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Acetylcholinesterase inhibitors and compounds promoting SIRT1 expression from Curcuma xanthorrhiza
Phytochemistry Letters 12 (2015) 215–219
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Acetylcholinesterase inhibitors and compounds promoting SIRT1 expression from Curcuma xanthorrhiza Chunmei Zhang a, Jianbo Ji b, Mei Ji a, Peihong Fan a,* a
Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China b Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2015 Received in revised form 31 March 2015 Accepted 10 April 2015 Available online 25 April 2015
Curcuma xanthorrhiza is a well-known traditional medicine with anti-inflammatory and anticancer activities, as well as protective effects against neurodegenerative disorders. A previous study revealed the acetylcholinesterase (AChE) inhibitory activity of some sesquiterpenoids from C. xanthorrhiza. In this study, further bioassay-guided isolation led to the identification of nine compounds for the first time from C. xanthorrhiza, including a new Guaiane-type sesquiterpene, zedoaraldehyde (1). Their structures were elucidated using NMR and MS techniques. The AChE inhibitory activities of compounds 1, 3, 4 and 7 were detected as minimum inhibitory quantities of 3, 4, 6 and 1 mg, respectively, using a TLC bioautography assay. Meanwhile, compounds 1, 3, 4 and 8 could promote SIRT1 expression by 1.37-, 1.71-, 1.73- and 1.27-fold, respectively, in HEK293 cell lines exposed to compounds at a concentration of 20 mM for 24 h. SIRT1 is becoming an important drug target for new therapies in the treatment of neurodegenerative diseases. This study indicates the potential of sesquiterpenoids from C. xanthorrhiza for use against Alzheimer’s disease. ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Curcuma xanthorrhiza Zedoaraldehyde Acetylcholinesterase inhibitory activity SIRT1 Alzheimer’s disease
1. Introduction The Curcuma genus (family Zingiberaceae) contains approximately 50 species found in Southeast Asia. Curcuma xanthorrhiza Roxb., commonly known as temu lawak or Javanese turmeric in Indonesia, is a well-known traditional medicinal plant with its anti-inflammatory (Ozaki, 1990) and anticancer (Park et al., 2008) activities as well as protective effects against liver damage (Lin et al., 1995). Terpenoids and curcuminoids were reported as its most abundant compounds (Uehara et al., 1992; Sukari et al., 2008). As part of our studies on C. xanthorrhiza, some novel sesquiterpenoids were reported (Zhang et al., 2014), and some of the sesquiterpenoids from C. xanthorrhiza showed acetylcholinesterase (AChE) inhibitory acitivities (Zhang et al., 2013). AChE inhibitors are important medications that have passed FDA approval for the treatment of mild to moderate Alzheimer’s
Abbreviations: AChE, acetylcholinesterase; SIRT1, silent information regulator two homologue 1; MIQ, minimum inhibitory quantity; PVDF, polyvinylidene fluoride. * Corresponding author. Tel.: +86 531 88382012. E-mail address: [email protected] (P. Fan).
disease (Lau and Brodney, 2008). The AChE inhibitory activities of sesquiterpenoids indicated the potential of C. xanthorrhiza in the therapy of Alzheimer’s disease. Discovering effective and safe AChE inhibitors from traditional medicines is an important way to prevent and treat Alzheimer’s disease. Further bioassay-guided investigation on C. xanthorrhiza led to the isolation of nine compounds for the first time, including one new Guaiane-type sesquiterpenoid 1, four of which showed AChE inhibitory activities. To further explore the potential of C. xanthorrhiza against Alzheimer’s disease, the SIRT1 (silent information regulator two homologue 1) promotion activities of the compounds were tested using HEK293 cell lines. SIRT1 is a member of the sirtuin family that possesses NAD+-dependent deacetylase activity and regulates a variety of cellular processes such as energy metabolism, cell-cycle progression and aging (Raghavan and Shah, 2012). SIRT1 is becoming an important drug target for new therapies in the treatment of neurodegenerative diseases (Huber and Superti-Furga, 2011; Braidy et al., 2012; Min et al., 2013). Small molecule SIRT1 activators or compounds that promote its expression could be candidates for the treatment of aging and age-related diseases (Bonda et al., 2011; Karagiannis and Ververis, 2012; Hubbard and Sinclair, 2014).
http://dx.doi.org/10.1016/j.phytol.2015.04.007 1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
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Table 1 1 H and 13C NMR data (CDCl3, 600 and 150 MHz, respectively) of 1 compared with zedoardiol (11 in Fig. 2, Zhang et al., 2014) (d in ppm, J in Hz). Position
Zedoaraldehyde
dH (multi., J in Hz) 1
Fig. 1. Key HMBCs of compound 1.
2. Results and discussion Compound 1 (Fig. 1) was isolated as a colorless needle crystal (MeOH). The molecular formula was established as C15H16O4 by HR-ESI-MS data ([M + H]+ m/z 261.1120, calcd. 261.1121 for C15H17O4), indicating eight degrees of unsaturation. The IR spectrum (ymax, cm 1) showed characteristic absorption bands for hydroxyl (3427 cm 1) and carbonyl (1763 and 1687 cm 1) groups. The single peak at d9.56 in the 1H NMR spectrum, which correlated with carbon d193.4 in the HSQC spectrum, indicated the presence of an aldehyde group. The 13C NMR spectrum showed 15 carbon signals (Table 1), which were assigned as two methyls (dC 9.2, 14.0), two methylenes (dC 25.8, 29.0), two aliphatic methanes (dC 38.5, 46.2), two olefinic methanes (dC 133.7, 143.1), four olefinic quaternary carbons (dC 124.4, 124.7, 139.6, 154.2), an oxygenated quaternary carbon (dC 84.5) and two carbonyl carbons (dC 193.4, 194.4) using the HSQC data. The degree of unsaturation suggested the presence of three rings in the structure. The above
2a 2b 3a 3b 4 5 6 7 8 9 10 11 12 13 14 15a 15b
3.03 (dt, 9.6, 2.2) 1.97–2.05 (m) 2.68–2.78 (m) 1.44–1.51 (m) 1.97–2.05 (m) 2.68–2.78 (m)
7.22 (d, 2.2)
7.38 2.23 1.07 9.56
(s) (s) (d, 6.7) (s)
Zedoardiol
dc 46.2 25.8 29.0 38.5 84.5 194.4 124.7 154.2 133.7 139.6 124.4 143.1 9.2 14.0 193.4
dH (multi., J in Hz) 3.16 (t, 9.0) 2.00–2.07 (m) 2.11–2.18 (m) 1.50–1.56 (m) 1.91–1.96 (m) 2.59–2.64 (m)
6.73 (s)
7.16 2.23 1.17 4.29 4.40
(s) (s) (d, 6.0) (d, 13.8) (d, 13.8)
dc 48.5 24.1 30.1 39.2 83.7 194.9 120.0 156.3 115.8 143.3 123.0 139.4 9.8 14.4 65.5
NMR data were similar to those of zedoardiol (11 in Fig. 2), a compound previously isolated from C. xanthorrhiza (Zhang et al., 2014). The major difference resulted from the appearance of an aldehyde group and disappearance of an oxygenated methylene (dC 65.5, Table 1) as well as the obvious downfield shift (17.9 ppm) for C-9 and up field shift (3.7 ppm) for C-10, which suggested that the substituent on C-10 in compound 1 was an aldehyde group instead of a hydroxymethylene group. This change was also confirmed by the correlations from H-15 to C-1, C-9 and C-10 as well as that from H-9 to C-15 in the HMBC spectrum. All other HMBC correlations support the deduced structure (Fig. 1). Compound 1 was thus identified as a new guaiane sesquiterpenoid, zedoaraldehyde. The relative configuration of C-4 was defined as
Fig. 2. Structures of compounds 1–11 from C. xanthorrhiza.
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respectively. For compound 8, a 1.27-fold increase in expression was observed. 3. Experimental 3.1. General experimental procedures
Fig. 3. SIRT1 expression in HEK239 cells exposed to compounds 1, 3, 4, 8 and 10 at a concentration of 20 mM for 24 h. C is representative of the control group. SIRT1 protein expression was evaluated using anti-SIRT1 antibodies as described in the Experimental section. One representative western blot from three independent experiments is shown, with b-actin serving as an internal control. Fold induction was calculated by Sirt1/b-actin (% control). Data are presented as the mean S.D. from triplicate independent experiments. The sign * means p < 0.05 versus the control group.
HRESI-MS data were acquired using a Thermofisher Scientific LTQ/Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionization interface. 1D and 2D NMR spectra were acquired at 600 MHz and 150 MHz for 1H and 13C NMR, respectively, on a Bruker-Avance 600 spectrometer in CDCl3, with d in ppm relative to TMS as an internal standard and J in Hz. HPLC separation was performed on an instrument consisting of an Agilent 1200 G1322A degasser, an Agilent 1200 G1311A quaternary pump, an Agilent 1100 G1315D DAD detector, and a ZORBAX SB-C18 5 mm column (9.4 250 mm for semi-preparative and 4.6 250 mm for analysis) (Agilent Technologies). Medium pressure liquid chromatography (MPLC) was carried out on a RP-18 column (15–25 mm; 350 mm 30 mm i.d.) using a slow MeOH–H2O gradient. TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with a 10% sulfuric acid–vanillin solution. 3.2. Reagents and antibodies
shown according to the similarity of the NMR data and same biosynthetic pathway with the previously isolated compound zedoardiol (11) from the same plant material, the relative configuration of which was established using X-ray single-crystal diffraction (Zhang et al., 2014). On the basis of NMR and MS data or by comparison with reference compounds, compounds 2–9 (Fig. 2) were identified as gweicurculactone (2) (Jiang et al., 1989), 13-hydroxygermacrone (3) (Shiobara et al., 1986), germacrone (4) (Yamazaki et al., 1988), gelchomanolide (5) (Kawabata et al., 1981), 8b-hydroxy -isogermafurenolide (6) (Friedrich and Bohlmann, 1988), a-curcumene (7) (Carter et al., 1939), 3-hydroxy-6-methylacetophenone (8) (Kozhevnikova et al., 2003), and dehydro-6-gingerdione (9) (Lee et al., 2011), respectively. All of these compounds were reported for the first time from C. xanthorrhiza Roxb. Among them, 8b-hydroxyisogermafurenolide (6) and 3-hydroxy-6-methylacetophe-none (8) were mentioned as by-products of synthetic reactions (Friedrich and Bohlmann, 1988; Kozhevnikova et al., 2003), but herein are reported for the first time as natural occurring compounds. Dehydro-6-gingerdione (9) was obtained from Zingiber officinale (Wang et al., 2011) but was isolated for the first time from Curcuma species in this work. The AChE inhibitory activities of the isolated compounds were tested using a TLC bioautography assay. The minimum inhibitory quantity (MIQ) values of compounds 1, 3, 4 and 7 were 3, 4, 6 and 1 mg, respectively (Fig. S5 in the supporting data). Compared with the positive control galanthamine (MIQ = 10 ng), an AChE inhibitor approved by FDA, these compounds exhibited moderate AChE inhibitory activities in vitro. Compounds 1, 3, 4, 8 and a previously isolated compound 10 (Fig. 2) were evaluated for their effects on SIRT1 expression in HEK293 cells. Before the test, the cytotoxicities of the compounds at different concentrations (12.5, 25.0, 50.0 and 100.0 mM) were first detected by MTT assays. The results indicated that all of these compounds have no cytotoxicity to HEK293 cells at a final concentration of 100 mM. Then, HEK293 cells were treated with the compounds at a concentration of 20 mM. After incubation for 24 h, the SIRT1 expression level was measured by western blot analysis. As demonstrated in Fig. 3, all of these compounds could increase SIRT1 expression. Sesquiterpenoids 1, 3, 4 and 10 enhanced SIRT1 expressions by 1.37-, 1.71-, 1.73- and 1.55-fold,
Petroleum ether, dichloromethane, ethylacetate, and methanol were purchased from the Fuyu Chemical Reagents Company (Tianjing, China). Methanol (HPLC grade) was from the Siyou Chemical Reagents Company (Tianjin, China), and ethylacetate (HPLC grade) was from the Aladdin Industrial Company (Shanghai, China). Milli-Q water (Millipore, Bedford, MA, USA) was used. Column chromatography: silica gel (Qingdao Haiyang Chemical Co., Ltd, Qingdao, China); ODS-A-HG gel (12 nm, 50 mm,YMC Co., Ltd, Kyoto, Japan); TLC bioautographic assay: silica gel 60 F254 Al sheets (Merck, Darmstadt, Germany). Acetylcholinesterase was from electric eel (EC 3.1.1.7); bovine serum albumin (BSA), Fast Blue B Salt, 1-naphylacetate, the positive reference compound galanthamine, and Tris base were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modification of Eagle’s medium (DMEM) was bought from Thermo (USA); FBS was purchased from Gibco (USA); the penicillin-streptomycin solution was bought from Ceres (China); phosphate buffered salts (PBS), dimethyl sulfoxide (DMSO) and MTT were bought from Sigma– Aldrich (USA); and the Bradford Protein Assay Kit was purchased from the Beyotime Institute of Biotechnology, China. The SIRT1, actin primary antibody and IgG HRP-conjugated second antibody used for western blotting analysis were purchased from the Cell Signaling Biotechnology Corporation (USA). The polyvinylidene fluoride (PVDF) membrane was bought from Millipore Corporation (USA); and the western ECL substrate kit was obtained from Bio-Rad (USA). 3.3. Plant material Curcuma xanthorrhiza Roxb. rhizome material (origin: Indonesia) was purchased from Dixa, CH-St. Gallen and was identified by Prof. Lan Xiang, Shandong University. A voucher specimen has been stored in the School of Pharmaceutical Sciences, Shandong University, Ji’nan, P.R. China. 3.4. Extraction and isolation The dried and powdered material (5.0 kg) of C. xanthorrhiza was percolated three times with 95% EtOH at r.t. The residue (193.0 g) was suspended with water and then was successively extracted
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with petroleum ether (3 500 ml), AcOEt (3 500 ml), and BuOH (500 ml). The petroleum ether soluble part (33.0 g) was subjected to CC (SiO2, 200–300 mesh, 8 70 cm; petroleum ether–AcOEt 100:0–0:100) to yield 15 fractions (Frs.1–15). Fr.3 (8.61 g) was subjected to repeated AgNO3 CC (SiO2, 200–300 mesh, 98 g; petroleum ether–AcOEt 100:0–0:100) to yield 4 (29 mg); Frs. 6–8 were subjected to RP-CC (MeOH–water 3:7–10:0) and provided 5 fractions (Frs.6–8-RP1-5). First Sephadex-LH-20 (MeOH–H2O) was used for separation and then HPLC was used for further purification. Frs.6–8-RP2 yielded: 1 (3.7 mg) and 8 (5.0 mg), while Frs.6–8-RP3 yielded: 3 (5.2 mg) and 6 (4.0 mg). Frs.9–11 were subjected to RP-CC (MeOH–water 3:7–10:0) to give 3 fractions (Frs. 9–11-RP1-3). Compound 5 (5.2 mg) was obtained by crystallization from Frs. 9–11-RP1. Frs. 9–11-RP2 were subjected to Sephadex-LH-20 (MeOH–H2O) twice and were further purified with HPLC (MeOH–H2O) to yield 2 (4.3 mg) and 7 (5.6 mg). Frs. 9–11-RP3 was treated in the same manner as Frs. 9–11-RP2 to yield 9 (7.7 mg).
150 MHz) d173.7 (C-12), 162.8 (C-7), 132.9 (C-4), 132.5 (C-10), 130.7 (C-1), 126.1 (C-11), 123.7 (C-5), 82.9 (C-8), 47.1 (C-9), 38.4 (C-3), 27.3 (C-6), 25.6 (C-2), 16.8 (C-15), 16.5 (C-14), 8.9 (C-13) (Kawabata et al., 1981).
3.4.1. Zedoaraldehyde (1) Pale yellow oil; [a]D24 42.90 (c 1.0, CH3OH); IR ymax 3427, 2971, 2931, 1763, 1687, 1048 cm 1; For 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) data, see Table 1; HRESIMS m/z 261.1120 ([M + H]+; calcd for C15H17O4, 260.1121).
3.4.7. a-Curcumene (7) Pale yellow oil; 1H NMR (CDCl3, 600 MHz) d7.11 (2H, m, H-3 and H-5), 7.08 (2H, m, H-2 and H-6), 5.10 (1H, t, J = 6 Hz, H-10), 2.66 (1H, m, H-7), 2.33 (3H, s, H-15), 1.88 (2H, m, H-9), 1.69 (3H, s, H-13), 1.58 (2H, m, H-8), 1.54 (3H, s, H-12), 1.22 (3H, s, H-14); 13C NMR (CDCl3, 150 MHz) d144.5 (C-1), 135.2 (C-4), 131.4 (C-11), 128.8 (C-3), 128.8 (C-5), 126.8 (C-2), 126.8 (C-6), 124.5 (C-10), 39.0 (C-7), 38.5 (C-8), 26.2 (C-9), 25.8 (C-13), 22.5 (C-14), 21.0 (C-15), 17.6 (C-12) (Carter et al., 1939).
3.4.2. Gweicurculactone (2) Colorless crystal; 1H NMR (CDCl3, 600 MHz) d6.92 (1H, s, H-6), 6.76 (1H, s, H-9), 3.10 (1H, m, H-4), 2.84 (1H, m, H-2a), 2.72 (1H, m, H-2b), 2.27 (3H, s, H-15), 2.01 (3H, s, H-13), 2.15 (1H, m, H-3a), 1.60 (1H, m, H-3b), 1.33 (3H, d, J = 6 Hz, H-14); 13C NMR (CDCl3, 150 MHz) d170.7 (C-12), 156.6 (C-5), 154.7 (C-8), 146.1 (C-7), 144.2 (C-10), 136.5 (C-1), 117.9 (C-6), 116.3 (C-9), 103.5 (C-11), 43.8 (C-4), 33.6 (C-2), 31.9 (C-3), 24.7 (C-15), 20.0 (C-14), 7.7 (C-13) (Jiang et al., 1989). 3.4.3. 13-Hydroxygermacrone (3) Colorless needle crystal; 1H NMR (CDCl3, 600 MHz) d5.01 (1H, d, J = 12 Hz, H-1), 4.66 (1H, d, J = 12 Hz, H-5), 4.32 (1H, d, J = 12 Hz, H12a), 4.21 (1H, d, J = 12 Hz, H-12b), 3.45 (1H, d, J = 10 Hz, H-9a), 2.97–3.04 (2H, m, H-6), 2.99 (1H, m, H-9b), 2.39 (1H, m, H-2a), 2.20 (1H, m, H-3a), 2.11 (2H, m, H-2b and H-3b), 1.84 (3H, s, H-13), 1.65 (3H, s, H-14), 1.45 (3H, s, H-15); 13C NMR (CDCl3, 150 MHz) d207.2 (C-8), 139.9 (C-11), 135.8 (C-4), 133.1 (C-1), 131.2 (C-7), 126.4 (C-10), 124.9 (C-5), 62.8 (C-12), 55.6 (C-9), 38.0 (C-3), 28.2 (C-6), 24.4 (C-2), 17.8 (C-13), 16.5 (C-14), 15.8 (C-15) (Shiobara et al., 1986). 3.4.4. Germacrone(4) Colorless crystal; 1 H NMR (CDCl 3 , 600 MHz) d 5.01 (1H, d, J = 12 Hz, H-1), 4.73 (1H, d, J = 12 Hz, H-5), 3.43 (1H, d, J = 12 Hz, H-9 a ), 2.97 (2H, m, H-7), 2.87 (1H, m, H-9 b ), 2.38 (1H, m, H-2 a ), 2.18 (1H, m, H-3 a ), 2.10 (2H, m, H-2 b and H-3 b ), 1.80 (3H, s, H-13), 1.73 (3H, s, H-12), 1.65 (3H, s, H-14), 1.46 (3H, s, H-15); 13 C NMR (CDCl 3 , 150 Hz) d 207.8 (C-8), 137.2 (C-11), 135.0 (C-4), 132.7 (C-1), 129.4 (C-7), 126.7 (C-10), 125.3 (C-5), 55.8 (C-9), 38.0 (C-3), 29.3 (C-6), 23.9 (C-2), 22.2 (C-13), 19.8 (C-12), 16.7 (C-14), 15.4 (C-15) (Yamazaki et al., 1988). 3.4.5. Gelchomanolide (5) Colorless crystal; 1H NMR (CDCl3, 600 MHz) d4.97 (1H, brd, J = 12 Hz, H-8), 4.86 (1H, brd, J = 12 Hz, H-1), 4.34 (1H, brd, J = 6 Hz, H-5), 3.41 (1H, d, J = 12 Hz, H-6a), 3.10 (1H, brd, J = 12 Hz, H-9a), 2.90 (1H, d, J = 12 Hz, H-6b), 2.19 (1H, m, H-2a), 2.11 (2H, m, H-2b and H-9b), 2.24 (1H, m, H-3a), 1.86 (1H, m, H-3b), 1.87 (3H, s, H-13), 1.62 (3H, s, H-14), 1.51 (3H, s, H-15); 13C NMR (CDCl3,
3.4.6. 8b-Hydroxy-isogermafurenolide (6) Colorless needle crystal; 1H NMR (CDCl3, 600 MHz) d5.70 (1H, dd, J = 18 Hz and 12 Hz, H-1), 4.99 (1H,d, J = 18 Hz, H-2a ), 5.01 (1H, d, J = 12 Hz, H-2b), 4.75 (1H, br s, H-3a), 5.02 (1H, br s, H-3b), 2.58 (1H, dd, J = 12 Hz and 3.5 Hz, H-6a ), 2.73 (1H, ddd, J = 12 Hz, 3.5 Hz and 1.3 Hz, H-6b), 2.06 (1H, dd, J = 12 Hz and 3.5 Hz, H-5), 2.14 (1H, d, J = 12 Hz, H-9a ), 1.77 (1H, d, J = 12 Hz, H-9b), 1.86 (3H, d, J = 1.3 Hz, H-13), 1.79 (3H, s, H-15), 1.29 (3H, s, H-14); 13C NMR (CDCl3, 150 MHz) d171.2 (C-12), 160.1 (C-8), 147.4 (C-1), 144.6 (C-4), 122.0 (C-11), 114.1 (C-3), 112.1 (C-2), 102.7 (C-7), 54.1 (C-5), 49.2 (C-9), 40.6 (C-10), 27.1 (C-6), 24.4 (C-15), 17.8 (C-14), 8.2 (C-13) (Friedrich and Bohlmann, 1988).
3.4.8. 3-Hydroxy-6-methylacetophenone (8) Colorless needle crystal; 1H NMR (CDCl3, 600 MHz) d7.52 (1H, s, H-2), 7.45 (1H, d, J = 6 Hz, H-5), 7.23 (1H, d, J = 6 Hz, H-6), 2.60 (3H, s, CH3), 2.34 (3H, s, Ph-CH3); 13C NMR (CDCl3, 150 MHz) d198.4 (C5 5O), 154.0 (C-1), 136.5 (C-4), 131.0 (C-3), 130.9 (C-6), 121.2 (C-5), 113.8 (C-2), 26.4 (Me), 16.0 (Ph- Me) (Kozhevnikova et al., 2003). 3.4.9. Dehydro-6-gingerdione (9) Yellow powder; 1H NMR (CDCl3, 600 MHz) d7.56 (1H, d, J = 12 Hz, H-7), 7.11 (1H, dd, J = 6 Hz and 18 Hz, H-6), 7.04 (1H, d, J = 18 Hz, H-2), 6.95 (1H, d, J = 6 Hz, H-5), 6.36 (1H, d, J= 12 Hz, H8), 5.85 (1H, s, OH), 5.64 (2H, s, H-10), 3.96 (3H, s, OMe), 2.38 (2H, t, J = 6 Hz, H-12), 1.68 (2H, m, H-13), 0.99 (3H, t, J = 6 Hz, H-14); 13 C NMR (CDCl3, 150 MHz) d200.0 (C-11), 178.2 (C-9), 147.6 (C-4), 146.8 (C-3), 139.8 (C-7), 127.9 (C-1), 122.6 (C-6), 120.5 (C-8), 114.7 (C-5), 109.4 (C-2), 100.5 (C-10), 55.9 (OMe), 42.0 (C-12), 19.0 (C-13), 13.9 (C-14) (Lee et al., 2011). 3.5. AChE activity assay The AChE inhibitory activities of the isolated compounds were determined using a TLC bioautography assay modified from a previous method (Fan et al., 2008). Briefly, the TLC plate with samples was sprayed with enzyme solution (1.5 U/mL in Tris– hydrochloric acid buffer at a pH 7.2) and dried. Then, the plate was sprayed with 1-naphthyl acetate solution (1.5 mg/mL in 40% EtOH) and dried again, followed by incubation at 37 8C for 20 min. For detection, a Fast Blue B Salt solution (0.5 mg/mL in water) was used to soak the plate for 20 s, and white zones on a purple background on the plate indicated the inhibitory activities of the samples. To ascertain detection limits of the active compounds, different volumes of 2.0 mg/mL solutions were applied onto the TLC plates, and the amount that produced the least visible white spot was noted as the minimum inhibitory quantity (MIQ). Galantamine (0.05 mg/mL) was used as a positive control.
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3.6. Cytotoxicity assay (MTT assay)
References
Cytotoxicities of compounds 1, 3, 4, 8 and 10 were assessed by 3-(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. HEK293 cells (5 103 cells/well) were incubated in 96-well microtiter plates for 24 h. After the addition of different concentrations (12.5, 25.0, 50.0 and 100.0 mM) of the test compounds or only medium, the plates were incubated for an additional 24 h. Three replicate wells were set for each concentration in the experiments. Then, 20 mL of MTT solution (final concentration of 5 mg/mL, freshly diluted in PBS before treatment) was added to each well and incubated for another 4 h. The medium was abandoned, and 150 mL/well DMSO was added. The amount of formazan was determined by measuring the absorbance at 490 nm with a microplate reader (Bio-Rad, USA).
Bonda, D.J., Lee, H.G., Camins, A., Palla`s, M., Casadesus, G., Smith, M.A., Zhu, X., 2011. The sirtuin pathway in ageing and Alzheimer disease: mechanistic and therapeutic considerations. Lancet Neurol. 10, 275–279. Braidy, N., Jayasena, T., Poljak, A., Sachdev, P.S., 2012. Sirtuins in cognitive ageing and Alzheimer’s disease. Curr. Opin. Psychiatr. 25, 226–230. Carter, F.D., Copp, F.C., Rao, B.S., Simmonsen, J.L., Subramaniam, K.S., 1939. Constituents of some Indian essential oils. XXVI. The structures of l-a- and -bcurcumenes. J. Chem. Soc. 1504–1509. Fan, P.H., Hay, A.E., Marston, A., Hostettmann, K., 2008. Acetylcholinesteraseinhibitory activity of linarin from Buddleja davidii, structure-activity relationships of related flavonoids, and chemical investigation of Buddleja nitida. Pharm. Biol. 46, 596–601. Friedrich, D., Bohlmann, F., 1988. Total synthesis of various elemanolides. Tetrahedron 44, 1369–1392. Hubbard, B.P., Sinclair, D.A., 2014. Small molecule SIRT1 activators for the treatment of aging and age-related diseases. Trends Pharmacol. Sci. 35, 146–154. Huber, K., Superti-Furga, G., 2011. After the grape rush: Sirtuins as epigenetic drug targets in neurodegenerative disorders. Bioorg. Med. Chem. 19, 3616–3624. Jiang, D.Q., Pu, Q.L., Huang, P., Huang, X.M., He, Y.Z., He, C.H., Zheng, Q.T., 1989. Studies on the chemical constituents of Curcuma kwangsiensis. Acta Pharm. Sinica 24, 357–359. Karagiannis, T.C., Ververis, K., 2012. Potential of chromatin modifying compounds for the treatment of Alzheimer’s disease. Pathobiol. Aging Age Relat. Dis. 2, 14980. Kawabata, J., Tahara, S., Mizutani, J., 1981. Studies on the chemical constituents of chloranthaceae plants. Part I. Isolation and structural elucidation of four sesquiterpenes from Chloranthus japonicus (Chloranthaceae). Biol. Chem. 45, 1447–1453. Kozhevnikova, E.F., Quartararo, J., Kozhevnikov, I.V., 2003. Fries rearrangement of aryl esters catalyzed by heteropoly acid. Appl. Catal. A 245, 69–78. Lau, L.F., Brodney, M.A., 2008. Therapeutic approaches for the treatment of Alzheimer’s disease: an overview. Top. Med. Chem. 2, 1–24. Lee, S.W., Lim, J.H., Kim, M.S., Jeong, J.H., Song, G.Y., Lee, W.S., Rho, M.C., 2011. Phenolic compounds isolated from Zingiber officinale roots inhibit cell adhesion. Food Chem. 128, 778–782. Lin, S.C., Lin, C.C., Lin, Y.H., Supriyatna, S., Teng, C.W., 1995. Protective and therapeutic effects of Curcuma xanthorrhiza on hepatotoxin-induced liver damage. Am. J. Chin. Med. 23, 243–254. Min, S.W., Sohn, P.D., Cho, S.H., Swanson, R.A., Gan, L., 2013. Sirtuins in neurodegenerative diseases: an update on potential mechanisms. Front. Aging Neurosci. 5, 53. Ozaki, Y., 1990. Antiinflammatory effect of Curcuma xanthorrhiza Roxb. and its active principles. Chem. Pharm. Bull. 38, 1045–1048. Park, J.H., Park, K.K., Kim, M.J., Hwang, J.K., Park, S.K., Chung, W.Y., 2008. Cancer chemoprotective effects of Curcuma xanthorrhiza. Phytother. Res. 22, 695–698. Raghavan, A., Shah, Z.A., 2012. Sirtuins in neurodegenerative diseases: a biologicalchemical perspective. Neurodegener. Dis. 9, 1–10. Shiobara, Y., Asakawa, Y., Kodama, M., Takemoto, T., 1986. Zedoarol, 13-hydroxygermacrone and curzeone, three sesquiterpenoids from Curcuma zedoaria. Phytochemistry 25, 1351–1353. Sukari, M.A., Rashid, N.Y., Tang, S.W., Rahmani, M., Lajis, N.H., Khalid, K., Yusuf, U.K., 2008. Chemical constituents and bioactivity of Curcuma xanthorrhiza Roxb. J. Ultra Sci. Phys. Sci. 20, 605–610. Uehara, S., Yasuda, I., Takeya, K., Itokawa, H., 1992. Terpenoids and curcuminoids of the rhizome of Curcuma xanthorrhiza. Roxb. Yakugaku Zasshi. 112, 817–823. Wang, Z., Wang, K.J., Cheng, C.S., Li, N., Wang, T.M., Di, L., 2011. Gingerol derivatives from the rhizomes of Zingiber officinale. Z Naturforsch B: J. Chem. Sci. 66, 740–744. Yamazaki, M., Maebayashi, Y., Iwase, N., Kaneko, T., 1988. Studies on pharmacologically active principles from Indonesian crude drugs. II. Hypothermic principle from Curcuma xanthorrhiza. Roxb. Chem. Pharm. Bull. 36, 2075–2078. Zhang, C.M., Fan, P.H., Li, M., Lou, H.X., 2014. Two new sesquiterpenoids from the rhizomes of Curcuma xanthorrhiza. Helv. Chim. Acta 97, 1295–1300. Zhang, C.M., Wang, J.D., Zhang, Y.R., Fan, P.H., 2013. Chemical constituents of Curcuma Xanthorrhiza Roxb. and their acetylcholinesterase inhibitoy activity. J. Shandong Uni. (Nat. Sci.) 48, 20–23.
3.7. Western blot analysis HEK293 cells were plated in 6-well plates at a density of 5 105 cells/well and grown at 37 8C. Cells were treated with compounds at a final concentration of 20 mM for 24 h. Then, cells were harvested, lysed, centrifuged. The supernatants were utilized for protein analysis and western blot analysis. The total protein concentration was determined using a Bradford Protein Assay Kit. Protein samples were denatured, and final stored at 80 8C for western blotting. Equal amounts of protein (30 mg) from cell lysates were loaded onto a 10% SDS-polyacrylamide gel and separated by electrophoresis. b-actin served as an internal control. Then, the separated proteins were electrophoretically transferred to a 0.45 mm PVDF membrane. The transferred PVDF membranes were incubated with anti-SIRT1 primary antibodies (1:1000) at 4 8C overnight. Bound antibodies were detected using a horseradish peroxidase-linked secondary antibody. Statistical analysis: All results were confirmed from a minimum of three independent experiments on replicate samples. Data are represented as the mean standard deviation (SD). Statistical significances were evaluated by the t-test. Data were considered significant at P < 0.05. Acknowledgements The authors thank the National Natural Science Foundation of China (grant no. 81473323, 81001616) and the China Postdoctoral Science Foundation (2014M551926) for their financial support of this work.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2015. 04.007.
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Acetylcholinesterase inhibitors and compounds promoting SIRT1 expression from Curcuma xanthorrhiza Chunmei Zhang a, Jianbo Ji b, Mei Ji a, Peihong Fan a,* a
Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China b Department of Pharmacology, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2015 Received in revised form 31 March 2015 Accepted 10 April 2015 Available online 25 April 2015
Curcuma xanthorrhiza is a well-known traditional medicine with anti-inflammatory and anticancer activities, as well as protective effects against neurodegenerative disorders. A previous study revealed the acetylcholinesterase (AChE) inhibitory activity of some sesquiterpenoids from C. xanthorrhiza. In this study, further bioassay-guided isolation led to the identification of nine compounds for the first time from C. xanthorrhiza, including a new Guaiane-type sesquiterpene, zedoaraldehyde (1). Their structures were elucidated using NMR and MS techniques. The AChE inhibitory activities of compounds 1, 3, 4 and 7 were detected as minimum inhibitory quantities of 3, 4, 6 and 1 mg, respectively, using a TLC bioautography assay. Meanwhile, compounds 1, 3, 4 and 8 could promote SIRT1 expression by 1.37-, 1.71-, 1.73- and 1.27-fold, respectively, in HEK293 cell lines exposed to compounds at a concentration of 20 mM for 24 h. SIRT1 is becoming an important drug target for new therapies in the treatment of neurodegenerative diseases. This study indicates the potential of sesquiterpenoids from C. xanthorrhiza for use against Alzheimer’s disease. ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Curcuma xanthorrhiza Zedoaraldehyde Acetylcholinesterase inhibitory activity SIRT1 Alzheimer’s disease
1. Introduction The Curcuma genus (family Zingiberaceae) contains approximately 50 species found in Southeast Asia. Curcuma xanthorrhiza Roxb., commonly known as temu lawak or Javanese turmeric in Indonesia, is a well-known traditional medicinal plant with its anti-inflammatory (Ozaki, 1990) and anticancer (Park et al., 2008) activities as well as protective effects against liver damage (Lin et al., 1995). Terpenoids and curcuminoids were reported as its most abundant compounds (Uehara et al., 1992; Sukari et al., 2008). As part of our studies on C. xanthorrhiza, some novel sesquiterpenoids were reported (Zhang et al., 2014), and some of the sesquiterpenoids from C. xanthorrhiza showed acetylcholinesterase (AChE) inhibitory acitivities (Zhang et al., 2013). AChE inhibitors are important medications that have passed FDA approval for the treatment of mild to moderate Alzheimer’s
Abbreviations: AChE, acetylcholinesterase; SIRT1, silent information regulator two homologue 1; MIQ, minimum inhibitory quantity; PVDF, polyvinylidene fluoride. * Corresponding author. Tel.: +86 531 88382012. E-mail address: [email protected] (P. Fan).
disease (Lau and Brodney, 2008). The AChE inhibitory activities of sesquiterpenoids indicated the potential of C. xanthorrhiza in the therapy of Alzheimer’s disease. Discovering effective and safe AChE inhibitors from traditional medicines is an important way to prevent and treat Alzheimer’s disease. Further bioassay-guided investigation on C. xanthorrhiza led to the isolation of nine compounds for the first time, including one new Guaiane-type sesquiterpenoid 1, four of which showed AChE inhibitory activities. To further explore the potential of C. xanthorrhiza against Alzheimer’s disease, the SIRT1 (silent information regulator two homologue 1) promotion activities of the compounds were tested using HEK293 cell lines. SIRT1 is a member of the sirtuin family that possesses NAD+-dependent deacetylase activity and regulates a variety of cellular processes such as energy metabolism, cell-cycle progression and aging (Raghavan and Shah, 2012). SIRT1 is becoming an important drug target for new therapies in the treatment of neurodegenerative diseases (Huber and Superti-Furga, 2011; Braidy et al., 2012; Min et al., 2013). Small molecule SIRT1 activators or compounds that promote its expression could be candidates for the treatment of aging and age-related diseases (Bonda et al., 2011; Karagiannis and Ververis, 2012; Hubbard and Sinclair, 2014).
http://dx.doi.org/10.1016/j.phytol.2015.04.007 1874-3900/ß 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
C. Zhang et al. / Phytochemistry Letters 12 (2015) 215–219
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Table 1 1 H and 13C NMR data (CDCl3, 600 and 150 MHz, respectively) of 1 compared with zedoardiol (11 in Fig. 2, Zhang et al., 2014) (d in ppm, J in Hz). Position
Zedoaraldehyde
dH (multi., J in Hz) 1
Fig. 1. Key HMBCs of compound 1.
2. Results and discussion Compound 1 (Fig. 1) was isolated as a colorless needle crystal (MeOH). The molecular formula was established as C15H16O4 by HR-ESI-MS data ([M + H]+ m/z 261.1120, calcd. 261.1121 for C15H17O4), indicating eight degrees of unsaturation. The IR spectrum (ymax, cm 1) showed characteristic absorption bands for hydroxyl (3427 cm 1) and carbonyl (1763 and 1687 cm 1) groups. The single peak at d9.56 in the 1H NMR spectrum, which correlated with carbon d193.4 in the HSQC spectrum, indicated the presence of an aldehyde group. The 13C NMR spectrum showed 15 carbon signals (Table 1), which were assigned as two methyls (dC 9.2, 14.0), two methylenes (dC 25.8, 29.0), two aliphatic methanes (dC 38.5, 46.2), two olefinic methanes (dC 133.7, 143.1), four olefinic quaternary carbons (dC 124.4, 124.7, 139.6, 154.2), an oxygenated quaternary carbon (dC 84.5) and two carbonyl carbons (dC 193.4, 194.4) using the HSQC data. The degree of unsaturation suggested the presence of three rings in the structure. The above
2a 2b 3a 3b 4 5 6 7 8 9 10 11 12 13 14 15a 15b
3.03 (dt, 9.6, 2.2) 1.97–2.05 (m) 2.68–2.78 (m) 1.44–1.51 (m) 1.97–2.05 (m) 2.68–2.78 (m)
7.22 (d, 2.2)
7.38 2.23 1.07 9.56
(s) (s) (d, 6.7) (s)
Zedoardiol
dc 46.2 25.8 29.0 38.5 84.5 194.4 124.7 154.2 133.7 139.6 124.4 143.1 9.2 14.0 193.4
dH (multi., J in Hz) 3.16 (t, 9.0) 2.00–2.07 (m) 2.11–2.18 (m) 1.50–1.56 (m) 1.91–1.96 (m) 2.59–2.64 (m)
6.73 (s)
7.16 2.23 1.17 4.29 4.40
(s) (s) (d, 6.0) (d, 13.8) (d, 13.8)
dc 48.5 24.1 30.1 39.2 83.7 194.9 120.0 156.3 115.8 143.3 123.0 139.4 9.8 14.4 65.5
NMR data were similar to those of zedoardiol (11 in Fig. 2), a compound previously isolated from C. xanthorrhiza (Zhang et al., 2014). The major difference resulted from the appearance of an aldehyde group and disappearance of an oxygenated methylene (dC 65.5, Table 1) as well as the obvious downfield shift (17.9 ppm) for C-9 and up field shift (3.7 ppm) for C-10, which suggested that the substituent on C-10 in compound 1 was an aldehyde group instead of a hydroxymethylene group. This change was also confirmed by the correlations from H-15 to C-1, C-9 and C-10 as well as that from H-9 to C-15 in the HMBC spectrum. All other HMBC correlations support the deduced structure (Fig. 1). Compound 1 was thus identified as a new guaiane sesquiterpenoid, zedoaraldehyde. The relative configuration of C-4 was defined as
Fig. 2. Structures of compounds 1–11 from C. xanthorrhiza.
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respectively. For compound 8, a 1.27-fold increase in expression was observed. 3. Experimental 3.1. General experimental procedures
Fig. 3. SIRT1 expression in HEK239 cells exposed to compounds 1, 3, 4, 8 and 10 at a concentration of 20 mM for 24 h. C is representative of the control group. SIRT1 protein expression was evaluated using anti-SIRT1 antibodies as described in the Experimental section. One representative western blot from three independent experiments is shown, with b-actin serving as an internal control. Fold induction was calculated by Sirt1/b-actin (% control). Data are presented as the mean S.D. from triplicate independent experiments. The sign * means p < 0.05 versus the control group.
HRESI-MS data were acquired using a Thermofisher Scientific LTQ/Orbitrap XL hybrid mass spectrometer equipped with an electrospray ionization interface. 1D and 2D NMR spectra were acquired at 600 MHz and 150 MHz for 1H and 13C NMR, respectively, on a Bruker-Avance 600 spectrometer in CDCl3, with d in ppm relative to TMS as an internal standard and J in Hz. HPLC separation was performed on an instrument consisting of an Agilent 1200 G1322A degasser, an Agilent 1200 G1311A quaternary pump, an Agilent 1100 G1315D DAD detector, and a ZORBAX SB-C18 5 mm column (9.4 250 mm for semi-preparative and 4.6 250 mm for analysis) (Agilent Technologies). Medium pressure liquid chromatography (MPLC) was carried out on a RP-18 column (15–25 mm; 350 mm 30 mm i.d.) using a slow MeOH–H2O gradient. TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with a 10% sulfuric acid–vanillin solution. 3.2. Reagents and antibodies
shown according to the similarity of the NMR data and same biosynthetic pathway with the previously isolated compound zedoardiol (11) from the same plant material, the relative configuration of which was established using X-ray single-crystal diffraction (Zhang et al., 2014). On the basis of NMR and MS data or by comparison with reference compounds, compounds 2–9 (Fig. 2) were identified as gweicurculactone (2) (Jiang et al., 1989), 13-hydroxygermacrone (3) (Shiobara et al., 1986), germacrone (4) (Yamazaki et al., 1988), gelchomanolide (5) (Kawabata et al., 1981), 8b-hydroxy -isogermafurenolide (6) (Friedrich and Bohlmann, 1988), a-curcumene (7) (Carter et al., 1939), 3-hydroxy-6-methylacetophenone (8) (Kozhevnikova et al., 2003), and dehydro-6-gingerdione (9) (Lee et al., 2011), respectively. All of these compounds were reported for the first time from C. xanthorrhiza Roxb. Among them, 8b-hydroxyisogermafurenolide (6) and 3-hydroxy-6-methylacetophe-none (8) were mentioned as by-products of synthetic reactions (Friedrich and Bohlmann, 1988; Kozhevnikova et al., 2003), but herein are reported for the first time as natural occurring compounds. Dehydro-6-gingerdione (9) was obtained from Zingiber officinale (Wang et al., 2011) but was isolated for the first time from Curcuma species in this work. The AChE inhibitory activities of the isolated compounds were tested using a TLC bioautography assay. The minimum inhibitory quantity (MIQ) values of compounds 1, 3, 4 and 7 were 3, 4, 6 and 1 mg, respectively (Fig. S5 in the supporting data). Compared with the positive control galanthamine (MIQ = 10 ng), an AChE inhibitor approved by FDA, these compounds exhibited moderate AChE inhibitory activities in vitro. Compounds 1, 3, 4, 8 and a previously isolated compound 10 (Fig. 2) were evaluated for their effects on SIRT1 expression in HEK293 cells. Before the test, the cytotoxicities of the compounds at different concentrations (12.5, 25.0, 50.0 and 100.0 mM) were first detected by MTT assays. The results indicated that all of these compounds have no cytotoxicity to HEK293 cells at a final concentration of 100 mM. Then, HEK293 cells were treated with the compounds at a concentration of 20 mM. After incubation for 24 h, the SIRT1 expression level was measured by western blot analysis. As demonstrated in Fig. 3, all of these compounds could increase SIRT1 expression. Sesquiterpenoids 1, 3, 4 and 10 enhanced SIRT1 expressions by 1.37-, 1.71-, 1.73- and 1.55-fold,
Petroleum ether, dichloromethane, ethylacetate, and methanol were purchased from the Fuyu Chemical Reagents Company (Tianjing, China). Methanol (HPLC grade) was from the Siyou Chemical Reagents Company (Tianjin, China), and ethylacetate (HPLC grade) was from the Aladdin Industrial Company (Shanghai, China). Milli-Q water (Millipore, Bedford, MA, USA) was used. Column chromatography: silica gel (Qingdao Haiyang Chemical Co., Ltd, Qingdao, China); ODS-A-HG gel (12 nm, 50 mm,YMC Co., Ltd, Kyoto, Japan); TLC bioautographic assay: silica gel 60 F254 Al sheets (Merck, Darmstadt, Germany). Acetylcholinesterase was from electric eel (EC 3.1.1.7); bovine serum albumin (BSA), Fast Blue B Salt, 1-naphylacetate, the positive reference compound galanthamine, and Tris base were purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modification of Eagle’s medium (DMEM) was bought from Thermo (USA); FBS was purchased from Gibco (USA); the penicillin-streptomycin solution was bought from Ceres (China); phosphate buffered salts (PBS), dimethyl sulfoxide (DMSO) and MTT were bought from Sigma– Aldrich (USA); and the Bradford Protein Assay Kit was purchased from the Beyotime Institute of Biotechnology, China. The SIRT1, actin primary antibody and IgG HRP-conjugated second antibody used for western blotting analysis were purchased from the Cell Signaling Biotechnology Corporation (USA). The polyvinylidene fluoride (PVDF) membrane was bought from Millipore Corporation (USA); and the western ECL substrate kit was obtained from Bio-Rad (USA). 3.3. Plant material Curcuma xanthorrhiza Roxb. rhizome material (origin: Indonesia) was purchased from Dixa, CH-St. Gallen and was identified by Prof. Lan Xiang, Shandong University. A voucher specimen has been stored in the School of Pharmaceutical Sciences, Shandong University, Ji’nan, P.R. China. 3.4. Extraction and isolation The dried and powdered material (5.0 kg) of C. xanthorrhiza was percolated three times with 95% EtOH at r.t. The residue (193.0 g) was suspended with water and then was successively extracted
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with petroleum ether (3 500 ml), AcOEt (3 500 ml), and BuOH (500 ml). The petroleum ether soluble part (33.0 g) was subjected to CC (SiO2, 200–300 mesh, 8 70 cm; petroleum ether–AcOEt 100:0–0:100) to yield 15 fractions (Frs.1–15). Fr.3 (8.61 g) was subjected to repeated AgNO3 CC (SiO2, 200–300 mesh, 98 g; petroleum ether–AcOEt 100:0–0:100) to yield 4 (29 mg); Frs. 6–8 were subjected to RP-CC (MeOH–water 3:7–10:0) and provided 5 fractions (Frs.6–8-RP1-5). First Sephadex-LH-20 (MeOH–H2O) was used for separation and then HPLC was used for further purification. Frs.6–8-RP2 yielded: 1 (3.7 mg) and 8 (5.0 mg), while Frs.6–8-RP3 yielded: 3 (5.2 mg) and 6 (4.0 mg). Frs.9–11 were subjected to RP-CC (MeOH–water 3:7–10:0) to give 3 fractions (Frs. 9–11-RP1-3). Compound 5 (5.2 mg) was obtained by crystallization from Frs. 9–11-RP1. Frs. 9–11-RP2 were subjected to Sephadex-LH-20 (MeOH–H2O) twice and were further purified with HPLC (MeOH–H2O) to yield 2 (4.3 mg) and 7 (5.6 mg). Frs. 9–11-RP3 was treated in the same manner as Frs. 9–11-RP2 to yield 9 (7.7 mg).
150 MHz) d173.7 (C-12), 162.8 (C-7), 132.9 (C-4), 132.5 (C-10), 130.7 (C-1), 126.1 (C-11), 123.7 (C-5), 82.9 (C-8), 47.1 (C-9), 38.4 (C-3), 27.3 (C-6), 25.6 (C-2), 16.8 (C-15), 16.5 (C-14), 8.9 (C-13) (Kawabata et al., 1981).
3.4.1. Zedoaraldehyde (1) Pale yellow oil; [a]D24 42.90 (c 1.0, CH3OH); IR ymax 3427, 2971, 2931, 1763, 1687, 1048 cm 1; For 1H NMR (CDCl3, 600 MHz) and 13 C NMR (CDCl3, 150 MHz) data, see Table 1; HRESIMS m/z 261.1120 ([M + H]+; calcd for C15H17O4, 260.1121).
3.4.7. a-Curcumene (7) Pale yellow oil; 1H NMR (CDCl3, 600 MHz) d7.11 (2H, m, H-3 and H-5), 7.08 (2H, m, H-2 and H-6), 5.10 (1H, t, J = 6 Hz, H-10), 2.66 (1H, m, H-7), 2.33 (3H, s, H-15), 1.88 (2H, m, H-9), 1.69 (3H, s, H-13), 1.58 (2H, m, H-8), 1.54 (3H, s, H-12), 1.22 (3H, s, H-14); 13C NMR (CDCl3, 150 MHz) d144.5 (C-1), 135.2 (C-4), 131.4 (C-11), 128.8 (C-3), 128.8 (C-5), 126.8 (C-2), 126.8 (C-6), 124.5 (C-10), 39.0 (C-7), 38.5 (C-8), 26.2 (C-9), 25.8 (C-13), 22.5 (C-14), 21.0 (C-15), 17.6 (C-12) (Carter et al., 1939).
3.4.2. Gweicurculactone (2) Colorless crystal; 1H NMR (CDCl3, 600 MHz) d6.92 (1H, s, H-6), 6.76 (1H, s, H-9), 3.10 (1H, m, H-4), 2.84 (1H, m, H-2a), 2.72 (1H, m, H-2b), 2.27 (3H, s, H-15), 2.01 (3H, s, H-13), 2.15 (1H, m, H-3a), 1.60 (1H, m, H-3b), 1.33 (3H, d, J = 6 Hz, H-14); 13C NMR (CDCl3, 150 MHz) d170.7 (C-12), 156.6 (C-5), 154.7 (C-8), 146.1 (C-7), 144.2 (C-10), 136.5 (C-1), 117.9 (C-6), 116.3 (C-9), 103.5 (C-11), 43.8 (C-4), 33.6 (C-2), 31.9 (C-3), 24.7 (C-15), 20.0 (C-14), 7.7 (C-13) (Jiang et al., 1989). 3.4.3. 13-Hydroxygermacrone (3) Colorless needle crystal; 1H NMR (CDCl3, 600 MHz) d5.01 (1H, d, J = 12 Hz, H-1), 4.66 (1H, d, J = 12 Hz, H-5), 4.32 (1H, d, J = 12 Hz, H12a), 4.21 (1H, d, J = 12 Hz, H-12b), 3.45 (1H, d, J = 10 Hz, H-9a), 2.97–3.04 (2H, m, H-6), 2.99 (1H, m, H-9b), 2.39 (1H, m, H-2a), 2.20 (1H, m, H-3a), 2.11 (2H, m, H-2b and H-3b), 1.84 (3H, s, H-13), 1.65 (3H, s, H-14), 1.45 (3H, s, H-15); 13C NMR (CDCl3, 150 MHz) d207.2 (C-8), 139.9 (C-11), 135.8 (C-4), 133.1 (C-1), 131.2 (C-7), 126.4 (C-10), 124.9 (C-5), 62.8 (C-12), 55.6 (C-9), 38.0 (C-3), 28.2 (C-6), 24.4 (C-2), 17.8 (C-13), 16.5 (C-14), 15.8 (C-15) (Shiobara et al., 1986). 3.4.4. Germacrone(4) Colorless crystal; 1 H NMR (CDCl 3 , 600 MHz) d 5.01 (1H, d, J = 12 Hz, H-1), 4.73 (1H, d, J = 12 Hz, H-5), 3.43 (1H, d, J = 12 Hz, H-9 a ), 2.97 (2H, m, H-7), 2.87 (1H, m, H-9 b ), 2.38 (1H, m, H-2 a ), 2.18 (1H, m, H-3 a ), 2.10 (2H, m, H-2 b and H-3 b ), 1.80 (3H, s, H-13), 1.73 (3H, s, H-12), 1.65 (3H, s, H-14), 1.46 (3H, s, H-15); 13 C NMR (CDCl 3 , 150 Hz) d 207.8 (C-8), 137.2 (C-11), 135.0 (C-4), 132.7 (C-1), 129.4 (C-7), 126.7 (C-10), 125.3 (C-5), 55.8 (C-9), 38.0 (C-3), 29.3 (C-6), 23.9 (C-2), 22.2 (C-13), 19.8 (C-12), 16.7 (C-14), 15.4 (C-15) (Yamazaki et al., 1988). 3.4.5. Gelchomanolide (5) Colorless crystal; 1H NMR (CDCl3, 600 MHz) d4.97 (1H, brd, J = 12 Hz, H-8), 4.86 (1H, brd, J = 12 Hz, H-1), 4.34 (1H, brd, J = 6 Hz, H-5), 3.41 (1H, d, J = 12 Hz, H-6a), 3.10 (1H, brd, J = 12 Hz, H-9a), 2.90 (1H, d, J = 12 Hz, H-6b), 2.19 (1H, m, H-2a), 2.11 (2H, m, H-2b and H-9b), 2.24 (1H, m, H-3a), 1.86 (1H, m, H-3b), 1.87 (3H, s, H-13), 1.62 (3H, s, H-14), 1.51 (3H, s, H-15); 13C NMR (CDCl3,
3.4.6. 8b-Hydroxy-isogermafurenolide (6) Colorless needle crystal; 1H NMR (CDCl3, 600 MHz) d5.70 (1H, dd, J = 18 Hz and 12 Hz, H-1), 4.99 (1H,d, J = 18 Hz, H-2a ), 5.01 (1H, d, J = 12 Hz, H-2b), 4.75 (1H, br s, H-3a), 5.02 (1H, br s, H-3b), 2.58 (1H, dd, J = 12 Hz and 3.5 Hz, H-6a ), 2.73 (1H, ddd, J = 12 Hz, 3.5 Hz and 1.3 Hz, H-6b), 2.06 (1H, dd, J = 12 Hz and 3.5 Hz, H-5), 2.14 (1H, d, J = 12 Hz, H-9a ), 1.77 (1H, d, J = 12 Hz, H-9b), 1.86 (3H, d, J = 1.3 Hz, H-13), 1.79 (3H, s, H-15), 1.29 (3H, s, H-14); 13C NMR (CDCl3, 150 MHz) d171.2 (C-12), 160.1 (C-8), 147.4 (C-1), 144.6 (C-4), 122.0 (C-11), 114.1 (C-3), 112.1 (C-2), 102.7 (C-7), 54.1 (C-5), 49.2 (C-9), 40.6 (C-10), 27.1 (C-6), 24.4 (C-15), 17.8 (C-14), 8.2 (C-13) (Friedrich and Bohlmann, 1988).
3.4.8. 3-Hydroxy-6-methylacetophenone (8) Colorless needle crystal; 1H NMR (CDCl3, 600 MHz) d7.52 (1H, s, H-2), 7.45 (1H, d, J = 6 Hz, H-5), 7.23 (1H, d, J = 6 Hz, H-6), 2.60 (3H, s, CH3), 2.34 (3H, s, Ph-CH3); 13C NMR (CDCl3, 150 MHz) d198.4 (C5 5O), 154.0 (C-1), 136.5 (C-4), 131.0 (C-3), 130.9 (C-6), 121.2 (C-5), 113.8 (C-2), 26.4 (Me), 16.0 (Ph- Me) (Kozhevnikova et al., 2003). 3.4.9. Dehydro-6-gingerdione (9) Yellow powder; 1H NMR (CDCl3, 600 MHz) d7.56 (1H, d, J = 12 Hz, H-7), 7.11 (1H, dd, J = 6 Hz and 18 Hz, H-6), 7.04 (1H, d, J = 18 Hz, H-2), 6.95 (1H, d, J = 6 Hz, H-5), 6.36 (1H, d, J= 12 Hz, H8), 5.85 (1H, s, OH), 5.64 (2H, s, H-10), 3.96 (3H, s, OMe), 2.38 (2H, t, J = 6 Hz, H-12), 1.68 (2H, m, H-13), 0.99 (3H, t, J = 6 Hz, H-14); 13 C NMR (CDCl3, 150 MHz) d200.0 (C-11), 178.2 (C-9), 147.6 (C-4), 146.8 (C-3), 139.8 (C-7), 127.9 (C-1), 122.6 (C-6), 120.5 (C-8), 114.7 (C-5), 109.4 (C-2), 100.5 (C-10), 55.9 (OMe), 42.0 (C-12), 19.0 (C-13), 13.9 (C-14) (Lee et al., 2011). 3.5. AChE activity assay The AChE inhibitory activities of the isolated compounds were determined using a TLC bioautography assay modified from a previous method (Fan et al., 2008). Briefly, the TLC plate with samples was sprayed with enzyme solution (1.5 U/mL in Tris– hydrochloric acid buffer at a pH 7.2) and dried. Then, the plate was sprayed with 1-naphthyl acetate solution (1.5 mg/mL in 40% EtOH) and dried again, followed by incubation at 37 8C for 20 min. For detection, a Fast Blue B Salt solution (0.5 mg/mL in water) was used to soak the plate for 20 s, and white zones on a purple background on the plate indicated the inhibitory activities of the samples. To ascertain detection limits of the active compounds, different volumes of 2.0 mg/mL solutions were applied onto the TLC plates, and the amount that produced the least visible white spot was noted as the minimum inhibitory quantity (MIQ). Galantamine (0.05 mg/mL) was used as a positive control.
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3.6. Cytotoxicity assay (MTT assay)
References
Cytotoxicities of compounds 1, 3, 4, 8 and 10 were assessed by 3-(4, 5-dimethylthiozol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. HEK293 cells (5 103 cells/well) were incubated in 96-well microtiter plates for 24 h. After the addition of different concentrations (12.5, 25.0, 50.0 and 100.0 mM) of the test compounds or only medium, the plates were incubated for an additional 24 h. Three replicate wells were set for each concentration in the experiments. Then, 20 mL of MTT solution (final concentration of 5 mg/mL, freshly diluted in PBS before treatment) was added to each well and incubated for another 4 h. The medium was abandoned, and 150 mL/well DMSO was added. The amount of formazan was determined by measuring the absorbance at 490 nm with a microplate reader (Bio-Rad, USA).
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3.7. Western blot analysis HEK293 cells were plated in 6-well plates at a density of 5 105 cells/well and grown at 37 8C. Cells were treated with compounds at a final concentration of 20 mM for 24 h. Then, cells were harvested, lysed, centrifuged. The supernatants were utilized for protein analysis and western blot analysis. The total protein concentration was determined using a Bradford Protein Assay Kit. Protein samples were denatured, and final stored at 80 8C for western blotting. Equal amounts of protein (30 mg) from cell lysates were loaded onto a 10% SDS-polyacrylamide gel and separated by electrophoresis. b-actin served as an internal control. Then, the separated proteins were electrophoretically transferred to a 0.45 mm PVDF membrane. The transferred PVDF membranes were incubated with anti-SIRT1 primary antibodies (1:1000) at 4 8C overnight. Bound antibodies were detected using a horseradish peroxidase-linked secondary antibody. Statistical analysis: All results were confirmed from a minimum of three independent experiments on replicate samples. Data are represented as the mean standard deviation (SD). Statistical significances were evaluated by the t-test. Data were considered significant at P < 0.05. Acknowledgements The authors thank the National Natural Science Foundation of China (grant no. 81473323, 81001616) and the China Postdoctoral Science Foundation (2014M551926) for their financial support of this work.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2015. 04.007.