Salmon Ovary PeptideSalmon Ovary Peptide

163 Kyoriki Toyama163 Kyoriki Toyama163 Kyoriki, Toyama163 Kyoriki, ToyamaKYOWA YAKUHIN CO., LTD.KYOWA YAKUHIN CO., LTD.

What is Salmon Ovary Peptide?（Wh t i O Pl t ?）（What is Ocean Placanta?）

Salmon Ovary PeptideO l bt i d ft t k ff f lOvary envelope obtained after takeoff of salmon roe

Salmons return to their home rivers from the open sea for 3 to 4Salmons return to their home rivers from the open sea for 3 to 4years.Low PCB, pesticides, and heavy metals storage※A.K.A.：Ocean Placenta

Meaning of the nameAll living things were born from oceanAll living things were born from oceanand evolved from fish to mammal,that is, from egg(egg capsule membrane) to fetus(placenta)

Manufacturing process of Salmon Ovary PeptideSalmon Ovary Peptide

Raw material：Ovary envelope usually disposed aftertakeoff of salmon roe

W hi Cutting & Enzyme Manufacturing Process (Patent）

Frozen salmon ovary envelope

Washing Cutting & fragmentation

Enzyme degradation

Mi filt tiReverse Freeze-

MicrofiltrationUltrafiltration

Reverse osmosis concentration

drying

Patent number:３６９１４９７

【 Manufacturing process of Salmon Ovary Peptide (1) 】

Salmon ovary envelope has been disposedafter takeoff of salmon roe, but it will becamethe ingredient (resouce) of Salmon Ovary

Peptide from now.Peptide from now.

Landing ofSalmon

Removing the ovary from Removed salmonfemale salmon Removed salmonovary

Manufacturing salmon roe S lManufacturing salmon roe Salmon ovaryenvelope

【 Manufacturing process of Salmon Ovary Peptide (2) 】Collected and frozen salmon overy envelope is concentrated with enzyme degradation after thawing, washing, dehydration. And it is finallypacked as SOP lyophilization product for health supplements and cosmetic materials.

Washed salmonovary envelope

Frozen salmon ovary envelope

Enzyme degradation of salmonFinal product: Enzyme degradation of salmonovary envelope

Final product:Salmon Ovary Peptide

Final Product : E h d l i d i d dEnzyme hydrolysis dried product

• Freeze-dried & purified product（OvaryFreeze dried & purified product（Ovary peptide）

Application of Salmon Ovary Peptide

Salmon Ovary PeptideSalmon Ovary Peptide

Food Health Supplement Cosmetic Pet FoodFood Health Supplement Cosmetic Pet Food

Products above field, especially for the purpose of the improvement effect of anti-aging or lifestyle

Salmon Ovary Peptide : PropertiesSalmon Ovary Peptide : PropertiesOrigin & Production：This product is a freeze-d i d tid bt i d f l fdried peptide obtained from ovary envelope of Salmoniformes Salmonidae by enzyme degradationdegradation.Aspect: Slightly yellow to yellow brownpowder with peculiar smellp p

Mean molecular weight ：６００～８００g

Merits of Salmon Ovary Peptide・First article material at home & abroad・Water-soluble and easy processing・Small MMW：600～800（oligopeptide）

cf. Pig placenta extracts, polymers, and foreign elements have large MMWselements have large MMWs.

・High safety No disease such as foot-and-mouth disease, the influenza No worries about pestisides and heavy metals（less than lipid 0 1%）lipid 0.1%）

・ Usable as beauty and sustainance materialslike animal (pig/horse) placenta extractsp g p

・Excellent effects for anti-aging and lifestyle improvement

Process PatentMethod for producing amino acid componentsby enzymatic hydrolysis of fish egg skin

International patentsIn China ＺＣ ０３１４８６９９．１C aIn Hong Kong ０４１０５５３１．１

In Korea ９２８９９／２００３In Korea ９２８９９／２００３

In U.S.A. １０／７３３．６２７

Safety Data of Salmon Ovary Peptidey y pAcute Toxicity Test

LD５０> 2,500mg/kg

NAD on performance status, weight and autopsyNAD on biochemical blood examination at autopsyp yNot containing residual pesticides or heavy metals

Safety Data of Salmon Ovary PeptideBHC UndetectedDDT UndetectedPesticides DDT UndetectedAldrin & Dieldrin UndetectedEndrin Undetected

Pesticides

Arsenic UndetectedCadmium UndetectedT t l M U d t t d

Heavy Total Mercury UndetectedStannum UndetectedTotal Chrome Undetected

ymetals

Total Chrome UndetectedCyanogen UndetectedProgesterone UndetectedHormone

Estradiol Undetected

Analysis Data of Salmon Ovary Peptide＜Food Ingredient＞ Items Results（/ １００ｇ）

W t ５ ２Water ５.２ｇ

Total nitrogen １４.９ｇ

Lipid ０.１ｇ

Ash ５.５ｇ

Na １.７ｇ

Dietary fiber ０.５ｇ

＜Minerals＞ Fe ３.３７ｍｇ

Ca ４９７ｍｇCa ４９７ｍｇ

Mg ３９.４ｍｇ

Cu １２７ｍｇ

Zn ０.０９ｍｇ

Mn ０.５１ｍｇ

Se １７２µｇ

<Vitamins group> Vitamin Ｂ２ ０.６１ｍｇ

Vitamin Ｂ６ ０ ３１ｍｇVitamin Ｂ６ ０.３１ｍｇ

Vitamin Ｂ１２ ２.４ｍｇ

Biotin ２６.４µｇ

Niacin equivalence １１.２ｍｇ

Niacin （Nicotine acid equivalent） ０.１６ｍｇ

<Other Amino Acids> Triptphan ６６０ｍｇ

Japan Food Research Laboratories

Amino acids ＳＯＰ Pig Placenta Human Placenta Salmon Collagen

Asparagine acid 7 25 7 29 4 91 5 25

Amino acid composition comparative chart between SOP & Animal Placenta Amino acid composition comparative chart between SOP & Animal Placenta ExtractsExtracts

Asparagine acid 7.25 7.29 4.91 5.25

Threonine 4.00 4.74 3.39 2.13

Serine 5.96 7.32 4.09 4.86

Glutamine acid 10.11 14.01 5.84 8.27

Proline 7.49 4.42 4.58 9.27

Glycine 21.38 11.52 9.34 36.52

Alanine 10.58 13.38 5.18 11.35

Cysteine 0.55

Valine 3.59 6.53 3.00 1.73

Methionine 2.06 1.97 1.16 1.58

Isoleucine 2.79 1.91 1.05

Leucine 5.13 3.88 4.23 1.95

Tyrosine 1.81 0.71 1.19 0.28

Phenylalanine 1.90 1.52 1.29

Histidine 1.32 1.91 1.30 0.98

Lysine 5.34 7.71 3.61 2.62

Arginine 5.44 3.89 2.88 5.22

Hydroxyproline 3.08 0.45 5.00

Tryptophan 0.32 0.45

Carnosine 0.33 0.43

（mol%）

Ornithine 0.39 0.65

Taurine 1.8

Phosphoserine 0.25

Hydroxyrysine 0.66

Amino acid composition comparative chart Amino acid composition comparative chart between SOP & Animal Placenta Extractsbetween SOP & Animal Placenta Extracts

Amino Acid Composition of SOP

Phe

His

Lys

Hyl

PseOrn

Hyp Asp ThrSer

GluLeu

Tyr

PheArg TrpHyp

Pro

Ala

Cyt

Val

Met

Ile

Gly

Amino Acid Composition of Pig Placenta Extract

Asp Thr

SerLeu

Tyr

Phe

His

Lys

TrpOrn

HypPse

HylArg

Amino Acid Composition of Human Placenta Extract

AspThr

SerTyr

Phe

His

Lys

HypArg

TrpHyl

Pse

Orn

Ser

Glu

ProAlaCyt

Val

Ile

Met

Ser

Glu

ProAlaMet

Ile

Leu

ProAlaGly

Ala

Cyt

Val Gly

Hepatoprotective activity ofS l O P tidSalmon Ovary Peptide

AST

500

600

ALT

200

250

100

200

300

400

IU/I

AST

50

100

150

IU/I

ALT

-100

0

100

St. STZ STZ＋Ovary -50

0St. STZ STZ＋Ovary

In Wister SPF rat（8-week-old）． n=8Dose of STZ(35mg/Kg) and Ovary(0.75g/day) for 3 weeks by oral

administrationadministrationRheology Functional Food Laboratories Co., Ltd.

Assessment on a genetic level of SOP’s protective effect against liver cells using cultured

hepatocytes of human origin (HepG2)NM_006121 Hs.80828 Keratin 1 (epidermolytic hyperkeratosis)BE778706 Hs 530381 Pim 3 oncogene

Enhanced expression Genes

BE778706 Hs.530381 Pim-3 oncogeneNM_000201 Hs.643447 Intercellular adhesion molecule 1 (CD54), human rhinovirus receptorNM_001227 Hs.9216 Caspase 7, apoptosis-related cysteine peptidaseNM_015675 Hs.110571 Growth arrest and DNA-damage-inducible, betaNM_005627 Hs.510078 Serum/glucocorticoid regulated kinaseNM_004045 Hs.125213 ATX1 antioxidant protein 1 homolog (yeast)

000 81 6686 G 1NM_000581 Hs.76686 Glutathione peroxidase 1AI078167 Hs.81328 Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alphaNM_004583 Hs.127764 RAB5C, member RAS oncogene familyNM_002162 Hs.75516 Intercellular adhesion molecule 3NM_005410 Hs.643494 Selenoprotein P, plasma, 1

Decreased expression genesNM_134268 Hs.95120 CytoglobinBF438173 Hs.9914 FollistatinAA164751 Hs.244139 Fas (TNF receptor superfamily, member 6)M55643 Hs.431926 Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105)BC021222 Hs.643509 Cyclin M2U25804 Hs 138378 Caspase 4 apoptosis-related cysteine peptidase

Decreased expression genes

U25804 Hs.138378 Caspase 4, apoptosis related cysteine peptidaseBE878463 Hs.488293 Epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)

NM_004417 Hs.171695 Dual specificity phosphatase 1NM_001186 Hs.154276 BTB and CNC homology 1, basic leucine zipper transcription factor 1NM_002133 Hs.517581 Heme oxygenase (decycling) 1BF676980 Hs.271264 Glutamate-cysteine ligase, catalytic subunitAW341649 Hs.492261 Tumor protein p53 inducible nuclear protein 1NM 004364 Hs 643434 CCAAT/enhancer binding protein (C/EBP) alphaNM_004364 Hs.643434 CCAAT/enhancer binding protein (C/EBP), alphaNM_012289 Hs.465870 Kelch-like ECH-associated protein 1BU683415 Hs.4055 Kruppel-like factor 6AF102988 Hs.170479 Phospholipase A2, group VI (cytosolic, calcium-independent)NM_004834 Hs.431550 Mitogen-activated protein kinase kinase kinase kinase 4NM_000389 Hs.370771 Cyclin-dependent kinase inhibitor 1A (p21, Cip1)

ResultsA protective effect of Salmon Ovary Peptide against liver cells was assessed on

a genetic level using cultured liver cells of human origin（HepG2）.The result showed increased expression of 6 genes; GADD45, SGK,NF B l t thi id 1 l t i P dNFκB, glutathione oeroxide 1, selenoprotein P andRab5C. And expression of Fas gene was decreased.The results suggests Salmon Ovary Peptide has the following effects.

（１）Further increase in cell’s original biologic defence mechanism （Increase expression of antioxidant enzymes, oxidized substances）

（２）Cytoprotection by inhibiting expression of oxidizer fmotive-related cell disorder factors

（３）Funcion to induce newly expression of cell survival factorsfactors

Effect of Salmon Ovary Peptideon blood sugar levelon blood sugar level

Blood Sugar Ave. Blood Sugar

140125

150

175

130

Nomal

STZ

STZ+O

75

100

125

mg/

dl

110

120

STZ+Ovary

Black P

mg/

dl0

25

50

0.0 0.5 1.0 1.5 2.0 2.5

90

100

In Wister SPF rat（8-week-old）． n=8Dose of STZ(35mg/Kg) and Ovary(0 75g/day) for 3 weeks by oral

Normal STZ STZ+Ovary Black PWeek

Dose of STZ(35mg/Kg) and Ovary(0.75g/day) for 3 weeks by oral administration

Rheology Functional Food Laboratories Co., Ltd.

ACE inhibition activity and scavenging & ti id t ti it f S l O P tidantioxidant activity of Salmon Ovary Peptide

Purified peptide ACE inhibition activity（mg／mL）

Scavenging & antioxidant activity（ l／ ）（µmol／g）

Sardine peptide ０．２６ －

Salmon Ovary Peptide

０．２２ ２．４Peptide

Antioxidant effect of Salmon Ovary Peptide

Antioxidative EffectAntioxidative Effect3

OvaryTocopherol

e )

1

2

Abs

orba

nce

O.D

. 500

nm

0 10 20 30 40 50 600

A (O

mg/assay

Inhibition of ferri-thiocyanate production by linoleic acid hydroperoxide

SOD-like activity & inhibition of tyrosinase activityinhibition of tyrosinase activity

SOD-like activity

40.0

50.0

tion r

ate %

） The figure shows SODThe figure shows SOD--like activity of like activity of Salmon Ovary Peptide (SOP) & pig Salmon Ovary Peptide (SOP) & pig placenta extract (PA). SOP has a placenta extract (PA). SOP has a i ifi t hi h ti it th PA ii ifi t hi h ti it th PA i

0 0

10.0

20.0

30.0

tivi

ty leve

l （in

hib

it

SOP

PA

significant higher activity than PA in significant higher activity than PA in low concentration.low concentration.In high concentration, PA has higher In high concentration, PA has higher activity, but the difference was onlyactivity, but the difference was only

0.0

0 50 100 150 200 250

Sample concentration(mg/ml)SO

D a

ct activity, but the difference was only activity, but the difference was only

several % , meaning both have almostseveral % , meaning both have almostsame activity.same activity.

Inhibition of tyrosinase activity

60708090

rat

e (

%) The figure shows inhibition of The figure shows inhibition of

tyrosinase activity of SOP and PA.tyrosinase activity of SOP and PA.SOP is less compared to PA but theSOP is less compared to PA but the

0102030405060

Inhib

itio

n

SOP

PA

SOP is less compared to PA, but the SOP is less compared to PA, but the inhibition activity of tyrosinase was inhibition activity of tyrosinase was confirmed.confirmed.

0

0 50 100 150 200 250

Sample concentration (mg/ml)

Inspection of fat deposition effect of Salmon Ovary Peptide using primary ratSalmon Ovary Peptide using primary rat

visceral adipocytes（VAC）4th day after administration of test product

Controls Salmon Ovary Peptide100mg/mL

In the negative control group, most fat cells became enlarged adult cells.In the group added 100 mg/mL of SOP, more fat cells were seen whichstored small fat droplets than the groups added 50mg/mL of SOP, and there are a few adult cells. In addition, enlarged liver cells were small in number.

NE addition for VAC（１）（１）

The negative contrasting group(seeding 8days, NEaddition 0hr )

Treatment group of ＳＯＰ (seeding 8days, NEaddition 0hr)

NE addition for VAC（（22））NE addition for VAC（（22））

The negative contrasting group(seeding 9days, NEaddition 15hrs )

Treatment group of ＳＯＰ(seeding 9days, NEaddition 15hrs)

The manifested amount of UCP-1

SOPは内臓脂肪細胞のUCP-1の発現量を上げるSOPは褐色脂肪細胞になるのを助けるSOPは褐色脂肪細胞になるのを助ける

1.80

2.00

1 20

1.40

1.60

ve e

xpre

ssio

n）

0.80

1.00

1.20

発現

量（re

lativ

0.20

0.40

0.60

UCP-1の

発

0.00

SOPなし SOP50μg SOP100μg SOP400μg

The activity of Salmon Ovary Peptide●Activity to reduce lifestyle risks●Activity to reduce lifestyle risks

Maintenance and improvement of liver function, improvement of blood sugar levels inhibition ofimprovement of blood sugar levels, inhibition of fat deposition, anti-high blood pressure

●Antioxidation●Antioxidation●Cosmetic efficacy（whitening）

●In addition:●In addition:Collagen-like amino acid composition which is different from animal placentadifferent from animal placenta（Containing many glycine and hydroxyproline）

We can also hope collagen-like activity!!??We can also hope collagen like activity!!??