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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
Biotechnologia Acta Т. 14, No. 6 , 2021
P. 71-79, Bibliography 34, Engl.
UDC: 579.262
https://doi.org/10.15407/biotech14.06.071
A. I. Olkhovska, K. О. Drobot, A. M. Shakhovsky, N. A. Matvieieva
Institute of Cell Biology and Genetic Engineering of the National Academy of Sciences of Ukraine, Kyiv
Artemisia absinthium L. plants are known as producers of substances with antioxidant properties. Among others, phenols and flavonoids are found in these plants. The synthesis of these bioactive compounds can be activated by genetic transformation. This process can be carried out even without the transfer of specific genes involved in the synthesis of flavonoids. Thus, “hairy” roots, obtained after Agrobacterium rhizogenes – mediated transformation, can produce a variety of valuable substances.
The aim of the study was to obtaine A. absinthium “hairy” roots with high phenolic content.
Methods. “Hairy” roots of plants were obtained by co-cultivation leaves with suspension of A. rhizogenes with pCB124 vector. The presence of transferred genes was confirmed by PCR. The reactions with AlCl3 and Folin-Ciocalteu reagent were used to determine the total flavonoids and phenols content. The antioxidant activity of extracts was evaluated by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity.
Results. PCR analysis detected the presence of bacterial rol genes and the absence of рСВ124 plasmid genes. Root lines differed in growth rate. “Hairy” roots were characterized by a higher phenolic content, particularly flavonoids (up to 4.784 ± 0.10 mg/g FW) compared to control (3.861±0.13 mg/g FW). Also, extracts from transgenic roots demonstrated higher antioxidant activity in the reaction with DPPH reagent (EC50 = 3.657 mg) when compared with extracts from control plants (EC50 = 6,716 mg).
Conclusions. Transformation of A. absinthium mediated by A. rhizogenes can be applied for obtaining transgenic root lines with increased phenolic content and higher antioxidant activity.
Key words. Artemisia absinthium L., Agrobacterium rhizogenes-mediated transformation, “hairy” roots of plants, flavonoids, phenolic compounds, antioxidant activity.
© Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, 2021
References
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ISSN 2410-776X (Online)
Biotechnologia Acta Т. 14, No. 6 , 2021
P. 60-70, Bibliography 24, Engl.
UDC: 543.544+547.655.6+577.1
https://doi.org/10.15407/biotech14.06.060
N. Kobylinska1, T. Bohdanovych2, V. Duplij2, I. Pashchenko3, N. Matvieieva1
1 Dumansky Institute of Colloid and Water Chemistry of of the National Academy of Sciences of Ukraine, Kyiv
2 Institute of Cell Biology and Genetic Engineering of of the National Academy of Sciences of Ukraine, Kyiv
3 Kyiv National University of Technologies and Design, Ukraine
Aim. The profiles of polyphenolic phytochemicals in extracts of “hairy” roots of Artemisia tilesii Ledeb. and Artemisia annua L. were studied. Analytical separation and quantification of main components in extracts were evaluated.
Methods. “hairy” roots were grown in vitro on Murashige and Skoog medium. High-performance chromatography coupled with different types of detection (photo diode array detection (DAD) and electrospray ionization with ultra-high resolution Qq-Time-of-Flight mass spectrometry) was used to identify and quantify the main biologically active components in ethanol extracts of “hairy” roots.
Results. The amount of flavonoids was 94.71–144.33 mg RE/g DW and 33.52–78.00 mg RE/g DW in “hairy” roots of A. annua and A. tilesii, respectively. In most samples of “hairy” roots, the amount of flavonoids was higher than the content in the control plant roots. The presence of Apigenin (0.168 ± 0.003 mg/L and 0.178 ± 0.006 mg/L), Quercetin (0.282 ± 0.005 mg/L and 0.174 ± 0.005 mg/L) in the extracts of A. annua and A. tilesii was shown by reverse-phase HPLC-DAD method. Chlorogenic acid, Kaempferol, and other flavonoids were detected.
Conclusions. The developed HPLC-DAD method demonstrated the high percentage of recovery, low limit of detection and quantification (9,11 ng/ml ≤ LOQ ≤16,51 ng/ml), accuracy and correctness. Thus, the method is suitable for the simultaneous quantification of phenolic acids and flavonoids in various plant extracts with short time and high efficiency.
Key words: Artemisia tilesii, Artemisia annua, polyphenols, flavonoids, “hairy” roots, reversed-phase HPLC with diode matrix detector.
© Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, 2021
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ISSN 2410-776X (Online)
Biotechnologia Acta Т. 14, No. 6 , 2021
P. 53-59, Bibliography 9, Engl.
UDC: 577.112.7: 612.115
https://doi.org/10.15407/biotech14.06.053
OBTAINING OF PLANT TISSUE CULTURE Scutellaria baicalensis GEORGI. AND ITS BIOCHEMICAL ANALYSIS
О. О. Poronnik 1, G. Yu. Myriuta 1, V. M. Anishchenko 2, R. V. Ivannikov 2, V. A. Kunakh 1
1 Institute of Molecular Biology and Genetics of the National Academy of Sciences of Ukraine, Kyiv
2 Gryshko National Botanical Garden of the National Academy of Sciences of Ukraine, Kyiv
Aim. To obtain a plant tissue culture of S. baicalensis as a possible source of biologically active compounds (BAC) with a wide range of pharmacological action.
Methods. Plant tissue culture, photocolorimetric method, reversed-phase high performance liquid chromatography (HPLC) method.
Results. Two stably productive plant tissue culture strains (16SB3 and 20SB4) of S. baicalensis were obtained from fragments of roots seedling on a specially developed agar nutrient medium 5С01. The yield of dry biomass from 1 liter of this medium per passage (21st day of growth) for strain 16SB3 is 25–30 g, for strain 20SB4 – 30–40 g. The total content of flavonoids in dry biomass was in terms of routine for strains 16SB3 and 20SB4 – 0.6–0.9 and 0.7–0.9 mg/g, respectively, and the yield of flavonoids – 18–27 and 21–36 mg/l of nutrient medium, respectively. BAC typical for plants in nature, in particular, flavonoids vogonin, baikalein, neobaikalein, skulkapfavon and their derivatives, were found in the studied biomass of both strains.
Conclusions. It was found that the biomass of the two strains of S. baicalensis plant tissue culture accumulated the same BAC, in particular, flavonoids, as do plants in natural conditions. The resulting plant tissue culture is promising as a possible source of Baikal skullcap BAC.
Key words: Scutellaria baicalensis Georgi., plant tissue culture, flavonoids, strains – producers of biologically active compounds.
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2021
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Biotechnologia Acta Т. 14, No. 6 , 2021
P. 44-52, Bibliography 28, Engl.
UDC: 577.112.7: 612.115
https://doi.org/10.15407/biotech14.06.044
O. K. Gulevskyy, Yu. S. Akhatova
Institute for Problems of Cryobiology and Cryomedicine of the National Academy of Sciences of Ukraine, Kharkiv
One of the modern transfusiology tasks is to preserve the properties of erythrocytes after hypothermic storage. Worsening of their functional state due to the storage leads to deterioration of the transfusion medium quality and a number of clinical problems. Plausible protective effects of a human cord blood low-molecular fraction (CBF) and the Actovegin drug were evaluated in the present study in order to use them as components of recovery medium.
Aim. The purpose of our study was to investigate the CBF and the Actovegin drug effect on erythrocytes morphology, energy balance, and oxygen transport function of erythrocytes after their hypothermic storage.
Materials and methods. In the research erythrocyte mass from human donor blood was used, which was stored in hypothermia for 7–21 days. Every 7 days, the CBF or Actovegin (final concentration 0.6 mg/ml) were added to aliquots of erythrocyte mass and incubated for 1 h at 37 °C, followed by further studies. CBF was obtained from human cord blood by ultrafiltration and lyophilization. The erythrocytes morphology was assessed using light microscopy. The content of ATP and 2.3-DPG was determined by the amount of inorganic phosphorus and the photoelectrocalorimetric method. The indicators of erythrocytes oxygen transport function (saturation, oxygen and carbon dioxide tension) were assessed using an analyzer of blood gases and electrolytes IL GEM Premier - 3000. The ratio of hemoglobin forms was studied by photometry.
Results. It was shown that both CBF and the Actovegin drug promoted to restore the morpho-functional characteristics of erythrocytes after 21 days of their storage at 2–4 °C. This was indicative as an increase in the normocytes number increase and restoration of oxygen tension, saturation, ATP and 2,3-DFG content, and normalization of the hemoglobin forms ratio. It was suggested that the mechanisms of the compared tested substances action were associated with ATP synthesis activation and 2,3-DFG formation.
Conclusions. The use of media with containing CBF or the Actovegin drug enabled to restore effectively the properties of erythrocytes disturbed during their prolonged storage.
Key words: cord blood, Actovegin, erythrocytes, hypothermia, recovery medium.
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2021
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- Details
- Hits: 638
ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
Biotechnologia Acta Т. 14, No. 6 , 2021
P. 37-43, Bibliography 32, Engl.
UDC: 577.112.7: 612.115
https://doi.org/10.15407/biotech14.06.037
LIMITED PROTEOLYSIS OF FIBRINOGEN BY PROTEASE OF Gloydius halys halys SNAKE VENOM
Palladin Institute of Biochemistry of of the National Academy of Sciences of Ukraine, Kyiv
Aim. One of the approaches for studying structure and functions of proteins is their limited proteolysis. Proteolytic fragments of macromolecules can preserve the biological activity and can be used for the study of their structural and functional peculiarities. Thus, the characterization of new proteolytic enzymes and determination of the specificity of their action can be of interest for exploration. In the present work, we focused on the action of protease from the venom of Gloydius halys halys on fibrinogen, the crucial protein of blood coagulation system.
Methods. Products of fibrinogen hydrolysis by protease from the venom of G. halys halys were studied by SDS-PAGE electrophoresis and western-blot analysis using monoclonal antibodies ІІ-5 Сand 1-5A targeted to 20–78 and 549–610 fragments of fibrinogen Aα-chain. Molecular weights of hydrolytic products were determined using MALDI-TOF analysis on Voyager DE PRO (USA). Sequence of hydrolytic products were predicted by «Peptide Mass Calculator» soft ware.
Results. SDS-PAGE showed that protease from the venom of Gloydius halys halys initially cleaved Аα-chain of fibrinogen molecule. Western-blot analysis confirmed that this protease specifically cleaves off fragment of C-terminal parts of Аα-chain with apparent molecular weight of 22 kDa. Cleaved fragment was identified by MALDI-TOF analysis as the 21.1 kDa polypeptide. "Peptide Mass Calculator" predicted that such a fragment corresponded to Аα414-610 residue of fibrinogen molecule. Thus, we showed that studied protease cleaved peptide bond AαK413-L414 with the formation of stable partly hydrolyzed fibrinogen desAα414-610.
Conclusions. The use of protease from the venom of Gloydius halys halys would allow obtaining the unique partly hydrolyzed fibrinogen des Aα414–610 that is suitable for the study of structure and functions of fibrinogen αС-regions.
Key words: fibrinogen, limited proteolysis, protease, fibrin polymerization, hemostasis.
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2021
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