Optimization of Ultrasonic-Assisted Extraction of Phenolic Compounds from Moringa Oleifera Leaves using Response Surface Methodology

 

Abdelhakim Benarima^1*, Salah Eddine Laouini¹, Bachir Ben Seghir², Yassine Belaiche¹, Mohammed ridha Ouahrani¹

¹University Hamma Lakhdar El Oued, Faculty of Technology/Department of Process Engineering, BP 789,
El-Oued 39000, Algeria.

²University 8 May 1945 Guelma, Faculty of Sciences and Technology/Department of Process Engineering,
BP 401, Guelma 24000, Algeria.

 

ABSTRACT:

Moringa Oleifera, or in other words the tree of life is considered one of the most useful plants in the world because it's rich in bioactive substances, which employing on Modern medical treatment, also can be used for many fields: food, pharmaceutical and cosmetics purposes. The present study consists of determine the optimal extraction conditions to improve the yield of the total phenolic content (TPC) from Moringa Oleifera leaves, by applied the response surface methodology (RSM) based on a Box–Behnken design (BBD) and the use of ultrasonic-assisted extraction as a new extraction technique. The results showed that the optimum extraction conditions were found at extraction time, 20 (min); extraction temperature, 42 (°C); solvent/solid ratio, 5 (ml/g), with the maximum yield of total phenolic content achieved in a quantity of 278.64 (mg GAE/g), which agreed with the predicted value 280.41 (mg GAE/g) of statistical method.

 

 

 

INTRODUCTION:

Recently, interest and research in biologically active substances from plant sources have increased.¹ As they are often healthy, effective and inexpensive also because they are considered as an alternative to the use of chemical drugs,² which may have adverse side effects on human, animal and environmental health. Among the most important of these plants, containing these bioactive substances is Moringa Oleifera.³

 

Moringa Oleifera is a plant from the Moringaceae family that grows in tropical and subtropical regions such as India, Kenya, Pakistan, Indonesia, Philippines and Uganda.^4,5 It is one of the most important medicinal and pharmaceutical plants which contains various useful nutrients such as potassium, iron, phosphorus, calcium, vitamins A, D and many essential amino acids.⁶ It is also used as a dietary supplement, treatment for malnutrition and traditional medicine in poor areas^6,7. Scientific research have shown that the extract of Moringa Oleifera plant has several biological activities such as anti-oxidant,^8,9 anti-Bacterial,¹⁰ anti-Inflammatory,^11,8 anti-atherosclerotic,¹² anti-microbial,^13,14 anti-obesity,¹⁵ anti-anthelmintic,^16,17 anti-HIV¹⁸ and anti-diabetic.¹⁹ The studies have shown that the existence of these vital roles may be due to the presence of biologically active elements, which mainly contain phenolic compounds.⁴

 

In recent years, numerous researchers have paid attention to bioactive substances, the most important of which are phenolic compounds that play a main role in protecting human health.²⁰ However, the amount and effectiveness of phenolic compounds vary even if they come from the same plant and from the same extraction technique, depending on applicable conditions such as extraction time, extraction temperature and solvent/solid ratio.⁶ Therefore, there are several methods based on economical, simple and environmentally friendly to extracting phenolic compounds using effective and safe techniques with high extraction yield.²⁰

 

There are many procedures used to extract phenolic compounds, like maceration extraction,²¹ microwave-assisted extraction,²² hot compressed water extraction,²³ solid-liquid extraction,²⁴ pressurized liquid extraction,²⁵ and soxhlet extraction.²⁶ One of the most important of these procedures is the ultrasonic-assisted extraction, which has been used in many studies,^27,28 it has been shown that the application of ultrasound-assisted extraction reduces problems of conventional extraction methods such as long extraction time and high consumption of solvents and energy.²⁰

 

To avoid these problems and to determine the optimal conditions, a response surface methodology (RSM) is applied, it’s a statistical method, which can evaluate and determine the effects of the experimental variables and their interactions with each other for predicting the optimal value during experiments.²⁹

 

This study aimed to determine the optimal conditions to increase the yield of total phenolic content (TPC) from Moringa Oleifera leaves by optimizing the experimental variables namely: solvent/solid ratio (ml/g); extraction time (min); extraction temperature (°C). We applied the statistical method (RSM) based on a Box–Behnken design (BBD) and using ultrasonic-assisted as effective extraction technology.

 

MATERIALS AND METHODS:

Materials:

All chemicals, solvents and reagents used in this study are of the analytical category. Ethanol, gallic acid, and Folin-Ciocalteu reagent are purchased from Scharlab S.L (Barcelona. Spain). Sodium carbonate is purchased from Biochem Chemopharma (France). The leaves of Moringa Oleifera are collected from the Technical Institute for the Development of Saharan Agronomy (TIDSA) Ouargla city in Algeria April 2019.

 

Plant material extraction:

The leaves are washed with distilled water several times and were dried at ambient temperature about (25-30°C) for approximately 72 hours. Finally, crushed in a fine mill to obtain a powder of 0.5mm of Moringa leaves. The Total phenolic content is extracted from Moringa Oleifera leaves using the "Ultrasonic-H" device (JP Selecta. Spain). Around of 10g was mixed with a 70% aqueous ethanol solution, in a solvent/solid ratio of 5, 6, 7ml/g, for 20, 40 and 60 minutes at three temperatures of 15, 30 and 45°C. After extraction process, the liquid extracted is filtered, evaporated to dry at 44°C, and stored at 6°C before to start the experiments.

 

Determination of total phenolic content:

The Folin-Ciocalteu test is used to determine the total phenolic content (TPC) of Moringa Oleifera leaves extracts. The reaction mixture contains 1ml of diluted Folin-Ciocalteu reagent (1:10), then 0.2ml of extracts is added, and after five minutes, 0.8ml of sodium bicarbonate solution (7.5%. w/v) is mixed with the above reaction. The absorbance is measured at 765nm using a UV visible spectrometer (Shimadzu UV-1800. Japan). Gallic acid is utilized as a reference standard, and the total content of phenol is expressed in equivalent milligrams of Gallic acid per gram of dry weight of extract (mg GAE/g DW).²⁶ All tests are repeated three times.

 

Experimental design and statistical analysis:

The statistical design of experiments shows an essential role through varying the same time all factors influence on a set of tests to explore the relation between these factors and the output response of the studied process. In this study, the Box-Behnken design (BBD) is used with three levels,³⁰ three variables and three central points, needing (15) experiments for the optimization of extraction conditions as shown in (Table 1), extraction time (X₁), extraction temperature (X₂) and solvent/solid ratio (X₃) are chosen as the independent variables, the total phenolic content is selected as the response for the combination of the independent variables given in (Table 2). All statistical analysis is performed using Design Expert (software. Trial Version 11. Stat-Ease Inc. Minneapolis. MN). In the last, the quadratic regression models for predicting the response variable are obtained from BBD (Equation) (1):

 

Where Y is the predicted response (total phenolic content),  is a constant. and  are the linear, quadratic and interactive coefficients of the model, respectively. Accordingly,  and  represent the levels of the independent variables, respectively.

 

Table.1: Experiment design levels for various parameters.

RESULTS AND DISCUSSION:

Table.2 shows experimental and predicted values of total phenolic content (mg GAE/g) with changing combination of the independent factors. We compared the predicted values with the experimental ones each time. The results show that these data are in practically close covenant.

 

Table.2: The Box–Behnken design matrix and the results for total phenolic content.

 

Model fitting:

The data obtained from Box-Behnken Design (BBD) is generated in a second-order polynomial response function model, which predicts the yield of TPC from Moringa Oleifera leaves, expressed by the coded variables (X₁, X_2, and X₃) in equation 2:

 

The ANOVA of the quadratic regression model shows the value of the adjusted determination coefficient (= 0.9012), while the value of the determination coefficient (R² = 0.9647), indicating a high level of correlation between the experimental and predicted values. All these results imply that the model, as evident from the response F value (15.9) and the probability value (P < 0.01), is adequate for predicting within the range of the variables applied. On the other hand, the coefficient values were also calculated and experimented for their importance, as shown in (Table 3). The calculated coefficient for the parameter optimization suggests that all the independent variables tested, with the exception X₁ along with the quadratic term (), are significant (P < 0.05) according to (Table 3). Results also show that the extraction temperature is the most significant impact factor on the yields of total phenolic content due to it having the greatest P-value (P < 0.01), followed by the solvent/solid ratio, while the extraction time is more than 0.01.

Table.3: Analysis of variance for the response surface quadratic model.

SS: sum of square, DF: degree of freedom, MS: mean square

 

Response surface analysis of TPC:

The characteristic of 3D response surfaces is a schematic interpretation of the relation between independent and dependent factors.³¹ It helps provide simple, easy understands and analysis of complex phenomena. As in figures (fig 1-2-3), that shows the effect of the three factors, extraction time (X₁); extraction temperature (X₂); solvent/solid ratio (X₃), on the extraction of total phenolic content.

 

Figure.1: Response surface plot of phenolic compounds between extraction temperature and extraction time on fixed solvent/solid ratio at 6 ml/g.

 

Figure.1 showed the relation between extraction temperature and extraction time at a fixed solvent/solid ratio at 6ml/g. Where it the extraction of phenolic compounds increased with increasing temperature from 15 to 45°C to reach its maximum value of 264.27mg, and all this at a time of 20 minutes, However, the extraction of phenolic compounds slightly decreased after 20 min, implying that the further increase in extraction time was not advantageous for the extraction because of the degradation of the produced phenolic compounds. A similar result was also obtained while extracting phenolic compounds from perilla leaves.³² Accordingly, it is good to find recovery time short, as this saves us time and cost, which makes the recovery process more economical.

 

Figure.2: Response surface plot of phenolic compounds between extraction time and solvent/solid ratio on fixed extraction temperature at 30°C.

 

Figure.2 shows the relation between the extraction time and the solvent/solid ratio at a constant recovery temperature of 30°C. Where the maximum production of phenolic compounds reached 271.23mg, that was at the solvent/solid ratio 5ml/g, but after these values, it began production is declining with a decrease in the solvent/solid ratio. Similar results reported while extracting phenolic compounds from pomegranate peel³³ and purple rice.³⁴ It may be the solvent/solid ratio 5ml/g is the peak, since then any higher amount of solvent will not change the driving force of the phenolic compounds in the solvent afterward, because the mass transfer is limited to the inner part of the plant material and thus reduces the recovery of phenolic compounds.

 

Figure.3: Response surface plot of phenolic compounds between extraction temperature and solvent/solid ratio on fixed extraction time at 40 min.

Figure.3 shows the relation between the extraction temperature and the solvent/solid ratio at a constant recovery time of 40 minutes. Were 276mg is the maximum value of phenolic compounds yield under these conditions: a solvent/solid ratio of 5ml/g and a temperature of 45°C. However, this value decreased by reducing the solvent/solid ratio and lowering the temperature, similar results reported while extracting phenolic compounds from Himanthalia elongata.³⁵ It can be said that this behavior makes sense the higher temperature could increase diffusion rate and phenolic compounds solubility as well as reduce solvent viscosity and surface tension. Leading to better extraction of phenolic compounds. Based on the results of linear and quadratic coefficients (Table 3), we concluded that the order of factors affecting the extraction of phenolic compounds was extraction temperature then solvent/solid ratio then extraction time.

 

Optimum conditions:

The optimal conditions obtained using the model were as follows: extraction time, 20 min; extraction temperature, 42°C; and solvent/solid ratio, 5ml/g. Under these conditions, the model predicted a maximum response of 280.41mg GAE/g, to optimize and validate the predicted mathematical model, experimental rechecking was performed under the same optimal conditions, where the resulting value is 278.64mg GAE/g. Experimental and predicted maximal values were nearly equal (Table 4). this is indicative that the model is well suited to extract phenolic compounds from the Moringa Oleifera leaves under the optimal conditions and that the model is well designed to predict the optimal extraction status.

 

Table.4: Optimum conditions, predicted and experimental value of response under these conditions.

 

CONCLUSION:

In this work, the ultrasonic-assisted technique was used for the extraction of phenolic compounds from Moringa Oleifera leaves. Response surface methodology (RSM) based Box-Behnken design (BBD) was applied to determine the optimal extraction conditions for maximized the yield of the total phenolic content (TPC) by optimizing the experimental variables such as solvent/solid ratio (ml/g); extraction time (min); extraction temperature (°C). The results showed that the optimal conditions to obtain the best extraction of total phenolic content (TPC) were determined as follows: extraction time, 20 (min); extraction temperature, 42 (°C); solvent/solid ratio, 5 (ml/g), where the yield achieved in a quantity of 278.64 (mg GAE/g), which matched with the predicted value 280.41 (mg GAE/g). This study indicates that Moringa Oleifera is a Very useful tree, which contains a high rate of phenolic compounds.

 

ACKNOWLEDGMENTS:

All thanks and gratitude to the ‎process engineering department of El-Oued University-Algeria for the efforts and contribution to achieving this study.

 

CONFLICT OF INTEREST:

The authors declare that there is no inconsistency for interests regarding the publication from this paper.

 

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Received on 08.04.2020                    Modified on 11.05.2020

Accepted on 03.06.2020                   ©AJRC All right reserved