Nghiên cứu cấu trúc và khảo sát hoạt tính sinh học của fucoidan và alginate từ hai loài rong nâu sargassum henslowianum và sargassum swartzii của việt nam (tt)

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I. Giíi thiÖu luËn ¸n
1. §Æt vÊn ®Ò



Trong php
        nên   
          

lý              
           


chung và các ionic polysaccharide    

         Chlorophytes), rong nâu
(Pheophytes  Rhodophytes 



         



de có
           
         
, Hot
tính sinh hc ca polysaccharide còn 




 



             
         


2
             ionic


            

 
-       Sargassum henslowianum 
Sargassum swartzii.
- fucoidan và alginate 
rong này.
- fucoidan và alginate.
-          trúc
fucoidan và alginate.
- fucoidan  
loài rong này.
2. §èi t-îng nghiªn cøu cña luËn ¸n

.

3. Nh÷ng ®ãng gãp míi cña luËn ¸n
3.1. Lần đầu tiên cấu trúc của fucoidan chiết tách từ rong nâu
Sargassum henslowianum đã được xác định    
chính 
α-L--
2, C-- 
C--4). Đây là một
fucoidan có cấu trúc mới 
fucoidan này cả 3 vị trí C-2, C-3 và
C-4 đều bị sulafte hóa.
3.2. tán xạ ánh
sáng (LS) và tán xạ tia X góc nhỏ (SAXS)cấu
trúc không gian 


3.3. Lần đầu tiên ở Việt Nam hoạt tính sinh học in vivo 
Sargassum henslowianum và rong nâu Sargassum swartzii
ã .
4. Bè côc cña luËn ¸n
28 trang 4 9 hình, 7 44 tài
o và 15  (2 trang),

3
      41      và
12 19 trang),
37 
(1 trang), 2

II. NỘI DUNG CỦA LUẬN ÁN
MỞ ĐẦU


Chương 1: TỔNG QUAN


 , tình hình khai thác và 
.
 Các i
 Tình hìn

Ch-¬ng 2
ĐỐI TƯỢNG vµ Ph-¬ng ph¸p nghiªn cøu
2.1. Nguyªn liÖu
Sargassum henslowianum 
 - .
+ Fucoidan Sargassum swartzii 
Sargassum swartzii 
2.2. Ph-¬ng ph¸p nghiªn cøu
C
 



 
ionic 
IR, NMR, MS,
LS, SAXS).
        
      và       
polysaccharide        
     



4


        

1. 


2.  
3. 


4. 
sáng (Light scattering-
X-ray scattering-SAXS).

    
íáính gâ
bào -7, SK-
.
Ch-¬ng 3: Thùc nghiÖm
3.1. Thu thập và phân loại rong biển
Hai 
Rong nâu Sargassum henslowianum t fucoidan, thu
hái  , th
Rong nâu Sargassum swartzii     và
fucoidan, thu hái Nha Trang  Khánh Hòa, th
Các mgiám  

 

trong bóng râm. -50
o

, 
3.2. Xác định thành phần hóa học của rong

3.3. Chiết tách các ionic polysaccharide
          
    
s
3.4. Xác định thành phần hóa học của ionic polysaccharide
3.4.1. Xác định hàm lượng sulfate

5
 pháp .
3.4.2. Xác định hàm lượng acid uronic
e.
3.4.3. Xác định thành phần đường
.
3.5. Các phương pháp để xác định cấu trúc
    ,
  
 và  
. Phép
 
     
-.
3.6. Thử hoạt tính sinh học

      
 
am.
Ch-¬ng 4
kÕt qu¶ vµ th¶o luËn
4.1. Thành phần hóa học của rong


 Sargassum henslowianum và
Sargassum swartzii 


Protein
Lipid
Sulfate
Alginate
Laminaran
Fucoidan
Sargassum
henslowianum
2,9
0,5
6,0
35,8
0,04
2,2
Sargassum
swartzii
7,0
0,5

5,9
29,5
0,29
0,82


d < 2%. Các loài rong khác
au. T
-

, 
 trên.

6
         


  % (Sargassum swartzii  % (Sargassum
henslowianum
  thì 2 loài rong Sargassum swartzii và
Sargassum henslowianum 




4.2. Cấu trúc hóa học của fucoidan từ rong nâu Sargassum
henslowianum

trên B 4.2         
Sargassum henslowianum 



 phân
Sargassum henslowianum
Hiệu suất
(% rong
khô)
Thành phần đường
(% mol)
Uronic
acid
(% khối
lượng)
Hàm lượng
Sulfate
(% khối
lượng)
Fuc
Man
Gal
Xyl
Glc
1,8
1
0,05
0,05
0,38
0,92
5,8
25,20

sulfate 

-1

235 cm
-1




cm
-1
-

-1
 
-2/C-.
s
   60ºC trong    60 phút     



7

Hình 4.1.   . henslowianum


1
H,

13
C-       
Sargassum henslowianum 

1
H-à
 
-5,5




-fucopyr-6

3
L-fucopyranose.

1
H-
13
C-NMR
        

           α-L-
fuα-L-
          α- L-

α-D-glucopy
 α-D-     -    
             

α- L-fucopyranose.

8


1
H-


13
C-

9
-Hình 4.4
m/z   
3
]
-
  m/z 225

3
-H
2
O]
-
m/z 
2
SO
3
]

-
.
m/z 549 gán cho[Fuc
2
(SO
3
)
3
]-m/z 405 và

3
]

và [FucGlu(SO
3
)
2
]

.
 ESI-    m/z      
Bérangère Tissot           
 m/z 
      -     m/z 139 là do nhóm
-α-L-
m/z 
             


ESI-m/z 


m/z 139 (
0,2
X), 225 (B
1
)
m/z 183 (
0,2

-2 và C-
m/z 

0,2
X
1
and
0,3
X
1
(Hình 4.6),
α- L-

0,2
A (m/z 
-α-L-
rong nâu Ascophyllum nodosum và Fucus evanescens.
-
3
]
-

m/z 405 (Hình 4.7) là
 
1
m/z 

1
m/z 225 là do
α-L-m/z 


heparin 
các ion
0,2
A
2
-
 -2-C-
0,2
A
2
m/z 345 và
0,2
X
0
ion có
-
0,2
A
1
và

3,5
A
1
m/z 



10

-sulfate 


-
3
]
-


-
2
SO
3
]
-
i m/z 389

11

-GluSO
3

]
-


0,2
A
1
, m/z 183
0,3
A
1
, m/z 153
O
O H
O H
O
O H
O H
O
CH
3
CH
3
O H
OSO
3
-
C
1
, m/z 243

B
1
, m/z 225



O
O H
O H
O
O H
O H
O
CH
3
CH
3
OSO
3
-
O H
Y
1
, m/z 243
Z
1
, m/z 225
0,2
X
1

, m/z 285




12


. henslowianum
-MS/MS
- 

             
Sargassum henslowianum 
     Thiên- có m    
α-L- -2, C- 
--4 và liên
-4).
4.3. Cấu trúc không gian của fucoidan từ rong nâu Sargassum
swartzii và phức của nó với protein (Bovine Serum Albumin BSA)
H

   Sargassum swartzii là ong các loài


           

Sargassum swartzii

SW



rong nâu Sargassum henslowianum 
           Sargassum
swartzii 4.
4fucoidan 
Sargassum swartzii
Thành phần đường
(% mol)
Uronic
acid
%
khối
lượng
Hàm
lượng
Sulfate
%
khối
lượng
Fucose
Galactose
Mannose
Xylose
Glucose
3,6
3,2
1,9
1
0,3

6,3
24,7

13

(q²+kc) × µm² × 10
3
0.00 3.00 6.00 9.00 12.00
Kc/R × g /mol
× 10
-6
8.0
11.0
14.0
17.0
20.0
23.0
M w(c): 1.243e+05 g /mol M w(q²): 1 .243e+05 g/mo l
A2: 1.4 23 e-06 mol dm ³/g ² Rg: 5.0 97e+01 nm
C:\A LV 50 00 W\Thuy\F061225.sta
Fucoidan in 0.1M NaCl
a
(q²+kc) × µm² × 10
3
0.00 3.00 6.00 9.00 12.00
Dapp II / µm²/s
9
12
15
18

21
Dz(c ): 1.143e+01 µm ²/s Dz(q ²): 1.1 43e+01 µm ²/s
kD: 4.205e-0 2 d m³/g C<S ²>: 1.102e-03 µm²
C:\ALV 5000W\Thuy\F0 61 225.s ta
Fuco ida n in 0 .1M N aC l

b

âu Sargassum swartzii a) SLS, b)DLS



SW



(static light scattering SLS) v       

fucoidan
Sargassum swartzii
tR
g
R
h
và


5.
5fucoidan 
nâu Sargassum swartzii

Dung môi
dn/dc
(ml/g)
Mw x 10
-3


R
g

(nm)
R
h

(nm)


(=R
g
/R
h
)
NaCl 0,1M
0,119
124
51,0
21,5
2,4

14
n
   


là

SW





0.
a và b).
4) cho




        
gc
 

          

gc
 

SW


Fucoidan tách Sargassum swartzii 


     


         
polysaccharide 
           


Albumin - Fucoidan (BSA-      

 
            

n


15
Trên hình 4.11 -
1a là phép o SLS và hình 4.11b là DLS.
(q²+kc) × µm² × 10
3
0.0 1.5 3.0 4.5 6.0
Kc/R × g/mol
× 10
-6
6.00

6.60
7.20
7.80
8.40
M w(c): 1.6 36e+05 g/m ol M w(q²): 1.647 e+ 05 g/m ol
A2: 7 .16 0e-08 mol d m³/g² Rg: 3.055e +0 1 n m
C:\ALV500 0W \Thuy \A- f74 b.s ta
A-F, pH 7.4, B uff+Fuc

(q²+kc) × µm² × 10
3
0.0 1.5 3.0 4.5 6.0
Dap p II / µm ²/s
39
42
44
47
49
Dz(c): 3 .92 8e+01 µm ²/s Dz(q ²): 3.9 24e+0 1 µ m² /s
kD : 1 .41 4e-02 dm ³/g C<S ²>: 2.201 e-04 µm ²
C:\ALV500 0W \Thuy \A-f74 b.s ta
A-F, pH 7.4, B uff+Fuc

 -
Fucoidan 
-
6.
6-
Fucoidan
pH

Dung môi

dn/dc
(ml/g)
Mw x
10
-3
(g/mol)
Rg
(nm)
Rh
(nm)


(Rg/Rh)

H
2
O
BSA
0,170
66,0
26,8
3,6
7,4
7,4


BSA
0,180

125,4
24,6
5,9
4,2


Fucoidan
BSA-
Fucoidan
0,160
164,7
30,5
6,3
4,8
4,0


BSA
0,164
186,5
20,9
6,5
3,2


Fucoidan
BSA-
Fucoidan
0,140
211,8

54,0
57,1
0,95

          
              

umin do trong

.

 


BSA và BSA-Fu    

16



B6
- 



 

         6   
BSA-



= 0,95 
ng



 


4.4. Cấu trúc hóa học của alginate từ rong nâu Sargassum swartzii
Sargassum swartzii

7.
7
Sargassum swartzii
Nhóm dao động
Hình dạng
Guluronic
cm
-1

Manuronic
cm
-1

-OH hóa tr (có liên kt
H)
Rng, rt mnh
3428,99
3428,99

-CH hóa tr
Nhn, trung
bình
2936,49
2936,49
COO- hóa tr
Nhn, rt mnh
1414,79
1630,24
C-O-C hóa tr
Nhn, rt mnh
1027,77
1086,61



              



17

-1


-1
.

Hình 4.12. 

 
          
Chemical Industries Ltd., Japan  


Chúng t
Sarrgasum swartzii. 
13
C-NMR
 
13
   







và 177,46
80-82
      nh
y


18
nuronate và guluronate khác nhau,


man             


13
B8 và có
.
8
13
C-
rong nâu Sargassum swartzii

C1
C2
C3
C4
C5
C6
acid D-mannuronic (M)
102,22
72,64
74,01
80,62
78,72
177,46
acid L-guluronic (G)
103,28
67,81
71,79
82, 56
69,95
177,65

Hình 4.13
1
H-. swartzii

1
H-
       -NMR - HSQC


1
H-    
13
C- 
carbon anomer G-      
-H1 
5,05



proton G--H5.

19

anomer M--ppm,,

anomer H

      





.

.swartzii

             β-D-
mannuronic và acid α-L-y

1
H-B9.
9
1
H-
rong nâu Sargassum swartzii

H1
H2
H3
H4
H5
H6
D-mannuronic acid (M)
4,65
3,97
3,73
3,89
3,71
-
L-guluronic acid (G)

5,05
3,89
4,02
4,12
4,45
-

20


1
H-

. a

1
H-Hình 4.16), cho
có 
-

MM
, F
MG
, F
GM
và F
GG

MGG
, F

GGG
, F
GGM

và F
MGM




Hình 4.16. Ph
1
H-NMR ca alginate t rong nâu Sargassum swartzii

21
M   ng, giá tr F
GG
và F
GGM
   n
 (I) cnh t giá tr tích phân ca các
pic) có th c tính bi công thc:
F
GG
=
I
MGG + GGG
I
MGG + GGG
+ I

GGM
+ I
MGM
+ I
MG
+ I
MM

MMMGMGMGGMGGGMGG
GGM
GGM
IIIII
I
F




 

MG
= F
GM
và F
MGG
= F
GGM
.

F

G
= F
GGG
+ F
MGG
+ F
GGM
+ F
MGM
.
F
MG
= F
GM
= F
GGM
+ F
MGM
.
F
GG
= F
GGG
+F
MGG
.
T l chui M có th c tính theo công thc:
F
G
+ F

M
= 1
T l M nh bng công thc:
G
M
=
G
M
F
F

Sargassum swartzii:
628,0
982,4
127,3
929,0466,0272,0188,0127,3
127,3



GG
F

038,0
982,4
188,0

GGMMGG
FF

59,0038,0628,0 
GGG
F

055,0
982,4
272,0

MGM
F

094,0
982,4
466,0

MGGM
FF

186,0
982,4
929,0

MM
F

F
G
= 0,721; F
M
= 0,279

387,0
721,0
279,0

G
M


22
Do vy, alginate t rong nâu Sargassum swartzii là giàu thành
ph
             
Sargassum -0,8. T l các mnh khi ca alginate
ng 4.10
Bng 4.10.  rong nâu S. swartzii
F
M

F
G

F
MM

F
GG

F
MG

F
MGG

F
GGG

F
MGM

F
GGM

0,279
0,721
0,186
0,628
0,094
0,038
0,590
0,055
0,038
t alginate giàu thành phnh
mt thông s cu trúc khác là giá tr trung bình n
G
c guluronic
trong block G theo biu thc:
GGM
MGMG
G
F

FF
n


1

038,0
055,0721,0 



= 17,53

 
phân  r 
GM
MG
FF
F




01 block, 
do  12  
Vi mu u:
093,0
279,0721,0
093,0





.
Giá tr này khnh alginate là mt co-polymer.
4.5. Kết quả thử hoạt tính sinh học
4.5.1. Hoạt tính gây độc tế bào

             
  Sargassum henslowianum và Sargassum swartzii  
-
50
=112,37 và 70,60 µg/ml,

50
=107,46 và 89,10 µg/ml, trên dòng

50
=107,89 và 89,
-
50
= 130,79 và 127,80 µg/ml, 
   
50
=76,71và 118,     
  n    

2

4.5.2. Hoạt tính hạ mỡ máu


 cSargassum
swartzii, Sargassum henslowianum      

23

c
 Sargassum swartzii và Sargassum henslowianum  
,56% và 21,
,25% v
   t      
Sargassum swartzii và Sargassum henslowianum 
,
   -      
Sargassum swartzii và Sargassum henslowianum 

      Sargassum henslowianum và
Sargassum swartzii 


          

   Sargassum henslowianum và Sargassum swartzii

           Sargassum
henslowianum 
 
     Sargassum henslowianum và Sargassum
swartzii 


4.5.3. Hoạt tính kháng vi sinh vật kiểm định
           
Sargassum henslowianum và Sargassum swartzii    
 
50
 g/ml.

KÕT LUËN

1.  xác 02 :
Sargassum henslowianum, Sargassum swartzii 
Thiên-Khánh Hòa. n

thô, laminaran, acid alginic và lipid.

24
2. 
   3       
fucoidan và alginate .
3. Lần đầu tiên cấu trúc của fucoidan chiết tách từ rong nâu
Sargassum henslowianum đã được xác định  
          
α-L-
-2, C--
-
-4). Đây là một fucoidan có cấu
trúc mới  
fucoidan này cả 3 vị trí C-2, C-3 và
C-4 đều bị sulafte hóa.
4. Sargassum swartzii


và  
tán xạ
ánh sáng (LS) và tán xạ tia X góc nhỏ (SAXS)
cấu trúc không gian       


5.             
Sargassum swartzii ron
acid man,387  

G
1 = 18. -

6. in vivo 
và in vitro Sargassum henslowianum và
rong nâu Sargassum swartzii. 
     Sargassum henslowianum và Sargassum
swartzii 

     Sargassum henslowianum và
Sargassum swartzii         

phì.
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