New Diarylheptanoids from the Rhizomes of Dioscorea spongiosa and Their Antiosteoporotic Activity

• Abstract
• Introduction
• Materials and Methods
• General
• Plant material
• Extraction and isolation
• Preparation of MTPA esters of 1 and 2
• Preparation of acetonide and bis(4-N,N-dimethylaminobenzoate) of 3
• Bone resorbing activity
• Results and Discussion
• References

Abstract

Bioassay-guided fractionation of the water extract of the rhizomes of Dioscorea spongiosa led to the isolation and identification of new diarylheptanoids, diospongins A - C, together with three known lignans. Their structures, including absolute stereochemistry, were determined by analyses of NMR data, chemical conversions and CD spectrum. The isolated compounds, except for diospongin A, exerted potent inhibitory activities on bone resorption induced by parathyroid hormone in a bone organ culture system.

Introduction

Rhizomes of Dioscorea spongiosa J. Q. Xi, M. Mizuno et W. L. Zhao (Dioscoreaceae) are used for the treatment of rheumatism, urethra, and renal infection in Chinese traditional medicine [1]. In a search for natural crude drugs having anti-osteoporotic activity, we found that the water extract of this plant displayed potent stimulatory activity (67 % at 400 μg/mL) on the proliferation of an osteoblast-like UMR106 cell line and complete inhibition on the formation of osteoclast-like multinuclear cells at a concentration of 200 μg/mL. Thus, a bioassay-guided fractionation of the water extract was conducted and resulted in the isolation of 21 glycosides [2]. In this paper, we report the isolation and structure elucidation of diospongins A - C (1 - 3) together with the inhibitory activities of the isolated compounds on bone resorption induced by parathyroid hormone (PTH) in a bone organ culture system.

Materials and Methods

General

Optical rotations were measured on a JASCO DIP-140 digital polarimeter at 25 °C. The CD spectrum was recorded on a JASCO J-805 spectrometer and IR spectra were recorded on a Shimadzu IR-408 spectrometer. NMR spectra were measured on a JEOL JNM-GX400 spectrometer with TMS as an internal standard. FAB-MS were measured on a JEOL JMS-700T mass spectrometer and glycerol was used as matrix. Analytical and preparative TLC were conducted on precoated Kieselgel 60F₂₅₄ and RP-18F₂₅₄ (Merck) plates.

Plant material

Rhizomes of D. spongiosa were purchased in March, 2000 at a market in Shenyang, People’s Republic of China, and identified by Professor Qishi Sun (Division of Pharmacognosy, Shenyang Pharmaceutical University). A voucher specimen (SPU 1129) is deposited at the herbarium of Shenyang Pharmaceutical University.

Extraction and isolation

The 90 % EtOH-H₂O fraction (20 g) was chromatographed on silica gel (6 × 30 cm) with CHCl₃-MeOH to give nine fractions: fr.1 (800 mL, CHCl₃-MeOH, 20 : 1), fr.2 (600 mL, CHCl₃-MeOH, 15 : 1), fr.3 (200 mL, CHCl₃-MeOH, 10 : 1), fr.4 (300 mL, CHCl₃-MeOH, 8 : 1), fr.5 (300 mL, CHCl₃-MeOH, 5 : 1), fr.6 (300 mL, CHCl₃-MeOH, 3 : 1), fr.7 (700 mL, CHCl₃-MeOH, 1 : 1), fr.8 (200 mL, CHCl₃-MeOH, 1 : 2), fr.9 (200 mL, CHCl₃-MeOH, 1 : 5). Fr. 3 (220 mg) was subjected to silica gel column (1 × 25 cm, 200 - 300 mesh) chromatography with CHCl₃-MeOH-H₂O (70 : 20 : 1) to yield four subfractions: fr. 3 - 1 (20 mL), fr. 3 - 2 (20 mL), fr. 3 - 3, (55 mL), fr. 3 - 4 (60 mL). Subfraction 3 - 3 (75 mg) was chromatographed on ODS (1 × 15 cm) with MeOH-H₂O (2 : 1) to give two fractions: fr. 3 - 3-1 (90 mL) and fr. 3 - 3-2 (20 mL). Fr. 3 - 3-2 (60 mg) was subjected to preparative TLC with MeOH-H₂O (5 : 2) to give diospongin A (1, 24.1 mg, R_f = 0.58), diospongin B (2, 17.3 mg, R_f = 0.42) and syringaresinol (6, 3.4 mg, R_f = 0.49) [3]. Subfraction 3 - 4 (80 mg) was also separated by reversed-phase preparative TLC with MeOH-H₂O (5 : 2) to give diospongin C (3, 18.7 mg, R_f = 0.76), piperitol (4, 4.3 mg, R_f = 0.62) [4] and sesaminone (5, 3.5 mg, R_f = 0.55) [5].

Preparation of MTPA esters of 1 and 2

The (S)-(-)- or (R)-(+)-MTPA esters of compounds 1 and 2 were prepared according to the procedure reported by Ohtani et al. [6].

Preparation of acetonide and bis(4-N,N-dimethylaminobenzoate) of 3

The monoacetonide 3a and diacetonide 3b of 3 were prepared according to the procedure reported by Rychnovsky et al., followed by separation on reversed-phase preparative TLC with MeOH-H₂O (5 : 2) (3a: R_f = 0.54; 3b: R_f = 0.32) [7]. Bis(4-N,N-dimethylaminobenzoate) 3c was prepared according to the procedure reported by Wiesler et al., followed by separation on normal-phase preparative TLC [CHCl₃-MeOH (20 : 1); R_f = 0.23] and then reversed-phase HPLC [MeOH-H₂O (2.7 : 1); t_R = 14.8 min] [8].

Diospongin A (1): Colorless amorphous solid, [α]_D ²⁵: -21.2° (c 0.8, CHCl₃); IR (CHCl₃): ν _max = 3450, 1690, 1610, 1495, 1450, 1055 cm^-1; HR-FAB-MS: m/z = 297.1523 [M + H]⁺ (calcd. for C₁₉H₂₁O₃ : 297.1491); ¹H- and ¹³C-NMR data, see Table [1].

Diospongin B (2): Colorless amorphous solid, [α]_D ²⁵: -23.4° (c 0.6, CHCl₃); IR (CHCl₃): ν _max = 3400, 1685, 1600, 1495, 1450, 1055 cm^-1; HR-FAB-MS: m/z = 297.1487 [M + H]⁺ (calcd. for C₁₉H₂₁O₃ : 297.1491); ¹H- and ¹³C-NMR data, see Table [1].

Diospongin C (3): Light yellow oil, [α]_D ²⁵: -45.5° (c 0.5, CHCl₃); IR (CHCl₃): ν _max = 3400, 1600, 1490 cm^-1; HR-FAB-MS: m/z = 317.1784 [M + H]⁺ (calcd. for C₁₉H₂₅O₄ : 317.1781); ¹H- and ¹³C-NMR data, see Table [1].

(R)-MTPA ester of 1 (1a): Light yellow oil; ¹H-NMR (CDCl₃): δ = 7.89 (2H, m, H-2′,6′), 7.53 (1H, m, H-4′), 7.40 (2H, m, H-3′,5′), 7.21 (2H, m, H-2′′,6′′), 7.19 (2H, m, H-3′′,5′′), 7.13 (1H, m, H-4′′), 5.61 (1H, t, J = 2.8 Hz, H-5), 4.48 (1H, dd, J = 13.2, 1.2 Hz, H-7), 4.41 (1H, m, H-3), 3.35 (1H, dd, J = 16.0, 5.2 Hz, H-2), 3.04 (1H, dd, J = 16.0, 6.8 Hz, H-2), 2.15 (1H, m, H-4), 1.94 (1H, m, H-6), 1.77 (1H, m, H-6), 1.68 (1H, m, H-4).

(S)-MTPA ester of 1 (1b): Light yellow oil; ¹H-NMR (CDCl₃): δ = 7.88 (2H, m, H-2′,6′), 7.49 (1H, m, H-4′), 7.37 (2H, m, H-3′,5′), 7.24 (2H, m, H-2′′,6′′), 7.18 (2H, m, H-3′′,5′′), 7.14 (1H, m, H-4′′), 5.48 (1H, br s, H-5), 4.60 (1H, dd, J = 12.0, 1.2 Hz, H-7), 4.32 (1H, m, H-3), 3.32 (1H, dd, J = 16.0, 6.8 Hz, H-2), 2.98 (1H, dd, J = 16.0, 6.8 Hz, H-2), 2.11 (1H, m, H-4), 2.02 (1H, m, H-6), 1.80 (1H, m, H-6), 1.66 (1H, m, H-4).

(R)-MTPA ester of 2 (2a): Light yellow oil; ¹H-NMR (CDCl₃): δ = 7.83 (2H, m, H-2′,6′), 7.51 (1H, m, H-4′), 7.39 (2H, m, H-3′,5′), 7.34 (2H, m, H-2′′,6′′), 7.28 (2H, m, H-3′′,5′′), 7.26 (1H, m, H-4′′), 5.32 (1H, m, H-5), 4.89 (1H, m, H-7), 4.29 (1H, m, H-3), 3.09 (1H, dd, J = 15.8, 6.8 Hz, H-2), 3.31(1H, dd, J = 15.8, 5.4 Hz, H-2), 2.46 (1H, m, H-6), 2.13 (1H, m, H-4), 1.91 (1H, m, H-6), 1.72 (1H, m, H-4).

(S)-MTPA ester of 2 (2b): Light yellow oil; ¹H-NMR (CDCl₃): δ = 7.84 (2H, m, H-2′,6′), 7.50 (1H, m, H-4′), 7.39 (2H, m, H-3′,5′), 7.34 (2H, m, H-2′′,6′′), 7.28 (2H, m, H-3′′,5′′), 7.26 (1H, m, H-4′′), 5.29 (1H, m, H-5), 5.07 (1H, m, H-7), 4.23 (1H, m, H-3), 3.33 (1H, dd, J = 15.8, 5.8 Hz, H-2),  2.94 (1H, dd, J = 15.8, 6.8 Hz, H-2), 2.62 (1H, m, H-6), 2.06 (1H, m, H-4), 2.00 (1H, m, H-6), 1.63 (1H, m, H-4).

Monoacetonide 3a: Colorless amorphous solid; ¹H-NMR (C₆D₆): δ = 7.45 (2H, d, J = 7.8 Hz, H-2′′,6′′), 7.31 (2H, d, J = 7.8 Hz, H-2′,6′), 7.24 (2H, d, J = 8.1, H-3′′,5′′), 7.20 (2H, m, H-3′,5′), 7.20 (1H, m, H-4′′), 7.13 (1H, m, H-4′), 4.93 (1H, dd, J = 8.5, 2.5 Hz, H-1), 4.83 (1H, dd, J = 9.2, 4.1 Hz, H-7), 3.79 (1H, ddt, J = 11.7, 9.0, 2.7 Hz, H-3), 3.53 (1H, ddt, J = 11.7, 9.2, 2.7 Hz, H-5), 1.87 (1H, dt, J = 14.2, 9.2 Hz, H-6), 1.75 (1H, ddd, J = 14.4, 9.0, 2.5 Hz, H-2), 1.57 (1H, ddd, J = 14.4, 8.5, 2.7 Hz, H-2), 1.45 (1H, ddd, J = 14.2, 4.1, 2.7 Hz, H-6), 1.36 (3H, s, CH₃), 1.17 (3H, s, CH₃), 0.91 (1H, dt, J = 12.9, 11.7 Hz, H-4), 0.67 (1H, dt, J = 12.9, 2.7 Hz, H-4); ¹³C-NMR (C₆D₆): δ = 145.7 (C-1′), 145.4 (C-1′′), 128.5 (C-3′,5′, C-3′′,5′′), 127.5 (C-4′), 127.2 (C-4′′), 126.3 (C-2′′,6′′), 125.9 (C-2′,6′), 98.8 [C(CH₃)₂], 74.9 (C-7), 70.9 (C-1), 69.5 (C-5), 66.7 (C-3), 46.2 (C-2), 44.9 (C-6), 36.9 (C-4), 30.2 (CH₃), 19.8 (CH₃); HR-FAB-MS: m/z = 357.2078 [M + H]⁺ (calcd. for C₂₂H₂₉O₄ : 357.2066).

Diacetonide 3b: Colorless amorphous solid; ¹H-NMR (C₆D₆): δ = 7.46 (6H, dd, J = 9.0, 2.4 Hz, H-2′,6′, H-4′, H-2′′,6′′, H-4′′), 7.27 (4H, m, H-3′,5′, H-3′′,5′′), 4.97 (1H, dd, J = 9.2, 6.5 Hz, H-1), 4.80 (1H, dd, J = 10.7, 3.5 Hz, H-7), 4.22 (1H, ddt, J = 14.2, 6.5, 2.0 Hz, H-3), 4.16 (1H, ddt, J = 10.0, 6.5, 3.0 Hz, H-5), 2.11 (1H, dt, J = 14.2, 6.5 Hz, H-4), 1.94 (1H, ddd, J = 14.4, 9.2, 6.5 Hz, H-2), 1.61 (1H, m, H-4), 1.59 (1H, m, H-6), 1.52 (1H, m, H-6), 1.50 (1H, m, H-2), 1.62 (3H, s, CH₃), 1.46 (6H, s, CH₃ × 2), 1.37 (3H, s, CH₃); ¹³C-NMR (C₆D₆) δ 143.6 (C-1′), 143.3 (C-1′′), 128.7 (C-3′,5′, C-3′′,5′′), 127.6 (C-4′), 127.4 (C-4′′), 126.2 (C-2′′,6′′), 126.1 (C-2′,6′), 100.7 [C(CH₃)₂], 98.8 [C(CH₃)₂], 71.5 (C-7), 68.7 (C-1), 66.1 (C-5), 63.2 (C-3), 42.8 (C-2), 40.7 (C-6), 39.6 (C-4), 30.6 (CH₃), 25.3 (CH₃), 25.0 (CH₃), 19.8 (CH₃); HR-FAB-MS: m/z = 397.2357 [M + H]⁺ (calcd. for C₂₅H₃₃O₄ : 397.2379).

Bis(4-N,N-dimethylaminobenzoate) 3c: Colorless amorphous solid; ¹H-NMR (C₆D₆): δ = 7.40 - 7.30 (10H, Ar-H), 6.09 (1H, dd, J = 9.0, 4.5 Hz, H-1), 5.31 (1H, m, H-3), 4.92 (1H, dd, J = 9.0, 4.1 Hz, H-7), 4.15 (1H, m, H-5), 3.00 (3H, s, N-CH₃), 2.47 (1H, ddd, J = 14.0, 9.0, 4.6 Hz, H-2), 2.40 (1H, ddd, J = 14.0, 9.0, 4.6 Hz, H-2), 2.02 (1H, dt, J = 14.4, 7.6 Hz, H-4), 1.89 (1H, dt, J = 14.4, 4.5 Hz, H-4), 1.81 (1H, dt, J = 14.1, 4.1 Hz, H-6), 1.79 (1H, dt, J = 14.1, 9.0 Hz, H-6); HR-FAB-MS: m/z = 629.3348 [M + Na]⁺ (calcd. for C₃₉H₄₆N₂O₄Na: 629.3355).

Bone resorbing activity

An assay method reported by Shigeno et al. [9] was used to evaluate bone resorbing activity, as presented previously [10]. In brief, the parietal bones of mice injected with ⁴⁵CaCl₂ were taken out to culture in Ham’s F-12 medium containing PTH and the tested samples for 6 days and the medium was changed every 3 days. After finishing the culture, bones were removed into EDTA-acetate buffer solution. ⁴⁵Ca released in changed medium and EDTA solution was measured by liquid scintillation counting. Bone resorption was obtained as the percentage of total ⁴⁵Ca that was released into the medium during the culture.

Results and Discussion

The water extract of rhizomes of D. spongiosa was chromatographed with a Diaion HP-20 column, using a H₂O-EtOH solvent system, to give four (H₂O and 30 %, 60 %, and 90 % EtOH-H₂O) fractions. They showed 11.2, 14.7, 86.7, and 89.5 % stimulation of the proliferation of osteoblast-like UMR106 cell line at a concentration of 200 μg/mL, and all showed 100 % inhibition of the formation of osteoclast-like multinuclear cells at the same concentration. On the other hand, only the 90 % EtOH-H₂O fraction inhibited the bone resorption induced by PTH in a bone organ culture system at a concentration of 440 μg/mL (82.8 % inhibition). Thus, this fraction was further separated by a combination of normal- and reversed-phase column chromatography and preparative TLC, to afford three diarylheptanoids, diospongins A - C (1 - 3), along with three known lignans, piperitol (4) [4], sesaminone (5) [5] and (+)-syringaresinol (6) [3].

Diospongin A (1) showed a quasi-molecular ion, corresponding to the molecular formula C₁₉H₂₀O₃ on HR-FAB-MS. Its IR spectrum displayed the absorptions of hydroxy (3450 cm^-1), conjugated carbonyl (1695 cm^-1) and aromatic ring (1610, 1495 cm^-1) functions. The ¹H- and ¹³C-NMR spectra of 1 revealed the presence of three oxymethines, three methylenes, two phenyl rings and a ketone carbonyl carbon (Table [1]). Extensive analysis of the 2D NMR spectra suggested that 1 should be a diarylheptanoid. The location of the carbonyl carbon was determined to be at C-1 by the HMBC correlations between the aromatic protons H-2′,6′ and the carbonyl carbon (δ _C = 198.4), while the HMBC correlations H-7/C-2¿,6¿ and H-2¿,6¿/C-7 confirmed the other phenyl ring at C-7. The correlations H-3/C-7 and H-7/C-3 indicated the presence of an ether linkage between C-3 and C-7. Thus, the structure of 1 was determined as 1,7-diphenyl-3,7-epoxy-5-hydroxy-1-heptanone. The large coupling constants between H-3 and H-4ax at δ _H = 1.67 (J = 11.2 Hz) and between H-7 and H-6ax at δ _H = 1.75 (J = 12.0 Hz) indicated that these protons should be axial, whereas H-5 was considered to be equatorial from the small coupling constants with H₂ - 4 and H₂ - 6 (each J = 3.0 Hz). The ROESY correlations H-3/H-7, H-3/H-4eq and H-4ax/H-6ax indicated that the pyran ring had a chair conformation, that H-3, H-4eq and H-7 are cis and that H-4ax and H-6ax are also cis. Finally, the absolute configuration at C-5 was determined to be S by the advanced Mosher method [6] (Fig. [1]). Thus, the structure of diospongin A was established as (3R,5S,7S)-1,7-diphenyl-3,7-epoxy-5-hydroxy-1-heptanone (1).

The molecular formula of diospongin B (2) was determined to be the same as that of 1 by HR-FAB-MS. The ¹H- and ¹³C-NMR spectra of 2 were very similar to those of 1, except for slight differences in splitting patterns of H-4ax, H-5, H-6ax and H-7 (Table [1]). Thus, 2 was considered to be a diastereomer of 1, which was confirmed by the analysis of its 2D NMR spectra. The coupling patterns of H-3, H-5 and H-7 indicated H-3 and H-5 to be axial and H-7 to be equatorial, while the ROESY correlations H-3/H-5, H-5/H-6eq and H-4ax/H-6ax revealed that the pyran ring should have a chair conformation. The analysis of the ¹H-NMR spectra of its α-methoxy-α-trifluoromethylphenylacetyl (MTPA) derivatives indicated the absolute configuration at C-5 to be S (Fig. [1]). Thus, the structure of diospongin B was established as (3S,5S,7S)-1,7-diphenyl-3,7-epoxy-5-hydroxy-1-heptanone (2).

The HR-FAB-MS of diospongin C (3) showed a quasi-molecular ion, consistent with the molecular formula C₁₉H₂₄O₄, and its IR spectrum showed the absorption of hydroxy group and aromatic ring. Its ¹H- and ¹³C-NMR spectra indicated the presence of four oxymethines, three methylenes and two monosubstituted benzene rings, suggesting that 3 was also a diarylheptanoid. The analysis of the 2D NMR spectra of 3, together with the molecular formula, suggested 3 to be an acyclic diarylheptanoid with four hydroxy groups at C-1, C-3, C-5 and C-7; i. e., 1,7-diphenylheptan-1,3,5,7-tetraol.

In order to assign the relative configuration of the acyclic chain moiety, monoacetonide 3a and diacetonide 3b were prepared with dimethoxypropane (DMP) and pyridinium p-toluenesulfonate (PPTS) in acetone [7], [11]. The ¹³C-NMR signals for the acetonide methyls revealed that 3a was a monoacetonide constructed from a syn-1,3-diol (δ _C = 19.8, 30.2), whereas 3b was a diacetonide formed from syn- (δ _C = 19.8, 30.6) and anti-1,3-diols  (δ _C = 25.0, 25.3), respectively. The ROESY correlations of the methyl protons (δ _H = 1.17) with H-3 and H-5 in 3a indicated the location of the syn acetonide to be at C-3 and C-5. The syn acetonide in 3b was located at C-5 and C-7 based on the ROESY correlations of the methyl protons (δ _H = 1.37) with H-5 and H-7, while the anti acetonide should be located at C-1 and C-3 by the ROESY correlation between the methyl protons at δ _H = 1.46 and H-1. Thus, the relative configuration of 3 was determined as 1,3-anti, 3,5-syn and 5,7-syn.

The bis(4-N,N-dimethylaminobenzoate) 3c was prepared in order to determine the absolute configuration of 3 [8]. Previously, it was reported that the CD spectra of dibenzoates of anti-diols showed a split Cotton effect in the region of the interchromophoric charge transfer, but those of syn-diols did not show a clear Cotton effect [12]. As can be seen in Fig. [2], the CD spectrum of 3c showed a negative Cotton effect at 323 nm (Δε -28.1) and a positive Cotton effect at 295 nm (Δε + 19.5). Thus, the two benzoyl groups in 3c should have an anti relationship, i. e., 3c is a 1,3-dibenzoate. Moreover, by applying the CD exciton chirality rule on acyclic 1,3-dibenzoates [13], the benzoyloxy groups at C-1 and C-3 possessed anticlockwise chirality (Fig. [2]), hence implying the 1S,3R absolute configuration. Thus, the structure of diospongin C was established as (1S,3R,5S,7S)-1,7-diphenylheptan-1,3,5,7-tetraol (3).

The known lignans, piperitol (4), [α]_D ²⁵: -68.3° (c 0.2, CHCl₃), sesaminone (5), [α]_D ²⁵: -30.6° (c 0.1, MeOH), and (+)-syringaresinol (6), [α]_D ²⁵: + 8.5° (c 0.1, MeOH), were identified by comparisons of their spectral data with those in the literature [3], [4], [5].

We evaluated all the compounds for inhibitory activity on bone resorption induced by PTH in a bone organ culture system [9], [10]. Elcitonin, a drug used clinically and employed here as a positive control [13], significantly inhibited bone resorption. All compounds, except for 1, showed significant activity and compounds 4 and 5 completely inhibited the ⁴⁵Ca release (Table [2]).

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Prof. Dr. Shigetoshi Kadota

Department of Natural Products Chemistry

Institute of Natural Medicine

Toyama Medical and Pharmaceutical University

2630 Sugitani

Toyama 930-0194

Japan

Phone: +81-76-434-7625

Fax: +81-76-434-5059

Email: kadota@ms.toyama-mpu.ac.jp

References

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  PubMedSearch in Google Scholar
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Prof. Dr. Shigetoshi Kadota

Department of Natural Products Chemistry

Institute of Natural Medicine

Toyama Medical and Pharmaceutical University

2630 Sugitani

Toyama 930-0194

Japan

Phone: +81-76-434-7625

Fax: +81-76-434-5059

Email: kadota@ms.toyama-mpu.ac.jp