Activation of the nuclear receptor PPARγ by metabolites isolated from sage (Salvia officinalis L.)

Journal of Ethnopharmacology 132 (2010) 127–133

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Activation of the nuclear receptor PPAR␥ by metabolites isolated from sage (Salvia officinalis L.) K.B. Christensen a,∗ , M. Jørgensen b , D. Kotowska c , R.K. Petersen c , K. Kristiansen c , L.P. Christensen a a b c

Institute of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Niels Bohrs Allé 1, DK-5230 Odense M, Denmark Institute of Molecular Medicine, University of Southern Denmark, J.B. Winsløws Vej 21-25, DK-5000 Odense C, Denmark Department of Biology, University of Copenhagen, Ole Maaløes vej 5, DK-2200 Copenhagen N, Denmark

a r t i c l e

i n f o

Article history: Received 28 May 2010 Received in revised form 20 July 2010 Accepted 30 July 2010 Available online 7 August 2010 Keywords: Salvia officinalis Abietane diterpenes Peroxisome proliferator-activated receptor (PPAR)␥ 12-O-methyl carnosic acid Epirosmanol ester of 12-O-methyl carnosic acid

a b s t r a c t Ethnopharmacological relevance: Salvia officinalis has been used as a traditional remedy against diabetes in many countries and its glucose-lowering effects have been demonstrated in animal studies. The active compounds and their possible mode of action are still unknown although it has been suggested that diterpenes may be responsible for the anti-diabetic effect of Salvia officinalis. Aim of the study: To investigate whether the reported anti-diabetic effects of Salvia officinalis are related to activation of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)␥ and to identify the bioactive constituents. Materials and methods: From a dichloromethane extract of Salvia officinalis able to activate PPAR␥ several major metabolites were isolated by chromatographic techniques. To assess bioactivity of the isolated metabolites a PPAR␥ transactivation assay was used. Results: Eight diterpenes were isolated and identified including a new abietane diterpene being the epirosmanol ester of 12-O-methyl carnosic acid and 20-hydroxyferruginol, which was isolated from Salvia officinalis for the first time, as well as viridiflorol, oleanolic acid, and ␣-linolenic acid. 12-O-methyl carnosic acid and ␣-linolenic acid were able to significantly activate PPAR␥ whereas the remaining metabolites were either unable to activate PPAR␥ or yielded insignificant activation. Conclusions: Selected metabolites from Salvia officinalis were able to activate PPAR␥ and hence, the antidiabetic activity of this plant could in part be mediated through this nuclear receptor. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Salvia officinalis L. (Lamiaceae) is a well-known medicinal and culinary herb widely used especially in the Mediterranean region. Preparations of Salvia officinalis leaves have been used traditionally as a remedy towards diabetes in many European countries as well as Morocco and Iran (Swanston-Flatt et al., 1991; Ziyyat et al., 1997; Jouad et al., 2001; Eidi et al., 2005; Tahraoui et al., 2007). Several studies have investigated the potential anti-diabetic properties of Salvia officinalis in relation to both type 1 and type 2 diabetes (Swanston-Flatt et al., 1989, 1990; Broadhurst et al.,

Abbreviations: DCM, dichloromethane; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; FXR, farnesoid X receptor; HDL, high-density lipoprotein; HPLC, high performance liquid chromatography; LBD, ligand binding domain; LDL, low-density lipoprotein; LXR, liver X receptor; PBS, phosphate buffered saline; PPAR, peroxisome proliferator-activated receptor; Rosi, rosiglitazone; TFA, trifluoroacetic acid; TLC, thin layer chromatography. ∗ Corresponding author. Tel.: +45 2135 6057; fax: +45 6550 7354. E-mail addresses: [email protected], [email protected] (K.B. Christensen). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.07.054

2000; Alarcon-Aguilar et al., 2002; Eidi et al., 2005; Lima et al., 2006). Aqueous ethanol extracts of Salvia officinalis have been found to significantly reduce blood glucose in healthy rats and to significantly diminish hyperglycaemia in mildly type 1 diabetic rats (Alarcon-Aguilar et al., 2002). In addition, Eidi et al. (2005) found that methanolic extracts of Salvia officinalis significantly decreased serum glucose in type 1 diabetic rats without affecting pancreatic insulin production. Metformin is often used in the clinical treatment of type 2 diabetes as it reduces liver glucose production as well as increases the action of insulin. Tea-infusions of Salvia officinalis have been shown to possess similar effects in vitro (Lima et al., 2006). Furthermore, an aqueous extract of Salvia officinalis has also been found to exhibit insulin-like activities (Broadhurst et al., 2000). Nuclear receptors such as the peroxisome proliferatoractivated receptors (PPARs), farnesoid X receptor (FXR), and the liver X receptors (LXRs) are potential therapeutic targets for many obesity-related disorders such as overt type 2 diabetes, atherosclerosis, and the metabolic syndrome (Tobin and Freedman, 2006). There are three subtypes of PPAR: ␣, ␦, and ␥, and natural and synthetic ligands for these receptors are fatty acids, eicosanoids,

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fibrates, and thiazolidinediones. PPAR␣ up-regulates genes that are involved in ␤-oxidation of fatty acids leading to decreased levels of free fatty acids and triglyceride levels in serum and increased levels of high-density lipoprotein (HDL). PPAR␦ is a regulator of genes involved in energy expenditure as well as lipid and glucose metabolism and activation improves the HDL/LDL ratio and lowers triglycerides in serum, reduces insulin resistance, and reduces size of adipose tissue. The two isoforms of PPAR␥ are highly involved in the control of lipid metabolism and adipocyte differentiation. Activation of PPAR␥ leads to a re-distribution of adipose tissue, an increased release of adiponectin and hence, an increased insulin sensitivity (Tobin and Freedman, 2006). Rau et al. (2006) presented a possible mode of action for Salvia officinalis in relation to diabetes as an extract of this was found to activate PPAR␥. Two abietane diterpenes that are major metabolites in sage: carnosol (1) and carnosic acid (3), were found to activate PPAR␥ and therefore could explain some of the observed bioactivity for the extract (Rau et al., 2006). In this study, we wanted to investigate further the metabolites that may be responsible for the glucose-lowering effect of Salvia officinalis with focus on the role of the abietane diterpenes. Extracts of Salvia officinalis as well as abietane diterpenes and other major metabolites isolated from the plant were tested for activation of PPAR␥. As PPAR␥ is highly involved in the control of glucose and lipid metabolism this nuclear receptor plays a significant role in regulation of insulin sensitivity and therefore metabolites activating PPAR␥ may at least in part explain the glucose-lowering effect of Salvia officinalis.

2. Materials and methods 2.1. Plant material Plants of Salvia officinalis L. (Lamiaceae) were cultivated at the Department of Horticulture, Research Centre Aarslev, Aarhus University, Denmark, and aerial parts (leaves and stems) of Salvia officinalis were harvested in August 2006 and stored at −25 ◦ C until use. Salvia officinalis plants were authenticated by Dr. Kai Grevsen, Department of Horticulture, Aarhus University, and a voucher specimen (22.06.2006 Salvia officinalis shoots) is deposited at the Department of Horticulture, Aarhus University, Denmark.

2.2. Chemicals and reagents Acetonitrile, dichloromethane (DCM), ethyl acetate (EtOAc), and n-hexane (CHROMASOLV high performance liquid chromatography (HPLC) grade, Sigma–Aldrich, Germany); trifluoroacetic acid (TFA, spectrophotometric grade, ≥99%, Sigma–Aldrich); dimethyl sulfoxide (DMSO, Biotech grade, 99.98%, Sigma–Aldrich); deionized water (prepared with a SG Ultra Pure Water System (Siemens, Germany)); CDCl3 or acetone-d6 with TMS as internal standard (Cambridge Isotop Laboratories, Inc., Andover, MA, USA); carnosol, carnosic acid, and oleanolic acid (Sigma–Aldrich). Normal phase flash column chromatography (Silica Gel 60, particle size 0.063–0.2 mm (70–230 mesh ASTM), Merck, Germany); thin layer chromatography (TLC) (silica gel aluminium cards (0.2 mm, 20 cm × 20 cm, F254 , Sigma–Aldrich). Analytical HPLC, LiChrospher 100 reversed-phase (RP) (C18) column (5 ␮m; 250 mm × 4.6 mm, Merck); preparative HPLC, Develosil ODS-HG-5 RP-C18 column (5 ␮m; 250 mm × 20 mm, Nomura Chemical Co., Japan). Dulbecco’s modified Eagle’s medium (DMEM, GibcoBRL, Life Technologies, Rockville, MD, U.S.A.); Dual-GloTM Luciferase assay (Promega, WI, U.S.A.); rosiglitazone (Rosi, Novo Nordisk® A/S, Denmark).

2.3. Apparatus 1D (1 H, 13 C, and DEPT) and 2D (HSQC, HMBC, COSY, and NOESY) NMR spectra were recorded on a Varian Unity Inova 500 spectrometer. ESI-MS spectra and accurate mass determination (HR-ESI-MS) were recorded on a high resolution QSTAR pulsar Qq-TOF mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, CA) in positive mode. Analytical HPLC was performed on a Merck-HITACHI-LaChrom HPLC system (Merck, Germany) consisting of a L-7100 HPLC pump, L-7200 autosampler with sample cooler, L-7300 column oven, and a L7450A diode array detector. Semi-preparative HPLC was carried out on a Dionex SemiPrep system (Dionex Denmark A/S, Denmark) consisting of an HPLC pump (P680), solvent rack (SOR-100), and a diode array detector (UVD340U). 2.4. Preparation of plant extracts 5 kg of frozen plant material (stems and leaves) were homogenized and subjected to a two-step sequential extraction procedure using n-hexane (15 L) and DCM (13 L). Extraction mixtures were allowed to stand for 20 h in the dark at 5 ◦ C with frequent shaking and were filtered before re-extraction. The extracts were dried under vacuum and 16.8 g of n-hexane extract and 36.0 g of DCM extract were obtained, giving w/w yields of the crude extracts from the starting material of 0.34% and 0.72%, respectively. 2.5. Isolation and characterization of compounds 2.5.1. Initial separation of plant extract The n-hexane and DCM extracts were tested for ability to activate PPAR␥ (see Section 2.6). In particular the DCM extract showed a significant ability to activate PPAR␥ yielding approximately 30fold activation relative to DMSO in a 104 times dilution of the extract (data not shown) in accordance with previous investigations (Christensen et al., 2009). Initial screening of the n-hexane and DCM extracts for abietane diterpenes was done by analytical HPLC. Mobile phase (A) water containing 500 ppm TFA and (B) CH3 CN was run at the following gradient: 10% B to 100% B in 60 min with a hold at 100% B for 10 min, then reduced to 10% B in 10 min and kept at 10% B for 10 min; flow rate 1 mL/min; injection volume 20 ␮L of a 10 mg/mL sample concentration in CH3 CN; detection wavelengths  = 210, 220, and 280 nm. The analytical HPLC profiles revealed that abietane diterpenes were mainly present in the DCM extract (data not shown). Consequently, the DCM extract was selected for further investigations. A flash column (∅ 100 mm) was packed with silica gel (800 g) and conditioned with n-hexane–EtOAc (50:50). The DCM extract (36.0 g) was re-dissolved in DCM (50 mL) and applied to the column and eluted using the following gradient system: 5% stepwise gradient increasing from 50 to 100% EtOAc in n-hexane (600 mL each) giving 58 fractions. Based on TLC, the fractions were combined into five fractions: A (5.5 g), B (12.5 g), C (4.1 g), D (1.6 g), and E (2.3 g). Different proportions of n-hexane–EtOAc eluents were used for TLC and all plates were inspected by UV light followed by visualization with vanillin (30 g vanillin, 500 mL ethanol, and 5 mL conc. H2 SO4 ). Abietane diterpenes were mainly present in fraction B according to TLC and analytical HPLC (data not shown) and therefore fraction B was selected for isolation of abietane diterpenes and other metabolites. Parts of fraction B were initially separated either by flash column chromatography or preparative HPLC (see Section 2.5.2). A flash column (∅ 40 mm) was packed with silica gel (125 g) and conditioned with n-hexane–EtOAc (75:25). A part of fraction B (3.3 g) was re-dissolved in 5 mL n-hexane–EtOAc (80:20) and applied to the column and eluted using the solvent mixture n-hexane–EtOAc

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starting with 75:25, 73:27, 70:30, 67:33, 65:35, and 60:40 (100 mL each) followed by a 10% stepwise gradient increasing to 100% EtOAc (100 mL each) giving 38 fractions. Based on TLC and analytical HPLC these were combined into six fractions B1 (0.2 g), B2 (0.65 g), B3 (0.56 g), B4 (0.88 g), B5 (0.10 g), and B6 (0.05 g). 2.5.2. Isolation of compounds 1–3 and 6–9 A part of fraction B (580 mg) was re-dissolved at a concentration of 9.8 mg/mL in CH3 CN and separated directly by semi-preparative HPLC. Mobile phase (A) water containing 500 ppm TFA and (B) CH3 CN was run at the following gradient: 0 min, 50% B; 60 min, 100% B; 75 min, 100% B; 95 min, 50% B and 110 min, 50% B; flow rate 5 mL/min; injection volume 2 mL; detection wavelengths  = 210, 220, and 280 nm. Direct separation of fraction B by semi-preparative HPLC resulted in carnosol (1; 91.4 mg), 20-deoxo carnosol (2; 27.4 mg), carnosic acid (3; 38.5 mg), 20hydroxyferruginol and 12-O-methyl carnosic acid (4 + 5; 71.7 mg), 11,12,20-trihydroxy-abieta-8,11,13-triene (6; 18.0 mg), viridiflorol (7; 6.1 mg), ␣-linolenic acid (8; 30.4 mg), as well as oleanolic acid (9; 62.9 mg). Compounds 4 and 5 were further separated by semipreparative HPLC to give 20-hydroxyferruginol (4; 2.6 mg) and 12-O-methyl carnosic acid (5; 40 mg). Mobile phase (A) water containing 500 ppm TFA and (B) CH3 CN with the following gradient: 0 min, 40% B; 70 min, 100% B; 80 min, 100% B; 90 min, 40% B and 100 min, 40% B; flow rate 5 mL/min; injection volume 2 mL of a 23.9 mg/mL sample concentration in CH3 CN; detection wavelengths  = 210, 220, and 280 nm. 2.5.3. Isolation of compounds 10 and 11 A part of fraction B4 (190 mg) was re-dissolved at a concentration of 38 mg/mL in CH3 CN and purified by semi-preparative HPLC. Mobile phase (A) water containing 500 ppm TFA and (B) CH3 CN was run at the following gradient: 0 min, 50% B; 60 min, 100% B; 75 min, 100% B; 95 min, 50% B and 110 min, 50% B; flow rate 5 mL/min; injection volume 2 mL; detection wavelengths  = 210, 220, and 280 nm, yielding an abietane diterpene being the epirosmanol ester of 12-O-methyl carnosic acid (10; 40.2 mg). Compound (10): colourless oil; UV max (nm): 230sh, 285. 1 H NMR (CDCl3 , 500 MHz) see Table 1. 13 C NMR (CDCl3 , 125 MHz) see Table 1. HR-ESI-MS m/z 697.3720 [M(C41 H54 O8 ) + Na]+ (calc. 697.3712), 369.2038 [C21 H30 O4 + Na]+ (base peak) (calc. 369.2042). Compound 10 was shown to be relatively unstable in mobile phase solution being hydrolysed within a few days at room temperature to 12-Omethyl carnosic acid (5), rosmanol, and epirosmanol in the ratio 2:1:1, as shown by analytical HPLC. A part of fraction B2 (210 mg) was re-dissolved at a concentration of 10 mg/mL in CH3 CN and purified by semi-preparative HPLC by the same method as described above to yield manool (11; 21 mg). 2.6. PPAR transactivation bioassay For analysis of PPAR␥-mediated transactivation, mouse embryonic fibroblasts (Hansen et al., 1999) were transiently transfected at 50–70% confluence using Metafectene (Biontex) (Christensen et al., 2009). For each well a total of 0.05 ␮g DNA (2.5 ng of Renilla normalization vector pRL-CVM + 30 ng of the Gal4-responsive Luciferase reporter vector + 15 ng of PPAR␥-ligand binding domain (LBD) expression vector pM-hPPAR␥-LBD) were used (Christensen et al., 2009). The medium in the plates was changed 6 h after transfection to 200 ␮L DMEM with antibiotics (62.5 ␮g/mL penicillin and 100 ␮g/mL streptomycin) containing either vehicle (0.1% DMSO), positive control (1 ␮M Rosi), plant extract dissolved in DMSO (103 , 104 , and 105 times dilutions of the extract stocks were used) or isolated compounds (tested in at least three concentrations ranging from 0.1 to 100 ␮M). After 18 h the cells were

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washed with phosphate buffered saline (PBS) (200 ␮L per well) and lysed with lysis buffer (20 ␮L per well). Photinus and Renilla activities were measured directly in the plate using a Dual-GloTM Luciferase assay system. All transient transfection experiments were done in triplicate and double determinations for each triplicate were carried out. Photinus activities were normalized to the corresponding Renilla activities to compensate for differences in transfection efficiency. Positive control (Rosi) gave 82–112-fold activation compared to vehicle (DMSO) in all experiments. 2.7. Statistical analysis For statistical analysis of variances, the general linear model procedure of the Statistical Analysis System (SAS Institute, Cary, NC) was used. Data were checked for outliers and ln transformed when necessary to fit normal distribution and uniform variances. Statistical significance was assessed by one-way analysis of variance. 3. Results and discussion 3.1. Identification of isolated metabolites Consecutive rounds of column chromatography and semipreparative HPLC of the DCM extract of the leaves of Salvia officinalis yielded seven abietane diterpenes (1–6, 10) including a new one consisting of two abietane skeletons. In addition, four other known metabolites were isolated from the DCM extract of Salvia officinalis and identified as viridiflorol (7), ␣-linolenic acid (8), oleanolic acid (9), and manool (11) by comparison with spectral data of authentic standards and/or previous reports (Buckwalter et al., 1975; Faure et al., 1991; Zhang et al., 1999; Seebacher et al., 2003). The known abietane diterpenes were identified as carnosol (1), 20-deoxo carnosol (2), carnosic acid (3), 20-hydroxyferruginol (4), 12-O-methyl carnosic acid (5), and 11,12,20-trihydroxy-abieta8,11,13-triene (6) (Fig. 1) by comparison with spectral data (UV, ESI-MS, and 1D and 2D NMR) of authentic samples and/or previous reports (Brieskorn and Fuchs, 1962; Linde, 1964; Inatani et al., 1982; Matsumoto and Usui, 1982; Gonzalez et al., 1988, 1991; Schwarz and Ternes, 1992; Cuvelier et al., 1994; Richheimer et al., 1996; Son et al., 2005). 20-Hydroxyferruginol was first isolated from Sequoia sempervirens (redwood) and shown to possess anti-tumour activity but has not previously been reported from Salvia officinalis (Son et al., 2005). Compound 10 was obtained as a colourless oil. The molecular formula of 10 was assigned as C41 H54 O8 based on HR-ESI-MS analysis (see Section 2). The 13 C NMR spectrum (Table 1) of 10 revealed the presence of 41 carbon atoms. The multiplicity of the carbon atoms was determined by analysis of DEPT-90 and DEPT135 experimental data and revealed the presence of eight methine, eight methylene, and nine methyl carbons of which one of the methyl groups (ı 61.8) was linked to an oxygen function, and finally sixteen quaternary carbons, including two carbonyl carbons (ı 178.7 and 175.3). Twelve of the carbon signals were in the aromatic region (ı 118.4–147.3) suggesting the presence of two aromatic rings in the structure of 10. From the 1 H–1 H-COSY, HSQC, HMBC, and NOESY experiments, all protons could be assigned to carbons, which confirmed the observations from the 13 C NMR spectrum and DEPT experiments and furthermore resolved the overlapping signals in the aliphatic region of the 1 H NMR spectrum (Table 1). The 1 H NMR spectrum of 10 showed characteristic signals of a diterpene containing two abietane skeletons with signals from two isopropyl groups [ı 1.16 (d, J = 6.8 Hz, H3 -16), 1.19 (d, J = 6.8 Hz, H3 -17), 3.16 (m, H-15) and ı 1.17 (d, J = 6.8 Hz, H3 -17 ), 1.19 (d, J = 6.8 Hz, H3 -16 ), 3.07 (sept, J = 6.8 Hz, H-15 )], geminal dimethyl

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Table 1 1 H and 13 C NMR spectral data and HMBC correlations of compound 10, and 1 H and 13 C NMR spectral data of compound 5. Position

Compound 10 ı

13

C (ppm)

1

35.0 t

2

20.0 t

3

41.6 t

4 5 6

34.2 s 54.4 d 18.6 t

7

32.0 t

8 9 10 11 12 13 14 15 16 17 18 19 20 21 1

134.5 s 125.6 s 46.9 s 147.3 s 142.3 s 139.2 s 118.4 d 26.3 d 23.4 q 23.7 q 32.6 q 21.7 q 175.3 s 61.8 q 27.1 t

2

18.8 t

3

37.9 t

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

30.8 s 51.3 d 75.5 d 70.0 d 124.5 s 124.3 s 48.4 s 142.7 s 141.8 s 134.4 s 121.7 d 27.2 d 22.0 q 22.5 q 31.2 q 21.5 q 178.7 s

Compound 5 ı H (ppm) [J = Hz] 1

␣ 1.20 m ␤ 3.67 br d (13.6 Hz) ␣ 1.58 m ␤ 2.33 m ␣ 1.58 m ␤ 1.30 m ␣ 1.58 m ␣ 1.78 br dd (4.1; 13.7 Hz) ␤ 2.16 ddd (4.1; 5.5; 13.7 Hz) ␣ 2.78 m ␤ 2.78 m

HMBC ( H → 1

13

C)

ı 1 H (ppm) [J = Hz]

34.1 t

␣ 1.19 m ␤ 3.55 br d (13.6 Hz) ␣ 1.56 m ␤ 2.20 m ␣ 1.54 m ␤ 1.28 ddd (4.2; 13.6; 13.6 Hz)

19.9 t C-1

41.4 t

C-6, 18, 19, 20

33.9 s 54.1 d 18.4 t

C-5, 10 C-5, 8, 9, 14 C-5, 8, 9, 14

31.9 t

6.48 s 3.16 m 1.16 d (6.8 Hz) 1.19 d (6.8 Hz) 0.96 s 0.98 s

C-7, 9, 12, 15 C-12, 13, 14, 16, 17 C-13, 15, 17 C-13, 15, 16 C-3, 4, 5, 19 C-3, 4, 5, 18

3.72 s ␣ 1.96 ddd (5.4; 14.0; 14.0 Hz) ␤ 3.14 m ␣ 1.45 m ␤ 1.62 m ␣ 1.30 m ␤ 1.04 m

C-12

C-10

␣ 1.84 s ␣ 4.22 d (3.1 Hz) ␣ 5.95 d (3.1 Hz)

C-7 , 18 , 19 , 20 C-8 , 9 , 20 C-5 , 8 , 9 , 14 , 20

6.92 s 3.07 sept (6.8 Hz) 1.19 d (6.8 Hz) 1.17 d (6.8 Hz) 0.38 s 0.72 s

C-7 , 9 , 12 , 15 C-12 , 13 , 14 , 16 , 17 C-13 , 15 , 17 C-13 , 15 , 16 C-3 , 4 , 5 , 19 C-3 , 4 , 5 , 18

groups at ı 0.96 (s, H3 -18), 0.98 (s, H3 -19) and ı 0.38 (s, H3 -18 ), 0.72 (s, H3 -19 ), respectively, and two aromatic methine protons at ı 6.48 (s, H-14) and ı 6.92 (s, H-14 ) (Nakatani and Inatani, 1984; Pukalskas et al., 2005). The upfield shifted H3 -18 and H3 -19 were determined to be geminal methyl groups similar to H3 -18 and H3 19 due to observed HMBC long-range couplings between the H3 -18 and H3 -19 protons and the carbons at ı 37.9 (C-3 ), 30.8 (C-4 ), 51.3 (C-5 ) as well as coupling to their own geminal neighbour carbon atoms. In addition, signals from one methoxy group were observed in the 1 H NMR spectrum at ı 3.72 (s, H3 -21) while signals at ı 4.22 (d, J = 3.1 Hz, H-6 ) and ı 5.95 (d, J = 3.1 Hz, H-7 ) were assigned to two methine protons attached to carbons bearing oxygen. Analysis of the 1 H–1 H-COSY spectrum revealed that the methine proton at ı 4.22 (H-6 ) was coupled with the methine protons at ı 5.95 (H-7 ) and ı 1.84 (H-5 ), respectively. These signals suggested that the partial structure of one of the abietane skeletons possess ␥-lactone functionality. Since a coupling of 3.1 Hz was observed between H-6 and H-7 , it was concluded that the methine proton at H-7 was in the ␣-position (Nakatani and

ı 13 C (ppm)

134.6 s 125.3 s 47.7 s 147.7 s 142.3 s 139.5 s 118.1 d 26.4 d 23.4 q 23.8 q 32.7 q 20.0 q 181.0 s 61.7 q

␣ 1.58 m ␣ 1.82 br d (13.7 Hz) ␤ 2.28 m ␣ 2.83 m ␤ 2.83 m

6.51 s 3.16 sept (6.8 Hz) 1.19 d (6.8 Hz) 1.21 d (6.8 Hz) 0.97 s 0.87 s 3.73 s

Inatani, 1984). The ␥-lactone configuration was further confirmed by a long-range coupling between H-6 and the carbonyl at ı 178.7 (C-20 ) in the HMBC spectrum. No coupling between H-5 and H-6 indicated cis configuration with a dihedral angle of 90◦ between these methine protons. These results established H-5 and H-6 to be in ␣-configuration (Nakatani and Inatani, 1984; Pukalskas et al., 2005). The HMBC experiment furthermore revealed cross-peaks from the aromatic proton H-14 to C-7 (ı 70.0), C-9 (ı 124.3), C-12 (ı 141.8) and C-15 (ı 27.2) suggesting that the isopropyl group was attached to C-13 of the aromatic ring. Its position was furthermore proven by HMBC correlations between H-15 and C-12 , C-13 , C-14 , C-16 and C-17 (Pukalskas et al., 2005). Based on these data, it was deduced that one of the abietane skeletons in 10 is epirosmanol, and this is in accordance with reported NMR data for epirosmanol (Nakatani and Inatani, 1984; Schwarz and Ternes, 1992; Cuvelier et al., 1994; Marrero et al., 2002). An HMBC correlation between H-7 and C-20 suggested that the two abietane skeletons in 10 were connected at this point via an ester linkage. This is in accordance with the downfield shift of H-7 in the 1 H

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131

Fig. 1. Structures of terpenoids isolated from the dichloromethane extract of Salvia officinalis in the present investigation.

NMR spectrum of 10 compared to epirosmanol clearly indicating the presence of an ester linkage at C-7 instead of a hydroxyl group (Nakatani and Inatani, 1984; Schwarz and Ternes, 1992). A striking resemblance was observed between the 1 H and the 13 C NMR spectrum of 12-O-methyl carnosic acid (5) and the second abietane moiety in compound 10. However, significant differences were observed for the carbonyl group at C-20, which had shifted further upfield in 10 (ı 175.3) compared to 5 (ı 181.0), and this can only be explained by the presence of an ester linkage at C-20 instead of a carboxylic acid. An HMBC correlation between H3 -21 and C-12 suggested that a methoxy group was attached to this aromatic carbon. This was also confirmed by the NOESY spectrum showing cross-peaks between H3 -21 and H3 -16 and H3 -21 and H3 -17, respectively, thus confirming that the second abietane skeleton in 10 is 12-O-methyl carnosic acid. This is also in accordance with the HR-ESI-MS spectrum of 10 showing a base peak ion at m/z 369.2039 [C21 H30 O4 + Na]+ corresponding to 12-O-methyl carnosic acid. In addition, compound 10 was found to be unstable in mobile phase solutions containing water and CH3 CN at room temperature being almost completely hydrolysed within days to 12-O-methyl carnosic acid, rosmanol, and epirosmanol in the ratio 2:1:1, as shown by analytical HPLC. This indicates that the decomposition of compound 10 proceeded via the generation of a secondary carbocation at C7 , which is relatively stable due to resonance stabilization by the aromatic ring. Thus, compound 10 was determined to be an epirosmanol ester of 12-O-methyl carnosic acid (Fig. 1) formed by a condensation between 12-O-methyl carnosic acid and epirosmanol by the loss of water, which is also fully in accordance with the UV spectrum of 10 with max at 285 nm and a peak shoulder at about 230 nm (Pukalskas et al., 2005). The analysis of sage extracts and fractions by analytical HPLC did not reveal the presence of epirosmanol and rosmanol, which indicates that compound 10, is not likely to be an artefact formed during extraction and isolation. Dimeric diterpenes have been isolated from other plant species (Rüedi et al.,

Fig. 2. Activation of PPAR␥ by 12-O-methyl carnosic acid (5). Results are shown as the average of 3 individual experiments (±SD) and each of the experiments were performed with 4 replica (n ≥ 10). Activation is given as fold activation relative to the vehicle DMSO (set to 1.00), and rosiglitazone (not shown) was used as positive control.

1981; Cerqueira et al., 2004; Drewes et al., 2006) but this is to the best of our knowledge the first example of a diterpene composed of two diterpene substructures connected via an ester linkage. 3.2. Activation of PPAR All isolated metabolites from sage (Salvia officinalis) were tested for the activation of PPAR␥ in three concentrations in the ␮M range. From this, it was found that the known PPAR␥ agonist ␣-linolenic acid was able to activate PPAR␥ and that 12-O-methyl carnosic acid (5) at 10 ␮M significantly activated PPAR␥ 6–7-fold relative to DMSO (data not shown and Fig. 2). Compound 5 has, to the best of

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our knowledge, not previously been reported as a PPAR␥ activator. Carnosol (1), carnosic acid (3), and oleanolic acid (9) were all very weak PPAR␥ activators with 2–3-fold activations relative to DMSO (data not shown). The remaining isolated metabolites were unable to activate PPAR␥. Rau et al. (2006) reported EC50 values for compounds 1 and 3 at 41.2 ± 5.9 ␮M and 19.6 ± 2.0 ␮M, respectively, for transactivation of PPAR␥ in Cos7 cells. The discrepancies between our results and the ones obtained by Rau et al. (2006) are most likely due to the use of different test systems as well as cell types. In our study, concentrations higher than 10 ␮M of compounds 1 and 3 were not more effective than 10 ␮M, and above 50 ␮M toxicity interfered with results (data not shown). Oleanolic acid (9) is a known PPAR␣ agonist and has been shown to exhibit antihyperlipidaemic as well as hypoglycaemic effects in vivo (Liu, 1995; Huang et al., 2005) but has not been found to significantly activate PPAR␥ in good accordance with our results. However, oleanolic acid has recently been shown to be a selective FXR modulator (Liu and Wong, 2010). FXR acts primarily as a sensor for bile acid and is thus highly involved in cholesterol homeostasis but also in lipid and glucose metabolism. Activation of FXR has been shown to for example improve HDL/LDL ratio and triglyceride levels in serum and decrease insulin resistance and hence oleanolic acid may contribute to the anti-diabetic effects of sage through this target. The metabolite profile of Salvia miltiorrhiza (Chinese sage) is similar to that of sage and recently it was shown that an extract of Salvia miltiorrhiza was able to lower plasma cholesterol, LDL, and triglycerides as well as increase HDL levels in hyperlipidaemic rats. The extract was subsequently identified as an FXR/LXR co-agonist (Ji and Gong, 2008), and oleanolic acid and other metabolites of terpenoid origin could be responsible for this effect. The two existing isoforms of LXR are involved in cholesterol homeostasis by acting as sensors for oxysterol levels and activation of LXR leads for example to improved glucose tolerance, insulin sensitivity, serum HDL/LDL ratio, and serum triglycerides levels (Tobin and Freedman, 2006). However, testing the n-hexane and DCM extracts of sage and isolated metabolites for LXR activation revealed that both the extracts and the isolated compounds do not activate LXR (data not shown). Hence, the activation of LXR does not seem to contribute to the anti-diabetic effects of sage. On the other hand carnosic acid and other abietane diterpenes from sage has been shown to significantly inhibit pancreatic lipase activity and carnosic acid has also been shown to suppress serum triglyceride elevation in oliveoil loaded mice (Ninomiya et al., 2004). Pancreatic lipase is well known to play an important role in lipid digestion and the effect of sage metabolites on pancreatic lipase activity could suggest that the regulation of the HDL/LDL ratio and the serum triglyceride level is important for the anti-diabetic effects of sage although this effect does not appear to be regulated through activation of FXR and LXR. In a previous study, four extracts of Salvia officinalis (n-hexane, EtOAc, DCM, and methanol) were found to significantly activate PPAR␥ relative to control. Furthermore, the extracts had no adipogenic potential, and three of them (EtOAc, DCM, and methanol) were able to positively affect insulin-dependent glucose-uptake (Christensen et al., 2009). The results of this and the present study suggest that extracts and metabolites of sage are also able to exert their beneficial effect on lipid and glucose homeostasis through PPAR␥-mediated pathways, although this warrants further investigations. 4. Conclusions Eleven metabolites were isolated from the traditional antidiabetic plant Salvia officinalis and of these, the epirosmanol ester of 12-O-methyl carnosic acid (10) was new and 20-hydroxyferruginol (4) was isolated from Salvia officinalis for the first time. Extracts of Salvia officinalis are able to activate PPAR␥ and in this study two

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