... Activity of Crambe Cordifolia. Syed Majid Bukhari, Nebojsa Simic, Hamid Latif Siddiqui and Viqar Uddin Ahmad ..... Ashraf, M.A., M.J. Maah and I. Yusoff, 2010.
World Applied Sciences Journal 22 (11): 1561-1565, 2013 ISSN 1818-4952 © IDOSI Publications, 2013 DOI: 10.5829/idosi.wasj.2013.22.11.184
Determination of Antioxidant Activity of Crambe Cordifolia 1
Syed Majid Bukhari, 1Nebojsa Simic, 2Hamid Latif Siddiqui and 3Viqar Uddin Ahmad Institute of Chemistry, NTNU, 7491 Trondheim Norway Institute of Chemistry, University of the Punjab Lahore, 54590, Pakistan 3 HEJ, Research Institute of Chemistry, University of Karachi-75270, Pakistan 1
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Abstract: In the present investigation, the antioxidant activity and free radical scavenging activity of Crambe cordifolia plant were spectrophotometrically studied. Free radical scavenging activity was determined by DPPH method and the obtained IC50 value was 1106.65 µg/mL. For comparison, the IC50 value of standard ascorbic acid was 21.69 µg/mL. Antioxidant activity was determined by phosphomolybdate method and the results showed that the plant had 205.73 µg per gram -tocopherol equivalent antioxidant content. Spectrophotometric determination of phenolic and flavonoid content was also carried out in terms of pyrocatechol equivalent and quercetin equivalent, respectively. Plant contained 10.60 µg pyrocatechol equivalent phenolic content and 1.43 µg quercetin equivalent flavonoid content per gram of plant material. The results indicated that Crambe cordifolia was a potent source of natural antioxidants. Key words: Free radical scavenging activity Anti-oxidant activity Crambe cordifolia Methanolic extract INTRODUCTION Our atmosphere contains around 21% of oxygen. It is not only the key element for plants in photosynthesis, but is also vital for animals and humans. Once oxygen is inhaled, it undergoes the process of reduction and results in the formation of several very reactive radicals, among which superoxide anion radical, singlet oxygen and hydroxyl radical are the important ones [1]. These reactive oxygen species have the tendency to cause peroxidation of membrane lipids and damage proteins, carbohydrates and nucleic acids [2]. Most living species have the natural defence system based on antioxidant activity, which protects them against these reactive oxygen species. The capacity of such protective systems decreases, as the process of aging goes on resulting in disturbance of the redox equilibrium system in the body. This disturbance is the cause of some major diseases, such as malaria, acquired immunity deficiency syndrome, heart disease, stroke, diabetes and cancer [3-5]. The most effective way to reduce the unwanted effects of that process is to use antioxidants as food supplements.
Phenolic content
Flavonoid content
Today there are two types of commercially available antioxidants: synthetic ones and those obtained from natural sources, mostly from plants. The synthetic antioxidants typically have phenolic groups as the main functionality and their variety is related to the number and nature of substituents. Those which are obtained from natural sources (e.g. plants), show much bigger diversity, with respect to their moieties. They can either be the nitrogen containing compounds, such as alkaloids, chlorophyll derivatives, amines and amino acids, or phenolic compounds such as tocopherols, flavonoids and phenolic acids, which can terminate the activity of free radicals by acting as oxygen scavengers [6, 7]. Flavonoids, a class of compounds with phenolic moiety, are considered to be the potent antioxidants. They can delay or inhibit the oxidation of lipids or other molecules by inhibiting the propagation of oxidative chain reactions [5, 8]. Ascorbic acid and carotenoids also act as good antioxidants in nature. There has been a great interest in finding natural antioxidants from plant materials in the recent years [9]. Genus Crambe is the largest genus in the family Brassiceae. Much work has been done upon different species of Crambe in order to evaluate their industrial and
Corresponding Author: Syed Majid Bukhari, Institute of Chemistry, NTNU, 7491 Trondheim Norway.
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pharmaceutical importance. For instance, Crambe orientalis can inhibit seed germination and shows phytotoxic effects [10]. Crambe abyssinica contains membrane active compounds [11]. According to another study, it also shows a good ratio of phenolic and flavonoid compounds [12]. Another member of this genus, Crambe cordifolia, contains amino acids [13], growth inhibitors [14] and is used as livestock feed [15]. It also contains some important flavonoids, such as acylated glycosides of kaempferol or quercetin. Typically, it contains kaempferol 3-(p-coumaroyl) glucoside-7, 4’-diglucoside and quercetin 3-feruloylglucoside-7, 4’-diglucoside. It also contains kaempferol, quercetin, glucose, kaempferol 4-glucoside, kaempferol 7-glucoside, quercetin 4’-glucoside and quercetin 7-glucoside [16]. The literature survey shows that there have not been many reported studies concerning antioxidant capacity in genus Crambe. This deficiency in experimental data motivated us to determine the antioxidant properties of Crambe cordifolia in order to evaluate its value as an accessible source of natural antioxidants.
Methods Free Radical Scavenging Activity: The free radical scavenging activity was determined by monitoring the ability of the extract to quench the free radical 1, 1-diphenyl-2-picryl-hydrazil [17-19]. The scavenging concentration of the extract was determined spectrophotometrically. The ability to quench 1, 1-diphenyl-2-picryl-hydrazil was shown in terms of IC50 value (the extract concentration required to inhibit the activity of DPPH by 50%) [20-22]. Seven different ethanol dilutions of the plant extract were made with concentrations 3200 µg/mL, 1600 µg/mL, 800 µg/mL, 400 µg/mL, 200 µg/mL, 100 µg/mL and 50 µg/mL. From each dilution 2.5 mL was mixed with 1.0 mL of 0.3 mM DPPH solution at room temperature. After 30 minutes the absorption was measured at 518 nm (Radical DPPH shows maximum absorption at 518 nm). Negative control was prepared by mixing 2.5 mL of ethanol with 1.0 mL of DPPH. Ethanol was used to calibrate the spectrophotometer. Percentage of inhibition for all of the dilutions was determined by the following equation. % inhibition = 100- [(Abssample – Absblank) x 100] / Abscontrol
MATERIALS AND MATHODS Plant Material: The plant Crambe cordifolia was collected from the place Nushki (about 143 km from the capital of Balochistan province) in the South Western region of Pakistan. Chemicals: Potassium acetate, 1, 1-diphenyl-2-picrylhydrazil, sodium phosphate, -tocopherol, quercetin, ammonium molybdate, folin- ciocaulteu reagent, pyrocatechol and aluminium nitrate were purchased from Sigma-Aldrich. Sulfuric acid and ascorbic acid were purchased from Merck. Sodium carbonate was purchased from Fluka. Extraction: Crushed plant parts (450 gram) were put in MeOH, left for two weeks at room temperature and then filtered. The methanol was evaporated from the filtrate and 150 gram of methanolic plant extract was collected (33.34%). Instrumentation: UV mini-1240 - Shimadzu Scientific spectrophotometer was used for all of the absorbance measurements.
where, Abssample is absorbance of each dilution; Absblank is absorbance of dilutions without adding DPPH; Abs control is absorbance of the solution of DPPH in ethanol. The graph percentage of inhibition vs. concentration was plotted and the IC50 value of the plant extract was compared with ascorbic acid as a standard. Antioxidant Activity Determination: The total antioxidant capacity of the extract was determined with phosphomolybdenum, using -tocopherol as standard [23, 24]. An aliquot of 0.2 mL (containing 1.0 mg) of the extract was combined with 2.0 mL of the reagent (0.6 M sulfuric acid, 28.0 mM sodium phosphate and 4.0 mM ammonium molybdate). The blank solution was made by mixing 2.0 mL of the reagent solution with the appropriate volume of the same solvent used to dissolve the sample. The tubes were capped and incubated in water bath at 95 °C for a period of 90 minutes. The sample and blank were left on the shelf for half an hour to cool down to room temperature. The absorbance of the sample was measured against blank solution at 695 nm. A tocopherol graph was plotted by using
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-tocopherol as standard and the total antioxidant activity of the plant extract was expressed as µg tocopherol equivalent. The equation of the plotted graph is given as: Y = 5.358x + 0.2427 where, Y = Absorbance and X = Concentration Total Phenolic Content Determination: Total soluble phenolic content of the plant extract was determined with Folin-Ciocalteu reagent using pyrocatechol as standard [25, 26]. The amount of 25 mg of the dry extract was dissolved in 20 mL of distilled water and the total volume was transferred to an erlenmeyer flask. It was diluted to 46 mL by adding distilled water. One mL of Folin-Ciocaulteu reagent was added to the extract solution in the flask and the mixture was shaken vigorously for 3 minutes, after which 3 mL of 2% sodium carbonate solution was added. The flask was covered with aluminium foil in order to protect the formed complex from light. The mixture was shaken occasionally at room temperature for 2 hours and then the absorbance was measured at 760 nm [21]. A standard curve was made by using different dilutions of pyrocatechol and the total soluble phenolic content in the extract was expressed as µg pyrocatechol equivalent. The Equation of Straight Line Graph Is Given As: Y = 0.0533x – 0.0994 where, Y = Absorbance and X = Concentration
Total flavonoid content determination: Quercetin was used as standard to determine the total flavonoid content of the plant extract [27-29]. The dry extract (10 mg) was dissolved in 1.0 mL of 80% ethanol. An aliquot of 0.5 mL was taken out of it and added to a test tube containing 4.3 mL of 80% ethanol, 0.1 mL of 1 M potassium acetate and 0.1 mL of 10% aluminium nitrate. The mixture was incubated at room temperature for 40 minutes and then the absorbance measured at 415 nm. The total flavonoid content in the plant extract was expressed as µg quercetin equivalents by using standard quercetin graph. The equation of the graph established is: Y = 0.0494x – 0.0026 where, Y = Absorbance and X = Concentration RESULTS AND DISCUSSION The free radical scavenging activity of different concentrations of Crambe cordifolia extract has been compared with the activity of ascorbic acid, which was used as a standard. The obtained results are shown in Table 1a and 1b. The IC 501 value for the plant extract and ascorbic acid was determined. The IC50 value of the methanolic extract of the plant is 1106.65 µg/mL as compared to 21.69 µg/mL of ascorbic acid. Concerning antioxidant activity, it was found that Crambe cordifolia contains 205.73 µg -tocopherol equivalent antioxidant content per milligram of the plant extract (Table 2). Total phenolic content of Crambe cordifolia is expressed as µg pyrocatechol equivalents. Total soluble phenolic content per milligram of the plant extract was found to be 10.60 µg pyrocatechol equivalents (Table 2).
Table 1a. 1, 1-diphenyl-2-picryl-hydrazil scavenging activity of Crambe cordifolia. The increase in concentration of methanolic extract of plant shows an increase in inhibition of the 1, 1-diphenyl-2-picryl-hydrazil radical Concentration (µg/mL) 50 100 200 400 800 1600 3200 1106.65 Percent inhibition 2.197 3.956 10.330 18.681 35.604 74.835 90.220 IC 50 Table
1b. 1, 1-diphenyl-2-picryl-hydrazil scavenging activity of Ascorbic acid. The increase in concentration of ascorbic acid shows an increase in percent inhibition of 1, 1-diphenyl-2-picryl-hydrazil radical Concentration (µg/mL) 1.25 2.50 5.00 10.00 20.00 40.00 21.69 Percent inhibition 9.56 12.09 15.93 25.38 47.25 86.70 IC50 *IC50 represents the concentration that is required for 50 percent inhibition of 1, 1-diphenyl-2-picryl-hydrazil Table 2: Total antioxidant activity, total phenolic content and total flavonoid content in methanolic extract of Crambe cordifolia. Total antioxidant activity Total phenolic content Total flavonoid content (µg -tocopherol equivalent/mg) (µg pyrocatechol equivalent/mg) (µg quercetin equivalent/mg) 205.73±0.049 10.60±0.006 1.43±0.004 *All values are expressed as mean ± standard error of three parallel measurements
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IC50 represents the concentration that is required for 50 percent inhibition of 1, 1-diphenyl-2-picryl-hydrazil.
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Flavonoid content was determined and expressed as µg quercetin equivalents. The flavonoid content in methanolic extract of Crambe cordifolia was 1.43 µg quercetin equivalents per milligram of the plant extract (Table 2). The obtained results, in terms of free radical scavenging ability, gave an IC50 value of 1106.65 µg/mL of the plant extract which was ca. 50 times weaker as compared to 21.69 µg/mL of ascorbic acid as standard. The total antioxidant activity of 205.73 µg -tocopherol equivalent per milligram of the plant extract also points out that the plant can be a good source of natural antioxidants. Crambe cordifolia contains 10.60 µg/mg of the total soluble phenolic content, which is a bit higher than 8.10 µg/mg determined in Crambe abyssinica [12]. On the other hand, the flavonoid content of Crambe cordifolia is 1.43 µg/mg of the plant extract, which is less than the reported flavonoid content (9.36 µg/mg) of Crambe abyssinica [12]. CONCLUSION
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As a conclusion, our first results suggest that Crambe cordifolia can be considered as a convenient and easily accessible source of natural antioxidants. Its exact composition is the logical goal of the next phase of our Crambe project.
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