A new triterpene glycoside from fruit of Phytolacca americana

Article abstract of DOI:10.1007/s10600-011-0052-9

Glycosides H and I, the structures of which were established by modern physicochemical analytical methods (PMR, ¹³C NMR, COSY, TOCSY, HMBC, MS) and acid-base hydrolysis, were isolated from the purified total saponins from fruit of Phytolacca americana containing at least 10 triterpene glycosides by rechromatography of enriched fractions over a column of silica gel. Glycoside H was a bidesmoside of phytolaccageninic acid, which was isolated earlier from cell culture of Phytolacca acinosa. Glycoside I was 3-O-(β-D-xylopyranosyl- (1 → 3)-β-D-galactopyranosyl-(1 → 3-β-D-xylopyranosyl)-28-O- β-D-glucopyranosyl phytolaccagenin, which was isolated by us for the first time.

Full text of DOI:10.1007/s10600-011-0052-9

Chemistry of Natural Compounds, Vol. 47, No. 5, November, 2011 [Russian original No. 5, September-October, 2011]
A NEW TRITERPENE GLYCOSIDE FROM FRUIT OF
Phytolacca americana
1*
1
1
M. Z. Getiya, M. A. Gabelaya, V. D. Mshvildadze,
UDC 547.918; 543.422
2
2
1
A. Pichette, S. Lavoie, and G. E. Dekanosidze
Glycosides H and I, the structures of which were established by modern physicochemical analytical methods
13
(PMR, C NMR, COSY, TOCSY, HMBC, MS) and acid-base hydrolysis, were isolated from the purified total
saponins from fruit of Phytolacca americana containing at least 10 triterpene glycosides by rechromatography
of enriched fractions over a column of silica gel. Glycoside H was a bidesmoside of phytolaccageninic acid,
which was isolated earlier from cell culture of Phytolacca acinosa. Glycoside I was 3-O-(ꢀ-D-xylopyranosyl-
(1ꢁ3)-ꢀ-D-galactopyranosyl-(1ꢁ3)-ꢀ-D-xylopyranosyl)-28-O-ꢀ-D-glucopyranosyl phytolaccagenin, which
was isolated by us for the first time.
Keywords: Phytolacca americana, American pokeweed, triterpene glycoside, sapogenin, phytolaccagenin.
The genus Phytolacca (pokeweed) (Phytolaccaceae) is represented in Georgia by one species, Phytolacca americana
L., which was imported from North America. This is a perennial herbaceous weed of 1–3 m height. Its fruit is ~7–8 mm in
diameter and is round without ribs when ripe [1].
American pokeweed is used in folk medicine to treat diseases such as rheumatism, syphilis, skin diseases, etc. [2]. Up
to 10 triterpenes were previously isolated and characterized chemically from various organs of pokeweed [3–6].
The isolation of triterpene saponins from ripe fruit of American pokeweed, the identification of a known glycoside,
and the elucidation of the structure of one new glycoside are described herein.
A comparative qualitative analysis by TLC of triterpene glycosides isolated from fresh ripe American pokeweed fruit
dried in air and in a microwave oven showed that the drying conditions did not affect the spectrum of these glycosides.
Rechromatography of enriched fractions of purified total saponins containing at least 10 triterpene glycosides designated
by us by the letters A, B, C, D, E, F, G, H, I, and J isolated glycosides H and I.
The products of total acid hydrolysis contained according to TLC the monosaccharides xylose, galactose, and glucose.
Glycosides H and I were not methylated by an ether solution of diazomethane. However, alkaline hydrolysis cleaved glucose.
The final structures of the isolated glycosides were elucidated using modern physicochemical analytical methods of
13
PMR, C NMR, and 1D and 2D experiments (Tables 1 and 2). According to the results, glycoside H was identified as a
bidesmoside and a trioside of phytolaccageninic acid that was isolated earlier from Phytolacca acinosa cell culture [6].
30
29
COOCH
3
20
17
OH
Glc
HO
HO
11
9
O
OH
OH
26
25
14H
28
HO
HO
HO
O
HO
O
O
1
O
O
O
Xyl 2
Gal
H
Xyl 1
5
27
O
H
OH
OH
OH
OH
24
OH
Glycoside I
1) Institute of Pharmaceutical Chemistry, I. G. Kutateladze Tbilisi State Medical University, Tbilisi, Ul. P. Saradzhishvili,
36, Georgia, e-mail: mgetia2004@yahoo.co.uk; 2) Department of Fundamental Science, University of Quebec at Chicoutimi,
555 University Boulevard, Canada. Translated from Khimiya Prirodnykh Soedinenii, No. 5, September–October, 2011,
pp. 671–673. Original article submitted March 3, 2011.
©
764
0009-3130/11/4705-0764 2011 Springer Science+Business Media, Inc.

13
TABLE 1. C NMR and PMR Spectra of the Aglycon Part of Glycoside I (CD OD, ꢂ, ppm, J/Hz)
3
C atom
C atom
ꢂC
ꢂH
ꢂC
ꢂH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
44.5 t
71.6 d
83.6 d
43.2 s
48.2 d
18.6 t
33.4 t
40.7 s
49.4 d
37.5 s
24.7 t
124.4 d
144.6 s
43.1 s
28.9 t
24.2 t
2.07 (1H, m) 1.15 (1H, m)
4.26 (1H, m)
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OMe
47.4 s
43.9 d
43.3 t
45.0 s
31.4 t
34.4 t
65.5 t
14.7 q
17.6 q
17.8 q
26.4 q
177.6 s
28.7 q
178.8 s
52.4 q
2.70 (2H, dd, J = 13.2, 3.2)
1.95 (1H, m) 1.69 (1H, m)
3.61 (1H, m)
1.31 (1H, m)
1.48 (2H, m)
1.60 (1H, m) 1.28 (1H, m)
2.00 (1H, m) 1.38 (1H, m)
1.72 (1H, m) 1.55 (1H, m)
3.60 (1H, m) 3.23 (1H, m)
0.92 (3H, s)
1.58 (1H, m)
1.28 (3H, s)
0.80 (3H, s)
1.17 (3H, s)
1.98 (2H, m)
5.32 (1H, m)
1.14 (3H, s)
3.70 (3H, s)
1.79 (1H, m) 1.09 (1H, m)
2.04 (1H, m) 1.78 (1H, m)
13
TABLE 2. C NMR and PMR Spectra of the Monosaccharide Part of Glycoside I (CD OD, ꢂ, ppm, J/Hz)
3
C atom
C atom
ꢂÑ
ꢂH
ꢂÑ
ꢂH
Glucose
Galactose
95.8 d
73.9 d
78.3 d
71.1 d
78.8 d
62.4 t
5.34 (1H, d, J = 7.4)
3.30 (1H, m)
105.1 d
72.2 d
84.2 d
69.9 d
76.7 d
62.6 t
4.60 (1H, d, J = 7.7)
3.79 (1H, m)
3.64 (1H, m)
4.05 (1H, d, J = 2.9)
3.60 (1H, m)
1ꢃ
2ꢃ
3ꢃ
1ꢃꢃꢃ
2ꢃꢃꢃ
3ꢃꢃꢃ
ꢃꢃꢃ
4
5ꢃꢃꢃ
6ꢃꢃꢃ
3.40 (1H, m)
3.34 (1H, m)
3.34 (1H, m)
ꢃ
4
5ꢃ
6ꢃ
3.81 (1H, m) 3.67 (1H, m)
3.79 (1H, m) 3.69 (1H, m)
Xylose 1
Xylose 2
106.2 d
74.6 d
87.5 d
69.9 d
66.4 t
4.43 (1H, d, J = 7.4)
3.47 (1H, t, J = 8.0)
3.52 (1H, m)
106.3 d
72.9 d
74.0 d
69.7 d
67.1 t
4.48 (1H, d, J = 7.1)
3.65 (1H, m)
3.55 (1H, m)
3.81 (1H, m)
3.86 (1H, dd, J = 11.6, 2.6)
3.57 (1H, m)
1ꢃꢃ
2ꢃꢃ
3ꢃꢃ
4ꢃꢃ
1ꢃꢃꢃꢃ
2ꢃꢃꢃꢃ
3ꢃꢃꢃꢃ
4ꢃꢃꢃꢃ
3.62 (1H, m)
3.90 (1H, dd, J = 11.8, 5.2)
3.25 (1H, m)
ꢃꢃ
ꢃꢃꢃꢃ
5
5
Glycoside I was a white amorphous powder. According to a peak for the pseudomolecular ion with m/z 1143.5202
+
for [M + Na] (HR-ESI-MS high-resolution mass spectrum), the molecular formula corresponded with C H O . Analysis
53 84 25
13
of the C and DEPT-135 NMR spectra showed that the compound contained nine quaternary C atoms and 24 methine,
14 methylene, and 6 methyl groups. Resonances of C atoms containing protons including four anomeric protons (ꢂ 5.34,
H
4.60, 4.48, and 4.43), five secondary C atoms (ꢂ 67.1, 66.4, 65.5, 62.6, 62.4), two carbonyl groups (ꢂ 178.8 and 177.6), and
C
C
two tri-substituted double bonds (ꢂ 144.6 and 124.4) were identified in the HSQC spectrum. HMBC and COSY spectra
C
confirmed the presence in the molecule of the aglycon phytolaccagenin [7].
The sites of attachment of the sugar moieties to the aglycon were established by HMBC and ROESY experiments; the
anomeric configurations, by coupling constants, the values of which were >7 Hz.
Based on the aforementioned, we proposed the structure of glycoside I as 3-O-(ꢀ-D-xylopyranosyl-(1ꢁ3)-ꢀ-D-
galactopyranosyl-(1ꢁ3)-ꢀ-D-xylopyranosyl)-28-O-ꢀ-D-glucopyranosyl phytolaccagenin, which was a new compound.
765

EXPERIMENTAL
Isolation and Purification. Purified total triterpene saponins were obtained by first treating air-dried ground fruit of
P. americana with CHCl to remove fats and slightly polar substances from the raw material and then extracting with EtOH
3
(96%). The EtOH was distilled off. The aqueous residue was extracted with EtOAc. The dried EtOAc extract was passed over
a column of Diaion LH-20 to produce three 3-4-component low-polarity, medium-polarity, and polar fractions. The polar
fraction was separated over a column of Kieselgel-60 (Merck) with elution by CHCl :CH OH:H O (26:14:3). The final
3
3
2
purification of the glycosides from ballast impurities was carried out using preparative chromatography on an Agilent 1100
series prep instrument (20 ꢄ 250 mm, Intertsil prep-ODS column using UV detection, MWD, wavelength 254 nm, bandwidth
100, H O 45%, MeOH 55%). Two glycosides H and I were isolated. The purity of these was checked on an analytical HPLC
2
(Agilent 1100, 6.0 ꢄ 250 mm, Intertsil prep-ODS column using DAD, UV-MSD, H O 45%, MeOH 55%).
2
1
NMR spectra were recorded on a Bruker Avance instrument at operating frequency 400 MHz for H and 100 MHz for
C. The structures of the glycosides were elucidated by analyzing H, C, COSY, TOCSY, HMBC, HSQC, and DEPT-135
13
1
13
spectra. Chemical shifts (ꢂ) are given in ppm relative to TMS.
TLC monitoring of sapogenins and monosaccharides was performed on silica gel plates (Silica gel 60 F , Merck)
254
using solvent systems CHCl :MeOH:H O (1, 26:14:3), BuOH:HOAc:H O (2, 4:1:5), CHCl :MeOH (3, 20:1),
3
2
2
3
CH Cl :MeOH:H O (4, 50:25:5). Aglycons were detected by phosphotungstic acid solution (25%) in EtOH; monosaccharides,
2
2
2
by anilinium biphthalate. Chromatograms were heated to 100-150°C after treatment with the detecting reagent.
TotalAcid Hydrolysis. Glycoside (3 mg) was placed into a flask, treated with HCl solution (3 mL, 10%), and heated
at 100°C for 4 h. Sapogenins were extracted with Et O. The aqueous part was neutralized by N,N-dioctylmethylamine (10%
2
in CHCl ). Sugars, i.e., glucose, galactose, and xylose, were identified in both instances on TLC with standards using system
3
4; sapogenins, system 3.
Alkaline Hydrolysis. Glycoside (5 mg) was placed into a flask, treated with aqueous KOH (5 mL, 5%), heated at
100°C for 90 min, and neutralized with HCl (10%) to pH 5. Prosapogenins were extracted by 1-BuOH and then analyzed on
TLC using systems 1 and 2.
Glycoside I, white amorphous powder, C H O , based on a peak for the pseudomolecular ion 1143.5202
53 84 25
+
25
[M + Na] (HR-ESI-MS high-resolution mass spectrum), [ꢅ] +4.87° (c 1, MeOH). Tables 1 and 2 present the NMR spectral
D
data.
ACKNOWLEDGMENT
The work was performed with partial financing by a Presidential Grant for Young Scientists (GNSF/PRES09_109_6-
40; No. 2-6/20).
REFERENCES
1.
2.
3.
4.
Flora of Georgia [in Russian], II, Vol. 4, Metsniereba, Tbilisi, 1978, p. 9.
Plant Resources of the USSR, Family Phytolaccaceae [in Russian], Nauka, Leningrad, 1985, pp. 175–176.
Y. Suga, Y. Maruyama, S. Kawanishi, and J. Shoji, Chem. Pharm. Bull., 26, No. 2, 520 (1978).
L. Wang, L. Bai, T. Nagasawa, T. Hasegawa, X. Yang, J.-I. Sakai, Y. Bai, T. Kataoka, S. Oka, K. Hirose,
A. Tomida, T. Tsuruo, and M. Ando, J. Nat. Prod., 71, 3535 (2008).
5.
M. Z. Getiya, V. D. Mshvildadze, S. Lavoie, J. Legot, A. Pichette, and G. E. Dekanosidze, Izv. Nats. Akad.
Nauk Gruzii, 34, No. 2, 202 (2008).
6.
7.
A. Strauss, S. M. Spengel, and W. Shaffner, Phytochemistry, 38, No. 4, 861 (1995).
W. S. Woo, S. S. Kang, K. Yamasaki, and O. Tanaka, Arch. Pharm. Res. 1, No. 1, 21 (1978).
766