UHPLC-MS Metabolome Fingerprinting: The Isolation of Main Compounds and Antioxidant Activity of the Andean Species Tetraglochin ameghinoi (Speg.) Speg.

The seriated extracts of petroleum ether (PE-E), dichloromethane (DCM-E) and methanol extracts (MeOH-E) from the aerial parts of the native South American plant Tetraglochin ameghinoi (Rosaceae), were evaluated regarding their antioxidant and antibacterial activities. The antioxidant properties were evaluated by free radical scavenging methods (DPPH and TEAC), ferric-reducing antioxidant power (FRAP) and lipoperoxidation in erythrocytes (LP), while the antibacterial activity was performed against Gram-positive and Gram-negative bacteria according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The chemical and biological analyses of this plant are very important since this bush is currently used in traditional medicine as a cholagogue and digestive. The polar MeOH-E showed the highest antioxidant activities (17.70 µg/mL in the DPPH assay, 381.43 ± 22.38 mM TE/g extract in the FRAP assay, 387.76 ± 91.93 mg TE/g extract in the TEAC assay and 93.23 + 6.77% in the LP assay) and it was selected for chromatographic isolation of its components. These components were found to be four acetophenones, including the new phloracetophenone glucoside: 4′,6′,-dihydroxy-2′-O-(6″-acetyl)-β-d-glucopyranosylacetophenone or IUPAC name: (6-(2-acetyl-3,5-dihydroxyphenoxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-yl)methyl acetate, whose structure was elucidated by NMR and MS methods. In addition, twenty-six compounds, including five of these acetophenone derivatives, two sugars, six flavonoids, eleven phenolic acids and two triterpenes, were identified based on UHPLC-OT-MS and PDA analysis on the MeOH-E. The results support the medicinal use of the plant.


Introduction
The numerous medicinal plants growing in the Andes of Argentina are collected annually by the inhabitants of these regions and are sold in popular local markets for their therapeutic properties. Indeed, a significant percentage of these species belonging to the genera Azorella, Baccharis, Tetraglochin and Senecio are used in local traditional medicine, which mostly aims to treat digestive and hepatic disorders, fevers, coughs and colds.
The  [1]. Recently, T. andina Ciald has been proposed by Acosta et al. [2] as a new species. The aerial parts of T. ameghinoi are employed as infusions and/or decoctions in Andean traditional medicine as cholagogues and digestives to treat hepatic disorders or bacterial infections and food-borne illnesses associated with enterobacteria. In addition, due to their medicinal properties, they are commercially exploited [3]. No previous reports about phytochemical studies and bioactive properties have been reported.
The use of HPLC or UHPLC coupled to hybrid state-of-the-art mass spectrometers, such as quadrupole orbitrap (Q-OT), is becoming a key tool for the rapid analysis of phenolics compounds in plant samples and other biological matrices. A considerable number of Andean species, mainly from Chile, have been recently analyzed using this technology [4][5][6][7][8][9].
The main goals and novelty of this work are the metabolome profiling using a hybrid high resolution mass spectrometer with an orbital trap (Q-Exactive Focus), the isolation of a main new acetophenone compound and the investigation of the antioxidant and antibacterial effects of extracts from the native medicinal plant T. ameghinoi, which have not yet been reported.

Results and Discussion
Seriated extracts (PE-E, DCM-E, EtOAc-E and MeOH-E extracts) prepared from aerial parts of T. ameghinoi were assessed in vitro for antioxidant and antimicrobial properties in addition to total content of phenolics and flavonoids. The following sections contain an explanation of the assays.

Total Phenolic and Flavonoids Content
The MeOH extract (Table 1) showed a high content of phenolic compounds (107 mg GAE/g extract) and among them, 20% corresponded to flavonoids (19 mg QE/g extract). Regarding the EtOAc-E, it is important to note the amount of phenolics (45 mg GAE/g extract) and the lack of any detected flavonoids.
During the last few decades, more than 9000 flavonoids have been identified in plants. The flavonoids have been recognized as chemical compounds with a preponderant role in the human diet, highlighted by their powerful antioxidant activities, which are vital for maintaining good human health and preventing associated diseases from a direct or indirect role from oxidative stress. The versatile health benefits of flavonoids include anti-inflammatory, antioxidant, hepatoprotective, antiproliferative and anticancer activity, free radical scavenging capacity, antihypertensive effects, coronary heart disease prevention and anti-human immunodeficiency virus functions [10,11]. Different flavonoids have been investigated for their potential antiviral activities and several of them exhibited significant antiviral properties in in vitro and even in vivo studies. [12].
The chemical nature of flavonoids depends on their structural class, degree of hydroxylation, glycosylation of hydroxyl groups or of the flavonoids nucleus, other substitutions and conjugations and the degree of polymerization [13].
Oxidative stress mainly caused by reactive oxygen species (ROS) damage normal organs, leading to a gradual loss of vital physiological function. Liver diseases are considered as serious problems, which can be caused by toxic chemicals, drugs, viral infiltration through ingestion or infection and the metabolic or drug/chemical-induced liver damage. Therefore, antioxidants can be used to protect the liver, which act on the inhibition of free radical generation and can decrease the oxidative damage by directly inhibiting the activity or expression of free radical generating enzymes or enhancing the activity or expression of intracellular antioxidant enzymes [16][17][18].
Polar extracts obtained from T. ameghinoi showed strong free radical scavenging activity in the DPPH, FRAP, ABTS and lipid peroxidation in erythrocytes assays ( Table 1). The non-polar extracts PE-E and DCM-E did not have any activity in the antioxidant assays, (Table 1), which is possibly due to the presence of waxes and fats and lack of no phenolic compounds. The DPPH assay is widely used for quickly assessing the ability of polyphenols to transfer labile H atoms to radicals, which is a likely mechanism of antioxidant protection [19]. MeOH and ethyl acetate extracts from T. ameghinoi showed high scavenging activities in the DPPH assay with IC 50 values of 17.0 and 45.0 µg/mL, respectively. This could be related the presence of hydrogen donating compounds, which are more likely to be present in polar extracts. The highest antioxidant capacity detected is consistent with the highest content of total phenolics detected in both extracts. Moreover, a positive significant Pearson's correlation (R 2 = 0.89 at p < 0.01) was found between the total phenolics content and the DPPH activity, which suggests that the phenolic compounds could be responsible for the antioxidant capacity of T. ameghinoi MeOH extract.
Regarding the FRAP assay, the MeOH-E and EtOAc-E showed stronger reducing power with values of 381 and 288 mM TE/g of the extract, respectively. Additionally, both extracts were the most active in the TEAC assay (387 and 154 mg TE/g extract, respectively).
Furthermore, as a cell-based model, lipid peroxidation in human erythrocytes was studied to evaluate the biological relevance of the antioxidant activities of T. ameghinoi polar extracts. The highest activity was found in the MeOH-E. The results showed that the MeOH-E prevented the hemolysis caused by the rupture of cell membranes induced by lipid peroxidation (>95%, at 100 µg/mL).
Flavonoids and phenolics of higher plants are known to be excellent antioxidants in vitro and numerous studies have suggested that dietary intake of plant polyphenol antioxidants may have positive effects in oxidative-stress related pathologies. The strong lipid peroxidation inhibition by T. ameghinoi polar extracts may be related to the presence of phenolic compounds [20][21][22].

Antibacterial Activity
The T. ameghinoi extracts were assayed and found to be antibacterial against the pathogenic bacteria Gram-negative strains (ATCC and clinical isolates of E. coli), and Gram-positive methicillin sensitive (MSSA) and methicillin resistant (MRSA) Staphylococcus aureus strains, such as S. aureus coagulase negative-502 and Streptococcus pyogenes-1. The DCM-E and MeOH-E showed very low activity against Gram-positive bacteria (MIC = 750 µg/mL), excluding the Streptococcus pyogenes-1 strain (Table 1), and null activity against the rest of the bacteria in the panel (MIC values > 1000 µg/mL). Regarding the PE-E, it was not active against any of the bacteria tested (MIC values > 1000 µg/mL, data not shown). Food-borne illnesses associated with Gram-positive bacteria, including the S. aureus enterotoxigenic strains, are a major public health issue worldwide [23].
The extracts did not exhibit relevant antimicrobial activities. The antimicrobial activity is considered very interesting in the case of MICs < 100 µg mL −1 for extracts and 10 µg mL −1 for isolated compounds [24].

UHPLC-OT Analysis of MeOH-E
Since the methanol (MeOH-E) extract showed the highest biological activity, this extract was selected for detailed chemical analysis. Twenty-nine compounds were detected and twenty-six compounds were identified based on the UHPLC OT-MS and PDA analysis on MeOH-E ( Figure 1 and Table 2). Several compounds were acetophenone derivatives (peaks 4, 5, 10, 12 and 15; Figure 2), two were sugars (peaks 1 and 2), several were flavonoids (peaks 16,[22][23][24][25], others were phenolic acids (peaks 6, 7, 9, 11, 13, 14, 17-20 and 28) and two were triterpenes (peaks 21 and 27). Figure 2 shows some of the biosynthetic relationships between the acetophenone compounds detected, while Figure S1 (Supplementary material) shows the full HR-MS spectra and structures of the compounds. The metabolomics identification is explained below in detail.    The choleretic activity in vivo of 4 ,6 -dihydroxy-2 -O-(β-D-glucopyranosyl) acetophenone, isolated from the ethyl acetate extract of Curcuma comosa rhizomes, has been reported [27]. Furthermore, the aglycone of this compound stands out for its powerful choleretic activity showing both high bile flow rate and high bile salt output, which leads to lower plasma cholesterol levels [28].
This can be explained by the increase in the activation of the hepatic 7 α-hydroxylase enzyme, which suggests that the phloracetophenone exerts a "forced" effect on the liver cholesterol, favoring its conversion into bile acids for its subsequent secretion [29]. The major phenolic compounds of T. ameghinoi, including phloracetophenone derivatives possessing choleretic activities, has medicinal uses in traditional Andean medicine to treat liver problems and its use as a cholagogue. was identified as gingerol [33]. The oxidative stress that results from an overproduction and accumulation of free radicals is the leading cause of several degenerative diseases, such as cancer, atherosclerosis, ageing, cardiovascular and inflammatory diseases. Polyphenols form an important class of naturally occurring antioxidants, having innumerable biological activities such as anticancer, antifungal, antibacterial, antiviral, anti-ulcer and anti-cholesterol. Among the various polyphenols, gallic acid has emerged as a strong antioxidant and an efficient apoptosis inducing agent, which is a naturally occurring low molecular weight triphenolic compound [34].
The liver performs a vital role in the metabolism, secretion, storage and detoxification of endogenous and exogenous substances. Oxidative stress and free radicals enhance the severity of hepatic damage, which can be overcome by antioxidants. Plant extracts can be the best source of such antioxidants and mediate hepatoprotective activities.
The hepatoprotective activity of ellagic acid in comparison to silymarin using paracetamol-induced acute liver damage supports the use of this active phytochemical against toxic liver injury, which may act by preventing the lipid peroxidation and augmenting the antioxidant defense system or regeneration of hepatocytes [35].  [7]. Quercetin galloyl glucosides suppress the oxidative metabolism in polymorphonuclear neutrophils at a comparable level to that of quercetin, although the latter was much stronger as an inhibitor of lipid peroxidation. The ortho arrangement of the two hydroxyls groups (free catechol grouping) in the B ring of the flavonoids, quercetin galloyl glucoside and quercetin, support their similar antioxidative properties. The activity-lowering effect of glucosidation at C-3 of the aglycone is cancelled out by the presence of the galloyl group, which is known for its antioxidative properties [37]. Some epidemiological studies have found a positive association between the consumption of foods containing kaempferol and a reduced risk of developing several disorders, such as cancer and cardiovascular diseases. Numerous preclinical studies have shown that kaempferol and their glycosides have a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial, anticancer, cardioprotective, neuroprotective, antidiabetic, anti-osteoporotic, estrogenic/antiestrogenic, anxiolytic, analgesic and anti-allergic activities [38].

Triterpenes
Pentacyclic triterpenoids, which are widely distributed in the plant kingdom, have been extensively reported to possess protective effects against drug-induced organ toxicities, including those of chemotherapeutic agents [40]. inhibitor [42], respectively. The anti-inflammatory effects of madecassic acid have been reported, which is thought to occur via the suppression of the NF-κB pathway in LPS-induced RAW 264.7 macrophage cells [43]. The results suggest that the anti-inflammatory properties of madecassic acid are caused by iNOS, COX-2, TNF-alpha, IL-1beta and IL-6 inhibition via the downregulation of NF-κB activation in RAW 264.7 macrophage cells.
For the structural identification of the compounds, 1 H-NMR spectra were recorded at 400 MHz and 13 CNMR were obtained at 125 MHz on a Bruker spectrometer (δ scale). The TMS was employed as the internal standard. The two-dimensional experiments (COSY, HSQC, HMBC and ROESY) were applied using standard sequences. The ESI-HRMSs spectra were recorded on an orbitrap (Q-OT) mass spectrometer. The melting points were determined on a Kofler hot stage apparatus (Electrothermal 9100) and were uncorrected. The IR spectra were recorded on a Nicolet Nexus FT-IR instrument. The identification and quantification of phenolic compounds was done by a UHPLC-Q-OT-HESI-MS/MS. A Thermo Scientific Dionex Ultimate 3000 UHPLC system controlled by the Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, USA), which was hyphenated with a Thermo high resolution Q Exactive focus mass spectrometer (Thermo, Bremen, Germany). Nitrogen (purity > 99.999%) obtained from a Zefiro nitrogen generator (Clantecnologica, Sevilla, España) was employed as both the collision and damping gas. All calibration and equipment parameters were set as previously reported . The LC parameters were as follows: the column used was a UHPLC C18 column (Acclaim, 150 × 4.6 mm ID, 5 µm, Restek Corporation, Bellefonte, PA, USA) operated at 25 • C. The detection was set at 254, 280, 320 and 440 nm, while the PDA from 200-800 nm was recorded. The mobile phases were 1% formic aqueous solution (A) and acetonitrile with 1% formic aqueous solution (B). The gradient program time (min), (% of B) was as follows: (0.00, 5); (5.00, 5); (10.00, 30); (15.00, 30); (20.00, 70); (25.00, 70); (35.00, 5) and 12 min for column equilibration. The flow rate was set at 1.00 mL min −1 , while the injection volume was 10 µL. The standards and extracts dissolved in methanol were maintained at 10 • C in the autosampler. The HESI II and other parameters for the Q-orbitrap instrument were optimized as previously reported [6].
The PE-E (2.8 g) was chromatographed on a silica gel column (length 30 cm, internal diameter 4.5 cm) with 1.2 L of PE:EtOAc (ethyl acetate) 70:30 v/v to 0:100 v/v gradient, followed by MeOH. Fourteen fractions of 100 mL each were obtained. The fractions yielded 535 mg of ursolic acid acetate.
The Fraction F5 (487 g) was applied to a silica gel column (length 50 cm, internal diameter 3 cm) and eluted with EtOAc:MeOH gradients. One hundred fractions of 10 mL each were obtained. After TLC comparison (silica gel, dichloromethane: methanol, 95:5 as the mobile phase; detection under UV light and spraying with p-anisaldehyde), the fractions with similar TLC patterns were combined.

Determination of Total Phenolics (TP) and Flavonoids (F) Content
The total phenolics (TP) and flavonoids (F) content of the extracts were determined by Folin-Ciocalteu and AlCl 3 colorimetric methods, respectively [47,48]. The TP were expressed as grams of gallic acid equivalents (GAE) per 100 g of extracts (g GAE/100 g extract). F were expressed as g of quercetin equivalents (QE) per 100 g of extracts on (g QE/100 g extracts). The values from triplicates were reported as the mean ± SD.

DPPH Scavenging Activity
Free radical scavenger activity on DPPH free radical scavenging effects were assessed by the procedure previously described in Reference [49]. The scavenging activities were evaluated at 517 nm in a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA). The analyses were performed in triplicate and values were reported as EC50 mean ± SD; being EC50, the extracts' concentration provided 50% of radicals scavenging activity. Quercetin was used as a reference compound.

Ferric-Reducing Antioxidant Power Assay (FRAP)
FRAP assay was performed in accordance to a previous study [50] with some modifications. Briefly, the FRAP solution was freshly prepared by mixing 10 mL of acetate buffer with a concentration of 300 mM at a pH of 3.6, 1 mL of ferric chloride hexahydrate with a concentration of 20 mM dissolved in distilled water and 1 mL of 2,4,6-tris(2-pyridyl)-s-triazine with a concentration of 10 mM dissolved in HCl with a concentration of 40 mM. A total of 10 µL of the sample solution was mixed with 190 µL of the FRAP solution and placed in 96-well microplates, which was performed in triplicate. The results were obtained by linear regression from a calibration plot obtained with Trolox (0-1 mmol L −1 ). All samples were analyzed in triplicate. The results were expressed as mM TE/g extract.

Trolox Equivalent Antioxidant Activity (TEAC) Assay
The TEAC assay was performed in accordance to a previous study [51] with minor modifications. Briefly, 10 µL of the sample or Trolox standard was mixed with 200 µL of ABTS •+ (dissolved in PBS). The vortex was mixed for 10 s and the absorbance at 734 nm after a 4 min reaction at 30 • C was measured. The results were obtained by linear regression from a calibration plot constructed with Trolox (0-1 mmol L −1 ) and are expressed in TEAC values [52]. The TEAC value of samples is equivalent to the concentration of a Trolox solution. All samples were analyzed in triplicate. The results were expressed as mg TE/g extract.

Lipid Peroxidation in Human Erythrocytes
The evaluation of lipid peroxidation in human erythrocytes was carried out as described by a previous study [49] with minor modifications. Human red blood cells obtained from healthy adult individuals were washed three times in cold phosphate buffered saline (PBS) by centrifugation at 3500 rpm. After washing, the cells were suspended in PBS, regulating the density to 1 mM hemoglobin in each reaction tube. The final cell suspension was incubated with different concentrations of the test compounds and dissolved in DMSO and PBS for 10 min at 37 • C. The final concentration of samples and controls in DMSO was 1%. After incubation, the cells were exposed to tert-butylhydroperoxide (1 mM) for 15 min at 37 • C under vigorous shaking. After this, the lipid peroxidation was determined indirectly by the TBARs formation. The results are expressed as a percentage of inhibition compared to the controls. Each determination was performed in quadruplicate.
An antibacterial susceptibility test was conducted, in which the minimal inhibitory concentration (MIC) values were determined using the broth microdilution method according to the protocols of the Clinical and Laboratory Standards Institute previously reported in a previous study [24]. The bacteria inoculum employed was 5 × 10 5 CFU/mL. The stock solutions of extracts in the DMSO were prepared to give serial two-fold dilutions to obtain the final concentrations of 0.98-1000 µg/mL. Cefotaxime (Argentia ® ) was included in the assays as a positive control. The plates were incubated for 24 h at 37 • C. The activity was evaluated at 620 nm using a Multiskan FC instrument. The MIC values were defined as the lowest extract concentrations showing no bacterial growth after the incubation time. Tests were done in triplicate.

Statistical Analysis
Determinations of TP, TF, TA, DPPH, FRAP, and TEAC were performed in triplicate and the results are expressed as mean values ± SD. The results were analyzed by one-way ANOVA and significant differences between mean values were determined by Duncan's test (p < 0.05). The statistical package InfoStat 26 was used for statistical analyses. Pearson's correlation analysis was also used.

Conclusions
The identification of the main antioxidant compounds by UHPLC Orbitrap (Q-OT) and the isolation of major acetophenones derivatives in addition to the strong free radical scavenging activity of this plant supports its ethnopharmacological use to treat hepatic disorders and as a cholagogue in Andean traditional medicine to a certain extent. Further investigations are required focused to determine the potential pharmacognostical/pharmacological effects of these native plant extracts as natural sources for the preparation of phyto-pharmaceutical products.

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