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Methods For Isolation of Marine-Derived Endophytic Fungi and Their Bioactive Secondary Products
Methods For Isolation of Marine-Derived Endophytic Fungi and Their Bioactive Secondary Products
Methods For Isolation of Marine-Derived Endophytic Fungi and Their Bioactive Secondary Products
Institut für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany. Correspondence should be addressed to
P.P. (proksch@uni-duesseldorf.de).
Marine-derived fungi have been shown in recent years to produce a plethora of new bioactive secondary metabolites, some
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols
of them featuring new carbon frameworks hitherto unprecedented in nature. These compounds are of interest as new lead
structures for medicine as well as for plant protection. The aim of this protocol is to give a detailed description of methods
useful for the isolation and cultivation of fungi associated with various marine organisms (sponges, algae and mangrove
plants) for the extraction, characterization and structure elucidation of biologically active secondary metabolites produced
by these marine-derived endophytic fungi, and for the preliminary evaluation of their pharmacological properties based on
rapid ‘in house’ screening systems. Some results exemplifying the positive outcomes of the protocol are given at the end.
From sampling in marine environment to completion of the structure elucidation and bioactivity screening, a period of at
least 3 months has to be scheduled.
INTRODUCTION
Terrestrial fungi have long been known as a rich source of bio- micro-organisms including bacteria, cyanobacteria, microalgae
logically active secondary metabolites. Since the discovery of and fungi have become an important source of new pharma-
penicillin by Sir Alexander Fleming in 1928, which resulted in cologically active metabolites. Recent reviews demonstrate the
a breakthrough in the treatment of bacterial infections, fungi importance of these organisms as potential sources of pharma-
have become an important source of drugs for the treatment of a ceutical leads6–25. Especially, marine fungi have shown promis-
variety of diseases. Beside other well-known antimicrobial agents ing potential as sources of new and bioactive natural products as
of fungal origin like fusidic acid and griseofulvin1, new semi suggested by the chemical diversity of their secondary meta
synthetic antifungal drugs like anidulafungin (Eraxis), caspafun- bolites6, even though sample collection and preparation as the
gin (Cancidas) and retapamulin (Altabax) are likewise derived first step might be more difficult for marine-derived fungi than
from fungal metabolites2,3. With the discovery of cyclosporine for terrestrial material because of difficulties inherent to the
isolated from Tolypocladium inflatum in 1971 an important step in collection in marine environment, such as the need for scuba
immunopharmacology was taken, and improvements in the field diving. Although the most famous group of bioactive compounds
of organ transplantation and treatment of autoimmune diseases obtained from marine-derived fungi are still the cephalosporines
are still in progress, like the introduction of substances such as with cephalosporine C, first isolated by G. Brotzu in 1945 from
the fungal-derived mycophenolic acid (Myfortic) to the market a marine strain of Acremonium chrysogenum26, there are also
in 2004 (ref. 2). more recent promising examples. These include halovir and
Further pharmacologically important fungal metabolites include several naturally occurring analogs, which are potent inhibitors
antilipidemic drugs collectively known as ‘statins’ with their parent of Herpes simplex viruses 1 and 2. Synthetic studies that were
compounds mevastatin and lovastatin isolated from Penicillium carried out for these substances allowed insight into structure–
citrinum and Aspergillus terreus, respectively. Statins reduce blood activity relationships22,27. The new alkaloid sorbicillactone A
cholesterol levels and are used for the treatment of coronary isolated from the sponge-derived fungus Penicillium chrysoge-
diseases2,4. num showed promising activity against leukemia cells without
Fungal metabolites are, however, not only indispensable for exhibiting notable cytotoxicity. Large-scale fermentation of
medicine but are also important for plant protection, as demon- the fungus yielded sufficient amounts of the compound for
strated by the discovery of the strobilurines that were first isolated preclinical investigations22,28. Overall, research on marine-
from Strobilurus sp. and served as lead compounds for synthetic derived fungi has led to the discovery of more than 270 new
fungicidals such as trifloxystrobin (Flint)5. natural products mainly from the early 1990s to mid 2002
However, ‘the rediscovery of high numbers of previously (with more than two-thirds of these compounds isolated from
described metabolites has to some extent precluded the study of sponge- and plant-derived fungi), whereas more than 330 new
traditional terrestrial sources of fungi’6 and recently, interest of metabolites were reported only in the 5-year period between 2002
natural product chemists and pharmacologists alike has turned and 2006 (refs. 6,12,22,29), including numerous compounds with
to so far less investigated habitats and ecological niches such as new carbon skeletons. This sharp rise in numbers indicates
the oceans. The oceans cover nearly three-quarters of the earth’s a growing interest in marine-derived fungi as sources of new
surface and can be considered a ‘soup’ of essentially all imaginable bioactive metabolites.
types of microbes6,7. Ecological niches, e.g. deep-sea hydrothermal The aim of this paper is to provide a detailed protocol on
vents, mangrove forests, algae and sponges, provide distinct the purification and cultivation of fungi from various selected
habitats for the isolation of specific micro-organisms 8. Marine marine macro-organisms (sponges, algae and mangrove plants),
Cell suspension
Several hours
8: Small-scale
Based on chemical
fermentation on rice or
screening/bioactivity results in large- Evaporate
in liquid medium for
scale fermentation of promising strains
screening purpose
8: Each 3– Residue ~1 h per l EtOAc
4 weeks
90% MeOH
+ n-Hexane
~1 h
8: Large-scale
9–11: 1–3 d 9–11: Extraction with EtOAc fermentation
90% MeOH n-Hexane
12–14: Fractionation and
isolation of secondary products
Figure 2 | Estimation of the time required for fermentation of fungi and
workup of extracts from liquid medium on a small-scale basis.
16: Bioassays
(cytotoxicity and antimicrobial activity)
12–16: At least
2–4 weeks limited and selective window of the actual diversity that is present
15: Structure elucidation
in a given sample36. Furthermore, it must be remembered that
Figure 1 | Schematic overview on important steps involved in the isolation different culture conditions might also result in different chemi-
of fungi from marine sources and in the isolation and identification of their cal patterns of metabolites formed, as already stated, in the
secondary metabolites.
OSMAC (‘one strain, many compounds’) concept that makes
deliberate use of the chemical plasticity of microorganisms as
a response to varying conditions of culture 37. However, after
as well as on the isolation, characterization and structure eluci conducting numerous fermentation experiments with endo-
dation of biologically active secondary metabolites produced phytic fungi using different culture conditions, we discovered
by these fungi when grown in liquid or on solid media. An the methods described in this protocol were most suitable for
overview including an approximate time schedule is shown our purposes, as they generally gave reproducible results regard-
in Figure 1. ing the isolation of endophytic fungi and regarding the patterns
In this protocol we describe the isolation of endophytic marine- of secondary metabolites that are produced by these fungi32,38–40.
derived fungi from inner parts of living tissues of marine inverte- In our hands, we have obtained optimum results when cultur-
brates, algae and mangrove plants. Endophytic fungi are referred to ing fungi on different solid or in liquid media at room tem-
as fungi that spend at least part of their lives inside healthy tissues perature (20–25 °C), followed by extraction of mycelia and
of host organisms regardless of whether the host is a plant or an growth media with organic solvents. Flow charts briefly describ-
animal30. For the isolation of marine-derived fungi, especially for ing the workup procedures of the respective fungal cultures
the isolation of fungi from sponges, alternative methods to those and the time needed for each step are shown in Figures 2–4.
described in this protocol are available. For example, instead of The crude extracts are then separated using various chromatographic
cutting pieces from surface-sterilized samples, it is also possible techniques, and the isolated bioactive compounds are analyzed
to drop water from a sponge on an agar slant or to excise an inner by HPLC–DAD for their purity followed by structure elucidation
piece of the tissue after thoroughly rinsing it with sterilized sea by MS and NMR using state of the art techniques. Chiral compounds
water without previous surface sterilization31–33. Likewise, several may be derivatized to determine their absolute configuration.
other media for successful isolation and fermentation of marine Chromatographic separation of fungal extracts usually proceeds
fungi, like potato dextrose agar, barley spelt solid substrate or solid guided by bioassays, thereby directing the isolation strategy
malt extract medium17,31 as well as the addition of specific nutri- towards the discovery of bioactive constituents. After the extraction
ents or of sub-lethal doses of fungicides to restrict fast-growing of cultures, the obtained extracts are treated in a similar manner
fungi34, have been described in the literature. Numerous fermen- as described earlier for extracts from marine macro-organisms.
tation conditions, such as shaking versus static cultures, applica- As the chromatographic separation, structure elucidation and
tion of different temperature regimes or employment of larger bioactivity screening for extracts derived from marine macro-
fermenters that can provide varying aeration17,31,35, have likewise organisms have been described in detail in a previous protocol41,
been reported. However, all conditions employed in cultivat- only those steps that differ from the described procedure are
ing marine-derived endophytic fungi suffer from limitations depicted in this protocol.
as different cultivation techniques usually select only a small Two examples taken from our group that focus on polyketides
fraction of the fungal community present, thereby providing a of fungal origin will be used to highlight the structural variability
Large-scale Fermentation
fermentation 3 Weeks on rice medium 4– 6 Weeks
~1 h Evaporate Evaporate ~1 h
90% MeOH n-Hexane
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Box
1 | IDENTIFICATION OF FUNGI
Taxonomic identification of the fungal strains is achieved by DNA amplification and sequencing of the fungal ITS region39.
For this purpose, a piece of fungal mycelium (0.5 cm2) is sampled from an agar plate, lyophilized in a freeze dryer and powdered in a
mixer mill after adding a tungsten carbide bead.
DNA isolation is carried out using the DNeasy Plant Mini Kit according to the manufacturer’s protocol. The procedure includes cell
lysis, digestion of RNA by RNAse A, removing of precipitates and cell debris, DNA shearing, DNA precipitation and purification. This is
followed by DNA amplification using Hot StarTaq Master Mix Kit and the primer pair ITS1 and ITS4 in an iCycler thermocycler. The PCR
product is loaded onto agarose gel (2% agarose in 100 ml TBE buffer, 10 µl SybrSafe). After electrophoresis at 70 V for 60 min, the band
corresponding to the desired PCR product (~size 550 bp) is removed from the gel slice using the PerfectPrep Gel Cleanup Kit following
the manufacturer’s protocol.
Pure PCR products are submitted for sequencing together with the primer ITS1 to a commercial service (e.g., SeqLab GmbH, Goettingen
and BMBF, Düsseldorf, Germany) and the base sequence is compared with publicly available databases (GenBank C, http://www.ncbi.nlm.
nih.gov/Genbank/, National Institute of Biotechnology Information, Bethesda, Maryland, USA) with the help of Blast-Algorithmus.
Box
2 | BIOACTIVITY SCREENING TESTS
Agar diffusion assay
According to the Bauer–Kirby test (DIN 59040), susceptibility disks are impregnated with 250 or 500 µg of crude extracts and 50 or
100 µg of pure compounds, respectively, and placed on agar plates that have been inoculated with either the standard bacterial strains
Bacillus subtilis DSM 22109 ( = ATCC 11774), Escherichia coli DSM 10290 ( = ATCC 15766), the yeast Saccharomyces cerevisiae DSM 1333
( = ATCC 9763) or the fungi Cladosporium herbarum DSM 63422 or Cladosporium curcumerinum DSM 62122.
The plates are checked for inhibition zones after 24 h of incubation (37 °C for bacteria, 27 °C for yeast) or after 7 d (22 °C for
fungi), respectively.
Negative controls are only treated with the respective solvents, positive controls are treated with penicillin G, streptomycin and
gentamycin (for bacteria) or nystatin (for fungi)33,47.
CYTOTOXICITY TESTS
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MATERIALS
REAGENTS • Chromatographic stationary phases: Different materials from different
• Bacto agar (BD, cat. no. 214010) suppliers are possible, in our laboratory material is used as described in
• Malt extract (Merck, cat. no. 105391) a previous protocol41
• Artificial sea salt (Sera, cat. no. 05420) • Laminar air-flow (Herasafe HS15, Heraeus)
• Chloramphenicol (Sigma, cat. no. C0378) • pH meter (420 Aplus, Orion)
• Sodium hydroxide (‘NaOH’, Sigma, cat. no. S5881) • Autoclave (Varioklav, H&P)
• Hydrochloric acid (‘HCl’, Sigma, cat. no. 258148) • Microcentrifuge (Biofuge pico, Heraeus)
• Yeast extract (Fluka, cat. no. 70161) • Freeze dryer (Lyovac GT2, pump Trivac D10E, Savant)
• Peptone (Merck, cat. no. 111931) • PCR machine (iCycler, Bio-Rad)
• Glucose (Caelo, cat. no. 5247) • UV transluminator (GVM 20, Syngene)
• Rice (parboiled, any supermarket brand) • Mixer mill (MixerMill MM30 Retsch, Haan, Germany)
• Glycerin (Roth, cat. no. 4043) • Power supply for electrophoresis (PowerPac 300, Bio-Rad)
• Penicillin G (Sigma, cat. no. P3032) • Ultra Turrax (T18 basic, IKA)
• Streptomycin (Sigma, cat. no. S6501) • Vacuum pump (type 4EKF 56CX-4, Greiffenberger Antriebstechnik)
• Gentamycin (Serva, cat. no. 22185) • Nitrogen generator (UHPN 3001, Nitrox)
• Nystatin (Sigma, cat. no. N4014) • Deep freezer ( − 80 °C, 86-Freezer, Forma Scientific)
• Antimicrobial detergent Melsept SF (B. Braun, cat. no. 18907) • Shaker SM 30 A (Edmund Bühler, cat. no. 6101 000)
• Tungsten carbide bead (Qiagen, cat. no. 69997) • Balance (BL1500, Sartorius)
• Agarose (Sigma, cat. no. A9414) • HPLC, MS and NMR: the equipment as described in a previous protocol is used41
• TBE buffer (Merck, cat. no. 106177) REAGENT SETUP
• DNeasy Plant Mini Kit (Qiagen, cat. no. 69104) Organic solvents for separation and purification Many organic solvents
• Hot StarTaq Master Mix Kit (Qiagen, cat. no. 203443) of varying polarities may be used for chromatographic separation and
• RNAse A (Invitrogen, cat. no. 12091-039) purification procedures, including acetone (! CAUTION highly flammable
• Primers: ITS1 and ITS4 (Invitrogen; individual order) and irritant), acetonitrile (ACN) (! CAUTION highly flammable and toxic),
• SYBR Safe DNA gel stain (Invitrogen, cat. no. S33102) dichloromethane (DCM) (! CAUTION harmful), ethanol (EtOH) (! CAUTION
• DNA loading buffer (Eppendorf, cat. no. 0032 006.850)
highly flammable), ethyl acetate (EtOAc) (! CAUTION highly flammable
• PerfectPrep Gel Cleanup Kit (Eppendorf, cat. no. 0032 007.740)
and irritant), n-hexane (! CAUTION highly flammable, irritant, harmful,
Solvents:
dangerous for the environment and toxic for reproduction), n-butanol
• Solvents for extraction and chromatographic separation (see Reagent
Setup) (n-BuOH) (! CAUTION flammable, harmful and irritant) and methanol
• Solvents for high performance liquid chromatography, HPLC, for (MeOH) (! CAUTION highly flammable and toxic). They all are of analyti-
measuring optical rotation, for NMR spectroscopy are used as described cal grade. ! CAUTION All organic solvents must be handled carefully. Wear
in a previous protocol41 protective clothing, safety glasses and gloves. They should be handled under a
• Spray reagents are used as described in a previous protocol41 fume hood and stored in a ventilated solvent cabinet.
• Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (‘Marfey’s reagent’, Fluka, Medium A for isolation of fungal strains from marine macro-organisms
cat. no. 42095)
and mangrove plants
• Standard amino acids (ICN, Sigma)
EQUIPMENT Bacto agar 15 g
• Sterile plastic petri dishes, diameter 9.4 cm (Greiner, cat. no. 633161) Malt extract 15 g
• Parafilm M (Marienfeld, cat. no. 74 038 10)
Artificial sea salt 24.4 g (for isolation from mangroves 10 g)
• Sterile tubes, 15 ml, 120 mm × 17 mm (Sarstedt, cat. no. 62553)
• Susceptibility paper disks (5 mm diameter, Oxoid) Chloramphenicol 0.2 g
• All micro-organisms are acquired from DSMZ–Deutsche Sammlung von pH 7.4–7.8 (adjusted with NaOH/HCl)
Mikroorganismen und Zellkulturen GmbH
Dem. water fill up to 1,000 g
• Artemia salina (Dohse Aquaristik, cat. no. 21350)
Yeast extract 3g The contents are combined in a flask big enough so that it is filled not more
than two-thirds, covered and autoclaved at 121 °C for 20 min. ! CAUTION
Malt extract 3g During autoclaving the flasks must not be tightly closed. The temperature of the
Peptone 5g autoclave has to be below 80 °C before it can be opened to prevent hot steam
from emerging. Heat protective gloves and safety glasses have to be worn.
Glucose 10 g
After cooling down to ~60 °C the medium is poured in sterile tubes, each
Artificial sea salt 24.4 g (for mangrove-derived fungi 10 g) with ~6 ml under aseptic conditions. The prepared flasks can be kept under
pH 7.2–7.4 (adjusted with NaOH/HCl) sterile conditions for up to 1 week before inoculation. CRITICAL All the
autoclaved media have to be stored under aseptic conditions to prevent
Dem. water ad 1,000 g
contamination with ubiquitous microbes from the environment.
PROCEDURE
Isolation and cultivation of fungi from marine organisms ● TIMING 6–9 weeks
1| Cut the samples (pieces of sponge tissue, algal biomass or mangrove leaves or other organs) into small segments
of approximately 1 cm × 1 cm and rinse three times with sterile sea water to eliminate adherent surface debris.
CRITICAL STEP It is crucial to maintain aseptic conditions by working under a laminar air-flow for the isolation,
purification, transfer and growth of the fungal cultures to prevent contamination by ubiquitous micro-organisms.
2| Immerse a piece of the sample in EtOH 70% (vol/vol) for 60–120 s for surface sterilization.
CRITICAL STEP If the treatment with EtOH is too short, the sterilization of the outer part is not complete; if the
sterilization time is too long, EtOH kills fungi in the inner parts of the tissue.
3| Dry the piece of tissue with sterile cotton cloth (or rinse with sterile artificial sea water) to stop the sterilization
with EtOH.
4| Streak the piece carefully over the surface of a first petri dish containing isolation medium A with sterilized tweezers,
then put it back onto sterile cotton cloth and cut it into smaller segments with a sterile razor blade (negative control).
5| Place the small pieces on a second petri dish containing isolation medium A so that the freshly cut edges are in direct
contact with the agar surface. Seal the agar dish with parafilm, label and store it at 20–25 °C. Cultures are kept between
20 °C and 25 °C under daylight. Fungal growth from the cut segments usually begins after 2–3 d until 14 d after the onset
of experiment.
? TROUBLESHOOTING
6| Usually different fungal strains will develop from one sample. Isolate the individual strains by transferring hyphal tips
growing out of the cut tissue pieces with a sterile loop onto a fresh petri dish containing medium B.
CRITICAL STEP For purification of the fungal strains this step might be repeated several times until the colony is deemed
pure following macroscopic and microscopic analysis.
Depending on the culture condition and different fungal strains, growth can be observed after 2–3 d. Pure strains must
grow for ~1–2 weeks before further workup.
8| For small-scale and large-scale fermentation, inoculate a pure fungal strain in a 1,000 ml Erlenmeyer flask containing
either 300 ml of liquid Wickerham’s medium or 210 g of solid rice medium. For this purpose, cut a strain that covers the
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surface of the inoculated petri dish into small pieces of ~1.5 cm × 1.5 cm and transfer these pieces with a sterile loop into
an Erlenmeyer flask containing the sterilized medium.
Cultivation is carried out at room temperature under static conditions and daylight. Depending on the fungal growth,
cultures on liquid medium are incubated for 3–4 weeks, while on rice for 4–6 weeks.
9| Bring the fermentation to an end by adding 250 ml EtOAc to the culture flask and leave the flask closed for at least
24 h. EtOAc will increase the wettability of the spores and decrease the number of spores, which will become airborne once
the flask is opened.
! CAUTION To prevent the distribution of fungal fragments or spores, flasks should only be opened under a laminar air-flow.
10| For long-term storage, transfer the pure fungal strains to 10-ml BD Falcon tubes containing ~5 ml MexA medium.
When growth can be observed (after ~3 d, depending on the fungal strain), freeze the fungal strain stepwise: first place
it in a fridge at 4 °C for 2 h, then in a freezer at − 20 °C for 2 h and finally place it in a deep freezer at − 80 °C.
PAUSE POINT Frozen pure fungal strains can be stored at − 80 °C.
For recovery from − 80 °C, thaw the frozen culture quickly in a water bath at 37 °C and transfer a small piece with a sterile
loop to a petri dish containing medium B.
! CAUTION To prevent contamination of the environment with viable material, equipment that has been in contact with
fungal material must be autoclaved at 134 °C for 15 min before reuse or disposal.
! CAUTION To prevent contamination of the environment with viable material, an antimicrobial detergent (Melsept SF)
has to be added to the medium and to the disrupted fungal cells and kept in it for at least 1 h before disposal. Auto-
claving is not possible at this stage because of the remaining traces of EtOAc that may cause explosion in the autoclave.
(C) Large-scale liquid medium (Fig. 3)
(i) Separate fungal mycelia from the culture media and cover the mycelia with ~5 liters of MeOH.
! CAUTION To prevent the distribution of fungal fragments or spores, flasks are only opened under a laminar air-flow.
The mycelia are left in MeOH overnight.
(ii) Disrupt and extract the cells for 10 min at 4000 µ min − 1 using an Ultraturrax.
(iii) Filter the mixture under vacuum using a Buchner funnel.
(iv) Repeat extraction in the same manner until exhaustion (2–3 times).
(v) Extract the culture media in the same manner as described for the extraction of small-scale cultures to obtain the
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EtOAc extract.
! CAUTION To prevent contamination of the environment with viable material, antimicrobial detergent has to be added
to the extracted mycelia and growth medium and kept in it for at least 1 h before disposal. Autoclaving of the residues
is not possible because of the remaining solvent.
(D) Large-scale rice medium (Fig. 4)
(i) The extraction of the large-scale rice media is carried out in the same manner as described for the small-scale cultures
using ~10 liters of EtOAc.
? TROUBLESHOOTING
13| In accordance with the diverse properties of the components of the fractions, two different procedures for purification
can be applied:
Box
3 | DERIVATIZATION METHODS
Determination of absolute stereochemistry by Mosher reaction has been described in detail in a protocol on isolation and
structural elucidation of metabolites from marine invertebrates38,41.
Determination of the absolute configuration of amino acids by Marfey’s analysis
Marfey’s reagent (FDAA = Nα-(2,4-dinitro-5-fluorophenyl)-L-alaninamide) is used as a reagent for derivatization of D- and L-amino acids
that are obtained after hydrolysis of cyclic or linear peptides to determine their absolute configuration. The obtained diastereoisomers
can be easily differentiated and identified by their retention times following HPLC analysis on RP columns and comparison with
commercially available D- and L-amino acids that have been treated in the same way47,49.
1. Mix 50 µl of 50 mM of each commercially available standard amino acid (D- or L-form) that is of interest in H2O, 20 µl 1 M NaHCO3
and 100 µl of 1% Marfey’s reagent in acetone and heat at 40 °C for 1 h.
2. Stop the reaction by addition of 10 µl of 2 M HCl.
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols
3. Freeze the derivatized product to − 80 °C, dry it in a freeze dryer, redissolve it in MeOH and analyze it by HPLC or by LC–MS.
4. Hydrolyze your isolated peptide (0.5–1 mg) in 1–2 ml 6 N-HCl at 110 °C for 24 h under N2 atmosphere until complete hydrolysis and
liberation of the amino acids.
5. Cool the hydrolysate containing the mixture of free amino acids, dry it and redissolve it in water to achieve a final concentration of
~50 µM. Proceed in the same way as applied to standard amino acids (see Steps 1–3).
6. Compare the retention times (HPLC or LC–MS) of the derivatized standard amino acids and of the derivatized amino acids obtained
following hydrolysis of the peptide to distinguish D- and L-amino acids.
For low- or medium-polar compounds refer to option (A), for polar compounds refer to option (B).
(A) Low- or medium-polarity fractions
(i) Fractions containing low- or medium-polar compounds are further fractionated and purified using MPLC methods like
VLC. Then, further purification proceeds by column chromatography (CC) using either normal or reversed stationary
phase and a suitable mobile phase to elute the components.
Refer to ref. 41 for advice on the choice of stationary phase and how to setup the experiment.
(B) Polar fractions
(i) Highly polar fractions contain water-soluble organic compounds. In our experience, a good procedure is to use
reversed phase CC (e.g., RP-18) or ion-exchange resin beds such as HP-20 eluted gradually from water to MeOH
to eliminate the remaining mineral salts and sugars present in these fractions. Chromatographic methods
are carried out as described in a previous protocol dealing with workup of extracts derived from marine
macro-organisms41.
? TROUBLESHOOTING
14| Continue the purification procedures until compounds of sufficient purity is obtained to allow structural elucidation.
15| Structure elucidation is carried out using various spectroscopic methods, mainly MS and NMR (1 d and 2 d). For the
elucidation of the absolute configuration of new chiral natural products, derivatization methods such as the preparation
of Mosher esters (see Box 3) or by using Marfey’s method (see Box 3) are sometimes necessary (alternatively, the
absolute configuration may be elucidated by X-ray crystallography or by CD spectroscopy followed by quantum chemical
calculations32).
16| Once the individual components are isolated in pure form and structurally identified, they should be subjected to
bioactivity testing.
17| Study the structure–activity relationships to identify optimized compounds which may serve as drug leads from natural
sources.
● TIMING
Steps 1–5: 15–30 min per isolate (growth ~1–2 weeks)
Step 6: 5 min per strain (growth ~1–2 weeks)
Step 7: see Box 1
Step 8: ~5 min per strain (growth ~3–5 weeks)
Steps 9 and 10: each ~5 min (plus 24 h extraction)
Step 11 A(i–iii): 45–60 min
Step 11 B/D(i): 5/30 min (plus 1 d for extraction)
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 1.
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4 Fungal growth on Insufficient surface sterilization If you are not sure how delicate the surface of your source
negative controls organism is, then try different times for surface sterilization,
e.g. 30, 60 and 120 s. The plates with growth on the respec-
tive negative controls, or those without any growth can be
discarded
6 No growth at all Time of surface sterilization too long, As described above
fungi residing in the tissue are killed
11 Insufficient separation Formation of an emulsion because of Leave standing overnight in the separation funnel; centrifu-
of the two phases extracted compounds gation also helps
13 No or insufficient Elution of the column is too fast Reduce the speed of the mobile phase (for Sephadex material
separation ~10 drops min − 1, silica < 20 drops min − 1, depending on the
size of the column)
Inappropriate stationary or mobile Always test on TLC to check stationary and mobile phase
phases before column chromatography
Column too small Combine all fractions again and find a better separation
system
13, 14 Loss of substance Adsorption on stationary phases Always keep some of your fraction to be able to repeat
separation steps
ANTICIPATED RESULTS 1
Fractions 3 and 4 from the obtained ten fractions were pooled and chromatographed over a Sephadex LH-20 column
with MeOH as eluent. From the obtained 17 fractions, fractions 6 and 7 contained the betaenone congeners, whereas the
anthraquinone derivatives were obtained from the yellow colored fractions 11, 12 and 13.
Purification of the betaenones was accomplished by chromatography on an RP-18 column. 10-Hydroxy-18-methoxybetaenone
(1) was obtained from fraction 7 with MeOH:H2O:TFA (95:5:0.1 vol/vol) as eluent and 10-hydroxy-18-N-2-naphthyl-N-phenyl
aminobetaenone (2) from fraction 6 with MeOH:H2O:TFA (90:10:0.1 vol/vol) as eluent.
The anthraquinone congeners were purified by semi-preparative HPLC on a C18 column using a gradient of H2O and
MeOH as follows: 0 min, 80% MeOH (vol/vol); 20 min, 90% MeOH (vol/vol); 25 min, 90% MeOH (vol/vol); and 30 min,
100% MeOH (vol/vol).
All compounds were readily identified by their spectroscopic data. Through-bond homonuclear and heteronuclear correla-
tions were used to establish assignments and atom connectivities. The relative stereochemistry of 10-hydroxy-18-methoxy
© 2010 Nature Publishing Group http://www.nature.com/natureprotocols
Alternaria metabolites
From the endophyte Alternaria sp. isolated from leaves of the Chinese mangrove plant Sonneratia alba, several natural
products, some of them structurally related to alternariol (6–11), perylene quinones and new compounds (12–17), were
isolated40.
The crude EtOAc extract obtained after fermentation of the fungus on solid rice medium was partitioned between n-hexane
and 90% (vol/vol) aqueous MeOH. After VLC of the MeOH extract using silica gel as stationary phase and a step gradient
employing CH2Cl2 and MeOH as solvent 6 7 8
systems, fraction 3 (80% (vol/vol) O O
CH2Cl2) was chromatographed over HO HOOC OH O OH O OH
Acknowledgments We are indebted to numerous previous coworkers and to 21. Verbist, J.F., Sallenave, C. & Pouchus, Y.F. Stud. Nat. Prod. Chem. 24, 979
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