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Research Article
 

In vitro Antioxidant and Anticholinesterase Activities of Colombian Plants as Potential Neuroprotective Agents



Erika A. Plazas, Monica C. Avila, Willman A. Delgado, Oscar J. Patino and Luis E. Cuca
 
ABSTRACT

Background and Objective: Neurodegenerative diseases are complex and heterogeneous group of multifactorial and progressive disorders, which constitute a worldwide health problem. Plants are a potential source of multi-target neuroprotective agents for the treatment of multifaceted diseases such as neurological disorders. In this study, in vitro antioxidant and anticholinesterase screening of 81 ethanolic extracts of 43 Colombian species (Lauraceae, Piperaceae, Rutaceae and Myristicaceae) was performed in order to determine their neuroprotective potential. Materials and Methods: The preliminary activity was tested using TLC-bioautography assay with 2,2-Diphenyl-1-picrylhydrazyl (DPPH•), β-carotene and acetylcholinesterase (AChE). The inhibitory effect (IC50) of the most active extracts in each assay was quantified by a microplate colorimetric test. Results: From the TLC-bioautography assay, almost 36 extracts showed radical scavenging capacity and antioxidant activity by β-carotene bleaching mainly of Lauraceae, Myristicaceae and Piperaceae. In addition, 13 extracts exhibited anticholinesterase activity and the most potential extracts were characterized by the presence of active alkaloids. Extracts of Lauraceae and Rutaceae were more active against enzymatic inhibition than DPPH assay. Five extracts of Zanthoxylum (Rutaceae) and Ocotea (Lauraceae) presented strong inhibition of AChE with IC50 lower than 50 μg mL–1. However, most of these were inactive against butyrylcholinesterase. Conclusion: The most active extracts showed the presence of isoquinoline alkaloids, this characteristic may be related with the strong AChE inhibition.

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Erika A. Plazas, Monica C. Avila, Willman A. Delgado, Oscar J. Patino and Luis E. Cuca, 2018. In vitro Antioxidant and Anticholinesterase Activities of Colombian Plants as Potential Neuroprotective Agents. Research Journal of Medicinal Plants, 12: 9-18.

DOI: 10.3923/rjmp.2018.9.18

URL: https://scialert.net/abstract/?doi=rjmp.2018.9.18
 
Received: October 03, 2017; Accepted: November 04, 2017; Published: February 09, 2018

INTRODUCTION

Neurodegenerative diseases (ND) comprise a diverse group of late-onset central nervous system (CNS) disorders, which affect more than 30 million people worldwide. The most common ND includes Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD)1. AD and PD are the most prevalent and high-cost disorders and constitute a rising public health problem due to high incidence and the low effectiveness of current treatments2.

The neurodegenerative disorders were characterized by the progressive and irreversible loss of neuronal cell populations from specific regions of the brain, with distinct morphological and pathophysiological features3. They have complex and multifactorial pathology; although, the specific mechanisms of neuronal death in ND were unknown currently, huge evidence suggested that many genetic and environmental factors play an important role in the pathophysiology4. In the last decades neuroscience has achieved significant advances in the understanding of the neurodegenerative process and basic biochemical pathways have been recognized in different pathogenic cascades5,6. The most common factors involve oxidative stress, metal dyshomeostasis, mitochondrial dysfunction, misfolding and dysfunctional trafficking of proteins7,8.

The reactive nitrogen and oxygen species (RNS and ROS) produced in the respiratory chain play important functions in the physiology of the CNS, mainly as biological messengers. Nevertheless, different exogenous factors such as high cholesterol diets, pollution and metal overexposure increase the imbalance between generation and elimination of RNS and ROS, triggering an oxidative response in nervous system. The brain is particularly susceptible to damage by oxidative stress due to their dependence on oxygen consumption and deficit of endogenous antioxidant systems9. The overproduction of nitric oxide (NO⋅) and RNS promote inflammatory process elicit mitochondrial, activated microglia, cytotoxicity and apoptosis; which finally lead to massive neuronal death. Thus, the oxidative stress is a key biochemical cascade associated whit neurodegeneration10,11. Therefore, the search of antioxidants is a promising strategy to the development of neuroprotective agents as prevention or clinical treatment12.

Due to the multifactorial nature of ND, the pharmacotherapy based on a single target has been insufficient, lead to ineffective or symptomatic drugs13. For these reason, the medicinal chemistry had proposed different pharmacological approaches to counteract these problems, as well as to improve the efficiency of current treatments. The use of combinations of drugs with different therapeutic mechanisms showed synergistic effects, however, might be disadvantageous due to increase the toxicity by biotransformation. Thus, emerge the multi-target directed ligands or MTDLs approach focused on development to a single agent capable to modulate different targets involved in the cascade of pathological events leading to a given disease14,15. The most extensive group of MTDLs agents explored in the last years comprises mainly cholinesterase inhibitors with some additional properties that increase the pharmacological profile16. The inhibition of cholinesterase is an important target strategy not only for AD, also for senile dementia, ataxia and PD. Actually, cholinesterase inhibitors were the only approved drugs for treating of AD as Galantamine and Rivastigmine, however, they only treat cognitive symptoms17. Natural products, one of the most promising sources of privileged scaffolds with multimodal characteristics for the treatment of neurodegenerative diseases18,19. Alkaloids and polyphenols are the secondary metabolites with greater neuroprotective potential against different biochemical pathways of ND diseases20-22.

The present study achieved an in vitro screening antioxidant and anticholinesterase activities of Colombian Lauraceae, Piperaceae, Rutaceae and Myristicaceae species, in order to select plant extracts for the subsequent isolation of secondary metabolites with neuroprotective potential.

MATERIALS AND METHODS

Plant material: Eighteen Lauraceae, fifteen Piperaceae, four Myristicaceae and five Rutaceae species were collected between August, 2015 and June, 2016, in different regions from Colombia. The plant material was identified by the Herbario Nacional Colombiano de la Universidad Nacional de Colombia, where voucher specimens have been deposited (Table 1).

Preparation of the extracts: Plant material of each species was separated into different parts (leaves, stems, flowers and fruits when it was possible) and was dried at room temperature. The powdered air-dried plant material was extracted by maceration with ethanol at room temperature. Resulting extracts were concentrated under vacuum at 40°C to obtain dry extracts.

Chemicals: Acetylcholinesterase from electric eel (E.C.3.1.1.7, Type VI-S), butyrylcholinesterase from horse serum (E.C.3.1.1.8), butyrylthiocholine iodide (BTCI), Fast Blue B salt, 1-Naphthyl acetate, dimethylsulfoxide (DMSO), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), β-carotene and bovine serum albumin (BSA) were purchased from Sigma-USA.

Table 1:
Extracts of Colombian plants tested using TLC-bioautography assay with DPPH, β-carotene and acetylcholinesterase

+: Active (Presence of spots with intensities comparable to positive controls), -: Non-active

Acetylthiocholine iodide (ATCI) and dithiobis-nitrobenzoic acid (DTNB) were obtained from Alfa Aesar. Sodium dihydrogen orthophosphate (NaH2PO4·2H2O), disodium hydrogen phosphate (Na2HPO4·2H2O), sodium bicarbonate (NaHCO3) and silica gel plates (silica gel 60 F254) were obtained from Merck (Darmstadt, Germany).

TLC bioautography: The preliminary activity of 81 extracts was evaluated by TLC-bioautography assay using silica gel as stationary phase and different mobile phases. The antioxidant potential was assessed by DPPH· scavenging and β-carotene bleaching test; the acetylcholinesterase inhibitory was carried out by Fast Blue assay. The extracts were solubilized in methanol at a final concentration of 10 mg mL–1 and 10 μL of these were spotted at the TLC plates. The plates were developed with two different mobile phases (hexane-ethyl acetate 8:2 and chloroform-methanol 9:1) to ensure that most compounds were separated and analyzed. Then, the developed plates were dried and sprayed with the reagents for each assay.

Antioxidant activity: A methanolic solution of the 0.2% DPPH radical was used, past 5 min after the plates were examined and the active compounds (free radical scavengers) appeared as yellow to white spots against to the purple plate. Additionally, two plates were sprayed with a β-carotene solution (0.05% in chloroform) and left at UV light (365 nm) until degradation of the reagent. Active compounds (β-carotene protectors) remained as yellow-orange spots on a white background. As a positive control, a stock solution of quercetin (1 mg mL–1) was used23,24.

Acetylcholinesterase inhibition: This was performed as by Komsta et al.25. with a few modifications. The plates were sprayed with enzyme solution (4 U mL–1), thoroughly dried and incubated at 36°C in a humid chamber for 30 min.

Subsequently the plates were sprayed with α-naphthyl acetate solution (2.5 mg mL–1) and re-incubated under the described conditions. Finally, the Fast Blue B solution (2.5 mg mL–1) was sprayed; and the characteristic purple coloration was developed. Potential AChE inhibitors were appeared as clear zones on a purple-colored background. Berberine and galantamine (1 mg mL–1) were used as positive controls in each plate.

The results of the AChE-TLC were compared with separate TLC plates with the same mobile phases spraying with Dragendorff reagent.

Microplate assays: The active extracts in TLC assays were tested in microdilution to determine the inhibitory concentration (IC50) according to following methodologies:

DPPH⋅ scavenging: A 0.1 mM solution of DPPH radical in methanol was used. From a stock solution (500 pmm) of each extract serial dilutions were made. DPPH solution of 195 μL was added to 5 μL of samples in methanol at different concentrations. The absorbance was measured at 517 nm at 0, 10 and 30 min. Positive controls were quercetin and ascorbic acid (100-1 ppm). Each measurement was made at least in triplicate. The inhibitory concentration (IC50) was calculated and expressed as VCEAC (mg L–1) in comparison with ascorbic acid26.

Anticholinesterase activity: The enzymatic activity of positive TLC-extracts was measured using an adaptation of the colorimetric Ellman’s method27 against AChE and BChE. The enzyme solution was prepared at 0.2 U mL–1 in phosphate buffer solution (0.1 M, pH 8 with 0.1% BSA). As substrate, ATCI or BTCI (15 mM) was used and Ellman et al.27 reactive (DTNB 3 mM) in phosphate buffer solution28,29. The extracts were solubilized in DMSO (10% in phosphate buffer) at final concentration of 500 μg mL–1 and serial dilutions were made. In a 96 well plate, 30 μL of samples, 125 μL of DTNB and 30 μL of enzyme were mixed, the plate was incubated at 36°C for 10 min and the absorbance was measured at 405 nm 3 times every 5 min. Then 30 μL of substrate was added and then absorbance was measured at 1 min intervals for 30 min. Assays were performed in triplicate and compared to the negative control. Berberine and galantamine (1 and 100 μg mL–1) were used as positive controls.

RESULTS AND DISCUSSION

TLC bioautography: According to the results of preliminary screening by TLC-bioautography (Table 1), among 36 extracts of 22 species showed compounds with DPPH radical scavenging capability, being the extracts with more antiradical metabolites mainly of the Lauraceae, Myristicaceae and Piperaceae families. Antiradical scavenging compounds were found to be predominant with medium to high polarities and were eluted with chloroform-methanol (Fig. 1). However, a few extracts of Piperaceae specifically, Piper pesaresanum, Piper artanthe and Piper bogotense exhibit nonpolar compounds with high radical scavenging capacity. The 36 extracts with radical scavenging capacity were also tested for antioxidant activity by β-carotene bleaching; in this test, the extracts presented similar results to those of the DPPH assay. Most of the extracts showed active spots, mostly in medium and low Rf regions (Fig. 1). The antiradical active nonpolar spots of P. pesaresanum, P. artanthe and P. bogotense were also active in the protection of β-carotene retained a strong orange color. From P. bogotense and P. pesaresanum different phenolic metabolites have been isolated, predominantly, benzoic acid derivatives, prenylated quinones, flavonoids and amides. In previous studies, the antioxidant activity of Piperaceae extracts has been related with the presence of flavonoids and derivatives of benzoic acid, consequently, this kind of compounds could be related with the antiradical activity observed in the results of this study30,31.

Fig. 1:
TLC-Bioautography of the most active antioxidant extracts

Fig. 2:
TLC-Bioautography of the most active anticholinesterase extracts

Also, was noted that all Ocotea discolor, Virola sebifera and P. bogotense exhibited very strong active polar compounds in both bioassays.

To detect the presence of phenolic antioxidant compounds the active extracts were analysed in TLC with the same mobile phases spraying with ferric chloride (2%) and anisaldehyde sulphuric acid (Fig. 1). In these results medium and high polarity active compounds (antiradical/antioxidant) were appeared as green, blue or purple spots by reaction with FeCl3; indicating the possible presence of free phenolic hydroxyls. On the other hand, with anisaldehyde reagent a variety of colours was observed, showing the presence of different metabolite scaffolds in the study extracts. Compounds that showed positive reaction with FeCl3 appeared as red, blue or purple spots with anisaldehyde, which confirmed the presence of phenolic type antioxidants in the most active extracts32. The highest antioxidant/radical scavenger potential by TLC was detected in the extracts of O. discolor, V. sebifera, Virola sp., Compsoneura sp., P. pesaresanum, P. artanthe and P. bogotense.

In the AChE inhibitory assay, 13 extracts of 7 species showed one or more inhibition zones on the purple background (Fig. 2). In the hexane mobile phase fewer and weakly active spots were observed. While, in chloroform mobile phase the strongest AChE inhibitory potential was detected. Some extracts of Rutaceae and Lauraceae showed the highest number of active spots, placed in the middle and high polarity. Particularly the most potent extracts as O. discolor, Z. monophyllum, Z. rhoifolium and Z. quinduensis exhibited white spots whose intensities comparable with the positive controls (berberine and galantamine) located at the end of the plate (Fig. 2).

Considering that Zanthoxylum and Ocotea species are characterized by the presence of alkaloids33,34 and this kind of metabolites were categorised by being cholinesterase inhibitors35 a TLC analysis spraying with Dragendorff was achieved to detect the possible presence (Fig. 2). The most active extracts (O. discolor, Z. monophyllum and Z. quinduensis) showed AChE inhibitory alkaloids; both genus have in common occurrence of isoquinoline alkaloids such as aporphine, protoberberine or benzophenanthridine. Some isoquinoline alkaloids have a neuroprotective potential against different targets36.

Microplate assays: DPPH⋅ scavenging assay is the most extensively used test for screening antioxidant activity in extracts, due to, easiness and low cost. In this assay, the antioxidant compounds reduced the radical to pale yellow hydrazine, which involved the measurement of decrease in absorbance proportional to concentration of free radical scavenger added to DPPH reagent solution37. The IC50 amount of antioxidant necessary to decrease by 50% the initial DPPH⋅ concentration, thus, lower IC50, indicated high antiradical scavenging efficiency. Therefore, according to the results (Table 2), the most active extracts were C. cinnamomifolium, N. membranaceae and O. longifolia which presented IC50 lower than 50 μg mL–1, all belonging to Lauraceae family. Some species of Lauraceae have shown antioxidant potential by DPPH* scavenging with variable inhibitory concentrations38,39; leaves and stems extracts of species of Ocotea genus like O. minor and O. ceanothifolia exhibited highly activity in DPPH assay40. In the present screening leaves extract of O. longifolia present the lowest IC50 (22.46 μg mL–1), this was the first report of antioxidant activity for the specie. In previous research of the authors, from the bark of O. longifolia were isolated eight secondary metabolites mainly terpenes and the chemical composition of leaves essential oil was studied; however, the radical scavenging capacity was not determined41. The leaves of O. longifolia were more active than bark, thus, could be supposed that active compounds in the leaves may be different from those previously isolated in the bark.

Table 2:
Antioxidant activity of selected extracts by DPPH
IC50 values represent the mean±SEM of three parallel measurements (p<0.05)

According to the profiles in TLC-bioautography (Fig. 1), both organs differ mainly in the most polar compounds (origin).

On the other hand, relating the bioautography and quantification results, the extracts with the highest potential in TLC (O. discolor, V. sebifera, Virola sp., Compsoneura sp., P. pesaresanum and P. artanthe) did not necessarily show lower IC50, which may be due to antagonistic effects in the total extracts.

Since the main limiting of the of DPPH⋅ is the steric accessibility with this test it is possible to determine mostly small compounds with radical scavenging capacity, hence its advisable to use another assay if it is necessary to determine the presence of large antioxidant compounds. For the purpose of this screening research, the results obtained were satisfactory with this test.

Data in Table 3 showed the inhibitory activity against cholinesterases was determine by Elman’s colorimetric test, the inhibition percentage was determined for each concentration and IC50 was calculated by log Probit analysis. Five extracts of three species showed strong inhibition of AChE (higher than 80%) at 250 μg mL–1 and IC50 lower than 50 μg mL–1. However, all extracts were inactive against BChE.

The active species against AChE (O. discolor, Z. quinduensis and Z. monophyllum) exhibited AChE inhibitory alkaloids in TLC-bioautography. Extract of bark to Z. monophyllum was the most powerful with an inhibitory concentration (10.2 μg mL–1) comparable with the positive controls.

Table 3:
Acetyl cholinesterase (AChE) and butyryl cholinesterase (BChE) inhibitory activity of selected extracts
aIC50 values determined by log-probit analysis 95% confidence interval in parentheses

In previous chemical studies of Z. monophyllum the presence of alkaloids, lignans, terpenes and coumarins has been reported42,43. From the bark of this specie were previously isolated protoberberine alkaloids as berberine and jathrorrhizine44 and recently they have been reported as AChE inhibitors45 which could explain the strong inhibitory activity of the extract.

From the Z. quinduensis in our research group were isolated some benzophenanthridine alkaloids44, including chelerythrine, which in recent studies showed inhibitory activity against AChE and BChE45.

Finally, from wood of Ocotea discolor were isolated 4 aporphine alkaloids: ocoxilonine, ocoteine, dicentrine and 1,2-Methylenedioxy-3,10,11-trimethoxyaporphine46, which didn’t reported previously for anticholinesterase activity. Therefore, to continue with the present research, currently being determined the inhibitory activity of these alkaloids and molecular docking studies were also performed to explore the detailed interaction with AChE.

The Genus Zanthoxylum and Ocotea are characterized by the occurrence of isoquinoline alkaloids and this feature seems to be related to AChE inhibitory activity. From this perspective, they can be considered promising for target isolation of potential multitarget therapeutic agents for the treatment of neurodegenerative diseases.

CONCLUSION

The C. cinnamomifolium, N. membranaceae and O. longifolia extracts, all belonging to Lauraceae family, showed high DPPH antiradical scavenging efficiency. Of these, O. longifolia leaves extract present the lowest IC50. On the other hand, five extracts of Zanthoxylum and Ocotea genus exhibited strong anticholinesterase activity against AChE, they were characterized by the presence of isoquinoline alkaloids. By comparison of antioxidant and anticholinesterase activities, was possible to identify at least five extracts which have activity in the both assays. Therefore, further studies should be conducted to isolate and characterize potential neuroprotective compounds.

SIGNIFICANCE STATEMENTS

This study discovers the possible relationship between the presence of isoquinolone alkaloids and anticholinergic activity in species of Ocotea and Zanthoxylum genus. Thus, they can be considered potential multitarget therapeutic agents for the treatment of neurodegenerative diseases. Therefore, which gives steadiness to the investigations in order to make rational isolation of active alkaloids and extend it with molecular docking studies to explore the detailed interaction with targets.

ACKNOWLEDGEMENT

The authors are thankful for the financial support provided by the Dirección de Investigación de Sede Bogotá, Universidad Nacional de Colombia (grant number 201010026356) at the project entitled "Búsqueda de agentes para el tratamiento del Alzheimer en especies vegetales de la flora colombiana (fase I)" to carried out this research.

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