Abstract

The aerial parts of Osteospermum vaillantii (DECNE) T.Norl., collected in Saudi Arabia, yielded four saponin glyco-sides, one of which was a new natural product. The struc-tures of the isolated compounds were elucidated using MS,1H, 13C NMR, 1H-1H COSY, HMQC, HMBC and HOHAHAexperiments.

Keywords: Osteospermum vaillantii, Asteraceae, saponinglycosides, NMR, MS.

Introduction

Osteospermum is the largest genus (ca. 70 species) in thesmall tribe Calendulaceae, family Asteraceae. Only a fewspecies of genus Osteospermum have been chemically in-vestigated. From different representatives of the genusOsteospermum, diterpenes of the sandaracopimarene typewere isolated (Bohlmann et al., 1973; Bohlmann & Zdero,1975). Several Osteospermum species were investigated(Bohlmann et al., 1983; Jakupovic et al., 1988) to affordtriterpenes, tridecapentayene, p-hydroxyacetophenone deriv-atives, three diterpenes from the rare cassane type, two san-daracopimarene derivatives, sesquiterpene glycosides, andthree trachylobane derivatives. We report here the first isola-tion of triterpene glycosides from the genus Osteospermum,one of which is new and the other three have been isolatedfrom other sources.

Materials and methods

Melting points were recorded on Gallen Kamp melting pointapparatus (England) and were uncorrected. IR spectra wererecorded on Pye Unicam Sp3–300. Optical rotation was measured at room temp. using a Perkin-Elmer 241 MC anda JASCO DIP-360 automatic polarimeter. 1H and 13C NMRspectra were recorded on a JEOL JNA-LA 400WB-FT

(Tokyo, Japan) or a Varian 300 (USA) spectrometer at400/100 or 300/75MHz, respectively. Atmospheric pressureionization mass spectra (API-MS) were recorded using a PESCIEX API III Biomolecular mass analyzer and EI-MS on aShimadzu PQ-5000. Flash silica gel 60 was used for columnchromatography (63–200mm, Merck, Darmstadt).

Plant material

The aerial parts of O. vaillantii (Decne) T. Norl. were col-lected in the Southern region of Saudi Arabia (Abha) inApril, 1997. A voucher specimen (# 13315) was deposited inthe herbarium of the College of Pharmacy, King Saud Uni-versity, Riyadh, Saudi Arabia. The plant material was kindlyidentified by Dr. Sultan Ul-Abedin, College of Pharmacy,King Saud University, Riyadh, Saudi Arabia.

Extraction and isolation

The dried ground aerial parts of O. vaillantii (1kg) weredefatted with ether followed by percolation with ethanol atroom temp. The ethanol extract (120g) was suspended in 500ml of water and shaken with ethyl acetate followed by n-butanol to give, on evaporation of solvents, 24 and 27gfractions, respectively. The n-butanol extract (10g) wasadsorbed on a small amount of silica gel and chro-matographed on a Si gel column (5.5 ¥ 15cm) using CHCl3-MeOH-H2O (4 :1 :0.1) as eluent and 120ml fractions werecollected. Three main fractions were collected: fr-A(1200–1800ml), fr-B (3250–5000ml) and fr-C (7800–8160ml). Crystallization of fraction A yielded compound 1 (430mg) as needles (MeOH/ether). Fraction B (1.4g) was puri-fied on a Si gel column (3.5 ¥ 18cm) using CHCl3-MeOH-H2O (13 :7 :0.6) as eluent (10ml fraction), to affordcompound 2 (230mg) which separated from the fraction B-1 eluted between 200–250ml. Fraction C (0.8g) was rechro-matographed on a Si gel column (2.5 ¥ 16cm) using

Accepted: March 27, 2001

Address correspondence to: Essam Abdel-Sattar, Pharmacognosy Department, College of Pharmacy, Cairo University, El-Kasr El-AiniStreet, Cairo 11562, Egypt. Fax: 0020-2-3624105, E-mail: abdel-sattar@excite.com

Saponin Glycosides from Osteospermum vaillantii

Essam Abdel-Sattar

Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, Egypt

Pharmaceutical Biology 1388-0209/01/3906-440$16.002001, Vol. 39, No. 6, pp. 440–444 © Swets & Zeitlinger

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Saponin glycosides from Osteospermum vaillantii 441

CHCl3-MeOH-H2O (3 :1.5 :0.3) as eluent and 10ml fractionswere collected. Compound 3 (43mg) was separated fromfraction C-1 eluted between 350–450ml, while compound 4 was crystallized from fraction C-2 eluted between 850–950ml as sandy crystals (15mg, MeOH/ether).

Acid and alkaline hydrolyses were performed on 5–10mg samples according to methods previously described by Pizza et al. (1987).

Compound 1. Needle crystals, mp 229–231°C,C42H68O13, [a]D 9.7° (c, 0.91, MeOH); IR nmax (KBr) cm-1:3420, 1730, 1490, 1070; 1H NMR (C5D5N-d5) d 0.81, 0.96,0.97, 0.98, 1.02, 1.30, 1.32 (7 Me groups), 3.36 (1H, dd, J =12.5, 4.3Hz, H-3), 4.86 (1H, d, J = 7.7Hz, H-1, Glc-I), 5.19(1H, d, J = 7.7Hz, H-1, Gal.), 5.48 (1H, br m, H-12); 13CNMR, see Tables 1 and 2; API-MS m/z (rel. int.): in positiveion mode 819 [M + K]+ (18), 803 [M + Na]+ (100), 457 [M+ H - 2 hexoses]+ (19), 439 [M + H - H2O - 2 hexoses]+ (9),in negative ion mode: 815 [M + 2H2O - H]- (46), 779 [M -H]- (97), 617 [M - H - hexose]- (44), 599 [M - H - hexose- H2O]- (38), 455 [M - H - 2 hexoses]- (34).

Compound 2. White amorphous powder, mp 204–205°C,C48H78O18, [a]D 4.3° (c, 0.18, MeOH); IR nmax (KBr) cm-1: 3400, 1730, 1460, 1080; 1H NMR (C5D5N-d5) d0.81, 0.86, 0.89, 0.96, 1.07, 1.25, 1.27 (7 Me groups), 3.33(1H, dd, J = 12, 4.4Hz, H-3), 4.84 (1H, d, J = 7.7Hz, H-1,Glc-I at C-3), 5.18 (1H, d, J = 7.7Hz, H-1, Gal), 5.41 (1H, br m, H-12), 6.29 (1H, d, J = 8.2Hz, H-1, Glc-III at C-28); 13C NMR, see Tables 1 and 2; API-MS m/z (rel. int.): inpositive ion mode 965 [M + Na]+ (100), 803 [M + Na -hexose]+ (6), in negative ion mode: 977 [M - H + 2H2O]-

(45), 779 [M - H - hexose]- (100), 599 [M - H - 2 hexoses- H2O]- (5).

Compound 3. White amorphous powder, mp 224–226°C,C54H88O13, [a]D 14.1° (c, 0.1, MeOH); IR nmax (KBr) cm-1:3400, 1740, 1460, 1080; 1H NMR (C5D5N-d5) d 0.82, 0.89,0.91, 2 ¥ 1.08, 2 ¥ 1.26 (7 Me groups), 3.27 (1H, dd, J =11.2, 4Hz, H-3), 4.82 (1H, d, J = 7.5Hz, H-1, Glc-I at C-3),5.28 (1H, d, J = 7.7Hz, H-1, Gal), 5.43 (1H, br m, H-12),5.67 (1H, d, J = 7.8 Hz, H-1, Glc-II), 6.32 (1H, d, J = 8.2Hz,H-1, Glc-III at C-28); 13C NMR, see Tables 1 and 2; API-MSm/z (rel. int.): in positive ion mode 1143 [M + K]+ (25), 1127 [M + Na]+ (100), in negative ion mode 1193 [M + 5H2O- H]- (13) 1141 [M + 2H2O - 2H ]- (45) 941 [M - H -hexose]- (100), 779 [M - H - 2 hexoses]- (13), 617 [M - H- 3 hexoses]- (5), 455 [M - H - 4 hexoses]- (18).

Compound 4. Sandy crystals, mp 191–192°C, C36H58O8,[a]D 12.8° (c, 0.09, MeOH); IR nmax (KBr) cm-1: 3400, 1730,1600, 1420, 1070; 1H NMR (C5D5N-d5) d 0.82, 0.92, 0.96,0.98, 2 ¥ 1.12, 1.3 (7 Me groups), 5.46 (1H, br m, H-12),6.32 (1H, d, J = 7.7Hz, H-1, Glc-III at C-28); 13C NMR, seeTables 1 and 2; API-MS m/z (rel. int.) in positive ion mode641 [M + Na]+ (9), 619 [M + H]+ (7), 479 [M + Na - hexose]+

(5), in negative ion mode: 671 [M - H + 3H2O]- (3), 455 [M- H - hexose]- (6).

Oleanolic acid: Mp 298–300°C, 1H NMR d 0.77, 0.78, 2¥ 0.91, 0.93, 0.98, 1.14 (7 Me), 2.83 (1H, dd, J = 13.7, 4.5

Hz, H-3), 5.27 (1H, m, H-12), 13C NMR, see Table 1. EI-MS:m/z 457 [M + H]+ (5).

Results and discussion

The aerial parts of the air-dried plant material were defattedwith ether followed by extraction with ethanol. The ethano-lic extract was fractionated between water and each of ethylacetate and n-butanol. The n-butanol extract was repeatedlychromatographed on Si gel columns to afford four saponinglycosides (1–4). Acid hydrolysis of compounds 1–4afforded a single aglycone, identified as oleanolic acid asconfirmed by comparison with an authentic sample (TLC, 1Hand 13C NMR).

Compound 4 had a molecular formula C36H58O8 and its IRspectrum showed the presence of bands at 3400, 1730 and1600cm-1. Basic hydrolysis of 4 (see experimental) yieldedoleanolic acid (TLC) and glucose (TLC). The spectral data(API-MS, 1H- and 13C NMR) are superimposable with thosedescribed for oleanolic acid 28-O-b-D-glucopyranoside pre-viously isolated from Panax japonicum (Cai et al., 1982) andreported here for the first time.

The IR spectrum of compound 1 showed bands at 3520 (-OH) and 1730 (C¨O) cm-1. The atmospheric pressure ion-ization (API) mass spectrum of 1 (positive ion mode) yieldedquasi molecular peaks at m/z 819 [M + K]+ and m/z 803 [M+ Na]+ indicating a molecular weight of 780 in agreementwith the molecular formula C42H68O13. Acid hydrolysis of 1afforded oleanolic acid, glucose and galactose (TLC). The 1Hand 13C NMR data showed two anomeric protons and carbons(dH 5.19, 4.86 and dC 106.3, 106.2) confirming the presenceof two sugar moieties (glucose and galactose, TLC). Thesugar protons were unambiguously assigned using 1H-1HCOSY, HMBC and HOHAHA experiments. The attachmentsof the glucose (Glc-I) unit to C-3 of the aglycone, and of thegalactose unit to C-3 of glucose (Glc-I), were confirmed byHMBC and by the observation of the downfield shift of therespective carbons to those of aglycone and methyl gluco-side (Agrawal, 1992). On this basis, compound 1 could beidentified as oleanolic acid 3-b-O-[O-b-D-galactopyra-nosyl](1Æ3)-b-D-glucopyranoside and was further con-firmed by comparison with the reported data for arvensosideB, a triterpenoid saponin previously isolated from Calendulaarvensis (Chemli et al., 1987; Pizza et al., 1987).

Basic hydrolysis of compound 2 yielded glucose and 1(TLC) indicating a similar structure with an additionalglucose moiety at C-28. API-MS (positive ion mode) of 2yielded quasi molecular peaks at m/z 965 [M + Na]+ and m/z977 [M + 2H2O - H]- (negative ion mode) indicating a molecular weight of 942, in agreement with the molecularformula C48H78O18. The presence of three anomeric protons and carbons (dH 6.29, 5.18, 4.84 and dC 106.4, 106.3, 95.7) indicated the presence of three sugar moieties(glucose and galactose). The glycosilation sites at C-3 and C-28 was ascertained similar to those of compounds 1 and 4.

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On the basis of the foregoing data, and by comparison withthe data of compounds 1 and 4, the structure of 2 was estab-lished as 3-b-O-[O-b-D-galactopyranosyl (1Æ3)-b-D-glu-copyranosyl] oleanolic acid 28-b-D-glucopyranosyl ester.This was further confirmed by comparison with the datareported for arvenoside A, a saponin glycoside previouslyisolated from Calendula arvensis (Chemli et al., 1987; Pizzaet al., 1987).

Compound 3 had a molecular formula of C54H88O23 asdeduced from quasi molecular peaks at m/z 1127 [M + Na]+

and 1143 [M + K]+ in API-MS (positive ion mode). The NMRspectral data of 3 showed the presence of four anomericprotons and carbons (dH 6.32, 5.67, 5.28, 4.82 and dC 105.2,104.9, 103.8, 95.7) indicating the presence of four sugar moi-eties (glucose and galactose, TLC). A b-glucopyranosyl (glc-II) moiety attached to C-28 was confirmed from the basic

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Saponin glycosides from Osteospermum vaillantii 443

Table 1. 13C NMR spectral data of aglycone moieties of com-pounds 1–4*.

C 1 2 3 4 Oleanolic acid

1 38.6 38.7 38.7 38.7 38.52 26.4 26.4 26.5 27.2 26.83 89.0 89.0 89.4 80.0 78.84 39.4 39.5 39.6 39.6 38.75 55.8 55.8 55.8 55.8 55.26 18.4 18.5 18.5 18.6 18.47 33.1 32.5 32.5 32.3 32.68 39.7 39.9 39.9 40.0 39.39 47.9 48.0 48.0 48.0 47.6

10 36.9 36.9 36.9 37.0 37.011 23.7 23.8 23.7 23.9 23.112 122.5 122.8 122.8 122.8 122.213 144.8 144.1 144.1 144.0 143.714 42.12 42.1 42.1 42.2 41.715 28.3 28.2 28.2 28.4 27.716 23.6 23.4 23.4 23.5 23.417 46.6 47.0 47.0 47.1 46.418 41.9 41.7 41.7 41.8 41.319 46.4 46.2 46.2 45.1 46.020 30.9 30.8 30.8 30.9 30.721 34.2 34.0 34.0 34.1 33.922 33.2 33.1 33.1 33.3 33.123 28.1 28.1 28.0 28.4 28.024 17.0 17.0 16.7 17.2 15.625 15.4 15.5 15.5 15.8 15.326 17.3 17.5 17.4 17.6 16.927 26.2 26.1 26.1 26.3 25.928 180.2 176.4 176.4 176.3 180.729 33.2 33.1 33.1 33.4 32.730 23.7 23.6 23.6 23.8 23.6

* Assignments were made on the basis of HMBC and HMQC.

Table 2. 13C NMR spectral data of sugar moieties of compounds1–4*.

C 1 2 3 4

Glc-I1 106.2 106.3 104.92 74.4 74.4 79.23 88.1 88.9 88.74 69.8 69.8 69.95 77.8 77.9 77.76 62.6 62.6 62.6Glc-II1 103.82 76.43 78.64 72.65 77.66 63.4Glc-III1 95.7 95.7 95.72 74.1 74.1 74.13 79.3 79.3 79.64 71.1 71.1 71.15 78.9 78.9 79.26 62.2 62.2 62.2Gal1 106.3 106.4 105.22 72.9 72.9 72.93 75.0 75.1 75.44 70.1 70.1 70.15 77.3 77.3 77.36 62.0 62.1 61.9

* Assignments were made on the basis of HMBC and HMQC.

hydrolysis and from the presence of anomeric signals at dH

6.32 (J = 8.2Hz) and dC 95.73 (see NMR data of 2 and 4).The C-3 as the other glycosilation site in the aglycone hasbeen ascertained by comparing the chemical shift of C-3(HMQC) with those of the aglycone and of compounds 1 and2 (see Table 1). Comparison of the 1H and 13C NMR data ofcompound 3 with those of 2 showed similar spectra with anadditional sugar moiety in 3, which was deduced as glucose(Glc-II). The additional glucose unit (Glc-II) was shown tobe attached to C-2 of the glucose moiety (Glc-I) due toobservation of the downfield shift of C-2 (+5ppm) relativeto the respective carbons in 1 and 2. Similarly, the galactosemoiety was shown to be attached to C-3 of Glc-I by obser-vation of a downfield shift (+10ppm) of C-3 relative tomethyl glucoside (Agrawal, 1992) and by comparison withthe data of compounds 1 and 2 (Table 2). On the basis of the foregoing data, the structure of compound 3 was establi-shed as 3-b-O-{[O-b-D-galactopyranosyl-(1Æ3)-b-D-glucopyranosyl-(1Æ2)]-b-D-glucooyrano-syl} oleanolic

acid 28-b-D-glucopyranosyl ester. This compound was iso-lated for the first time from nature.

Acknowledgement

The authors are indebted to Dr. Meselhy R. Meselhy, Phar-macognosy Department, College of Pharmacy, Cairo Uni-versity for running the NMR spectra and for valuablediscussions, and to Dr. Sultan Ul-Abedin, College of Phar-macy, King Saud University, Riyadh, Saudi Arabia for iden-tification of the plant material.

References

Agrawal PK (1992): NMR Spectroscopy in the structural eluci-dation of oligosaccharides and glycosides. Phytochemistry31: 3307–3330.

Bohlmann F, Weikgenannt G, Zdero C (1973): Neue diterpeneaus der tribus Calendulaceae. Chem Ber 106: 826–840.

Bohlmann F, Zdero C (1975): Notiz uber ein weiteres Diterpenaus Osteospermum subulatum DC. Chem Ber 108:362–363.

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444 E. Abdel-Sattar

Bohlmann F, Wallmeyer M, Jakupovic J, Ziesche J (1983): Diter-penes and sesquiterpenes from Osteospermum species.Phytochemistry 22: 1645–1651.

Cai P, Xiao Z, Wei J (1982): Studies on the chemical constituentsof Zhu Jie Shen (Panax japonicum). Zhongcaoyao 13: 1–2.Through C.A. 97, 107020 (1982).

Chemli R, Babadjamian A, Faure R, Boukef K, Balansard G, Vidal E (1987): Arvensoside A and B, triterpenoid

saponins from Calendula arvensis. Phytochemistry 26:1785–1788.

Jakupovic J, Zdero C, Paredels L, Bohlmann F (1988): Ses-quiterpene glycosides and other constituents fromOsteospermum species. Phytochemistry 27: 2881–2886.

Pizza, C, Zhong-Liang Z, De Tommasi N (1987): Plant metabo-lites. Triterpenoid saponins from Calendula arvensis. J NatProd 50: 927–993.

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