Send Orders of Reprints at Reprints@benthamscience.net Natural Ache Inhibitors from Plants and Their Contribution to Alzheimer's Disease Therapy

As acetylcholinesterase (AChE) inhibitors are an important therapeutic strategy in Alzheimer's disease, efforts are being made in search of new molecules with anti-AChE activity. The fact that naturally-occurring compounds from plants are considered to be a potential source of new inhibitors has led to the discovery of an important number of secondary metabolites and plant extracts with the ability of inhibiting the enzyme AChE, which, according to the cholinergic hypothesis, increases the levels of the neurotransmitter acetylcholine in the brain, thus improving cholinergic functions in patients with Alzheimer's disease and alleviating the symptoms of this neurological disorder. This review summarizes a total of 128 studies which correspond to the most relevant research work published during 2006-2012 (1 st semester) on plant-derived compounds, plant extracts and essential oils found to elicit AChE inhibition.


INTRODUCTION
Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with memory impairment and cognitive deficit. It is characterized by low levels of acetylcholine in the brain of AD patients. According to the cholinergic hypothesis, the inhibition of acetylcholinesterase (AChE), an enzyme that catalyzes acetylcholine hydrolysis, increases the levels of acetylcholine in the brain, thus improving cholinergic functions in AD patients. Furthermore, although the general consensus concludes that AChE inhibitors (AChEi) can alleviate AD symptoms, they neither delay nor reverse the disease progress. Most of the drugs currently available for the treatment of AD are AChEi: tacrine (1), donezepil (2), rivastigmine (3) and galanthamine (4), all of which have limited effectiveness and some kind of side effect [1]. Tacrine (1) and donepezil (2), both from synthetic origin, were the first drugs approved for the treatment of cognitive loss in AD patients by US-FDA in 1993 and 1996, respectively. Rivastigmine (3) was approved in 2000 (US-FDA) and was designed from the lead compound physostigmine, a natural AChEi alkaloid. Galanthamine (4), a natural alkaloid first obtained from Galanthus spp. was approved by US-FDA in 2001. Huperzine A (5), an alkaloid found in Huperzia spp., is an AChEi commercialized as a dietary supplement for memory support and it is used to treat AD symptoms in China. This alkaloid has been thoroughly studied with promising results yielded particularly from the evaluation of cognitive *Address correspondence to this author at the INQUISUR-CONICET, Departamento de Química, Universidad Nacional del Sur, Bahía Blanca, Argentina; Tel: 54 291 4595101 ext. 3538; Fax: 54 291 4595187; E-mail: apmurray@uns.edu.ar performance of animals as well as from studies on its efficacy, tolerance and safety.
Taking into account that inhibitors 3, 4 and 5 are related to natural products and that AChEi are an important therapeutic strategy for the treatment of AD, many research groups have focused their studies on naturally-occurring compounds from plants as potential sources of either new or more effective AChEi. These studies led to the discovery of an important number of secondary metabolites as well as plant extracts, both of which are characterized by their ability to inhibit AChE. On the other hand, the fact that a significantly relevant number of research papers has been recorded in this field during the last decades can be clearly attributed to the development of colorimetric methods which allow a rapid and facile screening of a large number of samples. Ellman's method is the most widely used for the detection of AChEi, even in complex mixtures, and for the quantification of anti-AChE inhibitory activity [2][3][4][5][6].
Several reviews on the newly discovered AChEi obtained from plants, fungus and marine organisms have also been published over the last years [7][8][9][10]. The majority of these AChEi belong to the alkaloid group, including indole, isoquinoline, quinolizidine, piperidine and steroidal alkaloids. On the other hand, several non-alkaloidal and potent AChEi have been obtained from natural sources, including terpenoids, flavonoids and other phenolic compounds. Interestingly, although literature demonstrates to be rich in the study on AChEi obtained from plants, this issue keeps on being the center of attention for research as confirmed by the increasing number of studies published every year. Therefore, the purpose of this review is to provide a comprehensive summary of the literature, particularly that published during 2006-2012 (1 st semester) on plant-derived compounds, plant extracts and essential oils which have been reported to inhibit AChE. Readers interested not only in previous findings but also in synthetic/semisynthetic AChEi or natural AChEi of fungal, marine or microbial origin are recommended to see the above-mentioned reviews [i.e. [7][8][9][10]. For the sake of brevity and in order to focus our attention on the most relevant findings, only those research papers reporting quantified results (IC 50 and/or percentage of inhibition at a given concentration) were included. Extracts or essential oils with IC 50 > 0.5 mg/ml were considered weakly active and were therefore not taken into account in the present review. With a few exceptions, only molecules with IC 50 < 50 M have been considered. Furthermore, unless otherwise stated, those results on AChE inhibition included in the present review refer to in vitro assays carried out with AChE from electric eel.
On the other hand, of the several alkaloids that were isolated from the active extracts of Esenbeckia leiocarpa (Rutaceae), leptomerine (9) and kokusaginine (10) with IC 50 values of 2.5 and 46 M, respectively, were observed to elicit AChE inhibitory activity [12]. The isolation of skimmianine (11), a furoquinoline alkaloid with very low AChE inhibitory activity, was also reported by the same authors. This alkaloid was observed in another Rutaceae, Zanthoxylum nitidum, exhibiting a moderate AChE inhibitory activity (IC 50 = 8.6 g/ml) [13]. Nelumbo nucifera is a well-known medicinal plant belonging to the Nelumbonaceae family which was studied due to its therapeutic potential [14]. N-methylasimilobine (12), an aporphine alkaloid with an IC 50 = 1.5 g/ml which was found to be a non-competitive inhibitor, was recently isolated from this plant [15]. In a random screening, two extracts of Beilschmiedia species were observed to exhibit AChE inhibition and a phytochemical study of B. alloiophylla and B. kunstleri revealed the presence of several alkaloids with IC 50 values ranging between 2.0 and 10.0 M [16]. The most potent AChEi were found to be 2-hydroxy-9methoxyaporphine (13), laurotetanine (14), liriodenine (15) and oreobeiline (16)  Research on plants from the genus Corydalis (Papaveraceae) which are used for the treatment of memory dysfunction in folk medicine reported the presence of benzylisoquinoline alkaloids with anti-AChE activity [7]. The ethanolic extract obtained from the tuber of C. turtschaninovii previously found to elicit AChE inhibition was selected to carry out a chemical study which led to the isolation of the isoquinoline alkaloids stylopine (22), epiberberine (23), pseudodehydrocorydaline (24), pseudocopsitine (25) and pseudoberberine (26). In the assay with mouse brain cortex as a source of AChE enzyme, the IC 50 values obtained for each of these alkaloids were 15.8, 6.5, 8.4, 4.3 and 4.5 M, respectively [17]. In addition, alkaloids 25 and 26, the two most active compounds, were found to elicit anti-amnesic activity [17,18]. Alkaloids with benzylisoquinoline skeleton from Corydalis species having aromatic methylenedioxy groups and a quaternary atom of nitrogen were observed to show the strongest AChE inhibition [7,17,18]. In a more recent work, six protoberberine alkaloids 23, 27 -31, were identified in rhizomes of Coptis chinensis which are traditionally used in Chinese medicine for the treatment of various diseases. Coptidis rhizomes and their alkaloids were reported to have cognitive-enhancing and neuroprotective effects and the analysis of the anti-AChE activity of these alkaloids showed that the IC 50 values of berberine (27), palmatine (28), jateorrhizine (29), coptisine (30) and groenlandicine (31) ranged between 0.44 and 0.80 M while that of epiberberine (23) was slightly higher (IC 50 = 1.07 M) [19]. Of these alkaloids, compounds 27, 30 and 31 were observed to have an aromatic methylenedioxy group. In this study groenlandicine (31) and berberine (27) were found to be the most active as BChE inhibitors and epiberberine (23) was observed to significantly inhibitsecretase (BACE1) [19].
The alkaloids (+)-canadaline (32)  and 12.4 M, respectively, were observed to elicit a moderate inhibitory activity when tested with AChE from human blood [20].
On the other hand, Stephania venosa (Menispermaceae), a Thai medicinal plant, was found to show a high AChE inhibitory activity. The ethanolic extract of S. venosa was subjected to bioassay-guided fractionation to identify AChEi [21]. The following moderately active quaternary protoberberine alkaloids could be isolated: stepharanine (34), cyclanoline (35) and N-methyl stepholidine (36) with IC 50 values of 14.10, 9.23 and 31.30 M, respectively. A similar fractionation approach was followed to identify the compounds responsible for AChE inhibition in Chelidonium majus (Papaveraceae) [22]. Three active constituents were identified, namely 8-hydroxydihydrochelerythrine (37), 8hydroxydihydrosanguinarine (38) and berberine (27). Compounds 37 and 38, with no previous record as AChEi, were found to elicit significant anti-AChE activity with an IC 50 = 0.61 and 1.37 M, respectively.
Taspine (39) was isolated from the alkaloid-enriched extract obtained from Magnolia x soulangiana (Magnoliaceae) [23]. This alkaloid was found not only to show a dosedependent and long-lasting inhibitory effect on AChE (IC 50 = 0.33 M) but also to be more potent than galanthamine (IC 50 = 3.2 M) although its inhibitory activity is comparable to that of tacrine (IC 50 = 0.22 M). Similar observations were obtained when the in vitro assay was performed with human AChE (IC 50 = 0.54 M). Compound 39 resulted to be inactive against BChE, acting as a selective AChEi.
Catharanthus roseus (Apocynaceae) is a plant mainly known as a source of vincristine and vinblastine, two alkaloids found in its leaves and appreciated as anticancer compounds. Several other compounds with biological importance can be also found in C. roseus. For example, the alkaloid serpentine (40), isolated from the roots of this plant, was reported to be a potent in vitro AChEi (IC 50 = 0.775 M) compared with physostigmine (IC 50 = 6.45 M) [24].
A bioassay-guided fractionation from the stems of Ervatamia hainanensis (Apocynaceae), a plant used in traditional Chinese medicine, allowed the isolation of several monoterpenoid indole alkaloids, some of them showing a potent AChE inhibitory activity [25]. For example, coronaridine (41) and voacangine (42), differing from each other only by the methoxy group attached to the aromatic ring, were observed to have an IC 50 = 8.6 and 4.4 M, respectively, these values being similar to that of galanthamine (3.2 M). On the other hand, 10-hydroxycoronaridine (43) was found to evidence a reduced AChE inhibition (IC 50 = 29 M), which was attributed to the introduction of a hydroxyl group to the aromatic ring. The indole alkaloids coronaridine (41) and voacangine (42), both detected in the stalks of Tabernaemontana australis (Apocynaceae), had been formerly identified as AChEi but no inhibition values were reported [26].
The genus Tabernaemontana is known for the wide variety of unusual bioactive indole alkaloids it produces. Among them, the bisindole alkaloids isolated from T. divaricata roots are an interesting example of new structures with potent AChE inhibitory activity. The crude alkaloid extract obtained from the root of T. divaricata was found to yield four bisindole alkaloids 44 -47 [27]. The analysis of AChE inhibition revealed that 19,20-dihydrotabernamine (44) and 19,20-dihydroervahanine A (45) strongly inhibit AChE, with an IC 50 = 0.227 and 0.071 M, respectively, thus showing that they are significantly more active than galanthamine (IC 50 = 0.594 M). The fact that inhibition was found to be higher for compound 45 than for compound 44 suggests that the introduction of a carbomethoxy group at C16' increases the enzymatic inhibition. In addition, taking into account that conodurine (46) and tabernaelegantine (47)  were found to show no activity in AChE, it was suggested that the substitution at C11' and C12' is relevant for AChE inhibitory activity [27].
Uncaria rhynchophylla (Rubiaceae) is a Chinese medicine herb used to treat epilepsy. The alkaloid fraction from U. rhynchophylla is known for its antiepileptic and neuroprotective effects. Geissoschizine methyl ether (48), a strong AChEi, as well as six other weakly active alkaloids were recently isolated from this herb [28]. The active compound 48 was observed to inhibit AChE in a reversible and non-competitive way with an IC 50 = 3.7 g/ml.
The study of AChE inhibitory activity of Brazilian apocynacea Himatanthus lancifolius, commonly known as "agoniada", led to the identification of active extracts in this plant and allowed the isolation of uleine (49), an active indole alkaloid, at a high concentration in the alkaloid fraction. The IC 50 value observed for this alkaloid was 0.45 M [29].
As to the Amaryllidaceae family, phytochemical research conducted in the last decades on this family revealed several alkaloids with moderate or potent inhibition of AChE [3,7,30]. In the search of new natural sources of galanthamine and other Amaryllidaceae alkaloids with anti-AChE activity, bulbs and leaves of Hippeastrum papilio collected in the South of Brazil were studied. Galanthamine (4), the already known alkaloids narwedine (50), haemanthamine (51), 11hydroxyvittatine (52), 8-O-demethylmaritidine (53) and vittatine (54) as well as the new alkaloid 11 -hydroxygalanthamine (55) were all isolated and of all of them galanthamine was obtained in significant amounts [31]. Compound 55 was observed to elicit AChE inhibition as other galanthamine-type alkaloids do, with an IC 50 = 14.5 M. Furthermore, because habranthine, epimer of 55, was observed to have an anti-AChE activity similar to that of galanthamine, it was concluded that configuration at C11 is unfavorable for the interaction with AChE [3,31]. Other potent AChEi, such as N-allylnorgalanthamine (56) and N-(14-methylallyl)norgalanthamine (57), were isolated from Leucojum aestivum, an amaryllidacea used for the industrial extraction of galanthamine [32]. N-alkylated galanthamine derivatives 56 and 57 were isolated together with galanthamine (4), epinorgalanthamine (58), narwedine (50) and lycorine (59), from the mother liquors obtained after the industrial production of galanthamine. Alkaloids 56 and 57, with IC 50 values of 0.18 and 0.16 M, respectively, resulted to be ten times more potent AChEi than galanthamine (IC 50 = 1.82 M).
Although benzylphenethylamine alkaloids were considered to belong exclusively to the Amaryllidaceae, some of them have been found to belong to other families [35]. A new example of this exception was found through the chemical investigation of Hosta plantaginea (Liliaceae) [ After the isolation of the potent AChEi huperzine A (5) from Huperzia serrata (Lycopodiaceae), several plants belonging to the genus Lycopodium have been investigated in an attempt to find alkaloids with unusual skeletons that could have AChE inhibitory activity [7,8,37]. Five new Lycopodium alkaloids, 11 -hydroxyfawcettidine (74), 2 ,11 -dihydroxyfawcettidine (75), 8 ,11 -dihydroxyfawcettidine (76), 2 -hydroxylycothunine (77) and 8hydroxylycothunine (78), with the fawcettimine skeleton were isolated from L. serratum, along with three known alkaloids, lycothunine (79), serratine (80) and serratanidine (81) [38]. AChE inhibitory activity was analyzed for the alkaloid lycoposerramine-H (82) previously isolated from L. serratum [39] and for compounds 74, 75, 78. Alkaloids 75 and 82 were observed to inhibit AChE with an IC 50 = 27.9 and 16.7 M, respectively, while 74 and 78 were observed to show no anti-AChE activity. In another study, three new alkaloids (83 -85) were isolated from L. carinatum, a species collected in Malasya [40]. Carinatumins A (83) and B (84) were observed to inhibit AChE from bovine erythrocytes with an IC 50 = 4.6 and 7.0 M, respectively, whereas carinatumin C (85) was observed to show no inhibition (IC 50 > 100 M). Alkaloids 83 and 84 were observed to exhibit an AChE inhibitory activity similar to that of huperzine A and huperzine B (IC 50 = 0.8 and 8.0 M). Alkaloids from L. casuarinoides were also isolated and three new compounds, lycoparins A-C (86 -88), were characterized, of which lycoparin C (88) was found to show a moderate AChE inhibitory activity (from bovine erythrocytes) with an IC 50 = 25 M [41]. Lycoparin A (86) and lycoparin B (87), both having a carboxylic acid at C-15 and one or two N-methyl groups, were found to show no inhibitory activity.
As to Sarcococca and Buxus species (Buxaceae), they are known to produce steroidal alkaloids, some of which were observed to evidence strong AChE inhibition [7,42,43]. New steroidal alkaloid AChEi from S. saligna and S. hookeriana were recently found. In the case of S. saligna, the study -which was a continuation of previous research [44,45]-of the bioactive steroidal alkaloids of this species allowed the isolation of five new compounds (89)(90)(91)(92)(93) and two already known bases (94 and 95) [46]. The new alkaloids 5,14-dehydro-N a -demethylsaracodine (89), 14dehydro-N a -demethylsaracodine (90) [43,49]. Three new triterpenoidal alkaloids, namely 17-oxo-3-benzoylbuxadine (106), buxhyrcamine (107) and 31-demethylcyclobuxoviridine (108) along with sixteen known compounds, all tested as AChEi, were isolated and characterized in a recent study on B. hyrcana collected from Iran [50]. Weak AChE inhibitory activity was observed for N b -dimethylcyclobuxoviricine The crude methanolic extract of B. natalensis, a plant used to improve memory in the elderly by traditional healers in South Africa, was found to elicit AChE inhibition (IC 50  However, all of them were weaker AChEi than galanthamine (IC 50 = 1.9 M). Compounds 127, 128, 129 and 130 were found to be stronger inhibitors on plasma BChE than galanthamine, the positive control [53].
In addition, the following steroidal alkaloids: conessine (131), isoconessimine (132), conessimin (133), conarrhimin (134) and conimin (135) were isolated in a bioassay-guided fractionation from the seeds of Holarrhena antidysenterica (Apocynaceae), a common Tibetan drug [54]. Compounds 131, 133, 134 and 135 were identified as active constituents against AChE. Conessimin (133) was found to be the strongest AChE inhibitor with an IC 50 = 4 M whereas conessine (131), conarrhimin (134) and conimin (135) were found to be moderate AChE inhibitors (IC 50 = 21 -28 M). These findings indicate that the elimination of the N-methyl group of pyrrolidine moiety induces a significant increase of activity while the cleavage of either one or two N-methyl groups at C-3 position reduces the inhibitory potency. Compound 133 was selected for a kinetic study through which it was demonstrated that its AChE inhibitory activity is both reversible and non-competitive. Molecular docking simulations of these compounds with AChE helped to understand their interactions with AChE and were consistent with the experimental results obtained [54].

NON-ALKALOIDAL COMPOUNDS WITH AChE INHIBITORY ACTIVITY
In spite of the fact that the majority of the most potent inhibitors known to date are alkaloids, several non-alkaloidal From the methanolic extract of Haloxylon recurvum (Chenopodiaceae), a plant used in Pakistan for the treatment of several neuronal disorders, four new C-24 alkylated sterols 149 -152 and five known sterols 153 -157 were isolated [59]. Compounds 149 -157 were analyzed as AChEi and were found to inhibit AChE in a concentrationdependent manner acting as non-competitive inhibitors. Haloxysterol B (150) and haloxysterol C (151), whose IC 50 values were 0.89 and 1.0 M, respectively, were found to be the most active AChE inhibitors. Their inhibitory activity was observed to be similar to that of galanthamine (IC 50 [60]. This bioassay-guided study is the first report of 4-phenylcoumarins as AChEi. In the past, some examples of xanthones with moderate AChE inhibitory activity were reported [7]. Further recent research introduced two new xanthones, 162 and 163, to this group of AChEi also with moderate inhibitory activity. Macluraxanthone (162) which was obtained from the root of Maclura pomifera (Moraceae) was found to elicit noncompetitive AChE inhibition (IC 50 = 8.47 M) [61]. Furthermore, docking studies yielded results supporting in vitro results. Triptexanthoside C (163) which was isolated from the methanolic extract of Gentianella amarella ssp. acuta (Gentianaceae) was observed to elicit AChE inhibition with an IC 50 = 13.8 M [62].
The methanol extract from roots of Morus lhou (Moraceae), a polyphenol-rich plant, was found to yield nine flavonoids (172 -180) of which eight showed AChE inhibition [65]. A new flavone, 5'-geranyl-4'-methoxy-5,7,2'-trihydroxyflavone (172), was identified as the most potent inhibitor (IC 50  Isoorientin (184) and isovitexin (185) were identified as the compounds responsible for the AChE inhibition observed in the extracts from flowers and rhizomes of Iris pseudopumila (Iridaceae) from Italy [67] On the other hand, a pterocarpan with moderate AChE inhibition was isolated from the polar extract of Zygophyllum eurypterum (Zygophyllaceae) collected in Pakistan. Atricarpan D [(-)-2,9-dimethoxy-4-(5-oxohexyl)pterocarpan] (186) was observed to inhibit AChE with an IC 50 = 20.5 M [68]. Interestingly, three other pterocarpans with similar structure were obtained along with atricarpan D but they were found to be inactive against AChE. Nevertheless, the four pterocarpans were all found to be BChE inhibitors. A study conducted on AChE and BChE inhibitory activity of coumarins and naphtoquinones obtained from Mansonia gagei (Sterculiaceae) proposed a novel class of cholinesterase inhibitor, mansonones or 1,2-naphtoquinones [69]. The level of cholinesterase inhibition observed in this study seemed to correlate to the presence of a fused pyran ring and a substituent at C-6 being present in the molecule. Mansonone E (187) was observed to be the most active AChE (IC 50 = 23.5 M) and BChE inhibitor. In several studies published during the period covered in the present review various phenolic compounds with different structural characteristics were reported as AChEi. Some of them are structurally simple such as gallic acid (188, IC 50 = 5.85 M) and ellagic acid (189, IC 50 = 45.63 M) [70]. Hopeahainol A (190), which was identified as a new compound isolated from Hopea hainensis, was observed to elicit a notable AChE inhibition (IC 50 = 4.33 M) with respect to huperzine A (IC 50 = 1.6 M), as a reversible mixed-type inhibitor [71].
The bioassay-guided fractionation of the extract from Terminalia chebula (Combretaceae) fruits allowed the isolation of 1,2,3,4,6-penta-O-galloyl--D-glucose (191) which demonstrated to be a significant AChE inhibitor (IC 50 = 29.9 M) [72]. This gallotanin which has been also isolated from other different sources and which is known by its diverse biological activities, was observed to exert good BChE inhibition and potent antioxidant activity (FRAP assay) in this study.
The bioassay-guided extraction of the stem bark of Knema laurina (Myristicaceae) yielded two active fractions (dichloromethane and hexane) which were subjected to chromatographic separation. That latter yielded five alkenyl phenol and salicylic acid derivatives 192 -196, of which 192 and 193 were new compounds [73]. Compounds 192, 195 and 196, all having salicylic acid moiety, were observed to strongly inhibit AChE with an IC 50 = 3.182, 2.172 and 0.573 M, respectively. Compounds 193 and 194, with no carboxyl moiety, were observed to be good AChE inhibitors (IC 50 = 17.224 and 13.114 M, respectively). These findings suggest that the acidic group is key to good AChE inhibition. It was also observed that anti-AChE activity dramatically decreased when the acidic and the phenolic hydroxy group were methylated. Two catechol alkenyls were isolated from the fruits of Semecarpus anacardium (Anacardiaceae), a species used in Ayurvedic medicine for retarding and treatment of memory loss [74]. Compounds 197 and 198 were identified as active components of the dichloromethane extract through a fractionation guided by the detection of AChE inhibition. Microplate assay revealed that these catechol alkenyls are moderate and weak selective AChEi. Compound 197, with a double bond in the aliphatic chain, was identified as a stronger inhibitor (IC 50  On the other hand, four structurally diverse AChEi were isolated from the polar extract of Nelumbo nucifera (Nelumbonaceae) stamens [75]. Cycloartenol (199), phydroxybenzoic acid (200), vanilloloside (201) and nuciferoside (202) were found to elicit good and noncompetitive inhibition against AChE with an IC 50 = 11.89, 20.07, 4.55 and 3.2 M, respectively. In the same study, compounds 199, 200 and 202 were observed to exert moderate BChE inhibition and compounds 199 -202 were found to show no inhibition against BACE1. Table 1 summarizes the studies published from 2006 to 2012 on plant extracts, fractions and essential oils that have been found to be good AChE inhibitors (IC 50 < 500 g/mL). Those plants included in other recent reviews were omitted [76,77]. Extracts and fractions under further phytochemical studies that led to the discovery of AChE inhibitors were also omitted. Whenever possible, reference is made to the      part of the plant used in each study reported. AChE inhibitory activity is reported in the same way as it was reported by authors and IC 50 values were chosen instead of inhibition percentages when both were available.