Antioxidants 13 00058

antioxidants
Article
Assessing the Antioxidant Properties, In Vitro Cytotoxicity and
Antitumoral Effects of Polyphenol-Rich Perilla leaves Extracts
Gigi Adam 1,2,† , Florina Daniela Cojocaru 3,† , Liliana Verestiuc 3,† , Oana Cioanca 1, * ,
Ingrid-Andrada Vasilache 4 , Ana-Maria Adam 2, *, Cornelia Mircea 1,† , Aurel Nechita 2 , Valeriu Harabor 2 ,
Bogdan Huzum 5 , AnaMaria Harabor 2 and Monica Hancianu 1
1 Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and Pharmacy, 16 Universitatii Street,
700115 Iasi, Romania; [email protected] (C.M.); [email protected] (M.H.)
2 Faculty of Medicine and Pharmacy, “Dunarea de Jos” University, 35 Al. I. Cuza Street,
800216 Galati, Romania; [email protected] (A.N.); [email protected] (V.H.);
[email protected] (A.H.)
3 Department of Biomedical Sciences, Faculty of Medical Bioengineering, University of Medicine and Pharmacy
“Grigore T. Popa” Iasi, 700454 Iasi, Romania; [email protected] (F.D.C.);
[email protected] (L.V.)
4 Department of Obstetrics and Gynecology, “Grigore T. Popa” University of Medicine and Pharmacy,
700115 Iasi, Romania
5 Department of Orthopaedic and Traumatology, Faculty of Medicine, “Grigore T. Popa” University of
Medicine and Pharmacy, 16 Universitatii Street, 700115 Iasi, Romania
* Correspondence: [email protected] (O.C.); [email protected] or
[email protected] (A.-M.A.)
† These authors contributed equally to this work.
Abstract: (1) Background: This study aimed to outline the antioxidant, antitumoral, and cytotoxic
proprieties of various types of Perilla frutescens extracts obtained from the leaves of the species.
(2) Methods: We determined total polyphenols, flavonoids and anthocyanins contents, as well as
the in vitro antioxidant, antitumoral, and cytotoxic actions in three types of ethanolic extracts (E1,
Citation: Adam, G.; Cojocaru, F.D.; E2, E3) and in three types of acetone: ethanol extracts (A1, A2, A3) of Perilla frutescens according
Verestiuc, L.; Cioanca, O.; Vasilache, to standardized procedures. (3) Results: We found that Perilla frutescens ethanolic extracts had
I.-A.; Adam, A.-M.; Mircea, C.; the highest total phenol and anthocyanins concentrations. The flavonoids concentration was not
Nechita, A.; Harabor, V.; Huzum, B.; statistically different between the extracts. The iron chelating capacity, hydroxyl radical scavenging
et al. Assessing the Antioxidant
capacity, superoxide anion radical scavenging capacity, and lipoxygenase inhibition capacity showed
Properties, In Vitro Cytotoxicity and
a significant increase with higher concentrations of Perilla frutescens extracts, particularly the ethanolic
Antitumoral Effects of Polyphenol-
extracts. Perillyl alcohol had greater cytotoxic capacity in the MG-63 cell line and E1 extract showed
Rich Perilla leaves Extracts.
similar significant cytotoxic effects in the A431 cell line. (4) Conclusions: Both ethanolic and acetone–
Antioxidants 2024, 13, 58. https://
doi.org/10.3390/antiox13010058
ethanol extracts from Perilla frutescens exhibited important antioxidant and antitumoral actions
in vitro, which proportionally increased with concentration. The cytotoxic threshold determined in
Academic Editor: Michał Swieca
this study for various types of extracts could help determine the best dosage with the maximum
Received: 21 November 2023 antioxidant and antitumoral potential. Our results could serve as a basis for further studies that will
Revised: 24 December 2023 investigate the cytotoxic effects of Perilla frutescens variants on various types of cancer cell lines.
Accepted: 27 December 2023
Published: 29 December 2023 Keywords: Perilla frutescens; extracts; antioxidant; antitumoral; cytotoxicity
Copyright: © 2023 by the authors.

1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article Perilla frutescens (L.) Britton var. frutescens is a medicinal herb that belongs to the
distributed under the terms and Lamiaceae family, with important antioxidant and antitumoral effects demonstrated both
conditions of the Creative Commons in vitro and in vivo [1,2]. Numerous types of extracts and components have been identified
Attribution (CC BY) license (https:// from this plant to date, each comprising a specific portfolio of biochemical activities and
creativecommons.org/licenses/by/ cytotoxic effects.
4.0/).
Antioxidants 2024, 13, 58. https://doi.org/10.3390/antiox13010058 https://www.mdpi.com/journal/antioxidants

Antioxidants 2024, 13, 58 2 of 23
A recent systematic review outlined 14 classes of Perilla frutescens active constituents:

alkaloids phenylpropane, terpenoids, polyphenol compounds, flavonoids, anthocyanins,
coumarins, carotenoids, neolignans, fatty acids, tocopherols, phytosterols, glucosides and
peptides [3]. These classes reunite a plethora of compounds that have antitumoral effects
by modulating various elements of the metabolic pathways or intercellular interactions
such as reactive oxidative species (ROS), nuclear factor kappa-light-chain-enhancer of
activated B cells (NF-κB), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), c-Jun
N-terminal kinases (JNK), yes-associated protein/WW domain-containing transcription
factor (YAP/TAZ), etc. [3,4].
Further studies outlined the antitumoral and cytotoxic properties of phenolic and
flavonoid compounds, which are abundant in water and ethanol extracts of Perilla
frutescens [5,6]. A recent study by Garcia et al. investigated the in vitro cytotoxic effects
of various parts of Perilla frutescens extracts (stems, leaves, and seeds) on prostate cancer
cells (DU-145) [7]. The authors demonstrated that the leaf extracts possessed the highest
amounts of phytochemicals compared to the other studied parts, and that the ethanolic
extract from the leaves exhibited the highest cytotoxic potential on DU-145 cells.
The antioxidant proprieties of Perilla frutescens extracts were demonstrated in nu-
merous trials and have an important impact throughout the evolution of metabolic or
degenerative disorders [8–10]. The metabolite profiles of Perilla frutescens extracts vary
depending on the species, the part of the plant used, the type of extract, and the method of
determination, providing numerous possibilities to study their antioxidant proprieties.
During inflammatory processes, a large amount of superoxide radicals, along with
other radicals, are produced by activated neutrophils and macrophages under the action
of NAD(P)H oxidase. These free radicals generated in the inflammatory focus can induce
local and general toxic phenomena. Anti-inflammatory drugs reduce the synthesis of
pro-inflammatory compounds, but generally do not have the ability to neutralize the pro-
oxidant compounds generated during the inflammatory process. Thus, compounds with
antioxidant action can represent an important therapeutic option [11–13].
Polyphenols, effective antioxidants identified from Perilla frutescens extracts, are sec-
ondary metabolites that have the ability to neutralize hydroxyl and superoxide radicals,
to block the oxidizing action of peroxynitrite that affects the structure of biologically ac-
tive molecules and to chelate pro-oxidizing transitional metals. The antioxidant action is
complemented by the anti-inflammatory, antibacterial, enzyme inhibitory and antimuta-
genic [14].
Flavonoids act as scavengers of free radicals due to their ability to donate hydrogen
atoms to radicals and stabilize them. The scavenger capacity of flavonoids depends on
their structure, on the number of hydroxyl groups in the structure, on the position of these
hydroxyl groups, and on the ability of the hydroxyl groups to give up hydrogen atoms [15].
The scavenging capacity of free radicals is conditioned by the presence of hydroxy
groups in the 3′ , 4′ positions of the B ring in the flavonoid structure. The hydroxy groups in
the 3′ , 4′ positions of the B ring also influence the ability of flavones to fix transition metals.
Methoxylation of hydroxy groups causes the disappearance of the scavenger action [16].
An important role is also played by the OH group in position 3 of ring C. The C2–C3
double bond conjugated with a keto group in position 4 determines the delocalization of
electrons in the B nucleus and increases the scavenger capacity of free radicals, while the
reduction of this bond will decrease the scavenger potential of the flavone without the
antiradical action disappearing [15].
The presence of the hydroxyl group in positions 3 (nucleus A) and 5 (nucleus C) and a
carbonyl group in position 4 (nucleus C) increases the scavenger capacity. In the absence of
o-dihydroxy groups in the structure of the B nucleus, the hydroxy groups in the A nucleus
are capable of inducing the scavenger action of free radicals [15]. The position of hydroxyl
groups in the structure of flavonoids is more important compared to their number.
The most active flavonoids are those that have hydroxyl groups in positions 3 and 4 of
nucleus B and/or hydroxyl groups in position 3 of nucleus C. Hydroxyl groups in nucleus
B also increase the stability of flavonoids. Flavones that do not have OH groups in the B
nucleus, but have an OH group in position 3 of the C nucleus and a keto group in position
4, have the scavenger action [10].
Glycosylation at the carbon atom increases the antioxidant character, and glycosylation
at the oxygen atom decreases the antioxidant capacity [14].
Flavones can inhibit enzymes that catalyze the synthesis of reactive oxygen species,
such as NADH oxidase, mitochondrial succinoxidase and microsomal monooxygenase.
They also increase the activity of antioxidant enzymes: superoxide dismutase, catalase,
glutathione peroxidase and glutathione reductase [17,18].
The cytotoxic effects of different varieties of Perilla frutescens and types of extracts
from these varieties are not extensively studied in the literature. Thus, in this study, we
aimed to characterize three varieties of Perilla frutescens species, namely Perilla frutescens
var. crispa f. purpurea, Perilla frutescens var. frutescens f. crispidiscolor, and Perilla frutescens
var. frutescens f. viridis, and their corresponding extracts (ethanolic or ethanol–acetone)
considering their chemical composition, as well as their in vitro antioxidant, cytotoxic and
antitumoral proprieties.
This study will comprise three parts corresponding to our primary objectives: (a) quan-
tification of the total flavonoid, polyphenols, and anthocyanin content of various types
of Perilla frutescens extracts; (b) determination of the in vitro antioxidant action of Perilla
frutescens extracts; (c) characterization of the in vitro cytotoxic and antitumoral activity of
Perilla frutescens extracts.
2. Materials and Methods

2.1. The Extraction Processes
The study focused on the chemical analysis of two types of extracts obtained by
processing the plant material harvested from three varieties of Perilla frutescens var. crispa f.
purpurea, Perilla frutescens var. frutescens f. crispidiscolor, and Perilla frutescens var. frutescens
f. viridis. The plant material consisted of leaves harvested in September 2020 at the
Vegetable Research and Development Station in Buzău, Romania, with the following
coordinates: 45◦ 09′ 30.2′′ northern latitude and 26◦ 49′ 37.9′′ eastern longitude. Cultivation
was carried out both in greenhouses and in the open field between May and September
2020. Taxonomic identification was provided by a specialized biologist, and the vegetal
material is currently deposited in the collection of conserved materials from the Vegetal
Morphoanatomy Laboratory at Biology Faculty of the “Alexandru Ioan Cuza” University,
Iasi, Romania.
The harvested leaves were dried to a constant weight over a period of three weeks.
They were placed on white paper sheets in a thin layer in a dark and well-ventilated room
at a temperature of 23 ◦ C. The plant material was used to obtain two types of extracts,
which were employed for both qualitative and quantitative chemical characterization of
polyphenolic compounds.
For the first type of extract, 2 g of pulverized plant material was placed in round-
bottom flasks and 50 mL of 70% ethanol was added. The mixture was subjected to reflux
extraction at a temperature of 70 ◦ C using a thermostatic water bath for a duration of 6 h, a
process repeated twice. The extraction products were filtered and then combined.
In the case of the second type of extract, 2 g of pulverized plant material was placed in
iodometric flasks, and 100 mL of a solvent mixture of acetone–ethanol (7:3) and 0.5 g of
citric acid was added. Each flask was equipped with a magnetic stir bar, tightly sealed, and
subjected to extraction on a magnetic stirrer for 6 h at room temperature. This process was
repeated twice. Following extraction, the obtained filtrates were combined.
The extracts obtained after combining the filtrates were transferred into porcelain
capsules and left at room temperature for concentration and drying. The resulting extracts
were utilized for both chemical and biological characterization.
We choose to study these types of extracts because ethanol is known for its high
extraction efficiency and its ability to extract bioactive compounds from various plant ma-
terials. It is a common choice for extracting phytochemicals, such as phenolic compounds,

flavonoids, and alkaloids. Also, acetone–ethanol mixtures can provide a balance between
polar and non-polar extraction, allowing for a more comprehensive extraction of a diverse
array of compounds.
2.2. Total Flavonoids, Phenols, and Anthocyanins Quantification

2.2.1. Total Flavonoids Quantification
Flavonoids were quantified as previously described, and their concentration was
expressed as rutoside equivalents (µg/mL). Briefly, 5 mL of each extract sample was
mixed with 5.0 mL of 100 g/L sodium acetate and 3.0 mL of 25 g/L aluminum chloride.
Methanol was added up to 25 mL in a graduated flask. The absorbance corresponding to the
yellow color of the complex was photometered at 430 nm (the corresponding wavelength
for optimal absorption for rutoside). The final results represent the average of three
determinations and the standard deviation.
2.2.2. Total Phenol Quantification

Total polyphenols were quantified according to the well-established methodology
using gallic acid as standard. For this, 40 microliters of the solution of each extract were
mixed with 3160 microliters of distilled water and 200 microliters of Folin–Ciocâlteu reagent.
After 5 min, 600 microliters of 20% sodium carbonate solution was added. After 2 h for
incubation in the dark, the absorbance of the mixture was read at λ = 716 nm against a
compensation liquid consisting of 40 µL extract solution, 3760 µL distilled water and 200 µL
Folin–Ciocâlteu reagent. This technique indicates the presence of free hydroxyl groups
that are available in all compounds found in the investigated sample. The final results
expressed as µg gallic acid/mL extract represent the average of three determinations ±
standard deviation.
Calibration curves in rutoside (concentration ranges 0.025–0.3 mg/mL, R2 = 0.9906)
and gallic acid (concentration ranges 0.039–2.5 mg/mL, R2 = 0.9969) were established in
parallel for each group of components, using the same methodology.
2.2.3. Total Anthocyanin Quantification

Starting from the method described by Giusti and Wrolstad in 2001, the total antho-
cyanin content was evaluated by using two dilutions of the same extract and changing
the pH between 1.0 and 4.5. It is known that the chemical form of anthocyanin radicals
(oxonium) has different absorption spectra when the acidic environment changes [19].
A quota from each extract was diluted to 10 mL in two volumetric flasks, adjusting
the pH to 1.0 (potassium chloride buffer), and the other to pH 4.5 (sodium acetate buffer),
mixing continuously. After an equilibration time (15–20 min), the absorbance of each
dilution was measured at 510 nm and then 700 nm against a blank of distilled water.
The difference between each measurement at both pH values represents the corrected
absorbance used for further calculation. The results were expressed as µg cyanidol/mL
extract and were calculated as the average of three determinations.
2.2.4. UHPLC-PDA Quantification and Identification of Extracted Compounds

In the present research, a Thermo Fischer UltiMate 3000 ultra-fast system (Thermo
Fisher Scientific Inc., Waltham, MA, USA) coupled with a multi-diode detector (PDA),
quaternary pump (LPG-3400 SD) with built-in four-channel degasser and an autosampler
(injecting variable amounts from 2 µL to 200 µL) from the same producer, was used.
The conditions for UHPLC-PDA included Kinetex C18 (150 × 4.6 mm, 100 Å) column
(Phenomenex, Torrance, CA, USA), and a gradient of 10 to 65 A (acetonitrile) in B (aqueous
solution of acetic acid 0.1%) over 25 min, a starting flow of 1 mL/min, then 0.8 mL/min.
Each sample was obtained from 4 mg of dry extract diluted to 1 mL (ethanol–water, 3:1),
from which 10 µL were injected for analysis. Simultaneous detection at three wavelengths
275 nm (flavonoids), 330 nm (polyphenolic acids), 520 nm (anthocyanins) was used.
The integration, identification and calculation of each peak was carried out with
Chromeleon™ 7.3 (Thermo Scientific™ Dionex™, Thermo Fisher Scientific Inc., Waltham,
MA, USA). A matching index of at least 950 (from a total of 1000) was used to identify
compounds based on UV spectra and retention time.
Aliquots (2–10 µL) of standard stock solutions (epicatechin, caffeic acid, rosmarinic
acid, chlorogenic acid, ferulic acid, luteolin, apigenin, quercetin-3-arabinoside, apigenin-7-
O-glucoside, and luteolin 7-O-glucoside) were used for calibration curves with a correlation
coefficient above 0.9989. The standard deviation was calculated at 0.02. The limit of
detection (LOD) and the limit of quantification (LOQ) of rutoside and rosmarinic acid were
calculated at 272 ng/mL and 182 ng/mL, respectively.
2.2.5. Chemicals and Materials

The reagents used for spectrophotometric determinations (sodium acetate, aluminum
chloride, Folin–Ciocalteu phenol reagent, reagent-grade sodium carbonate, sodium acetate
buffer, potassium chloride buffer) and solvents were bought from Merck (Darmstadt,
Germany)
All standards and solvents were of HPLC (Chromasolv) quality and were purchased
either from Sigma-Aldrich or from Merck (Darmstadt, Germany). The pure compounds
used as standards were obtained from Sigma-Aldrich (Darmstadt, Germany) and the purity
grade was at least 95%.
2.3. Iron Chelation Capacity Assessment

Principle of the Method: Fe2+ forms a pink-colored complex with ferrozine, exhibiting
maximum absorbance at 562 nm. The presence of a chelating agent in the reaction medium
results in reduced absorbance of the formed complex, leading to a decrease in the solution’s
color intensity [20,21]. The list of the reagents used for this procedure included:
- Dimethyl sulfoxide, DMSO (Merck, KgaA, Darmstadt, Germany);
- 0.1 M Acetate Buffer, pH 5.25;
- Iron (II) sulfate heptahydrate (Merck, KgaA, Darmstadt, Germany);
- 2 mM Iron (II) sulfate in 0.2 M HCl;
- Ferrozine (sodium salt of 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4′ ,4′ ’ disulfonic
acid) (Fluka, Sigma-Aldrich, Steinheim, Germany);
- 5 mM Ferrozine Solution;
- Hydrochloric Acid (Merck, Darmstadt, Germany);
- UV-VIS Spectrophotometer ABL & E Jasco V-550 (Tokyo, Japan);
- pH Meter Hanna Instruments pH210, Microprocessor pH-meter;
- Vortex Mixer Velp Scientifica (Usmate Velate, Italy);
- Ultra Clear TWF Water Purification System;
- Test samples—solutions obtained by dissolving dried extracts in DMSO—hydroalcoholic
extracts (E1, E2, E3), extracts in acetone: ethanol (7:3) acidified (A1, A2, A3); solution
concentration for analysis: 0.078125–10 mg/mL.
The procedure consisted of the following steps:
a. We mixed 0.2 mL of the test sample solution in ultrapure water, 0.74 mL of 0.1 M
acetate buffer (pH 5.25), and 0.02 mL of 2 mM iron sulfate in 0.2 M hydrochloric acid.
After 10–15 s of agitation, we added 0.04 mL of 5 mM ferrozine solution.
b. After 10 min of incubation in the dark, we measured the absorbance of the solution at
562 nm against a control prepared under the same conditions as the sample (ultrapure
water was used instead of iron sulfate solution).
c. Simultaneously, we prepared the control solution and its control: the control contained
0.2 mL of ultrapure water, 0.74 mL of 0.1 M acetate buffer (pH 5.25), 0.02 mL of 2 mM
iron sulfate in 0.2 M hydrochloric acid, and 0.04 mL of 5 mM ferrozine solution.
d. Gallic acid was used as a reference substance, and gallic acid solutions in DMSO were
processed under the same conditions as the ethanolic extract.
e. All determinations were carried out in triplicate, and results were expressed as the
mean of three determinations ± standard deviation.
The chelating capacity of the ferrous ion was calculated using the Formula (1):
% Activity = 100 × (Ac − Ap)/(Ac) (1)
where:
Ac is the absorbance of the control solution.
Ap is the absorbance of the sample solution.
For samples exhibiting a ferrous ion chelation capacity of over 50%, the CE50 value
was calculated and expressed in mg of sample/mL of final solution. CE50 was calculated
by considering the first value below 50% and the first value above 50% and interpolat-
ing linearly to determine the concentration of the antioxidant agent corresponding to
50% activity.
2.4. Determination of Hydroxyl Radical Scavenging Capacity

Principle of the Method: The hydroxyl radical, formed in the reaction between ferrous
ion and hydrogen peroxide, will hydroxylate salicylic acid, resulting in the formation of a
pink–violet compound with maximum absorbance at 562 nm [22]. A list of the reagents
used for this procedure included:
- Dimethyl sulfoxide (Merck, KgaA, Darmstadt, Germany);
- Ferrous sulfate heptahydrate (Merck, KgaA, Darmstadt, Germany);
- 1.5 mM ferrous sulfate solution in distilled water;
- 6 mM hydrogen peroxide solution in distilled water (Sigma-Aldrich, Steinheim,
Germany);
- 20 mM sodium salicylate solution in distilled water;
- Hanna Instruments pH210 Microprocessor pH-meter (Padova, Italy);
- Velp Scientifica Vortex agitator (Usmate Velate, Italy);
- Ultra Clear TWF water purification apparatus (Günzburg, Germany);
- ABL and E Jasco V-550 UV-VIS spectrophotometer (Tokyo, Japan);
- Test samples—solutions obtained by dissolving dry extracts in DMSO—hydroalcoholic
extracts (E1, E2, E3), extracts in acetone: ethanol 7:3 acidic mixture (A1, A2, A3);
concentration of analyzed solutions: 0.078125–10 mg/mL.
Over 0.225 mL of the sample solution in dimethyl sulfoxide, 0.750 mL of 1.5 mM
ferrous sulfate solution, 0.9 mL of 20 mM sodium salicylate solution, and 0.525 mL of 6 mM
hydrogen peroxide solution were added.
The mixture was kept at 37 ◦ C for 30 min, and after cooling to room temperature,
the absorbance of the sample (control) was read at 562 nm against the sample’s (control’s)
blank, in which the ferrous sulfate solution was replaced with distilled water. The positive
control was processed under the same conditions as the samples, but dimethyl sulfoxide
was used in place of the sample solution.
Gallic acid was used as a reference substance, and gallic acid solutions in DMSO were
processed under the same conditions as the ethanolic extract. All determinations were
performed in triplicate, and the results are expressed as the mean of three determinations
± standard deviation.
The hydroxyl radical scavenging capacity was calculated according to the Formula (1).
For the samples that exhibited a hydroxyl radical scavenging capacity of over 50%, the
CE50 value was calculated and expressed in µg of sample/mL of the final solution or µg of
gallic acid/mL. CE50 was calculated by considering the first value lower than 50% and the
first value higher than 50%, obtaining, through linear interpolation, the concentration of
the antioxidant solution that corresponds to a 50% activity.
2.5. Determining the Scavenging Capacity of the Superoxide Radical Anion

Principle of the Method: The superoxide radical generated by the reduced nicoti-
namide adenine dinucleotide-phenazine methosulfate system reduces nitroblue tetrazolium
to a violet-blue formazan compound with maximum absorbance at 560 nm [23].
The following reagents were used:
- Dimethyl sulfoxide (Merck, KgaA, Darmstadt, Germany);
- TRIS (Sigma-Aldrich, Steinheim, Germany);
- TRIS buffer pH 8—0.4845 g of TRIS dissolved in 180 mL of distilled water, adjusted to
pH 8 with 6M HCl solution, and completed with distilled water to 200 mL;
- Reduced nicotinamide adenine dinucleotide sodium salt (NADHNa2) (Sigma-Aldrich,
Steinheim, Germany);
- 557 µM solution of reduced nicotinamide adenine dinucleotide sodium salt (NADHNa2)
in TRIS buffer pH 8;
- nitroblue tetrazolium (Fluka, Steinheim, Germany);
- 108 µM solution of nitroblue tetrazolium in TRIS buffer pH 8;
- Phenazine methosulfate (Fluka, Sigma-Aldrich, Steinheim, Germany);
- 45 µM solution of phenazine methosulfate in TRIS buffer pH 8;
- pH meter Hanna Instruments pH210, Microprocessor pH-meter (Padova, Italy);
- Vortex mixer Velp Scientifica (Usmate Velate, Italy);
- Ultra Clear TWF water purification apparatus (Günzburg, Germany);
- UV-VIS spectrophotometer ABL & E Jasco V-550 (Tokyo, Japan);
extracts (E1, E2, E3), extracts in a mixed solution of acetone: ethanol 7:3 (A1, A2, A3);
concentration of the analyzed solutions: 0.078125–10 mg/mL.
Over 0.5 mL of sample solution in dimethyl sulfoxide (diluted solutions in dimethyl
sulfoxide) 0.5 mL of 557 µM NADHNa2 solution in TRIS buffer pH 8, and 0.5 mL of 108 µM
nitroblue tetrazolium solution in TRIS buffer pH 8 were added. The mixture was vortexed
for 5 s. To the mixture, 0.5 mL of 45 µM phenazine methosulfate solution in TRIS buffer
pH 8 was added, and it was allowed to stand for 5 min at room temperature. Afterward,
the absorbance of the sample (control) was measured against the sample control at 560 nm.
The positive control was processed under the same conditions as the samples, but dimethyl
sulfoxide was used instead of the sample solution.
Gallic acid was used as a reference substance, and solutions of gallic acid in DMSO
were processed under the same conditions as the ethanolic extract. All determinations were
performed in triplicate, and the results were expressed as the mean of three determina-
tions ± standard deviation.
The hydroxyl radical scavenging capacity was calculated according to the Formula (1).
For the samples that exhibited a superoxide radical scavenging capacity of over 50%,
the CE50 value was calculated and expressed in µg sample/mL final solution or µg gallic
acid/mL. The CE50 was calculated by considering the first value lower than 50% and
the first value higher than 50%, and then obtaining, through linear interpolation, the
concentration of the antioxidant solution corresponding to a 50% activity.
2.6. Determination of the Lipoxygenase Inhibition Capacity of Perilla frutescens Extracts

Principle of the Method: The active compounds present in the extracts block 15-
lipoxygenase by inhibiting the oxidation of linoleic acid and reducing absorbance at
234 nm [24].
The reagents used included:
- Dimethyl sulfoxide (DMSO) (Merck, KgaA, Darmstadt, Germany);
- 0.1 M borate buffer pH 9—Dissolve 6.2 g of boric acid in 950 mL of distilled water,
adjust to pH 9 with 1M NaOH, and make up to 1000 mL with distilled water;
- Linoleic acid (Sigma-Aldrich, Steinheim, Germany) 0.16 mM in 0.1 M borate buffer
pH 9;
- Soybean lipoxygenase (Sigma-Aldrich, Steinheim, Germany) in 0.1 M borate buffer

pH 9;
- UV-VIS spectrophotometer ABL & E Jasco V-550 (Tokyo, Japan);
- pH meter Hanna Instruments pH210, Microprocessor pH-meter (Padova, Italy);
- Vortex mixer Velp Scientifica (Usmate Velate, Italy);
- Ultra Clear TWF water purification system (Günzburg, Germany);
extracts (E1, E2, E3), extracts in a mixture of acetone: ethanol 7:3 (A1, A2, A3); concen-
tration of the analyzed solutions: 0.078125–10 mg/mL.
Firstly, 0.05 mL of 15-lipoxygenase solution in borate buffer pH 9 was treated with
0.05 mL of the diluted test solution in DMSO, and the mixture was left to stand for 10 min
at room temperature. Afterward, 2 mL of 0.16 mM linoleic acid solution in 0.1 M borate
buffer pH 9 was added. The absorbance of the solution was recorded at 234 nm within the
0–120 s interval. In parallel, a positive control was processed in which the test solution was
replaced with DMSO.
Gallic acid was used as a reference substance, and solutions of gallic acid in DMSO
were processed under the same conditions as the ethanolic extract. All determinations were
carried out in triplicate, and the results were expressed as the mean of three determina-
tions ± standard deviation.
The lipoxygenase inhibition capacity was calculated using the formula:
% Activity = (AEFI − AECI) × 100/AEFI (2)
where:
AEFI—represents the difference between the absorbance of the enzyme solution
without an inhibitor at 90 s and the absorbance of the same solution at 30 s;
AECI—represents the difference between the absorbance of the enzyme solution
treated with an inhibitor (test sample or gallic acid) at 90 s and the absorbance of the same
solution at 30 s.
For samples that exhibited a lipoxygenase inhibition capacity of more than 50%, the
CE50 value was calculated and expressed in µg of sample/mL of the final solution or µg of
gallic acid/mL. CE50 was calculated by taking into account the first value below 50% and
the first value above 50%, obtaining, through linear interpolation, the concentration of the
antioxidant solution corresponding to 50% activity.
2.7. In Vitro Cytotoxicity Tests and Antitumor Action of Perilla Leaves Extracts
For the study of material cytotoxicity, two human tumor cell lines were used: hu-
man osteosarcoma cells (MG-63 cell line from ATCC, Rockville, MD, USA) and tumor
keratinocytes (A431 cell line from Cell Service, Eppelheim, Germany). The cells were
separately incubated for 24 h (5% CO2 , 37 ◦ C, 95% relative humidity) in DMEM culture
medium enriched with 10% FBS and 1% P/S/N, in 96-well plates for the MTT assay
(2 × 103 cells/well for MG-63 and 3 × 103 cells/well for A431) and in 48-well plates for cell
morphology studies (8 × 103 cells/well for MG-63 and 1 × 104 cells/well for A431). After
24 h, the medium in the plates was replaced with fresh DMEM medium (with 10% FBS and
1% P/S/N) for the control, and with extracts prepared according to the protocol described
in the following paragraph.
For the in vitro cytotoxicity evaluation, an indirect contact method was used. A certain
amount (2 mg/mL stock solution) of each material was immersed in DMEM and 1% P/S/N
and left to shake (200 rpm, 37 ◦ C) for 24 h. Afterward, the stock solution was obtained by
passing the medium through 0.22 µm filters, and finally, 10% FBS was added. For each
material, the MTT test was performed for 6 different extract concentrations: 2 mg/mL,
1 mg/mL, 0.5 mg/mL, 0.2 mg/mL, 0.1 mg/mL, and 0.05 mg/mL.
For the MTT assay, the culture medium and extracts from the wells were replaced with
MTT working solution (5% MTT in DMEM without FBS and P/S/N). The culture plates
were incubated at 37 ◦ C for 2 h, during which viable cells reduced the tetrazolium salt to a
colored product called formazan, solubilized with DMSO.
The absorbance of the resulting formazan solution (blue–violet color) was measured
spectrophotometrically at λ = 570 nm using a plate reader (Tecan Sun-rise Plate Reader,
Tecan Trading AG, Männedorf, Switzerland). The spectrofotometric readings from the
experimental wells were reported relative to the control wells, where no extracts were
present. The calculated ratio represented cell viability (V):
abs extract
V = × 100 (3)
abs control
where:
abs extract—the absorbance of the extract;
abs control—the absorbance of the control.
The MTT assay was conducted at 24, 48, and 72 h, in triplicate, and analyzed by means
of two-way ANOVA followed by Bonferroni’s post hoc test. A p-value of less than 0.05 was
accepted as significant. We used the Compact Letter Display (CLD) system for coding the
statistical significance in the ANOVA test as follows: (a) variables with indistinguishable
means were assigned the same letter; (b) the variable with the highest mean (or average)
will be named “a”.
The Calcein-AM Cell Viability Assay was conducted at 72 h of contact. In the initial
stage, the culture medium in the wells was removed, and then the cells were washed twice
with HBSS containing calcium and magnesium, without phenol red.
Finally, a calcein solution was added (2 µL calcein per 1 mL HBSS with calcium and
magnesium, without phenol red), and the culture plate was incubated at 37 ◦ C, 5.5%
CO2 , and 96% relative humidity for 40 min. To study the cell morphology, an inverted
fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) was used, and
images were captured.
2.8. Statistical Analyses

Continuous data were compared among multiple groups using ANOVA analysis,
followed by Bonferroni’s post hoc test. Student’s t-test was used to compare the numerical
variables of two groups. A p-value less than 0.05 was considered statistically significant.
These analyses were performed using STATA SE (version 17, 2023, StataCorp LLC, College
Station, TX, USA).
3. Results
3.1. Quantification of the Total Phenols, Flavonoids and Anthocyanins from Ethanolic and Acetone:
Ethanol Extracts of Perilla frutescens
Following the drying process, six extracts were obtained, consisting of two with a
brown-violet color and four with a brown–green hue. These six dried extracts were coded
to facilitate their handling in the research, and the extraction yield was calculated. The
results are presented in Table 1. The 70% ethanol extracts exhibited the highest extraction
yields compared to those obtained using the solvent mixture.
The quantification of total phenols, flavonoids and anthocyanins in three types of
ethanolic extracts (E1, E2, E3) and in three types of a mixture solvents (acetone: ethanol)
extracts (A1, A2, A3) is presented in Table 2.
Our results indicated that the total phenols and anthocyanins concentrations were
significantly higher in the ethanolic extracts of Perilla frutescens. On the other hand, we
could not find any statistical difference regarding the flavonoids concentrations between
the ethanolic and acetone–ethanol extracts of Perilla frutescens varieties.
The results from the UHPLC analysis are presented in Table 3. Caffeic acid was
significantly higher in the ethanolic extracts of Perilla frutescens, with the highest amount
being identified in the E1 extract. Syringic acid, p-coumaric acid, ferulic acid, rosmarinic
acid, kaempferol, isorhamnetin, and pinocembrin were also found to be significantly higher
in ethanolic extracts. E1 extract had the highest amount of p-coumaric acid, isorhamnetin,
and pinocembrin, while E2 extract had the highest amount of syringic acid. E3 extract was
rich in ferulic acid and kaempferol.
Table 1. The coding of the extracts and their extraction yield.
Extraction Yield (%/Mean

Solvent Perilla frutescens Species Variety Extract Code
and Standard Deviation)
70% Ethanol Perilla frutescens var. crispa f. purpurea E1 58.22% (* 2.04: 1.18 ± 0.03) ab
70% Ethanol Perilla frutescens var. frutescens f. viridis E2 58.79% (* 2.03: 1.19 ± 0.01) a
Perilla frutescens var. frutescens f.
70% Ethanol E3 48.32% (* 2.02: 0.97 ± 0.01) c
crispidiscolor
Acetone: Ethanol (7:3) + Citric Acid Perilla frutescens var. crispa f. purpurea A1 38.51% (* 2.01: 0.77 ± 0.006) e
Acetone: Ethanol (7:3) + Citric Acid Perilla frutescens var. frutescens f. viridis A2 34.84% (* 2.04: 0.71 ± 0.02) f
Perilla frutescens var. frutescens f.
Acetone: Ethanol (7:3) + Citric Acid A3 44.47% (* 2.02: 0.90 ± 0.01) d
crispidiscolor
Table legend: * Dry mass. Means not sharing any letter are significantly different.
Table 2. Total phenols, flavonoids and anthocyanins quantification from ethanolic and ethanol–
acetone extracts.
Total Phenols Total Flavonoids (mg Anthocyanins (mg

Sample
(mg Gallic Acid/mL Extract) Rutoside/mL Extract) Cyanidol/mL Extract)
E1 2.15 ± 0.12 b 0.17 ± 0.02 a 0.10 ± 0.001 a
E2 2.36 ± 0.11 a 0.09 ± 0.002 c 0.007 ± 0.007 c
E3 2.13 ± 0.03 c 0.11 ± 0.01 bc 0.08 ± 0.001 b
A1 0.33 ± 0.02 d 0.11 ± 0.009 bcd 0.09 ± 0.001 d
A2 0.14 ± 0.03 f 0.08 ± 0.002 cf -
A3 0.28 ± 0.02 de 0.10 ± 0.001 bcdef 0.06 ± 0.001 e
Legend: E—ethanolic extract; A—acetone: ethanol extracts. Means not sharing any letter are significantly
different.
Table 3. Quantification of various compounds from Perilla frutescens extracts using UHPLC.
Identified Compound (µg/mL Extract)

Compound
E1 E2 E3 A1 A2 A3
Gallic Acid 0.249 a ab 0.095 ac - ad 0.103 ae
0.235 0.225
Catechin 1.526 c 2.025 ac 1.672 bc 1.388 ec 1.355 fc 1.581 dc
Chlorogenic Acid 1.15 bc 0.388 cd 4.448 a 0.376 cedf 0.633 ced 0.331 cef
Caffeic Acid 109.714 a 55.12 c 81.306 b 8.582 e 12.022 d 7.108 f
Epi-Catechin 1.014 abc 0.870 abc 0.912 abc 0.769 abce 0.653 bcef 1.030 abcd
Syringic acid 129.323 c 244.033 a 232.475 b 20.57 e 35.109 d 19.603 f
P-Coumaric Acid 11.163 a 10.069 b 7.097 c 2.222 f 2.25 fd 3.788 d
Ferulic Acid 2.423 abc 2.429 abc 2.551 abc - 0.538 e 0.722 d
Ellagic Acid 9.930 a 5.865b 4.807 c 3.421 e 3.677 d 2.731 f
Cinnamic Acid 70.465 a - 22.698 b 16.544 d 11.494 e 25.218 c
Rosmarinic Acid 67.665 a 10.351 c 14.871 b 1.098 f 3.177 e 9.004 d
Quercitin 19.663 a 3.912 c 16.828 b - - 12.243 d
Kaemferol 36.45 bc 38.12 bc 41.09 a 22.05 de 26.15 de 19.47 ef
Isorhamnetin 1.194 a 0.805 bc 0.745 bc 0.138 ef 0.101 def 0.263 de
Apigenin 3.400 c 8.361 b 10.656 a 2.677 e 4.061 d 1.313 f
Pinostrobin 31.827 a 28.35 b 24.081 cd 23.601 e - 24.320 cd
Pinocembrin 2.323 ab 0.972 c 2.302 ab 0.387 fd 0.447 def 0.424 def
Crysin 1.11 ce 4.214 a 3.239 b 1.449 de 1.354 cde 0.56 f
Table legend: Letters indicate statistical significance according to the Compact Letter Display system. Means not
sharing any letter are significantly different.
3.2. Determination of the In Vitro Antioxidant Action of Perilla frutescens Extracts

The results from the iron chelation capacity assessment are presented in Figure 1 and
Table 4. The iron chelation capacity increased with Perilla frutescens extracts concentrations,
and it was significantly higher in ethanolic extracts compared with other types of extracts
from this plant.
The results obtained for the evaluation of the active principles’ capacity, present in the
plant extracts, to neutralize the hydroxyl radical are presented in Table 4 and graphically
represented in Figure 2. The scavenging capacity of the hydroxyl radical increased with
the concentration of the analyzed Perilla frutescens extracts, and it was significantly higher
for ethanolic extracts in comparison with other types of extracts from this plant or with
gallic acid.
Table 4. Determination of the in vitro antioxidant action of Perilla frutescens extracts.
Iron Chelation Capacity Assessment—IC50 (µg/mL

Samples
Final Solution)
E1 166.36 ± 0.18 c
E2 386.69 ± 9.36 a
E3 297.01 ± 0.31 b
A1 811.88 ± 1.32 g
A2 1040.93 ± 0.17 e
A3 830.49 ± 0.82 f
Gallic acid 1163.15 ± 2.63 d
Determination of Hydroxyl Radical Scavenging
Samples
Capacity—IC50 (µg/mL final solution)
E1 122.62 ± 0.74 c
E2 154.58 ± 0.41 ab
E3 153.16 ± 1.11 ab
A1 156.96 ± 3.57 abe
A2 -
A3 -
Gallic acid 177.29 ± 0.66 d
Determination of Superoxide Anion Scavenging
Samples
Capacity—IC50 (µg/mL final solution)
E1 321.60 ± 9.12 c
E2 520.79 ± 16.90 ab
E3 470.08 ± 7.32 ab
A1 977.47 ± 39.37 e
A2 -
A3 1741.66 ± 29.87 d
Gallic acid 543.38 ± 15.43 af
Determination of the lipoxygenase inhibition
Samples
capacity—IC50 (µg/mL final solution)
E1 85.99 ± 1.85 c
E2 134.77 ± 2.49 a
E3 104.10 ± 5.05 b
A1 -
A2 -
A3 -
Gallic acid 28.85 ± 0.76 d
Table legend: IC50—Half maximal inhibitory concentration. Means that do not share any letter are significantly
different.
3.2. Determination of the In Vitro Antioxidant Action of Perilla frutescens Extracts
The results from the iron chelation capacity assessment are presented in Figure 1 and
Table 4. The iron chelation capacity increased with Perilla frutescens extracts concentra-
tions, and it was significantly higher in ethanolic extracts compared with other types of
extracts from this plant.
Antioxidants 2024, 13, x FOR PEER REVIEW 13

Figure 1. Graphical representation of the iron chelation capacity considering various concentrations
of Perilla
Figure frutescensrepresentation
1. Graphical extracts. of the iron chelation capacity considering various concentrations
of Perilla frutescens extracts.
Figure 2. The scavenging activity of hydroxyl radical in the analyzed Perilla frutescens extracts.
Figure 2. The scavenging activity of hydroxyl radical in the analyzed Perilla frutescens extracts
The results obtained from evaluating the capacity of the active principles present in
theThe
plantresults
extractsobtained
to neutralize the superoxide
from evaluatinganion radical areof
the capacity presented in Table
the active 4 and pres
principles
graphically represented in Figure 3. Our results indicated that the scavenging capacity of
the plant extracts to neutralize the superoxide anion radical are presented in Table
the superoxide anion radical was significantly higher for ethanolic extracts in comparison
graphically represented
with other types of extractsin Figure
from 3. Our
this plant results
or with gallicindicated
acid. that the scavenging capac
the superoxide
The resultsanion
obtainedradical was significantly
from evaluating higher
the capacity of the for ethanolic
active extracts
principles present in
in compa
with other types of extracts from this plant or with gallic acid.
plant extracts to inhibit lipoxygenase are presented in Table 4 and graphically represented
in Figure 4. The lipoxygenase inhibition capacity of the analyzed Perilla frutescens extracts
was significantly smaller compared to gallic acid. This inhibition capacity was higher for
ethanolic extracts in comparison with other types of extracts from this plant.
the plant extracts to neutralize the superoxide anion radical are presented in Table
graphically represented in Figure 3. Our results indicated that the scavenging capac
the superoxide anion radical was significantly higher for ethanolic extracts in compa
Antioxidants 2024, 13, 58
with other types of extracts from this plant or with gallic acid. 13 of 23
Antioxidants 2024, 13, x FOR PEER REVIEW 14 of 25

Figure 3. The graphical representation of the scavenging capacity of superoxide anion radicals in the
Figure 3. The graphical representation of the scavenging capacity of superoxide anion radic
analyzed Perilla frutescens extracts.
the analyzed Perilla frutescens extracts.
The results obtained from evaluating the capacity of the active principles prese
plant extracts to inhibit lipoxygenase are presented in Table 4 and graphically represe
in Figure 4. The lipoxygenase inhibition capacity of the analyzed Perilla frutescens ex
was significantly smaller compared to gallic acid. This inhibition capacity was high
ethanolic extracts in comparison with other types of extracts from this plant.
Figure 4. The graphical representation of the capacity of Perilla leaves extracts to inhibit lipoxygenase.
Figure 4. The graphical representation of the capacity of Perilla leaves extracts to inhibit lipoxygen-
3.3. Determination of In Vitro Cytotoxicity and Antitumor Effects of Perilla frutescens Extracts
ase.
The results from the MTT test using various types of Perilla frutescens extracts and
3.3. Determination
perillyl alcohol onof Incellular
the Vitro Cytotoxicity
lines MG-63and
are Antitumor
presented inEffects of 5Perilla
Figures and 6.frutescens Extracts
Theresults
The antitumoral
fromeffect
the was
MTTobserved for all
test using types oftypes
various extracts
of at ≥0.5 mg/mL.
Perilla The
frutescens cyto- and
extracts
compatibility with the cellular lines MG-63 was observed at concentrations of 0.2 mg/mL
perillyl alcohol on the cellular lines MG-63 are presented in Figures 5 and 6.
for all types of extracts. In particular, in case of A1–A3 extracts (Figure 5), for concentrations
of 0.5 mg/mL, 1 mg/mL and 2 mg/mL, the cell viability values were between 37% (48 h,
A3, 1 mg/mL) and 55% (24 h, A1, 1 mg/mL), indicating a cytotoxic behavior, while for
concentrations of 0.05 mg/mL, 0.1 mg/mL, and 0.2 mg/mL, the minimum values were 89%
(72 h, A1, 0.05 mg/mL) and the maximum values 114% (24 h, A2, 0.05 mg/mL), indicating
no toxicity.
As can be noted in Figure 6, E1–E3 have cytotoxic effects at concentrations of

0.5 mg/mL, 1 mg/mL and 2 mg/mL, the results indicating a decrease in the viability
of MG-63 cells, with values between 38% (72 h, E2) and 54% (24 h, E3). No significant
differences were observed between these three concentrations, meaning that at concentra-
tions higher than 0.5 mg/mL, extracts E1–E3 can be considered for the anti-tumor effect.
In contrast, for the concentrations of 0.05 mg/mL, 0.1 mg/L and 0.2 mg/mL, the extracts
did not show a cytotoxic effect, the cell viability values being between 87% (24 h, E2,
0.05 mg/mL) and 107% (48 h, E1, 0.2 mg/mL).
Perillyl alcohol showed an obvious cytotoxicity for 2 mg/mL and 1 mg/mL concen-
trations, while for 0.5 mg/mL and 0.2 mg/mL, close values were observed for the first two
contact times (24 and 48 h), but for 72 h, in the case of the concentration of 0.5 mg/mL, a
sudden decrease in the cell viability value was observed, reaching from 82% at 48 h to 43%
at 72 h. For the concentrations of 0.1 mg/mL and 0.05 mg/mL, it was observed that perillyl
alcohol4.had
Figure The no cytotoxic
graphical effect in contact
representation of the with MG-63
capacity cells.leaves extracts to inhibit lipoxygen-
of Perilla
ase. In Table 5, we present the results from ANOVA analysis with Bonferroni’s post hoc test
regarding the intergroup differences of cellular viabilities in MG-63 cell line. Our analysis
indicated
3.3. a statistically
Determination of In significant difference
Vitro Cytotoxicity and between
Antitumor groups
Effectsregarding
of Perilla the cellularExtracts
frutescens viability
of MG-63 cell lines when using E3 (p = 0.02), A1 (p = 0.02), and peryllyl alcohol (p < 0.001),
The results from the MTT test using various types of Perilla frutescens extracts and
the latter having the most pronounced cytotoxic effect on this type of cell line in the first 24
perillyl alcohol on the cellular lines MG-63 are presented in Figures 5 and 6.
and 48 h.
Figure 5. Mg-63 viability (%) in contact with E1–E3 Perilla frutescens extracts. Each value represents
the mean ±
the mean ± standard
standard error
error mean
mean (n
(n == 3).
3). ** pp <<0.01,
0.01,**
** pp <<0.001,
0.001,***
***pp <<0.0001
0.0001versus
versuscontrol
control (analyzed
(analyzed
by means of two-way ANOVA).
by means of two-way ANOVA).
Figure 6.
Figure 6. MG-63
MG-63 viability
viability (%)
(%) in
in contact
contact with
with A1–A3
A1–A3 Perilla
Perilla frutescens
frutescens extracts
extracts and
and perillyl
perillyl alcohol.
alcohol.
Each value represents the mean ± standard error mean (n = 3). * p < 0.01, ** p < 0.001, *** p < 0.0001
Each value represents the mean ± standard error mean (n = 3). * p < 0.01, ** p < 0.001, *** p < 0.0001
versus control (analyzed by means of two-way ANOVA).
The antitumoral effect was observed for all types of extracts at ≥0.5 mg/mL. The cy-
Table 5. Intergroup comparison of MG-63 celullar viability considering the cytotoxic concentrations
tocompatibility with the cellular lines MG-63 was observed at concentrations of 0.2 mg/mL
of Perilla frutescens extracts and Peryllyl alcohol.
for all types of extracts. In particular, in case of A1–A3 extracts (Figure 5), for concentra-
Extracts tions ofTime
0.5 mg/mL,
Frame 1 mg/mL and 2 mg/mL,
2 mg/mL the cell viability0.5values
1 mg/mL mg/mL were between 37% (48
Controls
h, A3, 1 mg/mL) and 55% (24 h, A1, 1 mg/mL), indicating a cytotoxic behavior, while for
E1 (mean and standard deviation) 24 h 53.50 ± 1.32 abc 48.98 ± 3.95 abc 47.03 ± 3.67 abc 100.00 ± 7.19 d
concentrations of 0.05 mg/mL, 0.1 abc mg/mL, and 0.2 mg/mL, abc the minimum values were 89%
E1 (mean and standard deviation) 48 h 47.33 ± 11.18 45.54 ± 3.82 41.33 ± 2.68 abc 100 ± 9.16 d
(72
E1 (mean and standard deviation) h, A1, 0.05
72 h mg/mL) and the
43.36 ± 1.94 maximum
abc values
40.99 ± 2.53 114%
abc (24 h, A2,
42.20 ± 1.990.05abc mg/mL),
100 ±indicating
20.64 d
no
E2 (mean and standard deviation) toxicity.24 h 48.74 ± 1.46 abc 47.76 ± 2.60 abc 49.84 ± 5.54 abc 100.00 ± 7.19 d
E2 (mean and standard deviation) As can be
48 h noted in Figure
39.70 ± 3.20 6,
abc E1–E3 have
43.05 ± 6.69cytotoxic
abc effects
42.50 ± 5.33at concentrations
abc 100 ± 9.16 ofd 0.5
mg/mL, 1 72
E2 (mean and standard deviation) mg/mL
h and40.24
2 mg/mL, abc
± 4.64 the results
45.68 ± abc
indicating
4.15 a38.12
decrease abc
± 2.56in the viability d
of MG-
100 ± 20.64
a a a
63 cells, with
E3 (mean and standard deviation) 24 hvalues between51.91 ± 38%
2.11 (72 h, 48.28
E2) and± 3.12
54% (24 h, E3).±No
53.69 4.21significant
100.00 ± 7.19 d
differences
E3 (mean and standard deviation) 48 h between44.21 ± threebc
5.9 concentrations, bc
41.26 ± 1.63 meaning 44.21 ± at bc 100 ± 9.16
6 concentrations d
were observed these that higher
E3 (mean and standard deviation) 72 h 43.64E1–E3 bc
± 4.69can be 39.18 bc
± 3.43 for the 45.99 ± 2.55 bc 100In± 20.64 d
than 0.5 mg/mL, extracts considered anti-tumor effect. contrast,
A1 (mean and standard deviation) 24 h 51.21 ± 8.12 a 55.34 ± 6.88 a 49.84 ± 8.37 a 100.00 ± 7.19 d
for the concentrations of 0.05 mg/mL, 0.1 mg/L andbc0.2 mg/mL, the extracts did not show
A1 (mean and standard deviation) 48 h 43.20 ± 3.86 bc 42.35 ± 4.41 40.09 ± 2.30 bc 100 ± 9.16 d
a cytotoxic effect, the cell viabilitybcvalues being between 87% (24 h, E2, 0.05 mg/mL) and
A1 (mean and standard deviation) 72 h 39.40 ± 2.02 39.71 ± 2.10 bc 43.72 ± 3.86 bc 100 ± 20.64 d
107%
A2 (mean and standard deviation)
(48 h, E1,
24 h
0.2 mg/mL).
45.994 ± 2.95 abc 47.46 ± 4.57 abc 47.55 ± 3.21 abc 100.00 ± 7.19 d
A2 (mean and standard deviation) Perillyl alcohol
48 h showed an
38.22 ± 1.76 obvious
abc cytotoxicity
44.60 ± 5.56 abc for 2 mg/mL
38.82 ± 4.53 and
abc 1 mg/mL concen-
100 ± 9.16 d
trations,
A2 (mean and standard deviation) while
72 h for 0.5 mg/mL and
42.20 ± 3.18 0.2
abc mg/mL, close
41.30 ± 5.59 values
abc were observed
43.64 ± 6.53 abc for
100 ± 20.64 two
the first d
contact times
A3 (mean and standard deviation) 24 h(24 and 45.1748 h),±but
6.06for 72 h,
abc in the
43.15 case of the
± 3.32 abc concentration
47.27 ± 6.18 abc of100.00
0.5 mg/ mL,d a
± 7.19
sudden decrease
A3 (mean and standard deviation) 48 h in the37.99
cell ± 4.90 abc
viability value
36.97was 3.03 abc
± observed, reaching
39.47 ± 2.56 abc
from 82% 100 at 48
± h9.16 d
to 43%
A3 (mean and standard deviation) 43.57 ± 2.45 abc abc
± 2.40.05 mg/mL, 44.402it±was abc ± 20.64 d
at 72 h. For72theh concentrations of 0.1 mg/mL 40.69and 1.66observed 100that perillyl
a a a
Perillyl alcohol (mean) alcohol had 24noh cytotoxic 57.53 ± 5.82 54.33 ± 9.36
effect in contact with MG-63 cells. 95.37 ± 7.03 100.00 ± 7.19 d
Perillyl alcohol (mean) 48 h 46.16 ± 2.23 bc 43.98 ± 4.62 bc 81.69 ± 10.88 b 100 ± 9.16 dhoc
In Table 5, we present the results from ANOVA analysis with Bonferroni s post
Perillyl alcohol (mean) 72 h 48.63 ± 2.43 bc 45.68 ± 4.37 bc 42.66 ± 2.15 c 100 ± 20.64 d
test regarding the intergroup differences of cellular viabilities in MG-63 cell line. Our anal-
Table
ysis legend: Letters
indicated indicate statistical
a statistically significance
significant according tobetween
difference the Compact Letter regarding
groups Display system.theMeans not
cellular
viability of MG-63 cell lines when using E3 (p = 0.02), A1 (p = 0.02), and peryllyl alcohol (p
Antioxidants 2024, 13, Antioxidants
58 2024, 13, x FOR PEER REVIEW 16 of 23 17 of
Since a cell line Since

is characterized
a cell line isby specific mechanisms
characterized by specific and behavior,and
mechanisms thebehavior,
six extracts
the six extra
Since a cell
and perillyl line is
alcohol characterized
testedbyinspecific mechanisms and behavior, the six extracts
and were alsoalcohol
perillyl werecontact with the
also tested in A431
contactcell linethe
with (tumor
A431keratinocytes);
cell line (tumor keratin
and perillyl alcohol were also tested in contact with the A431 cell line (tumor keratino-
the results are shown
cytes);intheFigures
results 7–10.
are shown in Figures 7–10.
cytes); the results are shown in Figures 7–10.
Figure(%)
Figure 7. A431 viability 7. A431 viability
in contact with(%) in contact
E1–E3 Perillawith E1–E3extracts.
frutescens Perilla frutescens
Each valueextracts. Each value
represents the represe
Figure 7. A431 viability
the (%)±instandard
mean contact error
with E1–E3
mean**(nPerilla
= 3). *frutescens
p <***
0.01, extracts.
** Each
p < 0.001, *** value represents
pcontrol
< 0.0001 versus control (analyz
mean ± standard error
the mean ± standard mean (n = 3). * p p p
p < 0.01, ** p < 0.001, *** p < 0.0001 versus control (analyzed by
< 0.01, < 0.001, < 0.0001 versus (analyzed
by error
means mean (n = 3). *ANOVA).
of two-way
means of two-way
by means of two-wayANOVA).
ANOVA).
Figure 8. A431 viability (%) in contact with A1–A3 Perilla frutescens extracts and perillyl alcohol. Each
value represents the mean ± standard error mean (n = 3). * p < 0.01, ** p < 0.001, *** p < 0.0001 versus
control (analyzed by means of two-way ANOVA).
Figure 8. A431 viability (%) in contact with A1–A3 Perilla frutescens extracts and perillyl alcohol.
Antioxidants 2024, 13, 58 Each value represents the mean ± standard error mean (n = 3). * p < 0.01, ** p < 0.001, *** p 17
< 0.0001
of 23
Figure 9. The structure and morphology of MG-63 cells cultured with extract A1–A3, E1–E3, perillyl
Figure 9. The structure and morphology of MG-63 cells cultured with extract A1–A3, E1–E3, perillyl
alcohol, and the control (marked or not with Calcein-AM).
The antitumoral effect was observed for all types of extracts at ≥0.2 mg/mL in com-
parison with perillyl alcohol, which exhibited an antitumoral effect at concentrations of
≥0.5 mg/mL. At 72 h of contact, cell viability values were below 9% for all analyzed
samples, which indicates the possible use of Perilla frutescens leaf extracts with a concentra-
tion higher than 0.5 mg/mL in the treatment of skin tumors. Supplementary tests were
necessary to accurately establish the dosage and mechanism of action of the extracts.
In Table 6, we present the results from ANOVA analysis with Bonferroni’s post hoc
test regarding the intergroup differences of cellular viabilities in A431 cell line. Our analysis
revealed that the cytotoxic activity was similar between various concentrations of E1
(p = 0.43), E2 (p = 0.36), E3 (p = 0.32) and A1 (p = 0.16) extracts at different timeframes. On
the other hand, our results indicated a statistically higher cytotoxic activity of A2 (p = 0.006)
and A3 (p < 0.001) extracts in the first 24 h of contact with A431 cell line. Moreover,
the cytotoxicity of perillyl alcohol was significantly higher in the 48–72 h time interval
(p < 0.001).
Figure 10. The structure and morphology of A431 cells cultured with extract E1–E3, A1–A3, perillyl
Figure 10. The structure and morphology of A431 cells cultured with extract E1–E3, A1–A3, perillyl
AsThe antitumoral
mentioned effect
above, wasextracts,
all six observed in for all types
contact of extracts
with the at ≥0.2mg/mL
MG-63 line, in com-
are not cytotoxic
at concentrations lower than or equal to 0.2 mg/mL, but in contact with A431, it wasof
parison with perillyl alcohol, which exhibited an antitumoral effect at concentrations
≥0.5 mg/mL.
observed that At
at a72concentration
h of contact, cell viability
of 0.2 mg/mL, values were below
the extracts 9% for all
contributed toanalyzed sam-
the decrease
ples, which indicates the possible use of Perilla frutescens leaf extracts with
in cell viability up to values of 46% in the case of extracts E1–E3, respectively, and 47% a concentration
inhigher
the casethan 0.5 mg/mL
of extracts in theAt
A1–A3. treatment
the otheroftwoskin tumors. concentrations,
analyzed Supplementary 0.1 tests were neces-
mg/mL and
sarymg/mL,
0.05 to accurately
extractsestablish
A1, A2 the
anddosage
A3 hadand mechanism of
a non-cytotoxic action of the extracts.
character.
InInthe
Table 6, we
figures present
below, thebe
it can results fromthat
observed ANOVA
all theanalysis with Bonferroni
data obtained for the Calceins postAM
hoc
test regarding the intergroup differences of cellular viabilities in A431 cell
assay are correlated with the data obtained in the MTT test. Thus, for the MG-63 cell line, in line. Our anal-
ysis
the revealed
case that E1–E3
of extracts the cytotoxic
and A1–A3,activity wasviability
where similar between various
values ranged concentrations
from 101% to 106%, of aE1
(p = 0.43), E2 (p = 0.36), E3 (p = 0.32) and A1 (p = 0.16) extracts at different
cell density similar to that in the control wells is observed. However, in the case of perillyl timeframes. On
the other
alcohol, hand,
where theour resultswas
viability indicated a statistically
82%, there are areashigher
that are cytotoxic activitywith
not populated of A2cells
(p =
0.006) 9).
(Figure and A3 (p < 0.001) extracts in the first 24 h of contact with A431 cell line. Moreover,
the In
cytotoxicity
the case ofofthe perillyl
A431 alcohol
cell line,was significantly
for extracts E3 andhigher
A1–A2in the 48–72
(Figure 9),hwhich
time interval
showed(p
< 0.001).values of approximately 98%, a comparable cell density can be observed compared
viability
to the control wells. For extract E1, which resulted in a 72 h cell viability, 82% (Figure 10)
cell density can be observed compared to the control.
Table 6. Intergroup comparison of A431 cellular viability considering the cytotoxic concentrations of
Perilla frutescens extracts and perillyl alcohol.
Extracts Time Frame 2 mg/mL 1 mg/mL 0.5 mg/mL Controls

E1 (mean and standard deviation) 24 h 13.84 ± 0.40 abc 13.76 ± 0.91 abc 14.40 ± 0.56 abc 100 ± 6.08 d
A1 (mean and standard deviation) 24 h 14.63 ± 1.38 abc 14.03 ± 0.43 abc 13.60 ± 0.90 abc 100 ± 6.08 d
A2 (mean and standard deviation) 24 h 12.95 ± 1.07 a 12.83 ± 0.77 a 14.40 ± 0.72 a 100 ± 6.08 d
A2 (mean and standard deviation) 48 h 8.71 ± 0.47 b 11.10 ± 1.98 b 9.78 ± 0.64 b 100 ± 9.58 d
A2 (mean and standard deviation) 72 h 4.81 ± 0.44 c 4.911 ± 0.75 c 4.73 ± 0.52 c 100 ± 8.91 d
A3 (mean and standard deviation) 24 h 15.37 ± 0.56 a 16.17 ± 0.43 a 28.90 ± 2.15 a 100 ± 6.08 d
A3 (mean and standard deviation) 48 h 9.46 ± 0.80 b 10.05 ± 1.65 b 9.52 ± 1.05 b 100 ± 9.58 d
A3 (mean and standard deviation) 72 h 4.82 ± 0.46 c 4.68 ± 0.29 c 5.51 ± 0.41 c 100 ± 8.91 d
Perillyl alcohol (mean and standard
24 h 15 ± 0.97 ab 13.88 ± 0.82 ab 13.43 ± 0.86 ab 100 ± 6.08 d
deviation)
48 h 11.50 ± 1.23 ab 10.20 ± 1.64 ab 17.78 ± 2.49 ab 100 ± 9.58 d
deviation)
72 h 5.39 ± 0.35 c 5.42 ± 0.25 c 8.76 ± 2.05 c 100 ± 8.91 d
deviation)
Table legend: Letters indicate statistical significance according to the Compact Letter Display system. Means not
4. Discussion
The present study aimed to quantify the total flavonoid, phenol, and anthocyanin con-
tent, to determine the in vitro antioxidant action, and to characterize the in vitro cytotoxic
and antitumoral activity of Perilla frutescens leaves extracts.
Our results indicated that the extraction yield was higher for ethanolic extracts com-
pared with solvent mixture extracts, both categories had an extraction yield of more than
30%, which indicates a performance comparable to others published in the literature [25,26].
The total phenols and anthocyanins concentrations were significantly higher in the
ethanolic extracts of Perilla leaves, but the flavonoids concentration did not significantly
differ between the two types of extracts. Zhao et al. investigated the total polyphenols
concentration from 44 species of Perilla frutescens using ultrasonic-assisted ethanol extrac-
tion (60%) and ultrasound-assisted cellulase hydrolysis, and demonstrated that the total
phenols concentration was significantly higher when using cellulase hydrolysis extracts
compared to ethanolic extracts [27].
The amount of caffeic acid in the ethanolic extracts of Perilla frutescens was substantially
higher, with the greatest concentration found in the E1 extract. Indeed, it was found that the
total concentration of caffeic acid varies among various strains of Perilla frutescens, although
less than rosmarinic acid, and this might be due to different genetic backgrounds of the
plant [28].
Additionally, it was discovered that ethanolic extracts had considerably greater levels
of syringic acid, p-coumaric acid, ferulic acid, kaempferol, isorhamnetin, and pinocembrin.
P-coumaric acid, isorhamnetin, and pinocembrin were most abundant in E1 extract, but
syringic acid was most abundant in E2. Kaempferol and ferulic acid were abundant in the
E3 extract. Other studies have confirmed the presence of these compounds in different
varieties of Perilla frutescens [29–31].
Polyphenolic compounds exhibit antioxidative properties through various mecha-

nisms. The hydroxyl groups serve as effective hydrogen donors and can engage with reac-
tive oxygen and nitrogen species, as discussed by Valentao et al. [32] and Heim et al. [33].
This interaction leads to a termination reaction that effectively halts the generation of free
radicals. Consequently, the initial reactive species transform into a radical form of the
antioxidant, characterized by significantly enhanced chemical stability compared to the
initial radical state.
The antioxidative capacity of phenolic compounds is also associated with their capabil-
ity to chelate metal ions involved in free radical production [34]. However, it is important
to note that phenols can, under certain circumstances, act as pro-oxidants. This can occur
through the chelation of metals that either maintain or enhance their catalytic activity or by
reducing metals, subsequently increasing their propensity to generate free radicals [35].
Moreover, the structural attributes of phenolic compounds make them prone to in-
teracting with proteins, owing to their hydrophobic benzenoid rings and the hydrogen-
bonding potential of phenolic hydroxyl groups. Consequently, phenolics possess the
capacity to act as antioxidants by inhibiting specific enzymes involved in radical gener-
ation, such as various cytochrome P450 isoforms, lipoxygenases, cyclooxygenase, and
xanthine oxidase [36].
In this study, we demonstrated that the iron chelation capacity, the scavenging capacity
of hydroxyl radical, the scavenging capacity of the superoxide anion radical, and the
lipoxygenase inhibition capacity increased with Perilla frutescens extracts concentrations,
and they were significantly higher in ethanolic extracts.
These results could be explained by the fact that the retrieval of antioxidant compounds
from botanical sources typically involves employing various extraction methodologies
tailored to their inherent chemistry and non-uniform distribution within the plant ma-
trix. Among these methods, solvent extraction stands out as the most commonly utilized
technique for the isolation of antioxidant compounds from plants [37].
However, it is crucial to note that the yields of extracted substances, the polyphenolic
contents, and the resultant antioxidant properties of plant materials are markedly influ-
enced by the choice of extracting solvent and the extraction method. This variation arises
due to the presence of diverse antioxidant compounds with distinct chemical properties
and polarities, which may or may not be soluble in a given solvent [37].
In practice, polar solvents are frequently selected for the recovery of polyphenols from
plant matrices. Of these, the most suitable options include aqueous mixtures, either hot or
cold, incorporating ethanol, methanol, acetone, and ethyl acetate [37].
As far as we know, the cytotoxic effects of Perilla frutescens extracts on tumoral cell
cultures have barely been studied. For example, alcoholic extracts (ethanol) from Perilla
frutescens leaves were shown to have antitumoral effects in vitro, inhibiting the adhesion,
proliferation and colony formation of human colon and lung tumor cells [38]. Moreover,
it was proved that isoegomaketone extracted from Perilla frutescens can induce in vitro
apoptosis in human melanoma cells [38] and in human breast tumor cells [39]. However,
to our knowledge, this was the first time Perilla frutescens extracts were tested on human
tumoral osteoblasts (MG-63 cell line) and human tumoral keratinocytes (A431 cell line).
The results indicated that in MG-63 cell line, the antitumoral effects were evident for
all extracts at concentrations ≥ 0.5 mg/mL. In contrast, cytocompatibility with the MG-63
cell line was observed at concentrations of 0.2 mg/mL for all extracts. Notably, perillyl
alcohol exhibited antitumoral effects at concentrations ≥ 0.5 mg/mL. In the case of the
MG-63 cell line, extracts E1–E3 and A1–A3, with viability values ranging from 101% to
106%, demonstrated cell densities comparable to those in the control group. However,
perillyl alcohol, with a viability of 82%, displayed areas devoid of cells.
Regarding the A431 cell line, cytocompatibility was observed at concentrations of
0.05 mg/mL for E1 and E3 extracts, 0.1 mg/mL for E2, A1, A2, and A3 extracts, and
0.2 mg/mL for perillyl alcohol. Moreover, extracts E3 and A1–A2, with viability values of
approximately 98%, exhibited cell densities similar to the control group. Conversely, extract
E1, resulting in a 72 h cell viability of 82%, displayed a reduced cell density compared to
the control.
These results provide additional data for researchers who are studying the effects
of herbal extracts on human tumoral osteoblasts and human tumoral keratinocytes. The
cytotoxic concentrations of Perilla frutescens extracts need to be determined in various cell
cultures, and our results showed that perillyl alcohol and various types of Perilla frutescens
extract could serve as potential cytotoxic agents in the studied cell lines.
Data from other studies confirmed the cytotoxic effects of perillyl alcohol on H520
(non-small cell lung cancer) [40], A549 (human lung cancer), HepG2 (human liver cancer)
cell lines [41], and BroTo (human tongue squamous cell carcinoma) [42] cell lines. On
the other hand, data on the cytotoxic effects of extracts from Perilla frutescens varieties a
are missing.
Further studies, on various type of oncogenic cell lines, and using different extracts of
Perilla frutescens could confirm our results. Overall, all ethanolic extracts exhibited higher
concentrations of phenols and antioxidant activity compared to mixed solvents extracts,
thus supporting their use in pharmacological practice.
5. Conclusions
The total concentrations of polyphenols and anthocyanins were significantly higher
in the ethanolic extracts of Perilla frutescens, while the concentration of flavonoids was not
significantly different between the two types of extracts.
In this study, we demonstrated that with increasing concentrations of Perilla frutescens
extracts, iron chelating capacity, hydroxyl radical scavenging capacity, superoxide anion
radical scavenging capacity, and lipoxygenase inhibition capacity increased and were
significantly higher for the ethanolic extracts.
The demonstrated cytotoxic effects of perillyl alcohol on MG-63 cell line and of E1
extract (Perilla frutescens var. crispa f. purpurea) on A431 cell line could serve as a basis for
further cytotoxicity studies.
Author Contributions: This paper was written as part of a doctoral program of G.A. at UMF “Grigore
T. Popa”. Conceptualization, G.A., F.D.C., L.V., O.C., A.-M.A., C.M. and M.H.; methodology, M.H.;
validation, I.-A.V., A.N., V.H., B.H. and A.H.; formal analysis, I.-A.V., A.N., V.H., B.H. and A.H.;
investigation, G.A., F.D.C., L.V., O.C., A.-M.A., C.M. and M.H.; resources, G.A.; data curation, I.-A.V.,
A.N., V.H., B.H. and A.H.; writing—original draft preparation, G.A., F.D.C., L.V., O.C., A.-M.A.,
C.M. and M.H.; writing—review and editing, G.A., F.D.C., L.V., O.C., A.-M.A., C.M. and M.H.;
visualization, G.A.; supervision, M.H.; project administration, M.H. All authors have read and agreed
to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to local policies.
Acknowledgments: We would like to offer our special thanks to Lecturer Lacramioara Ivanescu from
the Vegetal Morphoanatomy Laboratory at Biology Faculty of the “Alexandru Ioan Cuza” University,
Iasi, Romania, who provided the taxonomical identification and morpho-anatomical characterization
of Perilla frutescens.
Conflicts of Interest: The authors declare no conflicts of interest.
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Antioxidants 2024, 13, 58. https://doi.org/10.3390/antiox13010058 https://www.mdpi.com/journal/antioxidants

A recent systematic review outlined 14 classes of Perilla frutescens active constituents:

2. Materials and Methods

terials. It is a common choice for extracting phytochemicals, such as phenolic compounds,

2.2. Total Flavonoids, Phenols, and Anthocyanins Quantification

2.2.2. Total Phenol Quantification

2.2.3. Total Anthocyanin Quantification

2.2.4. UHPLC-PDA Quantification and Identification of Extracted Compounds

2.2.5. Chemicals and Materials

2.3. Iron Chelation Capacity Assessment

% Activity = 100 × (Ac − Ap)/(Ac) (1)

2.4. Determination of Hydroxyl Radical Scavenging Capacity

2.5. Determining the Scavenging Capacity of the Superoxide Radical Anion

2.6. Determination of the Lipoxygenase Inhibition Capacity of Perilla frutescens Extracts

- Soybean lipoxygenase (Sigma-Aldrich, Steinheim, Germany) in 0.1 M borate buffer

% Activity = (AEFI − AECI) × 100/AEFI (2)

2.8. Statistical Analyses

Table 1. The coding of the extracts and their extraction yield.

Extraction Yield (%/Mean

Total Phenols Total Flavonoids (mg Anthocyanins (mg

Identified Compound (µg/mL Extract)

3.2. Determination of the In Vitro Antioxidant Action of Perilla frutescens Extracts

Table 4. Determination of the in vitro antioxidant action of Perilla frutescens extracts.

Iron Chelation Capacity Assessment—IC50 (µg/mL

Antioxidants 2024, 13, x FOR PEER REVIEW 13

Antioxidants 2024, 13, x FOR PEER REVIEW 14 of 25

As can be noted in Figure 6, E1–E3 have cytotoxic effects at concentrations of

Since a cell line Since

Extracts Time Frame 2 mg/mL 1 mg/mL 0.5 mg/mL Controls

Polyphenolic compounds exhibit antioxidative properties through various mecha-

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