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
 OUTLINE
Chapter 1 : General Introduction

Chapter 2 : Distribution of phlorotannins in th

Ecklonia cava and comparison of pretr

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
 Chapter 1

General Introduction

3
Fig. 1. A community of Ecklonia cava at a depth of 5 meters

4
Fig. 2. Mature Ecklonia cava
5
Fig. 3 Ecklonia cava in natural environment
6
 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
• 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
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
Fig. 5. Life cycle of Ecklonia cava (Source: Maegawa, 1990)
10
Fig. 6. Morphological change of Ecklonia cava (Source: Maegawa, 1990)

11
Fig. 7. Procedure of isolation and culture of Ecklonia cava (Source: Wi et al. 2008)

12
Fig. 8. Mass culture technique for Ecklonia cava (Source: Wi et al. 2008)
13
Fig. 9. Aquaculture of Ecklonia cava (Source: Hwang et al. 2010)
14
Fig. 10. Biological properties of Eclonia cava and its proposed commercial use
(Source: Wijesinghe and Jeon, 2012)
15
 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
 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
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
 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
 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
Materials
&
Methods
21
 Collection of algae

: 2009 from coasts of the Busan, South Korea

 drying procedure

 First methods of treatments

 Second methods of treatments

 Temperature

 Time

22
 Drying procedure of the E. cava
First methods of Second methods of Temp Time
treatments treatments

Sunlight dry With/without salt Room temp (25°C) 72 hr

Shadow dry With/without salt Room temp (25°C) 72 hr

Oven dry With/without salt 60°C 24 hr

Lyophilize With/without salt -80°C 24 hr

Boiling 100°C 1/5 min

Steaming 100°C 5/30 min

23
 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

24
crude phlorotannins
 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
Results
26
 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
 Results

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

Lower
Retention Accuracy Precision LOD Correlation
LOQ
time (min) (%) (%) (µg/mL) coefficient (r)
(µg/mL)

Dieckol 32 92.64±6.95 3.92 7.25 24.15 0.996

Phlorofucofuroeckol-A 40 94.02±5.70 3.94 5.82 19.41 0.999

28
 Results

Structure of phlorotannins

Dieckol
Phlorofucofuroeckol-A
29
Results

30
Results

31
Results

32
Results

33
Results

34
Conclusions

35
• 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
 Chapter 3

Simple determination of phlorotannins

from hot water and solvent extracts
of three brown seaweed by RP-HPLC

37
Introduction

38
• 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
Ecklonia cava

40
Arame (荒布, Eisenia bicyclis)

41
Ecklonia stoloniferea

42
• Crude Phlorotannins can analysed by the
colorimetric method using phloroglucinol
as a standard agent

• Or simply in dry weight basis.

43
• 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
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
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
• 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
 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
Materials
&
Methods
49
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
 Preparation of standard
solution
• Purified dieckol and phlorofucofuroeckol-A
weighted, dissolved in 100% methanol and
diluted at 1mg/mL as stock solutions.
• Standard curve range at 0-750 µg/ml

51
Water Extraction of phlorotannins
Algal powder (1 g)

add 100 ml of boiled temperature distilled water (stirrer with magnetic stirrer)

Heat 100°C for 5 min

Remove seaweed debris

water extract was evaporated at 80OC to make 2 mL (stirrer with magnetic stirrer)

filtered through 0.45 µm cellulose acetate syringe filter

Inject to to the HPLC

52
Algal water extract → Filter through syringe filter → Water extract

53
 Solvent Extraction of crude
phlorotannins
Algal powder (1 g)

Shake by a shaker with methanol (4 ml) at room temp for 2 hours

add CHCl3 (8 ml) and shake vigorously by hand for 5 min

Filtratration through defat cotton

Add 3 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 (6 ml) and take the upper layer (2 times)

ethyl ether fraction was evaporated by nitrogen blower

54
crude phlorotannins
HPLC machine

55
 DPPH test

• The radical scavenging activities of the

three brown seaweed extracts were
measured using the DPPH (2, 2- diphenyl-
1-picrylhydrazyl) method.

56
Results
57
 Tab. 1. HPLC Validation parameters for the determination of phlorotannin
compounds

Lower Correlation
Retention Accuracy Precision LOD
LOQ coefficient
time (min) (%) (%) (µg/mL)
(µg/mL) (r)

Dieckol 32 92.64±6.95 3.92 7.25 24.15 0.996

Phlorofucofuroeckol-A 39 94.02±5.70 3.94 5.82 19.41 0.999

58
Fig. 2. HPLC chromatogram of E. cava by solvent extraction (A) and water extraction (B), E.
stolonifera by solvent extraction (C) and water extraction (D), and E. bicyclis by solvent
extraction (E) and water extraction (F). Peak 1 represents dieckol and peak 2 represents 59
phlorofucofuroeckol-A.
60
61
62
Tab. 2 The IC50 values of hot water extract of seaweed, dieckol, and
phlorofucofuroeckol-A for DPPH free radical scavenging activity. IC50 values
were determined by nonlinear regression analysis. Results are mean ± SE.
Different letters of a, b, c, d indicates significant differences by Duncan’s
multiple test at p < 0.05. ND = not determined.

63
Tab. 2 The IC50 values of hot water extract of seaweed, dieckol, and
phlorofucofuroeckol-A for DPPH free radical scavenging activity.

IC50 value (µg/ml) IC50 value (µM)

Dieckol 10.57 ±0 .30d 14.24± 0.41b

Phlorofucofuroeckol-A 11.15 ± 0.18d 18.51 ± 0.31b

E. cava 87.49 ± 3.28a ND

E. stolonifera 45.37 ± 1.32b ND

E. bicyclis 48.68 ± 3.78b ND

BHT 28.14 ± 0.32c 127.85 ± 1.46a

64
Conclusions

65
• After boiling, brown seaweed E. cava, E.
stolonifera and E. bicyclis released phlorotannins
of dieckol and phlorofucofuroeckol-A in the
water.
• Dieckol and phlorofucofuroeckol-A in the water
extracts were quantified by HPLC method and
compared with solvent extracts.
• DPPH radical scavenging activity of the three
seaweed and purified dieckol and
phlorofucofuroeckol-A were determined and
compared to commercial antioxidant BHT
• Purified dieckol and phlorofucofuroeckol-A
showed 9 fold and 7 fold more antioxidant
capacity compare to commercial antioxidant
BHT. 66
 Probable application
• Hot water extract of brown seaweed
particularly E. cava, E. stolonifera and E.
bicyclis might be a potential source of
polyphenols which can be utilized as an
water soluble, heat stable antioxidant-rich
functional food ingredient, polyphenol rich
beverage.
• Hot water extract of three brown seaweed
can be utilized for industrially dieckol and
phlorofucofuroeckol-A production.
. 67
 Chapter 4

Enhancement of phlorotannins from

the brown seaweed Ecklonia cava by
application of Methyl Jasmonate

68
Introduction

69
• Under various biotic and abiotic stresses, plant
can responds to produce defensive compounds
such as proteinase inhibitor to protect
themselves from stresses and also secondary
metabolites such as phenolic compounds.
• Several methods for inducing secondary
metabolites.
• Various elicitors such as chitosan, β-glucon
and plants hormonal chemicals such as
jasmonic acid (JA), methyl jasmonate (MeJA),
salicylic acid can induce secondary metabolites
in various plants.
70
Fig. 1. Chemical sructure of methyl jasmonate (MeJA)

71
• MeJA is classified by the U.S. Food and
Drug Administration as a Generally
Recognized As Safe (GRAS) substance.

• It have potential commercial applications

in post-harvest treatments for quality
maintenance by reducing decay and
enhancing antioxidant capacity by
increasing secondary metabolites.

72
• MeJA induced crude phlorotannins in brown
seaweed Fucus vesiculosus.

• To the best of our knowledge, so far no

report has been found on MeJA induced
individual phlorotannins enhancement in
brown seaweed.

73
 Objective

• Dose and time effects of MeJA on the

individual amount of phlorotannin of E.
cava tissue using RP-HPLC
• Determine the effect of MeJA on E. cava
tissue viability by triphenyltetrazolium
chloride (TTC) assay.

74
Materials
&
Methods
75
Sample preparation and culture
condition
• Matured thallus blades cut into 1 cm2 long
piece,
• Sonicated for 1 min to remove epiphytes.
• For 1 g of seaweed tissue, 25 mL of PES
media was added.

76
• MeJA dissolved in 100% ethanol (30 mg/mL).
• MeJA solution was applied at a concentration
from 0 to 10 µM for 24 hrs.
• To examine the time effect, E. cava treated
with 2 µM of MeJA solution between 0 to 72
hrs.
• Algal tissues incubated at 20°C, 40 µmolm-2s-
1 and 12h light: 12h dark cycle

77
 A B

Fig. 2. MeJA treatment in (A) multiroom incubator, (B) Lux meter for light intensity
determination (50 Lux = 1 µmolm-2s-1).
78
 Separation and identification of
phlorotannins

• Two major phlorotannins, dieckol and

phlorofucofuroeckol-A and two
phlorotannins from E. cava separated on a
preparative C18 column (22 mm i.d. × 25
cm) at flow rate 5.0 mLmin-1.

79
Results
80
Fig. 3. General HPLC profile of Ecklonia cava. Peak 1, 2, 3 and 4 represent the
dieckol, compound 2, compound 3 and phlorofucofuroeckol-A respectively.
81
• compound 2: light brown powder, FABMS
m/z (%) Found: 974.1183 (M+; bp),
997.1076 (M+Na), matching the formula
of C48H30O23.
• compound 3: light brown powder, FABMS
m/z (%) Found: 974.1175 (M+; bp),
matching the formula of C48H30O23.

82
Fig. 4. LR Fast Atom Bombardment Mass Spectrometry of phlorotannin compound 2

83
Fig. 5. HR Fast Atom Bombardment Mass Spectrometry of phlorotannin compound
2.
84
Fig .6.LR Fast Atom Bombardment Mass Spectrometry of phlorotannin compound
3.
85
Fig. 7. HR Fast Atom Bombardment Mass Spectrometry of phlorotannin compound 3.

86
Fig. 7. Probable structure of compound 3
87
Tab. 1. Validation parameters for the determination of phlorotannin compounds in
E. cava extract

Regression equation Accuracy Precision LOD LLOQ Correlation

(y) (%) (%) (µg/mL) (µg/mL) coefficient
(r2)

Dieckol (20353.14±133.28) 3.24 0.00479 0.0145 0.9995

95.7 ± 3.10
-(3744.33±2957.47)

compound 2 (32636.33±1582.22) 97.4±2.32 2.38 0.0008 0.00245 0.9989

+

(15829.22±7200.63)
compound 3 (32601.49±283.39) 99.2±3.44 3.47 0.0045 0.0135 0.9992
-(8163.13±4407.12)

Phlorofuco- (34316.87±355.87) 100.3 ± 2.12 3.94 0.0073 0.022 0.9999

furoeckol-A
-(883.27±841.84)

88
Fig. 8. Amount of crude phlorotannins after treatment with different
concentration of MeJA.

89
Fig. 9. Amount of dieckol (A), compound 2 (B), compound 3 (C) and
phlorofucofuroeckol-A (D) after treatment with different concentrations of MeJA,
quantifed by RP-HPLC. E. cava tissues were incubated 24 hrs at 20°C under 40
90
µmolm-2s-1 and 12h light: 12h dark cycle.
Fig. 10. Amount of crude phlorotannins over time course treated with 2 µM
MeJA.
91
Fig. 11. Amount of dieckol (A), compound 2 (B), compound 3 (C) and
phlorofucofuroeckol-A (D) over time course treated with 2 µM MeJA and
quantified by RP-HPLC. 92
Fig. 12. Determination of viability after treatment with different concentration of
MeJA by spectrophotometer.
93
Fig. 13. Determination of viability over time course treated with 2 µM MeJA
by spectrophotometer.
94
Conclusions
95
• For application of MeJA, optimum
concentration and time were determined.
• exogenous MeJA can enhance the crude
and individual phlorotannins such as
dieckol, phlorofucofuroeckol-A and two
phlorotannins matching the molecular
weight and formula to 974 and C48H30O23,
respectively.

96
 General
Conclusions
97
• The highest amounts of phlorotannins
observed in the mature thalli blade part of E.
cava.

• Different pretreatment techniques such as

drying and boiling influenced the amount of
phlorotannins.

98
• Hot water extraction was relatively easy, simple
and inexpensive method for phlorotannins
extraction and the recovery rate was also
satisfactory.
• Determined the antioxidant capacity of purified
dieckol, phlorofucofuroeckol-A, crude hot water
extracts of E. cava, Ecklonia stolonifera and
Eisenia bicyclis and compared with commercial
antioxidant BHT.

99
• For application of MeJA, optimum concentration
and time also determined.
• Exogenous MeJA can enhance the crude and
individual phlorotannins such as dieckol,
phlorofucofuroeckol-A and two phlorotannins
compounds matching the molecular weight
and formula to 974 and C48H30O23, respectively.

100
• These biological activities explore a new
opportunity for this alga to be employed as
heat stable, water soluble antioxidant rich
• functional foods and beverage
• Neutacuticals
• pharmaceuticals
• cosmoneutracutical ingredients

for industrial use.

101
102