Triterpenoid saponins from Madhuca butyracea

Pergamon

TRITERPENOID

Phytahemivrry, VoL 37. No. 3, pp. 827419, 1% Copyri@ Q 1994 Ekvim Scima Ltd Printed in Grclt Britain. AU ri@s twaved 0031~9422/94 $7.00+0.00

SAPONINS

FROM

MADHUCA

BUTYRACEA

XING-CONG Lr, Yu-QING LIU, DE-zu WANG, CHONG-RENYANG,* S. K. NIGAM~ and G. MISRA~

Laboratory of Phytochemistry, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, China; tNationa1 Botanical Research Institute, Lucknow 226 001, India (Received in revisedform 22 February 1994) Key Word Index-Madhuca

butyracea; Sapotaceae; seeds; triterpenoid

saponins; butyrosides C and

D.

Abstract-Further investigation of the seeds of Madhuca butyrocea butyrosides C and D whose structures were established by means glucuronopyranosyl protobassic acid 28-0-a-L-rhamnopyranosyl( ranosyl(l+2)-a+arabinopyranoside and 3-O+&glucuronopyranosyl apiofuranosyl( 14 3)-/?-D-xylopyranosyl( l-*4)-a-L-rhamnopyranosyl(

lNTRODUCIlON

In the preceding paper [ 1J, we reported the isolation and structure elucidation of two new triterpenoid saponins, butyrosides A and B, along with two known ones, Misaponin A and 16a-hydroxy Mi-saponin A from the seeds of Madhuca butyracea (Roxb.) Macbride. In a continuation of chemical examination of this plant, two further more polar triterpenoid saponins, named butyrosides C(1) and D(2) were isolated and their structures determined. RESULTS AND DISCUSSION

Butyrosides c(1) and D(2) were isolated in homogeneity by repeated column chromatography of the crude saponins on normal and reversed phase silica gel and finally with ion exchange resin. Comparison of their 13C and ‘H NMR signals due to aglycone moieties with those of saponins and related derivatives previously obtained from the title plant by ref. [l] indicated that 1 was a 3,28di-O-bisdesmoside of protobassic acid, with 2 being a 3,28di-0-bisdesmoside of 16a-hydroxy protobassic acid. On alkaline hydrolysis with 5% KOH, butyrosides C and D gave the corresponding prosapogenins 3 and 4, respectively. Compounds 3 and 4 showed quasimolecular ion peaks at m/z 679 [M(C,BH,,O,,)-HJand 695 [M(C,,H,,O,,)-HI-, respectively, in their negative FAB mass spectra. On acid hydrolysis with 5% HCl-dioxane (1: 1), compound 3 yielded protobassic acid and glucuronic acid, whereas 4 afforded 16a-hydroxy protobassic acid and glucuronic acid. Identification of protobassic acid and 16a-hydroxy protobassic acid was made by comparison of TLC behaviour with authentic *Author to whom correspondence should be addressed.

yielded two new triterpenoid saponins, namely of chemical and spectral analyses as 3-O-j-~1+3)-j&D-xylopyranosyl( 1-+4)-a-L-rhamnopy161-hydroxy protobassic acid 28-O-&~1-+2)-a-I_-arabinopyranoside, respectively.

samples which were obtained by the hydrolysis of butyrosides A and B [l]. In the ‘HNMR spectra of 3 and 4, they showed anomeric proton signals at 65.39 (d, J = 7.7 Hz) and 5.34 (d, J =6.4 Hz), respectively, diagnostic of p-configuration of glucuronic acid. In the 13C NMR spectra of 3 and 4, both showed a set of typical signals corresponding to a &+curonopyranosyl unit [2]. Therefore, the structures of 3 and 4 were readily assigned as protobassic acid 3-0-fl-glucuronopyranoside and 16a-hydroxy protobassic acid 3-O-fl-glucuronopyranoside, respectively. The sequence and interglycosidic linkages of 28-0sugar moiety of butyrosides c(1) and D(2) were established as follows. The negative FAB mass spectra displayed quasimolecular ion peaks at m/z 1235 CM(C~sH,@,s)W - and 12371M(C,,H,,O,,) - HI for saponins 1 and 2, respectively, and very typical fragment ions resulted from the cleavage of each sugar (see the Experimental section). In the 13C and ‘H NMR spectra of 1 and 2, besides one set of carbon signals corresponding to a B-glucuronopyranosyl unit and its anomeric proton signal, the remaining carbon signals of sugars and anomeric proton signals of 1 and 2 were fully superimposable on those of 28-O-sugar moiety of Mi-saponin A or 16a-hydroxy Mi-saponin A and butyrosides A or B, respectively, whose structures have been reported in our previous paper Cl]. This suggested that 1 should have the same 28-O-sugar moiety as that of Mi-saponin A or 16ahydroxy Mi-saponin A, and 2 possesses the same 28-Osugar moiety as that of butyrosides A or B. Finally, supposing the absolute configurations of sugars are the common forms in naturally occurring glycosides, i.e. Larabinose, D-xylose, t-rhamnose, D-apiose and D-ghcuronic acid, the structures of 1 and 2 should be ~-O-/?-Dglucuronopyranosyl protobassic acid 28-O-a-L-rhamnopyranosyl( l-+3+D-xylopyranosyl( 1+4~a+rhamno-

827

XING-CQNGLI et al.

828

the measurement of NMR spectra. It seems to be essential for such

saponins

possessing

a glucuronopyranosyl

unit

in the molecule to be deionized with ion exchange before subjected to spectral measurements.

resin

EXPERlMENTAL

Mps: uncorr. NMR spectra were recorded in pyridined, at 400 MHz for ‘H NMR and 100 MHz for “CNMR

(including DEPT) using TMS as int. standard. Isolation of saponins. The crude

saponins

were

separ-

frs as described in the preceding paper [ 11. The most polar fr. 8 (8.5 g) was chromatographed on silica gel with CHCI,-MeOH-H,O (15:lO:l) to give a mixture (1.8 g) mainly containing saponins 1 and 2. This mixture was further repeatedly chromatographed on reversed phase silica gel with 55% MeOH, followed by deionization with ion exchange resin (Amberlite MB-3) to furnish 1 (150 mg) and 2 (800 mg). Butyroside C (1). Powder from MeOH, mp 216-220” (dec.), C~l,Y - 20” (MeOH; ~0.80); FABMS (neg.)

ated

2

OH

into

Ho Table

1. 13C NMR chemical shifts of dine-d, (ppm)

compounds 1-4 in pyri-

. 3

4

1

2

1

46.5

2 3 4 5 6 7 8 9 10 11 12 13 14 IS 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

71.2 82.8 44.1 49.2 67.6 41.2 39.3 48.8 37.0 24.1 123.3 144.2 42.9 28.3 23.8 46.7 42.1 46.8 31.0 34.3 33.3 65.2 16.8 19.0 18.5 26.5 180.2 23.3 23.8 105.5 15.2 78.0 73.5 77.6 173.0

46.8 71.0 83.0 44.1 49.0 67.7 41.4 39.5 48.3 37. I 24.2 123.1 144.5 42.9 36.3 74.9 49.0 41.6 47.3 31.1 36.2 32.9 65.3 16.8 19.1 18.7 27.5 180.1 33.4 24.9 105.6 75.2 78. I 73.4 77.3 173.0

46.4 71.2 83.0 44.0 49.2 67.7 41.2 39.5 48.8 36.9 24.2 123.3 143.8 42.9 28.3 23.4 47.5 41.9 46.7 31.0 34.3 33.2 65.4 16.8 19.1 18.7 26.3 176.3 33.2 23.8 105.9 75.3 78.1 73.4 77.6 172.8

46.8 71.2 83.2

.’

>\

c\ COOH HO

-. RI

@

Rl 3 4

H OH

pyranosyl(l+2)-a-L-arabinopyranoside and 3-0-/&Dglucuronopyranosyl 16a-hydroxy protobassic acid 28-0fi-D-apiofuranosyl( l-+3)-/I-D-xylopyranosyf ( 1-r4)-a-Lrhamnopyranosyl-( I +2)-a-L-arabinopyranoside, respectively. It has to be pointed out that the arabinose ring in butyrosides C and D is predominantly in a ‘C, conformation depicted as shown, although it is predominantly in a 4Cr conformation in many naturally occurring glycosides. This has been described in platycodin-D [3] which possesses the same sugar sequence as that of butyrosides A, B and D at the carboxyl group of C-28. A significant feature for butyrosides C and D is that a /3-D-glucuronopyranosyl unit is attached to the hydroxy group of C-3 in place of a B-D-glucopyranosyl unit in the previously obtained four saponins from the title plant. Because of this, their polarities are much higher. In addition, the presence of a glucuronopyranosyl unit caused some troubles in the process of purification and

3-0-GlcA

1 2 3 4 5 6

44.1 49.0 67.9 41.4 39.7 48.4 37.0 24.3 123.5 143.9 42.9 36.3 74.2 49.9 41.6 47.2 31.0 36. I 33.3 65.5 16.8 19.2 18.9 27.5 176.2 33.3 25.1 105.8 75.3 78.1 73.5 77.5 173.0

Saponins from Madhuca Table 1. Continued 3

28-O-Ara

Rha

XYl

Rha

Api

4

1

2

93.4 75.5 70.4 66.2 63.1 101.3 72.1 72.1 83.6 68.7 18.4 106.8 76.1 83.6 69.4 67.3 102.7 72.5 12.7 74.2 69.9 18.7

93.5 75.6 70.3 66.1 63.1 101.4 72.2 72.7 83.5 68.8 18.4 106.7 15.3 85.1 69.5 67.0

111.3 77.9 80.4 15.3 65.5

m/z: 1235 [M-H]-, 1089 [M-Rha-HI-, 1059 Cl235 -GlcA-HI-, 957 [1089-Xyl-HI-, 811 [MRhax2-Xyl-HI-, 679 [M-Rhax2-Xyl-AraHJ- and 503 [M-Rhax2-Xyl-Ara-GlcA-HJ-. ‘H NMR: 60.88, 1.00, 1.26, 1.64,2.00,2.20 (3H each, s, H29,30,27,26,24,25, respectively), 1.66 (3H, d, J = 6.3 Hz, Rha Me), 1.71 (3H,d,J=5.2 Hz, Rha’Me),3.32(1H,brd, J= 12 Hz, H-18), 5.06 (lH, d, J=8.0 Hz, Xyl H-l), 5.18 (IH, br s, H-6a), 5.34 (lH, d, J=7.6 Hz, GlcA H-l), 5.50 (lH, br s, H-12), 5.68 (lH, br s, inner Rha H-l), 6.18 (lH, br s, terminal Rha H-l), 6.45 (lH, br s, Ara H-l). Butyroside D (2). Powder from MeOH, mp 213-215” (dec.), CalF -53” (MeOH; c 1.03); FABMS (neg.) m/z: 1237 [M-H]-, 1105 [M-Api-HI-, 1061 [M -GlcA-HI-, 973 [M-Api-Xyl-HI-, 929 [MApi-GlcA-HI-, 827 [M-Api-Xyl-Rha-HI-, 695 [M-Api-Xyl-Rha-Ara-H]and 519 [MApi-Xyl-Rha-Ara-GlcA-HI-; ‘HNMR: 61.00, 1.16, 1.65, 1.80, 1.97,2.27 (3H each, s, H-29, 30,26, 27,24, 25, respectively), 1.66 (3H, d, J=6.8 Hz, Rha Me), 3.60 (lH, m, H-18). 5.04(1H, d, J=6.4 Hz,Xyl H-l), 5.14, 5.16 (lHeach,brs,H-6a,H-l6/?),5.29(1H,d,J=7.4Hz,GlcA H-l), 5.70 (lH, br s, Rha H-l), 5.72 (lH, br s, H-12), 6.12 (lH, d, 5=2.3 HI Api H-l), 6.42 (lH, br s, Ara H-l).

butyracea

829

Alkaline hydrolysis of 1 and 2. A soln of saponin I (50 mg) in 5% KOH (2 ml) was heated at 95” for 8 hr. After cooling, the reaction mixt. was acidified with 36% AcOH to pH _ 4-5 and then directly subjected to CC on MCI gel CHP 20P eluting with H,O, 80% MeOH and MeOH, successively. The 80% MeOH eluate was coned to dryness to give 3 (25 mg). In a similar manner, saponin 2 (85 mg) yielded compound 4 (52 mg). Prosapogenin 3. Powder from MeOH, mp 215-218” (dec.), [a];’ +16” (MeOH; ~0.73); FABMS (neg.) m/z: 679 [M-H]-, 503 CM-GlcA-HI-; ‘HNMR: 60.91, 0.98, 1.31, 1.64,2.05, 2.24 (3H each, s, H-29, 30, 27,26, 24, 25, respectively), 3.31 (lH,dd,J= 13.3,3.7 Hz, H-18),4.96 (lH, br, d, J=2.8 HI H-2@, 5.17 (lH, br s, H-62), 5.39 (lH, d, J=7.7 Hz, GlcA H-l), 5.54 (lH, hr s, H-12). Prosapogenin 4. Powder from MeOH, mp 227-230 (dec.), [alA +lO” (MeOH; ~0.84); FABMS (neg.) m/z: 695 [M-H]-, 519 [M-GlcA-HI-; ‘HNMR: 61.02, 1.15, 1.67, 1.85,2.03, 2.26 (3H each, s, H-29,30, 26,27, 24, 25, respectively), 3.64(lH, dd, J= 14.0.3.8 Hz, H-18),4.95 (lH, br s, H-2a), 5.17, 5.23 (1H each, br s, H&Z. H-16B), 5.34 (lH, d, J=6.4 HI GlcA, H-l), 5.7alH, br s, H-12). Acid hydrolysis of 3 and 4, A soln of compounds 3 and 4 (about 3 mg) in 5% HCI-dioxane (1: 1, 0.3 ml) was individually heated at 90” for 6 hr. The reaction mixt. was directly examined with TLC (CHCI,-MeOH; 10: 1) and compared against authentic samples [ 11, protobassic acid was detected from 3 and 16a-hydroxy protobassic acid from 4 along with some by-products. On the other hand, the reaction mixt. was diluted with H,O and extracted with EtOAc. The Hz0 layer was neutralized with Amberlite MB-3, coned to dryness and then subjected to TLC analysis [CHCI,-MeOH-AcOH-H,O (7 : 3 : 1: 0.5); detection: aniline/phthalate] for identifying sugars. Glucuronic acid was detected with a R, value of 0.15 for both 3 and 4 after developing twice.

Acknowledgements-This study was supported in part by a grant from the Foundation of Laboratory of Phytochqnistry, Kunming Institute of Botany (KIB), Chinese A&demy of Sciences. The authors are grateful to Dr Ryoji Kasai (Hiroshima University, Japan) for performing negative FABMS spectra and Mr Y.-N. He (KIB) for recording NMR spectra.

REFERENCES

Nigam, S. K., Li, X.-C., Wang, D.-Z., Misra, G. and Yang, C.-R. (1992) Phytochemistry 31, 3169. Nie, R.-L., Morita, T., Kasai, R., Zhou, J., Wu, C.-Y. and Tanaka, 0. (1984) Planta Med. 322. Ishii, H., Tori, K., Tozyo, T. and Yoshimura, Y. (1984) J. Chem. Sot. Perkin Trans I 661.