comparative analysis of the natural products of three sea...
TRANSCRIPT
Comparative analysis of the natural products of three sea cucumber species: Holothuria grisea, Synaptula reciprocans and Holothuria manningi
By
Gabriela S. Vinueza-Hidalgo BSc. In Marine Ecology
A thesis presented for the degree of MSc. in Applied Marine and Fisheries Ecology
at the University of Aberdeen
Supervised by: Dr. Frithjof Küpper &
Dr. Rainer Ebel
University of Aberdeen
2013
STUDENT DECLARATION
I hereby declare this thesis is my own work and effort and that it has not been submitted anywhere for any degree application. Where other sources of information have been used, they have been acknowledged. Signature ___________________ Date _______________________
Comparative analysis of the natural products of three sea cucumber species: Holothuria grisea, Synaptula reciprocans and Holothuria manningi
Gabriela Stephanie Vinueza-Hidalgo*
Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen AB24 3UE,
Scotland, U.K
ABSTRACT
Holothurians have been used in food and medicine probably due to the chemical
compounds found within them. They are usually composed of triterpene glycosides, also
called saponins, which often play an ecological role in the environment, but also have
pharmacological and toxicological properties. This project explored the chemical
composition of three understudied holothurian species: Holothuria grisea, Synaptula
reciprocans and Holothuria manningi. LC-MS and NMR procedures were performed on
each of the species in an attempt to elucidate the chemical composition along with
bioassays to reveal the biological functions of the compounds. The analysis provided
evidence for the presence of saponins in H. grisea and H. manningi, but S. reciprocans
does not contain significant amounts of triterpene glycosides. The results have not
previously been reported thus corresponding to probably six new compounds between
the two species. The study was limited in its scope due to the material availability and
the complexity of the analysis. These results contribute the current literature in that they
provide a starting point for the chemical description of the studied species. Furthermore,
analysis utilizing more material and sophisticated NMR experiments with pure
compounds is required in order to make more concrete inferences and ecological
conclusions.
Holothurians, commonly known as sea cucumbers, are the second most numerous group of
echinoderms with more than 1500 described species1 - 3 and a worldwide distribution in all marine
climate zones and bioregions, from shallow waters to the deep ocean. Sea cucumbers have been
used as food and a source of medicine since ancient times4, especially in the Asian market.5 - 7 Known
as trepang or bêche-de-mer, they are considered a delicacy with aphrodisiac characteristics.2, 7 - 9 This
belief could be based on the medicinal properties that may be given by high protein content and
triterpene glycosides10; or, related to the so-called doctrine of signatures11, in which its properties are
associated to the physical appearance of an organism.
The triterpene glycosides, also called saponins (their name is derived from ability to form stable, soap-
like foams in aqueous solutions12) are formed by a hydrophobic part (known as aglycone) and
hydrophilic carbohydrate chain. These are typical secondary metabolites of plant origin13 that can also
be found in animals such as holothurians.14 Most of triterpene glycosides in sea cucumbers are
lanostane derivatives having aglycones belonging to a holostane type [3{3,205-dihydroxy-5a-
lanostano-18,20-lactone] (Figure 1).15 A carbohydrate chain including from two to six monosaccharide
units is linked to C-3 of aglycone,13 which may be triterpenoid or steroid.12 The sugar composition
often includes D-xylose, D-quinovose, 3-O-methyl-D-glycose, D-3-O-methylxylose and D-glucose15–16
but also other sugars. These are different types of sugars such as pentose (C5H10O5) or hexose
(C6H12O6) that can have a methyl group (i.e. methylated hexose, C7H14O6) or a hydrogen atom instead
of the hydroxyl group (i.e. deoxyhexose, C6H12O5). Different isomers (compounds with identical
formula but different stereochemistry) exist for these types of sugars, which usually make their
identification a difficult task. The same occurs with aglycones that have the same formula but differ in
the position of individual substituents (regiochemistry), in addition to the free-dimensional arrangement
of substituents in space (stereochemistry).
OO
OR
19
30 31
32 3
Figure 1. Holostane skeleton structure on which most aglycones from Holothurians are based. R corresponds to the terminal specific to each triterpene glycoside.
The discovery of triterpene glycosides and its complex structures in sea cucumbers have helped to
solve taxonomic problems17 because of their specificity for different taxonomic groups at species,
genus and sometimes for taxa and super-genus level.10, 13-14
The way a species responds to resources, natural enemies, and the physical environment determines
its ability to survive.18 At the same time, the scope of the organism’s response is determined by the
production of natural products that serve as protection against predators or that are of importance for
reproduction and feeding processes.19 In this matter, the chemicals found in holothurians are of
particular interest because of the pharmacological and toxicological properties20-22 including antifungal,
antitumor, hemolytic, cytostatic and immunomodulatory activities.9-10,18,23-29 In an ecological context,
saponins serve as chemical defense to prevent predation based on their deleterious characteristics for
most organisms.21 Among these systems are feeding deterrence29 and expulsion of the cuvierian
tubules (Figure 2).30 For instance, the tubules are expelled after disturbance and instantly become
sticky and immobilize the predator.31 Although the presence of secondary metabolites sometimes fulfill
a biological role in the environment26; it is not always clear what biological role these compounds play.
Figure 2. a) CT: Cuvierian tubules in holothurians are placed next to the cloaca and are expelled when disturbed. b) Illustration of expulsion and lengthening of Cuvierian tubules in Holothuria forskali. Adapted from Vandenspiegel & Jangoux, 1987; Flammang et al., 2002.
As a result, the research interest in the chemical composition of holothurians has much increased in
recent years. Thus, the discovery of new compounds, including bioactive substances, is progressing
fast.32 Nevertheless, many holothurian species remain underexploited and understudied.33 In
particular, investigation of holothurian triterpene glycosides is necessary to understand their role within
the marine ecosystem that may contribute to recognize distribution patterns, possible predators and
potential impacts of harvesting by humans.
The determination of the presence of secondary metabolites of potential value in holothurians relies on
combined procedures of isolation and elucidation techniques. Amongst the isolation procedures, the
most commonly applied methods for saponins are liquid-liquid separation and chromatography such
as column, thin layer (TLC) and high-performance liquid chromatography (HPLC).34 For structure
elucidation, mass spectrometry (MS) and nuclear magnetic resonance spectrometry (NMR) are the
most important techniques.35-36 The application of MS techniques is useful to detect and identify the
presence of chemicals in a mixture, for example at the level of the crude extracts. In order to
accomplish this, MS systems commonly include electrospray ionization (ESI), ion trap and UV
detection.34 ESI is a technique used to produce ions by imparting liquid droplets with small amounts of
energy into the molecule and thus inducing fragmentations.37 The droplets move along the spectra
based on the magnetic field and because of the ion trap, the existing ions in solution are moved into
gas phase. This process repeated several times generates charged ions that have either lost or
gained a small percentage of mass. As a result, these ions, called “quasi-molecular” or “pseudo-
molecular”, are suitable for mass analysis.38 The efficiency of ion formation depends on a molecule’s
ability to associate and carry a charge; this could either be in the negative or positive ionization mode.
The most common quasi-molecular ion is [M+H]+ and it is formed when a molecule easily dissociate to
form protonated molecular ions.37 However, some other major pseudo-molecular ions can also occur,
including [M+Na]+ or [M-H]-, depending on experimental conditions. In addition, the UV absorbance of
the molecule at particular wavelengths needs to be detected in order to produce peaks for each
possibly organic compound that can be later elucidated. It is important to note that while MS depends
on the ability of a compound to ionize (which is difficult to predict by inspection of the structure alone);
UV detection relies on the so-called chromophore of a molecule, which comprises (conjugated) double
bonds or heteroatoms (N or O) with non-bonding electrons (“lone pairs”). The LC-MS system used in
this study (ThermoFinnigan Orbitrap) comprises both MS and UV detection and so is able to detect a
compound as a “peak” as long as at least one of the two prerequisites is met.
The subject of this study are three holothurian species, Holothuria grisea (Selenka, 1867), Synaptula
reciprocans (Forskal, 1775) and Holothuria manningi (Pawson, 1978). Holothuria grisea is a widely
distributed holothurian occurring in the intertidal zone of the Gulf of Mexico 8, Brazil 2, Belize, Panama,
Colombia39, Florida, Texas, Puerto Rico, Lesser Antilles, Jamaica, West Africa, Venezuela40 and
Ascension Island.41 It has distinctive bright red and yellow pattern coloration and a maximum recorded
length of 25 cm.40
Synaptula reciprocans is a holothurian distributed throughout the tropical Indo-Pacific and is common
in the Red Sea.42 It has invaded the Mediterranean Sea following the opening of the Suez Canal.42
This immigration process from the Red Sea via the Suez Canal is known as Lessepsian invasion.43
Synaptula is one of the leading taxa with an established success and dispersal42, 44 in Eastern
Mediterranean. It has been recorded on the Coasts of Cyprus, Israel,44-45 Lebanon, Syria and
Turkey,46 and with casual findings in Greece.47 It is frequently found in shallow sublittoral waters 48 on
soft and hard substrates mostly covered with the green algae Caulerpa racemosa. 49 It has a sticky
rough cylindrical worm-like black body43 that reaches a maximum body length of 40 cm.48
On the other hand, Holothuria manningi is a native holothurian from Ascension Island in the South
Atlantic.50 However, it has occasionally been seen at St. Paul’s Rocks51 and in the continental shelf of
Brazil, especially in places where Panulirus echinatus occur and thus might predate on this species.52
This colonization is believed to have occurred by larval dispersal through water masses. Holothuria
manningi is dark brown in color that fades to lighter brown in the flanks. The species inhabits shallow
waters with calcareous sand and rocky bottoms.40
In contribution to the knowledge of the chemical composition of holothurians, a comparative
investigation has been carried out of these three understudied species from the Mediterranean Sea
and Ascension Island (tropical South Atlantic). Isolation methods in conjunction with mass
spectrometry were used to detect triterpene glycosides and to elucidate or propose tentative
structures. Moreover, assays were conducted to identify possible bioactivities. Altogether this will help
to understand if the chemical composition has an effect on their ecological role and distribution
patterns in marine ecosystems.
RESULTS AND DISCUSSION
1. General Isolation techniques. Kupchan isolation scheme53 was conducted (Figure 3) in
order to separate the compounds based on polarity. In this matter, the least polar to polar fractions
are: H2O, 2-Butanol, MeOH/H2O, DCM and hexane. After obtaining preliminary results by LC-MS,
further purification of the most promising fractions was achieved by flash chromatography such as
SephadexTM, BiotageTM or HPLCTM chromatography. Sephadex LH-20 is a manual column
chromatography in which a gel that swells based on solvents polarity is used to filtrate the particles by
size. The degree of gel swelling decreases with decreasing solvent polarity.54 The difference between
Sephadex and Biotage or HPLC chromatography relies in an automated system in which “air pressure
driven hybrid of medium pressure and short column chromatography […] has been optimized for
particularly rapid separations”.55 All isolation steps were guided by LC-MS results, meaning that the
purification of the most chemically interesting components was pursued.
Figure 3. Kupchan or liquid-liquid separation scheme to separate polar to non-polar organic compounds (left to right).
2. Holothuria manningi. After obtaining the Kupchan fractions, and based on LC-MS and NMR
data, the DCM fraction was identified as the one containing triterpene glycosides. Additional
separation by Sephadex LH-20 column chromatography was conducted to obtain purer compounds.
The total weights of each subfractions are shown in Figure 4.
Figure 4. Isolation Scheme for the DCM fraction of Holothuria manningi. The weight of each fraction is shown below each box.
Analysis of the LC-MS data provided evidence for the presence of a saponin with a molecular formula
of which was established as C41H62O13 at a retention time of 20.62 min. The pseudo-molecular ion
[M+H]+ was identified at m/z 763.4 in the LC-MS in the positive-ion mode, while two sugar moieties
were identified in the carbohydrate chain, a deoxyhexose (deoHex) and a pentose (Pent) based on
fragment ions resulting from the consecutive loss of the sugar moieties from the ([M+H]+) ion, i.e. at
m/z 617.4 ([M+H-deoHex]+) and 485.4 ([M+H-deoHex-Pent]+). In Figure 5, arrows indicate the
consecutive losses of monosaccharide units and the aglycone at m/z 485. The proposed chemical
structure and the collision-induced fragmentation of the sugar moieties are given in Figure 6.
Based on the available literature56 both the aglycone, as well as the aglycone connected to a pentose
might represent known compounds. While no hit was obtained for the diglycoside, thus it almost
certainly represents a new compound. By LC-MS analysis, the molecular formula of the aglycone was
established as C30H44O5, which gave three hits for sea cucumbers in MarinLit.56 On the one hand, it
could be 16-keto-holothurinogenin obtained from Actinopyga flammea (family Holothuriidae)57; while
equally possible are cucumechinol A or C, which both have a molecular weight of 484.66 and differ
only in the stereochemistry at the B/C-ring junction.58
A compound matching the molecular formula (C35O52O9) of the aglycone and a pentose, as detected
in the present study, has been described from Holothuria atra and Holothuria scabra (family
Holothuriidae), 3-O(Pentopyranosyl)-oxidoholothurinogenin with a molecular weight of 616.78.59 It
should be noted that MarinLit56 listed additional hits for this molecular formula, but as they occurred in
species from different phyla, (i.e. Porifera, Cnidaria and Dinophyta) these were deemed extremely
unlikely.
As stated above, MS alone only allows for establishing molecular formula, so it is very well possible
that the saponin detected in Holothuria manningi in the present study differs from the known
compounds both with regard to the aglycone or the sugar units. Clarification of this issue is only
possible by using sophisticated NMR experiments which, however, would require prior isolation of
individual saponins as pure compounds and are not applicable to compound mixtures. One of the
advantages of LC-MS as used in the present project is that this technique allow us to extract “clean”
mass spectra for any chromatographically resolved peak at particular retention time and thus enables
the analysis of a mixture of compounds.
Figure 5. Collision-induced fragmentation of peak at retention time 20.62 min from the DCM fraction of Holothuria manningi. Even though Holothuria manningi appears to have no close relationship to other species of the genus
Holothuria (Pawson, 1978), there is a similarity in the chemical composition to other species in the
same family; consequently, it confirms the specificity of triterpene glycosides for some taxonomic
groups that could suggest a parallel evolution.
- deoxyhexose
O
O
O
O
H
H
H
AglyconeHO
- Pentose
m/z 763.4
m/z 617.4
m/z 485.3
O
O
O
O
H
H
H
O
Chemical Formula: C41H62O13
Aglycone
O
OHHO
OO
OHHO
HOdeoHex
O
O
O
O
H
H
H
O
Chemical Formula: C35H52O9
Aglycone
O
OHHO
OH
Pent
Chemical Formula: C30H44O5
Pent
Figure 6. Possible chemical structure and collision-induced fragmentation of m/z 763.4 cation from Holothuria manningi. DeoHex: deoxyhexose, Pent: pentose. The aglycone corresponds to the known cucumechinol A, but this assignment is based on molecular weight only.
3. Holothuria grisea. The MeOH/H20 fraction was subjected to repeated chromatographic
purification including Biotage and Sephadex LH-20 chromatography. Several fractions were obtained
and a detailed scheme is shown in Figure 7. However, along with the separation process the UV
absorbance and ionization was not easily obtained. This is likely to occur given the limited amount of
material available for elucidation and it is why the Kupchan fraction MeOH/H2O was used to describe
the results below.
Figure 7. Isolation scheme for the MeOH/H2O fraction of Holothuria grisea. The weight of each fraction is shown
below each box.
As a result, the LC-MS data showed a mixture of compounds that could be seen into 5 peaks at
retention times of 16.78, 18.10, 19.86, 22.17 and 23.05 min, respectively (Figure 8).
Figure 8. LC-MS results for the MeOH/H2O fraction of Holothuria grisea. Five peaks were identified at retention times of 16.78, 18.10, 19.86, 22.17 and 23.05 min, respectively.
In the present study, the MS at retention time 16.78 min (Peak 1) exhibited the pseudo-molecular
([M+Na]+) ion peak at m/z 1417 as well as a methyl hexose loss from the ([M+Na-MeHex]+) at m/z
1241. No further fragmentation was observed from m/z 1241. Moreover, one additional pseudo-
molecular [M+H]+ ion peak at m/z 1395 was shown in the positive ion mode, resulting from the same
molecular weight, together with peaks resulting from the consecutive loss of six sugar moieties from
this [M+H]+ ion. The sugar moieties are most likely deoxyhexose (deoHex), Pentose (Pent),
methylated hexose (MeHex), and hexose (Hex). The first, fifth and sixth sugar moiety loss have been
identified as MeHex, deoHex or Hex and Pent, respectively (Figure 9a). However, the fragmentation
pattern from the second to the fourth sugar moieties losses can follow four possible alternative
pathways (Figure 9b). The total collision-induced fragmentation pattern is shown in Figure 9:
1) Orange arrows: 1219 ([M+H-MeHex]-), 1057 ([1219-Hex]-), 881 ([1057-MeHex]-), 749 ([881-Pent]-),
603 ([749-deoHex]-), 471 ([603-Pent]-); 2) Blue arrows: 1219 ([M+H-MeHex]-), 1057 ([1219-Hex]-), 911
([1057-deoHex]-), 749 ([911-Hex]-), 603 ([749-deoHex]-), 471 ([603-Pent]-); 3) Black arrows: 1219
([M+H-MeHex]-), 1057 ([1219-Hex]-), 911 ([1057- deoHex]-), 765 ([911-deoHex]-), 603 ([765-Hex]-),
471 ([603-Pent]-); 4) Pink arrows: 1219 ([M+H-MeHex]-), 1087 ([1219-Pent]-), 911 ([1087-MeHex]-),
765 ([911- deoHex]-), 603 ([765-Hex]-), 471 ([603-Pent]-). The simultaneous observation of these
alternative fragmentation pathways probably indicates that the peak(s) observed in the HPLC
chromatogram (Figure 8) rare not due to single compounds but in fact are derived from inseparable
mixtures of structurally closely related saponins (or other natural products, see below).
In fact, the sixth sugar moiety is directly attached to the aglycone at m/z 471. The suggested chemical
formula is C66H106O31 for the pseudo-molecular [M+H]+ ion peak at 1395.
Figure 9. Collision-induced fragmentation pattern at Retention time 16.78 for Holothuria grisea. a) Full MS of peak 1 b) Enlargement of the mass spectrum given in a). Orange arrows: -Hex at 1057, -MeHex at 881, -Pent at 749; Blue arrows: -Hex at 1057, - deoHex at 911, -Hex at 749; Black arrows: -Hex at 1057, - deoHex at 911, -deoHex at 765; Pink arrows: -Pent at 1087, -MeHex at 911, - deoHex at 765.
The chemical formula of the aglycone is C30H46O4, which corresponds to a compound that has been
previously isolated from the sea cucumber Pentacta quadrangularis (family Cucumariidae).60 It has
been recognized as philinopgenin B and the chemical structure is shown in Figure 10. Other
aglycones such as nebrosteroid L and phorbasterone C were also suggested by a search in the
database MarinLit based on them displaying the same mass. However, these correspond to sponges
and corals and thus it is more likely that the aglycone found for Holothuria grisea is the same occurring
in another species from the same class. Moreover, in H. grisea an aglycone with m/z 484 has been
previously identified as griseogenin. The main difference between the aglycone found in the current
project is an additional oxygen atom. Correspondingly, griseogenin has a chemical formula
C30H46O5.61
In addition, the sugar moieties are impossible to identify with certainty by MS alone given that there
are several different isomers sharing the same molecular mass. For example, glucose and galactose
have the molecular formula C6H12O6 but are stereoisomers, which differ in the arrangement of their
carbon, hydrogen and oxygen atoms in space. As a consequence, no definitive structural elucidation
can be achieved based on the mass spectrometry alone, but only with NMR experiments using
individual saponins as pure compounds.
HO
H3C CH3
CH3
CH3
O
OO
CH3 CH3
CH3
Chemical Formula: C30H46O4Molecular Weight: 470.69
Figure 10. Chemical structure of philinopgenin B that corresponds to the aglycone (m/z 471) identified in this study from Holothuria grisea. Note that the identity of the two compounds cannot be established with certainty.
In addition, peak 2 identified at retention time 18.10 min seems to follow the same fragmentation
pattern but differs from peak 1 in having three more CH2 groups, most likely in the aglycone. As a
result, the pseudo-molecular [M+Na]+ ion peak has been identified at m/z 1459. Consequently, peak 1
and 2 certainly are different compounds and most likely new compounds since no matches for the
chemical formulae were obtained in MarinLit.
Moreover, peak 3 at retention time 19.86 min showed the pseudo-molecular ion peaks at m/z 506
[M+H-H2O]+, 524 [M+H]+ and 546 [M+Na]+ in the positive ion mode (Figure 11a). The possible
chemical formula could either be C25H46N7O2 or C28H50N3O6. Peak 4 (Retention time 22.17 min)
exhibited similar pseudo-molecular ion peaks at m/z 482 [M]+, 508 [M+H]+ and 530 [M+Na]+ (Figure
11b) and in peak 5 (Retention time 23.05 min) only the [M+H]+ ion peak at m/z 510 could be seen
(Figure 11c). The main difference between these MS spectra lies in the presence of an additional
oxygen atom in peak 4 compared to peak 3, whereas in peak 5 there should be two additional
hydrogen atoms compared to 4. The presence of these groups confirm that peaks 3, 4 and 5 are
related compounds with different traces identified at several m/z. Likewise, the presence of 25 to 28
carbon units in this group of compounds and the completely different fragmentation pattern compared
to peaks 1 and 2 suggest that these are not saponins but a different class of secondary metabolites.
This can be concluded since most of the aglycones in triterpene saponins contain around 30 carbons
or more.62
Although some triterpene glycosides have been isolated from this species, the knowledge on the
constituents of Holothuria grisea remains fragmentary.63 In addition, previous studies on Holothuria
grisea have yielded two triterpene glycosides.56 These glycosides are 17-dehydroxyholothurinoside A
and griseaside A and includes the same sugar units observed in this analysis.63-64 It is important to
note that there is a contradiction in regard of griseaside A between Yi et al.64 and Sun et al.63
Furthermore, studies have also shown that holothurin A and B, among other secondary metabolites
are produced in this species.21, 65 In this regard, the chemical formulae here described have not been
previously reported, which strongly suggests that all five compounds described here are new.
Nonetheless, the complex mixture of triterpene glycosides and other secondary metabolites, did not
allow further separation and consequently chemical structures elucidation due to the low amounts of
available material and time constraints in the current project.