Neolignans and sesquiterpenes from leaves and embryogenic cultures of Ocotea Catharinensis (Lauraceae)

ARTICLE

Neolignans and sesquiterpenes from leaves and embryogenic cultures of Ocotea Catharinensis (Lauraceae)

Mariko Funasaki^I; Ana Luísa L. Lordello^II; Ana Maria Viana^III; Claudete Santa-Catarina^IV; Eny I. S. Floh^IV; Massayoshi Yoshida^I,V; Massuo J. KatoI,^*

^IInstituto de Química, Universidade de São Paulo, CP 26077, 05513-970 São Paulo-SP, Brazil

^IIDepartamento de Química, Universidade Federal do Paraná, CP 19081, 81531-990 Curitiba-PR, Brazil

^IIIDepartamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, 88040-900 Florianópolis-SC, Brazil

^IVInstituto de Biociências, Universidade de São Paulo, CP 11461, 05508-900 São Paulo-SP, Brazil

^VCentro de Biotecnologia da Amazônia, Av. Gov. Danilo de Matos Areosa, 690, 69075-351 Manaus-AM, Brazil

ABSTRACT

The extracts from leaves of Ocotea catharinensis Mez (Lauraceae) were found to contain fourteen neolignans and two sesquiterpenes: nine benzofuran types (including three new compounds 1e, 2f and 4b), one new seco-benzofuran type (3b), two bicyclo[3.2.1]octane types (including the new compound 5c), two new dimers of bicyclo[3.2.1]octane type (7a and 7b) and two sesquiterpenes (including a new humulanol 9). In addition, seven neolignans were also showed to occur in somatic embryos of O. catharinensis including one new bicyclo[3.2.1]octane type (4a).

Keywords: Ocotea catharinensis, benzofuran neolignans, bicyclo [3.2.1] octane neolignans, humulane sesquiterpene, somatic embryos

RESUMO

Foram isoladas de extratos de folhas de Ocotea catharinensis Mez (Lauraceae) quatorze neolignanas sendo nove benzofurânicas (incluindo três novas substâncias 1e, 2f e 4b), uma secobenzofurânica inédita (3b), duas biciclo[3.2.1]octânicas (incluindo a nova 5c), dois novos dímeros biciclo[3.2.1]octânicos (7a e 7b) e ainda dois sesquiterpenos (incluindo o novo humulanol 9). Nos embriões somáticos de O. catharinensis foram identificadas sete neolignanas incluindo uma nova neolignana biciclo[3.2.1]octânica (4a).

Introduction

Ocotea catharinensis (Lauraceae) is a woody plant species found in southern Atlantic forest in Brazil, which produces excellent quality of timber. The extensive logging over the past thirty years associated with difficulties for propagation has led its natural population to be significant decrease. Since O. catharinensis has been included as endangered species, a somatic embryogenic system was developed aiming to a massive propagation.^1,2

The Ocotea has been one of the most phytochemically investigated Lauraceous genus and their major secondary compounds were showed to be phenylpropanoidderived including several sub-classes of neolignans.³ Previous phytochemical studies carried out in leaves of O. catharinensis collected at Horto Florestal (Serra da Cantareira), São Paulo State, Brazil, reported the occurrence of benzofuran (1b, 1c, 1d, 2b, 2c, 2d, 2e and 2h) and bicyclo[3.2.1] octane (5a, 5b and 5d) neolignans.⁴ Representatives of both sub-classes of neolignans have also been previously isolated from barks and woods of a specimen collected in São Paulo State,^5,6 and also from wood and leaves of O. porosa ('imbuia') collected in Rio Grande do Sul State, southern Brazil.^7-9

This work describes the isolation and characterization of major secondary compounds from leaves collected at Vale do Itajaí, Santa Catarina State, Brazil and from embryogenic cultures developed from the same plant source. The extracts from leaves afforded seven new neolignans 1e, 2f, 3b, 4b, 5c, 7a, 7b, besides seven previously reported ones 1a,^6,10 1d,⁶2a,^6,10,112d,⁴2e,⁶2g,¹²5e.¹³ Additionally, a new sesquiterpene of humulane type (9), besides known spathulenol (8)^4,14 were isolated as well. The embryogenic cultures were shown to contain a new benzofuran neolignan 4a, six already known benzofuran 1c,^6,121d,⁶2a,^6,10,112b,^11,122e,⁶ and a bicyclo[3.2.1]octane neolignan 6.¹⁵

Figure 2

Results and Discussion

The defatted fraction of hexane extracts from O. catharinensis leaves and from O. catharinensis somatic embryos were submitted to flash chromatography followed by prep. TLC and/or circular chromatography (Chromatotron^®). This procedure yielded nine new compounds (1e, 2f, 3b, 4a, 4b, 5c, 7a, 7b and 9), besides armenin-B (1a),^6,10 1c,^6,12 5'-methoxyporosin (1d),⁶ ferrearin-C (2a),^6,10,11 ferrearin-B (2b),^11,12 2d,⁴ ferrearin-E (2e),⁶ ferrearin-G (2g),¹² 5e,¹³6¹⁵ and spathulenol (8).^4,14

The molecular formula of compound 1e was determined by HRESI as C₂₃H₃₀O₇. Its ¹H NMR spectrum was quite similar to that of armenin-B (1a) (C₂₁H₂₄O₆), previously isolated from Licaria armeniaca¹⁰ and from O. catharinensis.⁶ The only difference was assigned to the oxygenation pattern of the aromatic ring, which was determined as 3,4,5-trimethoxyphenyl in 1e instead of 3,4-methylenedioxyphenyl in 1a.

The ¹H NMR spectra of 2f (C₂₁H₂₆O₆) and of ferrearin-E (2e) (C₂₀H₂₄O₅) isolated from O. catharinensis⁶ were similar. The difference between both compounds consisted of an additional methoxyl group at β-position (C-5', δ 169.3) of 2f.

The molecular formula of compound 3b was determined by HRESI as C₂₂H₂₈O₆. All the ¹H NMR data was similar to that of a neolignan 3a (C₂₁H₂₆O₅) previously isolated from O. porosa.¹⁶ Nevertheless, the ¹H and ¹³C NMR spectra indicated that 3b contained a methoxyl group at C-3' (δ_H 3.58, δ_C 55.7) which was evidenced by the signal of C-3' at δ_C 152.8 instead of δ_C 132.7 in 3a. Its absolute configuration was determined as 8S,1'R, 5'R based on the signal of optical rotation ([α]_D ²¹ = -17º(MeOH, c = 0.92 g/100mL) and comparison with reported data for (-)-megaphone in which X-ray crystallographic studies was carried out.¹⁶

The compounds 4a and 4b were characterized as hexahydrobenzofuran neolignans by analysis of their ¹H and ¹³C NMR spectra and comparison with 4c which was previously isolated from O. porosa.⁹ The aromatic rings were determined as 3,4-methylenedioxyphenyl and 5-methoxy3,4-methylenedioxyphenyl for 4a and 4b, respectively. The relative stereochemistry between methyl and aryl groups was deduced from the anisotropic shielding effect caused by aromatic ring on cis methyl hydrogens as observed in the ¹H NMR spectra. The methyl (H-9) signals of 4a (trans) and 4b (cis) were observed at δ 0.84 (d, 6.8 Hz) and 0.57 (d, 7.5 Hz), respectively. The placement of hydroxyl group at C-2' and methoxyl group at C-3' were possible by comparison of ¹³C NMR data and those described for 4c.⁹

The molecular formula of compound 5c was determined by HRESI as C₂₁H₂₂O₆. Its ¹H NMR were similar to that observed for previously reported 5e (C₂₂H₂₄O₇) isolated from Aniba simulans,¹³ but with a 3,4-methylenedioxy for 5c instead of 5-methoxy-3,4-methylenedioxy observed for 5e.

Compounds 7a and 7b had their structures determined by analysis of their IR, MS and NMR spectra. The IR spectra of 7a/7b exhibited absorption bands at 1766 and 1714 cm^-1, assignable to two carbonyl groups for each compound. Their ¹H and ¹³C NMR spectra resembled those of 5c/5e, respectively. Nevertheless, the ¹³C NMR signals associated to the olefinic double bond at C5´-C6´ were replaced by signals of quaternary carbinolic and methine signals at δ 88.4/52.6 (7a) and δ 88.4/52.7 (7b), respectively. The EIMS of 7a/7b showed molecular ion at m/z 370 and 400, but the chemical ionization mass spectrometry (CIMS) provided a molecular ion peak (M + H)⁺ at m/z 741 and m/z 801, respectively. These molecular ions combined with ¹H and ¹³C NMR (PND and DEPT 135º), suggested a molecular formula C₄₂H₄₄O₁₂ and C₄₄H₄₈O₁₄ compatible with dimeric structures. Thus, the fragmentary ion peaks found at m/z 371 of 7a and 401 of 7b were assigned to the cleavage into monomers indicating that 7a and 7b were symmetric dimers of 5a and 5e. Based on similarities to 5a and 5e all ¹H and ¹³C NMR data were assigned with the aid of ¹H-¹H COSY and HETCOR spectra (Table 2). In order to determine the relative stereochemistry at the cyclobutane ring, NOESY spectra showed cross-peaks between H-8 and H-7', H-7' and H-6', H-6' and OMe-5'. Thus, among the cyclobutane syn-adducts at C-5' and C-6' four dimers would be expected (Figure 1). Dimers III and IV having cis configuration at the cyclobutane would involve a considerable steric hindrance between the bulky groups of bicyclooctane neolignan, which would prevent such arrangement. For these reason, dimers I and II having trans configuration were considered as the mostly probable structures.

The molecular formula of 9 (C₁₅H₂₆O) was deduced from MS and ¹³C NMR (PND and DEPT 135º) spectral data. The spectral characteristics of compound 9 were closely related to those of a-humulane¹⁷ except for the existence of a hydroxyl group (IR v_max/cm^-1: 3425) and two double bonds instead of three. All the ¹H and ¹³C NMR signalswere assigned by ¹H-¹H COSY and HETCOR techniques and were compatible with the structure depicted for humula-4,8-dien-2-ol (9).

The leaves of O. catharinensis collected in São Paulo and in Santa Catarina were showed to contain both benzofuran and bicyclo[3.2.1]octane type neolignans. The benzofuran neolignan 1d was the major compound in the leaves collected in São Paulo while bicyclo[3.2.1]octane neolignans 5e/5c were predominant in Santa Catarina specimen. The embryogenic cultures also produced both types of neolignans, but 1d was the major compound.

Experimental

General

Prep. TLC was carried out on silica gel PF-254 (Merck) and CC on silica gel 60H (0.005-0.045 mm) (Merck). Optical rotations were measured on Jasco Mod. DIP-370. ¹H and ¹³C NMR spectra were registered on a Bruker AC-200 spectrometers using CDCl₃ as solvent and TMS as internal standard. EIMS (70 eV) and CIMS (methane) were obtained on a HP 5988-A spectrometer. HRESIMS (positive mode) were recorded on a Bruker Daltonics microTOF. Elemental analysis were performed on a Perkin-Elmer CHN Elemental Analyser 2400. HPLC were performed on Gilson 321 using a reverse phase column (Supelco, C-18, 4.6 × 250 mm, 5 µm).

Plant material

The leaves of the O. catharinensis were collected in Brusque, Santa Catarina State, Brazil in June 1993. The species was collected and identified by Prof. Ademir Reis (Departamento de Botânica, UFSC). The voucher specimen (FLOR-17560) was deposited in the FLOR Herbarium (UFSC).

Initiation and multiplication of embryogenic cultures

Embryogenic cultures were initiated from mature zygotic embryos of O. catharinensis according to described methodology.^1,2

The somatic embryos produced at early cotyledonary stage (2-3 mm length) were inoculated in Woody Plant Medium (WPM, Sigma Co., USA) supplemented with 22.7 g ^L-1 sorbitol, 2 g ^L-1 Phytagel, 20 g ^L-1 sucrose and 400 mg ^L-1 glutamine (pH 5.8) and maintained at 25 ºC and with photon flux of 22 µmol m^-2 s^-1 provided by fluorescent tubes under 16 h photoperiod.^2,18 After four weeks cultivation the somatic embryos at mature stage (> 5 mm) were transferred to Petri dishes (6 cm), spread over two filter papers, and maintained at 25 ºC for 4 days for desiccation.

Extraction and isolation of the constituents from leaves

Dried and powdered leaves (395.0 g) were exhaustively extracted with hexane at room temp. Evaporation of the hexane under a reduced pressure gave a residue, which was partitioned between hexane and MeOH-H₂O (9:1). The hydroalcoholic phase was concentrated under reduced pressure yielding 2.4 g. This residue was submitted to flash chromatography column (silica gel, 150 g) and eluted with hexane-EtOAc mixtures at increasing polarities (7:3 to 0:1), yielding 120 fractions (30 mL each). Frs. 29-32 (33.0 mg) submitted to prep. TLC (silica gel, hexane-Et₂O, 4:1) gave spathulenol 8 (7.0 mg) and humula-4,8-dien-2-ol 9 (17.0 mg). Frs. 33-41 (76.0 mg) was also fractionated by prep. TLC (silica gel, cyclohexane-Me₂CO, 98:2) followed by prep. TLC (silica gel, hexane-CHCl₃-iso-PrOH, 94.5:5.0:0.5) and afforded 2d (5.5 mg) and 2a (4.9 mg). Frs. 44-59 (60.0 mg) submitted to prep. TLC (silica gel, cyclohexane-Me₂CO, 97:3) followed by prep. TLC (silica gel, CHCl₃-EtOAc-iso-PrOH, 94.5:5.0:0.5) gave 2g (3.0 mg) and 6 (6.0 mg). Fr. 60 (24.0 mg) fractionated by prep. TLC (silica gel, cyclohexane-Me₂CO, 98:2) yielded 5c (6.0 mg). Frs. 61-65 (440.0 mg) submitted to flash chromatography column (silica gel, CHCl₃EtOAc-iso-PrOH, 89:10:1), affording three sub-fractions (S₁-S₃). Prep. TLC of sub-fraction S₁ (70.0 mg) (silica gel, CHCl₃-EtOAc-iso-PrOH, 94.5:5.0:0.5) afforded 7a

(17.0 mg) and prep. TLC of sub-fraction S₃ (80.0 mg) (silica gel, CHCl₃-EtOAc-iso-PrOH, 94.5:5.0:0.5) yielded 5c (30.0 mg). Frs. 66-71 (272.0 mg) fractionated by prep. TLC [silica gel, cyclohexane-Me₂CO (97:3)] furnished 2e (6.0 mg). Frs. 77-79 (150.0 mg) submitted to chromatotron (silica gel, hexane-EtOAc-iso-PrOH, 69:30:1), to give two sub-fractions (S₄ and S₅). Prep. TLC of sub-fraction S₄ (14.0 mg) (silica gel, cyclohexane-Me₂CO, 95:5) gave 2a (5.0 mg) and 7b (4.0 mg) and prep. TLC of sub-fraction S₅ (98.0 mg) (silica gel, cyclohexane-Me₂CO, 95:5) gave 5e (70.0 mg). Frs. 80-81 (98.0 mg) submitted to prep. TLC (silica gel, CH₂Cl₂-Me₂CO, 95:5) yielded 4b (12.0 mg). Fr. 82-86 (500.0 mg) submitted to chromatotron (silica gel, hexane-EtOAc-iso-PrOH, 89:10:1) afforded 2e (7.2 mg) and two sub-fractions S₆ and S₇. Sub-fraction S₆ (400.0 mg) was further fractionated by flash chromatography column (silica gel, CH₂Cl₂-Me₂CO, 98:2) yielding 2f (12.0 mg) and 3b (9.0 mg). Sub-fraction S₇ (43.0 mg) was submitted to chromatotron (silica gel, CH₂Cl₂-Me₂CO, 95:5) to give 1a (24.0 mg). Frs. 105-110 (87.0 mg) was submitted to chromatotron (silica gel, CH₂Cl₂-Me₂CO, 96:4) yielding 1d and 1e (5.5 mg) as a mixture.

Extraction and isolation of the constituents from somatic embryos

The desiccated somatic embryos (400.0 g) were frozen in Me₂CO with dry ice, ground and extracted with 500 mL MeOH-H₂O (4:1). The hydroalcoholic extracts was concentrated and successively partitioned with hexane and CHCl₃ (3 × 200 mL, each). The CHCl₃ residue (360.0 mg) was submitted to flash chromatography column (silica gel, 150 g) and eluted with CH₂Cl₂-Me₂O at increasing polarity (1:1 to 0:1), affording 160 fractions (20 mL each). Fr. 1 (18.0 mg) submitted to prep. TLC (cyclohexane-Me₂CO, 95:5) yielded 2a (5.0 mg) and 6 (5.0 mg). Frs. 2-12 (7.0 mg) submitted to prep. TLC (cyclohexane-Me₂CO, 95:5) gave 2a (1.0 mg) and 2e (2.0 mg). Frs. 13-21 (20.5 mg) submitted to prep. TLC (cyclohexane-Me₂CO, 95:5) gave 2b (3.0 mg), 2a (4.0 mg) and 1c (3.0 mg). Frs. 22-54 (43.0 mg) purified by prep. TLC (CH₂Cl₂-Me₂O, 9:1) yielded 4a (3.0 mg) and 6 (4.0 mg). Frs. 145-158 (29.0 mg) fractionated by prep. TLC (CHCl₃-MeOH, 9:1) yielded 1d (7.0 mg).

(7S,8S,1'R,3'R)-3,4,5,3',5'-Pentamethoxy-4'-oxo-Δ^1,3,5,5',8'8.1',7.O.6'-neolignan (1e)

Viscous oil; IR (film) v_max/cm^-1: 3469, 2925, 1707, 1695, 1500, 1446, 1371, 1218, 1087, 1033, 925, 816, 772; ¹H and ¹³C NMR, see Table 1; HRESIMS m/z: 419.2090 [M+H]⁺ (calcd for C₂₃H₃₁O₇, 419.2071); EIMS (70 eV) m/z (rel. int.): 418(M⁺, 34), 388(19), 377(100), 349(87), 317(29), 285(13), 208(44), 181(22), 91(13).

rel(7R,8S,1'R,2'S)-2'-Hydroxy-3,4,5'-trimethoxy-3'-oxoΔ ^1,3,5,4',8'-8.1',7.O.2'-neolignan (2f)

Viscous oil; [α]_D ²¹ = - 130º(MeOH, c = 0.20 g/100mL); IR (film) v_max/cm^-1: 3435, 2936, 2849, 1739, 1664, 1588, 1511, 1457, 1251, 1142, 1012, 762; ¹H and ¹³C NMR, see Table 1; EIMS (70 eV) m/z (rel. int.): 374(M⁺, 7), 194(17), 167(100), 166(66), 165(76), 139(56), 95(17), 77(21), 69(18).

rel (8S, 1'R, 5'R)-3,4,3',5'-Tetramethoxy-7,2'-dioxoΔ^1,3,5,3',8'-8.1'-neolignan (3b)

Viscous oil; [α]_D ²¹ = - 17º(MeOH, c = 0.92 g/100mL); IR (film) v_max/cm^-1: 2957, 2914, 2860, 2348, 1739, 1620, 1511, 1457, 1371, 1263, 1229, 1144, 1023, 766; ¹H and ¹³C NMR, see Table 1; HRESIMS m/z: 389.1983 [M+H]⁺ (calcd for C₂₂H₂₉O₆, 389.1966); EIMS (70 eV) m/z (rel. int.): 388(M⁺, 4), 352(30), 339(23), 324(10), 316(3), 165(100), 137(6).

rel(7S,8S,1'R,2'S)-2'-Hydroxy-3,4-methylenedioxy-3',5'dimethoxy-Δ^1,3,5,3',8'-8.1',7.O.2'-neolignan (4a)

Viscous oil; ¹H NMR see Table 1.

rel(7R,8S,1'R,2'S)-2'-Hydroxy-3,4-methylenedioxy5,3',5'-trimethoxy-Δ^1,3,5,3',8'-8.1',7.O.2'-neolignan (4b)

Viscous oil; ¹H and ¹³C NMR, see Table 1.

rel(7S,8R,1'R,3'R)-4'-Hydroxy-3,4-methylenedioxy-3',5'dimethoxy-2',4'-dioxo-Δ^1,3,5,5',8'-8.1',7.3'-neolignan (5c)

Viscous oil; [α]_D ²¹ = -18º(MeOH, c = 3.75 g/100mL); IR (film) v_max/cm^-1: 1765, 1698, 1504, 1491, 1247, 1094, 1039. ¹H and ¹³C NMR, see Table 2; HRESIMS m/z: 371.1496 [M+H]⁺ (calcd for C₂₁H₂₃O₆, 371.1496); EIMS (70 eV) m/z (rel. int.): 370 (M⁺, 22), 329(80), 287(12), 269(9), 208(100), 149(82), 137(73), 77(31).

(7S,8R,1'R,3'R)-4'-Hydroxy-3,4-methylenedioxy-3',5',5trimethoxy-2',4'-dioxo-Δ^1,3,5,5',8'-8.1',7.3'-neolignan (5e)

Viscous oil; [α]_D ²¹ = - 29º(MeOH, c = 4.48 g/100mL); ¹H and ¹³C NMR, see Table 2; EIMS (70 eV) m/z (rel. int.): 400 (M⁺, 52), 359(21), 331(10), 288(14), 219(100), 208(45), 207(43), 193(18), 192(66), 180(53), 165(34), 137(23), 91(14), 77(16).

7a (Dimer of 5c)

Viscous oil; [α]_D ²¹ = + 220º(MeOH, c = 0.07 g/100mL); IR (film) v_max/cm^-1: 2922, 1766, 1714, 1513, 1453, 1137, 1094,1044; ¹H and ¹³C NMR, see Table 2; CIMS m/z (rel. int.): 741(M⁺, 1), 579(1), 419(15), 391(91), 371(100), 341(18), 209(28), 163(37), 57(27).

7b (Dimer of 5e)

Viscous oil; [α]_D ²¹ = + 57º(MeOH, c = 0.62 g/100mL); IR (film) v_max/cm^-1: 1767, 1714, 1505, 1491, 1445, 1240, 1039; ¹H and ¹³C NMR, see Table 2; CIMS m/z (rel. int.): 801(M⁺, 1), 609(18), 429(76), 419(85), 401(100), 371(11), 209(17), 193(11).

rel(8R)-Humulan-1,4-dien-8-ol (9)

Solid amorphous. Found: C, 80.89 %, H, 11.65% (C₁₅H₂₆O requires: C, 81.02%, H, 11.79%); IR (film) v_max/cm^-1: 3425, 2946, 1707, 1446, 1371, 1022, 762; ¹H and ¹³C NMR, see Table 3; EIMS (70 eV) m/z (rel. int.): 344(M⁺, 27), 303(9), 271(12), 189(100), 179(26), 178(76), 166(25), 165(66), 151(59), 137(19), 115(15), 107(27), 91(35), 77(33).

Acknowledgments

The authors acknowledge FAPESP, CNPq, CAPES and International Foundation for Science (Sweden) for financial support.

Supplementary Information

Supplementary information is available free of charge at http://jbcs.org.br, as PDF file.

Received: October 1, 2008

Web Release Date: March 20, 2009

FAPESP helped in meeting the publication costs of this article.

Supplementary Information

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Publication Dates

• Publication in this collection
  29 June 2009
• Date of issue
  2009

History

• Received
  01 Oct 2008
• Accepted
  20 Mar 2009