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Food Sci Nutr. 20 1 8;1–1 1 .
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www.foodscience-nutrition.com
1 | INTRODUCTION
Moringa oleifera is a pan- tropical plant having small- or medium-
sized perennial softwood tree with timber of low quality. It is the
best known and most widely cultivated species of a monogene-
ric genus plant family of Moringaceae. This plant is native to sub-
Himalayan regions of northern India and has been planted around
the world and naturalized in many locales (Martin, 2013). In Nigeria,
it is known by many native names such as “zogeli” in Hausa, “okwe
oyibo” in Igbo, “ewe ile,” “igi iyaanu,” or “ewe igbale” in Yoruba and
“dogalla” in Taroh (Fahey, 2005; Saalu et al., 2011). It is considered
one of the world’s most useful trees because almost every part of
the tree has some nutritional, medicinal, and other beneficial prop-
erties (Luqman, Srivastava, Kumar, Maurya, & Chanda, 2012). The
Received:1March2018
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Revised:1 2July2018
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Accepted:14July2018
DOI: 10.1002/fsn3.770
ORIGINAL RESEARCH
Drying alters the phenolic constituents, antioxidant properties,
α- amylase, and α- glucosidase inhibitory properties of Moringa
(Moringa oleifera) leaf
Adedayo O. Ademiluyi1 | Olubukola H. Aladeselu1 | Ganiyu Oboh1 | Aline A. Boligon2
ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,
provide d the original wor k is prope rly cite d.
©2018TheAuthors.Food Scien ce & Nutrition published by Wiley Periodicals, Inc.
1Functional Food s and Nutraceut icals
Unit, Department of Biochemistry, Federal
UniversityofTechnology,Akure,Nigeria
2Postgr aduate P rogramme in Pharmaceutical
Sciences, Universidade Federal de Santa
Maria, S anta Maria, RS , Brazil
Correspondence
AdedayoO.Ademiluyi,FunctionalFoods
and Nutraceuticals Unit, Department
of Bioche mistr y, Federal University of
Technology,Akure,P.M.B.704,Akure
340001,Nigeria.
Emails: ademiluyidayo@yahoo.co.uk;
aoademiluyi@futa.edu.ng
Abstract
Moringa oleifera leaf is a popular green leafy vegetable which has found its usefulness
in the preparation of traditional stews and soups. Like most green leafy vegetable
which are not around year- round, the leaf is usually dried and pulverized for storage
and easier handling, and despite the popularity of this processing technique, there is
dearth of information on how drying affects the health- promoting properties of the
leaves. Hence, this study sought to investigate the effect of some drying methods
(freeze- drying, sun, air and oven drying) on the phytoconstituents, antioxidant prop-
erties, and biological activities of moringa leaf. This study revealed that drying meth-
ods significantly altered the phytoconstituents (phenolics, flavonoids, vitamin C,
tannin, saponin, phytate, oxalate, alkaloid, cardenolides, and cardiac glycosides), an-
tioxidant capacities (reducing power, Fe2+chelating,ABTS•+, DPPH, and •OH scav-
enging abilities), and enzyme inhibitor y (α- amylase and α- glucosidase) effects of the
leaf, with freeze- drying being the most promising method for preserving the nutra-
ceutical properties of moringa leaf. However, for practical application, the order of
preference of the drying methods which ensures adequate retention of phytocon-
stituents and possibly biological activities of the leaf as observed in this study is
freeze- drying > air drying > sun dr ying > oven drying, in the order of decreasing
magnitude.
KEYWORDS
α-amylase, α-glucosidase, antioxidant properties, drying, Moringa oleifera leaf, phenolics
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ADEMILUYI Et AL.
medicinal proper ties have been attributed to phytochemical compo-
sitions of it s various parts: the roots, bark, leaf, flowers, fruits, and
seeds(Anwar,Latif,Ashraf,&Gilani,2007;Kumar,Mishra,Ghosh,&
Panda, 2010).
Moringa oleifera leaf belongs to the family of dark green leafy
vegetables, which are particularly rich in nutrients. The leaves
of Moringa are particularly good sources of proteins, calcium,
iron, β-carotene (converted to vitamin A in thehuman body), vi-
tamin C, and vitamin E (Zaku, Emmanuel, Tukur, & Kabir, 2015).
The most utilized component of Moringa is it s leaf, and like most
green leafy vegetables, the leaves are usually processed fresh or
driedandkeptforfutureusage.Greenleafyvegetablesareusually
processed into soups and stews in many communities in Nigeria
where they accompany a traditional starchy meal. “Efo- riro” is a
rich vegetable stew that is native to the Yoruba people of Western
Nigeria. This particular stew involves the use of many different
green leafy vegetables in it s preparation and often times, moringa
leaf is employed. The green leaves of Moringa are harvested and
carefully washed to remove dirt. The leaf is blanched before in-
corporation into the stew or could be included directly without
blanching. Furthermore, Moringa leaves are added to other vege-
table soups like “Okra,” “Egusi,” “Ugwu,” and “Spinach” (Babayeju
etal.,2014).InotherpartsofNigeria,theyoungleafiscommonly
cooked and eaten like spinach or used to make other soups and
salads. Furthermore, the leaf is often consumed raw, cooked or
dried and ground into fine powder which could be added to almost
any food such as pap (Ogi) and other cereal gruels as nutrient sup-
plement (Zaku et al., 2015).
Moringa have found usefulness in the folk medicine where the
infusions, decoctions, and concoctions of various par ts of this
plant are used in the treatment of several ailments such as cardiac
and circulatory stimulants; possesses antitumor, antipyretic, an-
tiepileptic, anti- inflammatory, antiulcer, antispasmodic, diuretic,
antihypertensive, cholesterol lowering, antioxidant, antidiabetic,
hepatoprotective, antibacterial, and antifungal activities (Anwar
etal., 2007). And these health-promoting effects have been at-
tributed to its constituent phytochemicals such as zeatin, quercetin,
β-sitosterol,caffeoylquinicacid,andkaempferol(Anwaretal.,2007;.
Furthermore, evidences are also pointing to antioxidant activity as
one of the main mechanism of action underlying the medicinal prop-
erties of moringa leaf (Pari & Kumar, 2002).
Moringa leaf, like most green leaf y vegetables in Nigeria, is not
around year- round; hence, they are usually processed dr y using
several local means such as sun drying, drying under shade, and
oven drying. These dried leaves are pulverized and applied directly
to soups as thickener and to several other food preparations. In an
unfortunate way, despite the popularity of these practices which is
mainly to ex tend the shelf- life as well as the handling of this green
leafy vegetable, there is dearth of information on the effect of these
various drying methods on the phytoconstituents, nutritional and
medicinal values of M. oleifera leaf. Hence, this study sought to in-
vestigate the effect of some drying methods (air- , oven- , sun- , and
freeze- dr ying) on some phy toconstituent s of moringa leaf and to
ascert ain how this affects its antioxidant proper ties and inhibitory
effects on α- amylase and α- glucosidase activities in vitro.
2 | MATERIALS AND METHODS
2.1 | Plant material
Fresh Moringa (M. oleifera) leaf was obtained from a local Moringa
plantation in Akure metropolis, Nigeria and authenticated at the
Department of Crop, Soil and Pest Management, Federal University
ofTechnology,Akure,Nigeria.
2.2 | Animals
Adultwistarstrainalbinoratsweighing220–260gwerepurchased
from the C entral Anima l House of Univer sity of Ibadan a nd were
maintained in wire mesh cages and fed with commercial rat chow
and water ad libitum. The animals were acclimatized under this con-
dition for 2 weeks prior to the study.
2.3 | Chemicals and reagents
Folin- Ciocalteu’s reagent, thiobarbituric acid, 1,10- phenanthroline,
deoxyribose,gallicacid,trichloroaceticacid(TCA),dinitrophenylhy-
drazine (DNPH), 1,1- diphenyl- 2 picrylhydrazyl (DPPH) radical, 2,2-
azino-bis(3-ethyl-benzothia-zoline-6-sulfonicacid)(ABTS),catechin,
epicatechin, quercetin, quercitrin, isoquercitrin, rutin, kaempferol, α-
amylase and α-glucosidaseweresourcedfromSigma-Aldrich,Inc.(St
Louis, MO). Methanol, formic acid, gallic acid, chlorogenic acid, caf-
feic acid and ellagic acids were purchased from Merck (Darmstadt,
Germany).Allotherchemicalsandreagentsusedwereofanalytical
grade and glass- distilled water was used.
2.4 | Sample preparation
The leaves were washed under running tap to remove dirt and
drained in a plastic sieve. Thereafter, a portion of the leaves was
freeze- dried, another portion was sun dried, the third portion was
air- dried at room temperature and the last portion was oven dried
(40°C).Allthedryingwascarriedouttoconstantweight.Thedried
samples were then pulverized and kept in air- tight containers prior
to analysis.
2.5 | Aqueous extract preparation
One gram e ach of the powdered samples was weighed and extra cted
in100mldistilledwaterfor24hronanorbitalshaker.The extract
was further filtered using Whatman filter paper (No. 1) and the fil-
trate obtained was centrifuged at 1,000 × g for 10 min. Thereaf ter,
the supernatant obtained was dried under vacuum using a rot atory
evaporatoranddriedextractobtainedwaskeptat−4°Cforsubse-
quent analysis (Oboh, Puntel, & Rocha, 20 07). The percentage yield
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ADEMILUY I Et AL.
oftheextractswere44.2%,45%,43.6%and43.7%forfreeze-dried,
oven dried, air- dried and sun dried samples, respectively.
2.6 | Total phenol determination
The total phenol content was determined according to the method of
Singleto n, Orthofer, and L amuela-R aventos (1999).A ppropriate dil u-
tions of the extracts were oxidized with 2.5ml 10% Folin-Ciocalteu’s
reagent(v/v)andneutralizedby2.0mlof7.5%sodiumcarbonate.The
reactionmixturewasincubatedfor40minat45°Candtheabsorbance
wasmeasuredat765nmin thespectrophotometer.Thetotal phenol
content was subsequently calculated as gallic acid equivalent.
2.7 | Total flavonoid determination
The total flavonoid content was determined using the method of
Meda, L amien, Romito, Millogo, and Nacou lma (2005). In brief, 0. 5 ml
of appropriately diluted sample was mixed with 0.5 ml methanol,
50 μl10%AlCl3, 50 μl1MPotassiumacetateand1.4mlwater,and
allowed to incubate at room temperature for 30 min. The absorb-
anceofthereactionmixturewasmeasuredat415nminthe spec-
trophotometer and the total flavonoid content was subsequently
calculated as quercetin equivalent.
2.8 | Vitamin C content determination
Vitamin C content of the extracts was determined using the
method of Benderitter, Maupoil, VeBriot, and Rochet te (1998).
In brief, 75 μl DNPH (2 g DNPH, 230 mg thiourea and 270 mg
CuSO4.5H2O in 100 ml of 5 M H2SO4) were added to 500 μl reac-
tion mixture (30 0 μl of appropriate dilution of the extracts with
100 μl13.3% TCAand water). This wassubsequently incubated
for 3hr at 37°C , then 0.5m l of 65%H2SO4 (v/v) was added to
the mixture and the absorbance was measured at 520 nm. The
vitamin C content was subsequently calculated as ascorbic acid
equivalent(AAE).
2.9 | Tannin content determination
The tannin content was determined according to the method of
Makkar and Goodchild (1996). In brief, 0.2g of the sample was
weighed int o 50ml sample bot tle and 10ml of 70% aq ueous ac-
etone was added and properly covered. The bottle was shaken for
2hrat30°Cand the solutionwas centrifuged at1,000×g before
the supernatant collected was stored in ice. Thereafter, 0.2 ml of
the solution was mixed with 0.8 ml of distilled water and 0.5 ml
of Folin- ciocalteu reagent was added (the same amount of Folin
reagent was added to 1 ml of 0.5 mg/ml standard tannic acid so-
lution). Then,2.5ml of 20%Na2CO3 was added and the solutions
vortexedandallowedtoincubatefor40minatroomtemperature.
The absorbance of the reaction mixture was measured at 725 nm
in a spectrophotometer. The tannin content was calculated as an
equivalent of tannic acid.
2.10 | Saponin content determination
The saponin content was determined using the method of Brunner
(1994).Inbrief,2gofthepowderedsamplewasweighedintoa250ml
beaker and 100 ml of isobutyl alcohol was added. The mixture was
shaken for 5 hr and the mix ture was filtered into 100 ml beaker con-
taining 20mlof 40% saturated magnesium carbonate (MgCO3) solu-
tion. The mixture obtained was also filtered to obtain a colorless clear
solution. Then, 1 ml of the colorless solution was pipetted into 50 ml
volumetricflask and 2ml of 5% iron (III)chloride(FeCl3) solution was
added and made up to the mark with distilled water (1 ml of 0.2 mg/
ml saponin solution was used as control). The mixture was incubated
at room temperature for 30 min and the absorbance was measured
at 380 nm in a spectrophotometer. The saponin content was subse-
quently expressed as standard saponin equivalent.
2.11 | Phytate content determination
The phytate content was determined according to Day and
Underwood(1986).Inbrief,4gofthepowderedsamplewassoaked
in100ml of 2% HClfor 3hr andfilteredthroughNo. 1 Whatman
filter paper.Thereafter,5ml of 0.3%ammoniumthiocyanate solu-
tion was added to 25 ml of the filtrate as indicator. In a subsequent
way, 53.5 ml of distilled water was added to give it the proper acidity
and this was titrated against iron (III) chloride solution that contained
1.95 mg of iron per milliliter until a brownish yellow color persisted
for 5 min. The phytate content was subsequently calculated using
the titer value obtained.
2.12 | Oxalate content determination
The oxalate content was determined using the method of Day
and Unde rwood (1986). In brief, 1g of the pow dered sampl e was
soaked in 75 ml of 1.5 N H2SO4 for 1 hr and filtered through a No.
1 Whatman filter paper. Then, 25 ml of the filtrate was titrated hot
(about 80–90°C) against 0.1MKMnO4 until a pink color persisted
for 15 s. The oxalate content was subsequently calculated using the
titer value obtained.
2.13 | Alkaloid content determination
The alkaloid content was determined according to the method
of Harbone (1973). Five grams of the powdered sample was
weighed an d 200ml of 10% acetic a cid in ethanol wa s added,
and the reaction mixture was incubated at room temperature for
4min.ThiswasfilteredusingNo.1Whatmanfilterpaperandthe
filtrate obtained was concentrated on a water bath to a quarter
of the original volume. Concentrated NH4OH (20 mM) was added
in drops to the concentrated filtrate until precipitation was com-
pleted. The whole solution was allowed to settle and the pre-
cipitate was collected, washed with dilute NH4OH (250 mM) and
filtered. The recovered residue was weighed and quantified as
the alkaloid.
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ADEMILUYI Et AL.
2.14 | Cardenolides content determination
The cardenolides content was determined according to the method
of Dantas-b arros, Foulquie r, Cosson, an d Jacquin-dubre uil (1993).
In brief, 0.5 g of finely powdered sample was weighed and 50 ml of
chloroformwas added.Thereafter,10mlof2%Na2CO3 was added
to remove any free acid. The reaction mixture was later transferred
into a 250 ml separating funnel and shaken thoroughly to allow the
layers to separate with five drops of acetic anhydride being added.
This mixture was filtered into 100 ml volumetric flask and made to
the mark with chloroform. The absorbance was measured at 510 nm
in a spectrophotometer. Standard cardenolide solution (10 mg/ml)
was used as control and the cardenolide content was subsequently
calculated as the equivalent of the standard solution.
2.15 | Cardiac content determination
The card iac glycoside conte nt was determined a ccording to Sofowora
(1993). One gram of the powdered sample was weighed, 50 ml chlo-
roform was added and vortexed for 1 hr. The reaction mixture was
filtered, followed by the addition of 10 ml of pyridine and 2 ml of
29%sodium nitroprusside and themixturewasshaken thoroughly
for 10min. The reafter, 3ml of 20% NaO H was added in or der to
develop a brownish color and the absorbance was read at 510 nm
in a spectrophotometer. Standard cardiac glycosides (Digitoxin) so-
lution (5 mg/ml) was used as the control and the cardiac glycoside
content was subsequently calculated as the equivalent of the stand-
ard solution.
2.16 | Antioxidant assays
Free radical scavenging ability was determined by assessing the abil-
ity of the moringa leaf extracts to bleach stable DPPH radical as re-
portedby Gyamfi, Yonamine,and Aniya (1999). Tocorrectfor the
limitations of DPPH assay (color interference and sample solubility),
the radi cal scavengi ng ability was f urther te sted using ABT S. The
assayprincipleisbasedonscavengingofABTS•+ formed from treat-
mentofABTS withsodium persulfate.This radical cation(ABTS•+)
isblueincolorandabsorbslightat734nm.ThebluecoloredABTS
radical is converted back to it s original colorless neutral form by anti-
oxidant molecules and the extent of bleaching is measured as trolox
equivalent antioxidant capacity (Re et al., 1999). The reducing prop-
erty(FRAP)oftheextractswasdeterminedbyassessingtheabilityof
the extract s to reduce Fe3+ to Fe2+ in solution as described by Oyaizu
(1986).Thehydroxylradical (•OH) scavenging assay was based on
the ability of the ex tracts to scavenge/prevent •OH production from
Fe2+/H2O2- induced decomposition of deoxyribose in solution. The
Fe2+ chelating ability of the extracts was determined using a modi-
fiedmethodofMinottiandAust(1987)withaslightmodificationby
Puntel, Nogueira, and Rocha (2005). Fur thermore, the ability of the
extracts to prevent both FeSO4 and sodium nitroprusside- induced
lipid peroxidation in rat’s pancreas and liver homogenates was stud-
ied (Ohkawa, Ohishi, & Yagi, 1979).
2.17 | α- Amylase inhibition assay
The α- amylase inhibitory activity was determined as described by
Worthington and Worthington (1993). Appropriate concentration
of the extracts and 50 μl of 20 mM sodium phosphate buffer (pH
6.9 with 6mM Na Cl) containin g pancreatic α- amylase (EC 3.2.1.1)
(0.5mg/ml)were incubated at25°C for10min. Then,50μl of 1%
starch solution (prepared in the same buffer) was added and reac-
tion mix ture was incub ated at 25°C for 10min. Th ereafter 20 0μl
ofdinitrosalicylicacid(DNSA)wasaddedandthereaction stopped
by incubating in a boiling water bath for 5 min. This was later cooled
to room temperature and diluted with 2 ml of distilled water, and
absorbancemeasuredat 540nm. The α- amylase inhibitory activity
wasexpressedaspercentage(%)inhibition.
2.18 | α- Glucosidase inhibition assay
The α- glucosidase inhibitory activity was determined as described
byApostolidis, Kwon,andShetty (2007). Appropriateconcentration
of the extracts and 100 μl of α- glucosidase (EC 3.2.1.20) solution in
100mMphosphatebuffer(pH6.9)wereincubatedat25°Cfor10min.
Thereafter, 50 μl of 5 mM p- nitrophenyl- α- d- glucopyranoside solution
(inthesamebuffer)wasadded.Themixturewasincubatedat25°Cfor
5min, before reading theabsorbanceat 405nm. The α- glucosidase
inhibitoryactivitywasexpressedaspercentage(%)inhibition.
2.19 | HPLC- DAD analysis
High-performance liquid chromatography (HPLC-DAD) was per-
formedwith aShimadzuProminenceAutoSampler(SIL-20A)HPLC
system (Shimadzu,Kyoto,Japan) andequipped with Shimadzu LC-
20ATreciprocatingpumpsconnectedtoaDGU20A5degasserwith
aCBM20Aintegrator,SPD-M20AdiodearraydetectorandLCsolu-
tion 1.22 SP1 soft ware. The quantif ication of phenolic compounds in
the differently dried moringa leaf was carried out using the method
described by Waczuk et al. (2015) with slight modifications. Reverse
phase chromatographic analyses were carried out under gradient
conditions using C18column (4.6mm×150mm)packed with5μm
diameter particles; themobile phases A andBwere Milli-Q water,
acidifi ed to pH 2.0 with 1% of pho sphoric acid an d methanol re-
spect ively, solvent grad ient was used as foll ows: 0–10min, 5% B;
10–25min,15%B;25–40min,30%;40–55min50%B;50–65min
70%B;65–80min,100%B,respectively.Theflowratewas0.6ml/
min and the injection volume was 50 μl.Quantifications werecar-
ried out by integration of the peaks using the external standard
method,at 254nmforgallicandellagicacids;280nmforcatechin
andepicatechin;327nm forchlorogenicandcaffeicacids;and366
for quercetin, kaempferol, and rutin.
2.20 | Statistical analysis
The results of the three replicate experiment s were pooled and
expressed as mean ± standard deviation (SD). A one-way analysis
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ADEMILUY I Et AL.
of variance was used to analyze the mean, and the post hoc treat-
ment was performed using Duncan multiple test. Significance was
accepted at p≤0.05.
3 | RESULTS
The effects of some drying methods on phytochemical composi-
tion of M. oleifera leaf revealed variations in their constituents. The
results of phenolics, flavonoid, vitamin C, tannin, saponin, phytate,
oxalate, alkaloid, cardenolides and cardiac glycosides content in the
differently dried moringa leaf are presented in Table 1. The result
revealed that freeze- dried sample had significantly (p < 0.05) the
highest composition of phytochemicals while oven- dried sample had
the lowest. However, irrespective of the drying method, phenolics,
flavonoids, vitamin C, and phytate were the most abundant while
tannin was the least abundant phytoconstituents in the moringa leaf.
The effect of the various dr ying methods on the DPPH free
radical scavenging abilit y of the moringa leaf was studied and the
result is presented in Table 2. The result revealed that aqueous
extracts scavenged DPPH radicals in a concentration- dependent
(0–330 mg/ml) pattern. However, drying altered the DPPH radical
TABLE1 Effects of drying on the phy tochemical constituents of Moringa oleifera leaf
Parameters Freeze- dried (mg/g) Air- dried (mg/g) Sun dried (mg/g) Oven dried (mg/g)
Phenolics 68.75±0.0 0d59.38±0.42c50.00 ± 0.00ab 46.88±1.42a
Flavonoid 62.50±0.89d58.33 ± 0.00cd 45.83±0.89b25.00 ± 0.00a
Vitamin C 52.94±0.31d41.17±0.31c35.29±0.63bc 23.53±0.60a
Tannin 0.06±0.03 0.05 ± 0.02 0.05 ± 0.03 0.05 ± 0.03
Phytate 70.26±2.40c89.82 ± 0.98d60.98±0.00ab 58.50±1.42a
Saponin 16.36±0.92c16.36±0.00c10.91 ± 0.82b7.27 ± 0.71a
Alkaloid 12.8 ± 1.71c13.4±0.00c5.00 ± 0.92a10.6±2.41b
Oxalate 9.96±0.84c9.09 ± 0.72c6.66±0.00a8.19±0.60b
Cardenolides 13.68±0.71b11.72 ± 1.90b12.53±2.40b8.17 ± 1.71a
Cardiac glycosides 17.36±1.31b16.72±1.91b14.79±2.81a14.79±1.82a
Note. Values represent mean ± standard deviation of triplicate experiments. Superscripts with different alphabets along the same row are significantly
(p < 0.05) different.
Parameters Freeze- dried Air- dried Sun dried Oven dried
Antioxidantproperties
DPPH (mg/ml)a251.42±1.03a275.88±0.14b337.91 ± 0.02d310.56±0.03c
ABTS•+ (mmol
TEAC/g)
1.25 ± 0.05b1.14±0.11ab 1.02 ± 0.00a0.98 ± 0.00a
FRAP(mg
AAE/g)
12.66±0.46c11.04±0.91bc 9.42±0.46b7.47±0.46a
•OH (m g/m l) a61.25±0.03a80.36±0.14b94.31±0.28c116.18±1.41d
Fe2+ Chelation
(mg /ml)a
73.14±0.03a85.39±0.04b142.75±0.03c197.89 ± 0.01d
IC50 for inhibition of Fe2+- induced lipid peroxidation (mg/ml)
Pancreas 26.14±1.86a31.92±0.14b32.52 ± 0. 28c35.09±0.06d
Liver 32.03±1.26a35.43±1.41c34.04±0.06b38.92 ± 0.03d
IC50 for inhibition of sodium nitroprusside- induced lipid peroxidation (mg/ml)
Pancreas 37.41±0.72a38.51 ± 0.02b44.26±0.06d42.53±0.02c
Liver 36.20±0.12a39.53 ± 0.28b40.68±0.02c42.23±0.04d
IC50 for enzymes inhibition (mg/ml)
α- A m y l a s e 64.29±0.52a73.47±0.81c69.90±0.14b81.82 ± 0.03d
α-Glucosidase 38.12 ± 0.71a42.52±0.14b46.16±0.09c51.27 ± 0.10d
Notes. Values represent mean ± standard deviation of triplicate experiments. Superscripts with differ-
ent alphabets along the same row are significantly (p < 0.05) different.
aRepresent the IC50values(theamountoftheextractscausing50%antioxidantorenzymeinhibitory
activity).
TABLE2 Effects of drying on the
DPPH and •OHscavengingability,ABTS•+
scavenging, and ferric reducing
antioxidantproperties(FRAP),Fe2+ -
chelating, inhibition of lipid peroxidation,
α- amylase, and α- glucosidase activities of
Moringa oleifera leaf
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ADEMILUYI Et AL.
scavenging ability of the leaves as freeze- dried sample had signifi-
cantly ( p < 0.05) the highest radical scavenging ability (251 mg/
ml). The effect of the drying methods on the antioxidant capacit y
of the moringa leaf was further studied using a moderately stable
nitrogen-centeredradicalspecie,ABTS•+; as presented in Table 2.
The result also showed that the various dr ying methods signifi-
cantly ( p < 0.05) altered the antioxidant capacity of the moringa
leaf in a manner similar to the DPPH radical scavenging ability.
This is evident by the fact that freeze- dried leaf with the highest
DPPHscavengingabilityalsohad the highest ABTS•+ scavenging
ability(1.25±0.05mmol TEAC/g).Furthermore,theferricreduc-
ing antioxidant property of the Moringa leaves was also studied
and expr essed as AAEs ( Table2). The fr eeze-dried leaf ha d the
highest (12.66±0.46mg AAE/g)reducingpowerwhilethe oven-
driedleafhadtheleast(7.47±0.46mgAAE/g).The effectofthe
drying methods on hydroxyl (OH*) radical scavenging ability of
the aqueous extracts of moringa leaf is also presented in Table 2.
The result showed that freeze- dried leaves had the highest •OH
scavengingability(61.25mg/ml) whileoven-driedleaves had the
least(116.18mg/ml). Moreover, the effect of the drying methods
on Fe2+ chelating ability of the moringa leaf was investigated and
the result is presented in Table 2. The result also revealed that
extracts of the differently dried Moringa leaves chelate Fe2+ in a
concentration- dependent (0–100 mg/ml) pattern. However, these
drying methods significantly (p < 0.05) altered the Fe2+ chelating
proper ty of the leaves; with freeze- dried leaf having the highest
Fe2+chelating ability(73.14mg/ml) while oven- dried leaf had the
least (197.89 mg/ml).
The results of lipid peroxidation presented in Table 2 as IC50 val-
ues showed that all the variously dried moringa leaf extracts inhibited
MDAproduction in both the pancreasand liver in a concentration-
(0–63mg/ml) dependent manner.However, drying methods signifi-
cantly (p<0.05)alteredthisproperty.Also,theresultsasrevealedin
Table 2 (IC50) showed that all the extracts of the differently dried mo-
ringa leaf exhibited α- amylase and α- glucosidase inhibitory properties
in a concentration- (0–80 mg/ml) dependent manner. Nevertheless,
significant (p < 0.05) alteration in the inhibition of these enzymes was
observed as affected by the different drying methods.
The result of some drying methods on phenolics and flavonoids
composition of the extracts of dried moringa leaf is presented in
Table 3. This revealed that gallic acid, catechin, epicatechin, chlo-
rogenic acid, caffeic acid, ellagic acid, quercetin, rutin, and kaemp-
ferol were the predominant phenolics constituents of the moringa
leaf. The result showed that there was variation in the phenolics
and flavonoids content of the extracts of dried moringa leaf sample.
However, gallic acid, chlorogenic acid, caffeic acid, and rutin were
the most abundant in phenolics and flavonoids composition of the
differently dried moringa leaf with freeze- dried sample having the
highest chlorogenic acid, caffeic acid and rutin constituents.
4 | DISCUSSION
Sun drying and air drying at room temperature are the most common
practices used in many parts of the world to preserve vegetables
for dry season consumption while freeze- dr ying and oven drying are
rarely used (Lyimo, Nyagwegwe, & Mukeni, 1991). However, these
processi ng techniques may sign ificantly affect the co ncentration and
bioavailability of some essential constituent s of the food. The phyto-
chemical analysis of the moringa leaf not only showed the presence
of phenolics, flavonoids, vitamin C, tannin, saponin, phytate, oxalate,
alkaloid, cardenolides and cardiac glycosides but also revealed a var-
iation in their concentration af ter being subjected to different drying
processes. Furthermore, drying significantly (p < 0.05) altered the
phytochemical constituents of the moringa leaf with the exception
of tannin content which was not altered by drying. The presence of
these phy tochemicals in the moringa leaf may contribute to its me-
dicinalvalue(Bakare,Magbagbeola,Akinwande,&Okunowo,2010;
Okwu & Morah, 20 07).
Phenolics are one of the most ef fective antioxidant constituents
of green leafy vegetables and studies have shown that the antiox-
idant properties of plant foods are directly proportional to their
TABLE3 Effects of drying on the phenolic constituents of Moringa oleifera leaf
Compounds
Freeze- dried Air- dried Sun dried Oven dried LOD LOQ
mg /g μg/m l
Gallicacid 43.19±0.02a58.35 ± 0.01b41.06±0.01a60.11±0.03bc 0.027 0.093
Catechin 6.08±0.01a7.13 ± 0.03a5.98 ± 0.01a29.76±0.01b0.009 0.034
Chlorogenic acid 79.53 ± 0.01c63.19±0.03a62.35±0.04a65.83±0.01b0.011 0.037
Caffeic acid 78.91 ± 0.03c62.81±0.02ab 78 .17 ± 0.03c58.72 ± 0.02a0.024 0.080
Ellagic acid 5.86±0.01a31.04±0.01c6.09±0.01a19.65±0.02b0.008 0.025
Epicatechin 43.37±0.04c27.76±0.01b18.63±0.01a28.95 ± 0.01b0.019 0.063
Rutin 91.05 ± 0.01c75.38±0.04b89.14±0.02c70.21 ± 0.03a0.023 0.076
Quercetin 17.83 ± 0.01a59.01 ± 0.01b62.17±0.03c19.87 ± 0.03a0.015 0.048
Kaempferol 43.90±0.02d40.11±0.01c9.58 ± 0.01a19.65±0.02b0.021 0.069
Note. Values represent mean ± standard deviation of triplic ate experiment s. Means followed by different letters along the same row are significantly
(p < 0.05) different.
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7
ADEMILUY I Et AL.
phenolic content (Chu, Sun, Wu, & Liu, 2002). Hence, the effect of
the different dr ying methods on the phenolic contents of the mor-
inga leaf was studied. The study revealed that the freeze- dried sam-
ple had the highest phenolic content while oven- dried sample had
the least. This is consistent with earlier repor t that thermal treat-
ments negatively impact the phenolic content of vegetables with a
concomitant reduction in their antioxidant activity (Ismail, Marjan,
&Foong,2004).Hence,theobserveddecreaseinthephenoliccon-
tents of the moringa leaf exposed to heat processes such as oven
and sun drying could be due to heat- induced degradation of phe-
nolic compounds (Oboh, Akinyemi, Ademiluyi, & Adefegha, 2010).
Furthermore, the flavonoid content of the differently dried moringa
leaf followed same trend with the phenolics. The observed high
flavonoid content of the freeze- dried leaf may be due to the fact
that some effective volatile compounds might have been destroyed
during the other dr ying processes employed. It has been reported
that heat treatment results in the degradation of flavonoids in veg-
etables(Mohd-Zainol,Abdul-Hamid,Abu-Bakar,&Pak-Dek,2009).
This might be responsible for the significant decrease (p < 0.05) ob-
served in the flavonoid content of the oven- dried leaf. Flavonoids
have been reported as one of the most numerous and widely
spreadgroupofphenolicsinhigherplants(Carini,Adlini,Furlanetto,
Stefani, & Facino, 2001; Schinella, Tournier, Prieto, Mordujovich de
Buschiazzo, & Rios, 2002). Consumption of flavonoid- rich foods has
been suggested to be effective in reducing the risk of coronary heart
diseases and other related diseases (Ok wu & Omodamiro, 20 05).
Ascorbicacid (vitamin C)has beendescribed asanantioxidant
found in plant foods that helps build up the body’s defenses against
freeradicals(Ajiboye,2009).Itisagoodreducingagentand exerts
its anti oxidant acti vities by elec tron donatio n (Oboh, 200 8). Also,
higher retention of vit amin C was observed in the freeze- dried sam-
ple, this may be due to the fact that the leaves were not exposed to
direct heat and air as vitamin C is rapidly oxidized on exposure to
heat and air. This suggest s that freeze- drying has an edge over sun
drying and oven drying in it s preservation of the vitamin C content
which could serve as a good dietary supplement for ascorbic acid.
The vitamin C content of the moringa leaf was higher than that of
some common green leafy vegetables consumed in Nigeria (Oboh,
Akindahunsi, &Oshodi, 2002; Yadav & Sehgal, 1997). Tanninsare
phytochemical compounds of sufficiently high molecular weight
containing sufficient hydrox yl and carboxyl groups which form ef-
fectively strong complexes with protein and other macromolecules.
They are well- known antioxidants in medicinal plants, foods, and
fruits with multifunctional properties beneficial to human health.
The tannin content was found to be very low and there was no sig-
nificant (p < 0.05) difference obser ved (Table 1) in all the samples
after treatment under the various dr ying methods. This may prob-
ably be due to little influence of drying methods on tannin content
(Akan ji, Ologhobo, Em iola, Adedej i, & Adedeji, 20 03). Kumari and
Jain(2012)reportedthattanninsareusuallypresentinlowamounts
in plants. It has been reported that tannins have antioxidative and
antidiabetic effects and this may be due to their binding ability with
physiologically relevant protein and carbohydrates, which results in
reduction in the bioavailability of carbohydrate and its digestive en-
zymes (i.e., α- amylase and α- glucosidase) (Kunyanga, Okoth, Imungi,
& Vellingiri, 2011). In addition, they also inhibit insulin degradation
and improve glucose utilization and may be relevant in the manage-
ment of diabetes (Kumar i & Jain, 2012). Further more, the tannin
content of the moringa leaf falls below the reported critical value of
7.3–9.0mg/gwhichcouldelicittoxicity(Aletor,1993).
Phytate is the primary storage form of phosphorus and inositols
in seeds, grains, a few tubers and fruits. The air- dried sample had
the highest phytate concentration while oven- dried sample had the
least. This may be attributed to the fact that heat reduces phytate
content in plant foods as a result of leaching which might affect
their extractabilit y. Oboh et al. (2002) reported that food process-
ing techniques such as thermal processing reduce phytate content in
plant foods. It has also been reported that phyt ate inhibits α- amylase
and this may prove useful in the management of hyperglycemia (Lee,
Park,Chung,&Cho,2006).Furthermore,theinteractionofphytate
with starch and divalent metals has been reported to result in low
glycemic index and also reduces the participation of iron in metal-
induced oxidative stress related with diabetes (Schlemmer, Frolich,
Prieto, & Grases, 20 09). Saponins are plant glycosides that form
soapy lathers when mixed or agitated with water, used in deter-
gents, fo aming agents, an d emulsifiers ( Attia-Ismail , 2015). These
groups of compounds are extremely diverse in biological activities
which are mostly related to their structure and sources. Saponins
significantly af fect feed intake and growth in animals (Das et al.,
2012) which might be related to its effect on protein digestibility
(Potter, Jimenez-Flores, Pollack, Lones, & Berber-Jimenez, 1993).
The values obtained for saponin content were higher than those re-
ported by Mbah, Ogbusu, and Eme (2012). However, as observed
from this study, both freeze- drying and air drying did not alter the
saponin content of the leaves, while both sun drying and oven drying
significantly (p < 0.05) reduced the saponin content. This observed
reduction in the saponin content may be attributed to heat- induced
degeneration involved in both dr ying processes. In like manner, this
finding was consistent with the findings of Mbah et al. (2012) where
both sun dr ying and oven drying significantly reduced the saponin
contents of Moringa leaves.
Alkaloids are the largest naturally occurring secondary sub-
stances with one or more nitrogen atom(s) in a heterocyclic ring.
They are widely employed in medicine because of their physiolog-
ical activities on humans and other animals (Imohiosen, Gurama,
&Lamidi, 2014). The value obtained for alkaloidin thisstudywas
higher thanthe 4.28mg/greportedbyOladeji, Taiwo,Gbadamosi,
Oladeji, and Ishola (2017) for M. oleifera leaf. This content of alkaloid
could be responsible for the slight bitter taste observed in the leaf.
However, this study shows that sun drying and oven drying reduced
the alkal oid content of the leaf. Oxalate occurs naturally in plants and
it is synthesized via incomplete oxidation of carbohydrate. The sun
dried leaf sample had the lowest oxalate content while the freeze-
dried sample had the highest content. The obser ved high oxalate
content in the freeze- dried compared to other samples processed
differently, could be due to the effect of heat on oxalate stability.
8
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ADEMILUYI Et AL.
In general, significant (p < 0.05) decrease in phytoconstituents
was obser ved for both sun and oven- dried samples; this may be at-
tributed to the effect of UV- radiation, speed and humidity of the
wind, as well as high temperatures involved in sun drying and moist
heat in oven- drying process. This is consistent with earlier findings
of Shilpi, Sabrina, and Nissreen (2011) where a decrease in phyto-
chemical content of edible Irish brown seaweed as temperature in-
crease (upon drying) was reported. Hence, the variations obser ved
in the phytochemical composition of the moringa leaf might be due
to the different heat- induced chemical modifications that took place
during the drying process.
DPPH is a relatively more stable nitrogen- centered free radical
donor that accepts an electron or hydrogen upon reduction to be-
comeastablediamagneticmolecule.Acompoundwithtendencyto
perform this reaction is an important factor in evaluating antioxidant
activity and therefore a free radical scavenger (Hu, Zhang, & Kitts,
2000). The trend obtained in this study agreed with the phytochem-
ical distribution in the moringa leaf and it is consistent with earlier
studies(Amic&Davidovic-Amic,2003;Chuetal.,2002).Moreover,
the ability of the aqueous ex tracts of the leaf to reduce Fe3+ to Fe2+
was also studied. This study revealed that drying process signifi-
cantly ( p < 0.05) affec ted the reducing power of the moringa leaf.
The pattern of alteration by the drying method is also similar to that
ofthe ABTS•+ scavenging ability with freeze- dried leaf having the
highest reducing power and the oven- dried leaf having the least. The
observed high reducing power of the freeze- dried leaf as compared
with others may be due to its high phytoconstituents. Reducing
agents are potent terminators of oxidation process, and two mech-
anisms available for this property include (a) electron transfer and
(b) hydrogen atom t ransfer (Rice-Evans, Miller, & Pagang a, 1996).
Likewise, the ability of the drying methods to alter the inhibition of
hydroxyl radical (•OH) produced from the degradation of deoxyri-
bose through the Fenton reaction by moringa leaf ex tracts was stud-
ied. This study revealed that drying significantly (p < 0.05) altered
the ability of moringa leaf to prevent •OH production in vitro. The
freeze- dried leaves had the highest •OH scavenging ability while
oven- dried leaves had the least. The trend in this result agreed with
the effect of various drying methods on the phytochemical contents
andantioxidantactivities(ABTS•+, DPPH and •OH scavenging abil-
ities and reducing power) of the moringa leaf earlier discussed. Iron
(Fe) is necessary for a lot of biochemical and physiological functions
in biological systems; however, free cytosolic and mitochondria Fe
can induce oxidative damage through its participation in reactions
leading to ROS production (Oboh & Rocha, 2007); this gives cre-
dence to the use of iron chelators as therapy for iron overload. The
in vitro total antioxidant analysis revealed that highest antioxidant
activity is found in freeze- dried moringa leaf with least in the oven-
dried leaf. This agreed with the phy tochemical distribution in the
leaf and is consistent with earlier studies where strong correlation
existed between phytochemical contents and antioxidant properties
of some plant foods (Chu et al., 2002; Hu et al., 2000).
Furthermore, peroxidation of membrane lipids and other macro-
molecules to give rise to reactive aldehydes and other electrophiles
leading to cell and tissue damage has been known as hallmark of oxi-
dative stress; hence determination and quantification of the reactive
aldehydes(suchas malondialdehyde,MDA) andotherelectrophiles
formed has been used as a measure of oxidative damage in a biolog-
ical systemandalso theextent of lipidperoxidation. Ability ofveg-
etable a nd plant extr acts to inh ibit the MDA produ ction has bee n
used as an indication of their antioxidant power in a biological system.
Hence, it is desirable to investigate the ef fect of the various drying
methods on the ability of the dried moringa leaf ex trac ts to prevent
both Fe2+andsodiumnitroprusside-inducedMDAproductioninrat
pancreas and liver homogenates in vitro. The pattern of alteration
of lipid peroxidation is similar to the antioxidant test carried out in
that, freeze- dried moringa leaf exhibited the highest inhibitory effect
and oven- dried leaf had the least. This also agreed with the result
obtained for phytoconstituents as affected by the drying methods.
Hence, it is safe to suggest that alteration in the phytoconstituents
as affected by drying methods could be principal modulator of the
antioxidant and possibly the biological proper ties of moringa leaf; as
previous studies have come to conclusion that antioxidant proper ties
of plant food are directly proportional to its phytoconstituents.
Pancreatic α- amylase catalyzes the breakdown of starch (poly-
saccharide) into disaccharides and oligosaccharides while intestinal
α- glucosidase is responsible for the breakdown of disaccharides to
glucose which is absorbed from small intestine into the blood cir-
culation. Inhibition of these enzymes has been adopted as dietar y
means for the control of postprandial hyperglycemia in diabetics.
Reports have shown the use of moringa leaf in the management of
diabetes(Jaiswal,Rai,Kumar,Mehta,&Watal,2009),withitseffect
linked to the inhibition of carbohydrate hydrolyzing enzymes such
α- amylase and α- glucosidase (Toma, Makonnen, Mekonnen, Debella,
&Addisakwattana, 2014). However,thereisdearth of information
on how drying affects these properties. Hence, the effect of dr ying
methods on the α- amylase and α- glucosidase ac tivities was inves-
tigated. The pattern of alteration of α- amylase and α- glucosidase
inhibitory properties is similar to the ones observed for the anti-
oxidant studies, this suggests that drying- induced alteration to the
phytoconstituents and this may also be responsible for the observed
enzyme inhibition pattern. Phytochemicals have been shown to be
responsible for the antidiabetic effect of many medicinal and plant
foods through their interac tion with critical enzymes involved in
carbohydratemetabolisms(Ademiluyi&Oboh,2012).Thisfunction
has been at tributed majorly to the phenolic compounds ( Toma et al.,
2014)andrecently,tosomealkaloids(Tiongetal.,2013).Hence,fac-
tors that negatively affect the amounts and bioavailability of these
compounds may significantly impact their antidiabetic properties.
Moringa leaf like many other green leafy vegetables contains
phytochemicals such as phenolics which have been attributed for
their health- promoting effect. However, repor ts are on the increase
about the effect of processing on the content and bioavailability of
this important class of plant phytochemicals in green leafy vegeta-
bles. Hence, to ascer tain the effect of the various dr ying methods
onthephenolicconstituentsofthetreatedmoringaleaf,HPLC-DAD
was employed. However, the effect of the drying methods on the
|
9
ADEMILUY I Et AL.
phenolic constituents of the leaf did not totally agree with the anti-
oxidant and enzyme inhibitory effect as the observed earlier trend
was not totally followed. However, the phenolics and flavonoids
constituents were significantly altered by the different drying meth-
ods used in this study. Chlorogenic acid, gallic acids, caffeic acid and
rutin had the highest peaks; these were the most abundant pheno-
lics and flavonoids composition in the dried moringa leaf. The freeze-
dried sample had the highest chlorogenic acid, caffeic acid and
rutin constituents; this is consistent with earlier findings whereby
freeze- dried moringa leaf had highest phenolics content. The high-
est phenolics composition (Figure 1a) in chlorogenic acid, caf feic
acid and rutin compounds may contribute to the highest α- amylase
and α- glucosidase inhibition, prevention of lipid peroxidation and
antioxidant proper ties obtained in the freeze- dried moringa leaf.
Chlorogenic acids are formed by the esterification of cinnamic acids,
such as caffeic, ferulic, and p- coumaric acids, with quinic acid. The
consumption of chlorogenic acids has a lot of health benefits (Oboh,
Agunloye,Akinyemi,Ademiluyi, & Adefegha, 2013). Gallicacid is a
trihydrox ybenzoic acid found both in free form and in esterif ied form
as part of hydrolyzable tannins (gallotannins and ellagitannins). It has
antifungal, antiviral, anticarcinogenic, anti- inflammatory, antioxidant
and antidiabetic properties (Shahrzad, Hodgson, & Narumi, 2001).
Caffeic acid has also been reported to increase glucose uptake in rat
myocytes(Cheng & Liu,2000).Quercetin is one of themost com-
mon flavonoids occurring mainly in glycosidic forms such as rutin
(Havsteen, 1983). Rutin exhibits antioxidants, antibacterial, antitu-
mor, anti- inflammatory, antidiarrheal, antiulcer, anticarcinogenic, an-
tidiabetic, antimyocardial protection, vasodilator, immunomodulator
andhepatoprotectiveactivities(Janbaz,Saeed,&Gilani,2002).
In conclusion, this study revealed that drying methods signifi-
cantly altered the phytoconstituents, antioxidant capacity, and en-
zyme inhibitory effect of moringa leaf with freeze- drying being the
most promising method. The observed alterations in these parameters
may be attributed to the fact that increase in temperature observed
in both sun and oven- dr ying processes could have resulted in UV- and
heat- induced destruction of some labile phytoconstituents, while the
time taken for air drying to be completed could have resulted in micro-
bial degradation of some valuable phytoconstituents. Hence, freeze-
drying appeared to be the best method of preservin g the nutraceutical
proper ties of moringa leaf. However, for practical application, the
order of preference of the drying methods which ensures adequate
retention of phytoconstituents and possibly biological activities of
moringa leaf as observed in this study is oven drying < sun drying < air
drying < freeze- drying, in the order of increasing magnitude.
CONFLICT OF INTEREST
The authors declare that they do not have any conflict of interest.
ETHICAL STATEMENT
This study was approved by the Institutional Research Ethical
Commit tee, Federal Uni versity of Technolog y,A kure, Nigeria an d
the handling and use of the animals were in accordance with NIH
Guide.
ORCID
Adedayo O. Ademiluyi http://orcid.org/0000-0001-8325-1304
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How to cite this article:AdemiluyiAO,AladeseluOH,Oboh
G,BoligonAA.Dryingaltersthephenolicconstituents,
antioxidant proper ties, α- amylase, and α- glucosidase
inhibitory properties of Moringa (Moringa oleifera) leaf. Food
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