Microwave-Assisted Extraction of Secondary Metabolites Using Ethyl Lactate Green Solvent from Ambrosia arborescens: LC/ESI-MS/MS and Antioxidant Activity

Alternative solvents are being tested as green solvents to replace the traditional organic solvents used in both academy and industry. Some of these are already available, such as ethyl lactate, cyrene, limonene, glycerol, and others. This alternative explores eco-friendly processes for extracting secondary metabolites from nature, thus increasing the number of unconventional extraction methods with lower environmental impact over conventional methods. In this context, the Peruvian Ambrosia arborescens was our model while exploring a microwave-assisted extraction (MAE) approach over maceration. The objective of this study was to perform a phytochemical study including UHPLC-ESI-MS/MS and the antioxidant activity of Ambrosia arborescens, using sustainable strategies by mixing both microwaves and ethyl lactate as a green solvent. The results showed that ethyl lactate/MAE (15.07%) achieved a higher extraction yield than methanol/maceration (12.6%). In the case of the isolation of psilostachyin, it was similar to ethyl lactate (0.44%) when compared to methanol (0.40%). Regarding UHPLC-ESI-MS/MS studies, the results were similar. Twenty-eight compounds were identified in the ethyl lactate/MAE and methanol/maceration extracts, except for the tentative identification of two additional amino acids (peaks 4 and 6) in the MeOH extract. In relation to the antioxidant assay, the activity of the ethyl lactate extract was a little higher than the methanol extract in terms of ORAC (715.38 ± 3.2) and DPPH (263.04 ± 2.8). This study on A. arborescens demonstrated that the unconventional techniques, such as MAE related to ethyl lactate, could replace maceration/MeOH for the extraction and isolation of metabolites from diverse sources. This finding showed the potential of unconventional methods with green solvents to provide eco-friendly methods based on green chemistry.


Introduction
Ambrosia arborescens Miller is an aromatic medicinal plant that belongs to the Asteraceae family.This species is common in Peru, Colombia, Ecuador, and Bolivia at altitudes between 2000 and 3500 m above sea level.It is commonly known as "marco" and its conservation status is common and abundant.Its leaves are used externally in traditional Plants 2024, 13, 1213 2 of 11 medicine for rheumatism and muscle pain [1].Extraction of secondary metabolites from plants requires various isolation techniques because the plants' metabolite content is generally low, and extraction techniques are often the bottleneck in natural product chemistry.These techniques can be divided into conventional and unconventional.The conventional techniques, which include maceration, percolation, and Soxhlet extraction, are the most widely used, and they are characterized by high cost, long extraction times, and the use of toxic organic solvents.Unconventional or modern techniques include ultrasound, microwave, enzyme extraction, supercritical fluid extraction, and high pressure, which are characterized by lower cost, shorter extraction time, and energy conservation [2].Chemat et al. (2012) propose the following definition for green extraction of natural products: "Green extraction is based on the discovery and development of extraction processes that reduce energy consumption, allow the use of alternative solvents and renewable natural products, and ensure a safe and high-quality extract or product".With this in mind, there are six principles for environmentally friendly extraction of natural products that all chemists should be aware of in order to follow an environmentally conscious path in natural product chemistry: 1. Innovate by selecting and using varieties of renewable plant resources.2. Use alternative solvents, especially water or agro-solvents.3. Reduce energy consumption through energy recovery and use of innovative technologies.4. Generate byproducts instead of waste involving bio-and agro-refining industry.5. Reduce operation units and promotion of safe, robust and controlled processes.6. Aim for a non-denatured and biodegradable extract without impurities [3].
Alternative solvents have been tested as green solvents to replace the traditional organic solvents used in both in academy and industry.A green solvent should be biodegradable, recyclable, non-toxic, non-volatile, cheap, and readily available, such as ethyl lactate, cyrene, limonene, glycerol, deep eutectic solvents, and water [2].Many natural product chemists base their research on considering the health of the planet.Thus, the implementation of ecopharmacognosy proposed by Cordell (2015) will be incorporated into the process of future natural product research [4].It is known that the main ecological challenge in phytochemistry is the use of solvents due to the risk of environmental pollution [4,5].This fact has opened up the search for more efficient processes in combination with more environmentally friendly solvents, whose negative impact is lower.In this sense, many pharmaceutical companies such as Pfizer, GlaxoSmithKline, AstraZeneca, and Sanofi have developed guidelines and new processes in this field.
Based on this context, and continuing our research on green chemistry applied to plant natural products, we now report the results of a phytochemical study of green extracts of Ambrosia arborescens Miller obtained using the green solvent ethyl lactate and microwaves.Finally, all extracts were verified by UHPLC/ESI/MS/MS.

Extraction Using Conventional or Non-Conventional Techniques
Powdered samples of A. arborescens (50 g) were extracted with MeOH and ethyl lactate, using conventional or non-conventional techniques.Maceration was used in the first case and microwaves in the second.Extraction with methanol and ethyl lactate (MAE) yielded 12.5% and 15.0%, respectively.These results suggest that ethyl lactate could replace methanol as an extraction solvent despite its higher cost.Thus, these results suggest that the non-conventional technique based on MAE is better than maceration.

Isolation
To isolate the main compound, crude extracts (CM and NCM) obtained after solvent evaporation were fractionated by both flash column chromatography (SiO 2 ) and CC (SiO 2 ).Psilostachyin 1 was isolated from methanol (maceration) and ethyl lactate (MAE) extracts, in yields of 0.40% and 0.44%, respectively.Psilostachyin was identified based on spectroscopic analysis ( 1 H-NMR).The chemical structure of psilostachyin is shown in Figure 1.This result shows that the isolation of psilostachyin with green solvents is comparable to that of Plants 2024, 13, 1213 3 of 11 methanol in terms of yield.On the other hand, the extraction of metabolites from plants usually requires the use of organic solvents such as hexane, dichloromethane, acetone, methanol, or their mixtures.Hexane is used for the extraction of lipophilic metabolites, and it is the first choice due to its low price.Methanol is preferred because of its polar compounds.However, hexane and methanol have negative effects on the environment and living organisms.An alternative to the use of organic solvents for extraction could be ethyl lactate and heptane or their mixtures [6,7].
extracts, in yields of 0.40% and 0.44%, respectively.Psilostachyin was identified based on spectroscopic analysis ( 1 H-NMR).The chemical structure of psilostachyin is shown in Figure 1.This result shows that the isolation of psilostachyin with green solvents is comparable to that of methanol in terms of yield.On the other hand, the extraction of metabolites from plants usually requires the use of organic solvents such as hexane, dichloromethane, acetone, methanol, or their mixtures.Hexane is used for the extraction of lipophilic metabolites, and it is the first choice due to its low price.Methanol is preferred because of its polar compounds.However, hexane and methanol have negative effects on the environment and living organisms.An alternative to the use of organic solvents for extraction could be ethyl lactate and heptane or their mixtures [6,7].

Nitrogen-Containing Compounds
Peak 4 and peak 6 were identified by HRMS and MS/MS, respectively.Fragments of amino acids were detected, including leucine-hexose (m/z 292.1420; 130.0875) or its isomer isoleucine-hexose and phenylalanine (m/z 164.0713; 147.0492) [8,9].These compounds were found in the methanolic extract and not in the ethyl lactate extract.

Hydroxybenzoic Acid and Its Derivatives
Peaks 7, 8, and 18 showed a deprotonated molecule at m/z 315.0745 (C 13 H 15 O 9 ), in almost all cases.In the MS/MS, ions at 153.0200 corresponding to dihydroxybenzoic acid fragments were observed, indicating the presence of dihydroxybenzoic acid hexosides.Vanillic acid hexoside was assigned to peak 9 based on the losses of the aglycone (m/z 167.0351).Galloyl dihexoside (peak 13) showed one molecular ion at m/z 493.1188 and its daughter ions at m/z 331.0668 (loss of hexoside) and m/z 169.0149 (loss of two hexosides) [8,9,14].All these hydroxybenzoic acids were found in both methanol and ethyl lactate extracts.

Antioxidant Activity
Flavonoid content, total phenolics, and antioxidant activity were evaluated by the Folin-Ciocalteau method and the ferric trichloride method, while antioxidant activity was expressed as FRAP, ferric reducing antioxidant power, ORAC, oxygen radical absorbance, and DPPH bleaching capacity (Table 2).MeOH extraction by maceration (CM) showed FRAP ferrous reduction ability (711.43 ± 23.2 mg/g extract), followed by ethyl lactate extraction by MAE (NCM, 705.22 ± 15.42 mg Trolox/g extract).In terms of DPPH quenching capacity, the extract of NCM (263.04 ± 2.9) was more effective than that of CM (180.47 ± 2.6).These results are in agreement with the ORAC assay (Table 2).Our results are in agreement with those from antioxidant plants, but lower in relation to fruits [15,16].As you can see in Table 2, the TPC of NCM (ethyl lactate/MAE) is almost twice as low as the TPC of CM (MeOH/maceration), while the TFC of NCM is 2.5 times lower than that of CM.In this sense, even though NCM uses a green solvent, it produces more yield than CM.These are general data and do not take into account the real extraction capacity of both methodologies.Moreover, the FRAP potency is similar between the two extracts, while ORAC capacity is higher for the NCM (715.38 ± 3.2) than for the CM (541.13 ± 4.8), with the same trend occurring in the case of the quenching of the DPPH radical (Table 2).Therefore, this means that the green NCM extract can differentiate between and extract more antioxidant compounds than the MeOH extract.In terms of yields, both extracts are comparable; however, the green extract was a little higher (15.0%), thus having more antioxidant power.
From the point of view of green extraction techniques and green isolation, the use or substitution of toxic solvents was minimal.In our case, psilostachyin was isolated from a 50 g sample of Peruvian A. arborescens using ethyl lactate (green solvent) with mixtures of n-heptane (GRAS solvent), representing a new transition from classical to green isolation in natural product chemistry.Although the yields were comparable to those of methanol, this is the first report of the application of green chemistry to A. arborescens.
The green extraction process is one important step for alternative phytochemical analysis in natural product chemistry.This process determines the isolation, identification, and composition of secondary metabolites prior to their assessment by analytical techniques such as LC-ESI/MS/MS.In recent years, a combination of unconventional techniques with green solvents has been used to promote a substantial reduction of toxic solvents in favor of producing higher quality extracts.A promising method includes MAE with ethyl lactate solvent; this extraction is simple, ecological, and low-energy and involves reduced time for extraction and low consumption of solvents for the extraction of compounds from natural sources.In this context, MAE has demonstrated itself to be an advantageous method for the extraction with the following influential factors: volume of the solvent, microwave power, extraction time, matrix, and temperature.All works reported in the literature include a combination of these factors to obtain caffeine and polyphenols from green tea; phenolic acids, flavonoid and its glycosides from Hibiscus sabdariffa; polyphenols and flavonoids from Cynara scolymus; pectins from orange peel; polyphenols in quercus shells; tannins and flavonoids from Myrtus communis leaves; polyphenols of stems, flowers, and seeds from Ocimum basilicum; and oleuropein from olive leaves [23,24].
In relation to the use of ethyl lactate as an alternative solvent, it shows a direct relationship with the green criterion, and it follows green chemistry principles.Ethyl lactate is biodegradable with an oral LD 50 over 2000 mg/kg, with properties such as low surface tension, low vapor pressure, and high boiling point.Thus, it can replace organic solvents such as acetone, xylene, toluene, methyl ethyl ketone, or other toxic solvents for many applications including paints, cleaning of graffiti, composites, and adhesives, among others [25].Therefore, the use of ethyl lactate is increasingly available in academic laboratories as alternative extracting agent to traditional solvents.In recent years, the use of ethyl lactate for extraction and separation has increased, showing the potential use of this solvent in natural product chemistry, such as for carotenoids and their congeners, tocopherol, sclareol, quercetin, rutin, caffeine, amino acids, and phenols from lichens [26][27][28][29][30][31][32][33].
The use of unconventional techniques, together with green solvents, could open new avenues for the extraction and isolation of secondary metabolites from plants, in order to provide eco-friendlier processes.

Maceration Extraction
The powdered sample of Ambrosia arborescens (200 mg) was added to 10 mL of methanol at room temperature with constant stirring (200 rpm) and left for 24 h.After centrifugation (9000× g, 30 min), the methanolic solution was evaporated in vacuum to yield 25.07 ± 0.4 mg of a dark extract (12.6 ± 0.21%).Extractions were performed in triplicate following previous work [34].

Microwave-Assisted Extraction (MAE)
The powdered samples of A. arborescens (200 mg) were placed in 10 mL of ethyl lactate.They were then put in a microwave reactor (Anton Parr, Buchs, Switzerland) with adjustable parameters (100 • C, 30 min, 15 W).After centrifugation at 9000× g for 30 min, the supernatant was evaporated under vacuum to yield a dark gummy extract (29.93 ± 1.2 mg; 15.0 ± 0.62%).Extractions were performed in triplicate following previous work [34].

Extraction and Isolation
Maceration and MAE in green solvents were used to prepare extracts, along with conventional organic solvents to fractionate and purify the main compound for comparative purposes.

UHPLC-APCI-HRMS/MS Instrument
The chemical identity of the target compounds was determined through a Dionex UltiMate 3000 HPLC system (Thermo Scientific, Waltham, MA, USA) connected to a highresolution tandem mass spectrometry (HRMS/MS) detector with atmospheric pressure chemical ionization (APCI) source (Impact HD mass spectrometer Bruker, Billerica, MA, USA) (HPLC-APCI-HRMS).Some 2 mg of extract (obtained of the point 3.3 and 3.4) was dissolved in methanol (2 mL) and then filtered (PTFE filter), and finally 10 µL was injected into the instrument, as previously described [34,36].

Polyphenol and Flavonoid Contents
The determination of total phenolic compounds (TPC) was performed following protocols previously described [8,36].Approximately 12 µL of the extract and 168 µL of 1% Folin-Ciocalteu reagent (Merck) were added to a well of a microplate reader.The mixture was allowed to react for 5 min, and then 120 µL of 10% Na 2 CO 3 was added.The mixture was incubated at room temperature for 30 min in darkness.Then, the absorbance was measured at 765 nm using a UV-visible multiplate reader (Synergy HTX, Billerica, MA, USA).The content of phenolic compounds was then expressed as micromoles of gallic acid per gram of dry weight (µmol GAE/g extract).The AlCl 3 method was used to determine the total flavonoid content.For this assay, 30 µL of sample (2 mg/mL) was added to 159 µL of 5% NaNO 2 .After a 5-min rest period, 18 µL of 10% AlCl 3 was added to the mixture.At the sixth minute, 18 µL of 1 M NaOH was added, and the absorbance was measured at 510 nm using a UV-visible multiplate reader (Synergy HTX, Billerica, MA, USA).The flavonoid content (TFC) was calculated using a quercetin standard calibration curve (25-150 ppm).Results were expressed as quercetin micromoles per gram of dry sample (µmol Q/g dry weight).

DPPH Test
The DPPH radical was measured by the decolorization method [8,36].Briefly, 9 µL of green or methanol extract (2 mg/mL) and 341 µL methanolic DPPH solution (400 µM) were adjusted with methanol to an absorbance of 1.10 ± 0.02 at 517 nm.The mixture was mixed and left to react in the dark at room temperature for 20 min, after which the time absorbance at 517 nm was measured.The percentage of discoloration of the DPPH radical was determined by measuring the change in absorbance at 517 nm, and the values obtained were converted to percentage inhibition of the DPPH moiety.A curve was plotted with different dilutions of the extract, and the results were expressed as IC 50 in µg/mL.

Ferric Reducing Antioxidant Power Test (FRAP)
The methodology was performed as published with slight modifications [8,36].Briefly, 10 µL of the solubilized extracts (2 mg/mL) were mixed with 290 µL of the FRAP solution, mixed in the wells of the microplate, and left to react in the dark at room temperature for 5 min.Absorbance measurement of the stained Fe-TPTZ complex was performed at 595 nm.The absorbance data were fitted to the Trolox standard curve equation (µmol/L).Results were calculated as Trolox equivalents (TE), in mg Trolox per gram dry weight (mg Trolox/g dry weight).

Oxygen Radical Absorbance Capacity (ORAC) Test
ORAC activity was determined according to the method previously described [8,36].Fluorescein (3 ′ ,6 ′ -dihydroxyspiro [2-benzofuran-3,9 ′ -xanthene]-1-one) was prepared as stock solution (4 µM) in 75 mM phosphate buffer pH 7.4, stored at 4 • C, and used as fluorescent probe.AAPH reagent (203.4 mg) was freshly prepared in 15 mL of 75 mM phosphate buffer and used to generate the peroxyl radical.Trolox was used as an internal standard.In all experiments, 150 µL of fluorescein was added to each well.The blank wells received 25 µL of phosphate buffer, the standards received 25 µL of Trolox dilution, and the samples received 25 µL of sample.Excitation was monitored at 485 nm and emission at 528 nm with a band pass of 20 nm on an HTX Multi-Mode Microplate Reader.Results were determined by a quadratic regression equation (Trolox/sample vs. fluorescence decay curves) and expressed as µM Trolox equivalents per 100 g of dry plant.

Statistical Analysis
Results were reported as the mean ± S.D. In all experiments, statistical differences between samples and controls were checked by one-way analysis of variance (ANOVA) followed by Dunnett's test.The significance level was set at p < 0.01.All statistical analyses were executed using GraphPad Prism 6 for Windows.

Conclusions
MAE was both used in combination with ethyl lactate to produce extracts and compared with a conventional method such as maceration to isolate and purify the main component of the Peruvian A. arborescens.Our study showed that the isolation of psilostachyin by MAE/ethyl lactate was comparable to maceration/methanol, based on the yield obtained (0.40-0.44%).In the metabolomic profile by UHPLC/ESI/MS/MS, the ethyl lactate and methanol extracts were similar in terms of secondary metabolites (in all, twenty-eight), except for the identification of two amino acids (peaks 4 and 6) in the MeOH extract.Finally, in relation to the antioxidant assay, the activity of the ethyl lactate extract was a little higher than the methanol extract in terms of ORAC (715.38 ± 3.2) and DPPH (263.04 ± 2.8).These unconventional techniques, together with the use of green solvents, could replace toxic solvents for the extraction and isolation of secondary metabolites from plants and provide more sustainable methods based on green chemistry.

Table 1 .
LC-ESI-MS/MS identification of secondary metabolites from Peruvian A. arborescens.

Table 1 .
LC-ESI-MS/MS identification of secondary metabolites from Peruvian A. arborescens.

Table 2 .
Total phenolic content, total flavonoid content, and antioxidant activity (FRAP, ferric reducing antioxidant power; ORAC, oxygen radical absorbance capacity; and DPPH bleaching capacity) of different extractions from Ambrosia.
All values are expressed as means ± SD (n = 3).* micrograms per gram of plant.Values in the same column marked with the same letter are not statistically different (p < 0.05).