3-Aminofurostane Alkaloids from Solanum paniculatum (“Jurubeba Verdadeira”) Roots 3-Aminofurostane Alkaloids from Solanum paniculatum (“Jurubeba Verdadeira”) Roots

The roots of Solanum paniculatum (Solanaceae) have extensively been used in folk medicine to treat liver infections and as a diuretic. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) were used for the profiling and structural characterization of alkaloids from the roots of S. paniculatum . Sixteen 3-aminofurostane alkaloids were characterized as novel compounds. In this study, three principal alkaloids were isolated in mixture, and their structures were established by different spectroscopic methods, including 1D, 2D nuclear magnetic resonance (NMR) experiments, and the high-resolution electrospray ionization (HR-ESI)-MS analysis. The isolated alkaloids were used to explore fragmentation pathways. Compound identification was based on the exact mass and fragmentation behaviors. Two compounds were identified as new natural compounds as: (25 R )-3 β -amino-furost-5-en-22 α ,26-diol O (26)- β -D-glucopyranoside (fatimagraine) and (25 R )-3 β -amino-furost-22-en,26-ol O (26)- β -D-glucopyranoside (bhattacharyyaine). The unambiguous assignments of 1 H and 13 C NMR data and chemical correction of the structure alkaloid jurubine are reported for the first time.


Experimental
General experimental procedures NMR spectra were obtained using Bruker DRX 500

3-Aminofurostane Alkaloids from Solanum paniculatum ("Jurubeba Verdadeira") Roots
Tania M. S. Silva, * ,a Telma M. G. Silva, a Maria F. Agra b and Celso A. Camara a (500 MHz for 1 H and 125 MHz for 13 C) and Bruker DPX300 (300 MHz for 1 H and 75 MHz for 13 C) spectrometers (Karlsruhe, Germany). Samples were prepared as solutions in CD 3 OD, and tetramethylsilane (TMS) was used as an internal reference. Thin layer chromatography (TLC) was performed with pre-coated silica gel 60 PF254 plates (0.25 mm, Merck, Darmstadt, Germany). A Strata Giga Tubes (SPE C18-E 70 g/150 mL, Phenomenex Inc., Torrance, USA) cartridge was employed to obtain alkaloids (Phenomenex Co., Torrance, CA, USA). Acetonitrile was obtained from Sigma (St. Louis, MO, USA). Milli-Q water (Billerica, USA) was used for the UPLC-QTOF-MS (ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry) analysis. The XEVO-G2XSQTOF mass spectrometer (Waters, Manchester, UK) was connected to the ACQUITY UPLC system (Waters, Milford, MA, USA) via an electrospray ionization interface (ESI). The chromatographic separation of compounds was performed on the ACQUITY UPLC with a conditioned autosampler at 4 °C using the Waters ACQUITY UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) (Waters, Milford, MA, USA). The mobile phase consisting of water with 0.1% formic acid in water (solvent A) and acetonitrile 0.1% formic acid (solvent B) was pumped at a flow rate of 0.4 mL min −1 . The gradient elution program was as follows: 0-5 min, 5-10% B; 5-9 min, 10-95% B. The injection volume was 5-10 μL. The MS analysis was performed on a Xevo G2 QTOF (Waters MS Technologies, Manchester, UK), a quadrupole time-of-flight tandem mass spectrometer coupled with an electrospray ionization source in positive ion mode. The scan range was from m/z 50 to 1200 for data acquisition. In addition, MS E experiments were carried, which allows both precursor and product ion data to be acquired in one injection. The source conditions were as follows: 3 kV capillary voltage; 120 °C source temperature; 450 °C desolvation temperature; 50 L h −1 cone gas flow rate; 800 L h −1 desolvation gas (N 2 ) flow rate, and 40 V cone voltage. All analyses were performed using the lockspray, which ensured accuracy and reproducibility. Leucine-enkephalin (200 pg mL −1 ) was used as a standard or reference compound to calibrate mass spectrometers during the analyses. All data acquisition and analyses were controlled using the Waters MassLynx v. 4.1 software.

Plant material
The roots of Solanum paniculatum were collected in Pernambuco State, Brazil, in the municipality of Recife in December 2012. The voucher specimen (53790) has been deposited at the Herbarium Vasconcelos Sobrinho of the Universidade Federal Rural de Pernambuco (UFRPE).

Extraction and isolation
S. paniculatum dried and powdered root (981.0 g) was extracted by maceration with ethanol:NH 4 OH 2% (9:1) at room temperature. After filtration, the combined extractive solutions were concentrated under reduced pressure to yield the crude extract (91.0 g). The ethanolic extract (50.0 g) was suspended with acetic acid 10% and filtered through a bed of Celite. The aqueous acid filtrate was basified with NH 4 OH and left standing overnight. This solution was extracted with butanol and concentrated to produce 12.8 g of the BuOH fraction. This fraction (5.6 g) was subjected to SPE C-18 with H 2 O, MeOH, and ethyl acetate as binary mixtures of increasing polarity, and yielded 62 fractions. Fractions 19-21 were eluted with 80% MeOH, and 35-42 with 100% MeOH resulting in the isolation of the compounds mixture 11/12 (7.8 mg), and 12/17 (43.7 mg).

Identification of isolated compounds
T h e a l k a l o i d a l f r a c t i o n wa s s u b j e c t e d t o UPLC-QTOF-MS E and was used for the profiling and structural characterization of alkaloids from the roots of S. paniculatum. Seventeen 3-aminofurostane alkaloids were characterized and three principal alkaloids were isolated in mixture. The isolated alkaloids were used to explore fragmentation pathways. The structures of compounds 11, 12, and 17 were identified by the combined use of 1D NMR ( 1 H and 13 C NMR), 2D-NMR ( 1 H-1 H COSY, HSQC, HMBC), and MS spectra. The unambiguous assignments of 1 H and 13 C NMR data of compound 12 (jurubine) are reported for the first time and involved a combination of homo-and heteronuclear 2D NMR techniques (Table 1)   suggesting that, most likely, they are deshielded by the cis-oriented OH-22 group. 28 The 25R configuration was determined on the basis of differences in chemical shifts of the geminal protons at H 2 -26 that exhibit pronounced dependence; the difference (Dab = da − db) between their chemical shifts (Dab ≤ 0.48 for 25R; Dab ≥ 0.57 for 25S) seems to be of general applicability for ascertaining the 25R/25S orientation of the 27-methyl group of furostanetype steroidal saponins. 27 Consequently, based on the results described above, the structure of compound 12 was established as (25R)-3β-amino-5α-furostane-22α,26-diol O(26)-β-D-glucopyranoside. Analogously, as a result of the inspection of the 1 H and 13 C NMR spectra of compound 12 (Table 1), the same gross structure of jurubine 29 was identified. Therefore, we argue that these molecules should differ only in the stereochemistry of one chiral carbon. This was readily identified as the C-25 carbon. Compound 12 have the 25R configuration as shown above, and jurubine has been reported to have the 25S configuration. 7 Since there are no NMR data in the literature that prove the absolute stereochemistry of C-25S for jurubine, it is possible that the correct structure of jurubine is presented in this work, i.e., jurubine has actually the C-25R configuration. Jurubine was only identified in nature in two plant species: roots of Solanum paniculatum 7 and Solanum torvum. 30 The unambiguous assignments of 1 H and 13 C NMR data of jurubine (12) are reported for the first time in this study.
The structure of compounds 11 and 17 was readily elucidated on the basis of their considerable similarities to compound 12. The (+)-HRESIMS spectrum of compound 11 exhibited a protonated molecular ion at   7 ) in accordance with the 13 C-APT spectra. A good level of similarity in the chemical shifts of the glucose was observed for the two compounds 11 and 17, however, with some differences in the aglycone. The molecular weight of compounds 11 and 17 that have mass units lesser than those of compound 12 indicates that 11 has two hydrogen atoms less, and 17 has one oxygen atom less than 12, respectively. The MS/MS experiment of compound 11 (Table 2) showed a profile similar to that of 12, with the difference in all ions fragments of only -2 Da due to the presence of a double bond between the C-5 and C-6 bonds of the molecule (Figure 1). The 1 H and 13 C NMR spectra of compound 11 (Table 3) (Table 2), with the only difference at ions fragments of −2 Da due to the presence of a double bond between the C-5 and C-6 bonds of the molecule (Figure 2). The 1 H and 13 C NMR spectra of compound 17 (Table 4) indicated a 25R-3β-aminofurostane-type steroidal alkaloid when compared with those of 11 and 12. The aglycone nature of compound 17 was also manifested by its 1 H and 13 C NMR, principally on the carbon and hydrogen values of the E-ring when compared with that in compound 12. The lack of finding spirostane alkaloids type compounds in the roots of S. paniculatum in this study, despite of   being reported in the literature, 6 may be due to the direct analysis of the alkaloidal fraction. According to Li et al., 25 conventional methods present disadvantages because of the cyclization of furostanol to spirostanol aglycone, which can yield several structures that are alternatives to that of the original compound. These probable artifacts have been previously reported as natural alkaloids. 6,8 Characterization the common characteristics for all compounds comprise glycosylation pattern at C-26 and furostanol-type aglycone structure. 31 The hydroxyl group was characterized by the neutral loss of 18 Da (loss of water). The presence of additional glycosyl residues was observed by the loss of 162 Da.
The evidence that all detected structures are glycoalkaloids was based on the protonated peak in the positive mode [M + H] + to the molecular ion with even mass (compounds contain one nitrogen atom). The furostanetype compounds with the 22-OH group (compounds 1-15 Table 2 shows the high-resolution mass data and fragments data in the positive ion mode of the 3-aminofurostane alkaloids from Solanum paniculatum roots. Their analyses and comparisons with structures reported in the literature 8 showed that out of the seventeen identified glycoalkaloid, only compound 12 (jurubine) had been previously reported, all other 16 are new compounds.
Each identified glycoalkaloid showed its pair with only one additional unsaturation such as compounds [M + H] + :   (Table 2).
Compounds 1, 2, 3, 4, 5, and 7 exhibited two hydroxyl groups, which may be attached at the carbons of the A-D rings (perhydrocyclopentenophenanthrene nucleus). Likewise, compounds 6, 8 and 10 showed only one hydroxyl group. Compounds 9 and 13 showed two more glycosides. Analyses of the mass spectra of these compounds supported the identification. The MS 2 experiment showed fragment ions characteristics of consecutive losses of water (−18 Da) and/or glucose (−162 Da). Compounds 1-8 and 10 eliminated at least two molecules of water whereas compounds 2, 3, 5, 7, 9 and 13 showed the loss of two glucose units. Compounds 11 and 12, and 17 were isolated and identified by 1 H and 13 C NMR.
The ESI-QTOF-MS/MS investigation of the fragmentation mechanism of each 3-aminofurostane alkaloids was performed, and the characteristic fragment ion derived from the fission of aglycone with crucial fragment ions are presented in Table 2  are derived from the sequential loss of sugar moieties, water, and ammonia groups from the molecular weight of protonated aglycone. The predominant ions were detected at m/z 399/397 and 381/379 (Figures 1, 2 and 4) (Figures 4  and 5). This pathway is attributable to the hydrogen transfer from C-16 to the carbonyl via the Mclafferty rearrangement. Table 2 summarizes the characteristic fragments observed in the MS/MS spectra of alkaloids from the S. paniculatum roots. Compounds 2 (m/z 772.4477) and 6 (m/z 612.4106) are representative of 3-amino-22-hydroxylated furostane alkaloids, with (2) and without (6) unsaturation at A-D rings, respectively. The typical MS/MS spectrum is shown in Figure 5. As an example, the fragmentation pathway proposed for 2 and 6 is a property that could be extended to other alkaloids. The scheme shown in Figure 4 summarizes the differences in fragmentation behavior that can be used for the assignment of 3-aminofurostane alkaloids according to the proposed stepwise analysis strategy of mass fragmentation spectra. The proposed fragmentation pathways of the new compounds 11, 12, and 16, 17 are presented in Figures 1 and 2, respectively.
Although shown to be a powerful tool for the assignment of 3-aminofurostane alkaloids, the presented method of mass spectra interpretation cannot completely solve the structure of newly detected molecules (positions of hydroxyl and sugar residues, unsaturation in the A-D rings, and stereochemistry). The correct order of sugar residues can only be partially determined, and the exact position of substituents/double bonds on the aglycones results are not completely clear. However, such information is very helpful in discriminating between the (often) large numbers of possible structures obtained.

Supplementary Information
The NMR and MS (11,12, and 17) spectra of isolated compounds are shown in the Supplementary Information, available free of charge at http://jbcs.sbq.org.br as PDF file.