Modelling and Optimization of Ultrasound Assisted Extraction of Polyphenols Using Response Surface Methodology

DOI: http://dx.doi.org/10.24018/ejers.2020.5.9.2115 Vol 5 | Issue 9 | September 202


I. INTRODUCTION
Polyphenolic compounds represent one of the major groups among secondary metabolite. They have a crucial role in plant growth, in addition to a very large variety of biological activities, as antioxidant activity, anti-microbial, anti-inflamatory, and others [1]. Those effects have a great interest in food, pharmaceutical and cosmetic industries. That is why the extraction of these natural biologically active compounds is an interesting and increasing area of research. In that regard, many solid-liquid extraction techniques have been used to extract polyphenols, from different plant parts [2]. They can be regrouped as conventional methods like maceration, infusion or decoction; and unconventional or advanced method like supercritical fluid extraction, Microwave-assisted extraction or Ultrasound-assisted removed without scratching the kernel. These were then cut in half lengthwise and dried to obtain a moisture content lower than 12% to avoid the growth of fungi. Dried the seeds were crushed, and lipids were extracted by the Soxhlet method with hexane as a solvent for 10 hours. The defatted flour was crush again to obtain a particle size of 0.8 mm.

B. Experimental setting 1) Experimental methodology and extraction procedure
The experimental set up is represented by an extraction unit (Fig. 1), a water bath to ensure the regulation of the extraction temperature and its circulation in the said unit, a magnetic stirrer and a magnetic bar, and a sonication system. The extraction cell consists of two elements: the inner part (a) which is where the extraction is carried out, and the outer part (b) functioning as a water bath which receives the internal part and where circulates the water at the desired temperature. The sonication system is represented by a 700W ultrasonic processor for a frequency of 20 kHz (Q700, QSonica Sonicators, Newtown, USA) with a 12.7 mm diameter probe.  (Table I). The energy input was controlled by setting the amplitude of the ultrasound probe with pulse durations of 5s on and 5s off, and the solid/liquid ratio was 1/20. The extraction solvent was a mixture of Methanol-Acetone-Water (54:23:23) [21]. The content of the slurry was centrifuged for 10 min, at 6000xg and 4 ° C, and the supernatant was collected. The same operation was repeated three times. The supernatants were collected, pooled and stored at 4° C. All treatments were carried out in triplicate.
A variable transformation, from coded to real variables values, is necessary to be able to implement the matrix of experiments. The use of coded values will facilitate the comparison of the effects of real variables, which are not always expressed in the same units.
The transformation used [22], [23] was: with: j the encoded variable, xj = its coded value; Uj = the real value of j corresponding to the coded value; U 0 j = the real value at the centre of the experimental domain, ΔUj is called "step" variation, Uj max is the real maximum value of j, and Uj min is the minimum.  2) Chemical analysis Gallic acid, Ascorbic acid, Ammonium Molybdate, Sodium phosphate and Sodium carbonate were purchased from SRL (Sisco research laboratories Pvt. Ltd., Mumbaï, Maharashtra, India), and Sulfuric acid was purchased from Merck (Mumbaï, Maharashtra, India). All chemicals and standard were of analytical grade.

a) Determination of the Total Phenolic Compounds (TPC) extraction yield
This determination was carried out according to the method described in [24], which is based on the principle of oxidoreduction using Folin-Ciocalteu reagent. Aliquot of 20 µL of the extract, made up to 500 µL with deionized water, was added to 250 µl of 10 times diluted Folin-Ciocalteu 34 solution, and 1.25 mL of sodium carbonate (20% w/v). After incubation for 40 min at room temperature away from light, the reading was taken at 725 nm. The content of total phenolic compounds was expressed as the equivalent mass of Gallic Acid per gram of defatted dry matter.

b) Evaluation of the Total Reducing Power (TRP) of the extract
The total reducing power of mango seed kernel extract was evaluated by the method developed by [25]. This method is based on the reduction of Mo (VI) Mo (V) by compounds present in the extract in acidic condition, with the formation of a green phosphate/molybdate complex. A volume of 0.05 mL of extract (3 repetitions) was mixed to 0.25 mL of methanol and 3 ml of the reagent solution (0.6 M sulfuric acid, sodium phosphate and 28 mM ammonium molybdate 4 mM). 0.3 mL of methanol was used for the control sample. The optical density was read at 695 nm and the total reducing power was expressed as an equivalent mass of Ascorbic Acid per gram of defatted dry matter.

3) Statistical analysis
The experimental design was generated and analysed by the software MINITAB 16 (Minitab, Ltd., Brandon Court, Unit E1-E2 Progress Way, Coventry, CV3 2TE, UK). The models obtained were validated using the following parameters: the determination coefficient (R 2 ), the adjusted R 2 , the Bias factor (Bf), the average absolute deviation (AAD) and the accuracy factor (Af). The R 2 is related to the variability of the response. The adjusted R 2 gives a measure of the variation around the mean explained by the model [26]. Bf and Af give the model precision and the AAD, the deviation of the model from the real values. They were calculated using the given formulas: A valid model should have R²adj = 80% [27], 0< AAD <0.3 [28] and 0.75 <Bf / Af <1.25 [29]. Graphic representations of the results were obtained using Sigmaplot version 12.5 (Systat Software Inc., 1735 Technology Drive, Suite 430, San Jose, CA 95110, USA). Table III presents the responses (mean ± SD) after the experiment and predicted by models of the extraction yield of total phenolic compounds (TPC) and total reducing power (TRP). The model equations were obtained using all three repetitions. The statistical analysis of these results allowed to derives the model equations (5) and (6), given in coded variables, representing the evolution of extraction yield (TPC) and the total reducing power (TRP) of the extract.    Table V shows the values obtained after calculating each validation parameter for both responses. Compared with the reference values for each parameter, the responses model equations are considered valid, that is to say, they follow and adequately explain the behaviour of each response within the field of study. As the main purpose of the study, an optimisation was done. The objective was to identify the best extraction conditions, within the study domain, to maximize the polyphenolic compounds extraction yield and the total reducing power. In that regard, a multi-response optimization was carried out, and the results are presented in Table VI.  Fig. 2 presents the variation of the polyphenolic compound extraction yield as a function of the number of extractions on the same material. 3 extractions have been done at the obtained optimal conditions. The extraction yield of each cycle and the cumulated values were compared. After the first cycle, the extraction yield was 73.51±3.24 mg GA/g. That amount is similar to the prediction from the multi-response optimization. The amount extracted from the residue, after the second and the third cycle, respectively, were 17.82±1.17 mg/g and 3.07±0.12 mg/g. A clear difference in extraction yield can be observed after every extraction, with the first having the best efficiency. The cumulative value of the 3 extraction yields doesn't show a significant difference between the total amount collected after the 2nd and the 3rd extraction cycle.  (Table IV). Among all quadratic effects, only the amplitude (X4 2 ) leads to increasing the extraction yield. This means that an excessive application of the other parameters will reduce the extraction yield, while the application of high amplitude value allows a great polyphenol release in the solvent. Both Time -Temperature interaction (X1*X2) and Stirring rate -Amplitude interaction (X3*X3) contribute to the extraction yield decrease, while Time -Stirring rate (X1*X3) and Temperature -Amplitude (X2*X4) contribute to its increase.

A. Modelling, validation and optimization
The results show an increase of the extraction yield with the ultrasound Amplitude (Fig. 3 b, d, f). As previously observed by others, the extraction yield was increased with the increase of ultrasonic power until the maximum [30]. Ultrasounds contribute to the cell disruption and the enhancement of the mass transfer in the boundary layer surrounding the solid particle. By providing the appropriate energy dissipation, ultrasound is an effective means to increase the mass transfer. The ultrasonic energy accelerates diffusion by enhancing the solid particle permeability to the solvent and hence facilitating the polyphenol release. Also, the lower energy barrier required to initiate diffusion is provided by ultrasonic energy, which may contribute to overcoming solute-solute and solute-matrix interactions [31]. By breaking the plant matrix cells with the cavitation effect, ultrasounds ease the penetration of the solvent inside the plant matrix cells. That penetration induces the plant cells to swell and their pores to be enlarged. As a result, the diffusion, and thus the mass transfer, of the targeted metabolites is improved [32], [33]. For biomolecules as polyphenols, extraction yield is highly time-dependent [34], [35]. A good definition of the extraction time will help to gain in time and save energy. In the present study, increasing extraction time improves the extraction yield (Fig. 3 a, d, e), by allowing polyphenols to diffuse from the mango seed kernel powder particles to the extraction solvent. The proportional increase of the extraction yield with extraction time has been observed by several authors [36]- [39]. However, long extraction time present a drawback. Since more polyphenols are extracted with a longer time, they are exposed longer to the action of other factors like temperature, light and oxygen [40]- [42] or other components they can react with. Then a reduction of the extraction yield can be observed, as we can see in the present work (Fig. 3). That reduction can also be explained by some minor decomposition of unstable polyphenols at high temperature under a long heating time [43].
The extraction temperature is a key factor in polyphenols extraction since it affects the physical and chemical characteristics of a product. As shown by the results (Fig. 3  a, b, c), the increase in extraction temperature leads to an increase in the extraction yield. Many authors explain it by the softening of the cell tissues and the weakening of the interaction between polyphenols with polysaccharides or proteins [44]. Besides, the extraction temperature also enhances the target compound solubility, the solvent diffusion rate and mass transfer, by reducing the solvent viscosity and surface tension [45]. It facilitates the access of the solvent to the deeper part of the solid particle, and therefore enhances the extraction efficiency by exposing more surface area of the sample to solvents used, and thus, enhance desorption and solubility of the polyphenols. A similar positive effect of temperature on total polyphenols recovery during extraction from various vegetal sources has already been observed [46]- [51]. On the other hand, higher temperatures, beyond 50-60°C in the present study (Fig. 3 a,  b, c), induced the destabilisation of phenolic compounds. This indicated that high temperature may have resulted in the degradation of some phenolic acids [45]. In the same vein, the increase in temperature beyond the observed limit induces denaturation of the cell membrane, hydrolysis of polyphenols, as well as reactions of polymerization and redox conditions which decrease their extraction yield [52]. The effect of polymerization reactions, between phenols by themselves, on their analytical quantification, has also been underlined by [46]. Some works present polymerization in phenols as a wide and common reaction [53], [54].
Despite his non-significant direct effect on the extraction yield, the stirring rate enhances the extraction yield. Also, its quadratic interaction as well as its interaction with the other parameters are significant and contribute to the increase of the extraction yield (Table IV) (Fig. 3 c, e, f). This observation is consistent with the mass transfer principle [55]. The extraction of polyphenols from plant matrix occurs within the solid particle till the surface, at the interface with the solvent, and by convective mass transfer across the boundary layer. Enhancing the extraction yield can be achieved by reducing the resistance to the convective mass transfer by increasing the Stirring rate. That increase will reduce the film thickness and, thus, the resistance transfer of the boundary layer at the surface of the particle. This leads to the increase of effective contact between the particles and the solvent, and the extraction yield. Same trends were observed by [56]. They point the mechanical action of the stirring which would help accelerate the extraction of the polyphenols from the particles and their dissolution. On the other hand, stirring rate, higher than 300-350 rpm in the present case (Fig.  3 c, e, f), reduce significantly the extraction yield. This is because, by facilitating the extraction thereof from the matrix, agitation allows the exposure of these molecules to the effects of other factors, as already mentioned. Fig. 4 illustrates the effect of two parameters at a time, on the total reducing power of the extract, the two others parameter are set at their optimal values, obtained from the statistical analysis of results.

C. Total reducing power
Among the significant factors and interactions (Table IV) (Table IV). These observations show the link between the antioxidant capacity of the plant extracts and their phenolic content [57].
As previously mentioned, the antioxidant activity is linked to the phenolic content of the extract. A similar observation has been made by other authors [58], [59], [18]. The interaction time-temperature (X1*X2), and time-stirring rate (X1*X3) follows the same trend as the polyphenols extraction yield, coupled with the temperature activation effect. Fig. 4e, shows the interaction temperature-stirring rate (X2*X3), reveals the importance of these factors because the lowest experimental conditions give a relatively high reducing power compared to other interactions. The stirring rate increases the effect of other factors studied, as with the extraction yield. It is for this reason that the antioxidant power profile as a function of the Stirring rate and temperature, and the Stirring rate and time are similar to the direct effects of the factors mentioned. Globally, the interaction between the ultrasonic wave amplitude and the other parameters lead to an increase in the total reducing power of the extract. This can be linked to the effect of these parameters on the release of the polyphenols in the medium. These observations are similar to those done by [39].

IV. CONCLUSION
From the modelling of the ultrasound-assisted extraction of polyphenols compounds from mango seed, except for the stirring rate, the other studied factors showed a significant effect on the extraction yield and the antioxidant activity. The developed models describe the operation with enough precision for prevision and control purposes. The multiresponses optimization provides operating conditions allowing to extract a maximum of phenolic compounds with high antioxidant activity, with a satisfactory exhaustion rate after two extraction cycles.