Among the numerous botanical species found in the Brazilian plant biome, Ximenia americana is widespread in the Northeast of Brazil, especially in the state of Ceará, where it is known as bush plum. Its bark, leaves and roots are used in popular medicine for the treatment of skin infections, hemorrhoids, stomach ulcers, gastric pain, among others. In this work, hexane and ethanolic extracts of X. americana seeds were subjected to silylation reactions followed by analysis of the silylated derivatives by gas chromatography-mass spectrometry (GC/MS). In the hexane extract, 18 substances were identified, where the most abundant and most important components were the Octadec-9-enoic (38.14%), (9 Z,12 Z)-Octadec-9,12-dienoic (18.83%), Ethanedioic (8.21%) and ( Z, Z, Z)-9,12,15-Octadecatrienoic (7.22%) acids. From the ethanol extract 18 substances were also identified having D-Sucrose (29.36%), L-Sorbose (9.19%), syllo-Inositol (8.34%) and D-Glucose (7.45%) as the main components. In addition, the presence of a steroid in hexane extract and triterpenes in ethanol extract was recorded. The constituents were identified using chromatographic and spectrometric methods, especially gas chromatography-mass spectrometry. To our knowledge, this is the first study of this nature from the oil of the X. american seeds.
Brazil is considered the country with the greatest biological diversity. It houses about 14% of the world’s plant diversity distributed in different biomes, mainly the Amazon region, the Atlantic Forest and Caatinga [
GC/MS analysis were performed on Shimadzu GCMS model QP2010SE Plus using a (5%-phenyl)-dimethyl- polysiloxane Rtx®-5MS capillary column (30 m × 0.25 mm × 0.25 µm) with film thickness 0.1 µm; the temperatures of the injector and detector were 260˚C and 300˚C, respectively; column conditions: 60˚C (0.5 min) to 260˚C (5 min) at 6˚C/min, then 12˚C/min to 300˚C (10 min) using He as carrier gas, flow rate 1.7 mL/min with split mode. The analysis with the mass detector was performed in the scan mode with analysis time of 52.21 minutes; EIMS data were recorded with electron impact ionization at 70 eV (1.5 KV voltage, analyzer quadrupole and ion source 200˚C), in the range 47 - 600 Da. 1H- and 13C-NMR spectra were recorded on Brucker DRX 500 spectrometer in C5D5N and are reported in ppm relative to using tetramethylsilane (TMS) as internal standard; IR spectra were recorded on Perkin-Elmer model Spectrum 100 FT-IR using a Universal Attenuated Total Reflectance (UATR). All solvents and reagents were of analytical grade and obtained from commercial sources (Synth and Merck). Silica gel 70 - 230 mesh (Merck) was used for column chromatography; silica gel Merck Kieselgel 60 (2 - 25 mm silica gel) with fluorescent indicator on aluminum support paper (Merck F254) was used for thin-layer chromatography (TLC) and UV light (254 and 365 nm) and vanillin/ perchloric acid/EtOH solution under heating, as developing agents. Mp’s were determined on digital Mettler Toledo, model FP62 apparatus and are uncorrected.
Seeds of X. americana were collected in Marrecas site, near the city of Acarape, Ceará, Brazil in 2014. A voucher specimen (N˚ 040411) is deposited at the Herbarium Prisco Bezerra of the Department of Biology, Federal University of Ceará, Fortaleza, Brazil.
On concentration in vacuo, the crude EtOH extract afforded a greenish-white precipitate, which was filtered. The precipitate after successive washes with MeOH resulted in a white solid (60 mg) amorphous soluble in pyridine, which was characterized as a triterpenes mixture: mp 209˚C - 212˚C.
The hexane fraction (2.1 g) of hexane extract was subjected to column chromatography on silica gel, eluted with increasing concentrations of EtOAc in hexane. After thin layer chromatography, the fractions 17 - 20 eluted with hexane-EtOAc 4:3, was rechromatographed on silica gel column eluted with increasing concentrations of CH2Cl2 in hexane. Thin layer chromatography brought together all fractions 16 - 44 (eluted with hexane-di- chloromethane 1:1), which were then characterized as triglycerides.
The extracts were submitted to a silylation reaction according to a known methodology [
In separate experiments, hexane and ethanol crude extracts of X. anericana seeds were successively partitioned into hexane, dichloromethane, ethyl acetate and methanol. Considering one of the main objectives of this study, namely the identification of oil components of the seed X. americana, using mainly gas chromatography coupled to mass spectrometry (GC/MS) and, without knowing the types of compounds present (hydrocarbons, alcohols, organic acids, esters of fatty acids, steroids, sugars, among others), preparation of volatile derivatives has become a basic requirement. Thus, the crude hexane was subjected to silylation reaction with BSTFA/ TMCS followed by obtaining a chromatogram (GC) of the crude reaction product. The structures of the components corresponding to 18 peaks in the chromatogram of total ion were identified by GC/MS from the fragmentation pattern observed in their mass spectra, and by the comparison to the mass spectra reported in the literature.
Among the identified compounds (
The MS of the TMS derivatives of these acids exhibit a characteristic profile fragmentation. Thus, their MS in addition to peaks due to [M]+ show peaks at m/z 73, 117, 132, 145 and 313 corresponding to fragments [CH3)3Si]+, [CH3)3SiCO2]+, [CH3)3SiCO2CH3]+, [CH3)3SiCO2C2H4]+ and [CH3)2SiCO2C15H31]+, respectively. In general, at 70 eV eims of these trimethylsilyl derivatives, m/z 73 [TMS]+ is registered as the base peak. Taken as an example, the mass spectrum (
The loss of methyl radical to give the ion at m/z 313 (M-15), occurs mainly from the silyl group [
Also, the mass spectra of silylated fatty acids containing a double bond showed virtually the same behavior. Even those containing more than one double bond, but showing differences, exhibited a similar profile. However, the peaks corresponding to the fragments at m/z 117, 132 and 145 in these latter cases were much less intense.
Scheme 1. Mass spectral fragmentation observed for TMS-hexadecanoic acid (palmitic acid).
Peak | Compounds | RT | % | Mass spectra, m/z (rel. intensity) |
---|---|---|---|---|
1 | Ethanedioic acid | 6186 | 8.21 | 73(29), 75(39), 90(28), 147(100) |
2 | Disiloxane, hexamethyl- | 7100 | 5.10 | 66(12), 73(22), 147(100), 148(17) |
3 | Pentasiloxane, dodecamethyl- | 8732 | 0.69 | 73(81), 147(100), 281(91), 369(18) |
4 | Glycerol | 10,957 | 0.76 | 73(100), 103(18), 117(19), 147(49), 205(22), 218(14) |
5 | n.i. | 12,230 | 1.89 | 73(66), 146(100), 205(62), 279(51), 367(100) |
6 | Nonadioic acid | 20,403 | 0.97 | 55(46), 73(100), 75(76), 117(28), 129(30), 317(21) |
7 | Hexadecanoic acid | 24,023 | 4.17 | 73(89), 75(62), 117(100), 129(40), 132(35), 145(26), 313(52), 328(5) |
8 | 9,12-Octadecadienoic acid (Z,Z)- | 26,297 | 18.83 | 73(100), 75(98), 117(20), 129(27), 132(4), 145(8), 337(39), 352(4) |
9 | cis-9-Octadecenoic acid | 26,358 | 38.14 | 73(100), 75(85), 117(65), 129(70), 132(19), 145(33), 339(47), 354(6) |
10 | trans-9-Octadecenoic acid | 26,440 | 1.70 | 73(100), 75(80), 117(80), 129(49), 132(15), 145(40), 339(67), 354(7) |
11 | Octadecanoic acid | 26,666 | 2.17 | 73(79), 75(58), 117(100), 129(38), 132(37), 145(27), 341(50), 356(4) |
12 | Linolenic acid | 27,887 | 7.22 | 73(80), 75(83), 79(100), 117(22), 129(19), 132(4), 145(10), 335(7), 350(3) |
13 | 9,12-Octadecadiynoic acid | 28,907 | 2.52 | 73(95), 75(100), 91(82), 105(84), 117(50), 119(58), 129(29), 132(7), 145(12), 148(57), 333(8), 348(4) |
14 | trans-Octadec-11-en-9-ynoic acid, 8-trimethylsilyloxy-, methyl ester | 29,526 | 1.24 | 73(100), 147(13), 217(12), 237(90), 365(4) |
15 | 1-Monooleoylglycerol | 32,741 | 0.90 | 55(55), 73(94), 103(48), 117(18), 129(100), 147(71), 201(20), 203(28), 205(12), 265(9), 397(40), 410(5), 485(6), 500(3) |
16 | Tetracosanoic acid | 33,449 | 0.58 | 73(100), 75(75), 117(79), 129(30), 132(34), 145(33), 201(12), 425(40), 440(8) |
17 | cis-15-Tetracosenoic acid | 37,198 | 1.13 | 73(100), 75(82), 117(62), 129(68), 132(22), 145(34), 423(32), 438(8) |
18 | β-Sitosterol | 39,343 | 3.04 | 43(58), 73(52), 129(100), 255(14), 357(42), 381(22), 396(40), 471(9), 486(14) |
19 | Lupeol | 43,736 | 0.74 | 73(50), 189(100), 203(52), 218(38), 279(10), 369(12), 393(9), 408(5), 483(4), 498(9) |
n.i.: no identified.
Steroids in general, exhibit a very similar fragmentation pattern. The mass spectrum of the constituent with retention time 39.343 in the chromatogram showed a molecular peak at m/z 486 and was identified as β-Sitosterol. The peaks at m/z 471, 396, 381, 357, 213 and 129, are the most representative, since they correspond to fragments of higher mass and are relatively more prominent peaks. The ion at m/z 471 (M-15) can occur in two paths (Scheme 2): fission of the C-Si bond (a) or the C10-C19 bond (b). Studies [
Scheme 2. Mass spectral fragmentation observed for TMS-β-Sitosterol.
another fragmentation (d) related to the M?90 results in one fragment at m/z 75, for which high-resolution mass measurements [
The crude EtOH extract was subjected to reaction silylation with BSTFA/TMCS followed by obtaining a chromatogram GC/MS of the crude reaction product. The structures of the components corresponding to 18 peaks in the chromatogram of total ion were identified by GC/MS from the fragmentation pattern observed in their mass spectra, and by the comparison to the mass spectra reported in the literature. Among the identified substances (
Peak | Compounds | RT | % | Mass spectra, m/z (rel. intesity |
---|---|---|---|---|
1 | 1,2-Bis(trimethylsiloxy)ethane | 6181 | 2.13 | 147(100), 73(58), 191(23) |
2 | Disiloxane, hexamethyl- | 7097 | 1.46 | 66(12), 73(22), 147(100), 148(14) |
3 | Glycerol | 10,958 | 5.58 | 73(100), 103(18), 117(19), 147(80), 205(38), 218(15) |
4 | n.i. | 14,239 | 1.41 | 172(100), 82(94), 73(22) |
5 | beta.-D-Galactofuranoside | 20,404 | 3.04 | 73(100), 217(82), 147(33) |
6 | D-Fructose | 20,856 | 1.48 | 73(100), 217(55), 147(25) |
7 | Mannonic acid, lactone | 20,982 | 3.08 | 73(100), 217(65), 147(27) |
8 | Sorbopyranose, L- | 21,091 | 9.19 | 73(100), 204(68), 217(33) |
9 | D-Galactose | 21,182 | 1.54 | 73(82), 204(100), 217(28), 147(51) |
10 | Inositol, scyllo- | 21,305 | 8.34 | 305(20), 318 (32), 217(40), 147(42), 191(22), 204(12) |
11 | alpha.-D-Glucopyranose | 22,212 | 5.15 | 204(100), 73(82), 191(40) |
12 | D-Mannitol | 22,916 | 6.27 | 73(100), 147(51), 319(42) |
13 | D-Glucose | 23,521 | 7.45 | 204(100), 191(41), 217(18) |
14 | Inositol, myo- | 25,082 | 1.62 | 305(56), 318(30), 217(62), 147(51), 191(30), 204(25) |
15 | 9,12-Octadecadienoic acid (Z,Z)- | 26,297 | 1.91 | 73(94), 75(100), 129(25), 337(34) |
16 | trans-9-Octadecenoic acid | 26,353 | 3.31 | 73(96), 75(100), 117(79), 129(68), 145(32), 339(42) |
17 | D(+)-Sucrose | 31,944 | 29.36 | 361(100), 73(80), 217(32), 147(23),103(20), 437(12), 540 |
18 | 14,17-Nor-3,21-dioxo-.beta.-amyrin, 17,18-didehydro-3-dehydroxy- | 43,022 | 1.79 | 73(100), 75(90), 117(55) |
19 | Silane, (9,19-cyclo-9.beta.-lanost-24-en-3. beta.-yloxy)trimethyl- | 43,309 | 5.89 | 73(100), 117(62), 75(57) |
n.i.: no identified.
The identification of these substances as trimethylsilyl derivatives by GC/MS can be made in view of some mechanistic proposals for characteristic fragments, according to published studies [
This two-carbon fragment, m/z 204, a common dominant peak of pyranose derivatives (aldo-, keto-, and glycosides), originates the most part, from C-2?C-3 and C-3?C-4 (aldopyranoses) [
Thus, by comparing with published data [
The peak at m/z 217 (Scheme 5) is characteristic of monosaccharides with furanose ring, which it is usually a very intense peak and, sometimes, the base peak. The relatively high intensity of this peak was observed in the mass spectrum of the component with retention time 20.856 (
Scheme 3. Formation of fragment m/z 204 via McLafferty type rearrangement.
Scheme 4. Formation of fragment at m/z 204 via the alternative paths.
Scheme 5. Formation of fragment m/z 217 in furanose monosaccharides.
involving rearrangement is seen in Scheme 5. In the spectrum (
Finally, by comparing the mass spectrum (
An intense peak at m/z 306 which is generally observed in the mass spectra of carbohydrates in their open forms [
At the concentration of the ethanolic extract of the seeds gave a precipitate which was recrystallized from MeOH. The 1H NMR spectrum of this recrystallized (60 mg) showed that it was still mixture of two or three closely related compounds: IR spectrum γ max 3230 (OH), 2918/2910 (Csp3-H), 1683 (C=O), 1029 (C?O) cm-1. The 1H NMR spectrum revealed the presence of various methyl groups, one of which (one singlet at 1.80 ppm) bonded to sp2 carbon; the spectrum also showed signs as broad triplet at 5.72 (1H), broad singlet and 5.51 (1H) and two broad singlet at 4.96 (1H) and 4.78 (1H) due to hydrogens mutually coupled. The first two were attributed to olefinic hydrogens and consistent with the presence of triterpenes of oleanane/ursano types [
Carbohydrate analysis has proven to be a powerful technique in the understanding of biological processes. In addition to the possibility of studying the source of carbohydrates and their rotation in the environment, analysis of specific stable compounds may be used to trace microbial biomass and to detect food adulteration or in studies
nutrition. The techniques used in the analysis have proved very useful, therefore, allowed the identification of various chemical constituents, mainly carbohydrates, from both crude extracts. To our knowledge, this is the first study from hexane and ethanol extracts of the seeds of X. ameriacana using silylated derivatives by gas chromatography-mass spectrometry. This study, the first with oil from the seeds of X. americana, enabled a possible use of this oil as human or animal nutraceutical ingredient.
The authors thank the Brazilian funding agencies FUNCAP, CAPES and CNPq for financial support.
Romézio Alves Carvalhoda Silva,Telma Leda Gomesde Lemos,Daniele AlvesFerreira,Francisco José QueirozMonte, (2016) Ximenia americana: Chemical and Spectral Studies of Extracts of Seeds. Analysis of Trimethylsilyl Derivatives by Gas Chromatography and Mass Spectrometry. American Journal of Analytical Chemistry,07,192-202. doi: 10.4236/ajac.2016.72016