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

Content

• General methods of isolation and purification; anticancer, agents of marine origin

Objective

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

• 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 includes

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  bio  assays  due  to endotoxins produced by the microorganisms

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

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

• Organisms itself 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

Desalting

• 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

Fractionation

• 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 phase are used with proper 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

Examples:

• 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

Cembranes

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

Sinularin

• 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

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

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

• Used as antineoplastic agent

Aplysistatin

• 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

Asperidol

• 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 – Dolastatins

• Obtained from sea hare, Dolabella auricularia

• Group of cyclic and linear peptides and depsipeptides - Dolastatins

• Compounds includes Dolastatin 10, Dolastatin H, isodolastatin etc

Polypropionates

• Auripyrone A and B from D. auricularia

• Ecteinascidin 743 from tunicates Ectenascida turbinate (Tunicates)

From Sponges

• Niphatesine D from Nimphates speices

• Globostellatic acids, isomalabaracane triterpenes from Stellatta globostellata

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