Natural Inhibitors of Cholinesterases: Chemistry, Structure–Activity and Methods of Their Analysis

This article aims to provide an updated description and comparison of the data currently available in the literature (from the last 15 years) on the studied natural inhibitors of cholinesterases (IChEs), namely, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These data also apply to the likely impact of the structures of the compounds on the therapeutic effects of available and potential cholinesterase inhibitors. IChEs are hitherto known compounds with various structures, activities and origins. Additionally, multiple different methods of analysis are used to determine the cholinesterase inhibitor potency. This summary indicates that natural sources are still suitable for the discovery of new compounds with prominent pharmacological activity. It also emphasizes that further studies are needed regarding the mechanisms of action or the structure–activity correlation to discuss the issue of cholinesterase inhibitors and their medical application.


Introduction
Cholinesterase inhibitors are chemical compounds that impair the activity of cholinesterases: AChE and BuChE. They reduce the hydrolysis of the neurotransmitters acetylcholine (ACh) (acetylcholinesterase inhibitors) and butyrylcholine (butyrylcholinesterase inhibitors), thereby increasing their levels in the body (brain, blood and nerve tissue). Naturally occurring cholinesterase inhibitors affect esterases in a reversible manner [1].
IChE drugs currently used in medicine are synthetically derived. The majority of them originate from natural substances. One of them, tacrine, was approved for treatment, and it has been used similarly to donepezil, galanthamine (1) and rivastigmine. Unfortunately, the first of them causes hepatotoxicity, while the others have side effects including insomnia, diarrhea, nausea and vomiting [2].
IChEs (BuChE and AChE) also show therapeutic activity when applied in treatments for myasthenia gravis, myopathies, disorders associated with peripheral nerve damage, impaired conduction of nervous stimuli, and diseases associated with dementia, such as vascular dementia and Alzheimer's and Parkinson's diseases [3][4][5].
The mechanisms causing Alzheimer's disease (AD) are not entirely understood. In patients with AD, marked decreases in neurotransmitter levels in the cells are observed. In particular, the concentration of acetylcholine is reduced, together with dopamine, glutamate, serotonin and norepinephrine [6].
Currently, due to the limited knowledge of effective methods of treating the causes of these disorders, therapies, as before, are mainly based on symptomatic treatments (except for Aduhelm ® Aducanumab, which underwent accelerated FDA (U.S. Food and Drug Administration) approval [7]). Studies indicate that an increase in the level of cholinergic transmission in patients with AD (increase in ACh) mitigates disease progression. It also has a beneficial effect on cognitive functions and improves the patient's mood, despite the flavonoids and coumarins (IBuChE) have become increasingly important as appropriate inhibitors, because they show strong inhibition of ChE and fewer side effects.
Therefore, the focus is on describing the correlation between the activity and structure of selected groups of compounds for which the results of AChE or BuChE inhibition were the most promising (Table 1) [13]. There are reports that suggest a correlation of the activity of a compound on AChE and BuChE with certain components of its structure.

Alkaloids
These metabolites are characterized by the presence of nitrogen in a negative oxidation state (proton acceptor), in most cases positioned in a heterocycle. This may affect the active site of cholinesterase [13,14].
Because of its use in therapeutics, galanthamine (1) may be considered the most important alkaloid inhibiting cholinesterases. It is applied in AD treatment or other neurological disorders. Amaryllidaceae plants are natural sources of galanthamine (1). Some species of Narcissus, Leucojum and Ungernia genera are particularly rich in this alkaloid. It can also be obtained synthetically. There were also attempts to obtain it through biosynthesis [15].
Galanthamine (1) has a strong inhibitory effect on both AChE and BuChE; however, it is more selective toward AChE. It reveals competitive inhibition; additionally, it has a modulating impact on the nicotinic acetylcholine receptor. Thanks to this effect, it also supports neuromuscular conduction [15][16][17]. There are many publications describing the inhibition of cholinesterase by galanthamine (1). Thus, it is often treated as a reference substance (Table 1). On the basis of research on the interaction between galanthamine (1) and AChE from Torpedo californica, it was found to bind in the active center of the enzyme. The interaction between the double bond present in the galanthamine (1) cyclohexene ring and Trp84 enzyme was observed [18].
Monoterpenoid indole alkaloids from Nauclea officinalis exhibit inhibitory activity against BuChE. The inhibitory impact of some of them (Table 1, Figure 1) is greater than that of galanthamine (1) [19]. Liew et al. (2015) [19], after performing molecular docking, speculate that the high value of cholinesterase inhibition exhibited by angustidine (2) [18] indicated that the inhibitory effect of lycorine-type alkaloids on AChE is due to an increase in the involvement of the lipophilic substituent in C-1 and C-2 acting as hydroxyl in galanthamine (1) (general structure of lycorine-type alkaloids (3)) ( Figure 1).  (1) and angustidine (2) and general structure of lycorine-type alkaloids (3).
According to Berkov et al. (2008), the alkaloids N-allyl-nor-galanthamine (4) and N-(14-methylallyl)-nor-galanthamine (5) isolated from the leaves of Leucojum aestivum L. demonstrated more potent inhibition of AChE than galanthamine (1) ( Table 1). It appears that the inhibitory activity of both compounds is due to the substitution of the N-methyl derivative (allyl or 14-methylallyl group). The compounds are characterized by the presence of a methoxyl substituent at C-9, and the nitrogen atom also has a substituent alkyl  (1) and angustidine (2) and general structure of lycorine-type alkaloids (3).
The structures of isoquinoline alkaloids of the protoberberine type (Table 1) are similar to the structure of acetylcholine, containing an anionic site-acetoxy-and simultaneously a cationic site (amine). As in the case of acetylcholine, this structure may enable the bonding of the acetoxy group to the serine hydroxyl group at the site of hydrolysis of the substrate located in the esteratic site of AChE. The cationic site may be an isoquinoline nitrogen atom [1]. Protoberberine-type alkaloids (e.g., berberine (7), dihydroberberine (8) and coptisine (9)) such as Amaryllidaceae alkaloids are characterized by the presence of substituent methoxy and hydroxy groups or methylenedioxy groups, but in different positions (at C-2, C-3 and C-9, C-10), as well as a positively charged nitrogen atom [23].
The structures of isoquinoline alkaloids of the protoberberine type (Table 1) are similar to the structure of acetylcholine, containing an anionic site-acetoxy-and simultaneously a cationic site (amine). As in the case of acetylcholine, this structure may enable the bonding of the acetoxy group to the serine hydroxyl group at the site of hydrolysis of the substrate located in the esteratic site of AChE. The cationic site may be an isoquinoline nitrogen atom [1]. Protoberberine-type alkaloids (e.g., berberine (7), dihydroberberine (8) and coptisine (9)) such as Amaryllidaceae alkaloids are characterized by the presence of substituent methoxy and hydroxy groups or methylenedioxy groups, but in different positions (at C-2, C-3 and C-9, C-10), as well as a positively charged nitrogen atom [23].
As noted by Song et al. (2021), the presence of a conjugated aromatic system in the B ring is responsible for the strong inhibitory activity (e.g., berberine (7), coptisine (9), epiberberine, jatrorrhizine and palmatine (Table 1)). The hydrogenation of this ring decreases the inhibitory activity of the alkaloid (e.g., dihydroberberine (8)), while the cyclization leading to the methylenedioxy group has no impact on this activity (e.g., coptisine (9)) [23] ( Figure 3). same moiety as in the case of galantamine. The stronger activity of sanguinine ( pared to galanthamine (1), N-allyl-nor-galanthamine (4) and N- (14-methylallyl)-no thamine (5) may be explained by the presence of a hydroxyl group at the C-9 carb is not due to a methoxy group as in their case. The stronger the directing effec hydroxyl substituent (compared to the methoxy group), the stronger the activatio aromatic ring in the electrophilic substitution reaction (Figure 2).  (4), N-(14-methylallyl)-nor-galan (5) and sanguinine (6).
The structures of isoquinoline alkaloids of the protoberberine type (Table 1) a ilar to the structure of acetylcholine, containing an anionic site-acetoxy-and sim ously a cationic site (amine). As in the case of acetylcholine, this structure may en bonding of the acetoxy group to the serine hydroxyl group at the site of hydrolys substrate located in the esteratic site of AChE. The cationic site may be an isoqu nitrogen atom [1]. Protoberberine-type alkaloids (e.g., berberine (7), dihydroberbe and coptisine (9)) such as Amaryllidaceae alkaloids are characterized by the pres substituent methoxy and hydroxy groups or methylenedioxy groups, but in diffe sitions (at C-2, C-3 and C-9, C-10), as well as a positively charged nitrogen atom [ As noted by Song et al. (2021), the presence of a conjugated aromatic system ring is responsible for the strong inhibitory activity (e.g., berberine (7), coptis epiberberine, jatrorrhizine and palmatine (Table 1)). The hydrogenation of this r creases the inhibitory activity of the alkaloid (e.g., dihydroberberine (8)), while clization leading to the methylenedioxy group has no impact on this activity (e.g. ine (9)) [23] (Figure 3).  (7), dihydroberberine (8) and coptisine (9).
In the case of alkaloids extracted from Lycopodium casuarinoides (lycoparins A (10), B (11) and C (12)), the structure is also important in the inhibitory activity. Only lycoparin C (12) showed such an ability (Table 1), whereas lycoparins A (10) and B (11) have poor activity (IC 50 > 200 µM) as a consequence of the occurrence of carboxylic acid at the C-15 and methyl substituents attached to N ( Figure 4) [24]. Strong inhibitory activity against AChE comparable to that of galanthamine (1) is demonstrated by indole alkaloids from Ervatamia hainanensis (coronaridine (13) and voacangine (14)). Due to the presence of the substituent voacangine (14), they have markedly increased AChE inhibition. This is because of the attachment of the methoxyl substituent to the phenyl group, while the substitution of 10-hydroxycoronaridine with a hydroxy group on the phenyl decreases the activity (Table 1) ( Figure 5) [25].
Research on the structure and inhibition led to the conclusion that furanocoumarins have more affinity for BuChE than AChE [13,14]. Cholinesterase-inhibiting coumarins are often found in the Apiaceae and Rutaceae families [26].
Research on the structure and inhibition led to the conclusion that furanocoumarins have more affinity for BuChE than AChE [13,14]. Cholinesterase-inhibiting coumarins are often found in the Apiaceae and Rutaceae families [26].
Research on the structure and inhibition led to the conclusion that furanocoumarins have more affinity for BuChE than AChE [13,14]. Cholinesterase-inhibiting coumarins are often found in the Apiaceae and Rutaceae families [26].
It is noted that the effect of compounds isolated from an extract of Citrus hystrix (6 -hydroxy-7 -methoxybergamottin (15) and 6 , 7 -dihydroxybergamottin (16)) against BuChE depends on the presence of a dioxygenated geranyl chain in their structures ( Figure 6) [27]. Strong inhibitory activity against AChE comparable to that of galanthami demonstrated by indole alkaloids from Ervatamia hainanensis (coronaridine ( voacangine (14)). Due to the presence of the substituent voacangine (14), they hav edly increased AChE inhibition. This is because of the attachment of the methoxyl uent to the phenyl group, while the substitution of 10-hydroxycoronaridine wi droxy group on the phenyl decreases the activity (Table 1) ( Figure 5) [25].
Research on the structure and inhibition led to the conclusion that furanoco have more affinity for BuChE than AChE [13,14]. Cholinesterase-inhibiting couma often found in the Apiaceae and Rutaceae families [26].

Diarylheptanoids
Diarylheptanoids are a group of natural compounds with structures based on a 1,7-diphenylheptane skeleton [30].

Diarylheptanoids
Diarylheptanoids are a group of natural compounds with structures based on a 1,7diphenylheptane skeleton [30].

Diarylheptanoids
Diarylheptanoids are a group of natural compounds with structures based on a 1,7diphenylheptane skeleton [30].
The bond-line formula of flavonoids is made of two aromatic rings linked to diphenylpropane in a C6-C3-C6 system. Most of them have an additional gamma-pyrone system (rings C) divided into types due to the different positions of the B ring, the oxidation number of the C ring and the presence of additional functional groups [13,14,32].  [32] studied the link between the binding affinities of flavonoids with AChE using a typical measurement-the fluorescence quenching method reported by Ryu et al. (2012) [33]. They checked 20 flavonoids (i.e., baicalin, genistein, chrysin, apigenin, formononetin, 7,8-dihydroflavone, puerarin, luteolin, rutin (36), fisetin, naringenin, daidzein, daidzin, myricetin, myricetrin, quercetin, quercetrin, kaempferol (35), kaempferide and baicalein). According to this research, it can be inferred that inhibitory The bond-line formula of flavonoids is made of two aromatic rings linked to diphenylpropane in a C6-C3-C6 system. Most of them have an additional gamma-pyrone system (rings C) divided into types due to the different positions of the B ring, the oxidation number of the C ring and the presence of additional functional groups [13,14,32]. Xie et al. (2014) [32] studied the link between the binding affinities of flavonoids with AChE using a typical measurement-the fluorescence quenching method reported by Ryu et al. (2012) [33]. They checked 20 flavonoids (i.e., baicalin, genistein, chrysin, apigenin, formononetin, 7,8-dihydroflavone, puerarin, luteolin, rutin (36), fisetin, naringenin, daidzein, daidzin, myricetin, myricetrin, quercetin, quercetrin, kaempferol (35), kaempferide and baicalein). According to this research, it can be inferred that inhibitory flavonoids form a complex with AChE. The presence of a hydroxyl group, especially in the A ring of the flavonoid, as well as the double bond between C-2 and C-3, increases the affinity of the enzyme (hydrogen bonds) and also increases the AChE inhibitory properties of flavonoids. Glycosylation, on the other hand, decreases the activity and affinity of flavonoids toward the enzyme in a manner that depends on the form of the attached sugar moiety (1-5-fold). The presence of a methoxy group affects the activity of a flavonoid differently depending on its type, and no correlation was observed here [32].
Analyzing the impact of the structure of flavonoids from Paulownia tomentosa fruits indicated that geranylated flavonoids at C-6 (e.g., diplacone (30)) ( Table 1) are pivotal against hAChE and BuChE. The lack of this moiety causes a clear decrease in inhibition (eriodictyol (31) (IC 50 = 1663 µM)). It has also been proved that dihydroflavonols (4 -O-methyldiplacol (32)) show stronger inhibition compared to flavones (4 -O-methyldiplacone (33)) ( Figure 11) [34]. flavonoids form a complex with AChE. The presence of a hydroxyl group, especially in the A ring of the flavonoid, as well as the double bond between C-2 and C-3, increases the affinity of the enzyme (hydrogen bonds) and also increases the AChE inhibitory properties of flavonoids. Glycosylation, on the other hand, decreases the activity and affinity of flavonoids toward the enzyme in a manner that depends on the form of the attached sugar moiety (1-5-fold). The presence of a methoxy group affects the activity of a flavonoid differently depending on its type, and no correlation was observed here [32]. Analyzing the impact of the structure of flavonoids from Paulownia tomentosa fruits indicated that geranylated flavonoids at C-6 (e.g., diplacone (30)) ( Table 1) are pivotal against hAChE and BuChE. The lack of this moiety causes a clear decrease in inhibition (eriodictyol (31) (IC50 = 1663 µM)). It has also been proved that dihydroflavonols (4′-Omethyldiplacol (32)) show stronger inhibition compared to flavones (4′-O-methyldiplacone (33)) ( Figure 11) [34]. Selected flavonoids have been studied (docking study) (galangin (34), kaempferol (35), quercetin, myricetin, fisetin, apigenin, luteolin and rutin (36)) [35]. The inhibitory potency of flavonoids toward BuChE depends on the presence and the location of OH groups in the structure. A sugar moiety causing steric hindrance reduces these properties. Galangin (34) showed the strongest activity, kaempferol (35) was proved to be weaker, and rutin (36) was the weakest ( Figure 12). Selected flavonoids have been studied (docking study) (galangin (34), kaempferol (35), quercetin, myricetin, fisetin, apigenin, luteolin and rutin (36)) [35]. The inhibitory potency of flavonoids toward BuChE depends on the presence and the location of OH groups in the structure. A sugar moiety causing steric hindrance reduces these properties. Galangin (34) showed the strongest activity, kaempferol (35) was proved to be weaker, and rutin (36) was the weakest (Figure 12).

Phenanthrenes
Phenanthrenes are a group of natural compounds with a structure based on the phenanthrene skeleton, occurring in the form of monomeric, dimeric or trimeric derivatives [36].
Phenanthrenes from Bletilla striata showed potent and selective inhibitory activity against BuChE [37]. A publication by Liu et al. (2022) described that the presence of sub-

Phenanthrenes
Phenanthrenes are a group of natural compounds with a structure based on the phenanthrene skeleton, occurring in the form of monomeric, dimeric or trimeric derivatives [36].
Phenanthrenes are a group of natural compounds with a structure nanthrene skeleton, occurring in the form of monomeric, dimeric or tri [36].
By testing acetone extracts of the roots of Salvia miltiorhiza Bunge, strong inhibitory activity against AChE for the diterpenes dihydrotanshinone I (39) (IC 50 = 1 µM) and cryptotanshinone (40) (IC 50 = 7 µM) and weak activity for tanshinone I (41) (IC 50 > 50 µM) and tanshionone IIA (42) (IC 50 > 140 µM) [38] (Table 1) were found by Ren et al. (2004). The authors suppose that the activity is probably a result of the existence of a dihydrofuran ring instead of a furan ring present in the compounds indicating weak inhibitory activity. Additionally, compounds containing an aromatic ring in their structures showed much higher activity than those that have a cyclohexane ring at this site [38]. However, the study by Zhou et al. (2011) showed quite different results [39]. Inhibitory activity was not observed in tanshinone IIA (42) or cryptotanshinone (40), but tanshinone I (41) and dihydrotanshinone I (39) showed strong activity. Both of these compounds are similar in terms of o-aromatic rings; they only differ in the presence or lack of a double bond in the furan ring. The authors suggest that for the inhibitory effect on AChE, the structure of the aromatic ring may be more important than the furan ring as was thought before (the presence or lack of a double bond) ( Figure 14) [39]. served in tanshinone IIA (42) or cryptotanshinone (40), but tanshinone I (41) and dihydro tanshinone I (39) showed strong activity. Both of these compounds are similar in terms o o-aromatic rings; they only differ in the presence or lack of a double bond in the fura ring. The authors suggest that for the inhibitory effect on AChE, the structure of the aro matic ring may be more important than the furan ring as was thought before (the presenc or lack of a double bond) ( Figure 14) [39].
In a more recent study by Urbain et al. (2008), the activity of xanthones of Gentianel amarella ssp. acuta was examined [42]. They exhibited weaker activity (also includin bellidin (44) and bellidifolin (43)), and only triptexanthoside C (45) reached significan results for activity against AChE (Table 1) [42]. This compound also has a methoxyl grou in its structure, which may influence the higher result of cholinesterase inhibition (Figur 15).
In the study by Urbain et al. (2004), xanthones isolated from Gentiana campestris exhibited inhibitory activity against AChE [41]. Bellidifolin (43) had the best result. It achieved a minimum inhibitory quantity on TLC identical to that of galanthamine (1) (0.03 nM), while weaker results were those of bellidin (44) (0.15 nM) and its bellidifolin glycosides: 8-O-β-glucopyranoside (nor-swertianolin) and 8-O-β-glucopyranoside (swertianolin) were even weaker (0.18 and 1.2 nM, respectively) [41]. The weaker inhibition of the enzyme by glycosides can probably be explained by steric hindrance and diverted hydrophobicity. On the other hand, xanthones containing an additional methoxyl group in the C-3 position showed stronger activity [41].
In a more recent study by Urbain et al. (2008), the activity of xanthones of Gentianella amarella ssp. acuta was examined [42]. They exhibited weaker activity (also including bellidin (44) and bellidifolin (43)), and only triptexanthoside C (45) reached significant results for activity against AChE (Table 1) [42]. This compound also has a methoxyl group in its structure, which may influence the higher result of cholinesterase inhibition ( Figure 15). In summary, the potential activity of an acetylcholinesterase inhibitor is influenced by the presence of hydroxyl and methoxyl groups in the molecule and also by the presenc of the cationic part of the structure of the compound (e.g., nitrogen in the heterocycli In summary, the potential activity of an acetylcholinesterase inhibitor is influenced by the presence of hydroxyl and methoxyl groups in the molecule and also by the presence of the cationic part of the structure of the compound (e.g., nitrogen in the heterocyclic system). The substrate-like structure of the inhibitor (or acetylcholine) indicates the competitive inhibition of the enzyme, and it is most beneficial in pharmacology. Large molecules, e.g., glycosidic forms of the tested compounds, were characterized by weaker AChE inhibitory activity due to their steric hindrance in the enzyme. The occurrence, different number and localization of double bonds, preferably in conjugated systems (diarylheptanoids and Amaryllidaceae alkaloids), are of utmost importance. With the increase in the number of conjugated double-bond systems, as well as the presence of substituents that polarize the aromatic system, the energy of the cation-π interaction increases, and thus, the binding energy of the inhibitor with the protein residue of the enzyme increases [43]. The presence of these substituents in the compounds was also significant in the inhibition against AChE. This may be related to the ability of BuChE to hydrolyze both butyrylcholine and acetylcholine [1,44]. The structure of the BuChE enzyme molecule enables the catalysis of large acyl groups, which the AChE molecule is not capable of. Hence, in the presented data (Table 1), there are many inhibitors that are inactive against AChE while demonstrating moderate or strong activity toward BuChE [1]. This may be due to the steric hindrance of the AChE enzyme due to the large branched structures of such compounds, as is demonstrated by the weaker activity of glycosides in relation to their aglycones (xanthonoids from Gentiana campestris) ( Table 1).
The review topic of natural cholinesterase inhibitors has been discussed in other publications, including [45][46][47]. Most of them are based on the description of results obtained for plant fractions and extracts or, in addition, for compounds isolated from them [45,46]. This article focuses on the comparison of particular isolated natural compounds' activities, considering both plant and animal origins (e.g., alkaloids from scorpions or sponges). Some of the previous reviews did not include this information [45,46]. The current review includes 20 groups (24 subgroups) of compounds; a total of 357 results for cholinesterase inhibition by natural compounds are listed, arranged alphabetically by compound group, species name and compound name. A total of 84 species or their varieties belonging to 44 families were examined. The current review shows, in tabular form, the results of the inhibition of both AChE and BuChE enzymes. The present summary is also characterized by the fact that the type of enzyme and the method used in the study are presented. This review shows that differences are significant and have an impact on the results of enzyme inhibition by the tested compounds. This paper focuses on the review of the results of studies on natural cholinesterase inhibitors tested using in vitro methods. The presented overview is also characterized by the description and consideration of the type of method used for the determination of cholinesterase inhibition, which has not been undertaken in other recent reviews, or they were limited to the modifications of colorimetric Ellman's method [46].
The data, mainly from the selected latest publications issued from 2008 to 2022, on cholinesterase inhibitors of natural origin are ordered in the table below ( Table 1). The following sources were used to prepare the review article database: Chemical Abstract (SciFinder), Reaxys and Science Direct (partially by authorized access), as well as sources directly obtained from the authors (ResearchGate GmbH)).          [48,114] Abbreviations in Table 1: nd-not determined; ns-not shown; a -inhibitory concentration for which enzyme activity is equal to half-maximal (IC 50 )/(IC 50 ) ± S.E.M.; b -percentage of inhibition against enzyme (xµg mL −1 -y%, xµM-y%); c -minimal inhibitory quantity (MIC); d -IC 50

Activity
A comparison of the activity of individual isolated compounds is presented in Table 1.
From the presented tabular comparison (Table 1) of the results of the conducted studies (values of IC 50 ), it follows that the compounds belonging to the alkaloid group exhibit the strongest activity against AChE and therefore are discussed in more detail. Fourteen of them have strong inhibitory activity with an IC 50 value < 1.5 µM, and forty-two have medium-strong activity below 20 µM ( Table 1).
The best AChE inhibition result in the entire table (Table 1) was obtained for sanguinine (IC 50 = 0.007 µM). This was confirmed in another independent study in which the compound was derived from a different plant material (IC 50 = 0.10 µM). Strong activity against AChE was also detected for five other Amaryllidaceae alkaloids (IC 50  The different values of the inhibition coefficient obtained for sanguinine (6) are probably due to the use of various origins of AChE in the two studies. Similarly, for the same Amaryllidaceae alkaloids, e.g., lycoramine, inconsistent results were observed, with potent activity against Electric eel AChE and inactivity or weak activity using human AChE (isolated from Narcissus jonquilla 'Pipit' and Narcissus poeticus 'Pink Parasol').
Stronger and more potent AChE inhibitory activity than galanthamine (1) was obtained for five other isoquinoline alkaloids of the protoberberine type (IC 50  Values of the IC 50 inhibition coefficient of AChE below 1.5 µM were demonstrated by alkaloids derived from the sponge Latrunculia biformis (discorhabdin G) and an alkaloid from Lycopodium, huperzine C, with a slightly weaker result than the known activity of huperzine A.
Similar moieties that are crucial for the binding of the inhibitor to the enzyme are also present in other strong and medium-strong inhibitors from other groups of compounds present in Table 1.
Lipophilic substituents will have a stronger affinity for the hydrophobic AChE ester part; hence, they show stronger binding to the enzyme and greater inhibition, which is described in more detail in the chemistry-structure-activity section [1,43].
A BuChE inhibition study showed inhibitory activity for thirty-one alkaloids with inhibition coefficients ranging from 10 to 50 µM and strong activity for alkaloids with inhibition coefficients below 10 µM. Alkaloids isolated from Nauclea officinalis (IC 50 = 1.03 µM, 4.98 µM, 7.70 µM), including angustidine (2), have the lowest inhibition coefficients of all the alkaloids listed in Table 1.
From the coumarin group, coumarins isolated from Mesua elegans (IC 50 = 0.70 µM, 1.06 µM, 3.06 µM) have the strongest ability to inhibit AChE. Four of them exhibit medium activity with an IC 50 value <10 µM. Their activity against BuChE has not been studied. However, other presented coumarins displayed in the results seem to show BuChE inhibition (bergapten, imperatorin (17)  Similarly, phthalates from Lonicera quinquelocularis had IC 50 = 1.65 µM and 3.43 µM for AChE and IC 50 < 10 µM for BuChE. Among diterpenes, dihydrotanshinon I and cryptotanshinone (40) showed strong inhibition against AChE, and triptexanthoside C (45) (xanthones) showed significant inhibition. Xanthonoids from Garcinia mangostana had IC 50 on AChE from 2.5 µM, with six compounds having IC 50 < 20 µM, and IC 50 on BuChE from 0.7 µM, with six compounds having IC 50 < 12 µM. Anthranoids from Psorospermum glaberrimum demonstrated medium-strong activity toward BuChE (9.25-13.30 µM) and weak activity toward AChE. A fatty acid from Lycopodiella cernua has shown high inhibition of AChE (0.22 µM). Remarkable results are also shown by polyphenols from Camellia sinensis var. assamica (caffeoylated catechin) against AChE, as well as by polyphenols from Vitis amurensis (heyneanol A, vitsin A), which had strong activity against AChE and BuChE. Medium-strong inhibition of AChE by creoside IV from Codonopsis pilosula and strong inhibition against BuChE by broussonin A (Anemarrhena asphodeloides) were found. High inhibition values against AChE and BuChE were also observed for sesquiterpene lactones from Amberboa ramosa (amberin, amberbin A and amberbin B). Sterols (leucisterol, haloxylon A and haloxylon B) from Haloxylon recurvum have shown strong inhibition of both BuChE and AChE. A strong inhibition value against AChE and medium inhibition against BuChE were shown by chromones from Sargassum siliquastrum and one of the diarylheptanoids: (−)-alpininoid B (23) ( Table 1). Terpenoids demonstrated strong (Lycopodiella cernua) or medium-strong (Lycopodiella cernua or Garcinia hombroniana) activity against AChE and BuChE. All results are presented in Table 1.

Analysis Methods
The studied compounds occur in materials of natural origin in the form of mixtures.
To determine the change in enzyme activity due to a particular compound, it is necessary to purify samples or even fractionate them. Studies of inhibitory activity toward cholinesterases in scientific reports are performed according to different methods and procedures. Nevertheless, in most cases, analyses are based on Ellman's assay [48]. A summary of analysis methods used in the selected studies of cholinesterase inhibition is presented in Table 1. The description of the most important one is presented below.
The method should be simple, quick to perform, sensitive and inexpensive [77,137]. The analysis methods are based on a colorimetric assay using chromatographic techniques, TLC and fluorimetric and spectrophotometric measurements.
These methods are based on measuring changes in parameters indicative of enzyme activity before and after the introduction of the inhibitor to the system. Even slight changes in temperature, incubation time, pH, the concentration of substrates and the enzyme and the presence of other interfering compounds (detergents and heavy metal ions) can affect the accuracy of the results.

The Colorimetric Method of Ellman (1961)
This procedure is based on the result of the color reaction between the formed prethiocholine and the DTNB color developer (5,5 -dithiobis-(2-nitrobenzoic acid). Thiocholine is the product of the enzymatic reaction between acetylthiocholine (ATCI) and ChE. The intensity of the color of the product measured colorimetrically allows the determination of changes in enzyme activity. In the presence of an inhibitor, the change is suppressed, and we observe a lower-intensity color or complete inhibition [48].

Spectrophotometric Modification of Ellman's Method
Ding et al. (2013) described a modification used to determine the inhibitory activity of flavonoids and ginkgolides B and C from the leaves of Ginkgo biloba against AChE and BuChE [111]. Only flavonoids inhibit AChE (results in Table 1). In the method of Park and Choi (1991), the supernatant from the brown planthopper maggot was prepared (which contains ChE) [110]; the homogenized supernatant (T = 4 • C, t = 30 min.) was prepared in phosphate buffer (pH = 7.0) and 0.1 % Triton X-100. Acetone solutions of the analyzed compounds and standard (chlorpyrifos) were mixed with the previously prepared solution containing the supernatant and analyzed in a 96-well microtiter plate after 1h. DTNB and ATCI were added. Then, the measurement of absorbance was performed (λ = 405 nm microplate reader). The activity is relative to the control reaction, assumed as 100 %, and to the test compounds replaced by the buffer. On the basis of the results, the IC 50 was determined [110].
The spectrophotometric modification of Ellman's method described by Senol et al. (2010) was used to verify the inhibition of the methanol extract and isolated compounds (imperatorin (17), xanthotoxin and bergapten) from the fruits of Angelica officinalis L. [99]. The inhibition of both cholinesterases was tested using an ELISA microplate reader; galanthamine (1) as a standard; AChE from electric eel; and BuChE from horse serum. The potent inhibition of BuChE was observed for both the extract (100 µg/mL-85.65 ± 1.49%) and each of the compounds (Table 1) [100]. Many of the compounds were tested by using various modifications of the spectrophotometric method; they differed in the incubation time, the equipment used, the concentration of reactants and the wavelength measurement. The inhibitors belong to different groups of compounds (Table 1).
Cholinesterase inhibitory activity was also identified by using a TLC technique. By comparing the methods performed using the microplate and TLC, as described in Rhee et al. (2001), it can be assumed that TLC methods are more sensitive [16]. Due to the advantages of the TLC approach (simple, inexpensive and accurate measurement), this review focuses on methods using this technique.

TLC Modification of Ellman's Method
The modification of Ellman's method has been described by Rhee et al. (2001) [16,48]. As a result of the disruption of ATCI by AChE, choline is formed, which constitutes a colored compound (5-thio-2-nitrobenzoate anion) with DTNB. The color intensity of the product is measured spectrophotometrically. The bands of the tested extract are developed on the TLC plate, and the band pattern is sprayed with a mixture of DTNB and then ATCI in Tris-HCl buffer (Trizma hydrochloride with bovine serum, pH = 8); the AChE enzyme is then applied (3 U mL −1 ; from electric eel). This results in a yellow background due to a diazo compound (5 min) with white trails, which indicates inhibition by the extract. The disadvantage of the method is the possibility of false-positive effects [16].
The modified method of Rhee et al. (2001) was used, inter alia, to evaluate the obtained compound (mahanimbine) and petroleum ether extract (10 mg mL −1 )) from Murraya koenigii. The plates were developed with a mobile phase (petroleum ether: CHCl 3 , 50: 50 (v/v)) and, after drying, were sprayed with DTNB/ATCI, followed by the implementation of the basic method. The enzyme activity was measured using a 96-well microplate reader [16,48,76]. The procedure described by Rhee et al. (2001) was also used to investigate the inhibitory activity against ChE by the extract and compounds (10-hydroxyinfractopicrin and infractopicrin) isolated from the toadstool Cortinarius infractus. For the measurement, the following compounds were used: AChE from bovine erythrocytes or equine serum BuChE and tacrine, physostigmine and galanthamine (1) as standards (>100 µM). The results were determined using a 96-well microplate reader [61].

TLC Bioautography by Marston
A properly made plate with applied spots of extracts was sprayed with a prepared mixture with the enzyme AChE or BuChE (T = 4 • C in Tris-hydrochloric acid, pH = 7.8, with bovine serum albumin as a stabilizer) and incubated (T = 37 • C, 20 min; increased humidity).
Then, in order to carry out the detection, a mixture containing, inter alia, Fast Blue Salt and alpha-naphthyl acetate prepared ex tempore was sprayed. After incubation (1-2 min.), a purple background due to the diazonium dye was obtained, while white spots indicated inhibition caused by the applied sample. The clear differences in the background color and band color indicate inhibition [101].
Then, it was sprayed with a solution of Fast Blue B salt. White spots demonstrating inhibition were clearly visible on the dark purple background due to the azo compound and appeared quickly (1 min), and they were very persistent (for 24 h). The advantage of this method is the decreased usage of the enzyme and the shortened time required for its incubation (10 min) compared to other methods. The method is highly sensitive and fast [77].
This validation method was performed by the author for the determination of the inhibition of Amaryllidaceae AChE isolated from extracts from Narcissus jonquilla 'Pipit' and Narcissus jonquilla 'Havera' and purified extracts of N. jonquilla 'Baby Moon', Crinum moorei and Scadoxus puniceus. This procedure manages to achieve high sensitivity. The inhibitory activity of the isolated alkaloid was demonstrated, and it was indicated that dihydrogalanthamine has greater inhibition, approximately 42% higher than galanthamine (1) [77]. With the application of this method, the activity of alkaloids present in the extract from Argemone mexicana L. roots was proved; it was weak for magnoflorine and strong for berberine (7), palmatine and galanthamine (1), isolated for the first time from the Papaveraceae family [138]. Additionally, a two-dimensional thin-layer chromatography/high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry (TLC/HPLC/DAD/MS) system has been developed for both qualitative and quantitative analyses of active AChE inhibitors in plant samples [139]. The method of bioautography by Mroczek confirmed the inhibition of AChE by Amaryllidaceae alkaloids and determined their numerous occurrences in three cultivars of Narcissus: N. jonquilla 'Baby Moon', N. 'Golden Ducat' and N. 'Cheerfulness'; the alkaloids were and identified both by using a TLC plate assay and by using TLC/HPLC/DAD/MS [140]. These methods have also been used to demonstrate AChE inhibitory activity and to qualitatively evaluate Lycopodiaceae alkaloids, and they were successfully used to study neuroprotective polyphenols from two species of Trifolium as well [141,142].

Fluorimetric Methods
These are fluorescent techniques (quenching) that measure enzyme-inhibitor binding affinities. This type of pathway has been chosen to demonstrate the activity of flavonoids from Paulownia tomentosa fruits with minor modifications to the spectrophotometric method of Ellman (1961). As a reference standard, physostigmine (eserine) was used (Table 1). In addition, using the fluorescence assay method (decrease), the affinity of the compounds with the relevant enzyme was studied.
The results were based on the dependency of the constant affinity rate, proportional to the inhibitory activity. Spectrophotometer measurements of the fluorescence emission were taken with a camera (M Series Multi-Mode Microplate Readers) (T = 18 • and 37 • C) as the solution was titrated with a predetermined amount of a solution of hAChE (phosphate buffer (pH 8.0) (5 U mL −1 )) with successive amounts of the tested flavonoids added. Studies have shown that the presence of a geranyl substituent at the C6 position in the structure of flavonoids is important for their ability to inhibit AChE [34].
The fluorimetric method was a part of the analysis of the Mangosteen seedcase extract outlined below [136]. To measure the compounds, the following steps were performed: the supernatant was centrifuged (12,000 rpm, 10 min.), a mixture with a buffer solution of ChE (5 µL) was added to the extract solution (20 µL), and the extract (CHCl 3 in MeOH) was incubated (T = 37 • C, t = 30 min.). The supernatant (2 µL) was analyzed using ultra-performance liquid chromatography coupled with a photo-diode array detector and quadrupole timeof-flight mass spectrometry (UPLC/PDA/QTOF/MS), and the result was compared with that of the analysis without the enzyme. In the chromatogram, the peaks of mangostanol, allanxanthone E, gudraxanthone, γ-mangostin, 8-deoxygartanin and α-mangostin vanished (results in Table 1), so those compounds show an affinity for the enzyme. Then, the inhibitory activity of both cholinesterases was measured using a modification of Ellman's method (Table 1). Using a fluorescence technique (quenching), affinity toward AChE was compared with γ-mangostin (Table 1) and 9-hydroxycalabaxanthone (IC 50 > 100 µM). The first compound gained a much higher score. The authors supposed that the significant inhibition of AChE can respond to the presence of more than one prenyl group [136].
The methods presented in this review for determining cholinesterase inhibition by the investigated compounds can be described as qualitative and quantitative ones. Those based on the TLC technique (TLC bioautography) are more suitable for demonstrating inhibition by particular compounds (qualitative), and they are more sensitive compared to spectrophotometric methods (modifications of Ellman's method). Nevertheless, they are not suitable for the determination of the inhibition coefficient, or it is difficult to measure. Therefore, they do not offer the possibility to compare the potency of inhibition among inhibitors. Both of these advantages are realized by methods based on a combination of the TLC technique (TLC bioautography) with more advanced techniques, such as HPLC/DAD/MS (high performance liquid chro-matography with photodiode array mass spectrometry), as mentioned in this article. Their use is increasingly observed in newer publications on cholinesterase inhibitors.

Conclusions
Reviewing the available publications, it can be concluded that methods for investigating cholinesterase inhibition have been mostly based on known procedures. These are generally modifications of previously used methods. They differ in parameters, which could affect the result of the activity of the enzyme and substrate, the incubation time, the method of analysis, the order of the addition of reactants and the type of assay.
It is important to study pure plant materials from respectively tested sources (heavy metal ions and detergents) and adequately purify the sample. The results (IC 50 ) of the same compound when determined relative to hAChE and eeAChE can differ [136]. In some of the publications, the type of cholinesterase used in the study is not described or this information is difficult to obtain.
It is only possible to compare the IC 50 values of specific compounds when determined under relatively similar conditions, using the same methods and compared to the same reference compound, thereby concluding potency. In most cases, one method is used, and results are rarely confirmed by using another method. An increased number of studies examining the inhibitory effects on both cholinesterases would be advantageous. It is beneficial to enhance the awareness and understanding of the subject of IChEs and activity measurement methods. Some of the studies did not include designations of activity toward both cholinesterases. It would be useful to use several reference substances in one study, which would allow for a better comparison of the available inhibition results.
However, more recent studies include the determination of the inhibition of both cholinesterases by the studied compounds and also attempt to analyze the structure and enzyme-inhibitor interaction, which is highly beneficial. This review reveals that inhibitors more potent than galanthamine (1), acting against both cholinesterases, are still being discovered. At the same time, compounds exhibiting potent selective activity against one of the cholinesterases have emerged. According to the established criteria in the study, strong activity against AChE was shown by 27 compounds, medium-strong was shown by 93 compounds, and weak activity was shown by 77 compounds, while against BuChE, strong, medium-strong and weak activity was shown by 43, 68 and 22 inhibitors, respectively. The largest group of compounds with a strong effect on both AChE and BuChE, as shown by the tabular comparison, were alkaloids. Compounds from this group demonstrated the most potent inhibition of AChE. Especially strong inhibition results against both cholinesterases were demonstrated for alkaloids from the Amaryllidaceae and Papaveraceae families. The most potent BuChE inhibition was demonstrated by compounds from various groups: alkaloids, coumarins, flavonoids, phenylpropanoids, polyphenols, phenanthrenes, phthalates, sterols and steroids, triterpenoids, xanthonoids and also lignans or phlorotannins. The presented review, as well as a summary of the results of the inhibitors' structure analysis, may be beneficial in the determination and planning of further stages of research for the presented compounds. These data may also be helpful in the search and synthesis of new semi-synthetic or synthetic derivatives, as well as new biologically active substances.
Work on finding compound derivatives with more specific, preferable features that we find in plant materials has yielded positive results. The ability to modify them allows for even better parameters of the drug, such as greater activity, a better match to the receptor, mitigated side effects, a longer duration of action or a favorable method of production. The integration of phytochemistry and cooperative disciplines of molecular modeling and chemical synthesis provides an opportunity to find effective drugs. The studies conducted continuously demonstrate that compounds of natural origin are still abundant and carry a lot of possible solutions.
The observed persistent deficiency of effective therapies for neurological diseases, including AD, requires researchers to further search for new therapeutic substances. The presented review, conducted for the period from 2008 to 2022 years, shows that the search for and analysis of natural cholinesterase inhibitors have not been exhausted yet. After summarizing in vitro studies, the conclusion emerges that the potential for the use of cholinesterase inhibitors in therapeutics has not been fully explored. Only some of them have been tested in vivo, and for several of them, clinical studies have been attempted. The results presented in this publication indicate that natural sources are a huge reservoir in the search for new therapeutic substances, including cholinesterase inhibitors.