plants

Article
Benefits of Cardamom (Elettaria cardamomum (L.) Maton)
and Turmeric (Curcuma longa L.) Extracts for Their Applications
as Natural Anti-Inflammatory Adjuvants
Gustavo R. Cárdenas Garza 1 , Joel H. Elizondo Luévano 2 , Aldo F. Bazaldúa Rodríguez 2 ,
Abelardo Chávez Montes 2 , Raymundo A. Pérez Hernández 1 , Ameyalli J. Martínez Delgado 1 ,
Sonia M. López Villarreal 1 , José Rodríguez Rodríguez 3 , Rosa M. Sánchez Casas 4 ,
Uziel Castillo Velázquez 4, * and Osvelia E. Rodríguez Luis 1, *

1 Faculty of Dentistry, Autonomous University of Nuevo León, Monterrey 64460, NL, Mexico;
[email protected] (G.R.C.G.); [email protected] (R.A.P.H.);
[email protected] (A.J.M.D.); [email protected] (S.M.L.V.)
2 Faculty of Biological Sciences, Autonomous University of Nuevo León, San Nicolás de los Garza 66455, NL,
Mexico; [email protected] (J.H.E.L.); [email protected] (A.F.B.R.);





 [email protected] (A.C.M.)
3 Tecnológico de Monterrey, Monterrey 64849, NL, Mexico; [email protected]
Citation: Cárdenas Garza, G.R.; 4 Faculty of Veterinary Medicine and Zootechny, Autonomous University of Nuevo León,
Elizondo Luévano, J.H.; Bazaldúa Monterrey 64460, NL, Mexico; [email protected]
Rodríguez, A.F.; Chávez Montes, A.; * Correspondence: [email protected] (U.C.V.); [email protected] (O.E.R.L.);
Pérez Hernández, R.A.; Martínez Tel.: +52-8113404390 (U.C.V.); +52-8183294230 (ext. 3117) (O.E.R.L.)
Delgado, A.J.; López Villarreal, S.M.;
Rodríguez Rodríguez, J.; Sánchez Abstract: The genus Zingiberaceae has been widely used for phytotherapeutic purposes in traditional
Casas, R.M.; Castillo Velázquez, U.; medicine throughout the world for its anti-inflammatory activity. Experimental studies have estab-
et al. Benefits of Cardamom (Elettaria lished that inflammation caused by chronic infections represents a risk factor for different forms
cardamomum (L.) Maton) and
of cancer. The objective of this study was focused on determining the anti-inflammatory capacity
Turmeric (Curcuma longa L.) Extracts
and cytotoxic activity of aqueous extracts of Elettaria cardamomum (cardamom) and Curcuma Longa
for Their Applications as Natural
(turmeric). The extracts were obtained by maceration and, through GC-MS/MS, a total of 11 different
Anti-Inflammatory Adjuvants. Plants
chemical components were determined in the aqueous extract of cardamom and 7 in the extract of
2021, 10, 1908. https://doi.org/
10.3390/plants10091908
turmeric. The main compounds found in cardamom and turmeric were α-terpinyl acetate (54.46%)
and β-turmerone (33.45%), respectively. RT-qPCR results showed significantly lower gene expression
Academic Editors: Seok-Geun Lee levels of innate inflammatory cytokines (IL-6 and TNF-α) compared to the control (LPS). Also, it was
and In Jin Ha observed that the extracts do not possess cytotoxic activity against different cell lines, where E. car-
damomum showed EC50 (µg/mL) of 473.84 (HeLa cells), 237.36 (J774A.1 cells), 257.51 (Vero E6 cells),
Received: 20 July 2021 and 431.16 (Balb/C peritoneal cells) and C. longa showed EC50 (µg/mL) of 351.17 (HeLa cells), 430.96
Accepted: 8 September 2021 (J774A.1 cells), 396.24 (Vero E6 cells), and 362.86 (Balb/C peritoneal cells). The results of this research
Published: 14 September 2021
suggest that natural extracts of E. cardamomum and C. longa possess anti-inflammatory effects and no
cytotoxic activity against HeLa, J774A.1, Vero E6, and Balb/C peritoneal cell lines. Finally, it was
Publisher’s Note: MDPI stays neutral
observed that the extracts also decreased nitric oxide (NO) production in peritoneal macrophages.
with regard to jurisdictional claims in
published maps and institutional affil-
Keywords: anti-inflammatory; Curcuma longa; cytokines; cytotoxic activity; Elettaria cardamomum;
iations.
extracts; inflammation; medicinal plants; natural products; phytochemicals

Copyright: © 2021 by the authors.

1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article
Nowadays, there is a tendency to incorporate phytotherapy as an efficient therapeu-
distributed under the terms and
tic option worldwide; the use of medicinal plants to treat various pathologies has been
conditions of the Creative Commons
reported since they possess active principles with different biological actions [1,2]. The
Attribution (CC BY) license (https:// World Health Organization (WHO) encourages the use of plants with a scientific basis
creativecommons.org/licenses/by/ for the therapy of systemic diseases [3]. These diseases can lead to serious complications,
4.0/).

Plants 2021, 10, 1908. https://doi.org/10.3390/plants10091908 https://www.mdpi.com/journal/plants

Plants 2021, 10, 1908 2 of 17

activating the inflammation process with infiltration of macrophages, neutrophils, lym-

phocytes, and plasma cells in the tissue, with the release of cytokines that contribute to
the repair of tissue damage [4]. The inflammation process occurs as a response to ag-
gression to repair and restore tissues [5]. During this period, cytokines, proteins that
regulate the function of immune system cells produced by lymphocytes, macrophages, or
monocytes and hematopoietic cells with pro-inflammatory or anti-inflammatory action,
are released [6]. Among the proinflammatory cytokines are tumor necrosis factor-alpha
(TNF-α), interleukin-1α (IL-1α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17
(IL-17), and, with antagonistic effects, interleukin-10 (IL-10) [7,8]. Current therapy involves
anti-inflammatory agents that, in some cases, may reveal adverse effects such as the pres-
ence of a peptic ulcer, gastrointestinal toxicity, myocardial infarction, heart failure, stroke,
or nephrotoxicity [9,10].
In traditional medicine, the antioxidant and pharmaceutical properties of some plants
belonging to the Zingiberaceae family, such as Elettaria cardamomum and Curcuma longa, have
been well studied, and have found applications in industries such as food, cosmetics, and
pharmaceuticals, among others; these capabilities are related to their diverse composition of
various phytochemical compounds, mainly phenolic compounds [11]. E. cardamomum (car-
damom), also known as “green or true cardamom”, is a perennial herbaceous plant, which
has been described as possessing compounds such as phenols, starch, tannins, terpenoids,
flavonoids, proteins, sterols [12], anthocyanins, and alkaloids, and it also possesses various
pharmacological properties, such as antioxidant, anti-inflammatory, anticancer, and antimi-
crobial activities [13]. C. longa, also called turmeric or curcuma, possesses different bioactive
components such as curcuminoids (curcumin) and essential oils (monoterpenes) [14], and
among its multiple therapeutic properties are anti-inflammatory, antihyperlipidemic, an-
timicrobial, and antiparasitic activity [15,16]. Its traditional use has been reported for the
treatment of diseases such as rheumatoid arthritis, multiple sclerosis, and psoriasis as it
manages to modulate the signaling of proinflammatory cytokines [17].
The main objective of this research focused on the evaluation of the effects of crude
aqueous extracts of E. cardamomum and C. longa on the inflammatory process through the
expression of interleukins by stimulated macrophages and their possible applications as
adjuvants in health improvement therapies.

2. Materials and Methods

2.1. Chemicals and Reagents
All chemicals and solvents were analytical grade. Antibiotic antimycotic solution
(100×) stabilized, diethyl polycarbonate (DEPC), dimethyl sulfoxide (DMSO), Dulbecco’s
Modified Eagle Medium (DMEM), fetal bovine serum (FBS), glycine buffer solution, Griess
reagent, guanidine thiocyanate, lipopolysaccharide (LPS) from Escherichia coli O26:B6
smooth strain, n-hexane, nystatin, RNAzol® RT, RPMI-1640 medium with L-glutamine,
SYBR® Green Quantitative RT-qPCR kit, and thiazolyl blue tetrazolium bromide (MTT)
were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). GoTaq® qPCR
Master Mix kit and ImProm-II™ Reverse Transcription System reverse transcription kit
was purchased from Promega (Promega® Corporation, Madison, WI, USA).

2.2. Plant Material and Extraction

E. cardamomum seeds (SKU: 209740-01) and C. longa rhizome (SKU: 205400-54) were
both purchased from Starwest Botanicals (Sacramento, CA, USA). Plant material was
purchased in powdered form. The extraction was performed with distilled water. Aqueous
extracts of both plants were obtained by the heat infusion method (100 ◦ C/100 rpm) for
30 min. For this purpose, 10 g of plant material were placed in 100 mL of distilled water
in a flat-bottomed ball flask. After the extraction time, the extracts were filtered, frozen
Plants 2021, 10, 1908 3 of 17

at −80 ◦ C, and, finally, lyophilized. The following formula was used to calculate the
extraction yield percentage [18]:
 
Final weight
% Yield = × 100 (1)
Initial weight

2.3. Qualitative Phytochemical Tests

The following phytochemical tests were performed to identify the functional groups
of each extract: Lieberman-Buchard (sterols, triterpenes), Shinoda (flavonoids), Baljet
(sesquiterpene lactones), sulfuric acid (quinones), and ferric chloride (tannins) [19], then
each extract was analyzed by gas chromatography coupled to GC/MS mass for identifica-
tion of the main components of its chemical composition [11].

2.4. GC and GC-MS/MS Analysis of the Aqueous Extracts

For chemical identification and quantification, 1 µL of diluted aqueous extract in
n-hexane (1:20, v:v) was analyzed using a gas chromatograph Varian Saturn 2100T coupled
to an MS/MS Saturn 2100 ion trap mass spectrometer (Agilent Technologies, Inc., Walnut
Creek, CA, USA). The chromatographic separation was performed with the capillary
column HP-5ms (30 m × 0.25 mm × 0.25 µm). The split injection mode was used (split
ratio 1:20), while helium-6 was used as the carrier gas with a flow rate of 1 mL min−1 . The
injector temperature was set to 250 ◦ C. The initial oven temperature was set to 70 ◦ C for
1 min, then the temperature was raised to 150 ◦ C at a rate of 5 ◦ C per min and held at
this temperature for 5 min. In addition, the column was heated up to 200 ◦ C at a rate of
10 ◦ C and, finally, kept at 200 ◦ C for 15 min. The total running time was 60 min. The mass
spectra were recorded in the SCAN mode in a range from 30 to 400 m/z using electron
ionization energy at 70 eV and the detector temperature was set to 150 ◦ C [11]. The GC-MS
structure analysis was performed using the National Institute Standard and Technology
(NIST) database; in addition, the results were compared with results from previous studies
available in the literature [20,21].

2.5. Gene Expression of Anti-Inflammatory Cytokines on Peritoneal Macrophages Stimulated with

Aqueous Extracts and Challenged with Lipopolysaccharide
To evaluate the anti-inflammatory effects of aqueous extracts of E. cardamomum and
C. longa, murine macrophages were challenged with lipopolysaccharide (LPS; Escherichia
coli serotype O26:B6 smooth strain) to induce a classical activation phenotype [22]. Relative
quantification of proinflammatory cytokine gene sequences IL-4, IL-6, IL-10, and TNF-α
induced by aqueous extracts of E. cardamomum and C. longa, as well as LPS, were measured
by real-time quantitative PCR (RT-qPCR) using SYBR® Green reagent.

2.5.1. Obtaining Peritoneal Cells

Five female Balb/C mice were inoculated intraperitoneally (IP) with 1 mL of DMEM,
then sacrificed by cervical dislocation, and a gentle massage of the abdomen was performed.
The culture medium with the cells in the peritoneal fluid was extracted and centrifuged
at 2700 rpm (4 ◦ C) for ten minutes, eliminating the supernatant and conserving the cell
pellet, which was homogenized with the aqueous extract individually (Table 1). Finally,
it was incubated for 30 min at 37 ◦ C (CO2 5%), centrifuged, and the supernatant was
eliminated [23]. Two washes were performed with phosphate buffer (PBS, ph 7.4) and then
homogenized with 500 µL of RNAzol® . As an inducer of inflammation in macrophages,
50 µg/mL of LPS was used as a positive control [24]. The concentrations of the extracts
were obtained from microbicide assays in which these concentrations were used (data
not shown).
Plants 2021, 10, 1908 4 of 17

Table 1. Concentrations of aqueous extracts evaluated.

Aqueous Extract Concentration (µg/mL)

E. cardamomum 100
C. longa 70

2.5.2. RNA Extraction

Extraction of total ribonucleic acid (RNA) from peritoneal macrophages was per-
formed from samples suspended in the cell lysis reagent RNAzol® , guanidine thiocyanate.
Subsequently, total RNA extraction was performed according to the manufacturer’s instruc-
tions. Once the total RNA pellet was obtained, it was suspended in 20 µL of nuclease-free
water obtained by DEPC treatment. The total RNA pellet obtained was quantified in
a NanoDrop™ ND-1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington,
DE, USA) at optical densities (OD) of 260 and 280 nm; for this purpose, total RNA was
suspended in 20 µL of nuclease-free water. The extraction purity and concentration showed
a variation of 1.98 and 2.09 and a concentration ranging from 485.45 to 940.39 ng/µL for
each of the triplicates [24].

2.5.3. Complementary DNA (cDNA) Synthesis

From the previously obtained total RNA, complementary DNA (cDNA) synthesis
was performed using the ImProm-II™ Reverse Transcription System reverse transcription
kit. For each reaction, 4 µL of ImProm-II™ 5X reaction buffer, 1 µL of the dNTP’s mixture
(0.5 mM of each), 2.4 µL of MgCl2 (25 mM), 1 µL of Oligo(dT)15 primer, 1 µL of ImProm-II™
transcriptase, 1500 ng of RNA quencher, and nuclease-free water were mixed to a final vol-
ume of 20 µL. The reactions were carried out in 200 µL capacity microtubes. The prepared
samples were incubated in a thermal cycler (Veriti® , Applied Biosystems® , Waltham, MA,
USA) to carry out cDNA synthesis, the reaction conditions used were as follows: 25 ◦ C
(5 min), 42 ◦ C (60 min), 70 ◦ C (15 min), and 4 ◦ C (∞). Once the retrotranscription was
completed, the concentration and purity of the cDNA obtained were determined using an
EPOCH™ microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA)
with the Gen5 software for microplate reading and data analysis. Finally, the products
obtained were stored at −80 ◦ C until further use.

2.5.4. Oligonucleotide Design

The design of oligonucleotides for the quantification of cytokines (IL-4, IL-6, IL-10,
and TNF-α) and endogenous genes (β-actin and Gpd-1) was performed from the mRNA
sequence of each gene, obtained from Genbank. (Table 2). The oligonucleotides for the
amplification of the eight selected sequences were designed using the Primer Quest™ Tool
online program from IDT™ (Integrated DNA Technologies, Inc., Coralville, IA, USA),
which was responsible for performing the synthesis of the oligonucleotides used in this
project. The oligonucleotides were designed according to the following specifications:
approximate size of 20 bp, melting temperature of 60 ◦ C, and a guanine-cytokine content
of 55%. The oligonucleotides obtained were analyzed using the PRIMER BLAST program
to verify their specificity with the sequence of interest.
Plants 2021, 10, 1908 5 of 17

Table 2. Sequences of oligonucleotides used for RT-qPCR.

Gene Genebank ID Oligonucleotides

F: TTG AGA GAG ATC ATC GGC ATT T -30
50
IL-4 NM_021283.2
R: 50 CTC ACT CTC TGT GGT GTT CTT C -30
F: 50 CTT CCA TCC AGT TGC CTT CT -30
IL-6 NM_031168.2
R: 50 CTC CGA CTT GTG AAG TGG TAT AG -30
F: 50 TTG AAT TCC CTG GGT GAG AAG -30
IL-10 NM_010548.2
R: 50 TCC ACT GCC TTG CTC TTA TTT -30
F: 50 CCT ACT GCT GAC CTT TCT TCT C -30
Gpd1 NM_010271.3
R: 50 GCC CTG AGG ACG ATA AAC TAT AA -30
F: 50 TTG TCT ACT CCC AGG TTC TCT -30
TNF-α NM_013693.3
R: 50 GAG GTT GAC TTT CTC CTG GTA TG -30
F: 50 - GAG GTA TCC TGA CCC TGA AGT A -30
β-actin NM_07393.5
R: 50 - CAC ACG CAG CTC ATT GTA GA -30
F: Forward; R: Reverse.

2.5.5. Real Time—qPCR (RT-qPCR)

Quantification of gene expression by RT-qPCR was calculated using the 2−∆∆Ct
method, which consisted of subtracting the threshold cycle (Ct) of the endogenous gene
(Gpd1) from the Ct of the gene of interest, thus obtaining the ∆Ct, then the average ∆Ct
of the control group was obtained and this was subtracted from the ∆Ct of each of the
biological samples, thus calculating the ∆∆Ct, and, finally, the formula 2−∆∆Ct was applied.
The Ct was determined based on the default baseline assigned by the system [25]. The
amplification reaction was performed using the GoTaq® qPCR Master Mix kit at a final
volume of 20 µL for each sample, including 10 µL of the GoTaq qPCR Mix (2x), 1 µL of the
oligonucleotide pair of interest that (100 µM), 1 µL of the cDNA of interest (75 ng), and,
finally, the reaction was completed with 8 µL of nuclease-free water. The qPCR reactions
were carried out in a 96-well PCR microplate (Axygen Scientific® , Union City, CA, USA)
coated with Platemax® UltraClear Sealing Film (Axygen Scientific® , Union City, CA, USA).
Each sample was placed in triplicate in the plate and, in addition, a control reaction was
included, which consisted of adding all the reagents used to perform the amplification
reaction except cDNA.

2.6. Index of Cytotoxicity of the Aqueous Extract of E. cardamomum and C. longa on Cell Cultures
For the evaluation of cytotoxicity of the aqueous extracts of E. cardamomum and C. longa,
three cell lines were used, cervical cancer (HeLa, ATCC CCL-2), mouse macrophages (J774A.1,
ATCC TIB-67), and African green monkey kidney (Vero E6, ATCC CRL-1586). All cells
were maintained in RPMI-1640 medium, which contained L-glutamine, 10% FBS, and
antibiotic/antifungal (penicillin, streptomycin, and amphotericin B), and were kept in
incubation at 37 ◦ C (5% CO2 ) in Corning® 25 cm2 cell culture flasks (Merck KGaA, Darm-
stadt, Germany). Medium changes were carried out every third day. Passages were made
when the cells reached approximately 80% confluence. Next, 100 µL of culture medium
containing 5 × 104 cells/well of each cell line were placed in a 96-well microplate and the
different serial concentrations of the aqueous extract of E. cardamomum and C. longa were
added, ranging from 3.125 to 200 µg/mL (200 µL final volume), which were then incubated
for 24 h. A positive control (nystatin 100,000 µL/mL) and negative control (cells without
treatment) were also included. Once the treatment time with the extracts was fulfilled,
the MTT colorimetric test was carried out [23]. The culture medium was extracted and,
subsequently, the plates were washed with PBS and 100 µL of tetrazolium salt MTT was
added in 0.25 mg/mL to the non-supplemented medium. The plates were incubated at
37 ◦ C/4 h. After that time, the supernatant was removed and 100 µL of DMSO + 20 µL
of glycine buffer was added and incubated for 30 min to allow the formazan crystals to
dissolve [26]. Subsequently, the absorbance was read on an EPOCH™ microplate reader at
570 nm and was analyzed with Gen5 software. Similarly, the same tests were performed
on mouse (Balb/C) peritoneal macrophages to observe changes or differences that could
Plants 2021, 10, 1908 6 of 17

be found between commercial and ex vivo cells. Percentage of cytotoxicity was calculated
according to Equation (2) and the IC50 was determined [18].
 
Abs of the sample
% Cytotoxicity = × 100 (2)
Abs control

2.7. Nitric Oxide Assay

To perform the nitric oxide (NO) assay, peritoneal cells were cultured for 22 h with
100 µg/mL of aqueous extract of E. cardamomum and 70 µg/mL of aqueous extract of
C. longa. As an inflammation model, 50 ng/mL of LPS from E. coli O26:B6 smooth strain
was used to induce a classical activation phenotype associated with an inflammatory
process by NO production, determined by nitrite accumulation in the supernatant using
Griess reagent [27].

2.8. Statistical Analysis

All results were expressed as mean value ± standard deviation. Each experiment was
performed in triplicate independently in at least three replicates. The IBM-SPSS software
(Ver. 22, IBM Corp., Armonk, NY, USA) was used for the statistical evaluation of the results
obtained. For that purpose, a one-way analysis of variance (ANOVA) at a 95% confidence
level and a Tukey post hoc test was applied. GraphPad Prism 6 was used to generate the
graphs. The half-maximal effective concentration (EC50 ) was determined by the Probit test.

3. Results
3.1. Phytochemical Tests
The aqueous extract of E. cardamomum was positive for sterols, triterpenes, flavonoids,
sesquiterpene lactones, and tannins. The extract of C. longa was positive for sterols, triter-
penes, flavonoids, and sesquiterpene lactones (Table 3). Both extracts were negative for
quinones. The extraction yields were 5.64% and 9.83%, respectively.

Table 3. Phytochemical tests.

Test Chemical Groups E. cardamomum C. longa

Lieberman Burchard Sterols, triterpenes + +
Shinoda Flavonoids + +
Baljet Sesquiterpene lactones + +
Sulfuric acid Quinones − −
Ferric chloride Tannins + −
Yield % 5.64 9.83
+ Positive reaction; − negative reaction.

3.2. Compound Identification

To identify the compounds of interest, GC-MS/MS-based phytochemical analysis
of the aqueous extracts was performed (Table 4); the analyses resulted in the identi-
fication of 11 major compounds in E. cardamomum, the α-terpinyl acetate, 2-((1R,4R)-
4-hydroxy-4-methylcyclohex-2-enyl) propan-2-yl acetate, 9-hexacosene, heneicosane, 8-
acetoxycarvotanacetone, geranyl oleate, γ-sitosterol, naphthalene, decahydro-4a-methyl-1-
methylene-7-(1-methylethenyl)-,[4aR-(4aα,7α,8aα)], α-terpineol, (Z)-3,7-dimethylocta-2,6-
dien-1-yl palmitate, and pentacosane (Figure 1a). In C. longa, β-turmerone, α-turmerone,
Ar-turmerone, 16-kauren-19-yl acetate, α-atlantone, β-sesquiphellandrene, and benzene
were identified (Figure 1b). The compounds identified in both aqueous extracts represent
the bioactive compounds previously reported for both plants [11].
Plants 2021, 10, x FOR PEER REVIEW 7 of 17

Plants 2021, 10, 1908 7 of 17

Table 4. Main chemical composition of aqueous extracts of two Zingiberaceae plants identified by GC‐MS/MS.

Plant Extract Chemical Compound R.T. P.A. % M.W. (g/mol)

Table 4. Main chemical composition of aqueous
α‐terpinyl extracts of two Zingiberaceae
acetate 10.27plants identified
54.46 by GC-MS/MS.
196.29
Plant Extract 2‐((1R,4R)‐4‐hydroxy‐4‐methylcyclohex‐2‐enyl)
Chemical Compound R.T. 3.55 P.A. % M.W. (g/mol)
11.31 212.28
propan‐2‐yl acetate
α-terpinyl acetate 10.27 54.46 196.29
9‐hexacosene
2-((1R,4R)-4-hydroxy-4-methylcyclohex-2-enyl) 26.96 3.52 364.69
11.31 3.55 212.28
γ‐sitoesterol
propan-2-yl acetate 29.93 3.38 414.71
9-hexacosene
Heneicosane 24.73 26.96 3.04 3.52 364.69
296.57
E. cadamomum γ-sitoesterol 29.93 3.38 414.71
8‐acetoxycarvotanacetone 12.93 2.57 210.27
(Cardamom)
E. cadamomum Heneicosane 24.73 3.04 296.57
Geranyl oleate
8-acetoxycarvotanacetone 27.24 12.93 2.28 2.57 418.70
210.27
(Cardamom)
(Z)‐3,7‐dimethylocta‐2,6‐dien‐1‐yl
Geranyl oleate palmitate 26.31 27.24 2.08 2.28 392.70
418.70
(Z)-3,7-dimethylocta-2,6-dien-1-yl
Naphthalene, palmitate
decahydro‐4a‐methyl‐1‐meth‐ 26.31 2.08 392.70
Naphthalene, decahydro-4a-methyl-1-methylene-7-(1-12.05 2.03 204.35
ylene‐7‐(1‐methylethenyl)‐, [4aR‐(4aα,7α,8aα)]‐
methylethenyl)-, 12.05 2.03 204.35
α‐terpineol
[4aR-(4aα,7α,8aα)]- 8.12 1.95 154.25
Pentacosane
α-terpineol 25.99 8.12 1.85 1.95 154.25
352.68
Pentacosane
β‐turmerone 15.51 25.99 33.45 1.85 352.68
218.33
α‐β-turmerone
turmerone 15.95 15.51 21.30 33.45 218.33
218.33
α-turmerone 15.95 21.30 218.33
Ar‐turmerone 15.57 19.85 216.32
C. longa
longa Ar-turmerone 15.57 19.85 216.32
C. 16‐kauren‐19‐yl acetate 27.08
16-kauren-19-yl acetate 27.08 18.72 18.72 330.26
330.26
(Turmeric)
(Turmeric)
α‐atlantone
α-atlantone 16.80 16.80 2.77 2.77 218.33
218.33
β-sesquiphellandrene
β‐sesquiphellandrene 13.84 13.84 1.98 1.98 204.35
204.35
Benzene 13.31 1.93 78.11
Benzene 13.31 1.93 78.11
R.T.: retention time; P.A. %: peak area percent; M.W.: molecular weight.
R.T.: retention time; P.A. %: peak area percent; M.W.: molecular weight.

(a)

Figure 1. Cont.
Plants 2021, 10, 1908 8 of 17
Plants 2021, 10, x FOR PEER REVIEW 8 of 17

(b)
Figure 1. Chromatograms
Chromatograms obtained from GC‐MS
GC-MS screening of the aqueous extracts of E. cadamomum seeds (a), and C. longa
roots (b).

3.3. Gene
3.3. Gene Expression
Expression of
of Anti‐Inflammatory
Anti-Inflammatory Cytokines
Cytokines
The gene
The gene expression
expression levels
levelsofofthe
theanti-inflammatory
anti‐inflammatorycytokines,
cytokines,Il-4 (p(p
Il‐4 < 0.01) and
< 0.01) Il-10
and Il‐
(p < 0.001), were analyzed to evaluate whether the aqueous extracts have an immunomodu-
10 (p < 0.001), were analyzed to evaluate whether the aqueous extracts have an immuno‐
latory effect on
modulatory the on
effect expression of cytokines
the expression that regulate
of cytokines or promote
that regulate or inflammatory processes
promote inflammatory
in peritoneal macrophages by using LPS as inflammation control (Table 5).
processes in peritoneal macrophages by using LPS as inflammation control (Table 5).
A significant increase in IL‐10 gene expression level (Figure 2a) was observed with
Table 5. Cytokines gene expression.
C. longa extract compared to E. cardamomum (p ≤ 0.05), as well as the same degree of sig‐
nificance compared to LPS (p ≤C.0.01);
Cytokine longa however, when IL‐4 gene expression was
E. cardamomum LPSanalyzed,
highly significant differences were observed between both aqueous extracts (p < 0.01) and
IL-4 5.07 ± 1.6 0.30 ± 0.185 0.00
highly significant
IL-6
differences2.21
(p ≤±0.001)
0.04
were also 1.05
observed
± 0.012between the 5.61
LPS±control
0.284
and
both extracts
IL-10 (Figure 2b). These results
1.84 ± 0.87 suggest that after induction
2.02 ± 0.026 with LPS, the extracts
0.90 ± 0.073
exhibitedTNF-α
a decrease in the inflammatory
1.46 ± 0.97 process produced by bacterial endotoxins
0.086 ± 0.070 4.59 ± 2.132(LPS),
therefore, we can consider those extracts as modulators in inflammatory processes in the
presence of Gram‐negative microorganisms.
A significant increase in IL-10 gene expression level (Figure 2a) was observed with
C. longa extract compared to E. cardamomum (p ≤ 0.05), as well as the same degree of
Table 5. Cytokines gene expression.
significance compared to LPS (p ≤ 0.01); however, when IL-4 gene expression was analyzed,
Cytokine highly significant
C. longadifferences were observed between both aqueous extracts
E. cardamomum LPS(p < 0.01) and
IL‐4 highly significant differences
5.07 ± 1.6 (p ≤ 0.001) were also
0.30 ± 0.185 observed between the LPS control and
0.00
IL‐6 both extracts (Figure
2.21 ± 0.04 2b). These results suggest that
1.05 ± 0.012 after induction with LPS, the extracts
5.61 ± 0.284
IL‐10 exhibited 1.84
a decrease
± 0.87 in the inflammatory process
2.02 ± 0.026produced by bacterial endotoxins
0.90 ± 0.073 (LPS),
TNF‐α therefore,1.46
we can consider
± 0.97 those extracts as modulators
0.086 ± 0.070 in inflammatory processes
4.59 ± 2.132 in the
presence of Gram-negative microorganisms.
Highly significant (p < 0.001) decreases in the gene expression levels of proinflamma‐
tory cytokines IL‐6 (Figure 2c) induced by aqueous extracts of both plants compared to
LPS were observed (Table 5). Similar results were obtained for TNF‐α (Figure 2d), which
is a potent proinflammatory interleukin, observing highly significant decreases (p ≤ 0.001)
for the treatments of each of the extracts with respect to the LPS control. The results pre‐
sented in Figure 2 and Table 5 reveal that the extracts evaluated decreased the gene ex‐
pression of proinflammatory cytokines, showing differences in expression in relation to
 Plants 2021, 10, x FOR PEER REVIEW 9 of

Plants 2021, 10, 1908 9 of 17

that produced by LPS, which makes them potential natural active ingredients to reduc
inflammatory processes.

(a) (b)

(c) (d)
Figure 2. Gene expression of pro‐inflammatory
Figure 2. Gene cytokines IL‐10cytokines
expression of pro-inflammatory (a), IL‐4 (b) IL‐6
IL-10 (a),(c),
IL-4and
(b)TNF‐α
IL-6 (c),(d) in TNF-α
and peritoneal
(d) inmacro‐
peritoneal macrophages stimulated by aqueous extracts of E. cardamomum and C. longa, challenged*** p <
phages stimulated by aqueous extracts of E. cardamomum and C. longa, challenged with LPS. * p < 0.05, ** p < 0.01,
0.001.
with LPS. * p < 0.05, ** p < 0.01, *** p < 0.001.

Highly3.4.significant
Cytotoxic (p
Activity of the
< 0.001) AqueousinExtract
decreases the gene expression levels of proinflamma-
tory cytokines ForIL-6the
(Figure 2c) induced
evaluation of the by aqueous extracts
cytotoxicity of aqueousof both plants
extracts of compared to
E. cardamomum and C
LPS were observed (Table 5). Similar results were obtained for TNF-α (Figure 2d), which
longa, three cell lines, HeLa, J774A.1, and Vero E6, and peritoneal cells of female Balb/is
a potent proinflammatory
mice were used interleukin,
(Table 6). Aobserving highlyrelationship
dose–response significant decreases (p ≤ 0.001)
was observed, as cytotoxicit
for the treatments of each of the extracts with respect to the LPS control. The results
presented in Figure 2 and Table 5 reveal that the extracts evaluated decreased the gene
expression of proinflammatory cytokines, showing differences in expression in relation to
Plants 2021, 10, 1908 10 of 17

that produced by LPS, which makes them potential natural active ingredients to reduce
inflammatory processes.

3.4. Cytotoxic Activity of the Aqueous Extract

For the evaluation of the cytotoxicity of aqueous extracts of E. cardamomum and C. longa,
three cell lines, HeLa, J774A.1, and Vero E6, and peritoneal cells of female Balb/C mice
were used (Table 6). A dose–response relationship was observed, as cytotoxicity enhanced
as the concentrations of the extracts increased. However, no high activity was observed
against the cell lines or in peritoneal cells at the concentrations tested; E. cardamomum
presented EC50 higher than 321.20 µg/mL on the three cell lines tested, while the EC50 of
C. longa was higher than 190.79 µg/mL. E. cardamomum was the most active on J774A.1
(EC50 237.36 µg/mL) and Vero E6 (EC50 257.51 µg/mL), while C. longa was more effective
on HeLa (EC50 351.17 µg/mL). No differences were observed between the activities of the
aqueous extracts when evaluated in peritoneal macrophages.

Table 6. Cell cytotoxicity assay.

Cell Cytotoxicity Percent (%)

Elettaria cardamomum Curcuma longa
Balb/C Balb/C
Concentration
HeLa J774A.1 Vero E6 Peritoneal HeLa J774A.1 Vero E6 Peritoneal
µg/mL
Cells Cells
3.125 8.26 a 4.11 a 4.14 a 11.73 a 2.18 a 11.74 a 8.34 a 2.18 a
6.25 10.17 a 10.28 b 4.57 a 14.07 a 8.23 b 14.06 a 10.18 a 8.23 b
12.5 13.03 a 13.73 b 8.39 b 14.33 a 9.59 b 14.35 a 19.03 b 9.59 b
25 13.51 a 22.26 c 12.71 b 16.23 a 13.85 b 16.24 a 21.76 b 13.85 b
50 19.47 b 31.31 c 13.04 b 17.60 a 14.63 b 17.62 ab 22.44 b 14.63 b
100 20.58 b 37.94 c 19.54 c 20.37 ab 22.16 c 20.39 ab 25.81 b 22.16 c
200 21.93 b 45.33 d 35.21 d 27.20 b 22.92 c 27.21 b 26.41 b 22.92 c
EC50 µg/mL 424.01 237.36 257.51 431.16 351.17 430.96 396.24 362.86
LL 372.09 190.79 191.02 287.35 321.20 381.60 349.78 269.66
UL 473.84 261.68 301.23 582.74 382.14 480.33 442.71 509.85
EC90 µg/mL 890.09 423.17 436.37 838.33 721.65 957.64 931.97 705.80
LL 811.96 412.91 402.75 690.10 705.80 934.98 905.02 596.56
UL 988.53 434.32 478.67 983.15 757.78 982.90 962.03 831.34
The table shows the mean EC50 and EC90 . The lower (LL) and upper limits (UL) are shown. Different letters in each column represent
significant differences between the groups analyzed via the Tukey post hoc test.

3.5. Nitric Oxide Depletion in Peritoneal Macrophages

NO production was measured as an indicator of inflammation in peritoneal macrophages.
Nitric oxide production averaged 22.12, 18.64, 10.91, and 7.45 µM nitrite for macrophages
stimulated with LPS, LPS plus E. cardamomum extract, E. cardamomum extract alone, and
without stimulation, respectively, while for C. longa, the results were 22.27, 16.97, 8.03, and
6.73 µM nitrite under the same treatments, showing significant differences (p < 0.001) when
macrophages were incubated with 50 ng/mL LPS to induce an activation state (Figure 3).
Plants 2021, 10, x FOR PEER REVIEW 11 of 17
Plants 2021, 10, 1908 11 of 17

Figure 3. Nitric oxide production in peritoneal macrophages stimulated with 100 µg/mL of aqueous
Figure 3.extracts of E. cardamomum,
Nitric oxide and
production in 70 µg/mL
peritoneal of C. longa, challenged
macrophages stimulatedwith
with50
100 μg/mLofofLPS.
µg/mL *** p < 0.001.
aqueous
extracts of E. cardamomum, and 70 μg/mL of C. longa, challenged with 50 μg/mL of LPS. *** p < 0.001.
4. Discussion
4. Discussion The use and evaluation of the traditional folk plants E. cardamomum and C. longa in
terms of their anti-inflammatory activities have been remarkable because of the positive
The use and evaluation of the traditional folk plants E. cardamomum and C. longa in
results obtained in several studies where these plants have been used to reduce the symp-
terms of their anti‐inflammatory activities have been remarkable because of the positive
toms of various chronic inflammatory diseases [28]. In the development of such research,
results obtained in several studies where these plants have been used to reduce the symp‐
numerous bioactive compounds present in its essential oils have been identified, which
toms of various chronic inflammatory diseases [28]. In the development of such research,
differ in proportions and presence depending on the region where the plant was collected,
numerous bioactive compounds present in its essential oils have been identified, which
the time of collection, or the type of extraction [13]. In the chemical characterization of
differ inE.proportions
cardamomum and presence
and C. longa, depending
a series of onmajor
the region where the
compounds wereplant was collected,
identified in the crude
the time of collection, or the type of extraction [13]. In the chemical
extracts of both plants, which largely comprise the composition of their essential characterization of E. oils,
cardamomum
showing their participation in the immune response by acting as mediators in theex‐
and C. longa, a series of major compounds were identified in the crude synthesis
tracts ofofboth
key plants,
factors which largely comprise
of the immune responsethe [29].composition of their essential oils, show‐
ing their participation
In the present in the immune response
investigation, by acting as
the compounds mediators
identified as in the synthesis
major of
in E. cardamomum
key factors of the immune response [29].
have been previously reported as components of its essential oils [30]. We determined
In the
theanti-inflammatory
present investigation, the of
activity compounds
the aqueous identified
extract of asthis
majorplant,in E. cardamomum
which coincides with
have been previously reported as components of its essential oils [30].
previous reports [13], and while this activity is justified by the compound 1,8-cineole, We determined the this
anti‐inflammatory activity of the aqueous extract of this plant, which
was not one of the majority compounds of the crude extract obtained by us. Instead, the coincides with pre‐
vious reports
combined [13],activity
and whileof thethis activity is9-hexacosene,
compounds justified by the compound
α-terpinol, and1,8‐cineole, this
linalool represents an
was notalternative
one of theoptionmajority for compounds of the crude
bioactive compounds withextract obtained by us.
anti-inflammatory Instead,
activity the from
derived
combined thisactivity of the compounds
plant. Previous 9‐hexacosene,
reports demonstrated theα‐terpinol, and linalool represents
activity of 9-hexacosene in reducing anthe size
alternative option for bioactive compounds with anti‐inflammatory activity
of edema in mouse ears, induced by dimethylbenzene [31]; on the other hand, the ability of derived from
this plant. Previous
α-terpinol toreports
suppress demonstrated
pro-inflammatory the activity of 9‐hexacosene
mediators generatinginan reducing
inhibition the of
size IL-6 has
of edema beenin identified
mouse ears, induced
[32]. Similarly,by dimethylbenzene
in previous works,[31]; on reported
it was the other that hand, the ability
linalool generates a
of α‐terpinol
reductionto suppress
of TNF-αpro‐inflammatory mediatorsof
in addition to an inhibition generating
neutrophilanactivation
inhibition[33]. of IL‐6 has
Regarding the
been identified
compound [32]. Similarly,
α-terpenyl in previous
acetate, the major works, it was reported
compound that linalool extract,
in our E. cardamomum generates it has not
a reduction
been of TNF‐α inassociated
previously addition towith an inhibition of neutrophil
anti-inflammatory activation
activity. Although [33]. Regarding
this compound has
the compound α‐terpenyl acetate, the major compound in our E. cardamomum
been previously reported in plants with anti-inflammatory activity [13,34–36], including extract, it
has notthe been previously
plant under study,associated
there with anti‐inflammatory
is a lack activity. Although
of evaluations demonstrating suchthis com‐ which
activity,
pound represents an opportunity
has been previously reportedto consider
in plants forwith
future research because,activity
anti‐inflammatory in E. cardamomum,
[13,34–36], this is
includinga majority
the plant compound
under study, of its essential
there oils.
is a lack of evaluations demonstrating such activity,
which representsThe compounds
an opportunity identified as major
to consider forinfuture
the aqueous
research extract
because, of C.inlonga have been
E. carda‐
momum, previously reportedcompound
this is a majority [37]. Meanwhile, the anti-inflammatory
of its essential oils. activity exhibited by this plant
The has been generally
compounds associated
identified as major within curcuminoids,
the aqueous extract specifically
of C. longacurcumin
have been [38].pre‐
However,
curcuminoids were not identified in our aqueous extract
viously reported [37]. Meanwhile, the anti‐inflammatory activity exhibited by this plant of C. longa; instead, turmerones
has been (AR, α, and β)
generally were identified
associated as major compounds,
with curcuminoids, specificallywhich have not
curcumin [38].received
However, extensive
investigation
curcuminoids were notin regards
identifiedto their
in ouranti-inflammatory
aqueous extract activity. In this
of C. longa; regard,
instead, there have been
turmerones
(AR, α,some
and β) reports of the anti-inflammatory
were identified as major compounds, propertieswhich of have
turmeric essential extensive
not received oils [29], where
Ar-turmerone and β-sesquiphellandrene have been reported as the main components
Plants 2021, 10, 1908 12 of 17

considered responsible for such activity [39], specifically the ability of Ar-turmerone to
inhibit the production of INF-γ and IL-2 [40]. Thus, it is possible to demonstrate the
ability to induce the expression of anti-inflammatory interleukins and inhibition of pro-
inflammatory interleukins, justifying the use of extracts from C. longa or E. cardamomum as
a treatment for inflammatory conditions.
Different research demonstrated the close relationship between food and health, high-
lighting the benefits of vitamins, minerals, fatty acids, probiotics, prebiotics, or phytochem-
icals in fighting various diseases [41]. Among them, the role of substances of plant origin,
such as carotenoids, phenolic compounds, alkaloids, nitrogen, and organosulfur com-
pounds, should be marked for their demonstrated influence on the immune system [42].
Some of the immune processes that take place when infections occur are mainly the
excessive production of proinflammatory cytokines, including IL-6, IFN-γ, IL-1b, and
TNF-α. Such expression has been closely related to apoptosis-inducing inflammatory
processes in animal models [25]. Neutrophil infiltration can lead to damage stimulated
by oxidative stress, which increases inflammation and activation of nuclear factor-kappa
beta (NF-kB)-dependent pathways. NF-kB induces the production of cyclooxygenase 2
(COX-2), which promotes the production of prostaglandins and other inflammatory agents
of the metabolic pathway involved in inflammatory diseases [43]. The presence of the
enzyme inducible nitric oxide synthase (iNOS) and COX-2 induces damage associated with
excessive production of reactive oxygen species (ROS) and suppression of the antioxidative
and defense system [44]. The regulation of iNOS for nitric oxide production will determine
whether this will be associated with damage or repair [24]. Some reports have indicated that,
in the case of inflammatory processes, chronic inflammation is largely due to uncontrolled
production of nitric oxide (NO) by mucosal cells—in this situation nitric oxide may not
be properly regulated by iNOS [45]. The arginine pathway plays an important role in
tissue repair as L-arginine is converted to an amino acid after tissue injury. Some studies
have indicated that arginine increases collagen deposition and tear strength (effects that
contribute to epithelial repair and arginase repair functions associated with inflammatory
processes); this enzyme inhibits nitric oxide synthesis, resulting in limited intracellular
L-arginine supply to produce reactive nitrogen species (RNS) [46], decreasing mucosal
damage, such as oral.
In a study conducted in hamsters in which an inflammatory process was induced
through local lacerations and a potent inflammatory agent such as 5-fluorouracil, it was ob-
served that daily topical treatment with extracts of Calendula officinalis on days 12 to 17 after
the inflammatory induction reduced the clinical severity of the disease in a concentration-
dependent manner, compared to animals treated only with the vehicle—this is another
example of how natural extracts are a good option in the management of lesions [47].
Another study demonstrated the anti-inflammatory effect of chamomile extracts by in-
hibiting the expression of IL-1β and TNF-α cytokines in an animal model, with data like
those observed in our research; however, they could also prove it at the histopathological
level, supporting the idea of the use of plant extracts as possible natural candidates in
the management of inflammatory processes [48]. In another study, the anti-inflammatory
effect of cardamom extracts was demonstrated by inhibiting the action of macrophages
producing proinflammatory cytokines such as Il-1β, TNF-α, and Il-8 through the effect
of LPS from Actinobacillus actinomycetemcomitans [13]. On the other hand, using a mouse
peritoneal macrophage model, the ability of an aqueous extract of cardamom to attenuate
IL-6 and TNF-α secretion was demonstrated [49]. It has been suggested that the anti-
inflammatory activity of cardamom extracts reported by other authors is related to the
presence of phytochemical agents in high amounts, in this case to 1,8-cineole (eucalyptol),
since this compound has also been shown to attenuate LPS-induced inflammatory signaling
pathways in the lung by alveolar macrophages [50].
The present investigation found a positive response of almost 6 relative units of expres-
sion for IL-6. The technique was proved valid by positive controls in alpaca enterocytes,
which indicated its scarce participation as an inducer of the acute phase of inflammation
Plants 2021, 10, 1908 13 of 17

where leukocytes participate—IL-6 has anti-inflammatory as well as pro-inflammatory

effects by regulating the expression of other pro-inflammatory cytokines and, in addition,
it induces the synthesis of glucocorticoids [51]. Quantification of cytokine and receptor
mRNA was performed by RT-qPCR, from which the results showed positive responses,
specifically of C. longa concerning IL-10, which is the interleukin responsible for regulating
and decreasing the inflammatory response produced by dendritic cells and macrophages,
and also for reducing the adaptive responses of CD4 T cells and IL-4, which is a cytokine
that acts as an anti-inflammatory by blocking the synthesis of IL-1, TNF-α, IL-6, and
macrophage inflammatory protein [52]. The proinflammatory activity of INF-γ, which is
produced in T cells and activated NK cells, was also observed. It potentiates the effects of
type I interferons released by Th1, which regulates leukocytes at the site of infection, giving
rise to inflammation [53]. As for IL-6, the results dictate a positive response since this
interleukin shows anti-inflammatory as well as pro-inflammatory activity [54]. In addition,
the extract also influenced the proinflammatory cytokine TNF-α, which also leads to the
recruitment of inflammatory cells.
Several in vitro studies in human cell lines have demonstrated the cytotoxic capacity
of several natural extracts [55,56] such as curcuminoids [16]. Different components of
Zingiberaceae plants can suppress the activity of some common mutagens and carcinogens
in various cell types in both in vitro and in vivo studies. The cytotoxic effects of methanol
extracts and their fractions (hexane, ethyl acetate, and water) of Alpinia mutica rhizomes
against six human carcinoma cell lines (KB, MCF7, A549, Caski, HCT116, HT29) and
the non-human fibroblast cell line (MRC 5) are currently known as a result of an in vitro
cytotoxicity assay [57]. In two studies on colon and prostate cancer, curcumin from C. longa
inhibited cell proliferation and tumor growth [58]. In addition, curcumin is a nonmutagenic
and nongenotoxic agent [15]. Although the cytotoxic activity of curcuminoids is known,
in this research, promising results were observed in terms of anti-inflammatory activity,
but no cytotoxic activity was observed in HeLa, J774A.1, Vero E6, and peritoneal cell lines,
as the EC50 values found were higher than 200 µg/mL and EC90 values were higher than
400 µg/mL, respectively, for all assays evaluated (Table 6).
Currently, there is a growing interest in both industry and scientific research on spices
and aromatic herbs due to their strong antioxidant powers and antimicrobial properties—
both natural and synthetic antioxidants are currently being used. In some of these studies,
microbicidal activity against Streptococcus mutans was observed with an increase of nitric
oxide in RAW 264.7 macrophages stimulated with extracts of Rosmarinus officinalis, Thymus
vulgaris, and C. longa [59]. Another study showed that curcumin at a dose of 0.5 ppm
induced polarization to an M2 phenotype or alternating activation with anti-inflammatory
characteristics in peritoneal macrophages, although at higher doses this phenotype was
lost and they observed increased expression at the level of Arg-1 transcripts, which is a
competitor of L-Arginine against iNOS enzymes [60]. When we stimulated macrophages
with LPS in the presence of the extracts, nitrite production decreased, in addition to the
fact that the extract alone induced little nitrite production without stimulation in both
aqueous extracts. These data suggest the anti-inflammatory effect of the extracts alone or
in combination with LPS (Figure 3). At the moment, the modulatory effects of turmeronole
A and B (plant components of the C. longa) on RAW 264.7 cells in inflammatory processes
are known—they significantly inhibited LPS-induced prostaglandin E (PgE) and NO
production, as well as the expression of iNOS and the gene encoding PgE. In addition,
turmeronols significantly inhibited the overexpression of transcripts encoding IL-1β, IL-6,
and TNF-α at the mRNA and protein level induced by LPS [61].
In addition, several investigations carried out with the main components in plants of
this family have demonstrated their antioxidant capacity against the DPPH radical in com-
parison with Trolox and ascorbic acid—among the most important ones are α-turmerone,
β-turmerone, and Ar-turmerone [62]. In an investigation to evaluate the hepatoprotective
effect of an extract of Amomum cardamomum on carbon-tetrachloride-induced liver damage
through antioxidant activity in rats, it was found to possess significant hepatoprotective
Plants 2021, 10, 1908 14 of 17

activity on acute liver injury, which could be derived from its antioxidant properties and the
decrease of liver cytochrome P450 [63]. In general, it can be stated that the pharmaceutical
properties of plants of the Zingiberaceae family are related to their chemical composition;
this is mainly due to the presence of phenolic compounds and other biologically active
constituents [11]. In addition to anticancer, antioxidant, and free radical scavenging effects,
these plants possess the capacity to indirectly increase glutathione levels, thus aiding in
the hepatic detoxification of mutagens and carcinogens and inhibiting the formation of
nitrosamines [58].
We think that the results presented in this manuscript may be sufficient to infer the
anti-inflammatory effects of the extracts in question, given that several investigations have
provided evidence such as those presented in this document—in the case of E. cardamomum,
some authors have mentioned the anti-inflammatory properties [13,49] and for C. longa,
some authors described these same effects through the suppression of NO and COX [64,65].

5. Conclusions
Under the experimental conditions analyzed and based on the results obtained in
the tests performed in this research, the anti-inflammatory effects of E. cardamomum and
C. longa were evidenced. The aqueous extracts of cardamom and turmeric showed elevated
expression of interleukins, suggesting their possible anti-inflammatory or immunomod-
ulatory action. As for the cytotoxic action, a dose–response relationship was observed,
since as the concentration increased, the cytotoxic activity increased. The concentrations
evaluated (from 3.125 to 200 µg/mL) showed no activity against HeLa, J774A.1 and Vero
E6 cell lines. In future research, we expect to continue with more studies to identify the
molecular mechanisms of action that favors the use of these plants as complementary alter-
natives in inflammatory processes in order to develop alternative or adjuvant therapies, as
well as safe applications of cardamom and turmeric, which could bring several benefits,
such as cost reduction compared to existing products, in addition to contributing to the
ethno-pharmacological development for the safe use of folk traditional plants.

Author Contributions: Conceptualization, O.E.R.L. and R.A.P.H.; correspondence, U.C.V. and

O.E.R.L.; methodology, S.M.L.V. and A.F.B.R.; software, J.R.R. and R.A.P.H.; validation, R.M.S.C.,
A.C.M., and A.F.B.R.; formal analysis, G.R.C.G. and A.C.M.; investigation, A.J.M.D.; resources,
S.M.L.V.; data curation, J.H.E.L. and U.C.V.; writing—original draft preparation, G.R.C.G. and
O.E.R.L.; writing—review and editing, O.E.R.L. and J.H.E.L.; visualization, S.M.L.V.; supervision,
J.R.R.; project administration and funding acquisition, O.E.R.L. and R.A.P.H. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by the National Council of Science and Technology (CONACYT,
Mexico), grant number 958507, as well as the Support Program for Scientific and Technological
Research (PAICYT-UANL 2021), and the Professional Development Program for Higher Education
(PRODEP). This publication was also supported by the Institutional Open Access Program (IOAP) of
the Tecnológico de Monterrey.
Institutional Review Board Statement: The laboratory animal study was conducted according to
the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of
the Faculty of Veterinary Medicine and Zootechny, Autonomous University of Nuevo León protocol
code 02/21-2021 (July of 2021).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this research are available on request from the
corresponding author.
Acknowledgments: We thank the personnel of the Laboratory of Analytical Chemistry (Faculty of
Biological Sciences, Autonomous University of Nuevo León) and the Laboratory of Microbiology
(Faculty of Dentistry, Autonomous University of Nuevo León) for their valuable technical assistance
during the present investigation.
Conflicts of Interest: The authors declare no conflict of interest.
Plants 2021, 10, 1908 15 of 17

References
1. Sánchez-Ramos, M.; Alvarez, L.; Romero-Estrada, A.; Bernabé-Antonio, A.; Marquina-Bahena, S.; Cruz-Sosa, F. Establishment
of a cell suspension culture of ageratina pichinchensis (Kunth) for the improved production of anti-inflammatory compounds.
Plants 2020, 9, 1398. [CrossRef] [PubMed]
2. Elizondo-Luévano, J.H.; Castro-Ríos, R.; Sánchez-García, E.; Hernández-García, M.E.; Vargas-Villarreal, J.; Rodríguez-Luis,
O.E.; Chávez-Montes, A. In vitro study of antiamoebic activity of methanol extracts of argemone mexicana on trophozoites of
entamoeba histolytica HM1-IMSS. Can. J. Infect. Dis. Med. Microbiol. 2018, 2018, 7453787. [CrossRef] [PubMed]
3. Frencken, J.E.; Sharma, P.; Stenhouse, L.; Green, D.; Laverty, D.; Dietrich, T. Global epidemiology of dental caries and severe
periodontitis—A comprehensive review. J. Clin. Periodontol. 2017, 44, S94–S105. [CrossRef]
4. Soehnlein, O.; Steffens, S.; Hidalgo, A.; Weber, C. Neutrophils as protagonists and targets in chronic inflammation. Nat. Rev.
Immunol. 2017, 17, 248–261. [CrossRef]
5. Konkel, J.E.; O’Boyle, C.; Krishnan, S. Distal consequences of oral inflammation. Front. Immunol. 2019, 10, 1403. [CrossRef]
6. Scannapieco, F.A.; Cantos, A. Oral inflammation and infection, and chronic medical diseases: Implications for the elderly.
Periodontology 2016, 72, 153–175. [CrossRef] [PubMed]
7. Rider, P.; Carmi, Y.; Cohen, I. Biologics for targeting inflammatory cytokines, clinical uses, and limitations. Int. J. Cell Biol. 2016,
2016, 9259646. [CrossRef] [PubMed]
8. Cardoso, E.M.; Reis, C.; Manzanares-Céspedes, M.C. Chronic periodontitis, inflammatory cytokines, and interrelationship with
other chronic diseases. Postgrad. Med. 2018, 130, 98–104. [CrossRef]
9. Dini, M. Peptic ulcer disease and non-steroidal anti-inflammatory drugs. Aust. Prescr. 2017, 40, 91–93.
10. Little, P. Non-steroidal anti-inflammatory drugs and covid-19. BMJ 2020, 368, m1185. [CrossRef] [PubMed]
11. Ivanović, M.; Makoter, K.; Razboršek, M.I. Comparative study of chemical composition and antioxidant activity of essential oils
and crude extracts of four characteristic zingiberaceae herbs. Plants 2021, 10, 501. [CrossRef]
12. Rajan, A.; Rajan, A.R.; Philip, D. Elettaria cardamomum seed mediated rapid synthesis of gold nanoparticles and its biological
activities. Open Nano 2017, 2, 1–8. [CrossRef]
13. Souissi, M.; Azelmat, J.; Chaieb, K.; Grenier, D. Antibacterial and anti-inflammatory activities of cardamom (Elettaria cardamomum)
extracts: Potential therapeutic benefits for periodontal infections. Anaerobe 2020, 61, 102089. [CrossRef]
14. Tanvir, E.M.; Hossen, M.S.; Hossain, M.F.; Afroz, R.; Gan, S.H.; Khalil, M.I.; Karim, N. Antioxidant properties of popular turmeric
(Curcuma longa) varieties from Bangladesh. J. Food Qual. 2017, 2017, 8471785. [CrossRef]
15. Soleimani, V.; Sahebkar, A.; Hosseinzadeh, H. Turmeric (Curcuma longa) and its major constituent (curcumin) as nontoxic and safe
substances: Review. Phyther. Res. 2018, 32, 985–995. [CrossRef] [PubMed]
16. Elizondo-Luévano, J.H.; Hernández-García, M.E.; Pérez-Narváez, O.A.; Castro-Ríos, R.; Chávez-Montes, A. Berberina, curcumina
y quercetina como potenciales agentes con capacidad antiparasitaria. Rev. Biol. Trop. 2020, 68, 1241–1249. [CrossRef]
17. White, C.M.; Pasupuleti, V.; Roman, Y.M.; Li, Y.; Hernandez, A.V. Oral turmeric/curcumin effects on inflammatory markers in
chronic inflammatory diseases: A systematic review and meta-analysis of randomized controlled trials. Pharmacol. Res. 2019, 146,
104280. [CrossRef]
18. Elizondo-Luevano, J.H.; Verde-Star, J.; González-Horta, A.; Castro-Ríos, R.; Hernández-García, M.E.; Chávez-Montes, A. In Vitro
Effect of Methanolic Extract of Argemone mexicana against Trichomonas vaginalis. Korean J. Parasitol. 2020, 58, 135–145. [CrossRef]
19. Parekh, J.; Chanda, S.V. In vitro antimicrobial activity and phytochemical analysis of some Indian medicinal plants. Turkish J. Biol.
2007, 31, 53–58.
20. Bekinbo, M.; Tariah FS, A.; Dapper, D. Comparative GC-MS determination of bioactive constituents of the methanolic extracts of
Curcuma longa rhizome and Spondias mombin leaves Bekinbo MT, Amah-Tariah FS and Dapper DV. J. Med. Plants Stud. 2020, 8,
1–6.
21. Masoumi-Ardakani, Y.; Mandegary, A.; Esmaeilpour, K.; Najafipour, H.; Sharififar, F.; Pakravanan, M.; Ghazvini, H. Chemical
composition, anticonvulsant activity, and toxicity of essential oil and methanolic extract of Elettaria cardamomum. Planta Med.
2016, 82, 1482–1486. [CrossRef]
22. Salvesen, Ø.; Reiten, M.R.; Espenes, A.; Bakkebø, M.K.; Tranulis, M.A.; Ersdal, C. LPS-induced systemic inflammation reveals an
immunomodulatory role for the prion protein at the blood-brain interface. J. Neuroinflam. 2017, 14, 106. [CrossRef]
23. Humberto, C.H.-M.; Ricardo, G.-F.; Patricia, T.-G.; Ramiro, Q.-L.; Mario, A.S.E.; Enriqueta, M.-C.; Reyes, T.-G.; Cristina, R.-P.
Antitumor activity of Pachycereus marginatus (DC.) Britton Rose extracts against murine lymphoma L5178Y-R and skin melanoma
B16F10 cells. J. Med. Plants Res. 2016, 10, 635–639. [CrossRef]
24. Castillo-Velázquez, U.; Aranday-Cortés, E.; Gutiérrez-Pabello, J.A. Alternative activation modifies macrophage resistance to
Mycobacterium bovis. Vet. Microbiol. 2011, 151, 51–59. [CrossRef] [PubMed]
25. Nevárez-Garza, A.M.; Castillo-Velázquez, U.; Soto-Domínguez, A.; Montes-de-Oca-Luna, R.; Zamora-Ávila, D.E.; Wong-González,
A.; Rodríguez-Tovar, L.E. Quantitative analysis of TNF-α IL-4, and IL-10 expression, nitric oxide response, and apoptosis in
Encephalitozoon cuniculi–infected rabbits. Dev. Comp. Immunol. 2018, 81, 235–243. [CrossRef] [PubMed]
26. Delgado, A.J.M.; Velázquez, U.C.; González, J.G.B.; Montes, A.C.; Villarreal, S.M.L.; García, L.E.V.; Casas, R.M.S.; Luis, O.E.R.
Evaluation of the Essential Oil of Citrus paradisi as an Alternative Treatment against Candida albicans. Open J. Stomatol. 2020, 10,
258–270. [CrossRef]
Plants 2021, 10, 1908 16 of 17

27. Adler, B.; Adler, H.; Jungi, T.W.; Peterhans, E. Interferon-α primes macrophages for lipopolysaccharide-induced apoptosis.
Biochem. Biophys. Res. Commun. 1995, 215, 921–927. [CrossRef]
28. Chandran, B.; Goel, A. A randomized, pilot study to assess the efficacy and safety of curcumin in patients with active rheumatoid
arthritis. Phyther. Res. 2012, 26, 1719–1725. [CrossRef]
29. Sandner, G.; Heckmann, M.; Weghuber, J. Immunomodulatory activities of selected essential oils. Biomolecules 2020, 10, 1139.
[CrossRef]
30. Noumi, E.; Snoussi, M.; Alreshidi, M.M.; Rekha, P.D.; Saptami, K.; Caputo, L.; De Martino, L.; Souza, L.F.; Msaada, K.; Mancini,
E.; et al. Chemical and biological evaluation of essential oils from cardamom species. Molecules 2018, 23, 2818. [CrossRef]
31. Githinji, C.G.; Mbugua, P.M.; Kanui, T.I.; Kariuki, D.K. Analgesic and anti-inflammatory activities of 9-Hexacosene and Stigmas-
terol isolated from Mondia whytei. Phytopharmacology 2012, 2, 212–223.
32. Nogueira, M.N.M.; Aquino, S.G.; Rossa, C.; Spolidorio, D.M.P. Terpinen-4-ol and alpha-terpineol (tea tree oil components) inhibit
the production of IL-1β, IL-6 and IL-10 on human macrophages. Inflamm. Res. 2014, 63, 769–778. [CrossRef] [PubMed]
33. Abe, S.; Maruyama, N.; Hayama, K.; Ishibashi, H.; Inoue, S.; Oshima, H.; Yamaguchi, H. Suppression of tumor necrosis
factor-alpha-induced neutrophil adherence responses by essential oils. Mediat. Inflamm. 2003, 12, 323–328. [CrossRef]
34. Hamdan, D.I.; Abdulla, R.H.; Mohamed, M.E.; El-Shazly, A.M. Chemical composition and biological activity of essential oils of
cleopatra mandarin (citrus reshni) cultivated in Egypt. J. Pharmacogn. Phyther. 2013, 5, 83–90. [CrossRef]
35. Djouahri, A.; Boualem, S.; Boudarene, L.; Baaliouamer, A. Geographic’s variation impact on chemical composition, antioxidant
and anti-inflammatory activities of essential oils from wood and leaves of Tetraclinis articulata (Vahl) Masters. Ind. Crops Prod.
2015, 63, 138–146. [CrossRef]
36. Valente, J.; Resende, R.; Zuzarte, M.; Gonçalves, M.J.; Lopes, M.C.; Cavaleiro, C.; Pereira, C.; Salgueiro, L.; Cruz, M.T. Bioactivity
and safety profile of Daucus carota subsp. maximus essential oil. Ind. Crops Prod. 2015, 77, 218–224. [CrossRef]
37. Angel, G.R.; Menon, N.; Vimala, B.; Nambisan, B. Essential oil composition of eight starchy Curcuma species. Ind. Crop. Prod.
2014, 60, 233–238. [CrossRef]
38. Niranjan, A.; Prakash, D. Chemical constituents and biological activities of turmeric (Curcuma longa L.)—A review. J. Food Sci.
Technol. 2008, 45, 109–116.
39. Liju, V.; Jeena, K.; Kuttan, R. An evaluation of antioxidant, anti-inflammatory, and antinociceptive activities of essential oil from
Curcuma longa L. Indian J. Pharmacol. 2011, 43, 526–531. [CrossRef] [PubMed]
40. Oh, S.; Han, A.R.; Park, H.R.; Jang, E.J.; Kim, H.K.; Jeong, M.G.; Song, H.; Park, G.H.; Seo, E.K.; Hwang, E.S. Suppression of
inflammatory cytokine production by ar-turmerone isolated from Curcuma phaeocaulis. Chem. Biodivers. 2014, 11, 1034–1041.
[CrossRef]
41. Guillamón, E. Effect of phytochemical compounds of the genus Allium on the immune system and the inflammatory response.
Ars Pharm. 2018, 59, 185–196. [CrossRef]
42. Freire-González, R.A.; Vistel-Vigo, M. Caracterizacion fitoquimica de la curcuma. Rev. Cuba. Quim. 2015, 27, 9–18.
43. Dávila-Martínez, C.; Castillo-Velázquez, U.; Soto-Domínguez, A.; Nevárez-Garza, A.M.; Arce-Mendoza, A.Y.; Hernandez-
Vidal, G.; Zamora-Avila, D.E.; Rodriguez-Tovar, L.E. Immunohistochemical localization of TNF-α and IL-4 in granulomas of
immunocompetent and immunosuppressed New Zealand white rabbits infected with Encephalitozoon cuniculi. Cytokine 2020,
130, 155055. [CrossRef] [PubMed]
44. Koppula, S.; Kumar, H.; Kim, I.S.; Choi, D.K. Reactive oxygen species and inhibitors of inflammatory enzymes, NADPH oxidase,
and iNOS in experimental models of parkinsons disease. Mediat. Inflamm. 2012, 823902. [CrossRef]
45. Baranipour, S.; Kadijani, A.A.; Qujeq, D.; Shahrokh, S.; Haghazali, M.; Mirzaei, A.; Asadzadeh-Aghdaei, H. Inducible nitric
oxide synthase as a potential blood-based biomarker in inflammatory bowel diseases. Gastroenterol. Hepatol. Bed Bench 2018, 11,
S124–S128. [CrossRef] [PubMed]
46. Witte, M.B.; Barbul, A. Arginine physiology and its implication for wound healing. Wound Repair Regen. 2003, 11, 419–423.
[CrossRef]
47. Yoshino, F.; Yoshida, A.; Nakajima, A.; Wada-Takahashi, S.; Takahashi, S.; Lee, M.C. Il Alteration of the redox state with reactive
oxygen species for 5-fluorouracil-induced oral mucositis in hamsters. PLoS ONE 2013, 8, 10–15. [CrossRef]
48. Curra, M.; Martins, M.A.T.; Lauxen, I.S.; Pellicioli, A.C.A.; Sant’Ana Filho, M.; Pavesi, V.C.S.; Carrard, V.C.; Martins, M.D. Effect
of topical chamomile on immunohistochemical levels of IL-1β and TNF-α in 5-fluorouracil-induced oral mucositis in hamsters.
Cancer Chemother. Pharmacol. 2013, 71, 293–299. [CrossRef]
49. Majdalawieh, A.F.; Carr, R.I. In vitro investigation of the potential immunomodulatory and anti-cancer activities of black pepper
(Piper nigrum) and cardamom (Elettaria cardamomum). J. Med. Food 2010, 13, 371–381. [CrossRef]
50. Yadav, N.; Chandra, H. Suppression of inflammatory and infection responses in lung macrophages by eucalyptus oil and its
constituent 1,8-cineole: Role of pattern recognition receptors TREM-1 and NLRP3, the MAP kinase regulator MKP-1, and NFκB.
PLoS ONE 2017, 12, e0188232. [CrossRef]
51. Adcock, I.M.; Mumby, S. Glucocorticoids. Handb. Exp. Pharmacol. 2017, 237, 171–196. [CrossRef]
52. Conlon, K.C.; Miljkovic, M.D.; Waldmann, T.A. Cytokines in the treatment of cancer. J. Interferon Cytokine Res. J. Int. Soc. Interferon
Cytokine Res. 2019, 39, 6–21. [CrossRef]
53. Billiau, A. Anti-inflammatory properties of Type I interferons. Antivir. Res. 2006, 71, 108–116. [CrossRef] [PubMed]
Plants 2021, 10, 1908 17 of 17

54. Holdsworth, S.R.; Can, P.Y. Cytokines: Names and numbers you should care about. Clin. J. Am. Soc. Nephrol. 2015, 10, 2243–2254.
[CrossRef]
55. Elizondo-Luévano, J.H.; Castro-Ríos, R.; Vicente, B.; Fernández-Soto, P.; López-Aban, J.; Muro, A.; Chávez-Montes, A. In vitro
antischistosomal activity of the argemone mexicana methanolic extract and its main component berberine. Iran. J. Parasitol. 2021,
16, 91–100. [CrossRef] [PubMed]
56. Elizondo-Luévano, J.H.; Castro-Ríos, R.; López-Abán, J.; Gorgojo-Galindo, O.; Fernández-Soto, P.; Vicente, B.; Muro, A.; Chávez-
Montes, A. Berberine: A nematocidal alkaloid from Argemone mexicana against Strongyloides venezuelensis. Exp. Parasitol. 2021, 220,
108043. [CrossRef] [PubMed]
57. Malek, S.N.A.; Phang, C.W.; Ibrahim, H.; Wahab, N.A.; Sim, K.S. Phytochemical and cytotoxic investigations of Alpinia mutica
rhizomes. Molecules 2011, 16, 583–589. [CrossRef]
58. Akram, M.; Ahmed, A.; Usmanghani, K.; Hannan, A.; Mohiuddin, E.; Asif, M. Curcuma longa and curcumin: A review article.
Rom. J. Biol. 2010, 55, 65–70.
59. Berbudi, A.; Rahmi, N.; Atik, N.; Wikayani, T.; Qomarilla, N.; Rahayu, N.S.; Putri, A.C. The administration of low-dose curcuma
longa extract induces m2 polarization in peritoneal macrophage culture. Res. J. Pharm. Technol. 2021, 14, 1079–1084. [CrossRef]
60. Figueira, L.W.; de Oliveira, J.R.; Camargo, S.E.A.; de Oliveira, L.D. Curcuma longa L. (turmeric), Rosmarinus officinalis L. (rosemary),
and Thymus vulgaris L. (thyme) extracts aid murine macrophages (RAW 264.7) to fight Streptococcus mutans during in vitro
infection. Arch. Microbiol. 2020, 202, 2269–2277. [CrossRef]
61. Okuda-Hanafusa, C.; Uchio, R.; Fuwa, A.; Kawasaki, K.; Muroyama, K.; Yamamoto, Y.; Murosaki, S. Turmeronol A and
turmeronol B from Curcuma longa prevent inflammatory mediator production by lipopolysaccharide-stimulated RAW264.7
macrophages, partially via reduced NF-κB signaling. Food Funct. 2019, 10, 5779–5788. [CrossRef] [PubMed]
62. Ibáñez, M.D.; Blázquez, M.A. Curcuma longa l. Rhizome essential oil from extraction to its agri-food applications. A review.
Plants 2021, 10, 44. [CrossRef] [PubMed]
63. Lim, D.W.; Kim, H.; Park, J.Y.; Kim, J.E.; Moon, J.Y.; Park, S.D.; Park, W.H. Amomum cardamomum L. ethyl acetate fraction
protects against carbon tetrachloride-induced liver injury via an antioxidant mechanism in rats. BMC Complement. Altern. Med.
2016, 16, 155. [CrossRef] [PubMed]
64. Lee, S.Y.; Cho, S.S.; Li, Y.C.; Bae, C.S.; Park, K.M.; Park, D.H. Anti-inflammatory Effect of Curcuma longa and Allium hookeri
Co-treatment via NF-κB and COX-2 Pathways. Sci. Rep. 2020, 10, 5718. [CrossRef]
65. Lee, T.K.; Trinh, T.A.; Lee, S.R.; Kim, S.; So, H.M.; Moon, E.; Hwang, G.S.; Kang, K.S.; Kim, J.H.; Yamabe, N.; et al. Bioactivity-based
analysis and chemical characterization of anti-inflammatory compounds from Curcuma zedoaria rhizomes using LPS-stimulated
RAW264.7 cells. Bioorg. Chem. 2019, 82, 26–32. [CrossRef] [PubMed]