Lasbela, U. J. Sci. Techl., vol. , pp. 92-100, 2015
ISSN 2306-8256
RESEARCH ARTICLE
Study of Phytochemistry and Antioxidant Activity of Hertia
intermedia (Boiss.) Flowers of Balochistan
Samiullah1*, Salma Khalid2, Rasool Bakhsh Tareen1, Naqeebullah khan1, Samina Aslam2,
Fariha Alam2, and Saleha Suleman2
1
2
University of Balochistan, Quetta, Pakistan
SBKWU, Quetta, Balochistan, Pakistan
Abstract:- Hertia intermedia (Boiss) O. Kuntze has been used as a traditional remedy for pain killer. The
sample was extracted with methanol and used for the evaluation of phytochemical constituents and its antioxidant
potential. The phytochemical compounds present in the sample were tannins, terpenoids, flavonoids, steroids,
coumarins and carbohydrates. Total phenolic and total flavonoid contents investigated for the tested sample were 211.8
mg tannic acid equivalent/g and 162.7 mg quercetin equivalent/g extract respectively. The antioxidant assays applied
for this study were DPPH free radical scavenging assay, Ferric-ion reducing antioxidant power assay, Reducing power
assay and Phosphomolybdate assay. The results on the basis of antioxidant capacity methods and high contents of
phenols and flavonoids of the flower extract suggest that the sample extract can be a valuable source of antioxidant.
Keywords: Hertia intermedia, methanolic extract, TFC, TPC, DPPH, FRAP, phosphomolybdenum,
alkaloids to terpenoids and acetogenins to
different phenol (Khan et al., 2011; Onuekwusi
et al., 2014). These phytochemicals differ in the
qualitative and quantitative distribution from
plant to plant and part to part of plants.
The phenolic compounds are found in
high concentrations as compared to the alkaloids
whereas alkaloids are present in high amounts in
storage tissues (roots, fruits and seeds) of plants
relative to the green leaves. The synthesis of
aromatic compounds (mostly phenols or their
oxygen-substituted derivatives) has been the
limitless ability of plants. The isolation of
12,000 secondary metabolites (most of the
natural products) has been reported so far that
serve as plant defense mechanism against
predation by microorganisms, insects and
herbivores Khan et al., 2011).
Biomolecules (protein, lipid, amino
acids and DNA) can be oxidized by the
accumulation of excess free radicals (FRs)
leading to cellular damage and can induce
various diseases (Basri and Setty , 2015).
Oxidative stress is caused by an imbalance
between reactive oxygen species (ROS) and
antioxidants (AO) and it has been related to
cancer, cardiovascular diseases(Kumar et al,
2013),ageing, atherosclerosis, arthritis, CNS
INTRODUCTION
An absolute resource of remedies has
been provided by the nature in the form of
medicinal plants to be used for the health care
and treatment and is the best companion of
pharmacy. The plants globally constitute a single
larger functional group and are most effective in
action as herbal drugs without any side effects
(Khan et al, 2011). According to an estimate,
just from 95 plant species through the world are
prescribed for 120 or so plant-based drugs. In
rural areas of many developing countries the use
of medicinal plants (or their parts) is well known
as folk medicines (Onuekwusi et al., 2014).
The studies of folk medicines should be
more intensified, in order to encourage the use
of herbal medicines and determination of their
valuable potentials (Khan et al, 2011). The two
classes of chemicals present universally in all
plants are primary and secondary metabolites.
The primary metabolites consist of proteins,
amino acids, sugars, purines and pyrimidines of
nucleic acids, chlorophylls etc., whereas
secondary bioactive chemicals include
*Corresponding author: sami435889@yahoo.com
92
Based on above apprehensions, the present study
is an effort to screen the methanolic extract of
flowers of H. intermedia for phytochemicals and
to assess the antioxidant and antibacterial
activities.
damage, degenerative diseases(Shahid-Ud-Daula
et al., 2015), Parkinson’s disease, cataracts and
inflammatory diseases. Antioxidant serves as
hydrogen donor or electron donor and help in
reducing the oxidative stress. Phenols have
antioxidant activity, which allow them to quench
FRs (Basri and Setty, 2015).
Synthetic
antioxidants
(such
as
butylated
hydroxyanisole
[BHA],
butlylatedhydroxytoluene [BHT], etc.) have
certain disadvantages and harmful side effects to
human health. In recent years, the novel
antioxidant substances derived from plant
tissuesare finding great interest for use in human
diet and drug industry (Shahid-Ud-Daula et al.,
2015).
The Gorgena of Mastung and Shabaan
of Quetta city of Balochistan province has
provided numerous herbal plants. Thus, there is
need to carry out the proper identification of the
wild species, their antioxidant activity and to
know their phytochemical constituents. This
valuable information will be essential later for
conservation purposes and advanced drug
manufacturing in Pakistan. We carried out a
research work on an indigenous plant namely
Hertia intermedia of Composite (also called
Asteracea) family. This indigenous plant locally
called Manguli has been used as a remedy of
pain-killer (for headache, stomach problems and
during menstrual cycle) in the hilly areas of
Pakistan (Tareen et al, 2010).
The Hertia genus belongs to the
Compositae family and Senecioneae tribe. The
genus Hertia consists of twelve species
distributed all over South- and North-Africa and
South West Asia. Hertia intermedia (Boiss.) O.
Kuntze is one of the species of the genus Hertia,
which grows wildly in hilly regions
ofBalochistan and Khyber Pakhtunkhua
provinces of Pakistan. It is also distributed
westward towards Iran. Hertia intermedia is also
called as Othonnopsis intermedia and found in
Balochistan mostly in Quetta, Koeie, Chaman,
Kanozai-Moorga, and Wazir, and is also found
in Kurram and the regions below Parachinar.
These are small shrubs, grow up to 30-50 cm,
with beautiful and attractive yellow flowers, and
are used as a remedy of painkiller in the hilly
areas of Pakistan (Akhgar et al, 2012; Malik et
al, 2011; Yasmeen et al., 2009).
MATERIALS AND METHODS
Reagents and Chemicals
The reagents and chemicals used were of
analytical grade and were products of MERCK
Darmstadt, Germany, Applichem Darmstadt,
Germany, Scharlab S.L., Spain, BDH Chemicals
Ltd, Poole, England, and SIGMA-ALDRICH St.
Louis, USA unless otherwise stated.
Sample Collection and Extract Preparation
The flowers of the plantused for this
study were collected from Gorgena and
Shabaan, Balochistan. H. intermedia was
taxonomically identified by Dr. Rasool Bakhsh
Tareen, Taxonomist and Dean of Life Sciences,
University of Balochistan, Quetta. The fresh
flowers of H. intermedia were screened to get
rid of the bad ones, washed, shade-dried at room
temperature (22±5) C for 20 days and was
extracted with methanol being soaked and
stirred at room temperature (21±5) C for one
month. The macerated sample was filtered prior
to evaporation of methanol below 50C under
reduced pressure through rotary evaporator
(IKA-WERKE GMBH & Co. KG Staufen,
Germany). Subsequently, the greenish-black (25
g concentrated extract) was stored below 4C
until further analysis.
Qualitative Tests of Phytochemicals
The crude methanolic extract of H.
intermedia flowers was subjected to different
qualitative chemical tests for the screening and
identification of secondary metabolites using
standard procedures of Sofowara (1993), Trease
and Evans (1989) and Harborne (1973) (10-12)
as illustrated in Table-1.
Quantitative Tests of Phytochemicals
Total Phenolic Content Estimation in the Extract
The total phenolic amount in the
methanolic extract was estimated by using
93
Folin-Ciocalteau (FC) reagent based method as
formerly described by Uddinet al.(2013)with
minor modifications. Briefly, to 0.5 ml of extract
Table-1. Qualitative analysis of phytochemical constituents for sample extract
Secondary Metabolites
Tannins
Terpenoids
Saponins
Flavonoids
Flavonoids
Alkaloids
Alkaloids
Alkaloids
Alkaloids
Steroids
Coumarins
Emodins
Phlobatannins
Anthraquinones
Cardiac Glycosides
Carbohydrates
Preliminary Test
Braymer’s Test:0.2 g extract + 5 ml H2O + 1-2
drops FeCl3 (0.1 %)
Salkowski Test:0.2 g extract + 2 ml chloroform
+ 2-3 drops conc. H2SO4
Frothing Test:0.2 g extract + 4 ml H2O
a) Shinoda Test: extract + 5-6 Mg fragments +
2-3 drops conc. HCl
b) Alkaline Reagent Test:
i. extract + 2ml NaOH (2%)
ii. soln. (i) + 2-3 drops dil. Acid
i. Picric acid test: 0.2 g extracts +3 ml hexane
+ shake + filter + 5 ml (2%) HCl + heat +
few drops picric acid
ii. Mayer’s and Wagner’s test: extract + 2 ml
(1%) HCl + heat gently + Mayer’s &
Wagner’s reagent
iii. Hager’s test: 2 ml extract + few drops
Hager’s reagent
iv. Dragendorff’s test: 2 ml extract + 0.2 ml
(1%) HCl + 1 ml Dragendorff’s reagent
Libermann-Burchard: extract + 2 ml
CH3COOH + cool + few drops H2SO4
2 ml extract + 3 ml (10%) NaOH
2 ml extract + 2 ml NH4OH + 3 ml C6H6
extract + 1 % HCl + boil
extract + 6 ml (1%) HCl + filter + 5 ml C6H6 +
filter + 2 ml (10%) NH3 solution + shake
Keller-Killiani Test: extract + 2 ml glacial acetic
acid + few drops FeCl3 + 1 ml H2SO4
Molisch Test: extract+ 5 ml H2O + few drops
Ethanolic α-naphthol (20%) + 1 ml conc. H2SO4
(1 mg/ml extract concentration), FC phenol
reagent (0.5 ml, ten times diluted) was added
and allowed to stand for 5 min at room
temperature. Subsequently, a 7 % (w/v) Na2CO3
solution (5 ml) was added in the mixture and
after keeping in dark for 90 min, the absrorbance
of developed color was measured at 750 nm.
Tannic acid (50-250 mg/L) solutions were used
as standard for extrapolation of calibration curve
(fig-1)by
using
the
linear
equation
y=0.0372x+0.1266 (R2=0.9985) and the TPC
was expressed as tannic acid equivalents (TAE
mg/g of compound extracted).
Observation
Green colored precipitates
Deep red coloration
Froth formation
Pink scarlet color
Intense yellow color
Yellow color vanishes
Yellow color precipitates
Turbidity appears
Yellow precipitates
Orange brown precipitates
Violet to blue steroidal ring at
interface
Yellow coloration
Red coloration
Red color precipitates
Pink, violet or red color
Brown ring at interface
Violet ring at interface
Aluminum chloride colorimetric assay was used
to measure total flavonoids described as per
(Kaur and Mondal, 2014) using quercetin as
positive control for the generation of calibration
curve (Onuekwusi et al., 2014). Precisely, an
aliquot of 1 ml of sample extract was mixed with
4 ml of distilled water and 0.3 ml of sodium
nitrite (5%) in a 10 ml volumetric flask. To this
0.3 ml of AlCl3 (10%) was added after
incubation of 5 min at room temperature. The
reaction mixture was further allowed to stand for
6 min and then 2 ml of sodium hydroxide (1M
solution) was added and immediately the total
volume was made up to 10 ml with distilled
water. Spectrophotometrically the absorbance of
the mixture was measured at 510 nm.
Total Flavonoid Content Estimation in the
Extract
94
Absorbance, 750 nm
0.35
reduction caused by DPPH radicals was
measured spectrophotometrically at 517 nm.
Control was prepared by adding DPPH (0.5 ml)
to 1.5 ml ethanol in place of flower extract. The
radical scavenging effect (RSE) was calculated
by using following equation and expressed in
RSE % values:
RSE (%) = [1 – ATE / ADS] * 100
Where ATE is the absorbance of test extract and
standard and ADS is the absorbance of DPPH
solutions in the absence of sample extract. A
greater anti-oxidant activity was indicated by the
lower absorbance values.
Absorban
ce
0.3
0.25
Linear
(Absorba
nce)
0.2
y =0.0372x+0.1266
R² = 0.9985
0.15
0.1
0.05
0
50
100
150
200
250
Concentration, mg/L
Fig-1. Calibration curve of Tannic acid
Ferric-ion Reducing Antioxidant Power (FRAP)
Activity
The ferric-ion reducing ability of plasma FRAP
method of Benzie and Strain (1996) with minor
modifications (Lahouar et al., 2014) was used to
determine the total antioxidant potential of
sample extract as a measure of anti-oxidant
power. In-brief, the freshly prepared FRAP
reagent consisted of 300 mM acetate buffer (pH
3.6), 10 mM TPTZ (2,4,6-tri[2-pyridyl] striazine in 40 mMHCl) plus 20 mM FeCl3.7H2O
in a ratio of 10:1:1 (v/v/v) and warmed to 37C
prior to use. A volume of 0.1 ml of sample
infusion at various concentrations (0.05-0.8
mg/L) and 0.3 ml of de-ionized water were
added to 3 ml of working FRAP reagent and the
reaction mixture was incubated at 37C in the
dark for 30 min. The increase in absorbance
was monitored at 593 nm due to the reduction of
colorless Fe (III)-TPTZ complex to a blue
colored Fe (II)-TPTZ complex in the presence of
antioxidants. The calibration curve was
constructed using various concentrations of
FeSO4.7H2O. The reaction signal given by a Fe+2
solutions was referenced to calculate the
reducing ability of the test extract. The resulting
FRAP values were expressed as mmol of Fe+2/g
of dried matter.
Absorbance, 510 nm
Based on the standard curve (fig-2) using the
following
equation:
y=0.0504x-0.042
(R2=0.9992) was used to estimate the total
flavonoids and results were expressed as
milligrams of quercetin equivalent (QE) per
gram of compound extracted.
0.25
0.2
y = 0.0504x - 0.042
R² = 0.9992
0.15
Absorba
nce
0.1
0.05
0
0.05 0.2
0.4
0.6
0.8
Linear
(Absorba
nce)
Concentration, mg/L
Fig-2.Calibration curve of Quercetin
Antioxidant Assays
Scavenging Ability of Stable DPPH Radical
The antioxidant property was appraised
by using 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging effect for both test extract
and standard based on the previously
documented method(Mohan et al., 2014). In this
assay, the natural antioxidant, ascorbic acid was
used as reference for comparison. Exactly, a
volume of 0.5 ml of freshly prepared DPPH
solution (0.004%) was mixed with 1.5 ml of
each concentration (0.05-0.8 mg/L) of test
extract. Prior to incubation for 25 minutes in
dark, the reaction mixture was shaken
vigorously. The reaction solution’s color
Reducing Power Antioxidant Assay
The ferric-reducing power of sample
extract and positive control (ascorbic acid) were
assessed by using the method described by
Thakralet al. (2010). Different concentrations of
the crude extract were mixed with a volume of
0.75 ml phosphate buffer (pH 6.6, 0.2 M) plus 1
% potassium ferricyanide (0.75 ml), followed by
95
recorded at 695 nm against blank. For blank
appropriate volume of solvent used for sample
was combined with 1 ml of reagent solution and
incubated under same conditions. The
antioxidant activity was expressed as mg of
equivalent of ascorbic acid, for unknown
composition of sample.
keeping the mixture for 20 min in a water bath at
50C. An equal volume of 10% TCA
(trichloroacetic acid) was added then to stop the
reaction and centrifuged for 10 min at 3000
r/min to collect the supernatant of the solution.
To 1.5 ml distilled water and 0.1 ml freshly
prepared FeCl3 solution (0.1%), 1.5 ml of
supernatant was mixed. The absorption maxima
was recorded at 700 nm: increase in absorbance
indicated stronger reducing power.
RESULTS AND DISCUSSION
Phytochemical characteristics
The phytochemical screening of
medicinal plants revealed the presence of
secondary metabolites which are known to
exhibit curative as well as physiologoical
properties(Yadav &Agarwala, 2011).
In table-2 the phytochemical characteristics of
H.
intermedia
(Boiss.)
flowers
are
summarized.From the results, it can be seen that,
tannins, terpenoids, flavonoids, steroids,
coumarins and carbohydrates were detected in
tested sample, whereas, saponins, alkaloids,
emodins, phlobatannins, anthraquinones and
cardiac glycosides were absent. The absence of
these phytocompounds in the flower extract
suggesting thereby the absence of therapeutic
properties associated with them.
Phosphomolybdenum(PM) Assay
The total antioxidant ability method is
based on the formation of a green
phosphomolybdenum [Mo (V)] complex due to
the reduction of Mo (VI) by the sample analyte
at acidic pH. The spectrophotometric method of
Prieto et al.(1999) was usedfor the quantitative
measurement of total antioxidant capacity. At
various concentrations, the sample solution (0.1
ml) was taken and combined with 1 ml of
molybdatereagent solution (0.6 M H2SO4, 28
mM sodium phosphate and 4 mM ammonium
molybdate mixture). Subsequently, the tubes
were capped and incubated for 90 min in water
bath at 95C. The reaction mixtures were
normalized to room temperature. After
normalizing, the absorbance of the mixture was
Table-2. The Phytochemical Screening of Methanolic extract of H. intermedia
Phytocompounds
Preliminary test
Methanolic extract
Tannins
Terpenoids
Braymer’s
Salkowski
Saponins
Frothing
(-)ve
Flavonoids
Shinoda
Alkaline-reagent test
(+)ve
(+)ve
Alkaloids
Picric acid
(-)ve
Mayer’s and Wagner’s
(-)ve
Hager’s
Dragendorff’s
(-)ve
(-)ve
Libermann-Burchard
(+)ve
Steroids
(+)ve
(+)ve
Coumarins
(+)ve
Emodins
Phlobatannins
Precipitate
(-)ve
(-)ve
Anthraquinones
Borntrager’s
(-)ve
Cardiac glycosides
Keller-Killiani
(-)ve
Carbohydrates
Molisch’s
(+)ve
(+)ve: Present; (-)ve: Absent
96
absorption is recorded by the blue coloration. It
is proportional to the total amount of phenolic
compounds originally present (Narayanaswamy
and Balakrishnan, 2011). The amount of TPC in
H. intermedia flowers was found to be 211.8 mg
TAE/g of dried matter reported in fig-3.
Secondary
metabolites
such
as
terpenoids have been reported to possess various
activities including antibacterial, antimalarial,
antiviral, anti-inflammatory and inhibition of
cholesterol synthesized in animals. Steroids are
responsible for wide variety of effects such as
cardiotonic activities (Wadood et al, 2013),
antibacterial properties (Yadav &Agarwala,
2011) and a natural alternative to treat age- and
disease-related muscle loss (Shahid-Ud-Daula et
al, 2015).Dietary flavonoids have been
recommended to oppose the coronary heartdisease (Wadood et al, 2013) and exhibit a vast
range of biological activities including
antiallergic, antithrombotic and vasodilatory
activites(Shahid-Ud-Daula et al, 2015).Studies
have revealed that tannins possess amazing
stringent properties including the healing of
wounds and inflamed mucous membranes.
Carbohydrates and coumarins are important
dietary supplements, contained in medicinal
plants are known to have valuable action on
immune system. The antimicrobial and antiinflammatory properties of coumarins can be
recommended to be helpful for hyperproliferative skin diseases (Yadav et al, 2014).
Hence, the identification of significant reservoir
of bioactive compounds via preliminary
phytochemical screening tests may lead to future
medicine bank.
mg/g dried matter
250
211.8
200
162.7
150
100.21
100
TPC
TFC
50
PM
0
TPC
TFC
PM
Methanolic extract
Fig-3. The amount of total phenols, total flavonoids and
phosphomolybdenum assay in H. intermedia flowers
Flavonoids are the naturally occurring
polyphenolic compounds including flavones,
flavonols, flavanols, chalcones, flavanones, isoflavonoids,
neo-flavonoids,
bi-flavonoids,
flavanonols and anthocyanins(Shahid-Ud-Daula
et al, 2015). Studies on derivatives of flavonoids
have revealed a wide range of biological effects,
which also inhibit enzymes (such as aldose
reductase and xanthine oxidase)(Mohan et al,
2014) and hinder the diffusion of free radicals,
thereby restricting the peroxidation reaction
(Shahid-Ud-Daula et al., 2015).
The total amount of flavonoid content
for methanolic extract was determined by the
aluminium chloride colorimetric assay using
quercetin as reference. Stable complexes of
aluminium chloride are formed with the C-4
keto groups and either the C-3/C-5 hydroxyl
group of flavones and flavonols and it also
forms liable complexes with ortho-dihydroxide
groups in A or B rings of flavonoids. Fig-3
demonstrates the total flavonoid content of
sample. It was found that TFC of the methnolic
extract of Hertia intermedia flowers was 162.7
mg QE/g of dried extract.The high contents of
phenols and flavonoids in the extracts are related
to the antioxidant activity by neutralizing the
free radicals (Mohan et al., 2014).
Total Phenolics and Total Flavonoids Content
Phenolic compounds (act as primary
antioxidants) are able to protect human body
from free radicals because their hydroxyl groups
possess
scavenging
property.
These
phytochemicals are derived from phenylalanine
and tyrosine(Saeed et al, 2012). They play an
important role in curing major diseases such as
diabetes, osteoporosis, neuro-degenerative and
cardiovascular diseases(Shahid-Ud-Daula et al,
2015)and their oxidative products confer
inhibitory
action
on
various
enzyme
systems(Aparadh et al, 2012).
Folin-Ciocalteu reagent was used to determine
the total amount of phenols using tannic acid as
standard. A mixture of phosphotungstic acid and
phosphomolybdic acid forming the reagent
oxidizes the phenols and hence, reduced to a
mixture of blue oxides of tungsten and
molybdenum. At 750 nm, a maximum
97
extract concentration (Patel et al, 2010). The
hydrogen donating ability of phenols and
flavonoids might be the reason of the antiradicalizing property of plant extract
(Narayanaswamy&Balakrishnan, 2011).
Antioxidant Assay
Antioxidant activity methods can be
categorized as single-electron transfer (ST) and
hydrogen-atom transfer (HT) based methods.
HT methods are mainly kinetic based and
includes a competitive reaction scheme among
antioxidant and substrate for free radicals
thermally generated whereas ST methods
measure the reduction of an oxidant which
decolorize when reduced (Phatak&Hendre,
2014).
100
% RSE
80
60
40
20
Free-radical scavenging assay
The free radicals contain one or more
unpaired electrons which make them highly
unstable. They extract electrons from other
molecules in order to gain stability. Therefore,
radical scavenging activities are essential in
biological systems due to the adverse role of free
radicals in a wide range of pathological
manifestations. The free radicals generated in
biological system are neutralized by antioxidants
which protect us from several diseases.
The widely accepted method for
determining antioxidant ability of plant extracts
is the assay based on the DPPH free radical. It is
the stable, organic nitrogen centered radical
which produces purple color in ethanol (Mohan
et al, 2014). In this assay the hydrogen donor is
an antioxidant which makes this method simple
and extremely sensitive. The disappearance of
DPPH radical in sample extract is monitored
with UV/Vis spectrophotometer.
The DPPH radical becomes a stable
diamagnetic molecule as it captures an electron
or absorbs a hydrogen atom. The antioxidants
reducing the DPPH radical results in
discoloration from deep purple to pale yellow.
The lower the absorbance of reaction mixture,
the greater is the discoloration of DPPH ethanol
solution that shows the notable free radical
scavenging ability (Morales and Paredes, 2014).
The RSE (%) values are illustrated in fig-4. The
results of this study suggest the increase in
concentration of extract and standard notably
increases the DPPH radical scavenging effect
and therefore, are said to be highly dependent on
0
0.05
0.2
0.4
0.6
0.8
Concentration, mg/L
Ascorbic acid
intermedia
H.H.
intermedia
Fig-4. The % radical scavenging effect (RSE) of
methanolic extract of H. intermedia flowers
FRAP activity
The FRAP method was used to assess
the antioxidant property as it is simple, rapid and
reliable method to perform. In addition, the
reaction is reproducible and linearly related to
molar concentration of the antioxidants present
(Uddin et al., 2013). Although FRAP assay was
found to react slowly with some antioxidants
(such as gluthathione) (Rabeta and NurFaraniza,
2013), but can still be used to determine
antioxidant capacity in vast range of biological
samples to pure compounds (Katalinic et al.,
2006).
Redox reactions can be explained as the
inactivation of oxidants by reductants, in which
one oxidant (reaction species) is reduced at the
cost of the oxidation of another antioxidant
(reductant). In the reaction medium the
antioxidant capacity of any substance is
measured as reducing ability by the FRAP assay
(Lahouar et al, 2014). Ferric reducing
antioxidant power of examined methanolic
extract of H. intermedia was 1.3 mMFeII/g
extract (Table-3).
98
Table-3.Quantitative values of PM and FRAP assay
S. No.
Antioxidant parameters
Line of regression
1.
Phosphomolybdenum assay
y=0.0333x+0.1205
2.
Ferric-reducing antioxidant potential
y = 0.0649x + 0.1483
a
Ascorbic acid equivalent
Measurement of reducing power
The reducing power (RP) of H.
intermedia methanolic extract was collated with
the standard ascorbic acid (Fig-5). The RP was
found to be correlated with increasing
absorbance at 700 nm with increasing
concentration. In this assay, the reduction of
FeIII-ferricyanide complex (yellow) to FeII
(Prussian-blue) formwould result by electron
donation by the sample extract (reducers) and
this is reflected in present study. The putative
reductants have been attributed to reduce the
oxidized
intermediates
of
peroxides
decomposition and act as primary or secondary
antioxidant substances (Mohan et al, 2014).
Ascorbic acid
Mo (V) is evaluated by phosphomolybdate
scavenging assay,thereby providing direct
estimation of antioxidant reducing capacity.It is
used to quantitatively assess the reduction
reaction degree among antioxidant, oxidant and
molybdenum ligand, by formation of green
complex without the involvement of free metal
ions (Phatak and Hendre, (2014).
CONCLUSION
The results obtained from different
antioxidant potential assays, support the view
that methanolic extract of H. intermedia flowers
has shown a significant source of potent
antioxidants. This may find efficient role as a
therapeutic agent in some of the diseases.
Besides this, it can also be considered as herbal
source in pharmacy and can be utilized in food
industry. However, further detailed studies on
screening of biological activities is required to
establish the scientific basis.
intermedia
H.H.
intermedia
1.6
Absorbance, 700 nm
Quantitative amount
100.21mg AAEa/g extract
1.3 mM Fe+2/g extract
1.4
1.2
1
0.8
ACKNOWLEDGEMENT
0.6
0.4
The authors are thankful to SBK
Women University and University of
Balochistan for providing assistance and
facilities to conduct this research.
0.2
0
0.05
0.2
0.4
0.6
0.8
Concentration, mg/L
REFERENCES
Fig-5. The reducing power of methanolic extract of H.
intermedia flowers
Akhgar, M. R., Ghazanfari, D. and Shariatifar, M., 2012.
Chemical Composition of the Essential Oil of
Hertia intermedia (Boiss.) O. Kuntze from Iran.
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