Chemistry Journal <strong>of</strong> Moldova. I. Lunga General, et al./Chem.J.Mold. Industrial and 2008, Ecological 3 (1), 89-93 Chemistry. 2008, 3 (1), 89-93STEROIDAL SAPONINS FROM THE SEEDS OF HYOSCYAMUS NIGER L.Irina Lunga a *, Pavel Kintia a , Stepan Shvets a , Carla Bassarello b , Sonia Piacente b , Cosimo Pizza baInstitute <strong>of</strong> Genetics and Physiology <strong>of</strong> Plants, Academy <strong>of</strong> Sciences <strong>of</strong> Moldova,Padurii 20, 2004, Chisinau, Moldova* lunga_irina@yahoo.com, tel. 373 22 555259, fax 373 22 556180bDepartment <strong>of</strong> Pharmaceutical Sciences, University <strong>of</strong> Salerno, Ponte Don Melillo, 84084, Fisciano, Salerno, ItalyAbstract: Ten steroidal saponins have been isolated form the seeds <strong>of</strong> Hyoscyamus niger L. for the first time andtheir structures have been elucidated. Seven saponins belong to spirostane series, two are furostane-type and oneis pregnane glycoside. Hyoscyamosides B, C and C 2are new steroidal saponins, which have never been reportedbefore in literature. Complete assignments <strong>of</strong> the 1 H and 13 C NMR chemical shifts for all these glycosides wereachieved by means <strong>of</strong> one- and two-dimensional NMR techniques, including 1H–1H COSY, HSQC, HMBC andROESY spectra.Keywords: Hyoscyamus niger L., steroidal saponins.IntroductionSteroidal saponins and pregnanes have established themselves as an important class <strong>of</strong> biologically activecompounds. Pharmacological studies in recent years have shown that these compounds possess antitumor [1-3], boneresorbing [4] and antifungal activities [5]. A number <strong>of</strong> such compounds have been isolated from the plants, especiallythe Solonaceae family [6].Hyoscyamus niger L. (Solonaceae) is a plant native to Europe and Asia and its leaves and seeds have been usedin traditional medicine for the treatment <strong>of</strong> nervous and convulsive diseases. Previous works have been focused onlyto the alkaloid constituents <strong>of</strong> this plant and it has never been reported about its steroidal saponins. This fact and ourinterest in secondary metabolites <strong>of</strong> Solonaceae family prompted us to perform the phytochemical investigation <strong>of</strong> theseeds <strong>of</strong> Hyoscyamus niger L. Our investigation yielded three new steroidal saponins for which we propose the nameshyoscyamosides B (1), C (2) and C 2(3) and seven known compounds - hyoscyamosides A (4), B 1(5), B 2(6), B 3(7), C1(8), E (9) and F1 (10).Results and discussionThe buthanol extract, containing steroidal saponins was chromatographed on silica gel column and also using prep.HPLC to afford ten compounds, 1-10. Among them, compounds 4-10 were identified as 3-O--D-galactopyranoside-(25R)-5-spirostan-3-ol (4) [7], 3-O--D-glucopyranosyl-(14)--D-galactopyranoside-(25 R)-5-spirostan-3ol(5) [7], 3-O--L-rhamnopyranosyl-(12)--D-glucopyranoside-(25R)- 5-spirostan-3-ol (6) [8], 3-O--Lrhamnopyranosyl-(12)--D-glucopyranoside-(25R)-spirost-5-en-3-ol(7) [8], 3-O--D-glucopyranosyl-(12)--D-glucopyranosyl-(14)-O--D-galactopyranoside-(25R)-5- spirostan-3-ol (8) [9], 3-O--D-glucopyranosyl-(14)-O--D-galactopyranoside-(25R)-26-O--D-glucopyranosyl-5-furostan-3,22,26-triol (9) [10], 3-O--Dglucopyranosyl-(12)--D-glucopyranosyl-(14)-O--D-galactopyranoside-(25R)-26-O--D-glucopyranosyl- 5furostan-3, 22, 26-triol (10) [10], on the basis <strong>of</strong> their spectroscopic data by comparison with literature data.The 1 H NMR spectrum <strong>of</strong> 1 showed signals for four steroidal methyl groups at 0.82 (3H, s, Me-18), 0.89 (3H,s, Me-19), 1.00 (3H, d, Me-21), 1.12 (3H, d, Me-27), two methine proton signals at 3.68 (1H, m, H-3) and 4.41 (1H,m, H-16) indicative <strong>of</strong> secondary alcoholic functions and two anomeric protons at 4.38 (1H, d, J = 7.5 Hz), 4.53(1H, d, J = 7.5 Hz), giving correlations with carbon signals at 103.5 and 105.0, respectively. The chemical shifts<strong>of</strong> all the individual protons <strong>of</strong> this sugar unit were ascertained from a combination <strong>of</strong> 1D-TOCSY and DQF-COSYspectral analysis, and the 13 C chemical shifts <strong>of</strong> their relative carbons could be assigned unambiguously from theHSQC spectrum. These data showed the presence <strong>of</strong> two sugars - -D-galactose and -D-glucose. The HMBC spectrumshowed key correlation peaks between the proton signal at 4.38 (H-1 gal) and the carbon resonance at 78.9 (C-3 <strong>of</strong> theaglycon) and at 4.53 (H-1 glc) and the carbon resonance at 80.7(C-3 gal) . On the basis <strong>of</strong> the above results the structure<strong>of</strong> 1 was determined as 3-O--D-glucopyranosyl- (13) - -D- galactopyranoside - (25 R)-5-spirostan - 3-ol andwas named by us hyoscyamoside B.The 1 H NMR spectrum <strong>of</strong> 2 showed identical aglycon moiety <strong>of</strong> compound 1 -(25R)- 5 spirostan-3-ol. Oneprimary alcoholic function at 67.7 (C-26) suggested the occurrence <strong>of</strong> a glycoside spirostanol skeleton. The 1 H NMRspectrum showed signals for three anomeric protons at 4.39 (1H, d, J = 7.5 Hz), 4.56 (1H, d, J = 7.5 Hz), and 4.69(1H, d, J = 7.5 Hz). 1D-TOCSY and DQF-COSY spectral analysis <strong>of</strong> the sugar moiety revealed the presence <strong>of</strong> one-galactopyranosyl unit ( 4.39) and two -glucopyranosyl units ( 4.56 and 4.69). The HMBC spectrum showed89
I. Lunga et al./Chem.J.Mold. 2008, 3 (1), 89-93key correlation peaks between the proton signal at 4.39 (H-1 gal) and the carbon resonance at 79.1 (C-3 <strong>of</strong> theaglycon), the proton signal at 4.56 (H-1 glc) and the carbon resonance at 78.1 (C-2 gal), the proton signal at 4.69(H-1 glcI) and the carbon resonance at 82.5 (C-3 gal). Thus, the structure <strong>of</strong> compound 2 was deduced as 3--{[-Dglucopyranosyl-(12)]-[-D-glucopyranosyl-(13)]--D-galactopyranoside}-(25-R)-5-spirostan-3-ol, which wenamed hyoscyamoside C.1H NMR spectrum <strong>of</strong> compound 3 displayed signals for three tertiary methyl groups at 0.90 (3H, s, Me-19),0.91 (3H, s, Me-18) and 2.28 (3H, s, Me-21), one olefinic proton at 6.91 (1H, m, H-16) together with three anomericprotons (1H, d, 4.40; 1H, d, 4.56; 1H, d, 4.69). On the other hand, the 13 C-NMR signals due to a total <strong>of</strong> 21 carbonsignals originating from the sapogenol were composed <strong>of</strong> three methyl groups at 12.2, 15.7, and 26.8, one oxygenbearingmethine carbon at 79.1 (C-3), one carbonyl group at 199.3 (C-20), two quaternary carbons at 35.7 and47.2, four methine carbons at 34.7, 45.9, 56.1 and 57.3, eight methylene carbons at d 21.3, 29.5, 30.0, 32.8, 32.9,35.0, 35.7 and 37.9, and two olefinic carbons at d 147.0 and 156.4. The HMBC disclosed the connectivities <strong>of</strong> the abovefunctional carbons to form a pregnane skeleton. Using 1D-TOCSY, COSY, HSQC and HMBC spectra the sugar chainwere characterized as -D-galactose, two -D-glucose. HMBC spectrum showed key correlation picks between sugarsanomeric protons and attached carbons at 4.40 (H-1 gal) and 79.1 (C-3 agl), 4.56 (H-1 glc) and 80.2 (C-4 gal), 4.69 (H-1 glcI)and 84.7 (C-2 glc). Thus, compound 3 was elucidated as 3-O-D-glucopyranosyl (12)]- -D-glucopyranosyl-(14)--D-galactopyranosyl-5-pregn-16-en-20-one-3-ol, for which we proposed the name hyoscyamoside C 2.MeMeOOHMeMeMeOOHMeMeMeROHRO1: R=Glc(1-3)Gal 7: R=Rha(1-2)Glc2: R=[Glc(1-3)][Glc(1-2)]Gal4: R=Gal5: R=Glc(1-4)Gal6: R=Rha(1-2)Glc8: R=Glc(1-2)Glc(1-4)GalOR 1MeOMeMe19Me1221Me18Me142017OH16O232625HMe2719ROHRO3H573: R=Glc(1-2)Glc(1-4)Gal 9: R=Glc(1-4)GalR 1=Glc10: R=Glc(1-2)Glc(1-4)GalR 1=Glc90
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