ANTIADIPOGENIC, FREE RADICAL SCAVENGING AND ANTI-INFLAMMATORY ACTIVITY OF NIGELLA SATIVA SEED FRACTIONS

ANTIOXIDANT, FREE RADICAL SCAVENGING AND ANTI-INFLAMMATORY ACTIVITY OF NIGELLA SATIVA SEED FRACTIONS

Ramineedu Keerthi, Reddy Sankaran Karunakaran and Meriga Balaji

Department of Biochemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.

ABSTRACT: Obesity and overweight have recently drawn greater attention due to their central role in the rapid increase of metabolic syndrome disorders. The research pursuit for exploring novel and effective therapeutic alternatives to deal with obesity ailments and oxidative stress has received immense interest. In this study, different solvent fractions of Nigella sativa (N. sativa; Black cumin) seeds were prepared, and their phytochemical analysis was done. Among different solvent fractions, the methanolic fraction of N. sativa (MFNS) contained a high level of polyphenols (451.77 ± 0.91 μg/mL) and flavonoids (401.24 ± 0.73 μg/mL). The GC-MS analysis of MFNS showed the presence of several glycosides, oils, and polyphenolic compounds, as mentioned in the results section. The effect of N. sativa seed fractions on inhibition of key digestive enzymes linked to adiposity such as α-amylase, α-glucosidase and pancreatic lipase was evaluated. The free radical scavenging activity of seed fractions was studied through standard antioxidant assays (DPPH and NO). The enzyme inhibitory effect of MFNS for α-amylase, α-glucosidase and pancreatic lipase was81.47%, 77.12%, and 84.26%, respectively, which were much higher than the rest of the N. sativa fractions. The MFNS at 250 μg/mL demonstrated considerable anti-inflammatory activity on RAW 264.7 macrophages, in terms of inhibition of LPS-induced nitric oxide production (33.02%) and membrane-stabilizing activity (84.26%). In conclusion, our data suggest that MFNS of N. sativa seeds is rich in several bioactive phytochemicals that qualify it as an effective therapeutic alternative to attenuate free radical stress, inflammation, and obesity ailments.

Keywords: Antioxidant, α-amylase, α-glucosidase, Nigella sativa, Pancreatic lipase

INTRODUCTION: Excess energy intake and a sedentary lifestyle are the primary causes of the growing incidence of obesity and associated ailments, generally referred to as “New world syndrome”. New world syndrome is similar to metabolic syndrome, including obesity, hypertension, type 2 diabetes, cardiovascular diseases (CVDs), etc. ¹.

Overweight and obesity lead to low-grade inflammation, oxidative stress, hormonal imbalances and certain forms of cancers ². Despite enormous global demand for anti-obesity drugs, very few drugs and formulations in the market are approved by the foods and drug administration (FDA) / European Medical Union (EMU). Some of the approved anti-obesity drugs like Sibutramine were later withdrawn due to considerable side effects ³.

As a result, there is a growing public inclination to favor natural product-based medications⁴. Therefore, the scientific community is exploring developing safe and cost-effective drugs/molecules from natural products ⁵.

In this context, the role of plant-based phytochemicals and, more particularly, nutraceutical-based products gained significance ⁶. Previous studies on functional foods highlighted their beneficial role in mitigating oxidative stress and other metabolic complications ⁷. Indian and Chinese traditional systems of medicine have emphasized the therapeutic effects of several plants and herbs in curing many human and animal diseases ⁸. Decades of scientific research have also reported the medicinal claim of certain plant-based natural and healthier alternatives to treat some metabolic disorders ⁹. Nigella sativa (N. sativa) is a member of the Ranunculaceae family and a widely used medicinal plant worldwide. Nigella seeds and oil have a long history of usage in various recipes and traditional systems of medicines. The seeds of N. sativa have been widely used to treat different diseases and ailments ¹⁰. Given the above, the present study was focused on preparing different solvent fractions of N. sativa seeds and evaluating their free radical scavenging efficacy, inhibitory activity on key digestive enzymes, and anti-inflammatory activity.

MATERIALS AND METHODS:

Chemicals and Reagents: Gries reagent, 2,2- diphenyl-2-picrylhydrazyl radical (DPPH), (2,2'- azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS), ascorbic acid, pancreatic α-amylase, pancreatic lipase, α-glucosidase, acarbose, 3,5-dinitrosalicylic acid (DNS) color reagent, orlistat, p-NPB (P-nitro phenyl butyrate), isopropyl alcohol, gallic acid, and quercetin were obtained from Sigma Aldrich, Bangalore. Folin-Ciocalteu’s phenol reagent and other chemicals used in this study were of analytical grade.

Plant Material Collection and Preparation of Fractions: Nigella sativa seeds were collected from the local market, dried, pulverized, and soaked in maceration chambers with different solvents (Hexane, Ethyl acetate, Hydroalcoholic, Methanolic, and Aqueous) at room temperature (RT). Crude solvent fractions of N. Sativa were filtered and concentrated in Rota-evaporator (Heidolf). The dark brown sticky material was collected and fractionated using silica gel in column chromatography with different eluting solvents. Preliminary phytochemical analysis of hexane, ethyl acetate, methanol, and aqueous fractions was done using the standard methods of Harborne ¹¹. Based on TLC and phytochemical analysis, the Methanolic fraction of Nigella sativa (MFNS) was used for GCMS/HRMS and other experiments.

Determination of Total Phenolic Content: Total phenolic compounds present in N. sativa seeds was estimated by using the Folin-Ciocalteu method ¹². Briefly, the sample solution (0.6 mL) at different concentrations (ranging from 100 to 500 µg/mL) was mixed with 2.58 mL of Folin-Ciocalteu’s phenol reagent. After 3 min, 0.3 mL of a saturated sodium carbonate solution was added to the mixture. The reaction mixtures were incubated at room temperature (25°C) for 20 min, and the absorbance was measured at 760 nm with a spectrophotometer. A dose-response linear regression curve was generated using the gallic acid standard absorbance. The concentration of phenolic compounds was expressed as gallic acid equivalents (mg of GAE/gm of a fraction). The estimation was performed in triplicate, and the results are expressed as mean ± SD.

Determination of Total Flavonoid Content: Total flavonoid content was estimated by the aluminium chloride method ¹². N. sativa seed fraction (0.5 µL) was mixed with 2.5 mL of distilled water and 150 μL NaNO2 solution (5 %). The contents were vortexed for a few seconds and kept at room temperature for 5 min. Then, 300 μL AlCl₃ (10 %), 1 mL NaOH (1 mM) and 550 μL of distilled water were added. The solution was mixed well and kept for 15 min. The absorbance of the sample was measured at 510 nm. The total flavonoid content was calculated using the quercetin standardization curve. The results were expressed as mg of quercetin equivalent (QE) per gram of sample.

Free Radical Scavenging Assays:

DPPH Assay: Free radicals scavenging activity of N. sativa fraction against 2,2-Diphenyl-1-picryl hydroxyl radical (DPPH) was determined spectrophotometrically at 517 nm according to the procedure of Baliyan¹³ with some modifications using vitamin C as a standard. Briefly, a 0.3 mM solution of DPPH was prepared in methanol, and 500 µL of this solution was added to 1 mL of the fraction (dissolved in 10% DMSO) at different concentrations (100-500 µg/mL).

These solutions were mixed and incubated in the dark for 30 min at room temperature. The free radical scavenging activity was calculated using the formula.

Absorbance = Control – Test × 100 / Control

Nitric Oxide (NO) Radical Scavenging Activity: Sodium nitroprusside (5 µM) in phosphate buffer (pH 7.4) was mixed with different concentrations (100, and 500 µg/mL) of N. sativa seed fractions and the tubes were incubated at 25 ºC for 120 min ¹⁴. A control experiment was conducted with an equal amount of solvent in an identical manner. At 5 min intervals, 0.5 mL of incubation solution was taken and diluted with 0.5 mL of Griess reagent (1% sulfanilamide, 0.1% N-naphthyl ethylenediamine dihydrochloride and 2% o-phosphoric acid dissolved in distilled water). The chromophore's absorbance formed during diazotization of nitrite with sulfanilamide and resultant N-naphthyl ethylenediamine dihydrochloride was read at 546 nm. Nitric oxide scavenging activity was calculated by the following equation.

Absorbance = Control – Test × 100 / Control

Key Digestive Enzyme Inhibition Assays:

In-vitro Pancreatic Lipase Assay: The ability of MFNS to inhibit the pancreatic lipase (PL) was measured using p-NPB (P-nitro phenylbutyrate) as the substrate following the procedure described by Duarte ¹⁵. An enzyme buffer was prepared by the addition of 30 µL (10 units) of PL in 10 mM MOPS and [1 mM EDTA, pH 6.8], 850 µL of Tris-buffer (100 mM Tris HCl and 5 mM CaCl₂, pH7.0). 100 µL of the test sample or orlistat was mixed with 880 µL of the enzyme buffer and incubated for 15 min at 37°C. A 20 µL of substrate solution (10 mM p-NPB) was added to the reaction. The enzymatic reaction was allowed to incubate for 15 min at 37°C. The lipase activity was determined by measuring the hydrolysis of P-NPB to P-nitro phenol at 405 nm using an ELISA reader. The inhibition of enzyme activity was calculated as follows

% Lipase inhibition = 1 (Absorbance of sample × 100) / Control

In-vitro α-Amylase Assay: The α-amylase activity was determined according to the method described by Karunakaranet al ¹⁶ with slight modifications. Briefly, 40 µL of α-amylase (5 U/mL) was mixed with 0.36 µL of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) and 0.2 µL of MFNS or acarbose. After incubation for 20 min at 37 °C, 300 µL of starch solution (1%) in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) was added, the mixture was re-incubated for 20 min, followed by the addition of 0.2 mL dinitrosalicylic acid (DNS). The contents were mixed well and kept in a boiling water bath for 5 min. The reaction mixture was diluted by adding 6 mL distilled water, and the absorbance was measured at 540 nm in a UV-Visible spectrophotometer. Acarbose was used as a positive control. All assays were carried out in triplicate.

% α - Amylase inhibition = 1 (Absorbance of sample × 100) / Control

In-vitro α-Glucosidase Assay: The α-glucosidase inhibitory activity of MFNS was determined according to the method described by Etsassala et al. ¹⁷, with slight modifications. Briefly, 250 µL of MFNSor acarbose at different concentrations (100-500 µg/mL) was incubated with 500 µL of 1.0 U/mL α-glucosidase solution in 100 mM phosphate buffer (pH 6.8) at 37 °C for 15 min. Thereafter, 250 µl of p-nitrophenyl D-glucoside solution (5 mM) in 100 mM phosphate buffer (pH 6.8) was added, and the mixture was further incubated at 37°C for 20 min. The absorbance of the released p-nitrophenol was measured at 405 nm, and the inhibitory activity was expressed as a percentage of the control without inhibitor. All assays were carried out in triplicate, and the calculation was done according to the following formula.

% α - Glucosidase inhibition = 1 (Absorbance of sample × 100) / Control

GC-MS/HRMS Analysis: Methanolic Nigella sativa (MFNS) fractions were analyzed using GC-MS (Gas Chromatography-Mass Spectroscopy) ¹⁸. The analysis was conducted with a JEOL GC MATE II, GC coupled with a quadruple double-focusing mass analyzer, and a photon multiplier tube detector was used HP 5 ms Column was used (30 m-0.25 mm internal diameter, 0.25 mm film thickness). The ultrapure helium was used as a carrier gas at a 1 mL/min flow rate and a linear velocity of 37 cm/s. The injector temperature was set at 250°C.

The initial oven temperature was 50°C, which was ramped up to 250°C at a rate of 10°C/min with a hold time of 3 min. Injections of 1mL sample were made in the split less mode. The mass spectrometer was operated in the electron ionization mode at 70 eV with the electron multiplier voltage at 1859 V. Other MS operating parameters were as follows: ion source temperature 230°C, quadrupole temperature 250°C, solvent delay 4 min, and scan range 50 to 600 amu. Compounds were identified by direct comparison of the retention times and mass fragmentation pattern with those from the National Institute of Standards and Technology library (NIST) Ver.2.1 MS data library and comparing the spectrum obtained through GC–MS compounds present in the plant fractions were identified.

Cell Culture Experiments: RAW 264.7 cells, a murine macrophage cell line, were obtained from NCCS Pune and cultured in Falcon plates (100 mm), grown at 37°C in 5% CO₂ in DMEM supplemented with 10% FBS, 2 mM glutamine, and 1,000 U/mL penicillin-streptomycin ¹⁹. The medium was changed on alternate days. The cells were passaged by trypsinization (Trypsin EDTA) to disrupt the cell monolayer at confluence while splitting RAW264.7 cells to continue cultures and plating the cells for the in-vitro assays.

Cell Viability Assay (MTT Assay): As described previously, the murine macrophage-like RAW 264.7 cells were cultured. Cells were seeded at a density of 10⁴ cells/well in 96-well plates, grown overnight followed by the pre-treatment with different concentrations of the EETP (50, 100, 150, 200 250 mg/mL) for 1 h before the addition of the LPS. RAW 264.7 cells viability was measured after 24 h of exposure to the MFNS with a colorimetric assay, based on the ability of mitochondria in viable cells to reduce MTT ²⁰. A concentration of 0.5 mg/mL of MTT solution was added to each well. After 4 h of incubation at 37°C, the medium was discarded and the formazan blue color formed in the cells were dissolved in dimethyl sulfoxide (DMSO). Optical density at 570nm was determined with a microplate reader (Bio-Rad).

Anti-Inflammatory Activity:

Quantification of NO in RAW 264.7 Cells: The levels of Nitic oxide production in the culture medium of 264.7 macrophages were measured using a standard procedure using the Griess reagent kit according to the manufacturer’s instructions. Cells were seeded onto a 96-well plate with 2 x 105 cells/well and allowed to adhere overnight. Then, the medium was removed and replaced with 0.2 mL of fresh medium alone or containing 0.5 mg/mL of fractions. After 1 h of incubation, LPS stimulation was performed. LPS was added at a 1 mg/mL concentration for 24 h. The cell-free culture medium was collected, 50 μLof it was used for NO determination, and the remainder was stored at -20°C for further use. The nitrite accumulated in the culture medium was measured as an indicator of NO production, based on the Griess reaction ²¹.

Briefly, 50 μL of culture supernatants were gently mixed with an equal volume of sulfanilamide solution (1% sulfanilamide in 5% phosphoric acid) and incubated in the dark at room temperature (RT) for 10 min. After the incubation, 50 μL of 0.1% n-naphthyl ethylenediamine dihydrochloride (NEDA) was added to the reaction and incubated in the dark at RT for another 10 min. The absorbance at 540 nm was measured in a microplate reader (Bio-Rad max 100). Nitrite concentration, an indicator of NO production, was calculated from a NaNO₂ standard curve.

Membrane Stabilization Assay: The membrane stabilization method has been used to prepare blood samples of human red blood cells (HRBC) to study in-vitro anti-inflammatory activity ²². The blood was collected from healthy volunteers who had not taken any Nonsteroidal anti-inflammatory drug (NSAIDs) for 3 weeks before the experiment and mixed with an equal volume of Alsever solution (2% dextrose, 0.8% sodium citrate, 0.5% citric acid, and 0.42% NaCl). All the blood samples were stored at 4°C for 24h before use. They were centrifuged at 3000 rpm for 5 min, and the supernatant was discarded. The cell suspension was washed with sterile saline solution (0.9% w/v NaCl) and centrifuged at 2500 rpm for 5 min. Centrifugation was re-peated till the supernatant was clear and colorless, and the packed cell volume (PCV) was measured. The cellular component was reconstituted to a 40% suspension (v/v) with phosphate-buffered saline (10 mM, pH 7.4) and was used in the assays.

Hypotonicity-Induced Hemolysis: To 0.5 mL of human RBC suspension, 1 mL of MFNS in phosphate buffer, and 2 mL hyposaline was added, incubated at 37°C for 30 min, and centrifuged at 3000 rpm for 20 min. The hemoglobin content of the supernatant solution was estimated spectrophotometrically at 560 nm. Ibuprofen (200 μg/mL) was used as reference standard. The percentage inhibition of hemolysis or membrane stabilization was calculated ²³.

% Inhibition of Hemolysis = 100 x (OD1-OD2/OD1)

Where, OD1 = Optical density of hypotonic-buffered saline solution alone.

OD2 = Optical density of test sample in hypo-tonic solution.

Statistical Analysis: Data are expressed as mean values ± standard deviation (SD). Comparisons between control and test groups were performed with (Tukey's honest significant difference multiple range post hoc test p < 0.05 IBM SPSS Ver.23). The experiments were performed in triplicate.

RESULTS AND DISCUSSION:

Preliminary Phytochemical Analysis: As shown in Table 1, among the tested fractions, a methanolic fraction (MFNS) was found to be rich in flavonoids, polyphenols, saponins, quinones, and glycosides followed by chloroform fraction (CFNS) and aqueous fraction (AqFNS). The presence of such phytochemicals might play a decisive role in the therapeutic activity of Nigella seeds.

TABLE 1: PHYTOCHEMICAL ANALYSIS OF NIGELLA SATIVA FRACTIONS

Total Polyphenols and Flavonoids Content: The results of total polyphenols (TPC) and total flavonoids content (TFC) in different solvent fractions of N. sativa are presented in Table 2. The content of phenolic and flavonoid compounds extracted from biological substances is dependent on location, soil, season, and extraction conditions. The Folin-Ciocalteu method determined the TPC and expressed it as milligram gallic acid equivalent (GAE) per 100 g of dry weight. Among different fractions of N. sativa seed, the methanolic fraction (MFNS) showed the highest yield of polyphenols (451.77 mg/g GAE), as shown in Table 2. Also, MFNS had higher flavonoids (401.24 mg/g QE) compared with the rest of the solvent fractions of N. sativa.

TABLE 2: TOTAL POLYPHENOL CONTENT AND TOTAL FLAVONOID CONTENT OF NIGELLA SATIVA FRACTIONS

According to Tungmunnithum et al., there is a correlation between polyphenolic, flavonoid content, and antioxidant capacities in medicinal plants since medicinal plants are generally a rich source of antioxidant compounds ²⁴. In the present context, the high content of polyphenols and flavonoids in MFNS might be crucial in the free radical scavenging and antioxidant capacity of N. sativa seeds, as demonstrated by DPPH assay Nitric oxide (NO)assays.

GC-MS & HRMS Analysis: Different fractions of N. sativa seeds were subjected to HRMS analysis, and their spectral data was depicted in Fig. 1.

In addition, GC-MS analysis of methanolic fractions of Nigella sativa seeds showed more than 20 phytoconstituents Table 3. The major components identified include thymoquinone, thymohydroquinone, rutin, astragalin, nigellidine, some fatty acids etc.

FIG. 1: HRMS ANALYSIS OF NIGELLA SATIVA FRACTIONS

TABLE 3: GC-MS ANALYSIS OF NIGELLA SATIVA FRACTIONS

Effect of N. sativa Seed Fractions on DPPH Radicals: In-vitro DPPH assay is extensively used to evaluate various components' free radical scavenging activity.

The results of the DPPH assay by different solvent fractions are presented in Fig. 2. Among different fractions, MFNS showed significantly higher inhibitory activity when compared with other solvent fractions in a dose depended manner. Maximum antioxidant capacity (88.26%) was observed at the concentration of 250 μg/mL.

These results are similar to previous studies by Baliyan et al ¹³. The degree of decoloration indicates the scavenging potential of the fraction due to hydrogen proton donation.

FIG. 2: EFFECT OF MFNS ON DPPH RADICALS

Nitric Oxide Scavenging Activity: Nitric oxide is a central agent in physiological and pathological conditions and, when reacted with macromolecules, may induce inflammation, muscle sclerosis, arthritis, ulcerative colitis, and other metabolic disorders ²⁵. The effect of N. sativa seed fractions against nitric oxide radicals was evaluated in this study. MFNS considerably reduced the release of nitric oxide radicals in a dose-dependent manner, and the maximum inhibition (78.66%) was noted at 250 μg/mL concentration, as shown in Fig. 3. Therefore, Nigella sativa seed fractions could alleviate nitric oxide-induced cellular derangements.

FIG. 3: EFFECT OF MFNS ON NITRIC OXIDE RADICALS

Effect of MFNS on Pancreatic Lipase Activity: Further, we evaluated the inhibitory effect of N. sativa seed fractions on lipase activity. In the digestive system, pancreatic lipase (PL) is the most active enzyme in the digestion of dietary fats and empowers absorption across the intestine. Control of pancreatic lipase is one of the significant marks to combat obesity and other metabolic disorders. Pancreatic lipase (PL) inhibitory activity is depicted in Fig. 4. MFNS has shown potent anti-lipase activity in a dose-dependent manner, which is higher than other solvent fractions. Maximum inhibition of PL (79.26%) was recorded at 250 μg/mL, which is nearer to the standard drug orlistat (81.92%). Bhardwaj et al., in their studies, reported the potential benefits of sitosterol derived from a plant species against hormone-sensitive lipase, adipogenesis, and lipolysis mechanism in rat adipocytes. Inhibiting lipase activity is an important therapeutic means in drug discovery to contain obesity and adipogenesis. Since lipase inhibits lipid digestion, it reduces lipid absorption and accumulation of triglycerides in adipose and other tissues ²⁶.

FIG. 4: PANCREATIC LIPASE INHIBITORY ACTIVITY OF MFNS. DATA ARE PRESENTED AS MEAN ± SD OF TRIPLICATE. DIFFERENT ALPHABETICAL SUPERSCRIPTS (^A*-D*) OVER THE ERROR BARS FOR GIVEN CONCENTRATIONS OF MFNS INDICATE SIGNIFICANT DIFFERENCES FROM EACH OTHER (P<0.05). MFNS: METHANOLIC FRACTION OF NIGELLA SATIVA

Effect of MFNS on α-Amylase and α-Glucosidase Activity: Elevation of blood sugar causes insulin resistance and diabetes, a metabolic disorder that leads to atherosclerosis, hypertension, other cardiovascular diseases, and obesity. A systematic approach for controlling diabetes would be to regulate post-prandial plasma glucose rise by inhibiting carbohydrate hydrolyzing enzymes (α-amylase, α-glucosidase) in the digestive tract. Among different solvent fractions of N. sativa, the MFNS showed the highest inhibition in a dose-dependent manner. Fig. 5 explains the pancreatic α-amylase inhibitory activity of MFNS. The maximum inhibition of α-amylase (81.47%) was recorded at 250 μg/mL of MFNS, which is higher than standard drug acarbose (83.53%). Fig. 6 explains the inhibitory activity of MFNS against the α-glucosidase activity. Among different fractions, the methanolic fraction was found to be a potential inhibitor against α-glucosidase when compared to other fractions. The maximum inhibition of 77.12% was recorded with MFNS at 250 μg/mL, close to the standard drug acarbose (79.45%). The obtained results are similar to the observations made by Salehi et al. and Gong et al., who reported that tea polyphenols might act as anti-nutritional factors on digestive enzymes. Inhibition of amylase and glucosidase significantly slows down carbohydrates digestion and thus reduces high calorie intake. These properties of medicinal plants may be considered in alternative adjuvant therapy with other oral hypoglycemic drugs for treating metabolic disorders ^{27, 28}.

FIG. 5: Α-AMYLASE INHIBITORY ACTIVITY OF MFNS. DATA ARE PRESENTED AS MEAN ± SD OF TRIPLICATE. DIFFERENT ALPHABETICAL SUPERSCRIPTS (^A*-D*) OVER THE ERROR BARS FOR GIVEN CONCENTRATIONS OF MFNS INDICATE SIGNIFICANT DIFFERENCES FROM EACH OTHER (P<0.05). MFNS: METHANOLIC FRACTION OF NIGELLA SATIVA

FIG. 6: α-GLUCOSIDASE INHIBITORY ACTIVITY OF MFNS. DATA ARE PRESENTED AS MEAN ± SD OF TRIPLICATE. DIFFERENT ALPHABETICAL SUPERSCRIPTS (^a*-d*) OVER THE ERROR BARS FOR GIVEN CONCENTRATIONS OF MFNS INDICATE SIGNIFICANT DIFFERENCES FROM EACH OTHER (p<0.05). MFNS: METHANOLIC FRACTION OF NIGELLA SATIVA

Inhibition of LPS-induced NO production: To evaluate the effects of Nigella sativa seed fraction on NO production, nitrite was measured as the stable metabolite accumulated in the culture supernatants.

Little production of NO occurs in unstimulated cells when compared with treated, but treatment with LPS induced a high release of NO into the culture medium. Treatment with a high concentration of MFNS resulted in considerable inhibition of the NO production compared with a low concentration of MFNS Fig. 7.

Thus, Nigella sativa seeds could contribute to good health by alleviating NO-induced inflammation and other cellular and molecular disturbances.

FIG. 7: EFFECT OF MFNS ON LPS-INDUCED NO PRODUCTION IN RAW 264.7 MACROPHAGES. DATA ARE SHOWN IN THE MEAN ± SD (n=3), p<0.05 SIGNIFICANTLY DIFFERENT FROM THE LPS GROUP.*INDICATES SIGNIFICANT DIFFERENCES BETWEEN CONTROL AND TREATED CELLS

Membrane Stability: The anti-inflammatory activity was determined by membrane stability test of the human red blood cells as shown in Fig. 8.

The MFNS showed a concentration-dependent activity, and the membrane protection percent was increased with an increase in the concentration of the test samples. At a 250 μg/mL concentration, MFNS showed a maximum of 81.01 ± 5.04% inhibition of RBC hemolysis, compared with standard Ibuprofen drug, which showed 85.23± 7.15 %.

FIG. 8. REPRESENTS THE PERCENTAGE OF MEMBRANE PROTECTION OF MFNS WITH REFERENCE TO IBUPROFEN 200 ??G/ML. THE RESULTS WERE EXPRESSED AS MEAN ± SD (n=3).*INDICATES SIGNIFICANT DIFFERENCES BETWEEN CONTROL AND TREATED CELLS

CONCLUSION: The results demonstrate that MFNS showed effective and dose-dependent free radicals scavenging activity, and inhibited α-amylase, α-glucosidase, and pancreatic lipase. MFNS also significantly inhibited nitric oxide production in RAW 264.7 macrophages and protected HRBC from hemolysis. Based on the above results, Nigella sativa seeds can be well considered as a potent anti-inflammatory, anti-adipogenic and free radicals scavenging agent.

ACKNOWLEDGEMENT: We are thankful to DST-FIST and ICMR for their support in carrying out this work.

CONFLICTS OF INTEREST: They declare that they have no conflicts of interest.

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    How to cite this article:

    Keerthi R, Karunakaran RS and Balaji M: Antioxidant, free radical scavenging and anti-inflammatory activity of Nigella sativa seed fractions. Int J Pharm Sci & Res 2022; 13(6): 2539-49. doi: 10.13040/IJPSR.0975-8232.13(6).2539-49.

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