A triterpenoid glycoside from Menyanthes trifoliata

Phytochemistry, Vol. 29, No. 12,pp. 3885-3887,1990 Printed in Great Britain.

0031-9422/90 $3.00+ 0.00 Pergamon Press plc

A TRITERPENOID GLYCOSIDE FROM MEN YANTHES ZBIGNIEW

JANECZKO,

J~ZEF

SENDRA,KRZYSZTOF KMIEC and

CARL

TRIFOLIATA

HEINZ

BRIESKORN*

Department of Pharmacognosy, Medical Academy of Krakcw, 10 Skaleezna str., 31-065 Krakbw, Poland; lInstitut fiir Pharmazie und Lebensmittelchemie der Universitlt am Hubland, D 8700 Wiirzburg, F.R.G. (Received in revised form 17 April 1990)

Key Word Index--Menyanthes trifoliata; Menyanthaceae; rhizomes; structure determination; betulinic acid; triterpenoid saponins; menyanthoside.

Abstract-The

structure of menyanthoside, one of the main saponin of Menyanthes trifoliata established to be 3-0-C /I-D-galactopyranosyl( 1~4)~-D-~ucuronopyranosyl]-28-~-[~-D-apiofuranosyl( glucopyranosyl]betulinic acid.

rhizomes, was 1+6)/?-D-

INTRODUCTION

Menyunthes

trifoliata (‘marsh trefoil’) is indigenous to Central and Eastern Europe, North America and Japan. Leaves of this plant have been reported to contain iridoids, secoiridoids, flavonoids, phenolic acids and catechins Cl], triterpenoids [Z], pectin and phytosterols [3], saponins [4] and alkaloids [5]. Rhizomes and roots contain pectins and inulin [6], as well as bitter glycosides which stimulate the appetite and increase the flow of digestive secretions. The plant is sometimes used as a bitter principle in digestive tonics and appetizers used in the treatment of various types of atonic dyspepsias [6]. Preliminary examination of the rhizomes showed the presence of triterpenoids giving positive tests for saponins. This paper deals with the isolation and the elucidation of the structure of menyanthoside (1). RESULTS AND DISCUSSION

Compound 1 exhibited a quasi-molecular ion at m/z 1088 in its FAB mass spectrum, indicating a M, of 1089, as well as signals at m/z 794, 63 1 and 455 corresponding to the sequential losses of a pento-hexosyl disaccharide and of hexosyl and uranic acid units. Broad and strong IR absorption bands at 1735 and 1610cm-’ suggested that 1 possesses an esterified carboxylic group and a free carboxylic function. The ‘H NMR spectrum of 1 showed two signals at 64.58 and 4.70 which can be attributed to the protons of the exocyclic methylene group as well as a three proton singlet at 61.64 corresponding to an isopropenylmethyl group in the aglycone moiety. The i3CNMR spectrum showed 53 carbon signals (Table 1). Acid hydrolysis of 1 yielded betulinic acid (mp, IR, MS and ‘H NMR) and galactose, glucose, apiose and glucuronic acid (GC) in the molar ratio 1: 1: 1: 1 [7]. The identity of the aglycone and of the sugars were confirmed by HPTLC [8]. Hydrolysis of 1 with 0.5 M NaOH followed by three days exposure of the product to acidified n-butanol afforded prosapogenin (2). The FAB mass spectrum of 2

1

R’ Gal

2

Gal

R2 Gal Bu

R’ Glc(6-1)Apiose H

showed a [M]’ at m/z 850 (M, 851) and an ion at m/z 455 corresponding to the elimination of one hexosyl, one uranic acid and an n-butyl group. The “CNMR spectrum showed 46 carbon signals (Table 1). The sugars obtained from the hydrolysate of 2 were galactose and glucuronic acid [7,8]. The presence of an n-butyl group was confirmed by 13C NMR analysis which revealed four carbon signals at 614.06, 20.07, 31.6 and 66.08. In addition the ‘HNMR spectrum now contained a triplet at 64.18 attributed to OCH, of the n-butyl chain and a triplet at 60.99 connected with protons of the terminal methyl group. The carboxyl carbon signals at 6170.6 and 179.9 suggested that one of them must be esterified by the n-butyl group and the second one must be free. Acid hydrolysis on HPTLC plates [8] of 2 methylated with diazomethane led to the identification of betulinic acid methyl ester. Hence the n-butyl group can be attached only to the carboxyl of glucuronic acid. The above results proved that 1 possesses a bisdesmosidic structure with two sugar units: galactosylo-glucuronic acid at position C-3 (“C NMR signal at 687.5) and glucosylo-apiose esterifield at position C-28 of betulinic acid. The last result was supported by the ‘HNMR analysis which revealed the

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Triterpenoid from Menyanthes trifoliata 106O(OHk ‘H NMR (DMSO): b0.72,0.77,0.84,0.93 (each s, 3H), 1.64 (3H, s, Me-29), 3-3.8 (sugar protons) 4.25 (lHd, J=7 Hz, anomeric proton of galactose), 4.32 (lH, d, J=7 Hz, anomeric proton of glucuronic acid) 4.58 (J = 1 Hz H,-30), 4.70 (lH, s, Hi,30), 4.82 (IH, d, J=4 Hz, anomeric proton of apiose), 5.36 (lH, d; J=8 Hz, anomeric proton of glucose); isC NMR (DMSO): see Table 1. FABMS (negative ion mode) (rel. int.) m/z [M-H](49), 794 [M - H - hex.-pent] - (27), 631 [M - H - bex.-pent.hex] - (28) and 455 [M - H - hex.-pent.-hex.-glucur. acid] - (32). Acid hydrolysis of 1. Compound (50 mg) 1 was refluxed with 5% methanolic HCl for 5 hr. After addition of H,O and filtration, the ppt. was purified by silica gel CC (solvent, b) to give crystals of aglycone (20 mg), which was identified on the basis of its physical and spectral data: mp 275-278”, [a]ioP’+ 10 (CHCI,). IR~~~crx-‘: 3425 (OH), 1690 (CO,H), 1635 (C=CH,), 880 (CMe=CH,); ‘HNMR (CDCI,): 60.75, 0.82, 0.93, 0.96, 0.97 (each3Hs,Mex5),1.69(3H,s,Me),3.18(1H,t,J=6,4Hz,H-3), 4.60,4.73 (each lH, two protons of methylene); MS 70eV, m/z (rel. int.): 456 EM]’ (42), 438 [M - H,O] + (20), 411 [M -CO,H]+ (lo), 248 [M-207-H]+ (44), 207 [M-248-H]+ (52), 189 [M-H,O-248-H]+ (100). Acetate: mp 27s282”, IR vzicm-‘: 1720 (CO,R), 1640 (C=CH,), 1250 (OAc), 880 (CMe =CH2), no OH band; MS 70 eV m/z (rel. int.): 498 [M]’ (8), 438 [M - HOAc] + (42). 189 [M - HOAc- H - 248]+ (82), 43 [AC] + (100). Me ester of acetate: mp 200-203”, IR v~:cm-‘: 1730 (CO,R), 1640 (C==CH,), 885 (CMe= CH,), 1252 (OAc) no OH band; MS 70eV, m/z (rel. int.): 512 [M]’ (4), 452 [M -CO,Me]+ (20), 497 [M-Me]+ (2), 399 [M-CO,Me-Ac -HI+ (8), 189 [M-CO,Me-AC-249]+ (84), 43 [AC]’ (100). The filtrate was neutralized with Dowex 1 ( x 4 OH form), evapd to dryness, silylated and analysed by GC. The following monosaccharides were identified: glucuronic acid, glucose, galactose, apiose in a molar ratio 1: 1: 1: 1 [7]. Alkaline hydrolysis ofl. Compound l(50 mg) was refluxed in 10 ml 0.5 aq. NaOH for 2 hr. The reaction mixture was adjusted to pH 6 with 2 M aq. HCI and then extracted with n-BuOH. The acidic BuOH phase was left aside for 3 days at room temp. TLC analysis of the organic phase (solvent a) showed spots at R, 0.8 (2) and 0.58 (1). Compound 2 was obtained in a pure state after CC on silica gel (solvent a), mp 240-243”, [a]:“” - 2” (MeOH; c 1). IRv;;cn-i: 3420 (OH), 1730 (CO,R), 1690 (CO,H), 1640 (CH=CH,) 890 (isopropenyl); ‘H NMR (200 MHz, MeOH): 60.83, 0.85, 0.90, 0.94, 0.95, (3H, each s, Me x 5), 0.91 (3H, t,
this manner were placed in a closed chamber which was filled with dry HCl. The chamber was then heated on the water bath at 80” for 1 hr. The plates were kept in a stream of warm air for 1 hr to remove HCI and then developed either with solvent a ( x 2) (for sugars) or solvent b (for aglycones). The following sugars were identified: 1 gave glucose, galactose, apiose and glucuronolactone; 2 gave galactose and glucuronolactone. Sugars were detected with aniline phtalate giving a brown colour; glucuronolactone gave a red colour. After spraying with cont. H,SO,, compound 1 gave betulinic acid only while 2 gave betulinic acid and hetulinic acid Me ester. Methylation of 1 and preparation of the partially methylated alditolacetates. Compound 1 (50 mg) was methylated by

Hakomori’s method [12] and the methylation products were chromatographed on a silica gel column (solvent c) giving per-Omethyl 3, mp 68-80”, [a]p” - 12.5”(MeOH; c l), IR v :t;crn-’ no free OH signal. The permethylated 3 was worked-up according to ref. [9] and the partly methylated alditol acetates were analysed by GC. The following alditol acetates were identified: 1,5-di-O-Ac-2,3,4,6-tetra-O-Me galacticol, [R, (min) 4.153, 1,5,6tri-O-Ac-2,3,4-tri-O-Me glucitol (R, 7.9) 1,5-di-O-AC 2,3,4 tri-OMe apitol (R, 1.95).The uranic acid derivative was not identified. LiAlH, reduction ofl. Compound 3 (30 mg) in 5 ml THF was reduced with LiAlH, [lo] and the residue purified on a silica gel column (solvent c) giving 20 mg amorphous compound 4: mp 1lO-113”, [a]i”+V (MeOH; ~0.8). Compound 4 was hydrolysed, reduced and acethylated as above giving (GC) 1,5-di-O-AC 2,3,4,6-tetra-O-Me galactic01 (R, 4.2) and 1,4,5,6-tetra-O-AC 2,3di-O-Me glucitol (R, 16.9). Acknowledgements-We are grateful to Dr A. Buschauer (Institut fur Pharmazie, FU. Berlin) for negative FABMS, ‘H, i3CNMR analysis and to Dr D. Scheiitzow (University of Wiirzburg) for ‘H and i3C NMR.

REFERENCES 1. Swiatek, L. (1984) Farmacja Polska. 5, 257. 2. Stabursvik, A. (1953) Acta Chem. Stand. 7,446. 3. Hoppe, H. A. (1975) Drogenkunde. Walter de Gruyter, Berlin. 4. Koczwara, M. (1949) Diss. Pharmaceut. 1, 65. 5. Rulko, F. (1972) Rocz. Chem. 43, 1831. 6. Keegan, P. Q. (1916) Chem. News. 113, 86. 7. Wulff, G. (1965) J. Chromat. 18, 285. 8. Kartnig, Th. and Ri, C. Y. (1973) Planta Med. 23, 379. 9. Jansson, P. E., Kenne, L., Liedgren, H., Lindberg, B. and Liinngren, J. (1976) Chem. Commun., limo. Stockholm 8, 1. 10. Okada, Y., Shibata, S., Ikekawa, T., Javellana, A. M. J. and Kamo, 0. (1987) Phytochemistry 26,2789. 11. Nakai, S., Takagi, N., Miichi, H., Hayashi, S., Nashimoto, N., Takemoto, T. and Kizu, H. (1984) Phytochemistry 23, 1703. 12. Hakomori, S. (1964) J. Biol. Chem. 55,205.