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

71

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

72

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).

73

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

74

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

75

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

76

has been obtained and analysed for the parent ion to determine the molecular mass

of the compound.

6.3.3 Fourier Transform Infrared Spectroscopy (FTIR)

FTIR is a technique is used to obtain infrared spectra of absorption,

emission, photoconductivity or Raman scattering of a solid, liquid or gas. An FTIR

spectrometer simultaneously collects spectral data in a wide spectral range. Dry

powder of the compounds were mixed with potassium chloride and analysed in the

Bruker FTIR instrument to detect the functional groups of the compounds.

6.3.4 UV Spectroscopy

Ultraviolet–visible spectroscopy or ultraviolet-visible spectrophotometry

(UV-Vis or UV/Vis) refers to absorption spectroscopy or reflectance spectroscopy in

the ultraviolet-visible spectral region. Compounds were dissolved DMSO at 1

mg/ml concentration and serially diluted in methanol and UV visible spectrum was

observed from 230 nm- 900 nm to identify lamda (λ ) max in one nm wave length

increment. The obtained UV spectrum the data was plotted against the concentration

vs wavelength.

6.3.5 NMR analysis

Nuclear Magnetic Resonance (NMR) is a physical phenomenon in which

nuclei in a magnetic field absorb and re-emit electromagnetic radiation. This energy

is at a specific resonance frequency which depends on the strength of the magnetic

field and the magnetic properties of the isotope of the atoms. About 20 mg of

samples were dissolved in 500µl of CDCL3 and the spectral analysis was done using

500 mHz for the following parameters; 1H,

13C, DEPT-135, COZY, HSQC and

HMBC, for purified compounds. The data was analyzed with the help of

experienced natural product chemist and structure and correlated with other

parameters.

6.4 Results

Solvent fraction

Solvent fractionation of the sponge, Jaspis penetrans was applied for the

solvent. Initially, 16.3 g of ethyl acetate fraction was obtained from 500 g of sponge.

77

Out of 16 g, 10.2 g was used for the solvent partition. And a total 9.2 g was obtained

from 10.2 g (Table 18).

Table 18. Solvent fractions Jaspis penetrans ethyl acetate extract

Fraction n-Hexane Chloroform Ethyl acetate n-Butanol H2O Total

Dry weight (g) 2.65 1.72 1.48 0.93 2.32 9.1

HDAC assay for fraction

All the five fractions were analyzed in HDAC IC50 assay with 2 different

concentration that showed chloroform extract exhibited maximum inhibition it

showed 84.3 % inhibition at 1000 µg and 45.61% inhibition at 300 µg. Similarly

ethyl acetate fraction showed good activity with 59.41% inhibition at 1000 µg and

17.14 % inhibition at 300 µg concentration (Table. 19 & Fig. 15).

IC50 determination was carried out for all the fractions (Table 19).

Chloroform fraction exhibited good dose dependent inhibition and showed 317.41µg

as IC50. Similarly, ethyl acetate fraction exhibited 527.65 µg as IC50. Hence both

fractions were pooled for the purpose of increasing the concentration of active

compound. In this assay, the used crude ethyl acetate fraction exhibited 517.99 µg.

Results indicates that solvent fraction are showing better activity than crude.

Table 19. HDAC assay for solvent fractions

% inhibition

Fractions

1000

(µg/ml)

300

(µg/ml)

n-Hexane 30.3 -2

Chloroform 84.3 45.61

Ethyl acetate 59.41 17.14

n-Butanol 20.01 5.31

H2O -6.1 -5.54

78

Fig 15. Percentage HDAC inhibition of solvent fraction.

Table 20. IC50 determination of solvent fractions

Fraction name HDAC assay IC50 (µg)

n-Hexane >1000

Chloroform 317.41

Ethyl acetate 527.65

n-Butanol >1000

H2O >1000

Extract-7 Crude 517.99

Table 21. Percentage inhibition of pooled fractions

% Inhibition of pooled the fractions

Fractions 111- 118

120-

126

138-

157

267-

276

158-

167

213-

234

252-

256

297-

313 SAHA

Conc.

(µg)

1000 µg 95.168 93.958 40.129 17.686 61.371 1.236 1.567 2.825 91.965 1

300 µg 60.462 58.678 17.964 2.821 30.04 9.123 2.786 2.088 47.559 0.3

79

Chromatographic purification of single compound

After successful selection of 2-active fractions, they were pooled and applied

in the silica column for the initial column purification. Totally 313 fraction were

collected and activity was analyzed for all fractions and found that fraction from 111

to 139 showed HDAC inhibition (Table 20). Further the identified fraction 111-118

a single compound, has been selected for structural elucidation. Similarly, from the

other fraction 7 was shown to be a single compound and so compound 7 also

considered as another compound (Plate. 10). Unfortunately compound-7 did not

inhibit the HDAC and not shown any anticancer activity. However, the compound-7

was taken for further structure elucidation.

Plate 10. TLC analysis of pure compounds

IC50 determination was performed for the pooled fractions of 111-118 (that inhibit

HDAC at 157.41 µg).

Table 22. IC50 determination of pure compounds

Compound name HDAC assay IC50 (µg)

Compound-7 Not determined

Compound- 111-118

111-118 157.41+12.3

SAHA (Std) 73+41 (nM)

80

Compound-7 structural characterization data are presented in the following

images:

HPLC analysis

Compound retention time as 2.492 min and it showed three other

contaminations in the chromatogram. Compound-7 peak showed 74.052 % purity.

Hence there are no major contamination and compound-7 has been included for

other analysis.

Fig. 16. HPLC analysis of Compound-7

UV-spectroscopic analysis of Compound-7

In the initial UV analysis, two peaks were observed for the compound.

Normally, this pattern will be observed in fluorescence compounds. So by fixing on

wavelength as excitation, again spectral wavelength analysis was performed. The

experiment revealed that compound has fluorescence property as shown by 2 peaks

at 320 & 440nm (Fig. 17-a&b).

81

(a)

Fig. 17 (a) UV spectra for compound-7 (UV 230-700nm)

(b)

Fig. 17 (b) UV spectra for compound-7 (ex-310)

82

Tra

nsm

itta

nce

[%

]

20

4

0

60

8

0

3775.7

5

371

2.1

9

3662.6

3

3629.2

6

343

4.1

6

3255.0

2

3129.6

3

2925.0

9

2861.8

2

1722.9

0

1627.2

7

1439.5

5

1381.4

9

1233.7

6

1176.0

0

1112.1

4

1040.0

7

979.4

4

752.9

7

695.2

9

628.3

4

598.3

7

556.8

9

493.2

4

453.

41

405.1

3

ESI-MS Analysis of Compound-7

ESI-MS analysis was performed for compound 7 and found that molecular

weight of the compound is 275.20 (Plate 11).

Plate 11. ESI-MS spectra of compound-7

3 5 00 3 0 00 2 5 00 2 00 0 1 5 0 0 1 0 0 0 5 0 0

W a ve n um b er c m- 1

C : \ P ro g ra m F ile s \ O P U S _ 65 \ M E A S \ M - P U R E -7 -. 0 M - P UR E -7 - S O L ID 2 8 / 0 5 / 2 0 1 3

P a g e 1 / 1

Plate 12. FTIR analysis of Compound-7

83

NMR analysis of Compound-7 (performed 1H proton,

13C and DEPT)

Plate 13. 1H Proton spectrum of Compound-7

84

Plate 14. 13

C NMR spectrum of Compound-7

85

Plate 15 DEPT NMR spectrum of Compound-7

86

Compound structure: Minor peaks could contribute the X groups.

Fig. 16 Predicted structure of Compound-7

The results of the structural analyses of the compound-111-118 are presented

through the following images

HPLC analysis

Compound retention time as 4.131 min and it showed three other

contaminations in the chromatogram. Compound 111-118 peak showed 81.23 %

purity. Hence there are no major contamination compound 111-118 has been taken

for further analysis.

Peak # Ret. Time Area Height Area % Height %

1 1.761 6523 3254 2.8809 2.8780

2 2.812 21612 12094 9.5451 10.6966

3 3.623 14363 8352 6.3435 7.3870

4 4.131 183921 89364 81.2304 79.0384

Total 226419 113064 100.00 100.00

Plate 17. HPLC analysis of Compound-111-118

87

UV spectroscopy analysis of Compound-111-118

In the Initial UV analysis lambda max was observed at 340 nm (Fig. 18).

Fig. 18 UV spectroscopic analysis of Compound 111-118

ESI-MS spectrum of compound 111-118

The ESI-MS analysis was performed for the compound 111-118 and found

that molecular weight of the compound was 1347.91 (Plate 18).

1347.91

Plate 18. ESI-MS spectra of compound 111-118

88

M M IX

Tra

ns

mit

tan

ce [

%]

0

20

4

0

60

8

0

10

0

3020

.15

2400

.16

1219

.83

786

.88

732

.34

669

.40

FTIR spectrum of Compound 111-118

3 5 0 0 3 0 0 0 2 5 0 0 2 0 0 0 1 5 0 0 1 0 0 0 5 0 0

W a ve n u m b e r c m - 1

C : \ P r o g r a m F i l e s \ O P U S _ 6 5 \ M E A S \ M M IX 1 1 1 - 1 1 8 . 0 1 1 1 - 1 1 8 L IQ U ID 2 7 / 0 5 / 2 0 1 3

P a g e 1 / 1

Plate 19. FTIR spectrum of Compound 111-118

Plate 20. 1H Proton NMR spectrum of Compound-111-118

89

Plate 21. 13

C NMR spectrum of Compound 111-118

Plate 22. DEPT NMR spectrum of Compound 111-118

90

Plate 23. COZY NMR spectrum of Compound 111-118

Plate 24. HMBC NMR spectrum of Compound 111-118

91

Plate 25. HSQC NMR spectrum of Compound 111-118

Structure of Compound 111-118

Summary:

The compound 111-118 displayed high degree of HDAC inhibition.

However, its molecular structure was highly complex as it did not form any crystals.

Hence, unable to arrive its predictive structure. However, the process of further

structural characterization studies of this molecule is in progress.

6.5 Discussion

A large number of bioactive molecules have been isolated from marine

organisms (Donia et al., 2003). The crude extracts having biological activity have

been used as a source for isolation of active molecule(s) through different

purification methods. The extract are subjected to several rounds of fractionation to

separate other molecules from bioactive molecule. The highly pure fraction with

potent biological activity is then subjected to structural analysis. One of the popular

approaches is to use combination of methods of purification, such as HPLC

92

separation and other chromatographic methods followed by determination of the

structure by Mass spectroscopic analysis, Nuclear magnetic resonance (NMR),

Infrared (IR) and X-ray crystallography.

The selection of an assay system to monitor fractionation is based on the

original activity of the extract. An effort is made whenever possible to use in vitro

systems to monitor activity since the test results can be obtained much more rapidly

than with in vivo testing. In most cases, the in vitro activity parallels with the in vivo

activity so that extracts, which were originally discovered as in vivo actives can be

followed by cell culture assay.

Many of the natural products isolated from marine invertebrates might share

their structural homology with compounds of microbial origin, leading to the

hypothesis that the marine compounds are actually produced by microorganisms

(e.g.microalgae, cyanobacteria and heterotrophic bacteria) living in association with

the invertebrates (e.g. sponges, tunicates, and soft corals). Marine invertebrates are

indeed largely sessile, filter-feeding organism that contain a complex assemblage of

symbiotic microorganism. Thus, the real producer of biologically active compounds

from marine invertebrates is always uncertain.

Innumerous bioactive compounds have been described and some of them

have reached the clinical space. However, it has often proven extremely difficult and

in some cases it is impossible, to provide from invertebrates or microalgae sufficient

amounts of many of these substances due to limited amounts found in the producing

organism, or to limited quantity of the organism itself, or to geographic, seasonal or

sexual variations in the amounts and in the nature of produced secondary

metabolites. On the other hand, the structural complexity of most of the interesting

bioactive substances precluded the development of commercially viable synthesis

(Alphonse Kelecom, 2002).

The chemical diversity found in one sponge may represent several different

classes of bioactive molecules that exhibit different and sometimes opposing

pharmacological activities. In many cases, the presence of a major non-selective

compound can mask the activity of minor selective compounds. Minor compounds

in many cases are present in crude extracts at concentrations that are below detection

93

thresholds. From a discovery standpoint, these problems can be addressed to a

certain point through the use of pre-fractionation strategies (Tim et al., 2008).

The structure elucidation of a natural product with high biological activity is

both stimulating and challenging. The first step in structure elucidation is to

ascertain what the skeleton of the molecule is, and this can often be narrowed down

by reference to phytochemical literature on related genera and species. A knowledge

of biosynthesis of secondary metabolites is very helpful in deducing the most logical

substitution patterns once the basic structural nucleus is established. Unfortunately,

not all the compounds isolated by activity-directed fractionation are novel and new.

Some known compounds are often detected. The classical method of structure

determination requires degradation of the molecule to establish the nucleus and

various transformation reactions combined with rigorous analysis of spectral data of

the derivatives. X-ray crystallographic studies are finally undertaken either on the

compound itself or a heavy atom containing derivative to establish the structure and

stereochemistry (Bhakuni & Rawat, 2005)

In the present study, two compounds have been purified using bioactivity

guided purification. Compound-7 is relatively simple molecule with the fluorescence

property and we could able to resolve the structure almost 90% with the help of

experts. In the other hand, compound 111-118 displayed target enzyme inhibition

and target modulation. Studies were conducted on regular structure elucidation

parameters including 2D-NMR. But still structure could not be revealed. It requires

additional studies including crystallography studies, circular dichroism (CD) and

further mass analysis experiments. Our future direction would be structural

characterization of this compound.

Recently, the 3D structural determination was available through X-ray

spectroscopy even if no other additional spectral information exist. An X-ray crystal

structure determination is the ultimate analysis. No other analytical technique is

currently available and can deliver such complete and unambiguous information

about the nature of the substance being investigated, but this technique has some

limitations. Obtaining good single crystals are important factor sometimes it may

take longer duration. Decomposition of the compound during crystallization trials

94

can be a difficulty with reactive compounds. In addition, generally the analysis

which done on a single crystal, which may not be representative of the target

compound. The conformational results apply to the solid state and may be different

to the molecular conformations present in solution, which is where most reactions

take place. The modern and highly advanced technology applied in NMR

spectroscopy and mass spectrometry provide unequivocal structural in formation for

the individually isolated compounds (Exarchou et al., 2005).

Advances in microscale spectroscopic techniques, particularly

microcryoprobe NMR, allow discovery and structure elucidation of new molecules

down to only a few nanomole. Newer methods for utilizing CD have pushed the

limits of detection to picomole levels. NMR and CD methods are complementary to

the task of elucidation of complete stereostructures of complex natural products.

Together, integrated microprobe NMR spectroscopy, microscale degradation and

synthesis are synergistic tools for discovery of bioactive natural products and have

opened new realm for discovery (Molinski, 2010).

The obtained/ isolated pure compound of Jaspis penetrans was subsequently

used for the evaluation of anticancer purpose.