method the MEAT showed
maximum activity, CEAT showed moderate and AEAT showed minimum activity. The strongest antioxidant activity of
MEAT could be due to the presence of flavonoids and phenols.
Keywords: Amaranthus tricolor, phenols, flavonoids, DPPH, p-NDA.
INTRODUCTION
Phenolic compounds are one of the main secondary
metabolites derived from pentose phosphate, shikimate
and phenyl propanoid pathways in plants1-3. They are
commonly found in non-edible and edible plants and
posses’ numerological biological effects4. They are
essential for reproduction and growth of plants. Phenolic
compounds posses’ redox properties, which allows acting
as hydrogen donors, reducing agents, metal chelators and
singlet oxygen quenchers and hence they are antioxidants.
Traditionally fruits, vegetables, tea and spices are used as
antioxidants. Some of these are commercially exploited
either as nutritional supplements or antioxidant additives.
The most common group of poly phenolics are flavonoids
that are ubiquitously found in leaves, flowering tissues,
woody parts such as stems and bark4. They posses free
radical scavenging, inhibition of hydrolytic and oxidative
enzymes and anti-inflammatory action because of
antioxidant activity5,6. Antioxidant is an important
property which posses the ability of protecting organisms
from damage caused by free radical-induced oxidative
stress7. The antioxidant activity of phenolics is because of
their redox property that allows them to act as hydrogen
donors, reducing agents, metal chelators and single oxygen
quenchers8. Flavonoids are also known for their free-
radical scavenging and antioxidant activities
Biological metabolism involved in various processes
produce reactive oxygen species or free radicals which are
harmful to living cells. Excess accumulation of these
radicals may cause asthma, cancer, liver diseases,
cardiovascular diseases, muscular degeneration and
inflammatory processess9, resulting into oxidative stress.
Oxidative stress is defined as imbalance between oxidants
and antioxidants and causes damage in all types of
biomolecules such as DNA, RNA, nucleic acid and
protein10. Hence, the balance between reactive species or
free radicals and antioxidants is believed to be a critical
concept for maintaining a good biological system.
Antioxidants act as free radical scavengers, reducing
agents, quenchers of singlet of age-related diseases which
could be due to the presence of various antioxidant
compounds oxygen molecule, and activators for
antioxidative enzyme to suppress the damage induced by
free radicals in biological system. Many researchers found
that consumption of plant products11 reduces the mortality,
especially, phenolics, which are the most reactive
compounds. Antioxidants present in plant products help in
the stimulation of cellular defense system and biological
system against oxidative damage.
A.tricolor (Amaranthaceae) is an ornamental plant
commonly known as ‘‘Red amaranth” or “Joseph’s coat”
cultivated throughout South-East Asia and many tropical
countries. It is highly nutritious and hence extensively used
as green leafy vegetable. A. tricolor L. is one of the
traditional medicines used in many folk claims and the
plant has been extensively used in ayurveda and sidda for
Sowjanya et al. / Determination of Total…
IJPPR, Volume 9, Issue 6: June 2017 Page 815
Table 2: Total phenolic, Non-tannin, Tannin &
Flavonoid content Present in MEAT, CEAT & AEAT.
Parameter
Unit
MEAT
CEAT
AEAT
Total
phenolic
content
mg of
GAE/gm of
extract
19.4
16.7
13.8
Non tannin
content
mg of
GAE/gm of
extract
12.2
10.8
9.3
Tannin
content
mg of
GAE/gm of
extract
7.2
5.9
4.5
Flavonoid
mg of
rutin/gm of
extract
4.5
3.8
3.2
treating menorrhagia, diarrhea, dysentery, haemorrhagic
colitis, bowel hemorrhages, cough and bronchitis. It is also
used externally as an emollient poultice or a mouth wash
to treat ulcerated conditions of the throat and mouth12.
The ethno-botanical properties of the plant were reported
as astringent13, hepatoprotective14, antinociceptive and
anti-inflammatory activities15, in-vitro antioxidant, anti-
amylase, anti-arthritic and cytotoxic activity16.The root
decoction along with Cucurbita moschata is used to
control haemorrhage following abortion17. The plant
decoction is taken internally to strengthen the liver and to
improve vision. Scientific study on the plant suggests that
it may inhibit calcium retention18. It was also reported to
posses’ antibacterial activity against urinary tract
pathogens of clinical origin19. The authors had undertaken
the present work to explore the antioxidant property of
leaves of A. tricolor.
In vitro antioxidant activity of plant extracts were carried
out using DPPH and p-NDA methods. DPPH (2, 2-
Diphenyl-1-picrylhydrazyl) is a rapid, simple and
inexpensive method to measure antioxidant capacity of
food. It involves the use of the free radical (DPPH), which
is widely used to test the ability of compounds to act as
free radical scavengers or hydrogen donors and to evaluate
antioxidant activity20. The DPPH assay method is based on
the reduction of DPPH, a stable free radical21. The free
radical DPPH with an odd electron gives a maximum
absorption at 517 nm (purple colour). When Antioxidants
react with DPPH, which is a stable free radical becomes
paired off in the presence of a hydrogen donor (e.g., a free
radical scavenging antioxidant) and is reduced to the
DPPHH and as consequence the absorbance’s decreased
from the DPPH22. Radical to the DPPH-H form, results in
decolorization (yellow colour) with respect to the number
of electrons captured23. More the decolorization more is
the reducing ability. This test has been the most accepted
model for evaluating the free radical scavenging activity of
any new drug24. When a solution of DPPH is mixed with
that of a substance that can donate a hydrogen atom, then
this gives rise to the reduced form (Diphenyl picryl
hydrazine; non radical) with the loss of this violet colour
(although there would be expected to be a residual pale
yellow colour from the picryl group still present)25.
MATERIALS AND METHODS
Plant material
The plant Amaranthus tricolor L was collected, identified
and authenticated by BSI, Coimbatore. The healthy leaves
were shade dried and powdered using electric blender to
get a coarse powder.
Extraction
The powdered leaf material was extracted by successive
solvent extraction using soxhlet apparatus. The solvents
were selected according to the increasing order of polarity.
Different solvents like chloroform (CEAT), methanol
(MEAT) & water (AEAT) were used for extraction and the
extracts were concentrated and preserved in a desiccator
for further study.
Phytochemical screening
The phytochemical screening for the crude extracts of
Amaranthus tricolor was carried out by standard
protocols26,27. The presence of alkaloids, glycosides,
saponins, carbohydrates, proteins, aminoacids, phenolic
compounds, flavonoids, steroids, tannins was analyzed.
Determination of Total Phenolic Content
The total phenolic content was determined using Folin
Ciocalteau reagent. A standard calibration curve was
prepared and the absorbance against concentration of
tannins at 725nm was estimated spectrophotometrically.
Gallic acid was used as a standard and the total phenolic
content was expressed as µg/ml gallic acid equivalents
(GAE). Concentration of 0.01, 0.02, 0.03, 0.04 and 0.05
mg/ml of gallic acid were prepared in methanol.
Concentration of 1mg/ml of plant extract was prepared in
methanol and 0.5ml of each sample were introduced into
test tubes and mixed with 0.5ml of a 1N dilute Folin-
Ciocalteau reagent and 2.5ml of 20% sodium carbonate.
The tubes were covered with parafilm and allowed to stand
for 40 minutes at room temperature and absorbance was
read at 725nm spectrophotometrically28.
Determination of Tannin Content
Tannin content was determined using insoluble polyvinyl-
polypyrrolidone (PVPP), which binds tannins29. Briefly
1ml of extract (1mg/ml) in which the total phenolics was
determined, was mixed with 100mg of PVPP, vortexed,
kept for 15min at 40C and then centrifuged for 10 min at
3000 rpm. In the clear supernatant non-tannin phenolics
were determined the same way as that of total phenolics.
Table 1: Preliminary phytochemical screening of
MEAT, CEAT & AEAT.
Name of the Test
MEAT
CEAT
AEAT
Carbohydrates
+
+
+
Proteins
+
+
+
Amino acids
+
+
+
Steroids
+
+
+
Cardiac
glycosides
+
+
+
Flavonoids
+
+
+
Alkaloids
+
+
+
Tannins &
phenolic
compounds
+
+
+
Sowjanya et al. / Determination of Total…
IJPPR, Volume 9, Issue 6: June 2017 Page 816
Tannin content was calculated as a difference between
total and non-tannin phenolic content.
Determination of Flavonoid Content
The aluminum chloride method was used for the
determination of the total flavonoid content of the sample
extracts30. Aliquots of extract solutions were taken and
made up the volume 3ml with methanol. Then 0.1ml AlCl3
(10%), 0.1ml Na-K tartarate and 2.8 ml distilled water
were added sequentially. The test solution was vigorously
shaken. Absorbance at 415 nm was recorded after 30
minutes of incubation. Rutin was used as a standard
compound in the range of 2-12 mg/ml concentration to
construct a standard curve.
Free Radical Scavenging Activity (DPPH Method)
The antioxidant activity of various extracts of A. tricolor
and ascorbic acid were assessed on the basis of radical
scavenging effect on the DPPH (2, 2-Diphenyl-1-
picrylhydrazyl) stable free radical. In this method different
concentrations of the crude extracts of Amaranthus
tricolor 100 to 500µg/ml concentrations of extracts were
prepared with methanol. 1ml of each prepared
concentration was mixed with 3 ml of DPPH (0.1 mM)
solution in methanol. The test tubes were incubated for one
hour at room temperature in dark and the absorbance is
measured at 517nm in UV-Visible Spectrophotometer31,32.
Ascorbic acid is used as a standard and the same
concentrations were prepared as to test solution. The
differences in absorbance between the test and the standard
was calculated and expressed as
% scavenging of DPPH radical Scavenging effect (%) =
(Ac-As)/Ac x100
Where, Ac is absorbance of control, As is absorbance of
sample or standard.
p-Nitroso dimethyl aniline radical scavenging method (p-
NDA)
Hydroxyl radical scavenging is measured by the inhibition
of p-NDA bleaching. Hydroxyl radicals generated through
Fenton reaction can bleach p-NDA specifically.
Scavenging activity was measured by the extent of
inhibition of bleaching in the presence and absence of the
Figure 1: Standard curve of different concentrations (mg/ml) of Gallic acid and their respective optical densities at
725nm.
Figure 2: Standard curve of different concentrations (mg/ml) of Rutin and their respective optical density at 506nm.
y = 0.0801x - 0.0109
R² = 0.9992
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
0 2 4 6 8 10 12 14
Absorbance(nm)
Different Concentrations of Rutin (mg/ml)
Standard curve of Rutin
Sowjanya et al. / Determination of Total…
IJPPR, Volume 9, Issue 6: June 2017 Page 817
extract solutions33. In this method to the different
concentrations of the crude extracts of Amaranthus
tricolor 500 to 1000µg, respectively dissolved in distilled
DMSO (or) any solvent, alcohol add ferric chloride
(0.1mM, 0.5ml), EDTA (0.1mM, 0.5ml), ascorbic
acid(0.1mM, 0.5ml), hydrogen peroxide(2mM, 0.5ml) and
p-nitroso dimethyl aniline (0.01mM, 0.5ml) in phosphate
buffer (PH 7.4, 20Mm) to make a total volume of 3ml.The
absorbance was measured at 700nm. Increased absorbance
of the reaction mixture indicated increased antioxidant
activity. Ascorbic acid was used as a reference standard.
The percentage of absorbance by this method can be
calculated by using the following formula.
p-NDA radical scavenging activity(%) = (Ac-As)/Ac x100
Where, Ac is absorbance of control, As is absorbance of
sample or standard.
RESULTS AND DISCUSSION
The present study revealed the presence of carbohydrates,
proteins, aminoacids, steroids, cardiac glycosides,
alkaloids, tannins and flavonoids. The results of
preliminary phytochemical screening was reported in
table:1. The presence of various phytoconstituents in plant
parts received attention because of their biological
activities. The presence of tannins and flavonoids in the
plants exhibited various biological activities like
antibacterial, antifungal, antioxidant and anthelmintic. The
total phenolic content in MEAT (19.4) is maximum, CEAT
(16.7) moderate and AEAT (13.8) is minimum. The tannin
content is 7.2, 5.9, 4.5 mg of GAE/gm of extract in MEAT,
CEAT and AEAT respectively. The total phenolic content
and tannin content were estimated through the standard
calibration curve of gallic acid (Fig:1).
Flavonoids as one of the most diverse and widespread
group of natural compounds are probably the most
important natural phenols. These compounds possess a
broad spectrum of chemical and biological activities
including radical scavenging properties. The flavonoid
content in MEAT is 4.5, CEAT is 3.8 and AEAT is 3.2 mg
of rutin/gm of extract. The flavonoid content was
estimated through the standard calibration curve of rutin
(Fig:2).The results of total phenolics, tannin and flavonoid
contents were represented in table:2.
The plant showed better antioxidant potential when
compare to standard ascorbic acid by DPPH scavenging
assay method. The leaf extracts strongly scavenge in dose
dependent manner (Fig:3). The IC50 values of MEAT,
CEAT, AEAT & ASA was found to be 290, 657, 830 &
130μg/ml respectively (table:3). In scavenging hydroxyl
radical by p-NDA method the MEAT showed maximum
activity, CEAT showed moderate activity and AEAT
showed minimum activity (Fig:4). The results were
reported in table:4.
Statistical analysis
Data were expressed as mean and standard deviation (SD).
Table 3: % inhibition of DPPH radical by MEAT, CEAT, AEAT & ASA.
Extract
Quantity in micrograms(µg/ml)
IC50
100
200
300
400
500
MEAT
32.58±0.85
42.37±0.22
51.55±1.27
57.34±1.06
63.92±0.54
290
CEAT
11.93±0.75
17.65±0.47
26.28±0.74
33.50±0.70
39.75±0.47
657
AEAT
5.70±0.47
9.75±0.53
15.72±0.63
19.61±0.36
23.37±0.35
830
ASA
48.88±0.36
59.83±0.65
72.93±0.95
81.86±0.55
85.96±0.25
130
Values are mean ± SD of triplicates
AT: Amaranthus tricolor (L); MEAT: Methanolic Extract of AT; CEAT: Chloroform Extract of AT; AEAT:
Aqueous Extract of AT; ASA: Ascorbic Acid
100 200 300 400 500
0
2 0
4 0
6 0
8 0
100
% i n h ib it i o n o f D P P H
C o n c e n tr a t i o n m c g / m l
A b s o r b a n c e
M E A T
C E A T
A E A T
ASA
Figure 3: Graphical representation of % inhibition of DPPH radical by MEAT, CEAT, AEAT & ASA.
Sowjanya et al. / Determination of Total…
IJPPR, Volume 9, Issue 6: June 2017 Page 818
Statistical analysis of parametric data for IC50 was carried
out using graph prism pad software.
CONCLUSION
The present study supports the use of A.tricolor as green
leafy vegetable, which may be due to its antioxidant
property. The presence of various phytochemicals was
responsible for high antioxidant activity. However, further
studies are required to confirm the same. The plant merits
further investigation to isolate its active constituents and to
establish the activity in animal models.
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Extract
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ASA
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64.16±0.35
>1000
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Extract of AT; ASA: Ascorbic Acid
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0
2 0
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% in h ib i ti o n b y p - N D A
C o n c e n t r a t io n m c g / m l
A b s o r b a n c e
M E A T
C E A T
A E A T
ASA
Figure 4: Graphical representation of % inhibition of p NDA radical by MEAT, CEAT & AEAT.
Sowjanya et al. / Determination of Total…
IJPPR, Volume 9, Issue 6: June 2017 Page 819
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