Chemical Fingerprinting and Biological Evaluation of the Endemic Chilean Fruit Greigia sphacelata (Ruiz and Pav.) Regel (Bromeliaceae) by UHPLC-PDA-Orbitrap-Mass Spectrometry

Greigia sphacelata (Ruiz and Pav.) Regel (Bromeliaceae) is a Chilean endemic plant popularly known as “quiscal” and produces an edible fruit consumed by the local Mapuche communities named as “chupón”. In this study, several metabolites including phenolic acids, organic acids, sugar derivatives, catechins, proanthocyanidins, fatty acids, iridoids, coumarins, benzophenone, flavonoids, and terpenes were identified in G. sphacelata fruits using ultrahigh performance liquid chromatography-photodiode array detection coupled with a Orbitrap mass spectrometry (UHPLC-PDA-Orbitrap-MS) analysis for the first time. The fruits showed moderate antioxidant capacities (i.e., 487.11 ± 26.22 μmol TE/g dry weight) in the stable radical DPPH assay, 169.08 ± 9.81 TE/g dry weight in the ferric reducing power assay, 190.32 ± 6.23 TE/g dry weight in the ABTS assay, and 76.46 ± 3.18% inhibition in the superoxide anion scavenging assay. The cholinesterase inhibitory potential was evaluated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). From the findings, promising results were observed for pulp and seeds. Our findings suggest that G. sphacelata fruits are a rich source of diverse secondary metabolites with antioxidant capacities. In addition, the inhibitory effects against AChE and BChE suggest that natural products or food supplements derived from G. sphacelata fruits are of interest for their neuroprotective potential.


Introduction
Plant-based foods, especially fruits, have a great impact on human health [1,2]. Indeed, there is a growing body of scientific literature describing curative effects of fruits against a broad spectrum of diseases including diabetes, obesity, neurodegenerative, gastric and cardiovascular disorders, and certain types of cancers [3][4][5][6]. Different types of secondary metabolites have been reported in fruits [4,[7][8][9]. These compounds are in part responsible for their biological properties.
HPLC or UHPLC coupled to mass spectrometry is a key technique for plant chemotaxonomy or comparative metabolic profiling. Q-Exactive type focus equipment uses a very rapid high-performance mass spectrometer for the detection of small organic molecules [12][13][14]. This is a dual high resolution with accurate mass (HRAM) spectrometer with an orbital trap (Orbitrap), a quadrupole (Q), and a high-performance collision cell (HCD), capable of producing high-resolution parent ions and diagnostic MS daughter fragments. The hyphenated ultrahigh performance liquid chromatography-photodiode array detection coupled with a Orbitrap mass spectrometry (UHPLC-PDA-Orbitrap-MS) approach is a key tool for the identification of secondary metabolites in plants and edible fruits [15][16][17].
In this work, we report the antioxidant activity, cholinesterase inhibitory potential plus the UHPLC-PDA-Orbitrap-MS fingerprinting from the endemic G. sphacelata fruits for the first time.

Metabolomic Analyses
In this study, the fingerprint was generated using UHPLC-PDA-Orbitrap-MS ( Figure 2) allowing the determination of several types of metabolites in the fruits of the Mapuche specie G. sphacelata (Table 1 Examples of full MS spectra and structures of the compounds identified are displayed in Figure  S1. The detailed identification is explained below: Molecules 2020, 25, x 3 of 20 performance mass spectrometer for the detection of small organic molecules [12][13][14]. This is a dual high resolution with accurate mass (HRAM) spectrometer with an orbital trap (Orbitrap), a quadrupole (Q), and a high-performance collision cell (HCD), capable of producing high-resolution parent ions and diagnostic MS daughter fragments. The hyphenated ultrahigh performance liquid chromatography-photodiode array detection coupled with a Orbitrap mass spectrometry (UHPLC-PDA-Orbitrap-MS) approach is a key tool for the identification of secondary metabolites in plants and edible fruits [15][16][17].
In this work, we report the antioxidant activity, cholinesterase inhibitory potential plus the UHPLC-PDA-Orbitrap-MS fingerprinting from the endemic G. sphacelata fruits for the first time.

Metabolomic Analyses
In this study, the fingerprint was generated using UHPLC-PDA-Orbitrap-MS ( Figure 2) allowing the determination of several types of metabolites in the fruits of the Mapuche specie G. sphacelata (Table 1 Examples of full MS spectra and structures of the compounds identified are displayed in Figure  S1. The detailed identification is explained below:

Total Phenolics, Flavonoid Contents, and Antioxidant Activity of G. sphacelata
The in vitro results of total phenolics, total flavonoid content, and antioxidant activity are summarized in Table 2. DPPH, ABTS, FRAP, and superoxide anion scavenging activity (O 2 − ) were used to measure the antioxidant potential in pulp and seed of G. sphacelata fruits. These in vitro assays are very simple and widely employed to calculate the antioxidant activities of plant and fruit extracts.
The results were compared with previous studies from our research as well as work on other Chilean fruits by other researchers [20,21]. The total phenolic contents of pulp of Chilean berries from G. sphacelata (45.44 ± 0.67 mg GAE/g dry weight, Table 2) was closer to previous studies reported for the blueberries high-bush type Vaccinium corimbosum L (45.86 ± 3.46 mg GAE/g) [20] and Chilean blackberries maqui (Aristotelia chilensis (Molina) Stuntz) (49.74 ± 0.57 mg GAE/g) [22]. In addition, our TPC values were almost twice the values for TPC (29 mg Q/g) of the Chilean blackberries Luma apiculata (DC.) Burret (commonly known as arrayán or palo colorado) [13]. The amount of TFC found, 35.57 ± 0.86 mg QE/g, was lower than those of Chilean berries Berberis microphilla G. Forst. (45.72 ± 2.68 mg QE/g) commonly known as calafate or michay [20]. In the DPPH assay, the trapping capacity of G. sphacelata (487.11 ± 26.22 µmol Trolox/g dry weight) was close to that obtained for the red Chilean berries Ugni molinae Turcz. (commonly known as murtilla, murta, murtillo, or uñi), (450 µmol Trolox equivalents, TE/g dry weight) which is considered as an average antioxidant level in the category of edible fruits [21]. The ABTS values (190.32 ± 6.23 µmol TE/g dry weight) were lower than those of numerous Latin American fruits, such as Chilean blackberries maqui with a trapping capacity of 254.8 ± 8.2 µmol TE/g dry weight, and Brazilian Acai (Euterpe oleraceae Mart.), with 211.0 ± 14 µmol TE/g dry weight [23], while the FRAP values (169.08 ± 9.81 µmol TE/g dry weight) were close to those of Acai (157.9 ± 8.7 µmol TE/g dry weight) [23], but lower than maqui (254.2 ± 2.6 mg TE/g dry weight) [22,23]. The superoxide anion scavenging activity (O 2 − ) of G. sphacelata (76.46 ± 3.18%) was higher compared to those established for the blueberries growing in Chile Vaccinium corimbosum L. (72.61 ± 1.91%) [20]. These values can classify these G. sphacelata fruits as moderate to high antioxidant small fruits such as plum, cherries, and strawberries [23].  SD (n = 3). The results are statistically compared with a positive control. The results were analyzed using one-way analysis of variance (ANOVA) and Tuckey test statistical analysis (p-values < 0.05 were regarded as significant). Values in the same column marked with the same letter are not significantly different (at p < 0.05).

In Vitro Cholinesterase Inhibitory Assay
The inhibition of key enzymes, such as AChE and BChE (linked to Alzheimer's disease), is an important metric for identifying novel and safe medicinal value from natural products, especially those found in edible fruits. Indeed, four of the approved anti-Alzheimer drugs are cholinesterase inhibitors.
For this reason, we tested the cholinesterase inhibitory potential of G. sphacelata against AChE and BChE. The results are shown in Table 3 and expressed as IC 50 values. Galantamine was used as a positive control (0.27 ± 0.03 µg/mL against AChE and 3.82 ± 0.02 µg/mL against BChE). G. sphacelata fruits showed promising results for both enzymes. In the AChE assay, the inhibition IC 50 for pulp was 4.49 + 0.08 µg/mL and for seeds was 4.38 ± 0.04 µg/mL. As for the BChE assay, the inhibition IC 50 for pulp was 73.86 ± 0.09 µg/mL and for seeds was 78.57 ± 0.06 µg/mL. No significant difference was observed. Some metabolites contained in G. sphacelata have been linked to counteraction against Alzheimer's disease in previous reports. The iridoid jatamanvaltrate H isolated from V. jatamansi showed moderate neuroprotective activity [24,25]. In the same way, the crude extract of V. jatamansi and its fractions have shown considerable activity against AChE [26]. Marrubiin exhibited potent antinociceptive effects in a dose-dependent manner [27]. Congestiflorone acetates, such as detected in this study, have shown significant inhibitory effects against AChE with an IC 50 value at 20.25 ± 0.55 µM [28]. On the other hand, the phenolic glucosyringic acid, did not show significant inhibitory effect on BChE (IC 50 > 100 µM) [29]. Catechin presents neuroprotective effects as evidenced by amyloid β-induced neurotoxicity by MTT, lactate dehydrogenase (LDH) release, and neutral red uptake assays [30], but insignificant inhibitory against AChE [31]. Recently, the inhibitory activity against BChE of a procyanidin B1 was reported [32]. To the best of our knowledge, this is the first scientific report regarding the cholinesterase inhibitory potential of G. sphacelata fruits.

Docking Studies
In order to get insights on the intermolecular interactions, the most abundant compounds according to the UHPLC chromatogram ( Figure 2) obtained from the pulp and seeds of G. sphacelata as well as the known cholinesterase inhibitor galantamine, were subjected to docking assays into the TcAChE catalytic site and hBChE catalytic site. In order to rationalize their pharmacological results analyzing their protein molecular interactions in the light of their experimental inhibition, activities are shown in Table 3. The best docking binding energies expressed in kcal/mol of each compound are shown in Table 4.  Table 3 showed that the flavonoid quercetin-3-O-glucoside-acetate, and the isoflavones lupinisoflavone and genistein-7-O-di-glucoside displayed the best binding energies of −9.46, −9.36, and −9.18 kcal/mol, respectively. These results suggest that the G. sphacelata pulp or seed extracts inhibitory activity over acetylcholinesterase are mainly due the compounds mentioned above, especially the flavonoid quercetin-3-O-glucoside-acetate.
Pulp and seeds extract presented considerably abilities to exert an inhibitory potency over the TcAChE enzyme (IC 50 = 4.49 ± 0.08 for pulp extract and IC 50 = 4.38 ± 0.04 for seeds extract) considering the known cholinesterase inhibitor galantamine (see Table 3). In this sense, Figure 3 shows the hydrogen bond interactions in a two-dimensional diagram of each main and most abundant compounds determined from both extracts into the TcAChE catalytic site to summarize the information.  Table 3 showed that the flavonoid quercetin-3-O-glucoside-acetate, and the isoflavones lupinisoflavone and genistein-7-O-di-glucoside displayed the best binding energies of −9.46, −9.36, and −9.18 kcal/mol, respectively. These results suggest that the G. sphacelata pulp or seed extracts inhibitory activity over acetylcholinesterase are mainly due the compounds mentioned above, especially the flavonoid quercetin-3-O-glucoside-acetate.
Pulp and seeds extract presented considerably abilities to exert an inhibitory potency over the TcAChE enzyme (IC50 = 4.49 ± 0.08 for pulp extract and IC50 = 4.38 ± 0.04 for seeds extract) considering the known cholinesterase inhibitor galantamine (see Table 3). In this sense, Figure 3 shows the hydrogen bond interactions in a two-dimensional diagram of each main and most abundant compounds determined from both extracts into the TcAChE catalytic site to summarize the information.

Butyrylcholinesterase (hBuChE) Docking Results
All binding energies obtained from docking assays over butyrylcholinesterase (hBChE) of the most abundant compounds in the pulp and the seeds extracts were shown to be poorer compared to those in TcAChE. These results are consistent with the less inhibitory activity of the extracts over this enzyme shown in Table 2 (IC50 = 73.86 ± 0.09 for pulp extract and IC50 = 78.57 ± 0.06 for seeds extract).
Just like in TcAChE, the flavonoid quercetin-3-O-glucoside-acetate exhibited the best binding energy profile, suggesting that this derivative could be the main responsible for the inhibitory activity over the hBChE.

Butyrylcholinesterase (hBuChE) Docking Results
All binding energies obtained from docking assays over butyrylcholinesterase (hBChE) of the most abundant compounds in the pulp and the seeds extracts were shown to be poorer compared to those in TcAChE. These results are consistent with the less inhibitory activity of the extracts over this enzyme shown in Table 2 (IC 50 = 73.86 ± 0.09 for pulp extract and IC 50 = 78.57 ± 0.06 for seeds extract).
Just like in TcAChE, the flavonoid quercetin-3-O-glucoside-acetate exhibited the best binding energy profile, suggesting that this derivative could be the main responsible for the inhibitory activity over the hBChE.
horse serum, 5,5 -dithiobis(2-nitrobenzoic) acid (DTNB), acetylthiocholine iodide, butyrylthiocholine chloride, and galantamine were purchased from Sigma-Aldrich Chemical Company (Santiago, Chile). G. sphacelata fruits were collected in November 2016 in Valdivia, XIV Region de Los Ríos, Chile. The fruits were identified by Jorge Macaya (Universidad de Chile) and a voucher specimen were kept at the Institute of Pharmacy of the Universidad Austral de Chile under number GS20161115.

Fruit Processing
Five g of pulp and seeds, separately, were chopped and lyophilized (FreeZone 2.5 L Labconco, USA). The material was extracted (20% w/v) with a mixture of ethanol-distilled water (1:1 v/v) as solvent (at 25 • C, for two h in an ultrasonic bath). The extract was filtered, and the solvent was evaporated under vacuum at 45 • C. The extract was frozen and lyophilized, until a yield of 327.3 and 125.8 mg of dark brown gums (6.54 and 2.51%) from pulp and seeds, respectively.

UHPLC-PDA-Orbitrap-MS
The Dionex Thermo Scientific Ultimate 3000 UHPLC system connected with a Thermo Q Exactive Focus machine was used as previously informed [33]. Samples were re-dissolved (2 mg/mL) in ethanol-distilled water (1:1 v/v) and 10 µL of filtered solution (PTFE filter) were injected in the instrument, as previously discussed [33].

Total Phenolic (TP) and Total Flavonoid (TF) Content
The total phenolic contents and total flavonoid content of the G. sphacelata fruits were measured using the Folin-Ciocalteu and FeCl 3 method previously described by our work with some modifications [35]. For TP, the results were expressed as mg of gallic acid equivalents per gram of dry fruit. While for the TF content results were presented as mg of quercetin equivalents per gram of dry fruit. The determination was performed using a Synergy HTX microplate reader (Biotek, Winoosky, VT, USA), in triplicate and reported as the mean ± SD.

DPPH Cation Radical Discoloration Test
The method previously reported by Brand-Williams et al., was used to determine radical DPPH scavenging activity. Briefly, 2 mL of the DPPH solution was added in 400 µL of the extract (2 mg/mL) and mixed and 1.10 ± 0.02 at 517 nm absorbance was adjusted with methanol. This homogenized mixture of fruit extract and DPPH solution was then kept in a dark environment for a period of 20 min at room temperature [36]. Finally, the absorbance was calculated at 517 nm. The inhibition percentage was measured by a given formula: where S.A. is sample absorbance and B.A. is used for blank absorbance.

Bleaching Test with the Cationic Radical ABTS •+
The ABTS •+ radical capacity was evaluated using the decolorization method described by Kuskoski et al., 2004 [37]. The ABTS solution (7 mM) and potassium persulfate (2.45 mM) solution were prepared and mixed in a ratio of 1:1. The resultant solution was incubated for 16 h. Using 96-well plates, 275 µL of the solution (absorbance 0.7) was mixed with 25 µL of samples/standard. The absorbances were measured at 734 nm after 6 min of incubation period at 30 • C using a Synergy HTX microplate reader. Results were expressed as µmol Trolox per milliliters.

Ferric Reduction Ability-Antioxidant Power Test (FRAP)
The FRAP test was performed with the previously described protocol by Benzie and Strain, with a slight modification [38]. Briefly, the FRAP solution (2 mL) mixed with 200 µL of extract (2 mg/mL), was stirred and kept in the dark for 5 min. The absorbance was measured at 595 nm.

Superoxide Anion Scavenging Assay
The assay was carried using xanthine oxidase and hypoxanthine, and the absorbances were measured at 560 nm according to the reported with some modifications [13]. The production of the enzyme xanthine oxidase of superoxide anion radical (O 2 − ) reduces the NBT (nitro blue tetrazolium) dye, producing a chromophore that absorbs at 520 nm. The superoxide anion trapping capacities of the extracts were measured using a Synergy HTX microplate reader (Biotek, CA, USA).

Cholinesterases (ChE) Inhibitory Activity
Ellman's method was used to determinate the inhibitory activity of G. sphacelata fruits [39]. DTNB was dissolved in Tris HCl (pH 8) containing 0.02 M of MgCl 2 and 0.1 M NaCl. The sample solution (50 mL dH 2 O, 2 mg L −1 ) was mixed with DTNB (125 mL), acetylcholinesterase (AChE; or BChE) (25 mL), while the blank sample as a control contained all the solutions except enzymes and was distributed in a 96-well microplate. The acetyl-thiocholine iodide (ATCI) or butyryl-thiocholine chloride (BTCl) (25 mL) was added to start the reaction. The reading was taken on a 405 nm absorbance after incubation at 25 • C for 15 min. The cholinesterase inhibitory activity was measured as IC 50 (µg mL −1 , concentration range 0.05 to 25 µg mL −1 ) by subtracting the absorbance of the sample from blank. Galantamine was used as a positive control. All data were collected in triplicate. In addition, 0.26 units/mL of each enzyme were used for the inhibition assays.

Docking Studies
The geometries and partial charges of several representative compounds contained in the extracts, as well as the known cholinesterases (TcAChE-hBChE) inhibitor Galantamine were fully optimized using the DFT method with the standard basis set PBEPBE/6-311 + g* [40,41]. All calculations were performed in the Gaussian 09W software.

Statistical Analysis
All the experiments were repeated five times to confirm the results and minimize the error and the data was presented as the mean of standard deviation. The results were analyzed using one-way analysis of variance (ANOVA) and Tuckey test statistical analysis (p-values < 0.05 were regarded as significant) using the origin Pro 9.0 software package (Origin lab Corporation, Northampton, MA, USA).

Conclusions
From the edible endemic Chilean G. sphacelata fruit seventy metabolites were detected using UHPLC-PDA-Orbitrap-MS analysis including phenolic acids, organic acids, sugar derivatives, catechins, proanthocyanidins, fatty acids, iridoids, coumarins, a benzophenone, flavonoids, and terpenes. G. sphacelata showed a good phenolic content and moderate antioxidant and enzyme inhibitory activities. This report could contribute for the better understanding of chemistry and biological activities in the genus Greigia. Furthermore, the list of compounds profiled with a cholinesterase inhibitory activity plus antioxidant potential make G. sphacelata fruits an interesting source of compounds with interesting properties to prepare functional foods or food derived supplements.