W-9422/91 $3.00+0.00 0 1991PergamonPressplc
Phytochemistry, Vol. 30, No. 3, pp. 927-931, 1991
Printedin GreatBritain.
FOUR TRITERPENOID SAPONINS FROM DRIED ROOTS OF G YPSOPHILA SPECIES DENISE FRECHET, BRUNO CHRIST,* BERTRAND MONEGIER DU SORBIER, HARTMUT FISCHER*
and MARC VUILHORGNE Rhone Poulenc Sante, Centre de Recherches de Vitry, 13 quai Jules Guesde - B.P. 14, F-94403 Vitry-sur-seine-Cedex; France; *A. Natterman and Cie. GmbH, Gruppe Rhone-Poulenc, Forschungszentrum K61n, Nattermannallee I, Postfach-350120, D-5000 Kijln 30. F.R.G. (Received in revisedform 10 July 1990) Key Word Index-Gypsophila paniculata;Gypsophila arrostii; Caryophyllaceae; soap root; Saponariae alba radix; root; saponins; triterpenoids; carbohydrates; quillaic acid; gypsogenin.
Abstract-Four new triterpenoid saponins were isolated from the roots of Gypsophila paniculata and G. arrostii. Their structures were elucidated using a combination of homo- and heteronuclear 2D NMR techniques, without having recourse to chemical degradation or modification. The saponins investigated are: 3-O-B-D-galactopyranosyl-( 1+2)[j-D-XylOpyranOSyl-( 1+3)]-j?-D-glucuronopyranosyl quillaic acid 2%O-B-D-glucopyranosyl-( 1+3)-[B-D-xylOpyranosyl-(l+4)]-cr-L-rhamnopyranosyl-(l-+2)-~-D-fucopyranoside; 3-O-j&D-galactopyranosyl-( 1+2)-D-D-xylopyranosyl-( 1+3)]+D-glucuronopyranosyl quillaic acid 28-O-fi-D-arabinopyranosyl-( l-+4)-fi-D-arabinopyranosyl-( 1+3)j?-D-xylopyranosyl-( 1+4)-a-L-rhamnopyranosyl-( 1-,2)-B-D-fucopyranoside; 3-O-/&D-gluCOpyranOSyl-(I +2)-B-D-glucuronopyranosyl gypsogenin 2%O-B-D-glucopyranosyl-( 1+3)-[b-D-XylOpyranOSyl-( 1+4)]-cc-L-rhamnopyranosyl3-O-D-D-xylopyranosyl-( 1+3)-[B-D-galactopyranosyl-( 1+2)]+D-glucuronopyranosyl (1+2)-/?-D-fucopyranoside; 2%0-/?-D-glucopyranosyl-( 1+3)-[B-D-xylopyranosyl-( 1+4)]-a-L-rhamnopyranosyl-( 1+2)$-D-fucopyrf;:;;;nin
INTRODUcTION
The roots of Gypsophila paniculata and G. arrostii (soap root, Saponariae alba radix) are known as saponin drugs and have been used as detergents and expectorants. Hitherto only gypsosid, a triterpenoid saponin containing gypsogenin and nine sugar moieties, has been isolated from these roots [l-5]. As part of our studies on the quantitation of saponins in soap roots by HPLC, we have isolated four new triterpenoid saponins. Their structures were completely elucidated by homo- and heteronuclear 2DNMR spectroscopy, without having recourse to chemical degradation or modifications. Surprisingly, none of the investigated samples were identified as gypsosid. Gypsosid is therefore absent from these roots, as no other fractions were detected by HPLC. RESULTS AND
DISCUSSION
The ethanolic extract of dried roots was fractionated as described in the Experimental. The isolated saponins, Gl-G4 showed single spots on TLC and single peaks by HPLC, respectively. The molecular formula of each saponin was determined by combining the results of mass spectroscopy, as well as 13C and ‘H NMR. These were identified as Gl (RP 68996)=C70H11003, (M, 1542, 7 sugars), G2 (RP 68997)=C,4H116040 (M, 1644, 8 sugars), G3 (RP 68998)=C,,H,,,O,, (M, 1394, 6 sugars), 64 (RP 68999)=C,oH,10036 (M, 1526, 7 sugars). Because the ‘H NMR signals of the aglycone moiety of each triterpene were well resolved at 400 MHz, their identificaPAY
30:3-N
tion is relatively straightforward. In the first step, a HOHAHA [6] experiment recorded with a mixing time of 14 msec allowed complete elucidation of the proton coupling network. This experiment is equivalent to a COSY [7], however, in that case the observed correlation cross peaks are in phase, thereby preventing the accidental nulling of overlapping peaks. Carbon resonances are assigned by means of a short range H< correlation [S], whereas the quaternary carbons are identified by means of a long-range proton-carbon correlation expe.riment. This latter experiment was recorded by the heteronuclear multiple bond correlation method [9] using ‘H detection, thereby increasing the sensitivity with regards to experiments using 13C detection. By comparison with literature data [IO], the aglycone was identified as quillaic acid in the case of Gl and G2 and gypsogenin for G3 and 64. Their 13C resonance assignments are listed in Table I. Elucidation of the structure of the carbohydrate chains was more challenging, given the extensive overlap of their NMR signals. In a first step, we proceeded to the identification of the sugar residues. ‘H subspectra of the various carbohydrate moieties were obtained from the rows corresponding to their anomeric proton resonances and to their other well resolved resonances (i.e. Me-6 for rhamnose and fucose, methylene-5 for xylose) in a HOHAHA experiment with a mixing time of 400 msec. Direct connectivity was determined from the results of the shorter mixing time (14 msec) HOHAHA experiment. Vicinal coupling constants can be extracted from the rows of these experiments with enough precision ( f 1 Hz)
D. FRECHETet al.
928
‘OR2
23 OH
R=
R’ Cl
XYl
a”
Rs
R4
OH
Glc
OH
HO
OH OH G2
XYl
G3
H
G4
XYl
GlC
&”
OH
H
H
Glc
H
Glc
HO
H H
OH
HO
to determine the relative stereochemistry
of each asymmetric centre and thus to identify the monosaccharides. The average coupling constant values thus measured for the constituent monosaccharide units of the four saponins are listed in Table 2. A partial assignment of 13C resonances was then obtained from the results of the short-range H-C correlation experiment. Given the extensive overlap of proton resonances, some ambiguities remained. These could however be resolved by the heteronuclear H-C relay experiment [ 111. From the anomeric proton resonances, relays could generally be observed to C-2 and C-3, even to C-4 in some cases. Relays from methyl resonances, well resolved geminal proton or even protons located in more crowded areas of the spectrum allowed complete assignment of the 13C resonances. The position of the carboxylic acid of glucuronic acid was provided by the heteronuclear multiple bond correlation experiment. ‘H and “C chemical shifts of all glycosidic residues of Gl-G4 are listed in Table 3. Comparison of the values of 13C chemical shifts with reference data [ 121 allowed identification of the position of substituted residues (underlined in Table 3). The next step was the determination of the linkages among the sugar residues and between the sugars and the triterpene. This information was provided by the longrange heteronuclear correlations arising from the anom-
eric protons. As the 13C resonances of each sugar moiety have been unambiguously assigned previously, interresidue correlations are easily identified. The long-range carbon correlations arising from the anomeric protons of the sugars of Gl are shown in Fig. 1. The linkages 1+2 between rhamnose (A) and fucose (B); l-+28 between fucose (B) and quillaic acid (T); l-+3 between glucose (E) and rhamnose (A); 1+3 between xylose (F) and glucuronic acid (G); 1+3 between glucuronic acid (G) and the terpene moiety (T) were unambiguously determined from this slice of the spectrum. Given the close proximity of the 13C resonances of C-2 of glucuronic acid and C-4 of rhamnose, the linkages l-+2 between galactose (C) and glucuronic acid (G) and 1+4 between xylose (D) and rhamnose (A) were confirmed by the observation of nonanomeric protons. H-2 of glucuronic acid (4.23 ppm) had a correlation to C- 1 of galactose (103 ppm), whereas H-4 of rhamnose (4.47 ppm) had a correlation to C-l of xylose (103.8 ppm). The same procedure was applied to G2-G4, thus allowing complete determination of the structures of these saponins. EXPERIMENTAL PIant panicdata
material. Commercial soap roots of Gypsophila and G. arrostii were from the China National Native
929
Triterpenoid saponins from Gypsophila spp. Table 1. rsC NMR chemical shifts (ppm) of aglycone moieties (in pyridine-d,) Gypsogenin 64 G3
Quillaic acid C 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
Cl
G2 37.25 24.10 83.10 54.05 47.55 19.50 31.80 39.30 45.95 35.15 22.65 121.05 143.45 41.05 35.25 72.90 48.25 40.50 46.30 29.60 34.90 30.65 208.50 9.80 14.70 16.35 26.00 174.80 32.00 23.55
31.25 24.15 83.00 54.00 47.80 19.45 32.00 39.35 46.00 35.15 22.65 120.95 143.45 41.10 35.25 73.10 48.25 40.65 46.45 29.55 35.00 30.40 208.40 9.90 14.75 16.40 26.05 174.80 31.95 23.55
37.25 24.00 82.35 53.90 47.60 19.45 31.60 39.25 46.85 35.25 22.65 121.35 143.00 41.15 27.10 22.55 50.60 40.95 45.40 29.70 33.00 31.30 208.20 9.85 14.65 16.40 24.90 175.25 32.00 22.75
36.30 23.25 82.25 53.05 46.85 18.60 30.65 38.35 45.95 34.35 21.75 120.50 142.80 40.25 26.20 21.60 48.15 40.05 44.50 28.75 32.05 30.40 207.70 9.05 13.75 15.45 24.00 174.35 31.10 21.85
Produce and Animal Byproducts Corporation, Native Produce Branch, Hebei, Peoples Republic of China. Extraction and separation. Dried powdered roots (100 g) were extracted with 200 ml EtOH-H,O (1: 1) by stirring for 24 hr. The plant material was filtered off and the extract was coned to ca 20 ml in uacuo. H,O (10 ml) was added and 3 ml portions of the extract separated on a RP-18 column (Lobar, size B) by a linear gradient from 30 to 100% MeOH during 8 hr, 3.5 hr after starting the gradient, 20 ml frs were collected and analysed by HPLC [column: Nucleosil 120X18 5 m 250 x 4 mm; eluent: 0.1% aq. H,PO,-MeCN (69: 31); temp. 35”; flow rate: 1 ml min-‘; detection wavelength 195 nm]. Frs having identical composition were combined. Four frs, containing the glycosides Gl-G4, were obtained. The MeOH was distilled off in uacuo. The glycosides from the remaining aq. phase were adsorbed on a RP-8 column (Lobar, size A). The column was washed with H,O and the saponins eluted with MeOH. The MeQH was evapd in ~acuo. The glycosides were redissolved in H,O contanining 20% MeCN and finally purified by semiprep. HPLC [column: Nucleosil 100X18 5 pm; 250x8 mm; eluent: 0.1% aq. H,PO,-MeCN linear gradient from 28 to 35% during 25 mm; temp.: ambient; flow rate 3 mlmin-t; detection wavelength 220 nm]. The MeCN was distilled off in vacua The saponins were then adsorbed on silica gel (Lichroprep RP 18) and the column was washed with H,O until complete elimination of H,PO,. The saponins were then eluted with MeOH, and this MeOH evapd in vacua. The purity was confirmed by TLC [precoated silicagel 60 plates; eluent: CHCI,-MeOH-H,QHOAc (32: 25 : 5 : 1); spray reagent: vanillin-H,SO.J. Mps uncorr. Gl (RP 68996). Amorphous powder, mp: 210-213”, TLC: R, 0.22, brownish spot by spraying with vanillin-H,SO, reagent, HPLC: R, 7.56 min. 62 (RP 68997). Amorphous powder, mp: 213-215”, TLC: R, 0.18, brownish spot, HPLC R, 8.93 min. 63 (RP 68998). Amorphous powder, mp: 207-21 l”, TLC: R, 0.32, greenish spot, HPLC: R, 12.38 min.
Table 2. Average vicinal and geminal proton coupling constants (Hz+ 1 Hz) JI-2
J,_,
J,-,
34-5
J,-,.
J,-,.
/7 Glur BXYf BGaf
8 8 8
8 8 8
8 8 2
8 2 2
9
11
j Glc j Fuc aRha f3 Ara
8 8 1 8
8 8 2 8
8 4 8 3
8 2 8 3
2
J,-,
J,-,.
J,-6.
*
*
*
3 6 6
7
12
12
*Not observed.
Table 3. “C and ‘H NMR chemical shifts (ppm) of sugar moieties (in pyridine-d,) (‘“C chemical shifts of substituted residues are underlined)
3-0Glur
1 2 3 4 5 6
102.55 77.55 85.15 70.05 76.00 170.65
4.86 4.32 4.20 4.42 4.42
102.65 77.35 :85 20 70.15 76.05 170.55
64
G3
G2
Gl
4.88 4.24 4.24 4.42 4.47
102.20 81.30 16.55 71.10 76.05 171.00
4.88 4.14 4.23 4.43 4.44
1-O 76.65 84.35 69.20 75.15 169.50
4.88 4.31 4.20 4.41 4.45
D. FRECHET et al.
930
Table 3. Continued Gl
64
63
G2
Xyl
1 2 3 4 5
103.80 14.25 77.40 69.65 66.10
5.27 3.94 4.07 4.09 3.64 4.23
103.85 74.10 77.35 69.70 66.10
5.27 3.93 4.08 4.10 3.67 4.24
102.95 73.15 74.45 68.75 65.20
5.26 3.93 4.07 4.08 3.65 4.22
Gal
1 2 3 4 5 6
1o3.00 72.50 74.25 69.10 75.50 60.80
5.48 4.37 4.10 4.49 3.97 4.40
102.95 72.50 74.30 69.15 75.40 60.70
5.49 4.38 4.10 4.50 3.99 4.42
102.50 71.65 73.40 68.20 74.60 59.90
5.49 4.38 4.10 4.48 3.99 4.42
Glc
1 2 3 4 5 6
28-OFuc 1 2 3 4 5 6
los.so 73.25 75.85 69.10 74.10 61.95
5.15 4.43 4.05 4.49 4.05 4.42 4.14
94.00 74.05 74.25 71.80 71.10 15.60
5.88 4.43 4.10 3.93 3.90 1.45
93 65 L 73.05 75.25 71.95 71.20 15.60
5.94 4.54 4.13 3.95 3.91 1.47
93.90 74.70 73.70 71.70 71.00 15.65
5.95 4.40 4.07 3.92 3.85 1.45
93.00 z&j L 73.20 70.80 70.15 14.70
5.95 4.40 4.08 3.94 3.87 1.46
Rha
1 2 3 4 5 6
100.55 69.90 81.60 7735 L 67.70 17.60
5.92 5.10 4.79 4.47 4.46 1.64
99.90 70.75 71.20 82.95 67.05 17.30
6.38 4.74 4.58 4.27 4.41 1.58
1oo.80 69.80 81.30 77.70 67.80 17.70
5.85 5.12 4.82 4.43 4.41 1.67
99.95 68.90 jO.4J 76.85 66.90 16.85
5.85 5.13 4.82 4.46 4.43 1.70
Xyl
1 2 3 4 5
103.80 74.80 77.90 70.05 66.00
5.43 3.90 4.05 4.12 3.43 4.20
105.25 74.50 85.80 67.80 65.70
5.06 3.93 3.97 4.03 3.42 4.18
104.05 74.65 78.10 70.10 65.95
5.38 3.88 4.01 4.10 3.42 4.09
103.15 73.75 77.20 69.20 65.05
5.40 3.89 4.03 4.13 3.43 4.17
Glc
1 2 3 4 5 6
104.25 74.05 77.35 70.15 77.10 61.80
5.30 3.93 4.06 4.01 3.89 4.16 4.45
104.10 74.25 77.40 70.95 77.05 61.15
5.29 3.95 4.06 3.98 3.87 4.18 4.46
103.20 73.35 76.55 70.05 76.15 61.05
5.31 3.96 4.07 3.98 3.89 4.18 4.40
Ara
1 2 3 4 5
104.25 72.00 73.10 77.35 65.40
5.07 4.38 4.09 4.32 3.80 4.47
Ara
1 2 3 4 5
105.60 71.85 73.35 68.30 65.90
4.98 4.41 4.09 4.22 3.73 4.25
Triterpenoid saponins from Gypsophiln spp. B(T2a
I
F
175
PPH
$1 91
4AS 70
OB2
75
G2&4 no
3
A300
A3
4 85
Q63
SO
95
100
n 5:s
5:6
514 PPR
512
510
931
the pulse repetition time was 2.26 set and the pulse angle 90”. Composite pulse decoupling of protons was performed during the relaxation delay, while broad-band irradiation was used during acquisition. r3C multiplicity was determined by J-modulated spin-echo experiment. The low field resonance of pyridined, was set at 148.7 ppm and was used as a chemical shift reference. HETCOR experiments were performed with a refocusing delay of 0.0033 sec. The matrix size was 4K by 128~. The aldehyde and pyridine resonances were folded over. HOHAHA experiments were performed with mixing times of 14 and 400 msec. (Matrix size 2K x 1K). Proton detected HMBC experiments were performed with mixing delays of 80 msec (matrix size: 2K x 256~). For heteronuclear relay experiments, the magnetization transfer was effected by the Hartman-Hahn mechanism. r3C decoupling was done by composite pulse decoupling using the GARPl [13] sequence during the entire acquisition period. The mixing time was set to 50 msec (memory size: 2K, 256 experiments). Mass spectra. Fast Atom Bombardment spectra (Xe, 8 keV negative mode, matrix: thioglycerol) were obtained on a triple stage quadrupole spectrometer. The fragmentation patterns observed by negative FABMS confirm these structure determinations: Cl: m/z 1541 [M-H]-, 1409 [M-H-132]-, 1379 [M-H-162]-, 955 [M-H-(132+162+146+.146)]-, 909 [M-H-(132+162+176+162)]-. GZ: m/z 1643 [M-H]-, 1511 [M-H-132]-, 1379 [M-H-132]-, 1379 [M-H -(132+132)]-, 955 [M-H-(132+132+132+146+146)]-, 909 [M-H-(162+132+176+132+132)]-. 63: m/z 1393 [M -H]-,1231[M-H-162]-,1055[M-H-(162+176)1-.64: m/z 1525 [M-H]-, 1393 [M-H-132]-, 1055 [M-H-(162 +176+132)]-,939[M-H-(162+132+146+146)]-,893[M -H-(162+176+162+132)]-.
105
a:8
Fig. 1. Slice extracted from the heteronuclear multiple bond _____>Z___~ correlation experiment (mixing time 80 msec) corresponamg to the anomeric proton resonances of saponin Gl A =a-rhamnose, B =g-fucose, C = /I-galactose, D = /?-xylose, E =/?-glucose, F = r% xylose, G =/?-ghtcuronic acid, T= terpene moiety. The numbering refers to the carbon position.
64 (RP 68999). Amorphous powder, mp: 2%218”, TLC: R, 0.28, greenish spot, HPLC: R, 13.38 min. NMR. ‘HNMR spectra were measured at 400 MHx, ‘“C
spectra at 100.6 MHZ A known amount of saponin (25 mg in the case. of Gl and 62 and 30 mg for G3 and G4) was dissolved in 0.5 ml pyridine-d,. All NMR experiments were performed at 50”. ‘H spectra were recorded with a spectral window of 4.4 MHz, 16K memory size, a 45” pulse, 16 scans and a 2.87 sec. repetition time. The low field resonance of pyridine-ds was set at 8.71 ppm and was used as a chemical shift reference. In the case of “C spectra, the spectral window was 26 MHz, the memory size 64K,
REFERENCES
1. Khorlin, A. Ya., Ovodov, Yu. S. and Kochetkov, N. K. (1962) Zh. Obshch. Khim. 32, 782.
2. Kochetkov, N. K., Khorlin, A. Ya. and Ovodov, Yu. S. (1963) Tetrahedron Letters 477. 3. Khorlin, A. Ya., Ovodov, Yu. S. and Ovodova, R. G. (1963) Izo. Akad. Nauk. SSSR Ser. Khim. 1522. 4. Kochetkov, N. K., Khorlin, A. Ya. and Ovodov, Yu. S. (1964) Izv. Akad. Nauk. SSSR Ser. Khim. 83. 5. Kochetkov, N. K., Khorlin, A. Ya. and Ovodov, Yu. S. (1964) Izv. Akad. Nauk. SSSR Ser Khim. 1436. 6. Bax, A. and Davis, D. (1985) J. Magn. Reson. 65, 355.
7. Bodenhausen, G., Kogler, H. and Ernst, R. R. (1984) J. Magn. Reson. 58, 370. 8. Bax, A. and Morris, G. (1918) J. Magn. Reson. 142, 501. 9. Bax, A. and Summers, M. F. (1986) J. Am. Chem. Sot. 108, 2093.
10. Wehrli, F. W. and Nichida, T. (1979) Prog. Chem. Org. Nat. Compd. 36,96.
11. Lerner, L. and Bax, A. (1986) .I. Magn. Reson. 69,375. 12. Nunex, H. A., Walker, T. E., Fuentes, R., O’Connor, J., Seriani, A. and Barker, R. (1977) J. Supramole. Strut. 6,535. 13. Shaka, A. J., Barker, P. B. and Freeman, R. (1985) J. Magn. Resort. 64, 547.
Phytochemistry, Vol. 30, No. 3, pp. 927-931, 1991
Printedin GreatBritain.
FOUR TRITERPENOID SAPONINS FROM DRIED ROOTS OF G YPSOPHILA SPECIES DENISE FRECHET, BRUNO CHRIST,* BERTRAND MONEGIER DU SORBIER, HARTMUT FISCHER*
and MARC VUILHORGNE Rhone Poulenc Sante, Centre de Recherches de Vitry, 13 quai Jules Guesde - B.P. 14, F-94403 Vitry-sur-seine-Cedex; France; *A. Natterman and Cie. GmbH, Gruppe Rhone-Poulenc, Forschungszentrum K61n, Nattermannallee I, Postfach-350120, D-5000 Kijln 30. F.R.G. (Received in revisedform 10 July 1990) Key Word Index-Gypsophila paniculata;Gypsophila arrostii; Caryophyllaceae; soap root; Saponariae alba radix; root; saponins; triterpenoids; carbohydrates; quillaic acid; gypsogenin.
Abstract-Four new triterpenoid saponins were isolated from the roots of Gypsophila paniculata and G. arrostii. Their structures were elucidated using a combination of homo- and heteronuclear 2D NMR techniques, without having recourse to chemical degradation or modification. The saponins investigated are: 3-O-B-D-galactopyranosyl-( 1+2)[j-D-XylOpyranOSyl-( 1+3)]-j?-D-glucuronopyranosyl quillaic acid 2%O-B-D-glucopyranosyl-( 1+3)-[B-D-xylOpyranosyl-(l+4)]-cr-L-rhamnopyranosyl-(l-+2)-~-D-fucopyranoside; 3-O-j&D-galactopyranosyl-( 1+2)-D-D-xylopyranosyl-( 1+3)]+D-glucuronopyranosyl quillaic acid 28-O-fi-D-arabinopyranosyl-( l-+4)-fi-D-arabinopyranosyl-( 1+3)j?-D-xylopyranosyl-( 1+4)-a-L-rhamnopyranosyl-( 1-,2)-B-D-fucopyranoside; 3-O-/&D-gluCOpyranOSyl-(I +2)-B-D-glucuronopyranosyl gypsogenin 2%O-B-D-glucopyranosyl-( 1+3)-[b-D-XylOpyranOSyl-( 1+4)]-cc-L-rhamnopyranosyl3-O-D-D-xylopyranosyl-( 1+3)-[B-D-galactopyranosyl-( 1+2)]+D-glucuronopyranosyl (1+2)-/?-D-fucopyranoside; 2%0-/?-D-glucopyranosyl-( 1+3)-[B-D-xylopyranosyl-( 1+4)]-a-L-rhamnopyranosyl-( 1+2)$-D-fucopyrf;:;;;nin
INTRODUcTION
The roots of Gypsophila paniculata and G. arrostii (soap root, Saponariae alba radix) are known as saponin drugs and have been used as detergents and expectorants. Hitherto only gypsosid, a triterpenoid saponin containing gypsogenin and nine sugar moieties, has been isolated from these roots [l-5]. As part of our studies on the quantitation of saponins in soap roots by HPLC, we have isolated four new triterpenoid saponins. Their structures were completely elucidated by homo- and heteronuclear 2DNMR spectroscopy, without having recourse to chemical degradation or modifications. Surprisingly, none of the investigated samples were identified as gypsosid. Gypsosid is therefore absent from these roots, as no other fractions were detected by HPLC. RESULTS AND
DISCUSSION
The ethanolic extract of dried roots was fractionated as described in the Experimental. The isolated saponins, Gl-G4 showed single spots on TLC and single peaks by HPLC, respectively. The molecular formula of each saponin was determined by combining the results of mass spectroscopy, as well as 13C and ‘H NMR. These were identified as Gl (RP 68996)=C70H11003, (M, 1542, 7 sugars), G2 (RP 68997)=C,4H116040 (M, 1644, 8 sugars), G3 (RP 68998)=C,,H,,,O,, (M, 1394, 6 sugars), 64 (RP 68999)=C,oH,10036 (M, 1526, 7 sugars). Because the ‘H NMR signals of the aglycone moiety of each triterpene were well resolved at 400 MHz, their identificaPAY
30:3-N
tion is relatively straightforward. In the first step, a HOHAHA [6] experiment recorded with a mixing time of 14 msec allowed complete elucidation of the proton coupling network. This experiment is equivalent to a COSY [7], however, in that case the observed correlation cross peaks are in phase, thereby preventing the accidental nulling of overlapping peaks. Carbon resonances are assigned by means of a short range H< correlation [S], whereas the quaternary carbons are identified by means of a long-range proton-carbon correlation expe.riment. This latter experiment was recorded by the heteronuclear multiple bond correlation method [9] using ‘H detection, thereby increasing the sensitivity with regards to experiments using 13C detection. By comparison with literature data [IO], the aglycone was identified as quillaic acid in the case of Gl and G2 and gypsogenin for G3 and 64. Their 13C resonance assignments are listed in Table I. Elucidation of the structure of the carbohydrate chains was more challenging, given the extensive overlap of their NMR signals. In a first step, we proceeded to the identification of the sugar residues. ‘H subspectra of the various carbohydrate moieties were obtained from the rows corresponding to their anomeric proton resonances and to their other well resolved resonances (i.e. Me-6 for rhamnose and fucose, methylene-5 for xylose) in a HOHAHA experiment with a mixing time of 400 msec. Direct connectivity was determined from the results of the shorter mixing time (14 msec) HOHAHA experiment. Vicinal coupling constants can be extracted from the rows of these experiments with enough precision ( f 1 Hz)
D. FRECHETet al.
928
‘OR2
23 OH
R=
R’ Cl
XYl
a”
Rs
R4
OH
Glc
OH
HO
OH OH G2
XYl
G3
H
G4
XYl
GlC
&”
OH
H
H
Glc
H
Glc
HO
H H
OH
HO
to determine the relative stereochemistry
of each asymmetric centre and thus to identify the monosaccharides. The average coupling constant values thus measured for the constituent monosaccharide units of the four saponins are listed in Table 2. A partial assignment of 13C resonances was then obtained from the results of the short-range H-C correlation experiment. Given the extensive overlap of proton resonances, some ambiguities remained. These could however be resolved by the heteronuclear H-C relay experiment [ 111. From the anomeric proton resonances, relays could generally be observed to C-2 and C-3, even to C-4 in some cases. Relays from methyl resonances, well resolved geminal proton or even protons located in more crowded areas of the spectrum allowed complete assignment of the 13C resonances. The position of the carboxylic acid of glucuronic acid was provided by the heteronuclear multiple bond correlation experiment. ‘H and “C chemical shifts of all glycosidic residues of Gl-G4 are listed in Table 3. Comparison of the values of 13C chemical shifts with reference data [ 121 allowed identification of the position of substituted residues (underlined in Table 3). The next step was the determination of the linkages among the sugar residues and between the sugars and the triterpene. This information was provided by the longrange heteronuclear correlations arising from the anom-
eric protons. As the 13C resonances of each sugar moiety have been unambiguously assigned previously, interresidue correlations are easily identified. The long-range carbon correlations arising from the anomeric protons of the sugars of Gl are shown in Fig. 1. The linkages 1+2 between rhamnose (A) and fucose (B); l-+28 between fucose (B) and quillaic acid (T); l-+3 between glucose (E) and rhamnose (A); 1+3 between xylose (F) and glucuronic acid (G); 1+3 between glucuronic acid (G) and the terpene moiety (T) were unambiguously determined from this slice of the spectrum. Given the close proximity of the 13C resonances of C-2 of glucuronic acid and C-4 of rhamnose, the linkages l-+2 between galactose (C) and glucuronic acid (G) and 1+4 between xylose (D) and rhamnose (A) were confirmed by the observation of nonanomeric protons. H-2 of glucuronic acid (4.23 ppm) had a correlation to C- 1 of galactose (103 ppm), whereas H-4 of rhamnose (4.47 ppm) had a correlation to C-l of xylose (103.8 ppm). The same procedure was applied to G2-G4, thus allowing complete determination of the structures of these saponins. EXPERIMENTAL PIant panicdata
material. Commercial soap roots of Gypsophila and G. arrostii were from the China National Native
929
Triterpenoid saponins from Gypsophila spp. Table 1. rsC NMR chemical shifts (ppm) of aglycone moieties (in pyridine-d,) Gypsogenin 64 G3
Quillaic acid C 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
Cl
G2 37.25 24.10 83.10 54.05 47.55 19.50 31.80 39.30 45.95 35.15 22.65 121.05 143.45 41.05 35.25 72.90 48.25 40.50 46.30 29.60 34.90 30.65 208.50 9.80 14.70 16.35 26.00 174.80 32.00 23.55
31.25 24.15 83.00 54.00 47.80 19.45 32.00 39.35 46.00 35.15 22.65 120.95 143.45 41.10 35.25 73.10 48.25 40.65 46.45 29.55 35.00 30.40 208.40 9.90 14.75 16.40 26.05 174.80 31.95 23.55
37.25 24.00 82.35 53.90 47.60 19.45 31.60 39.25 46.85 35.25 22.65 121.35 143.00 41.15 27.10 22.55 50.60 40.95 45.40 29.70 33.00 31.30 208.20 9.85 14.65 16.40 24.90 175.25 32.00 22.75
36.30 23.25 82.25 53.05 46.85 18.60 30.65 38.35 45.95 34.35 21.75 120.50 142.80 40.25 26.20 21.60 48.15 40.05 44.50 28.75 32.05 30.40 207.70 9.05 13.75 15.45 24.00 174.35 31.10 21.85
Produce and Animal Byproducts Corporation, Native Produce Branch, Hebei, Peoples Republic of China. Extraction and separation. Dried powdered roots (100 g) were extracted with 200 ml EtOH-H,O (1: 1) by stirring for 24 hr. The plant material was filtered off and the extract was coned to ca 20 ml in uacuo. H,O (10 ml) was added and 3 ml portions of the extract separated on a RP-18 column (Lobar, size B) by a linear gradient from 30 to 100% MeOH during 8 hr, 3.5 hr after starting the gradient, 20 ml frs were collected and analysed by HPLC [column: Nucleosil 120X18 5 m 250 x 4 mm; eluent: 0.1% aq. H,PO,-MeCN (69: 31); temp. 35”; flow rate: 1 ml min-‘; detection wavelength 195 nm]. Frs having identical composition were combined. Four frs, containing the glycosides Gl-G4, were obtained. The MeOH was distilled off in uacuo. The glycosides from the remaining aq. phase were adsorbed on a RP-8 column (Lobar, size A). The column was washed with H,O and the saponins eluted with MeOH. The MeQH was evapd in ~acuo. The glycosides were redissolved in H,O contanining 20% MeCN and finally purified by semiprep. HPLC [column: Nucleosil 100X18 5 pm; 250x8 mm; eluent: 0.1% aq. H,PO,-MeCN linear gradient from 28 to 35% during 25 mm; temp.: ambient; flow rate 3 mlmin-t; detection wavelength 220 nm]. The MeCN was distilled off in vacua The saponins were then adsorbed on silica gel (Lichroprep RP 18) and the column was washed with H,O until complete elimination of H,PO,. The saponins were then eluted with MeOH, and this MeOH evapd in vacua. The purity was confirmed by TLC [precoated silicagel 60 plates; eluent: CHCI,-MeOH-H,QHOAc (32: 25 : 5 : 1); spray reagent: vanillin-H,SO.J. Mps uncorr. Gl (RP 68996). Amorphous powder, mp: 210-213”, TLC: R, 0.22, brownish spot by spraying with vanillin-H,SO, reagent, HPLC: R, 7.56 min. 62 (RP 68997). Amorphous powder, mp: 213-215”, TLC: R, 0.18, brownish spot, HPLC R, 8.93 min. 63 (RP 68998). Amorphous powder, mp: 207-21 l”, TLC: R, 0.32, greenish spot, HPLC: R, 12.38 min.
Table 2. Average vicinal and geminal proton coupling constants (Hz+ 1 Hz) JI-2
J,_,
J,-,
34-5
J,-,.
J,-,.
/7 Glur BXYf BGaf
8 8 8
8 8 8
8 8 2
8 2 2
9
11
j Glc j Fuc aRha f3 Ara
8 8 1 8
8 8 2 8
8 4 8 3
8 2 8 3
2
J,-,
J,-,.
J,-6.
*
*
*
3 6 6
7
12
12
*Not observed.
Table 3. “C and ‘H NMR chemical shifts (ppm) of sugar moieties (in pyridine-d,) (‘“C chemical shifts of substituted residues are underlined)
3-0Glur
1 2 3 4 5 6
102.55 77.55 85.15 70.05 76.00 170.65
4.86 4.32 4.20 4.42 4.42
102.65 77.35 :85 20 70.15 76.05 170.55
64
G3
G2
Gl
4.88 4.24 4.24 4.42 4.47
102.20 81.30 16.55 71.10 76.05 171.00
4.88 4.14 4.23 4.43 4.44
1-O 76.65 84.35 69.20 75.15 169.50
4.88 4.31 4.20 4.41 4.45
D. FRECHET et al.
930
Table 3. Continued Gl
64
63
G2
Xyl
1 2 3 4 5
103.80 14.25 77.40 69.65 66.10
5.27 3.94 4.07 4.09 3.64 4.23
103.85 74.10 77.35 69.70 66.10
5.27 3.93 4.08 4.10 3.67 4.24
102.95 73.15 74.45 68.75 65.20
5.26 3.93 4.07 4.08 3.65 4.22
Gal
1 2 3 4 5 6
1o3.00 72.50 74.25 69.10 75.50 60.80
5.48 4.37 4.10 4.49 3.97 4.40
102.95 72.50 74.30 69.15 75.40 60.70
5.49 4.38 4.10 4.50 3.99 4.42
102.50 71.65 73.40 68.20 74.60 59.90
5.49 4.38 4.10 4.48 3.99 4.42
Glc
1 2 3 4 5 6
28-OFuc 1 2 3 4 5 6
los.so 73.25 75.85 69.10 74.10 61.95
5.15 4.43 4.05 4.49 4.05 4.42 4.14
94.00 74.05 74.25 71.80 71.10 15.60
5.88 4.43 4.10 3.93 3.90 1.45
93 65 L 73.05 75.25 71.95 71.20 15.60
5.94 4.54 4.13 3.95 3.91 1.47
93.90 74.70 73.70 71.70 71.00 15.65
5.95 4.40 4.07 3.92 3.85 1.45
93.00 z&j L 73.20 70.80 70.15 14.70
5.95 4.40 4.08 3.94 3.87 1.46
Rha
1 2 3 4 5 6
100.55 69.90 81.60 7735 L 67.70 17.60
5.92 5.10 4.79 4.47 4.46 1.64
99.90 70.75 71.20 82.95 67.05 17.30
6.38 4.74 4.58 4.27 4.41 1.58
1oo.80 69.80 81.30 77.70 67.80 17.70
5.85 5.12 4.82 4.43 4.41 1.67
99.95 68.90 jO.4J 76.85 66.90 16.85
5.85 5.13 4.82 4.46 4.43 1.70
Xyl
1 2 3 4 5
103.80 74.80 77.90 70.05 66.00
5.43 3.90 4.05 4.12 3.43 4.20
105.25 74.50 85.80 67.80 65.70
5.06 3.93 3.97 4.03 3.42 4.18
104.05 74.65 78.10 70.10 65.95
5.38 3.88 4.01 4.10 3.42 4.09
103.15 73.75 77.20 69.20 65.05
5.40 3.89 4.03 4.13 3.43 4.17
Glc
1 2 3 4 5 6
104.25 74.05 77.35 70.15 77.10 61.80
5.30 3.93 4.06 4.01 3.89 4.16 4.45
104.10 74.25 77.40 70.95 77.05 61.15
5.29 3.95 4.06 3.98 3.87 4.18 4.46
103.20 73.35 76.55 70.05 76.15 61.05
5.31 3.96 4.07 3.98 3.89 4.18 4.40
Ara
1 2 3 4 5
104.25 72.00 73.10 77.35 65.40
5.07 4.38 4.09 4.32 3.80 4.47
Ara
1 2 3 4 5
105.60 71.85 73.35 68.30 65.90
4.98 4.41 4.09 4.22 3.73 4.25
Triterpenoid saponins from Gypsophiln spp. B(T2a
I
F
175
PPH
$1 91
4AS 70
OB2
75
G2&4 no
3
A300
A3
4 85
Q63
SO
95
100
n 5:s
5:6
514 PPR
512
510
931
the pulse repetition time was 2.26 set and the pulse angle 90”. Composite pulse decoupling of protons was performed during the relaxation delay, while broad-band irradiation was used during acquisition. r3C multiplicity was determined by J-modulated spin-echo experiment. The low field resonance of pyridined, was set at 148.7 ppm and was used as a chemical shift reference. HETCOR experiments were performed with a refocusing delay of 0.0033 sec. The matrix size was 4K by 128~. The aldehyde and pyridine resonances were folded over. HOHAHA experiments were performed with mixing times of 14 and 400 msec. (Matrix size 2K x 1K). Proton detected HMBC experiments were performed with mixing delays of 80 msec (matrix size: 2K x 256~). For heteronuclear relay experiments, the magnetization transfer was effected by the Hartman-Hahn mechanism. r3C decoupling was done by composite pulse decoupling using the GARPl [13] sequence during the entire acquisition period. The mixing time was set to 50 msec (memory size: 2K, 256 experiments). Mass spectra. Fast Atom Bombardment spectra (Xe, 8 keV negative mode, matrix: thioglycerol) were obtained on a triple stage quadrupole spectrometer. The fragmentation patterns observed by negative FABMS confirm these structure determinations: Cl: m/z 1541 [M-H]-, 1409 [M-H-132]-, 1379 [M-H-162]-, 955 [M-H-(132+162+146+.146)]-, 909 [M-H-(132+162+176+162)]-. GZ: m/z 1643 [M-H]-, 1511 [M-H-132]-, 1379 [M-H-132]-, 1379 [M-H -(132+132)]-, 955 [M-H-(132+132+132+146+146)]-, 909 [M-H-(162+132+176+132+132)]-. 63: m/z 1393 [M -H]-,1231[M-H-162]-,1055[M-H-(162+176)1-.64: m/z 1525 [M-H]-, 1393 [M-H-132]-, 1055 [M-H-(162 +176+132)]-,939[M-H-(162+132+146+146)]-,893[M -H-(162+176+162+132)]-.
105
a:8
Fig. 1. Slice extracted from the heteronuclear multiple bond _____>Z___~ correlation experiment (mixing time 80 msec) corresponamg to the anomeric proton resonances of saponin Gl A =a-rhamnose, B =g-fucose, C = /I-galactose, D = /?-xylose, E =/?-glucose, F = r% xylose, G =/?-ghtcuronic acid, T= terpene moiety. The numbering refers to the carbon position.
64 (RP 68999). Amorphous powder, mp: 2%218”, TLC: R, 0.28, greenish spot, HPLC: R, 13.38 min. NMR. ‘HNMR spectra were measured at 400 MHx, ‘“C
spectra at 100.6 MHZ A known amount of saponin (25 mg in the case. of Gl and 62 and 30 mg for G3 and G4) was dissolved in 0.5 ml pyridine-d,. All NMR experiments were performed at 50”. ‘H spectra were recorded with a spectral window of 4.4 MHz, 16K memory size, a 45” pulse, 16 scans and a 2.87 sec. repetition time. The low field resonance of pyridine-ds was set at 8.71 ppm and was used as a chemical shift reference. In the case of “C spectra, the spectral window was 26 MHz, the memory size 64K,
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1. Khorlin, A. Ya., Ovodov, Yu. S. and Kochetkov, N. K. (1962) Zh. Obshch. Khim. 32, 782.
2. Kochetkov, N. K., Khorlin, A. Ya. and Ovodov, Yu. S. (1963) Tetrahedron Letters 477. 3. Khorlin, A. Ya., Ovodov, Yu. S. and Ovodova, R. G. (1963) Izo. Akad. Nauk. SSSR Ser. Khim. 1522. 4. Kochetkov, N. K., Khorlin, A. Ya. and Ovodov, Yu. S. (1964) Izv. Akad. Nauk. SSSR Ser. Khim. 83. 5. Kochetkov, N. K., Khorlin, A. Ya. and Ovodov, Yu. S. (1964) Izv. Akad. Nauk. SSSR Ser Khim. 1436. 6. Bax, A. and Davis, D. (1985) J. Magn. Reson. 65, 355.
7. Bodenhausen, G., Kogler, H. and Ernst, R. R. (1984) J. Magn. Reson. 58, 370. 8. Bax, A. and Morris, G. (1918) J. Magn. Reson. 142, 501. 9. Bax, A. and Summers, M. F. (1986) J. Am. Chem. Sot. 108, 2093.
10. Wehrli, F. W. and Nichida, T. (1979) Prog. Chem. Org. Nat. Compd. 36,96.
11. Lerner, L. and Bax, A. (1986) .I. Magn. Reson. 69,375. 12. Nunex, H. A., Walker, T. E., Fuentes, R., O’Connor, J., Seriani, A. and Barker, R. (1977) J. Supramole. Strut. 6,535. 13. Shaka, A. J., Barker, P. B. and Freeman, R. (1985) J. Magn. Resort. 64, 547.
