Phytochemistry,Vol. 30, No. 11, pp. 3717-3720,1991
0031-9422/91 $3.00+0.00 © 1991PergamonPress pie
Printed in Great Britain.
A BIDESMOSIDIC TRITERPENOID SAPONIN FROM SCHEFFLERA OCTOPHYLLA T. V. SUNG, J. PETER-KATALINIC*and G. ADAM~" Institute of Natural Products Chemistry, National Research Centre of Vietnam, Tu Liem, Hanoi, SR Vietnam; *Institute of Physiological Chemistry, University of Bonn, Germany; tInstitute of Plant Biochemistry, Halle/S., Germany
(Received in revisedform 1 February 1991) Key Word Index--Schefflera octophylla; Araliaceae; oleanonic acid; 3,28-bidesmosidic triterpene saponin; trisaccharide.
Abstract--A new 3,28-bidesmosidic triterpenoid saponin was isolated from the leaves of Schefflera octophylla together with a new trisaccharide and oleanonic acid. Based on spectroscopic data and chemical transformations, the structures of the new constituents were determined as 3-epi-betulinic acid 3-O-fl-D-glucopyranoside 28-O-[ct-L-rhamnopyranosyl(1--*4)-O-fl-D-glucopyranosyl(l~6)]-fl-D-glucopyranoside and Ct-L-rhamnopyranosyl(l~4)-O-fl-D-glucopyranosyl(1 ~ 6)-fl-D-glucopyranose.
INTRODUCTION
Schefflera octophylla is used in Vietnamese folk medicine as an antirheumatic agent and a tonic drug [1]. Some triterpenes and triterpene glycosides have been reported from its leaves [2-7]. In continuation of our studies on this plant we described the isolation and structure determination of a new bidesmosidic saponin: 3-epi-betulinic acid 3-O-fl-o-glucopyranoside 28-O-[~t-L-rhamnopyranosyl( 1 ~4)-O-fl-D-glucopyranosyl( 1 ~ 6)]-fl-D-glycopyranoside (3) and a new trisaccharide 8 together with oleanonic acid (1).
RESULTSAND DISCUSSION Flash chromatography combined with gel filtration on Sephadex LH-20 of the methanol extract afforded the new bidesmosidic triterpenoid saponin 3. Compound 3 showed IR absorptions for hydroxyl (3520), ester carbonyl (1730) and terminal methylene group (1640, 890 cm-1). Its FAB mass spectrum with thioglycerol as matrix revealed a molecular ion peak at m/z 1111.6 [M + Na] +, as well as peaks ~lue to the stepwise loss of sugars at m/z 948 [ M + N a - R h a ] +, 931.5 [ M + N a - G l c ] ÷, 639.3 [M + N a - (28-O-sugar residue)] ÷ and 460.2 [aglycone + Na] ÷. The 1H N M R spectrum exhibited signals of six tertiary methyls and one secondary methyl, the characteristic signals of lup-20(29)-ene derivatives having a carboxylic group at C-17 [61.69 (3H, s, H-30), 3.00 (1H, dt, H-19), 4.60 and 4.73 (~C=CH2)] [3, 8] and three anomeric glucose protons [64.26, d, J = 7 . 5 Hz; 4.38, d, J =7.5 Hz and 5.46, d, J = 7 . 5 Hz]. The methyl doublet at 61.26 (J = 6.3 Hz) indicated the presence of a rhamnose moiety. The 1aC N M R spectrum was very important for the structure elucidation of saponin 3, showing the presence of 54 carbon atoms with four sugar molecules (signals for anomeric C-atoms: 695.1, 101.5, 102.8, 104.4). The assignments was performed by the DEPT-experiments and comparison with the data of the related
compounds [5, 6]. Analysis of IH and lSCNMR spectrum of 3 in comparison with those of the other lup20(29)-ene-28-oic acid 28-O-glycosides, isolated recently from this plant [4, 5], confirmed that 3 has the same sugar residue at C-28, which is [~t-L-rhamnopyranosyl(1--*4)-Ofl-D-glucopyranosyl(1--*6)]-fl-D-glucopyranoside. Acid hydrolysis of 3 yielded 3-epi-betulinic acid (7) with aglycone and glucose and rhamnose as sugar components. The sugars were identified by TLC and PC, as well as by GC analysis of their peracetates. Alkaline hydrolysis of 3 afforded a prosapogenin 5 which contained a glucose molecule [anomeric proton at 64.84 (d, J = 7.9 Hz)]. Acetylation of 5 yielded the peracetate 6 which exhibited the typical signals in the 1H NMR spectrum at 64.47 (d, J = 8 Hz, anomeric proton), 2.00, 2.03, 2.04, 2.07 (each 3H, s, OAc) and 3.30 (1H, br s, H-3fl). The prosapogenin 5 and its peracetate 6 showed identical 1HNMR spectra and [~t]D values to those of 3-epi-betulinic acid 3-O-fl-D-glucopyranoside, isolated recently from this plant [6]. Acid hydrolysis of 6 provided 3-epi-betulinic acid (7), identified by comparison with an authentic sample, isolated from the same source and methyl-fl-D-glucopyranoside, identified by GC and TLC. The 3-O-glucosidic bond of 3 was supported by the downfield shift of C-3 in the ISCNMR spectrum (A6 =7.4 ppm) and highfield shift of C-2 (A6=4.8 ppm) in comparison with those of 3-epi-betulinic acid (7) [5, 9, 10]. Thus, the new saponin is 3-epi-betulinic acid 3-O-flD-glucopyranoside 28-O-[ct-L-rhamnopyranosyl(1--*4)O-fl-glucopyranosyl(1 ~6)]-fl-D-glucopyranoside (3). Further elution with chloroform-methanol-water (60: 35: 8) gave a mixture of l-epimeric trisaccharides 8 and 8a in a ratios of ~:fl=0.8:l as indicated by the 13C NMR data. The FAB mass spectrum of this mixture showed intense peaks at m/z 506 [ M + N H 4 ] + and 511 [ M + N a ] +. The signal at 61.24 (d, J=6.0 Hz) in the IH NMR spectrum indicated the presence of a rhamnose. The 13C N M R spectral data of the mixture of compounds 8 and 8a were similar to those of the sugar residue of the
3717
T.V. SUNGet al.
3718 29
the first natural occurrence of this compound in plants. The synthesis of the O-benzoyl derivatives has been reported [18]. The chloroform extract of dried leaves gave on repeated flash chromatography oleanonic acid (1) in 0.03 % yield. The EI mass spectrum of 1 exhibited the molecular ion peak at m/z 454.3470, C3oH46Oa calc. 454.3447 [M] +, two intense peaks at m/z 248 and 203 [248 - C O 2 H ] as well as peaks at m/z 191, 189 and 133, indicating that 1 could be an amyrin derivative having a free carboxylic group at C-17 [19-21]. The 1 H N M R spectrum contained a characteristic signal for a A t2oleanene derivative at 62.82 (dd, J = 14.4, 4 Hz, H-18). This was supported by the 13CNMR spectrum with signals at 3122.4 (C-12) and 143.6 (C-13) [22]. The presence of a ketone function at C-3 was shown by a signal at 6217.8 in the ~3CNMR spectrum, and by the absence of proton signals geminal to hydroxyl groups in the I H N M R spectrum [23, 24] as well as a positive Cotton effect (Ae2s 5 + 0.54). Direct comparison of I with an authentic sample, prepared by Jones' oxidation of oleanolic acid (2) established their identity. Oleanonic acid was first isolated from gum mastic [25] and as its methyl ester from Liquidambar orientalis [26].
30
12
COzH
R
~
~-~
1
2
R = O R = B-OH, ~x-H 29
25 ~ C 0 2 R 2
24
23
R1
3 4 5 6 "/
Rha ~ R10 "
R2
EXPERIMENTAL
Glc Rha4GIc6G1cGIcAc4 (Rha4Glc-6Glc)Ac9 Glc H GlcAc4 H H H
CH2ORI RI0 ~ , , ' 0
~
OR OR1
Glc
0~ . A tAa2 r, R~O" ~ ' ~ / / ' - ' \
6RI' 'R2 Glc 8 8a
9
R~
R~
H H Me
/3-OH a-OH fl-OH
Mps are uncorr. MS: 70 eV. 13C NMR: 100.6 MHz, IH NMR: 200 MHz. Chemical shifts (6) are given in ppm from TMS. TLC employed precoated silica gel plates 60 F254 (Merck). The following TLC solvent systems were used: for saponins and trisaccharides: CHCI3-MeOH-H20 (60:35:8) (FS 1), (70:30:3) (FS2); for free triterpenes: CHCI3-MeOH (49:1) (FS 4), (19: l) (FS 5) and petrol-EtOAc (7: 3) (FS 10), for sugars: n-BuOH-isoPrOH-H20 (5:3:1) (FS I 1). Spray reagents were for saponins and free triterpenes: vanillin-H2SO£ for sugars: aniline phthalate. Sephadex LH-20: Pharmacia Uppsala, Sweden. GC: column Chrompack CP Si119 CB. Conditions were: temp. programmed from 60 to 180° at 10° min- 1. Detector: FID; carrier gas: N 2 (0.7 bar). Isolation. Dried leaves (250 g) collected in the mountains of middle Vietnam were extracted with boiling CHCI 3 and MeSH successively in a Soxhlet for 10 hr. The solvents were distilled off under red. pres. and the residues subjected to chromatography. Repeated flash chromatography of the residue of the CHCI 3 extract (petrol-EtOAc, 4:1) gave oleanonic acid (1, 75mg, 0.33%) besides other triterpenes. Oleanonic acid (1). R s 0.48 (FS 10), [u]2o +36.1 ° (CHCI3; c 0.56). IR Ymax CHCI3 cm - 1. 1694 (C=O), 1456, 1384, 1362, 1020. MS m/z (rel. int.): 454.3470 C30H4603 calc. 454.3447 [M] + (6.5),408 [M-HCO2H] ÷ (8), 248 (100), 203 [248-CO2H] + (70), 191 (6), 189 (13), 133 (18) 119 (13). CD: Ae2ss +0.536 (MESH). 1H NMR (200 MHz, CDCI3): 30.79 (3H, s, H-26), 0.89 (3H, s, H-29), 0.91 (3H, s, H-30), 1.01 (3H, s, H-24), 1.02 (3H, s, H-25), 1.08 (3H, s, H23), 1.13 (3H, s, H-27), 2.82 (1H, dd, ./=14.4, 4Hz, H-18), 5.28 (1H, t, J=3.5 Hz, H-12). Flash chromatography of the MeSH extract (CHC13-MeOH-H20, 70:30:3) yielded a product, which was passed through Sephadex LH-20 (MESH, 600 ml) to give 3 (85 mg, 0.034%). Rf 0.34 (FS 2), powder mp 177-180°, [u]~o -30.9 ° (MESH; c 1.06). IRv~maB[cm-l: 3410 (OH), 1725 (ester carbonyl), 1640 (C=CH2). FABMS (positive ion) m/z (rel. int.): 1111.7 [M+Na] + (5.7), 948 [ M + N a - R h a ] + (0.4), 931.5 [M + Na - Glc] + (0.4), 639.3 [M + Na - (28-O-sugar chain)] + (0.5), 460.2 [aglycone+Na] + (1.2) 176.0 (100). 1HNMR (200 MHz, .
other triterpene glycosides isolated recently from this plant [5]. Acid hydrolysis of permethyl derivative 9 [11] gave the same sugars as those obtained from acid hydrolysis of the triterpene glycosides [4, 5]. They were methyl pyranosides of 2,3,4-tri-O-methylrhamnose, 2,3,4- and 2,3,6-tri-Omethylglucose (identified by GC). Therefore, the structure of the trisaccharides 8 and 8a should be identical with that of the 28-O-sugar chain of the saponin 3. Thus, the structures of the new compounds are ct-L-rhamnopyranosyl(1 -*4)-O-fl-n-glucopyranosyl(i ~ 6)-fl-D-glucopyranose (8) and its lct-epimer 8a. Although this trisaccharide moiety was found as the glycone in several plant saponins, especially those from Araliaceae [-8, 12-17] its isolation from Schefflera octophylla is, to our knowledge,
Triterpenoid saponin from Schefflera octophylla Table 1. 13CNMRspectraldataofcompound3(100.6MHz, in CD3OD) C
3
C
3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
35.2 21.9 82.5 38.3 51.1 19.2 34.4 42.1 50.8 38.0 21.9 26.7 39.3 43.6 30.8
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
32.8 57.9 50.5 48.3 151.7 31.5 37.5 29.2 22.9 16.9 16.6 15.3 176.2 110.4 19.5
1 2 3 4 5 6
Glc 1 95.1 73.6 79.3 70.8 77.9 69.4
Glc 2 104.2 75.0 76.8 77.7 76.6 61.8
Sugar moiety of 3 Rha 3-O-Glc 102.8 101.5 72.3 75.2 72.1 78.3 73.9 71.9 70.5 78.1 17.8 62.9
CD3OD): 60.85 (3H, s), 0.87 (3H, s), 0.94 (6H, s), 1.02 (3H, s) 1.26 (3H, d, J=6.3 Hz, Rham-Me), 1.69 (3H, s), 3.00 (1H, dt, J= 11.7, 5.4 Hz, H-19), 4.26 (1H, d, J = 7.5 Hz, anomeric proton), 4.38 (1H, d, J=7.5Hz, anomeric proton), 4.60, 4,73 (each 1H, br s, C=CH2), 5.46 (1H, d, J=7.5 Hz, anomeric proton), laCNMR (100.6 MHz, CD3OD): see Table 1. Peracetylation of compound 3. Compound 3 (15 mg) was acetylated with Ac20-pyridine (each 0.3 ml) at room temp. for 18 hr. The mixt. was evapd to dryness and chromatographed (CHCla) to give 4 (12.7 mg). R: 0.75 (FS 5), amorphous, [~t]~° -21.2 ° (CHCI3; c0.556). IRvma CHCI3 x cin-l: 1750 (ester carbonyl), 1640, 900 (C=CH2), 1220 (OAc). IHNMR (200MHz, CDC13): 60.80 (3H, s), 0.82 (3H, s), 0.87 (6H, s), 0.94 (3H, s), 1.12 (3H, d, J =6.1 Hz, Rha-Me), 1,66 (3H, s), 1.97-2.04 (27H, 3 x s, 9 x OAt), 2.05, 2.08, 2.10, 2.11 (each 3H, s), 2.90 (1H, t-like, br, H-19), 3.30 (1H, br s, H-3fl), 4.57, 4.71 (each 1H, br s, C=CH2), 4.85 (1H, d, J =7.7 Hz, anomeric proton), 5.64 (1H, d, J=7.7 Hz, anomeric proton). Alkaline hydrolysis of compound 3. Compound 3 (28 mg) was hydrolysed in MeOH with 5% KOH (5 ml) for 4 hr at 80°. The mixt. was passed through DOWEX 50 W × 4 (H ÷ form, MeOH) and evapd. The residue was dissolved in H20 and extracted with BuOH. The BuOH layer was evapd to dryness to give prosapogenin 5 (14mg), [~]2D° - 1 2 ° (MeOH; c0.325), t H N M R (200 MHz, pyridine-ds):/~0.74, 0.77, 0.81, 1.00, 1.24, 1.77 (each 3H, s, tert. Me), 4.77, 4.91 (each 1H, br s, C=CH2), 4.84 (1H, d, J=7.9 Hz, Glc H-I). Acid hydrolysis of compound 3. Compound 3 (20 mg) was hydrolysed with 5% HC1 (5 ml) as described for 6. 3-epi-Betulinic acid was identified as aglycone by TLC, IR and ~H NMR. The aq. layer showed the presence of the glucose and rhamnose by PC and TLC. After evapn and acetylation the glucose- and rhamnoseperacetates were identified by GC analysis.
3719
Peracetylation of prosapogenin 5. Compound 5 (5 mg) was acetylated with Ac20-pyridine (each 0.2 ml) for 7 hr at room temp. The mixt. was evapd to dryness to yield peracetate 6. R: 0.66 (FS 5), powder mp 200-201 ° (dec.), [~t]2° - 17.1° (CHCla; c 0.35). IR vC~el3 cm- 1:1740 (ester carbonyl), 1710 (CO2H), 1640, 890 (C---CH2), 1220 (OAc), 1085, 1035. 1HNMR (200MHz, CDCI3): 60.79, 0.81, 0.87, 0.90, 0.96, 1.67 (each 3H, s, tert. Me), 2.0, 2.03, 2.04, 2.07 (each 3H, s, OAc), 2.99 (IH, t-like, br, H-19), 3.30 (1H, br s, H-3fl), 4.47 (1H, d, J = 8 Hz, glucosyl-H-l), 4.58, 4.72 (each 1H, br s, C=CH2), Acid hydrolysis ofperacetate 6. Compound 6 (5 rag) was heated in MeOH with 5% HCI (1 ml) for 3 hr at 80°. The mixt. was passed through DOWEX 50 1 x 8 (OH- form) and evapd. The residue was dissolved in H20 and extracted with CHCI 3. The solvent was evapd to give 3-epi-betulinic acid (7), identified by comparison with an authentic sample. Examination of the residue of the water layer by GC and TLC showed the presence of fl-D-methylglucoside. Further elution with CHC13-MeOH-H20 (60:35:8) gave a mixt. of trisaccharides $ and ~ (625 rag, 0.25%), powder mp 148-149 °, [ct]2° -25.6 ° (MeOH; c 0.485). IR v ~ cm-l: 3340, 1060, 1030 (OH); FABMS (positive ion) m/z (rel. int.): 511 [ M + N a ] + (31), 506 [M +NH4] + (59), 309 [ M - G l c ] + (30), 325 [ M - R h a ] + (18). I H N M R (200MHz, CD3OD-D20): 61.24 (3H, d, J = 6 H z , Rha-Me), 4.52 (IH, d, J=7.6 Hz, anomeric proton), 5.12 (1H, br s, Rha-H-1). 13CNMR (50.3MHz, CD3OD-D20): 6104.1, 102.4, 97.7; 93.6, 78.9, 77.4, 76.5, 76.4, 76.1, 75.7, 74.9, 74.3, 73.4, 73.2, 70.0, 71.8, 71.0, 70.4, 69.8, 61.6, 17.8. Permethylation of compounds 8 and Sa. MeI (3.9 ml) was added to a soln of 8 and 8a (58 rag), t-BuONa (491 rag) and finely powdered NaOH (144 mg) in DMSO (4.7 ml). The mixt. was stirred at room temp. for 1 hr; poured into ice-water and extracted with Et20. The Et20 layer was washed with a satd NaCI soln, dried (Na2SO4) and evapd. Flash chromatography (CHC13-MeOH, 49:1) gave 9 (30 rag). R/0.44 (FS 4), oil, [~t]2° -29.2 ° (CHCI3; c 1.325). 1HNMR (200 MHz, CDC13): 61.26 (3H, d, J = 6 H z , Rha-Me), 4.11 (1H, d, J=7.8 Hz, anomeric proton), 4.31 (1H, d, J=7.8 Hz, anomeric proton), 4.94 (1H, d, J = 1.5 Hz, Rha-H-l). 13C NMR (22.63 MHz, CDC13): 317.6, 57.0, 57.8, 58.8, 59.5, 60.3, 60.4, 60.5, 60.9 (all q), 68.2 (d), 68.9 (t), 71.4 (t), 74.67, 74.64, 75.5, 77.3, 80.0, 81.0, 82.3, 83.9, 84.1, 84.3, 86.7, 97.5, 104.2, 104.3 (all d). Acid hydrolysis of permethyl 9. Compound 9 (5 rag) was hydrolysed in MeOH with 6% HC1 for 3 hr at 80°. The mixt. was evapd, diluted with H20 and extracted with CHCIv GC of the residue of the CHCI3 layer indicated the presence of methyl pyranosides of: 2,3,4-tri-O-methylrhamnose; 2,3,4- and 2,3,6-triO-methylglucose. Acknowledgements--T. V. Sung is indebted to the Alexander yon Humboldt Foundation for a research fellowship in the Institute of Organic Chemistry and Biochemistry, Bonn. We would like to thank Ing. Schmidt, University of Bonn, for the 1H and laC NMR spectra, Dr G. Eckhardt and Mr. Bressler (Bonn) for the mass spectra and Mr E. Gessi (University of Bonn) for the CD curves.
REFERENCES
1. Loi, D. T. (1977) Nhung Cay thuoc va vi thuoc Viet Nam (Glossary of Vietnamese Medicinal Plants) p. 817. Nha xuat ban KH KT (Science and Technical Publications). 2. Adam, G., Lischewski, M., Phiet, H. V., Preiss, A., Schmidt, J. and Sung, T. V. (1982) Phytochemistry 21, 1385. 3. Lischewski, M., Ty, Ph.D., Schmidt, J., Preiss, A., Phiet, H. V.
3720
T.V. SUNGet al.
and Adam, G. (1984) Phytochemistry 23, 1695. 4. Sung, T. V., Steglich, W. and Adam, G. (1990) Abstr. Int. Syrup. Biochemistry and Chemistry of Active Natural Substances, p. 80, Bonn, July 1990. 5. Sung, T. V., Steglich, W. and Adam, G. (1991) Phytochemistry 30 (in press). 6. Kitajima, J. and Tanaka, Y. (1989) Chem. Pharm. Bull. 37, 2727. 7. Kitajima, J., Shindo, M. and Tanaka, Y. (1990) Chem. Pharm. Bull. 38, 714. 8. Hahn, D.-R., Kasai, R., Kim, J.-H., Taniyasu, S. and Tanaka, O. (1984) Chem. Pharm. Bull. 32, 1244. 9. Agrawal, P. K., Jain, D. C., Gupta, R. K. and Thakur, R. S. (1985) Phytochemistry 24, 2479. 10. Tanaka, O. and Kasai, R. (1984) in Progress in the Chemistry of Organic Natural Products (Herz, W., Grisebach, H., Kirby, G. W. and Tamm, Ch., eds), p. 26. Springer. 11. Ciucanu, I. and Kerek, F. (1984) Carbohydr. Res. 131, 209. 12. Kochetkov, N. K. and Khorhin, A. J. (1966) Arzneimittelforsch. 16, 101. 13. Saito, S., Sumita, S., Tamura, N., Nagamura, Y., Nishida, K., Ito, M. and Ishiguro, I. (1990) Chem. Pharm. Bull. 38, 411. 14. Takai, M., Amagaya, S. and Oghihara, Y. (1977) J. Chem. Soc. Perkin Trans I 1801.
15. Shao, Ch.-J., Kasai, R., Xu, J.-D. and Tanaka, O. (1989) Chem. Pharnt Bull. 42, 311. 16. Mizutani, K., Ohtani, K., Kasai, R., Tanaka, O. and Matsuura, H. (1985) Chem. Pharm. Bull. 33, 2266. 17. Kasai, R., Oinaka, T., Yang, C.-R., Zhou, J. and Tanaka, O. (1987) Chem. Pharm. Bull. 35, 1486. 18. Takabe, S., Takeda, T. and Ogihara, Y. (1979) Carbohydr. Res. 76, 101. 19. Srivastava, S. K. and Jain, D. C. (1989) Phytochemistry 28, 644. 20. Karliner, J. and Djerassi, C. (1966) J. Org. Chem. 31, 1945. 21. Budzikiewicz, H., Djerassi, C. and Williams, D. H. (1964) Structure Elucidation of Natural Products by Mass Spectrometry VoL II, p. 122. Holden-Day, San Francisco. 22. Toh, K., Seo, S., Yoshimura, Y., Nakamura, M., Tomita, Y. and Ishii, H. (1976) Tetrahedron Letters 4167. 23. Nakanishi, K., Goto, T., Ito, S., Natori, S. and Nozoe, S. (1983) Natural Products Chemistry Vol. 3, p. 179. Kodansha, Tokyo. 24. Nakanishi, K., Goto, T., Ito, S., Natori, S. and Nozoe, S. (1974) Natural Products Chemistry VoL I, p. 365. Kodansha, Tokyo. 25. Seoane, E. (1956) J. Chem. Soc. 4158. 26. Huneck, S. (1963) Tetrahedron 19, 479.
0031-9422/91 $3.00+0.00 © 1991PergamonPress pie
Printed in Great Britain.
A BIDESMOSIDIC TRITERPENOID SAPONIN FROM SCHEFFLERA OCTOPHYLLA T. V. SUNG, J. PETER-KATALINIC*and G. ADAM~" Institute of Natural Products Chemistry, National Research Centre of Vietnam, Tu Liem, Hanoi, SR Vietnam; *Institute of Physiological Chemistry, University of Bonn, Germany; tInstitute of Plant Biochemistry, Halle/S., Germany
(Received in revisedform 1 February 1991) Key Word Index--Schefflera octophylla; Araliaceae; oleanonic acid; 3,28-bidesmosidic triterpene saponin; trisaccharide.
Abstract--A new 3,28-bidesmosidic triterpenoid saponin was isolated from the leaves of Schefflera octophylla together with a new trisaccharide and oleanonic acid. Based on spectroscopic data and chemical transformations, the structures of the new constituents were determined as 3-epi-betulinic acid 3-O-fl-D-glucopyranoside 28-O-[ct-L-rhamnopyranosyl(1--*4)-O-fl-D-glucopyranosyl(l~6)]-fl-D-glucopyranoside and Ct-L-rhamnopyranosyl(l~4)-O-fl-D-glucopyranosyl(1 ~ 6)-fl-D-glucopyranose.
INTRODUCTION
Schefflera octophylla is used in Vietnamese folk medicine as an antirheumatic agent and a tonic drug [1]. Some triterpenes and triterpene glycosides have been reported from its leaves [2-7]. In continuation of our studies on this plant we described the isolation and structure determination of a new bidesmosidic saponin: 3-epi-betulinic acid 3-O-fl-o-glucopyranoside 28-O-[~t-L-rhamnopyranosyl( 1 ~4)-O-fl-D-glucopyranosyl( 1 ~ 6)]-fl-D-glycopyranoside (3) and a new trisaccharide 8 together with oleanonic acid (1).
RESULTSAND DISCUSSION Flash chromatography combined with gel filtration on Sephadex LH-20 of the methanol extract afforded the new bidesmosidic triterpenoid saponin 3. Compound 3 showed IR absorptions for hydroxyl (3520), ester carbonyl (1730) and terminal methylene group (1640, 890 cm-1). Its FAB mass spectrum with thioglycerol as matrix revealed a molecular ion peak at m/z 1111.6 [M + Na] +, as well as peaks ~lue to the stepwise loss of sugars at m/z 948 [ M + N a - R h a ] +, 931.5 [ M + N a - G l c ] ÷, 639.3 [M + N a - (28-O-sugar residue)] ÷ and 460.2 [aglycone + Na] ÷. The 1H N M R spectrum exhibited signals of six tertiary methyls and one secondary methyl, the characteristic signals of lup-20(29)-ene derivatives having a carboxylic group at C-17 [61.69 (3H, s, H-30), 3.00 (1H, dt, H-19), 4.60 and 4.73 (~C=CH2)] [3, 8] and three anomeric glucose protons [64.26, d, J = 7 . 5 Hz; 4.38, d, J =7.5 Hz and 5.46, d, J = 7 . 5 Hz]. The methyl doublet at 61.26 (J = 6.3 Hz) indicated the presence of a rhamnose moiety. The 1aC N M R spectrum was very important for the structure elucidation of saponin 3, showing the presence of 54 carbon atoms with four sugar molecules (signals for anomeric C-atoms: 695.1, 101.5, 102.8, 104.4). The assignments was performed by the DEPT-experiments and comparison with the data of the related
compounds [5, 6]. Analysis of IH and lSCNMR spectrum of 3 in comparison with those of the other lup20(29)-ene-28-oic acid 28-O-glycosides, isolated recently from this plant [4, 5], confirmed that 3 has the same sugar residue at C-28, which is [~t-L-rhamnopyranosyl(1--*4)-Ofl-D-glucopyranosyl(1--*6)]-fl-D-glucopyranoside. Acid hydrolysis of 3 yielded 3-epi-betulinic acid (7) with aglycone and glucose and rhamnose as sugar components. The sugars were identified by TLC and PC, as well as by GC analysis of their peracetates. Alkaline hydrolysis of 3 afforded a prosapogenin 5 which contained a glucose molecule [anomeric proton at 64.84 (d, J = 7.9 Hz)]. Acetylation of 5 yielded the peracetate 6 which exhibited the typical signals in the 1H NMR spectrum at 64.47 (d, J = 8 Hz, anomeric proton), 2.00, 2.03, 2.04, 2.07 (each 3H, s, OAc) and 3.30 (1H, br s, H-3fl). The prosapogenin 5 and its peracetate 6 showed identical 1HNMR spectra and [~t]D values to those of 3-epi-betulinic acid 3-O-fl-D-glucopyranoside, isolated recently from this plant [6]. Acid hydrolysis of 6 provided 3-epi-betulinic acid (7), identified by comparison with an authentic sample, isolated from the same source and methyl-fl-D-glucopyranoside, identified by GC and TLC. The 3-O-glucosidic bond of 3 was supported by the downfield shift of C-3 in the ISCNMR spectrum (A6 =7.4 ppm) and highfield shift of C-2 (A6=4.8 ppm) in comparison with those of 3-epi-betulinic acid (7) [5, 9, 10]. Thus, the new saponin is 3-epi-betulinic acid 3-O-flD-glucopyranoside 28-O-[ct-L-rhamnopyranosyl(1--*4)O-fl-glucopyranosyl(1 ~6)]-fl-D-glucopyranoside (3). Further elution with chloroform-methanol-water (60: 35: 8) gave a mixture of l-epimeric trisaccharides 8 and 8a in a ratios of ~:fl=0.8:l as indicated by the 13C NMR data. The FAB mass spectrum of this mixture showed intense peaks at m/z 506 [ M + N H 4 ] + and 511 [ M + N a ] +. The signal at 61.24 (d, J=6.0 Hz) in the IH NMR spectrum indicated the presence of a rhamnose. The 13C N M R spectral data of the mixture of compounds 8 and 8a were similar to those of the sugar residue of the
3717
T.V. SUNGet al.
3718 29
the first natural occurrence of this compound in plants. The synthesis of the O-benzoyl derivatives has been reported [18]. The chloroform extract of dried leaves gave on repeated flash chromatography oleanonic acid (1) in 0.03 % yield. The EI mass spectrum of 1 exhibited the molecular ion peak at m/z 454.3470, C3oH46Oa calc. 454.3447 [M] +, two intense peaks at m/z 248 and 203 [248 - C O 2 H ] as well as peaks at m/z 191, 189 and 133, indicating that 1 could be an amyrin derivative having a free carboxylic group at C-17 [19-21]. The 1 H N M R spectrum contained a characteristic signal for a A t2oleanene derivative at 62.82 (dd, J = 14.4, 4 Hz, H-18). This was supported by the 13CNMR spectrum with signals at 3122.4 (C-12) and 143.6 (C-13) [22]. The presence of a ketone function at C-3 was shown by a signal at 6217.8 in the ~3CNMR spectrum, and by the absence of proton signals geminal to hydroxyl groups in the I H N M R spectrum [23, 24] as well as a positive Cotton effect (Ae2s 5 + 0.54). Direct comparison of I with an authentic sample, prepared by Jones' oxidation of oleanolic acid (2) established their identity. Oleanonic acid was first isolated from gum mastic [25] and as its methyl ester from Liquidambar orientalis [26].
30
12
COzH
R
~
~-~
1
2
R = O R = B-OH, ~x-H 29
25 ~ C 0 2 R 2
24
23
R1
3 4 5 6 "/
Rha ~ R10 "
R2
EXPERIMENTAL
Glc Rha4GIc6G1cGIcAc4 (Rha4Glc-6Glc)Ac9 Glc H GlcAc4 H H H
CH2ORI RI0 ~ , , ' 0
~
OR OR1
Glc
0~ . A tAa2 r, R~O" ~ ' ~ / / ' - ' \
6RI' 'R2 Glc 8 8a
9
R~
R~
H H Me
/3-OH a-OH fl-OH
Mps are uncorr. MS: 70 eV. 13C NMR: 100.6 MHz, IH NMR: 200 MHz. Chemical shifts (6) are given in ppm from TMS. TLC employed precoated silica gel plates 60 F254 (Merck). The following TLC solvent systems were used: for saponins and trisaccharides: CHCI3-MeOH-H20 (60:35:8) (FS 1), (70:30:3) (FS2); for free triterpenes: CHCI3-MeOH (49:1) (FS 4), (19: l) (FS 5) and petrol-EtOAc (7: 3) (FS 10), for sugars: n-BuOH-isoPrOH-H20 (5:3:1) (FS I 1). Spray reagents were for saponins and free triterpenes: vanillin-H2SO£ for sugars: aniline phthalate. Sephadex LH-20: Pharmacia Uppsala, Sweden. GC: column Chrompack CP Si119 CB. Conditions were: temp. programmed from 60 to 180° at 10° min- 1. Detector: FID; carrier gas: N 2 (0.7 bar). Isolation. Dried leaves (250 g) collected in the mountains of middle Vietnam were extracted with boiling CHCI 3 and MeSH successively in a Soxhlet for 10 hr. The solvents were distilled off under red. pres. and the residues subjected to chromatography. Repeated flash chromatography of the residue of the CHCI 3 extract (petrol-EtOAc, 4:1) gave oleanonic acid (1, 75mg, 0.33%) besides other triterpenes. Oleanonic acid (1). R s 0.48 (FS 10), [u]2o +36.1 ° (CHCI3; c 0.56). IR Ymax CHCI3 cm - 1. 1694 (C=O), 1456, 1384, 1362, 1020. MS m/z (rel. int.): 454.3470 C30H4603 calc. 454.3447 [M] + (6.5),408 [M-HCO2H] ÷ (8), 248 (100), 203 [248-CO2H] + (70), 191 (6), 189 (13), 133 (18) 119 (13). CD: Ae2ss +0.536 (MESH). 1H NMR (200 MHz, CDCI3): 30.79 (3H, s, H-26), 0.89 (3H, s, H-29), 0.91 (3H, s, H-30), 1.01 (3H, s, H-24), 1.02 (3H, s, H-25), 1.08 (3H, s, H23), 1.13 (3H, s, H-27), 2.82 (1H, dd, ./=14.4, 4Hz, H-18), 5.28 (1H, t, J=3.5 Hz, H-12). Flash chromatography of the MeSH extract (CHC13-MeOH-H20, 70:30:3) yielded a product, which was passed through Sephadex LH-20 (MESH, 600 ml) to give 3 (85 mg, 0.034%). Rf 0.34 (FS 2), powder mp 177-180°, [u]~o -30.9 ° (MESH; c 1.06). IRv~maB[cm-l: 3410 (OH), 1725 (ester carbonyl), 1640 (C=CH2). FABMS (positive ion) m/z (rel. int.): 1111.7 [M+Na] + (5.7), 948 [ M + N a - R h a ] + (0.4), 931.5 [M + Na - Glc] + (0.4), 639.3 [M + Na - (28-O-sugar chain)] + (0.5), 460.2 [aglycone+Na] + (1.2) 176.0 (100). 1HNMR (200 MHz, .
other triterpene glycosides isolated recently from this plant [5]. Acid hydrolysis of permethyl derivative 9 [11] gave the same sugars as those obtained from acid hydrolysis of the triterpene glycosides [4, 5]. They were methyl pyranosides of 2,3,4-tri-O-methylrhamnose, 2,3,4- and 2,3,6-tri-Omethylglucose (identified by GC). Therefore, the structure of the trisaccharides 8 and 8a should be identical with that of the 28-O-sugar chain of the saponin 3. Thus, the structures of the new compounds are ct-L-rhamnopyranosyl(1 -*4)-O-fl-n-glucopyranosyl(i ~ 6)-fl-D-glucopyranose (8) and its lct-epimer 8a. Although this trisaccharide moiety was found as the glycone in several plant saponins, especially those from Araliaceae [-8, 12-17] its isolation from Schefflera octophylla is, to our knowledge,
Triterpenoid saponin from Schefflera octophylla Table 1. 13CNMRspectraldataofcompound3(100.6MHz, in CD3OD) C
3
C
3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
35.2 21.9 82.5 38.3 51.1 19.2 34.4 42.1 50.8 38.0 21.9 26.7 39.3 43.6 30.8
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
32.8 57.9 50.5 48.3 151.7 31.5 37.5 29.2 22.9 16.9 16.6 15.3 176.2 110.4 19.5
1 2 3 4 5 6
Glc 1 95.1 73.6 79.3 70.8 77.9 69.4
Glc 2 104.2 75.0 76.8 77.7 76.6 61.8
Sugar moiety of 3 Rha 3-O-Glc 102.8 101.5 72.3 75.2 72.1 78.3 73.9 71.9 70.5 78.1 17.8 62.9
CD3OD): 60.85 (3H, s), 0.87 (3H, s), 0.94 (6H, s), 1.02 (3H, s) 1.26 (3H, d, J=6.3 Hz, Rham-Me), 1.69 (3H, s), 3.00 (1H, dt, J= 11.7, 5.4 Hz, H-19), 4.26 (1H, d, J = 7.5 Hz, anomeric proton), 4.38 (1H, d, J=7.5Hz, anomeric proton), 4.60, 4,73 (each 1H, br s, C=CH2), 5.46 (1H, d, J=7.5 Hz, anomeric proton), laCNMR (100.6 MHz, CD3OD): see Table 1. Peracetylation of compound 3. Compound 3 (15 mg) was acetylated with Ac20-pyridine (each 0.3 ml) at room temp. for 18 hr. The mixt. was evapd to dryness and chromatographed (CHCla) to give 4 (12.7 mg). R: 0.75 (FS 5), amorphous, [~t]~° -21.2 ° (CHCI3; c0.556). IRvma CHCI3 x cin-l: 1750 (ester carbonyl), 1640, 900 (C=CH2), 1220 (OAc). IHNMR (200MHz, CDC13): 60.80 (3H, s), 0.82 (3H, s), 0.87 (6H, s), 0.94 (3H, s), 1.12 (3H, d, J =6.1 Hz, Rha-Me), 1,66 (3H, s), 1.97-2.04 (27H, 3 x s, 9 x OAt), 2.05, 2.08, 2.10, 2.11 (each 3H, s), 2.90 (1H, t-like, br, H-19), 3.30 (1H, br s, H-3fl), 4.57, 4.71 (each 1H, br s, C=CH2), 4.85 (1H, d, J =7.7 Hz, anomeric proton), 5.64 (1H, d, J=7.7 Hz, anomeric proton). Alkaline hydrolysis of compound 3. Compound 3 (28 mg) was hydrolysed in MeOH with 5% KOH (5 ml) for 4 hr at 80°. The mixt. was passed through DOWEX 50 W × 4 (H ÷ form, MeOH) and evapd. The residue was dissolved in H20 and extracted with BuOH. The BuOH layer was evapd to dryness to give prosapogenin 5 (14mg), [~]2D° - 1 2 ° (MeOH; c0.325), t H N M R (200 MHz, pyridine-ds):/~0.74, 0.77, 0.81, 1.00, 1.24, 1.77 (each 3H, s, tert. Me), 4.77, 4.91 (each 1H, br s, C=CH2), 4.84 (1H, d, J=7.9 Hz, Glc H-I). Acid hydrolysis of compound 3. Compound 3 (20 mg) was hydrolysed with 5% HC1 (5 ml) as described for 6. 3-epi-Betulinic acid was identified as aglycone by TLC, IR and ~H NMR. The aq. layer showed the presence of the glucose and rhamnose by PC and TLC. After evapn and acetylation the glucose- and rhamnoseperacetates were identified by GC analysis.
3719
Peracetylation of prosapogenin 5. Compound 5 (5 mg) was acetylated with Ac20-pyridine (each 0.2 ml) for 7 hr at room temp. The mixt. was evapd to dryness to yield peracetate 6. R: 0.66 (FS 5), powder mp 200-201 ° (dec.), [~t]2° - 17.1° (CHCla; c 0.35). IR vC~el3 cm- 1:1740 (ester carbonyl), 1710 (CO2H), 1640, 890 (C---CH2), 1220 (OAc), 1085, 1035. 1HNMR (200MHz, CDCI3): 60.79, 0.81, 0.87, 0.90, 0.96, 1.67 (each 3H, s, tert. Me), 2.0, 2.03, 2.04, 2.07 (each 3H, s, OAc), 2.99 (IH, t-like, br, H-19), 3.30 (1H, br s, H-3fl), 4.47 (1H, d, J = 8 Hz, glucosyl-H-l), 4.58, 4.72 (each 1H, br s, C=CH2), Acid hydrolysis ofperacetate 6. Compound 6 (5 rag) was heated in MeOH with 5% HCI (1 ml) for 3 hr at 80°. The mixt. was passed through DOWEX 50 1 x 8 (OH- form) and evapd. The residue was dissolved in H20 and extracted with CHCI 3. The solvent was evapd to give 3-epi-betulinic acid (7), identified by comparison with an authentic sample. Examination of the residue of the water layer by GC and TLC showed the presence of fl-D-methylglucoside. Further elution with CHC13-MeOH-H20 (60:35:8) gave a mixt. of trisaccharides $ and ~ (625 rag, 0.25%), powder mp 148-149 °, [ct]2° -25.6 ° (MeOH; c 0.485). IR v ~ cm-l: 3340, 1060, 1030 (OH); FABMS (positive ion) m/z (rel. int.): 511 [ M + N a ] + (31), 506 [M +NH4] + (59), 309 [ M - G l c ] + (30), 325 [ M - R h a ] + (18). I H N M R (200MHz, CD3OD-D20): 61.24 (3H, d, J = 6 H z , Rha-Me), 4.52 (IH, d, J=7.6 Hz, anomeric proton), 5.12 (1H, br s, Rha-H-1). 13CNMR (50.3MHz, CD3OD-D20): 6104.1, 102.4, 97.7; 93.6, 78.9, 77.4, 76.5, 76.4, 76.1, 75.7, 74.9, 74.3, 73.4, 73.2, 70.0, 71.8, 71.0, 70.4, 69.8, 61.6, 17.8. Permethylation of compounds 8 and Sa. MeI (3.9 ml) was added to a soln of 8 and 8a (58 rag), t-BuONa (491 rag) and finely powdered NaOH (144 mg) in DMSO (4.7 ml). The mixt. was stirred at room temp. for 1 hr; poured into ice-water and extracted with Et20. The Et20 layer was washed with a satd NaCI soln, dried (Na2SO4) and evapd. Flash chromatography (CHC13-MeOH, 49:1) gave 9 (30 rag). R/0.44 (FS 4), oil, [~t]2° -29.2 ° (CHCI3; c 1.325). 1HNMR (200 MHz, CDC13): 61.26 (3H, d, J = 6 H z , Rha-Me), 4.11 (1H, d, J=7.8 Hz, anomeric proton), 4.31 (1H, d, J=7.8 Hz, anomeric proton), 4.94 (1H, d, J = 1.5 Hz, Rha-H-l). 13C NMR (22.63 MHz, CDC13): 317.6, 57.0, 57.8, 58.8, 59.5, 60.3, 60.4, 60.5, 60.9 (all q), 68.2 (d), 68.9 (t), 71.4 (t), 74.67, 74.64, 75.5, 77.3, 80.0, 81.0, 82.3, 83.9, 84.1, 84.3, 86.7, 97.5, 104.2, 104.3 (all d). Acid hydrolysis of permethyl 9. Compound 9 (5 rag) was hydrolysed in MeOH with 6% HC1 for 3 hr at 80°. The mixt. was evapd, diluted with H20 and extracted with CHCIv GC of the residue of the CHCI3 layer indicated the presence of methyl pyranosides of: 2,3,4-tri-O-methylrhamnose; 2,3,4- and 2,3,6-triO-methylglucose. Acknowledgements--T. V. Sung is indebted to the Alexander yon Humboldt Foundation for a research fellowship in the Institute of Organic Chemistry and Biochemistry, Bonn. We would like to thank Ing. Schmidt, University of Bonn, for the 1H and laC NMR spectra, Dr G. Eckhardt and Mr. Bressler (Bonn) for the mass spectra and Mr E. Gessi (University of Bonn) for the CD curves.
REFERENCES
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T.V. SUNGet al.
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