Antioxidant activity of chloroform extract of root parts of Inula racemosa was evaluated by measuring the scavenging activity of the extract on stable 2, 2-diphenyl-1-picryl hydrazyl radical (DPPH) exhibiting an interesting antioxidant profile. The reducing power was determined using a modification of Zou method. Metal chelating activity of the extract was determined at concentrations of 20, 30, 50 and 100 µg/mL, taking citric acid as standard. The extract displayed significant activity

Banday, Javid Ahmad

Bhat, Khursheed Ahmad

Chandra, Suresh

Lateef, Romaisa

English

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To Chemistry  Journal  Vol 1 No 2 (2018)  ISSN: 2581-7507                                              http://purkh.com/index.php/tochem 
179 
Antioxidant Activity of Chloroform Extract of  Inula Racemosa From Kashmir Himalayas 
Romaisa Lateef1, 2, Khursheed A Bhat2, Suresh Chandra3 and Javid A Banday1* 
 1Department of Chemistry, National Institute of Technology Srinagar-190006, India 
2Bioorganic Chemistry Division, Indian Institute of Integrative Medicine (CSIR) Srinagar-190005, India. 
3Genetic Resources & Agrotechnology, Indian Institute of Integrative Medicine (CSIR) Jammu-180001, India 
javidbanday@nitsri.net, romaiiim@gmail.com, kabhat@iiim.res.in,  schandra@iiim.res.in 
Abstract 
Antioxidant activity of chloroform extract of root parts of Inula racemosa was evaluated by measuring the 
scavenging activity of the extract  on stable 2, 2-diphenyl-1-picryl hydrazyl radical (DPPH),exhibiting an 
interesting antioxidant profile. The reducing power was determined using a modification of Zou method. 
Metal chelating activity of the extract was determined at concentrations of 20, 30, 50 and 100 µg/mL, taking 
citric acid as standard. The extract displayed significant activity. 
Key words: Inula racemosa, Chloroform extract, Antioxidant activity, Reducing power, Metal   chelating activity.  
Introduction 
Inula racemosa is distributed in temperate alpine Himalayas at an altitude of 1,500- 4,200 from Kashmir to 
Kumaon, Afghanistan to Central Nepal. It occurs wild among strong alpine scrub vegetation in the cold arid 
habitat of NW Himalayas between 2,700-3,500 m in the eastern Ladakh (Leh) region of Kashmir. Domesticated 
forms of this incipient cultigen are cultivated on borders of agricultural fields of wheat, barley and buckwheat 
both in Kashmir and Lahaul valley of Himachal Pradesh. (Wealth of India -Raw Materials, 1959) 
The roots of the plant have a dull brownish skin with yellowish colour inside. They possess a sweet and 
somewhat camphoraceous odour and have a bitter taste (Chopra RN, et al, 1956). Pushkarmool commercially 
is a very important medicinal plant of the North western Himalayas. The plant is used in Ayurveda as an 
expectorant and resolvent in indurations. Considered a ‘Rasayana’ (rejuvenator, immunomodulator) by 
Ayurvedic physicians, the drug according to Bhavaprakasha (Bhavaprakasha Bhavaprakash Nighantu, 1961) is 
bitter pungent in taste. When administered it mitigates vatakapha jwara (fever caused by vata pitta imbalance), 
sotha (swelling), arachi (anorexia), swasa (breathlessness) and parswasoola (pain in the sides of the chest). 
The root is medicinal and considered a specific for cough, dyspnea, asthma, pleurisy, tuberculosis and chest 
pain especially pre cordial pain. The aqueous extract of the fresh or dry roots is given orally in rheumatic pains 
and liver problems. Externally a paste or liniment is used for relieving pain. The root is also used in veterinary 
medicine as a tonic (Wealth of India-Raw Materials, 2002). The root forms an important ingredient of several 
polyherbal formulations for heart diseases and inflammatory conditions of spleen and liver (The Ayurvedic 
pharmacopoeia of India Part 1 Second edition, 1978). Along with Commiphora mukul, the drug combination 
called ‘pushkar guggulu’ is a popular anti obesity, hypolipidemic indicated in cardiac ailments. 
Inula racemosa is used in Chinese medicine for abdominal distension and pain, acute enteritis and bacillary 
dysentery (Tsarong, T.J, 1994). There are so many medicinal properties reported in literature pertaining to 
various Inula species. I. racemosa contain oxygenated allantolides e.g. 4-(15)-α-epoxy isotelekin and perhydro 
epoxyalantolide (Goyai R, et al, 1990). It also contains sesquiterpene lactones and lignans. (Tan RX, et al, 1998). 
I. britanica has been reported to contain polyphenolic constituents, exdate flavonoids (So RK, et al, 2002). 
Pulchellin C, Kaurane glycosides (Yu S, et al,1996) and aromatic esters (Anthonsem, et al,1971 ) . I. germanica 
has been reported to contain sesquiterpenoid lactones (Konovalova OA, et al, 1974). I. crithmoides contains 
methoxylated flavonols, chlorinated thymol derivatives, sesquiterpene lactones (Mohamed AM, et al, 1985). I. 
To Chemistry  Journal  Vol 1 No 2 (2018)  ISSN: 2581-7507                                              http://purkh.com/index.php/tochem 
180 
grantiodes contains triterpenes, sitosteryl glucosides, olefins, flavones, and fatty acids (Burdi DK, et al,1992) .I. 
caspica has been reported to possess incapsin, a new sesquiterpene lactone and britanin (Petrisheva; LP,1984) 
I. Indica contains germacranolides (Bhimsen AN, et al, 1981).I. royleana contain quinones, abietane 
triterpenoids, β-caryophyllene, exoxides, diterpenoids (Edwards OE, et al,1962). I.viscosa has been reported to 
contain sesquiterpenoids (Musa H, et al,1998), eucalptol, thymol content, eudesmane acids, oxygenated 
nerolidol esters, carboxy eudesmadiene (Lauro L,et al,1990). 
MATERIALS AND METHODS 
Plant Collection 
The plant material of Inula  racemosa was collected in the mid of July 2013 from Indian Institute of Integrative 
Medicine  (IIIM) Srinagar (Kashmir, India) and authenticated by Prof. A.R. Naqshi and Dr. Anzar Khuroo 
(University of Kashmir) and by comparison of the collected sample with that growing in Department of Plant 
Taxonomy, University of Kashmir. A voucher specimen (voucher number: IIIM 1125/13) of the plant was 
deposited in the herbarium of the Institute. 
Extraction 
The roots of Inula racemosa were obtained from the laboratory stock. The roots were dried and powdered. The 
root material of Inula racemosa (200 g) was extracted by soxhlet extraction apparatus using chloroform as the 
solvent. The soxhlet extraction was carried on for 24 h in two batches and the solvent was distilled off to 
obtain the crude extract of plant material. The chloroform extract was distilled to yield yellow oil (5 g).  
Chemicals  
2,2-diphenyl-1-picryl hydrazyl radical, Methanol, Chloroform,  α- tocopherol,  sodium phosphate buffer, 
potassium ferricyanide, trichloroacetic acid,  ferric chloride, BHT, ferrous chloride, citric acid (Qualigens Fine 
Chemicals, CDH Chemicals) were used during the study. 
Antioxidant Activity 
DPPH (1, 1-dihpenyl-2-picrylhydrazyl) Radical Scavenging Activity 
The scavenging activity of  the 1, 1-diphenyl-2-picryl hydrazyl radical (DPPH) radical was measured according 
to the standard method (Gani A,et al, 2015). The reaction mixture consists of 1 mL of 0.01%  methanolic 
solution of DPPH and 100 µL of sample solution with varying concentration (20-100 μg/mL). The volume was 
made up to 3 mL by methanol. The solution was incubated for 30 min in dark. The absorbance of sample and 
α- tocopherol as a standard was read at 517 nm. Percentage inhibition was calculated by using the formula: 
% inhibition = Acontrol - Asample/ Acontrol × 100 -------------------------------   (1) 
Where, Acontrol is the absorbance of the control and Asample is the absorbance of the extract. 
Reducing Power 
The reducing power was determined using a modification of Zou method (Oyetayo VO, et al, 2009). 100 µL 
extract of varying concentrations (20-100 μg/mL) was separately mixed with 0.2 M sodium phosphate buffer 
(pH 6.6, 2.5 mL) and 1% (w/v) aqueous potassium ferricyanide (2.5 mL). After incubated the solution for 20 min 
at 50oC, 10% (w/v) of trichloroacetic acid (2.5 mL) was added to the mixture, which was then centrifuged at 
3000 rpm for 10 min. The supernatant (2.5 mL) was diluted with deionized water (2.5 mL) and 0.1% (w/v) ferric 
chloride (0.5 mL) was added. The absorbance was measured at 700 nm and compared to BHT (standard).  
Metal Chelating (Fe+2) Activity 
To Chemistry  Journal  Vol 1 No 2 (2018)  ISSN: 2581-7507                                              http://purkh.com/index.php/tochem 
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Chelating ability was determined according to the standard method (Dinis TC, et al,1994).  The reaction 
mixture, containing 100 µL of sample with varying concentration (20-100 μg/mL), 0.5 mL ferrous chloride (2 
mM), 0.25 mL ferrozine (5 mM) and volume adjusted to 3 mL with methanol, vortexed  (mixed using a vortex 
mixer) and then incubated for 10 min at room temperature. The absorbance of the sample and citric acid 
standard was measured at 562 nm against the blank. 
% Chelation = [1− (Asample /Acontrol)×100] ------------------------------ (2) 
Where, Acontrol is the absorbance of the control and Asample is the absorbance of the extract. 
Results and Discussion 
Antioxidant Activity                      
Free radicals, at high concentration, mediate the damage of cell structures, nucleic acids, lipids and proteins. 
Examples of reactive oxygen species (ROS) induced DNA damage products involve single or double stranded 
DNA breaks, purine, pyrimidine or deoxyribose modifications. Such modifications of genetic material may 
represent the first step in mutagenesis, carcinogenesis and ageing. Free radical mediated DNA damage has 
been found in various cancer tissues. Thus, it has been hypothesized that free radical scavengers are molecules 
that could prevent or limit the damage provoked by free radicals (Valko M, et al, 2006).     
DPPH, in its radical form, has strong visible absorption and high molar extinction coefficient at 517 nm. On 
reaction with an antioxidant, the absorbance diminishes (Alkan M, et al,2008). 
The anti-oxidative effect of the extract was examined by its radical scavenging effects by measuring changes in 
absorbance of DPPH radical at 517 nm (Table 1, Fig 1). Results revealed that the DPPH scavenging ability 
shows the tendency to increase with the increase in concentration. The absorbance of sample and α- 
tocopherol, as a standard, was read at 517 nm. Percentage inhibition was calculated by using the formula: 
% inhibition = Acontrol - Asample/ Acontrol × 100 ------------------------------------ (3) 
Where, Acontrol is the absorbance of the control and Asample is the absorbance of the extract. 
          Table 1: DPPH scavenging activity (Absorbance) 
Concentration (µg/mL) Extract α-tocopherol 
20 20.91 28.70 
30 30.23 38.22 
50 46.40 50.29 
100 65.21 70.24 
0 20 40 60 80 100
-10
0
10
20
30
40
50
60
70
80
D
P
P
H
 S
ca
ve
ng
in
g 
ac
tiv
ity
 (
A
bs
or
ba
nc
e)
Concentration (µg/mL)
 Extract
 alpha tocopherol
 
Fig 1 DPPH scavenging activity of the extract at different concentrations 
To Chemistry  Journal  Vol 1 No 2 (2018)  ISSN: 2581-7507                                              http://purkh.com/index.php/tochem 
182 
Reducing Power  
In the reducing power assay, the more antioxidant compounds convert the oxidation form of iron (Fe+3) in 
ferric chloride to ferrous (Fe+2). The reducing capacity of compounds could serve as an indicator of 
antioxidant properties (Zhao H, et al, 2006) and the increasing absorbance suggests an increase in reducing 
power. Compounds  with  reducing  power  indicate  that  they  are  electron  donors  and  can  reduce  the  
oxidized  intermediates.  
The reducing power of the extract, at different concentrations, is presented in Table 2, Fig 2. The absorbance 
was measured at 700 nm and compared to butylated hydroxytoluene (BHT) as standard. The data revealed 
that the reducing power of the extract increases with the increase in concentration. 
                       Table 2: Reducing power (Absorbance) 
Concentration (µg/mL) Extract BHT 
20 0.12 0.54 
30 0.15 0.66 
50 0.35 0.75 
100 0.41 0.97 
0 20 40 60 80 100
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
R
ed
uc
in
g 
P
ow
er
 (A
bs
or
ba
nc
e)
Concentration (µg/mL)
 Extract
 BHT
 
Fig 2 Reducing power of the extract at different concentrations 
Metal Chelating Activity 
Direct reaction of a substance is not the only mechanism by which the antioxidants may display their activity. 
Secondary, preventive, or type 2, antioxidants act through numerous possible mechanisms. These antioxidants 
do not convert free radicals to more stable products, but slow the rate of oxidation by several different 
mechanisms. One of the most important mechanisms of action of secondary antioxidants is chelation of pro-
oxidant metals. Iron and other transition metals (copper, chromium, cobalt, vanadium, cadmium, arsenic, 
nickel) promote oxidation by acting as catalysts of free radical reactions. These redox-active transition metals 
transfer single electrons during changes in oxidation states. Chelation of metals by certain compounds 
decreases their pro-oxidant effect by reducing their redox potentials and stabilizing the oxidized form of the 
metal. Chelating compounds may also sterically hinder formation of the metal hydroperoxide complex 
(Reische D W, et al, 2008)). 
Chelating activity of the extract sample was determined at concentrations of 20, 30, 50 and 100 µg/mL, taking 
citric acid as standard. The oil displayed significant chelating ability,   which appeared to increase significantly 
with the increase in concentration. The absorbance of sample and citric acid standard was measured at 562 
nm against the blank. 
To Chemistry  Journal  Vol 1 No 2 (2018)  ISSN: 2581-7507                                              http://purkh.com/index.php/tochem 
183 
% Chelation = [1− (Asample /Acontrol)×100 --------------------------- (4)   
where Acontrol is the absorbance of the control reaction and Asample the absorbance in the presence of the 
sample.           
Table 3:  Metal chelating activity (Absorbance) 
Concentration (µg/mL) Extract Citric acid 
20 0.41 0.57 
30 0.50 0.83 
50 0.72 0.94 
100 0.86 0.93 
0 20 40 60 80 100
0.0
0.2
0.4
0.6
0.8
1.0
M
e
ta
l 
C
h
e
la
ti
n
g
 a
c
ti
v
it
y
 (
A
b
s
o
rb
a
n
c
e
)
Concentration (µg/mL)
 Extract
 Citric acid
 
Fig 3 Chelating activity of the extract at different concentrations 
Conclusion 
The extract displayed significant antioxidant activity at various concentrations on stable 2, 2-diphenyl-1-picryl 
hydrazyl radical (DPPH). The activity was found to increase with increase in concentration. Same trend was 
noticed in case of reducing power. Metal chelating activity of the extract was found to be very close to the 
standard, especially at concentration of 100 µg/mL.  
Acknowledgements 
Authors are thankful to Director NIT Srinagar for all possible support and facilities provided. One of the 
authors, Rumaisa is thankful to CSIR (India) for providing the financial assistance and to the Director Indian 
Institute of Integrative Medicine, (IIIM, CSIR) Jammu for providing the necessary facilities to carry the research 
work. 
 
 
 
 
To Chemistry  Journal  Vol 1 No 2 (2018)  ISSN: 2581-7507                                              http://purkh.com/index.php/tochem 
184 
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Antioxidant activity of chloroform extract of Inula Racemosa from Kashmir Himalayas

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Antioxidant activity of chloroform extract of Inula Racemosa from Kashmir Himalayas

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