FREE RADICAL SCAVENGING AND TOTAL ANTIOXIDANT CAPACITY OF ROOT EXTRACTS OF ANCHOMANES DIFFORMIS ENGL. (ARACEAE)

115-121.qxdActa Poloniae Pharmaceutica ñ Drug Research, Vol. 70 No.1 pp. 115ñ121, 2013 ISSN 0001-6837 Polish Pharmaceutical Society
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 dini- trogen 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, polysac- charides and DNA (2). Overwhelming literature evi-
dence has implicated the role of ROS or RNS in the progression of heart diseases, neurodegenerative diseases, cancer, aging process (4) and complica- tions in diabetes among others (5).
In recent years, there has been increasing sci- entific 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-
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
5School 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 Folin- Ciocalteu 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 µg/mL and 62.5 µg/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
115
* Corresponding author: e-mail: [email protected], phones: +2348057371917, +2348098160674
116 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 treat- ing 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 develop- ment 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), 1,1- diphenyl-2-picrylhydrazyl radical (DPPH) (Sigma- Aldrich Co.), sulfuric acid, sodium phosphate, ammonium molybdate, trichloroacetic acid (Sigma- Aldrich 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 succes- sively extracted on Soxhlet extractor with n- butanol, acetone and methanol for 3 h each. The extracts were filtered using Whatman filter paper no. 2, and concentrated on a B¸chi rotary evapora- tor 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 = C◊V/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).
Free radical scavenging and total antioxidant capacity of root extracts of... 117
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 equa- tion:
% 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 µg/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 µg/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 Studentís t-test and differences were consid- ered 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 investi- gated. 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 Methanol extract Acetone extract n-Butanol extract
Total phenolics (mg/100 g GAE)* 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.
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
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 consid- ering 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 DPPHï 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, DPPHï 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 statistical- ly 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 n- butanol extract because antioxidant capacity of ascorbic acid has been used as a reference standard with which plant extracts with potential antioxidants are compared (32).
Figure 3. Reducing power assay of extracts of A. difformis. Data are presented as the mean value ± standard deviation SD (n = 3)
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 provid- ing the facilities for conducting this research.
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FREE RADICAL SCAVENGING AND TOTAL ANTIOXIDANT CAPACITY OF ROOT EXTRACTS OF ANCHOMANES DIFFORMIS ENGL. (ARACEAE)

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