.
ABSRACT
Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-β-D-GlcpA and pentasaccharide β-D-Xylp-(1→3)-β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[β-D-Quip-(1→4)]-β-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.
The methanolic extract of the dried powdered roots of A. gypsophiloides was concentrated, and the crude mixture of saponins was precipitated from methanol by the addition of acetone and subjected to reversed-phase С18 HPLC. Compounds 1 and 2 (Figure 1) were isolated as white amorphous powders. Compound 1 exhibited in the HRMS (ESI) the [M + Na]+ peak at m/z 1681.7071, indicating a molecular weight compatible with the molecular formula C75H118O40. Compound 2 exhibited the [M + Na]+ peak at m/z 1665.7181, consistent with the molecular formula С75H118O39. GLC analysis of the acetylated (S)-2-octyl glycosides derived after full acid hydrolysis of compound 1 revealed the presence of D-galactose (D-Gal), L-arabinose (L-Ara), 6-deoxy-D-glucose (D-Qui), D-xylose (D-Xyl), L-rhamnose (L-Rha), D-fucose (D-Fuc), and D-glucuronic acid (D-GlcA). Similar investigation of compound 2 revealed the same sugar composition as for compound 1.
The structures of both compounds 1 and 2 were confirmed on the basis of their 1H NMR, 13C NMR, APT, COSY, TOCSY, ROESY, HSQC, and HMBC spectra. In accordance with the earlier reports [18] on structures of saponins from A. gypsophiloides, the aglycons of compound 1 and 2 were supposed to comprise quillaic acid (16-α-hydroxygypsogenin) and gypsogenin, respectively. This assumption was in good agreement with the detection of characteristic signals for six methyl groups in the 1H (Table 1) and 13C NMR (Table 2) spectra of 1 and 2. Furthermore, the presence of these aglycons was unambiguously confirmed by the good agreement between 13C NMR shifts of aglycon moieties of 1 and 2 and signals of aglycons for described bidesmosides comprising quillaic acid [21] and gypsogenin [21].
Analysis of COSY and TOCSY spectra of both 1 and 2 revealed the presence of the following residues: β-GlcpA (residue a), β-Galp (residue b), α-Arap (residue c), β-Fucp (residue d), β-Quip (6-deoxy-β-Glcp, residue e), α-Rhap (residue f), β-Xylp (residues g and h).
The HSQC spectrum confirmed the structures of the triterpene aglycon
and showed the positions of the substitutions within the oligosaccharide
fragments (Table 1 and Table 2). The ROESY spectra (identical for compounds 1 and 2)
disclosed the sequence of the residues in two oligosaccharides and
their location at the C-3 and C-28 of the aglycon. Thus, the location of
GlcA (residue a) at the position 3 of the triterpene was established from the presence of a correlation peak 1a/3Agl (Figure 2 and Figure 3). Correlation peaks 1b/2a and 1c/3a correspond to substitutions of the residue a by terminal b at the position 2 and by terminal c at the position 3. Esterification of the position 1 of Fuc (residue d)
with the carboxy group of the triterpene was unambiguously shown by the
high-field shift of C-1 (94.4 ppm), being indirectly confirmed with the
long-range correlation peak in the ROESY spectra 16Agl/3d. The sequence of the other residues was disclosed from the presence of the correlation peaks 1e/4d, 1f/2d, 1g/4f and 1g/4h (Figure 2). HMBC spectra finally confirmed the structure of the aglycons and the sequence of the residues. Thus, the correlation peak 1d/28Agl evidenced the location of Fuc (residue d) as the esterified substituent at C-28 of the triterpene (Figure 4).
The other inter-residue correlation peaks were in agreement with the
structure of oligosaccharides established from analysis of the ROESY
spectra.
Characteristic chemical shifts in the 13C NMR spectrum of 2 (δC 85.4 ppm for C-3 and δC 176.4 ppm for C-28 of the aglycon) evidence the bidesmosidic nature of the genin, which is glycosydated at C-3 and esterified to an oligosaccharide. The structures of both the trisaccharide and pentasaccharide fragments of compound 2 are similar to those established for compound 1. Thus the structure of 2 was elucidated as gypsogenin 28-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[6-deoxy-β-D-glucopyranosyl-(1→4)]-β-D-fucopyranosyl ester 3-O-α-L-arabinopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-glucuronopyranoside.
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ABSRACT
Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-β-D-GlcpA and pentasaccharide β-D-Xylp-(1→3)-β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[β-D-Quip-(1→4)]-β-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.
The methanolic extract of the dried powdered roots of A. gypsophiloides was concentrated, and the crude mixture of saponins was precipitated from methanol by the addition of acetone and subjected to reversed-phase С18 HPLC. Compounds 1 and 2 (Figure 1) were isolated as white amorphous powders. Compound 1 exhibited in the HRMS (ESI) the [M + Na]+ peak at m/z 1681.7071, indicating a molecular weight compatible with the molecular formula C75H118O40. Compound 2 exhibited the [M + Na]+ peak at m/z 1665.7181, consistent with the molecular formula С75H118O39. GLC analysis of the acetylated (S)-2-octyl glycosides derived after full acid hydrolysis of compound 1 revealed the presence of D-galactose (D-Gal), L-arabinose (L-Ara), 6-deoxy-D-glucose (D-Qui), D-xylose (D-Xyl), L-rhamnose (L-Rha), D-fucose (D-Fuc), and D-glucuronic acid (D-GlcA). Similar investigation of compound 2 revealed the same sugar composition as for compound 1.
The structures of both compounds 1 and 2 were confirmed on the basis of their 1H NMR, 13C NMR, APT, COSY, TOCSY, ROESY, HSQC, and HMBC spectra. In accordance with the earlier reports [18] on structures of saponins from A. gypsophiloides, the aglycons of compound 1 and 2 were supposed to comprise quillaic acid (16-α-hydroxygypsogenin) and gypsogenin, respectively. This assumption was in good agreement with the detection of characteristic signals for six methyl groups in the 1H (Table 1) and 13C NMR (Table 2) spectra of 1 and 2. Furthermore, the presence of these aglycons was unambiguously confirmed by the good agreement between 13C NMR shifts of aglycon moieties of 1 and 2 and signals of aglycons for described bidesmosides comprising quillaic acid [21] and gypsogenin [21].
Comp. | C-1 | C-2 | C-3 | C-4 | C-5 | C-6 | C-7 | C-8 | C-9 | C-10 | C-11 | C-12 | C-13 | C-14 | C-15 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
H-1 | H-2 | H-3 | H-5 | H-6 | H-7 | H-9 | H-11 | H-12 | H-15 | ||||||
1 | 38.3 | 25.2 | 85.3 | 55.9 | 48.3 | 20.7 | 32.8 | 40.4 | 47.0 | 36.3 | 23.9 | 122.7 | 144.1 | 42.2 | 35.9 |
1.53 | 2.28 | 4.06 | 1.37 | 1.40 | 1.62 | 1.75 | 1.91 | 5.37 | 2.04 | ||||||
0.91 | 1.97 | 1.01 | 1.49 | 1.86 | 1.89 | ||||||||||
2 | 38.2 | 25.1 | 85.4 | 56.0 | 48.2 | 20.8 | 32.6 | 40.2 | 47.8 | 36.3 | 23.8 | 122.8 | 144.1 | 42.5 | 28.7 |
1.51 | 2.29 | 4.10 | 1.43 | 1.43 | 1.62 | 1.66 | 1.87 | 5.37 | 1.81 | ||||||
0.94 | 1.97 | 1.08 | 1.48 | 1.82 | 1.42 | ||||||||||
Comp. | C-16 | C-17 | C-18 | C-19 | C-20 | C-21 | C-22 | C-23 | C-24 | C-25 | C-26 | C-27 | C-28 | C-29 | C-30 |
H-16 | H-18 | H-19 | H-21 | H-22 | H-23 | H-24 | H-25 | H-26 | H-27 | H-29 | H-30 | ||||
1 | 73.9 | 47.9 | 41.6 | 47.4 | 29.3 | 35.8 | 31.5 | 211.6 | 10.7 | 16.0 | 17.6 | 27.3 | 177.1 | 33.1 | 24.6 |
5.01 | 3.27 | 2.57 | 2.19 | 2.28 | 9.71 | 1.43 | 0.88 | 0.96 | 1.68 | 0.94 | 0.96 | ||||
1.24 | 1.26 | 2.04 | |||||||||||||
2 | 23.3 | 47.9 | 42.1 | 46.4 | 30.8 | 33.9 | 32.4 | 211.5 | 10.7 | 15.8 | 17.5 | 26.1 | 176.4 | 33.2 | 23.7 |
2.05 | 2.99 | 1.68 | 1.25 | 1.82 | 9.63 | 1.43 | 0.85 | 0.92 | 1.24 | 0.93 | 0.85 | ||||
1.75 | 1.17 | 1.14 | 1.66 | ||||||||||||
a1H NMR chemical shifts are italicized. |
Units, atoms | 1 | 2 | |||
---|---|---|---|---|---|
δC | δH (J) | δC | δH (J) | ||
→2,3)-GlcA (a) | |||||
1 | 103.4 | 4.83, d (7.8) | 103.4 | 4.82, d (7.3) | |
2 | 77.7 | 4.26 | 77.7 | 4.27 | |
3 | 85.0 | 4.30 | 85.0 | 4.31 | |
4 | 71.6 | 4.16 | 71.6 | 4.17 | |
5 | 77.7 | 4.26 | 77.7 | 4.27 | |
6 | 175.2 | 175.2 | |||
Gal (b) | |||||
1 | 103.2 | 5.33, d (7.7) | 103.2 | 5.33, d (7.5) | |
2 | 72.8 | 4.14 | 72.8 | 4.14 | |
3 | 74.4 | 4.09 | 74.5 | 4.08 | |
4 | 70.3 | 4.31 | 70.3 | 4.31 | |
5 | 76.5 | 3.97 | 76.5 | 3.97 | |
6(a, b) | 62.2 | 4.33, 4.17 | 62.1 | 4.35, 4.17 | |
Ara (c) | |||||
1 | 104.0 | 5.16, d (7.5) | 104.0 | 5.17, d (7.5) | |
2 | 72.4 | 4.23 | 72.4 | 4.23 | |
3 | 73.7 | 4.12 | 73.8 | 4.12 | |
4 | 69.3 | 4.28 | 69.4 | 4.28 | |
5(a, b) | 67.2 | 4.34, 3.95 | 67.2 | 4.34, 3.95 | |
→2,4)-Fuc (d) | |||||
1 | 94.4 | 5.78, d (8.1) | 94.5 | 5.80, d (8.1) | |
2 | 74.6 | 4.43 | 75.1 | 4.41 | |
3 | 76.3 | 4.20 | 76.0 | 4.19 | |
4 | 83.2 | 4.12 | 83.0 | 4.12 | |
5 | 71.9 | 4.03 | 71.8 | 4.02 | |
6 | 17.1 | 1.52 | 17.1 | 1.52 | |
Qui (e) | |||||
1 | 105.6 | 4.92, d (7.8) | 105.6 | 4.92, d (7.8) | |
2 | 75.6 | 3.81 | 75.6 | 3.80 | |
3 | 77.0 | 3.99 | 77.1 | 4.00 | |
4 | 76.1 | 3.53 | 76.1 | 3.53 | |
5 | 72.9 | 3.69 | 72.9 | 3.70 | |
6 | 18.2 | 1.51 | 18.2 | 1.51 | |
→4)-Rha (f) | |||||
1 | 101.2 | 6.01 s (<1) | 101.2 | 5.97 s (<1) | |
2 | 71.1 | 4.62 | 71.1 | 4.62 | |
3 | 71.8 | 4.41 | 71.8 | 4.43 | |
4 | 83.7 | 4.15 | 83.7 | 4.18 | |
5 | 68.3 | 4.26 | 68.7 | 4.28 | |
6 | 18.3 | 1.65 | 18.4 | 1.68 | |
→3)-Xyl (g) | |||||
1 | 106.1 | 5.06, d (8.5) | 105.9 | 5.09, d (7.7) | |
2 | 74.7 | 3.92 | 74.7 | 3.91 | |
3 | 86.5 | 4.02 | 86.4 | 4.01 | |
4 | 68.8 | 3.99 | 68.8 | 4.00 | |
5(a, b) | 66.2 | 4.18, 3.58 | 66.2 | 4.18, 3.58 | |
Xyl (h) | |||||
1 | 104.9 | 5.03, d (8.8) | 104.9 | 5.04, d (7.6) | |
2 | 74.7 | 3.92 | 74.7 | 3.91 | |
3 | 77.0 | 4.01 | 77.1 | 4.01 | |
4 | 70.2 | 4.09 | 70.2 | 4.09 | |
5(a, b) | 66.5 | 4.30, 3.69 | 66.5 | 4.30, 3.68 |
Characteristic chemical shifts in the 13C NMR spectrum of 2 (δC 85.4 ppm for C-3 and δC 176.4 ppm for C-28 of the aglycon) evidence the bidesmosidic nature of the genin, which is glycosydated at C-3 and esterified to an oligosaccharide. The structures of both the trisaccharide and pentasaccharide fragments of compound 2 are similar to those established for compound 1. Thus the structure of 2 was elucidated as gypsogenin 28-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[6-deoxy-β-D-glucopyranosyl-(1→4)]-β-D-fucopyranosyl ester 3-O-α-L-arabinopyranosyl-(1→3)-[β-D-galactopyranosyl-(1→2)]-β-D-glucuronopyranoside.
Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel
1Laboratory of Glycoconjugate Chemistry, N. D. Zelinsky
Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky
prospect 47, 119991 Moscow, Russian Federation
2Laboratory of NMR spectroscopy, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, 119991 Moscow, Russian Federation
3Institute of Immunology, Ministry of Health and Social Development of Russian Federation, Kashirskoe Chausseе, 24/2, 115478 Moscow, Russian Federation
4Laboratory of Pharmacological Researches N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, prospect Acad. Lavrent’eva, 9, 630090 Novosibirsk, Russian Federation
5Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences, 10 prospect Acad. Lavrent’eva, 630090 Novosibirsk, Russian Federation
6Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 prospect Acad. Lavrent’eva, 630090 Novosibirsk, Russian Federation
7G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russian Federation
2Laboratory of NMR spectroscopy, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, 119991 Moscow, Russian Federation
3Institute of Immunology, Ministry of Health and Social Development of Russian Federation, Kashirskoe Chausseе, 24/2, 115478 Moscow, Russian Federation
4Laboratory of Pharmacological Researches N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, prospect Acad. Lavrent’eva, 9, 630090 Novosibirsk, Russian Federation
5Institute of Cytology and Genetics Siberian Branch of the Russian Academy of Sciences, 10 prospect Acad. Lavrent’eva, 630090 Novosibirsk, Russian Federation
6Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 8 prospect Acad. Lavrent’eva, 630090 Novosibirsk, Russian Federation
7G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russian Federation
Corresponding author email
This article is part of the Thematic Series "Synthesis in the glycosciences II".
Guest Editor: T. K. Lindhorst
Beilstein J. Org. Chem. 2012, 8, 763–775.
http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-8-87//////
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