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

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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).

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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 (Bu.-H.) Nees. & Eb. Leaf, Bark Piper longum L. Fruit Crataeva religiosa Forst. Bark Piper nigrum L. Seed Croton tiglium L. Seed Plumbago zeylanica L. Root Cuminum cminum L. Seed Pterocarpus santalinus L. Stem Curcuma longa L. Root Rhus succedanea L. Fruit Curcuma longa L. (bitter) Root Santalum album L. Stem Daucus carota L. Root Stevia rebaudiana (Bertoni) Bertoni Solid Foeniculum vulgare Mill. Seed Styphnolobium japonicum (L.) schott Flower Gentiana kurroo Royle Stem Terminalia citrina (Gaertn) Roxb. Fruit Gloriosa sperba L. Root Tinospora cordifolia Miers Stem Glycyrrhiza glabra L. Stem Tinospora cordifolia Miers Bark Heliotropium indicum L. Leaf Trachyspermum ammi (L.) Sprague Seed Helleborus niger L. Root Tribulus terrestris Fruit Hydnocarpus kurzii (King) Warb. Fruit Valeriana officinalis L. & Maillefer Bulb Jasminum arborescens Roxb. Flower Vitex trifolia L. Leaf Kaempferia galangal L. Bud Zingiber officinale Roscoe Bulb Lepidium sativum L. Seed Mesua ferrea L. Flower Millingtonia hortensis Lf. Bark

2.4 The measurement of cytotoxicity by 3-(4,5- Japan) by measuring the absorbance at 562 nm with a dimethylthiazo-2-yl)-2,5-diphenyl-2H-tetrazolium bro- reference wavelength of 630 nm. mide (MTT) assay PC12 cells were cultured onto collagen-coated 2.5 Detection of phosphorylated proteins 96-well plates (2 × 106 cells/well) in serum-containing Each test sample was suspended in the serum-free medium for 2 days at 37°C in an atmosphere of 95% medium, and sonicated until fully emulsified. PC12 6 air/5% CO2. Culture medium was replaced with 50 μL cells were seeded at 2 × 10 cells/well onto collagen- of the serum-free medium containing each test agent coated 6-well plates (Corning Incorporated Life Sci- after washing with PBS, and the cells were cultured for ences, Lowell, MA) in the serum-containing medium, 48 hours. The cytotoxicity was determined by the and pre-cultured for 2 days at 37°C in an atmosphere of

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo- 95% air/5% CO2. The cells were then washed with lium bromide (MTT) reduction assay17). The cells were phosphate-buffered saline (PBS), and incubated with incubated with 0.25 ng of MTT/mL (final concentra- the above mentioned culture medium containing NGF tion) for 2 hours, and the reaction was stopped by add- or various concentration of C. tiglium methanol extract ing 50 μL of 50% (v/v) dimethylformamide (DMFA) shown in Figure 4 for 10 minutes at 37°C. The culture containing 20% (w/v) SDS. The amount of MTT plates were then placed on ice and each well was formazan product was determined photometrically washed with 3 mL of 2mM Tris-HCl buffer (pH 8.0) using a micro plate reader (Model Ultrospec Visible containing 0.33 M NaF and 6.25 M Na3VO4, and sub- Plate Reader II of Amersham Biosciences., Tokyo, sequently lysed with 150 μL of 20 mM Tris-HCl buffer

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(pH 8.0) containing 150 mM NaCl, 2 mM EDTA, 1% cubated for 3 hr in medium containing U0126, Nonidet P-40 (w/v), 1% sodium deoxycholate (w/v), p38MAPK, on JNK inhibitor at the concentration 0.1% sodium dodecyl sulfate (SDS) (w/v), 50 mM NaF, showed in Figure 5, cells were then washed with PBS, 0.1% aprotinin (w/v), 0.1% leupeptin (w/v), 1 mM and incubated with the medium containing NGF or C.

Na3VO4 and 1 mM phenylmethylsulfonylfluoride tiglium methanol extract at 100 μg/ml for six days at (PMSF). Cell lysates were collected by using a cell 37°C. The cells were then collected and analyzed for scraper, and centrifuged at 15000 x g for 30 minutes at expression of NFM. 4°C. The supernatant was collected, and the overall protein concentration was determined by a BCA Pro- 2.7 Statistical analysis tein Assay Reagent Kit (Pierce, Rockford, IL) with The results were expressed as mean ± standard BSA as a standard. deviation (SD). The significant difference between the Supernatant fluids containing proteins (20 μg) were groups compared were determined using analysis of mixed with lithium dodecyl sulfate (LDS) sample buf- variance (ANOVA) followed by Tukey-Kramer test. fer (Invitrogen, Carlsbad, CA) and incubated for 5 min- utes at 80°C. Proteins in samples were separated on 3. Results SDS-polyacrylamide gel electrophoresis, and the pro- teins in gels were electroblotted onto polyvinylidene 3.1 Yields of Extracts fluoride (PVDF) filters (Fluorotrans membrane W, 0.2 57 kinds of traditional medicinal plants in μm; Nihon Genetics, Tokyo, Japan). Immunoblotting Myanmar were extracted by hexane, ethyl acetate, and analysis was performed by using monoclonal antibod- methanol to obtain 117 extracts. Yields of 117 extracts ies against ERK1/2, phospho-ERK1/2, p38MAPK, from traditional medicinal plants in Myanmar were phospho-p38MAPK, stress-activated protein kinase/Jun summarized in Table 2. Yields from bark, bulb, flower, amino-terminal kinase (JNK/SAPK), phospho-JNK/ leaf, root, and stem were methanol extracts > ethyl ace- SAPK, Akt, phospho-Akt, neurofilament-M and β-actin tate extracts > hexane extracts, while extracts from fruit (Cell Signaling Technology, Lake Placid, NY) as pri- and seed were hexane extracts > methanol extracts > mary antibodies, followed by reaction with horseradish ethyl acetate extracts in turn (Figure 1). Because of peroxidase-conjugated anti-rabbit or anti-mouse immu- high content of lipids in seeds might induce high yields noglobulin G (IgG) antibodies from Promega Co. of seeds hexane extracts. (Fitchburg, WI, USA) as the secondary antibody. The blots were developed by the enhanced chemilumines- 3.2 Cytotoxic activity of each extract cence method (Hyperfilm-ECL plus, Amersham Biosci- Cytotoxic activity of each extract in PC12 cells ences Corp., Piscataway, NJ). was evaluated (Table 3). It was considered that, com- Image J software was used for densitometry calcu- pounds with lower polarity are more toxic, because lation18). For calculation of protein phosphorylation expedient mean value of 50% inhibitory concentration promotion, densitometry intensity ratios of total protein of cell survivability (IC50) of hexane, ethyl acetate, and and phosphorylated protein were measured. Immunos- methanol extracts were 57.7, 67.3, and 91.9 μg/ml, taining images of total protein are expressed in some respectively (Table 3). To individually, extracts from G. figures. When using images obtained in multiple exper- sperba and H. indicum showed significant cytotoxic iments, same sample (control) was used in all experi- effects, because IC50 of all three extracts of them were ments and contrast values of controls were adjusted on less than 20 μg/ml. Additionally, hexane and ethyl ace- Image J software. tate extract of A. paniculata (stem), A. indica, F. vul- gare, J. arborescens, M. oleifera, P. emblica, P. nigrum, 2.6 Treatment with Specific Inhibitors P. zeylanica, S. japonicum, and T. cordifolia showed Initially, PC12 cells were seeded at 2 × 106 cells/ cytotoxicity. well onto collagen-coated 6-well plate in the serum- containing medium, and pre-cultured for 2 days at 37°C in an atmosphere of 95% air/5% CO2. Cells were prein-

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Table 2. Yield of each extract.

Yield (%) Yield (%) Plant Name Ethyl Plant Name Ethyl Hexane Methanol Hexane Methanol acetate acetate Alpinia officinarum Hance. 0.7 1 3.4 Mimusops elengi L. 0.6 0.6 4.8 Alstonia scholaris (L.) R. Br. 3.1 0.2 3.6 Moringa oleifera Lamk. 0.1 0.1 1.1 Andrographis paniculata Nees. (stem) 0.7 1.6 6.2 Myoporum bontioides (Sieb. et Zucc.) A. Gray 0.1 3 5.1 Andrographis paniculata Nees. (leaf) 0.2 2.4 2.8 Myrica cerifera L. (Female) 0.3 1.6 33.5 Anethum graveolens L. 3 0.4 1.3 Myrica cerifera L. (Male) 0.2 2.1 27.1 Argemone mexicana L. 0.4 2.9 10.5 Nardostachys jatamansi (D. Don) DC. 6.2 3.2 5.8 Aristolochia indica L. 0.4 0.5 3.5 Nigella sativa L. 22.6 3.8 8.2 Baliospermum montanum (Willd.) Muell.-Arg. 0.1 0.2 1.1 Oroxylum indicum (L.) Benth. ex Kurz 0.1 2 3.8 Caesalpinia bonducella (L.) Fleming 11.3 0.4 2.3 Paramignya longispina Hook. f. 0.7 0.1 0.7 Centella asiatica (L.) Urban 0.2 1.4 1.7 Phyllanthus emblica L. 0.5 0.4 4 Cinnamomum obtusifolium (Roxb.) Nees. 1.3 1.7 13.9 Piper longum L. (stem) 0.8 0.5 3.3 Cinnamomum tamala (Bu.-H.) Nees. & Eb. 1.3 1 10.2 Piper longum L. (fluit) 2 1.2 1.5 Crataeva religiosa Forst. 0.3 0.4 0.7 Piper nigrum L. 6.5 7 8.7 Croton tiglium L. 9.3 1.4 5.8 Plumbago zeylanica L. 0.2 2.9 11.9 Cuminum cyminum L. 5.1 1.8 3.3 Pterocarpus santalinus L. 0.5 9.2 5.6 Curcuma longa L. 5 6.6 9.4 Rhus succedanea L. 0.2 7.7 5.4 Curcuma longa L. (bitter) 4 2.6 2.4 Santalum album L. 0 0.1 0.4 Daucus carota L. 1.4 2.5 14.3 Stevia rebaudiana (Bertoni) Bertoni 0 0.2 8.2 Foeniculum vulgare Mill. 4.8 1 2 Styphnolobium japonicum (L.) Schott 1.9 4.3 8.5 Gentiana kurroo Royle 6.8 2.6 5.7 Terminalia citrina (Gaertn) Roxb. 0.2 1.9 5.3 Gloriosa superba L. 1 4 1.4 Tinospora cordifolia Miers. (stem) 0.3 0.3 3.9 Glycyrrhiza glabra L. 0.3 2.6 13.9 Tinospora cordifolia Miers. (bark) 0.1 1.6 1.3 Heliotropium indicum L. 1.7 0.7 3.5 Trachyspermum ammi (L.) Sprague 6.5 2.8 1.9 Helleborus niger L. 0.7 0.7 17 Tribulus terrestris L. 8.2 0.4 1.5 Hydnocarpus kurzii (King) Warb. 36 3.9 6 Valeriana officinalis L. & Maillefer 0.7 4.7 4.7 Jasminum arborescens Roxb. 1.8 1.2 5.8 Vitex trifolia L. 3.9 6.2 3.1 Kaempferia galanga L. 2.1 1 1 Zingiber officinale Rosc. 2.8 2.9 4.9 Lepidium sativum L. 16 1.4 7 Mesua ferrea L. 6.5 5.4 10.2 Millingtonia hortensis L. f. 0.1 0.9 1.1

10.0 each extract could activate this signal pathway or not. 9.0 Extracts mentioned in Table 2 were evaluated for their 8.0 7.0 ability to induce phosphorylation of ERK1/2 (Table 4). 6.0 From the results in Table 4, ERK1/2 phosphorylation 5.0 Hexane 4.0 was induced by ethyl acetate extracts from C. bondu-

Yield (%) Yield Ethyl acetate 3.0 Methanol cella and O. indicum, hexane extracts from J. arbores- 2.0 cens and M. bontioides, hexane and ethyl acetate 1.0 0.0 extracts from P. santalinus, and all extracts from C. tig- lium. On the contrary, all of those extracts except extracts from C. tiglium and ethyl acetate extract from Figure 1. Part-specific yields of each solvent extracts. The O. indicum were cytotoxic (Table 3). Thus, we decided mean value of the amount of each extract was described. to examine neuronal differintiation induction by extracts from C. tiglium.

3.3 Induction of phosphorylation of ERK1/2 by each 3.4 Neuronal differentiation by C. tiglium methanol extract extract Activation of ERK1/2, necessary for neuronal dif- Intensity of neuronal differentiation induction by ferentiation, is one of the checkpoints to assess the acti- three extracts from C. tiglium was methanol > ethyl vation of the classical ras/MAPK cascade19), which is acetate > hexane in turn (data not shown). Neurite out- triggered by an engaged tyrosine kinase receptor or G growth induced by cultivation for 6 days with or with- protein-coupled receptor and results in proliferation out appropriate concentration of C. tiglium methanol and/or differentiation. Therefore, we tested whether extract or NGF was shown in Figure 2. NGF, used as a

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Table 3. Cytotoxicity of each extract.

IC50 (μg/ml) IC50 (μg/ml) Plant Name Ethyl Plant Name Ethyl Hexane Ethanol Hexane Ethanol acetate acetate Alpinia officinarum Hance. 92.3 >100 >100 Mimusops elengi L. >100 >100 >100 Alstonia scholaris (L.) R. Br. N.D. 94.7 >100 Moringa oleifera Lamk. 6.2 11.6 63.2 Andrographis paniculata Nees. (stem) 10.1 38.8 >100 Myoporum bontioides (Sieb. et Zucc.) A. Gray 89.7 88.3 >100 Andrographis paniculata Nees. (leaf) 61.4 >100 >100 Myrica cerifera L. (Female) 24.6 >100 74.4 Anethum graveolens L. 64.3 3.6 >100 Myrica cerifera L. (Male) >100 97.9 >100 Argemone mexicana L. 12.5 >100 >100 Nardostachys jatamansi (D. Don) DC. >100 >100 >100 Aristolochia indica L. 2.7 9.3 87.4 Nigella sativa L. >100 4.2 12.5 Baliospermum montanum (Willd.) Muell.-Arg. >100 70.2 >100 Oroxylum indicum (L.) Benth. ex Kurz >100 >100 >100 Caesalpinia bonducella (L.) Fleming 79.2 >100 >100 Paramignya longispina Hook. f. >100 >100 >100 Centella asiatica (L.) Urban 50.5 >100 >100 Phyllanthus emblica L. 13.2 31.1 >100 Cinnamomum obtusifolium (Roxb.) Nees. 28.5 75.7 >100 Piper longum L. (stem) 48.5 >100 >100 Cinnamomum tamala (Bu.-H.) Nees. & Eb. 27.3 >100 91.2 Piper longum L. (fluit) 90.8 66 >100 Crataeva religiosa Forst. >100 69.5 >100 Piper nigrum L. 24.2 34.8 68.7 Croton tiglium L. >100 90 >100 Plumbago zeylanica L. 9 7.5 >100 Cuminum cyminum L. 87.8 >100 >100 Pterocarpus santalinus L. 65.1 >100 >100 Curcuma longa L. >100 >100 >100 Rhus succedanea L. 24.6 >100 >100 Curcuma longa L. (bitter) 2.9 >100 >100 Santalum album L. >100 >100 >100 Daucus carota L. 32.2 20.2 >100 Stevia rebaudiana (Bertoni) Bertoni 17.3 82.8 >100 Foeniculum vulgare Mill. 11 34.6 94.9 Styphnolobium japonicum (L.) Schott 12 21.7 >100 Gentiana kurroo Royle 38.3 >100 >100 Terminalia citrina (Gaertn) Roxb. 21.3 >100 >100 Gloriosa superba L. 10.1 14.5 1< Tinospora cordifolia Miers. (stem) 27.4 23.6 >100 Glycyrrhiza glabra L. >100 68.4 >100 Tinospora cordifolia Miers. (bark) >100 >100 >100 Heliotropium indicum L. 7.5 4.3 19 Trachyspermum ammi (L.) Sprague 64.1 33.5 >100 Helleborus niger L. 86.5 60 73.3 Tribulus terrestris L. >100 13.8 >100 Hydnocarpus kurzii (King) Warb. >100 66.2 >100 Valeriana officinalis L. & Maillefer >100 24.5 93.5 Jasminum arborescens Roxb. 5 12.6 59.8 Vitex trifolia L. >100 26 >100 Kaempferia galanga L. 15.3 >100 >100 Zingiber officinale Rosc. 13.5 >100 >100 Lepidium sativum L. >100 >100 >100 Mesua ferrea L. 72.8 37.2 >100 Expedient average 57.7 67.3 91.9 Millingtonia hortensis L. f. 39.2 >100 >100

PC12 cells were treated with different concentrations (0–100 μg/ml) of each extract for 48 hours. Results are presented as 50% inhibitory concentration of survival (IC50). Expedient average was calculated by assuming that >100 is 100 and <1 is 1.

CTRL NGF

C. tiglium 3 10 30 100μg / mL

Figure 2. Neurite outgrowth by C. tiglium methanol extract. PC 12 cells were treated with NGF or C. tig- lium extract at appropriate concentration for 6 days. Arrows indicates neurite outgrowth.

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positive reference compound, significantly induced that expression of neurofilament-M (NFM) correlated neurite outgrowth. On the other hand, C. tiglium metha- with neurite outgrowth20), induction of NFM expression nol extract more than 10 μg/ml also induced neurite by C. tiglium methanol extract under the same condi- outgrowth dose dependently. Because it is confirmed tion as Figure 2 was measured (Figure 3). NGF as well

Table 4. Induction of phosphorylation of ERK1/2 by each extract.

Extraction solvent Extraction solvent

Plant Name Ethyl Plant Name Ethyl Hexane Ethanol Hexane Ethanol acetate acetate Alpinia officinarum Hance. × × × Mimusops elengi L. × × × Alstonia scholaris (L.) R. Br. × × × Moringa oleifera Lamk. × × × Andrographis paniculata Nees. (stem) × × × Myoporum bontioides (Sieb. et Zucc.) A. Gray ○ × × Andrographis paniculata Nees. (leaf) × × × Myrica cerifera L. (Female) × × × Anethum graveolens L. × × × Myrica cerifera L. (Male) × × × Argemone mexicana L. × × × Nardostachys jatamansi (D. Don) DC. × × × Aristolochia indica L. × × × Nigella sativa L. × × × Baliospermum montanum (Willd.) Muell.-Arg. × × × Oroxylum indicum (L.) Benth. ex Kurz × ○ × Caesalpinia bonducella (L.) Fleming × ○ × Paramignya longispina Hook. f. × × × Centella asiatica (L.) Urban × × × Phyllanthus emblica L. × × × Cinnamomum obtusifolium (Roxb.) Nees. × × × Piper longum L. (stem) × × × Cinnamomum tamala (Bu.-H.) Nees. & Eb. × × × Piper longum L. (fluit) × × × Crataeva religiosa Forst. × × × Piper nigrum L. × × × Croton tiglium L. ○ ○ ○ Plumbago zeylanica L. ○ ○ × Cuminum cyminum L. × × × Pterocarpus santalinus L. × × × Curcuma longa L. × × × Rhus succedanea L. × × × Curcuma longa L. (bitter) × × × Santalum album L. × × × Daucus carota L. × × × Stevia rebaudiana (Bertoni) Bertoni × × × Foeniculum vulgare Mill. × × × Styphnolobium japonicum (L.) Schott × × × Gentiana kurroo Royle × × × Terminalia citrina (Gaertn) Roxb. × × × Gloriosa superba L. × × × Tinospora cordifolia Miers. (stem) × × × Glycyrrhiza glabra L. × × × Tinospora cordifolia Miers. (bark) × × × Heliotropium indicum L. × × × Trachyspermum ammi (L.) Sprague × × × Helleborus niger L. × × × Tribulus terrestris L. × × × Hydnocarpus kurzii (King) Warb. × × × Valeriana officinalis L. & Maillefer × × × Jasminum arborescens Roxb. ○ × × Vitex trifolia L. × × × Kaempferia galanga L. × × × Zingiber officinale Rosc. × × × Lepidium sativum L. × × × Mesua ferrea L. × × × Millingtonia hortensis L. f. × × ×

Induction of phosphorylation of ERK1/2 was evaluated by imunoblot described in Material and methods section. ○ and × indicate significant induction of phosphoryla- tion and no significant difference, respectively.

Neurofilament-M

350 a 300

250

200 b b c 150 c c

100

50 Exression of NFM (% CTRL) of (% NFM of Exression

0 CTRL NGF 3 10 30 100 C. tiglium (μg / ml)

Figure 3. Expression of neurofilament-M promoted by NGF or C. tiglium extract. PC 12 cells were treated with NGF or C. tiglium extract at appropriate concentra- tion for 6 days. Expression of neurofilament-M was evaluated by imunoblot described in Material and methods section.

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total-ERK total-p38 3 phospho-ERK phospho-p38 30 2.5

ERK1 ERK2 Basal ) 2 20 of fold 1.5

10 uni t ( 1 0.5

0 nsi tometry 0 De

Densitometry Basal) Densitometry of(fold unit cont NGF 3 10 30 100 cont NGF 3 10 30 100 concentration (μg/ml) concentration (μg/ml) total-JNK total-ERK5 phospho-JNK30 phospho-ERK54 25 20 JNK1 JNK2 3

15 2 10 1 5

Densitometry Densitometry Basal) of(fold unit 0 0 cont NGF 3 10 30 100 Basal) Densitometry of(fold unit cont NGF 3 10 30 100 concentration (μg/ml) concentration (μg/ml)

total-Akt phospho-Akt

4 3 2 1 0

Densitometry Densitometry Basal) of(fold unit cont NGF 3 10 30 100 concentration (μg/ml)

Figure 4. Regulation of phosphorylation of MAPK and Akt by NGF or C. tiglium extract. PC 12 cells were treated with NGF or C. tiglium extract at appropriate concentration for twenty minutes. Induction of phosphorylation was evaluated by imunoblot described in Material and methods section.

as C. tiglium methanol extract (more than 30 μg/ml) ated (Figure 4). NGF induced phosphorylation of all significantly induced NFM expression. From the results kinases described in Figure 4, but C. tiglium methanol of Figure 2 and 3, it was considered that C. tiglium extract phosphorylated only ERK1/2, p38MAPK, and methanol extract induce neuronal differentiation in JNK. Thus, C. tiglium methanol extract may induce PC12 cells. neuronal differentiation via activation of one or more of ERK1/2, p38MAPK, and JNK2. 3.5 Effects of C. tiglium methanol extract on MAPKs Activation of ERK1/2 is one of a checkpoint of 3.6 Effects of inhibitors on phosphorylation of activation or inactivation of res/MAPK cascade. Addi- MAPKs by C. tiglium methanol extract tionally, it was confirmed that activation of ERK1/2 for Kinds of phosphorylated MAPKs by stimulation long period of time was necessary for neuronal differ- by NGF or C. tiglium methanol extract was different, entiation21). On the other hand, it was also reported that though C. tiglium methanol extract induced neuronal activation of p38MAPK and Akt were necessary for differentiation as well as NGF. Thus, it was deduced neurite outgrowth22) and drosophila controls the dorsal that neuronal differentiation mechanisms by C. tiglium closure of the embryo and larva’s synapse (neuromus- methanol extract was different from that of NGF. Sub- cular junction) formation by activation of JNK23). Even- sequently, for the purpose of clarify the MAPKs neces- tually, activation of MAPK and neuronal differentiation sary for neuronal differentiation, effects of inhibitors on are closely related. phosphorylation of MAPKs at neuronal differentiation For the purpose of estimation of action mecha- by C. tiglium methanol extract was evaluated. nisms of neuronal differentiation by C. tiglium metha- Expression of NFM in PC12 cells treated with nol extract, effects of it for MAPK and Akt were evalu- three inhibitors (MEK, p38MAPK, and JNK) followed

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Neurofilament-M Neurofilament-M Neurofilament-M 120 120 120

100 100 100

80 80 80

60 60 60

40 40 40

20 20 20 Densitmetry index (/NGF) Densitmetry Densitmetry index (/NGF) Densitmetry Densitmetry index (/NGF) Densitmetry

0 0 0 NGF ○○○ ○ NGF ○○○ ○ NGF ○ ○ ○ ○ C. tiglium ○○○ ○ C. tiglium ○ ○ ○ ○ C. tiglium ○ ○ ○ ○ U0126 p38 MAPK JNK (μM) 0 0 25 50 100 0 25 50 100 inhibitor 0 015 25 0 1 5 25 inhibitor 005 25 50 0 5 25 50 (μM) (μM)

Figure 5. Regulation of expression of neurofilament-M by MAPK inhibitors. PC 12 cells were treated with each inhibitor for 1 hour followed by treatment by NGF or C. tiglium extract at appropriate concentration for 6 days. Induction of neurofilament-M was evaluated by imunoblot described in Material and methods section. by cultivation with NGF or C. tiglium methanol extract kinases (ERK1/2 and JNK) were necessary for neuro- at 100 μg/ml for 6 days were shown in Figure 5. nal differentiation by NGF and C. tiglium methanol Because expression of NFM by NGF were down-regu- extract, respectively. lated by addition of all of 3 inhibitors, it was consid- Because among 171 extracts obtained from 57 spe- ered that phosphorylation of all of 3 kinases (ERK1/2, cies of traditional medicinal plants in Myanmar, only p38MAPK, and JNK) was necessary for neuronal dif- three kinds of C. tiglium extracts and ethyl acetate ferentiation by NGF. On the contrary, because addition extract of O. indicum, showed both low-cytotoxicity of p38MAPK inhibitor did not regulate the expression and induction of phosphorylation of ERK1/2, it was of NMF, it was considered that phosphorylation of understood that natural compound inducing neuronal p38MAPK was not related to neuronal differentiation differentiation is very rare. by C. tiglium methanol extract. From above mentioned Phorbol esters are contained in C. tiglium24) and results, it was considered that action mechanisms on various biological activities of them have been neuronal differentiation induced by C. tiglium methanol reported25–27), however reports concerning with neuro- extract may different from that by NGF. nal differentiation cannot be found. Thus, components, except phorbol esters, inducing neuronal differentiation 4. Discussion may be included in C. tiglium. NGF causes immediate activation of ERK, p38 In the present study, it was confirmed that C. MAPK, and JNK in PC12 cells28). Because, the neurite tiglium methanol extract showed low cytotoxicity and outgrowth induced by NGF is down-regulated by addi- high activity for phosphorylation of ERK1/2 among tion of inhibitors of these three MAPKs pathway or 171 extracts obtained from 57 kinds of traditional expression of dominant negative mutants29–31), it is medicinal plants in Myanmar. Additionally, it was understood that the MAPK cascades considerably take deduced that neuronal differentiation was induced by C. part in the induction of neuronal differentiation by NGF tiglium methanol extract because it up-regulated both in PC12 cells. Thus, the effect of NGF in the present neurite outgrowth and expression of NFM. On the other study was compatible with earlier reports. On the other hand, from the results using three MAPK inhibitors, it hand, because the neuronal differentiation by the C. was considered that phosphorylation of five kinases tiglium methanol extract did not need the activation of (ERK1/2, p38MAPK JNK, ERK5, and Akt) and two p38 MAPK, unlike NGF, it was presumed that action

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mechanism of neuronal differentiation of NGF and C. A. M.; Nordborg, C.; Peterson, D. A.; Gage, F. H. tiglium is different. (1998): “Neurogenesis in the adult human hippocam- pus”, Nature Medicine Vol. 4, No. 11, pp. 1313-7. In the meanwhile, ser-133 of CREB is phosphory- 6) Nakatomi, H.; Kuriu, T.; Okabe, S.; Yamamoto, S.; lated via activation of both ERK1/2 and p38MAPK in Hatano, O.; Kawahara, N.; Tamura, A.; Kirino, T.; PC12 cells by NGF. Additionally, phosphorylation of Nakafuku, M. (2002): “Regeneration of hippocampal CREB induced by NGF was completely regulated by pyramidal neurons after ischemic brain injury by recruit- ment of endogenous neural progenitors”, Cell Vol. 110, addition of PD98059 (MAK inhibitor) and SB203580 No. 4, pp. 429-41. 32) (p38 MAPK inhibitor) . Thus, it could be considered 7) Barbacid, M. 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