Trends Phytochem. Res.

5(1) 2021 44-52

Trends in Phytochemical Research (TPR)

Journal Homepage: http:/ / tpr.i au-sha h ro o d.ac.i r

Original Research Article

Phytochemical composition, antioxidant, and anti-inflammatory activities of essential

oil of Acmella uliginosa (Sw.) Cass. grown in North India Terai region of Uttarakhand

KANCHAN GAIROLA¹, SHRIYA GURURANI¹, RAVENDRA KUMAR², OM PRAKASH², SANJEEV AGRAWAL¹ AND SHIV
KUMAR DUBEY¹ *

¹Department of Biochemistry, College of Basic Sciences & Humanities, G. B. Pant University of Agriculture and Technology, Pantnagar-263145,
Uttarakhand, India
²Department of Chemistry, College of Basic Sciences & Humanities, G. B. Pant University of Agriculture and Technology, Pantnagar-263145,
Uttarakhand, India
ABSTRACT ARTICLE HISTORY

The plant Acmella uliginosa (Sw.) Cass. belonging to family Asteraceae was subjected to Received: 06 July 2020
hydro distillation for essential oil extraction and was chemically analyzed by GC/MS for its Revised: 22 January 2021
phytochemical composition. Thirty-five compounds were identified comprising 88.1% of Accepted: 07 March 2021
total essential oil composition. Limonene (12.2%) along with sesquisabinene isomer (7.3%) ePublished: 16 March 2021
and caryophyllene oxide (5.9%) were the prominent compounds of the essential oil. In-vitro
antioxidant activity of essential oil was investigated by DPPH free radical scavenging activity
and metal chelating activity having IC50 value of 326.77 ± 5.34 μL and 14.853 ± 0.106 μL KEYWORDS
,respectively, whereas reducing power activity having RP50 value of 14.011 ± 0.0446 μL. The
essential oil exhibited potent anti-inflammatory activity with IB50 value of 5.629 ± 0.0311 μL Acmella uliginosa (Sw.) Cass.
compared to standard Diclofenac sodium salt having IB50 value of 23.693 ± 0.306 μg/mL. The Anti-inflamatory activity
essential oil displayed exceedingly marked anti-inflammatory as well as antioxidant activities
Antioxidant activity
as compared to standard marketed drugs.
Essential oil
Limonene

© 2021 Islamic Azad University, Shahrood Branch Press, All rights reserved.

M
1. Introduction that have been exploited in both preventive and
curative medicine in traditional Indian medicinal system
edicinal plants are considered as an affluent for centuries (Bhutani and Gohil, 2010). In Uttarakhand,
source of traditional medicines, and used in more than 964 species have been documented for their
the preparation of modern medicine. The various medicinal properties (Kala, 2006) and in the
bioprospecting of useful natural products is a new way Trans Himalayan region, approximately 337 species of
of systematic and sustainable exploration of natural medicinal plants are reported so far (Kala and Mathur,
resources for the development of valuable products 2002). Kumaun and Garhwal region of Uttarakhand,
which includes all bioresources such as plants, animals, which is part of North India in the vicinity of the
and microorganism and is proved to be beneficial, not Himalayan region, is a rich repository of medicinal plants
only for pharmaceutical industries but also for the host and aromatic flowers might be an interesting region for
country and the native people in terms of knowledge, identification and exploration of other medicinal plants.
better education, and employment (Li et al., 2009). Acmella uliginosa (Sw.) Cass. from Asteraceae
Medicinal plants are a rich source of natural antioxidants (Compositae) family, is extensively distributed in
that are used in the prevention and treatment of various tropic and subtropic regions mainly in Brazil, Africa,
diseases. Correlating the geographical distribution and Indonesia, West-Indies, and Malaysia (Pandey et al.,
bioprospecting of flora and fauna, India is a treasure- 2007). This globally distributed plant is acclimatized in
trove in terms of biodiversity and has an abundant India and (known) acknowledged with a different name
number of herbal plants showing medicinal properties in the distinctive region such as “Akarkara” in Kumaun
Corresponding author: Shiv Kumar Dubey
Tel: +91-5944-233310; Fax: +91-5944-233473
E-mail address: shivdub@gmail.com, doi: 10.30495/tpr.2021.680495
 Gairola et al. / Trends in Phytochemical Research 5(1) 2021 44-52 45

and Garhwal region of Uttarakhand, “Gorokhbon” in level. Herbarium specimen of the plant was submitted
Bengal, “Subhangnenek” or “Butang Baju Siti Fatimah” at Govind Ballabh Pant University of Agriculture
in Peninsular Malaysia and “Jotang” in Indonesia. The and Technology, Pantnagar. The identification of A.
plant dwelling with pleiotropic medicinal properties is uliginosa (voucher number GBPUH-1018/1-8-2019) was
traditionally used for the treatment of various diseases in established by Dr. D.S. Rawat (Assistant Professor and
various parts of the world. The Plant being a rich source Plant Taxonomist), Department of Biological Science,
of secondary metabolites like isoflavones, flavonoids, College of Basic Sciences and Humanities, Govind
anthocyanins, catechins, polyphenols, flavonoids have Ballabh Pant University of Agriculture and Technology,
antimicrobial and antioxidant properties (Krishnaswamy Pantnagar.
et al., 1975; Dubey et al., 2013; Sana et al., 2014). A.
uliginosa is generally used in Malaysia by the Malay 2.1.2. Isolation of the essential oil
community to get relief from the pain caused due to
mouth ulcer, toothache, sore throat, and stomachache The sample of 4.94 kg fresh whole plant of A. uliginosa
(Etèka et al., 2010). The whole plant part of A. uliginosa was taken and subjected to hydro distillation for 3-4 h
contains a compound known as spilanthol. Spilanthol using Clevenger apparatus. The 1.9 mL of the essential
amid with saliva inducing effect acts as a powerful oil was separated from the aqueous solution and
insecticide and local anesthetic. It suppresses the desiccated over anhydrous sodium sulfate. The light
contraction in subcutaneous muscles especially of the yellow color essential oil was stored at low temperature
face and is used in anti-wrinkle products (Barbosa et (4 °C) in light protected bottle for further studies.
al., 2016). Spilanthes acmella dwells with the various
compounds because of which it is used as spices, 2.1.3. Analysis and identification of compounds with the
antiseptic, anti-bacterial, anti-fungal, anti-malarial help of gas chromatography-mass spectrometry (GC/
agent, and as a remedy for toothache, flu, cough, rabies MS)
diseases, and tuberculosis (Ramsewak et al., 1999).
Japanese usually use flower head as a spice appetizer. To analyze and identify the phytochemical composition
An extract of the plant is used as a flavoring material for of the essential oil, GC/MS was carried out at GCMS-
denitrifying and gum (Leng et al., 2011). The leaves of A. QP 2010 Plus equipment with carrier gas helium with
uliginosa are used in the treatment of skin disease and a pressure 73.3 kPa and split ratio was 10:1. During
leaf decoction is used for the treatment of diuretic and analysis, the total flow was 16.3 mL/min while the
lithotriptic conditions. It is also reported that the whole column flow rate of 1.21 mL/min. The linear velocity
plant part of A. uliginosa is used for the treatment of and purge flow were maintained at 40.1 cm/sec
dysentery (Rao et al., 2012). and 3 mL/min, respectively. Carrier gas saver, high-
Phytochemical estimation of medicinal plants plays pressure injection, and splitter hold were off and oven
a significant role in revealing the new sources for temperature was initially at 60 °C RAMP@ 3 °C/min up
therapeutically important compounds (El-Wahab et al., to 210°C (isotherm for 2 min) then 6 °C/min up to 280
2013). Bioactive compounds that are isolated from the °C (isotherm for 2 min), finally hold for 11 min, flame
plant materials have proven to be a beneficial source of thermionic detector (FTD).
metabolites which are difficult to get from other sources The individual compounds were identified by comparing
(Kinghorn, 2001; Samuelsson, 2004; Kharshiing, 2012). their Kovats indices (KI) of the peaks on Innowax fused
Analysis based on previous scientific documentation silica capillary column with literature values, matching
reveals that not much significant work has been against the standard library spectra, built up using pure
done on the chemical composition and biological substances and components of known essential oils.
activities of the essential oil of A. uliginosa from India. Further identification was carried out by comparison of
However, one report has been documented for the the fragmentation pattern of the mass spectra obtained
phytochemical composition of leaves of the plant from by GC/MS analysis with those stored in the spectrometer
Indonesia (Maimulyanti and Prihadi, 2016). The current database of NBS 54 K L, WILEY 8 libraries, and published
study analyses the phytochemical composition of the literature (Adams, 2007). Relative amounts of identical
essential oil from the whole plant part of A. uliginosa components were based on peak areas obtained
and investigates its antioxidant and anti-inflammatory without FID response factor correction.
properties.
2.1.4. Antioxidant activities
2. Experimental
2.1.4.1. DPPH (2,2’-Diphenylpicrylhydrazyl ) free radical
2.1. Materials and method scavenging activity

2.1.1. Plant collection and authentication DPPH free radical scavenging activity was analyzed with
the previously reported method (Brand-Williams et
The plant A. uliginosa was collected from the Terai al., 1995). The DPPH is a free and stable radical that
region of Uttarakhand in winters near sugarcane fields can accept hydrogen radical and electron to convert
located at the latitude of 29° N and longitude of 79° them into a stable molecule that is diamagnetic. A
E at an elevation of 243.8 m above the mean sea stock solution of 0.1 mM DPPH was freshly prepared
46 Gairola et al. / Trends in Phytochemical Research 5(1) 2021 44-52

in methanol and stored in the Amber bottle in dark at by adding methanol. The mixture was incubated at
4 °C. Test samples of the essential oil were prepared at room temperature for 10 min and the absorbance was
different concentrations of 50-250 μL. Then 0.1 mL of calculated at 562 nm (Pavithra and Vadivukkarasi, 2015).
each concentration of the essential oil was added with EDTA was taken as standard. The % inhibition of metal
2.9 mL of 0.1 mM DPPH solution and kept in dark for chelation of essential oil and standard were calculated
30 min incubation and subjected to measurement of using the given formula (Eqn.3):
absorbance at 517 nm using UV spectrophotometer.
Ascorbic acid was taken as standard. Percentage IC% =(A₀-At )/A₀ × 100 (Eqn.3)
scavenging activity of DPPH free radical was calculated
with the help of formula (Eqn.1): Where A0: Absorbance of control At: Absorbance of
test sample. The graph of % chelating activity against
Percentage scavenging(%) = (1-At/A0) x 100% (Eqn.1) concentration was plotted to calculate IC50 value of the
essential oil and standard. The decreasing value of IC50
Where A0: absorbance of the DPPH solution and indicates higher metal chelating activity.
At: absorbance of the test sample. The percentage
scavenging of DPPH free radical was then plotted 2.1.5. In-vitro anti-inflammatory activity
against the concentration of the test sample and the IC50
value was calculated. The term IC50 is the concentration Different concentrations of the essential oil (5 μL-25 μL)
of the sample in which 50% of DPPH free radicals were were prepared. About 2 mL of specific concentration
scavenged. The sample having less IC50 value implicates of the essential oil was added with 2.8 mL of freshly
better antioxidant activity. The IC50 value of sample was prepared 1 molar phosphate buffer with pH 6.4. To it,
also compared with standard i.e., ascorbic acid. 0.2 mL of egg albumin was added, and the final volume
was maintained to 5 mL. The mixture was left for
2.1.4.2. Reducing power activity incubation for 15 min at 37°C followed by incubation
for 5 min at 70°C. Absorbance was measured at 660
Reducing power activity was done with the help nm (Heendeniya et al., 2018). Diclofenac was taken as
of previously reported method (Yen et al., 2000). a standard. The protein denaturation % inhibition was
Different concentrations (5-25 μL) of the essential measured with the help of a formula that is given below
oil was prepared in hexane. About 2.5 mL of each (Eqn. 4):
concentration of the essential oil was added with the
2.5 mL of phosphate buffer (200 mM, 6.6 pH) and 2.5 Inhibition% =100 × (1-Vt/Vc) (Eqn.4)
mL of potassium ferricyanide (1% w/v). The mixture was
then incubated in the water bath for 20 min at (50 ± Where, Vt = Test sample absorbance and Vc= Control
1)°C. After incubation 2.5 mL trichloroacetic acid (10% absorbance
v/v in distilled water) was added and the mixture was
centrifuged at 50.54 g (650 rpm) for 10 min. The 5 mL of 2.2. Statistical analysis
supernatant was taken and mixed with 5 mL of distilled
water. About 1 mL of ferric chloride was added to the Statistical analysis was done by using SPSS16.00 software
mixture and absorbance of the resultant solution was for estimating the mean and standard deviation of
calculated at 700 nm using a UV spectrophotometer. triplicates of plant essential oil. All the results were
Gallic acid was used as a standard. Reducing power % subjected to the Duncan test for one way analysis
activity of the essential oil and standard was calculated (ANOVA) at 5% to test their significance (p<0.05). The
with the given formula (Eqn.2): significance and correlation of the essential oil were
done with the help of SPSS software.
Reducing power activity% = (A₀-At) ×100/ A₀ (Eqn.2)
3. Results and Discussion
Where, A₀: Absorbance of control, At: Absorbance of
test sample. The graph of reducing power % activity 3.1. Chemical composition of the essential oil
against concentration was plotted to calculate the RP50
value of the essential oil and standard. The decreasing The hydrodistillation of the fresh plant of A. uliginosa
value of RP50 indicates increasing reducing activity. gave light yellow colored essential oil having 0.037%
yield. Chromatographic analysis of the essential oil
2.1.4.3. Metal chelating activity through GC/MS led to the identification of a total
of 35 compounds as shown in (Table 1) and the
The principle of metal chelating activity is the metal chromatogram of the essential oil shown in (Fig. 1). The
chelating ability of ferrozine. Being a potent metal major compounds identified in the essential oil of A.
chelator ferrozine form a complex with ferrous ion and uliginosa were limonene (12.2%), sesquisabinene isomer
form red color. Different concentrations of the essential (7.4%), caryophylleneoxide (6%), (E)-caryophyllene
oil (5 μL-25 μL) were prepared and added with 0.05 mL (5.5%), 4-(1,5-dimethylhex-4-enyl) cyclohex-2-enone
of 2 mM FeCl₂· 4H₂O. To it, 0.2 mL of 5 mM of ferrozine (5.0%), myrcene (5.0%), trans-β-bergamotene (4.5%),
was added and the volume was maintained to 5 mL spathulenol (4.5%), 1-tetradecanol (2.6%), n-hexacosane
 Gairola et al. / Trends in Phytochemical Research 5(1) 2021 44-52 47

(2.4%), pentadecylic acid (2.4%) and sabinene (2.3%) Oxygenated sesquiterpenes comprised 13.6%, whereas
and others were present in minor amount. The oxygenated monoterpenes hydrocarbon comprised
compounds identified in the essential oil belong to the the least percentage of chemical class i.e., 1.7% and
class of monoterpenes hydrocarbons, sesquiterpenes the other compounds in total comprise 24.6% of
hydrocarbons, oxygenated sesquiterpenes, and total constituents as represented in (Table 2). The
oxygenated monoterpenes. Sesquiterpenes being literature data reveals that phthalate derivatives like
the dominant class of hydrocarbon comprises of phthalic acid, bis (6-methylheptyl) ester present in
28.7% compared to monoterpenes hydrocarbon trace amount (1.5%) in the extract might be probable
that comprises 19.5% of total compounds present. contaminants (Bianco et al., 2014; Venditti, 2020).

Table 1
Chemical composition of the essential oil of the whole plant of A. uliginosa (AUEO).
Chemical KI % Area Class of com-
S.N. Compounds formula (Kovats pounds*
index)
1 Sabinene C10 H16 969 2.3 MH
2 Myrcene C10H16 988 5.0 MH
3 Limonene C10 H16 1024 12.2 MH
4 trans-β-Ocimene C10H16 1050 0.7 OT
5 Z-Carveol C10H16O 1207 0.7 OM
6 Carvone C10 H14 O 1239 1.0 OM
7 β-Bourbonene C15H24 1380 0.5 SH
8 β-Elemene C15H24 1389 1.8 SH
9 cis-α-Bergamotene C15H24 1415 0.6 SH
10 α-Humulene C15H24 1489 1.3 SH
11 E-Caryophyllene C15 H24 1417 5.5 SH
12 Sesquisabinene isomer C15H24 1457 7.4 SH
13 Cyclopropyl 4-ethylphenyl carbinol C12H16O 1469 0.8 OT
14 2-Tridecanone C13H26O 1481 0.5 OT
15 Germacrene-D C15H24 1499 2.0 SH
16 β-Bisabolene C15H24 1505 4.4 SH
17 trans-β-Bergamotene C15 H24 1513 4.5 SH
18 E-Nerolidol C15H26O 1564 0.5 OS
19 Spathulenol C15H24O 1577 4.5 OS
20 Cadin-4-en-10-ol C15H26O 1598 0.8 OS
21 Caryophyllene oxide C15 H24 O 1606 6 OS
22 1-Tetradecanol C14H30O 1611 2.6 OT
23 Tetradecanal C14H28O 1611 1.1 OT
24 4-(1,5Dimethylhex-4-enyl) Cyclohex-2-enone C14H22O 1626 5.0 OT
25 3,7,11,15-Tertramethyl-, (R-(R*,R*-(E)-2-hexadecane C20H40O 1626 1.6 OT
1-ol
26 Epi-β-caryophyllene C15H24 1663 0.7 SH

27 Cuparenal C15H20O 1750 0.9 OS

28 Perhydrofarnesyl acetone C18H36O 1754 1.5 OT

29 Pentadecylic acid C15H30O2 1820 2.4 OT

30 9-Octadecyne C18H34 1828 2.0 OT

48 Gairola et al. / Trends in Phytochemical Research 5(1) 2021 44-52

Table 1 (Continued)
Chemical KI % Area Class of compounds*
S.N. Compounds formula (Kovats
index)
31 Humulene oxide C15H24O 2038 0.9 OS
32 n-Tetracosane C24H50 2400 0.6 OT
33 n-Pentacosane C25 H52 2500 1.9 OT

34 Phthalic acid, bis(6 methylheptyl) ester C24H38O4 2519 1.5 OT

35 n-Hexacosane C26 H54 2600 2.4 OT
88.1
*Class of compounds: MH=Monoterpenes hydrocarbon , SH=Sesquiterpenes hydrocarbon, OM=Oxygenated monoterpenes, OS=Oxygenat-
ed sesquiterpenes, OT=Others.

Fig. 1. Gas chromatogram of the essential oil of the whole plant of A. uliginosa.

Table 2
Classes of compounds present in the essential oil of the whole plant of A. uliginosa.
S. No. Classes composition % Peak area
1 Monoterpenes hydrocarbon (MH) 19.5
2 Sesquiterpenes hydrocarbon (SH) 28.7
3 Oxygenated monoterpenes (OM) 1.7
4 Oxygenated sesquiterpenes (OS) 13.6
5 Others (OT) 24.6
Total 88.1%
 Gairola et al. / Trends in Phytochemical Research 5(1) 2021 44-52 49

3.2. Antioxidant activities is disrupted which leads to the reduction in the intensity
of red color. The estimation of the chelating activity
The evaluation of the in-vitro antioxidant activity of of coexisting chelators can be depicted by measuring
the essential oil of the whole plant of A. uliginosa was color reduction. The most active extract inferred with
performed via three different methods i.e., 2,2-diphenyl- ferrous ferrozine complex suggests that the extract
1-picrylhydrazyl (DPPH) radical scavenging activity, the has chelating activity and captures ferrous ion before
metal chelating activity of Fe2+ and reducing power ferrozine (Ebrahimzadeh et al., 2008). The concentration
activity of Fe3+. All the activities were evaluated by of the essential oil considered for evaluation of metal
comparing the antioxidant activity of the essential oil chelating activity was 5-25 μL. The IC50 value of metal
with the standard antioxidant. chelating activity of A. uliginosa essential oil was 14.85
± 0.10 μL which is more significant in comparison
3.2.1. DPPH radical scavenging activity to standard EDTA having its IC50 value at 45.74 ±
0.67 µg/mL (Table 4). The IC50 value is defined as the
DPPH free radical method is to evaluate the antioxidant concentration of total antioxidant required to chelate
activity, based on the electron transfer that brings a metal ion by 50%. The essential oil is found to be a
change in violet color. DPPH is a free radical that remains potent antioxidant.
stable at room temperature and forms a diamagnetic
molecule by accepting electrons or free radicals. Table 4
DPPH because of the presence of its odd electron IC50 value of the metal chelating activity of the essential
has maximum absorbance at 517 nm. The antioxidant oil of the whole plant of A. uliginosa.
molecule leads to quenching of DPPH free radicals. The
freshly prepared DPPH solution fades and disappear Sample IC50 values Mean IC50
S. No.
from deep blue color to colorless or bleached that Name values
1 st
2 nd
3 rd
results into the decreasing in absorbance, hence more
the briskly the absorbance decreases, the antioxidant 1. AUEO 14.84 14.96 14.75 14.85±0.10 μL
will be more potent w.r.t. hydrogen-ion donating 45.74±0.67 µg/
capacity (Amarowicz et al., 2004; Lewis et al., 2018). The 4. EDTA 45.39 45.32 46.51
mL
DPPH free radical scavenging capacity of the essential AUEO: Acmella uliginosa essential oil, EDTA:
oil was evaluated and the competence of being a potent Ethylenediaminetetraacetic acid
antioxidant was tested in dose dependent manner.
The concentration range of the essential oil (50-250
μL) that was considered for antioxidant evaluation 3.2.3. Reducing power activity of Fe3+
possessed lesser inhibition activity on DPPH free radical
in comparison to the standard. The IC50 value is defined Reducing power indicates electron-donating capacity of
as the concentration of total antioxidants required to bioactive compounds and correlated with antioxidant
inhibit DPPH free radicals by 50%. The IC50 value of activity. On reduction Fe3+ form Prussian blue color which
DPPH free radical scavenging activity of the essential has its maximum absorbance at 700 nm. An increase
oil of A. uliginosa is 326.77 ± 5.34 μL which was less in in absorbance indicates higher reducing capacity. The
comparison to the standard ascorbic acid that has IC50 yellow color of test solution converts into green or blue
value of 51.10 ± 0.07 µg/mL (Table 3). color depending on the capacity of extract to reduce
Fe3+ to Fe2+. Higher the absorbance, higher the reducing
Table 3 power (Gülçin, 2015).The concentration of the essential
IC50 of DPPH free radical scavenging activity of the oil considered for evaluation of reducing power activity
essential oil of the whole plant of A. uliginosa. was taken in the range of 5-25 μL. The essential oil is
found to be a potent reducing agent. The RP50 value of
IC50 values Mean IC50 reducing power activity of A. uliginosa is 14.01 ± 0.04 μL
S. No. Samples
1 st
2nd 3rd values , whereas the standard gallic acid having its RP50 value
326.77 ± at 79.90 ± 1.60 µg/mL (Table 5).
1 AUEO 332.40 321.76 326.14
5.34 μL
Table 5
51.10 ±
2 AA 51.01 51.16 51.11 0.07 µg/ RP50 of the essential oil of the whole plant of A. uliginosa.
mL
AUEO: Acmella uliginosa essential oil, AA: Ascorbic acid Sample RP50 values Mean RP50
S. No.
Name 1st 2nd 3rd values
3.2.2. Metal chelating activity of Fe2+
1. AUEO 14.01 ± 0.04
13.96 14.01 14.05 μL
Ferrozine being the most common chelating agent
quantitatively form a complex with Fe2+ which 4. GA 79.90 ± 1.60
constitutively forms red color. In the presence of other 78.37 79.77 81.56 µg/mL
chelating agent formation of the ferrozine-Fe complex AUEO: Acmella uliginosa essential oil, GA: Gallic acid
50 Gairola et al. / Trends in Phytochemical Research 5(1) 2021 44-52

RP50 value is defined as the concentration of total (12.2%), caryophyllene (5.5%), and caryophyllene oxide
antioxidants required to reduce ferric ion into a ferrous (5.9%) in the higher amount as they are reported to
ion by 50%. have good anti-inflammatory activity (Tung et al., 2008;
Yu et al., 2017). The chemical compounds present in the
3.3 In-vitro anti-inflammatory activity essential oil revealed during the GC/MS analysis in the
present study of whole plant extract were compared with
Denaturation is the proclaimed cause of inflammation. the study of Maimulyanti and Prihadi (2016) performed
Protein loses tertiary structure into secondary structure on leaf extract of A. uliginosa from Indonesia based on
due to denaturation. Albumin protein undergoes the category and content of volatile component present
denaturation at physiological pH in presence of in the essential oil. The results of GC/MS in both the
phosphate-buffered saline salt. The anti-inflammatory analysis showed considerable variations in the chemical
activity is a measure of the ability of plant extract to composition of the essential oil of plant A. uliginosa.
inhibit denaturation. Inhibition of heat-induced albumin The major compound present in the essential oil in the
denaturation was studied (Leelaprakash and Dass, current study is limonene (12.2%) which is reported as
2011). The concentration of the essential oil considered a minor compound in the previous study i.e., 0.59%
for evaluation of anti-inflammatory activity was taken from Indonesia. Caryophyllene and caryophyllene
in the range of 5-25 μL. Essential oil is found to be a oxide was identified as a major compound in both the
potent anti-inflammatory agent. The IB50 value of the study but the percentage area of both the compounds
anti-inflammatory activity of A. uliginosa of essential oil were different. In a previous study from Indonesia, it
was 5.62 ± 0.03 μL, whereas the standard Diclofenac comprises 21.3% and 15.5% but in the present study
sodium salt having its IB50 value at 22.58 ± 0.404 µg/mL from India, it was found as 5.5% and 5.9%, respectively.
(Table 6). IB50 is the 50% inhibition of the denaturation However, 3-careen, β-pinene was not present in the
of the protein. essential oil in the present study but was part of the
The essential oil shows better and potent anti- essential oil in a previous study. Myrcene contributed
inflammatory activity in comparison to the standard to 4.9% of the total composition of the essential oil but
which may be attributed to the presence of limonene was absent in a previous study from Indonesia (Table 7).

Table 6
IB50 of anti-inflammatory activity of the essential oil of the whole plant of A. uliginosa.

IB50 values
S. No. Sample Name Mean IC50 values
1st 2nd 3rd
1 AUEO 5.62 5.60 5.66 5.62 ± 0.03 μL
2 DF 22.74 22.12 22.88 22.58 ± 0.404 µg/mL
AUEO: Acmella uliginosa essential oil, DF: Diclofenac sodium

Table 7
Comparative table of the major compounds of essential oil extract with previously reported research work.

% Area

Chemical compound Maimulyanti and

The present study (Uttarakhand,
S. No. Prihadi (2016) (In-
India)
donesia)

Essential oil (AUEO)

1 Caryophyllene 5.5 21.27
2 Caryophyllene oxide 5.9 15.49
3 3-Carene - 10.73
4 β-Pinene - 7.32
5 Sabinene - 2.30
6 Limonene 12.2 0.59
7 4-(1,5-Dimethylhex-4-enyl) Cyclohex-2-enone 5.0 -
8 Myrcene 4.9 -
9 trans-β-Bergamotene 4.5 -
10 β-Bisabolene 4.4 0.30
 Gairola et al. / Trends in Phytochemical Research 5(1) 2021 44-52 51

Limonene, which is a major component of the essential weed can also be used as an anti-inflammatory agent
oil of A. uliginosa, is traditionally used as a flavoring as it shows better activities compared to standard drugs
agent in cosmetics and food industries. Limonene available in the market.
is also utilized as an eco-friendly surfactant having
lower flammability due to its solvent properties, lower Conflict of interest
odor, and VOC (Volatile Organic Compound) that
make it suitable for treating contaminated surfaces in There is no conflict of interest declared by the author to
many industrial environments (Ciriminna et al., 2014). publish this manuscript.
Limonene acted as an antioxidant and was reported as
an excellent dietary source for cancer chemoprevention Acknowledgment
(Aggarwal and Shishodia, 2006). It has been reported to
reduce the risk of skin, stomach, mouth, breast, colon, Dr. D.S. Rawat, Assistant Professor Biological sciences
and lung cancer (Milind and Dev, 2012). Limonene at G.B. Pant University of Agriculture and Technology,
acts as a potent antioxidant, and anti-inflammatory Pantnagar, India is thankfully acknowledged for
agent, natural cholesterol-lowering agent, antibacterial identifying plant species.
and antifungal agent. It also acts as a mild appetite
suppressant and has an anti-anxiety effect (Mizrahi References
et al., 2006; Zheng et al.,1992). However, limonene is
known as an allergen in the essential oil and fragrances Adams, R.P., 2007. Identification of Essential Oil
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