Naslov (srp)

Biološka aktivnost izolovanih naftohinona i ekstrakata rizoma biljke Onosma visianii Clem

Autor

Vukić, Milena, 1990-, 27724391

Doprinosi

Vuković, Nenad, 1976-, 10962023
Tešević, Vele, 1963-, 12643943
Baskić, Dejan, 1970-, 13558631
Mladenović, Milan, 1984-, 19573607
Mihailović, Vladimir, 1984-, 14099303
Anđelković, Marija, 1977-, 24640359

Opis (srp)

Biljne vrste roda Onosma L se od davnina primenjuju kao prirodne boje. Cvet nekih vrsta se koriste kao povrće u ishrani dok se njihov koren u tradicionalnoj medicini koristi za lečenje rana, opekotina, otežanog disanja, promuklosti, hemoroida, bolova u abdomenu i čireva u želucu. Ipak, kada je reč o vrsti Onosma visianii Clem do sada nisu postojala naučna saznanja o hemijskom sastvu i farmakološkoj aktivnost njenog korena. Prvi zadatak ove disertacije sastojao se u izolovanju bioaktivnih naftohinonskih derivata iz korena biljke O. visianii Clem. Primenom različitih hromatografskih tehnika (stubne hromatografije-silika gel kao adsorbens, preparativne tankoslojne hromatografije-silika gel kao adsorbens, hromatografije na koloni Sephadex-LH20, preparativne tečne hromatografije (HPLC)) optimizovana je efikasna metoda za izolovanje naftohinonskih derivata iz ekstrakta dobijenog hladnom maceracijom primenom rastvaračke smeše petrol etar:metilen hlorid (1:1 v/v). Ovom metodom izolovano je sedam jedinjenja i to deoksišikonin, izobutirilšikonin, αmetilbutirilšikonin, acetilšikonin, β-hidroksiizovalerilšikonin, 5,8-O-dimetil izobutirilšikonin (prvi put identifikovan u prirodnom izvoru) i 5,8-O-dimetil deoksišikonin. Njihova strukturna karakterizacija izvršena je primenom UV-Vis, IR, 1H NMR i 13C NMR spektroskopskih tehnika, kao i upotrebom masene spektrometrije visoke rezolucije. Sledeći zadatak sastojao se u utvrđivanju najpogodnijeg rastvarača za ekstrakciju bioaktivnih komponenata korena biljke O. visianii Clem. Metodom hladne maceracije dobijeno je pet ekstrakata (acetonski, hloroformski, etil acetatni, metanolni i petrol etarski). Upotrebom HPLC analize utvrđeno je da ekstrakti hloroforma, acetona i etil acetata predstavljaju bolje rastvarače za preliminarnu ekstrakciju bioaktivnih derivata naftohinona izobutirilšikonina, α-metilbutirilšikonina i acetilšikonina, dok je za ekstrakciju 5,8-O-dimetil derivata poželjnija upotreba acetona ili hloroforma. U narednom koraku ispitana je i citotoksična aktivnost dobijenih ekstrakata na ćelijama kancera debelog creva i dojke primenom MTT testa i metoda za detekciju apoptoze i analize ćelijskog ciklusa. Dobijeni rezultati su pokazali značajno bolju aktivnost ekstrakata acetona, hloroforma i etil acetata pri čemu je citotoksični efekat ekstrakata u direktnoj korelaciji sa količinom individualnih komponenti naftohinonskih pigmenata. Ispitana je i biološka aktivnost izolovanih jedinjenja, najpre upotrebom MTT testa, kao i metodama za detekciju apoptoze i analize ćelijskog ciklusa na ćelijama kancera debelog creva i dojke. Naftohinonski derivati α-metilbutirilšikonin, acetilšikonin i β-hidroksiizovalerilšikonin pokazali su najjači citotoksični efekat pa je dalje ispitan i njihov efekat na markere oksidativnog stresa. Tretman ovim jedinjenjima izazvao je poremećaj oksidativne homeostaze ćelija raka debelog creva i dojke, povećavajući nivo superoksid anjon radikala, nitrita, oksidovanog i redukovanog glutationa. U sledećem koraku je ispitana i mogućnost interakcija ova tri naftohinona sa molekulima DNK i HSA. Rezultati dobijeni primenom UV-Vis i FL spektroskopskih tehnika kao i molekulske doking simulacije jasno ukazuju da se testirana jedinjenja vezuju u oblasti malog žleba molekula DNK kao i da imaju mogućnost da se čvrsto vežu za HSA, i sasvim bezbedno transportuju do ciljnih ćelija. Takođe, ispitana je i antibakterijska aktivnost svih izolovanih naftohinona. Dobijeni rezultati su pokazali da izolovani naftohinoni pokazuju dobru antibakterijsku aktivnost kako prema gram pozitivnim, tako i prema gram negativnim bakterijskim sojevima, pri čemu su α-metilbutirilšikonin i acetilšikonin pokazali najznačajniju aktivnost. Naftohinoni su po prirodi lipofilna jedinjenja na čiju stabilnost utiče i dejstvo kiseonika iz vazduha kao i temperatura ali i UV zračenje. U cilju povećanja bioraspoloživosti i farmaceutske efikasnosti naftohinona iskorišćena je mogućnost njihove enkapsulacije unutar hidrofobne šupljine molekula β-CD. Rezultati dobijeni primenom UV-Vis, IR i 1H NMR spektroskopije, kao i rendgenske difraktometrije praha (XPRD) i skenirajuće elektronske mikroskopije (SEM) potvrdili su da je dobijen binarni sistem acetilšikonin/β-CD. Test fazne rastvorljivosti pokazao je da je dobijen inkluzioni sistem AL-tipa, kao i da je odnos substrat/ligand 1:1 M/M. Kako bi potvrdili da li je enkapsulacijom povećana biološka aktivnost povećanjem bioraspoloživosti enkapsuliranog naftonihona, ispitano je citotoksično dejstvo enkapsuliranog i slobodonog acetilšikonina na ćelijama kancera debelog creva i dojke primenom MTT testa, klonogenog testa kao i metodama za detekciju apoptoze i analize ćelijskog ciklusa. Takođe, ispitan je i njihov efekat na produkciju intracelularnih reaktivnih kiseoničnih vrsta (ROS), kao i na ekspresiju ključnih apoptotskih proteina, i na inhibiciju autofagije. Dobijeni rezultati su ukazali da formiranje binarnog sistema značajno utiče na poboljšanje aktivnosti naftohinonskih derivata.

Opis (eng)

Since ancient times, plant species of the genus Onosma L have been used as natural colorаnts. Flowers of some species are used as a vegetable in nutrition, while their roots are used in traditional medicine in the treatment of wounds, burns, shortness of breath, hoarseness, hemorrhoids, abdominal pain and gastric ulcers. Nevertheless, for the plant species of Onosma visianii Clem, so far there has been no scientific knowledge of its root chemical composition and pharmacological activity. The first task of this dissertation was isolation of bioactive naphthoquinone derivatives from the roots of the plant O. visianii Clem. Using different chromatographic techniques (column chromatography-silica gel as adsorbent, preparative thin layer chromatography-silica gel as adsorbent, column chromatography Sephadex-LH20, preparative liquid chromatography (HPLC)), we optimized an efficient method for isolation of naphthoquinones from the extract obtained by cold maceration using a solvent mixture of petroleum ether:methylene chloride (1:1 v/v). Using this metode we isolated seven compounds: deoxyshikonin, isobutyrylshikonin, αmethylbutyrylshikonin, acetylshikonin, β-hydroxyisovalerylshikonin, 5,8-O-dimethyl isobutyrylshikonin (first time identified in a natural source) and 5,8-O-dimethyl deoxyshikonin. Тheir structural characterization was performed by UV-Vis, IR, 1H NMR and 13C NMR spectroscopic techniques, as well as using high resolution mass spectrometry. The next task was to determine the most suitable solvent for the extraction of the bioactive components from the roots of the O. visianii Clem. Five extracts (acetone, chloroform, ethyl acetate, methanol and petroleum ether) were obtained using cold maceration method. Performed HPLC analysis showed that chloroform, acetone, and ethyl acetate are better solvents for the preliminary extraction of isobutyrylshikonin, α-methylbutyrylshikonin and acetylshikonin, while the use of chloroform and acetone as solvents is preferable for extraction of 5,8-O-dimethyl derivatives. The cytotoxic activity of the obtained extracts was examined on colon and breast cancer cells using the MTT assay, methods for the detection of apoptosis and cell cycle analysis. Obtained results showed significantly better activity of acetone, chloroform and ethyl acetate extracts, suggesting that cytotoxic effect of the extracts are in direct correlation with the amount of individual components of the naphthoquinone pigments. Next, we examined the biological activity of the seven isolated naphthoquinone compounds. Initially we studied their cytotoxic effects on colon and breast cancer cells using the MTT assay, methods for the detection of apoptosis as well as cell cycle analysis. The naphthoquinone derivatives α-methylbutyrylshikonin, acetylshikonin and βhydroxyisovalerylshikonin showed the strongest cytotoxic activity so we additionally examined their effect on the oxidative stress markers. The results showed that treatment with these compounds caused a disruption of the oxidative homeostasis of colon and breast cancer cells, increasing the levels of superoxide anion radical, nitrite, oxidized and reduced glutathione. The following step was to examine the possibility of interactions of these three naphthoquinones with DNA and HSA molecules. The results using UV-Vis and FL spectroscopic techniques as well as molecular docking simulations clearly indicate that the tested compounds can bind in the region of the small groove of DNA molecule, and that can bind strongly to HSA and be transported safely to the target cells. Additionally, we studied antibacterial activity of seven isolated naphthoquinones. Obtained results showed that all isolated naphthoquinones exhibited potent antibacterial activity against both gram-positive and gram-negative bacterial strains, with αmethylbutyrylshikonin and acetylshikonin showing the most significant activity against all tested bacterial strains. Naphthoquinone are known as lipophilic compounds whose stability is affected by the action of oxygen from the air as well as temperature and UV radiation. In order to increase bioavailability and pharmaceutical efficiency of naphthoquinones, the possibility of their encapsulation within the hydrophobic cavity of the β-CD molecule was exploited. The results of UV-Vis, IR and 1H NMR spectroscopy, as well as X-ray powder diffractometry (XPRD) and scanning electron microscopy (SEM) showed that the acetylshikonin/β-CD binary system was obtained. The phase solubility test showed that the AL-type inclusion system was obtained, and that the substrate/ligand ratio was 1:1 M/M. In order to confirm whether encapsulation increased biological activity, the cytotoxic effect of encapsulated and free acetylshikonin on colon and breast cancer cells was examined using MTT assay, clonogenic assay as well as apoptosis detection methods and cell cycle analysis. Also, the effect on the production of intracellular reactive oxygen species (ROS) were examined, as well as their effect on expression of key apoptotic proteins, and the inhibition of autophagy. The obtained results indicate that the formation of a binary system significantly influences the enhancement of the activity of naphthoquinone derivatives. The results obtained represent a significant contribution to the pharmacological and phytochemical description of the Onosma visianii Clem plant and indicate the possible use of isolated naphthoquinone derivatives in the development of new antibacterial formulations, as well as in the development of new strategies against colon and breast cancer.Since ancient times, plant species of the genus Onosma L have been used as natural colorаnts. Flowers of some species are used as a vegetable in nutrition, while their roots are used in traditional medicine in the treatment of wounds, burns, shortness of breath, hoarseness, hemorrhoids, abdominal pain and gastric ulcers. Nevertheless, for the plant species of Onosma visianii Clem, so far there has been no scientific knowledge of its root chemical composition and pharmacological activity. The first task of this dissertation was isolation of bioactive naphthoquinone derivatives from the roots of the plant O. visianii Clem. Using different chromatographic techniques (column chromatography-silica gel as adsorbent, preparative thin layer chromatography-silica gel as adsorbent, column chromatography Sephadex-LH20, preparative liquid chromatography (HPLC)), we optimized an efficient method for isolation of naphthoquinones from the extract obtained by cold maceration using a solvent mixture of petroleum ether:methylene chloride (1:1 v/v). Using this metode we isolated seven compounds: deoxyshikonin, isobutyrylshikonin, αmethylbutyrylshikonin, acetylshikonin, β-hydroxyisovalerylshikonin, 5,8-O-dimethyl isobutyrylshikonin (first time identified in a natural source) and 5,8-O-dimethyl deoxyshikonin. Тheir structural characterization was performed by UV-Vis, IR, 1H NMR and 13C NMR spectroscopic techniques, as well as using high resolution mass spectrometry. The next task was to determine the most suitable solvent for the extraction of the bioactive components from the roots of the O. visianii Clem. Five extracts (acetone, chloroform, ethyl acetate, methanol and petroleum ether) were obtained using cold maceration method. Performed HPLC analysis showed that chloroform, acetone, and ethyl acetate are better solvents for the preliminary extraction of isobutyrylshikonin, α-methylbutyrylshikonin and acetylshikonin, while the use of chloroform and acetone as solvents is preferable for extraction of 5,8-O-dimethyl derivatives. The cytotoxic activity of the obtained extracts was examined on colon and breast cancer cells using the MTT assay, methods for the detection of apoptosis and cell cycle analysis. Obtained results showed significantly better activity of acetone, chloroform and ethyl acetate extracts, suggesting that cytotoxic effect of the extracts are in direct correlation with the amount of individual components of the naphthoquinone pigments. Next, we examined the biological activity of the seven isolated naphthoquinone compounds. Initially we studied their cytotoxic effects on colon and breast cancer cells using the MTT assay, methods for the detection of apoptosis as well as cell cycle analysis. The naphthoquinone derivatives α-methylbutyrylshikonin, acetylshikonin and βhydroxyisovalerylshikonin showed the strongest cytotoxic activity so we additionally examined their effect on the oxidative stress markers. The results showed that treatment with these compounds caused a disruption of the oxidative homeostasis of colon and breast cancer cells, increasing the levels of superoxide anion radical, nitrite, oxidized and reduced glutathione. The following step was to examine the possibility of interactions of these three naphthoquinones with DNA and HSA molecules. The results using UV-Vis and FL spectroscopic techniques as well as molecular docking simulations clearly indicate that the tested compounds can bind in the region of the small groove of DNA molecule, and that can bind strongly to HSA and be transported safely to the target cells. Additionally, we studied antibacterial activity of seven isolated naphthoquinones. Obtained results showed that all isolated naphthoquinones exhibited potent antibacterial activity against both gram-positive and gram-negative bacterial strains, with αmethylbutyrylshikonin and acetylshikonin showing the most significant activity against all tested bacterial strains. Naphthoquinone are known as lipophilic compounds whose stability is affected by the action of oxygen from the air as well as temperature and UV radiation. In order to increase bioavailability and pharmaceutical efficiency of naphthoquinones, the possibility of their encapsulation within the hydrophobic cavity of the β-CD molecule was exploited. The results of UV-Vis, IR and 1H NMR spectroscopy, as well as X-ray powder diffractometry (XPRD) and scanning electron microscopy (SEM) showed that the acetylshikonin/β-CD binary system was obtained. The phase solubility test showed that the AL-type inclusion system was obtained, and that the substrate/ligand ratio was 1:1 M/M. In order to confirm whether encapsulation increased biological activity, the cytotoxic effect of encapsulated and free acetylshikonin on colon and breast cancer cells was examined using MTT assay, clonogenic assay as well as apoptosis detection methods and cell cycle analysis. Also, the effect on the production of intracellular reactive oxygen species (ROS) were examined, as well as their effect on expression of key apoptotic proteins, and the inhibition of autophagy. The obtained results indicate that the formation of a binary system significantly influences the enhancement of the activity of naphthoquinone derivatives. The results obtained represent a significant contribution to the pharmacological and phytochemical description of the Onosma visianii Clem plant and indicate the possible use of isolated naphthoquinone derivatives in the development of new antibacterial formulations, as well as in the development of new strategies against colon and breast cancer.

Jezik

srpski

Datum

2020

Licenca

Ovo delo je licencirano pod uslovima licence
Creative Commons CC BY 2.0 AT - Creative Commons Autorstvo 2.0 Austria License.

CC BY 2.0 AT

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