Antioxidant potential of bitter cumin (Centratherum anthelminticum (L.) Kuntze) seeds in in vitro models
© Ani and Naidu; licensee BioMed Central Ltd. 2011
Received: 16 October 2010
Accepted: 20 May 2011
Published: 20 May 2011
Bitter cumin (Centratherum anthelminticum (L.) Kuntze), is a medicinally important plant. Earlier, we have reported phenolic compounds, antioxidant, and anti-hyperglycemic, antimicrobial activity of bitter cumin. In this study we have further characterized the antioxidative activity of bitter cumin extracts in various in vitro models.
Bitter cumin seeds were extracted with a combination of acetone, methanol and water. The antioxidant activity of bitter cumin extracts were characterized in various in vitro model systems such as DPPH radical, ABTS radical scavenging, reducing power, oxidation of liposomes and oxidative damage to DNA.
The phenolic extracts of bitter cumin at microgram concentration showed significant scavenging of DPPH and ABTS radicals, reduced phosphomolybdenum (Mo(VI) to Mo(V)), ferricyanide Fe(III) to Fe(II), inhibited liposomes oxidation and hydroxyl radical induced damage to prokaryotic genomic DNA. The results showed a direct correlation between phenolic acid content and antioxidant activity.
Bitter cumin is a good source of natural antioxidants.
KeywordsBitter cumin Centratherum anthelminticum polyphenolic compounds antioxidants DPPH ABTS reducing power liposomes oxidative DNA damage
In living systems, reactive oxygen species (ROS) constitute most important free radicals. ROS include not only oxygen radical, but, also some non radical derivatives of oxygen like H2O2 . ROS play a positive role in energy production, phagocytosis, and regulation of cell growth, cell signaling and synthesis of biologically important compounds. Oxidative stress is the result of an increased ROS production and/a decrease in their elimination. Based on the fact that ROS are dangerous for cells, tissues and organs it has been inferred that oxidative stress is the cause for number of disorders, including atherosclerosis, neural degenerative disease, inflammation, cancer and ageing [2–4]. The physiological role of antioxidants is to prevent damage to cellular constituents arising as a consequence of chemical reactions involving free radicals [5, 6]. The use of synthetic antioxidants in food has its beginning in the late 1940's when BHA was found to be effective antioxidant in fatty foods and toxicological studies proved it safe for food use. But, later there are serious concerns over the side effects of these synthetic antioxidants due to their carcinogenic potential [7, 8]. As a result there has been a general desire to replace the synthetic food additives with natural antioxidants [9, 10].
Phytochemicals are non-nutritive plant chemicals that have protective or disease preventive properties. There are thousands of phytochemicals falling into different groups and one of the most studied groups is phenolics. Plant phenolics are multifunctional and can act as reducing agents, metal chelators and singlet oxygen quenchers. Many studies have shown that phenolics are of great value in preventing the onset and/progression of many human diseases [11–13]. Therefore over the past few years a number of medicinal and food plants are extensively investigated for the presence and activity of polyphenols and other antioxidants [14–16]. There are several methods to determine the antioxidant activity of a biological sample each with its own advantages and disadvantages. These tests enable us to examine the possibility of a given biological sample could act as an antioxidant in one or more ways in vivo or in food substances.
Centratherum anthelminticum (L.) Kuntze (bitter cumin) is a member of Asteraceae family of the flowering plants. The seeds have a hot sharp taste; acrid, astringent to the bowls, antihelmintic; cure ulcers, used in skin diseases, leucoderma and fevers. Two novel and two known steroids were isolated respectively from benzene: acetone and ethanolic extracts of the seeds of C. anthelminticum . The plant has reported to possess antimicrobial , antifilarial [19, 20], post-coital anti-implantation  and insecticidal activities . Earlier we have reported an array of phenolic compounds, antioxidant activity in few model systems, antimicrobial and anti-hyperglycemic activity of bitter cumin [23, 24]. The present paper describes antioxidant studies with an emphasis on various in vitro antioxidant model systems.
1,1-Diphenyl -2-picryl hydrazyl (DPPH), 2,2 azinobis-3-ethyl benzothiazoline-6-sulfonic acid (ABTS), Butylated hydroxyl anisole (BHA), Ascorbic acid, α-Tocopherol, agarose, xylene cyanol, bromophenol blue, ethidium bromide, thiobarbituiric acid and tannic acid were purchased from Sigma chemicals (MO, USA). Bacillus genomic DNA was obtained from the Food Microbiology department of CFTRI. Folin-Ciocalteau reagent, was purchased from Sisco research laboratories (Mumbai, India). Ammonium molybdate, trichloroacetic acid, potassium ferricyanide and ferric chloride were purchased from Qualigens. All other chemicals and solvents used are of analytical grade.
The plant material was purchased from local market and authenticated by National Institute of Science Communication and Information Resources, New Delhi.
The seeds were hand sorted to remove stones and plant debris. The powdered seeds were defatted for 8 hours in Soxhlet's apparatus with hexane. The defatted powder was extracted with methanol:acetone:water (7:7:6) which is subsequently hydrolyzed with 2N HCl and extracted into ethyl acetate and named as Aqueous Methanol Acetone Extract -(AMAECA). The defatted powder was extracted with 80% methanol and termed as Aqueous Methanol extract (AMECA). The defatted bitter cumin seed powder was extracted with water and named as aqueous extract- (AECA) (1:10 w/v × 3) under continuous stirring at ambient temperature. The organic solvents were removed under vacuum in a rotavapour and water was removed by freeze drying. The solid extract obtained was stored at 4°C until use.
Estimation of total phenols
Total phenol content of the AMAECA, AMECA and AECA were estimated by using Folin-Ciocalteu reagent . 0.5 mL of the sample dissolved in MeOH was incubated with 2.5 mL of 10% FC reagent for 2 min at ambient temperature. To this 2.0 mL of 7.5% Na2CO3 was added and incubated for another 1 hour at ambient temperature. The absorbance of the color developed was measured at 765 nm against blank developed with 0.5 mL of solvent using a Shimadzu UV-Visible spectrophotometer (Model- 2100). The total phenolic content was expressed as tannic acid equivalents (TAE) in μg/mg of the extract, using a standard curve generated with tannic acid. Similarly, the total phenol content was also expressed as gallic acid equivalents (GAE) using a standard curve generated with gallic acid as standard.
In vitro antioxidant activity of bitter cumin extracts
DPPH radical scavenging assay
Where Ao is the absorbance of the control tube Ac is the absorbance of the tube with 'c' concentration of sample . All the experiments were performed in triplicates.
ABTS•+ scavenging assay
Generation of ABTS •+ radical  forms the basis of one of the spectrophotometric methods that have been applied to the measurement of the total antioxidant activity of various substances. The experiments were carried out using an improved ABTS•+ decolorization assay . ABTS radical cation (ABTS•+) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. The ABTS•+ solution was diluted to an absorbance of 0.7 ± 0.05 at 734 nm (Shimadzu UV-Vis Spectrophotometer) with ethanol. To one ml of ABTS•+ solution different concentrations of bitter cumin extracts/BHA were added. Absorbance was recorded at 1 min interval up to 7 minutes at 734 nm. All the experiments were performed in triplicates.
Phosphomolybdenum reducing assay
This assay is based on the reduction of Mo (VI) to Mo (V) by the sample analyte and the subsequent formation of a green phosphate/Mo (V) complex at acidic pH . The reagent solution consists of 0.6 M H2SO4, 28.0 mM sodium phosphate and 4.0 mM ammonium molybdate. An aliquot of 0.1 ml of sample was combined with 1 ml of reagent solution. The tubes were capped and incubated in a thermal block at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against a blank. The blank solution contained 1 ml of reagent solution and the solvent used for the sample, and it was incubated under the same conditions as the rest of the samples. All the experiments were performed in triplicates. The antioxidant capacity was expressed as equivalents of ascorbic acid. (μg/g extract).
Ferricyanide reducing assay
The reductive potential of the extract was determined according to the method of Oyaizu et al. 1986 . Different concentrations of sample in 0.5 ml of MeOH were mixed with 2.5 ml of Phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated for 20 minutes at 50° C. At the end of the incubation, 2.5 ml of 10% trichloroacetic acid was added to the mixture and centrifuged at 5000 rpm for 10 minutes. The upper 2.5 ml layer was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% ferric chloride, and the absorbance was measured at 700 nm . A higher absorbance of the reaction mixture indicated greater reducing power. Ascorbic acid and BHA were used as a positive control.
Liposome oxidation assay
The antioxidant activity of the extracts of bitter cumin and α-tocopherol in a liposome model system was determined according to the method of Duh and Yen 1997 . Egg lecithin (300 mg) was sonicated with 30 ml phosphate buffer (10 mM, pH 7.4) in an ultrasonic sonicator for 30 min to ensure proper liposome formation. Bitter extracts and standards were mixed with the sonicated solution (0.5 ml, 10 mg/ml) and incubated for 10 min. at room temperature. The oxidation of liposomes was initiated by adding FeCl3 (0.5 ml, 400 mM), and ascorbic acid (0.5 ml, 400 mM). The antioxidative action was measured by the method of Buege and Aust 1978 . The absorbance of the samples was determined at 535 nm after incubation for 1 h at 37°C. The results were expressed as nmol of malondialdehyde (MDA) formed per mg lipid and was calculated by using an extinction coefficient of 1.56 × 105 M_1 cm_1.
Oxidative DNA damage assay
Bacterial genomic DNA (2 μg) in phosphate buffered saline was incubated with different concentrations of bitter cumin and BHA for 15 minutes at ambient temperature. Oxidation was induced by treating DNA with 1 mM FeSO4 and 10 mM ascorbic acid. Positively controlled reaction was not treated with bitter cumin or oxidative stress and negatively controlled reaction mixture contained FeSO4 and ascorbic acid without any pretreatment with CA. The final reaction volume was 9 μl and the reaction mixture was incubated for 1 hour at 37°C. The reaction was stopped by adding 3 μl loading buffer (xylene-cyanol, 0.25%; bromophenol blue, 0.25%; and glycerol, 30%) and 9 μl of the reaction mixture was loaded on to an agarose gel (1%). The gel was run in TAE buffer initially for 1 h at 40 V followed by 2 hours run at 60 V. The gel was stained with ethidium bromide (1 μg/ml). DNA was visualized and photographed by a digital imaging system (Hero lab, GMBH, Germany).
All the experiments were done in triplicates and expressed as mean ± S.E.M. The differences in mean values were tested using one-way analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) was used to determine the significant differences amongst the test materials. Differences were considered to be significant at p ≤ 0.05.
Results and Discussion
Total phenol acids
Radical scavenging activity of bitter cumin phenols
Antiradical activity of bitter cumin extract (AMAECA)
IC 50 values (μg of AMAECA)
Phosphomolbdenum reducing power
Ferricyanide reducing Power
ABTS radical assay
DPPH radical assay
Liposomal lipid peroxidation
Reducing power of bitter cumin phenolics
Inhibition of liposome oxidation activity by bitter cumin phenols
Inhibition of oxidative damage to DNA by bitter cumin phenols
The results from various free radical scavenging systems revealed that bitter cumin extracts were strong antioxidants, with different magnitudes of potency in scavenging different ROS at microgram concentrations. The antioxidant activity of bitter cumin significantly correlated with total phenol content of bitter cumin extract. The phenol extract of bitter cumin contained an array of phenolic compounds which may be responsible for its antioxidant activity.
Authors are thankful to Dr. V. Prakash, Director and Dr. P.V. Salimath, Head of the Department of Biochemistry and Nutrition, Central Food Technological Research institute, Mysore for their constant encouragement and support. V. Ani is thankful to the Council of Scientific and Industrial Research (CSIR), New Delhi, for the award of Junior and Senior Research Fellowship. This work was partly supported by a project awarded to KAN by Department of Science and Technology (DST), New Delhi, India.
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