Research Article
Bruna da Silva Garais
Bruna da Silva Garais
Graduate Program in Food Science and Technology, Federal University of Southern Frontier, Laranjeiras do Sul, Parana, Brazil.
E-mail: brunagarais@gmail.com
Roberta Letícia Krüger
Roberta Letícia Krüger
Department of Food Engineering, Midwest State University, Guarapuava, Parana, Brazil.
E-mail: betakruger@yahoo.com.br
Leda Battestin Quast
Leda Battestin Quast
Corresponding Author
Graduate Program in Food Science and Technology, Federal University of Southern Frontier, Laranjeiras do Sul, Parana, Brazil.
E-mail: leda.quast@uffs.edu.br, Tel: +55 42 3635-8662
Abstract
This study aimed to evaluate the biological activity of clove (Syzygium
aromaticum) essential oil and clove extracts, obtained by
hydrodistillation, maceration, and ultrasound-assisted extraction. E. coli,
L. monocytogenes, S. enteritidis, and S. aureus strains were used to determine
the biological activity by the formation of inhibition zones. Eugenol (86.16
%), caryophyllene (7.14 %) and eugenyl acetate (4.84 %) were presented in clove
essential oil. The ultrasound-assisted essential oils and extracts exhibited
biological activity at the concentration of 100 µL/mL, with the highest
inhibition halos observed for all strains studied. The essential oil presented
greater inhibition for the concentration of 50 µL/mL. The chemical profile
analysis demonstrated the presence of groups of gallic acid compounds,
flavones, and their isomers. The total phenolic contents ranged from 0.101 and
0.290 mg GAE/g, with the highest values for the ultrasound-assisted extracts.
The results of DPPH varied from 8.15 to 65.24 %, with an emphasis on
ultrasound-assisted essential oil and extracts.
Abstract Keywords
Essential oil, bio-additive, antioxidant, antimicrobial, phenolic,
extract.
1. Introduction
Clove (Syzygium
aromaticum) is an aromatic plant with interest
in perfume, cosmetic, health, medical, flavoring and food industries, which presents
volatile compounds and antioxidants, that promotes biological activity relevant
to human health [1]. The use of bioactive
compounds presents great advantages over additives since they are easily
decomposed, are not a pollutant, and do not present phytotoxic or residual
properties. Bioactive compounds found in essential oils and plant extracts can
act concomitantly with other compounds with a proven effect on pathogens [2]. The physicochemical properties and the
composition of essential oils are directly affected by the extraction method.
Thus, the selection of an appropriate technique is necessary to produce plant
oils with high nutritional value, with no negative impact on yield and
commercial value [3].
Hydrodistillation is the most widely
used method for the extraction of essential oils on a laboratory scale [4]. In turn, maceration is a process widely used
to obtain extracts, especially for plants with active principles susceptible to
degradation at high temperatures. The ultrasound-assisted extraction generates a
small amount of organic solvent residues and allows greater efficiency in the
recovery of bioactive compounds from plants [5].
Spices such as cloves, cinnamon, and
mint are widely used in industry as flavoring and preserving agents. Clove (Syzygium
aromaticum) stands out from the others due to its antioxidant,
antimicrobial, antiseptic, and anesthetic properties [6,
7]. Considering the potential for industrial application of clove
essential oil, this study aimed to evaluate the biological activity and the
chemical profile of clove essential oil and clove extracts obtained by
different extraction methods.
2. Materials and methods
Dried clove buds in natura, from a
single lot, commercially produced in Brazil (Northeast Region, Latitude 13º 32' 17"
S, Longitude 39º 05' 55" W and Altitude 15m) were
used in this study. The clove essential oil was obtained by hydrodistillation
in a Clevenger apparatus using 150 g of ground clove and 500 mL of distilled
water in a 1 L round-bottom flask. The mixture was heated at 100 °C for 6 h
(from boiling) in triplicate. The clove essential oil was separated
manually by opening the hydrodistillation valve. Due to the density difference,
the first part removed is water and then the last part is essential oil. The
sample:solvent ratio and the extraction time were determined in preliminary
studies.
The extracts were obtained by three
methods: maceration, ultrasound-assisted extraction at 25 °C, and
ultrasound-assisted extraction at 40 °C.
For the maceration procedure, 20 g of
ground cloves and 80 mL of distilled water were placed in 500 mL Erlenmeyer
flasks, at 25 °C. The extraction times were 0, 6, 12, 24, 48, and 72 h. The
samples were stored protected from light until the moment of the filtration and
then stored at 4 °C.
The ultrasound-assisted extraction
(Kondortech® CD-4820) was performed in an environment at 25 ºC and 40 °C. For
that, 20 g of ground cloves and 80 mL of distilled water were placed in 500 mL
Erlenmeyer flasks, which were subjected to an ultrasound machine until
filtration. The ultrasound exposure times were 0, 5, 10, 15, 20, and 30 min.
The resulting extracts were stored at 4 °C.
Due to the conditions of the equipment and time of the experiments, the
maceration and ultrasound-assisted extraction tests were carried out only once.
The essential oil compounds were
identified in a gas chromatograph coupled to a mass spectrometer (Shimadzu®,
model GC anMS-QP2010 Ultra), equipped with a DB 5 ms fused silica capillary
column (5 % diphenyl, 95 % dimethylpolysiloxane)
of 30 m, with internal diameter of 0.25 mm and film thickness of 0.25 µm.
Helium was used as carrier gas at a linear velocity of 43 cm/s. The operation
conditions were: split mode (1:5) with an injection temperature of 210 °C;
interface at 210 °C; programmed column temperature: initial temperature 50 °C
held for 1 min, heating at a rate of 5 °C/min up to 130 °C, heating at a rate
of 10 °C/min up to 210 °C held for 5 min. The mass spectrometer was set for 35
to 600 m/z scanning, and 2 µL of the essential oil solution diluted in n-hexane
were injected, in triplicate. The compounds were identified by comparison of
the mass spectrum using the NIST11 and NIST11 databases.
The extracts obtained by maceration
(at 0, 6, 24, and 72 h) and ultrasound at 25 and 40 °C (at 0, 5, 15, and 30
min) were used for the characterization of the physicochemical profile by high
performance liquid chromatography (HPLC). For that, 300 µL of the extract was
diluted in 1 mL of distilled water. This dilution was necessary to adapt the
analysis within the HPLC operating parameters. The samples were
filtered through a 0.22 µm PTFE syringe filter. The analysis was conducted on a
liquid chromatograph coupled to a UFLC diode array detector (Shimadzu®). A
volume of 20 µL was injected, keeping the NST C18 column (250x4.6 mm, 5 µm) at
40 °C, mobile phase (phase A: 99.9 % water, 0.1 % methaneic acid; phase B: 99.9
% methanol, 0.1 % methaneic acid) with a flow rate of 1.2 mL/min, and an
average run time of 30 min.
The identification of the compounds (clove essential oil and
extracts) was performed using a calibration
curve with standard solutions of phenols ((+) catechin, (-) epicatechin,
caffeic acid, vanillic acid, p-coumaric acid, trans iso-ferric acid, (-)
resveratrol, myricetin, gallic acid, and quercitin). The quantification was
performed by simple area normalization. The analyses were performed in
triplicate at the Federal
University of Southern Frontier. The sample was
characterized for the moisture content, in triplicate, as described by Adolfo
Lutz Institute [8]. The extraction yield of
the essential oil was calculated by the ratio between the volume (mL) of
essential oil and the mass (g) of raw material used [9].
The essential oil and the extracts
obtained by maceration (0, 6, 24, and 72 h), and ultrasound at 25 and 40 °C (0,
5, 15, and 30 min) were used to determine the antimicrobial activity. E.
coli (ATCC 35218), L. monocytogenes (ATCC 7644), S. enteritidis
(ATCC 13076), and S. aureus (ATCC 25923) strains were used, from the
Strain Bank of the Microbiology Laboratory of the Midwestern State University,
which were cultured in tubes with 10 ml of Soy Trypticase Broth (TSB) for 24 h
at 37 °C.
Bacterial suspensions were adjusted
and compared to the 0.5 Mc Farland scale, and seeded in Petri plates containing
15 mL of Soy Trypticase Agar (TSA). After complete absorption of the inoculum
by the culture medium, 7 mm holes were drilled, and 20 µL of the filtered
extract was added. Sterile distilled water (20 µL) was used as a negative
control, and the gentamicin disk was used as a positive control. The plates
were kept at 4 °C for 3 h to allow diffusion of the extracts into the culture
medium before microbial growth and then incubated for 24 h at 37 °C.
The antimicrobial activity was
verified by the formation of inhibition halos (mm) around the holes. The assays
were performed in triplicate, and the results were presented by the arithmetic
mean of the inhibition halos [10].
The total phenolic content was
determined as described: for the extracts obtained by hydrodistillation,
maceration, and ultrasound-assisted extraction, an aqueous solution at 6 mg/mL
of the dried clove extract (oven with forced and renewal air at 60 °C until constant mass)
was prepared. For the essential oil, an ethanolic solution of the same
concentration was prepared. In 10 mL volumetric flasks, 100 μL of each aqueous
solution of the extracts was mixed with 2 mL of distilled water, 0.5 mL of
Folin-Ciocalteau's reagent, and 1.5 mL of 20 % (w/v) sodium carbonate solution.
The flasks were made up to 10 mL with distilled water, and stored in the dark
at 25 °C for 2 h. The absorbance of the samples was measured at 765 nm in a
spectrophotometer (Bel Photonics®, 2000 UV) using distilled water as a blank [11].
The results were analyzed using a
standard curve of gallic acid, using 100 μL of each dilution corresponding to
the concentrations of 0, 20, 30, 40, 50, 70, 90, 100, 200, 400, 600, 800, and
1000 µg/mL. The total phenolic concentration was calculated as mg gallic acid
equivalent (GAE) per g of clove and expressed as mean ± standard deviation.
The essential oil and the extracts
obtained by maceration (0, 6, 24, and 72 h), ultrasound at 25 and 40 °C (0, 5,
15, and 30 min) were used for the determination of antioxidant activity by the
DPPH (2,2-Diphenyl-1-picryl-hydrazyl) assay, in
triplicate. For the calibration curve, a Trolox 800 µmol/L solution was used and to
proceed with the analyses, a solution of
DPPH radical was prepared. The reading was carried out on a
spectrophotometer at 520 nm [12]. The
percentage of DPPH inhibition was calculated by converting to percentage of
antioxidant activity (AA %) (Eq 1).
where abssample is the absorbance of the sample for a given dilution, and absblank is the absorbance of the blank of the respective sample.
The results of chemical composition, antimicrobial and antioxidant analysis were analyzed using the software BioEstat 5.3®, with a confidence level of 95 %.
3. Results and discussion
The compounds identified by the
chromatographic analysis (Table 1) are commonly found in essential oils [13]. The amount and composition of essential oils
can be affected by several factors such as soil, harvest time, and methods of
harvesting, water stress, among others. Eugenol was the most abundant compound,
with a relative percentage area of 86.16 %, followed by caryophyllene (7.14 %)
and eugenyl acetate (4.84 %), which are considered typical compounds of clove
essential oil [14]. These three components are the main compounds present in
clove essential oil was reported by Haro-González et al., [1]. Eugenol possesses antibacterial activity,
mainly on E. coli, L. monocytogenes, and S. aureus [15, 16], which was verified in the antibacterial
activity analyses. It also has proven antioxidant activity, which reinforces
the potential of the biological activity of clove oil [17].
Eugenol, caryophyllene and eugenyl acetate are volatile compounds known
for their application in the pharmaceutical and chemical products,
insecticides, anti-inflammatory, wound healing, anti cancer
and application in food industries as baked foods, dairy products, processing
foods and packaging material [1].
Table 1. Chemical compounds of clove essential oil obtained by hydrodistillation
No. |
Compound |
Retention time (min) |
Relative percent area |
1 |
2-Heptanol
acetate |
9.80 |
0.27 (±
0.01) |
2 |
Beta-cis-Ozmene |
10.01 |
0.09 (± 0.00) |
3 |
2-Nonanone |
11.23 |
0.12 (±
0.00) |
4 |
Methyl
salicylate |
14.20 |
0.28 (± 0.03) |
5 |
Eugenol |
18.74 |
86.16 (±
0.18) |
6 |
Caryophyllene |
19.97 |
7.14 (± 0.05) |
7 |
Alpha-Humulene |
20.57 |
0.92 (±
0.01) |
8 |
Eugenyl
acetate |
21.59 |
4.84 (± 0.10) |
9 |
Caryophyllene
oxide |
22.60 |
0.18 (±
0.01) |
Compounds of the groups of gallic
acid, flavones, and their isomers were detected in the chromatographic analyses
(Table 2) of the extracts obtained by maceration and ultrasound, which
corroborates the literature data [18]. The
compounds gallic acid, theobromine, and myricetin were identified in the
chromatographic analysis. As shown in Table 3, a decrease in the concentration
of gallic acid was observed with increasing the extraction time, for all
extraction techniques. The higher concentrations of this compound were found in
the samples U(40 °C)5', U(40 °C)15', and U(40 °C)30', and the highest
concentration was observed in U(40 °C)5', corresponding to 798.58 µg/mL.
Table 2. Chemical compounds of clove extracts
No. |
Compound |
Retention time (min) |
[M-H]+
m/z |
[M-H]-
m/z |
Class |
1 |
Gallic Acid |
4.11 |
- |
169 |
Phenolic
Acid |
2 |
Isobiflorin |
8.9 |
- |
353 |
Isoflavone |
3 |
Biflorin |
10.02 |
- |
353 |
Flavone |
4 |
Calicosine-glycoside |
22.0 |
447 |
- |
Flavone |
5 |
Pratensein/Irilin B/Methylorobol-glycoside |
23.63 |
463 |
- |
Isoflavone |
6 |
Biochanin
A-7-O-glycoside |
25.27 |
445 |
- |
Isoflavone |
Table 3. Chemical compounds of clove extracts
Sample |
Mean
concentration (µg/mL) |
||
Gallic acid |
Theobromine |
Myricetin |
|
M6h |
651.80d (±
6.31) |
0.003a
(± 0.000) |
0.031d (± 0.001) |
M24h |
593.13e
(± 5.91) |
0.004a (± 0.001) |
0.037abc (±
0.001) |
M72h |
413.53f (±
8.83) |
0.004a
(± 0.000) |
0.031d (± 0.001) |
U(25 °C)5’ |
754.65b
(± 9.55) |
0.006a (± 0.001) |
0.033bcd (±
0.002) |
U(25 °C)15’ |
752.64b (±
3.37) |
0.004a
(±
0.001) |
0.039ab (± 0.001) |
U(25 °C)30’ |
654.26d
(± 3.48) |
0.005a (± 0.000) |
0.032cd (±
0.003) |
U(40 °C)5’ |
798.58a (±
4.48) |
0.004a
(± 0.000) |
0.040a (± 0.001) |
U(40 °C)15’ |
755.55b
(± 1.10) |
0.005a (± 0.000) |
0.036abcd (±
0.002) |
U(40 °C)30’ |
712.71c (±
2.42) |
0.004a
(± 0.000) |
0.033bcd (± 0.001) |
M: maceration; xh: extraction time in
hours; E.g.: M0h: maceration at time zero. U: Ultrasound; (x °C): extraction
temperature; x': extraction time in minutes; E.g.: U(25 °C)0': ultrasound at 25 °C at time zero. Values
with the same letter in the same column are not significantly different
(p<0.05). |
Among the antioxidant compounds,
polyphenols act in the sequestration of free radicals or as chelating agents of
metals, and gallic acid (3,4,5-trihydroxybenzoic acid) is an example of these
compounds. Scientific reports from the pharmaceutical and food industries have
shown that gallic acid has antioxidant, antiviral, antibacterial, and
antifungal properties [19].
No significant differences were
observed for the compound theobromine among the samples, for all extraction
methods, with relatively low values when compared to the other compounds.
Myricetin was found in small amounts in all samples
(3,5,7,3′,4′,5′-hexahydroxyflavone). It is a phenolic compound that presents
antioxidant activity, pro-oxidant, phytoestrogenic effects and anticarcinogenic
properties among others [20].
The moisture content of the clove
sample was 14.81 ± 0.47 % which meets the identity and quality standards
established by the Brazilian legislation [21], which
establishes the value of 16 % as the maximum moisture allowed for all clove
species.
The extraction yield (Table 4) for
the essential oil was 1.05 % (on a dry basis).
This value can oscillate depending on several factors, including the extraction
method, extraction time, and experimental errors. Water stress in plants can
impact their metabolism and cause alterations in their physiological processes,
such as reduced photosynthesis and transpiration, which directly interfere with
the production and composition of essential oils [22],
which may explain the high moisture content and low yield observed in
this study.
Table
4.
Extraction yield of clove extracts obtained by maceration and
ultrasound-assisted extraction at 25 and 40 °C
Sample |
Yield
(%) |
Sample |
Yield
(%) |
Sample |
Yield
(%) |
Essential
oil |
1.05 |
U(25 ºC)0’ |
1.45 |
U(40 ºC)0’ |
1.45 |
M0h |
1.45 |
U(25 ºC)5’ |
3.28 |
U(40 ºC)5’ |
3.35 |
M6h |
6.64 |
U(25 ºC)10’ |
4.19 |
U(40 ºC)10’ |
4.10 |
M12h |
6.88 |
U(25 ºC)15’ |
5.97 |
U(40 ºC)15’ |
6.01 |
M24h |
7.26 |
U(25 ºC)20’ |
7.33 |
U(40 ºC)20’ |
7.26 |
M48h |
7.30 |
U(25 ºC)30’ |
7.50 |
U(40 ºC)30’ |
7.74 |
M72h |
7.32 |
||||
Note: M:
maceration; xh: extraction time in hours; E.g.: M0h: maceration at time zero.
U: Ultrasound; (x °C): extraction temperature;
x': extraction time in minutes; E.g.: U(25 °C)0':
ultrasound at 25 °C at time zero |
The extracts obtained by ultrasound and maceration showed an increase in yield with increasing extraction time. Regarding the maceration procedure, the extraction yields varied between 1.45 and 7.32 % at 0 and 72 h of extraction, respectively. For the ultrasound-assisted extraction at 25 °C, the variation was 1.45 to 7.50 % at 0 and 30 min, respectively. In the same time interval, a variation of 1.45 to 7.74 % was observed for the ultrasound-assisted extraction at 40 °C. These results are higher than that observed for the essential oil, which demonstrates an advantage of the US extraction method once it does not expose the sample to high temperatures, with no degradation of compounds of interest, besides requiring less processing time and presenting a higher yield of the final product.
The results of the antimicrobial
analysis (Table 5) showed that the extracts obtained by maceration at 0 and 6
h, and by ultrasound-assisted extraction (25 °C and 40 °C) did not present
inhibition halos of bacterial growth at the concentration of 100 µL/mL, indicating
no detectable antimicrobial activity. The essential oils U (25 °C) 15', U(25
°C)30', U(40 °C)15', and U(40°C)30' presented the highest inhibition halos for S.
enteritidis, between 2.40 and 2.92 mm, with no significant difference among
them (p>0.05). Although Chesca et al., [23] reported
values of 14 and 15 mm, those authors used solvents containing toxic compounds,
which is not indicated for food production.
Table 5. Antimicrobial activities of clove extracts at 100 µL/mL and 50 µL/mL
Sample (100
µL/mL) |
Inhibition halo (mm) |
|||
S. enteritidis |
S. aureus |
E. coli |
L. monocytogenes |
|
Essential
oil |
2.40ab (± 0.14) |
1.85cd (± 0.07) |
2.10ab (± 0.14) |
1.75b (± 0.07) |
M24h |
0.25d
(± 0.00) |
- |
0.25c
(± 0.00) |
- |
M72h |
1.20c (± 0.14) |
1.00e (± 0.14) |
1.50b (± 0.14) |
1.10c (± 0.00) |
U(25
°C)15’ |
1.95bc
(± 0.21) |
1.52d
(± 0.03) |
1.78b
(± 0.18) |
- |
U(25
°C)30’ |
2.40ab (± 0.14) |
2.05bc (± 0.07) |
1.80b (± 0.14) |
1.25c (± 0.07) |
U(40
°C)15’ |
2.50ab
(± 0.14) |
2.28ab
(± 0.04) |
2.60a
(± 0.07) |
1.88ab
(± 0.04) |
U(40
°C)30’ |
2.92a (± 0.11) |
2.55a (± 0.21) |
2.67a (± 0.03) |
2.15a (± 0.21) |
Sample
(50 µL/mL) |
||||
Essential
oil |
1.25a (± 0.07) |
1.10a (± 0.09) |
1.45a (± 0.07) |
1.02a (± 0.01) |
M72h |
0.28c
(± 0.01) |
- |
0.30b
(± 0.00) |
- |
U(25
°C)30’ |
0.57b (± 0.01) |
0.52b (± 0.01) |
0.42b (± 0.01) |
0.10b (± 0.00) |
U(40
°C)30’ |
0.68b
(± 0.01) |
0.42b
(± 0.01) |
0.28b
(± 0.01) |
0.22b (± 0.01) |
M: maceration; xh: extraction time in
hours; E.g.: M0h: maceration at time zero. U: Ultrasound; (x °C): extraction
temperature; x': extraction time in minutes; E.g.: U(25 °C)0': ultrasound at 25
°C at
time zero. Values with the same letter in the same column are not
significantly different (p<0.05). |
For S. aureus, only the
treatment M24h showed no inhibition halo. The smallest halo was observed for
M72h (p<0.05) while the other samples presented halos ranging between 1.52
and 2.55 mm. Guimarães et al., [24] reported
the formation of an inhibition halo of 19 mm with the application of essential
oil, while the extract exhibited no antimicrobial activity. Xu et al., [25] also verified the inhibitory activity of
essential oil at concentrations of 25 % and 50 % on S. aureus, with
inhibition halos of 16.5 and 20.4 mm, respectively, much higher than the
concentration used in this study, which suggests that higher concentrations of
these extracts can lead to greater inhibition of microbial growth. The
essential oils U(40 °C)15' and U(40 °C)30' showed the highest inhibition halos
(p<0.05) for E. coli, between 2.10 and 2.67 mm. These values are low
when compared to those found by Guimarães et al., [24],
who reported a halo of 11 mm after the application of the extract;
however, the authors reported no growth inhibition with the application of
essential oil. For L. monocytogenes, the essential oils U(40 °C)15' and
U(40 °C)30' presented the highest inhibition halos (p<0.05), between 1.75
and 2.15 mm. Silveira [26] reported that
essential oil alone did not lead to halo formation for L. monocytogenes,
but an inhibition halo of 11.8 mm was observed when a polysaccharide film was
used, which highlights its potential in food preservation systems. As reported
by Petropoulos et al., [22], the
physiological processes of plants can be impacted by water stress, which
directly interferes with the composition of essential oils and extracts, and
can affect the antimicrobial capacity of essential oils and plant extracts. An
antimicrobial analysis with an extract concentration of 50 µL/mL was performed
with the extracts that presented antimicrobial activity at 100 µL/mL, and the
essential oils showed higher antimicrobial activity for all microorganisms. It
is known that eugenol, the most abundant compound in essential oils, has high
antibacterial activity, especially on E. coli, L. monocytogenes, S.
typhimurium, and S. aureus [15,16], which
indicates that the oil acts as an effective bacterial growth inhibition agent
even at lower concentrations. Therefore, essential oils are the most
recommended alternative for using low bioadditive concentrations, due to their
high eugenol levels.
The total phenolic content and DPPH antioxidant activity (% AA)
showed antioxidant activity of the essential oils and clove extracts (Table 6).
The total phenolic contents ranging from 0.101 to 0.290 mg GAE/g are relatively
low when compared to other essential oils and plant extracts, such as the
orange pomace (Citrus sinensis) extract evaluated by Benelli et al., [27], who reported total phenolics from 9 to 36 mg
GAE/g. The highest total phenolic contents were found in the
ultrasound-assisted extracts, which suggests the effectiveness of this
extraction method.
Table 6. Antioxidant activity of clove extracts obtained by different extraction methods
Sample |
Total phenolics (mg GAE/g) |
AA (%) (mg tocopherol/mL) |
Óleo |
0.226e (± 0.000) |
61.93b (± 0.001) |
M0h |
0.126l
(± 0.001) |
8.15j
(± 0.002) |
M6h |
0.101p (±0.000) |
15.65h (± 0.003) |
M12h |
0.119o
(±0.001) |
* |
M24h |
0.126l (±0.001) |
19.95g (± 0.002) |
M48h |
0.158j
(±0.001) |
* |
M72h |
0.130k (±0.001) |
30.87e (± 0.004) |
U(25
°C)0’ |
0.119no
(±0.000) |
9.47i
(± 0.011) |
U(25
°C)5’ |
0.203f (±0.001) |
20.80f (± 0.002) |
U(25
°C)10’ |
0.188h
(±0.001) |
* |
U(25
°C)15’ |
0.196g (±0.001) |
32.27e (± 0.004) |
U(25
°C)20’ |
0.264b
(±0.001) |
* |
U(25
°C)30’ |
0.290a (±0.001) |
58.43c (± 0.006) |
U(40
°C)0’ |
0.122mn
(±0.001) |
9.43i
(± 0.005) |
U(40
°C)5’ |
0.118o (± 0.002) |
21.17f (± 0.012) |
U(40
°C)10’ |
0.124lm
(±0.0011) |
* |
U(40
°C)15’ |
0.162i (±0.001) |
40.96d (± 0.005) |
U(40
°C)20’ |
0.234d
(±0.000) |
* |
U(40
°C)30’ |
0.248c (±0.001) |
65.24a (± 0.004) |
* sample volume insufficient for analysis. Values with the same letter in
the same column are not significantly different (p<0.05)
The antioxidant activity observed for the clove essential oils and extracts ranged between 8.15 and 65.24 %, which were similar but lower when compared to the values reported by El-Maati et al., [28] in aqueous and ethanolic extracts.
The highest antioxidant activities were observed for the essential oil and extracts obtained by ultrasound-assisted extraction, with a significant difference between the treatments. It was observed that there is a tendency for the content of total phenolic compounds to be higher in essential oil samples and in extracts obtained by ultrasound-assisted, and this tendency was observed in the largest inhibition halo for the microorganisms tested. It can be suggested that the antioxidant capacity of the extracts is related to the presence of gallic acid (Table 3), which was present in great quantities in the samples obtained using ultrasound-assisted extraction.
4. Conclusions
The extraction yields of the present
study showed the advantages of ultrasound-assisted extraction, once the sample
is not subjected to high temperatures, avoiding degradation of compounds of
interest, besides using less processing time, with a higher yield of the final
product.
For the concentration of 100 µL/mL,
the essential oils and the ultrasound-assisted extracts obtained at 25 and 40
°C showed satisfactory inhibition halos for all microorganisms studied.
Concerning the assay using the concentration of 50 µL/mL, the essential oil
presented the highest antimicrobial activity. The chemical profile analysis of
the clove extracts demonstrated the presence of compounds from the classes of
gallic acid, flavones, and their isomers. Higher DPPH antioxidant activities (%
AA) are observed for the essential oil and the extracts obtained by
ultrasound-assisted extraction, with a significant difference when compared to
the other treatments.
The results of this study have proven the biological activity of the essential oil and the extracts obtained by maceration and ultrasound-assisted extraction. The ultrasound-assisted extraction method stood out as the most recommended for the food industry due to numerous commercial advantages, including shorter processing time and lower production cost.
Abbreviations
ATCC- American Type Culture
Collection
E. coli - Escherichia coli
L. monocytogenes
- Listeria monocytogenes
S. aureus -
Staphylococcus aureus
S. enteritidis -
Salmonella enteritidis
S. typhimurium - Salmonella typhimurium
Authors’
contributions
Conceptualization, formal analysis, visualization, data analysis, manuscript draft writing, writing-review & editing, B.S.G.; Conceptualization, data curation, funding acquisition, project administration, resources, supervision, roles/writing- original draft, writing-review & editing, R.L.K.; Conceptualization, project administration, resources, supervision, roles/writing-original draft, writing-review & editing, L.B.Q.
Acknowledgements
Federal University of Southern Frontier (UFFS).
Funding
Federal University of Southern Frontier (UFFS)
Availability of data and materials
All
data will be made available on request according to the journal policy.
Conflicts of interest
The authors declare no
conflict of interest in this study.
References
1.
Haro-González
J.N.; Castillo-Herrera G.A.; Martínez-Velázquez M.; Espinosa-Andrews H. Clove
essential oil (Syzygium aromaticum L.
Myrtaceae): Extraction, chemical composition, food applications, and essential
bioactivity for human health. Molécules. 2021, 26, 2-25. https://doi: 10.3390/molecules26216387.
2.
Pimenta Neto A.A.; Gonçalves G.D.; Benjamin C.S.; Costa L.C.B.; Oliveira
R.S.; Oliveira S.M.A.; Luz E.D.M.N. Bioatividade
de óleos essenciais e extratos vegetais no controle de doenças causadas por
Phytophthora nicotianae em solanáceas. Summa Phytopathol. 2020, 46(3), 267–272.
https://doi.org/10.1590/0100-5405/215960.
3.
Shuai X.; Dai T.;
Chen M.; Liang R.; Du L.; Chen J.; Liu C. Comparative study on the extraction
of macadamia (Macadamia integrifolia) oil using different processing methods.
LWT - Food Sci. Tech. 2021, 154, 112614. https://doi.org/10.1016/j.lwt.2021.112614.
4.
Aramrueang, N.;
Asavasanti, S.; Khanunthong, A. Leafy Vegetables. Integrated Processing Technologies
for Food and Agricultural By-Products, 1 ed.; Acadmic Press: Cambridge, 2019.
5.
Silva R.S.;
Barbieri H.B.; Ferreira H.S.; Silva C.A.; Nebo L. Otimização da extração
assistida por ultrassom de compostos bioativos da espécie Caryocar brasiliense.
Res. Soc. Dev. 2021, 10(9),
1-13. https://doi.org/10.33448/rsd-v10i9.16493.
6.
Tunç M.T.; Koca
I. Ohmic heating assisted hydrodistillation of clove essential oil. Ind. Crops
Prod.. 2019, 141, 111763.
https://doi.org/10.1016/j.indcrop.2019.111763.
7.
Bhavaniramya S.;
Vishnupriya S.; Al-Aboody M.S.;
Vijayakumar R.; Baskaran D. Role of
essential oils in food safety: Antimicrobial and antioxidant applications,
Grain & Oil Sci. Tech. 2019, 2, 49–55. https://doi.org/10.1016/j.gaost.2019.03.001.
8.
IAL- Instituto Adolfo Lutz Métodos
físico-químicos para análise de alimentos, 4 ed, 2008.
9.
Chaves, F.C.M.;
Costa, J.S. Teor e rendimento de extrato
das folhas de três morfotipos de Arrabidaea chica (Bonpl.) B. Verl. em fução de
adubação orgânica em Manaus. In II Congresso Brasileiro de Recursos Genéticos,
Belém, Brasil, 24-28 September, 2012.
10.
Ostrossky A.;
Mizumoto M.K.; Lima M.E.L.; Kaneko T.M.; Nishikawa S.O.; Freitas B.R. Métodos
para avaliação da atividade antimicrobiana e determinação da concentração
mínima inibitória (CMI) de plantas medicinais. Braz. J. Pharmacognosy, 2008,
18(2), 301–307. https://doi.org/10.1590/S0102-695X2008000200026.
11.
Singleton V.L.;
Rossi J.A. Colorimetry of total phenolic with phosphomolybdic-phosphotungstic
acid reagentes. Am. J. Eno. Viticulture. 1965, 16, 144–158.
12. Molyneux, P. The use of the
stable free radical diphenylpicrylhydrazyl (DPPH) for estimating antioxidant
activity. Songklanakarin J. Sci. Technol. 2004, 26(2), 211–219.
13.
Amelia, B.;
Saepudin, E.; Cahyana, A.H.; Rahayu D.U.; Sulistyoningrum A.S.; Haib J. GC-MS analysis of clove (Syzygium aromaticum) bud essential oil from Java and Manado. In
Proceedings of the 2nd International Symposium on Current Progress
in Mathematics and Sciences, Depok, Jawa Barat, Indonesia, 1-2 November 2017.
14.
Oliveria, R.A.;
Reis, T.V.; Sacramento, C.K.; Duarte, L.P.; Oliveira, F.F.Constituintes
químicos voláteis de especiarias ricas em eugeno. Braz. J. Pharmacognosy, 2009,
19(3), 771–775. https://doi.org/10.1590/S0102-695X2009000500020.
15.
Sebaaly C.;
Haydar S.; Greige-Gerges H. Eugenol encapsulation into conventional liposomes
and chitosan-coated liposomes: A comparative study. J. Drug Delivery Sci. Tech.
2021, 67, 102942. https://doi.org/10.1016/j.jddst.2021.102942.
16.
Santana, M.S.;
Machado, E.C.L.; Stamford, T.C.M.; Stamford, T.L.M. Avanços em Ciência e Tecnologia de Alimentos,
1st ed.; Científica: Guarujá, Br, 2021.
17.
Pavithra, B. Eugenol - A review. J. Pharmatceutical Sci.
Res. 2014, 6(3), 153–154.
18.
Saviranta N.M.M.;
Julkunen-Tiitto R.; Oksanen E.; Karjalainen R.O. Leaf phenolic compounds in red clover (Trifolium pratense L.) induced by
exposure to moderately elevated ozone. Env. Pollution. 2010, 158(2), 440–446. https://doi.org/10.1016/j.envpol.2009.08.029.
19.
Nobre D.A.C.;
Macedo W.R.; Silva G.H.; Lopes L.S.; Jaimes E.H.L. Aplicación y efecto antioxidante del ácido
gálico sobre la calidad de semillas de trigo. Rev. Ciências Agra. 2019, 42,
22–29. https://doi.org/10.19084/RCA18184.
20.
Fu M.; Shen W.;
Gao W.; Namujia L.; Yang X.; Cao J.; Sun L.
Essential moieties of myricetins, quercetins and catechins for binding
and inhibitory activity against α-Glucosidase. Bioorg. Chem. 2021, 115, 105235.
https://doi.org/10.1016/j.bioorg.2021.105235.
21.
DIPOV -
Departamento de Inspeção de Produtos de Origem Vegetal, Anexo da Norma Interna
DIPOV 02/2019. Brasília: Ministério da
Saúde, 2019.
22.
Petropoulos S.A.;
Daferera D.; Polissiou M.G.; Passam, H.C.
The effect of water déficit stress on the growth, yield and composition
of essential oils of parsley. Scientia Hort. 2008, 115, 393–397. https://doi.org/10.1016/j.scienta.2007.10.008.
23.
Chesca, A.C.;
Tristão, D.S.; Tristão, M.S.; Almeida, R.N.; Begnini, M.L. Estudo comparativo da atividade antibateriana
do extrato de cravo-da-índia (eugenia caryophyllata thumb.) extraído por via
etanólica e metanólica. In: I Encontro de processos agroindustriais, Uberaba,
Brazil, 06 December, 2017.
24.
Guimarães, C.C.;
Ferreira, T.C.; Oliveira, R.C.F.; Simioni, P.U.; Ugrinovich, L.A. Atividade
antimicrobiana in vitro do extrato aquoso e do óleo essencial do alecrim
(Rosmarinus officinalis L.) e do cravo-da-índia (Caryophyllus aromaticus L.) frente a cepas de Staphylococcus aureus
e Escherichia coli. Rev. Bras. Bioc. 2017, 15(2), 83–89.
25. Xu
J.G.; Liu T.; Hu Q.P.; Ca, X.M. Chemical composition, antibacterial properties
and mechanism of action of essential oil from clove buds against Staphylococcus aureus. Molecules, 2016,
21(9), 1194. https://doi: 10.3390/molecules21091194.
26.
Silveira, Y.D.O.
Caracterização de filme polissacarídico obtido de casca de mandioca (manihot
esculenta) fincionalizado com óleo essencial de cravo-da-índia (Syzygium aromaticum). Thesis,
Universidade Federal de Minas Gerais, Minas Gerais, Brasil, 2018.
27.
Benelli P.; Riehl
C.A.S.; Smânia Jr A.; Smânia E.F.A.; Ferreira S.R.S. Biactive extracts of orange (Citrus sinensis L. Osbeck) pomace
obtained by SFE and low pressure techniques: Mathematical modeling and extract
composition. J. Superc. Fluids. 2010, 55, 132–141.
28.
El-Maati M.F.;
Mahgoub S.A.; Labib S.M.; Al-Gaby A.M.A.; Ramadan M.F. Phenolic extracts of
clove (Syzygium aromaticum) with
novel antioxidant and antibacterial activities. Eur. J. Intr. Med. 2016, 2,
494–504. https://doi.org/10.1016/j.eujim.2016.02.006.
This work is licensed under the
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License (CC BY-NC 4.0).
Abstract
This study aimed to evaluate the biological activity of clove (Syzygium
aromaticum) essential oil and clove extracts, obtained by
hydrodistillation, maceration, and ultrasound-assisted extraction. E. coli,
L. monocytogenes, S. enteritidis, and S. aureus strains were used to determine
the biological activity by the formation of inhibition zones. Eugenol (86.16
%), caryophyllene (7.14 %) and eugenyl acetate (4.84 %) were presented in clove
essential oil. The ultrasound-assisted essential oils and extracts exhibited
biological activity at the concentration of 100 µL/mL, with the highest
inhibition halos observed for all strains studied. The essential oil presented
greater inhibition for the concentration of 50 µL/mL. The chemical profile
analysis demonstrated the presence of groups of gallic acid compounds,
flavones, and their isomers. The total phenolic contents ranged from 0.101 and
0.290 mg GAE/g, with the highest values for the ultrasound-assisted extracts.
The results of DPPH varied from 8.15 to 65.24 %, with an emphasis on
ultrasound-assisted essential oil and extracts.
Abstract Keywords
Essential oil, bio-additive, antioxidant, antimicrobial, phenolic,
extract.
This work is licensed under the
Creative Commons Attribution
4.0
License (CC BY-NC 4.0).
Editor-in-Chief
Prof. Dr. Radosław Kowalski
This work is licensed under the
Creative Commons Attribution 4.0
License.(CC BY-NC 4.0).