Effects of age and extraction solvent on phytochemical content and antioxidant activity of fresh Moringa oleifera L. leaves

Abstract Antioxidant activity (AOA) and phytochemical content of Moringa oleifera Lam leaves were determined as a function of their age and extraction solvent. Fresh Moringa leaves aged 30, 45, and 60 days were harvested and extracted with three solvents; methanol, ethanol, and water. AOA of leaf extracts was measured using radical scavenging assays (DPPH, ABTS, antiperoxide activity [APA]) and reducing assays (FRAP and total antioxidant capacity [TAC]), and these were correlated with total polyphenols (TPC), total flavonoids (TFC), and chlorophyll contents of leaves. Significant variability (p < 0.05) in TPC and AOA of Moringa leaf extracts was observed with age and extraction solvent as well as their interaction. TPC and TFC increased with maturity, except in aqueous extract. The 60‐day‐old leaves showed highest TPC, TFC, and tocopherol contents with highest DPPH activity. On their part, 30‐day‐old leaves recorded better vitamin C, chlorophyll, and carotenoids with highest ABTS activity and APA. Methanol was best extraction solvent for TPC (4.6 g GAE/100 g DM) while ethanol was for flavonoids (1.8 g CE/100 g DM). Ethanol extracts exhibited the highest DPPH activity (53.3%–71.1%), while both ethanolic and methanolic extracts had similar and higher ABTS + activity (3.83–3.86 g AAE/100 g DM). Strong positive correlations (r ≥ 0.8; p < 0.05) were observed between chlorophyll content and DPPH, ABTS, and APA, suggesting that chlorophyll was the major contributor to AOA. TAC was highest in aqueous solvent. Free radical scavenging activity in Moringa leaves is positively correlated to chlorophyll, TFC, and TPC while reducing power is positively correlated to chlorophyll and TPC. AOA of fresh Moringa leaf extract is a function of its phytochemical content and is influenced by both the age of the leaves and the extraction solvent used. Methanolic and ethanolic extracts of 45‐day‐old Moringa leaves exhibited best antioxidant potentials.

Jetawattana, 2014), and neurodegeneration (Hannan et al., 2014). Therefore, they constitute a potential material for nutraceutical formulation. The mechanisms involved in these therapeutic properties of Moringa leaf extracts are linked to their antioxidant activity among others. It is therefore necessary to master parameters that can affect antioxidant activity of Moringa leaf extracts as several factors have been shown to affect the antioxidant activity of plant materials. These are intrinsic factors such as age and cultivar and extrinsic factors such as harvesting season, locality, extraction solvent, and postharvest treatment (Agamou et al., 2015;Tlili et al., 2014). The stage of maturity is an important factor that influences the compositional quality and the quantity of phytochemicals in vegetables; due to the evidence during maturation, several biochemical, physiological, and structural modifications occur (Siddiqui et al., 2013). This could account for the inconsistent results recorded in literature with regard to antioxidant activity of plant materials in relation to the stage of maturity. Dian-Nashiela, Noriham, Nooraain, and Azizah (2015) reported higher antioxidant activity in young Cosmos caudatus aqueous leaf extracts compared to mature and old leaves; Tlili et al. (2014) showed that antioxidant capacity varies significantly according to the ripening stage of Rhus tripartitum fruits. In the case of M. oleifera, it has been reported that the aqueous extract of mature leaves exhibits better antioxidant activity compared to young leaves (Sreelatha & Padma, 2009). It can thus be concluded that the optimal stage of maturity for antioxidant activity is plant-specific. On the other hand, influence of extraction solvent on the phytochemical content and antioxidant activity of vegetables is widely reported (Do et al., 2014;Lou, Hsu, & Ho, 2014;Siddhuraju & Becker, 2003). From these studies, it appears that the notion of the best extracting solvent varies widely from one plant to another and depends on the targeted class of phytochemicals. In view of preparing nutraceuticals from M. oleifera leaves, it is therefore important to determine which extraction solvent will be best suited for what class of phytochemicals and the corresponding antioxidant capacity. In addition, given the variability in phytochemical contents and antioxidant activity with age of the plant, determining the optimal age for these properties will ensure efficient exploitation of the antioxidant potential of M. oleifera leaves. This work therefore had as aim to study the combined effect of age and extraction solvent (methanol, ethanol and water) on the phytochemical contents and antioxidant activity of M. oleifera fresh leaf extracts as well as the relationship between phytochemical content and antioxidant activity.

| MATERIAL S AND ME THODS
Fresh M. oleifera leaves of different ages were harvested from our experimental garden in Ngaoundere, Adamawa Region, Cameroon.
The trees were pruned, and fresh leaves were harvested at 30, 45, and 60 days after pruning. The harvested leaves were immediately transported to the laboratory, sorted to remove extraneous material, washed with tap water, and drained. The leaves were subsequently used for determination of antioxidant nutrients (carotenoids, tocopherols, and vitamin C), phytochemicals, and antioxidant activity.

| Variation in phytochemical contents and antioxidant activity of Moringa oleifera leaves with age and extraction solvent
To study the effect of extraction solvent and age on phenolic content and antioxidant activity, fresh leaf samples were pounded in a porcelain mortar to a smooth paste; then 1 g of fresh sample was mixed with 20 ml of solvent (water, ethanol or methanol) in a conical flask. The conical flask was sealed with parafilm, and the mixture was stirred for 2 hr using a magnetic stirrer at room temperature (25 ± 2°C). The extracts were then centrifuged at 2500g for 30 min at 4°C (Anke DL-6000 B; China). The pellets were reextracted under the same conditions with 15 ml of solvent, and both supernatants were pooled and adjusted to 40 ml with the indicated solvent. All extracts were stored at 4°C until analysis.

| Total phenolic content
Total phenolic compounds in extracts were estimated as reported previously (Nobosse et al., 2017) using Folin-Ciocalteu's phenol reagent and gallic acid as standard. In brief, an aliquot (20 μl) of the extract was mixed with 0.2 ml Folin-Ciocalteu reagent (diluted in water 1:16 v/v) and 0.4 ml of 20% sodium carbonate solution. The tubes were vortexed for 15 s and allowed to stand for 40 min at 40°C for color development. Absorbance was recorded against a reagent blank at 760 nm using a UV-Vis spectrophotometer (Metertech SP8001; Germany). The total phenolic content was expressed as gallic acid equivalent (GAE) in g/100 g DM.

| Total flavonoid content
Flavonoids were determined according to the method described by Nobosse et al. (2017). Aliquots (100 μl) of Moringa extracts were mixed successively with 2.6 ml of deionized water and 0.15 ml of NaNO 2 (5%). After incubation at 25°C for 5 min, 0.15 ml AlCl 3 (10%) was added and the mixture was reincubated under the same conditions. At last, 1 ml of NaOH 1M was added and the absorbance was measured at 510 nm against a reagent blank. Catechin (0.01%) was used as standard, and the flavonoid content was expressed as catechin equivalent (CE) in g/100 g DM.

| Chlorophyll content
The chlorophyll content of each extract of M. oleifera leaves was estimated as described by Kaushal, Sharma, and Attri (2013). This consisted in the measurement of absorbance of ethanol, methanol, and aqueous extracts at 645 and 663 nm. Then, the chlorophyll content (mg/g) was calculated and further expressed in g/100 g DM as follows: where Abs645 and Abs663 are absorbance at 645 and 663 nm, respectively, V is the total volume of the extract in ml, and W is the initial mass of the sample.

| Total carotenoids and tocopherols
Carotenoids and tocopherols were extracted according to the modified AOAC, 970.64 spectrophotometric method (1974). Fresh Moringa leaves (1 g) were finely ground and extracted using 30 ml of hexane/ acetone (70/30 v/v) mixture followed by filtration on filter paper (Whatman No. 1). The procedure was repeated thrice, and all extracts were pooled. The extract was poured into a separating funnel, then 50 ml of 1% sodium chloride solution was added, and the mixture was shaken and allowed to rest. The bottom acetone phase was separated from the hexane fraction. This procedure was repeated thrice, and the hexane fraction containing carotenoids and tocopherols was recovered. At last, the absorbance of this fraction was measured at 450 nm for carotenoids and 270 nm for tocopherols using a UV-Vis spectrophotometer (Metertech SP8001, Germany). The carotenoids content was calculated as β-carotene equivalent using extinction coefficient of 2,592 (Rodriguez-Amaya, 2001), and the tocopherols content was calculated as α-tocopherol equivalent using extinction coefficient of 3,270 (Bell, John, Hughes, & Pham, 2014).

| Vitamin C (ascorbic acid)
Ascorbic acid was measured according to the method of AOAC (1990).
The principle is based on oxidoreduction reaction between ascorbic acid and 2,6-dichlorophenolindophenol (2,6-DIP) in acidic medium. For extraction of ascorbic acid, 1 g of fresh Moringa leaves was triturated in a mortar in the presence of few drops of 25% acetic acid. Then, 25 ml of 25% acetic acid was added and the mixture was vigorously vortexed for 10 min. The mixture was centrifuged at 3,500 rpm for 15 min at 0°C. The supernatant was filtered through Whatman No. 1 filter paper, and the total volume was made to 30 ml with 25% acetic acid. Total ascorbic acid content in Moringa leaves was evaluated by titrimetric assay. For this purpose, 2 ml of the aforementioned leaf extract was titrated with 2,6-DIP. The control test was realized using ascorbic acid standard 0.1% in place of the extract. The end of titration was reached when colorless ascorbic acid or extract solution turns pink in color. The results are expressed as mg ascorbic acid per 100 g DM.

| Antioxidant activity
Given that antioxidants have various mode of action such as radical scavenging and metal reduction, different assays based on these modes of action were used to determine the antioxidant activity.

| DPPH radical scavenging activity assay
The modified Brand-Williams, Cuvelier, and Berset (1995) method was used to measure the DPPH radical scavenging activity of M. oleifera leaf extracts. DPPH in ethanol is a stable radical, dark violet in color. Its color is bleached by its reaction with a hydrogen donor. For analyses, 0.1 ml of each extract was added to 2 ml of 100 μM DPPH solution. The reaction mixture was incubated for 30 min in the dark at 25°C, and the absorbance was read at 517 nm, against a reagent blank. Ascorbic acid and butylated hydroxyanisole (BHA) were used as reference standards. Antioxidant activity was expressed as percentage of DPPH radical scavenged by M. oleifera extract and calculated as follows: where A is the absorbance at 517 nm.

| ABTS+ radical scavenging activity
Experiments were performed according to Re et al. (1999) with slight modifications. ABTS was dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS + ) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12-16 hr before use. For the study of Moringa leaf extract radical scavenging ability, the ABTS + solution was diluted with ethanol to an absorbance of 0.700 ± 0.020 at 734 nm and equilibrated at room temperature (25 ± 2°C). A 50 μl aliquot of each extract was added to 3 ml of the diluted ABTS + solution, and the absorbance reading was taken 5 min after mixing using a spectrophotometer (Metertech SP8001, Germany). Ascorbic acid was used as reference, and results are expressed as ascorbic acid equivalent antioxidant capacity (AEAC).

| Antiperoxide activity
Antiperoxide activity was measured using the ferric thiocyanate assay as described by Miranda, Maureira, Rodríguez, and Vega-Gálvez (2009). Two milliliters of the extract, 2 ml of linoleic acid (2.51 g/100 ml in ethanol [95%]), 4 ml of 0.05 M phosphate buffer (pH 7.0), and 2 ml of distilled water were mixed in a 10 ml screw-top tube and maintained in a water bath at 40°C in the dark for 24 hr. A blank was prepared using 2 ml of ethanol solution containing linoleic acid, phosphate buffer (pH 7.0), and distilled water. After 24 hr of incubation, 0.1 ml of the mixture was added to 9.7 ml of 95% ethanol and 0.1 ml of 30% (w/v) ammonium thiocyanate. After 5 min, 0.1 ml of ferrous chloride (0.02 M in hydrochloric acid at 3.5 ml/100 ml) was added to the mixture and agitated. The absorbance of the mixture was measured at 500 nm using a UV-Vis spectrophotometer (Metertech SP8001, Germany). Butylated hydroxytoluene (BHT) solution was used as standard.
The antiperoxide activity of the extracts was calculated as: where Abs is the absorbance at 500 nm.

| Metal reducing ability
It was measured using the total antioxidant capacity (TAC) and FRAP assays.

| Total antioxidant capacity
The TAC was evaluated by the phosphomolybdenum assay as described by Prieto, Pineda, and Aguilar (1999). This assay is based on the reduction in Mo (VI) to Mo (V) by the sample analyzed and the subsequent formation of a green phosphate/Mo (V) complex at acidic pH. An aliquot of 0.1 ml of Moringa leaf extract was mixed with 3 ml of the reaction solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). For the blank, 0.1 ml of the corresponding extraction solvent was mixed with 3 ml of the reaction solution. After incubation in a water bath at 90°C for 90 min, the absorbance of the test sample was measured at 695 nm against the blank. Ascorbic acid was used as standard. The antioxidant activity was expressed as ascorbic acid equivalents (AAE/100 g DM).

| Ferric reducing/antioxidant power
The ability of M. oleifera leaf extracts to reduce iron was determined using the FRAP method as described by Yen and Chen (1995) and modified by Nobosse et al. (2017). An aliquot of extract (1 ml) was mixed with 1 ml of 0.2 M phosphate buffer (pH 6.6) and 1 ml of 1% K 4 Fe(CN) 6 and incubated for 20 min at 50°C. The mixture was further cooled and precipitated with 10% trichloroacetic acid solution.
After centrifugation at 3,500 rpm for 15 min, 1 ml of distilled water was added to 1 ml of the supernatant and 0.1 ml of 0.1% FeCl 3 solution was added and vortexed. The absorbance was read at 700 nm against a reagent blank. Ascorbic acid was used as reference.

| Statistical analysis
All determinations were carried out in three replicates. Means were compared using two-way analysis of variance (ANOVA) and separated by Duncan's multiple-range test using Statgraphics Centurion XVI software, and p < 0.05 was regarded as significant. SigmaPlot 11 was used for plotting graphs. The classification and discrimination of Moringa leaf extracts as well as the correlation between phytochemicals and antioxidant activity were established by principal component analysis (PCA) using XLSTAT software (XLSTAT 2007; Addinsoft, New York).

| RE SULTS
This study was carried out to determine the phytochemical content and antioxidant capacity of M. oleifera leaves as affected by their age and the type of extraction solvent used, as well as the relationship between phytochemical content and antioxidant activity.
Phytochemical (TPC, TFC) and chlorophyll contents were significantly (p < 0.05) affected by the age of the leaves and the extraction solvent as well as the interactions between them (Tables 1 and 2).
Total phenolic content ranged between 2.1 and 4.6 g GAE/100 g DM, and the flavonoids content ranged between 0.9 and 1.8 g CE/100 g DM (   The TAC on its part was highest in the aqueous extract of 60-day-old leaves ( Figure 2c). Irrespective of age, the TAC varied significantly (p < 0.05) with extraction solvent in the order methanol (5.11-5.35 g AAE/100 g DM) < ethanol (5.58-5.85 g AAE/100 g DM) < water (6.11-6.50 g AAE/100 g DM). There were no significant age-  was significantly (p < 0.05) lowest in 60-day-old leaves. As shown in  (Table 3), which shows significant (p < 0.05) positive correlations between DPPH with chlorophyll (r = 0.88) and total flavonoids (r = 0.80).
ABTS on the other hand shows strong significant (p < 0.05) positive correlation with TPC (r = 0.70) and chlorophyll (r = 0.99). APA is only significantly (p < 0.05) correlated with chlorophyll (r = 0.88) and to a lesser extent to TPC (r = 0.52) although not significant (p > 0.05).

| D ISCUSS I ON
Antioxidant activity (AOA) and phytochemical contents of M. oleifera leaves showed significant (p < 0.05) variation with both the age of the leaves and the extraction solvent as well as their interaction (Table 2).
Methanol was the best extraction solvent for total phenolic components of M. oleifera leaves, whereas flavonoids were best extracted in ethanol, with 60-day-old leaves having the highest TPC and flavonoid contents in each case. The lowest TPC and flavonoid contents were obtained with aqueous extracts. The increase in total phenolic and flavonoid content with age can result from their active biosynthesis and accumulation in the cells during plant growth (Kong et al., 2017). This influence of age corroborates the report by Sreelatha and Padma (2009) (Green & Fry, 2005), given that they are involved in cell division, plant growth, and photoprotection (Bartley & Scolnik, 1995;Smirnoff, 1996).
In addition to age and extraction solvent, the variation in AOA was also a function of the assay used. As secondary metabolites, phenolic compounds are widely distributed in fruits and vegetables and are considered the main actors for the antioxidant capacity of plants (Tlili et al., 2014). However, the present study shows that chlorophyll is a more potent radical scavenger and reducing agent than total phenolic compounds and flavonoids. Previous studies have shown that chlorophyll exhibits free radical scavenging and metal chelating activity (Hsu, Chao, Hu, & Yang, 2013;Khattab, Goldberg, Lin, & Thiyam, 2010) and improves resistance to oxidative stress in nematodes (Caenorhabditis elegans) (Wang & Wink, 2016) although the mechanism of its antioxidative activity is not clear.

| CON CLUS ION
The phytochemical content and antioxidant activity of fresh M. oleifera leaves are influenced by the age of the leaves and the extraction solvent used, as well as the interaction between them.
Worldwide, ethanol appears to be the most efficient solvent for the production of extracts with high flavonoid content, while methanol is better suited for production of polyphenol-rich extracts. Both ethanolic and methanolic extracts exhibited higher antioxidant TA B L E 3 Pearson correlation matrix of phytochemicals with antioxidant activity activity compared to water extracts. M. oleifera leaves aged 45 days are best suited for the production of extracts with the most potent antioxidant activity. The lowest antioxidant activity and phytochemical contents are obtained with aqueous extracts. AOA is a function of phytochemicals content. The free radical scavenging activity in M. oleifera leaves is positively correlated to chlorophyll, flavonoids, and total polyphenols while reducing power is positively correlated to chlorophyll and total polyphenols. However, chlorophyll is a more potent radical scavenger and reducing agent than total phenolic compounds and flavonoids.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L S TATEM ENT
Human testing and animal testing were not necessary in this study.