General methods of isolation and purification of anticancer agents of marine origin

General methods of isolation and purification of anticancer agents of marine origin

General methods of isolation and purification of anticancer agents of marine origin


At the end of this lecture, students will be able to:

• Discuss the general methods of isolation and purification

• Discuss the different sources of anticancer agents of marine origin

General methods of isolation and purification of anticancer agents

• Most of the compounds isolated so far are lipid soluble

• Recently many water-soluble compounds have been isolated and the important steps in the water-soluble compounds include

Preparation of extracts

• As the compounds are polar, aqueous media or strongly polar solvents like methanol are to be used for extraction

• One of the major problems is the bacterial and fungal growth which often degrades the active components

• Moreover they give false positive results in bioassays due to endotoxins produced by the microorganisms

• Addition of alcohol or if permissible fungicides like sodium azide will help to prevent bacterial growth

• If the heat does not destroy the activity, brief heating or autoclaving may alleviate the problem considerably

• Organisms themselves can be boiled prior to extraction

• Concentration of the aqueous extracts is also difficult as prolonged heat may cause destruction of activity; hence vacuum concentration or freeze drying is adopted


• It is the most important and often the most difficult process

• The presence of large amounts of salts interferes in chromatographic separation and also in bio assays

• Desalting gels or membranes used in biochemical preparations are not generally suitable as the difference in molecular size of the compounds and salts are not much different

• If the compounds are soluble and stable in methanol, crude desalting can be done by extracting the freeze-dried material with absolute methanol

• Desalting can be done with small molecular compounds (gels) with small matrices such as Sephadex G 10 or biogel P2

• If the compound is reasonably hydrophobic, nonionic resins such as XAD-2, XAD-7 polyethylene or polypropylene powder and porous polyethylene type resins can be used

• These resins often retain or retard the elution of organic molecules

• Adsorption on active charcoal is also sometimes effective for rough desalting


• Water soluble components are present in minute quantities

• Isolation of such components is very difficult

• Different methods which can be used are

Gel and ultrafiltration:

• These are effective for both rough and fine fractionation (through membranes)

Ion exchange chromatography:

• Used when the compounds are ionic and their stability on the resin and in buffer solutions are known

• Most effective method for the separation of water-soluble components

• Choice of resin is very important; strong resins may not be suitable

Reverse phase columns

• Various hydrophobic stationary phases are used withproper combination of solvents like methanol and acetonitrile and by using buffer

• For the separation of polar and ionic components the use of buffers with appropriate pH and ionic strength gives good results

• Major problem is the recovery of small amounts of compounds from the buffer solution and can be easily avoided by using volatile buffer

• Volatile buffer can be easily removed by vacuum evaporation or freeze drying


• Ammonium carbonate

• Ammonium acetate

• Pyridine- aetic acid

• Pyridine- aetic acid – picoline

• Pyridine- aetic acid – 2, 4, 6 collidine

Large amounts are required and hence used in final
purification stage

Molecular filtration and adsorption with pressure chromatography

• These are porous matrices which possess both molecular filtration, adsorption capabilities and withstand high pressure

• Examples are bonded silica with various pore size (TSK-125, TSK-250, TSK-400), Styrene divinyl benzene copolymers with adsorptive characters and pore characteristics (Hitachi gel 3000 series)

Combination of ion exchange and size exclusion chromatography

• Attachment of ion exchange of capabilities of matrices of various pore size provides very powerful separation effects; eg. DEAE sephadex and carboxy methyl cellulose

• Supports with functional groups on a variety of matrices are available

• In most cases the actual separation is done using ion exchange, size exclusion and hydrophilic and hydrophobic interaction

Common fractionation scheme for water-soluble compounds

• Freeze dried sample + Methanol

• Strongly acidic resin eluted with NaOH to give basic compounds

• Elute

• Made acidic and/ neutral

• Strongly basic resin; eluted with NaOH for acidic substances; neutral compounds retained

Anticancer or cytotoxic compounds

• National Cancer Institute (NCI) and National Sea Grant Office (NSGO) have discovered thousands of pure and semi pure compounds derived from marine origin with anticancer activities

• These compounds exhibited good activities in cell lines; besides, in vivo actions against both malignant tumors and leukemias in various animal models

• The various classes includes, macrolides, dipeptides and miscellaneous compounds etc


• Cembranoids – 14 membered cyclic diterpenes obtained from a wide variety of soft corals and contain exocyclic lactone as their integral part



• Sinularin is obtained from Sinulaira flexibilis

Crassin acetate

• It is obtained from Pseudoplexaura porosa (Caribbean gorgonian)

• Crassin acetate was observed to be comparatively inert to mammalian tissue system but extremely cytotoxic to human leukemic as well as Hella cells in vitro and mouse fibroblasts

Crassin acetate

Cytarabine (Ara-C, alexan, Arabitin, Aracytine, Cytarbel, Cytosar, U19920, CHX-3311, Aracytidine)

• Cytarabine is a synthetic compound exclusively based on the moieties present in the carribean sponges (spongosine, Spongogouridine)

• Cytarabine is indicated in both adult and childhood leukemia

• Used specifically in acute granulocytic leukemia; more potent when combined with thioguanine and daunorubacine

• Potent anti-neoplastic and antiviral agent

• Used in the treatment of acute myclogenous leukemia and human acute leukemia

Cytarabine (Ara-C, alexan, Arabitin, Aracytine, Cytarbel, Cytosar, U19920, CHX-3311, Aracytidine)

Fludarabine (2-Fluorovidarabine; 2F-Ara-A)

• Used as antineoplastic agent

Fludarabine (2-Fluorovidarabine; 2F-Ara-A)


• It is obtained from the Sea hare, Aplysia angasi

• Used as antineoplastic agent


Non lactonic cembranoid

• These don’t have lactone moiety and do posses cytotoxic actions

Geranylhydroquinone (Geroquinol, Geranyl-1,4 benzenediol)

• It is obtained from the chloroform extract of Alpidium species

• Found to be cytotoxic to leukemia and mammary carcinoma

• Employed as radio protective agent

Geranylhydroquinone (Geroquinol, Geranyl-1,4 benzenediol)


• It is obtained from gorgonian coral

• Antineoplastic agent


Macrolides – Bryostatins

• Obtained from bryozoans – Bugula neritina

• Also obtained from tunicates and sponges

• Triggers activation and differentiation of peripheral blood cells from lymphocytic leukemia patients

• Activates Protein kinase C (PKC)

Macrolides - Bryostatins

Macrolides – Dolastatins

• Obtained from sea hare, Dolabella auricularia

• Group of cyclic and linear peptides and depsipeptides – Dolastatins

• Compounds includes Dolastatin 10, Dolastatin H, isodolastatin etc

Macrolides – Dolastatins


• Auripyrone A and B from D. auricularia


• Ecteinascidin 743 from tunicates Ectenascida turbinate (Tunicates)

Ecteinascidin 743

From Sponges

• Niphatesine D from Nimphates speices

Niphatesine D

• Globostellatic acids, isomalabaracane triterpenes from Stellatta globostellata

Globostellatic acids

General methods of isolation and purification Summary

• Isolation – Preparation of extracts, desalting

• Purification – Gel and ultra-filtration, ion exchange chromatography, reverse phase, size exclusion chromatography

• Antitumours compunds – uncluse sinularin, crassin acetate, cytarabine, fludarabine, aplysiatatin, geranylhydroquinine, asperidol, bryostatins, dolostatins, polypropionates etcc

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