ent-Kaurane-Type Diterpenoids from a Cell Suspension Culture of the Liverwort Jungermannia subulata

• Abstract
• Introduction
• Materials and Methods
• General
• Plant material
• Cell suspension culture
• Extraction and isolation
• Oxidation of 3α-hydroxy-ent-16(S)-kauran-15-one (1)
• Oxidation of 4
• Catalytic hydrogenation of 3
• Oxidation and catalytic hydrogenation of 6
• X-ray crystallographic analyses of 3, 4, and 9
• Results and Discussion
• References

Abstract

A cell suspension culture of the liverwort Jungermannia subulata was established from callus tissue induced by culturing spores. From the suspension culture, three new ent-kaurane-type diterpenoids were isolated together with three previously reported ent-kaurane-type diterpenoids. Five ent-kaurane-type diterpenoids were isolated from the intact J. subulata, two of which were new compounds. Their structures were determined by spectroscopic methods, chemical reaction, and X-ray crystallographic analysis.

Introduction

The liverwort (Hepaticae) is placed in a special group considered to represent an early stage in the evolution of terrestrial plants. The liverwort contains several oil bodies, a characteristic organelle of the liverwort, in each cell of the gametophyte. It has been shown that a liverwort usually accumulates sesquiterpenoids and diterpenoids as well as esters of fatty acids and aromatic compounds as the major lipophilic constituents in their oil bodies [1], [2]. In spite of the phytochemical and biochemical interest in the chemical constituents of the bryophytes, investigations have always encountered problems when collecting a sufficient quantity of the plants and also with botanical homogeneity. Recently, cell culture techniques have been applied to study the metabolites of the liverwort [3], [4]. In many cases, the production of characteristic secondary metabolites is significantly decreased or lost in higher plant cell cultures. This phenomenon could be due to the dedifferentiation of plant tissues, because a number of secondary metabolites are synthesized and accumulated at specific tissues such as root, bark, and leaf expressing certain biosynthetic genes. In contrast to higher plants, the liverwort has a simple morphology and accumulates secondary metabolites in the oil bodies. This oil body is observed in liverwort cell cultures as well as in intact plant cells, suggesting that a liverwort cell culture retains the ability to produce secondary metabolite [5], [6].

Several studies have reported that the chemical constituents differ among the same species of liverwort collected in different places [7], [8]. This suggests that different environmental conditions affect the metabolite profile in the liverwort. A liverwort cell culture is grown under constant culture conditions, suggesting that the metabolite profile of the cell culture is reproducible. Therefore, cell cultures of liverwort can solve the collection problem from natural sources and can provide a constant metabolite profile.

The genus Jungermannia (Jungermanniaceae, liverwort) produces various unique sesquiterpenoids and diterpenoids [9]. Jungermannia species produce bioactive ent-kaurane-type diterpenoids as one of the characteristic metabolites in this genus. ent-Kaurane-type diterpenoids have been reported to posses various interesting biological activities such as induction of apoptosis [10], [11], [12], potent cytotoxicity [13], anti-inflammatory activity [14], antiplatelet aggregation [15], inhibition of superoxide anion generation [16], and anti-HIV activity [17]. Therefore, Jungermannia species are attractive pharmacognosy sources of bioactive kaurane-type diterpenoids. These reports prompted us to produce bioactive kaurane-type diterpenoids by cell culture technology.

In this paper, we discuss the isolation and structural characterization of three new ent-kauranes together with three known ent-kaurane-type diterpenoids [7], [18] from a cell suspension culture, and two new ent-kaurane-type diterpenes in addition to three known ent-kauranes [7], [19] from the intact plant (Fig. [1]).

Materials and Methods

General

All melting points were measured on a melting-point apparatus (Gallenkamp-Sanyo; Watford, UK) and are uncorrected. Optical rotations were measured with a SEPT-200 polarimeter (Horiba; Kyoto, Japan). UV spectra were recorded on a U-3210 spectrophotometer (Hitachi; Tokyo, Japan), and IR spectra on a model 1720 spectrometer (Perkin-Elmer Japan; Tokyo, Japan). ¹H- and ¹³C-NMR spectra were recorded on an ARX 400 spectrometer (Bruker Japan; Tukuba, Japan), with chemical shifts shown in δ values from TMS as the internal reference, with peak multiplicities quoted in Hz. Mass spectra were measured on a JMS-700 spectrometer (Jeol; Tokyo, Japan). Column chromatography was carried out on silica gel 60 (70 - 230 and 230 - 400 mesh; Merck Japan; Tokyo, Japan).

Plant material

The liverwort Jungermannia subulata A. Evans (Jungermanniaceae) was collected in a forest on the Kanmuri mountain in Hatsukaichi city, Hiroshima prefecture, Japan in 1997 and identified by Prof. Naoki Nishimura (Okayama University of Science). A voucher specimen (OUS JS0233) has been deposited in the herbarium of Okayama University of Science.

Cell suspension culture

Mature spores were removed from sporangia that had been surface-sterilized with 1 % NaOCl solution for 10 min. Callus tissue was induced by culturing the spores on solid Murashige and Skoog (MS) medium [20] containing 2 % glucose and 0.8 % agar. This callus was suspended in a liquid MSG medium [MS medium containing 4 % glucose, 0.1 % CaCO₃ and no phytohormones, pH 5.8] to establish a cell suspension culture. The suspension culture was routinely subcultured at 10 - 14 day intervals by pouring approximately 2 mL of cells into 100 mL fresh medium in a 300-mL conical flask. The flasks were placed on a gyratory shaker [120 rpm, 25 °C] under a continuous fluorescent light (3000 lux). Photographs of the cells were taken with an Olympus BX50 microscope.

Extraction and isolation

The cells were collected by filtering the suspension culture (9.6 L) during the growth phase. The whole cells (370 g) were dried in the shade for several days, and then cells were extracted twice with MeOH (1.5 L × 2) for a week at room temperature. The solvent was removed under reduced pressure. The resultant residue was suspended in water (1.5 L) and partitioned with n-hexane and EtOAc, respectively (1.5 L × 2).

The n-hexane extract (1.18 g) was first subjected to silica gel column chromatography (CC) [70 - 230 mesh, 6 × 100 cm, 1550 g], eluting with a gradient mixture of CHCl₃-EtOAc [(100 - 80 : 20 - 60 : 40 - 50 : 50, V/V) - EtOAc, each 2 L] to give six fractions. Fraction 2 (2200 - 2700 mL, 195 mg) was subjected to silica gel CC [230 - 400 mesh, 1.5 × 25 cm, 38 g, n-hexane-EtOAc (9 : 1, 250 mL)] to give four fractions. Fraction 2 - 2 (100 - 150 mL, 95.1 mg) and fraction 2 - 3 (150 - 200 mL, 60.3 mg) were purified by silica gel CC [230 - 400 mesh, 1.5 × 25 cm, 38 g, n-hexane-EtOAc (9 : 1, 250 mL) for fraction 2 - 2; n-hexane-acetone (8 : 2, 250 mL) for fraction 2 - 3] to give phytol (73.6 mg) and compound 2 (46.7 mg), respectively. Fraction 3 (2700 - 3200 mL, 125 mg) was subjected to silica gel CC [230 - 400 mesh, 1.5 × 25 cm, 38 g, benzene-acetone (96 : 4, 250 mL)] to give four fractions. Fraction 3 - 2 (80 - 150 mL, 70.8 mg) was further purified by silica gel CC [230 - 400 mesh, 1.5 × 25 cm, 38 g, n-hexane-EtOAc (8 : 2, 250 mL)] to give compounds 1 (30.4 mg), 3 (1.2 mg), 4 (8.3 mg), and pheophorbide A methyl ester (4.3 mg), respectively.

The EtOAc extract (0.53 g) was subjected to silica gel CC [230 - 400 mesh, 3 × 60 cm, 230 g] using a gradual increase of addition of acetone to CHCl₃ [(100 - 80 : 20 - 60 : 40 - 50 : 50, V/V)-acetone each 600 mL] to give seven fractions. Compounds 5 (1.5 mg) and 6 (2.6 mg) were isolated from fraction 4 (3200 - 3500 mL, 23.3 mg) and fraction 5 (3500 - 3700 mL, 12.8 mg), respectively, by repeated silica gel CC [230 - 400 mesh, 1 × 25 cm, 11 g] with benzene-acetone (1 : 1, 70 mL).

Air-dried J. subulata (100 g) was extracted with MeOH (1.5 L × 2) for a month. The MeOH extract was concentrated under vacuum and suspended in water. This aqueous suspension was extracted successively with n-hexane and EtOAc. The n-hexane extract (1.23 g) was divided into ten fractions by silica gel CC [70 - 230 mesh, 3 × 60 cm, 230 g] using an n-hexane-EtOAc gradient system [(100 - 80 : 20 - 60 : 40 - 50 : 50, V/V) - EtOAc, each 500 mL]. Fraction 2 (450 - 750 mL, 147.5 mg) was purified by silica gel CC [230 - 400 mesh, 1.5 × 25 cm, 38 g, n-hexane-EtOAc (9 : 1, 250 mL]] to give compound 7 (33.6 mg) and a triterpenoid, friedelin (19.5 mg). Silica gel CC [230 - 400 mesh, 1 × 30 cm, 13 g, benzene-EtOAc (15 : 1, 70 mL)] purification of fraction 3 (750 - 800 mL, 12.0 mg) resulted in the isolation of compound 9 (5.0 mg). From fraction 7 (1400 - 1500 mL, 40.0 mg), compound 3 (11.0 mg), together with β-sitosterol (4.4 mg), were purified by repeated silica gel CC [230 - 400 mesh, 1 × 25 cm, 11 g, n-hexane-EtOAc (9 : 1, 120 mL)]. Fractions 9 (1900 - 2000 mL, 39.5 mg) and 10 (2000 - 2200 mL, 27.3 mg) were separated by silica gel CC [230 - 400 mesh, 1.5 × 25 cm, 38 g, n-hexane-acetone (10 : 1, 120 mL) for fraction 9; n-hexane-EtOAc (9 : 1, 120 mL) for fraction 10] to yield compounds 8 (8.7 mg) and 10 (0.7 mg), respectively. The EtOAc extract (0.55 g) was subjected to silica gel CC [70 - 230 mesh, 2.5 × 30 cm, 81 g] with n-hexane-EtOAc [(100 - 80 : 20 - 60 : 40 - 50 : 50, V/V) - EtOAc, each 150 mL,] solvent gradient to give eleven fractions. Purification of fraction 3 (200 - 300 mL, 10.2 mg) and fraction 4 (300 - 350 mL, 6.2 mg) by silica gel CC [230 - 400 mesh, 1 × 25 cm, 11 g, n-hexane-EtOAc (85 : 15, 70 mL)] gave compounds 8 (1.3 mg) and 10 (0.7 mg), respectively.

15β-Hydroxy-ent-kaur-16-en-3-one (4): Crystalline needles; m. p. 129 - 132 °C; [α]_D ²⁵: -70.9 (c 0.2, CHCl₃); IR (CHCl₃): ν_max = 3600, 1730 cm^-1; ¹H- and ¹³C-NMR spectroscopic data: see Table [1]; EI-MS: m/z (%) = 302 [M] ⁺ (100), 287 (50), 284 (30); HR-EI-MS: m/z = 302.2248 for C₂₀H₃₀O₂ (Δmmu 0.2).

13α-Hydroxy-ent-kaur-16-ene-3,15-dione (5): Colorless crystals; m. p. 171 - 178 °C; [α]_D ²⁵: -132.3 (c 0.1, CHCl₃); IR (CHCl₃):  ν_max = 3440, 1735 cm^-1; ¹H- and ¹³C-NMR spectroscopic data: see Table [1]; EI-MS: m/z (%) = 318 [M] ⁺ (100), 301 (40), 267 (50), 261 (70); HR-EI-MS: m/z = 318.2197 for C₂₀H₃₀O₃ (Δmmu 0.2).

13α,15a-Dihydroxy-ent-kaur-16-en-3-one (6): Colorless crystals; m. p. 165 - 171 °C; [α]_D ²⁵: 27.5 (c 0.1, CHCl₃); IR (CHCl₃): ν_max = 3620, 1700 cm^-1; ¹H- and ¹³C-NMR spectroscopic data: see Table [1]; EI-MS: m/z (%) = 318 [M]⁺ (100), 300 (90), 285 (30), 137 (75); HR-EI-MS: m/z = 318.2195 for C₂₀H₃₀O₃ (Δ mmu 0.0).

7β-Hydroxykaur-16-en-15-one (9): Crystalline needles; m. p. 125 - 128 °C; [α]_D ²⁵: -75.0 (c 0.1, CHCl₃); IR (CHCl₃):  ν_max = 3625, 1740 cm^-1; UV (CHCl₃): λ_max (log ε) = 235 nm (3.79); ¹H-NMR (CDCl₃): δ = 0.83 (3H, s, H-19), 0.90 (3H, s, H-18), 1.11 (3H, s, H-20), 2.31 (1H, d, J = 10.0, H-14), 3.09 (1H, brs, H-13), 3.91 (1H, brs, H-7), 5.23 (1H, s, H-17), 5.97 (1H, s, H-17); ¹³C-NMR (CDCl₃): δ = 39.6 (t, C-1), 18.4 (t, C-2), 41.8 (t, C-3), 33.3 (s, C-4), 53.2 (d, C-5), 26.0 (t, C-6), 72.9 (d, C-7), 53.2 (s, C-8), 45.2 (d, C-9), 40.1 (s, C-10), 18.1 (t, C-11), 32.7 (t, C-12), 38.1 (d, C-13), 32.6 (t, C-14), 214.5 (s, C-15), 149.4 (s, C-16), 115.7 (t, C-17), 34.6 (q, C-18), 21.8 (q, C-19), 17.6 (q, C-20); HR-EI-MS: m/z 302.2240 [M]⁺ for C₂₀H₃₀O₂ (Δ mmu 0.6).

11α-Acetoxykaur-16-ene-3,15-dione (10): Colorless crystals; m. p. 221 - 224 °C; [α]_D ²⁵: -152.0 (c 0.1, CHCl₃); IR (CHCl₃): ν_max = 1700 cm^-1; UV (CHCl₃): λ_max (log ε) = 238 (3.59); ¹H-NMR (CDCl₃): δ = 1.04 (3H, s, H-19), 1.11 (3H, s, H-18), 1.17 (3H, s, H-20), 2.09 (3H, s, H-22), 2.48 (2H, dd, J = 7.4, 9.5 Hz, H-2), 5.42 (1H, d, J = 2.0, H-17), 6.09 (1H, d, J = 2.0, H-17); ¹³C-NMR (CDCl₃): δ = 38.5 (t, C-1), 33.8 (t, C-2), 217.0 (s, C-3), 47.1 (s, C-4), 53.6 (d, C-5), 19.7 (t, C-6), 32.0 (t, C-7), 54.6 (s, C-8), 49.9 (d, C-9), 38.3 (s, C-10), 20.6 (t, C-11), 35.3 (t, C-12), 84.2 (s, C-13), 40.1 (t, C-14), 206.5 (s, C-15), 147.7 (s, C-16), 116.2 (t, C-17), 27.0 (q, C-18), 20.9 (q, C-19), 17.4 (q, C-20), 22.0 (q,CH₃CO), 169.8 (s, CH₃CO); HR-EI-MS: m/z = 358.2124 [M]⁺ for C₂₂H₃₀O₄ (Δmmu 0.2).

Oxidation of 3α-hydroxy-ent-16(S)-kauran-15-one (1)

To a solution of 1 (3.1 mg) in Me₂CO (3 mL) was added one drop of Jones reagent (CrO₃, aq. H₂SO₄). After the mixture had been kept at room temperature for 3 h, it was diluted with H₂O and the product extracted with Et₂O. The Et₂O extract was washed, dried and evaporated to give a ketone (2.3 mg) of which spectra and physico-chemical values were identicalto those of 2.

Oxidation of 4

Compound 4 (2.4 mg) in Me₂CO (3 mL) was treated with an excess of Jones reagent at room temperature for 1 h. The product after extraction with Et₂O was evaporated to give 3 (1.3 mg).

Catalytic hydrogenation of 3

Compound 3 (1.1 mg) in EtOAc (3 mL) was hydrogenated in the presence of 10 % Pd-C for 21 h. Work-up as usual gave a dihydro-derivative (0.6 mg), whose spectral and physico-chemical values were identical to those of 2.

Oxidation and catalytic hydrogenation of 6

To a solution of compound 6 (1.8 mg) in Me₂CO (2 mL) was added one drop of Jones reagent. After 3 h, the mixture was diluted with H₂O and the product was extracted with Et₂O. The concentrated extract, without purification, was dissolved in EtOAc and hydrogenated in the presence of 10 % Pd/C for 24 h at 1 atm. Work-up of the reaction mixture gave 5 (0.7 mg).

X-ray crystallographic analyses of 3, 4, and 9

Crystal data of 3: C₂₀H₂₈O₂, MW 300.44, monoclinic, space group P2₁, a = 6.6018(6) Å, b = 10.597(1), c = 12.1167(9), β  = 92.873(8)°, V = 846.6(1) ų, D _X = 1.178 g/cm³, Z = 2.

Crystal data of 4: C₂₀H₃₀O₂, MW 302.46, monoclinic, space group C2, a = 18.59(1) Å, b = 7.431(1), c = 15.967(3), β = 130.55(2)°, V = 1676.1(8) ų, D _X = 1.198 g/cm³, Z = 4.

Crystal data of 9: C₂₀H₃₀O₂, MW 302.46, orthorhombic, space group P2₁2₁2₁, a = 10.865(2) Å, b = 24.868(3), c = 6.381(1), V = 1724.0(8) ų, D _X = 1.165 g/cm³, Z = 4.

The reflections of 4 were obtained on a Rigaku RAXIS-IV imaging plate area detector with graphite monochromated Mo-Kα radiation. The reflections of 3 and 9 were measured on a Rigaku AFC-7R diffractometer [graphite-monochromated Cu-Kα radiation (λ  = 1.54178 Å) for 3, graphite-monochromated Mo-Kα radiation (λ  = 0.71070 Å) for 9]. A total of 1468 reflections were obtained for 3 (1696 for 4, 1782 for 9), of which 1340 (1313 for 4, 1380 for 9) were unique (R _int = 0.092 for 3, R _int = 0.074 for 4, R _int = 0.774 for 9). These crystal structures were solved by direct methods using the program teXsan, a crystallographic software package of Molecular Structure Corporation. All atoms except hydrogen atoms were refined anisotropically by full-matrix least-squares methods on F ² using the above software package to give a final R-factor of 0.055 (R _W 0.054 for all data) for 3, 0.084 (R _W 0.079 for all data) for 4, and 0.066 (R _W 0.082 for all data) for 9, with a data-to-parameter ratio 6.41 for 3, 6.57 for 4 and 6.24 for 9, respectively. Crystallographic data of compounds 3, 4, and 9 have been deposited at the Cambridge Crystallographic Data Centre under the reference numbers CCDC 624 324 for 3, CCDC 624 325 for 4, and CCDC 624 326 for 9.

Results and Discussion

A cell suspension culture of Jungermannia subulata was grown in a liquid MSG medium under continuous light on a gyratory shaker at 25 °C. The cultured cells were green colored with small aggregated cells (Fig. [2] A). The growth curve of the cell culture and pH of the culture medium are indicated in Fig. [2] B. The cell growth entered the stationary phase on day 21. However, the pH of the medium dropped to pH 3.5 around day 15. Therefore, the cultured cells were collected by filtration during days 12 - 14 (pH range 5.0 - 5.8). The harvested cells were air-dried, and extracted with methanol and evaporated under vacuum. The methanol extract was suspended in H₂O and partitioned successively against n-hexane and ethyl acetate. The n-hexane and ethyl acetate extracts were subjected to chromatographic separation to give four ent-kaurane-type diterpenoids (compounds 1 - 4) from the former, and two ent-kaurenes (compounds 5 and 6) from the latter.

Compounds 1, 2, and 3 were identified to be 3α-hydroxy-ent-16(S)-kauran-15-one [7], ent-kaurane-3,15-dione, and ent-kaur-16-ene-3,15-dione [18], respectively, by a comparison of their spectral data with literatures. Compound 2 had been isolated from the cell culture of J. subulata, and its relative structure was confirmed by X-ray analysis [6]. However, the absolute configurations of 2 and 3 were just assumed to be the ent-form on the basis of the co-occurrence with other known ent-kauranes. In this study, the absolute configuration of 2 was determined to be ent-16(S)-kaurane-3,15-dione by oxidation of compound 1 to 2 with Jones reagent. The X-ray analysis of 3 was performed to confirm the relative structure of 3 deduced from spectral data, and gave the same relative structure as previously reported (see Fig. [4], ORTEP drawing). Then, the catalytic hydrogenation of 3 over Pd-C in EtOAc yielded a dihydro-derivative, of which the spectral and physico-chemical data were coincident with those of 2. Accordingly, the absolute configuration of 3 was determined to be ent-form.

Compound 4 was obtained as crystalline needles, and its molecular formula was found to be C₂₀H₃₀O₂ by HR-EI-MS. Its ¹H- and ¹³C-NMR spectra were similar to those of 3, except for the presence of another methine proton bearing a hydroxy group, instead of the disappearance of one carbonyl group in 3. By detailed analysis of the HMBC spectrum, the position of the hydroxy group was determined to be C-15, and its stereochemistry was assigned as the β-orientation on the basis of NOESY correlation (H-14/H-15 in Fig. [3]). Conclusive evidence for the structure of 4 was obtained by X-ray crystallographic analysis as shown in Fig. [4]. The absolute configuration was determined to be 15β-hydroxy-ent-kaur-16-en-3-one by Jones oxidation of compound 4 to 3. Although compound 4 has been reported as a reaction product [21], this is the first report from natural origins.

Compound 5 had the molecular formula of C₂₀H₃₀O₃ based on an HR-EI-MS peak at m/z = 318.2197. The IR absorptions (1735 and 3440 cm^{- 1}) confirmed the presence of carbonyl and hydroxy groups. The ¹H NMR spectrum exhibited a secondary methyl and three tertiary methyl protons. The ¹³C-NMR and HMQC spectra showed the signals of one oxygenated quaternary carbon (δ = 76.5) and two carbonyl carbons (δ = 217.3 and 221.5) as well as four methyls, seven methylenes, three methines and three quaternary carbons. The IR absorption (3440 cm^-1) and an oxygenated quaternary carbon signal (C-13: δ = 76.5) suggested that 5 contained a tertiary hydroxy group. These spectral data were similar to those of 2, indicating that compound 5 belongs to the kaurane-type of diterpenoid. The detailed analysis of HMBC and NOESY spectra (Fig. [3]) revealed the structure of 5 to be 13α-hydroxykaurane-3,15-dione.

Compound 6 with the molecular formula of C₂₀H₃₀O₃ (m/z = 318.2195 [M⁺]) had carbonyl and hydroxy groups on the basis of the IR spectrum (1700 and 3620 cm^-1). In comparison with 5, the NMR spectra of 6 showed the presence of an exo-methylene at C-17 [δ_H = 5.19 (1H, d, J = 3.0 Hz) and 5.21 (1H, d, J = 3.0 Hz)] and a secondary alcohol at C-15 [δ_H = 3.97 (brs) and  δ_C = 80.4 (d)] as well as the disappearance of a secondary methyl (C-17) and a carbonyl (C-15) signal observed in 5. The structure of 6 was elucidated to be 13α,15α-dihydroxykauran-3-one by HMBC and NOESY correlations (Fig. [3]). The structure was further supported by the formation of 5 from 6 by Jones oxidation and subsequent hydrogenation. The absolute stereochemistries of 5 and 6 were assumed to be the ent-form, on the basis of the co-occurrences with other isolated compounds.

The purification of the methanol extract from the intact plant resulted in the isolation of five ent-kaurane diterpenoids (compounds 3 and 7 - 10). Compounds 7 and 8 were identified as the known ent-kaur-16-en-15-one (7) [22], and 11β-hydroxy-ent-kaur-16-en-15-one (8) [7].

Compound 9 was crystalline, had the molecular formula of C₂₀H₃₀O₂ (m/z = 304.2240 [M]⁺), and its IR spectrum indicated the presences of hydroxy and carbonyl groups. The ¹H NMR spectrum displayed signals for three tertiary methyl, a secondary carbinyl, and an exo-methylene proton. The ¹³C-NMR and HMBC spectral data indicated that 9 was 7-hydroxykaur-16-en-15-one. Although 7α-hydroxy-ent-kaur-16-en-15-one was isolated from J. truncata [23], its spectral data were not consistent with those of 9. The NOESY correlation between carbonyl proton (H-7) and methylene protons at C-14 suggested that the hydroxy group at C-7 is in a β-orientation. The stereostructure of 9 was confirmed by X-ray analysis and determined to be 7β-hydroxykaur-16-en-15-one (Fig. [3]).

Compound 10 had the molecular formula of C₂₂H₃₀O₄ (m/z = 358.2124 [M]⁺). Its ¹H- and ¹³C-NMR spectra showed the presences of an acetyl [δ = 22.0 (q) and 169.8 (s)], an exo-methylene at C-17 [δ_H = 5.42 (1H, d, J = 2.0 Hz) and 6.09 (1H, d, J = 2.0 Hz)], two carbonyl groups (δ = 217.0 and 206.5), and an oxygenated quarternary carbon (δ = 84.2). These spectral data were similar to those of 5 except for the presence of an acetyl and an exo-methylene group in place of a hydroxy and a secondary methyl groups, suggesting that 10 is a kaurane-type diterpene. The analysis of its HMBC and NOESY spectra established the structure of 10 as 11α-acetoxykaur-16-ene-3,15-dione. Absolute configurations of compounds 9 and 10 were deduced to be of the ent-series on the basis of the co-occurrence of the known ent-kaur-16-ene-3,15-dione (3), ent-kaur-16-en-15-one (7) [22], and 11β-hydroxy-ent-kaur-16-en-15-one (8) [7] in the intact plant.

The kaurane-type diterpenoid is one of the characteristic metabolites in Jungermannia species. Compound 1 has been isolated from J. vulcanicola [7], and compounds 2 and 3 from J. truncata [18]. Compounds 7 and 8 have already been isolated from J. infusca [22] and J. truncata [7], respectively. The productivity of kaurane compounds from cell cultures was estimated to be at almost the same level as from the intact plant. The total amount of isolated kaurane-type diterpenoids was 90 mg from 1.71 g of hexane and ethyl acetate extracts from cell culture and 61 mg from 1.78 g of hexane and ethyl acetate extracts from intact plant. ent-Kaurene is biosynthesized from geranyl geranyl diphosphate by two enzymes, coparlyl diphosphate synthase and kaurene synthase in higher plants, and/or by bifunctional kaurene synthase in the moss Physcomitrella patens [24]. Therefore, cell cultures would express ent-kaurene synthetic genes to the same extent as the intact plant. In contrast to productivity, the metabolite profile of kaurane compounds in cell cultures differed in their oxidation-reduction status from that of intact plant. Tazaki et al. [6] have also reported the isolation of compound 3, together with three ent-kaurane-type diterpenoids, ent-kaur-16-ene, 15β-hydroxy-ent-kaur-16-ene, and ent-kaur-16-en-15-one, from cell cultures of J. subulata and also described the GC-MS identification of these four kaurenes in the intact plant collected at Hokkaido (Japan). These metabolites also differed from our result in oxidation-reduction status. The oxidation or reduction of the kaurene skeleton might be catalyzed by P450 oxido-reductases, monooxygenases, and NADH/NADPH-dependent reductases. These data indicate that the expression of these oxido-reductase genes differs between cell culture and intact plant, and is affected by the culture conditions or the growth environment. This will be the subject of further studies to reveal the culture conditions affecting metabolite profiles, such as a medium composition, light, temperature, and aeration.

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• 8 Matsuo A, Nakayama M, Hayashi S, Seki T, Amakawa T. A comparative study of the diterpenoids from several species of the genus Jungermannia . In: Claude S, editor Congress International de Bryologie, Bordeaux Nov. 21 - 23, Bryophytorum Bibliotheca, Vol. 13. Stuttgart; Schweizerbart Publishers 1977: 322-8.
  Search in Google Scholar
• 9 Asakawa Y. Chemosystematics of the Hepaticae.  Phytochemistry. 2004;  65 623-69.
  CrossrefPubMedSearch in Google Scholar
• 10 Nagashima F, Kondoh M, Kawase M, Simizu S, Osada H, Fujii M. et al . Apoptosis-inducing properties of ent-kaurene-type diterpenoids from the liverwort Jungermannia truncata .  Planta Med. 2003;  69 377-9.
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Suzuki I, Kondoh M, Harada M, Koizumi N, Fujii M, Nagashima F. et al . An ent-kaurene diterpene enhances apoptosis induced by tumor necrosis factor in human leukemia cells.  Planta Med. 2004;  70 723-7.
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Kondoh M, Nagashima F, Suzuki I, Harada M, Fujii M, Asakawa Y. et al . Induction of apoptosis by new ent-kaurene-type diterpenoids isolated from the New Zealand liverwort Jungermannia species.  Planta Med. 2005;  71 1005-9.
  Thieme ConnectPubMedSearch in Google Scholar
• 13 Nagashima F, Kasai W, Kondoh M, Fujii M, Watanabe Y, Braggins J E. et al . New ent-kaurene-type diterpenoids possessing cytotoxicity from the New Zealand liverwort Jungermannia species.  Chem Pharm Bull. 2003;  51 1189-92.
  CrossrefPubMedSearch in Google Scholar
• 14 Yeh S H, Chang F R, Wu Y C, Yang Y L, Zhuo S K, Hwang T L. An anti-inflammatory ent-kaurane from the stems of Annona squamosa that inhibits various human neutrophil functions.  Planta Med. 2005;  71 904-9.
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Yang Y L, Chang F R, Wu C C, Wang W Y, Wu Y C. New ent-kaurane diterpenoids with anti-platelet aggregation activity from Annona squamosa .  J Nat Prod. 2002;  65 1462-7.
  CrossrefPubMedSearch in Google Scholar
• 16 Yang Y L, Chang F R, Hwang T L, Chang W T, Wu Y C. Inhibitory effects of ent-kauranes from the stems of Annona squamosa on superoxide anion generation by human neutrophils.  Planta Med. 2004;  70 256-8.
  Thieme ConnectPubMedSearch in Google Scholar
• 17 Wu Y C, Hung Y C, Chang F R, Cosentino M, Wang H K, Lee K H. Identification of ent-16β, 17-dihydroxykauran-19-oic acid as an anti-HIV principle and isolation of the new diterpenoids annosquamosins A and B from Annona squamosa .  J Nat Prod. 1996;  59 635-7.
  CrossrefPubMedSearch in Google Scholar
• 18 Liu H J, Tseng S H, Wu J D, Wu C L. Kaurane-type diterpenoids from liverworts.  J Chin Chem Soc (Taipei). 1997;  44 385-9.
  PubMedSearch in Google Scholar
• 19 Nagashima F, Suzuki M, Asakawa Y. Seco-cuparane-type sesquiterpenoid from the Japanese liverwort Jungermannia infusca .  Phytochemistry. 2001;  56 807-10.
  CrossrefPubMedSearch in Google Scholar
• 20 Murashige T, Skoog F. A revised media for rapid growth and bioassay with tobacco cultures.  Physiol Plant. 1962;  15 473-97.
  CrossrefPubMedSearch in Google Scholar
• 21 Fraga B M, Guillermo R, Hernandez M G. The microbiological transformation of two 15β-hydroxy-ent-kaurene diterpenes by Gibberella fujikuroi .  J Nat Prod. 2004;  67 64-9.
  CrossrefPubMedSearch in Google Scholar
• 22 Matsuo A, Kodama J, Nakayama M, Hayashi S. ent-Kaurene diterpenoids from the liverwort Jungermannia infusca .  Phytochemistry. 1977;  16 489-90.
  CrossrefPubMedSearch in Google Scholar
• 23 Buchana M S, Connolly J D, Kadir A A, Rycroft D S. Sesquiterpenoids and diterpenoids from the liverwort Jungermannia truncata .  Phytochemistry. 1996;  42 1641-6.
  CrossrefPubMedSearch in Google Scholar
• 24 Hayashi K, Kawaide H, Notomi M, Sakigi Y, Matsuo A, Nozaki H. Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens .  FEBS Lett.. 2006;  580 6175-81.
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Hiroshi Nozaki

Department of Biochemistry

Okayama University of Science

1-1 Ridai-cho

Okayama City 700-0005

Japan

Phone: +81-86-256-9460

Fax: +81-86-256-9559

Email: nozaki@dbc.ous.ac.jp

References

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• 2 Matsuo A. Selected chemotaxonomic characteristics of liverwort sesquiterpenoids.  J Hattori Bot Lab. 1982;  53 295-304.
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• 3 Ohta Y, Kato K, Takeda R. Growth and secondary metabolites production in cultured cells of bryophytes. In: Chopra RN, Bhatla SC, editors Bryophyte development: physiology and biochemistry. Boca Raton; CRC Press 1990: 209-23.
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• 4 Tazaki H, Adam K P, Becker H. Five lignan derivatives from in vitro cultures of the liverwort Janesoniella autumnalis .  Phytochemistry. 1995;  40 1671-5.
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• 5 Matsuo A, Ono K, Hamasaki K, Nozaki H. Phaeophytins from a cell suspension culture of the liverwort Plagiochila ovalifolia .  Phytochemistry. 1996;  42 427-30.
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• 6 Tazaki H, Iwasaki T, Nakasuga I, Kobayashi K, Koshino H, Tanaka M. et al . ent-Kaurane-type deterpenoids produced cell culture of the liverwort Jungermannia subulata .  Phytochemistry. 1999;  52 1427-30.
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• 7 Nagashima F, Toyota M, Asakawa Y. Terpenoids from some Japanese liverworts.  Phytochemistry. 1990;  29 2169-74.
  CrossrefPubMedSearch in Google Scholar
• 8 Matsuo A, Nakayama M, Hayashi S, Seki T, Amakawa T. A comparative study of the diterpenoids from several species of the genus Jungermannia . In: Claude S, editor Congress International de Bryologie, Bordeaux Nov. 21 - 23, Bryophytorum Bibliotheca, Vol. 13. Stuttgart; Schweizerbart Publishers 1977: 322-8.
  Search in Google Scholar
• 9 Asakawa Y. Chemosystematics of the Hepaticae.  Phytochemistry. 2004;  65 623-69.
  CrossrefPubMedSearch in Google Scholar
• 10 Nagashima F, Kondoh M, Kawase M, Simizu S, Osada H, Fujii M. et al . Apoptosis-inducing properties of ent-kaurene-type diterpenoids from the liverwort Jungermannia truncata .  Planta Med. 2003;  69 377-9.
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Suzuki I, Kondoh M, Harada M, Koizumi N, Fujii M, Nagashima F. et al . An ent-kaurene diterpene enhances apoptosis induced by tumor necrosis factor in human leukemia cells.  Planta Med. 2004;  70 723-7.
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Kondoh M, Nagashima F, Suzuki I, Harada M, Fujii M, Asakawa Y. et al . Induction of apoptosis by new ent-kaurene-type diterpenoids isolated from the New Zealand liverwort Jungermannia species.  Planta Med. 2005;  71 1005-9.
  Thieme ConnectPubMedSearch in Google Scholar
• 13 Nagashima F, Kasai W, Kondoh M, Fujii M, Watanabe Y, Braggins J E. et al . New ent-kaurene-type diterpenoids possessing cytotoxicity from the New Zealand liverwort Jungermannia species.  Chem Pharm Bull. 2003;  51 1189-92.
  CrossrefPubMedSearch in Google Scholar
• 14 Yeh S H, Chang F R, Wu Y C, Yang Y L, Zhuo S K, Hwang T L. An anti-inflammatory ent-kaurane from the stems of Annona squamosa that inhibits various human neutrophil functions.  Planta Med. 2005;  71 904-9.
  Thieme ConnectPubMedSearch in Google Scholar
• 15 Yang Y L, Chang F R, Wu C C, Wang W Y, Wu Y C. New ent-kaurane diterpenoids with anti-platelet aggregation activity from Annona squamosa .  J Nat Prod. 2002;  65 1462-7.
  CrossrefPubMedSearch in Google Scholar
• 16 Yang Y L, Chang F R, Hwang T L, Chang W T, Wu Y C. Inhibitory effects of ent-kauranes from the stems of Annona squamosa on superoxide anion generation by human neutrophils.  Planta Med. 2004;  70 256-8.
  Thieme ConnectPubMedSearch in Google Scholar
• 17 Wu Y C, Hung Y C, Chang F R, Cosentino M, Wang H K, Lee K H. Identification of ent-16β, 17-dihydroxykauran-19-oic acid as an anti-HIV principle and isolation of the new diterpenoids annosquamosins A and B from Annona squamosa .  J Nat Prod. 1996;  59 635-7.
  CrossrefPubMedSearch in Google Scholar
• 18 Liu H J, Tseng S H, Wu J D, Wu C L. Kaurane-type diterpenoids from liverworts.  J Chin Chem Soc (Taipei). 1997;  44 385-9.
  PubMedSearch in Google Scholar
• 19 Nagashima F, Suzuki M, Asakawa Y. Seco-cuparane-type sesquiterpenoid from the Japanese liverwort Jungermannia infusca .  Phytochemistry. 2001;  56 807-10.
  CrossrefPubMedSearch in Google Scholar
• 20 Murashige T, Skoog F. A revised media for rapid growth and bioassay with tobacco cultures.  Physiol Plant. 1962;  15 473-97.
  CrossrefPubMedSearch in Google Scholar
• 21 Fraga B M, Guillermo R, Hernandez M G. The microbiological transformation of two 15β-hydroxy-ent-kaurene diterpenes by Gibberella fujikuroi .  J Nat Prod. 2004;  67 64-9.
  CrossrefPubMedSearch in Google Scholar
• 22 Matsuo A, Kodama J, Nakayama M, Hayashi S. ent-Kaurene diterpenoids from the liverwort Jungermannia infusca .  Phytochemistry. 1977;  16 489-90.
  CrossrefPubMedSearch in Google Scholar
• 23 Buchana M S, Connolly J D, Kadir A A, Rycroft D S. Sesquiterpenoids and diterpenoids from the liverwort Jungermannia truncata .  Phytochemistry. 1996;  42 1641-6.
  CrossrefPubMedSearch in Google Scholar
• 24 Hayashi K, Kawaide H, Notomi M, Sakigi Y, Matsuo A, Nozaki H. Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens .  FEBS Lett.. 2006;  580 6175-81.
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Hiroshi Nozaki

Department of Biochemistry

Okayama University of Science

1-1 Ridai-cho

Okayama City 700-0005

Japan

Phone: +81-86-256-9460

Fax: +81-86-256-9559

Email: nozaki@dbc.ous.ac.jp