Abstract
Context: Consumers have become more interested in beneficial effects of vegetables, fruits, and tea to protect their health.
Objective: The antioxidant potential and anticholinesterase activity of eleven edible plants were investigated.
Material and methods: The dichloromethane, ethanol and water extracts prepared from celery [Apium graveolens L. (Umbelliferae)], Jerusalem artichoke [Helianthus tuberosus L. (Compositae)], spinach [Spinacia oleracea L. (Chenopodiaceae)], chard [Beta vulgaris L. var. cicla (Chenopodiaceae)], purslane [Portulaca oleracea L. (Portulacaceae)], ispit, or borage [Trachystemon orientale (L.) G. Don (Boraginaceae)], garden rocket [Eruca sativa Mill. (Brassicaceae)], red cabbage [Brassica oleracea L. var. capitata f. rubra DC. (Cruciferae)], lime flower [Tilia tomentosa Moench (Tiliaceae)], cinnamon [Cinnamomum cassia Presl. (Lauraceae)], and rosehip [Rosa canina L. (Rosaceae)], were tested to determine their antioxidant and anticholinesterase activities by using CUPRAC (cupric reducing antioxidant capacity) and Ellman methods, respectively, for the first time.
Results: As a result, the dichloromethane, ethanol and water extracts of cinnamon showed the best antioxidant effect among the extracts of the tested plants. The ethanol extract of cinnamon exhibited 63.02% inhibition against acetylcholinesterase and 85.11% inhibition against butyrylcholinesterase (BChE) at 200 µg/mL concentration while the dichloromethane extract of garden rocket possessed the highest inhibition (91.27%) against BChE among all the tested extracts.
Discussion and conclusion: This study indicated that the ethanol extract of cinnamon may be a new potential resource of natural antioxidant and anticholinesterase compounds.
Introduction
Reactive oxygen species (ROS) are generated in specific organelles of cells under normal physiological conditions. In healthy humans, the production of ROS is balanced by the antioxidative defense mechanisms which include radical scavenging enzymes and cellular antioxidants. Various environmental pollutants (smog, cigarette smoke, pesticides, and herbicides), aging and diseases can increase the production of ROS and/or inhibit the antioxidative defense systems (CitationWong et al., 2006). These reactive oxygen radicals (superoxide, hydroxyl, peroxyl, alkoxy radicals) can cause oxidation of cell lipids and DNA damage that may lead to serious chronic diseases such as diabetes mellitus, cataracts, cancer, neurodegenerative, and cardiovascular diseases (CitationDudonné et al., 2009). Alzheimer’s disease (AD), being one of the neurodegenerative diseases, is an important health problem for elderly people. Since some of the clinical effects of herbs are closely related to their antioxidant activity, the use of antioxidants, especially plants and their constituents, in diet and as supplements, may be relevant in slowing AD progression and minimizing neuronal degeneration (CitationGu & Weng, 2001; CitationHowes et al., 2003).
Vitamins (β-carotene, vitamins C and E) and polyphenols (flavonoids, tannins, catechins) possess a wide range of biological effects such as antioxidant, antimicrobial, anti-inflammatory, and vasodilatory actions (CitationWong et al., 2006). They scavenge radicals and inhibit the chain initiation or break the chain propagation. Epidemiological studies show that a diet rich in vegetables, fruits and tea with high phenolic and vitamin contents can reduce the incidence of certain cancers, cardiovascular disease, and cerebrovascular disease and age-related degenerative brain disorders (CitationHertog et al., 1997; CitationPodsedek, 2007). For this reason, consumers have become more interested in beneficial effects of vegetables, fruits, and tea to protect their health.
In Turkey, the roots of celery, and Jerusalem artichoke, the aerial parts of spinach, chard, and purslane may be cooked in various ways. Ispit (vernacular name of the plant) is a perennial herb growing in Istanbul and Northern Turkey. In spring, its fresh aerial parts, with a spinach-like taste, are consumed traditionally for culinary use (CitationBaytop, 1984). Garden rocket and red cabbage may be eaten raw in salads. Lime flower and fruit of rosehip may be prepared as tea. The bark of cinnamon is commonly consumed in desserts, cakes, cookies, and tea.
The objectives of this study were to evaluate in vitro the antioxidant capacity and anticholinesterase activity of dichloromethane, ethanol and water extracts prepared from eleven edible plants such as celery [Apium graveolens L. (Umbelliferae)], Jerusalem artichoke [Helianthus tuberosus L. (Compositae)], spinach [Spinacia oleracea L. (Chenopodiaceae)], chard [Beta vulgaris L. var. cicla (Chenopodiaceae)], purslane [Portulaca oleracea L. (Portulacaceae)], ispit [Trachystemon orientale (L.) G. Don (Boraginaceae)], garden rocket [Eruca sativa Mill. (Brassicaceae)], red cabbage [Brassica oleracea L. var. capitata f. rubra DC. (Cruciferae)], lime flower [Tilia tomentosa Moench (Tiliaceae)], cinnamon [Cinnamomum cassia Presl. (Lauraceae)], and rosehip [Rosa canina L. (Rosaceae)]. The roots of celery, and Jerusalem artichoke, the aerial parts of spinach, chard, purslane, ispit, and garden rocket, the leaves of red cabbage, lime flower, the fruit of rosehip and the bark of cinnamon were used to prepare the extracts. In fact, there are some antioxidant activity studies on these eleven plant extracts (CitationYildirim et al., 2000; CitationPyo et al., 2004; CitationSu et al., 2007; CitationCho et al., 2008; CitationSacan et al., 2008; CitationHuang et al., 2009; CitationJagdish et al., 2009). In this study, the antioxidant activity of the selected plants was carried out for the first time using the cupric reducing antioxidant capacity (CUPRAC) method which was introduced and developed by CitationApak et al. (2004). To date, the antioxidant activity of ispit (Trachystemon orientale (L.) G. Don) and Jerusalem artichoke, and the anticholinesterase activity of these plants have not been reported before.
Materials and methods
Chemicals
Dichloromethane, ethanol, methanol, ammonium acetate (NH4OAc), sodium hydrogen phosphate (Na2HPO4), and sodium dihydrogen phosphate (NaH2PO4) (Riedel-de Haën), α-tocopherol (Aldrich), DTNB [5,5-dithiobis(2-nitro benzoic acid)], acetylcholinesterase (AChE), butyrylcholinesterase (BChE), copper (II) chloride dihydrate (CuCl2.2H2O) (Sigma), butylated hydroxytoluene (Merck), neocuproine (2,9-dimethyl-1,10-phenanthroline), galantamine hydrobromide (Sigma-Aldrich), acetylthiocholine iodide (Applichem), butyrylthiocholine iodide (Fluka) were purchased from the indicated sources. Distilled water was used.
Instrumentation
A Thermo pH-meter (USA), an Elma S15 ultrasonic bath (Germany), a vortex (LMS, Japan), and a BioTek Power Wave XS (USA) were used for the activity assays.
Plant materials
Fresh (celery, Jerusalem artichoke, spinach, chard, purslane, ispit, garden rocket, red cabbage) and dried (lime flower, cinnamon, rosehip) plant materials were purchased from a local market in Istanbul in March, 2009. Botanical identification was carried out by E. Akalin (Department of Pharmaceutical Botany, Faculty of Pharmacy, Istanbul University).
Preparation of extracts
The fresh samples were washed with water and damaged portions were removed. Then both fresh and dried plant materials (50 g) were chopped up and separated to three parts for macerating with 150 mL dichloromethane, ethanol, and water at room temperature three times (6 h × 3), individually. After filtration of each extract, the solvents were evaporated to dryness in vacuo, and the crude extracts were obtained, separately. The yield of the extracts varied from 0.11 to 5.22% (w/w) ().
Table 1. Yields of the extracts.
Activity tests
CUPRAC
CUPRAC of the extracts was determined according to the method described by CitationApak et al. (2004). All crude extracts were dissolved in distilled water to prepare their stock solution at 1000 µg/mL concentration. Aliquots of 61 mL of 1.0 × 10−2 M copper (II) chloride, 61 µL of NH4OAc buffer (1 M, pH 7.0), and 61 µL of 7.5 × 10−3 M neocuproine solution were mixed, x µL sample solution (2.5, 6.25, 12.5, and 25 µL) and (67 − x) µL distilled water were added to make the final volume 250 µL. The tubes were stopped, and after 1 h, the absorbance at 450 nm was measured against a reagent blank using a BioTek Power Wave XS.
Anticholinesterase activity
Acetyl and BChE inhibitory activities were measured by slightly modifying the spectrophotometric method developed by CitationEllman et al. (1961). Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates of the reaction and DTNB were used for the measurement of the anticholinesterase activity. All crude extracts were dissolved in ethanol to prepare their stock solution at 4000 µg/mL concentration. Aliquots of 150 mL of 100 mM sodium phosphate buffer (pH 8.0), 10 µL of sample solution and 20 µL AChE (or BChE) solution were mixed and incubated for 15 min at 25°C, and 10 µL of DTNB is added. The reaction was then initiated by the addition of 10 µL acetylthiocholine iodide (or butyrylthiocholine iodide). Final concentration of the tested solutions was 200 µg/mL. The hydrolysis of these substrates were monitored using a BioTek Power Wave XS by the formation of yellow 5-thio-2-nitrobenzoate anion as the result of the reaction of DTNB with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide or butyrylthiocholine iodide, at a wavelength of 412 nm. Ethanol was used as a solvent to dissolve the samples and controls.
Statistical analysis
The results were mean ± SD of three parallel measurements. All statistical comparisons were made by means of Student’s t-test, p values < 0.05 were regarded as significant.
Results and discussion
Numerous methods which are based on different mechanisms of reaction are available to determine the antioxidant activity of natural products. In this study, the antioxidant activity was evaluated by using CUPRAC method which is one of the electron transfer-based methods. Due to the lower redox potential of the CUPRAC reagent, reducing sugars and citric acid—which are not true antioxidants but oxidizable substrates in other similar assays—are not oxidized with the CUPRAC reagent. An advantage to other electron transfer-based assays as ABTS and Folin, CUPRAC values were acceptable in regard to its realistic pH close to the physiological pH (CitationYildiz et al., 2008).
The plant extracts which are prepared by using different solvents possess various kinds of secondary metabolites. In this study, the extracts of the tested plants were prepared with dichloromethane, ethanol, and water to understand which one is much more responsible for the antioxidant and anticholinesterase activities. Since these tested plants are edible, water was especially used as solvent. In addition, further phytochemical studies can be carried out on the active extract to isolate their active compounds.
As seen in , the dichloromethane extract of cinnamon exhibited the highest antioxidant activity among the tested dichloromethane extracts at all concentrations. The dichloromethane extracts of red cabbage and ispit possessed almost the same effect which was lower than that of the cinnamon extract and standards, BHT and α-tocopherol, but higher than that of the other dichloromethane extracts, at all concentrations. The ethanol extract of cinnamon showed higher antioxidant activity than α-tocopherol as well as than the other tested ethanol extracts at all concentrations (). The ethanol extract of ispit exhibited the best antioxidant effect among the other ethanol extracts, except for cinnamon extract. The cupric reducing antioxidant capacities of some water extracts were in an increasing order, cinnamon>red cabbage>purslane (). CitationDudonné et al. (2009) used DPPH, ABTS, FRAP, ORAC, and SOD assays to determine the antioxidant activity of the aqueous extract of cinnamon and they also found that it possessed very strong antioxidant effect. In addition, the methanolic extract of Cinnamomum verum barks exhibited reducing power, metal chelating, and free radical scavenging activities and total antioxidant activity in linoleic acid emulsion system (CitationMathew & Abraham, 2006). In this study, the ethanol, water and dichloromethane extracts of cinnamon bark showed good CUPRAC. Here, the phenolic compounds of cinnamon bark may be responsible for this activity as well as in the results of the studies mentioned above.
Table 2. Absorbance values of dichloromethane extracts of eleven plants, BHT and α-Toc in the CUPRAC assay†.
Table 3. Absorbance values of ethanol extracts of eleven plants, BHT and α-Toc in the CUPRAC assay†.
Table 4. Absorbance values of water extracts of eleven plants, BHT and α-Toc in CUPRAC assay†.
Twenty-three different anthocyanins, which were cyanidin derivatives highly conjugated with sugars and acylated groups, were the main constituents in red cabbage. Podsedek et al. found that red cabbage possessed strong O2·−, DPPH· and ABTS·− radical scavenging activities and inhibited a lipid peroxidation in linoleic acid emulsion (CitationPodsedek et al., 2006). In this study, the dichloromethane and water extracts of red cabbage showed moderate CUPRAC. The results of this electron transfer-based assay can also be related to the phenolic compounds, especially anthocyanins, which were the major contributor to the free radical scavenging activity of red cabbage.
The antioxidant potential of spinach, chard, purslane, garden rocket, lime flower, and rosehip was also determined by using different methods of previous studies (CitationYildirim et al., 2000; CitationPyo et al., 2004; CitationSu et al., 2007; CitationCho et al., 2008; CitationSacan et al., 2008; CitationHuang et al., 2009; CitationJagdish et al., 2009). In this study, none of the tested material exhibited CUPRAC.
The dichloromethane extracts of red cabbage (46.92% inhibition) and rosehip (49.55% inhibition) showed almost the same AChE inhibitory activity. They possessed moderate AChE inhibitory activity at 200 µg/mL (). Interestingly, although all the red cabbage extracts exhibited moderate activity against AChE, all the celery, Jerusalem artichoke, spinach, chard, purslane, ispit, garden rocket, lime flower extracts, the ethanol, and water extracts of rosehip were found to be inactive against AChE. On the other hand, the ethanol extract of cinnamon exhibited the highest AChE inhibitory activity (63.02% inhibition) among all the tested extracts while its dichloromethane and water extracts were inactive.
Table 5. Anticholinesterase activity of eleven plants at 200 µg/mL†.
The dichloromethane extract of garden rocket exhibited the highest BChE inhibitory activity (91.27% inhibition) among all the tested extracts, although it was inactive against AChE. The ethanol extract of cinnamon showed also strong BChE inhibitory activity (85.11% inhibition) similar to galantamine. Against BChE, almost all the dichloromethane extracts exhibited higher activity than the ethanol and water extracts, except for cinnamon. The non-polar compounds may be responsible for this activity. However, no such a relationship was observed for AChE inhibitory activity.
Cinnamomum species have been applied in folk medicine for their various activities such as anti-inflammatory, cytotoxic, antidiarrheal, antifungal, antipyretic, antimicrobial (CitationKuo et al., 2008; CitationLin et al., 2008; CitationRao et al., 2008). CitationKim et al. (2007) determined the ability of 27 herbs to protect PC12 rat pheochromocytoma and primary neuronal cells from beta-amyloid (1–42) insult by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide reduction assay and lactate dehydrogenase efflux assay. They found that Chinese cinnamon being one of the tested plants protected cells from beta amyloid (1–42) insult (CitationKim et al., 2007). CitationNgoc et al. (2009) also reported that a methanol extract of the twigs of Cinnamomum cassia exhibited a strong tyrosinase-inhibitory activity (>85% inhibition at 100 µg/mL). Tyrosinase inhibitors which may have antimelanin synthesis activity offer a potential treatment for Parkinson’s disease.
Conclusion
The present report describes that the ethanol extract of cinnamon possessed both strong CUPRAC and anticholinesterase activity, which can be highly related to its phenolic compounds. Phytochemical studies are needed to characterize the active constituents of the ethanol extract prepared from cinnamon bark. Further laboratory and clinical assays of the active compounds present in the ethanol extract of cinnamon are also required in order to better understand its antioxidant and anticholinesterase potential.
Declaration of interest
This work was supported by the Research Fund of the Istanbul University: Project number: BYP-976. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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