Cholinesterase Inhibitory Potential of Quercetin towards Alzheimer's Disease - A Promising Natural Molecule or Fashion of the Day? - A Narrowed Review.

Natural substances are known to have strong protective effects against neurodegenerative diseases. Among them, phenolic compounds, especially flavonoids, come to the fore with their neuroprotective effects. Since quercetin, which is found in many medicinal plants and foods, is also taken through diet, its physiological effects on humans are imperative. Many studies have been published up to date on the neuroprotective properties of quercetin, a flavanol derivative. However, there is no review published so far summarizing the effect of quercetin on the cholinesterase (ChE) enzymes related to the cholinergic hypothesis, which is one of the pathological mechanisms of Alzheimer's Disease (AD). However, ChE inhibitors, regardless of natural or synthetic, play a vital role in the treatment of AD. Although the number of studies on the ChE inhibitory effect of quercetin is limited, it deserves to be discussed in a review article. With this sensitivity, the neuroprotective effect of quercetin against AD through ChE inhibition was scrutinized in the current review study. In addition, studies on the bioavailability of quercetin and its capacity to cross the blood-brain barrier and how this capacity and bioavailability can be increased were given. Generally, studies containing data published in recent years were obtained from search engines such as PubMed, Scopus, and Medline and included herein. Consequently, quercetin should not be considered as a fashionable natural compound and should be identified as a promising compound, especially with increased bioavailability, for the treatment of AD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.

INTRODUCTION

Natural products have always been interesting to researchers to explore novel drug candidates. For the diseases threatening human health, many natural products have been served as the lead compounds to design novel drugs. Among them, quercetin as a well-known flavonoid derivative, a flavanol to be more specific, is commonly found in plant kingdom and food plants such as red wine, onions, green tea, apples, berries, Ginkgo biloba, St. John's wort, American elder, propolis, and many others [1-3]. Its chemical structure is described as 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one by the formal chemical name according to International Union of Pure and Applied Chemistry (IUPAC).

Quercetin as a popular bioflavonoid has been reported to display a wide range of desired biological activities such as anticancer, anti-inflammatory, immunomodulatory, antidiabetic, cardioprotective, neuroprotective, antihypertensive, chemopreventive, antioxidant, anti-obesity, antibacterial, wound healing, etc. [3-24]. Among all the above-mentioned biological activities of quercetin, special attention should be given to its potential neuroprotective impact [7, 8]. Results of numerous studies at in vitro, in vivo, in silico, and clinical levels performed against neurodegenerative diseases pointed out a notable protection by quercetin in several reviews [25-29]. This effect has been shown to emerge via many different complicated mechanisms, e.g. stimulating cellular defenses against oxidative stress such as through induction of nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) and induction of the antioxidant/anti-inflam-matory enzyme; paraoxonase-2 (PON2) and activating sirtuins (SIRT1) to induce autophagy [5, 29-31], degradation of learning and memory loss induced by inhibition of amyloid beta (Aβ) fibrils [32-34], increasing neurotrophin brain-derived neurotrophic factor in neurons [35, 36], inhibition of cholinesterases (ChEs) [37, 38], modulating the inflammatory responses involved in neurodegenerative diseases such as inhibition of Nitric Oxide (NO) excess production and inducible Nitric Oxide Synthase (iNOS) overexpression. The other mechanisms relevant to neuroprotective action of quercetin can be mentioned as downregulation of the overexpression of pro-inflammatory genes [e.g. Interleukin (IL)-1ß, Tumor Necrosis Factor (TNF)-α and cyclooxygenase (COX)-2] [39], regulating activity of kinases [40, 41], activation of apoptotic pathways affecting caspase-3 and Poly ADP Ribose Polymerase (PARP) expression [42], reducing the Gamma-Aminobutyric Acid (GABA)-activated currents in cortical neurons [43], inhibition of monoamine oxidase (MAO) and secretase enzyme families [44-47], halting the damaging toxic effects of L-dopa [48], inhibition of tyrosinase [49-51], attenuating radiation, traumatic or hypoxia-ischemia induced brain injury [52, 53], decreasing neuronal autophagy and apoptosis [54, 55], reversing mitochondrial dysfunction through such as the adenosine monophosphate-activated protein kinase/sirtuin 1 (AMPK/SIRT1) signaling pathway [56], increasing cellular NAD+/NADH and activation of peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α)-mediated pathways [57], regulating mitochondrial redox status [58]. On the other hand, potent hydroxyl (OH), hydrogen peroxide (H2O2), and superoxide anion (O2-) scavenging ability of quercetin was reported in numerous papers [59-62].

As aforementioned, cumulative evidence showed the neuroprotective potential of quercetin by diverse mechanisms of action. Among them, cholinergic system related to cognitive function through ChE enzyme family as well as Aβ in the form of neurofibrillary tangles and amyloid plaques plays a critical role in pathogenesis of Alzheimer’s Disease (AD) [63, 64] and inhibitors of ChEs have been considered to be the first-line symptomatic drugs for the treatment of AD [65-69]. AD, associated mainly with cognitive disability and memory loss, particularly influencing the elderly, is known to be a neurodegenerative disease with progressive character whose pathology has been described by two major mechanisms, i.e. cholinergic hypothesis and amyloid hypothesis [70, 71]. As the topic of the current review giving priority to the cholinergic hypothesis, the serine protease-type of “sister” enzymes, i.e. acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are linked to the regulation of cholinergic transmission among neurons [72]. Thus, deficit in the neurotransmitter acetylcholine (ACh) level in the synaptic gap between neurons or overexpression of ChE has been demonstrated to induce AD pathology. AChE functions to break down ACh and hence, its inhibition is an important target in drug design for effective treatment of AD [73]. On the other hand, BChE, the enzyme also closely allied with AChE, assists and co-regulates hydrolysis of ACh in cholinergic system as well as butyrylcholine [74, 75]. Actually, it should be noted that AChE and BChE possess various structural resemblances (50-55% species-dependent sequence similarity) to each other and the active gorge of BChE (Ser198, His438, and Glu325) and oxyanion hole (Gly116, Gly117, and Ala199) are matching to those of AChE [76].

Since no exact cure is available to stop AD, yet, inhibition of both ChEs is the target for developing new drugs towards the disease. In this regard, natural products have given hope as lead molecules. For instance, galanthamine, an alkaloid isolated from the bulbs of Galanthus woronowii Losinks. (snowdrop) as well as Leucojum species belonging to Amaryllidaceae has been in clinical use for AD treatment as the last generation ChE inhibitory drug [77-81]. Consistently, many flavonoids, being the most abundant class of natural products, have been identified with a promising ChE inhibitory activity [82-84]. Among those flavonoids, the present review aims to focus specifically on ChE inhibitory capacity of quercetin through scrutinizing the reported data obtained from the bibliographic databases, e.g. PubMed, Scopus, and Medline. In addition, capacity of quercetin to across Blood Brain Barrier (BBB) is touched.

ChE INHIBITORY EFFECT OF QUERCETIN

Although evidence indicated the strong neuroprotective effect of quercetin against not only AD, but also other neurological diseases, various mechanisms for its mentioned effect have been proposed. Nevertheless, only limited studies have been performed on ChE inhibitory potential of quercetin in a pure form as one of the mechanisms of action towards AD. On the other hand, more papers are available on ChE inhibitory effect of plant extracts containing with quercetin and its derivatives, e.g. Leiotulus dasyanthus (K. Koch) Pimenov & Ostr, Bauhinia forficata Link subsp. pruinosa (Vogel) Fortunato & Wunderlin, Davallia cylindrica Ching, Pistacia atlantica Desf., Zingiber officinale Roscoe, etc. [85-92].

In an earlier study [93], quercetin isolated from Agrimonia pilosa Ledeb was revealed to inhibit AChE with IC50 value of 19.8 μM, where dehydroevodiamine hydrochloride, the reference compound of natural origin, had IC50 value of 37.8 μM. This indicated that quercetin inhibited AChE twice effectively than the reference, whereas its anti-AChE capacity was weaker in comparison to that of tacrine (IC50 = 0.1 μM), a clinically used ChE inhibitor. The authors stated quercetin with therapeutic potential against AD.

In a screening study, some selected flavonoid derivatives, including galangin, kaempferol, quercetin, myricetin, fisetin, apigenin, luteolin, and rutin were reported to inhibit BChE of human origin reversibly [94]. Among them, the most selective and effective flavonoid against BChE was galangin, having 12 times higher selectivity against BChE than AChE. The results showed that a number of hydroxyl groups and their locations on the phenyl ring are quite imperative in inhibition. According to molecular docking experiments, the flavonoids tested were shown to bind to the active gorge of BChE through making multiple hydrogen bonds and pi-pi interactions. Quercetin was identified to have a reversible inhibition with K value of 38.3 ± 9.8 μM, while its binding pattern to BChE was shown to be opposite to that of galangin in docking simulations. Hydrogen binding lengths for quercetin were between 1.83 and 2.02 Å, while it interacted with Asp70, Glu197, O17, Gly115, and Tyr128 at the active site. Besides, the flavonoids did not display cytotoxicity in HepG2 and A549 cell lines, when tested up to 200 μM. Consistently, in our earlier work in which we screened a number of phenolic acids and flavonoids, 76.2 ± 0.99% inhibition against AChE of electric eel origin was exhibited by quercetin at 100 μg/mL, whereas it displayed 46.8 ± 1.35% of inhibition on BChE of horse serum origin [95]. Similar to our results, quercetin from Croton menyharthii Pax exhibited 56.6 ± 0.93% inhibition against AChE of electric eel (IC50= 41.6 ± 6.0 μg/mL) [96]. When compared to that of galanthamine (88.3 ± 2.0%, IC50= 0.3 ± 0.1 μg/mL), its effect was at mild level.

Conversely, quercetin from paper mulberry (Broussonetia papyrifera (L.) L’Her. ex Vent., syn. Morus papyrifera L.) was inactive against AChE (electric eel) (IC50 > 500 μM) [97]. In fact, some studies pointed out a good anti-BChE activity and a weak inhibition towards insect AChE by quercetin, while it did not exert a blocking effect on human AChE [98-100]. Relevantly, low ChE inhibitory activity of quercetin obtained from Ficus foveolata (Wall. ex Miq.) (syn. Ficus sarmentosa Buch.-Ham. ex Sm.) was reported [101]. It showed 18.8 ± 0.2% and 31.0 ± 0.1% of inhibition at 100 μg/mL against AChE (electric eel) and BChE (horse serum), respectively, while corresponding AChE and BChE inhibitory effect of galanthamine (the reference compound) was 95.6 ± 1.4 and 93.1 ± 0.4% at the same concentration [102]. In another study, quercetin was revealed to inhibit BChE (IC50= 19.08 μM) in a better capacity than AChE (IC50= 55.44 μM) [103]. However, the inhibitory effect was rather mild when compared to that of galanthamine (IC50= 0.59 μM).

According to docking data obtained with quercetin in the former work that we screened four phenolic compounds, several hydrogen bonds with the amino acid residues located at the anionic subsite of BChE were formed [104]. Besides, another hydrogen bond was observed between the hydroxyl group at 5th carbon of A ring as well as carbonyl group in C ring in quercetin structure and TYR133. Additionally, another hydrogen bond was detected between the hydroxyl group at 3rd carbon of A ring and TYR86 at the anionic region of BChE. In a relevant study in which 646 small natural molecules were screened for their AChE inhibitory potential through in silico experiments, caffeine, ascorbic acid, and gallic acid were identified as the strong AChE inhibitors [105]. Then the mentioned four molecules were subjected to in vitro AChE inhibition assay and confirmed again to be the active inhibitors of this enzyme in comparison to the reference drugs, i.e. donepezil and begacestat. Among them, quercetin and caffeine had the identical level of AChE inhibition to those of the references. Besides, the four active molecules did not display toxicity in hippocampal neurons. Therefore, predominantly quercetin and caffeine were concluded as disease-modifying drugs with encouraging therapeutic potential in AD. Quercetin was shown to inhibit AChE and BChE successfully, having IC50 values of 0.181 mM and 0.203 mM, respectively. According to this data, the compound seems to be a slightly better inhibitor of AChE [106]. At this point, it should be definitely noted once more that the use of different enzyme sources affects the biological activity of a compound.

In an in vivo study, quercetin was reported to prevent chlorpyrifos (an organophosphorus pesticide)-induced neurotoxicity through inhibition of AChE along with choline acyltransferase in female Wistar rats administrated at a dose of 50 mg/kg (body weight, b.w./day) during eight weeks [107]. Hence, it was stated to be a prophylactic neuroprotective agent against chlorpyrifos-induced neurotoxicity by the authors. In a similar study, diazinon-induced neurotoxicity was blocked by quercetin as well as in combination with curcumin in male albino rats, which were treated with different combinations of both compounds during four weeks [108]. Quercetin alone led to a decrease in AChE level from 133.62 IU/mL to 110.04 IU/mL, while combination of quercetin and curcumin caused a decline down to 129.62 IU/mL given at dose of 100 mg/kg (b.w./day). In another in vivo study, oral co-administration of quercetin with manganese at 10 and 20 mg/kg (b.w./day) for 45 sequential days led to a significant decrease in AChE level and improved the locomotor functions in adult male Wistar rats [109]. In more detail, when manganese was administrated alone in rats, excessive increase was determined in AChE activity in the hypothalamus, cerebrum, and cerebellum (101, 50, and 59%, respectively). Nevertheless, co-administration of quercetin significantly reversed the AChE activity in the above-mentioned brain areas of the rats. The diminution in AChE activity was 38 and 59% in the hypothalamus; 42 and 50% in cerebrum, and 33 and 38% in cerebellum for manganese + quercetin combination given at 10 and 20 mg/kg (b.w./day), respectively.

PERMEABILITY AND BIOAVAILABILITY OF QUERCETIN

Many flavonoids owe their neuroprotective effects to their ability to cross the BBB [110, 111]. However, their ability to cross BBB still remains as the main issue for most of them. Doubtlessly, hydrophobicity and lipophilicity play a critical role in transmembrane transport, bioavailability, toxicity, and biological activity of molecules. Passing a biological membrane for any drug or drug candidate is an essential step for their absorption and bioavailability. In this sense, Caco-2 cell line monolayer model is widely used as an in vitro model to determine drug transport and many studies have revealed that flavonoids are usually transported by passive diffusion [112-116]. In fact, Fang et al. demonstrated that the bilateral permeation of five groups of flavonoids was as follows; flavanones ≥ isoflavones > flavones ≥ chalcones > flavonols [117].

There have been a number of studies investigating BBB crossing capability of quercetin, a hydrophobic flavonol with low bioavailability (<2%) and low brain permeability [118]. For instance, 65.54% of quercetin was successfully able to across BBB in primary Brain Microvessel Endothelial Cell (BMVEC) and primary astroglia cell cultures [119]. In a study conducted on a number of flavonoid derivatives, i.e. puerarin, rutin, hesperidin, quercetin, genistein, kaempferol, apigenin, and isoliquiritigenin, their transmembrane transport was examined via the rat BBB cell and Caco-2 cell monolayer models [120]. The apparent permeability coefficients (Papp, pregnancy-associated plasma protein) of the flavonoids were calculated from the unilateral transport assays, where the Papp values were shown to be time-dependent. The results indicated that quercetin through the BBB cell layer model possessed the lowest permeation (Papp value 2.20 ± 0.34 × 10−6 cm/s) among the tested flavonoids. In Caco-2 cells, the data showed that permeation order of the eight flavonoids was genistein, isoliquiritigenin, kaempferol, apigenin, hesperidin, quercetin, puercetin, and rutin from the high to low, where Papp value of quercetin was established 16.23 ± 0.42 × 10−6 cm/s. Actually, Chabane et al. reported poor absorption of quercetin and naringenin in Caco-2 cells. Absorption of quercetin by passive diffusion was observed in a pH-dependent mechanism mediated by the Organic Anion Transporting Protein B (OATP-B) [121]. It was not connected to multidrug resistance associated protein (MRP)-1 substrate, but was the substrate of the MRP-2 efflux transporter and not P glycoprotein (Pgp). This finding may bring an explanation for low bioavailability of the compound. Quercetin was found to be a Pgp inhibitor [122-124]. On the other hand, in rat brain tissues, quercetin and its methylated derivative were analyzed by high-performance liquid chromatography (HPLC) after oral administration of quercetin at dose of 50 mg/kg (b.w.) using brain capillary endothelial cell line as the implementation of BBB model [125].

Nanostructures such as polymer nanoparticles, lipid nanoparticles, nanoliposomes, nanomicelles and carbon nanotubes are known to cause significant consequences, including tremendous stability and controlled release, elevated bioavailability, enhancement of therapeutic efficacy, improvement of pharmacokinetic, and marked ability of crossing the BBB for drugs and natural products such as quercetin having a hydrophobic character and poor solubility [126-128]. In recent years, diverse nanostructure forms of quercetin have been reported to help improving the bioavailability of quercetin particularly referring to its neurodefensive potential [129, 130]. In order to increase permeability of quercetin across BBB and inhibitory potential of Aβ aggregation towards AD, a nanoparticulated formulation of the compound sphered with RGV29 peptide was successfully implemented, which led to an improvement of delivery and neuroprotective effect of quercetin [131]. Another nanoparticle form of quercetin prepared with hydroxypropyl methylcellulose (HMPC) was tested in aluminum-induced neurodegeneration rat model [130]. The nanoparticulated quercetin administrated at 30 mg/kg (b.w.) led to improve neuronal degeneration parameters such as formation of amyloid peptides and neurofibrillary tangles, downregulation of tyrosine hydroxylase, astrogliosis and inhibition. As another example, quercetin-Solid Lipid Nanoparticle (SLN) form appreciably ameliorated aluminum-induced neurotoxicity [132]. In addition, this system caused meaningful improvement in behavioral and memory retention in animal models of dementia and AD. The SLN form of quercetin also exhibited neuroprotection in pentylenetetrazole (PTZ)-induced cognitive impairment of wild-type adult male Danio rerio (zebrafish) species at doses of 5 and 10 mg/kg through anti-inflammatory, antioxidant, and regulation of enzymes linked to cholinergic neurotransmission [133]. Relevantly, quercetin-SLN caused a reduction in AChE in the brains of zebrafish in that study.

Quercetin nanoparticles with gold-palladium core shell were reported to be effective to ameliorate autophagic dysfunction in AD using SH-SY5Y human neuroblastoma cells, which led to decrease degeneration due to Aβ toxicity [134]. Improving effect of superparamagnetic iron oxide-nanoparti-culated quercetin at the dose of 25 mg/kg (b.w.) given during 35 sequential days was described against diabetes-related memory deficit [135]. A similar result was obtained with nanoparticulation of quercetin again with superparamagnetic iron oxide, which led to augment bioavailability of quercetin in the brains of Wistar male rats [136]. Approximately, 10-fold increase was observed in the bioavailability of free quercetin. Liposome form of quercetin encapsulated with RMP-7, a bradykinin analogue, co-prepared with lactoferrin, was shown to pass the BBB without inducing robust toxicity and harming the tight junction [137]. Besides this drug delivery system decreased the neurotoxicity due to Aβ fibrils in SK-N-MC cells. In another work, quercetin nanoparticulated with polylactic-co-glycolic acid (PLGA), a biodegradable copolymer, was lowered toxicity induced by Aβ in SH-SY5Y cells and improved therapeutic efficacy in APP/PS1 mice in terms of learning and cognitive function.

CONCLUSION

Quercetin, as a popular dietary flavonoid-type of compound has attracted huge interest from scientists due to its desired biological effects on human health. At this point, a special part should be devoted to the neuroprotective properties of quercetin. In fact, a large number of action mechanisms referring to its neuroprotective potential have been elucidated due to complicated pathological features of neurodegeneration process. In many cases, quercetin was concluded to exert significant protection against neurodegenerative diseases, including AD. One of the principle pathological mechanisms of AD is known as cholinergic hypothesis and in this regard, ChE inhibitors related to AChE and BChE have been the most prescribed drug class for AD treatment. A good number of natural products have been identified with promising ChE inhibitory effect, including quercetin. Therefore, special attention was paid to ChE inhibition by quercetin in this context through the present review. Although most of the studies revealed the strong ChE inhibitory activity of quercetin, sometimes conflicting results have also been obtained. This contradiction may be due to various factors. However, the most important factor may be the difference in the enzyme source. The test methods used can also lead to differences in the inhibitory activity results. The reported data on ChE inhibitory potential of quercetin indicates that it has the ability so far to inhibit ChEs in in vitro, in vivo and in silico studies. Nevertheless, low bioavailability of quercetin stands as a big problem before us. As aforementioned, a wide variety of studies are being done to increase the bioavailability and BBB-passing ability of quercetin, which seems to be the most effective one being the nanoparticle form. Based on the current data available on quercetin, the question in the title of the current review can be answered that quercetin is not the fashion of the day. It can be considered one of the most effective and promising natural products and multifunctional natural agents against AD acting through a number of mechanisms related to neurodegeneration provided that its bioavailability and its ability to cross the BBB should be increased.