Inhibitory potential of some Romanian medicinal plants against enzymes linked to neurodegenerative diseases and their antioxidant activity
Gabriela Paun1, Elena Neagu1, Camelia Albu1, Gabriel Lucian Radu2
1 Centre of Bioanalysis, National Institute for Research-Development of Biological Sciences, Centre of Bioanalysis, 060031 Bucharest, Romania
2 Centre of Bioanalysis, National Institute for Research-Development of Biological Sciences, Centre of Bioanalysis, 060031 Bucharest; Department of Analytical Chemistry, Faculty of Applied Chemistry and Materials Science, Politehnica University of Bucharest, 060042 Bucharest, Romania
|Date of Submission||17-Sep-2014|
|Date of Acceptance||26-Nov-2014|
|Date of Web Publication||27-May-2015|
Prof. Gabriel Lucian Radu
National Institute for Research-Development of Biological Sciences, Centre of Bioanalysis, 296 Spl.Independentei, P. O. Box 17-16, Sector 6, 060031 Bucharest; Faculty of Applied Chemistry and Materials Science, Politehnica University of Bu-Charest, 313 Spl.Independentei, 060042 Bucharest
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Eryngium planum, Geum urbanum and Cnicus benedictus plants are an endemic botanical from the Romanian used in folk medicine. Objective: The extracts from three Romanian medicinal plants were investigated for their possible neuroprotective potential. Materials and Methods: Within this study, in vitro neuroprotective activity of the extracts of E. planum, G. urbanum, and C. benedictus plants were investigated via inhibition of acetylcholinesterase (AChE) and tyrosinase (TYR). Total content of phenolics, flavonoids, and proanthocyanidins, high-performance liquid chromatography profile of the main phenolic compounds and antioxidant activity were also determined. Results: Among the tested extracts, the best inhibition of AChE (88.76 ± 5.2%) and TYR (88.5 ± 5.2%) was caused by C. benedictus ethanol (EtOH) extract. The G. urbanum extracts exerted remarkable scavenging effect against 2,2-diphenyl-1-picrylhydrazyl (IC 50 , 7.8 ± 0.5 μg/mL aqueous extract, and IC 50 , 1.3 ± 0.1 μg/mL EtOH extract, respectively) and reducing power, whereas the EtOH extract of C. benedictus showed high scavenging activity (IC 50 , 0.609 ± 0.04 mg/mL), also. Conclusion: According to our knowledge, this is the first study that demonstrates in vitro neuroprotective effects of E. planum, G. urbanum and C. benedictus.
Keywords: Antioxidant, Cnicus benedictus, enzyme inhibition, Eryngium planum, Geum urbanum, neurodegenerative diseases
|How to cite this article:|
Paun G, Neagu E, Albu C, Radu GL. Inhibitory potential of some Romanian medicinal plants against enzymes linked to neurodegenerative diseases and their antioxidant activity. Phcog Mag 2015;11, Suppl S1:110-6
|How to cite this URL:|
Paun G, Neagu E, Albu C, Radu GL. Inhibitory potential of some Romanian medicinal plants against enzymes linked to neurodegenerative diseases and their antioxidant activity. Phcog Mag [serial online] 2015 [cited 2021 May 14];11, Suppl S1:110-6. Available from: http://www.phcog.com/text.asp?2015/11/42/110/157709
| Introduction|| |
Herbal infusions represent a rich source of phytochemicals, many of which have potent antioxidant activities and are consumed for their health enhancing properties. , Age-related neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD) are increasing in prevalence with the rise in longevity of populations world-wide. The cholinergic hypothesis is the most accepted theory to explain the pathogenesis of AD, and, therefore, the most prescribed drugs for the treatment of AD are the cholinesterase inhibitors.  On the other hand, tyrosinase (TYR) is involved in the neuromelanin-biosynthetic pathway and could be central to dopamine neurotoxicity, as well as contributing to the neurodegeneration associated with PD.  Thus, there is great interest in finding better acetylcholinesterase (AChE) and TYR inhibitors, particularly from edible plants sources, showing low toxicity, good brain penetration and high bioavailability.  Oxidative stresses a significant risk factor for age-associated cognitive decline and is widely considered to be a critical aspect in the complex pathogenesis of AD.  Among the dietary constituents, polyphenolics appear to a play a significant role as antioxidants in the protective effect of plant derived foods. 
Eryngium planum L. (Flat Sea Holly), a species that belongs to the Apiaceae family is widely spread throughout the Romanian territory. Eryngium species are of great value for their use in traditional European medicines, due to their content of phenolic acids, flavonoids, saponins, coumarin derivatives, essential oils and acetylenes. ,,
Geum urbanum L. (Common Avens) is a herbaceous plant in the rose family (Rosaceae), widely spread in the temperate zone of Europe. Both, the herb and underground parts have been used in folk medicine. In these raw materials, the main biologically active compounds are tannins and phenolic acids. 
Cnicus benedictus L. (Blessed Thistle), the sole species in the genus Cnicus, is a thistle-like plant in the family Asteraceae, native to the Mediterranean region. This herb contains alkaloids, cnicin, benedictin, mucilage, polyacetylene, triterpenoide, lignans, flavonoids, tannin, phytosterines and volatile oils. , This is a plant with antidepressant, anti-inflammatory, antiseptic, cardiac and antimicrobial properties.
For the selected plants, the phytochemical analysis and biological studies are poorly explored.
However, no study or analysis of the G. urbanum L., E. planum L. and C. benedictus L. extracts anti-cholinesterase or anti-TYR activities have previously been published.
| Materials and Methods|| |
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, USA). The medicinal plants were acquired from a local processing plant (SC STEF MAR SRL) of herbal teas in the dry form.
Preparation of the extracts
Ground plant material was separately extracted with 70% (v/v) ethanol (EtOH) and distilled water at 60°C (aqueous) by sonication for 1 h. The obtained extract was filtered under vacuum through no. 1 Whatman filter paper, stored at 4°C for further use. The herbal's mass concentration in the solvent was 100 g/L.
Phytochemical characterization of the extracts
Determination of total phenolic acids, flavonoids, and proanthocyanidins
The total phenolic content in extracts was determined using Folin-Ciocalteu reagent, as earlier described.  Gallic acid was used as a standard to perform the calibration. Briefly, an aliquot (0.5 mL) of extract was mixed with 0.5 mL of Folin-Ciocalteu reagent. The mixture was filtered, and 4.75 mL of 200 g/L sodium carbonate was added to 0.25 mL filtrate. Methanol (MeOH) was used as the blank, and total phenolic content was calculated from a calibration curve as gallic acid equivalents (GAEs) in mg/L of extracts.
The total flavonoid content was determined according Lin method (aluminum chloride based method), with slight modifications.  Briefly, 5 mL of extract was mixed with 7.5 mL of MeOH and after that the mixture is filtered. Then, 1 mL of the extract sample was transferred to a 5 mL volumetric flask and 1 mL of 10% sodium acetate solution, 0.6 mL of 2.5% aluminum chloride hexahydrate solution and 0.5 mL MeOH were added. After 15 min. the absorbance of the mixture was measured at 430 nm and the flavonoids content express in mg rutin equivalent (RE)/L of extract was calculated using the calibration curve obtained in the 0-120 mg/L concentration range.
Total proanthocyanidin content, expressed as epicatechin equivalents (CEs), was evaluated by a modified vanillin-HCl assay method. , Catechin is commonly used to standardize the vanillin reaction. The extract solution (0.5 mL) was mixed with 2.5 mL of glacial acetic acid/hydrochloric acid (5:1 v/v) solution and 2.5 mL of 1% vanillin in glacial acetic acid (w/v). After 30 min incubation in a 30°C water bath, the absorbance of the sample and control mixtures was measured at 500 nm against a reagent blank and their difference was used to determine total proanthocyanidins of the samples, expressed as mg CE/L sample.
High-performance liquid chromatography analysis
The experiments to adapt a method for the polyphenol's measurement were based on the previously high-performance liquid chromatography (HPLC) method for the measurement of these compounds in extracts.  The chromatographic measurements were performed using a complete HPLC Shimadzu system, by means of a Nucleosil 100-3.5 C18 column, Kromasil, 100 × 2.1 mm. The system was coupled to a mass spectrometry (MS) detector, liquid chromatograph mass spectrometer-2010 detector, equipped with an ESI interface. The mobile phase consists of formic acid 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 rate was: 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. ESI source and negative ionisation mode have been used. The rutin, ellagic acid, sinapic acid, epicatechin, chlorogenic acid, rosmarinic acid, quercetin 3-β-D-glucoside, genistein, daidzein, myricetin, quercetin, luteolin, kaempferol, gallic acid, caffeic acid, ferulic acid and p-coumaric acid were used as reference standard.
Enzyme inhibition assays
Acetylcholinesterase inhibitory activity
The AChE inhibitory activity was measured using an adaptation of the method described of Ingkaninan et al.  Acetylthiocholine iodide (AChl) as substrate of the reaction and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were used for the measurement of the anti-AChE activity. Briefly, 3000 μl of 50 mM Tris-HCl buffer (pH 8.0), 100 μL of sample solution at different concentrations (3 mg/mL, 1.5 mg/mL and 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 DTNB were added. The reaction was then initiated with the addition of 50 μL of AChl (15 mM). The hydrolysis of these substrates was monitored by the quantity of yellow 5-thio-2-nitrobenzoate anion formed as the result of the reaction of DTNB with thiocholine, at a wavelength of 405 nm.
Enzyme activity was calculated as a percentage of the average velocities compared to that of the assay using buffer instead of inhibitor (extract), based on 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. The experiments were carried out in triplicate.
Tyrosinase inhibition assay
The TYR activity was measured spectrophotometrically using l-DOPA as substrate.  TYR aqueous solution (100 μL, 0.5 mg/mL), plant extract (100 μL) and 1850 μL of 0.2M phosphate buffer (pH 7.0) were mixed and preincubated 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 after 3 min. The same reaction mixture without the plant extract but the equivalent amount of phosphate buffer served as the blank. Kojic acid (Sigma) was used as the reference. The percentage inhibition of TYR activity was calculaed as follows:
where ΔAcontrol is the change of absorbance at 475 nm without a test sample, and ΔAsample is the change of absorbance at 475 nm with a test sample.
2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and ferric reducing antioxidant power assay
2,2-diphenyl-1-picrylhydrazyl -radical scavenging assay
The free radical scavenging activity of the extracts was studied by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method - based on the decrease of the DPPH (Sigma-Aldrich) maximum absorbance in the antioxidant presence, with slight modification.  It can readily undergo reduction by an antioxidant (AH), which runs as the following reaction:
DPPH + AH → DPPH-H + A
100 μL of different concentrations of extract (0.3-3 mg/mL), were mixed with 1000 μL of the freshly prepared 0.25 mM DPPH in MeOH and 1900 μL MeOH. Absorbance at 516 nm was determined after 3 min. The decreasing of the DPPH radical absorption by the action of antioxidants could be used for measuring the antioxidative activity. The percentage of DPPH radical scavenging activity of the samples was calculated as follows:
A 0 = blank absorbance; A s = sample absorbance. Trolox and vitamin C were used as standard antioxidant.
The reductive potential of the extracts was determined according to the method of Oyaizu with some few changes.  Briefly, 0.1 mL of extract was mixed with 2.5 mL phosphate buffer (0.2 M, pH 6.6) and 2.5 mL potassium ferricyanide (K 3 Fe (CN) 6 ; 1%). The mixture was incubated at 50°C for 20 min. Then, 2.5 mL of 10% trichloroacetic acid were added, and the mixture was centrifuged at 3000 rpm for 10 min. The upper layer (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of 0.1% ferric chloride. Finally, the absorbance was measured at 700 nm against a blank, by ultra violet spectrophotometer (Thermo Scientific Evolution 260 Bio). Vitamin C was used as a control.
The measurements were performed in triplicate and for statistical processing Excel 2007 was used, standard deviation was <10%.
| Results and Discussion|| |
During the over studies on medicinal plants, in order to discover new enzyme inhibitors relevant to neurodegenerative diseases, we investigated enzyme inhibitory potential of E. planum, G. urbanum and C. benedictus extracts.
As neurodegenerative disease is also associated with inflammation, free radicals being one of the causes of the inflammatory process, the antioxidant activity of the extracts were also investigated. , In the last several years herbs rich in phenolic and flavonoids contents were given the prime importance due to their free radical scavenging capability.
As can be observed in [Table 1], the total phenolic acid contents of the different extracts as GAEs were found to be highest in EtOH extracts.
|Table 1: Bioactive compounds quantified in Eryngium planum, Geum urbanum and Cnicus benedictus extracts |
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The G. urbanum EtOH extract showed the highest total phenolics (1261.5 ± 31.3 mg GAE/L), whereas the phenolic contents of E. planum aqueous extract were much smaller (342.5 ± 16.2 mg GAE/L). The highest flavonoid content was confined to the C. benedictus EtOH extract (205.9 ± 9.5 mg RE/L) followed by the E. planum EtOH extract (116.1 ± 6.1 mg RE/L). The most abundant proantocyanidins amount was determined in the G. urbanum EtOH extract (60.1 ± 2.1 mg CE/L), followed by the C. benedictus EtOH extract (58.5 ± 1.9 mg CE/L).
The HPLC-MS method has been applied for the evaluation of the sample's polyphenolic profile. The contents of polyphenols and flavones in analyzed extracts are shown in [Table 2].
Elagic acid and epicatechin were the major identified compounds in G. urbanum ethanolic extract while rutin was the major compounds in an aqueous extract.
Rutin, rosmarinic acid, chlorogenic acid and quercetin 3-β-D-glucoside were the major identified compounds in E. planum ethanolic extract while ellagic acid was present in higher amounts in the aqueous extracts.
Sinapic acid and chlorogenic acid were the major identified compounds in C. benedictus ethanolic extract.
This is the first report of daidzein compound in G. urbanum, E. planum and C. benedictus extracts.
Rosmarinic acid and chlorogenic acid, which are known for their antioxidant activity, have been described for many Eryngium species. 
Inhibition of the cholinesterase enzyme family is vital in the fight against AD, while TYR inhibition is an important drug target for PD. The AChE inhibitory activity results of E. planum, G. urbanum, and C. benedictus extracts are presented in [Table 3]. According to our findings; all extracts displayed a concentration-dependent inhibitory factor against AChE and they especially showed stronger inhibition at concentration of 3 mg/mL (79.11 ± 3.0 χ 88.76 ± 5.2%). The E. planum and C. benedictus EtOH extracts were the most potent among all the extracts against AChE [Table 3]. Although this effect is significantly lower than pharmacological drugs used in neurological diseases, such as galantamine (IC 50 = 0.14 μg/mL),  this is a natural product with potential for daily consumption, without any side effects.
As shown in [Figure 1], the extracts showed inhibition against the TYR enzyme ranging between 36.4 ± 2.8% and 88.5 ± 5.2%. Among the tested extracts, the best inhibition against AChE (88.76 ± 5.2%) and TYR (88.5 ± 5.2%) was found to be C. benedictus EtOH extract, since it is richer in flavonoids compounds than the other studied extracts [Table 1].
|Figure 1: Tyrosinase inhibitory activity of the aqueous and ethanol extracts. EP: Eryngium planum; GU: Geum urbanum; CB: Cnicus benedictus|
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The studied herbs are rich in polyphenolic compounds. As one of the major constituent of C. benedictus, sinapic acid seems to be responsible in a large amount for this herb's neuroprotective effect. For instance, Lee et al. reported that sinapic acid improved, via its anti-oxidative and anti-inflammatory activities, the Aβ1-42 protein-related pathology, including neuronal cell death and cognitive dysfunction.  Therefore, preparations based on this substance could be very efficient in the AD treatment. Although memory enhancing effect of the C. benedictus is mainly attributed to sinapic acid, other polyphenolic compounds found in these plants have been also stated to exert neuroprotective effect. In fact, rutin, ellagic acid, genistein and daidzein identified in these species have shown to exhibit neuroprotection. ,,
The inhibitory potential of TYR and AChE might depend on the hydroxyl groups of the phenolic compounds extracts that could form a hydrogen bond to the active site of the enzyme, leading to lower enzymatic activity. Phenolic compounds such as ellagic acid, tannic acid and quercetin act as potent TYR and AChE inhibitors, as reported by other authors. ,,
Until now, no AChE and TYR inhibitory activity of G. urbanum and C. benedictus has been reported.
In this work, two methods were used to evaluate total antioxidant capacity of the different extracts: DPPH free radical scavenging assay [Figure 2]a and b and ferric-reducing antioxidant power assay [Figure 3]. In the present investigation, extracts of G. urbanum showed excellent inhibition rate of DPPH: 92 ± 3.7% aqueous extract (IC 50 , 7.8 ± 0.5 μg/mL) and 95.2 ± 4.2% alcoholic extract (IC 50 , 1.3 ± 0.1 μg/mL), respectively. The EtOH extract of C. benedictus showed high scavenging activity (84.1% DPPH inhibition; IC 50 , 0.609 ± 0.04 mg/mL) and the aqueous extract also showed a high scavenging activity (68.3% DPPH inhibition; IC 50 , 0.715 ± 0.05 mg/mL). The anti-radical activity of E. planum aqueous (IC 50 , 1.731 ± 0.12 mg/mL) and EtOH (IC 50 , 1.362 ± 0.11 mg/mL) extracts were reduced in comparison with the G. urbanum and C. benedictus extracts, but were comparable to the previously investigated antioxidant activity of other species of this genus. , The results indicate that the antioxidant activity of all concentrated extracts is higher than that of Trolox (6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) and the obtained results showed anti-oxidant activity depending on dosage. Significant antiradical activity of G. urbanum is obviously due to its high polyphenolic phytocompositions. Polyphenolic compounds are well known as effective free radical scavengers and antioxidants, resulting in a close correlation between the content of phenolic compounds and antioxidant activity. 
|Figure 2: Free radical scavenging activity of the aqueous (a) and ethanol (b) extracts and standard (Trolox) by the 2,2-diphenyl-1- picrylhydrazyl method. EP: Eryngium planum; GU: Geum urbanum; CB: Cnicus benedictus|
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|Figure 3: Antioxidant capacities of the aqueous and ethanol extracts and standard (ascorbic acid), using ferric reducing power method. EP: Eryngium planum; GU: Geum urbanum; CB: Cnicus benedictus|
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The reducing power of aqueous and EtOH extracts of the three herbs are presented in [Figure 3], in that, the graphs show, that the highest reducing potential is obtained from the aqueous and EtOH of G. urbanum, significantly higher than that of the standard (ascorbic acid). The reducing power of E. planum and C. benedictus extracts at the same concentration were lower than of G. urbanum extracts but were comparable to the activities of the ascorbic acid.
The results of the study demonstrate that the studied extracts may possess memory-enhancing effect by inhibiting AChE and TYR enzymes, also showing a great antioxidant activity.
| Conclusion|| |
Our findings revealed that, among the tested extracts, the EtOH extract of C. benedictus and G. urbanum showed high AChE (over 86%) and TYR inhibitory effects (over 63%). In addition, G. urbanum extracts appear to have significant antioxidant properties, which might be possibly associated with the polyphenols high content of the plant. Thus, these extracts could be beneficial in the therapy of degenerative diseases especially where the oxidative stress and cholinergic hypothesis are involved. To the best of our knowledge, we report our study on AChE and TYR inhibitory activity of E. planum, G. urbanum and C. benedictus as being the first of this sort.
| Acknowledgment|| |
This research was supported by the Romanian National Center for Program Management - PN 09-360101/2012 project.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]