Studies on chemical constituents of Dischidia acuminata Cost.

Le Minh Ha¹, Braca Alessandra², Chau Van Minh¹, Morelli Ivano², Pham Quoc Long¹, Nguyen Van Hoan¹

⁽¹⁾ Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology,

18- Hoang Quoc Viet, Hanoi- Vietnam.

⁽²⁾ Department of Bioorganic Chemistry and Biopharmaceutics, University of Pisa, Via Bonanno 33, 56126 Pisa, Italy

(Received, November, 19^th, 2004). 

Summary

Three alkyl glycosides: sec-butyl-b-d-glucopyranoside (1), 2-((b-d-apiofuranosyl-(1®6)-b-d-glucopyranosyl)oxy)butane (2), isopropyl-b-d-glucopyranoside (3), one monoterpene glycoside: 2,3-diol-2,6-dimethyl-7-octene-6-O-b-d-glucopyranoside (4) together with two flavonoid glycosides: vitexin 2^’’-O-rhamnopyranoside (5) and isovitexin 2^’’-O-galactopyranoside (6) have been isolated from Dischidia acuminata (Asclepiadaceae) whole plant. The structures of these compounds were elucidated by spectroscopic methods.

Keywords: Dischidia acuminata; alkyl glycosides; monoterpene glycoside; flavonoid glycosides.

I. Introduction

Dischidia acuminata Cost., is a climbing plant belonging to Asclepiadaceae family. It is widely distributed in mountainous areas of Vietnam. The whole herb is used in Vietnamese traditional and folk medicine for treating the symptoms of urinary cystitis, inflammation, and leucorrhoea [1]. To the best of our knowledge, the genus Dischidia has not been deeply investigated on its chemical constituents; only some steroids, triterpenoids, and flavonoids were isolated from the whole herb of D. formosana [2]. However, there are no studies on the chemical constituents of D. acuminata.

In this report, we describe the isolation and structural elucidation of secondary metabolites isolated from D. acuminata.

II. Results and Discussion

The whole herb of D.acuminata was extracted with n-hexane, CHCl₃, CHCl₃-MeOH 9:1, and MeOH, obtaining the respective residues R_H, R_C, R_CM, and R_M.

Fractionation of R_CM, by size-exclusion chromatography, and repeated column chromatography on a C-18 m-Bondapak column (30x7.8 mm, flow rate 2.0 ml/min) using MeOH-H₂O as eluent, led to the isolation of three alkyl glycosides and one monoterpene glycoside.

The residue R_M was partitioned between n-BuOH and water, obtaining the residue R_MButhat was submitted to Sephadex LH-20 column  chromatography using MeOH as eluent. Fractions obtained were finally purified by RP-HPLC chromatography using different percentage of MeOH-H₂O, yielding two flavonoid glycosides.

Compound 1 obtained as colourless needles. Its ESI-MS gave a molecular peak [M-H]^- atm/z 235, corresponding to the molecular formula C₁₀H₂₀O₆. The ¹³C-NMR spectrum showed 10 resonances of which 4 attributable to an alkyl moiety and 6 due to a sugar residue. The ¹H-NMR spectrum displayed signals of an alkyl moiety with two methyl groups at d 1.24 (3H, d,J=6.0 Hz, H-1), 0.95 (3H, t, H-4), one methylene at d 1.58 (2H, m, H-3), and one methine at d3.15 (1H, m, H-2). The glycosyl anomeric proton at d 4.33 (1H, d, J =8.0Hz) and the other proton signals of the sugar moiety in the spectral region between d 3.31 and 3.88 indicating the presence of a b-d-glucosyl moiety. This structure is further confirmed by its ESI MS spectrum in which there are, besides the molecular peak [M- H]^- at m/z 235, the peak due, to the loss of the 1-methylpropyl moiety [M-H-74]^- at m/z 161. Thus, the structure of 1 was identified as sec-butyl-b-d-glucopyranoside and it is in good agreement with the data reported in the literature [3]. This compound was isolated as major component from Acripeza reticulata Guer. (Orthoptera), and it possesses a bitter taste and may play an important part in defending the insect from aggressors [3].

Compound 2 obtained as colourless solid. The molecular formula of 2 was determined as C₁₅H₂₈O₁₀ from ESI-MS mass spectrometry at m/z 367 [M-H]^-. Comparison between the ¹H-NMR spectrum of 1 and 2 indicated that 2 differed for the presence of an additional anomeric proton resonances at d 5.01 (1H, d, J = 2 Hz, H-1) due to an additional sugar moiety. The ¹H- and ¹³C-NMR spectra revealed the presence of one apiofuranosyl moiety. The b-configuration of the glycosidic linkages was evident from the observed ¹³C-NMR chemical shifts for the anomeric carbon of glucose (d 103.8) and apiose (d 111.0). The downfield shifts of C-6^’ (68.8 ppm) suggested that the interglicosidic linkage is apiofuranosyl (1®6^’) glucopyranoside. Further evidence of the apiofuranosyl moiety was the peak at m/z 235 [M-H-132]^- in the ESI-MS spectrum. Therefore, compound 2 was identified as 2-((b-d-apiofuranosyl-(1®6)-b-d-glucopyranosyl)oxy)butane, which was reported before from Manihot esculenta [4].

Compound 3 obtained as colourless needles. The ¹H-NMR spectrum showed signals for two methyl groups at d 1.20 (3H, d, J=6.0 Hz, H-1) and 1.23 (3H, d, J=6.0 Hz, H-3). Comparison between the NMR data of 1 and 3 indicated that the difference between them was the absence in 3 of the signal of the methylene group at d 30.1 in the ¹³C-NMR spectrum. The sugar moiety was established to be a b-d-glucose (anomeric proton at d 4.33, 1H, d, J=8 Hz, for¹H-NMR and d 102.6 for ¹³C-NMR). These data are in good agreement comparing with those reported for isopropyl-b-d-glucopyranoside, which was isolated from the fruit of Foeniculum vulgare [5].

Compound 4 obtained as white powder. The ESI-MS showed a [M-H]^- peak at m/z 349, corresponding to a molecular formula C₁₆H₃₀O₈. Another prominent fragment due to the loss of a hexose unit was observed at m/z 187 [M-H-162]^-. The ¹H-NMR showed signals resonances of an alkene monosubstituted at d 5.02 (1H, dd, J = 12.0, 3.0Hz, H-8a), 5.20 ppm (1H, dd, J = 16.0, 3.0Hz, H-8b) and 5.92 ppm (1H, dd, J = 16.0, 12.0Hz, H-7). Signals for methyl groups at d1.14 (3H, s, H-1), 1.16 (3H, s, H-10) and 1.25 (3H, s, H-6) and for methine groups at d 2.04 (1H, m, H-3), 1.64 (2H, m, H-4), and 1.50 (2H, m, H-5) were also evident. An anomeric proton at d 4.34 (1H, d, J =8 Hz^’, H-1) and other proton signals of the sugar moiety between d 3.21-3.89 were attributed to the presence of a b-d-glucosyl moiety. The downfield shift of C-6 (90.6 ppm) suggested the glycosilation site. These data are in good agreement with those reported for 2,3-diol-2,6-dimethyl-7-octene-6-O-b-d-glucopyranoside, which was isolated fromSolenostemma argel [6].

Compound 5 obtained as yellow solid. In the ESI-MS spectrum, the quasi molecular peak was observed at m/z 577 [M-H]^-, corresponding to the molecular formula C₂₇H₃₀O₁₄. The ¹H-NMR spectrum displayed signals for two non-coupled aromatic hydrogen at d 6.60 (1H, s, H-3) and 6.30 (1H, s, H-6), two sets of ortho-coupled aromatic protons at d 7.98 (2H, d, J=8 Hz, H-2^’, H-6^’) and 6.94 (2H, d, J=8 Hz, H-3^’, H-5^’). These features are characteristic of a 7,5,4^’- trihydroxyflavone derivative [7]. The ¹H-NMR spectra revealed also two anomeric signals at d5.06 (1H, d, J=10.6 Hz, H-1) and 5.13 (1H, d, J=1.8 Hz, H-1) and one methyl doublet signal atd 0.68 (3H, d, J=6 Hz, H-6). The ¹³C-NMR chemical shifts of the two sugar units are consistent with one glucopyranosyl and one rhamnopyranosyl moiety. The downfield shift of C-8 (105.9 ppm), C-1^’’ (71.9 ppm), and C-2 (81.6 ppm) suggested the interglycosidic linkages and the C-subsitution at C-8 of ring A. Therefore, compound 5 was identified as vitexin 2^’’-O-rhamnopyranoside, which was reported before from Avena sativa [8].

Compound 6 obtained as yellow solid. The ESI-MS spectrum gave a molecular peak [M-H]^- atm/z 593, corresponding to the molecular formula C₂₇H₃₀O₁₅. Characteristic ¹H- and ¹³C-NMR chemical shift values and coupling constant data indicated that the structure was based on isovitexin (apigenin 6-C-b-d-glucopyranoside) [9]. Comparison between the ¹H-NMR and ¹³C-NMR spectra of sugar residues of compounds 5 and 6 indicated that 6 differed from 5 for the presence of a galactopyranosyl moiety instead of a rhamnose unit. Evidence for the C-substitution at carbon 6 of ring A was confirmed by the downfield shift of C-6 (104.4 ppm) in the ¹³C-NMR spectrum. All these data are in good agreement with those reported in the literature for isovitexin 2^’’-O- galactopyranoside, which was isolated from Secale cereale [10].

III. Experimental

3.1. Plant material

The whole herb of D. acuminata was collected in Tamdao National Botanical Garden, Vietnam, in February 2003. A voucher specimen is deposited in the Herbarium of Institute of Natural Products Chemistry, Vietnamese Academy of Science and Technology.

3.2. General experimental procedures

NMR spectra were recorded in CD₃OD using a Bruker AC 200 Spectrospin spectrometer, using TMS as internal standard. ESI-MS spectra were obtained from a Thermo Finnigan LCQ Advantage spectrometer. The following adsorbents were used for purification: TLC normal phase Kieselgel 60 F₂₅₄ (Merck 5554, 0.2 mm), and reversed phase Si gel RP18 F₂₅₄ (Merck 5559, 0.2 mm), CC: normal phase Si gel (Merck, 0.063-0.200 mm), reversed phase Si gel (LiChroprep RP-18, Merck 25-40 mm), RP-HPLC on a C-18 m-Bondapak column (30 x 7.8 mm, flow rate 2.0-2.5 ml/min), Pharmacia Fine  Chemicals Sephadex LH-20. The TLC chromatogram were visualized under UV at 254 and 366 nm and sprayed with solution of Ce(SO₄)₂ in H₂SO₄ 65% and NTS/PEG. All fractions were monitored by TLC with n-BuOH-CH₃COOH-H₂O (60:15:25) and CHCl₃-MeOH-H₂O (80:18:2) as eluents.

3.3. Extraction and isolation

4 kg of powdered dried whole herb of D. acu-minata were extracted three times with n-hexane, CHCl₃, CHCl₃-MeOH 9:1, and MeOH at room temperature. After removal of the solvents the residues R_H (400g), R_C (150g), R_CM (120g), and R_M (170g) were obtained.

R_CM was chromatographed on Sephadex LH-20 eluting with MeOH (fractions A-N). Fractions B and F were separately chromatographed on SPE RP-18 eluting with MeOH-H₂O (2:8) for fraction B and MeOH-H₂O (9:1) from fraction F, giving fractions B₁-B₁₆ and F₁-F₁₈, respectively. Fractions F₄ was chromatographed on RP-HPLC with MeOH-H₂O 15:85 as eluent, flow rate 2.5ml/min, giving compounds 1 and 2. Compounds 3 and 4 were obtained similarly from fractions F₂ with MeOH-H₂O 1:9 as eluent. R_M was partitioned with n-BuOH and water. After removal of the solvent, the residue R_MBu (10g) was chromatographed on Sephadex LH-20, with MeOH as eluent, giving fractions O-R. Fraction R was chromatographed on SPE RP-18 eluting with MeOH-H₂O (9:1) giving fractions R₁-R₁₈. Compounds 5 and 6were obtained from fraction R_2-4 by using RP-HPLC with MeOH-H₂O 35:65, flow rate 2.0 ml/min.

Compound 1:

Colourless needles (12 mg), mp.123-124^°C, C₁₀H₂₀O₆, ESI-MS m/z 235 [M-H]^-, 161 [M-H-74]^-. ¹H-NMR (CD₃OD): d (ppm) 0.95 (3H, t, H-4), 1.24 (3H, d, J=6.0 Hz, H-1), 1.56 (2H, m, H-3), 3.67 (1H, dd, J=12.0 and 5.0 Hz, H-6^’a), 3.75 (1H, m, H-2), 3.88 (1H, dd, J=12.0 and 3.0 Hz, H-6^’b), 4.33 (1H, d, J=8.0 Hz, H-1^’). ¹³C-NMR: d (ppm) 9.9 (C-4), 21.3 (C-1), 30.1 (C-3), 62.9 (C-6^’), 71.8 (C-4^’), 75.4 (C-2^’), 77.8 (C-5^’), 78.2 (C-3^’), 78.6 (C-2), 103.8 (C-1^’).

Compound 2:

Colourless solid (10 mg), mp. 114-115^°C, [a]_D²⁵ -64.9^° (c 0.09, H₂O),  C₁₅H₂₈O₁₀, ESI-MSm/z 367 [M-H]^-, 235 [M-H-132]^-. ¹H-NMR (CD₃OD): d (ppm) 0.95 (3H, t, H-4), 1.24 (3H, d,J=6.0 Hz, H-1), 1.47 (1H, m, H-3a), 1.61 (1H, m, H-3b), 3.58 (2H, s, H-5^’’), 3.72 (1H, m, H-2), 4.33 (1H, d, J = 8.0 Hz, H-1^’), 5.01 (1H, d, J=2.0 Hz, H-1^’’). ¹³C- NMR: d (ppm): 10.0 (C-4), 21.4 (C-1), 30.3 (C-3), 65.8 (C-5^’’), 68.8 (C-6^’), 71.9 (C-4^’), 75.0 (C-4^’’), 75.4 (C-2^’), 76.8 (C-2^’’), 78.1 (C-5^’), 78.2 (C-3^’), 78.3 (C-2), 79.0 (C-3^’’), 103.9 (C-1^’), 111.0 (C-1^’’).

Compound 3:

Colourless needles (15 mg), mp. 129-130^°C, [a]_D²³ -35.9^° (c 1.1, MeOH),  C₉H₁₈O₆, ESI-MS m/z 221 [M-H]^-. ¹H-NMR (CD₃OD): d (ppm) 1.20 (3H, d, J=6.0 Hz, H-1), 1.23 (3H, d,J=6.0 Hz, H-3), 3.67 (1H, dd, J=12.0 and 5.0 Hz, H-6^’a), 3.87 (1H, dd, J=12.0 and 3.5 Hz, H-6^’b), 4.04 (1H, m, H-2), 4.33 (1H, d, J=8.0 Hz, H-1^’). ¹³C-NMR: d (ppm) 22.1 (C-3), 23.8 (C-1), 62.9 (C-6^’), 71.7 (C-4^’), 72.6 (C-2), 75.2 (C-2^’), 77.9 (C-3^’), 78.5 (C-5^’), 102.6 (C-1^’).

Compound 4:

White powder (8.0 mg), [a]_D²⁰ -7.9^° (c 4.0, MeOH),  C₁₆H₃₀O₈, ESI-MS m/z 699 [2M-H]^-, 249 [M-H]^-, 187 [M-H-162]^-. ¹H-NMR (CD₃OD): d (ppm) 1.14 (3H, s, H-1), 1.16 (3H, s, H-9), 1.25 (3H, s, H-10), 3.66 (1H, dd, J=12.0 and 5.0Hz, H-6^’a), 3.68 (1H, m, H-3), 3.87 (1H, dd,J=12.0 and 3.0 Hz, H-6^’b), 4.34 (1H, d, J = 8.0 Hz, H-1^’), 5.02 (1H, dd, J=12.0, 3.0 Hz, H-8a), 5.20 (1H, dd, J=16.0, 3.0Hz, H-8b), 5.92 (1H, dd, J=16.0, 12.0Hz, H-7). ¹³C-NMR: d (ppm) 24.6 (C-4), 26.6 (C-9), 27.2 (C-1), 28.0 (C-10), 39.5 (C-5), 62.6 (C-6^’), 71.6 (C-4^’), 73.7 (C-2), 75.5 (C-2^’), 77.9 (C-3), 78.0 (C-5^’), 78.1 (C-3^’), 90.6 (C-6), 105.2 (C-1^’), 112.0 (C-8), 146.6 (C-7).

Compound 5:

Yellow solid (7.0 mg), mp. 206-207^°C, C₂₇H₃₀O₁₄, ESI-MS m/z 577 [M-H]^-. ¹H-NMR (CD₃OD): d (ppm) 0.68 (3H, d, J=6.0 Hz, CH₃-6^’’’), 2.48 (1H, m, H-5^’’’), 3.14 (1H, t, J=9.5 Hz, H-4^’’’), 3.42 (1H, dd, J=9.5, 2.5 Hz, H-3^’’’), 3.46 (1H, m, H-5^’’), 3.65 (1H, t, J=9.0 Hz, H-3^’’), 3.66 (1H,t, J=9.0 Hz, H-4^’’), 3.79 (1H, dd, J=12.0 and 5.0 Hz, H-6^’’b), 3.87 (1H, dd, J=2.5, 1.5 Hz, H-2^’’’), 3.98 (1H, dd, J=12.0, 3.0Hz, H-6^’’a), 4.28 (1H, dd, J=9.0, 7.5 Hz, H-2^’’), 5.06 (1H, d, J=7.5 Hz, H-1^’’), 5.13 (1H, d, J =1.5 Hz, H-1^’’’), 6.30 (1H, s, H-6), 6.60 (1H, s, H-3), 6.94 (2H, d, J=8.0 Hz, H-3^’, H-5^’), 7.98 (2H, d, J=8.0 Hz, H- 2^’, H-6^’). ¹³C NMR: d (ppm) 18.1 (C-6^’’’), 63.0 (C-6^’’), 70.0 (C-5^’’’), 71.9 (C-1), 72.2 (C-2), 72.4 (C-4), 73.5 (C-4), 73.7 (C-3), 78.1 (C-3^’’), 81.6 (C-2^’’), 82.9 (C-5^’’), 99.8 (C-6), 102.4 (C- 1^’’’), 103.6 (C-3), 105.7 (C-10), 105.9 (C-8), 117.0 (C-3^’, C-5^’), 123.5 (C-1^’), 130.1 (C-2^’, C-6^’), 157.9 (C-9), 162.7 (C-4^’), 163.2 (C-5), 164.2 (C-7), 166.7 (C-2), 184.1 (C-4).

Compound 6:

Yellow solid (9.0 mg), C₂₇H₃₀O₁₅, ESI MS m/z 593 [M-H]^-, 473 [M-H-120]^-, 431 [M-H-162]^-. ¹H- NMR (CD₃OD): d (ppm) 3.78 (1H, dd, J=12.0 and 5.0 Hz, H-6^’’a), 3.97 (1H, dd,J=12.0 and 3.0 Hz, H-6^’’b), 4.21 (1H, dd, J=8.5, 7.5 Hz, H-2^’’), 4.95 (1H, d, J=7.5 Hz, H-1^’’), 5.11 (1H, d, J=7.0 Hz, H-1^’’’), 6.71 (1H, s, H-8), 6.72 (1H, s, H-3), 6.96 (2H, d, J=8.0 Hz, H-3^’, H-5^’), 8.04 (2H, d, J=8.0 Hz, H-2^’, H-6^’). ¹³C-NMR: d (ppm) 61.8 (C-6^’’’), 62.7 (C-6^’’), 71.3 (C-4^’’’), 71.4 (C-1^’’), 72.8 (C-4^’’), 74.6 (C-2^’’’), 75.1 (C-3^’’’), 77.6 (C-5^’’’), 78.5 (C-3^’’), 80.3 (C-2^’’), 82.3 (C-5^’’), 95.3 (C-8), 102.7 (C- 3), 104.3 (C-1^’’’), 104.4 (C-6), 104.5 (C-10), 117.2 (C-3^’, C-5^’), 122.8 (C-1^’), 129.7 (C-2^’, C-6^’), 156.4 (C-9), 160.5 (C-5), 160.7 (C-4^’), 162.3 (C-7), 166.7 (C-2), 184.1 (C-4).

Acknowledgments

This work was supported by the Second Vietnamese - Italian Program of Cooperation in S&T (2002-2005).