Non-Alkaloid Cholinesterase Inhibitory Compounds from Natural Sources

Alzheimer’s disease (AD) is a severe neurodegenerative disorder of different brain regions accompanied by distresses and affecting more than 25 million people in the world. This progressive brain deterioration affects the central nervous system and has negative impacts on a patient’s daily activities such as memory impairment. The most important challenge concerning AD is the development of new drugs for long-term treatment or prevention, with lesser side effects and greater efficiency as cholinesterases inhibitors and the ability to remove amyloid-beta(Aβ) deposits and other related AD neuropathologies. Natural sources provide promising alternatives to synthetic cholinesterase inhibitors and many have been reported for alkaloids while neglecting other classes with potential cholinesterase inhibition. This review summarizes information about the therapeutic potential of small natural molecules from medicinal herbs, belonging to terpenoids, coumarins, and phenolic compounds, and others, which have gained special attention due to their specific modes of action and their advantages of low toxicity and high efficiency in the treatment of AD. Some show superior drug-like features in comparison to synthetic cholinesterase inhibitors. We expect that the listed phytoconstituents in this review will serve as promising tools and chemical scaffolds for the discovery of new potent therapeutic leads for the amelioration and treatment of Alzheimer’s disease.


Introduction
The research of novel drug candidates has shown that natural products such as plant extracts and plant-originated compounds have enormous potential to become drug leads with neuroprotective activity. Several non-alkaloid phytochemicals have been obtained from natural sources, including terpenoids, coumarins, flavonoids, and other phenolic compounds which have beneficial neuroprotective properties particularly in cholinesterase inhibition hence, they are potential drug candidates for the treatment of Alzheimer's disease (AD). Alzheimer's disease (AD), one of the leading causes of dementia, is an overwhelming neurodegenerative disease that particularly affects brain function, resulting in memory loss and impairment of language, emotional disturbance, personality changes, depression, behavioral problems, and judgment capacity [1,2]. Besides dementia, it is a major cause of death amongst old people. In the brains of Alzheimer's disease (AD) patients, key neuropathological features of pathological protein deposits such as insoluble The Supplementary Material (Tables S1-S4) gives the representative non-alkaloid naturally occurring compounds with useful anticholinesterase properties obtained mainly from plants as summarized below (Figure 1). Figure 1 gives a vivid indication of sources of starting material for cholinesterase inhibitory compounds and drugs. The compounds obtained from these plants have been explored to target pathological features in neurodegenerative diseases such as AD and can be also used as a starting point to design a new library of potent derivatives.

Terpenoids
Several terpenoids from natural sources have been reported as cholinesterase inhibitors . The compounds 1-11, as shown in Supplementary Material (Table S1), are of the Abietane-type diterpene skeletons isolated from Salvia austriaca, Salvia glutinosa, Caryopteris mongolica, and Perovskia atriplicifolia [14][15][16]. Between compounds 2-7, there is an -OH group on the side chain, except for compound 5 which has a methoxy (CH3O-) group on the side chain and has the highest AChE inhibition activity. The high activity could be due to the presence of this methoxy group. In the same way, compound 2 has good activity and possesses a methoxy group on ring C and has an IC50 of 27.9 ± 5.2 µ M compared to compound 5 with an IC50 of 20.8 ± 7.1 µ M. Compounds 8 to 11 are miltirone derivatives though with little structural differences, there is no significant difference in their cholin-

Terpenoids
Several terpenoids from natural sources have been reported as cholinesterase inhibitors . The compounds 1-11, as shown in Supplementary Material (Table S1), are of the Abietane-type diterpene skeletons isolated from Salvia austriaca, Salvia glutinosa, Caryopteris mongolica, and Perovskia atriplicifolia [14][15][16]. Between compounds 2-7, there is an -OH group on the side chain, except for compound 5 which has a methoxy (CH 3 O-) group on the side chain and has the highest AChE inhibition activity. The high activity could be due to the presence of this methoxy group. In the same way, compound 2 has good activity and possesses a methoxy group on ring C and has an IC 50 of 27.9 ± 5.2 µM compared to compound 5 with an IC 50 of 20.8 ± 7.1 µM. Compounds 8 to 11 are miltirone derivatives though with little structural differences, there is no significant difference in their cholinesterase inhibition activity. Compounds 12-19 are tanshinone derivatives [16]. Compounds 16 and 17 are the most active with percentage inhibitions of 6.19 ± 3.91% and 5.55 ± 3.03%, respectively at 10 µg/mL. This could be attributed to the conjugated double bond system in ring A which is particular to these two compounds. Compound 20, a monoterpene glycoside nuciferoside, shows very high activity with an IC 50 value of 3.20 ± 0.22 µM [17]. Compounds 21-36 are cycloartanes triterpenoids iso-lated from Cimicifuga dahurica and Nelumbo nucifera [17,18]. Amongst them, compound 25 is the most active with a percentage inhibition of 15.8 ± 4.3% and 14.0 ± 2.6% on AChE and BChE, respectively, at 100 µM. This could possibly be attributed to the absence of the double bond in ring B of this compound. Compounds 37-42 are lupane type triterpenoids isolated from Garcinia hombroniana and Xylia xylocarpa and they show relatively low activities [19,20]. The oleanane triterpenoids 43, 44, and 45 isolated from Xylia xylocarpa and Rhynchospora corymbose show low activities [19,21]. The sterols 46 and 47 from Rhynchospora corymbose show low activities as well as the monoterpenes 48, 49, and 50 from Pimpinella anisoides [21,22]. Sesquiterpene lactones from 51-64 isolated from Inula spp., Cynara cornigera, and Amberboa ramosa show good anticholinesterase activities [23][24][25]. Compounds 58-61 are amberbin C, amberin, amberbin A, and amberbin B, and have high anticholinesterase activity [24]. Amongst them, those possessing sugar moieties, amberin (IC 50 17.5 ± 0.01 µM and 2.7 ± 0.02 µM for AChE and BChE, respectively) and amberin B (IC 50 0.91 ± 0.015 µM and 2.5 ± 0.15 µM for AChE and BChE, respectively) are the most active and the structural difference between them is the interchange of the positions of an acetyl group and sugar moiety. The agarofuran derivatives 62-73 isolated from Euonymus japonicus and Maytenus disticha have low activities [25,26]. The taraxaranes 74, 75, and 76, oleananes 77, 78, and 79, as well as the ursane tritepenoids 80 and 81, have relatively low activities except for compound 74 with IC 50 values of 13.5 ± 0.95 µM and 10.6 ± 0.54 µM on AChE and BChE inhibitions [20,27,28]. Its relatively high activity could be attributed to the presence of the caffeoyl group at position 3. It can be concluded that amongst the terpenoids, sesquiterpenes are the most active compounds, especially sesquiterpene lactones.

Phenolic Compounds
Phenolic compounds from natural sources have shown anticholinesterase activity in several studies [34,. Phenolic compounds whose anticholinesterase activities have been reported are given in the Supplementary Material (Table S2). Compounds 1-19 are flavone derivatives with a double bond in ring C and a carbonyl at position 4 [34,[43][44][45][46][47][48][49][50][51][52][53]. There is no observable regular pattern of variation in activity. However, compounds with no hydroxyl group on position 3 show seemingly high cholinesterase inhibition activity, for example, compounds 4 and 5. However, compounds 16-19 do not have a hydroxyl group at position 3 but their activities are low and could be accounted for by the occurrence of methoxy groups on the other rings. There is an observable decrease in cholinesterase inhibition in flavones with methoxy substituents, for example, compounds 13 and 14, and compounds 7 and 8. This observation is not true for compounds 14 and 15 as 14 has a methoxy group on ring B but is more active than 15 without a methoxy group. This could be due to the absence of a substituent on ring B of compound 15. Between cirsilineol (18) and isothymusin (19), an additional hydroxy group on ring A causes a decrease in cholinesterase inhibition. For the flavonoid glycosides, compounds 20-33 [43,45,46,49,50,54,55], those with a sugar moiety at position 7, have higher activities than the others, for example, 27, 28, and 29 isolated from Achillea millefolium. If the sugar has substituents, as is the case of 32 and 33, the activity is further reduced. Compounds 34 to 38, isolated from Dodonaea viscosa, have isoprenyl substituents but, however, show no significant difference in their activities [51]. Rather, their activities are lower than their corresponding compounds without isoprenyl substituents. Compounds 39-41 have acetyl groups and their BChE inhibitory activity decreases with an increase in the number of acetyl groups [21]. The phenolic acid compounds 42-45, and compound 44 ferulic acid methyl ester have a good percentage of cholinesterase inhibition [17,18,57]. The presence of sugar substituents causes a decrease in cholinesterase inhibition as seen in compounds 46-48 [17,58], while an additional phenolic group causes an increase in cholinesterase inhibition as seen in compounds 49-64 [16,18,47,52,[59][60][61][62]. Amongst the biphenyl compounds, 58, 59, 60, and 61 isolated from Myristica cinnamomea have high activity, and in these compounds, the carbonyl function is adjacent to one of the phenyl groups (phenyl carbonyl). Isoflavones compounds 65-79 isolated from Iris pseudopumila, Maclura pomifera, and Belamcandae chinensis rhizoma have low activities [52,54,59,63]. Amongst them, methoxy substituents cause no significant change in cholinesterase inhibition while the presence of sugar molecules causes a decrease in this activity. For those with prenyl groups (75)(76)(77)(78)(79) isolated from Maclura pomifera, there was no observable effect due to the presence of the prenyls, but an -OH group on ring B caused an increase in activity between compounds 75 and 76. Catechin and its derivatives 79 to 84 isolated from Eugenia dysenterica and Orostachys japonicus had no good activity and no significant difference despite structural differences except between compound 82 and 83 where the additional benzoic acid substituent increased AChE and BChE inhibition activities [40,44,57]. This observation was similar for the flavanones 85 to 89, though the addition of sugar molecules caused an increase in AChE and BChE inhibitions in compound 90 compared to compound 85 [50,51,56,64]. The xanthones compound 95-101 isolated from Garcinia mangostana and Belamcandae chinensis rhizoma showed moderate to good AChE and BChE inhibition activities [19,66,67]. Evidently, an increase in the hydroxyl groups causes an increase in the cholinesterase inhibitory activity of these xanthones, while no significant difference in cholinesterase inhibition is observed for the prenyl groups. For the chalcones 102 to 105 isolated from Humulus lupulus, the activity decreases from compound 102 to 105 with a decrease in the number of hydroxyl (-OH) substituents [56].  [28,32,36,69,70]. This could be because it is less bulky, having only three benzoyl groups as compared to compounds 112 and 113 with five benzoyl groups and 110 and 111 with four benzoyl groups. The triflavanone Garcineflavanone A and biflavonol Garcineflavonol A isolated from Garcinia atroviridis both showed good percentage inhibition of cholinesterase. M. charantia extract showed many inhibitory activities, however, ligballinol a lignan found in extract showed relatively high activity. According to previous studies, not many lignans have been reported to exhibit cholinesterase inhibitory activity [85].
Other miscellenous compounds (Table S4) have equally shown interesting acetylcholinesterase and butyrylcholinesterase inhibitory activities .

Some Considerations on Terpenoids, Phenolic Compounds, and Coumarins as Cholinesterase Inhibitors
It is important to search for new therapies which are more effective than those currently existing, and which can both prevent neurodegenerative diseases such as AD and block the progression of these pathologies at their early stages, thereby reducing the socioeconomic costs involved in the management of AD and the patients [117]. Acetylcholine is a key neurotransmitter involved in cognitive activities, but its activity can be reduced by AChE and BChE which hydrolyze acetylcholine into choline and acetic acid causing the cholinergic neurotransmission to decrease. The development of many therapies for AD is based mainly on this cholinergic hypothesis, and the remediation of acetylcholine levels and cholinergic function in the central nervous system through the inhibition of cholinesterase enzymes (AChE and BChE) can eliminate the pathologies of AD. The classes of cholinesterase inhibitors discussed here are mainly terpenoids, phenolic compounds, and coumarins, and some of these compounds have shown high potency. In order to consider which classes are most suitable, based on the benefits and drawbacks, certain structural features of each class will be of great importance. Using chalcones as an example, it is believed that besides economical and cost-effective production, small molecular size and flexibility for modifications to improve lipophilicity necessary for blood-brain barrier permeability are important to consider for a preferred potential therapeutic candidate for AD [118]. Terpenoids are able to inhibit cholinesterases in different ways. It has been shown that 1,8-cineole, α-pinene, and camphor could inhibit AChE reversibly [119]. Certain tanshinone derivatives could be noncompetitive inhibitors of AChE and BChE in humans and are able to bind to the allosteric site of cholinesterases principally through hydrophobic interactions and also through hydrogen bonds with Tyr337 and Gly120 of AChE [120]. Carbonyl function in terpenes can bind by covalent arrangement to the free amino or sulfhydryl groups of the enzyme while phenolic hydroxyl groups bind proteins, leading to the conformational change of the enzyme [121]. Terpene alcohols and terpene hydrocarbon compounds possess identical cholinesterase inhibition while terpenoids with a ketone group exhibit stronger cholinesterase inhibition and an allylic group increases activity [121,122]. Amongst the terpenoids, monoterpenoids are the most promising because the inhibition of AChE has been shown to remedy AD by inhibiting amyloid-beta-induced neurotoxicity and also clearing it, tau-protein phosphorylation, and oxidative stress by boosting antioxidant defenses, neuroinflammation, restoration of mitochondrial function, initiation of processes with simultaneous inhibition of pro-apoptotic genes and proteins [123]. In the phenolic compounds, inhibitory activity is influenced by the position and number of hydroxyl and methoxyl groups bonded to the phenol ring, and the methoxy substitution on the phenol ring improves cholinesterase inhibitory activity and phenolic acids are capable of inhibiting the formation of amyloid β-peptide (Aβ) fibrils [124]. Phenolic compounds exert neuroprotective effects, though it is assumed that the transfer of polyphenols through the blood-brain barrier is limited, likewise, a considerable number of reports discuss the absorption and presence of phenolic acids in the brain [125]. Phenolic compounds are able to bind to the active sites of AChE or BChE resulting in the inhibition of these enzymes [126]. Aromatic ring moieties are suggested to be involved in the selection and stabilization of the positive charge of the quaternary group in the acetylcholine, and some of the flavonoids can induce modifications in the structure of cholinesterase enzymes blocking entrance into the active site and those with free OHgroups are also more potent than glycosylated ones. Phenolic compounds structurally similar to caffeic acid are capable of fitting into the gorge of the active site of AChE and are more potent [127]. In coumarins, it has been shown that those with larger substituents at position 7 have a higher inhibitory effect than those with small substituent groups at the same position, and equally, compounds that contain a coumarin nucleus and a long-chain substituent with some phenyl and aryl/benzyl-piperazines groups can be more potent inhibitors of cholinesterase [128,129]. The anticholinesterase activity of coumarins is mostly dependent on their binding ability on the enzyme, and this activity is greatly improved in the scaffolds with some cholinesterase inhibitory drugs [129]. Moreover, the structure of coumarins is highly modifiable through chemical means, thereby presenting them as suitable starting materials for the synthesis of drugs. Many cholinesterase drugs with a coumarin nucleus have been reported, making coumarin a priority pharmacophore for cholinesterase inhibitors. In a study in which 36 isolated compounds were classified and discussed according to their anti-AChE pharmacological potency, phenolic compounds and flavonoids were mostly found in the low activity zone of natural acetylcholinesterase inhibitors according to their ability to bind to the active site of acetylcholinesterase [130]. Coumarins and terpenoids occupied zones indicated as moderate to high activity and capacity of binding to the active site of acetylcholinesterase [130].
However, preclinical, clinical safety, selectivity, and toxicity of these compounds are not established, and classification of these compounds based on their benefits and drawbacks will be controversial and non-conclusive. However, a common point seems to be the overall size of the compound which should generally be small so as to be able to cross the blood-brain barrier and exerts its function.

Conclusions
Neurodegenerative disease is a generic term applied to a variety of conditions arising from a chronic breakdown and deterioration of the central nervous system (CNS) neurons. Many of these diseases exist, but Alzheimer's disease (AD) is the most prevalent. Alzheimer's disease (AD) patients present a progressive loss of cholinergic synapses in the brain regions associated with a decrease in the acetylcholine (ACh), a neurotransmitter, which appears to be a critical element in the development of dementia. Hence, AD and other forms of dementia could be treated by the use of agents that restore the level of acetylcholine through the inhibition of both major forms of cholinesterase: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Loizzo and co-workers postulated that AD causes and progression involves four relevant pathogenic events: primary events (genetic alterations, neuronal apoptosis-like processes leading to premature neuronal death and brain dysfunction), secondary events (β-amyloid deposition in senile plaques and brain vessels, neurofibrillary tangles due to the hyperphosphorylation of tau proteins, synaptic loss), tertiary events (neurotransmitter deficits, neurotrophic alterations, neuroimmune dysfunction, neuroinflammatory processes), and quaternary events (accelerated neuronal death due to excitotoxic reactions, alterations in calcium homeostasis, free radical formation, cerebrovascular dysfunction) [117]. The potential use of natural products in the treatment of neurodegenerative disorders has also been successfully demonstrated in the field of AD, and also to treat other forms of dementia including vascular dementia, Parkinson's dementia, dementia/Lewy body, and cognitive symptoms associated with multiple sclerosis and Down syndrome. However, much attention is focused on alkaloids while little is given to phenolics, terpenoids, and coumarins, and this review gives an update of representative non-alkaloid compounds with anticholinesterase activity.
Supplementary Materials: The following are available as supplementary material online; Table S1: Terpenoids as Acetyl and Butyryl cholinesterase inhibitors, Table S2: Phenolic compounds as Acetyl and Butyryl cho-linesterase inhibitors, Table S3: Coumarins as Acetyl and Butyryl cholinesterase  inhibitors and Table S4