General methods of isolation and purification , marine drug research and development: Problems and solutions

1. MARINE
NATURAL
PRODUCTS:
General methods
of isolation and
purification
Presented by- Jasmine Kaur
M pharm (Ist) year

2. INTRODUCTION
 Marine organisms provides variety of bioactive metabolites.
 These bioactive secondary metabolites are divided into:-
 Amino acids
 Peptides
 Nucleosides
 Alkaloids
 Terpenoids
 Sterols
 Saponins
 Polycyclic ethers
The ethanolic/ methanolic extracts of
marine organisms exhibits biological
activities could be mixture of
several class of compounds.

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. Methods to prevent Microbial Growth
• 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. Methods for Removal of salts : Desalting
 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 futhur 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.
 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.

7. Isolation techniques used in Marine natural products
 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

8. Ion- exchange chromatography (IEC)
 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.

9. Reverse phase columns
 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.

10. High/ Medium Pressure Chromatography
 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 Bioactives.

11. Combination of Ion- exchange and Size- exclusive
chromatography
 Attachment of Ion- 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.

12. Bioassay Directed fractionation
 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.  Land has great diversity with 17 phyla, but world’s oceans
consists 34 phyla ( more diversity as compared to land).
 Much of the species diversity is there especially on Indian
Ocean i.e. approximately 1000 species per square meter.
 Over the past 30 years, marine plants and animals have been
the focus of a worldwide effort to define the chemistry of
marine environment.
 Beginning in the mid-1980s, efforts have resulted in
applications of novel chemical substances found in sponges
and related colonial marine invertebrates.
 Over 2,500 structurally diverse compounds have been found
in marine plants and animals, and several of them have been
successfully interfaced with the pharmaceutical industry.

15.  Compounds from marine sources are novel both chemically
and pharmacologically.
 Although no marine drug has been developed , several of
them are in clinical and preclinical studies. Egs:- Bryostatin-
1, Dolastatin-10, and Spongistatin for treatment of cancer.
 Majority of Biodiversity in oceans is not plants and animals,
enormous microbial life is found in sea waters which is not
yet explored.
 In 1 millimeter of ordinary seawater contains 1 million
micro-organisms that are mostly uncultured and unknown.
 Terrestrial micro-organisms: 120 marketed drugs, same way
marine microbes investigation is warranted.

16. EXAMPLE -
 Micro-organism isolated from Polysyncraton lithostrotum
was reported to produce antibiotic “namenamicin”
 But instead organisms which were cultured in liquid media
and assayed for anti-microbial and DNA- damaging
activities. Potent activity was produced by another species
micromonospora.
 There was pilot plant scale fermentation of the organism
named M.lomaivitiensis, followed by bio-assay guided
frationation, led to the isolation of a unique dimeric bis-diazo
compound lomaiviticin A.
 But intial goal of isolation of antibiotic namenamicin was
not achieved, but instead new organism with potent activity
was obtained.

17. PROMBLEMS
 Marine microbes are sources of new chemical diversity
which are underexplored and there are wide range of new
opportunities exists for natural product based drug discovery.
 But drug discovery is facing hurdles of screening of
synthetic compound libraries and lead optimization.
 Challenge is to find ways to isolate and cultivate organisms
and thus realize their contributions to the treatment of human
disease.
 Major obstacle is difficulty of having them evaluated in a
wide range of targeted assays and scarcity of the compounds,
which are isolated in minute quantities insufficient to supply
a library for repeated rounds of bioassay.

18. SOLUTIONS
 Problem can sorted out by replacing traditional techniques
of assays by ultrahigh -throughout screening system (HTS)
for evaluation against the wide range of biological targets.
 By HTS libraries of crude extracts can be created which will
decrease time span involved in screening.
 Understanding the biological roles of marine natural
products should be encouraged.
 Too often, compounds with fascinating molecular structure
are discovered and put aside without sufficient attention to
their biological functions or mechanism of action which
should be given.

19.  Newer technologies that can be used to quickly link target
activity with a chemical species, i.e. bioactivity correlation
can be used.
 Furthermore, if the MOA correlates with a potential
therapeutic role, then attempts should be made to define
critical pharmacophore via synthesis, chemical degradation,
and/or modification.
 Eg- Hemiasterlin anti-tumor agent is an excellent example.

20. THANK YOU