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Journal of Cell Biology and Biochemistry Research Vol. 4(1), pp. 01 3-019, February, 2020
Available online at http://www.apexjournal.org
ISSN 2315-8870© 2020 Apex Journal International
Full Length Research
Free radical and reactive oxygen species scavenging
potentials of Luffa cylindrica leaf extracts
*Oluremi A. Saliu1, Adewumi M. Akanji2, Oluwafemi A. Idowu3, and Biodun N. Saliu4
1Department of Environmental Health Science, Faculty of Health Sciences, National Open University of Nigeria,
Abuja, Nigeria
2 Department of Biochemistry, Faculty of Life Sciences, University of Ilorin, Ilorin, Kwara State, Nigeria
3Department of Biochemistry, College of Natural and Applied Sciences, Oduduwa University, Ile-Ife, Osun State, Nigeria
4National Universities Commission, Abuja, Nigeria
Accepted 17 February, 2020; Published 20 February, 2020
Free radical and reactive oxygen species scavenging potentials of four solvent extracts of Luffa
cylindrica leaf was evaluated in vitro. Leaves of L. cylindrica were extracted with distilled water
(aqueous), methanol, ethylacetate, and hexane. The aqueous, methanolic and hexane extracts
effectively scavenged 1, 1-diphenyl-2-picrylhydrazyl (DPPH) in dose dependent manner with the
methanolic extract producing the highest DPPH scavenging effect of 96% at a concentration of 50
mg/ml. Also aqueous and methanolic extracts of L. cylindrica leaf showed a dose dependent 2, 2-azino-
bis (3-ethylbenzthiazone-6-sulphonic (ABTS) scavenging activity producing 98% and 99% scavenging
effect at 50 mg/ml respectively when compared with the synthetic antioxidant, butylated hydroxytoluene
(BHT). The four extracts (aqueous, methanolic, ethylacetate and hexane) expressed significant activity
(p < 0.05) for total antioxidant capacity, ferric ion reducing power and as well scavenged hydroxyl ion
radical and superoxide anion reactive oxygen species when compared with ascorbic acid. These results
establish Luffa cylindrica as an effective natural antioxidant agent and therefore accounts for the wide
use of the plant in treating different ailments.
Key words: Reactive oxygen species, Antioxidant, Ascorbic acid, Phenolics, Luffa cylindrica.
Introduction
Oxidative stress results from an imbalance between
reactive oxygen species (ROS) and endogenous
antioxidant system. ROS are molecules with unpaired
oxygen electrons such as superoxide anion (O2
-),
hydroxyl radical (OH-), singlet oxygen (1O2), Ozone (O3)
etc that induce physiological stress in the form of
oxidative stress resulting into aging and in the damage of
cells, tissues, DNA as well as the biological membrane.
Reactive oxygen species are generated endogenously (in
the living system) e.g. oxidative phosphorylation that
takes place in the mitochondrion during normal body
metabolism and exogenously from pollutants, drugs,
*Corresponding author email: remisaliu@yahoo.com
xenobiotics, radiation etc. ROS when generated interact
with other molecules in the body to gain stability by
forming a radical chain which induces oxidative stress
and thus subsequently causes the damage of vital cells
and DNA in the body. ROS have been implicated in the
pathogenesis of several diseases such as cancer
(Kinnula and Crapo, 2004), diabetes (Pietta et al., 1998),
cardiovascular diseases (Singh and Jialal, 2006),
immune deficiency disease (Middleton et al., 2000) and
other related diseases. Antioxidants however, are
molecules capable of preventing the deteriorative effects
caused by the ROS. They act by donating electrons to
ROS in order to terminate their radical chain reactions
and are therefore important in the prevention of human
diseases. Antioxidants may also be beneficial in
improving the wellbeing of life and may have a potential
014 J. Cell. Biol. Biochem. Res
for substantial savings in the cost of health care delivery.
There are natural and synthetic antioxidants, the former
is naturally present in food while the latter are industrially
produced and are mostly used as food additive.
Synthetic antioxidants such as butylated hydroxyl toluene
(BHT), butylated hydroxyl anisole (BHA) etc. are
suspected to be carcinogenic and are reported to have
side effect (Namiki,1990). Natural antioxidants like
ascorbic acid (Vitamin C), Vitamin E (tocopherol),
carotenoids, lycopene, carotene and phenolic
compounds (e.g. flavonoids) present in leafy vegetables,
fruits, leaves, seeds and all parts of plant are safer,
possess anti-cancer, anti-tumor, anti-mutagenic and
hepatoprotective properties (Ajila et al., 2007). In view of
this, there is high interest and demand in the use of
natural antioxidants especially those of plants origin due
to their presumed safety, nutritional and therapeutic value
(Ajila et al., 2007). Researchers have therefore grown
interest in examining plant extracts as a source of
cheaper and effective antioxidants thus Luffa cylindrica.
Luffa cylindrica (L.) Roem commonly called sponge
gourd, loofa, vegetable sponge, bath sponge or dish cloth
gourd, is a member of Cucurbitaceous family well
cultivated in Africa. In Nigeria, the local names of L.
cylindrica are kan-kan oyinbo (Yoruba), Ahia mmala
(Igbo), Ihion osa (Edo) and Sooso (Hausa). L. cylindrica
has great medicinal values, with the leaves reported to
have antiemetic and anti-inflammatory (Khan et al.,
2013), analgelsic (Salman et al., 2013), hepatoprotective
(Sharma et al., 2014), analgesic and antipyretic (Saliu et
al., 2019) activities. In this study, the leaf of the plant was
investigated for its free radical and reactive oxygen
species scavenging potential.
Materials and Methods
Reagents
Butylated hydroxyl toluene (BHT), hydrogen peroxide,
nitroblue tetrazolium (NBT) and α, α-diphenyl-β-
picrylhydrazyl (DPPH) were product of Sigma Chemical
Co., St. Loius, MO. All other reagents used were of
analytical grade and commercially obtained.
Collection of Plant Sample
Fresh leaves of Luffa cylindrica were collected in Suleja,
Niger State, Northern Nigeria. The authentication of the
plant leaves was done at National Institute for
Pharmaceutical Research and Development (NIPRD),
Abuja, Nigeria with voucher number assigned and
deposited in the herbarium of the institute.
Preparation of Leaf Extracts
The leaves of L. cylindrica were air-dried and pulverized
with an electrical blender (Mazeda Mill, MT 4100, Japan).
250 g of the pulverized leaves was macerated in 2.5 liters
of distilled water for 72 hours to give the aqueous extract.
Another 250 g of the pulverized leaves was successively
extracted with 2.5 liters each of hexane, ethyl acetate and
methanol solvents. All the resulting extracts were filtered
with Whatman No. 1 filter paper. The filtrates obtained
were concentrated in a rotary evaporator (RE-300B
model, China) at 65 – 70oC and dried to a constant
weight on a water bath to give a percentage yield of 23%,
13%, 10% and 4% for the aqueous, hexane, ethyl acetate
and methanol extracts respectively.
In vitro Free Radical and Reactive Oxygen
Scavenging Assay
1.1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging assay
1.1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity
of the four extracts (aqueous, etylacetate, n-hexane and
methanolic) were determined by the method described by
Burits and Bucar (2000). Briefly, to 2 ml of different
concentrations (10, 20, 30, 40 and 50 µg/ml) of the
extract, 4 ml of 0.004% methanol solution of DPPH was
added and the mixture was incubated for 30 minutes, the
absorbance was read against a blank containing only
methanol at a wavelength of 517 nm. Ascorbic acid was
used as the reference antioxidant compound. The
experiment was carried out in triplicates. DPPH radical
scavenging activity was calculated as percentage
inhibition (%I) using the expression:
% I = (Acontrol - Asample /Acontrol) x 100
Acontrol = absorbance of the control reaction (containing all
reagents except the test compound).
Asample = absorbance of the test compound, % I =
Percentage Inhibition
Superoxide anion scavenging assay
The method described by Robak and Gryglewski (1980)
was adopted to determine the superoxide anion
scavenging activity of the four extracts. Briefly,
superoxide anion radical was generated in a reaction
mixture containing 0.5 ml of nitroblue tetrazolium (NBT)
(0.3 mM), 0.5 ml NADH (0.936 mM) solution, 500 µl (1
mg/ml) of the extract and 0.5 ml of Tris–HCl buffer (16
mM, pH 8.0). The reaction was initiated by adding 0.5 ml
of phenazine methosulfate (PMS) solution (0.12 mM) to
the mixture and incubated at 25oC for 5 minutes. The
absorbance was measured at 560 nm against a blank
sample. L-ascorbic acid was used as the standard
antioxidant compound. The percentage inhibition of
superoxide generation was calculated from the
expression:
% I = (Acontrol - Asample /Acontrol) x 100
Acontrol = absorbance of the control reaction (containing all
reagents except the test compound).
Asample = absorbance of the test compound.
% I = Percentage Inhibition
Hydroxyl radical scavenging assay
The hydroxyl radical scavenging activity of the four
solvent extracts was determined in a Fenton reaction
(Fe3+-Ascorbic acid- EDTA H2O2 system) adopting the
method described by Kunchandy and Rao (1990). The
reaction mixture (1.0 ml) consisted of 100 µl of 2-
deoxyribose (28 mM in 20 mM KH2PO4-KOH buffer, pH
7.4), 500 µl of the different solvent extracts (1 mg/ml),
200 µl EDTA (1.04 mM) and 200 µl FeCl3 (1:1 v/v), 100 µl
of H2O2 (1.0 mM) and 100 µl ascorbic acid (1.0 mM)
which was incubated at 37oC for 1 hour and then cooled.
After cooling, 1 ml of thiobarbituric acid (1%) and 1.0 ml
of trichloroacetic acid (2.8%) were added and further
incubated for 20 minutes. The mixture was allowed to
cool and absorbance was measured at 532 nm against a
blank sample. Hydroxyl radical scavenging activity was
calculated using the expression:
% I = (Acontrol - Asample /Acontrol) x 100
Acontrol = absorbance of the control reaction (containing all
reagents except the test compound).
Asample = absorbance of the test compound.
% I = Percentage Inhibition
2, 2-azino-bis (3-ethylbenzthiazone-6-sulphonic acid
(ABTS) scavenging assay
The method described by Re et al. (1999) was used to
determine the 2, 2-azino-bis (3-ethylbenzthiazone-6-
sulphonic acid (ABTS) scavenging activity of the extracts.
ABTS•+ was produced by adding 7 mM aqueous stock
solution of ABTS+ to 2.4 mM potassium persulfate which
was allowed to stand in the dark for 12-16 hours at 25oC
(room temperature). The radical was stable in this form
for more than two days when stored in the dark at room
temperature. Prior to analysis, the solution was diluted in
ethanol (about 1:89 v/v) and equilibrated at 30oC to give
an absorbance of 0.7000 ± 0.02 at 734 nm. Then, 2 ml of
diluted ABTS•+ solution was added to 20 μl of the sample
at varying concentrations (10, 20, 30, 40, 50 mg/ml) and
incubated at room temperature. After 30 minutes of
incubation, the absorbance was recorded at 734 nm and
percentage of inhibition was calculated as stated in the
previous section above. Butylated hydroxyl toluene was
used as the reference antioxidant compound. The
Oluremi et al 015
experiment was performed in triplicates.
Ferric reducing power assay
Ferric reducing antioxidant power of the extracts
(aqueous, ethylacetate, n-hexane and methanolic) was
determined by the method described by Oyaizu (1986).
Briefly, 0.5 ml each of the extract and ascorbic acid
(reference antioxidant compound) prepared at
concentration of 5000 µg/ml were dissolved separately
into 0.5 ml phosphate buffer (pH 6.6) and 2.5 ml of 1%
potassium ferricyanide [K3Fe(CN)6] in a test tube. The
mixture was incubated at 50oC for 20 minutes. The
reaction was terminated by the addition of 0.5 ml of
trichloroacetic acid (TCA) and centrifuged at 3000 rpm
(1500 x g) for 10 minutes. 0.5 ml of the supernatant was
mixed with 2.5 ml of distilled water and 0.5 ml ferric
chloride. Absorbance was read at 700 nm. The
experiment was carried out in triplicates. Increased
absorbance of reaction mixture indicates increased
reducing ferric power.
Statistical Analysis
Data were expressed as mean ± standard error of mean.
Analysis of variance (ANOVA) was used to analyze data,
followed by Dunnet’s test for multiple comparisons. p-
values < 0.05 were considered to be statistically
significant.
Results
The phytochemical screening of the four extracts of Luffa
cylindrica leaf is depicted in Table 1. The four extracts
tested positive only to alkaloids while triterpenes, cardaic
glycoside, flavonoids, phenolics, tannins and saponins
were seen to be variably present among the extracts.
The four solvent extracts showed varying 1, 1-diphenyl-
2-picryhydrazyl (DPPH) inhibition. The DPPH scavenging
activities of ascorbic acid used as the reference
antioxidant compound and the extracts (aqueous,
methanolic and ethylacetate) were dose dependent with
the aqueous, methanolic and hexane extracts exhibiting
higher DPPH scavenging activity than ascorbic acid. The
result showed that the hexane extract of L. cylindtica leaf
exhibited the strongest inhibitory activity against DPPH at
all concentrations (10, 20, 30, 40 and 50 mg/ml) while the
ethylacetate extract showed the least inhibitory activity
against DPPH radical at these concentrations (Table 2).
Data are mean determinant of three replicates ± SEM.
(Values with different alphabets down the
column are significantly different).
The aqueous and methanolic extracts among other
extracts investigated scavenged 2, 2-azino-bis-3-
ethylbenzothiazoline-6-sulfonic acid (ABTS) radical in
dose-dependent manner. The two extracts (aqueous and
methanolic) expressed the strongest tendency to
016 J. Cell. Biol. Biochem. Res
Table 1: Phytoconstituents present in solvent extracts of Luffa cylindrica leaf
Secondary Aqueous Mathanolic Ethylacetate Hexane
Metabolites Extract Extract Extract Extract
Inference
Saponins + + - -
Tannins - + - -
Anthraquinones - - - -
Triterpenes - + + +
Phenolics + + - -
Flavonoids - + + -
Alkaloids + + + +
Phlobatannins - - - -
Cardaic Glycosides + + - +
Keys: + = Detected, - = Not detected
Table 2: DPPH scavenging activity of solvent extracts of Luffa cylindrica leaf % Inhibition
Extracts 10 mg/ml 20 mg/ml 30 mg/ml 40 mg/ml 50 mg/ml
Aqueous 73.69 ± 1.02a 77.12 ± 1.04a 78.96 ± 1.05a 79.92 ± 1.02a 79.92 ± 1.02a
Methanolic 57.56 ± 1.01b 67.96 ± 1.02b 80.51 ± 1.01a 87.80 ± 1.01b 96.18 ± 1.01b
Ethylacetate 14.05 ± 1.01c 16.19 ± 1.03c 26.00 ± 1.02b 37.89 ± 1.02c 65.38 ± 1.04c
Hexane 89.60 ± 1.01d 96.36 ± 1.03d 97.37 ± 1.03c 98.80 ± 1.01d 89.24 ± 1.03d
Ascorbic acid 49.51 ± 1.01e 57.26 ± 1.02e 63.01 ± 1.01d 65.33 ± 1.01e 68.75 ± 0.04e
DPPH = 1, 1 diphenyl-2-picryhydrazyl
scavenge ABTS at the concentrations of 10, 20 and 60
mg/ml than butylated hydroxyl toluene (BHT). The
strongest inhibition of ABTS radical was expressed by the
methanolic extract of L. cylindrica leaf. On a contrary, the
ethylacetate and hexane extracts expressed no
scavenging activity against ABTS radical (Table 3).
Data are mean determinates of three replicates ± SEM.
(Values with different alphabets down the column are
significantly different).
The aqueous, methanolic and ethylacetate extracts
exhibited a significant (p < 0.05) higher total antioxidant
capacity (TAC) than the reference antioxidant compound
(ascorbic acid). Among these three extracts, the
methanolic extract had the highest TAC followed by the
aqueous extract which also showed a promising capacity
while the hexane extract showed the least total
antioxidant capacity (Table 4). The four extracts
(aqueous, methanolic, ethylacetate and hexane)
exhibited scavenging activity against ferric ion radical.
The ferric reducing antioxidant power was significantly (p
< 0.05) higher in all the four extracts than as observed
with ascorbic acid. The aqueous extract expressed the
highest ferric reducing antioxidant power while the
methanolic extract showed the least (Table 4). Aqueous
and methanolic extracts showed a significantly (p < 0.05)
lower hydroxyl radical scavenging activity compared with
ascorbic acid. On the other hand, ethylacetate and
hexane extracts showed a significantly (p < 0.05) higher
hydroxyl radical scavenging activity when compared with
ascorbic acid. The ethylacetate extract expressed the
strongest hydroxyl radical scavenging activity (Table 4).
The ability of the extracts to scavenge superoxide ion
was significantly (p < 0.05) higher in methanolic,
ethylacetate and hexane extracts when compared with
the reference antioxidant compound (ascorbic acid). The
aqueous extract showed the least superoxide ion radical
scavenging activity than ascorbic acid and the other three
extracts (methanolic, ethylacetate and hexane) (Table 4).
Data are mean determinates of three replicates ± SEM.
(Values with different alphabets down the column are
significantly different).
Oluremi et al 017
Table 3: ABTS scavenging activity of leaf extracts of Luffa cylindrical % Inhibition
Extracts 10 mg/ml 20 mg/ml 30 mg/ml 40 mg/ml 50 mg/ml
Aqueous 93.73 ± 1.02a 94.68 ± 1.01a 95.04 ± 1.01a 97.31 ± 1.01a 98.86 ± 1.01a
Methanolic 96.30 ± 1.01b 97.31 ± 1.03b 98.87 ± 1.01b 99.44 ± 1.02b 99.70 ± 1.02a
Ethylacetate - - - - -
Hexane - - - -
BHT 61.50 ± 1.02c 76.87 ± 1.02c 90.19 ± 1.03c 96.64 ± 1.01a 98.15 ± 1.01a
BHT = Butylated hydroxyltoluene, ABTS = 2, 2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid
Table 4: In vitro antioxidant activity of leaf extracts of Luffa cylindrica
TAC FRAP OH- activity O2
- activity
Extracts (mg/100g) (µg/100g) (mg/100g) (µg/100g)
Aqueous 143.73±1.41a 498.97±1.27a 4.34±1.02a 19.14±1.28a
Methanolic 166.46±1.29b 376.43±1.30b 22.30±1.10b 120.88±1.26b
Ethylacetate 127.53±1.26c 490.84±1.09c 57.58±1.02c 90.90±1.06c
Hexane 79.01±1.11d 401.88±1.05d 39.85±1.01d 119.31±1.23b
Ascorbic acid 123.73±0.31e 318.27±1.04e 35.54±1.10d 84.75±1.28d
TAC = Total antioxidant capacity, FRAP = Ferric reducing antioxidant power, OH- = Hydroxyl radical,
O2
- = Superoxide radical
Discussion
The variation observed in the secondary metabolites
among the four extracts may be due to the polarity nature
of the solvents used for extraction. The secondary
metabolites reported in this study for the extracts of Luffa
cynidrica are known to be free radical scavenging
compounds. Phenolic compounds for example flavonoids
and phenolics belong to a class of antioxidants that act
as free radical terminators. The antioxidant activity of
phenolic compounds is attributed to their redox property
which makes them reducing agents, hydrogen donors
and singlet oxygen quenchers (Hassan et al., 2009).
Flavonoids have also been reported in several literatures
to possess antioxidant activity (Farounbi et al., 2002) due
to their metal ion chelating ability (Savita et al., 2011).
Therefore their presence in plant or as food additive
could help in preventing diseases caused by the free
radicals. DPPH radical assay is a fast and widely used
assay for assessing the antioxidant property of a
compound. It is a stable radical characterized with a
violet or purple colour which decolorizes in the presence
of an antioxidant and the decrease in absorbance of an
antioxidant agent under investigation for DPPH inhibition
indicates high DPPH radical scavenging activity of the
agent (Krishnaiah et al., 2012). The DPPH inhibition
expressed by the extracts indicate their ability to act as
reducing agents which could have been facilitated by the
secondary metabolites present in the extracts particularly
phenolics and flavonoids acting by donating hydrogen
atom to the radical DPPH (1, 1-diphenyl-1, 2-
picryhydrazyl) and converting it to (DPPH-H+) α, α-
diphenylhydrazyl, a non-radical form. Flavonoids and
phenolics are well documented in literature to be
responsible for the antioxidant activity expressed by most
plants Visioli et al., 1998; Lamson and Brignall, 2005;
Torres et al., 2006; Visioli et al., 1998). In ABTS radical
scavenging assay, a blue/green ABTS+ chromophore is
generated as a radical from the reaction of ABTS with
potassium persulfate and its reduction is measured
spectrophotometrically at 745 nm in the presence of
hydrogen–donating antioxidants. The ABTS radical
inhibition exhibited by the methanolic and aqueous
extracts may be due to the hydroxyl groups present
018 J. Cell. Biol. Biochem. Res
specifically in phenolics rather than flavonoids since the
ethylacetate also contain flavonoids but did not express
any scavenging activity towards ABTS. The use of
butylated hydroxyl toluene (BHT) as a synthetic
antioxidant reference compound in this study is to
investigate if L. cylindrica has the potential to serve as a
better substitutes to this synthetic antioxidant purported
to be carcinogenic in food additives. Mechanism such as
prevention of chain initiation, binding of transitional metal
ion or decomposition of peroxide attributed to phenolics
and flavonoids may be responsible for the total
antioxidant capacity exhibited by the extracts (El-Mastas
et al., 2006). Ferric ion take part in formation of hydroxyl
radical in the Fenton reaction and can also lead to
oxidative damage. Therefore preventing the oxidation
process of ferric ion can help prevent further reactions
that can induce oxidative damage. The reducing power
exhibited by the extracts may be in conjunction with the
phytoconstituents particularly phenolics and flavonoids
present in the methanolic and aqueous extracts which act
as reductants, reducing ferric ion from its radical form i.e.
Fe3+ to a non radical form i.e. ferrous ion (Fe2+). This
observation is in line with Gordon, (1990) who reported
that compound having reducing properties exert
antioxidant action by donating hydrogen atom in order to
break the free radical chain formed by reactive oxygen
species. The hydroxyl radical scavenging activity
exhibited by the extracts implies that the extracts could
prevent damage associated with hydroxyl radical on cells
and biological membranes. Hydroxyl radical is a powerful
reactive oxygen species in the biological system that can
penetrate into membrane and interact with the lipid
moieties of the membrane in induce damage (Halliwell
and Gutteridge, 1981; Khan et al., 2012). Superioxide
anion (O2-) radical serve as the major source of other free
radicals like singlet oxygen (O-), hydroxyl radical (OH-)
and hydrogen peroxide (H2O2) that induce oxidative
stress in the biological system (Pietta 2000). The
superoxide ion scavenging activity exhibited by the
extracts is an indication that Luffa cylindrica leaf is
capable of terminating radical chain reaction that can
further result to production of more deadly reactive
oxygen species that damage lipids, protein and DNA.
Conclusion
This study establishes Luffa cylindrica as an effective
natural antioxidant agent which may accounts for the
wide use of the plant in Africa for treating different
ailments. This antioxidant activity exhibited by L.
cylindrica leaf might be attributed to its phytochemical
constituents particularly the phenolic compounds.
Conflict of interest
The authors declare that there are no conflicts of interest.
Funding
This research did not receive any specific grant from
funding agencies in the public, commercial, or not-for-
profit sectors.
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