Abstract

Introduction

Nigella sativa is an annual flowering plant. It grows to 20–30 cm (7.9–11.8 inch) tall and has linear lanceolate leaves. The delicate flowers have 5-10 petals and the colors are usually yellow, white, pink, pale blue or pale purple. The fruit of plant is large and inflated capsule composed of 3-7 united follicles, that each of them has numerous seeds. The black colored seeds are flattened, oblong and angular, funnel shaped, with the length of 0.2 cm and 0.1 cm wide (1).

This plant is known by numerous names, for example black cumin (English), black caraway seeds (USA), shonaiz (Persian) and kalajira (Bangali) (2).

Chemical constituents

Extensive studies were done to identify the composition of the black cumin seed, the ingredients of N. sativa seed includes: fixed oil, proteins, alkaloid, saponin and essential oil.

The fixed oil (32-40 %) contains: unsaturated fatty acids which includes: arachidonic, eicosadienoic, linoleic, linolenic, oleic, almitoleic, palmitic, stearic and myristic acid as well as beta-sitosterol, cycloeucalenol, cycloartenol, sterol esters and sterol glucosides (3-5).

The volatile oil (0.4-0.45 %) contains saturated fatty acids which includes: nigellone that is the only component of the carbonyl fraction of the oil, Thymoquinone (TQ), thymohydroquinone (THQ), dithymoquinone, thymol, carvacrol, α and β-pinene, d-limonene, d-citronellol, p-cymene volatile oil of the seed also contains: p-cymene, carvacrol, t-anethole, 4-terpineol and longifoline (3, 4, 6).

Black cumin seed have two different forms of alkaloids: isoquinoline alkaloid that includes: nigellicimine, nigellicimine n-oxide and pyrazol alkaloid that includes: nigellidine and nigellicine (3, 4).

The nutritional compositions of N. sativa are vitamins, carbohydrates, mineral elements, fats and proteins that include eight or nine essential amino acids.

By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the fractionation of whole N. sativa seeds was done which shows the bands ranged from 94 to 100 kDa molecular mass (7).

Black cumin seeds also have saponin and alpha hederine and in trace amount has carvone, limonene and citronellol, as well as provide relatively good amounts of different vitamins and minerals such as Fe, Ca, K, Zn, P, Cu (3, 4).

Most of the pharmacological effects are due to quinine constituent, of which TQ is the mainly abundant. TQ possess anticonvulsant activity (8-10), antioxidant (11), anti-inflammatory (12), anti-cancer (13), antibacterial (14) and antifungal activity (15).

Traditional uses of folk remedies

N. sativa seeds have been used traditionally in middle eastern folk medicine as a treatment for various diseases for more than 2000 years ago (16).

The seeds were used as pungent appetizer, aromatic, thermogenic, diuretic, expectorant, purgative, stimulant, sudoriferous, sedative and carminative (17-23).

Black cumin seeds have a history of use in traditional Arabic herbal medicine to treat many diseases such as skin diseases, jaundice, gastrointestinal problems, anorexia, conjunctivitis, dyspepsia, rheumatism, diabetes, hypertension, intrinsic hemorrhage, paralysis, amenorrhea, anorexia, asthma, cough, bronchitis, headache, fever, influenza and eczema (17-23).

Pharmacological properties

In recent years huge number of studies have been carried out, acclaimed medicinal properties emphasized on different pharmacological effects of N. sativa seeds such as antioxidant (24), anti-tussive (25), gastroprotective (26), anti-anxiety (27), anti-ulcer (28), antiasthmatic (29), anti-cancer, anti-inflammatory, immunomodulatory and anti-tumor properties (30-32), hepatoprotective effect (33), also gastric ulcer healing (34), tumor growth suppression (35), men infertility improvement (36), cardiovascular disorders (37), memory improvement (38), stimulate milk production (39), protective effects on lipid peroxidation (40), antibacterial activity (41), anti dermatophyte (42), antiviral activity against cytomegalovirus (43), have been reported for this medicinal plant.

In this paper, we describe the antimicrobial effects of N. sativa and its constituents. Selected studies showing the different extracts and microorganisms tested in experimental models in vivo and in vitro (Figure 1) for antibacterial, antifungal, anti-schistosomiasis and antiviral also demonstrated in Tables ​Tables1,1, ​,2,2, ​,33 and ​and4,4, respectively.

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Figure 1

Different effects of Nigella sativa against microorganisms

Antibacterial activity

The antimicrobial properties of herbal plants and their extracts have been recognized since ancient times, while attempts to illustrate these qualities in the laboratory date back to the early 1900s (44). Now, the development of resistance via a pathogen to several of the usually used antibiotics provides a drive for additional attempts to find new antimicrobial agents to eradicate the infection and defeat the problems of resistance and side effects of the antimicrobial drugs that are currently used (45, 46).

The mechanism of the antimicrobial effect of N. sativa seeds has not been reported, its antimicrobial property could be attributed to the active constituents particularly TQ and melanin (47). Their broad spectrum of activity may be the reason of that the key processes of the organisms are affected (48).

In vitro studies for antibacterial activity

Concentration dependent inhibition of Gram-positive; Staphylococcus aureus and Gram-negative; Pseudomonas aeruginosa, Escherichia coli and pathogenic yeast Candida albicans in filter paper discs impregnated with ethyl ether extract of N. sativa (25-400 micrograms/disc) was observed, the extract showed antibacterial synergism with streptomycin and gentamicin and exhibited additive antibacterial action with doxycycline, spectinomycin, erythromycin, tobramycin, ampicillin, chloram-phenicol, nalidixic acid, lincomycin and sulfa-methoxazole trimethoprim combination (49).

To investigate the antibacterial effect of crude extracts of N. sativa, various bacterial isolates which included of 16 Gram-negative and 6 Gram-positive, these isolates, particularly Gram-negative bacteria, showed multiple resistance against antibiotics. Crude alkaloid and water extracts were the most effective extracts, and especially against Gram-negative isolates they were effective (45).

A population of 7.0 log CFU of each strain of Listeria monocytogenes was inoculated on duplicate plates having antibiotic medium one agar. Each discs (6 mm diameter), impregnated with 10 µl of black seed oil, or gentamicin (positive control).

N. sativa seed oil had a strong antibacterial activity against all the strains of L monocytogenes, yielding a significantly greater inhibition zone than that of gentamicin (P<0.01). The mean zones of inhibition produced by N. sativa seed oil and gentamicin were 31.50±1.0 and 14.80±0.50 mm, respectively (50).

Methicillin resistant Staph. aureus (MRSA) is one of the commonest pathogens encountered in laboratory and in clinic. All tested strains of MRSA were sensitive to ethanolic extract of N. sativa seeds at a concentration of 4 mg/discs, the extract had minimum inhibitory concentration (MIC) range of 0.2-0.5 mg/ml (46).

TQ has antibacterial activity that could be potentiated by antibiotics especially in case of Staph. aureus. In a study the antibacterial effect of TQ and HQ against Esch coli, Pseudo. aeruginosa, Shigella flexneri, Salmonella typhimurium, Salmonella enteritidis and Staph. aureus was investigated. Staph. aureus, was very susceptible to TQ, as, 3 and 6 µg/ml were sufficient to inhibit and kill the bacteria respectively. On the other hand, the concentration of THQ required to inhibit and kill Staph. aureus was 400 and 800 µg/ml respectively which is 100 times more than that of TQ. Gram-negative bacteria were less susceptible to TQ and THQ, their MIC and minimum bactericidal concentration (MBC) were in the range between 200 and 1600 μg/ml. When TQ and THQ combined with antibiotics (ampicillin, cephalexin, chloramphenicol, tetracycline, gentamicin, and ciprofloxacin) showed synergistic properties especially in case of Staph. aureus (14).

Chaieb and coworkers reported that, TQ had a significant bactericidal activity (MICs values in the range of 8 to 32 μg/ml) that is especially effective against Gram-positive cocci (Staph. aureus ATCC 25923 and Staphylococcus epidermidis CIP 106510) (51).

In MIC determination, TQ was active against all the strains that studied. The main antibacterial activity was seen against Streptococcus constellatus (MIC 4 µg/ml). The essential oil showed the strongest activity against Streptococcus mitis, Streptococcus mutans, Strep. constellatus and Gemella haemolysans (MIC 2.13 mg/ml), but it was not effective against Enterococcus faecalis and Enterococcus faecium (MIC > 8.5 mg/ml) (52).

By the disc diffusion method, TQ (150 µg/disk) was effective especially against Strep. mutans and Strep. mitis (zone of inhibition were: 24.5 ± 0.71 and 22 ± 1.41 mm, respectively). TQ showed also weak antibacterial activity against Entero. faecalis, Entero. faecium and Streptococcus salivarius (9 ± 0.00, 9.5 ± 0.71 and 9.5 ± 0.71 mm, respectively) (52).

The essential oil (2.43 mg/disc) have high activity against Strept. mitis, Streptococcus. oralis, Strep. mutans, Strep. constellatus and G. haemolysans with a zone of inhibition ranged from 13.5 to 15.5 mm, besides, it was not effective against Entero. faecalis, Entero. faecium and Strep. salivarius (52).

An obvious inhibition of the growth of Staph. aureus was seen at concentration of 300 mg/ml of N. sativa seeds compared with distilled water as control. The inhibitory effect was confirmed with azithromycin as positive control. The inhibitory effect may be due to TQ (47).

The aqueous extract of N. sativa seed showed less antibacterial effect compared to the methanol extract. At concentration of 20 mg/ml the methanol extract of seed was effective against Strep. pyogenes (10 mm zone of inhibition), and it induced 15 mm zone of inhibition at 100 mg/ml which in this concentration was effective against Streptococcus. pneumoniae, Pseudo. aeruginosa and Proteus vulgaris (53).

The aqueous extract of N. sativa seed at 100 mg/ml was effective against Pseudo. aeruginosa (20 mm zone of inhibition), Strep. pyogenes (15 mm zone of inhibition) and at concentration of 50 mg/ml exhibited modest effective against Strep. pyogenes (10 mm zone of inhibition), Klebsiella pneumoniae (11 mm zone of inhibition), Pro. vulgaris (12 mm zone of inhibition) (53).

Thermosensitive N-Isopropyl acryl amide-N-Vinyl 2-pyrollidone (NIPAAm-VP) co-polymeric micelle synthesized by radical copolymerization, and the extract of N. sativa entrapped in this polymeric system for checking the release of the bioactive compound and estimated its antibacterial effect (54).

N. sativa seed extract has been loaded into the polymeric micelle and its effectiveness has been evaluated against Gram-positive strain of Staph. aureus, Bacillus subtilis and a Gram-negative Esch. coli. N. sativa loaded polymeric micelles were hundred times more effective than the naked one (54).

These findings suggest that this thermosensitive polymeric system would more effectively release the drug in the body when there is an infection such as higher temperature conditions (54).

In vivo studies for antibacterial activity

In an animal study, it was shown that the methanol and chloroform extracts of N. sativa seed total extract (TE) as well as the essential oil (EO) caused a dose dependent antibacterial activity on Gram-positive and Gram-negative organisms. In this research, Staph. aureus and Esch. coli (0.1 ml from 10⁶ colony forming units/ml suspension) were injected intraperitoneally to male mice. After 24 hours infected mice were exposed to different doses of TE or EO. The specimens aspiration of intraperitoneal fluid were cultured on a soybean casein digest agar plate surface, finally it was observed that the EO and TE are effective against both Gram-positive and Gram-negative bacteria (41).

To assess whether treatment with TQ prior to or during inoculation of Esch. coli can prevents oxidative damage in an acute pyelonephritis (PYN) ascending obstructive rat model. TQ was injected intraperitoneally (10 mg / kg), 24 hr prior to bacteria inoculation and repeated at 24 hr intervals through the indicated time. The results showed that TQ diminished the oxidative damage that occurred in PYN. The protective effect of TQ in kidney tissue also was confirmed with histological examination, because of the releasing free radicals through PYN, these properties can attribute to antioxidant effect of TQ (55).

The methanol extract of N. sativa seed used to examine in vitro and in vivo antibacterial effects against pathogenic bacteria that cause mastitis in cows through the year 2010-11. The cows that have clinically confirmed mastitis were treated with local injection of the extract to the breast. For in vitro antimicrobial experiment, the extract was used against the pathogen that collected from infected breast. Agar dilution and disk diffusion methods were the technique of investigation. The results showed that the extract have significant in vitro and in vivo inhibitory effect (48).

In vitro antifungal activity

A study was done to investigate the antidermatophyte effects of ether extract of N. sativa seeds and TQ. In this research, the test was performed by using the agar diffusion method with serial dilutions of ether extract of N. sativa seed, TQ and griseofulvin. The species were used consists of eight species of dermatophytes, four species of Trichophyton rubrum and one each of Epidermophyton floccosum Trichophyton interdigitale, Microsporum canis, and Trichophyton menta-grophytes. The results showed that the MICs of the ether extract of N. sativa seed and TQ were in range of 10-40 and 0.125-0.250 mg/ml, respectively, whereas for griseofulvin was betwen 0.00095 to 0.01550 mg/ml (42).

Two novel defensins named Ns-D1 and Ns-D2 from seeds of N. sativa, were isolated, purified and sequenced. The Ns-D1 and Ns-D2 peptides exhibited strong divergent antifungal activity against a number of phytopathogenic fungi. The mechanism is due to expected interaction of defensins with specific sphingolipids on the fungal membranes (56).

The growth of Aspergillus parasiticus (CBS 921.7) and Aspergillus flavus (SQU 21) strains, and also the production of aflatoxin B1 by this fungus, was evaluated. The inhibition of aflatoxin B1 production by A. flavus and A. parasiticus with different concentrations of N. sativa oil (1, 2 and 3 ml/100 ml) were in the range of 49.7- 58.3% and 32-48% respectively, but different concentrations of N. sativa oil displayed no significant effect on the growth of other Aspergillus species. N. sativa oil might have metabolic effects on biosynthesis pathways of aflatoxin (57).

In a study to evaluate the antidermatophytic effects of N. sativa essential oil, microdilution and disc diffusion methods were used. N. sativa essential oil represented a significant antidermatophytic activity, that the maximum zone of inhibition was seen in Microsporum gypseum with inhibition zon of (IZ) 38 mm and activity index (AI): 1.90. Also NSO showed antidermatophytic activity against T. rubrum, Trichophyton simii (IZ:20 mm, AI: 1.33 and IZ:35 mm, AI: 1.09, respectively) when compared to standard. Whereas, it was seen that the inhibition effect against Chrysosporium tropicum (IZ: 26 mm, AI: 0.86) and Chrysosporium evolceanui (IZ: 25 mm, AI: 0.71) were slightly comparable to ketoconazole, the standard drug (AI = IZ of sample / IZ of standard) (58).

In vivo antifungal activity

When Candida albicans inoculated into mice will produce colonies in the liver, spleen and kidneys. In a study, 24 hours after inoculation, the infected mice were administered the aqueous extract of N. sativa seeds (6.6 ml/kg daily for the 3 days), the results showed markedly inhibition of the growth of these pathogens in all studied organs (59, 60).

The candidacidal effect is related to nitric oxide (NO) dependent pathway in rat neutrophils. NO is responsible for protection against pathogens that are living and proliferating in the intracellular environment of several different kinds of somatic cells (59, 60).

N. sativa seed extract has active ingredient (s), that may directly stimulate the granulocytes and monocytes to produce NO, that kills Can. albicans (59, 60).

The Can. albicans intravascular inoculation, produces colonies of the Can. albicans in the liver, spleen and kidneys (61).

Treatment of mice with the extract 24 hr after the inoculation produced a significant inhibitory effect on the growth of the organism in these organs. Histopathological examination also confirmed this finding, methanolic extract of N. sativa seeds was the most effective extract against different strains of Can. albicans, followed by the chloroform extract. The antifungal was not found with the aqueous extract (61).

A research was done to evaluate the effect of TQ against vaginal candidiasis in prednisolone induced immune suppressed mice. The mice were immunosuppressed via the subcutaneous injection of methyl prednisolone (150 mg/kg) on days 1 and 3 before the induction of infection. Then for inducing the infection via the vaginal route, a swab saturated with cell suspension of Can. albicans was used. To estimate the effect of TQ, 5 days later cream containing TQ administered once daily for 6 days. Treatment with TQ showed lower number of Can. albicans colonies as compared to control group. The reduction of yeast colonies was proportional to increase the TQ concentration. At the concentration of 10% TQ, the cream was able to successfully kill most of Can. albicans cells as well as all of the mold cells. Complete eradication of Rhizopus sp also was seen, at concentration of 2%, 4%, 6% and 8% TQ. The average numbers of colonies were 55, 21, 21 and 8 CFU/mouse. Histological analysis also confirmed the antifungal activity of TQ. The mechanism is attribute to that TQ lead to inactivate the plasma membrane protein of microorganism and loss of the function occurs followed by yeast cells death, whereas TQ had no effect on the vaginal cells (15).

Few studies have been conducted on the effects of antischistosomiasis, antiviral and antiparasite activity of N. sativa seed. In the following section we discuss these effects.

Antischistosomiasis activity

Two weeks treatment with N. sativa oil (NSO) in mice that infected with Schistosoma mansoni leads to diminish the number of Sch. mansoni worms in the liver and also reduced the whole number of ova deposited in both the liver and the intestine. Moreover, it augmented the number of dead ova in the intestinal wall and obviously diminished the granuloma diameters. The infected mice with Sch. mansoni produced a marked rising in the serum activity of L-alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), with a low increase in alkaline phosphatase (AP) level, while reduced serum albumin level. Administration of NSO partially be successful to correct the prior changes in ALT, GGT, AP activity, in addition to correct the albumin content in serum, when praziquantel administered with NSO, further reduces the dead ova number in comparison with praziquantel alone seen. These changes were related mostly with the ability of NSO to improve liver function and the immunological system of infected mice and partly to its antioxidant effects (62).

The protection is also due to the ability of NSO and TQ to reduce the cytogenetic damage caused by schistosomiasis infection (63).

The karyotype of the bone marrow and spleen cells of infected mice shown that the main chromosomal abnormalities were gaps, fragments and deletions on chromosome 2, 6, and some in chromosomes 13 and 14, compared to the control level. Decline in the percentage of chromosomal aberrations and the incidence of deletions and tetraploidy was observed in treated group with NSO or TQ. So that, N. sativa has therapeutic effects against Sch. mansoni (63).

The results of in vitro tests of N. sativa seeds against Sch. mansoni, miracidia, cercariae, and adult worms show its strong effects against all stages of the parasite and an inhibitory effect on egg laying of adult female worms. Besides, N. sativa seeds induced an oxidative stress against adult worms which shown with diminish in the activities of glutathione reductase, antioxidant enzymes, glutathione peroxidase, superoxide dismutase and enzymes of glucose metabolism, glucose-6-phosphate dehydrogenase and hexokinase. Disturbing of these enzymes of adult worms using N. sativa seeds could turn render the parasite vulnerable to damage by the host and may play a role in the anti-schistosomal potency (64).

The antioxidant and anti-schistosomal effects of the garlic extract (AGE) and NSO on normal and Sch. mansoni infected mice was studied. The results presented that, treatment with AGE and NSO prevented most of the hematological and biochemical changes and markedly improved the antioxidant capacity of schistosomiasis mice compared to the infected untreated ones (65).

Antiviral activity

Apoptosis is caused by viral infections leading to lymphocyte depletion in the host cell, and antioxidants can inhibit apoptosis which induced by viruses in addition to inhibit the viral replication in target cells, so antiviral and antioxidant effects can be linked together (66).

To investigate the antiviral effect of NSO, murine cytomegalovirus (MCMV) as a model were used. Intraperitoneal administration of NSO to mice completely inhibited the virus titers in spleen and liver on day 3 of infection. Viral load in the liver and spleen of the control had a high difference with NSO treated mice, 45×10⁴ vs. 7×10⁴ and 23×10³ vs. 3×10³, respectively. This antiviral effect accorded with raising the serum level of interferon-gamma and increased numbers of CD4+ helper T cells, suppressor function and numbers of macrophages. On the tenth day of infection, the virus titer was undetectable in spleen and liver of NSO treated mice, whereas it was detectable in control mice, so the in vivo treatment with N. sativa oil induced a remarkable antiviral effect against MCMV infection (43).

By nonspecific cells including natural killer cell (NK cells), and specific cells including CD4 and CD8 T cells, immunity produced to viral infection is controlled (67). The antiviral effect of the NSO is related with increasing response of CD4 cells (43).

In a research, the patient with hepatitis C virus (HCV) infection who was not eligible for IFN-α therapy, had received the capsule of NSO (450 mg) for three successive months, for three times in a day and the findings showed that the significantly decreased of the viral load and also improvement of the oxidative stress due to augmented total antioxidant activity, total protein and albumin, improved RBC and platelet counts in HCV patients. The augmented RBC count can attributed to the lowering of the membrane lipid peroxide level, leads to reduce the incidence of hemolysis (68).

Diminish the blood glucose levels, implying that it may provide a potential modulatory influence on HCV induced glucose intolerance, improve in the lower limb edema also was seen (68).

Antiparasite activity

The effects of N. sativa seeds in children who naturally infected with cestode worms investigated. A single oral administration of 40 mg/kg of ethanolic extract of N. sativa without side effect in the doses tested diminished the fecal eggs percentage (69).

1.25 g/kg of methanolic extract of N. sativa seeds (MENS) lead to suppression of Plasmodium yoelii infection (94%, P<0.05) whereas chloroquine, the choice drug, lead to 86%. So MENS is more effective than chloroquine for the treatment of Plas. yoelii infection. The antimalarial effect is due to MENS have antioxidant effect in Plasmodium infected mice, improving the oxidative status in red blood cells, and hepatocytes of infected mice was seen (70).

A 400 mg/kg of aqueous suspensions and oil emulsions of N. sativa seeds used for the treatment of coccidiosis in rabbits. The anticoccidial effects were seen with both treatments, but the more rapid antiparasite effect was seen with the N. sativa oil emulsion. Both treatment increased weight gain and decreased fecal oocyst shedding and histopathology the liver tissue improved remarkably. The improvements was included a significant reduction in infiltration of the inflammatory cells in the portal area, also the different stages of parasites in the bile ducts were diminished and bleeding between hepatic lobules disappeared, the hepatocytes returns to the natural radial arrangement and all severe symptoms disappeared. The N. sativa oil emulsion has higher concentrations of alkaloid nigellicine that has a deadly influence on parasites (71).

Conclusion

All findings discussed above indicate that N. sativa seeds have antimicrobial effects against different pathogens, including bacteria, viruses, schistosom and fungus.

Black cumin seed in traditional medicine and in recent years for the treatment of microbial diseases has been used without any reported side effects. Therefore, this plant can provide a valuable agent for microbial diseases. However, additional studies are required to evaluate and explore the specific cellular and molecular mechanisms of the antimicrobial effects of N. sativa, alone or in combination with other drugs.

References