The exploration research into medicinal functions of Aurantiochytrium and Botryococcus braunii using bioassay

Transcription

1 1 The exploration research into medicinal functions of Aurantiochytrium and Botryococcus braunii using bioassay Hiroko Isoda, 〇 Kazunori Sasaki, Midori Sakamaki, Shinya Takahashi, Makoto Watanabe, Mikihide Demura, Masaki Yoshida, Hideo Kigoshi, Masaki Kita, Kenichi Tominaga

2 Microalgae 2 Microalgae have served as important sources of functional materials, such as polyunsaturated fatty acids, polysaccharides, minerals, vitamins, enzymes and bioactive peptides. Moreover, microalgae are known to exhibit various biological and physiological activities including anti-oxidant, anti-coagulant, anti-viral and anti-tumor activities. Aurantiochytrium (mangrovei 18W-13a) Botryococcus braunii To the best of our knowledge, there have been, however, few reports exploring the physiological effects of Aurantiochytrium and Botryococcus braunii.

3 Extraction method 3 In vitro experiment 2 weeks Sterilized by filter RAW264.7 cells Aurantiochytrium Botryococcus braunii (freeze dry) Concentration PC12 cells In vivo experiment 99.5% EtOH The dried algae samples were dissolved in milliq water ICR mice 3

4 4 Anti-inflammatory effects of algae extracts

5 Inflammation 5 Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, blood vessels, and molecular mediators. Cancer Arthritis Aberrant Inflammation Neurological diseases Diabetes Alzheimer Inflammation is very important mechanism for the host defense. However, aberrant inflammation is associated with the development of chronic disease. Clinically, steroids are commonly used. However, major concerns are the undesirable side-effects caused by long term use of such drugs. Therefore, there has been increasing interests in the search of natural products possessing antiinflammatory potential.

6 Outline of experiments (Anti-inflammatory effect) 6 To investigate the anti-inflammatory effect of Aurantiochytrium, nitrite oxide (NO) production assays and inflammatory cytokine production on raw cells-treated with LPS were performed. Macrophage Inflammatory mediators RAW264.7 cells Murine macrophage cell line Produce inflammatory mediators by inflammatory stimulation such as lipopolysaccharide (LPS). Macrophages are the main pro-inflammatory cells. In inflammatory, its produce inflammatory mediators such as Nitric Oxide (NO) and inflammatory cytokines (TNF-α, IL-6). seeding 24h Sample treatment 24h LPS stimulation 12h Bio assay

7 Effect of Aurantiochytrium extracts on NO and inflammatory cytokines production 7 Aurantiochytrium extracts significantly inhibited the production of LPS-induced NO and inflammatory cytokines on RAW264.7 cells.

8 8 Anti-depressant-like effect of algae extracts

9 Depression 9 Depression is a common and life threatening psychiatric disorder with a high prevalence, affecting more than 121 million people worldwide. Side effects Loss of sleep, Loss of memory, Drug dependence, Dizzy visual defect etc Market size (Ten thousand yen)1111 Market size (Ten thousand yen) Market about anti-depressant drugs The size of the anti-depressant drugs market is increasing, reaching a market size of 15 million yen in Safe anti-depressants, from natural products, without side-effects are required Aurantiochytrium Cosmarium Trachelomonas Trachelomonas Desmodesmus Botryococcus

10 Experimental design (Anti-depressant-like effect) 10 Mice: male ICR mice Groups: control, (+) control and 2 treatment groups Treatment: 14 days Age: 5 weeks Administration: oral administration Tail Suspension Test (TST) Group 1: control, distilled water Group 2: positive control (imipramine 20 mg/kg) Group 3: Botryococcus braunii (100 mg/kg) Group 4: Aurantriochytrium (100 mg/kg) Forced Swimming Test (FST) Group 1: control, distilled water Group 2: positive control (imipramine 20 mg/kg) Group 3: Botryococcus braunii (100 mg/kg) Group 4: Aurantriochytrium (100 mg/kg) Acclimatization A measuring of mice s weight & Oral administration: Every days week weeks TST and FST

11 Tail suspension test (TST) 11 The tail was taped at 1 cm from the tip 50 cm acoustically and visually isolation TST: is a psychological stress used to investigate the anti-depressant-like effect of drugs. Mouse suspended by the tail shows alternate periods of agitation and immobility. The sum time of immobility periods during which mice remained immobile was measured in a test period of 6 min. A decrease in the duration of immobility is indicative of an anti-depressant-like effect of extract

12 Effect of Aurantiochytrium and Botryococcus on mice immobility time during TST 12 Immobility times (s) Control Imipramine Botryococcus Aurantiochytrium * P < 0.05, P < 0.01 Compared with control-mice (water-treated group) * Days Algae extracts-treated mice decreased their mobility behavior as compared to control mice at 5, 9, and 13 days. Confirmation of the anti-depressant-like activity of Aurantiochytrium and Botryococcus braunii 12

13 Forced swimming test (FST) ±1 C 19 cm 25 cm 14 cm FST: is a physical stress used to investigate the anti-depressant-like effect of drugs. The test is conducted in a cylinder filled with water. Animals are exposed to water for 5 min test. Two types of behavioral activity are quantified: climbing and immobility. A decrease in the duration of immobility is indicative of an anti-depressant-like effect of extract

14 Effect of Aurantiochytrium and Botryococcus braunii on mice immobility time during FST 14 immobility times (s) Control Imipramine Aurantiochytrium Botryococcus P < 0.01 Compared with control-mice (water-treated group) Days Algae extracts-treated mice decreased immobility time compared to control mice at 5, 9, and 13 days. Confirmation of the anti-depressant-like activity of the Aurantriochytrium and Botryococcus braunii.

15 15 Molecular mechanism analysis of anti-stress effect To investigate the molecular mechanisms underlying the anti-depressant-like effects of algae extracts, we performed microarray analysis, Real time PCR, and ATP measurement using mouse brain. Mouse Brain

16 Effects of Algae extracts on gene expression in brain of mice (Microarray) 16 Gene name Hold change (Imipramine) Hold change (Aurantiochytrium) Hold change (Botryococcus) Associated phenotype Shox Pitx Samd * 1.39 * 1.85 Lhx * Pnpt Arrdc * Rsrc * 1.49 * Ppp1r1b Neurogenesis Energy production Dopamine synthesis Administered Aurantriochytrium and Botryococcus induced significantly changed of neurogenesis-, energy metabolism-, and dopamine synthesis-related genes

17 Effects of Algae extracts on gene expression in brain of mice (PC and BDNF) 17 Energy metabolism Pyruvate Oxaloacetate Pyruvate Carboxylase (PC) Relative PC expression PC expression P < 0.01 Compared with control-mice TCA cycle NADH Citrate ADP NAD+ ATP Brain-derived neurotrophic factor (BDNF) Relative BDNF expression Control Aurantiochytrium Botryococcus BDNF expression Control Aurantiochytrium Botryococcus P < 0.01 Compared with control-mice Ø Our data showed that the treatment of FST-stressed ICR mice with Aurantriochytrium and Botryococcus significantly enhances the gene expression of PC and BDNF in brain.

18 Effects of Algae extracts on gene expression in brain of mice (TH) 18 Biosynthesis of neurotransmitter catecholamines Tyrosine hydroxylase (Th) Catecholamines Tyrosine L- DOPA Dopamine Noradrenaline Adrenaline Relative TH expression TH expression Control Aurantiochytrium Botryococcus Ø FST-stressed ICR mice treated with Aurantriochytrium and Botryococcus significantly increase TH expression in brain. Ø Our result suggested that treatment of Aurantriochytrium and Botryococcus enhance neurotransmitter production.

19 Effect of Aurantriochytrium and Botryococcus on ATP production in brain TST-stressed mice brain 2.5 FST-stressed mice brain Brain ATP Level * P < 0.05, P < 0.01 Compared with control-mice * Brain ATP Level FST * P < 0.05, Compared with control-mice * * Control Aurantiochytrium Botryococcus 0 Control Aurantiochytrium Botryococcus Ø TST and FST significantly decreased ATP levels in brain (2014). Ø Brain ATP level was significantly increased by Aurantriochytrium and Botryococcus in TST and FST.

20 Outline of Neuroprotective assay 20 PC12 cells: Neuronal model cell PC12 cells : 1x10 4 cell/well, DMEM+ F12 +10% HS +5% FBS +1% PS. Poly-l-lysine coated 96 well plate. Treatment with algae extracts. Neuroprotective assay Corticosterone: Main glucocorticoid of rodents High concentration of corticosterone Cellular damage seeding 24 h Sample treatment 1 h Corticosterone (stress hormone, 200 µm) 48 h Aurantiochytrium Botryococcus braunii MTT (5 mg/ml) 6h SDS (10%) 24h Absorbance measurement at 570 nm

21 Neuroprotective effect of algae extracts on Corticosterone-induced cell death Relative cell viabiity ## ## P < 0.01 Compared with non-treated cells ## P < 0.01 Compared with corticosteronetreated cells Control Corticosterone Sample (v/v) aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa - - Aurantiochytrium Botryococcus Corticosterone q Algae extracts showed a neuroprotective effect against corticosteroneinduced cell death by increasing the cell viability.

22 Conclusion (Anti-depressant-like effect) 22 Aurantiochytrium Neurogenesis Shox2 Pitx2 Samd4 Lhx9 Botryococcus braunii BDNF Neuroprotection Oral administration ICR mouse Energy production PC Pnpt1 Arrdc4 ATP production Dopamine synthesis Rsrc1 Ppp1r1b Th Dopaminergic functions Dopamine Noradrenaline Adrenaline Neuronal function Conclusion Anti-depressant-like effect Aurantiochytrium and Botryococcus braunii has anti-depressant like effect on mouse and this was through the enhancement of neurogenesis. Futhermore, promotion of ATP production and enhancement of dopaminergic functions were involved in the anti-depressant like effect

23 23 Thank you for your kind attention