J Nat Med DOI 10.1007/s11418-015-0919-3
NOTE
Phenolic compounds from the bark of Oroxylum indicum activate the Ngn2 promoter Rolly G. Fuentes1,4 • Midori A. Arai1 • Samir K. Sadhu2 • Firoj Ahmed3 Masami Ishibashi1
•
Received: 14 April 2015 / Accepted: 9 May 2015 Ó The Japanese Society of Pharmacognosy and Springer Japan 2015
Abstract A reporter gene assay that detects neurogenin 2 (Ngn2) promoter activity was utilized to identify compounds that induce neuronal differentiation. Ngn2 is a basic helix-loop-helix transcription factor that activates transcription of pro-neural genes. Using this assay system and an activity-guided approach, seven phenolic compounds were isolated from the methanol extract of Oroxylum indicum: 1 oroxylin A, 2 chrysin, 3 hispidulin, 4 baicalein, 5 apigenin, 6 baicalin, and 7 isoverbascoside. Compounds 1 and 2 induced an estimated 2.7-fold increase in Ngn2 promoter activity, whereas 3 increased the activity by 2.5fold. Furthermore, 1 and 2 enhanced neuronal differentiation of C17.2 cells, which are multipotent stem cells.
Graphical Abstract
Keywords
bHLH Flavonoids Neurite
Introduction
& Midori A. Arai [email protected] & Masami Ishibashi [email protected] 1
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
2
Pharmacy Discipline, Life Science School, Khulna University, Khulna 9208, Bangladesh
3
Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka 1000, Bangladesh
4
University of the Philippines Visayas Tacloban College, Tacloban City, Philippines
Neural stem cells are multipotent cells that have the ability to self-renew and differentiate into neurons, astrocytes, and glial cells [1]. The formation of new neurons, known as neurogenesis, is regulated by several signal-mediated reactions in which basic helix-loop-helix (bHLH) factors play a critical role [2–5]. Differentiation of stem cells into neurons is induced by bHLH activators such as neurogenin 2 (Ngn2) and mammalian achaete-scute homolog 1 (Mash1). However, their pro-neural function is inhibited by hairy and enhancer of split (Hes) factors such as Hes1, Hes3, and Hes5 [2]. In undifferentiated cells, the expression of Hes1 and Ngn2 oscillates. During this oscillation, Ngn2 expression is not sufficient to induce neural differentiation [6], and neurogenesis is induced only when the expression of Ngn2 is sustained [7]. Studies on neurogenesis are of particular interest because recent studies have shown that neurogenesis occurs not only in the developing brain but also in
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J Nat Med
Fig. 1 Reporter cell construct of the Ngn2 promoter luciferase-based assay system
the adult mammalian brain, suggesting the possibility for development of new strategies in regenerative medicine and treatment for neurodegenerative diseases. Several compounds have been reported to induce neuronal differentiation. These compounds increase the expression of some bHLH activators and decrease Hes1 expression [8–12]. Our group previously reported an assay system in which we stably transfected C3H10T1/2 cells with a luciferase reporter under control of the Ngn2 promoter (Fig. 1) [13]. With this assay, compounds that increase the activity of the Ngn2 promoter are considered candidate compounds that may induce neuronal differentiation. Here, we report our continued efforts in the identification of compounds that activate the Ngn2 promoter activity. Our screening process using this assay showed that the methanolic extract of Oroxylum indicum (Bignoniaceae) increases the activity of the Ngn2 promoter. Activityguided isolation of compounds from O. indicum resulted in isolation of six flavonoids: 1 oroxylin A, 2 chrysin, 3 hispidulin, 4 baicalein, 5 apigenin, 6 baicalin, and one phenylethanoid glycoside: 7 isoverbascoside (Fig. 2).
Results and discussion Ngn2 promoter activity was evaluated using our previously reported cell-based luciferase assay system [13], which utilizes C3H10T1/2 cells that are stably transfected with pGL4.20 containing an Ngn2 promoter. Using this assay system in our screening study, the bark extract of O. indicum activated Ngn2 promoter activity. The crude methanol extract of O. indicum (100 lg/mL) induced a two-fold increase. We then isolated the active constituents of O. indicum using an activity-guided approach. The methanol extract of O. indicum (13.9 g) was suspended in H2O:MeOH (9:1) and was partitioned successively with hexane, ethyl acetate, and butanol, leaving the residual water extract. Of these extracts, the hexane fraction (3.9 g) showed potent activity as it increased the relative Ngn2 promoter activity by 2.5-fold at 50 lg/mL. Fractionation of the hexane extract using chromatographic techniques [silica, octadecylbonded silica (ODS), and high-performance liquid chromatography (HPLC)] led to the isolation of oroxylin A (1) [14], chrysin (2) [15], hispidulin (3) [16], baicalein (4) [17], and apigenin (5) [18] (Fig. 2). The butanol fraction (2.9 g), which increased Ngn2 promoter activity by 1.5-
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Fig. 2 Structures of compounds 1–7
fold, was subjected to ODS column chromatography followed by HPLC and yielded baicalin (6) [19] and isoverbascoside (7) [20]. The identities of these compounds were determined by comparing their spectral values with values from the literature. The NMR spectroscopic values of 1 and 2 are shown in Table 1. Isolated compounds 1–6 were tested for Ngn2 promoter activity (Fig. 3). We did not determine the activity of 7 because it was isolated from an inactive fraction. We also determined the effect of the compounds on the viability of the reporter cells using the fluorometric microculture cytotoxicity assay [21], as the number of reporter cells affects luciferase activity measurement. All compounds induced Ngn2 promoter activity in a concentration-dependent manner, but their activity reached a plateau at their respective highest concentration. Compounds 1–3 had the strongest activity among the isolated compounds. At 10 lM, 1 and 2 increased Ngn2 promoter activity by 2.7fold, whereas 3 increased activity by 2.5-fold. Compound 4 showed a 2-fold increase in Ngn2 promoter activity at 10 lM, and 5 resulted in a 1.7-fold increase in activity at
J Nat Med
Table 1 NMR spectroscopic data for compounds 1 and 2 (DMSO-d6, 400 MHz) Position
1 1
H (d, ppm; J, Hz)
2 13
C (d, ppm)
1
H (d, ppm; J, Hz)
1 2 3 4
163.2 5.61 (1H, s)
5
104.8 182.2 152.6
6
94.4
7
157.8
8
6.96 (1H, s)
9
130.8
6.21 (1H, d, 2.4) 6.52 (1H, d, 2.4)
152.7
10
104.4
10
131.5
20
6.96 (1H, s)
8.06 (1H, d, 6.8)
126.4
8.05–8.07 (m)
0
3
7.62–7.55 (m)
129.1
7.54–7.63 (m)
40
7.62–7.55 (m)
132.0
7.54–7.63 (m)
50
7.62–7.55 (m)
129.1
7.54–7.63 (m)
60
8.06 (1H, d, 6.8)
126.4
8.05–8.07 (m)
6-OMe
3.75 (3H, s)
5-OH
12.92 (1H, s)
59.9 12.82 (1H, s)
15 lM. Interestingly, we were able to isolate 6, which exhibited a 1.9-fold increase in Ngn2 promoter activity at 50 lM. Compound 6 was reported previously to induce neuronal differentiation and increase gene expression of Ngn2 in C17.2 neural stem cells [9]. All compounds tested exhibited no cytotoxicity against the reporter assay cells. Adult neurogenesis is regulated by various factors such as cell intrinsic programs and environmental factors [22]. Thus, the fate of neural stem cells can be manipulated by regulating the pathway that is involved in neuronal fate specification. Ngn2 is a bHLH factor that promotes neuronal differentiation [3, 5, 7]. Using our cell-based luciferase assay system, we showed that 1 and 2 enhanced Ngn2 promoter activity, and we then determined their neuronal differentiation activity in C17.2 cells (Fig. 4), which are murine multipotent stem cells. Compound 1 induces adult neurogenesis in the hippocampal dentate gyrus region in mice [23]. On the other hand, 2 possesses neuroprotective activity [24] but, to the best of our knowledge, its neuronal differentiation activity has not been demonstrated. C17.2 cells were treated with either 1 or 2 at 20 lM for 7 days. Treated cells had dense, rounded cell bodies and a network of neurites on top of flat cells. Untreated control cells had some dense cell bodies on top of flat cells but showed no elongated neurites. The cells with dense, rounded cell bodies and extended neurites were presumed to be neurons based on
the morphological description of a previous study [25]. The results suggested that 1 and 2 enhanced neurite growth in C17.2 cells. Induction of the Ngn2 promoter by 1 and 2 may have participated in the stimulation of the neurite outgrowth in C17.2 cells. Overexpression of neurogenin in F11 neuroblastoma cells induces neurite outgrowth [26]. Our study did not show whether 1 and 2 promoted neuronal differentiation of C17.2 cells because the differentiation conditions used in this study do not favor astroglial cell formation. In conclusion, using the cell-based luciferase assay system, six flavonoids were identified that increased Ngn2 promoter activity. Oroxylin A and chrysin induced differentiated neurons with long neurites from C17.2 cells. Our study demonstrated that screening of compounds that regulate bHLH factors represents a good strategy to identify compounds with neuronal differentiation activity.
Experimental Plant materials The plant sample, O. indicum (bark), was collected from Bangladesh. A voucher specimen (KKB085) was deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Cell culture The Ngn2 reporter assay cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) with 10 % fetal bovine serum. C17.2 cells were purchased from DS Pharma Biomedical (Osaka, Japan) and were grown in proliferation medium consisting of DMEM (DS Pharma Biomedical, Osaka, Japan) supplemented with 10 % fetal bovine serum (BioWest, Nuaille´, France), 5 % horse serum (Gibco, Life Technologies, Tokyo, Japan), 2 mM glutamine, and 1 % antibiotic–antimycotic (Gibco). Cell cultures were incubated at 37 °C in 5 % CO2/95 % air. Activity-guided isolation of the constituents The dried bark of O. indicum (336 g) was extracted with MeOH to give a crude extract (13.9 g). The extract was resuspended in 10 % MeOH (300 mL) and partitioned with hexane, EtOAc, and BuOH (300 mL 9 3). The hexane fraction (3.9 g) was then subjected to silica gel column chromatography (u25 9 330 mm) and eluted with CHCl3:MeOH (0:1–1:1) to give nine fractions (1A–1I). Fraction 1C (40.3 mg), which was eluted with 100 %
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J Nat Med Fig. 3 Ngn2 promoter activity (bars) of compounds 1–6. Line graphs show the viability of the reporter cells when treated with 1–6. Data are from one experiment and are representative of two independent experiments (mean ± SD)
a
b
c
Fig. 4 Morphology of C17.2 cells after a 7-day treatment with a DMSO, b 1, or c 2. Phase-contrast images were obtained using an Olympus D72. Arrows Cells with dense, rounded cell bodies and elongated neurites
CHCl3, was subjected to ODS column chromatography (u15 9 250 mm) and eluted with H2O:MeOH (1:1–0:1) to yield 1 (13.4 mg). Fraction 1E (323 mg), which was eluted with 95:5 (CHCl3:MeOH), was subjected to silica gel
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column chromatography (u25 9 280 mm) and eluted with CHCl3:MeOH (1:0–0:1) to yield 1 (10.8 mg), 2 (6.8 mg), 3 (15.4 mg), and 4 (58.1 mg). Fraction 1F (1161.1 mg), which was eluted with 95:5 (CHCl3:MeOH), was subjected
J Nat Med
to ODS column chromatography (u20 9 270 mm) using H2O:MeOH (1:1–0:1) as the eluent to yield ten fractions (6A–6J). The MeOH-soluble fraction of 6E (11.0 mg) was then further purified with HPLC (Cosmosil 5C18-AR-II, u10 9 250 mm; eluent, 45 % MeCN; flow rate, 1.5 ml min-1) to obtain 5 (tR = 18.8 min; 1.4 mg). The BuOH fraction (2.8 g) was then subjected to ODS column chromatography (u30 9 270 mm) with H2O:MeOH (9:1–1:1) to yield fractions 8A-8I. Fraction 8D (92 mg), which was eluted with 20 % MeOH, was subjected to HPLC (Cosmosil 5C18-AR-II, u10 9 250 mm; eluent, 30 % MeOH; flow rate, 2 ml min-1) to yield 6 (tR = 16 min; 30.8 mg). Fraction 8G (30.2 mg), which was eluted with 40 % MeOH, was further purified with ODS chromatography (u30 9 120 mm) using 50 % MeOH to yield compound 7 (9.0 mg). Luciferase assay The Ngn2 promoter activity assay was conducted as described previously [13]. C3H10T1/2 cells (1 9 104) stably transfected with a plasmid containing the Ngn2 promoter were seeded in a 96-well plate. After 24 h of incubation, different concentrations of compounds were added and incubated for 24 h. Then, cells were lysed with Glo Lysis Buffer. Luciferase activity was measured using the BrightGlo Luciferase Assay System (Promega, Tokyo, Japan) according to the manufacturer’s recommendations. Relative Ngn2 promoter activity of the control (DMSO only) was defined as 100 %, and the activity of the other treatments was calculated accordingly. Cell proliferation assay Cell viability was determined using the fluorometric microculture cytotoxicity assay [21]. Reporter cells (1 9 104) were inoculated into 96-well plates for 24 h. Different concentrations of the compounds were then added and incubated for another 24 h. After incubation, fluorescein diacetate (Wako, Japan) in PBS buffer (10 lg/mL) was added, and fluorescence was detected after 1 h of incubation using Luminoskan (Ascent, http://www.thermoscientific.com). Neuronal differentiation experiment In brief, C17.2 cells (2 9 105) in a proliferation medium were seeded in a poly-L-lysine-coated 10-cm dish (Wako) and incubated for 24 h at 37 °C. Then, the medium was removed, and the cells were washed with DMEM only. The cells were then treated with the compounds prepared in differentiation medium [Neurobasal medium (Gibco) supplemented with 1 mM glutamine, 2 % B-27 without vitamin A (Gibco), and 1 % antibiotic–antimycotic (Gibco)]
for 7 days. Cell images were obtained under phase contrast using an Olympus DP72 (Olympus, Osaka, Japan). Acknowledgments We thank Prof. Dr. R. Kageyama for the Ngn2 plasmid. This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS).
References 1. Merkle FT, Alvarez-Buylla A (2006) Neural stem cells in mammalian development. Curr Opin Cell Biol 18:704–709 2. Kageyama R, Ohtsuka T, Hatakeyama J, Ohsawa R (2005) Roles of bHLH genes in neural stem cell differentiation. Exp Cell Res 306:343–348 3. Powell LM, Jarman AP (2008) Context dependence of proneural bHLH proteins. Curr Opin Genet Dev 18:411–417 4. Imayoshi I, Isomura A, Harima Y, Kawaguchi K, Kori H, Miyachi H, Fujiwara T, Ishidate F, Kageyama R (2013) Oscillatory control of factors determining multipotency and fate in mouse neural progenitors. Science 342:1203–1208 5. Imayoshi I, Kageyama R (2014) bHLH factors in self-renewal, multipotency, and fate choice of neural progenitor cells. Neuron 82:9–23 6. Shimojo H, Ohtsuka T, Kageyama R (2008) Oscillations in notch signaling regulate maintenance of neural progenitors. Neuron 58:52–64 7. Shimojo H, Ohtsuka T (2011) Kageyama R (2011) Dynamic expression of notch signaling genes in neural stem/progenitor cells. Front Neurosci 5:78 8. Katakura M, Hashimoto M, Shahdat HM, Gamoh S, Okui T, Matsuzaki K, Shido O (2009) Docosahexaenoic acid promotes neuronal differentiation by regulating basic helix-loop-helix transcription factors and cell cycle in neural stem cells. Neuroscience 160:651–660 9. Li M, Tsang KS, Choi ST, Li K, Shaw PC, Lau KF (2011) Neuronal differentiation of C17.2 neural stem cells induced by a natural flavonoid, baicalin. ChemBioChem 12:449–456 10. Takahashi J, Palmer TD, Gage FH (1999) Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adultderived neural stem cell cultures. J Neurobiol 38:65–81 11. Hsieh J, Nakashima K, Kuwabara T, Mejia E, Gage FH (2004) Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc Natl Acad Sci USA 101:16659–16664 12. Warashina M, Min KH, Kuwabara T, Huynh A, Gage FH, Schultz PG, Ding S (2006) A synthetic small molecule that induces neuronal differentiation of adult hippocampal neural progenitor cells. Angew Chem Int Ed Engl 45:591–593 13. Arai MA, Koryudzu K, Koyano T, Kowithayakorn T, Ishibashi M (2013) Naturally occurring Ngn2 promoter activators from Butea superba. Mol BioSyst 9:2489–2497 14. Kim DH, Jeon SJ, Son KH, Jung JW, Lee S, Yoon BH, Lee JJ, Cho YW, Cheong JH, Ko KH, Ryu JH (2007) The ameliorating effect of oroxylin A on scopolamine-induced memory impairment in mice. Neurobiol Learn Mem 87:536–546 15. Zeng YB, Yang N, Liu WS, Tang N (2003) Synthesis, characterization and DNA-binding properties of La(III) complex of chrysin. J Inorg Biochem 97:258–264 16. Nazaruk J, Gudej J (2003) Flavonoid compounds from the flowers of Cirsium rivulare (Jacq). Acta Pol Pharm 60:87–89 17. Pegg RB, Amarowicz R, Oszmian´ski J (2005) Confirming the chemical structure of antioxidative trihydroxyflavones from Scutellaria baicalensis using modern spectroscopic methods. Pol J Food Nutr Sci 14(55):43–50
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neurogenesis in the hippocampal dentate gyrus region of mice. Neurochem Res 35:1725–1732 24. Izuta H, Shimazawa M, Tazawa S, Araki Y, Mishima S, Hara H (2008) Protective effects of Chinese propolis and its component, chrysin, against neuronal cell death via inhibition of mitochondrial apoptosis pathway in SH-SY5Y cells. J Agric Food Chem 56:8944–8953 25. Lundqvist J, El Andaloussi-Lilja J, Svensson C, Gustafsson Dorfh H, Forsby A (2013) Optimisation of culture conditions for differentiation of C17.2 neural stem cells to be used for in vitro toxicity tests. Toxicol In Vitro 27:1565–1569 26. Kim S, Ghil SH, Kim SS, Myeong HH, Lee YD, Suh-Kim H (2002) Overexpression of neurogenin1 induces neurite outgrowth in F11 neuroblastoma cells. Exp Mol Med 34:469–475
NOTE
Phenolic compounds from the bark of Oroxylum indicum activate the Ngn2 promoter Rolly G. Fuentes1,4 • Midori A. Arai1 • Samir K. Sadhu2 • Firoj Ahmed3 Masami Ishibashi1
•
Received: 14 April 2015 / Accepted: 9 May 2015 Ó The Japanese Society of Pharmacognosy and Springer Japan 2015
Abstract A reporter gene assay that detects neurogenin 2 (Ngn2) promoter activity was utilized to identify compounds that induce neuronal differentiation. Ngn2 is a basic helix-loop-helix transcription factor that activates transcription of pro-neural genes. Using this assay system and an activity-guided approach, seven phenolic compounds were isolated from the methanol extract of Oroxylum indicum: 1 oroxylin A, 2 chrysin, 3 hispidulin, 4 baicalein, 5 apigenin, 6 baicalin, and 7 isoverbascoside. Compounds 1 and 2 induced an estimated 2.7-fold increase in Ngn2 promoter activity, whereas 3 increased the activity by 2.5fold. Furthermore, 1 and 2 enhanced neuronal differentiation of C17.2 cells, which are multipotent stem cells.
Graphical Abstract
Keywords
bHLH Flavonoids Neurite
Introduction
& Midori A. Arai [email protected] & Masami Ishibashi [email protected] 1
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
2
Pharmacy Discipline, Life Science School, Khulna University, Khulna 9208, Bangladesh
3
Department of Pharmaceutical Chemistry, University of Dhaka, Dhaka 1000, Bangladesh
4
University of the Philippines Visayas Tacloban College, Tacloban City, Philippines
Neural stem cells are multipotent cells that have the ability to self-renew and differentiate into neurons, astrocytes, and glial cells [1]. The formation of new neurons, known as neurogenesis, is regulated by several signal-mediated reactions in which basic helix-loop-helix (bHLH) factors play a critical role [2–5]. Differentiation of stem cells into neurons is induced by bHLH activators such as neurogenin 2 (Ngn2) and mammalian achaete-scute homolog 1 (Mash1). However, their pro-neural function is inhibited by hairy and enhancer of split (Hes) factors such as Hes1, Hes3, and Hes5 [2]. In undifferentiated cells, the expression of Hes1 and Ngn2 oscillates. During this oscillation, Ngn2 expression is not sufficient to induce neural differentiation [6], and neurogenesis is induced only when the expression of Ngn2 is sustained [7]. Studies on neurogenesis are of particular interest because recent studies have shown that neurogenesis occurs not only in the developing brain but also in
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J Nat Med
Fig. 1 Reporter cell construct of the Ngn2 promoter luciferase-based assay system
the adult mammalian brain, suggesting the possibility for development of new strategies in regenerative medicine and treatment for neurodegenerative diseases. Several compounds have been reported to induce neuronal differentiation. These compounds increase the expression of some bHLH activators and decrease Hes1 expression [8–12]. Our group previously reported an assay system in which we stably transfected C3H10T1/2 cells with a luciferase reporter under control of the Ngn2 promoter (Fig. 1) [13]. With this assay, compounds that increase the activity of the Ngn2 promoter are considered candidate compounds that may induce neuronal differentiation. Here, we report our continued efforts in the identification of compounds that activate the Ngn2 promoter activity. Our screening process using this assay showed that the methanolic extract of Oroxylum indicum (Bignoniaceae) increases the activity of the Ngn2 promoter. Activityguided isolation of compounds from O. indicum resulted in isolation of six flavonoids: 1 oroxylin A, 2 chrysin, 3 hispidulin, 4 baicalein, 5 apigenin, 6 baicalin, and one phenylethanoid glycoside: 7 isoverbascoside (Fig. 2).
Results and discussion Ngn2 promoter activity was evaluated using our previously reported cell-based luciferase assay system [13], which utilizes C3H10T1/2 cells that are stably transfected with pGL4.20 containing an Ngn2 promoter. Using this assay system in our screening study, the bark extract of O. indicum activated Ngn2 promoter activity. The crude methanol extract of O. indicum (100 lg/mL) induced a two-fold increase. We then isolated the active constituents of O. indicum using an activity-guided approach. The methanol extract of O. indicum (13.9 g) was suspended in H2O:MeOH (9:1) and was partitioned successively with hexane, ethyl acetate, and butanol, leaving the residual water extract. Of these extracts, the hexane fraction (3.9 g) showed potent activity as it increased the relative Ngn2 promoter activity by 2.5-fold at 50 lg/mL. Fractionation of the hexane extract using chromatographic techniques [silica, octadecylbonded silica (ODS), and high-performance liquid chromatography (HPLC)] led to the isolation of oroxylin A (1) [14], chrysin (2) [15], hispidulin (3) [16], baicalein (4) [17], and apigenin (5) [18] (Fig. 2). The butanol fraction (2.9 g), which increased Ngn2 promoter activity by 1.5-
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Fig. 2 Structures of compounds 1–7
fold, was subjected to ODS column chromatography followed by HPLC and yielded baicalin (6) [19] and isoverbascoside (7) [20]. The identities of these compounds were determined by comparing their spectral values with values from the literature. The NMR spectroscopic values of 1 and 2 are shown in Table 1. Isolated compounds 1–6 were tested for Ngn2 promoter activity (Fig. 3). We did not determine the activity of 7 because it was isolated from an inactive fraction. We also determined the effect of the compounds on the viability of the reporter cells using the fluorometric microculture cytotoxicity assay [21], as the number of reporter cells affects luciferase activity measurement. All compounds induced Ngn2 promoter activity in a concentration-dependent manner, but their activity reached a plateau at their respective highest concentration. Compounds 1–3 had the strongest activity among the isolated compounds. At 10 lM, 1 and 2 increased Ngn2 promoter activity by 2.7fold, whereas 3 increased activity by 2.5-fold. Compound 4 showed a 2-fold increase in Ngn2 promoter activity at 10 lM, and 5 resulted in a 1.7-fold increase in activity at
J Nat Med
Table 1 NMR spectroscopic data for compounds 1 and 2 (DMSO-d6, 400 MHz) Position
1 1
H (d, ppm; J, Hz)
2 13
C (d, ppm)
1
H (d, ppm; J, Hz)
1 2 3 4
163.2 5.61 (1H, s)
5
104.8 182.2 152.6
6
94.4
7
157.8
8
6.96 (1H, s)
9
130.8
6.21 (1H, d, 2.4) 6.52 (1H, d, 2.4)
152.7
10
104.4
10
131.5
20
6.96 (1H, s)
8.06 (1H, d, 6.8)
126.4
8.05–8.07 (m)
0
3
7.62–7.55 (m)
129.1
7.54–7.63 (m)
40
7.62–7.55 (m)
132.0
7.54–7.63 (m)
50
7.62–7.55 (m)
129.1
7.54–7.63 (m)
60
8.06 (1H, d, 6.8)
126.4
8.05–8.07 (m)
6-OMe
3.75 (3H, s)
5-OH
12.92 (1H, s)
59.9 12.82 (1H, s)
15 lM. Interestingly, we were able to isolate 6, which exhibited a 1.9-fold increase in Ngn2 promoter activity at 50 lM. Compound 6 was reported previously to induce neuronal differentiation and increase gene expression of Ngn2 in C17.2 neural stem cells [9]. All compounds tested exhibited no cytotoxicity against the reporter assay cells. Adult neurogenesis is regulated by various factors such as cell intrinsic programs and environmental factors [22]. Thus, the fate of neural stem cells can be manipulated by regulating the pathway that is involved in neuronal fate specification. Ngn2 is a bHLH factor that promotes neuronal differentiation [3, 5, 7]. Using our cell-based luciferase assay system, we showed that 1 and 2 enhanced Ngn2 promoter activity, and we then determined their neuronal differentiation activity in C17.2 cells (Fig. 4), which are murine multipotent stem cells. Compound 1 induces adult neurogenesis in the hippocampal dentate gyrus region in mice [23]. On the other hand, 2 possesses neuroprotective activity [24] but, to the best of our knowledge, its neuronal differentiation activity has not been demonstrated. C17.2 cells were treated with either 1 or 2 at 20 lM for 7 days. Treated cells had dense, rounded cell bodies and a network of neurites on top of flat cells. Untreated control cells had some dense cell bodies on top of flat cells but showed no elongated neurites. The cells with dense, rounded cell bodies and extended neurites were presumed to be neurons based on
the morphological description of a previous study [25]. The results suggested that 1 and 2 enhanced neurite growth in C17.2 cells. Induction of the Ngn2 promoter by 1 and 2 may have participated in the stimulation of the neurite outgrowth in C17.2 cells. Overexpression of neurogenin in F11 neuroblastoma cells induces neurite outgrowth [26]. Our study did not show whether 1 and 2 promoted neuronal differentiation of C17.2 cells because the differentiation conditions used in this study do not favor astroglial cell formation. In conclusion, using the cell-based luciferase assay system, six flavonoids were identified that increased Ngn2 promoter activity. Oroxylin A and chrysin induced differentiated neurons with long neurites from C17.2 cells. Our study demonstrated that screening of compounds that regulate bHLH factors represents a good strategy to identify compounds with neuronal differentiation activity.
Experimental Plant materials The plant sample, O. indicum (bark), was collected from Bangladesh. A voucher specimen (KKB085) was deposited at the Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Japan. Cell culture The Ngn2 reporter assay cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako, Osaka, Japan) with 10 % fetal bovine serum. C17.2 cells were purchased from DS Pharma Biomedical (Osaka, Japan) and were grown in proliferation medium consisting of DMEM (DS Pharma Biomedical, Osaka, Japan) supplemented with 10 % fetal bovine serum (BioWest, Nuaille´, France), 5 % horse serum (Gibco, Life Technologies, Tokyo, Japan), 2 mM glutamine, and 1 % antibiotic–antimycotic (Gibco). Cell cultures were incubated at 37 °C in 5 % CO2/95 % air. Activity-guided isolation of the constituents The dried bark of O. indicum (336 g) was extracted with MeOH to give a crude extract (13.9 g). The extract was resuspended in 10 % MeOH (300 mL) and partitioned with hexane, EtOAc, and BuOH (300 mL 9 3). The hexane fraction (3.9 g) was then subjected to silica gel column chromatography (u25 9 330 mm) and eluted with CHCl3:MeOH (0:1–1:1) to give nine fractions (1A–1I). Fraction 1C (40.3 mg), which was eluted with 100 %
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J Nat Med Fig. 3 Ngn2 promoter activity (bars) of compounds 1–6. Line graphs show the viability of the reporter cells when treated with 1–6. Data are from one experiment and are representative of two independent experiments (mean ± SD)
a
b
c
Fig. 4 Morphology of C17.2 cells after a 7-day treatment with a DMSO, b 1, or c 2. Phase-contrast images were obtained using an Olympus D72. Arrows Cells with dense, rounded cell bodies and elongated neurites
CHCl3, was subjected to ODS column chromatography (u15 9 250 mm) and eluted with H2O:MeOH (1:1–0:1) to yield 1 (13.4 mg). Fraction 1E (323 mg), which was eluted with 95:5 (CHCl3:MeOH), was subjected to silica gel
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column chromatography (u25 9 280 mm) and eluted with CHCl3:MeOH (1:0–0:1) to yield 1 (10.8 mg), 2 (6.8 mg), 3 (15.4 mg), and 4 (58.1 mg). Fraction 1F (1161.1 mg), which was eluted with 95:5 (CHCl3:MeOH), was subjected
J Nat Med
to ODS column chromatography (u20 9 270 mm) using H2O:MeOH (1:1–0:1) as the eluent to yield ten fractions (6A–6J). The MeOH-soluble fraction of 6E (11.0 mg) was then further purified with HPLC (Cosmosil 5C18-AR-II, u10 9 250 mm; eluent, 45 % MeCN; flow rate, 1.5 ml min-1) to obtain 5 (tR = 18.8 min; 1.4 mg). The BuOH fraction (2.8 g) was then subjected to ODS column chromatography (u30 9 270 mm) with H2O:MeOH (9:1–1:1) to yield fractions 8A-8I. Fraction 8D (92 mg), which was eluted with 20 % MeOH, was subjected to HPLC (Cosmosil 5C18-AR-II, u10 9 250 mm; eluent, 30 % MeOH; flow rate, 2 ml min-1) to yield 6 (tR = 16 min; 30.8 mg). Fraction 8G (30.2 mg), which was eluted with 40 % MeOH, was further purified with ODS chromatography (u30 9 120 mm) using 50 % MeOH to yield compound 7 (9.0 mg). Luciferase assay The Ngn2 promoter activity assay was conducted as described previously [13]. C3H10T1/2 cells (1 9 104) stably transfected with a plasmid containing the Ngn2 promoter were seeded in a 96-well plate. After 24 h of incubation, different concentrations of compounds were added and incubated for 24 h. Then, cells were lysed with Glo Lysis Buffer. Luciferase activity was measured using the BrightGlo Luciferase Assay System (Promega, Tokyo, Japan) according to the manufacturer’s recommendations. Relative Ngn2 promoter activity of the control (DMSO only) was defined as 100 %, and the activity of the other treatments was calculated accordingly. Cell proliferation assay Cell viability was determined using the fluorometric microculture cytotoxicity assay [21]. Reporter cells (1 9 104) were inoculated into 96-well plates for 24 h. Different concentrations of the compounds were then added and incubated for another 24 h. After incubation, fluorescein diacetate (Wako, Japan) in PBS buffer (10 lg/mL) was added, and fluorescence was detected after 1 h of incubation using Luminoskan (Ascent, http://www.thermoscientific.com). Neuronal differentiation experiment In brief, C17.2 cells (2 9 105) in a proliferation medium were seeded in a poly-L-lysine-coated 10-cm dish (Wako) and incubated for 24 h at 37 °C. Then, the medium was removed, and the cells were washed with DMEM only. The cells were then treated with the compounds prepared in differentiation medium [Neurobasal medium (Gibco) supplemented with 1 mM glutamine, 2 % B-27 without vitamin A (Gibco), and 1 % antibiotic–antimycotic (Gibco)]
for 7 days. Cell images were obtained under phase contrast using an Olympus DP72 (Olympus, Osaka, Japan). Acknowledgments We thank Prof. Dr. R. Kageyama for the Ngn2 plasmid. This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS).
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