www.elsevier.com/locate/intimp

International Immunopharmaco

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 cell-

and natural killer cell-mediated immune responses. Most importantly, both the oil and its active ingredients expressed anti-

microbial 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 Th1-

and 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; Anti-

inflammatory; 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,

1567-5769/$ - s

doi:10.1016/j.in

experimental all

peripheral blood

* Tel.: +1 843

E-mail addre

logy 5 (2005) 1749–1770

ee front matter D 2005 Published by Elsevier B.V.

timp.2005.06.008

ergic encephalomyelitis; FS, Fanconi syndrome; HHcy, hyperhomocysteinemia; i.p., intraperitoneal; i.v., intravenous; PBMC,

mononuclear cells; PHA, phytohemagglutinin; LPS, lipopolysaccharide; ROS, reactive oxygen species; STZ, streptozotocin.

792 7576; fax: +1 843 792 3200.

sses: salemm@musc.edu, mohamed_84@hotmail.com.

M.L. Salem / International Immunopharmacology 5 (2005) 1749–17701750

Contents

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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. N. sativa: botanical and historical background, and folk medicine . . . . . . . . . . . . . . . . . .

3. Ingredients of N. sativa seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1765

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 pro-

blems. A larger number of these plants and their

isolated constituents have shown beneficial therapeu-

tic effects, including anti-oxidant, anti-inflammatory,

anti-cancer, anti-microbial, and immunomodulatory

effects [1,3–9].

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 amaz-

ing herb with a rich historical and religious background

[10]. N. sativa is found wild in southern Europe, north-

ern Africa, and AsiaMinor. 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 ofN. sativa are the source

of the active ingredients of this plant. It is the black seed

referred to by the prophet Mohammed as having hea-

ling powers [10]. Black seed is also identified as the

curative black cumin in the Holy Bible and is described

as theMelanthion of Hippocrates and Discroides and as

the Gith of Pliny [12].

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770 1751

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 gas-

trointestinal 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.

3. Ingredients of N. sativa seeds

Four dolabellane-type diterpene alkaloids, nigella-

mines 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 nigel-

lone, 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

Thymoquinone (TQ)

O

O

Dithymoquinone (DTQ)

O

O

O

O

OH

Thymol (THY)OH

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]).

eight of the nine essential amino acids [17–21]. Frac-

tionation of whole N. sativa seeds using SDS–PAGE

shows a number of protein bands ranging from 94 to 10

kDamolecular 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 indi-

vidual phospholipid classes is phosphatidylcholine,

followed by phosphatidylethanolamine, phosphatidyl-

serine, 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].

4. Immunopharmacological properties of N. sativa

seeds

Several pharmacological properties of N. sativa,

including hypotensive, anti-nociceptive, uricosuric,

choleretic, anti-fertility, anti-diabetic, and anti-hista-

minic 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, anti-

tumor, and immunomodulatory properties of N.

sativa and its ingredients in the context of their

therapeutic potential.

4.1. Anti-oxidant properties

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 elec-

trically charged molecules that attack cells, tearing

through cellular membranes to react and create

M.L. Salem / International Immunopharmacology 5 (2005) 1749–17701752

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 even-

tually destroy them. ROS are produced mainly by

certain cells of immune system including macro-

phages (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 ap-

proach based on using medicinal plants in dAyurveda,TT 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 carcino-

ma 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 anti-

oxidant 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, unsaponi-

fiables, and phospholipids, as well as the initial per-

oxide values of crude oils [46]. Moreover, pre-

incubation 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-butylhy-

droquinone, also efficiently inhibited iron-dependent

microsomal lipid peroxidation in vitro in a concentra-

tion-dependent manner [48]. TQ also induced signif-

icant protection of isolated hepatocytes against tert-

butyl 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 pro-

tective action against superoxide anion radical either

generated photochemically, biochemically, or derived

from calcium ionophore, indicating to its potent su-

peroxide radical scavenger [50].

4.1.3. In vivo anti-oxidant activities

Both hepatoxicity and nephrotoxicity are associa-

ted 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, glu-

tathione (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), gen-

tamicin, 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 im-

provement 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

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770 1753

[55]. Moreover, treatment with N. sativa oil prevented

CCl4-induced liver fibrosis in rabbits with improve-

ment of the anti-oxidant status [56]. In gentamicine-

induced 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, inclu-

ding 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 anti-

oxidant enzymes [58]. Using gastric ulcer model in-

duced 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 in-

crease 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 find-

ings 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. Pro-

phylactic 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 triglyce-

rides, lipid peroxidation, cholesterol, and in the acti-

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 de-

creased creatinine clearance rate. Administration of

TQ with the drinking water to rats before and during

ifosfamide treatment ameliorated the severity of ifos-

famide-induced renal damage and improved most of

the alterations of biochemical parameters [64], inclu-

ding 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. Admin-

istration 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 hepatocel-

lular 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. man-

soni-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 dis-

cussed above, it is apparent that the crude oil of N.

sativa oil and its active constituents can lower oxida-

tive 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 pre-

clinical and clinical studies either as anti-cancer ther-

apy 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 chemo-

therapy with TQ treatment that might alleviate the

M.L. Salem / International Immunopharmacology 5 (2005) 1749–17701754

suppressive effects on the immune responses by che-

motherapy-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 ingre-

dients 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,Twhen 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 anti-

histaminic 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 contrac-

tion, 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; an-

tigen sensitized cells, compound 48/80 and the Ca-

ionophore A23187, was found to be mediated by

decreasing intracellular calcium by inhibition of pro-

tein 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 hista-

mine and serotonin receptors [72].

Preclinical and clinical studies have also shown

anti-histaminic effects for N. sativa seeds. Using gas-

tric ulcer model induced by oral administration of

ethanol, which caused a significant increase in muco-

sal histamine content, rat pretreated with N. sativa oil

before induction of ulcer induced a significant de-

crease 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 urethane-

anaesthetized 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 hista-

minergic mechanisms and indirect activation of mus-

carinic 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 thera-

peutic 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 dis-

cussed in Section 4.1, ROS, in particular NO, initi-

ates a wide range of toxic oxidative reactions causing

tissue injury. In addition to the ROS-induced inflam-

mation, inflammation is also mediated by two main

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770 1755

enzymes: cyclooxygenase (COX) and lipoxygenase

(LO) [75]. COX yields from arachidonic acid prosta-

glandines (PGE) and thrompoxane [76], while LO

catalysis the formation of leukotriens (LT). Both

PGE and LT function as the main mediators of aller-

gies 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 ingre-

dients on the production of these mediators. For in-

stance, TQ and the crude fixed oil of N. sativa

inhibited both COX and 5-LO pathways of arachido-

nate metabolism in rat peritoneal leukocytes stimulat-

ed 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 treat-

ment of calcium- or ionophore-stimulated polymor-

phonuclear 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 path-

ways 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 in-

flammatory 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 medi-

ated by T cells, while oxidative stress also plays a

central role in the onset and progression of this disease

[80]. When EAE animal received TQ, they showed

higher glutathione level, no perivascular inflammation

with no disease symptoms, compared with EAE un-

treated 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. Al-

though the pathogenesis of colitis remains poorly

understood, various mediators, such as eicosanoids,

leukotrienes, platelet activating factor and oxygen-

derived 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 in-

jection 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-coli-

tis drug [87]. The anti-colitis effects of TQ were

associated with reversed biochemical and histopatho-

logical changes towards the normal. This study sug-

gested 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 observa-

tions, 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 induc-

tion 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 sup-

pressed significantly both the cellular and fluid phases

M.L. Salem / International Immunopharmacology 5 (2005) 1749–17701756

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-lino-

lenic acid) fatty acids as substrates alternative to

arachidonic acid for oxidative metabolism, can mo-

dify monosodium urate crystal-induced acute inflam-

mation. 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 che-

mokines 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 ad-

hesion 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 en-

hancement 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 adhe-

sion 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 ingredi-

ents, in particular TQ, against murine colitis, EAE and

arthritis inflammatory diseases, that would be trans-

lated 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 in-

flammatory immune diseases, since these cytokines

have reciprocal inhibitory effects. In addition, more

attention is needed to test if TQ can modulate den-

dritic 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 recog-

nizes 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 antigen-

specific 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%

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770 1757

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 Scien-

tific Miracles of Quran and Sunnah, held in Islama-

bad, Pakistan [22]. Recently, a well-designed study

analyzed the immunomodulatory effects of the whole

extract of N. sativa seeds and their protein compo-

nents 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 compo-

nents did not show any significant stimulatory effect

on the PBMC responses to the T cell mitogens phy-

tohemagglutinin (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 cul-

tures, 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 con-

stituents 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 diabe-

tes significantly induced beneficial effect, coincided

with elevation in the phagocytic activity of peritoneal

Mf, and lymphocyte count in peripheral blood com-

pared with untreated diabetic hamsters [101], indica-

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 (18:3n-

3), and a small amount of stearidonic acid (18:4n-3)

[103]. The composition of the seeds reflects the recom-

mended 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 de-

crease 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 cell-

mediated immune response, N. sativa constituents

have shown a tendency to downregulate B cell-medi-

ated 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 con-

firmed 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 vaccina-

tion 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

M.L. Salem / International Immunopharmacology 5 (2005) 1749–17701758

this plant should be measured based on the nature of

the immune response mediating the disease. Because

PGE, LTB4, and mediators of oxidant stress down-

regulate lymphocyte proliferation [108,109], and be-

cause N. sativa oil significantly decreases the

production of these mediators, we suggest that the

immune-enhancing effect of N. sativa on cell-mediat-

ed 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 TH2-

type cells secreting IL-4, IL-5, IL-10, and IL-13. In-

deed, 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 pro-

teins 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 naı̈ve 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 sup-

pressed 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 mi-

togen-activated PBMC [22]. In a recent study, we

found that N. sativa oil exhibited a striking anti-

viral 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-inflam-

matory drugs express their effects through modulation

of DC functions. For instance, we have found that the

anti-inflammatory effects of estradiol, a natural anti-

inflammatory drug, on T cell-mediated immunity was

associated with inability of DCs of estradiol-treated

mice to induce optimal proliferation of antigen-sensi-

tized T cells in vitro [113,114]. In addition, we have

found that the anti-inflammatory effects of 5-aminoi-

midazole-4-carboxymide ribonucleoside, a novel syn-

thetic 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 toll-

like receptor 3 (TLR3) ligand poly(I:C) [119], or anti-

cancer drug such as cyclophosphamide [120], can

efficiently augments DCs function and as a conse-

quence 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 modula-

tion 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.

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770 1759

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 immuno-

modulatory effects, which as a consequence might

impact on the host-parasite interrelationship. Consis-

tent with this notion, the oil and active ingredients of

N. sativa seeds have been reported to exert anti-mi-

crobial activities, including anti-bacterial, anti-fungal,

anti-helminthic, and anti-viral effects [9,121–123].

Some of these anti-microbial effects have been attrib-

uted 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 non-

specific cells, including NK cells and Mf, and spe-

cific 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 main-

ly IFN-g are seminal factors in mediation the anti-

viral response. Interestingly, we found that the anti-

viral 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

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 en-

demic 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. mansoni-

infected mice with N. sativa oil induced reduction in

the number of S. mansoni worms in the liver, coin-

cided 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]. Administra-

tion of N. sativa oil to S. mansoni-infected mice

partially corrected the infection-caused alterations bio-

chemical and pathological in ALT, GGT, and AP

activities, as well as the albumin content in serum

[23,132]. In murine schistosomiasis, a variety of cyto-

kines are implicated as mediators of the granuloma-

tous inflammatory response. Accordingly, modulation

of cytokine levels can modify the intensity of the

inflammatory response. Since N. sativa seeds in-

creased the ratio of helper to cytotoxic T cells, and

enhanced Mf and NK cell activities in normal volun-

teers [100] and in MCMV-infected mice [9], and the

production of IL-3 [22,98] and IFN-g [9], its anti-

schistosome effect could in part be attributed to mod-

ulation 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, nema-

todes 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-

M.L. Salem / International Immunopharmacology 5 (2005) 1749–17701760

chia coli, Bacillus subtilis, Streptococcus faecalis,

Staphylococcus aureus, and Pseudomonas aerugi-

nosa, 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 ex-

tract 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 aque-

ous extract of N. sativa seeds using this model,

treatment of the infected mice daily for 3 days start-

ing 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 pos-

sess 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 inacti-

vated growth of these cells [99], suggesting that N.

sativa alone or in combination with oxidative stress is

effective anti-cancer agent. Studies attempted to de-

fine the anti-tumor mechanisms of the whole N. sativa

oil show that N. sativa extracts induced, in a concen-

tration-dependent manner, inhibition of the metasta-

sis-induced factors, including type 4 collagenase,

metalloproteinase, and serineproteinase inhibitors

[143], angiogenic protein-fibroblastic growth factor

[99], tissue-type plasminogen activator, urokinase-

type plasminogen activator, and plasminogen activa-

tor 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 metas-

tasis 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 in-

stance, the active ingredient extracted by ethyl-acetate

column chromatographic fraction 5 (CC-5), or a-

hedrin, 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 intra-

cellular GSH and disruption of mitochondrial mem-

brane 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

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770 1761

anti-apoptotic Bcl-2 protein. This indicates that the

anti-neoplastic effect of TQ is mediated by pro-apo-

ptotic 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 7,12-

dimethylbenz(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 carcino-

genesis induced by methylnitrosourea [138] or by 1,2-

dimethylhydrazine [149]. In the latter study, adminis-

tration of N. sativa oil given during the post-initiation

stage markedly decreased the total number of aberrant

crypt foci through anti-proliferative activity. In addi-

tion, a-hederin, another ingredient of the crude extract

of N. sativa oil, was also found to show in vivo anti-

tumor activity against leukemia and Lewis lung car-

cinoma [150], prolonging the life span of the tumor-

bearing 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 suppres-

sion of forestomach tumor induced by ben-

zo(a)pyrene [158]. Similarly, the same treatment

regimens of TQ significantly inhibited the tumor in-

cidence and tumor burden of 2-methyclonathrene in-

duced 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, adminis-

tration of N. sativa extract 30 days after subcutaneous

administration of methyclonathrene restricted fibro-

sarcoma tumor incidence to 33.3%, compared with

100% in control tumor-bearing mice [139], indicating

to therapeutic potentials. Furthermore, oral adminis-

tration of TQ to mice bearing Ehrlich ascites carcino-

ma 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 stan-

dard 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 as-

sociated with chemotherapy [33,38], radiotherapy

[34], and late stages in tumor-bearing hosts [37] is

mediated, at least in part, by NO produced by imma-

ture 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 anti-

tumor effects of TQ, it is also possible that its anti-

tumor 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 indi-

cate to the potential of the active ingredients of N.

sativa oil, in particular TQ, as a powerful chemopre-

ventive agents against several experimental cancer,

including fore-stomach, fibrosarcoma, colon, skin,

M.L. Salem / International Immunopharmacology 5 (2005) 1749–17701762

and hepatic tumors. In spite of these obvious anti-

tumor 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 stud-

ies 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. How-

ever, toxicity of medicinal plants is central for accep-

tance of their therapeutic application in human.

Unfortunately, very few studies have addressed the

possible toxicity of N. sativa seeds and their compo-

nents. 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 in-

creased 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 administra-

tion, and 2.06 ml/kg body with intraperitoneal admin-

istration. 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, includ-

ing ALT, AST, and GSH, and histopathological mod-

ifications (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 cho-

lesterol, triglyceride and glucose levels and the count

of leukocytes and platelets decreased significantly,

compared to control values, while hematocrit and he-

moglobin levels increased significantly. A slowing of

body weight gain was also observed in N. sativa-

treated 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. How-

ever, 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 20-

fold higher) than with intraperitoneal route [166], in-

dicating 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-inflamma-

tory 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 im-

portant 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-

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 Diet Toxicity Chicks [137]

0.2 g/day Oral Methylnitrosurea-induced colon cancer Rats [138]

Extract

6.6 ml/kg Oral Candidiasis infection Mice [134]

50 mg/kg Oral KBro3-induced toxicity Rats [58]

100 mg/kg Topical Skin carcinogenesis Mice [139]

100 mg/kg Oral Ehrlich ascites carcinoma Mice [140]

500 mg/kg Oral Carrageenan-induced oedema Mice [141]

100 mg/kg i.p. Nociceptive activities Mice [142]

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770 1763

ular TQ. These studies should be evaluated in differ-

ent 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 mechan-

isms of action of the active ingredients of N. sativa

seed, in particular TQ, in health and diseases at the

Table 2

Selected studies showing the different doses and routes of administrat

Dose Route Model

2 mg/kg i.p. MCMV (virus) infection

200 mg/kg Oral Colon Carcinoma

0.5–2 ml/kg Oral Gentamicin-induced toxicity

2.5, 5 mg/kg Oral Schistosoma mansoni infectio

180 mg/kg Diet Homeostasis

50 mg/kg i.p. Cisplatin-induced toxicity

4–32 Al/kg i.v. Urethane anaesthetization ind

2.5, 5 ml/kg Oral Ischemia/reperfusion-induced

800 mg/kg Oral CCl4-induced toxicity

400 mg/kg i.p. STZ-induced diabetes

100,400 Al/kg Oral Carrageenan-induced oedema

croton oil-induced ear oedem

0.2 ml/kg Oral Thyphoid immunization/Abs

0.2 ml/kg i.p. STZ-induced diabetes

0.2 ml/kg i.p CCl4-induced toxicity

2 g/kg Oral Ant-fertility against pregnanc

50,400 mg/kg Oral Nociceptive-induced insults

100 mg/kg Oral Methionine-induced HHcy

1 mg/kg Oral Blood homeostasis

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

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 neu-

trophils, 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

ion of N. sativa seed oil tested in experimental models in vivo

Animal Ref.

Mice [9]

Rats [149]

Rats [57]

n Mice [23]

Rats [151]

Rats [152]

uced respiratory pressure Guinea pigs [73]

gastric lesion Rats [63]

Rats [63]

Mice [101]

Rats [90]

a Rats

Mice [107]

Rats [153]

Rats [154]

y Rats [155]

Mice [156]

Rats [62]

Rats [157]

M.L. Salem / International Immunopharmacology 5 (2005) 1749–17701764

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 anti-

tumor 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 poten-

tials of TQ that is relevant to the nature of a partic-

ular 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, ex-

perimental conditions, nature of the purified ingredi-

ents and extracts, and the treatment schedules were

different (Tables 1– 3). Therefore, conducting collab-

orative research between different research institutes

(e.g., consortium) is highly recommended in order to

generate reproducible findings on the active ingredi-

ents from different laboratories. This will allow gen-

eralization of the effects of this plant gained in each

disease setting.

Table 3

Selected studies showing the different doses and routes of administration o

models in vivo

Agent dose Route Model

2.5–10 mg/kg Oral Nociceptive-induced insults

5 mg/kg Oral Ifosfamide-induced FS

10 mg/kg Oral Ehrlich ascites carcinoma

10 mg/kg Oral DOX-induced toxicity

0.01% Oral Benzo(a)pyrene-induced sto

0.01% Oral Methycholanthrene-induced

0.2 mg/kg i.v. Arterial blood pressure

1.6–6.4 mg/kg Oral Urethane anaesthetization in

5–100 mg/kg kg Oral Ischaemia/reperfusion-induc

100 mg/kg Oral Methionine-induced HHcy

5–10 mg/kg Oral Acetic acid-induced colitis

4–50 mg/kg i.p. CCl4-induced toxicity

78–103 mg/kg i.p. Determination of LD50=90

1 mg/kg i.v. Inflammation (EAE model)

100 mg/kg Oral CCl4-induced toxicity

10 mg/kg Oral DOX-induced toxicity

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

7. Conclusion

Scientific interest in medicinal plants has bur-

geoned due to increased efficiency of new plant-de-

rived 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 toxic-

ity 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 pharma-

cological 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 con-

tribute to its effectiveness. The original research arti-

cles published so far have shown the potential

immunomodulatory and immunotherapeutic poten-

tials 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 anti-

cancer properties of N. sativa oil or TQ. Since differ-

f TQ, the active ingredients of N. sativa seeds, tested in experimental

Animal Ref.

Mice [156]

Rats [64]

Mice [158]

Rats [61]

mach tumor Mice [158]

sarcoma Mice [159]

Rats [163]

duced respiratory pressure Guinea pigs [73]

ed gastric lesion Rats [26]

Rats [62]

Rats [87]

Mice [164]

mg/kg Mice [77]

Mice [81]

Rats [52]

Rats [50]

M.L. Salem / International Immunopharmacology 5 (2005) 1749–1770 1765

ent diseases are mediated by different mediators,

which sometimes exert opposing effects, the immu-

notherapeutic efficacy of ingestion or administration

of the whole seeds, oil or its purified constituents

should be measured by the nature of the disease.

Therefore, further studies are required to explore the

specific cellular and molecular targets of N. sativa

constituents in particular TQ.

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