Determination of Anti-Neurodegenerative Disease Activity of Plant Compounds

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1		Tomasz Tuzimski	--	4339	2022-07-27 12:52:12	\|
2	Reference format revised.	Lindsay Dong	-3 word(s)	4336	2022-07-28 06:22:23	\| \|
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This entry is adapted from the peer-reviewed paper 10.3390/molecules27103222

Neurodegenerative diseases, among which one of the most common is Alzheimer’s disease, are a multifactorial disease and therefore demand multiple therapeutic approaches. In the last few years, different active constituents from plants have been tested as potential drugs in neurodegenerative disease therapy. The availability, lower price and less toxic effects of herbal medicines compared with synthetic agents make them a simple and excellent choice in the treatment of neurodegenerative diseases. The empirical approach to discovering new drugs from the systematic screening of plant extracts or plant-derived compounds is still an important strategy when it comes to finding new biologically active substances.

anti-Alzheimer’s disease activity plant extract components enzyme activity inhibition IC50 values

1. Introduction

Neurodegenerative diseases are a heterogeneous group of disorders that are characterized by the progressive degeneration of the structure and function of the central nervous system or the peripheral nervous system. Common neurodegenerative diseases include Alzheimer’s disease and Parkinson’s disease. Alzheimer’s disease is a chronic progressive neurodegenerative disorder that leads to the selective deterioration of cholinergic neurons in the basal forebrain ^[1]. Patients with Alzheimer’s disease are characterized as having difficulties with cognition, such as loss of memory and reasoning disabilities due to a decrease in neuronal activity and decreased concentrations of neurotransmitters in intersynaptic space, causing poor synaptic transmission, which leads to a deficit in cholinergic neurotransmission in the central nervous system.

The pathogenicity of Alzheimer’s disease is complex and includes genetic and environmental factors and therefore demands multiple therapeutic approaches. Some of the more well-known processes involved in Alzheimer’s disease pathogenesis include cholinergic deficit, oxidative stress, inflammatory pathways (especially NFκB) and the hyperphosphorylation and aggregation of tau proteins and β and γ secretases responsible for APP processing ^[2]. The most important changes observed in the brain are a decrease in cortical levels of the neurotransmitter acetylcholine. In Alzheimer’s disease patients, acetylcholine has a very short half-life due to the presence of large amounts of acetylcholinesterase, an enzyme involved in the metabolic hydrolysis of acetylcholine at cholinergic synapses in the central and peripheral nervous system ^[3]. Alzheimer’s disease, which is the most common form of neurodegenerative disorder, is also the result of the accumulation of amyloid-β peptide into microscopic “plaques” and the twisting of tau proteins into strands of dead and dying neurons. The early stages of Alzheimer’s disease are also associated with inflammation and oxidative stress ^[4].

Plants are rich resources of different bioactive constituents that can be used for the treatment of several diseases. Especially, traditional medicinal plants have served as a repository of bioactive constituents, and they are the basis for new drug search. Therefore, the empirical approach to discover new drugs from the systematic screening of plant extracts or plant-derived substances remains an important strategy for finding new biologically active compounds. Currently, there are a large number of natural compounds of plant origin, such as alkaloids, terpenoids and phenolic compounds, with neuroprotective effects, which are considered for use in the treatment of neurodegenerative diseases, including Alzheimer’s disease or Parkinson’s disease.

Nowadays, in vitro and in vivo methods are used for screening compounds that can be potentially used for the treatment of neurodegenerative diseases. However, in vivo screening methods are time-consuming, waste a significant amount of human and material resources and usually consume large amounts of raw material ^[5]. The in vitro method is rapid and usually requires only a small amount of raw material, and it is suitable for the preliminary selection of active components from a variety of medicinal herbs and foods.

Cytotoxicity testing is also very important for safety assessment extracts from medicinal plants and for the search for new active compounds. Cytotoxicity testing is important when it comes to assessing and validating the safety of medicinal plants for traditional use, and it serves as a guide in the quest for novel active compounds. Investigations into the cytotoxicity of extracts and active compounds are evaluated in normal cell lines. For further investigation, extracts and their active components that exhibit the highest possible activity against Alzheimer’s disease and, at the same time, show the lowest toxicity should be selected.

1.1. Inhibition of Acetylcholinesterase Activity

Enzymes play several important roles in the homeostasis of living organisms, catalyzing important physiological reactions. In the control of diseases, it is possible to use the strategy of inhibiting the activity of a certain enzyme to improve the clinical condition of a specific disease. Cholinesterases include two types of enzymes, namely acetylcholinesterase and butyrylcholinesterase. Acetylcholinesterase preferentially hydrolyzes acetylcholine, while butyrylcholinesterase hydrolyzes butyrylcholine more efficiently than acetylcholine. Additionally, acetylcholinesterase is mostly of neuronal origin, while butyrylcholinesterase is primarily present in the blood and glial cells ^[6].

Acetylcholinesterase is a serine hydrolase enzyme whose main function is to modulate cholinergic signal transmission through the hydrolysis of acetylcholine. The enzyme catalyzes the hydrolysis of the neurotransmitter acetylcholine into the two inactive compounds choline and acetic acid ^[7]^[8].

Acetylcholinesterase is primarily responsible for the termination of the nerve impulse transmission at the cholinergic synapses through catalyzing the hydrolysis of the neurotransmitter acetylcholine and accelerates the aggregation of β-amyloid peptides. An important strategy for treating Alzheimer’s disease is to keep the levels of acetylcholine in the synaptic cleft by blocking the acetylcholinesterase. The inhibition of acetylcholinesterase in the brain leads to an increase in acetylcholine concentration and partly restores the substantial impairment of memory and cognitive dysfunctions. Furthermore, some research has shown that acetylcholinesterase induces amyloid fibril formation by interaction throughout the peripherical anionic site of the enzyme, forming highly toxic acetylcholinesterase–β-amyloid peptide complexes ^[9].

Most of the drugs currently used for treating Alzheimer’s disease (galanthamine, donezepil, tacrine, rivastigmine) are inhibitors of acetylcholinesterase activity ^[2].

One of the most important therapeutic strategies in the treatment of neurodegenerative diseases is controlling the level of acetylcholine, as a neurotransmitter in cholinergic synapses, through blocking the degradation of acetylcholine using acetylcholinesterase inhibitors ^[10]. Therefore, important issues for finding new efficient treatments of neurodegenerative diseases may be the discovery of effective acetylcholinesterase activity inhibitors.

1.2. Inhibition of Butyrylcholinesterase Activity

Butyrylcholinesterase is mainly involved in the breakdowns of butyrylcholine. The enzyme acetylcholinesterase predominates in the healthy brain, with butyrylcholinesterase considered to play a minor role in regulating brain acetylcholine levels. For this reason, under normal conditions, acetylcholine is dominantly hydrolyzed by acetylcholinesterase. However, when the level of acetylcholinesterase in cholinergic transmission declines, butyrylcholinesterase can play a function compensation role for acetylcholinesterase to some extent to maintain normal cholinergic pathways ^[6]. Butyrylcholinesterase activity progressively increases in patients with Alzheimer’s disease, while acetylcholinesterase activity remains unchanged or declines ^[11]. Both enzymes, therefore, represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral and global functioning characteristic of Alzheimer’s disease. The dual inhibition of acetylcholinesterase and butyrylcholinesterase is beneficial for Alzheimer’s disease patients, especially since butyrylcholinesterase replaces acetylcholinesterase in the acetyl choline catabolism in advanced Alzheimer’s disease patients ^[11].

Butyrylcholinesterase inhibitors also increase choline levels for the reduction in Alzheimer’s disease symptoms. In the later stages of Alzheimer’s disease, acetylcholinesterase activity is downregulated by up to 33–45% of normal values, while the activity of butyrylcholinesterase is improved by 40–90% in certain brain regions ^[12]. However, except for rivastigmine, which is a dual acetylcholinesterase-butyrylcholinesterase inhibitor, approved selective acetylcholinesterase inhibitor drugs are not suitable for late-stage Alzheimer’s disease since acetylcholine hydrolysis in the late stage of the disease mainly depends on butyrylcholinesterase but not acetylcholinesterase ^[6]. Butyrylcholinesterase has a multitude of hydrolyzing activities, not only nonspecific cholinesterase activity but also acylamidase and peptidase activities ^[13]. The peptidase activity of butyrylcholinesterase is important because it is believed to be involved in the development and progression of Alzheimer’s disease, as it is a causative factor in the production of β-amyloids.

1.3. Inhibition of Amyloid Fibrils Production

Neurodegenerative diseases are also characterized by the extraneuronal accumulation of β-amyloid peptide. The accumulation of aggregated proteins at neurons has been correlated with Alzheimer’s disease patients, who show the presence of amyloid fibrils in the brain, which indicates the relationship between amyloid fibrils and the disease ^[14]. The accumulation of this toxic peptide leads to the deposition of β-amyloid into plaques and is thought to drive a pathologic cascade, which culminates in neuronal death ^[15]. These fibrils accumulate in the brain cells and central nervous system of Alzheimer’s disease patients and contribute to the symptoms of dementia. In histopathological investigations, the extracellular formation of senile plaques and intraneuronal appearance of neurofibrillary tangles, which are, respectively, due to the accumulation of β-amyloid peptides and hyperphosphorylated tau, are observed ^[16]. The amyloidogenic pathway is the consequence of the cleavage of β-amyloid precursor protein by the two β-amyloid-forming β- and γ-secretases that release 39–43-amino acid-long β-amyloid peptides, which are key players in the progression of Alzheimer’s disease. The presence of large α-amylase units in postmortem entorhinal cortex from Alzheimer’s disease patients and nondemented controls were also determined. The inhibition of amyloid fibrils production by plant extracts and compounds isolated from plants have been tested much less frequently than the acetylcholinesterase and butyrylcholinesterase activity inhibition.

β secretase initiates the production of the toxic β-amyloid that plays a crucial role in Alzheimer’s disease pathogenesis. β secretase, widely known as β-site amyloid precursor protein cleaving enzyme 1, cleaves the amyloid precursor protein in the first step in β-amyloid peptide production. The inhibition of β secretase is a prime therapeutic target for lowering cerebral β-amyloid concentrations in Alzheimer’s disease, and currently, the clinical development of β secretase inhibitors is being pursued.

1.4. Inhibition of Monoamine Oxidases

Monoamine oxidases catalyze the oxidative deamination of pharmacologically important monoamine neurotransmitters and are present as two monoamine oxidase isoforms (monoamine oxidase A and monoamine oxidase B) in the outer mitochondrial membranes of most tissues, including the brain. Monoamine oxidase (MAO) catalyzes the oxidative deamination of biogenic and xenobiotic amines and has an important role in the metabolism of neuroactive and vasoactive amines in the central nervous system (CNS) and peripheral tissues. Monoamine oxidase is critically related to amyloid plaque formation in Alzheimer’s disease patients, and monoamine oxidase B is expressed at high levels in the brain of patients with Alzheimer’s disease ^[17]. However, excessive monoamine oxidase inhibition, which can occur as a result of an excessive intake of enzyme inhibitors, is also harmful. Serotonin toxicity may cause a serious pathological disorder resulting from hyperactivity of serotonin neurotransmitter as a result of an excessive accumulation of serotonin due to excessive monoamine oxidase inhibition.

1.5. Inhibition of Pancreatic Lipase

Emerging evidence indicates that elevated cholesterol and triglyceride levels precede Alzheimer’s disease pathology ^[18]. Obesity and high levels of plasma cholesterol in middle age are related to a higher risk of Alzheimer’s disease, and elevated plasma triglyceride levels also precede amyloid deposition in Alzheimer’s disease mouse models.

1.6. Inhibition of Tyrosinase

Tyrosinase is a copper enzyme that plays an essential role in melanin biosynthesis in skin and hair and has also been proposed to contribute to the formation of neuromelanin ^[19]. The levels of the enzyme striatal-enriched protein tyrosine phosphatase are also raised in several different neurodegenerative disorders, including Alzheimer’s disease, fragile X syndrome and schizophrenia. The enzyme striatal-enriched protein tyrosine phosphatase normally opposes the development of synaptic strengthening, and these abnormally high levels of active enzyme striatal-enriched protein tyrosine phosphatase disrupt synaptic function by removing phosphate groups from a number of proteins, including several glutamate receptors and kinases. Dephosphorylation results in the internalization of glutamate receptors and inactivation of kinases—events that disrupt the consolidation of memories ^[20].

1.7. Inhibition of Inflammatory Effects

Inflammation represents the defensive reaction of an organism to harmful exogenous or endogenous factors through the immune system. Inflammatory processes are involved in the onset and maintenance of many severe disorders including neurodegenerative diseases such as Alzheimer’s disease. Currently, increasing evidence highlights the important role of neuroinflammation in the degenerative process of neurodegenerative diseases. In Alzheimer’s disease, activated microglia and astrocytes are attracted and activated by β-amyloid plaques and release a series of pro-inflammatory mediators, resulting in inflammatory responses that may further damage neuronal cells, stimulate β-amyloid synthesis and increase microglial activation through a positive feedback loop ^[21]. Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation. It gives an anti-inflammatory effect under normal physiological conditions. On the other hand, NO is considered a pro-inflammatory mediator that induces inflammation due to overproduction in abnormal situations. NO is synthesized and released into endothelial cells by the help of nitric oxide synthases that convert arginine into citrulline, producing NO in the process.

1.8. Antioxidant Activity

Reactive oxygen species and oxygen-centric free radicals such as hydroxyl radical, superoxide radical and hydrogen peroxide cause tissue damage and then cell death, which can oxidize lipids, proteins and DNA. Humans are unavoidably and continually affronted by different environmental stresses caused by reactive oxygen species, which induce pathological processes of many neurodegenerative diseases ^[22]. Nervous systems are understood to be principally susceptible to oxidative stress due to their limited antioxidant capacity, the utilization of metabolic oxygen, the failure of their neurons to synthesize glutathione and their high lipid content.

2. Determination of Anti-Neurodegenerative Disease Activity of Plant Compounds

2.1. Alkaloids

Currently, the most commonly used drugs for the treatment of neurodegenerative diseases contain alkaloids as an active ingredient, whose action lies in the inhibition of acetylcholinesterase. For this reason, it is promising to evaluate other alkaloids from various families of plants. A large number of alkaloids have been isolated from plants, of which some are tested for their possible acetylcholinesterase inhibition potential. Most often as potential anti-neurodegenerative disease drugs, alkaloids belonging to the steroidal, isoquinoline and indole classes, have been considered. Among these natural products, alkaloids are considered to be the most promising candidates for the treatment of neurodegenerative diseases due to their complex nitrogen-containing structures. Alkaloids, especially in high concentrations, can exhibit toxic properties, but investigations of alkaloids toxicity have rarely been conducted. A number of acetylcholinesterase and butyrylcholinesterase activity inhibitors have been isolated from various plant extracts, as extensively described in the literature.

Indole alkaloids, geissoschizoline, geissoschizone, geissospermine and 3′,4′,5′,6′-tetradehydrogeissospermine isolated from stembarks of Geissospermum vellosii, a native tree of Brazil, were tested as acetylcholinesterase inhibitors by Ellman’s method performed in a 96-well plate ^[21]. The highest anti-cholinesterase activity was observed for 3′,4′,5′,6′-tetradehydrogeissospermine with IC₅₀ = 0.45 μM, but in cell viability tests (performed on mouse microglia N9 cell line), only geissoschizoline was not cytotoxic. For geissoschizoline in an experiment with Electrophorus electricus acetylcholinesterase, IC₅₀ = 5.86 μM, and with human acetylcholinesterase, IC₅₀ = 20.40 μM. Investigated indole alkaloids from Geissospermum vellosii were also evaluated as butyrylcholinesterase inhibitors ^[21]. IC₅₀ values obtained by modified Ellman method in experiments with equine serum butyrylcholinesterase were from 0.32 μM obtained for 3′,4′,5′,6′-tetradehydrogeissospermine to 82.98 μM for geissospermine. In experiments with human butyrylcholinesterase, IC₅₀ obtained for geissoschizoline was equal 10.21 μM. Both enzyme kinetic studies showed that geissoschizoline presented a mixed-type inhibition mechanism. The molecular docking simulation performed for acetylocholinesterase and butyrylcholinesterase shows that geissoschizoline interacts with both active site and peripheral anionic site, which suggest a dual site inhibitor profile. The anti-inflammatory activity of investigated alkaloids was also evaluated ^[21]. For the determination of anti-inflammatory activity, N9 cells cultured in 96-well plates were treated with lipopolysaccharide and geissoschizoline for 48 h. NO production was assessed by the determination of nitrite levels using the colorimetric Griess method ^[23]. An ELISA assay of inflammatory mediators was also performed. In the investigations, N9 cells cultured in 96-well plates were treated with lipopolysaccharide, physostigmine and geissoschizoline for 24 h. The levels of tumor necrosis factor alpha were determined in the cell supernatant, using enzyme immune-assay kits. Geissosquizoline showed an anti-inflammatory role, reducing the microglial release of NO and TNF-α at a concentration 20-fold lower than acetylcholinesterase IC₅₀ and 10-fold lower than butyrylcholinesterase IC₅₀. Indole alkaloid geissosquizoline, due to its multifunctional activity, can be useful in preventing neurodegeneration and restoring neurotransmission.

The extracts obtained from Ocotea percoriacea containing alkaloids isocorydine N-oxide, isocorydine N-oxide derivative, palmatine, roemerine and roemerine N-Oxide were in vitro tested for acetylcholinesterase activity inhibition. The determination of anti-cholinesterase activity was performed by modified Ellman method with the application of 96-well microplate ^[24]. Only hexane and dichlorometane fractions of Ocotea percoriacea extracts at concentration 1 mg/mL showed high inhibition activity after 30 min from the beginning of the reaction (83.28% and 92.09%, respectively). Ethanolic, buthanolic and aqueous fractions exhibited significantly lower activity. In silico studies performed for the alkaloids isolated from Ocotea percoriacea extracts suggest that the alkaloids bind as classical drugs such as tacrine and donepezil in the main binding sites and that they can be considered compounds with therapeutic potential.

Various another isoquinoline alkaloids have been tested as potentially acetylcholinesterase inhibitors. The anti-acetylcholinesterase activity of the alkaloids anonaine, glaucine and xylopine from Annona cherimola was evaluated using high-performance thin-layer chromatography coupled with mass spectrometry (HPTLC-MS) ^[25]. Chromatography was carried out on silica gel plates with a mobile phase that contains the enzyme substrate 1-naphthyl acetate. After separation, the mobile phase was removed, and an enzymatic solution in buffer at pH 7.8 was sprayed on a TLC plate. The liquid excess was quickly removed but not completely in order to keep the enzyme active. Next, the plate was incubated at 37 °C for 10 min. Immediately, a Fast Blue B salt aqueous solution was sprayed onto the plate to obtain a purple background, which contrasts with colorless inhibition zones. Acetylcholinesterase inhibitors were identified by direct analysis by means of a TLC-MS interface ^[26].

2.2. Essential Oils

Essential oils are mixtures of bioactive compounds that are synthesized by plants as secondary metabolites, such as terpenes and terpenoids. The compositions of essential oils are not only different in various plant species but can also be influenced by environmental factors such as soil type, climate and harvest time. In the literature, plants rich in essential oils have been proven to be a potential source of active acetylcholinesterase and butyrylcholinesterase inhibitory compounds, mainly because of the presence of monoterpenic and sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and phenylpropanoids ^[27].

The acetylcholinesterase inhibition activity of essential oils from Piper betle L. leaves was tested using the modified spectrophotometric method ^[28]. Essential oils of all the varieties, except Chhaanchi, significantly inhibited the acetylcholinesterase activity in a dose-dependent manner with IC₅₀ from 0.10 μg/mL for var. Ghanagete to 0.47 μg/mL for var. Bagerhati ^[28].

The extract obtained from blossoms of Citrus aurantium containing essential oils exhibited anti-acetylcholinesterase, inhibitory properties on the production of amyloid nanobiofibrils and antioxidant activity. The extract showed acetylcholinesterase inhibition with IC₅₀ = 42.8 mg/mL ^[14].

The multitargeted screening of anti-neurodegenerative disease potential was performed for Angelica purpurascens (Avé-Lall.) Gilli. fruit and roots extracts. The cholinesterase inhibition activity of essential oil from the extracts was detected using Ellman’s method ^[29]. The dichloromethane fraction of fruit exhibited the highest anti-acetylcholinesterase activity (39.86% inhibition at the concentration 20 mg/mL).

2.3. Phenolic Compounds

Plant phenols, commonly referred to as polyphenols or biophenols, have several biological activities such as antioxidant and neuroprotective effects and can be used in the prevention and/or management of diabetes and obesity. Phenolic compounds are well-acknowledged as potential metal chelation agents and inhibitors of lipid peroxidation. Various phenolic compounds were tested for acetylcholinesterase activity inhibition properties, most often simultaneously with antioxidant activity investigations. Among all compounds of plant origin, this group of compounds was most often tested for the activity of inhibiting acetylcholinesterase.

An extract from Tithonia diversifolia (Hemsl.) A. rich in phenolic acids (gallic acid, chlorogenic acid, caffeic acid and p-coumaric acid) and flavonoids (apigenin) exhibited anti-cholinesterase activity with IC₅₀ = 39.27 μg/mL ^[22]. The obtained results suggest that Tithonia diversifolia leaf has potential application as acetylcholinesterase activity inhibitor, exhibiting a stronger activity than the standard drug prostigmine (IC₅₀ = 50.02 μg/mL). The potential mechanism of the neuroprotective properties may be based on inhibiting cholinesterase activities and thwarting oxidative-stress-induced neurodegeneration. The extract also showed butyrylcholinesterase inhibition activity with IC₅₀ = 35.01 μg/mL ^[22]. The anti-butyrylocholinesterase activity of the extract was also higher than the activity of the standard drug prostigmine (IC₅₀ = 48.56 μg/mL). The extract obtained from Tithonia diversifolia additionally exhibited antioxidant activity determined by DPPH radical scavenging abilities with IC₅₀ = 41.05 μg/mL, ABTS acid radical scavenging with IC₅₀ = 33.51 μg/mL and iron chelation with IC₅₀ = 38.50 μg/mL ^[22].

Acetylcholinesterase inhibitory activities of extracts obtained from leaves and stem barks of Macaranga hurifolia Beille, Sterculia tragacantha Lindl. and Zanthoxylum gilletii were investigated using a modified Ellman method ^[30]. The highest acetylcholinesterase inhibition activity exhibited extract from Macaranga hurifolia. The antioxidant properties of these extracts were spectrophotometrically screened by different experiments as phosphomolybdenum, quenching of radicals (DPPH and ABTS), reduction potentials (FRAP and CUPRAC) and ferrous ion chelating ^[30]. An extract obtained from Sterculia tragacantha had the highest antioxidant activity. Radical scavenging assays also showed that the stem barks of all three investigated plants were better scavengers than leaf extracts.

2.4. Saponins

Saponins are steroid or triterpenoid glycosides, which exhibit several biological activities, e.g., immunostimulant, hypocholesterolaemic and anticarcinogenic, analgesic, anti-nociceptive, antioxidant, antifungal and antiviral properties. Some compounds belonging to the class of natural compounds have also been tested in terms of acetylcholinesterase activity inhibition.

The potential of acetylcholinesterase inhibition by saponins and ginsenosides from the stems and leaves of Panax ginseng extracts was determined by a modified Ellman method and by ultrafiltration analysis ^[31]. The samples of extract fractions were incubated in a solution consisting of 10 U/mL acetylcholinesterase and phosphate buffer at pH 7.6 for 0.5 h at 37 °C. After incubation, the binding mixture was filtered through an ultramembrane filter after being centrifuged. The filter was washed three times by centrifugation with phosphate buffer to remove the unbound compositions. The bound compounds were released by methanol. The most active fraction of Panax ginseng extract showed anti-acetylcholinesterase activity with IC₅₀ = 12.53 μg/mL. IC₅₀ values obtained for ginsenosides isolated from the plant were from 4.27 μg/mL for ginsenoside F1 to 20.00 μg/mL for ginsenoside Rc ^[31].

Polyacetylenes homopanaxynol, homopanaxydol, (9Z)-heptadeca-1, 9-diene-4,6-diyn-3-one and (8E)-octadeca-1,8-diene-4,6-diyn-3,10-diol obtained from Panax ginseng were tested as acetylcholinesterase inhibitors using the following procedure ^[32]. Samples in a dimethylsulfoxide solution at an appropriate concentration were diluted with an assay buffer (50 mM Tris–HCl buffer, pH = 7.8) in a 96-well microplate. An enzyme solution (2.0 U/mL) and solution of 1-naphthyl acetate (18 mM) were added to the mixture and incubated at 37 °C for 1 h. After incubation, 5% sodium dodecyl sulfate solution and Fast Blue B Salt solution (2 mM) were added. The absorbance was measured at 600 nm. The highest antiacetylcholinesterase was determined for (9Z)-Heptadeca-1,9-diene-4,6-diyn-3-one with IC₅₀ = 132 µM. The inhibitory activity of polyacetylenes from Panax ginseng were also evaluated as butyrylcholinesterase inhibitors by spectrophotometric method ^[32].

For the determination of the potential of acetylcholinesterase inhibition by Panax japonicus containing saponins, a PC12 cell model and acetylcholinesterase binding were applied ^[5]. Chikusetsusaponins V, Ib, IV, IVa and IVa ethyl ester were identified as acetylcholinesterase activity inhibitors. In the investigation, the protective effects of Panax japonicus leaf extract and its chemical constituent saponins against neuronal damage were determined by pretreating PC12 cells from rat pheochromocytoma with characteristics similar to midbrain dopamine neurons ^[5].

2.5. Other Compounds

The determination of acetylcholinesterase activity inhibition by anthocyanin from blueberry and purple potato extracts was performed by HPLC ^[33]. The procedure was based on an Ellman reaction performed before HPLC-DAD analysis. The chromatographic analysis was performed on a C18 column thermostated at 37 °C with mobile phase containing methanol–water–triethylamine (40:60:0.05, v/v/v). For the optimization of the experiment conditions, the effect of pH on enzyme activity, reaction temperature on enzyme activity, reaction time on enzyme activity, and acetylthiocholine iodide and acetylcholinesterase concentration on enzyme activity were investigated ^[33]. The scholars recommended the HPLC method especially for the evaluation of the acetylcholinesterase inhibitory activity in samples with deep color.

Lupeol long-chain alkanoic ester, lupeol β-hydroxy fatty acid esters 2c,d (laevigatins I and II) and lupeol and lupeol acetate isolated from the latex of Periploca laevigata were tested for acetylcholinesterase inhibition activity ^[34]. Methanol extract exhibited anti-acetylcholinesterase activity with IC₅₀ = 60.90 μg/mL. The highest inhibition activity was observed for lupeol with IC₅₀ = 38.31 μg/mL. The results obtained suggest that the triterpenic skeleton and the free secondary alcohol function at C-3 could be responsible of this activity, while the esterification of the alcohol function may decrease the inhibition of acetylcholinesterase ^[34].

Cyclohexanoids namely, speciosin U, speciosin V and speciosin W from the endophytic fungus Saccharicola sp. showed acetylcholinesterase inhibitory activities comparable to reference inhibitor galantamine ^[7]. The IC₅₀ value obtained for the most active compound speciosin U were 0.037 mg/mL and 0.026 mg/mL against acetylcholinesterase from human erythrocytes and from Electrophorus electricus, respectively. In the same experiments, the IC₅₀ values obtained for galantamine were 0.076 and 0.0047 mg/mL against acetylcholinesterase from human erythrocytes and from Electrophorus electricus, respectively. The obtained results indicated that Speciosin U possessed higher in vitro inhibitory activity, comparable to the reference inhibitor galantamine against acetylcholinesterase from human erythrocytes. Therefore, the compound may be a good candidate for further in vitro and in vivo investigations.

3. Conclusions

With the increasing burden of Alzheimer’s disease, it is essential to discover and develop new treatment options capable of preventing and treating the disease. In recent years, many studies have been described on the search for substances of plant origin with potential activity against neurodegenerative diseases. For the complex nature of Alzheimer’s disease, multi-target-directed ligands are increasingly being considered as promising therapeutic agents for treatment of the disease. Recently, multitarget therapeutic strategies have been devised to target acetylcholinesterase, butyrylcholinesterase and, for example, monoamine oxidase B. Because, in Alzheimer’s disease brain, the acetylcholinesterase activity is maintained or repressed, while the butyrylcholinesterase activity tends to increase, the discovery of drugs inhibiting both enzymes as well as that of selective butyrylcholinesterase inhibitors is advisable. Many plant extracts also exhibit dual cholinesterase inhibitor activity and antioxidant properties, which is a promising prospect for the treatment of neurodegenerative diseases.

Several Alzheimer’s disease targets are currently and intensively being investigated, divided in different hypotheses: mainly, cholinergic and β amyloid.

Most often, for in vitro investigations of acetylcholinesterase and butyrylcholinesterase activity inhibition, the spectrophotometric method firstly described by Ellman, currently most performed in 96-well microplates, is applied. Molecular docking is also a widely used technique in the search for new drugs, which reduces both the time and costs of lead investigations.

Further work is required to discover novel anti-Alzheimer’s disease active compounds and scale up the production of various biomolecules derived from these plants.

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Tomasz Tuzimski

Anna Petruczynik

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