Plants and Surgery: The Protective Effects of Thymoquinone on Hepatic Injury—A Systematic Review of In Vivo Studies

Multimodal treatment concepts including liver transplantation for hepatocellular carcinoma (HCC), extended resection methods and neoadjuvant chemotherapy for colorectal liver metastasis significantly improve patients’ outcome. However, surgery-induced hepatic ischemia-reperfusion injury (IRI) and chemotherapy-associated hepatotoxicity result in hepatocellular damage and compromised liver function. Activation of common key pathways in ischemic liver and hepatotoxic injury results in oxidative stress, inflammatory responses and apoptosis causing organ damage. Controlling liver damage before and during surgery is essential for the postoperative outcome. Nigella sativa has a long tradition as a natural remedy. In the essential oil, Thymoquinone (TQ) was identified as the main component and responsible for most of the therapeutic effects. Therefore, this systematic review aimed to summarize the hepatoprotective effects of TQ and its potential suitability to improve surgical outcome by reducing surgical ischemic injury and hepatotoxicity of neoadjuvant chemotherapy. The key findings can be summarized as TQ having strong antioxidant, anti-inflammatory, antifibrotic, anti-/proapoptotic and anticarcinogenic effects. Almost no side effects were reported irrespective of a large dose range, suggesting a wide therapeutic window. These results give rise to the expectation that TQ could evolve to a novel powerful drug to reduce hepatic injury.


Current Treatment Concepts and Hepatotoxicity
Surgical resection is the gold standard for curative treatment of colorectal liver metastasis [1] and hepatocellular carcinoma (HCC) [2]. However, only 10-20% of patients with liver metastasis and <30% with HCC are eligible for surgery due to the tumor size, location and/or the degree of liver function [3,4]. Those patients remarkably benefit from multimodal treatment concepts consisting of neoadjuvant chemotherapy for colorectal liver metastasis [5] as well as from extended resection methods [6] and liver transplantation for HCC [7,8].
Neoadjuvant chemotherapy is applied to downsize the tumor burden in the liver. Reduction of tumor mass may turn a primarily unresectable tumor into a resectable one, enabling a potentially curative resection for an increasing number of patients. Curative resection is the prerequisite for prolonging recurrence-free survival time [9].
Per contra, modern chemotherapy regimens such as Oxaliplatin and Irinotecan are associated with severe damage of the liver parenchyma, mainly leading to sinusoidal obstruction syndrome (SOS) and steatohepatitis [10]. Respectively, both injuries may increase the risk for post-hepatectomy morbidity [11].
The injury in SOS is caused by the damage to endothelial cells in the liver leading to their necrosis and extrusion into sinusoids which results in obstruction and congestion. Furthermore, stellate cells are activated and produce extracellular matrix and collagens. Due to loss of endothelial function, congestion and possibly direct toxicity of the implicated agents, hepatocellular necrosis is caused [10,12,13].
Steatohepatitis is due to an imbalance between triglyceride accumulation and elimination in the liver together with the activation of inflammatory cytokines. This results in interlobular inflammation and ballooning degeneration of hepatocytes [10,14].

Transplantation and IRI
In cases of HCC, liver resection can only be offered to patients with little impairment of hepatic function or to those needing only a minor resection. Liver transplantation is a curative option treating the tumor and the underlying liver disease [8]. Modern transplantation medicine includes well-developed surgical techniques, preservation strategies and immunosuppressive drugs making cadaveric or living-donor transplantations possible. Nevertheless, the inevitable hepatic IRI still remains a major problem in liver transplantation, potentially leading to hepatocellular dysfunction, aggravating liver rejection and ultimately contributing to the development of liver failure [15].
The mechanisms involved in the development of IRI are not fully understood. It is a dynamic process consisting of oxidative stress mediated by reactive oxygen species (ROS) leading to pro-inflammatory responses and hepatic damage upon reperfusion [16]. Scavenging of ROS and radicals in ischemic tissues and simultaneous suppression of the activated inflammatory cascade may reduce IRI.

Natural Remedies and Liver Protection
Toxic effects of modern allopathic drugs prompted the search for alternatives. Medicinal plants are a key resource for drug extraction [17]. A number of natural compounds with hepatoprotective properties promising to be safe alternatives to allopathic drugs with less side effects were identified. Three well-characterized examples are the grape skin component resveratrol, green tea catechins (GTCs) and tetrandin, a bis-benzylisoquinoline alkaloid isolated from the medicinal herb Stephania tetrandra S Moore. They have been tested for their ability to suppress pro-inflammatory immune responses and/or oxidative stress in ischemic liver [18][19][20].
Trans-resveratrol given before ischemia and before reperfusion at low doses protects against IRI mainly through maintenance of glutathione (GSH) levels and induction of superoxide dismutase (SOD) and catalase (CAT) enzymes [20]. GTCs have antioxidant properties in a dose-dependent manner via maintenance of manganese superoxide dismutase (MnSOD) and suppression of inflammation [19]. The agent tetrandine is anti-inflammatory and blocks calcium-channels. Furthermore, it can reduce oxidative stress by maintaining SOD activity [18].
Yet, in many cases, the actual mechanism of liver protection remains unclear. The agents could act as direct antioxidants, indirectly through nuclear respiratory factor 2 (Nrf2) activation or through unrecognized mechanisms [21]. However, none of the substances was implemented into clinical routine for different reasons. First, the bioavailability of most secondary plant extracts is very low. Second, their activity highly depends on concentration with many having contradictory effects at different concentrations. For instance, resveratrol is a pro-oxidant and may lead to liver damage when given at a high dose [21,22].
A recent study highlighted the hepatoprotective effect of Antrodia cinnamomea, a fungus species, in carbon tetrachloride (CCI4) induced hepatic injury in rats. The animals were either orally treated with an Antrodia cinnamomea water extract once per day or an emulsified extract for 5 days. The emulsified extract at 50 mg/kg significantly decreased the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-1 (IL-1), IL-6, nitric oxide (NO) and ROS levels. Additionally, cyclooxygenase-2 (COX-2) and caspase-3 protein expression was reduced [23]. This novel and intriguing data strengthens the need for further investigations in this field.
1.3. Characterization of Nigella sativa Seeds, Its Oil and Its Active Components N. sativa (Family Ranunculaceae) is a good example for a natural compound with a variety of beneficial properties. The bisexual plant is 20-90 cm tall with finely divided leaves and blue flowers on solitary long peduncles. It forms a fruit capsule which opens up when matured to expose the white trigonal seeds to the air, which then become black in colour [24,25].
N. sativa seeds contain numerous esters of unsaturated fatty acids with terpene alcohols. The currently known chemical composition of the seeds is depicted in Table 1.
The oil is one of the key components. An essential and a fixed oil can be extracted from the seeds [24][25][26]. The essential oil ranges from 0.4% to 2.5% [25,27]. It is cold-pressed from the seeds and is a yellow to dark amber liquid. The major active component in the essential oil is thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) with up to 50%, followed by p-cymene (40%), pinene (up to 15%) and traces of thymohydroquinone and dithymoquinone.
The fixed oil (36-38%) is produced by hydraulic expression of the seeds [26] and is rich in polyunsaturated fatty acids as well as some minor dihomolinolenic acids, phytosterols and tocopherols [28].
Alpha-hederin, a triterpene saponin, was also identified as an active component of N. sativa, but not much has been revealed about its amount in the seeds or its chemical stability. Further constituents of the seeds are unsaturated fatty acids, such as linoleic acid, oleic acid, eicodadienoic and dihomolinoleic acid. About 30% of the seeds consists of saturated fatty acids (stearic acid and palmitic acid) [24,25,29]. N. sativa has a long tradition in the Middle East as a part of an overall holistic approach to health. Commonly known as black seed, N. sativa is native to Southern Europe, North Africa and Southwest Asia. It is cultivated in many countries in the world like the Middle Eastern Mediterranean region, South Europe, India, Pakistan, Syria, Turkey and Saudi Arabia [30].
The use is wide spread in various traditional medical systems like Unani-Tibb, Ayurveda, Chinese medicine and Siddha [24]. Among Muslims, it has been defined as an ultimate remedy for all diseases except death due to a Prophetic hadith.

Toxicity Studies on N. sativa and Metabolism of TQ
N. sativa has a low profile of side effects and high LD 50 values in experimental studies prove its safety [35][36][37][38].
In 1965, El-Dakhakhani reported the LD 50 value of TQ as 10 mg/kg when intraperitoneally injected in rats [39]. Later, Mansour et al. indicated, that the LD 50 value in mice intoxicated with CCI4 is even higher. They reported a LD 50 dose of 90.3 mg/kg after intraperitoneal (ip) TQ application without specifying the cause of death [40]. In the same study, they did not observe any hepatotoxic effects when applying lower doses (4, 8, 12.5, 25 and 50 mg/kg) TQ. ALT, AST and lactate dehydrogenase (LDH) levels were not significantly elevated upon treatment [39][40][41]. This was confirmed in many other studies. Intraperitoneal TQ doses between 5 to 18 mg/kg were injected to mice and rats without side effects [40][41][42][43][44][45]. However, according to Mansour et al., the only dose that ameliorated hepatotoxicity of CCI4 was 12.5 mg/kg ip and side effects were reported at doses higher than 50 mg/kg ip (see Section 2.2) [40].
Badary et al. reported, that after oral TQ administration in healthy mice the LD 50 value is even higher (2400 mg/kg). At high doses, signs of toxicity were hypoactivity and difficulty in respiration [38,46]. However, Al-Ali et al. described lower LD 50 values after oral ingestion in mice (870.9 mg/kg) and in rats (794.3 mg/kg). The animals became more and more drowsy and dyspnoeic before death or they recovered after 24 h. On autopsy, both in the mice and rats receiving lethal doses of TQ, the heart, lungs, liver and kidneys as well as the peritoneum were congested, without any apparent sign of damage or necrosis. Probably, the cause of death was hypotension ultimately leading to shock [35].
Overall, these studies reveal strikingly different results between intraperitoneal and oral TQ administration (>8 to >25-fold difference in dose). The oral application seems to be better tolerated as signs of toxicity occurred after much higher doses than after intraperitoneal application.
According to Wang et al., this may be due to the fact that compounds with low intestinal absorption exhibit less toxicity in oral administration than in the injection route [47]. However, not much is revealed about the metabolism of TQ. Nagi et al. suggest, that TQ may undergo a reduction to dihydrothymoquinone [46]. This theory is strengthened by Mansour et al. They postulate that oral TQ may be transformed into less toxic metabolites in the gastrointestinal tract or into dihydrothymoquinone in the liver. In contrast, when intraperitoneally injected, a complete absorption of TQ into the systemic circulation occurs [40]. Khalife et al. proved, that TQ reacts with GSH, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) in physiological conditions. This induces the formation of glutathionyl-dihydrothymoquinone after rapid reaction with GSH and dihydrothymoquinone (DHTQ) after slow reaction time with NADH and NADPH. Interestingly, TQ showed lower scavenging activities than glutathionyl-dihydrothymoquinone and DHTQ. These results suggest, that the cellular antioxidant defense can possibly be modulated through the nonenzymatic metabolic activation of TQ [47].

Objective
Our systematic review aims for providing a comprehensive overview of in vivo experimental and human studies to explore the potential suitability of TQ for reducing hepatic injury in modern surgical treatment concepts.

Results
The search disclosed a total of 92 articles containing the keywords liver and TQ; 36 studies covering the period from 1999 to 2017 met our selection criteria ( Figure 1). The majority of the studies derived from eastern countries ( Table 2).

Study Protocols and Applied TQ Doses
We did not find any studies involving humans. The majority of the experiments were performed in rats (n = 24; 67%), followed by mice (n = 10; 28%) and rabbits (n = 2; 6%).
The mainly synthetically manufactured TQ was purchased from chemical companies. In most studies, animals received TQ orally (n = 30; 83%). In five studies, TQ was administered intraperitoneally (14%) and in one subcutaneously (3%).
A variety of injury models was used with the majority being hepatotoxic models using drugs such as cyclophosphamide, acetaminophen and methotrexate. Other models employed to investigate the hepatoprotective properties included steatosis, tumor, cholestasis, IRI, healthy animal and irradiation models (Table 3). Table 3. Hepatic injury models applied in the experiments (n = 36).

Effect of TQ
Our analysis revealed that all authors described a reduction of liver damage in TQ-treated animals, irrespective of the underlying etiology. Liver damage was assessed using histology and liver enzyme release. The beneficial effect was attributed to TQ having strong antioxidant, anti-inflammatory, antifibrotic, anti-and proapoptotic as well as anticarcinogenic effects (Tables 4-10, see Section 2.2.6).
Side effects were only reported in one study and consisted of oxidative stress leading to hepatic injury when higher doses of TQ (>50 mg/kg ip) were applied [40].

TQ and Inflammation
In a variety of hepatic injury models, concomitant treatment with TQ led to a reduction of a wide range of different inflammatory markers as described in the following paragraphs.
The majority of the authors attributed an important role to the TQ-induced inactivation of the tumor necrosis factor-α (TNF-α) cell signaling pathway. Within these authors, El-Sheikh et al. [56], Galaly et al. [72], Al-Malki et al. [53], Suddek et al. [61] and Hassanein et al. [58] performed their experiments on toxic hepatic injury models caused by drugs and chemicals, respectively Methotrexate, Gentamicin, Cisplatin, Tamoxifen and Titaniumdioxide nanoparticles. Helal et al. [59] provoked endotoxemia by lipopolysaccharide (LPS) application in the experimental animals. Awad et al. [62] applied a model of nonalcoholic steatohepatitis (NASH) induced by high-fat high-cholesterol diet. Beside the reduction of TNF-α, El-Sheikh et al. also showed a lower level of COX-2 [56]. Al-Malki et al. attributed a further role to the reduction of IL-1β levels [53]. In their steatosis model, Awad et al. reported an increase in IL10 along with TNF-α reduction [62].
Further parameters that seem to be involved in the development of the anti-inflammatory effect of TQ were described by Aras et al. [54], Mohany et al. [44] and Abd-Elbaset et al. [67]. Both, Aras et al. In addition, a decrease of TGF-ß1 and TGF-ßRII was analyzed [76].
Furthermore, a significant influence on the breakdown of the extracellular matrix through modulation of metalloproteinases was assigned to TQ. Three different ways were described: upregulation of matrix metallopeptidase 9 (MMP-9) [76], downregulation of tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression [73] and decrease of matrix metallo-proteinase-2 [62] which was observed in NASH-models. To summarize, TQ modulates different mechanisms involved in apoptosis and thereby the analyzed effects of TQ apparently vary from anti-to proapoptotic. However, taking into account, that NF-κB is also strongly involved in immune responses and the authors rather concentrated on this topic, the low NF-κB expression should be seen in the context of the anti-inflammatory effect of TQ which dominates the proapoptotic response.

TQ and Hepatic Malignancy
An important property of TQ is its direct and indirect anticarcinogenic effect in various tumor models. Our focus was on hepatic tumor models.
Ke X. et al. injected Hep3B cells subcutaneously in mice to create HCC models. They observed a normalization of the histology and a decrease of tumor volume when TQ was applied. This effect was explained by inhibition of Bcl-2 expression and repression of the Notch signaling pathway through downregulating NOTCH1 intracellular domain (NICD1). Furthermore, TQ inhibited cell growth by inducing cell cycle arrest through increased expression of p21 protein [77].
In N-nitrosodiethylamine (NDEA) induced HCC rats, Raghunandhakumar S. et al. reported that TQ greatly reduced liver injury markers, decreased tumor markers and prevented hepatic nodule formation through reduced neoplastic transformation of hepatocytes. Application of TQ had an anti-proliferative effect mediated by regulation of the G1/S phase cell cycle transition [78].
Sayed-Ahmed et al. also used NDEA-induced hepatocarcinogenesis models. Pathophysiologically, the initiation of hepatic cancer in these models is induced by decreased mRNA expression of GPx, CAT and GST. TQ application prevented the development of liver cancer by decreasing oxidative stress and preserving the activity and mRNA expression of antioxidant enzymes [64].
In conclusion, these promising properties of TQ can open new perspectives in liver cancer prevention and treatment.        · Mild hepatic injury (total score: 6) · ↓ ALT, ALP, total bilirubin · ↓ thiobarbituric acid reactive substance · ↑ GSH, GPx, GST, CAT · ↓ total nitrate/ nitrite

Summary
Our systematic review has summarized the effect of TQ on liver injury in experimental studies. This was done to analyze its potential suitability as a liver-protecting drug to improve IRI in transplantation as well as hepatic resection and optimize surgical outcome after hepatotoxic neoadjuvant chemotherapy. So far, no studies regarding the use of TQ in patients have been published despite the strong tradition in several countries.
For the in vivo studies, the key findings can be summarized as TQ having a strong antioxidant, anti-inflammatory, antifibrotic, anti-and proapoptotic and anticarcinogenic effect.
Despite the positive data, there are still some obstacles to be solved. First of all, in the reported studies, a wide range of doses was applied (ranging from 0.5 mg/kg/d [49,50] to 100 mg/kg/d [46,49]). Within this dose range, almost no side effects were reported, suggesting a wide therapeutic window. Yet, explanations of this striking broad dose range might be due to the differences in species and/or the route of administration [35,40,79]. According to toxicity studies (see Section 1.5), oral application of TQ seems to be better tolerated as signs of toxicity occurred at much higher doses than after intraperitoneal application. Overall, the LD 50 values reported in several studies after both, intraperitoneal injection and oral application, were much higher than the doses of TQ reported to have desirable effects.
Furthermore, as indicated in the reported experiments, TQ modulates various signaling pathways. However, the direct mechanism of action of TQ still remains unclear.
Therefore, for better data reproducibility, but also to improve drug safety and to optimize TQ application for the discussed indications, it is essential to explore the mechanisms and doses inducing benefits and side effects.

Methods
A systematic search was performed using two databases (Medline and Cochrane Library). The search was last actualized in June 2017. Inclusion criteria were full paper publications in peer reviewed journals reporting original works or systematic reviews.
The search string was (Thymoquinone OR Nigella sativa OR Nigella sat) AND (hepar OR hepatic OR hep* OR liver) with a filter for language (English) and study settings (humans, animals), without further restrictions.
The title/abstract screening was performed by two of the authors and all publications meeting the inclusion criteria were retrieved as full text. The review was performed in accordance with the PRISMA statement (Figure 1, [48]). The main data were extracted including the publication year, study authors and countries of origin, study settings and design, dose of TQ, LD 50 dose and route of application.
Furthermore, the outcome of TQ administration in hepatic injury was analyzed by recording the effects of TQ in respect to histological alterations, enzyme release and the postulated mechanisms of action. To classify the main properties, subgroups were defined. The outcomes of the review were not meta-analyzable due to the lack of homogeneity of the data.

Conclusions
In parallel to the development of novel and effective treatments in modern medicine, the number of drugs with hepatotoxic effects is also increasing. For this reason, substances reducing this toxicity are urgently needed.
Due to the wide therapeutic window with strong antioxidant and anti-inflammatory effects, TQ is a promising drug candidate for IRI. Furthermore, as a hepatoprotective drug with anti-proliferative properties and little reported side effects, TQ could enhance the tolerability and effectivity of neoadjuvant therapy prior to ablative liver surgery.
To date, no attempts have been made to test the potential of TQ at the clinical level. Therefore, a sequence of preclinical and clinical assessments and studies would be adequate.

Acknowledgments: No funds were received.
Author Contributions: Aysun Tekbas conceived the review, analyzed the data and wrote the paper; Aysun Tekbas and Jutta Huebner performed the title/abstract screening; Aysun Tekbas and Uta Dahmen designed the review; Uta Dahmen and Jutta Huebner contributed to the methods section; Uta Dahmen, Jutta Huebner and Utz Settmacher revised the review.

Conflicts of Interest:
The authors declare no conflict of interest.