Planta Med 2013; 79(08): 680-686
DOI: 10.1055/s-0032-1328460
Natural Product Chemistry
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Isolation and Identification of Constituents with Activity of Inhibiting Nitric Oxide Production in Raw 264.7 Macrophages from Gentiana triflora

Shufang Wang
1   Pharmaceutical Informatics Institute, Zhejiang University, Zijingang Campus, Hangzhou, P. R. China
,
Yimin Xu
1   Pharmaceutical Informatics Institute, Zhejiang University, Zijingang Campus, Hangzhou, P. R. China
,
Wei Jiang
1   Pharmaceutical Informatics Institute, Zhejiang University, Zijingang Campus, Hangzhou, P. R. China
,
Yufeng Zhang
1   Pharmaceutical Informatics Institute, Zhejiang University, Zijingang Campus, Hangzhou, P. R. China
› Author Affiliations
Further Information

Correspondence

Dr. Shufang Wang
Pharmaceutical Informatics Institute, Zhejiang University, Zijingang Campus
No. 866 Yuhangtang Road
Hangzhou 310058
P. R. China
Phone: +86 5 71 88 20 84 26   
Fax: +86 5 71 88 20 84 26   

Publication History

received 26 August 2012
revised 03 February 2013

accepted 10 March 2013

Publication Date:
18 April 2013 (online)

 

Abstract

Gentiana triflora is widely used to treat inflammation, jaundice, hepatitis, and rheumatism. In this study, three new compounds, including a benzo seven-membered ring compound, gentioxepine (1), two secoiridoid glucosides, (1S,5R,9R)-deglucosyltrifloroside (2) and (1S,5R,9R)-scabraside (3), together with seven known ones, (+)-syringaresinol (4), deglucogelidoside (5), 3,4-dihydro-1H,6H,8H-naphtho[1,2-c:4,5-c',d']dipyrano-1,8-dione (6), deglucoscabraside (7), 2-hydroxy-3-O-β-D-glucosyloxy benzoic acid methyl ester (8), gentiolactone (9), and trifloroside (10), were isolated from the ethanol extract of Gentiana triflora. Their structures were mainly confirmed on the basis of NMR, MS, IR, CD, and UV spectral evidences. Inhibiting activities of nitric oxide production of eight of the compounds isolated, as well as gentiopicroside, were evaluated in the macrophage cell line RAW 264.7. The results show that the three new compounds and compound 7 could significantly suppress lipopolysaccharide-induced production of NO, with IC50s of 2.2 µM, 37.5 µM, 17.6 µM and 6.9 µM, respectively. Among them, compounds 1, 3, and 7 showed stronger inhibitory activity than that of the clinically used drug indometacin. Other tested compounds exerted moderate inhibiting activities.


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Introduction

Gentiana, the largest genus of the Gentianaceae family, consists of over 400 species, which are widely distributed in many countries in the Northern hemisphere [1]. Among them, the roots and rhizomes of Gentiana manshurica Kitag, Gentiana scabra Bge, Gentiana triflora Pall. (G. triflora Pall.), and Gentiana rigescens Franch are officially listed in the Chinese Pharmacopoeia (2010 edition). Their common indications in the traditional Chinese medical system include treating inflammation and fungus infections, and stimulating oneʼs appetite and gastric secretion as well as protecting the liver [2]. Iridoid and secoiridoid glycosides are mainly constituents of Gentiana. Previous reports on Gentiana were mainly about Gentiana scabra Bge. There are few reports about the chemical constituents of G. triflora [3], [4].

Inflammation is a protective biological response to pathological and damaged cells in the human body [5]. The inflammatory process has been linked to human cancers in recent research [6]. Nitric oxide (NO) is a product of cells in the anti-inflammatory process. The prolonged and overproduction of NO plays an important role in several chronic diseases, for example, arthritis, myositis, and chronic hepatitis [6], [7]. NO is the major inflammatory mediator synthesized by inducible NO synthase (iNOS) [8]. The possible mechanism to suppress NO synthesis is the inhibition of gene expression and/or the inhibition of the iNOS enzyme. Thus, there may be great potential for the prevention and treatment of inflammation to suppress the signaling molecules of iNOS expression [9].

Previous reports have shown that the Gentiana species possesses anti-inflammatory activity [10], [11], [12], [13], [14]. Animal experiments showed that gentiopicroside possesses anti-inflammatory effects on acute and chronic inflammatory reactions [15], [16], [17], [18]. However, there are few reports regarding the anti-inflammatory activity of other compounds from G. triflora.

In this study, we explored the biological active constituents of G. triflora. We report the isolation and identification of ten compounds ([Fig. 1]), including three new chemicals (1, 2, and 3), from G. triflora. Inhibiting activities against lipopolysaccharide (LPS)-induced NO production in RAW 264.7 cells of 8 isolated compounds and gentiopicroside, which has been reported as an important component [19] and also identified in the extraction of this herb, were tested.

Zoom Image
Fig. 1 Structures of compounds 110 and gentiopicroside.

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Materials and Methods

General

1D and 2D-NMR spectra were recorded on a Bruker Ultrashield Plus 500 MHz (1H: 500 MHz, 13C: 125 MHz) spectrometer with tetramethylsilane (TMS) as an internal standard. Preparative HPLC was carried out with an Agilent-1100 system, photodiode array detector, and Zorbax SB-C18 column (21.2 × 250 mm, 7 µm). Optical rotations were performed on a JASCO 1010 polarimeter. IR spectra were recorded on a JASCO FT-4100 spectrophotometer. High-resolution fourier transform ion cyclotron resonance mass spectrometry (HR-FT-ICR-MS) was recorded on a Bruker Apex III spectrometer and HR-ESI-TOF-MS on a Waters Q-TOF premier mass spectrometer. Circular dichroism (CD) was recorded on a JASCO J-815 spectrometer. HPLC-PDA-MS measurement was performed on a Finigan LCQ DecaXPplus mass spectrometer, coupled with a photodiode array detector.

Preparative HPLC methanol and acetonitrile were purchased from Jiangsu Hanbon Science & Technology Co., Ltd. Chemicals of analytical grade, such as methanol, dicloromethane, and ethyl acetate, were purchased from Shanghai Lingfeng Chemical Reagents Co., Ltd.. Silica gel and diatomite were purchased from Qingdao Haiyang Silica Gel Co., Ltd.. Deionized water was purified by a Milli-Q Water Purification system (Milli-pore). Gentiopicroside (purity > 99 %) was purchased from Shanghai Winherb Medical S&T Development Co. Ltd.. Dulbeccoʼs modified Eagleʼs medium, fetal bovine serum, and the antibiotic mixture (penicillin-streptomycin) were purchased from Gibco Co.. Indometacin (purity ≥ 99 %), lipopolysaccharides, and DMSO were from Sigma (Sigma Chemical Co.). The purity (> 95 %) of the isolated compounds used for the biological assay was determined by the available spectroscopic techniques.


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Plant material

The roots and rhizomes of G. triflora were collected from Jilin province, China, in June 2011, and were authenticated by Professor Liurong Chen and Senior Laboratory Technician Jianxia Mo from the College of Pharmaceutical Sciences, Zhejiang University. A voucher specimen (LD-20110609) was deposited in the College of Pharmaceutical Sciences, Zhejiang University.


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Extraction and isolation

The air-dried and pulverized roots and rhizomes of G. triflora (6.5 kg) were soaked in 80 % ethanol (39 L) for 48 hours. The supernatant was separated and the residue was percolated with 80 % ethanol for 35 hours. The extracts were merged and concentrated by evaporation under reduced pressure. The residue was suspended in water and partitioned with ethyl acetate. The ethyl acetate portion (336.5 g) was purified by solid phase extraction using diatomite (400 g) as the stationary phase and was then eluted with a gradient of petroleum ether (7 L), CH2Cl2 (7 L), ethyl acetate (10 L), and CH3OH (4.5 L), respectively. Then, the CH2Cl2 fraction (145.4 g) was subjected to silica gel (200–300 mesh, 18 × 60 cm, 2 kg) column chromatography (CC) and eluted with a gradient of petroleum ether – CH2Cl2 – CH3OH (5 : 1 : 0, 2 : 1 : 0, 1 : 1 : 0, 0 : 1 : 0, 0 : 500 : 1, 0 : 200 : 1, 0 : 100 : 1, 0 : 50 : 1, 0 : 20 : 1, 0 : 10 : 1, 0 : 5 : 1, 0 : 2 : 1, 0 : 1 : 1, 0 : 0 : 1, each 27 L) to give 22 fractions (1–22). Fraction 2 (700 mg) was subsequently subjected to silica (80 g, 300–400 mesh, 2.0 × 45 cm) gel CC (petroleum ether/ethyl acetate, 500 : 1, 200 : 1, 100 : 1, 50 : 1, 30 : 1, 20 : 1, 10 : 1, 5 : 1, 3 : 1, 1 : 1, 0 : 1) to afford 24 subfractions (2–1–2–24), and fraction 2–15 was further purified by preparative HPLC (CH3OH/H2O, 73 : 27) to yield 1 (20 mg, 73 % CH3OH, t R 25 min). Fraction 4 (1 g) was subjected to silica gel (70 g, 300–400 mesh, 2.0 × 45 cm) CC (petroleum ether/acetoacetate, 15 : 1, 10 : 1, 8 : 1, 5 : 1, 3 : 1, 1 : 1, each 1.5 L) to give 6 (5 mg). Fraction 6 was purified by preparative HPLC (CH3OH/H2O, 42 : 58) to give 9 (83 mg, t R 14.2 min). Fraction 8 was then subjected to ODS CC (CH3OH/H2O, 30 : 70 to 90 : 10, 7 h) to afford 19 subfractions (8–1–8–19). Fraction 8–6 was purified by preparative HPLC (CH3CN/H2O 30 : 70) to give 4 (115 mg, t R 36 min). Fraction 8–12 was purified by preparative HPLC to give 5 (5 mg, CH3OH/H2O 53 : 47, t R 46 min) and 2 (233 mg, CH3OH/H2O 53 : 47, t R 58 min). Fraction 8–14 was purified by preparative HPLC repeatedly to get 7 (10 mg, CH3CN/H2O 60 : 40, t R 65 min). Fraction 18 was subjected to ODS CC (CH3OH/H2O, 30 : 70 to 90 : 10, 7 h) to afford 15 subfractions. Fraction 18–8 was applied to preparative HPLC to get 8 (20 mg, CH3CN/H2O 48 : 52, t R 8.5 min) and 3 (248 mg, CH3CN/H2O 73 : 27, 33 min). Fraction 18–8 was subjected to preparative HPLC to get 10 (500 mg, CH3CN/H2O 61 : 39, 49 min). Gentiopicroside was detected in the CH2Cl2 portion and identified by comparing its retention time, UV spectra, and MS spectra with those of the standard using HPLC-PDA-MS analysis. Therefore, it was not further isolated in this study.


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Inhibitory activity of compounds on NO production from RAW 264.7 cells

RAW 264.7 cells were cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM) supplemented with 10 % heat-inactivated FBS with penicillin G (100 units/mL) and streptomycin (100 µg/mL) in a humidified 5 % CO2/95 % air atmosphere at 37 °C. The cells were harvested with trypsin-EDTA and diluted with fresh medium to a suspension. The suspended cells were seeded in 96-well plates (2 × 104 cells/well) and allowed to adhere for 24 h. The stock solution of each test sample was dissolved in DMSO, and was then diluted with DMEM (final DMSO concentration is less than 0.1 %). Cytotoxicity of each compound was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric method. Briefly, after incubation with the test samples for 24 h, MTT solution (100 µL, 0.5 mg/mL in phosphate buffered saline [PBS]) was added to the wells. After 4 h further incubation, the medium was removed and DMSO (150 µL/well) was added to dissolve the formazan produced in the cells. The absorbance of the formazan solution was measured with a microplate reader at 550 nm.

To determine inhibitory activity of each compound, the cells were coincubated with fresh medium (150 µL/well) containing 200 ng/mL of LPS, together with the tested compounds at various concentrations (0.2–50.0 µM) for 24 h. For the positive control group, cells were coincubated with indometacin and LPS. Griess reagent was used to determine the NO production by measuring the accumulation of nitrite in the culture supernatant. Briefly, 100 µL of the supernatant from incubates were mixed with an equal volume of Griess reagent (0.5 % sulfanilamide and 0.05 % naphthylenediamide dihydrochloride in 2.5 % H3PO4) and vibrated at room temperature. The absorbance was measured with a microplate reader at 550 nm. Indomethacin was used as a positive control. Inhibition activity (%) was calculated usuing the following equation and IC50 values were calculated using the CalcuSyn program (Biosoft):

Inhibition activity (%) = A–B/A-C × 100

A–C: NO2 concentration (µM) [A: LPS (+), sample (−); B: LPS (+), sample (+); C: LPS (−), sample (−)]

The NO2 concentration in the medium was calculated from the calibration curve obtained by using different concentrations of sodium nitrite (NaNO2) in the culture medium as the standard.

The results are expressed as the means with SD of triplicate experiments.


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Supporting information

The NMR spectra for compounds 13 are available as Supporting Information.


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Results and Discussion

Compound 1 was obtained as a yellow powder with a molecular formula of C13H12O4 as established by the HR-ESI-TOF-MS m/z at 233.0799 [M + H]+ (calcd. for C13H13O4: 233.0808). UV, λ max (CH3OH) nm: 201, 239. The IR spectrum indicated the presence of an ester group (1713 cm−1), a conjugated carbonyl (1661 cm−1, 1624 cm−1) and a trisubstituted phenyl ring (889 cm−1, 848 cm−1, 770 cm−1).

The 1H-NMR (see [Table 1]) of compound 1 showed three aromatic proton signals at δ H 8.42 (1H, J = 2.3 Hz, d), 8.05 (1H, J = 8.5, 2.3 Hz, dd), and 7.23 (1H, J = 8.5 Hz, d), which were characteristic of a 1,3,4-substituted phenyl ring; an alkene proton signal at δ H 6.30 (1H, J = 1.2 Hz, d), a methylene signal at δ H 4.80 (s, 2H), a methoxy group at δ H 3.85 (3H, s), and a typical methyl resonance at δ H 2.04 (3H, J = 1.2 Hz, d). The 13C-NMR and distortionless enhancement by polarization transfer (DEPT) spectrum data (see [Table 1]) revealed that compound 1 contained 13 carbon resonances. Among them, signals at δ c 186.8 and 165.2 indicated that there was a ketone carbonyl and an ester carbonyl. Signals at δ c 72.8 (s), 52.3 (s), 22.5 (s) were assignable to an oxygenated methylene, a methoxy, and a methyl group, respectively. The remaining 8 signals were sp2 carbons. All of the assignments were made according to 1H-1H COSY, HSQC, and HMBC spectra ([Fig. 2]).

Zoom Image
Fig. 2 Key correlations in the HMBC and NOESY spectra of compounds 1, 2, and 3.

Table 11H and 13C (DEPT) NMR data of compound 1 (500 and 125 MHz, in DMSO).

Position

13C

1H

2

72.8 (CH2)

4.80 (s, 2H)

3

155.7 (C)

/

4

130.0 (CH)

6.30 (d, J = 1.2 Hz, 1H)

5

186.8 (C)

/

6

132.4 (CH)

8.42 (d, J = 2.3 Hz, 1H)

7

124.9 (C)

/

8

135.0 (CH)

8.05 (dd, J = 8.5, 2.3 Hz, 1H)

9

122.0 (CH)

7.23 (d, J = 8.5 Hz, 1H)

9a

162.3 (C)

/

6a

128.2 (C)

/

10

22.5 (CH3)

2.04 (d, J = 1.2 Hz, 3H)

11

165.2 (C)

/

12

52.3 (CH3)

3.85 (s, 3H)

In the HMBC spectra, correlations from H-2 [δ H 4.80 (s, 2H)] to C-9a (δ c 162.3), C-4 (δ c 130.0), C-3 (δ c 155.7), C-10 (δ c 22.5) and C-5 (δ c 186.8, a weak 4J correlation) and from H-4 [δ H 6.30 (d, J = 1.2 Hz, 1H)] to C-6a (δ c 128.2) and C-10 (δ c 22.5) indicated the existence of a seven-numbered oxo-heterocycle. Then, the correlations from H-6 [δ H 8.42 (d, J = 2.3 Hz, 1H)] to C-5 (δ c 186.8) and C-9a (δ c 162.3), and from H-9 [δ H 8.42 (d, J = 2.3 Hz, 1H)] to C-6a (δ c 128.2) confirmed the position of a seven-numbered oxo-heterocycle. The signals also showed the correlation between H-12 [δ H 3.85 (s, 3H)] and C-11 (δ c 165.2), which demonstrated the existence of a methyl benzoate. The correlations among H-9 [δ H 7.23 (d, J = 8.5 Hz, 1H)] and C-7 (δ c 124.9), H-8 [δ H 8.05 (dd, J = 8.5, 2.3 Hz, 1H)] and C-11 (δ c 165.2), and H-6 [δ H 8.42 (d, J = 2.3 Hz, 1H)] and C-11 (δ c 165.2) proved the position of the methyl ester. Consequently, the structure of compound was established as (Z)–methyl-3-methyl-5-oxo-2,5-dihydrobenzo[b]oxepine-7–carboxylate, unambiguously. It was named gentioxepine. It is the first benzo seven-membered ring compound isolated from Gentiana. Since chemicals of this skeleton had only been isolated from the fungus Marasmiellus ramealis (Bull. ex Fr.) Singer (which possesses a hydroxymethyl either at the C-3 or C-7 position) [20], this is the first time it has been found in natural plants.

Compound 2 was obtained as a white amorphous powder. [α]D 29 − 200 (c = 1.13, CH3OH); UV, λ max (CH3OH) nm: 221, 250, 331. Its molecular formula was determined to be C29H32O15 according to the HR-FT-ICR-MS: m/z 643.1601 [M + Na]+ (calcd. for C29H32O15Na: 643.1633) and 1D NMR spectra. The IR spectrum indicated the presence of a hydroxyl group (3468 cm−1), an ester group (1754 cm−1), a lactone group (1676 cm−1), a double bound (1624 cm−1) and a trisubstituted phenyl ring (905 cm−1, 842 cm−1, 754 cm−1). The 1H-NMR spectrum of 2 showed signals of three aliphatic acetoxyl groups: δ 1.99, 1.97, 1.90 (each 3H, s); a secoiridoid glucoside: δ 1.82–1.75, 1.75 − 1.64 (each 1H, m, C6-H2), 2.78–2.73 (1H, m, C9-H), 2.94–2.86 (1H, m, C5-H), 7.60 (1H, d, J = 2.3 Hz, C3-H); three aromatic aromatic protons: δ 7.05 (1H, d, J = 7.8 Hz, C4′′-H), 6.77 (1H, t, J = 8.0 Hz, C5′′-H); 7.30 (1H, d, J = 8.1 Hz, C6′′-H); and signals due to the acyl glucose moiety: δ 5.20 (d, J = 8.0 Hz, 1H, C1′-H). The 1C-NMR spectrum of 2 was similar to that of trifloroside (6) except for the absence of one sugar moiety. These facts show that 2 is deglucosyltrifloroside. This was further confirmed by HSQC and HMBC (as shown in [Fig. 2]). The planar structure of 2 was deduced as shown in [Fig. 1]. Then, a NOESY experiment was carried out on 2 to determine the stereochemistry of the molecule. In the NOESY spectrum ([Fig. 2]), the correlation between H-1 and H-5 indicated that H-1 and H-5 were in the same face of the ring system. Then a CD experiment was carried out on 2. The CD spectrum showed a negative couplet Cotton with a large amplitude at 247 nm {[θ 247] − 3.6 × 104}, which indicated that the Glc group was on the β face of the ring system [21]. From these data, the structure of 2 was elucidated as (1S,5R,9R)-deglucosyltrifloroside.

Compound 3 was obtained as a white amorphous powder with the molecular formula C40H44O20 as determined by the HR-FT-ICR-MS measurement: m/z 867.2296 [M + Na]+ (calcd. for 867.2318). [α]D 29 − 154 (c = 1.30, CH3OH); UV, λ max (CH3OH) nm: 201, 209, 235, 313. The IR absorption bands at 3447 cm−1 suggested the presence of a hydroxyl group. IR bands at 1728 cm−1 and 1624 cm−1 suggested a conjugated ester. And, 900 cm−1, 842 cm−1, 754 cm−1 present a trisubstituted aromatic ring. The 1H-NMR (see [Table 2]) of 3 showed signals of two aliphatic acetoxyl groups: δ 1.85, 2.01 (each 3H, s); a secoiridoid glucoside: δ 1.36, 1.64 (each 1H, C6-H2), 2.73–2.66 (1H, m, C9-H), 2.58–2.51 (1H, m, C5-H); eight aromatic aromatic protons: δ 7.23–7.86; signals due to the acyl glucose moiety: 5.75 (1H, t, J = 9.6 Hz, C3′-H); and an anomeric proton with a large coupling constant (J = 7.2 Hz) at δ 4.81 (1H, d, J = 7.2 Hz, C1′′′-H). The 1H-NMR spectrum of 3 was very similar to that of scabraside. According to the 13C-NMR, HSQC, and HMBC spectra ([Fig. 2]), the planar structure of 3 was determined as shown in [Fig. 1]. Its structure was further explored by NOESY and CD spectra. In the NOESY spectrum ([Fig. 2]), the correlation between H-1 and H-5 was observed, which indicated that H-1 and H-5 were in the same face of the ring system. According to the CD spectrum, compound 3 showed a negative couplet Cotton with a large amplitude at 245 nm {[θ 245] − 7.8 × 104}, which indicated that the Glc group was on the β face of the ring system [21]. The structure of compound 3 was finally determined as (1S,5R,9R)-scabraside.

Table 213C NMR data of compounds 2 (500 and 125 MHz, in CD3OD) and 3 (500 and 125 MHz, in DMSO-d 6), δ in ppm.

Position

2

3

13C

1H

13C

1H

1

98.7 (CH)

5.41 (1H, d, J = 1.7 Hz),

95.8 (CH)

5.34 (1H, s)

3

153.4 (CH)

7.60 (1H, d, J = 2.3 Hz)

150.6 (CH)

7.24 (1H, t, J = 3.1 Hz)

4

106.9 (C)

/

104.9 (C)

/

5

28.8 (CH)

2.94–2.86 (1H, m)

27.2 (CH)

2.58–2.51 (1H, m)

6

25.9 (CH2)

1.82–1.75 (1H, m)

24.0 (CH2)

1.64 (1H, d, J = 9.5 Hz)

1.75–1.64 (1H, m)

1.36 (1H, dd, J = 12.9, 3.6 Hz)

7

70.1 (CH2)

4.38–4.32 (1H, m)
4.50–4.44 (1H, m)

67.5 (CH2)

4.23–4.13 (1H, m)
3.76–3.61 (1H, m)

8

132.9 (CH)

5.56–5.49 (1H, m)

131.5 (CH)

5.42–5.36 (1H, m)

9

43.5 (CH)

2.78–2.73 (1H, m)

40.8 (CH)

2.73–2.66 (1H, m)

10

121.6 (CH2)

5.35–5.33 (1H, m)

120.7 (CH2)

5.33–5.27 (1H, m)

5.30 (dd, J = 10.3, 1.5 Hz, 1H)

5.23–5.20 (1H, m)

11

168.0 (C)

/

163.3 (C)

/

1′

97.7 (CH)

5.20 (1H, dd, J = 8.0, 0.7 Hz)

94.9 (CH)

5.43 (1H, d, J = 8.1 Hz)

2′

72.4 (CH)

5.04–5.00 (1H, m)

71.4 (CH)

5.16 (1H, dd, J = 9.6, 7.3 Hz)

3′

73.4 (CH)

5.58–5.53 (1H, m)

71.5 (CH)

5.75 (1H, t, J = 9.6 Hz)

4′

71.1 (CH)

5.39–5.34 (1H, m)

68.8 (CH)

5.33 (1H, m)

5′

73.0 (CH)

4.18–4.16 (1H, m)

71.2 (CH)

4.38–4.31 (1H, m)

6′

63.5 (CH2)

4.30 (1H, dd, J = 12.7, 4.4 Hz)

61.7 (CH2)

4.38–4.31 (1H, m)

4.23 (1H, dd, J = 12.3, 2.6 Hz)

4.23–4.13 (1H, m)

1′′

113.2 (C)

/

115.4 (C)

/

2′′

151.6 (C)

/

148.9 (C)

/

3′′

147.4 (C)

/

146.0 (C)

/

4′′

122.6 (CH)

7.05 (1H, d, J = 7.8 Hz)

123.1 (CH)

7.28 (1H, dd, J = 7.9, 0.9 Hz)

5′′

120.5 (CH)

6.77 (1H, t, J = 8.0 Hz)

118.9 (CH)

6.86 (1H, t, J = 8.0 Hz)

6′′

121.5 (CH)

7.30 (1H, dd, J = 8.1, 0.8 Hz)

120.8 (CH)

7.38 (1H, d, J = 7.4 Hz)

7′′

170.5 (C)

/

165.8 (C)

/

1′′′

/

/

101.8 (CH)

4.81 (1H, d, J = 7.2 Hz)

2′′′

/

/

73.3 (CH)

3.37–3.29 (1H, m)

3′′′

/

/

76.1 (CH)

3.21–3.16 (1H, m)

4′′′

/

/

69.8 (CH)

5.13–5.08 (1H, m)

5′′′

/

/

77.3 (CH)

3.37–3.29 (1H, m)

6′′′

/

/

60.8 (CH2)

3.76–3.61 (1H, m) 3.53–3.45 (1H, m)

1′′′′

/

/

128.6 (C)

/

2′′′′, 6′′′′

/

/

129.0 (CH)

7.84 (2H, t, J = 7.4 Hz)

3′′′′, 5′′′′

/

/

128.9 (CH)

7.48 (2H, t, J = 7.7 Hz)

4′′′′

/

/

133.8 (CH)

7.64 (1H, t, J = 7.4 Hz)

7′′′′

/

/

164.8 (C)

/

2′-COCH3

171.1 (C)

/

/

/

20.8 (CH3)

1.99 (3H, s)

/

/

3′-COCH3

171.5 (C)

/

169.5 (C)

/

20.6 (CH3)

1.90 (3H, s)

20.3 (CH3)

1.85 (s, 3H)

6′-COCH3

172.3 (C)

/

170.1 (C)

20.7 (CH3)

1.97 (3H, s)

20.5 (CH3)

2.01 (s, 3H)

The known compounds were confirmed by comparing their spectroscopic data with that in the literature. They were (+)-syringaresinol (4) [22], deglucogelidoside (5) [23], 3,4-dihydro-1H,6H,8H-naphtho[1,2-c:4,5-c',d']dipyrano-1,8-dione (6) [24], deglucoscabraside (7) [25], 2-hydroxy-3-O-β-D-glucosyloxy benzoic acid methyl ester (8) [25], gentiolactone (9) [26], and trifloroside (10) [27].

NO is the major inflammatory mediator synthesized by iNOS [8]. The concentration of nitrite that is accumulated in the cell culture medium is always measured indirectly as evidence of NO release. Macrophages were expected to be an origin of inflammation [8]. Thus, we tested the compounds toward NO production induced by LPS in macrophage-derived RAW 264.7 cells.

The inhibition effect against NO production was measured as the inhibitory rate against NO production when the tested compound was added along with LPS (200 ng/mL) compared to LPS (200 ng/mL) alone (0 %). All compounds were tested at various concentrations (0.2 to 50.0 µM) without cytotoxicity to the cells, according to the cytotoxicity test ([Tables 3] and [4]). The results showed that new compounds 1 and 3, along with compound 7, possessed the most potent inhibitory activity against NO production with IC50 values of 2.2 ± 1.4 µM, 17.6 ± 2.9, and 6.9 ± 1.7 µM, respectively, which were stronger than the positive control indometacin (IC50 31.6 ± 2.0 µM). The new compound 2 possessed a little weaker inhibitory activity than indometacin with an IC50 of 37.5 ± 8.3 µM. Above all, all the compounds tested, except for compound 9, showed that they were more sensitive in inhibiting NO production than gentiopicroside at various concentrations, which has been proven to be active in anti-inflammatory studies on animals. This shows that the isolated compounds have potential anti-inflammatory researches in the future.

Table 3 Inhibitory effects on NO production of compounds at various concentrationsa.

Compound

LPS (200 ng/mL)

0.2 (µM)

0.4 (µM)

0.8 (µM)

1.6 (µM)

3.1 (µM)

IC50 (µM)b

a The RAW 264.7 cells were incubated with 0.2–3.1 µM of the tested compounds in the presence of LPS (200 ng/mL) for 24 h. Each value represents mean ± SD of three determinations. b The IC50 value was calculated as the concentration of compound under which 50 % inhibition of nitrite production occurred compared to non-treated controls

1

24.3 ± 16.4

29.9 ± 19.2

30.6 ± 8.0

47.5 ± 15.6

59.5 ± 10.9

2.2 ± 1.4

Table 4 Inhibitory effects on NO production of compounds at various concentrationsa.

Compound

LPS (200 ng/mL)

3.1 (µM)

6.3 (µM)

12.5 (µM)

25.0 (µM)

50.0 (µM)

IC50 (µM)b

a The RAW 264.7 cells were incubated with 3.1–50.0 µM of the tested compounds in the presence of LPS (200 ng/mL) for 24 h. Each value represents mean ± SD of three determinations. b The IC50 value was calculated as the concentration of compound under which 50 % inhibition of nitrite production occurred compared to non-treated controls. c IC50 not detected

2

14.8 ± 6.7

19.8 ± 9.6

27.0 ± 3.8

39.6 ± 4.4

59.9 ± 4.4

37.5 ± 8.3

3

21.8 ± 1.3

22.5 ± 5.1

32.2 ± 2.9

59.4 ± 4.2

78.2 ± 4.2

17.6 ± 2.9

4

3.0 ± 9.7

10.2 ± 5.4

26.1 ± 4.1

36.5 ± 8.1

44.7 ± 10.3

-c

7

24.4 ± 4.3

33.3 ± 7.9

84.9 ± 11.0

91.7 ± 14.4

85.6 ± 10.8

6.9 ± 1.7

8

8.5 ± 5.2

11.5 ± 0.9

14.9 ± 2.9

17.1 ± 2.3

27.3 ± 5.4

9

3.6 ± 4.5

7.2 ± 6.7

12.3 ± 9.6

18.5 ± 4.1

37.8 ± 6.6

10

18.5 ± 6.5

23.2 ± 12.5

25.3 ± 9.9

36.1 ± 9.6

45.1 ± 14.6

Gentiopicroside

18.6 ± 17.4

24.5 ± 14.2

30.5 ± 9.5

34.0 ± 19.8

39.0 ± 7.0

Indometacin

19.8 ± 1.6

24.5 ± 1.4

39.8 ± 2.8

43.9 ± 2.4

56.7 ± 2.8

31.6 ± 2.0

In the present study, the structure-activity relationship of the isolated compounds may be summarized as follow: (1) The presence of a benzoyl group at the 2′-position and the different absolute structure of the 5-position may have resulted in the enhancement of inhibitory activity as shown in 2 versus 7, and 3 versus 10; and (2) The absolute structure of the 5-position and a glucoside in the 3′′-O-position may influence the inhibitory activity as shown in 2 and 10.

Ten compounds, including three new ones, have been isolated and identified from 80 % ethanol extracts of G. triflora. Compound 1 was the first benzo seven-membered ring compound isolated from this species. Compounds 2 and 3 were epimers of the known compounds deglucotrifloroside and scabraside, respectively. In fact, among the seven known compounds, 6 was isolated from G. triflora for the first time, and 5 was isolated from a natural plant for the first time. Their structures were determined using IR, UV, 1D and 2D NMR, MS, and CD experiments.

Eight of the isolated compounds and gentiopicroside were tested against LPS-induced NO production in RAW 264.7 cells. The results show that compounds 1, 3, and 7 possessed strong activity against the LPS-induced NO production in RAW 264.7 cells. These findings shed light on the research of active components with anti-inflammatory activity from G. triflora.


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Acknowledgements

The work is supported by the Fundamental Research Funds for the Central Universities (2011FZA7005) and the National Science and Technology Major Project of China (No. 2011ZX09307-002-01).


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Conflict of Interest

All authors declare that there are no conflicts of interest.

Supporting Information

  • References

  • 1 Georgieva E, Handjieva N, Popov S, Evstatieva L. Comparative analysis of the volatiles from flowers and leaves of three Gentiana species. Biochem Syst Ecol 2005; 33: 938-947
  • 2 Matsukawa K, Ogata M, Hikage T, Minami H, Shimotai Y, Saitoh Y, Yamashita T, Ouchi A, Tsutsumi R, Fujioka T, Tsutsumi KI. Antiproliferative activity of root extract from gentian plant (Gentiana triflora) on cultured and implanted tumor cells. Biosci Biotechnol Biochem 2006; 70: 1046-1048
  • 3 Inouye H, Ueda S, Nakamura Y, Inoue K, Hayano T, Matsumur H. Trifloroside, new secoiridoid glucoside from Gentiana triflora var. japonica . Tetrahedron 1974; 30: 571-577
  • 4 Jiang RW, Wong KL, Chan YM, Xu HX, But PP, Shaw PC. Isolation of iridoid and secoiridoid glycosides and comparative study on Radix Gentianae and related adulterants by HPLC analysis. Phytochemistry 2005; 66: 2674-2680
  • 5 Li C, Wang MH. Anti-inflammatory effect of the water fraction from hawthorn fruit on LPS-stimulated RAW 264.7 cells. Nutr Res Pract 2011; 5: 101-106
  • 6 Tamir S, Tannenbaum SR. The role of nitric oxide (NO center dot) in the carcinogenic process. Biochim Biophys Acta 1996; 1288: F31-F36
  • 7 Hsu CL, Chang FR, Tseng PY, Chen YF, El-Shazly M, Du YC, Fang SC. Geranyl flavonoid derivatives from the fresh leaves of Artocarpus communis and their anti-inflammatory activity. Planta Med 2012; 78: 995-1001
  • 8 Han Y, Jung HW, Lee JY, Kim JS, Kang SS, Kim YS, Park YK. 2,5-dihydroxyacetophenone isolated from Rehmanniae Radix Preparata inhibits inflammatory responses in lipopolysaccharide-stimulated RAW264.7 macrophages. J Med Food 2012; 15: 505-510
  • 9 Murakami A. Chemoprevention with phytochemicals targeting inducible nitric oxide synthase. Forum Nutr 2009; 61: 193-203
  • 10 Cao FH, Shao H, Li Q, Li JR, Li WQ, Li C. Anti-inflammatory activity of Gentiana striata Maxim. Nat Prod Res 2012; 26: 1038-1044
  • 11 Lim H, Son KH, Chang HW, Kang SS, Kim HP. Inhibition of chronic skin inflammation by topical anti-inflammatory flavonoid preparation, Ato Formula (R). Arch Pharm Res 2006; 29: 503-507
  • 12 Kwak WJ, Kim JH, Ryu KH, Cho YB, Jeon SD, Moon CK. Effects of gentianine on the production of pro-inflammatory cytokines in male Sprague-Dawley rats treated with lipopolysaccharide (LPS). Biol Pharm Bull 2005; 28: 750-753
  • 13 Yu FR, Yu FH, Li RD, Wang R. Inhibitory effects of the Gentiana macrophylla (Gentianaceae) extract on rheumatoid arthritis of rats. J Ethnopharmacol 2004; 95: 77-81
  • 14 Mathew A, Taranalli AD, Torgal SS. Evaluation of anti-inflammatory and wound healing activity of Gentiana lutea rhizome extracts in animals. Pharm Biol 2004; 42: 8-12
  • 15 Chen L, Liu JC, Zhang XN, Guo YY, Xu ZH, Cao W, Sun XL, Sun WJ, Zhao MG. Down-regulation of NR2B receptors partially contributes to analgesic effects of Gentiopicroside in persistent inflammatory pain. Neuropharmacology 2008; 54: 1175-1181
  • 16 Chen L, Wang HB, Sun XL, Sun WJ. Study on the analgesic and anti-inflammatory activities of Gentiopicroside. Tianran Chanwu Yanjiu Yu Kaifa 2008; 20: 903-906
  • 17 Li YQ. The main pharmacodynamics research of gentiopicroside [dissertation]. Shanxi: Xibei University; 2002
  • 18 Chen CX, Liu ZW, Sun ZR, Song CQ, Hu ZB. Studies on anti-inflammatory effect of gentiopicroside. Zhong Cao Yao 2003; 34: 49-51
  • 19 Song QS, Gao KB, Fu KZ. Isolation and identification of gentiopicroside from the roots of Gentiana triflora Pall. Zhong Yao Tong Bao 1987; 12: 36-59
  • 20 Holroyde JK, Orr AF, Thaller V. 3,7-Bis (hydroxymethyl)-1-benzoxepin-5 (2H)-one, a novel oxygen heterocyclic metabolite from cultures of the fungus Marasmiellus ramealis (Bull. ex Fr.) Singer. J Chem Soc [Perkin I] 1978; 1: 1490-1493
  • 21 Ikeda T, Hutchinson CR, Meier H, Tietze LF. Stereochemical correlations of secoiridoid aglucones. Tetrahedron Lett 1984; 25: 2427-2430
  • 22 Nie LY, Jin HZ, Yan L, Qin JJ, Zhang WD. Chemical constituents of Inula lineariifolia Turcz. Tianran Chanwu Yanjiu Yu Kaifa 2011; 23: 643-646
  • 23 Ikeshiro Y, Mase I, Tomita Y. A Secoiridoid Glucoside from Gentiana scabra var. buergeri . Planta Med 1990; 56: 101-103
  • 24 Kitanov GM, Spassov SL. A naphthodipyranodione from Gentiana asclepiadea . Phytochemistry 1992; 31: 1067-1068
  • 25 Ikeshiro Y, Tomita Y. A New bitter secoiridoid glucoside from Gentiana scabra var. buergeri . Planta Med 1983; 48: 169-173
  • 26 Kakuda R, Machida K, Yaoita Y, Kikuchi M. Studies on the constituents of Gentiana species. II. A new triterpenoid, and (S)-(+)- and (R)-(−)-gentiolactones from Gentiana lutea . Chem Pharm Bull 2003; 51: 885-887
  • 27 Tan RX, Wolfender JL, Zhang LX, Fuzzati W, Marston A, Hostettmann K. Acyl secoiridoids and antifungal constituents from Gentiana macrophylla . Phytochemistry 1996; 42: 1305-1313

Correspondence

Dr. Shufang Wang
Pharmaceutical Informatics Institute, Zhejiang University, Zijingang Campus
No. 866 Yuhangtang Road
Hangzhou 310058
P. R. China
Phone: +86 5 71 88 20 84 26   
Fax: +86 5 71 88 20 84 26   

  • References

  • 1 Georgieva E, Handjieva N, Popov S, Evstatieva L. Comparative analysis of the volatiles from flowers and leaves of three Gentiana species. Biochem Syst Ecol 2005; 33: 938-947
  • 2 Matsukawa K, Ogata M, Hikage T, Minami H, Shimotai Y, Saitoh Y, Yamashita T, Ouchi A, Tsutsumi R, Fujioka T, Tsutsumi KI. Antiproliferative activity of root extract from gentian plant (Gentiana triflora) on cultured and implanted tumor cells. Biosci Biotechnol Biochem 2006; 70: 1046-1048
  • 3 Inouye H, Ueda S, Nakamura Y, Inoue K, Hayano T, Matsumur H. Trifloroside, new secoiridoid glucoside from Gentiana triflora var. japonica . Tetrahedron 1974; 30: 571-577
  • 4 Jiang RW, Wong KL, Chan YM, Xu HX, But PP, Shaw PC. Isolation of iridoid and secoiridoid glycosides and comparative study on Radix Gentianae and related adulterants by HPLC analysis. Phytochemistry 2005; 66: 2674-2680
  • 5 Li C, Wang MH. Anti-inflammatory effect of the water fraction from hawthorn fruit on LPS-stimulated RAW 264.7 cells. Nutr Res Pract 2011; 5: 101-106
  • 6 Tamir S, Tannenbaum SR. The role of nitric oxide (NO center dot) in the carcinogenic process. Biochim Biophys Acta 1996; 1288: F31-F36
  • 7 Hsu CL, Chang FR, Tseng PY, Chen YF, El-Shazly M, Du YC, Fang SC. Geranyl flavonoid derivatives from the fresh leaves of Artocarpus communis and their anti-inflammatory activity. Planta Med 2012; 78: 995-1001
  • 8 Han Y, Jung HW, Lee JY, Kim JS, Kang SS, Kim YS, Park YK. 2,5-dihydroxyacetophenone isolated from Rehmanniae Radix Preparata inhibits inflammatory responses in lipopolysaccharide-stimulated RAW264.7 macrophages. J Med Food 2012; 15: 505-510
  • 9 Murakami A. Chemoprevention with phytochemicals targeting inducible nitric oxide synthase. Forum Nutr 2009; 61: 193-203
  • 10 Cao FH, Shao H, Li Q, Li JR, Li WQ, Li C. Anti-inflammatory activity of Gentiana striata Maxim. Nat Prod Res 2012; 26: 1038-1044
  • 11 Lim H, Son KH, Chang HW, Kang SS, Kim HP. Inhibition of chronic skin inflammation by topical anti-inflammatory flavonoid preparation, Ato Formula (R). Arch Pharm Res 2006; 29: 503-507
  • 12 Kwak WJ, Kim JH, Ryu KH, Cho YB, Jeon SD, Moon CK. Effects of gentianine on the production of pro-inflammatory cytokines in male Sprague-Dawley rats treated with lipopolysaccharide (LPS). Biol Pharm Bull 2005; 28: 750-753
  • 13 Yu FR, Yu FH, Li RD, Wang R. Inhibitory effects of the Gentiana macrophylla (Gentianaceae) extract on rheumatoid arthritis of rats. J Ethnopharmacol 2004; 95: 77-81
  • 14 Mathew A, Taranalli AD, Torgal SS. Evaluation of anti-inflammatory and wound healing activity of Gentiana lutea rhizome extracts in animals. Pharm Biol 2004; 42: 8-12
  • 15 Chen L, Liu JC, Zhang XN, Guo YY, Xu ZH, Cao W, Sun XL, Sun WJ, Zhao MG. Down-regulation of NR2B receptors partially contributes to analgesic effects of Gentiopicroside in persistent inflammatory pain. Neuropharmacology 2008; 54: 1175-1181
  • 16 Chen L, Wang HB, Sun XL, Sun WJ. Study on the analgesic and anti-inflammatory activities of Gentiopicroside. Tianran Chanwu Yanjiu Yu Kaifa 2008; 20: 903-906
  • 17 Li YQ. The main pharmacodynamics research of gentiopicroside [dissertation]. Shanxi: Xibei University; 2002
  • 18 Chen CX, Liu ZW, Sun ZR, Song CQ, Hu ZB. Studies on anti-inflammatory effect of gentiopicroside. Zhong Cao Yao 2003; 34: 49-51
  • 19 Song QS, Gao KB, Fu KZ. Isolation and identification of gentiopicroside from the roots of Gentiana triflora Pall. Zhong Yao Tong Bao 1987; 12: 36-59
  • 20 Holroyde JK, Orr AF, Thaller V. 3,7-Bis (hydroxymethyl)-1-benzoxepin-5 (2H)-one, a novel oxygen heterocyclic metabolite from cultures of the fungus Marasmiellus ramealis (Bull. ex Fr.) Singer. J Chem Soc [Perkin I] 1978; 1: 1490-1493
  • 21 Ikeda T, Hutchinson CR, Meier H, Tietze LF. Stereochemical correlations of secoiridoid aglucones. Tetrahedron Lett 1984; 25: 2427-2430
  • 22 Nie LY, Jin HZ, Yan L, Qin JJ, Zhang WD. Chemical constituents of Inula lineariifolia Turcz. Tianran Chanwu Yanjiu Yu Kaifa 2011; 23: 643-646
  • 23 Ikeshiro Y, Mase I, Tomita Y. A Secoiridoid Glucoside from Gentiana scabra var. buergeri . Planta Med 1990; 56: 101-103
  • 24 Kitanov GM, Spassov SL. A naphthodipyranodione from Gentiana asclepiadea . Phytochemistry 1992; 31: 1067-1068
  • 25 Ikeshiro Y, Tomita Y. A New bitter secoiridoid glucoside from Gentiana scabra var. buergeri . Planta Med 1983; 48: 169-173
  • 26 Kakuda R, Machida K, Yaoita Y, Kikuchi M. Studies on the constituents of Gentiana species. II. A new triterpenoid, and (S)-(+)- and (R)-(−)-gentiolactones from Gentiana lutea . Chem Pharm Bull 2003; 51: 885-887
  • 27 Tan RX, Wolfender JL, Zhang LX, Fuzzati W, Marston A, Hostettmann K. Acyl secoiridoids and antifungal constituents from Gentiana macrophylla . Phytochemistry 1996; 42: 1305-1313

Zoom Image
Fig. 1 Structures of compounds 110 and gentiopicroside.
Zoom Image
Fig. 2 Key correlations in the HMBC and NOESY spectra of compounds 1, 2, and 3.