Optimization of Phenolic Compound Extraction from Chinese Moringa oleifera Leaves and Antioxidant Activities

Rich in phenolic compounds,Moringa oleifera leaf extract (ME) exhibits signicant antioxidant activity both in vitro and in vivo. ME has already been widely used in elds of medicine, functional food, and cosmetics. Ultrasonic extraction (UE) method has been improved to be one of the most eective ways to extract phenols from M. oleifera leaves. e purpose of this study was to optimize ultrasonic extraction of phenols by response surface methodology (RSM). Four parameters were discussed, such as ethanol concentration, solvent-sample ratio, extraction temperature, and extraction time. Also, purication methods of the crude ME by organic solvent extraction and column chromatography were examined. Antioxidant activities of ME and each fraction were evaluated by DPPH, ABTS, and hydroxy radical-scavenging activities and reducing power.e phenol content of the puried ME reached up to 962.6mg RE/g, extremely higher than the crude extract 107.22± 1.93mg RE/g. e antioxidant activity of the puried ME was also signicantly improved. Furthermore, phenols were identied by using the HPLC-MS method, and the results showed that there were 6 phenolic acids and derivatives and 7 avonoids in ME. Quercetin-3-O-β-D-glucoside isolated from ME showed excellent DPPH and ABTS radical-scavenging abilities, which were comparable to VC.


Introduction
Moringa oleifera Lam. has been widely used as a nutritional supplement to reduce malnutrition and some ailments [1].M. oleifera was authorized as a new food resource by Ministry of Health of China in 2012 [2].Rich in phenolic acids and avonoids, M. oleifera extract exhibits signi cant antioxidant activity both in vitro and in vivo [3,4].Especially M. oleifera leaves have highest phenols and highest antioxidant activity, compared with roots, barks, owers, and seed [5].At present, the M. oleifera extract has been widely used in elds of medicine, functional food, and cosmetics [6][7][8].
ese are several heat-sensitive hydroxyl-type substituents existing in ME, such as kaempferol diglycoside and its acetyl derivatives [12,13].In the subcritical water/ethanol extraction method, these bioactive compounds may be destroyed by high temperature in subcritical conditions [14].In addition, microwave-assisted extraction always employs a temperature higher than 150 °C.e UE method has been proved to be the most e ective way to extract phenolic compounds from M. oleifera [3].It has been reported that there were signi cant di erences in the phenolic pro le, nutritional value, and antioxidant activity of M. oleifera from many di erent cultivars [15,16].
In this study, extraction conditions of phenolic compounds from Chinese M. oleifera leaves were optimized by using response surface methodology (RSM).Afterwards, the crude ME was further puri ed by organic solvent extraction and column chromatography.Antioxidant activity and total phenol content of ME and each fraction were evaluated.e antioxidant activity was evaluated in vitro by DPPH, ABTS, and hydroxyl radical-scavenging activity and reducing power.In addition, phenolic compounds were identified by using the HPLC-MS method.

Materials and Methods
2.1.Raw Materials and Chemicals.Samples of M. oleifera leaves were collected from the Moringa farm (100 km southeast of Dehong) (June 2016) in Yunnan Province (Southwest of China).M. oleifera leaves were dried in the open air and then ground to fine powder with a grinder.

Extraction Method Optimization
2.2.1.Extraction Method.M. oleifera leaves powder (50 g) was extracted with 70% aqueous ethanol (1.5 L) UE at 50 °C for 42 min, using an ultrasonic circulating extraction equipment (KQ-5200B, Gongyi Yuhua Co., Ltd.) at 300W.And then, the mixture was centrifuged at 3500 r/min for 15 min, concentrated by rotary evaporation at 50 °C, and further dried by the vacuum freeze-drying method.e resulting extract was stored at − 20 °C to avoid degradation until use.

Experiment Design of Response Surface Methodology.
e effect of extraction conditions on phenol yield was studied and optimized by RSM.A flour-factor three-level Box-Behnken design (BBD) was employed to discuss four independent variables: extraction time (X 1 ), extraction temperature (X 2 ), solvent/solid ratio (X 3 ), and ethanol concentration (X 4 ).Each factor was fixed at 3 levels (− 1, 0, and 1), with X 1 (30, 45, and 60 min), X 2 (40, 50, and 60 °C), X 3 (20 : 1, 30 : 1, and 40 : 1 mL/g), and X 4 (60, 70, and 80%).All experiments were conducted in triplicate, and the mean values were fitted to a second-order polynomial model equation as follows: where Y is the response (phenols content); X i and X j are independent variables; and β 0 , β i , β ii , and β ij are intercept, linear, quadratic, and cross-product terms regression coefficients, respectively.Analysis of variance (ANOVA) with 95% confidence interval was used for the analysis of the model and the optimization of extraction conditions of phenols.e regression coefficient (R 2 ) was used to test the adequacy of the model.Finally, experimental results and predicted values were compared to estimate the validity of the model.

Isolation
Method. 10 g of the crude ME was dissolved into 100 mL water.e obtained solution was fractionated with petroleum ether, ethyl acetate, and n-butanol, respectively, so that 4 different fractions were obtained as listed in Table 1.e n-butanol fraction was further purified by polyamide column (100-200 mesh) chromatography and eluted with a gradient of ethanol-water (0%, 30%, 50%, and 70% ethanol) to produce 4 fractions (BA, BB, BC, and BD) monitored by TLC.Fraction BB was separated over a Sephadex LH-20 column (MeOH/H 2 O) to produce 2 major fractions (BB1 and BB2).Fraction BB2 was purified over semipreparative HPLC (MeOH/H 2 O) to afford compound H.Each fraction was collected and measured by phenol content and antioxidant activity.

Phenol Content Determination.
e total flavonoid (TF) content was measured by a minor modification of the aluminum chloride colorimetric method [17].In brief, ME solution (500 μL and 1000 μg/mL) was mixed with aluminum chloride solution (500 μL, 2%, w/v).After incubating at 30 °C for 30 min, the absorbance was measured at 410 nm.Rutin was used as reference, and TF was expressed as mg of rutin equivalent gram of sample (mg RE/g).e total phenol (TP) content was determined by using the Folin-Ciocalteu method [18].In brief, ME solution (200 μL, 0.1 mg/mL in ethanol) was mixed with the Folin-Ciocalteu reagent (500 μL) and diluted 10 times.e mixture was left for 5 min at room temperature before being mixed with Na 2 CO 3 solution (800 μL, 60 mg/mL).After placing at room temperature and darkness for 2 h, the absorbance of the mixture was measured at 725 nm.Using gallic acid as reference, the concentration was recorded as mg of gallic acid equivalents gram of sample (mg GAE/g).
HPLC-MS analysis was performed on the HPLC system coupled to a G6310 mass spectrometer (Bruker Daltonik GmbH, Germany) equipped with electrospray ionization (ESI) ion source.e mass spectrometer was operated in the negative ion mode with a capillary voltage of 2.5 kV and a mass range of 100-1000 m/z.Nitrogen was used as the nebulizer and drying gas.e pressure of the nebulizer gas was 30.0 psi.e drying gas flow rate was 600.0 L/h, and the drying gas temperature was 350 °C.e DPPH radical-scavenging activity was measured as described in [19].In brief, DPPH solution (3.0 mL, 20 μM, ethanol as solvent) was mixed with the aqueous ME sample (1.0 mL).e mixture was kept in darkness for 30 min, and then the absorbance was measured at 517 nm.Vitamin C solution was prepared and used as an equivalent calibration standard.e radicalscavenging activity of each solution was calculated as the inhibition percentage with the following formula: where A 0 is the absorbance of the control and A 1 is the absorbance of the sample.
e mixture was kept in darkness for 2 h, and then the absorbance was measured at 732 nm. e radical-scavenging activity of each solution was calculated as the inhibition percentage with the following formula: where A 0 is the absorbance of the control and A 1 is the absorbance of the sample.

Hydroxyl Radical-Scavenging Activity.
e hydroxyl radical-scavenging activity was determined according to [21].e ME sample (1 mL) was mixed with FeSO 4 solution (1 mL, 9 mM), salicylic acid ethanol (70%) solution (1 mL, 9 mM), and H 2 O 2 solution (1 mL, 8.8 mM).e mixture was kept in darkness for 0.5 h, and then the absorbance was measured at 510 nm. e radical-scavenging activity of each solution was calculated as the inhibition percentage with the following formula: hydroxyl-scavenging activity (%) � where A 0 is the absorbance of the control and A 1 is the absorbance of the sample.
e mixture was kept at 50 °C for 20 min in water bath, after which trichloroacetic acid (2.5 mL, 10%) was added.And then, the mixture was centrifuged at 3000 r/min for 10 min.e upper layer of the solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl 3 (0.5 mL, 0.1%).e absorbance of the mixture was measured at 700 nm.Increased absorbance of the reaction mixture indicates increased reducing powder.

Statistical Analysis.
All experiments were repeated for triplication, and the results were expressed as mean-± standard deviation.ANOVA procedure and Duncan's multiple range method were used to evaluate the significant differences between treatments (p < 0.05).

Fitting the Model.
e experimental design and results of RSM are listed in Table 2. Based on the ANOVA results of total flavonoid (TF) content and total phenol (TP) content (Tables 3 and 4), two models were both remarkably significant (p < 0.0001) for TF and TP. e lack of fit of each model was not significant (0.8488 for TF and 0.2844 for TP), R 2 was close to 1 (0.9910 for TF, 0.9113 for TP), and adjusted R 2 was close to 1 (0.9821 for TF and 0.8226 for TP), indicating that both two models had good linear fitting.3 shows that the linear effect of ethanol concentration (X 1 ) and quadratic (X 2  1 , X 2 2 , X 2 3 , and X 2 4 ) had remarkably significant negative influence on TF (p < 0.001).Solvent-to-sample ratio (X 2 ) had significant positive influence on TF (p < 0.05), while extraction temperature (X 3 ) had significant negative influence (p < 0.05).According to the regression coefficient values (β), X 2  1 had a major influence, followed by

TF. Table
, and X 3 .e second-order polynomial equation of TF yield was expressed as follows:

4
Journal of Food Quality e interaction of ethanol concentration and solvent to sample ratio (X 1 X 2 ) had highly significant positive influence on TF (p < 0.01).Figure 1(a) shows the effect of ethanol concentration, solvent-to-sample ratio, and their interaction on TF yield at 50 °C and 45 min.e shape of contour plots was elliptical, which indicated that the interaction was significant [23].e maximum TF yield was achieved at a solvent-to-sample ratio of 30 : 1-35 : 1 and ethanol concentration of 65-70%.e TF yield gradually increased with the increase in solvent-to-sample ratio from 20 : 1 to 30 : 1.In some extent, increase in the solvent-to-sample ratio could enhance the TF yield.A relatively higher concentration gradient of solute between the inside and outside of the cell could help the solute to dissolve into the solvent.Appropriate ethanol concentration was also important for phenol extraction.Most phenols in ME are of medium and high polarity.Too high concentration will lead to decrease in the dissolution of phenols because of the dissolution of lipid soluble substances.Also, too low concentration will increase the dissolution of water-soluble impurities such as sugars and proteins, reducing the extraction rate of phenols.erefore, 70% ethanol was more suitable for phenol extraction, compared with other solutions.is result was in accordance with previous studies [11].
e interaction of ethanol concentration and extraction temperature (X 1 X 3 ) had a significant negative effect on TF yield (p < 0.05).As it could be seen in Figure 1(b), the ethanol concentration had more important influence on TF than extraction temperature.e maximum TF yield was achieved at an ethanol concentration of 70% and extraction temperature of 50 °C.
e interaction of ethanol concentration and extraction time (X 1 X 4 ) revealed a significant negative effect on TF (p < 0.05).As shown in Figure 1(c), the ethanol concentration had more important influence on TF than extraction time.Also, phenols almost kept constant with the increasing extraction time, which indicated that extraction time was an insignificant variable in RSM optimization of phenol extraction (p > 0.05).4, the linear effect of the solvent to sample ratio (X 2 ) exhibited remarkably a significant positive effect (p < 0.001).Ethanol concentration (X 1 ) and the quadratic (X 2  1 , X 2 2 , X 2 3 , X 2 4 ) exhibited a highly significant (p < 0.01) negative effect.TP depended mostly on and X 2 1 .e fitted second-order polynomial equation of TP yield was as follows:

TP. As shown in Table
e interactive effects of ethanol concentration and solvent-to-sample ratio (X 1 X 2 ) had highly significant positive effect (p < 0.01).As shown in Figure 1(d), the maximum   6 Journal of Food Quality TP yield was achieved at an ethanol concentration of 70% and solvent-to-sample ratio of 30 : 1. e interactive effects of ethanol concentration and extraction temperature (X 1 X 3 ) had remarkably significant positive influence (p < 0.001).As shown in Figure 1(e), at a lower level of ethanol concentration, TP yield gradually decreased with increasing extraction temperature.Also, at a lower level of extraction temperature, TP yield decreased as the ethanol concentration raised.
Table 3 shows that interactive effects (X 2 X 3 ) had a significant positive influence (p < 0.05).Figure 1(f ) shows that TP yield at low level of extraction temperature and high level of solvent-to-sample ratio was higher than that at a low level of solvent-to-sample ratio and a high level of extraction temperature.
ese indicated that solvent-to-sample ratio had more significant influence than extraction temperature.

Model Validation.
e optimum conditions for phenol extraction were predicted using Design Expert 10.0.4 : 70% of ethanol concentration, 30 : 1 of solvent-sample ratio, 50 °C of extraction temperature, and 42 min of extraction time.Afterwards, the model validation was evaluated.TF and TP yields were approximated at 4.83 ± 0.07% and 2.44 ± 0.10%, respectively, which did not show significant differences (p > 0.05) with the experimental value of TF yield (4.83%) and TP yield (2.44%).

Isolation of M. oleifera Leaf Extract.
e TF value of ME extracted by ethanol : water (70 : 30) was measured as 107.22 ± 1.93 mg RE/g.So total flavonoid quality of the crude ME (10 g) was calculated as 1.77 g.ME (10 g) was dissolved in water and then fractionated with petroleum ether, ethyl acetate, and n-butanol, respectively.TF of the obtained petroleum ether, ethyl acetate, n-butanol, and water fractions was measured, and total flavonoid quality and yield were also calculated, as shown in Table 1.Total flavonoid quality of n-butanol fraction was 1429.34 mg, much higher than that of other fractions.Most of the phenols in ME (80.75%) were enriched in n-butanol fraction, and TF of nbutanol fraction was 2.63 times of ME.It was indicated that n-butanol had a good enrichment effect on flavonoids.n-Butanol fraction was then subjected to a polyamide column and eluted with a gradient of ethanol-water (water and 30%, 50%, and 70% ethanol).TF of BB (30% ethanol fraction) and BC (50% ethanol fraction) fractions were 765.76 ± 6.47 and 713.94 ± 3.79 mg RE/g, respectively.
e total flavonoid quality of BB fraction (600.58 mg) was also higher than that of BC fraction (326.64 mg).BB fraction was then separated over a Sephadex LH-20 column so that BB1 and BB2 fractions were obtained.TF of BB2 fraction reached up to 962.6 ± 3.92 mg RE/g.BB2 fractions (237.81 mg) were purified over semipreparative HPLC (MeOH/H 2 O), and then compound H (158 mg) was obtained.

Antioxidant Activity of M. oleifera Leaf Extract and Each Fraction
3.5.1.DPPH-Scavenging Activity.DPPH and ABTS are two kinds of traditional free radical commonly used to evaluate the free radical-scavenging activity.As shown in Figure 2, the DPPH-scavenging activity of crude ME and each fraction increased with the increasing concentration.e scavenging activity of n-butanol and ethyl acetate fractions at 0.5 mg/mL were 92.62% and 90.27%, respectively, higher than crude ME (85.51%) and comparable to V C (96.82%).But the scavenging activity of petroleum ether and water fractions at 0.5 mg/mL was only 55.89% and 45.13%, respectively, rather lower than crude ME (85.51%).e scavenging activity of petroleum ether and water fractions at 1.0 mg/mL reached up to 89.43% and 82.40%, respectively.e results indicated that the DPPH-scavenging activity sequence was V C > nbutanol fraction > ethyl acetate fraction > petroleum ether fraction > water fraction (p < 0.05), with EC 50 of 0.020, 0.067, 0.082, 0.353, and 0.439 mg/mL, respectively.erefore, both n-butanol and ethyl acetate fractions showed excellent scavenging activity that may be due to its enrichment of active components.As shown in Figure 3, the DPPH-scavenging activity of BB, BC, and BD fractions increased rapidly with increasing concentration.e scavenging activity of BB, BC, and BD fractions at 0.025 mg/mL were all less than 50%, while that of BB, BC, and BD fractions at 0.2 mg/mL reached 93.37%, 91.87%, and 83.41%, respectively.ere was no significant difference on the DPPH-scavenging activity between BB and BC fractions at 0.5-1.0mg/mL (p > 0.05), which was a little lower than that of V C (p < 0.05).
DPPH-scavenging activity of compound H at 0.1 mg/mL reached 96.33%.Compound H showed excellent scavenging ability, with an EC 50 value of 0.022 mg/mL, comparable to V C (0.023 mg/mL).As shown in Figure 4, there was no significant difference of the DPPH scavenging ability between compound H and V C (p > 0.05).

ABTS-Scavenging Activity.
As shown in Figure 5, the ABTS radical-scavenging activity of each fraction increased with the increase in concentration.e scavenging activity of n-butanol and ethyl acetate fractions at 0.2 mg/mL reached 99.46% and 97.49%, respectively, higher than crude ME (77.82%) and comparable to V C (99.62%).With the same concentration, the scavenging activity of petroleum ether and water fractions was 42.22% and 33.50%, respectively.
ere was no significant difference of ABTS-scavenging activity between n-butanol fraction and V C at 0.2-1.0mg/ mL (p > 0.05), as well as between V C , BB, and BC fractions at 0.5-1.0mg/mL (p > 0.05).
e results indicated that the ABTS-scavenging activity sequence was V C > n-butanol Journal of Food Quality fraction > ethyl acetate fraction > petroleum ether fraction > water fraction, with an EC 50 value of 0.013, 0.036, 0.046, 0.181, and 0.285 mg/mL, respectively.e ABTS-scavenging activity of BB, BC, and BD fractions all increased with the increasing sample concentration significantly (p > 0.05) (Figure 6).ABTS-scavenging activity of BB, BC, and BD fractions at 0.1 mg/mL reached 96.84%, 92.95%, and 90.19%, respectively.
ere was no significant difference between BB and BC fractions and V C at 0.2-1.0mg/mL (p > 0.05).e scavenging activity decreased as V C > BB > BC > BD, with an EC 50 value of 0.009, 0.022, 0.026, and 0.030 mg/mL, respectively.
As shown in Figure 7, the radical-scavenging activity of compound H was slightly higher than V C , with an EC 50 value of 0.007 and 0.009 mg/mL, respectively.

Hydroxyl Radical-Scavenging Activity.
Hydroxyl radical (OH_) is a kind of reactive oxygen-free radical with strong oxidability, which can react with lipids, amino acids, sugars, and other substances.It is toxic to biological cells, DNA, and other macromolecules, thus causing pathological changes of the body.
As shown in Figure 8, the hydroxyl radical-scavenging activity of n-butanol and ethyl acetate fractions were remarkably higher than petroleum ether and water fractions (p < 0.05).
e hydroxyl radical-scavenging activity of nbutanol and ethyl acetate fractions at 1.5 mg/mL reached up to 94.46% and 80.68%, respectively, while that of petroleum ether and water fractions were both less than 20%.e hydroxyl radical-scavenging activity of n-butanol and ethyl acetate fractions increased steadily with the increase in concentration, but that of petroleum ether and water fractions both increased slightly.Figure 9 shows that BB, BC, and BD fractions all exhibited good hydroxyl radical-scavenging activity, and that of the three fractions all increased with the sample concentration gradually.
As shown in Figure 10, the hydroxyl radical-scavenging activity of V C was slightly higher than compound H, with an EC 50 value of 0.262 and 0.349 mg/mL, respectively.

Total Reducing Power.
e total reducing power was measured by the reduction of the Fe 3+ /ferriccyanide complex, which was reduced to its ferrous form by gaining an electron from antioxidants.
As shown in Figure 11, n-butanol and ethyl acetate fractions had better reducing power than petroleum ether and water fractions.Reducing powder of crude ME and each fraction decreased in order of V C > n-butanol fraction > ethyl acetate fraction > crude ME > petroleum ether fraction > water fraction.As shown in Figure 12, reducing power of V C , BB, BC, and BD fractions decreased in the same order with the hydroxyl radical-scavenging activity.As shown in Figure 13, the reducing power of V C was slightly better than that of compound H (p < 0.05).

Characterization of the M. oleifera Leaf Extract.
HPLC-DAD results of ME and isolated fractions are shown in Figures 14 and 15, respectively.A total of 14 representative peaks were obtained in ME. e HPLC-ESI-MS results of the compounds are summarized in Table 5.However, peak 1 was still unknown, needing further research.Peaks 2-7 were tentatively identified as phenolic acids, such as 3caffeoylquinic acid, 4-caffeoylquinic acid, coumaroylquinic acid isomers, and caffeoylquinic acid isomer.
Isolated fractions of crude ME were also analyzed by HPLC-ESI-MS.As shown in Figure 15

Conclusion
In the present study, the optimization of UE was established for improving the phenolic compounds from M. oleifera leaves.RSM was successfully applied to optimize the extraction process.e optimum extraction condition was as follows: ethanol concentration of 70%, solvent-to-sample ratio of 30 : 1, extraction temperature of 50 °C, and extraction time of 40 min, with a TF yield of 4.83% and TP yield of

4 X 4 :X 2 :
o lv e n ttos a m p le r a t io ( m L / g ) X 1 : e t h a n o l c o n c e n t r a t io n ( x t r a c t io n te m p e r a tu r e ( °C) X 1 : e t h a n o l c o n c e n tr a ti o n ( e x t r a c t io n t im e ( m in ) X 1 : e th a n o l c o n c e n tr a ti o n ( % ) s o lv e n ttos a m p le r a ti o ( m L / g ) X 1 : e t h a n o l c o n c e n t r a t io n ( % ) x tr a c ti o n te m p e r a tu r e ( °C) X 1 : e t h a n o l c o n c e n t r a t io n ( % )

X 3 :Figure 1 :
Figure 1: Interactive effect of extraction variables on (a-c) TF and (d-f ) TP.

Figure 13 :
Figure 13: Reducing power of compound H and V C .

Figure 14 :Figure 10 :Figure 11 :Figure 12 :
Figure 14: HPLC chromatogram profile of ME extracted by using the UE method.

Table 1 :
Total quality, TF purity, TF quality, and TF yield of isolated fractions.

Table 2 :
Response surface experimental design and results.

Table 5 :
HPLC-ESI-MS data of the compounds identified in ME. 44%.e crude ME obtained at the optimized conditions was then further isolated by organic solvent extraction and column chromatography.HPLC-DAD-MS results showed that there were 6 phenolic acids and derivatives and 7 flavonoids in ME.Antioxidant property results showed that the scavenging activity sequence was n-butanol fraction > ethyl acetate fraction > ME > petroleum ether fraction > water fraction.Quercetin-3-O-β-D-glucoside isolated from ME showed excellent DPPH and ABTS radical-scavenging abilities, which were comparable to V C .