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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

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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

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Anti-inflammatory effects of algae extracts

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• 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.

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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

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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)

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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)

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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)

****

*

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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

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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)

****

**

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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

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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

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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

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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

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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

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Thank you for your kind attention