Phytochemical analysis in Alternanthera brasiliana by LC-MS/MS and GC-MS

Abstract Alternanthera brasiliana is popularly known as ‘penicillin’ and used as an anti-inflammatory and for wound healing. The objective of this work was the phytochemical study of the species by analytical techniques such as LC-MS/MS and GC-MS. Twenty-seven compounds were identified, five by LC-MS and 22 by GC-MS analysis. All compounds identified by LC-MS are flavonoids. However, several classes were found in GC-MS analysis, such as hydrocarbons, diterpenes, monoterpenes, vitamin and carotenoid derivatives, phytosterols and triterpenes. In the analysis, it is suggested the presence of 27 substances, of which 23 are unpublished for the species, that reaffirms the importance of this study and the contribution to chemical knowledge.


Introduction
Alternanthera brasiliana (L.) KUNTZE is popularly known in Brazil as 'penicillin' and belongs to Amaranthaceae family. It is used in folk medicine for treatment of infections and wound healing (Caetano et al. 2002); it's also used as an ornamental plant due to the purple coloration that is characteristic of their leaves, when shade cultivated (Silva 2013). Antibacterial (Silva et al. 2011), antioxidant, photoprotective (Silva et al. 2014), wound healing (Barua et al. 2012) and antiproliferative (Brochado et al. 2003) activities have already been reported for this species.
Several chemical constituents have already been isolated and identified from A. brasiliana, among them flavonoids, most of which are glycosylated flavones (Brochado et al. 2003), betalains as betacyanins, which are pigments that guarantee the characteristic color of many species of Amaranthaceae family, various hydrocarbons, hydrocarbon derivatives and oxylipins (Silva et al. 2005;Deladino et al. 2017;Trapp et al. 2015). However, it is believed that species who grow in distinct habitats may have different chemical composition. Thus, this study sought to analyze the crude ethanolic extract of A. brasiliana, native from Brazilian Caatinga biome, by analytical techniques to determine its chemical constituents.

Results and discussion
From the analysis performed, the presence of five compounds is suggested from LC-MS/MS analysis and twenty-two from GC-MS. Of this total, only four constituents had been reported for the A. brasiliana species, the other twenty three are being described for the first time. The constituents were encoded from Ab-1 to Ab-27.

LC-MS/MS analysis
The total ion chromatograms (TIC) in negative and positive mode can be seen in Figure S1. All compounds identified in LC-MS analysis were flavonoids (Ab-1 to Ab-5). The identification was by comparison with the literature published and by fragmentation proposals. Information pertinent to identification of Ab-1 to Ab-5 can be seen in Table S1.
Ab-1, 2, 3, 4 and 5 presented similar fragmentations as can be seen in figures S2, S3, S4, S5 and S6 respectively. After the protonation or deprotonation of the molecule, the fragmentation follows with the exit of the outermost part of the disaccharide, always a rhamnose, with a neutral loss (À146 Da). After this loss, another fragmentation common to these molecules was the exit of the remaining portion of the disaccharide, always a glucose, also as a neutral loss (À162 Da), remaining the aglycone, with peaks registered in the spectra. Dehydrations were observed for Ab-1 to Ab-5 always after the exit of first portion of disaccharide, with a loss of 18 Da, and for Ab-1, 2 and 5, a second dehydration could easily happen after the first one (À18 Da). For Ab-1 to Ab-5, after the first dehydration, a Retro Diels-Alder segmentation occurred, with loss of a neutral fragment with minor mass (À102, À88 or À104 Da). Therefore, the peaks were recorded on the mass spectrum with the permanence of the radicals m/z 329 for Ab-1, m/z 313 for Ab-2, m/z 355 for Ab-3, m/z 343 for Ab-4, and m/z 325 for Ab-5, ending the fragmentation stages.
Another initial fragmentation pathway was observed for Ab-3 and Ab-5, starting with two dehydration in the outermost portion of the disaccharide (À36 Da). After that, a Retro Diels-Alder segmentation can occur in the remaining portion of the disaccharide, with loss of a neutral fragment (À86 Da, for both), leaving a radical with m/z 485 (Ab-3) and m/z 469 (Ab-5), with peaks recorded in the mass spectrum.

GC-MS analysis
In an attempt to identify the largest possible number of substances in A. brasiliana CEE, it was proceeded with GC-MS analysis. The chromatogram ( Figure S7) obtained revealed the presence of 44 peaks, of which 22 were identified (70.0% relative intensity) based on similarity index with other spectra in libraries available in the software.
From the analysis of Table S2 it can be seen that most of the compounds are hydrocarbons or their derivatives (33.44%), such as linoleic, myristic, palmitic and stearic acid. Next, there are the phytosterols (12.18%), which are represented by clionasterol, stigmast-7-ene-3-ol and stigmast-4-en-3-one. Attaining 9.30% of identified constitution are the diterpenes or their derivatives, as is the case of phytol, phytone and neofitadiene. The triterpenes are in 7.25% of the constitution and as representatives of this class were identified b-amyrin and a-amyrin. The last class with significant representation among the constituents identified is from vitamin derivatives (5.88%), which are a-tocopherol (vitamin E) and its acetylated derivative. Other compounds with minor representation are from the classes of monoterpenes or derivatives (1.46%, as neryl acetone) and the carotenoid derivative (0.49%, as dihydroactinidiolide).
As can be observed in the analysis and from the comparison with compounds already identified or isolated from A. brasiliana, it is possible to verify that, of the 22 substances identified, 20 are being reported for the first time for the species. Only n-hexadecanoic acid, also known as palmitic acid (major compound in chromatogram) and octadecanoic acid (stearic acid) had already been described.

Conclusions
The present study sought to analyze the crude ethanolic extract of A. brasiliana by different chromatographic analytical techniques in order to determine its major chemical constituents. From GC-MS analysis, 22 chemical constituents were identified, of which 20 were unpublished for the species. In the analysis by LC-MS/MS, 5 compounds were identified, 3 never before described for the species, which reaffirms the importance of the study and the contribution to the chemical knowledge.