Page 47 - JCTR-11-3

Journal of Clinical and
Translational Research Greek propolis use in COVID-19: Trial protocol

exceeding 10 days in COVID-19 patients, which may vary strong antioxidant activity comparable to quercetin.
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according to age, comorbidities, and available resources. 38 Mediterranean propolis is also notable for its diterpene
content, compounds with documented antibacterial and
6.2. Secondary study outcomes anti-inflammatory properties. 49,50

Body temperature will be measured orally using a Extraction will be conducted through ultrasound-
calibrated digital thermometer, as oral thermometry is a assisted extraction (UAE) at 100% amplitude for 30 min
widely accepted clinical method offering a practical balance at 58°C. Before extraction, crude propolis will be stored
between patient comfort and reliability. Assessments will at −20°C in the dark to preserve its bioactive integrity.
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be conducted on days 1 (baseline), 5, 7, 10, 14, 15, 16, The raw material will then be ground into powder,
and 17. If a patient’s body temperature returns to normal and UAE at 100% amplitude will be used as indicated
and remains so for 48 h, then no further temperature above, in a hydroalcoholic mixture (ethanol: water)
measurements will be conducted. 70:30 v/v, in a solvent-propolis ratio (w/v) 1:5. UAE
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The severity and duration of patients’ coughs will be combines mechanical and thermal effects that enhance
assessed through a cough Visual Analog Scale, which will be solvent penetration, disrupt cellular structures, and
validated for both acute and subacute cough conditions. 39,40 significantly improve the yield of polyphenols compared
In addition, a revised Leicester Cough Questionnaire to conventional maceration techniques. 52-55 Following
(LCQ) will be used for the assessment of cough-specific filtration and re-extraction, the combined supernatants
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QoL, altering its duration to 2 days (Figure A1). The time will be spray-dried, a method shown to preserve and even
point of the initial evaluation will be day 1 (baseline), enhance the antioxidant activity and phenolic content of
and the revised LCQ will be reassessed for patients every propolis compared to vacuum-drying alternatives. 56,57 This
second day for 20 days. If cough resolution occurs earlier, approach allows for alcohol-free, tasteless capsules that
the assessments will be ceased. Its responsiveness will be enhance palatability and enable indistinguishable placebos,
determined by calculating the effect size of the change supporting the integrity of the triple-blind design.
between baseline and cough resolution. The resulting powder will be encapsulated into hard
Other study outcomes include the assessment of the gelatin capsules delivering 800 mg/day over 10 days, based
safety of the intervention and mortality rates. For these on dosing used in prior COVID-19 clinical trials without
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outcomes, a calculation and percentage comparison of reported adverse events. Placebo capsules will be identical
adverse and serious adverse events, as well as mortality in appearance and composition, excluding active propolis,
rates among trial arms, will be conducted. The time frame as conducted by other researchers. 58
for these assessments will be set to 1 – 30 days.
7.2. Chemical characterization
7. Intervention, chemical characterization, Before formulation, raw propolis will undergo scanning
and quality assurance electron microscopy for morphological characterization

7.1. Intervention and screening for contaminants. Qualitative chemical
analysis will confirm the presence of key phytochemical
The intervention will consist of a standardized 20% (w/v) classes, including flavonoids, glycosides, phenols, and
hydroalcoholic extract of Greek poplar-type propolis, terpenoids. Balsam yield will be calculated gravimetrically
intended to be produced under Good Manufacturing using Popova’s validated protocol. Specifically, 2 mL of
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Practice conditions. Hydroalcoholic extracts outperform the extract will be evaporated in a vacuum oven to constant
other extraction types (e.g., aqueous, oil-based, weight, and the balsam yield will be calculated using the
supercritical) in terms of flavonoid and polyphenol yield, following formula in Equation I:
particularly when employing ethanol: water ratio of 70:30
with a solvent-to-propolis ratio of 5:1, which maximizes Yield (%) = (Weight of dry ethanolic extract/Weight of
bioactive content and yields wax-free tinctures. 42-48 crude propolis) × 100 (I)
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Propolis will be sourced from the Imathia region of This procedure, first proposed by Popova et al. and
Greece, known for its high phenolic content and bioactive subsequently used by others, ensures consistent estimation
flavonoids with demonstrated in silico activity against of extraction efficiency across batches.
SARS-CoV-2. This selection is further supported by the Quantification will focus on three principal
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findings of Kasiotis et al., who identified the Imathia bioactive groups: flavones/flavonols, flavanones/
region’s propolis as having the highest total phenolic dihydroflavonols, and total phenolics, reflecting the
and flavonoid content among Greek samples, along with taxonomic signature of poplar-type propolis. These
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Volume 11 Issue 3 (2025) 41 doi: 10.36922/jctr.24.00073