UHPLC-HESI-OT-MS-MS Biomolecules Profiling, Antioxidant and Antibacterial Activity of the “Orange-Yellow Resin” from Zuccagnia punctata Cav.

This research was designed to investigate the metabolite profiling, phenolics, and flavonoids content as well as the potential antioxidant and antibacterial, properties of orange-yellow resin from Zuccagnia punctata Cav (ZpRe). Metabolite profiling was obtained by a ultrahigh resolution liquid chromatography orbitrap MS analysis (UHPLC-ESI-OT-MS-MS). The antioxidant properties were screened by four methods: 2,2-diphenyl-1-picrylhydrazyl assay (DPPH), trolox equivalent antioxidant activity assay (TEAC), ferric-reducing antioxidant power assay (FRAP), and lipid peroxidation in erythrocytes (LP)). The antibacterial activity was evaluated according to the Clinical and Laboratory Standards Institute (CLSI) rules. The resin displayed a strong DPPH scavenging activity (IC50 = 25.72 µg/mL) and showed a percentage of inhibition of LP close to that of the reference compound catechin (70% at 100 µg ZpRe/mL), while a moderated effect was observed in the FRAP and TEAC assays. The resin showed a content of phenolic and flavonoid compounds of 391 mg GAE/g and 313 mg EQ/g respectively. Fifty phenolics compounds were identified by ultrahigh resolution liquid chromatography orbitrap MS analysis (UHPLC-PDA-OT-MS) analysis. Thirty-one compounds are reported for the first time, updating the knowledge on the chemical profile of this species. The importance of the biomolecules identified support traditional use of this endemic plant. Furthermore, additional pharmacological data is presented that increase the potential interest of this plant for industrial sustainable applications.


Introduction
The resins are nonvolatile products of plants, which include surface resins, naturally secreted by plants or internal resins, which can be obtained or collected from incisions. Their chemical composition includes flavonoids, terpenoids, and fatty substances that in some cases are protective barrier for the

Z. punctata Orange-Yellow Resin (ZpRe)
The orange-yellow resin was obtained by dipping fresh aerial parts (500 g; 4L of dichloromethane grade HPLC, 1 min; the extraction procedure was done three times), filtered and evaporated under reduced pressure to yield a semisolid yellow-orange resin (10 ± 1% yield w/w). The ZpRe was stored in a freezer at −40 • C until its use to bioassays, phenolics, and flavonoids identification/quantification as well as in ultrahigh resolution liquid chromatography orbitrap MS analysis (UHPLC-PDA-OT-MS) analysis.

LC Parameters and MS Parameters
Liquid chromatography was performed using an UHPLC C-18 column (150 × 4.6 mm Acclaim, ID, 2.5 µm; Thermo Fisher Scientific, Bremen, Germany) at 25 • C, hyphenated with a Thermo Q-Exactive MS focus (Thermo, Bremen, Germany) was used. The detection wavelengths were 330,280, 254, and 354 nm, and photodiode array detectors were set from 200-800 nm. Solvent delivery was performed at 1 mL/min using ultra-pure water supplemented with 1% formic acid (A) and acetonitrile with 1% acid formic (B) and a program starting with 5% B at zero time, then maintained 5% B for 5 min, then changing to 30% B within 10 min, then maintaining 30% B for 15 min, then going to 70% B for 5 min, then maintaining 70% B for 10 min, and finally returning to 5% B in 10 min. and keeping this condition for twelve additional minutes to achieve column stabilization before next injection of 20 µL. For the analysis, 5 mg of the resin was dissolved in 2 mL of methanol, filtered through a 200-µm PTFE (polytetrafluoroethylene) filter, and 20 µL was injected in the instrument. Standards and the resin dissolved in methanol were kept at 10 • C during storage in the autosampler. The HESI II and Orbitrap spectrometer (Thermo, Bremen, Germany) parameters were optimized as previously reported [16,17]. Additionally, relevant experimental parameters have been reported recently in detail [18] The Q-Exactive 2.3 SP 2, Xcalibur 2.4 and Trace Finder 3.3 (Thermo Fisher Scientific, Bremen, Germany) were used for UHPLC mass spectrometer control and data processing, respectively.

Total Phenolic (TP) and Flavonoid (F) Content
The total phenolics and flavonoid content of ZpRe was determined by employing total phenols assay by Folin-Ciocalteu reagent and flavonoids by AlCl 3 assay, both in microplate [18]. The total phenolic was expressed as milligrams of gallic acid equivalents (GAE) per gram of extracts (mg GAE/g ZpRe). Flavonoids were expressed as milligrams of quercetin equivalents (QE) per gram of extracts (mg QE/g ZpRe). The values were obtained using a Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA), and are showed as the mean ± standard deviation (SD). The Capacity of ZpRe to 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging (DPPH) was run by the following procedure: DPPH solution (20 mg/L) in methanol was mixed with ZpRe solution at concentrations of 1, 5, 10, 50, and 100 µg/mL [18]. The reaction progress absorbance of the mixture was monitored at 515 nm using a Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA). The percentage of the DPPH decoloration was proportional to the five antioxidant concentrations, and the concentration responsible for a decrease in the initial DPPH concentration by 50% was defined and calculated as EC 50 value, which is showed as the mean ± SD.

Ferric-Reducing Antioxidant Power Assay (FRAP)
The FRAP assay was run in microplate, as previously reported methodology [18,20]. Briefly, FRAP reagent and a methanolic solution of ZpRe resin (1 mg/mL) were mixed; simultaneously, a calibration curve was prepared by mixing FRAP reagent and Trolox solutions, at concentrations between 0 and 1 mmol/L. The absorbance values of mixtures were obtained in a Multiskan FC Microplate Photometer Results were obtained by linear regression from the FRAP-Trolox calibration plot and are showed in equivalent milligrams Trolox/g ZpReresin.

Trolox Equivalent Antioxidant Activity Assay (TEAC)
TEAC assay was run in microplate, as following the previously reported methodology [18,21]. Briefly, a ZpRe methanolic solution was mixed with 200 µL of ABTS, measuring their absorbance at 734 nm after 4 min. Results were obtained by linear regression from a calibration curve constructed with Troloxand are showed expressed as equivalent milligrams Trolox/g ZpRe resin.

Lipid Peroxidation in Erythrocytes
The ability of the ZpReresin at three concentrations (100, 250, and 500 µg/mL) and of catechin at a single concentration (100 µg/mL) to inhibit lipoperoxidation in erythrocytes (LP), induced by tert-Butyl hydroperoxide, was determined. Relevant technical aspects of the trial have been reported recently in detail [15,18]. The values obtained are expressed as percentages of lipid oxidation inhibition.

Antibacterial Susceptibility Testing
Minimum inhibitory concentration (MIC) of ZpRe and antibiotic Cefotaxime (Argentia ® , Buenos Aires, Argentina) was carried outby broth microdilution techniques, in according to CLSI [22]. The ZpRe was tested from 0.98 to 3000 µg/mL.using an inoculum of each bacterium adjusted to 5 × 10 5 cells with colony forming units (CFU)/mL. The absorbances at 620 nm were determined in a Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, MA, USA).

Statistical Analysis
The Duncan's test from InfoStat edition 2016 software (Universidad Nacional de Córdoba, Argentina) was run to determine potential significant differences (p < 0.05) in the carried out assays

UHPLC-PDA-OT-MS Analysis of the Orange-Yellow Resin From San Juan Province, Argentina
Fifty-one compounds were detected in ZpRe by UHPLC-PDA-OT-MS analysis, combining full mass spectra and MS n experiments, of which fifty were tentatively identified including flavonoids, chalcones, caffeic acid derivatives, coumaric acid esters, naphthoquinone, xanthene's derivatives, trichocethenes; vedelianin derivatives, and others. Several phenolics compounds from ZpRe were rapidly identified using available standards. Thirty-one not previously reported updated the chemical composition of this species. The identification of unknown phenolic compounds xanthene's characteristics of this bioactive plant was possible from comprehensive analysis of the full scan mass spectra, base peaks chromatograms, and data-dependent scan experiment, since the orbitrap provided high-resolution and accurate mass product ion spectra from precursor ions that are unknown before and within a single run.
The molecular formula was obtained through high resolution accurate mass analysis (HRAM) and matching with the isotopic pattern. The acquisition of the data in the UHPLC-PDA-OT-MS analysis was developed using electrospray negative mode, because compounds with a phenolic OH lose easily the proton in electrospray ionization, giving very good and diagnostic parent ions and fragments.
The metabolome identification of the 50 tentatively identified compounds is developed below, highlighting the relevant information of the 31 compounds that are new reports for the species (See Figures 1-3, Table 1, and Supplementary Material Figure S1 for some representative compounds and spectra S1 and S2). Fifty-one compounds were detected in ZpRe by UHPLC-PDA-OT-MS analysis, combining full mass spectra and MS n experiments, of which fifty were tentatively identified including flavonoids, chalcones, caffeic acid derivatives, coumaric acid esters, naphthoquinone, xanthene's derivatives, trichocethenes; vedelianin derivatives, and others. Several phenolics compounds from ZpRe were rapidly identified using available standards. Thirty-one not previously reported updated the chemical composition of this species. The identification of unknown phenolic compounds xanthene's characteristics of this bioactive plant was possible from comprehensive analysis of the full scan mass spectra, base peaks chromatograms, and data-dependent scan experiment, since the orbitrap provided high-resolution and accurate mass product ion spectra from precursor ions that are unknown before and within a single run.
The molecular formula was obtained through high resolution accurate mass analysis (HRAM) and matching with the isotopic pattern. The acquisition of the data in the UHPLC-PDA-OT-MS analysis was developed using electrospray negative mode, because compounds with a phenolic OH lose easily the proton in electrospray ionization, giving very good and diagnostic parent ions and fragments.
The metabolome identification of the 50 tentatively identified compounds is developed below, highlighting the relevant information of the 31 compounds that are new reports for the species (See Figures 1, 2 and 3, Table 1, and Supplementary Material Figure S1 for some representative compounds and spectra S1 and S2)

Total Phenolic and Flavonoid Contents and Antioxidant, and Antibacterial Activities
The orange-yellow resin from Z.punctata (ZpRe) displayed a stronger DPPH scavenging activity with an EIC50 25.72 µ g/mL, as well as an outstanding inhibition of lipid peroxidation in erythrocytes (70% percent at 100 µ g ZpRe/mL), this was comparable to the value shown by the reference    [7,10]; in the same way, peak 22 was assigned to 2-methyl-3-(3-hydroxy-4 -methoxyphenyl)-propyl caffeic acid ester, while peak 23 is supported by its mass properties as an isomer of compound 21; peak 28 was tentatively identified as recognized caffeic acid phenetyl ether; peak 42 was identified as 3,7-dimethyl-2,6-octadienyl caffeic acid ester; and peak 45 was identified as 3,7-dimethyl-2,6-octadienyl caffeic acid ester.

Total Phenolic and Flavonoid Contents and Antioxidant, and Antibacterial Activities
The orange-yellow resin from Z.punctata (ZpRe) displayed a stronger DPPH scavenging activity with an EIC 50 25.72 µg/mL, as well as an outstanding inhibition of lipid peroxidation in erythrocytes (70% percent at 100 µg ZpRe/mL), this was comparable to the value shown by the reference compound catechin (74% at 100µg/mL) ( Table 2) and to the values shown recently by others Andean species [14,15,18]. Phenolic antioxidant compounds acting as free radical scavengers can delay or inhibit lipid oxidation processes. This protection of polyunsaturated fatty acids against free radical damage may explain or supports phenolic compounds as a valuable natural product with potential to improve human health [36] Regarding FRAP and ABTS results, the ZpRe exhibited moderate effect in both trials. The ZpRe presented a high content of TP, with values of 391 mg GAE/g ZpRe, where as approximately eighty percent correspond to flavonoids (313 mg QE/g ZpRe). Table 2. Antioxidant assays and total phenolic and flavonoids content of ZpRe from Z. punctata. Results of antibacterial activity are depicted in Table S1 (Supplementary Material).The ZpRe showed activity against Gram-positive bacteria, including Staphylococcus aureus methicillin-resistant ATCC 43300, S. aureus methicillin-resistant-MQ-1, S. aureus methicillin-resistant-MQ-2, S. aureus methicillin-sensitive ATCC 25923 and Streptococcus pyogenes (MICs values were between 125 and 250 µg/mL). However, the ZpRe resin was not active against most of the other strains tested (MIC values >250 µg/mL).
However, the full UHPLC-MS identification of thirty one biomolecules for the first time in this species (peaks 2-7, 13-16, 19, 23, 25, 26, 31-33, 36-41, 43, 44, 46-51), some of them showing a broad spectra of pharmacological properties, including antioxidant and antimicrobial, canprovide additional and relevant support for the activities displayed by ZpRe resin. Figure 3 and Figure S2 show the structures of some newly reported compounds in the resin of this plant.
Pharmacological activities of naringenin (2), as therapeutic agent to treat different diseases, such as cancer, diabetes, cardiovascular diseases and neurological disorders, oxidative stress and inflammation have been extensively reported [37,38]. Additionally, antibacterial activity against Salmonella thypi, Staphylococcus aureus, and Escherichia coli ATCC as well as their antinociceptive and anti-inflammatory effect in mice model, have been also reported [39][40][41][42]. Furthermore, the free radical scavenging and antioxidant properties havebeen associated with improvements experienced by rats with diabetes type I treated with naringenin [43,44]. The protective effect against metabolic diseases of naringenin is supported by its ability to scavenging some free radicals, by its ability to induce antioxidant enzymes and targeting on phosphoinositide 3-kinase/protein Kinase B/nuclear factors [38]. These mechanisms are involved in the neuroprotective effect recently reported by Chandran et al. [45].
Shikonin (3) have demonstrated a broad spectrum of relevant biological activities such as, antioxidant, anti-inflammatory, antithrombotic, antimicrobial, wound healing effects, as well as neuroprotective effects against cerebral ischemia/reperfusion injury associated or supported to its antioxidant properties [46][47][48][49][50][51]. On the other hand, shikonin inhibited the proliferation of three human pancreatic cancer cell lines, and potentiated synergistically the cytotoxic effect of the gemcitabine a chemotherapeutic drug [52]. Results of antibacterial activity are depicted in Table S1 (Supplementary Material).The ZpRe showed activity against Gram-positive bacteria, including Staphylococcus aureus methicillin-resistant ATCC 43300, S. aureus methicillin-resistant-MQ-1, S. aureus methicillin-resistant-MQ-2, S. aureus methicillin-sensitive ATCC 25923 and Streptococcus pyogenes (MICs values were between 125 and 250 µ g/mL). However, the ZpRe resin was not active against most of the other strains tested (MIC values >250 µ g/mL).
Compound 25 (2-hydroxy-4-methoxychalcone) has been reported as antiangiogenic, antitumoral, glutathione S-transferase inhibitor, and as therapeutic agent to treat atherosclerosis. In addition, several epidemiological studies support the idea that regular consumption of fruits and vegetables rich in flavonoids reduces the risk of cardiovascular diseases [57][58][59].
In the other hand, the strong antibacterial activity of blestriarene B (36) and its antibacterial activity against Streptococcus mutans and Staphylococcus aureus (with MICs values of 12.5 and 6.25 µg/mL respectively) has been also reported [33].
Vedelianin (40) has been recently reported as a potent antiproliferative agent against several cancer cell lines [60,61]. The potential of morusin (49) against human colon rectal cancer and human cell lung cancer has been informed by Chang Lee et al. [62] and Park et al. [63]. Also, the cytotoxic activity of lupinifolin (46) in the cell line P-388 [35] was reported.
The tribenoside glyvenol, (38) was used clinically for hemorrhoidal disease associated with coagulation, inflammation, and wounds. Kikkawa et al. (2010) [64] reported that tribenoside interacts with epidermal cells and regulates the expression and localization of laminins to help reconstruct basement membranes in wound healing of hemorrhoids. Evidence exists to recommend the use of tribenoside as a fast, effective and safe option for the local treatment of low-grade hemorrhoids [65].
Caffeic acid phenethyl ester (CAPE, 28) is a bioactive compound of propolis and the exudate extract. It is known that CAPE possesses antioxidant, anti-inflammatory, anticancer, and cytotoxic properties and is a versatile therapeutically active polyphenol and an effective adjuvant of chemotherapy [67][68][69][70][71].

Conclusions
Fifty phenolics compounds were identified by ultrahigh resolution liquid chromatography orbitrap MS analysis (UHPLC-PDA-OT-MS). Thirty-one are reported for the first time, updating the knowledge of the chemical profile of this species. The importance of the biomolecules identified support its traditional use. Herein, more scientific data on bioactivity and chemistry is showed for this plant that increase its potential for sustainable applications and industrial interest that Z. punctata offers, a species that grows in semi-arid Andean areas.