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��ࡱ�>��	���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������[�	��7Tbjbj����	.dΐΐܞ
�������88{{����###8[|�\#�j3 �� � � � D#V#b#�������$Q���J,�uj#@#D#j#j#,�{{� � ����=�=�=j#{"� � ��=j#��=�=2h��rm� ������4(G7�#z;�j,���0�:j�=�"=j=�Xmm�
=��z`j#j#�=j#j#j#j#j#,�,��=jj#j#j#�j#j#j#j#��������������������������������������������������������������������=�j#j#j#j#j#j#j#j#j#8	A:	In vitro antioxidant and sub-acute toxicity studies of aqueous extract of White butterfly (Clerodendrum volubile) leaves
Olorunfemi Raphael Molehin*, Omotade Ibidun Oloyede

Department of Biochemistry, Faculty of Science, Ekiti State University Ado-Ekiti, P.M.B.5363 Ado-Ekiti, Nigeria

*Corresponding Author address: Department of Biochemistry, Faculty of Science, Ekiti State 
University, Ado-Ekiti P.M.B.5363 Ado-Ekiti Nigeria.Email: olorunfemi.molehin@eksu.edu.ng, molehin.olorunfemi@gmail.com Tel: +234-803-462-1267









Abstract
Objective: The purpose of the study is to investigate the potential antioxidant property and the level of safety of aqueous extract of�White butterfly (Clerodendrum volubile P. Beauv) (C. volubile) leaves. 
Methods: The radical scavenging activity of C. volubile extract against DPPH and ABTS+ radicals, in vitro was evaluated. Its reducing power was also measured using FRAP and lipid peroxidation method. In addition, sub-acute toxicity study in rats was carried out to establish the safe doses. 
Results: Data revealed that the extracts showed no signs of mortalities up to a dose level of 5000 mg/kg b.wt indicating that the LD50 is higher than 5g/kg. In vitro antioxidant studies revealed that C. volubile extract is rich in phenolics as typified by its total phenol and flavonoid contents. C. volubile extract showed a promising scavenging effect in DPPH, ABTS and ferric-reducing power assays in a dose-dependent manner. Hematological and biochemical parameters showed that C. volubile extract improved protein and blood levels preserved the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) when compared with the control group. The extract also demonstrated lipid lowering effect. The significant increases in urea and creatinine levels observed suggested disturbances of kidney functions.
Conclusion: Overall, the findings of this study indicate that C. volubile extract is non-toxic, safe at low dose levels and possesses antioxidant capacity. However, at a high dose, there is possibility that C. volubile extract could have toxic potential as reflected in weight increase in spleen, liver and kidney. 
Keywords: Clerodendrum volubile; antioxidant activity; free radical; sub-acute toxicity; Histopathology.




INTRODUCTION
The uses of herbal medicines are gaining increasing awareness globally because of their efficacy, safety and low price [1]. There is also a growing increase in the usage of herbal formulations because of the perceived belief that the products are natural and hence should be safe for treatment of diseases [2]. However, the assumptions that these herbal formulations are safe are not true as it may have potentially toxic constituents present in them either as natural or as contaminants [3]. Many of the plant-derived products used for therapeutic interventions does not meet the current standards for herbal drugs and are therefore not fully evaluated for their quality, safety and efficacy. The development of herbal medicinal products therefore requires tests for quality and toxicological safety [4]. Plant extracts should not only be efficacious but also safe for consumption. Hence, the need to monitor closely the screening of plants extracts for their activities against microorganisms or diseases and also the need to know their toxicity profiles [5].S  
Clerodendrum volubile P. Beauv (C. volubile) belongs to the family of Verbenaceae. It is a climbing shrub growing in deciduous forests across Africa [6]. Its common names among the Urhobo and Itsekiri tribes of the Niger-Delta of Nigeria are �Obenetete�, and �Marugbo� in Yoruba people of Ondo State [7-8]. In the southern part of Nigeria, which is densely dominated by the Ijaws, Urhobos and Itsekiris, it is regularly consumed as food and used in folklore medicine. The plant is effective in treatment of arthritis, diabetes, rheumatism, dropsy, swellings, edema, and gout. It is also used as an anti-abortifacient and sedative [8-9].
The reported pharmacological properties of C. volubile leaf include anti-inflammatory[8], suppression of oxidative burst[10], antihypertensive [11], antidiabetic [10] and cancer chemopreventive [12-13] activities. However, to the best of our knowledge, no toxicity studies have been carried out on the aqueous extracts of C. volubile leaves in experimental animals up till now. Considering the folkloric use of water extractable phytochemicals from C. volubile leaves in the management of various ailments, it is important to evaluate the toxicity profile of the aqueous extract of C. volubile in order to establish the safety dose for the plant uses. The present study therefore, aimed at evaluating the antioxidant and sub-acute toxicity profile of aqueous extracts of C. volubile in male wistar rats. 
MATERIALS AND METHODS
Plant material
Fresh leaves of C. volubile were purchased from Oja Oba Market in Akure Metropolis of Ondo State, Nigeria. The plant was identified in the Department of Biology Federal University of Technology, Akure (FUTA) Nigeria by Mr. A.A. Sorungbe. A voucher specimen (FUTA/BIO/0121) has been deposited at the University herbarium.
Preparation of the extract
The aqueous extract of C. volubile was prepared according to the method described by Adefegha and Oboh [14]. C. volubile was washed in distilled water, air dried and milled into fine powder using a laboratory mill with pore size of 0.5mm screen. Five hundred grams (500g) of C. volubile powder was soaked in 1000 ml distilled water for about 24 hours. The mixture was filtered; the filtrate was frozen and dried extract was obtained under pressure using a freeze drier. The extract was reconstituted in distilled water and stored in a refrigerator for subsequent analysis.
IN VITRO ANTIOXIDANT STUDIES
Determination of total phenol content 
The total phenol content was determined according to the method of Singleton et al. [15] Briefly, appropriate dilutions of the extracts were oxidized with 2.5 ml 10% Folin-Ciocalteau�s reagent (v/v) and neutralized by 2.0 ml of 7.5% sodium carbonate. The reaction mixture was incubated for 40 min at 45oC and the absorbance was measured at 765 nm in the spectrophotometer. The total phenol content was subsequently calculated as gallic acid equivalent.


Determination of total flavonoid content
The total flavonoid content of the extracts was determined using a slightly modified method reported by Meda et al. [16] Briefly, 0.5 ml of appropriately diluted sample extracts were mixed with 0.5 ml methanol, 50 �l 10% AlCl3, 50 �l 1 M potassium acetate and 1.4 ml water and allowed to incubate at room temperature for 30 min. The absorbance of the reaction mixture was subsequently measured at 415 nm and the total flavonoid content calculated as quercetin equivalent.
Determination of reducing power 
The reducing property of the extracts was determined by assessing the ability of the extracts to reduce FeCl3 solution as described by Oyaizu [17]. A 500 �l aliquot of the extracts was mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50oC for 20 min and then 2.5 ml of 10% trichloroacetic acid was added. This mixture was centrifuged at 801 � g for 10 min. 5 ml of the supernatant was mixed with an equal volume of water and 1 ml of 0.1% ferric chloride. The absorbance was measured at 700 nm and ferric reducing power was subsequently calculated using ascorbic acid equivalent. 
Determination of 2-azinobis (3-ethylbenzothiazoline-6-sulfonate radical (ABTS�) 
scavenging ability
The total antioxidant capacity was determined based on 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonate radical (ABTS�+) scavenging ability of the extracts according to the method described by Re et al [18]. ABTS�+ was generated by reacting ABTS aqueous solution (7 mM) with K2S2O8 (2.45 mM, final concentration) in the dark for 16 h and adjusting the absorbance at 734 nm to 0.700 with water. 0.2 ml of appropriate dilution of the extracts was added to 2.0 ml ABTS�+ solution and the absorbance was measured at 734 nm after 15 min. The trolox equivalent antioxidant capacity (TEAC) was subsequently calculated using trolox as the standard.

Determination of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging ability
The free radical scavenging ability of the extracts against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was evaluated as described by Gyamfi et al. ]19]. Briefly, an appropriate dilution of the extracts (1ml) was mixed with 1ml 0.4 mM DPPH radicals in methanolic solution. The mixture was left in the dark for 30 min, and the absorbance was taken at 516 nm. The control was carried out by using 2 ml DPPH solution without the test samples. The DPPH free radical scavenging ability was subsequently calculated as percentage of the control.

Lipid peroxidation assay
Preparation of tissue homogenate
Male albino rats weighing 160-180g were sourced from the breeding colony of Department of Medical Biochemistry, College of Medicine, Ekiti State University Ado-Ekiti Nigeria. Rats were maintained at 250C, on a 12h light/12 h dark cycle, with free access to food and water. They were acclimatized under these conditions for one week before the experiment. Rats were immobilized by cervical dislocation and the pancreatic tissue rapidly isolated, rinsed with cold saline and weighed on ice. The tissue was subsequently homogenized in cold saline (1/10 w/v) with about 10-up and -down strokes at approximately 1200rev/min in a Teflon glass homogenizer (Mexxcare, mc14 362, Aayu-shi Design Pvt. Ltd. India). The homogenate was centrifuged for 10 min at 3000 �g in a refrigerated centrifuge (KX3400C, KENXIN Intl. Co., Hong Kong) at 40C to yield a pellet that was discarded, and a low-speed supernatant (LSS), which was kept for lipid peroxidation assay [20]

Inhibition of lipid peroxidation and thiobarbituric acid reactions 
The ability of the extract to inhibit Fe2+-induced lipid peroxidation in pancreas homogenate was determined using thiobarbituric acid reactive species (TBARS) as described by Ohkawa et al. [21]. Briefly, 100 �L100 mL of rat pancreas homogenate fraction was mixed with a reaction mixture containing 30�L of 0.1 M Tris HCl buffer (pH 7.4), extract (0 100 L) and 30 L of the pro-oxidant solution (1 mM quinolinic acid [QA]). The volume was made up to 300 �L with water before incubation at 370C for 1h. The color reaction was developed by adding 300 �L, 8.1% sodium dodecyl sulfate to the reaction mixture containing S1; this was subsequently followed by the addition of 500 �L of acetic acid/ HCl (pH 3.4) and 500 �L 0.8% TBA. This mixture was incubated at 1000C for 1 h. TBARS produced was measured at 532 nm and expressed using MDA (malondialdehyde) equivalent.

Determination of sub-acute toxicity
Experimental Animals
Adult male Wistar rats weighing 120-145g used for this experiment were sourced from the breeding colony of the Department of Physiology, College of Medicine, Ekiti State University Ado-Ekiti, Nigeria. Rats were maintained at 250C, on a 12h light/12 h dark cycle, with free access to food and water. They were acclimatized under these conditions for two weeks prior to the commencement of the experiments. The study was conducted in accordance with the accepted principles outlined in the �Guide for the Care and Use of Laboratory Animals� prepared by the National Academy of Sciences and published by the National Institutes of Health and all efforts were made to minimize animal suffering and the number of animals used.
Sub-acute oral toxicity experiment
 A total of 25 rats were randomly divided into 5 groups of 5 animals each. Animals in Group 1 served as control and received distilled water orally. Animals in groups 2, 3, 4, and 5 were orally administered with 500, 1000, 1500 and 5000 mg/kg body weight respectively, once daily for 14 days following the modified method described by Cruz et al [22] . The body weights of all the rats were recorded daily throughout the experimental period. After 14 days of treatment, the rats were fasted overnight and anaesthetized in chloroform vapour and blood sample collected by cardiac puncture for biochemical analyses. 
Haematological and Biochemical Analyses
Haematological analysis was performed using an automatic haematological analyser (Mindray Auto hematology Analyser BC-5500). The parameters included: packed cell volume (PCV), red blood cell (RBC), white blood cells (WBC), haemoglobin (Hb), mean Corpuscular volume. (MCH), mean Corpuscular Hemoglobin (MCH) [23-24]. For biochemical analysis, blood was centrifuged at 3000rpm for 10min. Portions of uncoagulated blood were centrifuged at 3000rpm for 10mins. Serum was separated and stored below 4�C until determination of serum protein, serum creatinine, urea, total cholesterol, triglycerides, aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP), total bilirubin, creatinine kinase, lactate dehydrogenase which were determined through commercially available kits. The liver and kidney were collected for histopathological examination.

Data analysis
The results were pooled and expressed as mean � standard deviation (S.D.). One-way analysis of variance (ANOVA) was used to analyze the mean and the post hoc treatment was performed using Duncan multiple test. Significance was accepted at P < 0.05. IC50 (extract concentration causing 50% effectiveness) were calculated.
3.  Results
The phenolic content of the C. volubile leaf extract is shown in� HYPERLINK "http://www.sciencedirect.com/science/article/pii/S2225411017300081" \l "tbl1" Table�1. The total phenol content of the extract reported as gallic acid equivalent (GAE) was 7.35mg�GAE/g, while the total flavonoid content reported as quercetin equivalent was 6.57mg�QE/g. Also, the ferric reducing antioxidant property of the leaf extract is reported as ascorbic acid equivalent was 3.83 mg AAE/g. In addition, the ability of the extract to scavenge ABTS radical reported as trolox equivalent is 0.0000086 mmol TEAC. The DPPH free radical scavenging ability of the extract is presented in figure 1. The result revealed that the extract scavenged DPPH radicals in a concentration dependent manner at the concentrations tested (0.42�1.67mg/ml). The result revealed that the extract had a IC50 value of . The inhibition of Fe2+ - induced lipid peroxidation in isolated rat pancreas by the C. volubile leaf extracts was investigated and the result is presented in Figure 2. The result shows that the extract inhibited Fe2+-induced lipid peroxidation in rat pancreas in a dose dependent manner. 
The sub-acute toxicity of C. volubile aqueous leaf extract was undertaken on hepatic and kidney biochemical indices using rat model. As observed, the rats exposed to aqueous leaf extract of C. volubile for fourteen (14) days, short-term exposure caused a significant (P< 0.05) increase in their growth rate and relative body weights when compared to their corresponding control groups (Table 2). The highest concentration of the extract gave the highest body weight increment. The relative organ weight of rats is presented in Table 3. There is no significant (P >0.05) change in the relative weight of the heart and pancreas when compared with control while the liver and spleen only showed an increase in weight at the highest dose (5000mg/kg) of the extract though not statistically significant when compared with the control group. However, the weight of the kidney consistently and dose-dependently increased throughout the duration of the experiment (Table 3).
Table 4 shows the results of the biochemical parameters tested are in this study. There was no significant (P >0.05) difference in the serum activity of liver function marker enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) alkaline phosphatase (ALP) in the treated rats when compared with the control group. However, the extract treated rats had a significant (P <0.05) increase in the serum total protein as compared with the control group. Furthermore, a significant increase (P < 0.05) in the levels of serum urea and creatinine were observed when the experimental rats received higher doses of the extract of C. volubile ranging from 1000 to 5000 mg/kg body weight when compared with the control group. A significant decrease was observed in total bilirubin content in all the treated rats as compared with the control group.
The effect of the leaf extracts on heamatological parameters are presented in Table 5. PCV was not significantly affected by the extract at all the dose levels studied. However, significant increase in Hb level at 2000mg/kg and 5000mg/kg doses of C. volubile was observed while RBC content was significantly reduced at the dose of 5000mg/kg. A significant increase (p<0.05) in MCH and MCV which was dose dependent was oberved in rats given an oral dose of the extract in the groups 500, 1000 and 5000mg/kg body weight respectively when compared with the control group.
Examination of liver and kidney sections of the treated rats (500- 2000mg/kg body weight) showed no histological abnormalities when compared with control (untreated) rats. Figures 3A-D and 4A-D. However, the highest doses of 5000mg/kg body weight in liver revealed a moderate periportal cellular infiltration; the sinusoids are severely congested (figure 3E). Also, examination of the kidney revealed (figure 4E) shows there is a severe diffuse tubular necrosis at the renal cortex.

Discussion
Polyphenols are pharmacologically active components of plants which are capable of scavenging free radicals, chelating metal ions and inhibiting the activity of oxidizing enzymes in biological systems [25-27]. Several reports have attributed the health benefits of medicinal plants to their significant antioxidant properties due to the presence of phenolic compounds present in them      [13, 28-29] . Table 1 shows the total phenol and flavonoid contents of C. volubile leaves. The result revealed that C. volubile had a total phenol content of 7.35mg GAE/g and the total flavonoid content of 6.57mg QUE/g. These values are lower than those reported for different cultivars of raspberry extract [30]. However, the values of the phenolic content of C. volubile are higher than that of Celosia spp [29]. Various factors behind the differences in total phenolic content of different plant foods may include variation in cultivars, harvest and postharvest handling and storage conditions, and processing techniques during analytical determinations 31
The antioxidant activities of vegetables have been attributed to several mechanisms, some of which include prevention of chain initiation or radical scavenging ability, binding of transition metal ion catalysts, decomposition of peroxides, prevention of hydrogen abstraction and reductive capacity [28,32-33] . Phenolic compounds play a crucial role in scavenging and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides [34] C. volubile extracts (IC50 = 0.9� 0.01mg/ml) scavenged DPPH in a dose-dependent manner (Fig.1). This finding agrees with many earlier reports of positive correlations between total phenol content and DPPH free radical scavenging ability of several foods [32, 33, 35]. The free radical scavenging ability of the leaf extract was subsequently assessed using the moderately stable ABTS radical (ABTS�+), presented in Table 1. The results revealed that the C. volubile extract (0. 0000086 mmol TEAC/g) quenched ABTS radical. Similar results were reported in the ABTS radical scavenging abilities of other tropical vegetables [33].
The ability of the leaf extract to reduce Fe (III) to Fe (II) was reported as ascorbic acid equivalent (AAE). The result revealed that the C. volubile (3.83mg AAE/g) reduced Fe3+ to Fe2+ as shown in Table 1. Similar results were reported from previous studies on ferric reducing antioxidant property of some plant extracts [36-38].
In biological systems, lipid peroxidation (oxidative degradation of polyunsaturated fatty acid in the cell membranes) gives birth to a number of degradation products, such as malondialdehyde (MDA), which is found to be an important cause of cell membrane destruction and cell damage [39]. Lipid peroxidation in rat pancreas homogenate was induced with Iron II sulphate and the potential antioxidant effect of aqueous extract of Clerodendrum volubile was assessed. There was statistically significant increase (P< 0.05) in the formation of MDA contents of the pancreas (in vitro) as shown in figure 2. MDA is regarded as an important diagnostic index of lipid peroxidation in several tissue injuries [40]. The increased lipid peroxidation in the presence of Fe2+ could be attributed to the fact that Fe2+ can catalyze one electron transfer reaction that produces reactive oxygen species such as hydroxyl (OH.) radical which is formed from hydrogen peroxide (H2O2) through Fenton reaction [41]. The significant (p<0.05) reduction in MDA production by C. volubile extracts revealed that the extracts showed significant capacity to suppress the iron II sulphate induced-lipid peroxidation in a dose-dependent manner when compared with control. This result demonstrates the ability of C. volubile extracts to scavenge peroxyl radical generated by iron II sulphate. The potency of water extractable phytochemicals from the plant against lipid peroxidation in rat pancreas (in vitro) may have been influenced by the bilayer arrangement of lipids ((hydrophilic head embedded in hydrophobic tail) in the biological membrane [14]. In addition, the ability of the aqueous extract to inhibit lipid peroxidation induced by Fe2+ could be associated with their phenolic constituents by forming structural complexes with Fe2+ and thus chelating it from initiating the lipid peroxidation chain reaction. Therefore, this could further explain the biochemical mechanism underlying the use of C. volubile leaf for treatment of diabetes mellitus in traditional medicine.
Administration of herbal preparations without any standard dosage and coupled with non-availability of adequate scientific studies on their safety have raised a lot of concerns on their toxicity [42]. Toxicity studies in animals are commonly used to assess potential health risk in humans caused by internal adverse effects of chemical compounds/plant extracts [43]. These adverse effects may manifest in alterations on some biochemical parameters like enzymes, metabolic products, normal functioning and histomorphology of organs [44-45]. Aside from the long history of medicinal uses of plants, it is very essential to carry out toxicity evaluation on them in order to assess their safety/ right dosage. In the present study, we carried out sub-acute toxicity studies on the aqueous extract of the leaves of C. volubile in vivo. All the rats used for the sub-acute toxicity study appeared normal before, during and after the treatment. Mortality was not observed at any of the dose levels used in this study. The leaf extract had a positive effect on the weight status of the rats (table 2). The relative weights of rats given the leaf extract in all groups remained higher in contrast to those rats not given the leaf extract (control group) at the end of 14 day-treatment. All these combined, signify a better physical status of the rats and also suggested that even at the highest oral dose of the extract administered, the normal growth of rats were sustained. So the leaf extract did not impose any acute fluid loss, proteolysis and lipolysis on the test group and therefore no loss in body weight.
Relative organ weight may serve as indication of pathological and physiological status in man and animals. Toxic substances have been reported to have the ability to induce abnormal metabolic reactions that can affect primary organs such as heart, liver, spleen, kidney and lung [46]. Our data revealed no significant (P >0.05) change in the relative weight of the heart and pancreas when compared with control while the liver and spleen only showed an increase in weight at the highest dose (5000mg/kg) of the extract though not statistically significant when compared with the untreated group (Table 3). However, the weight of the kidney consistently and dose-dependently increased throughout the duration of the experiment. The non-significant changes in the weight of the heart and pancreas may suggest non-toxicity of the extract to these organs. However, precautions during the usage of this extract may be necessary, especially, in higher doses and in the kidney in particular. 
The C. volubile leaf extract improved the protein level as indicated by the significant increase (P < 0.05) in the total serum protein concentration of all the treatment groups when compared to the control group (Table 4). Protein is involved in the maintenance of osmotic pressure of the blood and body fluids, and transport of inorganic anions, fatty acids and drugs [47] . Therefore, increase in serum protein level would enhance the metabolism of substances that are transported by it. Indeed, the transaminases (AST and ALT) are well- known enzymes used as good indicators of liver function as biomarkers predicting possible toxicity [48-49].  Elevation in the levels of serum transaminase enzymes is highly indicative of hepatic impairment in animals [50]. C. volubile extracts did not cause a significant difference (P e" 0.05) in the activities of liver function marker enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) when compared with the control group (table 4). This is an indication that the aqueous extracts of C. volubile leaves did not induce any significant damage to the liver neither did it alter the functions of hepatocyte. Bilirubin is a useful index of the excretory function of the liver. It is conjugated with glucuronic acid in a reaction catalysed by bilirubin�UDP-glucuronyl transferase which renders it soluble and subsequently excreted into the bile. Elevated level of bilirubin is an indication of liver cell impairment [51]. The gradual decrease (Table 4.) in the serum levels of unconjugated bilirubin is pointing to increased ability of hepatocytes to conjugate bilirubin. Urea and creatinine are good indicators of renal function and their retention in the body indicates renal damage [52�54].  In this present study, significant increase (P < 0.05) in the levels of serum urea and creatinine were observed when the experimental rats received higher doses of the aqueous extract of C. volubile ranging from 1000 to 5000 mg/kg body weight when compared with the control group. The observed dose-related significant increase in urea and creatinine values suggests that the extract at high oral doses may induce renal dysfunction. In addition, non-significant increase in the weight of the kidney observed throughout the groups might also lend credence to the hypothesis that, the extract may have potential to induce kidney dysfunctions.
Furthermore, the effect of sub-acute oral administration of aqueous extracts of C. volubile on the haematological parameters is summarized in Tables 5. The haematological system is a sensitive target for toxic chemicals and can be used to determine the pathological status in humans and animals [55-56].  In this study, PCV was not significantly affected by the extract at all the dose levels studied. There was significant increase in Hb level at 2000mg/kg and 5000mg/kg doses of C. volubile while RBC content was significantly reduced at the dose of 5000mg/kg. A dose dependent significant increase (p<0.05) in MCH and MCV was oberved in rats given an oral dose of the extract in the groups 500, 1000 and 5000mg/kg body weight respectively when compared with the control group. Red blood cells have been shown to function in the maintenance of acid-base balance and in the transport of respiratory gases within the body. Its decrease could result in hypoxia or myocardial infarction [57-58]. However, hemoglobin concentration, a sensitive parameter for detection of anemia was not affected as there was significant rise in its level at higher doses of the extract. This indicates that the aqueous extracts of C. volubile leaves in rats are not causing any form of aneamia. 
Increase in lipid profile such as TC and TG in the Wistar rats may be useful indicators in investigating the influence of these extracts on metabolism of lipids and how animals and humans may be prone to coronary diseases from intake of preparations from these plants especially at higher doses. The results recorded in this study showed lipid-lowering effect of aqueous extracts of C. volubile with increase in dose. This could help in cholesterol degradation
Examination of liver and kidney sections of normal and extract-treated rats showed no histological abnormalities in rats treated with aqueous extract of C. volubile up till the 2000mg/kg doses figures 3A-D and 4A-D but at higher doses of 5000mg/kg reveals a moderate periportal cellular infiltration, the sinusoids are severely congested. Also a multifocal fatty infiltration of the liver parenchyma was observed. Likewise, in the kidney shows there is a severe diffuse tubular necrosis at the renal cortex. The observation of some histological abnormalities on the kidney may be as a result of the increase seen in the kidney weight. In addition, the biochemical parameters (urea and creatinine) altered did not reach a statistical significance level and so may be the reason for the kidney architecture being affected adversely.
Conclusions
In conclusion, this study provides useful data on the subacute oral toxicity profile of aqueous extracts of C. volubile that would be useful for future in vivo studies. Generally, the extract demonstrated non-toxicity at low dose, in sub-acute oral administrations. However, precautions need to be taken at its higher oral doses.

Conflict of Interest:  
The authors declare no conflict of interest.
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