STEROIDAL SAPONINS FROM SOLANUM UNGUICULATW (A*) RICH

Seven steroidal saponins were isolated for the first time fiom the green berries of Solanum unguiculatum. Their structures were determined by spectroscopic analysis as well as hydrolysis of the glycosides into the corresponding sapogenins, diosgenin, chlorogenin, diosgenin-3-O-P-Dgalactopyranoside, diosgenin-3-0P-D-glucopyranosyl-(1 + 4)P-Dgalactopyranoside, 3 P-hydroxyl-(25R) 5 a-spirostan-6-one (laxogenin) 3-0a-Lrhamnopyranosyl-(1 + 2)P-D-glucopyranoside, isonarthogenin-3-0a-Lrhamnopyranosyl-(1 + 2)-0-[ a-L-rhamnopyranosyl-( 1 + 4)P-D-glucopyranoside and 25(S) spirost-5-en-3 P, 17 a, 27 triol-3-0-[ a-L-rhamnopyranosyl(1 2)] [a-L-rhamnopyranosyl (1 -* 4)]P-D-glucopyranoside.


INTRODUCTION
The occurrence of steroidal saponins in Solanum plants is well d o c ~r n e n t e d ( ' ~~~' In our previous paper(17) we reported on the isolation and structure determination of new steroidal a1 kaloids fiom Solanum unguiculatum (A,) Rich.A survey of literature showed that no chemical work has been reported on the saponins fiom Solanum unguiculatum (A.) Rich.This small tree is widely distributed in Yemen, Sana'a region and its h i t s are known for their and ppm (13c-nmr).IR spectra were recorded on a Perkin-Elmer Model 377 spectrometer in KBr discs.FAB-MS spectra were recorded with an MS 2500 high resolution spectrometer (Kratos Manchester, UK) with 70 eV, an ion source temperature of 180°C and a direct inlet using sulphur glycerol as matrix.

EXTRACTION AND CHROMATOGRAPHY
The fresh h i t s were cut into pieces and exhaustively extracted with methanol (4 L) by cold maceration.The extract was concentrated to almost dryness under reduced pressure.The residue (20 g) was suspended in water (112 L) and extracted with CHC13 (2 L) and then with n-BuOH (2 L).The chloroform-soluble phase was concentrated under reduced pressure, the resulting residue (7 g.) was chromatographed over silica gel column.Elution being carried out with petroleum-ether, increasing the polarity with EtOAc, early petrol-EtOAc (9: 1) eluate fractions yielded compound 1 (50 mg) and petrol-EtOAc (3 : 7) eluate fractions yielded compound 2(100 mg).
The n-BuOH-soluble phase was concentrated under reduced pressure and the resulting residue (1 0 g) was chromatographed over silica gel column.Elution being carried out with chloroform then increasing the polarity with methanol.

RESULTS AND DISCUSSION
The residue remaining fiom methanolic extract of the green berries of S.
unguzculatum was partitioned between chloroform and Hz0 then between nbutanol and HzO.A series of chromatographic separations of the chloroformsoluble phase and n-butanol-soluble phase krnished compounds 1,2 and 3 -7 respectively.
Compound and 907 (M+L~)+ suggesting that MW is 900.The molecular formula of 7 was estimated as C45H72018 by 13cNMR (Table 1) and FABMS spectrometry.On Hz) showed that the glucosyl band of 7 had the j3-configuration, however, on the basis of the coupling constant of anomeric proton of rhamnose in the 'HNMR spectrum an a and j 3 nature could not be deduced, but a-rhamnose could be identified from its "CNMR chemical shift with C-5 of a-and 0-rhamnose

F
indicating the presence of saponins.As part of our contribution to the study of S. unguiculatum (A.) Rich we have now carried out a phytochemical study of the chloroform and n-butanol-soluble parts of the methanolic extract of S. unguiczdatum (A.) Rich.This resulted in the isolation of seven saponins.EXPERIMENTAL GENERAL MPs; were measured on a Fisher-Johns hot stage and are uncorr.Optical rotations were measured on a Zeiss polarimeter Model 53 187 using a sodium lamp.NMR spectra were measured in CDC13 and pyridine-ds and recorded at 300 MHz for 1 HNMR and 75 MHz for 1 3 ~~~~ (using TMS as internal standard) on a Varian XL300 (Darmstadt, Germany).Chemical shifts are expressed in 6 values ('H)
1 crystallized fiom chloroform-petrol mixture as colourless needles with mp.205OC, [ale -126*(CHC13).The 'HNMR(300 MHz, CDCl,)    spectrum suggested the presence of two secondary methyl groups at 6 0.78 (d, J=6.2Hz) and 1.0 1 (d, J=6.1 Hz), two tertiary methyl groups as singlet at 6 1.03 and an olefinic proton appearing at 5.34 as doublet (J=5.1 Hz)('').A comparison of the carbon shifts for 1 with those of d i o ~~e n i n ( ' ~" ~)led to the assignment of all the carbon shifts and elucidation of the structure and stereochemistry of 1 as diosgenin (Table1).The spectrum (75 MHz, CDCl3) of 1 showed the presence of signals appearing at 6 140.7, 12 1.3 are attributed to C-5 and C-6 respectively(21', the observed close similarity of the shifts for C-23, C-24, C-25, C-26 and C-27 in compound 1 with those in diosgenin are diagnostic ofthe equatorial orientation of the C-25 methyl in the 22 a-0-spirostane skeleton and thus settled the 22R and 25R configuration of compound 1.Diosgenin is thus shown to be (25R)-22a-O-spirost-5-ene-3P-01(1).Compound 2 showed molecular ion peak at m/z 430 [M-HIcorresponding to the molecular formula C27fi304 with prominent peaks at m/z 139 and 11 5 indicative of spiroketal moiety(22?The IR spectrum showed prominent hydroxyl band, as well as the characteristic bands in the finger print zone attributed to the spiroketal moiety.Analysis of the IR spectrum indicated that 2 was a 25R spirostan because the 900 cm-' band was more intense than 922 rn-1 b , The "c-nrnr spectrum confirmed this conclusion.The F-ring carbons of compound 2 exhibited resonances at 109.2 ppm (C-22), 3 1.5 (C-23), 28.8 (C-24), 30.2 (C-25), 66.8 (C-26) and 16.2 (C-27).The carbons carrying hydroxyls appeared at 71.7 and 69.2 ppm.Thus, compound 2 was identified as chlorogenin by comparison of its mp.and physical properties with the experimental values reported by Wall et al.'25' Compound 3 showed a molecular ion peak at m/z 575 [M (C33HS208)'Win the negative FABmass spectrum.Upon acid hydrolysis, 3 yielded 1 as aglycone and galactose.In the 'HNMR spectrum of 3, one anomeric proton signal was observed at 8 4.98 ( l y d, J=8Hz) diagnostic for the P-configuration of the sugar moiety.It was observed that the glycosylation at the hydroxyl group of C-3 of the aglycone resulted in a downfield shift (6.3 ppm) of (c-3)") Therefore, the structure of 3 was elucidated to be diosgenin-3-O-P-Dgalactopyranoside.Compound 4, demonstrated a molecular formula of C39&2013 determined from the negative FABMS.Upon acid hydrolysis 4 yielded 1 as aglycone with galactose and glucose in ratio 1 : 1 as sugar components.In the 'HNMR spectrum of 4 two anomeric proton signals were observed at 6 4.3 5 (1 H, d, J=8Hz) and 5.33 (IH, d,J=7.7 Hz), diagnostic of the P-configuration of the two sugars.Comparison of "CNMR spectrum of 4 with that of 3 indicated that the terminal glucose was attached at C-4 of the inner galactose.The "CNMR spectrum of 4 indicted that it was a 3-0-diglycoside of 1, for the signal of C-3 was shifted F.A. A b b a s : downfield at 77.7 where as the signals at C-2 and C-4 were displaced upfield at 30 and 39.1 respectively.Furthermore, a typical signal at 80 arising from the glycosylation at the hydroxyl of C-4' of galactose was confirmed.Based on the above evidence, the structure of 4 was proved to be diosgenin-3-O-P-Dglucopyranosyl-(1 --t 4)-j3-D-galactopyranoside, identical to Funkioside C isolated from Funkia ~v a t a ( ~~) .Compound 5 was assigned the molecular ion peaks at m/z 7 7 7 [ ~+ ~] + , 7 6 2 [ M + ~a + ~] + and 721[M-HI-corresponding to the molecular formula C39h2013.The IR spectrum indicated the existence of a hydroxyl group [3440 cm-'1, a carbonyl group [I705 cm-'1 and a (25R)-spiroacetal moiety (975, 9 1 5, 895 and 860 cm-I), intensity 9 1 5 < 895(23324).The existence of a carbonyl group was indicated .by the UV (Lax 284 nm) and 1 3 c ~M R (at 209.6) spectra.The 'HNMR spectrum exhibited signals for two tertiary methyl protons at 8 0.79 and 0.64, two secondary methyl protons at 6 1.15 (d, J= 6.9 Hz) and 0.7 (d, J=5.8 Hz) and two anomeric protons at 4.93 (d, J= 7.8 Hz) and 6.3 3 (br .s).The above data suggested 5 to be a (25R)-spirostanol disaccharide.On acid hydrolysis of 5 (2N hydrochloric acid in dioxane-Hz0 1 : l), 5 was hydrolysed to give D-glucose and L-rhamnose as the carbohydrate moieties and an aglycone identified as 3P- hydroxyl-(25R)-5a-spirostan-6-one (Laxogenin) by its physical and spectral data(28).The structure of the disaccharide was determined by comparison of the carbon assignments with those of reference methyl glycosides(29) indicated the presence of terminal a-L-rhamnose unit and an inner j3-D-glucopyranosyl unit joined at C-2. Accordingly, the structure of 5 was formulated as (25R)-3P-5a-Compound 6 is a major constituent [aE -76 (MeOH).The post.FABMS showed a molecular ion peak at m/z 885 (M+H)+, constituent with the molecular formula C45H72017.The 'HNMR spectrum exhibited three secondary methyl proton signals at 6 1.77 (3H, d, J=6.2Hz), 1.63 (3H, d, J=6.2 Hz), 1.16 (3H, d, J=6.9 Hz), two tertiary methyl proton signals at 6 1.06 and 0.85 and three anomeric proton signals at 8 6.38 (br,~), 5.84 (br,s) and 4.94 (d, J=7.4 Hz).The signals at 6 1.77 and 1.63 were due to 6-deoxyhexoses.The "CNMR spectrum showed 45 carbon resonances.The '27 was modified as hydroxyl-methyl.Acid hydrolysis of 6 gave Dglucose, L-rhamnose and (25S)-spirost-5-en-3P, 27-diol (is~nartho~enin)('~.").Thus, the structure of 6 was characterized as isonarthogenein-3-0-a-L-rhamnopyranosyl-(1 + 2)-0-[a-L-rhamnopyranosyl-(1 + 4)-P-D-glucopyranoside.Compound 7 gave a positive Liebermann-Burchard test and Molisch reactions but did not respond to the Ehrlich reagent.It possessed broad absorption IR bands at 3400 and 1040 cm-' indicating a glycosidic nature.The positive FABmass spectrum gave quasi molelcular ion peak at m/z 923 (M+N~)' acid hydrolysis, 7 afforded D-glucose and L-rhamnose as sugar moieties identified by TLC with authentic samples.The 'HNMR spectrum of 7 exhibited three anomeric protons of sugar components at 6 4.91 (d, J=6Hz), 5.82 (brs.) and 6.36 (brs.).Its ''CNMR spectrum exhibited three anomeric carbon resonances at 102.9, 102 and 100.3.Therefore, one glucosyl and two rhamnosyl units must be contained in saponin 7. Comparison of 13cNMR data of the sugar moieties in 7 with methyl P-D-glucopyranoside indicated that the C-2 and C-4 of the glucosyl moiety were shifted downfield to 78 and 78 from 74.8 and 70.3 respectively(32'.The significant glycosylation shifts clearly showed that two terminal rhamnopyranoses were linked to the C-2 and C-4 positions of the inner glucopyranose02'.The anomeric proton signal of the glucose of 7 at 4.9 (d, J=6.2

appearing at 6
69.4 and 73.5 respectivelf2).The C-5 signals of two rhamnose units in compound 7 were at 69.4 and 70.3 respectively, thus indicating that the two rhamnose units have the a -configuration.Also, CNMR spectrum of 7 exhibited a resonance at 690 of a quaternary carbon bearing an oxygen atom.In its 'HNMR spectrum, the appearance of a quartet at 62.29 (J=7.2Hz) due to H-20 methine proton which only coupled to the 21-Me protons, suggested the occurrence of an a-hydroxyl groups at C-17 position.In addition, the 1 3 ~~~~ chemical shifts of the carbons of 4 B, C, D and E rings of 7 were in accord with those of pennogenin glycoside(33' indicating that the aglycone of 7 possessed 17a hydroxyl-~5-spirostanol skeleton.The IR spectrum of 7 lacked the characteristic normal F-ring spirostene bands at 980, 920, 900 and 880 cm-l (')' indicating ring F substitution.By comparison of the "CNMR spectrum of 7 with that of pennogenin glycoside(33), C-27 of 7 shifted downfield to 664.4 fiom 17.2, thus, a hydroxyl group must be located at C-27 and this was also confirmed by the lack of secondary methyl protons of 6<1 in its 1 HNMR spectrum.The C-25 configuration of 7 was deduced to be S fiom its 'HNMR spectrum.The strong band at 993 cm-' in the IR spectrum corresponded to that at 995 cm-' in i~onartho~enin(~~' suggested the presence of hydroxymethyl on C-25 and revealed its 25 S-spirostanol behaviour.In the 'HNMR spectrum, the coupling constant of H-25 in the axial position and hydroxymethyl in the equatorial position(31'.In the ')CNMR spectrum of 7 the chemical shift of C-3 at 6 78.2 revealed that the compound was the 3-0-~l~coside(~~'.Thus 7 is 25(S) spirost-5-en-3 P, 1 7a, 27-triol-3 -0-[a-L-rhamnopyranosyl-( 1 --, 2)] [a-1,2 were done in CDC13 Spectra for compounds 3-7 were done in w d m e ds