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A bidesmosidic triterpene glycoside from the roots of Symphytum officinale
Pergamon
Phvmchemmrv. Vol. 36. No. 2. DD. 439443. 1994 Cop;nght 0 1994 El&icr Scicoa Ltd Rioted I” Great Britain. All rinhts tmcwcd m-9422/w $7.00+0.00
A BIDESMOSIDIC
TRITERPENE GLYCOSIDE FROM THE ROOTS SYMPH YTUM OFFICINALE
OF
MUSHTAQNOORWALA,FARYALVALI MOHAMMAD,VIQARUDDIN AHMAD* and BILGE SENERt H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan; tDepartment Pharmacy, Gazi University, Ankara, Turkey
of Pharmacognosy,
Faculty of
(Receivedin reoisedform 19 November 1993) Key Word
Index-Symphytum
oficinale;
Boraginaceae;
roots; triterpenoid
saponin; hederagenin;
structural elucidation; 2D NMR.
new bidesmosidic triterpenoidal saponin of hederagenin was isolated from the ethanolic extract of the roots of Symphytum oflcinale L. Its structure was elucidated by using ‘H NMR, ‘H-‘H COSY NMR, heteronuclear ‘H-13C correlated spectroscopy (heteroCOSY), ’ 3C NMR, DEPT, FAB mass spectrometry and chemical evidence as 3-O-cx-L-arabinopyranosyl]-hederagenin-28-O-[~-D-~ucopyranosyl-( l-+4)-/?-D-ghicopyranosyl-( l-+6)-/?-D-glucopyranosyl] ester. Abstract-A
INTRODUCTION
Symphytum oficinule
L. commonly known as comfrey is widely distributed in North Asia, England and Europe [ 11,and is found abundantly in Turkey. Within the family Boraginaceae, the genus Symphytum contains several species which are used in traditional medicine as home remedies for inflammatory, rheumatic and gastrointestinal diseases [2-53. Our systematic phytochemical investigations on the roots of Symphytum oficinale have resulted in the isolation and structure determination of two new triterpenoid saponins [6, 73. In continuation of our work on the chemical constituents of S. ojkinale, we report here the isolation and structure elucidation of a new triterpenoidal saponin (I). The aglycone of this saponin is hederagenin, which has been established by the ‘H and t3CNMR spectra of the aglycone. It is a bidesmosidic saponin with the oligosaccharide chain attached at C-3 and C-28 of the aglycone. The interglycosidic linkages, the position of attachment of the sugar chain to the aglycone, and the sequence of sugars in 1 has been determined by ‘H and 13C NMR spectra, interpreted with the aid of COSY and heteroCOSY spectra, as well as negative ion FAB mass spectrometry. RESULTS AND DISCUSSION
The UV spectrum of 1 had end absorption only at 202.8 nm showing the absence of conjugation. The IR spectrum exhibited the bands at 3350 (hydroxyl) and 1720 (C=O, ester) cm - ‘. On acid hydrolysis (HCl, MeOH), 1
*Author to whom correspondence
should be addressed.
yielded the aglycone that was characterized as hederagenin (2) by comparing with physical and spectral data reported in the literature [8,9]. The sugars obtained from the hydrolysates were identified as arabinose and glucose on PC and TLC by comparing with authentic samples. The 13C NMR spectral data indicated the j&D-pyranOSyl configuration for glucose and a-L-pyranosyl configuration for arabinose [lo]. The molecular mass and sequence of the sugars were deduced by FAB mass spectrometry (negative ion mode). The mass spectrum exhibited the pseudo-molecular ion peak at m/r 1089 [M -HI-, which together with ‘H and 13C NMR data allowed us to propose the molecular formula CS3Hs60Z3 for 1 indicating the presence of 11 double bond equivalents in the molecule. The fragment ion at m/z 927 [M - 162 - H] - was consistent with the loss of terminal glucose from the molecular ion. The fragment ion at m/z 765 was attributed to the loss of a glucose-glucose disaccharide unit from the molecular ion. The peaks at m/z 603 and 471 corresponded to the loss of a trisaccharide (3 glc) and a tetrasaccharide unit (3 glc + ara) from the molecular ion, respectively. The fragment at m/z 603 also indicated that a pentose moiety was attached directly to the aglycone. The 13CNMR spectrum of 1 (CD,OD, 100.613 MHz), showed the presence of 51 carbon atoms in the molecule (Table 1). Twenty-one carbon signals were seen for the sugar moieties, the signal at 678.0 was assigned to C-5” and C-5”“, and the signal at 678.1 was assigned to C-3 of Glc-I and Glc-III, indicating the presence of four monosaccharide moieties, corresponding to three hexoses and one pentose which corresponded to the appearance of four anomeric signals at 6106.2, 95.8, 104.7 and 106.2 assigned to Ara, Glc-I, Glc-II and Glc-III, respectively 439
M.
440
NOORWALA~~U~.
R’O
R’
R2
-Ara
-Glc(bl )Glc(4-1 )Glc
2
-H
-II
3
-Am
-11
1
An a-L-arabinopyranosyl, Glc
P-D-ghCOpyrPnOSyl
The remaining 30 carbon signals were due to pentacyclic triterpenoid aglycone. The assignments of all the carbon signals due to the aglycone were made by comparison with reported data of related compounds [I I]. Multiplicities of the carbon were determined by employing a DEPT pulse sequence [ 12, 131 with the last polarization pulse angles 45, 90 and 135^. This established that there were six methyl, I5 methylene and 24 methine carbon atoms. The number of quaternary carbons was detected by subtraction of carbons of DEPT from BB, in agreement with structure 1. The downfield C-3 signal at 683.6 and the upfteld carbonyl signal at S 178.1 of the aglycone in the 13CNMR spectrum suggested that the sugar moieties were attached at C-3 and C-28 of the aglycone [l I]. The unsaturation between C-12 and C-13 was revealed by the presence of easily recognizable signals at 6 123.8 and 144.9 assigned to these two carbons, respectively [14]. Moreover the 13C NMR spectrum showed the presence of six methyl groups at d 13.5, 16.7, 18.0, 24.1, 26.4 and 33.5. The ‘H NMR spectrum (400.13. CD,OD) of the intact saponin (1) showed the existence of six tertiary methyl groups for H,-24, H,-25, H,-26, H,-27, H,-29 and H,-30 characterized as singlets at 60.7 1,0.98,0.80, I. 16,0.88 and 0.90. These signals were correlated with C-24, C-25, C-26, C-27, C-29 and C-30 at 613.5, 16.7, 18.0, 26.4, 33.5 and 24.1 in the heteroCOSY spectrum [15]. Four anomeric proton signals were also observed at 64.32 (d, J = 6.60 Hz, H-l’), 4.34 (d, J=7.78 Hz, H-l”‘), 4.47 (d, J=7.56 Hz, H1”“). 5.35 (d, J=8.04 Hz, H-l”) supporting the z-configuration of L-arabinose and the ,!&configuration of Dglucose. These assignments were confirmed by i3C NMR assignments of the sugar moieties. In the ‘H- *3C COSY spectrum these signals were correlated with 6 106.2, 104.7, 106.2 and 95.8 peaks, respectively. The’H NMR spectrum also showed the presence of an olefinic proton resonance as a triplet at 65.25, characteristic for the A’*-
H in pentacyclic triterpenes. The signal at d 123.8 assigned to C-12 showed connectivity with H-12 at 55.25 in the heteroCOSY spectrum. The appearance of an ester absorption band in its IR spectrum (1720 cm-‘) and a carboxyl carbon signal (6 178. I), and one of the anomeric carbon signals (695.8) at rather high field in its 13C NMR spectrum strongly indicated that one of the sugars was linked to the C-28 carboxyl group of the genin in the ester form [ 16, 173, and the downfield signal of a methylene at 669.6 showed that two glucoses had (I -+6) linkage. The 13C NMR assignments also indicated that one arabinose and one glucose are terminal sugars [IO]. The alkaline hydrolysis [I81 of 1 alforded a prosapogenin (3), mp 256-258-, which exhibited an anomeric signal at 6 105.9 indicating the presence of one sugar moiety. The negative ion FAB mass spectrum of 3 showed a [M - H] ion peak at mlz 603. The other fragment was observed at m/i 471 which showed the loss of [M -H -atdbinose]from the [M-H] _ peak. The ‘H NMR spectrum of 3 in CD,OD+CDCI, (400.13 MHz) displayed an anomeric signal at 64.29 (d, J = 6.7 Hz, H-l’). A comparison of the ‘“C NMR spectrum of 3 with that of I revealed a loss of I5 resonance signals including the disappearance of three anomeric signals at 6 106.2. 104.7 and 95.8. It could therefore be suggested that I had a trisaccharide chain composed of three glucose units bonded to the C-28 carboxyl group by an ester linkage and one sugar moiety linked to C-3 of hederagenin by a glycosidic bond was arabinose. The disappearance of the downfield methylene signal at 669.6 and methine signal at 678.3 in the 13CNMR spectrum of 3 confirmed the presence of (l-6) [ I93 and (I -+4) [20] linkages between three glucose units attached to C-28 in 1. The structure of 3 has been established as 3-O-[a-L-arabinopyranosyl] hederagenin which was identical to leontoside A previously isolated from Leonticr ewrsmanni [21]. The FAB mass spectrum indicated the presence of one terminal pentose and one terminal hexose, and also an unbranched hexobiose. In the i3CNMR spectrum of I terminal Ara and Glc units were clearly observed (Table I) and all the carbon signals due to a sugar moiety were almost in agreement with the published data for similarly branched sugar moieties [l I]. The points of attachment of the sugar units were determined through 13CNMR chemical shifts in which the upfield shifts of P-carbons and the downfield shifts of z-carbons were characteristic for the establishment of interglycosidic linkages [22]. The downfield ’ 3C NMR chemical shift of C-6 due to Glc-I at 6 69.6, corresponding to the glycosidation shift of + 7.10 ppm in comparison to the reported values for methyl Glc [IO, 231 due to glycosidation at this position, thus disclosed that the middle B-D-glucopyranosyl (Glc-II) was attached to C-6 of the innermost fl-D-glucopyranosyl (Glc-I) unit. The glycosidic linkage at C-4 of Glc-II produced a downfield shift of + 7.45 ppm of this carbon atom (methine signal at S78.3) as compared to the methyl Glc [lo] and showed (I -4) linkage between Glc-III and Glc-II. The very small upheld shift of C-3 of Glc-II showed that C-4 of Glc-II was substituted. The chemical shifts of arabinose and Glc-
Table 1. NMR assignments and ‘H-I%
direct correlation (hetero-COSY) of saponin 1
‘H NMR (CDsOD, 400.13 MHz) Position Aglycone 1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Sugar moiety AlX (Terminal) 1’ 2 3 4 5 Glc I 1(, 2” 3” 4” 5” 6” Glc II 1I,, 2” 3,,, 4” 5”’ 6”’ Glc III (Terminal) 1”” 2”” 3,#,, 4”’ 5!,,I 6””
‘%NMR
(CD,OD, 100.613 MHz)
Chemical shift
Multiplicity
./ (Hz)
d
DEPT
‘H-l%
:
_-
-
CHz Ch
-.
(3.20) -_ -
39.6 26.3 83.6 43.9 48.3 19.0 33.1 40.8 49.1 37.8 23.7 123.8 144.9 43.1 29.0 24.6 48.1 42.6 47.3 31.6 35.0 33.5 65.2 13.5 16.7 18.0 26.4 178.1 33.5 24.1
CH C CH CH, CHz C CH C CH, CH C C CH, CH, C CH CH, C CH, CH, CH, Me Me Me Me C Me Me
3.60 (H-3) No coupling No coupling No coupling 1.90 (H-11) 5.25 (H-12) No coupling No coupling No coupling No coupling __ 3.58 (H-23) 0.71 (H-24) 0.98 (H-25) 0.80 (H-26) 1.16 (H-27) No coupling 0.88 (H-29) 0.89 (H-30)
(6.60) -
106.2 73.0 74.6 69.7 66.1
CH CH CH CH CH,
4.32 (H-l’) 3.54 (H-2’) 3.40 (H-3’) 3.78 (H-4’) 3.84 (H-5’)
(8.04) -
95.8 73.9 78.1 71.4 77.8 69.6
CH CH CH CH CH CH,
5.35 (H-l”) 3.32 (H-2”) 3.42 (H-3”) 3.44 (H-4”) 3.52 (H-S’) 4.10 (H-6”)
104.7 75.2 17.9 78.3 78.0 62.8
CH CH CH CH CH CHz
4.34 (H-l”‘) 3.21 (H-2”‘) 3.36 (H-3”‘) 3.34 (H-4”‘) 3.25 (H-5”‘) 3.84 (H-6”‘)
106.2 75.5 78.1 71.6 78.0 62.6
CH CH CH CH CH CH,
4.47 (H-l”“) 3.28 (H-2”“) 3.42 (H-3”“) 3.30 (H-4”“) 3.25 (H-S”‘) 3.82 (H-6”“)
3.60 + t t t 1.90 5.25 -
m _m t -
t t -
-
t t -
-
T 3.58 0.71 0.98 0.80 1.16 0.88 0.90
-
4.32 3.54 3.40 3.78 3.84
d
-
-
m s S S
s S S
m m
m m
5.35 3.32 3.42 3.44 3.52 4.10
d
4.34 3.21 3.36 3.34 3.25 3.84
d m m m m
-
4.47 3.28 3.42 3.30 3.25 3.82
d m m
(7.56)
tPeaks are not discernible.
m m m m
m
(7.78) -
m
m
-
m m
-
Correlation
Glycoside
from Symphytum oficinale
24), 0.80 (s, 3 x H-26), 0.88 (s, 3 x H-29), 0.90 (s, 3 x H-30), 0.98 (s, 3 x H-25). 1.16 (s, 3 x H-27). 4.32 (d, J =6.60 Hz, H-l’), 4.34 (d, 3=7.78 Hz, H-i”‘), 4.47 (d, J=7.56 Hz, Hl”“), 5.25 (t, H-12), 5.35 (d, J=8.04 Hz, H-l”), 13C NMR (CD,OD, 100.613 MHz): Table 1; FAB-MS negative ion mode m/z 1089 [M-H]-, 927 [M-H-glucose]-, 765 [M-H-2 x glucose]-, 603 [M-H-3 x glucose]-, 471 CM-H-3 x glucose-arabinosel-. Acid ~y~rofys~s of supon~n 1. Saponin 1 (12.5 mg) was refluxed with 10% HCl (5 ml) and MeOH (5 ml) on a boiling Hz0 bath for 3 hr. The reaction mixt. was coned under red. pres. to remove MeOH. It was then diluted with H,O and the hydrolysate was then extracted with CHCI,. The combined CHCI, layer was evapd to afford hederagenin (Z), which was crystallized from MeOH, mp 326”, identified by direct comparison with an authentic sample (co-TLC, mmp, IR, MS, ‘H NMR) [8]. Identification of the sugar moieties of 1. The aq. layer thus sepd was evapd under red. pres. with repeated addition of H,O to remove HCI. The residue obtained was compared with standard sugars on TLC (silica gel, H,O-MeOH-AcOH-EtOAc, 15: 15:20:65), which showed that the sugars were arabinose and glucose in saponin 1. Moreover, the identity of the monosaccharides was further confirmed by comparison with standard sugars on paper chromatography (Whatmann Filter paper No. 1, serrated edges along the lower descending ends) using the solvent system n-BuOH-py~dine-H~O (10: 3: 3) and developing time 48 hr. Spots were detected by spraying with freshly prepd aniline phthalate sugar reagent followed by heating. Two spots were present, whose R,s were identical to the R, of L-arabinose and Dglucose. AIkafine hydrofysjs of s~~n~n 1. Com~und 1 (20 mg) was refluxed with 2% NaOH (20 ml) in MeOH for 2 hr. After cooling, the reaction mixt. was slightly acidified with dilute HCI, and extracted with n-BuOH. The nBuOH extract was washed with H,O, evapd under red. pres. and crystallized with MeOH to yield prosapogenin 3, as a powder, mp 256-258” (lit. [28] mp 276-278”), [a]i6 +42” (pyridine; ~0.84) flit. [28] [a]g”+47&l” (pyridine)); which was identical with hederagenin 3-0-(xL-arabinopyranoside (leontoside-A) [I 1, 213. FAB-MS (negative ion mode) m/z 603 [M -HI-, 471 [M -arabinose -HI-.
443
3. Pahlow, M. (1985) in Dus gro3e Buch der HeiIp$?anzen. p. 75. Verlag Gr%fe und Unzer, Miinchen. 4. Hills, L. D. (1976) Comfrey: Past, Present and Future. Faber and Faber, London. 5. Schloss, L. (1979) Comfrey, Wiedergeburt einer Heilpj?anze. Selbstverlag Comfrey Vertrieb GmbH, Bergen/Chiemgau. 6. Ahmad, V. U., Noorwala, M., Mohammad, F. V., Sener, B., Gifani, A.U.H. and Aftab, K. (1993) Phytochemistry 32, 1003.
7. Ahmad, V. U., Noorwala, M., Mohammad, F. V. and Sener, B. (1993) J. Nut. Prod. 56, 329. 8. Buckingham, J. (1985) in Dictionary ofOrganic Compounds, Vol. 3, p. 164. Chapman and Hall, New York. 9. Tori, K., Seo, S., Shimaoka, A. and Tomita, Y. (1974) Tetrahedron
L..etters 4227.
10. Gorin, P. A. J. and Mazurek, M. (1975) Can. J. Chem. 53, 1212. 11. Li, X.-C., Wang, D.-Z., Wu, S.-G. and Yang, C.-R. (1990) Phytochemistry 29, 595. 12. Atta-ur-Rahman (1986) in Nuclear Magnetic Resonance, pp. 202-306. Springer, New York. Bendai, M. R. and Pegg, D. T. (1983) 1. Magn. Reson. 53, 272. Kitagawa, I., Taniyama, T., Hong, W. W. and Yoshikawa, M. (1988) Yakugaku Zasshi 108,538. Atta-ur-Rahman (1989) One and Two Di~~iona~ NMR Spectroscopy. Eisevier, Amsterdam. Ahmad, V. U., Uddin, S., Bano, S. and Fatima, I. (1989) Phytochemistry 28, 2169. 17. Shao, C. J., Kasi, R., Xu, J. D. and Tanaka, 0. (1989) Chem. Pharm. Bull. 37, 42. 18. Choi, J. S. and Woo, W. S. (1987) PIanta Med. 53,62. 19. Amagaya, S., Takeda, T., Ogihara, Y. and Yamasaki, K. (1979) J. Chem. Sot. Perkin Truns I 2044. 20. Mahato, S. B., Sahu, N. P., Roy, S. K. and Sen, S. (1991) Tetrahedron 47, 5215. 21. Agarwal, S. K. and Rastogi, R. P. (1974) Phytochemistry 13, 2623. 22. Lanzetta, R., Laonigro, G. and Parrini, M. (1984) Can. J. Chem. 62, 2874. 23. Seo, S., Tomita, Y. and Yoshimura, Y. (1978) J. Am. Chem. Sot. 100, 3331. 24. Shao, C.-J., Kasai, R., Xu, J.-D. and Tanaka, 0. (1988) Chem. Pharm. Bull. 36, 601. 25. Kohda, H., Tanaka, S. and Yamaoka, Y. (1990) Chem. Pharm. Bult. 38, 3380.
REFERENCES
1. Atta-ur-Rahman and Zaman, K. (1989) J. Ethnopharmucol. 26, 1. 2. Wichtl, M. (1984) in Teedrogen. p.70. Wissenschaftlithe Veria~g~lischaft m.b.H., Stuttgart.
PHYTO 36:2-M
26. Kouno, I., Tsuboi, A., Nanri, M. and Kawano, N. (1990) Phytochemistry 29, 338. 21. Higuchi, R. and Kawasaki, T. (1972) Chem. Pharm. Bull. 20, 2143.
28. Mzhel’ skaya, L. G. and Abubakirov, Khim. Prir. Soedin 3, 107.
N. K. (1967)
Phvmchemmrv. Vol. 36. No. 2. DD. 439443. 1994 Cop;nght 0 1994 El&icr Scicoa Ltd Rioted I” Great Britain. All rinhts tmcwcd m-9422/w $7.00+0.00
A BIDESMOSIDIC
TRITERPENE GLYCOSIDE FROM THE ROOTS SYMPH YTUM OFFICINALE
OF
MUSHTAQNOORWALA,FARYALVALI MOHAMMAD,VIQARUDDIN AHMAD* and BILGE SENERt H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan; tDepartment Pharmacy, Gazi University, Ankara, Turkey
of Pharmacognosy,
Faculty of
(Receivedin reoisedform 19 November 1993) Key Word
Index-Symphytum
oficinale;
Boraginaceae;
roots; triterpenoid
saponin; hederagenin;
structural elucidation; 2D NMR.
new bidesmosidic triterpenoidal saponin of hederagenin was isolated from the ethanolic extract of the roots of Symphytum oflcinale L. Its structure was elucidated by using ‘H NMR, ‘H-‘H COSY NMR, heteronuclear ‘H-13C correlated spectroscopy (heteroCOSY), ’ 3C NMR, DEPT, FAB mass spectrometry and chemical evidence as 3-O-cx-L-arabinopyranosyl]-hederagenin-28-O-[~-D-~ucopyranosyl-( l-+4)-/?-D-ghicopyranosyl-( l-+6)-/?-D-glucopyranosyl] ester. Abstract-A
INTRODUCTION
Symphytum oficinule
L. commonly known as comfrey is widely distributed in North Asia, England and Europe [ 11,and is found abundantly in Turkey. Within the family Boraginaceae, the genus Symphytum contains several species which are used in traditional medicine as home remedies for inflammatory, rheumatic and gastrointestinal diseases [2-53. Our systematic phytochemical investigations on the roots of Symphytum oficinale have resulted in the isolation and structure determination of two new triterpenoid saponins [6, 73. In continuation of our work on the chemical constituents of S. ojkinale, we report here the isolation and structure elucidation of a new triterpenoidal saponin (I). The aglycone of this saponin is hederagenin, which has been established by the ‘H and t3CNMR spectra of the aglycone. It is a bidesmosidic saponin with the oligosaccharide chain attached at C-3 and C-28 of the aglycone. The interglycosidic linkages, the position of attachment of the sugar chain to the aglycone, and the sequence of sugars in 1 has been determined by ‘H and 13C NMR spectra, interpreted with the aid of COSY and heteroCOSY spectra, as well as negative ion FAB mass spectrometry. RESULTS AND DISCUSSION
The UV spectrum of 1 had end absorption only at 202.8 nm showing the absence of conjugation. The IR spectrum exhibited the bands at 3350 (hydroxyl) and 1720 (C=O, ester) cm - ‘. On acid hydrolysis (HCl, MeOH), 1
*Author to whom correspondence
should be addressed.
yielded the aglycone that was characterized as hederagenin (2) by comparing with physical and spectral data reported in the literature [8,9]. The sugars obtained from the hydrolysates were identified as arabinose and glucose on PC and TLC by comparing with authentic samples. The 13C NMR spectral data indicated the j&D-pyranOSyl configuration for glucose and a-L-pyranosyl configuration for arabinose [lo]. The molecular mass and sequence of the sugars were deduced by FAB mass spectrometry (negative ion mode). The mass spectrum exhibited the pseudo-molecular ion peak at m/r 1089 [M -HI-, which together with ‘H and 13C NMR data allowed us to propose the molecular formula CS3Hs60Z3 for 1 indicating the presence of 11 double bond equivalents in the molecule. The fragment ion at m/z 927 [M - 162 - H] - was consistent with the loss of terminal glucose from the molecular ion. The fragment ion at m/z 765 was attributed to the loss of a glucose-glucose disaccharide unit from the molecular ion. The peaks at m/z 603 and 471 corresponded to the loss of a trisaccharide (3 glc) and a tetrasaccharide unit (3 glc + ara) from the molecular ion, respectively. The fragment at m/z 603 also indicated that a pentose moiety was attached directly to the aglycone. The 13CNMR spectrum of 1 (CD,OD, 100.613 MHz), showed the presence of 51 carbon atoms in the molecule (Table 1). Twenty-one carbon signals were seen for the sugar moieties, the signal at 678.0 was assigned to C-5” and C-5”“, and the signal at 678.1 was assigned to C-3 of Glc-I and Glc-III, indicating the presence of four monosaccharide moieties, corresponding to three hexoses and one pentose which corresponded to the appearance of four anomeric signals at 6106.2, 95.8, 104.7 and 106.2 assigned to Ara, Glc-I, Glc-II and Glc-III, respectively 439
M.
440
NOORWALA~~U~.
R’O
R’
R2
-Ara
-Glc(bl )Glc(4-1 )Glc
2
-H
-II
3
-Am
-11
1
An a-L-arabinopyranosyl, Glc
P-D-ghCOpyrPnOSyl
The remaining 30 carbon signals were due to pentacyclic triterpenoid aglycone. The assignments of all the carbon signals due to the aglycone were made by comparison with reported data of related compounds [I I]. Multiplicities of the carbon were determined by employing a DEPT pulse sequence [ 12, 131 with the last polarization pulse angles 45, 90 and 135^. This established that there were six methyl, I5 methylene and 24 methine carbon atoms. The number of quaternary carbons was detected by subtraction of carbons of DEPT from BB, in agreement with structure 1. The downfield C-3 signal at 683.6 and the upfteld carbonyl signal at S 178.1 of the aglycone in the 13CNMR spectrum suggested that the sugar moieties were attached at C-3 and C-28 of the aglycone [l I]. The unsaturation between C-12 and C-13 was revealed by the presence of easily recognizable signals at 6 123.8 and 144.9 assigned to these two carbons, respectively [14]. Moreover the 13C NMR spectrum showed the presence of six methyl groups at d 13.5, 16.7, 18.0, 24.1, 26.4 and 33.5. The ‘H NMR spectrum (400.13. CD,OD) of the intact saponin (1) showed the existence of six tertiary methyl groups for H,-24, H,-25, H,-26, H,-27, H,-29 and H,-30 characterized as singlets at 60.7 1,0.98,0.80, I. 16,0.88 and 0.90. These signals were correlated with C-24, C-25, C-26, C-27, C-29 and C-30 at 613.5, 16.7, 18.0, 26.4, 33.5 and 24.1 in the heteroCOSY spectrum [15]. Four anomeric proton signals were also observed at 64.32 (d, J = 6.60 Hz, H-l’), 4.34 (d, J=7.78 Hz, H-l”‘), 4.47 (d, J=7.56 Hz, H1”“). 5.35 (d, J=8.04 Hz, H-l”) supporting the z-configuration of L-arabinose and the ,!&configuration of Dglucose. These assignments were confirmed by i3C NMR assignments of the sugar moieties. In the ‘H- *3C COSY spectrum these signals were correlated with 6 106.2, 104.7, 106.2 and 95.8 peaks, respectively. The’H NMR spectrum also showed the presence of an olefinic proton resonance as a triplet at 65.25, characteristic for the A’*-
H in pentacyclic triterpenes. The signal at d 123.8 assigned to C-12 showed connectivity with H-12 at 55.25 in the heteroCOSY spectrum. The appearance of an ester absorption band in its IR spectrum (1720 cm-‘) and a carboxyl carbon signal (6 178. I), and one of the anomeric carbon signals (695.8) at rather high field in its 13C NMR spectrum strongly indicated that one of the sugars was linked to the C-28 carboxyl group of the genin in the ester form [ 16, 173, and the downfield signal of a methylene at 669.6 showed that two glucoses had (I -+6) linkage. The 13C NMR assignments also indicated that one arabinose and one glucose are terminal sugars [IO]. The alkaline hydrolysis [I81 of 1 alforded a prosapogenin (3), mp 256-258-, which exhibited an anomeric signal at 6 105.9 indicating the presence of one sugar moiety. The negative ion FAB mass spectrum of 3 showed a [M - H] ion peak at mlz 603. The other fragment was observed at m/i 471 which showed the loss of [M -H -atdbinose]from the [M-H] _ peak. The ‘H NMR spectrum of 3 in CD,OD+CDCI, (400.13 MHz) displayed an anomeric signal at 64.29 (d, J = 6.7 Hz, H-l’). A comparison of the ‘“C NMR spectrum of 3 with that of I revealed a loss of I5 resonance signals including the disappearance of three anomeric signals at 6 106.2. 104.7 and 95.8. It could therefore be suggested that I had a trisaccharide chain composed of three glucose units bonded to the C-28 carboxyl group by an ester linkage and one sugar moiety linked to C-3 of hederagenin by a glycosidic bond was arabinose. The disappearance of the downfield methylene signal at 669.6 and methine signal at 678.3 in the 13CNMR spectrum of 3 confirmed the presence of (l-6) [ I93 and (I -+4) [20] linkages between three glucose units attached to C-28 in 1. The structure of 3 has been established as 3-O-[a-L-arabinopyranosyl] hederagenin which was identical to leontoside A previously isolated from Leonticr ewrsmanni [21]. The FAB mass spectrum indicated the presence of one terminal pentose and one terminal hexose, and also an unbranched hexobiose. In the i3CNMR spectrum of I terminal Ara and Glc units were clearly observed (Table I) and all the carbon signals due to a sugar moiety were almost in agreement with the published data for similarly branched sugar moieties [l I]. The points of attachment of the sugar units were determined through 13CNMR chemical shifts in which the upfield shifts of P-carbons and the downfield shifts of z-carbons were characteristic for the establishment of interglycosidic linkages [22]. The downfield ’ 3C NMR chemical shift of C-6 due to Glc-I at 6 69.6, corresponding to the glycosidation shift of + 7.10 ppm in comparison to the reported values for methyl Glc [IO, 231 due to glycosidation at this position, thus disclosed that the middle B-D-glucopyranosyl (Glc-II) was attached to C-6 of the innermost fl-D-glucopyranosyl (Glc-I) unit. The glycosidic linkage at C-4 of Glc-II produced a downfield shift of + 7.45 ppm of this carbon atom (methine signal at S78.3) as compared to the methyl Glc [lo] and showed (I -4) linkage between Glc-III and Glc-II. The very small upheld shift of C-3 of Glc-II showed that C-4 of Glc-II was substituted. The chemical shifts of arabinose and Glc-
Table 1. NMR assignments and ‘H-I%
direct correlation (hetero-COSY) of saponin 1
‘H NMR (CDsOD, 400.13 MHz) Position Aglycone 1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Sugar moiety AlX (Terminal) 1’ 2 3 4 5 Glc I 1(, 2” 3” 4” 5” 6” Glc II 1I,, 2” 3,,, 4” 5”’ 6”’ Glc III (Terminal) 1”” 2”” 3,#,, 4”’ 5!,,I 6””
‘%NMR
(CD,OD, 100.613 MHz)
Chemical shift
Multiplicity
./ (Hz)
d
DEPT
‘H-l%
:
_-
-
CHz Ch
-.
(3.20) -_ -
39.6 26.3 83.6 43.9 48.3 19.0 33.1 40.8 49.1 37.8 23.7 123.8 144.9 43.1 29.0 24.6 48.1 42.6 47.3 31.6 35.0 33.5 65.2 13.5 16.7 18.0 26.4 178.1 33.5 24.1
CH C CH CH, CHz C CH C CH, CH C C CH, CH, C CH CH, C CH, CH, CH, Me Me Me Me C Me Me
3.60 (H-3) No coupling No coupling No coupling 1.90 (H-11) 5.25 (H-12) No coupling No coupling No coupling No coupling __ 3.58 (H-23) 0.71 (H-24) 0.98 (H-25) 0.80 (H-26) 1.16 (H-27) No coupling 0.88 (H-29) 0.89 (H-30)
(6.60) -
106.2 73.0 74.6 69.7 66.1
CH CH CH CH CH,
4.32 (H-l’) 3.54 (H-2’) 3.40 (H-3’) 3.78 (H-4’) 3.84 (H-5’)
(8.04) -
95.8 73.9 78.1 71.4 77.8 69.6
CH CH CH CH CH CH,
5.35 (H-l”) 3.32 (H-2”) 3.42 (H-3”) 3.44 (H-4”) 3.52 (H-S’) 4.10 (H-6”)
104.7 75.2 17.9 78.3 78.0 62.8
CH CH CH CH CH CHz
4.34 (H-l”‘) 3.21 (H-2”‘) 3.36 (H-3”‘) 3.34 (H-4”‘) 3.25 (H-5”‘) 3.84 (H-6”‘)
106.2 75.5 78.1 71.6 78.0 62.6
CH CH CH CH CH CH,
4.47 (H-l”“) 3.28 (H-2”“) 3.42 (H-3”“) 3.30 (H-4”“) 3.25 (H-S”‘) 3.82 (H-6”“)
3.60 + t t t 1.90 5.25 -
m _m t -
t t -
-
t t -
-
T 3.58 0.71 0.98 0.80 1.16 0.88 0.90
-
4.32 3.54 3.40 3.78 3.84
d
-
-
m s S S
s S S
m m
m m
5.35 3.32 3.42 3.44 3.52 4.10
d
4.34 3.21 3.36 3.34 3.25 3.84
d m m m m
-
4.47 3.28 3.42 3.30 3.25 3.82
d m m
(7.56)
tPeaks are not discernible.
m m m m
m
(7.78) -
m
m
-
m m
-
Correlation
Glycoside
from Symphytum oficinale
24), 0.80 (s, 3 x H-26), 0.88 (s, 3 x H-29), 0.90 (s, 3 x H-30), 0.98 (s, 3 x H-25). 1.16 (s, 3 x H-27). 4.32 (d, J =6.60 Hz, H-l’), 4.34 (d, 3=7.78 Hz, H-i”‘), 4.47 (d, J=7.56 Hz, Hl”“), 5.25 (t, H-12), 5.35 (d, J=8.04 Hz, H-l”), 13C NMR (CD,OD, 100.613 MHz): Table 1; FAB-MS negative ion mode m/z 1089 [M-H]-, 927 [M-H-glucose]-, 765 [M-H-2 x glucose]-, 603 [M-H-3 x glucose]-, 471 CM-H-3 x glucose-arabinosel-. Acid ~y~rofys~s of supon~n 1. Saponin 1 (12.5 mg) was refluxed with 10% HCl (5 ml) and MeOH (5 ml) on a boiling Hz0 bath for 3 hr. The reaction mixt. was coned under red. pres. to remove MeOH. It was then diluted with H,O and the hydrolysate was then extracted with CHCI,. The combined CHCI, layer was evapd to afford hederagenin (Z), which was crystallized from MeOH, mp 326”, identified by direct comparison with an authentic sample (co-TLC, mmp, IR, MS, ‘H NMR) [8]. Identification of the sugar moieties of 1. The aq. layer thus sepd was evapd under red. pres. with repeated addition of H,O to remove HCI. The residue obtained was compared with standard sugars on TLC (silica gel, H,O-MeOH-AcOH-EtOAc, 15: 15:20:65), which showed that the sugars were arabinose and glucose in saponin 1. Moreover, the identity of the monosaccharides was further confirmed by comparison with standard sugars on paper chromatography (Whatmann Filter paper No. 1, serrated edges along the lower descending ends) using the solvent system n-BuOH-py~dine-H~O (10: 3: 3) and developing time 48 hr. Spots were detected by spraying with freshly prepd aniline phthalate sugar reagent followed by heating. Two spots were present, whose R,s were identical to the R, of L-arabinose and Dglucose. AIkafine hydrofysjs of s~~n~n 1. Com~und 1 (20 mg) was refluxed with 2% NaOH (20 ml) in MeOH for 2 hr. After cooling, the reaction mixt. was slightly acidified with dilute HCI, and extracted with n-BuOH. The nBuOH extract was washed with H,O, evapd under red. pres. and crystallized with MeOH to yield prosapogenin 3, as a powder, mp 256-258” (lit. [28] mp 276-278”), [a]i6 +42” (pyridine; ~0.84) flit. [28] [a]g”+47&l” (pyridine)); which was identical with hederagenin 3-0-(xL-arabinopyranoside (leontoside-A) [I 1, 213. FAB-MS (negative ion mode) m/z 603 [M -HI-, 471 [M -arabinose -HI-.
443
3. Pahlow, M. (1985) in Dus gro3e Buch der HeiIp$?anzen. p. 75. Verlag Gr%fe und Unzer, Miinchen. 4. Hills, L. D. (1976) Comfrey: Past, Present and Future. Faber and Faber, London. 5. Schloss, L. (1979) Comfrey, Wiedergeburt einer Heilpj?anze. Selbstverlag Comfrey Vertrieb GmbH, Bergen/Chiemgau. 6. Ahmad, V. U., Noorwala, M., Mohammad, F. V., Sener, B., Gifani, A.U.H. and Aftab, K. (1993) Phytochemistry 32, 1003.
7. Ahmad, V. U., Noorwala, M., Mohammad, F. V. and Sener, B. (1993) J. Nut. Prod. 56, 329. 8. Buckingham, J. (1985) in Dictionary ofOrganic Compounds, Vol. 3, p. 164. Chapman and Hall, New York. 9. Tori, K., Seo, S., Shimaoka, A. and Tomita, Y. (1974) Tetrahedron
L..etters 4227.
10. Gorin, P. A. J. and Mazurek, M. (1975) Can. J. Chem. 53, 1212. 11. Li, X.-C., Wang, D.-Z., Wu, S.-G. and Yang, C.-R. (1990) Phytochemistry 29, 595. 12. Atta-ur-Rahman (1986) in Nuclear Magnetic Resonance, pp. 202-306. Springer, New York. Bendai, M. R. and Pegg, D. T. (1983) 1. Magn. Reson. 53, 272. Kitagawa, I., Taniyama, T., Hong, W. W. and Yoshikawa, M. (1988) Yakugaku Zasshi 108,538. Atta-ur-Rahman (1989) One and Two Di~~iona~ NMR Spectroscopy. Eisevier, Amsterdam. Ahmad, V. U., Uddin, S., Bano, S. and Fatima, I. (1989) Phytochemistry 28, 2169. 17. Shao, C. J., Kasi, R., Xu, J. D. and Tanaka, 0. (1989) Chem. Pharm. Bull. 37, 42. 18. Choi, J. S. and Woo, W. S. (1987) PIanta Med. 53,62. 19. Amagaya, S., Takeda, T., Ogihara, Y. and Yamasaki, K. (1979) J. Chem. Sot. Perkin Truns I 2044. 20. Mahato, S. B., Sahu, N. P., Roy, S. K. and Sen, S. (1991) Tetrahedron 47, 5215. 21. Agarwal, S. K. and Rastogi, R. P. (1974) Phytochemistry 13, 2623. 22. Lanzetta, R., Laonigro, G. and Parrini, M. (1984) Can. J. Chem. 62, 2874. 23. Seo, S., Tomita, Y. and Yoshimura, Y. (1978) J. Am. Chem. Sot. 100, 3331. 24. Shao, C.-J., Kasai, R., Xu, J.-D. and Tanaka, 0. (1988) Chem. Pharm. Bull. 36, 601. 25. Kohda, H., Tanaka, S. and Yamaoka, Y. (1990) Chem. Pharm. Bult. 38, 3380.
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PHYTO 36:2-M
26. Kouno, I., Tsuboi, A., Nanri, M. and Kawano, N. (1990) Phytochemistry 29, 338. 21. Higuchi, R. and Kawasaki, T. (1972) Chem. Pharm. Bull. 20, 2143.
28. Mzhel’ skaya, L. G. and Abubakirov, Khim. Prir. Soedin 3, 107.
N. K. (1967)