Furostanol-Type Steroidal Saponins from Cestrum laevigatum L .

Four new epimeric spirostanol and furostanol-type steroidal saponins, (25R,S)-5α-spirostan2α,3β-diol 3-O-β-D-glucopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D-galactopyranoside, (25R,S)-5α-spirostan-2α,3β-diol 3-O-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→4)β-D-galactopyranoside, 26-O-β-D-glucopyranosyl-(25R,S)-5α-furost-20-ene,2α,3β-diol 3-O-β-Dgalactopyranoside and 26-O-β-D-glucopyranosyl-(25R,S)-5α-furost-20-ene,2α,3β-diol 3-O-β-Dgalactopyranosyl-(1→4)-β-D-galactopyranoside, in addition to the known (25R,S)-5α-spirostan2α,3β-diol 3-O-β-D-galactopyranoside, were isolated from Cestrum laevigatum. Compounds were submitted to cytotoxic activity assays using colorectal adenocarcinoma (HCT-116), human promyelocytic leukemia (HL-60), ovarian carcinoma (OVCAR-8), glioma (SF-295) human cancer cell lines, and the antimicrobial activity was evaluated against Candida parapsilosis, C. albicans, C. krusei, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis.


Introduction
Steroidal saponins are constituted mainly by spirostanol and furostanol-type glycosides.Spirostanol glycosides are the largest group and comprises aglycones with the spirostan skeleton containing a sugar chain generally at C-3 position and a spiro-bicyclic acetal at C-22, while furostanol derivatives usually present a hemiacetal with a hydroxy or methoxy moiety at C-22 or yet a ∆ (20,22) -unsaturation, besides a glycosidic linkage at C-26.In both skeletons, C-25 is naturally found with either R or S configuration, or as inseparable epimeric mixtures. 1,2Furostanol are recognized as biogenetic precursors of spirostanol, since the 26-O glucoside unit can be enzymatically cleaved and a ring closure to 26-OH takes place with dehydration of 22-OH. 3So, the cooccurrence of spirostanol saponins with the correspondent furostanols, in innumerous plants, is the consequence of this ready conversion.
The isolation of bioactive steroidal saponins has been reported to several species of reputed poisonous plants belonging to the Solanaceae. 1,4,5Based on these findings, we have investigated Cestrum laevigatum, one of the most toxic plants lethal to mammals in the Brazilian livestock, among the group of common invasive species that cause liver damage. 6,7n this study, we have isolated epimeric mixtures of the new (25R,S)-5α-spirostan-2α,3β-diol 3  1).The cytotoxicity of all compounds was evaluated against four human tumor cell lines: colorectal adenocarcinoma (HCT-116), human promyelocytic leukemia (HL-60), ovarian carcinoma (OVCAR-8) and glioma (SF-295).The screening for antifungal activities was performed against Candida parapsilosis ATCC 22019, Candida albicans ATCC 10231 and Candida krusei ATCC 14243, while the antimicrobial activity was assayed against Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633.

Extraction and isolation
Stems of C. laevigatum (3.03 kg) were pulverized and extracted with hexane (3 × 5 L) at room temperature.The solvent was removed under reduced pressure to yield the hexane extract (0.3 g).The plant residue was then extracted with EtOH (3 × 6 L) to yield the corresponding EtOH extract (30.1 g), after evaporation of the solvent.
Roots (4.43 kg) were pulverized and extracted with hexane (3 × 7 L) at room temperature.The solvent was removed under reduced pressure to yield the hexane extract (0.45 g).The residue was extracted with EtOH (3 × 7 L) to yield the corresponding extract (22.5 g).

Acid hydrolysis, sililation and sugar analysis
The epimeric mixtures (ca.5.0 mg) were dissolved in 2 mol L −1 HCl (dioxane/H 2 O 1:1, 2 mL) and stirred at 90 °C for 2 h.After cooling, the reaction mixture was neutralized with solution of 1 mol L −1 NaOH, extracted with CH 2 Cl 2 , and the aqueous layer was evaporated to give a mixture of monosaccharides.The residue was dissolved in hexadimethyldisilazane/trimethylchlorosilan/pyridine (3:1:9), excess, and stirred at 70 °C for 60 min.The supernatants (3 μL) were analyzed by GC Agilent model 7890B/5977A GC/MSD (quadrupole), under the following conditions: CP-ChiraSil-L-Val column, 0.25 mm × 25 m; temperatures for detector and injector 150 and 200 °C, respectively; temperature of gradient system for the oven, 100 °C for 1 min and then raised to 180 °C, rate 5 °C min −1 kept for 5 min.The configurations of sugars were determined by comparison of the retentions times of the corresponding derivatives with those of standards treated simultaneously with same silylating reagents (L-rhamnose Rt:11.15,D-galactose Rt:16.02, and D-glucose Rt:16.57).Peaks of the hydrolysates were detected at Rt:11.20 (L-rhamnose), Rt:16.01 (D-galactose) and Rt:16.57(D-glucose).Co-injection of each hydrolysate with the respective standard gave single peaks.

In vitro antibacterial activity
Tests were performed according to the M02-A11 (CLSI, 2012) protocol with modifications. 8Wells with 6 mm diameter were made in the agar overlay of the Petri dish.To those wells, a volume of 20 μL (1000 μg mL −1 ) of the obtained compounds was applied.The plates were incubated for 20 h at 35 °C.The solvents and diluents used in the compounds dissolution were used as negative control.The assayed microorganisms used in this study were Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, and Bacillus subtilis ATCC 6633.The antibiotic disk used in antimicrobial sensitivity test was Cefepime (CPM) 30 μg (Specialized Diagnostics Microbiology, DME, São Paulo, SP, Brazil).

In vitro antifungal activity
The broth microdilution (BMD) antifungal susceptibility test was performed according to M27-A3 protocol using RPMI broth (pH 7.0) buffered with 0.165 mol L −1 3-(N-morpholino)propanesulfonic acid (MOPS; Sigma-Aldrich, St Louis, MO, USA). 9Compounds were dissolved in DMSO and tested at concentrations ranging from 1.95 to 1000 μg mL −1 .Fluconazole was used as positive control.The yeasts and compounds were incubated in 96-well culture plates at 35 °C for 24 h and the results were examined visually.The minimum inhibitory concentration (MIC) of each compound was determined as the concentration that inhibited 50% of fungal growth.The assayed microorganisms used in this study were Candida parapsilosis ATCC 22019, Candida krusei ATCC 14243 and Candida albicans ATCC 10231.

Results and Discussion
Compounds 1/2 were isolated as a pair of inseparable epimers, which showed only a single peak in reverse-phase (RP) HPLC and could not be separated by a variety of column chromatographies.This was evident from the observation of several signals of closely similar chemical shifts in the 13  Hz, H-1''') and 5.60 (d, J 7.9 Hz, H-1'').These data suggested for 1 a steroidal core structure containing three sugar units.
The 13 C NMR and distortionless enhancement by polarization transfer (DEPT) spectra displayed sixtythree signals, several of which related to oxygenated carbons with a high degree of overlapping.The presence of typical signals of spiroacetal carbons at d C 109.5 and 110.1 (C-22), and secondary methyl groups at d C 30.9 and 27.8 (C-25), suggested for 1/2 the structure of one mixture of steroidal saponins containing a spirostane skeleton as aglycone.The methyl groups at d C 16.9 and 17.6 (C-27) in axial and equatorial positions, respectively, besides the steric compression of the methyl group in axial position protecting the carbon at d C 26.7 (C-23) indicated the R/S epimeric pair at C-25.1][12] The relative configuration of the hydroxyl and sugar portion at C-2 and C-3 was established by comparison with the 13 C NMR spectral data described to the gitogenin and its 2β,3αdihydroxy isomer, through the characteristic chemical shifts relative to C-1 to C-5.The chemical shifts at d C 46.0 (C-1), 70.7 (C-2), 85.0 (C-3), 34.3 (C-4) and 44.9 (C-5) observed in 1/2 were in accordance with the 2α, 3β-orientation. 13ll proton and carbon signals were fully assigned through heteronuclear single-quantum correlation spectroscopy (HSQC) and heteronuclear multiple-bond correlation spectroscopy (HMBC) spectra analyses (Tables 1 and 2).The three sugar units were characterized by the HSQC correlations of the acetal carbon at d C 103.7 (C-1') with the anomeric proton at d The monosaccharides were identified as one glucose and two galactose units through NMR analyses followed by comparison with the literature data. 12,14The absolute configurations were determined as D, on the basis of the acid hydrolysis followed by sililation of the sugars and GC analysis.The β-anomeric configurations of the glucopyranosyl and galactopyranosyl moieties were supported by the relatively large vicinal coupling constant values (ca.8.0 Hz) observed for the anomeric protons.
The HMBC spectrum analysis allowed to establish the sequence of all sugar units by long-range correlations between the proton signal at d Compounds 3/4 was isolated as a yellow resin and showed only a single peak in RP HPLC.The support for an epimeric mixture with a spirostanol steroid skeleton came from the comparative analysis of its NMR data with those observed for compounds 1/2.
1][12][13][14][15] The β-orientations of glucose and galactose and the α-orientation of rhamnose were ascertained by the coupling constant values of the anomeric protons.A further coupling constant corresponding to a proton signal at d H 4.27 (t, J 9.5 Hz, H-4'') confirms the galactose unit.The absolute configuration of the glucose and galactose units was determined as D and that of rhamnose as L, through acid hydrolysis, followed by sililation and GC analysis.In addition, the quasimolecular ion peak at m/z 903.4979 (calcd.for 903.4953) in the HRESIMS, and the subsequent fragmentations signals at m/z 741.4476, 595.3871 and 433.3298 relative to the loss of the three sugar moieties, confirmed the proposed structure.
The attachment of the sugar moieties to C-3 of the aglycone was deduced by long-range correlations between the anomeric proton of the galactopyranosyl unit at d  Compounds 5/6, white solid, were isolated as one single peak by RP HPLC.The existence of several signals with closely similar chemical shifts in the NMR spectra led to the conclusion that this compound, as the other previously described, was also an epimeric mixture.
Inspection of the 1 H NMR spectrum also suggested a steroidal saponin for compounds 5/6, by the signals relative to methyl groups at d H 0.70 (s, H-18), 0.90 (s, H-19), 1.60 (s, H-21) and 0.95 (d, J 6.0 Hz, H-27), and the protons of the sugar units in the range of d H 3.2-3.9,besides the A typical ∆ 20,22 -furostanol skeleton was deduced after comparative analysis of the 13 C NMR data with those of compounds 1/2 and 3/4, that revealed the absence of the carbons relative to a spiroketal, and the presence of typical signals of unsaturated carbons at d C 105.2/105.3(C-20) and 153.0/153.1 (C-22), besides one oxymethylene at d C 75.9 (C-26). 16,17The presence of the 2α,3β-dioxy-decalin moiety was supported by the oxymethine carbons at d C 71.7 (C-2) and 85.1 (C-3).
1][12][13][14][15] Their β-orientations were as ascertained by the relatively large coupling constants of the anomeric protons.This was further confirmed by the precursor ion peak at m/z 757.4372 (calcd.for 757.4374) in the HRESIMS, and the subsequent loss of the two sugar units at m/z 595.3828 and 433.3308.
The attachment of the sugar moieties was deduced through the HMBC analysis by the correlation peaks observed for the anomeric proton of the glucose unit at d The values of ∆ ab = 0.34 and 0.46 were in agreement with those reported for 25R and 25S furostane-type steroidal saponins (∆ ab < 0.35 for 25R; ∆ ab > 0.45 for 25S). 17From the above mentioned data compound 5/6 was assigned as the new 26-O-β-D-glucopyranosyl-(25R,S)-5α-furost-20ene,2α,3β-diol-3-O-β-D-galactopyranoside.
Compounds 7/8 were isolated as white solid.As observed previously, the several signals with closely similar chemical shifts in the NMR spectra revealed it to be an epimeric mixture.
Comparison of the NMR data of 7/8 with those correspondents of 5/6 revealed that these compounds were quite similar, except for the presence of an additional hexose monosaccharide.Three anomeric protons at d H 4.22 (d, J 7.2 Hz, H-1'), 4.33 (d, J 7.2 Hz, H-1'') and 4.82 (m, H-1''') were observed in the 1 H NMR spectrum.In addition, the resonances of the aglycone moiety containing one glucose unit linked to C-26 appeared almost identical in the 13 C NMR, while some slight differences were detected among the resonances of the galactose that was linked to C-3 (Tables 1 and 2).In particular, the signals for C-4'' (d C 70.5) and for H-4'' (d H 3.82) of 5/6 were a lot more shielded than the corresponding C-4'' (d C 79.4) and H-4''(d H 4.02) on 7/8.These data suggested the additional sugar moiety to be linked at C-4''.It was identified as β-D-galactopyranosyl by comparison of its 13 C NMR chemical shifts with those reported for sugar moieties and by GC analysis. 14The quasimolecular ion peak at m/z 919.4902 (calcd.for 919.4902) in the HRESIMS, and the subsequent fragmentations at m/z 757.4390, 595.3831 and 433.3283, corroborated the above data.
H 4.92 (H-1') and the carbons at d C 85.6/85.2(C-3) in the HMBC spectrum.Moreover, the correlations between the anomeric proton of the glucopyranosyl unit at d Η 5.53 (H-1''') with the carbon at d C 76.5 (C-2'') of the rhamnopyranosyl, whose anomeric proton at d Η 6.38/6.28(H-1'') correlated with the carbons at d C 78.4/78.0(C-4') of the galactopyranosyl, established the sugar sequence (Figure 2).The nuclear overhauser effect spectroscopy (NOESY) spectrum provided certain information for the stereostructure assignments.This spectrum showed NOE correlations between the signals at d H 0.70 (H-19) and 3.91 (H-2), and between d H 1.05 (H-5) and 3.82 (H-3), and the junction of rings A/B was confirmed to be trans.
Η 4.22 (H-1') with the oxymethylene carbon at d C 75.9/75.8(C-26), and for the anomeric proton of the galactopyranosyl unit at d Η 4.22 (H-1'') with the carbon at d C 85.1 (C-3) of the aglycone.In addition, long range correlations were also detected for the proton at d Η 0.95 (H-27) with the carbons at d C 75.9 (C-26) and 32.2 (C-24), and for the proton at d Η 4.70 (H-16), 2.48 (H-17) and 1.71 (H-24) with the unsaturated carbons at d C 153.1/153.0(C-22).The C-25 configuration was assigned as 25R and 25S based on the observed difference of the 1 H NMR chemical shifts related to the H-26 geminal protons (∆ab = d a − d b ).
were recorded at 125 MHz; b and at 150 MHz; c solvent C 5 D 5 N; d solvent CD 3 OD; e show the same chemical shifts.
e a1 H NMR data recorded at 500 MHz; b and at 600 MHz; c solvent C 5 D 5 N; d solvent CD 3 OD; e show the same chemical shifts.

Table 4 .
Cytotoxicity against four human cancer cell lines a 95% of confidence interval; b positive control.Experiments were performed in triplicate.ND: Not determined.

Table 3 .
Antimicrobial activity Compound C. krusei MIC 50 / (μg mL −1 ) Geometric means of at least three halo determined on different days; b DMSO.NA: No activity. a