- Email: [email protected]
Saponins from Albizzia lucida
Phyrochemistry, Vol. 30, No. 12, pp. 41114115, 1991 Printedin Great Britain.
SAPONINS
0031-9422/91$3.00+0.00 Q 1991Pergamon Press plc
FROM
ALBIZZIA
LUCZDA
FULV~A ORSINI, FRANCESCAPELIZZONI and LUISELLAVEROTTA* Centro di Studio sulle Sostanze Organiche Naturali de1 CNR, Dipartimento di Chimica Organica e Industriale, via Venezian 21, 20133 Milano, Italy (Received4 March 1991)
Key Word Index--Albizzia
lucida; Leguminosae; Mimosaceae; seeds; echinocystic acid glycosides; cytotoxic
activity.
Abstract-Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-o-[fl-D-xylopyranosyl(l+2)-C+Larabinopyranosyl (1+6)] [/I-D-glucopyranosyl (l-+2)] /I-D-glucopyranosyl echinocystic acid; 3-O-[a-L-arabinopyranosyl (1+6)] [p-D-ghrcopyranosyl (l-+2)]-/I-D-glucopyranosyl echinocystic acid and 3-0-[b-D-xylopyranosyl (1+2)+-D-fucopyranosyl (1+6)-2-acetamido-2-deoxy-B-Dglucopyranosyl echinocystic acid, characterized as its methyl ester.
INTRODUCTION Albizziu lucida is frequently reported together with Albizzia bbbeck because both plants are similar: they are widespread in India and southern Africa and are hosts of the lac inset [ 1J. The seeds of the two plants and other Albizzia species were studied because of their lipid composition and their use as a food [2]. Recently we have isolated, from the seeds of A. lucida, a neurotoxin, 3-hydroxy-5-hydroxymethyl-4-methoxymethyl2-methylpyridine 3-O-jI-D-ghrcoside (5) [3]. We now report the isolation and the structure of three main saponins (l-3) from the seeds. Two of them showed cytotoxic activity in the brine shrimps test at a concentration lower than 50 ppm [4]. RISULTS AND DISCUSSION
The methanol extract of A. lucida was partitioned between n-butanol and water. After digestion with diethyl ether, the butanol extract (4% of the dried plant material) showed on TLC a sequence of several spots distributed between R, 0.1 and 0.6. One of them proved to be compound 5 which was first isolated from the aqueous extract [3]. The others, after spraying the TLC plate with sulphuric acid, appeared as plum coloured spots. The butanol extract was purified by repeated chromatographic steps involving gel filtration (Sephadex LH-20 and Fractogel TSK HW 40), reversed phase medium pressure liquid chromatography (RPMPLC) and DCCC. Three main compounds (l-3) were isolated. By acid hydrolysis (HCl-MeOH) of compound 1, the sapogenin echinocystic acid was isolated and identified from its physical data (TLC, ‘H NMR) and comparison with an authentic sample. Silylation of the hydrolysate [5] allowed the identification of glucose and arabinose in the ratio 2: 1 by capillary gas-chromatography in compari*Author to whom correspondence should be addressed. ~ sugars have been assumed D or L as in the natural series.
son with pure standards. The i3CNMR spectrum (52.4 MHz, pyridine-d,) of 1 confirmed the presence of a chain of three saccharidic units (anomeric carbons at 6 105.8, 105.4 and 105.3 typical for a /?-configuration for D-glucose and a-configuration for L-arabinose [6]),t linked to the C-3 hydroxyl group of echinocystic acid (6 89.6). The ‘H NMR spectrum (200 MHz, pyridine-d,) confirmed the oleanane skeleton (65.62, dd, Jiz-ll~ =J12_ll.=3 HgH-12;63.19,dd,J1sa-,9~=14,J,,,_,,, =4 Hz, H-18/?; 65.25, dd, J168-15.= J16,+158 = 3 Hz, H-16/3; seven singlet methyl groups at S 1.77,1.17,
1.13, 1.05, 1.02, 0.95 and 0.80). The anomeric protons appeared at 65.32 (d, J= 7 Hzk 64.91 (d, J= 7 Hz); 64.85 (d, J = 7 Hz). Compound 1 showed ions at m/z 951 [M + Na] + and 929 [M + H] + in the positive FAB mass spectrum. In the negative FAB mass spectrum a quasi molecular ion was observed at m/z 927 [M-H]and fragments at m/z 795 [M - 132 -HIand 765 [M - 162 -HI-. From the FAB mass spectrum it is noteworthy that 1 possesses a branched saccharidic chain as shown by the contemporary loss of the pentose unit (m/z 795) and the hexose unit (m/z 765) from the molecular ion. The “C NMR spectrum of 1, supported by a DEPT experiment [a showed a signal at 662.8 (t) characteristic of the hydroxymethylene group of a terminal glucose unit and a signal at 669.9 (t), attributed to the hydroxymethylene group of a glucose unit, downfield shifted by the linkage with the arabinopyranosyl moiety. The triplet at 666.6 was assigned to the hydroxymethylene of arabinose. The terminal glucose and arabinose were reasonably assigned in comparison with the data of methyl-~-D-glucopyranoside and methyl-a+arabinopyranoside [6]. As co&med by the glycosilation shifts [6] the 3-O-linked-p-Dglucopyranosyl unit had the two sugars linked in positions C-2 and C-6 (Table 1). From these considerations 1 was assigned as 3-0-[a+arabinopyranosyl-( 1+6)] [j?D-glucopyranosyl-(1+2)]-fl-D-glucopyranosyl echinocystic acid. The most polar compound 2 showed a quasi molecular ion at m/z 1059 [M-IIIin the negative FAB mass
4111
F. ORSINI et al.
4112
Table 1. ‘%NMR spectral data of sugar moieties in compounds 1,2, 4 and methyl glycopyranosides (pyridine-d,) Me-glycopyranoside C
R'O
H
3-O-GIC1 2 3 4 5 6 Ara1 2 3 4 5 Glc1 2 3 4 5 6 Xyl-
1 2 3 4 5
t0
WI
1
2
4
105.4 82.7 77.0 71.8 78.0 69.9
106.1 83.3 77.2 71.6 77.9 69.3
106.4 75.5 78.4 72.3 76.2 69.6
105.4 74.8 78.1 71.4 78.1 62.5
105.8 72.3 74.3 69.2 66.0
102.5 80.8 72.6 67.4 64.3
102.4 80.5 72.6 67.5 64.3
105.9 72.2 74.4 69.1 66.6
105.3 76.5 78.5 71.5 78.2 62.8
105.1 76.0 78.3 71.6 78.0 62.7 106.9 74.8 78.0 70.9 67.4
106.1 74.6 78.1 70.9 66.9
106.5 74.8 77.9 70.8 67.4
Me R=
H
NH I
6
CH,OMe GICO
,‘I I?
Me
N
s
CtI,OH
and fragments at m/z 927 [M-132-H]-, 897 [M-162-H]-, 795 [927-132-J-, 765, 633, 471 [C3,,H4s04 -H-J -. By acid hydrolysis (HCl-MeOH) it gave echinocystic acid and three sugars which were identified as glucose, xylose and arabinose units (anomeric carbons at 6 106.5,106.1,105.1 and 102.5), linked to the C-3 hydroxyl group of echinocystic acid (688.9). The analysis of the FAB mass spectrum and the 13C NMR signals in comparison with 1 showed that compound 2 brings a terminal /3-D-xylose unit, whereas the arrangement of the other sugars is the same as in 1. From the glycosylation shifts [7] the fi-D-xylopyranosyl moiety is linked to the C-2 of the a-L-arabinopyranosyl unit. In fact the signal at C-2 of r-L-arabinopyranose was shifted downfield (672.2-+80.8), whereas C-l and C-3 were shifted upfield (6 105.9-+102.5 and 674.4-+72.6) (Table 1). According to these results, compound 2 was assigned as 3-O-V-D-xylopyranosyl-( l-2)-cc+arabinopyranosyl-(l-+6)] [b-D-glucopyranosyl-(1+2)]-B-D-glucopyranosyl echinocystic acid. No chemical evidence could be ascertained to distinguish the oligosaccharidic chain linked at C-2 and C-6 of the 3-O-/?-D-glucopyranosyl unit in compounds 1 and 2. In fact we can reverse the substituents at C-2 with those at C-6 and obtain two compounds with the same spectral patterns as 1 and 2. To overcome this uncertainty, compound 2 was enzymatically hydrolysed with the hepato-pancreatic juice from Helix porn&a (P-glucosidase) [S] affording compound 4. The analysis of the r3CNMR spectrum (52.4 MHz, pyridine-d,) of 4 supported by a DEPT spectrum
Saponins from
experiment, with regard to the range 670-60 where the hydroxymethylene signal of the sugars are located, showed the presence of three triplets at 669.6,67.4 and 64.3. In compound 2 they were at the same values. No signal appeared near 663, thus verifying that the C-2 position of the 3-O+glucopyranosyl moiety is involved in the linkage with the terminal j-D-ghCOpyraUOsy1 unit and confirming the proposed structure for 2 and 1. The sequence of sugar components was confirmed by methylation analysis. Compound 2 was permethylated by the method described for monosaccharides [9], then hydrolysed with 3% HCl-methanol. Methyl 2,3,4,6-tetra-Omethylglucopyranoside, methyl 2,3,4-tri-O-methylxylopyranoside and methyl 3,4-di-O-methylarabinopyranoside were detected by TLC and capillary GC analysis in comparison with available standards. Compound 3 was isolated as the methyl ester, due to the difficulties in obtaining the pure compound as a free acid (see Experimental). In the positive FAB mass spectrum, compound 3 showed an ion at m/z 991 [M + Na +H]+. In the negative FAB mass spectrum it showed an ion at m/z 967 [M] -, corresponding to the molecular formula C,,,H,,NO,,, and fragments at m/z 835 [M - 132]- and 689 [835- 146]- which indicate successive elimination of a pentose and a deoxyhexose unit from the molecular ion. By acid hydrolysis (HCl-MeOH) of 3, the sapogenin echinocystic acid methyl ester was identified from its physical data (TLC and ‘HNMR) and by comparison with an authentic sample. Silylation of the hydrolysate [S] allowed the identification of D-xylose, Dfucose and D-glucosamine in the ratio 1: 1: 1 by capillary GC analysis of their persylil derivatives in comparison with pure standards. The 13C NMR spectrum (52.4 MHz, pyridine-d,) of 3 confirmed the presence of a chain of three saccharidic units (anomeric carbons at 6 107.1, 104.8,103.5, typical of a /?-configuration for the three sugars). This chain is linked to the C-3 hydroxy group of echinocystic acid methyl ester (S88.5). The ‘HNMR spectrum of 3 (200 MHz, pyridine-d,) showed the presence of signals at 68.87 (d, J=9 Hz, NHAc); 65.47 (dd, J12_11a=J1a_11B = 3 Hz, H-12k 63.68 (s, COzMe); 62.16 (s, NHAc). Seven singlet methyl signals appeared at 6 1.82, 1.22, 1.08, 1.03, 0.99, 0.91, 0.83; one doublet methyl group at 6 1.48 (d, J =7 Hz). A more accurate NMR analysis obtained by recording a proton spectrum at 500 MHz, completely resolved the zone of the anomeric protons (6 5.1-4.9) and allowed the assignement of all the chemical shifts and coupling constants, thus con8rming the configuration for the three saccharidic units (see Table 2). Moreover a HCcosy experiment [lo], unambiguosly assigned all the 13C NMR signals and most of the ‘H NMR spectrum (see Tables 2 and 3). The 13C NMR spectrum of compound 3, supported by a DEPT experiment [7], showed a signal at 6 70.0 (t), characteristic of the hydroxymethylene group of a N-acetyl-D-ghtcosamine involved in the fi-glycosidic linkage with the Dfucopyranosyl unit [8]. The terminal Dxylopyranosyl moiety is linked to the C-2 of the Dfucopyranose as demonstrated by the glycosylation shifts (672.0+82.3, C-2; 6105.4-tlO3.5, C-l) [6]. From these considerations, compound 3 was assigned the structure 30-[/I-D-xylopyranosyl( l-+2)-/I-D-fucopyranosyl( 1+6)]2-acetamido-2-deoxy-B_Dglucopyranosyl echinocystic acid. Fractionation of the n-butanol extract of A. lucida seeds was followed with the ‘brine-shrimps’ test [4], A
4113
Albizzia Zucida
Table 2. ‘H and 13CNMR assignments for compound 3 6 ‘H
Mult.* (J)
H
s r3c
8.87 5.47 5.06 5.06 5.02 4.98 4.53 4.42-4.30
d (9) dd (3, 3) d (8) d (8) dd (3, 3) d (8)
-
4.12 3.76 3.68 3.57 3.52 3.35 2.72 220 2.16 1.82 1.48 1.22 1.08 1.03 0.99 0.91 0.83
dd (12, 3)
-NH-AC 12 G-lt X-l? 168 F-l? G-2 x-5 F-2 x-2 G-6” F-3 F-5 G-Me 3a x-5 188
ddd (8, 8, 9) In
dq (7) id (11, 4) dd (12,4) dd (14, 5) dd (14, 14) In s i(7) s s s s s s
19a 2 -NH-AC 27 F-6 23 30 24 29 25 26
122.8 104.8 107.1 75.2 103.5 58.0 67.1 82.3 76.9 70.0 75.3 70.8 51.8 88.5 67.1 41.2 46.9 26.7 23.8 27.3 17.2 28.2 24.6 17.1 33.3 15.6 17.2
*Values in parentheses are ‘H-‘H splittings in the cases where these are clearly resolved. t G = N-acetyl-n-ghtcosamine, X = ~xylose., F = D-fUCOSC.
Table 3. r3C NMR spectral data of sugar moieties in compound 3 (pyridine-d,)
C
GloNAc1 2 3 4 5 6 Me(CG-NH-) Me(CG-NH-) Fuc2 3 4 5 Me Xyl1 2 3 4 5
3
Me-glycopyranoside [6]
104.8 58.0 75.9 72.5 75.8 70.0 23.8 170.2 103.5 82.3 75.3 72.3 70.8 17.3 107.1 76.9 77.5 71.4 67.1
105.4 72.0 75.2 72.6 71.3 17.2
4114
F. ORSINIet at.
fraction obtained from the Fractogel purification, containing mainly saponins l-3, showed LC,, < 100 ppm. The purified compounds 2,3 and a saponin (6), previously isolated by us from Albizzia anthebnintica root bark [8], were also tested: compounds 3 and 6 showed a considerable activity (LC,, ca 30 ppm).
Plant material was obtained from the botanical garden in Lucknow, India (March 1987). Mps: uncorr. Precoated Kieselgel 6OF,,,, RP-18 and RP-8 (Merck) were used for TLC. Spots were detected by spraying with H$O,,-MeOH (1:s) followed by heating. Gel filtration was carried out on Sephadex LH-20 (Pharmacia) and Fractogel TSK-HW 40 (Merck), eluting with MeOH. Kieselgel 60 (70-230 mesh, Merck) was employed for CC. RPMPLC was carried out at medium pressure with Buchi 685 columns (26 x460 mm filled with Lichroprep RP-18 40-63 w, Merck; and 36 x 460 mm filled with Lichroprep RP-8 25-40 pm, Merck). Droplet countercurrent chromatography (DCCC) was performed with CHCl,-MeOH-n-PrOH-H,O (5:6: 1:4f in the ascending mode (flow rate 0.2 ml mir-‘, 300 tubes 2 x 400 mm). NMR spectra were recorded on 200 and 500 MHz spectrometers. Samples were dissolved in py~dine-do and TMS was used as int. standard. FAB-MS samples were dissolved in a glycerol matrix. Sugars (silylated and methylated) were analysed on a WCOT CP Sil S-CB fused silica capillary column (Chrompack, 25 m x 0.32 mm, film thickness 0.11 pm, carrier H, 0.45 kgcm-‘; inj. and det. temp. 300”), prog. 140-+280” at 5” min-’ (silyl derivatives) and 80-250 at 4” min-’ (methyl derivatives). Extraction and isolation. Thinly minced dried seeds (482 g) were exhaustively extracted by percolation with MeOH (1.3 1)at room temp. The dried extract (60.1 s) was taken up in H,O (500 ml) and extracted with n-BuOH (4.50ml); the organic phase was then coned in uacuo to yield a crude mixt. (21.7 gf which was digested with Et,0 m order to obtain an extract free from the less polar ~rn~unds (20.4 g). This extract (4.7 g) was dissolved in MeOH and applied to a Sephadex LH-20 column (5 x 100 cm) eluting with MeOH (280 ml) at a flow rate of 0.25 mlmin-‘. Intermedtate frs were collected (4.071 8) and applied to another Sephadex LH-20 column (5 x 1OOcm) eluting with MeOH (300 ml) at a flow rate of 0.16 ml mm- I; frs 108-150 (2 ml) were evapd to dryness (517 mg) and a portion (205 mg) was submitted to DCCC at a flow rate of O.l6mlmin-‘; frs were collected according to their composition and afforded 77 mg of 5 and 37 mg of 2. Part of the digested n-BuOH extract (7.733 g) was dissolved in MeOH and applied to a Fractogel TSK-HW 40 column (2.5 x 80 cm) eluting with MeOH (480 ml) at a flow rate of 2 ml min- *; frs (7 ml) were collected according to their composition and taken to dryness. Frs 47-68 (965 mg) were submitted to DCCC at a flow rate of0.1.5 ml min-’ and afforded 1 (32 mg). Frs 4&46 (5.58 g) were purified by RPMPLC (RP-8 25-40 p) at a flow rate of 20 ml min-’ eluting with a gradient MeOH-H,O(3OOml6:4;6OOml7:3;2OOml8:2;4OOml9:1). Frs (15 ml) were collected according to their composition, taken to dryness (566 mg) and purified by RPMPLC (RP-18 40-63 m) eluting with MeOH-H,O (7: 3) at a flow rate of20 ml mir-‘. Frs 40-46 (199 mg) were taken to dryness, treated with ethereal CH,N,, then purified by flash chromatography on silica gel eluting with CHCl,-MeOH (3 : 1) affordin& after crystallization, 52 mg of compound 3. Compound 2.. Obtained from EtOAc-EtOH (4:lf as an amorphous powder, mp 275” (dec), [z]a5 - 14.75 (H,O, c 0.8); FAB-MS m/z (C,,H,,O,,): 1059 [M-H]-, 927 EM-132 -HI-, 897 [M-162-H]-, 795 [927-132]-, 765 [927
Table 4. 13CNMR spectral data of aglicone moieties in compounds l-4 (pyridine-d,)
C
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 COMe
1
2
38.9 26.8 89.6 39.7 56.0 18.7 33.6 40.0 47.4 37.2 24.0 122.7 145.2 42.3 36.2 74.8 49.0 41.6 47.3 31.2 36.3 33.4 28.3 17.0 15.8 17.7 27.4 180.0 33.6 24.9
38.9 26.8 88.9 39.6 56.0 18.6 33.0 39.9 47.2 37.1 23.9 122.5 145.1 42.1 36.2 75.5 48.9 41.4 47.2 31.1 36.2 32.9 28.2 17.5 15.7 17.5 27.3 180.0 32.9 24.8
3 acidt
Me-ester
38.6 38.7 26.6 26.7 88.4 88.5 39.3 39.4 55.9 55.9 18.5 18.6 33.4 33.3 39.9 39.9 47.1 47.2 37.0 37.1 23.7 23.9 122.4 122.8 145.0 144.5 42.0 41.9 36.2 36.0 75.2 75.2 48.8 49.0 41.3 41.2 47.1 46.9 31.0 30.9 36.2 36.0 32.9 32.6 28.1 28.2 17.0 17.1 15.5 15.6 17.4 17.3 27.3 27.3 180.0 177.8 33.4 33.3 24.7 24.6 51.8
4 38.9 26.8 88.7 39.6 56.0 18.6 33.5 40.0 47.3 37.2 23.9 122.5 145.1 42.1 36.2 75.7 48.9 41.5 47.3 31.1 36.2 32.9 28.3 17.1 15.7 17.6 27.4 180.2 33.4 24.8
t+ t d zi t t SI s
2 d S s t
d s d t s t t 4 4 4 4 4 S 4 4 s
*Carbon multiplicities were determined using DEPT experiments [7]. tTentatively assigned from the 13CNMR spectrum of the impure acid by comparison with the spectrum of the methyl ester.
-162]-, 633 [765-132]-, 471 [C,,H,,O,-HI-, 453 [Cs0H4s04-Hz0 - H] -; ‘H NMR (200 MHz, pyridine-d,); 3 Hz, H-12),5.34 (d, J=8 Hz, H-l 65.58(dd, Jlz_,,s=Jlr_l,g= Cl& 5.22 (dd, J1Ba-,k=J168_,sg=3H~. H-16/?), 5.11 (d, J =5Hz,H-1 Xyl),4.95(d, 1=7Hz),4.86fd, J=?Hz), 1.85, 1.26, 1.15, 1.10, 1.02, 1.00, 0.83 (s, CH,). ‘%NMR (52.4 MHz, pyridine-d,) see Tables 1 and 4. Acid hydrolysis o~cornpo~~ 2. A soln of 2 (4.8 mg) in 0.5 ml of 0.5 M HCI-MeOH, was refluxed for 7 hr under N,. The solvent was removed by flushing N,, then dried in vacua. The residue was taken up with H,O and extracted with CHCl,. Evapn of the solvent gave echinocystic acid, identified by comparison (‘HNMR, TLC) with an authentic sample. A soln of BSTFA-pyridine was added (0.5 ml) to the dried aq. residue and the mixt. warmed at 60‘ for 1 hr. This soln was directly analysed by capillary GC identifying glucose, xylose and arabinose in the ratio 2: 1: 1 by coinjeclion with appropnate standards. Permethylation and methanolysis of compound 2. Compound 2 (50 mg) was dissolved in I.5 ml of DMSO, 163 mg t-NaOBu (Merck), 30 mg dry powdered NaOH and then 0.1 ml MeI were added with stirring at room temp. After 2 hr, H,O was added
Saponins from Albizzia lucida and the mixt. extracted with CHCl, (3 x 2 ml). The organic phase was washed with HZ0 and evaporated to dryness (44 mg). The crude residue was treated with 3% HCl-MeOH (1 ml) and refluxed for 3 hr. Methyl 2,3,4,6-tetra-O-methyl+glucopyranoside, methyl 2,3,4-tri-0-methyl-D-xylopyranoside and methyl 3,4-di-O-methyl+arabinopyranoside were identified by TLC and capillary GC with appropriate standards. Enzymatic hydrolysis of compound 2. Compound 2 (100 mg) was hydrolysed with the hepatopancreatic juice of 10 snails (Helix pomatia) diluted with H,O (15 ml) and filtered. The soln was stirred at 36” for 20 hr; the mixt. was then extracted with nBuOH (2 x 15) to afford 81 mg, which were purified by CC on silica gel eluting with CHCl,-MeOH-H,O (14: 6: lk 43 mg of 4 were obtained. Compound 4. Crystallized from EtOAc-MeOH 9: 1, mp 225-226” (dec); [a];” -8.64 (MeOH; c 1); FAB-MS m/z (C&H7,,01,) negative mode: 897 [M-H]-, 765 [897-132]-, 633 [765- 132]- ‘HNMR (200 MHz, pyridine-d,): 65.63 (dd, 3 Hz, H-12), 5.26 (dd, J166-1sP=J16B-13a J12-11.=J12-11jl= = 3 Hz, H-16@, 5.18 (d, J = 5 Hz, H-l Xyl), 5.03 (d, J = 7 Hz), 4.93 (d, J=8 Hz), 1.91, 1.37, 1.22, 1.07, 1.03, 1.01, 0.91 (3, Me). ‘%NMR (52.4 MHz, pyridine-d,) see Tables 1 and 4. Compound 1. Crystallized from EtOAc, mp 246” (de& [cr]:’ -3.27 (MeOH; c 1); FAB-MS m/z (C.,,H,,Ols) negative mode: 927[M-H]-,795[M-132--~-,765[M--162-H]-;positive mode: 951 [M+Na]+, 929 [M +H] +, 455 [CsOHQs04 -H,O+H)+; ‘HNMR (2OOMHz, pyridine-d,) 65.62 (dd, ~12-11~=~1*-11~= 3 Hz, H-12), 5.32 (d, J = 7 Hz, H-l Glc), 5.25 (& J16B-15*=J16B-Isp= 3 Hz, H-168), 4.91 (d, 5=7 Hz), 4.85 (d,J=7Hz),3.19(dd,5=14,4Hz,H-l8#l), 1.77, 1.17, 1.13, 1.05, 1.02, 0.95, 0.80 (s, Me). 13CNMR (52.4 MHz, pyridine-d,) see Tables 1 and 2. Acid hydrolysis of compound 1. Compound 1 (4.9 mg) was treated under the same conditions as described above for 2 and echinocystic acid was again identified by comparison with an authentic sample (‘HNMR, TLC). After silylation (BSTFApyridine) the soln was analysed by capillary GC identifying glucose and arabinose in the ratio 2: 1 by co-injection with appropriate standards.
4115
Compound 3. Crystallized from EtOH, mp 254” (dec), [a];’ -0 (MeOH; cl) FAB-MS m/z (C,,H,,NO,,) negative mode: 967 CM]-, 835 [M - 132]-, 689 [M - 132 - 146]-; positive mode: 991 [M+Na+H]+. ’ H NMR (500 MHz, pyridine-d,) see Table 2. 13CNMR (52.4 MHz, pyridine-d,) see Tables 1 and 3. Acid hydrolysis of compound 3. Compound 3 (10 mg) was hydrolysed under the same conditions as described above for 1 and 2, but using HCl-MeOH at reflux for 22 hr. Echinocystic acid was again identified by comparison with an authentic sample (‘H NMR, TLC). After silylation the soln was analysed by capillary GC identifying fucose, xylose and glucose in the ratio 1: 1: 1 by co-injection with appropriate standards.
Acknowledgements-Financial assistance from the Minister0 della Universiti e della Ricerca Scientifica e Tecnologica of Italy is gratefully acknowledged. REFERENCES
1. Watt, J. M. and Breyer-Brandwijk, M. G. (1962) The Medicinal and Poisonous Plants of South and East Africa 2nd Edn, p. 556. Livingstone, Edinburgh. 2. Chowdhury, A. R., Banerji, R., Misra, G. and Nigam, S. K. (1984) .I. Am, Oil. Chem. Sot. 61, 1023. 3. Orsini, F., Pelizzoni, F., Pulici, M. and Verotta, L. (1989) Gazz. Chim. Ital. 119, 63. 4. Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jacobsen, L. B., Nichols, D. E. and McLaughlin, J. L. (1982) Planta Med. 45, 31. 5. Garibaldi, P., Verotta, L. and Gabetta, B. (1990) Phytochemistry 29, 2629. 6. Sea, S., Tomita, Y., Tori, K. and Yoshimura, Y. (1978) .I. Am. Chsm Sot. 100,333l.
7. Doddrell, D, M., Pegg, D. T. and Bendall, M. R. (1982) J. Magn. Reson. 48, 323. 8. Carpani, G., Orsini, F., Sisti, M. and Verotta, L. (1989) Phytochemistry 28, 863. 9. Ciukanu, I. and Kerek, F. (1984) Carbohydr. Res. 131, 209. 10. Bax, A. and Morris, G. A. (1981) J. Mugn. Reson. 42, 501.
SAPONINS
0031-9422/91$3.00+0.00 Q 1991Pergamon Press plc
FROM
ALBIZZIA
LUCZDA
FULV~A ORSINI, FRANCESCAPELIZZONI and LUISELLAVEROTTA* Centro di Studio sulle Sostanze Organiche Naturali de1 CNR, Dipartimento di Chimica Organica e Industriale, via Venezian 21, 20133 Milano, Italy (Received4 March 1991)
Key Word Index--Albizzia
lucida; Leguminosae; Mimosaceae; seeds; echinocystic acid glycosides; cytotoxic
activity.
Abstract-Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-o-[fl-D-xylopyranosyl(l+2)-C+Larabinopyranosyl (1+6)] [/I-D-glucopyranosyl (l-+2)] /I-D-glucopyranosyl echinocystic acid; 3-O-[a-L-arabinopyranosyl (1+6)] [p-D-ghrcopyranosyl (l-+2)]-/I-D-glucopyranosyl echinocystic acid and 3-0-[b-D-xylopyranosyl (1+2)+-D-fucopyranosyl (1+6)-2-acetamido-2-deoxy-B-Dglucopyranosyl echinocystic acid, characterized as its methyl ester.
INTRODUCTION Albizziu lucida is frequently reported together with Albizzia bbbeck because both plants are similar: they are widespread in India and southern Africa and are hosts of the lac inset [ 1J. The seeds of the two plants and other Albizzia species were studied because of their lipid composition and their use as a food [2]. Recently we have isolated, from the seeds of A. lucida, a neurotoxin, 3-hydroxy-5-hydroxymethyl-4-methoxymethyl2-methylpyridine 3-O-jI-D-ghrcoside (5) [3]. We now report the isolation and the structure of three main saponins (l-3) from the seeds. Two of them showed cytotoxic activity in the brine shrimps test at a concentration lower than 50 ppm [4]. RISULTS AND DISCUSSION
The methanol extract of A. lucida was partitioned between n-butanol and water. After digestion with diethyl ether, the butanol extract (4% of the dried plant material) showed on TLC a sequence of several spots distributed between R, 0.1 and 0.6. One of them proved to be compound 5 which was first isolated from the aqueous extract [3]. The others, after spraying the TLC plate with sulphuric acid, appeared as plum coloured spots. The butanol extract was purified by repeated chromatographic steps involving gel filtration (Sephadex LH-20 and Fractogel TSK HW 40), reversed phase medium pressure liquid chromatography (RPMPLC) and DCCC. Three main compounds (l-3) were isolated. By acid hydrolysis (HCl-MeOH) of compound 1, the sapogenin echinocystic acid was isolated and identified from its physical data (TLC, ‘H NMR) and comparison with an authentic sample. Silylation of the hydrolysate [5] allowed the identification of glucose and arabinose in the ratio 2: 1 by capillary gas-chromatography in compari*Author to whom correspondence should be addressed. ~ sugars have been assumed D or L as in the natural series.
son with pure standards. The i3CNMR spectrum (52.4 MHz, pyridine-d,) of 1 confirmed the presence of a chain of three saccharidic units (anomeric carbons at 6 105.8, 105.4 and 105.3 typical for a /?-configuration for D-glucose and a-configuration for L-arabinose [6]),t linked to the C-3 hydroxyl group of echinocystic acid (6 89.6). The ‘H NMR spectrum (200 MHz, pyridine-d,) confirmed the oleanane skeleton (65.62, dd, Jiz-ll~ =J12_ll.=3 HgH-12;63.19,dd,J1sa-,9~=14,J,,,_,,, =4 Hz, H-18/?; 65.25, dd, J168-15.= J16,+158 = 3 Hz, H-16/3; seven singlet methyl groups at S 1.77,1.17,
1.13, 1.05, 1.02, 0.95 and 0.80). The anomeric protons appeared at 65.32 (d, J= 7 Hzk 64.91 (d, J= 7 Hz); 64.85 (d, J = 7 Hz). Compound 1 showed ions at m/z 951 [M + Na] + and 929 [M + H] + in the positive FAB mass spectrum. In the negative FAB mass spectrum a quasi molecular ion was observed at m/z 927 [M-H]and fragments at m/z 795 [M - 132 -HIand 765 [M - 162 -HI-. From the FAB mass spectrum it is noteworthy that 1 possesses a branched saccharidic chain as shown by the contemporary loss of the pentose unit (m/z 795) and the hexose unit (m/z 765) from the molecular ion. The “C NMR spectrum of 1, supported by a DEPT experiment [a showed a signal at 662.8 (t) characteristic of the hydroxymethylene group of a terminal glucose unit and a signal at 669.9 (t), attributed to the hydroxymethylene group of a glucose unit, downfield shifted by the linkage with the arabinopyranosyl moiety. The triplet at 666.6 was assigned to the hydroxymethylene of arabinose. The terminal glucose and arabinose were reasonably assigned in comparison with the data of methyl-~-D-glucopyranoside and methyl-a+arabinopyranoside [6]. As co&med by the glycosilation shifts [6] the 3-O-linked-p-Dglucopyranosyl unit had the two sugars linked in positions C-2 and C-6 (Table 1). From these considerations 1 was assigned as 3-0-[a+arabinopyranosyl-( 1+6)] [j?D-glucopyranosyl-(1+2)]-fl-D-glucopyranosyl echinocystic acid. The most polar compound 2 showed a quasi molecular ion at m/z 1059 [M-IIIin the negative FAB mass
4111
F. ORSINI et al.
4112
Table 1. ‘%NMR spectral data of sugar moieties in compounds 1,2, 4 and methyl glycopyranosides (pyridine-d,) Me-glycopyranoside C
R'O
H
3-O-GIC1 2 3 4 5 6 Ara1 2 3 4 5 Glc1 2 3 4 5 6 Xyl-
1 2 3 4 5
t0
WI
1
2
4
105.4 82.7 77.0 71.8 78.0 69.9
106.1 83.3 77.2 71.6 77.9 69.3
106.4 75.5 78.4 72.3 76.2 69.6
105.4 74.8 78.1 71.4 78.1 62.5
105.8 72.3 74.3 69.2 66.0
102.5 80.8 72.6 67.4 64.3
102.4 80.5 72.6 67.5 64.3
105.9 72.2 74.4 69.1 66.6
105.3 76.5 78.5 71.5 78.2 62.8
105.1 76.0 78.3 71.6 78.0 62.7 106.9 74.8 78.0 70.9 67.4
106.1 74.6 78.1 70.9 66.9
106.5 74.8 77.9 70.8 67.4
Me R=
H
NH I
6
CH,OMe GICO
,‘I I?
Me
N
s
CtI,OH
and fragments at m/z 927 [M-132-H]-, 897 [M-162-H]-, 795 [927-132-J-, 765, 633, 471 [C3,,H4s04 -H-J -. By acid hydrolysis (HCl-MeOH) it gave echinocystic acid and three sugars which were identified as glucose, xylose and arabinose units (anomeric carbons at 6 106.5,106.1,105.1 and 102.5), linked to the C-3 hydroxyl group of echinocystic acid (688.9). The analysis of the FAB mass spectrum and the 13C NMR signals in comparison with 1 showed that compound 2 brings a terminal /3-D-xylose unit, whereas the arrangement of the other sugars is the same as in 1. From the glycosylation shifts [7] the fi-D-xylopyranosyl moiety is linked to the C-2 of the a-L-arabinopyranosyl unit. In fact the signal at C-2 of r-L-arabinopyranose was shifted downfield (672.2-+80.8), whereas C-l and C-3 were shifted upfield (6 105.9-+102.5 and 674.4-+72.6) (Table 1). According to these results, compound 2 was assigned as 3-O-V-D-xylopyranosyl-( l-2)-cc+arabinopyranosyl-(l-+6)] [b-D-glucopyranosyl-(1+2)]-B-D-glucopyranosyl echinocystic acid. No chemical evidence could be ascertained to distinguish the oligosaccharidic chain linked at C-2 and C-6 of the 3-O-/?-D-glucopyranosyl unit in compounds 1 and 2. In fact we can reverse the substituents at C-2 with those at C-6 and obtain two compounds with the same spectral patterns as 1 and 2. To overcome this uncertainty, compound 2 was enzymatically hydrolysed with the hepato-pancreatic juice from Helix porn&a (P-glucosidase) [S] affording compound 4. The analysis of the r3CNMR spectrum (52.4 MHz, pyridine-d,) of 4 supported by a DEPT spectrum
Saponins from
experiment, with regard to the range 670-60 where the hydroxymethylene signal of the sugars are located, showed the presence of three triplets at 669.6,67.4 and 64.3. In compound 2 they were at the same values. No signal appeared near 663, thus verifying that the C-2 position of the 3-O+glucopyranosyl moiety is involved in the linkage with the terminal j-D-ghCOpyraUOsy1 unit and confirming the proposed structure for 2 and 1. The sequence of sugar components was confirmed by methylation analysis. Compound 2 was permethylated by the method described for monosaccharides [9], then hydrolysed with 3% HCl-methanol. Methyl 2,3,4,6-tetra-Omethylglucopyranoside, methyl 2,3,4-tri-O-methylxylopyranoside and methyl 3,4-di-O-methylarabinopyranoside were detected by TLC and capillary GC analysis in comparison with available standards. Compound 3 was isolated as the methyl ester, due to the difficulties in obtaining the pure compound as a free acid (see Experimental). In the positive FAB mass spectrum, compound 3 showed an ion at m/z 991 [M + Na +H]+. In the negative FAB mass spectrum it showed an ion at m/z 967 [M] -, corresponding to the molecular formula C,,,H,,NO,,, and fragments at m/z 835 [M - 132]- and 689 [835- 146]- which indicate successive elimination of a pentose and a deoxyhexose unit from the molecular ion. By acid hydrolysis (HCl-MeOH) of 3, the sapogenin echinocystic acid methyl ester was identified from its physical data (TLC and ‘HNMR) and by comparison with an authentic sample. Silylation of the hydrolysate [S] allowed the identification of D-xylose, Dfucose and D-glucosamine in the ratio 1: 1: 1 by capillary GC analysis of their persylil derivatives in comparison with pure standards. The 13C NMR spectrum (52.4 MHz, pyridine-d,) of 3 confirmed the presence of a chain of three saccharidic units (anomeric carbons at 6 107.1, 104.8,103.5, typical of a /?-configuration for the three sugars). This chain is linked to the C-3 hydroxy group of echinocystic acid methyl ester (S88.5). The ‘HNMR spectrum of 3 (200 MHz, pyridine-d,) showed the presence of signals at 68.87 (d, J=9 Hz, NHAc); 65.47 (dd, J12_11a=J1a_11B = 3 Hz, H-12k 63.68 (s, COzMe); 62.16 (s, NHAc). Seven singlet methyl signals appeared at 6 1.82, 1.22, 1.08, 1.03, 0.99, 0.91, 0.83; one doublet methyl group at 6 1.48 (d, J =7 Hz). A more accurate NMR analysis obtained by recording a proton spectrum at 500 MHz, completely resolved the zone of the anomeric protons (6 5.1-4.9) and allowed the assignement of all the chemical shifts and coupling constants, thus con8rming the configuration for the three saccharidic units (see Table 2). Moreover a HCcosy experiment [lo], unambiguosly assigned all the 13C NMR signals and most of the ‘H NMR spectrum (see Tables 2 and 3). The 13C NMR spectrum of compound 3, supported by a DEPT experiment [7], showed a signal at 6 70.0 (t), characteristic of the hydroxymethylene group of a N-acetyl-D-ghtcosamine involved in the fi-glycosidic linkage with the Dfucopyranosyl unit [8]. The terminal Dxylopyranosyl moiety is linked to the C-2 of the Dfucopyranose as demonstrated by the glycosylation shifts (672.0+82.3, C-2; 6105.4-tlO3.5, C-l) [6]. From these considerations, compound 3 was assigned the structure 30-[/I-D-xylopyranosyl( l-+2)-/I-D-fucopyranosyl( 1+6)]2-acetamido-2-deoxy-B_Dglucopyranosyl echinocystic acid. Fractionation of the n-butanol extract of A. lucida seeds was followed with the ‘brine-shrimps’ test [4], A
4113
Albizzia Zucida
Table 2. ‘H and 13CNMR assignments for compound 3 6 ‘H
Mult.* (J)
H
s r3c
8.87 5.47 5.06 5.06 5.02 4.98 4.53 4.42-4.30
d (9) dd (3, 3) d (8) d (8) dd (3, 3) d (8)
-
4.12 3.76 3.68 3.57 3.52 3.35 2.72 220 2.16 1.82 1.48 1.22 1.08 1.03 0.99 0.91 0.83
dd (12, 3)
-NH-AC 12 G-lt X-l? 168 F-l? G-2 x-5 F-2 x-2 G-6” F-3 F-5 G-Me 3a x-5 188
ddd (8, 8, 9) In
dq (7) id (11, 4) dd (12,4) dd (14, 5) dd (14, 14) In s i(7) s s s s s s
19a 2 -NH-AC 27 F-6 23 30 24 29 25 26
122.8 104.8 107.1 75.2 103.5 58.0 67.1 82.3 76.9 70.0 75.3 70.8 51.8 88.5 67.1 41.2 46.9 26.7 23.8 27.3 17.2 28.2 24.6 17.1 33.3 15.6 17.2
*Values in parentheses are ‘H-‘H splittings in the cases where these are clearly resolved. t G = N-acetyl-n-ghtcosamine, X = ~xylose., F = D-fUCOSC.
Table 3. r3C NMR spectral data of sugar moieties in compound 3 (pyridine-d,)
C
GloNAc1 2 3 4 5 6 Me(CG-NH-) Me(CG-NH-) Fuc2 3 4 5 Me Xyl1 2 3 4 5
3
Me-glycopyranoside [6]
104.8 58.0 75.9 72.5 75.8 70.0 23.8 170.2 103.5 82.3 75.3 72.3 70.8 17.3 107.1 76.9 77.5 71.4 67.1
105.4 72.0 75.2 72.6 71.3 17.2
4114
F. ORSINIet at.
fraction obtained from the Fractogel purification, containing mainly saponins l-3, showed LC,, < 100 ppm. The purified compounds 2,3 and a saponin (6), previously isolated by us from Albizzia anthebnintica root bark [8], were also tested: compounds 3 and 6 showed a considerable activity (LC,, ca 30 ppm).
Plant material was obtained from the botanical garden in Lucknow, India (March 1987). Mps: uncorr. Precoated Kieselgel 6OF,,,, RP-18 and RP-8 (Merck) were used for TLC. Spots were detected by spraying with H$O,,-MeOH (1:s) followed by heating. Gel filtration was carried out on Sephadex LH-20 (Pharmacia) and Fractogel TSK-HW 40 (Merck), eluting with MeOH. Kieselgel 60 (70-230 mesh, Merck) was employed for CC. RPMPLC was carried out at medium pressure with Buchi 685 columns (26 x460 mm filled with Lichroprep RP-18 40-63 w, Merck; and 36 x 460 mm filled with Lichroprep RP-8 25-40 pm, Merck). Droplet countercurrent chromatography (DCCC) was performed with CHCl,-MeOH-n-PrOH-H,O (5:6: 1:4f in the ascending mode (flow rate 0.2 ml mir-‘, 300 tubes 2 x 400 mm). NMR spectra were recorded on 200 and 500 MHz spectrometers. Samples were dissolved in py~dine-do and TMS was used as int. standard. FAB-MS samples were dissolved in a glycerol matrix. Sugars (silylated and methylated) were analysed on a WCOT CP Sil S-CB fused silica capillary column (Chrompack, 25 m x 0.32 mm, film thickness 0.11 pm, carrier H, 0.45 kgcm-‘; inj. and det. temp. 300”), prog. 140-+280” at 5” min-’ (silyl derivatives) and 80-250 at 4” min-’ (methyl derivatives). Extraction and isolation. Thinly minced dried seeds (482 g) were exhaustively extracted by percolation with MeOH (1.3 1)at room temp. The dried extract (60.1 s) was taken up in H,O (500 ml) and extracted with n-BuOH (4.50ml); the organic phase was then coned in uacuo to yield a crude mixt. (21.7 gf which was digested with Et,0 m order to obtain an extract free from the less polar ~rn~unds (20.4 g). This extract (4.7 g) was dissolved in MeOH and applied to a Sephadex LH-20 column (5 x 100 cm) eluting with MeOH (280 ml) at a flow rate of 0.25 mlmin-‘. Intermedtate frs were collected (4.071 8) and applied to another Sephadex LH-20 column (5 x 1OOcm) eluting with MeOH (300 ml) at a flow rate of 0.16 ml mm- I; frs 108-150 (2 ml) were evapd to dryness (517 mg) and a portion (205 mg) was submitted to DCCC at a flow rate of O.l6mlmin-‘; frs were collected according to their composition and afforded 77 mg of 5 and 37 mg of 2. Part of the digested n-BuOH extract (7.733 g) was dissolved in MeOH and applied to a Fractogel TSK-HW 40 column (2.5 x 80 cm) eluting with MeOH (480 ml) at a flow rate of 2 ml min- *; frs (7 ml) were collected according to their composition and taken to dryness. Frs 47-68 (965 mg) were submitted to DCCC at a flow rate of0.1.5 ml min-’ and afforded 1 (32 mg). Frs 4&46 (5.58 g) were purified by RPMPLC (RP-8 25-40 p) at a flow rate of 20 ml min-’ eluting with a gradient MeOH-H,O(3OOml6:4;6OOml7:3;2OOml8:2;4OOml9:1). Frs (15 ml) were collected according to their composition, taken to dryness (566 mg) and purified by RPMPLC (RP-18 40-63 m) eluting with MeOH-H,O (7: 3) at a flow rate of20 ml mir-‘. Frs 40-46 (199 mg) were taken to dryness, treated with ethereal CH,N,, then purified by flash chromatography on silica gel eluting with CHCl,-MeOH (3 : 1) affordin& after crystallization, 52 mg of compound 3. Compound 2.. Obtained from EtOAc-EtOH (4:lf as an amorphous powder, mp 275” (dec), [z]a5 - 14.75 (H,O, c 0.8); FAB-MS m/z (C,,H,,O,,): 1059 [M-H]-, 927 EM-132 -HI-, 897 [M-162-H]-, 795 [927-132]-, 765 [927
Table 4. 13CNMR spectral data of aglicone moieties in compounds l-4 (pyridine-d,)
C
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 COMe
1
2
38.9 26.8 89.6 39.7 56.0 18.7 33.6 40.0 47.4 37.2 24.0 122.7 145.2 42.3 36.2 74.8 49.0 41.6 47.3 31.2 36.3 33.4 28.3 17.0 15.8 17.7 27.4 180.0 33.6 24.9
38.9 26.8 88.9 39.6 56.0 18.6 33.0 39.9 47.2 37.1 23.9 122.5 145.1 42.1 36.2 75.5 48.9 41.4 47.2 31.1 36.2 32.9 28.2 17.5 15.7 17.5 27.3 180.0 32.9 24.8
3 acidt
Me-ester
38.6 38.7 26.6 26.7 88.4 88.5 39.3 39.4 55.9 55.9 18.5 18.6 33.4 33.3 39.9 39.9 47.1 47.2 37.0 37.1 23.7 23.9 122.4 122.8 145.0 144.5 42.0 41.9 36.2 36.0 75.2 75.2 48.8 49.0 41.3 41.2 47.1 46.9 31.0 30.9 36.2 36.0 32.9 32.6 28.1 28.2 17.0 17.1 15.5 15.6 17.4 17.3 27.3 27.3 180.0 177.8 33.4 33.3 24.7 24.6 51.8
4 38.9 26.8 88.7 39.6 56.0 18.6 33.5 40.0 47.3 37.2 23.9 122.5 145.1 42.1 36.2 75.7 48.9 41.5 47.3 31.1 36.2 32.9 28.3 17.1 15.7 17.6 27.4 180.2 33.4 24.8
t+ t d zi t t SI s
2 d S s t
d s d t s t t 4 4 4 4 4 S 4 4 s
*Carbon multiplicities were determined using DEPT experiments [7]. tTentatively assigned from the 13CNMR spectrum of the impure acid by comparison with the spectrum of the methyl ester.
-162]-, 633 [765-132]-, 471 [C,,H,,O,-HI-, 453 [Cs0H4s04-Hz0 - H] -; ‘H NMR (200 MHz, pyridine-d,); 3 Hz, H-12),5.34 (d, J=8 Hz, H-l 65.58(dd, Jlz_,,s=Jlr_l,g= Cl& 5.22 (dd, J1Ba-,k=J168_,sg=3H~. H-16/?), 5.11 (d, J =5Hz,H-1 Xyl),4.95(d, 1=7Hz),4.86fd, J=?Hz), 1.85, 1.26, 1.15, 1.10, 1.02, 1.00, 0.83 (s, CH,). ‘%NMR (52.4 MHz, pyridine-d,) see Tables 1 and 4. Acid hydrolysis o~cornpo~~ 2. A soln of 2 (4.8 mg) in 0.5 ml of 0.5 M HCI-MeOH, was refluxed for 7 hr under N,. The solvent was removed by flushing N,, then dried in vacua. The residue was taken up with H,O and extracted with CHCl,. Evapn of the solvent gave echinocystic acid, identified by comparison (‘HNMR, TLC) with an authentic sample. A soln of BSTFA-pyridine was added (0.5 ml) to the dried aq. residue and the mixt. warmed at 60‘ for 1 hr. This soln was directly analysed by capillary GC identifying glucose, xylose and arabinose in the ratio 2: 1: 1 by coinjeclion with appropnate standards. Permethylation and methanolysis of compound 2. Compound 2 (50 mg) was dissolved in I.5 ml of DMSO, 163 mg t-NaOBu (Merck), 30 mg dry powdered NaOH and then 0.1 ml MeI were added with stirring at room temp. After 2 hr, H,O was added
Saponins from Albizzia lucida and the mixt. extracted with CHCl, (3 x 2 ml). The organic phase was washed with HZ0 and evaporated to dryness (44 mg). The crude residue was treated with 3% HCl-MeOH (1 ml) and refluxed for 3 hr. Methyl 2,3,4,6-tetra-O-methyl+glucopyranoside, methyl 2,3,4-tri-0-methyl-D-xylopyranoside and methyl 3,4-di-O-methyl+arabinopyranoside were identified by TLC and capillary GC with appropriate standards. Enzymatic hydrolysis of compound 2. Compound 2 (100 mg) was hydrolysed with the hepatopancreatic juice of 10 snails (Helix pomatia) diluted with H,O (15 ml) and filtered. The soln was stirred at 36” for 20 hr; the mixt. was then extracted with nBuOH (2 x 15) to afford 81 mg, which were purified by CC on silica gel eluting with CHCl,-MeOH-H,O (14: 6: lk 43 mg of 4 were obtained. Compound 4. Crystallized from EtOAc-MeOH 9: 1, mp 225-226” (dec); [a];” -8.64 (MeOH; c 1); FAB-MS m/z (C&H7,,01,) negative mode: 897 [M-H]-, 765 [897-132]-, 633 [765- 132]- ‘HNMR (200 MHz, pyridine-d,): 65.63 (dd, 3 Hz, H-12), 5.26 (dd, J166-1sP=J16B-13a J12-11.=J12-11jl= = 3 Hz, H-16@, 5.18 (d, J = 5 Hz, H-l Xyl), 5.03 (d, J = 7 Hz), 4.93 (d, J=8 Hz), 1.91, 1.37, 1.22, 1.07, 1.03, 1.01, 0.91 (3, Me). ‘%NMR (52.4 MHz, pyridine-d,) see Tables 1 and 4. Compound 1. Crystallized from EtOAc, mp 246” (de& [cr]:’ -3.27 (MeOH; c 1); FAB-MS m/z (C.,,H,,Ols) negative mode: 927[M-H]-,795[M-132--~-,765[M--162-H]-;positive mode: 951 [M+Na]+, 929 [M +H] +, 455 [CsOHQs04 -H,O+H)+; ‘HNMR (2OOMHz, pyridine-d,) 65.62 (dd, ~12-11~=~1*-11~= 3 Hz, H-12), 5.32 (d, J = 7 Hz, H-l Glc), 5.25 (& J16B-15*=J16B-Isp= 3 Hz, H-168), 4.91 (d, 5=7 Hz), 4.85 (d,J=7Hz),3.19(dd,5=14,4Hz,H-l8#l), 1.77, 1.17, 1.13, 1.05, 1.02, 0.95, 0.80 (s, Me). 13CNMR (52.4 MHz, pyridine-d,) see Tables 1 and 2. Acid hydrolysis of compound 1. Compound 1 (4.9 mg) was treated under the same conditions as described above for 2 and echinocystic acid was again identified by comparison with an authentic sample (‘HNMR, TLC). After silylation (BSTFApyridine) the soln was analysed by capillary GC identifying glucose and arabinose in the ratio 2: 1 by co-injection with appropriate standards.
4115
Compound 3. Crystallized from EtOH, mp 254” (dec), [a];’ -0 (MeOH; cl) FAB-MS m/z (C,,H,,NO,,) negative mode: 967 CM]-, 835 [M - 132]-, 689 [M - 132 - 146]-; positive mode: 991 [M+Na+H]+. ’ H NMR (500 MHz, pyridine-d,) see Table 2. 13CNMR (52.4 MHz, pyridine-d,) see Tables 1 and 3. Acid hydrolysis of compound 3. Compound 3 (10 mg) was hydrolysed under the same conditions as described above for 1 and 2, but using HCl-MeOH at reflux for 22 hr. Echinocystic acid was again identified by comparison with an authentic sample (‘H NMR, TLC). After silylation the soln was analysed by capillary GC identifying fucose, xylose and glucose in the ratio 1: 1: 1 by co-injection with appropriate standards.
Acknowledgements-Financial assistance from the Minister0 della Universiti e della Ricerca Scientifica e Tecnologica of Italy is gratefully acknowledged. REFERENCES
1. Watt, J. M. and Breyer-Brandwijk, M. G. (1962) The Medicinal and Poisonous Plants of South and East Africa 2nd Edn, p. 556. Livingstone, Edinburgh. 2. Chowdhury, A. R., Banerji, R., Misra, G. and Nigam, S. K. (1984) .I. Am, Oil. Chem. Sot. 61, 1023. 3. Orsini, F., Pelizzoni, F., Pulici, M. and Verotta, L. (1989) Gazz. Chim. Ital. 119, 63. 4. Meyer, B. N., Ferrigni, N. R., Putnam, J. E., Jacobsen, L. B., Nichols, D. E. and McLaughlin, J. L. (1982) Planta Med. 45, 31. 5. Garibaldi, P., Verotta, L. and Gabetta, B. (1990) Phytochemistry 29, 2629. 6. Sea, S., Tomita, Y., Tori, K. and Yoshimura, Y. (1978) .I. Am. Chsm Sot. 100,333l.
7. Doddrell, D, M., Pegg, D. T. and Bendall, M. R. (1982) J. Magn. Reson. 48, 323. 8. Carpani, G., Orsini, F., Sisti, M. and Verotta, L. (1989) Phytochemistry 28, 863. 9. Ciukanu, I. and Kerek, F. (1984) Carbohydr. Res. 131, 209. 10. Bax, A. and Morris, G. A. (1981) J. Mugn. Reson. 42, 501.