Porophyllum sp. pl. (Asteraceae): chemical compounds obtained by hydrodistillation and supercritical CO2 extraction procedures

Abstract Species of Porophyllum are characterized by the presence of essential oils, with reports of the occurrence of thiophenes. Essential oils have most often been obtained by hydrodistillation while thiophenes are isolated from nonpolar extracts and also by supercritical fluid extraction (SFE). In this study, essential oils from P. ruderale, P. lanceolatum, P. angustissimum and P. curticeps were obtained by hydrodistillation. Porophyllum lanceolatum, collected in greater quantity, was subjected to SFE under different pressures (90–250 bar). Except for P. lanceolatum, which showed high levels of aldehydes such as decanal (60.24%) and dodecadienal (12.14%), all species were rich in hydrocarbon monoterpenes. The fraction obtained by SFE at 90 bar, when analyzed by GC/MS, presented monoterpenes, with limonene as the main component (22.58%) while decanal constituted only 3% of the sample. All the fractions obtained by supercritical extraction contained compounds with chromatographic profile of thiophenes. The compounds, distinguished by intense fluorescence, were isolated by planar circular chromatography and subjected to 1H NMR analyses. The spectra exhibited the characteristic signals of thiophenes. The mass spectra also presented the fragments expected for these compounds. The present data confirm this genus as a producer of potentially active molecules that deserve deeper studies.


Introduction
Porophyllum (Asteraceae) is an American genus distributed from the southeastern United States to central Argentina, currently comprising about 30 species.In Brazil, there are nine species.Of these, seven occur in the south of the country (Carneiro and Ritter 2018).The species of this genus are known as the poreleaf plants.The large glands present in the leaves produce aromatic oils which give the plants a strong taste and pungent smell.Due to these characteristics, some species are valuable as edible plants.
Several species have medical properties in addition to their culinary usefulness.People in Mexico, Central America and South America, for example, utilize P. ruderale as medicine to heal stomach and liver illnesses, control cholesterol and blood pressure, and as anti-inflammatory agent (Conde-Hernández and Guerrero-Beltrán 2014;Vázquez-Atanacio et al. 2021;Pawłowska et al. 2022;Vázquez-Atanacio et al. 2022).Porophyllum tagetoides (Kunth) DC. is also used in traditional medicine to alleviate inflammatory conditions (Jimenez et al. 2012).Due to certain similarities with the traditional European Arnica montana L., various Asteraceae species, including P. ruderale, are commonly known as "arnica" in Brazil and utilized as anti-inflammatory remedies (de Athayde et al. 2021).
Excluding P. ruderale, examined for its thiophenes and essential oil, and P. angustissimum Gardner that was explored for its essential oil (Ferreira et al. 2012), the other species that occur in Brazil were not investigated.Therefore, the purpose of this study was to characterize the essential oils from the aerial parts of Porophyllum ruderale (Jacq.)Cass., Porophyllum lanceolatum DC., Porophyllum curticeps Malme and Porophyllum angustissimum Gardner obtained by hydrodistillation using gas chromatography coupled to mass spectrometry.Porophyllum lanceolatum, collected in higher amount, was also extracted by supercritical CO 2 and searched for the presence of thiophenes.

Plant material
The aerial parts of the Porophyllum spp.were collected in the Rio Grande do Sul state, South Brazil, and voucher specimens were deposited in the herbarium of Universidade Federal do Rio Grande do Sul (ICN).The collection data such as location, collection date, voucher and coordinates of the plants that were used as research material are described in Table 1.

Essential oils isolation
Fresh aerial parts were subjected to hydrodistillation in a Clevenger-type apparatus, for 4 h.The oils were collected and stored in the dark, at −20 °C, until the gas chromatography-mass spectrometry (GC/MS) analyses.The yields were expressed as volume (mL) of essential oil per 100 g of fresh plant material.

GC-MS analyses
For chemical analyses, the samples were diluted in ethyl ether at a ratio 2:100 (v/v).The chemical composition was determined by gas chromatography coupled with mass spectrometry (GC/MS) (Shimadzu QP5000) equipped with a capillary column of fused silica Durabond-DB5 (John Wiley & Sons Scientific, US, 30 m × 0.25 mm × 0.25 μm).The source temperature, quadrupole and injector were set at 230 °C, 150 °C and 220 °C, respectively, using a column temperature programming of 60 °C-300 °C at 3 °C/min.Ultrapure helium was used as the carrier gas at 80 kPa and flow of 1 mL/min.Scan time and mass range were 1 s and m/z 40-550 m/z, respectively, with electron energy of 70 eV.One microliter was injected in the split mode ratio of 1:20.The identification of compounds was based on the comparison of retention indices calculated by linear interpolation relative to retention times of a series of n-alkanes (C 8 -C 32 ), and their mass spectra with authentic samples and with data collected from the literature (Adams 2017) or by comparison with mass spectra recorded in the database of NIST 62 and NIST 12 (National Institute of Technology and Standards).The relative amounts of the components were calculated based on GC peak areas by normalization.The percentage areas were determined without using response factors.

Supercritical fluid extraction of Porophyllum lanceolatum
Extraction with supercritical carbon dioxide was performed in a 0.5 L high-pressure extraction vessel (internal diameter of 5.4 cm), in which the dried and powdered aerial parts of P. lanceolatum (50 g) were inserted.This extraction vessel is part of a pilot unit, described in detail by Scopel et al. (2013), which has pressure, temperature and flow controllers.Pressure values were monitored with a Novus 8800021600 digital transducer system, whose accuracy was ± 1.0 bar.Temperature controllers (PT-100) are positioned on the equipment to measure the temperature of the carbon dioxide inlet and inside the extraction vessel.The control software of this unit was developed using the Elipse SCADA platform developed by the company Elipse Software Ltd.The solvent used was supercritical carbon dioxide with a degree of purity equal to 99.9% (Air Products) which flowed with a flow rate of 1 kg h − 1 measured by a flowmeter test (Sitraus FC Massflo 2100-Siemens) with 0.1% accuracy.The flow of the solvent is carried out by a pump (Thar P − 200, USA).In the extraction experiments, there was no recycling of carbon dioxide.Using the extraction protocol reported in previous studies of the research group (Cargnin et al. 2010;Barros et al. 2011), the plant material was subjected to extraction at temperature of 40 °C and under successively increased pressures (90, 120, 150, 200 and 250 bar), collecting the samples of each pressure after 30 min.The extracts were solubilized with acetone to remove waxes.The acetone soluble fractions were evaporated to dryness.The fractions were analyzed by thin layer chromatography (TLC) on precoated TLC plates (Silica gel 60 F 254, Merck, Germany).The developed TLC plates were visualized at 254 and 365 nm under an ultraviolet lamp.Subsequently, the plates were sprayed with freshly prepared vanillin reagent (5 g vanillin in 9 mL methanol, 0.5 mL H 2 SO 4 and three drops of acetic acid) and heated (Adekunle 2007).

Centrifugal planar chromatography and spectroscopic analysis
Centrifugal planar chromatography (CPC) was carried out on silica gel 60 GF 254 (1 mm plates; Merck, Darmstadt, Germany) using a Chromatotron instrument (model 7924 T, Harrison Research, San Bruno, CA, USA).Analysis of the compounds was performed by TLC using the system mentioned above.Those with a similar profile were pooled and subjected to NMR analysis using a Bruker Ascend 400 (25 °C, 400 MHz for 1 H and 100 MHz for 13 C) spectrometer.Spectra were recorded in CDCl 3 (99.8%;Merck) referenced against a residual non-deuterated solvent (δH 7.27/δc 77.00).Mass spectra of the compounds were also obtained, with the same conditions used for the essential oils analysis.

Results and discussion
Porophyllum ruderale (pápalo) is an herbaceous annual plant that produces edible leaves that are consumed fresh; if they were cooked, they would lose their original flavor.Pápalo is also a part of the traditional medicine of original Mexican cultures.Porophyllum linaria (pipitza, also called pipicha, pepicha, chepiche) is an annual plant that presents aromatic and flavoring leaves.It has a strong taste similar to fresh cilantro with nuances of lemon and anise.It is mainly used as a fresh or dry spice in different Mexican regional dishes (Lazcano-Escobar et al. 2022).Aldehydes and terpenes such as nonanal, decanal, trans-pinene, β-myrcene and limonene were the major compounds found in this plant (Jimenez et al. 2012).The species P. lanceolatum, addressed in this study, according to Kinupp (2007), has a very pronounced flavor and when eaten raw, it causes an excess of gas (burping), therefore, its use is not recommended, at least in this way.This author also cites the use of P. obscurum (Spreng.)DC. as flavoring for soups in the region of Puna, Argentina.

Chemical composition of the essential oils
The essential oils were obtained from fresh aerial parts of Porophyllum species by hydrodistillation, and the essential oil content was determined as 0.2% for P. ruderale, P. angustissimum and P. lanceolatum.Porophyllum curticeps presented the lowest yield, less than 0.1%.The essential oils were analyzed by GC/MS (chromatograms shown in Figures S1-S4, Supplemental material) and the identified compounds with their percentages are presented in Table 2.
A total of 45 compounds were identified among the analyzed essential oils.Monoterpene hydrocarbons were detected as the most abundant chemical class in three of the four samples, accounting for 61.9%, 79.4% and 67.0% in P. ruderale, P. angustissimum and P. curticeps, respectively.The essential oil of P. lanceolatum was characterized mainly by aldehyde hydrocarbons.
There are no previous reports on the composition of essential oil and other components of P. lanceolatum and P. curticeps.Porophyllum angustissimum was investigated only once for its essential oil exhibiting myrcene (40.6%) as the main component accompanied by E-2-dodecenal (37.5%) and limonene (3.4%) (Ferreira et al. 2012).Differently, in the current analysis, β-pinene was the major component.It is noteworthy that E-2-dodecenal was absent from the sample analyzed.
Porophyllum ruderale, on the other hand, is the most well-known and has been the focus of several investigations.This plant is broadly distributed, from the southwestern United States to northern Argentina.Porophyllum ruderale subsp.ruderale, is more widely distributed, being predominantly tropical, extending from Costa Rica to northern Argentina.In Brazil, it occurs in all regions.The other subspecies, P. ruderale subsp.macrocephalum (DC.)R.R. Johnson, has a more northerly distribution, occurring from the extreme southwestern United States to northern Brazil, southern Peru and Bolivia, preferentially in undisturbed areas and at high altitudes (Carneiro and Ritter 2018).Except for variations in their reproductive organs, both subspecies are morphologically fairly similar.Raggi et al. (2015) compared the chemical composition of the two subspecies and observed that in the case of the subsp.macrocephalum, limonene (83.5%) tends to be the compound present in higher concentrations, while in the subsp.ruderale, E-β-ocimene (54.9%) appeared as the main component.According to the authors, these results suggest that the main component of the essential oil could differentiate the two P. ruderale subspecies.
These same authors reviewed the literature regarding the essential oil of P. ruderale and found that the subspecies analyzed were not mentioned.In some cases, the main compound was limonene while in others, the oil was dominated by E-β-ocimene.Nevertheless, some studies found other components, such as sabinene, as the main compounds (Loayza et al. 1999).Isosafrole and α-copaene were also reported but it is noteworthy to point out that these components were obtained through extraction with supercritical fluid (Conde-Hernández et al. 2017).
It is interesting to note that occasionally aldehydes such as decanal, dodecadienal and octadecanal appear in relatively significant concentrations (Neto et al. 1994;Bezerra et al. 2002;Fonsceca et al. 2006).Apparently, aldehydes seem to be the class of compounds that would confer a flavor similar to that of coriander, which makes the plant appreciated as a condiment.Therefore, it is curious that they are not always present in P. ruderale.
As stated before, P. ruderale taste and smell are reminiscent of those of cilantro, arugula and rue, and these characteristics could be attributed to the presence of aldehydes.In fact, the essential oil from the fresh leaves of Coriandrum sativum presents decenal, decanal, dodecenal and tetradecenal among the main components (Kumar et al. 2022).The essential oil of Eruca vesicaria, in turn, is dominated by erucin (an isothiocyanate derivative) and aldehydes such as (E)-12-tetradecenal and pentadecanal (Hichri et al. 2019).Finally, the odor reminiscent of rue described for pápalo is also justified since aldehydes, mainly undecanal, together with ketones, have already been identified as the major components of Ruta graveolens essential oil (Al-Shuneigat et al. 2015).

Supercritical fluid extraction of Porophyllum lanceolatum
Apart from the aldehydes, the contribution of thiophenes to the taste of the pápalo should not be overlooked.Throughout the years, there have been reports of thiophenes in a number of foods, including coffee and onions, among many others, and it is stated that they considerably enhance their flavor (Maga and Katz 1975).Although there are no reports on the role of the compounds that occur in Asteraceae,which can have one thiophene unit or made up of the union of up to five thiophene rings-in the taste of the plants, they could somehow influence the sensory properties.
Thiophenes constitute a small group of secondary metabolites characterized by one to five thiophene rings.These metabolites have been isolated from several Asteraceae genera as Artemisia, Echinops, Eclipta, Pluchea, Tagetes and Porophyllum, among some others.Less frequently they can be found in Apiaceae, genus Ferula, and in actinomycetes and fungi, as well.The compounds are also called acetylenic thiophenes because they are biosynthesized from fatty acids or polyacetylenes via acetylene intermediates.The pharmaceutical, and agrochemical industries might all benefit from the use of these metabolites due to their remarkable antibacterial, antiviral, anti-inflammatory, larvicidal, antioxidant, insecticidal, cytotoxic and nematicidal activities (Ibrahim et al. 2016(Ibrahim et al. , 2022)).
The isolation of thiophenes can be achieved by subjecting plant materials to extraction with nonpolar solvents such as n-hexane and petroleum ether.Nevertheless, the compounds have been selectively extracted by using supercritical fluid.These compounds were obtained from different parts of Tagetes patula by extraction with supercritical CO 2 without modifier co-solvent, at 30 MPa and 40 °C, in 60 min (Szarka et al. 2010).The same conditions were successfully applied to obtain thiophenes from Artemisia absinthium L. (Liu et al. 2019).Considering that thiophenes have already been reported for P. ruderale (Takahashi et al. 2011(Takahashi et al. , 2013)), the species P. lanceolatum, available in larger quantities, was extracted with supercritical CO 2 .Using conditions similar to those reported in literature, five fractions were obtained.The fractions were analyzed by thin layer chromatography using silica gel pre-coated plates as stationary phase and hexane: ethyl acetate 50:50 as mobile phase.The chromatogram was visualized under 365 nm ultraviolet light and later sprayed with vanillin/ sulfuric acid followed by heating.Some thiophenes appear as bluish spots under ultraviolet light and blue-green or blue with vanillin reagent (Adekunle 2007).
All fractions obtained by SFE showed compounds with the chromatographic profile of thiophenes.Nevertheless, the one obtained with a pressure of 90 bar, also presented components characteristic of essential oils.Therefore, this fraction was analyzed by GC (Figure S5 in Supplemental material).Volatile compounds are usually extracted at the lowest pressures.The analysis showed the presence of the following components: β-pinene (6.8%), myrcene (14.1%), limonene (22.6%), β-caryophyllene (3.1%), decanal (3.0%), heptadecanone (11.4%) and hexadecanoic acid (5.5%), in addition to several unidentified minor components, which were characterized as branched long-chain compounds (C26, C28).It appears that the volatile composition obtained with supercritical CO 2 is quite different from that obtained by hydrodistillation, where the major components were decanal and dodecadienal (Table 2).This disparity is mainly due to the different temperature conditions used in the two methods.
The only study with species of the genus Porophyllum in which supercritical CO 2 was used as solvent was carried out with P. ruderale.The essential oil obtained by this process presented isosafrole and α-copaene as the main compounds (Conde-Hernández et al. 2017).As far as it is known, these compounds were not found in oils obtained by hydrodistillation of this species.
The fractions obtained at 150, 200 and 250 bar, in addition to the compounds with chromatographic profile of thiophenes, presented several uncharacterized compounds.Thus, the fraction containing mostly thiophenes (120 bar) was selected to be fractionated.This fraction, showing the presence of strongly fluorescent compounds, was subjected to planar circular chromatography (Chromatotron®).The technique allowed the isolation of three compounds, in very small amounts.The compounds were subjected to NMR analyses.The spectra obtained, only one-dimensional due to the small amount available, did not allow the identification but it was possible to verify the presence of several signals in the aromatic region, characteristic of thiophenes (Ibrahim et al. 2016).
The compounds were also submitted to mass spectrometry.Although this method has been used for the identification and quantitative determination of thiophenes, the conditions used in this study did not allow to identify the compounds.Nevertheless, fragments expected for these compounds were observed (Figures S6 and S7 in Supplemental material).For example, the fragments at m/z 244, 215, 171, observed in the mass spectra of one of the isolated compounds, suggest the structure of a bithiophene.Other compound presented fragment at m/z 166, also suggestive of bithiophenes (Caniato et al. 1990).Actually, a bithiophene was already isolated from P. ruderale (Takahashi et al. 2011).

Conclusions
Several studies have been carried out with P. ruderale, a plant widely used as a flavoring ingredient and especially appreciated in Mexico for seasoning tacos.The majority of research has concentrated on essential oils, but compounds such as thiophenes have been reported.With the exception of this species, the genus has received little attention.
Among the species analyzed in this study, the presence of high concentrations of aldehydes as decanal and dodecadienal in the essential oil of P. lanceolatum is noteworthy.Additionally, compounds of the thiophene class were detected in this species.Even though it was not possible to determine the structure of the thiophenes, the data obtained are meaningful because they confirm this genus as a producer of potentially active molecules that deserve more in-depth investigation.

Table 1 .
collection data of testimonial samples of Porophyllum species collected in rio grande Do sul.

Table 2 .
Percentage composition of essential oils from fresh aerial parts of Porophyllum spp.
b ri: retention index calculated.c Values described by adams (2017).