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New Isoprenylated Phenolic Compounds from Morus laevigata by Meng Wang a ), Bang-Wei Yu b ), Mei-Hua Yu a ), Li-Xin Gao b ), Jing-Ya Li b ), He-Yao Wang b ), Jia Li b ), and Ai-Jun Hou* a ) a
) Department of Pharmacognosy, School of Pharmacy, Fudan University, 826 Zhang Heng Road, Shanghai 201203, P. R. China (phone/fax: þ 86-21-51980005; e-mail: [email protected]) b ) State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P. R. China
Two new isoprenylated flavonoids, laevigasins A and B (1 and 2, resp.), and one new isoprenylated 2arylbenzofuran, leavigasin C (3), together with eight known compounds, 4 – 11, were isolated from the twigs of Morus laevigata. Their structures were elucidated by spectroscopic methods. Laevigasin A (1) showed significant inhibitory effect on a-glucosidase in vitro. Notabilisin E (5), taxifolin (10), and hultenin (11) inhibited PTP1B phosphatase activity in vitro.
Introduction. – The genus Morus (Moraceae) is well-known and abundant in China and has important economic and medicinal value. Plants of this genus are rich in isoprenylated phenolics, such as flavonoids and 2-arylbenzofurans, some of which exhibited antioxidant and anti-inflammatory activities and inhibition on COXs and HIF-1 [1 – 5]. In the course of our search for structurally and biologically interesting compounds from plant resources, we investigated three Morus species, M. nigra, M. notabilis, and M. yunnanensis. A series of new isoprenylated flavonoids, biflavonoids, and 2-arylbenzofuran dimers were isolated, and some compounds showed adipogenesis-promoting and tyrosinase-inhibiting activities [6 – 8]. As a continuing research on this genus, studies on the bioactive constituents of M. laevigata Wall. ex Brandis were carried out. The plant M. laevigata is widely distributed in Yunnan Province of China. While there have been some chromatographic studies on the metabolite profiles of its leaves and fruits that focused on flavonoids and phenolic acids [9 – 12], to the best of our knowledge, no secondary metabolites have been isolated from this plant until now. Fractionation of an EtOH extract of its twigs afforded two new isoprenylated flavonoids, laevigasins A and B (1 and 2, resp.), and one new isoprenylated 2arylbenzofuran, leavigasin C (3), as well as eight known compounds, notabilisins D, E, and A (4 – 6, resp.) [7], 3’,4’,5,7-tetrahydroxy-3-methoxy-6-geranylflavone (7) [13], gemichalcone A (8) [14], sanggenol F (9) [15], taxifolin (10) [16], and hultenin (11) [17] (Fig. 1). Protein tyrosine phosphatase 1B (PTP1B) and a-glucosidase have been regarded as important targets for the treatment of type 2 diabetes [18] [19]. Discovery of effective PTP1B inhibitors is getting wide attention, and the a-glucosidase inhibitors are often used as first-line antidiabetes drugs. Some of the isolated compounds were tested for PTP1B and a-glucosidase-inhibitory effects. Compound 1 showed significant inhibition on a-glucosidase in vitro. Compounds 5, 10, and 11 inhibited PTP1B activity Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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Fig. 1. Structures of compounds 1 – 11
in vitro. We herein report the structural elucidation and biological evaluation of these compounds.
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Results and Discussion. – Laevigasin A (1), obtained as yellow gum, was assigned the molecular formula of C26H30O9 by HR-ESI-MS at m/z 485.1812 ([M ¢ H] ¢ ; calc. 485.1817). The IR absorptions of 1 implied the presence of OH (3355 cm ¢ 1), C¼O (1652 cm ¢ 1), and aromatic ring (1606, 1451 cm ¢ 1) moieties. The UV absorption maxima at 260 and 340 nm resembled those of flavonol derivatives [20]. The 1H-NMR spectrum (Table) provided signals of a H-bonded OH group at d(H) 12.77 (s, 1 H), two meta-coupled aromatic H-atoms (ring A) at d(H) 6.42 and 6.24 (2 br. s, 1 H each), two ortho-coupled aromatic H-atoms (ring B) at d(H) 6.91 and 6.86 (2 d, J ¼ 7.3, 1 H each), and a MeO group at d(H) 3.68 (s, 3 H). Moreover, the 1H- and 13C-NMR data for two quaternary C-atoms (d(C) 135.7, 73.1), an olefinic CH group (d(H) 5.22 (br. t, J ¼ 7.0, 1 H); d(C) 123.5), an O-bearing CH group (d(H) 3.22 (br. d, J ¼ 10.0, 1 H); d(C) 78.4), three CH2 groups (d(H) 3.44 (br. s, 2 H), 2.21 – 2.10, 1.98 – 1.82 (2 m, 1 H each), 1.61 – 1.50, 1.38 – 1.20 (2 m, 1 H each); d(C) 26.8, 37.4, 30.4), and three Me groups (d(H) 1.48 (br. s, 3 H), 1.09 (s, 6 H); d(C) 16.3, 25.6, 24.9) were observed, which were associated with a geranyl-derived 6,7-dihydroxy-3,7-dimethyloct-2-en-1-yl group. The 13C-NMR spectrum exhibited 26 C-atom signals, corresponding to a flavonol skeleton with a C10 isoprenoid and a MeO group. The HMBC of MeO/C(3) assigned the MeO group to be located at C(3) (Fig. 2). The 6,7-dihydroxy-3,7-dimethyloct-2-en-1-yl group was confirmed by the HMBC cross-peaks CH2(9)/C(10,11); Me(12)/C(10,13); CH2(13)/ C(10,11,14); and Me(17,18)/C(15). This group was located at C(2’) based on the HMBCs CH2(9)/C(1’,2’,3’). The two ortho-coupled aromatic H-atoms at d(H) 6.91 and 6.86 and the two meta-coupled aromatic H-atoms at d(H) 6.42 and 6.24 were assigned to H¢C(6’), H¢C(5’), H¢C(8), and H¢C(6), respectively, as supported by the HSQC spectrum of 1 and the HMBCs shown in Fig. 2. The ROESY correlation of H¢C(10)/ CH2(13) indicated an (E)-configuration for C(10)¼C(11) bond. The absolute configuration at C(15) was assigned using an in situ dimolybdenum CD method [21 – 23]. According to the empirical rule proposed by Snatzke, the observed induced CD (ICD) curve at ca. 300 nm showing the same sign with the O¢C¢C¢O torsion angle in the favored conformation allowed the assignment of the absolute configuration. The absolute configuration at C(15) was assigned as (S) by the ICD spectrum of the metal
Fig. 2. Key HMBCs (H ! C) of compound 1
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Fig. 3. a) Conformations of the Mo 2þ 2 complex of 1, b) CD spectrum of 1 (black, in MeOH) and ICD spectrum of the Mo 2þ 2 complex of 1 (red, in DMSO)
complex of 1 in DMSO, which showed a positive Cotton effect at 305 nm (Fig. 3). Thus, the structure of 1 (laevigasin A) was assigned as 2-{2-[(2E,6S)-6,7-dihydroxy-3,7dimethyloct-2-en-1-yl]-3,4-dihydroxyphenyl}-5,7-dihydroxy-3-methoxy-4H-1-benzopyran-4-one (Fig. 1). Laevigasin B (2), obtained as pale yellow gum, was assigned the molecular formula of C25H26O7 by HR-ESI-MS at m/z 437.1590 ([M ¢ H] ¢ ; calc. 437.1606). The IR spectrum showed absorptions for OH (3359 cm ¢ 1), C¼O (1631 cm ¢ 1), and aromatic ring (1580, 1452 cm ¢ 1) moieties. The 1H-NMR spectrum (Table) displayed signals of 1,3,4-trisubstituted benzene ring (ring B) at d(H) 7.06 (d, J ¼ 2.0, 1 H), 6.92 (dd, J ¼ 8.2, 2.0, 1 H), and 6.86 (d, J ¼ 8.2, 1 H), an aromatic singlet at d(H) 5.89 (s, 1 H), and a geranyl-derived pyranoid moiety at d(H) 6.64 (d, J ¼ 10.2, 1 H), 5.62 (d, J ¼ 10.2, 1 H), 5.12 (br. t, J ¼ 7.0, 1 H), 2.14 – 2.06 (overlap, 2 H), 1.79 – 1.67 (m, 2 H), 1.64 (br. s, 3 H), 1.56 (br. s, 3 H), and 1.41 (s, 3 H). Furthermore, the 1H-NMR signals for an AX spin system (ring C) at d(H) 5.05 and 4.64 (2 d, J ¼ 11.7, 1 H each) were characteristic of H¢C(2) and H¢C(3) of a flavanonol derivative, which were consistent with the 13 C-NMR signals at d(C) 84.5 (C(2)) and 73.1 (C(3)). The HMBCs H¢C(9)/C(7); H¢C(10)/C(6); H¢C(8)/C(4a,6,7,8a) (Fig. 4), together with a comparison of the 13 C-NMR data with those of analogs from Desmodium caudatum [24], revealed that the pyranoid ring was attached to C(6) and C(7). The 2,3-trans-configuration was assigned by the coupling constant between H¢C(2) and H¢C(3) (J ¼ 11.7). The absolute configurations at C(2) and C(3) were established as (2R) and (3R) by the CD data with Cotton effects at 333 (þ) and 295 (¢) nm, which were consistent with those of related
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Table. 1H- and 13C-NMR (400 and 100 MHz, resp.; in ( D6 )acetone) Data of 1 – 3. d in ppm, J in Hz. Position
1
2
d( H ) 2 3 3a 4 4a 5 6 7 7a 8 8a 1’ 2’ 3’ 4’ 5’ 6’ 9 10 11 12 13 14 15 16 17 18 5-OH 3-MeO 6-MeO a
6.24 (br. s)
6.42 (br. s)
6.86 (d, J ¼ 7.3) 6.91 (d, J ¼ 7.3) 3.44 (br. s) 5.22 (br. t, J ¼ 7.0) 1.48 (br. s) 2.21 – 2.10 (m), 1.98 – 1.82 (m) 1.61 – 1.50 (m), 1.38 – 1.20 (m) 3.22 (br. d, J ¼ 10.0) 1.09 (s) 1.09 (s) 12.77 (s) 3.68 (s)
3
d(C )
d( H )
160.5 140.2
5.05 (d, J ¼ 11.7) 4.64 (d, J ¼ 11.7)
d(C )
179.6 106.2 163.2 99.4 164.8 94.5 158.1 123.2 129.0 144.4 147.3 113.1 122.5 26.8 123.5 135.7 16.3 37.4
5.89 (s)
7.06 (d, J ¼ 2.0)
6.86 (d, J ¼ 8.2) 6.92 (dd, J ¼ 8.2, 2.0) 6.64 (d, J ¼ 10.2) 5.62 (d, J ¼ 10.2) 1.41 (s) 1.79 – 1.67 (m)
30.4
2.14 – 2.06 a )
78.4 73.1 25.6 b ) 24.9 b )
5.12 (br. t, J ¼ 7.0) 1.64 (br. s) 1.56 (br. s)
60.6
84.5 73.1
d( H )
d(C )
198.7 101.8 158.6 103.3 163.3
6.93 (s)
156.8 102.4 125.5 104.4
96.5 163.5 129.6 115.9 145.8 146.6 116.1 120.8 115.8 126.3 81.7 27.5 42.3
3.66 (br. d, J ¼ 7.0)
148.1 145.0 119.3 148.5 24.1
23.3 124.7 132.2 25.8 17.6
7.00 (s)
1.91 (br. s) 2.12 – 1.98 a ) 2.12 – 1.98 a )
133.4 103.9 159.8 103.6 159.8 103.9 123.1 136.1 16.4 40.4 27.3
5.05 (br. t, J ¼ 7.0)
125.0
1.51 (br. s) 1.56 (br. s)
131.7 17.6 25.7
6.89 (d, J ¼ 1.8) 6.38 (t, J ¼ 1.8) 6.89 (d, J ¼ 1.8) 5.42 (br. t, J ¼ 7.0)
3.84 (s)
b
) Signals overlapped. ) Signals may be exchangeable.
Fig. 4. Key HMBCs (H ! C) of compound 2
61.5
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flavanonol derivatives [24] [25]. Thus, the structure of 2 (laevigasin B) was assigned as (7R,8R)-8-(3,4-dihydroxyphenyl)-7,8-dihydro-5,7-dihydroxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H,6H-benzo[1,2-b:5,4-b’]dipyran-6-one (Fig. 1). Laevigasin C (3), pale yellow amorphous powder, was assigned the molecular formula of C25H28O5 by HR-ESI-MS at m/z 407.1845 ([M ¢ H] ¢ ; calc. 407.1864). The IR spectrum showed absorptions for OH (3375 cm ¢ 1) and aromatic ring (1600, 1445 cm ¢ 1) moieties. The 1H-NMR spectrum (Table) displayed resonances of a 1,3,5trisubstituted benzene ring (ring B) at d(H) 6.89 (d, J ¼ 1.8, 2 H) and 6.38 (t, J ¼ 1.8, 1 H), an olefinic H-atom at d(H) 7.00 (s, 1 H), an aromatic singlet at d(H) 6.93 (s, 1 H), and a MeO group at d(H) 3.84 (s, 3 H). The presence of a geranyl group [26] was indicated by the 1H- and 13C-NMR signals at d(H) 5.42 (br. t, J ¼ 7.0, 1 H), 5.05 (br. t, J ¼ 7.0, 1 H), 3.66 (br. d, J ¼ 7.0, 2 H), 2.12 – 1.98 (overlap, 4 H), 1.91, 1.56, 1.51 (3 br. s, 3 H each), and at d(C) 24.1 (C(8)), 123.1 (C(9)), 136.1 (C(10)), 16.4 (C(11)), 40.4 (C(12)), 27.3 (C(13)), 125.0 (C(14)), 131.7 (C(15)), 17.6 (C(16)), 25.7 (C(17)). These data exhibited that 3 was a geranylated 2-arylbenzofuran. The HMBCs MeO/C(6) and CH2(8)/C(6,7,7a) indicated that the MeO and geranyl groups were located at C(6) and C(7), respectively (Fig. 5). Thus, the structure of 3 (laevigasin C) was deduced as 5-{7-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-5-hydroxy-6-methoxy-1-benzofuran-2yl}benzene-1,3-diol (Fig. 1). Compounds 1 – 5, 7, 8, 10, and 11 were evaluated in vitro for the inhibition on aglucosidase. Compound 1 showed significant inhibitory activity against a-glucosidase with an IC50 value of 17.95 1.97 mm, whereas other compounds were not active. Acarbose was used as positive control (IC50 347.12 68.61 mm). Compounds 5, 10, and 11 were tested in vitro for the inhibition on PTP1B enzymatic activity. They showed significant inhibitory effects on PTP1B with IC50 values of 0.87 0.09 mm (for 5), 5.30 0.63 mm (for 10), and 1.04 0.16 mm (for 11), respectively. Oleanolic acid, an effective natural PTP1B inhibitor [27], was used as positive control in this test (IC50 2.98 0.35 mm). In summary, chemical investigations on the twigs of M. laevigata afforded two new isoprenylated flavonoids, laevigasins A and B (1 and 2, resp.), and one new isoprenylated 2-arylbenzofuran (3), as well as eight known flavonoids, 4 – 11. While isoprenylated phenolics have already been isolated from the genus Morus [1 – 8], this is the first report of such compounds in M. laevigata. Furthermore, this genus as a
Fig. 5. Key HMBCs (H ! C) of compound 3
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promising source for discovering compounds against obesity and type 2 diabetes deserves further studies. This study was supported by the National Natural Science Foundation of China (Nos. 81222045, 81125023, and 21072035), the Science and Technology Commission of Shanghai Municipality (11431920101), and the Shu Guang Project (No. 12SG02) from Shanghai Municipal Education Commission and Shanghai Education Development Foundation.
Experimental Part General. TLC: Precoated silica gel GF254 plates (10 – 40 mm; Qingdao Haiyang Chemical Co., Ltd., P. R. China). Column chromatography (CC): silica gel H (200 – 300 mesh; Qingdao Haiyang Chemical Co., Ltd., P. R. China), ODS gel (50 mm; YMC Co., Japan), Sephadex LH-20 (GE Healthcare Amersham Biosciences, Sweden), and Diaion HP-20 (Mitsubishi Chemical Co., Japan). HPLC: Agilent 1200 (Agilent Technologies, USA), YMC C18 column (150 10 mm, 5 mm, YMC Co., Japan). Optical rotations: Nicolet iS5 polarimeter. UV Spectra: Hitachi U-2900 spectrophotometer; lmax (log e) in nm. IR spectra: Nicolet Avatar-360 spectrometer; ˜n in cm ¢ 1. CD spectra: Jasco J-810 spectropolarimeter. NMR Spectra: Varian Mercucy Plus 400 MHz spectrometers, in (D6 )acetone; d in ppm rel. to the residual solvent signals, J in Hz. ESI- and HR-ESI-MS: Agilent 1100 LC/MSD and Bruker Daltonics Apex III mass spectrometer, resp.; in m/z. Plant Material. The twigs of M. laevigata were collected in Mengzi County, Yunnan Province, P. R. China, in June 2008. The plant material was identified by Prof. Xun Gong, Kunming Institute of Botany, and a voucher specimen (TCM 2008 – 06 – 03 Hou) has been deposited with the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Fudan University. Extraction and Isolation. The milled, air-dried twigs of M. laevigata (8.0 kg) were extracted with 95% EtOH under reflux (2 30 l, 2 h each). The solvent was evaporated under reduced pressure to give a residue (300 g), which was suspended in H2O (1.2 l) and extracted successively with CHCl3 (1.0 l) and AcOEt (1.0 l). The AcOEt extract (17 g) was subjected to CC (SiO2 ; CHCl3/MeOH 40 : 1, 20 : 1, 15 : 1, and 10 : 1) to afford six fractions, Frs. 1 – 6. Fr. 3 (2.8 g) was fractionated by CC (SiO2 ; CHCl3/MeOH 20 : 1 and 15 : 1) to yield compound 11 (60 mg). Fr. 4 (3.4 g) was separated by CC (SiO2 ; CHCl3/MeOH 40 : 1, 30 : 1, 20 : 1, and 10 : 1) to give three fractions, Frs. 4a – 4c. Fr. 4b (650 mg) was subjected to CC (SiO2 ; CHCl3/MeOH 20 : 1 and 15 : 1), followed by CC (Sephadex LH-20; MeOH) to afford compound 10 (50 mg). Fr. 4c (100 mg) was purified by CC (Sephadex LH-20; MeOH), followed by prep. HPLC (MeOH/H2O 6 : 4, 1.0 ml/min, 210 nm) to provide compound 1 (tR 35 min; 5 mg). The CHCl3 extract (130 g) was subjected to CC over Diaion HP-20 eluted with 80% and 90% EtOH. The 80% EtOH fraction (80 g) was fractionated by CC (SiO2 ; petroleum ether (PE)/AcOEt 8 : 1, 6 : 1, 4 : 1, 2 : 1, 1 : 1, 1 : 2, and 1 : 4) to afford nine fractions, Frs. 1 – 9. Fr. 6 (2.1 g) was subjected to CC (SiO2 ; PE/acetone 7 : 1, 4 : 1, and 2 : 1) to afford five subfractions, Frs. 6a – 6e. Fr. 6d (120 mg) was separated by CC (ODS; MeOH/H2O 5.5 : 4.5) and prep. HPLC (MeOH/H2O 7.5 : 2.5, 1.0 ml/min, 210 nm) to yield compound 3 (tR 27 min; 4 mg). Fr. 7 (3.3 g) was fractionated by CC (SiO2 ; PE/acetone 8 : 1, 6 : 1, and 4 : 1) to give six subfractions, Frs. 7a – 7f. Part of Fr. 7c (80 mg) was subjected to CC (Sephadex LH-20; MeOH), followed by CC (ODS; MeOH/H2O 7 : 3) to afford compound 6 (4 mg). Fr. 7f (89 mg) was separated by CC (Sephadex LH-20; MeOH) to provide compounds 4 (6 mg) and 5 (5 mg). Fr. 8 (7.3 g) was subjected to CC (Sephadex LH-20; MeOH), followed by CC (SiO2 ; CHCl3/MeOH 200 : 1, 150 : 1, and 120 : 1) to give eight subfractions, Frs. 8a – 8h. Fr. 8h (75 mg) was fractionated by prep. HPLC (MeOH/H2O 4 : 1, 1.0 ml/min, 210 nm) to afford compounds 7 (tR 15 min; 6 mg) and 8 (tR 18 min; 6 mg). Fr. 9 (3.4 g) was subjected to CC (Sephadex LH-20; MeOH) to afford five subfractions, Frs. 9a – 9e. Fr. 9d (30 mg) was separated by CC (ODS; MeOH/H2O 7 : 3) to afford compounds 2 (6 mg) and 9 (5 mg). Laevigasin A ( ¼ 2-{2-[(2E,6S)-6,7-Dihydroxy-3,7-dimethyloct-2-en-1-yl]-3,4-dihydroxyphenyl}-5,7dihydroxy-3-methoxy-4H-1-benzopyran-4-one; 1). Yellow gum. [a] 25 D ¼ þ 27.8 (c ¼ 0.09, MeOH). UV (MeOH): 260 (4.21), 340 (3.90). IR (KBr): 3355, 2970, 2926, 1652, 1606, 1451, 1361, 1294, 1221, 1169. 1H-
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and 13C-NMR: see the Table. ESI-MS (neg.): 485.1 ([M ¢ H] ¢ ). HR-ESI-MS (neg.): 485.1812 ([M ¢ H] ¢ , C26H29O ¢9 ; calc. 485.1817). Laevigasin B ( ¼ (7R,8R)-8-(3,4-Dihydroxyphenyl)-7,8-dihydro-5,7-dihydroxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H,6H-benzo[1,2-b:5,4-b’]dipyran-6-one; 2). Pale-yellow gum. [a] 25 D ¼ þ 12.5 (c ¼ 0.24, MeOH). UV (MeOH): 275 (3.57, sh), 369 (3.43). CD (MeOH): 333 ( þ 1.51), 295 ( ¢ 4.82). IR (KBr): 3359, 2970, 1631, 1580, 1452, 1384, 1277, 1093. 1H- and 13C-NMR: see the Table. ESI-MS (neg.): 437.1 ([M ¢ H] ¢ ). HR-ESI-MS (neg.): 437.1590 ([M ¢ H] ¢ , C25H25O ¢7 ; calc. 437.1606). Laevigasin C ( ¼ 5-{7-[(2E)-3,7-Dimethylocta-2,6-dien-1-yl]-5-hydroxy-6-methoxy-1-benzofuran-2yl}benzene-1,3-diol; 3). Pale-yellow amorphous powder. UV (MeOH): 215 (4.40), 318 (4.36). IR (KBr): 3375, 2980, 1600, 1445, 1384, 1155, 1091, 1046. 1H- and 13C-NMR: see the Table. ESI-MS (neg.): 407.2 ([M ¢ H] ¢ ). HR-ESI-MS (neg.): 407.1845 ([M ¢ H] ¢ , C25H27O ¢5 ; calc. 407.1864). Determination of the Absolute Configuration at C(15) in 1. According to the published procedure [21] [22], compound 1 (1.5 mg) and Mo2(OAc)4 (7.5 mg) were mixed in DMSO and kept for 30 min to form a stable chiral metal complex for CD measurement. Then, the CD spectrum was recorded. The sign of the diagnostic ICD curve at ca. 300 nm was correlated to the absolute configuration at C(15) in 1 [21 – 23]. Assay of a-Glucosidase Activity. The procedure was performed according to the reported methods with slight modifications [28] [29]. Briefly, the substrate, 4-nitrophenyl a-glucopyranoside (1 mm, 50 ml, pNPG; Sigma), was hydrolyzed to 4-nitrophenol by a-glucosidase from Saccharomyces cerevisiae (0.0125 U/ml, 100 ml; Sigma). The substrate and enzyme were dissolved in 50 mm sodium phosphate buffer (pH 6.8). All tested compounds and the positive control acarbose (Melonepharma) were dissolved in DMSO and diluted with 50 ml sodium phosphate buffer. Samples with seven concentrations ranging from 300 to 0.3 mm and a-glucosidase were pre-incubated at 378 for 15 min. The increment in absorption at 405 nm due to the hydrolysis of pNPG by glycosidase was monitored on Flexstation III instrument (Molecular Devices, CA). All experiments were performed in triplicate. The extent of aglucosidase inhibition is expressed as IC50 , which was calculated with Graphpad prism 5. Preparation of PTP1B Protein and Assay of PTP1B Activity. The procedure was the same as that reported previously [30]. Briefly, the recombinant plasmid was transformed into Escherichia coli BL21CodonPlus (DE3) (Stratagene) for expression. BL21-CodonPlus (DE3) cells containing the recombinant plasmid were grown in 1 l Luria-Bertani (LB) medium in the presence of ampicillin (100 mg/l) by shaking at 378, and the protein expression was induced by adding isopropyl b-d-thiogalactopyranoside (IPTG) to 500 nm when the cell density reached optical density at 600 nm (OD600) of 0.4 – 0.6. The protein samples purified by GSTrap FF column were analyzed by 10% reducing SDS-PAGE ( ¼ sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel, and the protein concentration was determined by the Bradford method with bovine serum albumin (BSA). A colorimetric assay to measure inhibition against PTP1B was performed in 96-well plates. The tested compounds were dissolved in DMSO with the initial concentration of 1 mg/ml, then serially diluted by 3-fold for six times. The compound soln. was diluted by 50-fold into the assay system, and the final concentration of DMSO was 2%. Each sample was treated in three replicate wells. The assays were carried out in a final 100 ml mixture containing 50 mm 3(morpholin-4-yl)propane-1-sulfonic acid (MOPs), pH 6.5, 2 mm 4-nitrophenyl phosphate (pNPP, Calbiochem), 30 nm GST-PTP1B, and 2% DMSO. The catalysis of pNPP was continuously monitored on a SpectraMax 340 microplate reader (Molecular Devices, CA) at 405 nm for 3 min at 308. We used oleanolic acid as the positive control and DMSO as the negative control. The IC50 value was calculated from the nonlinear curve fitting of the percent inhibition vs. the inhibitor concentration ([I]) using the following equation: % inhibition ¼ 100/{1 þ (IC50/[I])k}, where k is the Hill coefficient. REFERENCES [1] [2] [3] [4]
T. Nomura, T. Fukai, Y. Hano, Stud. Nat. Prod. Chem. 2003, 28, 199. S.-J. Dai, Z.-B. Ma, Y. Wu, R.-Y. Chen, D.-Q. Yu, Phytochemistry 2004, 65, 3135. N. T. Dat, X. Jin, K. Lee, Y.-S. Hong, Y. H. Kim, J. J. Lee, J. Nat. Prod. 2009, 72, 39. Y.-X. Tan, R.-Y. Yan, H.-Q. Wang, R.-Y. Chen, D.-Q. Yu, Planta Med. 2009, 75, 249.
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[5] J. M. Rollinger, A. Bodensieck, C. Seger, E. P. Ellmerer, R. Bauer, T. Langer, H. Stuppner, Planta Med. 2005, 71, 399. [6] X. Hu, J.-W. Wu, X.-D. Zhang, Q.-S. Zhao, J.-M. Huang, H.-Y. Wang, A.-J. Hou, J. Nat. Prod. 2011, 74, 816. [7] X. Hu, J. Ji, M. Wang, Q.-S. Zhao, H.-Y. Wang, A.-J. Hou, Bioorg. Med. Chem. Lett. 2011, 21, 4441. [8] X. Hu, J.-W. Wu, M. Wang, M.-H. Yu, Q.-S. Zhao, H.-Y. Wang, A.-J. Hou, J. Nat. Prod. 2012, 75, 82. [9] C.-Y. Lin, H.-L. Lay, Sci. Hortic-Amsterdam 2013, 162, 285. [10] W. Song, H.-J. Wang, P. Bucheli, P.-F. Zhang, D.-Z. Wei, Y.-H. Lu, J. Agric. Food Chem. 2009, 57, 9133. [11] T. Mahmood, F. Anwar, M. Abbas, N. Saari, Int. J. Mol. Sci. 2012, 13, 4591. [12] A. A. Memon, N. Memon, D. L. Luthria, M. I. Bhanger, A. A. Pitafi, Pol. J. Food Nutr. Sci. 2010, 60, 25. [13] D. Lee, K. P. L. Bhat, H. H. S. Fong, N. R. Farnsworth, J. M. Pezzuto, A. D. Kinghorn, J. Nat. Prod. 2001, 64, 1286. [14] M.-I. Chung, M.-H. Lai, M.-H. Yen, R.-R. Wu, C.-N. Lin, Phytochemistry 1997, 44, 943. [15] T. Fukai, Y.-H. Pei, T. Nomura, C.-Q. Xu, L.-J. Wu, Y.-J. Chen, Phytochemistry 1998, 47, 273. [16] H. Imamura, H. Kurosu, T. Takahashi, Mokuzai Gakkaishi 1967, 13, 295. [17] T. Sasaya, Enshurin Kenkyu Hokoku 1965, 24, 177. [18] B. J. Goldstein, A. Bittner-Kowalczyk, M. F. White, M. Harbeck, J. Biol. Chem. 2000, 275, 4283. [19] M. Koyama, R. Wada, H. Mizukami, H. Sakuraba, H. Odaka, H. Ikeda, S. Yagihashi, Metabolism. 2000, 49, 347. [20] Q.-J. Zhang, D.-Z. Li, R.-Y. Chen, D.-Q. Yu, Chin. Chem. Lett. 2008, 19, 196. [21] L. D. Bari, G. Pescitelli, C. Pratelli, D. Pini, P. Salvadori, J. Org. Chem. 2001, 66, 4819. [22] S.-H. Dong, C.-R. Zhang, L. Dong, Y. Wu, J.-M. Yue, J. Nat. Prod. 2011, 74, 1042. [23] L.-B. Zhang, J. Ji, C. Lei, H.-Y. Wang, Q.-S. Zhao, A.-J. Hou, J. Nat. Prod. 2012, 75, 699. [24] H. Sasaki, Y. Kashiwada, H. Shibata, Y. Takaishi, Phytochemistry 2012, 82, 136. [25] S. Rosselli, M. Bruno, A. Maggio, G. Bellone, C. Formisano, C. A. Mattia, S. Di Micco, G. Bifulco, Eur. J. Org. Chem. 2007, 2504. [26] Y.-Q. Shi, T. Fukai, H. Sakagami, W.-J. Chang, P.-Q. Yang, F.-P. Wang, T. Nomura, J. Nat. Prod. 2001, 64, 181. [27] Y.-N. Zhang, W. Zhang, D. Hong, L. Shi, Q. Shen, J.-Y. Li, J. Li, L.-H. Hu, Bioorg. Med. Chem. 2008, 16, 8697. [28] D.-S. Lee, S.-H. Lee, FEBS Lett. 2001, 501, 84. [29] E. Tsujii, M. Muroi, N. Shiragami, A. Takatsuki, Biochem. Biophys. Res. Commun. 1996, 220, 459. [30] L. Shi, H.-P. Yu, Y.-Y. Zhou, J.-Q. Du, Q. Shen, J.-Y. Li, J. Li, Acta Pharmacol. Sin. 2008, 29, 278. Received June 1, 2014
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New Isoprenylated Phenolic Compounds from Morus laevigata by Meng Wang a ), Bang-Wei Yu b ), Mei-Hua Yu a ), Li-Xin Gao b ), Jing-Ya Li b ), He-Yao Wang b ), Jia Li b ), and Ai-Jun Hou* a ) a
) Department of Pharmacognosy, School of Pharmacy, Fudan University, 826 Zhang Heng Road, Shanghai 201203, P. R. China (phone/fax: þ 86-21-51980005; e-mail: [email protected]) b ) State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P. R. China
Two new isoprenylated flavonoids, laevigasins A and B (1 and 2, resp.), and one new isoprenylated 2arylbenzofuran, leavigasin C (3), together with eight known compounds, 4 – 11, were isolated from the twigs of Morus laevigata. Their structures were elucidated by spectroscopic methods. Laevigasin A (1) showed significant inhibitory effect on a-glucosidase in vitro. Notabilisin E (5), taxifolin (10), and hultenin (11) inhibited PTP1B phosphatase activity in vitro.
Introduction. – The genus Morus (Moraceae) is well-known and abundant in China and has important economic and medicinal value. Plants of this genus are rich in isoprenylated phenolics, such as flavonoids and 2-arylbenzofurans, some of which exhibited antioxidant and anti-inflammatory activities and inhibition on COXs and HIF-1 [1 – 5]. In the course of our search for structurally and biologically interesting compounds from plant resources, we investigated three Morus species, M. nigra, M. notabilis, and M. yunnanensis. A series of new isoprenylated flavonoids, biflavonoids, and 2-arylbenzofuran dimers were isolated, and some compounds showed adipogenesis-promoting and tyrosinase-inhibiting activities [6 – 8]. As a continuing research on this genus, studies on the bioactive constituents of M. laevigata Wall. ex Brandis were carried out. The plant M. laevigata is widely distributed in Yunnan Province of China. While there have been some chromatographic studies on the metabolite profiles of its leaves and fruits that focused on flavonoids and phenolic acids [9 – 12], to the best of our knowledge, no secondary metabolites have been isolated from this plant until now. Fractionation of an EtOH extract of its twigs afforded two new isoprenylated flavonoids, laevigasins A and B (1 and 2, resp.), and one new isoprenylated 2arylbenzofuran, leavigasin C (3), as well as eight known compounds, notabilisins D, E, and A (4 – 6, resp.) [7], 3’,4’,5,7-tetrahydroxy-3-methoxy-6-geranylflavone (7) [13], gemichalcone A (8) [14], sanggenol F (9) [15], taxifolin (10) [16], and hultenin (11) [17] (Fig. 1). Protein tyrosine phosphatase 1B (PTP1B) and a-glucosidase have been regarded as important targets for the treatment of type 2 diabetes [18] [19]. Discovery of effective PTP1B inhibitors is getting wide attention, and the a-glucosidase inhibitors are often used as first-line antidiabetes drugs. Some of the isolated compounds were tested for PTP1B and a-glucosidase-inhibitory effects. Compound 1 showed significant inhibition on a-glucosidase in vitro. Compounds 5, 10, and 11 inhibited PTP1B activity Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich
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Fig. 1. Structures of compounds 1 – 11
in vitro. We herein report the structural elucidation and biological evaluation of these compounds.
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Results and Discussion. – Laevigasin A (1), obtained as yellow gum, was assigned the molecular formula of C26H30O9 by HR-ESI-MS at m/z 485.1812 ([M ¢ H] ¢ ; calc. 485.1817). The IR absorptions of 1 implied the presence of OH (3355 cm ¢ 1), C¼O (1652 cm ¢ 1), and aromatic ring (1606, 1451 cm ¢ 1) moieties. The UV absorption maxima at 260 and 340 nm resembled those of flavonol derivatives [20]. The 1H-NMR spectrum (Table) provided signals of a H-bonded OH group at d(H) 12.77 (s, 1 H), two meta-coupled aromatic H-atoms (ring A) at d(H) 6.42 and 6.24 (2 br. s, 1 H each), two ortho-coupled aromatic H-atoms (ring B) at d(H) 6.91 and 6.86 (2 d, J ¼ 7.3, 1 H each), and a MeO group at d(H) 3.68 (s, 3 H). Moreover, the 1H- and 13C-NMR data for two quaternary C-atoms (d(C) 135.7, 73.1), an olefinic CH group (d(H) 5.22 (br. t, J ¼ 7.0, 1 H); d(C) 123.5), an O-bearing CH group (d(H) 3.22 (br. d, J ¼ 10.0, 1 H); d(C) 78.4), three CH2 groups (d(H) 3.44 (br. s, 2 H), 2.21 – 2.10, 1.98 – 1.82 (2 m, 1 H each), 1.61 – 1.50, 1.38 – 1.20 (2 m, 1 H each); d(C) 26.8, 37.4, 30.4), and three Me groups (d(H) 1.48 (br. s, 3 H), 1.09 (s, 6 H); d(C) 16.3, 25.6, 24.9) were observed, which were associated with a geranyl-derived 6,7-dihydroxy-3,7-dimethyloct-2-en-1-yl group. The 13C-NMR spectrum exhibited 26 C-atom signals, corresponding to a flavonol skeleton with a C10 isoprenoid and a MeO group. The HMBC of MeO/C(3) assigned the MeO group to be located at C(3) (Fig. 2). The 6,7-dihydroxy-3,7-dimethyloct-2-en-1-yl group was confirmed by the HMBC cross-peaks CH2(9)/C(10,11); Me(12)/C(10,13); CH2(13)/ C(10,11,14); and Me(17,18)/C(15). This group was located at C(2’) based on the HMBCs CH2(9)/C(1’,2’,3’). The two ortho-coupled aromatic H-atoms at d(H) 6.91 and 6.86 and the two meta-coupled aromatic H-atoms at d(H) 6.42 and 6.24 were assigned to H¢C(6’), H¢C(5’), H¢C(8), and H¢C(6), respectively, as supported by the HSQC spectrum of 1 and the HMBCs shown in Fig. 2. The ROESY correlation of H¢C(10)/ CH2(13) indicated an (E)-configuration for C(10)¼C(11) bond. The absolute configuration at C(15) was assigned using an in situ dimolybdenum CD method [21 – 23]. According to the empirical rule proposed by Snatzke, the observed induced CD (ICD) curve at ca. 300 nm showing the same sign with the O¢C¢C¢O torsion angle in the favored conformation allowed the assignment of the absolute configuration. The absolute configuration at C(15) was assigned as (S) by the ICD spectrum of the metal
Fig. 2. Key HMBCs (H ! C) of compound 1
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Fig. 3. a) Conformations of the Mo 2þ 2 complex of 1, b) CD spectrum of 1 (black, in MeOH) and ICD spectrum of the Mo 2þ 2 complex of 1 (red, in DMSO)
complex of 1 in DMSO, which showed a positive Cotton effect at 305 nm (Fig. 3). Thus, the structure of 1 (laevigasin A) was assigned as 2-{2-[(2E,6S)-6,7-dihydroxy-3,7dimethyloct-2-en-1-yl]-3,4-dihydroxyphenyl}-5,7-dihydroxy-3-methoxy-4H-1-benzopyran-4-one (Fig. 1). Laevigasin B (2), obtained as pale yellow gum, was assigned the molecular formula of C25H26O7 by HR-ESI-MS at m/z 437.1590 ([M ¢ H] ¢ ; calc. 437.1606). The IR spectrum showed absorptions for OH (3359 cm ¢ 1), C¼O (1631 cm ¢ 1), and aromatic ring (1580, 1452 cm ¢ 1) moieties. The 1H-NMR spectrum (Table) displayed signals of 1,3,4-trisubstituted benzene ring (ring B) at d(H) 7.06 (d, J ¼ 2.0, 1 H), 6.92 (dd, J ¼ 8.2, 2.0, 1 H), and 6.86 (d, J ¼ 8.2, 1 H), an aromatic singlet at d(H) 5.89 (s, 1 H), and a geranyl-derived pyranoid moiety at d(H) 6.64 (d, J ¼ 10.2, 1 H), 5.62 (d, J ¼ 10.2, 1 H), 5.12 (br. t, J ¼ 7.0, 1 H), 2.14 – 2.06 (overlap, 2 H), 1.79 – 1.67 (m, 2 H), 1.64 (br. s, 3 H), 1.56 (br. s, 3 H), and 1.41 (s, 3 H). Furthermore, the 1H-NMR signals for an AX spin system (ring C) at d(H) 5.05 and 4.64 (2 d, J ¼ 11.7, 1 H each) were characteristic of H¢C(2) and H¢C(3) of a flavanonol derivative, which were consistent with the 13 C-NMR signals at d(C) 84.5 (C(2)) and 73.1 (C(3)). The HMBCs H¢C(9)/C(7); H¢C(10)/C(6); H¢C(8)/C(4a,6,7,8a) (Fig. 4), together with a comparison of the 13 C-NMR data with those of analogs from Desmodium caudatum [24], revealed that the pyranoid ring was attached to C(6) and C(7). The 2,3-trans-configuration was assigned by the coupling constant between H¢C(2) and H¢C(3) (J ¼ 11.7). The absolute configurations at C(2) and C(3) were established as (2R) and (3R) by the CD data with Cotton effects at 333 (þ) and 295 (¢) nm, which were consistent with those of related
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Table. 1H- and 13C-NMR (400 and 100 MHz, resp.; in ( D6 )acetone) Data of 1 – 3. d in ppm, J in Hz. Position
1
2
d( H ) 2 3 3a 4 4a 5 6 7 7a 8 8a 1’ 2’ 3’ 4’ 5’ 6’ 9 10 11 12 13 14 15 16 17 18 5-OH 3-MeO 6-MeO a
6.24 (br. s)
6.42 (br. s)
6.86 (d, J ¼ 7.3) 6.91 (d, J ¼ 7.3) 3.44 (br. s) 5.22 (br. t, J ¼ 7.0) 1.48 (br. s) 2.21 – 2.10 (m), 1.98 – 1.82 (m) 1.61 – 1.50 (m), 1.38 – 1.20 (m) 3.22 (br. d, J ¼ 10.0) 1.09 (s) 1.09 (s) 12.77 (s) 3.68 (s)
3
d(C )
d( H )
160.5 140.2
5.05 (d, J ¼ 11.7) 4.64 (d, J ¼ 11.7)
d(C )
179.6 106.2 163.2 99.4 164.8 94.5 158.1 123.2 129.0 144.4 147.3 113.1 122.5 26.8 123.5 135.7 16.3 37.4
5.89 (s)
7.06 (d, J ¼ 2.0)
6.86 (d, J ¼ 8.2) 6.92 (dd, J ¼ 8.2, 2.0) 6.64 (d, J ¼ 10.2) 5.62 (d, J ¼ 10.2) 1.41 (s) 1.79 – 1.67 (m)
30.4
2.14 – 2.06 a )
78.4 73.1 25.6 b ) 24.9 b )
5.12 (br. t, J ¼ 7.0) 1.64 (br. s) 1.56 (br. s)
60.6
84.5 73.1
d( H )
d(C )
198.7 101.8 158.6 103.3 163.3
6.93 (s)
156.8 102.4 125.5 104.4
96.5 163.5 129.6 115.9 145.8 146.6 116.1 120.8 115.8 126.3 81.7 27.5 42.3
3.66 (br. d, J ¼ 7.0)
148.1 145.0 119.3 148.5 24.1
23.3 124.7 132.2 25.8 17.6
7.00 (s)
1.91 (br. s) 2.12 – 1.98 a ) 2.12 – 1.98 a )
133.4 103.9 159.8 103.6 159.8 103.9 123.1 136.1 16.4 40.4 27.3
5.05 (br. t, J ¼ 7.0)
125.0
1.51 (br. s) 1.56 (br. s)
131.7 17.6 25.7
6.89 (d, J ¼ 1.8) 6.38 (t, J ¼ 1.8) 6.89 (d, J ¼ 1.8) 5.42 (br. t, J ¼ 7.0)
3.84 (s)
b
) Signals overlapped. ) Signals may be exchangeable.
Fig. 4. Key HMBCs (H ! C) of compound 2
61.5
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flavanonol derivatives [24] [25]. Thus, the structure of 2 (laevigasin B) was assigned as (7R,8R)-8-(3,4-dihydroxyphenyl)-7,8-dihydro-5,7-dihydroxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H,6H-benzo[1,2-b:5,4-b’]dipyran-6-one (Fig. 1). Laevigasin C (3), pale yellow amorphous powder, was assigned the molecular formula of C25H28O5 by HR-ESI-MS at m/z 407.1845 ([M ¢ H] ¢ ; calc. 407.1864). The IR spectrum showed absorptions for OH (3375 cm ¢ 1) and aromatic ring (1600, 1445 cm ¢ 1) moieties. The 1H-NMR spectrum (Table) displayed resonances of a 1,3,5trisubstituted benzene ring (ring B) at d(H) 6.89 (d, J ¼ 1.8, 2 H) and 6.38 (t, J ¼ 1.8, 1 H), an olefinic H-atom at d(H) 7.00 (s, 1 H), an aromatic singlet at d(H) 6.93 (s, 1 H), and a MeO group at d(H) 3.84 (s, 3 H). The presence of a geranyl group [26] was indicated by the 1H- and 13C-NMR signals at d(H) 5.42 (br. t, J ¼ 7.0, 1 H), 5.05 (br. t, J ¼ 7.0, 1 H), 3.66 (br. d, J ¼ 7.0, 2 H), 2.12 – 1.98 (overlap, 4 H), 1.91, 1.56, 1.51 (3 br. s, 3 H each), and at d(C) 24.1 (C(8)), 123.1 (C(9)), 136.1 (C(10)), 16.4 (C(11)), 40.4 (C(12)), 27.3 (C(13)), 125.0 (C(14)), 131.7 (C(15)), 17.6 (C(16)), 25.7 (C(17)). These data exhibited that 3 was a geranylated 2-arylbenzofuran. The HMBCs MeO/C(6) and CH2(8)/C(6,7,7a) indicated that the MeO and geranyl groups were located at C(6) and C(7), respectively (Fig. 5). Thus, the structure of 3 (laevigasin C) was deduced as 5-{7-[(2E)-3,7-dimethylocta-2,6-dien-1-yl]-5-hydroxy-6-methoxy-1-benzofuran-2yl}benzene-1,3-diol (Fig. 1). Compounds 1 – 5, 7, 8, 10, and 11 were evaluated in vitro for the inhibition on aglucosidase. Compound 1 showed significant inhibitory activity against a-glucosidase with an IC50 value of 17.95 1.97 mm, whereas other compounds were not active. Acarbose was used as positive control (IC50 347.12 68.61 mm). Compounds 5, 10, and 11 were tested in vitro for the inhibition on PTP1B enzymatic activity. They showed significant inhibitory effects on PTP1B with IC50 values of 0.87 0.09 mm (for 5), 5.30 0.63 mm (for 10), and 1.04 0.16 mm (for 11), respectively. Oleanolic acid, an effective natural PTP1B inhibitor [27], was used as positive control in this test (IC50 2.98 0.35 mm). In summary, chemical investigations on the twigs of M. laevigata afforded two new isoprenylated flavonoids, laevigasins A and B (1 and 2, resp.), and one new isoprenylated 2-arylbenzofuran (3), as well as eight known flavonoids, 4 – 11. While isoprenylated phenolics have already been isolated from the genus Morus [1 – 8], this is the first report of such compounds in M. laevigata. Furthermore, this genus as a
Fig. 5. Key HMBCs (H ! C) of compound 3
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promising source for discovering compounds against obesity and type 2 diabetes deserves further studies. This study was supported by the National Natural Science Foundation of China (Nos. 81222045, 81125023, and 21072035), the Science and Technology Commission of Shanghai Municipality (11431920101), and the Shu Guang Project (No. 12SG02) from Shanghai Municipal Education Commission and Shanghai Education Development Foundation.
Experimental Part General. TLC: Precoated silica gel GF254 plates (10 – 40 mm; Qingdao Haiyang Chemical Co., Ltd., P. R. China). Column chromatography (CC): silica gel H (200 – 300 mesh; Qingdao Haiyang Chemical Co., Ltd., P. R. China), ODS gel (50 mm; YMC Co., Japan), Sephadex LH-20 (GE Healthcare Amersham Biosciences, Sweden), and Diaion HP-20 (Mitsubishi Chemical Co., Japan). HPLC: Agilent 1200 (Agilent Technologies, USA), YMC C18 column (150 10 mm, 5 mm, YMC Co., Japan). Optical rotations: Nicolet iS5 polarimeter. UV Spectra: Hitachi U-2900 spectrophotometer; lmax (log e) in nm. IR spectra: Nicolet Avatar-360 spectrometer; ˜n in cm ¢ 1. CD spectra: Jasco J-810 spectropolarimeter. NMR Spectra: Varian Mercucy Plus 400 MHz spectrometers, in (D6 )acetone; d in ppm rel. to the residual solvent signals, J in Hz. ESI- and HR-ESI-MS: Agilent 1100 LC/MSD and Bruker Daltonics Apex III mass spectrometer, resp.; in m/z. Plant Material. The twigs of M. laevigata were collected in Mengzi County, Yunnan Province, P. R. China, in June 2008. The plant material was identified by Prof. Xun Gong, Kunming Institute of Botany, and a voucher specimen (TCM 2008 – 06 – 03 Hou) has been deposited with the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Fudan University. Extraction and Isolation. The milled, air-dried twigs of M. laevigata (8.0 kg) were extracted with 95% EtOH under reflux (2 30 l, 2 h each). The solvent was evaporated under reduced pressure to give a residue (300 g), which was suspended in H2O (1.2 l) and extracted successively with CHCl3 (1.0 l) and AcOEt (1.0 l). The AcOEt extract (17 g) was subjected to CC (SiO2 ; CHCl3/MeOH 40 : 1, 20 : 1, 15 : 1, and 10 : 1) to afford six fractions, Frs. 1 – 6. Fr. 3 (2.8 g) was fractionated by CC (SiO2 ; CHCl3/MeOH 20 : 1 and 15 : 1) to yield compound 11 (60 mg). Fr. 4 (3.4 g) was separated by CC (SiO2 ; CHCl3/MeOH 40 : 1, 30 : 1, 20 : 1, and 10 : 1) to give three fractions, Frs. 4a – 4c. Fr. 4b (650 mg) was subjected to CC (SiO2 ; CHCl3/MeOH 20 : 1 and 15 : 1), followed by CC (Sephadex LH-20; MeOH) to afford compound 10 (50 mg). Fr. 4c (100 mg) was purified by CC (Sephadex LH-20; MeOH), followed by prep. HPLC (MeOH/H2O 6 : 4, 1.0 ml/min, 210 nm) to provide compound 1 (tR 35 min; 5 mg). The CHCl3 extract (130 g) was subjected to CC over Diaion HP-20 eluted with 80% and 90% EtOH. The 80% EtOH fraction (80 g) was fractionated by CC (SiO2 ; petroleum ether (PE)/AcOEt 8 : 1, 6 : 1, 4 : 1, 2 : 1, 1 : 1, 1 : 2, and 1 : 4) to afford nine fractions, Frs. 1 – 9. Fr. 6 (2.1 g) was subjected to CC (SiO2 ; PE/acetone 7 : 1, 4 : 1, and 2 : 1) to afford five subfractions, Frs. 6a – 6e. Fr. 6d (120 mg) was separated by CC (ODS; MeOH/H2O 5.5 : 4.5) and prep. HPLC (MeOH/H2O 7.5 : 2.5, 1.0 ml/min, 210 nm) to yield compound 3 (tR 27 min; 4 mg). Fr. 7 (3.3 g) was fractionated by CC (SiO2 ; PE/acetone 8 : 1, 6 : 1, and 4 : 1) to give six subfractions, Frs. 7a – 7f. Part of Fr. 7c (80 mg) was subjected to CC (Sephadex LH-20; MeOH), followed by CC (ODS; MeOH/H2O 7 : 3) to afford compound 6 (4 mg). Fr. 7f (89 mg) was separated by CC (Sephadex LH-20; MeOH) to provide compounds 4 (6 mg) and 5 (5 mg). Fr. 8 (7.3 g) was subjected to CC (Sephadex LH-20; MeOH), followed by CC (SiO2 ; CHCl3/MeOH 200 : 1, 150 : 1, and 120 : 1) to give eight subfractions, Frs. 8a – 8h. Fr. 8h (75 mg) was fractionated by prep. HPLC (MeOH/H2O 4 : 1, 1.0 ml/min, 210 nm) to afford compounds 7 (tR 15 min; 6 mg) and 8 (tR 18 min; 6 mg). Fr. 9 (3.4 g) was subjected to CC (Sephadex LH-20; MeOH) to afford five subfractions, Frs. 9a – 9e. Fr. 9d (30 mg) was separated by CC (ODS; MeOH/H2O 7 : 3) to afford compounds 2 (6 mg) and 9 (5 mg). Laevigasin A ( ¼ 2-{2-[(2E,6S)-6,7-Dihydroxy-3,7-dimethyloct-2-en-1-yl]-3,4-dihydroxyphenyl}-5,7dihydroxy-3-methoxy-4H-1-benzopyran-4-one; 1). Yellow gum. [a] 25 D ¼ þ 27.8 (c ¼ 0.09, MeOH). UV (MeOH): 260 (4.21), 340 (3.90). IR (KBr): 3355, 2970, 2926, 1652, 1606, 1451, 1361, 1294, 1221, 1169. 1H-
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and 13C-NMR: see the Table. ESI-MS (neg.): 485.1 ([M ¢ H] ¢ ). HR-ESI-MS (neg.): 485.1812 ([M ¢ H] ¢ , C26H29O ¢9 ; calc. 485.1817). Laevigasin B ( ¼ (7R,8R)-8-(3,4-Dihydroxyphenyl)-7,8-dihydro-5,7-dihydroxy-2-methyl-2-(4-methylpent-3-en-1-yl)-2H,6H-benzo[1,2-b:5,4-b’]dipyran-6-one; 2). Pale-yellow gum. [a] 25 D ¼ þ 12.5 (c ¼ 0.24, MeOH). UV (MeOH): 275 (3.57, sh), 369 (3.43). CD (MeOH): 333 ( þ 1.51), 295 ( ¢ 4.82). IR (KBr): 3359, 2970, 1631, 1580, 1452, 1384, 1277, 1093. 1H- and 13C-NMR: see the Table. ESI-MS (neg.): 437.1 ([M ¢ H] ¢ ). HR-ESI-MS (neg.): 437.1590 ([M ¢ H] ¢ , C25H25O ¢7 ; calc. 437.1606). Laevigasin C ( ¼ 5-{7-[(2E)-3,7-Dimethylocta-2,6-dien-1-yl]-5-hydroxy-6-methoxy-1-benzofuran-2yl}benzene-1,3-diol; 3). Pale-yellow amorphous powder. UV (MeOH): 215 (4.40), 318 (4.36). IR (KBr): 3375, 2980, 1600, 1445, 1384, 1155, 1091, 1046. 1H- and 13C-NMR: see the Table. ESI-MS (neg.): 407.2 ([M ¢ H] ¢ ). HR-ESI-MS (neg.): 407.1845 ([M ¢ H] ¢ , C25H27O ¢5 ; calc. 407.1864). Determination of the Absolute Configuration at C(15) in 1. According to the published procedure [21] [22], compound 1 (1.5 mg) and Mo2(OAc)4 (7.5 mg) were mixed in DMSO and kept for 30 min to form a stable chiral metal complex for CD measurement. Then, the CD spectrum was recorded. The sign of the diagnostic ICD curve at ca. 300 nm was correlated to the absolute configuration at C(15) in 1 [21 – 23]. Assay of a-Glucosidase Activity. The procedure was performed according to the reported methods with slight modifications [28] [29]. Briefly, the substrate, 4-nitrophenyl a-glucopyranoside (1 mm, 50 ml, pNPG; Sigma), was hydrolyzed to 4-nitrophenol by a-glucosidase from Saccharomyces cerevisiae (0.0125 U/ml, 100 ml; Sigma). The substrate and enzyme were dissolved in 50 mm sodium phosphate buffer (pH 6.8). All tested compounds and the positive control acarbose (Melonepharma) were dissolved in DMSO and diluted with 50 ml sodium phosphate buffer. Samples with seven concentrations ranging from 300 to 0.3 mm and a-glucosidase were pre-incubated at 378 for 15 min. The increment in absorption at 405 nm due to the hydrolysis of pNPG by glycosidase was monitored on Flexstation III instrument (Molecular Devices, CA). All experiments were performed in triplicate. The extent of aglucosidase inhibition is expressed as IC50 , which was calculated with Graphpad prism 5. Preparation of PTP1B Protein and Assay of PTP1B Activity. The procedure was the same as that reported previously [30]. Briefly, the recombinant plasmid was transformed into Escherichia coli BL21CodonPlus (DE3) (Stratagene) for expression. BL21-CodonPlus (DE3) cells containing the recombinant plasmid were grown in 1 l Luria-Bertani (LB) medium in the presence of ampicillin (100 mg/l) by shaking at 378, and the protein expression was induced by adding isopropyl b-d-thiogalactopyranoside (IPTG) to 500 nm when the cell density reached optical density at 600 nm (OD600) of 0.4 – 0.6. The protein samples purified by GSTrap FF column were analyzed by 10% reducing SDS-PAGE ( ¼ sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel, and the protein concentration was determined by the Bradford method with bovine serum albumin (BSA). A colorimetric assay to measure inhibition against PTP1B was performed in 96-well plates. The tested compounds were dissolved in DMSO with the initial concentration of 1 mg/ml, then serially diluted by 3-fold for six times. The compound soln. was diluted by 50-fold into the assay system, and the final concentration of DMSO was 2%. Each sample was treated in three replicate wells. The assays were carried out in a final 100 ml mixture containing 50 mm 3(morpholin-4-yl)propane-1-sulfonic acid (MOPs), pH 6.5, 2 mm 4-nitrophenyl phosphate (pNPP, Calbiochem), 30 nm GST-PTP1B, and 2% DMSO. The catalysis of pNPP was continuously monitored on a SpectraMax 340 microplate reader (Molecular Devices, CA) at 405 nm for 3 min at 308. We used oleanolic acid as the positive control and DMSO as the negative control. The IC50 value was calculated from the nonlinear curve fitting of the percent inhibition vs. the inhibitor concentration ([I]) using the following equation: % inhibition ¼ 100/{1 þ (IC50/[I])k}, where k is the Hill coefficient. REFERENCES [1] [2] [3] [4]
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