Nigella Plants – Traditional Uses, Bioactive Phytoconstituents, Preclinical and Clinical Studies

Nigella is a small genus of the family Ranunculaceae, which includes some popular species due to their culinary and medicinal properties, especially in Eastern Europe, Middle East, Western, and Central Asia. Therefore, this review covers the traditional uses and phytochemical composition of Nigella and, in particular, Nigella sativa. The pharmacological studies reported in vitro, in vivo, and in humans have also been reviewed. One of the main strength of the use of Nigella is that the seeds are rich in the omega-6 fatty acid linoleic acid and provide an extra-source of dietary phytochemicals, including the bioactive thymoquinone, and characteristics saponins, alkaloids, and flavonoids. Among Nigella species, N. sativa L. is the most studied plant from the genus. Due to the phytochemical composition and pharmacological properties, the seed and seed oil from this plant can be considered as good candidates to formulate functional ingredients on the basis of folklore and scientific knowledge. Nonetheless, the main limations are that more studies, especially, clinical trials are required to standardize the results, e.g. to establish active molecules, dosage, chemical profile, long-term effects and impact of cooking/incorporation into foods.


INTRODUCTION TRADITIONAL USES OF NIGELLA
Latest ethno-pharmacological studies showed that Nigella species are among the most usually used for traditional and folk medicinal practices. Among them, N. sativa is probably the best-known species of the Nigella genus and it has been used in many parts of the world as a natural medicine. The traditional use of N. sativa dates from the 1st century A.D.; Pliny the Elder recommended N. sativa as a digestive and an ingredient of antidotes curing snake bites and scorpion stings (Dönmez and Mutlu, 2004), or even at least till Tutankhamen kingdom (Mrozek-Wilczkiewicz et al., 2016). Today, the seed powder of N. sativa is recommended at 0.5-4 g in the Pharmacopoeia of India , which is used as a stimulant to ease bowel and indigestion problems and as carminative. It has also been administered to manage pain during menstruation and diabetes in India and Bangladesh (Esakkimuthu et al., 2016;Hossan et al., 2018). Similarly, N. sativa is widely used in traditional medicine of Algeria for the treatment of diabetes and also to treat high blood pressure (BP) (Bouzabata, 2013).
Moreover, according to the Bedouins (Egypt), the wooden stem is used to treat jaundice, while seeds are used to treat BP as before, as well as heart diseases, etc. (El-Seedi et al., 2013) (Table 1). Similar uses have been reported in Iranian traditional medicine (Ghazeeri et al., 2012;Amiri and Joharchi, 2013;Bahmani et al., 2016b), in Pakistan (Khan et al., 2014;Yaseen et al., 2015;Aziz et al., 2017) and in Morocco (Eddouks et al., 2002;El-Hilaly et al., 2003;Khabbach et al., 2012;Jamila and Mostafa, 2014;Teixidor-Toneu et al., 2016), where N. sativa seeds and leaves are orally ingested, consumed as a powder, herbal tea, as decoction or as inhalant. In Pakistan N. sativa is also applied to manage lactation, bacterial diseases, etc. (Khan et al., 2014;Aziz et al., 2017), while in Morocco the seeds are recommended to deal with otolaryngological, urological, and nephrological ailments as well as to treat pathologies of the respiratory and skeleton-muscular systems, allergy and hypersensibility (Eddouks et al., 2002;El-Seedi et al., 2013;Jamila and Mostafa, 2014). The anti-rheumatic and analgesic properties of N. sativa combined with honey have been reported (Khabbach et al., 2012). In addition, seeds infusion is used to treat malaria in the Malaysian traditional medicine (Al-Adhroey et al., 2010), as well as seeds (fresh, dried, and powdered forms) and leaves in Ethiopia (Alrawi et al., 2017). The use of N. sativa is still more widespread, being recognized as panacea for its healing properties in Qatar (Alrawi et al., 2017) and Bangladesh (Jennings et al., 2015). Some of the latter uses are also common in Mauritius, Nepal, Turkey, Thailand, Lebanon, and Palestine (Ghazeeri et al., 2012;Al-Ramahi et al., 2013;Sreekeesoon and Mahomoodally, 2014;Guler et al., 2015;Jennings et al., 2015;Kunwar et al., 2015;Bahmani et al., 2016a;Neamsuvan et al., 2016;Alrawi et al., 2017;Ahmed et al., 2018). Furthermore, in Bangladesh and Libano N. sativa seeds are used as a spice and food preservative, directly consumed after being ground, while Nigella oil also can be applied topically (Jennings et al., 2015). Similarly, seeds of N. sativa (also named Nigella glandulifera Freyn and Sint.) are consumed in some regions of China and frequently added to "naan" (a crusty pancake). Its water decoction is used in the Uighur's traditional medicine for the treatment of numerous disorders as the other species (Table 1) (Zhao et al., 2013).
Other Nigella species with a wide range of medicinal properties are N. damascena and N. ciliaris. In Central Europe, the use of N. damascena dates from Bronze Age but it cannot reliably assign any ethnobotanical relevance and its origin is unclear; it has never grown in the wild in central Europe (Heiss and Oeggl, 2005). Alternatively, N. damascena seeds are used for example in Sicilian folk medicine as a galactogogue (Geraci et al., 2018). Other uses are as emmenagogue, vermifugue, and disinfectant (Heiss and Oeggl, 2005). This plant is also used as an helmintic agent and to treat hematuria, and skin diseases in the Serbian medieval medicine (Jarić et al., 2014). The former use has also been reported in Epirus (Greece) (Vokou et al., 1993). Traditionally, N. damascena is used for treating trachoma in Tunisia and Italy (Leporatti and Ghedira, 2009). Besides its use as herbal remedy, N. damascena is used as a condiment in several regions (Heiss and Oeggl, 2005), including in Morocco (Khabbach et al., 2011).
In the folklore medicine of Palestine and Iran, N. ciliaris seeds are used for abdominal pain, to facilitate delivery and to treat menstrual cycle related problems, respectively (Ali-Shtayeh et al., 2015) (Bahmani et al., 2015). In Turkey, Meriç Town, dried flowers of N. arvensis are used as a winter tea (Kartal and Güneş, 2017). On the other hand, in Iran (Kerman), the seed powder of N. arvensis mixed with other seeds are administrated to enhance male potency and to improve memory and intelligence (Khajoei Nasab and Khosravi, 2014). The seeds of both Nigella species are used for the treatment of cancer in Palestine (Jaradat et al., 2016a;Hamarsheh et al., 2017).

PHYTOCONSTITUENTS
Nigella genus is widely used for their culinary and medicinal properties especially in the Eastern Europe, Middle East, Western and Central Asia. The plants are mainly consumed for their seeds and seed oil, as commented before. The main constituents of N. sativa seeds reported in the literature are fixed oil (27-40%), proteins (16-19%), characterized mainly by the amino acids arginine, glutamic acid, leucine, lysine; minerals (1.79-3.74%), like Cu, Zn, P, and Fe; carbohydrates (28.5-33.7%), and solubre dietary fibers (5.5-8.9%) (Al-Naqeep et al., 2009;Tiruppur Venkatachallam et al., 2010;Kooti et al., 2016;El-Naggar et al., 2017;Saxena et al., 2017). Concerning the phytochemical composition of Nigella seeds, the most interesting plant part, it is constituted of alkaloids, terpenes and phenolic compounds (Agradi et al., 2001). Although the number of studies on the chemical composition and pharmacological properties of N. sativa is continuously increasing (Bourgou et al., 2010b), there are other interesting species for their culinary and medicinal properties, as commented before. Thus, this section details phytochemicals found in N. sativa and other Nigella plants.

N. damascena
Antihelmintic (for children) and to treat haematuria and skin diseases (itchiness and eczema) also reported as major compounds from the seed oils, extracted by n-hexane, of different N. sativa genotypes from India (Saxena et al., 2017). Other study have shown that fixed oils from N. sativa seed from Turkey and Egypt obtained by supercritical CO 2 extraction were similar in fatty acid composition of saturated fatty acids (16%), mainly constituted by palmitic acid (4) and stearic acid (5); monounsaturated acids (23%), mainly oleic acid and cis-vaccenic acid (6), and polyunsaturated acids (58%), mainly linoleic acid (1) and eicosadienoic acid (7) (Piras et al., 2013) (Figure 1). In this work the ratio omega-6/omega-3 ranged between 180 and 221 considering linoleic and alphalinolenic acid.

Volatile Oil Phytochemicals
Volatile oils are derived from plant tissues and are characterized to evaporate under room temperature and failure to saponify.

Alkaloids
The bio-potential of nitrogen-containing heterocycles has been recently revised (Thakral and Singh, 2019). In plants, alkaloids contain one or more nitrogen atoms and usually are situated in some cyclic system. Similarly, these nitrogen-containing heterocycles, such as isoquinoline alkaloids and their N-oxides, can be a source of leads for drug discovery (Dembitskya et al., 2015). This included compound such as nigellimine (23) and nigellimine-N-oxide (24) from N. sativa seeds. This species also contains other type of alkaloids such as nigellicine (25), and nigellidine-4-O-sulfite (26) (Figure 3). Nigellidine (27) and its derivative methyl nigellidine, higenamine (28), and nigeglanine (29) were also characterized in N. sativa seeds (Atta Ur et al., 1995;Yun et al., 2014). Among them, methyl nigellidine, nigeglanoside and nigelloside could be used as markers to differentiate both species (Yun et al., 2014).

Saponins
Other studies also suggest the presence of phenolic compounds and triterpenes like saponins in the seeds of N. sativa and other Nigella species (Ali et al., 2008;Atta Ur, 1985;Atta Ur, 1992;El-Naggar et al., 2017;Kooti et al., 2016;Tiruppur Venkatachallam et al., 2010;Zribi et al., 2014). Saponins are a heterogeneous group of glycosides, which have one or more hydrophilic moieties combined with a lipophilic triterpene or steroid derivative. In particular, triterpene saponins are  highly characteristic compounds in Nigella seeds. Besides alphahederin (30) (Boubertakh et al., 2013), other relative saponins are: Figure 3). The latter were reported in the seeds of N. sativa and exhibit a broad spectrum of bioactivities (Chen et al., 2018;Dönmez and Mutlu, 2004). Others also found in the latter species are: Figure 3), with anticancer properties reported in vitro (Tian et al., 2006).

Factors that Affect the Phytochemical Composition
Phytoconstituents of Nigella may vary even within the same species and this is related to many factors such as growing and climatic conditions, location, different organs of the plants and the extraction methods used (Edris, 2010;Manju et al., 2016;Saxena et al., 2017) (Tables 2 and 3). Secondary metabolites, compounds which are not directly related to the development, growth and reproduction of plants but they have significant performance in chemical communication, primary defense against biotic and abiotic stress (Sarkar and Shetty, 2014) and also in epigenetic memory. The chemical composition may vary also during development stage and this was confirmed by Zribi et al. (2014) who demonstrated that the total phenolics, flavonoids, flavonols and flavones, alkaloids and proanthocyanidins contents of Tunisian and Indian N. sativa aqueous extracts were the highest in the vegetative stage. Moreover, the aerial parts from the two varieties were richer in total phenolics and flavonoids, including flavonols, flavones and proanthocyanidins, than seeds. Salinity is also another factor that greatly influenced the phenolic composition of Nigella seeds; e.g. generally the content of certain phenolic compounds decreased, including the major one vanillic acid (45), while the content of trans-cinnamic acid (53),  (54) increased. This was related with a decrease of the antioxidant activity (Bourgou et al., 2010a). Today, this is essential information since water scarcity and salinization of arable lands may occur in a future scenario exposed to drastic changes caused by climate (Selim et al., 2019), affecting not only crop growth but also its phytochemical content and antioxidant properties. This means that a high variability in the content of phytochemicals can be found in Nigella plant parts, and in particular in N. sativa seeds, and from a functional point of view each essential oil/extract should be further characterized in order to elucidate the real active molecules inside for further standardization and quality control.

Antioxidant Activity and Effects on Oxidative Stress
Some studies suggest that N. sativa oil and extracts have antioxidant properties and can modulate oxidative stress, which could be beneficial for oxidative stress related diseases/ disorders. In this sense, oils and solvent extracts from N. sativa seeds possessed antioxidant activity in vitro (Toma et al., 2015;Mohammed et al., 2016;Abedi et al., 2017;Singhal et al., 2017), ex vivo (Ghoreyshi et al., 2020), in vivo (Mahmoudi et al., 2018;Rasoli et al., 2018) and in humans (Nikkhah-Bodaghi et al., 2019). Moreover, in vivo regular intake of ethanolic extract of N. sativa seed (400 mg/kg) lowered the lipid peroxidation, and enhanced catalase activity in Wistar rats (Rasoli et al., 2018). Another work suggested that hydroalcoholic and hexane extracts of N. sativa seed as well as thymoquinone may counteract oxidative stress caused by high-fat diets (HFDs), e.g. malondialdehyde (MDA)  Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 625386 levels decreased, while the activity of catalase enzyme and serum total antioxidant capacity increased (Mahmoudi et al., 2018). Concerning other Nigella species, N. damacena 70% ethanolic extract (seeds) has shown antioxidant properties in vitro, even higher generally than N. sativa seeds (Toma et al., 2015); the former contained higher amounts of quercetin derivatives. Also, the fixed oil from the seeds of another species, Nigella unguicularis Spenner, showed favorable oxidant/antioxidant balance and blood lipids profile when it was administered to rats (1 ml/kg orally for 4 weeks) (Kökdil et al., 2005). However, the responsible active compounds were not determined in the latter case. As commented before, thymoquinone can have a protective role against oxidative stress (Woo et al., 2013). Thymoquine has shown antioxidant properties in vitro, in particular using the oxygen radical absorbance capacity (Tesarova et al., 2011). Essential oil extracted from N. sativa seeds with higher thymoquinone content showed stronger antioxidant activity than other essential oils (Abedi et al., 2017). In this context, Usta and Dede (2017) assessed role of thymoquinone on oxidative DNA damage and NF-κB levels in diabetic rats. The results revealed that the oxidative DNA damage (8 hydroxy-2-deoxyguanosine) and NF-κB levels were insignificantly lowered after treatment. Moreover, it also reduced glycosylated hemoglobin, glucose levels and aspartate aminotransferase (AST), alanine aminotransferase and gammaglutamyl transpeptidase activities. In recent in vitro and in vivo studies, thymoquinone in combination with iron oxide nanoparticles showed attenuation in genetic and oxidative damage, and caused an enhancement in the levels of antioxidant enzymes (Ansari et al., 2019).
Nonetheless, other Nigella phytochemicals constituents have antioxidant activity, which is not strictly dependent on the Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 625386 thymoquinone content (Bordoni et al., 2019). Thymohydroquinone (11) has also shown scavenging properties in vitro, while no effects were found for dithymoquinone (10) (Tesarova et al., 2011). Trans-anethole (14), isolated from the essential oil of N. sativa seeds by thin-layer chromatography, also showed free radical scavenging properties using several methods (Burits and Bucar, 2000). Moreover, the phenolic nature of thymol (13) and carvacrol (12) makes them to possess higher antioxidant activity than other volatile constituents in oils (see reviews by (Salehi et al., 2018;Sharifi-Rad et al., 2018;Salehi et al., 2019). In an antioxidant activity guided fractionation of the essential oil from N. sativa seeds it was found that thymoquinone 11) (51%), thymol (25%) and carvacrol (8%) were the main antioxidant compounds (Kazemi, 2015). Moreover, in oxidative stress induced experimental subjects, thymol (12) (thymol 10 mg/kg) has been shown significant impact on sperm quality via increasing the spermatozoa concentration and motility, and lowering the MDA level. It also decreases the dead sperm ratio and enhances the glutathione concentrations in the testicles, liver and kidney tissues (Fangfang et al., 2017). The antioxidative properties of thymol (12) and carvacrol (13), through improving the antioxidants, suppressing lipid peroxidation markers and ameliorating oxidative stress, have also been shown in other in vivo studies (Bakir et al., 2016;Samarghandian et al., 2016;Saravanan and Pari, 2016).
It should be notice that polar extracts from Nigella could have a wide spectrum of other phenolic compounds, which are antioxidant in nature (see section 4.5), and thus it should be taken into account in further studies. As an example, free and bound phenolic compounds, which were associated to N. damascena and N. arvensis proteins from the seeds, have shown antioxidant properties in vitro (Alu'datt et al., 2016). As noted before, N. damascena seeds contain quercetin derivatives, including quercetin aglycone, hyperoside and quercitrin, as well as the total phenolic content showed enhanced antioxidant properties (Toma et al., 2015). Furthermore, total saponins from N. sativa seeds increased the plasma superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and decreased MDA level compared to control group in D-galactose-induced aging model (6-24 mg/kg). These seeds also showed (2,2-diphenyl-1picrylhydrazyl) DPPH radical scavenging properties (Dönmez and Mutlu, 2004).

Anti-cancer Properties
Nigella sativa has anti-proliferative, pro-apoptotic, anti-oxidant, cytotoxic, anti-mutagenic, and anti-metastatic effects, underlined by several mechanisms reviewed by (Majdalawieh and Fayyad, 2016). Studies in cell lines, including in human lung (A-549 cells), epithelial cervical (HeLa and SiHa) and Michigan Cancer Foundation-7 (MCF-7) breast cancer cells, have shown that the administration of N. sativa alcoholic seed extracts, seed oil and nanoemulsions markedly lowered the cell viability, inducing apoptotic cell death, and/or altered the cellular morphology, with IC 50 values between 0.41-82 μL/mL in some cases (Shafi et al., 2009;Hasan et al., 2013;Al-Sheddi et al., 2014;Periasamy et al., 2016;Butt et al., 2019). Among these studies, Hasan et al. (2013) revealed the presence of thymoquinone as the potential active Nigella constituent. Concerning in vivo studies, supplementation of different doses of N. sativa ethanolic extract (seed) on daily basis (150, 250, 350 mg/kg), thymoquinone (20 mg/kg), and daily dose of silymarin (100 mg/kg) prevented from the hepatocellular carcinoma proliferation provoked by diethylnitrosamine through multiple pathways: reduction in p-EGFR and p-ERK1/2, deactivation of EGFR/ERK1/2 signaling, down regulation of target genes (c−fos, PCNA, and Bcl2), suppression of cell proliferation, reduction in alpha-fetoprotein (AFP) and hepatic enzymes, and enhancement in the levels of antioxidant enzymes (Shahin et al., 2018). Other in vivo studies suggested a protective effect of N. sativa seeds oil and thymoquinone against breast carcinogens (Linjawi et al., 2015) with dosages between 1 and 10 mg/kg. The water extract of N. sativa has shown immunomodulatory and anti-tumor effects on Ehrlich ascites carcinoma in mouse model (Aikemu et al., 2013), but its composition was not characterized.

Cardioprotective Properties
Nigella sativa has a high potential for the prevention and treatment of cardiovascular diseases, while little is known about the cardiovascular properties of other Nigella species. As an example, N. sativa seeds powder improved lipid profile and prevented atherosclerosis in hypercholesterolemic rabbits when it was supplied in their diet (5%). It significantly decreased fatty streak formation, total cholesterol and low-density lipoprotein (LDL) (Asgary et al., 2013). A recent study also suggests that diabetic rats treated with an ethanolic extract from N. sativa seeds (100-400 mg/kg) for six weeks reduced serum glucose and lipids, while it improved atherogenic index of plasma, vasoreactivity, endothelial dysfunction, and vascular inflammation (Abbasnezhad et al., 2019). Nigella sativa (seed, oil and extracts) has a potential role in the management of hypertension, as shown ex vivo (Hebi et al., 2016;Abbasnezhad et al., 2019) and in vivo studies (Khattab and Nagi, 2007;Leong et al., 2013;Enayatfard et al., 2018) due to vasorelaxant properties. It also may increase endothelial nitric oxide (NO) synthesis, attenuate cardiovascular effects of the vasoconstrictor angiotensin II, and inhibit the parasympathetic tone (Khattab and Nagi, 2007).
Nigella sativa seed oil used in the study by Asgary et al. (2013) contained predominantly terpenoids, including p-cymene 9) (37.3%) and thymoquinone 8) (13.7%). In other studies, N. sativa seeds extracts contained thymoquinone (8), which were standardized (0.05-0.06% extract weight) (Enayatfard et al., 2018;Abbasnezhad et al., 2019). In fact, thymoquinone has demonstrated very good cardioprotective benefits and antihypertensive effects (Enayatfard et al., 2018). Liu et al. (2019) reported that thymoquinone has protective effects against cardiac damage in BALB/c mice via multiple mechanisms including reduction in intestinal histological alterations, suppressions of p62, NLRP3, IL-1β, TNF-α, caspase-1, IL-6 and 18, as well as MCP-1 expressions, inhibition of troponin-T levels in serum, enhancement in ATP, improvement in IL-10 and beclin 1 levels, and decrement in phosphatidylinositide 3-kinase level. Convincingly, thymoquinone effectively modulates pyroptosis, autophagy, and pro-inflammatory markers in cardiac stress (Liu et al., 2019). Likewise, thymoquinone reverted doxorubicineinduced cardiotoxicity when administered to mice. of the administration of thymoquinone (10-20 mg/kg p.o) significantly ameliorated oxidative stress markers involving lipid peroxidation, creatine kinase (CK)-MB, lactate dehydrogenase (LDH), and AST. Alongside, meaningful improvement in antioxidant enzymes like SOD, catalase, glutathione reductase, and glutathione-S-transferase has also been noticed (Alam et al., 2018). Another recent study reported by Atta et al. (2018) showed the protective role of thymoquinone on diabetes-caused cardiac complications in Wistar male rats. Lu et al. (2018) reported that thymoquinone (8) provides shield against myocardial ischemia/reperfusion (I/R) injury in isolated rat hearts and neonatal rat cardiomyocytes models via: improving left ventricular function, lowering myocardial infarct size, attenuating mitochondrial oxidative damage, producing LDH, and lowering MDA levels and H 2 O 2 concentrations, alongside improving antioxidant enzymes levels. Additionally, the cardioprotective role of this compound was also linked to the up-regulation of sirtuin 1 (SIRT1) expression and inhibition of p53 acetylation (Lu et al., 2018). Thymoquinone 8) showed prophylactic effect from myocardial I/R injury in Langendorff perfused rat hearts. It encouraged autophagy, favored cardiac function, lowered infarct size, LDH and CK-MB levels, and suppressed oxidative stress (Xiao et al., 2018). Finally, Salahshoor and their co-workers highlighted that the supplementation of thymoquinone (4.5-18 mg/kg) in mice significantly increased the mean diameter of central hepatic vein, blood serum NO level, and liver enzymes level, while it decreased the liver weight (Salahshoor et al., 2018). Two other interesting molecules are again carvacrol (12) and thymol (13). The latter was a cardioprotective agent against carotid tissue of hypercholesterolemic rats. The supplementation of thymol (24 mg/kg) in experimental subjects prevents from cardio complications through several mechanisms such as reduction in low density lipoprotein, triglycerides, increment in high-density lipoprotein level, reduction in apoptotic proteins and inflammatory expressions, phosphorylation of p38 (p-p38) and the protein expression of cleaved caspase-3 (Bayatmakoo et al., 2017). In another work, thymol (13) administration (7.5 mg/kg) reversed changes produced by isoproterenol in rats. It also enhanced the caspase-8 and 9, as well as Fas genes expression while lowered Bcl-xL gene expression in myocardium (Meeran et al., 2016). Among other effects, previous investigation revealed that multiple pathways are involved in this effect: a lowered serum cardiac troponin-Y, lysosomal thiobarbituric acid reactive substances (TBARS), high sensitivity C-reactive protein (hsCRP), and lowered activities of β-glucuronidase, β-galactosidase, cathepsin-B and D activities in lysosomes. Thymol down-regulated the proinflammatory cytokines involving IL-6 and 1β genes expression and TNF-α in myocardium of rats, and lowered heart weight, left ventricular hypertrophy and increased

Antidiabetic
Nigella sativa could play a role against diabetes and obesity, and thereby it could be an interesting agent to treat/prevent the metabolic syndrome. In cell in vitro assays, the ethanol extract from N. sativa seeds has shown antidiabetic activity using adipocytes (Benhaddou-Andaloussi et al., 2008;Benhaddou-Andaloussi et al., 2010). In particular, it was able to activate the AMPK pathway and the insulin signaling pathway, as well as it acted as an agonist of the peroxisome proliferator-activated receptor (PPARγ) (Benhaddou-Andaloussi et al., 2010), which plays a role in the regulation of metabolism. The seed oil from N. sativa, which contained compounds such as thymoquinone (8), anethole (14), p-cymene (9), saturated and unsaturated fatty acids, had also antidiabetic properties in vivo. In this concern, N. sativa oil (500 mg/kg/day) was co-administered orally along with high-fructose water (20%, w/v) for 45 days. It was able to reduce fructose-induced insulin resistance by reduction of hepatic insulin-degrading enzyme protein and activation of insulin receptor signaling. It also decreased body weight (BW), serum lipids, and glucagon (Elseweidy et al., 2018). In addition, n-hexane and petroleum ether from the seeds of N. sativa has shown inhibitory properties against protein tyrosine phosphatase 1B, with half maximal inhibitory concentration values of 33.15 and 18.50 μg/mL, respectively. This could be another mechanism of action since this enzyme is involved in the down regulation of insulin and leptin signaling and thus this plant could have antidiabetic potential (Xin et al., 2010).
Among the bioactive constituents of Nigella, the antidiabetic and antiobesity properties of thymoquinone have been also revealed. The administration (20 mg/kg/day) to diet-induced obesity mice was able to reduce decrease fasting blood glucose (FBG) and insulin levels, and enhanced glucose tolerance and insulin sensitivity. Moreover, this compound decreased the level of liver triglycerides and serum cholesterol, increased protein expression of phosphorylated Akt, decreased serum levels of inflammatory markers, and decreased NADH/NAD + ratio. It was related to the capacity of thymoquinone to increase insulin sensitivity in insulin-resistant HepG2 cells through a SIRT1-dependent mechanism (Karandrea et al., 2017). Glycemic parameters were momentously controlled by the administration of thymoquinone (8) to the diabetic rats (Usta and Dede, 2017). El-Aarag et al. (2017) also investigated the effects of this compound to boost antidiabetic properties of metformin in streptozotocin-induced diabetes in male rats. Their investigation unveiled that negative impacts of streptozotocin were corrected and normal biochemical functions were restored by combined treatment of thymoquinone (8) and metformin. This combination also up-regulated the expression level of glucose transporter-2 (Glut-2). The elevated MDA level was reduced in liver homogenates of the thymoquinone (8) treated rats (Zribi et al., 2014). Thymoquinone (8) in nano-formulation (20-80 mg/kg) showed significant decrease in blood glucose and HbA1c levels and showed a dose dependent antihyperglycemic effect. It can result in better antihyperglycemic effect in type-2 diabetic rats than the non-capsulated compound (Rani et al., 2018). In another research, intraperitoneal administration of thymoquinone (8) (50 mg/kg) resulted in amelioration of dyslipidemia, hypoinsulinemia, hyperglycemia, impaired antioxidant defense system and upregulation of the expression of PPAR-γ and GLUT4 genes in diabetic rats (Moneim et al., 2018).
Concerning thymol (13), intragastric administration (40 mg/kg/ day) for the subsequent 5 weeks momentously lowered kidney weight and blood and urinary markers of kidney injury by HFDinduced nephropathy in diabetic mice (Saravanan and Pari, 2016). It further reduced the HbA1c, leptin and adiponectin levels in the mice receiving thymol treatment. The plasma triglyceride, cholesterol, LDL and free fatty acids were reported to be significantly lowered and HDL was momentously elevated in thymol treated mice (Saravanan and Pari, 2016). It also represented beneficial effects on HFD-induced cognitive deficits via activating Nrf2/HO-1 signaling and improving hippocampal insulin resistance (Fangfang et al., 2017). The neuroprotective effect of carvacrol (12) has been also demonstrated on diabetesassociated cognitive deficit in a rat model of diabetes treated with this compound (25-100 mg/kg, 7 weeks). It prevented behavioral, biochemical, and molecular changes associated with diabetes in a dose-dependently way (Deng et al., 2013).

Antiobesity
Nigella sativa fruit and seed extracts and oil (33% thymoquinone) have shown antiobesity potential using in vitro and in vivo studies, and the mechanisms include the inhibition of pancreatic lipase and α-amylase (15-100%, depending on the extract and enzyme), proinflammatory cytokine production in pre-adipocytes (a model of low-grade inflammation in Simpson-Golabi-Behmel syndrome human) and weight loss, by positively affecting the uncoupling protein-1 (UCP-1), which the index protein of the brown adipose tissue used in the obesity studies (Buchholz and Melzig, 2016;Mahmoudi et al., 2018;Bordoni et al., 2019).
The latter in vivo study, which was performed in mice feed with HFD, suggested that the weight loss effect was less dependent on tymoquinone, while other phenolic compounds may affect (Mahmoudi et al., 2018). Alternatively, studies on female C57BL/ 6 mice, which were also subjected to HFD and supplemented with thymoquinone (10-20%), showed that this compound ameliorated obesity-induced metabolic dysfunction and impaired positive effects on ovarian and mammary gland metabolic functions (Harphoush et al., 2019). In another context, obesity markers were significantly down-regulated while momentous control over obesity-induced changes in biochemical profile of the body by thymol (13) treatment. It also modified fat and glucose metabolism in such a way to favor the control over obesity in mice partly via hypolipidemic, hypoglycemic, hypo-insulinemic, hypoleptinemic, and pancreatic lipase inhibition action (Haque and Ansari, 2018). This compound has also been endorsed to possess anti-obesity properties by controlling obesity markers in HFD induced obese male Wistar rats. They recorded significant reduction in body and liver weights, visceral fat pad weight, food intake, leptin and inhibition of pancreatic lipase (Haque et al., 2014). Among molecular mechanisms, Choi et al. (2017) assessed the role of thymol (13) in 3T3-L1 white adipocytes via promoting mitochondrial biogenesis, enhancing expression of a core set of brown fat-specific markers as well as enhancing the protein levels of PPARγ, PPARδ, pAMPK, pACC, HSL, PLIN, CPT1, ACO, PGC-1α, and UCP1. It also augmented lipolysis, thermogenesis and fat oxidation. In the case of carvacrol (12), it can act by reduction of autophagy (essential for adipocyte maturation) and on carbohydrate-responsive element-binding protein ChREBP activity, reducing adipogenic differentiation (Spalletta et al., 2018). The supplementation of this compound in the diet (0.1%) may prevent diet-induced obesity by modulating gene expressions involved and protein associated with the signaling cascades in adipogenesis, as well as inflammation (Cho et al., 2012). Overall, it seems that N. sativa has an interesting profile of phytochemicals, which can act in different ways and require attention.

Neuroprotection
The role of N. sativa and its components as promising neuropharmacological agents (Beheshti et al., 2016) in facitilating learning and memory have been recently reviewed (Sahak et al., 2016). In rats, 1 ml/kg of N. sativa oil for 14 days depleted reactive oxygen species (ROS)/NO levels, improved neurogenic proteins, acetylcholinesterase (AChE) activities and neuro-cognitive markers depletions in chlopyrifos exposure (Imam et al., 2018). Moreover, N. sativa oil improved neurocognicives indices such as Morris water maze. In other study performed in rats, the hydroalcoholic extracts of N. sativa seeds (200 and 400 mg/kg, 5 days) were used to investigate its effects on memory and brain tissues oxidative damage in penthylenetetrazole-induced repeated seizures (Vafaee et al., 2015). The results showed a beneficial effects of N. sativa on learning and memory impairments (Morris water maze) and improved antioxidant effects in the rat brain (lower MDA levels).

HEALTH-PROMOTING EFFECTS: CLINICAL TRIALS IN HUMANS
Clinical studies have mostly confirmed some effects of the seeds of N. sativa and their derivatives (seed extract and seed oil) obtained in the aforementioned in vitro and in vivo animal studies. Particularly, in the on line database about clinical trials "www.clinicaltrials.gov", there are 16 studies on N. sativa. Among them, the status of four studies on blood lipids, obesity and diabetes management has been completed, while other two were about palmer arsenical keratosis and asthma. The administration of N. sativa seeds (or seeds oil/extract) was alone or with other herbal substances (e.g., Curcuma longa extract), with different dosages and up to 60 days. Some of the results were positive, e.g. in total cholesterol and asthma, and subject of publications (Qidwai et al., 2009;Koshak et al., 2017). Furthermore, in the following section the results of other clinical studies regarding preventive and relieving effects of N. sativa on metabolic disorders and risk factors (diabetes, obesity and hypertension), as well as other effects are shown. In Table 4, we have summarized these effects. The effects of other Nigella plants are also commented.

Metabolic Syndrome
The possibility of application of N. sativa seed in patients with metabolic syndrome was firstly investigated by Najmi et al. (2008). They found that the administration with N. sativa seed oil (2.5 mL twice a day for 6 weeks) in patients with metabolic syndrome significantly decreased FBG and LDL and increased HDL levels. The same group of authors (Najmi et al., 2012) analyzed the effect of supplementation with powdered N. sativa seed (500 mg/day for two months) in patients with low glycemic control (glycate hemoglobin-HbA1C was lower than 7%). As a result, in the intervention group significant lowering of FBG, postprandial blood glucose (PPBG) and HbA1c was observed.
Conversely, a recent cross-over study (2 months treatment, 2 weeks washout period) evaluated the effects of N. sativa, as a form of traditional bread spiked with seeds (2 g), on FBG, BP and anthropometric indices (BW, waist circumference or WC, and BMI body mass index) in patients with metabolic syndrome. It found no significant effect between the mean of changes of parameters in the beginning and end of study (time effect), with the exception of diastolic BP (Mohtashami, 2019).

Diabetes
Oxidation and inflammation are important factors connected with occurrence of various chronic diseases, such as diabetes mellitus type 2 (Pouvreau et al., 2018). Due to the antioxidant, antiobesity and antidiabetic effects of N. sativa seeds and their constituents, this plant species can offer potential in prevention and treatment of type 2 diabetes. A recent review suggests that N. sativa can improve glycemic stages and lipid profile in diabetes . Hosseini et al. (2013) investigated anti-hyperglycemic effect of N. sativa seed oil in type II diabetic patients. As a result of supplementation with 5 mL of oil/day for three months, blood levels of fasting and 2 h PPBG, as well as HbA1c were significantly decreased compared to placebo group. This hypoglycemic effect can be result of an insulin senzitation and stimulation of pancreatic beta-cell function, resulting in intensified activity and consequent decrease in glucose level. Moreover, Kaatabi et al. (2015) reported pronounced antidiabetic activity after three-month application of N. sativa powdered seed (2 g/day) in combination with oral hypoglycemic agent in patients with type 2 diabetes. In this study, N. sativa received group showed significant reduction of FBG, HbA1C, and TBARS, and at the same time noticeable increase of the total antioxidant capacity, SOD, and glutathione levels were recorded in the intervention group.  investigated effect of N. sativa seed oil application (3 g/day) on glucose metabolism and lipid concentrations in patients with type 2 diabetes. According to this study, FBG, HbA1C, and levels of triglycerides (TG) and LDL significantly changed in the intervention group compared to the placebo one. On the other hand, insulin level and its resistance decreased and HDL increased in the intervention group, but after adjusting for confounder factors, these parameters were not significant. Overall, the potential antidiabetic mechanisms of N. sativa could be mediated through a change in the oxidative status (either via upregulation of endogenous antioxidants or reduction of oxidative species), reduction of inflammation, and improvement of lipid profiles (Yimer et al., 2019). Furthermore, a recent meta-analysis confirms that supplementation with N. sativa could be a suitable choice to manage the complications of type 2 diabetes, including FBS (−17.84 mg/dL), HbA1c (−0.71%), total cholesterol (−22.99 mg/dL), and LDL (−22.38 mg/dL) (Daryabeygi-Khotbehsara et al., 2017).

Hypercholesterolemia and Obesity
Nigella sativa powder supplementation (1 g/day) for sixty days caused momentous reductions in concentrations of LDL, TG levels and enhancement in HDL level in hypercholesterolemic patients (Tasawar et al., 2011). Moreover, a double-blind, randomized, placebo-controlled 4-weeks trial showed that the administration of N. sativa seeds (1g/day) can reduce total cholesterol and LDL. Thus, it can be interesting as lowering lipid agent in hyperlipidaemic subjects (Pelegrin et al., 2019). Nigella sativa oil (3 g/day for 8 weeks) was also tested in a lowcalorie diet on cardiometabolic risk factors in obese women.
Compared to the placebo group, in the N. sativa treated group, weight (−6.0%) and WC (−6.9%) decreased. It also favored a reduction in triglyceride and LDL levels (Mahdavi et al., 2015). Recently, a recent meta-analysis (literature till June 2017) has been performed on the effects of supplementation with N. sativa on some anthropometric indices in adult subjects. It indicated that N. sativa supplementation exerts a moderate effect on reduction in BW (−2.11 kg), BMI (−1.16 kg/m 2 ) and WC (−3.52 cm) (Namazi et al., 2018). Nonetheless, other metaanalysis performed till January 2018 suggested that N. sativa supplementation have only an effect on BW (−1.76 kg) and BMI (−0.85 kg/m 2 ) in adults compared to placebo (Mousavi et al., 2018).

Hypertension
There are several clinical studies reporting positive effect of N. sativa application on hypertension. Dehkordi and Kamkhah (2008) demonstrated that application of N. sativa seeds extract (100 and 200 mg for eight weeks) in patients with mild hypertension led to significant reduction of systolic and diastolic BP compared to placebo. At the same time, a significant decrease in total and LDL cholesterol was observed and no other complications caused by the treatment were found. Similar results were obtained for N. sativa seed oil. Fallah  exhibited that its application in a dose 2.5 mL two times per day for 8 weeks reduced systolic and dyastolic BP in healthy volunteers. Authors suggested that the exhibited effect could be attributed to the activity of thymoquinone (8), one of main constituents of the volatile oil from N. sativa seed, as commented before.
Some of the cardiovascular benefits evidenced in vitro and now in vivo through risk factors, such as blood lipids, and BP, could be related to the presence of omega-6 fats as linoleic acid, which is the major fatty acid of the seed oil. Nonetheless, the cardiovascular health benefits of linoleic acid are controversial (Hooper et al., 2018;Marklund et al., 2019), and the positive effects of minor phytochemicals remains unclear.
Other Effects of N. sativa

Effect of N. sativa Supplementation in Patients with Hashimoto's Thyroiditis (HT)
HT is one of the most common human autoimmune diseases influencing the thyroid glands and an organ-specific T-cell mediated disease (Chistiakov, 2005). The disease is ten times more frequent in women than in men and it affects 2% of general population. HT is associated with serious alterations in composition and the transport of lipoproteins. Several clinical studies have demonstrated the efficacy of N. sativa application in patients with HT. According to the study conducted by Farhangi et al. (2016), the application of powdered N. sativa seeds (2 g) for eight weeks significantly decreased BW and BMI compared to placebo. Also, a positive effect has been achieved on thyroid function. A decrease in the serum concentrations of thyroid stimulating hormone (TSH) and anti-thyroid peroxidase (anti-TPO) has been shown, while serum concentrations of T3 increased in N. sativa treated group. In the same study, significant reduction of vascular endothelial growth factor (VEGF) was noted in the intervention group. It has been observed that its concentration grows during pathological states characterized by increased TSH secretion .
These results were confirmed by Tajmiri et al. (2016) and Farhangi et al. (2018), who showed lower BMIs in the group of patients with HT after the treatment with N. sativa seeds. This positive effect was exhibited through hypolipidemic effect: lowering serum LDL cholesterol and TG concentrations, while raising the level of HDL cholesterol. Beside this effect, Tajmiri et al. (2016) showed that treatment with N. sativa seed powder for eight weeks reduced levels of serum TSH, anti-TPO and IL-23, while level of serum T3 was increased. Kolahdooz et al. (2014) studied effect of N. sativa seed oil on abnormal semen quality in infertile man. These authors showed that the daily application of 5 mL for two months significantly improved sperm count, morphology and motility and volume of semen, pH, and number of round cells. The exact mechanism of the exhibited effect is not determined, but it is likely related to strong antioxidant activity of the oil. Namely, it is well known that high level of oxidative stress contributes to decreased semen quality (Schulte et al., 2010).

Effect of N. sativa Supplementation on Patients with Allergic Rhinitis
The potential of application of N. sativa seeds and its oil in patients with allergic rhinitis has been also confirmed in clinical studies. Nikakhlagh et al. (2011) showed in prospective and double blind clinical trial that the oral administration of N. sativa seed oil (6 mg/kg daily) for 30 days in patients with allergic rhinitis significanly decreased the severity of respiratory symptoms, such as the presence of the nasal mucosal congestion, nasal itching, runny nose, sneezing attacks, turbinate hypertrophy, and mucosal pallor. Moreover, Rezaeian and Amoushahi Khouzani (2018) investigated the effect of N. sativa nasal spray in randomized clinical study. In this study patients in the intervention group received 2 puffs/day of N. sativa nasal spray (i.e. 1 g/day of N. sativa) and in the placebo group received 2 puffs/day of sodium chloride spray (0.65%). Lund-McKay, Lund Kennedy, and Sino-Nasal Outcome Test-22 scores were used and the outcomes were significantly lower in the intervention group compared to the placebo group. These findings are in line with previous reports about the antihistamine properties of N. sativa (Boskabady and Sheiravi, 2002;Kanter et al., 2006).

Effect of N. sativa Supplementation on Patients with Skin Diseases
Several clinical trials demonstrated positive effects of N. sativa application in resolving symptoms related to some skin diseases. Ghorbanibirgani et al. (2014) showed positive effects of 6 months topical application of N. sativa oil on vitiligo lesions compared to fish oil according to Vitiligo Area Scoring Index. In another study, Yousefi et al. (2013) investigated the therapeutic potential of 2% ointment topically applied (twice/day during 4 weeks) in patients with hand eczema compared to eucerin and betamethasone. According to scores obtained using Hand Eczema Severity index and Dermatology Life Quality Index, they showed that N. sativa ointment could have the same efficacy as betamethasone, while it is more efficient compared to eucerin. Jawad et al. (2014) investigated effect of various preparations of N. sativa in psoriasis treatment. A first group has been receiving Nigella ointment topically (10% w/w, twice daily), the second group took crude pulverized plant (capsules with 500 mg three times daily), while the third group received the combination of both treatments. The inspected reaction has been estimated following the Psoriasis Area and Severity Index score, while MDA serum level was used as indicator of the oxidative stress. After 12 weeks of treatment ointment reached total healing of psoriatic lesions, with good response in 65% of patients, and a relapse rate of 31% four weeks after cessation of treatment. Oral doses of N. sativa developed good response in 50% of patients, with a relapse rate of 50% observed four weeks after application. The combination of ointment and oral doses gained best results-total cure of lesions, and good responses in 85% of patients, with a relapse rate of 18%. All of these positive effects of N. sativa in dermatological diseases could be attributed to its antimicrobial, antioxidant, immunomodulatory, and anti-inflammatory effects.

Effect of Nigella sativa Supplementation in Patients with Rheumatic Artritis
The efficacy of black cumin oil in patients with rheumatoid arthritis (RA) was evaluated in female patients diagnosed with RA. Application of N. sativa oil in capsules (500 mg) twice daily exhibited improvement in disease activity score compared to placebo. Respectively, a pronounced improvement was demonstrated in amount of inflamed joints and occurrence of morning stiffness (Gheita and Kenawy, 2012). These results were confirmed by Hadi et al. (2016) with higher doses of N. sativa oil (1 g of oil/day, divided in two capsules). They showed that after 8 weeks of application in patients with RA noticeable decrease of serum MDA and NO was observed in the intervention group, while at the same time the serum level of anti-inflammatory cytokine IL-10 was increased (Hadi et al., 2016). Azizi et al. (2019) compared the effects of topical application of N. sativa oil and diclofenac gel in patients with osteoartritis, one of the most common diseases in aging population. In this study, pain score has been expressed as average number obtained using Knee injury and Osteoarthritis Outcome Score scale. It has been observed that first ten days there has been no significant differences in placebo and the intervention group. Still, after 21 days of application better pain relief effect was observed after treatment with N. sativa oil compared to diclofenac gel.

Effects on Memory, Attention and Cognition
In a randomized study with 20 healthy humans, the effects of 500 mg of N. sativa seeds (twice a day for 9 weeks) on memory, attention, and cognition were studied. The results showed an improvement in the score of the tests studied, without an Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 625386 alteration of the biochemical markers of cardiac, liver and kidney functions (Bin Sayeed et al., 2013).

Effects of Other Nigella Species
Among other herbs, there are several studies on the clinical use of N. ciliaris during childbirth and postpartum (Ali-Shtayeh et al., 2015), against diabetes (Ali-Shtayeh et al., 2012) and hypertension (Ali-Shtayeh et al., 2013), as well as N. arvensis against psoriasis (Jaradat et al., 2016b) and cancer (Jaradat et al., 2016a), which were performed in Palestine using questionnaires. Some patients seem to be satisfied with the outcomes of herbal therapy (Ali-Shtayeh et al., 2012;Ali-Shtayeh et al., 2013), but the authors did not reveal the herb treatment that was successful to manage the latter diseases/conditions. In any case, clinical evidences are still lacking and no studies have been found in the abovementioned database https://clinicaltrials.gov/.

CONCLUSION
Nigella seeds and extracts have a wide range of bioactivities, which have been demonstrated in vitro and in vivo. Nonetheless, N. sativa is the most studied species probably due to their popularity in folklore medicine. In all cases, future perspectives should be oriented to perform a better characterization of the constituents of the extracts since most studies were only focused on determining the concentration of thymoquinone, but other phytochemicals could be present as in this review more than 80 compounds have been reported. This is also important to remark that bioavailability could affect the way that Nigella components act in vivo, e.g., some studies administered Nigella products orally, while other intravenously. When referring to clinical trials, it seems that the supplementation of N. sativa can exert some effects on diabetes, obesity, and hypertension, among others. Nonetheless, the administration way (seeds, powder, oil, etc.), the dose and the treatment period are highly variable and should be further established from a therapeutic point of view, as well the chemical and phytochemical composition. This should be further study in order to formulate functional ingredients/nutraceuticals to promote the health benefit and how should be added into food. Moreover, besides the pleiotropic health applications of Nigella, it can be useful in nanotechnology applications as well as to modulate the activity of other therapeutic agents.
It is important to highlight that the culinary and medicinal use of some Nigella seeds as spice, condiment or infusion (e.g., N. sativa, N. damascena, and N. arvensis) suggests that their consumption is safe. The seeds are characterized by a low toxicity degree (Ali and Blunden, 2003) and no serious side effects in clinical trials (Namazi et al., 2018), but more studies were focused on N. sativa. In fact, toxicity studies performed in animal models also suggest a wide of margin of safety for using N. sativa fixed oil, with high LD 50 value (28.8 ml of oil/kg BW administered orally to mice) (Zaoui et al., 2002), in powder form (Dollah et al., 2013) and as extracts (Vahdati-Mashhadian et al., 2005).