New oligomeric proanthocyanidins from Alhagi pseudalhagi

Article abstract of DOI:10.1007/s10600-010-9615-4

Two new oligomeric proanthocyanidin glucosides were isolated from the aerial part and roots of Alhagi pseudalhagi. Their structures and relative configurations were elucidated as 7-O-β-D-Glc p→⁶ galloyl-(+)catechin-(4α-8)-(+)-catechin-(4α-8)-(-

Full text of DOI:10.1007/s10600-010-9615-4

Chemistry of Natural Compounds, Vol. 46, No. 3, 2010
NEW OLIGOMERIC PROANTHOCYANIDINS FROM Alhagi pseudalhagi
D. F. Alimova, Z. A. Kuliev,† S. Z. Nishanbaev,
A. D. Vdovin, N. D. Abdullaev, and S. F. Aripova*
UDC 547.972
Two new oligomeric proanthocyanidin glucosides were isolated from the aerial part and roots of Alhagi
6
pseudalhagi. Their structures and relative configurations were elucidated as 7-O-ꢀ-D-Glcpꢁgalloyl-(+)-
6
catechin-(4ꢂ-8)-(+)-catechin-(4ꢂ-8)-(–)-epigallocatechin and 7-O-ꢀ-D-Glcpꢁgalloyl-(+)-catechin-[(4ꢂ-
8)-(+)-catechin]3-(4ꢂ-8)-(–)-epigallocatechin-3-O-gallate.
Keywords: Alhagi pseudalhagi, Fabaceae, oligomeric proanthocyanidins, isolation, structure, NMR spectroscopy.
We isolated 12 pure compounds from the total extractable compounds in the aqueous alcohol extract of Alhagi
pseudalhagi (Bieb.) Fisch by using column chromatography over silica gel and microcrystalline cellulose in addition to gel
chromatography over Sephadex LH-20 [1]. Of these, six compounds were identified as monomeric catechins; four, known
dimeric proanthocyanidins [2]. Two compounds were believed to be oligomeric acylglycosylated proanthocyanidins that we
called alhacin (I) and alhacidin (II) on the basis of UV and IR spectra.
COOH
COOH
CH OR
2
OH
O
OH
OH
HO
OH
OH
4: R = H
5: R = Galloyl
HO
OH
HO
OH
OH
OH
4, 5
1
3
2
OH
OH
5'
1'
HO
O
+
R O
1
7
O
2
4
OH
-
OH
OH
3'
a
OH
OH
5
OH
OH
+
OH
H
OH
6
HO
O
C
B
OH
HO
O
n
OH
A
OH
O
b, c, d
OH
OH
C H SH
6 5
OR
OH
HO
OH
7: R = H
10: R = Galloyl
+
OH
OH
7, 10
H
e
OR
OH
OH
OH
HO
O
S
I, II
OH
Raney Ni
HO
O
OH
CH I
3
OH
OH
Permethylate
OH
+
OH
H
9
2,3,4-Tri-O-Me-D-glucopyranose
8
6
6
______
†Deceased.
I: R = H, R = GlcpꢁGalloyl; n = 1; II: R = Galloyl, R = GlcpꢁGalloyl; n = 3
1
1
S. Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan,
Tashkent, fax: (99871) 120 64 75, e-mail: salima_aripova@mail.ru. Translated from Khimiya Prirodnykh Soedinenii, No. 3,
pp. 297–301, May–June, 2010. Original article submitted February 25, 2008.
©
352
0009-3130/10/4603-0352 2010 Springer Science+Business Media, Inc.

13
TABLE 1. Chemical Shifts of Resonances in the C NMR Spectrum of I (ppm)
of fragments
e
ꢄC
C atom
a
b
Glucose
Galloyl
Ñ-2
83.41
73.54a
38.81
96.03
96.03
81.84
73.81a
38.81
79.31
65.41
–
Ñ-3
Ñ-4
C-6
96.03
96.03
Ñ-8
107.44
102.74
153.84–155.50
130.64
114.94
143.83
143.83
115.21
118.40
107.44
102.74
153.84–155.50
130.64
109.61b
145.02
133.80
145.02
109.61b
Ñ-10
Ñ-5, 7, 9
102.74
153.84–155.50
130.64
114.94
143.83
143.83
115.21
118.40
Ñ-1
ꢃ
102.74
72.81a
77.34
70.52
75.53
63.10
119.91
109.61b
145.02
138.84
145.02
109.01b
168.38
C-2
ꢃ
C-3
ꢃ
C-4
ꢃ
C-5
ꢃ
C-6
ꢃ
-COO-
______
Resonances denoted by the same superscripts may be reversed.
22
The proanthocyanidin alhacin was optically active, [ꢂ] +13.2° (c 0.37, EtOH), and decomposed at 290–300°C
D
without melting. Its molecular weight (MW ~ 1210–1220) and composition (C H O ) were determined by sedimentation
58 52 29
equilibrium in an ultracentrifuge. Gel filtration over a calibrated column of Sephadex LH-20 confirmed these results.
Several chemical transformations were carried out in order to determine the monomer composition and elucidate the
structure. Alkaline cleavage of alhacin under a N atmosphere formed three compounds that were identified using
2
physicochemical properties as phloroglucinol (1) and protocatechoic (2) and gallic (3) acids [3–5].
Acid hydrolysis of alhacin formed (-)-epigallocatechin (7), cyanidin (6), ꢀ-glucose (4), and galloylglucose (5) [6, 7].
Mild thiolytic cleavage in the presence of thiophenol and acetic acid produced from the “lower” block of alhacin
(–)-epigallocatechin (7); from the “upper” block, thioether 8, which was destroyed catalytically in the presence of Raney
nickel [8]. The resulting compound was identified as (+)-catechin (9).
Thus, the chemical analysis showed that proanthocyanidin alhacin was a glycosylated oligomeric proanthocyanidin
consisting of (+)-catechin and (–)-epigallocatechin.
13
In fact, the C NMR spectrum (Table 1) of alhacin that was taken with full proton decoupling exhibited resonances
characteristic of epigallocatechin, catechin, ꢀ-glucose, and gallic acid. Abroad resonance at 153.84–155.50 ppm was assigned
to C-5, C-7, and C-9. A resonance at 96.03 ppm belonged to unsubstituted C-6 and C-8 of phloroglucinol. Resonances at
109.01 and 109.61 ppm (C-2ꢃ and C-6ꢃ), 145.03 (C-3ꢃ and C-5ꢃ), and 133.80 (C-4ꢃ) were indicative of a gallocatechin block
whereas resonances at 114.94, 115.21, 118.40, and 143.84 were assigned to C-2ꢃ, C-5ꢃ, C-6ꢃ and C-3ꢃ, and C-4ꢃ, respectively,
and showed that alhacin had a catechin block. Resonances at about 81-83 (C-2) showed that the upper blocks of the
proanthocyanidin had the 2,3-trans orientation [6-13].
It is known that the resonance for C-10 appears at greater than 100.0 ppm if C-4–C-8 interflavane bonds are present.
For C-4–C-6 interflavane bonds, this resonance shifts to even stronger field [14].
Based on our results (chemical shift of C-10 of 102.74 ppm), it was found that alhacin had C-4–C-8 interflavane
bonds.
Resonances for glucose C-1, C-3, and C-5 at 102.74, 77.34, and 75.53 ppm, respectively, showed that the anomeric
center had the ꢀ-configuration. A resonance at 63.10 for a substituted glucose C-6 proved that the carbohydrate was acylated
in the 6-position [15, 16].
A D-glucose acylated by gallic acid in the 6-position was bonded to the aglycon by a ꢀ-glycoside bond. Mild
thiolytic cleavage of alhacin was consistent with the lack of a sugar in the lower block of the molecule. Considering this and
the steric hindrance in the “middle” block of the proanthocyanidin, we hypothesized that the most likely site of addition of the
acylated glucose was C-7 of the upper block.
353

13
TABLE 2. Chemical Shifts of Resonances in the C NMR Spectrum of II (ppm)
ꢄC of fragments
C atom
a
b, c, d
e
Glucose
Galloyl
Ñ-2
Ñ-3
83.11
73.21*
38.44
96.70
96.70
102.30
154.20
131.01
114.63a
144.33
144.33
115.90
119.10
81.63–82.34
72.80*
38.44
77.01
69.64
26.70
Ñ 4
-
C-6
96.70
96.70
Ñ-8
107.63
102.30
154.20
130.01
115.14a
144.33
144.33
115.90
119.10
107.63
102.30
154.20
131.01
108.94
144.33
133.60
144.33
108.94
Ñ-10
Ñ 5, 7, 9
-
Ñ 1ꢃ
-
102.30
73.21
77.01
71.72
75.82
63.20
121.73
109.83
144.33
139.10
144.33
109.83
167.21
C 2ꢃ
-
ꢃ
C-3
C-4ꢃ
C-5ꢃ
C-6ꢃ
-COO-
______
a
Resonances denoted by the same superscripts may be reversed.
*Resonance not observed because of overlap with the solvent resonance.
Thus, the chemical transformations and the physicochemical and spectral data reported above suggested for alhacin
6
the structure and relative configuration 7-O-ꢀ-D-Glcpꢁgalloyl-(+)-catechin-(4ꢂ-8)-(+)-catechin-(4ꢂ-8)-(–)-epigallocatechin.
22
Proanthocyanidin alhacidin (II) was optically active, [ꢂ] +21.9° (c 0.29, EtOH); mp 290–300°C (dec.),
D
13
MW = 1935–1945, C H O . The physicochemical properties and spectral parameters (UV, IR, C NMR) of II (Table 2)
95 80 45
were very similar to those of I. The difference was that II had a higher molecular weight. Based on this, proanthocyanidin II
also consisted of (+)-catechin and (–)-epigallocatechin blocks and had an acylated glucose.
13
The C NMR of II had resonances at 77.01 and 26.70 ppm for C-2 and C-4 of the heterocycle. This showed that
the lower block had a galloyl substituent in the C-3 position. There was also a C-4–C-8 interflavane bond (CS of C-10,
102.30 ppm). Chemical shifts of C-2 and C-4 of the upper and middle blocks indicated that II had the 2,3-trans configuration
and lacked an ester bond in the C-3 position [17].
The most probable location of the acylated glucose was C-7 of the upper block of II according to the same data as
for I.
Both proanthocyanidins (I and II) gave permethylates with an excess of methyliodide in the presence of NaH. These
were hydrolyzed to form the same product, 2,3,4-tri-O-methyl-D-glucopyranose.
6
Thus, proanthocyanidin alhacidin had the structure and relative configuration 7-O-ꢀ-D-Glcpꢁgalloyl-(+)-catechin-
[(4ꢂ-8)-(+)-catechin]3-(4ꢂ-8)-(–)-epigallocatechin-3-O-gallate.
EXPERIMENTAL
UV spectra of proanthocyanidins and their derivatives in EtOH were taken on a Perkin–Elmer Lambda-16 instrument;
IR spectra, in KBr disks on a Perkin–Elmer System 2000 FTIR instrument. Optical activity was determined using a Zeiss
13
polarimeter. C NMR spectra of compounds in deuteroacetone and deuterated water mixtures were recorded on a Bruker AM
400/100 MHz instrument. Molecular weights were determined on a MOM 3170 ultracentrifuge using gel chromatography
over a calibrated column of Sephadex LH-20. PC and TLC on Silufol UV-254 plates were used to identify compounds and
determine their purity. We used the following solvent systems: n-BuOH:AcOH:H O 40:12:28 (1), CHCl :EtOAc 1:2-4 (2),
2
3
n-BuOH:HCOOH (85%):H O 9.5:1:2 (3), HCl (2 N) (4), n-BuOH:Py:H O 6:4:3 (5). Detection used vanillin (1%) in alcoholic
2
2
H SO (5%); aqueous FeCl and K [Fe(CN) ] solutions (1%) (1:1); and anilinium phthalate.
2
4
3
3
6
Extraction and Isolation of Proanthocyanidins. Ground air-dried aerial part of A. pseudalhagi (6.0 kg) was extracted
with 80% EtOH (6 ꢅ 20 L). The resulting EtOH extracts were combined. The EtOH was vacuum distilled at 45–50°C. The
354

remaining thick extract (503.4 g, 8.9% of the air-dried raw material) was diluted with distilled water (1:1 v/v) and fractionated
successively according to polarity of the organic solvents Et O (500 ꢅ 4), EtOAc (500 ꢅ 4), and n-BuOH (500 ꢅ 4).
2
The Et O, EtOAc, and n-BuOH extracts were evaporated to afford 43.5 g (0.72%), 35.2 (0.58), and 127.6 (2.12),
2
respectively, of low-molecular-weight relatively slightly polar and polar total extracts. The remaining aqueous layer was
evaporated in a porcelain dish on a water bath with constant stirring and dried. The resulting thick residue was ground to
afford 297.1 g (4.95%) of total high-molecular-weight light-brown proanthocyanidins.
Separation of Proanthocyanidins. The n-BuOH extract (127 g) was mixed with cellulose (127 g), placed on a
column of microcrystalline cellulose (140 ꢅ 6 cm, 1700 g), and eluted with CHCl , CHCl :EtOAc, EtOAc, EtOAc:(CH ) CO,
3
3
3 2
(CH ) CO, and (CH ) CO:H O. Fractions of 100–150 mLwere collected. The elution was monitored using TLC. Homogeneous
3 2
3 2
2
fractions (according to TLC) were combined and rechromatographed over a column of Sephadex LH-20 (140 ꢅ 3 cm, 158 g).
22
Proanthocyanidin I, 0.820 g, C H O , MW 1210–1220, [ꢂ] +132° (c 0.37, EtOH). UV spectrum (ꢆ , nm):
58 52 29
D
max
–1
225, 229, 253, 271; ꢆ
200. IR spectrum (
, cm ): 3589, 3388, 2936, 1719, 1609, 1513, 1450, 1271, 1165, 1077.
min
max
13
Table 1 lists the C NMR spectrum.
22
Proanthocyanidin II, 0.875 g, C H O , MW 1935–1945, [ꢂ] +21.9° (c 0.28, EtOH). UV spectrum (ꢆ , nm):
95 80 45
–1
D
max
201, 222, 275; ꢆ
200. IR spectrum (
, cm ): 3387, 2936, 1702, 1606, 1510, 1450, 1365, 1242, 1076, 1042.
min
max
13
Table 2 lists the C NMR spectrum.
Alkaline Cleavage of I. A 20-mL four-necked round-bottomed flask was charged with I (75 mg) and purged slowly
with N . KOH solution (50%, 5 mL) was added. The mixture was constantly stirred. The lower part of the flask was
2
immersed into a bath with a low-melting metallic alloy at 150–160°C that was heated over five minutes to 230°C. The mixture
was rapidly cooled by immersing the flask into icewater acidified with H SO (20%). The contents of the flask were diluted
2
4
with water and extracted with EtOAc. The EtOAc extract was dried over anhydrous Na SO . The solvent was distilled off.
2
4
+
The solid was chromatographed over a column of polyamide to afford three compounds with M 126, mp 218–219°C, R 0.64
f
+
+
(PC, system 3) (phloroglucinol); M 154, mp 200°C (dec.), R 0.72 (PC, system 3) (protocatechoic acid); and M 170,
f
mp 220–221°C, R 0.56 (PC, system 3) (gallic acid) [7, 18].
f
Alkaline cleavage of II (80 mg) was carried out by the method described for I to afford three compounds that were
identified as phloroglucinol (1) and protocatechoic (2) and gallic acids (3).
Acid Cleavage of I. Compound I (90 mg) was dissolved in EtOH (4 mL), treated with HCl (1.5 mL, 2 N), and
refluxed under N on a water bath for 2 h. The mixture was diluted with water and extracted with EtOAc. The extract was
2
washed with NaHCO solution and dried over anhydrous Na SO . The solvent was distilled off. The solid was chromatographed
3
2
4
over a column of Sephadex LH-20 with elution by EtOH (60%) to afford a compound (6 mg) of composition C H O ,
15 14
7
21
mp 215–216°C, [ꢂ] –56° (c 0.41, MeOH), R 0.42 (system 2) [(-)-epigallocatechin (7)] [7, 18].
D
f
Paper chromatography of the hydrolysate detected cyanidin (6), R 0.69 (system 4); glucose, R 0.51 (system 5); and
f
f
24
a compound with mp 135–137°C, [ꢂ] –26.5° (c 0.21, acetone) [galloylglucose (5)].
D
Acid cleavage of II (85 mg) was carried out analogously to that of I to afford a compound (5 mg) of composition
20
C H O , mp 210–211°C, [ꢂ] –135° (c 0.06, MeOH:H O, 1:1), R 0.6 (system 2) [(-)-epigallocatechin-3-O-gallate (10)].
22 18 10
D
2
f
Paper chromatography of the hydrolysate detected cyanidin (6), R 0.69 (system 4); glucose, R 0.51 (system 5); and a compound
f
f
24
with mp 135–137°C, [ꢂ] –26.5° (c 0.21, acetone) [galloylglucose (5)].
D
Thiolytic Cleavage of Proanthocyanidins. Cleavage of I. Compound I (180 mg) and thiophenol (4 mL) were
mixed, treated with HOAc (3 mL) in EtOH (10 mL), and left at room temperature for 48 h. The course of the reaction was
monitored by TLC. The reaction mixture was condensed. The resulting oily residue was chromatographed over Sephadex
21
LH-20 (elution by EtOH) to afford a compound (7 mg), C H O , mp 215–216°C, [ꢂ] –56° (c 0.41, MeOH), R 0.42
15 14
7
D
f
(system 2) [(-)-epigallocatechin (7)] and an amorphous substance (50 mg) of total thioethers [7, 18].
Cleavage of II. Compound II (190 mg) was cleaved and the reaction products were purified as described above. The
reaction mixture was chromatographed over Sephadex LH-20 (60%) to afford a compound (15 mg) of composition C H O ,
22 18 10
20
mp 210–211°C, [ꢂ] –135° (c 0.06, MeOH:H O, 1:1), R 0.6 (system 1) [(–)-epigallocatechin-3-O-gallate (10)] and a thioether
D
2
f
mixture (68 mg).
Cleavage of Thioethers I and II. Thioethers (68 mg) were mixed with an EtOH:HOAc mixture (3.5 mL, 9:1),
treated with catalyst (Raney Ni), and held at 50°C for 1 h. The reaction mixture was filtered. The filtrate was condensed and
chromatographed over Sephadex LH-20 (elution by 80% EtOH) to afford a compound of composition C H O , mp 178–
15 14
6
20
179°C, [ꢂ] +18.6° (c 0.2, acetone:H O, 1:1), R 0.64 (system 1) [(+)-catechin (9)].
D
2
f
355

Methylation of I. A solution of I (80 mg) in DMSO (10 mL) was stirred, treated with NaH (0.10 g), held at room
temperature for 1 h, treated dropwise with MeI (5 mL), stored for another 4 h, poured into icewater (30 mL), and extracted
with CHCl . The extract was worked up with sodium thiosulfate, washed with water, and dried over anhydrous Na SO . The
3
2
4
solvent was distilled off. The solid was methylated another five times. The final product was purified by chromatography to
afford the amorphous permethylate (63 mg).
Hydrolysis of the Permethylate of I. The resulting permethylate (63 mg) was dissolved in aqueous MeOH (5 mL,
50%) containing H SO (5%), heated for 8 h on a water bath, cooled, neutralized with BaCO , and filtered to remove the
2
4
3
precipitate of BaSO . The filtrate was evaporated to dryness. The solid was purified by column chromatography to afford the
4
methylated carbohydrate (28 mg) that was identified by comparison with an authentic sample of 2,3,4-tri-O-methyl-D-
glucopyranose.
Methylation of II. The methylation was carried out analogously as above. The resulting permethylate (58 mg) was
hydrolyzed to afford 2,3,4-tri-O-methyl-D-glucopyranose.
Enzymatic Hydrolysis of I and II. The glycoside (20 mg) was dissolved in H O (10 mL), treated with ꢀ-glucosidase,
2
placed into a thermostat, and held at 30°C for 6 h. Polyphenols were precipitated by adding lead acetate solution. Paper
chromatography of the filtrate detected glucose (R 0.51, system 5).
f
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