- Email: [email protected]
Molluscicidal steroidal saponins and lipid content of Agave decipiens
Fitoterapia 70 Ž1999. 371]381
Molluscicidal steroidal saponins and lipid content of Aga¨e decipiens M.M. Abdel-Gawad, M.M. El-SayedU , E.S. Abdel-Hameed Laboratory of Medicinal Chemistry, Theodor Bilharz Research Institute, Giza, Egypt
Received 29 September 1998; accepted Žrevised. 23 November 1998
Abstract GLC analysis of the petrol extract of the leaves of Aga¨ e decipiens revealed that n-hexacosane was the main hydrocarbon, b-sitosterol the main sterol and oleic acid the main fatty acid. Two spirostanol and two furostanol saponins have been isolated from the methanolic extract. The structures of these saponins were established as 3-O-a-L-rhamnopyranosyl-Ž1 ª 2.-w a-L-rhamnopyranosyl-Ž1 ª 4.x-b-D-glucopyranosyl-26-Ob- D -glucopyranosyl-22 a-methoxy- Ž25R .-furost-5-ene-3b,26-diol Ž1 ., neoruscogenin 1-O-b-D-glucopyranosyl-Ž1 ª 3.-w a-L-rhamnopyranosyl-Ž1 ª 2.x-b-D-glucopyranosyl-Ž1 ª4.b-D-galactopyranoside Ž2., 1-O-a-L-rhamnopyranosyl-Ž1 ª 2.-w a-L-rhamnopyranosyl-Ž1 ª 4 .x-b- D -glucopyranosyl-26-O-b- D -glucopyranosyl-22-O-methylfurosta-5,25 Ž27 . ] diene1b,3b,22,26]tetraol Ž3 . and neohecogenin 3-O-b-D-glucopyranosyl-Ž1 ª 3 .-w b-Dxylopyranosyl-Ž1 ª 3.-b-D-xylopyranosyl-Ž1 ª 2.x ] b-D-glucopyranosyl-Ž1 ª 4.-b-D ]galactopyranoside Ž4.. Saponins 2 and 4 showed high molluscicidal activity against B. alexandrina snails ŽLC 90 s 13 and 6 ppm, respectively., whereas saponins 1 and 3 were inactive up to 50 ppm. Q 1999 Elsevier Science B.V. All rights reserved. Keywords: Aga¨ e decipiens; Steroidal saponins; Molluscicidal activity; Hydrocarbons; Sterols; Fatty acids
U
Corresponding author.
0367-326Xr99r$ - see front matter Q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 9 9 . 0 0 0 5 7 - X
372
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
1. Introduction The family Agavaceae with more than 480 species has a distribution in tropical and subtropical dry climate regions through the world. The occurrence of steroidal constituents in several Agavaceae plants, especially those belonging to the genera Aga¨ e and Yucca, is well documented w1]3x. A review of literature showed that the leaves of different Aga¨ e species contain a number of steroidal saponins and sapogenins w4]7x. No study seems to have been made on saponins of Aga¨ e decipiens Baker. In the same time, the leaves of this plant showed high molluscicidal activity against Biomphalaria alexandrina snail, the intermediate host of Schistosoma mansoni in Egypt w8x. The present paper provides an account of the isolation and structure elucidation of certain molluscicidal saponins as well as the lipid content of this plant.
2. Experimental 2.1. Plant material A. decipiens leaves were collected in June 1996 from the Orman Garden. The plant was kindly identified by Eng. Badia H. Diwan. ŽAgriculture Eng. of Orman Garden.. 2.2. Extraction and isolation Shade-dried powdered leaves Ž2 kg. were defatted four times with petrol Ž60]808C. Ž4 = 6 l. and the petrol extract was evaporated to dryness Ž6.5 g.. The defatted leaves were extracted with MeOH and the extract was evaporated under reduced pressure to dryness. The residue Ž200 g. was dissolved in water and extracted successively with CHCl 3 , EtOAc and n-BuOH to yield the corresponding fractions Ž2.5 g, 12 g and 40 g, respectively.. 2.3. Analysis of the lipidic fraction The petrol extract Ž6.5 g. was saponified with 10% KOH in MeOH w9,10x. About 500 mg of fatty acids fraction was methylated w11,12x and GLC analyzed w13,14x. 2.4. Isolation of the saponins The n-BuOH extract Ž40 g. was Si-gel CC eluting with CHCl 3 and gradient with MeOH and finally with MeOH. Fractions eluted with CHCl 3-MeOH 95:5 were collected and purified by recrystallization several times with MeOH to yield saponin 1 Ž100 mg.. Fractions eluted with CHCl 3-MeOH 85:15 and 75:25 were collected and subjected to PTLC Ž n-PrOH-EtOAc-H 2 O 4:3:2. to give saponin 2 Ž200 mg. and 3 Ž110 mg.. Fractions eluted with CHCl 3-MeOH 70:30 were collected
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
373
and Si-gel CC eluting with a step-wise gradient of CHCl 3-MeOH and finally with MeOH. Fractions eluted with CHCl 3-MeOH 60:40 were collected and purified on PTLC to give saponin 4 Ž170 mg..
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
374
Table 1 13 C-NMR chemical shifts Ž d, ppm. of the aglycone moieties of saponins 1]4 Ž100 MHz, DMSO-d6 . Carbon
1
2
3
4
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 OMe
36.2 30.1 77.5 39.1 140.0 121.4 31.9 31.3 45.2 36.2 21.2 39.5 40.6 56.2 32.1 80.5 62.5 16.10 19.10 40.6 15.9 112.1 30.3 28.7 34.2 75.3 17.20 46.4
82.57 37.84 68.74 41.05 139.71 121.20 31.56 32.39 49.45 41.16 23.9 40.42 40.10 55.84 32.33 80.62 64.09 16.07 15.51 41.05 14.68 109.12 33.41 29.709 143.32 64.9 108.65 ]
82.51 37.50 68.3 41.9 139.5 121.5 31.5 32.6 49.40 42.4 24.7 40.3 40.1 55.5 31.5 80.02 63.50 16.40 14.60 40.20 15.90 112.10 31.50 27.80 146.0 71.8 111.2 46.3
37.2 31.5 76.08 37.7 44.7 28.3 31.3 31.9 55.5 36.7 37.2 ] 55.5 55.8 31.7 79.2 52.9 16.02 18.12 42.4 13.4 108.6 26.3 25.5 26.9 65.8 16.02 ]
Saponin 1. White powder, mp 271]2728C Rf 0.37 ŽCHCl 3- MeOH 9:1.. IR max ŽKBr.: 3412 ŽOH., 1636 ŽCHsCH., 1026 ŽC}O}C. cmy1 , no spiroketal absorption; 1 H-NMR Ž270 MHz, DMSO-d6 .: d 0.67 Ž3H, s, H-18., 0.85 Ž3H, d, H-27., 0.95 Ž3H, s, H-19., 1.15 Ž3H, d, H-21., 1.50 ŽMe of Rha., 3.12 Ž3H, s, OMe., 4.48 Ž1H, d, Glc, H-1., 4.91 Ž1H, d, Glc, H-1., 4.95 Ž1H, d, Rha, H-1., 5.10 Ž1H, d, Rha, H-1. and 5.35 Ž1H, C-6.; 13 C-NMR: see Tables 1 and 2; CI-MS: mrz 1063 wMqq Hx, 1032 wMq-MeOHx, 885 wMq-MeOH-Rhax, 869 wMq-MeOH-Glcx, 737 wMq-MeOH-2 = Rhax, 575 wMq-MeOH-2 = Rha-Glcx and 413 wMq-MeOH-2 = Rha-2 = Glcx. Saponin 2. White powder, mp 255]2578C, Rf 0.65 Ž n-PrOH-EtOAc-H 2 O 4:3:2.; IR max ŽKBr.: 3389 ŽOH., 2928, 2369, 1609, 1563, 1448, 1072, 980, 919, 890 and 669 cmy1 ; 1 H-NMR Ž270 MHz, DMSO-d6 .: d 0.72 Ž3H, s, H-18., 0.87 Ž3H, d, H-21., 1.10 Ž3H, s, H-19., 1.70 ŽMe of Rha., 4.81 and 4.78 ŽH 2-27., 4.70 Ž1H, d, Gal, H-1., 4.90 Ž1H, d, Glc, H-1., 5.00 Ž1H, d, Glc, H-1., 5.10 Ž1H, d, Rha, H-1. and 5.35 Ž1H, d, H-6.; 13 C-MNR: see Tables 1 and 2; CI-MS: mr z 1079 wMqq 17x, 1061 wMqq Hx, 915 wMq-Rhax, 899 wMq-Glcx, 753 wMq-Rha-Glcx, 591.4 wMq-Rha-2 = Glcx and 429 wMq-Rha-2 = Glc-Galx.
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
375
Table 2 13 C-NMR chemical shifts Ž d, ppm. of 3-O-sugar moieties of saponins 1]4 Ž100 MHz, DMSO-d6 . Carbons
1
2
100.80 81.00 76.10 77.00 76.80 61.80
3
4
Glc
1 2 3 4 5 6
103.50 79.80 76.20 81.90 76.30 62.10
Gal
1 2 3 4 5 6
102.40 72.62 75.10 80.10 75.20 61.30
100.50 72.50 75.30 79.60 75.00 60.30
Glc Ž1 ª 4.
1 2 3 4 5 6
103.50 81.32 82.60 70.60 72.30 62.20
101.50 81.10 83.50 70.90 76.00 62.50
Glc Ž1 ª 3.
1 2 3 4 5 6
104.1 75.20 77.30 71.30 77.60 62.60
Glc Ž1 ª 2.
1 2 3 4 5 6
Rha Ž1 ª 2.
1 2 3 4 5 6
102.00 70.50 72.60 74.00 69.00 18.50
Rha Ž1 ª 4.
1 2 3 4 5 6
102.50 71.60 72.20 74.10 69.00 18.90
102.30 75.20 77.10 70.90 76.80 62.10 102.50 71.60 72.50 74.30 69.70 18.30
102.50 71.60 72.10 74.20 69.30 18.30 101.50 72.80 72.30 74.10 69.10 18.50
376
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
Table 2 Ž Continued. Xyl Ž1 ª 2.
1 2 3 4 5
104.31 75.20 78.10 71.60 67.20
Xyl Ž1 ª 3.
1 2 3 4 5
105.50 75.10 76.50 70.90 66.60
26-O-GLc
1 2 3 4 5 6
105.50 75.20 78.80 71.80 77.80 62.30
104.50 75.10 77.90 71.20 78.20 62.60
Saponin 3. White powder, mp 258]260 o C, Rf 0.52 Ž n-PrOH-EtOAc-H 2 O 4:3:2.; IR max ŽKBr.: 3405 ŽOH., 2927ŽCH., 1425, 1367, 1278, 1074, 663 cmy1 ; 1 H-NMR Ž270 MHz, DMSO-d6 .: d 0.75 Ž3H, s, H-18., 0.90 Ž3H, d, H-21., 1.0 Ž3H, s, H-19., 1.55 Ž3H, d, Me of Rha. 1.65 Ž3H, d, Me of Rha., 3.24 Ž3H, s, OMe., 4.70 Ž1H, d, Glc, H-1., 4.90 Ž1H, d, Glc, H-1., 5.00 Ž1H, d, Rha, H-1., 5.10 Ž1H, d, Rha, H-1., 5.34 and 5.02 ŽH-27., and 5.60 Ž1H, d, H-6.; 13 C-NMR: see Tables 1 and 2; CI-MS: mrz 1077 wMqq Hx, 1046.6 wMq-MeOHx, 898.5 wMq-MeOH-Rhax, 737 wMq-RhaGlcx, 591 wMq-2 = Rha-Glcx, and 429 wMq-2 = Rha-2 = Glcx. Saponin 4. White powder, mp 248]250 o C, Rf 0.50 Ž n-PrOH-EtOAc-H 2 O 4:3:2.; IR max ŽKBr.: 3419 ŽOH., 2930, 1704 Žcarbonyl group on a six membered ring., 1455, 1376, 1231, 1158, 1040, 919 and 896 Ž25S, 919 ) 896 spiroketal. cmy1 ; 1 H-NMR Ž270 MHz, DMSO-d6 .: d 0.75 Ž3H, s, H-19., 0.95 Ž3H, d, H-27., 1.01 Ž3H, s, H-18., 1.25 Ž3H, s, H-21., 4.47 Ž1H, d, Gal, H-1., 4.78 Ž1H, d, Glc, H-1., 4.95 Ž1H, d, Glc, H-1., 5.09 Ž1H, d, Xyl, H-1. and 5.15 Ž1H, d, Xyl, H-1.: 13 C-NMR: see Tables 1 and 2; CI-MS: mrz 1211.9 wMqq 29x, 1181.9 wMqq Hx, 1050 wMq-Xylx, 887 wMq-Xyl-Glcx, 753.5 wMq-2 = Xyl-Glcx, 591 wMq-2 = Xyl-2 = Glcx and 429 wMq-2 = Xyl-2 = Glc-Galx. 2.5. Acid hydrolysis of saponins 1–4 Each saponin Ž20 mg. was hydrolyzed with 2N HCl in MeOH Ž20 ml. for 5 h. The mixture was diluted with water and extracted with CHCl 3 . The CHCl 3 extract was concentrated and each aglycone was identified on the bases of spectral data. Saponin 1 gave diosgenin, saponins 2 and 3 gave neoruscogenin and saponin 4 gave neohecogenin.
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
377
2.6. Molluscicidal assay Biomphalaria alexandrina snails were collected in Egypt from the irrigation canal in Abou]Rawash, Giza Governorate and maintained in dechlorinated tap water in the laboratory conditions Ž25 " 28C and pH 7.0]7.7.. Tests were performed in duplicate using 10 snails for each test. Compounds were initially dissolved in a small amount of absolute ethanol then the desired dilution was prepared with dechlorinated tap water. The snails were exposed to different dilutions for 24 h followed by 24 h in dechlorinated tap water as recovery period. Procedures and statistical analysis were carried out according to the WHO and Litchfield and Wilcoxon protocols w15]17x.
3. Results and discussion The lipid content of the leaves of A. decipiens was studied. Twelve compounds Žhydrocarbons and sterols. were identified from the unsaponifiable matter ŽTable 3.. GLC analysis of the methylated fatty acids ŽTable 4. revealed the presence of 18 components, 14 of which were identified. Table 3 GLC analysis of the unsaponifiable fraction of A. decipiens leaves Peak no.
RRta
Peak area Ž%.
Identification
1 2 3 4 5 6 7 8 9 10 11 12 13
0.042 0.228 0.443 0.540 0.540 0.556 0.576 0.615 0.654 0.719 0.754 0.874 1
1.906 2.506 0.009 0.190 0.226 2.229 0.341 4.396 12.216 0.121 0.053 8.149 67.569
n-Tetradecane n-Hexadecane n-Heneicosane n-Docosane n-Tricosane Unknown n-Tetracosane n-Pentacosane n-Hexacosane n-Octacosane Squalene Stigmasterol b-Sitosterol
a
RRt, retention time relative to b-sitosterol ŽRt s 20.700..
The crude saponins from the BuOH fraction of the MeOH extract were subjected to repeated silica gel column and preparative TLC to afford compounds 1–4. Compounds 1 and 3 were easily deduced to be furostanol saponins while compounds 2 and 4 were spirostanol saponins on the basis of the color reaction with Ehrlich’s reagent and detailed spectroscopic investigation. Saponin 1 showed broad IR absorption bands at 3412 and 1026 cmy1 indicating
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
378
Table 4 GLC analysis Žas methyl esters. of the fatty acids from the saponifiable fraction of A. decipiens leaves a Peak no.
RRt
Peak areaŽ%.
Fatty acid
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
0.242 0.308 0.388 0.507 0.542 0.607 0.688 0.756 0.836 0.888 1.000 1.052 1.134 1.212 1.258 1.386 1.531 1.668
0.085 0.108 0.855 0.367 5.000 0.354 4.294 1.369 12.652 2.813 20.668 13.257 9.445 9.050 4.640 1.150 2.832 8.292
Caprylic acid Unknown Capric acid Unknown Lauric acid Lauroleic acid Myristic acid Myristolic acid Palmitic acid Palmitoleic acid Oleic acid Linoleic acid Linolenic acid Arachidic acid Unknown Heicosanoic acid Unknown Behenic acid
a
RRt, retention time relative to oleic acid methyl ester ŽRt s 7.317..
its glycosidic nature w18,19x. Saponin 1 was believed to be 22]methoxy furostanol saponin by Ehrlich’s test and the 1 H- and 13 C-NMR w dH 3.12 Ž3H, s ., d C 112.10 ŽC., 46.40 ŽMe.x w20,21x. The characteristic furostanol carbon signals and four anomeric carbon atom signals at d 100.80, 102.00, 102.50 and 105.50 for the sugar moieties were present in the 13 C-NMR spectrum ŽTable 2.. The signal at d 75.30 ppm was assigned to the glycosidated at C-26 and the downfield signal at 77.50 ppm indicated that the other glycosidation linkage was at C-3 w18,20x. This was confirmed by the absence of the characteristic spiroketal bands in the IR spectrum w22]24x. 1 H-NMR spectrum revealed the presence of four methyl groups at d 0.67, 0.85, 0.95 and 1.15 for methyls attached to C-18, C-27, C-19, and C-21, respectively w18,19x. The signal at d 1.50 was due to the methyl group of 6-deoxyhexopyranose and the signals of four anomeric protons of the sugar moieties appeared at d 4.48, 4.91, 4.95 and 5.10 w21]23x. The CI-MS spectrum of saponin 1 showed an ion at mrz 1063 ŽMqq H.. The loss of MeOH was reflected in the presence of a peak at mrz 1032. Fragment ions at mrz 885 wMq-MeOH-Rhax and 869 ŽMq-MeOH-Glc. indicated loss of one rhamnosyl and one glycosyl units respectively w18,21x. Another fragment at mrz 737 wMq-MeOH-2 = Rhax reflected the loss of another rhamnosyl unit and indicated that the two rhamnosyl units are terminals. The peaks at 575 ŽMq-MeOH- 2 = Rha- Glc. and 413 wMq- MeOH-2 = Rha]2 = Glcx indicated that the aglycone of this saponin was directly attached to hexose unit Žglucose. w21]23x. Acid hydrolysis of saponin 1 yielded D-glucose, L-rhamnose and diosgenin as aglycone. Also the 13 C-NMR chemical shifts of the sugar moiety at C-3 suggested that the two terminal rhamnose units were attached at C-2 and C-4 of the inner glucose and this was confirmed by the shifts of C-2 and C-4 of the inner
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
379
glucose at downfield d 81.00 and 77.0 w24]27x. Thus, the structure of saponin 1 was determined to be 3-O-a-L-rhamnopyranosyl-Ž1 ª 2.-w a-L-rhamnopyranosylŽ1 ª 4.x-b-D-glucopyranosyl-26-O-b-D-glucopyranosyl-22a-methoxy-Ž25R.-furost-5ene-3b, 26-diol. The IR spectrum of saponin 2 exhibited absorption bands due to hydroxyl groups and the glycosidic linkage at 3389 and 1072 cmy1 w27x. On comparison between 1 Hand 13 C-NMR spectra of saponin 2 with those of saponin 1, the signals due to the secondary methyl group C-27, which were observed at d H 0.85 Ž3H, d . and d C 17.20 in saponin 1, were replaced by the signals assignable to an exomethylene group at d H 4.81 and 4.78 and d C 143.32 ŽC. and 108.65 ŽCH 2 . in saponin 2 w29]32x. Also the 1 H-NMR spectrum showed signals for three typical steroid methyl groups at d 0.72, 0.87 and 1.10. An olefinic proton and four anomeric proton signals were also observed at d 5.35, 4.70, 4.90, 5.00 and 5.10, respectively. The spirostanol structure of this saponin was suggested by the presence of a quaternary carbon signal at 109.12 assignable to C-22 of the spirostanol skeleton in the 13 C-NMR spectrum w30,32x. The CI-MS spectrum of saponin 2 gave a molecular ion at mrz 1061 wMqq Hx. The fragment ions at mrz 915 wMq-Rhax, mrz 899 wMq-Glcx and 753 wMq-Rha-Glcx indicated that the rhamnosyl and glucosyl units are terminals. Other fragments at mrz 591.4 wMq-Rha-2 = Glcx and mrz 429 wMq-Rha-2 = Glc-Galx assigned to the loss of one rhamnosyl unit, two glucosyl units and galactosyl unit indicated that the galactosyl unit is directly attached to the aglycone moiety w35,36x. Acid hydrolysis of saponin 2 gave neoruscogenin w30]33x. The sugar residues were D-galactose, D-glucose and L-rhamnose. In the 13 C-NMR spectrum, the signal of C-4 due to inner galactose shifted downfield to d 80.10, suggested it to be linked to the inner glucose unit. Also, it was deduced that the terminal glucose and the terminal rhamnose units are attached to C-2 and C-3 of the inner glucose where the signals due to the two carbon atoms in 13 C-NMR spectrum were shifted downfield to d 81.32 and 82.60, respectively, whereas the other carbon signals of this glucose remained almost unaffected w29]31x. The tetrasaccharide was concluded to be linked at C-1 hydroxyl of the aglycone because in the 13 C-NMR spectrum of saponin 2 the signal due to C-1 of the aglycone was shifted to a lower field at d 82.57, whereas the chemical shift of this carbon in neoruscogenin appears at d 73.20 w32,33x. Therefore, the structure of saponin 2 was established as neoruscogenin 1-O -b- D -glucopyranosyl- Ž 1 ª 3 . - w a- L rhamnopyranosyl-Ž1 ª 2.x-b-D-glucopyranosyl-Ž1 ª 4.-b-D-galactopyranoside. Saponin 3 was shown to be a 22-methoxy furostanol saponin by Ehrlich’s reagent w20x and the 1 H- and 13 C-NMR spectra w d H 3.24 Ž3H, s ., d C 112.10 ŽC. and 46.30 ŽMe.x w20,21x. The 1 H-NMR spectrum of this saponin showed signals of three methyls at d 0.75 Ž s, 18-Me., 0.90 Ž d, 21-Me. and 1.0 Ž s, 19-Me. and an exomethylene group at d 5.34 and 5.02 ŽH 2-27. beside the presence of an olefinic proton signal of H-6 at d 5.60. This was confirmed by the presence of a pair of olefinic carbon signals 139.50 ŽC. and 121.50 ŽCH. in 13 C-NMR spectrum w28,30x. The 1 Hand 13 C-NMR spectra displayed four anomeric protons signals at d 4.70, 4.90, 5.00 and 5.10, and four anomeric carbons at d 103.50, 102.50, 101.50 and 104.50 indicating that saponin 3 contained two glucosyl units and two rhamnosyl units
380
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
w30,31x. CI-MS spectrum of saponin 3 showed a molecular ion at mrz 1077 wMqq Hx. Fragments at mrz 1046.6 wMq-MeOHx and mrz 898.5 wMq-MeOH-Rhax resulted from the elimination of one molecule of methanol and one rhamnosyl unit indicating that the rhamnose moiety is terminal. Other fragments at mrz 737 wMq-Rha-Glcx and mrz 591 wMq-2 = Rha-Glcx indicated loss of two rhamnosyl and one glucosyl units. Fragment at mrz 429 wMq-2 = Rha-2 = Glcx demonstrated that the aglycone moiety is directly linked to one glucose unit and the other glucose unit is attached to C-26 of the aglycone w29,31x. The trisaccharide was shown to be linked at C-1 of the aglycone where signal due to C-1 in 13 C-NMR was shifted to lower field at d 82.51 whereas the signal of this C-atom in neoruscogenin appears at d 73.2 w32x. The signals of C-2 and C-4 of the inner glucose were shifted downfield to d 79.8 and 81.9, respectively indicating that the two terminal rhamnose units are linked in these positions w29]33x. The above 1 H-NMR data and inspection of 13 C-NMR spectrum allowed identification of the aglycone of saponin 3 as neoruscogenin w28]31x. Thus, the structure of saponin 3 was shown to be 1-O-a-L-rhamnopyranosyl-Ž1 ª 2.-w a-L-rhamnopyranosyl-Ž1 ª 4.x-b-Dglucopyranosyl-26-O-b-D-glucopyranosyl-22-O-methylfurosta-5, 25Ž27. ]diene-1b, 3b, 22, 26-tetraol. The IR spectrum of saponin 4 indicated the existence of hydroxyl groups Ž3419 cmy1 ., a carbonyl group on a six membered ring Ž1704 cmy1 . and the characteristic absorption bands of Ž25S.-spiroketal at 919 and 896 cmy1 with the absorption at 919 cmy1 being of greater intensity than at 896 cmy1 w27,34x. Also, the Ž25S. -spirostan skeleton of saponin 4 was suggested by the occurrence of a resonance at d 108.6 ŽC-22. and diagnostic shifts at d 26.3 ŽC-23., 25.5 ŽC-24., 26.9 ŽC-25. and 65.8 ŽC-26. in the 13 C-NMR spectrum w26]34x. The S-orientation of the C-27 methyl was further confirmed by its 13 C-NMR signal at d 16.02 w34,35x. The downfield shift Žq5.18., as compared to neohecogenin Ž d 70.9. for C-3, indicated that the sugar chain is attached at C-3 of the aglycone w27,34x. The 1 H-NMR spectrum of saponin 4 showed signals attributable to C-19 and C-27 methyl groups at 0.75 and 0.95, the C-18 and C-21 methyls at 1.01 and 1.25 beside the presence of five anomeric proton signals at d 4.47, 4.78, 4.95, 5.09 and 5.15 indicative of the presence of five sugar units w34]36x. This was confirmed by the presence of five anomeric carbon signals in the 13 C-NMR at d 100.50, 101.50, 102.30, 104.31 and 105.50. The MS fragments at mrz 1050 wMq-Xylx and mrz 887 wMq-Xyl-Glcx indicated that both one xylose and one glucose are terminal. Other fragments at mrz 753.5 wMq-2 = Xyl-Glcx and mrz 591 wMq-2 = Xyl-2 = Glcx demonstrated the loss of two xylose and one glucose units. Fragment at mrz 429 wMq-2 = Xyl-2 = Glc]Galx indicated that the aglycone Žneohecogenin. is directly attached to the galactose moiety w35]37x. The glycosidic linkage of the pentasaccharide moiety was established by 13 C-NMR. The C-2 and C-3 of the inner glucose were shifted downfield at d 81.10 and 83.50, respectively indicating that the glycosidation linkages occurred at the two carbon atoms. The C-3 of the inner xylose shifted downfield at d 78.10 indicated that the terminal xylose is linked at this carbon. Also, C-4 of the inner galactose was shifted downfield at 79.60 indicating that the glycosidation linkage occurs at C-4 w27]34x. On the basis of the above data, saponin
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
381
4 was assigned to be neohecogenin 3-O-b-D-glucopyranosyl-Ž1 ª 3.-wb-Dxylopyranosyl-Ž1 ª 3.-b-D-xylopyranosyl-Ž1 ª 2.x ]b-D-glucopyranosyl-Ž1 ª 4.-b-Dgalactopyranoside. The results of the biological tests confirmed that monodesmosidic saponins 2 and 4 have strong molluscicidal activity against B. alexandrina snails ŽLC 90 s 13 and 6 ppm, respectively. within 24 h, whereas the bidesmosidic saponins 1 and 3 are inactive up to 50 ppm.
References w1x w2x w3x w4x w5x w6x w7x w8x w9x w10x w11x w12x w13x w14x w15x w16x w17x w18x w19x w20x w21x w22x w23x w24x w25x w26x w27x w28x w29x w30x w31x w32x w33x w34x w35x w36x w37x
Mahato SB, Ganguly AN, Sahu NP. Phytochemistry 1982;21:959. Blunden G, Yi Y, Jewers K. Phytochemistry 1978;17:1923. Kishor N, Sati OP. J Nat Prod 1990;53:1557. Sati OP, Pant G. J Nat Prod 1985;48:395. Sharma SC, Sati OP. Phytochemistry 1982;21:1820. Sati OP, Rana U, Chaukiyal DC. J Nat Prod 1987;50:263. El-Sayed MM. Bull Pharm Assiut University 1997;20:147. Abdel-Gawad MM, El-Sayed MM, Abdel-Hameed ES. The 9 th Int. Conf. of pesticide chemistry. 2 August. London: Westminster, 1998. Farag RS, Ahmed AI, Rashed SE, Ewies MA. J Agr Res 1980;9:248. Khalifa TI, Ammer HA, Azizi M, Hosny J. Az J Nat Prod 1989;3:23. Johnson AR, Dovenpart JB. Biochemistry and methodolgy of lipids. New York: A division of John Wiley and Sons, Inc, 1971:35. Marrison WR, Smith JM. J Lip Res 1964;5:60. Bayer E. Gas chromatography, 5th ed, vol I. London: Longmans, Green and Co, 1967:2. Fathy MM, Abdel- Megid RM. Zag J Pharm Sci 1995;4:121. WHO, . W H O 1953;65:33. WHO, . W H O Monogr Ser 1965;50:124. Litchfield TT, Wilcoxon F. J Pharmacol Exp Ther 1949;96:99. Wall ME, Eddy CR, McClennan ML, Klumpp MA. Anal Chem 1952;24:1337. Eddy CR, Wall LE, Scott MK. Anal Chem 1952;25:266. Kawasaki T, Komori T, Miyahara K, Nahara T, Hosokawa I, Mihashi K. Chem Pharm Bull 1974;22:2175. Hirai Y, Konishi T, Sanada S, Ida Y, Shoji J. Chem Pharm Bull 1982;30:3476. Kiyosawa S, Hotoh M, Komori T, Nohara T, Hosokawa I, Kawasaki. Chem Pharm Bull 1968;16:162. Sati OP, Pant G. J Nat Prod 1985;48:390. Shao Y, Poobrasert O, Kennely E et al. Planta Med 1997;63:258. Aquino R, Behar I, De Simone F, D’Agostina M, Pizza C. J Nat Prod 1986;49:1096. Liang ZZ, Aquino R, De Simone F, Dini A, Schettino O, Pizza C. Planta Med 1987;53:344. Wu G, Jiang S, Jiang F, Zhu D, Wu H, Giang C. Phytochemistry 1996;42:1677. Mimaki Y, Takaashi Y, Kuroda M, Sashida Y. Phytochemistry 1997;45:1229. Yu BY, Qiu SX, Zaw K et al. Phytochemistry 1996;43:201. Mimaki Y, Kuroda M, Kameyawa A, Yokosuka A, Sashida Y. Phytochemistry 1998;47:1351. Mimaki Y, Kanmoto T, Sashida Y, Nishino A, Satomi Y, Nisheno H. Phytochemistry 1996;41:1405. Pourrat H, Lamaison JI, Gramin JC, Remuson R. Ann Pharm Francaises 1982;40:451. Gonzalez AG, Francisco CG, Friere R, Hernandez R, Salazar JA, Suarez E. Phytochemistry 1975;14:2257. Li XC, Wang DZ, Yang CR. Phytochemistry 1990;29:3899. Wang Y, Ohtani K, Kasai R, Yamasaki K. Phytochemistry 1996;42:1417. Li XC, Wang DZ, Yang CR. Phytochemistry 1990;29:3893. Wang Y, Ohtani K, Kasai R, Yamasaki K. Phytochemistry 1997;45:811.
Molluscicidal steroidal saponins and lipid content of Aga¨e decipiens M.M. Abdel-Gawad, M.M. El-SayedU , E.S. Abdel-Hameed Laboratory of Medicinal Chemistry, Theodor Bilharz Research Institute, Giza, Egypt
Received 29 September 1998; accepted Žrevised. 23 November 1998
Abstract GLC analysis of the petrol extract of the leaves of Aga¨ e decipiens revealed that n-hexacosane was the main hydrocarbon, b-sitosterol the main sterol and oleic acid the main fatty acid. Two spirostanol and two furostanol saponins have been isolated from the methanolic extract. The structures of these saponins were established as 3-O-a-L-rhamnopyranosyl-Ž1 ª 2.-w a-L-rhamnopyranosyl-Ž1 ª 4.x-b-D-glucopyranosyl-26-Ob- D -glucopyranosyl-22 a-methoxy- Ž25R .-furost-5-ene-3b,26-diol Ž1 ., neoruscogenin 1-O-b-D-glucopyranosyl-Ž1 ª 3.-w a-L-rhamnopyranosyl-Ž1 ª 2.x-b-D-glucopyranosyl-Ž1 ª4.b-D-galactopyranoside Ž2., 1-O-a-L-rhamnopyranosyl-Ž1 ª 2.-w a-L-rhamnopyranosyl-Ž1 ª 4 .x-b- D -glucopyranosyl-26-O-b- D -glucopyranosyl-22-O-methylfurosta-5,25 Ž27 . ] diene1b,3b,22,26]tetraol Ž3 . and neohecogenin 3-O-b-D-glucopyranosyl-Ž1 ª 3 .-w b-Dxylopyranosyl-Ž1 ª 3.-b-D-xylopyranosyl-Ž1 ª 2.x ] b-D-glucopyranosyl-Ž1 ª 4.-b-D ]galactopyranoside Ž4.. Saponins 2 and 4 showed high molluscicidal activity against B. alexandrina snails ŽLC 90 s 13 and 6 ppm, respectively., whereas saponins 1 and 3 were inactive up to 50 ppm. Q 1999 Elsevier Science B.V. All rights reserved. Keywords: Aga¨ e decipiens; Steroidal saponins; Molluscicidal activity; Hydrocarbons; Sterols; Fatty acids
U
Corresponding author.
0367-326Xr99r$ - see front matter Q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 6 7 - 3 2 6 X Ž 9 9 . 0 0 0 5 7 - X
372
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
1. Introduction The family Agavaceae with more than 480 species has a distribution in tropical and subtropical dry climate regions through the world. The occurrence of steroidal constituents in several Agavaceae plants, especially those belonging to the genera Aga¨ e and Yucca, is well documented w1]3x. A review of literature showed that the leaves of different Aga¨ e species contain a number of steroidal saponins and sapogenins w4]7x. No study seems to have been made on saponins of Aga¨ e decipiens Baker. In the same time, the leaves of this plant showed high molluscicidal activity against Biomphalaria alexandrina snail, the intermediate host of Schistosoma mansoni in Egypt w8x. The present paper provides an account of the isolation and structure elucidation of certain molluscicidal saponins as well as the lipid content of this plant.
2. Experimental 2.1. Plant material A. decipiens leaves were collected in June 1996 from the Orman Garden. The plant was kindly identified by Eng. Badia H. Diwan. ŽAgriculture Eng. of Orman Garden.. 2.2. Extraction and isolation Shade-dried powdered leaves Ž2 kg. were defatted four times with petrol Ž60]808C. Ž4 = 6 l. and the petrol extract was evaporated to dryness Ž6.5 g.. The defatted leaves were extracted with MeOH and the extract was evaporated under reduced pressure to dryness. The residue Ž200 g. was dissolved in water and extracted successively with CHCl 3 , EtOAc and n-BuOH to yield the corresponding fractions Ž2.5 g, 12 g and 40 g, respectively.. 2.3. Analysis of the lipidic fraction The petrol extract Ž6.5 g. was saponified with 10% KOH in MeOH w9,10x. About 500 mg of fatty acids fraction was methylated w11,12x and GLC analyzed w13,14x. 2.4. Isolation of the saponins The n-BuOH extract Ž40 g. was Si-gel CC eluting with CHCl 3 and gradient with MeOH and finally with MeOH. Fractions eluted with CHCl 3-MeOH 95:5 were collected and purified by recrystallization several times with MeOH to yield saponin 1 Ž100 mg.. Fractions eluted with CHCl 3-MeOH 85:15 and 75:25 were collected and subjected to PTLC Ž n-PrOH-EtOAc-H 2 O 4:3:2. to give saponin 2 Ž200 mg. and 3 Ž110 mg.. Fractions eluted with CHCl 3-MeOH 70:30 were collected
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
373
and Si-gel CC eluting with a step-wise gradient of CHCl 3-MeOH and finally with MeOH. Fractions eluted with CHCl 3-MeOH 60:40 were collected and purified on PTLC to give saponin 4 Ž170 mg..
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
374
Table 1 13 C-NMR chemical shifts Ž d, ppm. of the aglycone moieties of saponins 1]4 Ž100 MHz, DMSO-d6 . Carbon
1
2
3
4
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 OMe
36.2 30.1 77.5 39.1 140.0 121.4 31.9 31.3 45.2 36.2 21.2 39.5 40.6 56.2 32.1 80.5 62.5 16.10 19.10 40.6 15.9 112.1 30.3 28.7 34.2 75.3 17.20 46.4
82.57 37.84 68.74 41.05 139.71 121.20 31.56 32.39 49.45 41.16 23.9 40.42 40.10 55.84 32.33 80.62 64.09 16.07 15.51 41.05 14.68 109.12 33.41 29.709 143.32 64.9 108.65 ]
82.51 37.50 68.3 41.9 139.5 121.5 31.5 32.6 49.40 42.4 24.7 40.3 40.1 55.5 31.5 80.02 63.50 16.40 14.60 40.20 15.90 112.10 31.50 27.80 146.0 71.8 111.2 46.3
37.2 31.5 76.08 37.7 44.7 28.3 31.3 31.9 55.5 36.7 37.2 ] 55.5 55.8 31.7 79.2 52.9 16.02 18.12 42.4 13.4 108.6 26.3 25.5 26.9 65.8 16.02 ]
Saponin 1. White powder, mp 271]2728C Rf 0.37 ŽCHCl 3- MeOH 9:1.. IR max ŽKBr.: 3412 ŽOH., 1636 ŽCHsCH., 1026 ŽC}O}C. cmy1 , no spiroketal absorption; 1 H-NMR Ž270 MHz, DMSO-d6 .: d 0.67 Ž3H, s, H-18., 0.85 Ž3H, d, H-27., 0.95 Ž3H, s, H-19., 1.15 Ž3H, d, H-21., 1.50 ŽMe of Rha., 3.12 Ž3H, s, OMe., 4.48 Ž1H, d, Glc, H-1., 4.91 Ž1H, d, Glc, H-1., 4.95 Ž1H, d, Rha, H-1., 5.10 Ž1H, d, Rha, H-1. and 5.35 Ž1H, C-6.; 13 C-NMR: see Tables 1 and 2; CI-MS: mrz 1063 wMqq Hx, 1032 wMq-MeOHx, 885 wMq-MeOH-Rhax, 869 wMq-MeOH-Glcx, 737 wMq-MeOH-2 = Rhax, 575 wMq-MeOH-2 = Rha-Glcx and 413 wMq-MeOH-2 = Rha-2 = Glcx. Saponin 2. White powder, mp 255]2578C, Rf 0.65 Ž n-PrOH-EtOAc-H 2 O 4:3:2.; IR max ŽKBr.: 3389 ŽOH., 2928, 2369, 1609, 1563, 1448, 1072, 980, 919, 890 and 669 cmy1 ; 1 H-NMR Ž270 MHz, DMSO-d6 .: d 0.72 Ž3H, s, H-18., 0.87 Ž3H, d, H-21., 1.10 Ž3H, s, H-19., 1.70 ŽMe of Rha., 4.81 and 4.78 ŽH 2-27., 4.70 Ž1H, d, Gal, H-1., 4.90 Ž1H, d, Glc, H-1., 5.00 Ž1H, d, Glc, H-1., 5.10 Ž1H, d, Rha, H-1. and 5.35 Ž1H, d, H-6.; 13 C-MNR: see Tables 1 and 2; CI-MS: mr z 1079 wMqq 17x, 1061 wMqq Hx, 915 wMq-Rhax, 899 wMq-Glcx, 753 wMq-Rha-Glcx, 591.4 wMq-Rha-2 = Glcx and 429 wMq-Rha-2 = Glc-Galx.
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
375
Table 2 13 C-NMR chemical shifts Ž d, ppm. of 3-O-sugar moieties of saponins 1]4 Ž100 MHz, DMSO-d6 . Carbons
1
2
100.80 81.00 76.10 77.00 76.80 61.80
3
4
Glc
1 2 3 4 5 6
103.50 79.80 76.20 81.90 76.30 62.10
Gal
1 2 3 4 5 6
102.40 72.62 75.10 80.10 75.20 61.30
100.50 72.50 75.30 79.60 75.00 60.30
Glc Ž1 ª 4.
1 2 3 4 5 6
103.50 81.32 82.60 70.60 72.30 62.20
101.50 81.10 83.50 70.90 76.00 62.50
Glc Ž1 ª 3.
1 2 3 4 5 6
104.1 75.20 77.30 71.30 77.60 62.60
Glc Ž1 ª 2.
1 2 3 4 5 6
Rha Ž1 ª 2.
1 2 3 4 5 6
102.00 70.50 72.60 74.00 69.00 18.50
Rha Ž1 ª 4.
1 2 3 4 5 6
102.50 71.60 72.20 74.10 69.00 18.90
102.30 75.20 77.10 70.90 76.80 62.10 102.50 71.60 72.50 74.30 69.70 18.30
102.50 71.60 72.10 74.20 69.30 18.30 101.50 72.80 72.30 74.10 69.10 18.50
376
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
Table 2 Ž Continued. Xyl Ž1 ª 2.
1 2 3 4 5
104.31 75.20 78.10 71.60 67.20
Xyl Ž1 ª 3.
1 2 3 4 5
105.50 75.10 76.50 70.90 66.60
26-O-GLc
1 2 3 4 5 6
105.50 75.20 78.80 71.80 77.80 62.30
104.50 75.10 77.90 71.20 78.20 62.60
Saponin 3. White powder, mp 258]260 o C, Rf 0.52 Ž n-PrOH-EtOAc-H 2 O 4:3:2.; IR max ŽKBr.: 3405 ŽOH., 2927ŽCH., 1425, 1367, 1278, 1074, 663 cmy1 ; 1 H-NMR Ž270 MHz, DMSO-d6 .: d 0.75 Ž3H, s, H-18., 0.90 Ž3H, d, H-21., 1.0 Ž3H, s, H-19., 1.55 Ž3H, d, Me of Rha. 1.65 Ž3H, d, Me of Rha., 3.24 Ž3H, s, OMe., 4.70 Ž1H, d, Glc, H-1., 4.90 Ž1H, d, Glc, H-1., 5.00 Ž1H, d, Rha, H-1., 5.10 Ž1H, d, Rha, H-1., 5.34 and 5.02 ŽH-27., and 5.60 Ž1H, d, H-6.; 13 C-NMR: see Tables 1 and 2; CI-MS: mrz 1077 wMqq Hx, 1046.6 wMq-MeOHx, 898.5 wMq-MeOH-Rhax, 737 wMq-RhaGlcx, 591 wMq-2 = Rha-Glcx, and 429 wMq-2 = Rha-2 = Glcx. Saponin 4. White powder, mp 248]250 o C, Rf 0.50 Ž n-PrOH-EtOAc-H 2 O 4:3:2.; IR max ŽKBr.: 3419 ŽOH., 2930, 1704 Žcarbonyl group on a six membered ring., 1455, 1376, 1231, 1158, 1040, 919 and 896 Ž25S, 919 ) 896 spiroketal. cmy1 ; 1 H-NMR Ž270 MHz, DMSO-d6 .: d 0.75 Ž3H, s, H-19., 0.95 Ž3H, d, H-27., 1.01 Ž3H, s, H-18., 1.25 Ž3H, s, H-21., 4.47 Ž1H, d, Gal, H-1., 4.78 Ž1H, d, Glc, H-1., 4.95 Ž1H, d, Glc, H-1., 5.09 Ž1H, d, Xyl, H-1. and 5.15 Ž1H, d, Xyl, H-1.: 13 C-NMR: see Tables 1 and 2; CI-MS: mrz 1211.9 wMqq 29x, 1181.9 wMqq Hx, 1050 wMq-Xylx, 887 wMq-Xyl-Glcx, 753.5 wMq-2 = Xyl-Glcx, 591 wMq-2 = Xyl-2 = Glcx and 429 wMq-2 = Xyl-2 = Glc-Galx. 2.5. Acid hydrolysis of saponins 1–4 Each saponin Ž20 mg. was hydrolyzed with 2N HCl in MeOH Ž20 ml. for 5 h. The mixture was diluted with water and extracted with CHCl 3 . The CHCl 3 extract was concentrated and each aglycone was identified on the bases of spectral data. Saponin 1 gave diosgenin, saponins 2 and 3 gave neoruscogenin and saponin 4 gave neohecogenin.
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
377
2.6. Molluscicidal assay Biomphalaria alexandrina snails were collected in Egypt from the irrigation canal in Abou]Rawash, Giza Governorate and maintained in dechlorinated tap water in the laboratory conditions Ž25 " 28C and pH 7.0]7.7.. Tests were performed in duplicate using 10 snails for each test. Compounds were initially dissolved in a small amount of absolute ethanol then the desired dilution was prepared with dechlorinated tap water. The snails were exposed to different dilutions for 24 h followed by 24 h in dechlorinated tap water as recovery period. Procedures and statistical analysis were carried out according to the WHO and Litchfield and Wilcoxon protocols w15]17x.
3. Results and discussion The lipid content of the leaves of A. decipiens was studied. Twelve compounds Žhydrocarbons and sterols. were identified from the unsaponifiable matter ŽTable 3.. GLC analysis of the methylated fatty acids ŽTable 4. revealed the presence of 18 components, 14 of which were identified. Table 3 GLC analysis of the unsaponifiable fraction of A. decipiens leaves Peak no.
RRta
Peak area Ž%.
Identification
1 2 3 4 5 6 7 8 9 10 11 12 13
0.042 0.228 0.443 0.540 0.540 0.556 0.576 0.615 0.654 0.719 0.754 0.874 1
1.906 2.506 0.009 0.190 0.226 2.229 0.341 4.396 12.216 0.121 0.053 8.149 67.569
n-Tetradecane n-Hexadecane n-Heneicosane n-Docosane n-Tricosane Unknown n-Tetracosane n-Pentacosane n-Hexacosane n-Octacosane Squalene Stigmasterol b-Sitosterol
a
RRt, retention time relative to b-sitosterol ŽRt s 20.700..
The crude saponins from the BuOH fraction of the MeOH extract were subjected to repeated silica gel column and preparative TLC to afford compounds 1–4. Compounds 1 and 3 were easily deduced to be furostanol saponins while compounds 2 and 4 were spirostanol saponins on the basis of the color reaction with Ehrlich’s reagent and detailed spectroscopic investigation. Saponin 1 showed broad IR absorption bands at 3412 and 1026 cmy1 indicating
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
378
Table 4 GLC analysis Žas methyl esters. of the fatty acids from the saponifiable fraction of A. decipiens leaves a Peak no.
RRt
Peak areaŽ%.
Fatty acid
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
0.242 0.308 0.388 0.507 0.542 0.607 0.688 0.756 0.836 0.888 1.000 1.052 1.134 1.212 1.258 1.386 1.531 1.668
0.085 0.108 0.855 0.367 5.000 0.354 4.294 1.369 12.652 2.813 20.668 13.257 9.445 9.050 4.640 1.150 2.832 8.292
Caprylic acid Unknown Capric acid Unknown Lauric acid Lauroleic acid Myristic acid Myristolic acid Palmitic acid Palmitoleic acid Oleic acid Linoleic acid Linolenic acid Arachidic acid Unknown Heicosanoic acid Unknown Behenic acid
a
RRt, retention time relative to oleic acid methyl ester ŽRt s 7.317..
its glycosidic nature w18,19x. Saponin 1 was believed to be 22]methoxy furostanol saponin by Ehrlich’s test and the 1 H- and 13 C-NMR w dH 3.12 Ž3H, s ., d C 112.10 ŽC., 46.40 ŽMe.x w20,21x. The characteristic furostanol carbon signals and four anomeric carbon atom signals at d 100.80, 102.00, 102.50 and 105.50 for the sugar moieties were present in the 13 C-NMR spectrum ŽTable 2.. The signal at d 75.30 ppm was assigned to the glycosidated at C-26 and the downfield signal at 77.50 ppm indicated that the other glycosidation linkage was at C-3 w18,20x. This was confirmed by the absence of the characteristic spiroketal bands in the IR spectrum w22]24x. 1 H-NMR spectrum revealed the presence of four methyl groups at d 0.67, 0.85, 0.95 and 1.15 for methyls attached to C-18, C-27, C-19, and C-21, respectively w18,19x. The signal at d 1.50 was due to the methyl group of 6-deoxyhexopyranose and the signals of four anomeric protons of the sugar moieties appeared at d 4.48, 4.91, 4.95 and 5.10 w21]23x. The CI-MS spectrum of saponin 1 showed an ion at mrz 1063 ŽMqq H.. The loss of MeOH was reflected in the presence of a peak at mrz 1032. Fragment ions at mrz 885 wMq-MeOH-Rhax and 869 ŽMq-MeOH-Glc. indicated loss of one rhamnosyl and one glycosyl units respectively w18,21x. Another fragment at mrz 737 wMq-MeOH-2 = Rhax reflected the loss of another rhamnosyl unit and indicated that the two rhamnosyl units are terminals. The peaks at 575 ŽMq-MeOH- 2 = Rha- Glc. and 413 wMq- MeOH-2 = Rha]2 = Glcx indicated that the aglycone of this saponin was directly attached to hexose unit Žglucose. w21]23x. Acid hydrolysis of saponin 1 yielded D-glucose, L-rhamnose and diosgenin as aglycone. Also the 13 C-NMR chemical shifts of the sugar moiety at C-3 suggested that the two terminal rhamnose units were attached at C-2 and C-4 of the inner glucose and this was confirmed by the shifts of C-2 and C-4 of the inner
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
379
glucose at downfield d 81.00 and 77.0 w24]27x. Thus, the structure of saponin 1 was determined to be 3-O-a-L-rhamnopyranosyl-Ž1 ª 2.-w a-L-rhamnopyranosylŽ1 ª 4.x-b-D-glucopyranosyl-26-O-b-D-glucopyranosyl-22a-methoxy-Ž25R.-furost-5ene-3b, 26-diol. The IR spectrum of saponin 2 exhibited absorption bands due to hydroxyl groups and the glycosidic linkage at 3389 and 1072 cmy1 w27x. On comparison between 1 Hand 13 C-NMR spectra of saponin 2 with those of saponin 1, the signals due to the secondary methyl group C-27, which were observed at d H 0.85 Ž3H, d . and d C 17.20 in saponin 1, were replaced by the signals assignable to an exomethylene group at d H 4.81 and 4.78 and d C 143.32 ŽC. and 108.65 ŽCH 2 . in saponin 2 w29]32x. Also the 1 H-NMR spectrum showed signals for three typical steroid methyl groups at d 0.72, 0.87 and 1.10. An olefinic proton and four anomeric proton signals were also observed at d 5.35, 4.70, 4.90, 5.00 and 5.10, respectively. The spirostanol structure of this saponin was suggested by the presence of a quaternary carbon signal at 109.12 assignable to C-22 of the spirostanol skeleton in the 13 C-NMR spectrum w30,32x. The CI-MS spectrum of saponin 2 gave a molecular ion at mrz 1061 wMqq Hx. The fragment ions at mrz 915 wMq-Rhax, mrz 899 wMq-Glcx and 753 wMq-Rha-Glcx indicated that the rhamnosyl and glucosyl units are terminals. Other fragments at mrz 591.4 wMq-Rha-2 = Glcx and mrz 429 wMq-Rha-2 = Glc-Galx assigned to the loss of one rhamnosyl unit, two glucosyl units and galactosyl unit indicated that the galactosyl unit is directly attached to the aglycone moiety w35,36x. Acid hydrolysis of saponin 2 gave neoruscogenin w30]33x. The sugar residues were D-galactose, D-glucose and L-rhamnose. In the 13 C-NMR spectrum, the signal of C-4 due to inner galactose shifted downfield to d 80.10, suggested it to be linked to the inner glucose unit. Also, it was deduced that the terminal glucose and the terminal rhamnose units are attached to C-2 and C-3 of the inner glucose where the signals due to the two carbon atoms in 13 C-NMR spectrum were shifted downfield to d 81.32 and 82.60, respectively, whereas the other carbon signals of this glucose remained almost unaffected w29]31x. The tetrasaccharide was concluded to be linked at C-1 hydroxyl of the aglycone because in the 13 C-NMR spectrum of saponin 2 the signal due to C-1 of the aglycone was shifted to a lower field at d 82.57, whereas the chemical shift of this carbon in neoruscogenin appears at d 73.20 w32,33x. Therefore, the structure of saponin 2 was established as neoruscogenin 1-O -b- D -glucopyranosyl- Ž 1 ª 3 . - w a- L rhamnopyranosyl-Ž1 ª 2.x-b-D-glucopyranosyl-Ž1 ª 4.-b-D-galactopyranoside. Saponin 3 was shown to be a 22-methoxy furostanol saponin by Ehrlich’s reagent w20x and the 1 H- and 13 C-NMR spectra w d H 3.24 Ž3H, s ., d C 112.10 ŽC. and 46.30 ŽMe.x w20,21x. The 1 H-NMR spectrum of this saponin showed signals of three methyls at d 0.75 Ž s, 18-Me., 0.90 Ž d, 21-Me. and 1.0 Ž s, 19-Me. and an exomethylene group at d 5.34 and 5.02 ŽH 2-27. beside the presence of an olefinic proton signal of H-6 at d 5.60. This was confirmed by the presence of a pair of olefinic carbon signals 139.50 ŽC. and 121.50 ŽCH. in 13 C-NMR spectrum w28,30x. The 1 Hand 13 C-NMR spectra displayed four anomeric protons signals at d 4.70, 4.90, 5.00 and 5.10, and four anomeric carbons at d 103.50, 102.50, 101.50 and 104.50 indicating that saponin 3 contained two glucosyl units and two rhamnosyl units
380
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
w30,31x. CI-MS spectrum of saponin 3 showed a molecular ion at mrz 1077 wMqq Hx. Fragments at mrz 1046.6 wMq-MeOHx and mrz 898.5 wMq-MeOH-Rhax resulted from the elimination of one molecule of methanol and one rhamnosyl unit indicating that the rhamnose moiety is terminal. Other fragments at mrz 737 wMq-Rha-Glcx and mrz 591 wMq-2 = Rha-Glcx indicated loss of two rhamnosyl and one glucosyl units. Fragment at mrz 429 wMq-2 = Rha-2 = Glcx demonstrated that the aglycone moiety is directly linked to one glucose unit and the other glucose unit is attached to C-26 of the aglycone w29,31x. The trisaccharide was shown to be linked at C-1 of the aglycone where signal due to C-1 in 13 C-NMR was shifted to lower field at d 82.51 whereas the signal of this C-atom in neoruscogenin appears at d 73.2 w32x. The signals of C-2 and C-4 of the inner glucose were shifted downfield to d 79.8 and 81.9, respectively indicating that the two terminal rhamnose units are linked in these positions w29]33x. The above 1 H-NMR data and inspection of 13 C-NMR spectrum allowed identification of the aglycone of saponin 3 as neoruscogenin w28]31x. Thus, the structure of saponin 3 was shown to be 1-O-a-L-rhamnopyranosyl-Ž1 ª 2.-w a-L-rhamnopyranosyl-Ž1 ª 4.x-b-Dglucopyranosyl-26-O-b-D-glucopyranosyl-22-O-methylfurosta-5, 25Ž27. ]diene-1b, 3b, 22, 26-tetraol. The IR spectrum of saponin 4 indicated the existence of hydroxyl groups Ž3419 cmy1 ., a carbonyl group on a six membered ring Ž1704 cmy1 . and the characteristic absorption bands of Ž25S.-spiroketal at 919 and 896 cmy1 with the absorption at 919 cmy1 being of greater intensity than at 896 cmy1 w27,34x. Also, the Ž25S. -spirostan skeleton of saponin 4 was suggested by the occurrence of a resonance at d 108.6 ŽC-22. and diagnostic shifts at d 26.3 ŽC-23., 25.5 ŽC-24., 26.9 ŽC-25. and 65.8 ŽC-26. in the 13 C-NMR spectrum w26]34x. The S-orientation of the C-27 methyl was further confirmed by its 13 C-NMR signal at d 16.02 w34,35x. The downfield shift Žq5.18., as compared to neohecogenin Ž d 70.9. for C-3, indicated that the sugar chain is attached at C-3 of the aglycone w27,34x. The 1 H-NMR spectrum of saponin 4 showed signals attributable to C-19 and C-27 methyl groups at 0.75 and 0.95, the C-18 and C-21 methyls at 1.01 and 1.25 beside the presence of five anomeric proton signals at d 4.47, 4.78, 4.95, 5.09 and 5.15 indicative of the presence of five sugar units w34]36x. This was confirmed by the presence of five anomeric carbon signals in the 13 C-NMR at d 100.50, 101.50, 102.30, 104.31 and 105.50. The MS fragments at mrz 1050 wMq-Xylx and mrz 887 wMq-Xyl-Glcx indicated that both one xylose and one glucose are terminal. Other fragments at mrz 753.5 wMq-2 = Xyl-Glcx and mrz 591 wMq-2 = Xyl-2 = Glcx demonstrated the loss of two xylose and one glucose units. Fragment at mrz 429 wMq-2 = Xyl-2 = Glc]Galx indicated that the aglycone Žneohecogenin. is directly attached to the galactose moiety w35]37x. The glycosidic linkage of the pentasaccharide moiety was established by 13 C-NMR. The C-2 and C-3 of the inner glucose were shifted downfield at d 81.10 and 83.50, respectively indicating that the glycosidation linkages occurred at the two carbon atoms. The C-3 of the inner xylose shifted downfield at d 78.10 indicated that the terminal xylose is linked at this carbon. Also, C-4 of the inner galactose was shifted downfield at 79.60 indicating that the glycosidation linkage occurs at C-4 w27]34x. On the basis of the above data, saponin
M.M. Abdel-Gawad et al. r Fitoterapia 70 (1999) 371]381
381
4 was assigned to be neohecogenin 3-O-b-D-glucopyranosyl-Ž1 ª 3.-wb-Dxylopyranosyl-Ž1 ª 3.-b-D-xylopyranosyl-Ž1 ª 2.x ]b-D-glucopyranosyl-Ž1 ª 4.-b-Dgalactopyranoside. The results of the biological tests confirmed that monodesmosidic saponins 2 and 4 have strong molluscicidal activity against B. alexandrina snails ŽLC 90 s 13 and 6 ppm, respectively. within 24 h, whereas the bidesmosidic saponins 1 and 3 are inactive up to 50 ppm.
References w1x w2x w3x w4x w5x w6x w7x w8x w9x w10x w11x w12x w13x w14x w15x w16x w17x w18x w19x w20x w21x w22x w23x w24x w25x w26x w27x w28x w29x w30x w31x w32x w33x w34x w35x w36x w37x
Mahato SB, Ganguly AN, Sahu NP. Phytochemistry 1982;21:959. Blunden G, Yi Y, Jewers K. Phytochemistry 1978;17:1923. Kishor N, Sati OP. J Nat Prod 1990;53:1557. Sati OP, Pant G. J Nat Prod 1985;48:395. Sharma SC, Sati OP. Phytochemistry 1982;21:1820. Sati OP, Rana U, Chaukiyal DC. J Nat Prod 1987;50:263. El-Sayed MM. Bull Pharm Assiut University 1997;20:147. Abdel-Gawad MM, El-Sayed MM, Abdel-Hameed ES. The 9 th Int. Conf. of pesticide chemistry. 2 August. London: Westminster, 1998. Farag RS, Ahmed AI, Rashed SE, Ewies MA. J Agr Res 1980;9:248. Khalifa TI, Ammer HA, Azizi M, Hosny J. Az J Nat Prod 1989;3:23. Johnson AR, Dovenpart JB. Biochemistry and methodolgy of lipids. New York: A division of John Wiley and Sons, Inc, 1971:35. Marrison WR, Smith JM. J Lip Res 1964;5:60. Bayer E. Gas chromatography, 5th ed, vol I. London: Longmans, Green and Co, 1967:2. Fathy MM, Abdel- Megid RM. Zag J Pharm Sci 1995;4:121. WHO, . W H O 1953;65:33. WHO, . W H O Monogr Ser 1965;50:124. Litchfield TT, Wilcoxon F. J Pharmacol Exp Ther 1949;96:99. Wall ME, Eddy CR, McClennan ML, Klumpp MA. Anal Chem 1952;24:1337. Eddy CR, Wall LE, Scott MK. Anal Chem 1952;25:266. Kawasaki T, Komori T, Miyahara K, Nahara T, Hosokawa I, Mihashi K. Chem Pharm Bull 1974;22:2175. Hirai Y, Konishi T, Sanada S, Ida Y, Shoji J. Chem Pharm Bull 1982;30:3476. Kiyosawa S, Hotoh M, Komori T, Nohara T, Hosokawa I, Kawasaki. Chem Pharm Bull 1968;16:162. Sati OP, Pant G. J Nat Prod 1985;48:390. Shao Y, Poobrasert O, Kennely E et al. Planta Med 1997;63:258. Aquino R, Behar I, De Simone F, D’Agostina M, Pizza C. J Nat Prod 1986;49:1096. Liang ZZ, Aquino R, De Simone F, Dini A, Schettino O, Pizza C. Planta Med 1987;53:344. Wu G, Jiang S, Jiang F, Zhu D, Wu H, Giang C. Phytochemistry 1996;42:1677. Mimaki Y, Takaashi Y, Kuroda M, Sashida Y. Phytochemistry 1997;45:1229. Yu BY, Qiu SX, Zaw K et al. Phytochemistry 1996;43:201. Mimaki Y, Kuroda M, Kameyawa A, Yokosuka A, Sashida Y. Phytochemistry 1998;47:1351. Mimaki Y, Kanmoto T, Sashida Y, Nishino A, Satomi Y, Nisheno H. Phytochemistry 1996;41:1405. Pourrat H, Lamaison JI, Gramin JC, Remuson R. Ann Pharm Francaises 1982;40:451. Gonzalez AG, Francisco CG, Friere R, Hernandez R, Salazar JA, Suarez E. Phytochemistry 1975;14:2257. Li XC, Wang DZ, Yang CR. Phytochemistry 1990;29:3899. Wang Y, Ohtani K, Kasai R, Yamasaki K. Phytochemistry 1996;42:1417. Li XC, Wang DZ, Yang CR. Phytochemistry 1990;29:3893. Wang Y, Ohtani K, Kasai R, Yamasaki K. Phytochemistry 1997;45:811.