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Journal of the Indian Chemical Society 99 (2022) 100723
Available online 21 September 2022
0019-4522/© 2022 Indian Chemical Society. Published by Elsevier B.V. All rights reserved.
A comparative study of selected vitex species for phenolics estimation along
with their antioxidant and herbicidal activities
Monika Tewari, Sonu Kumar Mahawer, Ravendra Kumar
*
, Om Prakash
Department of Chemistry, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar, 263145, Uttarakhand, India
ARTICLE INFO
Keywords:
Vitex species
Phenolics
Antioxidant activity
Herbicidal activity
ABSTRACT
This study aims to quantitative, qualitative estimation of phenolics, antioxidant (DPPH free radical scavenging,
nitric oxide radical scavenging, superoxide anion scavenging, reducing power and metal chelating activity), and
herbicidal properties of methanol extract of Vitex species {(Vitex negundo L. collected from Haldwani (VNH),
Bhimtal (VNB) and Salari (VNS); Vitex agnus-castus L. collected from Pantnagar (VACP); Vitex trifolia L. collected
from Pantnagar (VTP)}, Kumaun region, Uttarakhand, India. Herbicidal activity of methanol extracts of Vitex
species was evaluated against Raphanus sativus at various concentrations for 120 h. The results revealed that the
phenolic content varies from species to species as well as their location. HPLC proling of Vitex species was
carried out to identify the presence of different phenolic acids viz: vanillic acid, ferulic acid, p-coumaric acid, etc.
In methanol extracts of Vitex species. VTP showed the highest DPPH (IC
50
=6.84
μ
g/mL), nitric oxide (IC
50
=
2.86
μ
g/mL) and superoxide radical scavenging (IC
50
=3.12
μ
g/mL) activity. The maximum reducing power was
observed in VNH (RP
50
=28.22
μ
g/mL) and the highest ion chelating capacity in VNB (IC
50
=6.80
μ
g/mL). The
effective herbicidal activity was shown by VNS, resulting in complete inhibition of seed germination. Based on
the heatmap clustering selected species are divided into two clusters. The rst cluster comprises VNB and VTP
are there whereas in the second cluster there are two sub-clusters including VNS only in one sub-cluster and VNH
and VACP in the second sub-cluster.
1. Introduction
Antioxidants are capable of protecting the biological systems against
oxidative stress [1]
.
They terminate Reactive oxygen species (ROS)
chain reactions by removing free radical intermediates and inhibit
oxidation reactions by being oxidized themselves. The formulations with
antioxidants are currently being used for the prevention and treatment
of complex diseases such as Alzheimer’s and cancer [2]. Because of the
toxicity and side effects of synthetic antioxidants like butylated
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT),
plant-derived antioxidants are gaining importance, nowadays. Plants
have a wide variety of free radical scavenging molecules like terpenoids,
phenolic acids, quinones, coumarins, lignans and tannins, nitrogen
containing compounds (alkaloids, amines, betalains), vitamins, and
other endogenous metabolites which are potential antioxidants proved
by epidemiological and in vitro studies [3,4]. Phenolics, in particular
polyphenolics, are the most commonly identied as an allelopathic
compounds. The dietary role, medicinal importance, herbicidal poten-
tial, and phytotoxicity of polyphenolics have been reported.
Polyphenolic compounds like catechin, ferulic acid, etc. Have been
found to be signicantly phytotoxic [5]. Because of the pesticidal
properties of the plant extracts, their formulations with pesticides have
been recommended as good for agricultural practices [6].
Phenols are a member of a group of aromatic chemical compounds
with weakly acidic properties and are characterized by a hydroxyl (OH)
group attached directly to an aromatic ring. The presence of phenols is
considered to be potentially toxic to the growth and development of
pathogens [7]. The structural classes of phenolic compounds include
polyphenolic (hydrolysable and condensed tannins) and monomers such
as ferulic acid and catechol [8]. Quinones are aromatic rings with two
ketone substitutions. They are ubiquitous and are characteristically
highly reactive. These compounds, being colored, are responsible for the
browning reaction in cut or injured fruits and vegetables and are an
intermediate in the melanin synthesis pathway in human skin. The
natural quinine pigments range in color from pale yellow to almost black
and there are over 450 known structures. In addition to providing a
source of stable free radicals, quinones are known to complex irrevers-
ibly with nucleophilic amino acids in proteins often leading to
* Corresponding author.
E-mail address: ravichemistry.kumar@gmail.com (R. Kumar).
Contents lists available at ScienceDirect
Journal of the Indian Chemical Society
journal homepage: www.journals.elsevier.com/journal-of-the-indian-chemical-society
https://doi.org/10.1016/j.jics.2022.100723
Received 19 April 2022; Received in revised form 22 August 2022; Accepted 4 September 2022
Journal of the Indian Chemical Society 99 (2022) 100723
2
inactivation of the protein and loss of function [9]. Flavonoids are
15-carbon compounds generally distributed throughout the plant
kingdom. They are known to be synthesized by plants in response to
microbial infection and have been found in vitro to be effective against a
wide array of microorganisms [10]. Flavone with the molecular formula,
C
15
H
10
O
2
, is a commonly found plant avonoid. Flavonoids are potent
water-soluble super antioxidants and free radical scavengers, which
prevent oxidative cell damage, have strong anti-cancer activity, and
protects against all stage of carcinogens. Flavonoids in the body are
known to reduce the risk of heart diseases [11].
Vitex is the largest genus in the family Verbenaceae distributed all
over the world. The Vitex species are deciduous shrubs. Traditionally
some of its species are being used for rheumatic pains, sprains, inam-
mation, as anti-tubercular, anticancer, diuretic, respiratory infections, in
migraine, premenstrual problems, anti-fungal, and insecticidal. Several
Vitex species used in medicine include Vitex negundo Linn., Vitex agnus-
castus Linn. and Vitex trifolia. V. agnus-castus (chaste tree) is widespread
on river banks and on shores in the Mediterranean region, Southern
Europe, and Central Asia. V. negundo chiey occurs in Pakistan, India,
and Sri Lanka whereas, V. trifolia occurs in Asian countries and Vietnam
[12]. Most of the species of the genus Vitex are used therapeutically in
ancient Indian systems of medicine especially, Ayurveda and Siddha. It
includes approximately 270 known species of trees and shrubs in trop-
ical and subtropical regions, although a few species are also found in
temperate zones. There are about twelve species available in India with
medicinal value [13]. The major diagnostic characters of the genus Vitex
include their form (trees or shrubs); leaves (opposite, palmately com-
pound with three to ve pinnae, margins entire or seldom dentate),
presence or absence of a petiole; inorescence terminal, racemose or
axillary; and owers bisexual, slightly zygomorphic or zygomorphic
[14]. The genus Vitex is important in traditional medicine and has been
utilized for many years as an integral part of herbal medicines. These
species contain a variety of potentially bioactive molecules such as iri-
doids, avonoids, diterpenoids, derivatives, and phytosteroids. Most of
these species possess analgesic, anti-inammatory, antimicrobial, anti-
oxidant, hepatoprotective, antihistamine, and antiasthmatic properties.
The plant extracts of Vitex species in Europe and Asia illustrate its ef-
cacy and safety to treat current fatal diseases such as cancer, bacterial
infections, premenstrual syndrome, inammatory, immune related dis-
eases or lymphomas, central nervous system dysfunction, allergy, and
snakebite [15]. In view of the pharmacological activity and industrial
potential of the use of extracts, it is essential to check the phytochemi-
cals in the extracts of Vitex species.
With this background the current study was carried out concerning
different Vitex species i.e., Vitex agnus-castus, Vitex negundo, and Vitex
trifolia grown in different climatic regions around the Kumaun region,
Uttrakhand, India carried with the objectives as (1) phytochemical
analysis of the methanolic extract of vitex species, (2) assessment total
antioxidant capacity by the common methods (DPPH radical scavenging
activity, Nitric oxide radical scavenging activity, Superoxide anion
scavenging activity, Reducing power assay, Metal chelating activity)
and herbicidal activities of three selected vitex species from 5 different
regions, and correlation determination among antioxidant activity and
types of phenolic compounds with the help of heatmap clustering and to
clear that what type of phenolic compounds are governing for what type
of biological activity.
2. Materials and methods
2.1. Plant material
The plant material was collected from different regions of Kumaun,
Uttarakhand, India in the month of October (Table 1). The plant mate-
rial was taxonomically identied and authenticated by Dr. D. S. Rawat,
Assistant Professor (Plant Taxonomist), Department of Biological Sci-
ences, College of Basic Sciences & Humanities, Govind Ballabh Pant
University of Agriculture and Technology, Pantnagar, Uttarakhand
(India). The Herbarium specimen was deposited in the department for
future reference.
2.2. Extraction of plant material
The collected fresh leaves of VNH, VNB, VNS, VACP, and VTP were
washed thoroughly and kept for drying followed by grinding in a kitchen
mixture and grinder to form their powder. Then the powdered material
(100 gm) was subjected to extraction in methanol by cold percolation
method, individually, the extraction was performed three times and the
nal organic extracts were pooled for considering the one sample
extract. The prepared extracts were ltered through muslin cloth and
evaporated till it gets solidied under vacuum and nally, the yield of
extracts was recorded. The yield (w/w) of methanolic extracts were
found 10.66% (VNH), 13.46% (VNB), 12.47% (VNS), 10.56% (VACP)
and 11.86% (VTP). The extracts were stored in glass vials at 4 ◦C until
use.
2.3. Quantitative estimation of phenolics
The chemical assay of methanolic extract of Vitex species (VNH,
VNB, VNS, VACP, and VTP) was studied quantitatively by spectropho-
tometer (Thermo Scientic EVOLUTION 201 series) in terms of total
phenols, avanoids, and orthodihydroxy phenols and the concentration
of these samples were measured with the help of standard calibration
curve by the relation between absorbance and concentration of the
sample.
2.3.1. Total phenolics and avonoids content
The total phenolics of MeOH extracts were determined by the Folin-
Ciocalteu method modied by Shetty et al. [16]. The results were
expressed as mg of catechol per gram of sample in dried weight (DW).
The avonoid content of the extracts was measured using an aluminium
chloride colorimetric technique modied slightly from Nguyen and
Eun’s (2011) method [17]. The concentration of total avonoids was
measured in mg per 100 g of DW.
2.3.2. Estimation of orthodihydric phenols
Ortho-dihydric phenol content was determined according to Arnow’s
reagent method [18]. The intensity of the pink color developed was
measured by recording the absorbance at 515 nm and the results were
expressed in mg per 100 g of DW.
2.4. Qualitative estimation of phenolic acids
For qualitative estimation of phenolic acids in the tested samples, the
HPLC retention time is taken into consideration. The retention time of
standard phenolic acids was compared to the sample.
2.4.1. Phenolic acids
The crushed material (20 gm) was treated with 2 N HCl (125 mL) and
reuxed in a water bath at 100 ◦C for 45 min, cooled, and ltered. The
ltrate was extracted with diethyl ether, using a separating funnel. The
ether layer was collected and evaporated to obtain the nal product. The
material was re-dissolved in a known amount of HPLC grade methanol
(5 mL) before analysis. The sample was ltered through 0.5
μ
m organic
Table 1
Plant species used in experiment and location(s) of their collection.
S. No. Plant Species Location of collection
1. Vitex negundo L. Haldwani (VNH), Bhimtal (VNB) and Salari (VNS)
2. Vitex agnus-castus L. Pantnagar (VACP)
3. Vitex trifolia L. Pantnagar (VTP)
M. Tewari et al.
Journal of the Indian Chemical Society 99 (2022) 100723
3
lters (Millipore) before injecting on to the HPLC column. A 10
μ
L
sample was injected on the C-18 HPLC column for analysis using a micro
syringe of 25
μ
L tted with a blunt end type needle [19].
2.4.2. HPLC analysis of phenolic acids
For identication of phenolic acids in plant samples, gallic acid,
chlorogenic acid, p-hydroxy benzoic acid, vanillic acid, caffeic acid,
ferulic acid, p-coumaric acid, and p-benzoic acid were used as standard.
HPLC analysis was performed on a Waters 600, Quaternary gradient
HPLC system with microprocessor controller, 2996 photodiode array
detector, and 700 E recorder. The mobile phase was ltered through a
0.45
μ
, 47 mm Nylon-66 lter and degassed before use. The following
operational conditions of HPLC were maintained during the study:
Column C-18 (ODS) non-polar analytical column (4.6 ×250 mm)
Mobile phase 2% acetic acid in water-methanol (82:18 v/v) at a ow rate of 1
mL/min.
Detector PDA at 254 nm (xed wavelength)
Pressure 2300 psi.
Injection
volume
10
μ
L
Flow rate 1.0 mL/min
Run time 30 min
2.5. Antioxidant activity
To check the in-vitro antioxidant property the methanol extracts of
different Vitex species (VNH, VNB, VNS, VACP, and VTP) were subjected
following methods practiced and reported earlier.
2.5.1. DPPH radical scavenging activity
The free radical scavenging activity of extract on DPPH is analyzed
according to the method described by Anjum et al. [20]. In brief,
different amounts of the tested samples (20–100
μ
g/mL) were added to
5 mL of a 0.004% methanolic solution of DPPH. The sample was incu-
bated for 30 min at room temperature and the reduction of DPPH free
radical was measured at 517 nm using a UV–Vis spectrophotometer The
activity was expressed in 50% inhibition concentration (IC
50
; the con-
centration at which 50% of the total DPPH free radicals were inhibited),
calculated using the formula as follows:
IC
50
=[(Absorbance
(control)
- Absorbance
(sample)
) / Absorbance
(control)
] ×100
2.5.2. Nitric oxide radical scavenging activity
The nitric oxide radical scavenging activity of different methanolic
extracts was determined by the method described by Kumar et al. [21].
Briey, 2 mL of sodium nitroprusside (SNP) (10 mM) in phosphate
buffer saline (PBS) pH 7.4 was mixed with different concentrations of
samples (20–100
μ
g/mL) and incubated at 25 ◦C for two and a half hours
followed by addition of 1 mL of Griess reagent (1% sulphanilamide,
0.1% naphthyl ethylenediamine dichloride and 2 mL orthophosphoric
acid). Inhibition of NO⋅ was recorded at 546 nm using a UV–Vis spec-
trophotometer and the NO⋅ scavenging activity was expressed in IC
50
.
and calculated using the same formula as in DPPH scavenging activity.
2.5.3. Superoxide anion scavenging activity
The superoxide anion scavenging activity of different methanolic
extracts was determined by the method described by Kumar et al. with
slight modications [21]. In brief, a volume of 1 mL of nitro blue
tetrazolium (NBT) solution (100
μ
M of NBT in 100 mmol/L phosphate
buffer, pH =7.4), 1 mL of NADH (468
μ
mol in 100 mM/L phosphate
buffer, pH =7.4) solution and varying concentration of methanol ex-
tracts (20–100
μ
g/mL) were mixed. The reaction was initiated by the
addition of 100
μ
L of Phenazinemethosulphate solution (PMS) (60 mM
of PMS in 100 mM/L phosphate buffer, pH =7.4) and the reaction
mixture was incubated at 30 ◦C for 15 min scavenging or superoxide
anion was measured at 560 nm absorbance in a UV–Vis spectropho-
tometer and the superoxide anion reduction activity was expressed in
IC
50
and calculated using the same formula as in DPPH scavenging
activity.
2.5.4. Reducing power assay
The reducing power of different methanolic extracts was determined
by the method developed earlier and recently used by the method
described by Anjum et al. [20] was followed to determine the reducing
power of the extracts. Varying concentrations of tested sample (20–100
μ
g/mL) were mixed with 2.5 mL of phosphate buffer (200 mM, pH =
6.6) and 2.5 mL of 1% potassium ferricyanide, K
3
[FeCN
6
]. The mixtures
were incubated for 20 min at 50 ◦C followed by the addition of a volume
of 2.5 mL of trichloroacetic acid. the reaction mixture was centrifuged at
650 rpm for 10 min and 1 ml supernatant solution was mixed with 5 ml
distilled water and 1 mL of 0.1% ferric chloride and the absorbance of
the resultant solution was measured at 700 nm on the UV–Vis spectro-
photometer. The reducing capacity was expressed in IC
50
and calculated
using the same formula as in DPPH scavenging activity.
2.5.5. Metal chelating activity
The chelation of Fe
2+
was evaluated by adding 0.1 mL of 2 mM
FeCl
2
.4H
2
O, 0.2 mL of 5 mM ferrozine and 4.7 mL of methanol was in
different concentrations (20–100
μ
g/mL) of extract of Vitex species [22].
After incubating for 10 min the absorbance at 562 nm was measured in a
Thermo Scientic UV spectrophotometer. All the readings were taken in
triplicate and EDTA (0.01 mM) was used as the standard. The metal
chelating activity was expressed in IC
50
and calculated using the same
formula as in the DPPH scavenging activity.
2.6. Herbicidal activity
Herbicidal activity of methanol extract of Vitex species was evaluated
at different concentrations viz. 500 ppm, 1000 ppm, and 2000 ppm. For
this activity, pretreatment of seed includes surface sterilization with
95% ethanol for 15 s. After pretreatment, ten seeds were taken in each
Petri plate with three replicates for each concentration of test extract.
The Petri plates were layered with two ordinary lter papers each on
which 7 mL of test solutions of different compounds of varying con-
centrations (500 ppm, 1000 ppm, and 2000 ppm) were poured. A
mixture of distilled water and ethanol (30:1, 7 mL) was taken as control.
The radish seeds were allowed to germinate at 25 ◦C in an incubator
with 12 h of photoperiod. After 120 h, the number of seeds germinated
in each Petri plate was counted and percent seed germination inhibition
(SGI) values were calculated. The standard used for comparing the
herbicidal activity is pendimethalin.
2.7. Statistical analysis
SPSS 17.0 for Windows was used for statistical analysis (SPSS Inc.,
United States). To evaluate the signicance (p 0.05), the data was
studied using a thorough analysis of variances. All data were presented
as a mean standard deviation. For chemical analyses, antioxidant
testing, and herbicidal actions, statistical comparisons were made using
the Student’s t-test between three independent duplicates. Heatmapper
web server (http://www.heatmapper.ca) [23] was used to create an
interactive heat map that visualized all data (species, total phenol, a-
vonoids, orthodihydric phenols, DPPH radical scavenging activity, nitric
oxide radical scavenging activity, superoxide anion scavenging activity,
reducing power assay, Metal chelating and herbicidal actions at
different concentrations viz. 500 ppm, 1000 ppm, 2000 ppm).
M. Tewari et al.
Journal of the Indian Chemical Society 99 (2022) 100723
4
3. Results and discussion
3.1. Quantitative estimation of phenolics
3.1.1. Total phenolic content
Total phenolic content extracted from the selected plant species is
given in Table 2. The measured phenolic content ranged from 7.5 ±
0.30 mg to 14.93 ±0.472 mg GAE/g dry weight of the extract. The
maximum phenolic contents were found in VACP (14.93 ±0.472 mg
GAE/g dry weight of extract) whereas, the total phenolic content of the
extracts among Vitex species was observed in the following descending
order: VACP >VTP >VNS >VNB >VNH.
3.1.2. Flavonoids
A perusal of Table 2 revealed that the highest avonoid content was
found in VNB (46.33 ±0.577 mg CNE/g dry weight of extract) while the
lowest in VTP (32.83 ±1.15 mg CNE/g dry weight of extract). Total
avonoid content of the different plants’ extracts decreased in the
following descending order: VNB >VACP >VNS >VNH >VTP.
3.1.3. Orthodihydric phenol
The orthodihydric phenol content found in various extracts ranged
from 1.37 ±0.12 to 3.22 ±0.11 mg/g (CLE) among which VNH (i.e.,
1.37 ±0.12 mg/g) had the highest orthodihydric phenol content fol-
lowed by the VNB (2.48 ±0.06)>VNS (1.74 ±0.06)>VTP (1.70 ±
0.06)>VACP (1.37 ±0.12). Catechol is taken as standard and is
expressed as Catechol Equivalent per gram dry weight of plant extract.
The results of total orthodihydric phenol content are shown in Table 2.
3.2. Qualitative estimation of phenolic acids
To know the qualitative proling of phenolic acids the individual
phenolic acids were analyzed by HPLC. The results have been depicted
in Table 3. About 14, 12, 14, 17, and 9 compounds were found in the leaf
samples of VNH, VNB, VNS, VACP, and VTP, respectively. Among these,
vanillic acid was identied in VNH and VNB, whereas gallic acid in VNB,
VNS, and VACP, caffeic acid in VNS, VACP, and VTP, ferulic acid in all
the samples except VTP and p-coumaric acid in all the samples was
identied by HPLC.
Phytochemical studies of Vitex negundo, Vitex trifolia, and Vitex agnus-
castus have been reported to possess several types of compounds, such as
volatile oils, lignans, avonoids, terpenoids, steroids, iridoids, pheno-
lics, ecdysteroids, avonoids, and lignans [13]. A signicant amount of
phenolics and avonoids have been reported in leaves, fruits, and
owers of Vitex species viz. Vitex negundo, Vitex trifolia and Vitex
agnus-castus [24–34]. Caffeic acid has been reported in Vitex negundo L.
by HPLC–QTOF-MS method [35]. In the present study V. negundo and
Vitex agnus-castus extracts also revealed the presence of caffeic as
analyzed by HPLC. Similarly, the phenolic content, avonoid content,
and orthodihydric phenol content have also been identied in meth-
anolic extract of other Vitex species viz. VNH, VNB, VNS, VACP and VTP.
3.3. Antioxidant activity
3.3.1. 2,2′-diphenylpicrylhydrazyl (DPPH) radical scavenging activity
The free radical scavenging activity of the leaves methanolic extracts
of Vitex species was tested through the DPPH method and the results are
presented in Table 4. In the DPPH method, the antioxidants react with
the stable free radical i.e., 2,2-diphenyl-1-picrylhydrazyl (deep violet
color), and convert it into 2,2-diphenyl-1-picrylhydrazine with discol-
oration. The discoloration indicates the scavenging potentials of the
sample antioxidant such as phenolic compounds [36]. In the present
study the methanolic extracts of leaves of different Vitex species
collected from different places were able to decolorize DPPH and the
free radical scavenging potentials (IC
50
) of the extracts of were found to
be in the order of VTP (IC
50
=6.84
μ
g/mL) >VNB (IC
50
=6.99
μ
g/mL)
>VNS (IC
50
=7.76
μ
g/mL) >VNH (IC
50
=12.86
μ
g/mL) >VACP (IC
50
=14.25
μ
g/mL). The IC
50
of methanol extract of V. trifolia, Pantnagar
(IC
50
=6.84
μ
g/mL) was signicantly lower than that of V. negundo and
Table 2
Phenolic content in the methanolic extracts from leaves of different Vitex
species.
S.
No.
Sample
Name
Total phenol
(mg/g)
Flavonoids
(mg/g)
Orthodihydric phenol
(mg/g)
1 VNH 7.50 ±0.30 35.16 ±0.28 3.22 ±0.11
2 VNB 10.66 ±0.05 46.33 ±0.57 2.48 ±0.06
3 VNS 11.93 ±0.40 37.33 ±1.04 1.74 ±0.06
4 VACP 14.93 ±0.47 39.66 ±2.25 1.37 ±0.12
5 VTP 14.46 ±0.05 32.83 ±1.15 1.70 ±0.06
{(Vitex negundo L. collected from Haldwani (VNH), Bhimtal (VNB) and Salari
(VNS); Vitex agnus-castus L. collected from Pantnagar (VACP); Vitex trifolia L.
collected from Pantnagar (VTP)}.
Table 3
Phenolic acid proling of standards by HPLC.
S.
No.
Name of standard
phenolic acids
Retention
Time
VNH VNB VNS VACP VTP
1 p-coumaric acid 2.430 + + + + +
2 gallic acid 4.220 nd + + + nd
3 p-hydroxy
benzoic acid
5.448 nd nd nd nd nd
4 chlorogenic acid 5.756 nd nd nd nd nd
5 vanillic acid 6.540 + + nd nd nd
6 caffeic acid 6.773 nd nd + + +
7 ferulic acid 8.895 + + + + nd
8 p-benzoic acid 13.756 nd nd nd nd nd
nd =not detected.
Table 4
IC50 value of methanolic extracts from leaves of different Vitex species.
S.
No.
Plant
extracts
IC
50
of various antioxidant activities
DPPH radical scavenging
activity (
μ
g/mL) ±SD
NO radical scavenging
activity (
μ
g/mL) ±SD
Superoxide anionradical
scavenging activity (
μ
g/mL) ±SD
Reducing power activity of
Fe
+3
(
μ
g/mL) ±SD
Metal chelating activity of
Fe
+2
(
μ
g/mL) ±SD
1 VNH 12.86 ±0.051 4.21 ±0.009 4.16 ±0.001 28.22 ±0.79 8.39 ±0.013
2 VNB 6.99 ±0.004 4.01 ±0.003 4.04 ±0.002 34.88 ±0.15 6.80 ±0.004
3 VNS 7.76 ±0.023 4.16 ±0.004 3.16 ±0.001 34.62 ±0.33 9.80 ±0.059
4 VACP 14.25 ±0.188 2.97 ±0.002 4.05 ±0.003 57.38 ±0.13 6.99 ±0.010
5 VTP 6.84 ±0.015 2.86 ±0.003 3.12 ±0.001 43.46 ±0.33 9.29 ±0.021
6. Ascorbic
acid
2.68 ±0.002 64.41 ±0.110 4.73 ±0.003 – –
7. Gallic acid – – – 6.78 ±0.08 –
8. Na
2
EDTA – – – – 5.64 ±0.006
{(Vitex negundo L. collected from Haldwani (VNH), Bhimtal (VNB) and Salari (VNS); Vitex agnus-castus L. collected from Pantnagar (VACP); Vitex trifolia L. collected
from Pantnagar (VTP)}; Na
2
EDTA =sodium salt of ethylene di-amine tetra acetic acid.
M. Tewari et al.
Journal of the Indian Chemical Society 99 (2022) 100723
5
V. agnus-castus which revealed higher antioxidant activity.
3.3.2. Nitric oxide (NO) radical scavenging activity
Nitric oxide or reactive nitrogen species, formed by reaction with
oxygen or superoxide such as NO
2
, N
2
O
4
, N
3
O
4,
and NO
3
−
are very
reactive. These compounds are responsible for altering the structural
and functional behavior of many cellular components. Incubation of
solutions of sodium nitroprusside in phosphate buffer saline at 25 ◦C for
2 h resulted in linear time-dependent nitrite production, which is
reduced by the tested methanolic extracts of Vitex leaves. This may be
due to the antioxidant principles in the extract, which compete with
oxygen to react with nitric oxide thereby inhibiting the generation of
nitrite [37]. In present study, the order of nitric oxide radical scavenging
activity was observed in the order of VTP (IC
50
=2.86
μ
g/mL) >VACP
(IC
5
=2.97
μ
g/mL) >VNB (IC
50
=4.01
μ
g/mL) >VNS (IC
50
=4.16
μ
g/mL)>VNH (IC
50
=4.21
μ
g/mL) whereas the IC
50
of ascorbic acid
was 64.41
μ
g/mL. The variations in NO radical scavenging potential as
indicated by IC
50
values are recorded in Table 4.
3.3.3. Superoxide anion radical scavenging activity
Superoxide anion radical is one of the strongest ROS among the free
radicals and gets converted to other harmful reactive oxygen species
such as hydrogen peroxide and hydroxyl radical, damaging bio-
molecules which result in chronic diseases. In the current investigation,
we assessed in vitro superoxide anion scavenging activity of methanolic
extracts of different Vitex species, and the results were compared with
standard antioxidant ascorbic acid. Among different species of Vitex the
order of superoxide anion radical scavenging activity was observed as
VTP (IC
50
=3.12
μ
g/mL)>VNS (IC
50
=3.16
μ
g/mL)>VNB (IC
50
=4.04
μ
g/mL)>VACP (IC
50
=4.05
μ
g/mL)>VNH (IC
50
=4.16
μ
g/mL)
compared to the IC
50
of ascorbic (IC
50
=4.73
μ
g/mL). The variations in
NO radical scavenging potential have been calculated as IC
50
values and
are being recorded in Table 4. In this system, superoxide anion derived
from dissolved oxygen by PMS-NADH coupling reaction reduces NBT.
The decrease of absorbance at 560 nm with antioxidants indicates the
consumption of superoxide anion in the reaction mixture [38].
3.3.4. Reducing power
Reducing power is associated with antioxidant activity and serves as
a signicant reection of the antioxidant activity. Compounds with
reducing power indicate that they are electron donors and can reduce
the oxidized intermediates of lipid peroxidation processes so that they
can act as primary and secondary antioxidants. In this assay, the yellow
color of the test solution changes to various shades of green and blue
depending on the reducing power of each compound. The presence of
reducers causes the conversion of the Fe
3+
/ferricyanide complex used in
this method to the ferrous form [39]. The maximum reducing power was
observed in VNH (RP
50
=28.22
μ
g/mL) while the minimum reducing
power is shown by VACP (RP
50
=57.38
μ
g/mL). The RP
50
values of
different methanolic extracts were observed in the order of VNH (RP
50
=28.22
μ
g/mL) >VNS (RP50 =34.62
μ
g/mL) >VNB (RP
50
=34.88
μ
g/mL) >VTP (RP
50
=43.46
μ
g/mL)>VACP (RP
50
=57.38
μ
g/mL)
compared to the RP
50
of gallic acid (RP
50
=6.78
μ
g/mL). The detailed
description of data related to this activity has been recorded in Table 3.
3.3.5. Ferrous ion-chelating activity
The transition metal, iron is capable of generating free radicals from
peroxides by Fenton reaction and may be implicated in human cardio-
vascular disease. Thus, the ability to chelate transition metals is
considered to be an important antioxidant mode of action. The chelating
agents forming
σ
-bonds with a metal ion are effective as secondary an-
tioxidants as they reduce the redox potential thereby stabilizing the
oxidized form of the metal ion. Furthermore, it has been reported that
non-avonoid polyphenolics can reduce iron and form Fe
2+
polyphenol
complexes that are inert in nature [40]. Na
2
EDTA is a known metal ion
chelator; and therefore, the chelating effect of Vitex species was
compared with it. The percentage of metal chelating capacity of Vitex
extracts and positive control Na
2
EDTA was recorded in the order of
EDTA (IC
50
=5.64
μ
g/mL) >VNB (IC
50
=6.80
μ
g/mL) >VACP (IC
50
=
6.99
μ
g/mL) >VNH (IC
50
=8.39
μ
g/mL) >VTP (IC
50
=9.29
μ
g/mL)>
VNS (IC
50
=9.80
μ
g/mL) from 20 to 100
μ
g/mL concentration. The
variations in the metal chelating ability of different extracts indicated by
IC
50
values are recorded in Table 4.
Free radicals such as singlet oxygen and hydrogen peroxide are
generated by an oxidation process that ultimately led to damage to the
body cells and causes oxidative stress. Thus, for the survival of all life
forms detoxication of reactive oxygen species is highly essential.
Antioxidant compounds are capable of mitigating the negative effects of
oxidative stress as they are efcient scavengers of free radicals. They can
be either natural or synthetic, however, synthetic antioxidants are
considered harmful to health. Therefore, there is a need to look for new
natural sources with potential pharmaceutical and antioxidant capa-
bilities [41]. Phenolic compounds are commonly found in both edible
and inedible plants and various plant parts. They have been reported to
have multiple biological effects, including antioxidant activity. The
antioxidant activity of phenolic compounds is mainly due to their redox
properties, which play an important role in adsorbing and neutralizing
free radicals, quenching singlet and triplet oxygen, or decomposing
peroxides [42]. Flavonoids are very effective anti-oxidants. The
different mechanisms which provide antioxidative properties to avo-
noids are scavenging of free radicals, chelation of metal ions, such as
iron and copper, and inhibition of enzymes responsible for free radical
generation [28].
Aweng et al. [31] analyzed the methanolic extract of Vitex trifolia
var. simplicifolia for antioxidant activity and reported the presence of
phenolic compounds. Similarly, Devi and Singh [43] also investigated
the total phenols content and antioxidant activity in Vitex trifolia leaves
extract. Rashed evaluated phytoconstituents and antioxidant activity of
aerial parts of Vitex agnus castus for different extracts and found that the
antioxidant potential of V. agnus-castus aerial parts ethyl acetate extract
is due to the presence of phenolic compounds (tannins and avonoids)
[44]. Likewise, Maltas¸ et al. also analyzed the total avonoid content,
phenolic content, and antioxidant activity of methanolic extract of Vitex
agnus-castus [45]. In another study, the antioxidant properties of the
extracts were evaluated using different antioxidant tests. The results
revealed that the methanolic extract of Vitex agnus-castus exhibited
stronger antioxidant activity as it is rich in phenolic compounds. Like-
wise, Zargar et al. also reported antioxidant activity of methanol extract
from Vitex negundo leaf using DPPH, free radical scavenging capacity,
FRAP, and β-carotene-linoleic acid assays [46]. Saklani et al. suggested
that TPC and TFC presented in the V. negundo and V. trifolia leaf extracts
could be an important source of antioxidant molecules [33]. In the
present investigation, the methanol extract of Vitex species viz: VNH,
VNB, VNS, VACP, and VTP possesses a signicant amount of total phe-
nolics, avonoids, and ortho-dihydric phenols. The methanolic extract of
Vitex species exhibited excellent antioxidant properties. Hence, it can be
inferred that the antioxidant activity in methanolic extracts of Vitex
species might be due to the presence of a good amount of phenolic
content in the extracts. Dietary phenolic acids which occur ubiquitously
in plants, play a major role as protective in oxidative stress conditions.
Phenolic acids such as ferulic acid, caffeic acid, p-coumaric acid, gallic
acid, chlorogenic acid, and rosmarinic acid, readily absorbed through
intestinal tract walls, are benecial to human health due to their po-
tential antioxidants and avert the damage of cells resulting from
free-radical oxidation reactions [47]. HPLC proling of Vitex species was
carried out to identify the presence of different phenolic acids viz:
vanillic acid, ferulic acid, p-coumaric acid, caffeic acid, gallic acid, and
gallic acid.
3.4. Herbicidal activity
Redish (Raphanus sativus) has been subjected to be tested for
M. Tewari et al.
Journal of the Indian Chemical Society 99 (2022) 100723
6
herbicidal activity of plant extracts since it has a rapid and good
germination percentage [48,49]. The seed germination inhibition ac-
tivity of the methanol extracts of Vitex species (VNH, VNB, VNS, VACP,
and VTP) was studied on radish a (Raphanus sativus) seed at three
different concentrations (500 ppm, 1000 ppm, and 2000 ppm) and the
activity was compared with standard herbicide, pendimethalin (3,
4-dimethyl-2,6-dinitro-N-pentan-3-yl-aniline) which is a pre and post-
emergence herbicide to control annual grasses and certain broadleaf
weeds. After 120 h of experiment, seed germination inhibition values of
methanolic extracts at different concentrations were recorded and the
mean seed germination values are depicted in Table 5.
The methanolic extracts of Vitex species showed marked herbicidal
activity against germination of radish (Raphanus sativus) seeds. The seed
germination inhibition activity of different extracts of Vitex species was
found to be dose dependent i.e., increase signicantly with increasing
concentrations. At lower concentration, percent germination inhibition
was recorded in the order of pendimethalin >VNS >VACP >VNH >
VTP >VNB (Table 5). The methanolic extract of VNS was found to be the
most effective at the 2000 ppm concentration completely inhibiting the
seed germination. Its activity was at par with that of standard pendi-
methalin for all the dose levels. It was evident from the results that VNS
(SGI =93.33–100%) showed better herbicidal activity than VNH (SGI =
56.56–96.66%) and VNB (SGI =13.33–93.33%). Highest SGI was
recorded in the case of VNS whereas, it was recorded lowest in the case
of CNB at all the test concentrations. As compared to different species,
V. negundo showed better seed germination inhibition activity as
compared to V. agnus-castus and V. trifolia. From preliminary screening,
it was found that the extract of V. negundo, V. agnus-castus, and V. trifolia
showed signicant inhibition of germination in the seeds of Raphanus
sativus.
It has been reported that phenolics and terpenoids present in plants
are responsible for the inhibition of germination as these are found to
have inhibitory effects on the growth of nitrogen xing and nitrifying
bacteria [50,51]. The results of the present study resemble similar to the
study done by Azizuddin and Choudhary on the herbicidal potential of
the methanolic extract of V. agnuscastus against Lemna aequinoctialis
Welv [52]. They revealed that V. agnus-castus has excellent phytotoxicity
at the highest (500
μ
g/mL) tested concentration and caused 82.5% in-
hibition of L. aequinoctialis Welv. Phenolic acids such as catechin,
p-coumaric acid, ferulic acid, and phloridzin have been reported as a
phytotoxic agents in Chrysanthemoides monilifera subsp. monilifera
(Boneseed) [5]. Some of these phenolic acids were also identied in the
present investigation (see Fig. 1).
Table 5
Mean percent seed germination inhibition values of methanolic extracts from
different Vitex species at different concentration.
S.No. Extracts/Standard Dose (ppm)
500 ppm 1000 ppm 2000 ppm CD at 5%
1 Pendimethalin 100.00 100.00 100.00 00.00
2 VNH 56.66 83.33 96.66 11.54
3 VNB 13.33 73.33 93.33 11.54
4 VNS 93.33 96.66 100 –
5 VACP 66.66 93.33 96.66 11.54
6 VTP 16.66 83.33 96.66 11.54
{(Vitex negundo L. collected from Haldwani (VNH), Bhimtal (VNB) and Salari
(VNS); Vitex agnus-castus L. collected from Pantnagar (VACP); Vitex trifolia L.
collected from Pantnagar (VTP)}.
Fig. 1. HPLC- chromatograms for phenolic acid proling in methanol extracts of different Vitex species. (Vitex negundo L. collected from Haldwani (VNH), Bhimtal
(VNB) and Salari (VNS); Vitex agnus-castus L. collected from Pantnagar (VACP); Vitex trifolia L. collected from Pantnagar (VTP)}.
M. Tewari et al.
Journal of the Indian Chemical Society 99 (2022) 100723
7
3.5. Heatmap clustering
Based on chemical constituents, antioxidant activities, and herbi-
cidal activities, all the ve species of Vitex were visualized within the
heatmap (Fig. 2). All the species are clearly divided into two main
clusters. In the rst cluster only two species viz. VNB and VTP are there
whereas in the second cluster there are two sub-clusters comprising VNS
only in one sub-cluster whereas VNH and VACP are in the second sub-
cluster. VACP in the second cluster) shows high DPPH radical scav-
enging activity, superoxide anion scavenging activity, reductive power
assay, and total phenol content. These two Antioxidant activities in
VACP are suggested to be governed by phenolic compounds. In the same
cluster, VNH also shows a similar activity as VACP in terms of DPPH
radical scavenging activity, superoxide anion scavenging activity with
lesser total phenol content but highest orthodihydric phenols which
suggest that these two types of antioxidant activities may govern spe-
cically by orthodihydric phenols at some constant. Based on the heat
map clustering data it is revealed that VNS (separate subcluster in the
second cluster) is showing the highest herbicidal activities at all the
concentrations (500 ppm, 1000 ppm, and 2000 ppm).
4. Conclusion
The methanolic extracts of different Vitex species were studied
quantitatively for their biochemical assay in terms of their total phenols,
avonols, and orthodihydric phenolic content. The methanol extract of
Vitex species was found to possess a signicant amount of total pheno-
lics, avonoids, and ortho-dihydric phenols. HPLC proling of Vitex
species was carried out to identify the presence of different phenolic
acids viz: vanillic acid, ferulic acid, p-coumaric acid, caffeic acid, gallic
acid, and gallic acid. The methanolic extract of Vitex species exhibited
excellent antioxidant properties and herbicidal activity. The results of
the present study suggested that the leaves of Vitex species (Vitex
negundo L., Vitex agnus-castus L., Vitex trifolia L.,) could be potent source
of natural antioxidants because of their phenolic, avonoid, and
orthodyhydric phenol contents and also an opportunity to develop
phytotoxic agent to promote sustainable agriculture. Further studies
should be directed toward the extensive in vivo antioxidant activities and
eld trials to measure the phytotoxicity of the Vitex species and the
relationship of individual phenolic compounds to antioxidants with
different mechanisms and isolation, screening, and characterization of
individual compounds responsible for antioxidant properties and her-
bicidal potential to authenticate their probable uses as sources of natural
antioxidants and plant based herbicidal agent.
Authors contribution
MT-literature survey, experimental work, SKM- manuscript writing,
RK-research design, OP- manuscript drafting.
Funding
There is no specic nancial grant received from any funding
agencies for this work.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this review article.
Acknowledgment
The author hereby acknowledges Dr. D.S. Rawat, Department of
Biological Sciences, College of Basic Sciences and Humanities, G.B.P.U.
A. & T., Pantnagar in identifying the plant specimen. Thanks to
Advanced Instrumentation Research Facility, J.N.U., New Delhi for
facilitating GC-MS analysis of the plant sample.
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