Isorhamnetin and Quercetin Derivatives as Anti-Acetylcholinesterase Principles of Marigold (Calendula officinalis) Flowers and Preparations

Marigold (Calendula officinalis L.) is one of the most common and widespread plants used medicinally all over the world. The present study aimed to evaluate the anti-acetylcholinesterase activity of marigold flowers, detect the compounds responsible and perform chemical analysis of marigold commercial products. Analysis of 23 varieties of C. officinalis flowers introduced into Siberia allowed us to select the Greenheart Orange variety due to the superior content of flavonoids (46.87 mg/g) and the highest inhibitory activity against acetylcholinesterase (IC50 63.52 µg/mL). Flavonoids, isorhamnetin and quercetin derivatives were revealed as potential inhibitors with the application of high-performance liquid chromatography (HPLC) activity-based profiling. Investigation of the inhibitory activity of isorhamnetin glycosides demonstrated the maximal potency for isorhamnetin-3-O-(2′′,6′′-di-acetyl)-glucoside (IC50 51.26 μM) and minimal potency for typhaneoside (isorhamnetin-3-O-(2′′,6′′-di-rhamnosyl)-glucoside; IC50 94.92 µM). Among quercetin derivatives, the most active compound was quercetin-3-O-(2′′,6′′-di-acetyl)-glucoside (IC50 36.47 µM), and the least active component was manghaslin (quercetin-3-O-(2′′,6′′-di-rhamnosyl)-glucoside; IC50 94.92 µM). Some structure-activity relationships were discussed. Analysis of commercial marigold formulations revealed a reduced flavonoid content (from 7.18–19.85 mg/g) compared with introduced varieties. Liquid extract was the most enriched preparation, characterized by 3.10 mg/mL of total flavonoid content, and infusion was the least enriched formulation (0.41 mg/mL). The presented results suggest that isorhamnetin and quercetin and its glycosides can be considered as potential anti-acetylcholinesterase agents.


Introduction
Cognitive-mental deficiency is one of the key signs of the disturbances of higher nervous activity in many disease states, including neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, neuroinfections and others. The clinical picture of cognitive mnestic deficits manifests in disturbance of concentration, a decrease in motivation and malfunction of abstract and The present research is aimed at chemical examination of 23 varieties of C. officinalis flowers introduced into Siberia and determination of their acetylcholinesterase inhibiting activity, detection of the most active compounds responsible for the manifestation of anti-acetylcholinesterase activity with the use of high-performance liquid chromatography (HPLC) activity-based profiling and revealing the active compound content in marigold flower commercial samples.

Chemical Composition and Anti-Acetylcholinesterase Potential of 23 Varieties of C. officinalis Flowers
Based on known data of the chemical composition of C. officinalis flowers, we investigated the most evident correlations between the parameters of compound content and the values of anti-acetylcholinesterase inhibition. For this purpose, the total extracts of flowers of 23 varieties of C. officinalis introduced into Siberia were analyzed to determine the content of essential oil, carotenoids, triterpenoids, flavonoids, phenylpropanoids and polysaccharides, as well as the index of 50% inhibition of acetylcholinesterase in in vitro experiments (Table 1). Table 1. Chemical composition and anti-acetylcholinesterase activity (AChA) of total extracts of 23 varieties of C. officinalis flowers (mg/g dry weight (DW) ± standard deviation (SD)) 1 . The total essential oil content in the varieties analyzed was from 0.32 (Cardinal) to 3.04 mg/g (Jiga-Jiga) dry extract weight. Variations of carotenoid and triterpene content were 2.63 (Touch of Red) to 11.39 mg/g (Rose Surprise) and 10.28 (Flame Dancer) to 65.70 mg/g (Egypt Sun), respectively. The basic phenolic groups of total extracts of C. officinalis flowers were flavonoids and phenylpropanoids with content values of 10.52 (Jiga-Jiga) to 46.87 mg/g (Greenheart Orange) and 6.07 (Golden Prince) to 33.47 mg/g (Golden Imperator), respectively. The concentration of polysaccharide components in C. officinalis flowers extracts varied from 11.09 (Rose Surprise) to 44.15 mg/g (Honey Cardinal).
The range of acetylcholinesterase inhibitory value (IC 50 ) of total extracts of 23 varieties of C. officinalis flowers was from 223.9 µg/mL for the least effective sample, the Jiga-Jiga variety, to 63.5 µg/mL for the most active sample, the Greenheart Orange variety. The inhibitory activity of a Turkish sample of C. officinalis was lower, reaching 22.37% at a dose of 1000 µg/mL for methanolic extract [6]. To understand the correlation among all of the studied chemical parameters and biological potential, linear regression analysis was used ( Figure 1). The highest correlation was observed between total flavonoid content and anti-acetylcholinesterase activity (r 2 = 0.6717). No other class of phytocomponents demonstrated appropriate correlations due to the low r 2 value: essential oil (0.0601), carotenoids (0.0018), triterpenoids (0.0023), phenylpropanoids (0.1152) and polysaccharides (0.0603). Previously, flavonoids were demonstrated to have good correlative dependency with the anti-acetylcholinesterase activity of natural extracts of Smallanthus sonchifolius [25], propolis [26] and Garcinia parvifolia [27]. (Japan) [17], 2.0-35.1 mg/g (Estonia) [18]. The triterpenoid content of C. officinalis flowers may reach levels of 20 mg/g (Germany) [19], 20.53 mg/g (Poland) [20] or 25.98-40.82 mg/g (Italy) [21].
Previously declared data about the content of flavonoids in C. officinalis flowers collected in different places were 2.1-6.8 mg/g (Estonia) [22], 2.5-8.8 mg/g (Bratislava) [23], 6.3-7.9 mg/g (Brazil) [24] and 18.3-36.3 mg/g (Italy) [15]. This demonstrates the good ability of the Siberian cultivars of C. officinalis to concentrate the bioactive components in flowers. The range of acetylcholinesterase inhibitory value (IC50) of total extracts of 23 varieties of C. officinalis flowers was from 223.9 µg/mL for the least effective sample, the Jiga-Jiga variety, to 63.5 µg/mL for the most active sample, the Greenheart Orange variety. The inhibitory activity of a Turkish sample of C. officinalis was lower, reaching 22.37% at a dose of 1000 µg/mL for methanolic extract [6]. To understand the correlation among all of the studied chemical parameters and biological potential, linear regression analysis was used ( Figure 1). The highest correlation was observed between total flavonoid content and anti-acetylcholinesterase activity (r 2 = 0.6717). No other class of phytocomponents demonstrated appropriate correlations due to the low r 2 value: essential oil (0.0601), carotenoids (0.0018), triterpenoids (0.0023), phenylpropanoids (0.1152) and polysaccharides (0.0603). Previously, flavonoids were demonstrated to have good correlative dependency with the anti-acetylcholinesterase activity of natural extracts of Smallanthus sonchifolius [25], propolis [26] and Garcinia parvifolia [27].

Flavonoid Profile of C. officinalis Flowers' Extract and High-Performance Liquid Chromatography (HPLC) Activity-Based Profiling of Acetylcholinesterase Inhibitors
According to the preliminary stage of the study, the Greenheart Orange variety of C. officinalis flowers was selected and investigated as the most active sample with anti-acetylcholinesterase activity and superior flavonoid content. In order to examine the phenolic profile of the selected marigold, its 60% ethanol extract from flowers was subjected to a previously developed microcolumn reversed-phase HPLC procedure with ultraviolet (UV) detection (MC-RP-HPLC-UV) [28]. From the comparison of retention times, UV and mass spectrometry (MS) data with reference substances, 12 flavonoids and five phenylpropanoids were detected ( Figure 2, Table 2).

Flavonoid Profile of C. officinalis Flowers' Extract and High-Performance Liquid Chromatography (HPLC) Activity-Based Profiling of Acetylcholinesterase Inhibitors
According to the preliminary stage of the study, the Greenheart Orange variety of C. officinalis flowers was selected and investigated as the most active sample with anti-acetylcholinesterase activity and superior flavonoid content. In order to examine the phenolic profile of the selected marigold, its 60% ethanol extract from flowers was subjected to a previously developed microcolumn reversed-phase HPLC procedure with ultraviolet (UV) detection (MC-RP-HPLC-UV) [28]. From the comparison of retention times, UV and mass spectrometry (MS) data with reference substances, 12 flavonoids and five phenylpropanoids were detected ( Figure 2, Table 2).
To identify the compounds of interest in C. officinalis flower extract with high acetylcholinesterase inhibitory activity, the extract investigated was submitted to HPLC activity-based profiling. This technique is a miniaturized and highly effective approach for localization and characterization of bioactive natural products with minute amounts of injected extracts [31][32][33]. This technique combines the speed and separation power of HPLC with the structural information of online spectroscopy and miniaturized bioassays. For the detection of acetylcholinesterase inhibitors in C. officinalis flower extract, the procedure of small-scale semi-preparative microfractionation by reversed-phase HPLC was used. This yielded 60 microfractions of 30 s each that were transferred to a deep-well microtiter plate.
Then, microfractions were dried, redissolved in buffer solution and subjected to post-chromatographic reaction with an acetylcholinesterase/α-naphthyl acetate/Fast Blue B salt model system to evaluate the anti-acetylcholinesterase activity. The anti-acetylcholinesterase activity of the microfractions after post-column derivatization is shown in Figure 2B. Major inhibition was observed in Fractions xxv, xxvi, xxx, xxxviii and xiv, which displayed the highest anti-cholinesterase activity potential with inhibition values of 14.2%, 16.1%, 8.5%, 16.9% and 9.8%, respectively, while the activity of the other fractions was not significantly different from zero. The data obtained showed considerable acetylcholinesterase inhibiting activity of the fractions containing flavonoids. The majority of compounds eluted in the most active fractions were derivatives of isorhamnetin like typhaneoside (Fraction xxvi), calendoflavoside (Fraction xxx), narcissin (Fraction xxxviii) and isorhamnetin-3-O-(6 -acetyl)-glucoside (Fraction xlv), while only calendoflavobioside was a derivative of quercetin (Fraction xxv). The fractions containing other derivatives of quercetin are characterized as inhibitors of moderate power, and caffeoylquinic acids do not show a pronounced mode of action.

Acetylcholinesterase Inhibitory Activity of C. officinalis Flavonoids
For the further detailed studies, samples of the individual flavonoids previously isolated from C. officinalis flowers were used [28,[34][35][36]. The acetylcholinesterase inhibition assay was performed using a spectrophotometric method [37].

Acetylcholinesterase Inhibitory Activity of C. officinalis Flavonoids
For the further detailed studies, samples of the individual flavonoids previously isolated from C. officinalis flowers were used [28,[34][35][36]. The acetylcholinesterase inhibition assay was performed using a spectrophotometric method [37].    The inhibitory activity of isorhamnetin glycosides expressed as IC 50 was from 51.26-98.45 µM with maximal potency for 3-O-(2 ,6 -di-acetyl)-glucoside and minimal potency for 3-O-(2 ,6di-rhamnosyl)-glucoside; the latter was the dominant compound in the plant object investigated ( Table 3). The isorhamnetin inhibition power was highest (24.18 µM), demonstrating the negative influence of 3-O-glycosylation on the anti-acetylcholinesterase activity of flavonoids. However, according to the literature data, the hydroxyl group at position C-3 is not involved in the hydrogen bonding with acetylcholinesterase. Hydroxylation at these positions is important for metal chelation, antioxidant effect and the prevention of Aβ aggregation [38][39][40]. Attaching a rhamnosyl moiety to an isorhamnetin skeleton resulted in the formation of a more active compound compared with a glucosyl analogue (IC 50  Quercetin glycosides were 20-35% more active than the same analogues of isorhamnetin, and the general character of the structure-activity dependence was close. The high potency of quercetin and its glucosides to inhibit acetylcholinesterase has been described previously in many works [41][42][43]. Information about isorhamnetin derivatives is not so common. However, the results obtained allow us to conclude that isorhamnetin and its glucosides are natural components with anti-acetylcholinesterase potency. Previously, some authors mentioned that all flavonols possess a similar binding pattern in the active site of acetylcholinesterase [40]. The general interactions were found to be between the flavonol skeleton and enzyme active sites. Interaction of the A-ring-involved functional groups was described as between the hydroxyl group at the C-7 position and Asp74 or Tyr72 residues, forming a hydrogen bond [44]. Hydroxylation of the B-ring at C-3 and C-4 may also form a hydrogen bond with the residues Ser203 and Gly121 and often with Gly122. The possibility of interaction between the C-4-keto function of C-rings and the residue Phe295 was shown. The structural differences between quercetin and isorhamnetin are only in the methoxy group in the 3 -position in the B-ring of the latter compound. Based on these data, it can be concluded that both substances may decrease the activity of acetylcholinesterase by binding to its active sites.
Given the widespread use of preparations from C. officinalis in therapeutic practice, we also investigated the qualitative and quantitative content of phenolic compounds in four medicinal forms, including commercial ethanol-containing forms (liquid extract and tincture) and aqueous forms (decoction and infusion) as frequently applied home-made preparations.
The qualitative composition of the analyzed preparations from C. officinalis was similar to those of native plant material (Table 5). This indicates the safety of the componential profile of the analyzed preparations within the manufacturing process. The most enriched liquid formulation was liquid extract, characterized by 3.10 mg/mL of total flavonoid content. Tincture, decoction and infusion are dosage forms prepared by low technology, which affects the composition of the resulting product. The content of flavonoids in tincture, decoction and infusion was significantly lower (0.70, 0.57 and 0.45 mg/mL, respectively) than in liquid extract. In all liquid preparations, a predominance of the quercetin derivative calendoflavobioside and isorhamnetin derivative typhaneoside was observed. Information on the acceptable intake of various liquid preparations [45] allowed us to calculate values for maximal daily consumption of flavonoids after the application of the mentioned marigold preparations. The results obtained showed that despite the archaic character of aqueous preparations of C. officinalis, their application maximized the intake values of flavonoids compared with ethanol formulations. Thus, the intake from daily dosage of marigold decoction (142.50 mg/day) increased flavonoid consumption by 45-times compared to a daily dose of tincture (3.15 mg/day). Despite the high content of flavonoids in liquid extract, daily uptake (9.30 mg/day) is 15.3-times lower than for consumption of a daily dose of decoction.
These data demonstrate the possibility of adequate substitution of liquid extract or tincture by infusions or decoctions when it is not appropriate to administer ethanol-containing formulations (children's therapy, allergy to ethanol, etc.).

Chemicals
The following chemicals were purchased from Extrasynthese (Lyon,

Sample Preparation for the Extraction of Total Phytochemicals and Anti-acetylcolinesterase Acitivity Determination
For preparation of total extracts of twenty three varieties of C. officinalis with maximal content of basic groups of compounds (essential oils, carotenoids, triterpenoids, flavonoids, phenylpropanoids and polysaccharides), plant material was extracted by following solvents, as 96% ethanol for extraction of essential oils, carotenoids and triterpenoids; 60% ethanol as an optimal solvent for flavonoids and phenylpropanoids; water as an optimal solvent for polysaccharides. Accurately-weighed C. officinalis plant sample (100 g) was placed in a conical flask. Then, 1500 mL of the 96% ethanol solution were added, and the mixture was extracted twice in an ultrasonic bath for 90 min at 45 • C. The extracted solution A was filtered through a cellulose filter. The plant residue was repeatedly extracted by 60 % ethanol and water in the same conditions receiving extracts B and C, accordingly. Finally extracts A, B and C were combined and evaporated in vacuo until dryness using a rotary evaporator. The total extracts were stored at 4 • C until further chemical composition analysis and anti-acetylcholinesterase activity microplate assay.

Chemical Composition Analytical Methods
The essential oil content was determined gravimetrically after hydrodistillation in a Clevenger-type apparatus for 150 min [46]. The concentration of carotenoids was estimated as β-carotene equivalent using the spectrophotometric method at 450 nm in preliminary saponified extracts [47]. The total triterpenoid content was determined by HPTLC-densitometric analysis after acidic hydrolysis in 7% HCl/acetone media as oleanolic acid equivalents [48]. The flavonoid content was estimated as narcissin equivalents after spectrophotometric procedure after 5% AlCl 3 addiction [49]. The phenylpropanoid content was determined by the colorimetric Arnow method using 3-O-caffeoylquinic acid as the standard [50]. The polysaccharides content was determined by the spectrophotometric anthrone-sulfuric acid method with galactose as the standard [51].

Anti-Acetylcholinesterase Activity Microplate Assay
The acetylcholinesterase inhibition assay was performed using a spectrophotometric microplate assay [37]. . The method was realized in 96-well plates. The microplate was read by a Bio-Rad microplate reader Model 3550 UV. Enzyme activity and inhibition were quantified by determination of the absorbance at 600 nm after the formation of the purple-colored diazonium dye as a percentage. A control sample was considered to have 100% was carried out using the same volume of DMSO instead of tested compound (or plant extract). The percentage of inhibition was calculated relative to a control sample, for which the anti-acetylcholinesterase activity was assessed under identical conditions, but in the absence of the test compound, using the expression: where A CE is the absorbance at 600 nm of the control sample with enzyme; A C is the absorbance at 600 nm of the control sample without enzyme; A TE is the absorbance at 600 nm of the test compound (or plant extract) with enzyme; A T is the absorbance at 600 nm of the test compound (or plant extract) without enzyme. Linear equation indicating the correlation between the common logarithm of the compound concentration (µM) and percentage of acetylcholinesterase inhibition (%) was build, and from which the IC 50 values (concentration that inhibits 50% of acetylcholinesterase activity) of tested compounds (or plant extracts) were extrapolated. For preparation of 60% ethanol extract, used for MC-RP-HPLC-UV and MC-RP-HPLC-UV-ESI-MS analysis, accurately-weighed C. officinalis plant sample of Greenheart Orange variety (100 g) was placed in a conical flask. Then, 1500 mL of the 60% ethanol solution were added, and the mixture was extracted twice in an ultrasonic bath for 90 min at 45 • C. The extracted solutions were filtered through a cellulose filter and evaporated in vacuo until dryness using a rotary evaporator. For the preparation of 60% ethanol extract solution, an accurately weighed dry extract of C. officinalis (10 mg) was placed in an Eppendorf tube; 1 mL of 60% ethanol was added; and the mixture was weighed again. Then, the sample was extracted in an ultrasonic bath for 10 min at 40 • C. After cooling, the resultant extract was filtered through a 0.22-µm polytetrafluoroethylene (PTFE) syringe filter before injection into the HPLC system for analysis.
For the preparation of the sample solution of commercial of marigold tea batches (samples 01-16), accurately-weighed plant sample (1 g) was placed in conical flasks. Then, 100 mL of the 60% ethanol solution were added, and the mixture was extracted in an ultrasonic bath for 30 min at 45 • C. The extracted solution was filtered through a 0.22-µm PTFE syringe filter before injection into the HPLC system for analysis.
For the preparation of the decoction, accurately-weighed C. officinalis plant Sample 15 (1 g) was placed in conical flasks. Then, 100 mL of distilled water was added, and the sample was heated on a hotplate and boiled for 10 min. The mixture was left to stand at room temperature for 15 min, then filtered under reduced pressure and made up to 100 mL in a volumetric flask. The resultant decoction was filtered through a 0.22-µm PTFE syringe filter before injection into the HPLC system for analysis.
For the preparation of infusion, accurately-weighed C. officinalis plant Sample 15 (1 g) was placed in conical flasks. Then, 100 mL of boiled distilled water was added. The sample was then stirred for 40 min. Then, the mixture was filtered under reduced pressure and made up to 100 mL in a volumetric flask. The resultant infusion was filtered through a 0.22-µm PTFE syringe filter before injection into the HPLC system for analysis.

Microcolumn Reversed-Phase High-Performance Liquid Chromatography with Electrospray Ionization Mass Spectrometry Detection (MC-RP-HPLC-ESI-MS) Conditions
MC-RP-HPLC-ESI-MS experiments were performed on an Econova MiLiChrom A-02 microcolumn chromatograph (Novosibirsk, Novosibirsk Oblast, Russia) coupled with triple-quadrupole electrospray ionization mass-spectrometer LCMS 8050 (Shimadzu, Columbia, MD, USA). LC conditions were the same as described in Section 3.5. MS conditions were: ionization mode, ESI (negative for phenylpropanoids and positive for flavonoids); electrospray ionization (ESI) interface temperature, 300 • C; desolvation line temperature, 250 • C; heat block temperature, 400 • C; nebulizing gas flow (N 2 ), 3 L/min; heating gas flow (air), 10 L/min; heating gas flow (N 2 ), 10 L/min; collision-induced dissociation gas (Ar) pressure, 270 kPa; Ar flow, 0.3 mL/min; capillary voltage, 3 kV. The full scan mass covered the range from m/z 100 up to m/z 1900. . Enzyme activity and inhibition were quantified by determination of the absorbance at 600 nm after the formation of the purple-colored diazonium dye as a percentage. The percentage of inhibition was calculated relative to a control sample, for which the anti-acetylcholinesterase activity was assessed under identical conditions, but in the absence of the test compound.

Statistical Analysis
All experiments were done in triplicates, and the results were given as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with a post hoc least significant difference test was used to determine significance (p ≤ 0.05) with Statistica 5.5 software (Dell Technologies Inc., Round Rock, TX, USA) together with the correlation matrix with the use of elementary statistics.

Conclusions
In this study, we investigated the anti-acetylcholinesterase potential of C. officinalis flowers introduced into Siberia based on their chemical composition, which has not been reported previously. The effect of total flavonoid content on the anti-acetylcholinesterase activity of ethanolic extracts was significant. HPLC activity-based profiling data suggested that the quercetin and isorhamnetin derivatives possess strong and moderate anti-acetylcholinesterase activity, respectively. Structural characteristics that may contribute to the understanding of the bioactivity of quercetin and isorhamnetin glycosides were identified. The presence of a rhamnosyl moiety and acetyl groups at the 2 -and/or 6 -position of the carbohydrate function of a flavonoid skeleton has an influence on their anti-acetylcholinesterase properties. In addition, the flavonoid content of 16 commercial batches of marigold flowers and four liquid preparations was analyzed. The levels of isorhamnetin and quercetin derivatives detected in samples of marigold tea were 6.57-17.86 and 0.61-2.08 mg/g, respectively, and in liquid preparations were 0.37-2.62 and 0.08-0.48 mg/mL, respectively. On the basis of this study, it can be concluded that quercetin and isorhamnetin glycosides from C. officinalis flowers have the potential to be promising candidates for the development of anti-acetylcholinesterase agents.