2.

Materials and Methods

2.1 Plant Material
Hyptis suaveolens fresh leaves were
collected in Nakhon Ratchasima region:
(15◦07’41.6’N 101◦33’47.8’E.) of Northeast and
Phitsanulok region: (16◦50’35.9’N
100◦32’23.7’E.) of Northern, Thailand,
representing a typically different environments,
during September and October 2023. The plant
specimens were identified by the Forest
Herbarium, Department of National Parks,
Wildlife and Plant Conservation, Bangkok,
Thailand.

2.2 Extraction of essential oil

Two kilograms of fresh leaves H. suaveolens
of each location were reduced their size and
subjected to steam distillation method with a
Clevenger apparatus containing about 10 L of
distilled water. The time used for distillation was
boiled for 3 h. The essential oil was dried using
anhydrous sodium sulfate and after filtration was
stored in an amber bottle at 4 °C until use for
further use. The essential oil yield was
determined in mL relative to 100 g of fresh
matter. The steam distillation process was done
in triplicates.
2.3. Gas chromatography/mass
spectrometry (GC–MS) analyses.
 The chemical composition of the essential
oils were analyzed using a gas chromatograph
coupled with the mass spectrometry (GC-MS)
model Agilent 7890A GC system Agilent
Technologies/ Agilent 7000D mass spectrometer
system Agilent Technologies, the column used
was the Agilent HP-5 MS capillary column (30 m x
0.250 mm x 25 μm ). The analysis conditions
were as follows: injector temperature of 250◦C,
injection volume of 1.0 μL, injection ratio in split
mode 1:50. The oven temperature was held at
50◦C for 1 min and then programmed from 50◦C
to 230◦C at 4 ◦C/min and held for 6 min. The
carrier gas was helium with a flow rate of 1.2
mL/min. Mass spectra were taken at 70 eV with
the mass range from m/z 40–400 amu.
Identification of chemical components was based
on analysis of the chromatograms obtained for
essential oil, by means of evaluating the
retention indices (RI) in comparison with
standards of n-alkane mixtures (C8-C20) and the
mass spectral data of each peak using computer
library (Wiley-14 and NIST-17 Mass Spectral
Library) and comparison of these data with the
literature (Adams, R.P. Identification of Essential
Oil Components by Gas Chromatography/Mass
Spectrometry; Allured Publishing: Carol Stream,
IL, USA, 2007). The analyses were performed in
triplicate. Standard deviations (SDs) did not
exceed 2% of the obtained values of each
compound.

2.4 Antibacterial activity

2.4.1 Microbial strains
The antibacterial activity of the essential oil
was tested against three bacterial strains such as
Staphylococcus aureus DMST 8840,
Staphylococcus epidermis DMST 15505 and
Escherichia coli DMST XXXX were obtained from
Culture Collection for Medical Microorganisms,
Department of Medical Sciences, Thailand. For
antibacterial testing fresh inoculums was
prepared for each bacterial strain and incubated
at 37◦C in agar nutrient for 24 h. The final
bacterial cell concentration was adjusted in a
spectrophotometer at 625 nm using 0.85% (m/v)
sterile saline and adjusted to the 0.5 McFarland
(about 1.5 × 108 CFU/mL).
2.4.2 Agar diffusion method
This procedure was performed according to
Clinical and Laboratory Standards Institute
method (CLSI M02-A11, 2012). The 90 mm Petri
plates were prepared by pouring 25 mL Mueller
Hinton Agar (MHA) for S. aureus, S. epidermidis
and E. coli allowing the medium to solidify. The
plates were then dried, uniformly spread, and
allowed to dry. Paper discs (9.0 mm diameter)
were impregnated with 50 µL of the H.
suaveolens essential oil and 1,8-cineole, β-
caryophyllene used for standard reference (10
µL/disc). Each test paper disk was placed on each
test medium plate using forceps with slightly
press for firm contact between test disk and test
media. The plates were incubated at 37 °C for 24
h and their antibacterial activity was evaluated
for each strain by measuring the diameter of the
inhibition halo (in millimeters), according to the
antibiogram ruler. To evaluate the repeatability
of the results, three replicates for each tested
sample and each strain were performed.
Dimethylsuloxide (DMSO) was used as negative
control and Ampicilin (10 µg/disk) was used as
positive control under the same conditions of
tested oils.
2.4.3 Determination of minimum inhibitory
concentration (MIC)
The minimum inhibitory concentration (MIC)
able to inhibit the growth of bacteria of the
essential oils was determined by serial
microdilution in 96-well microplates according to
the broth microdilution method (Clinical
Laboratory Standards Institute. Methods for
Dilution Antimicrobial Susceptibility Test for
Bacteria That Grow Aerobically;Approved
Standard M7-A10; Clinical Laboratory Standards
Institute: Wayne, PA, USA, 2015).
Essential oil was dissolved in a mixture of
Mueller Hinton Broth (MHB) medium and DMSO
to reach a concentration of 700 µL/mL and were
diluted by using the same mixture to reach
different concentration. (200.0 to 0.097 µL/mL),
using the known density of H.suaveolens EO at
0.880 g/mL at 20◦C as a conversion factor, these
percentage values corresponded to a range from
176.00 to 0.086 mg/mL. Experiments were
performed by using sterrile 96-well microplates
were filled 50 µL of MHB medium was added to
each well. Then, 50 µL of the essential oil at
different concentrations were added to each well.
After serial dilution, 50 µL of bacterial suspension
with 0.5 McFarland dilution was added to each
well to achieve a concentration of approx. 1x10 8
CFU/mL. Plates were incubated at 37◦C for 24 h.
The MIC was determined the lowest
concentration of the essential oil visually as the
preventing microbial growth (absence of
turbidity). Negative control group was treated
only with 20%DMSO. The MIC values of 1,8-
cineole and β-caryophyllene were determined in
the same way as HSEOs. All tests were
performed in triplicate.
2.4.4 Determination of minimum
bactericidal concentration (MBC)
For the determination of minimum
bactericidal concentration (MBC), we used the
minimum inhibitory concentration (MIC),
collecting 10 µl individually from the wells in
which bacterial growth was not observed, from
the MIC well and a well following this one
(showing no turbidity), seeding on Mueller Hinton
agar plates. The plates were incubated at 37 °C
for 24 h for subsequent reading of MBCs,
considering the bacterial show no detectable
colonies on individual agar plates as determining
factors for MBC (Clinical and Laboratory
Standards Institute 2003). Experiments were
done in triplicate. The MBC values of 1,8-cineole
and β-caryophyllene were determined in the same
way as HSEOs.
2.4.5 Determination of ratio MBC/MIC
The ratio MBC/MIC was calculated to
determine the efficacy and the
bactericidal/bacteriostatic effect of essential oil
on the bacterial growth of strains tested. If the
MBC/MIC is ≤4, then the effect is bactericidal ,
while if MBC/MIC is >4, then the effect is
bacteriostatic. (Goly et al., 2015, Mihin et al.,
2019)
*Goly et al., 2015 K.R.C. Goly, Y. Soro, A. Dadié,
A.B.B. Kassi, M. Djé Antibacterial activity of
essential oils and extracts from the leaves of
Hyptis suaveolens and Lippia multiflora on multi-
resistant bacteria Rasayan J. Chem., 8 (4) (2015),
pp. 396-403
**Mihin et al., 2019 H.B. Mihin, M.K. Somda, D.
Kabore, S. Sanon, A.Y. Akakpo, A.S. Traore, A.S.
Ouattara Biopreservation of meat by using
antimicrobial properties of essential oil from
Laggera aurita in Burkina Faso Adv. Nutr. Food
Sci., 2019 (2) (2019), pp. 1-13

2.5 Statistical Analysis

For statistical analysis, data were obtained
from three triplicates of each sample, and
expressed as means ± SD using Microsoft Excel
and SPSS statistical software version 21
(Chicago, IL, USA)
Statistical analysis
Statistical data were reading values of inhibition
zones (in diameter) and concentration values
(MIC &MBC) analyzed using SPSS, version 21
according to CLSI. Each experiment values are
expressed as mean ± S.D. Statistical significance
was determined by student’s t-test.Values with p
< 0.05 were considered significant