Page 33 - Book of Abstracts

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

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EVALUATION OF ACID-INDUCED HEMOLYSIS OF EQUINE ERYTHROCYTES EXPOSED
TO EXTRACTS FROM LEAVES OF SOME COELOGYNE SPECIES LINDL. (ORCHIDACEAE)
Lyudmyla Buyun , Halyna Tkachenko , Natalia Kurhaluk , Maryna Opryshko ,
1
2
2
1
Lyudmyla Kovalska , Oleksandr Gyrenko
1
1
1 M.M. Gryshko National Botanic Garden, National Academy of Science of Ukraine, Kyiv, Ukraine;
E-mail.: buyun@nbg.kiev.ua
2 Institute of Biology and Earth Sciences, Pomeranian University in Słupsk, Poland
In the present study, by using equine erythrocytes as an in vitro model, the membrane-
disruptive effects of extracts obtained from leaves of the Coelogyne genus have been explored
to gain fresh insight into the extract toxicology. The leaves and pseudobulbs of orchids, i.e. C.
flaccida Lindl., C. huettneriana Rchb.f., C. speciosa (Blume) Lindl., C. fimbriata Lindl., C. tomentosa
Lindl., C. ovalis Lindl., C. cristata Lindl., C. viscosa Rchb.f., C. asperata Lindl., C. pandurata Lindl.,
C. assamica Linden & Rchb.f., C. brachyptera Rchb.f., C. rochussenii de Vriese cultivated under
glasshouse conditions, were sampled at M.M. Gryshko National Botanical Garden (Kyiv,
Ukraine). Freshly collected leaves were washed, weighed, crushed, and homogenized in 0.1 M
phosphate buffer (pH 7.4) (in proportion 1:19, w/w) at room temperature. Blood was drawn
from the jugular vein of the animals. A volume of 0.1 ml of the plant extract was added to 1.9 ml
of equine erythrocytes. For positive control, phosphate buffer was used. After incubating the
mixture at 37 °C for 60 min with continuous stirring, it was centrifuged at 3,000 rpm for 5 min.
The acidic resistance of erythrocytes was induced by 0.5M HCl, and erythrocyte hemolysis was
measured spectrophotometrically.
According to the results from the protective effect of extracts obtained from leaves of
selected Coelogyne species, the incubation time of 1 h was chosen for assessing the HCl-induced
hemolysis in the equine erythrocyte suspension. At 1 h of incubation, the maximum percent of
hemolyzed erythrocytes of untreated erythrocytes comprised (8.3 ±0.64 %). The results
indicated that all extracts led to an increase in hemolysis percentage (ranged from 21.7 to 73.5
%, respectively). When erythrocytes were incubated with C. flaccida and C. pandurata extracts,
the hemolysis level was maximal (73.5 and 67.5 %, respectively) compared to the untreated
control. In the meantime, the treatment of C. assamica and C. tomentosa extracts caused a non-
significant increase in the formation of hemolyzed erythrocytes by approximately 21.7–24.1 %,
respectively. However, C. asperata, C. viscosa, C. fimbriata, C. ovalis, and C. cristata had a
significant increase in hemolysis level in the extract-treated erythrocytes (ranged 49 to 59 %,
p<0.05). The current results are in agreement with our previously obtained data, including
assessment of antioxidant activity of extracts obtained from leaves and pseudobulbs of selected
species from the Coelogyne genus. Further, our results suggest that high doses of extracts might
be a good candidate for inducing oxidative stress in cells, e.g., for targeting the diverse forms of
tumor cells, while low doses of extracts could exhibit antioxidant properties. Future dose-
dependent studies will be performed to determine the optimal dose for an exhibition of
antioxidant properties of these extracts. Moreover, it is well evidenced that there is a significant
discrepancy between data obtained in vitro and in vivo experiments. Nevertheless, the results
obtained from in vitro bioassay could be considered as a predictor of in vivo response.
Keywords: Coelogyne, leaves, extracts, HCl-induced hemolysis, equine erythrocytes.

Acknowledgments
We are grateful to the Polish National Commission for UNESCO for supporting our study. Since 1999 the
whole collection of tropical and subtropical plants (including orchids), maintained both ex situ and in
vitro, has the status of a National Heritage Collection of Ukraine and is supported through State funding.

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5 International Scientific Conference Agrobiodiversity for Improving the Nutrition, Health, Quality of Life and |32
Spiritual Human Development
November 3 2021
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