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Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts
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Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts Elena Neagu a, Gabriela Paun a, Camelia Albu a, Gabriel-Lucian Radu b,∗ a b
Centre of Bioanalysis, National Institute of Research and Development for Biological Sciences, 296 Splaiul Independentei, 060031 Bucharest 6, Romania Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Str. Gh. Polizu Nr. 1–7, 011061 Bucharest 1, Romania
a r t i c l e
i n f o
Article history: Received 11 June 2014 Revised 23 January 2015 Accepted 24 January 2015 Available online xxx Keywords: Acetylcholinesterase inhibitory activity Tyrosinase inhibitory activity Antioxidant activity Alchemilla vulgaris Filipendula ulmaria Neurodegenerative disorders
a b s t r a c t Medicinal plants represent an important source of active biological compounds that could be used for new drugs development. The present work aims the assessment of two Romanian medicinal plants that were not well studied Alchemilla vulgaris and Filipendula ulmaria with respect to their neuroprotective potentiality. The aqueous extracts (10% mass) and ethanolic extracts (10% mass and 70% (v/v) ethanol) of A. vulgaris and F. ulmaria were screened to evaluate their acetylcholinesterase and tyrosinase inhibitory effects as well as their antioxidant activity. The antioxidant activity was determined using two methods, namely 2,2-diphenyl1-picrylhydrazyl (DPPH) and reducing power assay. The total polyphenolic content determined in the tested compounds was between 88.00 and 112.33 μg/mL, flavones between 360.00 and 862.00 μg/mL and proanthocyanidins between 77.66 and 130.00 μg/mL. The acetylcholinesterase inhibitory activity was determined to be between 77.03 and 98.39% (at the highest used dose –3 mg/mL), the tyrosinase inhibitory activity was found to be between 60.00 and 90.65% (at 3 mg/mL) and the DPPH radical inhibition between 73.90 and 93.49%. These findings support the use of these medicinal plants in the treatment of neurodegenerative disorders such as Parkinson, Alzheimer. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is one of the most common forms of dementia that affects approximately 10% of the population over 65 year old population [1]. AD has become a major issue especially in the developed countries due to the increase in the old-age population. The AD pathogenesis has not been yet fully elucidated, however the ‘‘cholinergic hypothesis’’ is the well-accepted theory [2]. The cholinergic hypothesis is based on the deficit the neurotransmitter called acetylcholine that represents the substrate for acetylcholinesterase (AChE) [3]. Therefore, the main therapeutic approach to address AD is the AChE inhibition [4]. Now, several kinds of AChE inhibitors such as donepezil, galantamine and rivastigmine are available for the symptomatic treatment of patients with mild to moderate AD [1]. However, these compounds have been reported to have the problems associated with the gastrointestinal disturbances and bioavailability [5]. Recent studies have demonstrated that phytochemicals are effective acetylcholinesterase
∗
Corresponding author. Tel.: +40 214023802; fax: +40 214023802. E-mail address: [email protected] (G.-L. Radu).
inhibitors having little or no side effects and therefore could be used as dietary intervention in controlling this disease [6,7]. As reactive oxygen species (ROS) have been described as contributors to cellular aging and neuronal damage and they also play an important role in the pathogenesis of various diseases, including neurodegenerative disorders, cancer and atherosclerosis stroke, Alzheimer’s disease, vascular dementia, etc. [8,9], it would be very advantageous if anti-AD drug candidates possess AChE inhibitory activities along with antioxidant ones [10]. Among the phytochemicals, flavonoids together with flavonols and phenols represent important and interesting classes of biologically active compounds that are effective in the protection of various cell types from oxidative injury [11]. Parkinson disease (PD) represents another neurodegenerative disease, resulting from dopaminergic neurons deficiency in the brain. Tyrosinase is a copper-containing enzyme involved in the melanin synthesis in skin and hair and may contribute to neuromelanin formation [12]. Therefore tyrosinase inhibition has become a popular target in drug development and research for PD [13]. Alchemilla vulgaris (lady’s mantle) belongs to the Rosaceae family, with astringent, anti-hemorroidal and anti-diarrheal properties. The plant infusion is used externally in the cases of stomatitis and wound healing [14].
http://dx.doi.org/10.1016/j.jtice.2015.01.026 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026
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Filipendula ulmaria (meadowsweet) belongs to the Rosaceae family and is a wild flower having astringent, diuretic (due to flavonoids), sub-tonic and antirheumatic properties [15]. The aim of the present work was to screen the acetylcholinesterase and tyrosinase inhibitory effects as well as the antioxidant activity of aqueous extracts (10% mass) and ethanolic extracts (10% mass and 70% (v/v) ethanol) of A. vulgaris and F. ulmaria from Romania. Moreover the total polyphenolic content, the flavonoid and proanthocyanidins content were determined and the results were correlated to the results obtained for the enzymatic inhibitory effects (acetylcholinesterase and tyrosinase) and the results obtained for antioxidant activity. Our research results indicate that the tested plants could have important neuroprotective effects due to their acetylcholinesterase and tyrosinase inhibitory and antioxidant activities.
consisted of formic acid (to improve ionization and resolution) in water (pH 3.0) as solvent A and formic acid in acetonitrile (pH 3.0) as solvent B. The polyphenolic compounds separation was performed using binary gradient elution: 0 min 5% solvent B; 0.01–20 min 5– 30% solvent B; 20–40 min 30% solvent B; 40.01–50 min 30–50% solvent B; 50.01–52 min 50-5% solvent B. The flow rates were: 0–5 min 0.1 mL/min; 5.01–15 min 0.2 mL/min; 15.01–35 min 0.1 mL/min; 35.01–50 min 0.2 mL/min; 50–52 min 0.1 mL/min.
2. Materials and methods
2.7.1. DPPH radical scavenging activity DPPH radical scavenging activity was determined by measuring the decrease in the DPPH maximum absorbency at 517 nm after 3 min, using Bondet V. method with slight modifications [20]. The percentage of DPPH radical scavenging activity of the samples was calculated as follows:
2.1. Materials All chemicals and solvents were purchased from Sigma Chemical Company (Sigma–Aldrich, Germany), Fluka (Switzerland), Roth (Carl Roth GmbH, Germany) and deionized water was used for all the performed analysis (Millipore, Bedford, MA).
2.7. Antioxidant assays The antioxidant activity was measured using following two methods.
radical scavenging activity (%) =
AB − AA × 100 AB
2.2. Preparation of the extracts
where AB = control absorbance and AA = sample absorbance.
The plant material was purchased from a national producer (Fares Orastie) of herbal infusions in dry and already packed forms and sold in supermarkets, drug stores and herbal shops. The aqueous extracts (10% mass) were made as follows: 10 g of each dried plant was soaked and stirred with 100 mL of distilled water at 60 °C, then the mixtures were sonicated for 1 h at room temperature. The herbal extracts were then filtered through a Whatman filter. 70% ethanolic extract (10% mass) were obtained as follows: 10 g plant dried were mixed with 100 mL of 70% (v/v) EtOH, the mixtures were sonicated for 1 h at room temperature and then filtered.
2.7.2. Reducing power activity Reducing power activity (iron (III) to iron (II) reduction) was determined according to a previously described procedure [21]. Sample extracts (0.1 mL) were mixed with 2.5 mL of 200 mM/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide and incubated at 50 °C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid (w/v) was added and then the mixture was mixed with 2.5 mL deionized water and 0.5 mL of 0.1% ferric chloride. The absorbance was measured spectrophotometrically at 700 nm.
2.3. Determination of polyphenols content Polyphenols content was made by spectrophotometry at 760 nm wavelength using Folin–Ciocalteu method [16]. The polyphenols content was expressed in μg gallic acid equivalents (GAE)/mL of extract. 2.4. Determination of flavones content Flavones content was analyzed using aluminum chloride colorimetric method [17]. The flavones content express in μg rutin equivalent (RE)/mL of extract. 2.5. Determination of proanthocyanidins Proanthocyanidins was carried out using the vanillin assay in glacial acetic acid [18] with slight modifications. The absorbance was read at 500 nm. The results were expressed as μg catechin equivalents/mL of extract.
2.8. Acetylcholinesterase inhibition activity Acetylcholinesterase inhibition activity was measured using the method described by Ingkaninan et al. [22]. Briefly, 3 ml of 50 mM Tris–HCl buffer (pH 8.0), 100 μL of sample solution at different concentrations (3 mg/mL, 1.5 mg/mL, 0.75 mg/mL) and 20 μL AChE (6 U/mL) solution were mixed and incubated for 15 min at 30 °C, and 50 μL of 3 mM 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) were added. The reaction was then initiated by the addition of 50 μL of acetylthiocholine iodide (AChl) (15 mM). The hydrolysis of this substrate was monitored by the formation of yellow 5-thio-2nitrobenzoate anion as the result of the reaction of 5,5 -dithiobis-(2nitrobenzoic acid (DTNB) with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide, at a wavelength of 405 nm. The enzymatic activity was calculated as a percentage of the velocities compared to that of the assay using buffer instead of inhibitor (extract), using the formula (E − S)/E × 100, where E is the activity of enzyme without test sample, and S is the activity of enzyme with test sample.
2.6. HPLC analysis 2.9. Tyrosinase inhibition assay The chromatographic measurements were performed using a complete HPLC SHIMADZU system, using a Nucleosil 100-3.5 C18 column, KROMASIL, 100 × 2.1 mm. The system was coupled to a MS detector, LCMS-2010 detector (liquid chromatograph mass spectrometer), equipped with an ESI interface. The mobile phase was subjected to ultrasounds in order to eliminate the dissolved air and then filtrated using a PTFE 0.2 μm membrane. The used method was published by Cristea et al. [19] for polyphenols identification. The mobile phase
The tyrosinase (EC1.14.1.8.1, Sigma) activity was measured spectrophotometrically using l-DOPA as substrate [23]. Tyrosinase aqueous solution (100 μL, 0.5 mg/mL), plant extract (3 mg/mL, 1.5 mg/mL, 0.75 mg/mL) and 1850 μL of 0.2 M phosphate buffer (pH 7.0) were mixed and incubated at 30 °C for 15 min, and then L-DOPA solution (50 μL, 10 mM) was added and the absorbance at 475 nm was measured (3 min). The same reaction mixture without the plant extract
Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026
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but the equivalent amount of phosphate buffer served as blank. The percentage inhibition of tyrosinase activity was calculated as follows:
%Tyrosinase inhibition =
ΔAcontrol − ΔAsample × 100 ΔAcontrol
where Acontrol is the change of absorbance at 475 nm without a test sample, and A sample is the change of absorbance at 475 nm with a test sample. 2.10. Statistical analysis The tests were carried out in triplicate and the software Microsoft Office Excel 2007 was used for the statistical analysis, the standard deviation (STDV) being less than 10%. 3. Results and discussions 3.1. The determination of total polyphenolic, flavones and proanthocyanidins content The total polyphenolic content expressed as gallic acid equivalents found in our samples varied between 88.00 and 112.33 μg/mL. The highest amount of polyphenols (112.33 μg/mL) and proanthocyanidins being found in the A. vulgaris ethanolic extract. The highest amount of flavones, 862.00 μg/mL, was found in the F. ulmaria ethanolic extract (Table 1). The obtained results are in agreement with the results previously reported by other scientists F. ulmaria samples from Portugal revealed high content in phenolics and flavonoids [24]. 3.2. HPLC analysis The HPLC-MS method has been applied for the evaluation of polyphenolic profile of different plant extract samples using the SIM
3
mode, the corresponding peaks of the rutin, ellagic acid, sinapic acid, epicatechin, chlorogenic acid, rosmarinic acid, quercetin 3β -D-glucoside, genistein, daidzein, myricetin, quercetin, luteolin, kaempferol, gallic acid, caffeic acid, ferulic acid and p-coumaric fragment ions being obtained. Under the optimum chromatographic conditions the compounds of interest were fairly resolved. The elution order of phenolic classes analyzed by this method was: flavanol, followed by hydroxycinnamic acid, proanthocyanidins and finally the flavonol classes. Values for polyphenolic compounds in samples of plant extracts obtained with this method are shown in Table 2. The F. ulmaria ethanolic extract contains high concentrations of rutin (218.0 μg/mL), chlorogenic acid (106.7 μg/mL) and quercetin 3-β -D-glucoside (118.0 μg/mL), while the A. vulgaris ethanolic extract contains high amounts of ellagic acid (996.6 μg/mL) and higher amounts of flavones than F. ulmaria ethanolic extracts. Both plant extracts have similar amounts of sinapic acid. The phenolic composition of F. ulmaria flowers was previously studied, but with plant material from other European countries [25,26]. These flowers revealed the presence of a number of polyphenolic constituents including salicylates (e.g. spiraein, salicylic acid and methyl salicylate), flavonols (e.g. spiraeoside, hyperoside, rutin, kaempferol 4-O-glucosides, quercetin-4-O-D-galactopyranoside and quercetin-3-O-glucopyranoside) and ellagitannins (tellimagrandins I and II, and rugosin D) [25,26]. Alchemilla species as members of Rosaceae family produce flavonoids, phenolic acids and tannins. Literature survey revealed information concerning total flavonoid and tannin content rather than detailed chemical composition, only few species being phytochemically investigated such as isoquercetin, rutin, quercetin-3-O-α -D-arabinofuranoside (avicularin) that seem to be characteristic for A. vulgaris [27].
Table 1 The active biological compounds content (polyphenols, flavones, proanthocyanidins) from the tested extracts. Sample
Polyphenols (GAE μg/mL)
Proanthocyanidins (catechine μg/mL)
Flavones (rutin μg/mL)
Alchemilla vulgaris
Aqueous extract (10% mass) 70% Ethanolic extract (10% mass)
94.66 ± 8.12 112.33 ± 6.17
96.33 ± 7.37 130.00 ± 8.12
485.60 ± 8.97 497.00 ± 2.41
Filipendula ulmaria
Aqueous extract (10% mass) 70% ethanolic extract (10% mass)
88.00 ± 4.25 103.00 ± 5.32
77.66 ± 6.23 99.66 ± 2.62
360.00 ± 6.89 862.00 ± 4.67
The data represent the means ± SD of triplicate samples of three independent experiments. Table 2 HPLC values for polyphenolic compounds in plant extracts. Compound [m/z]− Name
Alchemilla vulgaris 70% Ethanol μg/mL
Alchemilla vulgaris Water μg/mL +
Filipendulaulmaria 70% Ethanol μg/mL
Filipendulaulmaria Water μg/mL
Flavanols
Epicatechin 289
0.5
12.8
+
Flavonols
Quercetin 301 Rutin 609 Myricetin 317 Kaempferol 285
133.7 6.4 + +
+ 28.5 + +
132.8 218.6 + +
− − + −
227.7
38.8
Ellagitannins
Elagic acid 301
996.6
15.1
Phenolic acids
Gallic acid 169
+
+
Hydroxy cinnamic acids
p-Coumaric acid 163 Sinapic acid 223 Caffeic acid 179 Ferulic acid 193
4.8 34.6 − −
Hydroxy cinnamic acid esters
Rosmarinic acid 359 Chlorogenic acid 353
5.1 11.8 20.2
Flavonoid glucosides
Quercetin 3-β -D-glucoside 463
Flavones
Luteolin 285
4.2
Isoflavones
Genistein 269 Daidzein 253
4.9 2.9
5.4
+
− 1.0 − −
+ 34.5 + −
− − − −
+ 14.3
+ 106.7
− −
1.8
118.7
−
+
−
+ 2.3 1.5
2.0 2.5
4.8 4.6
Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026
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Fig. 1. Radical scavenging activity. ∗∗p < 0.01 compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗∗p < 0.001 compared the activity of the ethanolic extracts with that of the aqueous extracts.
3.3. Antioxidant activity determination There are few data in the scientific literature about the tested plants, the reported results being especially related to their antioxidant activity and total polyphenolic content. 3.3.1. DPPH radical scavenging activity The DPPH test is based on the ability of plant extracts to donate a hydrogen atom or electron to the stable radical DPPH [28]. The assay evaluates the capacity of the plant extracts to scavenge free radicals in solution. The obtained results are presented in Fig. 1. The ethanolic plant extracts showed a higher percent of DPPH radical scavenging activity than the aqueous plant extracts. The DPPH radical scavenging activity proportionally with the concentration of the analyzed sample, the highest DPPH radical scavenging activity value being obtained for the F. ulmaria ethanolic extract for the first two sample concentrations 93.49% (at 3 mg/mL) and 80.51% (at 1.5 mg/mL) respectively. The highest amounts of flavones, polyphenols and proanthocyanidins were obtained for the extract at 3 mg/mL. It was previously demonstrated that there is a clear correlation between the amount of plant polyphenols and their antioxidant character [29]. High values of DPPH radical scavenging activity percent were also obtained for A. vulgaris ethanolic extract namely 87.95% (at 3 mg/mL) and 80.71% (at 1.5 mg/mL). In fact flavonoids have antioxidant activity that may explain the high values found [30]. The previous reports indicate that A. vulgaris extracts possess strong antioxidant and protective effects and seems to be a good candidate as natural additive in food, cosmetics and pharmaceutical industries [31]. The A. vulgaris extracts have high antioxidant activity correlated to the tannin content [32]. The studied F. ulmaria extracts showed better results than F. ulmaria extracts from Portugal (50% at 0.047 mg/mL) [24] and F. ulmaria from France (35.0% for water fraction and 59.4% for methanol fraction at 0.1 mg/mL [33]. 3.3.2. Reducing power activity The test allows pointing out the reducing capacity of the tested samples that indicates their potential antioxidant activity. The plant extracts ability to reduce Fe3+ to Fe2+ was investigated using ascorbic acid as standard, the results being showed in Fig. 2. The obtained results are correlated to the results obtained using the DPPH assay, namely the ethanolic extracts had a higher reducing power capacity than the aqueous extracts, and the A. vulgaris extracts had a slightly higher reducing power capacity than the F. ulmaria extracts, the highest value being obtained for the Alchemilla ethanolic extract, this being correlated to the high polyphenolic content and
proanthocyanidins amount determined in this extract type. The reducing power of F. ulmaria ethanolic extracts might be related to their phenolic compounds content, especially to rutin and quercetin that have high hydrogen and electron-donating ability [34]. A correlation between flavones content and reducing power was highlighted at F. ulmaria ethanolic extracts (70% EtOH) from Portugal [24]. 3.4. Acetylcholinesterase and tyrosinase inhibition activity Numerous researches on medicinal plants try to find new enzymatic inhibitors appropriate for neurodegenerative diseases. The inhibition of an enzyme from cholinesterases class is important in fighting against Alzheimer disease [35], while inhibition of tyrosinase is an important target in finding new drugs against Parkinson disease. In this context, the present study investigates enzyme inhibitory potential of two medicinal plants from Romania – A. vulgaris and F. ulmaria – which there is no data in the literature in this regard. The results obtained regarding the acetylcholinesterase inhibitory activity of the aqueous and ethanolic extracts of these plants are presented in the Table 3. The results are similar for the two tested plant extracts, the ethanolic extracts having an acetylcholinesterase inhibitory activity more pronounced than the aqueous extracts, the inhibition values being >96 % for the highest sample concentration used (3 mg/mL) and the highest inhibition value being obtained for the F. ulmaria ethanolic extract (98.3%), this extract being the one containing the highest content of flavones. These compounds are known to have high biochemical activities the inhibition of acetylcholinesterase (AChE) being one of them [36, 37]. The high content in polyphenols and flavones in A. vulgaris ethanolic extracts could be explained as high percentage of acetylcholinesterase inhibition. The values obtained for tyrosinase inhibition were higher than 71%, with no significant difference between the two plant extracts, slightly higher for ethanolic extracts, similarly to the acetylcholinesterase inhibitory activity, and they are decreasing proportionally with the decrease in sample concentration (Table 4). The highest tyrosinase inhibition percentage was obtained for F. ulmaria ethanolic extract – 90.65% (as for acetylcholinesterase inhibition activity). Our experimental data showed that the ellagic and the chlorogenic acids (used as standards) had the highest ACh inhibition degree. In the case of the ethanolic extract of A. vulgaris the high value of the ACh inhibition could be explained by the presence of a high quantity of ellagic acid, while the high value of ACh inhibition for the hydroalcoholic F. ulmaria extract could be explained by the presence of a high amount of chlorogenic acid and quercetin (Tables 2 and 3). The
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Fig. 2. Reducing power of Alchemilla vulgaris and Filipendula ulmaria aqueous and ethanolic extracts. ∗p < 0.05 compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗p < 0.01 compared the activity of the ethanolic extracts with that of the aqueous extracts. Table 3 AChE inhibition (%). Sample
AChE inhibition (%) 3 mg/mL
Alchemilla vulgaris
Aqueous extract (10% mass) 70% Ethanolic extract (10% mass) Aqueous extract (10% mass) 70% Ethanolic extract (10% mass) – – – – –
Filipendula ulmaria Galanthamine Elagic acid Quercetin Chlorogenic acid Rutin
84.56 96.50 77.03 98.30 – 75.61 62.73 75.34 59.46
1.5 mg/mL ∗
± ± ± ±
5.64 4.93∗ 3.82∗∗ 3.91∗∗
± ± ± ±
1.32 7.61 2.39 3.65
71.36 78.56 65.43 79.56 99.98 60.28 51.62 54.11 31.23
± ± ± ± ± ± ± ± ±
0.75 mg/mL ∗∗
3.54 2.45∗∗ 4.19∗∗ 6.39∗∗ 2.02 5.36 3.23 5.24 2.57
50.76 56.83 44.27 66.62
± ± ± ±
5.78∗ 3.25∗ 2.56∗∗ 3.45∗∗
The data represent the means ± SD of triplicate samples of three independent experiments. ∗ p < 0.05, compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗ p < 0.01, compared the activity of the ethanolic extracts with that of the aqueous extracts. Table 4 Tyrosinase inhibition (%). Sample
Tyrosinase inhibition (%) 3 mg/mL
Alchemilla vulgaris Filipendula ulmaria Kojic acid Elagic acid Quercetin Chlorogenic acid Rutin
Aqueous extract (10% mass) 70% Ethanolic extract (10% mass) Aqueous extract (10% mass) 70% Ethanolic extract (10% mass) 98.7 ± 4.23 – – – –
60.00 71.55 87.88 90.65 94.2 45.77 62.60 58.09 47.23
± ± ± ± ± ± ± ± ±
1.5 mg/mL 2.78∗∗ 4.39∗∗ 7.89∗∗∗ 3.67∗∗∗ 2.87 1.71 3.65 2.31 6.54
53.21 59.12 68.54 79.82 89.7 25.42 33.45 41.45 32.30
± ± ± ± ± ± ± ± ±
6.78∗ 2.51∗ 4.49∗∗ 5.91∗∗ 5.24 1.52 2.59 2.83 1.37
0.75 mg/mL 39.21 45.12 54.25 58.31
± ± ± ±
5.29∗∗ 4.26∗∗ 3.54∗∗ 2.98∗∗
The data represent the means ± SD of triplicate samples of three independent experiments. ∗ p < 0.05 compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗ p < 0.01 compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗∗ p > 0.05 compared the activity of the ethanolic extracts with that of the aqueous extracts (at this concentration there is not statistical significance).
experimental data showed that the chlorogenic acid and quercetin (used as standards) had the highest Tyr inhibition fact that could explain the inhibitory activity of F. ulmaria extracts. Ellagic acid had the smallest activity and even if it is present in high amounts in the A. vulgaris extracts the Tyr inhibition of this extract was very small (Tables 2 and 4). Several studies have shown that flavonoids and other phenolic compounds possess anti-acetylcholinesterase activity [38]. Previous studies have shown that ellagic acid acts as potent tyrosinase and acethylcholinesterase inhibitor [39–41] and it has neuroprotective effects and also could play an important role in the pathogenesis of Alzheimer’s disease [42].
The polyphenols found in F. ulmaria hydroalcoholic extracts are rutin, quercetin, chlorogenic acid that could explain the high inhibition values of acetylcholinesterase (AChE) and tyrosinase: rutin is believed to exhibit significant pharmacological activities, including anti-oxidation, anti-inflammation, anti-diabetic, anti-adipogenic, tyrosinase and acethylcholinesterase inhibitor [39–41], neuroprotective and hormone therapy [43]. Quercetin and rutin have been reported to exert numerous pharmacological activities, such as freeradical scavenging, effects on immune and inflammatory cell functions, and could have benefits in Alzheimer’s disease (AD) by mitigating cellular damage induced by reactive oxygen species (ROS) [44]. Chlorogenic acid (CGA) is a polyphenolic component that in addition
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to anti-oxidant activities associated with free radical scavenging [45] also has neuroprotective effects on hydrogen peroxide and amyloid beta (Aβ ) [46]. Potent cholinesterase inhibitory effect of quercetin was demonstrated by in vitro and in silico studies [47]. Sinapic acid present in similar amounts in both plants, has anti-apoptotic, antiinflammatory and radical-scavenging properties and therefore can be used as effective treatment for Alzheimer disease [48]. 4. Conclusions Two Romanian less known medicinal plants, A. vulgaris and F. ulmaria, were evaluated for their acetylcholinesterase and tyrosinase inhibitory effects as well as the antioxidant activity. The A. vulgaris and F. ulmaria extracts showed high acetylcholinesterase inhibitory effects: 98.30% ± 3.91 — F. ulmaria, 96.50% ± 2.93 — A. vulgaris, high tyrosinase inhibitory effects: 90.65% ± 3.67 — F. ulmaris, 71.55% ± 4.39 — A. vulgaris and high antioxidant activities: 93.49% ± 2.01 — F. ulmaria and 87.95% ± 1.78 — A. vulgaris, as well as considerable amounts of flavones (F. ulmaria), polyphenols and proanthocyanidins (A. vulgaris) that make them extremely useful in the therapy of degenerative diseases especially where the oxidative stress and cholinergic hypothesis are involved. Acknowledgment This research was supported by the Romanian National Center for Program Management – PN 09-360101/2012 project. References [1] Racchi M, Mazzuchelli M, Porrello E, Lanni C, Govoni S. Acetylcholinesterase inhibitions: novel activities of old molecules. Pharmacol Res 2004;50:441–51. [2] Mukherjee PK, Kumar V, Mal M, Houghton PJ. Acetylcholinesterase inhibitors from plants. Phytomedicine 2007;14:289–300. [3] Orhan I, Ustun O. Determination of total phenol content, antioxidant activity and acetylcholinesterase inhibition in selected mushrooms from Turkey. J Food Comp Anal 2011;24:386–90. [4] Orhan G, Orhan I, Sener B. Recent developments in natural and synthetic drug research for Alzheimer’s disease. Lett Drug Des Discov 2006;3:268–74. [5] Schulz V. Ginkgo extract or cholinesterase inhibitors in patients with dementia; what clinical trial and guidelines fail to consider. Phytomedicine 2003;10(Suppl. 4):74–9. [6] Orhan I, Sener B, Choudhary MI, Khalid A. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal plants. J Ethnopharmacol 2004;91:57–60. [7] Conforti F, Statti GA, Tundis R, Loizzo MR, Menichini F. In vitro activities of Citrus medica L. cv. diamante (diamante citron) relevant to treatment of diabetes and Alzheimer’s disease. Phytother Res 2007;21:427–33. [8] Zawia NH, Lahiri DK, Cardozo-Pelaez F. Epigenetics, oxidative stress and Alzheimer’s disease. Free Radic Biol Med 2009;46:1241–9. [9] Confortia F, Sosa S, Marrelli M, Menichini F, Statti G, Uzunov D, et al. In vivo antiinflammatory and in vitro antioxidant activities of Mediterranean dietary plants. J Ethnopharmacol 2007;116:144–51. [10] Senol FS, Orhan I, Celep F, Kahraman A, Dogan M, Yilmaz G, et al. Survey of 55 Turkish Salvia taxa for their acetylcholinesterase inhibitory and antioxidant activities. Food Chem 2010;120:34–43. [11] Zou Y-P, Lu Y-H, Wei D-Z. Protective effects of a flavonoidsrich extract of Hypericum perforatum L. against hydrogenperoxide-induced apoptosis in PC12 cells. Phytother Res 2010;24(Suppl. 1):S6–S10. [12] Bao K, Dai Y, Zhu Z-B, Tu F-J, Zhang W-G, Yao X-S. Bioorg Med Chem 2010;18:6708. [13] Asanuma M, Miyazaki I, Ogawa N. Dopamine- or L-DOPA-induced neurotoxicity: the role of dopamine quinone formation and tyrosinase in a model of Parkinson’s disease. Neurotoxicol Res 2003;5:165–76. [14] Ecaterina D., Raducanu D. Terapie naturista. Ed. Stiintifica, Bucuresti; 1992. [15] Costantinescu C. Plantele medicinale in apararea sanatatii, Editia a VI-a, Ed. RECOOP; 1978. [16] Singleton VL, Orthofer R, Lamuela-Raventos RM. Methods Enzymol 1999;299:152–78. [17] Lin JY, Tang CY. Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation. Food Chem 2007;101:140–7. [18] Butler LG, Price ML, Brotherton JE. Vanilin assay for proanthocyanidins (condensed tannins) modification of the solvent for estimation of the degree of polymerization. J Agricult Food Chem 1982;30:1087–9. [19] Cristea V, Deliu C, Oltean B, Brummer A, Albu C, Radu GL. Soilless cultures for pharmaceutical use and biodiversity conservation. Acta Hortic 2009;843:157–64.
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Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts Elena Neagu a, Gabriela Paun a, Camelia Albu a, Gabriel-Lucian Radu b,∗ a b
Centre of Bioanalysis, National Institute of Research and Development for Biological Sciences, 296 Splaiul Independentei, 060031 Bucharest 6, Romania Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Str. Gh. Polizu Nr. 1–7, 011061 Bucharest 1, Romania
a r t i c l e
i n f o
Article history: Received 11 June 2014 Revised 23 January 2015 Accepted 24 January 2015 Available online xxx Keywords: Acetylcholinesterase inhibitory activity Tyrosinase inhibitory activity Antioxidant activity Alchemilla vulgaris Filipendula ulmaria Neurodegenerative disorders
a b s t r a c t Medicinal plants represent an important source of active biological compounds that could be used for new drugs development. The present work aims the assessment of two Romanian medicinal plants that were not well studied Alchemilla vulgaris and Filipendula ulmaria with respect to their neuroprotective potentiality. The aqueous extracts (10% mass) and ethanolic extracts (10% mass and 70% (v/v) ethanol) of A. vulgaris and F. ulmaria were screened to evaluate their acetylcholinesterase and tyrosinase inhibitory effects as well as their antioxidant activity. The antioxidant activity was determined using two methods, namely 2,2-diphenyl1-picrylhydrazyl (DPPH) and reducing power assay. The total polyphenolic content determined in the tested compounds was between 88.00 and 112.33 μg/mL, flavones between 360.00 and 862.00 μg/mL and proanthocyanidins between 77.66 and 130.00 μg/mL. The acetylcholinesterase inhibitory activity was determined to be between 77.03 and 98.39% (at the highest used dose –3 mg/mL), the tyrosinase inhibitory activity was found to be between 60.00 and 90.65% (at 3 mg/mL) and the DPPH radical inhibition between 73.90 and 93.49%. These findings support the use of these medicinal plants in the treatment of neurodegenerative disorders such as Parkinson, Alzheimer. © 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
1. Introduction Alzheimer’s disease (AD) is one of the most common forms of dementia that affects approximately 10% of the population over 65 year old population [1]. AD has become a major issue especially in the developed countries due to the increase in the old-age population. The AD pathogenesis has not been yet fully elucidated, however the ‘‘cholinergic hypothesis’’ is the well-accepted theory [2]. The cholinergic hypothesis is based on the deficit the neurotransmitter called acetylcholine that represents the substrate for acetylcholinesterase (AChE) [3]. Therefore, the main therapeutic approach to address AD is the AChE inhibition [4]. Now, several kinds of AChE inhibitors such as donepezil, galantamine and rivastigmine are available for the symptomatic treatment of patients with mild to moderate AD [1]. However, these compounds have been reported to have the problems associated with the gastrointestinal disturbances and bioavailability [5]. Recent studies have demonstrated that phytochemicals are effective acetylcholinesterase
∗
Corresponding author. Tel.: +40 214023802; fax: +40 214023802. E-mail address: [email protected] (G.-L. Radu).
inhibitors having little or no side effects and therefore could be used as dietary intervention in controlling this disease [6,7]. As reactive oxygen species (ROS) have been described as contributors to cellular aging and neuronal damage and they also play an important role in the pathogenesis of various diseases, including neurodegenerative disorders, cancer and atherosclerosis stroke, Alzheimer’s disease, vascular dementia, etc. [8,9], it would be very advantageous if anti-AD drug candidates possess AChE inhibitory activities along with antioxidant ones [10]. Among the phytochemicals, flavonoids together with flavonols and phenols represent important and interesting classes of biologically active compounds that are effective in the protection of various cell types from oxidative injury [11]. Parkinson disease (PD) represents another neurodegenerative disease, resulting from dopaminergic neurons deficiency in the brain. Tyrosinase is a copper-containing enzyme involved in the melanin synthesis in skin and hair and may contribute to neuromelanin formation [12]. Therefore tyrosinase inhibition has become a popular target in drug development and research for PD [13]. Alchemilla vulgaris (lady’s mantle) belongs to the Rosaceae family, with astringent, anti-hemorroidal and anti-diarrheal properties. The plant infusion is used externally in the cases of stomatitis and wound healing [14].
http://dx.doi.org/10.1016/j.jtice.2015.01.026 1876-1070/© 2015 Taiwan Institute of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026
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Filipendula ulmaria (meadowsweet) belongs to the Rosaceae family and is a wild flower having astringent, diuretic (due to flavonoids), sub-tonic and antirheumatic properties [15]. The aim of the present work was to screen the acetylcholinesterase and tyrosinase inhibitory effects as well as the antioxidant activity of aqueous extracts (10% mass) and ethanolic extracts (10% mass and 70% (v/v) ethanol) of A. vulgaris and F. ulmaria from Romania. Moreover the total polyphenolic content, the flavonoid and proanthocyanidins content were determined and the results were correlated to the results obtained for the enzymatic inhibitory effects (acetylcholinesterase and tyrosinase) and the results obtained for antioxidant activity. Our research results indicate that the tested plants could have important neuroprotective effects due to their acetylcholinesterase and tyrosinase inhibitory and antioxidant activities.
consisted of formic acid (to improve ionization and resolution) in water (pH 3.0) as solvent A and formic acid in acetonitrile (pH 3.0) as solvent B. The polyphenolic compounds separation was performed using binary gradient elution: 0 min 5% solvent B; 0.01–20 min 5– 30% solvent B; 20–40 min 30% solvent B; 40.01–50 min 30–50% solvent B; 50.01–52 min 50-5% solvent B. The flow rates were: 0–5 min 0.1 mL/min; 5.01–15 min 0.2 mL/min; 15.01–35 min 0.1 mL/min; 35.01–50 min 0.2 mL/min; 50–52 min 0.1 mL/min.
2. Materials and methods
2.7.1. DPPH radical scavenging activity DPPH radical scavenging activity was determined by measuring the decrease in the DPPH maximum absorbency at 517 nm after 3 min, using Bondet V. method with slight modifications [20]. The percentage of DPPH radical scavenging activity of the samples was calculated as follows:
2.1. Materials All chemicals and solvents were purchased from Sigma Chemical Company (Sigma–Aldrich, Germany), Fluka (Switzerland), Roth (Carl Roth GmbH, Germany) and deionized water was used for all the performed analysis (Millipore, Bedford, MA).
2.7. Antioxidant assays The antioxidant activity was measured using following two methods.
radical scavenging activity (%) =
AB − AA × 100 AB
2.2. Preparation of the extracts
where AB = control absorbance and AA = sample absorbance.
The plant material was purchased from a national producer (Fares Orastie) of herbal infusions in dry and already packed forms and sold in supermarkets, drug stores and herbal shops. The aqueous extracts (10% mass) were made as follows: 10 g of each dried plant was soaked and stirred with 100 mL of distilled water at 60 °C, then the mixtures were sonicated for 1 h at room temperature. The herbal extracts were then filtered through a Whatman filter. 70% ethanolic extract (10% mass) were obtained as follows: 10 g plant dried were mixed with 100 mL of 70% (v/v) EtOH, the mixtures were sonicated for 1 h at room temperature and then filtered.
2.7.2. Reducing power activity Reducing power activity (iron (III) to iron (II) reduction) was determined according to a previously described procedure [21]. Sample extracts (0.1 mL) were mixed with 2.5 mL of 200 mM/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide and incubated at 50 °C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid (w/v) was added and then the mixture was mixed with 2.5 mL deionized water and 0.5 mL of 0.1% ferric chloride. The absorbance was measured spectrophotometrically at 700 nm.
2.3. Determination of polyphenols content Polyphenols content was made by spectrophotometry at 760 nm wavelength using Folin–Ciocalteu method [16]. The polyphenols content was expressed in μg gallic acid equivalents (GAE)/mL of extract. 2.4. Determination of flavones content Flavones content was analyzed using aluminum chloride colorimetric method [17]. The flavones content express in μg rutin equivalent (RE)/mL of extract. 2.5. Determination of proanthocyanidins Proanthocyanidins was carried out using the vanillin assay in glacial acetic acid [18] with slight modifications. The absorbance was read at 500 nm. The results were expressed as μg catechin equivalents/mL of extract.
2.8. Acetylcholinesterase inhibition activity Acetylcholinesterase inhibition activity was measured using the method described by Ingkaninan et al. [22]. Briefly, 3 ml of 50 mM Tris–HCl buffer (pH 8.0), 100 μL of sample solution at different concentrations (3 mg/mL, 1.5 mg/mL, 0.75 mg/mL) and 20 μL AChE (6 U/mL) solution were mixed and incubated for 15 min at 30 °C, and 50 μL of 3 mM 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB) were added. The reaction was then initiated by the addition of 50 μL of acetylthiocholine iodide (AChl) (15 mM). The hydrolysis of this substrate was monitored by the formation of yellow 5-thio-2nitrobenzoate anion as the result of the reaction of 5,5 -dithiobis-(2nitrobenzoic acid (DTNB) with thiocholine, released by the enzymatic hydrolysis of acetylthiocholine iodide, at a wavelength of 405 nm. The enzymatic activity was calculated as a percentage of the velocities compared to that of the assay using buffer instead of inhibitor (extract), using the formula (E − S)/E × 100, where E is the activity of enzyme without test sample, and S is the activity of enzyme with test sample.
2.6. HPLC analysis 2.9. Tyrosinase inhibition assay The chromatographic measurements were performed using a complete HPLC SHIMADZU system, using a Nucleosil 100-3.5 C18 column, KROMASIL, 100 × 2.1 mm. The system was coupled to a MS detector, LCMS-2010 detector (liquid chromatograph mass spectrometer), equipped with an ESI interface. The mobile phase was subjected to ultrasounds in order to eliminate the dissolved air and then filtrated using a PTFE 0.2 μm membrane. The used method was published by Cristea et al. [19] for polyphenols identification. The mobile phase
The tyrosinase (EC1.14.1.8.1, Sigma) activity was measured spectrophotometrically using l-DOPA as substrate [23]. Tyrosinase aqueous solution (100 μL, 0.5 mg/mL), plant extract (3 mg/mL, 1.5 mg/mL, 0.75 mg/mL) and 1850 μL of 0.2 M phosphate buffer (pH 7.0) were mixed and incubated at 30 °C for 15 min, and then L-DOPA solution (50 μL, 10 mM) was added and the absorbance at 475 nm was measured (3 min). The same reaction mixture without the plant extract
Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026
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but the equivalent amount of phosphate buffer served as blank. The percentage inhibition of tyrosinase activity was calculated as follows:
%Tyrosinase inhibition =
ΔAcontrol − ΔAsample × 100 ΔAcontrol
where Acontrol is the change of absorbance at 475 nm without a test sample, and A sample is the change of absorbance at 475 nm with a test sample. 2.10. Statistical analysis The tests were carried out in triplicate and the software Microsoft Office Excel 2007 was used for the statistical analysis, the standard deviation (STDV) being less than 10%. 3. Results and discussions 3.1. The determination of total polyphenolic, flavones and proanthocyanidins content The total polyphenolic content expressed as gallic acid equivalents found in our samples varied between 88.00 and 112.33 μg/mL. The highest amount of polyphenols (112.33 μg/mL) and proanthocyanidins being found in the A. vulgaris ethanolic extract. The highest amount of flavones, 862.00 μg/mL, was found in the F. ulmaria ethanolic extract (Table 1). The obtained results are in agreement with the results previously reported by other scientists F. ulmaria samples from Portugal revealed high content in phenolics and flavonoids [24]. 3.2. HPLC analysis The HPLC-MS method has been applied for the evaluation of polyphenolic profile of different plant extract samples using the SIM
3
mode, the corresponding peaks of the rutin, ellagic acid, sinapic acid, epicatechin, chlorogenic acid, rosmarinic acid, quercetin 3β -D-glucoside, genistein, daidzein, myricetin, quercetin, luteolin, kaempferol, gallic acid, caffeic acid, ferulic acid and p-coumaric fragment ions being obtained. Under the optimum chromatographic conditions the compounds of interest were fairly resolved. The elution order of phenolic classes analyzed by this method was: flavanol, followed by hydroxycinnamic acid, proanthocyanidins and finally the flavonol classes. Values for polyphenolic compounds in samples of plant extracts obtained with this method are shown in Table 2. The F. ulmaria ethanolic extract contains high concentrations of rutin (218.0 μg/mL), chlorogenic acid (106.7 μg/mL) and quercetin 3-β -D-glucoside (118.0 μg/mL), while the A. vulgaris ethanolic extract contains high amounts of ellagic acid (996.6 μg/mL) and higher amounts of flavones than F. ulmaria ethanolic extracts. Both plant extracts have similar amounts of sinapic acid. The phenolic composition of F. ulmaria flowers was previously studied, but with plant material from other European countries [25,26]. These flowers revealed the presence of a number of polyphenolic constituents including salicylates (e.g. spiraein, salicylic acid and methyl salicylate), flavonols (e.g. spiraeoside, hyperoside, rutin, kaempferol 4-O-glucosides, quercetin-4-O-D-galactopyranoside and quercetin-3-O-glucopyranoside) and ellagitannins (tellimagrandins I and II, and rugosin D) [25,26]. Alchemilla species as members of Rosaceae family produce flavonoids, phenolic acids and tannins. Literature survey revealed information concerning total flavonoid and tannin content rather than detailed chemical composition, only few species being phytochemically investigated such as isoquercetin, rutin, quercetin-3-O-α -D-arabinofuranoside (avicularin) that seem to be characteristic for A. vulgaris [27].
Table 1 The active biological compounds content (polyphenols, flavones, proanthocyanidins) from the tested extracts. Sample
Polyphenols (GAE μg/mL)
Proanthocyanidins (catechine μg/mL)
Flavones (rutin μg/mL)
Alchemilla vulgaris
Aqueous extract (10% mass) 70% Ethanolic extract (10% mass)
94.66 ± 8.12 112.33 ± 6.17
96.33 ± 7.37 130.00 ± 8.12
485.60 ± 8.97 497.00 ± 2.41
Filipendula ulmaria
Aqueous extract (10% mass) 70% ethanolic extract (10% mass)
88.00 ± 4.25 103.00 ± 5.32
77.66 ± 6.23 99.66 ± 2.62
360.00 ± 6.89 862.00 ± 4.67
The data represent the means ± SD of triplicate samples of three independent experiments. Table 2 HPLC values for polyphenolic compounds in plant extracts. Compound [m/z]− Name
Alchemilla vulgaris 70% Ethanol μg/mL
Alchemilla vulgaris Water μg/mL +
Filipendulaulmaria 70% Ethanol μg/mL
Filipendulaulmaria Water μg/mL
Flavanols
Epicatechin 289
0.5
12.8
+
Flavonols
Quercetin 301 Rutin 609 Myricetin 317 Kaempferol 285
133.7 6.4 + +
+ 28.5 + +
132.8 218.6 + +
− − + −
227.7
38.8
Ellagitannins
Elagic acid 301
996.6
15.1
Phenolic acids
Gallic acid 169
+
+
Hydroxy cinnamic acids
p-Coumaric acid 163 Sinapic acid 223 Caffeic acid 179 Ferulic acid 193
4.8 34.6 − −
Hydroxy cinnamic acid esters
Rosmarinic acid 359 Chlorogenic acid 353
5.1 11.8 20.2
Flavonoid glucosides
Quercetin 3-β -D-glucoside 463
Flavones
Luteolin 285
4.2
Isoflavones
Genistein 269 Daidzein 253
4.9 2.9
5.4
+
− 1.0 − −
+ 34.5 + −
− − − −
+ 14.3
+ 106.7
− −
1.8
118.7
−
+
−
+ 2.3 1.5
2.0 2.5
4.8 4.6
Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026
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Fig. 1. Radical scavenging activity. ∗∗p < 0.01 compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗∗p < 0.001 compared the activity of the ethanolic extracts with that of the aqueous extracts.
3.3. Antioxidant activity determination There are few data in the scientific literature about the tested plants, the reported results being especially related to their antioxidant activity and total polyphenolic content. 3.3.1. DPPH radical scavenging activity The DPPH test is based on the ability of plant extracts to donate a hydrogen atom or electron to the stable radical DPPH [28]. The assay evaluates the capacity of the plant extracts to scavenge free radicals in solution. The obtained results are presented in Fig. 1. The ethanolic plant extracts showed a higher percent of DPPH radical scavenging activity than the aqueous plant extracts. The DPPH radical scavenging activity proportionally with the concentration of the analyzed sample, the highest DPPH radical scavenging activity value being obtained for the F. ulmaria ethanolic extract for the first two sample concentrations 93.49% (at 3 mg/mL) and 80.51% (at 1.5 mg/mL) respectively. The highest amounts of flavones, polyphenols and proanthocyanidins were obtained for the extract at 3 mg/mL. It was previously demonstrated that there is a clear correlation between the amount of plant polyphenols and their antioxidant character [29]. High values of DPPH radical scavenging activity percent were also obtained for A. vulgaris ethanolic extract namely 87.95% (at 3 mg/mL) and 80.71% (at 1.5 mg/mL). In fact flavonoids have antioxidant activity that may explain the high values found [30]. The previous reports indicate that A. vulgaris extracts possess strong antioxidant and protective effects and seems to be a good candidate as natural additive in food, cosmetics and pharmaceutical industries [31]. The A. vulgaris extracts have high antioxidant activity correlated to the tannin content [32]. The studied F. ulmaria extracts showed better results than F. ulmaria extracts from Portugal (50% at 0.047 mg/mL) [24] and F. ulmaria from France (35.0% for water fraction and 59.4% for methanol fraction at 0.1 mg/mL [33]. 3.3.2. Reducing power activity The test allows pointing out the reducing capacity of the tested samples that indicates their potential antioxidant activity. The plant extracts ability to reduce Fe3+ to Fe2+ was investigated using ascorbic acid as standard, the results being showed in Fig. 2. The obtained results are correlated to the results obtained using the DPPH assay, namely the ethanolic extracts had a higher reducing power capacity than the aqueous extracts, and the A. vulgaris extracts had a slightly higher reducing power capacity than the F. ulmaria extracts, the highest value being obtained for the Alchemilla ethanolic extract, this being correlated to the high polyphenolic content and
proanthocyanidins amount determined in this extract type. The reducing power of F. ulmaria ethanolic extracts might be related to their phenolic compounds content, especially to rutin and quercetin that have high hydrogen and electron-donating ability [34]. A correlation between flavones content and reducing power was highlighted at F. ulmaria ethanolic extracts (70% EtOH) from Portugal [24]. 3.4. Acetylcholinesterase and tyrosinase inhibition activity Numerous researches on medicinal plants try to find new enzymatic inhibitors appropriate for neurodegenerative diseases. The inhibition of an enzyme from cholinesterases class is important in fighting against Alzheimer disease [35], while inhibition of tyrosinase is an important target in finding new drugs against Parkinson disease. In this context, the present study investigates enzyme inhibitory potential of two medicinal plants from Romania – A. vulgaris and F. ulmaria – which there is no data in the literature in this regard. The results obtained regarding the acetylcholinesterase inhibitory activity of the aqueous and ethanolic extracts of these plants are presented in the Table 3. The results are similar for the two tested plant extracts, the ethanolic extracts having an acetylcholinesterase inhibitory activity more pronounced than the aqueous extracts, the inhibition values being >96 % for the highest sample concentration used (3 mg/mL) and the highest inhibition value being obtained for the F. ulmaria ethanolic extract (98.3%), this extract being the one containing the highest content of flavones. These compounds are known to have high biochemical activities the inhibition of acetylcholinesterase (AChE) being one of them [36, 37]. The high content in polyphenols and flavones in A. vulgaris ethanolic extracts could be explained as high percentage of acetylcholinesterase inhibition. The values obtained for tyrosinase inhibition were higher than 71%, with no significant difference between the two plant extracts, slightly higher for ethanolic extracts, similarly to the acetylcholinesterase inhibitory activity, and they are decreasing proportionally with the decrease in sample concentration (Table 4). The highest tyrosinase inhibition percentage was obtained for F. ulmaria ethanolic extract – 90.65% (as for acetylcholinesterase inhibition activity). Our experimental data showed that the ellagic and the chlorogenic acids (used as standards) had the highest ACh inhibition degree. In the case of the ethanolic extract of A. vulgaris the high value of the ACh inhibition could be explained by the presence of a high quantity of ellagic acid, while the high value of ACh inhibition for the hydroalcoholic F. ulmaria extract could be explained by the presence of a high amount of chlorogenic acid and quercetin (Tables 2 and 3). The
Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026
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Fig. 2. Reducing power of Alchemilla vulgaris and Filipendula ulmaria aqueous and ethanolic extracts. ∗p < 0.05 compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗p < 0.01 compared the activity of the ethanolic extracts with that of the aqueous extracts. Table 3 AChE inhibition (%). Sample
AChE inhibition (%) 3 mg/mL
Alchemilla vulgaris
Aqueous extract (10% mass) 70% Ethanolic extract (10% mass) Aqueous extract (10% mass) 70% Ethanolic extract (10% mass) – – – – –
Filipendula ulmaria Galanthamine Elagic acid Quercetin Chlorogenic acid Rutin
84.56 96.50 77.03 98.30 – 75.61 62.73 75.34 59.46
1.5 mg/mL ∗
± ± ± ±
5.64 4.93∗ 3.82∗∗ 3.91∗∗
± ± ± ±
1.32 7.61 2.39 3.65
71.36 78.56 65.43 79.56 99.98 60.28 51.62 54.11 31.23
± ± ± ± ± ± ± ± ±
0.75 mg/mL ∗∗
3.54 2.45∗∗ 4.19∗∗ 6.39∗∗ 2.02 5.36 3.23 5.24 2.57
50.76 56.83 44.27 66.62
± ± ± ±
5.78∗ 3.25∗ 2.56∗∗ 3.45∗∗
The data represent the means ± SD of triplicate samples of three independent experiments. ∗ p < 0.05, compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗ p < 0.01, compared the activity of the ethanolic extracts with that of the aqueous extracts. Table 4 Tyrosinase inhibition (%). Sample
Tyrosinase inhibition (%) 3 mg/mL
Alchemilla vulgaris Filipendula ulmaria Kojic acid Elagic acid Quercetin Chlorogenic acid Rutin
Aqueous extract (10% mass) 70% Ethanolic extract (10% mass) Aqueous extract (10% mass) 70% Ethanolic extract (10% mass) 98.7 ± 4.23 – – – –
60.00 71.55 87.88 90.65 94.2 45.77 62.60 58.09 47.23
± ± ± ± ± ± ± ± ±
1.5 mg/mL 2.78∗∗ 4.39∗∗ 7.89∗∗∗ 3.67∗∗∗ 2.87 1.71 3.65 2.31 6.54
53.21 59.12 68.54 79.82 89.7 25.42 33.45 41.45 32.30
± ± ± ± ± ± ± ± ±
6.78∗ 2.51∗ 4.49∗∗ 5.91∗∗ 5.24 1.52 2.59 2.83 1.37
0.75 mg/mL 39.21 45.12 54.25 58.31
± ± ± ±
5.29∗∗ 4.26∗∗ 3.54∗∗ 2.98∗∗
The data represent the means ± SD of triplicate samples of three independent experiments. ∗ p < 0.05 compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗ p < 0.01 compared the activity of the ethanolic extracts with that of the aqueous extracts. ∗∗∗ p > 0.05 compared the activity of the ethanolic extracts with that of the aqueous extracts (at this concentration there is not statistical significance).
experimental data showed that the chlorogenic acid and quercetin (used as standards) had the highest Tyr inhibition fact that could explain the inhibitory activity of F. ulmaria extracts. Ellagic acid had the smallest activity and even if it is present in high amounts in the A. vulgaris extracts the Tyr inhibition of this extract was very small (Tables 2 and 4). Several studies have shown that flavonoids and other phenolic compounds possess anti-acetylcholinesterase activity [38]. Previous studies have shown that ellagic acid acts as potent tyrosinase and acethylcholinesterase inhibitor [39–41] and it has neuroprotective effects and also could play an important role in the pathogenesis of Alzheimer’s disease [42].
The polyphenols found in F. ulmaria hydroalcoholic extracts are rutin, quercetin, chlorogenic acid that could explain the high inhibition values of acetylcholinesterase (AChE) and tyrosinase: rutin is believed to exhibit significant pharmacological activities, including anti-oxidation, anti-inflammation, anti-diabetic, anti-adipogenic, tyrosinase and acethylcholinesterase inhibitor [39–41], neuroprotective and hormone therapy [43]. Quercetin and rutin have been reported to exert numerous pharmacological activities, such as freeradical scavenging, effects on immune and inflammatory cell functions, and could have benefits in Alzheimer’s disease (AD) by mitigating cellular damage induced by reactive oxygen species (ROS) [44]. Chlorogenic acid (CGA) is a polyphenolic component that in addition
Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026
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to anti-oxidant activities associated with free radical scavenging [45] also has neuroprotective effects on hydrogen peroxide and amyloid beta (Aβ ) [46]. Potent cholinesterase inhibitory effect of quercetin was demonstrated by in vitro and in silico studies [47]. Sinapic acid present in similar amounts in both plants, has anti-apoptotic, antiinflammatory and radical-scavenging properties and therefore can be used as effective treatment for Alzheimer disease [48]. 4. Conclusions Two Romanian less known medicinal plants, A. vulgaris and F. ulmaria, were evaluated for their acetylcholinesterase and tyrosinase inhibitory effects as well as the antioxidant activity. The A. vulgaris and F. ulmaria extracts showed high acetylcholinesterase inhibitory effects: 98.30% ± 3.91 — F. ulmaria, 96.50% ± 2.93 — A. vulgaris, high tyrosinase inhibitory effects: 90.65% ± 3.67 — F. ulmaris, 71.55% ± 4.39 — A. vulgaris and high antioxidant activities: 93.49% ± 2.01 — F. ulmaria and 87.95% ± 1.78 — A. vulgaris, as well as considerable amounts of flavones (F. ulmaria), polyphenols and proanthocyanidins (A. vulgaris) that make them extremely useful in the therapy of degenerative diseases especially where the oxidative stress and cholinergic hypothesis are involved. Acknowledgment This research was supported by the Romanian National Center for Program Management – PN 09-360101/2012 project. References [1] Racchi M, Mazzuchelli M, Porrello E, Lanni C, Govoni S. Acetylcholinesterase inhibitions: novel activities of old molecules. Pharmacol Res 2004;50:441–51. [2] Mukherjee PK, Kumar V, Mal M, Houghton PJ. Acetylcholinesterase inhibitors from plants. Phytomedicine 2007;14:289–300. [3] Orhan I, Ustun O. Determination of total phenol content, antioxidant activity and acetylcholinesterase inhibition in selected mushrooms from Turkey. J Food Comp Anal 2011;24:386–90. [4] Orhan G, Orhan I, Sener B. Recent developments in natural and synthetic drug research for Alzheimer’s disease. Lett Drug Des Discov 2006;3:268–74. [5] Schulz V. Ginkgo extract or cholinesterase inhibitors in patients with dementia; what clinical trial and guidelines fail to consider. Phytomedicine 2003;10(Suppl. 4):74–9. [6] Orhan I, Sener B, Choudhary MI, Khalid A. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of some Turkish medicinal plants. J Ethnopharmacol 2004;91:57–60. [7] Conforti F, Statti GA, Tundis R, Loizzo MR, Menichini F. In vitro activities of Citrus medica L. cv. diamante (diamante citron) relevant to treatment of diabetes and Alzheimer’s disease. Phytother Res 2007;21:427–33. [8] Zawia NH, Lahiri DK, Cardozo-Pelaez F. Epigenetics, oxidative stress and Alzheimer’s disease. Free Radic Biol Med 2009;46:1241–9. [9] Confortia F, Sosa S, Marrelli M, Menichini F, Statti G, Uzunov D, et al. In vivo antiinflammatory and in vitro antioxidant activities of Mediterranean dietary plants. J Ethnopharmacol 2007;116:144–51. [10] Senol FS, Orhan I, Celep F, Kahraman A, Dogan M, Yilmaz G, et al. Survey of 55 Turkish Salvia taxa for their acetylcholinesterase inhibitory and antioxidant activities. Food Chem 2010;120:34–43. [11] Zou Y-P, Lu Y-H, Wei D-Z. Protective effects of a flavonoidsrich extract of Hypericum perforatum L. against hydrogenperoxide-induced apoptosis in PC12 cells. Phytother Res 2010;24(Suppl. 1):S6–S10. [12] Bao K, Dai Y, Zhu Z-B, Tu F-J, Zhang W-G, Yao X-S. Bioorg Med Chem 2010;18:6708. [13] Asanuma M, Miyazaki I, Ogawa N. Dopamine- or L-DOPA-induced neurotoxicity: the role of dopamine quinone formation and tyrosinase in a model of Parkinson’s disease. Neurotoxicol Res 2003;5:165–76. [14] Ecaterina D., Raducanu D. Terapie naturista. Ed. Stiintifica, Bucuresti; 1992. [15] Costantinescu C. Plantele medicinale in apararea sanatatii, Editia a VI-a, Ed. RECOOP; 1978. [16] Singleton VL, Orthofer R, Lamuela-Raventos RM. Methods Enzymol 1999;299:152–78. [17] Lin JY, Tang CY. Determination of total phenolic and flavonoid contents in selected fruits and vegetables, as well as their stimulatory effects on mouse splenocyte proliferation. Food Chem 2007;101:140–7. [18] Butler LG, Price ML, Brotherton JE. Vanilin assay for proanthocyanidins (condensed tannins) modification of the solvent for estimation of the degree of polymerization. J Agricult Food Chem 1982;30:1087–9. [19] Cristea V, Deliu C, Oltean B, Brummer A, Albu C, Radu GL. Soilless cultures for pharmaceutical use and biodiversity conservation. Acta Hortic 2009;843:157–64.
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Please cite this article as: E. Neagu et al., Assessment of acetylcholinesterase and tyrosinase inhibitory and antioxidant activity of Alchemilla vulgaris and Filipendula ulmaria extracts, Journal of the Taiwan Institute of Chemical Engineers (2015), http://dx.doi.org/10.1016/j.jtice.2015.01.026