INHIBITION OF LIPID PEROXIDATION AND IN-VITRO ANTIOXIDANT CAPACITY OF AQUEOUS, ACETONE AND METHANOL LEAF EXTRACTS OF GREEN AND RED Acalypha wilkesiana Muell Arg.

Abstract

Keywords: Acalypha wilkesiana (A.w.) antioxidant lipid peroxidation phenolic free-radical scavenging.

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ARTICLE INFO ABSTRACT
Keywords:
Acalypha wilkesiana (A.w.)
antioxidant
lipid peroxidation
phenolic
free-radical scavenging.
Original article
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International Journal of Biological & Medical Research
INHIBITION OF LIPID PEROXIDATION AND IN-VITRO ANTIOXIDANT CAPACITY OF
AQUEOUS, ACETONE AND METHANOL LEAF EXTRACTS OF GREEN AND RED
Acalypha wilkesiana Muell Arg.
a b c
MO. DIDUNYEM , BO. ADETUYI , IA. OYEWALE
aDepartment of Chemical Science, Faculty of sciences, Olusegun Agagu University of science and technology (OAUSTECH), Okitipupa, Ondo State, Nigeria.
bDepartment of Biochemistry, Faculty of natural sciences, Joseph Ayo Babalola University (JABU), Ikeji-Arakeji, Osun State, Nigeria.
cDepartment of Chemical Sciences, Faculty of sciences, Olusegun Agagu University of science and technology (OAUSTECH), Okitipupa, Ondo State, Nigeria.
INTRODUCTION:
Plant based approach in the management of free radical-
induced diseases is continually attracting scientific attentions.
Various experimental reports have implicated involvement (in
part or full) of free radicals such as hydrogen peroxide (H2O2),
superoxide anion (O2._), hydroxyl radical (.OH), nitric oxide (NO)
etc. in the pathogenesis of various diseases such as cancer, malaria,
neurodegeneration etc (Gutteridge, 1994). Free radicals, being
dual in role could either be beneficial as are well understood to be
involved in the normal physiological functioning of the body cells
at low concentration or detrimental as excessive production could
mediate damage to cellular components such as DNA and RNA,
lipids and proteins, triggering structural and functional
deterioration of vital organs, presented as diseases (Sen et al.,
2010). The antioxidant defense system of the body naturally helps
to scavenge and prevent the deleterious effect of free radicals. Such
antioxidants produced by the body include glutathione and
enzymes (e.g. superoxide dismutase, catalase etc). Proper
physiological functioning of the cellular environment is normally
necessitated by a balance between free radicals and antioxidants.
Should free radicals production overwhelm the body's antioxidant
defense, oxidative stress is triggered, at which level the quest for
external sources of antioxidants becomes imperative. Butylated
hydroxytoluene, butylated hydroxyanisole and a number of
synthetic antioxidants are reported to be harmful to human health
(Lobo et al., 2010). It is thus scientifically advantageous to embrace
the use of less or non-toxic, effective natural components with
antioxidant values (Lobo et al., 2010).
Folkloric medicine practices involve the use of natural
products and plants with various pharmacological actions.
Medicinal advantages of these plants have been explained by
re se arch er s a s b ei ng own ed t o the ir a nti oxid at ive
phytoconstitutions such as phenolics and flavonoids (Sulaiman
and Balachandran, 2012). Acalypha wilkesiana is one among
numerous plants with antioxidant properties. It belongs to the
family of Euphorbiaceae and the genus Acalypha. It is alternatively
called fire dragon, copper leaf or Jacob's coat (Makoshi et al., 2016).
It is an outdoor plant, native to Fiji and Islands in the south pacific
but has gained availability in various regions of the world
International Journal of
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Int J Biol Med Res
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Objective: The therapeutic potency of a medicinal plant is widely implicative of its anti-
oxidative power. This study investigated the phytoconstitution, phenolic contents and the
antioxidant potency (in-vitro) of aqueous, acetone and methanol leaf extracts of green and red
Acalypha wilkesiana (A.w.) Method: Phytochemical screening was carried out as well as
investigation of the antioxidant activity of the extracts by estimating the level of phenolics,
ferric reducing antioxidant power (FRAP), diphenyl-2-picryl-hydrazyl (DPPH), 2,2-azino-
biz(3-ethylthianzoline-6-sulfonic acid) (ABTS), nitric oxide (NO) as well as lipid peroxidation
inhibitory potencies. Results: Preliminary phytochemical screening revealed the presence of
flavonoid, saponin, alkaloid and carotenoid in the extracts. The highest total phenol content
was observed in the aqueous extracts of red (29.72 ± 3.39mg/g) and green A.w. (22.56 ±
0.66mg/g). Total flavonoids content and FRAP results also followed the same trend; (344.60 ±
7.01mg/g and 706.46 ± 1.04mmol/g respectively) for the red A.w. and (339.37 ± 9.50mg/g and
679.14 ± 0.45mmol/g respectively) for the green A.w. meanwhile the highest total flavonol
content was observed in the methanol extract of red A.w. with a value of 213.19 ± 2.44mg/g. The
highest inhibitory effect on DPPH radicals, ABTS radicals and lipid peroxidation were
expressed by the aqueous extract of red Acalypha wilkesiana with IC50 values of 0.59mg/ml,
0.64mg/ml and 0.62mg/ml respectively, followed by the aqueous extract of green Acalypha
wilkesiana with IC50 values of 0.60mg/ml, 0.68mg/ml and 0.78mg/ml respectively. The
aqueous extracts of green and red A. w. exhibited the highest inhibition towards NO with IC50 of
0.42mg/ml and 0.43mg/ml respectively. Conclusion: Results from this study suggest that
aqueous extracts of these plants (with a higher potency observed in the red) possess high
antioxidant levels.
* Corresponding Author : MO. DIDUNYEMI
om.didunyemi@osustech.edu.ng
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7090
including America, Asia and tropics of Africa (Forcados et al.,
2016). Acalypha wilkesiana reportedly possess antidiabetic and
hypocholesterolemic properties (Ikewuchi and Ikewuchi, 2010),
antibacterial properties (Din et al., 2013) and antioxidant
properties (Anokwuru et al., 2015). Acalypha wilkesiana (Mull
Arg) species is presented to nature in two colors; the green and
the red (or coppery green + red splashes) given the same specific
naming and both are reportedly potent medicinally. This
research tends to question the colorational difference by
investigating to know and compare the antioxidant power of
both plants using different solvents for extraction.
MATERIALS AND METHODS
Plant Materials
Leaves of green and red Acalypha wilkesiana were collected
in March, 2019 from the Engineering and Engineering
technology department/campus, Olusegun Agagu University of
Science and Technology (OAUSTECH), Ondo State, Nigeria and
authenticated at the department of plant biology of the
university.
Chemicals
2, 2' – azino - bis - (3 – ethylbenzothiazoline – 6 - sulphonic
acid) (ABTS), 2, 2 – Diphenyl – 1 -picrylhydrazyl (DPPH), ferric
chloride (FeCl3), thiobarbituric acid (TBA) aluminum chloride,
Folin-Ciocalteu reagent, sodium carbonate (Na2CO3), potassium
persulphate (K2S2O8), trichloroacetic acid (TCA), gallic acid,
quercetin and ascorbic acid were products of Sigma Co. (St.
Louis, MO, USA). Sulphuric acid used, methanol, acetone and
butanol were of analytical grade and purchased from Merck Co.
(Darmstadt, Germany).
Preparation of plant extracts
Collected leaves of Acalypha wilkesiana were washed
properly and kept under shade for two weeks until dried. Leaves
were then ground to powder using an electric blender and 350g
immersed in 1000ml of distilled water/methanol/acetone for 48
hours at room temperature. The extracts obtained were then
subjected to rotary evaporator-aided dryness, weighed and
stored at 4oC for subsequent analysis. Percentage yield was
calculated using the formula;
Yield (%) = (W1 / W2) * 100
Where W1 = weight of extract residue obtained after solvent
removal
W2 = weight of ground sample taken
complex where the depth of the coloration is directly
proportional to the concentration of the phenolic compounds.
Briefly, 0.2ml of extract was mixed with 1ml of Folin-Ciocalteu
reagent, 1ml of saturated sodium carbonate Na2CO3 (7.5%) was
added after 4 minutes. Mixture was thereafter allowed to stand
at room temperature for 120 minutes after which absorbance
was read at 760 nm and total polyphenols in different extract
expressed as mg of gallic acid equivalent per gram of extract (mg
GAE/g).
Total Flavonoid Content Determination
Flavonoids quantification of aqueous, methanolic and
acetone extracts was done by the aluminium chloride reagent
(AlCl3) method as described by Ayoola et al. (2008). 1ml of
extract dissolved in corresponding solvent was added to 1ml of
2% AlCl3 in methanol. Mixture was allowed to incubate at room
temperature for 10 minutes and absorbance was measured at a
wavelength of 430nm in a u.v. visible spectrophotometer. Total
flavonoid was expressed as mg of quercetin equivalent per gram
(mg QE/g).
Total Flavonol Determination
The method described by Yermakov et al. (1987) was used in
the determination of the flavonol contents of the extracts. 2ml of
sample in ethanol was mixed with 2ml of 2% Aluminium
trichloride (AlCl3) and 6ml of 5% sodium acetate. Flavonol
content was estimated from the calibration curve of quercetin
prepared by mixture of 2ml of varying concentration of
quercetin with 2ml of 2% AlCl3 and 6ml of sodium acetate
(C2H3NaO2). Mixtures stood for 150 minutes incubation time at
20oC and absorbance was then read at 440nm.
Ferric Reducing Antioxidant Power (FRAP) estimation
FRAP assay was carried out using a modified method of
Benzie and Strain (1996). This assay method leverages on the
ability of the extract to reduce ferric tripyridyltriazine (Fe (III) -
TPTZ) complex to ferrous tripyridyltriazine (Fe (II) - TPTZ) at a
low pH. A blue coloration of produced Fe (II) - TPTZ was read at
593nm. FeSO4 was used in generating calibration curve and
ascorbic acid was reference control. Briefly, 1.5ml of newly
prepared FRAP solution [25ml of 300 mmol acetate buffer pH
3.6, 2.5ml of 10 mmol 2,4,6 - tripyridyltriazine (TPTZ) in 40
mmol HCl and 2.5ml of 20 mmol ferric chloride (FeCl3.6H2O)
solution] and thoroughly mixed with 1ml of the extract. Mixtures
were incubated for half an hour at 37oC and absorbance read at
593nm. FRAP values were expressed as mmol Fe2SO4
equivalent per gram of extract (mmol/g)
Diphenyl – 2 – picryl - hydrazyl (DPPH) Radical
Scavenging Activity
2, 2 - diphenyl-1-picryl-hydrazyl (DPPH) is a characteristic
stable free radical owned to delocalization of spare electron over
the molecule giving rise to a deep violet color. Any substrate (AH)
capable of donating a hydrogen atom to DPPH will yield a
reduced form, with loss of the characteristic violet color. Assay of
extracts' ability to scavenge DPPH was carried out according to
the method of Gyamfi et al. (1999). Briefly 50ul of different
concentrations (0.25, 0.50, 0.75 and 1.0mg/ml) was mixed with
DPPH solution (2.4mg in 100ml methanol). Mixture was then
allowed to stand for 30 minutes at room temperature.
Absorbance (of Control and Samples) was measured at
wavelength 517nm using gallic acid as standard. Inhibition of
DPPH in percentage was calculated thus;
Table 1: Percentage yield of extraction
Total Phenolics content Determination
Using gallic acid as standard, Folin-Ciocalteu colorimetric
method was adopted in the determination of total phenol
contents of the extracts as described by Yafang et al. (2011). This
method relies on the transfer of electron from phenolic
compound, in an alkaline medium forming a bluish coloration
constituted by a phosphotungstic/phosphomolybdenum
MO. DIDUNYEM 7089-7094 et al. Int J Biol Med Res. 2020; 11(3):

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Percentage inhibition of DPPH = (Ac – As) / Ac * 100
Where Ac = Absorbance of Control [DPPH + methanol]
As = Absorbance of sample [DPPH + extract/ standard]
Inhibitory concentration of 50% DPPH radical (IC50) was
calculated as extracts' effective concentration scavenging half
population of DPPH free radical
2, 2'- azino–bis (3 – ethylbenzothiazoline – 6 – sulfonic acid)
[ABTS] Radical Scavenging Power
The assay method of Re et al. (1999) was employed in
determining the ABTS free radical scavenging ability of the
extracts. Reacting ABTS stock solution (7mmol) with potassium
persulfate - K2S2O8 (2.45mmol, final concentration) in the dark
for 16 hours was used in generating ABTS radical cations
(ABTS.+) and absorbance was adjusted to 0.700 with ethanol at
734nm. Ascorbic acid was used as standard. 0.2ml of varying
concentrations (0.25, 0.50, 0.75 and 1mg/ml) of extracts in
DMSO were added to 2ml of ABTS.+ solution, mixture was
allowed to stand for 15 minutes after which absorbance was read
at 734nm. Results were expressed as percentage inhibition of
ABTS using the formula
% ABTS inhibition = (Ac – As) / Ac *100
Where, Ac = Absorbance of control
As = Absorbance of sample
Inhibitory concentration of 50% ABTS radical (IC50) was
calculated as extracts' effective concentration inhibiting half
population of ABTS free radical
Nitric Oxide Scavenging Ability Determination
Sodium nitroprusside is understood to decompose in
aqueous solution at physiological pH to yield nitric oxide (NO).
Interaction of nitric oxide with oxygen leads to production of
nitrite ion which is normally quantified using Griess reagent.
Scavengers of nitric oxide do compete with O2 consequently
leading to reduced generation of nitrite ion. Briefly 10 mmol
sodium nitroprusside in phosphate buffered saline was mixed
with varying concentrations (0.25, 0.50, 0.75 and 1.0mg/ml) of
the extracts and incubated for 150 minutes at room temperature.
Assay control contained same amount of reaction mixture but
with distilled water in place of extract. Following the incubation
time, 0.5ml of griess reagent [1% sulfanilide, 2% H3PO4 and
0.1% N (1-naphthyl ethylene diamine dihydrochloride)] was
introduced and absorbance reading was done at 546nm
wavelength. Ascorbic acid was used as standard and result was
calculated as
% NO Inhibition = (Ac – As) / Ac * 100
Where Ac = Absorbance of control
As = Absorbance of sample
Inhibitory concentration (IC50) was also calculated as
extracts' effective concentration inhibiting half population of NO.
Inhibition of Lipid Peroxidation assay
Egg yolk homogenate was the lipid rich medium in the
modified thiobarbituric acid reactive species (TBARS) assay
adopted here to measure the lipid peroxide formed as described
by Ruberto et al. (2000). 500ul of 10% v/v egg yolk homogenate
was added to 100ul of sample, content volume was made up to
1ml with distilled water. The reaction mixture received an
addition of 0.05ml of Fe2SO4 and incubated at 37oC for 30
minutes. 1.5ml of acetic acid was added after which 1.5ml TBA in
SDS was also introduced. The resulting mixture was vortex
mixed and heated for 60 minutes at 95oC. It was allowed to cool
and 5ml of Butanol was added and entire mixture centrifuged for
10 minutes at 3000 revmin-1. Absorbance of the supernatant
was measured at 532nm and inhibition (in percentage) was
calculated with the formula;
% inhibition of lipid peroxidation = (Ac – As) / Ac * 100
Where; Ac = Absorbance of control
As = Absorbance of sample
Inhibitory concentration (IC50) was calculated as extracts'
effective concentration inhibiting lipid peroxidation by half.
Phytochemical Screening
Qualitative phytochemical screening for the presence of
alkaloids, saponins, tannins, anthraquinones, flavonoids,
carotenoids, cardiac glycosides, steroids etc were carried out
using standard methods of Sofowora (1993) and Trease and
Evans (2002).
Statistical Analysis
Assays were carried out in triplicates and results expressed
as mean ± standard deviation. Data were analyzed using one way
ANOVA. Differences in mean values were ascertained by Duncan
multiple range test on graph pad prism (Graph pad software Inc.
San Diego, USA). Values were considered statistically significant
at P < 0.05. IC50 values were calculated following extrapolation
from linear regression.
Results:
Table 2: Phytoconstituents of aqueous, acetone and
methanol extracts of Acalypha wilkesiana
Table 3: Polyphenolic contents and Ferric reducing
antioxidant power of Aqueous, acetone and methanol leaf
extracts of green and red Acalypha wilkesiana (A. w.)
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Figure 1: Percentage scavenging ability of DPPH radical of
aqueous, acetone and methanol leaf extracts of Acalypha
wilkesiana. (Values are expressed as mean ± SD, n = 3)
Table 4: IC50 values of DPPH inhibition of aqueous, acetone
and methanol leaf extracts of Acalypha wilkesiana.
Table 6: IC50 values of NO inhibition of aqueous, acetone
and methanol leaf extracts of Acalypha wilkesiana.
Table 8: IC50 values of “inhibition of lipid peroxidation” of aqueous,
acetone and methanol leaf extracts of Acalypha wilkesiana.
DISCUSSION
Scientific investigations into the fundamentals of medicinal plants
have revealed close proximity between their therapeutic potencies and
antioxidative phytoconstituents. Implicated phytochemicals, generally
reflecting antioxidant activity of plants are polyphenols and carotenoids
(Jovanovic et al., 1994). Acalypha wilkesiana are reportedly antidiabetic
Table 5: IC50 values of ABTS inhibition of aqueous, acetone
and methanol leaf extracts of Acalypha wilkesiana.
MO. DIDUNYEM 7089-7094 et al. Int J Biol Med Res. 2020; 11(3):

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and hypocholesterolemic (Ikewuchi and Ikewuchi, 2010), antibacterial
(Gotep et al., 2010), hepatoprotective at a safe dose of 100mg/kg
(Ikewuchi et al., 2011; Ogbuehi et al., 2014), antimalarial (Ogbuehi et al.,
2014) etc. These documented efficacies are at least in part due to the rich
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(Ogbuehi et al., 2014) with no exceptions to flavonoid and carotenoid.
This was supportedly revealed by our findings of the presence of
flavonoid and carotenoid as part of the major phytochemicals in the
tested extracts (table 2).
We investigated and compared the antioxidant potency of the green
and red Acalypha wilkesiana species using established methods. We
understand that Acalypha wilkesiana (Muell Arg) species is presented as
green colored and red colored (or coppery green with prominent red
splashes) and both have been individually investigated for their
therapeutic values, however, to the best of our knowledge there is yet to
be a comparative antioxidant report on them.
The antioxidant power of a plant is directly related to its phenolic
content due to the OH group, facilitating the donation of hydrogen to
unstable free radicals (Ayoola et al., 2008; Hegazy and Ibrahim, 2012).
We thus quantified the total phenolics present in the aqueous, methanol
and acetone extracted leaves of both Acalypha wilkesiana plants. The
aqueous extracts of both plants had the highest total phenolic content
with a significantly higher (p < 0.05) value in the red Acalypha
wilkesiana. Methanol extracts were next, with same result trend as
observed in the aqueous extracts. Flavonoid content was also
consequently highest in the aqueous extract followed by the methanol
extract and acetone extracts - the least, with the red extracts having
higher values than their counterparts. Flavonoids are active water
soluble antioxidants (Flora, 2009; Oseni & Okoye, 2013), inhibiting
peroxidation and mopping up reactive oxygen species, its hydrophilicity
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aqueous extracts. Contrastingly the flavonol content of the methanol
extract of red and green Acalypha wilkesiana was highest of the six
extracts, suggesting that the major flavonoid content of the aqueous
extracts might possibly not be of the flavonol origin as opposed to the
case in the methanol extracts (table 3).
We investigated the Ferric Reducing Antioxidant Power (FRAP) of
the extracts. A good antioxidant is believed to sufficiently reduce Fe3+ to
Fe2+ due to the ability of phenolic compound to donate hydrogen. The
FRAP value of the extracts were significantly different (p<0.05) from one
another with the aqueous extracts expressing the highest value, followed
by the methanol extracts. In each case (aqueous and acetone), the red
showed a higher potency in reduction of Fe3+ except for methanol
extracts where the green Acalypha wilkesiana had a higher value (table
3)
DPPH radical scavenging ability is one of the scientifically adopted
standards in screening the antioxidant strength of samples (Lee et al.,
2003) as unstable DPPH radicals accept electron from donor to become
sta ble. DPPH radi cals were presented a gain st f our di fferen t
concentrations of each extracts of the two plants. Values were expressed
as percentage inhibition (figure 1) and inhibitory strength of extracts
were reflected by their IC50 values. The aqueous extracts of the red and
green Acalypha wilkesiana showed the highest inhibition of DPPH with
IC50 values of 0.59 mg/ml and 0.60 mg/ml respectively (table 4), the
methanol extracts showed the second highest inhibitory potency and the
acetone extracts showed the least efficacy towards DPPH having the
highest IC50 values (1.29 mg/ml and 1.40 mg/ml for the green and red
respectively).
A more flexible free radical scavenging model allowing for
screening of both non polar and polar samples (Re et al, 1999; Oboh &
Omorege, 2011) was also adopted in our antioxidant experimentation;
the ABTS (2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonate) assay. All
samples were further investigated for their scavenging abilities towards
ABTS radicals and result obtained for each extract was concentration
dependent with the aqueous extract being comparable with the standard
Ascorbic acid (figure 2). The efficacy of the extracts based on their IC50
values was as follows; (aqueous red > aqueous green) > (methanol red >
methanol green) > (acetone red > acetone green) (table 5). In each case,
red Acalypha wilkesiana proved to be more promising. Efficacies of the
extracts correlated well with the amount of phenolics expressed.
Nitric oxide, an important signaling molecule transmitting signals
to cells in the immune and nervous systems of the body, owned to its
minute size - enhancing its permeability through membranes of cells
facilitating its signaling function and the possession of a free radical
confers higher reactivity on it than other signaling molecules. The body
synthesizes nitric oxide through the help of the enzyme nitric oxide
synthase from L-arginine, via metabolism to Citrulline and via a five
electron oxidative reaction (Knowles et al., 1989). NO is also generated
from macrophages, neurons, as well as endothelial cells, regulating a
number of processes physiologically. Production in excess of NO is
associated with modifications (structurally and functionally) of cell
components. Anti radical agents are capable of quenching nitric oxide
generated experimentally from nitroprusside in aqueous solutions via a
competition with oxygen. Aqueous extracts of red and green Acalypha
wilkesiana showed promising results towards nitric oxide with the
lowest IC50 (0.43mg/ml and 0.42mg/ml respectively), followed by
methanol extract with the acetone extracts having the highest IC50
values (table 6) and least inhibition of NO.
The inhibition of lipid peroxidation effect of the extracts was also
investigated. A good antioxidant specie should capable donate electron
to unstable free radicals inhibiting them from stealing free electrons
from lipids in the cell membranes and consequently preventing oxidative
degradation of lipids. Polyunsaturated fatty acids from egg yolk react
with oxygen to form malondialdehyde which react with thiobarbituric
acid producing a pink coloration. Our extracts were tested against lipid
peroxidation, and concentrations capable of inhibiting lipid oxidation by
50% was calculated in mg/ml. Aqueous extracts showed the best
potency against lipid peroxidation with IC50 of 0.62 mg/ml and 0.78
mg/ml for the red and green plant respectively. Methanol extracts
displayed the second best efficacy and acetone extracts the least (table 8)
with the red extracts showing better results for aqueous and acetone
extracts.
CONCLUSION
The results of the lipid peroxidation assay showed that Acalypha
wilkesiana contain potent inhibitory agents especially the aqueous and
methanolic extracts, which is in conformity with the in-vivo reports of
Ogbuehi et al. 2014. From the results of total phenolic and anti radical
assays, the aqueous extracts of green and red Acalypha wilkesiana
obviously proved to be most potent antioxidatively with a higher efficacy
from the red plant. We thus suggest that aqueous extract of green and red
Acalypha wilkesiana can be adopted in the management of free radical
induced diseases.
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