New Flavonoids and other Constituents from Ouratea hexasperma ( Ochnaceae )

O estudo fitoquímico do extrato metanólico de galhos de Ouratea hexasperma (Ochnaceae) forneceu dois novos flavonóides, 7-O-β-D-glicopiranosil-6-(3-metilbut-2-enil)-5,4’diidroxiflavanonol (6-β,β-dimetilalil-7-O-β-D-glucopiranosil-aromadendrina) e 7-O-β-Dglicopiranosil-6-(3-metilbut-2-enil)-3,5,4’-triidroxiflavona (6-β,β-dimetilalil-7-O-β-Dglucopiranosil-kaemperol) além de uma mistura de sitosterol e estigmasterol, lupeol, 3-O-β-Dglicopiranosil-sitosterol e o ácido 2,4-diidroxifenilacético. As estruturas dessas substâncias foram estabelecidas através de análise dos espectros de IV, EM-IES e RMN das substâncias naturais e dos derivados peracetílico e éter dimetílico da nova flavona, além de comparação com dados da literatura.


Introduction
Previous phytochemical and pharmacological investigations on Ouratea species (Ochnaceae) have shown the presence of terpenoids, isoflavonoids, flavonoid glycosides, and more frequently biflavones which are considered as chemical markers for this genus. 1][10][11] The present paper reports the results of an additional phytochemical study of the extract from the branches of O. hexasperma, describing the identification of two new prenylflavonoid glucosides, including some known compounds.

General procedures
Melting points have not been corrected.IR spectra were recorded on a Perkin-Elmer 1605 FT-IT spectrophotometer. 1 H (200.0 MHz) and 13 C (50.3 MHz) NMR spectra were recorded on a Brüker AC 200 spectrometer using DMSO-d 6 , CD 3 OD or CDCl 3 with TMS as internal standard.HRESI mass spectra were obtained with a Brüker Daltonics UltrOTOF-Q, Billerica, MA, spectrometer using (H 2 O, Ar as CAD), CE 20 eV for MS and 45 eV for MS/MS in negative mode.Electron Ionization Mass Spectra (LREI-MS) of 3 was taken using a gas chromatograph coupled to a mass spectrometer (GC-MS) on a Varian Saturn 2000 using an ion trap at 70 eV.The Advanced Chemistry Development (ACD/ Labs) software V8. 14 for Solaris 1994-2007 ACD/Labs was used for prediction of 1 H and 13 C NMR chemical shifts of 3. Column chromatography 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%), Lieberman-Burchard and/or Godin reagents, or exposure to iodine vapor.

Extraction and isolation
The dried and powdered branches of O. hexasperma (410.0 g) were extracted with methanol at room temperature.The solvent was removed under vacuum furnishing a residue (44.0 g).The crude methanol residue (30.0 g) was filtered on silica gel with CH 2 Cl 2 and EtOAc, yielding, after removal of the organic solvent, two residues named OHMC (196.0 mg) and OHMAc (15.5 g).The residue OHMC was fractionated by CC on silica gel eluted with a mixture of CH 2 Cl 2 :EtOAc with increasing polarity to EtOAc (100%), and the resulting fractions grouped according to their TLC profile.Fractions 2-10 yielded a hydrocarbon mixture, Fr 15-25 furnished a mixture of steroids, and Fr 27-40 yielded lupeol (300 mg).Fraction OHMAc (10.0 g) was fractionated on a silica gel column, eluted with CH 2 Cl 2 :MeOH with increasing polarity to 100% MeOH.Thirthy sub fractions were collected, and assembled according to their composition, as checked by TLC.The sub fractions 2-4 yielded a mixture of sitosterol and stigmasterol (25.0 mg); fractions 6-7 furnished a residue (mp 308-310 o C, 40.0 mg) insoluble in chloroform, that was identified as sitosterol glycoside, from the formation of its acetyl derivative (Ac 2 O:Pyridine).Fractions 8-9 were purified by preparative TLC using CH 2 Cl 2 :MeOH (8:2) to yield compound 3 (gum, 5.0 mg).Fr 11-15 was submitted to Sephadex LH-20 column employing methanol as eluent.First pure fractions were grouped and crystallized from ethyl acetate to give 1 (mp 172-173 o C, 90.0 mg), whereas the last fractions yielded a mixture of 1 and 2. Fr 20-25 was submitted to Sephadex LH-20 CC using methanol as eluent, to furnish a mixture of 1 and 2.Last chromatographic fractions yielded 2 as an yellow solid (mp 179-180 o C, 80.0 mg).The flavone 2 was treated with diazomethane to yield 2a (gum, 12 mg), and with Ac 2 O/pyridine (1:1) to form the peracetyl derivative 2b (gum, 20 mg; Table 1).

Figure 1 .
Figure 1.Structures of the new flavonoids and 2,4-dihydroxyphenylacetic acid isolated from O. hexasperma and the derivates 2a and 2b.

Figure 2 .
Figure 2. Proposed fragments for the principal peack detected in ESI-MS of 1 and 2.