1 Introduction

Isoprenylated flavonoids are a group of natural products with diverse structures and important bioactivities. A large number of new compounds have been isolated mainly from species of the Leguminosae, Moraceae, and Euphorbiaceae families, and some of the compounds showed antibacterial, antioxidant, anti-HIV, antidiabetic, and tyrosinase-inhibiting effects [1]. The genus Macaranga Thou. (Euphorbiaceae) comprises about 300 species mainly distributed in the tropical regions of Africa, Asia, Australia, and the Pacific islands [2]. The leaves of some Macaranga species have been used as folk medicine for the treatment of swellings, cuts, sores, boils, and bruises [3]. Macaranga denticulata (Bl.) Muell. Arg. is a tree with rich resources in Hainan and Xishuangbanna areas of China. Its roots have been used as traditional Chinese medicine against icteric hepatitis, eczema, and epigastric pain [4]. Previous phytochemical studies on this plant resulted in the isolation of isoprenylated flavonoids and diterpenylated flavonoids or stilbenes, some of which showed antioxidant, acetylcholinesterase-inhibiting, and antiangiogenic activities [5–7].

Protein tyrosine phosphatase 1B (PTP1B) has been regarded as a promising target for treating type 2 diabetes and obesity [8]. Discovery of effective PTP1B inhibitors is one of our research interests [9–12], and a few of isoprenylated phenolics including flavonoids and Diels–Alder adducts were found to have significant PTP1B inhibitory effects [9, 10]. In our continuing search for natural PTP1B inhibitors from plants, chemical investigations of M. denticulata were carried out. Fractionation of the ethanol extract afforded nine isoprenylated flavonoids, including three new C-methylated and isoprenylated chalcones, dentichalcones A–C (1–3), together with six known compounds, (2E)-1-(5,7-dihydroxy-2,2,6-trimethyl-2H-benzopyran-8-yl)-3-(4-methoxyphenyl)-2-propen-1-one (4), (2E)-1-(5,7-dihydroxy-2,2-dimethyl-2H-benzopyran-8-yl)-3-phenyl-2-propen-1-one (5), laxichalcone (6), macarangin (7), bonanniol A (8), and bonannione A (9) (Fig. 1). The isolated compounds were tested in vitro for inhibition on PTP1B enzymatic activity. Compounds 4–9 showed significant inhibitory effects. This is the first report of C-methylated and isoprenylated chalcones from the genus Macaranga. Herein, we describe the structural elucidation and biological evaluation of these compounds.

Fig. 1
figure 1

Structures of compounds 1–9

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2 Results and Discussion

Dentichalcone A (1) was assigned the molecular formula C21H20O5 by HREIMS with an m/z 352.1308 [M]+ (calcd for C21H20O5, 352.1311). The IR spectrum showed absorptions for OH (3417 cm−1), carbonyl (1625 cm−1), and aromatic (1605, 1514, and 1445 cm−1) moieties. The 1H NMR spectrum (Table 1) displayed a hydrogen-bonded hydroxyl signal at δ H 14.62 (1H, s, OH-2′), two trans-coupled olefinic protons at δ H 8.08 (1H, d, J = 15.6 Hz, H-α) and 7.76 (1H, d, J = 15.6 Hz, H-β), resonances of a 1,4-disubstituted benzene moiety at δ H 7.61 (2H, d, J = 8.6 Hz, H-2, 6) and 6.95 (2H, d, J = 8.6 Hz, H-3, 5), a methyl group at δ H 2.05 (3H, s, H3-7′), and signals of a 2,2-dimethylpyran ring at δ H 6.70 (1H, d, J = 10.0 Hz, H-1″), 5.59 (1H, d, J = 10.0 Hz, H-2″), and 1.56 (6H, s, H3-4″, 5″). The 13C NMR spectrum (Table 1) exhibited 21 carbon signals: three methyls, eight sp 2 methines, and ten quaternary carbons including a carbonyl, eight sp 2, and one oxygenated sp 3. These NMR spectroscopic data indicated that 1 was a chalcone derivative with an isoprenoid and a C-methyl group. By interpretation of the HMBC and NOESY spectra (Fig. 2), the structure of 1 was established. The HMBC cross-peaks of H-α/C-γ, C-1 and H-β/C-α, C-γ, C-1, C-6 verified the presence of chalcone skeleton. The hydroxyl group at δ H 14.62 were assigned to OH-2′ by the HMBC correlations of OH-2′/C-1′, C-2′, C-3′. The methyl group at δ H 2.05 (H3-7′) was located at C-3′ by the HMBC correlations of H3-7′/C-2′, C-3′, C-4′. The 2,2-dimethylpyran group was fused at C-5′ and C-6′, as deduced from the HMBC correlations of H-1″/C-4′, C-5′, C-6′ and H-2″/C-5′, together with the key NOESY correlations of H3-4″, 5″/H-α and H-2, 6. Thus, the structure of 1 was elucidated as (2E)-1-(5,7-dihydroxy-2,2,6-trimethyl-2H-benzopyran-8-yl)-3-(4-hydroxyphenyl)-2- propen-1-one and named dentichalcone A.

Table 1 1H and 13C NMR spectroscopic data of compounds 1–3 (in acetone-d 6)
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Fig. 2
figure 2

Selected HMBC and NOESY correlations of compounds 1–3

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Dentichalcone B (2) was assigned the molecular formula C21H22O5 by HREIMS (m/z 354.1465 [M]+; calcd for C21H22O5, 354.1467). Comparison of its NMR spectroscopic data (Table 1) with those of 1 showed that 2 was a dihydrochalcone derivative of 1. This was confirmed by some diagnostic signals at δ H 3.40 (2H, t, J = 7.6 Hz, H2-α) and 2.89 (2H, t, J = 7.6 Hz, H2-β) and at δ C 46.6 (C-α), and 30.4 (C-β), and 207.3 (C-γ) and by the HMBC correlations of H2-α/C-γ, C-1 and H2-β/C-α, C-γ, C-1, C-6 (Fig. 2). Thus, the structure of 2 was elucidated as 1-(5,7-dihydroxy-2,2,6-trimethyl-2H-benzopyran-8-yl)-3-(4-hydroxyphenyl)propan-1-one and named dentichalcone B.

Dentichalcone C (3) was assigned the molecular formula C21H20O5 by HRESIMS (m/z 351.1237 [M–H]−; calcd for C21H19O5, 351.1238). The 1H and 13C NMR spectra indicated the presence of a chalcone skeleton with a hydrogen-bonded hydroxyl and a methyl group, which was similar to that of 1. Its NMR spectra also displayed signals of a monosubstituted benzene ring [δ H 7.86 (2H, br d, J = 7.3 Hz, H-2, 6), 7.44 (3H, m, H-3, 4, 5); δ C 136.7 (C-1), 130.8 (C-4), 129.7 (C-2, 6), and 129.5 (C-3, 5)] and resonances of a 2-hydroxymethyl-2-methylpyran moiety [δ H 6.79 (1H, d, J = 10.0 Hz, H-1″), 5.52 (1H, d, J = 10.0 Hz, H-2″), 3.90 and 3.61 (each 1H, d, J = 11.9 Hz, H-4″a, b), 1.57 (3H, s, H3-5″); δ C 119.7 (C-1″), 122.5 (C-2″), 81.5 (C-3″), 66.5 (C-4″), 22.7 (C-5″)]. The planar structure of 3 was further constructed by the HMBC spectrum (Fig. 2). The stereochemistry at C-3″ could not be assigned by the available data. Thus, the structure of 3 was elucidated as (2E)-1-[5,7-dihydroxy-2-(hydroxymethyl)-2,6-dimethyl-2H-benzopyran-8-yl]-3-phenyl-2-propen-1-one and named dentichalcone C.

The known compounds were identified as (2E)-1-(5,7-dihydroxy-2,2,6-trimethyl-2H-benzopyran-8-yl)-3-(4-methoxyphenyl)-2-propen-1-one (4) [13], (2E)-1-(5,7-dihydroxy-2,2-dimethyl-2H-benzopyran-8-yl)-3-phenyl-2-propen-1-one (5) [14], laxichalcone (6) [15], macarangin (7) [7], bonanniol A (8) [16], and bonannione A (9) [16] (Fig. 1) by comparison of their spectroscopic data with those reported. Compound 4 was a new natural product, which was previously reported as a synthetic molecule [13].

All the isolated compounds were tested in vitro for the inhibitory effects on PTP1B. Compounds 4–9 showed inhibition with IC50 values ranging from 14.0 ± 1.2 to 48.8 ± 5.5 μM (Table 2). Oleanolic acid, an effective natural PTP1B inhibitor [17], was used as the positive control (IC50 = 2.6 ± 0.6 μM).

Table 2 Inhibitory activities of compounds 4–9 against PTP1B
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In summary, this is the first report of C-methylated and isoprenylated chalcones from the genus Macaranga. This class of compounds is distributed limitedly in the family Euphorbiaceae, and only Mallotus philippinensis was reported to produce C-methylated or both C-methylated and isoprenylated chalcones [18–21]. The Macaranga and Mallotus genera are monophyletic sister groups in the family Euphorbiaceae, which show a remarkable resemblance in their phylogeny, habit, and geographical distribution [22]. The present study indicates that the two genera also have some similarity in their secondary metabolites. Furthermore, isoprenylated flavonoids as potent PTP1B inhibitors for the therapy of obesity and type 2 diabetes need further studies.

3 Experimental Section

3.1 General Experimental Procedures

Optical rotation was measured on a JASCO P-1030 digital polarimeter. UV spectra were recorded on a Hitachi U-2900 spectrophotometer. IR spectra were measured on a Nicolet Avatar-360 spectrometer with KBr pellets. NMR spectra were obtained on Varian Mercucy Plus 400 instruments. Chemical shifts were reported with TMS as internal standard or with respect to acetone-d 6 (δ H 2.04, δ C 206.0 ppm). EIMS (70 eV) and HREIMS were recorded on an Agilent 5973N and a Waters Micromass GCT mass spectrometer, respectively. ESIMS and HRESIMS were performed on an Agilent 1100 LC/MSD and a Bruker Daltonics ApexIII mass spectrometer, respectively. Semi-preparative HPLC was performed on an Agilent 1200 (Agilent Technologies, Palo Alto, CA, USA) and a Sepax Amethyst C18 column (150 × 10 mm, 5 μm, Sepax Techologies, Inc., Newark, DE, USA), using a UV detector set at 210 nm. Column chromatography (CC) was performed on silica gel (200-300 mesh, Yantai Institute of Chemical Technology, Yantai, People’s Republic of China), Diaion HP-20 (Mitsubishi Chemical Co., Tokyo, Japan), and Sephadex LH-20 gel (GE Healthcare Amersham Biosciences, Uppsala, Sweden). Fractions were monitored by TLC analysis run on precoated silica gel GF254 plates (10–40 μm, Yantai Institute of Chemical Technology, Yantai, People’s Republic of China).

3.2 Plant Material

The twigs and leaves of M. denticulata were collected in Hekou County, Yunnan Province, People’s Republic of China, in April 2011. The plant material was identified by Dr. Qin-Shi Zhao, Kunming Institute of Botany, Chinese Academy of Sciences, and a voucher specimen (TCM 11-04-15 Hou) has been deposited at the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Fudan University.

3.3 Extraction and Isolation

The milled, air-dried twigs and leaves of M. denticulata (5.0 kg) were percolated with 95 % EtOH at room temperature (60 L). The filtrate was evaporated under reduced pressure to give a residue (500 g), which was suspended in H2O and extracted with CH2Cl2 (4 × 1 L). The CH2Cl2 extract (140 g) was subjected to CC on Diaion HP-20 eluted with 90 % EtOH. The 90 % EtOH fraction (95 g) was separated by CC on silica gel eluted with a gradient of petroleum ether–EtOAc (1:0, 10:1, 5:1, 1:1, 1:2) to give fractions A–J. Fraction D was separated by CC on Sephadex LH-20 eluted with CHCl3–MeOH (1:1) to afford fractions D1–D4. Fraction D4 was chromatographed over silica gel eluted with a gradient of petroleum ether–Me2CO (10:1, 5:1) to afford fractions D4.1–D4.5. Fraction D4.3 was purified on a Sephadex LH-20 column eluted with MeOH to provide 4 (100 mg). Fraction E was chromatographed by silica gel eluted with a gradient of CH2Cl2–Me2CO (40:1, 2:1) to afford fractions E1–E8. Fraction E4 was separated on silica gel eluted with a gradient of petroleum ether–Me2CO (12:1, 10:1) to give fractions E4.1–E4.5. Fraction E4.5 was chromatographed by semi-preparative HPLC (CH3OH–H2O, 90:10, flow rate 1 mL/min) to afford 6 (10 mg). Fraction F was isolated by CC over Sephadex LH-20 eluted with CHCl3–MeOH (1:1) to afford fractions F1–F5. Fractions F2 and F4 were chromatographed over silica gel eluted with a gradient of petroleum ether–EtOAc (10:1, 2:1) to afford fractions F2.1–F2.6 and F4.1–F4.4, respectively. Fractions F2.2, F2.4, and F4.4 were purified by semi-preparative HPLC at flow rate 1 mL/min to afford 2 (4 mg; CH3OH–H2O, 83:17), 9 (10 mg; CH3OH–H2O, 90:10), and 7 (12 mg; CH3OH–H2O, 91:9), respectively. Fraction F4.2 was separated by CC on Sephadex LH-20 eluted with CH3OH to provide 1 (15 mg). Fraction G was separated by CC on silica gel eluted with a gradient of CH2Cl2–Me2CO (30:1, 2:1) to give fractions G1–G7. Fractions G2, G3, and G4 were chromatographed by semi-preparative HPLC at flow rate 1 mL/min to yield 5 (2 mg; CH3OH–H2O, 88:12), 3 (3 mg; CH3OH–H2O, 80:20), and 8 (12 mg; CH3OH–H2O, 88:12), respectively.

3.4 Dentichalcone A (1)

Red, amorphous powder; UV (MeOH) λ max (log ε) 229 (4.43), 244 (4.42) (sh), 289 (4.30), 368 (4.66) nm; IR (KBr) ν max 3417, 2972, 2915, 1625, 1605, 1541, 1514, 1445, 1167, 830, 536 cm−1; 1H NMR and 13C NMR data, see Table 1; EIMS m/z 352 [M]+ (30), 337 (64), 217 (100), 91 (15), 77 (7); HREIMS m/z 352.1308 [M]+ (calcd for C21H20O5, 352.1311).

3.5 Dentichalcone B (2)

Yellow, amorphous powder; UV (MeOH) λ max (log ε) 222 (3.79), 284 (3.82) nm; IR (KBr) ν max 3419, 2975, 2924, 1638, 1606, 1515, 1427, 1218, 1165, 1133, 827, 548 cm−1; 1H NMR and 13C NMR data, see Table 1; EIMS m/z 354 [M]+ (49), 339 (100), 233 (35), 219 (33), 191 (36), 107 (47), 91 (16), 77 (21), 65 (8), 43 (8); HREIMS m/z 354.1465 [M]+ (calcd for C21H22O5, 354.1467).

3.6 Dentichalcone C (3)

Red, amorphous powder; [α] 25D −32.9 (c 0.30, MeOH); UV (MeOH) λ max (log ε) 224 (4.33), 240 (4.26), 287 (4.30), 346 (4.41) nm; IR (KBr) ν max 3420, 2975, 2915, 1633, 1595, 1456, 1348, 1171, 1130, 701, 575 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 351 [M–H]−; HRESIMS m/z 351.1237 [M–H]− (calcd for C21H19O5, 351.1238).

3.7 Assay of PTP1B Activity

The bioassay procedure was the same as that reported previously [10, 23]. The result of PTP1B inhibition was expressed as IC50, which was calculated with Prism 4 software (Graphpad, San Diego, CA).