Flavanones from Vernonia diffusa *

Isolaram-se do extrato metanólico da madeira de Vernonia diffusa duas flavanonas: hesperidina e a nova flavanona, 3’-metilhesperetina (homoesperetina) e sacarose que foi identificada como derivado acetilado. A homoesperetina foi identificada como a aglicona obtida da hidrólise do novo glicosidio natural 7-rutinosilhomoesperetina. Do extrato em diclorometano foi identificada a mistura de sistosterol e estigmasterol. Foi preparado o novo derivado octaacetilhesperidina. As determinações estruturais foram realizadas através da análise dos dados espectrométricos de RMN de H e C incluindo experiências de DEPT, HxH-COSY, HxC-COSY e NOEDIFF.


Introduction
Vernonia diffusa Less belongs to the Vernoneae tribe which has many endemic genera occurring in Brazil.Some of them are used as adornment, medicinal or as pasture's bee.This species is a tree widely distributed in the plains and in the Serra do Mar's forest in São Paulo, Rio de Janeiro, Paraná and Santa Catarina.
A review on the previous phytochemical investigations of this genus revealed the study concerned 138 Vernonia species among which 38 are from Brazil.The chemical constituents found are triterpenes, steroids, and lignoids, but the more frequent compounds are sesquiterpenoid lactones and flavonoids 1 .These two classes of compounds have been used as taxonomic markers of this genus.The literature has presented many publications of pharmacological activities of sesquiterpene lactones and flavonoids which have been isolated from Vernonia species 1,2,3 .In this first chemical investigation of V. diffusa we report the isolation and identification of two flavanones besides sitosterol, stigmasterol and sucrose.Eriodictiol is the only flavanone isolated from Vernonia species so far (V.hindei) 4 .
The mixture of compounds 1 and 2 was recognized by analysis of the 1 H and 13 C-NMR spectral data and comparison with the literature 5 .The relative percentage of 1 (45%) and 2 (55%) was deduced from 1 H-nmr integrals of the signals registered for H-22/23 (in 2) and H-6 (in 1 + 2).
The carbohydrate 3 was identified by comparative analysis with the literature data described for peracetylated sucrose (3a) 6,7 .
Spectral and TLC analysis of the hydrolysis products of a crystalline fraction (VDM-2) allowed us to identify the aglycone as a flavanone similar to 6 but with two methoxy groups [δH 3.89 and 3.81(s, 3H)], 3'-methylhesperetin (7).The identification of this flavanone was done by comparison of 1 H and 13 C-NMR data with those of hesperetin (6)  and hesperidin (4).The presence of the methoxy groups at C-3'and C-4' in 7 was confirmed by an upfield shift of the signals (δC-2' 110.6 and δC-5' 112.6), when compared with that of the same carbon of 4 and 6 (δC-2' 114.4 and δC-5' 112.0).This can be attributed to the γ-effect of the methyl group of the methoxy function at C-3'.Analysis of the aqueous fraction from the hydrolysis by thin layer chromatography (TLC) allowed the identification of the carbohydrates glucose and rhamnose by comparison with authentic samples using the literature methodology 10 .The analysis of 1 H-NMR and 13 C-NMR spectra data of VDM-2 led to identification of the signals of 4 and the additional chemical shifts similar to those of 7.This observation led us to propose the presence of homoesperetin-7-O-rutinoside (5), which is the new natural substance that yields 7 upon hydrolysis of VDM-2.The value δCH2 66.14 for CH2-OR in the 13 C-NMR spectra (PND and DEPT) led us to discard the possibility of two monoglycosylflavanones and to confirm the connection 1''' → 6'' in the rutinoside moiety of 5.

General experimental procedure
Melting points were determined using a kofler hot stage instrument and are uncorrected; NMR spectra were measured in DMSO-d6, D3CCOCD3, or CDCl3 using TMS as the internal standard, employing a Bruker AC-200 ( 1 H: 200 MHz, 13 C: 50.3 MHz); C.C. was run with Silica gel S (Riedel, 0.0032-0.0063mm); TLC was performed on Silica gel 60 F 254 (Merck).

Plant material
Vernonia diffusa Less, Compositae, was collected in Barra do Piraí, RJ, and authenticated by Dr a Marilena de Menezes Silva Conde (IB-UFRRJ).A voucher specimen is available for inspection in the herbarium of Instituto de Biologia, UFRRJ-RJ, Brazil.

Extraction and isolation
The powdered wood (4.0 kg) was extracted by maceration with CH2Cl2 and methanol.The solvent was removed under vacuum to yield 9.1 g of CH2Cl2 and 45.02 g of methanolic residue.The CH2Cl2 residue (2.0 g) was fractionated in C.C. of Silica gel with dichloromethane as solvent, gradually enriched with MeOH, to afford 200 fractions of 20 mL.The fractions 80-120 were crystallized from dichloromethane/hexane (1:1) to yield a mixture of 1 and 2 (190 mg).The methanolic residue (20.0 g) was precipitated from MeOH/AcOEt (1:1) to yield a solid (400 mg, mp 261°), named VDM-1, that was identified as 4. The soluble part was fractionated in florisil with EtOAc and MeOH.EtOAc fraction yielded KCl (81.0 mg) and a mixture of aliphatic acids.The MeOH fraction was fractionated in a column of silica gel in AcOEt/MeOH (7:3) increasing the polarity to neat MeOH.This procedure afforded 69 fractions.Fractions 5-30 yielded an amorphous material (200 mg), named VDM-2 whose 1 H and 13 C-NMR spectra revealed a mixture of glycoside similar to 4. The analysis of the acid hydrolysis products 10 led us to identify the flavanone homoesperetin (7) in the chloroform fraction and the carbohydrates glucose and rhamnose in the aqueous fraction which is in agreement with 5 as natural substance in VDM-2.Fractions 35-64 (160.0 mg) yielded impure 4 and fractions 65-69 (45.0 mg) were acetylated with Ac2O and pyridine.The solution was kept at room temp for 24 h and usual work-up gave a mixture of acetates (45.0 mg).This mixture was fractionated on a silica gel column (AcOEt : MeOH, 7:3) to yield 4a (10 mg) and 3a + 4a (30 mg).The same procedure was done to prepare the acetyl derivatives 4a (80.0 mg, gum) from 4 (100.0mg).

Acid hydrolysis of VDM-2
VDM-2 (100 mg) was dissolved in 10 mL of MeOH-H2O (1:1) with concentrated HCl (1.0 mL) and the solution was kept under reflux for 3 hr.The mixture was extracted with CHCl3.The CHCl3 layer was washed with H2O, dried over anhydrous Na2SO4 and concentrated under reduced pressure, filtered in silica gel, to yield 7 (30.0mg).The H2O of the solution was evaporated to dryness under reduced pressure after addition of acetone.The sugars were identified as D-glucose and L-rhamnose by comparison with authentic samples by thin layer chromatography (TLC) using silica gel S (Riedel) impregnated with 5% of NaOAc as adsorbent and EtOAc-isoPrOH-H2O (35:39:26) as eluent.Spots were visualized by spraying with a freshly prepared solution of diphenylamine (4% in EtOH), aniline (4% in EtOH) and concentrated phosphoric acid (5:4:1), after heating for 10 min.