Biotechnological Potential of Eugenol and Thymol Derivatives Against Staphylococcus aureus from Bovine Mastitis

Article abstract of DOI:10.1007/s00284-021-02344-9

Bovine mastitis is an infectious disease that affects the mammary gland of dairy cattle with considerable economic losses. Staphylococcus aureus is the main microorganism involved in this highly contagious process, and the treatment is only using antibiotics. Currently, the search for new treatment and/or compounds is still in need due to microbial resistance. In this work, we evaluated the potential of eugenol and thymol derivatives against S. aureus strains from bovine mastitis. On that purpose, nine derivatives were synthesized from eugenol and thymol (1–9), and tested against 15 strains of S. aureus from subclinical bovine mastitis. Initially, the strains were evaluated for the biofilm production profile, and those with strong adherence were selected to the antimicrobial sensitivity determination in the Minimum Inhibitory Concentration (MIC) assays. Herein the compounds toxicity was also evaluated by in silico analysis using Osiris DataWarrior software. The results showed that 60% of the strains were considered strongly adherent and three strains (S. aureus 4271, 4745 and 4746) were selected for the MIC tests. Among the nine eugenol and thymol derivatives tested, four were active against the evaluated strains (MIC = 32?μg?mL^?1) within CLSI standard values. In silico analysis showed that all derivatives had cLopP < 5, cLogS > ??4 and TPSA < 140 ?2, and similar theoretical toxicity parameters to some antibiotics currently on the market. These molecules also showed negative drug-likeness values, pointing to the originality of these structures and theoretical feasibility on escaping of resistance mechanism and act against resistant strains. Thus, these eugenol derivatives may be considered as promising for the development of new treatments against bovine mastitis and future exploring on this purpose.

Full text of DOI:10.1007/s00284-021-02344-9

Current Microbiology (2021) 78:1846–1855
https://doi.org/10.1007/s00284-021-02344-9
Biotechnological Potential of Eugenol and Thymol Derivatives Against
Staphylococcus aureus from Bovine Mastitis
Daiana O. S. Nunes1,4 · Rafaelle Vinturelle1,4 · Francislene J. Martins1,5 · Thiago F. dos Santos3 ·
Alessandra Leda Valverde2,3 · Carlos Magno R. Ribeiro3 · Helena C. Castro1,5 · Evelize Folly1,4,6
Received: 15 May 2020 / Accepted: 11 December 2020 / Published online: 16 April 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Bovine mastitis is an infectious disease that afects the mammary gland of dairy cattle with considerable economic losses.
Staphylococcus aureus is the main microorganism involved in this highly contagious process, and the treatment is only
using antibiotics. Currently, the search for new treatment and/or compounds is still in need due to microbial resistance. In
this work, we evaluated the potential of eugenol and thymol derivatives against S. aureus strains from bovine mastitis. On
that purpose, nine derivatives were synthesized from eugenol and thymol (1–9), and tested against 15 strains of S. aureus
from subclinical bovine mastitis. Initially, the strains were evaluated for the bioflm production profle, and those with strong
adherence were selected to the antimicrobial sensitivity determination in the Minimum Inhibitory Concentration (MIC)
assays. Herein the compounds toxicity was also evaluated by in silico analysis using Osiris DataWarrior® software. The
results showed that 60% of the strains were considered strongly adherent and three strains (S. aureus 4271, 4745 and 4746)
were selected for the MIC tests. Among the nine eugenol and thymol derivatives tested, four were active against the evalu-
ated strains (MIC=32 µg mL−1) within CLSI standard values. In silico analysis showed that all derivatives had cLopP<5,
cLogS>− 4 and TPSA<140 Å2, and similar theoretical toxicity parameters to some antibiotics currently on the market.
These molecules also showed negative drug-likeness values, pointing to the originality of these structures and theoretical
feasibility on escaping of resistance mechanism and act against resistant strains. Thus, these eugenol derivatives may be
considered as promising for the development of new treatments against bovine mastitis and future exploring on this purpose.
Introduction
caused by the colonization of the secretory tissue of the
udder by several types of pathogenic agents [1]. It is char-
acterized as an infammatory process of the mammary gland
that decreases the milk production and alters its composi-
tion [2]. It can be classifed as clinical mastitis, in which the
Bovine mastitis is an infection that presents high economic
importance, since it is present in most of productive herds
worldwide. It is a disease with multifactorial aspects and
4
* Evelize Folly
Laboratório de Estudos em Pragas e Parasitos (LEPP), UFF,
evelizefolly@yahoo.com.br
IB, Department of Cellularand Molecular Biology, Instituto
de Biologia, Niterói, RJ, Brazil
Helena C. Castro
5
hcastrorangel2@gmail.com
Laboratório de Antibióticos, Bioquímica, Ensino e
Modelagem Molecular (LABIEMOL), Universidade Federal
Fluminense, Outeiro de São João Batista s/no, Centro,
Niterói, RJ 24020-141, Brazil
1
Programa de Pós-Graduação em Ciências e Biotecnologia,
Instituto de Biologia, Universidade Federal Fluminense,
Outeiro de São João Batista s/no, Centro, Niterói,
RJ 24020-141, Brazil
6
Instituto Nacional de Ciências e Tecnologia—Entomologia
Molecular (INCT-EM), Rio de Janeiro, Brazil
2
Programa de Pós-Graduação em Química, Instituto de
Química, Universidade Federal Fluminense, Outeiro de São
João Batista s/no, Centro, Niterói, RJ 24020-141, Brazil
3
Instituto de Química, Departamento de Química Orgânica,
Universidade Federal Fluminense, Outeiro de São João
Batista s/no, Centro, Niterói, RJ 24020-141, Brazil
Vol:.(1234567890)
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Biotechnological Potential of Eugenol and Thymol Derivatives Against Staphylococcus aureus…
1847
symptoms are visible to the naked or subclinical eye, but
auxiliary tests are necessary for full diagnosis [3].
Materials and Methods
Compounds
Several microorganisms have been associated with mas-
titis by colonizing the mammary gland canal of the infected
animal [4, 5]. In terms of economic losses, it is the most
common and devastating disease that afects dairy cattle as
it reduces not only quality but also quantity of milk produced
worldwide. The control and prevention of this infection are
a challenge, despite the continuous eforts from the part of
ranchers, due especially to resistant strains [6–8].
General
All solvents and reagents used were supplied by E. Merck
or Aldrich Co. TLC: silica gel 60 F254 plates (0.25 mm;
Merck). Column chromatography (CC): silica gel 60
(0.040–0.063 mm; Merck). Melting points were deter-
mined on a Fischer-Johns apparatus: uncorrected. 1H-NMR
(500 MHz) and 13C-NMR (125 MHz) spectra: Varian 7 T
instrument in CDCl3 (Me4Si as internal standard); chemi-
cal shifts are in ppm and coupling constants are given in
Hz. IR spectra were recorded on flms or KBr pellets with a
Perkin Elmer 1420 spectrometer. The HRMS/MS analysis
was done with mass spectrometer operating in positive ion
modes (m/z 100–1000). The capillary voltage applied was
4500 V, end plate ofset 500 V, nitrogen was employed as the
nebulizer gas (1 bar), dry heater temperature 200 °C, dry-
ing gas (8 L/min) and collision cell energy 8 eV. The mass
spectrometer was programmed to perform acquisition in auto
MS/MS mode (number of precursors 4) in experiments with
diferent collision energy between 18 and 45 eV for all m/z
range analyzed.
Bacteria of the genus Staphylococcus are considered
important agents that cause bovine mastitis, especially the
Staphylococcus aureus species, which are known to cause
contagious mastitis [9, 10]. This disease is transmitted to
other animals of the herd, mainly through milking proce-
dures [3–8]. Due to its highly contagious profle, these bac-
teria cause infection for more than 30 days by colonization
in udder wounds, in the hands of milkers and/or in the mam-
mary gland. The production of a thermostable toxin, which
remains active even in pasteurized milk, also contributes to
the pathogenesis [11–13].
The most common method of treatment of bovine mas-
titis is the use of antibiotics. However, the inefciency is
observed in approximately 10–30% of the cases, especially
for S. aureus caused disease [14, 15]. It is worth noting that
the presence of antibiotic residues in milk is also an impor-
tant parameter evaluated in the dairy industry. Thus, the
rational use of antimicrobials in dairy herds is essential due
to its impact in the fnal production [16].
Preparation of Compound 1
(1,3‑bis(4‑allyl‑2‑Methoxyphenoxy)Propane)[20]
In view of its economic impact and the difculty in treat-
ment due to microbial resistance, it is necessary the search
for new therapeutic alternatives [17]. In this context, the
products derived from the secondary metabolism of plants
have been pointed as biotechnology options due to their
therapeutic properties. The current literature showed that
molecules derived from plant metabolism such as trans-
cinnamaldehyde (TC), eugenol, carvacrol and thymol are
efective against microorganisms and present promising
results against the major pathogens causing bacterial mas-
titis. These molecules are consistently pointed as potential
alternatives or adjuvant to antibiotics in the prevention and
treatment of bovine mastitis [18, 19].
In a 25 mL flask, 5.0 mL of acetone and 2.2 mmol
(304.0 mg) of K2CO3 were added, followed by 2.1 mmol
(621.3 mg) of eugenol. The reaction mixture was allowed
to stir for 5 min, then 1 mmol (295.84 mg) of 1,3-dii-
odopropane was added dropwise. The reaction was left
to refux with stirring for 9 h. The resulting mixture was
evaporated to dryness and the residue (0.378 g) was purifed
by silica chromatographic column with a mixture of ethyl
acetate:hexane (1:9) as eluent. Compound 1 was obtained
in 49% yield (181 mg, 0.5 mmol); M.p. 78 °C; IR (cm−1):
2919, 1513, 1227; 1H-NMR (CDCl3, 500 MHz)δ: 6.85 (d,
2H, J=8.26 Hz), 6.69 (m, 4H), 5.95 (m, 2H), 4.21 (t, 4H,
J=6.26 Hz), 3.83 (s, 6H), 3.32 (d, 4H, J=6.68 Hz), 2.32
(quint, 2H, J=6.26 Hz); [21] 13C-NMR (CDCl3, 126 MHz)
δ: 149.70, 146.96, 137.90, 133.22, 120. 73, 115.78, 113.92,
112.68, 66.27, 56.15, 40.02, 29.59 [21].
Thus, the objective of this work was to evaluate the bio-
technological potential of nine eugenol and thymol deriva-
tives against S. aureus strains from bovine mastitis.
Preparation of Compound 2
(1,10‑bis(4‑Allyl‑2‑Methoxyphenoxy)Decane [20]
In a 25 mL fask, 10 mL of acetone and 4.4 mmol (609 mg)
of K2CO3 and then 4.2 mmol (688.8 mg) of eugenol were
1 3

1848
D. O. S. Nunes et al.
added, this mixture was left under stirring for about 10 min,
then 2 mmol (576 mg) of 1.10-dibromodecane was added
dropwise. The reaction was left to refux with stirring for 7 h
and 30 min. The resulting mixture was evaporated to dry-
ness and the residue (0.8412 g) was purifed by silica chro-
matographic column with a mixture of ethyl acetate:hexane
(1:9) as eluent. Compound 2 was obtained in 58% yield;
M.p. > 200 °C; IR (cm−1): 2918, 2852, 1516, 1259, 793;
1H-NMR (CDCl3, 500 MHz)δ: 6.74 (d, 2H, J=8.52), 6.63
(m, 4H), 5.89 (m, 2H), 4.99 (m, 4H), 3.91 (t, 4H, J=6.90),
3.78 (s, 6H), 3.26 (d, 4H,J=6.69), 1.75 (m, 4H), 1.37 (m,
4H), 1.27 (m, 8H); 13C-NMR (CDCl3, 126 MHz)δ: 149.61,
147.17, 137.95, 132.86, 120.66, 115.75, 113.41, 112.62,
69.42, 56.18, 40.03, 29.46, 26.19; HRESI–MS [M-Na]+m/z
489.2971 (calculated. for m/z C30H42NaO4, 489.2975).
3.33 (d, 2H, J=6.68 Hz); [24] 13C-NMR (CDCl3, 126 MHz)
δ: 149.08, 147.57, 137.86, 132.81, 120.57, 115.76, 112.06,
111.46, 56.11, 55.97, 39.97 [23].
Preparation of Compound 5 (O‑Ethyleugenol) [22]
In a 100 mL fask connected to the refux apparatus con-
taining 10 mmol (1.64 g) of eugenol in anhydrous acetone
(60 mL) was added 29 mmol (4.008 g) of K2CO3. The mix-
ture was stirred in an oil bath for 1 h at 50–80 °C. Then,
17 mmol (1.852 g) of ethyl bromide was added to the reac-
tion solution and refuxed for an additional 19 h. The result-
ing mixture was evaporated to dryness in a rotary evaporator
and the residue (1.5056 g) was purifed by silica chroma-
tographic column with a mixture of ethyl acetate:hexane
(1:5) as eluent. Compound5 was obtained in 42% yield; yel-
low oil; IR (cm−1): 3076, 2978, 2904, 1511, 1258, 1230,
911, 802, 747; 1H-NMR (CDCl3, 500 MHz)δ: 6.80 (d, 1H,
J=7.92 Hz), 6.70 (m, 2H), 5.96 (m, 1H), 5.07 (m, 2H), 4.07
(q, 2H, J=7.00 Hz), 3.85 (s, 3H), 3.33 (d, 2H, J=6.68 Hz),
1.44 (t, 3H, J = 7.00 Hz); 13C-NMR (CDCl3, 126 MHz)δ:
149.43, 146.82, 137.88, 132.83, 120.58, 115.72, 113.10,
112.34, 64.58, 56.04, 39.98, 15.04 [22].
Preparation of Compound 3
(1,9‑bis(4‑Allyl‑2‑Methoxyphenoxy)Nonane) [20]
In a 25 mL fask, 10 mL of acetone and 4.4 mmol (609 mg)
of K2CO3 and then 4.2 mmol (688.8 mg) of eugenol were
added, this mixture was left under stirring for about 10 min,
then 2 mmol (576 mg) of 1,9-dibromononane was added
dropwise. The reaction was left to refux for 7 h and 30 min
with stirring. The resulting mixture was evaporated to dry-
ness and the residue was purifed by silica chromatographic
column with a mixture of ethyl acetate:hexane (1:9) as elu-
ent. Compound 3 was obtained in 32% yield; M.p.>200 °C;
IR (cm−1): 2917, 2849, 1515, 1232, 796; 1H-NMR (CDCl3,
500 MHz)δ: 6.73 (d, 2H, J = 8.47 Hz), 6.63 (m, 4H), 5.8
(m, 2H), 4.99 (m, 4H), 3.91 (t, 4H, J = 6.89 Hz), 3.77 (s,
6H), 3.26 (d, 4H, J=6.69 Hz), 1.75 (m, 4H), 1.37 (m, 4H),
1.28 (m, 6H); 13C-NMR (CDCl3, 126 MHz) δ: 149.17,
147.17, 137.96, 132.88, 120.67, 115.76, 113.43, 112.63,
69.42, 56.19, 40.03, 29.70, 29.56, 29.46, 26.19; HRESI–MS
[M-Na]+m/z 475.2819 (calculated. for m/z C29H40NaO4,
475.2818).
Preparation of Compound 6 (O‑Acetyleugenol) [25]
In a 25 mL fask with refux condenser, were added 6 mmol
(0.98 g) of eugenol, 20 mmol (2.04 g) of acetic anhydride
and acetic acid (2 mL). Then the mixture was stirred at
refux for 2.5 h. Then it was cooled to room temperature and
a mixture of H2O and ice was added under magnetic stirring.
The organic phase was separated and then washed with H2O
(2×20 mL), 10% NaOH (2×20 mL) and H2O (2×20 mL).
The reaction crude was obtained after removal of the sol-
vent in a rotary evaporator, and purifed by silica chroma-
tographic column with a mixture of ethyl acetate:hexane
(1:5) as eluent. Compound 6 was obtained in 82% yield;
1
M.p.31 °C; IR (cm−1): 3076, 2840, 1762, 1185; H-NMR
Preparation of Compound 4 (O‑Methyleugenol)[22]
(CDCl3, 500 MHz)δ: 6.93 (d, 1H, J = 7.98 Hz), 6.79 (s,
1H), 6.76 (d, 1H, J=7.93 Hz), 5.95 (m, 1H), 5.10 (m, 2H),
3.81 (s, 3H), 3.37 (d, 2H, J = 6.51 Hz), 2.29 (s, 3H); 13C-
NMR (CDCl3, 126 MHz) δ: 169.13, 150.84, 138.98, 137.96,
136.99, 122.48, 120.61, 116.14, 112.71, 55.80, 40.04, 20.65
[26].
In a 100 mL fask connected to the refux apparatus con-
taining 10 mmol (1.64 g) of eugenol in anhydrous acetone
(60 mL) was added 29 mmol (4.008 g) of K2CO3. The mix-
ture was stirred in an oil bath for 1 h at 50–80 °C. Then,
17 mmol (2.413 g) of methyl iodide was added to the reac-
tion medium and refuxed for a further 9 h. The resulting
mixture was evaporated to dryness in a rotary evaporator
and the residue (1.2722 g) was purifed by silica chroma-
tographic column with a mixture of ethyl acetate:hexane
(1:5) as eluent. Compound 4 was obtained in 63% yield; yel-
low oil; IR (cm−1): 2935, 2834, 1638, 1512; [23] 1H-NMR
(CDCl3, 500 MHz) δ: 6.80 (d, 1H, J = 7.97 Hz), 6.72 (m,
2H), 5.96 (m, 1H), 5.07 (m, 2H), 3.87 (s, 3H), 3.85 (s, 3H),
Preparation of Compound 7 (O‑Acetylthymol) [25]
In a 25 mL fask with refux condenser, were added 6 mmol
(0.9013 g) of thymol, 20 mmol (2.04 g) of acetic anhy-
drous and acetic acid (2 mL). Then the mixture was stirred
at refux for 2 h. Then, it was cooled to room tempera-
ture and a mixture of H2O and ice was added under mag-
netic stirring. The organic phase was separated and then
1 3

Biotechnological Potential of Eugenol and Thymol Derivatives Against Staphylococcus aureus…
1849
washed with H2O (2×20 mL), 10% NaOH (2×20 mL) and
H2O (2 × 20 mL). The reaction crude was obtained after
removal of the solvent in a rotary evaporator, and purifed
by silica chromatographic column with a mixture of ethyl
acetate:hexane (1:5) as eluent. Compound 7 was obtained
in 83% yield; yellow oil; IR (cm−1): 2963, 1759, 1199,
Bacterial Strains
A total of 15 strains of S. aureus from subclinical bovine
mastitis isolated from herds of the states of Minas Gerais,
São Paulo and Rio de Janeiro (Brazil) were characterized
phenotypically and genotypically and provided by the
researcher PhD Aparecida Vasconcelos Paiva Brito, from the
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA).
They were designated by 4026, 4027, 4074, 4075, 4098,
4099, 4271, 4272, 4293, 4313, 4384, 4523, 4745, 4746, and
4784.
1
816; [27] H-NMR (CDCl3, 500 MHz) δ: 7.19 (d, 1H,
J = 7.91 Hz), 7.02 (d, 1H, J = 7.94 Hz), 6.80 (s, 1H), 2.97
(m, 1H), 2.31 (s, H, J = 6.94 Hz); [27] 13C NMR (CDCl3,
126 MHz) δ: 169.95, 148.07, 137.19, 136.74, 127.35,
126.64, 122.92, 27.34, 23.22, 21.10, 21.00 [28].
The strains were evaluated for the susceptibility to anti-
biotics amicacin, gentamicin, tobramycin, oxacillin, peni-
cillin, sulfonamide, chloramphenicol, oxytetracycline and
tetracycline by means of the Disk Difusion Test, as recom-
mended by Clinical and Laboratory Standards Institute CLSI
[31] using 5 mm flter paper discs (Laborclin®, Brazil). For
vancomycin, the assay was used to determine the Minimum
Inhibitory Concentration (MIC). According to the recom-
mendation, resistant strains have MIC ≥16 μg mL−1; with
intermediate sensitivity between 4 and 8 μg mL−1 and sus-
ceptible strains≤2 μg mL−1 [32].
Preparation of Compound 8 (O‑Methylthymol) [22]
In a 100 mL fask connected to the refux apparatus, a solu-
tion of 10 mmol thymol (1.502 g) in anhydrous acetone
(60 mL) was added 29 mmol (4.008 g) of K2CO3. The mix-
ture was stirred in an oil bath for 1 h at 50–80 °C. Then,
17 mmol (2.413 g) of methyl iodide was added to the
reaction solution and refuxed for an additional 8 h. The
resulting mixture was evaporated to dryness in a rotary
evaporator and the residue was purifed by silica chroma-
tographic column with a mixture of ethyl acetate:hexane
(1:5) as eluent. Compound 8 was obtained in 50% yield;
colourless oil; IR (cm−1): 2958, 2866, 1502, 1462, 1256,
1191, 1161, 1042;1H-NMR (CDCl3, 500 MHz) δ: 7.09
(d, 1H, J = 7.66 Hz), 6.74 (d, 1H, J = 7.67 Hz), 6.67 (s,
1H), 3.81 (s, 3H), 3.27 (m, 1H), 2.33 (s, 3H), 1.19 (d,
6H, J = 6.92 Hz); 13C-NMR (CDCl3, 126 MHz) δ: 156.83,
136.47, 134.20, 125.98, 121.26, 111.59, 55.49, 26.60,
22.99, 21.55 [29].
Determination of Bioflm Formation
The 15 strains of S. aureus were spiked on BHI agar and
incubated for 24 h at 37 °C. After this period, one colony
from each plate was transferred to test tubes containing
Tryptic Soy Broth (TSB) enrichment broth plus 1% glucose.
The tubes were kept under vigorous agitation (250–300 rota-
tions per minute) for 20 h at 37 °C until massive growth
was achieved. After this time, the cultures were diluted 1:
100 using TSB broth with 1% glucose. Then, 200 μL of
these dilutions were transferred to 4 wells of a fat bottom 96
well plate (Nunc, ThermoFisher Scientifc, United States of
America) and incubated for 20 h at 37 °C without shaking.
The optical density (OD) was frst read through the micro-
plate reader (SpectraMax 190, Microplate Reader, Molecular
Devices, Sunnyvale, CA) at a wavelength of 540 nm. The
supernatant was removed and the wells were washed gen-
tly twice with 0.85% saline for non-removal of the adhered
cells. Fixation was carried out in an oven at 65–70 °C for
about 1 h and 30 min, until the plate was completely dried.
Next, 200 μL of violet crystal dye was added to the wells
for 1 min. This dye was then discarded and the wells were
washed with sterile water and the plates were again dried in
an oven. The second OD reading of each well was carried
out. The tests were performed in triplicate. The methicillin
resistant strain (BMB 9393) was used as a positive control
for bioflm production.
Preparation of Compound 9 (O‑Ethylthymol) [22]
In a 100 mL fask connected to the refux apparatus, a solu-
tion of 10 mmol (1.502 g) thymol in anhydrous acetone
(60 mL) was added 29 mmol (4.008 g) of K2CO3. The mix-
ture was stirred in an oil bath for 1 h at 50–80 °C. Then,
17 mmol (1.852 g) of ethyl bromide was added to the reac-
tion solution and refuxed for an additional 8 h. The result-
ing mixture was evaporated to dryness in a rotary evapora-
tor and the residue was purifed by silica chromatographic
column with a mixture of ethyl acetate:hexane (1:5) as
eluent. Compound 9 was obtained in 48% yield; colour-
less oil; IR (cm−1): 2960, 1505, 1255, 1047, 807; 1H-NMR
(CDCl3, 500 MHz) δ: 7.08 (d, 1H, J=7.67 Hz), 6.72 (d, 1H,
J=7.72 Hz), 6.65 (s, 1H), 4.02 (q, 2H, J=6.97 Hz), 3,29
(m, 1H), 2.31 (s, 3H), 1.41 (t, 3H, J = 6.97 Hz), 1.20 (d,
6H, J=6.91 Hz); 13C-NMR (CDCl3, 126 MHz) δ: 156.16,
136.26, 134.23, 125.92, 121.04, 112.50, 63.59, 26.71, 22.85,
21.42, 15.10 [30].
The biofilm unit (UB) was calculated according to
Amaral et al. [33] and classifed as follows:• DO ≤0.24—
Non-adherent; • 0.24 > OD < 0.46—Weakly adherent; •
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D. O. S. Nunes et al.
0.46 > OD < 0.919—Moderate adherent; • DO ≥ 0.92—
Strongly adherent. The strains that were considered to be
the strongest adherent were selected for the other tests.
The MBC, the concentration of the drug capable of inhib-
iting 99.9% of bacterial growth, was carried out after deter-
mining the MIC. We added 10 μL of the wells corresponding
to the MIC and 2×MIC concentrations in BHI agar plates.
After 24 h of incubation at 37 °C, colonies were counted.
Tests were performed in triplicates for the validation of the
results.
Disc Difusion Assay
This assay was performed according to the established by
CLSI [31]. The antibacterial vancomycin (Sigma-Aldrich®,
Germany) was used as a positive control for the S. aureus
strains. Dimethylsulphoxide (DMSO) was used for the dilu-
tion and as negative control under identical conditions to
the tested derivatives. The direct inoculation method was
carried out for the species of S. aureus considered strongly
adherent in the previous test. Bacterial inocula correspond-
ing to the 0.5 scale of MacFarland were seeded on Müeller
Hinton agar plates, with the aid of sterile swab. Then, the
disks impregnated with the compounds at a concentration of
5 mg mL−1 were fxed on the surface of this agar. The plates
were incubated at 35 °C for 24 h and checked for growth
inhibition halo.
In Silico Analysis of Toxicological
and Physicochemical Parameters
In silico analysis of the derivatives was performed using
the Osiris DataWarrior® program from Acetilion Pharma-
ceuticals Ltda., Version 5.0 available at http://www.organ
ic-chemistry.org/prog/peo/. This program allows the cal-
culation of theoretical physico-chemical and toxicological
parameters, aiming to evaluate the tumorogenicity, muta-
genicity, efects on reproduction and irritating efects, and
parameters such as drug-likeness, which help in the selection
of new drug candidates.
Results
Minimal Inhibitory Concentration (MIC) and Minimal
Bactericidal Concentration (MBC)
Synthesis of Eugenol and Thymol Derivatives
After the identifcation of the active derivatives by the disc
difusion test, a quantitative analysis (MIC) was performed
using broth microdilution test comparing with the positive
controls (vancomycin, eugenol and thymol).
Nine compounds derived from eugenol and thymol were
synthesized using classical methods of phenolic alkylation
[20, 22]. Compounds designated by 1, 2, 3, 4, 5, 6 were
derived from eugenol and 7, 8 and 9 from thymol (Fig. 1).
Compounds 2 and 3 were obtained and describe in this work
for the frst time.
The bacterial inoculum was prepared in 0.85% saline
solution and adjusted to 1.5 × 108 colony forming units
(CFU) mL−1 and diluted in Müeller Hinton broth to a fnal
concentration of 5×105 CFU mL−1. The experiments were
performed in triplicate on a 96 well fat bottom microtiter
plate (TPP-Europe, Switzerland) and three controls were
used in each experiment, including (a) the culture medium
with the compound and without the bacterial inoculum, (b)
the culture medium without inoculum or compound to be
tested (sterility control of the culture medium) and (c) the
culture medium inoculated without the compound (growth
control).
Evaluation of Bioflm Production by Mastitis S.
aureus Strains
In the assay for evaluation of bacterial bioflm production,
optical density values ranged from 0.58, by strain S. aureus
4075, to 3.51 for S. aureus 4746. Overall, the strains of S.
aureus 4271, 4745 and 4746 were the strongest bioflm
producers, whereas the strains 4074, 4075, 4384 and 4784
showed lower production, when compared to the positive
control (Table 1).
Serial dilution of the compounds in Müeller Hinton broth
was performed. Whereas derivatives of eugenol and thymol
were tested at concentrations of 256 to 2 µg mL−1; the posi-
tive controls vancomycin, eugenol and thymol were tested at
concentrations of 25 to 2 μg mL−1 for the frst one and 256
to 2 μg mL−1 for the others. Each well of the 96-well plate
remained at 100 μL volume, and then added 100 μL aliquot
of the appropriately diluted bacterial inoculum.
Among all the strains tested, approximately 60% (n=9)
were classifed as strongly adherent whereas 40% (n = 6)
showed moderate adherence. No non-adherent or poorly
adherent strains were observed on the samples tested. The
strains of code 4271, 4745 and 4746 were the ones that
presented the highest bioflm production among the tested
ones, being classifed as strongly adherent and presenting
values of bioflm production comparable to the values of the
strain used as positive control. Thus, these three strains were
selected for the subsequent tests.
After 24 h of incubation at 37 °C, MIC was defned as the
lowest concentration of the derivative capable of inhibiting
visible growth of the bacterial culture. This procedure was
performed according to CLSI Protocol M07-A9 [32].
1 3

Biotechnological Potential of Eugenol and Thymol Derivatives Against Staphylococcus aureus…
1851
Fig. 1 Eugenol, Thymol and its derivatives 1–6 and 7–9, respectively
Table 1 Classifcation of S. aureus strains in the bioflm production
Table 2 Evaluation of antibacterial susceptibility by disk difu-
sion method against strains of S. aureus 4271, S. aureus 4745 and S.
aureus 4746
according to that described by de Amaral et al. [33]
S. aureus strains
Bioflm unit (BU)
Classifcation
Compounds
Halo (mm)
4026
4027
4074
4075
4098
4099
4271
4272
4293
4313
4384
4523
4745
4746
4784
C+
0.81
1.05
0.59
0.58
0.90
1.46
3.16
1.25
1.19
2.70
0.8
1.38
3.22
3.51
0.78
2.5
Moderate
Strong
Moderate
Moderate
Moderate
Strong
Strong
Strong
Strong
Strong
Moderate
Strong
Strong
Strong
Moderate
Strong
S. aureus 4271
S. aureus 4745
S.
aureus
4746
Thymol
Eugenol
Vancomycin
DMSO
7
20
17
0
6
16
15
0
6
16
15
0
1
2
3
4
5
6
7
8
9
0
0
0
21
18
0
10
8
0
0
0
20
15
0
10
11
10
0
0
0
19
15
0
10
10
9
8
C+,Positive control (S.aureus BMB 9393 (MRSA) strain)
Disc Difusion Assay
vancomycin, eugenol and thymol (Table 2). Therefore,
these compounds were evaluated in assays for determining
Minimum Inhibitory Concentration (MIC) and Minimal
Bactericidal Concentration (MBC) and comparison with
the controls.
The antibacterial profile was detected for compounds
4, 5, 7, 8 and 9 against S. aureus 4271, S. aureus 4745
and S. aureus 4746 on disc difusion assays, similar to
1 3

1852
D. O. S. Nunes et al.
Determination of Minimum Inhibitory
Concentration (MIC) and Minimal Bactericidal
Concentration (MBC)
Determination of In Silico Toxicity of Active
Compounds
All active derivatives on disc difusion assays were submit-
ted to the in silico evaluation of toxicological and physi-
cal–chemical parameters. According to the Osiris DataWar-
rior® program, it is estimated that 80% of the drugs on the
market have solubility values higher than − 4, which was
also observed for all derivatives tested, thus inferring the
same biodisponibility (Table 4).
The active derivatives were evaluated to determine the
lowest concentration of the compound capable of visually
inhibiting bacterial growth in culture medium (Table 3).
Thus, among the evaluated derivatives, the derivative 4
was the most promising (MIC of 32 µg mL−1) against S.
aureus 4271, 4745 and 4746 strains; whereas eugenol,
thymol, 5, 7, 8 and 9 derivatives were not active within
the values of Clinical and Laboratory Standards Insti-
tute (CLSI) that provides standards and guidelines for
medical professional (Table 3). The MBC for derivative
4 ranged from 32 µg mL−1, against the strains S. aureus
4271 and 4746, to 64 µg mL−1 against the S. aureus 4745
(not shown).
The eugenol and thymol derivatives demonstrated Top-
ographical Polar Surface Area (TPSA) values lower than
those observed for vancomycin, close to 4, 5 and 7 values,
whereas cLogP values were higher than that found for that
antibiotic. Our data suggested that eugenol and thymol
derivatives are more hydrophobic than vancomycin and can
be easier absorbed.
The OsirisDataWarrior program evaluates the risk of tox-
icity by locating risk chemical groups within the molecule
structure. According our in silico analysis, the derivatives
4 and 5 were theoretically mutagenic whereas 5 was also
considered tumorigenic and irritant, 4 have reproductive
efects and 7 was also considered irritant. Similarly, Thymol
was considered mutagenic, tumorigenic, irritant and likely
to cause reproductive efects, whereas Eugenol, also used
as positive control, was also pointed as irritant (Table 4).
Computational testing help on reducing the time and cost of
searching for new antibacterials by applying computational
knowledge and somewhat simplifying the process of discov-
ering new active compounds [34]. Vancomycin antibacterial
was not associated with the theoretical toxic efects of the
program, but is considered in clinical practice as nephro-
toxic. This parameter of renal toxicity is not included in the
software database [35]. Therefore, far from establishing the
real safetyness or risk of these molecules, these theoreti-
cal data are useful to prioritize these molecules for future
exploring.
Table 3 Comparison of the Minimal Inhibitory Concentration (MIC)
obtained with the synthetic derivatives of eugenol and thymol against
strains of S. aureus 4271, S. aureus 4745 and S. aureus 4746
Compounds
MIC (µg mL−1
)
S. aureus 4271
S. aureus 4745
S. aureus 4746
Thymol
Eugenol
Vancomycin
>256
>256
2
>256
>256
2
>256
>256
2
4
5
7
8
9
32
32
32
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
MICMininal inhibitory concentration
Table 4 Evaluation of
Compounds Toxicological parameters
Physical–chemical parameters
the toxicological and
pharmacokinetic parameters
of the eugenol and thymol
derivatives analyzed by the
Osiris DataWarrior program
Mutagenicity Tumori- Efect in
Irritant cLogP cLogS TPSA (A2) Molecular
weight (Da)
genicity reproduc-
tion
4
5
7
8
+
+
−
−
−
−
+
−
−
+
−
−
−
−
+
−
+
−
−
−
−
−
+
−
−
+
+
−
−
+
+
−
2.55 − 2.36 18.46
2.95 − 2.66 18.46
3.18 − 3.13 26.3
178.23
192.26
192.25
164.224
178.24
164.20
159.22
1447.00
3.12 − 2.84
3.52 − 3.14
9.23
9.23
9
EUGENOL
THYMOL
VANCO
2.27 − 2.05 29.46
2.84 − 2.54 20.23
− 6.75 − 9.42 530.40
Vanco,Vancomycin; −,Negative;+,Positive
1 3

Biotechnological Potential of Eugenol and Thymol Derivatives Against Staphylococcus aureus…
1853
The drug-likeness parameters were also calculated based
on the scores of the fragments present in these molecules
under investigation and compared to the commercially avail-
able drugs. Since positive values indicate the similarity of
fragments to commercial drugs, the negative values found
for these derivatives pointed to the originality of these struc-
tures (Fig. 2). Thus, these negative results can be considered
as a favorable feature, since these diferences may suggest
diferent mechanisms of action for these molecules com-
pared to commercially available drugs. It is noteworthy to
mention that various action mechanisms were reported by
Zorzet as positive points for new compounds, so that they
can delay the emergence of resistance [36].
factors and the production of certain proteins, which help in
the adhesion of these bacteria whereas reduces the suscepti-
bility of these organisms to the antibiotics [38, 39].
According to Aslantas and Demir [40], bioflm formation
not only helps in the adhesion of bacteria and colonization
of the mammary gland tissue in the avoidance of phago-
cytosis and adverse conditions in the host, but also in the
permanence of the infectious state. These and other authors
observed several antibiotic resistance rates among the strains
analyzed in their studies with diferent genes involved [38].
Among the nine derivatives tested, derivative 4 was with
the most potential antibacterial profle against the three
strains evaluated (MIC=32 µg mL−1 against S. aureus 4271,
4745 and 4746), whereas eugenol, thymol and derivatives 5,
7, 8 and 9 were not active within the CLSI values against the
S aureus strains tested. The structural analysis of compounds
4, 5 and 6 showed that the substitution of a methoxy group
for ethoxy in 5 or acetate in 6 reduced the activity of these
compounds.
Discussion
Bioflm production plays an important role in the establish-
ment of infection and colonization of the glandular mam-
mary tissue in bovine mastitis. It is directly related to the
establishment and maintenance of infection in host tissues.
The bioflm confers resistance to these pathogens against the
treatment using antibiotics also allowing the proliferation
when it is mature or/and the conditions inside the matrix
are unfavorable. The resistance provided by the formation
of the bioflm is related to the physical and chemical difu-
sion barrier formed by the exopolysaccharide matrix, which
hinders the antimicrobial action. It also contributes to drug
resistance due to: (a) an environment that antagonize anti-
biotic action, (b) the activation of stress responses that lead
to physiological changes in the bacterium, (c) the slower
growth of these microorganisms due to nutrient limitation
and (d) the absence of antimicrobial targets [37].
Silva et al. [41] evaluated extracts of Salvia ofcinalis and
Plectranthus ornatus, which contained eugenol and thymol
among their main components. The authors verifed MIC
values of 300 µg mL−1 and 1200 µg mL−1 against S. aureus
3993 and 4125, for both extracts, respectively, but with val-
ues highest and with less potential than our derivative 4. It
is worth mentioning that Apolónio et al. [42] did not verify
the development of antimicrobial resistance after exposing
S. aureus ATCC 6838 to eugenol, which may be pointed as
an additional, favorable and feasible characteristic for our
derivatives.
Silva et al. [43] synthesized eugenol derivatives from
addition reactions to the double bond of the allyl or esteri-
fcation group on the hydroxyl group with carboxylic acids.
The compound designated by code 16, obtained by addition
reaction to the double bond, exhibited a 10 mm inhibition
halo against S. aureus and was considered to have a mod-
erate efect to this species. However, this compound was
not active under the working conditions in the MIC assay.
The authors verifed that the reactions of esterifcation or
acetylation in the hydroxyl produced compounds with lower
antimicrobial activity than that observed for eugenol, which
may justify the results obtained in this research.
Our studies demonstrated a strong bioflm production in
60% of the evaluated S. aureus strains. This production is
one of the most important mechanisms developed for the
survival of this species and is directly related to genetic
Vacomycin
Eugenol
Tymol
The evaluation of toxicological and physical–chemi-
cal parameters showed that all derivatives had cLopP< 5,
cLogS > − 4 and TPSA < 140 Å2. Derivatives were con-
sidered mutagenic (4 and 5); tumorigenic (5); irritating (5
and 7) or with possible efect on reproduction, similar to our
antibiotic controls. In addition, all derivatives showed nega-
tive drug-likeness values, indicating that they difer from the
compounds available on the market with theoretical potential
for avoiding resistance bacterial mechanism.
9
8
7
5
4
-8
-6
-4
-2
0
2
4
6
The literature reported that compounds derived from
the secondary metabolism of plants may be associated
Fig. 2 Evaluation of fragments of the tested derivatives calculated by
using Osiris DataWarrior program
1 3

1854
D. O. S. Nunes et al.
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the improvement of this activity when used in synergism.
Zheng and colleagues [44] observed that more promising
results were observed when using two combinations: thy-
mol and carvacrol and thymol, carvacrol and eugenol. Thus,
our derivatives may be more active for use as antimicrobi-
als if combined and it is important to consider that these
compounds can also serve as the basis for the synthesis of
new substances that may be even more active and less toxic
than derivative 4. These perspectives make derivative 4 even
more promising to be known and further explored.
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