Acta Poloniae Pharmaceutica Drug Research, Vol. 70 No.1 pp.

115121, 2013

ISSN 0001-6837
Polish Pharmaceutical Society

FREE RADICAL SCAVENGING AND TOTAL ANTIOXIDANT CAPACITY

OF ROOT EXTRACTS OF ANCHOMANES DIFFORMIS ENGL. (ARACEAE)
ABUBAKAR B. ALIYU1*, MOHAMMED A. IBRAHIM2, ALIYU M. MUSA3, AISHA O. MUSA4,
JOYCE J. KIPLIMO5 and ADEBAYO O. OYEWALE1
Departments of 1Chemistry, 2Biochemistry, 3Pharmaceutical and Medicinal Chemistry, 4Biological Sciences,
Ahmadu Bello University, Zaria, Nigeria
5
School of Chemistry, Faculty of Science and Agriculture, University of KwaZulu-Natal,
Westville campus, Durban, 4000, South Africa
Abstract: Antioxidants activities from plants sources have attracted a wide range of interest across the world
in recent times. This is due to growing concern for safe and alternative sources of antioxidants. The free radical scavenging activity using 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), reducing power assay, total
antioxidant capacity of the phosphomolybdenum method and the total phenolics content using the FolinCiocalteu reagent were carried out on the acetone, n-butanol and methanol root extracts of Anchomanes difformis. The results of the total phenolics content expressed in mg/100 g of gallic acid equivalent (GAE) showed
that the n-butanol extract has significantly (p < 0.05) higher phenolics content (381 1.13) than the methanol
and acetone extracts. All the extracts displayed strong concentration dependent radical scavenging activity. It
was also observed that the n-butanol extract showed higher activity of 70.87% and 78.59% at low concentrations of 31.25 g/mL and 62.5 g/mL, respectively, than methanol and acetone extracts. The results also
showed that the n-butanol extract has strongest reducing ability which is comparable to that of gallic acid at all
the concentrations tested. Phytochemical screening on the extracts revealed the presence of flavonoids,
saponins, and tannins. The results suggest that n-butanol extract of the plant is very rich in antioxidant compounds worthy of further investigations.
Keywords: Anchomanes difformis, DPPH, phytochemicals, reducing power, total phenolics

dence has implicated the role of ROS or RNS in the

progression of heart diseases, neurodegenerative
diseases, cancer, aging process (4) and complications in diabetes among others (5).
In recent years, there has been increasing scientific interest in antioxidants compounds especially from plants. This is informed by the emerging
evidence of protective roles of vegetables and plant
foods against cancer and other neurodegenerative
diseases on one hand and the growing concern of
health implications associated with the synthetic
antioxidants currently used as food additives on the
other. Antioxidant compounds are free radical scavengers because they inhibit or delay the oxidation of
substrate by free radicals thereby resulting in significant prevention of lipid peroxidation in biological
systems. Phenolic and polyphenolic compounds
constitute the main class of natural antioxidants
present in plants, foods, and beverages (6). These
compounds, including flavonols, quecertin, cate-

Oxygen derived free radicals or reactive oxygen species (ROS) as well as reactive nitrogen
species (RNS) which include hydroxyl (.OH), superoxide (O2), nitroxide (NO.), peroxyl (ROO.), peroxy
nitrite (OONO.), hypochlorous acid (HOCl), hydrogen peroxide (H2O2), nitrous acid (HNO2) and dinitrogen trioxide (N2O3) are generated during normal
metabolism and energy production in the body (1).
They are produced to help the normal healthy tissues
perform physiological roles such as signaling molecules, regulation of signal transduction and gene
expression, activation of receptor and nuclear transduction among others (2). But when these ROS or
RNS are present in higher concentration beyond the
antioxidant capacity of a biological system, due to
metabolic and other environmental factors; it gives
rise to an imbalance known as oxidative or nitrosative
stress (3); a situation that mediates damage to biological molecules such as lipids, proteins, polysaccharides and DNA (2). Overwhelming literature evi-

* Corresponding author: e-mail: aliyubabando@gmail.com, phones: +2348057371917, +2348098160674

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ABUBAKAR B. ALIYU et al.

chins and anthocyanins, exhibit similar structural

chemistry whose functions among others is to
strengthen the oxidative stability of foods and
human systems due to their redox properties, which
can play significant roles in neutralizing free radicals, quenching singlet oxygen or decomposing
hydroperoxides (7-9).
Anchomanes difformis is an important medicinal plant that grows in the tropical zones in various areas of Africa (10). The plant root is commonly known as chakara (Hausa) in Northern
Nigeria and ishu ag (Yoruba) in South Western
Nigeria (11). Ethnomedicinal information from
herbalist in Zaria city, revealed that a decoction of
the root is used to treat diabetes, cough and throat
related problems (personal communication).
Locally peeled root soaked in water is used in treating cases of dysentery in South Western Nigeria
(12). Disengomoka et al. (13) reported the use of
powdered root mixed with palm oil as remedy for
respiratory diseases in children in Zaire (DRC),
where as in Benin republic, the root is used as
diuretic, to treat diabetes, oral and anal lesions,
tuberculosis and malaria (14). Antimicrobial properties of the plant have been reported (12, 15).
Phytochemical studies revealed the presence of
organic acids, amino acids, heterosides and
polyphenols (10, 16). It is important, therefore, to
evaluate the antioxidant properties of root extracts
of A. difformis in view of the widely use in traditional medicine. This will help to justify some of
the claims and understand the potentials of the
plant for maximum utilization, because antioxidant
substances could play a crucial role in the development of new chemotherapeutic agents for the treatment of some of the diseases mentioned.
Previously, we reported the antioxidant potentials
of some medicinal plants (17-19) widely used in
Northern Nigerian traditional medicine in our
efforts to bridge the gap between medicinal plants,
free radicals, diseases and human health. In this
study, we report the free radical scavenging, total
antioxidant activity and total phenolics content of
acetone, n-butanol and methanol root extracts of A.
difformis.
MATERIALS AND METHODS
Chemicals and reagents
Deionized water, gallic acid (Fluka, UK), 1,1diphenyl-2-picrylhydrazyl radical (DPPH) (SigmaAldrich Co.), sulfuric acid, sodium phosphate,
ammonium molybdate, trichloroacetic acid (SigmaAldrich Co.), anhydrous ferric chloride, potassium

ferricyanide, anhydrous sodium carbonate, phosphate buffer, ascorbic acid and all other chemicals
were of analytical grade BDH Chemical Laboratory
(England, UK).
Plant material and extraction
The whole plant Anchomanes difformis was
collected in the month of September, 2010 at
Dakace village along Jos road, Zaria. It was
authenticated at the herbarium of the Department
of Biological Sciences, Ahmadu Bello University,
Zaria. A voucher specimen (No. 900283) was
deposited there. The root sample was air dried and
pulverized to powder. A 200 g of it was successively extracted on Soxhlet extractor with nbutanol, acetone and methanol for 3 h each. The
extracts were filtered using Whatman filter paper
no. 2, and concentrated on a Bchi rotary evaporator at 45OC, which afforded 5.6 g, 12.2 g and 23.5
g of n-butanol, acetone and methanol extracts,
respectively.
Phytochemical screening of extracts
Phytochemical screening was carried out on
the three extracts to detect the presence of secondary
metabolites such as alkaloids, flavonoids and tannins according to procedures described by Sofowora
(20).
Total phenolics content using Folin-Ciocalteu
reagent
The total phenolics content of the extracts were
determined using the method of Macdonald et al.
(21) with slight modifications. Calibration curve
was prepared by mixing ethanol solution of gallic
acid (1 mL; 0.025-0.400 mg/mL) with 5 mL of
Folin-Ciocalteu reagent (diluted tenfold) and sodium carbonate (4 mL, 0.7 M). Absorbance values
were measured at 765 nm using a UV-VIS spectrophotometer
(UVmini-1240,
Shimadzu
Corporation, Kyoto, Japan) and the standard curve
was plotted. One milliliter of each of the extract
solutions in methanol (5 g/L) was also mixed with
the reagents above and after 30 min the absorbance
was measured to determine the total phenolic contents. All determinations were carried out in triplicate. The total phenolics components in the extracts
in gallic acid equivalents (GAE) were calculated by
the following formula: T = CV/M; where T = total
phenolic contents, milligram per gram of sample
extract, in GAE; C = the concentration of gallic acid
established from the calibration curve, mg/mL; V =
the volume of extract, milliliter; M = the weight of
sample extract (g).

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Free radical scavenging and total antioxidant capacity of root extracts of...

Measurement of free radical scavenging activity

using DPPH method
The free radical scavenging activity (antioxidant capacity) of the plant extracts on the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) was
evaluated by the method reported by Shirwaikar et
al. (22). In this assay, a volume of 1.5 mL of
methanol solution of the extract at different concentrations was mixed with 0.5 mL of the methanol
solution of DPPH (0.1 mM). An equal amount of
methanol and DPPH without sample was served as a
control. After 30 min of reaction at room temperature in the dark, the absorbance was measured at 517
nm against methanol as a blank using a UV-VIS
spectrophotometer (UVmini-1240) as mentioned
above. The percentage free radical scavenging activity was calculated according to the following equation:
% scavenging activity = [(Ac-As) / Ac] 100
where Ac = absorbance of control and As =
absorbance of sample.
Total antioxidant capacity
The total antioxidant capacity of the extracts
was evaluated by the phosphomolybdenum method
according to the procedure described by Prieto et al.
(23). A 0.3 mL of extract was combined with 3 mL
of reagent solution (0.6 M sulfuric acid, 28 mM
sodium phosphate and 4 mM ammonium molybdate). The tubes containing the reaction solution
were incubated at 95OC for 90 min. Then, the
absorbance of the solution was measured at 695 nm
using a UV-VIS spectrophotometer (UVmini-1240)
against blank after cooling to room temperature.
Methanol (0.3 mL) in the place of extract was used
as the blank. The total antioxidant activity is
expressed as the number of gram equivalent of
ascorbic acid. The calibration curve was prepared by
mixing ascorbic (1000, 500, 250, 125, 62.5 and
31.25 g/mL) with methanol.

Reducing power assay

The total reducing power of each extract was
determined according to the method described previously (24). Volumes of 2.5 mL of different concentrations of the extracts (1000, 500, 250, 125, 62.5 and
31.25 g/mL) were mixed with 2.5 mL phosphate
buffer solution (0.2 M, pH = 6.6) and 2.5 mL of 1%
potassium ferriccyanide [K3Fe(CN) 6] in test tubes.
The mixture was placed in a water bath at 50OC, for
20 min. Then, 2.5 mL of 10% trichloroacetic acid
was added to the mixture and mixed thoroughly. A
volume of 2.5 mL of this mixture was then added to
2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%
solution) and allowed to stand for 10 min. Then, the
absorbance of this mixture was measured at 700 nm
using a UV-VIS spectrophotometer (UVmini-1240);
the higher the absorbance of the reaction mixture, the
greater the reducing power. Ascorbic acid was used
as a positive control. All these procedures were done
in triplicate.
Statistical analysis
The experiments were carried out in triplicate
and results are given as the mean standard deviation. The data in all the experiments were analyzed
(Microsoft Excel 2007) for statistical significance
using Students t-test and differences were considered significant at p < 0.05.
RESULTS AND DISCUSSION
Anchomonas difformis is commonly used to
traditionally treat many diseases (12, 14) whose
pathogenesis are, among other factors, linked to
oxidative stress. However, information on antioxidant potentials of this plant that could be relevant in
the treatment of such diseases has not been investigated. In this study, we report the antioxidant potentials of A. difformis root extracts.
Phytochemical examination revealed the presence of flavonoids, saponins and tannins in all the

Table 1. Total phenolics and phytochemical screening of root extracts of A. difformis.

Phenolics/phytochemicals
Total phenolics (mg/100 g GAE)*

Methanol extract

Acetone extract

n-Butanol extract

358 1.20

336 2.52

381 1.13

Alkaloids

Flavonoids

Saponins

Anthraquinones

Tannins

*Data are presented as the mean standard deviation SD (n = 3), + = present, - = absent, GAE = gallic acid equivalents.

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ABUBAKAR B. ALIYU et al.

extracts but no alkaloids and anthraquinones. The

extracts were found to have phenolic contents of
358, 336 and 381 mg/100 g GAE for methanol, acetone and n-butanol extracts, respectively (Table 1).
All three extracts showed significant free radical
scavenging activity, with n-butanol showing the
highest activity at concentrations of 31.25 and
62.5%. At highest concentration tested there is no

significant (p = 0.05) difference in activity between

the extracts and gallic acid (Fig. 1).
Phytochemicals are currently receiving
increased attention because of interesting new findings regarding their biological activities (25). The
flavonoids, saponins and tannins detected in these
extracts could implicate these classes of phytochemicals as important bioactive agents of the root parts

Figure 1. DPPH radical scavenging activity of extracts of A. difformis. Data are presented as the mean value standard deviation SD (n = 3)

Figure 2. Total antioxidant capacity of extracts of A. difformis. Data are presented as mean value standard deviation SD (n = 3)

Free radical scavenging and total antioxidant capacity of root extracts of...

119

Figure 3. Reducing power assay of extracts of A. difformis. Data are presented as the mean value standard deviation SD (n = 3)

of this plant and might be involved in the therapeutic action of this plant part. The high phenolics content of these extracts indicates high antioxidant
potentials because the phenolics constituents can
react with active oxygen radicals such as hydroxyl
radical (26), superoxide anion radical (27) and lipid
peroxy radical (28). The literature reports showed
that there is high correlation between antioxidant
activity and phenolics content (29). The n-butanol
extract was found to possess significantly (p = 0.05)
higher phenolics content than other extracts and
although correlation analysis was not performed,
our data also suggest high correlation between total
phenolics content and antioxidant potential considering the higher antioxidant activity observed with
the n-butanol extract than other extracts in most of
the assays.
The free radical chain reaction is widely
accepted as a common mechanism of lipid peroxidation. Radical scavengers may directly react with
and quench peroxide radicals to terminate the peroxidation chain reactions (30) which are important
in the pathogenesis of various diseases. Assay
based upon the use of DPPH radicals is among the
most popular spectrophotometric methods for
determination of the antioxidant capacity of plant
extracts, foods, beverages and vegetable extracts
because the radical compounds can directly react
with antioxidants. Additionally, DPPH scaveng-

ing method has been used to evaluate the antioxidant activity of compounds due to the simple,
rapid, sensitive, and reproducible procedures (31).
The present results suggest that the extracts are
apparently good free radical scavengers (especially
of those of peroxy type) and probably have the
ability to inhibit autoxidation of lipids and could
thus be beneficial in the treatment of various diseases where lipid peroxidation is an important
mechanism for pathogenesis.
The total antioxidant capacity (TAC) was
based on the reduction of Mo(VI) to Mo(V) by the
extract and subsequent formation of green phosphate/Mo(V) complex at acid pH. It evaluates both
water-soluble and fat-soluble antioxidants (total
antioxidant capacity). The results indicate higher
TAC (expressed as ascorbic acid equivalent) of the
methanol and acetone extracts, respectively, at low
concentration, but the differences are not statistically significant (p = 0.05) from that of n-butanol
extract. It was, however, observed that the n-butanol
extract possesses significant total antioxidant capacity equivalent to 90 mg/g ascorbic acid at higher
concentration (Fig. 2). This suggests the potential
comparable antioxidant constituents of the nbutanol extract because antioxidant capacity of
ascorbic acid has been used as a reference standard
with which plant extracts with potential antioxidants
are compared (32).

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ABUBAKAR B. ALIYU et al.

It was suggested that the electron donating

capacity, reflecting the reducing power of bioactive
compounds, is associated with antioxidant activity.
Antioxidants can be reductants, and inactivation of
oxidants by reductants can be described as redox
reactions in which one reaction species is reduced at
the expense of the oxidation of the other (30). The
presence of reductants, such as antioxidant substances in the samples, causes the reduction of the
Fe3+/ferricyanide complex to the ferrous form. The
reducing power of the extracts increased with
increasing concentration, which suggests that the
electron donating ability of the extracts is concentration dependent.
The significantly higher absorbance values of
n-butanol extract than gallic acid at lower concentrations suggests that the n-butanol extract, especially at such concentrations, has high redox potentials
and can acts as reducing agent, hydrogen donor and
singlet oxygen quencher (33). Although we detected
the same classes of phytochemicals in the extracts,
the quantitative difference in antioxidant activity
could results from difference in the concentration of
the phytochemicals. The reducing power of the
extracts followed the order: n-butanol > acetone >
methanol (Fig. 3).
Our results suggest that the n-butanol extract
contains more antioxidant agents than the methanol
and acetone extracts. It was concluded that A. difformis root contains some antioxidant agents that
could be relevant in the therapeutic action of this
plant part. These findings warrant further studies on
the isolation and characterization of the bioactive
compounds responsible for the antioxidant activity
observed herein.
Acknowledgment
The authors acknowledge the contribution of
Ahmadu Bello University, Zaria, Nigeria for providing the facilities for conducting this research.
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Received: 13. 01. 2012