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Molecular modelling analysis of the metabolism of thymoquinone
Fazlul Huq and Ehsanul Hoque Mazumder
Discipline of Biomedical Science,
School of Medical Sciences,
Faculty of Medicine, Cumberland Campus, C42,
The University of Sydney,
Lidcombe, NSW, Australia.
Phone: +61 2 9351 9522; Fax: +61 2 9351 9520
Email: F.Huq@usyd.edu.au
Abstract
Thymoquinone (TQ) is the major biologically active component (about 54%) of volatile oil of black
seed that has been shown to exert anti-inflammatory, antioxidant and anti-neoplastic effects both in
vitro and in vivo. TQ induces apoptosis, disrupts mitochondrial membrane potential and triggers the
activation of caspases 8, 9 and 3 in myeloblastic leukaemia HL-60 cells. Although TQ acts as
antioxidant and is found to inhibit inflammation in animal models and culture systems, it can also
cause glutathione depletion thus acting as a prooxidant. It has been suggested that TQ may be
metabolized to reactive species and increase oxidative stress that contributes to the depletion of
antioxidant enzymes and damage to DNA in hepatocytes treated with high concentrations of the
compound. Molecular modelling analyses based on molecular mechanics, semi-empirical (PM3) and
DFT (at B3LYP/6-31G* level) calculations show that TQ and its metabolic products have LUMO-
2
HOMO energy differences ranging from 3.8 to 5.4 eV indicating that the compounds would be
moderately inert kinetically with THY being most inert.
The molecular surfaces of TQ and DTQ are found to possess significant amounts of positively charged
electron-deficient regions so that they may be subject to nucleophilic attacks by glutathione and
nucleobases in DNA, thus causing cellular toxicity due to glutathione depletion and DNA damage due
to oxidation of nucleobases.
Key words: Thymoquinone, black seed, antioxidant, apoptosis, molecular modelling
Introduction
Over the recent years, there has been a growing interest in tumour active phytochemicals because they
are relatively non-toxic, inexpensive and available in forms that can be easily ingested [1]. Nigella
sativa Linn, also known as black seed or black cumin, caraway seed, habbat-ul-baraka (meaning the
blessed seed) has been used to treat many different ailments including asthma, rheumatoid arthritis,
stomach aches, cancer and cough for thousands of years [2]. In the Middle East it is incorporated in
diets and everyday life style as it is regarded as a part of holistic approach to health. Traditionally,
there is a common Islamic belief that black seed is a universal healer that provides remedy for all
ailments except death and ageing [3]. Black seed was discovered in Tutankhamen’s tomb, implying
that it played an important role in ancient Egyptian practices as well.
Thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone) is the major biologically active
component (about 54%) of volatile oil of black seed. TQ has been shown to exert anti-inflammatory,
antioxidant and anti-neoplastic effects both in vitro and in vivo [1]. Many investigations have shown
that the growth inhibitory effects of TQ are specific to cancer cells [4, 5]. It induces apoptosis, disrupts
3
mitochondrial membrane potential and triggers activation of caspases 8, 9 and 3 in myeloblastic
leukaemia HL-60 cells. Growth inhibition by TQ was found to be associated with inhibition of DNA
synthesis [6] and that of cell cycle arrest [5]. TQ significantly reduces proliferation of mouse
papilloma cells and mouse spindle cells [1]. Treatment of mouse papilloma cells with 30 M TQ was
found to cause a 22% increase in the number of cells in the G
1
phase within 24 h [1]. The TQ-induced
G
1
arrest was associated with a sharp increase in p16 protein levels as early as 2 h after treatment; the
increased level was sustained for up to 24 h [7]. The increase in expression of p16 protein is
considered to be of special significance as it increases the sensitivity of tumour to chemotherapeutic
drugs.
When more aggressive mouse spindle cancer cells were treated with TQ, it caused G
2
/M arrest (38%
increase) that was accompanied with a decrease in the expression of cyclin B1 protein. TQ has also
been found to suppress the proliferation of several human colon cancer cell lines including Caco-2,
LoVo, HT-29, DLD-1 and HCT-116 cells. It modulates the Bax/Bcl2 ratio by upregulation of
proapoptotic Bax and down-regulation of antiapoptotic Bcl2 proteins in p53-null myeloblastic-
leukemia HL-60 cells during apoptosis [8].
TQ has been found to inhibit inflammation in animal models and culture systems [4]. Administration
of TQ to rats has been found to protect against transient forebrain ischemia-induced damage in the
hippocampus [10]. It acts as a powerful antioxidant with greater ROS scavenging capacity than some
other natural flavavnoids including those isolated from olive oil and silibinin [9].
TQ is found to be equally sensitive to both the multi-drug resistant forms of human pancreatic
adenocarcinoma, uterine sarcoma and leukemic cancer cell lines and their parental controls whereas
commonly used anti-neoplastic agents such as doxorubicin and etopside are 10 times less active to the
resistant forms [6]. Orally administered TQ has been found to potentiate antitumour activity of
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cisplatin and prevent cisplatin-induced nephrotoxicity in mice and rats [11]. In primary rat hepatocyte
cultures TQ has been found to exert cyto- and genotoxic effects in a concentration dependent manner;
it induced significant antiproliferative effects at 20 M and acute cytotoxicity at higher concentrations
[12]. It has been suggested that TQ may be metabolized to reactive oxygen species (ROS) and increase
oxidative stress that contributes to the depletion of antioxidant enzymes and damage to DNA in
hepatocytes treated with high concentrations of the compound [13, 14]. When TQ was orally
administered to mice, LD
50
was found to be 2.4 g/kg [15]. At high doses (2-3 g/kg) hypoactivity and
difficulty in respiration, and a significant reduction in tissue (liver, kidney and heart) glutathione
content were observed 24 h after administration. It is thought that TQ like other quinones can undergo
redox cycling so that it is metabolized in vivo to hydroquinones or semiquinone radicals by cellular
oxidoreductases leading to the formation of ROS.
Besides TQ, black seed is also found to contain dithymoquinone (DTQ), thymohydroquinone (THY)
and thymol [16]. These compounds are likely to be metabolites of TQ. Indeed under physiological
conditions, TQ is slowly reduced to thymol (THY) and dihydrothymoquinone (THQ). THQ reacts
rapidly with glutathione (GSH) to form glutathionyl-dihydrothymoquinone (THQSG) [17]. TQ may
also exist as dimymoquinone (DTQ). Several authors have reported that in vivo protection against
oxidative damage provided by TQ may be due to the combined action of TQ and THQ. In this regard
THQSG may also be playing an important role, since it has antioxidant activity similar to that of THQ.
The mechanism of metabolic activation of TQ remains unclear. However, it has been suggested that
the reduction of TQ may be catalysed by different cellular reductants possibly according to
mechanisms similar to those reported for the activation of other quinones such as menadione and
CoenzymeQ that have reduction poptential close to that of TQ [17].
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O
O
CH
3
CH(CH
3
)
2
OH
OH
CH
3
CH(CH
3
)
2
OH
OH
CH
3
CH(CH
3
)
2
SG
CH
3
CH(CH
3
)
2
OH
TQ
O
O
CH
3
O
O
CH
3
CH
3
CH
3
CH
3
DTQ
THQSG
THY
THQ
Figure 1 Products formed from TQ under physiological conditions
In this study, molecular modelling analyses have been carried out using the program Spartan ’08 [19]
of TQ and its likely metabolic products, with the aim of providing a better understanding of their
relative toxicity. Previous studies have shown that xenobiotics and their metabolites which are
kinetically labile and abound in electron-deficient regions on the molecular surface tend to induce
cellular toxicity due to glutathione depletion and cause DNA damage due to oxidation of nucleobases
in DNA [20, 21]. The work was carried out in the Discipline of Biomedical Science, School of
Medical Sciences, The University of Sydney during February to May 2009.
6
Computational methods
The geometries of TQ, and its likely metabolites THY, THQ, THQSG and DTQ have been optimised
based on molecular mechanics, semi-empirical and DFT calculations, using the molecular modelling
program Spartan ’08. Molecular mechanics calculations were carried out using MMFF force field.
Semi-empirical calculations were carried out using the routine PM3. DFT calculations were carried at
B3LYP/6-31G* level. In optimization calculations, a RMS gradient of 0.001 was set as the
terminating condition. For the optimised structures, single point calculations were carried out to give
heat of formation, enthalpy, entropy, free energy, dipole moment, solvation energy, energies for
HOMO and LUMO. The order of calculations: molecular mechanics followed by semi-empirical
followed by DFT ensured that the structure was not embedded in a local minimum. To further check
whether the global minimum was reached, some calculations were carried out with improvable
structures. It was found that when the stated order was followed, structure corresponding to the global
minimum or close to that could ultimately be reached in all cases. Although RMS gradient of 0.001
may not be sufficiently low for vibrational analysis, it is believed to be sufficient for calculations
associated with electronic energy levels [20].
Results and discussion
Table 1 gives the total energy, heat of formation as per PM3 calculation, enthalpy, entropy, free
energy, surface area, volume, dipole moment, and energies of HOMO and LUMO as per both PM3
and DFT calculations for TQ, THY, THQ, THQSG and DTQ. Figures 2-6 give the regions of negative
electrostatic potential (greyish-white envelopes) in (a), HOMOs (where red indicates HOMOs with
high electron density) in (b), LUMOs in (c), and density of electrostatic potential on the molecular
surface (where red indicates negative, blue indicates positive and green indicates neutral) in (d) as
applied to optimised structures of TQ, THY, THQ, THQSG and DTQ.
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The LUMO-HOMO energy differences for TQ, THY, THQ, THQSG and DTQ from DFT calculations
are found to range from 370.3 kJ/mol (3.8 eV) to 569.6 kJ/mol (5.9 eV), indicating that TQ and its
metabolites would be neither extremely inert nor extremely labile kinetically. The most labile
compound would be TQ itself and the most inert metabolite would be THY.
The solvation energies of TQ, THY, THQ, THQSG and DTQ obtained from PM3 calculations are
found to range from -10.5 to -97.3 kJ mol
-1
, indicating that the compounds would vary greatly in their
solubility in water. Whereas the conjugation product THQSG would be highly soluble in water, the
parent compound TQ is expected to be insoluble in water but soluble in fat.
In the case of TQ and DTQ, the electrostatic potential is found to be more negative around the
carbonyl oxygen atoms, indicating that the positions may be subject to electrophilic attack. In the case
of THY and THQ, the electrostatic potential is found to be more negative around the hydroxyl oxygen
atoms, indicating that the positions may be subject to electrophilic attack. In the case of THQSG, the
electrostatic potential is found to be more negative around various oxygen atoms, indicating that the
positions may be subject to electrophilic attack.
In the case of TQ, THY, THQ, THQSG and DTQ, both the HOMOs with high electron density and
LUMOs are found to be located close to the non-hydrogen atoms of the phenyl ring, indicating the
possibility of occurrence of electronic excitation from the HOMOs with high electron density to the
LUMOs.
5
Table 1 Calculated thermodynamic and other parameters of TQ and its metabolites
Molecule
Calculation
type
Total energy
(kJ mol
-1
)
Heat of
formation
(kJ mol
-1
)
Enthalpy
(kJ mol
-1
K
-1
)
Entropy
(J mol
-1
K
-1
)
Free energy
(kJ mol
-1
)
Solvation
energy (kJ
mol
-1
)
Area
(Å
2
)
Volume
(Å
3
)
Dipole
moment
(debye)
HOMO
(kJ/mol)
LUMO
(kJ/mol)
LUMO-HOMO
(kJ/mol)*
TQ
PM3
-244.4
-244.4
554.9
450.7
420.7
-10.5
201.5
181.1
1.1
-1034.4
-146.9
887.5 (9.2)
DFT
-1414418.4
555.7
452.1
420.9
-7.4
202.9
182.0
0.2
-680.6
-310.3
370.3 (3.8)
THY
PM3
-206.6
-206.6
603.5
429.7
475.4
-13.7
196.8
177.7
1.2
-867.0
26.8
893.8 (9.3)
DFT
-1220137.5
-184.6
604.1
437.6
473.6
-13.0
198.3
178.8
1.4
-551.8
17.8
569.6 (5.9)
THQ
PM3
-389.2
-389.2
621.2
449.2
487.3
-31.2
204.0
184.6
0.2
-830.0
15.2
845.2 (8.8)
DFT
-1417339.7
617.4
457.1
481.2
-31.2
205.5
185.6
0.1
-500.9
16.0
516.9 (5.4)
THQSG
PM3
-1236.2
-1236.2
1234.2
847.9
981.4
-97.3
426.7
395.4
3.5
-878.0
-74.2
803.8 (8.4)
DFT
-4557498.0
1231.6
851.3
977.9
-96.2
425.9
395.9
3.9
-555.0
-74.8
480.2 (5.0)
DTQ
PM3
-421.6
-421.6
1116.1
665.1
917.5
-16.5
351.7
343.7
0.5
-1028.4
-98.5
929.9 (9.6)
DFT
-2828702.0
11155.2
667.5
913.6
-14.2
356.4
346.7
0.9
-650.9
-256.5
394.4 (4.1)
* The numbers in parentheses are the equivalent values in electron volt
6
O
O
CH
3
CH(CH
3
)
2
(a) (b)
(c) (d)
Figure 2 Structure of TQ giving in: (a) the electrostatic potential (greyish envelope denotes negative
electrostatic potential), (b) the HOMOs, (where red indicates HOMOs with high electron density) (c)
the LUMOs (where blue indicates LUMOs) and in (d) density of electrostatic potential on the
molecular surface (where red indicates negative, blue indicates positive and green indicates neutral).
7
CH
3
CH(CH
3
)
2
OH
(a) (b)
(c) (d)
Figure 3 Structure of THY giving in: (a) the electrostatic potential (greyish envelope denotes negative
electrostatic potential), (b) the HOMOs, (where red indicates HOMOs with high electron density) (c)
the LUMOs (where blue indicates LUMOs) and in (d) density of electrostatic potential on the
molecular surface (where red indicates negative, blue indicates positive and green indicates neutral).
8
OH
OH
CH
3
CH(CH
3
)
2
(a) (b)
(c) (d)
Figure 4 Structure of THQ giving in: (a) the electrostatic potential (greyish envelope denotes negative
electrostatic potential), (b) the HOMOs, (where red indicates HOMOs with high electron density) (c)
the LUMOs (where blue indicates LUMOs) and in (d) density of electrostatic potential on the
molecular surface (where red indicates negative, blue indicates positive and green indicates neutral).
9
OH
OH
CH
3
CH(CH
3
)
2
SG
(a) (b)
(c) (d)
Figure 5 Structure of THQSG giving in: (a) the electrostatic potential (greyish envelope denotes
negative electrostatic potential), (b) the HOMOs, (where red indicates HOMOs with high electron
density) (c) the LUMOs (where blue indicates LUMOs) and in (d) density of electrostatic potential on
the molecular surface (where red indicates negative, blue indicates positive and green indicates
neutral).
10
O
O
CH
3
O
O
CH
3
CH
3
CH
3
CH
3
(a) (b)
(c) (d)
Figure 6 Structure of DTQ giving in: (a) the electrostatic potential (greyish envelope denotes negative
electrostatic potential), (b) the HOMOs, (where red indicates HOMOs with high electron density) (c)
the LUMOs (where blue indicates LUMOs) and in (d) density of electrostatic potential on the
molecular surface (where red indicates negative, blue indicates positive and green indicates neutral).
The overlap of HOMO with high electron density and region of negative electrostatic potential at
some positions, gives further support to the idea that the positions may be subject to electrophilic
attack. It was stated earlier that Orally administered TQ potentiates antitumour activity of cisplatin and
prevents cisplatin-induced nephrotoxicity in mice and rats. The effects may be mediated by
antioxidant role played by TQ.
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The molecular surfaces of TQ and DTQ are all found to possess significant amounts of electron-
deficient (blue) regions so that they may be subject to nucleophilic attacks such as those by glutathione
and nucleobases in DNA. Reaction with glutathione can induce cellular toxicity by compromising the
antioxidant status of the cell whereas that with nucleobases in DNA can cause DNA damage. The
reaction with glutathione and the resulting oxidative stress may explain why TQ in large doses can
cause acute toxicity.
Conclusion
Thymoquinone (TQ) is the major biologically active component (about 54%) of volatile oil of black
seed that has been shown to exert anti-inflammatory, antioxidant and anti-neoplastic effects both in
vitro and in vivo. It induces apoptosis, disrupts mitochondrial membrane potential and triggers
activation of caspases 8, 9 and 3 in myeloblastic leukaemia HL-60 cells. Molecular modelling analyses
based on molecular mechanics, semi-empirical (PM3) and DFT (at B3LYP/6-31G* level) calculations
show that TQ and its products have LUMO-HOMO energy differences ranging from 3.8 to 5.4 eV
indicating that the compounds would be moderately inert kinetically with THY being most inert. The
molecular surfaces of TQ and DTQ are found to possess significant amounts of electron-deficient
(blue) regions so that they may be subject to nucleophilic attacks such as those by glutathione and
nucleobases in DNA. Reaction with glutathione can induce cellular toxicity by compromising the
antioxidant status of the cell whereas that with nucleobases in DNA can cause DNA damage. The
acute toxicity from large doses of TQ may be associated with its reaction with glutathione.
Abbreviations
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TQ: Thymoquinone; 2-isopropyl-5-methyl-1,4-benzoquinone
DTQ: Dithymoquinone
THY: Thymol
THQ: Dihydrothymoquinone
GSH: Glutathione
THQSG: Glutathionyl-dihydrothymoquinone
LD
50
:
Dose that kills 50% of the tested group
DFT: Density functional theory
LUMO: Lowest unoccupied molecular orbital
HOMO: Highest occupied molecular orbital
Acknowledgments
Fazlul Huq is grateful to the Discipline of Biomedical Science, School of Medical Sciences, The
University of Sydney for the time release from teaching.
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