Distribution, determination and enhancement of phlorotannins from brown seaweed Ecklon ia cava

1. Distribution, determination and enhancement of phlorotannins from brown seaweed Ecklonia cava Muhammad Tanvir Hossain Chowdhury Department of Biotechnology, The Graduate School of Pukyong National University 22 May 2012

2. OUTLINE Chapter 1 :General Introduction • Chapter 2 : Distribution of phlorotannins in the brown alga Ecklonia cava and comparison of pretreatments for extraction Chapter 3 : Simple determination of phlorotannins from hot water and solvent extracts of three brown seaweed by RP-HPLC Chapter 4 :Enhancement of phlorotannins from brown seaweed Ecklonia cava by application of Methyl Jasmonate 2

3. Chapter 1 General Introduction 3

4. Fig. 1. A community of Ecklonia cava at a depth of 5 meters 4

5. Fig. 2. Mature Ecklonia cava 5

6. Fig. 3 Ecklonia cava in natural environment 6

7. Ecklonia cava • E. cava is distributed only in the temperate costal areas of the Korean peninsula and Japan. • Forms dense populations in clear waters. • Utilized as food, fertilizer and animal feed. • Underutilized marine bio-resource because of its bitter taste. 7

8. E. cava occupies sub-littoral deep water, 4-25 m or more. • Grows attached to solid substratum anchored into place by fibrous holdfasts • Forms 1-3 m in height dense population, and called marine forest. 8

9. Fig. 4. Biometric parameters used for E. cava sporophytes. (Source: Sarisawa, 2002). PL = Plant length, BL = Primary Blade length, SL= Stipe length, SD= Stipe diameter, BW= Primary blade width, BtL= Longest bladelet length, NBt= Number of bladelet, GR= Number of growth rings 9

10. Fig. 5. Life cycle of Ecklonia cava (Source: Maegawa, 1990) 10

11. Fig. 6. Morphological change of Ecklonia cava (Source: Maegawa, 1990) 11

12. Fig. 7. Procedure of isolation and culture of Ecklonia cava (Source: Wi et al. 2008) 12

13. Fig. 8. Mass culture technique for Ecklonia cava (Source: Wi et al. 2008) 13

14. Fig. 9. Aquaculture of Ecklonia cava (Source: Hwang et al. 2010) 14

15. Fig. 10. Biological properties of Eclonia cava and its proposed commercial use (Source: Wijesinghe and Jeon, 2012) 15

16. Phlorotannins • Marine algal polyphenols, phlorotannins, only found in brown algae • Synthesized via acetate-malonate pathway • Formed by polymerization of phloroglucinol (1,3,5-trihydroxybenzene) • Concentrated within the outer cortical layers, and the mitotic, meristematic and meiotic sporogenous tissues 16

17. Function of phlorotannin in brown algae Defense against • Herbivore and microbes • Allelopathic activity against epibionts • Harmful effects of UV radiation • Structural compounds in cell wall hardening 17

18. Biological activity of phlorotannin • Antioxidant activity • Anti-diabetic activity • Anti-cancer activity • Matrix metalloproteinases inhibitory activity • Tyrosinase inhibitory activity • Anti-inflammatory activity • Anti-allergic activity • Anti-plasmin activity • Bactericidal activity • HIV-1 reverse transcriptase and protease inhibitory activity 18

19. Chapter 2 Distribution of phlorotannins in the brown alga Ecklonia cava and comparison of pretreatments for extraction Chowdhury MTH, Bangoura I, Kang JY, Park NG, Ahn DH, Hong YK (2011) Distribution of phlorotannins in the brown alga Ecklonia cava and comparison of pretreatments for extraction. Fish Aquat Sci 14:198-204 19

20. Objectives • To determine the distribution of phlorotannins among the Laminariaceous brown algae, E. cava in different body parts • Compare the different pretreatment for phlorotannins extraction. 20

21. Materials & Methods 21

22. Collection of algae : 2009 from coasts of the Busan, South Korea • drying procedure • First methods of treatments • Second methods of treatments • Temperature • Time 22

23. Drying procedure of the E. cava 23

24. Extraction of phlorotannins Algal powder (0.5 g) Shake by a shaker with methanol (2 ml) at room temp for 2 hours add CHCl3 (4 ml) and shake vigorously by hand for 5 min Filtratration through defat cotton add 1.5 ml deionized water and shaking by hand for 5 min the mixture partition between upper and lower layers, the upper layer corresponding of non lipid fraction collect extracted twice with ehyl ether (3 ml) and take the upper layer (2 times) ethyl ether fraction was evaporated by nitrogen blower crude phlorotannins 24

25. Purification of phlorotannins • Measured quantitatively by HPLC • HPLC system consisted of a Waters 600 pump, a UV detector and an C 18 column. • Flow rate of 1.0 mL/min • Linear gradient of 30-100% methanol • The UV detector was set at 290 nm 25

26. Results 26

27. Production of phlorotannins Fig. 1. HPLC profile of phlorotannins from the brown seaweed E. cava. 1 indicates a peak of dieckol at 32 min of retention time and 2 indicates a peak of phlorofucofuroeckol-A at 40 min retention time. 27

28. Results Tab. 1. HPLC Validation parameters for the determination of phlorotannin compounds 28

29. Results Structure of phlorotannins Dieckol Phlorofucofuroeckol-A 29

30. Results 30

31. Results 31

32. Results 32

33. Results 33

34. Results 34

35. Conclusions 35

36. Highest amount of crude phlorotannins, dieckol and phlorofucofuroeckol-A observed in mature thalli tissue. • 90% dieckol and phlorofucofuroeckol-A extracted from Shadow dry pretreatment compare to lyophilized tissue • Washing with fresh water reduce the phlorotannins content. • Steam, boiling parameters is not good for the yield of phlorotannins. 36

37. Chapter 3 Simple determination of phlorotannins from hot water and solvent extracts of three brown seaweed by RP-HPLC 37

38. Introduction 38

39. Traditionally Korean and Japanese people drink brown seaweed soup as for healthy food. • Consumption of polyphenol-rich foods or beverages prevent diseases like • Cardiovascular disease and Cancer • Biological properties of polyphenols depend on their bioavailability • Indirect evidence of polyphenols absorption through the gut barrier is increase the antioxidant capacity of the plasma after the consumption of polyphenol-rich food. 39

40. Ecklonia cava 40

41. Arame (荒布, Eisenia bicyclis) 41

42. Ecklonia stoloniferea 42

43. Crude Phlorotannins can analysed by the colorimetric method using phloroglucinol as a standard agent • Or simply in dry weight basis. 43

44. Total phlorotannins consist of a complex set of different types of individual phlorotannins. • Some phlorotannins are highly soluble in water and the others are soluble in organic solvents. • So, RP-HPLC can offer a suitable tool for quantitative analysis of phlorotannins. 44

45. Structure of Dieckol and activity • Dieckol inhibit the MMP-2 and 9 expression, • ACE inhibitory activity • Antibacterial activity • Protective effects on UV-B radiation induced cell damage • Improvement of memory 45

46. Structure of phlorofucofuroeckol A and activity • Hyaluronidase inhibitory activity • Anti-allergic activity • Anti-oxidant activity • anti-inflammatory activity • Preventive effect s on diabetic complications • Improvement of memory 46

47. There was no report on the analytical method for simultaneous determination of dieckol and phlorofucofuroeckol-A from the brown seaweed water extract and chemical extract by HPLC. 47

48. Objectives • Validate the HPLC method for the analysis of two major phlorotannins, dieckol and phlorofucofuroeckol-A from water and organic solvent extracts of E. cava, E. stolonifera and E. bicyclis in a single run for use of brown seaweed water extract as a polyphenol rich functional food ingredient. • Compare the amounts of dieckol and phlorofucofuroeckol-A from different extract • Determine the antioxidant capacity of crude extract and purified phlorotannins compounds. 48

49. Materials & Methods 49

50. Sample collection and preparation • E. cava, E. stolonifera and E. bicyclis were collected from Korean coast in December 2010. • Dieckol and phlorofucofuroeckol-A were isolated by RP-HPLC with purity over 99% from the seaweed, E. cava and identified by FABMS and 1H and 13C-NMR data. 50