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��ࡱ�>��	�����������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������U	��@�bjbj�n�n	4���a��ap ��������	�	����   8X�� &(���������!'#'#'#'#'#'#'$�*�|-FG'������G'����'�$�$�$�F	��!'�$�!'�$�$�&�&�������c��������#�&
'�'0&(�&,�-+#x�-�&�-�&4���$�����G'G'�$���&(�������������������������������������������������������������������������-����������	B�:	Antihyperlipidemic activities and Hematological Properties of Ethanol Extract of Blighia sapida Koenig Bark in Alloxan-induced Metabolic Syndrome in Rats 
Oluwafemi Adeleke Ojo1,2*, Adebola Busola Ojo3, Basiru Olaitan Ajiboye1, Oluwatosin Debbie Imiere1, Babatunji Emmanuel Oyinloye1 
1 Phytomedicine, Biomedical Toxicology and Diabetes Research Laboratories, Department of Biochemistry, Afe Babalola University, Ado-Ekiti, Nigeria.
2 Department of Biochemistry, University of Ilorin, Ilorin, Ado-Ekiti, Nigeria.
3 Department of Medical Biochemistry, Afe Babalola University, Ado-Ekiti, Nigeria.
Abstract
Blighia sapida (BS) has been shown to be rich sources of antioxidant, thus, we evaluated effects of B. sapida Koenig stem bark ethanol extract (BSE) on lipid metabolism and hematological indices in diabetes rats. Forty-two male rats were divided into six groups of seven rats each. Diabetes was elicited by intraperitoneal injection of alloxan (65 mg/kg body weight) for twenty-eight days and orally administered with glibenclamide (5 mg/kg), B. sapida extract (50, 100 and 150 mg/kg body weight (bw) once every day for 28 days. Serum lipid profile, markers of hepato-renal toxicity and hematological indices were examined using automated analyzer.  Data were analyzed using one-way ANOVA and p < 0.05 were considered to be statistically different. Diabetic untreated animals showed considerably elevated total cholesterol p < 0.05, significantly increase AST, ALT, ALP, urea and creatinine compared to control. Triglycerides, LDL-c, VLDL-c, AI and CRI decreased with extract administration and HDL-c increased considerable compared to untreated diabetic rats. Furthermore, significant lower hemoglobin (Hb) levels, packed cell volume (PCV), red blood cells (RBCs) levels, white blood cells (WBCs) compared with normal animals. These changes were returned to normal after the administration of extract 50, 100 and 150 mg/kg body weight. Hence, these effects were most prominent in the animals treated with 150 mg/kg body weight of B. sapida bark. This indicates that B. sapida stem bark possess anti-hyperlipidemic activity and improved the biochemical parameters within the hematological profile of diabetic rats.
Keyword: Blighia sapida, antihyperlipidemic, haematological profile, diabetes

Introduction
Diabetes mellitus (DM) is a metabolic disturbance defined by hyperglycemia caused by lack of insulin or disruption of insulin signaling as a result of lack of hypoglycemic agent or insensitivity of insulin hormone. DM is related to aberrant metabolism of macromolecules [1, 2, 3]. The illness happens as a results of exocrine gland �-cells injury, resulting in decreased secretion of insulin. It might conjointly arise once the receptors are resilient to the roles of insulin [4]. Hyperglycemia reoccurrence during diabetes leads to body proteins being glycated, that successively results in complications affecting body organs and arteries [5]. Stiffening of red blood cells could be liable for and or related to, large vessel disease in diabetes state [6]. Management option for DM is predicated on insulin therapy and oral hypoglycemic agents that have several facet effects [7]. In diabetes state, the effects and locations of involvement in biochemical activity are varied and elevated serum total triglyceride level, increase level of transaminase, creatinine and urea are of concerned [6]. Additional major issue, that confounds diabetic state, leading to death is hyperlipidemia [8]. Different ways to this fashionable pharmacotherapy of diabetes mellitus are desperately required, attributed to the lack of accessible therapies to manage all the pathological basis of the ailment, in addition to the mammoth cost and poor accessibility for several populations in the developing world [6]. Therefore, plants use as medical aid for diabetes is inspired and acclaimed, though, a number of them are lacking scientific examination. 
	Blighia sapida K.D. Koenig, also known as �Akee apple� in English, belongs to the plant family called Sapindaceae and it is noted for its highly characteristic reddish fruits. The species in this family include B. sapida K.D. Koenig, B. unijugata Baker, and B. welwitschii (Hiern) Radlk. It is recognized as �isin� in Yoruba, �gwanja kusa� in Hausa and �okpu� in Igbo [9]. B. sapida K.D. Koenig is distributed throughout Nigeria [10]. B. sapida could be a therapeutic herbal plant ordinarily utilized by trado-medical healers in Nigeria, and highly appreciated in West Africa for the management of various diseases like diabetes mellitus [11]. The fruit has inhibitory effect against �-glucosidase and �-amylase as reported by Kazeem et al. [12]. B. sapida root extract has been shown to possess normoglycemic impact [13]. The stem bark has been shown to ameliorate pancreatic �-cell dysfunction in diabetic rats [11]. Therefore, this study was focused on analyzing the effects of Blighia sapida K.D. Koenig stem bark on antihyperlipidemic and hematological parameters in diabetic rats.
















Methods
Plant material
Fresh stem bark peelings of B. sapida Koenig was obtained at a farm in the suburbs of Abeokuta, Ogun State, Nigeria. The plant was identified and authenticated by a senior taxonomist at the university herbarium with herbarium approval number (UHAE/2016/020). All plant names in this manuscript are formatted according to the latest revision in "The Plant List" ( HYPERLINK "http://www.theplantlist.org" www.theplantlist.org).
Plant extracts preparation
Stem bark was air-dried in the laboratory at temperature (25 � 2�C), pulverized using an electrical blender and the powders obtained stored till further use. The small grained sample (100 g) was extracted with solvent combination of 70% ethanol for 48 h. The extract was filtered and thereafter evaporated to dryness using rotary evaporator [14]. The concentrated extract was stored at 4 �C until further analysis. The extraction yield is as follows.
������������� Weight of the extract Percentage yield = �������������    x 100 % [15]������������� Weight of powdered stem bark 
The percentage yield of the extraction was 19.1 %.
Experimental Animals
Six-week-old male Wistar rats with an initial mean body weight of 150 � 50 g were obtained from Animal house Afe Babalola University, Ado-Ekiti, Ekiti State, Nigeria. The animals were divided into six groups of five each and adapt to investigational condition for two weeks. The animals were housed in clean metabolic cages that provided free access to water and rat pellets (Ladokun Feeds, Nigeria). The principles of Animal Care (Public Health Services, 1986) was monitored through the duration (for twenty-eight days) of the experiment. The handling of animals was conformed to the standards of National Institute of Health (NIH publication 85-23, 1985) for experimental animal maintenance [16]. The ethical committee of the Afe Babalola University approved this study with approval number (12/SCI03/015). The rats in this study followed the rules of the established Animal Ethical Committee of Afe Babalola University.
Animal grouping
The animals were randomly divided into six groups of 7 animals namely; 
Normal control group (distilled water), 
Diabetic untreated group (65 mg/kg Alloxan, intraperitoneal) 
Diabetic + glibenclamide (5 mg/kg bw) 
Diabetic + combination-1 (50 mg/kg bw BSE, oral gavage) 
Diabetic + combination-2 (100 mg/kg bw BSE, oral gavage) 
Diabetic + combination-3 (150 mg/kg bw BSE, oral gavage) 
Dose dependent studies were previously carried out in our laboratory with three different doses of BSE (50, 100 and 150 mg/kg body weight) based on ethnobotanical survey conducted by [11].
Induction of diabetes
In induction of diabetes, after acclimatization period, alloxan (65 mg/kg body weight) was dissolved in sterile physiological saline and intraperitoneal injected (5.6 mL) into the animals in the diabetic control, diabetic + glibenclamide (5 mg/kg body weight), diabetic + 50 mg/kg body weight of BSE (2.5 mL), diabetic + 100 mg/kg body weight of BSE (5.5 mL), diabetic + 150 mg/kg body weight of BSE (8.5 mL) groups to induce pancreatic �-cell dysfunction were done in morning after the rats have been fasted overnight, while the animals in normal control group received a similar volume of physiological saline. Forty-eight hours after induction, fasting blood glucose (FBG) of all rats was measured by collecting blood from the tail vein using a portable glucometer. Animals with a FBG level e" 200 mg/dl were considered as diabetic while animals with a FBG level < 200 mg/dl were disqualified [17].
Collection of blood
Under di ethyl anaesthesia, the neck area of the rats was quickly cleared to expose the jugular veins. Blood samples from each animal was collected through their jugular vein and preserved until further processing. The blood sample was collected into a dry tube and allowed to clot for 30 min before centrifuging at 3500 rpm for 10 min to collect the serum for further study [18].
Biochemical parameters
Serum lipid concentrations, in additional to aspartate and alanine aminotransferases and alkaline phosphatase as well as urea and creatinine concentrations were assayed using commercially available kits (DRG Diagnostics, USA) according to manufacturer�s protocol.
Low density lipoprotein-cholesterol was estimated according to equation as shown below:
LDL-cholesterol (mg/dl) = TC- HDL- (TG/5)
Whereas, TG/5 is equivalent to the concentration of very low density lipoprotein. VLDL means very low density lipoprotein, TG means triacylglycerol
Atherogenic index (AI) was calculated and expressed as:
Atherogenic index (AI) =   TC-HDL-cholesterol
         HDL-cholesterol.
Coronary artery risk index (CRI) was deduced using the formula below.
Coronary artery risk index (CRI) = TC (mg/dl)
        HDL-cholesterol (mg/dl)
Whereas, HDL means high density lipoprotein-cholesterol, TC means total cholesterol

Haematological analysis
The haematological parameters like packed cell volume (PCV), hemoglobin (Hb), WBC count and WBC percentage composition, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular volume (MCV) was analysed by means of an automated analyzer (Sysmex K-2 IN, Japan). Total white blood cell counts (WBC) was analysed by the haemocytometer method, whereas smears was ready and marked by using Leishman technique and numbered by the longitudinal counting method to resolve differential count. Packed cell volume (PCV) was analyzed by the micro-haematocrit method while haemoglobin (Hb) levels were assessed by the cyano methaemoglobin method [19]. 
Data analysis
Data are presented as the mean � SD (n=7). The data were analyzed by one-way analysis of variance via a statistical software package (SPSS, Version 20.0, IBM Corporation, NY, USA) one-way ANOVA using Duncan multiple range post-hoc test (DMRT). Values were considered to be significantly different at p < 0.05. 










Results
	The effect of Blighia sapida stem bark ethanol extract on fasting blood glucose level of alloxanized rats is presented in Table 1. Fasting blood glucose levels in all animals induced with alloxan was significantly increased, compared to the normal control group, p < 0.05. The fasting blood glucose levels was however, reduced considerably in diabetic + 50 mg/kg body weight of BSE, diabetic + 100 mg/kg body weight of BSE and diabetic + 150 mg/kg body weight of BSE groups in addition to glibenclamide treated animals. 
Serum lipid concentrations, calculated atherogenic index (AI) and coronary risk index (CRI) scores are displayed in Table 2. High serum concentrations of TC, TG and LDL-cholesterol levels in addition to analyzed AI and CRI scores with progressive decrease in serum HDL-cholesterol was observed within diabetic untreated group compared to the normal control. Administration with B. sapida stem bark ethanol extract to diabetic animals considerably and dose-dependently abridged TC, TG and LDL-cholesterol levels, AI and CRI in 50 ,100 and 150 mg/kg bw of BSE and glibenclamide groups compared with the diabetic untreated. Although considerably increase in serum HDL-cholesterol level was observed in the B. sapida K.D. Koenig stem bark ethanol extract treated groups compared with the diabetic untreated.
	Serum ALT, AST, ALP, urea and creatinine levels are presented in Table 3. Concentrations of serum ALT, AST, ALP, urea and creatinine levels were considerably increased within the diabetic untreated compared with the normal control. On the other hand, administration of B. sapida stem bark ethanol extract to diabetic animals progressively ameliorated these changes in 50, 100 and 150 mg/kg bw of BSE and glibenclamide treated groups. 
	Hemoglobin (Hb) levels, packed cell volume (PCV) count, red blood cells (RBCs), white blood cells (WBCs), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), neutrophils (N), lymphocyte (L), monocytes (M) and eosinophils (E) are displayed in Table 4. Hemoglobin (Hb) levels, packed cell volume (PCV), red blood cells (RBCs) levels, white blood cells (WBCs), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), neutrophils (N), lymphocyte (L), monocytes (M) and eosinophils (E) were evidently reduced in diabetic untreated compared with normal control. Though, administration of 50-150 mg/kg bw of BSE groups demonstrated a considerably increase on the biochemical parameters mentioned above. Glibenclamide treatment conjointly considerably increase these biochemical parameters. 














Discussion
In this study, we examined the effect of oral administration of B. sapida Koenig stem bark ethanol extract in diabetic rats through twenty-eight days� post-treatment period. Aberrant lipid metabolism, resulting in amassing of LDL-C, VLDL and total cholesterol in addition to reduced HDL-cholesterol, is often related to diabetes mellitus [20]. Elevated levels of LDL, VLDL, atherogenic index, coronary artery index and total cholesterol are thought of as main menace for cardiovascular disease (CVD). On the contrary, elevated HDL-cholesterol that functions in the transport of cholesterol from the periphery to the liver reduces the chance of CVD [21]. Oral intervention of B. sapida stem bark ethanol extract and the standard drug both averts dyslipidemia, decreases the chance of developing atherogenesis and coronary artery disease and amplified serum HDL-cholesterol level. This is in accordance with newly published studies [8, 22, 23, 24].
Vital reductions in haemoglobin (Hb) levels, packed cell volume (PCV), red blood cells (RBCs), white blood cells (WBCs), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), neutrophils (N), lymphocyte (L), monocytes (M) and eosinophils (E) were observed (p < 0.05) in the diabetic untreated rats. Substantial significant (p < 0.05) increase in haematological parameters occurred upon administration of the B. sapida stem bark ethanol extracts at 50 - 150 mg/kg doses compared to the diabetic untreated rats. Constant trend was observed in the levels of parameters mention above for glibenclamide treated rats. Though these effect were more pronounced in the 150 mg/kg bw of BSE group [6, 25]. This may be as a result of the presence of metabolites like saponins, phenols and alkaloids [26 27, 28]. Blood examination could be a great means of evaluating the well-being standing of animals because it performs significant physiological and biological roles in organisms [29]. However, in diabetic state, the extra glucose present reacts with haemoglobin to produce glycated haemoglobin. This therefore shown that diabetic untreated rats displayed aberrations in the haematological profiles. A number of these aberrations may result in destruction of developed red blood cells resulting in the small Hb counts accompanied with the drop in the RBC and PCV [30, 31]. Extremely low values of RBC, Hb and hematocrit might specify anemia [25, 30]. Furthermore, modulatory effect and confined toxicity might ensue as noted within the lymphocytes and neutrophils of the diabetic untreated animals. Administration of the B. sapida stem bark ethanol extract stimulates positive changes in the haematological profile of the diabetic rats. Hence, upsurge in RBC by the extract is a sign of its restorative impact on alloxan-induced anemia whereas the alteration in level of lymphocytes by the extract could specify an anti-infection activity [27, 31].
Conclusion
The results from this study indicates that B. sapida ethanol stem bark extract possesses robust anti-diabetic activity via improving dyslipidemia and ameliorate anemic condition in diabetic rats. Though, these result is based on diabetic rats and may not be applicable in human. Hence, ethanol extract of B. sapida K.D. Koenig stem bark could be a possible anti-diabetic natural product with no significant side effects. Further studies to isolate the bioactive principles using HPLC, complete safety assessment as well as activities on metabolizing enzymes and pro-inflammatory biomarkers should be carried out in vivo. 
Statement
Acknowledgment
Authors acknowledge the laboratory assistant of the Department of Biochemistry, Afe Babalola University.
Statement of Ethics
The handling of animals was conformed to the standards of National Institute of Health (NIH publication 85-23, 1985) for experimental animal maintenance. The ethical committee of the Afe Babalola University approved this study with approval number (12/SCI03/015). The rats in this study followed the rules of the established Animal Ethical Committee of Afe Babalola University.
Disclosure Statement
Authors declare no conflict of interest.
Funding Sources
This study did not receive any external funding.
Authors Contribution 
OAO design the study, ODI carried out the study, ABO wrote the manuscript, ABO and ODI carry out analysis and interpretation of data, BOA and BEO assisted with and supervised the manuscript writing, BEO did the first proof reading and BOA and BEO did the second proof reading. OAO, BOA and BEO supported the manuscript preparation, made conceptual contributions on data analysis, manuscript drafting and critically revised the manuscript. The authors have read and approved the final manuscript.
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Table 1: Effects of ethanol extract Blighia sapida stem bark on the fasting blood glucose level of alloxan-induced diabetic rats 
GroupsInitial fasting blood glucose level (mg/dl)Fasting blood glucose at 48 h after induction (mg/dl)Final fasting blood glucose at 21 days after induction (mg/dl)Control87.25 � 0.14a88.45 � 0.11a88.46 � 1.12aDiabetic control87.48  � 1.32a291.45 � 4.21b355.78 � 7.56dDiabetic + glibenclamide86.02  � 1.76a344.21 � 3.45e91.12  � 1.10aDiabetic + 50 mg/kg B.S86.21  � 1.52a387.65 � 3.12e105.10 � 3.20cDiabetic + 100 mg/kg B.S87.50  � 1.20a376.21 � 3.24d98.96 � 1.24bDiabetic + 150 mg/kg B.S87.85 � 1.49a356.24 � 3.23d90.01 � 1.10aData are presented as the mean � SEM of 7 animals. Values with different superscript letters (a-e) along a column for a given parameter are significantly different from each other between groups at P < 0.05. 



Table 2: Effect of administration of ethanol extract of Blighia sapida stem bark on Serum lipid profiles, atherogenic and coronary risk indices of alloxan-induced diabetic rat
GroupsTC (mg/dl)TG (mg/dl)LDL (mg/dl)VLDL (mg/dl)HDL (mg/dl)AICRIControl56.28�1.04a40.21�0.02a19.39�0.01a8.04�0.01a28.84�0.01a0.95�0.02a1.95�0.01aDC95.84�0.12e104.26�0.14d64.91�0.21d20.85�0.12d10.08�0.02e8.51�1.01d9.51�1.22dDC + glibenclamide62.20�0.14d53.12�1.12c33.39�1.11c10.62�0.04c18.19�0.30d2.42�0.33c3.42�0.31cDiabetic rats + 50mg/kg BSE64.32�1.20c54.36�1.18c33.33�1.08c10.87�0.38c20.12�0.14c2.20�0.41c3.19�0.58cDiabetic rats + 100mg/kg BSE69.92�1.10b44.10�0.06b34.98�0.048.82�0.25a25.12�0.06b1.74�0.30b2.78�0.28bDiabetic rats + 150mg/kg BSE55.89�1.48a40.82�1.06a18.74�0.05a8.16�0.01a28.98�1.01a0.93�0.01a1.93�0.02aData are presented as the mean � SEM of 7 animals. Values with different superscript letters (a-e) along a column for a given parameter are significantly (P < 0.05) different from each other. TC, Total cholesterol; TG, Triglyceride;Q_o�������������				�	�	

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Table 3: Effect of administration of ethanol extract of Blighia sapida stem bark on haematological parameters of alloxan-induced diabetic rat 
ParametersControlDiabetic controlDC + GlibenclamideDiabetic rats + 50 mg/kg BSEDiabetic rats + 100 mg/kg BSEDiabetic rats + 150 mg/kg BSEPCV (%)55.10�1.01a27.10�1.01e42.00�0.10d48.10�1.01c51.20�1.04b55.84�2.02aHb (g/dl)11.98�0.50a6.14�0.02e8.20�0.20d9.58�0.32c10.62�0.04b11.64�0.52aWBC (x w3/�l)2.42�0.22a0.45�0.14e1.26�0.28d1.69�0.46c2.00�0.07b2.44�0.84aN (%)49.20�1.12a26.20�2.10e36.21�0.14d39.40�1.10c46.24�0.12b49.62�0.45aL  (%)30.11�0.01a20.20�0.12d24.03�0.12c27.20�1.12b28.20�1.02b30.44�1.20aM (%)8.01�0.14a4.28�0.18d6.01�0.19c6.99�0.14c7.46�0.12b8.46�0.26aE (%)3.01�0.11a0.92�0.13d1.49�0.16c1.94�0.08c2.46�0.02b3.68�0.04aRBC (xw11/l)3.20�0.31a0.80�0.04d1.45�0.02c1.64�0.42c2.48�0.12b3.48�0.06aMCHC (g/dl)34.10�0.06a22.10�0.04d28.41�1.10c29.10�0.12c32.41�0.02b34.46�0.12aMCV (fl)79.01�1.10a54.12�0.16e63.49�0.10d69.10�0.52c75.96�0.48b79.60�0.09aMCH (pg)35.11�1.20a24.01�0.06e28.42�0.04d30.20�0.44c32.69�0.16b35.64�1.10aData are presented as mean � SEM of 7 animals.  Values with different superscript letters (a-e) along a column for a given parameter are significantly (P < 0.05) different from each other group of animals. Haemoglobin (Hb), packed cell volume (PCV), red blood cells (RBCs), white blood cells (WBCs), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), neutrophils (N), lymphocyte (L), monocytes (M) and eosinophils (E).












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