ORIGINAL ARTICLE INTRODUCTION. Lucky L Nwidu 1, Ekramy Elmorsy 2, Oboma I Yibala 3, Wayne G Carter 4, ABSTRACT

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1 ORIGINAL ARTICLE Hepato-Protetive and Antioxidant Effets of Spondias Mombin Leaf and Stem Extrats upon Carbon Tetrahloride-Indued Hepatotoxiity and Oxidative Stress Luky L Nwidu 1, Ekramy Elmorsy 2, Oboma I Yibala 3, Wayne G Carter 4, 1 Department of Experimental Pharmaology and Toxiology, Faulty of Pharmaeutial Sienes, University of Port Harourt, Choba, East West Road, Rivers State, Nigeria. 2 Department of Forensi Mediine and Clinial Toxiology, Faulty of Mediine, Mansoura University, Egypt. 3 Department of Medial laboratory Siene, Faulty of Basi Medial Sienes, College Health Sienes, Niger Delta University, Wilberfore Island, Bayelsa State, Nigeria. 4 Shool of Mediine, University of Nottingham, Royal Derby Hospital Centre, Derby, UK. ABSTRACT Objetives: Spondias mombin is used in folk mediine in Nigeria for the treatment of hepatitis. This study omparatively evaluates the in vivo hepato-protetive and antioxidant effets of Spondias mombin leaf (SML) and Spondias mombin stem (SMS) methanoli extrats in a rat model of hepatotoxiity. Methods: 42 rats were equally divided into seven groups of six animals eah. Group A reeived water, Group B water, Groups C and D reeived SML at 500 and 1000 mg/ kg bw, respetively, Groups E and F reeived SMS 500 and 1000 mg/kg bw, respetively, and Group G reeived silymarin at 100 mg/kg. All extrats and drugs were administered daily by oral gavage for a total of seven days, and then for Groups B to G aute hepatotoxiity was indued by administering CCl 4. After 48 hours rats were sarified and assayed for histologial and biohemial indies of hepatotoxiity. Results: CCl 4 treatment indued liver injury, with signifiantly inreased levels of markers of hepatoellular injury alanine aminotransferase (ALT), aspartate transaminase (AST) total bilirubin (TBIL) and onjugated bilirubin (CBIL), as well as a signifiant redution of total irulatory protein. SML or SMS plant extrats at 500 and 1000 mg/kg prior to CCl 4 treatment signifiantly ameliorated liver injury, and dereased the levels of ALT, AST, TBIL, and CBIL. SML or SMS extrats signifiantly inreased ellular levels of glutathione, the ativities of atalase and superoxide dismutase, and signifiantly dereased thiobarbituri aid reative substanes. Conlusion: This study provides preliminary evidene supporting the potential benefit of Spondias mombin for treatment of xenobioti-indued hepatotoxiity. Key words: Antioxidant, hepato-protetion, hepatotoxiity, oxidative stress, spondias mombin Correspondene: Luky Nwidu, Department of Pharmaology, Faulty of Pharmay, Niger Delta University, Yenegoa, Nigeria. meneluky@yahoo.om Aess this artile online Website: Quik Response Code: INTRODUCTION Liver diseases ontribute markedly to the global burden of diseases and are major auses of illness and death worldwide. [1-4] Liver diseases remain a publi health hallenge, for whih the development of new pharmaeutial treatments are required. The evaluation of the hepatoprotetive benefits of mediinal plants using laboratory animals is a useful initial step in determining drug safety of new biomoleules. [5-9] Natural produts from ethnomediine provide safe and effetive alternative treatments for hepatotoxiity. Many previous reports have assoiated these hepato-protetive effets with endogenous phytoextrats or phyto-ompounds that are rih in natural antioxidants. [5-15] Hene an inreasing number of bioative ompounds and plant extrats have been evaluated for hepato-protetive and antioxidant effets against hepatotoxin-indued liver damage. [16] The phenoli ompounds ommonly found in both edible and traditional mediinal plants are inriminated with multiple biologial ativities, inluding This is an open aess artile distributed under the terms of the Creative Commons Attribution NonCommerial ShareAlike 3.0 Liense, whih allows others to remix, tweak, and build upon the work non ommerially, as long as the author is redited and the new reations are liensed under the idential terms. For reprints ontat: invoie@jblinpharm.org Cite this artile as: Nwidu L, Elmorsy E, Yibala OI,Carter WG. Hepatoprotetive Effets of Hydromethanoli Leaf and Stem Extrats of Spondias mombin in. J Basi Clin Pharma 2017;8:S11-S19. S Journal of Basi and Clinial Pharmay

2 free radial savenging ativity. [18-20] It has been suggested that natural antioxidants in food, suh as phenoli ompounds or flavonoids, might play an essential role in the prevention of oxidative stress-related disorders and diseases, and in the redution of premature mortality. [21-22] Flavonoids are ertainly ubiquitous in the epidermal ells of plant parts suh as the flowers, leaves, stems, roots, seeds, and fruits, and exist in glyosidi and non-gluosidi forms. [23] Spondias mombin L. (Anaardiaeae) is ommonly known as (English), akika (Yoruba), ijikara (Igbo), tsadarmaser (Hausa), habbuh (Fulani), nsukakara (Efik) and atoa (Ashanti). [24] It is a deiduous eret tree, whih grows up to meters in height with a trunk m wide. [25-26] Spondias mombin is ommonly found in the tropial Amerias, inluding the West Indies, and has been naturalized in parts of Afria, inluding Ghana, and some parts of Asia. [26] In ethnomediine, Spondias mombin plant parts inluding the stem-bark, leaves, and roots have been used for the treatment of various onditions. Spondias mombin possesses anti-mirobial [27,28], and anti-viral ativities [29], with leaves used, for example, for their anti-inflammatory [30], anthelminti ativity [31], haematini [32], and sedative [33] ativities, and stem-bark has an anti-myobaterial [34] ativity Phytohemial sreening indiates that the Spondias mombin leaf (SML) ontains tannins, saponins, alkaloids, flavonoids and phenols. [35] The leaves are also rih in asorbi aid, niain, and ontain riboflavin and thiamine. [35] Although the hepatoprotetive effets of SML and Oimum gratissimum have been evaluated in rats after intoxiation with dimethyl nitrosamine, [36] the effets of SML or SMS on CCl 4 -indued hepatotoxiity has yet to be assessed. Hene the aim of this study was to establish if SML and SMS methanoli extrats are hepato-protetive to CCl 4 -indued hepatotoxiity in rats. MATERIALS AND METHODS General reagents and hemials Carbon tetrahloride, silymarin, diethyl ether and methanol were purhased from Sigma-Aldrih, St. Louis, Missouri, USA. Randox Diagnosti kits for serum alanine aminotransferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP), onjugated bilirubin (CBIL) and total bilirubin (TBIL) were purhased from Randox Laboratories Ltd. London, UK. Other hemials and solvents were of the highest (analytial) grade ommerially available and obtained from either Sigma-Aldrih or Merk, UK. Colletion and plant validation Fresh leaves and stem of Spondias mombin L. were olleted from Obafemi Awolowo University ampus during the month of January, The plant was identified and authentiated by Dr. Oladele Adekunle, a Taxonomist with the Forestry Department of the University of Port Harourt. A speimen of SML (20015) and SMS (20016) were deposited at Forestry Department of the University of Port Harourt, Nigeria. Preparation of plant extrat Fresh leaves and stem-bark of Spondias mombin L. were weighed, air dried and powdered. The powdered leaves (300g) and powdered stem (300g) were extrated using a old extration method (maeration) using methanol as solvent. The SML and SMS powder were soaked in one litre of 50% methanol for a period of 72 hours during whih the mixture was shaken twie daily to promote extration. The solvent was filtered over a layer of gauge and then the filtrate evaporated to dryness in vauo at 55 C. The weight of the dried leaf extrat was 21.3g, and for the stem, 9.4g. The yield obtained was 7.1% and 3.1% for SML and SMS extrats, respetively. The extrat was stored in a refrigerator for up to four weeks for use in assays. Phytohemial sreening The SML and SMS extrats were quantitatively assayed for the presene of phytohemials suh as saponins, tannins, alkaloids, terpenoids, ardia glyosides and flavonoids using the following standard proedures. To detet reduing sugars Fehling s Test: Fehling s solution A (1 ml) and Fehling s solution B (1 ml) were mixed with 1 ml of SML or 2 ml of SMS and heated in a boiling water bath for 10 minutes. The appearane of yellow and then brik red preipitate was indiative of the presene of reduing sugars. Benedit s test: An equal volume (2 ml) of Benedit s solution and SML or SMS extrats were mixed in a test tube and heated in a boiling water bath for 10 minutes. The hanges in olour to yellow, green and red refleted the presene of reduing sugars. To detet alkaloids: 10g of eah of the dry extrats were mixed with 20 ml of dilute hydrohlori aid. The mixture was shaken well and then filtered. The filtrate was used for the following tests. Mayer s test: To 3 ml of the filtrates, 1mL of Mayer s reagent (potassium meruri iodide) was added. The appearane of a white preipitate was indiative of the presene of alkaloids. Wagner s test: To 3 ml of the filtrates, 1ml of Wagner s reagent (iodine in potassium iodide) was added. The appearane of a reddish brown preipitate indiated the presene of alkaloids. Dragendorff s test: To 3 ml of the filtrates, 1 ml of Dragendorff s reagent (potassium bismuth iodide) was added. The appearane of a brik red preipitate showed the presene of alkaloids. To detet glyosides Borntrager s test: To test tubes ontaining 2 ml of either extrat, 2 ml of dilute sulphuri aid was added, and the mixture boiled for 5 min and then filtered. To the filtrate, an equal volume of hloroform was added and mixed. The organi layer was separated and then ammonia added. The presene of a pinkish red olour within the ammonia layer was indiative of the presene of anthraquinone glyosides. Keller-killiani test: To test tubes ontaining 2 ml of either extrat, 1 ml of glaial aeti aid, 3 drops of 5% (w/v) ferri hloride, and onentrated sulphuri aid were added. The disappearane of the reddish-brown olour at the juntion of the two layers, and bluishgreen olouration within the upper layer was onsistent with the presene of ardia glyosides. To detet flavonoids Shinoda test: To either of the dry extrats (2g), 5 ml of ethanol (95% v/v), 5 drops of hydrohlori aid and 0.5g of magnesium turnings were added. The generation of a pink olouration to the solution suggested the presene of flavonoids. To detet saponins Foam Test: To 2g of either extrat 20 ml of water was added and the mixture shaken vigorously and observed for persistent foaming, indiative of the presene of saponins. To detet tannins and phenolis Ferri hloride test: To 3 ml of either extrat, 3 ml of 5% (w/v) ferri hloride solution was added. Formation of a blue-blak olour S0012

3 suggested the presene of tannins and phenols. Lead aetate test: To 3mL of either extrat, 3 ml of lead aetate solution was added. The generation of a white preipitate indiated tannins and phenols were present. Experimental animals Forty-two healthy Wistar rats of average weight ( g) of either sex (21 male, 21 female) were purhased from the Animal House of the Department of Pharmaology, Faulty of Pharmay, Niger Delta University, Bayelsa State. The animals were alimatized for one week prior to experimentation. All the animals were fed on a standard how diet and given aess to water ad libitum. Experimental tehniques and protools used in this study follow the Guide to the are and use of animals in researh and teahing [37] as adopted and approved by Niger Delta University, Bayelsa State, Nigeria, Institutional Animal Care and Use Committee (NDUAEC) on 20/02/2015 via an approved irular No. NDU/2014/007. Aute toxiity study To asertain an approximate LD 50 value for a small mammal, Albino mie (25-30g) of either sex were used. Animals were divided into eight groups of three animals per group. Doses of 100, 500, 1000, 2000, 3000, 4000, and 5000 mg/kg were administered intraperitoneally per animal. The treated animals were monitored for 24 hours for mortality and behavioural hanges onsistent with toxiity. [38,39] Experimental design A total of 42 animals were weighed and divided into seven groups of six animals eah (3 male, 3 female). The protool used for indution of hepatotoxiity was as follows: Group A (normal ontrol: reeived distilled water (0.2 ml/kg bw by oral dosing), Group B reeived distilled water (0.2 ml/kg bw by oral dosing), Groups C and D reeived SML at 500 and 1000 mg/kg bw, respetively, dissolved in distilled water, Groups E and F reeived SMS 500 and 1000 mg/kg bw, respetively, dissolved in distilled water, and Group G reeived silymarin as a 100 mg/kg suspension in distilled water. All of the extrats and drugs were administered daily by oral gavage for a total of seven days. On the seventh day, Groups B to G were treated with a mixture of freshly prepared CCl 4 in liquid paraffin (2ml/kg bw, 1:1 intraperitoneally) one hour after administration of the last dosing. Body weights of all rats were reorded daily throughout the seven days of treatment. After 48 hours rats were anesthetized using diethyl ether prior to sarifie. Blood was obtained by ardia punture into an EDTA vautainer for determination of hematologial parameters of the blood samples using an Automated Hematologial Analyzer, SYSMEX KX21 (SYSMEX Corporation, Japan). The heamoglobin onentration (Hb), paked ell volume (PCV), red blood ell ount (RBC), mean orpusular hemoglobin (MCH), mean orpusular hemoglobin onentration (MCHC), mean orpusular volume (MCV), white blood ell ount (WBC), and platelet ount (PLC) were determined. For biohemial assessment, blood was spun at 3000 rpm for 10 minutes at 4ºC to separate serum into vautainer vials and stored at 4ºC until used for analyses. Livers were immediately extrated and perfused with ie-old saline (0.9% sodium hloride) before utilisation for further analyses. Measurement of biohemial parameters The serum olleted was used to determine Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP), onjugated bilirubin (CBIL), total bilirubin (TBIL) and total protein (TP) using Randox diagnosti kits. These analyses were performed at the Department of Chemial Pathology, Niger Delta Teahing Hospital (NDUTH), Okolobiri, Bayelsa state, Nigeria. Measurement of hepati antioxidant enzymes Liver tissues from experimental animals were perfused with ieold saline and transported from Niger Delta University, Faulty of Pharmay Pharmaology Laboratory on dry ie to the Shool of Mediine, University of Nottingham, Royal Derby Hospital Centre, Derby, UK and stored at -80ºC until required. Liver piees (100 mg) were died and homogenized in 100 ml of 5 mm Tris/HCl buffer (ph 7.4), 1 mm EDTA and omplete mini protease inhibitor oktail (Rohe). Homogenates were then entrifuged at 10,000 rpm at 4ºC for 10 minutes and the lear supernatant used for the estimation of antioxidant parameters (glutathione (GSH), superoxide dismutase (SOD), atalase (CAT), and also measurements of thiobarbituri reating substanes (TBARS). Glutathione levels GSH levels were determined based on the published method of Ellman et al. [40] Homogenate (0.2 ml) was mixed with 25% trihloroaeti aid (TCA) and entrifuged at 3000 rpm for 10 min. The supernatant (~0.2 ml) was mixed with 10 mm of DTNB in the presene of phosphate buffer (0.1 M, ph 7.4) and the absorbane read at 420 nm. Catalase ativity measurements The determination of atalase assay was performed based upon the method of Aebi. [41] The assay relies upon the ultraviolet absorption of hydrogen peroxide that an be measured at 240 nm. Assays were performed in the presene of 50 mm phosphate buffer. Hydrogen peroxide deomposition was monitored in a 96 well Quartz plate using a Spetramax (Thermofisher) miroplate reader. Catalase ativity was expressed as units/mg protein. Superoxide dismutase (SOD) ativity measurements Liver ytosoli SOD ativity was measured aording to the method of Kakkar et al. [42] Cytosol (0.05 ml) was mixed with sodium pyrophosphate buffer (0.052 M, ph 8.3, 1.2 ml), phenazine methosulphate (0.186 mm, 0.1 ml), nitroblue tetrazolium hloride (0.3 mm, 0.3 ml), and NADH (0.78 mm, 0.2 ml). The reation was stopped after 90s by the addition of glaial aeti aid. Colour intensity of the hromogen was extrated in butanol (2.0 ml) with vigorous shaking. The mixture was then entrifuged at 3000 rpm for 10 min and the supernatant extrated and the absorbane at 560 nm determined using a Spetramax miroplate reader. Determination of thiobarbituri reative substanes (TBARS) Lipid peroxidation was determined spetrophotometrially by measuring the level of lipid peroxidation produt, malondialdehyde (MDA), as desribed by Draper and Hadley. [43] MDA reats with Table 1: Phytohemial onstituent of SML and SMS Extrats EXTRACT Phytohemials Observations SML Extrat SMS Extrat Reduing sugars Reddish brown preipitate upon heating + + Cardia glyosides Brik red preipitate + + Saponins Persistent froth unbroken upon standing Tannins Blue blak preipitate Flavonoid Resultant solution turns yellow (+) to (+++)=deteted in moderate to abundant quantities. S0013

4 thiobarbituri aid (TBA) to form a red/pink oloured omplex that absorbs maximally in aid solution at 532 nm. Spetrophotometri measurements were reorded at 532 nm using a Spetramax miroplate reader. Histopathologial studies Portions of rat liver from eah rat of eah group were ut into piees of approximately 6 mm 3 size and fixed in phosphate buffered 10% formaldehyde solution. Liver piees were embedded in paraffin wax before thin setions of 5 µm thikness ut and then stained with hematoxylin-eosin (H and E). These thin liver setions were made into permanent slides and examined using a high-resolution mirosope after whih photomirographs were taken. Statistial analysis All statistial measures were performed using PRISM 5 (GraphPad Software In., San Diego, California USA). Unless speified otherwise results are expressed as means ± standard error of mean (SEM). One way analysis of variane (ANOVA) was used to ompare group data, followed by Tukey s multiple omparisons test. A p value of <0.05 was onsidered signifiant. RESULTS AND DISCUSSION Phytohemial studies Preliminary phytohemial sreening of the SML and SMS extrat revealed the presene of alkaloids, reduing sugars, saponins, and tannins in both extrats [Table 1]. The SMS extrat ontained visually more phytohemials with respet to saponins and tannins than the SML extrat, but for other qualitative assays, both extrats were similar. Aute toxiity study No lethality was observed in mie after a single dose (p.o.) of either SML or SMS ( mg/kg bw). From aute toxiity testing, no PARAMETERS A Table 2: Effet of Spondias mombin (leaf) and (stem) on hematologial parameters B C D E F G PCV 42 ± ± ± 3.5 ns 42 ± 3.9 ns 40 ± 6.2 ns 44 ± 6.5 ns 49 ± 3.1 ns HB 12 ± ± ± 1.8 ns 11 ± 0.7 ns 12 ± 0.8 ns 13 ± 2.3 ns 13 ± 0.6 ns WBC 13 ± ± ± 0.4 ns 11 ± 7.7 ns 11 ± 4.2 ns 8 ± 2.3 ns 13 ± 4.5 ns PLT 468 ± ± ± 1.4 ns 284 ± 316 ns 500 ± 288 ns 446 ± 306 ns 642 ± 322 ns RBC 7 ± ± ± 0.0 ns 6 ± 1.1 ns 7 ± 0.8 ns 7 ± 0.9 ns 8 ± 0.3 ns MCV 63 ± ± ± 7.1 ns 66 ± 4.4 ns 62 ± 2.6 ns 63 ± 0.8 ns 65 ± 4.2 ns MCH 17 ± ± ± ± 0.5 ns 17 ± 0.7 ns 19 ± 1.4 ns 18 ± 1.2 ns MCHC 27 ± ± ± ± 2.1 ns 26 ± 2.1 ns 30 ± 2.5 ns 27 ± 2.1 ns NEU 32 ± ± ± ± 14 ns 52 ± 13 ns 39 ± 7.2 ns 38 ± 5.4 ns LYM 63 ± ± ± ± 15 ns 42 ± 14 ns 54 ± 9.3 ns 54 ± 7.3 ns MEB 6 ± ± ± ± 2.1 ns 6 ± 3.5 ns 8 ± 3.1 ns 7 ± 4.3 ns Values represent mean ± Standard Error of mean (S.E.M) n=6. Results are displayed relates to positive ontrol values and ns p>0.05; Statistial analysis was done using one way ANOVA. Group A reeived 0.2 ml/kg distil water; B reeived CCl 4 1ml/kg; C and D reeived 500 mg/kg and 1000 mg/kg of SML+1 ml CCl 4 eah respetively; E and F reeived 500 and 1000 mg/kg of SMS+1ml CCl 4 eah respetively; G reeived 100 mg/kg of silymarin. Monoytes, eosinophil and basophils (MEB). Table 3: Effet of Spondias mombin (leaf) and Spondias mombin (stem) on biohemial parameters Treatment Control Healthy Control Positive S. mombin Dose mg/kg ALT AST ALP CBIL TBIL TP body weight (U/L) (U/L) (U/L) (μmol/l) (μmol/l) ± ± ± ± ± ± ml/kg CCl ± ± 0.6 C 97.2 ± ± ± ± 0.2 CCL ± 0.9 ns 77.4 ± 2.0 ns 48.2 ± 2.5 * 1.8 ± 0.2 ns 9.8 ± 0.4 ns 81.4 ± 2.5 * (leaf) SML CCL ± ± ± ± ± ± 0.1 S. mombin (stem) SMS CCL CCL ± ± ± 2.2 ns 52.4 ± 1.5 ns 1.4 ± 0.2 ns 9.6 ± 0.5 ns 35.2 ± ± ± ± ± ± 0.2 Silymarin CCL ± ± ± ± ± ± 0.05 Values represent mean ± Standard Error of mean (S.E.M) n=6; Signifiant results of extrats and pure drug are displayed relative to positive ontrol values; and positive ontrol displayed relative health ontrol; results with signifiant hanges from ontrols marked with alphabets (a, b, ). For signifiane: a p<0.05; b P<0.01 and P< Statistial analysis was done using one-way ANOVA. TP: Total protein; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline phosphatase; CB: onjugated proteins; TB: Total bilirubin. Figure in braket represents the alulated perentage hepatoprotetion. S0014

5 sign of toxiity was observed hours after extrat administration. Consequently, we adopted a dose range of 1/10 th and 1/5 th of the maximal dose examined (5000 mg/kg bw) for both extrats for bioassays. Weight evaluation The effet of CCl 4, SML and SMS on the body weights of rats throughout the experimental ourse is presented in Figure 1. There were signifiant hanges in the weight of the rats of eah group through the time ourse of the experiment (0-7 days) (two-way ANOVA, p<0.0001). However, when onsidering the effet of treatment, there were no signifiant differenes in body weight among the studied groups when ompared to the CCl 4 treated group (p=0.506). There was a 3.5% inrease in body weight with 500 mg/kg SMS, and this inreased to 4.3 % at 1000 mg/kg. Effet of SML and SMS on haematologial parameters The effets of SML and SMS on hematologial parameters are shown in Table 2. Extrats at either 500 or 1000 mg/kg did not have any signifiant effet on the hematologial indies evaluated exept SMS at 500 mg/kg whih indued a signifiant (p<0.05) hange in PCV when ompared to the CCl 4 intoxiated group. Effet of SML and SMS on hepati biomarkers To assess the hepato-protetive effet of pre-treatment with SML (500, 1000 mg/kg) and SMS (500, 1000 mg/kg) on CCl 4 -indued hepatotoxiity, serum ALT, AST, ALP, total protein (TP), onjugated bilirubin (CBIL), and total bilirubin (TBIL) levels in rats were determined [Table 3]. Hepatoellular toxiity indued by CCl 4 signifiantly (p< ) inreased ALT (102%), AST (58%), ALP (27%), and TBIL (62%) and signifiantly (p<0.001) dereased TP (54%) when ompared to the negative ontrol. The perentage hepato-protetion alulated for SML extrats at 500 and 1000 mg/kg was for ALT: 7% and 32%; AST: 54% and 91%; CBIL: 75% and 88%; TBIL: 62% and 41%, respetively, when ompared to the CCl 4 treated group. For SMS at 500 and 1000 mg/kg the hange of ALT was 4% and 26%; AST: 47% and 83%; CBIL: 8% and 31%; and TBIL: 59 % and 55 %, respetively, when ompared to the CCl 4 treated group. The perentage redution of TP (54%) was signifiant (p<0.001) following intoxiation with CCl 4 but pre-treatment with plant extrats inreased the perentage hepato-protetion for TP. SML at 500 and 1000 mg/kg inreased TP by 97% and 116% while SMS at 500 and 1000 mg/kg triggered a 111% and 119% inrease, respetively, ompared to the CCl 4 treated group. Silymarin (100 mg/kg), a known antioxidant, signifiantly (p<0.001) inreased the level of TP and signifiantly (p< ) dereased the levels of ALT, AST, ALP, CBIL, and TBIL by 116%, 22%, 90%, 92%, 22%,19%, and 36% respetively, ompared to the CCl 4 intoxiated group. Histopathologial analysis The effets of SML and SMS at 500 and 1000 mg/kg, and silymarin at 100 mg/kg on liver histology of CCl 4 -intoxiated rats are presented in Figure 2. The setion of the ontrol rat s liver shows normal sinusoidal spaes with a non-ongested portal trat and differentiated blood vessels and viable hepatoytes [Figure 2A]. Liver setions of rats intoxiated with CCl 4 indiated marked extensive nerosis, fatty aumulation, ballooning degeneration, gross mononulear infiltration, and loss of ellular boundaries of the hepatoytes [Figure 2B]. Pretreatment with SML at 500 mg/kg showed extensive amelioration of the histopathologial arhiteture trunated by CCl 4 intoxiation. However, prominent miro-vesiles with degenerating lipid ells (lipoid nerosis) were still present suggesting that hepatoyte insult (by CCl 4 ) was not yet fully resolved at this dose of SML [Figure 2C]. Following pre-treatment with SML at 1000 mg/kg liver tissue with areas of fibrosis and loalized areas of mild nerosis were still present, indiating that SML is only partially hepato-protetive at this dosage [Figure 2D]. Hepatoytes of rats treated with 500 mg/kg SMS showed miro-vesiles and hepatoytes with hyperhromati nulei indiating that this dose is not fully hepato-protetive [Figure 2E]. SMS at 1000 mg/kg showed infiltration of inflammatory ells mostly neutrophils along the portal trat and abundant mitoti bodies of the hepatoytes, indiating ell regeneration and hepato-protetion. After pre-treatment with Silymarin at 100 mg/kg, liver tissue displayed a loalized inflammatory response, and areas of fibrosis admixed with mitoti bodies; demonstrating healing by fibrosis, indiative of hepatoprotetion. Effet of SML and SMS on hepati antioxidants and enzyme markers The levels of the liver antioxidant GSH, and enzymes CAT and SOD were dereased by 48%, 59%, and 30%, respetively, and TBARS were inreased by 67% following intoxiation with CCl 4 [Figure 3]. By ontrast, GSH levels were inreased signifiantly (p<0.05) by 42% with pre-treatment with SML (1000 mg/kg), signifiantly (p<0.01) by 50% after pre-treatment with SMS (1000 mg/kg), and signifiantly (p<0.001) by 74% after pre-treatment with silymarin (100 mg/kg). Similarly, both CAT and SOD enzyme levels inreased signifiantly (p<0.05) by 28% and 18% after SML (1000 mg/kg), and were signifiantly (p<0.05) inreased by 41% and 20% after SMS (1000 mg/kg) pre-treatment, respetively. DISCUSSION Environmental hemial-indued hepatotoxiity is a major publi health onern. Natural antioxidants ameliorate the effets of freeradial indued oxidative stress. The effets of SML and SMS on the weights of rats, their hematologial indies, hepati enzymes, and the hepati antioxidant system were examined to reveal whether biomoleules present in these plants ould offer hepato-protetion from CCl 4 -indued ellular insult. Animals treated with SML showed no signifiant hanges in body weights but those treated with SMS revealed a dose-dependent but not signifiant inrease in body weight. An inrease of the body weights of laboratory animals following a sub-aute toxiity study of the aqueous extrat of Enantia hlorantha has been published [44]. However, in ontrast a derease of body weights in rats following administration of an ethanoli stem-bark extrat of the same plant has also been reported. [45] Body weight hanges may provide an indiator of drug effets, and have been used by others for assessment of responses to Spondias mombin drug therapy. [33] This observed effet on body weights of the animals treated with the stem extrat may be mediated through drug effets on the appetite entre within the hypothalamus. The inrease in body weights we observed orroborates earlier reports of Spondias mombin-indued redution of weight and appetite. [33] Neither the toxiant, CCl 4, nor the assessed hepato-protetive agents, indued signifiant hanges to hematologial parameters suggesting no aute adverse effets on hematopoiesis. Dereased levels in platelet ounts have been impliated in the severity of liver irrhosis. [46] However, platelets up regulation was observed only with the lower dose of SML but this effet was not signifiant. The omparative ability of methanoli extrats (500 and 1000 mg/kg -1 ) of SML and SMS on hepati lipid aumulation in fatty liver diseases and resolution of aute intoxiation were examined. Following pre-treatment of rats with SML and SMS for 7 days before hallenging with toxi insult, the alterations observed in the moleular arhiteture of the hepatoytes after CCl 4 were partially restored. This damage to the histopathologial arhiteture of hepatoytes by nerosis and membrane lipid peroxidation are ommon [47-48] aetiology mediated by CCl 4 -indued liver damage. S0015

6 Weight variation(g) a a aa b b a a b ba 0 Control CCl4 SML 500 SML 1000 SMS 500 SMS1000 Silym 100 Day 0 Day 4 Day 1 Day 5 Day 2 Day 6 Day 3 Day 7 Figure 1: The effet of CCL 4, SML and SMS on rats' weight through the ourse of experiment. Regarding to the effet of time, Two-way ANOVA results showed that there were signifiant hanges in the weight of the rats of eah group through the ourse of the experiment (0-7 days) (p-value<0.0001). However, onsidering the effet of treatments, Two-way ANOVA showed no signifiant (p=0.506) hange among the studied groups when ompared to CCL 4 treated group. Bonferroni post-test showed the differenes between the weights of the rats of eah group in the different days in omparison to day 0 (before the experiments). Data is shown as M ± SD. (a) Means p<0.05, (b) means p<0.01 while () means p< Figure 2: Effets of SML and SMS extrat on markers of oxidative stress. Oxidative stress markers (GST, CAT, SOD and TBARS) were measured from homogenised liver samples. Histograms are results displayed relative to CCl4 treated ontrol values, with signifiant hanges from Controls marked with asterisks. For signifiane: *p<0.05;**p<0.01;***p<0.001.gst: glutathione; CAT: atalase; SOD: Sodium dismutase; TBARS: Thiobarbituri aid reative substanes. S0016

7 Figure 3: Photomirograph of liver setions of rats H and E 400. A: mirograph of hepatoytes of rat treated with 0.2 ml/kg distil water showing normal liver tissues with prominent hepatoytes, hepati artery, portal trat, normal blood vessel and hepatoytes. B: mirograph of hepatoyte from rodent treated with CCL4 shows marked distortion of hepatoytes morphology with areas of omplete nerosis denoting CCL4 administered is grossly hepatotoxi at the onentration and route of administration. C: mirograph of hepatoyte of rat treated with CCL4 plus 500 mg/kg of SML shows hepatoytes tissue with prominent miro vesiles with degenerating lipid ells (lipoid nerosis) revealing that hepatoytes injury due to CCL4 not fully unresolved at this dose of SML. D: mirograph of hepatoyte of rat treated with CCL4 plus 1000 mg/kg methanoli SML showing a liver tissue with areas of fibrosis and loalized area of mild nerosis indiating that SML is hepatoprotetive at this partiular dosage; E. mirograph of hepatoytes of rats treated with CCL4 plus 500 mg/kg of methanoli SMS showing liver tissue with miro vesiles and hepatoytes with hyper hromati nulei; dosage not quite hepatoprotetive; F: mirograph of hepatoyte from rat treated with CCL4 plus 1000 mg/kg methanoli SMS showing infiltration of inflammatory ells mostly neutrophils along the portal trat and abundant mitoti bodies, indiating ell regeneration therefore substane is hepatoprotetive at this dosage; G: mirograph of liver setion from hepatoyte treated standard drug, silymarin (100 mg/kg) showing liver tissue with loalized inflammatory response and also areas of fibrosis admixed mitoti bodies; indiating healing by fibrosis and substane is hepatoprotetive. S0017

8 The toxiant CCl 4 indued hepatotoxiity with signifiantly inreased levels of AST, ALT, ALP, CBIL and TBIL. Upregulation in the levels of serum aminotransferases are surrogates of liver injury. Hepati damage aused after CCl 4 treatment triggers both an inrease in serum transaminases and alkaline phosphatase ativity. [49] Both doses of plant extrats were signifiant (p< ) at mediating effetive redution of CBIL, TBIL and ALP ompared to the positive ontrol (CCl 4 ). The SMS extrat (1000 mg/kg) provided higher hepato-protetivity than the SML for the redution of CBIL, TBIL, and ALP. The higher doses of both extrats produed higher hepato-protetive effets when assayed for ALT and AST levels than the lower doses, with SMS more hepatoprotetive than SML. The level of TP was signifiantly (p<0.001) redued following aute indution of hepatotoxiity with CCl 4, but pre-treatment with SML and SMS signifiantly (p<0.001) normalized the TP level ompared with the intoxiated rats. The mehanism of CCl 4 intoxiation of laboratory animals inludes reative oxygen speies (ROS) generation and depletion of glutathione to indue oxidative stress. [50] Administration of CCl 4 generates the free radial CCl 3 whih auses hepati damage due to the ativation of the NADPH-Cyt P 450 system of liver endoplasmi retiulum [51] leading to generation of the more reative radial, trihloromethyl peroxy (CCl 3 O 2 ) whih provokes lipid peroxidation, disruption of Ca 2+ homeostasis and apoptosis. [52] These funtional and morphologi hanges in the ellular membrane and death of hepatoytes results in leakage of hepati enzymes. The oxidation of fatty aids by free trihloromethyl radial (CCl 3 ) liberates lipid peroxides. [51] These free radials are also a likely induer of oxidative stress within a milieu defiient in antioxidants. The mehanisms of defense against free radials inludes mobilization of radial savengers and hain terminators suh as vitamins C and E, and antioxidants suh as glutathione (GSH), and redox regulatory enzymes atalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx). Therefore the tannins, saponins, alkaloids, flavonoids, phenols and asorbi aid reported to be abundant within the SML extrats of Spondias mombin [34] might play a role in this regard. This neessitated an evaluation of the effet of SML and SMS on GSH levels, CAT and SOD ativities, and the levels of TBARS, markers of lipid peroxidation. Our results demonstrate that the marked elevation in TBARS indued by CCl 4 was redued by SML and SMS signifiantly and dose dependently. Depletion in liver GSH is related frequently to hepati fatty infiltration in different experimental models [16,17,35], and the pathophysiologial onsequenes of GSH deline have been widely investigated. The redution of serum GSH aompanying CCl 4 intoxiation may relate to free radial generation. Hene the pre-treatments with SML and SMS that mediated inreased GSH levels may well be a refletion of plant hemials that savenge free radials. Indeed, serum GSH level is a sensitive biomarker of antioxidant status playing a pivotal defensive role against oxidative insults as an endogenous savenger of free radials. [54,55] The five-fold dereased GSH levels in CCl 4 intoxiated rats was signifiantly ountered, and in a dose-dependent manner, by pre-treatment with SML and SMS. This implies that SML and SMS ould improve antioxidant potential in the irulation by elevating GSH onentration thereby depressing oxidative-indued damage. Certainly the liver is reported to maintain GSH even under the ondition of elevated lipid peroxidation due to a supportive and ompensatory mehanism. [55,56] The role of SOD as an antioxidant is to onvert superoxide to hydrogen peroxide thereby proteting against the pervasive harmful effets of superoxide. The inreased ativity of SOD after SML or SMS pretreatment ompared to the derease observed with CCl 4 intoxiated rats, might also be in part responsible for the plant extrats hepatoprotetive mehanism. The elevation of CAT ativity deteted after the SML and SMS pre-treatments may be due to inreased prodution of hydrogen peroxide, sine CAT is a H 2 O 2 savenger. Colletively, we hypothesize that the elevation of SOD and CAT ativities and GSH levels in Spondias mombin treated groups will augment the endogenous antioxidant system, via biomoleules diretly present within SML and SMS extrats. The restorative effets upon liver ytoarhiteture of silymarin after CCl 4 treatment may leave the liver with sar tissue, but extensive fibrosis was not observed with SMS at 1000 mg/kg. Hene ellular regeneration may have arisen via ativation of liver stem ells. The mehanisti intervention of amelioration of CCl 4 -indued damage by SML and SMS might also be due to membrane stabilization to prevent leakage of ellular ontents, in keeping with other hepato-protetive studies inluding the use of Vernonia amygdalina, [57] Rumex rispus, [58] Chrysophyllum albidum [53] as well as Oimum gratiissimum and Spondias mombin. [35] CONCLUSION Both SML and SMS extrats are thought to display hepato-protetive effets through a stabilizing effet on hepatoyte ell membranes, promoting repair of injured hepati tissues, enhanement of radial savenging effets, and augmentation of antioxidant systems limiting oxidative insults. These hepato-protetive effets provide the premise for further evaluation of the promising therapeuti potential of Spondias mombin to ounter live damage or disease that is mediated by free radials. A protetive effet of plant extrats against CCl 4 - indued liver damage has been asribed to the presene of endogenous onstituents inluding flavonoids, tannins, triterpenoids and alkaloids. [59,60] Flavonoids represent the most ommon and extensively distributed groups of plant polyphenols and do serve as free-radial savengers and super antioxidants to prevent oxidative ell damage. [61] The presene of flavonoids and saponins in the leaves of SML has been reported. [62] Spondias mombin derived antioxidants, partiularly polyphenols, ould ontribute to the antioxidant ativities and hepatoprotetion. [63,64] However, further investigation is required to isolate the bioative agents and further haraterize the biohemial mehanisms responsible for antioxidant and hepato-protetive ativities of SML and SMS extrats. This work is ongoing in our laboratory and may well lead to the identifiation of a substane or substanes of potential linial benefit to ounter liver damage and diseases. Dislosure The authors report no onflit of interest assoiated with the prodution or publiation of this work. Funding and aknowledgements The authors dislosed reeipt of the following finanial assistane for the researh, authorship and publiation of this artile: a Niger Delta University 3 month s Sabbatial fellowship and International Fellowship from the University of Nottingham, UK to Dr Luky Legbosi. Dr Ekramy ElMorsy was also funded by an International Fellowship from the University of Nottingham, UK. Dr Wayne Carter was supported by the University of Nottingham. REFERENCES 1. Rehm J, Samokhvalov AV, Shield KD. Global burden of aloholi liver diseases. J. Hepatolo 2013;59: Byass P. The global burden of liver disease: a hallenge for methods and for publi health, BMC Med 2014;12: Mokdad AA, Lopez AD, Lozano R, Mokdad AH, Stanaway J, Murray CJL, et al. Liver irrhosis mortality in 187 ountries between 1980 and 2010: a systemati analysis. BMC Med 2014;12:145.. 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