Polyphenolic constituents of cynomorium songaricum Rupr. and antibacterial effect of polymeric proanthocyanidin on methicillin-resistant staphylococcus aureus

Article abstract of DOI:10.1021/jf301621e

Oligomeric and polymeric flavan-3-ols were obtained by chromatographic fractionation of extracts from Cynomorium songaricum Rupr. The structure of the polymeric constituent, cynomoriitannin, was characterized using spectral and chemical data. Results from acid-catalyzed degradation indicated that cynomoriitannin is a polymeric proanthocyanidin predominantly composed of epicatechin, together with low proportions of epicatechin-3-O-gallate and catechin as extension units. The terminal unit was chiefly composed of catechin, with an admixture of epicatechin. Size exclusion chromatographic analysis demonstrated a mean polymerization degree of 14. Two new phloroglucinol adducts (cynomoriitannin-phloroglucinol adducts A and B) obtained by acid-catalyzed degradation of cynomoriitannin in the presence of phloroglucinol were characterized using spectral analyses. Six oligomeric flavan-3-ols were also identified as follows: procyanidin B3, catechin-(6'-8)-catechin, catechin-(6′-6)-catechin, epicatechin-(4β-8)- epicatechin-(4β-8) -catechin, epicatechin-(4β-6)-epicatechin-(4β-8)-catechin, and arecatannin A1, respectively. These flavan-3-ols were isolated from C. songaricum. This is the first time that this procedure has been described. The antibacterial activity of the fractions and constituents was tested against methicillin-resistant Staphylococcus aureus (MRSA). The crude acetone-water (7:3) extract had moderate activity against MRSA. Cynomoriitannin was the most effective of the plant constituents against MRSA.

Full text of DOI:10.1021/jf301621e

Article
pubs.acs.org/JAFC
Polyphenolic Constituents of Cynomorium songaricum Rupr. and
Antibacterial Effect of Polymeric Proanthocyanidin on Methicillin-
Resistant Staphylococcus aureus
Shangwu Jin,†,‡ Eerdunbayaer,‡ Airi Doi,‡ Teruo Kuroda,‡ Guixia Zhang,† Tsutomu Hatano,*,‡
and Guilin Chen*,†
†College of Life Sciences, Inner Mongolia University, Hohhot, Inner Mongolia, China
‡Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Tsushima, Okayama 700-8530, Japan
ABSTRACT: Oligomeric and polymeric flavan-3-ols were obtained by chromatographic fractionation of extracts from
Cynomorium songaricum Rupr. The structure of the polymeric constituent, cynomoriitannin, was characterized using spectral and
chemical data. Results from acid-catalyzed degradation indicated that cynomoriitannin is a polymeric proanthocyanidin
predominantly composed of epicatechin, together with low proportions of epicatechin-3-O-gallate and catechin as extension
units. The terminal unit was chiefly composed of catechin, with an admixture of epicatechin. Size exclusion chromatographic
analysis demonstrated a mean polymerization degree of 14. Two new phloroglucinol adducts (cynomoriitannin-phloroglucinol
adducts A and B) obtained by acid-catalyzed degradation of cynomoriitannin in the presence of phloroglucinol were
characterized using spectral analyses. Six oligomeric flavan-3-ols were also identified as follows: procyanidin B3, catechin-(6′-8)-
catechin, catechin-(6′-6)-catechin, epicatechin-(4β-8)- epicatechin-(4β-8)-catechin, epicatechin-(4β-6)-epicatechin-(4β-8)-
catechin, and arecatannin A1, respectively. These flavan-3-ols were isolated from C. songaricum. This is the first time that this
procedure has been described. The antibacterial activity of the fractions and constituents was tested against methicillin-resistant
Staphylococcus aureus (MRSA). The crude acetone−water (7:3) extract had moderate activity against MRSA. Cynomoriitannin
was the most effective of the plant constituents against MRSA.
KEYWORDS: proanthocyanidin, Cynomorium songaricum Rupr., antibacterial effect, methicillin-resistant Staphylococcus aureus,
phloroglucinol adducts
oligomeric flavan-3-ols and described the structural character-
ization of polymeric PA. We also investigated the antibacterial
activity of these extracts against MRSA.
INTRODUCTION
■
Proanthocyanidins (PAs) are oligomers and polymers of flavans
that are widely distributed in the plant kingdom. Previous
studies have demonstrated the antibacterial and other
pharmacological effects of PA oligomers and their gallate
esters.1 Epigallocatechin gallate and its oxidative products have
been shown to have antibacterial activity against methicillin-
resistant Staphylococcus aureus (MRSA), and they markedly
decrease the minimum inhibitory concentrations (MICs) of
oxacillin and other antibiotics.2−4 Condensed procyanidins
isolated from the fruit peel of Zanthoxylum piperitum have also
been shown to be synergistic with β-lactams against MRSA.5
Cynomorium songaricum Rupr. is an obligate root parasitic
plant that mainly grows in Northwestern China and Central
Asia. It is a source of healthy food and nutrients6 and has been
widely used in traditional Chinese medicine. A previous report
indicated that procyanidins obtained from the methanol extract
of this plant possessed potent superoxide (SOD)-like and α-
glucosidase inhibitory activity. These properties have been
shown to increase with increasing degrees of polymerization.7
Polymeric procyanidin, which is mainly composed of
epicatechin, was reported to have the most potent activities
among them. These procyanidins have also been reported to
act as inhibitory properties against HIV-1 protease in water
extracts of C. songaricum.8 However, to date, the oligomeric and
polymeric flavan-3-ols from C. songaricum have not been
adequately characterized. In this study, we identified six
MATERIALS AND METHODS
■
General Procedures. Electrospray ionozation (ESI)-MS spectra
were recorded using a Bruker amaZon ETD spectrophotometer in the
negative-ion mode. Circular dichroism (CD) spectra were measured
on a JASCO J-720 W spectrophotometer. 1H and 13C nuclear
magnetic resonance (NMR) spectra were recorded on a Varian
INOVA AS 600 instrument (600 MHz for H and 150 MHz for 13C)
1
at 27 °C, in acetone-d6 or acetone-d6 containing 10% D2O (v/v),
unless mentioned otherwise. Chemical shifts were expressed as δ
(ppm) relative to tetramethylsilane, based on solvent signals (acetone-
d6: δH 2.04 and δC 29.8).
Diaion HP-20 (Mitsubishi Chemicals, Tokyo, Japan), MCI gel
CHP-20P (Mitsubishi Chemicals), and Toyopearl HW-40 (Tosoh,
Tokyo, Japan) were used for column chromatography. Normal-phase
high-performance liquid chromatography (HPLC) was performed at
room temperature on a YMC-Pack SIL A003 (YMC, Kyoto, Japan)
column (4.6 mm i.d. × 250 mm) with n-hexane−MeOH−
tetrahydrofuran−formic acid (55:33:11:1, v/v/v/v) containing 450
mg/L oxalic acid as the solvent.
Received: April 15, 2012
Revised: June 30, 2012
Accepted: June 30, 2012
Published: July 2, 2012
© 2012 American Chemical Society
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Figure 1. Structures of compounds isolated from C. songaricum.
Analytical reversed-phase HPLC was carried out on a YMC-Pack
ODS A302 (4.6 i.d. × 150 mm, YMC) column eluted with 0.01 M
H3PO4−0.01 M KH2PO4−MeOH (9:9:2, v/v/v) at 40 °C unless
mentioned otherwise. Preparative reversed-phase HPLC was per-
formed on a YMC-Pack ODS A324 (10 mm i.d. × 300 mm, YMC)
column. HPLC-UV analyses detection was undertaken at 280 nm.
Materials. Fresh stems of C. songaricum were collected in a
marginal zone of Hobq Desert, Inner Mongolia, China (39.83 N, 108.7
E), on May 7, 2008. The voucher specimen was kept in the Herbarium
of Inner Mongolia University. The plants were dried at room
temperature in a dark space.
The MRSA strain (OM 623) used in this study was a clinical isolate
from Okayama University Hospital (Okayama, Japan). Catechin,
epicatechin, and phloroglucinol were purchased from Sigma (St. Louis,
MO). Solvents used for HPLC were purchased from Merck
(Darmstadt, Germany), and those for LC-MS were from Sigma.
Oxacillin, used in the bacterial experiments, was purchased from
Sigma.
HP-20 (5 cm i.d. × 30 cm) column, and adsorbed compounds were
successively eluted with H2O, 20, 40, 60, and 100% aqueous MeOH,
and 70% aqueous acetone to give the corresponding H2O (28.8 g), 20
(1.53 g), 40 (5.49 g), 60 (3.22 g), and 100% (0.11 g) aqueous MeOH,
and 70% aqueous acetone (47 mg) fractions, respectively.
Separation of each of the fractions was monitored with normal- and
reversed-phase HPLC. The 40% aqueous MeOH fraction was
subjected to column chromatography over Toyopearl HW-40 (2.2
cm i.d. × 35 cm) with 70% aqueous EtOH and then with 70% aqueous
acetone to give fractions I−V. Fraction II (112.4 mg) was
chromatographed on MCI gel CHP-20P (1.1 cm i.d. × 30 cm) with
0% → 10% → 20% → 30% → 100% MeOH in water, to give
(+)-catechin (compound 1, 19.3 mg) and (−)-epicatechin (compound
2, 2.3 mg) (Figure 1). Fraction III (239.3 mg) was fractionated by
column chromatography on MCI gel CHP-20P (1.1 cm i.d. × 30 cm)
with 0% → 10% → 15% → 20% → 30% → 100% MeOH in water, to
give procyanidin B1 (compound 3, 7.2 mg), procyanidin B3
(compound 4, 8.4 mg), catechin-(6′-8)-catechin (compound 5, 1.7
mg), and catechin-(6′-6)-catechin (compound 6, 1.4 mg). Fraction IV
(224.1 mg) was chromatographed on a MCI gel CHP-20P (1.1 cm i.d.
× 30 cm) with 0% → 10% → 15% → 20% → 30% → 100% MeOH in
water, to give epicatechin-(4β-8)-epicatechin- (4β-8)-catechin (com-
Purification of Oligomeric and Polymeric Flavan-3-ols from
C. songaricum. The stems of C. songaricum (100 g) were
homogenized in 70% aqueous acetone (v/v, 3 L) at room temperature.
The filtered homogenate was concentrated to 500 mL at 40 °C under
reduced pressure. The concentrated solution was applied to a Diaion
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Figure 2. 13C NMR spectra of cynomoriitannin (10). The signals indicated by 2T, 3T, and 4T are of the terminal unit. The spectrum also suggested
the presence of gallate structure in 10. The signal at δ 49.8 is due to the residual MeOH in the sample.
pound 7, 8.6 mg). Fraction V (224.1 mg) was subjected to a MCI gel
CHP-20P (1.1 cm i.d. × 30 cm) column with 0% → 10% → 15% →
20% → 30% → 100% aqueous MeOH, and factions were further
purified by preparative HPLC to give epicatechin-(4β-6)-epicatechin-
(4β-8)-catechin (compound 8, 3.4 mg) and a tetrameric procyanidin,
arecatannin A1 (compound 9, 5.3 mg). The eluate in 70% acetone
from the Toyopearl HW-40 column (fraction V) was a polymeric PA,
cynomoriitannin (compound 10, 2.46 g).
Epicatechin-(4β-6)-epicatechin-(4β-8)-catechin (8). Compound 8
20
was an off-white amorphous powder, [α]D +117.8° (MeOH, c 1.0).
ESI-MS m/z: 865 [M − H]−. 1H NMR: δ 6.96−6.58 (9H, br), 6.08−
5.92 (4H, br), 4.89−4.72 (3H, br), 4.55−4.45 (2H, br), 4.01−3.87
(3H, br), 2.63 (br), 2.50 (br). 13C NMR: δ 159.0−155.2 (9C), 145.4−
144.8 (6C), 131.9−130.6 (3C), 119.4−119.0 (3C), 115.8−114.7
(6C), 107.8−106.8 (2C), 101.3−100.4 (3C), 97.0−95.4 (4C), 81.8,
77.0−76.2 (2C), 72.1−71.2 (2C), 67.3, 37.1 (2C), 27.7.
Procyanidin B3 (4). Compound 4 was an off-white amorphous
Arecatannin A1 (9). Compound 9 was an off-white amorphous
20
1
20
powder, [α]D −192.8° (MeOH, c 1.0). H NMR: Major rotamer, δ
6.77 (d, J = 1.8 Hz, H-2U′), 6.76 (d, J = 7.8 Hz, H-5U′), 6.65 (d, J = 9
Hz, H-2L′), 6.62 (d, J = 2.4 Hz, H-2L′), 6.47 (dd, J = 1.8, 7.8 Hz, H-
6U′), 6.22 (dd, J = 1.8, 7.8 Hz, H-6L′), 6.15 (brs, H-6L), 5.90 (d, J = 2.4
Hz, H-6U), 5.78 (d, J = 2.4 Hz, H-8U), 4.58 (d, J = 7.2 Hz, H-2L), 4.39
(m, 2H, H-3U, 4U), 4.24 (m, H-2U), 3.80(m, H-3L), 2.69 (dd, J = 6,
15.6 Hz, H-4La), 2.49 (dd, J = 7.8, 16.2 Hz, H-4Lb). Minor rotamer, δ
7.01 (d, J = 1.8 Hz, H-2L′), 6.95 (d, J = 1.8 Hz, H-2U′), 6.83 (dd, J =
1.8, 8.4 Hz, H-6L′), 6.79 (dd, J = 1.8, 7.8 Hz, H-6U′), 6.75 (d, J = 7.8
Hz, H-5L′), 6.67 (d, J = 7.8 Hz, H-5U′), 6.03 (s, H-6L), 5.99 (d, J = 2.4
Hz, H-6U), 5.83 (d, J = 2.4 Hz, H-8U), 4.68 (d, J = 7.8 Hz, H-2L), 4.51
(m, 2H, H-3U, 4U), 4.34 (m, H-2U), 4.01 (m, H-3L), 2.86 (dd, J = 5.4,
16.2 Hz, H-4La), 2.56 (dd, J = 7.8, 16.2 Hz, H-4Lb).
powder, [α]D +127.2° (MeOH, c 1.0). ESI-MS m/z: 1153 [M −
H]−. 1H NMR: δ 6.98−6.65 (12H, br), 6.01−5.93 (5H, br), 5.14−4.86
(4H, br), 4.65−4.56 (3H, br), 4.07−3.88 (4H, br), 2.68−2.66 (1H,
br), 2.56 (dd, J = 5.4, 16.2 Hz). 13C NMR: δ 158.5−154.3 (12C),
145.9−144.8 (8 C), 133.1−130.2 (4C), 120.1−118.7 (4C), 115.9−
114.4 (8C), 107.3−106.0 (3C), 100.7 (3C), 98.6, 96.8−95.0 (5C),
81.4, 76.9−76.4 (3C), 72.7−71.1 (3C), 67.4, 37.1−36.7 (3C), 27.5.
Cynomoriitannin (10). Compound 10 was a light-brown
amorphous powder. 13C NMR: δ 29−37 (C-4), 70−81 (C-3, C-2),
95−110 (C-4a, C-6, C-8), 113−120 (C-2′, C-5′, C-6′), 130−132 (C-
1′), 143−147 (C-3′, C-4′), 152−159 (C-5, C-7, C-8a) (Figure 2).
Size Exclusion Chromatography (SEC). The molecular weight
distribution of cynomoriitannin as the polymeric flavan-3-ol fraction
was characterized by SEC9 using TSK gel Super AW3000 column (6.0
mm i.d. × 150 mm; Tosoh, Japan) with N,N-dimethylformamide
(DMF) containing 0.5% (v/v) 3 M HCOONH4 as a mobile phase at
40 °C. The flow rate was maintained at 0.5 mL/min, and the elution
was monitored at 280 nm. (+)-Catechin (1) [retention time (tR), 4.95
min), procyanidin B1 (3) (tR 4.67 min), epicatechin-(4β-8)-
epicatechin-(4β-8)-catechin (7) (tR 4.43 min), and arecatannin A1
(9) (tR 4.23 min) were used as molecular weight markers. The
equation (Log M = −0.863 × tR + 6.6269, R2 = 0.985) was obtained to
describe that relationship between M and tR. The number average
molecular weight (Mn) and weight average molecular weight (Mw) of
cynomoriitannin were calculated from its SEC profile, based on this
equation.
Catechin-(6′-8)-catechin (5). Compound 5 was an off-white
20
amorphous powder, [α]D −204.8° (MeOH, c 1.0). ESI-MS m/z:
577 [M − H]−. 1H NMR (CD3OD): δ 6.81 (brs, H-2U′), 6.70 (d, J =
1.8 Hz, H-2L′), 6.68 (d, J = 7.8 Hz, H-5L′), 6.63 (brs, H-5U′), 6.58 (dd,
J = 1.8, 7.8 Hz, H-6L′), 6.10 (brs, H-6L), 5.88 (d, J = 2.4 Hz, H-8U),
5.80 (d, J = 1.8 Hz, H-6U), 4.75 (d, J = 6.6 Hz, H-2U), 4.70 (d, J = 6.0
Hz, H-2L), 3.98 (m, H-3L), 3.95 (m, H-3U), 2.72 (dd, J = 5.4, 16.8 Hz,
H-4Lb), 2.66 (dd, J = 4.7, 16.2 Hz, H-4Ub), 2.58 (dd, J = 5.4, 16.2 Hz,
H-4La), 2.40 (dd, J = 6.6, 16.2 Hz, H-4Ua).
Catechin-(6′-6)-catechin (6). Compound 6 was an off-white
20
amorphous powder, [α]D −30.4° (MeOH, c 1.0). ESI-MS m/z:
577 [M − H]−. 1H NMR (CD3OD): δ 6.81 (br s, H-2U′), 6.74 (d, J =
2.4 Hz, H-2L′), 6.70 (d, J = 8.4 Hz, H-5L′), 6.63 (dd, J = 2.4, 8.4 Hz,
H-6L′), 6.55 (br s, H-5U′), 6.08 (br s, H-6L), 5.90 (d, J = 2.4 Hz, H-
8U), 5.88 (d, J = 2.4 Hz, H-6U), 4.85 (d, J = 4.8 Hz, H-2U), 4.52 (d, J =
6.6 Hz, H-2L), 4.11 (m, H-3U), 3.99 (m, H-3L), 2.93 (dd, J = 6.0, 16.2
Hz, H-4Lb), 2.58 (2H, m, H-4Ub, 4La), 2.48 (dd, J = 6.0, 16.2 Hz, H-
4Ua).
Acid-Catalyzed Degradation of Cynomoriitannin in the
Presence of Phloroglucinol. Cynomoriitannin (1 mg) was treated
with 1% HCl in EtOH (0.2 mL) in the presence of phloroglucinol (1
mg) overnight at room temperature as described previously.9 LC-MS
on an ODS column (YMC-Triart C18, 100 mm × 2.0 mm, 3 μm) was
performed for the identification of degradation products. Elution was
undertaken with two solvents, A (1% aqueous formic acid) and B
(acetonitrile−water−formic acid, 50:49:1, v/v/v) using the following
gradient elution program: initially 5% B, 5−7% B over 10 min, 7% B
for 5 min, then 7−35% B over 35 min, and 35−40% B over 10 min.
Eluted materials were monitored with UV at 280 nm and with ESI-MS
detector in the negative-ion mode. The formation of the catechin-
phloroglucinol adduct ([M − H]−, m/z: 413), the epicatechin−
Epicatechin-(4β-8)-epicatechin-(4β-8)-catechin (7). Compound 7
20
was an off-white amorphous powder, [α]D +63.8° (MeOH, c 1.0).
ESI-MS m/z: 865 [M − H]−. 1H NMR: δ 6.98−6.66 (9H, br), 6.02−
5.90 (4H, br), 5.06−4.71 (5H, br), 4.10−3.94 (3H, br), 2.69 (br s, H-
4La), 2.58 (dd, J = 7.2, 16.8 Hz, H-4Lb). 13C NMR: δ 157.4−155.2
(9C), 145.4, 145.2 (3C), 145.0, 144.8, 132.1 (3C), 119.1−118.7 (3C),
115.8, 115.6, 115.5 (2C), 114.9, 114.5, 109.6, 107.3, 105.2, 100.3, 97.0
(2C), 96.4, 95.8 (2C), 81.7, 76.7 (2C), 72.8, 71.5, 67.6, 36.8 (2C).
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phloroglucinol adduct ([M − H]−, m/z: 413), the (epi)catechin
dimer−phloroglucinol adduct ([M − H]−, m/z: 701), catechin ([M −
H]−, m/z: 289), and the (epi)catechin trimer−phloroglucinol adduct
([M-H]−, m/z: 989) were demonstrated by their molecular weights
and also by HPLC comparisons with isolated products.
7.8 Hz, H-6′), 6.38 (d, J = 1.2 Hz, H-6), 6.37 (d, J = 1.8 Hz, H-8), 5.96
(2H, s, H-3″, 5″), 5.92 (2H, brs, H-3‴, 5‴), 5.18 (s, H-2), 4.61 (d, J =
1.2 Hz, H-4), 3.95 (t, J = 1.2 Hz, H-3). 13C NMR: δ 158.2 (C-4‴),
157.6 (C-4″), 157.4 (2C, C-2‴, 6‴), 157.1 (C-5), 156.6 (C-8a), 156.3
(2C, C-2″, 6″), 145.2−144.9 (2C, C-3′, 4′), 133.9 (C-7), 132.3 (C-1′),
118.8 (C-6′), 115.4−115.0 (2C, C-2′, 5′), 110.8 (C-8), 110.3 (C-6),
109.2 (C-1″), 109.0 (C-1‴), 107.0 (C-4a), 96.0 (C-3‴, 5‴), 95.3 (C-
3″, 5″), 76.4 (C-2), 72.5 (C-3), 36.8 (C-4).
The HPLC profile of the products (Figure 3) indicated the presence
of the following compounds in the reaction mixture: catechin-(4α-2)-
Cynomoriitannin-phloroglucinol B (14). Compound 14 was a
20
light-brown amorphous powder, [α]D +16.8° (MeOH, c 1.0). ESI-
MS m/z: 685 [M − H]−, CD (MeOH) [θ] (nm): −6.6 × 104 (202),
+3.8 × 103 (217), −2.9 × 104 (222), −2.8 × 104 (226), −1.3 × 104
(239), +1.4 × 104 (287). 1H NMR: δ 6.98 (d, J = 2.4 Hz, H-2U′), 6.73
(d, J = 8.4 Hz, H-5U′), 6.65 (dd, J = 2.4, 7.8 Hz, H-6U′), 6.51 (d, J =
8.4 Hz, H-5L′), 6.24 (br s, H-2L′), 6.05 (d, J = 7.8 Hz, H-6L′), 5.93 (d,
J = 2.4 Hz, H-8U), 5.92 (br s, H-6L), 5.90 (d, J = 2.4 Hz, H-6U), 5.89
(br s, H-3″, 5″), 5.11 (br s, H-2U), 4.57 (d, br, J = 8.4 Hz, H-β), 4.41
(d, br, J = 7.8 Hz, H-4U), 4.13 (br s, H-α), 3.93 (br s, H-3U), 2.76 (t,
br, J = 12 Hz, H-γa), 2.46 (br, H-γb). 13C NMR: δ 160.9 (C-7L), 158.6
(C-4″), 157.9 (2C, C-2″, 6″), 157.4 (C-5U), 157.2 (2C, C-7U, 8aU),
156.7 (C-8aL), 153.6 (C-5L), 145.2 (2C, C-4U′, 4L′), 144.9 (C-3U′),
143.4 (C-3L′), 137.9 (C-1L′), 132.4 (C-1U′), 119.0 (C-6U′), 118.9 (C-
6L′), 115.6 (C-5L′), 115.4 (C-5U′), 115.1 (C-2U′), 115.0 (C-2U′),
108.6 (C-1″), 104.3 (2C, C-4aL, 8L), 102.0 (C-4aU), 96.3 (C-6L), 95.7
(2C, C-8U), 95.1 (3C, C-6U, 3″, 5″), 91.6 (C-β), 76.4 (C-2U), 72.7 (C-
3U), 48.4 (C-α), 36.8 (C-4U), 32.3 (C-γ).
Estimation of MICs. The antibacterial effects of the isolated
procyanidins against MRSA were estimated using a liquid micro-
dilution method.10 Briefly, MRSA strains were cultured overnight at 32
°C in cation-supplemented Mueller−Hinton broth (CSMHB; Difco,
Detroit, MI) containing CaCl2 (50 μg/mL) and MgCl2 (25 μg/mL).
They were then diluted with 0.85% NaCl and plated at 104 CFU well−1
on 96-well plates. The cell suspensions in the wells were incubated at
32 °C for 24 h in the absence or presence of serially diluted test
compounds. The MICs of the compounds were defined as the lowest
concentrations at which the culture lacked turbidity after incubation.
Figure 3. HPLC profile of degradation mixture of cynomoriitannin at
280 nm.
phloroglucinol (15, 20.7 min), epicatechin-(4β-2)-phloroglucinol (11,
22.0 min), (+)-catechin (1, 30.4 min), 3-O-galloylepicatechin-(4β-2)-
phloroglucinol (12, 36.2 min), and (−)-epicatechin (2, 39.6 min),
which were confirmed by comparisons with the authentic samples.
The molar ratios of the products were calculated based on the peak
area relative to that of (+)-catechin.
Isolation and Characterization of Phloroglucinol Adducts.
Cynomoriitannin (100 mg) was treated with phloroglucinol (100 mg)
in 1% HCl-EtOH (20 mL) overnight at room temperature. After
evaporation of the solution to dryness under reduced pressure, the
residue was subjected to column chromatography on Toyopearl HW-
40 (30 cm × 1.1 cm) with 70% ethanol, and the eluent was monitored
with RP-HPLC. Fractions were purified by preparative HPLC. In
another experiment, 500 mg of the sample was treated in an analogous
way, to give the following compounds.
RESULTS AND DISCUSSION
■
Purification of Oligomeric and Polymeric Flavans
from C. songaricum and Structure of Cynomoriitannin.
The 70% acetone extract of C. songaricum was subjected to
column (Diaion HP-20, 40% MeOH) fractionation and was
further purified by column chromatography (Toyopearl HW-
40C and MCI gel CHP-20P) and by preparative HPLC to yield
compounds 1−9 and a polymeric PA named cynomoriitannin
(10). This is the first time that compounds 4−9 have been
isolated from C. songaricum.
Epicatechin-(4β-2)-phloroglucinol (11). Compound 11 was an off-
white amorphous powder, [α]D20 +112.4° (MeOH, c 1.0). ESI-MS m/
z: 413 [M − H]−, CD (MeOH) [θ] (nm): −6.3 × 105 (203), +9.1 ×
105 (214), +4.6 × 104 (236), −1.7 × 104 (270). 1H NMR: δ 6.98 (d, J
= 1.2 Hz, H-2′), 6.76 (d, J = 8.4 Hz, H-5′), 6.73 (dd, J = 1.8, 8.4 Hz,
H-6′), 6.04 (d, J = 1.8 Hz, H-8), 6.03 (d, J = 2.4 Hz, H-6), 5.92 (brs,
H-3″, 5″), 5.06 (brs, H-2), 4.60 (d, J = 1.2 Hz, H-4), 4.04 (brs, H-3).
13C NMR: δ158.7−157.8 (6C, C-5, 7, 8a, 2″, 4″, 6″). 145.4 (C-4′ or
3′), 145.2 (C-3′ or 4′), 132.4 (C-1′), 119.2 (C-6′), 115.5 (C-2′ or 5′),
115.2 (C-5′ or 2′), 106.9 (C-1″), 100.6 (C-4a), 96.4−95.9 (4C, C6, 8,
3″, 5″), 77.0 (C-2), 72.5 (C-3), 36.9 (C-3).
Compounds 1−9 were identified using NMR, MS spectra,
and comparisons with the literature.11−15 The structure of
Cynomoriitannin (10) was characterized on the basis of 13C
NMR, SEC, and acid-catalyzed degradation analyses. The 13C
NMR spectrum showed signals assignable to A-ring (C-4a, -5,
-6, -7, -8, and -8a), C-ring (C-2, -3, and -4), and B-ring (C-1′−
C-6′) carbons (see Figure 2), indicating that compound 10 is a
PA polymer mainly composed of (−)-epicatechin, as described
previously.16
PAs can be depolymerized under acidic conditions, releasing
terminal subunits as flavan monomers and extension subunits as
flavanyl cation intermediates. The cation intermediates are
trapped by a nucleophilic reagent to form stable adducts.17
Thus, the constituent monomeric units of cynomoriitannin can
be investigated by acid-catalyzed degradation in the presence of
an excess amount of phloroglucinol, which is odorless,
environmentally friendly, and regarded to be a better trapping
reagent than benzyl mercaptan.18
Epicatechin-3-O-gallate-(4β-2)-phloroglucinol (12). Compound
20
12 was an off-white amorphous powder, [α]D +54.4° (MeOH, c
1.0). ESI-MS m/z: 565 [M − H]−, CD (MeOH) [θ] (nm): −4.4 ×
105 (205), +4.9 × 105 (213), +3.1 × 105 (236), −7.4 × 104 (289). 1H
NMR: δ 7.01 (2H, s), 6.96 (d, J = 1.8 Hz, H-2′), 6.73 (dd, J = 1.8, 8.4
Hz, H-6′), 6.71 (d, J = 7.8 Hz, H-5′), 6.07 (d, J = 1.8 Hz, H-8), 5.98
(d, J = 2.4 Hz, H-6), 5.95 (2H, br s, H3‴, 5‴), 5.42 (s, H-2), 5.19 (t, J
= 1.8 Hz, H-3), 4.58 (d, J = 1.6 Hz, H-4). 13C NMR: δ 167.2 (CO),
158.2−157.2 (6C, C-5, 7, 8a, 2‴, 4‴, 6‴), 145.9 (2C, C-3″, 5″),
145.4−145.3 (2C, C-3′, 4′), 139.1 (C-4″), 131.1 (C-1′), 118.8 (C-6′),
115.5 (C-2′ or 5′), 114.7 (C-2′ or 5′), 109.9 (2C, C-2″, 6″), 105.8 (C-
1‴), 100.5 (C-4a), 96.5−95.3 (4C, C-6, 8, 3‴, 5‴), 75.4 (C-2), 75.0
(C-3), 34.0 (C-4).
Cynomoriitannin-phloroglucinol A (13). Compound 13 was an
off-white amorphous powder, [α]D20 +102.6° (MeOH, c 1.0). ESI-MS
m/z: 521 [M − H]−, CD (MeOH) [θ] (nm): +6.3 × 105 (212), +4.5
1
× 105 (234), −3.7 × 104 (279). H NMR (acetone-d6-D2O, 9:1): δ
6.98 (d, J = 2.4 Hz, H-2′), 6.73 (d, J = 7.8 Hz, H-5′), 6.66 (dd, J = 2.4,
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Figure 4. Structures of degradation products from cynomoriitannin.
Figure 5. Degradation of cynomoriitannin (10) in the presence of phloroglucinol.
Three phloroglucinol adducts were identified based on LC-
MS data and by comparing of HPLC retention time peaks
observed with authentic samples. These were epicatechin-(4β-
2)-phloroglucinol (11), epicatechin-3-O-gallate-(4β-2)-phloro-
glucinol (12), and catechin-(4α-2)-phloroglucinol (15) (Figure
4). These adducts were derived from the extension units in the
PA structure, and the relative molar ratio was estimated as
10.2:0.98:0.94 on the basis of the HPLC analysis. HPLC
analysis of the degradation mixture also exhibited the presence
of (+)-catechin (1) and (−)-epicatechin (2) in the proportion
4.5:1. The extension units were mainly composed of
(−)-epicatechin together with trace amounts of epicatechin-3-
O-gallate and (+)-catechin. The terminal units were composed
of (+)-catechin and smaller amounts of (−)-epicatechin.
SEC techniques have been used by others to the determine
molecular weight distribution of polymeric compounds, which
are routinely summarized as number average (Mn) and weight
average (Mw) molecular weights. SEC analysis of PAs has
traditionally been carried out on methylated or acetylated
derivatives to reduce interactions between the hydroxyl groups
of PA and the SEC gels.19 However, this method of
derivatization is time-consuming and often results in poor
recovery due to side reactions.
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In the present study, SEC analysis of cynomoriitannin was
performed on the free phenolic form, using DMF containing
0.5% (v/v) of 3 M HCOONH4 as the mobile phase. The
molecular weight distribution analysis based on the elution
profile of cynomoriitannin indicated that the number (Mn) and
weight (Mw) average molecular weight were 4.4 × 103 and 6.6
× 103, respectively. This Mn value corresponds to PA 14-mer
structure taking into account the constituent proportion. The
structure of cynomoriitannin is shown in Figure 5.
phloroglucinol structure (δ 109.2) (Figure 6). The CD
spectrum (Figure 7) of compound 13 exhibited a positive
Identification of New Degradation Products from
Cynomoriitannin. Cynomoriitannin-phloroglucinol A (13)
was isolated as an off-white amorphous powder. The [M − H]−
ion peak at m/z 521 in ESI-MS indicated the molecular formula
C27H22O11. The 1H NMR spectrum of 13 showed a set of ABX
spin systems (δ6.98, d, J = 1.8 Hz; 6.73, d, J = 7.8 Hz and 6.67,
dd, J = 1.8, 8.4 Hz) and a pair of meta-coupled proton signals
(δ6.38 and 6.37, each d, J = 1.5 Hz), as well as two 2H singlets
at δ5.97 and 5.92, indicating the presence of two phloroglucinol
groups in its structure (Table 1). The signal pattern of the
Figure 6. Important HMBC correlations of compound 13.
1
Table 1. H and 13C NMR Assignments and HMBC
Correlations for Compound 13
compd 13 (acetine-d6, 27 °C)
carbon
no.
carbon δ (151
Hz)
proton δ (600 Hz)
5.18 (s)
HMBC
3, 1′, 2′, 6′
2
76.4
3
72.5
3.95 (m, J < 1 Hz)
4.61 (d, J = 1.2 Hz)
2, 4, 4a, 1‴
2, 3, 4a, 1‴, 2‴, 6‴
4
36.8
4a
5
108.9
157.2
110.3
133.9
110.9
156.7
132.3
115.4
144.9 or 145.2
144.9 or 145.2
115.0
118.8
109.2
156.3
95.3
Figure 7. CD spectrum of degradation products of cynomoriitannin.
6
6.37 (d, J = 1.8 Hz)
6.38 (d, J = 1.8 Hz)
5, 8, 1″
Cotton effect at 220−240 nm with a noticeable amplitude,
indicating the R configuration at epicatechin C-4. The assigned
structure suggested that compound 13 was a side reaction
product from acid-catalyzed degradation.
7
8
6, 8a, 1″
8a
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
4‴
5‴
6‴
Cynomoriitannin-phloroglucinol B (14) was obtained as a
light-brown amorphous powder. The [M − H]− ion peak at m/
z 685 in ESI-MS indicated the molecular formula C36H30O14.
6.98 (d, J = 1.8 Hz)
6.73 (d, J = 7.8 Hz)
2, 3′, 4′, 6′
3′, 4′
1
The H NMR spectrum (Figure 6 and Table 2) of compound
14 showed a set of signals forming a typical ABX spin system (δ
6.98, d, J = 2.0 Hz; 6.73, d, J = 8.4 Hz; 6.65, dd, J = 2.0, 8.4 Hz),
two ortho-coupled doublets (δ 6.65, d, J = 8.0 Hz; 6.05, d, J =
8.0 Hz), and a broad singlet at δ 6.24 forming another ABX
system. These two ABX systems of two flavan-3-ol B-rings in
compound 14 were also indicated by the 1H−1H COSY
spectrum. The 13C NMR spectrum showed aliphatic carbon
signals at δ 91.6 and 48.4. These chemical shifts were markedly
different from those of the ordinary flavan-3-ols and
procyanidins, and the presence of a 2-benzyl-2,3-dihydroben-
zofuran structure in compound 14 was assumed based on
comparison with previous data.20 As shown in Table 2, an
epicatechin structure was proposed on the basis of 13C signals
from the HSQC spectrum and the structure of compound 14.
The HMBC spectrum showed correlations of H-4U (δ 4.41)
with C-4aU (δ 102.0), C-7L (δ 160.9), and C-8aL (δ 156.7),
indicating that the dihydrobenzofuran and epicatechin
structures were connected by a C−C linkage at C-4aU. On
the other hand, a HMBC correlation of one of the meta-
coupled doublets at δ 5.90 with C-4aU and an aromatic singlet
at δ 5.92 with C-5L (δ153.6), shown by the arrows in Figure 8,
substantiated that the dihydrobenzofuran unit was connected to
C-4 of the upper epicatechin unit. On the basis of the
composition of the polymeric PA structure of compound 10, it
6.67 (dd, J = 8.4, 1.8 Hz) 5′
5.97 (brs)
5.97 (brs)
1″, 2″, 4″
1″, 4″, 6″
157.4
95.3
156.3
106.9
157.6
96.0
5.92 (brs)
5.92 (brs)
158.2
96.0
157.6
aliphatic protons was similar to those of compound 11.
However, the 13C NMR spectrum showed a downfield shift of
C-4a (δ 107.0) relative to that of compound 11, indicating a
difference in the A-ring structure. Compound 13 had an
additional phloroglucinol group on the A-ring of epicatechin-
(4β-2)-phloroglucinol (11). In the HMBC spectrum, H-4 (δ
4.61), H-6 (δ 6.38), and H-8 (δ 6.37) were correlated with C-
4a (δ 107.0), and in turn, H-6, H-8, and the phloroglucinol 2H
singlet at δ 5.96 were correlated with C-1 of the other
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1
Table 2. H and 13C NMR Assignments, COSY, and HMBC
Correlations for Compound 14
compd 14 (acetone-d6, 27 °C)
carbon δ
carbon
no.
(151 Hz)
(150 Hz)
proton δ
(600 Hz) COSY
HMBC
2
76.4
5.11 (brs) 3U, 4U C-1′ , 2′U,
6′UU,
3
4
72.7
36.8
3.93 (brs) 2U, 4U
4.41 (d, J = 2U, 3U C-2U, 4a,
7.8 Hz)
5U, 7L,
8L, 8aL,
4a
5
102.0
157.4
95.1
6
5.90 (d, J = 8U
2.4 Hz)
C-4a, 5U
Figure 8. Important HMBC correlations of compound 14.
7
8
157.2
95.7
two continuous epicatechin extension units during the
degradation process. Compounds with similar structures have
also been isolated from hot water extracts of Unicaria gambir
and are thought to be derivatives of catechin.20−22
Antibacterial Activity of Extracts and Oligomeric and
Polymeric Flavan-3-ols Obtained from C. songaricum.
The antibacterial effects of the crude extract, Diaion fractions,
and cynomoriitannin on MRSA are shown in Table 3. The
5.93 (d, J = 6U
C-7U, 8aU
upper unit
2.4 Hz)
8a
1′
2′
157.2
132.4
115.1
6.98 (d, J = 6′U
C-3′U, 6′U
C-3′U, 4′U
2.4 Hz)
3′
4′
5′
144.9
145.2
115.4
6.73 (d, J = 6′U
8.4 Hz)
Table 3. Inhibition Activity (MIC and Inhibition
Concentration: μg/mL) against MRSA for Extracts, Diaion
Fractions, and Cynomoriitannin from C. songaricum
6′
119.0
6.65 (dd, J 2′U,
= 2.4,
7.8 Hz)
5′U
α
β
48.4
91.6
4.13 (brs)
β
C-2″, 6″
a
extracts
MIC (μg/mL)
effective concn (μg/mL)
4.41 (d, J = α,γa
7.8 Hz)
crude acetone extract
512
512
64
64
512
8
b
deposit
γ
32.3
2.76(t, br, J α, γb
= 12 Hz)
c
H2O fraction
>1024
256
c
2.46 (br)
γa
20% MeOH fraction
c
4a
5
104.3
153.6
96.3
40% MeOH fraction
128
16
16
64
128
8
c
60% MeOH fraction
128
c
6
5.92 (brs)
100% MeOH fraction
256
c
7
160.9
104.3
156.7
137.9
115.0
143.8
145.2
115.6
70% acetone fraction
cynomoriitannin
256
lower unit
8
64
8a
1′
2′
3′
4′
5′
a
Concentration at which an inhibitory effect on the proliferation of the
bacterial strain was observed. Precipitate obtained by concentration
of the filtrate from the plant homogenate. These fractions were
obtained by column chromatography of crude acetone extract with the
solvents indicated.
b
c
6.24 (brs)
6.51 (d, J =
8.4 Hz)
C-3′L, 4′L
6′
118.9
6.05 (d, J =
crude aqueous acetone extract exhibited moderate antibacterial
activity against MRSA OM 623 strain (MIC 512 μg/mL).
Among the Diaion fractions, 40 and 60% MeOH fractions had
the lowest MICs (128 μg/mL). The monomeric and oligomeric
flavans obtained from the 40% MeOH fraction had MICs >
1024 μg/mL and exhibited limited inhibitory effects on the
growth of MRSA at lower concentrations (data not shown).
Previous studies have reported that procyanidin dimers
generally have weak antibacterial effects on MRSA.23 However,
cynomoriitannin was found to have a MIC of 64 μg/mL and
exhibited higher antibacterial activity than a procyanidin
polymer from Z. piperitum.5 It also reduced the MIC of
oxacillin against MRSA strains by 64−512-fold or more.
Further investigation of antibacterial effects of cynomoriitannin
is now in progress.
7.8 Hz)
1″
2″
3″
4″
5″
6″
108.6
157.9
95.1
5.89 (brs)
5.89 (brs)
phloroglucinol
unit
158.6
95.1
157.9
was proposed that the dihydrobenzofuran unit was formed
from an epicatechin unit. The configuration at C-β of the
dihydrobenzofuran unit and the R configuration at C-4U (rather
than the S configuration) were thus inferred as shown in Figure
8.20 On the other hand, the absence of a noticeable Cotton
effect in the 220−240 nm of the CD suggests the existence of
an S configuration at C-α.
These findings suggest that PA polymers are effective against
MRSA, acting either directly or by restoring the antibacterial
effects of coadministered antibiotics. Thus, PA polymers may
The structure of cynomoriitannin-phloroglucinol B may be
present in the polymeric PA, and it can also be formed from
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Journal of Agricultural and Food Chemistry
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gallate, a bioactive tea polyphenol, on incubation in neutral solution.
Heterocycles 2004, 63, 1547−1554.
(5) Kusuda, M.; Inada, K.; Ogawa, T. O.; Yoshida, T.; Shiota, S.;
Tsuchiya, T.; Hatano, T. Polyphenolic constituent structures of
Zanthoxylum piperitum fruit and the antibacterial effects of its
polymeric procyanidin on methicillin-resistant Staphylococcus aureus.
Biosci., Biotechnol., Biochem. 2006, 70, 1423−1431.
(6) Ma, L. J.; Chen, G. L.; Jin, S. W.; Wang, C. X. The anti-aging
effect and the chemical consttuents of Cynomorium songaricum Rupr.
Acta Hortic. 2006, 765, 23−30.
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3-ol contents, anti-oxidative and α-glucosidase inhibitory activities of
Cynomorium songaricum. Food Chem. 2010, 118, 116−119.
(8) Ma, C. M.; Nakamura, N.; Miyashiro, H.; Hattori, M.;
Shimotohno, K. Inhibitory effects of constituents from Cynomorium
songaricum and related triterpene derivatives on HIV-1 protease. Chem.
Pharm. Bull. 1999, 47, 141−145.
(9) Kimura, Y.; Ito, H.; Kawaji, M.; Ikami, T.; Hatano, T.
Characterization and antioxidative properties of oligomeric proantho-
cyanidin from prunes, dried fruit of Prunus domestica L. Biosci.,
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(10) Shiota, S.; Shimizu, M.; Mizushima, T.; Ito, H.; Hatano, T.;
Yoshida, T.; Tsuchiya, T. Marked reduction in the minimum inhibitory
concentration (MIC) of beta-lactams in methicillin-resistant Staph-
ylococcus aureus produced by epicatechin gallate, an ingredient of green
tea (Camellia sinensis). Biol. Pharm. Bull. 1999, 22, 1388−1390.
(11) Hatano, T.; Hemingway, R. W. Conformational isomerism of
phenolic procyanidins: Preferred conformations in organic solvents
and water. J. Chem. Soc. Perkin Trans 2 1997, 1035−1043.
(12) Sang, S. M.; Cheng, X. F.; Stark, R. E.; Rosen, R. T.; Yang, C. S.;
Ho, C. T. Chemical studies on antioxidant mechanism of tea catechins:
analysis of radical reaction products of catechin and epicatechin with
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Nakajima, N. Systematic synthesis of four epicatechin series
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form the basis of developing anti-MRSA drugs and alternative
therapies. PA itself is a widely used food additive but is not
microbiologically active. Other derivatives of PAs are found in
grapes, apples, and cacao.24−26 The relationships between
structure and antimicrobial activity of PAs against MRSA may
be an important area for further research.
In summary, six oligomeric procyanidins, procyanidin B3 (4),
catechin-(6′-8)-catechin (5), catechin-(6′-6)-catechin (6),
epicatechin-(4β-8)-epicatechin-(4β-8)-catechin (7), epicate-
chin-(4β-6)-epicatechin-(4β-8)-catechin (8), and arecatannin
A1 (9), were isolated from C. songaricum. The polymeric PA,
cynomoriitannin, was shown to be composed mainly of
epicatechin together with low proportions of epicatechin-3-O-
gallate and catechin in the extension units. The terminal unit
consists chiefly of catechin, with an admixture of epicatechin.
The mean degree of polymerization was 14. Two new
phloroglucinol adducts from the degradation mixture of
cynomoriitannin were isolated and identified as cynomor-
iitannin-phloroglucinol A (13) and cynomoriitannin-phloroglu-
cinol B (14) based on spectral analyses. Preliminary
investigation of the antibacterial activity indicated that the
crude extract had moderate effect on MRSA. Cynomoriitannin
obtained from the extract showed the most effective inhibition
against MRSA (MIC 128 μg/mL) among the purified
compounds.
AUTHOR INFORMATION
Corresponding Author
■
*Fax: +81-86-251-7936. E-mail: hatano@pharm.okayama-u.ac.
jp (T.H.). Tel: +86-471-4992577. Fax: +86- 471-4992435. E-
mail: guilinchen@yahoo.com.cn (G.C.).
Funding
This study was supported, in part, by grants from National Key
Technology R&D Program of China (2011BAI07B07), Science
and Technology Innovation Fund of Inner Mongolia
Autonomous Region of China, Intractable Infectious Diseases
Research Project Okayama (IIDTPO), and “211 project” for
postgraduate student program of Inner Mongolia University,
China.
(15) Saito, A.; Mizushina, Y.; Tanaka, A.; Nakajima, N. Versatile
synthesis of epicatechin series procyanidin oligomers, and their
antioxidant and DNA polymerase inhibitory activity. Tetrahedron
2009, 65, 7422−7428.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(16) Czochanska, Z.; Foo, L. Y.; Newman, R. H.; Porter, L. J.
Polymeric proanthocyanidins: Stereochemistry, structural units, and
molecular weight. J. Chem. Soc. Perkin Trans. 1 1980, 2278−2286.
(17) Hemingway, R. W.; McGraw, G. W. Kinetics of acid-catalyzed
cleavage of procyanidins. J. Wood Chem. Technol. 1983, 3, 421−425.
(18) Kennedy, J. A.; Jones, G. P. Analysis of proanthocyanidin
cleavage products following acid-catalysis in the presence of excess
phloroglucinol. J. Agric. Food Chem. 2001, 49, 1740−1746.
(19) Kennedy, J. A.; Taylor, A. W. Analysis of Proanthocyanidins by
High-Performance Gel Permeation Chromatography. J. Chromatogr., A
2003, 995, 99−107.
■
The Varian INOVA 600AS NMR instrument used in this study
is the property of the SC-NMR laboratory of Okayama
University.
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