Aqueous fraction of Alstonia boonei de Wild leaves suppressed inflammatory responses in carrageenan and formaldehyde induced arthritic rats

Biomedicine & Pharmacotherapy 86 (2017) 95–101

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Original article

Aqueous fraction of Alstonia boonei de Wild leaves suppressed inflammatory responses in carrageenan and formaldehyde induced arthritic rats Omowumi O. Akinnawo, God’swill N. Anyasor* , Odutola Osilesi Department of Biochemistry, Benjamin S. Carson (Snr.) School of Medicine, Babcock University, Ilishan-Remo, Ogun State, PMB, 21244 Ikeja, Nigeria

A R T I C L E I N F O

Article history: Received 28 October 2016 Received in revised form 23 November 2016 Accepted 30 November 2016 Keywords: Alstonia boonei Anti-Inflammation Antioxidant Phytochemicals

A B S T R A C T

Alstonia boonie de Wild is an ethnomedical plant used as therapy against inflammatory disorders. This study evaluated the most active anti-inflammatory and anti-oxidant fraction of A. boonei leaves using in vitro and in vivo models. Quantitative phytochemical analysis, anti-protein denaturation and hypotonicity-induced hemolysis of human red blood cell membrane (HRBC), radical scavenging activity assays, carrageenan and formaldehyde-induced inflammation models were carried out. Results showed that aqueous and ethyl acetate fractions of 70% methanol extract of A. boonie leaves contained high quantities of total phenolic and flavonoid compounds compared with hexane and butanol fractions. Aqueous fraction of A. boonie leaves significantly (P < 0.05) inhibited heat-induced protein denaturation, stabilized hypotonicity-induced hemolysis of HRBC, scavenged DPPH, NO
and H2O2 radicals in a concentration-dependent manner compared with other fractions in vitro. In addition, orally administered 50–250–mg/kg body weight (b.w.) aqueous fraction of A. boonei leaves suppressed carrageenan-induced rat paw edema thickness by 74.32%, 79.22% and 89.86% respectively at 6th h in a dose-dependent manner comparable with animals treated with standard diclofenac sodium (88.69%) in vivo. Furthermore, investigation of formaldehyde-induced inflammation in rats showed that 50–250 mg/ kg b.w. aqueous fraction of A. boonei reduced plasma alanine aminotransferase and aspartate aminotransferase activities. Aqueous fraction of A. boonei also suppressed eosinophils, monocytes and basophils, total white blood cell, total platelet, neutrophil and lymphocyte counts and modulated plasma lipid profile compared with control group. Aqueous fraction of A. boonei leaves exhibited substantial active anti-inflammatory and antioxidant activities. Hence, an aqueous fraction of A. boonei leaves could be channeled towards pharmaceutical drug development. In addition, this study provided scientific insight to account for the traditional use of A. boonei leaves in ethnomedical practice. © 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction

Abbreviation: ALT, alanine aminotransferase; AST, aspartate amino transferase; BUHREC, babcock university health, research ethics committee; COX-2, cyclooxygenase 2; DPPH, 1,1-deiphenyl 2-picryl hydrazyl; HRBC, human red blood cell membrane; EMB, eosinophils, monocytes and basophils; GC–MS, gas chromatography mass spectrometry; HDL, high density lipoprotein; IC50, fifty percent inhibitory concentration; LDL, low density lipoprotein; NRC, national research council; NSAIDs, non-steroidal anti-inflammatory drugs; OECD, organization for economic cooperation and development; RT, retention time; SEM, standard error of mean; TP, total protein; VLDL, very low density lipoprotein; WBC, white blood cell. * Corresponding author. E-mail addresses: [email protected] (O.O. Akinnawo), [email protected] (G.N. Anyasor), [email protected] (O. Osilesi). http://dx.doi.org/10.1016/j.biopha.2016.11.145 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.

Medicinal plants are known to contain important bioactive compounds that could be harnessed for the discovery and development of novel therapeutic drugs. These drugs of plant origin could possess therapeutic advantage against inflammatory and oxidative stress related diseases with minimal side effects than conventional drugs [1]. Inflammation is considered primarily as a physiologic defence mechanism which protects the body system against infections, toxins, allergens or other noxious stimuli [2]. However, persistent inflammation may act as an etiologic factor for many chronic debilitating musculoskeletal disorders [3]. In addition, upregulation of reactive oxygen and nitrogen species, prostaglandins, histamines, bradykinins, leukotrienes and polymorphonuclear

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cells had been implicated in the pathogenesis of several inflammatory diseases including rheumatoid arthritis, Alzheimer’s, Parkinson, arteriosclerosis and aging [4]. Currently, disorders associated with inflammation are managed through the implementation of various intervention strategies aimed at suppressing pro-inflammatory mediators. However, application of steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) by patients with inflammatory disorders have been associated with severe adverse effects. Hence, there is a need to discover and develop an alternative anti-inflammatory drug of plant origin with minimal or no side effects. Numerous scientific investigations have verified that herbal remedies elicit maximum therapeutic benefits with minimum side effects [1]. In addition, medicinal plants are generally more acceptable to ethnomedical practitioners when compared to the expensive synthetic medicines and majority of the world population especially in developing countries depends on herbal medicines to meet their health requirements since synthetic medicine are usually faced with the challenge of adulteration which influences its efficacy [5]. Alstonia boonei de Wild of the family Apocynaceae, commonly called “devil tree” consist of about 40–60 species widely used ethno-medically for the treatment of several inflammatoryassociated disorders [6]. It is found mostly in tropical and subtropical Africa, Southeast Asia, Central America and Australia [7]. A. boonei is a large deciduous evergreen tree with a height of about 45 m and diameter of 1.2 m. In the South-Western Nigeria it is called “Awun”, South-Eastern Nigerian calls it “Egbu-ora” and “Ukpukunu” by South-Central Nigeria [6]. A. boonie leaves and stem bark extracts have been used in ethnomedical practice as therapy for the treatment of arthritis and rheumatism [8]. The stem bark of A. boonei has been reported to possess potent neuroleptic and anxiolytic properties in mice [9]. It also contains minerals like calcium, phosphorous, iron, sodium, potassium and magnesium and phytochemicals including alkaloids, tannins, saponins, flavonoids, cardiac glycoside and vitamin C [6]. However, there is little or no scientific validation of the use of A. boonei leaves extract in the management of inflammatory disorders. Therefore, this study was designed to evaluate the anti-inflammatory and antioxidant fraction of Alstonia boonei leaves using in vitro and in vivo models with the objective to proffer scientific explanation to the ethnomedical use of this plant. 2. Materials and methods 2.1. Plant collection and identification Fresh leaves of A. boonei were collected from a farm in Ondo State, Nigeria. It was authenticated by Professor E.B. Esan, Plant Breeding and Biotechnology, Department of Basic Sciences and voucher sample with number FHI 107254 has been deposited at the Forestry Research Institute of Nigeria, Ibadan, Nigeria. 2.2. Animals Sixty male albino rats (Wistar strains), weighing between 150 and 250 g were purchased from an inbred colony, Babcock University Animal Facility. Animals were acclimatized at 28  5  C with a 55  5% relative humidity in a standard wire mesh wooden cage under 12 h light/dark cycle for 14 d prior to experimentation. Animals were provided with commercial pelleted rat chow and water. All animal experimental protocols were in conformity with the National Institute of Health/ National Research Council (NRC) Regulations on Laboratory Animal Care and Use Guidelines [10]. Institutional ethical

approval with identification number BUHREC065/14 was obtained from the Babcock University Health, Research Ethics Committee (BUHREC). Acute toxicity testing was in line with the median lethal dose of the test fraction in rats according to the Organization for Economic Cooperation and Development (OECD) guidelines 425 [11]. 2.3. Preparation of plant extract A. boonei leaves were thoroughly washed to remove debris, oven-dried at 35  C and subsequently pulverized using Warring blender. Pulverized A. boonei leaves (300 g) were soaked with 1800 ml 70% methanol in ratio 1:8 (w/v) and mixed intermittently for 48 h. Subsequently, the suspension was filtered using Whatman No. 1 filter paper. The obtained filtrate was concentrated using a rotary evaporator (RE52-3 model, LIDA Instrument) at 45  C. The concentrate was immediately reconstituted in distilled water in a ratio of 1:2 (concentrate: distilled water) and further partitioned using successive solvent partitioning method as follows: hexane, butanol, ethyl acetate and the remaining fraction was considered as an aqueous fraction. The different solvent fractions were concentrated using rotary evaporator at 40–45  C and stored in a refrigerator until further use. 2.4. Quantitative phytochemical analysis Total phenolic content was determined according to the method of Singleton et al. [12]. Flavonoid content was determined using the complex aluminum chloride method as described by Ordonez et al. [13]. Tannin content was determined according to the modified vanillin-HCl methanol method as described by Noha et al. [14]. Percentage saponins and alkaloids were determined using gravimetric methods as described by Okwu and Josiah [15] and Onyilagba and Islam [16] respectively. 2.5. In vitro assays for anti-inflammatory and antioxidant determination Stabilization of hypotonicity-induced hemolysis of human red blood cell membrane (HRBC) by A. boonei fractions was carried out using the method described by Sadique et al. [17]. Anti-thermal induced protein denaturation by the fractions was performed using the method described by Sakat et al. [18] with minor modifications. DPPH (1,1-diphenyl 2-picryl hydrazine) radical scavenging activity was carried out according to the method described by Cox et al. [19]. Determinations of nitric oxide radical and hydrogen peroxide scavenging activities were carried out using the methods adopted by Ebrahimzadeh et al. [20] and Karunakaran and Kumaran [21] respectively. 2.6. Assay for anti-inflammatory activity (in vivo models) 2.6.1. Model I: carrageenan-induced paw edema Effect of A. boonei fraction on carrageenan-induced rat paw edema was carried out using the protocol described by Shashank et al. [22]. Thirty rats were randomly assigned into six groups of 5 rats each. Right hind paw thickness of each rat was initially measured using a micrometer screw gauge, this was followed by the oral administration of different doses of A. boonei leaves aqueous fraction and diclofenac sodium to experimental animals in line with the study protocol. Rat paw edema was induced by injecting 0.1 ml 1% (w/v) carrageenan suspension in 0.9% normal saline subcutaneously into the subplantar region of the right hind paw, one hour after oral administration of test drugs. The increase in paw thickness was measured for 6 h after carrageenan injection.

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Experimental design was as follows: Group 1: Normal; rats not induced with edema, administered 0.5 ml 0.9% NaCl. Group 2: Control (untreated); rats induced with edema, administered 0.5 ml 0.9% NaCl. Group 3: Rats induced with edema, treated with 10 mg/kg body weight (b.w.) diclofenac sodium. Group 4: Rats induced with edema, treated with 50 mg/kg b.w. aqueous fraction of A. boonei leaves. Group 5: Rats induced with edema, treated with 150 mg/kg b.w. aqueous fraction of A. boonei leaves. Group 6: Rats induced with edema, treated with 250 mg/kg b.w. aqueous fraction of A. boonei leaves. Anti-inflammatory activity (AA) in percentage was calculated according to the following formula AAð%Þ ¼

ðCt  CoÞcontrol  ðCt  CoÞtreated  100 ðCt  CoÞcontrol

Where Ct: right hind paw thickness (mm) at time t, Co: right hind paw thickness (mm) before carrageenan injection. (Ct  Co) control: increase in paw size after carrageenan injection to control rats at time t. (Ct  Co) treated: increase in paw size after carrageenan injection to treated (reference or sample drug) rats at time t. 2.6.2. Model II: formaldehyde-induced inflammation in rat Effect of A. boonie leaves aqueous fraction on formaldehydeinduced inflammation in rat was carried out using the protocol described by Eun-Michoi and Jae-Kwan [23]. Thirty rats were assigned into six groups of five rats viz. normal group (0.5 ml 0.9% NaCl), control group induced with edema using 0.02 ml 2% v/v formaldehyde and orally administered with 0.5 ml 0.9% NaCl, standard group (10 mg/kg b.w. diclofenac sodium) and test groups of 50, 150 and 250 mg/kg b.w. aqueous fraction of A. boonie leaves induced edema by injecting 0.02 ml formaldehyde (2%v/v) solution into the left hind paw of the rats beneath the plantar aponeurosis on the first and third day 30 min after oral administration of the test drugs. Treatment lasted for 7 d. At the end of treatment period, rats were made to fast overnight, anesthetized by using petroleum ether, sacrificed and blood sample obtained by cardiac puncture into lithium heparinised tubes for biochemical assays: plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and lipid profile assays using the methods of Reitman and Frankel [24] and Henry [25] respectively. In addition, hematological parameters were assessed at the Haematological Unit, Babcock University Teaching Hospital using auto-analyser (Swelab Alfa 3- Part Hematology Analyser by Boule Medicals). 2.7. Statistical analysis Statistical analysis was carried out with the aid of SPSS for windows; SPSS Inc., Chicago, standard version 17.0 to determine the difference between mean using One-Way Analysis of Variance (ANOVA). This was followed by post hoc analysis using least significance difference (LSD) analytical test. Nonlinear regression was carried out to determine fifty percent inhibitory concentration (IC50) for test fractions using GraphPad Prism 7.00. Graphical presentations were plotted using Microsoft Excel 2013 version; Microsoft Office Suite 2013. P < 0.05 and 0.01 were considered

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significant. All values were expressed as mean  SEM of triplicate readings. 3. Results 3.1. Quantitative phytochemical analysis of A. boonei leaves Table 1 showed that aqueous and ethyl acetate fractions of A. boonie leaves had significantly high (P < 0.05) total phenol and flavonoid contents when compared with the other fractions. In addition, A. boonie leaves contained 51.34  0.43% saponins, 19.78  0.21% tannins and 4.20  0.20% alkaloids. 3.2. Evaluation of anti-inflammatory activity of A. boonei leaves fractions in vitro Table 2 showed that 20–100 mg/mL fractions of A. boonei leaves and diclofenac sodium inhibited heat-induced protein denaturation in a concentration dependent manner. Aqueous fraction (IC50 = 34.71  0.25 mg/mL) exhibited a significantly (P < 0.05) high inhibition of heat-induced protein denaturation when compared with the other test fractions. This was followed by ethyl acetate fraction (IC50 = 64.33  0.66 mg/mL) > hexane fraction (IC50 = 95.12  1.15 mg/mL) > butanol fraction (IC50 = 127.80  0.25 mg/mL). Furthermore, 50–250 mg/mL fractions of A. boonei leaves and diclofenac sodium stabilized hypotonicity-induced hemolysis of human red blood cell membrane (HRBC) in a concentration dependent manner. Aqueous fraction (IC50 = 61.89  0.16 mg/mL) exhibited a significantly (P < 0.05) high stabilization of hypotonicity-induced hemolysis of HRBC. This was followed by ethyl acetate fraction (IC50 = 149.30  1.12 mg/mL) > hexane fraction (IC50 = 228.10  0.16 mg/mL) > butanol fraction (IC50 = 258.90  0.09 mg/ mL). 3.3. Evaluation of anti-oxidant activity of fractions of A. boonei leaves in vitro Table 2 showed that 10–100 mg/mL fractions of A. boonei leaves and ascorbic acid scavenged DPPH, NO
and H2O2 radicals in a concentration dependent manner. Aqueous fraction (IC50 = 37.29  0.02 mg/mL) exhibited a significantly (P < 0.05) high DPPH radical scavenging activity followed by ethyl acetate fraction (IC50 = 66.08  1.03 mg/mL) > hexane fraction (IC50 = 72.88  1.00 mg/mL) > butanol fraction (IC50 = 122.90  1.03 mg/mL). Furthermore, aqueous fraction (IC50 = 12.06  0.59 mg/mL) exhibited a significantly (P < 0.05) high NO
scavenging activity. This was followed by hexane fraction (IC50 = 59.65  0.01 mg/mL) > ethyl acetate fraction (IC50 = 65.47  1.26 mg/mL) > butanol fraction (IC50 = 103.10  2.30 mg/mL). In addition, aqueous fraction (IC50 = 51.37  1.66 mg/mL) exhibited a significantly (P < 0.05) high H2O2 scavenging activity followed by ethyl acetate fraction

Table 1 Quantitative phytochemical determination of A. boonie leaves. Parameters

A. boonei leaves fractions (1 mg/ml) Ethyl acetate

Aqueous

Hexane

Butanol

Total phenol (mg GAE/g) 0.98  0.00a 0.81  0.01b 0.70  0.01 0.42  0.01 Total flavonoid (mg QE/ 0.25  0.00a 0.14  0.00b 0.07  0.00 0.07  0.00 g) GAE – indicates Gallic acid equivalent; QE-indicates Quercetin equivalent. Values were expressed as mean  SEM; (n = 3). a and b symbols indicate significantly (P < 0.05) higher compared with other fractions.

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Table 2 Anti-inflammatory and antioxidant activities of A. boonei leaf fractions in vitro. IC50 Inhibition of protein denaturation (20–100 mg/mL)

Stabilization of HRBC (50–250 mg/mL)

DPPH Scavenging activity (10–100 mg/mL)

Nitric oxide scavenging activity (10–100 mg/mL)

H2O2 Scavenging activity (25–250 mg/mL)

Diclofenac

23.90  0.50a

54.61  0.01a

23.57  1.03a

12.71  1.30a

94.94  0.96b

Aqueous Ethyl acetate Hexane Butanol

34.71  0.25b 64.33  0.66c

61.89  0.16b 149.30  0.12c

37.29  0.02b 66.08  1.03c

12.06  0.59a 65.47  1.26c

51.37  1.66a 146.62  1.20c

95.12  1.15d 127.80  0.25e

228.10  0.16d 258.90  0.09e

72.88  1.00d 122.90  1.03d

59.65  0.01b 103.10  2.30d

155.1  0.96d 221.3  1.67e

Test samples

A. boonei leaves fractions

IC50 indicates fifty percent inhibitory concentration; Different small letters indicates significantly different at P < 0.05.

(IC50 = 146.62  1.20 mg/mL) > hexane fraction (IC50 = 155.1  0.96 mg/mL) > butanol fraction (IC50 = 221.3  1.67 mg/mL). 3.4. Evaluation of anti-inflammatory activity of fractions of A. boonei leaves in vivo 3.4.1. Carrageenan and formaldehyde induced inflammation models Fig. 1 showed that orally administered 50, 150 and 250 mg/kg b. w. aqueous fraction of A. boonei leaves and 10 mg/kg diclofenac sodium significantly (P < 0.05) suppressed carrageenan-induced rat paw edema thickness from 3rd to 6th h compared with untreated control group in a dose dependent manner. Percentage inhibition of carrageenan-induced rat paw edema thickness at the 6th h by 50, 150 and 250 mg/kg b.w. aqueous fraction of A. boonei leaves and 10 mg/kg b.w. diclofenac sodium were 74.32%, 79.22%, 89.86% and 88.69% respectively. Data in Fig. 2 showed that the 50, 150 and 250 mg/kg b.w. aqueous fraction of A. boonei leaves treated rats induced inflammation using formaldehyde rats had significantly (P < 0.05) reduced ALT activity (89.2  2.58 U/L, 84.2  0.54 U/L and 67.8  0.83 U/L respectively) and AST (227.6  1.14 U/L, 200.4  0.89 U/L and 170.8  1.09 U/L respectively) compared with untreated control group ALT (133.8  1.78 U/L) and AST (235.8  0.83 U/L) in a dose-dependent manner. Furthermore, 250 mg/kg b.w. aqueous fraction of A. boonei leaves had a significantly (P < 0.05) reduced plasma ALT and AST activities compared with the standard diclofenac drug ALT (75.2  1.30 U/L) and AST (198.6  0.54 U/L).

Data in Table 3 showed that 50, 150 and 250 mg/kg b.w. aqueous fraction of A. boonei leaves and 10 mg/kg diclofenac sodium treated formaldehyde-induced arthritic rats significantly (P < 0.05) reduced the level of EMB (10.62  0.35%, 8.52  0.61%, 5.34  0.36% and 6.82  0.38% respectively), WBC (8.80  0.73  109/L, 8.24  0.55  109/L, 6.20  0.21 109/L and 6.70  0.38  109/L respectively), total platelet (552.40  2.50  109/L, 457.20  1.48  109/L, 321.20  0.44  109/L and 299.40  1.51 109/L), neutrophil (6.94  0.36  109/L, 5.26  0.38  109/L, 3.41  0.09  109/L and 3.5  0.14  109/L) and lymphocyte (6.46  0.20  109/L, 4.76  0.20  109/L, 2.68  0.31 109/L and 3.12  0.22  109/L) compared with untreated control group EMB (11.61  0.33%), WBC (10.98  0.37  109/L), total platelet (766.20  1.64  109/L), neutrophil (8.5  0.60  109/L) and lymphocyte (7.32  0.44  109/L). 3.5. Lipid profile assessment in formaldehyde-induced inflammation rat model Data in Fig. 3 showed that 50, 150 and 250 mg/kg aqueous fraction of A. boonei leaves and 10 mg/kg diclofenac sodium treated formaldehyde-induced arthritic rats had significantly (P < 0.05) reduced concentrations of plasma total cholesterol (114.4  4.50 mg/dL, 128.4  3.36 mg/dL, 100.2  2.94 mg/dL and 107.2  3.96 mg/dL respectively), LDL-cholesterol (29.12  4.88 mg/dL, 38.44  4.97 mg/dL, 17.28  2.47 mg/dL and 24.24  6.05 mg/dL respectively), VLDL-cholesterol (11.28  0.71 mg/dL, 10.96  0.93 mg/dL, 10.52  0.70 mg/dL and

Fig. 1. Percentage inhibition of carrageenan-induced rat paw edema treated with 50–250 mg/kg b.w. aqueous fraction of A. boonei leaves.

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Fig. 2. Effects of 50–250 mg/kg b.w. A. boonei leaves on plasma ALT and AST activities in rats induced with arthritis using formaldehyde. Different small letters denote significantly different at P < 0.05.

Table 3 Effects of 50–250 mg/kg b.w. aqueous fraction of A. boonei leaves on hematological parameters of formaldehyde-induced arthritic rats. Hematological Parameters

Normal

Control (Untreated)

Diclofenac Sodium (10 mg/kg)

A. boonie (50 mg/kg)

A. boonei (150 mg/kg)

A. boonie (250 mg/kg)

EMB count (%) WBC count (10^9/L) Total platelet count (10^9/L) Neutrophil count (10^9/L) Lymphocyte count (10^9/L)

5.48  0.27 6.22  0.27 229.60  1.30 1.46  0.08 2.44  0.23

11.61  0.33e 10.98  0.37c 766.20  1.64e 8.5  0.60d 7.32  0.44d

6.82  0.38b 6.70  0.38a 299.40  1.51a 3.5  0.14a 3.12  0.22a

10.62  0.35d 8.80  0.73b 552.40  2.50d 6.94  0.36c 6.46  0.20c

8.52  0.61c 8.24  0.55b 457.20  1.48c 5.26  0.38b 4.76  0.20b

5.34  0.36a 6.20  0.21a 321.20  0.44b 3.41  0.09a 2.68  0.31a

EMB indicates eosinophil, monocytes and basophil; WBC indicates white blood cell. Different small letters across rows indicate significantly different at P < 0.05.

Fig. 3. Effects of 50–250 mg/kg b.w. on plasma lipid profile of rats induced with arthritis using formaldehyde. Different small letters denote significantly different at P < 0.05.

9.96  0.88 mg/dL respectively) and triglycerides (56.4  3.57 mg/ dL, 54.8  4.65 mg/dL, 52.6  3.50 mg/dL and 49.8  4.43 mg/dL respectively) when compared with untreated control group total cholesterol (140  4.47 mg/dL), LDL-cholesterol (85.96  7.52 mg/ dL), VLDL-cholesterol (18.2  1.17 mg/dL) and triglycerides (91  5.87 mg/dL). Furthermore, 50, 150 and 250 mg/kg b.w. aqueous fraction of A. boonei leaves and 10 mg/kg b.w. diclofenac sodium treated formaldehyde-induced arthritic rats had significantly (P < 0.05) elevated concentrations of plasma HDL-cholesterol (74  1.58 mg/dL, 77.8  6.09 mg/dL, 72.4  2.0 mg/dL and

73  3.16 mg/dL respectively) when compared with untreated control group (35.8  6.30 mg/dL). 4. Discussion The understanding that medicinal value of plant materials lie within their constituent phytochemicals has resulted in an intensified efforts by investigators to study phytochemicals possessing anti-inflammatory and antioxidant properties to be used as therapy against chronic disorders and infectious diseases

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[26]. In this present study, quantitative phytochemical screening of A. boonei leaves showed an appreciable quantity of total phenolic and flavonoid contents in aqueous and ethyl acetate fractions. Phenolic and flavonoids are known to possess anti-inflammatory and antioxidant properties [27]. This could be partly due to the presence of polar functional groups in the polyphenolic structures with the capacity to perhaps scavenge free radicals. In addition, A. boonei leaves also contained tannins, saponins, and alkaloids. Previous study had shown that tannins and saponins are immunomodulatory agents which could hasten wound healing and inflamed mucous membrane [28]. Alkaloids have also been documented to possess analgesic, antispasmodic and anti-inflammatory properties [29]. This detected phytochemicals could account for the ethnomedical use of A. boonei leave extracts as therapy in the treatment of several inflammatory disorders This present study showed that aqueous fraction of A. boonei leaves exhibited high inhibitory and stabilization effects on heatinduced protein denaturation and hypotonicity-induced hemolysis of HRBC respectively when compared with the other fractions. This suggested that the aqueous fraction of A. boonei leaves could possess anti-inflammatory property. The denaturation of tissue proteins in the body system is one of the well-documented causes of inflammation [30]. Hence, agents that could prevent protein denaturation would serve as a promising candidate for antiinflammatory drug development. Conventional anti-inflammatory drugs including salicylic acid have been reported to inhibit protein denaturation [18]. In addition, anti-inflammatory compounds possess the capacity to stabilize red blood cell membrane against hypotonicity-induced hemolysis [31]. They also play an important role in limiting inflammatory responses through the prevention of lysosomal release of pro-inflammatory factors [5]. Similar outcomes have been reported for plant extracts exhibiting an antiinflammatory effect in vitro [18,32]. Investigation of the antioxidant property of A. boonei leaves showed that the aqueous fraction exhibited high DPPH, H2O2 and NO
radical scavenging effects than the other studied fractions. This could indicate that the aqueous fraction of A. boonei leaves possesses compounds with the higher antioxidant property. This seems to support the observation that aqueous fraction of A. boonei contained appreciable quantities of phenolic and flavonoids. Previous study had shown that phyto-compounds with free radical scavenging activity donate hydrogen ion(s) to stabilize the tissue-damaging reactive oxygen species and also terminates free radical initiated deleterious chain reactions [33]. This chain reaction could also induce and sustain inflammatory responses [34]. Furthermore, H2O2 and NO
had been implicated as part of pro-inflammatory mediators released from activated neutrophils and macrophages in vivo [35]. These pro-inflammatory mediators have been shown to propagate inflammatory signals by inducing upregulation of cytokines, tumor necrosis factor and interferon-g [36]. Hence, phytochemicals that could neutralize H2O2 and NO
could also reduce the progression of inflammatory responses. It was therefore deduced at this stage that the aqueous fraction of A. boonie leaves might be the most active anti-inflammatory and antioxidant fraction than the other studied fractions. In vivo anti-inflammatory study showed that 50, 150 and 250 mg/kg b.w. aqueous fraction of A. boonei leaves suppressed carrageenan-induced rat paw edema thickness in a dose-dependent manner and the result was comparable with standard 10 mg/ kg b.w. diclofenac sodium. This suggests that the aqueous fraction of A. boonei leaves possesses anti-inflammatory property in vivo. Other researchers have reported that medicinal plant extracts with anti-inflammatory property exhibited a similar effect in the carrageenan-induced arthritis model in vivo [37,38]. Previous study had shown that the early phase (1–2 h) of the carrageenan inflammatory model is mainly mediated by pro-inflammatory

mediators including histamine, serotonin and increased prostaglandin synthesis around the localized tissue damage while the later phase (3rd h) is mediated by bradykinin, leukotrienes, polymorphonuclear cells and sustained by prostaglandins produced by cyclooxygenase 2 (COX-2) through a feedback mechanism. The later phase is believed to be sensitive to most conventional anti-inflammatory drugs [37]. In this present study, an aqueous fraction of A. boonei leaves suppressed carrageenaninduced paw edema from the 3rd to 6th h which indicates that the anti-inflammatory mechanism of action of A. boonie leaf may be related to prostaglandin inhibition. This could explain the folkloric use of A. boonei leaf in the treatment of rheumatoid arthritis. Furthermore, investigation of the formaldehyde-induced arthritis model showed that 50, 150 and 250 mg/kg b.w. aqueous fraction of A. boonei leaves reduced plasma ALT and AST activities in animals induced with arthritis using formaldehyde compared with untreated control groups. This indicated that aqueous fraction of A. boonei leaves could possess hepatoprotective property against formaldehyde-induced hepatic damage. Xing-Jiu et al. [39] had reported that elevation of plasma ALT and AST activities serves as a biomarker for liver damage due to leakage of these enzymes into the blood stream. In this present study, the hepatoprotective property of aqueous fraction of A. boonie leaves may be related to its antioxidant and anti-inflammatory properties. In addition, the aqueous fraction of A. boonie leaves suppressed the EMB, total WBC, TP, neutrophil and lymphocyte counts in animals induced with inflammation using 2% (v/v) formaldehyde. This indicated that aqueous fraction of A. boonei leaves could possess immunomodulatory effect against infiltrating immunological cells to the localized site of tissue damage. Previous study reported that conventional anti-inflammatory drugs exhibit an immunomodulatory action against infiltrating cells at the localized site of inflammation [40]. More so, lipid profile analysis showed that aqueous fraction of A. boonie leaves reduced plasma total cholesterol, triglycerides, LDL-cholesterol and VLDL-cholesterol concentrations in animals induced with inflammation using formaldehyde while it elevated HDL-cholesterol concentrations. This suggested that the aqueous fraction of A. boonei leaves could also possess hypocholesterolemic property with the capacity to interfere with the progression lipid associated inflammatory response. Previous studies had associated inflammation with cardiovascular events [41]. In addition, rheumatoid arthritis patients have been reported to have altered lipoprotein pattern with a decrease in plasma HDL-cholesterol concentration [42]. Compounds that could alter lipoprotein pattern associated with inflammatory disorders may confer protection against inflammatory related disease [43]. 5. Conclusion The data from this study showed that the aqueous fraction of A. boonei leaves exhibited an anti-inflammatory effect in vivo. It also provided some insight into the anti-inflammatory mechanism of action which may be through inhibition of prostaglandins biosynthesis, neutralization of reactive oxygen and nitrogen species, hepatoprotective, immunomodulatory and hypocholesterolemic effects. Therefore, the aqueous fraction of A. boonei leaves could be harnessed as a source of anti-inflammatory drug(s). However, further study is required to fully elucidate the molecular basis for the anti-inflammatory and antioxidant properties of aqueous fraction of A. boonei leaves. Conflict of interest The authors declare that there are no known conflicts of interest associated with this work and there has been no significant

O.O. Akinnawo et al. / Biomedicine & Pharmacotherapy 86 (2017) 95–101

financial support for this work that could have influenced its outcome.

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