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Hiroko Isoda, 〇Kazunori Sasaki, Midori Sakamaki, Shinya Takahashi, Makoto Watanabe, Mikihide Demura, Masaki Yoshida, Hideo Kigoshi, Masaki Kita, Kenichi Tominaga
The exploration research into medicinal functions of Aurantiochytrium and Botryococcus braunii using bioassay
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)
To the best of our knowledge, there have been, however, few reports exploring the physiological effects of Aurantiochytrium and Botryococcus braunii.
Microalgae
Botryococcus braunii
3
3
99.5% EtOH
・Aurantiochytrium・Botryococcus braunii
(freeze dry)
2 weeksSterilizedby filter
Extraction method
Concentration
The dried algae samples weredissolved in milliQ water
In vivo experiment
RAW264.7 cells
PC12 cells
In vitro experiment
ICR mice
4
Anti-inflammatory effects of algae extracts
5
• However, aberrant inflammation is associated with the development of chronic disease.
AberrantInflammation
Cancer
Alzheimer
Diabetes
Neurologicaldiseases
Arthritis
• However, major concerns are the undesirable side-effects caused by long term use of such drugs.• Clinically, steroids are commonly used.
Therefore, there has been increasing interests in the search of natural products possessing anti-inflammatory potential.
• Inflammation is very important mechanism for the host defense.
Inflammation
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.
6Outline of experiments (Anti-inflammatory effect)
Inflammatorymediators
Macrophage
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).
seedingSampletreatment
24h
LPSstimulation
Bio assay24h 12h
To investigate the anti-inflammatory effect of Aurantiochytrium, nitrite oxide (NO) production assays and inflammatory cytokine production on raw 264.7 cells-treated with LPS were performed.
7Effect of Aurantiochytrium extracts on NO and inflammatory cytokines production
• Aurantiochytrium extracts significantly inhibited the production of LPS-induced NO and inflammatory cytokines on RAW264.7 cells.
8
Anti-depressant-like effect of algae extracts
9Depression
Side effects
Market about anti-depressant drugs
The size of the anti-depressant drugsmarket is increasing, reaching a marketsize of 15 million yen in 2022.
Loss of sleep, Loss of memory, Drug
dependence, Dizzyvisual defect … etc
Depression is a common and lifethreatening psychiatric disorder with ahigh prevalence, affecting more than 121million people worldwide.
Safe anti-depressants, from natural products, without side-effects are required
Cosmarium Trachelomonas Trachelomonas Desmodesmus BotryococcusAurantiochytrium
8009001000110012001300140015001600
1 2
Marketsize
(Ten
thou
sand
yen
)
Mar
ket s
ize
(Ten
thou
sand
yen
)111
1
2013 2022
10
A measuring of mice’s weight & Oral administration: Every days
Tail Suspension Test (TST)Group 1: control, distilled waterGroup 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 waterGroup 2: positive control (imipramine 20 mg/kg)Group 3: Botryococcus braunii (100 mg/kg)Group 4: Aurantriochytrium (100 mg/kg)
0 1 5 9 13
1 week
0 7
TST and FST
2 weeks2 weeks
Acclimatization
Mice: male ICR mice Age: 5 weeksGroups: control, (+) control and 2 treatment groups Administration: oral administrationTreatment: 14 days
♂
Experimental design (Anti-depressant-like effect)
11
A decrease in the duration of immobility is indicative of an anti-depressant-like effect of extract
TST: is a psychological stress used to investigatethe anti-depressant-like effect of drugs. Mousesuspended by the tail shows alternate periods ofagitation and immobility. The sum time ofimmobility periods during which mice remainedimmobile was measured in a test period of 6 min.
50 c
m
The tail was taped at 1 cm from the tip
acoustically and visually isolation
Tail suspension test (TST)
12
12
Effect of Aurantiochytrium and Botryococcus on mice immobility timeduring TST
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
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14
Imm
obili
ty ti
mes
(s)
Days
Control Imipramine Botryococcus Aurantiochytrium
** **
****
****
* P < 0.05, ** P < 0.01Compared with control-mice (water-treated group)
****
*
13
FST: is a physical stress used to investigate theanti-depressant-like effect of drugs. The test isconducted in a cylinder filled with water. Animalsare exposed to water for 5 min test. Two types ofbehavioral activity are quantified: climbing andimmobility .
14 cm
19 c
m
25 c
m
25 ±1°C
Forced swimming test (FST)
A decrease in the duration of immobility is indicative of an anti-depressant-like effect of extract
14
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.
Effect of Aurantiochytrium and Botryococcus braunii on mice immobilitytime during FST
0
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14
imm
obili
ty ti
mes
(s)
Days
Control Imipramine Aurantiochytrium Botryococcus
****
****
****
** P < 0.01Compared with control-mice (water-treated group)
****
**
15
MouseBrain
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.
Molecular mechanism analysis of anti-stress effect
16
Gene name Hold change (Imipramine)
Hold change (Aurantiochytrium)
Hold change (Botryococcus)
Associated phenotype
Shox2 2.41 ** 3.02 ** 3.09 **
NeurogenesisPitx2 1.84 ** 2.38 ** 2.37 **
Samd4 1.50 * 1.39 * 1.85 **
Lhx9 1.69 ** 1.63 ** 1.39 *
Pnpt1 1.06 2.07 ** 2.05 **Energy production
Arrdc4 -1.08 -1.23 * -1.33 **
Rsrc1 1.63 ** 1.51 * 1.49 *Dopamine synthesis
Ppp1r1b -3.83 ** -8.16 ** -5.54 **
Effects of Algae extracts on gene expression in brain of mice (Microarray)
Administered Aurantriochytrium and Botryococcus induced significantly changed of neurogenesis-, energy metabolism-, and dopamine synthesis-related genes
17
PyruvatePyruvate
Carboxylase (PC)
TCA cycle
NADHNAD+
ATP
ADP
Effects of Algae extracts on gene expression in brain of mice (PC and BDNF)
Ø Our data showed that the treatment of FST-stressed ICR mice with Aurantriochytriumand Botryococcus significantly enhances the gene expression of PC and BDNF in brain.
Energy metabolism
Oxaloacetate
Citrate
0
0.5
1
1.5
2
Control Aurantiochytrium Botryococcus
Rel
ativ
e B
DN
F ex
pres
sion
0
0.5
1
1.5
2
Control Aurantiochytrium Botryococcus
Rel
ativ
e PC
exp
ress
ion
** **
** **
BDNF expression
PC expression
Brain-derived neurotrophic factor
(BDNF)
** P < 0.01Compared with control-mice
** P < 0.01Compared with control-mice
18Effects of Algae extracts on gene expression in brain of mice (TH)
0
0.5
1
1.5
2
Control Aurantiochytrium Botryococcus
Rel
ativ
e TH
exp
ress
ion
TH expression
Ø FST-stressed ICR mice treated with Aurantriochytrium and Botryococcussignificantly increase TH expression
in brain.
Ø Our result suggested that treatment of Aurantriochytrium and Botryococcus enhance
neurotransmitter production.
**
**
Dopamine Noradrenaline AdrenalineL- DOPATyrosine
Tyrosine hydroxylase (Th)Biosynthesis of neurotransmitter catecholamines
Catecholamines
19
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.
Effect of Aurantriochytrium and Botryococcus on ATP production in brain
FST
0
0.5
1
1.5
2
2.5
Control Aurantiochytrium Botryococcus
Bra
in A
TP L
evel
0
0.5
1
1.5
2
2.5
Control Aurantiochytrium Botryococcus
Bra
in A
TP L
evel**
*
* *
TST-stressed mice brain FST-stressed mice brain* P < 0.05, ** P < 0.01Compared with control-mice
* P < 0.05, Compared with control-mice
20
PC12 cells : 1x104 cell/well,DMEM+ F12 +10% HS +5%FBS +1% PS. Poly-l-lysinecoated 96 well plate. Treatmentwith algae extracts .
Neuroprotective assay
MTT (5 mg/ml)
seeding
Sampletreatment
24 h
Absorbance measurement at 570 nm
1 h
6h
SDS (10%)
Corticosterone(stress hormone, 200 µM)
48 h
24h
PC12 cells: Neuronal model cell
High concentration of corticosterone
Cellular damage
Corticosterone:Main glucocorticoid ofrodents
Aurantiochytrium Botryococcus braunii
Outline of Neuroprotective assay
21Neuroprotective effect of algae extracts on Corticosterone-induced cell death
q Algae extracts showed a neuroprotective effect against corticosterone-induced cell death by increasing the cell viability.
0
0.2
0.4
0.6
0.8
1
1.2
Control Corticosterone Aurantiochytrium Botryococcus
Rel
ativ
e ce
ll vi
abiit
y
**
##
##** P < 0.01Compared with non-treated cells
## P < 0.01Compared with corticosterone-treated cells
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaSample (v/v) --Corticosterone
-+ + +
Aurantiochytrium Botryococcus
22Conclusion (Anti-depressant-like effect)
BDNF
Oral administration Dopamine synthesisRsrc1
Ppp1r1bTh
PCPnpt1
Arrdc4
ATP productionNeuroprotection
Neuronal function
Aurantiochytrium andBotryococcus 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
Conclusion
ICR mouse
DopamineNoradrenaline
Adrenaline
Anti-depressant-like effect
Dopaminergic functions
Aurantiochytrium Botryococcus braunii
Neurogenesis
Energy production
Shox2Pitx2
Samd4Lhx9
23
Thank you for your kind attention