Biochemical Systematics and Ecology 96 (2021) 104262
3
indicates that starfish saponins would be involved in chemical defense,
digestion and reproduction.
Since 1981, no further investigation of E. sepositus saponins has been
reported. In this study, we investigated the specific distribution of saponins compounds in the various body components of E. sepositus
collected on the Algerian coast. Our approach was to determinate the
heterogeneity inter-organs, sexual difference and seasonal variation of
the saponins distribution, by analyzing separately saponins extracts of
five different body components (stomach, pyloric caeca, gonads, oral
body wall and aboral body wall) from both sexes (males and females)
collected at different seasons (winter, spring, summer and autumn).
Recent studies have demonstrated that mass spectrometry (MS and
MS/MS analysis) procedures represent very valuable techniques in
respect to fragmentation mechanism and rapid determination of native
saponins in extract mixture (Van Dyck et al., 2009, 2010a, 2010b;
Demeyer et al., 2014; Grauso et al., 2019). Electrospray ionization
tandem mass spectrometry (ESI-MS) has been established as a powerful
tool for the profiling of saponin mixtures, which may somewhat avoid
the time-consuming steps in the isolation of saponins (Schoepke et al.,
1996).
In this study, high resolution mass spectrometry experiments (HRESI-MS and HR-ESI-MS/MS) were used to detect and analyze saponins. A
semi-quantitative study was performed to compare total saponin contents between different organs, sexes and seasons.
2. Materials and methods
2.1. Biological materials
Individuals of Echinaster (Echinaster) sepositus (Retzius, 1783) were
sampled monthly (September 2017 to September 2018), between 5 and
10 m in depth, on the rocky shore the Algerian coast. The site (36
◦
49
′
17
′′
N, 3
◦
0
′
37
′′
E) is located in the Bay of Algiers (Pointe Pescade). The individuals of starfish were separately transported in plastic bags containing sea water. At the laboratory, they were rapidly dissected.
Identification of sexes was performed according to Riesgo et al. (2011),
based on a color of the gonads being dark red in females and yellow in
males. The organs of both sexes (stomach, pyloric caeca, gonads, oral
body wall and aboral body wall) were recovered, weighted, freeze-dried,
ground and stored in dark waterproof tubes inside a dissector until use.
2.2. Saponins extraction and purification
The saponins were extracted from the different organs of both sex
(stomach, pyloric caeca, gonads and bodywall: aboral face and oral face)
of the starfish E. sepositus by microwave assisted extraction using a
multimode microwave oven (NN–S674MF, Samsung, Malaysia). The
operating conditions selected for microwave-assisted extraction were:
25 min, 60% methanol, liquid-solid ratio 44 mL.g
− 1
, and microwave
power 177 W (Dahmoune et al., 2019). The extract obtained at the end
of microwave irradiation was filtered with a Büchner flask through No. 1
Whatman paper under vacuum. The filtrate was evaporated at low
pressure in a double boiler at 30
◦
C using a rotary evaporator (RE301 –
Stuart). The residues were recovered and dissolved in milli-Q water
(aqueous crude extract) to undergo different purification steps. The
extracts were purified according to the method reported in the literature
(Van Dyck et al., 2009). The aqueous crude extract was partitioned by
using the liquid-liquid extraction method. Three organic solvents,
n-hexane (0.09 D), dichloromethane (1.60 D) and chloroform (1.04 D)
were conducted respectively to partition crude extracts. Saponins were
maintained in methanol phase (1,70 D) for each one of these extractions
(Lide, 2009). In fact, the aqueous crude extracts were dried by using
rotary evaporation (RE301 – Stuart, UK). Then dry extracts were solubilized in 20 ml of methanol 90% followed by liquid-liquid partitioning
in n-hexane (v/v) to eliminate lipids and fatty acid. The methanol
fractions were recovered and adjusted with water corresponding to 20%
of the methanolic volume. After that, this later was mixed with an equal
volume of dichloromethane. After decantation, the dichloromethane
fraction was removed and the hydromethanolic phase was adjusted with
40% of water. Then, the liquid-liquid partitioning procedure for a
remaining hydromethanolic phase was continued by adding in a separatory funnel a chloroform (v/v) that was stirred and left at rest for 3 h
minimum. After the sample decantation process, the lower phase was
removed and the resulting hydromethanolic upper phase was evaporated under rotary evaporation to evaporate the solvents. Then, the
obtained dry extracts were dissolved in 5 ml distilled water, which was
transferred to chromatography column packed with Amberlite XAD-4
(Sigma- Aldrich St. Louis, MO, USA). The column was then washed
with 100 ml of distilled water to remove any organic salts. The absorbed
fraction saponins were eluted with 100 ml of methanol.
Finally, the obtained methanolic fractions were evaporated to
remove the methanol solvent; the dry extracts were then dissolved in 5
ml of distilled water. The resulting aqueous solution obtained previously
was mixed in a separatory funnel with 5 mL iso-butanol solvent that was
blended and left at rest for 2 h. After this period, the upper saponins-rich
phase was separated from lower phases, then, the iso-butanol solvent
was evaporated in a rotary evaporator. Finally, the dry recovered extract
was dissolved in distilled water and lyophilized.
2.3. Mass spectrometry analysis
High resolution mass spectrometry experiments (HR-ESI-MS and HRESI-MS/MS) were performed on a Thermo Fisher Scientific Q-Exactive
mass spectrometer, in both positive and negative ionization modes by
direct infusion using Electrospray ionization source (ESI+/-).
MS and MS/MS experiments were performed at the higher resolution
of the instrument (140,000 at m/z 200). Elementary composition of the
ions was determined using instrument software (Xcalibur). The saponin
extracts were prepared using a concentration of 10–20 μg mL
− 1
and the
flow rate was 5–10 μL min
− 1
.
ESI conditions parameters were as follows: spray voltage 3–4 kV,
capillary temperature 300
◦
C, sheath gas flow rate 10–80, auxiliary gas
flow 0–10, SLens RF Level 100.
The single-stage ESI–MS spectra were recorded by scanning
150–2000 m/z mass range.
For the ESI–MS/MS experiments, the interest ions were massselected with a window of 0.4 D. The [M+Na]
+
of each ion was
selected as a precursor and they were submitted to collision energy.
Optimized conditions were obtained by decrease of 2/3 intensity of the
parent ion to obtain the better MS/MS fragment ions.
The relative intensities are calculated by comparing the peak intensity of each saponin ion to the most intense saponin ion signal (set at
100%).
2.4. Semi-quantitative study
A semi-quantitative approach has been realized in order to obtain
data on the total saponin content in E.sepositus. The aim of this approach
was to quantify the concentration of the whole saponin mixture, for each
body compartment of each sex collected in different seasons. The saponins amount was determinate according to the Food Standard Code
(Kwon et al., 2003). The round bottom flask was dried at 105
◦
C, cooled
in desiccators, and weighed to a constant weight before its freeze-drying.
Once the lyophilization was completed, the flasks were weighed again.
The difference in weights corresponds to the total saponin amount of the
sample (mg Tsap ). The saponins were recovered in the form of white
residues.
The total saponins compound yield (TSC) was expressed in mg.g
− 1
and calculated from the total saponin amount (T sap ) and starfish powder
dry, according to the following equation (Eq. (1)):
B. Dahmoune et al.
3
indicates that starfish saponins would be involved in chemical defense,
digestion and reproduction.
Since 1981, no further investigation of E. sepositus saponins has been
reported. In this study, we investigated the specific distribution of saponins compounds in the various body components of E. sepositus
collected on the Algerian coast. Our approach was to determinate the
heterogeneity inter-organs, sexual difference and seasonal variation of
the saponins distribution, by analyzing separately saponins extracts of
five different body components (stomach, pyloric caeca, gonads, oral
body wall and aboral body wall) from both sexes (males and females)
collected at different seasons (winter, spring, summer and autumn).
Recent studies have demonstrated that mass spectrometry (MS and
MS/MS analysis) procedures represent very valuable techniques in
respect to fragmentation mechanism and rapid determination of native
saponins in extract mixture (Van Dyck et al., 2009, 2010a, 2010b;
Demeyer et al., 2014; Grauso et al., 2019). Electrospray ionization
tandem mass spectrometry (ESI-MS) has been established as a powerful
tool for the profiling of saponin mixtures, which may somewhat avoid
the time-consuming steps in the isolation of saponins (Schoepke et al.,
1996).
In this study, high resolution mass spectrometry experiments (HRESI-MS and HR-ESI-MS/MS) were used to detect and analyze saponins. A
semi-quantitative study was performed to compare total saponin contents between different organs, sexes and seasons.
2. Materials and methods
2.1. Biological materials
Individuals of Echinaster (Echinaster) sepositus (Retzius, 1783) were
sampled monthly (September 2017 to September 2018), between 5 and
10 m in depth, on the rocky shore the Algerian coast. The site (36
◦
49
′
17
′′
N, 3
◦
0
′
37
′′
E) is located in the Bay of Algiers (Pointe Pescade). The individuals of starfish were separately transported in plastic bags containing sea water. At the laboratory, they were rapidly dissected.
Identification of sexes was performed according to Riesgo et al. (2011),
based on a color of the gonads being dark red in females and yellow in
males. The organs of both sexes (stomach, pyloric caeca, gonads, oral
body wall and aboral body wall) were recovered, weighted, freeze-dried,
ground and stored in dark waterproof tubes inside a dissector until use.
2.2. Saponins extraction and purification
The saponins were extracted from the different organs of both sex
(stomach, pyloric caeca, gonads and bodywall: aboral face and oral face)
of the starfish E. sepositus by microwave assisted extraction using a
multimode microwave oven (NN–S674MF, Samsung, Malaysia). The
operating conditions selected for microwave-assisted extraction were:
25 min, 60% methanol, liquid-solid ratio 44 mL.g
− 1
, and microwave
power 177 W (Dahmoune et al., 2019). The extract obtained at the end
of microwave irradiation was filtered with a Büchner flask through No. 1
Whatman paper under vacuum. The filtrate was evaporated at low
pressure in a double boiler at 30
◦
C using a rotary evaporator (RE301 –
Stuart). The residues were recovered and dissolved in milli-Q water
(aqueous crude extract) to undergo different purification steps. The
extracts were purified according to the method reported in the literature
(Van Dyck et al., 2009). The aqueous crude extract was partitioned by
using the liquid-liquid extraction method. Three organic solvents,
n-hexane (0.09 D), dichloromethane (1.60 D) and chloroform (1.04 D)
were conducted respectively to partition crude extracts. Saponins were
maintained in methanol phase (1,70 D) for each one of these extractions
(Lide, 2009). In fact, the aqueous crude extracts were dried by using
rotary evaporation (RE301 – Stuart, UK). Then dry extracts were solubilized in 20 ml of methanol 90% followed by liquid-liquid partitioning
in n-hexane (v/v) to eliminate lipids and fatty acid. The methanol
fractions were recovered and adjusted with water corresponding to 20%
of the methanolic volume. After that, this later was mixed with an equal
volume of dichloromethane. After decantation, the dichloromethane
fraction was removed and the hydromethanolic phase was adjusted with
40% of water. Then, the liquid-liquid partitioning procedure for a
remaining hydromethanolic phase was continued by adding in a separatory funnel a chloroform (v/v) that was stirred and left at rest for 3 h
minimum. After the sample decantation process, the lower phase was
removed and the resulting hydromethanolic upper phase was evaporated under rotary evaporation to evaporate the solvents. Then, the
obtained dry extracts were dissolved in 5 ml distilled water, which was
transferred to chromatography column packed with Amberlite XAD-4
(Sigma- Aldrich St. Louis, MO, USA). The column was then washed
with 100 ml of distilled water to remove any organic salts. The absorbed
fraction saponins were eluted with 100 ml of methanol.
Finally, the obtained methanolic fractions were evaporated to
remove the methanol solvent; the dry extracts were then dissolved in 5
ml of distilled water. The resulting aqueous solution obtained previously
was mixed in a separatory funnel with 5 mL iso-butanol solvent that was
blended and left at rest for 2 h. After this period, the upper saponins-rich
phase was separated from lower phases, then, the iso-butanol solvent
was evaporated in a rotary evaporator. Finally, the dry recovered extract
was dissolved in distilled water and lyophilized.
2.3. Mass spectrometry analysis
High resolution mass spectrometry experiments (HR-ESI-MS and HRESI-MS/MS) were performed on a Thermo Fisher Scientific Q-Exactive
mass spectrometer, in both positive and negative ionization modes by
direct infusion using Electrospray ionization source (ESI+/-).
MS and MS/MS experiments were performed at the higher resolution
of the instrument (140,000 at m/z 200). Elementary composition of the
ions was determined using instrument software (Xcalibur). The saponin
extracts were prepared using a concentration of 10–20 μg mL
− 1
and the
flow rate was 5–10 μL min
− 1
.
ESI conditions parameters were as follows: spray voltage 3–4 kV,
capillary temperature 300
◦
C, sheath gas flow rate 10–80, auxiliary gas
flow 0–10, SLens RF Level 100.
The single-stage ESI–MS spectra were recorded by scanning
150–2000 m/z mass range.
For the ESI–MS/MS experiments, the interest ions were massselected with a window of 0.4 D. The [M+Na]
+
of each ion was
selected as a precursor and they were submitted to collision energy.
Optimized conditions were obtained by decrease of 2/3 intensity of the
parent ion to obtain the better MS/MS fragment ions.
The relative intensities are calculated by comparing the peak intensity of each saponin ion to the most intense saponin ion signal (set at
100%).
2.4. Semi-quantitative study
A semi-quantitative approach has been realized in order to obtain
data on the total saponin content in E.sepositus. The aim of this approach
was to quantify the concentration of the whole saponin mixture, for each
body compartment of each sex collected in different seasons. The saponins amount was determinate according to the Food Standard Code
(Kwon et al., 2003). The round bottom flask was dried at 105
◦
C, cooled
in desiccators, and weighed to a constant weight before its freeze-drying.
Once the lyophilization was completed, the flasks were weighed again.
The difference in weights corresponds to the total saponin amount of the
sample (mg Tsap ). The saponins were recovered in the form of white
residues.
The total saponins compound yield (TSC) was expressed in mg.g
− 1
and calculated from the total saponin amount (T sap ) and starfish powder
dry, according to the following equation (Eq. (1)):
B. Dahmoune et al.