Specialized Graduate School Science & Technology Convergence Dept. of Marine-Bio. Conbergence Science / Marine Bioprocess Researh Center Pukyong National University, Busan

“To lose one’s property is to lose a little.

To lose one’s honor is to lose much.

But, to lose one’s health is to lose everything.”

2 The relationship between mortality rate and the consumption frequency of fish and shellfish

Intake frequency of fish and shellfish Cause of death Everyday Often Sometimes Never Neuronal death 1.0 1.08 1.10 1.10 Heart diseases 1.0 1.09 1.13 1.24 Hyperplasia 1.0 1.55 1.89 1.79 Liver cirrhosis 1.0 1.21 1.30 1.74 Stomach cancer 1.0 1.04 1.04 1.44 Liver cancer 1.0 1.03 1.16 2.62 Uterine cancer 1.0 1.28 1.71 2.37 Total death 1.0 1.07 1.12 1.32 Number of investigative subjects 1,412,740 2186,368 203,945 28,943

Institute of preventive cancer in Japan (45;3, 1992) Investigation period: 17 years 3 Development of oriental medicine from marine organism

Traditional Oriental-Chinese Medicinal Database

There are number of terrestrial plants and animals cited in the book on oriental medicine

Plants : 7815 species Animals: 1050 species

Marine organism

Plants : 40 species Coral : 12 species Animals : 134 species Below 10 species are utilized at present

There is a great deal of interest and potential to make use of these marine bio-resources through marine biotechnology research, for the development of drugs which could be used in various medicinal applications.

4 Ecklonia cava

1. Classification: Ecklonia cava is a brown alga (Laminariaceae)

2. Habitat: Jeju island in Korea.

3. Efficacy: Antitumor, MMPs inhibitory, Anti-inflammation, Anti-HIV Anti-osteoarthritis, Antioxidant activity

4. Active Ingredients: Phlorotannins such as eckol, 6,6’-bieckol , dieckol phlorofucofuroeckol

5 Phlorotannin compounds isolated from Ecklonia cava

Fucodiphloroethol G Dieckol

6,6’- bieckol 7-phloroeckol

Phlorofucocuroeckol-A Dioxinodehydroeckol

6 Biological activities of Ecklonia cava…

Matrix metalloproteinase inhibitory activity Anti-inflammatory activity Anti-HIV activity Anti-asthma activity Anti-allergy activity

7 Matrix metalloproteinase (MMP)

 Matrix metalloproteinase (MMP)

 Secreted or transmembrane endopeptidases  MMP Substrates: fibrillar and non-fibrillar collagens, fibronectin, laminin, elastin and basement membrane glycoproteins Calcium and zinc dependent endopeptidase

 MMP functions: - Metastasis - Angiogenesis - Arthritis - Wound healing - Periodontal disease - Osteoporosis Ca Zn - Wrinkle formation - Chronic inflammation

MMPs are a group of endopeptidase that mainly involve in the degradation of cellular matrix 8 Matrix metalloproteinase (MMP)

9 MMP inhibitors in Clinical development

FDA approved

10 Preliminary screening of MMP inhibitors from algae extracts

DCL : Doxycycline, positive control

Weight EtOH Scientific name yield (%) (g) Extract (g) DCL 1. Codium 1 1.63 0.0942 5.78 7 contractum 2. Ulva 10 0.127 1.27 conglobata Blank 2 6 3.Scinaia 2 0.031 1.55 okamurae 5 3 4.Ecklonia cava 10 0.9467 9.47 4 5. Ulva pertusa 4.5 0.0758 1.68 6.Enteromorpha 3.5 0.0134 0.38 compressa 7.Enteromorpha 2.63 0.1555 5.91 linza

Only collagenase treated blank group showed complete digestion of gelatin. Ecklonia cava extract showed strong inhibitory effect on gelatin digestion, same with doxycycline.

11 Effects of EC extract on MMP-2 and MMP-9 activities in HT1080 cells

PMA (Phorbol miristic acetate) : Stimulator of MMP

Ecklonia cava extract suppressed the expression and activation of MMP-2 and 9 dose-dependently in human fibrosarcoma cell.

12 Effects of eckol on PMA-induced protein expression of MMP-2 and 9 in HT1080 human fibrosarcoma

HO OH

EK 0 0 1 10 50 100 μM OH O O OH PMA - + + + + + MMP-2 O O HO OH OH MMP-9 OH O O OH β-actin HO O 4 OH Eckol PMA (Phorbol myristate acetate) : Stimulator of MMP

Eckol inhibits the expression of MMP-2 and 9 dose-dependently in human fibrosarcoma cells.

13 Effect of eckol on cell invasion in the HT1080 3D culture

0 h Blank 36 h 1 µM 36 h 10 µM

36 h Blank 36 h 50 µM 36 h 100µM

After 36 hours of eckol treatment, the highest concentration treatment showed the lower cell invasion. Eckol strongly suppressed tumor cell invasion due to inhibition of the degradation of cell matrix. Eckol, has great potential as natural MMP inhibitor in medicinal application. 14 Signalling pathway that lead to inflammation by NF-κB activation

NATURE REVIEWS IMMUNOLOGY 2005, 5; 749-759 15 Effect of eckol on LPS-induced NO and PGE2 production in RAW 264.7 mouse macrophage

50 180 # # 160 40 140 ** * ** 120 30 100

*** (pg/ml)

2 80 *** 20 NO (µM)NO

PGE 60 *** 10 40

20

0 0 EK (µM) 0 0 1 10 50 100 EK (µM) 0 0 1 10 50 100 LPS (1 µg/ml) - + + + + + LPS (1 µg/ml) - + + + + +

Nitric oxide (NO) and prostagradin E2 are the key mediators of early phase inflammation. Eckol decrease NO and PGE2 levels dose dependently.

16 Effects of eckol on LPS-induced protein (a) and mRNA (b) expressions of iNOS and COX-2 in RAW 264.7 macrophage

EK 0 0 1 10 50 100 μM LPS - + + + + + iNOS

COX-2 (a) β-actin

iNOS

COX-2 (b) GADPH

Eckol suprpessed the COX-2 and iNOS expression

17 Effects of eckol on NF-κB nuclear localization in LPS-stimulated RAW 264.7 macrophage under confocal microscope

 NF-κB is a protein complex that controls the transcription of many inflammatory proteins.

 NF-κB translocation to nucleus cause the transcription of these pre-imflammatory Blank FF 10 µM EK 10 µM cytokines and enzymes.

 100 uM eckol stronlgly suppress NF-κB translocation to nucleus.

 Eckol inhibit inflammation via suppressing the nuclear LPS EK 100 µM translocation of NF-kB.

18 Anti-HIV activity

19 Anti-retroviral agents for HIV

Four anti-HIV targets

Entry stage

Transcription of HIV RNA

Transcription of HIV DNA

Exit of newly formed viruses

20 HIV Entry

HIV-1 gp41

HIV-1 gp120 Envelope Glycoprotein

CD4

CXCR4 T-Cell Surface Gp120 of HIV-1 binds to CD4 cell surface receptor and CXCR4 co-receptor.

21 HIV Entry

HIV-1 gp41

gp120

CD4

CXCR4

T-Cell Surface First, Gp120 firstly binds to CD4 cell surface receptor.

22 HIV Entry

HIV-1 gp41

gp120

CD4

CXCR4

T-Cell Surface Gp120 binds to CXCR4 co-receptor, then, conformational change was happened in gp41.

23 HIV Entry

HIV-1

gp41

gp120

CD4

CXCR4

T-Cell Surface And eventually gp41 anchors the cell membrane.

24 HIV Entry

HIV-1 RNA

HIV-1 HIV-1 Nucleocapsid

T-Cell Surface Finally, the membrane of HIV and the host cell fuses, then, nucleocapsid of HIV-1 is transferred into the host cell.

25 Anti-HIV 1 activity of 6,6’-bieckol derived from Ecklonia Cava

Syncytia Formation 6,6’-bieckol from Ecklonia cava

Uninfected C8166 cells HIV-1RF infected C8166 cells forming syncytia

When an infected cell starts producing HIV proteins, the HIV envelope proteins migrate to the cell surface and neighbouring cells bind each other, forming syncytia.

26 Effects of 6,6’-bieckol on HIV-1 induced syncytia formation in C8166 cells

I. Blank (I) II. HIV-1RF infected control

Fluorescent microscope images of C8166 cells acutely infected with HIV-1.

Cells were labeled with a fluorescent agent after a III. 6,6’-bieckol 25 µM IV. 6,6’-bieckol 50 µM 3-day incubation period.

When bieckol was treated, syncytia formation was clearly inhibited. 27 Effect of 6,6’-bieckol on syncytia formation(A) and inhibition of HIV- 1 RT activity(B) in co-culture system

. RT: reverse transcriptase

6,6'-bieckol significantly inhibited the syncytia formation. In addition, the compound inhibited the RT-activity of HIV in the co-culture system. 6,6'-bieckol protects cells from HIV infection probably via inhibition of HIV gene expression. 28 Anti-asthma activity

29 Anti-asthma effects of Ecklonia cava extract in induced mouse asthma model Induced mouse asthma model Non- ovalbumin(OVA)

EC. extract

Normal tissue Induce asthma Allergic-reaction of respiratory tract • Cytokines

• IgE

• NF-κB

To evaluate the anti-asthma activity of EC extracts, we studied effects of EC extract on mucus secretion, allergic-reaction of respiratory tract and suppression of inflammatory markers

30 Ecklonia cava extracts inhibits pathological changes in lung tissues of asthma mouse

Blank Control EC. Extract OVA - OVA + OVA +

Control showed the mucus cell hypertrophy in asthmatic lung. EC. extract acts against the occlusion by reducing the inflammation in the tissue.

31 Effects of Ecklonia cava extracts on the recruitment of inflammatory cells into BAL(A) and airway responsiveness (B) in asthma mouse

(A) (B) 1200 50 Total cells SalineBlank OVAControl

/ml) 1000 EC. 4 Macrophages 40 OVA+EC Eosinophils 800 30 600 20 400

10 % Baseline Penh 200 0

Cells in in fluidBAL Cells x ( 10 0 Blank대조군 Control천식군 EC extract감태 0 2.5 5 10 50 Methacholine (mg/ml)

The number of macrophages and eosinophils increased in BAL fluids of control groups. But the cells in BAL fluid decreased in the EC extracts treated group. 32 Suppression of Th2 cytokine (A) and Ig E antigen (B) by EC. extract in asthma mouse model

(A) (B) 350 SalineBlank

300 Contro OVAl 1.5 250 OVA+ECEC.

200 1 specific IgE 150 - 100 0.5

Cytokines (pg/ml) Cytokines 50 Serum OVA Serum (OD units) 0 0 Blank Control EC extract IL-4 IL-5 TNF-α Saline OVA OVA+EC

The Th2 cytokines, IL-4, IL-5 and TNF-a, are important in the initiation and propagation of allergic inflammatory responses. The administration of EC extracts reduced the concentrations of IL-4, IL-5 and TNF-a compared with that of the control mouse. 33 Anti-allergy effect

34 Allergic mechanisms

The cross-linking of FcεRI with allergen-IgE complexes causes the release of inflammatory mediators such as histamines and prostaglandins from activated mast cells, which contributes to the allergic responses in asthma,.

35 Effects of dieckol on FcεRI mRNA and protein expression

(A) Blank 5 μM 10 μM 25 μM 50 μM FcεRI α chain

β-acitn

(B) Blank 5 μM 10 μM 25 μM 50 μM

FcεRI α chain

β-acitn

Dieckol showed the reduction of FcεRI α chain gene (A) and protein levels (B) in a dose-dependent manner.

36 Effects of dieckol on binding between IgE and FcεRI

80 85.66%

Blank Cell number Cell The cross linking between Fcε R1 with allergen-IgE is mainly 100 101 102 103 responsible for the allergic 100 μM 50 μM 80 80 reactions. 17.69% 21.14% At the highest concentration, the

Cell number Cell dieckol showed the strongest

Cell number Cell inhibition.

Relative cell number cell Relative 100 101 102 103 100 101 102 103 This data suggest that dieckol may 25 μM 12.5 μM 80 80 be useful for the prevention of 56.43% 66.56% allergic-IgE induced allergic reactions by inhibiting the release

Cell number Cell of histamine. Cell number Cell

100 101 102 103 100 101 102 103

Fluorescent intensity 37 In-vivo anti-atopic effects of EC extract

Positive control : Betamethasone 17,21,-dipropionate A

B atopic dermatitis Positive control EC extract

Dermal administration of Ecklonia cava extract remarkably reduced atoptic dermatitis in mice.

38 Anti-HIV Antioxidant

Ecklonia cava

Anti-allergic Anti-cancer

Anti-asthma Anti-osteoarthritis 39

Development of Marine Nutraceuticals

☞ There are various kind of bioactive substances in marine organism. But it is very difficult to develop them as a drug for human.

☞ There are extremely small quantities of bioactive substances and their structures are highly complex. It is very difficult to synthesize them. ☞ There are many latent bioactive materials in marine organism and seafood processing by-products such as proteins, lipids, carbohydrates and so forth. However, they are not ready to be used in their natural form.

☞ For the bioconversion of these latent bioactive materials, membrane bioreactor technology has several advantages.

☞ Specially, this technology is capable of producing bioactive compounds in massive scale, controlling important factors like molecular weight, continuous production and economical feasibility.

40 Potential ways of utilizing marine bioresources

Biological materials Food Medicine Marine vitamins Muscle

Head Water-soluble Bone/Skin calcium Collagen Intestine

Enzymes Microbial genes Microorganisms

Unutilized byproducts contain valuable bioresources and can be converted into value-added products as functional foods and nutraceuticals.

41 Total fish catch and processing statistics in Korea

Year 2010 (Ton) 2011 (Ton)

Total fish catch 3,348,184 3,244,288 Fresh fish consumption 591,866 421,828

Processed raw fish (processing rate) 2,030,474 (82%) 2,163,926 (87%)

Products (yield %) 1,510,474 (60%) 1,318,132 (53%)

Byproducts * (%) 965,714 (40%) 1,168,910 (47%)

* Byproducts : fish skin, fish bone, fish head, fish intestine, (crustacean shells, shellfish shells)

In South Korea, almost one million tons of seafood byproducts are generated annually from fish processing plants. Only a small percentage is recovered as value-added products. Therefore, there is a need to find methods utilizing these materials efficiently and more meaningfully.

42 Bioconversion of latent bioactive materials into active forms using bioconversion techniques

If we can change these latent bioactive materials into active forms using bioconversion techniques, they can perform remarkable functions in our body.

43 Structures of Chitin, Chitosan & Cellulose

Chitin Å° ƾ CH OH H NHCOCH3 2 H NHCOCH3 O H H O O H OH H OH H H H H When the degree of deacetylation in chitin is over OH H H O O H H O O 70%. The term, chitosan is prefered. H NHCOCH CH2OH 3 CH2OH Celluose is insoluble in water. Since the chemical structure of chitin and chitosan Å°Åä»êChitosan CH OH backbone are very similar to that of cellulose. H NH2 2 H NH2 O H H O O H Chitin and chitosan are also insoluble in water. OH H OH H H H H OH H H O O H H O O H NH CH2OH 2 CH2OH

¼¿·ê·Î¿À½ºCellulose H OH CH2OH H OH O H H O O H OH H OH H H H H OH H H O O H H O O H OH CH2OH CH2OH

44 ● Chitosan is known to have important functional properties in many area such as medicinal material, biopolymer, food, medicine and agriculture. Its application for industry is restricted because of poor solubility and absorption through the human intestine. ● Chemical and enzymatic methods are widely used in COS production approaches. Among them, chemical hydrolysis is used more commonly in the industrial-scale production.

● However, chemical hydrolysis has some drawbacks to be commercialized, due to the development of some toxic compounds, higher risk associated with the environmental pollution and lower production yields. ● Lack of proper technology for the large-scale manufacturing of COS with desired MW makes it difficult for human use in past years.

● The enzymatic processes are generally carried out in batch reactors and are preferable over chemical methods. However, the higher cost associated with hydrolytic enzymes reduce the application of the enzyme methods.

● Therefore, reuse of enzyme is recommended and sequential development is achieved in continuous production of COS in large scale.

45 Continuous production of chitooligosaccharides (COS) using an enzyme membrane reactor system

Pump Thermometer Stirrer Concentrate UF membrane Pump Drying

Chitooligosaccharides Enzyme reactor Supplement Reservoir tank tank

Water bath Pump

The entire reaction mixture is pumped continuously through the membrane module, where the product of the reaction, small enough to permeate through the membrane are removed, while the enzyme and other compounds rejected by membrane are recycled back to the reactor for further reaction.

46 Continuous production of COS using the membrane reactor system

한 Reactor 외

생물반 여

응기 과

막

$ 30 $150

Cross section of Outer wall surface the membrane of the membrane

Hallow fiber cartridge contains a large number of hollow fiber. Each hollow fiber has UF membrane which allow small particles to permeate while large particle could not permeate. 47 Molecular weight distribution profiles of different chitosan oligosaccharides obtained from membrane reactor system

COS I: 5~10 kDa COS II: 5~1 kDa COS III: <1 kDa 0.8 6.6 kDa 5.8 kDa COS I COS III COS II 8.5 kDa 1.5 Hexamer 0.6 3.7 kDa Pentamer

0.4 1.0 Heptamer

0.2 0.5 Absorbance at570 nm Absorbance Absorbance at570 nm Absorbance

0.0 0.0

20 40 60 80 100 20 40 60 Fraction number Fraction number

48 In vivo absorption of COS Rats were fed with FITC-labeled chitosan After 30 min, epithelium of duodenum and jejunum was dissected and observed through a (original magnification: ×100) confocal microscope

Duodenum (3.8 kDa) Jejunum (3.8 k Da) Duodenum (7.5 kDa) Jejunum (7.5 k Da)

Duodenum (13 kDa) Jejunum (13 k Da) Duodenum (22 kDa) Jejunum (22 k Da)

Absorption of COS was significantly increased by their low MW

49 Biological activities of chitooligosaccharides (COS)

Anti-microbial activity Anti-tumor activity Antioxidant activity Enzyme inhibitory activity (MMP, β-secretase, ACE)

50 Antibacterial activities of chitosan and COSs

Bactericidal activity (%) Bacteria Chitosan COS I COS II COS III MW 5-10 kDa MW 1-5 kDa MW < 1 kDa

Streptococcus mutans 100 ± 0 100 ± 0 99 ± 0 99 ± 0 Micrococcus luteus >99 ± 0 70 ± 0 67 ± 3 63 ± 7 Staphylococcus aureus 100 ± 0 97 ± 3 95 ± 0 93 ± 9 Bacillus subtilis 98 ± 2 63 ± 5 60 ± 5 63 ± 7 Escherichia coli O-157 >99± 0 71 ± 3 56 ± 4 60 ± 2 Escherichia coli >98± 0 98 ± 5 62 ± 5 51 ± 7 Salmonella typhi >99 ± 0 91 ± 0 88 ± 0 89 ± 0 Pseudomonas aeruginosa 68 ± 3 47 ± 5 35 ± 5 22 ± 8

Chitosan showed stronger bactericidal effects on all bacteria tested except Pseudomonas aeruginosa. The three oligosaccarides have also significantly inhibiting the growth of the bacteria although their effects were lower than that of chitosan.

51 MICs of chitosan and COSs against several virulent bacteria

0.5

Chitosan 0.4 COS I COS II COS III 0.3 MIC(%) 0.2

0.1

0 Str. mutans St. aureus B. subtilis M. luteus St. epidermidis

COS I: 5~10 kDa COS II: 1~5 kDa COS III: <1 kDa

MIC value of chitosan were less than 0.06%. COS were able to inhibit same species effectively. All samples MIC values for Str. mutants were below 0.04% unrelated to molecular weight.

Streptococcus mutants which induces dental caries was completely inhibited by COS, suggesting that gargling with a COS solution, preventing our teeth from decaying. 52 Antifungal effects of chitosan and COSs prepared with different molecular weights

(a) Aspergillus niger (b) Alteraria mali

Chitosan Chitosan COS I COS I

Control Control

COS II COS III COS II COS III

COS I: 5~10 kDa COS II: 5~3 kDa COS III: <1 kDa

No fungal growth was visible in cultures treated with chitosan. COS also has an antifungal effect. The higher Mw of COS, the greater antifungal effect.

53 Biological activities of chitooligosaccharides (COS)

Anti-microbial activity Anti-tumor activity Antioxidant activity Enzyme inhibitory activity (MMP, AchE)

54 Effect of COSs on the growth of implanted sarcoma 180 and uterine cervix carcinoma tumor cells in mice

Sarcoma 180 tumor cells Uterine cervix carcinoma tumor cells Sample Dose Number Tumor weight Inhibition Tumor weight Inhibition (mg/Kg/day) of mice (mg) rate (%) (mg) rate (%)

Control 12 1032.5 ± 839.5 912.2 ± 612.1 COS I 50 12 1147.0 ± 933.9 - 965.2 ± 839.7 - (5~10kDa) 20 12 901.0 ± 741.7 12.7 803.3 ± 641.8 11.9 10 12 795.5 ± 384.8* 22.9 772.7 ± 592.2 15.3 COS II 50 12 345.2 ± 218.6* 66.6 240.5 ± 202.5* 73.6 (1~5kDa) 20 12 665.6 ± 340.1 35.5 352.3 ± 331.1 61.4 10 12 739.5 ± 351.8 28.4 669.5 ± 562.3 26.6 COS III 50 12 904.0 ± 510.3 12.4 665.0 ± 477.9 27.1 ( < 1kDa) 20 12 874.8 ± 516.6 15.3 841.1 ± 602.1 7.8 10 12 973.5 ± 417.1 5.7 879.9 ± 650.3 3.5 * P<0.05 versus the control group

Among three kinds of COSs tested, COS II effectively inhibited the growth of both types of tumor. Therefore it is clear that MW of COS is very important for anticancer activity.

55 Comparison of COSs on survival effect of BALB/c mice implanted subcutaneously with sarcoma 180 cell

16 Control COS I 14 COS II COS III 12 10 8

6 88.5% 4 Number of of Number survival mouse 2 69.7% 30.4% 0 10 15 20 25 30 35 40 45 50 Survival days

There were no survivors in the control group after 30days, however the groups treated with COS II exhibited extremely higher survival prolongation rates 88.5%.

56 Effects of COSs on the gene expression of MMP-1 in human dermal fibroblasts

G3PDH

MMP-1

Intergrated density value of MMP-1 PCR products

250000

200000

150000

100000

50000

0 Blank Control COS <1KDa COS 1KDa~3KDaCOS 3KDa~5KDa COS 5KDa~10KDa

MMP-1 mRNA expression clearly exhibited that COS with 3-5 kDa is effective in controlling the expression of MMP-1 compared to the other MW of COS.

57 Effects of COSs on MMP-9 in human dermal fibroblasts

BK PMA 50 100 500 1000 µg/ml

MMP-9

activity(%) 9 MMP - Intensitive value (%) value Intensitive

PMA (Phorbol miristic acetate) : Stimulator of MMP

MMP-9 is also a very important enzyme that activates specially during metastasis of cancer. We identified that MMP-9 can also be inhibited by COS.

58 Biological activities of chitooligosaccharides (COS)

Anti-microbial activity Anti-tumor activity Antioxidant activity Enzyme inhibitory activity (MMP, β-secretase, AchE)

59 Free Radicals and Reactive Oxygen Species

Antioxidants

Living organisms Foods

• Inflammation • Rancidity • Diabetes • Arthritis • Off-flavor • Parkinsonism • Structural instability • Cancer • Shorter shelf-life • Aging

60 Effect of COSs with different MW on scavenging of radicals using electron spin resonance (ESR)

Control COS < 1 kDa COS 1-3 kDa (100 ug/ml) Hydroxyl radical

Superoxide radical

Carbon-Centered radicals

COS bearing below 1 kDa molecular weight could scavenge hydroxyl, superoxide and alkyl radicals better than that of 1-3 kDa COS.

61 Effect of COSs on inhibition of intracellular generation of ROS in melanoma cells

6000 (C) 100 µg/ml 5000

4000 ** Melanoma (B16F1) ** cells were treated with 3000 ** ** COS and intracellular ** formation of ROS was 2000 ** ** assessed using 1000 ** oxidation sensitive dye, DCFH-DA( 2′,7′- DCF intensity fluorescence 0 dichlorofluorescin diacetate) 0 30 60 90 120 Time(min)

Low MW COS, generation of intracellular radicals was greatly reduced with time.

62 Inhibition of DNA oxidative damage by COS

COS <1 kDa (µg/ml) Genomic DNA from H2O2 10 50 100 500 BLK (2 mM) B16F1 cells was pre- treated with COS and exposed to hydroxyl radicals.

After 30 min, DNA was electrophorased on a 1% agarose gel and visualized with EtBr

DNA damage was protected by low MW COS which was dose dependent.

63 Protective effect of COS on the damage of pancreas cancer cells induced by streptozotocin.

Control

STZ

COS

NIT-1 cells : pancreas cancer cell

Photomicrograph of NIT-1 cells DNA migration in different experimental groups (× 200).

STZ induces pancreas cancer cells to be damaged by oxidative stress and undergo DNA migration. However, COS addition protects pancreas cancer cells from STZ-induced cell damage due to its anti- oxidant effect. 64 Development of Bio-active Peptides

65 Schematic diagram of the three-step recycling membrane reactor for the preparation of bioactive peptides from fish processing byproducts

PCV PCV PCV

pHIC pHIC pHIC NaOH FI NaOH FI NaOH FI T T T

PI PI PI Protein solution FSEH SSEH TSEH Heater Heater Heater

This system enables consecutive digestion of protein using different specific enzymes and simultaneous separation of products, based on their MW using UF membranes.

66 ACE inhibitory peptides purified from different fish protein hydrolysates using UF membrane system

Protein source Peptide IC50 (uM) AP skin collagen Gly-Pro-Leu 2.65 a AP skin collagen Gly-Pro-Met 17.13 a AP frame protein Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala 14.7 b YF skin collagen Met-Ile-Phe-Pro-Gly-Ala-Gly-Gly- Pro-Glu-Leu 10.75 c YF frame protein Arg-Pro-Asp-Phe-Asp-Leu-Glu- Pro-Pro-Try 15.02 d Soy protein Pro-Try-Gly-Ala-Glu 22.56 e Spinach protein Leu-Pro-Leu-Gly-Pro 18.76 f

IC50: Half maximal (50%) AP: Alaska pollack, YF: Yellowfin sole inhibitor concentration

Angiotensin I converting enzyme (ACE) plays an important physiological role in regulating blood pressure.

67 Effect of ACE inhibitory peptides on systolic blood pressure of spontaneously hypertensive rat (SHR)

(Alaska pollack skin gelatin peptide) : Gly-Pro-Leu

Peptide

- Single oral administration was performed with the dose of 10 mg/kg of body weight - Systolic blood pressure was measured at 0, 1, 2, 3, 6 and 9 h after the administration.

The peptide exhibited a similar potency to Captopril in reduction of the systolic blood pressure of SHR.

68 Antioxidative peptides purified from different fish protein hydrolysates using UF membrane system

Fish protein derived peptide sequences that are capable of acting as antioxidants.

69 Antioxidant activity of purified peptide from hoki skin collagen in linoleic acid oxidation system

RSP

Radical-scavenging peptide (RSP) purified from Hoki skin collagen

His-Gly-Pro-Leu- Gly-Pro-Leu (797 Da)

Antioxidant activity of purified peptide was higher than α-tocopherol in linoleic acid oxidation system.

70 Protective effect of the purified peptide from oyster hydrolysate against hydroxyl-radical-induced DNA damage

Purified peptide from oyster hydrolysate Leu-Lys-Gln-Glu-Leu-Glu-Asp-Leu-Leu-Glu-Lys-Gln-Glu (1.60 kDa)

FeSO4 & H2O2 Blank Control 45.6 uM 22.8 uM 5.7 uM

OC DNA : Open circular DNA, SC DNA : Super coiled DNA

DNA damage was protected in the presence of purified peptide dose dependently.

71 Water soluble calcium from fish bone

Fish processing byproducts

$ 1 / kg

Homogenization Extract of fish crude Treatment of fish crude intestinal enzyme intestine enzyme

Water Soluble Calcium

$ 50~100 / kg

72 Calcium ion transferring and influx activity using confocal laser scanning microscope

Confocal Laser Scanning Microscope image

Commercial Casein phosphopeptide Soluble Ca from Products (CPP) fish bone

Water soluble calcium showed strong calcium transferring and influx activity in Osteoblast like CPP which is a famous commercial calcium accelerator.

73 Measurements of the strength of femur after 4 week diets

14

12

10

8

6

Strength of femur (kg) femur of Strength 4

2

0 A B C D

A, lack of Ca; B, Commercial products; C, soluble Ca from fish bone; D, soluble Ca + P-peptides

Water soluble calcium treatment group showed enhanced strengths of femurs compared to commercial products. Furthermore water soluble calcium and CPP mixed treatment group showed slight synergy effect.

74 Bone densities of femur after 4 week diets using SEM image

Lack of Ca Soluble Ca from fish bone

We can identify the remarkable increasing of bone density in water soluble calcium treatment group.

75 Hair growth effect

76 Hair growth effect of IGF-1 in hair dermal papilla cells

Hair cycle

Source: Relevant Research, Inc.International Society of Hair Restoration Surgery, 2013

Insulin-like growth factor-1 (IGF-1)

Sites of action of androgenetic alopecia treatment drugs Promotion of forliferation of hair papilla cell

Release of growth factors such as IGF-1, Baldness prevention and HGF, FGF-7. etc. improvement

In the hair roots, CGRP acts on the hair papilla cells, and the hair paliia cell produces IGF-1, to promote cell division of the hair pailla cell, to grow hair , and to promote hair restoraton!77 777 Effect of marine alga extract on dermal papilla cells, hair shaft elongation and hair follicles

(a) (b) μg/ml

Control E.cottonii (c)

DAPI IGF-1 DAPI IGF-1 Control E.cottonii

DAPI : 4'-6-Diamidino-2-phenylindole staining Effect of marine alga extract on dermal papilla cells (a)hair shaft elongation (b) and hair follicles gene expression (c) fluorcent image of DAPI and IGF-1 staining in hair follicals 78 Growing phase induction effect of marine alga extract on C57BL/6 mice

5 weeks after treatment

Extract treated group show a clear induction of growing phase and increase in hair shaft elongation which means that E.cottonii extract has restored the hair growth cycle. 79

Effect of marine alga extract on human subjects in clinical tests

1. Hair density

Tonic containing 1% of seaweed extract 0 day 16 Week

Control

Sample E. cottonii : Hydroxydihydrobovolide

Sample treated area showed elevated hair density compared to the control. 80 Effect of marine alga extract on human subjects in clinical tests

2. Hair diameter

0 day 16 week

Control

m) μ Diameter(

Sample 16 week

Sample Control

Hair diameter is also increased by sample treatment compared to control group. 81 Effect of marine alga extract on human subjects in clinical tests

3. Hair growth rate

Tonic containing 1% of seaweed extract 0 day 16 week

Control

Sample

16 week

Sample Control Hair shafts are longer compared to control group. This tonic can be produced as a commercial product for hair loss. 82 Erectile Dysfunction

83 Mechanism of Sexual Dysfunction

60

50

40

30

20

10

0 Erectile dysfunction dysfunction (%) incidence Erectile

The Global Study of Sexual Attitudes and Behaviors (GSSAB), 2011

-NO activates the guanylate cyclase which converts GTP to cGMP cGMP induces erection

84 84 Effect of Urechis unicinctus on NO and GTP production for an erection of the penis

A) NO assay Urechis unicinctus

- lives in soil or mud - eaten raw with salt and sesame oil in Korea DDL (Asp-Asp-Leu)

3000 1.2 B) C) 2500 1.0

2000 0.8

0.6 1500 p

0.4 1000

0.2 500 NO production (%) GTP [Phosphate] [Phosphate] (μM) GTP 0.0 0 Blank Control 개불 Blank Control 개불 Blank Control Urechis unicinctus Blank Control Urechis unicinctus Nitric Oxide production in Urechis unicinctus using the NO assay GTP production in Urechis unicinctus using the ATP assay Urechis unicinctus enhances the NO and GTP production

85 Effect of Urechis unicinctus on eNOS and iNOS production in vascular endothelial cells

eNOS and iNOS are important modulators of NO production related to erection. Sample treatment increased both eNOS and iNOS expression in a dose dependant manner. At the concentration of 1000 ug/ml, expression eNOS was almost 6 times higher than control.

86 In-vivo improving sexual dysfunction effects of Urechis unicinctus extract

Evaluation of sexual dysfunction

Mount Glooming Ejaculation

number number number

Mount Glooming Ejaculation Mount Glooming Ejaculation Mount Glooming Ejaculation

Control 500 mg/kg Cialis

Sexual dysfunction effects of U. unicinctus extract and cialis treatment group were almost similar. This extract could replace synthetic drugs. Because it is safe to use and shows similar effects to commerical drugs. 87 Clinical tests of Urechis unicinctus extract

A randomized, placebo-controlled, double-blind, single-center clinical trial to evaluate the efficacy and safety of Urechis unicinctus extract(UU104) compared to placebo with male hypogonadism

- AMS score (Aging Males’ Symptom Score) variation - Total testosterone variation

- IIEF (International Index of Erectile Function) variation - IPSS (International Prostate Symptom Score) variation

Oral administration of Urechis unicinctus extract shows better activity than control group 88 89 Edited Books

Marine Bioenergy / CRC press Taylor and Francis Group - December 1, 2014

Handbook of Anticancer Drugs from Marine Origin / Springer

Functional Marine Biomaterials - Properties and Application / Woodhead publishing

Marine Algae Extracts / Willey

Handbook of Marine Microalge / Academic Press

Marine Carbohydrates / Academic Press

Marine Omics / CRC Press

Marine Microorganisms : Interaction and analysis of bioactive compounds / CRC Press

Marine Glycobiology : Principle and Application / CRC Press

Encyclopedia of Marine Biotechnology / Willey

90 Bioactive materials from marine organism

Functional Food Anti-hypertension food Anti-asthma food

Hair growth tonic Liver Functional Food Anti-diabetes mellitus food Functional Cosmetics

92 Bioactive materials from marine organism

Microfluidics chip Antihypertensive food Erectile Dysfunction food Sleep improve food

Marine time hair treatment Marine time skin whitening & wrinkle free

93 Summary

Marine organisms live in an extreme environment and there produce novel fascinating and unique bioactive compounds which could not be produced by terrestrial organisms.

We have investigated and developed various marine derived bioactive compounds for variety of biological activities such as anti-inflammatory, anti-H I V, anti-asthma, anti-allergy, sexual dysfunction, hair growth effect and sleep promoting effect. So much attention has been paid on marine bioactive compounds to treat chronic diseases which can be developed as commercial products.

Chemical synthesis of natural products will be an alternative for industrial commercial applications and those that cannot synthesized marine bioactive compounds can be used as oriental medicine

Finally much attention to be paid on marine organism collections for the development of high value added products in industrial scale.

93 Location: south east of korea.

Pukyong National Univeristy

Busan

Area: 765.94 Km2 Population: 3,586,079 (‘12.12.31) Fire festival

Gwanganbridge Haundae beach

Gwangan beach 96 Jagalchi market

Shinsegae department store

APEC house Film festival BIFF Post Doctors

Dr. Kyong-Hwa Dr. J. Venkatesan Dr. Yong-Xin Li Dr. Fatih Dr. Mani Dr. Vo Thanh Dr. Ngo Dai Kang Karadeniz Sang Hung Prof. Se-Kwon Kim

Lab Scholars

Ta Van Yong-Sang Kim Soyeon Kim Babuka Se-Hun Oh Alarm Jun Ho Hyoung Woo Sung Han Ding Su Kyoung Heo Quang

98