Chemical Engineering and Processing - Process Intensification 163 (2021) 108359
2
obtain the maximum yield from the low amount of the primary matrix.
In this regard, this study aims at enhancing the extraction yield of saponins mixture, from the starfish Echinaster sepositus collected in
Algerian coasts, through applying microwave-assisted extraction (MAE)
technique and optimizing its conditions.
The interest in saponins of this species is justified by their unusual
structure, belonging to a rare structure group of marine glycosides
which discovered by Italian scientists more than 40 years ago [6,7]. This
unique compound containing a trisaccharide chain cyclized between C-3
and C-6 of the Δ7-3β,6β-dihydroxysteroid aglycone (Fig. 5). On the other
hand, starfish saponins are of high pharmaceutical interest due to their
anti-bacterial, anti-fungal, anti-viral and anti-tumor properties [8–12].
To optimize the parameters affecting the MAE of saponins from
starfish, the Box–Behnken experimental design (BBD) using response
surface methodology (RSM) was applied. BBD is one of the most efficient
experiment designs, because it does not allow the combinations for
which all factors are simultaneously at their highest or lowest levels.
Thus, it is used to avoid experiments performed under extreme conditions. In this study, the following factors have been evaluated: the solvent concentration (X1), irradiation time (X2), microwave power (X3)
and liquid-solid ratio (X4). Based on the response surfaces, optimal
conditions of the microwave pretreatment have been determined.
Finally, the study has been complemented with a variance analysis
(ANOVA), to determine the statistically significant factors.
To the best of our knowledge, there is no available literature detail
with the optimization of green procedure for the extraction of total saponins compound (TSC) from starfish.
Therefore, the main objectives of the current study can be listed as
follows:
• Optimization of MAE procedure by response surface methodology
(RSM) for the extraction of total saponins compounds from
E. sepositus;
• Studying comparatively the optimized MAE, UAE and the reference
CSE methods;
• Using high-resolution mass spectrometry (HR-ESI-MS) to identify the
saponins mixture obtained by MAE method.
• Evaluation of the hemolytic power of the three saponins extracts
(MAE, UAE and CSE).
• Evaluation of the antioxidant capacity power of the three saponins
extracts (MAE, UAE and CSE) by their ability to scavenge
electrochemically-generated superoxide radical (O 2
• −
).
2. Materials and methods
2.1. Biological material
E. sepositus (Retzius, 1783) individuals were collected in summer
2018 from Algerian sea coast between 5–10 m of depth. In total, 28
individuals as samples weighed about 1724 g were collected and were
transported in plastic bags containing sea water. In the laboratory, the
starfish individuals were freeze-dried (Alpha-1-2 LD plus freeze-dryer).
The lyophilized samples obtained were ground with an electrical
grinder and they were stored in dark waterproof tubes inside a desiccator until use.
2.2. Experimental work design and statistical modelling
The type of solvent, solvent concentration, irradiation time, microwave power and liquid-to-solid ratio were firstly studied separately in
single-factor experiments to reduce the number of the total experiments
work, and to determine the appropriate interval for each parameter in
the design of experiment for microwave assisted extraction - MAE
(Table 1). In this preliminary study, the experimental work has been
carried out by varying an experimental parameter while all the other
parameters were kept constant at their middle values.
In the MAE, the type and concentration of solvent effect were studied
for a constant value of liquid-solid ratio (20 mL g
− 1
), microwave power
(200 W) and irradiation time (5 min). The irradiation time effect was
evaluated for a MeOH concentration of 50 %.
The irradiation time was set at 3 min in the trials to investigate the
microwave power influence. This latter was set at 200 W in the trials to
investigate the ratio liquid-to-solid parameter. The response surface
methodology (RSM) based on Box–Behnken design (BBD) of threelevels-four-factor experimental was assessed to maximize the saponins
yield extracted from E. sepositus using MAE process (Table 2). The design
requires a number of experiment trials according to N = 2k(k − 1) + C 0 ,
where k is the number of factors and C 0 is the number of center points
and each independent factor is studied at three level (-1, 0 and +1)
reference.
The data was analyzed by Minitab.17 with a standard least square in
order to obtain RSM. The ANOVA F test was assessed to adjust models
and the coefficient significance at a p-value less than 0.05 (Table 3).
The model used for data regression analysis is corresponding to a
second-order polynomial equation (Quadratic model) (Eq. (1)). This
equation is then used to predict the optimal extraction conditions.
Y = B 0 +
∑ k
i=1
B i X i +
∑ k
i=1
B ii X
2
i +
∑ k
i>1
B ij X i X j + E
(1)
where Y represents the total saponin compounds (TSC); β 0 is a constant
coefficient; B i ,B ii and B ij are the coefficients of the linear, quadratic and
interactive terms, respectively, and X i and X j represent the actual independent variables (i–j), E is an error.
Additional experiments of extraction were carried out at the optimal
conditions, and the results obtained were compared to the values predicted by the regression model in order to validate the model.
Finally, the extract obtained at the optimal conditions was compared
to ultrasound assisted extraction (UAE) and conventional solvent
extraction method (CSE). The effects of microwave, ultrasonic and
Table 1
Single-factor experiment results for MAE of saponins from E. sepositus. Results are reported as means ± SD. Same letters in the same column refer to means not
statistically different according to ANOVA and Tukey’s test. TSC, total saponin compound; T sap , saponin total; SP, starfish powder.
Solvent
Methanol
concentration (%)
Irradiation time (min)
Microwave power (W)
Liquid-to-solid ratio (mL/g)
Type
TSC
(mg Tsap /g SP )
% (V/V)
TSC
(mg Tsap /g SP )
min
TSC (mg Tsap /g SP )
W
TSC
(mg Tsap /g DM )
ml/g
TSC
(mg Tsap /g SP )
70 %MeOH
24± 0.00
A
20
8.00 ± 0.00
D
1
39.89 ± 0.18
E
100
39.79 ± 0.18
B
10
11.98 ± 0.01
D
70 % EtOH
19.66±0.57
B
30
13.33 ± 2.30
C
2
48.00 ± 0.00
C,B
200
46.63 ± 0.12
A
20
23.87 ± 0.14
C
H 2 O
12 ± 0.00
C
40
18.66 ± 0.30
B
3
55.85 ± 0.26
A
300
32.35 ± 0.20
C
30
36.00 ± 0.25
B
50
24.00 ± 0.00
A
4
49.47 ± 0.23
B
400
24.67 ± 0.58
D
40
39.89 ± 0.18
A
60
24.00 ± 0.00
A
5
42.97 ± 0.20
D
500
16.14 ± 0.47
E
50
39.48 ± 0.45
A
70
24.00 ± 0.00
A
6
35.28 ± 0.68
F
80
24.00 ± 0.00
A
8
22.34 ± 0.10
G
90
22.6 ± 2.3
A
B. Dahmoune et al.
2
obtain the maximum yield from the low amount of the primary matrix.
In this regard, this study aims at enhancing the extraction yield of saponins mixture, from the starfish Echinaster sepositus collected in
Algerian coasts, through applying microwave-assisted extraction (MAE)
technique and optimizing its conditions.
The interest in saponins of this species is justified by their unusual
structure, belonging to a rare structure group of marine glycosides
which discovered by Italian scientists more than 40 years ago [6,7]. This
unique compound containing a trisaccharide chain cyclized between C-3
and C-6 of the Δ7-3β,6β-dihydroxysteroid aglycone (Fig. 5). On the other
hand, starfish saponins are of high pharmaceutical interest due to their
anti-bacterial, anti-fungal, anti-viral and anti-tumor properties [8–12].
To optimize the parameters affecting the MAE of saponins from
starfish, the Box–Behnken experimental design (BBD) using response
surface methodology (RSM) was applied. BBD is one of the most efficient
experiment designs, because it does not allow the combinations for
which all factors are simultaneously at their highest or lowest levels.
Thus, it is used to avoid experiments performed under extreme conditions. In this study, the following factors have been evaluated: the solvent concentration (X1), irradiation time (X2), microwave power (X3)
and liquid-solid ratio (X4). Based on the response surfaces, optimal
conditions of the microwave pretreatment have been determined.
Finally, the study has been complemented with a variance analysis
(ANOVA), to determine the statistically significant factors.
To the best of our knowledge, there is no available literature detail
with the optimization of green procedure for the extraction of total saponins compound (TSC) from starfish.
Therefore, the main objectives of the current study can be listed as
follows:
• Optimization of MAE procedure by response surface methodology
(RSM) for the extraction of total saponins compounds from
E. sepositus;
• Studying comparatively the optimized MAE, UAE and the reference
CSE methods;
• Using high-resolution mass spectrometry (HR-ESI-MS) to identify the
saponins mixture obtained by MAE method.
• Evaluation of the hemolytic power of the three saponins extracts
(MAE, UAE and CSE).
• Evaluation of the antioxidant capacity power of the three saponins
extracts (MAE, UAE and CSE) by their ability to scavenge
electrochemically-generated superoxide radical (O 2
• −
).
2. Materials and methods
2.1. Biological material
E. sepositus (Retzius, 1783) individuals were collected in summer
2018 from Algerian sea coast between 5–10 m of depth. In total, 28
individuals as samples weighed about 1724 g were collected and were
transported in plastic bags containing sea water. In the laboratory, the
starfish individuals were freeze-dried (Alpha-1-2 LD plus freeze-dryer).
The lyophilized samples obtained were ground with an electrical
grinder and they were stored in dark waterproof tubes inside a desiccator until use.
2.2. Experimental work design and statistical modelling
The type of solvent, solvent concentration, irradiation time, microwave power and liquid-to-solid ratio were firstly studied separately in
single-factor experiments to reduce the number of the total experiments
work, and to determine the appropriate interval for each parameter in
the design of experiment for microwave assisted extraction - MAE
(Table 1). In this preliminary study, the experimental work has been
carried out by varying an experimental parameter while all the other
parameters were kept constant at their middle values.
In the MAE, the type and concentration of solvent effect were studied
for a constant value of liquid-solid ratio (20 mL g
− 1
), microwave power
(200 W) and irradiation time (5 min). The irradiation time effect was
evaluated for a MeOH concentration of 50 %.
The irradiation time was set at 3 min in the trials to investigate the
microwave power influence. This latter was set at 200 W in the trials to
investigate the ratio liquid-to-solid parameter. The response surface
methodology (RSM) based on Box–Behnken design (BBD) of threelevels-four-factor experimental was assessed to maximize the saponins
yield extracted from E. sepositus using MAE process (Table 2). The design
requires a number of experiment trials according to N = 2k(k − 1) + C 0 ,
where k is the number of factors and C 0 is the number of center points
and each independent factor is studied at three level (-1, 0 and +1)
reference.
The data was analyzed by Minitab.17 with a standard least square in
order to obtain RSM. The ANOVA F test was assessed to adjust models
and the coefficient significance at a p-value less than 0.05 (Table 3).
The model used for data regression analysis is corresponding to a
second-order polynomial equation (Quadratic model) (Eq. (1)). This
equation is then used to predict the optimal extraction conditions.
Y = B 0 +
∑ k
i=1
B i X i +
∑ k
i=1
B ii X
2
i +
∑ k
i>1
B ij X i X j + E
(1)
where Y represents the total saponin compounds (TSC); β 0 is a constant
coefficient; B i ,B ii and B ij are the coefficients of the linear, quadratic and
interactive terms, respectively, and X i and X j represent the actual independent variables (i–j), E is an error.
Additional experiments of extraction were carried out at the optimal
conditions, and the results obtained were compared to the values predicted by the regression model in order to validate the model.
Finally, the extract obtained at the optimal conditions was compared
to ultrasound assisted extraction (UAE) and conventional solvent
extraction method (CSE). The effects of microwave, ultrasonic and
Table 1
Single-factor experiment results for MAE of saponins from E. sepositus. Results are reported as means ± SD. Same letters in the same column refer to means not
statistically different according to ANOVA and Tukey’s test. TSC, total saponin compound; T sap , saponin total; SP, starfish powder.
Solvent
Methanol
concentration (%)
Irradiation time (min)
Microwave power (W)
Liquid-to-solid ratio (mL/g)
Type
TSC
(mg Tsap /g SP )
% (V/V)
TSC
(mg Tsap /g SP )
min
TSC (mg Tsap /g SP )
W
TSC
(mg Tsap /g DM )
ml/g
TSC
(mg Tsap /g SP )
70 %MeOH
24± 0.00
A
20
8.00 ± 0.00
D
1
39.89 ± 0.18
E
100
39.79 ± 0.18
B
10
11.98 ± 0.01
D
70 % EtOH
19.66±0.57
B
30
13.33 ± 2.30
C
2
48.00 ± 0.00
C,B
200
46.63 ± 0.12
A
20
23.87 ± 0.14
C
H 2 O
12 ± 0.00
C
40
18.66 ± 0.30
B
3
55.85 ± 0.26
A
300
32.35 ± 0.20
C
30
36.00 ± 0.25
B
50
24.00 ± 0.00
A
4
49.47 ± 0.23
B
400
24.67 ± 0.58
D
40
39.89 ± 0.18
A
60
24.00 ± 0.00
A
5
42.97 ± 0.20
D
500
16.14 ± 0.47
E
50
39.48 ± 0.45
A
70
24.00 ± 0.00
A
6
35.28 ± 0.68
F
80
24.00 ± 0.00
A
8
22.34 ± 0.10
G
90
22.6 ± 2.3
A
B. Dahmoune et al.