Page 103 - Book of Abstracts

5 International Scientific Online Conference DOI: https://doi.org/10.15414/2021.9788055224015

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IN VITRO HEMOLYSIS ASSESSMENT OF THYME ESSENTIAL OIL AND ITS PROTECTIVE
EFFECT ON OXIDATIVE DAMAGE OF HUMAN ERYTHROCYTES
1
Maryna Opryshko , Myroslava Maryniuk , Oleksandr Gyrenko , Lyudmyla Buyun ,
1
1
1
Halyna Tkachenko , Natalia Kurhaluk
2
2
1 M.M. Gryshko National Botanic Garden, National Academy of Science of Ukraine, Kyiv, Ukraine;
E-mail.: maryna.opryshko@meta.ua
2 Institute of Biology and Earth Sciences, Pomeranian University in Słupsk, Poland
As the hemolytic activity of any compounds is considered as an indicator of general
cytotoxicity toward normal healthy cells, the main objective of this study was to evaluate the
dose-dependent hemolytic/antihemolytic potential of thymus essential oil (TEO) using a
human erythrocyte model.
The venous blood (10–20 ml) was obtained from normal volunteers via venipuncture (7
females aged 23–36-years old). The Research Ethics Committee of the Regional Medical
Commission in Gdańsk (Poland) approved the study (KB-31/18). Human erythrocytes from
citrated blood were isolated by centrifugation at 3,000 rpm for 10 min and washed two times
with 4 mM phosphate buffer (pH 7.4) and then re-suspended using the same buffer to the
desired hematocrit level. The blood sample was incubated with TEO (Etja, Elbląg, Poland),
respectively (final concentrations were 20 and 10 μg/mL) for 15 min at 25 °C. Samples were
removed at 20 min of storage for analysis. The untreated erythrocyte samples were used as the
control sample. The osmotic-induced hemolysis of erythrocytes was measured
spectrophotometrically with different 0.1–0.9 % NaCl solutions. The assay is based on the
measuring of the percent of erythrocytes disintegration into hemolytic reagent action (0.1–0.9
% NaCl). The absorbance was read at 540 nm. The disintegration of erythrocytes (%) at
different NaCl solutions was expressed as a curve.
It was observed that, TEO in final concentration 20 μg/mL caused a significant increase of
hemolysis in the samples with 0.9–0.5 % NaCl solutions: 13.3 % vs. 4.1 % (0.9 % NaCl), 15.4 %
vs. 4.3 % (0.8 % NaCl), 16.7 % vs. 5.6 % (0.7 % NaCl), 17.8 % vs. 5.7 % (0.6 % NaCl), and 58 %
vs. 18.2 % (0.5 % NaCl). The increase of hemolysis was 3.3-fold (0.9 % NaCl), 3.6-fold (0.8 %
NaCl), 3-fold (0.7 % NaCl), 3.1-fold (0.6 % NaCl), 3.2-fold (0.5 % NaCl), respectively. On the
contrary, after incubation in solutions of 0.4–0.1 % NaCl, adding of TEO to the human
erythrocytes caused the decrease of hemolysis (by 14.9 %, 10.5 %, 7.1 %, and 4.7 %,
respectively). Treatment of the human erythrocytes by TEA in final concentration 10 μg/mL
caused a statistically significant increase of hemolysis in the samples with 0.9–0.6 % NaCl
solutions: 5.8 % vs. 1.2 % (0.9 % NaCl), 6.9 % vs. 1.2 % (0.8 % NaCl), 9.3 % vs. 1.5 % (0.7 %
NaCl), and 13.3 % vs. 2 % (0.6 % NaCl). The increase of hemolysis was 4.8-fold (0.9 % NaCl),
5.6-fold (0.8 % NaCl), 6.4-fold (0.7 % NaCl), and 6.8-fold (0.6 % NaCl), respectively. In the
sample with 0.5–0.1 % NaCl, a difference between the percentage of hemolysis in samples after
adding TEO to the erythrocytes suspension compared to the untreated samples was non-
significant (decrease of hemolysis by 2.5 %, 3.3 %, 2.9 %, and 0.1 %). In the sample with 0.5–
0.1 % NaCl, TEO added to human erythrocyte suspension in final concentration of 10 μg/mL
exhibited anti-hemolytic activity. Thus, TEO screened in this study possessed good anti-
hemolytic activities using the human erythrocytes model. The interest in the formulation of
pharmaceuticals, nutraceuticals, and cosmeceuticals based on thymol is due to several studies
that have evaluated the potential therapeutic uses of this compound for the treatment of
disorders affecting the respiratory, nervous, and cardiovascular systems. Moreover, this
compound also exhibits antimicrobial, antioxidant, anti-carcinogenesis, anti-inflammatory, and
antispasmodic activities, as well as a potential as a growth enhancer and immunomodulator.

Keywords: hemolysis, erythrocytes, thymus essential oil, antioxidants.

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Spiritual Human Development
November 3 2021
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