International Journal of
Molecular Sciences
Article
Bridging the Chemical Profile and Biological Activities of a
New Variety of Agastache foeniculum (Pursh) Kuntze Extracts
and Essential Oil
Fănică Bălănescu 1,2 , Andreea Veronica Botezatu 2, * , Fernanda Marques 3 , Anna Busuioc 2 ,
Olivian Marincaş 4 , Costel Vînătoru 5 , Geta Cârâc 2 , Bianca Furdui 2 and Rodica Mihaela Dinica 2, *
Citation: Bălănescu, F.; Botezatu,
A.V.; Marques, F.; Busuioc, A.;
Marincaş, O.; Vînătoru, C.; Cârâc, G.;
Furdui, B.; Dinica, R.M. Bridging the
Chemical Profile and Biological
Activities of a New Variety of
Agastache foeniculum (Pursh) Kuntze
Extracts and Essential Oil. Int. J. Mol.
Sci. 2023, 24, 828. https://doi.org/
10.3390/ijms24010828
Academic Editor: Amélia M. Silva
Received: 26 November 2022
Revised: 25 December 2022
Accepted: 28 December 2022
Published: 3 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Int. J. Mol. Sci. 2023, 24, 828. https://doi.org/10.3390/ijms24010828
1 Faculty of Medicine and Pharmacy, “Dunărea de Jos” University of Galati, 35 A.I. Cuza Street,
800010 Galati, Romania
2 Faculty of Sciences and Environment, Department of Chemistry Physical and Environment, “Dunărea de Jos”
University of Galati, 111 Domnească Street, 800201 Galati, Romania
3 Departamento de Engenharia e Ciências Nucleares (DECN), Centro de Ciências e Tecnologias Nucleares,
Instituto Superior Técnico, University of Lisbon, Campus Tecnológico e Nuclear, Estrada Nacional 10,
Km 139.7, Bobadela, 2695-066 Boticas, Portugal
4 National Institute for Research and Development of Isotopic and Molecular Technologies, 67-103 Donat Street,
400293 Cluj-Napoca, Romania
5 Plant Genetic Resources Bank for Vegetables, Floriculture, Aromatic and Medicinal Plants Buzău,
56 Nicolae Bălcescu Street, 120187 Buzau, Romania
* Correspondence: andreea.botezatu@ugal.ro (A.V.B.); rodica.dinica@ugal.ro (R.M.D.)
Abstract: This study investigated the phytochemical content of alcoholic extracts and essential
oil of a new variety of medicinal plants, Agastache foeniculum (Pursh), which Kuntze adapted for
cultivation in Romania, namely “Aromat de Buzău”. The essential oil was investigated by GC-
MS, while the identification and quantification of various compounds from alcoholic extracts were
performed by HPLC-DAD. The total phenol and flavonoid contents of the extracts were evaluated
by using standard phytochemical methods. The antioxidant activities of ethanol, methanol extracts,
and essential oil of the plant were also assessed against 2,20 -diphenyl-1-picrylhydrazyl (DPPH• ),
2,20 -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS•+ ), and by ferric reducing power
(FRAP) using spectroscopic methods. Cyclic voltammetry was used to evaluate the antioxidant
capacity of the essential oil. The concentrations of phenolic compounds were higher in methanolic
extract compared to ethanolic extract. A significant correlation was found between total phenol
and total flavonoid contents (r = 0.9087). Significant high correlations were also found between the
total phenolic compounds and the antioxidant activities of the extracts (r ≥ 0.8600, p < 0.05). In
addition, the extracts and essential oil showed good antioxidant and xanthine oxidase inhibitory
activities. Estragole was detected as the major constituent of the essential oil (94.89%). The cytotoxic
activity of the essential oil was evaluated by the MTT assay. At lower concentrations (1 µg/mL)
high cytotoxicity against MCF-7 breast cancer cells was observed but not on the non-tumoral dermal
fibroblasts (HDF) which indicated selectivity for cancer cells and suggests the presence of biologically
active components that contribute to the observed high cytotoxic effect. Findings from the present
study offer new perspectives on the use of A. foeniculum as a potential source of bioactive compounds
and a good candidate for pharmaceutical plant-based products.
Keywords: Agastache foeniculum; xanthine oxidase inhibitors; antioxidants; bioactive compounds
1. Introduction
Several studies have associated many diseases such as cancer or gout with increased
oxidative stress, resulting from an imbalance between reactive oxygen species (ROS) and
antioxidants [1]. Cancer is considered the second cause of death worldwide, after ischemic
heart disease and stroke, according to the world health organization (WHO) statistics
https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2023, 24, 828 2 of 22

on global health [2]. Oxidative stress, by inducing gene mutations and activating pro-
oncogenic signaling, can generate carcinogenesis and the progression of cancer cells [3]. In
the treatment of several diseases, the aim is to reduce the accumulation of ROS that occurs
as a result of metabolic disorders [1,4–6]. Inhibition of xanthine oxidase (XO) reduces both
vascular oxidative stress and circulating uric acid levels, which helps reduce the risk of
gout [7]. XO inhibitors (XOIs) work by blocking the biosynthesis of uric acid from purines
in the body and can act either at the purine binding site such as allopurinol or at the
cofactor site like benzimidazole [8,9]. Known XO inhibitors such as allopurinol, oxypurinol,
and febuxostat have been widely used for the treatment of hyperuricemia and gout [7].
However, due to their side effects, the identification of new non-purine selective xanthine
oxidase inhibitors is sought [10].
Over the centuries, empirical knowledge about the benefits of medicinal plants in
alternative medicine has been passed from generation to generation [11,12]. The health
benefits of chemical compounds from natural sources have gained a growing interest
in modern medicine. More and more herbal products are used as sources of bioactive
compounds, such as in herbal teas, food supplements with herbal powders, essential
oils (EOs), extracts obtained in different solvents, purified isolated organic compounds,
and in synergistic drug combinations consisting of classic target synthetic molecules with
compounds from natural sources [13,14]. In the food industry, antioxidants are added
to food, in order to prevent the deterioration of its taste, smell, and color [15]. Previous
studies have reported that the consumption of synthetic antioxidants was related to possible
adverse effects such as skin allergies, gastrointestinal tract problems, and increased risk
of cancer [15]. Therefore, it is necessary to find compounds from natural sources with
therapeutic action to circumvent the severe side effects of synthetic drugs [16]. The use
of compounds from natural sources has many advantages, such as chemical diversity,
targeting multiple host sites through various mechanisms, displaying high biological
specificity, and low side effects [17,18].
Many plants of the Lamiaceae family are used in traditional medicine as herbal products,
but also in the food industry as flavorings [19,20]. The genus Agastache is part of the
Lamiaceae family and includes 22 species of perennial ornamental and medicinal plants [21].
Agastache foeniculum (Pursh) Kuntze with fragrant leaves and purple flowers is widely
used for ornamental purposes, flavoring sweets, and other foods due to its appearance
and pleasant aromas similar to anise [22]. This plant is used to produce herbal teas that
are especially preferred by native Americans for therapeutic purposes such as relieving
various symptoms of colds, fevers, coughs, heart disease, inflammation, and pain [23].
This herbaceous and aromatic perennial plant is native to North America [24] and was
mentioned in other studies as anise hyssop, Lophantus anisatus, blue giant hyssop, flagrant
giant hyssop, or lavender giant hyssop [22,25,26].
So far, in the literature, several studies have represented A. foeniculum phytochemical
content and its pharmacological properties. The phytochemical profile of these plants
includes non-volatile metabolites belonging to several classes such as flavones and flavone-
glycosides (a rare dimeric malonylflavone—agastachin, agastachoside, acacetin, apigenin,
tilianin, myricetin, luteolin) [22,27], phenolic compounds (rosmarinic acid, caffeic acid) [28],
lignans (agastenol, agastinol) [29], terpenoids including triterpenoids (betulin, betulinic
acid, maslinic acid, oleanolic acid, β-amyrin, ursolic acid, corosolic acid, α-amyrin), diter-
penes (agastaquinone, agastol), and sterols [22,30,31]. Volatile metabolites were reported in
their chemical composition such as estragole, pulegone, eugenol, methyleugenol, menthone,
isomenthone, and spathulenol [20,30,32].
In the current context, taking into account the worldwide largest number of diseases
that affect humans such as gout, cardiovascular diseases, diabetes or cancer [2,33–35], the
adapted cultivation of various plants from other geographical regions is very important to
properly reduce the costs of cultivating, processing, and transport, and also to make more
plant products with therapeutic applications [36] available. This innovative, economical,
and sustainable approach is also able to promote the impact of natural compounds from
 that affect humans such as gout, cardiovascular diseases, diabetes or cancer [2,33–35], the
adapted cultivation of various plants from other geographical regions is very important
to properly reduce the costs of cultivating, processing, and transport, and also to make
more plant products with therapeutic applications [36] available. This innovative,
economical, and sustainable approach is also able to promote the impact of natural
Int. J. Mol. Sci. 2023, 24, 828 3 of 22
compounds from plants on human health. Based on the antioxidant potential of the main
components identified in the EO and extracts from A. foeniculum, this paper aimed to
evaluate the phytochemical characterization of a new Romanian plant variety A.
plants on human
foeniculum health.
(Pursh) Basednamely
Kuntze, on the antioxidant
“Aromat de potential
Buzau” of the mainregistered
(AdB) components inidenti-
2018
fied in the EO and extracts from A. foeniculum,
(https://istis.ro/en/catalog-oficial/, accessed on this15
paper aimed to2022),
September evaluate the phytochem-
which has been
ical characterization
adapted of acultivation
to the climatic new Romanian plant variety
conditions A. foeniculum
in Romania (Pursh) Kuntze, namely
by the Research-Development
“Aromat de Buzau” (AdB) registered in 2018 (https://istis.ro/en/catalog-oficial/,
Station for Vegetable Growing Buzău. Moreover, this study sought to evaluate accessed
the
on 15 September 2022), which has been adapted to the climatic cultivation conditions
biological properties of the alcoholic extracts and EO from this plant, such as antioxidant, in
Romania by the Research-Development Station
xanthine oxidase inhibitory, and cytotoxic activities. for Vegetable Growing Buzău. Moreover,
this study sought to evaluate the biological properties of the alcoholic extracts and EO from
this
2. plant, such as antioxidant, xanthine oxidase inhibitory, and cytotoxic activities.
Results
2.1. Morphological and Structural Characteristics of A. foeniculum AdB Using Confocal Laser
2. Results
Scanning Microscopy
2.1. Morphological and(CLSM)
Structural Characteristics of A. foeniculum AdB Using Confocal Laser
Scanning Microscopy
The present (CLSM)
study shows morphological and structural characteristics of the leaves
and The
stems of a plant
present study adapted to cultivationand
shows morphological in structural
Romania by confocal laser
characteristics scanning
of the leaves
microscopy.
and stems ofThe sections
a plant through
adapted the stems in
to cultivation and leaves of
Romania bythe maturelaser
confocal plantscanning
of the newmi-
variety
croscopy.of The
A. foeniculum AdB indicated
sections through the stemshealthy plantoftissues,
and leaves withplant
the mature normal morphological
of the new variety
and
of A.structural
foeniculum characteristics,
AdB indicated as observed by confocal
healthy plant tissues,laser
withscanning microscopy (Figure
normal morphological and
1). Microscopic images of fresh cross-sectional samples through
structural characteristics, as observed by confocal laser scanning microscopy the A. foeniculum
(Figurestem
1).
showed cortical
Microscopic parenchyma
images under the epidermis,
of fresh cross-sectional samples with large
through thepolygonal cellsstem
A. foeniculum (58.61–84.78
showed
µm). Theparenchyma
cortical medullary areaunder wastherich in vascular
epidermis, elements
with (T) with acells
large polygonal thick secondary wall
(58.61–84.78 µm).
with spiral ornaments.
The medullary area wasThe richthick secondary
in vascular cell wall
elements is important
(T) with in the transport
a thick secondary wall withof
organic compounds
spiral ornaments. synthesized
The in the cell
thick secondary assimilating parenchyma
wall is important (Figure
in the 1a,b).ofThe
transport leaf
organic
section
compounds of A. foeniculumin the
synthesized presents a veryparenchyma
assimilating compact assimilating
(Figure 1a,b).tissue
The leafwith small
section of
A. foeniculumspaces,
intercellular presentspolygonal
a very compact
cells ofassimilating
approximatelytissue15with
± 2 small
µm, and intercellular
thick cellspaces,
walls
polygonal
(Figure 1c).cells of approximately 15 ± 2 µm, and thick cell walls (Figure 1c).

(a) (b) (c)

Figure
Figure 1.
1. Sections
Sections of
of (a,b)
(a,b) stem
stem and
and (c)
(c) leaves
leaves of
of A.
A. foeniculum
foeniculum AdB
AdB observed using CLSM.
observed using CLSM.

Profiling of Chemical Compounds in A. foeniculum AdB EO by GC-MS

2.2. Profiling
Hydrodistillation of
Hydrodistillation of the
the A. foeniculum
foeniculum AdB
AdB plant
plant gave
gave an odorous pale yellow EO
yield of
with a yield 1.86±±0.64%
of 1.86 0.64%(v/w),
(v/w),considering
considering three
three independent
independent extraction
extraction procedures
procedures (n
=(n3).
= 3).
The total ion chromatogram recorded by GC–MS of the EO from A. foeniculum, which
contained seven identified compounds, is shown in Figure 2, and the chemical composition
is presented in Table 1.
 91.50 MS
90 RMD-03-01

ESTRAG
Int.
80 J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 23

ESTRAGOLE
85
75

80
70

75
65

70
60 The total ion chromatogram recorded by GC–MS of the EO from A. foeniculum,
Relative Abundance
Abundance

65
Int.
55 J. Mol. Sci. 2023, 24, 828 which contained seven identified compounds, is shown in Figure 2, and the chemical
4 of 22
60
50
composition is presented in Table 1.
Relative Abundance
Abundance

55
45
RelativeRelative

50
40
RT:
RT:5.72
5.72- –
20.21
20.21
45
35 10.82 NL:
100 4.09E6
m/z=

CARYOPHYLLENE
40

METHYL-EUGENOL
30 95 90.50-
LIMONENE
35 91.50 MS
25 90 RMD-03-01

CARYOPHYLLENE
METHYL-EUGENOL
30
20

ESTRAGOLE
85
LIMONENE

25
15 80
20
10 75 8.16
155
70
13.57 13.97
100 5.89 6.58 7.23 7.48 8.07 8.41 9.04 9.25 9.85 10.12 11.59 12.07 12.76 14.43 14.75 14.93 15.93 16.66 16.94 18.00 18.77 19.53
65 8.16
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
5
60 TimeTime (min)
(min) 13.57 13.97
7.23 7.48 8.07 11.59 12.07 12.76 14.43 14.75 14.93 15.93
Relative Abundance

5.89 6.58 8.41 9.04 9.25 9.85 10.12 16.66 16.94 18.00 18.77 19.53
Relative Abundance

0
6 7 55 8 9 10 11 12 13 14 15 16 17 18 19 20

50 TimeTime (min)
(min)
Figure 2. Total ion chromatogram of the EO from A. foeniculum AdB using GC–MS.
45

40
FigureThe
2. Total
mainioncomponents
chromatogram of the EO
detected infrom A. foeniculum
A. foeniculum AdBestragole,
were using GC–MS.
limonene, methyl
35

eugenol, and caryophyllene (Figures S1–S8), together with other volatile compounds

CARYOPHYLLENE
METHYL-EUGENOL
30
The1). main components detected inonA. A. foeniculum wererevealed
estragole,different
limonene,chemical
methyl
LIMONENE

25 (Table Previous investigations foeniculum

20 eugenol, and caryophyllene (Figures S1–S8),
compositions of the EOs in which the major compounds detected were methylchavicol, together with other volatile compounds
15 (Table 1). Previous
β-caryophyllene, limonene, investigations
menthone,on A. foeniculum
linalool, silvestrene,revealed or thymoldifferent chemical
[22,37]. Therefore,
10 compositions 8.16 of the EOs in which the major compounds
there is diversity in the chemical composition of Agastache species, which is mainly detected were methylchavicol,
5
5.89
β-caryophyllene,
reflected
6.58 7.23 7.48 8.07in8.41
the limonene,
9.04 EOs
9.25 9.85 and
10.12
menthone,
biological linalool,
11.59 12.07activities
12.76 due silvestrene,
13.57 13.97
14.43to
14.75several
14.93
or thymol
15.93 environmental,
16.66 16.94
[22,37].
18.00
Therefore,
cultivation,
18.77 19.53
0
6 there
7 is diversity
8
and harvesting factors. 9 in the
10 chemical
11 12 composition
13 14 of Agastache
15 16 species,
17 which
18 is
19 mainly 20
Time (min)
reflected in the EOs and biological Time (min)
activities due to several environmental, cultivation,
and
Table harvesting
1. The chemical factors.
Figure 2. composition of the EO from
Total ion chromatogram of theA. EOfoeniculum
from A.AdB. foeniculum AdB using GC–MS.
Figure 2. Total ion chromatogram of the EO from A. foeniculum AdB using GC–MS.
Table 1. The Molecular
Rtchemical composition of the EO from A. foeniculumFormula
AdB. &
No. Table 1.Constituent m/z of the EO from A. foeniculum AdB.
The chemical composition Concentration (%)
(min.) The main components detected in A. foeniculum
Molecular Weight were estragole, limonene, methyl
Rt
eugenol,Constituent
and caryophyllene Molecular Formula
(Figures S1–S8), Molecular &
together with other
No. m/z & volatile
Concentration (%)compounds
No. Rt (min.)(min.)
(Table Constituent m/z Molecular
1). Previous investigations Weight Formula
on A. foeniculum Concentration
revealed different (%)
chemical
Molecular Weight
compositions of the EOs in which the major compounds detected were methylchavicol,
C10H16
1 β-caryophyllene, limonene, 136
8.16 menthone, linalool, silvestrene, or 2.91 thymol [22,37].
± 0.65 b Therefore,
(MW:
there is diversity in the chemical composition 136.23)
of Agastache species, which is mainly
C10H16 b 2.91 ± 0.65 b
1 8.16
1 8.16
reflected Limonene 136 136
in the EOs and biological activities C 10 H 16 (MW: 136.23)
2.91 ± 0.65
(MW: due to several environmental, cultivation,
136.23)
and harvesting factors.
Limonene
Limonene
. Mol. Sci. 2023, 24, x FOR PEER REVIEW Table 1. The chemical composition of the EO from A. foeniculum AdB. 5 of 23

. Mol. Sci. 2023, 24, x FOR PEER REVIEW Rt Molecular Formula & 5 of 23
2 10.82
2 No.
10.82 Constituent
148148 m/z C10HC1210OH12 O(MW: 148.20)
94.89 ± Concentration
1.02 94.89
a ± 1.02(%) a
(min.) Molecular
(MW: 148.20) Weight
Methylchavicol
Methyl chavicol C10H12O
23 12.07 Unknown 0.40 ±±0.28 d
10.82 (Estragole)
(Estragole) 148 94.89 1.02 a
4 12.76 Unknown
Methyl chavicol (MW: 148.20) 0.04 ±± 0.28
0.02 df
3 3
12.07 12.07 Unknown
Unknown 0.40 0.40 ± 0.28 d
4 12.76 (Estragole)
Unknown C10H16 0.04 ± 0.02 f
4 12.76 1 Unknown
8.16 136 2.910.04±± 0.02b f
0.65
(MW: 136.23)
C10H12O2
5 12.97 164 0.01 ± 0.01 g
Limonene (MW:
C10H164.20)
12O2
5 12.97
5 12.97 Eugenol 164 164 0.01 ± 0.01 g 0.01 ± 0.01 g
C10 H12 O2 (MW: 164.20)
(MW: 164.20)
Eugenol
Eugenol

C11H14O2 C10H12O f 0.04 ± 0.01af

6 13.18
6 13.182 178 178
10.82 148 C11 H14 O2 (MW: 178.23)
0.04 ± 0.0194.89 ± 1.02
(MW:
C11H178.23)
14O2(MW: 148.20)
6 13.18 Methyl Methyl chavicol
178 0.04 ± 0.01
Methylisoeugenol
isoeugenol f
(Estragole) (MW: 178.23)
Methyl isoeugenol

C11H14O2
7 13.57 178 0.73 ± 0.04 c
(MW:
C11H178.23)
14O2
7 13.57 Methyl eugenol 178 0.73 ± 0.04 c
(MW: 178.23)
Methyl eugenol

C15H24
8 13.98 204 0.74 ± 0.02 c
 C10H12O2
5 12.97 Eugenol 164 0.01 ± 0.01 g
(MW: 164.20)
Eugenol C10H12O2
5 12.97 164 0.01 ± 0.01 g
(MW: 164.20)
Eugenol
C11H14O2
6
Int. J. Mol. Sci. 2023, 24, 828 13.18 178 0.04 ± 0.01 f 5 of 22
(MW: 178.23)
C11H14O2
6 13.18 Methyl isoeugenol 178 0.04 ± 0.01 f
(MW: 178.23)
Methyl isoeugenol C11H14O2
6 13.18Table 1. Cont. 178 0.04 ± 0.01 f
(MW: 178.23)
Methyl isoeugenol
No. Rt 7
(min.)13.57 Constituent
C11HMolecular
14O2 Formula &
178 m/z Molecular 0.73 ± 0.04Concentration
Weight
c (%)
(MW: 178.23)
Methyl eugenol C11H14O2
7 13.57 178 0.73 ± 0.04 c
(MW: 178.23)
Methyl eugenol C11H14O2
7 13.57
7 13.57 178 178 0.73 ± 0.04 c 0.73 ± 0.04 c
C11 H14 O2 (MW: 178.23)
(MW: 178.23)
Methyleugenol
Methyl eugenol

C15H24
8 13.98 204 0.74 ± 0.02 c
(MW: 204.35)
C15H24
8 13.98
8 13.98 204 204 0.74 ± 0.02 c 0.74 ± 0.02 c
C15 H24 (MW: 204.35)
Caryophyllene (MW: 204.35)
C15H24
8 13.98 204 0.74 ± 0.02 c
Caryophyllene
Caryophyllene (MW: 204.35)

Caryophyllene

C15H24
9 14.75
9 14.75 204 204 0.23 ± 07 e 0.23 ± 07 e
C15 H24 (MW: 204.35)
(MW: 204.35)
C15H24
9 14.75 204 0.23 ± 07 e
(MW: 204.35)
C15H24
9 14.75 Germacrene 204 0.23 ± 07 e
Germacrene DD (MW: 204.35)
The values followed by the same letter (a, b, c, d, e, f, g) in the same column show no statistically significant
Germacrene D
The values followed by(pthe
differences same
< 0.05) letter (a,
according to b,
thec,analysis
d, e, f,ofg)variance
in the same column
(ANOVA). Eachshow nothe
value is statistically
mean of three replicates ±
significant differences (p < 0.05)
standard deviation (SD).according to the analysis of variance (ANOVA). Each value is the
The Germacrene same D
meanvalues followed
of three by the
replicates ± standard letter (a, b, c,
deviation d, e, f, g) in the same column show no statistically
(SD).
significant differences (p < 0.05) according to the analysis of variance (ANOVA). Each value is the
The values
mean followed
of three Theby main
replicates ±the components
same
standard letter (a, b, detected
deviation c, d, e, f, g)
(SD). ininA.the
foeniculum
same columnwere estragole,
show limonene, methyl
no statistically
2.3. Total Phenolic
significant eugenol,and(pFlavonoid
differences and< 0.05) Contentsto the
caryophyllene
according (Figures
analysisS1–S8), together
of variance with Each
(ANOVA). othervalue
volatile
is thecompounds
mean of
By three replicates
ultrasound-assisted
(Table 1). ± standard
Previous deviation
extraction,
investigations
2.3. Total Phenolic and Flavonoid Contents (SD).
methanolic
on A. extracts
foeniculum were obtained
revealed with
different a yield
chemical ofcompositions
11.062 ± 0.945%of theandEOsethanolic
in whichextracts
the major with a yield ofdetected
compounds 7.212 ± 0.686%, considering three
were methylchavicol, β-caryophyllene,
2.3. By ultrasound-assisted
Total Phenolic and extraction,
Flavonoid Contents methanolic extracts were obtained with a yield of
independentlimonene,
extractions.menthone,
Total linalool,
phenolicsilvestrene,
(TPC) andor flavonoid
thymol [22,37].(TFC)Therefore,
contentsthere were is diversity in
11.062 ± 0.945% and ethanolic extracts with a yield of 7.212 ± 0.686%, considering three
determined the
bothchemical
By ultrasound-assisted composition
for methanolic (MeOH)
extraction, of Agastache species,
and ethanolic
methanolic extracts whichobtained
(EtOH)
were is mainly
extracts fromreflected
with the inofthe EOs and
dried
a yield
independent extractions. Total phenolic (TPC) and flavonoid (TFC) contents were
stem,
11.062 flowers,biological
± 0.945% and activities
and leaves
ethanolicof A.due to several
foeniculum.
extracts environmental,
with a Results
yield ofare
7.212 ±cultivation,
summarized inand harvesting
Table
0.686%, considering The factors.
2.three
determined both for methanolic (MeOH) and ethanolic (EtOH) extracts from the dried
methanolic extract was characterized by the highest phenolic and
independent extractions. Total phenolic (TPC) and flavonoid (TFC) contents were flavonoid contents.
stem, flowers, 2.3. and
Totalleaves
Phenolic ofand
A. Flavonoid
foeniculum. Results are summarized in Table 2. The
Contents
determined both for methanolic (MeOH) and ethanolic (EtOH) extracts from the dried
methanolic extract was characterized by the highest phenolic and flavonoid contents.
Table
stem, 2.flowers, By ultrasound-assisted
Total phenolic
and and flavonoid
leaves of A. contents extraction,
foeniculum. Resultsmethanolic
of methanolic extracts
and summarized
are ethanolic were
extracts
in of A.obtained
Table 2. Thewith a yield
foeniculum
AdB; means
methanolic ± SD.
of 11.062 ± 0.945% and ethanolic extracts with aandyield of 7.212 ± 0.686%, considering
Table 2. Totalextract
phenolic was
andcharacterized by the
flavonoid contents of highest phenolic
methanolic and ethanolic flavonoid
extracts ofcontents.
A. foeniculum
three independent extractions. Total phenolic (TPC) and flavonoid (TFC) contents were
AdB; means ± SD. Extraction TPC TFC
Table 2.Samples
Totaldetermined
phenolic andboth for
flavonoid
Yield
methanolicof(MeOH)
(%) contents(mg
andand
methanolic
GAE/g DW)
ethanolic
ethanolic(EtOH) extracts
extracts
(mg QE/g of A. from the dried stem,
foeniculum
DW)
AdB; means ±flowers,
SD. and Extraction
leaves of A. foeniculum. TPC
Results are summarized TFC
in Table 2. The methanolic
MeOH
Samples 11.062 ± 0.945 a 485.084 ± 0.052 a 367.32 ± 0.008 a
extract was Yield (%) b by the
characterized (mghighest
GAE/g phenolic
DW) and (mg
flavonoid QE/g DW)
contents.
EtOH 7.211 ± 0.686
Extraction 403.918 ± 0.057 b
TPC 355.94 ± 0.007 b
MeOH
Samples 11.062 ± 0.945 a 485.084 ± 0.052 a 367.32TFC
± 0.008 a
EtOH Yield
7.211 (%) (mg
± 0.686and flavonoid
b GAE/g
403.918 DW)
± 0.057 b (mg QE/g
355.94 DW)b
± 0.007
Table 2. Total phenolic contents of methanolic and ethanolic extracts of A. foeniculum
MeOH 11.062 ± 0.945 a 485.084 ± 0.052 a 367.32 ± 0.008 a
AdB; means ± SD.
EtOH 7.211 ± 0.686 b 403.918 ± 0.057 b 355.94 ± 0.007 b
TPC TFC
Samples Extraction Yield (%)
(mg GAE/g DW) (mg QE/g DW)
MeOH 11.062 ± 0.945 a 485.084 ± 0.052 a 367.32 ± 0.008 a
EtOH 7.211 ± 0.686 b 403.918 ± 0.057 b 355.94 ± 0.007 b
TPC: total polyphenol content; TFC: total flavonoid content; MeOH: methanolic extract; EtOH: ethanolic extract;
GAE: gallic acid equivalents; DW: dry weight; QE: quercetin equivalents. The values followed by different letters
(a, b) in the same column show statistically significant differences (p > 0.05). Each value is the mean of three
replicates ± SD.
Int. J. Mol.
Int. J.Sci.
Mol.2023,
Sci. 24, x FOR
2023, PEER REVIEW
24, 828 6 of 236 of 22

TPC: 2.4.
totalIdentification
polyphenol content; TFC:
of Bioactive total flavonoid
Compounds in thecontent;
ExtractsMeOH:
from A.methanolic
foeniculumextract; EtOH:
AdB through
ethanolic extract; GAE: gallic acid equivalents; DW: dry weight; QE: quercetin equivalents. The
HPLC-DAD
values followed by different letters (a, b) in the same column show statistically significant
In this work, the phytochemical profile of alcoholic extracts from A. foeniculum AdB
differences (p > 0.05). Each value is the mean of three replicates ± SD.
was investigated by HPLC-DAD analysis (Figure 3). The chromatographic study of the
extracts showed
2.4. Identification that the
of Bioactive methanolic
Compounds and
in the ethanolic
Extracts fromextracts revealed
A. foeniculum AdBhigh quantities of
through
quercetin and genistein. The quantification of each compound in the studied extracts is
HPLC-DAD
presented in Table 3.
In this
In work, the phytochemical
the methanolic profile
extracts, the of alcoholic
concentrations ofextracts from A.detected
all compounds foeniculum AdB
were higher
was investigated
compared to ethanolic extracts. Genistein and quercetin were found in all samplesthe
by HPLC-DAD analysis (Figure 3). The chromatographic study of tested,
extracts showed
while that was
tannic acid the methanolic
quantifiable and
onlyethanolic extracts
in the sample revealed
extracted withhigh quantities
methanol. of acid,
Caffeic
quercetin and genistein.
p-coumaric The quantification
acid, quercetin, hyperoside, andof each compound
rutin in theinstudied
were identified higher extracts is
concentrations
presented in Table extracts.
in methanolic 3.

(a)

(b)
Figure 3. HPLC–DAD
Figure 3. HPLC–DAD chromatograms
chromatograms of of
thethe
samples:
samples:(a)(a)A.A. foeniculum-
foeniculum- methanolic extract (Ta—
methanolic extract
(Ta—tannic acid,Ca—caffeic
tannic acid, Ca—caffeic
acid,acid, Da—daidzein,
Da—daidzein, H—hyperoside,
H—hyperoside, R—rutin,R—rutin, Q—quercetin,
Q—quercetin, N—naringenin,
N—naringenin,
G—genistein); G—genistein); (b) ethanolic
(b) A. foeniculum- A. foeniculum- ethanolic acid,
extract (Ta—tannic extract (Ta—tannic
H—hyperoside, acid,
Q—quercetin,
H—hyperoside, Q—quercetin, N—naringenin, G—genistein).
N—naringenin, G—genistein).

In the methanolic extracts, the concentrations of all compounds detected were

higher compared to ethanolic extracts. Genistein and quercetin were found in all samples
tested, while tannic acid was quantifiable only in the sample extracted with methanol.
Caffeic acid, p-coumaric acid, quercetin, hyperoside, and rutin were identified in higher
concentrations in methanolic extracts.

Table 3. Bioactive compounds contained in ethanolic (EtOH) and methanolic (MeOH) extracts of A.
foeniculum AdB.
Int. J. Mol. Sci. 2023, 24, 828 7 of 22

Table 3. Bioactive compounds contained in ethanolic (EtOH) and methanolic (MeOH) extracts of
A. foeniculum AdB.

Content (µg/g dw)

Chemical Name Chemical Structure
MeOH EtOH
Phenolic Acids
p-Coumaric acid C9 H8 O3 62.079 ± 0.436 a 8.982 ± 0.093 b
Caffeic acid C9 H8 O4 59.014 ± 0.103 a 31.320 ± 0.145 b
Tannins
Tannic acid C76 H52 O46 72.061 ± 0.077 a -
Flavonols
Rutin C27 H30 O16 76.409 ± 0.100 a 56.701 ± 0.111 b
Quercetin C15 H10 O7 1073.637 ± 0.130 a 704.148 ± 0.150 b
Hyperoside C21 H20 O12 98.693 ± 0.190 a 58.892 ± 0.105 b
Flavanones
Naringenin C15 H12 O5 43.683 ± 0.114 a 30.242 ± 0.121 a
Isoflavones
Genistein C15 H10 O5 3171.823 ± 0.218 a 2229.999 ± 0.256 b
MeOH: methanolic extract; EtOH: ethanolic extract; dw: dry weight. The values followed by the same letter (a,
b) in the same row show no statistically significant differences (p < 0.05) according to the analysis of variance
(ANOVA). Each value is the mean of three replicates ± SD.

2.5. In Vitro Antioxidant Activities

Free radicals are produced in biological systems and are also found as exogenous
and are known to influence various chronic degenerative diseases such as cardiovascular
disease, inflammatory diseases, aging, carcinogenesis, or arthritis [6,38]. Antioxidant
compounds protect cells against oxidative stress through a mechanism of intervention on
one of the three major stages of the oxidative process mediated by free radicals, namely
initiation, propagation, and termination. These antioxidants are found naturally in many
foods, and the balance between oxidants and antioxidants in the body can have a significant
effect on human health [39].

2.5.1. 2,20 -Diphenyl-1-Picrylhydrazyl (DPPH) Radical Scavenging Activity

Plants with a high content of secondary metabolites show antioxidant activity due to
their redox properties and chemical structures. The MeOH extract, EtOH extract, and EO of
A. foeniculum AdB showed strong antioxidant activity against the investigated free radicals
(Figure 4). The phenolic content could justify the antioxidant activity of the alcoholic
extracts from A. foeniculum AdB. The percentage of inhibition of DPPH radicals by the
extracts did not vary significantly in time (from 15 min up to 1 h), unlike the EO where it
was found that the same concentration of EO increases its DPPH radical inhibition activity
in time (Figure 4c).

Int. J. Mol. Sci. 2023, 24, 828
their redox properties and chemical structures. The MeOH extract, EtOH extract, and EO
of A. foeniculum AdB showed strong antioxidant activity against the investigated free
radicals (Figure 4). The phenolic content could justify the antioxidant activity of the
alcoholic extracts from A. foeniculum AdB. The percentage of inhibition of DPPH radicals
by the extracts did not vary significantly in time (from 15 min up to 1 h), unlike the EO
where it was found that the same concentration of EO increases its DPPH radical 8 of 22
inhibition activity in time (Figure 4c).

Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 23

(a) (b)
100
100
Inhibition of DPPH (%)

80
Inhibition of DPPH (%)
80
15 min
60 15 min 30 min
60
30 min
45 min
40 45 min
40 1h
1h
20
20

0
0
0 10 20 30 40 50
0 100 200 300 400 500
Concentration (µg/mL) Concentration (µM)

(c) (d)
Figure
Figure 4.4. DPPH
DPPHscavenging
scavengingactivity
activity of methanolic
of (a) (a) methanolic extract,
extract, (b) ethanolic
(b) ethanolic extractsextracts
from A.from A.
foenicu-
foeniculum AdB, (c) EO from A. foeniculum AdB, and (d) gallic acid. Each value is the
lum AdB, (c) EO from A. foeniculum AdB, and (d) gallic acid. Each value is the mean of threemean of three
replicates ± SD.
replicates ± SD.

2.5.2. ABTS [2,2 0 -Azinobis-(3-ethylbenzothiazoline-6-sulfonic Acid)] Scavenging

[2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic Scavenging Activity
Activity
Powerful antioxidants
antioxidantsprevent
prevent biomolecules
biomolecules (proteins,
(proteins, sugars,
sugars, nucleicnucleic acids,
acids, polyun-
polyunsaturated
saturated lipids) fromlipids) from suffering
suffering oxidativeoxidative damage free
damage through through free radical-mediated
radical-mediated reactions.
reactions.
Discoloration Discoloration
of the ABTSofsolution
the ABTS solution isbydetermined
is determined by of
the percentage theinhibition
percentage of
of the
ABTS •+ radical
inhibition of thecation
ABTSas •+ radical cation
a function as a function
of sample of sample
concentration andconcentration andThese
time (Figure 5). time
(Figure 5). These
plant extracts and EOplant extracts
were foundand EO antioxidant
to have were foundeffects
to have ABTS•+ effects
antioxidant
against radical against
cations
that may
ABTS •+ justifycations
radical and therefore
that may encourage
justify andtheir use in medicine.
therefore encourage their use in medicine.

2.5.3. FRAP Scavenging Activity

100 The assay consists of the reduction
100 of ferric to ferrous ions at low pH, providing
a colored complex. Ferric tripyridyltriazine (TPTZ 2,4,6-Tris(2-pyridyl)-s-triazine) has a
Inhibition of ABTS (%)

Inhibition of ABTS (%)

80 yellow color that, upon reduction 80 to ferrous form by antioxidants, turns to a violet-blue
color. The change in absorbance
15 min is proportional to the total ferric-reducing power15ofminthe
60 60
antioxidants in the sample.
30 min The MeOH extract demonstrated higher total ferric-reducing
30 min
40
power (45.721 ± 0.014 45µM min
Fe(II) equivalents/g
40
of extract) when compared to the45EtOH min
extract (39.483 ± 0.017 µM Fe(II) equivalents/g of extract), which indicates that the MeOH
1h 1h
20 extract contains a higher amount of20antioxidant compounds (Table 4).

0 0
0 0.2 0.4 0.6 0.8 1 0 1 2 3 4 5
Concentration (mg/mL) Concentration (mg/mL)

(a) (b)
100 100
ABTS (%)

80
ABTS (%)

75

60 15 min 15 min
 2.5.2. ABTS [2,2′-Azinobis-(3-ethylbenzothiazoline-6-sulfonic Acid)] Scavenging Activity
Powerful antioxidants prevent biomolecules (proteins, sugars, nucleic acids,
polyunsaturated lipids) from suffering oxidative damage through free radical-mediated
reactions. Discoloration of the ABTS solution is determined by the percentage of
inhibition of the ABTS•+ radical cation as a function of sample concentration and time
Int. J. Mol. Sci. 2023, 24, 828 9 of 22
(Figure 5). These plant extracts and EO were found to have antioxidant effects against
ABTS•+ radical cations that may justify and therefore encourage their use in medicine.

100 100
Inhibition of ABTS (%)

Inhibition of ABTS (%)

80 80

15 min 15 min
60 60
30 min 30 min
40 45 min 40 45 min
1h 1h
20 20

0 0
0 0.2 0.4 0.6 0.8 1 0 1 2 3 4 5
Concentration (mg/mL) Concentration (mg/mL)

(a) (b)
100 100
Inhibition of ABTS (%)

80
Inhibition of ABTS (%) 75

60 15 min 15 min

30 min 50 30 min
40
45 min 45 min

1h 25 1h
20

0 0
0.00 1.00 2.00 3.00 4.00 5.00 0 10 20 30 40 50 60
Concentration (µg/mL) Trolox Concentration (µM)

(c) (d)

Figure 5. ABTS scavenging activity of (a) MeOH extract; (b) EtOH extract; (c) EO, from A. foeniculum
AdB; and (d) Trolox. Each value is the mean of three replicates ± SD.

Table 4. IC50 values for antioxidant assays.

FRAP
DPPH ABTS (µM Fe(II) Unit of
Sample
IC50 Value IC50 Value Equivalents/g of Measurement
Extract)
MeOH 76.368 ± 0.002 b 98.274 ± 0.002 b 45.721 ± 0.014 a µg/mL
EtOH 94.986 ± 0.002 a 244.261 ± 0.003 a 39.483 ± 0.017 a µg/mL
EO 12.943 ± 0.001 c 0.3356 ± 0.002 d µg/mL
Trolox 32.562 ± 0.002 c µM
Gallic acid 15.614 ± 0.012 d µg/mL
The values followed by the same letter (a, b, c, d) in the same column show no statistically significant differences
(p < 0.01). Each value is the mean of three replicates ± SD.

Pearson’s correlation coefficients between the means of phytochemical contents (TPC,

TFC) and antioxidant activities (DPPH• , ABTS•+ , FRAP) were computed and reported in
Table 5. A statistically high significant correlation (p < 0.01) coefficient was found between
total flavonoid content and free radical scavenging activity against DPPH• (r = 0.9972)
and ABTS•+ (r = 0.9813). Correlation coefficients were also very high among the three
antioxidant activity values (DPPH• , ABTS•+ , FRAP) (r ≥ 0.8600).
Int. J. Mol. Sci. 2023, 24, 828 10 of 22

Table 5. Pearson correlation matrix between phytochemical contents and antioxidant activities of A.
foeniculum extracts.

TPC a TFC b DPPH•c ABTS•+ d FRAP e

TPC 1
TFC 0.9087 * 1
DPPH• 0.9373 * 0.9972 ** 1
ABTS•+ 0.9720 ** 0.9813 ** 0.9929 ** 1
FRAP 0.7356 0.9275 * 0.9057 * 0.8600 1
aTotal phenolic content, b Total flavonoid content, c IC50 in DPPH assay, d IC50 in ABTS assay, e FRAP assay (µM
Fe(II) equivalents); * Correlation is significant at the 0.05 level; ** Correlation is significant at the 0.01 level.

2.6. Electrochemical Evaluation of Antioxidant Capacity by Cyclic Voltammetry

Fresh samples of 5 µL of EO in 20 mL methanol were prepared and analyzed by open
circuit voltage (OCV), after 24 h and 5 d (samples were kept in the fridge). The evolution
of the potential (E) of the samples registered for 10 min can be observed in Figure 6. The
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 23
potential of the sample analyzed after 24 h showed a difference of about +30 mV in the
positive zone compared with the fresh sample. In the next 30 min and further, no noticeable
differences were observed, but an increase in the potential was registered. Results indicate
Results
the indicateofthe
availability availability
electronic of electronic
exchanges between exchanges
chemicalbetween chemical
components fromcomponents
the sample
fromthey
since the sample sincewith
are systems theydynamic
are systems with that
evolution dynamic evolution
have an thatvalue
increasing haveofan increasing
E to 100 mV
value30ofmin.
after E to 100 mV after 30 min.

0.08
after 5 days

0.07
|Ewe/V vs. Ag/AgCl / NaCl (sat'd)|

0.06
after 24 h

0.05

0.04

0.03

0.02 fresh

0.01
0 200 400 600
tim e/s

(a) (b)
Figure 6.
Figure 6. (a)
(a) The
Theevolution
evolutionofofthe
thepotential
potential(E)(E)
byby
OCV registered
OCV for for
registered 10 min of A.offoeniculum
10 min AdB
A. foeniculum
EO with time; (b) Cyclic voltammograms of A. foeniculum AdB EO registered at 24 h and 5 d (E = ± 2
AdB EO with time; (b) Cyclic voltammograms of A. foeniculum AdB EO registered at 24 h and 5 d
V/Ag/Ag Clsat, at 100 mVs−1).
(E = ± 2 V/Ag/Ag Clsat , at 100 mVs−1 ).

The cyclic
The cyclic voltammograms
voltammogramsof of the
the standard
standard compound
compoundeugenol,
eugenol, aa natural
natural compound
compound
with known antioxidant properties, and A. foeniculum EO are shown in Figure 7.7.In
with known antioxidant properties, and A. foeniculum EO are shown in Figure In both
both
samples, at ca. 0.5 V/Ag/AgCl sat, the cyclic voltammograms showed biocompounds that
samples, at ca. 0.5 V/Ag/AgClsat, the cyclic voltammograms showed biocompounds that
improve the
improve the potential
potential intensity.
intensity. In In the
the sample
sample with
with the
the EO
EO from
from A.A. foeniculum
foeniculum AdB,
AdB, aa
maximum anodic current of 20–22 µA was registered at the potential between 1.5 tilltill
maximum anodic current of 20–22 µA was registered at the potential between 1.5 1.91.9
V.
V.. The
The half-wave
half-wave potential
potential (E 1 )(E
of½)eugenol
of eugenol waswas registered
registered around
around 1.85 1.85 V/Ag/AgCl
V/Ag/AgCl sat .sat.
2

50

40
<I>/µA

30

20

10

Int. J. Mol. Sci. 2023, 24, 828
The cyclic voltammograms of the standard compound eugenol, a natural com
with known antioxidant properties, and A. foeniculum EO are shown in Figure 7.
samples, at ca. 0.5 V/Ag/AgClsat, the cyclic voltammograms showed biocompou
improve the potential intensity. In the sample with the EO from A. foeniculum
maximum anodic current of 20–22 µA was registered at the potential11between
of 22 1.
V.. The half-wave potential (E½) of eugenol was registered around 1.85 V/Ag/AgC

50

40

<I>/µA
30

20

10

0
0 0.5 1 1.5 2
Ewe/V vs. Ag/AgCl / NaCl (sat'd)

Figure7. 7.
Figure Cyclic
Cyclic voltammograms
voltammograms of A. foeniculum
of A. foeniculum AdBand
AdB EO (blue) EOstandard
(blue) and standard
compound compound
eugenol
(black),
(black), E=E ±=2±2 V/Ag/Ag
V/Ag/Ag Cl, at
Clsat sat,100 100 −mVs
at mVs 1. −1.

2.7. Xanthine Oxidase (Xo) Inhibitory Activity of the EO and Alcoholic Extracts from
2.7.
A. Xanthine Oxidase (Xo) Inhibitory Activity of the EO and Alcoholic Extracts from A
foeniculum
foeniculum
Xanthine oxidase is involved in purine degradation in humans by forming xanthine
from hypoxanthine and is ultimately converted to uric acid by an enzymatic reaction cat-
Xanthine oxidase is involved in purine degradation in humans by forming x
alyzed by xanthine oxidase. Uric acid is eliminated in the urine, but excessive uric acid for-
from hypoxanthine
Int. J. Mol. Sci. 2023, 24, x FOR PEER mation
REVIEW can consequently leadandtoishyperuricemia
ultimately and converted to The
gout [8,10]. uricEOacid
from by an11enzymatic
of 23
A. foeniculum
catalyzed
AdB by xanthine
demonstrated oxidase.ofUric
a high inhibition acidoxidase
xanthine is eliminated
activity inin the(84.077
vitro urine,±but excessive u
0.031%)
formation can consequently lead to hyperuricemia and gout [8,10]. The
at a concentration of 20 µg/mL, which is comparable with that of 30 µg/mL allopurinol, a EO
well-known
foeniculum
30µg/mL XOAdBinhibitor
allopurinol, [8]. The EOXO
demonstrated
a well-known and the alcoholic
a inhibitor
high extracts
inhibition
[8]. The demonstrated
EOofandxanthine significant
oxidase
the alcoholic extractsactivity
inhibitory activities
demonstrated at concentrations
significant inhibitory higher than 2.5 µg/mL
activities atofconcentrations (p < 0.001)
higher and
than higher
2.5 µg/mLthan(p with
(84.077
0.25 mg/mL
± 0.031%)
(p < 0.001),
at a concentration 20 µg/mL, which is comparable
< 0.001) and higher thanrespectively
0.25 mg/mL (Figure 8). respectively (Figure 8).
(p < 0.001),

*
100.00
*
XO inhibitory activity (%)

80.00
*
*
*

60.00
* * *
40.00
* * *
20.00 * * *
0.00
0.00

Samples

Figure 8.8.Xanthine
Figure Xanthine oxidase (XO)
oxidase inhibitory
(XO) activity
inhibitory of allopurinol,
activity extracts,extracts,
of allopurinol, and A.
and EO from EOfoeniculum
from A.
AdB evaluated
foeniculum AdBat varying concentrations.
evaluated at varying concentrations. mean ±are
Results areResults SDmean
of three
± SDreplicates
of three per condition.
replicates per
*condition.
Significantly
* Significantly differentt-tests,
different (Dunnett 2-sided)
(Dunnett from
t-tests, the control
2-sided) (without
from the inhibitor)
control (withoutconditions
inhibitor)
conditions
(p < 0.001). (p < 0.001).

2.8.
2.8. Cytotoxic
Cytotoxic Activity
Activity of
of the
the EO
EO from
from A.
A. foeniculum
foeniculum
In the search for new medicines for
In the search for new medicines for treating cancer,
treating the compounds
cancer, isolated
the compounds from plants
isolated from
could be better alternatives to the current chemotherapeutics that present
plants could be better alternatives to the current chemotherapeutics that present poor selectivity
poor
selectivity and high toxicity, hoping to get lesser side effects and to spare healthy cells
and tissues [2]. Certain components of medicinal plants have proved to be alternative
approaches in the fight against cancer, either administered alone or combined with
chemotherapeutic drugs to improve the treatment efficacy. As an interesting example,
 Int. J. Mol. Sci. 2023, 24, 828 12 of 22

and high toxicity, hoping to get lesser side effects and to spare healthy cells and tissues [2].
Certain components of medicinal plants have proved to be alternative approaches in the
fight against cancer, either administered alone or combined with chemotherapeutic drugs to
improve the treatment efficacy. As an interesting example, the cytotoxic activity of estragole
from the EO from A. foeniculum AdB was tested against MCF-7 breast cancer cell lines [23].
As shown, estragole exhibited a dose-dependent cytotoxic effect in the MCF-7 cells showing
IC50 values of 74 µg/mL [23]. Our studies with the EO from the Romanian A. foeniculum
AdB and the MCF-7 cell lines indicated a higher cytotoxic effect against MCF-7 breast
cancer cell lines at lower concentrations (1 µg/mL) which suggested the presence of other
biologically active components that contributed to the high cytotoxic effect observed. In
addition, using the normal cells, the human dermal fibroblasts, the cytotoxic effect seemed
to indicate improved selectivity for the breast cancer cells, at concentrations higher than
0.2 µg/mL (Figure 9). The EO demonstrated statistically significant cytotoxic activities
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 12 of 23
against MCF-7 cells at concentrations higher than 0.2 µg/mL (p < 0.001). Further studies
will be carried out to evaluate the cytotoxicity of the alcoholic extracts.

120
MCF-7
Cellular viability (% Control)

100
HDF
80 *
*
60
* *
40

20 *
*
0

Figure 9. Cytotoxic activity of A. foeniculum EO against MCF-7 and HDF cell lines. The IC50 values
Figure
found9.using
Cytotoxic activity ofPrism
the GraphPad A. foeniculum
softwareEO against
(vs. MCF-7
5.0) were and HDF
MCF-7, 0.24 cell lines.
± 0.09 The IC50
µg/mL, andvalues
HDF,
found using the GraphPad Prism software (vs. 5.0) were MCF-7, 0.24 ± 0.09 µg/mL, and HDF, 0.47 ±
0.47 ± 0.1 µg/mL. Control = cells without treatment. Data are mean ± SD of two independent
0.1µg/mL. Control = cells without treatment. Data are mean ± SD of two independent experiments
experiments with at least four replicates per condition. * Significantly different (Dunnett t-tests,
with at least four replicates per condition. * Significantly different (Dunnett t-tests, 2-sided) from
2-sided)
the from the basal
basal conditions conditions (p < 0.001).
(p < 0.001).
3. Discussion
3. Discussion
Human diseases are becoming more aggressive and resistant to classical drugs, there-
foreHuman
the demand diseases aretreatments
for new becomingismore aggressive
increasingly aiming and resistantnew
to explore to sources
classicalofdrugs,
natural
therefore the demand for new treatments is increasingly aiming
multi-target drugs [40]. Plants are an increasingly explored sources in the search to explore new sources
for new
ofdrugs.
natural multi-target
According drugs studies,
to previous [40]. Plants are an of
the efficacy increasingly explored with
natural compounds sources in the
antioxidant
search for new drugs. According to previous studies, the efficacy
activity for therapeutic purposes was demonstrated, and future studies may expand theirof natural compounds
with antioxidant
applications activitytherapies
in clinical for therapeutic
[4]. purposes was demonstrated, and future studies
may expand their applications in
This study investigated the chemical clinical therapies [4]. and biological properties of two
composition
alcoholic extracts, and EO from the A. foeniculum AdB and
This study investigated the chemical composition biological
variety. properties
The sections of two
through the
alcoholic extracts, and EO from the A. foeniculum AdB variety.
stems and leaves from the mature plant adapted to cultivation in Romania observed The sections through theby
stems
CLSMand leaves from
indicated theplant
healthy mature plantwith
tissues, adapted
normalto cultivation
morphological in Romania observed
and structural by
charac-
CLSM indicated
teristics. The yield healthy
of EO fromplant A.tissues, withAdB
foeniculum normal morphological
is similar to other results and reported
structuralin
characteristics. The yield
the literature [22]. of EOcomponent
The main from A. foeniculum
detectedAdB is similar
in the EO from to A.
other results reported
foeniculum AdB was
inestragole
the literature [22]. The main component
(1-allyl-4-methoxybenzene), detected in analog,
an allylbenzene the EO afrom A. foeniculum
colorless liquid with AdB an
was estragole (1-allyl-4-methoxybenzene), an allylbenzene analog, a colorless
odor of anise and sweet taste. In this study, we found a slightly different chemical composi- liquid with
an odor
tion of anise
of the and sweet
EO compared to taste.
other In this study,
studies, wesome
but also foundcommona slightly different
chemical chemical
components.
composition
Variations inofthethe EO compared
phytochemical to other studies,
composition occur due buttoalso
the some common
geo-climatic chemicalof
conditions
components.
their growth,Variations
maturity at inthe
thetime
phytochemical
of collection,composition occur due
or species variation [36].to Charles
the geo-climatic
et al. [41]
conditions
reported the of analysis
their growth, maturityofatAgastache
of 19 varieties the timespecies
of collection, or species
for EO content andvariation
26 compounds[36].
Charles et al. [41]The
were identified. reported
main the analysis of
constituents of 19
thevarieties
oil from ofA.Agastache
foeniculumspecies
included for methylchavi-
EO content
and
col,26 compoundsbornyl
spathulenol, were acetate,
identified. The main
γ-catenin, constituents of α-limonene,
β-caryophyllene, the oil from and A. foeniculum
α-cadinol.
included methylchavicol, spathulenol, bornyl acetate, γ-catenin, β-caryophyllene,
α-limonene, and α-cadinol. The GC-MS analysis of the EO of 14 different A. foeniculum
species from Canada prairies detected over 50 compounds, and methylchavicol was the
major constituent (96 ± 2%) [42]. Skakovskii et al. [43] reported the composition rich in
limonene, isomenthone, pulegone, menthone methylchavicol, and methyleugenol of the
Int. J. Mol. Sci. 2023, 24, 828 13 of 22

The GC-MS analysis of the EO of 14 different A. foeniculum species from Canada prairies
detected over 50 compounds, and methylchavicol was the major constituent (96 ± 2%) [42].
Skakovskii et al. [43] reported the composition rich in limonene, isomenthone, pulegone,
menthone methylchavicol, and methyleugenol of the EO from A. rugosa (Fish. et Mey)
O. Kuntze, which was determined by using 1 H and 13 C NMR analysis. Ivanov et al. [22]
identified by GC-MS in the EO of A. foeniculum eight main components: estragole, eugenol,
methyl isoeugenol, sylvestrene, 1-octen-3-ol acetate, β-caryophyllene, spathulenol, and
caryophyllene oxide. In another study, a total of 37 components of the EO obtained from
aerial parts of A. rugosa were determined by GC-FID and GC-MS, and the main identified
compounds were methyleugenol, estragole, eugenol, thymol, pulegone, limonene, and
caryophyllene [37].
In the present study, the phenolic acids and flavonoids compounds from the alcoholic
plant extracts were evaluated by HPLC-DAD analysis. Phenolic compounds accumulate in
various plant tissues and cells during ontogenesis and, respectively, under the influence of
various environmental stimuli, being involved in many interactions of plants with their bi-
otic and abiotic environment [44]. The chromatographic analysis of the extracts showed that
the methanolic and ethanolic extracts revealed high quantities of quercetin and genistein.
In the MeOH extract, the concentration of all compounds detected was higher compared
to EtOH extracts, these results being similar to others from the literature [45]. Genistein
and quercetin were found in high concentrations in both extracts, while tannic acid was
quantifiable only in the MeOH extract. Caffeic acid, p-coumaric acid, quercetin, hyperoside,
and rutin were identified in both extracts. Overall, the results of all analyzes confirmed
that the methanolic extract presented a richer chemical composition and better biological
activities than the ethanolic extract. The main compounds in the chemical composition of
the aqueous and organic extracts of various Agastache species presented in other several
studies were flavonoids (hesperitin, quercetin, tilianin), flavones (acacetin, 7-O-glucosyl
acacetin, diosmetin 7-O-β-D-(6”-O-malonyl)-glucoside, luteolin 7-O-β-D-glucoside), ter-
penes (limonene, linalool, eugenol, ursolic acid, oleanolic acid, estragole, β-amirin), organic
acids (malic acid, butanoic acid, hexadecanoic acid), esters (butanoic acid-hexane-dioctyl
ester, hexanedioc-dioctyl ester, 6-octen-1-ol-3,7-dimethyl propionate, ethyl palmitate) [46].
At the flowering stage of Agastache species, two glucosylflavones, namely isoagastachoside
and agastachin, were detected in the chemical composition of their methanolic extract [30].
Strilbytska et al. [26] detected in A. foeniculum leaf water extracts by HPLC-MS analysis the
presence of 24 compounds with various previously described biological properties such
as antioxidant, neuroprotective, antidiabetic, antineoplastic, or cardioprotective. Among
these compounds, genistein and caffeic acid were also identified in our present study.
Our results showed a significant correlation between the total polyphenol and flavonoid
contents (r = 0.90, p < 0.05) and the ability to inhibit the DPPH• (r ≥ 0.93, p < 0.05) and
ABTS•+ (r ≥ 0.97, p < 0.01) free radicals, while the power to reduce Fe3+ to Fe2+ demon-
strated a good correlation with the total flavonoid contents (r = 0.93, p < 0.05) and DPPH•
radical inhibition activity (r = 0.90, p < 0.05). The correlations are similar to others from the
literature [47,48]. The increased activity of reducing free radicals by natural compounds
also justifies the results of this study in which extracts from A. foeniculum AdB with a high
total content of flavonoids showed high antioxidant activities. The cyclic voltammetry
studies demonstrate that the electrochemical oxidation of the A. foeniculum AdB samples is
strongly related to the structure of the electroactive chemical compounds. The observed
electrochemical process supports the good antioxidant activity of the EO from A. foeniculum
AdB. Several studies have demonstrated a relationship between the high concentration
of flavonoids and modulating cellular redox homeostasis processes such as ROS scaveng-
ing [6,49]. The plant extracts and EO from this study demonstrated antioxidant effects that
may justify and encourage some of their uses for several disease prevention and further
clinical studies.
The potential biological activities of Agastache species differ between subspecies, as
each has a varied chemical profile. The variation in the composition of EOs and extracts
Int. J. Mol. Sci. 2023, 24, 828 14 of 22

of Agastache medicinal plants occurs due to their genetic variations, the stages of plant
growth, geo-climatic conditions, nitrogen fertilizers, irrigation regimes, and maturity stage
at the time of collection [36]. Due to their bioactive components, Agastache species could
be promising therapeutic agents for human health with antioxidant, anti-inflammatory,
analgesic, antimicrobial, antihypertensive, vasorelaxant, antiviral, nutraceutical, anticancer,
and anti-diabetic properties [22,23,46].
In the search for new XOIs, it was shown in the current study that the extracts and
EO from A. foeniculum, at low concentrations, exerted XO inhibition efficacy similar to
allopurinol, a well-known XO inhibitor [10]. The main compound from alcoholic extracts
that demonstrated XO inhibitory activities in other studies was quercetin. Flavonoids,
phenols, and their glycosides have been presented in other studies to show inhibitory
effects on XO [7,50,51]. These types of chemical constituents can also have an active role in
the XO inhibitory effects of plant extracts in vivo due to their multiple-targets properties.
The cytotoxic activity of the EO from A. foeniculum AdB was evaluated in the MCF-7
breast cancer cells and the normal fibroblasts, HDF. The EO consists of multiple compounds
and each compound potentially enhances or modifies the effects of others [52]. This study
used the EO with all its constituents, a complex phytochemical mixture, to maximize the
potential anticancer effect and to properly assess the potential risks to healthy cells and
tissues. Our studies with the EO from the Romanian A. foeniculum AdB on MCF-7 breast
cancer cell lines indicated a high cytotoxic effect at low concentrations (<0.5 µg/mL) which
means that a plethora of biologically active components contributed to the high cytotoxic
effect observed. With the HDF cells only at concentrations > 0.5 µg/mL, loss of viability
was observed, but not as extensively as that observed for the cancer cells. Other studies
reported that agastinol and agastenol, lignans detected in Agastache species whole plant
extract, inhibited etoposide-induced apoptosis in U937 leukemia cells [29]. Estragole, the
main component of the EO, has demonstrated muscle relaxant, anticonvulsant, anesthetic,
bradycardic, vasoactive, anti-inflammatory, antioxidant, anticancer, and antimicrobial
properties [53]. In humans, estragole usually enters the body as a component of herbal
teas or as a food that has been seasoned with herbs that contain many other substances,
such as flavonoids or anethole, that have a protective roles and thus reduce the possible
harmful effects of pure estragole. The European Union has established maximum levels
for estragole, as well as other naturally occurring compounds in plants such as methyl
eugenol and safrole, in finished foods that have been flavored with flavors and/or food
ingredients in which these constituents naturally occur [54]. According to the FEMA
(Flavor and Extract Manufacturers Association) expert panel, these flavors continue to
meet the criteria for FEMA GRAS (“generally recognized as safe”) [55]. Experimentally,
estragole showed no toxicity in mice that consumed food containing this compound in
low doses but possibly hepatocarcinogenic in animal experiments at high doses when
given as a pure compound [56,57]. This perspective also increases when recent studies are
taken into account, showing that long-term bone administration of 100 mg/kg estragole
had no toxic effects in mice, probably because at low exposures they are preferentially
detoxicated by biotransformation of ring substituents [53,56]. Anise hyssop EO showed a
strong antioxidant capacity and also antimicrobial activity against Staphylococcus aureus,
Curtobacterium flaccumfaciens, Listeria monocytogenes, Bacillus subtilis, Salmonella species.,
Escherichia coli, and Pneumonia vulicans [21]. The EO from A. foeniculum, rich in methyl
chavicol, 1,8-cineole, 1-octen-3-ol, 3-octanone, and germacrene D, showed toxicity against
two important coleopteran pests (Oryzaephilus surinamensis L. and Lasioderma serricorne F.)
of stored-food products [58].
These results suggest that the extracts and EO from A. foeniculum may have several
biological activities due to their chemical composition. This study intends to provide new
contributions to the pharmacologically relevant effects of the alcoholic extracts and EO
from A. foeniculum such as antioxidant and xanthine oxidase inhibitory activities, as well as
the cytotoxic properties of the EO on cancer cells, which can be considered as evidence of
the effectiveness of this medicinal plant.
Int. J. Mol. Sci. 2023, 24, 828 15 of 22

4. Materials and Methods

4.1. General
All reagents were purchased from Merck (Merck KGaA, Darmstadt, Germany). The
reference standards were purchased from Merck (Merck KGaA, Darmstadt, Germany), and
Fluka
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW(Honeywell Fluka™, Fluka, Germany). 15 of 23
Plant material. Agastache foeniculum (Pursh) Kuntze “Aromat de Buzău” (also known
as Lophantus anisatus) was obtained from the Vegetable Research and Development Station
in Buzău, Romania. Dr. Costel Vînătoru (Plant Genetic Resources Bank for Vegetables,
Vegetables, Floriculture, Aromatic and Medicinal Plants, Buzău, Romania) proposed the
Floriculture, Aromatic and Medicinal Plants, Buzău, Romania) proposed the acclimatiza-
acclimatization of A. foeniculum and purchased seeds belonging to this species from
tion of A. foeniculum and purchased seeds belonging to this species from several sources,
several sources, the main companies being Agrosemens (Z.A. du Verdalaï, 105 rue du
the main companies being Agrosemens (Z.A. du Verdalaï, 105 rue du chemin de fer, 13790
chemin de fer, 13790 ROUSSET, Peynier, France) and Rühlemann (Rühlemann’s Kräuter
ROUSSET, Peynier, France) and Rühlemann (Rühlemann’s Kräuter & Duftpflanzen, Auf
dem& Duftpflanzen,
Berg 2, 27367,Auf dem Berg
Horstedt, 2, 27367,From
Germany). Horstedt,
a large Germany).
number of From a large
assessed number of
genotypes,
assessed genotypes, after the evaluation, one of these proved great adaptability
after the evaluation, one of these proved great adaptability and was genetically stabilized and was
genetically
after breeding. stabilized after source
The selected breeding. The selected
of seed procurement,sourcefrom of seed procurement,
an original fromofan
genotype
original genotype of North-American origin,
North-American origin, was Rühlemann (https://www.kraeuter-und-duftpflanzen.de/ was Rühlemann
(https://www.kraeuter-und-duftpflanzen.de/pdf/Ruehlemanns-Kraeuterkatalog-2022.pd
pdf/Ruehlemanns-Kraeuterkatalog-2022.pdf, accessed on 20 December 2022). After com-
f, accessed
pleting on 20 December
the acclimatization and 2022). After the
breeding, completing
researchthe wasacclimatization
completed with and
thebreeding,
obtainingthe
research was completed with the obtaining of the A.foeniculum
of the A.foeniculum “Aromat de Buzau” variety. At the same time, the cultivation “Aromat de of
Buzau”
the
variety.
other At the
varieties wassame time, the
stopped, cultivation
knowing of the
that this oneother varieties wasand
is entomophilous stopped, knowing
can easily that
lose its
this one is This
authenticity. entomophilous and canwas
new plant variety easily lose its
adapted to authenticity. This new in
the climatic conditions plant variety
Romania was
and
adapted topassed
successfully the climatic conditions
the testing stage forinhomologation
Romania andand successfully
patentingpassed the testing
in the Official stage
Catalog
of cultivated plant varieties in Romania from 2018 with plant variety ISTIS (State Institute in
for homologation and patenting in the Official Catalog of cultivated plant varieties
forRomania
Testing andfromRegistration
2018 with plant variety patent
of Varieties) ISTIS (State
no. 00536Institute
by theforVegetable
Testing and Registration
Research and
of Varieties)Station
Development patentinno. 00536
Buzău, by the (https://istis.ro/en/catalog-oficial/,
Romania Vegetable Research and Development Station
accessed on in
15Buzău,
SeptemberRomania
2022).(https://istis.ro/en/catalog-oficial/, accessed on 15 September 2022).
For
For this
this study,
study, the the plants
plants (stems,
(stems, leaves,
leaves, flowers)
flowers) were harvested
were harvested at the flowering
at the flowering stage
instage
August in 2020
Augustand2020
wereand were
further further
used used
for this for this
research research
(Figure 10).(Figure
A voucher10). specimen
A voucher
specimen
(ISTIS (ISTISwas
no. 00536) no. deposited
00536) wasatdeposited at the
the Vegetable Vegetable
Research andResearch and Development
Development Station in
Station
Buzău, in Buzău, Romania.
Romania.

Figure
Figure 10.10.
A.A. foeniculum
foeniculum AdB
AdB plant
plant used
used in in
thethe present
present study.
study.

Confocal
Confocallaser
laserscanning
scanningmicroscopy
microscopy was usedused ininthis
thisstudy
studytoto characterize
characterize plant
plant cells
cells
andand tissues.
tissues. ThisThis analysis
analysis waswas performed
performed withwith
the the Zeiss
Zeiss A system
A system LSMLSM 710 710 (Carl
(Carl Zeiss
Zeiss Microscopy
Microscopy GmbH,GmbH,07740 07740
Jena,Jena, Germany)
Germany) equipped
equipped with awith a diode
diode laser
laser (405 (405Ar-laser
nm), nm),
Ar-laser (458, 488, 514 nm), DPSS laser (diode pumped solid state e 561 nm),
(458, 488, 514 nm), DPSS laser (diode pumped solid state e 561 nm), and HeNe-laser (633 and HeNe-
laser
nm).(633 nm).microscopic
Glass Glass microscopic slidesA.
slides with with A. foeniculum
foeniculum AdB AdBstem stem
and and
leaf leaf sections
sections were
were observed using a Zeiss AxioObserver Z1 inverted microscope
observed using a Zeiss AxioObserver Z1 inverted microscope equipped with equipped with 40×40×
apochromatic
apochromatic objective (1.4 aperture)
objective and FS38,
(1.4 aperture) andFS15,
FS38,andFS15,
FS49 filters.
and FS49 The image
filters.acquisition
The image
was made with
acquisition wasthe following
made parameters:
with the in-line scan in-line
following parameters: mode, scan
average
mode,method,
average average
method,
average number 4, speed 6, and 12-bit depth. The images of A. foeniculum plant cells were
analyzed with ZEN 2012 SP1 software (Black Edition).

4.2. General Experimental Procedures

Int. J. Mol. Sci. 2023, 24, 828 16 of 22

number 4, speed 6, and 12-bit depth. The images of A. foeniculum plant cells were analyzed
with ZEN 2012 SP1 software (Black Edition).

4.2. General Experimental Procedures

4.2.1. Essential Oil Extraction
The air-dried stem, leaves, and flowers were ground to a fine powder with a grinder
(Heinner, Grinder Optim150, 150 W, 50/60 Hz, Shenzhen, China). Powdered plant material
(50 g) was subjected to hydrodistillation with Clevenger-type apparatus for 180 min. The
distillate was extracted with diethyl ether and dried over anhydrous sodium sulfate. The
EO was then stored in sealed brown vials at −20 ◦ C before chemical analysis. The results
were presented by considering three independent extraction procedures (n = 3).

4.2.2. Gas Chromatography-Mass Spectrometry (GC-MS)

The separation and identification of volatile organic compounds (VOCs), from the
analyzed oil samples, were performed by using a Trace GC Ultra Gas Chromatograph
from Thermo Scientific (Thermo Fisher Scientific Inc., Bremen, Germany) coupled with
Thermo Electron Polaris Q Mass Spectrometer, equipped with an electron ionization source
operating at 70 eV. For the chromatographic separation of the target compounds, a DB-
5MS (30 m × 0.25 mm ID, 0.25 µm film thickness) capillary column (5% diphenyl, 95%
dimethylpolysiloxane) was used. The injection volume of each diluted oil sample was
2 µL. The oven temperature program was 40 ◦ C for 3 min, which was then increased from
40 to 300 ◦ C with 10 ◦ C min−1 , and kept at 300 ◦ C for 10 min. The injector and transfer
line were 250 ◦ C and 300 ◦ C, respectively. The carrier gas was helium with a flow rate of
1.5 mL min−1 . The constituents of the samples were identified and confirmed based on
two different approaches: linear retention indices (LRIs) determined relative to a series
of n-alkanes (C8–C20) and by comparison of the mass spectra with those available in the
commercial libraries (NIST 2011) [59].

4.2.3. Ultrasound-Assisted Extraction with Solvents (Methanol, Ethanol)

The air-dried stem, leaves, and flowers were ground to a fine powder with a grinder
(Heinner, Grinder Optim150, 150 W, 50/60 Hz, Shenzhen, China). For the extraction of
phytochemical compounds, methanol and ethanol were used as solvents, and to obtain a
maximum yield, ultrasound-assisted extraction was used. The powdered plant material
(10 g) was extracted in alcoholic solvents (100 mL) for 120 min in a temperature-controlled
ultrasonic bath (40–50 ◦ C, Bandelin Sonorex Ultrasonic, Bandelin, Berlin, Germany, oper-
ating frequency 35 kHz, with digital timer and temperature control). The extracts were
filtered through Whatman No. 4 filter paper, evaporated under a vacuum to dryness by
using a rotary evaporator (RE100-Pro, DLAB Scientific Inc., Riverside, CA 92501, USA),
and stored at 4 ◦ C until analyzed. The extraction yields were determined as below [60]:

Extraction yield (%) = weight of extract (g)/weight of plant material (g) × 100 (1)

4.2.4. Total Phenolic Content

Total phenolic content was determined according to the literature with Folin–Ciocalteu
reagent [61]. Gallic acid was used as a reference standard, and the results were ex-
pressed as milligram gallic acid equivalents per g dry weight of extract (mg GAE/g
DW). All experiments were performed in triplicate. All data are expressed as the mean
± standard deviation.

4.2.5. Total Flavonoid Content

Total phenolic content was determined according to the literature with 10% AlCl3 [61,62].
Quercetin was used as a reference standard, and the results were expressed as milligram
quercetin equivalents per g dry weight of extract (mg QE/g DW). All experiments were
performed in triplicate. All data are expressed as the mean ± standard deviation.
Int. J. Mol. Sci. 2023, 24, 828 17 of 22

4.2.6. Profiling of Bioactive Compounds by HPLC-DAD

High-performance liquid chromatography coupled with a diode array detector (HPLC-
DAD) analysis was performed using an L-3000 high-performance liquid chromatography
system (Rigol Technologies, INC, Beijing, China). In the chromatographic analysis, the
Kinetex EVO C18 column (150 × 4.6 mm, 5 µm particle size) with an injection volume of
10 µL was used.
The extracts were analyzed after solubilizing 10 mg of each dry extract in methanol/
water (80:20, v/v) and the phenolic compounds were separated on an EVO C18 column
(150 × 4.6 mm, 5 µm particle size), at a temperature of 30 ◦ C at 1 mL/min, with a linear gra-
dient consisting of water-trifluoroacetic acid (99.9:0.1, v/v) and acetonitrile/trifluoroacetic
acid (99.9:0.1, v/v) [63]. The standards used for HPLC-DAD: p-coumaric acid CAS No:
501-98-4, caffeic acid CAS No: 331-39-5, rutin CAS No.: 153-18-4, quercetin CAS No.:
849061-97-8, hyperoside CAS No.: 482-36-0, tannic acid CAS No.: 1401-55-4, genistein CAS
No.: 446-72-0, and naringenin CAS No.: 67604-48-2, were purchased from Sigma-Aldrich
(Merck KGaA, Darmstadt, Germany), dissolved in methanol/water, and used in HPLC
analysis according to previous works [62,64,65]. The phenolic compounds were identified
by combining the following information: elution order, retention time in the C18 column,
comparison with standard solutions analyzed under the same conditions, and quantified by
using analytical curves of the analyzed compounds [62,64]. The compound contents were
expressed as mg/g of dried weight, considering three independent extraction procedures
(n = 3).

4.3. Antioxidant Assays

4.3.1. In Vitro-Antioxidant Assays
In the present study, four methods were used to evaluate the antioxidant activity of
the alcoholic extracts and EO from A. foeniculum AdB. For each assay, standard compounds
were used as positive controls. All details of the antioxidant assays were published in
previous studies [61,64,66]. All the analyses were performed in triplicate, and the results
were reported as mean ± SD.
The antioxidant activity of the extract was determined by the DPPH assay, as previ-
ously described [61]. Briefly, 100 µL of each extract (2–1000 µg/mL) was mixed with 100 µL
DPPH solution and incubated in the dark at room temperature for 1 h. The absorbance of
the mixture was measured at 517 nm with a microplate reader with 96-well plates (Tecan
Pro 200, Tecan Trading AG, Männedorf, Switzerland). Gallic acid is known for its antioxi-
dant potential and was used as a positive control [67]. The ability of the sample to scavenge
DPPH radical was determined with the following equation:

DPPH scavenging effect = (ODControl − ODSample )/ODControl × 100 (2)

where ODControl represents the absorbance of the control, and ODSample represents the
absorbance of the sample.
The scavenging activity of each sample against 2,20 -azinobis-3-ethylbenzothiazoline-6-
sulfonic acid radical was carried out according to the Busuioc et al. method [64]. Trolox
was used as a positive antioxidant reference control [65]. The ABTS radical cation was
produced by the reaction between ABTS solution (7 mM) and K2 S2 O8 aqueous solution
(15 mM) for 12 h, in dark conditions at room temperature. Then, the ABTS radical cation
solution was diluted with deionized water to obtain an absorbance of 0.70 ± 0.02 at 734 nm.
The ABTS radical cation solution (100 µL) was added to 100 µL extract solution of various
concentrations (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg/mL). After each 15 min of incubation
in dark conditions, the absorbance at 734 nm was determined with a microplate reader with
96-well plates (Tecan Pro 200, Tecan Trading AG, Männedorf, Switzerland). The scavenging
activity of the samples against radical ABTS was evaluated by the following Equation (3):

ABTS scavenging activity = (ODControl − ODSample )/ODControl × 100 (3)

Int. J. Mol. Sci. 2023, 24, 828 18 of 22

where ODControl represents the absorbance of the control, and ODSample represents the
absorbance of the sample.
The IC50 values were determined from the relationship curve of radical scavenging
activity versus concentrations of the respective sample curve. A lower IC50 value means
higher antioxidant activity. The IC50 value is a parameter commonly used to measure the
antioxidant activity of various samples. It is evaluated as the concentration of antioxidants
necessary to decrease the initial DPPH concentration by 50% [68].
Determination of antioxidant activity by FRAP assay was carried out according to
the procedure previously described in the literature [65], by monitoring the reduction of
Fe3+ -tripyridyl triazine (TPTZ) to blue-colored Fe2+ -TPTZ measured at 593 nm. The FRAP
reagent was freshly prepared by adding a 10:1:1 (v/v/v) ratio of 0.3 M acetate buffer (pH 3.6),
10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3 . The assay was performed by mixing 50 µL
of sample in a 96-well microplate and then adding 200 µL of FRAP reagent. After 15 min
of incubation and shaking at 37 ◦ C, the absorbance was read at 593 nm with a microplate
reader with 96-well plates (Tecan Pro 200, Tecan Trading AG, Männedorf, Switzerland).
FRAP was expressed as µM FeSO4 equivalents/g of extract. All samples were analyzed
in triplicate.

4.3.2. Electrochemical Evaluation of Antioxidant Capacity by Cyclic Voltammetry

The EO from A. foeniculum AdB was electrochemically evaluated for its antioxi-
dant potential by using SP-150 Biologic potentiostat/galvanostat (Bio-Logic SAS, Claix,
France). The experiments were conducted at room temperature with an electrochemi-
cal cell equipped with three electrodes: platinum wire (counter electrode), Ag/AgClsat
(E = 0.194 V/NHE) (reference electrode), and glass carbon electrode (working electrode).
For this assay, the applied potential was E = ±2 V vs. Ag/AgClsat and the scan rate was set
at 100 mVs−1 . The working electrode was polished with alumina and diamond slurries
(BASi® polishing kit), followed by cleaning with ethanol after each measurement. All
measurements were conducted in triplicate.

4.4. Xanthine Oxidase Inhibitory Activity Assays

This assay was established as previously described [9], with a few modifications, and
adapted to the microplate. This method is based on monitoring uric acid formation in
the xanthine oxidase system [9]. Freshly prepared substrate (0.6 mM xanthine dissolved
in 0.1 N NaOH), and enzymatic (0.25 XO units/mL in 0.1 M phosphate buffer, pH 7.5)
solutions were used in this assay. The EO from A. foeniculum AdB of various concentrations
was dissolved in 1% DMSO in phosphate buffer (pH 7.5). The extracts were dissolved in
phosphate buffer at various concentrations. The assay mixture consisted of 40 µL of test
solution, with 40 µL phosphate buffer (pH 7.5), 20 µL of enzyme solution (0.25 U/mL),
and xanthine solution (100 µL). The reaction was initiated by adding the enzyme with or
without inhibitors. Allopurinol was used as a positive control because it is a well-known
XO inhibitor [10]. Changes in the absorbance of the mixtures were spectrophotometrically
determined at 295 nm with a microplate spectrophotometer (Tecan Pro 200, Tecan Trading
AG, Männedorf, Switzerland), at 30 s intervals for 3 min, and 25 ◦ C. Determination of the
inhibition activity (I) was made by the following Equation (4):

% inhibition = (ODControl − ODSample )/ODControl × 100 (4)

where ODControl represents the absorbance of the control sample without inhibitor, and
ODSample represents the absorbance of the sample.
The results were statistically analyzed by comparing the xanthine oxidase inhibition
values obtained at baseline (control) with those obtained in different treatments by analysis
of variance (ANOVA) followed by the Dunnett test (multiple comparisons with one control)
with p < 0.05.
Int. J. Mol. Sci. 2023, 24, 828 19 of 22

4.5. Cytotoxic Activities

The effect of the EO from A. foeniculum AdB on the cellular viability of MCF-7 (ATCC)
and HDF (Sigma-Aldrich) cell lines was measured using the MTT assay, as previously
reported [38]. Cells were grown in DMEM + Glutamax I medium (MCF-7) supplemented
with 10% FBS and 1% antibiotics or fibroblast growth medium (HDF) without supplements.
For the assays, cells were seeded in 96-well plates and left for 24 h to adhere in a 5% CO2
incubator at 37 ◦ C. After the media was removed and 200 µL of complete media with
serial dilutions of the A. foeniculum EO were applied to each well. After 24 h of incubation,
the supernatant was removed, MTT (0.5 mg/mL PBS) was added, and the plates were
incubated for 3 h at 37 ◦ C. The purple formazan formed was dissolved in 200 µL DMSO.
Optical density was measured at 570 nm using a microplate reader (Power Wave Xs, Bio-
Tek, Winooski, VT, USA). The results were statistically analyzed by comparing the HDF
and MCF-7 values obtained at baseline (control) with those obtained in different treatments
by analysis of variance (ANOVA) followed by the Dunnett test (multiple comparisons with
one control) with p < 0.05.

4.6. Statistical Analysis

The experiments were performed in triplicate, and the data presented represent the
average of the three determinations ± standard deviation (SD). The IC50 values were found
using the GraphPad Prism software (vs. 5.0). The data were statistically analyzed using IBM
SPSS Statistics software (version 29, IBM Corp., New York, NY, USA). Statistical evaluation
of the obtained data was assessed by one-way analysis of variance (ANOVA) followed
by the Duncan multiple range test to find out significant differences (p ≤ 0.05). Pearson’s
correlation coefficients were computed to identify the relationship between antioxidant
activities, total polyphenols, and flavonoid contents, and a statistical correlation was
considered significant at the 0.05 level (p < 0.05). The results from the biological assays
were statistically analyzed by analysis of variance (ANOVA) followed by the Dunnett test
(multiple comparisons with one control) to find out significant differences (p ≤ 0.05) among
the obtained data.

5. Conclusions
This study is the first to investigate the phytochemical profiling and in vitro biological
properties of the EO and alcoholic extracts from the A. foeniculum “Aromat de Buzau”
variety. The EO and alcoholic extracts showed good antioxidant properties and inhibitory
potential against xanthine oxidase, a critical enzyme involved in pathologies like gout. The
EO also revealed selective cytotoxic activity against breast cancer MCF-7 cells compared
to normal HDF fibroblasts; however, further in vivo and clinical studies are needed. This
plant is a source of bioactive compounds that could be good candidates in therapeutic
applications to treat human diseases and may find use as raw material for the nutraceutical,
food, pharmaceutical, and cosmetic industries, and may be synergistically used with other
antioxidants and chemotherapeutic agents.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/ijms24010828/s1.
Author Contributions: Conceptualization, R.M.D., F.B. and A.V.B.; methodology, R.M.D., B.F., G.C.,
O.M. and F.M.; software, O.M., F.M. and F.B.; validation, R.M.D., A.V.B., O.M., F.M., G.C. and A.B.;
formal analysis, A.V.B., A.B. and F.B.; investigation, A.V.B., A.B., F.B. and R.M.D.; resources, R.M.D.,
B.F., G.C. and C.V.; data curation, R.M.D., F.B., G.C., A.V.B., F.M. and A.B.; writing—original draft
preparation, A.V.B., F.B. and R.M.D.; writing—review and editing, A.V.B., F.B., R.M.D., F.M., G.C.
and A.B.; visualization, R.M.D., A.V.B. and F.B.; supervision, R.M.D.; project administration, A.V.B.
and R.M.D.; funding acquisition, R.M.D., F.M. and O.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Int. J. Mol. Sci. 2023, 24, 828 20 of 22

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors would like to thank “Dunarea de Jos” University, ECEE and MORAS
infrastructure, Centro de Ciências e Tecnologias Nucleares/Departamento de Engenharia e Ciências
Nucleares (DECN), Instituto Superior Técnico, Universidade de Lisboa and the National Institute for
Research and Development of Isotopic and Molecular Technologies Cluj-Napoca, and Ionna Organic,
Vinatori, Galati, for the material and technical support through which part of this work was achieved.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.

References
1. Souto, E.B.; Sampaio, A.C.; Campos, J.R.; Martins-Gomes, C.; Aires, A.; Silva, A.M. Chapter 2—Polyphenols for Skin Cancer:
Chemical Properties, Structure-Related Mechanisms of Action and New Delivery Systems. In Bioactive Natural Products; Atta-ur-
Rahman, Ed.; Studies in Natural Products Chemistry; Elsevier: Amsterdam, The Netherlands, 2019; Volume 63, pp. 21–42.
2. Khan, T.; Ali, M.; Khan, A.; Nisar, P.; Jan, S.A.; Afridi, S.; Shinwari, Z.K. Anticancer Plants: A Review of the Active Phytochemicals,
Applications in Animal Models, and Regulatory Aspects. Biomolecules 2020, 10, 47. [CrossRef] [PubMed]
3. Luo, M.; Zhou, L.; Huang, Z.; Li, B.; Nice, E.C.; Xu, J.; Huang, C. Antioxidant Therapy in Cancer: Rationale and Progress.
Antioxidants 2022, 11, 1128. [CrossRef] [PubMed]
4. Bhuyan, U.; Handique, J.G. Chapter 6—Plant Polyphenols as Potent Antioxidants: Highlighting the Mechanism of Antioxidant
Activity and Synthesis/Development of Some Polyphenol Conjugates. In Bioactive Natural Products; Atta-ur-Rahman, Ed.; Studies
in Natural Products Chemistry; Elsevier: Amsterdam, The Netherlands, 2022; Volume 75, pp. 243–266.
5. Rehman, M.U.; Abdullah; Khan, F.; Niaz, K. Chapter 1—Introduction to Natural Products Analysis. In Recent Advances in Natural
Products Analysis; Sanches Silva, A., Nabavi, S.F., Saeedi, M., Nabavi, S.M., Eds.; Elsevier: Amsterdam, The Netherlands, 2020;
pp. 3–15. ISBN 978-0-12-816455-6.
6. Xi, X.; Wang, J.; Qin, Y.; You, Y.; Huang, W.; Zhan, J. The Biphasic Effect of Flavonoids on Oxidative Stress and Cell Proliferation
in Breast Cancer Cells. Antioxidants 2022, 11, 622. [CrossRef] [PubMed]
7. Nile, S.H.; Nile, A.S.; Keum, Y.S.; Sharma, K. Utilization of Quercetin and Quercetin Glycosides from Onion (Allium cepa L.) Solid
Waste as an Antioxidant, Urease and Xanthine Oxidase Inhibitors. Food Chem. 2017, 235, 119–126. [CrossRef] [PubMed]
8. Shukor, N.A.A.; Ablat, A.; Muhamad, N.A.; Mohamad, J. In Vitro Antioxidant and in Vivo Xanthine Oxidase Inhibitory Activities
of Pandanus amaryllifolius in Potassium Oxonate-Induced Hyperuricemic Rats. Int. J. Food Sci. Technol. 2018, 53, 1476–1485.
[CrossRef]
9. Huang, C.Y.; Chang, Y.Y.; Chang, S.T.; Chang, H.T. Xanthine Oxidase Inhibitory Activity and Chemical Composition of Pistacia
chinensis Leaf Essential Oil. Pharmaceutics 2022, 14, 1982. [CrossRef] [PubMed]
10. Vickneson, K.; George, J. Xanthine Oxidoreductase Inhibitors. In Reactive Oxygen Species; Handbook of Experimental Pharmacol-
ogy; Springer: Cham, Switzerland, 2021; Volume 264, pp. 205–228. [CrossRef]
11. Marrelli, M. Medicinal Plants. Plants 2021, 10, 1355. [CrossRef]
12. Park, C.H.; Yeo, H.J.; Baskar, T.B.; Park, Y.E.; Park, J.S.; Lee, S.Y.; Park, S.U. In Vitro Antioxidant and Antimicrobial Properties of
Flower, Leaf, and Stem Extracts of Korean Mint. Antioxidants 2019, 8, 75. [CrossRef]
13. Atanasov, A.G.; Zotchev, S.B.; Dirsch, V.M.; Orhan, I.E.; Banach, M.; Rollinger, J.M.; Barreca, D.; Weckwerth, W.; Bauer, R.;
Bayer, E.A.; et al. Natural Products in Drug Discovery: Advances and Opportunities. Nat. Rev. Drug Discov. 2021, 20, 200–216.
[CrossRef]
14. Michela, A.; Montone, I.; Papaianni, M.; Malvano, F.; Capuano, F.; Capparelli, R.; Albanese, D. Lactoferrin, Quercetin, and
Hydroxyapatite Act Synergistically against Pseudomonas fluorescens. Int. J. Mol. Sci. 2021, 22, 9247.
15. Xu, X.; Liu, A.; Hu, S.; Ares, I.; Martínez-Larrañaga, M.R.; Wang, X.; Martínez, M.; Anadón, A.; Martínez, M.A. Synthetic Phenolic
Antioxidants: Metabolism, Hazards and Mechanism of Action. Food Chem. 2021, 353, 129488. [CrossRef] [PubMed]
16. Lourenço, S.C.; Moldão-Martins, M.; Alves, V.D. Antioxidants of Natural Plant Origins: From Sources to Food Industry
Applications. Molecules 2019, 24, 4132. [CrossRef] [PubMed]
17. Fadaka, A.O.; Sibuyi, N.R.S.; Martin, D.R.; Klein, A.; Madiehe, A.; Meyer, M. Development of Effective Therapeutic Molecule
from Natural Sources against Coronavirus Protease. Int. J. Mol. Sci. 2021, 22, 9431. [CrossRef] [PubMed]
18. Balanescu, F.; Busuioc, A.C.; Botezatu, A.V.D.; Gosav, S.; Avramescu, S.M.; Furdui, B.; Dinica, R.M. Comparative Study of Natural
Antioxidants from Glycine max, Anethum graveolens and Pimpinella anisum Seed and Sprout Extracts Obtained by Ultrasound-
Assisted Extraction. Separations 2022, 9, 152. [CrossRef]
19. Karpiński, T.M. Essential Oils of Lamiaceae Family Plants as Antifungals. Biomolecules 2020, 10, 103. [CrossRef]
20. Lawson, S.K.; Satyal, P.; Setzer, W.N. The Volatile Phytochemistry of Seven Native American Aromatic Medicinal Plants. Plants
2021, 10, 1061. [CrossRef]
Int. J. Mol. Sci. 2023, 24, 828 21 of 22

21. Hashemi, M.; Ehsani, A.; Hassani, A.; Afshari, A.; Aminzare, M.; Sahranavard, T.; Azimzadeh, Z. Phytochemical, Antibacterial,
Antifungal and Antioxidant Properties of Agastache Foeniculum Essential Oil. J. Chem. Health Risks 2017, 7, 95–104.
22. Ivanov, I.G.; Vrancheva, R.Z.; Petkova, N.T.; Tumbarski, Y.; Dincheva, I.N.; Badjakov, I.K. Phytochemical Compounds of Anise
Hyssop (Agastache foeniculum) and Antibacterial, Antioxidant, and Acetylcholinesterase Inhibitory Properties of Its Essential Oil.
J. Appl. Pharm. Sci. 2019, 9, 72–78. [CrossRef]
23. Lashkari, A.; Najafi, F.; Kavoosi, G.; Niazi, S. Evaluating the In Vitro Anti-Cancer Potential of Estragole from the Essential Oil of
Agastache foeniculum [Pursh.] Kuntze. Biocatal. Agric. Biotechnol. 2020, 27, 101727. [CrossRef]
24. Fuentes-Granados, R.G.; Widrlechner, M.P.; Wilson, L.A. Inheritance Studies of Aromatic Compounds in Agastache Foeniculum
(Pursh) Kuntze. J. Essent. Oil Res. 2000, 12, 581–594. [CrossRef]
25. Talebi, S.F.; Saharkhiz, M.J.; Kermani, M.J.; Sharafi, Y.; Raouf Fard, F. Effect of Different Antimitotic Agents on Polyploid Induction
of Anise Hyssop (Agastache foeniculum L.). Caryologia 2017, 70, 184–193. [CrossRef]
26. Strilbytska, O.M.; Zayachkivska, A.; Koliada, A.; Galeotti, F.; Volpi, N.; Storey, K.B.; Vaiserman, A.; Lushchak, O. Anise Hyssop
Agastache foeniculum Increases Lifespan, Stress Resistance, and Metabolism by Affecting Free Radical Processes in Drosophila.
Front. Physiol. 2020, 11, 596729. [CrossRef] [PubMed]
27. Itokawa, H.; Suto, K.; Takeya, K. Structures of Isoagastachoside and Agastachin, New Glucosylflavones Isolated from Agastache
rugosa. Chem. Pharm. Bull. 1981, 29, 1777–1779. [CrossRef]
28. Fathiazad, F.; Hamedeyazdan, S. A Review on Hyssopus officinalis L.: Composition and Biological Activities. Afr. J. Pharm.
Pharmacol. 2011, 5, 1959–1966. [CrossRef]
29. Lee, C.; Kim, H.; Kho, Y. Agastinol and Agastenol, Novel Lignans from Agastache rugosa and Their Evaluation in an Apoptosis
Inhibition Assay. J. Nat. Prod. 2002, 65, 414–416. [CrossRef]
30. Zielińska, S.; Matkowski, A. Phytochemistry and Bioactivity of Aromatic and Medicinal Plants from the Genus Agastache
(Lamiaceae). Phytochem. Rev. 2014, 13, 391–416. [CrossRef]
31. Jovanović, A.A.; Ðord̄ević, V.B.; Zdunić, G.M.; Pljevljakušić, D.S.; Šavikin, K.P.; God̄evac, D.M.; Bugarski, B.M. Optimization
of the Extraction Process of Polyphenols from Thymus serpyllum L. Herb Using Maceration, Heat- and Ultrasound-Assisted
Techniques. Sep. Purif. Technol. 2017, 179, 369–380. [CrossRef]
32. Ebadollahi, A. Chemical Constituents and Toxicity of Agastache foeniculum (Pursh) Kuntze Essential Oil against Two Stored-Product
Insect Pests. Chil. J. Agric. Res. 2011, 71, 212–217. [CrossRef]
33. Baker, R.E.; Mahmud, A.S.; Miller, I.F.; Rajeev, M.; Rasambainarivo, F.; Rice, B.L.; Takahashi, S.; Tatem, A.J.; Wagner, C.E.; Wang,
L.F.; et al. Infectious Disease in an Era of Global Change. Nat. Rev. Microbiol. 2022, 20, 193–205. [CrossRef]
34. Watkins, K. Emerging Infectious Diseases: A Review. Curr. Emerg. Hosp. Med. Rep. 2018, 6, 86–93. [CrossRef]
35. Al-Ishaq, R.K.; Abotaleb, M.; Kubatka, P.; Kajo, K.; Büsselberg, D. Flavonoids and Their Anti-Diabetic Effects: Cellular Mechanisms
and Effects to Improve Blood Sugar Levels. Biomolecules 2019, 9, 430. [CrossRef] [PubMed]
36. Svitlana, K.; Vladimir, V.; Inna, M. Perspectives Culture of the Lophanthus anisatus Benth. and Peculiarities of Its Ontogenesis in
the Conditions of the Lowland Zone of Transcarpathian. Ecol. Evol. Biol. 2020, 5, 29. [CrossRef]
37. Li, H.Q.; Liu, Q.Z.; Liu, Z.L.; Du, S.S.; Deng, Z.W. Chemical Composition and Nematicidal Activity of Essential Oil of Agastache
rugosa against Meloidogyne incognita. Molecules 2013, 18, 4170–4180. [CrossRef] [PubMed]
38. Hernández-Rodríguez, P.; Baquero, L.P.; Larrota, H.R. Flavonoids: Potential Therapeutic Agents by Their Antioxidant Capacity.
In Bioactive Compounds; Elsevier Inc.: Amsterdam, The Netherlands, 2018; ISBN 9780128147757.
39. Pastor-Villaescusa, B.; Rodriguez, E.S.; Rangel-Huerta, O.D. Polyphenols in Obesity and Metabolic Syndrome. In Obesity Oxidative
Stress and Dietary Antioxidants; Academic Press: Cambridge, MA, USA, 2018; pp. 213–239. [CrossRef]
40. Venugopala, K.N.; Tratrat, C.; Chandrashekharappa, S.; Attimarad, M.; Sreeharsha, N.; Nair, A.B.; Pottathil, S.; Venugopala, R.;
Al-Attraqchi, O.H.A.; Morsy, M.A.; et al. Anti-Tubercular Potency and Computationally- Assessed Drug-Likeness and Toxicology
of Diversely Substituted Indolizines. Indian J. Pharm. Educ. Res. 2019, 53, 545–552. [CrossRef]
41. Charles, D.J.; Simon, J.E.; Widrlechner, M.P. Characterization of Essential Oil of Agastache Species. J. Agric. Food Chem. 1991,
39, 1946–1949. [CrossRef]
42. Mazza, G.; Kiehn, F.A. Essential Oil of Agastache foeniculum, a Potential Source of Methyl Chavicol. J. Essent. Oil Res. 1992,
4, 295–299. [CrossRef]
43. Skakovskii, E.D.; Kiselev, W.P.; Tychinskaya, L.Y.; Schutova, A.G.; Gonsharova, L.W.; Spiridowish, E.W.; Bovdey, N.A.; Kiselev,
P.A.; Gaidukevich, O.A. Characterization of the Essential Oil of Agastache rugosa by NMR Spectroscopy. J. Appl. Spectrosc. 2010,
77, 329–334. [CrossRef]
44. Bayguinov, P.O.; Oakley, D.M.; Shih, C.C.; Geanon, D.J.; Joens, M.S.; Fitzpatrick, J.A.J. Modern Laser Scanning Confocal
Microscopy. Curr. Protoc. Cytom. 2018, 85, 1–17. [CrossRef]
45. Borges, A.; José, H.; Homem, V.; Simões, M. Comparison of Techniques and Solvents on the Antimicrobial and Antioxidant
Potential of Extracts from Acacia dealbata and Olea europaea. Antibiotics 2020, 9, 48. [CrossRef]
46. Palma-Tenango, M.; Sánchez-Fernández, R.E.; Soto-Hernández, M. A Systematic Approach to Agastache mexicana Research:
Biology, Agronomy, Phytochemistry, and Bioactivity. Molecules 2021, 26, 3751. [CrossRef]
47. Igbinosa, O.O.; Igbinosa, I.H.; Chigor, V.N.; Uzunuigbe, O.E.; Oyedemi, S.O.; Odjadjare, E.E.; Okoh, A.I.; Igbinosa, E.O.
Polyphenolic Contents and Antioxidant Potential of Stem Bark Extracts from Jatropha curcas (Linn). Int. J. Mol. Sci. 2011,
12, 2958–2971. [CrossRef] [PubMed]
Int. J. Mol. Sci. 2023, 24, 828 22 of 22

48. Araújo, M.G.d.F.; Hilário, F.; Vilegas, W.; dos Santos, L.C.; Brunetti, I.L.; Sotomayor, C.E.; Bauab, T.M. Correlation among
Antioxidant, Antimicrobial, Hemolytic, and Antiproliferative Properties of Leiothrix spiralis Leaves Extract. Int. J. Mol. Sci. 2012,
13, 9260–9277. [CrossRef] [PubMed]
49. Kim, T.Y.; Leem, E.; Lee, J.M.; Kim, S.R. Control of Reactive Oxygen Species for the Prevention of Parkinson’s Disease: The
Possible Application of Flavonoids. Antioxidants 2020, 9, 583. [CrossRef] [PubMed]
50. Zhang, C.; Wang, R.; Zhang, G.; Gong, D. Mechanistic Insights into the Inhibition of Quercetin on Xanthine Oxidase. Int. J. Biol.
Macromol. 2018, 112, 405–412. [CrossRef]
51. Liu, D.; Wang, D.; Yang, W.; Meng, D. Potential Anti-Gout Constituents as Xanthine Oxidase Inhibitor from the Fruits of Stauntonia
brachyanthera. Bioorg. Med. Chem. 2017, 25, 3562–3566. [CrossRef]
52. Sharifi-Rad, J.; Sureda, A.; Tenore, G.; Daglia, M.; Sharifi-Rad, M.; Valussi, M.; Tundis, R.; Sharifi-Rad, M.; Loizzo, M.; Ademiluyi,
A.; et al. Biological Activities of Essential Oils: From Plant Chemoecology to Traditional Healing Systems. Molecules 2017, 22, 70.
[CrossRef]
53. Lima, C.C.; de Holanda-Angelin-Alves, C.M.; Pereira-Gonçalves, Á.; Kennedy-Feitosa, E.; Evangelista-Costa, E.; Bezerra, M.A.C.;
Coelho-de-Souza, A.N.; Leal-Cardoso, J.H. Antispasmodic Effects of the Essential Oil of Croton Zehnteneri, Anethole, and
Estragole, on Tracheal Smooth Muscle. Heliyon 2020, 6, e05445. [CrossRef]
54. European Commission Regulation (EC) No 1334/2008 on Flavourings. Off. J. Eur. Union 2008, L 354/34, 34–50.
55. Eisenbrand, G.; Cohen, S.M.; Fukushima, S.; Gooderham, N.J.; Guengerich, F.P.; Hecht, S.S.; Rietjens, I.M.C.M.; Rosol, T.J.;
Davidsen, J.M.; Harman, C.L.; et al. FEMA GRAS Assessment of Natural Flavor Complexes: Eucalyptus Oil and Other Cyclic
Ether-Containing Flavoring Ingredients. Food Chem. Toxicol. 2021, 155, 112357. [CrossRef]
56. Bristol, D.W. NTP 3-Month Toxicity Studies of Estragole Administered by Gavage to F344/N Rats and B6C3F1 Mice. Toxic Rep.
Ser. 2011, 82, 1–111.
57. Gori, L.; Gallo, E.; Mascherini, V.; Mugelli, A.; Vannacci, A.; Firenzuoli, F. Can Estragole in Fennel Seed Decoctions Really Be
Considered a Danger for Human Health? A Fennel Safety Update. Evid.-Based Complement. Altern. Med. 2012, 2012, 860542.
[CrossRef] [PubMed]
58. Ebadollahi, A.; Safaralizadeh, M.; Pourmirza, A.; Gheibi, S. Toxicity of Essential Oil of Agastache foeniculum (Pursh) Kuntze to
Oryzaephilus surinamensis L. and Lasioderma serricorne F. J. Plant Prot. Res. 2010, 50, 215–219. [CrossRef]
59. NIST/EPA/NIH Mass Spectral Database; SRD Program; National Institute of Standards and Technology: Gaithersburg, MD, USA,
2011.
60. Gonfa, T.; Teketle, S.; Kiros, T. Effect of Extraction Solvent on Qualitative and Quantitative Analysis of Major Phyto-Constituents
and in-Vitro Antioxidant Activity Evaluation of Cadaba rotundifolia Forssk Leaf Extracts. Cogent Food Agric. 2020, 6, 1853867.
[CrossRef]
61. Balanescu, F.; Mihaila, M.D.I.; Cârâc, G.; Furdui, B.; Vînătoru, C.; Avramescu, S.M.; Lisa, E.L.; Cudalbeanu, M.; Dinica, R.M.
Flavonoid Profiles of Two New Approved Romanian Ocimum Hybrids. Molecules 2020, 25, 4573. [CrossRef]
62. Cudalbeanu, M.; Ghinea, I.O.; Furdui, B.; Dah-Nouvlessounon, D.; Raclea, R.; Costache, T.; Cucolea, I.E.; Urlan, F.; Dinica, R.M.
Exploring New Antioxidant and Mineral Compounds from Nymphaea alba Wild-Grown in Danube Delta Biosphere. Molecules
2018, 23, 1247. [CrossRef]
63. Chisté, R.C.; Mercadante, A.Z. Identification and Quantification, by HPLC-DAD-MS/MS, of Carotenoids and Phenolic Com-
pounds from the Amazonian Fruit Caryocar villosum. J. Agric. Food Chem. 2012, 60, 5884–5892. [CrossRef]
64. Busuioc, A.C.; Botezatu, A.V.D.; Furdui, B.; Vinatoru, C.; Maggi, F.; Caprioli, G.; Dinica, R.M. Comparative Study of the Chemical
Compositions and Antioxidant Activities of Fresh Juices from Romanian Cucurbitaceae Varieties. Molecules 2020, 25, 5468.
[CrossRef]
65. Wandjou, J.G.N.; Mevi, S.; Sagratini, G.; Vittori, S.; Dall’acqua, S.; Caprioli, G.; Lupidi, G.; Mombelli, G.; Arpini, S.; Allegrini, P.;
et al. Antioxidant and Enzyme Inhibitory Properties of the Polyphenolic-Rich Extract from an Ancient Apple Variety of Central
Italy (Mela Rosa Dei Monti Sibillini). Plants 2020, 9, 9. [CrossRef]
66. Cudalbeanu, M.; Furdui, B.; Cârâc, G.; Barbu, V.; Iancu, A.V.; Marques, F.; Leitão, J.H.; Sousa, S.A.; Dinica, R.M. Antifungal,
Antitumoral and Antioxidant Potential of the Danube Delta Nymphaea Alba Extracts. Antibiotics 2020, 9, 7. [CrossRef]
67. Gao, J.; Hu, J.; Hu, D.; Yang, X. A Role of Gallic Acid in Oxidative Damage Diseases: A Comprehensive Review. Nat. Prod.
Commun. 2019, 14. [CrossRef]
68. Rivero-Cruz, J.F.; Granados-Pineda, J.; Pedraza-Chaverri, J.; Pérez-Rojas, J.M.; Kumar-Passari, A.; Diaz-Ruiz, G.; Rivero-Cruz, B.E.
Phytochemical Constituents, Antioxidant, Cytotoxic, and Antimicrobial Activities of the Ethanolic Extract of Mexican Brown
Propolis. Antioxidants 2020, 9, 70. [CrossRef] [PubMed]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.