Two new monoterpene glycosides from Qing Shan Lu Shui tea with inhibitory effects on leukocyte-type 12-lipoxygenase activity

Article abstract of DOI:10.3390/molecules18044257

We evaluated the inhibitory effect of 12 Chinese teas on leukocyte-type 12-lipoxygenase (LOX) activity. Tea catechins such as epigallocatechin gallate have been known to exhibit leukocyte-type 12-LOX inhibition. Qing Shan Lu Shui, which contains lower catechin levels than the other tested teas, suppressed leukocyte-type 12-LOX activity. To characterize the bioactive components of Qing Shan Lu Shui, leukocyte-type 12-LOX inhibitory activity-guided fractionation of the aqueous ethanol extract of the tea was performed, resulting in the isolation of two new monoterpene glycosides: liguroside A (1) and B (2). The structures of compounds 1 and 2 were characterized as (2E,5E)-7- hydroperoxy-3,7-dimethyl-2,5-octadienyl-O-(α-L-rhamnopyranosyl) -(1 →3')-(4' -O-transp- coumaroyl)-β-D-glucopyranoside and (2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-octa-dienyl- O-(α-L- rhamnopyranosyl)-(1 →3')-(4' -O-cis-p-coumaroyl) -β-D-glucopyranoside, respectively, based on spectral and chemical evidence. Ligurosides A (1) and B (2) showed inhibitory effects on leukocyte-type 12-LOX activity, with IC₅₀ values of 1.7 and 0.7 μM, respectively.

Full text of DOI:10.3390/molecules18044257

Molecules 2013, 18, 4257-4266; doi:10.3390/molecules18044257
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Two New Monoterpene Glycosides from
Qing Shan Lu Shui Tea with Inhibitory Effects on
Leukocyte-Type 12-Lipoxygenase Activity
Hideyuki Ito 1,2,*, Akemi Otsuki 2, Hitomi Mori 1, Peng Li 1, Mai Kinoshita 2, Yuki Kawakami 2,
Hideaki Tsuji 2, Ding Zhi Fang 3 and Yoshitaka Takahashi 2
1
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University,
1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan;
E-Mails: ph19139@s.okayama-u.ac.jp (H.M.); pnen3yvi@s.okayama-u.ac.jp (P.L.)
2
Faculty of Health and Welfare Sciences, Okayama Prefectural University, 111 Kuboki, Soja,
Okayama 719-1197, Japan; E-Mails: hb24001a@fhw.oka-pu.ac.jp (A.O.);
hb24002x@fhw.oka-pu.ac.jp (M.K.); kawaka@fhw.oka-pu.ac.jp (Y.K.);
htsuji@fhw.oka-pu.ac.jp (H.T.); ytaka@fhw.oka-pu.ac.jp (Y.T.)
3
Department of Biochemistry and Molecular Biology, West China School of Preclinical and
Forensic Medicine, Sichuan University, 17 Section 3, South Renmin Road, Chengdu 610041,
China; E-Mail: dzfang@scu.edu.cn
* Author to whom correspondence should be addressed; E-Mails: hito@fhw.oka-pu.ac.jp or
hito@cc.okayama-u.ac.jp; Tel./Fax: +81-866-94-2086.
Received: 18 February 2013; in revised form: 7 April 2013 / Accepted: 8 April 2013 /
Published: 11 April 2013
Abstract: We evaluated the inhibitory effect of 12 Chinese teas on leukocyte-type
12-lipoxygenase (LOX) activity. Tea catechins such as epigallocatechin gallate have been
known to exhibit leukocyte-type 12-LOX inhibition. Qing Shan Lu Shui, which contains
lower catechin levels than the other tested teas, suppressed leukocyte-type 12-LOX
activity. To characterize the bioactive components of Qing Shan Lu Shui, leukocyte-type
12-LOX inhibitory activity–guided fractionation of the aqueous ethanol extract of the tea
was performed, resulting in the isolation of two new monoterpene glycosides: liguroside A
(1) and B (2). The structures of compounds 1 and 2 were characterized as (2E,5E)-7-
hydroperoxy-3,7-dimethyl-2,5-octadienyl-O-(α-L-rhamnopyranosyl)-(1″→3′)-(4′″-O-trans-
p-coumaroyl)-β-D-glucopyranoside and (2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-octa-dienyl-
O-(α-L-rhamnopyranosyl)-(1″→3′)-(4′″-O-cis-p-coumaroyl)-β-D-glucopyranoside, respectively,

Molecules 2013, 18
based on spectral and chemical evidence. Ligurosides A (1) and B (2) showed inhibitory
4258
effects on leukocyte-type 12-LOX activity, with IC50 values of 1.7 and 0.7 μM, respectively.
Keywords: Qing Shan Lu Shui; Ligustrum robustum; liguroside; monoterpene glycoside;
leukocyte-type 12-lipoxygenase; Chinese tea; catechin
1. Introduction
Oxidative modification of low-density lipoprotein is the first key step in the development of
atherosclerosis, and the role of leukocyte-type 12-lipoxygenase (LOX) in this process has been
established using leukocyte-type 12-LOX-knockout mice [1,2]. We previously reported that guava leaf
extracts showed anti-atherogenic activity by inhibiting leukocyte-type 12-LOX activity [3]. Chinese
teas derived from various kinds of plants are drunk for health benefits in parts of South China such as
the province of Sichuan. Tea catechins such as epigallocatechin gallate and epicatechin gallate are
known to inhibit several enzyme activities and to show inhibitory effects on inflammation and
cardiovascular disease [4]. In the present study, we investigated the isolation and characterization of
active constituents showing inhibitory effects on the leukocyte-type 12-LOX activity from Chinese teas.
2. Results and Discussion
We evaluated the inhibitory effects of the 12 Chinese teas listed in Table 1 on leukocyte-type
12-LOX activity. Among the 12 Chinese tea extracts, Zhuya, Man Jian, Ganlu, Longjin Cha, Emei
Mao Jian, Tie Guan Yin, and Qing Shan Lu Shui inhibited the conversion of arachidonic acid to
12-HPETE, which is catalyzed by leukocyte-type 12-LOX, with IC50 values of less than 20 µg/mL
(Table 1). Tea catechins such as epigallocatechin gallate are known to inhibit leukocyte-type 12-LOX
activity [5]. However, these inhibitors, except for those in Qing Shan Lu Shui, contained high amount
of catechins (Table 1). Qing Shan Lu Shui [Ligustrum robustum (Roxb.) Blume] of the family
Oleaceae contains quinic acid derivatives, flavonoids, and monoterpene glycosides, which have been
reported to show antiviral and anti-inflammatory activities and inhibitory effects against the hemolysis
of red blood cells induced by free radicals [6–10]. To characterize ingredients having inhibitory effects
on leukocyte-type 12-LOX from Qing Shan Lu Shui, the dried tea leaves were extracted with 50%
aqueous ethanol followed by ethyl acetate. Leukocyte-type 12-LOX inhibitory assay–guided
fractionation of the 30% aqueous ethanol soluble portion of the ethyl acetate extract was carried out
using reversed-phase HPLC, to yield fractions showing potent inhibitory activity at a retention time of
24–26 min (Figure 1). The active fractions were further purified by preparative HPLC to furnish two
compounds, namely, ligurosides A and B.

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Table 1. Inhibitory effect on leukocyte-type 12-LOX activity and catechin contents of 12 Chinese teas.
EGCg
Total Catechins
(mg/g dried leaves)
IC50
(μg/mL)
2.4
Chinese Teas
Zhuya
Man Jian
Ganlu
87.4
38.4
61.3
63.3
58.9
18.6
0.6
20.8
16.3
<0.1
<0.1
<0.1
136.5
63.4
100.7
100.6
95.5
39.1
0.7
41.3
51.4
0.4
6.7
6.9
9.0
Longjin Cha
Emei Mao Jian
Tie Guan Yin
Qing Shan Lu Shui
Puer San Cha
Tou Cha
13.5
16.6
19.3
20.0
21.2
26.8
38.1
>100
Tian Cha
Hainan Kudig
Qin Hua
0.1
0.1
EGCg: Epigallocatechin gallate.
Figure 1. HPLC profile of Qing Shan Lu Chui extract and leukocyte-type 12-LOX activity
of each fraction.
Liguroside A (1) was obtained as an off-white amorphous powder. Its molecular formula was
determined as C31H44O14 on the basis of an HRESIMS peak at m/z 639.2665 [M−H]− (calcd. for
1
C31H43O14, 639.2658). The H-NMR spectrum of 1 showed AA′BB′-type protons at δ 7.55 and 6.89
(each 2H, d, J = 8.4 Hz) and trans olefin protons at δ 7.64 and 6.35 (each 1H, d, J = 15.6 Hz) in the
aromatic proton region, corresponding to a trans-p-coumaroyl group. Proton signals due to a vinyl
(δ 5.37), 2 mutually coupled vinyl (δ 5.67, 5.61), oxygenated methylene (δ 4.33, 4.21), a methylene
(δ 2.75), a vinyl methyl (δ 1.67), and 2 tertiary methyl (δ 1.28) groups were observed in the aliphatic
proton region. The presence of β-glucose and α-rhamnose residues was suggested by two anomeric
proton signals at δ 4.40 (d, J = 7.2 Hz) and δ 5.29 (d, J = 1.8 Hz), and a secondary methyl proton
signal at δ 1.09 (d, J = 6.6 Hz) (Table 2). The 13C-NMR and HSQC spectra for 1 displayed 31 carbon
signals (listed in Table 2) due to 13 sp2 carbons, including an ester carbonyl carbon, 10 oxygenated
methines, three methylenes, four methyls including a vinyl, and an oxygenated tertiary carbons, which
were quite similar to those of p-coumaroyl, glucosyl, and rhamnosyl units in kudingoside B (3) [11]
(Figure 2). The stereochemistry of sugars in 1 confirmed D-glucose and L-rhamnose on the basis of

Molecules 2013, 18
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HPLC with an optical rotatory detector after acid hydrolysis of 1. The locations of sugar and acyl units
in 1 were established by an HMBC experiment (Figure 3).
Table 2. 1H- and 13C-NMR spectral data of ligurosides A (1) and B (2) in acetone-d6+D2O
(δH: 600 MHz; δC: 150 MHz).
1
2
Position
δH (J in Hz)
δC
δH (J in Hz)
δC
Monoterpene
1
4.21 dd (7.2, 12)
4.33 dd (6, 12)
5.37 dd (6, 7.2)
65.0
4.20 dd (7.2, 12)
4.32 dd (6, 12)
5.36 dd (6, 7.2)
65.9
2
121.4
138.7
42.2
122.2
139.6
43.1
3
4
2.75 d (6.6)
5.61 dt (15.6, 6.6)
5.67 d (15.6)
2.74 d (6.6)
5.61 dt (15.6, 6.6)
5.66 d (15.6)
5
136.7
127.0
80.6
137.6
127.8
81.4
6
7
8
1.28 s
1.28 s
1.67 s
24.1
1.27 s
1.27 s
1.65 s
24.9
9
24.1
24.9
10
15.6
16.5
Glucosyl
1′
4.40 d (7.2)
3.42 dd (7.2, 9)
3.87 t (9)
101.4
75.3
78.7
69.4
75.1
61.5
4.38 d (8.4)
3.41 dd (8.4, 9)
3.83 t (9)
102.3
76.1
79.7
70.1
75.8
62.4
2′
3′
4′
4.89 t (9)
4.86 t (9)
5′
3.52 m
3.48 m
6′
3.59 m, 3.52 m
3.59 m, 3.50 m
Rhamnosyl
1″
5.29 d (1.8)
3.86 m
100.9
71.1
71.3
72.7
68.5
17.6
5.28 d (1.8)
3.87 m
101.9
71.9
72.2
73.6
69.4
18.3
2″
3″
3.52 m
3.54 dd (3, 9.6)
3.32 t (9.6)
4″
3.30 t (9)
5″
3.62 dd (6.6, 9)
1.09 d (6.6)
3.65 dd (6.6, 9.6)
1.14 d (6.6)
6″
Coumaroyl
1′″
126.1
130.2
115.8
159.9
115.8
130.2
145.5
114.2
166.2
127.2
134.1
115.7
159.8
115.7
134.1
145.9
115.9
166.0
2′″
7.55 d (8.4)
6.89 d (8.4)
7.79 d (8.4)
6.83 d (8.4)
3′″
4′″
5′″
6.89 d (8.4)
7.55 d (8.4)
7.64 d (15.6)
6.35 d (15.6)
6.83 d (8.4)
7.79 d (8.4)
6.95 d (13.2)
5.78 d (13.2)
6′″
7′″
8′″
9′″

Molecules 2013, 18
Figure 2. Structures of ligurosides A (1) and B (2), and kudingoside B (3).
4261
Figure 3. Key HMBC Correlations of liguroside A (1).
In particular, the anomeric proton signal at δ 4.40 of glucose was correlated through three-bond
coupling with C-1 resonance at δ 65.0 of a monoterpene unit. The signal of H-1 (δ 4.21 and 4.33) of
the monoterpene unit was also correlated with the anomeric carbon (δ 101.4) of glucose. The other
anomeric proton (rhamnose) at δ 5.29 displayed a correlation with the C-3′ carbon at δ 78.7 of glucose,
and H-3′ at δ 3.87 of glucose was also correlated with the anomeric carbon at δ 100.9 of rhamnose.
The H-4′ (δ 4.89) of glucose was correlated with H-7′′′ (δ 7.64) of p-coumaroyl unit through three-
bond coupling with a common ester carbonyl carbon (δ 166.2). The L-rhamnopyranosyl-(1′′→3′)-(4′′′-
O-trans-p-coumaroyl)-D-glucopyranosyl moiety was thus allocated to the C-1 position of the
monoterpene system. The 13C resonances of the monoterpene unit of 1 were close to those of 3, except
for C-7 (δ 80.6 for 1 and δ 71.1 for 3) and the tertiary methyl (C-8 and 9) (δ 24.1 for 1 and δ 30.0 for 3)
carbon signals [11] (Table 2). The remarkable downfield shift of C-7 and upfield shift of C-8 and C-9
resonances implied that the hydroxyl group of C-7 position in 3 for 1 is replaced by the hydroperoxy
group [12,13]. The molecular formula of 1 calculated by HRESIMS analysis supports this suggestion.
Furthermore, the reduction of 1 with triphenylphosphine yielded 3 [11], confirming the presence of a
hydroperoxy group in the molecule. Consequently, the structure of liguroside A (1) was determined
to be (2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-octadienyl-O-(α-L-rhamnopyranosyl)-(1′′→3′)-(4′′′-O-
trans-p-coumaroyl)-β-D-glucopyranoside, as depicted in Figure 2.
Liguroside B (2), an off-white amorphous powder, exhibited a pseudomolecular ion peak at m/z
639.2641 [M−H]− (calcd. for C31H43O14, 639.2658) in the HRESIMS, establishing its molecular
13
formula as C31H44O14. The 1H-, C-NMR, MS, and UV spectral data of 2 were almost superposed on
1
those of 1, except for the coupling constant between H-7′′′ and H-8′′′ (J = 13.2 Hz) in the H-NMR
spectrum. This indicates that the stereochemistry at C-7′′′ and C-8′′′ in 2 displays cis configuration.

Molecules 2013, 18
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Liguroside B (2) was thus assigned to be a cis isomer at the p-coumaroyl moiety of 1, namely,
(2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-octadienyl-O-(α-L-rhamnopyranosyl)-(1′′→3′)-(4′′′-O-cis-p-
coumaroyl)-β-D-glucopyranoside (Figure 2). The cis isomer of the p-coumaroyl moiety, which often
exists in flavonoids, coumarins, and terpenoids, is well documented to be converted to its trans isomer
under light conditions, such that cis and trans isomers occur as an equilibrium mixture [6,14,15]. In
this study, we isolated each isomer as liguroside A (1, trans isomer) and liguroside B (2, cis isomer)
under dark conditions; we also elucidated these structures.
We evaluated the inhibitory effects of the new compounds isolated from Qing Shan Lu Chui on
leukocyte-type 12-LOX activity. Ligurosides A (1) and B (2) exhibited significant inhibitory effects,
with IC50 values of 1.7 and 0.7 μM, respectively. The inhibitory potency of liguroside A was
comparable to that of quercetin (IC50 value of 1.6 μM) used as a positive control [3]. p-Coumaric acid,
as a part of the catabolites believed to be produced from ligurosides by intestinal microflora after Qing
Shan Lu Chui consumption showed no inhibition on leukocyte-type 12-LOX activity. These results
suggested that the inhibitory effects of ligurosides A and B can be attributed to the monoterpene unit
with the hydroperoxy group.
3. Experimental
3.1. General
Twelve Chinese teas (Zhuya, Man Jian, Ganlu, Longjin Cha, Emei Mao Jian, Tie Guan Yin, Qing
Shan Lu Shui, Puer San Cha, Tou Cha, Tian Cha, Hainan Kudig, and Qin Hua) were purchased at a
specialty tea market in Sichuan, China. Optical rotations were recorded on a Jasco DIP-1000
instrument. UV spectra were measured on a Jasco V-530 spectrometer. 1H- and 13C-NMR spectra were
measured in acetone-d6 on a Varian NMR System 600 MHz (600 MHz for 1H-NMR and 150 MHz for
13C-NMR) instrument; chemical shifts are given in δ (ppm) values relative to that of the solvent
(δH: 2.04; δC: 29.8) on a tetramethylsilane scale. The standard pulse sequences programmed for the
instrument were used for each 2D-NMR experiment (1H-1H COSY, NOESY, HSQC, and HMBC).
The JCH value was set at 8 Hz in the HMBC experiment. Mass spectra were obtained on a Bruker
MicrOTOF II spectrometer using ESI source in negative-ion mode. HPLC was performed on a Waters
Alliance 2625 separations module (Milford, MA, USA). Reversed-phase HPLC was conducted on a
250 × 4.6 mm i.d. Inertsil ODS-3 column (GL Sciences, Inc., Tokyo, Japan) developed with 0.01%
acetic acid (solvent A) and 0.01% acetic acid–methanol (solvent B) at 40 °C and a flow rate of 1.0
mL/min. Detection was effected at 200–700 nm. A gradient was applied as follows: the proportion of
solvent B in the eluent increased from 30% to 100% (t = 50 min) and 100% (t = 60 min).
3.2. Quantification of Catechins in Extracts of Chinese Teas
Dried tea leaves (1 g) were extracted with 50% aqueous ethanol (20 mL) for 30 min. After filtration
and evaporation in vacuo of the extract, the residue was re-dissolved with 50% aqueous ethanol (10 mg/mL),
centrifuged (12,000 rpm, 4 °C, 5 min), filtered (0.45 μm), and injected into the HPLC system.
Quantification of tea catechins [(+)-catechin, (−)-catechin, epicatechin, gallocatechin, gallocatechin
gallate, epicatechin gallate, epigallocatechin, epigallocatechin gallate, and catechin gallate obtained

Molecules 2013, 18
4263
from Wako Pure Chemical Industries, Ltd., Osaka, Japan] was performed on the basis of the absolute
calibration curve method. HPLC was performed on a Waters Alliance 2695 separations module.
Quantitative HPLC was conducted on a 250 × 4.6 mm i.d. Inertsil ODS-3 column (GL Sciences, Inc.)
eluted with 0.05% phosphoric acid (solvent A) and 0.05% phosphoric acid–acetonitrile (solvent B) at
40 °C and a flow rate of 1.0 mL/min. Detection was effected at 280 nm. A gradient was applied as
follows: the proportion of solvent B in the eluent increased from 5% to 10% (t = 15 min) and 45%
(t = 40 min). The calibration curves were linear and had mean correlation coefficients of ≥0.99.
3.3. Leukocyte-type 12-LOX Inhibitory Assay
Porcine leucocyte-type 12-LOX was partially purified from J774A.1 cells overexpressing the
enzyme [16]. The partially purified leukocyte-type 12-LOX was preincubated for 5 min at 30 °C with
indicated concentrations of inhibitors in a standard 200-μL reaction mixture containing 100 mM
Tris-HCl buffer (pH 7.4), and then incubated with 25 μM arachidonic acid (5 nmol/5 μL of ethanol
solution) for 5 min at 30 °C. The products were reduced by the addition of glutathione peroxidase (0.1 U)
and 5 mM glutathione, followed by incubation for a further 20 min. After acidification of the reaction
mixture by the addition of 50 mM HCl, 0.5 nmol of 15-HEDE was added as an internal standard for
LOX reaction. The products extracted with ice-cold diethyl ether were analyzed by reverse-phase
HPLC, using a Waters Alliance system equipped with a COSMOSIL 5C18-MS-II column (5-μm
particle, 250 × 4.6 mm i.d.; Nacalai, Kyoto, Japan) with a solvent system of methanol–water–acetic
acid (80:20:0.01, v/v) at a flow rate of 1 mL/min, as previously described [17]. Absorption at 235 nm
was continuously monitored, using a Waters 2489 UV/Visible detector. The products that
co-chromatographed with authentic 12-HETE and 15-HETE in the leucocyte-type 12-LOX reaction
were quantified by comparison of the areas of the peaks with that of an internal standard. Linear
relationship was confirmed by calibration curves (five-point measurements) for conjugated dienes of
produced HETEs, as well as internal standards. Protein concentration was determined using a
bicinchoninic acid protein assay kit with bovine serum albumin as a standard.
3.4. Extraction and Isolation
The dried leaves of Qing Shan Lu Shui (1 g) were extracted with 50% aqueous ethanol (20 mL) for
30 min. After filtration and evaporation of the extract, the residue was partitioned between water (2 mL)
and ethyl acetate (2 mL × 2). The organic layer was evaporated, and then, the resulting residue (110 mg)
was re-dissolved with 30% aqueous ethanol (11 mL). The aqueous ethanol extract was purified by
preparative HPLC to afford liguroside A (1) (retention time 31.5 min, 8.5 mg) and liguroside B (2)
(retention time 37.6 min, 3.1 mg). HPLC was performed on a Waters Alliance 2695 separations
module. Reversed-phase HPLC in an isocratic condition was conducted on a 250 × 4.6 mm i.d. Inertsil
ODS-3 column (GL Sciences, Inc.) developed with methanol–water–acetic acid (47:53:0.01) at 40 °C
and a flow rate of 1.0 mL/min. Detection was effected at 200–700 nm.
Liguroside A (1), An off-white amorphous powder; [α]1D9 −81.2° (c 0.3, MeOH); UV λmax (MeOH) nm
1
13
(log ε): 316 (4.40); H- and C-NMR, see Table 2; HRESIMS m/z 639.2665 [M−H]−, calcd. for
C31H44O14-H, 639.2658.

Molecules 2013, 18
4264
Reduction of 1 with Triphenylphosphine, A methanol solution (1 mL) of 1 (1 mg) was stirred for 12 h
at ambient temperature with triphenylphosphine (Sigma-Aldrich, Tokyo, Japan) (1 mg). The reaction
mixture was evaporated and purified by Bond Elut C18 cartridge column to yield 3 (0.3 mg) [10],
HRESIMS m/z 623.2723 [M−H]−, C31H44O13-H, requires 623.2709.
Acid Hydrolysis of 1. A solution of 1 (3 mg) in 1% H2SO4 (3 mL) was heated in a boiling water bath
for 1 h. The reaction mixture was extracted with ethyl acetate. The ethyl acetate extract was analyzed
by normal phase HPLC [250 × 4.6 mm i.d. YMC-Pack SIL-003 column (YMC Co., Ltd., Kyoto, Japan)
with n-hexane–methanol–tetrahydrofuran–formic acid (55:33:11:1) containing oxalic acid (450 mg/L)
at ambient temperature and a flow rate of 1.5 mL/min] to detect p-coumaric acid (2.9 min). The
aqueous layer was neutralized with Diaion SA-20AP and evaporated. The residue was analyzed by
HPLC equipped with an optical rotation detector (Jasco OR-2090, Jasco International Co., Tokyo,
Japan), which demonstrated the retention time of the sugars to be identical with those of authentic
D-glucose (9.3 min) and L-rhamnose (6.7 min). HPLC was performed on a Jasco Borwin system with a
250 × 4.6 mm i.d. TSK-gel Amide-80 column (Tosoh Co., Tokyo, Japan) developed with acetonitrile-
water (82:18) at ambient temperature and at a flow rate of 1.0 mL/min.
Liguroside B (2), An off-white amorphous powder; [α]1D9 −88.8° (c 0.18, MeOH); UV λmax (MeOH) nm
1
13
(log ε): 315 (4.81); H- and C-NMR, see Table 2; HRESIMS m/z 639.2641 [M−H]−, calcd. for
C31H44O14-H, 639.2658.
4. Conclusions
We have investigated the inhibitory effects of 12 commercial Chinese teas on leukocyte-type
12-LOX activity. Catechins such as epigallocatechin gallate have been known to inhibit leukocyte-type
12-LOX. Indeed, tested catechin-rich teas were found to attenuate the activity of the enzyme.
However, Qing Shan Lu Shui, which contains lower levels of catechin among the tested teas,
suppressed 12-LOX activity. Bioassay-guided fractionation of Qing Shan Lu Shui led to the isolation
of two new monoterpene glycosides, named liguroside A (1) and B (2). Compounds 1 and 2
were determined to be (2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-octadienyl-O-(α-L-rhamnopyranosyl)-
(1′′→3′)-(4′′′-O-trans-p-coumaroyl)-β-D-glucopyranoside and (2E,5E)-7-hydroperoxy-3,7-dimethyl-2,5-
octadienyl-O-(α-L-rhamnopyranosyl)-(1′′→3′)-(4′′′-O-cis-p-coumaroyl)-β-D-glucopyranoside, respectively,
on the basis of spectral and chemical evidence. Ligurosides A (1) and B (2) showed inhibitory effects
on leukocyte-type 12-LOX activity, with IC50 values of 1.7 and 0.7 μM, respectively, while
p-coumaric acid corresponding to the gut microbial metabolite of ligurosides showed no effect. These
results implied that the inhibitory effect of ligurosides A and B on leukocyte-type 12-LOX activity is
attributed to the monoterpene moiety having a hydroperoxy group. Although further details about the
activity of ligurosides A and B are needed, our findings suggest that Qing Shan Lu Shui consumption
might be beneficial in the prevention of atherosclerosis.

Molecules 2013, 18
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Acknowledgments
The authors are grateful to the SC-NMR Laboratory of Okayama University and the MS Laboratory
of Faculty of Agriculture, Okayama University for providing the facilities to conduct our experiments.
We thank Miyuki Kaneda and Hiroaki Sasaki of School of Pharmacy, Shujitsu University for analysis
of sugars using HPLC with an optical rotatory detector.
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Sample Availability: Samples of the compounds are not available from the authors.
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