PHYTOTHERAPY RESEARCH Phytother. Res. 29: 1304–1310 (2015) Published online 9 June 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.5378
Tyrosinase and Cholinesterase Inhibitory Potential and Flavonoid Characterization of Viola odorata L. (Sweet Violet) Ilkay Erdogan Orhan,1* Fatma Sezer Senol,1 Sinem Aslan Erdem,2 I. Irem Tatli,3 Murat Kartal2,4 and Sevket Alp5 1
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100, Ankara, Turkey Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, 06100, Ankara, Turkey 4 Department of Pharmacognosy, Faculty of Pharmacy, and Center of Phytotherapy Education, Research and Practice, Bezmialem Vakif University, 34093, Istanbul, Turkey 5 Department of Landscape Architecture, Faculty of Agriculture, Yuzuncu Yıl University, 65080, Van, Turkey 2 3
Inhibitory potential of the dichloromethane, ethyl acetate, ethanol, and aqueous extracts of Viola odorata L. (VO) was investigated against tyrosinase (TYR) and cholinesterases by microplate assays. The antioxidant activity was tested using six in vitro assays. Only the ethanol extract inhibited TYR (80.23 ± 0.87% at 100 μg mL1), whereas none of them were able to inhibit cholinesterases. The extracts were more able to scavenge NO radical (31.98 ± 0.53–56.68 ± 1.10%) than other radicals tested, and displayed low to moderate activity in the rest of the assays. HPLC analysis revealed that the aqueous extract of VO contained a substantial amount of vitexin (18.81 ± 0.047 mg g1 extract), while the ethanol extract also possessed rutin (1.31 ± 0.013 mg g1 extract) and vitexin (4.65 ± 0.103 mg g1 extract). Furthermore, three flavonoids (rutin, isovitexin, and kaempferol-6-glucoside) were isolated from the ethanol extract. This is the first report on TYR inhibitory activity of VO as well as presence of vitexin and isovitexin in this species. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Viola odorata; tyrosinase; cholinesterase; antioxidant activity; flavonoid; HPLC.
INTRODUCTION The genus Viola L. (Violaceae), which has approximately 400 flowering species throughout the world, comprises 33 taxa in Turkey (Dinc et al., 2007). Among them, Viola odorata L. (sweet violet, garden violet, VO), native to Europe and Asia, is cultivated largely because of its great commercial value resulted from its essential oil content with a sweet, powdery, and woody-floral scent used popularly in cosmetic industry, production of perfumes, toiletry, and flavorings in soufflés, cream, and similar desserts (Chawia, 2000). The blue-violet or whitish flowers are also used for ornamental purposes as well as extensively as a garnish and in chocolates. Newly opened violet flowers may be used to decorate salads or in stuffings for poultry or fish. In the USA, this French violet syrup is used to make violet scones and marshmallows (Monadi and Rezaie, 2013). VO, known as ‘kokulu menekşe’ in Turkish, has been also recorded to be used in traditional medicines for various purposes. For instance, this species has been used to treat cough in Anatolian folk medicine (Akaydin et al., 2013), expectorant, sedative, sweating stimulation, as laxative/purgative, and against bronchitis in Portugal (Neves et al., 2009). The plant is also used as herbal tea in India for anti-aging, anti-stress, appetizer, and stimulant purposes (Naithani et al., 2006). * Correspondence to: Ilkay Erdogan Orhan, Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
Polyphenol oxidases (PPOs), found in plants, fungi, and other organisms, are a copper-containing enzyme family with dinuclear copper centers, and the monophenol oxidase type of PPOs is known as tyrosinase (TYR, EC 1.14.18.1) (Mayer, 2006). The main function of PPOs is to catalyze browning reactions in vegetables and fruits resulting from the oxidation and dehydrogenation reactions of polyphenols present in the plants, which finally leads to formation of reddishbrown ortho-quinones as well as in negative influence on their color, taste, flavor, and nutritional value. Because browning causes major agricultural and economic problems, inhibition of TYR is quite important in the food industry. Additionally, inhibitors of TYR are popularly used in skin-whitening products in cosmetics industry as the demand grows toward these particular cosmetic agents. On the other hand, inhibition of cholinesterase enzyme family consisting of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is the most effective approach toward the treatment of Alzheimer type of dementia as it has become a major health problem especially in developed countries because of higher population of the elderly (Orhan et al., 2011). Although there has been extensive research going on discovery of new cholinesterase and TYR inhibitory compounds of both synthetic and natural origins, a great need still remains for new inhibitors of these enzymes because of either side effects or low efficacy of current inhibitors of AChE and BChE. Besides, the number of the present TYR inhibitors is quite limited, and new inhibitors are in demand particularly for cosmetics industry. On this purpose, in continuation of our studies Received 12 March 2015 Revised 26 April 2015 Accepted 28 April 2015
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on finding new TYR inhibitors from plants, we have now aimed to investigate inhibitory potential of VO against TYR and along with its antioxidant activity in various in vitro assays by high-throughput screening method using enzyme-linked immunosorbent assay microplate reader. HPLC analysis has been carried out on the extracts of VO in order to quantify phenolic acids and flavonoid derivatives.
MATERIALS AND METHODS Plant material. The samples of VO were collected from the town of Ercis of Van province, Turkey, in 2010. The plant was identified by Dr Fazli Ozturk from Yuzuncu Yıl University (Van, Turkey) and the voucher specimen is kept in the Herbarium of Yuzuncu Yıl University (coded as F1399).
Extraction procedure. The air-dried and powdered aerial parts of VO (200 g) were extracted sequentially with dichloromethane (DCM), ethyl acetate (EtOAc), ethanol (75%, EtOH), and distilled water (H2O). The macerates obtained with DCM, EtOAc, and EtOH were evaporated in vacuo until dryness, while the H2O macerate was lyophilized. The extracts were kept in the freezer until the experiments were performed. Yield percentages (w/w) of the extracts are as follows: 1.29% for the DCM extract; 10.52% for the EtOAc extract; 5.70% for the EtOH extract; 9.87% for the H2O extract.
Isolation procedure of Viola odorata 1, 2, and 3. The EtOH extract (508.5 mg) was first dissolved in H2O, and the water-soluble portion was fractionated over a LiChroprep C18 column (Merck, Opfikon-Glattbrugg, Switzerland; C-18, Sepralyte, 40 μm, VLC, 150 g) (Fig. 1). Employment of H2O and methanol (MeOH) (0–100% MeOH) afforded five main fractions (Frs. A–E).
Figure 1. Isolation scheme of Viola odorata 1, 2, and 3. Copyright © 2015 John Wiley & Sons, Ltd.
Purification of fraction A (98.5 mg) by silica gel column chromatography [Merck, 230–400 mesh, 30 g, CHCl3/ MeOH (85:15 → 70:30)] furnished VO-1 (5.3 mg), VO-2 (5.4 mg), and VO-3 (3.2 mg). Structure elucidation of Viola odorata 1, 2, and 3. Structure elucidation of the isolated compounds VO 1, 2, and 3 from fraction A was carried out by spectral techniques using 1H nuclear magnetic resonance (NMR) and 13C NMR, and detailed data was compared with the literature (Crews et al., 1998). The structures of compounds VO-1, VO-2, and VO-3 were identified as rutin, isovitexin, and kaempferol-6-glucoside, respectively (Fig. 2). Quantification of rutin and vitexin in the extracts by HPLC. Analyses were performed using an Agilent Technologies 1200 Series high pressure liquid chromatography (HPLC), including a binary pump, a vacuum degasser, an autosampler, and a diode array detector. Chromatographic separations were performed on Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm). A mobile phase consisting of two eluents (acetonitrile and 40 mM formic acid) was used for separation with a gradient elution at a flow rate of 1 mL min1. All solvents were filtered through a 0.45-μm Millipore filter before use. Detection wavelength was set at 254 nm for rutin and 330 nm for vitexin. The injection volume was 20 μL. Standard solutions of rutin (1–100 μg mL1) and vitexin (1–100 μg mL1) whose standards were purchased from Sigma-Aldrich Co. (Germany) were prepared in methanol. Five different concentration levels of rutin and vitexin were subjected to regression analysis to calculate calibration equation and correlation. Each injection was achieved in triplicate to see the reproducibility of the detector response at each concentration level. Identification of rutin and vitexin was made by comparing their retention time and ultraviolet (UV) spectra with those of pure standards. All the calculations concerning the quantitative analysis were performed with external standardization by measurement of peak areas.
Figure 2. Structures of Viola odorata 1, 2, and 3. Phytother. Res. 29: 1304–1310 (2015)
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Enzyme inhibition assays Cholinesterase inhibition. The AChE and BChE inhibitory activity was measured by slightly modified spectrophotometric method of Ellman et al. (1961). Electric eel AChE (Type-VI-S; EC 3.1.1.7, Sigma, St. Louis, MO, USA) and horse serum BChE (EC 3.1.1.8, Sigma, St. Louis, MO, USA) were used, while acetylthiocholine iodide and butyrylthiocholine chloride (Sigma, St. Louis, MO, USA) were employed as the substrates of the reaction. 5,5′-Dithio-bis(2-nitrobenzoic)acid (DTNB; Sigma, St. Louis, MO, USA) was used for the measurement of the anticholinesterase activity. Briefly, in this method, 140 μL of sodium phosphate buffer (pH 8.0), 20 μL of DTNB, 20 μL of test solution, and 20 μL of AChE/BChE solution were added by multichannel automatic pipette (Gilson Pipetman, Paris, France) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of 10 μL of acetylthiocholine iodide/butyrylthiocholine chloride. Hydrolysis of acetylthiocholine iodide/butyrylthiocholine chloride was monitored by the formation of the yellow 5-thio-2nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, catalyzed by enzymes at 412 nm utilizing a 96-well microplate reader (VersaMax Molecular Devices, Sunnyvale, CA, USA). The measurements and calculations were evaluated by using Softmax PRO 4.3.2.LS software. Percentage of inhibition of AChE/BChE was determined by the comparison of reaction rates of the samples relative to the blank sample (ethanol in phosphate buffer pH = 8) using the formula (ES)/E × 100, where E is the activity of enzyme without test sample and S is the activity of enzyme with test sample. The experiments were carried out in triplicate. Galanthamine (Sigma, St. Louis, MO, USA), the anticholinesterase alkaloid-type of drug obtained from the bulbs of snowdrop (Galanthus sp.), was used as the reference. Tyrosinase inhibition. Inhibition of tyrosinase (TYR) (EC 1.14.1.8.1; 30 U, mushroom tyrosinase, Sigma) was determined using the modified dopachrome method with L-DOPA as substrate (Masuda et al., 2005). The assays were conducted in 96-well microplate using enzyme-linked immunosorbent assay microplate reader (VersaMax Molecular Devices, USA) to measure absorbance at 475 nm. An aliquot of the extracts dissolved in DMSO with 80 μL of phosphate buffer (pH 6.8), 40 μL of tyrosinase, and 40 μL of L-DOPA were put in each well. Results were compared with control (DMSO). Baicalein (Sigma, St. Louis, MO, USA) was used as the reference. The percentage tyrosinase inhibition (I%) was calculated as follows: I % ¼ Absorbance ðAÞcontrol –AbsorbanceðAÞsample =Absorbance ðAÞcontrol 100
Antioxidant activity by radical-formation methods DPPH radical scavenging activity. The stable 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was determined by the method of Blois (1958). Copyright © 2015 John Wiley & Sons, Ltd.
The samples (30 μL) and the reference, which is dissolved in ethanol (75%), were mixed with 2700 μL of DPPH solution (1.5 × 104 M). The remaining DPPH amount was measured at 520 nm using a Unico 4802 UV-visible double beam spectrophotometer (Unico, Dayton, NJ, USA). Gallic acid (Sigma, St. Louis, MO, USA) was employed as the reference. Inhibition of DPPH in percent (I%) was calculated as given below: I% ¼ Ablank Asample =Ablank 100 where Ablank is the absorbance of the control reaction (containing all reagents except the test sample), and Asample is the absorbance of the extracts/reference. Analyses were run in triplicate, and the results were expressed as average values with SEM (standard error of the mean). DMPD radical scavenging activity. The assay is based on reduction of the purple-colored radical DMPD+ (N, N-dimethyl-p-phenylendiamine). According to the method (Schlesier et al., 2002), a reagent comprising 100 mM DMPD, 0.1 M acetate buffer (pH = 5.25), and 0.05 M ferric chloride solution, which led to the formation of DMPD radical, was freshly prepared, and the reagent was equilibrated to an absorbance of 0.900 ± 0.100 at 505 nm. Then, the reagent was mixed up with 50 μL of the extract dilutions, and absorbance was taken at 505 nm using a Unico 4802 UV-visible double beam spectrophotometer. Quercetin was employed as the reference, and the experiments were performed in triplicate. The results were calculated according to the same formula given for DPPH radical scavenging test and expressed as average values with SEM. Nitric oxide radical scavenging activity. The scavenging activity of the extracts against nitric oxide (NO) was assessed by the method of Marcocci et al. (1994). Briefly, the extract dilutions were mixed with 5 mM sodium nitroprusside and left to incubate for 2 h at 29 °C. An aliquot of the solution was removed and diluted with Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the occurred chromophore was measured at 550 nm using a Unico 4802 UV-visible double beam spectrophotometer. Inhibition of NO radical in percent (I%) was calculated as given in the following: I% ¼ Ablank Asample =Ablank 100 where Ablank is the absorbance of the control reaction (containing all reagents except the test sample), and Asample is the absorbance of the extracts. Analyses were run in triplicates, and the results were expressed as average values with SEM. Quercetin was the reference in this test. Antioxidant activity by metal-related and reducing power methods Metal-chelating capacity. The metal-chelating capacity of the extracts through ferrous ion was estimated by the method of Chua et al. (2008). Briefly, dilutions of the extracts were incubated with 2 mM FeCl2 solution. The reaction was initiated by the addition of 5 mM ferrozine into the mixture and left standing at ambient temperature for 10 min. The absorbance of the reaction mixture Phytother. Res. 29: 1304–1310 (2015)
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was measured at 562 nm using a Unico 4802 UV-visible double beam spectrophotometer. The ratio of inhibition of ferrozine-Fe2+ complex formation was calculated as follows: I% ¼ Ablank Asample =Ablank 100 where Ablank is the absorbance of the control reaction (containing only FeCl2 and ferrozine), and Asample is the absorbance of the extracts/reference. Analyses were run in triplicate, and the results were expressed as average values with SEM. The reference was employed as ethylenediamine tetraacetic acid in this assay.
Table 1. Tyrosinase inhibitory activity (percentage ± standard error of the mean) of the extracts of Viola odorata at 100 μg mL1 Inhibition against tyrosinase Extracts Viola odorata-DCM Viola odorata-EtOAc Viola odorata-EtOH Viola odorata-H2O Alpha-kojic acid2
(Percentage ± SEM1) 13.52 ± 2.73**** 27.38 ± 2.08**** 80.23 ± 0.87* 15.35 ± 2.85**** 78.89 ± 0.09
SEM, standard error of the mean. Standard error mean (n = 3). 1 2 Reference for tyrosinase inhibition tested at 100 μg mL . *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. 1
Ferric-reducing antioxidant power assay. Ferric-reducing antioxidant power assay (FRAP) of the samples was tested using the assay of Oyaizu (1986). Different concentrations of the extracts were mixed with 2500 μL of phosphate buffer (pH 6.6) and 2500 μL of potassium ferricyanide. Later, the mixture was incubated at 50 °C for 20 min and, then, trichloroacetic acid (10%) was added. After the mixture was shaken vigorously, this solution was mixed with distilled water and ferric chloride (0.1%). After 30 min of incubation, absorbance was read at 700 nm using a Unico 4802 UV-visible double beam spectrophotometer. Analyses were achieved in triplicate. Increased absorbance of the reaction meant increased reducing power and compared with that of chlorogenic acid (Sigma, St. Louis, MO, USA) as the reference. Phosphomolibdenum-reducing antioxidant power assay. In order to perform phosphomolibdenum-reducing antioxidant power (PRAP) assays on the extracts, each dilution was mixed with 10% phosphomolybdic acid solution in ethanol (w/v) (Falcioni et al., 2002). The solution was subsequently subjected to incubate at 80 °C for 30 min, and the absorbance was read at 600 nm using a Unico 4802 UV-visible double beam spectrophotometer. Analyses were run in triplicate. Increased absorbance of the reaction meant increased reducing power and compared with that of quercetin as the reference. Statistical analysis of data. Data obtained from in vitro enzyme inhibition and antioxidant experiments were expressed as the mean standard error (±SEM). Statistical differences between the reference and the sample groups were evaluated by analysis of variance (one way). Dunnett’s multiple comparison tests were used as post hoc tests. A p < 0.05 was considered to be significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).
RESULTS AND DISCUSSION Among the VO extracts evaluated, the ethanol extract showed a marked inhibition (80.23 ± 0.87%) against TYR comparable with that of the reference (78.89 ± 0.09%) (Table 1), whereas all of the extracts were inactive against cholinesterases. Although radical scavenging activity of the extracts was found to be none to low against DPPH and DMPD, a modest level of NO radical scavenging effect was observed with the extracts ranging between 31.98 ± 0.53% and 56.68 ± 1.10% (Fig. 3). The highest metal-chelating capacity was Copyright © 2015 John Wiley & Sons, Ltd.
observed with the VO-DCM extract (39.67 ± 5.54%), while the VO-EtOAc extract displayed better values in FRAP and PRAP assays (Table 2). Herein, we also report that cholinesterase inhibitory effect of the VO extracts was tested, although all of the extracts were revealed to be devoid of any inhibition against AChE and BChE. Based on the data on high TYR inhibitory effect of the VO-EtOH extract and antioxidant activity of the VOH2O extract, we subjected these extracts to HPLC analysis in order to identify its phenolic acid and flavonoid content by comparing the standards available in our laboratory. The results that we obtained through HPLC analysis indicated that the extract did not contain rosmarinic acid, gallic acid, and p-coumaric acid as well as the following flavonoid derivatives; quercitrin, luteolin, luteolin-7-diglucoside, apigenin, quercetin, and kaempferol. On the other hand, presence of vitexin was determined in the VO-EtOH (4.65 ± 0.103 mg g1 extract) and the VO-H2O extract (18.81 ± 0.047 mg g1 extract). Besides, the EtOH extract also contained rutin (1.31 ± 0.013 mg g1 extract) (Fig. 4). Thus, our HPLC analysis revealed that the plant can be considered as a prolific source of vitexin in particular. In continuation with our present work, the routine chromatographic techniques were applied to the VO-EtOH extract, which led to the isolation of three flavonoids [rutin (VO-1), isovitexin (VO-2), and kaempferol-6-glucoside (VO-3)] (Figs. 1 and 2). Up to date, Viola species have been reported to contain a number of flavonoids and other phenolic substances including rutin (Lamaison et al., 1991), violarvensin, violanthin (Carnat et al., 1998), isoschaftoside, schaftoside, neoschaftoside, vicenin-2, apigenin 6-C-α-Larabinopyranosyl-8-C-β-D- xylopyranoside, apigenin 6-C-β-D-xylopyranosyl-8-C-α-L-arabinopyranoside, isoorientin, isocarlinoside (Xie et al., 2003), luteolin, apigenin, quercetin, isorhamnetin, hyperoside, hesperidin, isoferulic acid, ferulic acid, ellagic acid, dicoumarin, catechol, and arbutin (Bubenchikov and Goncharov, 2005). Despite the previous reports on flavonoid content of Viola species, we have come across only a single study carried out on VO characterizing its individual phenolic substances. The flowering tops of VO collected from Italy was shown to contain rutin using HPLC by Karioti et al. (2011), which is consistent in our current data. Phytother. Res. 29: 1304–1310 (2015)
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100 90 80 70 60 50 40 30 20 10 0
(A) DMPD radical scavenging activity
(B) DPPH radical scavenging activity 100 90 80 70 60 50 40 30 20 10 0
100 90 80 70 60 50 40 30 20 10 0
(C) NO radical scavenging activity
(D) Iron-chelating capacity 1
Figure 3. Antioxidant activity graphs (A–D) of Viola odorata extracts tested at 100 μg mL reference).
Besides, they did not detect apigenin and luteolin in their VO sample in accordance again with our HPLC data. Consequently, presence of isovitexin (VO-2) and kaempferol-6-glucoside (VO-3) was demonstrated for the first time in VO as we isolated in our study. The VO has been mostly searched for its antioxidant activity in addition to a few other pharmacological activities such as antipyretic (Khattak et al., 1985), antitumoral (Perwaiz and Sultana, 1998), and antiinflammatory (Koochek et al., 2003). In a study by Ebrahimzadeh et al. (2010), the flower extracts of VO from Iran were found to display weak antioxidant effect in DPPH and NO radical scavenging, and FRAP assays, which is consistent again with our antioxidant results. Similarly, the aqueous
(The last column in each graph belongs to the
flower extracts of the VO samples gathered from two locations in Serbia exerted only moderate DPPH radical scavenging effect (27.35 and 43.39%) at 100 μg mL1 (Stojkovic et al., 2011), while the EtOH and dichloromethane/methanol extracts prepared from VO collected in the Czech Republic had almost no xanthine oxidase inhibitory effect (IC50 = 200 μg mL1) as compared with that of the reference (allopurinol, IC50 = 3.8 μMol L1), which was stated to be correlated with its low polyphenol content (Havlik et al., 2010). According to our literature survey, TYR inhibitory activity of VO has never been studied up to now. Because the VO-EtOH extract possessed a remarkable inhibition against this enzyme (80.23 ± 0.87%) and was
Table 2. Ferric-reducing antioxidant power and phosphomolibdenum-reducing antioxidant power of the extracts of Viola odorata at 100 μg mL1 Extracts Viola odorata-DCM Viola odorata-EtOAc Viola odorata-EtOH Viola odorata-H2O Chlorogenic acid (for FRAP) Quercetin (for PRAP)
FRAP1 (absorbance at 700 nm ± SEM2)
PRAP1 (absorbance at 600 nm ± SEM)
0.557 ± 0.40**** 0.631 ± 0.03**** 0.626 ± 0.22**** 0.350 ± 0.25**** 3.547 ± 0.006
0.455 ± 0.17** 0.461 ± 0.19** 0.356 ± 0.01** 0.317 ± 0.18**** 0.819 ± 0.001
FRAP, ferric-reducing antioxidant power; PRAP, phospomolibdenum-reducing antioxidant power; SEM, standard error of the mean. Higher absorbance indicates greater antioxidant activity. 2 Standard error mean (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. 1
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Figure 4. The HPLC chromatograms of vitexin (A) and the Viola odorata-H2O extract (B).
shown to contain vitexin and rutin in considerable amount as well as isovitexin and kaempferol-6glucoside, its TYR inhibitory potency might be suggested to be related to the flavonoids. Among them, we have recently showed that the ability of rutin to inhibit TYR was at an ignorable level (1.79 ± 0.54% and 100 μg mL1) (Senol et al., 2014). On the other hand, vitexin and isovitexin were previously determined with mixed-type of TYR inhibitory effect (IC50 = 6.3 and 5.6 mg mL1), while IC50 value of kaempferol and kaempferol-7-O-β-glucopyranoside was found to be 25 and 161.54 μM, respectively (Loizzo et al., 2012). Because of very little amount of the isolated compounds herein most of which were used up in the NMR techniques for their structure determination, we were regrettably not able to determine TYR inhibitory activity of vitexin and isovitexin in the current study. However, as aforementioned, the previous studies clearly showed the inhibitory potential of these compounds against TYR to some extent, which may lead to the following assumption that vitexin, isovitexin, and kaempferol derivatives found in this plant species might be most likely contributing to TYR inhibitory action of VO. In summary, the findings obtained herein evidently established that the VO-EtOH extract has outshined with a marked anti-TYR effect, whereas the extracts displayed a low to moderate level of antioxidant activity based on various mechanisms in vitro. It should be also Copyright © 2015 John Wiley & Sons, Ltd.
noted that none of the VO extracts possessed anticholinesterase activity. We suggest that TYR inhibitory effect of VO-EtOH extract could be attributed to its flavonoid derivatives, vitexin, isovitexin, and kaempferol, in particular. Additionally, in the current study, we disclose the presence of vitexin by HPLC and the isolation of isovitexin and kaemferol-6-glucoside from VO for the first time. In conclusion, this study provides a base for further identification of individual constituents from VO in addition to rutin, vitexin, isovitexin, and kaempferol6-glucoside as possible natural agents useful for both anti-browning purpose and skin-whitening cosmetics.
Acknowledgements This study was by supported by the grant presented to I. E. Orhan through the Young Woman Scientist Award in Life Sciences provided by L’Oreal Turkey Branch and the Turkish Academy of Sciences for the bioactivity and phytochemical studies on VO. F. S. Senol expresses her sincere thanks to the Scientific and Technological Research Council of Turkey (TUBITAK) for the scholarship provided for her PhD program.
Conflict of Interests The authors have declared that there is no conflict of interest. Phytother. Res. 29: 1304–1310 (2015)
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Loizzo MR, Tundis R, Menichini F. 2012. Natural and synthetic tyrosinase inhibitors as antibrowning agents: an update. Compr Rev Food Sci Food Saf 11: 378–398. Marcocci I, Marguire JJ, Droy-Lefaiz MT, Packer L. 1994. The nitric oxide scavenging properties of Ginkgo biloba extract. Biochem Biophys Res Commun 201: 748–755. Masuda T, Yamashita D, Takeda Y, Yonemori S. 2005. Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica. Biosci Biotechnol Biochem 69: 197–201. Mayer AM. 2006. Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 67: 2318–2331. Monadi A, Rezaie A. 2013. Evaluation of sedative and preanesthetic effects of Viola odorata Linn. extract compared with diazepam in rats. Bull Environ Pharmacol Life Sci 2: 125–131. Naithani V, Nair S, Kakkar P. 2006. Decline in antioxidant capacity of Indian herbal teas during storage and its relation to phenolic content. Food Res Int 39: 176–181. Neves JM, Matos C, Moutinho C, Queiroz G, Gomes LR. 2009. Ethnopharmacological notes about ancient uses of medicinal plants in Trás-os-Montes (northern of Portugal). J Ethnopharmacol 124: 270–283. Orhan IE, Orhan G, Gurkas E. 2011. An overview on natural cholinesterase inhibitors – a multi-targeted drug class – and their mass production. Mini Rev Med Chem 11: 836–842. Oyaizu M. 1986. Studies on products of browning reactionsantioxidative activities of products of browning reaction prepared from glucosamine. Jpn J Nutr 44: 307–315. Perwaiz S, Sultana S. 1998. Antitumorigenic effect of crude extract of Viola odorata on DMBA- induced two stage skin carcinogenesis in the Swiss albino mice. Asia Pac J Pharmacol 13: 43–45. Schlesier K, Harvat M, Bohm V, Bitsch R. 2002. Assessment of antioxidant activity by using different in vitro methods. Free Radic Res 36: 177–187. Senol FS, Bahadir Acikara O, Saltan Citoglu G, Orhan IE, Dall’Acqua S, Ozgokce F. 2014. Prospective neurobiological effects of the aerial and root extracts and some pure compounds of randomly selected Scorzonera species. Pharm Biol 52: 873–882. Stojkovic D, Glamočlija J, Ćirić A, Šiljegović J, Nikolić M, Soković M. 2011. Free radical scavenging activity of Viola odorata water extracts. J Herbs Spices Med Plants 17: 285–290. Xie C, Veitch NC, Houghton PJ, Simmonds MSJ. 2003. Flavone Cglycosides from Viola yedoensis Makino. Chem Pharm Bull 51: 1204–1207.
Phytother. Res. 29: 1304–1310 (2015)
Tyrosinase and Cholinesterase Inhibitory Potential and Flavonoid Characterization of Viola odorata L. (Sweet Violet) Ilkay Erdogan Orhan,1* Fatma Sezer Senol,1 Sinem Aslan Erdem,2 I. Irem Tatli,3 Murat Kartal2,4 and Sevket Alp5 1
Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey Department of Pharmacognosy, Faculty of Pharmacy, Ankara University, 06100, Ankara, Turkey Department of Pharmaceutical Botany, Faculty of Pharmacy, Hacettepe University, 06100, Ankara, Turkey 4 Department of Pharmacognosy, Faculty of Pharmacy, and Center of Phytotherapy Education, Research and Practice, Bezmialem Vakif University, 34093, Istanbul, Turkey 5 Department of Landscape Architecture, Faculty of Agriculture, Yuzuncu Yıl University, 65080, Van, Turkey 2 3
Inhibitory potential of the dichloromethane, ethyl acetate, ethanol, and aqueous extracts of Viola odorata L. (VO) was investigated against tyrosinase (TYR) and cholinesterases by microplate assays. The antioxidant activity was tested using six in vitro assays. Only the ethanol extract inhibited TYR (80.23 ± 0.87% at 100 μg mL1), whereas none of them were able to inhibit cholinesterases. The extracts were more able to scavenge NO radical (31.98 ± 0.53–56.68 ± 1.10%) than other radicals tested, and displayed low to moderate activity in the rest of the assays. HPLC analysis revealed that the aqueous extract of VO contained a substantial amount of vitexin (18.81 ± 0.047 mg g1 extract), while the ethanol extract also possessed rutin (1.31 ± 0.013 mg g1 extract) and vitexin (4.65 ± 0.103 mg g1 extract). Furthermore, three flavonoids (rutin, isovitexin, and kaempferol-6-glucoside) were isolated from the ethanol extract. This is the first report on TYR inhibitory activity of VO as well as presence of vitexin and isovitexin in this species. Copyright © 2015 John Wiley & Sons, Ltd. Keywords: Viola odorata; tyrosinase; cholinesterase; antioxidant activity; flavonoid; HPLC.
INTRODUCTION The genus Viola L. (Violaceae), which has approximately 400 flowering species throughout the world, comprises 33 taxa in Turkey (Dinc et al., 2007). Among them, Viola odorata L. (sweet violet, garden violet, VO), native to Europe and Asia, is cultivated largely because of its great commercial value resulted from its essential oil content with a sweet, powdery, and woody-floral scent used popularly in cosmetic industry, production of perfumes, toiletry, and flavorings in soufflés, cream, and similar desserts (Chawia, 2000). The blue-violet or whitish flowers are also used for ornamental purposes as well as extensively as a garnish and in chocolates. Newly opened violet flowers may be used to decorate salads or in stuffings for poultry or fish. In the USA, this French violet syrup is used to make violet scones and marshmallows (Monadi and Rezaie, 2013). VO, known as ‘kokulu menekşe’ in Turkish, has been also recorded to be used in traditional medicines for various purposes. For instance, this species has been used to treat cough in Anatolian folk medicine (Akaydin et al., 2013), expectorant, sedative, sweating stimulation, as laxative/purgative, and against bronchitis in Portugal (Neves et al., 2009). The plant is also used as herbal tea in India for anti-aging, anti-stress, appetizer, and stimulant purposes (Naithani et al., 2006). * Correspondence to: Ilkay Erdogan Orhan, Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330, Ankara, Turkey. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
Polyphenol oxidases (PPOs), found in plants, fungi, and other organisms, are a copper-containing enzyme family with dinuclear copper centers, and the monophenol oxidase type of PPOs is known as tyrosinase (TYR, EC 1.14.18.1) (Mayer, 2006). The main function of PPOs is to catalyze browning reactions in vegetables and fruits resulting from the oxidation and dehydrogenation reactions of polyphenols present in the plants, which finally leads to formation of reddishbrown ortho-quinones as well as in negative influence on their color, taste, flavor, and nutritional value. Because browning causes major agricultural and economic problems, inhibition of TYR is quite important in the food industry. Additionally, inhibitors of TYR are popularly used in skin-whitening products in cosmetics industry as the demand grows toward these particular cosmetic agents. On the other hand, inhibition of cholinesterase enzyme family consisting of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is the most effective approach toward the treatment of Alzheimer type of dementia as it has become a major health problem especially in developed countries because of higher population of the elderly (Orhan et al., 2011). Although there has been extensive research going on discovery of new cholinesterase and TYR inhibitory compounds of both synthetic and natural origins, a great need still remains for new inhibitors of these enzymes because of either side effects or low efficacy of current inhibitors of AChE and BChE. Besides, the number of the present TYR inhibitors is quite limited, and new inhibitors are in demand particularly for cosmetics industry. On this purpose, in continuation of our studies Received 12 March 2015 Revised 26 April 2015 Accepted 28 April 2015
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on finding new TYR inhibitors from plants, we have now aimed to investigate inhibitory potential of VO against TYR and along with its antioxidant activity in various in vitro assays by high-throughput screening method using enzyme-linked immunosorbent assay microplate reader. HPLC analysis has been carried out on the extracts of VO in order to quantify phenolic acids and flavonoid derivatives.
MATERIALS AND METHODS Plant material. The samples of VO were collected from the town of Ercis of Van province, Turkey, in 2010. The plant was identified by Dr Fazli Ozturk from Yuzuncu Yıl University (Van, Turkey) and the voucher specimen is kept in the Herbarium of Yuzuncu Yıl University (coded as F1399).
Extraction procedure. The air-dried and powdered aerial parts of VO (200 g) were extracted sequentially with dichloromethane (DCM), ethyl acetate (EtOAc), ethanol (75%, EtOH), and distilled water (H2O). The macerates obtained with DCM, EtOAc, and EtOH were evaporated in vacuo until dryness, while the H2O macerate was lyophilized. The extracts were kept in the freezer until the experiments were performed. Yield percentages (w/w) of the extracts are as follows: 1.29% for the DCM extract; 10.52% for the EtOAc extract; 5.70% for the EtOH extract; 9.87% for the H2O extract.
Isolation procedure of Viola odorata 1, 2, and 3. The EtOH extract (508.5 mg) was first dissolved in H2O, and the water-soluble portion was fractionated over a LiChroprep C18 column (Merck, Opfikon-Glattbrugg, Switzerland; C-18, Sepralyte, 40 μm, VLC, 150 g) (Fig. 1). Employment of H2O and methanol (MeOH) (0–100% MeOH) afforded five main fractions (Frs. A–E).
Figure 1. Isolation scheme of Viola odorata 1, 2, and 3. Copyright © 2015 John Wiley & Sons, Ltd.
Purification of fraction A (98.5 mg) by silica gel column chromatography [Merck, 230–400 mesh, 30 g, CHCl3/ MeOH (85:15 → 70:30)] furnished VO-1 (5.3 mg), VO-2 (5.4 mg), and VO-3 (3.2 mg). Structure elucidation of Viola odorata 1, 2, and 3. Structure elucidation of the isolated compounds VO 1, 2, and 3 from fraction A was carried out by spectral techniques using 1H nuclear magnetic resonance (NMR) and 13C NMR, and detailed data was compared with the literature (Crews et al., 1998). The structures of compounds VO-1, VO-2, and VO-3 were identified as rutin, isovitexin, and kaempferol-6-glucoside, respectively (Fig. 2). Quantification of rutin and vitexin in the extracts by HPLC. Analyses were performed using an Agilent Technologies 1200 Series high pressure liquid chromatography (HPLC), including a binary pump, a vacuum degasser, an autosampler, and a diode array detector. Chromatographic separations were performed on Eclipse XDB-C18 column (150 mm × 4.6 mm, 5 μm). A mobile phase consisting of two eluents (acetonitrile and 40 mM formic acid) was used for separation with a gradient elution at a flow rate of 1 mL min1. All solvents were filtered through a 0.45-μm Millipore filter before use. Detection wavelength was set at 254 nm for rutin and 330 nm for vitexin. The injection volume was 20 μL. Standard solutions of rutin (1–100 μg mL1) and vitexin (1–100 μg mL1) whose standards were purchased from Sigma-Aldrich Co. (Germany) were prepared in methanol. Five different concentration levels of rutin and vitexin were subjected to regression analysis to calculate calibration equation and correlation. Each injection was achieved in triplicate to see the reproducibility of the detector response at each concentration level. Identification of rutin and vitexin was made by comparing their retention time and ultraviolet (UV) spectra with those of pure standards. All the calculations concerning the quantitative analysis were performed with external standardization by measurement of peak areas.
Figure 2. Structures of Viola odorata 1, 2, and 3. Phytother. Res. 29: 1304–1310 (2015)
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Enzyme inhibition assays Cholinesterase inhibition. The AChE and BChE inhibitory activity was measured by slightly modified spectrophotometric method of Ellman et al. (1961). Electric eel AChE (Type-VI-S; EC 3.1.1.7, Sigma, St. Louis, MO, USA) and horse serum BChE (EC 3.1.1.8, Sigma, St. Louis, MO, USA) were used, while acetylthiocholine iodide and butyrylthiocholine chloride (Sigma, St. Louis, MO, USA) were employed as the substrates of the reaction. 5,5′-Dithio-bis(2-nitrobenzoic)acid (DTNB; Sigma, St. Louis, MO, USA) was used for the measurement of the anticholinesterase activity. Briefly, in this method, 140 μL of sodium phosphate buffer (pH 8.0), 20 μL of DTNB, 20 μL of test solution, and 20 μL of AChE/BChE solution were added by multichannel automatic pipette (Gilson Pipetman, Paris, France) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of 10 μL of acetylthiocholine iodide/butyrylthiocholine chloride. Hydrolysis of acetylthiocholine iodide/butyrylthiocholine chloride was monitored by the formation of the yellow 5-thio-2nitrobenzoate anion as a result of the reaction of DTNB with thiocholines, catalyzed by enzymes at 412 nm utilizing a 96-well microplate reader (VersaMax Molecular Devices, Sunnyvale, CA, USA). The measurements and calculations were evaluated by using Softmax PRO 4.3.2.LS software. Percentage of inhibition of AChE/BChE was determined by the comparison of reaction rates of the samples relative to the blank sample (ethanol in phosphate buffer pH = 8) using the formula (ES)/E × 100, where E is the activity of enzyme without test sample and S is the activity of enzyme with test sample. The experiments were carried out in triplicate. Galanthamine (Sigma, St. Louis, MO, USA), the anticholinesterase alkaloid-type of drug obtained from the bulbs of snowdrop (Galanthus sp.), was used as the reference. Tyrosinase inhibition. Inhibition of tyrosinase (TYR) (EC 1.14.1.8.1; 30 U, mushroom tyrosinase, Sigma) was determined using the modified dopachrome method with L-DOPA as substrate (Masuda et al., 2005). The assays were conducted in 96-well microplate using enzyme-linked immunosorbent assay microplate reader (VersaMax Molecular Devices, USA) to measure absorbance at 475 nm. An aliquot of the extracts dissolved in DMSO with 80 μL of phosphate buffer (pH 6.8), 40 μL of tyrosinase, and 40 μL of L-DOPA were put in each well. Results were compared with control (DMSO). Baicalein (Sigma, St. Louis, MO, USA) was used as the reference. The percentage tyrosinase inhibition (I%) was calculated as follows: I % ¼ Absorbance ðAÞcontrol –AbsorbanceðAÞsample =Absorbance ðAÞcontrol 100
Antioxidant activity by radical-formation methods DPPH radical scavenging activity. The stable 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was determined by the method of Blois (1958). Copyright © 2015 John Wiley & Sons, Ltd.
The samples (30 μL) and the reference, which is dissolved in ethanol (75%), were mixed with 2700 μL of DPPH solution (1.5 × 104 M). The remaining DPPH amount was measured at 520 nm using a Unico 4802 UV-visible double beam spectrophotometer (Unico, Dayton, NJ, USA). Gallic acid (Sigma, St. Louis, MO, USA) was employed as the reference. Inhibition of DPPH in percent (I%) was calculated as given below: I% ¼ Ablank Asample =Ablank 100 where Ablank is the absorbance of the control reaction (containing all reagents except the test sample), and Asample is the absorbance of the extracts/reference. Analyses were run in triplicate, and the results were expressed as average values with SEM (standard error of the mean). DMPD radical scavenging activity. The assay is based on reduction of the purple-colored radical DMPD+ (N, N-dimethyl-p-phenylendiamine). According to the method (Schlesier et al., 2002), a reagent comprising 100 mM DMPD, 0.1 M acetate buffer (pH = 5.25), and 0.05 M ferric chloride solution, which led to the formation of DMPD radical, was freshly prepared, and the reagent was equilibrated to an absorbance of 0.900 ± 0.100 at 505 nm. Then, the reagent was mixed up with 50 μL of the extract dilutions, and absorbance was taken at 505 nm using a Unico 4802 UV-visible double beam spectrophotometer. Quercetin was employed as the reference, and the experiments were performed in triplicate. The results were calculated according to the same formula given for DPPH radical scavenging test and expressed as average values with SEM. Nitric oxide radical scavenging activity. The scavenging activity of the extracts against nitric oxide (NO) was assessed by the method of Marcocci et al. (1994). Briefly, the extract dilutions were mixed with 5 mM sodium nitroprusside and left to incubate for 2 h at 29 °C. An aliquot of the solution was removed and diluted with Griess reagent (1% sulfanilamide in 5% H3PO4 and 0.1% naphthylethylenediamine dihydrochloride). The absorbance of the occurred chromophore was measured at 550 nm using a Unico 4802 UV-visible double beam spectrophotometer. Inhibition of NO radical in percent (I%) was calculated as given in the following: I% ¼ Ablank Asample =Ablank 100 where Ablank is the absorbance of the control reaction (containing all reagents except the test sample), and Asample is the absorbance of the extracts. Analyses were run in triplicates, and the results were expressed as average values with SEM. Quercetin was the reference in this test. Antioxidant activity by metal-related and reducing power methods Metal-chelating capacity. The metal-chelating capacity of the extracts through ferrous ion was estimated by the method of Chua et al. (2008). Briefly, dilutions of the extracts were incubated with 2 mM FeCl2 solution. The reaction was initiated by the addition of 5 mM ferrozine into the mixture and left standing at ambient temperature for 10 min. The absorbance of the reaction mixture Phytother. Res. 29: 1304–1310 (2015)
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was measured at 562 nm using a Unico 4802 UV-visible double beam spectrophotometer. The ratio of inhibition of ferrozine-Fe2+ complex formation was calculated as follows: I% ¼ Ablank Asample =Ablank 100 where Ablank is the absorbance of the control reaction (containing only FeCl2 and ferrozine), and Asample is the absorbance of the extracts/reference. Analyses were run in triplicate, and the results were expressed as average values with SEM. The reference was employed as ethylenediamine tetraacetic acid in this assay.
Table 1. Tyrosinase inhibitory activity (percentage ± standard error of the mean) of the extracts of Viola odorata at 100 μg mL1 Inhibition against tyrosinase Extracts Viola odorata-DCM Viola odorata-EtOAc Viola odorata-EtOH Viola odorata-H2O Alpha-kojic acid2
(Percentage ± SEM1) 13.52 ± 2.73**** 27.38 ± 2.08**** 80.23 ± 0.87* 15.35 ± 2.85**** 78.89 ± 0.09
SEM, standard error of the mean. Standard error mean (n = 3). 1 2 Reference for tyrosinase inhibition tested at 100 μg mL . *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. 1
Ferric-reducing antioxidant power assay. Ferric-reducing antioxidant power assay (FRAP) of the samples was tested using the assay of Oyaizu (1986). Different concentrations of the extracts were mixed with 2500 μL of phosphate buffer (pH 6.6) and 2500 μL of potassium ferricyanide. Later, the mixture was incubated at 50 °C for 20 min and, then, trichloroacetic acid (10%) was added. After the mixture was shaken vigorously, this solution was mixed with distilled water and ferric chloride (0.1%). After 30 min of incubation, absorbance was read at 700 nm using a Unico 4802 UV-visible double beam spectrophotometer. Analyses were achieved in triplicate. Increased absorbance of the reaction meant increased reducing power and compared with that of chlorogenic acid (Sigma, St. Louis, MO, USA) as the reference. Phosphomolibdenum-reducing antioxidant power assay. In order to perform phosphomolibdenum-reducing antioxidant power (PRAP) assays on the extracts, each dilution was mixed with 10% phosphomolybdic acid solution in ethanol (w/v) (Falcioni et al., 2002). The solution was subsequently subjected to incubate at 80 °C for 30 min, and the absorbance was read at 600 nm using a Unico 4802 UV-visible double beam spectrophotometer. Analyses were run in triplicate. Increased absorbance of the reaction meant increased reducing power and compared with that of quercetin as the reference. Statistical analysis of data. Data obtained from in vitro enzyme inhibition and antioxidant experiments were expressed as the mean standard error (±SEM). Statistical differences between the reference and the sample groups were evaluated by analysis of variance (one way). Dunnett’s multiple comparison tests were used as post hoc tests. A p < 0.05 was considered to be significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).
RESULTS AND DISCUSSION Among the VO extracts evaluated, the ethanol extract showed a marked inhibition (80.23 ± 0.87%) against TYR comparable with that of the reference (78.89 ± 0.09%) (Table 1), whereas all of the extracts were inactive against cholinesterases. Although radical scavenging activity of the extracts was found to be none to low against DPPH and DMPD, a modest level of NO radical scavenging effect was observed with the extracts ranging between 31.98 ± 0.53% and 56.68 ± 1.10% (Fig. 3). The highest metal-chelating capacity was Copyright © 2015 John Wiley & Sons, Ltd.
observed with the VO-DCM extract (39.67 ± 5.54%), while the VO-EtOAc extract displayed better values in FRAP and PRAP assays (Table 2). Herein, we also report that cholinesterase inhibitory effect of the VO extracts was tested, although all of the extracts were revealed to be devoid of any inhibition against AChE and BChE. Based on the data on high TYR inhibitory effect of the VO-EtOH extract and antioxidant activity of the VOH2O extract, we subjected these extracts to HPLC analysis in order to identify its phenolic acid and flavonoid content by comparing the standards available in our laboratory. The results that we obtained through HPLC analysis indicated that the extract did not contain rosmarinic acid, gallic acid, and p-coumaric acid as well as the following flavonoid derivatives; quercitrin, luteolin, luteolin-7-diglucoside, apigenin, quercetin, and kaempferol. On the other hand, presence of vitexin was determined in the VO-EtOH (4.65 ± 0.103 mg g1 extract) and the VO-H2O extract (18.81 ± 0.047 mg g1 extract). Besides, the EtOH extract also contained rutin (1.31 ± 0.013 mg g1 extract) (Fig. 4). Thus, our HPLC analysis revealed that the plant can be considered as a prolific source of vitexin in particular. In continuation with our present work, the routine chromatographic techniques were applied to the VO-EtOH extract, which led to the isolation of three flavonoids [rutin (VO-1), isovitexin (VO-2), and kaempferol-6-glucoside (VO-3)] (Figs. 1 and 2). Up to date, Viola species have been reported to contain a number of flavonoids and other phenolic substances including rutin (Lamaison et al., 1991), violarvensin, violanthin (Carnat et al., 1998), isoschaftoside, schaftoside, neoschaftoside, vicenin-2, apigenin 6-C-α-Larabinopyranosyl-8-C-β-D- xylopyranoside, apigenin 6-C-β-D-xylopyranosyl-8-C-α-L-arabinopyranoside, isoorientin, isocarlinoside (Xie et al., 2003), luteolin, apigenin, quercetin, isorhamnetin, hyperoside, hesperidin, isoferulic acid, ferulic acid, ellagic acid, dicoumarin, catechol, and arbutin (Bubenchikov and Goncharov, 2005). Despite the previous reports on flavonoid content of Viola species, we have come across only a single study carried out on VO characterizing its individual phenolic substances. The flowering tops of VO collected from Italy was shown to contain rutin using HPLC by Karioti et al. (2011), which is consistent in our current data. Phytother. Res. 29: 1304–1310 (2015)
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100 90 80 70 60 50 40 30 20 10 0
(A) DMPD radical scavenging activity
(B) DPPH radical scavenging activity 100 90 80 70 60 50 40 30 20 10 0
100 90 80 70 60 50 40 30 20 10 0
(C) NO radical scavenging activity
(D) Iron-chelating capacity 1
Figure 3. Antioxidant activity graphs (A–D) of Viola odorata extracts tested at 100 μg mL reference).
Besides, they did not detect apigenin and luteolin in their VO sample in accordance again with our HPLC data. Consequently, presence of isovitexin (VO-2) and kaempferol-6-glucoside (VO-3) was demonstrated for the first time in VO as we isolated in our study. The VO has been mostly searched for its antioxidant activity in addition to a few other pharmacological activities such as antipyretic (Khattak et al., 1985), antitumoral (Perwaiz and Sultana, 1998), and antiinflammatory (Koochek et al., 2003). In a study by Ebrahimzadeh et al. (2010), the flower extracts of VO from Iran were found to display weak antioxidant effect in DPPH and NO radical scavenging, and FRAP assays, which is consistent again with our antioxidant results. Similarly, the aqueous
(The last column in each graph belongs to the
flower extracts of the VO samples gathered from two locations in Serbia exerted only moderate DPPH radical scavenging effect (27.35 and 43.39%) at 100 μg mL1 (Stojkovic et al., 2011), while the EtOH and dichloromethane/methanol extracts prepared from VO collected in the Czech Republic had almost no xanthine oxidase inhibitory effect (IC50 = 200 μg mL1) as compared with that of the reference (allopurinol, IC50 = 3.8 μMol L1), which was stated to be correlated with its low polyphenol content (Havlik et al., 2010). According to our literature survey, TYR inhibitory activity of VO has never been studied up to now. Because the VO-EtOH extract possessed a remarkable inhibition against this enzyme (80.23 ± 0.87%) and was
Table 2. Ferric-reducing antioxidant power and phosphomolibdenum-reducing antioxidant power of the extracts of Viola odorata at 100 μg mL1 Extracts Viola odorata-DCM Viola odorata-EtOAc Viola odorata-EtOH Viola odorata-H2O Chlorogenic acid (for FRAP) Quercetin (for PRAP)
FRAP1 (absorbance at 700 nm ± SEM2)
PRAP1 (absorbance at 600 nm ± SEM)
0.557 ± 0.40**** 0.631 ± 0.03**** 0.626 ± 0.22**** 0.350 ± 0.25**** 3.547 ± 0.006
0.455 ± 0.17** 0.461 ± 0.19** 0.356 ± 0.01** 0.317 ± 0.18**** 0.819 ± 0.001
FRAP, ferric-reducing antioxidant power; PRAP, phospomolibdenum-reducing antioxidant power; SEM, standard error of the mean. Higher absorbance indicates greater antioxidant activity. 2 Standard error mean (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. 1
Copyright © 2015 John Wiley & Sons, Ltd.
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Figure 4. The HPLC chromatograms of vitexin (A) and the Viola odorata-H2O extract (B).
shown to contain vitexin and rutin in considerable amount as well as isovitexin and kaempferol-6glucoside, its TYR inhibitory potency might be suggested to be related to the flavonoids. Among them, we have recently showed that the ability of rutin to inhibit TYR was at an ignorable level (1.79 ± 0.54% and 100 μg mL1) (Senol et al., 2014). On the other hand, vitexin and isovitexin were previously determined with mixed-type of TYR inhibitory effect (IC50 = 6.3 and 5.6 mg mL1), while IC50 value of kaempferol and kaempferol-7-O-β-glucopyranoside was found to be 25 and 161.54 μM, respectively (Loizzo et al., 2012). Because of very little amount of the isolated compounds herein most of which were used up in the NMR techniques for their structure determination, we were regrettably not able to determine TYR inhibitory activity of vitexin and isovitexin in the current study. However, as aforementioned, the previous studies clearly showed the inhibitory potential of these compounds against TYR to some extent, which may lead to the following assumption that vitexin, isovitexin, and kaempferol derivatives found in this plant species might be most likely contributing to TYR inhibitory action of VO. In summary, the findings obtained herein evidently established that the VO-EtOH extract has outshined with a marked anti-TYR effect, whereas the extracts displayed a low to moderate level of antioxidant activity based on various mechanisms in vitro. It should be also Copyright © 2015 John Wiley & Sons, Ltd.
noted that none of the VO extracts possessed anticholinesterase activity. We suggest that TYR inhibitory effect of VO-EtOH extract could be attributed to its flavonoid derivatives, vitexin, isovitexin, and kaempferol, in particular. Additionally, in the current study, we disclose the presence of vitexin by HPLC and the isolation of isovitexin and kaemferol-6-glucoside from VO for the first time. In conclusion, this study provides a base for further identification of individual constituents from VO in addition to rutin, vitexin, isovitexin, and kaempferol6-glucoside as possible natural agents useful for both anti-browning purpose and skin-whitening cosmetics.
Acknowledgements This study was by supported by the grant presented to I. E. Orhan through the Young Woman Scientist Award in Life Sciences provided by L’Oreal Turkey Branch and the Turkish Academy of Sciences for the bioactivity and phytochemical studies on VO. F. S. Senol expresses her sincere thanks to the Scientific and Technological Research Council of Turkey (TUBITAK) for the scholarship provided for her PhD program.
Conflict of Interests The authors have declared that there is no conflict of interest. Phytother. Res. 29: 1304–1310 (2015)
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Phytother. Res. 29: 1304–1310 (2015)
