Export quality edible Nigella sativa seed oil, was procured from a local store. Pure thymoquinone, limonene, 2,2-diphenyl-1-picryl hydrazyl, 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid, brain extract type VII and 1,1,3,3-tetramethoxypropane were purchased from Sigma-Aldrich Inc., USA, while thiobarbituric acid, 2-deoxy-2-ribose, sodium nitroprusside, sulphanilamide, napthylene diamine dihydrochloride, potassium nitrite, triphenyl phosphine and butylated hydroxyl toluene were purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. Folin–Ciocalteu reagent was procured from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Sodium dodecyl sulfate was purchased from Bio-Rad Laboratories USA. Hemoglobin assay kit was purchased from Ranbaxy Diagnostics, New Delhi, India. Rat chow was procured from Ashirwad Industries, Chandigarh, India. Other chemicals and reagents used in this work were of analytical grade.
Methanolic extract (ME) and volatile oil (VO) fractions from Nigella sativa seed oil
ME from NS seed oil was basically extracted as reported in our previous experiment (Ahmad and Beg 2013a). In brief, 10 g of NS seed oil was added to 100 ml of pure methanol and then stirred for 100 min at ambient temperature. Then this was kept at 45°C till the methanolic layer has evaporated completely. On the other hand, a procedure of steam distillation was used for the extraction of VO from NS seed oil. The procedure for VO extraction was employed essentially in the same method as used by Kanter et al. (2006) with minor modification. First of all, 12 g of NS seed oil was taken in a distillation flask, and then 400 ml of distilled water was added to it. The temperature of distillation unit was set to boiling point. Then 150 ml of the distillate containing VO was carefully collected in a dark glass bottle. For extraction of VO from the distillate, 50 ml of diethyl ether was added to it, and anhydrous sodium sulphate was used to remove moisture present in the sample. Thus diethyl ether extract containing VO was evaporated by keeping it at 40°C. The resulting ME and VO residues were flushed with nitrogen and kept in dark colored bottles at 4°C.
Total phenolic contents (TPC) in NS oil, and its ME and VO
With slight modification, total phenolic contents of the NS seed oil and its ME and VO fractions were determined in triplicate by using the Folin–Ciocalteu reagent (Yu et al. 2002a). The reaction mixture contained several concentrations of NS oil, ME or VO extracts in DMSO-saline, 125 μl of the Folin–Ciocalteu reagent, and 375 μl of 20% sodium carbonate in a total volume of 2.5 ml. After 2 h of incubation at ambient temperature, the absorbance at 765 nm was measured. The TPC was calculated by using gallic acid as standard.
Free radical scavenging activities
2,2-Diphenyl-1-picryl hydrazyl (DPPH•) scavenging activity
With minor modification, 2,2-diphenyl-1-picryl hydrazyl scavenging activities of NS seed oil, ME, VO fractions, pure TQ and LMN were determined in triplicate by the method of Mensor et al. (2001). The reaction mixture contained several concentrations of NS seed oil, ME, VO fractions or pure TQ in methanol, whereas LMN was dissolved in Tween 80-PBS. The reaction was started by the addition of freshly prepared methanolic solution of DPPH• in a total volume of 3.5 ml, mixed thoroughly and allowed to react in dark at ambient temperature. After 30 min the absorbance of sample at 518 nm was read. The concentration dependency of the above antioxidant fractions and compounds was done by plotting the percent of DPPH• remaining against each level of antioxidants by using standard DPPH•. The percent antioxidant activity of the above antioxidants was calculated according to the following formula:
Hydroxyl radical (•OH) scavenging activity
The scavenging of OH radical for the test drugs was done by the method of Halliwell et al. (1987). Briefly, one ml of the reaction mixture contained 2.8 mM 2-deoxy-2-ribose dissolved in 200 mM of KHPO4–K2HPO4 buffer pH 7.4, and 20 μM FeCl3 and 0.104 mM EDTA (1:1 v/v), 1.0 mM H2O2, 1.0 mM ascorbic acid and several concentrations of NS seed oil, ME, VO, pure TQ or LMN dissolved in Tween 80-PBS. The reaction mixture was mixed and incubated for 1 h at 37°C, followed by the addition of 1.0 ml of 1% thiobarbituric acid prepared in 50 mM NaOH and 1.0 ml of 2.8% TCA. The samples were then boiled for 20 min at 100°C, cooled to room temperature and the absorbance was recorded at 532 nm against a reagent blank in a Beckman DU 640 spectrophotometer. A control blank without test fractions or compounds was used to determine the percentage inhibition of deoxyribose degradation by the above test fractions or compounds.
2,2-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) scavenging activity
With slight change, the concentration-dependent scavenging efficiencies of NS seed oil, ME, VO, TQ and LMN against ABTS•+ were evaluated by the method of Re et al. (1999), where ABTS radical cation was produced by mixing equal volumes of 7 mM ABTS and 2.45 mM potassium persulfate solutions and allowed them to react for 12 h at ambient temperature in the dark. The reaction mixture in triplicate contained several concentrations of NS seed oil, ME, VO or pure TQ dissolved in methanol, whereas LMN was dissolved in Tween 80 and 10 mM phosphate buffer saline pH 7.4 and 500 μl of 60-fold diluted methanolic solution of ABTS•+ in a total volume of 1.1 ml. The samples were mixed thoroughly and allowed to react in dark at room temperature. The absorbance was taken at 734 nm after 7 min against methanol or distilled water using the Beckman DU 640 spectrophotometer. A control blank lacking test fractions or compounds, was used to calculate their percent ABTS•+ scavenging capacities.
Nitric oxide (NO•) scavenging activity
Nitric oxide scavenging activities of several concentrations of NS seed oil, ME, VO, TQ and LMN were determined according to the procedure of Marcocci et al. (1994). When sodium nitroprusside is allowed to dissolve in aqueous medium nitric oxide is spontaneously generated from it at physiological pH which interacts with oxygen to produce nitrite ions that can be estimated by the use of Greiss reagent. Scavengers (antioxidants) of nitric oxide compete with oxygen leading to reduced production of nitric oxide. One ml of the reaction mixture contained 500 μl of various concentrations of the samples dissolved in DMSO-saline was mixed with 5 mM sodium nitroprusside prepared in 10 mM potassium phosphate buffer pH 7.4 then incubated at 25°C for 150 min. At the end of the incubation, the samples from the above were allowed to react with 1 ml of Greiss reagent containing equal volume of solutions A (2% sulfanilamide and 4% H3PO4) and B (0.2% naphthylethylenediamine dihydrochloride). The absorbance of the chromophore formed during the diazotization of nitrite with sulfanilamide and subsequent coupling with naphthylethylenediamine was read at 542 nm in the Beckman DU 640 spectrophotometer. Their percent NO• scavenging capacities were calculated by using standard potassium nitrite.
The concentration-dependent ferrous (Fe+2) chelating capacities of NS seed oil, ME and VO extracts were determined as described by Yu et al. (2002b). The reaction mixture contained 46.296 μM FeSO, 250 μl of different concentrations of the samples dissolved in 1% SDS, 3.7037 mM Tris–HCl buffer pH 7.4, 1.185 mM 2,2’-bipyridyl prepared in 37.037 mM hydrochloride and 106.592 mM hydroxylamine hydrochloride in the total volume 5.4 ml, was made by adding 2.5 ml of ethanol. A control blank without test samples was conducted in an identical manner. The absorbance of sample was read at 522 nm against distilled water and used to calculate Fe+2 chelating capacity using a standard curve prepared with EDTA.
Nonenzymatic lipid peroxidation
The inhibition of nonenzymatic lipid peroxidation in phospholipid liposomes, prepared from type VII Folch bovine brain extract, by four concentrations of ME, VO or TQ, was carried out essentially the same as previously reported method of Houghton et al. (1995). Phospholipid liposomes were prepared from brain extract Type VII from bovine brain by mixing with 200 mM of KHPO4–K2HPO4 buffer pH 7.4 (5 mg/mL) and stored at 4°C for 6 days. It was then sonicated under cooling with ice until a milky solution was obtained. One ml of the reaction mixture contained 500 μl of phopholipid suspension, 3 mM sodium phosphate buffer saline pH 7.4 containing different concentrations of the test samples dissolved in Tween 80 and PBS, 1 mM FeCl3 and 1 mM ascorbic acid to start peroxidation. The reaction mixture was mixed and incubated for 1 h at 37°C. At the end of incubation, 1.0 ml of 1% TBA prepared in 50 mM NaOH, 1.0 ml of 2.8% TCA and 0.1 ml of 2% BHT prepared in ethanol were added to the reaction mixture. The samples were boiled for 20 min at 100°C, cooled to room temperature then 2.5 ml of n-butanol was added. The reaction mixtures were then centrifuged at 3500 rpm for 5 min. The absorbance was recorded at 532 nm against a reagent blank in the Beckman DU 640 spectrophotometer. All reagents were prepared freshly. A control blank without test samples was conducted in an identical manner. The inhibition of malondialdehyde (MDA) formation by the test fractions and compound was represented as a percentage of the control (minus antioxidant) value.
Animals and treatments
Approval of this experimental study in animals was obtained from the Board of Studies of Biochemistry department and Ethics Committee of Jawahar Lal Nehru Medical College, A.M.U. Healthy male Wistar rats and their weights 180–210 g, from inbred colony maintained by the central animal facility of Jawahar Lal Nehru Medical College, were used. Standard rat chow and water were available for these animals ad libitum. The test fractions, 10% ME, 2% VO from NS seed oil, and the compounds 1% TQ and 20% LMN suspensions for the treatment of rats were prepared according to Kanter (2008); Altan et al. (2007); El Gazzar et al. (2006) with slight change by dissolving in DMSO (12.5% final concentration) and finally homogenized with saline. To induce hyperlipidemia, an atherogenic suspension consisted of (w/v) 0.5% cholesterol, 3% coconut oil and 0.25% cholic acid that is 5 mg cholesterol, 30 mg coconut oil and 2.5 mg cholic acid per ml was prepared by mixing in a Potter-Elvehjem homogenizer. Rats were randomly assigned into the following different treatment groups:
Normolipidemic control (NLP-C): This normal control group containing five albino rats was orally administered one ml of saline containing 12.5% DMSO twice per day.
Hyperlipidemic control (HLP-C): The four rats in hyperlipidemic control group were orally given 0.5 ml of saline containing 12.5% DMSO, before the administration of 0.5 ml of the above prepared atherogenic suspension twice per day, with no drug intervention.
Hyperlipidemic ME (HLP-ME): Before administration of atherogenic suspension, four rats in this treated group received one ml of the saline suspension containing 100 mg of ME orally in two equal doses (morning and evening) of 0.5 ml each, for 30 days.
Hyperlipidemic VO (HLP-VO): Before administration of atherogenic suspension, four rats in this treated group received one ml of the saline suspension containing 20 mg of VO orally in two equal doses (morning and evening) of 0.5 ml each, for 30 days.
Hyperlipidemic TQ (HLP-TQ): Before administration of atherogenic suspension, four rats in this treated group received one ml of the saline suspension containing 10 mg of TQ orally in two equal doses (morning and evening) of 0.5 ml each, for 30 days.
Hyperlipidemic LMN (HLP-LMN): Before administration of atherogenic suspension, four rats in this treated group received one ml of the saline suspension containing 200 mg of LMN orally in two equal doses (morning and evening) of 0.5 ml each, for 30 days.
Blood collection and erythrocyte preparation
At the end of treatment, blood was drawn from cardiac puncture of anaesthetized, and overnight fasted rats in each group and collected in heparinised tubes. And it was mixed gently by inversion 2–3 times and incubated at 4°C for 2–3 h. Centrifugation of blood was performed at 2,500 rpm for 30 min to separate plasma and buffy coat. The packed erythrocytes from blood thus, obtained were resuspended in physiological saline and centrifuged again at 1,500 rpm for 10 min at 4°C, and this procedure was done two times more. The procedure described by Lakshmi and Rajagopal (1998) was employed for hemolysate preparation from a portion of packed erythrocytes.
Preparation of liver homogenate
Livers from each rat were blotted. And the livers of each group were cut into small pieces. With the help of a waring blender, 10 g of wet tissue was homogenized with 90 ml of chilled 0.1 M sodium phosphate buffer, pH 7.4, containing 1.17% KCl. The homogenate was centrifuged at 1,000 rpm for 10 min at 4°C. A portion of the homogenate obtained from liver samples in each group was aliquoted and stored at −20°C for future use. Other necessary steps and precautions were taken for the sample preparation and its storage.
Determination of hydrogen peroxide-induced MDA release
For the determination of MDA released in intact erythrocytes, the procedure of Cynamon et al. (1985) was employed. On the other hand, the determination of basal MDA content in erythrocyte hydrolysate was performed according to the method of Stocks and Dormandy (1971). The concentrations of MDA in these samples were calculated by taking a standard malondialdehyde (Liu et al. 1982).
Determination of conjugated diene (CD), lipid hydroperoxide (LOOH) and MDA in liver
Lipid contents were extracted from liver, according to the method of Folch et al. (1957). In Beckman DU 640 spectrophotometer, the absorbance of lipid residues dissolved in 1.5 ml of cyclohexane was taken at 234 nm against a cyclohexane blank, and their CD concentration was determined by using a molar extinction coefficient of 2.52 × 104 M−1 cm−1. The method of Nourooz-Zadeh et al. (1996) for LOOH quantification from liver homogenates was used, and hydroperoxide contents were determined by using a molar absorption coefficient of 4.3 × 104 M−1 cm−1. The method of Ohkawa et al. (1979) was used for the determination of lipid peroxides in liver homogenate, and malondialdehyde was used to quantify MDA concentration (Liu et al. 1982).
The method of Bradford (1976) was used to quantify protein content present in liver homogenate by using bovine serum albumin as standard. Liver homogenates were first precipitated with 10% TCA, and then the protein pellets were dissolved in 0.5 N NaOH. Suitable aliquots of the animal samples then were used for protein determination.
For statistical analysis of the data, one way analysis of variance was used, then Tukey’s Kramer multiple comparison test was employed. For statistical determination, P values < 0.05 were considered as significant.
Note: In treated groups of animals, the efficacy of test samples was expressed in terms of reduction in percent or it may be called as percent restoration value in relation to reduction. It can be calculated using the below equation: