Parasitology ResearchFounded as Zeitschrift für Parasitenkunde

© Springer-Verlag 2010

10.1007/s00436-010-2204-4

Original Paper

Antimalarial and antioxidant activities of methanolic extract of Nigella sativa seeds (black cumin) in mice infected with Plasmodium yoelli nigeriensis

Valeelat O. Okeola1, Oluwatosin A. Adaramoye1, Chiaka M. Nneji2, 3, Catherine O. Falade2, 3, E. Olatunde Farombi1 and Olusegun G. Ademowo2, 3

(1)

Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria

(2)

Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria

(3)

Department of Pharmacology and Therapeutics, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Nigeria

Olusegun G. Ademowo

Email: ademowo_g@yahoo.com

Email: ogademowo@comui.edu.ng

Received: 27 August 2010Accepted: 27 November 2010Published online: 14 December 2010

Abstract

The antimalarial and antioxidant activities of methanolic extract of Nigella sativa seeds (MENS) were investigated against established malaria infection in vivo using Swiss albino mice. The antimalarial activity of the extract against Plasmodium yoelli nigeriensis (P. yoelli) was assessed using the Rane test procedure. Chloroquine (CQ)-treated group served as positive control. The extract, at a dose of 1.25 g/kg body weight significantly (p < 0.05) suppressed P. yoelli infection in the mice by 94%, while CQ, the reference drug, produced 86% suppression when compared to the untreated group after the fifth day of treatment. P. yoelli infection caused a significant (p  < 0.05) increase in the levels of red cell and hepatic malondialdehyde (MDA), an index of lipid peroxidation (LPO) in the mice. Serum and hepatic LPO levels were increased by 71% and 113%, respectively, in the untreated infected mice. Furthermore, P. yoelli infection caused a significant (p  < 0.05) decrease in the activities of superoxide dismutase, catalase, glutathione-S-transferase and the level of reduced glutathione in tissues of the mice. Treatment with MENS significantly (p < 0.05) attenuated the serum and hepatic MDA levels in P. yoelli-infected mice. In addition, MENS restored the activities of red cell antioxidant

enzymes in the infected mice to near normal. Moreover, MENS was found to be more effective than CQ in parasite clearance and, in the restoration of altered biochemical indices by P. yoelli infection. These results suggest that N. sativa seeds have strong antioxidant property and, may be a good phytotherapeutic agent against Plasmodium infection in malaria.

Introduction

Malaria still represents, in spite of enormous control efforts, one of the most important parasitic diseases in humans worldwide. It remains a major public health problem in Africa and is responsible for the annual death of two to four million people, most of whom are children (Butler et al. 1997; Pasvol 1998). Plasmodium falciparum, the most lethal etiological agent for human malaria, is becoming increasingly resistant to standard antimalarial drugs which necessitate a continuous effort to search for new drugs, particularly with novel modes of action (Muregi et al. 2003). However, many communities who live in endemic areas make use of local remedies from plants in their local environments to treat malaria. Plants have invariably been a rich source for new drugs, especially antimalarial drugs such as quinine and artemisinin, which were originally obtained from plants before there chemical structures are used as templates for the synthetic products in market (Gessler et al. 1994). It is estimated that 122 drugs from 94 plant species have been discovered through ethnobotanical leads (Fabricant and Farnsworth 2001). Quite a number of studies have been undertaken to evaluate the in vivo antimalarial properties of several plants (Ajaiyeoba et al. 2006). Therefore, scientific investigation of plants used in traditional herbal remedies for diseases will contribute to both knowledge and a healthy society.

Nigella sativa L. (NS) (black cumin), commonly known as black seed belongs to the Ranunculaceae family. This seed has been used in many of the Middle East and Far East countries as natural remedy. NS has been used for the treatment of asthma (El-Tahir et al. 1993) and as an anti-tumour agent (El-Daly 1998), bactericide (Mouhajir et al. 1999), anticestode and antinematode (Mahmoud et al. 2002), anti-inflammatory (Mutabagani and El-Mahdy 1997), analgesic (Khanna et al. 1993), anti-diabetic (El-Shabrawy and Nada 1996), antiulcerogenic (El-Dakhakhny et al. 2000) and diuretic (Zaoui et al. 2000), lactogogue and vermifuge. In folk medicine, NS is used to treat cough, bronchitis, headache, rheumatism, fever, eczema and dyslipidemia (Zaoui et al. 2002). Furthermore, the black seeds are important as a carminative and spice and, they are used as a condiment in bread and other dishes (Burits and Bucar 2000).

In our effort to find new plants with antimalarial activity, we have studied the possible in vivo antimalarial and antioxidant activities of methanolic extracts of defatted N. sativa seeds in mice infected with Plasmodium yoelli nigeriensis.

Materials and methods

Chemicals

Chloroquine diphosphate, glutathione, thiobarbituric acid, trichloroacetic acid, 5,5 -dithio-bis (2-′nitrobenzoic acid), sulphosalicyclic acid, 1-chloro-2,4-dinitrobenzene (CDNB) were purchased from SIGMA Chemical Co. USA. Potassium chloride (KCl) and dihydrogen phosphate were purchased from BDH Chemical Ltd., England. All other reagents were of analytical grade and purest quality available.

Preparation of plant extract

Dried black seed of N. sativa was purchased at Agbowo market, Ibadan, Nigeria and were identified at the herbarium of the Forestry Research Institute of Nigeria, Ibadan. The N. sativa seed was extracted according to the method described by Al-Naggar et al. (2003). Briefly, the powdered seed (1 kg) was defatted with n-hexane and, the defatted seed was extracted in methanol using soxhlet apparatus. The methanolic extract was evaporated to dryness in vacuum with a rotatory evaporator to obtain the methanolic extract of defatted N. sativa (MENS). Stock solution of MENS was prepared with corn oil and administered to experimental animals according to their body weights.

Animals and parasite

Animals (adult Swiss albino mice) were obtained from the animal house of the Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, Nigeria. The animals were housed in well-aerated plastic cages, fed with standard mouse cubes (Ladokun feeds, Nigeria, Ltd.) and supplied with clean drinking water ad libitum. P. yoelii nigeriensis, used in this study was a donation to the laboratory of one of us (OGA) by Malaria Research and Reference Reagent Resource Centre. The parasites were maintained in animals by serial passages of blood collected from a patent donor mouse to a naïve recipient.

Study of the course of infection and antimalarial activity

The course of infection following intraperitoneal inoculation in mice was studied in each experimental mouse that received 107 parasitized red blood cells in 0.2-ml inoculum. Thin blood films were prepared from the tail vein of infected mice, fixed with methanol and stained with 10% Giemsa stain for 30 min and then rinsed with tap water. Parasitemia was monitored daily using ×100 objective of a light microscope. In vivo antimalarial activity against P. yoelli infection in mice was done according to Rane's test as described by Elufioye and Agbedahunsi (2004). The Rane's test relies on the ability of a standard inoculum of P. yoelli to kill the recipient mouse within 12 days of inoculation. Extension of survival beyond 12 days is regarded as activity.

Study design

Mice weighing between 20 and 25 g were distributed into five groups of ten animals each. Three groups of animals were infected intraperitoneally with standard inoculum (107 P. yoelii-infected parasitized red blood cells) and treated with MENS, chloroquine (CQ) and corn oil (Vehicle, negative control group). Two other groups were uninfected mice that were treated separately with MENS and corn oil. MENS was administered at a dose of 1.25 g/kg body weight (Al-Naggar et al. 2003), while CQ was given at dose of 10 mg/kg body weight (Ogunbayo et al. 2006) for the first 2 days and, at a dose of 5 mg/kg body weight for the last 3 days. Both MENS and CQ were given once a day for five consecutive days after infection was established. The control animals received equivolume of corn oil (vehicle) and, all administration was by oral gavage.

The mice were sacrificed 24 h after the administration of last dose of the drugs. All experiments were conducted without anaethesia and the protocol conforms to the guidelines of the National Institute of Health (NIH publication 85-23, 1985) for laboratory animal care and use. Whole blood from each animal was collected into heparinized bottles, stored at 4°C and, the red cell was used to assay for glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities, while the serum was used to determine the extent of lipid peroxidation. Liver was excised and rinsed in ice-

cold 1.15% KCl, dried and weighed. The liver samples were homogenised in four volumes of 0.1 M phosphate buffer, pH 7.4 using a Potter Elvehjem homogenizer and centrifuged at 10,000×g for 15 min to obtain postmitochondrial supernatant fraction, which was used to assay for the levels of lipid peroxidation (LPO), GSH, SOD, CAT and glutathione-S-transferase (GST).

Biochemical assays

The rate of lipid peroxidation was assessed by measuring the red cell and hepatic malondialdehyde (MDA) levels using the method of Varshney and Kale (1990). GST activity was determined by the method of Habig et al. (1974), the method is based on the rate of conjugate formation between GSH and CDNB. Reduced GSH level was assayed by measuring the rate of formation of chromphoric product in a reaction between DTNB and free sulphydryl groups (such as reduced glutathione) at 412 nm as described by Jollow et al. (1974). SOD was measured by the nitro blue tetrazolium reduction method of McCord and Fridovich (1969). CAT activity was assayed by measuring the rate of decomposition of hydrogen peroxide at 240 nm as described by Aebi (1974). Protein was estimated using the Biuret method as described by Gornall et al. (1949).

Statistical analysis

The results were expressed as mean ± standard deviation (SD) of ten mice per group. Data were analysed using one-way analysis of variance (ANOVA) followed by post hoc Duncan's multiple range test for analysis of biochemical data using SPSS (12.0) statistical software. Values were considered statistically significant at p < 0.05.

Results

A progressive increase in average percentage parasitaemia was observed in P. yoelii-infected mice, with a maximum of 36% average parasitaemia by day 5 (post infection). However, the results showed that MENS and CQ were able to suppress parasitaemia considerably by 85% and 81%, respectively, at day 6 (post infection). Maximum suppression of 94% was obtained in MENS treated group at day 8 (post infection) (Table 1). This implies that MENS has slightly better antimalarial effect than CQ in the P. yoelii-infected mice. This observation was corroborated by 100% survival of mice in MENS-treated group against 90% in CQ-treated group (data not shown). A significant (p < 0.05) increase in the serum and hepatic MDA levels (lipid peroxidation index) was observed in P. yoelii-infected mice as parasitemia increased. Precisely, serum and hepatic MDA were elevated by 71% and 113% in infected mice when compared to control (Fig. 1 and Table 2). Furthermore, the red cell and hepatic enzymic and non-enzymic antioxidant indices, SOD, CAT, GST and GSH levels were significantly (p < 0.05) decreased with increase in parasitemia in untreated malaria group when compared to normal (Table 2 and Figs. 2, 3 and 4). Specifically, P. yoelii infection decreased red cell SOD, CAT and GSH levels by 46%, 67% and 74%, respectively, while hepatic SOD, CAT, GSH and GST were decreased by 40%, 63%, 69% and 64%, respectively, relative to normal. MENS administration significantly (p < 0.05) decreased the red cell and hepatic MDA levels of P. yoelii-infected mice (Fig. 1 and Table 2). In addition, administration of MENS significantly (p < 0.05) ameliorated the activities of red cell SOD and CAT as well as the hepatic SOD and GST of P. yoelii-infected mice (Table 2 and Figs. 2 and 3). Furthermore, MENS treatment significantly (p < 0.05) augmented the levels of both red cell and hepatic GSH of P. yoelii-infected mice (Table 2 and Fig. 2). However, MENS produced no significant (p > 0.05) effect on the level of hepatic CAT inhibited by P. yoelii infection (Fig. 4).

Table 1

Effect of methanolic extract of N. sativa (MENS) and chloroquine (CQ) on established malaria infection in mice

Treatment

% Parasitaemia/days of treatment

3 4 5 6 7 8

%P %S %P %S %P %S %P %S %P %S %P %S

UI 13 ± 0.1 0.0 16 ± 0.4 0.0 36 ± 0.4 0.0 65 ± 0.6 0.0 74 ± 0.7 0.0 83 ± 0.8 0.0

MENS + M 11 ± 0.2 15.4 21 ± 0.6 −31.3 26 ± 0.6 27.8 10 ± 0.3a 84.6a 8 ± 0.2a 89.2a 5 ± 0.6a 94a

CQ + M 12 ± 0.2 7.7 25 ± 0.6 −56.3 35 ± 0.3 2.8 12 ± 0.2 81.5 12 ± 0.6 83.8 12 ± 0.2 85.5

Values are reported as mean ± S.D. (n = 10)

Samples on day 3 were collected before commencement of treatment

%P, percentage parasitaemia;%S, percentage suppression; UI, untreated infected mice; MENS + M, infected mice treated with N. sativa; CQ + M, infected mice treated with chloroquine

aSignificantly different from corresponding values in days 3, 4 and 5 (p < 0.05)

Fig. 1

Effect of methanolic extract of N. sativa (MENS) on the level of postmitochondrial fraction (PMF) lipid peroxidation (LPO) of Plasmodium yoelli-infected mice. Single asterisk, significantly different from normal (p < 0.05); N, Normal (uninfected mice); UI, untreated infected mice; NNS, normal mice treated with N. sativa; MENS + M, Plasmodium yoelli-infected mice treated with N. sativa; CQ + M, Plasmodium yoelli-infected mice treated with chloroquine

Table 2

Effect of methanolic extract of N. sativa (MENS) and chloroquine (CQ) on red cell antioxidant profiles of Plasmodium yoelli-infected mice

GroupingRed cell

Serum LPO (nmol/mg protein)GSH (μg/ml) SOD (U/mg protein) CAT (U/mg protein)

N 0.53 ± 0.07 13.6 ± 4.6 0.81 ± 0.24 22.5 ± 7.0

UI 0.14 ± 0.05a 7.4 ± 2.2a 0.27 ± 0.07a 38.4 ± 6.3a

NNS 0.56 ± 0.11 12.1 ± 3.7 0.73 ± 0.19 19.3 ± 7.6

MENS + M 0.39 ± 0.09 11.6 ± 2.9 0.67 ± 0.20 28.4 ± 5.5

CQ + M 0.17 ± 0.06a 6.5 ± 1.8a 0.34 ± 0.06a 43.0 ± 9.1a

Values are reported as mean ± S.D. (n = 10)

N, normal (uninfected mice); UI, untreated infected mice; NNS, normal mice treated with N. sativa; MENS + M, Plasmodium yoelli-infected mice treated with N. sativa; CQ + M, Plasmodium yoelli-infected mice treated with chloroquine

aSignificantly different from normal (p < 0.05)

Fig. 2

Effect of methanolic extract of N. sativa (MENS) on the levels of postmitochondrial fraction (PMF), reduced glutathione (GSH) and glutathione-S-transferase (GST) of Plasmodium yoelli-infected mice. Single asterisk, significantly different from normal (p < 0.05). N, normal (uninfected mice); UI, untreated infected mice; NNS, normal mice treated with N. sativa; MENS + M, Plasmodium yoelli-

infected mice treated with N. sativa; CQ + M, Plasmodium yoelli-infected mice treated with chloroquine

Fig. 3

Effect of methanolic extract of N. sativa (MENS) on the activity of postmitochondrial fraction (PMF), superoxide dismutase (SOD) of Plasmodium yoelli-infected mice. Single asterisk, significantly different from normal (p < 0.05). N, normal (uninfected mice); UI, untreated infected mice; NNS, normal mice treated with N. sativa; MENS + M, Plasmodium yoelli-infected mice treated with N. sativa; CQ + M, Plasmodium yoelli-infected mice treated with chloroquine

Fig. 4

Effect of methanolic extract of N. sativa (MENS) on the activity of postmitochondrial fraction (PMF) catalase (CAT) of Plasmodium yoelli-infected mice. Single asterisk, significantly different from normal (p < 0.05); N, normal (uninfected mice); UI, untreated infected mice; NNS, normal mice treated with N. sativa, MENS + M, Plasmodium yoelli-infected mice treated with N. sativa; CQ + M; Plasmodium yoelli-infected mice treated with chloroquine

Discussion

The present study focused on the following issues: (a) can N. sativa (NS) counter oxidative stress induced by P. yoelli infection? and (b) can co-administration of N. sativa decrease or inhibit P. yoelli growth assessed by changes in percentage parasitaemia in the red cell of mice? Thus, we investigated these issues in mice to detect the protective role of methanolic extract of N. sativa seeds (MENS) and, evaluated the P. yoelli-induced oxidative stress using biochemical markers (activities of SOD, CAT and GST, as well as GSH and MDA levels).

The results of our study showed that treatment with MENS reduced the subsequent P. yoelli-induced oxidative stress demonstrated by decrease in lipid peroxidation (reduced MDA), improvement in antioxidant enzyme status and increase in detoxifying ability (GST) of the hepatocytes. Also, the in vivo antimalarial activity of MENS on established P. yoelii infection showed that it suppressed the growth of the parasites by 94%, against 85% obtained in CQ-treated group. This result confirms the antimalarial activity of MENS in the mice.

Clinical and experimental data suggest that reactive oxygen species (ROS), such as superoxide radical and hydrogen peroxide, which do not cause any damage under physiological conditions, but at high concentrations can cause cellular damages are produced during Plasmodium infection (Farombi et al. 2003). In fact, it has been documented that in Plasmodium infection excessive production of free radicals or oxidants is possible and may impair antioxidant system. (Rodrigues and Gamboa 2009). Therefore, the observed elevation in MDA values of infected mice in this study is in concordance with those findings (Farombi et al. 2003; Rodrigues and Gamboa 2009). Increased MDA implies increase in ROS levels, which are cellular renegades, and can wreak havoc in biological systems by tissue damage, altering biochemical compounds, corroding cell membranes and killing out rightly (Wiseman and Halliwell 1996). This claim was further supported by decrease in red cell and hepatic activities of SOD and CAT, and hepatic GST of infected mice, which indicate that excess ROS probably inactivate these antioxidant enzymes. This observation is in line with the findings of Areekul and Boonme (1986) that linked the reduced activity of catalase in P. berghei infection to excessive ROS generation. We assessed the effect of MENS on lipid peroxidation and antioxidant enzymes of the infected mice. Administration of MENS reversed the increase in MDA levels to a considerable extent when compared to untreated infected mice, and also increased the activities of red cell SOD and CAT as well as hepatic SOD of infected mice, thereby confirming the antioxidant role of MENS in Plasmodium infection. The glutathione cycle represents one of the major systems for avoiding the deleterious effects of free radicals. Plasmodium infection has been associated with GSH decrease, which may reflect the consumption of GSH by the overproduction of ROS or increase in oxidised form of GSH, which could lead to inadequate antioxidative function of red cells (Lüersen et al. 2000). In the present study, we observed significant decrease in the levels of red cell and hepatic GSH of infected mice. Similarly, significant decrease in the activity of hepatic GST was observed in the infected mice. Our results showed that MENS significantly augmented the Plasmodium yoelli-induced decrease in hepatic GST activity as well as the depletion of GSH. These results further confirm antioxidant activity of MENS in Plasmodium-infected mice. Previous experimental investigations have supported the antioxidant role of N. sativa under different conditions. For example, this plant was shown to protect against oxidative damage in isolated rat hepatocytes (Daba and Abdel-Rahman 1998). It was reported that fixed oil of this plant inhibited membrane lipid peroxidation and eicosanoid generation in leukocytes (Houghton et al. 1995). Also, Türkdoğan et al.

(2003) observed the therapeutic effectiveness of this plant in the prevention of liver fibrosis and cirrhosis. They suggested that hepatoprotective property of N. sativa may be through immunmodulatory and antioxidant activities. The antioxidant activity of N. sativa extract may be due to the presence of active compounds among which are thymoquinone, alkaloids and saponins (Lautenbacher 1997), which has been linked to the wide range of pharmacological activities of this plant (Daba and Abdel-Rahman 1998; Al-Naggar et al. 2003).

In conclusion, the present study suggests that N. sativa has antimalarial property with a mechanism of action based on the amelioration of oxidative status in red cell and hepatocytes of infected mice. Therefore, N. sativa could be a candidate for consideration as a new antimalarial agent. Further studies should explore N. sativa as a prototype for an antimalarial aimed at the P. falciparum chloroquine-resistant parasites which are rather frequent worldwide.

References

Aebi H (1974) Methods of enzymatic analysis. In: Bergmeyer HV (ed) catalase estimation. Verlag Chemic, New York, pp 673–684

Ajaiyeoba EO, Abiodun OO, Falade MO, Ogbole NO, Ashidi JS, Happi CT, Akinboye DO (2006) In vitro cytotoxicity studies of 20 plants used in Nigerian antimalarial ethnomedicine. Phytomedicine 13:295–298PubMed CrossRef

Al-Naggar TB, Gomez-Serranillos MP, Carretero ME, Villar AM (2003) Neuropharmacological activity of Nigella Sativa L. extracts. J Ethnopharmacol 88:63–68PubMed CrossRef

Areekul S, Boonme Y (1986) Catalase activity in red cell and liver of mice infected with Plasmodium berghei. Southeast Asian J Trop Med Public Health 17:48–52PubMed

Burits M, Bucar F (2000) Antioxidant activity of Nigella sativa L. essential oil. Phytother Res 14:323–328PubMed CrossRef

Butler D, Maurice J, O'Brien C (1997) Time to put malaria control on the global agenda. Nature 386:535–536PubMed CrossRef

Daba MH, Abdel-Rahman MS (1998) Hepatoprotective activity of thymoquinone in isolated rat hepatocytes. Toxicol Lett 95:23–29PubMed CrossRef

El-Dakhakhny M, Barakat M, Abd-El-Halim M, Aly SM (2000) Effects of Nigella sativa oil on gastric secretion and ethanol induced ulcer in rats. J Ethnopharmacol 72:299–304PubMed CrossRef

El-Daly ES (1998) Protective effect of cysteine and Vitamin E, Crocus sativus and Nigella sativa extracts on cisplatin-induced toxicity in rats. J Pharm Belg 53:87–95PubMed

El-Shabrawy OA, Nada SA (1996) Biological evaluation of multicomponent tea used as hypoglycemic in rats. Fitoterapia 67:99–102

El-Tahir KEH, Ashour MMS, Al-Harbi MM (1993) The respiratory effects of the volatile oil of the black seed (Nigella sativa) in guinea-pigs: elucidation of the mechanism(s) of action. Gen Pharmacol 24:1115–1122PubMed

Elufioye TO, Agbedahunsi JM (2004) Antimalarial activities of Tithonia diversifolia (Asteraceae) and Crossopteryx febrifuga (Rubiaceae) on mice in vivo. J Ethnopharmacol 93:167–171PubMed CrossRef

Fabricant DS, Farnsworth NR (2001) The value of plants used in traditional medicine for drug discovery. Environ Health Perspect 109:69–75PubMed CrossRef

Farombi EO, Shyntum YY, Emerole GO (2003) Influence of chloroquine treatment and Plasmodium falciparum malaria infection on some enzymatic and non-enzymatic antioxidant defense indices in humans. Drug Chem Toxicol 26:59–71PubMed CrossRef

Gessler MC, Nkunya MH, Mwasumbi LB, Heinrich M, Tanner M (1994) Screening Tanzanian medicinal plants for antimalarial activity. Acta Trop 56:65–77PubMed CrossRef

Gornall AG, Bardawill CJ, David MM (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751–766PubMed

Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione-S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem 249:7130–7139PubMed

Houghton PJ, Zarka R, Heras B, Hoult RS (1995) Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Med 61:33–36PubMed CrossRef

Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR (1974) Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3, 4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacol 12:251–271CrossRef

Khanna T, Zaidi FA, Dandiya PC (1993) CNS and analgesic studies on Nigella sativa. Fitoterapia 64:407–410

Lautenbacher LM (1997) Schwarzkümmelöl. Dtsch Apoth Ztg 137:68–69

Lüersen K, Walter RD, Müller S (2000) Plasmodium falciparum infected red blood cells depend on a functional glutathione de novo synthesis attributable to an enhanced loss of glutathione. Biochem J 346:545–552PubMed CrossRef

Mahmoud MR, El-Abhar HS, Saleh S (2002) The effect of Nigella sativa oil against the liver damage induced by Schistosoma mansoni infection in mice. J Ethnopharmacol 79:1–11PubMed CrossRef

McCord JM, Fridovich I (1969) Superoxide dismutase, an enzymatic function for erythrocuperin. J Biol Chem 244:6049–6055PubMed

Mouhajir F, Pedersen JA, Rejdali M, Towers GHN (1999) Antimicrobial thymohydroquinones of Moroccan Nigella sativa seeds detected by electron spin resonance. Pharm Biol 37:391–395CrossRef

Muregi FW, Chhabra SC, Njagi EN, Langàt-Thoruwa CC, Njue WM, Orago AS, Omar SA, Ndiege IO (2003) In vitro antiplasmodial activity of some plants used in Kisii, Kenya against malaria and their chloroquine potentiation effects. J Ethnopharmacol 84:235–239PubMed CrossRef

Mutabagani A, El-Mahdy SAM (1997) A study of the anti-inflammatory activity of Nigella sativa L. and thymoquinone in rats. Saudi Pharm J 5:110–113

Ogunbayo OA, Adisa RA, Ademowo OG, Olorunsogo OO (2006) Incidence of chloroquine induced oxidative stress in the blood of rabbit. Intl J Pharmacol 2:121–125CrossRef

Pasvol G (1998) The treatment of falciparum malaria in African children. Afr Health 20:19–20PubMed

Rodrigues JR, Gamboa ND (2009) Effect of dequalinium on the oxidative stress in Plasmodium berghei-infected erythrocytes. Parasitol Res 104:1491–1496PubMed CrossRef

Türkdoğan MK, Ozbek H, Yener Z, Tuncer I, Uygan I, Ceylan E (2003) The role of Urtica dioica and Nigella sativa in the prevention of carbon tetrachloride-induced hepatotoxicity in rats. Phytother Res 17:942–946

Varshney R, Kale RK (1990) Effects of calmodulin antagonists on radiation-induced lipid peroxidation in microsomes. Int J Radiat Biol 58:733–743PubMed CrossRef

Wiseman H, Halliwell B (1996) Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J 313:17–29PubMed

Zaoui A, Cherrah Y, Lacaille-Dubois MA, Settaf A, Amarouch H, Hassar M (2000) Diuretic and hypotensive effects of Nigella sativa in the spontaneously hypertensive rat. Therapie 55:379–382PubMed

Zaoui A, Cherrah Y, Alaoui K, Mahassine N, Amarouch H, Hassar M (2002) Effects of Nigella sativa fixed oil on blood homeostasis in rat. J Ethnopharmacol 79:23–26PubMed CrossRef