Spireasalicin, a New Acylated Quercetin Glycoside from Spiraea salicifolia

Article abstract of DOI:10.1007/s10600-017-2197-7

A new acylated quercetin glycoside and 36 known compounds were isolated from flowering runners of Spiraea salicifolia L. (Rosaceae). The structures of the new glycoside quercetin-3-O-[6″-(4?-hydroxy-2?-methylenebutyroyl)]-β-D-glucopyranoside (spireasalicin, 1) and the 36 known compounds were elucidated using UV, IR, and NMR spectroscopy and mass spectrometry. The biological screening of the isolated compounds showed that 1 inhibited α-glucosidase. The contents of several phenolic compounds in S. salicifolia organs were studied and found to be unevenly distributed in the separate plant parts.

Full text of DOI:10.1007/s10600-017-2197-7

DOI 10.1007/s10600-017-2197-7
Chemistry of Natural Compounds, Vol. 53, No. 6, November, 2017
SPIREASALICIN, A NEW ACYLATED QUERCETIN
GLYCOSIDE FROM Spiraea salicifolia
*
D. N. Olennikov and N. I. Kashchenko
A new acylated quercetin glycoside and 36 known compounds were isolated from flowering runners of
Spiraea salicifolia L. (Rosaceae). The structures of the new glycoside quercetin-3-O-[6ꢀꢀ-(4ꢀꢀꢀ-hydroxy-2ꢀꢀꢀ-
methylenebutyroyl)]-ꢁ-D-glucopyranoside (spireasalicin, 1) and the 36 known compounds were elucidated
using UV, IR, and NMR spectroscopy and mass spectrometry. The biological screening of the isolated
compounds showed that 1 inhibited ꢂ-glucosidase. The contents of several phenolic compounds in
S. salicifolia organs were studied and found to be unevenly distributed in the separate plant parts.
Keywords: Spiraea salicifolia, Rosaceae, quercetin-3-O-[6ꢀꢀ-(4ꢀꢀꢀ-hydroxy-2ꢀꢀꢀ-methylenebutyroyl)]-ꢁ-D-
glucopyranoside, spireasalicin, anti-glucosidase activity, HPLC.
Diabetes mellitus is a metabolic disorder characterized by abnormal post-prandial elevation of the blood glucose
level. Recently, research on diabetes treatment has been directed more and more at the discovery of efficacious inhibitors of
ꢂ-glucosidase, the key enzyme in carbohydrate metabolism. Previously, we showed that plants of the family Rosaceae
(Comarum, Filipendula, Potentilla) are sources of phenolic compounds with anti-glucosidase activity [1–3]. A promising
plant species of this family is Spiraea salicifolia L., which is used in Tibetan medicine (‘om bu) to treat diabetes-like conditions
and gastrointestinal diseases [4]. Few chemical studies of this species were reported. The leaves are known to contain
cinnamoyl glycosides [5] and lignans and triterpenes [6]. The biological activity of S. salicifolia has not been studied. Our
screening studies established that extracts of S. salicifolia inhibited ꢂ-glucosidase [7]. Research on flowering runners of
S. salicifolia isolated 37 compounds including the new quercetin glycoside 1. Its anti-glucosidase activity was determined.
The distribution of phenolic compounds in separate organs was studied.
OH
OH
2'
O
8
HO
O
O
5'
OH
O
1'''
4'''
6
6''
O
5'''
3
O
OH
OH
1'' HO
OH
1
–
According to HR-ESI-MS, 1 had the formula C H O (561.318 [M – H] , calcd 561.480). The ESI-MS spectrum
26 26 14
–
exhibited a quasi-molecular ion with m/z 561 that corresponded to deprotonated [M – H] and ions with m/z 463 and 301 that
were indicative of sequential cleavage of fragment A with m/z 98 and glucose {[(M – H) – 98 – 162] }. The principal products
–
from acid hydrolysis of 1 were quercetin and D-glucose. The UV spectrum was typical of quercetin glycosides [8]. The IR
–1
–1
spectrum of 1 showed bands for ester (ꢃ
1710 cm ) and conjugated carbonyls (ꢃ
1652 cm ) [9].
max
max
Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, 6 Sakhꢀyanovoi St.,
Ulan-Ude, 670047, fax: 7 (3012) 43 47 43, e-mail: olennikovdn@mail.ru. Translated from Khimiya Prirodnykh Soedinenii,
No. 6, November–December, 2017, pp. 883–888. Original article submitted January 31, 2017.
©
1038
0009-3130/17/5306-1038 2017 Springer Science+Business Media New York

13
TABLE 1. PMR (500 MHz) and C NMR (125 MHz) Spectra of 1 (MeOH-d , ꢄ, ppm, J/Hz)
4
C atom
C atom
ꢄH
ꢄC
ꢄH
ꢄC
2
3
4
5
6
7
8
9
10
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
–
–
–
–
158.4
135.4
179.5
161.3
100.2
165.4
95.0
157.8
106.3
122.0
112.7
145.3
146.2
116.0
7.53 (1Í, dd, J = 8.1, 2.0)
5.10 (1Í, d, J = 8.0)
3.57 (1H, m)
123.3
101.6
76.1
78.7
71.8
75.2
64.5
6ꢀ
1ꢀꢀ
2ꢀꢀ
3ꢀꢀ
4ꢀꢀ
5ꢀꢀ
6ꢀꢀ
3.50 (1H, m)
3.43 (1H, m)
3.87 (1H, m)
6.19 (1Í, d, J = 2.0)
–
6.30 (1Í, d, J = 2.0)
4.18 (1Í, dd, J = 12.0, 6.1)
4.45 (1Í, dd, J = 12.0, 2.1)
–
–
–
–
169.2
139.4
35.1
60.4
129.5
1ꢀꢀꢀ
2ꢀꢀꢀ
3ꢀꢀꢀ
4ꢀꢀꢀ
5ꢀꢀꢀ
–
7.92 (1Í, d, J = 2.0)
2.43 (2Í, t, J = 7.1)
3.65 (2Í, t, J = 7.1)
5.68 (1H, s)
–
–
6.91 (1Í, d, J = 8.1)
6.15 (1H, s)
UV spectroscopy with diagnostic additives found that 1 was characterized by unsubstituted hydroxyls on C-5, C-7,
C-3ꢀ, and C-4ꢀ. Thus, fragment A was an acyl moiety on the carbohydrate substituent. The nature of the acyl moiety was
determined by analyzing the alkaline hydrolysis products of 1, which showed a compound with molecular mass 116 that was
identified using NMR and MS data as 4-hydroxy-2-methylenebutyric acid [10].
13
PMR and C NMR spectra of 1 were similar to those of quercetin-3-O-ꢁ-D-glucopyranoside (isoquercitrin) [11]
13
with the exception of additional resonances in the C NMR spectrum for ester carboxyl (ꢄ 169.2), a pair of alkene C atoms
>C=CH (ꢄ 139.4, 129.5), saturated –CH – (ꢄ 35.1), and a terminal –CH OH (ꢄ 60.4) [12]. The PMR spectrum also
2
C
2
2
contained two additional strong-field resonances for saturated –CH –CH – (ꢄ 2.43, 3.65) and weak-field vinyl =CH resonances
2
2
2
(ꢄ 5.68, 6.15) [13] (Table 1). HMBC correlations between inequivalent H-6ꢅ protons of glucopyranose (ꢄ 4.18, 4.45) and the
carboxyl of 4-hydroxy-2-methylenebutyric acid (ꢄ 169.2) and a weak-field shift of the glucopyranose C-6ꢅ resonance (ꢄ 64.5)
[8] were consistent with a substituted carbohydrate C-6ꢅ. Thus, the results elucidated the structure of 1 as quercetin-3-O-
[6ꢅ-(4ꢅꢀ-hydroxy-2ꢅꢀ-methylenebutyroyl)]-ꢁ-D-glucopyranoside, which we called spireasalicin.
The 4-hydroxy-2-methylenebutyric acid moiety was also found in the natural glycosides 6-tuliposide A [6-O-(4ꢀ-
hydroxy-2ꢀ-methylenebutyroyl)-ꢁ-D-glucopyranoside] from S. thunbergii Siebold ex Blume [14] and 1-O-cinnamoyl-6-O-
(4ꢅ-hydroxy-2ꢅ-methylenebutyroyl)-ꢁ-D-glucopyranoside and 1-O-(4ꢀ-hydroxy-2ꢀ-methylenebutyroyl)-6-O-cinnamoyl-ꢁ-D-
glucopyranoside from S. thunbergii [12] and S. prunifolia Siebold & Zucc. [15]. Until now, flavonoids containing this type of
acyl substituent were not observed.
Besides 1, the known compounds 2–37 were also isolated from runners of S. salicifolia. They were identified using
UV, IR, and NMR spectroscopy and mass spectrometry as kaempferol-3-O-(6ꢅ-caffeoyl)-ꢁ-D-glucopyranoside (2), kaempferol-
3-O-(6ꢅ-caffeoyl)-ꢁ-D-galactopyranoside (3), quercetin-3-O-(6ꢅ-caffeoyl)-ꢁ-D-glucopyranoside (4), quercetin-3-O-(6ꢅ-
caffeoy)-ꢁ-D-galactopyranoside (5) [16], tiliroside (6) [17], helichrysoside (7) [18], astragalin (8), trifoliin (9), isoquercitrin
(10), hyperoside (11), quercitrin (12), miquelianin (13), kaempferol-4ꢀ-glucoside (14), spireoside (15) [19], nicotiflorin (16),
rutin (17) [11], caffeic acid (18), 3-O-caffeoylquinic acid (19) [20], 4-O-caffeoylquinic acid (20), 1-O-caffeoyl-ꢁ-D-
glucopyranoside (21), cinnamic acid (22), p-coumaric acid (23), 4-methoxycinnamic acid (24) [5], 6-O-cis-p-coumaroyl-ꢁ-D-
glucopyranoside (25),6-O-trans-p-coumaroyl-ꢁ-D-glucopyranoside (26), 6-O-cis-4-methoxycinnamoyl-ꢁ-D-glucopyranoside
(27), 6-O-trans-4-methoxycinnamoyl-ꢁ-D-glucopyranoside (28), 1-O-trans-cinnamoyl-6-O-(4ꢅ-hydroxy-2ꢅ-
methylenebutyroyl)-ꢁ-D-glucopyranoside (29) [12], protocatechoic acid (30), gallic acid (31) [20], 5-O-galloylquinic acid
(32), (+)-catechin (33), (–)-epicatechin (34) [21], and procyanidins B (35), B (36), and C (37) [22]. Previously, leaves of
1
2
1
S. salicifolia afforced 11 and 22–24 [5]. Compounds 2–10, 12–21, and 25–37 were detected in this plant species for
the first time.
1039

TABLE 2. ꢂ-GlucosidaseActivity 50% Inhibitory Concentration (IC ) of Several Flavonoids from S. salicifolia, g/mL ꢇ SD
50
Aglyconeb
Carbohydrate parta
kaempferol
> 350
quercetin
–
207.10 ꢇ 8.69
273.57 ꢇ 11.21 (
10
291.64 ꢇ 11.95 (11)
3- -Glc
3- -Gal
O
>350 ( )
>350 (9)
O
p
p
8
)
–
ꢀꢀ
3- -(6 -HMB)-Glc
O
ꢇ
254.31 7.62 (1)
p
3- -(6ꢀꢀ-Caf)-Glc
O
302.07 ꢇ 12.38 (2)
42.97 ꢇ 1.93 (4)
p
ꢀꢀ
3- -(6 -Caf)-Gal
ꢇ
ꢇ
308.16 13.25 (3)
40.69 1.66 (5)
O
p
p
> 350 (6)
3- -(6ꢀꢀ-Cou)-Glc
331.14 ꢇ 14.23 (7)
O
Acarbosec
ꢇ
273.11 12.28
______
a
b
Caf = caffeoyl; Cou = p-coumaroyl; Galp = galactopyranose; Glcp = glucopyranose; HMB = 4ꢀ-hydroxy-2ꢀ-methylenebutyroyl;
c
compound number shown in parentheses; “–“, not determined (compound missing); reference compound.
An examination of the occurrence in the genus Spiraea of phenolic glycosides containing an acyl substituent on the
carbohydrate found that flavonoid acylglycosides 2, 3, and 5 were also observed in flowers of S. cantoniensis Lour. [16]; 6, in
fruit of S. formosana Hayata [23] and runners of S. canescens D. Don [24]. Phenolic glycosides 25–29 were detected as
constituents in the aerial part of S. canescens [24], S. prunifolia [15], and S. thunbergii [12]. Acylation of phenolic glycosides
is probably a systematic chemical feature of the genus Spiraea.
Studies of the biological activities of the isolated compounds with respect to ꢂ-glucosidase showed that several
flavonoids were very potent inhibitors of the enzyme (Table 2). The 50% inhibitory concentrations (IC ) for phenylpropanoids
50
18–29, benzoic acids 30–32, catechins 33 and 34, and procyanidins 35–37 were ꢆ400 g/mL, which indicated that they were
not efficacious. The degree of ꢂ-glucosidase inhibition increased from kaempferol to quercetin for both the aglycones and
glycosides. This indicated that substitution of flavonoid ring B was very important for the manifestation of biological activity.
The activities of the glycoside derivatives decreased if a carbohydrate moiety (glucose, galactose) was added to the flavonoid
C-3 position. However, a C-6ꢅ p-coumaroyl or caffeoyl substituent on the sugar helped to increase the inhibition efficacy with
caffeoyl being the most potent. Quercetin glycosides 4 (IC 42.97 g/mL) and 5 (IC 40.69 g/mL) were the most
50
50
efficacious ꢂ-glucosidase inhibitors. Their activities were significantly greater than that of the reference compound acarbose
(IC 273.11 g/mL). The efficacy of 1 (IC 254.31 g/mL) as the 4-hydroxy-2-methylenebutyryl derivative of glycoside 10
50
50
was similar to that of the starting compound (IC 273.57 g/mL) and acarbose.
50
HPLC-UV was used for quantitative characterization of S. salicifolia flowering runners as a source of phenolic
ꢂ-glucosidase inhibitors. An analysis of S. salicifolia organs showed that the various compound groups were unevenly distributed
in the plant (Table 3). Flowers of S. salicifolia were capable of accumulating flavonoids (42.40 mg/g) and less catechins and
phenylpropanoids. The principal flavonoids of the flowers were quercitrin (12, 11.70 mg/g), hyperoside (11, 8.01 mg/g), and
isoquercitrin (10, 7.43 mg/g). Quercetin derivatives were responsible for ~92% (38.91 mg/g) of the total flavonoids in flowers.
Flavonoid monosides dominated over biosides. The contents of glucosides, galactosides, and rhamnosides were similar.
The total content of acylated glycosides 2, 3, and 5 was 5.22 mg/g or 12.3% of the total flavonoids. The flavonoid concentrations
in S. salicifolia leaves were lower (23.40 mg/g) with 10 (7.55 mg/g), 3 (5.53 mg/g), and 11 (4.56 mg/g) being dominant.
The principal flavonoid species were distributed similarly in leaves and flowers. The flavonoid contents in S. salicifolia stems
were the least at 5.37 mg/g. Glycoside 1 was found in S. salicifolia flowers and leaves. The accumulation of phenylpropanoids
in S. salicifolia was greater in leaves (48.11 mg/g) than in flowers (21.86 mg/g). The principal compounds of this group in
S. salicifolia leaves was 1-O-caffeoyl-ꢁ-D-glucopyranoside (21, 38.46 mg/g); in flowers, 3-O-caffeoylquinic acid
(19, 13.42 mg/g). Benzoic acids (30 and 31) and derivative 32 were detected only in S. salicifolia flowers (2.19 mg/g), where
(+)-catechin (33, 21.81 mg/g) and catechins in general (30.14 mg/g) also localized. Stems of S. salicifolia were characterized
by larger concentrations of catechins (10.88 mg/g) than leaves (5.57 mg/g). In general, it is noteworthy that the various organs
of S. salicifolia runners typically had high contents of phenolic compounds. However, flowers and leaves had the highest
contents of the target group of acylated flavonoid glycosides (1–3 and 5) and could be recommended as a new medicinal raw
material.
1040

TABLE 3. Contents of Several Phenolic Compounds (PCs) in S. salicifolia Organs, mg/g of Air-dried Mass ꢇ SD
Compound
Flowers
Leaves
Stems
Flavonoids (Fl)
< 0.01
2.19 ꢇ 0.04
< 0.01
0.86 ꢇ 0.02
1
2
0.18 ꢇ 0.00
< 0.01
3
ꢇ
ꢇ
1.30 0.03
5.53 0.11
5
1.73 ꢇ 0.04
7.43 ꢇ 0.14
8.01 ꢇ 0.15
11.70 ꢇ 0.26
1.12 ꢇ 0.02
1.91 ꢇ 0.05
3.62 ꢇ 0.08
0.64 ꢇ 0.02
7.55 ꢇ 0.15
4.56 ꢇ 0.09
1.08 ꢇ 0.02
0.11 ꢇ 0.00
1.07 ꢇ 0.02
< 0.01
0.21 ꢇ 0.00
2.80 ꢇ 0.05
1.22 ꢇ 0.02
0.44 ꢇ 0.01
< 0.01
10
11
12
13
15
17
0.17 ꢇ 0.00
0.16 ꢇ 0.00
Phenylpropanoids (PP)
1.51 ꢇ 0.03
18
19
20
21
22
24
1.34 ꢇ 0.04
1.09 ꢇ 0.02
ꢇ
ꢇ
ꢇ
13.42 0.28
7.14 0.14
2.14 0.04
1.93 ꢇ 0.05
4.82 ꢇ 0.11
0.18 ꢇ 0.00
0.17 ꢇ 0.00
0.25 ꢇ 0.00
38.46 ꢇ 0.76
0.48 ꢇ 0.01
0.27 ꢇ 0.00
1.00 ꢇ 0.02
2.25 ꢇ 0.04
Benzoic acids (BA)
Catechins (C)
30
31
32
ꢇ
0.93 0.02
< 0.01
< 0.01
< 0.01
0.36 ꢇ 0.01
ꢇ
0.90 0.02
33
34
21.81 ꢇ 0.43
8.33 ꢇ 0.16
5.57 ꢇ 0.11
< 0.01
6.95 ꢇ 0.14
3.93 ꢇ 0.07
42.40
21.86
2.19
30.14
96.59
23.40
48.11
< 0.01
5.57
5.37
6.48
< 0.01
10.88
22.73
ꢊFl
ꢊPP
ꢊBA
ꢊC
77.08
ꢊPC
EXPERIMENTAL
Runners of S. salicifolia were collected during flowering in the vicinity of Kyren (Tunkinskii District, Republic
of Buryatia, Russia; July 20, 2015; 51°40ꢀ48ꢅ N, 102°11ꢀ54ꢅ E, 741 m above sea level). The species was determined by
Dr. T. A. Aseeva (IGEB, SB, RAS). Raw material was dried in a convection oven (50°C) to moisture content ꢈ5%. Column
chromatography (CC) was performed over SiO , RP-SiO , polyamide (Sigma-Aldrich, St. Louis, MO, USA), and Sephadex
2
2
LH-20 (GE Healthcare, Little Chalfont, UK). Spectrophotometry was carried out on an SF-2000 spectrophotometer
(OKB Spektr, St. Petersburg, Russia). MS analyses used LCMS-8050 TQ-mass spectrometer (Shimadzu, Columbia, MD,
USA) with electrospray ionization (ESI) in negative-ion mode at capillary potential –30 to –60 V, field potential 5.0 kV,
ion-source temperature 250°C, and molecular mass scan range 100–2000 Da. NMR spectra were recorded on a VXR 500S
NMR spectrometer (Varian, Palo Alto, CA, USA). Preparative HPLC was performed on a Summit liquid chromatograph
(Dionex, Sunnyvale, CA, USA).
Extraction and Fractionation. A weighed portion of dried and ground raw material (1400 g) was extracted (2ꢉ)
with EtOH (60%, 1:20) in an ultrasonic bath (100 W, 35 kHz) at 40°C for 2 h. The resulting extract was filtered off and
concentrated in vacuo (40°C) to an aqueous residue that was extracted with hexane and n-BuOH. The solvents were removed
to produce hexane (SF-1, 36 g), n-BuOH (SF-2, 283 g), and aqueous fractions (SF-3, 204 g). Anti-glucosidase activity of
the fractions showed that fraction SF-2 had the greatest inhibitory activity with IC 163.27 ꢇ 6.52 g/mL. The activities of
50
1041

SF-1 and SF-3 were <400 g/mL. Fraction SF-2 (250 g) was investigated further by separation over polyamide (CC, 2.5 kg)
with elution successively by H O (fraction SF-2/1, 26 g), EtOH (30%, SF-2/2, 49 g; 70%, SF-2/3, 134 g), and NH (0.5%) in
2
3
EtOH (90%, SF-2/4, 37 g). Fraction SF-2/1 was not investigated further because it was inactive. Fraction SF-2/2 (40 g) was
chromatographed over SiO (CC, 4 ꢉ 60 cm) using a EtOAc–Me CO gradient (100:0ꢋ60:40) to produce subfractions
2
2
SF-2/2-01–SF-2/2-09. Subfractions SF-2/2-04 and SF-2/2-05 were rechromatographed over SiO (CC, 2 ꢉ 30 cm) using a
2
EtOAc–EtOH gradient (100:0ꢋ70:30) to isolate procyanidins B (63 mg, 35) [22], B (40 mg, 36) [22], and C (15 mg, 37)
1
2
1
[22]. Analogous separation of subfraction SF-2/2-06 produced 1-O-caffeoyl-ꢁ-D-glucopyranoside (1264 mg, 21) [5],
(+)-catechin (107 mg, 33) [21], and (–)-epicatechin (27 mg, 34) [21]. Separation of subfractions SF-2/2-08 and SF-2/2-09
over SiO (CC, 2 ꢉ 40 cm, Me CO–H O, 100:0ꢋ60:40) isolated rutin (142 mg, 17) [11], protocatechoic acid (84 mg, 30)
2
2
2
[20], gallic acid (20 mg, 31) [20], and 5-O-galloylquinic acid (54 mg, 32) [21]. Fraction SF-2/3 (125 g) was chromatographed
over SiO (CC, 8 ꢉ 70 cm) using a hexane–EtOAc–Me CO gradient (100:0:0ꢋ60:40:0ꢋ0:70:30) to produce subfractions
2
2
SF-2/3-01–SF-2/3-16.
Next, subfractions SF-2/3-01 and SF-2/3-02 were separated over SiO (CC, 1 ꢉ 30 cm) using a hexane–EtOAc
2
gradient (100:0ꢋ80:20) to isolate tiliroside (14 mg, 6) [17], cinnamic acid (30 mg, 22) [5], p-coumaric acid (10 mg, 23) [5],
and 4-methoxycinnamic acid (14 mg, 24) [5]. Subfractions SF-2/3-03 and SF-2/3-04 were chromatographed over SiO (CC,
2
2 ꢉ 30 cm, EtOH–H O, 95:5ꢋ70:30) and RP-SiO (CC, 1 ꢉ 18 cm, H O–MeCN, 100:0ꢋ10:90) to isolate kaempferol-4ꢀ-
2
2
2
glucoside (10 mg, 14) [19], spireoside (207 mg, 15) [19], 6-O-cis-p-coumaroyl-ꢁ-D-glucopyranoside (52 mg, 25) [12], and
6-O-trans-p-coumaroyl-ꢁ-D-glucopyranoside (75 mg, 26) [12]. Subfractions SF-2/3-06 and SF-2/3-07 were separated over
SiO (CC, 3 ꢉ 30 cm, hexane–EtOAc, 100:0ꢋ70:30), Sephadex G-10 (CC, 2 ꢉ 90 cm, EtOH–H O, 95:5ꢋ60:40), RP-SiO
2
2
2
(CC, 1 ꢉ 20 cm, H O–MeCN, 100:0ꢋ20:80), and preparative HPLC [LiChrospher RP-18 column (250 ꢉ 10 mm, # 10 m,
2
Supelco, Bellefonte, PA, USA); mobile phase H O (A) and MeCN (B) in gradient mode (%B): 0–40 min, 40–60%; 40–60 min,
2
60–90%; flow rate 1 mL/min; column temperature 30°C; UV detector at 350 nm] to produce kaempferol-3-O-(6ꢅ-caffeoyl)-ꢁ-
D-glucopyranoside (30 mg, 2) [16], kaempferol-3-O-(6ꢅ-caffeoyl)-ꢁ-D-galactopyranoside (31 mg, 3) [16], and helichrysoside
[quercetin-3-O-(6ꢅ-p-coumaroyl)-ꢁ-D-glucopyranoside; 28 mg, 7) [18].
Subfraction SF-2/3-08 was separated analogously to afford 1 (spireasalicin, 18 mg), 6-O-cis-4-methoxycinnamoyl-
ꢁ-D-glucopyranoside (47 mg, 27) [12], 6-O-trans-4-methoxycinnamoyl-ꢁ-D-glucopyranoside (29 mg, 28) [12], and
1-O-trans-cinnamoyl-6-O-(4ꢅ-hydroxy-2ꢅ-methylenebutyroyl)-ꢁ-D-glucopyranoside (15 mg, 29) [12]. Subfraction
SF-2/3-09 yielded quercetin-3-O-(6ꢅ-caffeoyl)-ꢁ-D-glucopyranoside (14 mg, 4) [16], quercetin-3-O-(6ꢅ-caffeoyl)-ꢁ-D-
galactopyranoside (36 mg, 5) [16], and nicotiflorin (25 mg, 16) [11]. Subfractions SF-2/3-10–SF-2/3-15 were separated using
CC over SiO (2 ꢉ 30 cm, hexane–EtOAc, 85:15ꢋ60:40), Sephadex G-10 (2 ꢉ 90 cm, EtOH–H O, 95:5ꢋ50:50), and
2
2
RP-SiO (1 ꢉ 20 cm, H O–MeCN, 100:0ꢋ40:60) to produce astragalin (35 mg, 8) [19], trifoliin (27 mg, 9) [19], isoquercitrin
2
2
(180 mg, 10) [19], hyperoside (183 mg, 11) [19], quercitrin (1541 mg, 12) [19], and miquelianin (48 mg, 13) [19].
Fraction SF-2/4 (35 g) was chromatographed over Sephadex G-10 (CC, 2 ꢉ 90 cm, EtOH–H O, 95:5ꢋ20:80) and RP-SiO
2
2
(CC, 1 ꢉ 20 cm, H O–MeCN, 100:0ꢋ40:60) to isolate caffeic acid (102 mg, 18) [20], 3-O-caffeoylquinic acid (915 mg, 19)
2
[20], and 4-O-caffeoylquinic acid (26 mg, 20) [5].
–
–
Spireasalicin (1), Ñ Í Î . HR-ESI-ÌS m/z 561.318 ([M – Í] ; calcd 561.480). (–)ESI-ÌS m/z: 561 [M – Í] ,
26 26 14
–
–
463 [(M – Í) – 98] , 301 [(M – Í) – 98 – 162] . UV spectrum (ÌåÎÍ, ꢌ , nm): 256, 267 sh, 356; +AlCl 276, 412;
max
3
+AlCl /HCl 270, 298, 401; +NaOAc 270, 323, 395; +NaOAc/H BO 261, 300, 391; +NaOMe 271, 331, 411. IR spectrum
3
3
3
–1
13
(ꢃ , cm ): 3425, 1710, 1652, 1602. Table 1 lists the PMR and C NMR spectra.
max
Acid Hydrolysis of 1. Compound 1 (2 mg) was dissolved in TFA (5%, 10 mL) and heated at 110°C (2 h).
The hydrolysate was concentrated in vacuo and dissolved in MeOH. The resulting hydrolysate was chromatographed over
polyamide (CC, 20 g) with elution successively by H O (100 mL, eluate I) and EtOH (90%, 250 mL, eluate II). The resulting
2
eluates were concentrated in vacuo and analyzed by HPLC (conditions 1, monosaccharides as 3-methyl-1-phenyl-2-pyrazolin-
5-ones [25]; conditions 2, phenolic compounds). Glucose was detected in eluate I (t 12.53 min); quercetin, in eluate II
R
(t 12.66 min). Eluate I was also analyzed as before to determine if the monosaccharides were D- or L-isomers [26].
R
Alkaline Hydrolysis of 1. Compound 1 (7 mg) was dissolved in NaOH (5 mL, 1 M) and thermostatted at 50°C (4 h).
The resulting solution was neutralized with AcOH and extracted with Et O (3 ꢉ 30 mL). The organic layer was
2
concentrated in vacuo. The resulting residue was dissolved in hexane and chromatographed over SiO (CC, 1 ꢉ 10 cm) using
2
a hexane–EtOAc gradient (100:0ꢋ70:30). The fraction eluted by hexane–EtOAc (85:15) afforded a compound (1.1 mg) that
was identified by NMR and mass spectrometry as 4-hydroxy-2-methylenebutyric acid [10].
1042

–
1
4-Hydroxy-2-methylenebutyric acid, Ñ Í Î . HR-ESI-ÌS m/z 115.127 ([M – Í] ; calcd 115.109). Í NMR
5
8 3
spectrum (500 MHz, ÌåÎÍ-d , ꢄ, ppm, J/Hz): 6.18 (1Í, s, =ÑÍ ), 5.63 (1Í, s, =ÑÍ ), 3.58 (1Í, t, J = 7.0, Í-4), 2.40 (1Í, t,
4
2
2
13
J = 7.0, Í-3). C NMR spectrum (125 MHz, ÌåÎÍ-d , ꢄ, ppm): 170.5 (Ñ-1), 140.6 (Ñ-2), 129.1 (=ÑÍ ), 60.9 (Ñ-4), 34.8
4
2
(Ñ-3).
HPLC. Conditions 1: ProntoSIL-120-5-C18 AQ column (2 ꢉ 75 mm, # 5 m, Metrohm AG); mobile phase:
CH COONH (100 mM, pH 4.5) (A) and MeCN (B); gradient mode (%B): 0–20 min, 20–26%; flow rate 150 L/min; column
3
4
temperature 35°C; UV detector at 250 nm. Conditions 2: ProntoSIL-120-5-C18 AQ column (2 ꢉ 75 mm, # 5 m, Metrohm
AG); mobile phase LiClO (0.2 M) in HClO (0.006 M) (A) and MeCN (B); gradient mode (%B): 0–9 min, 0–80%; 9–15 min,
4
4
80–100%; flow rate 100 L/min; column temperature 35°C; UV detector at 270 nm. Quantitative analysis was performed on
a Milichrom A-02 microcolumn liquid chromatograph (EcoNova, Novosibirsk, Russia); ProntoSIL-120-5-C18 AQ column
(2 ꢉ 75 mm, # 5 m, Metrohm AG, Herisau, Switzerland); mobile phase: LiClO (0.2 M) in HClO (0.006 M) (A) and MeCN
4
4
(B); gradient mode (%B): 0–40 min, 5–80; 40–43 min, 100; flow rate 100 L/min; column temperature 35°C; UV detector at
270 (benzoic acids, catechins), 320 (phenylpropanoids), and 356 nm (flavonoids). The contents of pure constituents were
calculated from calibration curves constructed using commercial samples of isoquercitrin; hyperoside; quercitrin; miquelianin;
spireoside; rutin; caffeic acid; 3-O- and 4-O-caffeoylquinic acids; cinnamic, 4-methoxycinnamic, protocatechoic, and gallic
acids; (+)-catechin; and (–)-epicatechin (Sigma-Aldrich). Astragalin (Sigma-Aldrich) was used for analysis of kaempferol-3-
O-(6ꢅ-caffeoyl)glucoside and kaempferol-3-O-(6ꢅ-caffeoyl)-ꢁ-D-galactopyranoside; hyperoside, quercetin-3-O-(6ꢅ-caffeoyl)-
ꢁ-D-galactopyranoside; and caffeic acid, 1-O-caffeoyl-ꢁ-D-glucopyranoside; and gallic acid, 5-O-galloylquinic acid.
The difference in molecular masses of the compounds was considered in the calculations when using external standards.
The results were reported as the averages of three parallel determinations (ꢇstandard deviation, SD).
Anti-glucosidase activity of the fractions and compounds was studied by a microplate spectrophotometric method
using ꢂ-glucosidase from Saccharomyces cerevisiae (3.2.1.20; type I; ꢆ10 U/mg; Sigma-Aldrich, St. Louis, MO, USA) [3].
Results were expressed as the IC ( g/mL) values. Solutions of the compounds in normal saline (0.9% NaCl) were used. The
50
reference compound was acarbose (ꢆ94%, Sigma-Aldrich).
ACKNOWLEDGMENT
The work was supported financially by the RFBR and Administration of the Republic of Buryatia under scientific
project No. 16-43-030857.
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