Immunomodulatory and therapeutic properties of the Nigella sativa L. seed

International Immunopharmacology 5 (2005) 1749 – 1770 www.elsevier.com/locate/intimp

Review

Immunomodulatory and therapeutic properties of the Nigella sativa L. seed Mohamed Labib Salem * Department of Surgery, Section of Surgical Oncology, Medical University of South Carolina, Charleston, SC 29425, United States Received 26 April 2005; received in revised form 13 June 2005; accepted 14 June 2005

Abstract A larger number of medicinal plants and their purified constituents have been shown beneficial therapeutic potentials. Seeds of Nigella sativa, a dicotyledon of the Ranunculaceae family, have been employed for thousands of years as a spice and food preservative. The oil and seed constituents, in particular thymoquinine (TQ), have shown potential medicinal properties in traditional medicine. In view of the recent literature, this article lists and discusses different immunomodulatory and immunotherapeutic potentials for the crude oil of N. sativa seeds and its active ingredients. The published findings provide clear evidence that both the oil and its active ingredients, in particular TQ, possess reproducible anti-oxidant effects through enhancing the oxidant scavenger system, which as a consequence lead to antitoxic effects induced by several insults. The oil and TQ have shown also potent anti-inflammatory effects on several inflammation-based models including experimental encephalomyelitis, colitis, peritonitis, oedama, and arthritis through suppression of the inflammatory mediators prostaglandins and leukotriens. The oil and certain active ingredients showed beneficial immunomodulatory properties, augmenting the T celland natural killer cell-mediated immune responses. Most importantly, both the oil and its active ingredients expressed antimicrobial and anti-tumor properties toward different microbes and cancers. Coupling these beneficial effects with its use in folk medicine, N. sativa seed is a promising source for active ingredients that would be with potential therapeutic modalities in different clinical settings. The efficacy of the active ingredients, however, should be measured by the nature of the disease. Given their potent immunomodulatory effects, further studies are urgently required to explore bystander effects of TQ on the professional antigen presenting cells, including macrophages and dendritic cells, as well as its modulatory effects upon Th1and Th2-mediated inflammatory immune diseases. Ultimately, results emerging from such studies will substantially improve the immunotherapeutic application of TQ in clinical settings. D 2005 Published by Elsevier B.V. Keywords: Nigella sativa; Thymoquinone; Colitis; Encephamolyelitis; Arthritis; Anti-diabetic; Anti-oxidant; Toxicity; Anti-histaminic; Antiinflammatory; Anti-tumor; Anti-microbial; Bacteria; Virus; Fungus; Schisosoma; Immunity

Abbreviations: Abs, antibodies; CCL4, carbon tetrachloride; Con A, concanavalin-A; DCs, dendritic cells; DOX, doxorubcin; EAE, experimental allergic encephalomyelitis; FS, Fanconi syndrome; HHcy, hyperhomocysteinemia; i.p., intraperitoneal; i.v., intravenous; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinin; LPS, lipopolysaccharide; ROS, reactive oxygen species; STZ, streptozotocin. * Tel.: +1 843 792 7576; fax: +1 843 792 3200. E-mail addresses: [email protected], [email protected]. 1567-5769/$ - see front matter D 2005 Published by Elsevier B.V. doi:10.1016/j.intimp.2005.06.008

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Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. sativa: botanical and historical background, and folk medicine . . . . . . . Ingredients of N. sativa seeds . . . . . . . . . . . . . . . . . . . . . . . . . . Immunopharmacological properties of N. sativa seeds . . . . . . . . . . . . . 4.1. Anti-oxidant properties . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. Oxidant stress system and toxicity . . . . . . . . . . . . . . . 4.1.2. In vitro anti-oxidant activities . . . . . . . . . . . . . . . . . . 4.1.3. In vivo anti-oxidant activities . . . . . . . . . . . . . . . . . . 4.2. Anti-histaminic properties . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Anti-inflammatory properties . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Inflammatory mediators . . . . . . . . . . . . . . . . . . . . . 4.3.2. In vitro anti-inflammatory effects of N. sativa seed components 4.3.3. In vivo anti-inflammatory effects of N. sativa seed components 4.4. Immunomodulatory properties . . . . . . . . . . . . . . . . . . . . . . 4.5. Anti-microbial properties . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.1. Anti-viral effect . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.2. Anti-helminthic effects . . . . . . . . . . . . . . . . . . . . . 4.5.3. Anti-bacterial effects. . . . . . . . . . . . . . . . . . . . . . . 4.6. Anti-tumor properties. . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6.1. In vitro anti-tumor effects . . . . . . . . . . . . . . . . . . . . 4.6.2. In vivo anti-tumor effects . . . . . . . . . . . . . . . . . . . . 5. Potential toxicity of N. sativa seeds . . . . . . . . . . . . . . . . . . . . . . . 6. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Interest in medicinal plants has burgeoned due to increased efficiency of new plant-derived drugs and the growing interest in natural products. Because of the concerns about the side effects of conventional medicine, the use of natural products as an alternative to conventional treatment in healing and treatment of various diseases has been on the rise in the last few decades. The use of plants as medicines dates from the earliest years of man’s evolution [1,2]. Medicinal plants serve as therapeutic alternatives, safer choices, or in some cases, as the only effective treatment. People in separate cultures and places are known to have used the same plants for similar medical problems. A larger number of these plants and their isolated constituents have shown beneficial therapeutic effects, including anti-oxidant, anti-inflammatory, anti-cancer, anti-microbial, and immunomodulatory effects [1,3–9].

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2. N. sativa: botanical and historical background, and folk medicine Among the promising medicinal plants, N. sativa, a dicotyledon of the Ranunculaceae family, is an amazing herb with a rich historical and religious background [10]. N. sativa is found wild in southern Europe, northern Africa, and Asia Minor. It is a bushy, self-branching plant with white or pale to dark blue flowers. N. sativa reproduces with itself and forms a fruit capsule which consists of many white trigonal seeds. Once the fruit capsule has matured, it opens up and the seeds contained within are exposed to the air, becoming black in color [11]. The seeds of N. sativa are the source of the active ingredients of this plant. It is the black seed referred to by the prophet Mohammed as having healing powers [10]. Black seed is also identified as the curative black cumin in the Holy Bible and is described as the Melanthion of Hippocrates and Discroides and as the Gith of Pliny [12].

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Historically, it has been recorded that N. sativa seeds were prescribed by ancient Egyptian and Greek physician to treat headache, nasal congestion, toothache, and intestinal worms, as well as a diuretic to promote menstruation and increase milk production [10,13]. The seeds of N. sativa, known as black seed, black cumin or bHabatul-Barakah,Q have long been used in folk medicine in the Middle and Far East as a traditional medicine for a wide range of illnesses, including bronchial asthma, headache, dysentery, infections, obesity, back pain, hypertension and gastrointestinal problems [11,14]. Its use in skin condition as eczema has also been recognized worldwide [10]. Externally, the seeds can be ground to a powder, mixed with a little flour as a binder, and applied directly to abscesses, nasal ulcers, orchitis, and rheumatism.

eight of the nine essential amino acids [17–21]. Fractionation of whole N. sativa seeds using SDS–PAGE shows a number of protein bands ranging from 94 to 10 kDa molecular mass [22]. Monosaccharides in the form of glucose, rhamnose, xylose, and arabinose, are also found. N. sativa seeds are rich in the unsaturated and essential fatty acids. Chemical characteristics, as well as fatty acid profile of the total lipids, revealed that the major unsaturated fatty acid is linoleic acid, followed by oleic acid [17,18,23–25]. The major separate individual phospholipid classes is phosphatidylcholine, followed by phosphatidylethanolamine, phosphatidylserine, and phosphatitdylinisitol, respectively [17, 18,26]. The seeds contain carotene which is converted by the liver to vitamin A [18]. The N. sativa seeds are also a source of calcium, iron, and potassium [27].

3. Ingredients of N. sativa seeds

4. Immunopharmacological properties of N. sativa seeds

Four dolabellane-type diterpene alkaloids, nigellamines A (1) (1), A (2) (2), B (1) (3), and B (2) (4), have been isolated from the seeds of N. sativa [15,16]. By HPLC analysis of N. sativa oil, thymoquinone (TQ), dithymquinone (DTQ), which is believed to be nigellone, thymohydroquinone (THQ), and thymol (THY), are considered the main active ingredients [17] (Fig. 1). N. sativa seeds contain other ingredients, including nutritional components such as carbohydrates, fats, vitamins, mineral elements, and proteins, including

Several pharmacological properties of N. sativa, including hypotensive, anti-nociceptive, uricosuric, choleretic, anti-fertility, anti-diabetic, and anti-histaminic have been reported [28], however, it is not the focus of this article. This article will focus on the anti-oxidant, anti-inflammatory, anti-microbial, antitumor, and immunomodulatory properties of N. sativa and its ingredients in the context of their therapeutic potential. 4.1. Anti-oxidant properties

O

O Thymoquinone (TQ)

O

O

O O Dithymoquinone (DTQ) OH

OH Thymol (THY)

OH Thymohydroquinone (THQ)

Fig. 1. Chemical structure of the active ingredients: TQ, DTQ, THY, and THQ, in the oil of N. sativa L seed (quoted from Ref. [17]).

4.1.1. Oxidant stress system and toxicity Oxidative damage to biological structures has been implicated in the toxicity-induced pathophysiology of several diseases, in particular cardiovascular disease and cancer [29]. The cause of this oxidative damage has been reported to be due to the shift in the balance of the pro-oxidant (free radicals) and the anti-oxidant (scavenging) mediators, where pro-oxidant conditions dominate either due to the increased generation of the free radicals caused by excessive oxidative stress, or due to the poor scavenging capability in the body [30]. Free oxygen radicals, including O2, OH, and NO (collectively known as oxidative stress), are electrically charged molecules that attack cells, tearing through cellular membranes to react and create

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havoc with the nucleic acids, proteins, and enzymes present in the body [31]. The attacks by ROS cause damage to cell structure and function and can eventually destroy them. ROS are produced mainly by certain cells of immune system including macrophages (Mf) and neutrophils [32]. It has recently reported that suppression of immune cell function associated with chemotherapy [33], radiotherapy [34], infection [35,36] and in tumor-bearing hosts [37] is mediated by production of NO produced by immature myeloid cells that are massively generated under these conditions [38–40]. The central role of ROS in mediating the pathology in several diseases has stimulated interest in the possible role of natural anti-oxidant agents in preventing the development of these diseases. It has been reported that the health promotive, disease preventive and rejuvenation approach based on using medicinal plants in dAyurveda,T T an ancient Indian systems, is due to the anti-oxidant effects of these plants [41]. One of the potential properties of N. sativa seeds is the ability of one or more of its constituents to reduce toxicity due to its anti-oxidant activities. Of the studies that have been performed to evaluate the different effects of N. sativa, majority (more than 35) of the studies have confined to address its antitoxic properties both in vitro and in vivo. 4.1.2. In vitro anti-oxidant activities In vitro studies show that N. sativa seed extract induces inhibition of the hemolytic activities of snake and scorpion venoms [42], protects erythrocytes against lipid peroxidation, protein degradation, loss of deformability, and increased osmotic fragility caused by H2O2 [43]; and protects laryngeal carcinoma cells, from programmed cell death (apoptosis) induced by lipopolysaccharide (LPS) or cortisol [44]. These results indicate to the antitoxic effects of N. sativa seed components that could be attributed to its anti-oxidant properties. Several in vitro studies confirm this hypothesis. For instance, essential oil obtained from six different extracts of N. sativa seeds and from a commercial fixed oil showed antioxidant effects with almost identical qualitative effects. Differences, however, were mainly restricted to the quantitative composition [45]. The crude N. sativa oil and its fractions (neutral lipids, glycolipids, and phospholipids) showed potent in vitro radical

scavenging activity that is correlated well with their total content of polyunsaturated fatty acids, unsaponifiables, and phospholipids, as well as the initial peroxide values of crude oils [46]. Moreover, preincubation of peritoneal Mf with aqueous extract or the boiled fraction of the extract of N. sativa seeds caused a dose-dependent decrease in NO production when activated with LPS of E. coli [47]. Interestingly, TQ and a synthetic structurally-related tert-butylhydroquinone, also efficiently inhibited iron-dependent microsomal lipid peroxidation in vitro in a concentration-dependent manner [48]. TQ also induced significant protection of isolated hepatocytes against tertbutyl hydroperoxide induced toxicity evidenced by decreased leakage of ALT and AP [49]. In addition, TQ in a dose- and time-dependently manner, reduced nitrite production, a parameter for NO synthesis, and decreased both gene expression and protein synthesis levels of iNOS in supernatants of LPS-stimulated Mf without affecting the cell viability [26]. Stimulation of polymorphonuclear leukocytes with TQ showed protective action against superoxide anion radical either generated photochemically, biochemically, or derived from calcium ionophore, indicating to its potent superoxide radical scavenger [50]. 4.1.3. In vivo anti-oxidant activities Both hepatoxicity and nephrotoxicity are associated with alteration in the levels and activities of certain mediators such as l-alanine aminotransferase (ALT), alkaline phosphatase (AP), lipid peroxide (LPD), and the oxidant scavenger enzyme system including, glutathione (GSH) and superoxide dismutase (SOD). The anti-oxidant effects of N. sativa have been examined using different hepatic and kidney toxicity in vivo murine models induced by tert-butyl hydroperoxide, carbon tetrachloride (CCl4), doxorubcin (DOX), gentamicin, methionine, potassium bromate (KBrO3), cisplatin, or Schistosoma manson infection. 4.1.3.1. In vivo anti-oxidant activities of N. sativa seed oil. In CCl4-induced toxicity, N. sativa oil protected against hepatotoxicity coinciding with improvement in serum lipid profile [51,52], decreasing the elevated serum K and Ca levels, ameliorating the reduced RBC, WBC, PCV, and Hb levels [53,54], decreasing the elevated LPD and liver enzyme levels, and increasing the reduced anti-oxidant enzyme levels

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[55]. Moreover, treatment with N. sativa oil prevented CCl4-induced liver fibrosis in rabbits with improvement of the anti-oxidant status [56]. In gentamicineinduced toxicity, treatment with N. sativa oil produced a dose-dependent amelioration of the biochemical and histological indices of nephrotoxicity, coincided with the increase in the scavenger defense system, including GSH concentration and the total anti-oxidant status in renal cortex [57]. In KBrO3-mediated renal oxidative stress prophylaxis of rats orally with N. sativa extract resulted in a significant decrease in renal LPD and oxidative stress that coincided with marked recovery of renal glutathione content and antioxidant enzymes [58]. Using gastric ulcer model induced in rats by oral administration of ethanol that causes a significant reduction in free acidity and glutathione level, pretreatment of rats with N. sativa before induction of ulcer induced a significant increase in glutathione level, mucin content, and free acidity with a protection ratio of 53.56% as compared to the ethanol group [59]. Taken together, these findings show the potential antitoxic effect of N. sativa seeds in form of crude extracts or oil mediated by their anti-oxidant properties. 4.1.3.2. In vivo anti-oxidant activities of TQ. Prophylactic treatment of mice with TQ 1 h before CCl4 injection ameliorated hepatotoxicity of CCl4 as evidenced by the significant reduction of the elevated levels of serum enzymes, and significant increase of the hepatic GSH content [45,60]. Treatment of mice with the other volatile oil constituents, p-cymene or alpha-pinene, however, did not induce any changes. The effect of TQ on the nephrotoxicity, cardiotoxicity, and oxidative stress induced by DOX in rats shows that its administration counteracted the development of nephrotic hyperlipidemia, and hyperproteinuria; and restored the biomarker’s values of oxidative stress towards normal [61]. The pathogenesis in hyperhomocysteinemia (HHcy), including gastric lesion, liver fibrosis, and cardiotoxicity, is known to be linked with free radical formation associated with higher risks of coronary, cerebral and peripheral vascular disease. Interestingly, oral pretreatment of rats with either crude N. sativa oil or TQ protected against methionine-induced HHcy through amelioration of the plasma levels of triglycerides, lipid peroxidation, cholesterol, and in the acti-

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vities of the anti-oxidant status [62]. Also, when rats were subjected to ischaemia/reperfusion, injection of N. sativa oil or TQ tended to normalize the level of LDH, GSH, and SOD; TQ showed higher effect than that induced by the oil [63]. Fanconi syndrome (FS), induced by ifosfamide, is characterized by wasting off glucose, electrolytes and organic acids, along with elevated serum creatinine and urea, as well as decreased creatinine clearance rate. Administration of TQ with the drinking water to rats before and during ifosfamide treatment ameliorated the severity of ifosfamide-induced renal damage and improved most of the alterations of biochemical parameters [64], including renal GSH depletion and LPD accumulation. Schistosoma mansoni infection induces marked alteration in the liver function due to the heavy worm and egg burden deposited in the liver. Administration of N. sativa oil markedly reduced the worm and egg burden, coincided with partial amelioration of the schistosoma-induced liver fibrosis and changes in ALT, GSH, AP activities in serum [23], suggesting that the anti-schistosomal effect of N. sativa oil might be induced partly by its anti-oxidant effect. Similarly, treatment with N. sativa oil decreased the hepatocellular necrosis, degeneration and advanced fibrosis in CCl4-induced liver fibrosis in rabbits [56]. S. mansoni infection also induces a genotoxic effect, causing a significant increase in the incidence of chromosomal aberrations [65]. Interestingly, treatment of S. mansoni-infected mice with N. sativa oil or purified TQ induced a protective effect on the infection-induced genotoxicity evidenced by reduction in the percentage of chromosomal aberrations and the incidence of chromosome deletions and tetraploidy [66]. Coupling the fact that N. sativa seeds have been used in folk medicine with its antitoxic findings discussed above, it is apparent that the crude oil of N. sativa oil and its active constituents can lower oxidative stress-mediated toxicity induced accidentally by environmental or infectious factors, or by anti-cancer drugs. For instance, chemotherapy, cyclophosphamide and other anti-cancer drugs, is currently used in preclinical and clinical studies either as anti-cancer therapy or in combination with cancer immunotherapy [67]. Since chemotherapy induces massive expansion of the immature granulocytes, which produce large amount of NO, it might be feasible to follow chemotherapy with TQ treatment that might alleviate the

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suppressive effects on the immune responses by chemotherapy-induced NO. 4.2. Anti-histaminic properties Histamine is released by body tissues, creating allergic reactions associated with conditions such as bronchial asthma. There is an indication from the traditional use of N. sativa seeds that its active ingredients have a substantial impact on the inflammatory diseases mediated by histamine. It was found from four decades that DTQ dimer isolated from N. sativa seed’s volatile oil, under the name of dNigellone,T when given by mouth to some patients suffering from bronchial asthma, it suppressed symptoms in the majority of patients [13]. Following this study, nigellone was administered to children and adults in the treatment of bronchial asthma with effective results and with no sign of toxicity. In a clinical study, treatment of patients with allergic diseases, including allergic rhinitis, bronchial asthma, atopic eczema, with N. sativa oil decreased the IgE, and eosinophil count, endogenous cortisol in plasma and urine [68], indicating to effectiveness of N. sativa oil as adjuvant for the treatment of allergic diseases. Indeed, the anti-allergic effect of N. sativa seed components could be attributed to its anti-histaminic effects. In vitro studies support this notion. Aqueous extract of N. sativa has shown relaxant and antihistaminic effects on precontracted guinea pig tracheal chains. This effect was observed in the presence of both ordinary and calcium free Krebs solution, but with no effect in the absence of KCl induced contraction, suggesting that the calcium channel blocking effect of this plant does not contribute to its relaxant effect [69]. In addition, the potent inhibitory effect of nigellone on histamine release from rat peritoneal mast cells, stimulated by different secretagogues; antigen sensitized cells, compound 48/80 and the Caionophore A23187, was found to be mediated by decreasing intracellular calcium by inhibition of protein kinase C, a substance known to trigger the release of histamine [70,71]. Moreover, by investigating its effect on the guinea pig isolated tracheal zig-zag preparation, TQ caused a concentration-dependent decrease in the tension of the tracheal smooth muscle precontracted by carbachol [72]. Moreover, TQ totally abolished the pressor effects of histamine and seroto-

nin on the guinea pig isolated tracheal and ileum smooth muscles. These effects of TQ were suggested to be mediated, at least in part, by inhibition of lipoxygenase products of arachidonic acid metabolism and possibly by non-selective blocking of the histamine and serotonin receptors [72]. Preclinical and clinical studies have also shown anti-histaminic effects for N. sativa seeds. Using gastric ulcer model induced by oral administration of ethanol, which caused a significant increase in mucosal histamine content, rat pretreated with N. sativa oil before induction of ulcer induced a significant decrease in gastric mucosal histamine content with a protection ratio of 53.56% as compared to the ethanol group [59]. In contrast to the relaxant effect observed above for TQ, another study showed a stimulant effect. In this study, the effect of the volatile oil of N. sativa on the respiratory system of the urethaneanaesthetized guinea pig was compared to those of TQ [73]. Both the respiratory rate and the intratracheal pressure were increased, in a dose-dependent manner, by the i.v. administration of the oil mediated via release of histamine with direct involvement of histaminergic mechanisms and indirect activation of muscarinic cholinergic mechanisms [73]. On the other hand, i.v. administration of TQ induced significant increases in the intratracheal pressure without any effect in the respiratory rate. Taken together, it seems that different active ingredients of N. sativa oil possess different impacts on the histamine release. The active ingredient nigellone of the crude extract of N. sativa seeds acts as calcium channel blocker(s), which might explain the beneficial traditional therapeutic uses of N. sativa toward diarrhea, asthma and hypertension. 4.3. Anti-inflammatory properties 4.3.1. Inflammatory mediators Progression and persistence of acute or chronic state of inflammation are mediated by a number of mediators, including eicosinoids, oxidants, cytokine, and lytic enzymes secreted by the inflammatory cells macrophages and neutrophils [74]. As discussed in Section 4.1, ROS, in particular NO, initiates a wide range of toxic oxidative reactions causing tissue injury. In addition to the ROS-induced inflammation, inflammation is also mediated by two main

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enzymes: cyclooxygenase (COX) and lipoxygenase (LO) [75]. COX yields from arachidonic acid prostaglandines (PGE) and thrompoxane [76], while LO catalysis the formation of leukotriens (LT). Both PGE and LT function as the main mediators of allergies and inflammation. 4.3.2. In vitro anti-inflammatory effects of N. sativa seed components Several in vitro studies reproducibly reported the inhibitory effects of N. sativa oil and its active ingredients on the production of these mediators. For instance, TQ and the crude fixed oil of N. sativa inhibited both COX and 5-LO pathways of arachidonate metabolism in rat peritoneal leukocytes stimulated with calcium ionophore A23187, as shown by dose-dependent inhibition of thromboxane B2, LTC4 and LTB4, respectively; TQ showed higher effects [77,78]. Both substances also inhibited non-enzymatic peroxidation in brain phospholipid liposomes; again TQ was about ten times more potent. Interestingly, however, the inhibitory effect of the fixed oil of N. sativa on eicosanoid generation and lipid peroxidation was greater than that of TQ, suggesting that other components, such as unsaturated fatty acids, may contribute also to the anti-eicosanoid and anti-oxidant activities of N. sativa oil. Furthermore, in vitro treatment of calcium- or ionophore-stimulated polymorphonuclear leukocytes (neutrophils) with either crude extract of N. sativa, nigellone, or TQ produced a concentration dependent inhibition of 5-LO products and 5-hydroxy-eicosa-tetra-enoic acid production [79]. Thus, inhibition of both COX and 5-LO pathways is key factors mediating the anti-inflammatory effects of the crude oil of N. sativa and its active ingredients. 4.3.3. In vivo anti-inflammatory effects of N. sativa seed components Components of N. sativa have also been shown appreciated anti-inflammatory effects in several inflammatory diseases, including experimental allergic encephalomyelitis (EAE), colitis, and arthritis. EAE is an autoimmune demyelinating disease of the central nervous system that is widely accepted as an animal model for the human multiple sclerosis that is mediated by T cells, while oxidative stress also plays a central role in the onset and progression of this disease

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[80]. When EAE animal received TQ, they showed higher glutathione level, no perivascular inflammation with no disease symptoms, compared with EAE untreated animals. These data reveal the therapeutic potential of TQ in EAE model [81] and indicate to its possible efficacy in treatment of multiple sclerosis in humans. Ulcerative colitis is another inflammatory disease that is characterized by cycles of acute inflammation, ulceration and bleeding of the colonic mucosa. Although the pathogenesis of colitis remains poorly understood, various mediators, such as eicosanoids, leukotrienes, platelet activating factor and oxygenderived free radicals have been implicated in the pathogenesis of this disease [82]. Treatment with anti-inflammatory [83,84] or anti-oxidant agents has been shown to ameliorate the disease symptoms [85,86]. In a recent study, the effects of TQ on the acetic acid-induced colitis in rats by intracolonic injection of 3% acetic acid showed that pretreatment of animals for 3 days with TQ led to complete protection against acetic acid-induced colitis with a comparable or even higher effects than sulfasalazine, an anti-colitis drug [87]. The anti-colitis effects of TQ were associated with reversed biochemical and histopathological changes towards the normal. This study suggested that the anti-colitis effect of TQ is due to its anti-oxidant and anti-histaminic activities. Further studies are required to define the therapeutic impact of TQ on another form of colitis, as well as to explore the underlying mechanisms. It has been observed for a long time that the N. sativa oil has an anti-inflammatory effect relieving the effects of arthritis [28]. Consistent with these observations, recent studies have reported also that externally in an ointment form, the anti-inflammatory activity of the black seed was found to be the same range as that of other similar commercial products without induction of skin allergy [88]. Injection of emuslion of N. sativa oil induced significant reduction in endotoxin shock in response to LPS [89] and did markedly inhibit oedema induced by carrageenan or croton oil [90]. Similar to the anti-inflammatory effects of N. sativa seed extracts, the black currant seed oil also inhibited subcutaneous air pouch formed in Sprague– Dawley rats induced by monosodium urate crystals [91]. The black currant seed oil enriched diet suppressed significantly both the cellular and fluid phases

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of inflammation (polymorphonuclear leukocyte and exudates accumulation). In contrast, administration of normal chow or of a diet enriched in safflower oil containing the normal ratio of polyunsaturated fatty acid (PUFA) did not influence monosodium urate crystal-induced inflammation in this model [91]. The findings indicate that a diet, which provides both n-6 (gammalinolenic acid) and n-3 (alpha-linolenic acid) fatty acids as substrates alternative to arachidonic acid for oxidative metabolism, can modify monosodium urate crystal-induced acute inflammation. In this regard, we have found recently that injection of both n-3 and n-6 polyunsaturated fatty acids induces higher anti-inflammatory responses than the effect obtained after treatment with either of them alone [92]. Similar to the black currant seed oil, N. sativa seeds contain both n-6 and n-3 fatty acids, it thus might also induce similar anti-inflammatory effects on monosodium urate crystal-induced acute inflammation. However, this hypothesis needs to be tested. Intensity of inflammatory immune responses is controlled by recruitment of inflammatory cells into inflammatory lesions. This process is tightly governed by expression of certain inflammatory chemokines, such as MCP-1 (CCL2), MIP-1a (CCL3), MIP-1h (CCL4), and RANTES (CCL5) [93,94]; and adhesion molecules, such as LFA-1, CD62L and CD44, by the inflammatory cells, and ICAM-1 and VCAM-1 by the endothelial cells [95]. Given the central role of chemokines and adhesion molecules in orchestrating the immune response, interference with the expression of these mediators substantially alter the quality of the immune response, leading to either enhancement or inhibition of the ongoing immune response. Thus, one potential mechanism that might mediate the inhibitory effect of N. sativa on inflammatory immune responses is an alteration of trafficking of the inflammatory cells via modulating expression of chemokines and/or adhesion molecules. Even though there is no reported study that addressed the effect of N. sativa on the chemokines or adhesion molecules, the inhibition of the inflammatory cytokines IL-1, TNF-a and enhancement of the chemokine IL-8 by N. sativa might give an indication of this effect. Given the potent and reproducible anti-inflammatory effects of N. sativa seeds on different inflammatory disease models, future studies are required to explore the

effects of single and combined active ingredients of N. sativa on the expression of chemokines and adhesion molecules by immune cells. This will enhance our knowledge on the therapeutic potentials of this plant. Taken together, these findings suggest a potential therapeutic effect of N. sativa and its active ingredients, in particular TQ, against murine colitis, EAE and arthritis inflammatory diseases, that would be translated to the clinical settings of these diseases in humans. However, it still remains unknown if the anti-inflammatory effects discussed above are merely attributed to non-specific inhibitory effects on Mf and neutrophils, or also involve inhibitory effects on T cell populations. Further studies, therefore, should be precisely designed to dissect the impact of TQ on the cytokines that drive Th1- and Th2-mediated inflammatory immune diseases, since these cytokines have reciprocal inhibitory effects. In addition, more attention is needed to test if TQ can modulate dendritic cells (DCs). Indeed, we are currently testing if TQ can bias the post-vaccination T cell responses toward Th1 or Th2 cytokines, as well as its impact on DC maturation. 4.4. Immunomodulatory properties Generation of effective immunity requires both innate immunity that recognizes pathogen associated molecular patterns and adaptive immunity that recognizes specific antigens [96]. Innate immunity consists of non-specific cells, including Mf, granulocytes, NK cells, and DCs. Adaptive immunity is comprised of a humoral arm mediated by B cells that secrete antigenspecific antibodies, and cellular arm mediated by CD4+ (helper) and CD8+ (cytolytic) T cells [97]. CD4+ T helper cells are responsible for orchestrating an immune response, whereas cytolytic CD8+ T cells are the killer cells that traffic to sites of infection or cancer and lyse infected or tumor cells. Together, these two types of effector T lymphocytes play critical roles in eliminating infections and controlling cancer. One of the precious properties of N. sativa is the immunomodulatory effects of its constituents. Studies begun just over a decade ago suggest that if it is used on an ongoing basis, N. sativa can enhance immune responses in human. The majority of subjects who treated with N. sativa oil for 4 weeks showed a 55%

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increase in CD4 to CD8 T cells ratio, and a 30% increase in natural killer (NK) cell function. The results have been presented by A. E1-Kadi and O. Kandil to the 1st International Conference on Scientific Miracles of Quran and Sunnah, held in Islamabad, Pakistan [22]. Recently, a well-designed study analyzed the immunomodulatory effects of the whole extract of N. sativa seeds and their protein components in vitro [22,98]. By investigating the in vitro effects of the whole and soluble fractions of N. sativa seeds on human peripheral blood mononuclear cells (PBMC) response to different mitogens, the components did not show any significant stimulatory effect on the PBMC responses to the T cell mitogens phytohemagglutinin (PHA), or concanavalin-A (Con A). By contrast, the components expressed stimulatory effect on the PBMC response to pooled allogeneic cells [98]. Furthermore, in mixed lymphocyte cultures, four different purified proteins of N. sativa showed stimulatory effects. By contrast, a uniformly suppressive effect of the four fractions was noticed when lymphocytes were activated with the B cell mitogen PWM [22]. Consistent with the stimulatory effects of N. sativa oil on proliferation of T cells, its ethyl-acetate column chromatographic fraction and water fraction enhanced the proliferative response to ConA, but again not to the B cell mitogen LPS [99]. These findings indicate that certain constitutions of N. sativa oil possess potent potentiating effects on the cellular (T cell-mediated) immunity, while other constituents possess suppressor effects on B cell-mediated (humoral) immunity. These findings suggest also that the stimulatory effects of N. sativa on the cellular immunity are dependent on the nature of the immune (e.g. ConA versus allogenic) response. In line with the in vitro enhancing effects of N. sativa on the T cell immunity, in vivo studies confirm these effects. For instance, 1 week oral administration of aqueous extracts of N. sativum seeds increased (about 2-fold) the number of splenic NK cells, and their cytotoxicity against YAC-1 tumor targets when compared with control NK cells [100]. In addition, oral administration of N. sativa oil commenced 6 weeks after induction of streptozotocin (STZ)-induced diabetes significantly induced beneficial effect, coincided with elevation in the phagocytic activity of peritoneal Mf, and lymphocyte count in peripheral blood compared with untreated diabetic hamsters [101], indica-

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ting to the potential of N. sativa oil to enhance functions of cells of innate immunity, including Mf and NK cells, as well as cellular immunity. Another example for enhancing immunity by N. sativa is its ability to ameliorate age-associated decline in T cell functions. Nutritional supplementation can enhance the immune response in elderly humans by changing both the total amount and the type of dietary lipids [102]. N. sativa oil is rich in the n-6 PUFA a-linoleic acid (18:3n-6), the n-3 PUFA a-linolenic acid (18:3n3), and a small amount of stearidonic acid (18:4n-3) [103]. The composition of the seeds reflects the recommended optimal dietary intake of n-3 and n-6 fatty acids, i.e., it has a ratio of n-3 to n-6 fatty acids of 1 to 4 or 5 [104]. Dietary supplementation with the N. sativa oil has found to improve the immune response of healthy elderly subjects, which is mediated by a change in the factors closely associated with T cell activation [105]. Delayed type hypersensitivity (DTH) skin tests have been widely used as an in vivo assay to determine cell-mediated immune function, and a decrease in DTH, is associated with increased morbidity and mortality [106]. Treatment with N. sativa oil significantly increased the total diameter of indurations after 24 h of DTH induction in response to specific antigens (tetanus toxoid and T. mentagrophytes), when compared with presupplementation measurements or to the placebo group [106]. In contrast to its enhancing effect of on the T cellmediated immune response, N. sativa constituents have shown a tendency to downregulate B cell-mediated immunity based on the results obtained from the in vitro experiments discussed earlier where N. sativa proteins suppressed PBMC responses to the B cell mitogens LPS and PWM [22,99]. One study confirmed this hypothesis in vivo, where the effect of the volatile oil of N. sativa seeds was studied on the antigen-specific response induced by vaccinating rats with the typhoid TH antigen. In that study, treatment with N. sativa oil induced about 2-fold decrease in the antibody production in response to typhoid vaccination as compared to the control rats [107]. Thus, based on the in vitro and in vivo data, it is likely that N. sativa constituent may enhance cellular immunity, while suppress humoral immunity. Further studies, however, are required to validate this hypothesis, and to define the components responsible for each effect. Therefore, the immunomodulatory effects of

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this plant should be measured based on the nature of the immune response mediating the disease. Because PGE, LTB4, and mediators of oxidant stress downregulate lymphocyte proliferation [108,109], and because N. sativa oil significantly decreases the production of these mediators, we suggest that the immune-enhancing effect of N. sativa on cell-mediated immunity might be, at least in part, due to its ability to reduce these inflammatory mediators. Quality and quantity of cytokines are critical in initiation and execution of immunity. A variety of experiments have shown that excessive or insufficient production of cytokines may significantly contribute to the pathophysiology of a range of disease responses and are thought to be decisive for pathological or physiological consequences [110]. After activation, CD4 T helper cells differentiate into either TH1-type cells, secreting IL-2, IL-12, IFN-g and TNF-a, or TH2type cells secreting IL-4, IL-5, IL-10, and IL-13. Indeed, the balance between TH1 and TH2 cytokines is critical for the orientation of the inflammatory response toward cell-mediated or humoral-mediated responses. Thus, any factors that can interfere with TH1/TH2 axis might affect the outcome of the response [97]. By investigating the effects of N. sativa seed proteins on cytokine production by humans PBMC, the proteins enhanced the production of IL-3 and IL-1 by lymphocytes when cultured with or without allogeneic cells [98], suggesting the stimulatory effects of N. sativa seed proteins on the naı¨ve cells itself. However, under the same culture conditions, crude extract of N. sativa seeds or their soluble fractions did not show any effect on the production of IL-2 and IL-4 [98]. Interestingly, even though N. sativa proteins suppressed the production of IL-8 in non-activated PBMC, they did enhance its production by these cells when stimulated with PWM, a B cell mitogen. Of note, stimulatory effect of whole N. sativa and their fractionated proteins was also noticed on the production of TNF-a by either non-activated or mitogen-activated PBMC [22]. In a recent study, we found that N. sativa oil exhibited a striking antiviral effect against murine cytomegalovirus infection coincided with elevation of IFN-g in serum, which lasted for a prolonged time [9]. Thus, it is apparent that the effect of N. sativa on cytokine production depends on the nature and doses of ingredients and the nature of cytokines itself. Further studies, thus, are

required to explore the modulatory effects of N. sativa seed products on both TH1 and TH2 cytokines in well-defined in vitro and in vivo model systems. These studies would allow better understanding the mode of action of N. sativa on the inflammatory diseases, and as a consequence to design appropriate approaches for its therapeutic regimens. Functional immune response requires interaction between innate and adaptive immunity. Among the mediators that link these two arms of immunity are DCs, which are the most efficient at processing and presenting antigen to T cells and play a critical role in the activation and/or regulation of pathogenic T cell populations [111]. Mature DCs, through their IL-12 and TNF-a, induce the development of effector T cells, while immature DCs induce the development of regulatory T cells that suppress the activation of effector T cell responses [112]. Several anti-inflammatory drugs express their effects through modulation of DC functions. For instance, we have found that the anti-inflammatory effects of estradiol, a natural antiinflammatory drug, on T cell-mediated immunity was associated with inability of DCs of estradiol-treated mice to induce optimal proliferation of antigen-sensitized T cells in vitro [113,114]. In addition, we have found that the anti-inflammatory effects of 5-aminoimidazole-4-carboxymide ribonucleoside, a novel synthetic anti-inflammatory drug, on EAE inflammatory model is mediated by a direct inhibitory effects on DCs-T cells cross talk [115]. In contrast, we have shown that adjuvant (stimulatory) effects of cytokines such as IL-12 [116], GM-CSF [117], and IL-15 [118], or of agents with cytokine-like effects such as the tolllike receptor 3 (TLR3) ligand poly(I:C) [119], or anticancer drug such as cyclophosphamide [120], can efficiently augments DCs function and as a consequence the T cell immunity. Thus, modulation of the functional status of DCs can markedly impact on the quality and quantity of the immune responses. Thus, another potential mechanism that might mediate the anti-inflammatory effects of N. sativa is the modulation DC functions. To the best of our knowledge, so far there are no published studies that address the influence of N. sativa products on either phenotype or functions of DCs. Therefore, future studies should give attention to explore the effects of N. sativa on the phenotype, function, and cytokine production of DCs both in human volunteers and experimental models.

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This will further enhance our understanding of the immunomodulatory effects of N. sativa for better immunotherapeutic applications. 4.5. Anti-microbial properties The findings discussed above indicate that N. sativa seed constituents possess potential immunomodulatory effects, which as a consequence might impact on the host-parasite interrelationship. Consistent with this notion, the oil and active ingredients of N. sativa seeds have been reported to exert anti-microbial activities, including anti-bacterial, anti-fungal, anti-helminthic, and anti-viral effects [9,121–123]. Some of these anti-microbial effects have been attributed to the immunomodulatory effects of N. sativa seed components. 4.5.1. Anti-viral effect Murine cytomegalovirus (MCMV) is a herpes virus that causes disseminated and fatal disease in immunodeficient animals [124] similar to that caused by human cytomegalovirus in immunodeficient humans [125]. In our own experience, we have found that in vivo treatment with N. sativa oil induced a striking anti-viral effect against MCMV infection [9], indicating a promising therapeutic potential of N. sativa oil as an anti-viral remedy. Immunity generated toward viral infection is controlled by both the nonspecific cells, including NK cells and Mf, and specific cells including CD4 and CD8 T cells [126]. Each cell population plays a central anti-viral role at a certain time point post infection, where NK cells and Mf are important during the early phase, while T cells are crucial for clearance of the virus at late stages [127]. Mediators produced by these cells mainly IFN-g are seminal factors in mediation the antiviral response. Interestingly, we found that the antiviral effect of the N. sativa oil is associated with enhancing response of CD4 and CD8 cells, and Mf [9], augmenting their ability of IFN-g production that is known to render mice more resistance to MCMV infection [128,129]. It has been reported that viral infection induces apoptosis leading to lymphocyte depletion in the host, and that anti-oxidant agents can inhibit virus-induced apoptosis as well as the viral replication in target cells [130]. Eventually, the anti-oxidant effect of the N. sativa oil may represent

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another mechanism that contributes to its anti-viral activity. Indeed, the anti-viral effects of N. sativa against MCMV infection open a new avenue for a novel anti-viral remedy. However, further studies are required to confirm this effect in other viral models, as well as to define which active ingredients exerting such anti-viral effects. 4.5.2. Anti-helminthic effects Schistosomiasis, a tropical parasitic disease, is endemic in the third world countries. Protection from this disease is mediated by both cellular and humoral immunity. Although vaccine trials have been tested, chemotherapy is still the only choice regimen to the human host [131]. N. sativa seed extracts and TQ have shown potential protective effects against S. mansoni infection [66]. Treatment of S. mansoniinfected mice with N. sativa oil induced reduction in the number of S. mansoni worms in the liver, coincided with a decrease in the egg burden in both the liver and the intestine. Importantly, the oil showed additive effects with praziquental, the drug of choice for the treatment of schistosomiasis [23]. Administration of N. sativa oil to S. mansoni-infected mice partially corrected the infection-caused alterations biochemical and pathological in ALT, GGT, and AP activities, as well as the albumin content in serum [23,132]. In murine schistosomiasis, a variety of cytokines are implicated as mediators of the granulomatous inflammatory response. Accordingly, modulation of cytokine levels can modify the intensity of the inflammatory response. Since N. sativa seeds increased the ratio of helper to cytotoxic T cells, and enhanced Mf and NK cell activities in normal volunteers [100] and in MCMV-infected mice [9], and the production of IL-3 [22,98] and IFN-g [9], its antischistosome effect could in part be attributed to modulation of the immune response to schistosome eggs trapped in the liver. Similar to its anti-schistosome effects, the essential oil from the seeds of N. sativa showed pronounced anti-helminthic activity even in 1:100 dilution against tapeworms, earthworms, nematodes and cestode [121,133]. 4.5.3. Anti-bacterial effects In addition to its anti-viral and anti-helminthic effects, N. sativa showed also anti-bacterial activity against several bacterial strains, including Escheri-

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chia coli, Bacillus subtilis, Streptococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa, as well as against the pathogenic yeast Candida albicans and fungus [122,134–136]. In an earlier study, DTQ showed anti-bacterial effect against the Gram-positive bacteria [135]; and diethyl ether extract caused concentration-dependent inhibition of the Gram-positive bacteria Staphylococcus aureus, and of Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli. Furthermore, the ether extract showed synergistic and additive anti-bacterial effect with several antibiotics [122]. Importantly, the extract proved to be more effective against the drug resistant bacteria, including V. cholera, E. coli and all strains of Shigella dysentriae [136]. Even though in vitro treatment of human PBMC with the soluble fractions of N. sativa seeds had no effect on the bacterial phagocytosis or killing activities of these cells when cultured with Staphylococcus aureus [98], in vivo treatment with the N. sativa seed diethyl ether extract successfully eradicated a non-fatal subcutaneous staphylococcal infection in mice when injected at the site of infection [122]. This might indicate that the bactericidal activity of N. sativa seed components observed in vivo is mediated by different host factors. Inoculum of Candida albicans into mice produces colonies of the organism in the liver, spleen, and kidneys. By studying anti-fungal effect of the aqueous extract of N. sativa seeds using this model, treatment of the infected mice daily for 3 days starting 24 h after inoculation of C. albicans markedly inhibited the growth of the fungus in all organs studied [134]. All the findings discussed above show that N. sativa seed constituents possess anti-microbial effects againist different pathogens, including bacteria, viruses, helminths, and fungus. These findings are of a great practical significance, since N. sativa seeds have been traditionally and clinically used in Middle and Far Eastern countries without any reported undesirable effects. It may thus be valuable as a co-therapeutic agent against different microbes. However, further studies are required to assess and explore the specific mechanisms of the anti-microbial effects of N. sativa, alone or in combination with other drugs, and on other bacterial, viral, and parasitic models in order to measure and validate its potentail therapeutic effects.

4.6. Anti-tumor properties 4.6.1. In vitro anti-tumor effects In vitro and in vivo studies indicate that both the oil and the active ingredients of N. sativa seeds possess anti-tumor effects. By investigating effect of the volatile oil of N. sativa seeds on different human cancer cell lines, the oil expressed marked cytotoxic effects against a panel of human cancer cell lines [107]. Exposure of MCF-7 breast cancer cells to aqueous and alcohol extracts alone or in the presence of descending potency for H2O2 completely inactivated growth of these cells [99], suggesting that N. sativa alone or in combination with oxidative stress is effective anti-cancer agent. Studies attempted to define the anti-tumor mechanisms of the whole N. sativa oil show that N. sativa extracts induced, in a concentration-dependent manner, inhibition of the metastasis-induced factors, including type 4 collagenase, metalloproteinase, and serineproteinase inhibitors [143], angiogenic protein-fibroblastic growth factor [99], tissue-type plasminogen activator, urokinasetype plasminogen activator, and plasminogen activator inhibitor type 1 [144]. Because tumor cells to ensue their metastasis produce these factors, it can be suggested that the anti-tumor effects of N. sativa oil might be mediated through anti-angiogenic effects through inhibition of local tumor invasion and metastasis in vivo. In addition to the anti-tumor effects of the whole extract of N. sativa, TQ, DTQ, and other active ingredients also showed cytotoxic effects. For instance, the active ingredient extracted by ethyl-acetate column chromatographic fraction 5 (CC-5), or ahedrin, expressed ant tumor effects against different cancer cell lines with selectivity against hepatocellular carcinoma, leukemic cell, Lewis lung carcinoma [99], and leukemia cells through a rapid depletion of intracellular GSH and disruption of mitochondrial membrane potential with subsequent increase in the production of reactive oxygen species [145]. Both TQ and DTQ were equally cytotoxic against different human tumor cells lines, including the pancreatic adenocarcinoma, human uterine sarcoma and human leukemic [140,146], triggering their apoptosis through arresting the growth of these cells in G1 phase of the cell cycle [147] associated with increase in the gene and protein expression of p53 and inhibition of the

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anti-apoptotic Bcl-2 protein. This indicates that the anti-neoplastic effect of TQ is mediated by pro-apoptotic effects modulated by Bcl-2 protein and is linked to and dependent on p53. 4.6.2. In vivo anti-tumor effects The reported in vitro anti-tumor effects of the N. sativa oil and its active ingredients have also been confirmed in vivo in different tumor models. For instance, topical application of N. sativa inhibited two-stage initiation/promotion anthracene/croton oil skin carcinogenesis induced in mice by 7,12dimethylbenz(a)anthracene//croton oil in mice, where the onset of papilloma formation was delayed, and the mean number of papillomas was reduced [139]. The active principle fatty acids derived from N. sativa, completely inhibited the growth of Ehrlich ascites carcinoma and Dalton’s lymphoma ascites cells [140]. Moreover, oral feeding with N. sativa extract suppressed hepatic tumor in rat induced by diethylnitrosamine or by partial hepatectomy [148]. Furthermore, N. sativa oil suppressed colon carcinogenesis induced by methylnitrosourea [138] or by 1,2dimethylhydrazine [149]. In the latter study, administration of N. sativa oil given during the post-initiation stage markedly decreased the total number of aberrant crypt foci through anti-proliferative activity. In addition, a-hederin, another ingredient of the crude extract of N. sativa oil, was also found to show in vivo antitumor activity against leukemia and Lewis lung carcinoma [150], prolonging the life span of the tumorbearing mice. The anti-tumor effects of N. sativa oil might be attributed to the effect of TQ, since administration of TQ in drinking water resulted in significant suppression of forestomach tumor induced by benzo(a)pyrene [158]. Similarly, the same treatment regimens of TQ significantly inhibited the tumor incidence and tumor burden of 2-methyclonathrene induced soft tissue fibrosarcoma [159] associated with reduction in hepatic lipid peroxides and increased enzyme contents and activities of GST and GSH. Using the same fibrosarcoma tumor model, administration of N. sativa extract 30 days after subcutaneous administration of methyclonathrene restricted fibrosarcoma tumor incidence to 33.3%, compared with 100% in control tumor-bearing mice [139], indicating to therapeutic potentials. Furthermore, oral adminis-

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tration of TQ to mice bearing Ehrlich ascites carcinoma xenograft significantly enhanced the anti-tumor effect of ifosfamide, coincided with less body weight loss and mortality rate [64,158,159]. Interestingly, TQ protects against doxorubicin-induced cardiotoxicity without compromising its anti-tumor activity [160]. These observations demonstrate that TQ, in addition to its prophylactic and therapeutic anti-tumor effects, can be a potential chemotherapeutic adjuvant to standard chemotherapy. This might lower the does of standard chemotherapeutic drugs, while augmenting their anti-tumor efficacy. As discussed in Section 4.1 above, it became known that suppression of immune cell function associated with chemotherapy [33,38], radiotherapy [34], and late stages in tumor-bearing hosts [37] is mediated, at least in part, by NO produced by immature Ly6G+CD11b+ granulocytes that are massively generated under these conditions [38–40]. Therefore, it is possible that the anti-tumor effects reported for N. sativa oil and TQ are mediated by their abilities to scavenging the NO produced by these cells. The impact of N. sativa ingredient, in particular TQ, on these cells in the tumor-bearing hosts needs to be explored. In addition, since chemotherapy induces massive expansion of the immature granulocytes, which produce large amount of NO, it might be feasible to follow chemotherapy with TQ treatment that might alleviate the suppressive effects on the immune responses by chemotherapy-induced NO. In addition to the possible anti-oxidant mediating antitumor effects of TQ, it is also possible that its antitumor effects if mediated by the ability to suppress PEG and LT. Higher levels of these inflammatory mediators have been reported to correlate with tumor progression in vivo [161], and several drugs that are able to block the eicosanoid signaling, both COX-1 and COX-2 pathways, are being tested now in clinical trials [161,162]. However, the possibility that both the anti-oxidant and anti-inflammatory effects of TQ mediate its anti-tumor effects needs to be directly tested by using mice that are knock out for these mediators. Taken together, the findings of these studies indicate to the potential of the active ingredients of N. sativa oil, in particular TQ, as a powerful chemopreventive agents against several experimental cancer, including fore-stomach, fibrosarcoma, colon, skin,

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and hepatic tumors. In spite of these obvious antitumor effects of N. sativa oil and TQ, it is still remain to know if these effects are immune-mediated through modulation of anti-tumor immune responses. CD8+ T cells mainly mediate anti-tumor immune responses, while CD4+ cell help is also required for the optimal anti-tumor immune response. Therefore, further studies are required to define tumor-specific CD8+ T cell responses under the effects of TQ. This will allow to insight if TQ can also be beneficial for anti-tumor immunotherapeutic approaches.

5. Potential toxicity of N. sativa seeds All the information discussed above reveal the beneficial immunotherapeutic potentials of the crude oil and extracts of N. sativa seeds and its active ingredient TQ toward several disease settings. However, toxicity of medicinal plants is central for acceptance of their therapeutic application in human. Unfortunately, very few studies have addressed the possible toxicity of N. sativa seeds and their components. In an earlier study, aqueous extract of the seeds of N. sativa was administered orally to male Sprague– Dawley rats for 14 days, and the possible toxicity was evaluated by measuring changes in the levels of the key hepatic enzymes, and histopathological changes [165]. Serum gamma-glutamyl transferase and alanine aminotransferase concentrations were significantly increased after treatment with N. sativa extract with no evident pathological changes [165]. In another study, potential toxicity of the fixed oil of N. sativa seeds was investigated in mice and rats through determination of LD50 values and examination of possible biochemical, hematological and histopathological changes [166]. LD50 values, obtained by single doses (acute toxicity) in mice, were 28.8 ml/kg body with oral administration, and 2.06 ml/kg body with intraperitoneal administration. Chronic toxicity was studied in rats treated daily with an oral dose of 2 ml/kg body wt. for 12 weeks. Changes in key hepatic enzymes levels, including ALT, AST, and GSH, and histopathological modifications (heart, liver, kidneys and pancreas) were not observed in rats treated with N. sativa oil after 12 weeks of treatment. Of note, however, the serum cholesterol, triglyceride and glucose levels and the count of leukocytes and platelets decreased significantly,

compared to control values, while hematocrit and hemoglobin levels increased significantly. A slowing of body weight gain was also observed in N. sativatreated rats compared to control animals. Consistent with this non-toxic effect of N. sativa, it has been reported recently that treatment of Fischer 344 rats with the crude oil of N. sativa for 14 weeks did not induce pathological changes in the liver, kidneys, spleen, or other organs [149] nor the biochemical parameters of blood and urine as well as body weight gain. Further analysis on the potential toxicity of N. sativa seeds revealed that feeding Hibro broiler chicks diet containing 20 or 100 g/kg N. sativa seed ground for 7 weeks did not adversely affect growth [137]. Taken together, the parameters emerged from these studies indicate that N. sativa is not toxic, as evidenced by high LD50 values, hepatic enzyme stability and organ integrity, suggesting a wide margin of safety for the therapeutic doses of N. sativa fixed oil. However, the changes in hemoglobin metabolism and the fall in leukocyte and platelet count must be taken into consideration. In addition, the route of administration of N. sativa seems to be crucial for its toxicity, since the LD50 was higher with oral administration (a 20fold higher) than with intraperitoneal route [166], indicating that oral intake is safer than the systemic one. The changes in hemoglobin metabolism and the fall in leukocyte and platelet count observed after N. sativa treatment discussed above might be due to the effect of one of its constituents. It is possible that TQ induced these effects, given that TQ is considered as the most potent active ingredient with anti-inflammatory effects. This seems to be a working hypothesis since intraperitoneal administration of different doses of the TQ (4, 8, 12.5, 25 and 50 mg/kg) did not alter CCl4-induced changes in biochemical parameters, while its higher doses were lethal; the LD50 of TQ was 90.3 mg/kg [164]. TQ was effective only when injected before CCl4 at a dose of 12.5 mg/kg. The results of this study indicate that TQ at certain doses (e.g., 12.5 mg/kg, intraperitoneally) may play an important role as anti-oxidant and may efficiently act as a protective agent against chemically-induced hepatic damage. In contrast, higher doses of TQ might induce oxidative stress leading to hepatic injury. Indeed, future studies should give more attention to define any possible toxicity for the whole extract and oil of N. sativa as well as their active ingredients, in partic-

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Table 1 Selected studies showing the different doses and routes of administration of N. sativa seed grains and extracts tested in experimental models in vivo Dose

Route

Model

Animal

Ref.

Grains 20, 200 g/kg 0.2 g/day

Diet Oral

Toxicity Methylnitrosurea-induced colon cancer

Chicks Rats

[137] [138]

Extract 6.6 ml/kg 50 mg/kg 100 mg/kg 100 mg/kg 500 mg/kg 100 mg/kg

Oral Oral Topical Oral Oral i.p.

Candidiasis infection KBro3-induced toxicity Skin carcinogenesis Ehrlich ascites carcinoma Carrageenan-induced oedema Nociceptive activities

Mice Rats Mice Mice Mice Mice

[134] [58] [139] [140] [141] [142]

ular TQ. These studies should be evaluated in different animal species, and after different doses, routes, and administration period.

6. Future perspectives Further research both in human and in animal models are urgently required to explore the mechanisms of action of the active ingredients of N. sativa seed, in particular TQ, in health and diseases at the

cellular and molecular levels. For instance, it remains unclear if the anti-inflammatory effects of TQ are mediated by (1) modulation of COX-1 and/or COX-2 pathways, (2) non-specific inhibitory effects on cells of innate immunity, including Mf and neutrophils, and/or specific inhibitory effects on adaptive immunity components, including CD4 and CD8 T cells, (3) biasing the immune response from TH-1 inflammatory type to TH-2 anti-inflammatory type, (4) induction of regulatory (tolereogenic) DCs that have been shown to suppress inflammatory

Table 2 Selected studies showing the different doses and routes of administration of N. sativa seed oil tested in experimental models in vivo Dose

Route

Model

Animal

Ref.

2 mg/kg 200 mg/kg 0.5–2 ml/kg 2.5, 5 mg/kg 180 mg/kg 50 mg/kg 4–32 Al/kg 2.5, 5 ml/kg 800 mg/kg 400 mg/kg 100,400 Al/kg

i.p. Oral Oral Oral Diet i.p. i.v. Oral Oral i.p. Oral Oral i.p. i.p Oral Oral Oral Oral

Mice Rats Rats Mice Rats Rats Guinea pigs Rats Rats Mice Rats Rats Mice Rats Rats Rats Mice Rats Rats

[9] [149] [57] [23] [151] [152] [73] [63] [63] [101] [90]

0.2 ml/kg 0.2 ml/kg 0.2 ml/kg 2 g/kg 50,400 mg/kg 100 mg/kg 1 mg/kg

MCMV (virus) infection Colon Carcinoma Gentamicin-induced toxicity Schistosoma mansoni infection Homeostasis Cisplatin-induced toxicity Urethane anaesthetization induced respiratory pressure Ischemia/reperfusion-induced gastric lesion CCl4-induced toxicity STZ-induced diabetes Carrageenan-induced oedema croton oil-induced ear oedema Thyphoid immunization/Abs STZ-induced diabetes CCl4-induced toxicity Ant-fertility against pregnancy Nociceptive-induced insults Methionine-induced HHcy Blood homeostasis

Abs, CCl4, HHcy, i.p., i.v., MCMV, and STZ, see abbreviations.

[107] [153] [154] [155] [156] [62] [157]

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responses, and (5) modulation of the regulatory cells including CD4+CD25+ and Ly6G+CD11b+ cells, which are known to suppress inflammatory T cell responses. It also remains to define if the anti-tumor effects of TQ are due to direct effects on the tumor or due to immune-mediated effects. Most of the antitumor effects of TQ have been tested in the absence of vaccination protocols, therefore, it will be of great interest to test its anti-tumor effects in the setting of vaccination or adoptive immunotherapy, particularly following chemotherapy. Such studies are important to take advantage of the immunotherapeutic potentials of TQ that is relevant to the nature of a particular disease. Of note, most of the published studies have been carried out in laboratories in the Middle and Far East may be due the popularity, historical, religious, and traditional use of N. sativa in these countries. In spite of the reproducibility of the effects of biological properties of N. sativa, the doses, experimental conditions, nature of the purified ingredients and extracts, and the treatment schedules were different (Tables 1– 3). Therefore, conducting collaborative research between different research institutes (e.g., consortium) is highly recommended in order to generate reproducible findings on the active ingredients from different laboratories. This will allow generalization of the effects of this plant gained in each disease setting.

7. Conclusion Scientific interest in medicinal plants has burgeoned due to increased efficiency of new plant-derived drugs, growing interest in natural products, and rising concerns about the side effects of conventional medicine. Before being considered for clinical trials in humans, the active ingredients of these plants should be identified and must show tolerable levels of toxicity in several animal models. Today, there are at least 120 distinct chemical substances derived from plants that are considered as important drugs currently in use in one or more countries in the world. More than 150 studies conducted since 1959 confirmed the pharmacological effectiveness of N. sativa seed constituents. N. sativa seed is a complex substance of more than 100 compounds, some of which have not yet been identified or studied. A combination of fatty acids, volatile oils and trace elements are believed to contribute to its effectiveness. The original research articles published so far have shown the potential immunomodulatory and immunotherapeutic potentials of N. sativa seed active ingredients, in particular TQ. The immunotherapeutic efficacy of TQ is linked to its antitoxic, anti-histaminic and anti-inflammatory properties. These effects with its immunomodulatory properties can explain the anti-microbial and anticancer properties of N. sativa oil or TQ. Since differ-

Table 3 Selected studies showing the different doses and routes of administration of TQ, the active ingredients of N. sativa seeds, tested in experimental models in vivo Agent dose

Route

Model

Animal

Ref.

2.5–10 mg/kg 5 mg/kg 10 mg/kg 10 mg/kg 0.01% 0.01% 0.2 mg/kg 1.6–6.4 mg/kg 5–100 mg/kg kg 100 mg/kg 5–10 mg/kg 4–50 mg/kg 78–103 mg/kg 1 mg/kg 100 mg/kg 10 mg/kg

Oral Oral Oral Oral Oral Oral i.v. Oral Oral Oral Oral i.p. i.p. i.v. Oral Oral

Nociceptive-induced insults Ifosfamide-induced FS Ehrlich ascites carcinoma DOX-induced toxicity Benzo(a)pyrene-induced stomach tumor Methycholanthrene-induced sarcoma Arterial blood pressure Urethane anaesthetization induced respiratory pressure Ischaemia/reperfusion-induced gastric lesion Methionine-induced HHcy Acetic acid-induced colitis CCl4-induced toxicity Determination of LD50 = 90 mg/kg Inflammation (EAE model) CCl4-induced toxicity DOX-induced toxicity

Mice Rats Mice Rats Mice Mice Rats Guinea pigs Rats Rats Rats Mice Mice Mice Rats Rats

[156] [64] [158] [61] [158] [159] [163] [73] [26] [62] [87] [164] [77] [81] [52] [50]

CCl4, DOX, EAE, FS, and HHcy, i.p., and i.v., see abbreviations.

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770

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