6″-Galloylpicein and other phenolic compounds from Arctostaphylos uva-ursi

Article abstract of DOI:10.1007/s10600-013-0491-6

Phenolic compounds from leafy shoots of A. uva-ursi (Ericaceae) were studied. The new phenolic glycoside 6″-galloylpicein and 40 known compounds were isolated. Roots of A. uva-ursi afforded 16 compounds. A C-glycoside of bergenin was found for the first time in the family Ericaceae. The dominant components of A. uva-ursi leaves according to HPLC were arbutin, (+)-catechin, and corilagin; of stems, picein and (+)-gallocatechingallate; of roots, (-)-epicatechin, (-)-epicatechingallate, and (+)-catechin.

Full text of DOI:10.1007/s10600-013-0491-6

Chemistry of Natural Compounds, Vol. 49, No. 1, March, 2013 [Russian original No. 1, January–February, 2013]
6ꢀ-GALLOYLPICEIN AND OTHER PHENOLIC
COMPOUNDS FROM Arctostaphylos uva-ursi
*
D. N. Olennikov and G. V. Chekhirova
UDC 582.912.4:547.918
Phenolic compounds from leafy shoots of A. uva-ursi (Ericaceae) were studied. The new phenolic glycoside
6ꢀ-galloylpicein and 40 known compounds were isolated. Roots of A. uva-ursi afforded 16 compounds.
A C-glycoside of bergenin was found for the first time in the family Ericaceae. The dominant components of
A. uva-ursi leaves according to HPLC were arbutin, (+)-catechin, and corilagin; of stems, picein and
(+)-gallocatechingallate; of roots, (–)-epicatechin, (–)-epicatechingallate, and (+)-catechin.
Keywords: Arctostaphylos uva-ursi, Ericaceae, 6ꢀ-galloylpicein, phenolic glycosides, catechins, tannins, HPLC.
Leaves of Arctostaphylos uva-ursi (L.) Spreng. (Ericaceae) are an official plant raw material and are recommended
for use as a diuretic and disinfectant for inflammatory diseases of urinary pathways [1]. The leaves (gyaꢁ kyi ma in Tibetan)
were used in Buryatiya Tibetan medical practice for diseases related to increased stomach acidity (gastritis, heartburn), as an
antipyretic for measles and a sedative for neurasthenia, and as powders in agents for diseases of the endocrine system (Basedow’s
disease) [2]. Chemical studies showed the presence in the aerial part of A. uva-ursi of various groups of phenolic compounds
(simple phenols, phenolic glycosides, flavonoids, procyanidins, tannins) [3–5], triterpenes [6], polysaccharides [7], lipids [8],
and essential oil [9]. Data on the composition of the plant roots are limited to reports of the presence in them of unedoside
[10]. Usable reserves of A. uva-ursi within the Republic of Buryatiya are greater than 1,000 t/year [11]. Therefore, this region
was recommended for inclusion among the principal sites for collection of this plant species. Leafy plant shoots were proposed
as medicinal raw material in addition to the traditionally used leaves. This necessitated additional chemical studies.
The goal of our work was to study the composition of phenolic compounds from leafy shoots and roots of A. uva-ursi
growing in Buryatiya.
Fractionation and chromatographic separation of substances extracted from leafy shoots of A. uva-ursi by column
chromatography (CC) over polyamide, SiO , Sephadex LH-20, and preparative HPLC isolated the new phenolic glycoside 1
2
and 40 known compounds that were identified as arbutin (2), methylarbutin (3), 6ꢀ-galloylarbutin (4), picein (5), pyroside (6),
gallic acid (7), 1,6-di-O- (8), 3,4,6-tri-O- (9), and 1,2,3,4,6-penta-O-galloylglucoses (10), (+)-catechin (11), (–)-epicatechin
(12), (–)-epigallocatechin (13), (+)-gallocatechingallate (14), (–)-epigallocatechingallate (15), (–)-epicatechingallate (16),
(–)-epigallocatechinmethylgallate (17), (–)-epigallocatechindigallate (18), procyanidins B1 (19) and B2 (20), corilagin (21),
chebulagic acid (22), caffeic acid (23), 2-O- (24), 3-O- (25), 4-O- (26), 1,3-di-O- (27), and 3,5-di-O-caffeylquinic acids (28),
cinnamic (29), 2-hydrocinnamic (30), 2-methoxycinnamic (31), ferulic (32), isoferulic acids (33), quercetin (34) and its
glycosides hyperoside (35), 6ꢀ-galloylhyperoside (36), isoquercitrin (37), avicularin (38), quercitrin (39), rutin (40), and
quercetin-3-O-gentiobioside (41).
Compound 1 was an amorphous white powder. The molecular formula according to HR-ESI-MS was C H O
21 22 11
+
{m/z 473.361 ([M + Na] ; calcd 473.389)}. The acid hydrolysis products of 1 contained an equimolar mixture of
p-hydroxyacetophenone, gallic acid, and glucose. Treatment of the compound with tannase formed picein (5) and gallic
acid. This indicated that the structure of 1 was probably a picein derivative substituted by gallic acid in the carbohydrate.
The site of attachment of the gallic acid of 1 was determined by exhaustive methylation by dimethylsulfate, subsequent
formolysis, and analysis of the methylation products by GC/MS. It was established that the hydrolysis products contained
2,3,4-tri-O-Me-Glcp, which indicated that the glucopyranose was acylated only in the C-6 position because the
p-hydroxyacetophenone group was located on C-1. The PMR spectrum exhibited two resonances of a p-substituted benzene
ring (A B -type) as 2H doublets at 7.15 and 8.03 ppm that were assigned to H-2, H-6 and H-3, H-5, respectively (Table 1).
2
2
Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, 670047, Ulan-Ude,
Ul. Sakhꢁyanovoi, 6, fax: (3012) 43 47 43, e-mail: oldaniil@rambler.ru. Translated from Khimiya Prirodnykh Soedinenii,
No. 1, January–February, 2013, pp. 5–10. Original article submitted November 27, 2012.
©
0009-3130/13/4901-0001 2013 Springer Science+Business Media New York
1

13
TABLE 1. PMR (500 MHz) and C NMR (125 MHz) Spectra of 1 and 5 (DMSO-d , ꢃ, ppm, J/Hz)
6
ꢃÍ
ꢃÑ
C atom
1
5
1
5
-Hydroxyacetophenone
–
p
1
2
3
4
5
6
7
8
–
132.8
121.5
119.3
163.0
120.3
121.5
202.9
27.5
132.9
121.7
119.2
162.8
119.2
121.7
202.3
27.4
8.03 (2H, d, J = 9.2)
7.15 (2H, d, J = 9.2)
–
7.15 (2H, d, J = 9.2)
8.03 (2H, d, J = 9.2)
–
7.92 (2H, d, J = 9.2)
7.12 (2H, d, J = 9.2)
–
7.12 (2H, d, J = 9.2)
7.92 (2H, d, J = 9.2)
–
2.63 (3H, s)
2.60 (3H, s)
Gallic acid
1ꢁ
–
–
–
–
–
–
–
–
122.3
109.8
145.3
139.8
145.3
109.8
168.5
–
–
–
–
–
–
–
2
ꢁ
3
ꢁ
4
ꢁ
5
ꢁ
7.25 (2H, s)
–
–
–
6ꢁ
7.25 (2H, s)
–
7
ꢁ
-Glucopyranose
ꢂ
1
2
3
4
5
6
4.78 (1H, d, J = 7.4)
3.31–3.69 (4H, m)
4.81 (1H, d, J = 7.4)
102.7
75.2
78.9
72.4
76.0
65.8
102.5
75.3
78.8
72.0
78.2
62.3
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
3.29–3.82 (4H, m)
3.85 (1H, dd, J = 2.1, 12.2)
4.04 (1H, dd, J = 6, 12.2)
3.89 (1H, dd, J = 2.1, 12.2)
4.02 (1H, dd, J = 6, 12.2)
OH
HO
OH
6'
2'
7'
O
O
6''
O
OH
HO
HO
O
1
3
1''
3''
O
CH
5
1
3
The presence of a 3H singlet at 2.63 ppm showed that the acyl substituent on the benzene ring was an acetyl. Resonances
of the carbohydrate part were located in the range 3.31–4.78 ppm and included a doublet at 4.78 ppm belonging to the
anomeric proton. The positions and multiplicities of protons in the range 3.31–3.69 ppm were characteristic of glucosyl
13
methine protons. The presence of a gallic acid group was confirmed by a 2H singlet at 7.25 ppm. The C NMR spectrum was
similar to that of 5 and contained additional resonances due to the presence of the galloyl group in the compound (109.8,
122.3, 139.8, 145.3, 168.5 ppm) (Table 1). A weak-field shift of the C-6ꢀ resonance (+3.5 ppm) relative to that of 5 confirmed
that the substituent was located in this position. A strong-field shift was observed for the resonance of C-5ꢀ (–2.2 ppm),
indicating that neighboring atom C-6ꢀ was substituted. The position of the resonance for glucopyranose C-1ꢀ (102.7 ppm) in
13
the C NMR spectrum and the size of the spin–spin coupling constant of the resonance for the anomeric proton (J = 7.4 Hz)
were characteristic of the ꢂ-configuration of the carbohydrate.
Thus, the results indicated that 1 was 4-hydroxyacetophenone-4-O-ꢂ-(6ꢀ-galloyl)glucopyranoside (6ꢀ-galloylpicein),
was a new natural compound, and was the first acylated picein derivative.
Shoots and leaves of A. uva-ursi had previously afforded 2, 3, 4 [12], 5 [10], 7, 10 [13], 11 [5], 20 [14], 21 [15], 23
[16], 34, 35, 37–41 [4], and 36 [17]. Compounds 6, 8, 9, 12–19, 22, 24–33 were found for the first time in this plant.
Compounds 17, 18, 22, 30, 31, and 33 were isolated for the first time from the family Ericaceae.
2

a,b
TABLE 2. Content of Phenolic Compounds in Morphological Groups of A. uva-ursi
Compound
Leaves
Stems
Roots
Simple phenols, mg/g
82.40
+
Arbutin
6ꢁꢁ-Galloylarbutin
18.69
Tr.
0.81
–
c
12.32
–
2.66
–
68.12
+
0.89
–
1.07
Tr.
0.03
1.05
Picein
6ꢁꢁ-Galloylpicein
Gallic acid
Bergenin
Galloylglucoses, mg/g
1,6-Di- -galloylglucose
4.93
10.09
23.52
4.19
3.22
2.82
0.07
0.18
Tr.
O
3,4,6-Tri- -galloylglucose
O
1,2,3,4,6-Penta- -galloylglucose
O
Catechins, mg/g
(+)-Catechin
(–)-Epicatechin
(–)-Epigallocatechin
(–)-Epicatechingallate
(+)-Gallocatechingallate
(–)-Epigallocatechingallate
(–)-Epigallocatechinmethylgallate
(–)-Epigallocatechindigallate
64.40
Tr.
15.76
–
7.96
23.92
+
17.38
10.24
9.19
5.48
26.44
5.59
+
1.45
8.18
–
2.89
–
–
+
–
Tr.
–
Tannins, mg/g
Corilagin
Chebulagic acid
57.51
15.07
15.03
4.12
0.66
Tr.
Phenylpropanoids, ꢄg/g
Caffeic acid
354.78
31.73
95.20
23.57
Tr.
861.36
3578.24
202.55
195.17
Tr.
79.33
Tr.
Tr.
–
–
–
–
–
Tr.
–
–
–
2- -Caffeylquinic acid
O
3- -Caffeylquinic acid
37.77
141.87
245.63
262.87
3152.60
Tr.
Tr.
76.23
Tr.
O
4- -Caffeylquinic acid
O
1,3-Di- -caffeylquinic acid
O
3,5-Di- -caffeylquinic acid
Cinnamic acid
O
2-Hydroxycinnamic acid
2-Methoxycinnamic acid
Ferulic acid
Isoferulic acid
497.69
–
Flavonoids, mg/g
Quercetin
Hyperoside
Isoquercitrin
Avicularin
Quercitrin
Rutin
Tr.
2.92
0.62
0.91
0.20
4.69
1.06
336.78
Tr.
1.12
0.64
0.50
0.24
2.82
1.51
202.23
–
Tr.
–
–
–
–
–
Quercetin-3- -gentiobioside
O
Total content of identified compounds,
mg/g, including:
simple phenols
galloylglucoses
catechins
tannins
phenylpropanoids
flavonoids
15.73
97.38
38.54
112.04
72.58
5.84
87.70
10.23
74.32
19.15
4.00
2.96
0.25
12.52
0.66
Tr.
10.40
6.83
Tr.
______
a
b
c
Of air-dried raw material mass; raw material I; –, absent; +, present; Tr.: trace < 10 ꢄg/g.
3

TABLE 3. Content of Phenolic Compounds in Shoots of A. uva-ursi, mg/g, of Air-Dried Raw Material Mass
Raw material sample
Compound
II
III
IV
V
VI
VII
VIII
IX
Arbutin
Picein
Gallic acid
52.28
8.68
2.60
80.59
7.47
2.71
79.61
12.32
1.61
81.29
7.18
2.56
88.13
7.12
2.67
86.56
6.92
2.27
67.86
8.65
3.16
64.34
8.55
2.35
1,6-Di- -galloylglucose
3.58
9.80
3.91
3.23
7.49
4.15
3.84
3.95
4.01
8.49
2.71
7.87
O
3,4,6-Tri- -galloylglucose
11.16
13.89
34.31
17.11
55.32
14.46
240.93
90.77
28.96
51.42
69.78
11.56
15.99
35.71
17.63
55.45
14.30
245.82
91.03
31.70
53.34
69.75
11.95
15.04
34.78
17.43
54.85
14.55
250.36
97.92
30.83
52.21
69.40
11.46
15.52
35.91
17.22
53.37
13.49
246.67
95.75
30.93
53.13
66.86
O
1,2,3,4,6-Penta- -galloylglucose
12.14
29.77
15.34
48.05
14.18
196.42
63.56
25.52
45.11
62.23
15.65
48.35
8.71
53.09
14.01
244.07
93.54
26.37
57.06
67.10
21.89
31.87
11.23
62.79
16.65
236.60
79.67
34.39
43.10
79.44
11.71
20.00
10.99
49.79
8.05
186.36
75.24
22.29
30.99
57.84
O
(+)-Catechin
(–)-Epicatechin
Corilagin
Chebulagic acid
Total content including:
simple phenols
galloylglucoses
catechins
tannins
HPLC was used to study the quantitative composition of phenolic compounds in stem and leaves of A. uva-ursi
(Table 2). The dominant groups of compounds in leaves were catechins (112.04 mg/g), simple phenols (97.38 mg/g), and
tannins (72.58 mg/g). Simple phenols (87.70 mg/g) and catechins (74.32 mg/g) typically dominated the stems. The nature of
the accumulation of pure compounds in morphological groups showed that high concentrations of arbutin (2, 82.40 mg/g),
(+)-catechin (11, 64.40 mg/g), and corilagin (21, 57.51 mg/g) were noted in leaves; picein (5, 68.12 mg/g) and
(+)-gallocatechingallate (14, 26.44 mg/g), in stems. Afeature of the phenylpropanoid complex was the dominance of cinnamic
acid derivatives (29, 30, 31), the total contents of which were 3.98 and 3.15 mg/g for leaves and stems, respectively. The
principal component of the caffeic acid derivatives was 3,5-di-O-caffeylquinic acid (28, 262.87–861.36 mg/g). The flavonoids
of A. uva-ursi included mono- and biosides of quercetin and were dominated by rutin (40, 2.82–4.69 mg/g), hyperoside (35,
1.12–2.92 mg/g), and quercetin-3-O-gentiobioside (41, 1.06–1.15 mg/g). The total content of unidentified phenolic compounds
in leaves was 336.78 mg/g; in stems, 202.23 mg/g.
Quantitative analysis of A. uva-ursi shoots for the presence of the dominant compounds in eight batches of raw
material from various regions of the Republic of Buryatiya showed that the content of simple phenols in them could be 63.56–
97.92 mg/g; of galloylglucoses, 22.29–34.39; of catechins, 30.99–57.06; and of tannins, 57.84–79.44 mg/g (Table 3). Arbutin
(2, 52.28–88.13 mg/g), corilagin (21, 48.05–62.79), and (+)-catechin (11, 20.00–48.35) were the species marker components
with the highest contents.
The study of phenolic compounds from A. uva-ursi roots led to the isolation of 16 compounds including 2, 5, 7, 8–12,
16, 17, 21–23, 29, 35, and bergenin (42). The presence of C-glycoside 42, which is characteristic of species from the family
Saxifragaceae (Bergenia, Saxifraga, etc.), was established for the first time for the family Ericaceae. According to HPLC,
the dominant components of A. uva-ursi roots were catechins (–)-epicatechin (12, 8.18 mg/g), (–)-epicatechingallate
(16, 2.89 mg/g), and (+)-catechin (11, 1.45 mg/g) (Table 2). The total contents of phenolic glycosides, galloylglucoses, and
tannins were 15.73, 0.25, and 0.66 mg/g, respectively.
EXPERIMENTAL
Leafy shoots of A. uva-ursi were collected near Ulan-Ude (28 Aug., 2011; 51°86ꢁ85ꢀ N, 107°56ꢁ06ꢀ E; raw material
No. I); on Barsk ridge (Mukhorshibir Region; 3 Aug. 2009; 51°32ꢁ04ꢀ N, 107°53ꢁ83ꢀ E; II); in Goryachinsk village (Pribaikal
Region; IGEB SB RAS experimental plantation; 7 Sept. 2010; 52°98ꢁ55ꢀ N, 108°29ꢁ03ꢀ E; III); in Goryachinsk village (Pribaikal
Region, wooded zone; 7 Sept. 2010; 52°98ꢁ63ꢀ N, 108°29ꢁ31ꢀ E; IV); in Pykhta gap (near Ulan-Ude; 15 Sept. 2011; 51°82ꢁ37ꢀ
N, 107°70ꢁ00ꢀ E; V); in Verkhnyaya Berezovka village (near Ulan-Ude; 9 Sept. 2012; 51°80ꢁ90ꢀ N, 107°57ꢁ92ꢀ E; VI); in
Mostovka town (Pribaikal Region; 29 Aug. 2011; 52°07ꢁ70ꢀ N, 107°00ꢁ08ꢀ E, VII); in Kucheger town (Kurumkan Region; 27
Aug. 2011; 54°88ꢁ52ꢀ N, 110°99ꢁ36ꢀ E; VIII); and in Ust-Barguzin village (Barguzin Region; 29 Aug. 2012; 53°40ꢁ91ꢀ N,
4

109°02ꢁ09ꢀ E, IX). Roots of A. uva-ursi were collected near Ulan-Ude (28 Aug. 2011; 51°86ꢁ85ꢀ N, 107°56ꢁ06ꢀ E). The
species was determined by Dr. Pharm. Sci. T. A. Aseeva (IGEB SB RAS). Samples of A. uva-ursi were preserved in the
IGEB SB RAS Herbarium (No. AEr/ae-14/52-24/0811, -16/22-17/0809, -21/14-54/0910, -21/14-55/0910, -11/63-05/0911,
-18/53-33/0912, -15/54-28/0811, -15/54-30/0811, and -20/47-25/0812).
Polyamide (Woelm), silica gel (SiO , Sigma), and Sephadex LH-20 (Pharmacia) were used for CC. Spectrophotometry
2
studies were carried out on an SF-2000 spectrophotometer (OKB Spektr); MS analysis, in an MAT 8200 high-resolution mass
spectrometer (Finnigan). NMR spectra were recorded on a VXR 500S NMR spectrometer (Varian). GC/MS analysis was
performed on a 5973 N chromatography mass spectrometer with an MSD 5973 N mass-selective detector (Agilent Technologies)
with a diffusion pump, an HP-5ms capillary column (30 m/250 ꢄm/0.25 ꢄm), He carrier gas (1 mL/min), vaporizer temperature
280°C, column 50°C (2 min), 50–200°C (4°C/min), 200–280°C (20°C/min), 280°C (isothermal, 5 min), ion source 170°C,
interface between the GC and mass-selective detector 280°C, ionizing-electron energy 70 eV, sample volume 1 ꢄL with stream
division 20:1.
Extraction and Fractionation. Ground leafy shoots (980 g, raw material I) was extracted with EtOH (70%) on a
boiling-water bath (1:20, 5ꢅ). The EtOH extract was concentrated to an aqueous residue that was extracted with hexane,
CHCl , EtOAc, and BuOH to afford five fractions: hexane (30.97 g, 3.2% yield of air-dried raw material mass), CHCl
3
3
(32.73 g, 3.3%), EtOAc (167.22 g, 17.1%), BuOH (189.14 g, 19.3%), and aqueous residue (124.46 g, 12.7%). An interfacial
solid (3.23 g, 0.33%) precipitated during treatment of the concentrated extract with hexane. It was identified as ursolic acid
and ꢆ-amyrin in a 1:9.2 ratio according to GC/MS.
The EtOAc fraction (104 g) was separated beforehand over a preconditioned polyamide cartridge (300 g) [18] with
elution by H O, EtOH (40% and 98%), and NH (0.5%) in EtOH. The aqueous fraction was chromatographed over Sephadex
2
3
LH-20 (2.5 ꢅ 70 cm) using CHCl :EtOH (100:0ꢇ70:30) and afforded arbutin (22.42 g, 2), methylarbutin (21 mg, 3),
3
6ꢀ-galloylarbutin (54 mg, 4) [12, 15], pyroside (6ꢀ-acetylarbutin, 14 mg, 6) [19], and gallic acid (42 mg, 7) [20]. Fractions
eluted by EtOH (40% and 98%) were separated over polyamide (4 ꢅ 80 cm, H O:EtOH, 100:0ꢇ4:98) and SiO (3 ꢅ 60 cm;
2
2
hexane:EtOAc, 100:0ꢇ70:30; EtOAc:Me CO, 100:0ꢇ70:30) and by preparative HPLC (conditions 1). The isolated compounds
2
were identified as 1,6-di-O- (92 mg, 8), 3,4,6-tri-O- (101 mg, 9), and 1,2,3,4,6-penta-O-galloylglucoses (117 mg, 10) [21];
(+)-catechin (57 mg, 11) [20]; (–)-epicatechin (5 mg, 12); (–)-epigallocatechin (62 mg, 13); (+)-gallocatechingallate (37 mg,
14) [22]; corilagin (2.87 g, 21) [23]; chebulagic acid (219 mg, 22) [24]; cinnamic (14 mg, 29), o-coumaric (2-hydroxycinnamic,
3 mg, 30), 2-methoxycinnamic (6 mg, 31), ferulic (19 mg, 32), and isoferulic acids (12 mg, 33) [25]; quercetin (7 mg, 34) [20];
hyperoside (quercetin-3-O-galactoside, 53 mg, 35) [26]; 6ꢀ-galloylhyperoside [quercetin-3-O-(6ꢀ-galloyl)-galactoside, 7 mg,
36] [27], isoquercitrin (quercetin-3-O-glucoside, 11 mg, 37) [28]; avicularin (quercetin-3-O-arabinoside, 17 mg, 38), and
quercitrin (quercetin-3-O-rhamnoside, 5 mg, 39) [26]. The fraction eluted by NH (0.5%) in EtOH was chromatographed over
3
polyamide (4 ꢅ 90 cm, H O:EtOH, 100:0ꢇ4:98) to isolate caffeic acid (14 mg, 23) [20] and 2-O- (9 mg, 24), 3-O- (8 mg, 25),
2
4-O- (8 mg, 26), 1,3-di-O- (5 mg, 27), and 3,5-di-O-caffeylquinic acids (63 mg, 28) [29].
The BuOH fraction (125 g) was separated analogously. The aqueous fraction obtained after elution of the polyamide
cartridge underwent CC over SiO (3 ꢅ 60 cm; hexane:EtOAc, 100:0ꢇ70:30; EtOAc:Me CO, 100:0ꢇ70:30) and preparative
2
2
HPLC (conditions 1) to yield picein (7.56 g, 5) [19] and 1 (14 mg). The alcohol-soluble eluates contained
(–)-epigallocatechingallate (17 mg, 15), (–)-epicatechingallate (21 mg, 16), (–)-epigallocatechinmethylgallate (5 mg, 17) [30],
(–)-epigallocatechindigallate (9 mg, 18) [31], procyanidins B1 (33 mg, 19) and B2 (20 mg, 20) [32], rutin (115 mg, 40) [28],
and quercetin-3-O-gentiobioside (51 mg, 41) [33].
Fractionation and chromatographic separation of A. uva-ursi root (320 g) extract isolated 2 (22 mg), 5 (37 mg), 7
(5 mg), 8 (9 mg), 9 (14 mg), 10 (4 mg), 11 (22 mg), 12 (31 mg), 16 (15 mg), 17 (4 mg), 21 (37 mg), 22 (6 mg), 23 (4 mg), 29
(3 mg), 35 (2 mg), and bergenin (97 mg, 42 ) [34].
6ꢀ-Galloylpicein (1). C H O . UV spectrum (MeOH, ꢈ , nm): 216, 268, 280sh. HR-ESI-MS m/z 473.361
21 22 11
+
max
+
+
+
+
[M + Na] . FAB -MS m/z 451 [M + H] , 298 [(M – galloyl) + H] , 136 [(M – galloyl – glucose) + H] (100%). Table 1
13
presents the PMR spectrum (500 MHz, DMSO-d ) and C NMR spectrum (125 MHz, DMSO-d ).
6
6
Acid Hydrolysis. Compound 1 (5 mg) was dissolved in TFA(10 mL, 1%) and heated at 100°C for 6 h. The TFAwas
removed in vacuo in the presence of MeOH. The hydrolysate was dissolved in MeOH (5 mL) and analyzed by HPLC (conditions
2, phenolic compounds; conditions 3, carbohydrates).
Enzymatic Hydrolysis. Compound 1 (2 mg) and tannase (10 mg) from A ficuum (Sigma, 150 U/g) were dissolved in
acetate buffer (5 mL, pH 5.0), incubated at 38°C for 6 h, heated on a boiling-water bath for 15 min, and centrifuged (6000 g,
5

20 min). The supernatant was extracted with EtOAc (5 ꢅ 5 mL). The organic extracts were combined and concentrated. The
dry solid was dissolved in MeOH (5 mL) and analyzed by HPLC (conditions 2).
Methylation. A mixture of 1 (4 mg), Me SO (100 ꢄL), and anhydrous Na CO (150 mg) in Me CO (2 mL) was
2
4
2
3
2
heated at 90°C for 4 h. Solid Na SO was filtered off. The mixture was poured into ice water. The resulting precipitate was
2
4
centrifuged, washed until neutral, and dried in vacuo. Formolysis and hydrolysis of the permethylate were carried out as
before [35]. The hydrolysate was analyzed by GC/MS.
HPLC. Conditions 1: 600E liquid chromatograph (Waters), Lichrospher RP-18 column (4.6 ꢅ 200 mm, # 5 ꢄm,
Merck Millipore), mobile phase H O (A), MeCN (B), gradient regime (%B in A) 0–60 min linear gradient 7–40% B,
2
60–90 min 40–60% B, flow rate 1 mL/min, column temperature 40°C, ꢈ 270 nm. Conditions 2: Milikhrom A-02 microcolumn
liquid chromatograph (Ekonova), ProntoSIL-120-5-C18 AQ column (2 ꢅ 75 mm, # 5 ꢄm, Metrohm AG), mobile phase
(4.1 M LiClO in 0.1 M HClO ):H O 5:95 (A), MeCN (B), gradient regime (%B in A) 0–7.6 min 7–22%, 7.6–8.6 min
4
4
2
22–25%, 8.6–12 min 25–27%, 12–17 min 27–100%, 17–20 min 100–7%, flow rate 150 ꢄL/min, column temperature 35°C,
sample volume 2 ꢄL, ꢈ 216, 278, 330, 360 nm. Conditions 3: Milikhrom A-02 microcolumn liquid chromatograph (Ekonova),
Separon 5-NH column (2 ꢅ 75 mm, # 5 ꢄm, Tessek Ltd.), mobile phase 75% MeCN, isocratic regime (0–20 min), flow rate
2
100 ꢄL/min, column temperature 35°C, sample volume 4 ꢄL, ꢈ 190 nm.
ACKNOWLEDGMENT
The work was supported financially by the SB RAS Project “Centers of New Medical Technologies.”
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