Results and Discussion From the trichomes isolated from green fruits of Solanum crinitum

O estudo fitoquímico de Solanum crinitum Lam forneceu quatro flavonóides: tilirosídeo (1), astragalina (2), kaempferol (3) e biochanina A-7-O-b-D-apiofuranosil-(1→5)-b-D-apiofuranosil(1→6)-b-D-glucopiranosideo (7), ácido 4-hidroxibenzoico (12), e quatro derivados do ácido cinâmico: cise transcumárico (10 e 11), cise trans-cumarato de etila (8 e 9), isolados de tricomas do fruto. Do extrato metanólico de frutos verdes foram isolados três alcalóides esteroidais glicosilados: solamargina (13), 20-epi-solamargina (14) e solasonina (16). Os derivados 3,5,7,4’-tetra-O-metil-kaempferol (4), 3,7,4’-tri-O-metil-kaempferol (5), 3,7,4’-tri-O-metil-5-Oacetil-kaempferol (6), peracetil-epi-solamargina (15) e peracetil-solasonina (17) foram sintetizados e estão sendo registrados pela primeira vez na literatura. As estruturas foram definidas através de análise de dados espectrométricos.

6][17][18] The first report of 1 in Solanum species was made by Souza et al. 14 Kaempferol and astragalin have been isolated from some Solanum species. 8The treatment of 3 with diazomethane yielded the methyl derivatives 4 and 5, described in the literature, 19 and the derivative 6 was obtained by treating 5 with Ac 2 O/Pyridine.The 2D NMR spectra, including NOESY experiments, of these derivatives were used to confirm the proposed structure of 3 and to carry out the complete assignment of the 1 H and 13 C chemical shifts of 6 (see Experimental).
The 1 H and 13 C NMR spectra of compound 7 revealed characteristic resonances of the isoflavonoid biochanin A besides additional signals for three sugars unities, one glucopyranoside and two apiofuranosides.Comparison of these data with those of glycosides isolated from Dalbergia nigra 20 and Andira anthelmia, 21    (7).This is the first report of this apiofuranosyl derivative in Solanum species.
2][23][24][25] The integration of the 1 H NMR signals allowed us to calculate the approximate relative percentage of both compounds: 36.16% of 8 and 63.84% of 9.
The fractions containing compounds 13, 14 and 16 showed a positive test for alkaloids.The detailed analysis of the 1 H and 13 C NMR spectra of the isolated compounds allowed to identify characteristic signals corresponding to the same aglycone as that of the steroidal spirazolane-type alkaloid in the three glycoalkaloids: four quaternary carbon atoms, including one linked to oxygen and nitrogen atoms:  1) having a trisaccharide moiety (three anomeric carbon atoms: d C 100.6 to 100.3) attached to the oxygen atom of carbon CH-3, with d C 78.5 to 78.0 (Table 1 and 2), which is significantly higher when compared with the 13 C chemical shift of the methyl carbon CH-3 sustaining free hydroxyl group (about d C 71).The 1 H and 13 C chemical shifts of the trisaccharide moieties of 13 and 14, had practically the same values (Table 2)   15 = m/z 868.5058), besides additional peaks compatible with loss of sugar moieties, were used to confirm the molecular formula of these isomeric compounds (13 and 14).The hydrogen and carbon atoms signals observed in the 1 H and 13 C NMR spectra of 13-17 (Tables 1 and 3) were also assigned with aid of the homonuclear 2D 1 H-1 H-COSY and heteronuclear 2D HMQC ( 1 H-13 C-COSY-1 J CH ) and HMBC ( 1 H- 13 C-COSY-n J CH , n = 2 and 3), allowing to identify the 1 H chemical shifts of the methyl group signals of the aglycone and of the trisaccharide moieties (Table 3 and 4 1.Comparative analysis of these data was used to suggest the two stereoisomers H-20b ( 13) and H-20a (14), since the 22aN and 22bN possibilities were eliminated considering the absence of 13 C signal corresponding to a methylene carbon CH 2 -23 at about d C 27 which is consistent with the 13 C NMR chemical shift of the 22bN stereoisomer (Figure 1).7][28][29][30] In order to compare the relative stabilities of the epimeric structures 13/14, a molecular modeling study was implemented using the Spartan 06 for Linux program (Wavefunction, Inc.), see Supplementary Information. 31he complete 1 H and 13 C chemical shift assignments of the signals of CH 3 , CH 2 , CH and C observed in the 1 H (including 1 H-1 H-COSY) and 13 C ({ 1 H} and DEPT) NMR spectra (including 2D experiments HMQC and HMBC) (Tables 1, 2, 3 and 4) and comparison with values described in the literature for solamargine (13) [26][27][28][29] and solasonine (16) 28,31 led to the proposition of the structures (25R) en (solasonine, 16) for the three steroidal glycoalkaloids isolated from this plant (Figure 1).The natural alkaloids 14 and 16 were treated with Ac 2 O/pyridine to yield the peracetyl derivatives 15 and 17.The 1 H and 13 C NMR spectral data of these peracetyl derivatives (15 and 17), obtained through the analysis of extensive 1D and 2D NMR experiments (Tables 1-4), were also used to confirm the postulated structures to 14 and 16.The analysis of 1D and 2D NMR spectra was also used to make the complete hydrogen and carbon-13 chemical shift assignments for the alkaloid, the new 16-epi-solamargine ( 14) and for the two peracetyl derivatives 15 and 17.

Experimental
General procedure Melting points have not been corrected.IR, NMR and mass spectra were recorded on the same equipments used in previous papers. 21,32Column chromatography was carried out with silica gel (Vetec and Aldrich 0.05-0.20 mm) and Sephadex LH-20 (Sigma, USA); silica gel F254 G (Vetec) was used for preparative TLC; aluminum backed (Sorbent) silica gel plates W/UV254 were used for analytical TLC, with visualization under UV (254 and 366 nm), with AlCl 3 -ETOH (1%), Liebermann-Burchard and/or Godin reagents, or exposure to iodine vapor.

Extraction and isolation
The trichomes (9.7 g) were isolated by scraping the green fruits with a glass slide and were subsequently extracted with CHCl 3 and MeOH in an ultrasound bath to furnish the CHCl 3 extract (310.0 mg) and MeOH (3.0 g) residues.The CHCl 3 residue was chromatographed on a sephadex CC, using MeOH as eluent, and 13 fractions were collected and analyzed by TLC and 1 H NMR spectroscopy.Fractions 7-8 yielded astragaline (1, 10.0 mg) and fractions 9-10 yielded the tilirozide (2, 25.0 mg).The methanolic extract was chromatographed on a silica gel column (col A) and 20 fractions were collected and analyzed by TLC plate.Fractions 5-8 (2.0 g) were dissolved in methanol and addition of CH 2 Cl 2 yielded a precipitate that was separated by filtration to afford a solid (1.6 g) that was identified as kaempferol (3, mp 282-284 o C) and the mother liquor (AM-5-8).The reaction of kaempferol (65.0 mg) with diazomethane yielded 16.1 mg of 4 (mp 128-130 o C) and 36.6 mg of 5 (mp 140-142 °C) that were separated by preparative TLC.20.3 mg of 5 were treated with Ac 2 O/ pyridine (1:1) to afford 6 (14.8 mg, mp 166-168 °C).The mother liquor (AM 5-8, 360.0 mg) was submitted to silica gel CC and fractions 8-12 yielded a mixture of 8 and 9 (1.2 mg, gum).Fraction 10 (850 mg) from col A was chromatographed on a silica gel column and 130 fractions of 50 mL were collected.Fractions 32-40 yielded 35.5 mg of a material corresponding to the mixture of 10, 11 and 12. Fractions 94-114 (102.3 mg) were chromatographed on a silica gel column and fractions 3-5 yielded 7 (9.5 mg) after filtration on Sephadex LH20.
The green fruits (2.6 g) of Solanum crinitum Lam were powdered and extracted with ethanol + acetic acid (2%) and 900 mL of solution was obtained.900 mL of acetic acid (10%) were added, and the solution was left to stand and chill overnight.The solution was filtered under vacuum using Hirsch funnel.NH 4 OH (pH 9-10) was added to the filtrate which was allowed to stand overnight in the fridge affording a precipitate (96.4 g) corresponding to the glycoalkaloids fraction was obtained.90 g of the glycoalkaloids was adsorbed on silica gel and applied on a silica gel column, eluted with hexane, dichloromethane, ethyl acetate, acetone and methanol.14 fractions of 500 mL were collected.Fraction 8 (1.0 g), collected with ethyl acetate, was chromatographed in a silica gel CC and fractions analyzed by silica gel TLC plate.Fraction 84 (52.6 mg) from this column was filtered on a Sephadex LH20 column, eluted with methanol, and the alkaloid solamargine 13 (268-270 °C) was obtained.Fraction 10 (8.67 g) was extracted with methanol and the alkaloids were detected with Dragendorff and Libermann Burchard.This fraction was chromatographed on a silica gel column using CH 2 Cl 2 /MeOH (3:1) as initial eluent.79 fractions were collected and were analyzed by TLC plate and reunited in group.Fractions 24-29 (1.2 g) were filtered on a Sephadex LH-20 column and the epi-solamargine (14, 225.8 mg, 238-250 °C) was obtained.Acetic anhydride:pyridine (1:1) treatment of 14 (44.5 mg) yielded 44.5 mg of the peracetyl derivative 15 (mp 128-130 °C).Fractions 39-44 (1.3 g) were filtered on Sephadex LH20 to isolate solasonine (16, 93.5 mg, mp 244-246 °C).Treatment of 16 (79.1 mg) with acetic anhydride:pyridine (1:1) gave the peracetyl-solasonine (17, 27.6 mg, mp 148-150 °C).

Figure 1 .
Figure 1.Comparative analysis of the chemical shifts corresponding to the hydrogen (d H ) and carbon (d C ) atoms CH-16, CH-17, CH-20, CH 3 -21, C-22, CH 2 -23 and CH 2 -26 of 13 and 14, including comparison of the d C these carbon atoms and of the CH 2 -24 to reveal the structural distinction between solamargine and b-solamarine.