0031 9422192 $5.00+ 0.00 C 1991PergamonPressplc

Phytochemistry, Vol.31,No. 1,pp. 227.231,1992 Printedin Great Britain.

TRITERPENOIDS

T. V.

SUNG,

AND THEIR GLYCOSIDES FROM THE BARK OF SCHEFFLERA OCTOPH YLLA C. LAVAUD,*A. PORZEL,~ W. STEGLIcHt and G. ADAMS

Institute of Natural Products Chemistry, National Research Centre of SRV, Hanoi, Vietnam; *FacultCde Pharmacie, UA au CNRS no 492, Reims, France; tInstitute of Organic Chemistry and Biochemistry, University Bonn, Germany; $,Institute of Plant Biochemistry, Halle/Saale,.Germany (Received in revisedform

Key

Word

Index-Schejjka

octophylla;

28 March

1991)

Araliaceae; bark; triterpenoids; triterpenoid saponins; asiatic acid;

asiaticosid. Abstract-A

new triterpene and its glycosides were isolated from the bark of Scheflera octophylla together with asiatic acid and asiaticoside. Based on spectroscopic data, especially 2DNMR, and chemical transformations the structures of the new compounds were determined as 3u-hydroxy-urs-12-ene-23,28-dioic acid and 3a-hydroxy-urs-12-ene-23,28dioic acid 28-0-[a-L-~hamnopyranosyl(l+4)-O-/?-D-glucopyranosy~ (l-*6)]-/?-D-glucopyranoside. For the first time asiaticoside was isolated from a plant other than Centella asiatica.

INTRODUCTION Scheflera octophylla, a plant widespread in southeast Asia, is used in Vietnamese folk medicine as a tonic drug and an antirheumatic agent Cl]. In earlier communications, the isolation of a series of free as well as glycosidic lupane type triterpenes from leaves of this plant has been reported [2-4]. From the bark, long chain fatty acid esters of 3a-hydroxy-lup-20(29)-ene-23,28-dioic acid besides oleanolic acid were detected [S]. We now report the isolation and structure elucidation of a new triterpenoid and its saponin together with asiatic acid and asiaticoside from the same source.

RESULTS

AND DI!XUSSlON

Repeated flash chromatography of a methanol extract from the bark of Scheflera octophylla gave the free terpenoid and the saponin fraction. The saponin fraction was separated followed by gel filtration, elution then afforded asiaticoside (1) in 0.05% yield, needles. Compound 1 showed in FAB-mass spectrum with thioglycerol as matrix, peaks at m/z 976.6 [M+NHJ+ and 813 [M +NH,-Rha]+. Its ‘H NMR spectrum showed signals at 62.24 (d, J= 11.0 Hz, H-18) and 5.24 (t-like, H-12) in addition to six methyl signals (four singlets, a broad singlet and a doublet) indicating the presence of a A”ursene skeleton. The sugar part of the ‘H NMR spectrum showed besides the three doublets of the anomeric protons(65.29d,J=7.9Hz,Glc’-1;4.84,d,J=lSHz,Rha-1; 4.37, d, J=7.9 Hz, Glc-1) many overlapping multiplets whit. prevented an unambiguous assignment even by performing an ordinary COSY experiment. However, the 2D HOHAHA experiment allowed an easy identification of the three sugar spin systems. Combined use of the information extracted from the COSY and the HOHAHA experiment led to unequivocal assignments for all individual sugar signals of 1 (Table 1).

The ROESY cross peaks, which were observed between Glc’-l/Glc’3, Glc-l/GIc-3 and Rha-3/Rha-5, respectively, showed the spatial proximity of these 1,3-diaxially oriented protons in agreement with the results of the singlecrystal X-ray investigation of asiaticoside [6] which gave a 4C, chair conformation for both B-D-glUCOSC residues and a ‘C, chair conformation for the r+rhamnose. Assignments of “C NMR signals were done on the basis of heteronuclear shift correlations via one bond coupling (HMQC) and 2.3J,_,,,,, (HMBC); both 2D experiments were performed in inverse mode. The data (Table 2) were in good agreement with those published by Mahato et al. [6] for asiaticoside, with exception of C-29, which we assigned to 6 17.7 ([6]: 623.7) because of its one-bond correlation to the proton doublet at 60.89 (from the COSY spectrum this doublet was known to couple to H19 at 61.39, H-19 showed also a correlation to H-18, doublet at 62.24). On alkaline hydrolysis compound 1 yielded an aglycone identified as asiatic acid (2) [6-8] besides methyl asiatate (3) as minor product. Compound 2 was methylated with diazomethane to give 3 and acetylated to give acetate 4. Asiaticoside was first found in Centella asiatica (Umbelliferae) [S, 91, a plant used in India and Madagascar for the treatment of leprosy [lo] and in Vietnam for liver diseases [l]. Its occurrence in SchefJlera octophylla is the first isolation of this compound from a natural source other than Centella asiatica. Further elution gave the main saponin 5 in 0.075% yield. Compound 5 showed quasi-molecular ion peaks at m/z 979.7 [M + Na]+ and 974.8 [M + NH,] •I in the FAB mass spectrum. Its IR spectrum contained absorptions for hydroxyl (3400) and ester carbonyl groups (1720cm- ‘). Acid hydrolysis of 5 provided aglycone 6 besides glucose and rhamnose as sugar components, identified by TLC and PC. Alkaline hydrolysis of 5 also afforded 6, establishing that the sugar chain in 5 was linked to the aglycone via an ester bond. This was

227

228

T.V. SUNGet al. Table 1. ‘H NMR spectral data of compounds 1 and 5 (S:ppm, J: Hz, 300 MHz, in CD,OD)

C0,R2 **

1 2 3

4

R’ fj H H AC

R’ a-L-RhaiS-D-Cic”P-D-Glc’

H Me H ?Z = =

C02RS 21

H

1

2 3 12 18 19 23 Me-24 Me-25 Me-26 Me-27 Me-29 Me-30 Glc’ 1 2 3 4 5 6a 6b Glc

3.61 ddd (10/9.5/4)*

1

23

5 6

R’ H H

7H

8

9 10

II 12

AC H H H AC

R2 H H

R’ a-L-Rha4-8-D-GIc6-8-D-Glc’

H

H

Me

H Me Me Me H

Me e-L-Rha4-8-D-GlC6-8-D-Glc’

H Me H

2 3 4 5 6a 6b Rha 1 2 3 4 5 Me

5

3.34 d (9.6)t 5.24 br t (3.4) 2.24 d (11.0) 1.38 m 3.46 d (10.8)/3.26 d (10.8)$ 0.71 s 1.05 s 0.84 s 1.13 s 0.90 d (6.6) 0.97 br s

1.12 s 0.99 s 0.83 s 1.14 s 0.89 d (6.5) 0.98 br s

5.29 d (7.9) 3.31 m 3.39 m 3.42 m 3.49 m 4.06 dd (12.2/1.7) 3.77 dd (11.8/5.1)

5.28 d (7.8) 3.32 m 3.4 m 3.4 m 3.51 m 4.08 br dd (11.7/1.7) 3.76 dd (11.7/H)

4.37 d (7.9) 3.23 t (8.4) 3.45 t (8.8) 3.53 t (9.0) 3.29 m 3.80 br d (11.5) 3.65 dd (12.2/4.1)

4.38 d (7.8) 3.23 t (8.5) 3.45 f (9) 3.54 t (9) 3.28 m 3.81 br d (12.5) 3.65 dd (12.5/3.2)

4.84 d (1.5) 3.83 dd (3.1/7) 3.62 dd (9.4/3.4) 3.40 t (9.4) 3.96 dq (9.516.2) 1.27 d (6.1)

4.85 d (1.6) 3.83 dd (3.3/1.6) 3.63 dd 9.513.5) 3.40 t (9.5) 3.96 dq (9.516.2) 1.26 d (6.3)

3.67 br s8 5.24 br t (3.4) 2.24 d (11.2) 1.39 m

*H-2B;tH-3a; $CH,H,OH; §H-3fi.

supported by the ‘H NMR spectrum of 5 (Table l), which showed three anomeric protons at 64.38, 4.85 and 5.28, the latter chemical shift indicating that one of the sugars is connected to the genin via a carboxylic acid group. Comparison of the 2D HOHAHA spectrum of5 with that of 1 confirmed the identity of the sugar residues of both compounds. This was proved by a 2D ROESY spectrum of 5, which showed, besides intrasugar cross peaks, one structurally important correlation between Glc-1 (64.38) and Glc’-6 (63.77) indicating that the two glucopyranosyl residues are connected by a 1+6 linkage. Furthermore, the HMBC experiment showed long range couplings between Glc-H-1 (64.38) and Glc’-C-6 (669.6), Glc-H-4 (63.54) and Rha-C-l (6102.9) as well as Glc’-H-1 (65.28) and C-28 of the genin (6 178.0). Therefore, the sugar chain was determined to be [a-L-rhamnopyranosyl(1 --*4)-O+Dglucopyranosyl( 1 -+ 6)-0-/I-D-glucopyranosidelinked to the carboxylic acid function at C-28. The ‘H NMR spectrum as well as the 13CNMR spectrum of 5 (Tables 1 and 2) showed characteristic signals for urs-12-ene derivatives [11,12] with two carboxylic acid functions. A three-bond correlation in the HMBC spectrum between the methyl

protons at C-24 (61.12) and the carboxylic acid signal at S 183.8 led to the assignment C-23 for the latter. Another three-bond correlation from the same protons to the methine signal at 673.4 showed C-3 of the genin to be hydroxylated. A ROESY cross peak betwen Me-24 (S 1.12) and H-3 (83.67 br s) indicated this proton to be in the /?-position with a 3a-OH substitution, Thus the new saponin is 3u-hydroxy-urs-12ene-23,28-dioic acid 28-0-[cc-L-rhamnopyranosyl (1 + 4)-0-/I-D-glu~opyrano syl (1 -+ 6)]-@@ucopyranoside (5). The location of the ester bond was also determined by methylation of 5 with diazomethane into methyl ester 9, which upon mild alkaline hydrolysis with 5% methanolic KOH [13, 143 yielded monomethylester 10 besides small amounts of diester 11.The EI mass spectrum of 10with a base peak at m/z 248 (retro-Diels-Alder fragment) indicated the presence of a CO,H-28 group. Therefore, the sugar chain of 5 must be linked at C-28. The EI mass spectrum of aglycone 6 contained peaks at m/z 486.3346 (C30H460s [Ml’), 248 (base peak), 203,189 and 133 indicating that 6 was an amyrin derivative with a free carboxylic group at C-l 7 [ 15, 161. The H-3 signal of

Triterpenoids from Scheflera Table 2. ‘“CNMR

C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Glc’ 1 2 3 4 5 6 Glc 1 2 3 4 5 6 Rha 1 2 3 4 5 6

spectral data of compounds 75.5 MHz, (in CD,OD)

1

5

48.3 69.7 78.2 44.1 48.4 19.1 33.6 41.0 49.3 39.0 24.5 127.0 139.3 43.4 29.3 25.3 49.5 54.1 40.2 40.4 31.5 37.6 66.4 14.0 17.9 18.1 24.0 178.0 17.6 21.6

33.8 25.8 73.4 51.8 46.3 22.1 33.9 41.4 49.2 37.8 24.3 127.1 139.2 43.4 29.3 25.3 49.7 54.1 40.4 40.2 31.6 37.7 183.8 18.4 16.6 18.2 24.1 178.0 17.7 21.6

95.8 73.8 78.1 71.0 77.9 69.6

95.9 73.8 78.2 71.1 77.9 69.6

104.5 75.3 76.7 79.6 76.8 61.9

104.4 75.3 76.7 79.5 76.8 61.9

102.9 72.4 72.2 73.8 70.7 17.8

102.9 72.4 72.2 73.8 70.6 17.8

1, 5 and 6

6 (50.3 MHz) 33.8 25.9 73.6 51.8 45.4 22.2 33.4 41.2 48.9 37.7 24.2 126.7 139.7 43.4 30.8 25.3 48.7 54.3 40.4 40.3 31.8 37.9 181.7 18.1 16.3 17.9 24.3 179.8 17.7 21.6

the ‘HNMR spectrum appeared as a broad singlet at 63.73 indicating Qe axial (a) position for the OH-3 group [17]. Upon acetylation 6 yielded acetate 12. The free terpenoid fraction was acetylated with acetic anhydride-pyridine and then flash-chromatographed to give the acetates 4 and 12 in 0.025 and 0.015% yield, respectively. The ‘H NMR, IR and mass spectra and R,

octophylla

229

of compounds 4 and 12, respectively, were identical with those of the products obtained from hydrolysis of saponins 1 and 5 followed by acetylation. This is an indirect proof for the occurrence of the new compound 3~ hydroxy-urs-12-ene-23,28-dioic acid (6) and asiatic acid (2) in the methanol extract of the bark of Schefle*a octophylla. These two compounds (6 and 2) have been identified by TLC in the methanol extract. However, they cannot be isolated in the pure forms by chromatography on silica gel and on Sephadex LH 20.

EXPERIMENTAL

General. Mps: uncorr. MS ionization energy: 70eV. 13CNMR: 50.3 and 75.5 MHz. ‘H NMR: 200 and 300 MHz. TLC employed precoated silica gel plates 60 F,,, (Merck). The following TLC solvent systems were used: for saponins: CHCl,MeOH-H,O (60: 35:s) (FSl) and (70:30:3) (FS2); for sapogenins and triterpenoids: CHCl,-MeOH (19:l) (FS5) and (9:l) (FS3); for monosaccharides: n-BuOH-iso-PrOH-H,O (5:3:1) (FSll). Spray reagents were for saponins and triterpenoids: vanillin-H,SO,; for the sugars: aniline phthalate and orcine reagent. For flash chromatography silica gel 60, 40-63 pm (Merck and Baker) was used. Sephadex LH 20: Pharmacia Uppsala, Sweden; Lobar-Fertig&le LiChroprep RP-8, Size B (Merck). Isolation. Dried finely powdered bark of Scheflera octophylla (200g) was extracted with hot MeOH for 15 hr at 70” and the extract was filtered and evapd. The residue was dissolved in H,O and extracted with petrol. The aq. layer was evapd to dryness and subjected to chromatography. Elution with CHCl,-MeOH (19:l) afforded the free triterpenoid frs (200 mg), which were acetylated with A@-pyridine (each 1 ml) for 18 hr at room temp. The mixture was evapd to dryness and the residue was repeatedly flash chromatographed (CHCl,-MeOH, 19: 1) to give acetates 4 (50mg, 0.025%) and 12 (26mg, 0.015%). Compounds 4 and 12 were identical with those obtained from alkaline hydrolysis of saponins 1 and 5 followed by acetylation (Ac,O-pyridine). Further elution with CHCl,-MeOH-H,O (60: 35 :8) gave the saponin frs, which were chromatographed on a Labar LiChroprep RP-8 (40-63 pm) column followed by Sephadex LH 20 to afford l(lO0 m& 0.05% yield) and 5 (150 mg, 0.075% yield). Asiaticoside (1). R, 0.59 (FSl). Needles mp 23&232” (dec.) (MeOH). [a]$‘-15’ (MeOH, c 1.60). (lit. [6]: mp. 23&232” (dec.); [a&-- 16” (MeOH)). FAB-MS (positive ion) m/z (rel. int.): 976.6 [M +NHJ+ (6.2), 830.5 [M+NH,-CsHlOO.,]+ (0.4), 813 [M+NH,-Rha]+ (0.3), 635.4 (1.6), 453.3 (14), 232.1 (Sl), 181.0(78), 126.1(100). ‘HNMRseeTable 1. “CNMR seeTable 2. Alkaline hydrolysis of asiaticoside (1). Compound 1 (53 mg) was hydrolysed in MeOH with 5% KOH (6 ml) for 3 hr at 80”. The soln was passed through Dowex 50W x 4, evapd and the residue was dissolved in H,O. The ppt. was filtered and flash chromatographed (CHCl,-MeOH, 19: 1) to give asiatic acid (2, 18 mg) and methyl asiatate (3, 5 mg). Asiatic acid (2). R, 0.46 (FS3). Mp 242-244” (MeGH) (lit. [7]: mp 238-242”). [a]i’+46” (MeOH, c 0.26) (lit. [9]: [a]o+Sl” (EtOH; c 2.06)). IR ~2; cm-‘; 3410 (OH), 1690 (CO,H). MS m/z (rel. int.): 488 [M]‘(Sh 473 [M-Me]+ (3), 470 [M-H,O]+ (4), 442 [M-HCO,H]+ (2),248(100),203 [248-CO,H]+ (67), 191 (14), 173 (9), 133 (48). ‘H NMR (3OOMH2,CDCl,-MeOH): 60.81 (6H, s, H,-24 and H,-26), 0.86 (3H, d, 5=6.8 Hz, H,-29), 0.94 (3H, br d, H,-30), 1.02 (3H, s, H,-25), 1.09 (3H, s, H,-27), 2.20 (br d,J=ll Hz, H-18), 3,34(lH,d,.I=9,5Hz, H-3a), 3.35(1H,d,J = 10.9 Hz, H,-23), 3.55 (lH, d, J = 10.9 Hz, H,-23), 3.72 (lH, ddd,

Triterpenoids from Scheflera octophylla 12. Seo, S., Tomita, Y. and Tori, K. (1975) Tetrahedron Letters 7. 13. Hikada, K., Ito, M., Matsuda, Y., Kohda, H., Yamasaki, K. and Yamahara, J. (1987) Phytochemistry X,2023. 14. Gao, F., Chen, F., Tanaka, T., Kasai, R., Seto, T. and Tanaka, 0. (1985) Chem. Pharm. Bull. 33,37. 15. Karliner, J. and Djerassi, C. (1966) J. Org. Chem. 31, 1945.

231

16. 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.

17. De la Torte, M. C., Bruno, M., Piozzi, F., Savano, G., Rodriguez, B. and Arnold, N. A. (1990) Phytochemistry 29, 668.

Triterpenoids and their glycosides from the bark of Schefflera octophylla.

A new triterpene and its glycosides were isolated from the bark of Schefflera octophylla together with asiatic acid and asiaticoside. Based on spectro...
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