New Flavonoid Glucuronides from the Aerial Part of Scutellaria intermedia

Article abstract of DOI:10.1007/s10600-017-2079-z

The aerial part of the plant Scutellaria intermedia Popov yielded five glucuronides, three of which were identified as chrysin 7-O-β-D-glucuronide, wogonin 7-O-β-D-glucuronide, and baicalein 7-O-β-Dglucuronide (baicalin). IR, UV, PMR, and ¹³C N

Full text of DOI:10.1007/s10600-017-2079-z

DOI 10.1007/s10600-017-2079-z
Chemistry of Natural Compounds, Vol. 53, No. 4, July, 2017
NEW FLAVONOID GLUCURONIDES FROM THE AERIAL
PART OF Scutellaria intermedia
A. M. Karimov,1,3 T. N. Slobodyanyuk, and E. Kh. Botirov2*
2
The aerial part of the plant Scutellaria intermedia Popov yielded five glucuronides, three of which were
identified as chrysin 7-O-ꢀ-D-glucuronide, wogonin 7-O-ꢀ-D-glucuronide, and baicalein 7-O-ꢀ-D-
1
3
glucuronide (baicalin). IR, UV, PMR, and C NMR spectral data and acid hydrolysis of the two new
glucuronides established their structures as 5,7,2ꢁ-trihydroxyflavone 2ꢁ-O-ꢀ-D-glucuronopyranoside (4) and
scutevulin 2ꢁ-O-ꢀ-D-glucuronopyranoside (5).
Keywords: Scutellaria intermedia, flavone glycosides, 5,7,2ꢁ-trihydroxyflavone 2ꢁ-O-ꢀ-D-glucuronopyranoside,
scutevulin 2ꢁ-O-ꢀ-D-glucuronopyranoside.
Many species of the genus Scutellaria L. (skullcap) exhibit broad spectra of biological activity and are used in
science-based and folk medicine [1–3]. Several of the 32 Scutellaria species indigenous to Uzbekistan are used in folk
medicine to treat epilepsy, allergies, nervousness, hypertension, and other diseases [2, 4]. Plants of this genus have varied
chemical compositions. Flavonoids, phenylpropanoids, phenolic acids, iridoids, clerodane-type diterpenoids, steroids,
triterpenes, lignans, alkaloids, phytosterols, tanning agents, essential oils, and other classes of natural compounds have now
been isolated from them [1–7].
Modern pharmacological research confirmed that extracts and individual flavonoids from plants of the genus Scutellaria
possessed antitumor, hepatoprotective, antioxidant, anti-inflammatory, anticonvulsant, antibacterial, and antiviral activity
[
8–10]. The biological activity of Scutellaria flavonoids has prompted unwavering interest in them and an increasing number
of scientific publications. Greater than 320 flavonoids have now been isolated from various species. The list is constantly
being updated with new compounds [5–7].
Flavonoids from plants of the genus Scutellaria were studied systematically during our search for new biologically
active compounds and their accessible sources. The present work focused on the aerial part of S. intermedia Popov growing
on rocky and stony slopes, cliffs, and gravel beds of the western Tian-Shan and Pamir-Alay mountains [11]. The plant was
collected during flowering (May 2015) in the foothills of Pap District, Republic of Uzbekistan. Information about flavonoids
from this plant was missing in the literature available to us.
The EtOAc fraction of the EtOH extract of the aerial plant part was separated by column chromatography using
various absorbents and was isolated eight flavonoids. Spectral data and direct comparisons with authentic samples identified
them as chrysin, oroxylinA, 7-O-methylnorwogonin, 5,7,2ꢁ-trihydroxyflavone, scutevulin (5,7,2ꢁ-trihydroxy-8-methoxyflavone),
2
(S)-5,7,2ꢁ-trihydroxyflavanone, baicalein, and apigenin [12].
Flavonoids 1–5 were isolated from the n-BuOH fraction. Three of them were identified based on spectral data and
acid hydrolysis as the known flavonoids chrysin 7-O-ꢀ-D-glucuronide (1) [5, 7, 13], wogonin 7-O-ꢀ-D-glucuronide (2)
5, 7, 14], and baicalein 7-O-ꢀ-D-glucuronide (baicalin, 3) [5–7, 15].
[
1
) S. Yu. Yunusov Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan,
Tashkent, 100170, Uzbekistan; 2) Surgut State University, 1 Lenina St., Surgut, 628412, Russia, e-mail: botirov-nepi@mail.ru;
) Namangan State University, 316 Uichi St., Namangan, 716001, Uzbekistan; translated from Khimiya Prirodnykh Soedinenii,
No. 4, July–August, 2017, pp. 543–546. Original article submitted September 26, 2016.
3
6
38
0009-3130/17/5304-0638 ©2017 Springer Science+Business Media New York

TABLE 1. 13C NMR Spectra of Flavonoids 4, 4a, 5, and 5a (100 MHz, DMSO-d , ꢄ, ppm)
6
C atom
4
4a
5
5a
C atom
4
4a
5
5a
2
3
4
5
6
7
8
9
161.2
109.8
182.3
157.9
98.5
164.1
94.1
161.1
103.5
120.6
155.7
161.8
109.3
182.5
161.6
99.6
166.3
94.5
158.3
103.6
117.6
158.0
160.4
109.7
181.7
156.5
99.1
157.0
127.3
149.5
103.2
120.1
154.5
161.5
108.3
181.9
156.5
99.2
157.3
127.2
150.2
103.8
117.4
156.6
3ꢁ
4ꢁ
5ꢁ
6ꢁ
1ꢁꢁ
2ꢁꢁ
3ꢁꢁ
4ꢁꢁ
5ꢁꢁ
115.8
132.3
122.3
128.9
99.2
72.8
75.4
71.3
75.7
117.4
133.0
119.1
128.8
115.2
132.2
121.7
128.4
99.0
72.6
75.2
71.5
75.4
117.7
132.5
119.2
128.6
1
0
1
ꢁ
6ꢁꢁ
OCH3
170.1
170.3
61.1
2ꢁ
60.8
R
2
O
2'
R
1
HO
9
O
7
3
10
5
OH
O
4
, 4a, 5, 5a
4
5
: R = H, R = GlcAp; 4a: R = R = H
1
2
1
2
: R = OCH , R = GlcAp; 5a: R = OCH , R = H
1
3
2
1
3
2
Flavonoid 4, C H O , had a UV spectrum (ꢂ 254, 260, 268, 326 nm) characteristic of flavone derivatives.
max
2
1
18 11
Spectra taken with NaOAc and AlCl indicated that free phenolic hydroxyls were present in the flavone 5- and 7-positions
3
[
15]. The IR spectrum of flavonoid 4 exhibited absorption bands for hydroxyls, carboxylic C=O, and ꢃ-pyrone in addition to
aromatic C=C bonds. The PMR spectrum of 4 in DMSO-d showed resonances for protons of a 5,7,2ꢁ-trisubstituted flavone,
6
a chelated 5-OH (12.84 ppm), an anomeric proton (ꢄ 5.12 ppm, 1H, d, J = 7.4 Hz), and other carbohydrate protons. The TLC
1
3
mobility and IR, PMR, and C NMR spectral data (Table 1) were consistent with a flavonoid glycoside for 4. The carbonyl
–
1
absorption band at 1742 cm in the IR spectrum suggested that the carbohydrate part was a uronic acid. In fact, acid hydrolysis
of 4 produced 5,7,2ꢁ-trihydroxyflavone (4a) and D-glucuronic acid. Resonances of the D-glucuronic-acid C atoms appeared
1
3
in the C NMR spectrum at 99.2 (C-1ꢁꢁ), 72.8 (C-2ꢁꢁ), 75.4 (C-3ꢁꢁ), 71.3 (C-4ꢁꢁ), 75.7 (C-5ꢁꢁ), and 170.1 ppm (C-6ꢁꢁ) [15–17].
1
3
The bonding site of the carbohydrate was found by comparing C NMR spectra of the compound and 5,7,2ꢁ-trihydroxyflavone
Table 1) and their UV spectra. Therefore, flavonoid 4 had the structure 5,7,2ꢁ-trihydroxyflavone 2ꢁ-O-ꢀ-D-glucuronopyranoside
(
and was a new and previously unreported glycoside of 5,7,2ꢁ-trihydroxyflavone.
Flavonoid 5 was a yellow amorphous compound, C H O . Its IR spectrum had absorption bands for hydroxyls,
2
2 20 12
two carbonyls, and aromatic C=C bonds. Its UV spectrum (ꢂmax 223, 275, 316, 345 nm) was characteristic of flavone derivatives.
Bathochromic shifts of the absorptions upon adding NaOAc and AlCl were consistent with free phenolic hydroxyls in the
3
5
- and 7-positions. The PMR spectrum of 5 had resonances for 5,7,8,2ꢁ-tetrasubstituted flavone protons, a methoxyl, phenolic
1
3
hydroxyls in the 5- and 7-positions, an anomeric proton, and other carbohydrate protons. Resonances in the C NMR
spectrum at 99.0 (C-1ꢁꢁ), 72.6 (C-2ꢁꢁ), 75.2 (C-3ꢁꢁ), 71.5 (C-4ꢁꢁ), 75.4 (C-5ꢁꢁ), and 170.3 (C-6ꢁꢁ) were indicative of
a ꢀ-D-glucuronopyranosyl moiety in flavone glycoside 5 [15, 17]. This was confirmed by acid hydrolysis that produced
scutevulin (5,7,2ꢁ-trihydroxy-8-methoxyflavone, 5a) and D-glucuronic acid. The bonding site of the glucuronic acid to the
phenolic 2ꢁ-OH was established by comparing C NMR spectra of scutevulin and flavone glycoside 5. Thus, 5 was a new
natural glycoside of scutevulin and had the structure scutevulin 2ꢁ-O-ꢀ-D-glucuronopyranoside.
1
3
Flavone glycosides glycosylated at the C-2ꢁ hydroxyl are rather common in plants of the genus Scutellaria [5, 7].
639

EXPERIMENTAL
UV spectra were recorded in EtOH on an SF-2000 spectrophotometer. IR spectra were taken on a PerkinElmer
1
3
+
Spectrum FTIR spectrometer. PMR and C NMR spectra were recorded on a Unity-400 , Bruker AV-4 spectrometer at
1
3
operating frequency 400 MHz (PMR) and 100 MHz ( C NMR). Chemical shifts are given in ppm on the ꢄ-scale. TLC was
performed on Silufol UV-254 plates. Spots of flavonoids on TLC plates were viewed under UV light in a UFS-254/365
chromatographic irradiator and were also detected by treatment with NH vapor, NaOH (0.5%) in EtOH, and vanillin (3%) in
3
EtOH mixed with conc. HCl (4:1 ratio). Column chromatography used KSK SiO 100/160 ꢅm, Woelm polyamide, and
2
Sephadex LH-20 (Pharmacia). TLC used Sorbil PTSKh-P-A-UV plates and solvent systems 1) CHCl –EtOAc (9:1);
3
2
) CHCl –EtOH (9:1); 3) CHCl –EtOAc–EtOH (6:1:3); 4) n-BuOH–EtOH–H O (5:3:2); and 5) n-BuOH–Py–H O (6:4:3).
3 3 2 2
Extraction and Isolation of Flavonoids. Air-dried ground aerial part of S. intermedia (1.13 kg) was extracted (5ꢆ)
with EtOH (85%) at room temperature. The obtained extract was condensed in vacuo to 0.5 L, treated with an equal volume
of distilled H O, shaken, and worked up sequentially with petroleum ether, EtOAc, and n-BuOH. The aqueous residue was
2
acidified with H SO solution (5%) and extracted (2ꢆ) with n-BuOH. The n-BuOH extracts from before and after acidification
2
4
were combined. The extracts were evaporated in vacuo to afford petroleum-ether (7.2 g), EtOAc (21.8 g), and n-BuOH
fractions (34.8 g).
A part of the EtOAc fraction (20.0 g) was chromatographed over a column of silica gel (600 g) using a petroleum
ether–EtOAc gradient (80:20–0:100) to produce five subfractions (E1-E5). Rechromatography of subfraction E1 (2.38 g)
over a column of silica gel using a hexane–EtOAc gradient (90:10–20:80) isolated chrysin (82.0 mg), oroxylin A (106.0 mg),
and 7-O-methylnorwogonin (269.8 mg). Subfraction E2 (4.25 g) was chromatographed over a column of silica gel using
a CHCl –PrOH gradient (98:2–70:30) to isolate from separate fractions 5,7,2ꢁ-trihydroxyflavone (49.6 mg) and scutevulin
3
(
135.2 mg). Subfraction E3 (3.82 g) was separated by preparative TLC using solvent system 2 to obtain 2(S)-5,7,2ꢁ-
trihydroxyflavanone (21.4 mg). Subfraction E4 (2.89 g) was separated over a column of Sephadex LH-20 using an
EtOH–H O gradient (95:5–20:80) to isolate baicalein (34.3 mg). Rechromatography of subfraction E5 (1.42 g) over an SiO
2
2
column with gradient elution by n-hexane–EtOAc (98:2–30:70) produced apigenin (25.3 mg).
The combined n-BuOH fraction (34.0 g) was also chromatographed over an SiO column using an EtOAc–EtOH
2
gradient (90:10–70:30) to produce four fractions. Subfraction B1 (4.37 g) was separated over a column of Sephadex LH-20
with elution by EtOH–H O (95:5–10:90) to isolate 1 (358.5 mg) and 2 (176.7 mg). Subfraction B2 (6.48 g) was
2
rechromatographed over a polyamide column using CHCl –PrOH (98:2–0:100) to isolate 3 (214.1 mg). Subfraction B3
3
(
5.85 g) was separated over a column of Sephadex LH-20 with elution by EtOH–H O (95:5–10:90) to isolate 4 (34.3 mg) and
2
5
(46.8 mg). Subfraction B4 (7.31 g) was chromatographed over a column of Sephadex LH-20 with elution by EtOH–H O
2
(
95:5–10:90) to isolate 4 (12.6 mg) and 5 (19.3 mg).
Acid Hydrolysis of Glucuronides. Glycosides 1–5 were hydrolyzed on a water bath using HCl solution (20%) for
5
–6 h. The aglycon precipitated on cooling and was filtered off and recrystallized. The filtrate was evaporated in vacuo to
a syrupy residue. Paper chromatography with an authentic sample detected D-glucuronic acid (system 5).
Chrysin 7-O-ꢀ-D-glucuronide (1), C H O , mp 219–221ꢇÑ. UV spectrum (EtOH, ꢂ , nm): 270, 305. Í NMR
1
2
1
18 10
max
spectrum (DMSO-d , ꢄ, ppm, J/Hz): 5.18 (1H, d, J = 6.8, H-1ꢁꢁ), 6.48 (1H, br.s, H-6), 6.90 (1H, br.s, Í-8), 7.06 (1Í, s, Í-3),
6
7
.50–7.60 (3Í, m, Í-3ꢁ, 4ꢁ, 5ꢁ), 8.00–8.11 (2Í, m, Í-2ꢁ, 6ꢁ), 12.60 (1Í, s, 5-ÎÍ). Acid hydrolysis of 1 produced chrysin and
D-glucuronic acid.
Wogonin 7-O-ꢀ-D-glucuronide (2), C H O , mp 194–196ꢇÑ. UV spectrum (EtOH, ꢂmax, nm): 275, 345.
2
2 20 11
1
Í NMR spectrum (DMSO-d , ꢄ, ppm, J/Hz): 3.78 (3Í, s, 8-ÎÑÍ ), 5.32 (1Í, d, J = 7.2, Í-1ꢁꢁ), 7.06 (1Í, s, Í-3), 7.12 (1Í,
6
3
s, Í-6), 7.60 (3Í, m, Í-3ꢁ, 4ꢁ, 5ꢁ), 8.00 (2Í, m, Í-2ꢁ, 6ꢁ), 12.79 (1Í, s, 5-ÎÍ). Acid hydrolysis of 2 produced wogonin and
D-glucuronic acid.
Baicalin (baicalein 7-O-ꢀ-D-glucuronide) (3), C H O , mp 220–222ꢇÑ. UV spectrum (EtOH, ꢂmax, nm): 245,
2
1 18 11
1
2
77, 313. Í NMR spectrum (DMSO-d , ꢄ, ppm, J/Hz): 5.24 (1Í, d, J = 7.1, Í-1ꢁꢁ), 6.98 (1Í, s, Í-3), 7.09 (1Í, s, Í-8), 7.6
6
(
3Í, m, Í-3ꢁ, 4ꢁ, 5ꢁ), 8.0 (2Í, m, Í-2ꢁ, 6ꢁ), 12.70 (1Í, s, 5-ÎÍ). Acid hydrolysis of 3 produced baicalein and D-glucuronic acid.
,7,2ꢁ-Trihydroxyflavone 2ꢁ-O-ꢀ-D-glucuronopyranoside (4), amorphous yellow compound, C H O ,
5
2
1 18 11
[
(
(
ꢈ] –65.3ꢇ (ñ 0.13, Me CO). UV spectrum (EtOH, ꢂmax, nm): 254, 260, 268, 326; (+NaOMe): 246, 254, 262, 272, 373;
D 2
+NaOAc): 255, 263, 271, 357; (+AlCl ): 246, 253, 264, 276, 340; (+AlCl /HCl): 242, 248, 257, 265, 280, 335. IR spectrum
3
3
–1
1
KBr, ꢉmax, cm ): 3471 (OH), 1742 (carboxylic C=O), 1660 (ꢃ-pyrone C=O), 1624, 1578 (aromatic C=C bonds). Í NMR
spectrum (400 MHz, DMSO-d , ꢄ, ppm, J/Hz): 3.16–3.50 (m, H-2ꢁꢁ–H-4ꢁꢁ), 3.76 (1H, d, J = 9.0, H-5ꢁꢁ), 5.12 (1H, d, J = 7.4,
6
640

H-1ꢁꢁ), 6.16 (1H, d, J = 1.8, H-6), 6.43 (1H, d, J = 1.8, H-8), 6.97 (1H, s, H-3), 7.14 (1H, m, H-5ꢁ), 7.29 (1H, d, J = 8.0, 1.8,
H-3ꢁ), 7.46 (1H, m, H-4ꢁ), 7.85 (1H, dd, J = 8.0, 1.8, H-6ꢁ), 10.68 (1H, s, 7-OH), 12.84 (1H, s, 5-OH). Table 1 presents the
1
3
C NMR spectrum. Acid hydrolysis of 4 produced 5,7,22-trihydroxyflavone (4a) and D-glucuronic acid.
Scutevulin 2ꢁ-O-ꢀ-D-glucuronopyranoside (5), yellow crystalline compound, C H O , mp 278–279ꢇÑ (dec.),
2
2 20 12
[
(
ꢈ] –70.4ꢇ (ñ 0.09, Me CO). UV spectrum (EtOH, ꢂmax, nm): 223, 275, 316, 345; (+NaOMe): 230, 258, 282, 320, 344;
+NaOAc): 263, 281, 344, 377; (+AlCl ): 223, 252, 284, 294, 336, 345, 397; (+AlCl /HCl): 225, 247, 282, 294, 332, 345, 398.
IR spectrum (KBr, ꢉ , cm ): 3476 (OH), 1742 (carboxylic C=O), 1665 (ꢃ-pyrone C=O), 1615, 1574 (aromatic C=C bonds).
Í NMR spectrum (400 MHz, DMSO-d , ꢄ, ppm, J/Hz): 3.24–3.43 (m, H-2ꢁꢁ–H-4ꢁꢁ), 3.84 (3H, s, OCH ), 3.92 (1H, d, J = 9.0,
D 2
3
3
–
1
max
1
6
3
H-5ꢁꢁ), 5.23 (1H, d, J = 7.2, H-1ꢁꢁ), 6.34 (1H, s, H-6), 7.06 (1H, s, H-3), 7.28 (1H, m, H-5ꢁ), 7.32 (1H, d, J = 8.0, 1.8, H-3ꢁ), 7.52
(
1H, m, H-4ꢁ), 7.82 (1H, dd, J = 8.0, 1.8, H-6ꢁ), 10.78 (1H, s, 7-OH), 12.54 (1H, s, 5-OH). Table 1 presents the 13C NMR
spectrum. Acid hydrolysis of 5 produced scutevulin (5a) and D-glucuronic acid.
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