Archives of

1. Introduction
The increasing prevalence of antibiotic-resistant hospital infections is one of the problems that patients and physicians encounter. The number of effective and available antibiotics for the treatment of these infections is continuously decreasing [1]. Nosocomial infections have always been among the major health and treatment problems and recently they have become more important, posing a serious challenge to the health systems of countries. The increased number of hospitals, the emergence of new infections, increasing microbial resistance, and the need for various medical services have made the occurrence of hospital infections inevitable. Therefore, the control of hospital infections is now considered a global priority [2]. Therefore, many efforts have been made to find new compounds as suitable alternatives to existing antibiotics [3]. Plants produce different types of bioactive substances that introduce them as a rich source of medicinal substances [4].
Due to the serious side effects of consumed antibiotics and the resistance that pathogenic bacteria have acquired against them, there is a growing scientific consensus that the inappropriate use of antibiotics to treat infectious diseases causes the development of bacterial antibiotic resistance. As a consequence, the use of natural antimicrobial agents is now considered important because it helps reduce the disadvantages of traditional medicine [5]. In medical science, much attention has been paid to extracts and compounds of plant origin with biological properties [6]. The use of medicinal plants has been growing in the past few years due to their fewer side effects compared to chemical drugs. Plant extracts are among the sources of antibacterial compounds against pathogenic bacteria [7,8].
Date palm (Phoenix dactylifera L.) is a monocotyledonous and tropical plant that is a member of the palm family [9]. Dates contain minerals such as iron, potassium, zinc, and vitamins such as A, B, and C [10,11]. Carbohydrates, amino acids, and fatty acids are also abundantly found in them [10]. This fruit contains large amounts of antioxidant, antimutagenic, anthocyanin, phenolic compounds, and free acids [7]. Dates contain sucrose, glucose, polyphenol compounds, water, proteins, salts, fat, mineral salts (such as iron, potassium, manganese, and zinc), vitamins (A,
Evaluation of Antimicrobial Properties of Bam Date Kernel Extract and Investigation of the Structure of Extract Evaluation of antimicrobial properties of bam date kernel extract
B, C), and various enzymes (such as polyphenol oxidase,
invertase, etc) [10,11]. Since ancient times, both date fruits
and kernels have been used in various traditional and folk
medicinal systems where the date palm is growing [12,13].
Based on current knowledge, it has been considered
that phenolic compounds in date extracts are mainly
responsible for the antimicrobial activities reported in the
literature. This is believed to be caused by the ability of
these chemicals to bind to the bacterial cell wall, which
also explains their greater effectiveness against Grampositive
bacteria [14,15]. In addition, phenolic compounds
produce hydrogen peroxide, which minimizes microbial
growth [16]. Rutin, catechins, and sinapic acid are
common flavonoids in food sources that have various
properties, including anti-bacterial activity [17-19].
Considering the increasing need to find new antibacterial
compounds and to investigate the applications of these
compounds, the purpose of this research was to prove
the antibacterial activity of the extract of Mazafati date
kernel from Bam city on several hospital bacteria whose
antibacterial effect has not been proven until now.
2. Materials and Methods
2.1. Sample collection
Packaged Mazafati dates from Bam city were prepared and
after separating the kernels from the fruit, the date kernels
were completely washed with tap water for 5 minutes to
remove possible surface contamination. Then, 200 g of
date kernels was crushed into a powder and mixed with
200 mL of acetone. Then, it was placed in a shaker at 130
rpm for 48 hours at room temperature. After 48 hours, the
homogenized mixture was filtered twice with Whatman
No. 1 filter paper. Next, the solvent was separated from
the extract by a rotary evaporator, and with the help of
a vacuum pump (vacuum distillation), the remaining
acetone was removed from the solution. Afterwards, the
date kernel extract was dried in an oven. Then, the date
kernel extract was hydrated in a nutrient broth, passed
through a 0.22 μm filter, and stored at -80°C for further
tests. The final concentration of date kernel extract was
1000 mg/mL [20].
2.2. Bacterial pathogens
In this research, hospital pathogens (Staphylococcus aureus
ATCC 12600, Escherichia coli ATCC 10536, Pseudomonas
aeruginosa ATCC 27853, Acinetobacter baumannii ATCC
BAA-747, and Salmonella enterica CCM 3807) were used
to investigate the antibacterial effect of date kernel extract.
The pathogens were purchased from the Scientific and
Industrial Research Organization of Iran.
2.3. Antibacterial Effect
First, a suspension of the tested strains with turbidity
equal to 0.5 McFarland standard was prepared and
uniformly inoculated on Mueller-Hinton agar (MHA)
culture medium. Then, using a sterile Pasteur pipette,
wells with a diameter of 6 to 8 mm were created in MHA
culture medium at regular intervals. Date fruit extract at
concentrations of 500 and 1000 mg/mL was poured into
the well. Then, the plates were incubated for 24 hours at a
temperature of 37 °C and the activity of date kernel extract
on the tested bacteria was determined by measuring the
diameter of the inhibition zone [21].
2.4. Minimum inhibitory concentration (MIC)
The broth microdilution method was used to measure the
MIC. According to the references, serial dilutions were
prepared in a 96-well microplate with a volume of 200 μL
for each well, so that the first well contained the highest
concentration and the last well contained the lowest
concentration of date kernel extract. Next, from the 24-
hour culture of the tested bacteria, a bacterial suspension
of 0.5 McFarland standard was prepared and 20 μL of the
final inoculation suspension was added to each well. One
well was considered a positive control (containing 10 μL
of microbial suspension in the well without date kernel
extract) and one well was a negative control (containing
200 μL of medium without bacterial inoculation and date
seed extract). Then, the microplates were incubated at
37°C for 24 hours. After the incubation, bacterial growth
was evaluated in terms of turbidity. The MIC value was
defined as the lowest concentration of date kernel extract
at which no macroscopic growth was detectable. The
results were recorded by ELISA readers (Hiperion MRP
4 + , Germany) [22].
2.5. Minimum bacterial concentration (MBC)
First, 10 μL of the content of the wells in which the
inhibitory concentration of bacterial growth was observed
was poured on the plates containing MHA, and the plates
were incubated for 24 hours at 37 °C. Then, the plates
were examined after 24 hours. The amount of MBC was
determined based on the minimum concentration of
the antimicrobial substance that prevents the growth of
bacteria on the plate [22].
2.6. Analysis of extract structure by HPLC
The structure of the extract was investigated using a
high-performance liquid chromatography (HPLC) device
(Agilent HPLC 1260 Infinity II). The device had a fourchamber
pump, a sampler, and an automatic detector. The
column type was Zorbax Eclipse, the size of the column
was 150 × 4.6 mm, and the injection volume was 20 μL.
Electrospray ionization settings included: temperature of
30 °C, electrospray ionization of 4500 volts, curtain gas
with 8 psi pressure, and CEM detector (2300 V). A 1%
solution of formic acid in methanol was prepared with
volume ratios of 10:90, 25:75, 60:40, and 70:30. The mobile
phase flow rate was 1 mL/min. The phenolic compounds
in the extract were identified by comparing the inhibition
Monajjemi et al
12 Arch Hyg Sci. Volume 13, Number 1, 2024
time of the compounds with the standard compound and
their amount was expressed as a percentage [23].
2.7. Statistical analysis
All experimental procedures were performed with three
repetitions. Microsoft Excel was used for statistical and
graphical analysis of the data. The data were analyzed by
one-way analysis of variance and expressed as mean and
standard deviation.
3. Results
3.1. Antimicrobial effect of date kernel extract
The MIC of date kernel extract for Escherichia coli,
Salmonella enterica, and Pseudomonas aeruginosa was
500 mg/mL and for Staphylococcus aureus, it was 400
mg/mL. However, a decreasing trend was observed for
Acinetobacter baumannii.
MBC test results were negative for the tested
concentrations. The antimicrobial effects of the extract were
investigated at concentrations of 500 and 1000 mg/mL.
MIC and MBC data and a comparison of the
antimicrobial effect of the examined concentrations are
shown in Table 1.
3.2. Structural investigation of date kernel extract by
HPLC
Based on the results, 7 effective compounds including
gallic acid, catechin, chlorogenic acid, rutin, vanillin,
quercetin, and sinapic acid were identified in date kernel
extract. The findings showed that rutin was the most
effective composition of date kernel extract, followed by
catechin, sinapic acid, chlorogenic acid, vanillin, gallic
acid, and quercetin, respectively. The effective compounds
in date kernel extract are listed in Table 2. The chemical
composition of date kernel extract is given in Figure 1.
4. Discussion
The antimicrobial activity of date kernel extract was
determined by the well diffusion method, the MIC, and
the MBC. The results of this study showed that the acetone
extract of date kernel had antimicrobial capacity against S.
aureus, A. baumannii, S. enterica, E. coli, and P. aeruginosa.
Moreover, the HPLC analysis showed the presence of
gallic acid, catechin, chlorogenic acid, rutin, vanillin,
quercetin, and sinapic acid in the date kernel extract.
In previous studies, the presence of various compounds
including gallic acid [24], chlorogenic acid [25], and rutin
[26] and their antimicrobial effects have been proven.
The antimicrobial effect of date kernel extract in the
present study can be attributed to the presence of these
compounds in the extract and it can be stated that the date
kernel has various valuable antimicrobial compounds.
In the study conducted by Shariati et al, the
antimicrobial activity of date fruit and kernel extracts was
investigated against S. aureus strains and it was found that
none of the acetone and ethanol extracts of date fruit had
an antimicrobial effect on the above-mentioned bacteria
[3]. In the study by Shariati et al, date fruit extract had no
antimicrobial effect, but date kernel extract, like the extract
used in the present study, showed the antimicrobial effect
against the investigated pathogens, including S. aureus.
Celik et al evaluated the antimicrobial activity of date
kernels (Fructus dactylus). They found that aqueous
and methanol extracts of date kernels had high phenolic
and flavonoid compounds, and this extract had high
antibacterial activity against 11 bacteria. The MIC was in
the range of 12.50 to 250 mg/mL [27]. In both studies,
date kernel extract showed a strong antimicrobial effect,
and the common results prove the value of examining and
determining the content of date kernels in future research.
Aljazy et al investigated the effect of date kernel extract
on several hospital pathogens including A. baumannii,
Bacillus subtilis, E. coli, Klebsiella pneumoniae, P.
aeruginosa, Proteus mirabilis, S. aureus, and Streptococcus
pyogenes and showed that the ethanolic extract was the
most effective extract compared to other extracts in
preventing the growth of pathogens [28]. In the present
Table 1. Antimicrobial activity of date kernel extract on bacteria
Bacterial Strains MIC (mg/mL)
MBC (mm)
500 (mg/mL) 1000 (mg/mL)
Escherichia coli 500 20 30
Salmonella enterica 400 13 22
Pseudomonas aeruginosa 500 11 17
Staphylococcus aureus 500 21 31
Acinetobacter baumannii N/A 10 15
N/A, Not-available.
Table 2. Chromatogram characteristics of date kernel extract with HPLC
Compound mg/L Retention time (min) Width (min) Area (mA.s) Height (mA) Area (%)
Gallic acid 1.49 3.23 0.8 239.13 31.26 20.32
Catechin 24.1 7.3 0.20 115.22 9.75 11.10
Chlorogenic acid 5.18 10.9 0.21 109.88 7.11 9.77
Rutin 31.67 13.1 0.19 95.39 8.84 7.53
Vanillin 5.3 14.4 0.16 327.39 27.65 28.30
Quercetin 1.03 15.1 0.15 62.56 6.23 6.66
Sinapic acid 19.44 16.9 0.22 95.38 6.19 7.8
Arch Hyg Sci. Volume 13, Number 1, 2024 13
Evaluation of antimicrobial properties of bam date kernel extract
study, the acetone extract of date kernel was used, and the
comparison of the results of Aljazy et al (28) with ours
shows that both types of extracts have strong antimicrobial
properties.
In a study, Al-Shwyeh investigated the antimicrobial
properties of date (P. dactylifera L) and showed that due
to the presence of phenolic compounds, antioxidants,
benzoic acid, cinnamic acid, and flavonoids, date fruit had
strong biological activities against several gram-positive
and gram-negative bacterial pathogens and it showed the
best activity against E. coli bacteria [29]. In both studies,
the antimicrobial properties of date fruit and kernel
extracts were shown, and it can be said that dates have
different bioactive compounds with strong antimicrobial
effects. However, in the study by Shariati et al (3), date
kernel extract had no antibacterial effect.
Alrajhi et al investigated the antibacterial activity of date
palm extract (P. dactylifera) in a study. The study aimed
to evaluate the active compounds of date seeds in terms
of their antimicrobial activity. The extraction of date seed
powder was performed using high-polarity solvents such
as hexane and ethyl acetate by cold extraction method. The
isolates were later tested for antibacterial activity against
Gram-positive and Gram-negative bacteria including
P. aeruginosa, E. coli, K. pneumoniae, Proteus vulgaris,
P. mirabilis, methicillin-resistant S. aureus (MRSA),
and Enterococcus faecalis using the agar well diffusion
method. The potential antibacterial activity of the ethyl
acetate extract was observed against a large number of
gram-positive pathogens [30]. In the study by Alrajhi
et al, the obtained date extract showed antimicrobial
activity against a wide range of hospital pathogens like the
present study, and the findings of both studies confirm
the antimicrobial properties of date extract.
Boudghane et al investigated the antimicrobial properties
of different extracts of Algerian date seeds. Based on the
results of this research, the methanolic extract had the
highest percentage of phenolic and tannin compounds
and this extract showed the highest antimicrobial activity
[31]. Bhaskaracharya et al in a systematic review of the
antibacterial activity of polyphenol extract of date kernel
(P. dactylifera L) showed that similar to other fruit
processing industry by-products, date kernels, waste
from date processing industry, are rich in extractable
polyphenols. The rich polyphenolic content suggests
that date kernel extracts can be a cost-effective source of
antimicrobial agents; however, their antibacterial activity
is not well understood [32]. In the present study and
studies conducted by Boudghane et al and Bhaskaracharya
et al, date kernel extract, which has different metabolites,
showed antimicrobial properties against the investigated
pathogens, indicating the importance of using the
basic compounds of this plant in dealing with hospital
pathogens.
5. Conclusion
Today, due to the increase in bacterial resistance in various
types of pathogens, there is a need for research to find new
compounds with antibacterial properties. According to
the results of the experiments conducted in this research,
it can be claimed that the extract of Mazafati date kernel
from Bam city has an antibacterial effect on the tested
pathogens, including E. coli, S. aureus, A. baumannii, S.
enterica, and P. aerogenes. Therefore, by conducting more
research on the date kernel extract and comparing it with
the antimicrobial properties of other plant extracts, some
compounds can be found to combat antibiotic resistance
of pathogens.
Acknowledgments
We would like to thank the staff of Lab Microbiology, School of
Medicine, Qom University of Medical Sciences for providing the
necessary equipment to carry out the research.
Authors’ Contribution
Conceptualization: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi.
Data curation: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi.
Formal analysis: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi.
Funding acquisition: Maryam Tabibi, Alireza Rasouli, Ali
Monajjemi.
Investigation: Maryam Tabibi, Alireza Rasouli, and Ali Monajjemi.
Methodology: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi,
Gholam Ali Jafari, Amir Arsalan Asgari, Fateme Kheiri.
Project administration: Maryam Tabibi, Alireza Rasouli, Ali
Monajjemi.
Resources: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi.
Figure 1. Comparison of the amount of date extract compounds
1.49
24.1
5.18
31.67
5.3
1.03
19.44
-10
0
10
20
30
40
Gallic acid Catechin Chloregenic
acid
Rutin Vanilin Quercetin Sinapic acid
mg/L
Date seed extract
Monajjemi et al
14 Arch Hyg Sci. Volume 13, Number 1, 2024
Software: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi
Supervision: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi.
Validation: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi, Gholam
Ali Jafari, Amir Arsalan Asgari, Fateme Kheiri.
Visualization: Maryam Tabibi, Alireza Rasouli, Ali Monajjemi.
Writing–original draft: Maryam Tabibi, Alireza Rasouli, Ali
Monajjemi.
Writing–review & editing: Maryam Tabibi, Alireza Rasouli, Ali
Monajjemi.
Competing Interests
The authors declared no conflict of interests regarding the
publication of the current article.
Ethical Approval
This study was approved by the ethics committee of Qom University
of Medical Sciences (Research Code: 3336).
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or non-profit sectors.
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