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Antioxidant and antiacetylcholinesterase activities of chard (Beta vulgaris L. var. cicla)
Food and Chemical Toxicology 48 (2010) 1275–1280
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Antioxidant and antiacetylcholinesterase activities of chard (Beta vulgaris L. var. cicla) Ozlem Sacan 1, Refiye Yanardag * Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar-Istanbul 34320, Turkey
a r t i c l e
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Article history: Received 13 January 2010 Accepted 16 February 2010
Keywords: Chard Antioxidant activity Antiacetylcholinesterase activity Free radicals Scavenging activity
a b s t r a c t Plants have been used for many years as a source of traditional medicine to treat various diseases and conditions. Many of these medicinal plants are also excellent sources for phytochemicals, many of which contain potent antioxidant and antiacetylcholinesterase activities. Chard (Beta vulgaris L. var. cicla) is widely spread in Turkey and used as an antidiabetic in traditional medicine. In the present study, the antioxidant activity and acetylcholinesterase inhibitor capacity of chard were examined. In addition, proline level of chard was determined. The antioxidant activity of water extract of chard was evaluated using different antioxidant tests. The results were compared with natural and synthetic antioxidants. The results suggest that chard may provide a natural source of antioxidant and antiacetylcholinesterase activities and proline content. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Oxygen is critical for life on earth. It is produced by plants during photosynthesis, and is necessary for aerobic respiration for animals. The oxygen consumption inherent in cell growth leads to the generation of a series of reactive oxygen species (ROS). They are continuously produced by the body’s normal use of oxygen such as respiration and some cell-mediated immune functions. ROS play an important role in energy production, phagocytosis, regulation of cell growth and intercellular signaling or synthesis of biologically important compounds in humans. Production of ROS exceeded was ascertained to play multiple important roles in tissue damage and loss of function in a number of tissues and organs (Verma et al., 2009). However, ROS may also be very harmful as they can readily attack biomolecules such lipids, proteins, and DNA, leading to various disorders, e.g. arthritis, diabetes, inflammation, arteriosclerosis, cancer, genotoxicity, and neurological disorders such as Alzheimer’s disease (Shukla et al., 2009). Plants and their products are rich Abbreviations: AChE, acetylcholinesterase; AD, Alzheimer disease; ABTS, 2,20 azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DMPD, N,N-dimethylp-phenylenediamine 2HCl; DPPH, 2,2-diphenyl-1-picryl-hydrazyl; DTNB, 5,50 -dithiobis (2-nitrobenzoic acid); EC50, efficiency concentration; ferrozine, 3-(2pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine; NADPH2, reduced nicotinamide adenine dinucleotide phosphate; ROS, reactive oxygen species; TCA, trichloroacetic acid; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. * Corresponding author. Tel.: +90 212 4737037; fax: +90 212 4737180. E-mail addresses: [email protected], [email protected] (O. Sacan), refi[email protected], [email protected] (R. Yanardag). 1 Tel.: +90 212 4737070/17790; fax: +90 212 4737180. 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.02.022
sources of phytochemicals and have been found to possess a variety of biological activities including antioxidant potential (Verma et al., 2009). In recent decades, various extracts of plants have been of great interest as sources of natural products. This interest has increased considerably in finding naturally occurring antioxidants for use in foods, cosmetics or medicinal materials to replace synthetic antioxidants, such as tertiary butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT) butylated hydroxyanisole (BHA), and propylgallate, which are being restricted due to their carcinogenicity. Recently, various phytochemicals especially phenolic compounds found in vegetables, fruits, and medicinal plants have received increasing attention for their potential role in prevention of human diseases. The human can use antioxidants either as dietary, food supplement or as a drug (Abdel-Hameed, 2009). Proline is one of the 20 amino acids found in proteins. Pentose phosphate pathway is regulated by synthesis of cytosolic proline which oxidizes NADPH2. The role of proline in proline-linked pentose phosphate pathway in plants is studied and it is concluded that plants with high proline content exhibit high concentration of phenolic compounds (Shetty, 2004). Therefore, proline content of edible plants may be accepted as a measure of its antioxidant capacity. Acetylcholine (ACh) is one of the most important neurotransmitter in human. Inhibition of acetylcholinesterase (AChE), the key enzyme in the breakdown of ACh, is considered one of the treatment strategies against several neurological disorders such as, Alzheimer’s disease (AD), senile dementia, ataxia, myasthenia gravis and Parkinson’s disease (Mukherjee et al., 2007). Several AChE inhibitors (such as tacrine, donepezil, rivastigmine and galanthamine) were
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used for treatment of AD. Some of the drugs approved for therapeutic use show hepatotoxicity (Knapp et al., 1994) and gastrointestinal disturbances (Schulz, 2003), consequently there have been a continuous search for new drugs. Therefore, the search for new AChE inhibitors is of great interest for AD treatment. Swiss chard (Beta vulgaris species) is a herbaceous biennial leafy vegetable cultivated in many parts of the world for its year round availability, low cost and wide use in many traditional dishes (Gao et al., 2009). The leaves can be used in salads or cooked like spinach, and the stems are usually chopped and cooked like celery. The plant is more robust and easier to grow than spinach and celery. The leaves of chard contain nutritionally significant concentrations of Vitamins A, C and B, calcium, iron and phosphorus (Pyo et al., 2004). Chard (Beta vulgaris L. var. cicla: Chenopodiaceae) has been indicated to have hypoglycemic properties (Bolkent et al., 2000). Beta vulgaris L. species are used as a popular folk remedy for liver and kidney diseases, for stimulation of the immune and hematopoietic systems, and as a special diet in the treatment of cancer (Kanner et al., 2001). Phytochemical screenings of chard have revealed the presence of some fatty acids (palmitic, stearic, oleic, linoleic and linolenic acids), phospholipids, glycolipids, polysaccharides, ascorbic acid, folic acid, pectin, saponins, flavonoids, phenolic acids (Bolkent et al., 2000), betalains (Kugler et al., 2004) and apigenin (Gao et al., 2009). The antioxidant activity and phenolic compounds of the methanolic extracts, including other Beta vulgaris subspecies have been studied (Pyo et al., 2004), but limited studies have been reported on the antioxidant activity of Beta vulgaris subspecies cycla aqueous extracts. The present study was undertaken to examine antioxidant and antiacetylcholinesterase activities of water extract of chard through various in vitro models. Possible relation between phenolic content and antioxidant activity was also discussed. Additionally, determination of proline levels of the extract as indicative of antioxidant capacity was aimed.
2. Materials and methods 2.1. Chemicals 2-Deoxy-D-ribose, b-carotene, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine), 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), proline, N,N-dimethyl-p-phenylenediamine 2HCl (DMPD), (+) catechin hydrate were purchased from Fluka Chemical Co. (Buchs, Switzerland). 2,2-diphenyl-1-picrylhydrazyl (DPPH), a-tocopherol and pyrocatechol, rutin, 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB), were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Trichloroacetic acid (TCA), ferric chloride, ninhydrine, glacial acetic acid, and Tris were obtained from Merck. All other reagents were of analytical grade.
2.2. Plant materials Chard (Beta vulgaris L. var. cicla) leaves were collected from Istanbul, Turkey and identified by Prof. Dr. Neriman Özhatay (Faculty of Pharmacy, Istanbul University). Plant materials were washed with distilled water and dried at room temperature. The dried plant was stored in 20 °C until used.
2.3. Preparation of extracts Dried chard leaves (50 g) were extracted by adding 500 ml distilled water and boiled for 30 min. The extract was then filtered and evaporated to dryness under reduced pressure and controlled temperature (40–50 °C) in a rotary evaporator. The water extract yielded a dark brown solid residue weighing 15.59 g (31.18%) and was kept at 20 °C. The extract was dissolved in distilled water and used for the assessment of antioxidant and antiacetylcholinesterase activities and proline content.
2.4. Determination of anthocyanin content Anthocyanin content of the dried leaves was determined according to method of Padmavati et al. (1997) modified by Chung et al. (2005). The dried leaves (25 mg/ml) were mixed with acidified methanol (1% HCl/methanol) for 24 h at 4 °C in the dark, and then centrifuged at 1000g for 15 min. The anthocyanin concentration in the supernatant was measured spectrophotometrically at 530 and 657 nm and the absorbance values were indicated as A530 and A657. The extinction coefficient of 31.6 M1 cm1 was used to convert the absorbance values into anthocyanin concentration. The concentration was calculated using the following equation:
Anthocyanin concentration ðlmol=gÞ ¼ ½ðA530 0:33 A657 Þ=31:6 ½volume ðmlÞ=weight ðgÞ: Results were expressed as the average of triplicates. 2.5. Determination of total phenolic compounds Total phenolics in chard extract were determined with Folin–Ciocalteau reagent, according to the method of Slinkard and Singleton (1977) with some modifications. Briefly, 0.1 ml of the chard extract (20–100 lg/ml) was transferred into test tubes and their volumes made up to 4.6 ml with distilled water. After addition of 0.1 ml Folin–Ciocalteau reagent (previously diluted 3-fold with distilled water) and 0.3 ml 2% Na2CO3 solution, tubes were vortexed and then allowed to stand for 2 h with intermittent shaking. The absorbance was measured at 760 nm in a spectrophotometer. The total phenolic compounds in the chard extract were calculated as mg of pyrocatechol equivalent from the calibration curve and as mg pyrocatechol equivalents per mg of extract. The data were presented as the average of triplicate analyzes. 2.6. Determination of total flavonoid content Total flavonoid content was determined by using a method described by Sakanaka et al. (2005) using catechin as standard flavonoid compound. Briefly, 0.25 ml of the extract (1000 lg/ml) or (+)-catechin standard solution (20–100 lg/ml) was mixed with 1.25 ml of distilled water in a test tube, followed by the addition of 75 ll of a 5% sodium nitrite solution. After 6 min, 150 ll of a 10% aluminium chloride solution was added and the mixture was allowed to stand for a further 5 min before 0.5 ml of M NaOH was added. The mixture was brought to 2.5 ml with distilled water and mixed well. The absorbance was measured immediately at 510 nm using a spectrophotometer. Results were expressed as the average of triplicates. The results were expressed as the mean (±SD) mg of (+)-catechin equivalents per mg of extract. 2.7. Proline content Proline analysis was performed according to Bates (1973). Briefly, 50 mg extract was homogenized in 10 ml sulphosalicylic acid (3%) and filtrated through filter paper. Two milliliters of the filtrate were mixed with 2 ml of acid ninhydrin solution (1.25 g ninhydrin + 30 ml glacial acetic acid + 20 ml 6 M H3PO4) and 2 ml of glacial acetic acid and kept at 100 °C for 1 h. Then the reaction was stopped by transferring the mixture to an ice bath. Four milliliters of toluene were added to the mixture and vortexed for 15–20 s. The toluene phase was aspirated and absorbance at 520 nm was measured using pure toluene reference. A calibration curve was prepared with pure proline. Results were expressed as lg proline/gram extract. 2.8. Antiacetylcholinesterase activity The enzymatic activity was measured using an adaptation of the method described by Ingkaninan et al. (2003). Tris–HCl buffer (325 ll) (50 mM, pH 8), 100 ll of extract, with different concentrations and 25 ll of an enzyme solution containing 0.28 U/ml were incubated during 15 min. Subsequently, 75 ll 15 mM acetylcholine iodide (AChI) solution, and 475 ll. DTNB (3 mM) solution was added and the final mixture was incubated for 30 min at room temperature. The absorbance of the mixture was measured at 405 nm. A control mixture was performed without addition of the extract. Results were expressed as the average of triplicates. The percent inhibition was calculated using the following equation: Acetylcholinesterase inhibition (%) = (A0 Aı)/A0 100 A0 is the absorbance of the control A1 is the absorbance of the sample
2.9. DPPH radical scavenging activity The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the chard extract was measured according to the procedure described by Brand-Williams et al. (1995). Appropriate dilution series (0.25–1 mg/ml) were prepared for
O. Sacan, R. Yanardag / Food and Chemical Toxicology 48 (2010) 1275–1280 each aqueous extract in methanol 0.1 ml of each dilution was added to 3.9 ml of a 6 105 M methanolic solution of DPPH followed by vortexing. The mixture was shaken vigorously and allowed to stand in the dark at room temperature for 30 min. The decrease in absorbance of the resulting solution was then measured spectrophotometrically at 517 nm against methanol. All measurements were made in triplicate and averaged. The DPPH radical scavenging activity was calculated using the following equation:
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DMPD+ scavenging activity (%) = (A0 A1/A0) 100 A0 is the absorbance of the initial concentration of DMPD+ A1 is the absorbance of the remaining concentration of DMPD+ in the presence of chard.
2.14. Statistical analysis DPPH radical scavenging activity (%) = (A0 A1/A0) 100 A0 is the absorbance of the control A1 is the absorbance of the sample
2.10. ABTS+ scavenging activity The ABTS+ scavenging activity of the chard extract was measured according to the procedure described by Arnao et al. (2001). The stock solutions included 7.4 mM ABTS+ solution and 2.6 mM potassium persulfate solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted by mixing 1 ml ABTS+ solution with 60 ml methanol to obtain an absorbance of 1.1 ± 0.02 units at 734 nm using the spectrophotometer. Fresh ABTS+ solution was prepared for each assay. Chard extracts (150 ll) were allowed to react with 2850 ll of the ABTS+ solution for 2 h in a dark condition. Then the absorbance was taken at 734 nm using the spectrophotometer. The ABTS+ scavenging activity was calculated using the following equation: ABTS radical scavenging activity (%) = (A0 A1/A0) 100 A0 is the absorbance of the control A1 is the absorbance of the sample
2.11. Hydroxy radical scavenging activity The effect of extract on hydroxyl radicals was assayed by using the deoxyribose method (Chung et al., 1997). The reaction mixture contained 0.45 ml of 0.2 M sodium phosphate buffer (pH 7.4), 0.15 ml of 10 mM 2-deoxyribose, 0.15 ml of 10 mM FeSO4-EDTA, 0.15 ml of 10 mM hydrogen peroxide, 0.525 ml of distilled water and 0.075 ml of extract solution in a tube. The reaction was started by the addition of hydrogen peroxide. After incubation at 37 °C for 4 h, the reaction was stopped by adding 0.75 ml of 2.8% TCA and 0.75 ml 1.0% of thiobarbituric acid. The mixture was boiled for 10 min, cooled in an ice bath and then measured at 520 nm. Hydroxyl radical scavenging activity was calculated in the following equation: Hydroxyl radical scavenging activity (%) = (A0 A1/A0) 100 A0 is the absorbance of the control reaction A1 is the absorbance of sample
2.12. Reducing power The reducing power of the chard extract was determined according to the method described by Oyaizu (1986). Different amounts of extracts (20–100 lg) in 1 ml of distilled water were mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide (1%) and then the mixture was incubated at 50 °C for 30 min. Afterwards, 2.5 ml of TCA (10%) was added to the mixture to stop the reaction, then the mixture was centrifuged at 3000 rpm for 10 min. The supernatant (2.5 ml) was mixed with 2.5 ml distilled water and 0.5 ml FeCl3 (0.1%), and then absorbance was measured at 700 nm in a spectrophotometer. The reducing power of the tested samples increased with the absorbance values.
2.13. DMPD+ scavenging activity DMPD+ scavenging activity was performed according to Fogliano et al. (1999). DMPD+ (100 mM) was prepared by dissolving 209 mg of DMPD+ in 10 ml of deionized water and 1 ml of this solution was added to 100 ml of 0.1 M acetate buffer (pH 5.3), and the colored radical cation (DMPD+) was obtained by adding 0.2 ml of a solution of 0.05 M ferric chloride. The absorbance of this solution, which is freshly prepared daily, is constant up to 12 h at room temperature. Different concentrations of chard (10–30 lg/ml) were added in test tubes and the total volume was adjusted with distilled water to 0.5 ml. Ten minutes later, the absorbance was measured at 505 nm. One milliliter of DMPD+ solution was directly added to reaction mixture and its absorbance at 505 nm was measured. The buffer solution was used as a blank sample. The DMPD+ scavenging activity was calculated using the following equation:
Results were expressed as mean ± standard deviation of triplicate analyzes. The correlation coefficient (r2) between the parameters tested was established by regression analysis.
3. Results and discussion 3.1. Anthocyanins, total phenolic, total flavonoid and proline content Anthocyanins possess well known pharmacological properties and strong biological functions such as antiinflammatory, antitumor, antimutagenic and antioxidant activities (Kong et al., 2003). The anthocyanin content was found to be 0.47 ± 0.03 lmol/g for chard extract (Table 1). It was reported that some pharmacological activities of polyphenol compounds, like anthocyanosides may attributed to their antioxidant properties (Martin-Aragon et al., 1998). Phenolics or polyphenols are secondary plant metabolites that are ubiquitously present in plants and plant products. Polyphenols are marker of the nutritional quality of foods. Polyphenols are known for their antioxidant activity as radical scavengers and possible beneficial roles in human health, such as reducing the risk of cancer, cardiovascular disease, other pathologies (Selappan et al., 2002). Plants containing high phenolic compounds can be a good source of antioxidants. For this reason, this information has led to the determination of the total phenolic content of the sample under study. It was determined that there was 31.09 ± 3.34 lg pyrocatechol equivalent of phenolic compounds in the 1 mg of the water extracts of chard (Table 1). A high correlation was observed between the total phenolic content and hydroxyl radical scavenging activity (r2 = 0.9931), ABTS+ scavenging activity (r2 = 0.9628), DPPH radical scavenging activity (r2 = 0.9596), DMPD+ scavenging activity (r2 = 0.9392) and reducing power (r2 = 0.9596) of the extract. These data are in accordance with others, which have shown that a high total phenolic content increases antioxidant activity, and that there is a linear correlation between phenolic content and antioxidant activity (Holasova et al., 2002). These results indicate that the higher antioxidant activity of chard extract may be associated with its total phenolic content. Flavonoids are natural phenolic compounds and well known antioxidants. Therefore dietary intake of flavonoid-containing foods was suggested to be of benefit for the preservation from free radical damage. The concentration of flavonoids in the extracts was expressed as lg of epicatechin equivalents per mg of the extract, as shown in Table 1. It was determined that there was 11.88 ± 1.46 lg epicatechin equivalent of phenolic compounds in 1 mg of the water extracts of chard (Table 1). Wu et al. (2003) has demonstrated that some amino acids also have antioxidant properties. Proline is one of these antioxidant amino acids. Proline is intracellular nonenzymatic ROS scavenging molecule (Xu et al., 2009). Proline provides protecting against stress by maintaining redox homeostasis (Hoque et al., 2008) and scavenging free radicals and ROS (Sharma and Dietz, 2006). In this study, the proline contents increased with increasing concentration and high proline contents (427.74 ± 24.41 lg/g extract) were seen in 10 mg water extract (Table 1). 3.2. Acetylcholinesterase inhibitor activity Plants have been used traditionally to enhance cognitive function and to alleviate other symptoms associated nowadays with
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Alzheimer’s disease (Howes and Houghton, 2003). In this study, AChE inhibitor activity of chard was found to increase dosedependently, the results were expressed as EC50 values, calculated from the regression equations prepared from the concentrations of the samples. A high AChE inhibition (20.98 ± 5.25%) was seen in 6 lg/ml (Table 1). EC50 values were found 14.43 ± 2.76 lg/ml. Sterols, terpenoids, oils, flavonoids, alkaloids and other phenolic compounds have been shown to possess antiacetylcholinesterase properties (Ji and Zhang, 2008), these constituents, flavonoids in particular, may have a particular contribution in the occurence of antioxidant and antiacetylcholinesterase activity of chard. This result is similar to the work of Lin et al. (2008) who reported this activity in the aqueous extract of Radix paeoniae rubra and other plants. Adsersen et al. (2006) have reported that Rosmarinus officinalis water extract showed low AChE inhibition with an average 12% value and Mentha spicata 3% inhibition of 0.1 mg/ml, respectively. It was evident that chard extract had a stronger effect on AChE inhibition compared to various plant extracts in literature (Adsersen et al., 2006; Lin et al., 2008). Also, Delwing et al. (2003) have shown that proline inhibited AChE activity from rat cerebral cortex in vivo and in vitro. The data suggest that the inhibitor effect of proline on AChE activity is associated with oxidative stress. In this study, in vitro inhibition of acetylcholinesterase by chard extract is reported for the first time. The results indicate that chard extract may offer great potential for the treatment of AD. 3.3. DPPH radical scavenging activity Antioxidant properties, especially radical scavenging activities, are very important due to the deleterious role of free radicals in foods and in biological systems. Excessive formation of free radicals accelerates the oxidation of lipids in foods and decreases food quality and consumer acceptance (Min, 1998). The model of scavenging the stable DPPH radical is a widely used method to evaluate antioxidant activities in relatively short time as compared to other methods. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. Fig. 1 shows, the dose response curves of DPPH radical scavenging activity of the extracts from chard. The extracts were capable of scavenging DPPH radicals in a concentration-dependent manner. BHA and BHT were used as references for radical scavengers. The scavenging activity of chard extract, BHA, and BHT on DPPH radicals increased between 20–100 lg/ml and were 85.03 ± 0.77%, 81.61 ± 5.96%, and 47.55 ± 4.04% at a concentration of 100 lg/ml, respectively. Chard extract and BHA showed similar DPPH radical scavenging activity, while BHT was a considerably less effective DPPH radical scavenger. The DPPH radical scavenging activity was correlated with phenolic compounds (r2 = 0.9596). These results indicated that the radical scavenging capacity of the water extract might be mostly related to concentration of phenolic hydroxyl groups. DPPH scavenging activity is best presented by EC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution. A higher DPPH radical scavenging activity was associated with a lower EC50 value. EC50 values for chard extracts, BHA and BHT on DPPH radical scavenging activity were found as 23.85 ± 1.63,
47.61 ± 0.91 and 80.83 ± 0.72 lg/ml. Pyo et al. (2004) have reported that Beta vulgaris subspecies cycla methanol extract showed the highest activity at 500 lg/ml (87.00 ± 1.2%). This value was lower than our result (85.03 ± 0.77%, at a concentration of 100 lg/ml). For this reason, in our study, chard extract had a stronger effect on the DPPH radicals compared to Beta vulgaris subspecies cycla. 3.4. ABTS+ scavenging activity ABTS+ scavenging activity is also one of the most commonly used methods to evaluate the antioxidant activity. The ABTS+ scavenging activity of water extract of chard compared to rutin are shown in Fig. 2. ABTS+ scavenging activity increased with increasing concentration, reaching 18.56 ± 1.77% at 400 lg/ml and this value was much lesser than that of the positive controls, rutin 99.77 ± 0.01% at a same concentration. EC50 values for chard extracts, and rutin on ABTS radical scavenging activity were found as 1093.14 ± 52.02, and 113.70 ± 3.24 lg/ml, respectively. A high correlation was observed between ABTS radical scavenging activity and phenolic compounds (r2 = 0.9628). 3.5. Hydroxyl radical scavenging activity Hydroxyl radical is the most reactive among ROS and possesses the shortest half-life period. Hydroxyl radical causes oxidative damage to DNA, proteins, lipids (Moncada et al., 1991). The effect of chard on inhibition of free radical-mediated deoxyribose damage was assessed by means of the Fe2+ dependent DNA damage assay. The Fenton reaction generates hydroxy radical, which degrades DNA deoxyribose, using Fe2+ salts as a catalyst. Hydroxy radical may attack DNA either at the sugar or the base, giving rise to toxic products. Fig. 3 shows, the dose response curves of radical scavenging activities of the extract and reference antioxidants on the hydroxyl radicals and can be formed from superoxide anion and hydrogen peroxide in the presence of metal ions such as copper or iron. Chard extract scavenged hydroxyl radicals by 12.03 ± 1.37% at 20 lg/ml and 20.24 ± 6.28% at 60 lg/ml. Trolox and BHA exhibited good scavenging activity of 54.31 ± 4.30% and 52.00 ± 3.43% at a concentration of 60 lg/ml, respectively. The hydroxyl radical scavenging effect of water extract (20.24 ± 6.28%) was nearly equal to that of ascorbic acid (26.47 ± 2.86%) with EC50 values of water extract (157.94 ± 41.31 lg/ml) lower than that of Trolox (46.76 ± 4.17 lg/ml), ascorbic acid (116.04 ± 4.75 lg/ml) and BHA (51.54 ± 2.24 lg/ml). The hydroxyl scavenging radical activity in water extract was correlated with phenolic contents (r2 = 0.9931). 3.6. Reducing power The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity (Bhandari and Kawabata, 2004). Fig. 4 shows, the extent of the reduction, in terms of absorbance values at 700 nm. The reducing power of water extract (0.069 ± 0.003) was not concentration dependent and was found to be below those of tocopherol (0.294 ± 0.001), ascorbic acid (0.432 ± 0.003) and BHA (0.538 ± 0.001) at 100 lg/ml. The water extract showed lower reducing power than the standards. Reduc-
Table 1 Total anthocyanins, total phenolic compounds (PC, as pyrocatechol equivalents), total flavonoids (as epicatechin equivalents) and proline contents, and antiacetylcholinesterase (AChE) activity in water extract from chard. Extract
Water
Anthocyanins (lg mol/g extract) 0.47 ± 0.03
PC (lg pyrocatechol/ mg extract) 31.09 ± 3.34
Flavonoids (lg catechin/mg extract) 11.88 ± 1.46
Proline (lg/g extract) 427.74 ± 24.41
AChE Inhibition % (6 lg extract/ml)
EC50 (lg extract/ml)
20.98 ± 5.25
14.43 ± 2.76
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ing power of extract and standards decreased in order of BHA > ascorbic acid > a-tocopherol > water extract. The total phenolic contents were correlated with reducing power (r2 = 0.9922) for water extract. The results demonstrate the electron donor properties of chard extract for neutralizing free radicals by forming stable products.
DPPH radical scavenging activity (%)
90 80 70 Chard BHA BHT
60 50
3.7. DMPD+ scavenging activity
40 30 20 10 0 0
20
40
60
80
100
120
Concentration μg/ml Fig. 1. DPPH radical scavenging activity of the water extract from chard. BHA and BHT were used as reference antioxidant. Values are means ± SD (n = 3).
100
0.6 Chard α-Tocopherol
80
0.5
Ascorbic Acid BHA
60
Absorbance (700nm)
ABTS radical scavenging activity (%)
120
The antiradical capacity was analyzed by DMPD+ assay. The principle of the DMPD+ assay is that at acidic pH and in the presence of a suitable oxidant solution. DMPD can form a stable and colored radical cation (DMPD+). This reaction is rapid, and the end point, which is stable, is taken as a measure of the antioxidative efficiency. Therefore, this assay reflects the ability of radical hydrogen-donors to scavenge the single electron from DMPD+. This method ensures low cost and high reproducible analysis (Fogliano et al., 1999). As shown in Fig. 5, chard extract was an effective DMPD+ radical scavenger in a concentration-dependent manner. Water extract scavenged DMPD+ radicals by 34.05 ± 0.19% at 10 lg/ml and 47.51 ± 0.30% at 50 lg/ml. Cysteine and Trolox exhibited good scavenging activity of 99.89 ± 0.00% and 96.53 ± 0.16% at a concentration of 50 lg/ml, respectively. EC50 value was found
Chard Rutin 40
20
0 0
50
100
150
200
250
300
350
400
0.4
0.3
0.2
0.1
450
Concentration (μg/ml) 0
Fig. 2. ABTS radical scavenging activity of the water extract from chard. Rutin was used as reference antioxidant. Values are means ± SD (n = 3).
0
20
40
60
80
100
120
Concentration (μg/ml) Fig. 4. Reducing power of the water extract from chard. Values are means ± SD (n = 3).
120
50
DMPD radical scavenging activity (%)
Hydroxyl radical scavenging activity (%)
60
Chard
40
BHA Ascorbic Acid Trolox
30
20
10
100
80
Chard Trolox Cysteine
60
40
20
0 0
10
20
30
40
50
60
70
Concentration (μg/ml)
0 0
10
20
30
40
50
60
Concentration (μg/ml) Fig. 3. Hydroxyl radical scavenging activity of the water extract from chard. BHA, ascorbic acid and Trolox were used as reference antioxidants. Values are means ± SD (n = 3).
Fig. 5. DMPD radical scavenging activity of the water extract from chard. Trolox and cysteine were used as reference antioxidant. Values are means ± SD (n = 3).
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9.87 ± 0.27 lg/ml for cysteine and 10.38 ± 0.28 lg/ml for Trolox and 45.21 ± 0.75 lg/ml for chard extract. A high correlation was observed between DMPD+ scavenging activity and phenolic compounds (r2 = 0.9392). 4. Conclusion Numerous epidemiological and clinical studies have proven that the consumption of foods and beverages rich in antioxidant phytochemicals can have a beneficial effect upon human health by potentially affecting the etiology of chronic diseases mediated through reactive oxygen species such as free radicals. The studies to date on chard extract suggested that they might be used as an important natural antioxidant source due to its high levels of phenolics, flavonoids and proline. According to this study, a strong correlation has been observed between total phenolic content and antioxidative activity, which shows that the phenolic content might be responsible for the antioxidant activity. It can be concluded that water extract of chard in the way which they are consumed as a food stuff in Turkey, can be used as an accessible source of natural antioxidants with consequent health benefits. It is well known that free radicals are one of the causes of several diseases, such as Parkinson’s disease, Alzheimer type dementia etc. The great acetylcholinesterase inhibitor activity together with the antioxidant activity indicates that chard extract can be used in the prevention of neurodegenerative diseases such as AD and Parkinsons’s diseases. Thus, it can be concluded that water extract can also be used as an accessible source of natural antioxidants and antiacetylcholinesterase with consequent health benefits. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments The authors would like to thank Professor Neriman Özhatay (Faculty of Pharmacy, Istanbul University) for the identification of chard. This study was supported by the Research Fund of Istanbul University, Project No: UDP-3972/24062009. References Abdel-Hameed, E.S., 2009. Total phenolic contents and free radical scavenging activity of certain Egyptian Ficus species leaf samples. Food Chem. 114, 1271– 1277. Adsersen, A., Gauguin, B., Gudiksen, L., Jager, A.K., 2006. Screening of plants used in Danish folk medicine to treat memory dysfunction for acetylcholinesterase inhibitor activity. J. Ethnopharmacol. 104, 418–422. Arnao, M.B., Cano, A., Acosta, M., 2001. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem. 73, 239–244. Bates, S.L., 1973. Rapid determination of free proline for water stres studies. Plant Soil 39, 205–207. Bhandari, M.R., Kawabata, M., 2004. Organic acid, phenolic content and antioxidant activity of wild yam (Dioscorea spp.) tubers of Nepal. Food Chem. 88, 163–168. Bolkent, S ß ., Yanardag˘, R., Tabakog˘lu-Og˘uz, A., Özsoy-Saçan, Ö., 2000. Effects of chard (Beta vulgaris L var. cicla) extract on pancreatic B cells in streptozotocindiabetic rats: a morphological and biochemical study. J. Ethnopharmacol. 73, 251–259. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm.-Wiss. Technol.-Food Sci. Technol. 28, 25–30. Chung, S.K., Osawa, T., Kawakishi, S., 1997. Hydroxyl radical scavenging effects of species and scavengers from brown mustard (Brassica nigra). Biosci. Biotechnol. Biochem. 61, 118–123. Chung, Y.-C., Chen, S.-J., Hsu, C.-K., Chang, C.-T., Chou, S.-T., 2005. Studies on the antioxidative activity of Graptopetalum paraguayense E. Walhter. Food Chem. 91, 419–424. Delwing, D., Chiarani, F., Delwing, D., Bavaresco, C.S., Wannmacher, C.M.D., Wajner, M., Wyse, T.S., 2003. Proline reduces acetylcholinesterase activity in cerebral cortex of rats. Metab. Brain Dis. 18, 79–86.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Antioxidant and antiacetylcholinesterase activities of chard (Beta vulgaris L. var. cicla) Ozlem Sacan 1, Refiye Yanardag * Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar-Istanbul 34320, Turkey
a r t i c l e
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Article history: Received 13 January 2010 Accepted 16 February 2010
Keywords: Chard Antioxidant activity Antiacetylcholinesterase activity Free radicals Scavenging activity
a b s t r a c t Plants have been used for many years as a source of traditional medicine to treat various diseases and conditions. Many of these medicinal plants are also excellent sources for phytochemicals, many of which contain potent antioxidant and antiacetylcholinesterase activities. Chard (Beta vulgaris L. var. cicla) is widely spread in Turkey and used as an antidiabetic in traditional medicine. In the present study, the antioxidant activity and acetylcholinesterase inhibitor capacity of chard were examined. In addition, proline level of chard was determined. The antioxidant activity of water extract of chard was evaluated using different antioxidant tests. The results were compared with natural and synthetic antioxidants. The results suggest that chard may provide a natural source of antioxidant and antiacetylcholinesterase activities and proline content. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Oxygen is critical for life on earth. It is produced by plants during photosynthesis, and is necessary for aerobic respiration for animals. The oxygen consumption inherent in cell growth leads to the generation of a series of reactive oxygen species (ROS). They are continuously produced by the body’s normal use of oxygen such as respiration and some cell-mediated immune functions. ROS play an important role in energy production, phagocytosis, regulation of cell growth and intercellular signaling or synthesis of biologically important compounds in humans. Production of ROS exceeded was ascertained to play multiple important roles in tissue damage and loss of function in a number of tissues and organs (Verma et al., 2009). However, ROS may also be very harmful as they can readily attack biomolecules such lipids, proteins, and DNA, leading to various disorders, e.g. arthritis, diabetes, inflammation, arteriosclerosis, cancer, genotoxicity, and neurological disorders such as Alzheimer’s disease (Shukla et al., 2009). Plants and their products are rich Abbreviations: AChE, acetylcholinesterase; AD, Alzheimer disease; ABTS, 2,20 azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; DMPD, N,N-dimethylp-phenylenediamine 2HCl; DPPH, 2,2-diphenyl-1-picryl-hydrazyl; DTNB, 5,50 -dithiobis (2-nitrobenzoic acid); EC50, efficiency concentration; ferrozine, 3-(2pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine; NADPH2, reduced nicotinamide adenine dinucleotide phosphate; ROS, reactive oxygen species; TCA, trichloroacetic acid; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. * Corresponding author. Tel.: +90 212 4737037; fax: +90 212 4737180. E-mail addresses: [email protected], [email protected] (O. Sacan), refi[email protected], [email protected] (R. Yanardag). 1 Tel.: +90 212 4737070/17790; fax: +90 212 4737180. 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.02.022
sources of phytochemicals and have been found to possess a variety of biological activities including antioxidant potential (Verma et al., 2009). In recent decades, various extracts of plants have been of great interest as sources of natural products. This interest has increased considerably in finding naturally occurring antioxidants for use in foods, cosmetics or medicinal materials to replace synthetic antioxidants, such as tertiary butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT) butylated hydroxyanisole (BHA), and propylgallate, which are being restricted due to their carcinogenicity. Recently, various phytochemicals especially phenolic compounds found in vegetables, fruits, and medicinal plants have received increasing attention for their potential role in prevention of human diseases. The human can use antioxidants either as dietary, food supplement or as a drug (Abdel-Hameed, 2009). Proline is one of the 20 amino acids found in proteins. Pentose phosphate pathway is regulated by synthesis of cytosolic proline which oxidizes NADPH2. The role of proline in proline-linked pentose phosphate pathway in plants is studied and it is concluded that plants with high proline content exhibit high concentration of phenolic compounds (Shetty, 2004). Therefore, proline content of edible plants may be accepted as a measure of its antioxidant capacity. Acetylcholine (ACh) is one of the most important neurotransmitter in human. Inhibition of acetylcholinesterase (AChE), the key enzyme in the breakdown of ACh, is considered one of the treatment strategies against several neurological disorders such as, Alzheimer’s disease (AD), senile dementia, ataxia, myasthenia gravis and Parkinson’s disease (Mukherjee et al., 2007). Several AChE inhibitors (such as tacrine, donepezil, rivastigmine and galanthamine) were
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used for treatment of AD. Some of the drugs approved for therapeutic use show hepatotoxicity (Knapp et al., 1994) and gastrointestinal disturbances (Schulz, 2003), consequently there have been a continuous search for new drugs. Therefore, the search for new AChE inhibitors is of great interest for AD treatment. Swiss chard (Beta vulgaris species) is a herbaceous biennial leafy vegetable cultivated in many parts of the world for its year round availability, low cost and wide use in many traditional dishes (Gao et al., 2009). The leaves can be used in salads or cooked like spinach, and the stems are usually chopped and cooked like celery. The plant is more robust and easier to grow than spinach and celery. The leaves of chard contain nutritionally significant concentrations of Vitamins A, C and B, calcium, iron and phosphorus (Pyo et al., 2004). Chard (Beta vulgaris L. var. cicla: Chenopodiaceae) has been indicated to have hypoglycemic properties (Bolkent et al., 2000). Beta vulgaris L. species are used as a popular folk remedy for liver and kidney diseases, for stimulation of the immune and hematopoietic systems, and as a special diet in the treatment of cancer (Kanner et al., 2001). Phytochemical screenings of chard have revealed the presence of some fatty acids (palmitic, stearic, oleic, linoleic and linolenic acids), phospholipids, glycolipids, polysaccharides, ascorbic acid, folic acid, pectin, saponins, flavonoids, phenolic acids (Bolkent et al., 2000), betalains (Kugler et al., 2004) and apigenin (Gao et al., 2009). The antioxidant activity and phenolic compounds of the methanolic extracts, including other Beta vulgaris subspecies have been studied (Pyo et al., 2004), but limited studies have been reported on the antioxidant activity of Beta vulgaris subspecies cycla aqueous extracts. The present study was undertaken to examine antioxidant and antiacetylcholinesterase activities of water extract of chard through various in vitro models. Possible relation between phenolic content and antioxidant activity was also discussed. Additionally, determination of proline levels of the extract as indicative of antioxidant capacity was aimed.
2. Materials and methods 2.1. Chemicals 2-Deoxy-D-ribose, b-carotene, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulfonic acid)-1,2,4-triazine (ferrozine), 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), proline, N,N-dimethyl-p-phenylenediamine 2HCl (DMPD), (+) catechin hydrate were purchased from Fluka Chemical Co. (Buchs, Switzerland). 2,2-diphenyl-1-picrylhydrazyl (DPPH), a-tocopherol and pyrocatechol, rutin, 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB), were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Trichloroacetic acid (TCA), ferric chloride, ninhydrine, glacial acetic acid, and Tris were obtained from Merck. All other reagents were of analytical grade.
2.2. Plant materials Chard (Beta vulgaris L. var. cicla) leaves were collected from Istanbul, Turkey and identified by Prof. Dr. Neriman Özhatay (Faculty of Pharmacy, Istanbul University). Plant materials were washed with distilled water and dried at room temperature. The dried plant was stored in 20 °C until used.
2.3. Preparation of extracts Dried chard leaves (50 g) were extracted by adding 500 ml distilled water and boiled for 30 min. The extract was then filtered and evaporated to dryness under reduced pressure and controlled temperature (40–50 °C) in a rotary evaporator. The water extract yielded a dark brown solid residue weighing 15.59 g (31.18%) and was kept at 20 °C. The extract was dissolved in distilled water and used for the assessment of antioxidant and antiacetylcholinesterase activities and proline content.
2.4. Determination of anthocyanin content Anthocyanin content of the dried leaves was determined according to method of Padmavati et al. (1997) modified by Chung et al. (2005). The dried leaves (25 mg/ml) were mixed with acidified methanol (1% HCl/methanol) for 24 h at 4 °C in the dark, and then centrifuged at 1000g for 15 min. The anthocyanin concentration in the supernatant was measured spectrophotometrically at 530 and 657 nm and the absorbance values were indicated as A530 and A657. The extinction coefficient of 31.6 M1 cm1 was used to convert the absorbance values into anthocyanin concentration. The concentration was calculated using the following equation:
Anthocyanin concentration ðlmol=gÞ ¼ ½ðA530 0:33 A657 Þ=31:6 ½volume ðmlÞ=weight ðgÞ: Results were expressed as the average of triplicates. 2.5. Determination of total phenolic compounds Total phenolics in chard extract were determined with Folin–Ciocalteau reagent, according to the method of Slinkard and Singleton (1977) with some modifications. Briefly, 0.1 ml of the chard extract (20–100 lg/ml) was transferred into test tubes and their volumes made up to 4.6 ml with distilled water. After addition of 0.1 ml Folin–Ciocalteau reagent (previously diluted 3-fold with distilled water) and 0.3 ml 2% Na2CO3 solution, tubes were vortexed and then allowed to stand for 2 h with intermittent shaking. The absorbance was measured at 760 nm in a spectrophotometer. The total phenolic compounds in the chard extract were calculated as mg of pyrocatechol equivalent from the calibration curve and as mg pyrocatechol equivalents per mg of extract. The data were presented as the average of triplicate analyzes. 2.6. Determination of total flavonoid content Total flavonoid content was determined by using a method described by Sakanaka et al. (2005) using catechin as standard flavonoid compound. Briefly, 0.25 ml of the extract (1000 lg/ml) or (+)-catechin standard solution (20–100 lg/ml) was mixed with 1.25 ml of distilled water in a test tube, followed by the addition of 75 ll of a 5% sodium nitrite solution. After 6 min, 150 ll of a 10% aluminium chloride solution was added and the mixture was allowed to stand for a further 5 min before 0.5 ml of M NaOH was added. The mixture was brought to 2.5 ml with distilled water and mixed well. The absorbance was measured immediately at 510 nm using a spectrophotometer. Results were expressed as the average of triplicates. The results were expressed as the mean (±SD) mg of (+)-catechin equivalents per mg of extract. 2.7. Proline content Proline analysis was performed according to Bates (1973). Briefly, 50 mg extract was homogenized in 10 ml sulphosalicylic acid (3%) and filtrated through filter paper. Two milliliters of the filtrate were mixed with 2 ml of acid ninhydrin solution (1.25 g ninhydrin + 30 ml glacial acetic acid + 20 ml 6 M H3PO4) and 2 ml of glacial acetic acid and kept at 100 °C for 1 h. Then the reaction was stopped by transferring the mixture to an ice bath. Four milliliters of toluene were added to the mixture and vortexed for 15–20 s. The toluene phase was aspirated and absorbance at 520 nm was measured using pure toluene reference. A calibration curve was prepared with pure proline. Results were expressed as lg proline/gram extract. 2.8. Antiacetylcholinesterase activity The enzymatic activity was measured using an adaptation of the method described by Ingkaninan et al. (2003). Tris–HCl buffer (325 ll) (50 mM, pH 8), 100 ll of extract, with different concentrations and 25 ll of an enzyme solution containing 0.28 U/ml were incubated during 15 min. Subsequently, 75 ll 15 mM acetylcholine iodide (AChI) solution, and 475 ll. DTNB (3 mM) solution was added and the final mixture was incubated for 30 min at room temperature. The absorbance of the mixture was measured at 405 nm. A control mixture was performed without addition of the extract. Results were expressed as the average of triplicates. The percent inhibition was calculated using the following equation: Acetylcholinesterase inhibition (%) = (A0 Aı)/A0 100 A0 is the absorbance of the control A1 is the absorbance of the sample
2.9. DPPH radical scavenging activity The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of the chard extract was measured according to the procedure described by Brand-Williams et al. (1995). Appropriate dilution series (0.25–1 mg/ml) were prepared for
O. Sacan, R. Yanardag / Food and Chemical Toxicology 48 (2010) 1275–1280 each aqueous extract in methanol 0.1 ml of each dilution was added to 3.9 ml of a 6 105 M methanolic solution of DPPH followed by vortexing. The mixture was shaken vigorously and allowed to stand in the dark at room temperature for 30 min. The decrease in absorbance of the resulting solution was then measured spectrophotometrically at 517 nm against methanol. All measurements were made in triplicate and averaged. The DPPH radical scavenging activity was calculated using the following equation:
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DMPD+ scavenging activity (%) = (A0 A1/A0) 100 A0 is the absorbance of the initial concentration of DMPD+ A1 is the absorbance of the remaining concentration of DMPD+ in the presence of chard.
2.14. Statistical analysis DPPH radical scavenging activity (%) = (A0 A1/A0) 100 A0 is the absorbance of the control A1 is the absorbance of the sample
2.10. ABTS+ scavenging activity The ABTS+ scavenging activity of the chard extract was measured according to the procedure described by Arnao et al. (2001). The stock solutions included 7.4 mM ABTS+ solution and 2.6 mM potassium persulfate solution. The working solution was prepared by mixing the two stock solutions in equal quantities and allowing them to react for 12 h at room temperature in the dark. The solution was then diluted by mixing 1 ml ABTS+ solution with 60 ml methanol to obtain an absorbance of 1.1 ± 0.02 units at 734 nm using the spectrophotometer. Fresh ABTS+ solution was prepared for each assay. Chard extracts (150 ll) were allowed to react with 2850 ll of the ABTS+ solution for 2 h in a dark condition. Then the absorbance was taken at 734 nm using the spectrophotometer. The ABTS+ scavenging activity was calculated using the following equation: ABTS radical scavenging activity (%) = (A0 A1/A0) 100 A0 is the absorbance of the control A1 is the absorbance of the sample
2.11. Hydroxy radical scavenging activity The effect of extract on hydroxyl radicals was assayed by using the deoxyribose method (Chung et al., 1997). The reaction mixture contained 0.45 ml of 0.2 M sodium phosphate buffer (pH 7.4), 0.15 ml of 10 mM 2-deoxyribose, 0.15 ml of 10 mM FeSO4-EDTA, 0.15 ml of 10 mM hydrogen peroxide, 0.525 ml of distilled water and 0.075 ml of extract solution in a tube. The reaction was started by the addition of hydrogen peroxide. After incubation at 37 °C for 4 h, the reaction was stopped by adding 0.75 ml of 2.8% TCA and 0.75 ml 1.0% of thiobarbituric acid. The mixture was boiled for 10 min, cooled in an ice bath and then measured at 520 nm. Hydroxyl radical scavenging activity was calculated in the following equation: Hydroxyl radical scavenging activity (%) = (A0 A1/A0) 100 A0 is the absorbance of the control reaction A1 is the absorbance of sample
2.12. Reducing power The reducing power of the chard extract was determined according to the method described by Oyaizu (1986). Different amounts of extracts (20–100 lg) in 1 ml of distilled water were mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml potassium ferricyanide (1%) and then the mixture was incubated at 50 °C for 30 min. Afterwards, 2.5 ml of TCA (10%) was added to the mixture to stop the reaction, then the mixture was centrifuged at 3000 rpm for 10 min. The supernatant (2.5 ml) was mixed with 2.5 ml distilled water and 0.5 ml FeCl3 (0.1%), and then absorbance was measured at 700 nm in a spectrophotometer. The reducing power of the tested samples increased with the absorbance values.
2.13. DMPD+ scavenging activity DMPD+ scavenging activity was performed according to Fogliano et al. (1999). DMPD+ (100 mM) was prepared by dissolving 209 mg of DMPD+ in 10 ml of deionized water and 1 ml of this solution was added to 100 ml of 0.1 M acetate buffer (pH 5.3), and the colored radical cation (DMPD+) was obtained by adding 0.2 ml of a solution of 0.05 M ferric chloride. The absorbance of this solution, which is freshly prepared daily, is constant up to 12 h at room temperature. Different concentrations of chard (10–30 lg/ml) were added in test tubes and the total volume was adjusted with distilled water to 0.5 ml. Ten minutes later, the absorbance was measured at 505 nm. One milliliter of DMPD+ solution was directly added to reaction mixture and its absorbance at 505 nm was measured. The buffer solution was used as a blank sample. The DMPD+ scavenging activity was calculated using the following equation:
Results were expressed as mean ± standard deviation of triplicate analyzes. The correlation coefficient (r2) between the parameters tested was established by regression analysis.
3. Results and discussion 3.1. Anthocyanins, total phenolic, total flavonoid and proline content Anthocyanins possess well known pharmacological properties and strong biological functions such as antiinflammatory, antitumor, antimutagenic and antioxidant activities (Kong et al., 2003). The anthocyanin content was found to be 0.47 ± 0.03 lmol/g for chard extract (Table 1). It was reported that some pharmacological activities of polyphenol compounds, like anthocyanosides may attributed to their antioxidant properties (Martin-Aragon et al., 1998). Phenolics or polyphenols are secondary plant metabolites that are ubiquitously present in plants and plant products. Polyphenols are marker of the nutritional quality of foods. Polyphenols are known for their antioxidant activity as radical scavengers and possible beneficial roles in human health, such as reducing the risk of cancer, cardiovascular disease, other pathologies (Selappan et al., 2002). Plants containing high phenolic compounds can be a good source of antioxidants. For this reason, this information has led to the determination of the total phenolic content of the sample under study. It was determined that there was 31.09 ± 3.34 lg pyrocatechol equivalent of phenolic compounds in the 1 mg of the water extracts of chard (Table 1). A high correlation was observed between the total phenolic content and hydroxyl radical scavenging activity (r2 = 0.9931), ABTS+ scavenging activity (r2 = 0.9628), DPPH radical scavenging activity (r2 = 0.9596), DMPD+ scavenging activity (r2 = 0.9392) and reducing power (r2 = 0.9596) of the extract. These data are in accordance with others, which have shown that a high total phenolic content increases antioxidant activity, and that there is a linear correlation between phenolic content and antioxidant activity (Holasova et al., 2002). These results indicate that the higher antioxidant activity of chard extract may be associated with its total phenolic content. Flavonoids are natural phenolic compounds and well known antioxidants. Therefore dietary intake of flavonoid-containing foods was suggested to be of benefit for the preservation from free radical damage. The concentration of flavonoids in the extracts was expressed as lg of epicatechin equivalents per mg of the extract, as shown in Table 1. It was determined that there was 11.88 ± 1.46 lg epicatechin equivalent of phenolic compounds in 1 mg of the water extracts of chard (Table 1). Wu et al. (2003) has demonstrated that some amino acids also have antioxidant properties. Proline is one of these antioxidant amino acids. Proline is intracellular nonenzymatic ROS scavenging molecule (Xu et al., 2009). Proline provides protecting against stress by maintaining redox homeostasis (Hoque et al., 2008) and scavenging free radicals and ROS (Sharma and Dietz, 2006). In this study, the proline contents increased with increasing concentration and high proline contents (427.74 ± 24.41 lg/g extract) were seen in 10 mg water extract (Table 1). 3.2. Acetylcholinesterase inhibitor activity Plants have been used traditionally to enhance cognitive function and to alleviate other symptoms associated nowadays with
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Alzheimer’s disease (Howes and Houghton, 2003). In this study, AChE inhibitor activity of chard was found to increase dosedependently, the results were expressed as EC50 values, calculated from the regression equations prepared from the concentrations of the samples. A high AChE inhibition (20.98 ± 5.25%) was seen in 6 lg/ml (Table 1). EC50 values were found 14.43 ± 2.76 lg/ml. Sterols, terpenoids, oils, flavonoids, alkaloids and other phenolic compounds have been shown to possess antiacetylcholinesterase properties (Ji and Zhang, 2008), these constituents, flavonoids in particular, may have a particular contribution in the occurence of antioxidant and antiacetylcholinesterase activity of chard. This result is similar to the work of Lin et al. (2008) who reported this activity in the aqueous extract of Radix paeoniae rubra and other plants. Adsersen et al. (2006) have reported that Rosmarinus officinalis water extract showed low AChE inhibition with an average 12% value and Mentha spicata 3% inhibition of 0.1 mg/ml, respectively. It was evident that chard extract had a stronger effect on AChE inhibition compared to various plant extracts in literature (Adsersen et al., 2006; Lin et al., 2008). Also, Delwing et al. (2003) have shown that proline inhibited AChE activity from rat cerebral cortex in vivo and in vitro. The data suggest that the inhibitor effect of proline on AChE activity is associated with oxidative stress. In this study, in vitro inhibition of acetylcholinesterase by chard extract is reported for the first time. The results indicate that chard extract may offer great potential for the treatment of AD. 3.3. DPPH radical scavenging activity Antioxidant properties, especially radical scavenging activities, are very important due to the deleterious role of free radicals in foods and in biological systems. Excessive formation of free radicals accelerates the oxidation of lipids in foods and decreases food quality and consumer acceptance (Min, 1998). The model of scavenging the stable DPPH radical is a widely used method to evaluate antioxidant activities in relatively short time as compared to other methods. DPPH is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. Fig. 1 shows, the dose response curves of DPPH radical scavenging activity of the extracts from chard. The extracts were capable of scavenging DPPH radicals in a concentration-dependent manner. BHA and BHT were used as references for radical scavengers. The scavenging activity of chard extract, BHA, and BHT on DPPH radicals increased between 20–100 lg/ml and were 85.03 ± 0.77%, 81.61 ± 5.96%, and 47.55 ± 4.04% at a concentration of 100 lg/ml, respectively. Chard extract and BHA showed similar DPPH radical scavenging activity, while BHT was a considerably less effective DPPH radical scavenger. The DPPH radical scavenging activity was correlated with phenolic compounds (r2 = 0.9596). These results indicated that the radical scavenging capacity of the water extract might be mostly related to concentration of phenolic hydroxyl groups. DPPH scavenging activity is best presented by EC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution. A higher DPPH radical scavenging activity was associated with a lower EC50 value. EC50 values for chard extracts, BHA and BHT on DPPH radical scavenging activity were found as 23.85 ± 1.63,
47.61 ± 0.91 and 80.83 ± 0.72 lg/ml. Pyo et al. (2004) have reported that Beta vulgaris subspecies cycla methanol extract showed the highest activity at 500 lg/ml (87.00 ± 1.2%). This value was lower than our result (85.03 ± 0.77%, at a concentration of 100 lg/ml). For this reason, in our study, chard extract had a stronger effect on the DPPH radicals compared to Beta vulgaris subspecies cycla. 3.4. ABTS+ scavenging activity ABTS+ scavenging activity is also one of the most commonly used methods to evaluate the antioxidant activity. The ABTS+ scavenging activity of water extract of chard compared to rutin are shown in Fig. 2. ABTS+ scavenging activity increased with increasing concentration, reaching 18.56 ± 1.77% at 400 lg/ml and this value was much lesser than that of the positive controls, rutin 99.77 ± 0.01% at a same concentration. EC50 values for chard extracts, and rutin on ABTS radical scavenging activity were found as 1093.14 ± 52.02, and 113.70 ± 3.24 lg/ml, respectively. A high correlation was observed between ABTS radical scavenging activity and phenolic compounds (r2 = 0.9628). 3.5. Hydroxyl radical scavenging activity Hydroxyl radical is the most reactive among ROS and possesses the shortest half-life period. Hydroxyl radical causes oxidative damage to DNA, proteins, lipids (Moncada et al., 1991). The effect of chard on inhibition of free radical-mediated deoxyribose damage was assessed by means of the Fe2+ dependent DNA damage assay. The Fenton reaction generates hydroxy radical, which degrades DNA deoxyribose, using Fe2+ salts as a catalyst. Hydroxy radical may attack DNA either at the sugar or the base, giving rise to toxic products. Fig. 3 shows, the dose response curves of radical scavenging activities of the extract and reference antioxidants on the hydroxyl radicals and can be formed from superoxide anion and hydrogen peroxide in the presence of metal ions such as copper or iron. Chard extract scavenged hydroxyl radicals by 12.03 ± 1.37% at 20 lg/ml and 20.24 ± 6.28% at 60 lg/ml. Trolox and BHA exhibited good scavenging activity of 54.31 ± 4.30% and 52.00 ± 3.43% at a concentration of 60 lg/ml, respectively. The hydroxyl radical scavenging effect of water extract (20.24 ± 6.28%) was nearly equal to that of ascorbic acid (26.47 ± 2.86%) with EC50 values of water extract (157.94 ± 41.31 lg/ml) lower than that of Trolox (46.76 ± 4.17 lg/ml), ascorbic acid (116.04 ± 4.75 lg/ml) and BHA (51.54 ± 2.24 lg/ml). The hydroxyl scavenging radical activity in water extract was correlated with phenolic contents (r2 = 0.9931). 3.6. Reducing power The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity (Bhandari and Kawabata, 2004). Fig. 4 shows, the extent of the reduction, in terms of absorbance values at 700 nm. The reducing power of water extract (0.069 ± 0.003) was not concentration dependent and was found to be below those of tocopherol (0.294 ± 0.001), ascorbic acid (0.432 ± 0.003) and BHA (0.538 ± 0.001) at 100 lg/ml. The water extract showed lower reducing power than the standards. Reduc-
Table 1 Total anthocyanins, total phenolic compounds (PC, as pyrocatechol equivalents), total flavonoids (as epicatechin equivalents) and proline contents, and antiacetylcholinesterase (AChE) activity in water extract from chard. Extract
Water
Anthocyanins (lg mol/g extract) 0.47 ± 0.03
PC (lg pyrocatechol/ mg extract) 31.09 ± 3.34
Flavonoids (lg catechin/mg extract) 11.88 ± 1.46
Proline (lg/g extract) 427.74 ± 24.41
AChE Inhibition % (6 lg extract/ml)
EC50 (lg extract/ml)
20.98 ± 5.25
14.43 ± 2.76
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ing power of extract and standards decreased in order of BHA > ascorbic acid > a-tocopherol > water extract. The total phenolic contents were correlated with reducing power (r2 = 0.9922) for water extract. The results demonstrate the electron donor properties of chard extract for neutralizing free radicals by forming stable products.
DPPH radical scavenging activity (%)
90 80 70 Chard BHA BHT
60 50
3.7. DMPD+ scavenging activity
40 30 20 10 0 0
20
40
60
80
100
120
Concentration μg/ml Fig. 1. DPPH radical scavenging activity of the water extract from chard. BHA and BHT were used as reference antioxidant. Values are means ± SD (n = 3).
100
0.6 Chard α-Tocopherol
80
0.5
Ascorbic Acid BHA
60
Absorbance (700nm)
ABTS radical scavenging activity (%)
120
The antiradical capacity was analyzed by DMPD+ assay. The principle of the DMPD+ assay is that at acidic pH and in the presence of a suitable oxidant solution. DMPD can form a stable and colored radical cation (DMPD+). This reaction is rapid, and the end point, which is stable, is taken as a measure of the antioxidative efficiency. Therefore, this assay reflects the ability of radical hydrogen-donors to scavenge the single electron from DMPD+. This method ensures low cost and high reproducible analysis (Fogliano et al., 1999). As shown in Fig. 5, chard extract was an effective DMPD+ radical scavenger in a concentration-dependent manner. Water extract scavenged DMPD+ radicals by 34.05 ± 0.19% at 10 lg/ml and 47.51 ± 0.30% at 50 lg/ml. Cysteine and Trolox exhibited good scavenging activity of 99.89 ± 0.00% and 96.53 ± 0.16% at a concentration of 50 lg/ml, respectively. EC50 value was found
Chard Rutin 40
20
0 0
50
100
150
200
250
300
350
400
0.4
0.3
0.2
0.1
450
Concentration (μg/ml) 0
Fig. 2. ABTS radical scavenging activity of the water extract from chard. Rutin was used as reference antioxidant. Values are means ± SD (n = 3).
0
20
40
60
80
100
120
Concentration (μg/ml) Fig. 4. Reducing power of the water extract from chard. Values are means ± SD (n = 3).
120
50
DMPD radical scavenging activity (%)
Hydroxyl radical scavenging activity (%)
60
Chard
40
BHA Ascorbic Acid Trolox
30
20
10
100
80
Chard Trolox Cysteine
60
40
20
0 0
10
20
30
40
50
60
70
Concentration (μg/ml)
0 0
10
20
30
40
50
60
Concentration (μg/ml) Fig. 3. Hydroxyl radical scavenging activity of the water extract from chard. BHA, ascorbic acid and Trolox were used as reference antioxidants. Values are means ± SD (n = 3).
Fig. 5. DMPD radical scavenging activity of the water extract from chard. Trolox and cysteine were used as reference antioxidant. Values are means ± SD (n = 3).
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9.87 ± 0.27 lg/ml for cysteine and 10.38 ± 0.28 lg/ml for Trolox and 45.21 ± 0.75 lg/ml for chard extract. A high correlation was observed between DMPD+ scavenging activity and phenolic compounds (r2 = 0.9392). 4. Conclusion Numerous epidemiological and clinical studies have proven that the consumption of foods and beverages rich in antioxidant phytochemicals can have a beneficial effect upon human health by potentially affecting the etiology of chronic diseases mediated through reactive oxygen species such as free radicals. The studies to date on chard extract suggested that they might be used as an important natural antioxidant source due to its high levels of phenolics, flavonoids and proline. According to this study, a strong correlation has been observed between total phenolic content and antioxidative activity, which shows that the phenolic content might be responsible for the antioxidant activity. It can be concluded that water extract of chard in the way which they are consumed as a food stuff in Turkey, can be used as an accessible source of natural antioxidants with consequent health benefits. It is well known that free radicals are one of the causes of several diseases, such as Parkinson’s disease, Alzheimer type dementia etc. The great acetylcholinesterase inhibitor activity together with the antioxidant activity indicates that chard extract can be used in the prevention of neurodegenerative diseases such as AD and Parkinsons’s diseases. Thus, it can be concluded that water extract can also be used as an accessible source of natural antioxidants and antiacetylcholinesterase with consequent health benefits. Conflict of interest statement The authors declare that there are no conflicts of interest. Acknowledgments The authors would like to thank Professor Neriman Özhatay (Faculty of Pharmacy, Istanbul University) for the identification of chard. This study was supported by the Research Fund of Istanbul University, Project No: UDP-3972/24062009. References Abdel-Hameed, E.S., 2009. Total phenolic contents and free radical scavenging activity of certain Egyptian Ficus species leaf samples. Food Chem. 114, 1271– 1277. Adsersen, A., Gauguin, B., Gudiksen, L., Jager, A.K., 2006. Screening of plants used in Danish folk medicine to treat memory dysfunction for acetylcholinesterase inhibitor activity. J. Ethnopharmacol. 104, 418–422. Arnao, M.B., Cano, A., Acosta, M., 2001. The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem. 73, 239–244. Bates, S.L., 1973. Rapid determination of free proline for water stres studies. Plant Soil 39, 205–207. Bhandari, M.R., Kawabata, M., 2004. Organic acid, phenolic content and antioxidant activity of wild yam (Dioscorea spp.) tubers of Nepal. Food Chem. 88, 163–168. Bolkent, S ß ., Yanardag˘, R., Tabakog˘lu-Og˘uz, A., Özsoy-Saçan, Ö., 2000. Effects of chard (Beta vulgaris L var. cicla) extract on pancreatic B cells in streptozotocindiabetic rats: a morphological and biochemical study. J. Ethnopharmacol. 73, 251–259. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm.-Wiss. Technol.-Food Sci. Technol. 28, 25–30. Chung, S.K., Osawa, T., Kawakishi, S., 1997. Hydroxyl radical scavenging effects of species and scavengers from brown mustard (Brassica nigra). Biosci. Biotechnol. Biochem. 61, 118–123. Chung, Y.-C., Chen, S.-J., Hsu, C.-K., Chang, C.-T., Chou, S.-T., 2005. Studies on the antioxidative activity of Graptopetalum paraguayense E. Walhter. Food Chem. 91, 419–424. Delwing, D., Chiarani, F., Delwing, D., Bavaresco, C.S., Wannmacher, C.M.D., Wajner, M., Wyse, T.S., 2003. Proline reduces acetylcholinesterase activity in cerebral cortex of rats. Metab. Brain Dis. 18, 79–86.
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