Transcript Document 7607897

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
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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, 425 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)
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Fig. 7. Procedure of isolation and culture of Ecklonia cava (Source: Wi et al. 2008)
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Fig. 8. Mass culture technique for Ecklonia cava (Source: Wi et al. 2008)
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Fig. 9. Aquaculture of Ecklonia cava (Source: Hwang et al. 2010)
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Fig. 10. Biological properties of Eclonia cava and its proposed commercial use
(Source: Wijesinghe and Jeon, 2012)
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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
•
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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
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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
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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
treatments
Second methods of
treatments
Temp
Time
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
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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
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
Retention
time (min)
Accuracy
(%)
Precision
(%)
LOD
(µg/mL)
Lower
LOQ
(µg/mL)
Correlation
coefficient (r)
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
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Arame (荒布, Eisenia bicyclis)
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Ecklonia stoloniferea
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• 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
crude phlorotannins
54
HPLC machine
55
DPPH test
• The radical scavenging activities of the
three brown seaweed extracts were
measured using the DPPH (2, 2- diphenyl1-picrylhydrazyl) method.
56
Results
57
Tab. 1. HPLC Validation parameters for the determination of phlorotannin
compounds
Retention
time (min)
Accuracy
(%)
Precision
(%)
LOD
(µg/mL)
Lower
LOQ
(µg/mL)
Correlation
coefficient
(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.
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61
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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
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BHT.
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.
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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.
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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.
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• 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).
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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
(y)
Dieckol
(20353.14±133.28)
Accuracy
(%)
Precision LOD
LLOQ Correlation
(%)
(µg/mL) (µg/mL) coefficient
(r2)
3.24
0.00479
0.0145
0.9995
97.4±2.32
2.38
0.0008
0.00245
0.9989
99.2±3.44
3.47
0.0045
0.0135
0.9992
100.3 ± 2.12
3.94
0.0073
0.022
0.9999
95.7 ± 3.10
-(3744.33±2957.47)
compound 2
(32636.33±1582.22)
+ (15829.22±7200.63)
compound 3
(32601.49±283.39)
-(8163.13±4407.12)
Phlorofucofuroeckol-A
(34316.87±355.87)
-(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
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