70 6. BIOACTIVITY GUIDED PURIFICATION OF COMPOUNDS AND THEIR STRUCTURAL CHARACTERIZATION 6.1 Introduction Today, descriptions of nearly 20,000 marine derived compounds can be found in the literature and/or in commercial databases (Ebada et al., 2008). Some of these structures are extremely significant and examples to underscore this point include ziconotide (Prialt) and ET-743 (trabectedin or Yondelis), which are now available as clinical therapeutics (Ebada et al., 2008). The classical way to work on natural-product-containing extracts is often labor intensive and time consuming (Ebada et al., 2008). Many drug discovery programs based on screening of extract libraries, bioassay guided isolation and dereplication/structure elucidation are now following the high throughput screening method (HTS) as an important filter (Koehn & Carter, 2005) Surprisingly, only few investigators have followed the high throughput approach for generating extracts as far as natural resources is concerned. Several years ago it began to de-emphasize the classic Kupchan extraction scheme. But Thale method involves standard solvent partitioning (SSP), in favor of the pressurized liquid extraction system called accelerated solvent extraction (ASE). The ASE apparatus is now widely used in marine environmental studies and terrestrial-based natural products research, as several comparative studies have validated its use as being both time and cost effective. However, there are no reports specifically describing the benefits or problems of employing ASE for the rapid discovery of bioactive marine natural products (Tyler et al., 2010). Majority of the biologically active natural products have been isolated using bioactivity-guided fractionation (Pezzuto et al., 1997). In bioactivity-guided fractionation, the extract of an organism or a mixture of unknown molecules is fractionated and simultaneously biological activities of purified fractions are tested to determine the active fraction in each step of purification. In this process, extract of an organism having large number of molecules is initially separated into two or major parts based on their solubility in aqueous and organic solvents or a combination of organic and aqueous solvents. Then the bioactive sample is further
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70
6. BIOACTIVITY GUIDED PURIFICATION OF COMPOUNDS
AND THEIR STRUCTURAL CHARACTERIZATION
6.1 Introduction
Today, descriptions of nearly 20,000 marine derived compounds can be
found in the literature and/or in commercial databases (Ebada et al., 2008). Some of
these structures are extremely significant and examples to underscore this point
include ziconotide (Prialt) and ET-743 (trabectedin or Yondelis), which are now
available as clinical therapeutics (Ebada et al., 2008).
The classical way to work on natural-product-containing extracts is often
labor intensive and time consuming (Ebada et al., 2008). Many drug discovery
programs based on screening of extract libraries, bioassay guided isolation and
dereplication/structure elucidation are now following the high throughput screening
method (HTS) as an important filter (Koehn & Carter, 2005) Surprisingly, only few
investigators have followed the high throughput approach for generating extracts as
far as natural resources is concerned.
Several years ago it began to de-emphasize the classic Kupchan extraction
scheme. But Thale method involves standard solvent partitioning (SSP), in favor of
the pressurized liquid extraction system called accelerated solvent extraction (ASE).
The ASE apparatus is now widely used in marine environmental studies and
terrestrial-based natural products research, as several comparative studies have
validated its use as being both time and cost effective. However, there are no reports
specifically describing the benefits or problems of employing ASE for the rapid
discovery of bioactive marine natural products (Tyler et al., 2010).
Majority of the biologically active natural products have been isolated using
bioactivity-guided fractionation (Pezzuto et al., 1997). In bioactivity-guided
fractionation, the extract of an organism or a mixture of unknown molecules is
fractionated and simultaneously biological activities of purified fractions are tested
to determine the active fraction in each step of purification. In this process, extract
of an organism having large number of molecules is initially separated into two or
major parts based on their solubility in aqueous and organic solvents or a
combination of organic and aqueous solvents. Then the bioactive sample is further
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purified into small fractions using chromatographic methods and HPLC, etc. Purified
fractions in each step of purification are subjected to test their biological activity. This
procedure is also useful to detect any modifications in the nature of bioactive
component due to the purification, which may lead to the loss of its bioactivity.
Further, it is also useful to select and make changes in the process of purification so us
to purify the active molecule without significant changes in its activity.
The active extract at early stages is fractionated by solvent partitions which
eliminates much of the weight of inactive material, although the active fractions
from these partitions are still exceedingly complex chemically. A typical solvent
partitions of an active extract is suggested by Suffness & Douros (1979).
In spite of available advanced isolation technologies, still isolation of marine
natural products are very challenging task for researchers. Marine natural products are
diverse and often difficult and expensive to synthesize. The amount of metabolite found
in the source organisms is rarely enough to get through preclinical trials. Increasing the
amount of compound by a massive harvest of the source organism is rarely a viable
option because of the disastrous ecological impact. This supply issue has to be
overcome in order to meet the requirements of the demand for those compounds that
become successful drugs. In some cases, it may be possible to chemically synthesize the
compound but most of the time, the complexity of the molecules or the costs involved
preclude this approach. Under this scenario, accurate isolation methodologies,
compound handling techniques, miniaturized screening and analytical systems have to
be evolved that would boost the natural product discovery and development.
Hence, the present attempt on the bioactivity guided isolation of active fraction
form the sponge, Jaspis penetrans and its purification and structural elucidation.
6.2 Materials and methods
6.2.1 Extraction and separation scheme
The objective was to separate the compounds in two stages. Stage 1 is to
separate the fraction initially from less polar solvent to high polar solvent. This
initial step would provide us first step purification. This initial method is mainly
performed by separating funnel with different partitioning method (Fig. 14). The
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crude ethyl acetate extract was applied through this process. At every stage of
solvent extraction thin layer chromatography (TLC) analysis was carried out and
major compounds were recorded. Subsequently, the solvent fractions were tested in
HDAC assay to identify the active fractions. The active fractions were then used for
chromatography evaluation for purifying the active compounds.
6.2.2 Solvent partitioning
Initially solvent partition was carried out using n-hexane: water. A small
portion (10-20 ml) of water was added to make crude as suspension. Once crude
become suspension, 500 ml of n-hexane was added to the separating funnel and then
entire mixture was vigorously shaked for 3-4 time at 2-3 min interval and the funnel
was allowed to stand for 1 h without any disturbance. Once the layers were
separated, the upper layer (n-hexane) was carefully removed from the separating
funnel. This extraction procedure was repeated two more time and n-Hexane
fractions were pooled and concentrated using rotary evaporator and stored at -20°C
for the bioassay screening (Fig. 14).
Fig 14. Solvent partition and chromatographic purification scheme (Adopted from
Ebada et al., 2008).
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6.2.3 Chloroform extraction
After n-hexane extraction was completed, the remaining crude suspension
was added with 500 ml of chloroform in to the separating funnel and vigorously
shaked for 2 min and allowed to stand without any disturbance for 60 min. In case of
chloroform the lower layer was collected and further two more additional extractions
was carried out using 500 ml each with chloroform. Chloroform fractions were
pooled and concentrated using rotary evaporator and stored at -20°C for bioassay
screening.
6.2.4 Ethyl acetate extraction
After chloroform extraction was completed, the remaining crude suspension
was added with 500 ml of ethyl acetate in to the separating funnel and vigorously
shacked for 2 min and allowed to stand without any disturbance for 60 min. In case
of chloroform the lower layer was collected and further two more additional
extractions was carried out using 500 ml each with ethyl acetate. All the three
fractions were pooled and concentrated using rotary evaporator and stored at -20°C
for bio assay screening.
6.2.5 n-Butanol extraction
After ethyl acetate extraction was completed, the remaining crude suspension
was added with 500 ml of n-Butanol in to the separating funnel and vigorously
shacked for 2 min and allowed to stand without any disturbance for 60 min. In case
of n-Butanol, the lower layer was collected and two more additional extraction was
carried out using 500 ml each with n-Butanol. All the three fractions were pooled
and concentrated using rotary evaporator and stored in -20°C for bio assay
screening. Remaining water extract was also air dried and stored at -20°C for assay.
6.2.6 Bioassay screening (HDAC assay)
Bioassay (HDAC assay) was performed as described in Chapter 5 (Section 5.1.2).
6.2.7 Chromatographic purification of active extract
Since the bio-activity and compounds patterns in TLC was similar it was
decided that chloroform and ethyl acetate fractions were combined and used for
compound isolation. Completely dried fractions were mixed together and it was
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further mixed with about 2 g of silica matrix and then kept in hot air oven at 45°C
for 1 h to remove the moisture content of the components.
6.2.7.1 Column packing
Glass column with 60 cm length and 30 mm diameter was purchased from a
local glass blower, Silica gel matrix mesh size 230 – 400 µm (Merck Co., USA),
solvents including Methanol, Chloroform, Ethyl acetate, n-Hexane were purchased
from the authorized Merck Co.,
Silica matrix was taken in to a 100 ml beaker and n-hexane was added to the
suspension mixed well and carefully poured in to the vertically fixed column and it
was allowed to settle and further silica gel was mixed with n-Hexane and poured in
to the column until it reaches the desires column height. Once column matrix height
was finalized, the column was washed with 3 bed volume of n-hexane. Once column
was ready the lower stop cock was closed with little amount for solvent in the
column. Care was taken to maintain the solvent levels in the column.
6.2.7.2 Sample application and column purification
Moisture free silica gel mixed fractions were briefly mixed with n-hexane
and immediately applied in to the column. Once sample matrix was settled the n-
hexane was applied initially with two bed volumes and it was mixed with
chloroform to increase the polarity and factions were collected. All individually
collected fractions were analysed in TLC for the compound separation. In the course
of time polarity of the solvent was slowly increased once chloroform become major
portion then chloroform and methanol was mixed and used as mobile phase for the
compound purification. Fractions were collected until TLC showed compound
peaks.
Individual compound fractions were pooled and evaporated for the individual
and less than 3 compound mix to carry out the separation in the gel filtration matrix.
6.2.7.3 Gel filtration chromatography (Sephadex LH-20)
Sephadex LH-20 matrix was purchased from GE Health Care, Sweden.
Sephadex™ LH-20 was prepared by hydroxypropylation of Sephadex G-25, a bead-
formed dextran medium, and has been specifically developed for gel filtration of
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natural products, such as steroids, terpenoids, lipids and low molecular weight
peptides, in organic solvents. Matrix was allowed to swell in methanol for 3 h and
packed in to the smaller column side (30 cm and 1.5 cm diameter). Once martix was
washed with solvent 1-2% of the bed volume, the purified fraction was applied and
eluted using methanol as eluent. Fractions were collected and analyzed in TLC.
Fractions were pooled and solvent was evaporated using rotary evaporator and taken
for the structural analysis.
6.3 Structural characterization
Methods
6.3.1 Purity analysis by HPLC
The purified and TLC verified compounds were initially tested for their
purity using reverse phase HPLC analysis. C18 column was used as stationary phase
and methanol: water (9:1) ratio was used as mobile phase (Shimadzu LC2010A,
Japan). Once purity was confirmed by HPLC, the molecules were taken for further
analysis.
One mg of both compounds was weighed and dissolved in DMSO and
further it was diluted in methanol to get the concentration of 1µg/ml stock. Initially
20 µl samples were injected in to the HPLC C18 column and both the compounds
were detected by UV. The methanol: water (9:1) was used as the mobile phase.
Percentage of purity was calculated after subtraction of contamination peaks in the
chromatogram.
6.3.2 Electrospray Ionisation Mass Spectrometry (ESI- MS)
ESI uses electrical energy to assist the transfer of ions from solution into the
gaseous phase before they are subjected to mass spectrometric analysis. Ionic
species in solution can thus be analysed by ESI-MS with increased sensitivity.
Neutral compounds can also be converted to ionic form in solution or in gaseous
phase by protonation or cationisation (e.g. metal cationisation), and hence it can be
studied by ESI-MS.
To determine the mass of the compound the 2-3 mg of compounds were
dissolved in chloroform and directly injected in to the ESI MS. The mass spectrum
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has been obtained and analysed for the parent ion to determine the molecular mass