Untargeted Metabolomics by Using UHPLC–ESI–MS/MS of an Extract Obtained with Ethyl Lactate Green Solvent from Salvia rosmarinus

: Salvia rosmarinus (Lamiaceae), previously known as Rosmarinus ofﬁcinalis , is a plant cultivated worldwide, native to the Mediterranean region. Its leaves are traditionally used for cooking. This species possesses numerous biological activities, including antioxidant, antimicrobial, anticancer, anti-inﬂammatory, and hepatoprotective properties. These biological properties are due to the presence of phenolic compounds, including rosmarinic acid and phenolic diterpenoids, such as carnosic acid and carnosol. In this study, we investigated the chemical composition of a green extract obtained by maceration with ethyl lactate for the ﬁrst time. Seventy-ﬁve compounds were tentatively identiﬁed by UHPLC–ESI–MS/MS, including six organic acids, six cinnamic acid derivatives, ﬁve fatty acids, eighteen ﬂavonoids, and thirty-eight terpenoids. Thus, abietane-type diterpenoids from the ethyl lactate extract were the predominant diterpenoids in the Chilean S. rosmarinus species, in contrast to the Chinese species, in which labdane and isopimarane-type diterpenoids were found for the ﬁrst time. Finally, our study conﬁrms that the extraction of S. rosmarinus with green ethyl lactate as a solvent is efﬁcient and sustainable for the identiﬁcation of ﬂavonoids, phenols, and terpenoids from leaves.


Introduction
Organic solvents are widely used for dissolving, diluting, and dispersing waterinsoluble substances, and as a medium for organic synthesis and extraction in natural product chemistry [1].Despite the warnings against their use due to exposure and environmental pollution, their continued use is inevitable.In recent decades, many researchers have focused on reducing the use of volatile organic solvents by introducing green solvents into their processes [2].Green solvents have shown to be promising candidates and good alternatives to petrochemical solvents because they are derived from crops and are environmentally friendly [3].Ethyl lactate (EL) is considered to be a green solvent derived from corn.Chemically, it is the ester of lactic acid.EL is biodegradable, non-corrosive, non-carcinogenic, and non-ozone depleting.It is mainly used in the food, pharmaceutical, and cosmetic industries.Therefore, if it is not possible to replace volatile organic solvents, their use should be optimized, and one should try to recycle them [4][5][6][7].However, to our knowledge, there are very few studies on the use of ethyl lactate as an extracting solvent in natural product chemistry.Therefore, we decided to use ethyl lactate for the extraction of secondary metabolites.
UHPLC, coupled with orbitrap technology as a mass analyzer, provides high resolution, sensitivity, high mass accuracy, and a powerful separation of metabolites in natural extracts; therefore, it is the most commonly used form in metabolomic studies.It can also determine the elemental composition of parent and daughter ions in the structural elucidation of organic compounds [20,21].For R. officinalis, some LC-MS/MS reports have previously been published, showing the presence of flavonoids and their glycosides, phenols, phenolic diterpenoids, and pentacyclic triterpenoids (Table 1).Leaves Supercritical fluid 29 LC-Q/TOF-MS [32] This study aims to determine and evaluate the chemical composition of an S. rosmarinus extract, obtained by maceration in the green solvent ethyl lactate using UHPLC-ES-MS/MS to initiate the transition from toxic organic solvents to green solvents.

Plant Material
Salvia rosmarinus L., was collected in Talca, VII Region, Chile.Leaves were dried at room temperature in darkness.S. rosmarinus was identified by Prof. O. Garcia, and a voucher specimen (N • RO-2015/1215) was preserved in the Laboratory of Herbarium of Extreme Natural Products of the University of Chile.

UHPLC-ESI-MS/MS Conditions for Analysis
The Ultimate 3000 UHPLC system (Thermo Scientific Dionex), equipped with a quaternary pump and Ultimate 3000 series TCC-3000RS column compartments, with a Ultimate 3000 series WPS-3000RS autosampler and a PDA detector controlled by Chromeleon 7.2 software (Thermo Fisher Scientific, Waltham, MA, USA, and Dionex Softron GmbH Part of Thermo Fisher Scientific, Bremen, Germany), coupled with a high-resolution Thermo Q Exactive focus mass spectrometer (Thermo, Bremen, Germany) were employed for analysis.The chromatographic unit was coupled with the MS with a heated electrospray ionization source II (HESI II).Nitrogen (purity > 99.999%) was used as both a collision and damping gas.Mass calibration was performed once a week, in both negative and positive modes, to ensure a working mass accuracy of 5 ppm or less.Caffeine and N-butylamine (Sigma Aldrich, Saint Louis, MO, USA) were the calibration standards for positive ions and buspirone hydrochloride, while sodium dodecyl sulfate and taurocholic acid sodium salt (Sigma Aldrich, Saint Louis, MO, USA) were used for mass-spectrometer calibration.These chemicals were dissolved in a mixture of acetic acid, acetonitrile, water, and methanol (Merck, Darmstadt, Germany) and were infused using a Chemyx Fusion 100 syringe pump (Thermo Fisher Scientific, Bremen, Germany).XCalibur 3.0 (Thermo Fisher Scientific, Bremen, Germany) and Trace Finder 3.2 (Thermo Fisher Scientific, San José, CA, USA) software were used for UHPLC control and data processing, respectively.Q Exactive 2.0 SP 2, from Thermo Fisher Scientific, was used to control the mass spectrometer.
The HESI parameters included a sheath gas flow rate of 75 units, an auxiliary gas flow rate of 20, a capillary temperature of 400 • C, an auxiliary gas heater temperature of 500 • C, a spray voltage of 2500 V (for ESI), and an S-lens at RF stage 30.Full scan data in the positive and negative regions were acquired at a resolving power of 70,000 FWHM (full width at half maximum) at m/z 200.A scan range of m/z 100-1000 was chosen for the compounds of interest: the automatic gain control (AGC) was set at 3 × 10 6 and the injection time to 200 ms.The scan rate was set to 2 scans s −1 .External calibration was obtained with a calibration solution in positive and negative polarity.For confirmation, a targeted MS/MS analysis was performed using the mass inclusion list with a time window of 30 s, with the Orbitrap spectrometer operating in both positive and negative modes at 17,500 FWHM (m/z 200).The AGC target was set to 2 × 10 5 , and the maximum injection time was 20 ms.The precursor ions were filtered through the quadrupole, which operated with an isolation window of m/z 2. The pre-vacuum, high vacuum, and ultra-high vacuum were maintained at about 2 mbar, from 10 5 and below 10 10 mbar, respectively.The higher energy collisional dissociation (HCD) cell was operated at 30 kV.Detection was based on the calculated exact mass and the retention time of the compounds.The mass tolerance window was set at 5 ppm for both modes.

Metabolomic Profiling Using UHPLC-ESI-MS/MS
Ethyl lactate was chosen because there is little information on an environmentally friendly extraction agent and to reduce the negative effects of the toxic organic solvents used.The high-resolution, accurate mass via Orbitrap used in this study yielded the identification and preliminary characterization of seventy-five compounds (Figure 1; Table 2), including organic acids, cinnamic acids, flavonoids, and terpenoids.As shown in Table 1, the solvents previously used in LC/MS studies are methanol, or their mixtures.Here, ethyl lactate was used for the first time for S. rosmarinus.(full width at half maximum) at m/z 200.A scan range of m/z 100-1000 was chosen for the compounds of interest: the automatic gain control (AGC) was set at 3 × 10 6 and the injection time to 200 ms.The scan rate was set to 2 scans s −1 .External calibration was obtained with a calibration solution in positive and negative polarity.For confirmation, a targeted MS/MS analysis was performed using the mass inclusion list with a time window of 30 s, with the Orbitrap spectrometer operating in both positive and negative modes at 17,500 FWHM (m/z 200).The AGC target was set to 2 × 10 5 , and the maximum injection time was 20 ms.The precursor ions were filtered through the quadrupole, which operated with an isolation window of m/z 2. The pre-vacuum, high vacuum, and ultra-high vacuum were maintained at about 2 mbar, from 10 5 and below 10 10 mbar, respectively.The higher energy collisional dissociation (HCD) cell was operated at 30 kV.Detection was based on the calculated exact mass and the retention time of the compounds.The mass tolerance window was set at 5 ppm for both modes.

Metabolomic Profiling Using UHPLC-ESI-MS/MS
Ethyl lactate was chosen because there is little information on an environmentally friendly extraction agent and to reduce the negative effects of the toxic organic solvents used.The high-resolution, accurate mass via Orbitrap used in this study yielded the identification and preliminary characterization of seventy-five compounds (Figure 1; Table 2), including organic acids, cinnamic acids, flavonoids, and terpenoids.As shown in Table 1, the solvents previously used in LC/MS studies are methanol, or their mixtures.Here, ethyl lactate was used for the first time for S. rosmarinus. .

Fatty Acids
Five fatty acids were detected and tentatively identified.Peaks  ) were not identified.Many toxic organic solvents have been identified as causing negative environmental impacts, pollution, and potential harm to humans.Fortunately, natural product extraction processes consider environmental safety by using a combination of environmentally friendly technology and solvents.The use of ethyl lactate as a solvent in the extraction of natural products for the untargeted analysis of extracts is not widely used, but it is considered to be a potential green solvent for the extraction of hydrophilic and lipophilic phytonutrients [40].In some studies, ethyl lactate was used as a solvent for the decaffeination of green tea, preserving the content of catechins [41,42].In addition, it has shown a higher extraction capacity of α-mangotin in Garcinia mangostana [43], carotenoids in dried tomatoes, luteolin, and β-carotene from powders of white corn and carrots [44].Other studies have shown that ethyl lactate is an efficient solvent for the extraction of polyphenols, such as caffeic acid, protocatechuic acid, kaempferol, quercetin, chrysin, orientin, and apigenin, as well as the alkaloid lupamine, which has high antioxidant and antibacterial activity [45].Many toxic organic solvents have been identified as causing negative environmental impacts, pollution, and potential harm to humans.Fortunately, natural product extraction processes consider environmental safety by using a combination of environmentally friendly technology and solvents.The use of ethyl lactate as a solvent in the extraction of natural products for the untargeted analysis of extracts is not widely used, but it is considered to be a potential green solvent for the extraction of hydrophilic and lipophilic phytonutrients [40].In some studies, ethyl lactate was used as a solvent for the decaffeination of green tea, preserving the content of catechins [41,42].In addition, it has shown a higher extraction capacity of α-mangotin in Garcinia mangostana [43], carotenoids in dried tomatoes, luteolin, and β-carotene from powders of white corn and carrots [44].Other As shown in Table 1, the analysis of LC/MS/MS, previously performed on methanolic extracts of R. officinalis leaves, showed the presence of the following: organic acids, including quinic acid, syringic acid, vanillic acid, gallic acid, protocatechuic acid, and hydroxybenzoic acid with their hexosides [22][23][24][25]27,28]; cinnamic acid derivatives, such as caffeic acid and its derivatives, chlorogenic acids, p-coumaric acid, salvianolic acid B, and rosmarinic acid and its hexoside [22][23][24][25][26][27][30][31][32]; flavonoids, such as luteolin and its derivatives, nepitrin, apigenin and its hexosides, acacetin, quercetin and its derivatives, eriodictyol, and kaempferol and its hexosides [22][23][24][25][26][27][28][29][30][31][32]; some phenolic diterpenes or their derivatives, including carnosic acid, rosmanol and its derivatives, epirosmanol, rosmadial, and carnosol [22][23][24][25][26][27][28][29]; and triterpenes, such as oleanolic acid, ursolic acid, betulinic acid, asiatic acid, and micromeric acid [22,23,[30][31][32].In this study, the qualitative analysis of the green ethyl lactate extract revealed the presence of 6 organic acids, 6 cinnamic acid derivatives, 19 flavonoids, 37 phenolic diterpenes, and 1 triterpene.One group of abundant secondary metabolites were diterpenoids, including abietane skeletons, especially carnosol and carnosic acid, and polyphenolic compounds, such as luteolin and rosmarinic acid.Although the secondary metabolites in S. rosmarinus from different sites were almost similar, the profiles of flavonoids, phenolic compounds, and terpenoids showed great differences.Therefore, the use of a green solvent, such as ethyl lactate proved to be efficient for qualitative analysis, as the 75 metabolites were tentatively identified in negative mode.
Some studies based on green strategies have been applied to R. officinalis.Wang et al. [57] studied ultrasound-assisted extraction coupled with high-speed countercurrent chromatography (HSCCC) separation using hydrophobic deep eutectic solvents (DESs).They found that, among the studied DESs, D,L-menthol: D,L-lactic acid, 1:2 was the best extraction agent, but not as a HSCCC solvent.Similarly, Vladimir-Knezevic et al. [58] showed that DESs, such as choline chloride (ChCl): ethylene glycol (EG), 1:3 at 50% water, gave the same yields as 70% ethanol for phenolic acid extraction.Meanwhile, 70% ethanol was most effective for flavonoid extraction from R. officinalis compared to water, 70% ChCl: EG and 50% ChCl: EG.Kessler et al. [59] compared two extraction methods from Portuguese R. officinalis: hydrodistillation (HD) and supercritical fluid extraction (SFE)-CO 2 .They demonstrated and confirmed that the essential oils from the SFE-CO 2 extraction were higher than those obtained from the HD extraction and, after defining the safety profile, they can be used to improve bread odor with these green extracts.Finally, Chen et al. [60] reported that they used SFE-CO 2 as the first step of the purification of R. officinalis.Then, the remaining solid was subjected to an isolation process, which revealed the presence of labdane (six) and isopimarane (five) diterpenoids for the first time.Among them, seven diterpenoids were identified as new diterpenoids after nuclear magnetic resonance (NMR) and MS/MS analyses.In addition, rosmarinusin J; M; O; labda-8( 14), 12E, 15-triene-18-acid and (E)-geranylferulic acid showed cytoprotective activity against H 2 O 2 -induced oxidative damage to SH-SY5Y cells.In this study, neither labdane nor isopimarane diterpenoids were found in the Chilean species.
Our study confirms that the extraction of S. rosmarinus with the green solvent ethyl lactate is efficient and sustainable for the identification of flavonoids, phenols and terpenoids from the leaves.

Conclusions
Green solvents are a good alternative to toxic organic solvents because they are environmentally friendly.Among them, ethyl lactate, which is considered a green solvent, is biodegradable, non-corrosive, non-carcinogenic, and non-ozone depleting.In this work, a green extract of the plant Salvia rosmarinus, formerly known as Rosmarinus officinalis, was prepared by maceration as a conventional technique, in combination with ethyl lactate Then, the chemical composition of this extract was investigated for the first time by UHPLC-ESI-MS/MS.The obtained results showed that seventy-five compounds were tentatively identified by untargeted metabolomics study, including six organic acids, six cinnamic acid derivatives, five fatty acids, eighteen flavonoids, one triterpene, and thirty-seven phenolic diterpenes.This result shows that the extraction of phenolic diterpenoids with ethyl lactate is better than that with toxic organic solvents (Table 1).Many diterpenoids, such as hydroxyrosmanol, hydroxyrosmadial, hydroxyepirosmanol, hydroxycarnosic acid, salvicanaric acid methyl ester, acetoxycarnosic acid, hydroxydeoxocarnosol, desoxycarnosic acid, and prenylated dihydroxycarnosic acid, were detected for the first time in this species.Further isolation efforts should be made to confirm the molecular structures of these diterpenoids.Finally, ethyl lactate could be used for the extraction of secondary compounds as an alternative to toxic solvents to enable more sustainable extraction processes.

*
First report in the specie S. rosmarinus.

Table 1 .
Metabolomic profiles of R. officinalis samples by using LC-MS reported.

Table 2 .
Metabolomic profiles of ethyl lactate extract from S. rosmarinus.
* First report in the specie S. rosmarinus.