Inhibition of Aβ-42 production

The load of amyloid plaques (Aβ-42) in the neuronal cells will be reduced by slowing the process.

Ferulic acid (1), existing in Ferula communis [23], inhibits Aβ aggregation with an IC₅₀ value of 9.30 µM [43] (Table 1). Curcumin (2), isolated from the rhizome of Curcuma longa, exhibits an IC₅₀ value of 0.20 µM [44] (Table 1). Rosmarinic acid (3), derived from Lamiaceae, inhibits Aβ aggregation with an IC₅₀ value of 20.3 µM [45] (Table 1). These results suggest that one of the structural requirements to express inhibitory activity on these compounds is the position of the phenolic hydroxyls, which should be in a para (p) position relative to the aliphatic side (Figure 2).

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Figure 2. Phenolic compounds that inhibit the formation of Aβ-42 plaques (1–3)

Resveratrol (4), existing in the skin of grapes, blueberries, raspberries, mulberries, and peanuts, promotes intracellular degradation of Aβ, via a mechanism that involves the proteasome, with an IC₅₀ value of around 30.0 µM [46] (Table 1). 3,4-Di-O-caffeoylquinic acid (5) is present in Andrographis paniculata, which exists in peninsular India, Sri Lanka, as well as in different regions of Southeast Asia, China, America, the West Indies, and Christmas Island [47]. It presents inhibitory effects on Aβ-42 aggregation, exhibiting an IC₅₀ value of 4.7 µM [48]. 3,5-Di-O-caffeoylquinic acid (6), isolated from Chrysanthemum spp. and Arctium spp. [49], 4,5-di-O-caffeoylquinic acid (7), isolated from Ipomoea batatas L. [50], 1,4,5-tri-O-caffeoylquinic acid (8), existing in Arnica montana and Arnica chamissonis [51], 3,4,5-tri-O-caffeoylquinic acid (9), isolated from Brasilian propolis [52] and 3,4,5-tri-O-caffeoylquinic acid methyl ester (10), existing in Lonicera japonica Thunb [53] also exhibit inhibitory activity on Aβ-42 aggregation with IC₅₀ values of 16.4, 0.1, 2.2, 0.3 and 3.0 µM, respectively [48] (Table 1). On the other hand, compounds like caffeic acid (11) existing in Eucalyptus globulus [54] and chlorogenic acid (12) (Figure 3), found in the bamboo Phyllostachys edulis [55], were slightly active with IC₅₀ values of 32.8 and 92.9 µM, respectively [48] (Table 1). From Orobanche minor were isolated acteoside (13), cistanoside D (14), methylacteoside (15), oraposide (16), 3'''-O-methylcrenatoside (17), methyloraposide (18), isoacteoside (19), isocrenatoside (20) and hydroxytyrosol (21) (Figure 3), which presented Aβ aggregation inhibitory activity with IC₅₀ values of 11.3, > 100, > 100, 8.2, 28.4, > 100, 33.5, 27.4 and 92.0 µM, respectively [56] (Table 1).

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Figure 3. Phenolic compounds that inhibit the formation of Aβ-42 plaques (4–21)

These results show the importance of a caffeoyl group for the inhibitory activity on Aβ-42 aggregation. Indeed, by auto-oxidation, the catechol unit is susceptible to turn into an O-quinone, which might form a covalent bond with some residues of Aβ-42. Such modification may destabilize the β-sheet structure, turning it into amyloidogenic polypeptides. Thus, compounds with two or three catechol units like 4,5-di-O-caffeoylquinic acid (7), 3,4,5-tri-O-caffeoylquinic acid (9) or 3,4,5-tri-O-caffeoylquinic acid methyl ester (10) inhibit β-sheet formation of Aβ-42 while caffeic acid (11) and chlorogenic acid (12), with only one catechol unit, are slightly active [48] and methylacteoside (15) and methyloraposide (18) with no hydroxyl groups on the benzene rings are inactive. On the other hand, the presence of more than one caffeoyl group on the same side of the cyclohexane molecule decreases the inhibitory activity on Aβ-42 aggregation due to steric reasons.

Being BACE1, the enzyme involved in the rate-limiting step in the production of Aβ-42 plaques, the inhibition of this protease will reduce the load of the Aβ-42 plaques in the neuronal cells by slowing the process [57].

Syringaldehyde (22), benzoic acid (23), phthalic acid (24), urolithin B (25) and (+)-usnic acid (26) (Figure 4), isolated from the lichen Xanthoparmelia somloensisthe (Gyel.) Hale collected around Mongolian Shilajit, exhibit IC₅₀ values for the inhibition of BACE1 of 34.9, 309.3, 133.4, 35.6 and 43.1 µM [58] (Table 1). Benzoic acid (23) and phthalic acid (24) with no hydroxyl group on the benzene rings are very weak inhibitors (IC₅₀ > 100 µM). These results suggest that the presence of hydroxyl groups on the benzene ring is essential for the inhibition of BACE1.

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Figure 4. Phenolic compounds that inhibit the formation of Aβ-42 plaques (22–26)

From the Chinese herbal medicine Fraxinus rhynchophylla Hance, esculetin (27) was extracted. The inhibition of the activities of BACE1 presents IC₅₀ values of 7.67 µM [59] (Table 1). These results suggest that the presence of two hydroxyl groups at the ortho position of a benzene ring as well as being part of a very planar molecule increases the activity for the inhibition of BACE1.

3,4-Di-O-caffeoylquinic acid (5), isolated from Andrographis paniculata which exists in peninsular India, Sri Lanka, as well as, in different regions of Southeast Asia, China, America, the West Indies, and Christmas Island, exhibits an IC₅₀ value of 3.30 µM [47] (Table 1) as BACE1 inhibitory activity.

Bergenin (28) isolated from the bark of Bergenia ligulate, Japan [60], 11-O-p-hydroxybenzoylbergenin (29), and 11-O-protocatechoylbergenin (30) (Figure 5), isolated from Bergenia ciliata (Haw) Sternb., Asia [61], present inhibitory activity of BACE1 with IC₅₀ values of > 400, 23.8 and 0.6 µM [60] (Table 1), suggesting again that the presence of two hydroxyl groups at the ortho position of a benzene ring as 11-O-protocatechoylbergenin (30), as well as being part of a very planar group of a molecule like the benzoyl group, increases the activity for the inhibition of BACE1.

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Figure 5. Phenolic compounds that inhibit the formation of Aβ-42 plaques (27–30)

1,2,3-Trigalloyl glucopyranoside (31) isolated from Euphorbia prostrata, acetonyl geraniin (32) from Euphoria longana Lam., helioscopinin A (33) and helioscopinin B (34) from Euphorbia helioscopia L., furosin (35) from Erodium moschatum, rugosin E (36), from Euphorbia supina, euphorscopin (37) from Euphorbia helioscopia L. and jolkinin (38) (Figure 6) from Euphorbia jolkinii existing in Shangri-La, the Southwest China [62], exhibited strong inhibition of BACE1 with IC₅₀ values of 9.43, 0.71, 0.99, 0.41, 17.77, 0.06, 2.50 and 54.93 µM [63] (Table 1). Rugosin E (36) was the most potent (IC₅₀ = 0.06 µM) [63], which is the only compound with two 4,4',5,5',6,6'-hexahydroxydiphenoyl (HHDP) groups linked to the 4,6-positions of the glucopyranose core. 1-Galloyl glucopyranose (39), 1,6-digalloyl glucopyranose (40), 2,6-digalloyl glucopyranose (41) and 1,2,3,4,6-pentagalloyl glucopyranose (42), from Euphorbia helioscopia L. collected in Fukuoka, Japan [63]; 1,2,6-trigalloyl glucopyranose (43) from Euphorbia supina Rafin collected in Fukuoka, Japan [64]; 2-galloyl galactose (44), corilagin (45), elaeocarpusin (46), geraniin (47) and helioscopin B (48) from Euphorbia helioscopia L. collected in Fukuoka, Japan [62, 64]; 1,2,6-trigalloyl allose (49), 1,3,6-trigalloyl allose (50) and 1,2,3,6-tetragalloyl allose (51) from Euphorbia fischeriana, Japan [65] (Figure 7); Bixanin (52) existing in Macaranga sinensis (Baill.) Müll.Arg., China [66]; 3-O-galloylshikimic acid (53) existing in Arbutus unedo L. Mediterranean fringe [67]; 5-pyrogallo-O-quinic acid (54) existing in Camellia sinensis L., Taiwan, China [68]; α-viniferin (55) existing in Shorea ovalis Blume, Indonesia [69] and schizandrin (56) (Figure 8) existing in Schisandra chinensis Turcz. (Baill.), Korea, [70] are very weak inhibitors of BACE1 (IC₅₀ > 350 µM) [63] (Table 1). These results suggest the importance of having HHDP groups linked to the 1,6- or 4,6-positions of the glucopyranose core. Having those linkages of the HHDP groups, which are very planar, there is no steric hindrance of the other groups of the molecule to the hydrogen bonds between the hydroxyl groups of the benzene rings and the active centres of BACE1. Comparing the activities of acetonyl geraniin (32) and geraniin (47) it can be concluded that the inhibitory activity of the former (IC₅₀ = 0.71 µM) is due to the acetonyl group and not to the hydroxyl groups linked to benzene ring of the HHDP group.

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Figure 6. Phenolic compounds that inhibit the formation of Aβ-42 plaques (31–38)

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Figure 7. Phenolic compounds that inhibit the formation of Aβ-42 plaques (39–51)

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Figure 8. Phenolic compounds that inhibit the formation of Aβ-42 plaques (52–56)

Inhibition of NFTs production

Rosmarinic acid (3) (Figure 2), isolated from Rosmarinus officinalis L., collected in Talca, Chileyet known., inhibits τ-protein in concentrations ranging from 10 µM to 100 µM, in a dose-dependent manner. This compound (3) exhibits an IC₅₀ value of 7.7 µM [71] (Table 1). Rosmarinic acid (3) has two catechol moieties, suggesting that the presence of a catechol moiety might be important for the activity as an inhibitor of τ-protein. Indeed, τ-protein is a natural unstructured protein, and its exact structure is not yet known. However, the structure of the fibril-forming hexapeptide motif of τ-protein—³⁰⁶VQIVYK³¹¹—has been resolved by X-ray crystallography [71]. Molecular docking analysis concluded that rosmarinic acid (3) docking was done inside the cylindrical cavity, which is formed by paired ³⁰⁶VQIVYK³¹¹ β-sheets, in which compound (3) enters with no steric hindrance. As rosmarinic acid (3) has an elongated shape, aromatic and polar groups, and a negatively charged group, it might inhibit τ-aggregation by establishing chemical interactions with the fibril-forming hexapeptide VQIVYK. Thus, the aromatic rings of rosmarinic acid (3) stay packed against apolar side chains of Val309 and form several hydrogen bonds with glutamine and lysine side chain groups on both sides of the steric zipper. The most important interaction is the salt link between the carboxylate group of rosmarinic acid and two Lys311 ammonium ions from ³⁰⁶VQIVYK³¹¹ fibers [71].

As described above, another promising strategy to combat AD is the inhibition of protein kinases [10–23]. The most important protein kinase involved in τ-protein phosphorylation is GSK3β.

The compound gastrodin (57) (Figure 9), isolated from the orchid Gastrodia elata and from the rhizome of Galeola faberi [72], suppresses the activity of GSK3β in the brain of mice treated with a water solution of gastrodin (57) and administered it orally in a dosing volume of 10 mL/kg at doses of 150 mg/kg for nine consecutive months [73].

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Figure 9. Phenolic compounds that inhibit the NFTs production (57 & 58)

Salidroside (58) (Figure 9), a phenol glycoside compound found in plants of the Rhodiola genus, also decreases GSK3β activity after mice treatment by administering water solutions of the compound [74]. These results show the need for a phenol or benzyl group for the inhibition of GSK3β.

Inhibition of pro-inflammatory factors

From the flowering aerial parts of Phagnalon saxatile (L.) Cass. which were collected, in Cefalù, Capo Playa (Sicily), Southern Italy, caffeic acid (11), chlorogenic acid (12), methylchlorogenic acid (59) (Figure 10), were isolated, exhibiting inhibitory effect against NO production with IC₅₀ values of 40.0, 49.6 and 34.9 µM [75], respectively (Table 1).

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Figure 10. Phenolic compounds that inhibit the pro-inflammatory factors (59–63)

(E)-1,7-diphenylhept-4-en-3-one (60), 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylheptan-3-one (61), (4Z,6E)-5-hydroxy-1,7-diphenylhepta-4,6-dien-3-one (62) and p-hydroxycinnamic acid (63) (Figure 10) isolated from Alpinia officinarum Hance rhizomes at Saudi Arabia, reduce the expression of ROS by showing activity on single electron transfer (SET) mechanisms suitable for both hydrophobic and hydrophilic compounds (SET-OH/H) or on hydrogen atom transfer (HAT) mechanisms as scavenging peroxyl radicles (HAT-OOR). The inhibition to produce free radicals by the SET-OH/H mechanism presents IC₅₀ values of 149.8, 14.81, > 250 and 29.94 µM, respectively (Table 1). On the other hand, the antioxidant potential following the HAT-OOH mechanism of the compounds estimated as Trolox equivalents presented 0.28, 1.74, 0.33 and 3.20 mM TE/mM compound (Table 1). In both mechanisms 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylheptan-3-one (61) and p-hydroxycinnamic acid (63) showed the most powerful antioxidant effects [76], suggesting the need of an hydroxyl group on the para position for the inhibition of ROS production.

Inhibition of acetylcholinesterase and butirylcholinesterase

From Carthamus tinctorius L. flowers from Jeddah, Saudi Arabia, was isolated p-hydroxybenzoic acid (64), which displays inhibition of AChE with IC₅₀ values of 150.6 µM [77]. 2-Hydroxy-4-methoxybenzaldehyde (MBALD) (65) from roots of Hemidesmus indicus H. indicus and 4-hydroxy-3-methoxybenzaldehyde (vanillin) (66) (Figure 11) from pod extracts of Vanilla planifolia exhibit inhibitory activity of AChE with IC₅₀ values of 47.0 and 37.0 µM [78]. These results suggest that the aldehyde and the hydroxy group on MBALD (65) might be linked by intramolecular hydrogen bonding, not allowing any other hydrogen bonding with other molecules. When comparing the inhibition activities of p-hydroxybenzoic acid (64) and vanillin (66), it can be concluded that the methoxy group increases the inhibition activity by hydrophobic interactions with the enzyme.

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Figure 11. Phenolic compounds that inhibit AChE and BuChE (64–66)

Methyl syringate (67) from Taraxacum formosanum, Taiwan, China [79]; p-hydroxyphenylpyruvic acid (68) from Agave angustifolia Haw, Mexico [80]; salicylic acid (69) from willow (Salix acmophylla Boiss.), found worldwide [81]; p-hydroxyphenylacetic acid (70) (Figure 12) found in olive oil [82]; p-hydroxybenzoic acid (64) found in Vitex agnus-castus, Mediterranean region [83]; homovanillic acid (71) found in Aloe greatheadii, Africa [84]; nordihydroguaiaretic acid (72), found in Larrea tridentata (Creosote bush), an abundant plant of Mexican and US-American deserts [85]; protocatechuic acid (73), found in Boswellia dalzielii, Africa [86]; m-hydroxybenzoic acid (74) which exists at Senna alata leaves, Brasil [87]; vanillic acid (75) found in Camellia sinensis (L.) Kuntze (green tea), China [88]; syringic acid (76) found in Ardisia elliptica Thunb, China [89]; homogentisic acid (77) isolated from the Indonesian fungus, Penicillium citrinum [90]; gentisic acid (78) (Figure 12) extracted from the wine of the berries of Vitis vinifera L. cv. Vidal, China [91]; gallic acid (79) existing in Quercus robur L., Poland [92]; ethyl p-hydroxybenzoate (80) existing in Tetragonia tetragonoides (Pall.) Kuntze, New Zealand [93]; and ethyl vanillate (81) existing in Sisymbrium officinale (L.) Scop., collected in the sub-Mediterranean region of South Croatia near Split [94] exhibit inhibition of AChE with IC₅₀ values of 5.50, 5.84, 6.07, 6.24, 6.36, 6.45, 6.47, 6.50, 6.68, 6.79, 6.96, 7.16, 8.02, 9.32, 31.38 and 34.19 µmol/µmol AChE [95].

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Figure 12. Phenolic compounds that inhibit AChE and BuChE (67–81)

These results indicate that when O-hydroxybenzoic acid (salicylic acid) (69) is compared with the other isomers, p-hydroxybenzoic acid (64) and m-hydroxybenzoic acid (74), salicylic acid (69) shows the highest inhibition of AChE (IC₅₀ = 6.07 µmol/µmol AChE) and p-hydroxybenzoic acid (64) shows a slightly lower inhibition (IC₅₀ = 6.36 µmol/µmol AChE) [95].

It was noticed by Docking Simulation that both isomers were characterized by hydrogen bonds and hydrophobic interactions within the anionic binding site and the peripheral anionic site (PAS). m-Hydroxybenzoic acid (74) is less active than the other two isomers (IC₅₀ = 6.68 µmol/µmol AChE). By Docking Simulation, it was realized that in this compound, the interactions with AChE were mainly through hydrogen bonds. These results suggest that the hydrophobic phenol-AChE interactions may be obstructed by the specific location of the hydroxyl groups [95].

Protocatechuic acid (3,4-dihydroxybenzoic acid) (73), with an additional hydroxyl group, presents an IC₅₀ value of 6.50 µmol/µmol AChE. By Docking Simulation, it was realized that the interaction was almost identical to binding of the m-hydroxybenzoic acid (74) to AChE, with another hydroxyl group forming an additional hydrogen bond. However, as hydrogen bonding is a directed link, one of them is weaker than the other one. The isomer of 3,4-dihydroxybenzoic acid (73), 2,5-dihydroxybenzoic acid (gentisic acid) (78), exhibits a significantly lower activity (IC₅₀ = 8.02 µmol/µmol AChE). Indeed, the interactions of gentisic acid (78) with AChE are very similar in nature to m-hydroxybenzoic acid (74). However, due to the presence of another hydroxyl group linked to carbon 2 of gentisic acid (78), the binding within the anionic binding site is decreased.

The presence of three hydroxyl groups in the aromatic ring of gallic acid (79) resulted in a weaker activity of the inhibitor (IC₅₀ = 9.32 µmol/µmol AChE) compared to protocatechuic acid (73), with a similar pattern of interaction with the enzyme. In the case of the trihydroxy-derivative, there were interactions that weakened the binding of the complex (lower binding constant) related to the stress of the phenolic acid molecule.

The influence of the presence of methyl groups on the interactions with AChE was also analysed. Vanillic acid (4-hydroxy-3-methoxybenzoic) (75) showed weaker interactions of the hydrogen bond type than protocatechuic acid (73), due to the blocking of the hydroxyl group with a methyl substituent.

In the presence of two methyl groups, as in syringic acid (76) (Figure 12), hydrogen interactions prevailed in the complex when compared to hydrophobic interactions. However, in this case, the oxygen of the methoxy groups may also take part in a series of hydrophobic phenol-AChE interactions. As a result, the activity of syringic acid as an AChE inhibitor increases (IC₅₀ = 6.96 µmol/µmol AChE) when compared to gallic acid (79) (Figure 12). However, the inhibitory activity of AChE of methyl derivative of syringic acid (76), methyl syringate (67), was very high (IC₅₀ = 5.50 µmol/µmol AChE) suggesting that after the interaction of this compound with AChE the methyl group of the ester blocks the active centre of the enzyme completely and the hydrolysis of the ACh is impracticable [95].

When the interactions with AChE of ethyl derivatives like ethyl-p-hydroxybenzoate (80) and ethyl vanillate (81) (Figure 12) are analysed, they are characterized by a low binding constant. The interactions presented by p-hydroxybenzoic acid (64) are like the ones presented by the ethyl derivative ethyl-p-hydroxybenzoate (80). However, the higher stresses due to bond rotation, caused by the presence of the ethyl substituent and the loss of some interactions by hydrogen bonding, explain the difference in activity presented by these phenolic compounds. Vanillic acid (75) and its ethyl derivative ethyl vanillate (81) show similar interactions when analysed by calorimetry (ITC) and Docking Simulation, however, the binding constant was lower for ethyl vanillate (81) explaining the weaker inhibition of AChE (IC₅₀ = 34.19 µmol/µmol AChE) presented by this compound suggesting the existence of some fewer and weaker hydrogen bonds between ethyl vanillate (81) and AChE. The derivatives of homohydroxyphenylacetic acids have also been analysed by calorimetry (ITC) and Docking Simulation. The activity of p-hydroxyphenylacetic acid (70) (IC₅₀ = 6.24 µmol/µmol AChE) was like the one presented by p-hydroxybenzoic acid (64), but the type of interactions was different. The acetic derivative showed more hydrophobic interactions at the anionic binding site, and there were practically no repulsive forces. Similarly, homogentisic acid (77), when compared to gentisic acid (78), shows a higher activity as AChE inhibitor (IC₅₀ = 7.16 µmol/µmol AChE), homovanillic acid (71) exhibits a higher activity than vanillic acid (75), p-hydroxyphenylpyruvic acid (68) presents a higher activity than p-hydroxyphenylacetic acid (70), which might be due to the greater number of significant interactions with AChE.

Nordihydroguaiaretic acid (72), a benzodiol dimer, due to its complex structure and the presence of two aromatic groups, may interact with AChE through many amino acids. However, due to the repulsive forces and stresses resulting from the rotation around the bonds, the interaction between the compound and AChE becomes weaker [95].

From Vanda roxburghii, found all over Bangladesh, syringaldehyde (22), dihydroconiferyl dihydro-p-coumarate (82) and gigantol (83) (Figure 13) were isolated. All of them were very weak or inactive as inhibitors of AChE with IC₅₀ values of > 1,000, 357.9 and > 1,000 µM, respectively [96]. Syringaldehyde (22) is inactive. Indeed, as already mentioned, phenolic compounds with a structure like syringic acid (76) may have an alkyl group linked to the side chain, like methyl syringate (67), to block the active centre of the enzyme completely, and the hydrolysis of the AChE may be impossible [95].

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Figure 13. Phenolic compounds that inhibit AChE and BuChE (82–89)

From the Chinese herbal medicine Fraxinus rhynchophylla Hance, esculetin (27) was extracted. The inhibition of the activities of AChE and BuChE presents IC₅₀ values of 6.13 and 8.66, respectively [59]. These results suggest that the presence of the two hydroxyl groups at the ortho position, as well as being an aglycone with no sugar moieties, makes the molecule very active.

Olivetol (84) (Figure 13), found in several Parmeliaceae lichens like Hypogymnia physodes, Evernia prunastri and Parmelia sulcata, in Serbia [97], presents activity as an inhibitor of AChE and BuChE with IC₅₀ values of 0.005 and 0.006 µM, respectively [98]. As already mentioned above when two hydroxyl groups are on meta (m) substitution the interactions with AChE are mainly through hydrogen bonds, which on this compound are very strong as the two hydroxyl groups are well separated and the side chain, being aliphatic, where carbons are linked only by a sigma bond (σ), allows the entrance of the compound in the deep gorge of AChE.

Comparing phenolic compounds like 3,5-dihydroxybenzoic acid (85), present in Artemisia vulgaris L. and Artemisia alba Turra [99], vanillic acid (75), found in the roots of Angelica sinensis [100], hydroquinone (86) (Figure 13), found in Agaricus hondensis mushrooms [101] and chlorogenic acid (12), found in the bamboo Phyllostachys edulis [55], present weak inhibitory activity of AChE with IC₅₀ values of > 1,000, 923, 260 and 410 µM, respectively [102], suggesting that the inhibitory activity of AChE depends on the number of hydroxyl groups and their distribution on the ring as well as of the aliphatic side chain.

In Thymus vulgaris L. of central Dalmatia, Croatia, several phenolic compounds as thymohydroquinone (87), carvacrol (88), and thymol (89) (Figure 13) which show some or no inhibitory activity of AChE. The IC₅₀ values of them are 240.6, 419.4 and > 1,000 µM [103, 104] (Table 1). These results show again that the number of hydroxyl groups is important for the inhibition activity of AChE and their para position on the ring, as well as the steric effect of the isopropyl group.

3,3'-Di-O-methylellagic acid (90), 3,3',4'-tri-O-methylellagic acid-4-O-β-D-xylopyranoside (91), 3,3',4'-tri-O-methylellagic acid-4-O-β-D-glucopyranoside (92), and 3,3'-di-O-methylellagic acid-4-O-β-D-glucopyranoside (93) (Figure 14) exist in Terminalia macrocarpa (Combretaceae) plant collected in Ngaoundere, Cameroon. The inhibitory activity of AChE exhibits IC₅₀ values of 141.6, 128.6, 118.0, and 129.1 µM. Against BuChE, the IC₅₀ values are 152.8, > 200, 148.5, and 182.4 µM [105] (Table 1). Therefore, the presence of a sugar moiety doesn’t change the inhibition of AChE for these types of compounds.

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Figure 14. Phenolic compounds that inhibit AChE and BuChE (90–98)

From fresh aerial parts of Anisacanthus virgularis (Salisb.) Nees gathered from Giza, Egypt, were extracted five furofuranoid-type lignans: anisacanthin (94), pinoresinol (95), epipinoresinol (96), phillyrin (97) and pinoresinol-4-O-β-D-glucoside (98) (Figure 14). These compounds show AChE inhibition with IC₅₀ values of 0.09, 0.29, 0.24, 0.28, and 0.64 µM [106] (Table 1). All these molecules are active as inhibitors of AChE suggesting that the activity is due again to the aliphatic side chain, exhibiting anisacanthin (94) a higher activity as it is longer and more flexible the allowing the entrance of the compound in the wide and deep gorge of AChE.

Tannic acid (penta‐m‐digalloyl‐glucose) (99) (Figure 15) is a natural polyphenolic compound found in some galls of Quercus species, Rhus species, beverages (red wine, tea, coffee), fruits, and vegetables. It presents inhibitory activity of AChE and BuChE with IC₅₀ values of 0.12 and 0.09, respectively [107] (Table 1), suggesting that the several gallic acid moieties might interact with the enzyme AChE.

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Figure 15. Phenolic compounds that inhibit AChE and BuChE (99)

Curcumin (2) isolated from Curcuma longa, Ásia, presents inhibition of AChE with an IC₅₀ value of 58.08 µM [108] (Table 1). Suggesting that, as the molecule is very planar, it has difficulty in entering the gorge of the enzyme.

(E)-1,7-diphenylhept-4-en-3-one (60), 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylheptan-3-one (61) and (4Z,6E)-5-hydroxy-1,7-diphenylhepta-4,6-dien-3-one (62) isolated from the rhizomes Alpinia officinarum Hance rhizomes, Saudi Arabia, exhibit inhibition of AChE with IC₅₀ values of 277.8, 190.7, and 194.5 µM, respectively [76]. 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylheptan-3-one (61) also presented activity against BuChE with IC₅₀ values of 252.0 µM. All three other compounds were inactive against BuChE [76]. These results suggest that the presence of one hydroxyl group in a benzene ring and a flexible side chain increases the binding capacity of phenolic compounds to AChE and BuChE.

From Salvia L. species (Lamiaceae) existing at Asia [109], rosmarinic acid (3), salvianolic acid B (100), salvianolic acid A (101), carnosic acid (102), carnosol (103) and danshensu salt (104) (Figure 16) were extracted. The inhibition of AChE exhibits IC₅₀ values of > 150, > 150, > 150, 95.8, 33.7, and >>150 (not active) µM [109] (Table 1), respectively. These compounds for the inhibition of BuChE exhibit IC₅₀ values of 33.5, 14.6, 97.7, 12.4, 11.9, and 109.8 µM [109] (Table 1), respectively. It is interesting to notice that only carnosic acid (102) and carnosol (103) present two hydroxyl groups in an ortho-position. As already mentioned, by Docking Simulation was realized that the interaction between AChE and phenolic compounds with ortho hydroxyl groups is characterized by strong hydrogen bonds and hydrophobic interactions within the anionic binding site and PAS, explaining the increase of activity of these compounds for the inhibition of AChE.

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Figure 16. Phenolic compounds that inhibit AChE and BuChE (100–106)

Eugenol (105), existing in Cinnamomum zeylanicum Blume, India [110], and butylated hydroxyl toluene (106) (Figure 16), existing in the green algae Botryococcus braunii, Taiwan, China [111], exhibit activity as inhibitors of AChE with IC₅₀ values of 8.69 and 9.16 µM, respectively. They also exhibit inhibition of BuChE with IC₅₀ values of 8.86 and 9.00 µM, respectively [112]. These results suggest that the bulky t-butyl groups at the ortho-position relative to the hydroxyl group don’t prevent the hydrogen bonding of the hydroxyl group on butylated hydroxyl toluene (106).

From the dried roots of Harpagophytum procumbens (Pedaliaceae), Africa, were isolated martinoside (107), cinnamic acid (108), acteoside (13), isoacteoside (109), decaffeoylverbascoside (110) (Figure 17), and p-hydroxycinnamic acid (63) (Figure 10). They exhibit AChE inhibitory activity with IC₅₀ values of > 100, 78.5, 19.9, 21.9, 16.1, and 68.5 µM [113], respectively. The inhibition of BuChE presents IC₅₀ values of > 100, > 100, 35.0, 29.7, 46.0, and > 100 µM [113] (Table 1). It is interesting to compare the inhibition of AChE of martinoside (107) (IC₅₀ > 100 µM) with the ones presented by acteoside (13) (IC₅₀ = 19.9 µM), isoacteoside (109) (IC₅₀ = 21.9 µM) and decaffeoylverbascoside (110) (IC₅₀ = 16.1 µM). As already mentioned, the interaction is almost identical to the binding of the hydroxyl group at m-position to AChE with another hydroxyl group at p-position, forming an additional hydrogen bond with Glu199.

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Figure 17. Phenolic compounds that inhibit AChE and BuChE (107–111)

Lavandulifolioside (111) (Figure 17) isolated from Leonurus japonicus L. (Lamiaceae), Korea, presents inhibitory activity of AChE with an IC₅₀ value of 301 µM [114]. The hydrolysate of lavandulifolioside (111), caffeic acid (11) (Figure 3), exhibits AChE inhibitory activity with IC₅₀ value of 179.9 µM [75] suggesting that lavandulifolioside (111) with four hydroxyl groups linked to benzene rings cannot use all of them to link to the enzyme by steric reasons.

From Fallopia dentatoalata (Fr. Schm.) Holub, China, Lapathoside B (112), Vanicoside B (113), Lapathoside A (114), Smilaside J (115), and Smilaside G (116) (Figure 18) were extracted. They display inhibition of AChE with IC₅₀ values of > 100, 32.3, 30.6, 56,0, and > 100 µM [115] (Table 1). The inhibition of BuChE exhibits IC₅₀ values of 10.9, 7.5, 2.7, 10.1, and 17.1 µM [115] (Table 1). These results suggest that the feruloyl group produces on the side groups steric effects hampering the formation of hydrogen bonds on the side substituents.

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Figure 18. Phenolic compounds that inhibit AChE and BuChE (112–119)

4-O-Caffeoyl quinic acid (117) and 4,5-di-O-caffeoyl quinic acid (118) (Figure 18) were isolated from Acanthopanax henryi Harms, China. They display inhibition of AChE with IC₅₀ values of 80.2 and 62.6 µM [116] (Table 1). The latter, having two more hydroxyl groups linked to benzene rings, is more active; however, the increase in activity is not proportional to the increase in hydroxyl groups of the benzene rings.

From Phyllanthus niruri leaves, Malaysia, isocorilagin (119) (Figure 18), this compound presents inhibition of AChE and BuChE with IC₅₀ values of 0.49 and 4.20 μM, respectively [117]. Molecular docking analysis revealed that this compound, by the formation of hydrogen bonds with residues at the entrance of the AChE active site, effectively blocks it. With BuChE, the compound completely docked inside and occupied the active site of the enzyme [117].