Albizia zygia ( DC . ) Macbr . Hydroethanol Root Extract Exerts Anti-Oedemic and In Vivo Antioxidant Activities in Animal Models

Article history: Received on: 30/01/2017 Accepted on: 26/03/2017 Available online: 30/04/2017 Various parts of Albizia zygia (DC.) Macbr. (Family: Leguminosae-Mimosoideae) are used traditionally in the management of arthritis and pain. The present study evaluated the anti-inflammatory and in vivo antioxidant effects of the hydroethanolic root extract of Albizia zygia in animal models. Oedema was induced by the injection of carrageenan (2% suspension) into the right footpads of 7-day old chicks. The footpad thickness was measured hourly for 5 h. Carrageenan was also administered into rat paw subcutaneous tissues, which subsequently induced oxidative stress and inflammation at the injected site. After 5 h the rats were sacrificed and inflamed paw tissues isolated for antioxidant enzyme analysis. The extract (30-300 mg/kg, p.o.) and the positive control, diclofenac (3-30 mg/kg, i.p.), significantly attenuated pedal oedema when given either preemptively or curatively. The extract also augmented the endogenous expression of superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) as well as reduced the activity of myeloperoxidase (MPO) and malondialdehyde (MDA) level at the inflamed site. The results suggest that the hydroethanolic extract of Albizia zygia exerts in vivo anti-inflammatory activity after oral administration and also has antioxidant properties which may contribute to its activity.


INTRODUCTION
Inflammation is a biological response of an organism's immune system to cellular and vascularized tissue damage (Nathan, 2002).Inflammation when gone awry, initiates an innocent bystander attack on the host cells within hours after trauma.The local release of toxic metabolites and enzymes destroy uninjured tissue (Stahel et al., 2007).Tissue injury can generate reactive oxygen species (ROS) by releasing transition metal ions from damaged cells, activating phagocytes or generating NO .(Aruoma, 1998).The phagocytes also generate O 2 .-,H 2 O 2 and HOCl to kill pathogens.The overproduction of these ROS can further worsen tissue damage (Aruoma, 1998).
The methanol stem bark extract is very active in malaria and sleeping sickness (Arbonnier, 2004;Abdalla et al., 2012).Since there is scanty scientific documentation on the efficacy of A. zygia roots in the traditional management of inflammation, the present study validates such folkloric use of the plant.

Plant material collection
Albizia zygia roots were collected from the campus of Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana (6°40ʹ31.8ʺN;1°34ʹ44.1ʺW) in the month of January, 2015.The root sample was authenticated by Dr. George Henry Sam in the Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences, KNUST.A voucher specimen (no: KNUST/H/M/2016/R001) has been preserved in the Faculty herbarium.

Preparation of extract
The roots were room-dried for 2 weeks, and then pulverized into fine powder.The powder was cold macerated with 70 % ( v / v ) ethanol.The hydro-alcoholic supernatant was filtered and then concentrated to a brown syrupy mass under reduced pressure at 50°C in a rotary evaporator (R-210, BUCHI, Switzerland).The extract was further dried in a hot air oven at 50°C and stored at 4°C until use.The final product, of yield 9.03 % ( w / w ), is subsequently referred to as AZE or extract in this study.

Animals
Cockerels (Gallus gallus; strain Shaver 579, Akropong Farms, Kumasi, Ghana) were obtained one-day post-hatch and housed in stainless steel cages (34 cm × 57cm × 40 cm) at a population density of 12-13 chicks per cage.Feed (Agricare Ltd, Kumasi, Ghana) and water were available ad libitum through 1-qt gravity-fed feeders and waterers.Overhead incandescent illumination was provided with room temperature at 29°C.Chicks were tested at 7 days of age.Group sample sizes of six were utilized throughout the study.
Sprague-Dawley rats (100-200 g) of both sexes acquired from Noguchi Memorial Institute of Medical Research (NMIMR), University of Ghana, Accra were grouped (n=6) in stainless steel colony cages.Animals had free access to chow (Agricare Ltd, Kumasi, Ghana), and were provided water ad lib.The animals were humanely handled in all experiments according to the internationally accepted principles concerning animal protection in experimental protocols (EU Directive of 2010; 2010/63/EU).In addition, ethical approval was granted by the Ethics Committee of the Department (FPPS/PCOL/010/2015).

Carrageenan-induced pedal oedema test
The acute anti-oedemic effect of AZE was investigated in the carrageenan-induced oedema model in chicks (Roach and Sufka, 2003;Abotsi et al., 2012).A volume of 10 μl carrageenan suspension (2 % w / v ) was administered into the chicks' right footpads.Dorso-ventral ipsilateral pedal thickness was measured initially (0 h) and at every hour post-carrageenan administration for 5 h (using electronic callipers Z22855, Milomex Ltd, Bedfordshire, UK) (Murayama et al., 1991).Oedema was calculated as the normalized percentage change in foot thickness from time 0 h using the equation: % change in pedal thickness=[(V t -V 0 )/V 0 ×100]; where, V 0 is carrageenan preadministration pedal thickness (0 h) and V t is carrageenan postadministration pedal thickness (at time t).

In vivo antioxidant assay
The assay of endogenous antioxidants was carried out according to the protocol prescribed by Halici et al. (2007).Intraplantar carrageenan administration was made into the subcutaneous paw tissues of rats, which subsequently induced oxidative stress at the inflamed site.Rats were assigned into eight groups (n=3) with respective drug treatments as follows: experimental naïve control (normal saline; 10 ml/kg; i.p.), vehicle control (2% w / v tragacanth, 10 ml/kg; p.o.), AZE suspended in 2 % tragacanth (30-300 mg/kg, p.o.) or diclofenac (3-30 mg/kg, i.p.).General intraplantar introduction of 100 μl carrageenan (1% w / v ), either 30 min (i.p.) or 1 h (p.o) post-drug administration, induced oedema in the right hind paw.Five hours after carrageenan administration, rats were sacrificed by cervical dislocation.Oedematous paw tissues were harvested and immediately stored at -80 o C for subsequent enzyme analysis.A volume of 4.5 ml of TNG buffer at pH 7.4 (150 mM Tris HCl, 150 mM NaCl, 1% Triton X 100, 10% glycerol and protease inhibitor cocktail) was added to each sample, and homogenized on ice using a homogenizer (Ultra-Turrax T-25; IKA-Labortechnik, Staufen, Germany) at 24000 ×g for 15 min.Each sample was centrifuged at 4000 ×g for 20 min, decanted and preserved for enzyme analysis.Sample protein content was calculated using the Bradford method (Bradford, 1976).All subsequent microtiter plate readings were carried out in triplicates.

Superoxide dismutase (SOD) assay
The activity of superoxide dismutase (SOD) was measured according to the method of Misra and Fridovich (1972).A volume of 500 μl homogenate was centrifuged in 150 μl of icecold chloroform and 750 μl ethanol (96 % v/v) at 2000 ×g for 20 min.Successive additions of 1 ml carbonate buffer (0.1 M; pH 10.2) and 0.5 ml EDTA (0.6 mM) were made to 500 μl of supernatant aliquot.A 1.3 mM adrenaline solution of volume 0.05 ml was then added to initiate adrenochrome formation.A blank solution containing all reagents except tissue homogenate was processed in a similar manner.A volume of 150 µl was then dispensed into a 96-well plate.Absorbance was read spectrophotometrically at 480 nm using Synergy H1 Multi-mode Reader (BioTek Technologies, Winooski, VT, USA).The percentage inhibition of adrenaline autoxidation was calculated as: Specific SOD activity was expressed in units per mg protein, where 1 unit is the enzyme quantity needed to prevent 50 % of the autoxidation of adrenaline at 25 °C; calculated using the formula:

Catalase (CAT) assay
The test principle is based on the propensity of CAT to hydrolyse H 2 O 2 , thereby inhibiting the dichromate in acetic acid reduction to chromic acetate by H 2 O 2 (Sinha, 1972).Successive additions of 0.4 ml H 2 O 2 (1.18 M) and 1 ml phosphate buffer (0.01 M; pH 7.0) were made to 0.1 ml of homogenate and incubated for 5 min (25 o C).A 2 ml dichromate-acetic acid mixture (containing 3 parts glacial acetic acid and 1 part 5% potassium dichromate) was then added to terminate the reaction.A volume of 150 µl of the mixture was pipetted into a 96-well plate.Chromogenate absorbance was calorimetrically read at 620 nm.
Specific CAT activity was expressed as units per mg protein based on the molar extinction coefficient of H 2 O 2 , 39.4 M-1 cm-1 at 620 nm.One unit is the enzyme quantity needed to hydrolyse 1 mmol of H 2 O 2 /min in a neutral pH (25 °C), i.e.

Reduced glutathione (GSH) assay
The concentration of GSH in inflamed paw was measured by the procedure stated by Ellman (1959).To 100 µl of the homogenate was added 2.4 ml 0.02 M EDTA; solution was then cooled at 4 °C for 10 min.Additions of 2 ml H 2 O and 0.5 ml of 50 % w / v TCA were made to the mixture and centrifuged at 3000 ×g for 5 min.About 50 μl of 10 mM DTNB solution and 2 ml of Tris buffer (0.4 M; pH 8.9) were then thoroughly mixed with 1 ml of the supernatant and the reaction incubated for 5 min (25 o C).A reaction mixture was repeated also for the blank.The wells of 96well plate were filled with 150 µl of mixture and absorbance was spectrophotometrically read at 412 nm.GSH concentration was expressed in µmol per mg protein, and determined using the curve y = 0.0004x + 0.0026.

Myeloperoxidase (MPO) assay
Enzyme concentration was determined spectrophotometrically by a modified o-dianisidine method (Şenoğlu et al., 2009).An assay mixture-consisting of 5 ml 0.02 M o-dianisidine + 3 ml H 2 O 2 (0.01 M) + 3 ml phosphate buffer (0.1 M; pH 6.0) and made up to 30 ml-was freshly prepared.About 125 µl aliquot of it was dispensed into a 96-well microtiter plate and topped up with 0.01 ml of tissue homogenate.The absorbance increase was straightway read at 460 nm in 60 s cycles for 600 s.MPO specific activity was expressed in units per mg protein, where 1 unit increases absorbance by 0.001 per 60 s.

Lipid peroxidation assay
MDA levels were determined by a modified protocol (Heath et al., 1968).To a 1 ml volume of homogenate was mixed 3 ml of the mixture (3 ml of 20 % TCA containing 0.5% TBA) in a test tube.It was heated at 95°C for 30 min, cooled immediately and then centrifuged at 5000 ×g for 10 min.Absorbance was initially read at 532 nm and then read again at 600 nm to correct for nonspecific absorbance.The molar extinction coefficient of MDA-TBA abduct, 155 mM -1 cm -1 , was used to determine the levels of MDA from the equation:

Analysis of data
Statistical analyses and ED 50 determinations were done using GraphPad Prism for Windows version 6 (GraphPad Software, San Diego, CA, USA).The time course curves were analyzed by two-way repeated measures analysis of variance (ANOVA).The mean treatment differences at each time period were compared by Tukey's post hoctest.Subsequent area under the curves (AUCs) were then calculated (arbitrary units) to determine the total treatment effect.One-way ANOVA was used to analyse the AUC mean differences; pair-wise comparisons were done using Tukey's post hoc test.P<0.05 between treatments was considered statistically significant.

Phytochemical screening
Alkaloids, tannins, flavonoids, saponins, glycosides and terpenoids were found to be present in AZE.

Carrageenan-induced pedal oedema test
The introduction of 10 μl of 2% carrageenan suspension into pedal tissues of chicks effectuated an acute local oedema.Oedema peaked at 2-3 h in all paws (Figures 1 and 2), correlating with the findings of treatments across the time courses of AZE (30-300 mg/kg; p.o.) and diclofenac (3-30 mg/kg, i.p.) (Roach and Sufka, 2003).Figures 1(a, c) and 2(a, c) represent the time course of effects of the respective treatments.The column graphs of AZE and diclofenac are represented in Figure 1(b, d) for preemptive and Figure 2(b, d) for curative treatments respectively.

Superoxide dismutase assay
The vehicle-treated group showed a marked reduction in SOD activity at the inflamed site compared to that in the naïve subjects (Table 1).AZE (100 and 300 mg/kg; p.o.)-treated rats had a significant increase in SOD activity (F 4, 10 =13.91,P=0.0004).A dose-dependent effect was obtained for diclofenac (F 4, 10 =21.71,P<0.0001).

Catalase assay
The intraplantar carrageenan administration induced an acute local inflammation in the rat paw with resultant oxidative stress.The oxidative stress caused a marked reduction in CAT levels in negative control group compared to the naïve group (Table 1).On the other hand, AZE (100 and 300 mg/kg; p.o.) and diclofenac (3-30 mg/kg; i.p.) increased CAT activity (F 7, 16 =16.24,P<0.0001).The lowest dose of AZE produced no significant change in CAT activity but a dose-dependent effect was seen with diclofenac.

Myeloperoxidase assay
MPO activity increased in the negative control group relative to naïve group (Table 1).AZE and diclofenac pretreatments significantly reversed the increased MPO concentration in their respective groups (F 5, 11 =3.221, P=0.0493).The significant effects were seen in only AZE 100 mg/kg (p.o.) and diclofenac 30 mg/kg (i.p.)

Lipid peroxidation assay
Oxidative stress increases lipid peroxidation with a resultant increase in malondialdehyde generated from the decomposition of lipid hydroperoxides.The measurement of MDA levels revealed an increased concentration in negative control group relative to the naïve group (Table 1).The administration of AZE (30-300 mg/kg, p.o.) and diclofenac (3-30 mg/kg, i.p.) significantly and dose-dependently reduced MDA levels (F 7, 16 =10.47, P<0.0001).

DISCUSSION
This present study has established that oral administration of the hydroethanolic root extract of Albizia zygia has anti-inflammatory and antioxidant properties in animals.
Carrageenan-induced pedal oedema in chicks is simple to conduct, reproducible and highly reliable.Overdependence on rodent models is curtailed by replacing rodents with a less sentient species, chicks (Fereidoni et al., 2000;Roachand Sufka, 2003).
Carrageenan administration produces a local inflammation with no systemic effects and no antigenicity.Pedal oedema in chicks lasts approximately 6 h, peaking 2 h post-carrageenan injection.Subcutaneous injection of carrageenan induces a biphasic inflammation characterized by oedema, erythema and pain.Proinflammatory mediators-histamine, serotonin, bradykinin, tachykinins, complement and ROS are responsible for inducing the inflammation (Morris, 2003).Prostaglandins then induce the last phase of the oedema; as a result of the action of inducible forms of nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) (Posadas et al., 2004).Preemptive and curative administration of AZEto chicks significantly suppressed the acute local oedema in both treatment protocols.The anti-oedemic effects of AZE may possibly be attributable to the inhibition of synthesis, release and/or activity of the inflammatory mediators.The precise mechanism by which AZE suppresses inflammation however needs to be established.AZE was compared to the standard antiinflammatory analgesic, diclofenac.It is a non-selective COX inhibitor which prevents the synthesis of the arachidonic acid metabolites such as prostaglandins, thromboxanes and leukotrienes (Gan, 2010).The body's generation of ROS helps maintain homeostasis in the cells of a healthy tissue.They also serve as signaling molecules (Devasagayam et al., 2004).Oxidative imbalance abounds in inflamed tissues after trauma.The relative diminution of endogenous antioxidants increase cellular susceptibility to oxidative damage (Wang et al., 2004;Valerio et al., 2009).Free radicals possess short half-lives and are less concentrated; as such oxidative damage is best assessed by measuring the amount of products and enzymes produced (Hovatta et al., 2010).SOD converts O 2 .
-to H 2 O 2 , and then either CAT or GPx converts the H 2 O 2 to oxygen and water.GSH and its congeners detoxify peroxynitrites, hydroperoxides and electrophiles such as reactive aldehydes (malondialdehyde, MDA).MPO is used by neutrophils to synthesize HOCl during phagocytic lysis of foreign bodies (Zhu et al., 2008;Şenoğlu et al., 2009;Halliwell, 2012).Histamine, serotonin and eicosanoids mediate the early phase of the carrageenan-induced paw oedema whilst polymorphonuclear infiltration is responsible for the delayed inflammatory phase.Neutrophil-derived OH, H 2 O 2 and O 2 .-,MPO and MDA concentrations are augmented whilst antioxidant levels are reduced in the paw tissue (Bilici et al., 2002;Halici et al., 2007).Pretreatment of the rats with AZE significantly elevated the activities/levels of the preventive enzyme antioxidants (SOD and CAT) and the scavenger, chain breaking or repair antioxidant, GSH, at the inflamed site.AZE also reduced the formation of MDA and the pro-inflammatory enzyme MPO.Similar results were obtained for diclofenac.The anti-inflammatory effect of AZE may at least partly be as the result of the prevention of oxidative damage.

CONCLUSION
In conclusion, the hydroethanol extract of the roots of Albizia zygia demonstrated anti-oedemic and in vivo antioxidant activity in animals.The results validate the folkloric use of A. zygia in the treatment of inflammatory disorders.

Table 1
Effect of AZE and diclofenac on the levels of SOD, CAT, MPO, GSH and MDA.