Marine natural products m. Pharmacy in Pharmacognosy

1. GENERAL METHOD OF ISOLATION AND
PURIFICATION
BY : GAVADE GANU CHANDRAKANT (M.PHARM
1ST YEAR )
SUBJECT NAME :ADVANCED PHARMACOGNOSY

2.  Marine organisms provide variety of bioactive
metabolites.
 These bioactive secondary metabolites are divided
into:-
 Amino acids
 Peptides
 Nucleosides
 Alkaloids
 Terpenoids
 Sterols
 Saponins
 Polycyclic ether

3.  Chemical nature of complex mixtures of several
class of compounds is not known, it is not
possible to follow any specific technique for
separation of constituents.
 In that case broad separation of the mixture can
be achieved by fractionation with organic
solvents.
 Methanolic/ ethanolic extracts are successively
extracted with hexane, chloroform, ethyl acetate.
 Extracts can be divided into water soluble and
water insoluble fractions.
 lipophilic compounds are present in hexane and
chloroform soluble fractions.
 Compounds extracted in hexane, benzene,
chloroform are generally esters, ethers,
hydrocarbons of terpenoids, sterols, fatty acids
etc.

4.  The isolation of pure compounds from hexane,
chloroform soluble fraction is comparatively easier
than from water soluble fraction.
 Due to the presence of various water soluble salts
associated with marine organisms the separation
of water soluble components becomes difficult.
 In water soluble extracts there are chances of
microbial growth development because of which
we get false results. Because of endotoxins
produced by microbes.
 Eg- Anti-tumour activity detection in which
lipopolysaccharides(endotoxins) produced by
microbes interfers.

5.  There are 3 methods by which problem can be
avoided:-
 Addition of alcohols or small amount of
immiscible organic solvent like n-butanol and
toulene. (Inhibits microbes)
 If the compounds are not heat senstive in that
case heating or autoclaving can be done.
 Eg- Nereistoxin (Insecticide from sea weed) is
heated to prevent microbes
 Aqueous extract can be freeze dried to remove
this problem.

6.  Desalting by methanol- Freeze dried extract can be
desalted conveniently and effectively by use of
absolute methanol. For this we need to extract with
absolute methanol and then removal of solvent is
done.
 This process is repeated 3-4 times. By this process
majority of salt is removed further desalting
process becomes easier.
 Then by use of Gel matrixes (Sephadex G-10, Bio-
Gel P2) material is filtered. If the components are
hydrophobic we use ionic resins or matrices such
as XAD- 2, XAD-7, polyethylene or polypropylene
powder and porous polyether type resins.

7.  Filtration method- It is rarely used as filtration
through small pore membranes gives imperfect
separation of salt.
 Adsorption method- It is the method in which
adsorption on active charcoal is there but it
does not provide effective desalting

8.  Mixture of substances (Bioactives from marines)
are resolved by standard chromatographic
techniques over SiO2, Al2O3, HPLC etc.
 Isolation Techniques :
 Ion- exchange chromatography
 Reverse-phase columns
 High/ Medium pressure chromatography on
porous materials
 Combination of ion-exchange and size exclusion
chromatography
 Bioassay directed fractionation

9.  Ion- exchange chromatography is the most
effective method of separating water soluble
compounds, if already ionic character of the
compounds and their stability on the resin and
in buffer solutions are known.
 It is important to known ionic character and
their stability as many compounds they
decompose on the H+ form of strongly acidic
resin or OH- form of strongly basic resins.
 In that case we use medium acidic and basic
type of resins which are available.

10.  By this wide range of polarity can be separated
by reverse phase columns with various
hydrophobic stationary phase with proper
combination of organic solvents such as methyl
alcohol, acetonitrile, and buffers.
 By this biochemical analysis of almost all the
compounds is possible.
 Problem associated with this technique is that
marine sample size is limited hence separation
on reverse phase columns is usually done for
final purification and fine separation.

11.  Columns are used for preparative purpose like
C15 or C8 column.
 These materials are mostly porous matrices,
which possess both molecular filtration and
adsorption capabilities and withstand high
pressure.
 TSK-125, TSK-250, and TS-400 are some of
materials which are bonded silica with various
pore sizes for separation of various molecular
sizes used for marine Bioactive.

12.  Attachment of lon- exchange capabilities of matrices of
various pore sizes provides a very powerful separation
capability. Eg carboxymethylcellulose
 In most cases the separation is due to the combination
of three principles i.e. ion-exchange resin, size
exclusion, or hydrophilic/ hydrophobic interactions.
 Selection of a proper matrix is important for
separation Generally compounds with basic characters
are separated on cation- exchange resin.
 Compounds with acidic functional groups on anion-
exchange resins.
 Strongly acidic or basic resins are also widely used to
separate neutral and amphoteric compounds.

13.  The selection of any assay system to monitor
fractionation is based on the original activity of
the extract.
 In vitro systems are used to monitor activity
since the test results can be obtained much
more rapidly than with invivo testing, also cost
of bioassay is less. Eg- For anti-cancer activity
we use cell lines (P388) for in-vitro bioassay
fractionation studies.
 In vivo studies are used when in-vitro systems
extracts are not giving active results.

14. THANK YOU