Three new secoiridoid glycosides from the rhizomes and roots of Gentiana scabra and their anti-inflammatory activities

Article abstract of DOI:10.1080/14786419.2015.1013955

Three new (1-3) and 17 known (4-20) iridoid and secoiridoid glycosides were isolated from a methanol extract of the rhizomes and roots of Gentiana scabra. Their chemical structures were elucidated from 1D and 2D NMR, IR absorption, and HR-ESI-MS spectra, as well as comparisons of these data with reported values. The effects of the isolated compounds on lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells were investigated. Compounds 6, 10 and 20 exhibited significant inhibitory effects on LPS-induced IL-12 p40 and IL-6 production with IC₅₀ values of 1.62-14.29 M. Compound 10 also showed a strong inhibitory effect on the LPS-stimulated production of TNF- with an IC₅₀ value of 10.45 M.

Full text of DOI:10.1080/14786419.2015.1013955

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Natural Product Research: Formerly
Natural Product Letters
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Three new secoiridoid glycosides from
the rhizomes and roots of Gentiana
scabra and their anti-inflammatory
activities
Wei Lia, Wei Zhoubc, Sohyun Kimd, Jung-Eun Kood, Yuna Kima,
Young-Sang Kohd, Sang Hee Shime, Jin Yeul Maf & Young Ho Kimb
a School of Biotechnology, Yeungnam University, Gyeongsan,
Gyeongbuk 712-749, South Korea
b College of Pharmacy, Chungnam National University, Daejeon
305-764, South Korea
c College of Pharmacy, Yanbian University, Yanji 133002, P.R.
China
Click for updates
d School of Medicine and Brain Korea 21 PLUS Program, Institute
of Medical Science, Jeju National University, Jeju 690-756, South
Korea
e College of Pharmacy, Duksung Women's University, 33, Samyang-
ro 144-gil, Dobong-gu, Seoul 132-714, South Korea
f Korea Institute of Oriental Medicine, Center of Herbal Medicine
Improvement Research, Daejeon 305-811, South Korea
Published online: 04 Mar 2015.
To cite this article: Wei Li, Wei Zhou, Sohyun Kim, Jung-Eun Koo, Yuna Kim, Young-Sang Koh, Sang
Hee Shim, Jin Yeul Ma & Young Ho Kim (2015): Three new secoiridoid glycosides from the rhizomes
and roots of Gentiana scabra and their anti-inflammatory activities, Natural Product Research:
Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1013955
To link to this article: http://dx.doi.org/10.1080/14786419.2015.1013955
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Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2015.1013955
Three new secoiridoid glycosides from the rhizomes and roots of Gentiana
scabra and their anti-inflammatory activities
Wei Lia, Wei Zhoubc, Sohyun Kimd, Jung-Eun Kood, Yuna Kima, Young-Sang Kohd, Sang
Hee Shime*, Jin Yeul Maf* and Young Ho Kimb*
aSchool of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, South Korea; bCollege of
Pharmacy, Chungnam National University, Daejeon 305-764, South Korea; cCollege of Pharmacy, Yanbian
University, Yanji 133002, P.R. China; dSchool of Medicine and Brain Korea 21 PLUS Program, Institute of
Medical Science, Jeju National University, Jeju 690-756, South Korea; eCollege of Pharmacy, Duksung
Women’s University, 33, Samyang-ro 144-gil, Dobong-gu, Seoul 132-714, South Korea; fKorea Institute of
Oriental Medicine, Center of Herbal Medicine Improvement Research, Daejeon 305-811, South Korea
(Received 5 December 2014; final version received 28 January 2015)
Inhibition
O
O
O
O
O
O
HO
OH
O
O
AcO
OH
O
O
O
AcO
HO
HO
O
O
HO
O
O
O
O
OH
IL-12 p40
IL-6
O
R2
O
OR1
AcO
OAc
R1 R2
O
1
2
3
H
Ac
H
H
H
Ac
6
HO
COOH
O
HO
HO
O
O
OH
OH
O
O
O
AcO
HO
O
HO
O
O
O
O
AcO
HO
O
O
HO
OH
10
20
TNF-α
Gentiana scabra Bunge
Three new (1–3) and 17 known (4–20) iridoid and secoiridoid glycosides were
isolated from a methanol extract of the rhizomes and roots of Gentiana scabra. Their
chemical structures were elucidated from 1D and 2D NMR, IR absorption, and HR-
ESI-MS spectra, as well as comparisons of these data with reported values. The effects
of the isolated compounds on lipopolysaccharide (LPS)-stimulated bone marrow-
derived dendritic cells were investigated. Compounds 6, 10 and 20 exhibited significant
inhibitory effects on LPS-induced IL-12 p40 and IL-6 production with IC50 values of
1.62–14.29 mM. Compound 10 also showed a strong inhibitory effect on the LPS-
stimulated production of TNF-a with an IC50 value of 10.45 mM.
Keywords: Gentiana scabra; Gentianaceae; secoiridoid glycoside; anti-inflammatory
1. Introduction
The rhizomes and roots of Gentiana scabra Bunge (Gentianaceae), commonly known as
‘Longdan’ in Chinese herbal medicine, have been used to treat inflammation, anorexia,
indigestion and gastric infections (Ikeshiro & Tomita 1983). Pharmacological research has
shown that G. scabra protects the liver, inhibits liver dysfunction and promotes gastric acid
secretion, which makes it a popular ingredient in Chinese herbal medicines and health products
(Zhang et al. 2010). The chemical constituents of G. scabra including iridoids, secoiridoids,
iridoid glycosides, secoiridoid glycosides, triterpenoids, flavonoids, xanthones and alkaloids
*Corresponding authors. Email: shshim29@naver.com; jyma@kiom.re.kr; yhk@cnu.ac.kr
q 2015 Taylor & Francis

2
W. Li et al.
have been reported previously (Ikeshiro & Tomita 1983; Ikeshiro et al. 1990; Tan et al. 1996;
Bergeron et al. 1997; Kakuda et al. 2001, 2002; Kim et al. 2009). Secoiridoid glycosides are
primary functional components of G. scabra and exhibit smooth muscle relaxing, antibacterial
and free radical-scavenging activities (Rojas et al. 2000; Kumarasamy et al. 2003). In this study,
20 iridoid and secoiridoid glycosides were isolated from the rhizomes and roots of G. scabra.
The anti-inflammatory effects of isolated compounds on LPS-induced expression of the pro-
inflammatory cytokines IL-6, IL-12 p40 and TNF-a in bone marrow-derived dendritic cells
(BMDCs) were investigated.
2. Results and discussion
Three new (1–3) secoiridoid glycosides and 17 known (4–20) compounds were isolated from a
methanol extract of the rhizomes and roots of G. scabra. The isolated compounds were identified
as 2,3-deacetyltrifloroside (1), 3-deacetyltrifloroside (2), 2-deacetyltrifloroside (3), trifloroside
(4; Kim et al. 2009), 4000-O-b-D-glucopyranosyltrifloroside (5; Kim et al. 2009), deglucosyltri-
floroside (6; Wang et al. 2013), scabraside (7; Kim et al. 2009), 4000-O-b-D-glucopyranosylscab-
raside (8; Kim et al. 2009), gelidoside (9; Tan & Kong 1997), deglucosylscabraside (10; Ikeshiro
& Tomita 1983), gentiopicroside (11; Kim et al. 2009), 60-O-b-D-glucopyranosylgentiopicroside
(12; Kakuda et al. 2001), 30-O-b-D-glucopyranosylgentiopicroside (13; Takeda et al. 1999), 40-
O-b-DD-glucopyranosylgentiopicroside (14; Kakuda et al. 2001), 8-epikingiside (15; Garcia
et al. 1989), loganin (16; Ikeshiro & Tomita 1983), caryoptoside (17; Damtoft 1992),
secologanoside (18; Basaran et al. 1988), gentianaside (19; Kyoya et al. 2005) and 400-O-b-D-
glucopyranosyllinearoside (20; Bergeron et al. 1997). The chemical structures of these
compounds were elucidated based on 1D and 2D NMR spectroscopic analyses, MS spectra and
comparisons of these data with reported values. Notably, compounds 6, 9, 15, 17, 18 and 20 were
isolated from G. scabra for the first time.
Compound 1 was isolated as a white powder. The molecular formula was established as
C31H38O18 by a quasimolecular-ion peak [M þ Na]þ at m/z 721.1925 (calcd for 721.1950) in the
HR-ESI-MS spectrum. The IR spectrum of 1 contained absorption bands characteristic of a
hydroxyl group (3412 cm21), an ester group (1760 cm21) and an aromatic moiety (1641 and
1588cm21). The1HNMR spectrumcontainedthreesignalscorrespondingtoaromaticprotonsatdH
6.68 (t, J ¼ 8.1 Hz, H-500), 7.55 (dd, J ¼ 8.1, 1.2 Hz, H-600) and 7.65 (dd, J ¼ 8.1, 1.2 Hz, H-400);
double bonds at dH 5.07 (m, H-10), 5.36 (ddd, J ¼ 16.0, 9.1, 7.7 Hz, H-8), and 7.88 (d, J ¼ 2.2 Hz,
H-3); an acetal proton at dH 5.67 (brs, H-1); two anomeric protons at dH 5.30 (d, J ¼ 7.5 Hz, H-10)
and 5.56 (d, J ¼ 7.5 Hz, H-1000) and methyl protons at dH 1.91 (s, 60-COCH3). The 13C NMR and
DEPTspectraof 1 contained signals correspondingtothree carbonylgroups[dC 164.7 (C-11), 169.3
(C-700), 170.3 (60-COCH3)], six aromatic carbon atoms [dC 114.2 (C-100), 118.8 (C-500), 122.3 (C-400),
123.0(C-600),147.0(C-300),152.5(C-200)], twodoublebonds[dC 105.2(C-4),120.0(C-10), 132.3(C-
8), 152.1 (C-3)], two anomeric carbon atoms [dC 100.5 (C-10), 102.6 (C-1000)], ten sugar moiety
carbon atoms [dC 62.1–78.8] and a methyl carbon atom at dC 20.4. The above-mentioned data are
consistent with compound 1 as a secoiridoid glycoside similar to trifloroside (4). However,
compound 1 lacks the two acetyl groups at C-20 and 30 of compound 4. HMBC correlations between
dH 4.42/4.66 (H-60) and dC 170.3 (60-COCH3) indicated that the acetyl group was connected to C-60
of glucose. Enzymatic hydrolysis of 1 produced D-glucose as a sugar residue, identified by GC and
consistent with reported results. The b-D-configurations of the glucose residue were determined
from the coupling constant (7.5Hz) of the anomeric proton. Basis on the structure determined from
these data, compound 1 was named 2,3-deacetyltrifloroside.
Compounds 2 and 3 were obtained as white powder. The molecular formulae of 2 and 3 were
C33H40O19 based on a pseudomolecular ion peak [M þ Na]þ at m/z 763.2024 in their HR-ESI-
MS spectra (calcd for C33H40O19Na, 763.2056). The 1H and 13C NMR spectra of 2 and 3 were

Natural Product Research
3
similar to those of 1. However, 2 and 3 both contained two acetyl groups attached to the glucose
moiety. The difference between compounds 2 and 3 was revealed in their 13C NMR and DEPT
spectra. Compound 2 contains a glucose moiety indicated by peaks at dC 64.0 (C-60), 68.5 (C-30),
71.8 (C-40), 74.5 (C-50), 75.5 (C-20) and 96.9 (C-10). In contrast, the 13C NMR spectra of
compound 3 contained peaks at dC 62.6 (C-60), 72.1 (C-30), 72.3 (C-20), 72.5 (C-50), 73.9 (C-40)
and 96.9 (C-10). These data show that the acetyl groups of 2 and 3 are located at different
positions on the glucose moiety. The HMBC spectrum of 2 showed correlations between dH
4.40/4.95 (H-60) and dC 170.1 (60-COCH3), and 5.80 (H-20) and dC 169.7 (20-COCH3), indicating
acetyl groups on the C-20 and C-60 atoms of glucose. The HMBC spectrum of 3 showed
correlations between dH 4.44/4.62 (H-60) and dC 170.3 (60-COCH3), and 5.83 (H-30) and dC 168.9
(30-COCH3), indicating acetyl groups on the C-30 and C-60 atoms of glucose. Thus, based on the
structures elucidated from these data, compounds 2 and 3 were named as 3-deacetyltrifloroside
and 2-deacetyltrifloroside, respectively (Figure 1).
The effects of various concentrations (1, 2, 5 and 10 mM) of compounds 1–20 on the
secretion of cytokines IL-6, IL-12 p40 and TNF-a were evaluated. BMDCs were incubated in
48-well plates and treated for 1 h with the isolated compounds prior to stimulation with LPS
(10 ng/mL). Supernatants were harvested 18 h after stimulation. SB203580, an inhibitor of p38
kinase, was used as a positive control (Lee et al. 1994) and inhibited IL-6, IL-12 p40 and TNF-a
production with IC50 values of 3.5, 5.0 and 7.2 mM, respectively. Compounds 6, 10 and 20
significantly inhibited the production of IL-6 and IL-12 p40 production, with IC50 values ranging
from 1.62 to 14.29 mM (Figure 2). In particular, compound 10 was the most effective for IL-12
p40 inhibition and was more potent than the positive control. Compound 10 also showed a strong
inhibitory effect on LPS-stimulated production of TNF-a with an IC50 value of 10.45 mM.
However, several of the isolated compounds had no effects at the concentrations tested
(IC50 . 100 mM). The anti-inflammatory effects of secoiridoid glycosides from the Gentiana
O
O
O
O
O
O
O
O
11
7
4
6
3
5
10
8
HO
HO
OH
OH
1''
O
O
OH
O
O
9
AcO
AcO
AcO
1
O
O
O
RO
O
R O
3
O
O
O
1'''
HO
O
O
O
7''
HO
O
HO
O
O
OH
OH
1'
OR
AcO
R O
AcO
2
O
O
OAc
1
R
H
Ac
H
Ac Ac
Ac Ac
R
R
1
2
3
7
8
R = H
R = Glc
6
1
H
H
Ac
H
H
H
H
2
3
4
5
O
O
O
COOCH
3
O
O
O
Glc
O
O
O
O
O
O
O
OH
AcO
O
HO
R O
HO
1
O
O
HO
O
O
O
OH
HO
OH
O
O
HO
HO
R O
O
3
AcO
AcO
O
O
R O
2
HO
HO
O
OH
15
O
O
OH
AcO
R
R
R
3
1
2
OAc
O
11
12
13
14
H
H
H
H
9
10
Glc
H
H
Glc
H
H
H
Glc
HO
COOH
COOCH
COOCH
COOH
HO
3
3
O
HO
O
HO
HO
O
O
HOOC
HO
OH
O
O
HO
HO
HO
HO
HO
OH
HO
O
O
O
O
O
O
O
HO
O
OH
O
HO
O
OH
O
O
O
O
O
HO
HO
HO
HO
HO
HO
HO
HO
OH
OH
OH
17
OH
18
20
16
19
O
Figure 1. Structures of compounds 1–20 from the rhizomes and roots of G. scabra.

4
W. Li et al.
(a) 100
(b) 100
80
60
40
20
0
80
60
40
20
0
10
1
2
5 10
1
2
5
10
1
2
5
10
10
1
2
5
10
1
2
5
10
1
2
5
10
Comp.20
Comp.20
Comp.6
Comp.6
Pos.
Comp.10
Pos.
Comp.10
Concentration (μM)
Concentration (μM)
Figure 2. Effect of compounds 6, 10 and 20 on IL-12 p40 (A) and IL-6 (B) production by LPS-stimulated
BMDCs at the concentration of 1.0, 2.0, 5.0 and 10.0 mM. The data were presented as inhibition rate (%)
compared with the value of vehicle-treated DCs. SB203580 was used as positive control (Pos.).
genus have been reported recently, and compound 10 significantly suppress the LPS-induced
production of nitric oxide (Wang et al. 2013).
The observed anti-inflammatory activities and structural features of compounds 1–10
provide information regarding structure–function relationships. Compounds 6 and 10 showed
stronger activities than compounds 1–5 and 7–9. This may be attributed to the fact that C-300 was
not bound to a glucosyl group. This suggests that the 2,3-dihydroxy benzoyl group on the C-40
atom of the glucose moiety plays an important role in the anti-inflammatory activity. These also
provide information that can be used to evaluate the structure–function relationship of other
secoiridoid glycosides. This study is the first to show that iridoid and secoiridoid glycosides from
G. scabra inhibit the production of pro-inflammatory cytokines IL-12 p40, IL-6 and TNF-a. The
data presented herein suggest that the various iridoid and secoiridoid glycosides in G. scabra
contribute to its observed anti-inflammatory effect.
3. Experimental
3.1. General experimental procedures
Optical rotations were determined using a Jasco DIP-370 automatic polarimeter (Easton, USA).
The FT-IR spectra were measured using a Jasco Report-100 infrared spectrometer. The NMR
spectra were recorded using a JEOL ECA 600 spectrometer (1H, 600 MHz; 13C, 150 MHz).
High-resolution electrospray ionisation mass spectra (HR-ESI-MS) were obtained using an
Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Santa Clara, USA). Column
chromatography was performed using a silica gel (Kieselgel 60, 70–230, and 230–400 mesh,
Merck, Darmstadt, Germany), YMC RP-18 resins, and thin-layer chromatography was
performed using pre-coated silica gel 60 F254 and RP-18 F254S plates (both 0.25 mm, Merck).
3.2. Plant material
Dried rhizomes and roots of G. scabra were purchased from herbal company, Naemome Dah,
Ulsan, Korea in December 2013 and identified by one of the authors (Prof. Young Ho Kim).

Natural Product Research
5
A voucher specimen (CNU 13109) was deposited at the Herbarium of College of Pharmacy,
Chungnam National University.
3.3. Extraction and isolation
Dried rhizomes and roots (2.5 kg) of G. scabra were extracted with MeOH (10 L £ 3) under
reflux. The MeOH extract (670.0 g) was suspended in water and partitioned with CHCl3 and n-
BuOH. The n-BuOH fraction (190.0 g) was subjected to silica gel (8 cm £ 30 cm) column
chromatography with CHCl3–MeOH–H2O (12:1:0, 6.5:1:0, 4:1:0, 2.5:1:0.1) to give four
fractions (Fr. 1A–1D). The fraction 1B (15.0 g) was subjected to YMC (3 cm £ 80 cm) column
chromatography with a MeOH–H2O (0.05:1 to 0.95:1) elution solvent to give eight sub-
fractions (Fr. 1B-1–1B-8). The fraction 1B-5 was subjected to silica gel (2.5 cm £ 80 cm)
column chromatography with CHCl3–MeOH (10:1 to 7:1) elution solvent to give compounds 6
(1.1 g), 10 (127.0 mg) and 11 (2.2 g). The fraction 1C (17.0 g) was subjected to YMC (3 cm
£ 80 cm) column chromatography with a MeOH–H2O (0.05:1 to 1:1) elution solvent to give
12 sub-fractions (Fr. 1C-1–1C-12). The fraction 1C-1 was separated using a silica gel
(1.5 cm £ 80 cm) column chromatography with CHCl3–MeOH (8.5:1 to 8:1) elution solvent to
give compounds 1 (13.0 mg), 15 (22.0 mg) and 16 (1.5 g). The fraction 1C-3 was separated using
an YMC (1 cm £ 80 cm) column chromatography with a MeOH–acetone–H2O (0.25:0.25:1 to
0.4:0.4:1) elution solvent to give compounds 4 (60.0 mg) and 17 (15.0 mg). The fraction 1C-5
was separated using an YMC (1 cm £ 80 cm) column chromatography with a MeOH–acetone–
H2O (0.2:0.2:1 to 0.3:0.3:1) elution solvent to give compounds 2 (11.0 mg) and 3 (5.0 mg). The
fraction 1C-6 was separated using a silica gel (1.5 cm £ 80 cm) column chromatography with
CHCl3–MeOH (14:1 to 10:1) elution solvent to give compounds 5 (100.0 mg) and 8 (420.0 mg).
The fraction 1D (10.0 g) was subjected to YMC (3 cm £ 80 cm) column chromatography with a
MeOH–H2O (0:1 to 1:1) elution solvent to give 9 sub-fractions (Fr. 1D-1–1D-9). The fraction
1D-4 was separated using a silica gel (1 cm £ 80 cm) column chromatography with CHCl3–
MeOH–H2O (6.5:1:0.05 to 4:1:0.1) elution solvent to give compounds 12 (70.0 mg), 13
(20.0 mg) and 14 (2.5 mg). The fraction 1D-5 was separated using a silica gel (1 cm £ 80 cm)
column chromatography with CHCl3–MeOH–H2O (6.5:1:0.1 to 4:1:0.1) elution solvent to give
compounds 18 (6.0 mg), 19 (8.5 mg) and 20 (15.0 mg). The fraction 1D-6 was separated using an
YMC (2 cm £ 80 cm) column chromatography with a MeOH–acetone–H2O (0.4:0.4:1) elution
solvent to give compounds 7 (360.0 mg) and 9 (280.0 mg).
3.3.1. 2,3-Deacetyltrifloroside (1)
25
White powder; C31H38O18; ½aꢀD : 213.02 (c ¼ 0.1, MeOH); IR (KBr): nmax 3412, 1760, 1641,
1588 cm21; 1H NMR (pyridine-d5, 600 MHz): 5.67 (1H, brs, H-1), 7.88 (1H, d, J ¼ 2.2 Hz, H-3),
2.87 (1H, m, H-5), 1.42 (1H, m, H-6a), 1.30 (1H, m, H-6b), 4.16 (1H, ddd, J ¼ 12.5, 11.0,
3.0 Hz, H-7a), 3.76 (1H, t, J ¼ 10.6 Hz, H-7b), 5.36 (1H, ddd, J ¼ 16.0, 9.1, 7.7 Hz, H-8), 2.61
(1H, m, H-9), 5.07 (2H, m, H-10), 5.30 (1H, J ¼ 7.5 Hz, H-10), 4.39 (1H, t, J ¼ 9.0 Hz, H-20),
4.20 (1H, m, H-30), 5.80 (1H, t, J ¼ 9.6 Hz, H-40), 4.25 (1H, m, H-50), 4.66 (1H, dd, J ¼ 12.3,
4.8 Hz, H-60a), 4.42 (1H, m, H-60b), 7.65 (1H, dd, J ¼ 8.1, 1.2 Hz, H-400), 6.68 (1H, t, J ¼ 8.1 Hz,
H-500), 7.55 (1H, dd, J ¼ 8.1, 1.2 Hz, H-600), 5.56 (1H, J ¼ 7.5 Hz, H-1000), 4.10 (1H, m, H-2000),
4.28 (1H, m, H-3000), 4.26 (1H, m, H-4000), 4.06 (1H, m, H-5000), 4.48 (1H, m, H-6000a), 4.32 (1H, m,
H-6000b), 1.91 (3H, s, 60-OCOCH3). 13C NMR (pyridine-d5, 150 MHz): 97.6 (C-1), 152.1 (C-3),
105.2 (C-4), 27.5 (C-5), 24.8 (C-6), 67.7 (C-7), 132.3 (C-8), 42.7 (C-9), 120.0 (C-10), 164.7 (C-
11), 100.5 (C-10), 74.8 (C-20), 72.6 (C-30), 72.4 (C-40), 74.6 (C-50), 62.9 (C-60), 114.2 (C-100),
152.5 (C-200), 147.0 (C-300), 122.3 (C-400), 118.8 (C-500), 123.0 (C-600), 169.3 (C-700), 102.6 (C-1000),

6
W. Li et al.
74.6 (C-2000), 78.3 (C-3000), 70.9 (C-4000), 78.8 (C-5000), 62.1 (C-6000), 170.3 (60-OCOCH3), 20.4 (60-
OCOCH3); HR-ESI-MS: m/z 721.1925 [M þ Na]þ (calcd for C31H38O18Na, 721.1950).
3.3.2. 3-Deacetyltrifloroside (2)
25
White powder; C33H40O19; ½aꢀD : 212.85 (c 0.1, MeOH); IR (KBr): nmax 3410, 1755, 1631,
1579cm21; 1H NMR (pyridine-d5, 600 MHz): 5.56 (1H, brs, H-1), 7.80 (1H, d, J ¼ 2.2 Hz, H-3),
2.89(1H, m, H-5), 1.38(1H, m, H-6a), 1.28(1H, m, H-6b), 4.20 (1H, ddd, J ¼ 12.5, 11.0, 3.0 Hz, H-
7a), 4.06 (1H, m, H-7b), 5.28 (1H, ddd, J ¼ 16.0, 9.1, 7.7 Hz, H-8), 2.49 (1H, m, H-9), 5.00 (2H, m,
H-10), 5.40 (1H, J ¼ 7.5 Hz, H-10), 5.80 (1H, t, J ¼ 9.0 Hz, H-20), 4.31 (1H, m, H-30), 5.41 (1H, m,
H-40), 4.26(1H, m, H-50), 4.95(1H, m, H-60a), 4.40(1H, m, H-60b), 7.64(1H, dd, J ¼ 8.1, 1.2 Hz, H-
400), 6.62 (1H, t, J ¼ 8.1 Hz, H-500), 7.51 (1H, dd, J ¼ 8.1, 1.2 Hz, H-600), 5.56 (1H, J ¼ 7.5 Hz, H-
1000), 4.19 (1H, m, H-2000), 4.28 (1H, m, H-3000), 4.26 (1H, m, H-4000), 4.04 (1H, m, H-5000), 4.48 (1H, m,
H-6000a), 4.32 (1H, m, H-6000b), 2.00 (3H, s, 20-OCOCH3), 1.91 (3H, s, 60-OCOCH3). 13C NMR
(pyridine-d5, 150 MHz): 97.2 (C-1), 151.4 (C-3), 105.9 (C-4), 27.7 (C-5), 24.6 (C-6), 68.1 (C-7),
131.7 (C-8), 42.0(C-9), 120.3 (C-10), 164.7 (C-11), 96.9(C-10), 75.5(C-20), 68.5(C-30), 71.8(C-40),
74.5 (C-50), 64.0 (C-60), 114.0 (C-100), 152.5 (C-200), 147.1 (C-300), 122.2 (C-400), 118.7 (C-500), 122.9
(C-600), 169.6 (C-700), 102.8 (C-1000), 74.9 (C-2000), 78.3 (C-3000), 70.9 (C-4000), 78.8 (C-5000), 62.1 (C-6000),
169.7(20-OCOCH3), 20.4(20-OCOCH3), 170.1(60-OCOCH3), 20.5(60-OCOCH3);HR-ESI-MS: m/
z 763.2024 [M þ Na]þ (calcd for C33H40O19Na, 763.2056).
3.3.3. 2-Deacetyltrifloroside (3)
25
White powder; C33H40O19; ½aꢀD : 212.02 (c ¼ 0.1, MeOH); IR (KBr): nmax 3427, 1763, 1633,
1581 cm21; 1H NMR (pyridine-d5, 600 MHz): 5.54 (1H, brs, H-1), 7.84 (1H, d, J ¼ 2.2 Hz, H-3),
2.95 (1H, m, H-5), 1.41 (1H, m, H-6a), 1.29 (1H, m, H-6b), 4.22 (1H, ddd, J ¼ 12.5, 11.0,
3.0 Hz, H-7a), 4.10 (1H, m, H-7b), 5.30 (1H, ddd, J ¼ 16.0, 9.1, 7.7 Hz, H-8), 2.50 (1H, m, H-9),
5.00 (2H, m, H-10), 5.35 (1H, J ¼ 7.5 Hz, H-10), 4.47 (1H, m, H-20), 5.83 (1H, t, J ¼ 9.6 Hz, H-
30), 5.50 (1H, t, J ¼ 9.0 Hz, H-40), 4.28 (1H, m, H-50), 4.62 (1H, dd, J ¼ 12.3, 4.8 Hz, H-60a),
4.44 (1H, m, H-60b), 7.63 (1H, dd, J ¼ 8.1, 1.2 Hz, H-400), 6.66 (1H, t, J ¼ 8.1 Hz, H-500), 7.51
(1H, dd, J ¼ 8.1, 1.2 Hz, H-600), 5.54 (1H, J ¼ 7.5 Hz, H-1000), 4.24 (1H, m, H-2000), 4.28 (1H, m,
H-3000), 4.25 (1H, m, H-4000), 4.03 (1H, m, H-5000), 4.44 (1H, m, H-6000a), 4.32 (1H, m, H-6000b), 1.98
(3H, s, 30-OCOCH3), 1.89 (3H, s, 60-OCOCH3). 13C NMR (pyridine-d5, 150 MHz): 97.0 (C-1),
151.3 (C-3), 106.1 (C-4), 27.8 (C-5), 24.6 (C-6), 68.1 (C-7), 131.7 (C-8), 42.0 (C-9), 120.3 (C-
10), 164.8 (C-11), 96.9 (C-10), 72.3 (C-20), 72.1 (C-30), 73.9 (C-40), 72.5 (C-50), 62.6 (C-60), 114.2
(C-100), 152.4 (C-200), 147.1 (C-300), 122.3 (C-400), 118.7 (C-500), 122.9 (C-600), 169.7 (C-700), 102.7
(C-1000), 74.5 (C-2000), 78.2 (C-3000), 70.9 (C-4000), 78.9 (C-5000), 62.1 (C-6000), 168.9 (30-OCOCH3),
20.6 (30-OCOCH3), 170.3 (60-OCOCH3), 20.3 (60-OCOCH3); HR-ESI-MS: m/z 763.2024
[M þ Na]þ (calcd for C33H40O19Na, 763.2056).
3.4. Enzymatic hydrolysis
Compounds 1–3 (each 3.0 mg) was mixed with b-glucosidase (3.0 mg) in water (1.0 mL) and
was shaken in a water bath at 378C for 12 h. After this, the reaction mixture was concentrated and
then subjected to silica gel (1.0 cm £ 15.0 cm, 40–63 mm) column chromatography with
CHCl3–MeOH (15:1, 60 mL) and CHCl3–MeOH–H2O (7:3:0.5, 60 mL) to afford aglycone and
a sugar fraction. The sugar fraction was concentrated to dryness using N2 gas. The resulting
residue was dissolved in dry pyridine (0.1 mL), and then L-cysteine methyl ester hydrochloride
in pyridine (0.06 M, 0.1 mL) was added to the solution. After heating the reaction mixtures at
608C for 2 h, 0.1 mL of trimethylsilylimidazole solution was added. Heating at 608C was

Natural Product Research
7
continued for a further 1.5 h. The dried product was partitioned with n-hexane and H2O (0.1 mL
each), and the organic layer was analysed using gas chromatography (GC): DB-5 capillary
column (0.32 mm £ 30 m); FID detector; column temp., 2108C; injector temp., 2708C; detector
temp., 3008C; carrier gas He (2 mL/min). Under these conditions, standard sugars gave peaks at
tR (min) ¼ 14.12 and 12.24 for L- and D-glucose, respectively. The peaks of the hydrolysate of
1–3 were detected at tR (min) ¼ 12.21, 12.20 and 12.23, respectively, which was identified as D-
glucose by comparison with the retention time of the authentic samples after treatment with
trimethylsilylimidazole in pyridine.
3.5. Cell culture
BMDCs were grown from wild-type C57BL/6 mice (Orient Bio Inc., Seongnam-si, South
Korea) as previously described. All animal procedures were approved and performed according
to the guidelines of the Institutional Animal Care and Use Committee of Jeju National
University (#2010-0028). Briefly, the mouse tibia and femur was obtained by flushing with
Dulbecco’s modified Eagle’s medium to yield bone marrow cells. The cells were cultured in
RPMI 1640 medium containing 10% heat-inactivated foetal bovine serum (FBS; Gibco,
New York, NY, USA), 50 mM b-ME, 2 mM glutamine supplemented with 3% J558L hybridoma
cell culture supernatant containing granulocyte–macrophage colony stimulating factor. The
culture medium was replaced with fresh medium every second day. At day 6 of culture, non-
adherent cells and loosely adherent DC aggregates were harvested, washed and resuspended in
RPMI 1640 supplemented with 5% FBS.
3.6. Cytokine production measurements
BMDCs were incubated in 48-well plates in 0.5 mL containing 1 £ 105 cells per well, and then
treated with the isolated compounds 1–20 at the indicated concentration for 1 h before
stimulation with 10 ng/mL LPS from Salmonella minnesota (Alexis, New York, NY, USA).
Supernatants were harvested 18 h after stimulation. Concentrations of murine TNF-a, IL-6, IL-
12 p40 in the culture supernatants were determined by using ELISA (BD PharMingen, San
Diego, CA, USA) according to the manufacture’s instructions. The data are presented as
means ^ SD of at least three independent experiments performed in triplicate.
4. Conclusion
In our study, 20 iridoid and secoiridoid glycosides were isolated from a methanol extract of the
rhizomes and roots of G. scabra. Compounds 6, 9, 15, 17, 18 and 20 were isolated from
G. scabra for the first time. Moreover, this study is the first to show that iridoid and secoiridoid
glycosides from G. scabra inhibit the production of pro-inflammatory cytokines IL-12 p40, IL-6
and TNF-a. The data presented herein suggest that the various iridoid and secoiridoid glycosides
in G. scabra contribute to its observed anti-inflammatory effect.
Supplementary material
Supplementary material relating to this paper is available online, alongside Figures S1–S19 and Table S1.
Funding
This study was supported by the Priority Research Center Program (2009-0093815) through the National
Research Foundation of Korea (NRF) and a grant [grant number K14050] awarded to the Korean Institute
of Oriental Medicine funded by the Ministry of Education, Science and Technology (MEST), Republic of
Korea.

8
W. Li et al.
References
Basaran A, Akdemir Z, Yuruker A, Calis I. 1988. Iridoid and secoiridoid glucosides from Lonicera etrusca. Fitoterapia.
59:389–392.
Bergeron C, Marston A, Gauthier R, Hostettmann K. 1997. Iridoids and secoiridoids from Gentiana linearis.
Phytochemistry. 44:633–637.
Damtoft S. 1992. Iridoid glucosides from Lamium album. Phytochemistry. 31:175–178.
Garcia J, Lavaitte S, Gey C. 1989. 8-Epikingiside and its vanillate ester, isolated from Gentiana pyrenaica.
Phytochemistry. 28:2199–2201.
Ikeshiro Y, Mase I, Tomita Y. 1990. A secoiridoid glucoside from Gentiana scabra var. Buergeri. Planta Med.
56:101–103.
Ikeshiro Y, Tomita Y. 1983. A new bitter secoiridoid glucoside from Gentiana scabra var. Buergeri. Planta Med.
48:169–173.
Kakuda R, Iijima T, Yaoita Y, Machida K, Kikuchi M. 2001. Secoiridoid glycosides from Gentiana scabra. J Nat Prod.
64:1574–1575.
Kakuda R, Iijima T, Yaoita Y, Machida K, Kikuchi M. 2002. Triterpenoids from Gentiana scabra. Phytochemistry.
59:791–794.
Kim JA, Son NS, Son JK, Jahng Y, Chang HW, Jang TS, Na M, Lee SH. 2009. Two new secoiridoid glycosides from the
rhizomes of Gentiana scabra Bunge. Arch Pharm Res. 32:863–867.
Kumarasamy Y, Nahar L, Sarker SD. 2003. Bioactivity of gentiopicroside from the aerial parts of Centaurium erythraea.
Fitoterapia. 74:151–154.
Kyoya T, Kikuchi M, Kakuda R, Yaoita Y, Kikuchi M. 2005. A new iridoid diglycoside from Gentianae Scabrae Radix.
Nat Med. 59:178–180.
Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter
SW. 1994. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature.
372:739–746.
Rojas A, Bah M, Rojas JI, Gutierrez DM. 2000. Smooth muscle relaxing activity of gentiopicroside isolated from
Gentiana spathacea. Planta Med. 66:765–767.
Takeda Y, Masuda T, Honda G, Takaishi Y, Ito M, Ashurmetov OA, Khodzhimatov OK, Otsuka H. 1999. Secoiridoid
glycosides from Gentiana olivieri. Chem Pharm Bull. 47:1338–1340.
Tan RX, Kong LD. 1997. Secoiridoids from Gentiana siphonantha. Phytochemistry. 46:1035–1038.
Tan RX, Wolfender JL, Zhang LX, Ma WG, Fuzzati N, Marston A, Hostettmann K. 1996. Acyl secoiridoid and
antifungal constituents from Gentiana macrophylla. Phytochemistry. 42:1305–1313.
Wang S, Xu Y, Jiang W, Zhang Y. 2013. Isolation and identification of constituents with activity of inhibiting nitric oxide
production in Raw 264.7 macrophages from Gentiana triflora. Planta Med. 79:680–686.
Zhang HL, Xue SH, Pu F, Tiwari RK, Wang XY. 2010. Establishment of hairy root lines and analysis of gentiopicroside
in the medicinal plant Gentiana macrophylla. Russ J Plant Physiol. 57:110–117.