.


 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.

Figure 1: Saponins from A. gypsophiloides 1, R = OH and 2, R = H.

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].

Table 1: ¹H and ¹³С NMR data (δ, ppm) of the triterpene units of compounds 1 and 2 (500 MHz, pyridine-d₅/D₂O 1:1).^a

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.

Table 2: ¹H and ¹³С NMR data (δ, ppm; J, Hz) for carbohydrate units of compounds 1 and 2 (500 MHz, pyridine-d₅/D₂O 1:1).

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

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.

Figure 2: Part of a 2D ROESY spectrum of compound 1. The corresponding parts of the ¹H NMR spectrum are shown along the axes. Arabic numerals refer to atoms in sugar residues denoted by letters, as shown for compounds 1 and 2. Slashes are used for the designation of inter-residual interactions.

Figure 3: Key ROESY (dashed line) correlations for compound 1.

Figure 4: Part of the HMBC spectrum of compound 1. ¹H and ¹³C NMR spectra are shown along the horizontal and vertical axes, respectively. Arabic numerals before a slash refer to protons and after a slash refer to carbons in sugar residues denoted by letters, as shown for compounds 1 and 2.

Characteristic chemical shifts in the ¹³C NMR spectrum of 2 (δ_C 85.4 ppm for C-3 and δ_C 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

Elena A. Khatuntseva¹, Vladimir M. Men’shov¹, Alexander S. Shashkov², Yury E. Tsvetkov¹, Rodion N. Stepanenko³, Raymonda Ya. Vlasenko³, Elvira E. Shults⁴, Genrikh A. Tolstikov⁴, Tatjana G. Tolstikova⁴, Dimitri S. Baev⁴, Vasiliy A. Kaledin⁵, Nelli A. Popova⁵, Valeriy P. Nikolin⁵, Pavel P. Laktionov⁶, Anna V. Cherepanova⁶, Tatiana V. Kulakovskaya⁷, Ekaterina V. Kulakovskaya⁷ and Nikolay E. Nifantiev¹

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

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
//////