STUDY OF SULFATED

POLYSACCHARIDES FROM
BROWN ALGAE FOR ITS
PROPERTIES (anti-cancer,
anti-oxidant, ...)
        Under the Guidance of​ Presented by:   
         Prof Dr. K. KATHIRAVAN 
         MOHAMED ASLAM SHARIFF HS
         Head of the Department​
          M.SC BIOTECHNOLOGY 
         Department of Biotechnology​
          
         University of Madras​
         Guindy Campus​
         Chennai-25​
RECAP OF 1ST
REVIEW  
  Work flow 
Selection of Macro algae

Identification ( Taxonomical or DNA bar coding )

Shade drying and Powdering

Hot / Acid / Alkaline/  Extraction

Partial Purification ( Estimation of SPs )

Purification of SPs (Ion exchange, column Chromatography)

Active Fractions ( Lyophilization and storage )

Characterization of Purified SPs

              UV-Vis spectroscopy          
              FT-IR                                
NMR 1H, 13C       
             MS Analysis of purified SPs
             HPLC Analysis                   
 Selection & Collection of Macroalgae 

 In this process we selected which algae we going to work

with 
 This was done by referring the large pool of scientifical
article from the reputed journal like PUBMED, Springer
link, sciencedirect, researchgate, google scholar etc..
 For collection we selected Rameswaram Tamil Nadu snice
it is located in gulf of mannar a very rich source of
marine algae 
Processing the Algae

 Firstly we wash the algae in sea water to

remove any debris and epiphytes
 Later in distilled water to make sure the is
no salt left in the surface of algae 
 After that we keep the washed algae in big
container for sedimenting excess water 
 Then only our algae is ready for the shade
Dring 
 For 5 day the algae was  shade dried to
ensure the algae was in perfect condition for
the pulverization (powdering) 
Note: not to dry the algae in direct sun light 
Pulverization 

 “Pulverization” (comminution,
crushing, grinding) is the process
of applying an external force to
a (solid) material of a certain
size to destroy it and reduce it
into pieces that are smaller than
the original size. Pulverization
has long been done for many
materials.
 Here we use
electrical blender for pulverizatio
n of dried algae 
 The pulverization process was
very succesfull we get very
fine powder of brown algae
Extraction of
 polysaccharides 
 The extraction procedure of K. Arunkumar
et al 2020 was followed with minor
modifications 
 Thirty gram of dried seaweeds was soaked
in 250 mL of 0.3 N KOH solutions for 6 hrs
in the shaker, then the KOH solution was
removed and freshly prepared 0.3N KOH
solution was added again then keep it in
shaker for another 6 hrs to ensure the
proper depigmentation,
 Calculation of 0.3N KOH

 To Calculate the amount of KOH needed to prepare 0.3N solution

 Formula:
 Weight of KOH (in grams) = Normality (N) x Equivalent weight (EW) x Volume
(in liters)
The equivalent weight of KOH is 56.1 g/mol.
So, to prepare 1 liter of 0.3N KOH solution, the weight of KOH required would be:
Weight of KOH = 0.3 x 56.1 x 1 = 16.83 grams
Weigh out 16.83 grams of KOH using a weighing scale.
 To the depigmented
brown algae powder
250ml distilled water
was added and kept in
the shaker for 1 hour
to wash out the
remaining KOH
solution this step was
repeated twice 
 After washing the sample pH
was 10.38 this was neutralized
by adding 0.1N HCl solution 
 To the neutralized
sample 0.1N HCl was added to
the sample to reach 1:50 ratio
(30:1500 w/v) and extracted
by heating at 80 °C for 1 h.
 0.1N HCL calculation 

 To prepare 1 liter of 0.1N HCl solution from 37% concentrated HCl, the volume
of concentrated HCl required would be:
 Formula 
          N1V1=N2V2
      (Where N1 is normality of conce HCL, V1 is volume of conce HCl,N2 is 0.1N, V2
is 1 liter)
          12 * N1=0.1*1000
          N1=100/12
          N1=8.33
 So 8.33 ml of conce HCl in 1 liter of distilled water
 After the hot acid water extraction the
content is allowed to cool down in room
temperature. Then it was centrifuged at
6000 rpm for 15 mins in 25 degree Celsius.
With utmost care the supernatant was
transfer in the beaker and the volume was
measured for alginate precipitation 
 The alginate precipitation was done by
adding 1% of cacl2 then overnight
incubation in 4 degree Celsius 
 The 1% CaCl2 treated supernatant was again centrifuged
at 9000 RPM for 20 mins in 4 degree Celsius to remove the
precipitated of alginate 
 To the supernatant 1:1 ration of ice cold ethanol was
added to precipitate the Fucoidan and this was incubated
in the –20 degree Celsius freezer over night 
 Then we need to centrifuged it at 9000 rpm 20mins 4
degree Celsius the pellet was collected and dialysis
against distilled water for 24 hours 
FUTURE WORK