A sinapic acid ester from Boreava orientalis

Pergamon

~3~-~2~93)~7-0

Pkyrochemistry, Vol. 35, No. 6, pp, 1481-1484, 1994 Copyright Q 1994 Ekwier S&u% Ltd Pnntcd in Great Britain. Ail rights IWWXI 0031~9422/W WCWO.00

A SINAPIC ACID ESTER FROM BORLEAJ4 ORZENTALZS AKIYO SAKUSHIMA,MAKSUT CO$KUN,* MEKIN TANKER* and NEVIN TANKER* Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobatsu, Hokkaido, 061-02, Japan; *Department of Pha~acognosy and Pharma~nti~ Botany, Faculty of Pharmacy, University of Ankara, Ankara, Turkey (Received 11 August 1993) Key Word Index-Boreoa

disinapoyl)glucopyranose;

orient&; Cruciferae; 6-O-fl-D-(2’-O-sinapoyl)ghtcopyranosyl sinapic acid ester; gentiobiose ester, structural elucidation.

fl-~-(1,2-O-

Abstract-A new sinapic acid ester, 6-O-/I-D-(2’-O-sinapoyl)glucopyranosyl fi-D.(l,2-@disinapoyl)glucopyranose, was identified from fruits of Boreauu orientah Structural elucidation was carried out on basis of UV, mass, ‘H and ’ 3C NMR spectral data, including 2D shift correlation and selective INEPT experiments.

Boreava orient&s (Cruciferae) is a herb widely distributed in Turkey. The fruits of this plant are used in traditional medicine against coughs and also in skin disease [l]. Previously, protein, fatty acids and glucosinolates were reported from the fruits of the plant [l, 23. From the methanol extract of the fruits of this plant, we isolated a new gentiobiose derivative. A considerable number of oligosaccharides conjugated with hydroxycinnamoyl groups have been found to occur in many higher plants [3], some of which have pha~acolo~c~ and biological activities [4]. The occurrence of conjugated substances containing sucrose as the core sugar, however, is limited to several groups of plants, e.g. the Polygonaceae [S], Polygalaceae [6] and Cruciferae [7].

OR’

1

R’

Ra

sinapoyi

H

XP sinapoyl

Ac

was identified by GC with an authentic sample, however, the aglycone decomposed. Basic hydrolysis of 1 with 0.5 The dried fruits of B. orientalis were successively ex- M NaOH afforded sinapic acid. A disaccharide, identical to gentiobiose by chromatographic behaviour and staintracted with petroleum and methanol. The methanol ing, was found in the aqueous layer. extract was treated as described in the Experimental. The The molecular weight of 1 was 960amu as shown by n-butanol and water extracts were subjected to column FAB-mass spectrum in negative ion mode of 1 (a quasichromatography over gel filtration on Sephadex LH-20 molecular ion at m/z 959 [M-H] -). The fragment ions and G-10 with water and methanol gradient as a solvent. 547 [M-H-412]and 341 Compound 1 was rechromato~aph~ on Sephadex LH- at m/z 753 [M-H-206-, which were due to the loss of sinapoyl 20 to give a pale yellow amorphous powder, C&H,, O,, , [M-H-618]-, from HR-FAB-mass spectrum, mp 155-158”, exhibiting a moieties [lo], were indicative of three sinapoyl units. The fragment pattern was similar to that of a-~-(3-0positive ferric chloride reaction. a-D-(4-O-acetyl-6-O-sinapoyl) The infrared spectrum of 1 suggested the presence of sinapoyl)fructofuranosyl hydroxyl(3408 cm-‘), conjugated ester (1710 cm-‘), o&/J- glucopyranose [8,9], whereas absence of fragment ions at unsaturated acid (1632cm-‘) and aromatic ring (1606 m/z 797 [M- 162]- and 779 [H- 178]-, which would correspond to the elimination of a hexose residu% suggesand 1516 cn-‘). The UV spectrum of 1 showed absorpted esteritication of sinapoyl groups on each of tion maxima at 238 and 328 nm. The bathochromic shifts two hexosyl units. The peak appearing at m/z 591 w of the absorption maxima on the addition of NaOMe -206-162]was due to the elimination of a hexose unit were similar to those of a-D-(3-O-sinapoyl)fructofurwith a sinapoyl moiety. It was supported by the presence a-D~4-~-a~tyi-6-~-~na~yI)~~opyranoside anosyi of m/z 537 of peraeetate of 1, which corresponds to the [8,9]. Gn acidic hydro~ys~ of I with 3% HCl, ~-glucose RESULTSAND DISCUSSION

1481

A. SAKUSHIMA et al.

1482

elimination of peracetylated terminal hexose with a sinapoyl moiety. The ester locations in the sugar system were determined by the detailed analysis of ‘H and 13CNMR spectra including ‘H-‘H COSY, ‘?-‘H COSY, DEPT and selective INEPT experiments. Assignments of proton and carbon signals were achieved by combination of ‘H-‘HCOSY and ‘3C-1HCOSY. The ‘HNMR spectrum of 1 showed the presence of the typical proton signals of three sinapoyl moieties, which were six aromatic methoxyls, a singlet equivalent to six aromatic protons and an olefinic proton attributable to three E double bonds (Table 1). Coupling constants and chemical shifts of the remaining signals were similar to those reported for gentiobiose. However, the signals attributed to H-2 and H-2’ of glucose core were shifted downfield ca l-2.0 ppm when compared with gentiobiose, as expected for esterification [ll]. Chemical shifts of the 13CNMR spectrum were in good agreement with values estimated for gentiobiose esterified with sinapoyl groups at C-l, C-2 and C-2’ (Table 2) [l 1, 121. Therefore, 1 is 6-0-D-~-(2’-@ sinapoyl)glucopyranosyl B-D-(1,2-O-disinapoyl)glucopyranose, a new natural product.

EXPERIMENTAL

‘HNMR spectra were run on a 400 MHz instrument and 13CNMR spectra on 100 MHz instrument in

CD,OD. Chemical shifts are given in 6 relative to TMS as int. standard. EI-MS were obtained by direct inlet, electron energy 70 eV, ion source temp. 320” and FABMS of negative ion mode were measured using Xe, ion gun at 7 kV and glycerol as matrix. MeOH solns of the sample were injected into a Shimadzu 6 AD HPLC instrument provided with a 250 x 4 mm i.d. Nucleosil5 Cis column(Nomura Chemicals). UV detector was equipped with a 330nm filter. H,O-MeOH-AcOH(2100: 1050:140, solvent A) was used as the solvent system. The flow-rate was 1 ml min- ’ and pressure drop 95 kgcme2. GC was carried out on Shimazdu GC-6 AM machine equipped with a hydrogen flame ionization detector. Material. Plants of B. orientalis were collected near Ankara in 1990. A voucher specimen is retained in the Ankara oniversitesi Eczacilik Fakiiltesi herbaryumu(AEF). Isolation. Dried fruits (1.0 kg) were extracted with petrol and then MeOH. The methanolic extract was dissolved in H,O and extracted successively with Et,O, CHCI,, EtOAc and n-BuOH. The n-BuOH and H,O extracts (3 and 18 g) were subjected to CC over Sephadex LH-20 with H,O and MeOH gradient as a solvent. The fr. eluted with 30% aq. MeOH was rechromatographed using Sephadex LH-20 and G-10 with H,O-MeOH gradient. The high polarity fr. containing 1 was rechromatographed using Sephadex G-10 and purified by rechromatography to give 60 mg of 1.

Table 1. ‘H NMR (400 MHz, CD,OD) spectral data of 1 H

Gentiobiose*

1

glc-1 glo2 glc-3 gIc-4 glc-5 glc-6a b

4.57 (d, J = 8.3) 3.19 (d, d, J=8.3,9.0) 3.40-3.44 (tunes.) 3.68-3.70 (tunes.) 3.54 (unres.) 3.77 (d, J = 11.2) 4.17(d,.J=11.2) 4.42 (d, J = 7.8) 3.26 (d, d, J = 7.8) 3.41-3.46 (unres.) 3.40-3.47 (urines.) 3.30-3.35 (unres.) 3.68 (d, J = 12.2) 3.87 (d, .I = 12.2)

5.78 (d, J = 8.3) 5.04 (d, d, J = 9.3) 3.63-3.73 3.85-3.91 (tunes.) 3.41-3.46 (unres.) 3.79 (mires.) 4.16(d,J=11.2) 4.72 (d, J = 7.8) 4.86 (d, d, J = 9.3) 3.80-3.91 (unres.) 3.41-3.43 (unres.) 3.29-3.34 (unres.) 3.87 (unres.) 3.64 (unres.)

;::t glc-3’ $:;J glc-6’a b 10, l”‘, 1”” 2”, 2”‘, 2”” 3”, 3”‘, 3”” 4” 4”’ 4”” 5U:5”‘: 511!1 6”, 6”‘, 6”” 7”17”’?7”” 8”98”’I 8”” OMe

6.86 (s), 6.74 (s), 6.69 (s) -

6.86 (s), 6.74 (s), 6.69 (s) 7.67(d,J=15.9), 7.51 (d,J=15.6) 6.5O(d,J=16.1), 6.31 (d,.J=15.6) 6.17 (d, J= 16.1) 3.78 (s), 3.77 (s)

AI1 assignments were made by 2D COSY experiments. In parentheses coupling constant (J) values in I-Ix. *Measured in CD,OD + D,O (1: 1).

A sinapic acid ester from Boreava orientalis Table 2. 13C NMR spectral data of 1 and reference compounds (400 MHz, CD,OD, d-value) Gentiobiose (Reference value) Carbon 1 2 3 4 5 6 1’ 2 3 4’ 5 6

98.0 75.9 77.7 71.4 76.8 70.0 104.4 74.9 77.6 71.1 77.4 62.5

(97.2) (75.5) (77.1) (71.7) (76.1) (70.2) (103.8) (74.5) (77.1) (71.1) (77.1) (62.5)

1 (ppm)

A

DEF’T

93.8 74.2 75.8 71.3 78.0 69.4 102.7 75.2 76.2 71.6 78.8 62.6

-4.2 -1.7 -1.9 -0.1 +1.2 -0.6 -1.7 +0.3 -1.4 +0.5 +1.4 +O.l

CH CH CH CH CH CH, CH CH CH CH CH CH,

Sinapic acid

1

1”9 1”’, 1”” 2” I 2”’, 2”” 6”6”‘6”” 3”: 3”‘: 3”” 50, 5”‘, 5””

126.9 107.0 149.7 _

126.8, 126.4, 126.2 107.0, 106.9, 106.9 -

4” , 4”’ , 4”” 7” 7”’ 7”” 8”’ 8”” 8”” y 9”’ 9””

139.7 147.3 116.6 171.1 57.0

139.8, 148.9, 116.1, 168.5, 56.8

Oicle

’

149.2 139.5, 147.8, 115.3, 168.1,

139.3 147.5 114.4 166.8

*Measured in CD,OD + D,O (1: 1).

6-0-P-D-(2’-O-Sinapoyl)8lucopyranosyl

B-D-(1,2-G-&powder, mp 155158”, HR-NI-FAB-MS m/z: 959.28449 [M required 959.28211. TLC [silica -HI- %J%I% gel, EtOAc-MeCOEt-HCGGH_C,H6H,O (4: 3: 1: 12, upper layer; system A)], R, 0.38. HPLC (solvent A): R, (min) = 13.82. UV JMeoHnm (E):226, 238 (40 750), 328 (44 102), + MeONa; 238,265 (sh), 334,399. NI-FAB-MS ni/z: 959 [M=(C,,H,,O,,)-HI-, 753 [M-H-206]-, 735 [M-H -sinapic acid]-, 547 [M-H -206 -sinapic acid]-, 341 [M-H-206x 2 -sinapic acid]-, 305 [M -H - 206 - sinapic acid x 2]-. PI-FAB-MS m/z: 737 [M+H-sinapic acid]+, 207 [CirHirOJ+. ‘H and r3CNMR data: Tables 1 and 2. Basic hydrolysis of 1. Compound l(4 mg) was stirred with 0.5 M NaOH (4 ml) at room temp. for 24 hr. The reaction mixt. was adjusted to pH 6.0 with dilute HCl and then extracted with EtOAc. The EtOAc extract was washed with Hz0 and then evapd to dryness in vacua. The extract was subjected to CC on silica gel and afforded sinapic acid. The pale brown powder cochromatographed with an authentic sample of sinapic acid by HPLC: R&in); 8.41 (solvent A). IR VPB’cm- ‘: 3380 (OH), 2928 (CH), 1666 (C=G), 1626 (C=(J), 1518 (aromatic C=C), 1468 (aromatic C=C), 1434, 1388 (OMe), 1298, 1270, sinapoyl)glucopyranose (1). Pale yellow amorphous

1483

1226 1122 (C-O) UV Au*” nm: 328. ‘HNMR (in CD&,,: 67.65 (H, h, J = 16.0 Hz, -CI+CH-CO-), 6.77 (2H, s, aromatic 2, H-5), 6.29 (lH, d, J= 16.0 Hz, -CH =CIJ-CO-). EI-MS m/z (%): 224[M=(C,,H,,O,)]+ (lOO), 209 [M-Me]+(30.0), 193 [M-OMe]+ (2.4), 181 [M-Me-CO]+ (14.4), 163 (13.8). The aq. layer was evapd to dryness in uacuo. Sugars were then extracted with pyridine and gentiobiose was identified by co-TLC [silica gel, EtOAc-MeOH-HzO-HOAc (65 : 15: 15 : 20), R, 0.26 (sucrose 0.3511 and GC {R,(min); 6.36 [TMSi derivatives of gentiobiose, (sucrose R,; 3.45 )], 1.5% SE-52 on Chromsorb-W(60-80 mesh), N, flow rate 60 ml mitt-‘, column temp. 260”, a glass column (2 m x 3 mm i.d.)} with an authentic sample. Detection of TLC was sprayed with 10% H,SO, and then heated in the oven. Acidic hydrolysis ofl. Compound l(1 mg) was refluxed with 3% HCl(3 ml) for 2 hr. The reaction mixt. was extracted with EtOAC. The aq. layer was treated as described above. Glucose was identified by GC. GC R,(min); 13.5, 14.7 DMSi derivatives of D-glucose, 3% OV-17 on Shimalite-W(80-100 mesh), column temp. 150-220” (3” min- ‘), Nz(30 ml min- ‘), inj. and det. temp. 280’=]. Acetylation of 6-o-fi-D-(2’-O-sinapoyo glucopyranosyl P-D-(1,2-O-disinupoy~)glucopyranose (1). Compound 1

(5 mg) was treated with AczO and pyridine. The product was purified by CC on silica gel to give the peracetylated derivative la, mp ill-115”, as a powder. IR vKBrcm-i: 2924 (CI-I), 1758 (-COO-), 1634 (C=C), 1598(aromatic C==C),1552 (aromatic C=C), 1464 (aromatic C==C), 1372 (OMe), 1234 (C-O), 1134 (C-O). ‘HNMR (in CD&): Gaglycons moiety 7.69,7.60,7.53 (3H each, d, J = 16.0 Hz, -CI+CH-CO), 6.85, 6.74, 6.69 (3H each, s, H-2, 2, 2”, 6, 6’ and 6’1, 6.48, 6.27, 6.24 (3H each, d, 5=16.0Hz, -CH=CIJ-CO), 3.80, 3.81, 3.83 (9H, s, phenohc-Ohle), 2.29, 2.30, 2.31 (9H, s, phenolic-&CO). Sugar moiety: 5.86, 4.66 (2H, d, J=7.8Hz, glc-1 and H-l’), 5.27, 5.13 (2H, m, glc-2 and H-2’), 5.10-5.01 (4H, m, glc-3,4, 3’ and H-4’), 3.70-3.67, 3.81-3.78 (2H, m, glc-5 and H-5’), 4.27-4.22,4.14-4.12, 3943.92, 3.70-3.67 (4H, m, glc-6,,, and H-6, & 1.86, 1.96, 1.98,2.02, 2.08 (15H, s, alcoholicmC0). i3C NMR (in CDCl,): 6aglycone moiety 130.9, 130.8, 130.5 (C-l, 1’ and l”), 105.0, 104.9 (C-2,2’, 2”, 6, 6 and 6”), 152.5, 152.4 (C-3, 3’, 3”, 5, 5’ and 5”), 132.7, 132.1, 132.1 (C-4, 4, 4”), 147.1, 146.3, 145.8 (C-7, 7’ and 7”), 117.5, 116.5 (C-8, 8’, 8”), 165.3, 165.0, 164.4 (C-9, 9’ and 9”). Sugar moiety: 100.9, 91.9 (glc-1, l’), 70.3, 72.5 (glc-2, 2’), 72.5,70.9 &k-3,3’), 68.5,68.6 &k-4,4’), 71.9,74.7 (glc5, S), 61.8, 67.4 (glc-6, 6’) [gentiobiose peracetate: 100.6, 91.6,73.9,72.9,72.7,71.9,70.9,70.3,68.5,68.4,67.5,61.9]. MeCO: 170.7, 170.3, 170.1, 169.5, 169.4, 168.3, 20.7, 20.6, 20.6,20.4. EI-MS m/z(%): 1047 [M -C,,H,,O,(sinapoyl moiety)] + (LO), 903 [M-C,,H,,O,-2 xC,H,O -MeCOOHl+ (6.4), 787, 680, 621 (2.6), 555 (2.2X 537 [Cz5Hz90i3 (terminal sugar moiety)]+ (3.4), 512 (13.3), 494 [C,,H,,0i3-MeCO]+ (11.3), 249 [Cr3Hr305 (acetylated sinapoyl moiety)]+ (64.7), 207 [Cr3Hi305 -CzHzO]+ (lOO.O), 169 [CsH,04 (sugar moiety)]’ (49.6), 137, 109 (54.8).

A. SAKUSHIMA

1484

Acknowledgement-The

authors thank Prof. Dr Sansei Nishibe for his support and encouragement in the preparation of this manuscript. REFERENCES 1.

Tanker, M. and Yenen, M. (1978) Ankara Ecz. FaK.

Met. 8, 1. 2. Tanker, M., Et-tan, M., Coskun, M., Sariseker, N. and Yurdesin, T. (1983) Doga Bilim Dergisi. Clit. 7, 99. 3. Lahlaub, M. F., Gross, G. A., Sticher, O., Winkler, T. and Schulten, H. R. (1986) Planta Med. 52, 352. 4. Sato, T., Kozima, S. and Kobayashi, K. (1985) Yakugaku Zasshi 105, 1131.

5. Fufuyama, Y., Sato, T., Miura, I., Asakawa, Y. and Takemoto, T. (1983) Phytochemistry 22, 549.

et al.

6. Hamburger, M. and Hostettmann, K. (1985) Phytochemistry 24, 1793. 7. Linscheid, M., Wendisch, D. and Strack, D. (1980) 2. Naturforsch. 35C, 907. 8. Bashir, A., Hamburger, M., Mosonthi, J. D. and Hostettmann, K. (1993) Phytochemistry 32, 741. 9. Tommasi, N. D., Piacente, S., Simone, F. D. and Pizza, C. (1993) J. Nat. Prod. 56, 134. 10. Sakushima, A., Hisada, S., Nishibe, S. and Brandenberger, H. (1985) Phytochemistry 24, 325. 11. Yoshimoto, W., Itatami, Y. and Tuda, Y. (1980) Chem. Pharm. Bull. 28, 2065. 12. Usi, T., Yamaoka, N., Matsuda, K., Tuzimura, K., Sugiyama, H. and Seto, S. (1973) J.C.S. Perkin Trans I 2425.