Dose of Fucoxanthin 3.5 Mg (5.6 Μmol)/Day/Mouse for 1 Month （Equal to 760 G Per Kg Bw of Raw Kombu)

Searching for new functional properties in traditional foods of Japan and South America

November 8, 2010 Kazuki Kanazawa Most of functional factors in food are non-nutrients.

Nutrients (proteins, sugars and lipids) undergo decomposition to produce energy (catabolism) in liver. This indicates that nutrients cannot exhibit bio-functions.

Contrarily, non-nutrients do not undergo catabolism, and therefore can exist in mostly their unchanged forms in food and can exhibit diverse bio-functions. However, most of non-nutrients undergo a different metabolism, conjugations, in intestinal surface cells.

The conjugations occur on the functional groups, masking with glucuronic acid and/or sulfate, and then invalidate active non-nutrients. A strategy for finding out biofunctional factors

To find out endogenously active functional non-nutrients, difficult forms to undergo the conjugations should be searched according to the following strategy:

1) Compounds retaining the activity even after the conjugations 2) A hard chemical to undergo conjugations 3) Unchanged chemical during absorption process 4) Metabolically activated chemicls when absorbed 5) Available chemicls after de-conjugation with endogenous β-glucuronidase 6) Compounds that can interact with intestinal surface cells and transfer immune signals without absorption The second idea Propolis includes a hard chemical to be conjugated, artepillin C

Conjugate Bee collects from enzymes Baccharis dracunculifolia (chilca)

Prenyls monolayers in Caco-2 artepillin Bioavailability of Free of; Conjugated of; □; Artepillin C △ ○; Ferulic acid ; p Artepillin C Ferulic acid p -Coumaric acid -Coumaric acid C C

Phenolics (nmol/well) 0.3 0.6 0.9 1.2 1.5 0.2 0.4 0.6 0.8 1.0 1.0 2.0 5.0 3.0 4.0 0 0 0

△

○ □

Serosal D N D N D N In thecellIn 30

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D N D N D N 60

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D N

90 Incubation time (min) Incubation

D N D N D N *** 120 ***

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0.1 0.2 0.3 0.4 0.6 0.8 1.0 0.2 0.2 0.3 0.4 0.1 0.5 0 0 0

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Serosal D N *** ▼ ** ◆ ▲ ▲ ▼ ◆ D N 30 ▼ ▲ ◆ D N ■ ● ● ■ *** D N ■ ●

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Conjugated of; Free of; ▼; Kaempferide ▲; Kaempferol ■; Isosakuranetin ◆ ●; Naringenin Naringenin ; Chrysin Kaempferol Kaempferide Isosakuranetin Chrysin Artepillin C can Enter HepG2 Cells and Suppress Oxidative Stress

3 - HepG2 cells Exposed to O2

cells) cells) ○ ○ 7 ○ ○ 2 Artepillin C (µM) Lipid peroxides ○ Artepillin C (nmol/mg protein) △Conjugated Artepillin C 1 0 0.468+0.013 5 0.426+0.010* Artepillin C (nmol/10 Artepillin 10 0.446+0.006* 0 E △ △ △ △ 0 30 60 90 120 150 20 0.392+0.013* Incubation time (min) 8-OHdG/2’-dG X 10-5

0 8.22+1.089 H

O O 5 7.14+0.411 10 5.35+0.724* 20 5.23+0.176* O H Mixed Polyphenols 7.18+0.815 Mixed polyphenol + 10 µM of Artepillin C 4.86+0.481* Speculation Artepillin C daily P. O. ddY mice (♂) 6-weeks old 1st day 8th day 28th day

Azoxymethane (AOM) 10 mg/kg subcutaneously

Artepillin C inhibits formation of aberrant crypt foci Treatment Number of ACF % of Control ACS/Focus Negative control (non-treatment) 0 0 0 Positive control (vehicle alone) 63.25 + 16.5 100 1.50 + 0.18 80 mg/Kg propolis 38.4 + 14.5* 60.8 1.40 + 0.20 160 mg/Kg propolis 35.6 + 6.8* 56.3 1.57 + 0.14 10 mg/Kg artepillin C 35.8 + 15.2* 56.6 1.49 + 0.19 n=5, p<0.05 Artepillin C Induces G0/G1 Arrest in WiDr

Proliferation G0/G1 (%) G0/G1 ■ 0.1% DMSO ● 25 µM 250 ■ S ▲ 50 µM ■ ● 200 ◆ 75 µM ● G2/M ▼ 100 µM ▲ ■ ▲ ◆ ■ ▲● ◆ ◆ ▼ 100 ◆●▲ ▼ ▼ Artepillin C ▼◆●▲■ ▼ (µM) G0/G1 (%) S (%) G2/M (%)

0 h 0 61.6 ±0.8 33.0 ±1.7 5.4 ±1.0 0 0 24 48 72 24 h 0 61.6 ±4.8 29.0 ±4.3 9.4 ±2.2 Incubation time (h) 25 62.5 ±1.7 27.8 ±0.6 9.7 ±1.0 50 68.8 ±0.4 22.1 ±0.4 9.1 ±0.5 100 82.1 ±3.2 13.4 ±2.3 4.5 ±1.0

48 h 0 63.2 ±1.4 29.9 ±1.3 6.9 ±0.1 25 77.9 ±0.6 17.1 ±0.5 5.0 ±0.5 50 80.3 ±0.2 14.4 ±0.5 5.3 ±0.7 100 86.9 ±0.2 6.7 ±0.3 6.4 ±0.1 TGF-β DNA oxidation

Growth factor Rac ATR ATM P Ras proteosome GF-receptor P P P MAPkinase PI3K （JNK） MAP kinase cascade P Smad2/3 P （ERK） P Ser15 P Thr68 P cascade Smad4 P P PIP3 MDM2 Chk2 P Smad2/3 p53 P Ser20 P P FKHR P P PDK P Ser473 Thr308 P Akt Bax INK4 family p15 p21Cip1 p27Kip1 p18 p19 P P Bad GSK-3β p16 Apoptosis

Thr14 P P Cyclin D Thr160 P CDK2 Tyr15 P P Ser123 Apoptosis CDK4/6 Cyclin E cdc25A De-phospjorylation Proteosome INK family Retinoblastoma Proteosome DP E2F Gene expression

P P P P Check point Retinoblastoma G1 phase

S phase Artepillin C Regulates Cell-Cycle Related Proteins

Artepillin C 48 h

0 25 50 100 (µM) P Rb-C fusion protein (Ser 780)

P Rb-C fusion protein (Ser 807/811)

0 h 24 h 48 h

0 25 50 75 100 0 25 50 75 100 (µM)

Cyclin D1

Cyclin D2

Cyclin D3

CDK4

β-actin In up-stream signals

Artepillin C 48 h Control (0.1% DMSO) 50 µM Artepillin C 0 25 50 100 (µM) 0 6 12 24 36 48 6 12 24 36 48 (h)

Cip1/p21 Cip1/p21 1.0 1.73 1.89 2.78 Kip1/p27 Fold (BAS/28S)

0 h 24 h 48 h Kip1/p27 0 25 50 75 100 0 25 50 75 100 (µM) 1.0 1.28 1.27 0.67 Fold (BAS/28S) Cip1/p21

Kip1/p27 28S β-actin

18S In Cip1/p21-deleted cells 80 Vehicle alone

50 µM artepillin C 60 100 µM artepillin C

Distribution (%) Distribution 20

G0/G1 S G2/M Regulatory events by Artepillin in Colon p21waf p19 p18 ink4a kip1 p16 p15 p57 p27

CDK4 CDK2 Cyclin D Cyclin E

Phosphorylation p

p E2F Rb Rb active E2F p p G0/G1 arrest G0 phase G1 phase G1/S transition Proliferation In Human trial

Artepillin C 20.4 µmol (6.12 mg) 8 Ferulic acid 0.3 µmol p-Coumaric acid 3.4 µmol Kaempferol 8.6 µmol x dG

5 6 Naringenin 1.8 µmol

4 3 Capsules per day 8-OHdG/10 For 3 month 2

0 Basal Final Basal Final n=15 n=15 n=15 n=15 Placebo Propolis Dosed amounts were too small 3) Unchanged chemical during absorption process

OCOCH fucoxanthin HO 3 . Japanese kelp Kombu O

O HO In intestinal surface cells

OH fucoxanthinol HO . O Fucoxanthin Contents O In liver of animal Fucoxanthin per HO Brown algae not human 100 g fresh Wakame (Undaria pinnatifida) 11.1 mg OH amarouciaxanthin A HO Arame (Eisenia bicyclis) 7.7 mg . Hondawara (Sargassum fulvellum) 6.5 mg O Kombu (Laminalia japonica) 17.7 mg OH Hijiki (Hizikia fusiformis) 2.2 mg O Hashimoto et al., Br. J.Nutr. (nmol/g) (nmol/g) (nmol/g) 100 200 300 100 200 300 200 400 600 800 0 0 0 0 0 0 Time dose (hr) after 5 5 5 10 10 10 Single dose of fucoxanthin 0.1 mg(160nmol)/mouse Single offucoxanthin dose 15 15 15 kidney liver spleen 20 20 20

25 25 25

(nmol/g) (nmol/g) (nmol/g) 400 600 100 200 300 200 20 40 60 0 0 0 0 0 0 5 5 20 Time dose (hr) after 10 10 40 15 15 lung heart heart tissue tissue Adipose 60 20 20

25 25 80 (nmol/l) (nmol/l) 100 200 300 Fucoxanthin was not detectable. not was Fucoxanthin Amarouciaxanthin A Fucoxanthinol 20 40 60 80 0 0 0 0 5 5 Time dose (hr) after 10 10 （●） 15 15 serum cells blood Red （○） 20 20

25 25 Fucoxanthin Fucoxanthinol 8 80 Amarouciaxanthin A Fucoxanthin-fed mouse

6 60

4 40 mol/l mol/l mol/l mol/l µ µ 2 20 Control 0 0 Serum Liver Lung Kidney Heart Spleen Adipose

Dose of fucoxanthin 3.5 mg (5.6 µmol)/day/mouse for 1 month （equal to 760 g per kg bw of raw kombu)

In another experiments (20 times more), F344 fed rats on 0, 17.5, 35, 70 mg/day/mouse of fucoxanthin for 90 days and found NOAEL was more than 70 mg/day/mouse. 60 No abnormality; more than 8 mg/day for 5 weeks in humans

40 Pharmaceutical parameter in human 20 Cmax 44.2 nml/L 0 Tmax 4 h 0 5 10 15 20 25 t1/2 7.0 h Time after ingestion (h) AUC(∞) 663.7 nmol/L・h Fucoxanthinol (nmol/l) Fucoxanthinol (nmol/l) NoA bloodaccumulation profile of 18 volunteers indicates with 31 mg that produces no side effects and no toxicity. Amarouciaxanthin A Fucoxanthin cis-isomer of fucoxanthinol Fucoxanthinol Internal Standard chemicals standard In humans fucoxanthin and amarouciaxanthin A were In mouse 4 h after dose under the detection limits

In human 4 h after dose

In human before ingestion 0 10 20 30 Fucoxanthin in drinking water ddY mice (♂) 1st day 8th day 28th day 6-weeks old Subcutaneous injection of AOM treatment ACF %

Negative control 0 0 Positive control 85 + 26 100 Fucoxanthin 50 ppm 60 + 10 70 Fucoxanthin 100 ppm 55 + 21* 65 Two step skin cancer 100 Spontaneous hepatic cancer

20 Detectable tumor % tumor Detectable

0 Fucoxanthin showed the strongest activity to prevent form colon, skin and hepatic cancers among carotenoids. 250 Fucoxanthin (µM) 0 200

150 25 G0/G1 arrest 100 % of control % of control 50 G0/G1 75 100 50 Fucoxanthin

0 S 0 24 48 72 96 G2/M Culture time (h)

Sub-G1(%) G0-G1(%) S (%) G2/M (%) Time Control Fucoxanthin Control Fucoxanthin Control Fucoxanthin Control Fucoxanthin

24h 62.5±3.4 83.7±2.4* 30.1±4.1 11.9±1.9 7.5±0.9 4.25±0.7

48h 69.5±2.7 95.3±0.7* 24.5±1.9 3±0.5 5.8±0.5 1.7±0.4 72h 2.8±0.1 29.3±1* 81.8±0.6 61.2±1 5.3±0.6 3.1 8.7±0.4 4±0.1 96h 4.9±0.1 45.5±1* 78.2±2 48.2±1.9 5.3±0.2 1.8±0.3 9.9 2.2±0.3

Fucoxanthin induced cell cycle arrest at G0/G1 phase in human WiDr cells Fucoxanthin Regulates G1 Cell Cycle Related Proteins

Fucoxanthin ( M) Fucoxanthin (µM) µ 75 0 25 50 75 0 25 50 Phospho-Rb Cdk4 (ser 780) Cyclin D1 Phospho-Rb (ser807/811) Cyclin D2 Rb Cyclin D3

β-actin β-actin

Fucoxanthin (µM) 0 25 50 75 p21 p27

β-actin

Das et al., BBA, 2005 In Cip1/p21（-/-）cells

24 h

wild p21(-/-) G /G : 54.2±1.1 G /G : 48.1±0.1 p21 0 1 0 1 S: 36.3±0.7 S: 45.0±0.3 G2/M: 9.7±1.8 G2/M: 6.7±1.8 Vehicle control control Vehicle

G0/G1: 78.9±1.7 G0/G1: 33.6±1.2 S: 11.2±0.1 S: 56.1±0.2 A target protein is G2/M: 9.8±1.6 G2/M: 6.7±1.6

p21 for fucoxanthinol M) µ (25 Fucoxanthin In HL-60 cells Phase-contrast Fluorescent microscopy microscopy M µ 0 M µ 2

No effects of 4 µM M on peripheral blood µ mononuclear cells of 4 healthy volunteers Was not candidate for a ligand Fas TNFR1, DR5, DR4

C-FLIP Reduction of ΔΨm 50 50 40 40 50 Pro-Caspase8 Pro-Caspase8 Pro-Caspase8 Pro-Caspase8 30 30 40 cells (%) cells cells (%) cells 1 1 20 20 30

10 (%) cells

10 1 Sub-G Sub-G 20 0 0 0 0.01 0.1 1 10 Fucoxanthinol ‐ ＋＋＋

Sub-G 10 CHX (ng/ml) CHX (ng/ml) ‐ ‐ 0.01 0.1 0 Protein synthesis did not participate in Fucoxanthinol ‐ ＋＋＋＋ NAC (mM) ‐ ‐ 1 2 4

Mitochondrial pathway did not participate in

Apoptosis Fucoidan 3 hours Caco-2 cells Mineral oil

Apicsal LPS 3 hours RAW264.7 IL-8 cells basolatral GAPDH

** Caco-2 ** RAW Another 3 hours later, 14 * determined IL-8 in 2.0 ** 12 Caco-2 and TNF-α 1.6 10 in RAW 8 1.2 (ng/ml) α 0.8 - 6

(fold increase) (fold 4 0.4 TNF 2

IL-8 mRNA expression mRNA IL-8 0 0 LPS (100 pg/ml) Budesonide (1 µM) Fucoidan (500 µg/ml) Fucoidan modulates over response of macrophages (R) against LPS and Caco-2 (L)

Tanoue et al., Biochemical Biophysical Research Communications, 2008. Raw kombu (Japanese kelp) extract residual fibers

Remove Remove Arsenic Arsenic Salts Salts A part of Iodine A part of Iodine Anti-cancer Reduction of BBA.1726,328,2005 Fucoxanthin Fucoidan Anti-osteoporosisThank you for kind attentionAllergy Reconstruction BBRC. 2008 JAFC.2010 Anti-obesity Wrapping fucoxanthin No waste BBRC.332,392,2005 with fucoidan is super kombu parts

Functional foods being easy to eat

We are making a novel functional food