Phenolic Profile and Cholinesterase Inhibitory Properties of Three Chilean Altiplano Plants: Clinopodium gilliesii (Benth.) Kuntze [Lamiaceae], Mutisia acuminata Ruiz & Pav. var. hirsuta (Meyen) Cabrera, and Tagetes multiflora (Kunth) [Asteraceae]

This research aimed to identify the phenolic profile and composition of the aerial parts of three native species used in traditional medicine in the Andean Altiplano of northern Chile: Clinopodium gilliesii (Benth.) Kuntze [Lamiaceae] (commonly known as Muña-Muña), Mutisia acuminata Ruiz & Pav. var. hirsuta (Meyen) Cabrera [Asteraceae] (commonly known as Chinchircoma), and Tagetes multiflora (Kunth), [Asteraceae] (commonly known as Gracilis), as well as to evaluate their potential inhibitory effects against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Polyphenolic enriched-extracts (PEEs) of the species were prepared and analyzed and the main components were quantified using HPLC-DAD. In total, 30 phenolic compounds were identified and quantified in all species, including simple phenolics, hydroxycinnamic acids, flavan-3-ols (monomers and polymers), flavanones, and flavonols. In addition, other main phenolics from the extracts were tentatively identified by ESI-MS-MS high-resolution analysis. T. multiflora extract showed the greatest anti-AChE and BChE activity in comparison with C. gilliesii and M. acuminata extracts, being the anti-AChE and BChE activity weak in all extracts in comparison to galantamine control. To comprise to better understand the interactions between cholinesterase enzymes and the main phenolics identified in T. multiflora, molecular docking analysis was conducted.


Introduction
The Andean Altiplano is a highland plateau region (above 3700 m.a.s.l.) located in west-central South America, in the geographical limit between northwest Argentina, south-  (1)(2)(3)(4)(5) indicate the total content (including compounds not identified by standards) expressed as 1 total simple phenolics content is expressed as mg p-hydroxybenzoic acid equivalents/g PEE; 2 total hydroxycinnamic acids content is expressed as mg 3-O-caffeoylquinic acid equivalents/g PEE; 3 total flavan-3-ol monomers and polymers content is expressed as mg (+)-catechin equivalents/g PEE; 4 total flavanones content is expressed as mg naringenin equivalents/g PEE; 5 total flavonols content is expressed as mg quercetin-3-O-glucoside equivalents/g PEE.

Clinopodium gilliesii Kuntze [Lamiaceae]
The HPLC-DAD chromatogram of the PEEs of C. gilliesii is shown in Figure 1a and the total content of compounds is shown in Table 1. Two main compounds were detected as simple phenolic derivatives, vanillic acid and p-hydroxybenzoic acid, compounds 3 and 4. The compounds were identified by matching with commercial standards such as vanillic acid and p-hydroxybenzoic acid (p-HBA), respectively. The total content of simple phenols derivatives was 13.80 ± 0.45 mg of p-HBA equivalents/g PEE. Vanillic acid was the major phenolic with 12.37 mg of p-HBA equivalents/g PEE. Both phenolic acids have been described in species belonging to the Lamiaceae family and with high content in the genus Clinopodium [27].
Compounds 6 and 8 presented the same retention time and UV profile of the commercial standards and were identified as 3-O-caffeoylquinic acid (3CQA) and trans-caffeic acid, respectively. Compound H 1 was tentatively identified as a coumaroylquinic acid derivative due to its strong absorption of band I at 323 nm and the UV spectrum characteristic of coumaroylquinic acids [28]. The total content of hydroxycinnamic acids (HCA) was 4.27 ± 0.35 mg of 3CQA equivalents/g PEE. Cabana et al. (2013) [15] reported the presence of caffeoylquinic acid derivatives, including 3CQA, in agreement with our results.
Flavonoids such as flavan-3-ols, flavanones, and flavonols were the main compounds in the PEE. Compounds 5 and 7 were identified by matching with standards as (+)-catechin (CAT) and B-type procyanidin dimer, respectively. Compounds F 1 , F 5 , F 8 , F 10 , F 12 , F 16, and F 17 were identified as flavan-3-ol derivatives following the UV profile of band II, with λ max at 280 nm and spectra similar to that of catechin [29]. The total content of flavan-3-ol derivatives was 70.00 mg of CAT equivalents/g PEE. This high content of flavan-3-ol derivatives was also reported in other studies with this species, reporting high content of epigallocatechin-3-O-gallate and epicatechin-3-O-gallate [15]. Compounds naringin (19) and naringenin (NAR) (26) were identified in this extract by matching with commercial standards. Compounds F 2 , F 3 , F 4 , F 6 , F 7 , F 9 , F 11 , F 13 , and F 14 were tentatively identified as flavanones. Compounds F 2 , F 3 , F 4, and F 6 presented a λ max at 286-289 with a characteristic shoulder at 330 nm in agreement with the UV spectra reported for eri-odictyol [30] and previous reports with this species [15]. Compounds F 7 , F 9 , F 11 , F 13, and F 14 presented a λ max at 283-285 nm with a characteristic shoulder at 330 nm and were tentatively identified as naringenin derivatives following the UV spectra [30]. The lower retention time suggests that these peaks could present extra glycosyl units. The flavanone derivatives were the main compounds in the PEE with a total content of 93.08 mg of NAR equivalents/g PEE. Similar results were found in other studies with polar extracts of C. gilliesii reporting high content of flavonoids with a predominant presence of naringenin and eriodictyol [15,17,31]. Compounds 9, 13, and 28 were identified by matching with standards such as luteolin-7-O-glucuronide, luteolin-7-O-glucoside, and luteolin, respectively. Compounds F 15 , F 18 , F 19, and F 20 presented similar UV spectra to Luteolin derivatives reported in the literature [15,30], with strong absorption of the band I between 342 and 369 nm and with a shoulder at 265 nm being identified as luteolin derivatives.  (27). The main flavonols in the extract were luteolin and quercetin derivatives and the total content was 48.39 mg of Q3G equivalents/g PEE. Previous studies reported high content of luteolin derivatives in polar extracts of this species [15,17,31]. Compounds 6 and 8 presented the same retention time and UV profile of the commercial standards and were identified as 3-O-caffeoylquinic acid (3CQA) and trans-caffeic acid, respectively. Compound H1 was tentatively identified as a coumaroylquinic acid derivative due to its strong absorption of band I at 323 nm and the UV spectrum characteristic of coumaroylquinic acids [28]. The total content of hydroxycinnamic acids (HCA) was 4.27 ± 0.35 mg of 3CQA equivalents/g PEE. Cabana et al. (2013) [15] reported the presence of caffeoylquinic acid derivatives, including 3CQA, in agreement with our results.  The HPLC-DAD chromatogram of the PEE of M. acuminata is shown in Figure 1b and the total content of compounds is shown in Table 1. Compound 10 was identified by matching with the standard salicylic acid. The compounds S 1 , S 2 , and S 3 were identified as simple phenols derivatives. The compounds S 1 , S 2 , and S 3 presented UV spectra similar to arbutin derivatives according to the literature [32,33]. The QTOF analysis of the PEE of M. acuminata showed the presence of the aglycone arbutin (hydroquinone) ( Figure S1); however, the identity remains to be confirmed. The total content of simple phenols derivatives was 66.99 mg of p-HBA equivalents/g PEE. Compounds S 1 , S 2 , and S 3 were the major simple phenols in the extract. Previous studies with extracts of M. acuminata also reported a high content of arbutin [18,32] Compounds 2, 6, 8, 14, and 18 were identified by matching with standards such as 5-O-caffeoylquinic acid (2), 3-O-caffeoylquinic acid (3CQA) (6), trans-caffeic acid (8), 1,5-dicaffeoylquinic acid (14), and 3,5-dicaffeoylquinic (18) in agreement with previous reports [13,28,34]. The total content of caffeoylquinic derivatives was 117.07 mg 3CQA equivalents/g PEE. 3CQA was the main compound in the PEE of M. acuminata with 90.03 mg/g PEE. High content of 3CQA also was reported in other polar extracts obtained from M. friesiana [35].
Compound 11 was identified by matching with commercial standards such as pinocembrin. Compounds F 1 , F 2 , F 3 , F 4 , F 5 , and F 6 were tentatively identified as flavanones. Compound F 1 presented UV spectra similar to the pinocembrin standard and the literature reports [30] suggesting that it could be a glycosylated pinocembrin derivative of the lower retention time. Compound F 2 presented a strong absorption of band II at 287 nm with a UV spectrum characteristic of tetrahydroxyflavanone derivatives [30]. Compounds F 3 , F 4 , F 5 , and F 6 presented λ max at 282-287 with a characteristic shoulder at 310 nm in agreement with the UV spectra reported for trihydroxy methoxyflavanone derivative (F 3 ), dihydroxy dimethoxyflavanone derivative (F 4 ), and methoxyflavanone derivatives (F 5 and F 6 ) [30]. The QTOF analysis of the PEE ( Figure S1) shows signals in agreement with the tentative identification of F 3 , F 4 , F 5 , and F 6 . However, the presence of these compounds remains to be confirmed. Our results showed a total content of 144.01 mg NAR equivalents/g PEE. Compound F 3 was the main compound with 74.66 mg NAR equivalents/g PEE. In a study of different Haplopappus spp. (Asteraceae), high content of dihydroxy and trihydroxy dimethoxyflavanones was reported [36].
Compounds 15, 17, 20, 23, and 29 were identified by matching with standards such (23), and quercetin (29). The total flavonol content was 24.43 mg Q3G equivalents/g PEE. Compound 15 was the main flavonol with 17.80 mg Q3G equivalents/g PEE. Our results are in agreement with previous studies that reported a high content of quercetin-3-O-glucuronide in polar extracts from M. acuminata [11,13] and M. friesiana Cabrera [Asteraceae] [35]. Pelargonidin was detected in the extract, but at concentrations below the quantification limit.

Tagetes multiflora Kunth [Asteraceae]
The HPLC-DAD chromatogram of the PEE of T. multiflora is shown in Figure 1c and the total content of compounds is shown in Table 1. Compounds 1 and 3 were identified as gallic acid (1) and vanillic acid (3) by matching with commercial standards. The total content of simple phenolic acids was 13.45 mg of p-HBA equivalents/g PEE. Vanillic acid was the major phenolic with 7.17 mg of p-HBA equivalents/g PEE Compounds 6 and 8 were identified by matching with standards such as 3-O-caffeoylquinic acid and trans-Caffeic acid, respectively. Compound H 1 was tentatively identified as caffeoylquinic acid derivative since it presented λ max at 323 nm with a UV spectrum characteristic of coumaroylquinic acids derivatives [28]. The total content of HCAs was 11.74 mg 3CQA equivalents/g PEE. Our results are in agreement with the high content of gallic acid and caffeic acid that was found in fractions of methanolic extracts from T. lucida [37].  (29), and isorhamnetin (30) was confirmed by matching with commercial standards. The total flavonol content was 276.90 mg Q3G equivalents/g PEE. In addition, compounds F 1 , F 2 , and F 3 presented λ max 356-369 nm and UV spectra similar to quercetagetin derivatives according to the literature [30,[38][39][40]. The compound F 1 was the main compound present in this species with 156.72 mg Q3G equivalents/g PEE. The QTOF analysis of the PEE of T. multiflora suggests the tentative identity of this compound as quercetagetin-hexoside derivative ( Figure S2). In addition, Luteolin-7-O-glucoside, Kaempferol-3-O-glucoside, and Isorhamnetin were the other main flavonols with a content of 11.18, 8.00, and 8.08 mg naringenin equivalents/g PEE, respectively. Several studies have described a wide variety of glycosylated flavonoids present in the Tagetes species [41], but the high content of quercetagetin derivatives has been pointed out as a molecular marker of this genus [38][39][40]. Compound F 4 presented UV spectra similar to the luteolin standards and the literature reports [30]. Compounds F 5 and F 6 presented λ max 347 nm and UV spectra similar to patuletin derivatives according to the literature [30]. Traces of pelargonidin were detected at concentrations below the quantification limit. So far, only the chemical composition of the essential oils from T. multiflora has been studied [19]. Abdala (2003) [42] studied the aerial parts of T. gracilis and reported flavonoids such as quercetagetin-7-O-glucoside, quercetagetin-3-O-glucoside, quercetagetin, quercetin-7-Oglucoside, quercetin-5-O-glucoside, quercetin-7-O-glucoside, luteolin-7-O-glucoside, and luteolin and suggested that this species is probably related to T. multiflora and could present similar type of flavonoids. However, to the best of our knowledge, no information can be found in the literature regarding the chemical characterization of polar extracts from T. multiflora, being this study the first report of the phenolic composition.

Cholinesterase Inhibitory Activities
Cholinesterase inhibitors are one of the most common approaches for the management of Alzheimer s disease (AD) [24,43]. They can prevent the breakdown of acetylcholine (Ach), increasing the levels and improving the brain's cognitive function [24,43]. However, most of them are linked with side effects [23,24,43]. Therefore, the search for new inhibitors from medicinal plants is an important task to find novel and safe medicines for AD [23,24,43]. Among many natural products that have effects on the AchE and BchE activities, polyphenols, phenolic acids, and stilbenes have been tested in vitro and ex vivo, showing inhibitory effects via a hydrogen bond, hydrophobic, and π-π mechanism of interaction [44]. In this study and given the diversity and content of phenolic compounds identified, the in vitro inhibitory effects of the PEE from C. gilliesii, M. acuminata, and T. multiflora was investigated.
Under our experimental conditions, the highest AChE inhibitory activity was obtained for T. multiflora (IC 50 298.17 ± 32.04 µg/mL) followed by C. gilliesii (IC 50 527.84 ± 32.00 µg/mL), while the PEE of M. acuminata was not active against AChE (IC 50 > 1000 µg/mL). In the BChE inhibition assays, all PEE inhibited the enzyme activity. T. multiflora showed an IC 50 value of 583.52 ± 25.53 µg/mL, while M. acuminata presented an IC 50 of 668.15 ± 39.07 µg/mL, and C. gilliesii an IC 50 of 725.209 ± 27.62 µg/mL. The reference compound Galantamine showed IC 50 values of 0.57 ± 0.05 µg/mL for AChE and 8.08 ± 0.02 µg/mL for BChE. A simple trend can be observed regarding the total content of polyphenols and the inhibitory activity of the extracts against both enzymes, in agreement with previous studies that reported a correlation between flavonoids and acetylcholinesterase inhibitory activity [20]. Compared with results in the literature among other Chilean Altiplano plants, the observed effects herein can be considered weak. The cholinesterase inhibitory activity of Artemisia copa Phil. (Asteraceae) was evaluated, showing IC 50 values of 3.92 ± 0.08 µg/mL and 44.13 ± 0.10 µg/mL) against AChE and BChE, respectively [24].
[Lamiaceae] showed less than 15% inhibitory activity at 1000 µg/mL [47]. In another study, the acetone, methanol, and water extracts of Clinopodium vulgare spp showed 1.34 ± 0.01, 1.08 ± 0.03, and 0.25 ± 0.01 mg galantamine equivalents/g of the extract against AChE, while against BChE, only the acetone extract was reported as active, with 0.93 ± 0.23 mg galantamine equivalents/g of extract. [48]. From the Algerian plant Clinopodium nepeta (L.) Kuntze [Lamiaceae], only the dichloromethane extract showed IC 50 values against AChE (170.1 ± 1.58 µg/mL), while the ethyl acetate and dichloromethane extracts were inhibitors of BChE, with IC 50 values of 187.8 ± 1.57 and 73.06 ± 0.83 µg/mL, respectively [49]. Among Tagetes genus, the ethanolic extract obtained from orange and yellow flowers of T. erecta was investigated against AChE, showing IC 50 values > 1000 µg/mL for both cultivars [50]. No investigations have been reported regarding cholinesterase inhibition for species of the Mutisia genus. To the best of our knowledge, this is the first report concerning the inhibitory capacity of C. gilliesii, M. acuminata, and T. multiflora species against these enzymes.

Molecular Docking
Although our results of IC 50 calculated for the PEE from C. gilliesii, M. acuminata, and T. multiflora are higher than that achieved by galantamine (reference drug), the PEE can be considered active, given that the extracts are complex mixtures of compounds, including structures that can be inactive. For a better understanding of the effects observed, and to study if the main individual compounds present in these species could have effects on AChE and BChE, an in silico study was carried out. Since the PEE from T. multiflora presented the best AChE and BChE inhibitory activities, the interactions of AChE and BChE with the main compounds present in this extract were evaluated. The docking analysis showed that all the compounds analyzed presented hydrogen bonding (HB) interactions within the active site of both AChE and BChE enzymes. Compounds present in their structure hydroxyl groups can generate this interaction with side chains or backbones of some residues. In BChE, six of the analyzed compounds presented HB with the Gly115 and Tyr128 residues, located in the oxyanion hole and anionic site, respectively.
On the other hand, the tested compounds showed interactions with residues located in the peripherical anionic site of AChE, around the entrance to the active site cavity. About 86% of the studied compounds showed HB with the Phe295 residue, followed by Asp74, which interacted with four compounds. Unlike BChE, the ligand-AChE complexes exhibited π-π stacking-type interactions with residues such as Trp286, Tyr341, His447, and Phe338 (Table 2).  Figure 2 shows the binding mode of luteolin-7-O-glucoside (13) and kaempferol-3-O-glucoside (22) with AChE and BChE, respectively. Both compounds present π-π stacking interactions with residues Trp286 and Tyr341 located on the periphery of the active site. In addition, the two compounds show HB contacts with the amino acids Tyr72, bond contacts with Asp74 in AChE and Tyr128 within the active site of BChE. The lack of π-π stacking interaction in BChE is because the residues of this active site are mainly aliphatic, while AChE has several amino acids with aromatic side chains [53]. Likewise, these additional interactions can make ligand-AChE complexes more stable than BChE complexes, consistent with the calculated binding energy, evidenced in Table 3.

Standards and Reagents
From Sigma-Aldrich (St. Louis, MO, USA): Amberlite ® XAD7HP, gallic acid, vanillic In the same way, compounds 13 and 22 shared similar interactions in the BChE active site, as HB with residues Tyr128 and Gly115. The binding mode of the rest of the compounds is shown in Figure S3. Compared to other studies, Kuppusamy highlighted that molecular docking of several flavonoids confirms the presence of HB and pi stacking interactions in the AChE binding site with residues such as Tyr124, Trp286, Ser293, Phe295, and Tyr341, which coincide with the present study [51]. Khan et al. [52] described the interactions of quercetin in the active site of cholinergic enzymes, highlighting hydrogen bond contacts with Asp74 in AChE and Tyr128 within the active site of BChE. The lack of π-π stacking interaction in BChE is because the residues of this active site are mainly aliphatic, while AChE has several amino acids with aromatic side chains [53]. Likewise, these additional interactions can make ligand-AChE complexes more stable than BChE complexes, consistent with the calculated binding energy, evidenced in Table 3.

Plant Material and Preparation of Extracts
The aerial parts from C. gilliesii (Muña-Muña), M. acuminata (Chinchircoma), and T. multiflora (Gracilis) were collected in March of 2019 in Putre, Región de Arica y Parinacota, Chile. The samples were identified and deposited in our laboratory herbarium (voucher numbers: Cg-032019, Ma-032019 and Tm-032019). The samples were cleaned with water and dried in an oven at 35 • C. A total of 10 g of dried aerial parts for each species were ground and extracted with 100 mL of EtOH using an ultrasonic water bath (UC-60A Biobase, Guanzhou, China). To clean-up crude extracts and to make polyphenolic enriched-extracts (PEEs), an Amberlite ® XAD7HP resin was used. The column was washed with water and compounds were desorbed with MeOH. The PEEs were evaporated under reduced pressure and freeze dried for further analyses.

HPLC-DAD Analysis and Quantification
The HPLC-DAD system used was Shimadzu equipment (Shimadzu Corporation, Kyoto, Japan) consisting of an LC-20AT pump, an SPD-M20A UV diode array detector, a CTO-20AC column oven, and LabSolution software. A MultoHigh ® RP18 (250 × 4.6 mm, 5 µm of particle size) column (CS-Chromatographie Service GmbH, Langerwehe, Germany). The column temperature was set at 30 • C. The HPLC analyses were performed using a linear PEEs from each species was 20 µL at a concentration of 5 mg/mL. The compounds were monitored at 280, 320, 354, and 520 nm, and UV spectra from 200 to 600 nm were recorded for peak characterization.
The compounds in each PEEs were identified by matching retention times and UV-vis spectra with commercial standards and literature related to the species in this study. The UV absorbance profile of compounds without matching was used for the tentative identification of groups of compounds. Each peak UV profile was assessed and compounds were classified and quantified by the typical absorption band I of the B-ring cinnamoyl structure and band II of the A-ring benzoyl or benzene structure [54,55], as follows: simple phenols derivatives, flavan-3-ol monomers and polymers, and flavanones were identified by the absorption band II with λ max at 280 nm; hydroxycinnamic acid derivatives were identified by the absorption band I with λ max at 320-329 nm; flavonol derivatives were identified by the absorption band I with λ max at 354-370 nm; and anthocyanin compounds were identified by the strong visible absorption band at 500-520 nm. Quantification of total simple phenols derivatives (S), hydroxycinnamic acids (H), and flavonoids (F), such as flavan-3-ol monomers and polymers, flavanones, flavonols, and anthocyanins was carried out using external calibration curves with commercial standards of p-hydroxybenzoic acid

Molecular Docking
The crystallographic structures of AChE in complex with donepezil (PDB ID: 4EY7, Resolution: 2.3 Å) [58] and BChE in complex with tacrine (PDB ID: 4BDS, Resolution: 2.1 Å) [59] were obtained from the Protein Data Bank (PDB) (http://www.rcsb.org (accessed on 28 October 2022)). These protein structures were prepared for molecular docking using the Preparation Wizard module from suite Maestro [60]. The three-dimensional structures of the compounds were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov (accessed on 28 October 2022)) and prepared through the Ligprep module [60] under the OPLS-AA force field [13] to generate the minimized energy conformations. Molecular docking studies were performed using Glide from suite Maestro. Glide uses a series of hierarchical filters to find the best ligand binding posse in a protein grid space [61]. The original co-crystallized ligands' location was used as the grid box center, which was refined and set up to 20 Å. Initially, the ligands were docked using the standard precision algorithm (SP), and the best-ranked poses were re-docked with the extra precision algorithm (XP). The glide score, interactions, and visually inspected were used as a criterion for selecting the best poses.

Statistical Analysis
The results are expressed as mean values ± standard error using GraphPad Prism 8 (GraphPad Software Corporation, La Jolla, CA, USA). One-way analysis of variance (ANOVA; repeating three times each measurement of sample solutions) (p < 0.05) was used for the comparison of means.

Conclusions
This is the first report on the identification and characterization of phenolic compounds from three Chilean Altiplano plants: Clinopodium gilliesii, Mutisia acuminata, and Tagetes multiflora. In this study, 30 phenolic compounds were tentatively identified, including simple phenolics, hydroxycinnamic acids, flavan-3-ols (monomers and polymers), flavanones, and flavonols. The three PEEs showed mild inhibitory activity against cholinesterase enzymes, with higher IC 50 values achieved by T. multiflora extract. Docking analysis for main selected compounds tentatively identified in T. multiflora, including gallic acid, vanillic acid, luteolin-7-O-glucoside, quercetin-3-O-arabinoside, quercetin, isorhamnetin, and kaempferol-3-O-glucoside, indicated that hydrogen bonding interaction and π-π stackingtype interactions were predominant in the experiments, partially explaining the observed effects. The main individual compounds inhibited the AD-associated enzymes (AChE and BChE) through π-π stacking interactions with Trp286 and Tyr341 residues located on the periphery of the active site in both enzymes. This is the first evidence about the potential neuroprotective effect of phenolic compounds from C. gilliesii, M. acuminata, and T. multiflora.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12040819/s1, Methodology S1: High-Resolution Mass Spectrometry; Figure S1: High-resolution Mass Spectrometry analysis of the main compounds present in PEE from the aerial parts of Mutisia acuminata; Figure S2: High-resolution Mass Spectrometry analysis of the main compounds present in PEE from the aerial parts of Tagetes multiflora; Figure S3: Predicted binding modes for some studied compounds within the AChE and BChE active sites.

Conflicts of Interest:
The authors declare no conflict of interest.