In vitro antioxidant activity of two different extracts of Moringa oleifera leaves from Caribbean St. Lucia Island

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Acta Hortic. 1306. ISHS 2021. DOI 10.17660/ActaHortic.2021.1306.17
Proc. II International Symposium on Moringa
Eds.: R. Kleynhans et al.
143
In vitro antioxidant activity of two different extracts
of
Moringa oleifera
leaves from Caribbean St. Lucia
Island
J. Fe j é r a, I. Kron, D. G ru ľová and A. E li aš o vá
Department of Ecology, University of Prešov in Prešov, 17 novembra 1, 081 16 Prešov, Slovak Republic.
Abstract
In this study we evaluated ethanol and hot water extracts from young leaves of
moringa plants growing on the island of St. Lucia. The dry matter, total phenol content,
antioxidant activity against superoxide radicals, hydroxyl radicals and FRAP were
evaluated. Content of dry matter (DM), total phenols (mg GAE L-1) and total phenols
calculated to DM (mg GAE g-1 DM) were statistically significantly higher in the ethanol
extract in comparison to the hot water extract. Antioxidant activity against superoxide
and hydroxyl radical were comparable for both extracts, but the antioxidant activity
calculated on the dry matter was significantly higher for the hot water extract. Ferric
reducing ability of plasma (FRAP) assay was in both cases (determined in µmol L-1 and
recalculated in µmol L-1 g-1 DM) statistically significantly higher for the hot water
extract in comparison to the ethanol extract. Finally, secondary metabolites extracted
by hot water (probably phenolic acids) have (relatively) higher antioxidant activity
than polyphenols in the 70% ethanol extract.
Keywords: antioxidant activity, hot water extract, ethanol extract, leaves
INTRODUCTION
Moringa oleifera Lam. (moringa) is a tree growing in tropical and subtropical regions.
All parts of moringa are commonly used as a food (source of proteins, vitamins and minerals),
and have a beneficial effect in the treatment of various diseases, including cancer (Charoensin,
2014; Aré valo-Hı́jar et al., 2018; Khor et al., 2018; Uphadek et al., 2018; Sandeep et al., 2019a,
b).
When evaluating health beneficial effects of various parts of medicinal plants and fruits,
many authors used different solvents or their combination to extract secondary metabolites
(Lapornik et al., 2005; Kim et al., 2011; Stankovič, 2011; Santos and Goncalves, 2016). The
main metabolites, on which research is focused, are simple phenolic acids and polyphenolic
compounds (flavonoids, tannins, lignans, coumarins, lignins) (Jahromi, 2019). Leaves are the
main producer of such compounds and moringa leaves are cooked and edible, therefore we
were curious whether the hot water extract would reveal the same antioxidant activity on
comparison to the 70% ethanol extract.
MATERIAL AND METHODS
Plant material and extracts preparation
Dried leaves (in the dryer at 40°C) from juvenile plants (five months old) of moringa
from the Caribbean island of Saint Lucia (October 2018) were received directly from the
company Moringa Caribbean Ltd. Ten grams of powder were dissolved in 100 mL of 70%
ethanol. The extraction was carried out for 72 h at room temperature. The obtained extracts
were filtered over filter KA 1-M (very fast). The dry matter (DM) content was determined in
the fil Jozef Fejér, Ivan Kron, Daniela Gruľová and Adriana Eliašová trates (Fejér et al., 2019).
The hot water extracts (“tea”) of moringa leaves were prepared by weighing 1.0 g of dried
crushed leaves and extracting in 100 mL of boiling water. The extraction time was 15 min.
aE-mail: jozef.fejer@unipo.sk

144
After cooling and filtration the extracts were used for analysis.
Superoxide anion radical scavenging activity
The assay was inspired by the work of Fridovich (1970). Phosphate buffer (PB, sodium
dihydrogen phosphate dihydrate and disodium hydrogen phosphate dodecahydrate, 0.05 mol
L-1 at pH 7.4) was used with 0.1 mmol L-1 Na2EDTA. Hypoxanthine (HX, 0.4 mmol L-1, Alfa Aesar
a Johnson Matthey Company) was dissolved in the phosphate buffer. Then, 0.01 g xanthine
oxidase (XO, Sigma-Aldrich, with an activity of 0.11 units mg-1 solid or 0.713 units mg-1
protein) was dissolved in 20 mL of phosphate buffer. Nitro blue tetrazolium chloride (NBT, 5
mmol L-1, Sigma-Aldrich) in phosphate buffer was used as an indicator of superoxide radicals.
Antioxidant activity of tested samples against superoxide radicals was compared with the
antioxidant activity of salicylic acid (SA) 10 mmol L-1 in the phosphate buffer. The solutions
were pipetted (volumes are in µL) into test tubes (in duplicate) according to Table 1.
Table 1. Reaction mixture composition (μL) in the superoxide anion radical scavenging
activity determination.
Test tube
PB
HX
XO
NBT
Sample, SO
“0”
2550
200
-
50
-
“X0”
2350
200
200
50
-
“0-SO”
2500
200
-
50
50
“SO”
2300
200
200
50
50
“0-S1-2”
2500
200
-
50
50
“S1-2”
2300
200
200
50
50
PB = phosphate buffer, HX = hypoxanthine, XO = xanthine oxidase, NBT = nitro blue tetrazolium
chloride, SO = sodium salicylate, S = sample of extract, 1 = ‘ethanol extract’, 2 = ‘hot water extract’.
Solutions were mixed well and incubated in a water bath at 38°C for 40 min. After
incubation and cooling, the absorbance of the solutions was determined in a 1-cm cell at 560
nm using a spectrophotometer. The antioxidant activity of each sample, expressed as
percentage of inhibition (POI), was calculated by the formula:
 = {[(0) – (0)] − [() – (0 −)]} . 100
[A(X0) – A(0)]
All the determinations of antioxidant activity against superoxide radicals in the samples
were performed at least four times.
Hydroxyl radical scavenging activity
The assay was inspired by the paper of Gutteridge (1984). Phosphate buffer (PBS,
NaH2PO4/Na2HPO4) 0.05 mol L-1, pH 7.4 was used with 0.1 mol L-1 NaCl and 9 mmol L-1 2-
deoxyribose. The 3 mmol L-1 ferrous sulfate heptahydrate was dissolved in 100 mL DDW
(double distilled water) with the addition of 0.1 mL concentrated sulfuric acid to prevent
oxidation of Fe(II) and hydrolysis of Fe(III). Hydrogen peroxide (10 mmol L-1) was dissolved
in 100 mL DDW with addition of 0.1 mL concentrated sulfuric acid to prevent
disproportionation of hydrogen peroxide. Thiobarbituric acid (TBA) 1 g was dissolved in 100
mL of 50 mmol L-1 sodium hydroxide solution. Trichloroacetic acid (TCA) 5.6 g was dissolved
in 100 mL DDW. The antioxidant activity of samples tested against hydroxyl radicals generated
by the Fenton reaction was compared with the antioxidant activity of gallic acid (GA) 10 mmol
L-1 in the PBS. The solutions were pipetted (volumes are in µL) into test tubes (in duplicate)
as shown in Table 2.

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Table 2. Reaction mixture composition (μL) in the hydroxyl radical scavenging activity.
Test tube
PBS
Fe(II)
H2O2
Sample, GA
“100”
980
10
10
-
“0-GA”
990
10
-
10
“GA”
970
10
10
10
“0-S1-2”
980
10
-
10
“S1-2”
970
10
10
10
PBS = phosphate buffer, GA = gallic acid, S = sample of extract, 1 = ‘ethanol extract’, 2 = ‘hot water extract’.
Solutions were mixed well and incubated in a water bath at 38°C for 40 min. Then, 500
µL of TBA solution were added into each test tube, which was stoppered, mixed well and
incubated in a boiling water bath for 10 min. After boiling and uncorking, 500 µL of TCA
solution was added into each test tube, mixed well and cooled down in a beaker with tap water.
The absorbance of the solutions was determined in a 1-cm cell at 532 nm against DDW using
a spectrophotometer. The antioxidant activity of each sample, expressed as POI, was
calculated by the formula:
 ={A(100) − [A(Sx) – A(0 −Sx)]} × 100
A(100)
All determinations of antioxidant activity against hydroxyl radicals in the samples were
performed at least four times.
Ferric reducing ability of plasma (FRAP) assay
The working FRAP reagents and other reagents were prepared according to Benzie and
Strain (1996). The only change was in the increase of hydrochloric acid concentration to 50
mmol L-1 for dissolving of 10 mmol L-1 TPTZ (2,4,6-tripyridyl-s-triazine). Aqueous solutions
of iron (II) sulfate heptahydrate in the concentration range 0-900 mmol L-1 were used for
calibration at 600 nm (r=0.9997). The assay was performed manually at room temperature.
The solutions were pipetted (volumes are in µL) into test tubes (in duplicates) as shown in
Table 3.
Table 3. Reaction mixture composition (μL) in the FRAP assay.
Test tube
Working
solution
DDW
Ethanol
70% (v/v)
Fe2+
(500 mmol L
-1
)
Sample
“Blank”
2000
300
-
-
-
“Blank-Ethanol”
2000
250
50
-
-
“Standard-Fe2+”
2000
250
-
50
-
“Sample S1”
2000
250
-
-
50
“Sample S2”
2000
250
-
-
50
DDW = double distilled water, S = sample of extract, 1 = ‘ethanol extract’, 2 = ‘hot water extract’.
Solutions were mixed well and the absorbance at 600 nm was recorded after 5 min with
the spectrophotometer. A Gallic acid solution with concentration of 10 mmol L-1 was used for
comparison. The FRAP of the samples (in µmol L-1) was calculated by the formula:
FRAP = (Sample – Blank) × 500
(Standard – Blank)
All determinations of FRAP in the samples were performed at least four times. All
solutions were used on the day of preparation.

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Total phenols
The total phenolic content of the ethanol extracts of leaves was determined with the
Folin-Ciocalteu reagent (FCR, Merck) according to a procedure described by Singleton et al.
(1999) with slight modifications. The working solution was prepared by mixing 1 volume of
FCR with 9 volumes of 5% sodium carbonate solution. The solutions were pipetted (volumes
are in µL) into test tubes (in duplicate) as shown in Table 4.
Table 4. Reaction mixture composition (μL) in the total phenolics determination.
Test tube
Working
solution
DDW
GA
(500 mg L
-1
)
Sample
“Blank”
2000
150
-
-
“Standard-GA”
2000
-
150
-
“Sample S1”
2000
-
-
150
“Sample S2”
2000
-
-
150
DDW = double distilled water, GA = gallic acid, S = sample of extract, 1 = ‘ethanol extract’, 2 = ‘hot water extract’.
The solutions were mixed well, and after 2 min, the absorbance was determined at 765
nm with the spectrophotometer. The amounts of polyphenols in the samples were calculated
as gallic acid equivalents (GAE). All determinations of total polyphenols in the samples were
performed at least four times. All solutions were used on the day of preparation.
Statistical analysis
The statistical was used to perform a multifactorial analysis of variance (ANOVA), and
the Tukey test was used post-hoc where there were significant differences between the
means. A 95% confidence interval was used for the statistical analysis.
RESULTS AND DISCUSSION
The results of the dry matter extracts, total phenolic content, antioxidant activity
against superoxide radical, hydroxyl radical and FRAP are shown in the Table 5. In the cases
of total phenolic content and antioxidant activities we applied also the correction by the dry
matter in the extracts. Such values much better express the relative antioxidant activity
related to the content of phenols, which are different in the plant extracts. The amount of dry
matter in the extracts depended on the solvent and time of extraction.
Table 5. The dry matter, total phenolics content, antioxidant activity against superoxide
radical, hydroxyl radical and FRAP of two moringa leaves extracts.
Parameter
Ethanol extract
Hot water extract
p
DM (g L-1)
15.75±0.22a
3.64±0.01b
p˂0.001
Phenols (mg GAE L-1)
727.50±66.72a
41.64±0.36b
p˂0.001
Phenols (mg GAE g-1 DM)
46.18±4.24a
11.44±0,10b
p˂0.001
POI - Superoxide (%)
60.52±5.40a
63.62±1.70a
p=0.289
POI - Superoxide (% g-1 DM)
3.84±0.34b
17.48±0.47a
p˂0.001
POI - Hydroxyl (%)
65.77±1.27a
66.04±2.08a
p=0.851
POI - Hydroxyl (% g-1 DM)
4.17±0.08b
18.14±0.57a
p˂0.001
FRAP (µmol L-1)
7499.47±484.24b
9514.81±224.54a
p=0.005
FRAP (µmol g-1 DM)
476.06±30.74b
2613.96±61.69a
p˂0.001
Data represent the mean ± s.d. (standard deviation); a, b values followed differences between extracts; significantly in p<0.05,
according the multiple range % (ANOVA), test LSD 95 (least significant differences).
DM = dry matter; GAE = gallic acid equivalents; POI = percentage of inhibition.
The content of dry matter, total phenolics (mg GAE L-1) and total phenolics calculated
(mg GAE g-1 DM) were significantly higher in the ethanol extract compared to the hot water
extract. The antioxidant activity against superoxide and hydroxyl radicals expressed as

147
percentage of inhibition (POI) was compared for both extracts. However, the antioxidant
activity corrected by the dry matter was significantly higher for the hot water extract
(p<0.001).
The ferric reducing ability of plasma (FRAP) was in both cases (determined in µmol L-1
and recalculated in µmol L-1 g-1 DM) significantly higher for the hot water extract compared to
ethanol extract (p=0.005, resp. p<0.001) (Table 5).
Ademiluyi et al. (2018) showed the strong effect of the moringa leaf drying method on
the total phenolic value. They found from 47 to 69 mg GAE g-1 DM. The amount of phenolic
substances depends on the extraction agent. Pari et al. (2007) reported the amount of
phenolics (118 mg g-1) in methanol extracts of moringa leaves from India, while 71.1-76.3 mg
GAE g-1 DM were reported in moringa leaves from Mexico by Castillo-Ló pez et al. (2017). In
our study, leaf extracts from young plants contained 11.44 mg GAE g-1 DM (hot water extract)
and 46.18 mg GAE g-1 DM (70% ethanol extract).
There are many references in the literature about antioxidant activity by DPPH method
(Pari et al., 2007; Santos et al., 2012; Charoensin, 2014). Kalpna et al. (2011) reported
antioxidant activity against superoxide and hydroxyl radicals in chloroform, acetone and
methanol extracts (IC50 value μg mL-1 >1000). Fejér et al. (2019) found high antioxidant
activity of crushed leaf ethanol extracts against superoxide radical (83.6%, resp. 3.0%
calculated on the DM and against hydroxyl radical 60.7%, resp. 2.2% calculated on the DM).
In this study the ethanol leaf extract of young plants showed activity against superoxide
radical 60.52 and 3.84% calculated on the DM, respectively. Antioxidant activity of the hot
water extract was 63.62 and 17.48% calculated on the DM, respectively. The antioxidant
activity against hydroxyl radical of the ethanol extract was determined 65.77 and 4.17%
calculated on the DM, respectively. The hot water extract showed 66.04% antioxidant activity
and 18.14% calculated on the DM, respectively. Ferric reducing ability of plasma (FRAP) assay
was statistically significantly higher in the hot water extract on comparison to the ethanol
extract (Table 5).
One can speculate that during the hot water extraction mainly lower molecular weight
(phenolic acids) and other compounds are extracted. Those compounds can easily exchange
electrons in the FRAP and superoxide assays and react with hydroxyl radicals (see bold values
in Table 5). Ethanol (70%) is less polar than water, but is more efficient in the cell wall’s
degradation (Lapornik et al., 2005) of moringa leaves therefore higher molecular weight (like
flavonoids – Jahromi, 2019) and less polar compounds are likely extracted. Those compounds
show significantly lower antioxidant activity in spite of higher contents of polyphenolic
compound and dry matter (see Table 5).
CONCLUSIONS
Leaves of moringa from the St. Lucia Island were extracted with hot water for 15 min
and 70% ethanol for 3 days. Test of antioxidant activity against superoxide and hydroxyl
radicals and FRAP assay were performed. The results showed that the hot water extract have
higher antioxidant activity compared to the ethanol extracts. Short cooking does not damage
plant metabolites with antioxidant activity (probably phenolic acids).
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