GC-MS and HPLC-ESI-MS-MS Characterization of Sanchezia oblonga (Acanthaceae) Extracts

The genus Sanchezia (Acanthaceae) comprises neotropical herbs and shrubs with showy flowers. Sanchezia oblonga (syn. S. nobilis) is a shrub of the rainforests of central and south America. The ethanolic extracts of leaves and stems from S. oblonga were analyzed by GC-EI-MS and RPHPLC-DAD-ESI-MS/MS. Fatty acids (free and esterified) and phytosterols were detected by the former method. Benzyl alcohol glycosides (21 and 25), sinapic acid glycoside esters (29 and 31), ethyl rosmarinate (24), sinapic acid-O-glucoside (28), dihydrosinapic acid-O-glucoside (26), catechin-O-arabinoside (36), in addition to flavonols glycosides (23, 32, 33 and 35) and rosmarinic acid-3’-O-glucoside (34) were detected by RPHPLC-DAD-ESI-MS/MS. Three new compounds, detected only in leaves, were tentatively identified as phenylpropane glyceride derivatives 1-O-coumaroyl-2-hydroxy propanal (20) and 1-O-coumaroyl-2-O-glycosyl propanal (22, 30). Compounds 20, 22 and 30 from S. oblonga are similar with phenylpropane glycerides present in red sorghum (Sorghum bicolor L. (Moench) and Lilium longiflorum Thunb. It is noteworthy that S. oblonga could be used in cooking as a complement after more detailed studies. Sorghum grain foods exhibit potential health benefits against chronic diseases related to over-nutrition. Lilium longiflorum possess flower buds and bulbs that are used for both culinary and medicinal purposes in many parts of the world. Studies on chemical composition and biological activity of the genus Sanchezia are scarce. The presence of phytosterols and flavonol glycosides were recently reported in leaves from this species. However, the chemical profile of the extracts analyzed in this work differs from that previously reported for aerial parts of S. nobilis (sin. S. oblonga). Further studies, including statistical methods, such as principal component analysis and hierarchical cluster analysis will be needed to evaluate chemical markers for this species.


Introduction
Species of Sanchezia Ruiz & Pav. (Acanthaceae) are neotropical herbs and shrubs. Several species of Sanchezia, such as S. parvibracteata, S. nobilis and S. speciosa, are cultivated as ornamentals in tropical areas and botanical gardens due to their showy leaves and conspicuous bright and colorful flowers. Recently, this genus was revised by Leonard and Smith (1964); among 58 species, over half were newly described (Tripp and Koenemann, 2015). There are few reports on chemical constituents and pharmacological activity of Sanchezia species. Therefore, there is no exhaustive identification of the basic chemical constituents and comprehensive quality control of this genus. S. speciosa contains cardiac and flavonoid glycosides and their extracts exhibited antioxidant and anti-inflammatory activities (Bui Thanh et al., 2017). Extracts of the same species exerted cytotoxicity in human epithelial cervical cancer cell lines (Shaheen et al., 2017, Parvin et al., 2015. detector, CTO-20A column oven and SIL 20AC autosampler (Shimadzu Corporation Kyoto, Japan). All the operations, acquisitions and data analyses were controlled by the Shimadzu CBM-20A software. Separations were carried out using a C18 RP Luna Phenomenex reverse phase column (4.6 x 250 mm i.d., 5 µm of particle size) protected with a security guard cartridge (Gemini C18, 4.0 × 2.0 mm i.d.). The mass spectrometer was an ion trap with atmospheric pressure ionization method through electrospray ionization interface (ESI) operating in the scan MS mode from m/z 100 to 1500. The mobile phase was composed of eluent A (0.1% aq. formic acid) and eluent B (methanol) at the constant flow rate 1.0 mL min −1 and constant temperature of the column oven at 40 °C. The following elution program, based on concentrations of the B solvent, was used: 0 min, 20%; 10 min, 40%; 20 min, 60%; 30 min, 80%; 40 min, 100%; 50 min, 20%. The time for the whole run was 60 min, 10 minutes were needed for equilibration of the column. Helium was used as the collision and nitrogen as the nebulizing gas, respectively. Nebulization was aided with a coaxial nitrogen sheath gas provided at pressure of 27 psi. Mass spectra were acquired in negative mode with ion spray voltage at 3.0 kV, capillary temperature at 300C, capillary voltage at 45 V and drying gas flow 6 L/min. Collision induced dissociation spectra were obtained in the ion trap using helium as the collision gas, with voltage ramping cycles from 0.5 to 1.3 V. Data were analyzed by an HP Chemistation System and Bruker Daltonics Data Analysis.

Analyses of the Leaf Extract by Fourier Transform Infrared Spectrometry (FT-IR)
Fourier transform infrared spectrometry is used for analysis, because it represents a non-invasive analytical tool allowing a fast and simultaneous qualitative and quantitative characterization of natural products and their constituents. The infrared (IR) spectrum of the ethanolic extract of leaves exhibited bands at 3369,3012,2925,2854,1737,1640,1454,1409,1378,1346,1240,1162 and 1073 cm -1 . The maximum absorptions were attributed to the following functional groups: at 3369 -3200 cm -1 (stretching of free OH, as sharp peak), 2925 -2854 cm -1 (C-H aliphatic stretching), 1737 cm -1 (C=O ester bond) and 1640 cm -1 (C=C aromatic). The C-O stretching characteristic from esters appeared in the region 1346 -1240 cm -1 . The peak at 1737 cm -1 assigned to the C=O stretching vibration means that some carbonyl compounds exist in the leaves of S. Oblonga. Bands from 1240 to 1162 were attributed to C-O stretching of phenolics and asymmetric C-C-O stretching of esters. The bands between 1454-1409 cm -1 are attributed to the asymmetric in-plane bending of -CH 3 , which is also the same spectral region reflecting to the phenyl (C=C bonds). The absorption bands that were attributed to C=C stretching vibration at 1640 cm-1 and the band at 1378 cm-1 referred to C-O stretching vibration of phenyl groups were observed indicating the presence of phenolic compounds. FT-IR analysis results revealed the presence of fatty acids and its respective ethyl esters and phenolic compounds, such as phenylpropane glycerides.

Compounds Detected in Leaves and Stems by RPHPLC-ESI-MS/MS Analyses
The identification of compounds 19-36 (Table 2)  The glycosyl and alkyl residues are weak chromophores, so these substituents did not change significantly, the maximum absorption coefficients and absorption wavelengths of compounds 19, 21-23 and 25-36 (Table 2), indicating that the loss of 132, 146, 162 and 176 Da, in these compounds corresponds to arabinose, rhamnose, glucose and glucuronide, respectively.

Compounds Detected by RPHPLC-ESI-MS/MS Analyses of Leaf Ethanolic Extract
Compounds 20, 22, 29, 30, 31, 35 and 36 were detected only in ethanolic extract from leaves (Table 2). Compounds 20, 22 and 30 are new and their identification was based on UV and mass spectra, which were compared with that phenylpropane glycerides isolated from sorghum grain (Kang et al., 2016, Kadam et al., 2018Nguyen et al., 2015, andSvensson et al., 2010) and Lilium longiflorum Thunb. (Munafo Jr and Gianfagna, 2015). Sorghum is the fifth most-produced cereal in the world and the major producers are USA, Mexico, Nigeria, Sudan and India. Sorghum grain foods is used in many parts of Africa, Asia and the semi-arid tropics world-wide and exhibited potential health benefits against chronic diseases related to over-nutrition. Compound 20 (Rt -3.6 min) exhibited UV maximum absorption at 310 nm, characteristic of coumaric acid derivatives. p-Coumaroylglycolic acid was reported for Lepidium sativum (Kadam et al., 2018). Caffeoylglycolic acid methyl ester was isolated from the grains of Sorghum bicolor (L.) Moench var. hwanggeumchal Kang et al., 2016;Nguyen et al., 2015). 2-O-coumaroylglycerol and 1-O-coumaroyl-2-O-glucosylglycerol were detected in hydromethanolic extracts (50:50) of brown, red and white sorghum whole grains (Kang et al., 2016;and Svensson et al., 2010). Feruloyl-caffeoylglycerol was found in Ananas comosus L. leaves and sorghum grain (Wu et al., 2016). Sorghum grains in the diet promoted cardiovascular health and exhibited beneficial effects for weight control (Kang et al., 2016). Caffeoylglycolic acid methyl ester and 1-O-caffeoylglycerol isolated from Sorghum bicolor, showed inhibitory potential on nitric oxide production (Salazar-López et al., 2018). Lilium longiflorum, an attractive ornamental plant, possesses flower buds and bulbs that are used for both culinary and medicinal purposes in many parts of the world. This species contains significant amounts of phenylpropanoid glycerol glucosides, which may contribute to plant pathogen defense, ultraviolet/high-intensity visible light (UV/high light) protection, and use in traditional medicine (Munafo Jr and Gianfagna, 2015).  (Kang et al., 2016;Zerbib et al., 2018;Karar and Kuhnert, 2015), compound 36 was tentatively identified as catechin arabinoside.

Compounds Detected by RPHPLC-ESI-MS/MS Analyses of Leaf and Stem Ethanolic Extracts
Compounds 19, 25, 27 and 28 were detected in ethanolic extracts from leaves and stems (Table 2). The mass spectrum of 19, (Rt -2.7 min) exhibited [M -H]at m/z 341, and base peak at m/z 179 (deprotonated caffeic acid), produced by the loss of glucose moiety (162 Da) (Karar and Kuhnert, 2015;Liang et al., 2017). Compared with its elution order and MS/MS fragmentation pattern, compound 19 was assigned as caffeic acid glucoside.
Regarding the RPHPLC-ESI-MS/MS analyses (Table 2) (Table 2). It is noteworthy that ethyl rosmarinate (24) and rosmarinic acid-3'-O-glucoside (34) were unique in the ethanolic extract of stems. Differences in gene expression between plant parts is well known. For example, plant polyphenol profile varied widely ontogenetically and among organs of Geranium sylvaticum (Tuominem and Salminem, 2017).
There have been few chemical studies about genus Sanchezia. In this study a hyphenated chromatographic analysis of ethanolic extracts of leaves and stems of S. oblonga was performed. Although the isolation of constituents is necessary for their identification by NMR methods, it is possible to observe that the results are different from those previously obtained for this species (Ellah et al., 2014). Table 2 lists various synapic acids and their esters, phenylpropane glycerides derivatives and rosmarinic acid derivatives, which had not been reported previously for this species. In this work, phytosterols and kaempferol glycosides were detected only in stems. However, a recent work reported that phytosterols were isolated from n-hexane and ethylacetate extract of the leaves , while kaempferol glycosides were isolated from ethylacetate and aqueous extracts of leaves of S. nobilis (syn. S. oblonga) .
The differences in chemical composition between the extracts analyzed in the present work and the previously published papers regarding S. nobilis can be explained by two possibilities: a) they may correspond to the same species but to different varieties or chemotypes; b) differences may be due to climate, season or growing conditions (Liu et al., 2015).
S. oblonga may turn out an interesting source of bioactive substances. Phytosterols exhibited antibacterial and antifungal activity (Aldini et al., 2014), are active against leukemic cell lines (Suttiarporn et al., 2015) and useful in the treatment of gastrointestinal inflammatory diseases by association with systemic and local metabolic anti-inflammatory drugs (Burčová et al., 2018). Stigmasterol inhibited tumor endothelial cells and cholangiocarcinoma (Saeidnia et al., 2014, Kangsamaksin et al., 2017. Sterols inhibited cholesterol absorption and exhibited trypanocidal and mosquito larvicidal activity (Ghosh et al., 2013). Hydroxycinnamic acids and their derivatives promote a variety of health benefits, e.g. reducing obesity and adverse health complications (Alam et al., 2016), as well as exhibiting antibacterial, antifungal (Guzman, 2014), antityrosinase, UV protection, anti-aging and anti-inflammatory effects (Alam et al., 2016).

Conclusion
Leaves and stems of Sanchezia oblonga contain a variety of biologically active compounds, such as phytosterols, flavonol glycosides, benzyl alcohol glycosides, sinapic acid glycoside esters, phenylpropane glycerides and rosmaric acid derivatives, which were detected using GC-EI-MS and RPHPLC-DAD-ESI-MS/MS. These techniques revealed that ethanolic extracts of leaves and stems contain distinct chemical profiles. The leaves contain phenylpropane glycerides and can be used in cooking as supplement. Comparison of the current results with previous studies suggests the possibility of chemical polymorphisms within species. Further studies are needed to explore this possibility. Compounds should be isolated and identified by NMR methods. Statistical methods, principal component analysis and hierarchical cluster analysis can be applied to evaluate intrinsic quality and identify chemical markers, which is useful for the chemical standardization of this species.