Isolation and identification of the phenolic compounds from the roots of Sanguisorba officinalis L. and their antioxidant activities

Article abstract of DOI:10.3390/molecules171213917

Four phenolic compounds were isolated from the roots of Sanguisorba officinalis L. by silica gel column chromatography and preparative HPLC. On the basis of chemical and spectroscopic methods, their structures were identified as methyl 4-O-β-D-glucopyranosy- 5-hydroxy-3-methoxylbenzoate (1), 3,3',4'-tri-O-methylellagic acid (2), fisetinidol-(4a-8)- catechin (3), and (+)-catechin (4). Compound 1 is a new phenolic glycoside and compounds 2 and 3 were isolated from the Sanguisorba genus for the first time. Compounds 1-4 were also assayed for their antioxidant activities using the DPPH free radical assay.

Full text of DOI:10.3390/molecules171213917

Molecules 2012, 17, 13917-13922; doi:10.3390/molecules171213917
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Isolation and Identification of the Phenolic Compounds from
the Roots of Sanguisorba officinalis L. and
Their Antioxidant Activities
Shuang Zhang, Xin Liu, Zi-Long Zhang, Lu He, Zhe Wang and Guang-Shu Wang *
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China
*
Author to whom correspondence should be addressed; E-Mail: wgs@jlu.edu.cn;
Tel.: +86-431-8561-9706.
Received: 26 September 2012; in revised form: 15 November 2012 / Accepted: 16 November 2012 /
Published: 23 November 2012
Abstract: Four phenolic compounds were isolated from the roots of Sanguisorba officinalis L.
by silica gel column chromatography and preparative HPLC. On the basis of chemical and
spectroscopic methods, their structures were identified as methyl 4-O-β-D-glucopyranosy-
5
-hydroxy-3-methoxylbenzoate (1), 3,3′,4′-tri-O-methylellagic acid (2), fisetinidol-(4α-8)-
catechin (3), and (+)-catechin (4). Compound 1 is a new phenolic glycoside and compounds
and 3 were isolated from the Sanguisorba genus for the first time. Compounds 1–4 were
2
also assayed for their antioxidant activities using the DPPH free radical assay.
Keywords: Sanguisorba officinalis; isolation; antioxidant activities; phenolic glycoside;
fisetinidol-(4α-8)-catechin; ellagic acid; catechin
1
. Introduction
Sanguisorba officinalis L. (Rosaceae) is a perennial plant widely distributed in China, and its roots
have been used as a traditional Chinese medicine for the treatment of hemostasis and inflammation [1].
Until now, thirty two phenolic compounds, including tannins and flavonoids, were isolated from
S. officinalis L. [2–7]. Pharmacological studies on its hemostatic and anti-inflammatory properties
have been reported [8,9], but the molecular level mechanisms of these activities have not been reported
until now. In order to study the mechanism of hemostasis, we have carried out the isolation and
identification of bioactive constituents of the roots of S. officinalis L. In a previous paper [10], we have
reported the terpenoid constituents from the roots of S. officinalis L. As another part of our study, we

Molecules 2012, 17
13918
report in the present study the isolation and identification of a new phenolic glycoside 1, together with
three phenolic compounds 2, 3 and 4, and their antioxidant activity.
2
. Results and Discussion
2
.1. Isolation and Identification of Compounds 1–4
Compound 1 was obtained as a colorless amorphous powder, which produced a positive reaction to
FeCl reagent. HR-MS(ESI) indicated the molecular formula of 1 to be C H O . Its IR spectrum
3
15 20 10
indicated the presence of hydroxyl, carbonyl and aromatic groups. Acid hydrolysis of 1 afforded sugar
1
component identified as D-glucose by TLC comparison with an authentic sample. The H-NMR
6
spectrum (DMSO-d ) showed the presence of two aromatic protons at δ 7.03 (s, 1H) and 6.92 (s, 1H),
two methyl groups at δ 3.77 (s, 3H) and 3.79 (s, 3H), and one β-glucopyranose unit from the anomeric
1
3
6
proton at δ 4.83 (d, J = 6.0 Hz). The C-NMR spectrum (DMSO-d ) showed fifteen carbon signals,
among which six are assigned to one sugar unit, nine to the aglycone moiety, and the nine aglycone
moiety carbon signals were attributed to two methyl signals, two methine signals and five quaternary
1
13
carbons by DEPT and HMQC spectra. By analyzing the H-and C-NMR data along with the reported
data [11], the aglycone moiety was identified as the derivative of gallic acid. The correlations of one
methyl protons at δ 3.77 to C-3 at δ 153.0 and another methyl protons at δ 3.79 to C-7 at δ 166.2 in the
HMBC spectrum indicated that one methyl group is connected to C-3 through oxygen and another
methyl group to C-7 through oxygen. The HMBC correlation H-1' at δ 4.83 to C-4 at δ 138.8 revealed
that the linkage position with the glucose unit is at C-4. The complete assignment of the signals of
1
3
compound 1 was based on DEPT C-NMR and 2D NMR of H-H COSY, HMQC and HMBC. All the
1
13
data of H, C, and HMBC NMR of compound 1 see Table 1, and key correlations and the structure of
compound 1 see Figure 1. Therefore, the structure of compound 1 was elucidated as methyl 4-O-β-D-
glucopyranosy-5-hydroxy-3- methoxybenzoate.
1
13
Table 1. H-NMR (400 MHz), C-NMR (100 MHz), HMQC and HMBC data of methyl
4
-O-β-D-glucopyranosy-5-hydroxy-3- methoxylbenzoate (DMSO-d
6
,  ppm).
H
No.
C
H
HMBC
H→C)
No.
C
HMBC
(
(H→C)
aglycone
glc
1 104.5
1
2
125.0
102.9
4.83 (d, 1H, J = 6.0 Hz)
138.8
6.92
111.8, 138.8,
166.2
2
74.0
3.27 (m, 1H)
(
(
s, 1H)
3
4
5
6
153.0
138.8
152.6
111.8
3
4
5
6
76.5
69.6
77.3
60.7
3.22 (m, 1H)
3.20 (m, 1H)
3.13 (m, 1H)
7.03
102.9, 138.8,
166.2
3.49 (m, 1H),
s, 1H)
3.62 (d-like, 1H, J = 10.4 Hz)
7
166.2
56.2
51.9
3
7
-OCH3
-OCH3
3.77
3.79
153.0
166.2
1
1
All assignments based on extensive 1D and 2D NMR experiments (HMQC, HMBC, H- H COSY).

Molecules 2012, 17
13919
Using similar methods as described above, compounds 2–4 were identified as 3,3′,4′-tri-O-
methylellagic acid (2) [12], fisetinidol-(4α-8)-catechin (3) [13], and (+)-catechin (4) [14], respectively.
Figure 1. The key HMBC correlations of methyl 4-O-β-D-glucopyranosy-5-hydroxy-3-methoxylbenzoate.
OH
H C
3
O
6
'
4
'
3
2
O
5
'
O
O
HO
4
1
1
'
HO
O
7
2
'
3
'
OH
CH3
5
6
HO
Note: Arrows point from proton to carbon.
2
.2. Antioxidant Activity of Compounds 1–4
Compounds 1–4 were next assayed for their antioxidant activity with the DPPH free radical assay,
and the results are shown in Table 2. The data proved that fisetinidol-(4α-8)-catechin showed the
strongest antioxidant activity.
Table 2. The antioxidant assay data of the isolated compounds.
Compound
IC50 (ug/mL)
720 ± 7.3
Methyl 4-O-β-D-glucopyranosy-5-hydroxy-3-methoxylbenzoate (1)
3
,3′,4′-tri-O-Methylellagic acid (2)
Fisetinidol-(4α-8)-catechin (3)
+)-Catechin (4)
Note: All values are averages of at least three runs in Table 2.
820 ± 7.3
12.3 ± 0.2
38.2 ± 0.5
(
3. Experimental
3.1. General
IR spectra were recorded on a FT-IR 5DX Nicolet/Nicolet Magna IR-560 spectrometer (Thermo
1
13
Scientific, Osaka, Japan). H- and C-NMR spectra were recorded on a Bruker AV-400 spectrometer
Zürich, Switzerland). HR-ESI-MS were recorded on a Bruker microOTOF-Q II mass spectrometer.
Prep. HPLC was performed on a Shimadzu LC-10A equipped with a SPD-10A detector and Gemini 5 μm
18 110A column (250 mm × 10.00 mm, 5 μm, flow rate: 3.0 mL/min). The bioactivities were
(
C
measured on WFZ UV-2100 ultraviolet visible spectrophotometer (Unico Shanghai Instrument
Company Limited, Shanghai, China), using the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH,
Sigma-Aldrich, Shanghai, China). The roots of S. officinalis L. were collected in Tong-Hua County in
Jilin Province, China. They were identified by Prof. Jing-Min Zhang of the School of Pharmaceutical
Sciences, Jilin University, Changchun, China.

Molecules 2012, 17
.2. Extraction and Isolation
The air-dried the roots of S. officinalis (4.0 kg) were extracted with 70% EtOH (ca. 20 L, 24 h,
13920
3
room temperature). The EtOH extract was concentrated under reduced pressure, and the viscous
concentrate (420 g) was passed through a D101 polyporous resin column eluting successively with
H O, 30% EtOH, 70% EtOH, and 95% EtOH, and by vacuum distillation recovery, four fractions were
2
obtained. The 30% ethanol eluate was further chromatographed repeatedly on silica gel columns and
then purified by preparative RP-HPLC with CH OH–H O (30:70) to yield the new compound 1 (20 mg).
3
2
The 95% ethanol eluate was further chromatographed repeatedly on silica gel columns eluted with
CHCl –MeOH–EtOAc–H O (3:1:4:2.5, 3:1:7:1.5) to yield compound 2 (200 mg). The 70% EtOH
fraction was subjected to silica gel column chromatography eluted with a stepwise gradient mixture of
CHCl –MeOH (9:1; 6:1; 3:1), and finally with MeOH alone, and four fractions I–IV were obtained.
Fraction IV was further subjected to a silica gel column eluted with CHCl –MeOH–EtOAc–H O
3
2
3
3
2
(6.5:5:4:1.7), and three fractions (A, B, C) were obtained. Fraction A was applied to a ODS-A (50 μm,
1
2 nm, YMC, Kyoto, Japan) column eluted with a stepwise gradient mixture of MeOH–H
2
O (2:3; 3:2;
4
:1) to yield compound 3 (230 mg). Fraction C was first separated by a ODS-A (50 μm, 12 nm, YMC,
Kyoto, Japan) column eluted with a stepwise gradient mixture of MeOH–H O (2:3; 3:2; 4:1), and then
2
2
purified by preparative HPLC using MeOH–H O (80:20) to yield compound 4 (20 mg).
Methyl 4-O-β-D-glucopyranosy-5-hydroxy-3-methoxylbenzoate (1): Colorless amorphous powders,
+
3
produced a positive reaction to FeCl reagent, m.p. 212–214 °C. HRESIMS, m/z: 383.0948 [M+Na]
−1
(
calcd for 383.0949). IR (KBr) max cm : 3375 (OH), 1703 (C=O), 1578, 1502, 1403 (aromatic C=C),
1
13
1052 (C–O–C). H and C-NMR: See Table 1.
3
,3′,4′-Tri-O-methylellagic acid (2): Pale yellow amorphous powder, produced a positive reaction to
+
1
3 6
FeCl reagent. ESIMS, m/z: 345 [M+H] . H-NMR (DMSO-d ) δ: 10.46 (1H, s, C4-OH), 7.53 (1H, s,
5
-H), 7.60 (1H, s, 5′-H), 4.07 (3H, s, C3-OCH ), 4.09 (3H, s, C3′-OCH ), 4.01 (3H, s, C4′-OCH ).
3 3 3
1
3
C-NMR (DMSO-d
58.3 (C-7), 111.9 (C-1′), 141.5 (C-2′), 140.8(C-3′), 153.8 (C-4′), 107.5 (C-5′), 113.4 (C-6′), 158.5 (C-7′);
1.0 (C3-OCH ), 61.3 (C3′-OCH ), 56.7 (C4′-OCH ).
6
) δ: 111.6 (C-1), 141.0 (C-2 ), 140.2 (C-3), 152.6 (C-4), 111.2 (C-5), 112.5 (C-6),
1
6
3
3
3
Fisetinidol-(4α-8)-catechin (3): yellow amorphous powder produced a positive reaction to FeCl
3
+
1
reagent. HRESIMS, m/z: 563.1536 [M+H] ; (calcd for C30
H
27
O11, 563.1548). H-NMR (DMSO-d
6
) δ:
2
.41 (dd, J = 16.0 and 9.2 Hz, H-4F), 2.84 (dd, J = 16.0 and 5.2 Hz, H-4F), 3.76 (m, H-3F), 4.32 (d,
J = 8.5 Hz, H-4C), 4.35 (d, J = 8.5 Hz, H-2C), 4.45 (t, J = 8.5 Hz, H-3C), 4.48 (d, J = 8.4 Hz, H-2F),
6
2
.05 (dd, J = 8.0 and 1.5 Hz, H-6′E), 5.92(s, H-6D), 6.06 (d, J = 2.4 Hz, H-8A), 6.17 (dd, J = 8.0 and
.4 Hz, H-6A), 6.48 (d, J = 8.0 Hz, H-5A), 6.65 (dd, J = 8.0 and 2.0 Hz, H-6′B and H-6′E), 6.66 (d,
J = 8.0 Hz, H-5′E and H-5A), 6.68 (d, J = 8.0 Hz,H-5′B), 6.76 (d, J = 2.0 Hz, H-2′B), 6.80 (d, J = 2.0 Hz,
1
3
H-2′E). C-NMR (DMSO-d ) δ: 28.8 (C-4F), 40.0 (C-4C), 66.9 (C-5F), 67.9 (C-3C), 81.4 (C-2F),
6
8
2.9 (C-2C), 95.9 (C-6D), 98.6 (C-4aF), 101.9 (C-8A), 106.5 (C-8D), 108.0 (C-6A), 114.7 (C-2′E),
14.8 (C-5′E), 115.0 (C-2′B), 115.2 (C-5′B),118.2 (C-4aC), 118.4 (C-6′E), 119.6 (C-6′B), 128.5
1
(C-5A), 130.9 (C-1′E), 131.2 (C-1′B), 144.8 (C-3′B and C-3′E), 144.9 (C-4′B and C-4′E), 153.8 (C-7D),
1
54.2 (C-8aA), 154.7 (C-8aD), 155.0 (C-5D), 156.6 (C-7A).

Molecules 2012, 17
13921
(
+)-Catechin (4): Pale yellow amorphous powder, produced a positive reaction to FeCl reagent.
3
+
1
ESIMS, m/z: 291 [M+H] . H-NMR (DMSO-d
6
) δ: 2.35 (1H, dd, J = 15.9, 7.8 Hz, H-4), 2.66 (1H, dd,
J = 15.9, 4.8 Hz, H-4), 3.81 (1H, m, H-3), 4.48 (1H, d, J = 7.3 Hz, H-2), 5.69 (1H, s, H-8), 5.89 (1H,
1
3
s, H-6), 6.59 (1H, d, J = 7.8 Hz, H-6′), 6.68 (1H, d, J = 7.8 Hz, H-5′), 6.72 (1H, s, H-2′). C-NMR
DMSO-d ) δ: 27.8 (C-4), 66.3 (C-3), 81.0 (C-1), 93.8 (C-8), 95.1 (C-6), 99.0 (C-4a), 114.5 (C-2′),
15.0 (C-5′), 118.4 (C-6′), 130.6 (C-1′), 144.8 (C-3′,4′), 155.3 (C-8a), 156.1 (C-5), 156.4 (C-7).
(
6
1
3
.3. Acid Hydrolysis of 1
Solution of 1 (1.0 mg) in 0.5 M H SO (2.0 mL) was heated under reflux for 3 h. After cooling, the
2
4
reaction mixture was diluted with H
2
3
O, neutralized with BaCO , then filtered. The solution was
partitioned with EtOAc to give two layers. The aqueous layer was evaporated and then subjected to
TLC analysis with authentic sugar samples using n-BuOH–MeOH–CHCl –HOAc (12.5:4.5:9:1.5:1,
detection with aniline-phthalic acid). Compounds 1 afforded D-glucose (R = 0.30).
3
f
3.4. Bioactivity Assay
The antioxidant activity of compounds 1–4 were assessed according to their DPPH scavenging
ability. Reaction mixtures, containing 0.5 mL of the relevant compound (dissolved in EtOH) and 2.5 mL
of a 100 µM DPPH ethanolic solution, were added to 96-well microtiter plates and incubated at 37 °C
for 30 min. Absorbances were measured at 515 nm. Percent inhibition was determined by comparison
with an EtOH-treated control group. IC50 values denote the concentration of samples required to
scavenge 50% of the DPPH free radicals.
4
. Conclusions
Compound 1 is a new phenolic glycoside and compounds 2 and 3 were isolated from the
Sanguisorba genus for the first time. Compounds 1–4 were assayed for their antioxidant activity with
DPPH free radicals, and the data proved that fisetinidol-(4α-8)-catechin showed the strongest
antioxidant activity.
Acknowledgments
This work was supported by National Science and Technology Major Project of China
(
2009ZX09502-011). The authors gratefully thank Jing-Min Zhang (School of Pharmaceutical
Sciences, Jilin University) for the identification of plants.
References
1.
The Editorial Board of Zhong Hua Ben Cao of State Administration of Traditional Chinese
Medicine of the People’s Republic of China. Zhong Hua Ben Cao 4, 1st ed.; Scientific and
Technical Publishers: Shanghai, China, 1999; p. 281.
2
.
Nonaka, G.; Tanaka, T.; Nishioka, I. Tannins and related compounds. Part 3. A new phenolic
acid, sanguisorbic acid dilactone, and three new ellagitannins, sanguiins H-1, H-2, and H-3, from
Sanguisorba officinalis. J. Chem. Soc. Perkin. Trans. 1 1982, 4, 1067–1073.

Molecules 2012, 17
13922
3
.
.
Nonaka, G.; Tanaka, T.; Nita, M.; Nishioka, I. A dimeric hydrolyzable tannin, sanguiin H-6 from
Sanguisorba officinalis L. Chem. Pharm. Bull. 1982, 30, 2255–2257.
4
Tanaka, T.; Nonaka, G.; Nishioka, I. Tannins and related compounds. XXVIII: Revision of the
structures of sanguiins H-6, H-2, and H-3 and isolation and characterization of sanguiin H-11,
a novel tetrameric hydrolysable tannin, and seven related tannins, from Sanguisorba officinalis.
J. Chem. Res. 1985, 6, 176–177.
5
.
.
Tanaka, T.; Nonaka, G.; Nishioka, I. 7-O-galloyl-(+)-catechin and 3-O-galloylprocyanidin B-3
from Sanguisorba officinalis. Phytochemistry 1983, 22, 2575–2578.
6
Tanaka, T.; Nonaka, G.; Nishioka, I. Tannins and related compounds XVI. Isolation and
characterization of six methyl glucoside gallates and a gallic acid glucoside gallate from
Sanguisorba officinalis L. Chem. Pharm. Bull. 1984, 32, 117–121.
7
8
9
1
1
.
.
.
Cheng, D.L.; Cao, X.P.; Zhou, P.X.; Yang, P. Isolation and identification of flavonoid compounds
in Sanguisorba officinalis. Chin. Tradit. Herb. Drugs 1995, 26, 570–571.
Yu, B.B.; Zhong, F.X.; Dong, X. Progress on chemical ingredient of Sanguisorba officinalis L.
Chin. J. Inf. TCM 2009, 16 (Suppl.), 103–105.
Xia, H.M.; Sun, L.L.; Sun, J.Y.; Zhong, Y. Progress on chemical ingredient and pharmacological
activity of Sanguisorba officinalis L. Food Drug 2009, 11, 67–69.
0. Sun, W.; Zhang, Z.L.; Liu, X.; Zhang, S.; He, L.; Wang, Z.; Wang, G.S. Terpene glycosides from
the roots of Sanguisorba officinalis L. and their hemostatic activities. Molecules 2012, 17, 7629–7636.
1. Ding, Y.; Liang, C.; Nguyen, H.T.; Choi, E.M.; Kim, J.A.; Kim, Y.H. Chemical constituents
from Acer mandshuricum and their effects on the function of osteoblastic MC3T3-E1 cells.
Bull. Korean Chem. Soc. 2010, 31, 929–933.
1
1
1
2. Shi, X.H.; Du, X.L.; Kong, L.Y. Studies on chemical constituents in roots of Euphorbia
soongarica (In Chinese). Zhongguo Zhong Yao Za Zhi 2006, 31, 1503–1506.
3. Duan, W.G.; Ohara, S.; Hashida, K.; Makino, R. Condensed tannins from steamed Acacia
mearnsii bark. Holzforschung 2005, 59, 289–294.
4. Sun, Y.; Xu, B.C.; Xu, D.P.; Gu, W.Y. Study on several phenolic compounds in grape seeds.
J. Henan Univ. Technol. (Nat. Sci. Ed.) 2006, 27, 73–77.
Sample Availability: Samples of the compounds 2 and 3 are available from the authors.
©
2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(