DOI 10.1007/s10600-015-1276-x Chemistry of Natural Compounds, Vol. 51, No. 2, March, 2015
TWO ISORHAMNETIN GLYCOSIDES FROM Arthrocnemum glaucum THAT INHIBIT ADIPOGENESIS IN 3T3-L1 ADIPOCYTES
Yukiko Sekii,1 Junkyu Han,1,2 Hiroko Isoda,1,2 Mohamed Bouaziz,3 Abdelhafidh Dhouib,3 Sami Sayadi,3 and Hideyuki Shigemori1*
In our search for bioactive compounds from North African plants [1, 2], we found that the Tunisian medicinal plant Arthrocnemum glaucum Delile inhibited adipogenesis in 3T3-L1. A. glaucum, which belongs to the Chenopodiaceae, is a densely branched robust plant that grows wild in the Sahara Desert of Tunisia, especially in the Mediterranean Coastal region. It was previously reported that some alkaloids and some sterols were isolated from A. glaucum [3–6]. In this research, the bioassay-guided fractionation of this plant implied that the bioactive components are two isorhamnetin glycosides, isorhamnetin-3-O-[apiosyl(1o6)]glucosyl-7-O-rhamnoside (1) and isorhamnetin-3-O-rutinoside (2). In this paper, we report the isolation and structure elucidation of the two isorhamnetin glycosides 1 and 2 from aerial parts of this plant and their antiadipogenesis activity. The aerial part of A. glaucum was collected at the Sahara Desert of Tunisia. The voucher specimen (TPM-20080401) is maintained at Alliance for Research on North Africa (ARENA), University of Tsukuba and Environmental Bioprocesses Laboratory at Biotechnology Center of Sfax. The leaves were dried in the shade at 25qC. The dried leaves of A. glaucum (100 g) were extracted with MeOH. The MeOH extracts were partitioned between EtOAc and H2 O, and the H2O layer was partitioned with n-BuOH. The n-BuOH-soluble portion (1.33 g) was subjected to ODS column chromatography and purified by reversed-phase HPLC or gel-filtration HPLC to afford two known flavonol glycosides, isorhamnetin-3-O[apiosyl(1o6)]glucosyl-7-O-rhamnoside (1, 0.0085%) and isorhamnetin-3-O-rutinoside (2, 0.0027%). Structure elucidation of 1 and 2 was through spectroscopic data, mainly 1H and 13C NMR spectra and ESI-MS spectra. Full assignment of signals in the NMR spectra of 1 and 2 was confirmed by 2D NMR experiments (1H–1H COSY, NOESY, HMQC, and HMBC) and comparison with reported data [7, 8]. OH
HO HO
OCH3 3' OH
O O
7
9
O
1' 2
OCH3 OH
3'
5'
HO
7
9
O
2
HO 4 5
10
OH
3
O
O HO
OH OH
O
5
10
OH
4
3
5' 1'
HO
O
O
O
O
O O
HO HO OH OH 1
OH OH
O
OH
2
1) Graduate School of Life and Environmental Sciences, University of Tsukuba, 305-8572, Tsukuba, Japan, fax: +81 29 853 4603, e-mail:
[email protected]; 2) Alliance for Research on North Africa, University of Tsukuba, 305-8572, Tsukuba, Japan; 3) Laboratory of Environmental Bioprocesses, Sfax Biotechnology Center, University of Sfax, BP”1177” 3013, Tunisia. Published in Khimiya Prirodnykh Soedinenii, No. 2, March–April, 2015, pp. 295–297. Original article submitted July 20, 2013. 338
0009-3130/15/5102-0338 2015 Springer Science+Business Media New York
Isorhamnetin-3-O-[apiosyl(1o6)]glucosyl-7-O-rhamnoside (1) showed a pseudomolecular ion peak at m/z 779 [M + Na]+ in the ESI-MS. The 13C NMR data indicated that the molecule possesses one unsaturated carbonyl carbon, three acetal carbons, 12 aromatic carbons, two olefin carbons, one quaternary oxy carbon, nine oxymethine carbons, three oxymethylene carbons, one methyl carbon, and one methoxy carbon. The 1H NMR data showed five aromatic proton signals (G 6.18, 6.37, 6.94, 7.64, and 7.95) in the flavonol skeleton [9] and one methoxy proton signal (G 4.06). The HMBC correlations of H-8 to C-7, H-6 to C-8, H-2c to C-6c, H-5c to C-3c, and H-6c to C-1c and C-2 indicated the presence of the flavonol skeleton in 1. An HMBC correlation between the methoxy proton and the aromatic carbon (G 150.2, C-3c) revealed that the aglycone of 1 was isorhamnetin. In addition, characteristic signals of the sugar moiety (GH 3.2–5.6) and anomeric proton signals were observed, suggesting 1 to be an isorhamnetin glycoside. The 1H–1H COSY connectivities of Glc-H-1 to Glc-H-6, Api-H-1 to Api-H-4, and Rha-H-1 to Rha-H-6, and the NOESY correlations indicated the presence of glucose, apiose, and rhamnose. Three anomeric proton signals in the 1H NMR data (G 5.55, 5.49, and 4.54) were in good agreement with the previously reported data [10]. An HMBC correlation between Glc-H-6 and Api-C-1 indicated that Glc-6 was connected to Api-1. Moreover, an HMBC correlation between Glc-H-1 and C-3 (G 135.2) showed that Glc-1 was connected to C-3. The glucose residue was identified as the E-anomer by the chemical shifts (GH 5.55 and GC 101.7 for H-1 and C-1, respectively) and the 1H–1H coupling constant (J1,2 = 7.7 Hz). The 13C–1H coupling constant (1JCH = 159.4 Hz) was in good agreement with published data [11]. The linkage of rhamnose was proved to be at C-7 because of the low-field shifts of C-6 (G 101.5) and C-8 (G 96.3) (glycosylation shift). The 13C–1H coupling constant (1JCH = 166.3 Hz) is characteristic of the D-anomer [12]. On the other hand, the anomeric carbon resonance of the apiose was located at G 111.3, which is characteristic of the E-anomer [13]. On the basis of the above results and 2D NMR data, 1 was confirmed to be isorhamnetin-3-O-[apiosyl(1o6)]glucosyl-7-O-rhamnoside [7]. Isorhamnetin-3-O-rutinoside (2) showed a pseudomolecular ion peak at m/z 647 (M + Na)+in the ESI-MS. The 1 H and 13 C NMR spectral data were very similar to those of rutin [14], except for the presence of a methoxy signal at G 3.99. The HMBC correlations of H-8 to C-6 and C-10, H-6 to C-8 and C-10, H-2c to C-4c and C-6c, H-5c to C-1c and C-3c, and H-6c to C-2c and C-4c revealed the linkage of the flavonol skeleton. The HMBC correlation between the methoxy proton and the aromatic carbon (G 149.5, C-3c) revealed that the aglycone of 2 was isorhamnetin. The 1H–1H COSY connectivities of Glc-H-1 to Glc-H-6 and Rha-H-1 to Rha-H-6 and the 13C NMR data indicated the presence of glucose and rhamnose. An HMBC correlation between Rha-H-1 and Glc-C-6 revealed that Glc-6 was connected to Rha-1. Moreover, an HMBC correlation between Glc-H-1 and C-3 (G C132.0) showed that Glc-1 was connected to C-3. The glucose residue was identified as the E-anomer by the chemical shifts (G H 5.25 and GC 103.4 for H-1 and C-1, respectively), the 1 H– 1 H coupling constant (J1, 2 = 7.4 Hz), and the 13C–1H coupling constant (1JCH = 166.6 Hz of Glc-C-1) [11]. For the rhamnose residue, the 13C–1H coupling constant (1J CH = 171.1 Hz of Rha-C-1) is characteristic of the D-anomer [12]. On the basis of the above results and 2D NMR data, 2 was confirmed to be isorhamnetin-3-O-rutinoside [8]. The 3T3-L1 JCRB 9014 cells were maintained in Dulbeccocs modified Eagle medium, supplemented with 10% fetal bovine serum, 50 U mL–1 penicillin, and 50 Pg mL–1 streptomycin at 37qC with 5% CO2. The cellular lipid content was assessed by lipid staining with Oil Red O. Staining was quantified at 505 nm after solubilization using an Adipogenesis Assay Kit. Berberine chloride was used as a positive control [15]. The secretion of adiponectin from 3T3-L1 cells was determined by measuring the adiponectin concentration of the culture medium using the Mouse/Rat Adiponectin ELISA kit. The MeOH extract of this plant was preliminarily subjected to biological tests for any inhibitory effect on adipogenesis, and it showed inhibitory activity. Thus, compounds 1 and 2 were further examined for any inhibitory effect on adipogenesis using 3T3-L1 preadipocytes. Compounds 1 and 2 exhibited inhibitory activity (52.7% and 54.8%, at 50 PM) on adipogenesis (Fig. 1). The secretion of adiponectin from 3T3-L1 cells comparing the control with treated cells were119% (for 1) and 124% (for 2) (data not shown). In this research, we found that an extract of A. glaucum inhibited adipogenesis in 3T3-L1adipocytes, and the bioassayguided fractionation of this plant implied that the antiadipogenesis components are the two isorhamnetin glycosides 1 and 2. Compounds 1 and 2 were firstly isolated from A. glaucum. Compound 1 possesses an apiose for substructure, and very few flavonol glycosides containing apiose have been reported [12]. Although compound 2 has been known topossess various bioactivities [16, 17], there are no reports of bioactivities in compound 1. Therefore, this is the first report of compounds 1 and 2 inhibiting adipogenesis in 3T3-L1 adipocytes.
339
% of control
100 *
80
* *
60
*
*
*
*
40 20 0
Control
1 5 PM
2
Ber
25 PM
50 PM
Fig. 1. Effect of compounds 1 and 2 and berberine chloride (Ber) on adipogenesis assay. Results represent means ± SD of triplicate samples. *significant difference (p < 0.001) vs control ACKNOWLEDGMENT This work was partially supported by the JST-JICA Science and Technology Research Partnership for Sustainable Development (SATREPS) and grant-in-aids for the Ministry of Education, Science, Sports, and Culture of Japan.
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