Flavanones from Vernonia diffusa

Abstracts

From the methanolic extract of the wood of Vernonia diffusa two flavanones were isolated and identified: hesperidin and a new flavanone, 3'-methylhesperetin (homoesperetin) and sucrose, which was identified as its acetyl derivative. The homoesperetin was identified as the aglicone obtained in the hydrolysis of the new natural flavanone glycoside, homoesperetin-7-O-rutinoside. From the dichloromethane extract, a mixture of sitosterol and stigmasterol was isolated together with a mixture of aliphatic acids. The new octaacetylhesperidin derivative was also prepared. Structural determination was made by ¹H and 13C-NMR spectrometric data including DEPT, ¹Hx¹H-COSY, and ¹Hx13C-COSY and NOEDIFF techniques.

Vernonia diffusa; compositae; homoesperetin; flavanones

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 ¹H e 13Cincluindo experiências de DEPT, ¹Hx¹H-COSY, ¹Hx13C-COSY e NOEDIFF.

Article

Flavanones from Vernonia diffusa*

Mário Geraldo de Carvalho*^a, Patricia Miranda da Costa^a, and Heber dos Santos Abreu^b

^a Curso de Pós-Graduação em Química Orgânica, Departamento de Química, I.C.E.Curso de Pós-Graduação em Química Orgânica, Departamento de Química, I.C.E. Universidade Federal Rural do Rio de Janeiro, 23851-970 Seropédica - RJ, Brazil;

^b Departamento de Produtos Florestais, Instituto de Florestas, Universidade Federal Rural do Rio de Janeiro

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 ¹H e ¹³Cincluindo experiências de DEPT, ¹Hx¹H-COSY, ¹Hx¹³C-COSY e NOEDIFF.

From the methanolic extract of the wood of Vernonia diffusa two flavanones were isolated and identified: hesperidin and a new flavanone, 3'-methylhesperetin (homoesperetin) and sucrose, which was identified as its acetyl derivative. The homoesperetin was identified as the aglicone obtained in the hydrolysis of the new natural flavanone glycoside, homoesperetin-7-O-rutinoside. From the dichloromethane extract, a mixture of sitosterol and stigmasterol was isolated together with a mixture of aliphatic acids. The new octaacetylhesperidin derivative was also prepared. Structural determination was made by ¹H and ¹³C-NMR spectrometric data including DEPT, ¹Hx¹H-COSY, and ¹Hx¹³C-COSY and NOEDIFF techniques.

Keywords: Vernonia diffusa, compositae, homoesperetin, flavanones

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 flavonoids¹. 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.

Results and Discussion

Our phytochemical investigation of V. diffusa by chromatographic fractionation of the wood's dichloromethane and methanol extracts yielded a mixture of sitosterol (1) and stigmasterol (2), sucrose (3), the known flavanone hesperidin (4) and two new flavanones, 3'-methylhesperetin (7), named homoesperetin and its 7-O-rutinoside (5).

The mixture of compounds 1 and 2 was recognized by analysis of the ¹H and ¹³C-NMR spectral data and comparison with the literature⁵. The relative percentage of 1 (45%) and 2 (55%) was deduced from ¹H-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.

Glycoside 4 was identified by analysis of its spectroscopic data [¹H-NMR and ¹³C-NMR including 2D NMR experiments ¹Hx¹H-COSY and ¹Hx¹³C-COSY, ⁿJ_CH (n = 1, 2 and 3, COLOC)] and comparison with those previously reported for the hesperidin (hesperetin-7-rutinoside, 4)^8,9. The hydrolysis and comparison with authentic samples using the same procedure described in the literature¹⁰ (see experimental) were used to identify the carbohydrates L-rhamnose and D-glucose of the rutinoside unit. The connection (1"'® 6") of this moiety was confirmed by the chemical shift of the CH₂-6" (d_C 66.4 ppm). The presence of a OCH₃ group at 4' position was confirmed by irradiation at d_H 3.89 (s, 3H) of 4a which resulted in a 9% NOE at H-5'doublet (d_H 6.92, d, 8.0 Hz,) and by the cross peak of OCH₃ (d_H 3.89) and H-5'(d_H 6.92) with C-4'(d_C 140.0, ^2,3J_CH) in the ¹Hx¹³C-COSY spectra. The same spectrum shows a cross peak of H-2' [d_H 7.12 (s)] with d_C 121.0 (C-2', ¹J_CH) and 161.2 (C-3', ²J_CH) which were used to confirm the location of the hydroxyl group at C-3'. The ¹H and ¹³C-NMR data of the new octaacetyl derivative 4a are cited in the experimental part.

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 [d_H 3.89 and 3.81(s, 3H)], 3'-methylhesperetin (7). The identification of this flavanone was done by comparison of ¹H and ¹³C-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 (d_C-2' 110.6 and d_C-5' 112.6), when compared with that of the same carbon of 4 and 6 (d_C-2' 114.4 and d_C-5' 112.0). This can be attributed to the g-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 methodology¹⁰. The analysis of ¹H-NMR and ¹³C-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 d_CH2 66.14 for CH₂-OR in the ¹³C-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.

Experimental

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 (¹H: 200 MHz, ¹³C: 50.3 MHz); C.C. was run with Silica gel S (Riedel, 0.0032-0.0063 mm); 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 ¹H and ¹³C-NMR spectra revealed a mixture of glycoside similar to 4. The analysis of the acid hydrolysis products¹⁰ 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 (100.0 mg).

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 (30.0 mg). 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.

Octaacetylhesperidin (4a): gum, IR (film, n_max^NaCl) cm^-1: 2970, 2860, 1740, 1650, 1607, 1500, 1450, 1380; 1240, 1050; ¹H-NMR (CDCL₃, d): 7.22 (br d, 8.0 Hz, H-6'), 7.10 (br s, H-2'), 6.92 (d, 8.0Hz, H-5'), 6.40 (d, 2.2Hz, H-8), 6.24 (d, 2.2 Hz, H-6), 5.34 (dd, 10.0; 3.1 Hz, H-2), 5.22 (m, H-3"), 5.18 (m, H-1", 3"', 4"'), 5.12 (m, H-4"), 5.10 (m, H-2"'), 4.95 (t, 9.8Hz, H-2"), 4.59 (br s, H-1"'), 3.9 (s, OCH3), 3.8-3.9 (m, H-5", 5"'), 3.6 (m, H-6"), 2.92 (dd, 18.6; 10.0Hz, H-3ax), 2.6 (dd, 18.6; 3.1Hz, H-3-eq), 1.9-2.1, 2.26, 2.30 (s, H₃C-CO), 1.1 ( d, 6.0Hz, H-6'"); ¹³C-NMR(CDCl₃, d): 188.5(C-4), 179.0-169.0 (O-C=O), 163.5 (C-7), 161.8 (C-5), 161.2 (C-3'), 151.8 (C-9), 140.0 (C-4'), 130.7 (C-1'), 124.7 (C-6'), 121.0 (C-2'), 109.5 (C-10), 105.8 (C-6), 102.1 (C-8), 97.9 (C-1"'), 97.5 (C-1"), 73.2 (C-5"), 72.3 (C-3"), 70.8 (C-2"'and C-4"'), 69.2 (C-2"0, 68,9 (C-3"'), 68.6 (C-4"), 66.5 (C-5"'), 66.4 (C-6"), 55.8 (OCH₃), 20.1-20.4 (CH₃-CO), 17.5 (C-6"').

Homoesperetin-7-O-rutinoside (5): (mixture with 4), ¹H-NMR (DMSO-d₆, d): 12.3(HO-5), 6.92 (br s, H-6',5'), 6.95 (s, H-2'), 6.10 (br s, H-6, 8), 5.50 (dd, 10.0; 3.10 Hz, H-2), 3.1-3.6 (m, H-C-O), 4.90 (d, 6.0 Hz, H-1"), 4.60 (br s, H-1"'), 3.82, 3.80 (s, OCH₃), 2.70-3.20 (m, 2xH-3), 1.07 (d, 6.0Hz, H-6'"); ¹³C-NMR(CDCl₃, d): 197.1(C-4), 165.2 (C-7), 162.6 (C-5), 163.1 (C-9), 147.0 (C-3'), 146.0 (C-4'), 130.0(C-1'), 118.0 (C-6'), 112.0(C-5'), 110.5 (C-2'), 96.4 (C-6), 95.7 (C-8), 100.0 (C-1"'), 99.5 (C-1"), 78.6 (C-3"), 76.3 (C-5"), 73.0(C-4"'), 70.4 (C-2"'), 70.3 (C-3"'), 69.6 (C-4"), 69.2 (C-2"), 68.4 (C-5"'), 66.1 (C-6"), 55.8 (2xOCH₃), 17.9 (C-6"').

Homohesperetin (7): gum; IR (film, n_max^NaCl) cm^-1:3476 2917, 1648, 1607, 1521, 1277, 1205;¹H-NMR (DMSO-d₆, d): 12.2 (br s, HO-5), 7.9 (br s, 7-O-H), 6.96 (br d, 8.0 Hz, H-2', H-5', 6'), 5.90 (br s, H-6, 8), 5.40 (br d, 12.0; H-2), 3.81 (s, OCH₃), 3.84 (s, OCH₃), 3.06 (dd, 14.0; 12.0Hz, H-3ax), 2.76 (dd, 14.0; 3.0 Hz, H-3-eq). ¹³C-NMR (DMSO-d₆, d): 195.9 (C-4), 165.6 (C-7), 163.0 (C-5), 163.5 (C-9), 146.9 (C-3'), 145.7 (C-4'), 130.0 (C-1'), 118.2 (C-6'), 112.7 (C-5'), 110.6 (C-2'), 103.7 (C-10), 95.6 (C-6), 96.7 (C-8), 55.8 and 55.9 (OCH₃). EIMS m/z (rel. Int.): 316(37, M^.+), 191(25), 164(100), 152(22), 151(80), 137(30), 124(20).

Acknowledgments

The authors are grateful to CNPq for research fellowships to P.M.C. and to M.G. de C. We thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Financiadora de Estudos e Projetos (FINEP-PADCT) for grants. We also thank to Dr. V.M. Runjaneck (DQuim, UFRRJ) for revision of the english.

Received: April 30, 1998

• 1. Costa, P.M. da. Tese de Mestrado; Departamento de Química; Universidade Federal Rural do Rio de Janeiro; Brazil, 1996, p. 5-20.
• 2. Kiesel ,W. Pol. J. Pharmacol. Pharm 1975, 27, 461.
• 3. Bruneton, J.; Pharmocognosy, Phytochemistry, Medicinal Plants; Lavoisier Tec & Doc., N.Y., 3^nd Ed, 1995, p 267 and 501.
• 4. Jacobs, H.; Bunbury, M.; McLean, S. J. Nat. Prod 1986, 49, 1164.
• 5. Goulart, M.O. F.; Sant'Ana, A.E.g.; Lima; R.A. de; Cavalcante, S. H.; Carvalho, M. G. de; Braz-Filho, R. Química Nova 1993, 16, 95.
• 6. Nishida, T.; Enzell, C.R.; Morris, G.A. Magn. Reson. Chem. 1986, 24, 179.
• 7. Descartes, G.; Muller, G.; Mentech, J. Carbohydr. Res. 1984, 134, 313.
• 8. Ikan, R. Natural Products, A Laboratory Guide; 2^nd Ed., Academic Press Inc; 1991, p. 9.
• 9. Agrawal, P.K.; Bansal, M.C. The Flavonoids in Carbon-13 NMR of Flavonoids; (Agrawal, P.K., ed.); Elsevier, Amsterdan, 1989, p. 316.
• 10. Simăo, S.M.; Carvalho, M.G. de; Braz-Filho, R.; Magalhăes, H.G. Química Nova 1995, 18, 34.

Publication Dates

• Publication in this collection
  23 Oct 2002
• Date of issue
  1999

History

• Received
  30 Apr 1998