# Optimization of Microwave-Assisted Extraction Method for Increased Extraction Yield and Total Phenol Content from Moringa Leaves (Moringa oleifera Lam.) var. PKM 1

Authors: Sandeep Gunalan; Arumugam Thangaiah; Janavi Gnanaguru Janaki; Anitha Thiyagarajan; Senthil Kuppusamy; Lakshmanan Arunachalam
Journal: Advances in Agriculture
 (2022)

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## Abstract

Moringa (Moringa oleifera Lam.) is an incredible plant with a storehouse of essential nutrients such as proteins, vitamins, minerals, and phytochemicals such as phenols, flavonoids, glycosides, and sterioids. From the qualitative screening, phytochemical was highly present in hydroethanolic extracts. Through hydroethanolic extraction, the recovery of these biomolecules could be achieved throughadvanced novel extraction methods such as microwave-assisted extraction. The main aim of this experimental trial is to optimize three independent factors including power (500 W–700 W), temperature (30°C–50°C), and extraction time (20 min–40 min) consisting of two levels of dependent variables such as extraction yield (EY) and total phenol content (TPC) using Response Surface Methodology (RSM) constructed by Central Composite Design (CCD) with 20 trial runs. The optimized extraction method recorded 14.64 to 17.65% of extraction yield and 63.36 to 76.40 mg GA/gram of total phenol content. As a result of the optimization of microwave-assisted extraction, the recovery of biomolecules from the dried moringa leaves is achieved by increasing the extraction temperature and time with the microwave power of the instrument.


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## Body

## 1. Introduction

In recent days, various biomolecules of human interest were extracted from plant parts as the cheapest and most effective components for the manufacture of various chemical drugs [1]. Many secondary metabolites present in the plants have medicinal value and can be tapped by various pharma industries for the manufacture of nutraceutical products that could be consumed on daily basis [2, 3]. Regular intake of chemical drugs causes various side effects in human beings and the environment that can be overcome by extraction of similar phytocomponents from plant sources to cure serious diseases such as cancer, hypertension, and overweight [4–6]. Various secondary metabolites that have a wide range of beneficial effects on human beings were found in Moringa (M. oleifera Lam.) in dried or dehydrated leaves than in fresh leaves [7, 8].Moringa is a tree found to be grown all over the world with its native to the sub-Himalayan tract of India, Pakistan, Bangladesh, and Afghanistan, and later it was distributed to various continents of the world with wide adaptability to all types of soil and stress. Moringa is one of the power-packed nutrient-rich and medicinally important crops containing major nutrients such as iron, calcium, phosphorus, carotenoids, and important amino acids [9, 10]. All these beneficial phytocomponents from the moringa leaves could be used to cure several diseases and alleviate malnutrition through the extraction process. Direct consumption of moringa leaves shows reduced assimilation of phytonutrients and this could be overcome by consuming biomolecules through suitable extraction method [11]. The quality and quantity of the biomolecules were influenced by the extraction methodologies, temperature, time, and other various factors [10].Extraction of biomolecules from the plant source can be recovered through various extraction methods such as conventional and nonconventional methods that includes maceration, Soxhlet, pressurized solvent extraction, Supercritical fluid extraction, ultrasound-assisted extraction, and microwave-assisted extraction [12]. During the process of extraction, biomolecules are extracted from the solid plant matrix through the solvent-liquid phase by the leaching principle [13]. The recovery of these phytonutrients can be enhanced by imposing external energy on the plant samples in the form of heat, microwave, and ultrasonic waves through novel extraction methods that lack conventional extraction methods [14]. The main objective of the experiment is to evaluate the efficiency of microwave-assisted extraction method in extracting moringa biomolecules from leaves by optimizing the extraction power, temperature, and time concerning the extraction yield, and total phenol content in response to the above-mentioned independent factors using response surface methodology (RSM) based on central composite design (CCD). The RSM statistical design is to evaluate and optimize the interaction between the independent and dependent variables.


## 2. Materials and Methods

### 2.1. Solvents and Reagents

The solvents and reagents essential for the extraction process and other biochemical analyses such as 99% pure ethyl alcohol, Folin-Ciocalteu reagent, sodium carbonate, sodium hydroxide, aluminium chloride, methanol, sodium nitrate, potassium acetate, and standards such as quercetin, ascorbic acid, catechol, and gallic acid were purchased from Hi-Media, Merk, Central Drug House, and Sigma-Aldrich.


### 2.2. Plant Sample Preparation

Moringa leaves of PKM 1 variety were harvested from the organic field at the western block of Horticultural College and Research Institute, Periyakulamand fresh leaves are cleaned, washed, and dried using a solar cabinet dryer at 40 ± 2°C until the leaves crumble. Dried leaves were grounded using a commercial-grade hammer mill and sieved using a 250μm sieve. Pulverized moringa dried leaves are stored in dark and refrigerated conditions for further analysis.


### 2.3. Microwave-Assisted Extraction

Microwave-assisted extraction (MAE) was performed using ETHOS™ X equipped with dual 950 W magnetron for a total power output of 1900 W manufactured by Milestone, Italy. Extraction of biomolecules from the moringa leaf samples were carried out by adding 3 g moringa powder with 30 ml of 70% ethanol in high-pressure TFM (tetrafluoroethylene) microwave vessels provided with the equipment. After extraction with RSM-generated differential runs for optimization, the moringa extracts were centrifuged and concentrated using a rotary vacuum evaporator (Equitron-ROTEVA 66 series, Medica Instrument Mfg. Co., Mumbai) and stored at 4°C for further analysis.


### 2.4. Determination of Extraction Yield

The extraction yield of each experimental run was estimated based on the method described by Cho et al. [15]. The mass of the dried moringa extracts after rotary evaporation was calculated, and the extraction yield obtained was expressed as percentages using the following equation:(1)Yield%=WeightofthelyophilizedextractgWeightoftheplantsampletakenforextraction×100.


### 2.5. Determination of Total Phenol Content in Extracts

Total phenolic content in the moringa leaf extract from different runs of the experimental trial was estimated by spectrophotometric method using a Folin-Ciocalteau reagent with slight modifications as described by Rakesh et al. [16]. In brief, dried moringa leaf extracts were rediluted using deionized water at a concentration of about 1 mg per ml. Using a micropipette 100 μl of the prepared extract was pipetted into the 96 well microplate and further diluted with 400 μl distilled water to the mixture 1 : 1 diluted FC reagent was added. The microplate containing a mixture of experimental runs were incubated under dark condition for 1 hour to develop greenish-blue colour. The absorbance of the extracts was read using a Bio-Rad microplate reader in triplicates at 650 nm. 500 ml distilled water with FC reagent without plant extract was considered blank, and gallic acid is taken as a standard to estimate the amount of phenol content in the extracts.


### 2.6. GC-MS Profiling of Moringa Leaf Extract

The phytochemical profiling of moringa leaf extracts from microwave-assisted extraction (MAE) were performed using Trace GC ultrachromatograph system (Thermo Fischer Scientific, Austria) coupled to Thermo Scientific DSQ II quadruple MS. The freeze-dired MAE moringa leaf extract from optimized parameters was resuspended in HPLC grade methanol (99.9% Pure) in the ratio of 1 mg/ml of methanol and sonicated using a water bath sonicator. The sonicated moringa extracts were filtered using a 0.45μm membrane filter and introduction into the equipment by Tri-plus RSH headspace autosampler. The methanolic extract was made to pass through a 5% phenyl methyl silicone fused silica capillary column (TG-SQC, 15 m in length, 0.25 mm I.D., and 0.25 μm film thickness) with ionizing energy of 70 eV and pure helium gas (99.99% pure) as carrier gas (Flow rate: 1 ml/min; Split flow: 10 ml). Column temperature was programmed with 50°C as the initial temperature for 1 min and progressively raised to 150°C at 25°C/min rate. Using Xcalibur software, spectra pattern and chromatograms were compared with the National Institute of Standards and Technology’s library of NIST11.LIB database. Unknown phytochemicals present in the optimized MAE moringa extracts were identified using NIST library.


### 2.7. Experimental Design

The experimental design of the microwave-assisted extraction method was performed from the central composite design by studying the interactions between the independent and dependent variables by RSM [17]. The effect of the independent factor (Table 1) was X1 power of the instrument (500 W to 700 W), X2 extraction temperature (30°C to 50°C), and X3 extraction time (20 min to 40 min). Two response variables were taken as dependent factors to represent quantitative (extraction yield) and qualitative (total phenol content) attributes of MAE from dried moringa leaves. The RSM Experimental design was generated by DESIGN EXPERT v13.0 software to develop a quadratic model that fits the experimental data to draw response D plots and optimization of MAE extraction process.Table 1
Independent factors and its range set in experiment design.
Input variablesSymbolLevelsXi−10+1Power WX1500600700Temperature (°C)X2304050Time (min)X3203040Two levels of response variables were selected, and 20 extraction runs were carried out to predict the optimization process. Estimation of pure error at the sum of the square was carried out in triplicates at the centre of the experiment, and the interactions between the dependent and independent variables and their responses were presented in Table2. From the experimental values, a quadratic model was fitted and regression equations were calculated.(2)Y=β0∑i=0nβiXi+∑i=0nβiiXi2+∑i∑i=0nβijXiXj+r,where Y represents the responses such as extraction yield (%), total phenol content (mg GA/g), and constant (β0). Further, Xi and Xj represent the input independent variables with the number of independent parameters (n) involved in the experiment. The coefficient of the regression model, linear, quadratic, and interaction terms were denoted by βi, βii, and βij, respectively. The interactive effects of the variables were represented as three-dimensional surface plots by keeping other variables constant, and the significance of p0.05, R square, and adjusted R square values were calculated. According to Sin et al. [18], the value of R2 should be greater than 0.90 for the best fit of the regression model.Table 2
Optimization of extraction parameters for moringa leaf powder using MAE by RSM.
RunFactorsExtraction yield (%)Total phenol content (mg GA/g)X1 (W)X2 (°C)X3 (min)ExperimentalPredictedExperimentalPredicted1600403017.5117.5475.7375.872700302016.0516.0969.4569.643500504015.0415.0165.1064.944600503016.1716.2669.9770.375700502015.1115.1765.4065.666750403016.2616.2170.3570.157600403017.3817.5475.1675.878700304016.1816.2370.0170.29500502015.6715.6367.8167.6510700504016.0415.9669.3969.0411600403017.5517.5475.9075.8712500304015.3615.3166.4866.2613500302016.5116.671.4171.7914600303017.0116.8873.5573.0215600404017.1417.2574.1274.6616400403014.6414.6663.3663.4417600402017.6517.576.475.7318600403017.6117.5476.2575.8719600403017.6417.5476.3175.8720600403017.4717.5475.6375.87


### 2.8. Statistical Analysis

Data analysis of the extraction yield and total phenol content was carried out using SPSS 27.0 under a completely randomized design (CRD).


## 2.1. Solvents and Reagents

The solvents and reagents essential for the extraction process and other biochemical analyses such as 99% pure ethyl alcohol, Folin-Ciocalteu reagent, sodium carbonate, sodium hydroxide, aluminium chloride, methanol, sodium nitrate, potassium acetate, and standards such as quercetin, ascorbic acid, catechol, and gallic acid were purchased from Hi-Media, Merk, Central Drug House, and Sigma-Aldrich.


## 2.2. Plant Sample Preparation

Moringa leaves of PKM 1 variety were harvested from the organic field at the western block of Horticultural College and Research Institute, Periyakulamand fresh leaves are cleaned, washed, and dried using a solar cabinet dryer at 40 ± 2°C until the leaves crumble. Dried leaves were grounded using a commercial-grade hammer mill and sieved using a 250μm sieve. Pulverized moringa dried leaves are stored in dark and refrigerated conditions for further analysis.


## 2.3. Microwave-Assisted Extraction

Microwave-assisted extraction (MAE) was performed using ETHOS™ X equipped with dual 950 W magnetron for a total power output of 1900 W manufactured by Milestone, Italy. Extraction of biomolecules from the moringa leaf samples were carried out by adding 3 g moringa powder with 30 ml of 70% ethanol in high-pressure TFM (tetrafluoroethylene) microwave vessels provided with the equipment. After extraction with RSM-generated differential runs for optimization, the moringa extracts were centrifuged and concentrated using a rotary vacuum evaporator (Equitron-ROTEVA 66 series, Medica Instrument Mfg. Co., Mumbai) and stored at 4°C for further analysis.


## 2.4. Determination of Extraction Yield

The extraction yield of each experimental run was estimated based on the method described by Cho et al. [15]. The mass of the dried moringa extracts after rotary evaporation was calculated, and the extraction yield obtained was expressed as percentages using the following equation:(1)Yield%=WeightofthelyophilizedextractgWeightoftheplantsampletakenforextraction×100.


## 2.5. Determination of Total Phenol Content in Extracts

Total phenolic content in the moringa leaf extract from different runs of the experimental trial was estimated by spectrophotometric method using a Folin-Ciocalteau reagent with slight modifications as described by Rakesh et al. [16]. In brief, dried moringa leaf extracts were rediluted using deionized water at a concentration of about 1 mg per ml. Using a micropipette 100 μl of the prepared extract was pipetted into the 96 well microplate and further diluted with 400 μl distilled water to the mixture 1 : 1 diluted FC reagent was added. The microplate containing a mixture of experimental runs were incubated under dark condition for 1 hour to develop greenish-blue colour. The absorbance of the extracts was read using a Bio-Rad microplate reader in triplicates at 650 nm. 500 ml distilled water with FC reagent without plant extract was considered blank, and gallic acid is taken as a standard to estimate the amount of phenol content in the extracts.


## 2.6. GC-MS Profiling of Moringa Leaf Extract

The phytochemical profiling of moringa leaf extracts from microwave-assisted extraction (MAE) were performed using Trace GC ultrachromatograph system (Thermo Fischer Scientific, Austria) coupled to Thermo Scientific DSQ II quadruple MS. The freeze-dired MAE moringa leaf extract from optimized parameters was resuspended in HPLC grade methanol (99.9% Pure) in the ratio of 1 mg/ml of methanol and sonicated using a water bath sonicator. The sonicated moringa extracts were filtered using a 0.45μm membrane filter and introduction into the equipment by Tri-plus RSH headspace autosampler. The methanolic extract was made to pass through a 5% phenyl methyl silicone fused silica capillary column (TG-SQC, 15 m in length, 0.25 mm I.D., and 0.25 μm film thickness) with ionizing energy of 70 eV and pure helium gas (99.99% pure) as carrier gas (Flow rate: 1 ml/min; Split flow: 10 ml). Column temperature was programmed with 50°C as the initial temperature for 1 min and progressively raised to 150°C at 25°C/min rate. Using Xcalibur software, spectra pattern and chromatograms were compared with the National Institute of Standards and Technology’s library of NIST11.LIB database. Unknown phytochemicals present in the optimized MAE moringa extracts were identified using NIST library.


## 2.7. Experimental Design

The experimental design of the microwave-assisted extraction method was performed from the central composite design by studying the interactions between the independent and dependent variables by RSM [17]. The effect of the independent factor (Table 1) was X1 power of the instrument (500 W to 700 W), X2 extraction temperature (30°C to 50°C), and X3 extraction time (20 min to 40 min). Two response variables were taken as dependent factors to represent quantitative (extraction yield) and qualitative (total phenol content) attributes of MAE from dried moringa leaves. The RSM Experimental design was generated by DESIGN EXPERT v13.0 software to develop a quadratic model that fits the experimental data to draw response D plots and optimization of MAE extraction process.Table 1
Independent factors and its range set in experiment design.
Input variablesSymbolLevelsXi−10+1Power WX1500600700Temperature (°C)X2304050Time (min)X3203040Two levels of response variables were selected, and 20 extraction runs were carried out to predict the optimization process. Estimation of pure error at the sum of the square was carried out in triplicates at the centre of the experiment, and the interactions between the dependent and independent variables and their responses were presented in Table2. From the experimental values, a quadratic model was fitted and regression equations were calculated.(2)Y=β0∑i=0nβiXi+∑i=0nβiiXi2+∑i∑i=0nβijXiXj+r,where Y represents the responses such as extraction yield (%), total phenol content (mg GA/g), and constant (β0). Further, Xi and Xj represent the input independent variables with the number of independent parameters (n) involved in the experiment. The coefficient of the regression model, linear, quadratic, and interaction terms were denoted by βi, βii, and βij, respectively. The interactive effects of the variables were represented as three-dimensional surface plots by keeping other variables constant, and the significance of p0.05, R square, and adjusted R square values were calculated. According to Sin et al. [18], the value of R2 should be greater than 0.90 for the best fit of the regression model.Table 2
Optimization of extraction parameters for moringa leaf powder using MAE by RSM.
RunFactorsExtraction yield (%)Total phenol content (mg GA/g)X1 (W)X2 (°C)X3 (min)ExperimentalPredictedExperimentalPredicted1600403017.5117.5475.7375.872700302016.0516.0969.4569.643500504015.0415.0165.1064.944600503016.1716.2669.9770.375700502015.1115.1765.4065.666750403016.2616.2170.3570.157600403017.3817.5475.1675.878700304016.1816.2370.0170.29500502015.6715.6367.8167.6510700504016.0415.9669.3969.0411600403017.5517.5475.9075.8712500304015.3615.3166.4866.2613500302016.5116.671.4171.7914600303017.0116.8873.5573.0215600404017.1417.2574.1274.6616400403014.6414.6663.3663.4417600402017.6517.576.475.7318600403017.6117.5476.2575.8719600403017.6417.5476.3175.8720600403017.4717.5475.6375.87


## 2.8. Statistical Analysis

Data analysis of the extraction yield and total phenol content was carried out using SPSS 27.0 under a completely randomized design (CRD).


## 3. Results and Discussion

### 3.1. The Impact of Power, Temperature, and Time on Extraction Yield (EY%)

The effect of MAE parameters on the extraction yield of moringa leaf extracts was presented in Table3. The extraction yield varied from 14.64% to 17.65% in 20 experimental treatments. The mean values of the extraction yield from the moringa-dried leaves were 16.50%. The F value of the model for extraction yield was 150.64, which implies that the model is significant. The extraction yield response of the experimental extraction runs was influenced by A, B, AB, A2, and B2, and the p values were found to be significant at 0.05 in this condition. On the other hand, the R2 value of 0.9927 was found to fit with the predicted and actual values of the trial. The predicted R2 value was 0.9598 states that the adjusted R2 value (0.9861) was in logical agreement. The 3D plots of the cumulative effect of extraction temperature and power was presented in Figure 1. From the results obtained, it is found that at the elevated temperature (50°C) and power (700 W) there is a significant decrease in the extraction yield, and at the level of moderate temperature (30°C) there was a maximum yield of biomolecules (17.64%) from the moringa leaves at 600 W of operation power. These results were similar to the findings of Chen et al. 2017 in moringa, [10, 19] in pectin yield from orange peels, and [20] in Stevia.Table 3
Regression coefficients and ANOVA estimated for extraction yield of MAE moringa leaf extract.
SourceSum of squaresdfF valuep valueModel18.429150.64<0.0001SignificantA-power0.1688112.420.0055B-temperature0.9486169.82<0.0001C-time0.1513111.130.0075AB0.000810.05890.8132AC1.01174.20<0.0001BC0.2178116.030.0025A27.661563.92<0.0001B22.971218.57<0.0001C20.081415.990.0344Residual0.135910Lack of fit0.090552.000.2330Not significantPure error0.04535Cor total18.5619R20.9927AdjustedR20.9861PredictedR20.9598Figure 1
3D and 2D plots of extraction yield against power and temperature.As the extraction temperature and power increased above 50°C and 700 W, the yield was decreased to the effect of high energy in the form of microwaves produced from the magnetrons in the instrument. At a lower level of temperature (30–40°C) with 500 W–600 W microwave power, extraction yield was recorded higher and it might be due to the increase in the diffusion rate of solvent into the plant matrix which leads to the leaching of metabolites into the solvent phase. Due to these reasons, a better extraction yield was obtained from the optimum temperature and power of the experimental runs and biochemical analysis.


### 3.2. The Impact of Power, Temperature, and Time on Total Phenol Content

The microwave-assisted extraction of moringa-dried leaves through hydroethanolic extraction shows a better yield of phenolic compounds. Among the 20 runs, the total phenol content ranged from 63.36 mg GA/g to 76.40 mg GA/g with independent variables ranging in power, temperature, and time of extraction. The quantity of total phenol content in each run shows significant differences, and the mean values of TPC for the experimental trial were recorded as 71.39 mg GA/g. The modelF value of 144.12 with a p value <0.0001 showed that the model is significant and also the p values of AC, A2, and B2 were less than 0.0001 showing that intercepts were significant (Table 4). The R squared value (0.9923) shows that predicted and actual values fit well. As the predicted R squared value was 0.9586, it is in reasonable agreement with the adjusted R square value (0.9855). The influence of response factors from the independent factors of the instrumental parameters was represented in a 3D plot by combining the extraction temperature and power (Figure 2). Maximum total phenol content of about 76.31 mg GA/g was obtained at optimal power of 600 W at 40°C for 30 min of extraction time and also with an increase in temperature and power above 50°C and 700 W, respectively, showed a decrease in total phenol content. This might be mainly due to the raise in temperature, and the influence of microwave energy in destroying the phenolic compounds as they are temperature sensitive leading to the oxidation of phenolic derivatives [21, 22].Table 4
Regression coefficients and ANOVA estimated for total phenol content of MAE moringa leaf extract.
SourceSum of squaresdfF valuep valueModel343.389144.12<0.0001SignificantA-power3.13111.840.0063B-temperature17.5166.12<0.0001C-time2.88110.890.0080AB0.01210.04540.8356AC18.57170.16<0.0001BC3.99115.070.0030A2142.911539.81<0.0001B255.741210.53<0.0001C21.4715.560.0401Residual2.6510Lack of fit1.7451.910.2478Not significantPure error0.91065Cor total346.0319R20.9923AdjustedR20.9855PredictedR20.9586Figure 2
3D and 2D plots of total phenol content against power and temperature.Increased yield of phenolic compounds from the moringa leaves might be achieved by rupturing of the plant cells along with solvent diffusion into the matrix caused due to the vibrations of the water molecules present in the liquid solvent phase by the interactions of microwaves. These results concerning the maximized yield of total phenol content were similar to the findings of Chen et al. [23] and Rodríguez et al. [10] in moringa, Dahmoune et al. [24] in Myrtus communis, and Karami et al. [25] in licorice roots.


### 3.3. Experimental Validation of the Optimum Conditions with RSM

A desirable approach was employed for the maximum recovery of biomolecule yield and phenol content by generating RSM experimental models with optimization of the independent parameters such as power, temperature, and duration of extraction. The plot of the experimental responses and predicted values showed no significant deviation between the corresponding values (Figure3). Based on the point prediction of the optimized design of the experimental trial showed the highest mean extraction yield (16.50%) and total phenolic compound (71.39 mg GA/g) extraction were near the predicted mean values (17.53% and 75.87 mg GA/g) of the trial run with 95% confidence at respective parameters 600 W power, 40°C and 30 min of extraction period of microwave-assisted extraction methodology.Figure 3
Linear regression plots of the predicted and experimental values for extraction yield (a) and total phenol content (b).
(a)(b)


### 3.4. GC-MS Profiling of Unknown Phytocompounds in Moringa Extracts from Microwave-Assisted Extraction Methodology

The GC-MS analysis of moringa leaf extract from the microwave-assisted extraction method resulted in thirty-one major compounds. The mass spectra of the unknown phytomolecules were characterized using the NIST library database. Important phytocompounds detected using GC-MS were listed in Table5. Based on the previous research outcomes of [29, 31, 32, 36], it is perceived that microwave-assisted extraction of moringa leaves posses various medicinal values such as antihypercholesterolemia, antiarthritic, analgesic, antianginal, antihypertensive, antibacterial, and antifungal properties.Table 5
List of identified compounds in moringa leaf extract from microwave-assisted extraction.
Name of the compoundMolecular weight (g/mol)Molecular formulaRetention time (min)UsesReferenceThieno(2,3-c)furan-3-carbonitrile, 2-amino-4,6-dihydro-4,4,6,6-tetramethyl-124.16C6H4OS2.39Antibacterial, analgesic, antianginal, and nonopoid effectKadhim et al. [26]Arsenous acid, tris(trimethylsilyl) ester342.49C9H27AsO3Si36.79——2-Methyl-3,5-dinitrobenzyl alcohol, tert-butyldimethylsilyl ether326.42C14H22N2O5Si34.3Antimicrobial effectAbd El-Karim [27]Ethyl iso-allocholate436.31C26H44O535.83Potent inhibitor for dihydropteroate synthaseMalathi and Ramaiah [28]5H-Cyclopropa(3,4)benz(1,2-e)azulen-5-one, 9,9a-bis(acetyloxy)-, 7b-trihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-(1aR-(1aà,1bá,2á,4aá,7aà,7bà,8à,9á,9aà))-,1,1a,1b,2,4a,7a,7b,8,9,9a-decahydro-2,4a406.5C22H30O736.23Antioxidant and antibacterialMajumder et al. [29]9-Desoxo-9-x-acetoxy-3,8,12-tri-O-acetylingol536.6C28H40O1036.91Anti-inflammatory and antibacterial effectShareef et al. [30]9-Desoxy-9x-chloroingol 3,7,8,12-tetra-acetate555.1C28H39ClO939.029,12,15-Octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z)-496.9C27H52O4Si240.65Antimicrobial, anti-inflammatory, anticancer, antioxidant, and hypercholesterolemia activityHaber et al. [31] and Good et al. [32]Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate530.9C35H62O341.67Antioxidant activityNeal-Kluever [33].psi.,.psi.-Carotene, 1,1′,2,2′-tetrahydro-1,1′-dimethoxy-601.0C42H64O243.06Antioxidant activityImhoff and Pfennig [34]Propanoic acid, 2-(3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl)-430.6C27H42O447.18Antimicrobial and antitumourKadhim et al. [26](22S)-21-Acetoxy-6á,11á-dihydroxy-16à,17à-propyl methylen edioxypregna-1,4-diene-3,20-dione418.5C24H34O647.93Anti-inflammatory steroid compound to treat eczema and rheumatismAroonrerk and Kamkaen [35]


## 3.1. The Impact of Power, Temperature, and Time on Extraction Yield (EY%)

The effect of MAE parameters on the extraction yield of moringa leaf extracts was presented in Table3. The extraction yield varied from 14.64% to 17.65% in 20 experimental treatments. The mean values of the extraction yield from the moringa-dried leaves were 16.50%. The F value of the model for extraction yield was 150.64, which implies that the model is significant. The extraction yield response of the experimental extraction runs was influenced by A, B, AB, A2, and B2, and the p values were found to be significant at 0.05 in this condition. On the other hand, the R2 value of 0.9927 was found to fit with the predicted and actual values of the trial. The predicted R2 value was 0.9598 states that the adjusted R2 value (0.9861) was in logical agreement. The 3D plots of the cumulative effect of extraction temperature and power was presented in Figure 1. From the results obtained, it is found that at the elevated temperature (50°C) and power (700 W) there is a significant decrease in the extraction yield, and at the level of moderate temperature (30°C) there was a maximum yield of biomolecules (17.64%) from the moringa leaves at 600 W of operation power. These results were similar to the findings of Chen et al. 2017 in moringa, [10, 19] in pectin yield from orange peels, and [20] in Stevia.Table 3
Regression coefficients and ANOVA estimated for extraction yield of MAE moringa leaf extract.
SourceSum of squaresdfF valuep valueModel18.429150.64<0.0001SignificantA-power0.1688112.420.0055B-temperature0.9486169.82<0.0001C-time0.1513111.130.0075AB0.000810.05890.8132AC1.01174.20<0.0001BC0.2178116.030.0025A27.661563.92<0.0001B22.971218.57<0.0001C20.081415.990.0344Residual0.135910Lack of fit0.090552.000.2330Not significantPure error0.04535Cor total18.5619R20.9927AdjustedR20.9861PredictedR20.9598Figure 1
3D and 2D plots of extraction yield against power and temperature.As the extraction temperature and power increased above 50°C and 700 W, the yield was decreased to the effect of high energy in the form of microwaves produced from the magnetrons in the instrument. At a lower level of temperature (30–40°C) with 500 W–600 W microwave power, extraction yield was recorded higher and it might be due to the increase in the diffusion rate of solvent into the plant matrix which leads to the leaching of metabolites into the solvent phase. Due to these reasons, a better extraction yield was obtained from the optimum temperature and power of the experimental runs and biochemical analysis.


## 3.2. The Impact of Power, Temperature, and Time on Total Phenol Content

The microwave-assisted extraction of moringa-dried leaves through hydroethanolic extraction shows a better yield of phenolic compounds. Among the 20 runs, the total phenol content ranged from 63.36 mg GA/g to 76.40 mg GA/g with independent variables ranging in power, temperature, and time of extraction. The quantity of total phenol content in each run shows significant differences, and the mean values of TPC for the experimental trial were recorded as 71.39 mg GA/g. The modelF value of 144.12 with a p value <0.0001 showed that the model is significant and also the p values of AC, A2, and B2 were less than 0.0001 showing that intercepts were significant (Table 4). The R squared value (0.9923) shows that predicted and actual values fit well. As the predicted R squared value was 0.9586, it is in reasonable agreement with the adjusted R square value (0.9855). The influence of response factors from the independent factors of the instrumental parameters was represented in a 3D plot by combining the extraction temperature and power (Figure 2). Maximum total phenol content of about 76.31 mg GA/g was obtained at optimal power of 600 W at 40°C for 30 min of extraction time and also with an increase in temperature and power above 50°C and 700 W, respectively, showed a decrease in total phenol content. This might be mainly due to the raise in temperature, and the influence of microwave energy in destroying the phenolic compounds as they are temperature sensitive leading to the oxidation of phenolic derivatives [21, 22].Table 4
Regression coefficients and ANOVA estimated for total phenol content of MAE moringa leaf extract.
SourceSum of squaresdfF valuep valueModel343.389144.12<0.0001SignificantA-power3.13111.840.0063B-temperature17.5166.12<0.0001C-time2.88110.890.0080AB0.01210.04540.8356AC18.57170.16<0.0001BC3.99115.070.0030A2142.911539.81<0.0001B255.741210.53<0.0001C21.4715.560.0401Residual2.6510Lack of fit1.7451.910.2478Not significantPure error0.91065Cor total346.0319R20.9923AdjustedR20.9855PredictedR20.9586Figure 2
3D and 2D plots of total phenol content against power and temperature.Increased yield of phenolic compounds from the moringa leaves might be achieved by rupturing of the plant cells along with solvent diffusion into the matrix caused due to the vibrations of the water molecules present in the liquid solvent phase by the interactions of microwaves. These results concerning the maximized yield of total phenol content were similar to the findings of Chen et al. [23] and Rodríguez et al. [10] in moringa, Dahmoune et al. [24] in Myrtus communis, and Karami et al. [25] in licorice roots.


## 3.3. Experimental Validation of the Optimum Conditions with RSM

A desirable approach was employed for the maximum recovery of biomolecule yield and phenol content by generating RSM experimental models with optimization of the independent parameters such as power, temperature, and duration of extraction. The plot of the experimental responses and predicted values showed no significant deviation between the corresponding values (Figure3). Based on the point prediction of the optimized design of the experimental trial showed the highest mean extraction yield (16.50%) and total phenolic compound (71.39 mg GA/g) extraction were near the predicted mean values (17.53% and 75.87 mg GA/g) of the trial run with 95% confidence at respective parameters 600 W power, 40°C and 30 min of extraction period of microwave-assisted extraction methodology.Figure 3
Linear regression plots of the predicted and experimental values for extraction yield (a) and total phenol content (b).
(a)(b)


## 3.4. GC-MS Profiling of Unknown Phytocompounds in Moringa Extracts from Microwave-Assisted Extraction Methodology

The GC-MS analysis of moringa leaf extract from the microwave-assisted extraction method resulted in thirty-one major compounds. The mass spectra of the unknown phytomolecules were characterized using the NIST library database. Important phytocompounds detected using GC-MS were listed in Table5. Based on the previous research outcomes of [29, 31, 32, 36], it is perceived that microwave-assisted extraction of moringa leaves posses various medicinal values such as antihypercholesterolemia, antiarthritic, analgesic, antianginal, antihypertensive, antibacterial, and antifungal properties.Table 5
List of identified compounds in moringa leaf extract from microwave-assisted extraction.
Name of the compoundMolecular weight (g/mol)Molecular formulaRetention time (min)UsesReferenceThieno(2,3-c)furan-3-carbonitrile, 2-amino-4,6-dihydro-4,4,6,6-tetramethyl-124.16C6H4OS2.39Antibacterial, analgesic, antianginal, and nonopoid effectKadhim et al. [26]Arsenous acid, tris(trimethylsilyl) ester342.49C9H27AsO3Si36.79——2-Methyl-3,5-dinitrobenzyl alcohol, tert-butyldimethylsilyl ether326.42C14H22N2O5Si34.3Antimicrobial effectAbd El-Karim [27]Ethyl iso-allocholate436.31C26H44O535.83Potent inhibitor for dihydropteroate synthaseMalathi and Ramaiah [28]5H-Cyclopropa(3,4)benz(1,2-e)azulen-5-one, 9,9a-bis(acetyloxy)-, 7b-trihydroxy-3-(hydroxymethyl)-1,1,6,8-tetramethyl-(1aR-(1aà,1bá,2á,4aá,7aà,7bà,8à,9á,9aà))-,1,1a,1b,2,4a,7a,7b,8,9,9a-decahydro-2,4a406.5C22H30O736.23Antioxidant and antibacterialMajumder et al. [29]9-Desoxo-9-x-acetoxy-3,8,12-tri-O-acetylingol536.6C28H40O1036.91Anti-inflammatory and antibacterial effectShareef et al. [30]9-Desoxy-9x-chloroingol 3,7,8,12-tetra-acetate555.1C28H39ClO939.029,12,15-Octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z)-496.9C27H52O4Si240.65Antimicrobial, anti-inflammatory, anticancer, antioxidant, and hypercholesterolemia activityHaber et al. [31] and Good et al. [32]Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate530.9C35H62O341.67Antioxidant activityNeal-Kluever [33].psi.,.psi.-Carotene, 1,1′,2,2′-tetrahydro-1,1′-dimethoxy-601.0C42H64O243.06Antioxidant activityImhoff and Pfennig [34]Propanoic acid, 2-(3-acetoxy-4,4,14-trimethylandrost-8-en-17-yl)-430.6C27H42O447.18Antimicrobial and antitumourKadhim et al. [26](22S)-21-Acetoxy-6á,11á-dihydroxy-16à,17à-propyl methylen edioxypregna-1,4-diene-3,20-dione418.5C24H34O647.93Anti-inflammatory steroid compound to treat eczema and rheumatismAroonrerk and Kamkaen [35]


## 4. Conclusion

In response to surface methodology, the output in terms of quantitative variables such as extraction yield (%) and quantitative variables such as total phenol content (mg GA/g) was high at optimized parameters of microwave-assisted extraction with operation power of 600 W for 30 min of extraction with 40°C of extraction temperature. On running the extraction process with the abovementioned optimized parameters through RSM, the maximum extraction yield was recorded at about 17.64%, and total phenol content of 76.40 mg GA/g was obtained. Microwave-assisted extraction is a user-friendly technology that allows to extraction wide range of secondary metabolites and other biomolecules of medicinal and nutritional value in a very short period. When compared to other conventional methods of extraction, MAE paves the way to extract a wide range of compounds with less utilization of solvents at lower extraction temperatures. These phytocomponents could be further incorporated into human supplementary and could be further fortified in food products to supply all essential phytonutrients through their daily intake. Extracted moringa leaf extract after encapsulation with suitable stable wall materials can be often incorporated in nutritional interventions either as enrichment or also used to develop novel functional food products such as an energy bar, energy drink, and extruded products (noodles and pasta). The developed products will be highly suitable for commercialization and alleviate nutritional deficiency problems, especially in vulnerable groups in the community.



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*Source: 7370886-2022-12-27.xml*