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Acta Bioethica 2016; 22 (2): 1086-1095
THE EFFECT OF THYMOQUINONE ON CONCENTRATION
OF HUMAN
MU
-OPIOID RECEPTORS MEDIATED BY
CHRONIC MORPHINE TREATMENT IN OPIOID RECEPTOR
EXPRESSING CELL (U87 MG)
1Liyana Hazwani Mohd Adnan, 1Nasir Mohamad, 1Khairi Che Mat, 1Nor Hidayah Abu Bakar,
2Khamsah Suryati Mohd, 1Mohd Izuddin Mansor.
ABSTRACT Morphine and Thymoquinone (TQ) are both opioid receptor stimulating compounds although they have different
pharmacological origins. Morphine is a natural opioid derivative whereas TQ is one of the main pharmacologically active
compounds from Nigella sativa oils. Recently, a great deal of attention had been focused on the potential roles of TQ in opio id
dependence therapy by focusing on mu-opioid receptor, a primary site of action for morphine’s effects. This study was carried
out to study the effects of TQ on protein expression of mu-opioid receptors mediated by chronic morphine treatment in opioid
receptor expressing cell line (U87 glioblastoma cells). U87 cells was grown in tissue culture flasks with RPMI 1640 medium
containing 1 mmol/L L-glutamine, supplemented with 10% (v/v) fetal bovine serum (FBS), and 1% (w/v) penicillin/streptomycin.
The cell viability was assessed by the trypan blue dye and manually counted using a haemocytometer. The MTT assay was
used to determine the cytotoxic effects of Morphine and TQ. The protein concentration of human mu-opioid receptor (MOR)
level in the cells was determined using the Cusabio® ELISA Kit. Data obtained from this assay indicated that 35 µM morphine
and 60.9 µM TQ gives maximum MOR protein concentration. Co-treatment of morphine with TQ and methadone increased the
MOR protein concentration (*P < 0.05). These finding suggest that TQ could possibly reduce the tolerance and dependence at
cellular level by increasing MOR protein concentration. However, it needs to be further confirmed at molecular level and in-
vivo study..
KEYWORDS: Morphine, thymoquinone, mu-opioid receptors protein concentration, opioid receptor expressing cell (U87
MG).
ABBREVIATIONS: TQ = thymoquinone, MOR = mu-opioid receptor, MG = monoglioma, ELISA = Enzyme-linked
immunosorbent assay.
1 Faculty of Medicine, Universiti Sultan Zainal Abidin (UniSZA), 20400, Kuala, Terengganu,Terengganu, Malaysia.
2 Faculty of Bioresources and Food Industry, University Sultan Zainal Abidin (UniSZA), Gong Badak Campus, 21300, Kuala
Terengganu, Terengganu, Malaysia.
Corresponding Author: Faculty of Medicine, Universiti Sultan Zainal Abidin (UniSZA), 20400, Kuala, Terengganu,Terengganu,
Malaysia.
THE EFFECT OF THYMOQUINONE ON CONCENTRATION… Nasir Mohamad et al.
1087
INTRODUCTION
Opioid receptors have been classified into three subtypes; m, δ and κ based upon a characteristic pharmacology using
highly selective ligands (Yu
et al.,
2003; Al-Hasani and Bruchas, 2011). Mu-opioid receptor (MOR) is one of the opioid receptor
subtypes that belong to the G protein-coupled receptor super family (Law
et al.,
2000; Koch and Hollt, 2008; Gretton and
Droney, 2014;). It has high affinity of binding with morphine and other opioid peptides and is critical for the rewarding effects
of heroin and morphine (Kosten and George, 2002; Yu
et al.,
2003, McDonald and Lambert, 2005). It is well known that chronic
administration of morphine and heroin will cause the development of opioid dependence and tolerance (Waldhoer
et al.,
2004;
Christie 2008; Berger and Whistler, 2010; Morgan and Christie, 2011). Several in vitro and in vivo studies revealed that the
development of opioid tolerance in long-term exposure with morphine will lead to decrease in protein concentrations. Changes
in MOR number in response to chronic opioid treatment have long been speculated to directly contribute to receptor
desensitization and the development of opioid tolerance (Connor
et al.,
2004; Koch and HÖllt, 2008).
Thymoquinone (IUPAC name: 2-Isopropyl-5-methylbenzo-1, 4-quinone); TQ is an aromatic ketone found in many
medicinal plants. It is known to be an active phytochemical constituent in seeds of Nigella sativa (NS) or black cumin. Most
properties of the whole NS seeds or their extracts are mainly attributed to quinone constituents, of which thymoquinone is
more abundant compound (Mahfouz
et al.,
1960; Filippo D’Antuono
et al.,
2002; Parvardeh and Fatehi 2003).
Recently, a great deal of attention has been given to this pharmacologically active quinone. TQ has been reported for its
many therapeutic potential in a number of medical conditions (Ghayur
et al.,
2012). Currently, TQ had been shown to
attenuates the development of morphine-induced dependence in mice by inhibiting brain oxidative stress and increase the
expression of brain inducible nitric oxide (NO) synthase (Abdel-Zaher
et al.,
2013). Thymoquinone also has potential chemical
effect mimicking opioid, especially on the mechanisms of opioid dependency and tolerance.
It’s an opioid receptor stimulating compound with 45% ligand displacement at mu-opioid receptor (Nutten
et al.,
2012),
which is receptor that is critical for morphine’s rewarding effects. Although TQ were claimed to have the potential to reduce
the opioid tolerance and dependence, there is no report on cellular mechanisms for chronic opioid treatment.
In this study, the effects of TQ on the viability of opioid receptor expressing cells (U87 MG), the optimum concentration of
TQ on the maximum MOR protein concentration level and the ability of TQ to increase protein concentration of the MOR were
investigated. We showed that TQ were not toxic to the cell lines. TQ had also been able to increase MOR protein concentration
in U87 MG cells after co treatment with morphine for 6h. Similar results were observed when cells were TQ-co treated with
morphine and methadone for 12h.
MATERIALS AND METHOD
MATERIALS
Opioid receptor expressing cells (U87 cells) were a gift from Animal Tissue Culture Lab, Faculty of Food Sciences and
Bioresources, UniSZA. RPMI 1640 medium, fetal bovine serum (FBS), and penicillin/streptomycin were obtained from Gibco
(Invitrogen, USA). Dimethyl sulfoxides (DMSO) were purchased from Merck (Germany). Thymoquinone (>99% pure) and 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder was purchased from Sigma-Aldrich (USA). Morphine
sulphate and methadone hydrochloride were purchased from Pharmaserv. The Cusabio® human mu-opioid receptor (MOR)
ELISA Kit were purchased from Cusabio Biotech (China).
DOSE RESPONSE STUDY (MTT ASSAY)
The dose response study of TQ, and morphine were evaluated using the colorimetric MTT assay. Briefly, 2×105cells/ml
were seeded on a 96-well plate in 100 µl culture medium per well. The cells were plated in triplicate. A serial dilution of TQ
(6090, 3045, 1522.5, 761.25, 380.625, 190.3125, and 95.15625 µM) and morphine (3500, 1750, 875, 437.5, 218.75, 109.375 and
54.69 µM) were prepared in different concentrations All dilutions were transferred to the cells in the 96-well plate and
THE EFFECT OF THYMOQUINONE ON CONCENTRATION… Nasir Mohamad et al.
1088
incubated for 72 h. Subsequently, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml) were
added to the cells in the dark and incubated for 4 h, covered with aluminium foil. After incubation, DMSO (100 µl) was added to
each well to dissolve the formazan crystals formed, and absorbance was read at a wavelength of 570 nm as measurement
wavelength and 630 nm as reference wavelength using the Tecan ELISA micro plate reader. The potency of cell growth
inhibition for the test agents were expressed as the half maximal (50%) inhibitory concentration, IC50. The amount of color
produced is directly proportional to the number of viable cell. Cell viability rate was calculated as the percentage of MTT
absorption as follows:
% Survival = (mean experimental absorbance/mean control absorbance) × 100.
CELL CULTURE AND TREATMENTS
The opioid receptor expressing cells (U87 MG) was used to measure the effect of TQ on MOR protein concentrations. U87
MG cells was grown in tissue culture flasks with RPMI 1640 medium containing 1 mmol/L L-glutamine, supplemented with
10% (v/v) fetal bovine serum (FBS), and 1% (w/v) penicillin/streptomycin. U87 MG cells were grown as a mono- layer in a
humidified incubator supplemented with 5% CO2 at 37°C. Morphine and TQ dose response studies were conducted by MTT
assay. In order to determine the optimum concentration of TQ on MOR protein expression, U87 MG cells were treated by
removing the growth media and replacing with fresh growth media containing the following final concentrations of morphine:
701, 350, 175, 35 and 17.5 µM and TQ; 1218, 609, 304.5, 60.9, and 30.45 µM. The optimum time-course of TQ on MOR protein
expression level were later conducted by incubating U87 MG with morphine and TQ for 3h, 6h, 12h, 24h and 48 h. As a negative
control, cells were treated with an equivalent volume of vehicle in growth media. The treatment groups were vehicle, morphine
alone (35 µM), morphine and methadone (162 µM), morphine and TQ (60.9 μM), and morphine, methadone and TQ. Finally, the
supernatants were harvested for measuring the MOR level.
MEASUREMENT OF HUMAN MOR PROTEIN CONCENTRATION
Treated and non-treated U87 MG cells in 96 well plates were assayed for the MOR concentration after incubation with
morphine and TQ using the kit. Briefly, the medium was removed and cells were washed with PBS for three times to remove
remaining drugs. The cells were scraped off from the plate and stored at -20°C in 1XPBS. After two freeze-thaw cycles, the cell
lysate were centrifuged at 5000 x g, 4°C for 5 minutes. The supernatants were collected and assayed directly by incubating th e
samples in the antibody-coated wells for 2 hours at 37°C. Later, Biotin Antibody (1x) were added and incubated for 1 hour at
37°C. Subsequently, the wells were later washed and HRP-avidin 1x was added to each well and further incubated for 1 hour at
37°C. After washing for several times, substrate was added to the wells and incubate for 15-30 minutes at 37°C. The reactions
were stopped by adding stop solution and the results were determined using a micro plate reader set to 450nm.
STATISTICAL ANALYSIS
The cell viability was calculated using a computer software program developed by Microsoft Excell 2007 based on the
optimal density readings at 570 nm. Results were presented as means ± SDs. Statistical analysis was done using one way
analysis of variance (ANOVA) for grouped comparison followed by Dunnet’s test and unpaired student’s t-test for comparing
unpaired samples. P-values less than 0.05 were considered statistically significant. The percentages of cell viability were
presented graphically in the form of graph, using a computer software program of Graph Pad Prism 6.
RESULTS
IN VITRO CELL PROLIFERATION ASSAY
The cytotoxicity of TQ and morphine on the opioid receptor expressing cells (U87 MG) were determined using MTT assay.
The dose response curve was plotted by plotting the percentage growth of cells vs. concentration of the compound. As shown
THE EFFECT OF THYMOQUINONE ON CONCENTRATION… Nasir Mohamad et al.
1089
in Fig. 1A, the IC50 value of TQ was 87.1 µM and considered as non-toxic to the opioid receptor expressing cells. Thus, the TQ
concentrations used for subsequent experiments were 1218, 609, 304.5, 60.9, and 30.45 µM. Morphine was also observed to be
non-toxic to the cells (Fig. 1B) with IC50 value of 39.81 µM. Hence, the subsequent concentrations used were 701, 350, 175, 35
and 17.5 µM.
A B
Fig. 1: The cytoxicity effect of TQ and morphine on U87 MG cells. The cells were treated with a range of concentration
54.69-6090 µM and incubated for 72h. The IC50 value of (A) thymoquinone was 87.1 µM and (B) morphine was 39.81 µM. Data
represent the mean ± SD of three independent experiments.
INVESTIGATION ON THE OPTIMUM CONCENTRATION OF TQ ON MOR PROTEIN CONCENTRATION
In order to find the optimum concentration of TQ that gives the maximum concentration of protein MOR, U87 MG cells
were incubated with fresh growth media containing the following final concentrations of morphine: 701, 350, 175, 35 and 17.5
µM and TQ; 1218, 609, 304.5, 60.9, and 30.45 µM. The MOR concentrations were later determined using ELISA kits. Based on
figure 2, TQ at 60.9 µM and 35 µM morphine gives maximum MOR protein concentrations thus these concentrations were
chosed used for subsequent co-treated experiments. MOR protein concentrations from the cell were significantly reduced
after treating with morphine at 175 µM and 701 µM as compared to the control. TQ also significantly decreased MOR protein
concentrations but at higher concentration of TQ (1218 µM) as compared to the control.
THE EFFECT OF THYMOQUINONE ON CONCENTRATION… Nasir Mohamad et al.
1090
Fig. 2: Investigation on the optimum concentration of TQ and morphine on human MOR protein concentration. U87 MG
cells were treated with either vehicle (cell culture medium) or with a range of concentration 701, 350, 175, 35 and 17.5 µM of
morphine and TQ; 1218, 609, 304.5, 60.9, and 30.45 µM for 3 hours. Data are the mean ± SD. One way analysis of variance
(ANOVA) was used to determine significance between groups followed by Dunnet’s test. *P < 0.05 compared to control (0 µM).
(TQ = thymoquinone).
INVESTIGATION ON THE OPTIMUM TIME COURSE OF TQ ON MOR CONCENTRATION
Chronic exposure to morphine desensitizes the MOR. To further examine the ability of TQ to potentially increase a
desensitized MOR after chronic morphine treatment was examined. Cells were incubated in 5% CO2 at 37°C for 3, 6, 12, 24, or
48 hours (Byrne
et al.,
2012). A desensitization time course indicated that U87 MG cells treated with 35 µM morphine for 12
hours had a significant decreased in the concentration of MOR (0.30060) compared to the control (0.4482). Interestingly, after 3
hours of morphine treatment, the MOR concentration was increased at 6 hours (0.506) compared to the control. However, MOR
concentration again decreased at 12 hours (0.3006) and 24 hours (0.275) of morphine treatment compared to control (Fig. 3).
THE EFFECT OF THYMOQUINONE ON CONCENTRATION… Nasir Mohamad et al.
1091
Fig. 3: The effects of morphine on MOR protein concentration in U87 MG cells. U87 MG cells were treated with either
vehicle (cell culture medium) or morphine (35 µM) for 3, 6, 12, 24 and 48 hours. Data are the mean ± SD. A student’s t-test was
used to determine significance. *P < 0.05 compared to control.
Further incubation with morphine at 48 hours however prevents MOR desensitization manifested with an increase in
MOR protein concentration at 48 hours (0.746). TQ 60.9 µM significantly decreased MOR protein concentrations at 6 hours
(0.3205) and 12 hours (0.30045) compared to the control before increasing it again after 24 hours treatment (1.4126) and
subsequently decreased after 48 hours.
Fig. 4: The effects of TQ on MOR protein concentration in U87 MG cells. U87 MG cells were treated with either vehicle
(cell culture medium) or TQ (60.9 µM) for 3, 6, 12, 24 and 48 hours. Data are the mean ± SD. A student’s t-test was used to
determine significance. *P < 0.05 compared to control.
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In order to examine the ability of TQ to increase MOR protein concentration after desensitization, U87 MG cells were co-
treated with morphine (35 µM) and TQ (60.9 µM), morphine, methadone (162 µM) and TQ for 3, 6, 12, 24, or 48 hours. Untreated
control cells exhibited similar MOR protein concentration (0.4482). Co-treatment of TQ with morphine showed a significant
increase in MOR protein concentration at 6h and 12h compared to the cells treated with morphine alone (Figure 5). Meanwhile,
co-treatment of TQ with morphine and methadone also significantly increased the MOR protein concentration but at 24h as
compared to the cells treated with morphine alone. Interestingly, MOR protein concentrations in the cells co-treated with
morphine and TQ were the highest and the effects were more sustained along desensitization time-course compared to the
other groups (**P < 0.05).
Fig. 5: The effects of co-treatment of TQ on MOR protein concentration in U87 MG cells. U87 MG cells were treated with
either morphine alone or co-treated with TQ (60.9 µM) and methadone (162 µM) for 3, 6, 12, 24 and 48 hours. Data are the mean
± SD. One way analysis of variance (ANOVA) was used to determine significance between groups followed by Dunnet’s test. *P
< 0.05 compared to morphine. (MOR = Morphine, TQ = thymoquinone, MET= methadone)
DISCUSSION
Nigella Sativa Linn (NS) belongs to family Ranunculaceae. The herb is widely known in different parts of the world and its
seeds are used as condiment. In subcontinent it is known as ‘kalonji’ and its Arabic name is ‘Habatul Sauda’. In the west it is
known as “Black Cumin”. There is a Hadith of Hazrat Muhammad (PBUH) that, ‘black seed is treatment of every disease but
death’. (Huong
et al.,
2009). In the present study, the potential of thymoquinone, a bioactive compound from NS as a drug
substitution therapy for chronic morphine treatment on a regulation of MOR protein concentration in U87 MG cells was
investigated. It is well understood that, repeated exposure to drugs of abuse alters the amounts, and even the types of genes
expressed in specific brain regions (Nestler, 2004). In this aspect, altered expression of genes can mediate changes in the
function of individual neurons and the larger neural circuits within which the neurons operate. Activation of any of the three
opioid receptor subtypes produces common cellular actions. The most commonly reported actions include inhibition of
adenylyl cyclase activation of a potassium conductance, inhibition of calcium conductance, and an inhibition of transmitter
release (Williams
et al.,
2001). Other observations studied in the actions of opioids are the activation of protein kinase C (PKC),
the release of calcium from extracellular stores, the activation of the mitogen-activated protein kinase (MAPK) cascade, and
the realization that receptor trafficking plays an important role in receptor function (Berger and Whistler, 2010).
THE EFFECT OF THYMOQUINONE ON CONCENTRATION… Nasir Mohamad et al.
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In the present study, the effects of thymoquinone on protein concentration of MOR in U87 MG cells were examined. We
first identify the optimum concentration of thymoquinone that can give maximum MOR protein concentration and the
supplement effects of thymoquinone on morphine MOR desensitization time course and MOR protein concentration. The
opioid effect was attributed largely to the mu-receptor rather than the δ-receptor (Yu
et al.,
1990). Therefore, U87 MG cells are
useful to study the efficacy and tolerance of narcotic analgesics. Based on the dose response study, several ranges of
concentrations of thymoquinone and morphine were tested in order to know the optimum concentrations that give the
maximum MOR protein concentrations from U87 MG cells. Our results showed that thymoquinone and morphine at
concentrations 60.9 µM and 35 µM gives maximum MOR protein concentrations from U87 MG cells.
We then examined the effects of morphine on MOR protein concentrations in U87 MG cells. U87 MG cells were treated
with either vehicle (cell culture medium) or morphine (35 µM) for 0 (control), 3, 6, 12, 24 or 48 hours. In this study, treatment
with morphine showed decreased in MOR protein concentrations in U87 MG cells after 3h treatment, confirming previous
studies showing decreased radio ligand binding of opioid ligands (Horner and Zadina, 2004) or MOR mRNA down regulation
(Yu
et al.,
2003) in the same or other neuroblastoma cell lines after chronic morphine exposure. A decrease in MOR protein
concentration are known to be the important adaptive changes induced by chronic exposure to opiate and is rela ted to
tolerance and dependence which have been demonstrated at the level of individual neurons with chronic morphine exposure
such as in U87 MG cells, which express abundant MOR (Yu
et al.,
2003; Mohan
et al.,
2010). Further incubation with morphine
at 48 h however prevents MOR desensitization manifested with an increase in MOR protein concentration at 48 h (0.746). This
phenomena are in line with a study reported by Eisinger and friends that chronic morphine treatment inhibits opioid receptor
desensitization and internalization (Eisinger
et al.,
2002).
TQ was reported to have potential as a substitution therapy for chronic morphine treatment (Abdel-Zaher
et al.,
2013). We
also studied the effect of co-treatment of TQ with morphine on MOR protein concentration after 48 h treatment. The result of
the present studies showed that co treatment of 60.9 µM TQ with morphine significantly increased MOR protein concentration
at 6h and 12h as compared to the cells treated with morphine alone. The results of this study indicated that TQ has a lower
opioid effect starting from 6h onwards. It is probably due to the properties of TQ itself as calcium channel blocker. Calcium
channel blocker had been investigated to have the capability to reduce opioid agonist-induced downregulation of μ-opioid
receptors and hence can prevent the magnitude of tolerance (Lee and Yoburn 2000). Co treatment of TQ with Methadone also
had been investigated in this study. The result showed that co treatment of TQ with methadone significantly increased MOR
protein concentration but at 24h in U87 MG cell. These results also suggested that methadone can prevent morphine-induced
MOR desensitization (Liu
et al.,
1999). Methadone and morphine are similar opioid receptor agonists but methadone has a
lower dependence potential than morphine and is effectively used in the treatment of opioid addiction, whereas morphine
induces dependence (Berger and Whistler, 2010).
Previous report has shown that TQ is an opioid receptor stimulating compound with 45% ligand displacement at mu-
opioid receptor (Nutten
et al.,
2012). Based on these ligands displacement, TQ has the potential to be used as an agent for
opioid addiction treatment. The present study has the limitation to understand the effect of TQ on cAMP level a fter chronic
morphine treatment. Therefore, it will be interesting to study further the effect of TQ on the desensitization of mu-opioid
receptor, cAMP level and cAMP-response element binding protein (CREB). In summary, these findings suggest that TQ could
potentially attenuate the tolerance and dependence of chronic morphine treatment by increasing the MOR protein
concentration starting from 6h onwards. We also were able to show the optimum concentration of TQ that can increase the
MOR protein at cellular level and proven non-toxic to the opioid receptor expressing cells (U87 MG). The findings also provide
an understanding of the effect of TQ on the cellular basis of substitution therapy for opioid addiction. It also provides an insight
into the mechanisms of dependence and tolerance, thereby facilitating further studies on the design and development of highly
effective and addictive substitute drugs for opioid addiction therapy.
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ACKNOWLEDGMENTS
This study was supported by Niche Research Grant Scheme (NRGS) from the Ministry of Education (MOE), Malaysia
RR057-1and Fundamental Research Grant (FRGS), RR076.
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