molecules

Communication
In Vitro Antimelanoma Properties of Verbena officinalis Fractions
Rabia Nisar 1 , Sanjay Adhikary 2 , Saeed Ahmad 1 and Mohammad Abrar Alam 2, *

1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The Islamia University of Bahawalpur,

Bahawalpur 63100, Pakistan
2 Department of Chemistry and Physics, College of Sciences and Mathematics, Arkansas State University,
Jonesboro, AR 72404, USA
* Correspondence: malam@astate.edu

Abstract: Verbena officinalis is commonly used in traditional medicine to treat many ailments. Extracts
of this plant are therapeutic agents for the potential treatment of different diseases, including colorectal
and liver cancers, but have not been explored for their anti-melanoma potential so far. The goal of
the current work was to prepare a methanolic extract and fractionate it using hexane, chloroform,
ethyl acetate, butanol, and acetone to get semi-purified products. These semi-purified fractions were
studied for their potency against melanoma cell lines. The three potent fractions (HA, VO79, and
EA3) demonstrated 50% inhibition concentration (IC50 ) values as low as 2.85 µg/mL against the LOX
IMVI cell line. All three fractions showed similar potency in inhibiting the growth of the B16 cells, a
murine melanoma cell line. Based on high-resolution mass spectrometry (HRMS) data, for the first
time, we report on lupulone A from this plant. LC-MS data also indicated the presence of hedergonic
acid, serjanic acid, and other compounds in V. officinalis extracts.

Keywords: Verbena officinalis; melanoma; cytotoxicity; medicinal plant; LC-MS

Citation: Nisar, R.; Adhikary, S.;

Ahmad, S.; Alam, M.A. In Vitro 1. Introduction
Antimelanoma Properties of Verbena Humans have been utilizing plants therapeutically for a long time. The World Health
officinalis Fractions. Molecules 2022, Organization (WHO) estimates that almost 80% of the world’s population relies on medic-
27, 6329. https://doi.org/10.3390/ inal plants to cover their basic healthcare needs [1]. Traditional medications made from
molecules27196329 plants are considered more effective and safer clinically when compared to synthetic enti-
Academic Editors: Heiko Lange and ties [2]. Approximately 25% of the medications prescribed worldwide are originated from
Margherita Brindisi plants [3]. Medicinal plants containing a rich source of bioactive compounds are the most
common source of novel drug discovery and are particularly useful as antimicrobial and
Received: 20 August 2022
anticancer therapeutics [4]. A recent analysis found that around 50% of approved anticancer
Accepted: 21 September 2022
drugs between 1940 to 2014 were obtained from natural sources or directly derived from
Published: 26 September 2022
them [5].
Publisher’s Note: MDPI stays neutral Verbena officinalis, a plant from the Verbenaceae family, is commonly called pigeons’
with regard to jurisdictional claims in grass, herb of grace, or vervain. This plant is popularly called bitter herb or Kori-booti
published maps and institutional affil- in Pakistan. It is mostly found in Asia, North Africa, and Europe. This plant is fairly
iations. distributed near water in cultivated fields and wastelands in the western and northern
regions of Pakistan. It is a perennial erect herb, which grows to a height of about 25–100 cm,
having toothed and lobed leaves. The delicate spikes hold elegant, silky, pale purple, or
pink flowers [6]. In the traditional herbal system of medicines, V. officinalis has been em-
Copyright: © 2022 by the authors.
ployed for the treatment of many ailments such as gastric diseases, abrasion, skin burns [7],
Licensee MDPI, Basel, Switzerland.
wounds, thyroid problems, rheumatic pain [8], asthma and cough [9], amenorrhea, en-
This article is an open access article
teritis, acute dysentery [10], expectorant and diuretic [11]. V. officinalis studied for its new
distributed under the terms and
conditions of the Creative Commons
important bioactivities including analgesic and anti-inflammatory [12,13], antioxidant [14],
Attribution (CC BY) license (https://
antifungal [15], anticonvulsant [6], antibacterial [16,17], anticancer [18,19], neuroprotec-
creativecommons.org/licenses/by/ tive [10], antidepressant [20], antiproliferative [21], urolithiasis [22], and antitumor [23]
4.0/). effects. V. officinalis has a pool of bioactive metabolites, including flavonoids [24], sterols

Molecules 2022, 27, 6329. https://doi.org/10.3390/molecules27196329 https://www.mdpi.com/journal/molecules

 new important bioactivities including analgesic and anti-inflammatory [12,13],
antioxidant [14], antifungal [15], anticonvulsant [6], antibacterial [16,17], anticancer
[18,19], neuroprotective [10], antidepressant [20], antiproliferative [21], urolithiasis [22],
Molecules 2022, 27, 6329 2 of 8
and antitumor [23] effects. V. officinalis has a pool of bioactive metabolites, including
flavonoids [24], sterols and triterpenoids [25], phenylethanoid glycosides [21], iridoids
[26] and ursolic acid [27], which further explains the folk use of this plant [6]. In addition,
and triterpenoids
the species nowadays [25], phenylethanoid
is recognized glycosides
as a valuable [21], iridoids
cosmetic plant, [26] and due
mainly ursolic
to acid
the [27],
which further explains the folk use of this plant [6]. In addition, the species
presence of essential oils. The vervain herb is characterized by high variability in chemical nowadays is
recognized as a valuable cosmetic
composition depending on its origin [28]. plant, mainly due to the presence of essential oils. The
vervain herb
Melanoma is is characterized
the by high
most fatal and variability
aggressive skinincancer
chemicalthatcomposition depending
accounts for 3% of allon its
origin [28].
malignant cancer [29]. Melanoma has a high potential for metastasis and invasion, which
Melanoma is the most fatal and aggressive skin cancer that accounts for 3% of all
accounts for about 75% of death related to skin cancer worldwide [30]. It is the 5th and 7th
malignant cancer [29]. Melanoma has a high potential for metastasis and invasion, which
most prevalent cancer in American men and women, respectively [31]. In the United
accounts for about 75% of death related to skin cancer worldwide [30]. It is the 5th and
States, the expected number of new cases and deaths in 2022 will be 99,780 and 7,650,
7th most prevalent cancer in American men and women, respectively [31]. In the United
respectively [32]. Antimelanoma properties of plant extracts such as Aloysia citrodora
States, the expected number of new cases and deaths in 2022 will be 99,780 and 7650,
essential oil inhibit melanoma cell growth and migration by targeting HB-EGF-EGFR
respectively [32]. Antimelanoma properties of plant extracts such as Aloysia citrodora
signaling [33]. The aim of this study was to analyze the important secondary metabolites
essential oil inhibit melanoma cell growth and migration by targeting HB-EGF-EGFR
of V. officinalis for the evaluation of their antimelanoma potential. Until now, this is the
signaling [33]. The aim of this study was to analyze the important secondary metabolites
first anti-melanoma study of V. officinalis extracts.
of V. officinalis for the evaluation of their antimelanoma potential. Until now, this is the first
anti-melanoma study of V. officinalis extracts.
2. Results and Discussion
2.1. In2.Vitro
Results and Discussion
Antimelanoma Studies of Different Fractions
2.1. In Vitro
Initially, we Antimelanoma Studiesextracts
tested the different of Different
of V.Fractions
officinalis at 50 µg/mL against the LOX
IMVI cells, a melanoma
Initially, cell
we tested theline. Acetone
different andof n-butanol
extracts V. officinalisfractions
at 50 µg/mL didagainst
not show any IMVI
the LOX
cells, a for
cytotoxicity melanoma cell line.cell
this melanoma Acetone
line atand
thisn-butanol fractions
concentration did1).
(Figure notTherefore,
show anywe cytotoxicity
did
for thisthese
not pursue melanoma cell line at
two fractions ofthis
theconcentration (Figureof
methanolic extract 1).V.Therefore,
officinalis we did not
further. Thepursue
these
extracts of two fractions
hexane, of the methanolic
chloroform, and ethyl extract
acetate of V. officinalis
showed > 70%further.
growthThe extracts at
inhibition of 50
hexane,
µg/mLchloroform, and ethyl
concentration. We acetate
furthershowed >70% growth
fractionated inhibition
the extract at 50 µg/mL
of chloroform by concentration.
column
We further
chromatography. fractionated the extract of chloroform by column chromatography.

FigureFigure Flow for

1. chart
1. Flow chart
thefor the melanoma
melanoma activeactive fractions
fractions of V. officinalis.
of V. officinalis.

All these fractions were tested against LOX IMVI cell line at 50 µg/mL concentration.
All these fractions were tested against LOX IMVI cell line at 50 µg/mL concentration.
Extracts showing >70% growth inhibition were tested against the cell lines at five serial
Extracts showing > 70% growth inhibition were tested against the cell lines at five serial
dilutions. We found three potent fractions (HA, VO79, and EA3) with 50% inhibition
dilutions. We found three potent fractions (HA, VO79, and EA3) with 50% inhibition
concentration (IC ) as low as 2.8 µg/mL against the LOX IMVI cell line (Table 1). The
concentration (IC50) as 50 low as 2.8 µg/mL against the LOX IMVI cell line (Table 1). The
fraction VO79 inhibited the growth of melanoma cell lines (SK MEL 28, LOX IMVI, and SK
fraction VO79 inhibited the growth of melanoma cell lines (SK MEL 28, LOX IMVI, and
MEL 5) with the IC50 values in the range of 6.2 to 11.6 µg/mL. This fraction inhibited the
SK MEL 5) with the IC50 values in the range of 6.2 to 11.6 µg/mL. This fraction inhibited
growth of a murine melanoma cell line (B16 cell line) with an IC50 value of 7.0 µg/mL. The
the growth of a murine melanoma cell line (B16 cell line) with an IC50 value of 7.0 µg/mL.
HA fraction was found to be the most potent isolate of the V. officinalis to inhibit the growth
The HA fraction
of LOX IMVIwas found
cell lines.toThe
be the
EA3most potent
fraction isolate
was veryof the V. officinalis
efficient to inhibit
in inhibiting the of
the growth
SK-MEL-28 and LOX IMVI cell lines with the IC50 values of 4.8 and 3.3 µg/mL, respectively.
All three fractions inhibited the growth of the B16 cell line effectively, with IC50 values as
low as 6.2 µg/mL. These results are very significant as the positive controls is 3–4 times
less potent than these fractions against the melanoma cell lines except for LOX IMVI cell
line against cisplatin.
 growth of LOX IMVI cell lines. The EA3 fraction was very efficient in inhibiting the
growth of LOX IMVI cell lines. The EA3 fraction was very efficient in inhibiting the
growth of SK-MEL-28 and LOX IMVI cell lines with the IC50 values of 4.8 and 3.3 µg/mL,
growth of SK-MEL-28 and LOX IMVI cell lines with the IC50 values of 4.8 and 3.3 µg/mL,
respectively. All three fractions inhibited the growth of the B16 cell line effectively, with
respectively. All three fractions inhibited the growth of the B16 cell line effectively, with
IC50 values as low as 6.2 µg/mL. These results are very significant as the positive controls
IC50 values as low as 6.2 µg/mL. These results are very significant as the positive controls
Molecules 2022, 27, 6329 is 3–4 times less potent than these fractions against the melanoma cell lines except for LOX3 of 8
is 3–4 times less potent than these fractions against the melanoma cell lines except for LOX
IMVI cell line against cisplatin.
IMVI cell line against cisplatin.
Table 1. IC50 values of the HA, VO79, EA3 fractions (µg/mL) against murine (B16) and human (SK
Table
Table 1.50 IC
1. IC 50 values
values of theof the VO79,
HA, HA, VO79, EA3 fractions
EA3 fractions (µg/mL)(µg/mL) against murineand
(B16) and (SK
human
MEL 28, LOX IMVI, and SK MEL 5) melanoma cell lines. NB:against murine
IC50 values are (B16)
presentedhuman
with mean
MEL (SK
28,MEL
LOX 28, LOXand
IMVI, IMVI, and SK
SK and
MEL MEL 5) melanoma
5) melanoma cell lines.cell
NB:lines. NB: IC50
IC50 values arevalues are presented
presented with meanwith
± Standard deviation. Taxol cisplatin are positive controls.
± Standard
mean ±deviation. Taxol and cisplatin
Standard deviation. Taxol andare positive
cisplatin arecontrols.
positive controls.
Fraction IC50 (µg/mL)
Fraction ICIC50 (µg/mL)
50 (µg/mL)
Fraction B16 SK MEL 28 LOX IMVI SK MEL 5
B16
B16 SKSKMELMEL28 28 LOX
LOX IMVI
IMVI SKSKMEL MEL5 5
HA 7.6± 1.1 10.6 ± 1.0 2.8 ± 0.2 8.0 ± 0.1
HA HA 7.6±
7.6±±1.1
1.1 10.6 ±
10.6± ±1.0 2.8 ±
2.8±±0.2 8.08.0
± 0.1
± 0.1
VO79 7.0 0.8 7.2 0.71.0 6.2 0.10.2 9.6 ± 0.0
VO79
VO79 7.0
7.0 ±±0.8
0.8 7.2
7.2±±0.70.7 6.2
6.2± ±
0.10.1 9.69.6
± 0.0
± 0.0
EA3
EA3
6.2 ± 0.8
6.2 ±±0.8
0.8
4.8 ± 1.0
4.8±±1.01.0
3.3 ± 0.0
3.3± ±
6.2 ± 0.1
± 0.1
EA3 6.2 4.8 3.3 0.00.0 6.26.2
± 0.1
Taxol
Taxol (µM)
(µM) 27.4
27.4 ±±4.5
4.5 27.3
27.3±±2.812.81 32.1
32.1±± 1.1
1.1 19.8 ± 3.1
19.8 ± 3.1
Taxol (µM) 27.4 ± 4.5 27.3 ± 2.81 32.1 ± 1.1 19.8 ± 3.1
Cisplatin
Cisplatin 24.3 ±±1.0
24.3 1.0 5.4±±
5.4 0.30.3 27.127.1 ± 3.1
± 3.1
Cisplatin 24.3 ± 1.0 5.4 ± 0.3 27.1 ± 3.1
2.2.2.2. Phytochemical
Phytochemical Analysis
Analysis of V.ofofficinalis
V. officinalis by HR-ESI-MS
by HR-ESI-MS
2.2. Phytochemical Analysis of V. officinalis by HR-ESI-MS
TheThe three
three potent
potent fractions
fractions werewere subjected
subjected to HR-EST-MS
to HR-EST-MS analysis.
analysis. TheThe
datadata
werewere
The threewith
compared potent fractions
online weresuch
databases, subjected
as NIST to Chemistry
HR-EST-MS analysis.and
WebBook ThePubChem.
data wereThe
compared with online databases, such as NIST Chemistry WebBook and PubChem. The
compared
structure with online databases,
of metabolites
13 metabolites such as NISTbelonging
V. officinalis
of officinalis Chemistry toWebBook
different and PubChem. groups
phytochemical The
structure of 13 of V. belonging to different phytochemical groups
structure of 13 metabolites of V. officinalis belonging to different phytochemical groups
waswas putatively
putatively assigned
assigned using
using mass mass spectrometry
spectrometry (Table
(Table 2). 2).
We We have
have found
found the the bis(2-
bis(2-
wasethylhexyl)phthalate
putatively assigned and using mass spectrometry
lupulone A using (Table 2). We
high-resolution have
mass found the bis(2-
spectrometry (HRMS)
ethylhexyl)phthalate and lupulone A using high-resolution mass spectrometry (HRMS)
ethylhexyl)phthalate
data (Supplementary andMaterials).
lupulone A using high-resolution mass spectrometry (HRMS)
data (Supplementary Materials). ThisThis is the
is the firstfirst report
report indicating
indicating the the presence
presence of lupolone
of lupolone
dataA(Supplementary
in this plant. Materials). This is the first report indicating the presence of lupolone
A in this plant. ThisThis result
result is very
is very significant,
significant, as lupulones
as lupulones are are known
known to show
to show anticancer
anticancer
A inproperties
this plant.[34].
ThisTriterpenoids
result is very weresignificant,
the as lupuloneschemical
representative are known to show
class anticancer
consisting of seven
properties [34]. Triterpenoids were the representative chemical class consisting of seven
properties [34]. Triterpenoids were
compounds—hederagonic or the representative
glycyrrhetinic acid chemical
(V4), class consisting of seven
(2α,3β)-2,3-dihydroxyurs-12-en-28-
compounds—hederagonic or glycyrrhetinic acid (V4), (2α,3β)-2,3-dihydroxyurs-12-en-28-
compounds—hederagonic or glycyrrhetinic acid (V4), (2α,3β)-2,3-dihydroxyurs-12-en-28-
oic oic
acidacid
or or hederagenin(V5),
hederagenin (V5), gypensapogenin
gypensapogenin AA (V6),
(V6), momordicinin
momordicinin(V8), (V8),camarolide
camarolide (V9),
oic ursonic
acid or or hederagenin (V5), gypensapogenin A (V6), momordicinin (V8), camarolide
(V9), ursonic or moronic acid (V10), and serjanic acid (V12). Three compounds (V3, V11,and
moronic acid (V10), and serjanic acid (V12). Three compounds (V3, V11,
(V9), ursonicnot or moronic acid (V10), and serjanic acid (V12). Three compounds (V3, V11,
andV13)
V13)diddid notmatch
matchthethestructures
structuresofofthe theonline
onlinedatabase.
database.
and V13) did not match the structures of the online database.
Table
Table 2. Tentative
2. Tentative identification
identification of the
of the phytochemicals
phytochemicals of V. V. officinalis
of officinalis extracts.
extracts.
Table 2. Tentative identification of the phytochemicals of V. officinalis extracts.
No No Probable Probable MF MF m/z
m/z Class
Class Structure m/z
m/z Ref
Ref.
No Compounds Probable MF m/z
(found) Class Structure m/z
(cald) Ref
Compounds (found) (cald)
Compounds (found) (cald)
V1 V1 Bis(2- C HC24OH38O 391.2812
Bis(2-ethylhexyl) 391.281 Phthalate
Phthalate 391.284
391.2842 [35]
V1 Bis(2- 24 38 4
C24H38O 391.281 Phthalate 391.284 [35][35]
ethylhexyl)
phthalate 4 2 2
ethylhexyl) 4 2 2
phthalate
phthalate

HA HA
HA
V2 Lupulone
V2 A
Lupulone A C26 HC OH436
26
3626 36O 413.2665
413.266 β-Bitter
β-Bitter acids 413.2686
413.268 [34][34]
44 5 acids 6

V3Vo7
Vo7 Unknown
V3
V3 Unknown C26 HC
Unknown C OH
3026
26 30O
H330 O 391.23
391.23
391.23 --- -- --- -- -
Vo79 V4 9 Hederagonic acid C30 H46 O34 471.34 Triterpenoids 471.3468 [36,37]
9 3
or
Glycyrrhetinic acid
V5 (2α,3β)-2,3- C30 H48 O4 473.36 Triterpenoids 473.3625 [38]
Dihydroxyurs-12-en- [39,40]
28-oic acid
or
Hederagenin
 V4 acid
Hederagonic C 4 s 8 ]
V4 Hederagonic C3030H
H4646O O 471.34
471.34 Triterpenoid
Triterpenoid 471.346
471.346 [36,37
[36,37
V4
V4 Hederagonic
or
Hederagonic C 30
C30H H 46O 471.34 Triterpenoid 471.346 [36,37
acid 4 46O 471.34 Triterpenoid
ss 471.346
88 [36,37
]]
acid 4
acid
Glycyrrhetinic
acid 4
ss 88 ]]
or
or
4
or
acid
or
Glycyrrhetinic
Glycyrrhetinic
Glycyrrhetinic
Glycyrrhetinic
V5
Molecules 2022, 27, 6329 acid
(2α,3β)-2,3-
acid C30H48O 473.36 Triterpenoid 473.362 [38] 4 of 8
acid
acid
V5 Dihydroxyurs-
(2α,3β)-2,3- C 4 s 5 [39,40
V5 (2α,3β)-2,3- C3030H
H4848O O 473.36
473.36 Triterpenoid
Triterpenoid 473.362
473.362 [38]
[38]
V5
V5 (2α,3β)-2,3-
12-en-28-oic
(2α,3β)-2,3- C 30
C30H H 48O 473.36 Triterpenoid 473.362 [38]
]
Dihydroxyurs- 4 48O 473.36 Triterpenoid
ss 473.362
55 [38]
[39,40
Dihydroxyurs- 4 [39,40
Dihydroxyurs-
acid
Table 2. Cont. 44
Dihydroxyurs- ss 55 [39,40
[39,40
12-en-28-oic
12-en-28-oic ]]
12-en-28-oic
or
12-en-28-oic ]]
acid
acid
No Probable acid
Hederagenin
acid MF m/z Class Structure m/z Ref.
or
or
Compounds
V6 or
Gypensapogeni
or C30H42O (found)
435.32 Triterpenes (cald)
435.325 [41]
Hederagenin
Hederagenin
Hederagenin
n A
Hederagenin 7
V6 V6
V6 Gypensapogeni
Gypensapogenin A
Gypensapogeni C30 HC
4230
C O
30H
H224242O
O 435.32
435.32
435.32 Triterpenes
Triterpenes
Triterpenes 435.325
435.3257
435.325 [41]
[41][41]
V6
V6 Gypensapogeni
Gypensapogeni C 30H42O
C30H 435.32 Triterpenes 435.325 [41]
nn A 2 42O 435.32 Triterpenes 435.325
77 [41]
A 2
nA
n A 2
2 77

V7 Fistuloate A C30H44O 437.34 Aromatic 436.67 [42]

V7 Fistuloate A C 2
H compounds
V7 V7 Fistuloate
Fistuloate A A C3030OH4444O
O 437.34
437.34 Aromatic
Aromatic 436.67
436.67 [42]
[42]
V7
V7 Fistuloate
Fistuloate A C30 HC
A 30H
C4430 H244O 437.34
437.34 Aromatic
Aromatic 436.67
436.67 [42][42]
2 44O 437.34 Aromatic
compounds 436.67 [42]
V8 Momordicinin C30H 2 46O 439.35 compounds
Triterpenoid
com- 439.357 [43]
2
2
compounds
compounds
2 pounds
s 0
V8
V8 Momordicinin
Momordicinin C
C3030H
H4646O
O 439.35
439.35 Triterpenoid
Triterpenoid 439.357
439.357 [43]
[43]
V8 V8 Momordicinin
Momordicinin
V8 C 30H46O
MomordicininC30 HC4630O
H 439.35
439.35 Triterpenoid
Triterpenoids 439.357
439.3570 [43][43]
2246O 439.35 Triterpenoid
ss 439.357
00 [43]
2
2
2 ss 00

V9 Camarolide C30H44O 453.33 Triterpenoid 453.336 [44]

EA3 V9 Camarolide C 3 s 3
EA3 V9 Camarolide C3030HH4444O
O 453.33
453.33 Triterpenoid
Triterpenoid 453.336
453.336 [44]
[44]
V9 V9 Camarolide C30 HC
Camarolide
V9 Camarolide C4430
30OH344
H 44O 453.33
453.33 Triterpenoid
Triterpenoids 453.336
453.3363 [44][44]
3 O 453.33 Triterpenoid
ss 453.336
33 [44]
EA3
EA3
3
EA3
3
3 ss 33
EA3

V1 Ursonic acid C30H46O 455.35 Triterpenoid 455.351 [45,46

V1 0 or 3 s 9 ]
V10 UrsonicUrsonic
V1 acid
Ursonic acid
acidC30 HCC30H46O
4630OH346O 455.35
455.35
455.35 Triterpenoid
Triterpenoids
Triterpenoid 455.351
455.3519
455.351 [45,46
[45,46]
[45,46
V1
V1 Ursonic
Moronic acid
acid C 30H46O 455.35 Triterpenoid 455.351 [45,46
00 or Ursonic
or
or
acid C30H 3 46O
3
455.35 Triterpenoid
ss 455.351
99 [45,46
]]
00
V1
Moronic acid
Moronic
or
Unknown
or acid C31H 3 48O
3 485.36 ss- - 99- ]]-
Moronic acid
V11 Moronic acid
1Unknown acidC31 H48 O44 485.36
V1
V1
Moronic
Unknown
Unknown C
C3131H
H4848OO 485.36
485.36 --- --- -- - -- -
V1
V1
V1 Unknown
Serjanic
Unknown acid C
C
C 31
31 H
H
H 48
48OO
O 485.36
501.36
485.36 --
Pentacyclic -- --
501.357 -- [47]
[47]
V12 Serjanic
11 acid C31 H48 O44548
31 501.36 Pentacyclic 501.3574
112
V1 Serjanic
4
45 triterpenes
triterpenes 4
V1 Serjanic acid
acid C3131H
C H4848OO 501.36
501.36 Pentacyclic
Pentacyclic 501.357
501.357 [47]
[47]
V1
V1 Serjanic acid
Serjanic acid C 31H48O
C31H 501.36 Pentacyclic 501.357 [47]
22 5 48O 501.36 Pentacyclic
triterpenes 501.357
44 [47]
5 triterpenes
22 5
5 triterpenes
triterpenes 44

V13 V1 Unknown C34 HC

Unknown 34H46O
46 O5
511.34
511.34 -- -- -- - -
V13 Unknown C 5
V1 Unknown C3434H
H4646OO 511.34
511.34 -- -- -- --
V1
V1 Unknown
Unknown CC34
34H46O
H O 511.34
511.34 -- -- -- --
33 3. Materials 5
and
5
46
Methods
33 3. Materials and55Methods
3.1. Chemicals, Instruments, and Software
3.1.
3.
3. Chemicals,
Materials
Materials Instruments,
and
and Methods
Methods and Software
3. Chemicals
3. Materials
Materials and
and and silica: Methanol, n-hexane, chloroform, ethyl acetate, n-butanol, nor-
Methods
Methods
3.1.
3.1.malChemicals
Chemicals,
Chemicals, and gel
silica:
phase Instruments,
Instruments,
silica and
mesh Methanol,
and Software
Software
size n-hexane,
(70–230); chloroform,
Instrument: ethyl acetate,
preparative n-butanol,
TLC columns, Rotary
3.1.
3.1.
normalChemicals,
Chemicals,
phase Instruments,
Instruments,
silica gel meshand
and Software
Software
size (70–230); Instrument: preparative
TM5 TLC columns, Rotary VT,
Chemicals
evaporator,
Chemicals and and silica:
Shimadzu
and silica: Methanol,
IT-TOF
silica: Methanol, mass n-hexane, chloroform,
spectrometer,
Methanol, n-hexane, Cytation
n-hexane, chloroform,
chloroform, ethyl acetate,
(BioTek,
ethyl acetate, n-butanol,
Winooski,
acetate, n-butanol,
n-butanol,
Chemicals
evaporator, Shimadzu IT-TOF TM5ethyl
normal Chemicals
USA),
normal phase
and
phase andgel
silica
GraphPad
silica silica:
gel mesh
Prism
mesh 9mass
Methanol,
size
size
spectrometer,
n-hexane,
(70–230);
(software).
(70–230);
Cytation
chloroform,
Instrument:
Instrument: preparative
preparative
(BioTek,
ethyl
TLC
TLC
Winooski,
acetate,
columns,
columns,
VT,
n-butanol,
Rotary
Rotary
normal
USA),
normal phase
and
phase
evaporator, silica
GraphPad gel mesh
Prism
silica gel mesh
Shimadzu IT-TOF 9size
size (70–230);
(software).
(70–230);
mass Instrument:
Instrument:
spectrometer, preparative
preparative
Cytation TLC columns,
TLC columns,
TM5 (BioTek, Rotary
Rotary
Winooski, VT,
evaporator,
evaporator, Shimadzu
Shimadzu IT-TOF
IT-TOF mass
mass spectrometer,
spectrometer, Cytation
Cytation
TM5 (BioTek,
TM5 (BioTek, Winooski,
Winooski, VT,
VT,
USA),3.2.and
Collection
evaporator, GraphPadof Plant
Shimadzu Materials
IT-TOF
Prism 9 mass
(software).spectrometer, CytationTM5 (BioTek, Winooski, VT,
USA),
USA), and
and GraphPad
GraphPad Prism
Prism 9 (software).
3.2. Collection
USA), and of Plant
TheGraphPad
whole Materials
Prism
plant 99 (software).
of V. (software).
officinalis was collected during the flowering season, November
3.2.
3.2. Collection
2017
Collection of
to March
of Plant Materials
2018,
Plant from tehsil Gojra, district Toba Tek Singh Pakistan (Figure 2). The
Materials
3.2. taxonomic
3.2. Collection of
Collection of PlantofMaterials
Plant
status Materials
the plant was verified by Dr. Zaheer-ud-Din Khan, Government College
University, Lahore, Pakistan. A voucher number 3514 of the plant specimen was deposited
in the department of Botany, Government College University, Lahore, Pakistan.
 The whole plant of V. officinalis was collected during the flowering season, November
2017 to March 2018, from tehsil Gojra, district Toba Tek Singh Pakistan (Figure 2). The
taxonomic status of the plant was verified by Dr. Zaheer-ud-Din Khan, Government
Molecules 2022, 27, 6329 5 of 8
College University, Lahore, Pakistan. A voucher number 3514 of the plant specimen was
deposited in the department of Botany, Government College University, Lahore, Pakistan.

Figure 2.2. Verbena officinalis (06-21-2018, Chak NO. 363 JB, Tehsil Gojra, District Toba Tek
Figure Tek Singh,
Singh,
Division Faisalabad 56000).
Division Faisalabad 56000).

3.3.
3.3. Extraction
Extraction andand Fractionation
Fractionation
A
A whole
whole plant
plant was
was shade
shade dried
dried and
and then
then ground
ground into
intoaacoarse
coarsepowder.
powder.Pulverized
Pulverized
powder
powder (10 (10 kg)
kg) was
was macerated
macerated inin 80%
80% aqueous
aqueous methanol
methanol for for two
two weeks
weeks under
under normal
normal
conditions
conditionswithwithoccasional
occasionalshaking.
shaking. The
The methanolic
methanolic extract
extract was
was filtered
filtered and
and concentrated
concentrated
at ◦ C in a vacuum using a rotary evaporator under reduced pressure to yield a dry
at 40
40 °C in a vacuum using a rotary evaporator under reduced pressure to yield a dry
crude
crude extract
extract (460
(460 g).
g). The
The dry
dry methanolic
methanolic extract of V.
extract of V. officinalis
officinalis was
was suspended
suspended in in 11 LL of
of
distilled water and extracted with hexane, chloroform, ethyl acetate,
distilled water and extracted with hexane, chloroform, ethyl acetate, n-butanol n-butanol and acetone
and
successively to obtain different
acetone successively to obtainsolvent fractions.
different Eachfractions.
solvent fraction was Eachthen concentrated
fraction was then by
using a rotary evaporator and weighed. All extracts were then stored in sealed
concentrated by using a rotary evaporator and weighed. All extracts were then stored in containers
in a refrigerator
sealed containers forinfurther purification
a refrigerator and biological
for further evaluation.
purification and biological evaluation.
3.3.1. Hexane Fraction
3.3.1. Hexane Fraction
After fractionating the methanolic crude extract using hexane solvent, 2 g of hexane
After fractionating the methanolic crude extract using hexane solvent, 2 g of hexane
fraction (HA) was produced as a yellow color oil.
fraction (HA) was produced as a yellow color oil.
3.3.2. Isolation of EA3 from the Ethyl Acetate Fraction
3.3.2. Isolation of EA3 from the Ethyl Acetate Fraction
The ethyl acetate fraction (100 g) was obtained by performing extraction of the crude
The ethyl
methanolic acetate
extract withfraction
ethyl (100 g) was
acetate obtained
solvent. by performing
A greenish powder extraction of the crude
was obtained from
methanolic extract with ethyl acetate solvent. A greenish powder was obtained
the ethyl acetate extract, which was recrystallized from methanol to obtain a white from the
pow-
ethyl
der acetate extract, which was recrystallized from methanol to obtain a white powder
(EA3).
(EA3).
3.3.3. Isolation of VO79 from the Chloroform Fraction with Column Chromatography
3.3.3.The
Isolation of VO79
chloroform from(80
fraction theg)Chloroform Fraction
was subjected with Column
to column Chromatography
chromatography using a wet
technique, and elution
The chloroform was started
fraction withsubjected
(80 g) was hexane, chloroform, and chloroform-methanol
to column chromatography using a wet
solvent system.
technique, Elutionwas
and elution wasstarted
startedwith
at pure hexane,
hexane, and the polarity
chloroform, was increased by
and chloroform-methanol
10% by adding
solvent system.chloroform
Elution wasuntil it reached
started at pure 100% chloroform.
hexane, The polarity
and the polarity was increased
was increased by 10%
gradually by adding methanol with a 10% increment in the solvent system
by adding chloroform until it reached 100% chloroform. The polarity was increased until 100%
methanol. This whole process has resulted in the elution of 21 sub-fractions. The fractions
eluted at the solvent system chloroform: methanol (9:1 to 7:3) was obtained in a significant
quantity (11 g). It was again subjected to column chromatography. VO79 fraction was
eluted with hexane: chloroform (1:9 to 0:1) solvent system with a 2% polarity increment.
Molecules 2022, 27, 6329 6 of 8

3.3.4. N-Butanol Fraction

V. officinalis dried methanolic extract fractionated using n-butanol as the solvent,
yielding a 95 g n-butanol fraction.

3.3.5. Acetone Fraction

Dried methanolic extract was fractionated with acetone solvent, affording an acetone
fraction (29 g).

3.4. Cytotoxicity Studies

The Resazurin assay was performed to assess the cytotoxicity of the compounds
as we described previously [48,49]. A 96-well plate was seeded with 6000 cells/well
and incubated for 24 h in the presence of growth media. After 24 h of incubation, cells
were treated with six two-fold serial dilutions of fractions from 25 (µM) to 0.78 µM. Each
dilution was treated in triplicate, and another 24 h incubation was performed. After 24 h of
treatment, resazurin dye was added at a final concentration of 25 µg/mL, and again the
cells were incubated for 4 h. Finally, the fluorescence intensity was measured at 560 nm
excitation and 590 nm emission using CytationTM 5 (BioTek, Winooski, VT, USA). The
non-linear regression of viable cells and treatment concentration was performed using a
GraphPad Prism 9 (San Diego, CA, USA) to calculate the 50% inhibition concentration
(IC50 ) of fractions.

4. Conclusions
For the first time, we have revealed the anti-melanoma properties of V. officinalis
extracts. These mixtures of compounds are potent growth inhibitors in different melanoma
cell lines with IC50 values at low micromolar concentrations. These potent fractions are very
significant as these fractions of multiple compounds can be further separated into individual
compounds, which can be significantly more potent than the mixtures. We have putatively
characterized these fractions by using HRMS. The potent cytotoxic properties of these
fractions warrant further separation and anti-melanoma studies of V. officinalis extracts.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules27196329/s1, Figure S1: ESI high-resolution mass
spectrum of HA extract of V. officinalis; Figure S2. LC chromatogram of VO79 isolated from the
chloroform extract of V. officinalis; Figure S3. ESI mass spectrum of VO79 at retention time 3.37 min;
Figure S4. ESI mass spectrum of VO79 at retention time 8.21 min; Figure S5. ESI mass spectrum of
VO79 at retention time 9.53 min; Figure S6. LC chromatogram of EA3 isolated from the ethyl acetate
extract of V. officinalis; Figure S7. ESI mass spectrum of EA3 at retention time 5.41 min; Figure S8. ESI
mass spectrum of EA3 at retention time 5.58 min; Figure S9. ESI mass spectrum of EA3 at retention
time 6.23 min; Figure S10. ESI mass spectrum of EA3 at retention time 6.52 min; Figure S11. ESI mass
spectrum of EA3 at retention time 6.86 min; Figure S12. ESI mass spectrum of EA3 at retention time
7.13 min; Figure S13. ESI mass spectrum of EA3 at retention time 7.55 min; Figure S14. ESI mass
spectrum of EA3 at retention time 7.55 min; Figure S15. ESI mass spectrum of EA3 at retention time
8.23 min; Figure S16. ESI mass spectrum of EA3 at retention time 8.59 min; Figure S17. ESI mass
spectrum of EA3 at retention time 10.23 min
Author Contributions: S.A. (Saeed Ahmad) and R.N. selected the plant and extracted the com-
pounds/fractions, and M.A.A. got the mass spectrometry data and conceptualized the anti-melanoma
studies. R.N. wrote the manuscript and M.A.A. edited it for the final draft. S.A. (Sanjay Adhikary)
carried out the cytotoxicity studies. All authors have read and agreed to the published version of
the manuscript.
Funding: This publication was made possible by the Research Technology Core of the Arkansas
INBRE program, supported by a grant from the National Institute of General Medical Sciences,
(NIGMS), P20 GM103429 from the National Institutes of Health.
Institutional Review Board Statement: Not applicable.
Molecules 2022, 27, 6329 7 of 8

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to thank the Arkansas INBRE program, supported by
a grant from the National Institute of General Medical Sciences (NIGMS), P20 GM103429 from the
National Institutes of Health for the mass spectrometry data, and the Arkansas Biosciences Institute
for the infrastructure for cytotoxicity studies. Rabia Nisar would like to thank International Research
Support Initiative Program by Higher Education Commission Pakistan.
Conflicts of Interest: The authors declare no conflict of interest.

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