dc.description | 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. | sr |
dc.description | 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. | en |