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Microbes & Immunity Hyphaene thebaica honey as a multitarget therapeutic product

inflammation encompass anti-inflammatory medications 2.2. Assessment of antibacterial activities
and immunosuppressants, which unfortunately can induce The microbial strains used in the current investigation
side effects such as gastrointestinal ulcers, cardiovascular were Pseudomonas aeruginosa strain 27853, Staphylococcus
toxicity, hormonal imbalances, and disruptions to the aureus strain BAA-1026, Escherichia coli strain 25922,
body’s normal functions. 23,24 Therefore, it is of paramount Streptococcus strain 49619, Klebsiella quasipneumoniae
importance to explore the incorporation of natural anti- strain 700603, Haemophilus influenzae strain 49247, and
inflammatory substances into treatment regimens to Bacillus subtilis strain 6633, which were purchased from
enhance pharmacological response and mitigate adverse the American Type Culture Collection (ATCC; Manassas,
effects. Recent studies have demonstrated promising VA, USA). The minimum inhibitory concentration (MIC)
results in utilizing natural products such as various types and minimum bactericidal concentration (MBC) of DH
of honey for the treatment of ulcers. 25 samples in 96-well, flat-bottomed micro-titration plates,
The antioxidant properties of honey play a critical role in were determined as described previously. 31
lowering the levels of ROS generated during inflammatory 2.3. Cell culture
processes. Elevated ROS levels are associated with the
development of serious health conditions, including The MDA-MB-231 cell line (European Collection
cardiovascular, muscular, metabolic, neurodegenerative, of Authenticated Cell Cultures [ECACC] catalog
and cancer-related diseases. The antioxidant capacity of no. 92020424) is supplied by the ECACC, which is part
honey primarily stems from its phenolic compounds, of the UK Health Security Agency and the THP-1 human
especially flavonoids, which effectively neutralize ROS and monocytic cell line (ATCC: TIB-202) were cultured in
interact with metals. Furthermore, flavonoids contribute Dulbecco’s Modified Eagle Medium (DMEM; Biological
to regulating enzymes, thereby boosting their antioxidant Industries, Israel) supplemented with 10% fetal calf serum,
activity and influencing various biological functions. 26-28 1% non-essential amino acids, 1% glutamine, 100 U/mL
penicillin, and 10 μg/mL streptomycin to maintain optimal
Hyphaene thebaica and Doum honey (DH) are growth conditions.
extensively utilized throughout the Mediterranean region
due to its health-enhancing properties, such as anti- 2.4. Evaluation of cytotoxic and cytostatic effects
infection, anti-inflammatory, and anticancer attributes. 29,30 In the cytotoxicity experiments, 20,000 MDA cells per
Scientific evidence on the health advantages of DH remains 100 μL of media were seeded in 96-well plates and
largely unexplored, given that studies on this specific incubated for 24 h. Subsequently, they were exposed to 0
honey variety are not as comprehensive as those on the – 4000 μg/mL of DH for 24 h. The cytotoxic impacts were
more frequently researched types such as Manuka honey. then evaluated using an MTT assay.
H. thebaica L., commonly known as the Doum plant, is
a desert tree native to regions such as the Mediterranean In the cytostatic experiments, 5,000 MDA cells per
region, Sub-Saharan Africa, and West India. 29,30 The 100 μL of media were seeded in 96-well plates and
precise cellular and molecular mechanisms underlying incubated for 24 h. These cells were then exposed to a
the traditionally asserted antibacterial, antioxidant, anti- range of honey concentrations, from 0 to 4000 μg/mL, over
inflammatory, and anticancer properties of the Palestinian a period of 72 h. The MTT assay was employed to assess
DH remain largely unexplored. This study is designed the cytostatic effects, following the methodology outlined
to assess these effects and analyze the polyphenolic by Abu-Farich et al. 32
composition of DH. 2.5. Scratch assay

2. Materials and methods MDA cells were seeded at a density of 400,000 cells per well
in a 12-well plate (Corning Costar Corporation, Corning,
2.1. DH sample collection and preparation
NY, USA) with 2.5 mL of culture medium and incubated
The DH samples, which were collected from the Jordan for 24 h in a humidified atmosphere containing 5% CO₂ at
Valleys in 2021, were obtained from a reputable supplier 37°C. After incubation, monolayer formation in each well
known as “Honey Spring,” situated in Tulkarem in the was confirmed under a microscope. Scratches were then
Northern West Bank. Following their purchase, these created across the monolayers using sterile 200 μL pipette
samples were sealed in cans for preservation and stored tips. The culture medium was removed, and each well was
under ambient conditions in a moisture-free environment. washed 4 times with DMEM devoid of serum and additives.
They remained in this unopened state until further Following this, 4 mL of either 4 mg/mL DH solution or
experimental procedures. culture medium (for untreated control) was added to the

Volume 2 Issue 1 (2025) 80 doi: 10.36922/mi.4994