Coronarin D, a Metabolite from the Wild Turmeric, Curcuma aromatica, Promotes the Differentiation of Neural Stem Cells into Astrocytes

Plants in the genus Curcuma have been widely used as traditional medicines in Asian countries. These plants contain bioactive compounds with neuroprotective properties or activities that increase neural stem cells (NSCs) and neurons. However, bioactive components in Curcuma that promote the differentiation of NSCs into astrocytes have not yet been reported. Here, the effects of Curcuma extracts on the in vitro differentiation of embryonic stem-cell-derived NSCs were evaluated. The extract of the wild turmeric, Curcuma aromatica, strongly promoted the differentiation of NSCs into astrocytes. Bioassay-guided isolation yielded coronarins C (1) and D (2), as well as (E)-labda-8(17),12-diene-15,16-dial (3) as the bioactive compounds. Coronarin D (2) markedly promoted the differentiation of NSCs into astrocytes up to approximately 4 times (3.64 ± 0.48) and increased the expression level of GFAP at the mRNA and protein level, while compounds 1 and 3 exhibited only weak effects, suggesting that the 15-hydroxy-Δ12-γ-lactone moiety is important for bioactivity. Moreover, compound 2 increased the number of pSTAT3-positive cells, suggesting that compound 2 promoted astrocytic differentiation through JAK/STAT signaling pathway.


■ INTRODUCTION
Plants in the genus Curcuma in the Zingiberaceae family have been widely used as traditional medicines in Asian countries, especially India and China. 1 Many bioactive components have been isolated from Curcuma species, including Curcuma longa (turmeric) and Curcuma aromatica (wild turmeric). 2 Curcumin, 3 demethoxycurcumin, and bisdemethoxycurcumin are considered anti-inflammatory, neuroprotective, and antioxidant curcuminoids. 4 In addition to curcuminoids, a wide variety of terpenoids with antibacterial, antitumor, or other pharmacological properties form another class of bioactive components from the genus Curcuma. 5−7 Among many bioactivities of Curcuma, the neuroprotective property of Curcuma has attracted the attention of researchers, and curcumin has been shown to be neuroprotective through its antioxidative, anti-inflammatory, and anti-protein aggregating properties. 8 Curcumin also inhibits neuroinflammation involved in the progression of neurodegenerative diseases by reducing the expression of inflammatory cytokines, including IL-1β, IL-6, and TNF-α. 9 In addition to its neuroprotective activities, some Curcuma compounds were reported to affect the proliferation and differentiation of neural stem cells (NSCs). Curcumin stimulates the proliferation 10 or differentiation of NSCs into neurons, 11 and the aromatic compound turmerone, another major Curcuma component, was shown to increase the number of NSCs and promote neuronal differentiation. 12 NSCs are distributed in brain regions such as the hippocampus and the lateral ventricles, and provide neurons and glial cells, such as astrocytes and oligodendrocytes, throughout the life span. 13−15 Dysfunction of neural cells such as NSCs, neurons, and glial cells are deeply involved in neurodegenerative diseases such as Alzheimer's disease, 16 Parkinson's syndrome, 17 and depression. 18 Therefore, the proliferation and differentiation of NSCs are potential targets for neuroprotective medicines and supplements.
Although numerous bioactive compounds promoting neuronal differentiation have been discovered in Curcuma, there are yet no reports of compounds promoting astrocytic differentiation. Bioassays using astrocytes or NSCs derived from pluripotent stem cells have been recognized as a new approach for studying neurogenesis or neurodegenerative diseases in vitro from the viewpoint of animal welfare. 19 Some researchers have developed neural differentiation methods using NSCs derived from pluripotent stem cells 20,21 and have used them to test neural toxicity or neuroprotective activity. 22,23 Therefore, in this study, Curcuma components promoting astrocytic differentiation of NSC derived from mouse embryonic stem cells (ESCs) were searched for. The successful isolation, identification, and characterization of coronarin D and its analogues as bioactive substances in C. aromatica as well as their activities on astrocytic differentiation of NSCs are described.

■ MATERIALS AND METHODS
Experimental Equipment for Structure Elucidation. All NMR spectra were acquired using Avance 400 or 600 MHz NMR spectrometer (Bruker Corporation, Billerica, MA). Liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS) data were obtained using a Shimadzu UFLC XR liquid chromatography apparatus (Shimadzu Corporation, Kyoto, Japan) equipped with a TripleTOF 4600 system (AB Sciex LLC, Framingham, MA).
Extraction and Isolation. The processed products of C. aromatica (tablets, 110 g; Nakazen Corporation, Okinawa, Japan) were first extracted with MeOH. This extract was subjected to octadecylsilyl (ODS) flash column chromatography (⌀2.0 cm × 3.0 cm) using a 3)] to give compounds 1 (fr.6-7) and 2 (fr.  as the active substances. The fresh rhizomes of C. aromatica (520 g wet weight) were extracted with MeOH. The extract was subjected to ODS flash giving compound 3 as the other active substance. Purification schemes for compounds 1−3 are described in Figure S1.
In Vitro Differentiation of ESCs into NSCs. NSCs were induced from mouse ESCs using a previously reported method, with some modifications. 24,25 Briefly, embryoid bodies (EBs) were formed with the hanging drop method using 7500 ESCs in 20 μL of medium in the absence of LIF for 3 days. The obtained EBs were transferred to a lowadhesion plate (Corning Inc., Corning, NY) and cultured in Neuron Culture Medium (Fujifilm Wako Pure Chemical Corporation) supplemented with 20 ng/mL rhEGF (R&D Systems, Minneapolis, MN) and 20 ng/mL rhFGF-2 (R&D Systems) for 96 h. Thereafter, the EBs were transferred to matrigel (BD Biosciences, Franklin Lakes, NJ)coated dishes and incubated in NSC maintenance medium, MACS NeuroMedium (Miltenyi Biotec, Bergisch Gladbach, Germany) containing 2% MACS NeuroBrew-21 (Miltenyi Biotec), 1% P/S, 20 ng/mL rhEGF, and 20 ng/mL rhFGF-2, for 20 days. Finally, NSCs that migrated radially outward from the EBs were collected and cryopreserved until use in the in vitro NSC differentiation assay.
In Vitro NSC Differentiation Assay. NSCs induced from ESCs were seeded into each well of matrigel-coated 96-well plates (Corning Inc.) at a density of 1 × 10 4 cells/well and cultured for 72 h in the NSC maintenance medium. Then, the medium was replaced with fresh NSC maintenance medium or NSC differentiation medium, DMEM/Ham's F-12 (1:1) (Fujifilm Wako Pure Chemical Corporation) containing 2% MACS NeuroBrew-21, 1% P/S, and 1% FBS, for 72 h. During this step, test samples dissolved in DMSO (Fujifilm Wako Pure Chemical Corporation) were added to the medium at a 1000-fold dilution. The differentiation rate of NSCs into astrocytes was calculated using the immunocytochemistry method described below. The bioactivity of the sample for NSC differentiation was evaluated by comparing the differentiation rate with that of the control (0.1% DMSO).
Immunocytochemistry. Cells were washed with phosphatebuffered saline (PBS) (Takara Bio, Shiga, Japan) twice and incubated with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corporation) at 4°C for 30 min. After PBS washing, the cells were incubated with PBS containing 5% skim milk (Fujifilm Wako Pure Chemical Corporation) and 0.2% Triton X-100 (Thermo Fisher Scientific) at 4°C for 30 min. Then, the cells were treated with 5% skim milk solution containing primary antibody anti-GFAP (an astrocyte marker, mouse monoclonal antibody, 1:500, Merck Millipore) at 4°C overnight. After washing with 5% skim solution, the cells were incubated with 5% skim milk solution containing secondary antibody Alexa Fluor 488conjugated anti-mouse IgG (1:1000, Thermo Fisher Scientific) at room temperature for 2 h. After washing with 0.2% Triton X-100 solution, 0.2% Triton X-100 solution containing Hoechst 33342 (1:1000; Dojindo, Kumamoto, Japan) was added to visualize the nuclei, and the fluorescent cell images were obtained under the microscope (IX71, Olympus Corporation, Tokyo, Japan). The obtained images were analyzed by CellProfiler software, 26 and the ratio of the number of GFAP-positive cells to that of the control was calculated as the rate of NSC differentiation into astrocytes.
Western Blotting. Whole proteins of cells were extracted with sample buffer solution (Nacalai Tesque, Kyoto, Japan), incubated at 95°C for 5 min, and then centrifuged at 15,000 rpm for 5 min at 4°C. Supernatants were subsequently separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) with a gradient gel (Atto, Tokyo, Japan), followed by electrophoretic transfer onto PVDF membrane (Merck Millipore). After the blotting, the membranes were blocked in Blocking One (Nacalai Tesque) for 45 min and then incubated with primary antibodies [anti-GFAP mouse monoclonal antibody (1:2500, Merck Millipore) and anti-ACTB mouse monoclonal antibody (1:2500, Santa Cruz, Dallas, TX)] at 4°C overnight, followed by incubation at room temperature for 2 h with HRPconjugated secondary antibodies (Santa Cruz). The Can Get Signal Immunoreaction Enhancer Solution Kit (Toyobo) was used as an antibody diluent for the signal enhancement. Signal was detected with LAS-4000 (GE Healthcare, Chicago, IL) using Chemi-Lumi One L (Nacalai Tesque), and signal intensities were calculated with ImageQuant TL Software (GE Healthcare).
Flow Cytometry. Cells were fixed with 4% paraformaldehyde and blocked in PBS with 5% skim milk and 0.2% Triton X-100 at 4°C for 30 min, respectively. Then, the cells were reacted at room temperature for 2 h with the antibodies of anti-GFAP mouse monoclonal antibody Statistical Analysis. In Figure 4A,B, one-way ANOVA was used to evaluate statistical differences among each independent group, and Tukey's multiple comparison test was used to assess differences between two groups using EZR, 27 a graphical user interface for statistical analysis software R (R Foundation for Statistical Computing, Vienna, Austria). In Figures 4C−E and S15, Student's t-test was used to    Inspection of the 13 C NMR spectrum of 3 indicated the existence of two aldehyde groups (δ C 197. 47, 193.74). In addition to this observation, database search with SciFinder identified compound 3 as (E)-labda-8(17),12-diene-15,16-dial ( Figure 3). 29 Compound 2 obtained from fresh rhizomes of C. aromatica also showed the promoting activity of differentiation of NSCs into astrocytes, but compound 3 had no significant effects ( Figure 3B,C).
Evaluation of Astrocytic Differentiation of NSCs by Compound 2. The effects on the differentiation of NSCs were investigated quantitatively for compound 2. Effects at three concentrations (3.75, 7.5, and 15 μM) of 2 were calculated based on the fluorescence of the microscopic images ( Figures 4A  and S14). This revealed that compound 2 increased the ratio of GFAP-positive cells in a dose-dependent manner. Since the total number of cells was not decreased by any concentration of compound 2 ( Figure 4B), compound 2 was not cytotoxic towards NSCs within the range of the concentrations tested.
To validate the activity of compound 2, the effects of 15 μM of compound 2 on mRNA and protein expression level of GFAP were examined. Consistent with the result of the in vitro NSC differentiation assay, the expression level of GFAP was increased by treatment with compound 2 in mRNA and protein levels ( Figure 4C,D). Flow cytometry analysis also revealed that compound 2 treatment enhanced the rates of GFAP-positive cells ( Figure 4E). In this analysis, the rate of pSTAT3-positive cells compared to the control condition (1.50 ± 0.259%) was increased (21.9 ± 1.39%, Figure 4E) in the cells treated with compound 2. It is known that pSTAT3 activates transcription of GFAP. 30 The elevated level of pSTAT3 caused by compound 2 may play some roles in the promotion of astrocytic differentiation. In contrast, the increases of rates of GFAP or pSTAT3-positive cells by the treatment with compound 2 were not observed in the NSC maintenance medium ( Figure S15), which indicates that compound 2 may promote astrocytic differentiation as an assistant in NSCs differentiation medium through JAK/STAT3 signaling.

■ DISCUSSION
This is the first report identifying coronarin D (2) in the wild turmeric C. aromatica, as the bioactive compound promoting the astrocytic differentiation of NSCs. Compound 2 is a labdane diterpene. While many labdane diterpenes have been isolated from various plants in the Zingiberaceae family, such as the Ginger lily Hedychium coronarium, 31−35 they have never been previously reported from C. aromatica. Compounds 1 and 2 were previously isolated from the rhizomes of H. coronarium 28 and 3 from the seeds of Alpinia galanga. 29 Compound 2 has also been reported from the rhizomes of Amomum maximum 36 and Curcuma amada. 37 Biological activities of labdane diterpenes include cytotoxicity against V-79 cells, 28 anti-inflammatory activity, 38 inhibition of vascular permeability, NO production, 39 and inhibition of hexosaminidase release in RBL-2H3 cells. 40 The reported bioactivity for compound 1 is the inhibition of the proliferation of A-549 cells, 41 while antibacterial 42,43 and anti-inflammatory activities 44 have been reported for compound 2. Compound 3 shows inhibition against α-glucosidase, lipase, 45 and the growth of Gram-negative bacteria. 46 Curcumin 3 is regarded as the major bioactive compound in C. aromatica, but in this study, coronarin D (2) was identified as another bioactive substance with a strong ability to promote astrocytic differentiation. Coronarin C (1), an isomer of 2, showed only a weak tendency for promoting the astrocytic differentiation of NSCs. Compounds 1 and 2 differ only in the position of the double bond (Δ 12 vs Δ 13 ), but this difference significantly affects the activity. In addition, (E)-labda-8(17),12diene-15,16-dial (3) showed only a weak activity for astrocytic differentiation, suggesting that the 15-hydroxy-Δ 12 -γ-lactone moiety in compound 2 is essential for the bioactivity.
GFAP is a microfilament protein almost in astrocytes in brain tissue, used for the identification of astrocytes in vivo, and GFAPpositive astrocytes display a typically stellate morphology. 19 In the series of experiments, it was found that compound 2 increased the rate of GFAP-positive stellate astrocytes and GFAP expression at the mRNA and protein levels. Flow cytometry analysis showed that compound 2 increased the number of pSTAT3-positive cells. In the JAK-STAT signaling pathway, phosphorylated STAT3 plays the role of the key transcription factor that promotes astrocytic differentiation. 47,48 This suggests that compound 2 may promote astrocytic differentiation by activating JAK-STAT signaling pathway and phosphorylating STAT3. This pathway is activated when some cytokines (e.g., IL-6 or EGF) bind to receptors such as GP130 or EGFR. 49 An affinity test of compound 2 with these receptors or a comprehensive expression profiling analysis related to JAK-STAT signaling pathway may help to clarify the mechanism of action for compound 2.
The relationship between the number of astrocytes in the cerebral cortex and various neurological diseases has garnered increasing attention. Accumulating reports indicate that inflammatory reactions caused by decreased number of normal astrocytes can lead to the development of Alzheimer's disease, 50 vulnerability to stress, 51 and depressive symptoms. 52 Therefore, bioactive compounds that modulate the astrocytic differentiation of NSCs may have potential in the treatment or prevention of neurodegenerative diseases. For example, AMP-N 1 -oxide contained in royal jelly has been reported to promote astrocytic differentiation 53 and piceatannol found in the seeds of passion fruit promotes the proliferation and differentiation of NSCs into astrocytes. 54 Coronarin D (2) is an additional example of a food component that promotes astrocytic differentiation, and it may be a promising lead compound for the treatment of various neurological diseases or as a supplement for dementia prevention.