2. Nigella sativa: The Main Source of Thymoquinone
Medicinal plants have been used since the dawn of time and are used in the preparation of herbal medicines because they are considered to be safer than modern allopathic drugs. One of these plants is
Nigella sativa, commonly known as black seed or black cumin, which is native to southern Europe, North Africa and south-west Asia, and is grown in many countries around the world
[18].
Nigella sativa is an annual plant that grows to a height of 45 cm and has long, lineal-lanceolate leaves that measure 2–2.5 cm. Flowering and fruiting occurs from January to April.
Nigella sativa flowers are pale blue, 2–2.5 cm in diameter, and are solitary on long peduncles. Seeds of this plant are small dicotyledonous, trigonous, angular, regulose-tubercular, 2–3.5 × 1–2 mm, black on the outside and white inside
[19].
Nigella sativa seeds contain several biologically active compounds, and their phytochemical composition varies depending on several factors, such as the growing region or maturity stage. Compounds isolated from
Nigella sativa seeds belong to several chemical classes. The most notable is the family of terpenes and terpenoids, the most important of which is thymoquinone and its derivatives
[20]. TQ accounts for about 25% of the volatile oil of
Nigella sativa [21]. Other sources from which TQ is isolated are plants such as
Monarda didyma,
Monarda media wild,
Monarda menthifolia,
Satureja hortensis,
Satureja montana,
Thymus pulegioides,
Thymus serpyllum,
Nepeta leucophylla,
Tetraclinis articulata,
Juniperus cedrus,
Callitris quadrivalvis and
Thymus vulgaris [22,23,24][22][23][24]. However,
Nigella sativa is the most commonly reported source of TQ in the literature
[22]. The crude oil and thymoquinone extracted from its seeds and oil have many beneficial health properties that have been investigated in several research projects for their biological activity and potential therapeutic effects
[18].
3. Characterization, Pharmacognostic Isolation and Purification of Thymoquinone
The molecular weight of TQ is 164.204 g/mol and its chemical formula is C
10H
12O
2. TQ concentrations in seed oil have been reported between 18–25 µg/mL
[24]. TQ is a member of the monoterpene class of compounds, which are formally derived from the condensation of two isoprene units. Secondary metabolism in plants leads to the formation of these compounds, which can be isolated using steam distillation or solvent extraction of plant parts
[24]. Several methods have been described for the isolation and purification of TQ from plant material. These methods primarily involve supercritical fluid extraction, hydrodistillation, soxhlation and chromatographic techniques
[23,25][23][25]. Using thin-layer chromatography on silica gel, the yellow crystalline molecule TQ was isolated
[23]. Comparing two methods for the extraction of TQ from oil, the percentage obtained was 3% for extraction using hydrodistillation, as opposed to 48% for extraction using the Soxhlet method. A TQ-rich fraction was obtained using supercritical fluid extraction
[23,26,27][23][26][27]. Ghanavi et al.
[28] proposed a method for the isolation and purification of TQ from
Nigella sativa seeds that has the potential for use in industrial applications, including a consideration in production for pharmaceutical purposes. They followed a two-step procedure for the TQ extraction: first, maceration with methyl tert-butyl ether, followed by liquid–liquid extraction with methanol, which effectively removed most of the impurities. Next, preparative high-performance liquid chromatography (HPLC) was performed to separate and purify the TQ. The results of the HPLC analysis showed that the purity of the collected TQ was 97%, while the gas chromatography–mass spectrometry results identified that the purity of the obtained TQ was 97%. Several studies concluded that organic solvent, supercritical CO
2, or subcritical water extraction is a better method for separating TQ from herbal materials than water or steam distillation. Volatile compounds from
Monarda didyma and
Monarda fistulosa extracted with supercritical CO
2 were much richer in TQ
[29]. Other research found that the best solvent for TQ extraction from
Nigella sativa was benzene, suggesting that the choice of the most efficient TQ extraction method may depend on the type of starting plant material
[30]. There have also been published studies indicating that TQ is obtained from thymol by biotransformation using
Synechococcus sp. However, the reported yield from this process was low
[31]. Recently, the use of electrospun nanofibers as a sorbent for TQ extraction has been proposed by Nejabati et al.
[32].
4. Properties and Pharmacological Features of Thymoquinone
TQ is present in tautomeric forms, such as the keto or enol form, or in mixtures of them
[21,33][21][33]. Furthermore, due to its hydrophobic nature, the bioavailability and drug formulation of TQ are limited. In addition, the solubility of TQ in aqueous solutions varies from 549 to 669 µg/mL after 24 h to approximately 665 to 740 µg/mL after 72 h, and it depends on time
[21,34][21][34]. The routes of administration of TQ include the oral, sub-acute, sub-chronic, intraperitoneal and intravenous routes
[21,35][21][35]. After oral administration, liver enzymes may be elevated. This is due to metabolic activity, which reduces TQ to hydroquinone
[21,36][21][36].
Toxicity data are also available for TQ, which are essential when considering its pharmaceutical potential. The lethal dose (LD
50) varies depending on the route of administration, the carrier used and the model organism, but the LD
50 of TQ after oral administration to mice was 870.9 mg/kg and 104.7 mg/kg after intraperitoneal injection
[24,37][24][37]. A mean LD
50 of 790 mg/kg and 57 mg/kg for oral and intraperitoneal administration, respectively, was reported for rats, with signs of toxicity, such as hypoactivity and respiratory depression
[21,38][21][38]. TQ administered by intraperitoneal injection showed signs of toxicity associated with acute pancreatitis, and TQ administered orally showed signs of transient toxicity. Deaths were reported after doses of 500 mg/kg bw due to complications associated with intestinal obstruction. At the 300 and 500 mg/kg dose levels, signs of weight loss, diarrhea, mild abdominal distension and respiratory distress were observed in 34% of the rats within 48 h of administration. Subsequently, the rats regained weight and signs of toxicity began to disappear by the fifth day of the experiment
[23,39][23][39]. In a study by Mansour et al.
[40], rats and mice were given intraperitoneal injections at doses ranging from 5 mg/kg to 12.5 mg/kg and no toxicity was observed
[40]. Other studies conducted by Kanter et al.
[41] showed that the oral administration of 100 mg/kg or less of TQ also had no toxicological effect
[41].
The pharmacokinetics of thymoquinone have been studied, using different dosing regimens of TQ administered by intraperitoneal, intravenous or intragastric routes, for their efficacy in disease models. Overall, it has been characterized that the usual doses of TQ tested are 5 mg/kg (intravenous) and 20 mg/kg (oral)
[25]. This dose was used in a study by Alkharfy et al.
[42] who investigated the plasma pharmacokinetic behavior of the intravenous and oral bioavailability of thymoquinone in a rabbit model. They found that the estimated clearance (CL) after intravenous administration was 7.19 ± 0.83 mL/kg/min and the volume of distribution at the steady state (Vss) was 700.90 ± 55.01 mL/kg. During the subsequent oral administration, the CL/F and Vss/F values were 12.30 ± 0.30 mL/min/kg and 5109.46 ± 196.08 mL/kg, respectively
[42]. These parameters remained associated with an elimination half-life (t
1/2) of 63.43 ± 10.69 and 274.61 ± 8.48 min for intravenous and oral administration, respectively
[42]. The proposed t
1/2 absorption was approximately 217 min. Compartmental analysis showed a t
1/2a of ~8.9 min and a t
1/2b of ~86.6 min. The predicted total bioavailability of TQ was maintained at ~58% for a lag time of ~23 min. They also found that binding to proteins was greater than 99%
[42]. Alkharfy et al.
[42] showed that TQ has high bioavailability, but oral administration is associated with rapid elimination and relatively slow absorption. Also, Ahmad et al.
[39] used a dose of TQ 5 mg/kg intravenously and 20 mg/kg orally administered to rats. They found that after oral administration, the maximum plasma concentration (C
max) of thymoquinone was 4.52 ± 0.092 μg/mL in male rats and 5.22 ± 0.154 μg/mL in female rats (
p = 0.002). Similarly, after intravenous administration, the C
max was 8.36 ± 0.132 μg/mL in males and 9.51 ± 0.158 μg/mL in females (
p = 0.550)
[39]. The area under the plasma concentration–time curve after oral administration was 47.38 ± 0.821 μg/mL–h in females and 43.63 ± 0.953 μg/mL–h in males (
p = 0.014)
[39]. The results indicated that there were no sex differences in the pharmacokinetics of TQ.
The beneficial properties of thymoquinone are due to the presence of a lipophilic quinine moiety in its structure, which allows the molecule easy access to cellular and subcellular structures, as well as targeting intracellular transcription factors and kinases
[43]. Despite the promising safety profile and high protein binding of TQ, its bioavailability is limited due to certain physicochemical properties. TQ is photosensitive and even brief exposure can lead to substantial degradation, whatever the solution acidity and solvent used
[21]. Furthermore, TQ is unstable under alkaline conditions, with stability decreasing with increasing pH
[34]. The solubility of TQ in aqueous media is <1.0 mg/mL at room temperature. In addition to its hydrophobic nature, TQ is characterized by slow absorption, rapid metabolism, rapid elimination and low physicochemical stability, which limits its pharmaceutical applications
[23,24][23][24]. A study by Salmani et al.
[34] shows a very low stability profile of TQ in all aqueous solutions, with rapid degradation that varied with the type of solvent. The study of degradation kinetics showed a significant effect of pH on the degradation process. Also, light sensitivity may limit the applicability of TQ
[25]. To increase the bioavailability of TQ, researchers propose solutions based on the use of nanoformulations, i.e., liposomes, solid lipid nanoparticles, niosomes, nanostructured lipid carriers and nanoemulsions
[24,44,45,46,47][24][44][45][46][47].
5. Preclinical and Clinical Studies on Thymoquinone and Its Main Source Nigella sativa
Nigella sativa has several promising pharmacological properties, mainly related to the presence of TQ, which has been confirmed in preclinical and clinical studies. Biologically active compounds from
Nigella sativa have been shown to have antioxidant, antimicrobial, anti-inflammatory, antidiabetic, hepatoprotective, antiproliferative, proapoptotic, antiepileptic and immunomodulatory activities, among others
[48,49,50,51,52,53,54][48][49][50][51][52][53][54]. Black seed could also be used for the prevention of some diseases, such as cardiovascular disease, diabetes, gastrointestinal diseases or wound healing
[20,55,56,57,58,59][20][55][56][57][58][59]. In clinical studies,
Nigella sativa and its constituents, including TQ, have demonstrated antimicrobial, antioxidant, anti-inflammatory, anticancer and anti-diabetic properties, as well as therapeutic effects on metabolic syndrome and gastrointestinal, neural, cardiovascular, respiratory, urinary and reproductive disorders
[54]. Recent clinical trial reports suggest the potential use of
Nigella sativa in complementary treatment and prevention of conditions such as coronary artery disease, diabetic peripheral neuropathy,
Helicobacter pylori infection and polycystic ovary syndrome
[60,61,62,63][60][61][62][63]. The safety of thymoquinone-rich black cumin oil was evaluated in a phase I clinical trial and provided satisfactory results
[64]. Also, the antihyperglycemic activity of TQ was evaluated in a diabetic mouse model and patients
[65]. Another pilot clinical trial proved that thymoquinone has anti-epileptic effects in children with refractory seizures
[66].
Particular attention is currently being paid to research into the anticancer effects of TQ. Thymoquinone has anticancer properties that have been confirmed in preclinical studies of many types of cancer, including ovarian, colon, laryngeal, breast, leukemia, lung and osteosarcoma
[67,68,69,70,71,72,73,74,75,76][67][68][69][70][71][72][73][74][75][76]. Currently, there is only one clinical trial evaluating the chemopreventive effect of thymoquinone on potentially malignant oral lesions in the clinicaltrials.gov database (accessed 14 November 2023). In addition, an Arabian phase I trial evaluated the clinical activity of thymoquinone in patients with advanced refractory malignant disease. TQ was found to be safe and well-tolerated in patients up to 10 mg/kg/day, but there was no significant anticancer activity found
[77].
6. Brief on the Anticancer Properties of Thymoquinone
In vitro and in vivo studies have shown that TQ exerts tumorigenic effects in a variety of ways, including modulation of the epigenetic machinery and effects on proliferation, the cell cycle, apoptosis, angiogenesis, carcinogenesis and metastasis
[43,78,79,80][43][78][79][80]. Thymoquinone also shows hepatoprotective and renal protective properties against drug cytotoxicity, affecting key enzymes involved in detoxification, i.e., aspartate transaminase, alanine transaminase, etc.
[81]. It is also promising that TQ has low toxicity to normal cells, as confirmed by several studies, including studies on normal mouse kidney cells, normal human lung fibroblasts and normal human intestinal cells
[82,83,84,85,86][82][83][84][85][86].
The ability of TQ to inhibit proliferation and influence apoptosis of cancer cells has been confirmed in studies on several cancer models, in addition to those mentioned above, this includes prostate and squamous cell carcinoma
[87,88,89][87][88][89]. References show that TQ exerts its effect by influencing the expression of specific genes, including the upregulation of apoptotic mediators and the downregulation of transcription factors related to cytokine production
[43]. TQ induces a pro-apoptotic effect by acting through numerous molecular mechanisms, such as the activation of c-Jun N-terminal kinases (JNK) and p38, as well as the phosphorylation of nuclear factor-κB (NF-κB) and the reduction of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K) activities
[90,91][90][91]. TQ has also been shown to downregulate the PI3K/PTEN/Akt/mTOR and WNT/β-catenin pathways, which are critical for tumorigenesis
[92,93][92][93]. TQ also interrupts metastasis by downregulating the epithelial to mesenchymal transition (EMT) transcription factors twist-related protein 1 (TWIST1) and E-cadherin
[94]. For example, Zhang et al.
[95] showed that TQ can suppress invasion and metastasis in bladder cancer cells by reversing EMT through the WNT/β-catenin pathway
[95,96][95][96].
Oxidative stress is one of the factors in cancer development, as it leads to DNA damage
[80]. The anticancer properties of TQ are also linked to its effects on oxidative stress, as TQ has been shown to act as an antioxidant at low concentrations. Higher concentrations, however, induce apoptosis of cancer cells through the induction of oxidative stress
[90]. Thymoquinone acts by inducing cytoprotective enzymes, resulting in the protection of cells from cellular damage caused by oxidative stress. Thymoquinone upregulates the expression of genes encoding specific enzymes, such as catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase and glutathione peroxidase, whose role is to protect against reactive oxygen species
[43].
TQ has antitumor activity by reducing inflammation and acting as an immunomodulator. Inflammatory mediators and enzymes are known for their role in cancer development and progression
[80]. TQ has the ability to downregulate NF-κB, interleukin-1β, tumor necrosis factor alpha, cyclooxygenase-2 (COX-2,) matrix metalloproteinase 13 (MMP-13), prostaglandin E2 (PGE2), the interferon regulatory factor, which are associated with inflammation and cancer development. What is more, TQ increases the activity of natural killer cells, which are essential in the body’s defense against cancer cells
[43].
Although the exact mechanism of TQ’s anticancer action is not known and fully characterized, it is generally possible to distinguish several points at which TQ acts. The general outline of the anticancer nature of thymoquinone is summarized in
Figure 2.
Figure 2.
Overview of the mechanisms of action of thymoquinone in antitumor therapy.
7. Thymoquinone a Promising Candidate for the Treatment of Colorectal Cancer
7.1. Cytotoxic Effect of Thymoquinone on Colorectal Cancer Cells
The effect of thymoquinone on CRC has been studied in both cell lines and animal models. In general, the half-maximal inhibitory concentration (IC
50) is the most commonly used indicator of a compound’s potency on cell viability and the evaluation of resistance. Available study reports were reviewed to determine whether TQ has the ability to inhibit CRC cells. The IC
50 values of thymoquinone in selected colorectal cancer lines are summarized in
Table 1.
Table 1.
IC
50
values of thymoquinone in selected colorectal cancer lines.