Journal of Essential Oil Bearing Plants

ISSN: 0972-060X (Print) 0976-5026 (Online) Journal homepage: http://www.tandfonline.com/loi/teop20

Chemical Analysis and Antioxidant Activity of

Essential Oils of Two Morphotypes of Lippia alba
(Mill.) N.E. Br. ex Britton & P. Wilson (Verbenaceae)

Archana Joshi, Om Prakash, A.K. Pant, Ravendra Kumar & M.S. Negi

To cite this article: Archana Joshi, Om Prakash, A.K. Pant, Ravendra Kumar & M.S. Negi (2018)
Chemical Analysis and Antioxidant Activity of Essential Oils of Two Morphotypes of Lippia�alba
(Mill.) N.E. Br. ex Britton & P. Wilson (Verbenaceae), Journal of Essential Oil Bearing Plants, 21:3,
687-700

To link to this article: https://doi.org/10.1080/0972060X.2018.1486232

Published online: 31 Jul 2018.

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 TEOP 21 (3) 2018 pp 687 - 700 687
ISSN Print: 0972-060X
ISSN Online: 0976-5026

Chemical Analysis and Antioxidant Activity of Essential Oils of Two Morphotypes

of Lippia alba (Mill.) N.E. Br. ex Britton & P. Wilson (Verbenaceae)

Archana Joshi 1, Om Prakash 1*, A.K.Pant 1*, Ravendra Kumar 1 and M.S. Negi 2

Department of Chemistry, College of Basic Sciences and Humanities

1

2
Medicinal Plants Research and Development Centre (MRDC) G.B. Pant University of
Agriculture and Technology, Pantnagar, U.S. Nagar-263145, Uttarakhand, India
Received 12 September 2017; accepted in revised form 21 May 2018

Abstract: Lippia alba (Mill. ) N.E.Br.ex Britton & P.Wilson is an aromatic shrub of Verbenaceae family.
L. alba exhibits variability among its different accessions, showing difference in morphology and in the
composition of essential oil. The objective of this study was to chemically characterize and evaluate the
antioxidant activity of essential oil of two L. alba morphotypes. The essential oils were isolated by hydrodistillation
using a Clevenger apparatus and the identification was done through GC/GC-MS analysis. The antioxidant
activity was evaluated using DPPH radical scavenging, Iron chelating activity, FRAP assay, Super oxide radical
scavenging and Nitric oxide radical scavenging activity. More than eighty constituents were identified in both
the oil’s. Both the oils were characterized by a high amount of oxygenated monoterpenes (80.8 %) and (82.0 %).
The major constituent in both the morphotypes was linalool. The other major constituents found in type-I were
eucalyptol (4.2 %), neral (1.1 %), geranial (1.5 %), E-caryophyllene (3.1 %), germacrene-D (3.3 %), germacrene-
B (1.6 %), 3E,5E-2,6-dimethyl-3,5,7-octatriene-2-ol (2.1 %), 3E,5Z-2,6-dimethyl-3,5,7-octatriene-2-ol (3.8 %) whereas
in type-II were eucalyptol (4.7 %), β-citronellol (1.3 %), neral (2.3 %), geranial (2.8 %), β-caryophyllene (2.3 %),
germacrene-D (3.0 %), germacrene-B (1.4 %), carryophyllene oxide (1.1 %), 3E,5E-2,6-dimethyl-3,5,7-octatriene-
2-ol (1.6 %), 3E,5Z-2,6-dimethyl-3,5,7-octatriene-2-ol (2.9 %). Both the morphotypes exhibited good antioxidant
activity but the activity, was found higher in type- II L. alba morphotype than type-I. The chemical composition
and antioxidant activity suggest L. alba as a potential source of natural linalool and further its use in
ethanopharmacological applications.

Key words: Lippia alba, linalool, essential oil, antioxidant activity.

Introduction but can reach up to 2 metres. The leaves are mem-

The members of family Verbenaceae (Lamiales)
are distributed throughout the world, particularly
in tropical and subtropical countries, and may be
classified into 34 genera of trees, shrubs and herbs.
The genus Lippia, consists approximately 200
species 1,2. Five species of Lippia including L.
alba have been reported to occur in India 3,4. It
holds the prime position among a diversity of aro-
matic plants being cultivated world over. The shrub
is aromatic, and normally 1 to 1.5 metres of height,
*Corresponding authors (Om Prakash, A.K.Pant)
E-mail: < oporgchem@gmail.com, anilpant54@gmail.com >
branaceous, petiolate, pubescent with a strong
flavour and with the typical lemon smell. Its limbs
have variable forms with pointed apex, cuneiform
or decumbent base, and serrated or crenated bor-
der.
Lippia alba has five scientific synonyms (Lan-
tana alba Mill., Lantana geminata (Kunth)
Spreng., Lippia geminata Kunth, Lippia gemi-
nata var. microphylla Griseb. and Lippia globi-
flora var. geminata (Kunth) Kuntze), besides four
© 2018, Har Krishan Bhalla & Sons
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700
homonymies and infraspecific taxa-Lippia alba
f. alba, Lippia alba f. intermedia Moldenke,
1982, Lippia alba var. carterae Moldenke, 1961,
Lippia alba var. globiflora (L’H´er) Moldenke,
1970 5.
This species found to be promising for
ethanobotanical applications in pharmaceutical,
aromatic, and perfumery industries and in agri-
cultural chemical industry because of its proven
antifungal, insecticidal, and repellent properties 6.
The essential oil of L. alba has been found as a
potential source of several commercially impor-
tant terpenoid compounds 7. It is commonly em-
ployed in the form of a leaf infusion or decoction
in the treatment of colds, bronchitis, coughs,
asthma, and stomach and intestinal disorders 8.
Several pharmacological aspects have been as-
cribed to the genus Lippia including,
antidysenteric, stimulant, antirheumatic, anti-
inflammatory, hypertension and analgesic activi-
ties. It is more often used as a tea, antipyretic and
to heal wounds. The alcoholic infusion rubbed
upon the chest is found to be effective against
colds 9,10.
The analgesic, anti-inflammatory, sedative and
antioxidant effects of this plant have been associ-
ated to essential oil constituents, like citral,
myrcene and limonene 11-13. Sedative effects, are
attributed to non-volatile flavonoids and iridoids
14, 15
. year old Lippia alba plants cultivated in herbal
A large variations in the essential oils composi-
tion have been observed, subjected to the origin
of the plant material, plant stage and the plant part
selected for extraction of the essential oil 16.This
may be due to environmental effects, different
extraction methods, as well as due to genetic vari-
ability in these plants 17. The rich pharmacologi-
cal potential of L. alba is related to the large
chemical variability of its essential oils composi-
tion. The variability is so common that Matos sug-
gested a classification into different chemotypes
18,19,20
. Currently twelve chemotypes have been
recognized, which are : citral 21, linalool 22, carvone
21
, limonene 23, γ-terpinene 24, citral-myrcene 18,
citral-limonene 18, citral-β-caryophyllene 25, citral-
germacrene-D 16, carvone-limonene 18, 1,8-cin-
eol-camphor 26, 1,8-cineol-limonene 16 and li-
monene-piperitone 27 chemotypes. The genetic
688
diversity among L. alba species has been con-
firmed through cytogenetic studies of reproduc-
tive behavior, molecular screening and DNA con-
tent 28. The ethnopharmacological importance and
considerable biodiversity of L. alba, led to a com-
parative study of the morphological and chemical
characteristics of its morphotypes 29, 30. Keeping
in mind the above statement the aim of the present
study was to compare the two different morpho-
logical accession of L. alba species for their es-
sential oil composition and concomitant antioxi-
dant activity.
Materials and methods
Plant material
The L. alba morphotypes were collected from
a cultivated farm of Medicinal Plant Research and
Development Centre (MRDC) of the G.B.Pant
University, Pantnagar. The morphotypes with red
flowers (Type-I) and purple flowers (Type-II)
were identified and authenticated by Dr. D.S.
Rawat, Plant Taxonomist, G.B.Pant University,
Pantnagar. The voucher specimens were depos-
ited in the Department for future reference. Fresh
aerial parts were collected for the extraction of
essential oils.
Cultivation condition’s
The plant sample was obtained from the three
garden in silty clay loam soil belonging to order
Mollisol. The plants were provided with 15 tonne/
ha FYM (Farm Yard Manure) at the time of plan-
tation and were also provided with 5 tonne/ha
FYM annually during winter season for proper
growth and development. The plants were spaced
at 50 × 50 cm row to row and plant to plant dis-
tance. The plants were also subjected to periodic
harvesting at 3 months interval for obtaining herb-
age.
Essential oil extraction
Fresh aerial parts were crushed and subjected
to a Clevenger apparatus for hydrodistillation for
six hour. The essential oils were extracted in
dichloromethane followed by drying over anhy-
drous Na2SO4.The yield of essential oils obtained
were 0.72 % and 0.80 % (w/v) in type-I and type-
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700
II respectively. The oils were stored at 4°C in a
refrigerator for future analysis.
GC-MS analysis
GC/MS analysis of the different essential oil
samples were performed using a GC MS-QP
2010, in the following conditions: Column DB-5
(30 m × 0.25 mm i.d.; 0.25 μm film thickness;
J&W Scientific, USA); carrier gas: Helium, with
a flow rate of 1 mL/min; injection temperature :
250°C; Oven temperature programme : initial tem-
perature 80°C, isothermal for 2 min, RAMP 7°C
/ min, final temperature 280°C and isothermal for
10 min. Ionization mode : EI(70Ev), mass range:
40-6500 amu.The compounds were identified with
the help of NIST-MS, FFNSC Wiley Library and
comparing the data with literature reports and GC
retention indices 31. Compounds of the essential
oil and relative percentage composition are com-
piled. Linear retention indices were calculated
using a homologous series of n-alkanes (C8-C25)
under the same temperature-programmed condi-
tions.
The components were identified by compari-
son with linear retention indices (RI) from litera-
ture 32 mass spectra with those of NIST mass
spectral library 33.
Antioxidant activity
DPPH radical scavenging activity
DPPH radical scavenging activity was deter-
mined by following the reported protocol with slight
modifications 34-36. In brief, the assay mixture con-
tained 5 mL of 0.004 % methanol solution of
DPPH and different amount of essential oil at 5-
25 μg/ml concentrations. The solutions were rap-
idly mixed and scavenging capacity was measured
spectrophotometrically using Thermo Scientific
Evolution 201 series by monitoring the decrease
in absorbance at 517 nm. Catechin and BHT were
used as positive control while reaction mixture
(DPPH radical solution) minus extract solution
was taken as control. Inhibition of free radical by
DPPH in percent (IC %) was calculated by us-
ing the
IC % = A0 - As / A0 × 100
Where, A 0 = Absorbance value of Control
689
sample As = Absorbance value of test sample
IC % = Inhibitory concentration.
Percent inhibition was plotted against concen-
trations and the standard curve was drawn using
standard antioxidant (catechin and BHT) to cal-
culate the IC50 values for standard and essential
oils. A lower IC50 value indicated more radical
scavenging activity.
Metal chelating activity
The metal chelating activity of Fe2+ was exam-
ined by the methods, recently being practiced 37,
based on the principle of the Fe2+ chelating ability
the absorbance of ferrous iron-ferrozine complex
formed was measured at 562 nm. 0.1 mL of 2
mM FeCl2.4H2O, 0.2 mL of 5 mM ferrozine and
4.7 mL of methanol was added to different con-
centrations of tested samples (5 - 25 μg/mL). Af-
ter incubation, the absorbance of test samples was
measured at 562 nm. The IC % was calculated
as:
IC % = A0 - As / A0 × 100
Where, A 0= Absorbance value of Control
sample As = Absorbance value of Test sample
IC % = Inhibitory concentration.
Percent inhibition was plotted against concen-
trations and the standard curve was drawn using
standard antioxidant (citric acid and EDTA) to
calculate the IC50 values for standard and essen-
tial oils.
Reducing power
The reducing power of essential oils was de-
termined by the method developed earlier and re-
cently being used 38. In brief varying concentra-
tions of essential oils (5-25 μg/mL) were mixed
with 2.5 mL of phosphate buffer (200 mM, pH=
6.6) and 2.5 mL of 1 % potassium ferricyanide,
K3[FeCN6]. After 20 minute incubation at 50 ±
1ºC, 2.5mLof trichloroacetic acid was added to
the mixtures, followed by centrifugation at 650
RPM for 10 min. The upper layer (1.0 mL) was
mixed with 5 mL distilled water and 1 mL of 0.1
% ferric chloride and absorbance of the resultant
solution were measured at 700 nm using UV spec-
trophotometer (Thermo Scientific Evolution 201
series). All the readings were taken as triplicate
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700
and BHT was used as the standard.
The reducing power of samples was calculated
using the formula given below:
Reducing activity % = A0 - As / A0 × 100
Where, A0 = Absorbance value of control sample
As = Absorbance value of test sample.
Percent inhibition was plotted against concen-
trations and the standard curve was drawn using
standard antioxidant (BHT) to calculate the RP50
values for standard and essential oils. The lower
RP50 value indicates greater reducing power abil-
ity.
Assay of nitric oxide scavenging activity
Nitric oxide (NO) was generated from sodium
nitroprusside (SNP) and was measured by the
Griess reagent. It is based on the principal that
SNP in aqueous solution at physiological pH spon-
taneously generates NO, which interacts with
oxygen to produce nitrite ions that can be esti-
mated by the use of Griess Reagent. Scavengers
of NO compete with oxygen leading to reduced
production of NO. 2 mL of SNP (10 mM) in phos-
phate buffer saline (PBS) pH 7.4 was mixed with
different concentrations of essential oil (5-25 μg/
mL) dissolved in acetone and incubated at 25°C
for two and half hours. The samples were re-
acted with 1 mL of Griss reagent (1 % sulph-
anilamide, 0.1 % naphthylethylenediamine dichlo-
ride and 2 ml orthophosphoric acid). Pink colour
will arise. Absorbance was reset at 546 nm.
Ascorbic acid was taken as standard 39.
Nitric oxide scavenging activity was calculated
by following equation.
IC % = A0 - As / A0 × 100
Where, A0= Absorbance value of control sample
As = Absorbance value of test sample IC = In-
hibitory concentration.
Percent inhibition was plotted against concen-
trations.The standard curve was drawn using
standard antioxidant (ascorbic acid) to calculate
the IC50 values for standard and essential oils.
Super oxide radical scavenging activity
1 mL of Nitroblue terazolium (156 Mm), 1 mL
Nicotinamide adenine dinucleotide (reduced) (468
690
Mm) and 0.1 mL of phenanzine methosulphate
solution (PMS) in 0.1 M of phosphate buffer so-
lution (pH 7.4) were added to different concen-
trations of essential oil (5 - 25 μg/ml) then incu-
bated at 25°C for 5 min and absorbance was read
at 560 nm against blank containing all reagent ex-
cept PMS. Ascorbic acid was taken as standard
40
.
Super oxide radical scavenging activity was cal-
culated by following equation.
Superoxide radical scavenged (%) = IC % =
A0 - As / A0 × 100
Where, A0= Absorbance value of control sample
As = Absorbance value of test sample IC % =
Inhibitory concentration.
Percent inhibition was plotted against concen-
trations.The standard curve was drawn using
standard antioxidant (ascorbic acid) to calculate
the IC50 values for standard and essential oil.
Statistical analysis
All experiments were performed thrice and the
results averaged data were expressed as mean ±
SD. Linear regression analysis was used to cal-
culate IC50 for each essential oils.
Results and discussion
Phytochemical analysis
Both the essential oils (type-I and II) were ana-
lyzed using GC-MS. Over 71 and 82 constituents
were identified in type -I and in type -II respec-
tively. The dominating compound was linalool in
both the morphotypes constituting 65.7 % and 63.8
% of the total oil respectively. The other major
constituents in type-I were eucalyptol (4.2 %),
neral (1.1 %), geranial (1.5 %), β-caryophyllene
(3.1 %), germacrene-D (3.3 %), germacrene-B
(1.6 %), 3E,5E-2,6-dimethyl-3,5,7-octatriene-2-ol
(2.1 %), 3E,5Z-2,6-dimethyl-3,5,7-octatriene-2-ol
(3.8 %) whereas in type-II were eucalyptol (4.7
%), β-citronellol (1.3 %), neral (2.3 %), geranial
(2.8 %), β-caryophyllene (2.3 %), germacrene-
D (3.0 %), germacrene-B (1.4 %), carryophyllene
oxide (1.1 %), 3E,5E-2,6-dimethyl-3,5,7-octa-
triene-2-ol (1.6 %), 3E,5Z-2,6-dimethyl-3,5,7-
octatriene-2-ol (2.9 %). The detailed comparison
of major and minor constituents in essential oil of
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700
morphotypes I and II are recorded in table 1.
Though linalool was the major compound in both
the oils, however some minor differences could
be seen in their quantitative make up. Constitu-
ents like n-hexanol,1-octen-3-ol, cis -arbusculone,
trans-pinocarveol, menthone, myrtenal, citronellyl
formate, trans linalool oxide acetate, aroma-
dendrene, α-himachalene, γ-amorphene, guaiyl ac-
etate were completely absent in type-I, whereas
type-II devoid of lilac aldehyde, terpinen-4-ol, E-
nerolidol. Constituents such as cis-linalool oxide,
isogeranial, β-citronellol, nerol, neral, geranial,
neryl acetate, caryophyllene oxide were found to
be slightly higher in type- II. According to results
both the morphotypes can be characterized as lina-
lool chemotype. In terms of class composition both
the oils of type -I and type -II were characterized
by high content of oxygenated monoterpenes (80.8
%) and (82.0 %) and low content of monoterpe-
nes hydrocarbons (2.1 %) and (2.2 %), while in
sesquiterpenoids oxygenated sesquiterpenes con-
Table 1. Essential oil profile of L.alba morphotypes
691
tributed 3.7 % and 3.8 % and sesquiterpene hy-
drocarbons contributed 11.3 % and 8.9 % of
the total oil respectively in type -I and type -II.
Comparison of oil composition of both the
morphotypes revealed variations in their compo-
sition in terms of the minor constituents as well as
small quantitative variations in major constituents.
Two different chemotype of L.alba viz. citral
and citral, lomonene and carvone has been re-
ported from Brazil 41. In another report on the
essential oil of L. alba from Brazil, the main char-
acterized components were the isomeric monot-
erpenes nerol/geraniol (27.09 %) and citral (21.87
%), as well as 6-methyl-5-heptene-2-one (11.98
%) and E-caryophyllene (9.25 %) 42.
Literature search also revealed that essential
oil of L. alba growing in northtern part of India
contained linalool (65.2 %), 1,8-cineole (6.6 %),
geraniol (2.8 %), terpinen-4-ol (2.7 %), α-terpin-
eol (1.6 %) as the major components 43. The re-
sults were somewhat similar to the oil reported

% Composition
No. Compound KI % Type I % Type II

1 4-Methyl-3-penten-2-one 798 t 0.1

2 trans-2-Hexenal 855 t t
3 3-Hexen-1-ol 858 t 0.1
4 n-Hexanol 867 - t
5 2-Methyl butyl acetate 880 t -
6 Isobutyl isobutyrate 895 0.1 t
7 α-Thujene 931 t t
8 α-Pinene 938 t t
9 Unidentified 0.1 -
10 5-Ethyl-2,4-dimethyl-2-heptane 972 0.2 0.2
11 Sabinene 973 0.7 0.8
12 1-Octen-3-ol 977 - 0.1
13 5-Methyl ,hept-6-ene-2-one 981 0.1 0.3
14 Myrcene 988 0.2 0.2
15 Eucalyptol 1034 4.2 4.7
16 E-α-Ocimene 1041 0.1 0.1
17 E-β-Ocimene 1051 1.0 1.0
18 cis-Arbusculone DB5-524 1053 - 0.4
19 Isoamyl isobutyrate 1064 0.1 t
20 cis-Linalool oxide 1067 t 0.2
21 trans-Linalool oxide 1088 0.2 0.1
21 Linalool 1098 65.7 63.8
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700 692
table 1. (continued).

% Composition
No. Compound KI % Type I % Type II

23 α-Pinene oxide 1103 0.2 0.1

24 p-Mentha-1,3,8-triene 1114 t t
25 trans-Pinocarveol 1129 - 0.1
26 Cosmene 1130 0.1 0.1
27 Menthone 1150 - 0.2
28 Lilac aldehyde 1154 t -
29 Pinocarvone 1164 0.1 0.1
30 p-Mentha-1,5-dien-8-ol 1166 0.2 0.2
31 Terpinen-4-ol 1176 0.1 -
32 cis-3-Hexenyl butyrate 1183 - 0.3
33 Isogeranial 1184 0.1 0.1
34 α-Terpineol 1189 0.4 0.5
35 Myrtenal 1193 - t
36 E-2-Hexenyl butanoate 1195 0.4 0.3
37 3E,5E-2,6-Dimethyl-3,5,7-octatriene-2-ol 1208 2.1 1.6
38 3E,5Z-2,6-Dimethyl-3,5,7-octatriene-2-ol 1209 3.8 2.9
39 β-Citronellol 1217 0.3 1.3
40 2-Hydroxy -1,8-cineol 1228 0.2 0.1
41 Nerol 1229 0.1 0.2
42 Neral 1235 1.1 2.3
43 E-Citral/geranial 1269 1.5 2.8
44 Citronellyl formate 1282 - 0.4
45 trans-Linalool oxide acetate 1283 - 0.1
46 Myrtenyl acetate 1328 t t
47 Citronelyl acetate 1356 - t
48 Eugenol 1359 0.1 0.2
49 Neryl acetate 1365 0.4 0.6
50 α-Copaene 1376 0.1 0.1
51 β-Elemene 1380 1.0 0.6
52 Neranyl acetate 1393 0.1 0.1
53 E-Caryophyllene 1415 3.1 2.3
54 Aromadendrene 1431 - 0.1
55 γ-Elemene 1434 0.3 0.3
56 Citronellyl propionate 1445 - 0.1
57 α-Himachalene 1450 - t
58 α-Humulene 1454 0.4 0.3
59 β-Farnesene 1459 1.0 0.4
60 Germacrene-D 1480 3.3 3.0
61 Citronellyl isobutyrate 1485 - 0.1
62 Neryl isobutyrate 1487 0.2 0.2
63 γ-Amorphene 1491 - 0.1
64 Bicyclogermacrene 1494 0.2 0.1
65 α-Muurolene 1502 0.1 0.1
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700 693
table 1. (continued).

% Composition
No. Compound KI % Type I % Type II

66 Germacrene-A 1512 0.3 0.3

67 γ-Cadinene 1513 0.1 0.1
68 Cubebol 1520 0.4 0.4
69 Bourbonanol DB5-1681 1520 0.1 0.1
70 δ-Cadinene 1524 0.2 0.1
71 Hedycaryol 1559 0.1 0.1
72 Germacrene-B 1561 1.6 1.4
73 E-Nerolidol 1565 t -
74 Z-Nerolidol 1572 0.7 0.4
75 Copaene-15-ol 1574 0.2 0.2
76 Germacrene D-4-ol 1576 0.1 0.1
77 Caryophyllene oxide 1578 0.8 1.1
78 β-Copaen-4-α-ol 1579 0.3 0.3
79 Spathulenol 1582 0.1 0.1
80 Humulene epoxideII 1606 0.1 0.1
81 Unidentified 0.1 -
82 Caryophylladienol II 1637 0.1 0.1
83 Cubenol 1643 t t
84 Unidentified - 0.3
85 2E,6Z-farnesal 1719 0.2 0.2
86 Guaiyl acetate 1736 - t
87 α-Costol 1778 0.1 0.2
88 β-Costol 1778.4 0.1 0.1
89 14-hydroxy-α-murrolene 1780 0.1 0.2
Monoterpene Hydrocarbons 2.1 2.2
Oxygenated monoterpenes 80.8 82.0
Sesquiterenes Hydrocarbons 11.3 8.9
Oxygenated Sesquiterpenes 3.7 3.8
Other 1.9 2.6
Total identified 99.8 99.7

KI: Kovat Index; t: trace (< 0.05 %)

from Brazilian species having 78.9 % linalool, 6.5
% 1,8-cineole, 3.5 % germacrene-D and 2.7 %
β-caryophyllene as the major constituents 22.
However our results show lower yields of lina-
lool, 1,8-cineole and other constituents. In another
published report from North India geranial (22.2
%) and neral (14.2 %) have been reported as
major constituents 44.
Similarly in another study on the essential oil of
L. alba from northern India the oil produced by
γ-irradiation was found to be rich in linalool (61.5-
67.7 % ) along with neral (0.3-3.3 %), geranial
(1.9-4.0 %), 1,8-cineole (0.3-3.6 %) 45. Interest-
ingly the other published work on essential oil com-
position of L.alba from north India was completely
different with myrcene (26.4 %) and geranial (9.8
%) as the major component 46. One more report
from northern India revealed, geranial (15.57 %),
myrthenal and myrthenol (9.89 %), neral (9.44
%), geraniol (7.36 %), 2,6-octadiene-1-ol-3,7-
dimethylacetate (6.87 %), 1-octan-3-ol(4.66 %),
6-methyl-5-hepten-2one (4.60 %), citronellol
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700
(2.63 %), linalool (2.20 %), 3-pinen-2-ol (2.19 %),
β-myrcene (4.19 %), caryophyllene oxide (4.52
%), farnesol (1.35 %), and β-caryophyllene (3.09
%) as major compounds 47. These distinct chemi-
cal composition points to a species with a great
variability in their chemical composition and fur-
ther research is necessary to confirm whether this
is due to geological or pedological conditions of
the collection site or due to expression of genes
according to climatic conditions.
Antioxidant activity
The antioxidant potential of L.alba essential oil
was evaluated using numerous assays. The first
step in these examinations is the screening of the
potential activity by different in vitro tests. Each
of those is based on one feature of the antioxi-
dant activity, such as the ability of scavenging free
radicals, the electron transfer ability, the chelat-
ing of transition metal ions (TMI), etc. However,
in order to get relevant data, a single method for
testing antioxidant activities of plant products is
not recommended due to their complex composi-
tion 48. Therefore, the antioxidant activity of the
tested essential oil has been evaluated in a series
of in vitro tests. In the present study, we exam-
ined the antioxidant property of L.alba using five
different assays. FRAP assay was performed to
evaluate the reducing antioxidant activity, whereas
DPPH, superoxide and NO radical scavenging
assays were conducted to investigate the radical
Table 2. Antioxidant activity of essential oils of L.alba morphotypes
694
scavenging activity of the essential oils. Iron
chelating activity was done to evaluate chelating
potential of the essential oils.
DPPH radical scavendging activity
In the DPPH assay, the ability of the investi-
gated essential oils to act as donors of hydrogen
atoms or electrons in transformation of DPPH
into its reduced form DPPH-H was investigated.
DPPH radical scavenging activity was studied for
essential oil at selected dose levels.Lower IC 50
value indicated higher antioxidant activity. Both
the assessed essential oils were able to reduce
the stable, purple-colored radical DPPH to yel-
low colored DPPH-H reaching 50 % of reduc-
tion of the two samples studied, the type II had
the higher free radical scavenging activity (IC50
= 21.05 ± 0.06) than type I (IC50 = 26.8 ± 0.04)
compared to standard catechin (13.67 ± 0.0) and
BHT (9.28 ± 0.1). The IC50 values are given in
Table 2. The DPPH radical scavenging activity
was observed minimum at lower dose level, how-
ever increases as a function of concentration and
was maximum at highest dose level.
Reducing power
The morphotypes showed different reducing ac-
tivity. On the basis of the results obtained for the
values of investigated system it can be seen that
the greatest reducing ability was shown by Type
II ( RP50 = 10.50 ± 0.63) than Type I (RP50 =

Sample/ DPPH Reducing Metal Super Oxide Nitric Oxide

Standard (IC50 μL/mL) Power Chelating Scavenging Scavenging
(RP50 μL/mL) (IC50 μL/mL) (IC50 μL/mL) (IC50 μL/mL)

Type I 26.8±0.04 15.99±1.23 12.13±0.44 20.85±0.41 22.16±0.85

Type II 21.05±0.06 10.5±0.63 8.1±0.17 12.46±0.11 14.01±0.06
BHT 9.28±0.09 8.95±0.20 - - -
Catechin 13.67±0.00 - - - -
Ascorbic acid - - - 9.84±0.92 11.26±0.80
EDTA - - 5.56±0.07 - -
Citric acid - - 4.04±0.63 - -

DPPH: 2,2-diphenyl-1-picrylhydrazyl
RP: reducing power
BHT: butylated hydroxyl toluene
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700
15.99 ± 1.23) essential oil and this ability is a bit
weaker compared to the one exhibited by the syn-
thetic antioxidant tert-butylated hydroxytoluene
(BHT) (RP 50 = 8.95 ± 0.20).
Metal chelating activity
Ferrozine, a chelating reagent, was used to in-
dicate the presence of chelator in the reaction
type. Ferrozine form a complex with free Fe3+
but not with Fe2+. In presence of chelating agents,
the complex formation between ferrous and
ferrozine is disturbed, resulting in decrease of the
colour of the complex. Measurement of colour
reduction therefore allows estimating the metal
chelating activity of the coexisting chelator 49. In
present study type II and I exhibit chelating activ-
ity with IC 50 values of (IC50 = 8.10 ± 0.17) and
(IC50 = 12.13 ± 0.44) respectively in comparison
to standards citric acid (IC50 = 4.04 ± 0.63) and
EDTA (IC 50 = 5.56 ± 0.07) (Table 2). Dose de-
pendency was observed during the metal chelat-
ing activity.
Superoxide radical scavenging activity
Superoxides are produced from molecular oxy-
gen due to oxidative enzymes of body as well as
via non enzymatic reactions such as auto-oxida-
tion by catecholamines. In the PMS/NADH-NBT
system, superoxide anion derived from dissolved
oxygen by PMS/NADH coupling reaction reduces
NBT. The decrease in the absorbance at 560 nm
with antioxidants thus indicates the consumption
of superoxide anion in the reaction mixture. The
essential oils exhibit superoxide radical scaveng-
ing activity in a dose dependent manner. Type- II
exhibited greater superoxide radical scavenging
activity (IC50 = 12.46 ± 0.11 μg/ml) followed by
type- I (IC50 = 20.85 ± 0.41) compared to the
standard, ascorbic acid (IC 50 = 9.84 ± 0.92)
(Table-2). The maximum activity was observed
at highest dose level.
Nitric oxide radical scavenging activity
The NO radical scavenging activity of essen-
tial oils in a dose-dependent manner at 546 nm
has been presented with respect to the standard
antioxidant ascorbic acid. Both the morphotypes
show significant scavenging activity. Type -II ex-
hibited more scavenging activity (IC50 = 14.01 ±
695
0.06 μg/ml) than type I (IC50 = 22.16 ± 0.85) as
compared to standard ascorbic acid (IC50 = 11.26
± 0.80). Both the oils exhibited dose dependent
NO radical scavenging activity with minimum at
lower dose level and maximum at highest dose.
The IC 50 values has been presented in table 2
and fig. 1.
An antioxidant can be defined as: “any substance
that, prevent the oxidative deterioration of sub-
stances or inhibits the oxidation of the substrate”.
The antioxidants obtained from plants have been
found more functional towards improving the shelf
life of food products and providing health promo-
tion 50.
The chemical complexity of essential oils, often
a mixture of dozens of compounds with different
functional groups, polarity and chemical behaviour,
could lead to diversified antioxidant activity de-
pending on the test employed 51. Antioxidant ac-
tivities of essential oils from aromatic plants are
mainly attributed to their chemical structure 52.
Antioxidant properties are very important in coun-
teracting the deleterious role of free radicals in
foods or biological systems. Excessive formation
of free radicals accelerates the oxidation of lipids
in foods, impairs food quality and consumer ac-
ceptance 53.The DPPH is a stable free radical,
which has been widely accepted as a tool for es-
timating the free radical scavenging activities of
antioxidants. The IC50 values of essential oil in
present investigation were calculated and com-
pared with that of standards and the antioxidant
potential was justified on the basis of their IC50
values. The lower IC50 value indicated stronger
ability of the oil to act as a DPPH scavenger while
the higher IC50 value indicated a lower scaveng-
ing activity. Free radicals cause auto-oxidation of
unsaturated lipids in food, and the antioxidant activ-
ity of the EOs could be attributed to their hydro-
gen donating ability 54. There is a consensus in
the literature that essential oils rich in monoterpe-
nes with aromatic rings attached to hydroxyl
groups (phenolics) have good antioxidant activity,
while oils rich in sesquiterpene hydrocarbons have
weak action. The main constituents of the essen-
tial oil obtained in our study are oxygenated monot-
erpenes (80.8 % & 82 %) respectively, which is
consistent with the observed activity. Solano et
al. reported a good antiradical activity of L. alba
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700
Fig. 1. Antioxidant activity of essential oils of L.alba morphotypes
essential oil with IC50 = 12.45 mg/mL and 15.6
Trolox eq μmol/g in DPPH and TEAC assay 55.
Puertas-Mejia et al. reported that L. alba essen-
tial oil samples from Colombia showed a low IC50
= 0.28 kg oil/mmol for DPPH and also displayed
lower activity against the ABTS radical (14.4
mmol Trolox/kg oil) 56. Our results from different
assays revealed moderate to good antioxidant
activity in two morphotypes.
The antioxidant efficiency of the L.alba essen-
tial oil has been attributed mainly to its dominanat
component linalool or it may also be caused by
the synergic effect of minor components as well
as by the interaction among the compounds. Lina-
lool, a monoterpene alcohol, is a component of
many natural aromatic plants. Linalool has been
found to have biological activities, including anal-
gesic, anti-inflammatory and antioxidant effects.
The role of linalool as antioxidant was confirmed
by the report where linalool reduced the levels of
nuclear factor-erythroid 2, a regulator of antioxi-
dant stress 57. In other report, linalool have been
found to be effective as an antioxidant in guinea
pig brains injected with H2O2 58, one of the major
reagents used in antioxidant studies. Also, in male
Wistar rats, linalool decreased oxidative stress by
696
modulating malondialdehyde, a marker for lipid
peroxidation and increased glutathione content 59.
The minor presence of α-terpineol, β-citronellol,
nerol, eugenol in addition to germacrene D,
germacrene B, β-caryophyllene and citral isomers
can also be responsible for the remarkable anti-
oxidant activity of the two morphotypes. The an-
tioxidant potential of these compounds have been
supported by various studies 60-64. Type-II
morphotype exhibited higher antioxidant activity
than Type -I. Since the quantity of linalool is al-
most similar in both the morphotypes,the higher
activity of Type -II can be attributed to the pres-
ence of minor compounds which are however
absent in Type-I.
Conclusions
Previous studies have shown that many spe-
cies of medicinal and aromatic plants exhibit sig-
nificant morphological and phytochemical variabili-
ties 41. The most common example is of Lantana
camara (Verbeneace) which has many morpho-
types with different coloured flowers 65. The major
component in the essential oil of L.alba morpho-
types in our study was linalool. Both the morpho-
types exhibit moderate to good radical scaveng-
 Archana Joshi et al., / TEOP 21 (3) 2018 687 - 700
ing and chelating activity. However it was found
that type II exhibit higher antioxidant activity. The
antioxidant activity in the essential oil of both the
morphotypes may be because of linalool. This
statement can be supported by the published re-
port on the antioxidant activity of linalool 66. How-
ever the higher antioxidant activity in type II might
be because of synergetic effects of other minor
constituents present in Type II morphotype.
The monoterpenoid linalool is used widely in the
flavour,fragrance, agrochemical and pharmaceu-
tical industries. Linalool is largely used in food and
cosmetics for its floral and sweet lemony aroma.
Hence this study concludes that essential oil from
L.alba could serve as an important bioresource
697
to extract natural linalool. Further, the antioxidant
and radical scavenging activity of the oil points
towards its strong protective role against oxida-
tive diseases. The antioxidant potential indicates
a possible use of L.alba essential oil as a natural
antioxidant, food supplement and as potential phar-
maceutical application. Also cultivation of this shrub
can be a good source of revenue generation for
local people besides its academic importance.
Acknowledgement
Medicinal Plant Research and Development
Centre (MRDC) of G.B.Pant University of Agri-
culture and Technology, Pantnagar and AIRF,JNU
are thankfully acknowledged.

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