aina and oyedapo (4) - obafemi awolowo university scholars · 236 steroids, saponins, tannins and...

Ife Journal of Science vol. 15, no. 2 (2013)

IN VITRO INVESTIGATIONS INTO THE ANTIOXIDANT AND ANTI-INFLAMMATORY POTENTIALS OF THE FRACTIONS AND ETHANOLIC

EXTRACT OF CYCLOSORUS AFER (CHRIST.) CHING, STALKS.

Aina, Oluwatoyin Imoleayo and Oyedapo, Oluokun OluboadeDepartment of Biochemistry, Obafemi Awolowo University, Ile-Ife, Nigeria.

Corresponding Author: Prof. O. O. Oyedapo ([email protected])(Received: April, 2013; Accepted: June, 2013)

ABSTRACT

This study was designed to evaluate the in vitro antioxidant and anti-inflammatory activities of the ethanolic extract (EECA) and the fractions of Cyclosorus afer. In addition, the phytochemical constituents, total phenolic and flavonoid contents were identified and quantified respectively. The assessment of the antioxidant and anti-inflammatory potentials using the DPPH and hydroxyl radicals, ferric reducing power (FRAP) activities, inhibition of lipid peroxidation, and stabilization of erythrocyte membranes and inhibition of denaturation of albumin were employed. Phytochemical screening gave positive tests for the presence of flavonoids, alkaloids, saponins, cardiac glycosides, steroids, xanthoprotein and tannins. The total phenolic content of the ethanolic extract was 17.62 ± 0.43 mg/g and fractions ranged between 4.40 ± 0.27 mg/g and 81.64 ± 0.38 mg/g TAE (tannic acid equivalent) while the flavonoid content showed that the EECA contained 0.98 ± 0.09 mg/g and its fractions ranged between 0.39 ± 0.01 mg/g and 4.51 ± 0.07 mg/g QE (Quercetin Equivalent). The fractions exhibited significant, potent and appreciable antioxidant and anti-inflammatory activities which were concentration-dependent. The mode of action of the fractions was both monophasic and biphasic at all the concentrations assayed and was comparable to that of standard non-steroidal anti-inflammatory drugs used as positive control. In conclusion, the study revealed that the fractions of the stalks of C. afer possessed a number of bioactive molecules (flavonoids, tannins and saponins) that exhibited beneficial effects in the management of oxidative and inflammatory conditions hence could be employed as medicinal plants.

Key words: Cyclosorus afer, Antioxidants, Phytochemicals, Screening, DPPH, Phenolics, Peroxidation.

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INTRODUCTIONThe broad range of effects of free radicals in biological systems has drawn the attention of many scientists and many reports had supported the use of antioxidant supplementation in reducing the level of oxidative stress (Aruoma and Cuppett, 1997). In recent times, there has been an upsurge of interest in the therapeutic potentials of medicinal plants due to their well-known abilities to scavenge free radicals (i.e. antioxidant power) (Hou et al., 2003; Kukic et al., 2006). The large generation of free radicals, particularly reactive oxygen species and their high activity play important roles in the progression of a great number of pathological disorders l ike inflammation, atherosclerosis, stroke, heart disease, diabetes mellitus, multiple sclerosis, cancer, Parkinson's and Alzheimer's diseases (Mensor et al., 2001; Ozgen et al., 2006). Although many plant species have been investigated in the search for novel antioxidants (Chu 2000; Koleva et al., 2002) but generally, literature contains few reports on biological activities and chemical composition of lower plants (Bode and

Oyedapo 2011). Like other groups of plants, pteridophytes elicit medicinal potentials and many of them like male ferns have been employed medicinally from the time of Theophrastus and Dioscorides. However, only a limited number has been subjected to experimental studies for bioactivity and identification of bioactive molecules (Paul et al., 2011). Ferns are vascular plants with real roots (Alston, 1959) and the anatomy of their leaves corresponds quite close to that of seed plants. They synthesize diverse arrays of secondary metabolites with diverse biological activities such as protection against predators, antimicrobial, antioxidant as well as anti-inflammatory. The extract of Cyathea phalerata has been demonstrated to exhibit antioxidant and hepatoprotective potentials (Mariana et al., 2008), fractions (ethylacetate, butanol and aqueous) of Blechnum orientale have been confirmed to possess strong antioxidant, antibacterial and anticancer properties (How et al., 2010). Phytochemical screening and HPLC studies of the acetone extract of C. parasitica, (L.) H. Lev. revealed the presence of alkaloids, flavonoids, terpenoids,

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steroids, saponins, tannins and sugars. The extracts exhibited antibacterial activity against various bacteria species (Paul et al., 2011, Asim and Amal, 2011, Ajay and Purshotam, 2011, Pauline et al., 2012).

Cyclosorus afer a fern is an ornamental plant commonly called Omu (Yoruba), Imo-osun (Oyo), it grows in a marshy to waterlogged areas, and common in South Western (rainfall) belt of

Nigeria (Plate 1). It consists of various species such as C. striatus, C. interruptus, C. lenormandii, C. sandwicensis and C. afer (Alston 1959, Oloyede et al., 2011). Unlike other species of Cyclosorus, literature surveys indicate dearth of scientific information on the biological activities of C. afer. As such, this study investigated the chemical composition, antioxidant and anti-inflammatory potentials of the fractions of stalks of C. Afer.

Plate 1: Photograph of Cyclosorus afer showing the leaves and stalks

MATERIALS AND METHODS

Chemicals and ReagentsAll the reagents used were of analytical grade and procured from various sources. Thiobarbituric acid, 2-deoxy-D-ribose, DPPH (2,2-Diphenyl-1-picrylhydrazyl), bovine albumin, butylated hydoxytoluene (BHT), L-ascorbic acid were obtained from Sigma Fine Chemical Limited, while hydrogen peroxide, sodium potassium

tartarate, sodium dihydrogen phosphate, disodium hydrogen orthophosphate, copper tetraoxosulphate (vi) salt, sodium hydroxide and others were obtained from British Drug House (BDH) Chemical Limited, Poole, England.

Plant MaterialsFresh stalks of C. afer were collected along Opa river bank, at the main Campus of Obafemi

Aina and Oyedapo: In Vitro Investigations into the Antioxidant and Anti-inflammatory Potentials...

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Awolowo University, Ile-Ife, and along Ibadan/Ife Road. The plant was identified and authenticated by Mr. G.A. Ibhanesebor at IFE Herbarium, Obafemi Awolowo University, Ile-Ife, Nigeria.

Preparation of Ethanolic Extract Ethanolic extract (EE) of the stalk (3.62 kg) of Cyclosorus afer, was prepared according to the procedure of Mariana et al. (2008) using 80 % ethanol (15 L) for 72 h. The filtrate was concentrated to dryness on a rotatory evaporator (Edwards Vacuum Pump) under reduced pressure

oat 40 C to obtain a dark brown crude extract termed (ethanolic extract).

Phytochemical Screening of EEPreliminary phytochemical screening of the EE was carried out based on a procedure of Oyedapo et al., (1999), Trease and Evans (2002) and Sofowora (2006) for the presence of steroids, phlobatannins, tannins, flavonoids, saponins, alkaloids, anthraquinone, triterpenes, cardiac glycosides and xanthoproteins.

Solvent-Solvent Partitioning of EEThe extract was partitioned according to the method described by Tauheeda et al. (2011). Typically, EECA (30.5 g) was dissolved in hot distilled water (100 ml), filtered and partitioned sequentially with n-hexane (200 ml × 3), chloroform (200 ml × 3), dichloromethane (200 ml × 3), ethylacetate (200 ml × 6) and n-butanol (200 ml × 4). The fractions (HF, CF, DCMF, EAF, nBF and AqF) were concentrated to dryness rotatory evaporator under reduced

opressure at 40 C.

Biochemical AnalysesE s t i m a t i o n o f To t a l P h e n o l i c s ConcentrationsThe total phenolic concentrations in the extract and fractions were determined using Folin- Ciocalteu's phenol reagent reaction method (Singleton et al., 1999) as modified slightly by Bode and Oyedapo (2011). The reaction mixtures consisted of extracts/fractions (0.2 ml, 1 mg/ml), distilled water (0.8 ml), Folin- Ciocalteau's Phenol reagent (1.5 ml) and followed by incubation at room temperature for 15 min. To the reaction mixture was added 10% (w/v) NaHCO (1.5 ml) 3

and further incubated for 1.5 h in the dark. The

absorbance was read at 720 nm against reagent blank. The total phenolics in the EECA and fractions were expressed as mg/ g (TAE) tannic acid equivalent.

Estimation of Total FlavonoidsThe aluminum chloride reaction method was used for the quantification of flavonoid concentrations (Sun et al., 1999). The reaction mixtures consisted of extract/fraction (0.2 ml) in ethanol, distilled water (2.8 ml), 5% (w/v) NaNO (0.3 ml), 10% 2

(w/v) AlCl (0.3 ml) and 4% (w/v) NaOH (4 ml). 3

The reaction mixture was left at room temperature for 15 min., before the absorbance was read at 500 nm against the reagent blank. The flavonoid content of the EECA/ fractions was expressed as mg/g (QE) quercetin equivalent.

Assay of DPPH Free-Radical Scavenging Activity 2, 2'-diphenyl-2-picryl hydrazyl radical (DPPH) was used for determination of free radical-scavenging activity of the EECA and fractions using the method of Blois (1985) as reported by Cakir et al. (2003) with ascorbic acid as standard. The percentage scavenging activity was calculated using the expression:

Percentage Scavenging Activity = X 100

Assay of Hydroxyl Radical Scavenging ActivityThe hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extracts for hydroxyl radicals

3+generated from the Fe /ascorbate/EDTA/H O 2 2

system according to the method of Halliwell et al. (1987). The reaction mixture contained 1 ml (3.0 mM deoxyribose, 0.1 mM EDTA, 2 mM H O , 0.1 2 2

mM L-Ascorbic acid, 0.1 mM FeCl .6H O in 10 3 2

mM phosphate buffer, pH 7.4) and various concentrations of the extracts (50-350 µg/ml).

oThe reaction mixtures were incubated at 37 C for 1 h, followed by the addition of 1 ml of 1 % (w/v) TBA (in 0.25 N HCl) and 1.0 ml 10 % (w/v) TCA. The reaction mixtures were heated in boiling

owater bath at 100 C for 20 min and the pink chromogen (malondialdehyde-(TBA) adduct) 2

was extracted into 1.0 ml of butan-1-ol and the absorbance was read at 532 nm against reagent blank.

Abscontrol - Abssample

Abscontrol


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The percentage inhibition was calculated using the expression:

Percentage inhibition = X 100

Assay of Reducing Antioxidant PowerThe ability of the EECA and its fractions to reduce iron from the form (III) to the form (II) was assessed using the method of Chu et al. (2000). Different concentrations of the extracts were separately mixed with 2.5 ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of 1 % (w/v) potassium hexacyanoferrate [K Fe-(CN) ]. The reaction 3 6

mixtures were incubated at 50 °C for 20 min., followed by the addition of 2.5 ml of 10 % (w/v) trichloroacetic acid, the reaction mixtures were centrifuged at 2000 rpm for 10 min. The upper layer (2.5 ml) was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1 % FeCl . The absorbance 3

was read at 700 nm against reagent blank. The different absorbance obtained was plotted against the concentrations, owing to the fact that the higher the absorbance, the higher the reducing power of the extract.

Assay of Anti-lipid Peroxidation ActivityThe assay of anti-lipid peroxidation was carried out according to the thiobarbituric acid reaction method of Yoshiyuki et al. (1981) and Masao et al. (1993) with ascorbic acid as standard. Ten percent liver homogenate (0.25 ml) was added to 0.1 ml of Tris- HCl buffer, pH 8.2, 0.05 ml of ascorbic acid, 0.05 ml of FeSO and varying concentrations of 4

the extracts. The reaction mixtures were incubated o

at 37 C for 1 h. After incubation, 0.5 ml of 0.1 N

HCl, 0.2 ml of 9.8 % SDS, 0.9 ml of distilled water and 2.0 ml of 0.67% TBA were added sequentially. The reaction mixtures were heated in boiling

owater at 100 C for 30 min., cooled and 2.0 ml of butan-1-ol was added and later centrifuge at 3000 rpm for 10 min. The supernatant was collected and measured at 532 nm against reagent blank.The percentage inhibition was estimated using the expression:

Rate of inhibition (%) = X 100 where A = Abs of MDA produced in the absence of extract (control); B = Abs of MDA produced in the presence of extract.

Assay of Membrane Stabilizing ActivityThe membrane stabilizing activity assay method was based on the procedure described by Oyedapo et al. (2004). The assay mixture consisted of hyposaline (1 ml), 0.1 M phosphate buffer, pH 7.4 (0.5 ml), varying concentrations of EECA/ fractions (50 - 350 µg/ml) and 0.5 ml of 2% (v/v) erythrocyte suspension in a total volume of 3 ml. The control was prepared as above without the drug while the drug control (3 ml) lacked erythrocyte suspension. The standard anti-inflammatory drug for the assay was ibuprofen. The reaction mixtures were incubated at 56° C for 30 min. The absorbance of the released haemoglobin was read at 560 nm against reagent blank. The percentage membrane stability was estimated using the expression: Percentage membrane stability

Abscontrol - Abssample

Abscontrol

A - B

A

100 - Abstest drug - Absdrug control

Absblood control= X 100

The blood control represented 100% lysis.

Assay of Inhibition of Denaturation of Albumin ActivityDifferent concentrations of EECA/ fractions were assayed for the ability to inhibit denaturation of albumin following the method of Mizushima and Kobayashi (1968) with slight modification. The reaction mixtures contained 0.5 ml (1.5 mg/ml albumin) and different concentrations of

othe extracts, followed by incubation at 37 C for 20

omin. The reaction mixtures were heated at 57 C for 3 min and 2.5 ml of 0.5 M phosphate buffer, pH 6.3 was added. From each of the reaction mixtures, 1 ml was pipetted into clean dried test tubes in triplicates followed by the addition of Copper-Alkaline reagent (1 ml) and 1.0 ml of Folin-Ciocateu's Phenol reagent (1:10). The


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oreaction mixtures were incubated at 55 C for 10 min. The tubes were cooled and the absorbance was read at 650 nm against reagent blank.The quantity of protein left was calculated using the expression:

X 100

The percentage inhibition was calculated using the expression

X 100

Statistical AnalysisThe results represented mean of 3 determinations ± standard deviation (SD).Differences between mean values were determined by one way ANOVA using Graph Pad Prism GraphicalStatistical package version 5 and comparisons were done by the Dunnett test. Differences were considered to be significant if p < 0.05.

RESULTSYields of EECA/ FractionsA total of 3.62 kg of stalks of C. afer was extracted in a total of 15L of 80 % (v/v) ethanol. The yields of EECA and fractions were 49.46 g (0.014% of the starting material). The yields of the fractions were as follows: HF (1.08 g), CF (0.35 g), DCMF (0.21 g), EAF (1.87 g), nBF (3.04 g) and AqF (19.3 g).

Phytochemical Constituents. Phytochemical screening revealed the presence of flavonoids, alkaloids, amino acid derivatives, saponins, cardiac glycosides, steroids, xanthoprotein and tannins.

Concentrations of Phenolic and Flavonoid Contents of the EE and Fractions of C. aferThe phenolic and flavonoid contents of the EECA and its fractions are presented in Table 1. It was noted that EAF contained the highest amount of phenolics followed by nBF while EAF also contained the highest quantity of flavonoids, and followed by CF and nBF respectively.

DPPH-Radical Scavenging ActivityIn Fig. 1a, b is the result of the DPPH scavenging activity of the extract/ fractions. It was observed that EAF gave the highest DPPH scavenging activity (75.17 ± 0.06%), while HF had the least potency (17.07 ± 0.06%).

Hydroxyl Radical Scavenging ActivityThe inhibition of hydroxyl radical by EECA/ fractions (Fig. 2a, b) revealed that the EECA (37.37 ± 1.65%) had the highest potency while CF (17.94 ± 1.32%) had the least.

Anti-Lipid Peroxidation ActivityThe EECA/ fractions exhibited protection

2+ against lipid peroxidation induced by Fe in a concentration (50-350 µg/ml) dependent manner (Fig. 3a, b). It showed that EAF (44.51 ± 0.37%) offered the highest protection but of all the fractions, only EAF and AqF to some extent compared favourably with ascorbic acid used as standard which gave percentage inhibition of 65.18 ± 0.07%. Antioxidant Power ActivityFig. 4 showed that the reducing power of the EECA/ fractions of C. afer increased and correlated well with increase in concentration. However, EAF showed a higher reduction power than BHT used as standard.

RBC Membrane Stability ActivityThe stability effects of the EECA/ fractions of C. afer on red blood cell membranes exposed to both heat and hypotonic lyses is represented in Fig. 5. In vitro assessment on membrane stability showed that they inhibited heat and hypotonic-induced lyses of red blood cells to varying degrees with EAF (70.30 ± 1.52%) offering the highest protection. The mode of protection exhibited by the fractions was both monophasic and biphasic, with EAF and AqF comparing favourably with ibuprofen (75.94 ± 1.31%).

Inhibition of Denaturation of AlbuminThe ability of the EECA/ fractions and a known anti-inflammatory drug (ibuprofen) to inhibit denaturation of albumin is represented in Fig. 6. The results revealed that there was inhibition at the different concentrations (50-350 µg/ml). It was observed that AqF (58.26 ± 0.54%) offered

Abstest - Absblank

Absstandard - Absblank

Quantity of protein left

Total protein


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the highest protection and compared favourably with ibuprofen (78.21 ± 0.75%).

Table 1: Summary of the Phenolic and Flavonoid Contents of the EECA and Fractions of Cyclosorus afer.

Extract/ fractions Total Phenolic Total Flavonoid(mg/g TAE/ g extract) (mg/g QE/ g extract)

Ethanolic 17.62 ± 0.43 0.98 ± 0.09 HF 4.40 ± 0.27 0.39 ± 0.01 CF 43.45 ± 0.58 1.79 ± 0.06 DCMF 17.34 ± 0.32 0.72 ± 0.03 EAF 81.64 ± 0.38 4.51 ± 0.07 nBF 52.76 ± 0.64 1.36 ± 0.04 AqF 15.20 ± 0.38 0.56 ± 0.01

Each value represented the mean ± SD n = 3 determinations. HF: (Hexane fraction), CF: (Chloroform fraction), DCMF: (Dichloromethane fraction), EAF: (Ethyl acetate fraction), nBF: (n-Butanol fraction) and AqF: (Aqueous fraction)

0 50 100 150 200 250 300 3500

20

40

60

80

100Ascorbic acid

HF

CF

DCMF

EAF

Concentration (mg/ml)

% D

PP

H-R

adic

al S

cave

ngin

gA

ctiv

ity

Figure 1a: Percentage DPPH-Radical Scavenging Activity of HF, CF, DCMF and EAF.

0 50 100 150 200 250 300 3500

20

40

60

80

100nBF

AqF

Ethanolic extract

Ascorbic acid


% D

PP

H-R

adic

al S

caven

gin

gA

ctiv

ity

Figure 1b: Percentage DPPH-Radical Scavenging Activity of nBF, AqF and EE.Each value represented mean ± SD of n = 3 determinations. EAF: (Ethyl acetate Fraction), nBF: (Butanol Fraction), AqF: (Aqueous Fraction), and EE: (Ethanolic extract).


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0 50 100 150 200 250 300 3500

10

20

30

40 HF

CF

DCMF

EAF

ASCORBIC ACID

Concentration (mg/ml

% H

ydro

xyl

Rad

ical

Sca

ven

gin

g A

ctiv

ity

Figure 2a: Percentage Inhibition of Hydroxyl Radical Potential of HF, CF, DCMF and EAF.

0 50 100 150 200 250 300 3500

10

20

30

40

50nBF

AqF

Ethanolic Extract

Ascorbic Acid


% H

ydro

xyl

Rad

ical

Sca

ven

gin

g A

ctiv

ity

Figure 2b: Percentage Inhibition of Hydroxyl Radical Potential of nBF, AqF and EE.Each value represented the mean ± SD of n = 3 determinations. HF: (Hexane Fraction), CF: (Chloroform Fraction), DCMF: (Dichloromethane Fraction), EAF: (Ethylacetate Fraction), nBF: (Butanol Fraction), AqF: (Aqueous Fraction), and EE:(Ethanolic extract).

0 50 100 150 200 250 300 3500

20

40

60

80HF

CF

DCMF

EAF

Ascorbic Acid


% A

nti

-Lip

id P

erox

ida

tio

n

Figure 3a: Percentage Anti-Lipid Peroxidation in the Presence of HF, CF, DCMF and EAF.


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0 50 100 150 200 250 300 3500

20

40

60

80nBF

AqF

Ethanolic Extract

Ascorbic Acid

Concentration ( mg/ml)

% A

nti

-Lip

id P

erox

idat

ion

Figure 3b: Percentage Anti-Lipid Peroxidation in the Presence of nBF, AqF and EE. Each value represent the mean ± SD of n = 3 determinations. HF: (Hexane Fraction), CF: (Chloroform Fraction), DCMF: (Dichloromethane Fraction), EAF: (Ethylacetate Fraction, nBF: (Butanol Fraction), AqF: (Aqueous Fraction), and EE: (Ethanolic extract).

0 50 100 150 200 250 300 350

0.0

0.1

0.2

0.3

0.4

BHT

EE

HF

CF

DCMF

EAF

BF

AqF


Abs

@ 7

00nm

Figure 4: Ferric Reducing Power of the EECA and Fractions of C. afer.Each value represented the mean ± SD of n = 3 determinations. HF: (Hexane Fraction), CF: (Chloroform Fraction), DCMF: (Dichloromethane Fraction), EAF: (Ethyl acetate Fraction), nBF: (Butanol Fraction), AqF: (Aqueous Fraction), EE: (Ethanolic Extract), and BHT:( Butylated Hydroxy Toluene).


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Figure 5: Membrane Stabilizing Profiles of the EECA and its Fractions.Each value represented mean ± SD of n = 3 determinations. HF: (Hexane Fraction), CF: (Chloroform Fraction), DCMF: (Dichloromethane Fraction), EAF: (Ethyl Acetate Fraction), nBF: (Butanol Fraction), and AqF: (Aqueous Fraction).

Figure 6: Percentage Inhibition of Albumin Denaturation of the EECA and its Fractions.Each value represented the mean + SD of n = 3 determinations. HF: (Hexane Fraction), CF: (Chloroform Fraction), DCMF: (Dichloromethane Fraction), EAF: (Ethyl Acetate Fraction), nBF: (Butanol Fraction), and AqF: (Aqueous Fraction).


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DISCUSSION.Studies have supported the use of antioxidant supplementation in reducing the level of oxidative stress and in slowing or preventing the development of complications associated with diseases ( ). However, many synthetic antioxidant components have shown to elicit toxic and/or mutagenic effects, which have shifted the attention towards natural antioxidants. The medicinal potentials of many plants have been attributed to the antioxidant properties of their phytochemicals. In this study, phytochemical screening revealed the presence of saponins, flavonoids, tannins, alkaloids, amino acid derivatives, cardiac glycosides, steroids and xanth protein in the EECA of C. afer, which is in agreement with the earlier observations of Asim and Amal (2011) and Pauline et al. (2011) in their investigations on the biological activities of Cyclosorus parasitica.

Phytochemical analyses of the extract of C. afer and its fractions revealed that the plant is rich in phenolic compounds i.e. both tannins and flavonoids. It was observed that ethylacetate fraction (EAF) contained the highest amounts of both phenolics and flavonoids (Table 1). It was not quite surprising therefore that EAF exhibited highest activity in all the assays in this study. Earlier studies (Egemole et al., 2007; Oyedapo et al., 1999; Bode and Oyedapo, 2011) revealed that ethylacetate is normally employed for the extraction of phenolics especially flavonoids for biochemical investigations.

Chu et al. (2000) reported that, total antioxidant activities of plant extracts cannot be evaluated with a single method due to the complex nature of phytochemicals. A number of assays have been introduced. Each method relates to the generation of a different radical, acting through a variety of mechanisms and the measurement of a range of end points at a fixed time point or over a range. Two types of approach have been taken, first is the inhibition assays in which the extent of the scavenging by hydrogen- or electron-donation of a pre-formed free radical is the marker of antioxidant activity. Secondly, the assays involving the presence of antioxidant system during the generation of the radical (Rice-Evans et al., 1996).

Devasagayam et al., 2004

The DPPH radical scavenging assay (an inhibition assay) is a rapid, simple and convenient method to investigate the ability of the phenolic components in extracts to act as donors of hydrogen atoms or electrons (Mensor et al., 2001, Stoileva et al., 2007). Numerous plant constituents have been demonstrated to exhibit free radical scavenging or antioxidants activity. Figure 1a, b showed the rate of hydrogen donating ability of the EECA and fractions of C. afer with only HF showed very poor radical scavenging, although the observed activity was concentration dependent. The radical scavenging activity of these fractions correlated well with their phenolic contents which according to the earlier report of Skerget et al. (2005) that the antioxidant activity of plant materials closely correlated with the content of their phenolic contents. Phenolics are well recognized as potential antioxidants and free radical scavengers due to their hydroxyl groups (Dorman et al., 2003; Majid et al., 2004). The findings of this study are consistent with literature data that showed that several extracts, fractions and flavonoids isolated from different plants are able to donate hydrogen atoms.

Hydroxyl radical is the principal contributor of tissue injury; it is an extremely reactive free radical making it a very dangerous and harmful with a very short in vivo half-life (Valko et al., 2007). It has been implicated as a highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells (Yasuda et al., 2000). Hydroxyl radicals are generated by direct addition of Fe (II) salts to a reaction mixture containing phosphate buffer. The C. afer stalk extracts were found to be powerful scavengers of hydroxyl radicals employing deoxyribose assay as shown in Figure 2a, b. It was noted that nBF gave the highest hydroxyl radical scavenging activity followed by DCMF, while HF and EAF gave the same percentage inhibition activity. The antiradical and scavenging activity of these extracts could be attributed to flavonoids which usually contain the high metal chelating activity (Dorman et al., 2003; Oboh et al., 2007).

The ability of the EECA and its fractions to reduce iron (III) to (II) was assessed using the method of Chu et al. (2000). This assay measures


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the reducing ability of antioxidants against oxidative effects of reactive oxygen species. Increasing absorbance indicates an increase in reductive ability (Riaz et al., 2011). The reducing power of the EE and its fractions increased with increase in the amount of the extracts (Fig. 4) and this reduction might serve as a significant indicator of their potential antioxidant activity. However, the reduction power of EAF was relatively more pronounced than that of BHT that was used as standard. The reducing powers of different fractions were in the following order: EAF > EECA > nBF > CF > DCMF > AqF > HF.

Membrane lipids are particularly susceptible to lipid peroxidation (LP) due to the presence of polyunsaturated fatty acids. Since membranes form the basis of many cellular organelles like mitochondria, plasma membranes, endoplasmic reticulum, lysosomes, peroxisomes, etc, the damage caused by LP is highly detrimental to the functioning of the cell and its survival. It has been implicated in the pathogenesis of a number of diseases and clinical conditions. Hence, agents that can interact with secondary radicals formed during peroxidation and scavenging them, would be effective in inhibiting LP (Lakshmi et al., 2005). The degree of LP was assayed by estimating the thiobarbituric acid-reactive substances (TBARS) (Yoshiuki et al., 1981, Masao et al., (1993). It involved spectrophotometric measurement of the pink pigment produced through reaction of thiobarbituric acid (TBA) with malondialdehyde (MDA) and other secondary lipid peroxidation products. The EECA and its fractions exhibited

2+ protection against LP induced by Fe in a concentration dependent manner (Fig. 3a,b). Flavonoids and other phenolic compounds (hydroxylcinnamic derivatives, tannins etc) of plant origins have been reported as scavengers and inhibitors of lipid peroxidation (Formica and Regelson, 1995). Therefore, the ability of the extract/fractions to inhibit lipid peroxidation could be attributed to the phenolic contents.

Anti-inflammatory agents elicit their effects through a spectrum of different modes of action and various models are often employed to test or screen anti-inflammatory drugs or agents which included cotton pellet granulation in animals, platelet aggregation (Kumar and Sadique, 1987;

Oyedapo et al., 1999), stabilization of erythrocyte membranes (Sadique et al., 1989; Oyedapo et al., 1997, 1999). Erythrocytes have been used as a model by a number of researchers for the study of interaction of drugs with membranes (Horie, et al., 1979; Oyedapo and Famurewa, 1995). Membrane stabilizing activity of RBC membrane exhibited by some drugs, serves as a useful in vitro method for assessing the anti-inflammatory activity of various compounds (Naibi et al., 1985). In vitro assessment of the effects of the EECA and fractions of C. afer on membrane stabilization showed that it inhibited heat and hypotonic induced lyses of red blood cells. The study demonstrated capability of the extracts to stabilize red blood cell membrane, which is an indication of the extracts' ability to prevent rupture, or haemolysis in hypotonic-stress induced condition.

The result (Fig. 5) showed that EAF and EECA exhibited potent and appreciable membrane stabilizing activities. The mode of action of the fractions was both monophasic and biphasic. The anti-inflammatory effect of the EECA and the different fractions could be attributed to the presence of flavonoidal and saponin entities as shown in the preliminary phytochemical screening. It has been demonstrated that saponins and sapogenins were known to cause significant anti-inflammatory and analgesic effects. Studies have established that both saponins and flavonoids possess the abilities to bind to the cellular macromolecules on the surfaces of the membranes thereby preventing the interactions of inflammatory agents from the cell surfaces (Sur et al., 2001; Coi et al., 2005).

Denaturation of proteins is a well-documented cause of inflammation (Sachin et al., 2010). Under normal conditions, white blood cells (WBC) circulate in the blood stream waiting to be called by damaged tissues to the site of injury or infection. Movement of the leukocytes into the damaged tissue from blood requires the cells to squeeze between endothelial cells that line the blood vessels wall. Increase in WBC population occurs at the site of inflammation and this could mean that either there was migration of WBCs to the site or increase in the population of the white cells pre-existing at the site of inflammation.


Leukocyte aggregation at the site of inflammation is a fundamental event in the inflammatory process (Umapathy et al., 2010). There was inhibition of heat induced albumin denaturation at the different concentrations (Figure 6), showing that there was decrease in the migration of WBC count by the EECA and fractions of C. afer to the site of inflammation. The ability of the EECA of C. afer, and the different fractions to inhibit thermal protein denaturation might also contribute to its anti-inflammatory properties. Polyphenols are well known natural products known to possess several notable biological properties including anti-oxidant and anti-inflammatory (Bhattacharya, 2011). In this study, the in vitro anti-inflammatory activity of C. afer could be attributed to its polyphenols content which might be due to the synergistic effect with other phytoconstituents rather than single constituent.

In conclusion, this study revealed that the stalk of C. afer possessed a number of bioactive molecules that could be beneficial in the management of oxidative and inflammatory related conditions. Hence, the plant could be regarded as an excellent source of natural antioxidants and a free radical scavenger, which could be employed as medicinal plants. However, there is need to carry out toxicological analysis of the extract and fractions on laboratory animals.

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