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Induction of Neuronal Differentiation by Extracts from 57 Kinds of Traditional Medicinal Plants in Myanmar

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Induction of Neuronal Differentiation by Extracts from 57 Kinds of Traditional Medicinal Plants in Myanmar J. Res. Inst. Sci. Tech.,Induction Nihon Univ. of Neuronal No. 139 pp.Differentiation 1–11 by Extracts from 57 Kinds of Traditional Medicinal Plants in Myanmar Induction of Neuronal Differentiation by Extracts from 57 Kinds of Traditional Medicinal Plants in Myanmar Atsuyoshi NISHINA1*, Kei YOSHII1, Makoto FUKATSU2, Yasunori KUSHI1, Yusuke SUZUKI1 and Motohiko UKIYA1 Abstract Aging is becoming one of a big problem in advanced countries, thus development of medicine treat- ing or preventing dementia is carried out since incidence of the disease is raised higher along with aging. We tried to find the components inducing neuronal differentiation from 57 kinds of traditional medicinal plants in Myanmar. From the results of evaluation of cytotoxicity and induction of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in PC12 cells by 171 extracts from 57 species of tradi- tional medicinal plants in Myanmar, only three kinds of Croton tiglium L. extracts and ethyl acetate extract of Oroxylum indicum (L.) Benth. ex kurz, showed both of low-cytotoxicity and phosphorylation of ERK1/2. It was deduced that neuronal differentiation was induced by C. tiglium methanol extract, because it up-regulated both neurite outgrowth and expression of neurofilament-M (NFM). On the other hand, from the results using three mitogen-activated protein kinase (MAPK) inhibitors, it was considered that phosphorylation of five kinases (ERK1/2, p38MAPK, c-jun N-terminal kinas (JNK), ERK5, and Akt) and three kinases (ERK1/2, p38MAPK, and JNK) were necessary for neuronal differentiation by NGF and C. tiglium methanol extract, respectively. Water soluble polymer such as proteins is not contained in C. tig- lium methanol extract, though the neuronal differentiation induction was shown by it. Therefore, there is a possibility that the taken active components from C. tiglium are carried by blood flow followed by trans- portation to the brain. Key Words : traditional medicinal plants in Myanmar, neuronal differentiation, Croton tiglium L., neurofilament-M, mitogen-activated protein kinase 1. Introduction spheres from brain striatum of adult mice by addition of epidermal growth factor (EGF). And they showed Neurodegenerative diseases such as Alzheimer dis- that the neutrospheres were divided and grown fol- ease cause dementia via accumulation of malign pro- lowed by differentiation for neurons or astrocytes4). teins such as aggregated β-amyloid in central neurons. Eriksson5) reported that undifferentiated neutrospheres In addition, neural networks are injured by lengthy neu- in adult brain were pluripotential neural stem cells ral cell death followed by appearance of symptoms which had ability to differentiate into neurons or neuro- such as memory impairment or affective disorder. glial cells such as astrocytes or oligodendrocytes etc. It was once confirmed by Cajal in 1928 that the Namely, it was confirmed that neuronal differentiation cranial nervous system is constructed by division and was produced in adult brain. In addition, it was reported migration of neural cells at fetal period, number of cells that neurons were reproduced from progenitor cells is retained while maturing until around 20 years old. existing near the damage part of the rat global cerebral Subsequently, number of cells are decreased along with ischemia model by injection of growth factor such as aging or injury1). In addition, it was considered that, fibroblast growth factor-2 (FGF-2) or EGF, and learn- damaged central nervous systems are nonrenewable2). ing functions and memory of global cerebral ischemia However, after the discovery of the neuronal regenera- model rats were remarkably improved compared with tion by the canary hippocampus by Nottebohm in the untreated group6). Above-mentioned result suggests that 1980’s3), Reynolds and Weiss of Canada found neutro- replenishment and recovery of the function of neurons 1 Department of Materials and Applied Chemistry, College of Science and Technology, Nihon University 2 Department of Biotechnology and Material Chemistry, Nihon University Junior College * Corresponding author Received 6 May 2016, Accepted 18 October 2016 – 1 – A. NISHINA et al. are also possible in adults’ neural systems by differen- 2. Materials and methods tiation of neural stem cells. Growth factors―polypeptides consist of about 120 2.1 Plant materials and Reagents amino acid residue homodimers promoting neuronal Traditional medicinal plants in Myanmar (57 spe- differentiation―activate Trk family high-affinity and cies) (Table 1) were purchased commercially in p75 low-affinity receptors on the neuronal cells’ mem- Yangon. The voucher specimen has been deposited in brane followed by regulation of growth, differentiation, Pathein University. All solvents and reagents used in mature, neurotransmission, and death of neural progen- isolation were purchased from Sigma (St. Louis, MO) itor cells7). Among such compounds, nerve growth fac- and Wako Pure Chemical Industries (Osaka, Japan). tor (NGF) and brain-derived neurotrophic factor 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-2H-tetrazo- (BDNF) are frequently studied8). NGF promotes neurite lium bromide (MTT) were purchased from Sigma. outgrowth and maintains functions of fetal neurons, and Hexane, chloroform, methanol, and trifluoroacetic acid its stimulation is necessary for survival of neurons. were obtained from Wako Pure Chemical. U0126 [Spe- NGF also takes part in regulation of proliferation and cific inhibitors of mitogen-activated protein kinase the differentiation of neural stem cells, and the clinical kinase (MAPKK)/extracellular signal-regulated kinase application of NGF was tried to reproduce damaged (ERK) kinase (MEK)], p38MAPK inhibitor, and c-jun neural cells9). However, because neurotrophic factors N-terminal kinase (JNK) inhibitor were purchased from such as NGF are large-molecular weight polypeptides, Calbiochem (Sun diego, CA). it is easy to metabolize and hardly to pass the blood- brain barrier after administration in vivo. In addition, 2.2 Solvent Fractionation the clinical application of NGF has not succeeded Plant materials were grinded and the components because it accompanies side effects including neuro- were fractionated. In brief, 100 g of each plant material pathic pain10,11). Then, low-molecular compounds have powder was immersed in 500 mL of hexane for 24 h at effects on proliferation and differentiation of neural sys- room temperature. The solvent containing the extracts tems as well as neurotrophic factors is widely studied. was filtrated through a filter paper (5C; Whatman, The PC12 cells are the cell line isolated from rat Brentford, UK) and the filtrate was evaporated to dry- adrenal medulla derived pheochromocytoma by Greene ness to prepare hexane extract. The residue was then and Tischler in 1976 and widely used as a model of stirred in 500 mL of chloroform at room temperature neural progenitor cells12). The major features of the for 24 h, and the filtrate was dried in vacuo to prepare PC12 cells are growth arrest and neurite outgrowth chloroform extract. Then, methanol extract was along with neuronal differentiation by NGF-stimula- obtained in the same manner15). The mean value of the tion. In addition, secretion of neurotransmitters such as amount of each extract was calculated. dopamine was induced by depolarization by stimulation of acetylcholine13). Moreover, implantation in the brain 2.3 Cell culture of encapsulated PC12 cells caused dopamine secretion In the present study, we used rat pheochromocy- followed by improvement of symptoms of Parkinson’s toma PC12 cells, because previous reports suggested disease14). Thus, PC12 cells are the suitable model sys- that this cell line is a useful tool as various models of tem for evaluation of neuronal differentiation by low- neurological dysfunctions16). PC12 cells were cultured molecular neurotrophic factor-like compounds. as described previously15). In brief, the cells were main- Considering above-mentioned problems, in the tained in Dulbecco’s modified Eagle’s medium present study, we tried to evaluate effects of induction (DMEM; Sigma) supplemented with 10% heat-inacti- of neuronal differentiation by extracts from 57 kinds of vated horse serum (HS; Gibco BRL, Grand Island, NY) traditional medicinal plant in Myanmar for discovering and 5% heat-inactivated fetal bovine serum (FBS, of new drug seeds from natural resources using PC12 Sanko Junyaku, Co., Ltd., Tokyo, Japan) (serum-con- cells. taining medium) or in DMEM supplemented with 1% bovine serum albumin (BSA) (serum-free medium). – 2 – Induction of Neuronal Differentiation by Extracts from 57 Kinds of Traditional Medicinal Plants in Myanmar Table 1. Tested traditional medicinal plants in Myanmar. Botanical name Part Botanical name Part Alpinia officinarum Hance. Root Mimusops elengi L. Flower Alstonia scholaris (L.) R. Br. Bark Moringa oleifera Lamk. Bark Andrographis paniculata Nees. Stem Myoporum bontioides A. Gray Stem Andrographis paniculata Nees. Leaf Myrica cerifera L. (female) Bark Anethum graveolens L. Seed Myrica cerifera L. (male) Bark Argemone mexicana L. Flower Nardostachys jatamansi (D. Don) DC. Root Aristolochia indica L. Leaf Nigella sativa L. Seed Bacopa Monnieri (L.) pennell Root Oroxylum indicum (L.) Benth. ex kurz Bark Baliospermum montanum (Willd.) Muell.-Arg. Stem Paramignya longispina Hook.f. Root Caesalpinia bonducella (L.) Fleming Seed Phyllaunthus emblica L. Fruit Cinnamomum obtusifolium (Roxb.) Nees. Stem Piper longum L. Stem Cinnamomum tamala
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