HRGC-MS Analysis of Terpenoids from Maytenus ilicifolia and Maytenus aquifolium ( “ Espinheira Santa ” )

Neste trabalho é descrita a identificação de alguns constituintes químicos minoritários da “espinheira santa” (Maytenus ilicifolia e Maytenus aquifolium, Celastraceae), planta medicinal amplamente utilizada no Brasil. Através do acoplamento entre cromatografia gasosa de alta resolução e espectrometria de massas (HRGC-MS), foi possível identificar principalmente triterpenóides e esteróides presentes na “espinheira santa”, vários dos quais relatados pela primeira vez em ambas as espécies de Maytenus.


Introduction
"Espinheira santa" is the popular name of two species, Maytenus aquifolium Martius and Maytenus ilicifolia Martius (Celastraceae).These medicinal plants are also known as "cancorosa", "cancerosa", "salva-vidas", "sombra de touro", "espinho de Deus" and others 1 , and are found in Brazil, Chile, Uruguay, Paraguay and Bolivia.In Brazil their utilization is mainly in stomach ulcers and gastritis treatment, due to the presence of both triterpenes friedelan-3-ol and friedelin as well as polyphenolic compounds 2,3 .In Paraguay and Bolivia, they are utilized by indian tribes and rural populations as a fertility regulating agent 4 , due to the alkaloid maytensine 5 .
Previous work showed by HRGC-FID (high resolution gas chromatography -flame ionization detector) the predominance of friedeline and friedelan-3-ol in terpene fraction from Maytenus ilicifolia and Maytenus aquifolium leaves 2,3 , which indicates that these compounds may hide minor terpenes in conventional phytochemical study (extraction of plant material followed by purification of each individual compound, for spectroscopic and spectrometric analysis).
In this work, the characterization of further triterpenes from the extracts obtained by direct extraction with ethyl acetate from these two Maytenus species was done by HRGC-MS (high resolution gas chromatography-mass spectrometry), by MS fragmentation data acquisition both by SCAN and SIM mode.In the first, the mass/charge ratio scanning was changed, and in the latter the characteristic terpenoid ions were monitored, followed by molecular ion selective monitoring.

Extraction of plant material
which contained the neutral compounds, was washed with 30 mL of water and concentrated in a rotatory evaporator to 30 mL, being then centrifuged during 10 min at 6000 rpm, to remove the suspended particles.The solvent was evaporated to dryness, giving a residue, which was dissolved in CHCl 3 for GC-MS analysis.Figure 1 summarizes the procedure of the extraction process.

Chromatographic analysis
HRGC-MS analysis was performed on a Hewlett-Packard 5890 gas chromatograph, with a split injector (1:50) at 280 °C and a Hewlett-Packard 5970 mass selective detector (MSD), with the GC-MS interface temperature at 280 °C.The injection volume was 2 µL.Hydrogen was employed as carrier gas, at a pressure of 60 kPa.A HP-1 25 m x 0.25 mm x 0.33 µm methylpolysiloxane cross-linked capillary column was employed with temperature programming from 100 °C (held for 2 min) to 280 °C (held for 30 min) at a ratio of 4 °C/min.

Results and Discussions
Figures 2 and 3 show the total ion chromatograms (TIC) of the leaves extracts obtained with ethyl acetate from Maytenus aquifolium and Maytenus ilicifolia, respectively.The chromatographic profile was similar for both species, differing only in the relative concentration of some compounds; this similarity had been observed in HRGC-FID analysis 2 .Most of the extracted compounds had retention times between 36 and 56 min and were identified as being terpenoids.
For the characterization of the compounds detected in TIC/HRGC-MS, a process which increased the confidence of the identification of compounds by the mass spectrometry was applied.Firstly, the fragmentogram obtained for each compound was compared with the fragmentation data base of the mass spectrometer, for obtaining a list of the 10 most probable substances (tentative identification).Sweep-   ing was done by using the SCAN mode, in which the values of m/z between 50 and 480 daltons were chosen.It was observed that, at the beginning of each probable fragmentogram, that the fragments of the compounds with t R 40 to 56 min had the same fragmentation between 50 and 120 daltons, and that differences appeared only after 120 daltons.
The samples were analyzed again using the SCAN mode, but with the sweeping range set between m/z 120 and 550.In this new sweeping, several fragments that showed lower intensity (5 to 10% abundance) and have not been useful for comparation with standards library data, turned to 100% abundance, therefore participating in the comparison with NBS library (around 40,000 compounds).This provided alternatives for the more probable compounds, with a higher "match quality" score for the analyzed compounds.
The MS data of the compounds found in Maytenus samples were studied by comparison with the "Eight Peak Index" data 6 .This allowed the conclusion that, within the retention timespan from 40 to 50 min, most of the compounds belonged to the terpenoids class.Thereafter, the SIM mode (selective ion monitoring) was utilized for monitoring the terpenoids characteristic ions.Table 1 reports the monitored ions for some pentacyclic terpenoids, as indicated by Djerassis data concerning terpenoid fragmentations 7 .
The suggested method was checked by the confirmation of the identity of some proposed structures through the injection of authentic standards.Table 2 gives the retention times and molecular ion obtained for those standards.

Conclusions
The utilization of mass spectrometry was effective for the identification of the minor terpenoids from Maytenus ilicifolia and Maytenus aquifolium.Most of these compounds are reported for the first time in these Maytenus species.Their low concentration in plant material and the similarity of skeletons justify the fact that these compounds were not previously reported in conventional phytochemical studies of Maytenus ilicifolia or Maytenus aquifolium 9 .
The importance of the study of terpenoids is due to the biological activity of some of these compounds.In the case of "espinheira santa", the major triterpenoids friedelan-3-ol and friedelin were shown 9 to be related to activity against stomach ulcers and gastritis.The present study, which reveals the complexity of the terpenoidal fraction of "espinheira santa", suggest that the relative contribution of the minor components on the pharmacological activity should be evaluated.

Table 1 .
List of characteristic ions of some triterpenoids, utilized for the SIM mode analysis of Maytenus extracts.

Table 2 .
Data of standards utilized for identification of compounds in Maytenus extracts.
For chromatographic conditions, see Experimental.