Triterpene glycosides from Kalopanax septemlobum. 1. Glycosides A, B, C, F, G1, G2, I2, H, and J from leaves of Kalopanax septemlobum var. maximowichii introduced to Crimea

Article abstract of DOI:10.1007/s10600-005-0110-2

Eight known glycosides of hederagenin and the new triterpene glycoside 3-O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→2) -O-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl-(1→4) -O-6-O-acetyl-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl ester of hederagenin were isolated by chromatographic methods from leaves of Kalopanax septemlobum var. maximowichii introduced to Crimea. The known 3-O-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl-(1→4) -O-6-O-acetyl-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl ester of hederagenin was observed for the first time in Kalopanax septemlobum. 2005 Springer Science + Business Media, Inc.

Full text of DOI:10.1007/s10600-005-0110-2

Chemistry of Natural Compounds, Vol. 41, No. 2, 2005
TRITERPENE GLYCOSIDES FROM Kalopanax septemlobum.
1. GLYCOSIDES A, B, C, F, G , G , I , H, AND J FROM
1
2
2
LEAVES OF Kalopanax septemlobum VAR. maximowichii
INTRODUCED TO CRIMEA
V. I. Grishkovets,1 D. A. Panov,1
V. V. Kachala,2 and A. S. Shashkov2
UDC 547.918:543.422
β
→
α
Eightknownglycosidesofhederageninandthenewtriterpeneglycoside3-O- -D-xylopyranosyl-(1 3)-O- -
→
α
α
→
β
L-rhamnopyranosyl-(1 2)-O- -L-arabinopyranosyl-28-O- -L-rhamnopyranosyl-(1 4)-O-6-O-acetyl- -D-
→
β
glucopyranosyl-(1 6)-O- -D-glucopyranosyl ester of hederagenin were isolated by chromatographic
Kalopanax septemlobum maximowichii
introduced to Crimea. The known
methods from leaves of
var.
α
α
→
β
→
3-O- -L-arabinopyranosyl-28-O- -L-rhamnopyranosyl-(1 4)-O-6-O-acetyl- -D-glucopyranosyl-(1 6)-O-
β
Kalopanax septemlobum.
-D-glucopyranosyl ester of hederagenin was observed for the first time in
Key words: Kalopanax septemlobum var. maximowichii, Araliaceae, triterpene glycosides, hederagenin glycosides.
Representativesofthe
genusaredistributedin southern Primorskii territory, southern Sakhalin, South Kuril
Kalopanax
islands, Japan, Korea, and northeastern and central China and have been studied in detail [1-7]. Nevertheless, we repeated a
comparison of the glycoside composition of various organs of the two varieties var. (Nakai)
Kalopanax septemlobum
typicum
Pojark and var.
(Van Houtte) Hara, which were introduced on the southern shore of Crimea (Nikitskii Botanical
maximowichii
Garden) for a number of reasons. Thus, the
genus is currently considered to be monotypic [8]. It includes only the
Kalopanax
(Thunb.) Koidz. {synonyms
species
(Thunb.) Nakai,
Miq., Koidz.,
K. autumnalis
K. septemlobum
Sieb. et Zucc.,
K. pictum K. ricinifolium
Seem., Seem., et al. [9]}. However, the
Acanthopanax ricinifolium
Panax ricinifolium
broad synonymy in the names of this species and the existence of varieties that are often taken as separate species
and (= and ) make it difficult to understand with which plant material
Brassaiopsis ricinifolia
K. septemlobum
K. pictum K. septemlobus K. pictus
researchers worked since external morphological features are not indicated in the articles. The varieties
of
maximowichii K. septemlobum
and
typicum
differ primarily in the shape of the leaves (slightly palmatilobate for the former and deeply
palmatilobate with thorns on the petiole for the latter).
Preliminarytwo-dimensional TLC [10] ofthe leafglycosidesofboth varietiesshowedfundamentaldifferences between
them. First of all it has been observed that hederagenin glycosides dominate substantially in the variety (blue-
maximowichii
violet chromatographic bands) whereas oleanolic acid glycosides (pink chromatographic bands) occur only in trace amounts.
However, the fraction of oleanolic acid glycosides from the variety consists of half or even more in addition to other
typicum
hederagenin glycosides. It was even more unexpected that acylated glycosides occur in significant quantities in the
variety. The amount of these for different glycosides is half and more of their unacetylated analogs whereas only
maximowichii
trace amounts of acylated glycosides are found in the
variety.
typicum
Several researchers studied previously triterpene glycosides from roots and stems of
. The glycoside
Kalopanax
composition was given for leaves [4, 5]. Differences in the glycoside composition of leaves from plants from various habitats
were noted [5]. It is possible that results from investigations of two
not assigned to one variety or the other.
varieties were given [4, 5]. However, they were
Kalopanax
1) V. I. Vernadskii Tauris National University, 95007, Ukraine, Simferopol′, pr. Vernadskogo, 4, e-mail:
vladgri@ukr.net; 2) N. D. Zelinskii Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, B-334, Leninskii
pr., 47. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 156-159, March-April, 2005. Original article submitted
November 29, 2004.
0009-3130/05/4102-0194 ©2005 Springer Science + Business Media, Inc.
194

In the present article, we describe the isolation and structure determination for several glycosides from leaves of
Kalopanax septemlobum var. maximowichii, which was introduced on the southern shore of Crimea. The total glycosides were
separated by preparative chromatography over silica gel to afford glycoside fractions A-L.
Fractions A (1), B (2), C (3), F (4), H (8), and J (9) were chromatographicallypure glycosides whereas fractions G and
I were separated into glycosides with similar chromatographic mobilities G (5), G (6), I , and I (7) by rechromatography of
1
2
1
2
the fractions over the high-performance microsphere silica gel Silpearl.
12
.
COOR2
28
3
R1O
CH2OH
23
R1
1 - 9
R2
H
H
H
1: α-L-Arap→
2: α-L-Rhap→2α-L-Arap→
3: β-D-Xylp→3a-L-Rhap→2a-L-Arap→
4: α-L-Arap→
←β-D-Glcp6←(β-D-Glcp6←OAc)4←α-L-Rhap
←β-D-Glcp6←β-D-Glcp4←α-L-Rhap
5: α-L-Arap→
6: α-L-Rhap→2α-L-Arap→
7: α-L-Rhap→2α-L-Arap→
8: β-D-Xylp→3a-L-Rhap→2a-L-Arap→
9: β-D-Xylp→3a-L-Rhap→2a-L-Arap→
←β-D-Glcp6←(β-D-Glcp6←OAc)4←α-L-Rhap
←β-D-Glcp6←β-D-Glcp4←α-L-Rhap
←β-D-Glcp6←(β-D-Glcp6←OAc)4←α-L-Rhap
←β-D-Glcp6←β-D-Glcp4←α-L-Rhap
Glycosides 1, 2, 3, 5, 7, and 9 were identical by TLC with authentic samples of the hederagenin glycosides 3-O-α-L-
arabinopyranoside, 3-O-α-L-rhamnopyranosyl-(1→2)-O-α-L-arabinopyranoside, 3-O-β-D-xylopyranosyl-(1→3)-O-α-L-
rhamnopyranosyl-(1→2)-O-α-L-arabinopyranoside, and their 28-O-α-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-
(1→6)-O-β-D-glucopyranosyl esters, respectively, that were isolated previouslyfrom various
species, for example, from
Hedera
13
[11] and
[12]. Furthermore, C NMR spectra of 1, 2, 3, 5, 7, and 9 were identical
H. canariensis
Acanthopanax sieboldianus
to those published earlier [11, 12]. Glycosides 1-3 and 5-7 were observed previously in leaves of
[4, 5]; 9 was
Kalopanax
isolated only from
stems [7].
Kalopanax
Glycosides 4, 6, and 8 are converted bymild ammonolysis intothe glycosides described above, 5, 7, and 9, respectively.
Obviously 4, 6, and 8 are acyl derivatives of 5, 7, and 9, respectively. According to TLC, 4 coincided with an authentic sample
of 3-O-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl-(1→4)-O-6-O-acetyl-β-D-gluccopyranosyl-(1→6)-O-β-D-
glucopyranosyl ester of hederagenin, which was isolated from leaves of
[13]; 6, with 3-O-α-L-
Hedera rhombea
rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl-28-O-α-L-rhamnopyranosyl-(1→4)-O-6-O-acetyl-β-D-glucopyranosyl-(1→6)-
O-β-D-glucopyranosyl ester of hederagenin that was isolated previously by us from leaves of
identity of these glycosides also follows from the complete coincidence of their C NMR spectra (Tables 1 and 2) with those
[14]. The
Hedera canariensis
13
in the literature. Glycoside 4 was observed in
for the first time.
K. septemlobum
13
The C NMR spectrum of 8 (Tables 1 and 2) shows two additional signals for an acyl fragment when compared with
that of 9. Considering the values of the chemical shifts, these signals (170.7 and 20.7 ppm) represent an acetate. This acyl
fragment is located on the C-6 hydroxyl of the inner glucose in the triose fragment on the aglycon carboxyl. This is consistent
with the shifts of the signals for the C atoms of this glucose unit, namely, C-6 from 61.3 to 63.7 ppm and C-5 from 77.1 to
73.8 ppm compared with 9. Furthermore, after assigning signals of the aglycon, a disubstituted hederagenin, and the triose
fragment on C-3 of the aglycon by comparison with 9, the remaining signals of the acetylated triose fragment coincided with
those ofglycosides 4and6. Thus, 8isthe28-O-α-L-rhamnopyranosyl-(1→4)-O-6-O-acetyl-β-D-glucopyranosyl-(1→6)-O-β-D-
glucopyranosyl ester ofhederagenin 3-O-β-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→2)-O-α-L-arabinopyranoside
and is a new triterpene glycoside.
195

TABLE 1. 13C Chemical Shifts for Aglycons of Glycosides 1-9
C atom
1-3
4-9
C atom
1-3
4-9
1
2
39.0
26.2
81.0
43.5
47.8
18.2
32.9
39.8
48.2
36.9
23.8
122.6
144.7
42.1
28.3
39.0
26.2
81.1
43.5
47.7
18.2
32.8
39.9
48.2
36.9
23.9
122.9
144.1
42.2
28.3
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
23.7
46.7
42.0
46.4
30.9
34.3
33.2
63.8
13.8
16.0
17.5
26.1
180.1
33.2
23.8
23.4
47.0
41.7
46.2
30.8
34.0
32.6
63.9
13.9
16.2
17.6
26.1
176.5
33.1
23.8
3
4
5
6
7
8
9
10
11
12
13
14
15
______
Here and in Table 2, average chemical shifts are given for the compounds.
The deviations for individual compounds are less than ±0.1 ppm.
TABLE 2. 13C Chemical Shifts for Carbohydrates of Glycosides 1-9
C atom
1, 4, 5
C atom
2, 6, 7
C atom
3, 8, 9
C atom
5, 7, 9
C atom
4, 6, 8
Ara-1
106.7
73.2
74.8
69.7
66.9
Ara-1
104.3
75.9
74.5
69.3
65.5
Ara-1
104.7
75.1
75.6
69.8
66.3
Glc-1
95.7
73.8
78.6
70.8
78.0
69.4
Glc-1
95.6
73.8
78.6
70.8
78.1
69.4
2
3
4
5
2
3
4
5
2
3
4
5
2
3
4
5
6
2
3
4
5
6
Rha-1
101.7
72.3
72.5
74.2
69.8
18.6
Rha-1
101.3
72.0
82.9
73.0
69.7
18.6
Glc-1
104.9
75.3
76.5
78.4
77.1
61.3
Glc-1
104.7
75.1
76.4
79.3
73.8
63.7
20.7
170.7
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
OAc
OAc
Xyl-1
107.5
75.1
78.3
71.1
67.4
Rha-1
102.8
72.5
72.7
73.9
70.4
18.6
Rha-1
102.9
72.4
72.6
73.9
70.7
18.5
2
3
4
5
2
3
4
5
6
2
3
4
5
6
196

TABLE 3. 1H Chemical Shifts for Carbohydrates of Glycoside 8
H atom
Chemical shift
H atom
Chemical shift
Ara-1
5.05
4.58
4.03
4.13
4.24
3.66
Glc-1
2
6.19
4.09
4.18
4.21
4.10
4.64
4.32
2
3
3
4
4
5e
5a
5
6A
6B
Rha-1
6.30
4.86
4.73
4.43
4.67
1.54
Glc-1
2
4.98
3.92
4.10
4.07
3.82
4.62
4.52
1.93
2
3
4
5
6
3
4
5
6A
6B
OAc
Xyl-1
5.30
4.02
4.12
4.13
4.21
3.63
Rha-1
5.50
4.55
4.47
4.28
4.79
1.68
2
3
2
3
4
5
6
4
5e
5a
The structures of the carbohydrate fragments of 8 were solved by an independent analysis of several two-dimensional
(2D) NMR spectra: COSY, TOCSY, ROESY, HSQC, and HMBC. Based on an analysis of the COSY and TOCSY spectra,
beginning with the easilyinterpreted signals for the anomeric protons, signals of the skeleton protons were completelyassigned
for each monosaccharide unit (Table 3). An analysis of the ROESY spectrum confirmed the types of glycoside bonds and the
attachment sites of the carbohydrate chains to the aglycon since structurally informative interglycoside cross-peaks between
signals of the anomeric protons and the skeletal protons of other monosaccharide units or the aglycon are easily followed in it.
An analysis of the HSQC spectrum for glycosylation effects on C atoms compared with chemical shifts of unsubstituted
carbohydrate units also confirmed the types of glycoside bonds. An analysis of the HMBC spectrum revealed the site of the
acetyl from the cross-peak between the carbonyl C atom and the H-6 protons of the inner glucose unit.
We note that the variety maximowichii described by us, according to our data, contains a significant quantity of
acetylated glycosides and is obviouslyidentical tothe Kalopanax varietygrowing in Japan that has been described [5]. However,
it differs in glycoside composition from the variety growing in China that, according to the literature [4], lacks acetylated
glycosides. It is curious that the Japanese variety is called [5] K. pictus whereas these same authors call the Chinese variety
K. septemlobus [4] although they present no systematic basis for this (for example, external morphological features).
EXPERIMENTAL
NMR spectra were obtained on a Bruker DRX-500 instrument. Standard Bruker programs were used for 2D COSY,
TOCSY, ROESY, HSQC, and HMBC experiments. The mixing time (spin-locking) in the 2D ROESY experiments was 200
ms. We used solutions of the glycosides in pyridine-d with added TMS as internal standard.
5
TLC analysis and separation monitoring were performed on Silufol plates (Czech Rep.). Spots of triterpene
glycosides and their aglycons were detected in the chromatograms by alcoholic (10%) phosphotungstic acid with added
p-hydroxybenzaldehyde (2%) with subsequent heating at 100-120°C. Sugars were detected using alcoholic (10%) anilinium
acid phthalate with subsequent heating at 100-150°C.
197

Two-dimensional TLC of extracts and pure fractions was performed on Silufol, Sorbosil, Polygram, and Merck plates
using neutral solvents in one direction and alkaline solvents in the perpendicular direction. The neutral solvent system was a
CHCl :CH OH:H O (100:40:7) mixture; alkaline, CHCl :CH OH:NH OH (25%) (100:40:10).
3
3
2
3
3
4
Total acid hydrolysis was carried out by treating glycoside (1 mg) with dioxane (0.1 mL) and aqueous CF CO H
3
2
(0.1 mL, 2 N) and heating for 2 h at 100°C. The aglycon was extracted with benzene (0.5 mL). The resulting extract was
analyzed byTLC using benzene:acetone (4:1) or CHCl :CH OH:NH OH (25%) (100:20:1) with authentic samples ofaglycons.
3
3
4
Sugar in the hydrolysate was identified byTLC using CHCl :CH OH:NH OH(100:40:10) with authentic samples ofrhamnose,
3
3
4
arabinose, glucose, galactose, xylose, and glucuronic acid.
Alkaline hydrolysis was performed by treating glycoside (2 mg) with KOH (0.2 mL, 10%) in H O:CH OH (1:1) and
2
3
heating at 100°C for 2 h. The resulting solution was neutralized with aqueous H SO (1 N) until slightly acidic. Progenins
2
4
were extracted with butanol. The butanol extract was analyzed by TLC using CHCl :CH OH:H O (100:40:7).
3
3
2
Mild alkaline hydrolysis (ammonolysis) was carried out by dissolving glycoside (2 mg) in aqueous-alcoholic (1:1,
0.2 mL, 10%) ammonia and storing at 20°C for 2-3 h. The solution was neutralized with KU-2-8 cation-exchanger in the
+
H -form. The filtrate was analyzed by TLC using CHCl :CH OH:H O (100:40:7).
3
3
2
Isolation of Glycosides. Leaves of K. septemlobum var. maximowichii that were collected in Nikitskii Botanical
Garden in October during shedding (60 g, air-dried mass) were thoroughly ground and treated with benzene (3×300 mL). The
defattedsolidwasextractedwith isopropanol (80%, 4×400 mL). The combined extracts wereevaporatedtoaffordtotalextracted
substances (20 g) that were dissolved in water-saturated butanol (700 mL) and washed with water (3×200 mL). Evaporation
of butanol gave purified total glycosides (10 g).
Separation of Glycosides. Total glycosides (10 g) were chromatographed over silica gel L (1 kg, 40-100 µm) with
gradient elution by CHCl :isopropanol (10:1→1:1) saturated with water to give fractions of glycosides A (50 mg), B (500 mg),
3
C (800 mg), D (610 mg), E (300 mg), F (50 mg), G (490 mg), H (750 mg), I (880 mg), J (850 mg), K (860 mg), and L
(350 mg).
Fractions A, B, C, F, H, and J gave pure glycosides 1 (20 mg), 2 (300 mg), 3 (650 mg), 4 (30 mg), 8 (300 mg), and
9 (350 mg) after additional chromatographic purification in water-saturated CHCl :isopropanol systems of the appropriate
3
polarity. Fraction G (490 mg) was rechromatographed over silica gel Silpearl (250 g) with elution by water-saturated
CHCl :isopropanol (3:1) to give 5 (50 mg) and 6 (300 mg). Fraction I (880 mg) was rechromatographed over Silpearl silica
3
gel (500 g) with elution by water-saturated CHCl :isopropanol (2:1) to give I (350 mg) and 7 (200 mg).
3
1
TLC identified hederagenin in the total acid hydrolysates of all glycosides and arabinose in 1; rhamnose and arabinose
in 2; xylose, rhamnose, and arabinose in 3; glucose, rhamnose, and arabinose in 4-7; xylose, rhamnose, arabinose, and glucose
in 8 and 9. Alkaline hydrolysis of 4 and 5 gives 1, 6, and 7; then 2, 8, and 9; finally 3. Ammonolysis of 4 gives 5; of 6, 7; of
8, 9.
13
Tables 1 and 2 give the C chemical shifts for 1-9. Table 3 lists the chemical shifts for protons in the carbohydrate
parts of 8.
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