INTRODUCTION:
Recently, there has been a renewed interest in the
plant secondary metabolites for their potential preventive effects on chronic
diseases such as cardiovascular disease and cancer. Therefore, the isolation,
identification and quantification of phytochemicals in food and assessment of
their potential health benefits have been highlighted: However, in vitro and in
vivo studies have shown that action of certain chemicals can be obtained by
eating certain plants. Thus, extraction of the active ingredient is essential
if it is to have a prophylactic or therapeutic value in humans1.
Phenolic
compounds are common dietary phytochemicals found in fruits, vegetables and
plant seeds2. The main feature of polyphenols is that they are very
powerful antioxidants. Indeed, they are able to trap free radicals and activate
other antioxidants in3,4 the body.
Ammosperma
cinereum is a fairly gracile plant of
the Brassicaceae family, it is localized only in southern Algeria. It is
endemic to North Africa, annual and spontaneous5.
To
date, no work has been done on the chemical composition or the optimization of
extraction conditions, agronomic and zoological studies are cited in the
literature.
The
total phenols, flavonoids and antioxidant activity of aqueous methanol extract
of Ammosperma cinereum can be affected by many factors. To surmount this
problem, the optimization of extraction conditions was undertaken using
multivariate statistical techniques. Among the most relevant techniques used in
analytical optimization, figures the response surface methodology (RSM).
The
objective of this study was to optimize the extraction conditions, including
the proportion of methanol, the temperature and the extraction time, to improve
the antioxidant activity, the content of total phenols and flavonoids of the
Ammosperma cinereum plant aerial part by the response surface methodology using
the Box Behnken design.
MATERIEL
AND METHODS:
Plants:
Ammosperma cinereum was collected in its natural habitat. Harvesting is carried out in the
region of Oued Souf (Latitude: 33° 21'21 ''North, Longitude: 6° 51'47 ''East Algeria). The
botanical identification of the species was made by the botanist EDDOUD Mr.
Amar (Univ kasdi merbah Ouargla). The harvest of the plant material was
performed May 17,2017 at the stage of full bloom. The collected portions were
dried at ambient temperature and sheltered from light.
Preparation of extracts:
Series of extractions were carried out by maceration
of a quantity (0.5g) of vegetable matter obtained above in a volume (5mL) of
extraction solvent to determine the methanol percentage (X1)%
Temperature (X2)°C and the time (X3) min. extraction.
Determining the content of phenolic compounds:
The total polyphenol content of the extracts was determined
using the Folin-Ciocalteu6, 7. 150µL of each extract (1mg/mL) were
added to test tubes, to which 750 uL of Folin-Ciocalteu at (10%) are added.
After 10 minutes, 2000µL of sodium carbonate was added (Na2CO3)
to 7.5% (w / v). After 30 minutes of incubation in the dark, reading the
absorbance of each solution was determined at 765 nm using a UV-VIS
spectrophotometer (UNICAM). All measurements are repeated three times. The
total phenolic content (TPC) is expressed as mg equivalent gallic acid per mg
of extract (EAG/mg of extract).
Determination of the flavonoid content:
The extracts flavonoids content was determined
according to the method of aluminum trichloride7, 8; A 1.5 mL of
each extract (1 mg/ml), are added 1.5mL of aluminum trichloride (AlCl3)
to 20% (m/v) in distilled water. After incubation in the dark for 30 minutes at
room temperature, the assay was performed by UV/Visible spectrophotometry at
430 nm. All operations are repeated three times. The flavonoid concentration
(TF) is expressed as mg ER (equivalent of Rutin) per mg of extract.
Antioxidant activity:
The antioxidant capacity of each extract concentration of 1mg/ml, was
determined in vitro by the inhibition test (O2.-)
according to the method of H. Li et al. (2007)9 with some
modifications. The test is based on self-oxidation of pyrogallol (benzene-1,2,3-triol)
which is used as anion source (O2.-).
4.5mL
of the phosphate buffer solution (pH = 8.2) were added in a tube, 0.5mL of each
extract. The reaction was initiated by adding 10mL (45mM) of pyrogallol. The
absorbance of reaction mixture was measured at 320 nm every 30 seconds3.
The rate of inhibition of the autoxidation of pyrogallol is calculated by using
the following equation:
I%
= [ΔA0 - dA / ΔA0]
x 100
Where:
ΔA0 is the speed of the self-oxidation of
pyrogallol in the absence of antioxidant, ΔA is the speed of the
self-oxidation of pyrogallol in the presence of antioxidant.
Method
Answer area:
Box-Behnken
design:
The
method of response surfaces (MSI) consists of a collection of mathematical and
statistical approaches to achieve the optimization of a process. Among the most
widely used designs, is the Box-Behnken (BBD), which is effective and
especially suitable for testing of three variables of three levels10.
This method is used to optimize the extraction factors, namely the methanol
percentage (%), temperature (°C) and the extraction time (min.). Each of these
three coded levels -1, 0, and 1 (low, medium, and high, respectively) (Table
1).The response values are the polyphenol content (Y1),
flavonoids (Y2) and antioxidant activity (Y3). The
experimental design has fifteen experimental trials that included twelve point
factorial and three central points. The combinations of variables in these
tests are described in Table 2.
Multiple linear regression and other statistical
analyzes were performed using Minitab 17® software. Experimental data have been
adjusted by the second-order polynomial model according to the following
equation:
where
is the expected response,
is the constant of the model; And 
are independent variables; and 
are linear coefficients; 
,
are cross-product coefficients; and '
are the quadratic coefficients [11]. The goodness of
fit of the equation 'model polynomial was expressed by the ratio R2[12]
The
linear coefficients, quadratic, and interaction were determined by least
squares regression, followed by analysis of variance (ANOVA). Values
of P <0.05 were considered significant.
Table
1: The independent variables and their levels encoded for 'Box-Behnken Design'
|
|
Varying levels, coded
|
|
independent variables
|
-1
|
0
|
1
|
|
X1 (%)
|
60
|
80
|
100
|
|
X2 (° C)
|
20
|
40
|
60
|
|
X3 (min)
|
20
|
60
|
100
|
Model Verification:
The extraction conditions were numerically optimized
for maximum content of TPC, TF and with high antioxidant activity based on
regression analysis and plots of 3D surface (studs area) of the independent
variables. Responses were determined under normal conditions of extraction.
Finally, predictable values were compared with experimental ones
to assess the validity of the model.
RESULTS
AND DISCUSSION:
In this study, the relationship between the response
functions and process variables, has been identified by design box Behnken and
registered by three factors. In addition, the extraction conditions of total
polyphenols, flavonoids and antioxidant activities were optimized.
The
Table 2 illustrates the results of planned and experimentally measured
responses for the 15 tests according to the experimental design. The
performance of TPC and TF extends EAG 12.66mg/g extract (X1 = 100%;
X2 = 60°C; X3 = 60 min) to 38.29 mg EAG / g of extract (
X1 = 80%; X2 = 40 ° C; X3 = 60 min), and 15.71
mg ER / g of extract (X1 = 60%; X2 = 40°C; X3
= 20 min) to 31.64 ER mg / g extract (X1 = 100%; X2 =
20°C; X3 = 60 min) respectively.
In
terms of antioxidant activity, the ability of trapping of superoxide anion
radical I% stretched from 36.94% (X1 = 60%; X2 = 60°C; X3
= 60 min) to 72.79% (X1 = 100%; X2 = 60°C; X3
= 60 min). Based on these data, the extraction process was optimized to obtain
the desirable responses to the fullest.
Statistical
analysis and model fitting:
The
matrix of the Box-Behnken design applied with experimental and predicted
responses for TPC, TF and I% are shown in Table 2. The results of the analysis
of variance (ANOVA) showed a significant result for the model. Overall, the
experimental data were well fitted by polynomial models of the second order (R2
= 0.986, 0.962 and 0.985 for TPC, TF and I% respectively). Moreover, a close
relationship between the experimental values and the predicted
values indicate that the developed model is highly satisfactory.
Effect
of extraction conditions on the total content of polyphénols:
The
percentage of methanol (60-100%) has a significant influence on the TPC level
in the linear first-order (p <0.003) and second order (p <0.001). The
regression coefficients for this factor were negative values, indicating that
increasing the ratio of methanol resulted in a decrease of the TPC content;
when the methanol concentration was increased by 60% to 100%, a reduction of
the phenol content of from 38.29 to 12.66 mg EAG / g extract was observed (Fig.
1). Since extraction and separation of the phenolic compounds are largely
dependent on the polarity of solvents and compounds, a combination of alcohol
and water is more effective than alcohol alone to remove phenolic compounds13,
14. This tendency to the reduction of extraction capacity with increased
alcohol ratio in the solvent has been reported in black tea extracts where the
hydro-alcoholic extracts 50% have the highest content of TPC, followed
respectively by 80 and 100% aqueous alcohol15. Further, in
optimizing the extraction of phenolic compounds from plant Mangifera Pajang and
Kosterm peels, the content in the extracts TPC began to decrease after reaching
the lower maximum operating conditions of 68% acidified aqueous methanol and 38
min of extraction time16.
With regard to the extraction temperature, the TPC
increases with increase in temperature to some extent and then decreases (Fig.
1 and Fig. 3). An increase in extraction temperature can break the bonds of the
phenolic matrix and influence the membrane structure of plant cells, making
them less selective due to the coagulation of lipoproteins17. The
dielectric constant of water decreases and the solvent property and ability to
change to a higher temperature, resulting in a better extraction of phenolic
compounds18. A higher temperature may also increase the phenolic
content by increasing phenolic solubility, diffusion rate, the rate of
extraction, the decrease in viscosity and surface tension of the solvent19.
However, an increase in extraction temperature could degrade phenolic
compounds, because of the interference of the stability of the compounds,
caused by chemical and enzymatic degradation or reaction with other herbal
ingredients reducing the effectiveness extraction20. Pompeu D. R et al (2009)21 reported a maximum temperature of 58°C to obtain the
highest phenolic content of Euterpe oleraeceae fruit. While Gan, C. et al (2011)22 have obtained a higher content of phenolics in the pod
stinking beans at a temperature of 35°C. In addition, the extraction time used
was 100 minutes. Increasing the extraction temperature will be useful to
increase the extraction efficiency and to reduce the extraction time23.
Figure 3 illustrates the influence of time and
temperature on the extraction of TPC. The linear effect of duration and
quadratic effect of temperature have been clearly expressed. Note that the two
factors (time and temperature) act independently on the TPC (Table 4).
The mass transfer of the plant material and solvent
are related to the duration and temperature. For a long high temperature
extraction period, the negative quadratic effect became significant. A higher
extraction temperature above 36.9°C showed no significant improvement in the
extraction of TPC. This can be attributed to thermal degradation at high
temperature antioxidants, favoured by a long extraction24, indicating
that the extracts contain heat sensitive compounds.
Effect
of extraction conditions on flavonoid content and antioxidant activity:
According
to the regression coefficients (Table 4) and three figures 1, 2 and 3, it is
found that the duration and the methanol
proportion positively affect the extraction of TF and antioxidant activity.
The
TF and I% increased significantly with increasing extraction time of 20 minutes
to 60 minutes. After 60 min, there was a slight decrease in TF and I%. This
could be due to the decomposition of active compounds during the extraction
process when extended25, 26, 27.
Whereas when the proportion of methanol increases from
60% to 100%, the TF and I% increase (Fig. 2). The significant (p <0.05) TF
and I% values increase with an increased content of methanol was
expected and consistent with the findings of Yu J et al (2005)28 according
to which a majority of phenolic compounds were extracted at higher rate in
methanol. Thus, our results suggest that a higher concentration of methanol
increases significantly the extraction of these phenolic compounds (flavonoids)
that could effectively eliminate free radicals.
At high temperatures, extraction improves both the
solubility of the solute and the diffusion coefficient (decreased solvent viscosity and increase the mobility of the
molecules), but its elevation cannot
indefinitely increase the yield of phenol extraction. In extreme temperatures, the yield may be reduced by
chemical and thermal degradation of certain polyphenols, which explains the
decrease in antioxidant activity29, favoured
by degradation of certain heat-sensitive
compounds24, 30.
CONCLUSION:
The
Box-Behnken design has been successfully used to optimize the extraction of
polyphenols, flavonoids and antioxidant activity of the aerial part of
Ammosperma cinereum by maceration. The optimum conditions to maximize yields of
each response have been proposed to be 36.9°C for 66.8 min with 93.1% methanol.
In addition, reconciliation between the predicted responses and experimental
results shows a satisfactory predictive ability of developed mathematical
models. These proposed conditions present significant advantages in terms of
short treatment time and low temperature.
CONFLICTS OF INTEREST:
The manuscript represents valid work, neither this
manuscript nor one with substantially similar content under my authorship has
been published or being considered to publication elsewhere. I confirm that
work is an accurate representation of the trial results.
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