Marine organisms collection and isolation of marine natural products (MNPs)

MarineOrganisms
CollectionAnd Isolation
of MNPs.
2/15/2020 1Dr. Mosa E. O. Ahmed
Dr. Mosa E. O. Ahmed
As. Professor of Pharmacognosy
Facultyof Pharmacy
AlneelainUniversity

Introduction
• Almost 75% of the earth’s surface is covered by oceans and this huge quantity of marine
environment is a fertile area presenting a large opportunity for research.
• Land has great diversity with 17 phyla, but oceans consists of 34 phyla (more diversity as
compared to land).
• Of the estimated 153,000 known natural products, 30,000 compounds are obtained from
marine organisms and there are still many more to be discovered.
• Approximately half of the novel MNPs reported in literature are biologically active.
• 60-70 years of activity in the NMP field has resulted in 4,796 genera/species being
recorded in the MarinLit database as sources of novel MNPs.
Ana Zivanovic. Marine natural products isolation, screening and analogue synthesis, Doctor thesis, school of chemistry, University of Wollogong,
2012. http://ro.uow.edu.au/thesis/3692.

Introduction…cont
 Production ofantibacterialcompounds in marine
organisms is related to thefact thatmarinesessile organisms
receive theirdietary uptake by filteringseawater, which
containsa high concentrationof bacteria.
 Sponges occasionally grow together which creates competition for a
place and the sponge that produces more chemicals to harm the other
sponge has the advantage. The chemicals involved in this type of defense
are mostly found to be active againsthuman cancer.

MNPs in clinicaltrails

MarineOrganisms
2/15/2020 Dr. Mosa E. O. Ahmed 6

Collectionand Storage of MarineOrganisms
 Handling of marine organisms during collection is highly critical.
Satyajit D. Sarker. Natural Products Isolation Second Edition, 2006. human press inc. 353-378
 Information on the organism and the place of collection should becarefully
recordedto facilitate the re-collection and the subsequent taxonomic identification.
 Measures should be taken to avoid decomposition of compounds.

An exampleofa collection recordsheet.

Voucher specimens
• Voucherspecimens for taxonomic purposes should be prepared by taking a small
(e.g.,2–5 cm) section of tissue and preserving it in a solution of 10% formalin in
seawater.
• Algae specimens areusually preservedin a solution of 5% formalin in seawater.
• Specimens should berepresentative of the entire organism and include as much
tissue relevant to taxonomy as possible, e.g.,for sponges, both the exo- and
endosome areessential for accurate identification.
 For tunicatesandsoftcorals, oftenpart oftheorganism or wholeorganism (if nottoo
large) mustbe collected,includingthe‘‘root.’’ Afterthe specimenreaches thelab,formalin
solution shouldbe decantedandreplaced by70%ethanolfor long-term storage..

Marineorganisms collection
• The amount of the organism to be collected is usuallydetermined in
view of its abundance.
• An ideal sample size is 1–2 kg wet weight (100–200 g dry weight).
• Complete harvesting of the organism should be avoided. If only a
single large organism is available,a part of it may be collected.
• The sample shouldbe lyophilized immediately after collection to
prevent anychemical degradation.
• If this is not possible, the sample should be kept at –20oC to 0oC until
freeze drying.

Marineorganisms collection…cont.
• An alternative approach is to fix the samples by immersing
them in a mixture of ethanol–water (50:50 v/v) for approx 24
h after which the liquid is discarded.
• Damp organisms are then placed in high-density polyethylene
bottles (Nalgene® 2 L wide-mouth containers are best) and
shipped back to the home lab atambient temperature (27oC)
• Samples preserved in this wayusually remain in good
condition for up to 2 weeks in tropical conditions withno
significant loss of secondary metabolic content.
• The a addition of MeOH should occur immediately after
samples reach the lab.

Extractionof MNPs
• Three extraction strategies are widely used in thefield of
marinenaturalproducts.
• The choice of a method depends on:
The aim of the isolation process.
The facilitiesavailable.
The intrinsic advantages and disadvantages of the procedures.

1. Maceration:
• Thismethod involves maceration of the sample with solvent, followed
by filtration or centrifugation.
• Thesamplesare usuallycut intosmallpiecesor ground into fine
particlesto facilitatesolventpenetration.
• Stirring or sonicationcan be appliedtoincreasethediffusionrate.
• In mostcases,MeOH or EtOHis thesolventofchoice.

2. National cancerinstitute(NCI) method:
• Frozensamples areground with dry ice (CO2) and extractedwith water at 4oC.
• Theaqueous extract is removedbycentrifugation and lyophilized.
• Thedry marcis then successively extractedwith MeOH–CH2Cl2 (1:1 v/v),
followed by MeOH (100%).
• Theorganic extracts are combined and concentrated under vacuum.
• This method is highly efficient. Moreover, lyophilization of aqueous extracts
eliminates the risks of bumping and heat degradation.

3. SCF protocol:
• Super critical fluids (SCFs) have the advantages of low viscosity, superior mass
transfer properties, and good solvation power.
• They also have the ability to penetrate micro porous materials.
• Although this method offers a fast and effective way for extraction and subsequent
solvent removal, it needs sophisticated equipment and some experimentation to
choose the bestorganic modifier.

Fractionationof MarineExtracts
• Marine extractsare extremelycomplex,andcomprise mixturesof
neutral,acidic, basic,lipophilic,and amphiphiliccompounds.
• The nature ofthecompound(s) of interestmaydifferaccordingto
theaimof theproject.
• Thereis nogeneralfractionationprocedure thatcan servefor all
situations.
• Despitetherecent advances in separationtechnology,experience
stillplays an indispensablerolein theisolationofmarinenatural
products.

Four stages of fractionationprocedure:
(1) Collectionof informationabout:
– The chemical content profile.
– The biological activity of the extract.
– The nature of compounds of interest.
– The type of impurities.
• Someinformationcan be revealed by exact taxonomic
identificationof the organismunder investigation.

Stage (1) Collection of information… cont.
• Literaturesearches can provide information about
compounds previously isolated from the species.
• Further information can be gainedby carrying out:
– Biologicaltesting.
– TLCanalysis.
– Mass spectrometry(MS).
– NMRexperiments.

Stage (2): Dereplication:
• Dereplication, used to identify extractsthat contain only known
compounds as early as possible before elaborate fractionation steps are
undertaken.
• This process relies mainly on the availabilityof comprehensive databases
for know compounds. including those containing information on:
– Thesource of the organism.
– Taxonomic identification.
– Extraction methods.
– Thedifferent chromatographic and spectroscopic characteristics of the
isolated compounds.

Stage (3): Removal ofthe bulk ofunwanted materials
• The aim of thethird stageis oftento remove thebulkof
unwantedmaterials, e.g., fatsand salts usingfairly low-
resolution separation steps, e.g.:
– liquid–liquid partition
– SPE
– SEC.

• Achieved according to a modified method developed by Kupchan.
• It canbe used for defatting and desalting as well.
The useof Sephadex LH-20 and MeOH: CH2Cl2 (1:1 v/v)as eluent is oneof the
commonly usedprocedures. Fats and large non-polar organic compounds are
usually eluted first.
Other commonprocedures involve the use of an SPE cartridgecontaining C18
silica and MeOH/ H2Oas a washing liquid.
SOLVENTPARTITIONING
DEFATTING

Modified Kupchan’spartition scheme.

Desaltingbymethanol-:
• Freezedriedextract can be desaltedconveniently and effectivelybyuse of
absolute methanol.
• For this we need toextract withabsolutemethanol and thenremovalofsolvent
is done. This process is repeated3- 4 times.
• Bythis process majorityofsalt is removed furtherdesalting process becomes
easier.
• Thenbyuse ofGel matrixes(Sephadex G-10,Bio- Gel P2) materialis filtered.
• If the components are hydrophobic we use ionic resins ormatricessuch as
XAD- 2, XAD-7,polyethylene or polypropylene powder andporouspolyether
type resins.
DESALTING:

Stage (4): purification:
• The fourthstage usually involves high-resolutionseparation
steps, e.g., HPLC withtheaim of purifying interesting
compounds to a degree thatenables thesubsequentstructure
elucidation.

General approach forfractionation of marine extracts

Difficulties in the Isolation of Marine Natural Products
 Taxonomicinformation can facilitateliterature searches on compounds
produced by the species under investigation and of course their methods of
purification.
 This hassome impact on the selection of the best purification scheme
for new metabolites.
 Incorrect or incomplete taxonomic assignments can lead to difficulties if
assumptions are made about the chemistry that an organism may contain.
1. TaxonomicUncertainty:

 The presence of a highlypotent metabolitein trace amounts
can complicate theextraction and isolation process.
 One example is the isolation of only 10.7mg of the highly potent
antitumor macrolide spongistatin 4 from about 2.5 ton of the South
African marine sponge Spirastrellaspinispirulifera.
2. Small Quantities of Metabolites:

About 2.5 ton ofthe South African marine sponge Spirastrella spinispirulifera areneeded
to isolate 10.7mg of this compound (spongistatin 4 ).

Marine extracts may contain extremely labile compounds.
Decomposition of these compounds may occurat any step during the purification
process.
Heat, light, air, andpH are among other factors that may lead
to the degradation of compounds.
Materials usedfor separation may also activate some reactions:
Alumina can catalyze the aldol condensation, rearrangement, hydration
and dehydration reactions,.
Silica can enhance oxidation, rearrangement, and N- and O-
demethylation.
Somesolvents such as acetone, methanol (MeOH),ethylene glycol, and
dimethylformamide (DMF) may give rise to adducts.
The slightly acidic nature of some NMR solvents (e.g.,CDCl3) may cause
degradation of highly pH-sensitive compounds.
3. Instability ofMetabolites:

 Thereare many difficultiesassociated withthe isolation and
purification ofwater-solublecompounds.
 In the case of aqueous solutions, a problem is bacterial and
fungalgrowth, which oftendegrades the active components or
gives false results in bioassays because of endotoxinsproduced
by the micro-organisms.
4. Purification ofWater-Soluble Compounds:

There are 3 methods by which problem can be avoided:-
Addition ofalcohols orsmall amount ofimmiscible organic solvent like n-
butanol and toulene. (Inhibits microbes)
 If the compounds are not heat sensitive in that case heating or autoclaving can
bedone. Eg- Nereistoxin (Insecticide from sea weed)is heated to prevent
microbes.
Aqueous extract can befreezedried to removethis problem.
Methods to prevent Microbial Growth:

 Ultraviolet (UV) detection is the preferred detection technique for
HPLC analysisof naturalproducts because of its ease of use and high
sensitivity.
 However, one shortcoming of UV detectors is the inability to detect
compounds that lack UV chromophores.
Moreover, some of the solvents used in normal-phase
chromatography are themselves strong absorbers of UV light, which
means that low detector wavelength settings are not possible.
5. Compounds Lacking UVChromophores:

Mixture of substances (Bioactivefrom marines) are resolved by standard
chromatographic techniques over SiO2, Al2O3, HPLC, etc.
Isolation techniques used in MNPs
 Ion- exchange chromatography
 Reverse –phase columns
 High/ Medium pressure chromatography on porous materials
 Combination of ion-exchangeand size exclusionchromatography
 Bioassay directed fractionation
Isolation Techniques:

 Ion-exchangechromatography is the most effective method
of separating water soluble compounds, if already ionic
character of the compounds and their stability on theresin and
in buffer solutionsare known.
 It is important to known ionic character and theirstability as
many compounds they decompose on theH+ form of strongly
acidic resin or OH form of strongly basic resins.
 In thatcase we use mediumacidic and basic type of resins
whichare available.
Ion- exchange chromatography (IEC)

 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 asmethyl alcohol, acetonitrile,
and buffers.
 By this biochemical analysisof almost all the compounds is
possible.
 Problem associated with this technique is thatmarine sample size
is limited hence separation on reverse - phase columns is usually
done for final purification and fine separation.
Reversephase columns:

 Columns are used for preparative purpose likeC15 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 silicawith various pore sizes for separation of various
molecular sizes used for marine bioactive compounds.
High/ Medium Pressure Chromatography

Attachment of ion- exchangecapabilities of matrices of various pore sizes
provides a very powerful separation capability. Eg –carboxymethylcellulose.
In most cases the separation is due tothe combination of three principles i.e.
ion-exchange resin, size exclusion, or hydrophilic/hydrophobic interactions.
Selection of a propermatrix is important for separation
Generally compounds with basic charactersare separated on cation exchange
resin.
Compounds with acidic functional groups on anion –exchangeresins.
Strongly acidic orbasic resins are also widely usedto separate neutral and
amphoteric compounds.
CombinationofIon-exchangeandSize- exclusive chromatography

 The selection of any assaysystem to monitor fractionation is based
on the original activityof the extract.
 In vitro systems are used to monitor activity since the test results
can be obtained much more rapidly thanwith in vivo testing, also cost
of bioassay is less. E.g., For anti-canceractivity we use cell lines (P388)
for in-vitrobioassay fractionation studies.
 In vivo studies are used when in-vitro systems extracts are not
giving active results.
Bioassay Directed fractionation:

MNPs in clinicaltrails

2/15/2020 Dr. Mosa E. O. Ahmed 41

Marine organisms collection and isolation of marine natural products (MNPs)

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