Hydrolyzed tannins from Geranium pusillum

Article abstract of DOI:10.1023/A:1025418401804

The polyphenolic compound 1-O-galloyl-3,6-hexahydroxybiphenyl-D- galactopyranoside (pusilagin) was isolated from the aerial part. The tannin structure was established using acid hydrolysis and IR, PMR, and ¹³C NMR spectral methods.

Full text of DOI:10.1023/A:1025418401804

Chemistry of Natural Compounds, Vol. 39, No. 3, 2003
HYDROLYZED TANNINS FROM
Geranium pusillum
K. B. Kobakhidze and M. D. Alaniya
UDC 547.56+547.985
The polyphenolic compound 1-O-galloyl-3,6-hexahydroxybiphenyl-D-galactopyranoside (pusilagin) was
isolated from the aerial part. The tannin structure was established using acid hydrolysis and IR, PMR, and
13C NMR spectral methods.
Key words:
L., tannin monomer, pusilagin.
Geranium pusillum
The genus
L. is represented by 23 species in the flora of Georgia. These are widely used in folk medicine
Geranium
as hemostatic and wound-healing agents [1].
Species of are known to be rich in flavonoids and hydrolyzed tannins [2]. We previously reported the
Geranium
isolation and identification of phenolic acids and flavonoids from the aerial part [3, 4]. This article presents results from a study
of polyphenols of . The air-dried aerial part was exhaustively extracted with ethanol (70%). Phenolic compounds
G. pusillum
were extracted from the aqueous residue byethylacetate. The ethylacetate fraction was separated over a polyamide column with
1
subsequent purification over Sephadex LH-20 to afford , which was an ellagotannin [5].
1
Compound
forms platelike light-brown crystals, mp 202-206°C. The UV spectrum exhibits maxima at 218 and
-1
270 nm. The IRspectrum has absorption bands at 3200 (OH), 1695 (>C=O), and 1610 and 1580 cm , indicative of its phenolic
OH
nature.
HO
OH
OH
HO
C
O
O
C
O
C
O
CH
2
HO
HO
O
O
O
HO
HO
OH
OH
1
+
H
COOH
CH OH
2
O
O
OH
HO
O
HO
HO
OH
HO
OH
OH
OH
OH
OH
O
O
2
3
4
The PMR spectrum contains signals at 7.04 ppm for galloyl and at 6.65 and 6.68 ppm for hexahydroxybiphenyl
13
(HHBP). Resonances from 6.35 to4.15 ppm were assigned to carbohydrate protons. The PMR and C NMR spectra confirmed
1
the structure of (Table 1).
I. G. Kutateladze Institute of Pharmaceutical Chemistry, Academy of Sciences of Georgia, fax (99532) 25 00 26.
Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 204-205, May-June, 2003. Original article submitted January 6,
2003.
0009-3130/03/3903-0262$25.00 ©2003 Plenum Publishing Corporation
262

TABLE 1. 13C NMR Data for Pusilagin (CD3OD)
C
CS, ppm
C
CS, ppm
Galactose
HHBP
1
2
3
4
95.06
69.47
71.64
65.04
76.22
62.50
ring B
1
2
3
4
5
6
125
110.22
145.66
138.20
145.33
117.23
168.55
5
6
Galloyl
1
120.65
111.0
7 (C=O)
ring C
2.6
3.5
4
7 (C=O)
146.41
140.43
166.72
1
2
3
4
5
6
125.54
108.37
145.24
137.70
146.08
116.72
170.14
7 (C=O)
Compound 1 was hydrolyzed by H SO (5%) togive gallic (2) and ellagic (3) acids and D-galactose (4) in a 1:1:1 ratio.
2
4
Quantitative determination of gallic acid used a colorimetric method [6]; of ellagic, gravimetric [7]; of D-galactose, semimicro
sugar determination [8].
The experimental data and a comparison with the literature leads to the conclusion that the tannin isolated by us has
a structure analogous to corilagin but with D-galactose instead of D-glucose in the carbohydrate part. An analogous compound
has not been described in the literature. We named it pusilagin.
EXPERIMENTAL
Tannin was isolated and purified using polyamide prepared by the literature method [9] and Sephadex LH-20
(Pharmacia, Sweden). Paper chromatography (PC) used FN012 grade paper (Germany) and the following solvent systems: n-
butanol:acetic acid:water (4:1:2, 1), pyridine:benzene:butanol:water (3:1:5:3, 2), acetic acid (5%, 3; 15%, 4). The melting point
was determined on a Kofler block. The UV spectrum was recorded on a SF-16 spectrophotometer; IR spectrum, on a UR-20
13
1
13
apparatus in KBr disks; PMR and C NMR, on a Bruker Avance DRX-400 ( H, 400 MHz; C, 100.6 MHz) in CD OD.
3
Isolation. Air-dried and ground raw material (500 g) from the aerial part of G. pusillum L. collected during massive
flowering in Goriisk region of Georgia was exhaustively extracted with ethanol (70%, 1:10 ratio). After alcohol was removed,
the aqueous fraction was purified of lipophilic compounds with CHCl (1:1 ratio) until the CHCl layer was colorless. Phenolic
3
3
compounds were extracted from the aqueous fraction with ethylacetate(1:1). Thecombinedfractionswereevaporatedtodryness
in vacuum to afford the total polyphenols (16 g, 3.2%). A portion of these (2.6 g) was chromatographed over a polyamide
column (d = 3 cm, h = 53 cm) with elution first by water and then by aqueous methanol (with increasing methanol
concentration). The course of the separation was monitored byPC using system 1. Fractions eluted by15% methanol contained
mainlyone compound with R 0.39 ± 0.1. The fractions were combined, concentrated, and rechromatographed over a Sephadex
f
LH-20 column with elution by water to afford the pure compound (0.094 g, 0.116% yield based on air-dried raw material).
Compound 1 forms light-brown plates; mp 202-206°C; soluble in diluted methanol, ethanol, and acetone; R 0.39 ±
f
0.1 (system 1); 0.41 (system 3); 0.50 (system 4).
Found (%): C 49.7, H 3.45, O 43.9; C H O .
27 22 18
Calc. (%): C 51.1, H 3.47, O 45.42; MW 634.
263

-1
UV spectrum (λ, nm): 218, 270. IR spectrum (ν, cm ): 3200 (OH), 1695 (>C O), 1610 (phenyl), 1580 (–C=C–), 1200
(–C–O–C–).
PMR spectrum (δ, ppm, J/Hz): 6.35 (1H, d, J = 1.6, H-1 of D-galactose), 3.98 (1H, br.s, H-2), 4.80 (1H, br.s, H-3), 4.46
(1H, br.s, H-4), 4.51 (1H, t, J = 9.9, H-5), 4.95 and 4.15 (2H, t, J = 11, dd, J = 8, J = 11, CH -galactose), 7.04 (1H, s, H-2 of
2
galloyl), 6.68 (1H, s, H-5 of HHBP), 6.65 (1H, s, H-5 of HHBP).
13
Table 1 contains the C NMR data.
Acid Hydrolysis. A solution of 1 (0.25 g) was treated with H SO (10 mL, 5%) and boiled for 3 h. The reaction
2
4
mixture was cooled. The resulting precipitate of ellagic acid was filtered off, washed with water, and dried over anhydrous
CaCl . Yield 0.1 g (83.3% of its content in C H O ). Gallic acid was determined photocolorimetricallyas 0.052 g or 86.7%
2
27 22 18
of its content in C H O . Galactose in the hydrolysate was determined by a semimicro sugar analysis [8]. Treatment of the
27 22 18
hydrolysate with KMnO formed copper oxide (141 mg), which corresponds to galactose (62 mg, 88.83% of its content in
4
C H O ).
27 22 18
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