Diarylheptanoid glycosides from Tacca plantaginea and their effects on NF-κB activation and PPAR transcriptional activity

Diarylheptanoid glycosides from Tacca plantaginea and their effects on NF-κB activation and PPAR transcriptional activity

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6681–6687 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Let...

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Bioorganic & Medicinal Chemistry Letters 22 (2012) 6681–6687

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Diarylheptanoid glycosides from Tacca plantaginea and their effects on NF-jB activation and PPAR transcriptional activity Tran Hong Quang a,b, Nguyen Thi Thanh Ngan a, Chau Van Minh b, Phan Van Kiem b, Pham Hai Yen b, Bui Huu Tai a,b, Nguyen Xuan Nhiem a,b, Nguyen Phuong Thao a,b, Hoang Le Tuan Anh b, Bui Thi Thuy Luyen a, Seo Young Yang a, Chun Whan Choi a, Young Ho Kim a,⇑ a b

College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam

a r t i c l e

i n f o

Article history: Received 8 June 2012 Revised 24 August 2012 Accepted 27 August 2012 Available online 13 September 2012 Keywords: Tacca plantaginea Diarylheptanoid glycoside Plantagineoside NF-jB-luciferase assay PPRE-luciferase assay GAL-4-PPAR chimera assay

a b s t r a c t In the screening search for NF-jB inhibitory and PPAR transactivational agents from medicinal plants, a methanol extract of the whole plant of Tacca plantaginea and its aqueous fraction showed the significant activities. Bioassay-guided fractionation combined with repeated chromatographic separation of the aqueous fraction of the methanol extract of T. plantaginea resulted in the isolation of two new diarylheptanoid glycosides, plantagineosides A (1) and B (2), an unusual new cyclic diarylheptanoid glycoside, plantagineoside C (3), and three known compounds (4–6). Their structures were determined by extensive spectroscopic and chemical methods. Compounds 3–6 significantly inhibited TNFa-induced NF-jB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC50 values ranging from 0.9 to 9.4 lM. Compounds 1–6 significantly activated the transcriptional activity of PPARs in a dose-dependent manner, with EC50 values ranging from 0.30 to 10.4 lM. In addition, the transactivational effects of compounds 1–6 were evaluated on three individual PPAR subtypes, including PPARa, c, and b(d). Compounds 1–6 significantly enhanced the transcriptional activity of PPARb(d), with EC50 values in a range of 11.0– 30.1 lM. These data provide the rationale for using T. plantaginea and its components for the prevention and treatment of inflammatory and metabolic diseases. Ó 2012 Elsevier Ltd. All rights reserved.

Taccaceae is a small plant family, containing only the genus Tacca, which includes approximately 20 species. They are all herbal plants and are distributed predominately in tropical regions of Asia, the Pacific islands, and Africa.1 Taccalonolides,2–4 diarylheptanoids and diarylheptanoid glucosides,5 and steroids and steroidal glycosides,6,7 have been isolated from some Tacca species. Tacca plantaginea is a perennial plant that mainly grows in the south of Vietnam and China. Its rhizomes have been used in the traditional medicine as an analgesic, antipyretic, and anti-inflammatory agent to treat incised wounds and furuncles.1 Previous investigations of the chemical components of T. plantaginea have shown the presence of taccalonolide2,3 and winthanolide steroids.8,9 Although, T. plantaginea has been reported as a rich source of steroids, no diarylheptanoid have yet been isolated from this plant. The current study reports the isolation and structural elucidation of three new diarylheptanoid glycosides (1–3) from whole plants of T. plantaginea. NF-jB (nuclear factor-kappaB) is a transcription factor that plays important roles in the immune system. NF-jB regulates the ⇑ Corresponding author. Tel.: +82 42 821 5933. E-mail address: [email protected] (Y.H. Kim). 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.08.099

expression of cytokines, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), growth factors, inhibitors of apoptosis, and effector enzymes in response to ligation by T-cell receptors, B-cell receptors and members of the Toll-like receptor/IL-1 receptor superfamily.10–12 NF-jB plays an important role in the transcriptional regulation of numerous cytokines and adhesion molecules. Therefore, NF-jB is the most extensively studied transcription factor associated with the immune system. The activation of NF-jB by various stimuli, including inflammatory cytokines such as tumor necrosis factor a (TNFa) and IL-1, T-cell activation signals, growth factors, and stress inducers causes transcription at jB sites that are involved in a number of diseases, such as inflammatory disorders and cancer.13,14 As a result, the inhibition of NF-jB signaling has become a therapeutic target for the treatment of inflammatory diseases and cancer. In this study, the effects of compounds isolated from T. plantaginea on TNFa-induced NF-jB transcriptional activity in human hepatocarcinoma (HepG2) cells were evaluated using an NF-jB-luciferase assay (see Supplementary data). Peroxisome proliferator-activated receptors (PPARs) are ligandactivated transcription factors of the nuclear hormone receptor superfamily, of which three PPAR isoforms have been identified:

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PPARa, c, and b(d). PPARs play an important role in regulating the expression of genes involved in the regulation of glucose, lipid, and cholesterol metabolism, cell growth and differentiation by binding to specific peroxisome proliferator response elements (PPREs) in the enhancer sites of regulated genes.15–18 Activation of PPARs results in anti-inflammatory effects in several cell types, including smooth muscle cells, endothelial cells, and macrophages.19,20 PPARs act as anti-inflammatory agents by interfering with the transcriptional pathways involved in inflammatory responses, such as the modulation of NF-jB signaling.21,22 Therefore, discovery of PPAR agonists has become a target for the prevention and treatment of obesity, obesity-induced inflammation, insulin resistance, dyslipidemia, and cardiovascular disease. Thus, the effects of compounds isolated from T. plantaginea on the transcriptional activity of PPARs in HepG2 cells were assessed using a PPRE-luciferase assay (see Supplementary data). Although the PPAR subtypes share a high level of sequence and structural homology, each has distinct physiological functions and exhibits a unique tissue expression pattern.23 PPARa is highly expressed in metabolically active tissues, including liver, brown adipose tissue, muscle, and heart.24 PPARa regulates the expression of numerous genes involved in fatty acid metabolisms24 and inflammatory responses in the cells.25 PPARa is activated by a variety of natural and synthetic agonists, including unsaturated fatty acids and eicosanoids, and fibrate drugs, respectively. PPARc is expressed in adipose tissue, skeletal muscle, colon, and lung.26 PPARc target genes are related to adipocyte differentiation, lipid storage, and glucose metabolism.27 Unsaturated fatty acids and some eicosanoids are endogenous agonists, whereas thiazolidinediones (anti-diabetic drugs) are synthetic agonists of PPARc. PPARc agonists have demonstrated therapeutic potential for the treatment of obesity, diabetes, cardiovascular disease, inflammation, and cancer. PPARb(d) is expressed in a variety of tissues, particularly metabolically active sites such as the liver, muscle, and fat; however its role in metabolic syndromes has yet to be elucidated.28,29 PPAR b(d)-specific agonists have been reported to suppress hepatic glucose output, increase glucose elimination, and inhibit the release of free fatty acids from adipocytes, and thereby regulating glucose metabolism and insulin sensitivity.30 Currently, PPARb(d) is a pharmacological target for the treatment of metabolic disorders associated with metabolic syndromes, including dyslipidemia, obesity, and insulin resistance.31 The use of two synthetic selective agonists, GW501516 and GW610742, in various cell line models and animals, and the concomitant development of new genetically modified mouse models, are helping to unravel the role of PPARb(d) in lipid and glucose homeostasis and inflammation. Therefore, to understand how the isolated compounds modulate PPAR transcriptional activity, we further examined the transactivational effects of the isolated compounds on individual PPAR subtypes, PPARa, c, and b(d) using GAL-4-PPAR chimera assays (see Supplementary data). The whole plants of T. plantaginea were collected in Bach Ma National Garden, Thua Thien Hue Province, Vietnam in April 2010. The plant material was identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. A voucher specimen (IMBC100484) was deposited at herbarium, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST). A MeOH extract (150 g) of the whole plants of T. plantaginea was suspended in H2O and successively extracted with n-hexane, CH2Cl2, and EtOAc. The aqueous fraction was subjected to multiple chromatographic steps over silica gel and reversed-phase C18 to provide compounds 1–6.32 Comparison of the NMR and MS data with those reported previously led to the identification of known compounds: (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)heptane 3-O-b-Dglucopyranoside (4),5 (3R,5R)-3,5-dihydroxy-1-(3,4-dihydroxy-

phenyl)-7-(4-hydroxyphenyl)-heptane 3-O-b-D-glucopyranoside (5),5 and (3R,5R)-3,5-dihydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane 3-O-b-D-glucopyranoside (6)5 (Fig. 1). Acid hydrolysis led to the isolation of two products, 2a and 6a, as the aglycones of 2 and 6, respectively.33 Compound 6a was identified as (3R,5R)-3,5dihydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane based on comparison of its 1H and 13C NMR spectroscopic data with those of previously reported compound.5 Compound 1 was obtained as a white, amorphous powder.34 The HRESIQTOFMS of 1 exhibited a pseudo-molecular ion peak at m/z 689.2218 [M+Cl] (Calcd for C31H42O15Cl, 689.2212), which is consistent with the molecular formula of C31H42O15. The IR spectrum revealed a hydroxyl absorption band at 3365 cm1, a ketone band at 1705 cm1, and aromatic ring bands at 1593, 1508, and 1433 cm1. The 1H NMR spectrum of 1 showed signals characteristic of two ABX spin systems [dH 6.66 (1H, d, J = 2.4 Hz, H-20 ), 7.04 (2H, each d, J = 7.8 Hz, H-50 and H-500 ), 6.55 (1H, dd, J = 7.8, 2.4 Hz, H-60 ), 6.64 (1H, d, J = 2.4 Hz, H-200 ), 6.53 (1H, dd, J = 7.8, 2.4 Hz, H-600 )] revealing the presence of two trisubstituted aromatic rings (Table 1). The 1H NMR spectrum also contained signals at dH 4.69 (1H, d, J = 7.8 Hz, H-1000 ) and 4.67 (1H, d, J = 7.8 Hz, H-10000 ), corresponding to two anomeric protons and indicating that 1 possessed two monosaccharide moieties. These monosaccharide units were identified as D-glucose using acid hydrolysis followed by GC analysis.33 The 13C NMR and DEPT spectra indicated the presence of 31 carbon atoms, including seven methylene, 17 methine, and seven quaternary carbons. The 13C NMR spectrum indicated the presence of a heptane chain [dC 213.2 (C-3), 44.8 (C-2), 43.4 (C4), 35.8 (C-7), 31.8 (C-5), 30.1 (C-1), and 24.1 (C-6)] and two trisubstituted aromatic rings (Table 1). Two monosaccharide moieties were attached at C-40 and C-400 as indicated by HMBC correlations from H-1000 to C-40 (dC 144.9) and from H-10000 to C-400 (dC 144.7). A carbonyl group was positioned at C-3 based on 1H–1H COSY and HMBC correlations (Fig. 2), and comparisons with the data reported previously.35 Finally, the structure of compound 1 was established as 1,7-bis(3-hydroxy-4-O-b-D-glucopyranosylphenyl)heptan-3-one, named plantagineoside A. Compound 2 was isolated as a white, amorphous powder.34 Its molecular formula was identified as C31H44O15 based on an ion peak at m/z 691.2386 [M+Cl] (Calcd for C31H44O15Cl, 691.2369) in the HRESIQTOFMS. The IR spectrum showed a hydroxyl absorption band at 3361 cm1 and aromatic ring bands at 1593, 1508, and 1432 cm1. The 1H, 13C NMR, and DEPT spectra of 2 showed the presence of two identical 3,4-dihydroxyphenyl rings and seven carbon signals corresponding to the heptane chain [dC 71.5 (C-3), 40.1 (C-2), 38.0 (C-4), 36.1 (C-7), 32.5 (C-6), 32.3 (C-1), and 26.2 (C-5)] (Table 1). Signals corresponding to two anomeric protons at dH 4.69 (2H, each d, J = 7.8 Hz, H-1000 and H-10000 ) were observed in the 1H NMR spectrum of 2, revealing the presence of two single sugar units. Acid hydrolysis of 2 with 5% HCl gave D-glucose and its aglycone (2a).33 These two glucose moieties were connected at C-40 and C-400 based on HMBC correlations from H-1000 to C-40 (dC 144.7) and from H-10000 to C-400 (dC 144.7) (Fig. 2). By comparing NMR spectroscopic data of 2a with those of reported compound,34,36 the planar structure of 2a was identified as 1,7-bis(3,4-dihydroxyphenyl)heptan-3-ol. The stereochemistry at C-3 of 2a was determined to be S by comparison of its optical rotation value ½aD +3.59 (c 1.5, MeOH) with that of a diarylheptanoid analogue (3R)-1,7-diphenylheptan-3-ol ½aD 7.3 (c 1.7, MeOH).37 Based on the above analyses, the structure of 2 was identified as (3S)1,7-bis(3-hydroxy-4-O-b-D-glucopyranosyl-phenyl)heptan-3-ol, named plantagineoside B. Compound 3 was obtained as a white, amorphous powder.34 Its molecular formula was determined to be C25H32O11 based on a pseudo-molecular ion peak at m/z 543.1655 [M+Cl] (Calcd for C25H32O11Cl, 543.1633) in the HRESIQTOFMS. The IR spectrum

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T. H. Quang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6681–6687 O HO

HO 5'''

HO HO

OR1

1 3'

7

1'

3

O

5

1''

3''

Glc I

OH

OH 5''''

HO

1''' 3'''

OH

4'

4''

3''''

O

HO

R 5O

OH

OH

HO HO HO

OH

Glc II

1

O

O O

OH

OH

Glc I

R4

R OH

O

1''''

O

R2

3

3

OR6 4

5

6

R1

R2

R3

R

2

H

H

OH

OH

2a

H

H

OH

OH

H

H

4

Glc

OH

H

H

H

H

5

Glc

OH

OH

H

H

H

6

Glc

OH

OH

OH

OH

OH

6a

H

OH

OH

OH

OH

OH

R

R

Glc I Glc II

Figure 1. Structure of compounds 1–6, 2a and 6a.

Table 1 1 H and 13C NMR data for compounds 1–3 Position

1 dCa,b 30.1

2.67 (br d, 6.6)

32.3

2

44.8

2.63 (br d, 6.6)

3 4

213.2 43.4

5

c

3

dHa,c (mult., J)

dCa,b

dHa,c (mult., J)

78.6

4.18 (dd, 12.0, 1.8)

40.1

2.61 (m) 2.46 (m) 1.62 (m)

40.3

2.35 (br s)

71.5 38.0

3.46 (m) 1.41 (m)

76.4 40.3

31.8

1.45 (br s)

26.2

76.2

6

24.1

1.45 (br s)

32.5

1.42 (m) 1.28 (m) 1.52 (m)

eq: 2.04 (dddd, 1.8, 2.4, 4.2, 12.0) ax: 1.46 (ddd, 11.4, 11.4, 12.0) 4.00 (dddd, 4.8, 4.8, 10.8, 10.8) eq: 2.22 (dddd, 1.8, 1.8, 4.2, 12.0) ax: 1.31 (ddd, 11.4, 11.4, 12.0) 3.41 (m)

7

35.8

2.40 (br s)

36.1

2.45 (m)

31.9

Glc II 10000 20000 30000 40000 50000 60000 a

dCa,b

1

10 20 30 40 50 60 100 200 300 400 500 600 Glc I 1000 2000 3000 4000 5000 6000

b

2

dHa,c (mult., J)

138.2 117.0 148.1 144.9 119.0 120.8 139.3 117.0 148.0 144.7 118.9 120.7

6.66 (d, 2.4)

7.04 (d, 7.8) 6.55 (dd, 7.8, 2.4) 6.64 (d, 2.4)

7.04 (d, 7.8) 6.53 (dd, 7.8, 2.4)

139.7 117.1 148.1 144.7 119.0 120.8 139.5 117.0 148.0 144.7 118.9 120.8

6.68 (d, 1.8)

7.06 (d, 8.4) 6.58 (dd, 8.4, 1.8) 6.66 (d, 1.8)

7.07 (d, 8.4) 6.56 (dd, 8.4, 1.8)

104.4 74.7 77.3 71.1 77.9 62.2

4.69 3.46 3.47 3.39 3.36 3.87 3.71

(d, 7.8) (dd, 8.4, 7.8) (dd, 9.0, 8.4) (dd, 9.0, 8.4) (m) (dd, 12.0, 2.4) (dd, 12.0, 2.4)

104.5 74.7 77.4 71.1 78.0 62.2

4.69 3.46 3.47 3.39 3.36 3.87 3.71

(d, 7.8) (dd, 8.4, 7.8) (dd, 9.0, 8.4) (dd, 9.0, 8.4) (m) (dd, 12.0, 1.8) (dd, 12.0, 4.8)

104.5 74.7 77.3 71.0 77.9 62.2

4.67 3.46 3.47 3.46 3.36 3.85 3.70

(d, 7.8) (dd, 8.4, 7.8) (dd, 9.0, 8.4) (dd, 9.0, 8.4) (m) (dd, 12.0, 2.4) (dd, 12.0, 2.4)

104.5 74.7 77.4 71.1 78.0 62.2

4.69 3.46 3.47 3.46 3.37 3.87 3.71

(d, 7.8) (dd, 8.4, 7.8) (dd, 9.0, 8.4) (dd, 9.0, 8.4) (m) (dd, 12.0, 1.8) (dd, 12.0, 4.8)

39.1

135.3 114.7 146.0 144.1 116.0 118.8 135.0 116.6 146.1 145.7 116.2 120.7 102.2 75.0 77.9 71.1 78.0 62.8

1.81 1.68 2.58 2.53

(m) (m) (m) (m)

6.84 (d, 2.4)

6.71 (d, 8.4) 6.68 (dd, 8.4, 2.4) 6.59 (d, 2.4)

6.63 (d, 8.4) 6.48 (dd, 8.4, 2.4) 4.41 3.12 3.24 3.25 3.32 3.85 3.63

(d, 8.4) (dd, 9.6, 8.4) (dd, 9.6, 8.4) (dd, 9.0, 8.4) (m) (dd, 12.0, 1.8) (dd, 12.0, 5.4)

Recorded in methanol-d4. 150 MHz. 600 MHz. J values are in Hz.

contained absorptions corresponding to hydroxyl groups at 3354 cm1 and aromatic rings at 1606, 1525, and 1447 cm1. In the 1H NMR spectrum, the observation of signals at [dH 6.84 (1H, d, J = 2.4 Hz, H-20 ), 6.71 (1H, d, J = 8.4 Hz, H-50 ), 6.68 (1H, dd,

J = 8.4, 2.4 Hz, H-60 ), 6.59 (1H, d, J = 2.4 Hz, H-200 ), 6.63 (1H, d, J = 8.4 Hz, H-500 ), and 6.48 (1H, dd, J = 8.4, 2.4 Hz, H-600 )] revealed the presence of two similar 1,3,4-trisustituted benzene rings (Table 1). The 1H NMR spectrum further showed a signal

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HO

OH

O

HO HO

HO

OH

O

OH OH

O

OH

O 1 OH

HO

HO

OH

O

HO HO

HO

OH

O

O

OH O

OH OH

2 HO

OH

OH

HO HO HO

O

O O

OH

OH 3 COSY HMBC 1

1

Figure 2. Selected H– H COSY and HMBC correlations for compounds 1–3.

corresponding to an anomeric proton at dH 4.41 (1H, d, J = 8.4 Hz, H-1000 ) indicating the presence of a monosaccharide moiety. This sugar was identified as D-glucose by acid hydrolysis.33 The 13C NMR and DEPT spectra displayed 25 carbon signals, including six methylene, 13 methine, and six quaternary carbons. The 13C NMR spectrum indicated the presence of seven carbon signals of the heptane chain, including three oxygenated methine groups at dC 78.6 (C-1), 76.4 (C-3), and 76.2 (C-5) and four aliphatic methylene carbon at dC 40.3 (C-2, C-4), 39.1 (C-6), and 31.9 (C-7), suggesting 3 possessed a six-membered cyclic ether structure such as a pyrane skeleton (Table 1). This suggestion was confirmed by the observation of an HMBC cross-peak from H-1 [dH 4.18 (1H, dd, J = 12.0, 1.8 Hz)] to C-5 (Fig. 2). The signal at dH 4.18 (H-l) was coupled to methylene signals at dH 1.46 and 2.04 (H-2) with coupling constants of 12.0 and 1.8 Hz, respectively, indicating an axial orientation of H-1.38 The hydroxyl group attached to H-3 was determined to be equatorial, based on the coupling constants of H-3 (J = 4.8, 4.8, 10.8, 10.8 Hz).38 ROESY correlations between H-1/H-5 and between H-5/H-3 confirmed the axial orientation of H-5 (Fig. 3). Wtype H-H long-range coupling between H-2eq (dH 2.04) and H-4eq (dH 2.22) confirmed the chair conformation of the six-membered ring (Table 1).38 Furthermore, an HMBC correlation between the anomeric proton H-1000 [dH 4.41 (1H, d, J = 8.4 Hz)] and C-3 indicated that the glucose moiety was connected at C-3 (Fig. 2). Based

HO

OH

OH

H

HO HO HO

H 1'''

OH

H

2

O

H

on these data, the structure of 3 was established as 1,5-epoxy-3hydroxy-1-(3,4-dihydroxyphenyl)-7-(3,4-dihydroxyphenyl)heptane 3-O-b-D-glucopyranoside, and named plantagineoside C. HepG2 cells were first transfected with NF-jB luciferase reporter plasmids. After treatment with 10 ng/mL TNF-a, the luciferase activity increased five-fold, representing an increase in transcriptional activity compared to untreated cells. The compounds were pretreated with transfected HepG2 cells at various concentrations, followed by stimulation with TNF-a. The results showed that compounds 3–6 significantly inhibited TNFa-induced NF-jB transcriptional activity in a dose-dependent manner (Fig. 4), with IC50 values ranging from 0.9 to 9.4 lM (Table 2). Among the compounds tested, the inhibitory effect of compound 5 was most

O

1

O

4

3

H H

5

H

H

Figure 3. Selected ROESY correlations for compound 3.

OH

Figure 4. Effects of compounds 1–6 on the TNFa-induced NF-jB luciferase reporter activity in HepG2 cells. The values are mean ± SD (n = 3). aStimulated with TNFa. b Stimulated with TNFa in the presence of 1–6 (0.1, 1, and 10 lM), and sulfasalazine. SFZ: sulfasalazine, positive control (10 lM). Statistical significance is indicated as ⁄ (p <0.05) and ⁄⁄(p <0.01) as determined by Dunnett’s multiple comparison test.

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T. H. Quang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6681–6687 Table 2 Inhibitory effects of compounds 1–6 on the TNFainduced NF-jB transcriptional activity Compound

IC50 (lM)

3 4 5 6 Sulfasalazine

8.0 ± 0.5 6.2 ± 0.3 0.9 ± 0.1 9.4 ± 0.4 0.9 ± 0.1

The values are mean ± SD (n = 3). Compounds 1 and 2 were inactive at tested concentrations (IC50 >10 lM).

Figure 6. PPARa transactivational activity of compounds 1–6 in HepG2 cells. (): vehicle group. CPF: ciprofibrate, positive control (1 lM). Significantly different from vehicle group: ⁄(p <0.05) and ⁄⁄(p <0.01).

Figure 5. PPARs transactivational activity of compounds 1–6 in HepG2 cells. (): vehicle group. BZF: benzafibrate, positive control (1 lM). Significantly different from vehicle group: ⁄(p <0.05) and ⁄⁄(p <0.01).

noticeable, and was comparable to that of the positive control, sulfasalazine (IC50 = 0.9 lM). This primary finding provides support for further studies of these compounds for the development of novel anti-inflammatory agents. PPARs are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid and thyroid hormone receptors.39 PPARs regulate gene expression by binding, as heterodimers with retinoid X receptors (RXRs), to specific response elements (PPREs) in the promoter regions of target genes.40 Currently, PPARs are therapeutic targets in the design and development of agonists for the treatment of type 2 diabetes and metabolic syndromes. The effects of compounds 1–6 on the PPARs activation were evaluated using a PPRE-luciferase reporter assay. The results showed that compounds 1-6 significantly activated PPARs transcriptional activity in a dose-dependent manner (Fig. 5), with EC50 values ranging from

Figure 7. PPARc transactivational activity of compounds 1–6 in HepG2 cells. (): vehicle group. TGZ: troglitazone, positive control (1 lM). Significantly different from vehicle group: ⁄(p <0.01).

0.30 to 10.4 lM (Table 3). Remarkably, compound 5 displayed the most potent effect, even higher than that of the positive control, benzafibrate (IC50 = 1.04 lM). The activity of compound 4 was also noticeable (IC50 = 1.2 lM), and was comparable to that of the positive control. Based on these preliminary data, to find out how specifically the compounds modulate PPAR transcriptional activity, the PPAR transactivational effects of the compounds were further examined on individual PPAR subtypes, including PPARa, c, and b(d) using a GAL-4-PPAR chimera assay (see Supplementary data). At a concentration of 10 lM, compounds 1, 2, 5, and 6 significantly activated PPARa transcription by 1.4, 1.3, 1.4, and 1.3-fold compared with the vehicle group, respectively (Fig. 6). Of the

Table 3 PPARs, a, c, and b(d) transactivational activities of compounds 1–6 EC50 (lM)

Compound

1 2 3 4 5 6 Benzafibrate Ciprofibrate Troglitazone L-165041

PPARs

Gal4/PPARa-LBD

Gal4/PPARc-LBD

Gal4/PPARb(d)-LBD

7.0 ± 1.0 10.4 ± 1.5 5.2 ± 1.3 1.2 ± 0.5 0.30 ± 0.06 9.9 ± 1.7 1.04 ± 0.13

47.1 ± 5.2 >50a >50 >50 42.9 ± 6.8 >50

>50 33.1 ± 3.9 >50 >50 >50 >50

11.0 ± 1.8 13.1 ± 2.7 30.1 ± 1.8 29.3 ± 3.6 15.6 ± 1.3 23.1 ± 2.4

0.90 ± 0.11

EC50: the concentration of a tested compound that gave 50% of the maximal reporter activity. a A compound was considered inactive with EC50 >50 lM. The values are mean ± SD (n = 3).

0.72 ± 0.03 0.65 ± 0.06

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Figure 8. PPARb(d) transactivational activity of compounds 1–6 in HepG2 cells. (): vehicle group. L-165041, positive control (1 lM). Significantly different from vehicle group: ⁄(p <0.05) and ⁄⁄(p <0.01).

compounds tested, only compound 2 noticeably upregulated PPARc transcription at 10 lM, exhibiting an enhancement of 1.5fold over the vehicle group (Fig. 7). In terms of the transactivational effects on PPARb(d) (Fig. 8), compounds 1–6 showed dose-dependent activities, with EC50 values in a range of 11.0–30.1 lM (Table 3). These data indicate that the effects of the diarylheptanoid glycosides on PPAR transcription focus predominantly on PPARs and PPARb(d), which are important for the regulation of glucose, lipid metabolism and inflammation. This finding might be important for the discovery and development of PPARs and PPARb(d) specific agonists from diarylheptanoid glycoside compounds. Cell viability, as measured by the MTS colorimetric assay (see Supplementary data), showed that compounds 1–6 had no significant cytotoxicity in HepG2 cells at tested concentrations (data not shown). In conclusion, these results provide a scientific support for the therapeutic use of the whole plant of T. plantaginea and warrant further studies to develop new agents for the prevention and treatment of inflammatory and metabolic diseases. Acknowledgments This work was financially supported by Vietnam National Foundation for Science and Technology Development (NAFOSTED, No. 104.01.2012.22) and the Priority Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0093815), Republic of Korea. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012. 08.099. References and notes 1. Bich, D. H.; Chung, D. Q.; Chuong, B. X.; Dong, N. T.; Dam, D. T.; Hien, P. V.; Lo, V. N.; Mai, P. D.; Man, P. K.; Nhu, D. T.; Tap, N.; Toan, T. In The medicinal plants and animals in Vietnam; Hanoi Science and Technology Publishing House: Hanoi, 2004; Vol. 1, p 990. 2. Shen, J.; Chen, Z.; Gao, Y. Phytochemistry 1996, 42, 891. 3. Yang, J.-Y.; Zhao, R.-H.; Chen, C.-X.; Ni, W.; Teng, F.; Hao, X.-J.; Liu, H.-Y. Helv. Chim. Acta 2008, 91, 1077. 4. Muehlbauer, A.; Seip, S.; Nowak, A.; Tran, V. S. Helv. Chim. Acta 2003, 86, 2065. 5. Yokosuka, A.; Mimaki, Y.; Sakagami, H.; Sashida, Y. J. Nat. Prod. 2002, 65, 283. 6. Yokosuka, A.; Mimaki, Y.; Sashida, Y. J. Nat. Prod. 2002, 65, 1293. 7. Yokosuka, A.; Mimaki, Y.; Sashida, Y. J. Nat. Prod. 2003, 66, 876. 8. Liu, H. Y.; Ni, W.; Xie, B. B.; Zhou, L. Y.; Hao, X. J.; Wang, X.; Chen, C. X. Chem. Pharm. Bull. 2006, 54, 992.

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The whole plants of T. plantaginea (3 kg) were extracted with MeOH. After concentration, the MeOH extract (150 g) was suspended in H2O and then partitioned successively with n-hexane, CH2Cl2, and EtOAc to give n-hexane (A), CH2Cl2 (B), EtOAc (C) and aqueous (D) fractions, respectively. Fraction D was chromatographed on a column of highly porous polymer (Diaion HP-20) and eluted with H2O and MeOH, successively to give four fractions (D1–D4). Fraction D2 was column chromatographed (CC) over silica gel, eluting with MeOH in CH2Cl2 (0–100%, step-wise) to provide four subfractions (D2.1–D2.4). Subfraction D2.2 was separated by an YMC reverse-phase (RP) CC, using MeOH–H2O (1:2) as eluents, and further purified by CC over silica gel, eluting with CH2Cl2–MeOH–H2O (5:1:0.1) to obtain 4 (18 mg) and 5 (30 mg). Subfraction D2.4 was purified by YMC RP CC, eluting with MeOH–H2O (1:1.5) to give 3 (10 mg) and 6 (80 mg). Fraction D4 (4.3 g) was chromatographed over silica gel, eluting with CH2Cl2–MeOH–H2O (4:1:0.1) to provide three subfractions (D4.1–D4.3). Subfraction D4.2 was separated by an YMC RP CC, using MeOH–H2O (1:2) as eluents, and further purified by CC over silica gel, eluting with CH2Cl2–MeOH–H2O (3:1:0.1), to obtain 1 (55 mg) and 2 (20 mg). 33. Compound 1–3 and 6 were heated in 5% HCl at 80 °C for 5 h. After drying under reduced pressure, the residue was partitioned between EtOAc and H2O, and chromatographed over silica gel to give the aglycone and sugar, respectively. The sugar in the aqueous layer was analyzed by silica gel TLC by comparison with standard sugars. The solvent system was CH2Cl2–MeOH–H2O (2:1:0.2), and spots were visualized by spraying with H2SO4 10%, then heated at 150 °C for 5 min. The Rf value of glucose by TLC was 0.30. The result was confirmed by GC analysis. The aqueous layer was evaporated to dryness to give a residue, and was dissolved in anhydrous pyridine (100 mL) and then mixed with a pyridine solution of 0.1 M L-cysteine methyl ester hydrochloride (100 mL). After warming at 60 °C for 2 h, trimethylsilylimidazole solution was added and warmed at 60 °C for 2 h. The mixture was evaporated in vacuo to give a dried product, which was partitioned between n-hexane and H2O. The n-hexane layer was filtered and analyzed by GC. The absolute configuration of the monosaccharide was confirmed to be D-glucose by comparison of the retention time of persilylated monosaccharide derivative with those of standard samples. 34. Plantagineoside A (1): white, amorphous powder; ½a25 D 62.4 (c 0.3, MeOH); FT-IR (CH3CN) mmax: 3365, 2929, 1705, 1593, 1508, 1433, 1277, 1070, and 1 + 802 cm ; ESI MS: m/z 677 [M+Na] ; HRESIQTOF MS: m/z 689.2218 [M+Cl] (Calcd for C31H42O15Cl, 689.2212); 1H (methanol-d4, 600 MHz) and 13C NMR data (methanol-d4, 150 MHz), see Table 1. Plantagineoside B (2): white, amorphous powder; ½a25 D 56.5 (c 0.3, MeOH); FT-IR (CH3CN) mmax: 3361, 2929, 1593, 1508, 1432, 1277, 1070, and 802 cm1; + ESI MS: m/z 679 [M+Na] ; HRESIQTOF MS: m/z 691.2386 [M+Cl] (Calcd for C31H44O15Cl, 691.2369); 1H NMR data (methanol-d4, 600 MHz) and 13C NMR data (methanol-d4, 150 MHz), see Table 1. (3S)-1,7-Bis(3,4-dihydroxyphenyl)heptan-3-ol (2a): white, amorphous powder; ½a25 D +3.59 (c 1.5, MeOH); FT-IR (CH3CN) mmax: 3361, 2932, 1604, 1 1525, 1445, 1359, 1282, 1244, 1113, and 810 cm ; ESI MS: m/z 355 [M+Na]+; 1 H NMR (methanol-d4, 400 MHz): d 6.51 (2H, each d, J = 8.0 Hz, H-50 and H-500 ), 6.47 (1H, d, J = 2.0 Hz, H-20 ), 6.45 (1H, d, J = 2.0 Hz, H-200 ), 6.35 (1H, dd, J = 8.0, 2.0 Hz, H-60 ), 6.33 (1H, dd, J = 8.0, 2.0 Hz, H-600 ), 3.35 (1H, m, H-3), 2.44 (1H, m, H-1a), 2.33 (1H, m, H-1b), 2.29 (2H, m, H2-7), 1.50 (2H, m, H2-2), 1.40 (2H, m, H2-6), 1.30 (1H, m, H-5a), 1.28 (2H, m, H2-4), and 1.17 (1H, m, H-5b). 13C NMR data (methanol-d4, 100 MHz): d 32.3 (C-1), 40.5 (C-2), 71.7 (C-3), 38.2 (C-4), 26.2 (C-5), 32.9 (C-6), 36.2 (C-7), 135.7 (C-10 ), 116.6 (C-20 ), 146.1 (C-30 ), 144.2

T. H. Quang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6681–6687 (C-40 ), 116.3 (C-50 ), 120.7 (C-60 ), 135.4 (C-100 ), 116.5 (C-200 ), 146.0 (C-300 ), 144.1 (C-400 ), 116.2 (C-500 ), and 120.7 (C-600 ). Plantagineoside C (3): white, amorphous powder; ½a25 D 5.7 (c 0.17, MeOH); FT-IR (CH3CN) mmax: 3354, 2924, 1606, 1525, 1447, 1363, 1284, 1069, and 1 + 815 cm ; ESI MS: m/z 531 [M+Na] ; HRESIQTOF MS: m/z 543.1655 [M+Cl] (Calcd for C25H32O11Cl, 543.1633); 1H NMR data (methanol-d4, 600 MHz) and 13 C NMR data (methanol-d4, 150 MHz), see Table 1. (3R,5R)-3,5-Dihydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane (6a): white, + 1 amorphous powder; ½a25 D +5.2 (c 0.2, MeOH); ESI MS: m/z 371 [M+Na] ; H 0 NMR (methanol-d4, 400 MHz): d 6.49 (2H, each d, J = 8.0 Hz, H-5 and H-500 ), 6.46 (2H, each d, J = 2.0 Hz, H-20 and H-200 ), 6.34 (2H, each dd, J = 8.0, 2.0 Hz, H60 and H-600 ), and 3.64 (2H, m, H-3 and H-5). 13C NMR data (methanol-d4,

35. 36. 37. 38. 39. 40.

6687

100 MHz): d 32.3 (C-1), 41.3 (C-2), 68.7 (C-3), 45.6 (C-4), 68.7 (C-5), 41.3 (C-6), 32.6 (C-7), 135.4 (C-10 ), 116.3 (C-20 ), 146.2 (C-30 ), 144.2 (C-40 ), 116.6 (C-50 ), 120.7 (C-60 ), 135.4 (C-100 ), 116.3 (C-200 ), 146.2 (C-300 ), 144.2 (C-400 ), 116.6 (C-500 ), and 120.3 (C-600 ). Giang, P. M.; Son, P. T.; Matsunami, K.; Otsuka, H. Chem. Pharm. Bull. 2006, 54, 139. Lee, K. K.; Bahler, B. D.; Hofmann, G. A.; Mattern, M. R.; Johnson, R. K.; Kingston, D. G. J. Nat. Prod. 1998, 61, 1407. Ohta, S. Bull. Chem. Soc. Jpn. 1986, 59, 1181. Kikuzaki, H.; Nakatani, N. Phytochemistry 1996, 43, 273. Evans, R. M. Science 1988, 240, 889. Murphy, G. J.; Holder, J. C. Trends Pharmacol. Sci. 2000, 21, 469.