¹ Department of Anatomy, Faculty of Basic Medical Sciences, Edo State University Uzairue, Edo State, Nigeria;

² Department of Anatomy, College of Medicine, University of Lagos, Lagos State, Nigeria;

³ Department of Anatomy, Faculty of Basic Medical Sciences, Chukwuemeka Odomegwu Ojukwu University, Uli Anambra state, Nigeria;

⁴ Department of Physiology. Faculty of Basic Medical Sciences, Madonna University, Elele. River State, Nigeria

Corresponding Author: Idaguko C. Anna, Department of Anatomy, Faculty of Basic Medical Sciences, Edo State University Uzairue, Edo State, Nigeria. Tel: + 234-(0)805-491-9134. ORCID- 0000-0002-0799-527X; E-mail: annachi67@yahoo.com.

Running title: Haematology and glucose evaluation of C. Albidum

	ABSTRACT
	INTRODUCTION
	MATERIALS AND METHODS
	RESULTS
	DISCUSSION
	CONCLUSION
	ACKNOWLEDGEMENT
	ABBREVIATIONS
	CONFLICTS OF INTEREST
	REFERENCES

	ABSTRACT

Aim: This study investigated the outcome of administration of the methanolic root bark extract of Chrysophyllum albidum orally on normal blood glucose levels and haematological indices in rats. Materials and methods: Twenty adult rats were grouped into 4 groups of 5 rats each. The control group (A) was administered distilled water of 0.5 ml while the treatment groups B, C and D received 200, 400 and 600 mg/kg body weight of methanolic root bark extract of C. albidum respectively for 21 days. Before the commencement of the experiment, blood glucose was checked for all the groups. Blood was collected at the end of the test period to analysed changes in blood glucose levels and haematological parameters. Results: The RBC, PCV, Hb, MCH, MCV, lymphocytes, monocytes, basophils were not significantly (P<0.05) different in all the extract doses when compared with the control group. Furthermore, WBC, eosinophils, neutrophils and platelets counts were significantly increased (P<0.05) in all the groups treated with the extract in a dose-dependent form when compared to that of the control. Also, a significant (P<0.05) reduction in blood glucose in groups administered with 400 and 600 mg/kg body weight of the C. albidum relative to the control group was noted. However, a significant (P<0.05) increase in body weight of all groups treated with the extract when compared with the control group was also noted. Conclusion: The results suggest that methanolic root bark extract of C. albidum may be used to boost thrombopoiesis and also has hypoglycemic properties.

	INTRODUCTION

   Blood is very essential to human health because of its involvement in various physiological functions in the body such as respiration, nutrition, immune defense, water, acid-base balance, digestion, and thermoregulation etc. It serves as an important biomarker that is useful in determining and understanding the pathogenesis of certain medical conditions such as malaria, anaemia, inflammation, rhesus disease, cancer etc. (1-3).

   The use of medicinal plants in treating certain diseases is a common practice especially among the traditional societies around the world including those in Nigeria (4). Over the years, mankind has discovered varieties of medicinal plants that have been of help in alleviating or in the treatment of different diseases (5). Some of these plants have also served as models from which some synthetic drugs have been developed. As the use of natural plants gains more attention in recent times for the treatment of diseases, it becomes very important to understand the effect of medicinal plants on hematological parameters (6). Analysing hematological parameters is a necessary tool in medicine, which has led to the understanding of deleterious effect of certain foreign compounds especially those that results from environmental toxicants. It is also an important biomarker used in understanding the various changes that occur in the body such as metabolic product, morphology of the organs, enzymes and even the hematology (7).

   Studies have reported the haematological benefits of some plants such as Hibiscus sabdariffa Calyx, Telfairia occcidentalis Aloe ferox, Agave sisalana, and Gunnera perpensa etc. (8-10). However, oral administration of medicinal drugs have been reported to alter the haematological parameter values either negatively or positively (11). Most of these negative or positive effects have been reported by scientific studies but their effects have not been fully investigated.

   Chrysophyllum albidum is of the family of Sapotaceae and every parts of the plant has various ethno- medical uses (12). The roots, barks, and leaves all have medicinal benefits. One of the benefits of the bark is that it cures malaria and yellow fever (13). The plant has relevant biological activities, including; antiplatelet, antioxidant, antifertility, antidiabetic, hypoglycemic and hypolipidemic (14-18).

   Despite the various benefits associated with the use of medicinal plants caution must be taken to understand the likely toxicological potentials of such plants. Hence, this research was embarked upon with the aim of studying the effect of the methanolic root bark extract of Chrysophyllum albidum on the normal blood glucose level and hematological parameters in rats.

	MATERIALS AND METHODS

   Collection of plant material

   The root barks of Chrysophyllum albidum were collected from Isiaku Isuofia, Aguata Local Government Area of Anambra State in Nigeria. Authentication of the plant was done in the Department of Pharmacognosy, Madonna University, Elele, Rivers State. A voucher specimen was kept in the herbarium. The procedures for the “Guide for the Care and Use of Laboratory Animals” was used for the study (19). Approval for this study was gotten from the Institute Animal Ethics Committee of Madonna University, Elele, Rivers State.

   Preparation of the extract

   Fresh root bark of Chrysophyllum albidum was washed in tap water and were cut into pieces and dried for four weeks. The root bark was ground in a grinding machine to powder. Thereafter, 400 g of the pulverized root bark of Chrysophyllum albidum was macerated into 1000 mL of methanol (BDH Chemical Limited, England) at room temperature and stirred constantly for 4 days. The mixture was filtered with porcelain cloth and Whatman No. 1 filter paper, the filtrate was further concentrated using a rotary evaporator (Buchi R-215 Switzerland) at the temperature of 400C. The extract was then placed in an oven to get dried. The resulting extract was kept in a bottle and stored for future use.

   Experimental Animals

   Twenty healthy male Sprague Dawley rats weighing about 150-170 g were acquired from the Anatomy Department animal house in University of Port Harcourt, River State. All the rats were acclimatised for a period of two weeks; and were fed with rat chow and had free access to water ad libitum. The animals were kept in well-ventilated cages under controlled environmental conditions of temperature (25 ± 5°C), relative humidity 50 ± 5% and 12 hours light/dark cycle in the animal house of Madonna University, Elele Campus, Rivers State.

   Administration of extract

   The rats were grouped into four groups of five rats each. Group A which served as the control was administered with 0.5 ml of distilled water, while groups B, C and D received methanolic root bark extract of C. albidum i.e. 200, 400 and 600 mg/kg body weight respectively for 21 days using an oropharyngeal cannula. The weights of the rats were measured using Mettler Toledo weighing balance with model number-AL204, Switzerland before, during and at the end of the study. On the 21st day the animals from each group were sacrificed using diethyl ether.

   Acute toxicity (LD50) studies

   The LD₅₀ determination for C. albidum was carried out using Lorke method (20). In experiment one, three groups of four rats each received orally C. albidum; 10, 100 and 1000 mg/kg body weight respectively while, the fourth group (control) had 1 mL of normal saline. The rats were monitored for any signs or symptoms of toxicity within 24 hours and death for the next 72 hours. In experiment two, three groups of four rats were treated with 1500, 3000 and 5000 mg/kg orally of C. albidum respectively. The fourth group (control) received 1 mL of normal saline. The rats were also monitored for toxicity effects for 24 hour and death for the next 72 hours.

   Biochemical Assay

   Blood glucose test was conducted at the commencement and end of the study. The rats were fasted overnight, blood samples were drawn from the tail vein and blood glucose was analysed using a glucometer and test strips (Prestige, U.S.A).

   Determination of haematological parameters

   Using heparinized capillary tube, 2 mL of blood was drawn from the medial canthus of the eyes. Platelet count, red blood cells, haemoglobin concentration, mean cell volume, mean corpuscular haemoglobin, packed cell volume, white blood cell count and its differential counts (basophils, monocytes, lymphocytes, neutrophils, and eosinophils) were conducted using the methods of Ochei and Kolhaktar., and Cheesbrough (21, 22).

   Statistical analysis

   The data obtained were subjected to analysis using the GraphPad Prism, version 5.0 (GraphPad Software, La Jolla, CA, USA). Analysis of variance (ANOVA) followed by Bonferroni post-test was done on the data. Hence, P < 0.05 was regarded to be statistically significant. The results obtained were expressed as mean ± SD.

	RESULTS

   Acute toxicity studies

   The doses of C. albidum extract had no known behavioral change in any of the activities of the rats, after its administration. In this study, no mortality was observed and the LD50 for the root bark of C. albidum was above 2000 mg/kg.

   Body weight

   After the 21 days treatment with C. albidum; the control group rats had a normal body weight gain, while the weight gained in the extract treated groups was significantly (P<0.05) increased when compared to the control group. The weight gain in the C. albidum extract treated groups were dose dependant as shown in Table 1.

   Blood glucose level

   The blood sugar levels of the control and experimental animals before and after 21 days of treatment (Table 2). Following administration of C. albidum at doses of 400 and 600 mg/kg body weight, the blood glucose levels were significantly (P<0.05) reduced when compared to the control.

   Haematology parameter

   The changes in (HB, RBC, PCV, MCV, MCH) levels of the C. albidum treated groups were not statistically significant when compared with that of the control group (Table 3). There was a significant (P <0.05) increase in WBC, neutrophils, eosinophils, and platelets levels in groups B, C, and D that received 200, 400 and 600 mg/kg body weight of the extract respectively when compared to the control group. The extracts at all dosages did not have any significant effect on the levels of lymphocyte, monocytes and basophil as shown in Table 4.

	DISCUSSION

   The oral safety and nontoxic nature of the methanolic extract of the root bark of C. albidum at selected doses was confirmed where the doses used did not result in death of the rats. As reported by (23) any drug or compound that has oral LD50 that is greater than 1000 mg/kg, may be regarded as having minimal toxicity and can be regarded as harmless. This agrees with the result that the methanolic root bark extract of C. albidum was observed to be safe up to the dose of 2000 mg/kg body weight.

   The findings of this study showed that there was a significant increase in the weight of rats in the C. albidum group when compared to the control. Therefore, the increase in body weight may be as a result of the present of tannins in the extract, as reported by (24) that increase in body mass is as a result of the tannins present in medicinal plants. This agrees with the report by (15) that treatment with root bark extract of C. albidum results in increase in weight of rats.

   Serum fasting blood glucose level was not affected at a 200 mg/kg BW dose of the extract but was significantly decreased in doses of (400 mg/kg BW and 600 mg/kg BW) as shown table II, this could mean that the root bark extract at the doses of 400 and 600 mg/kg BW respectively has hypoglycemic potentials. The hypoglycaemic activity of C. albidum may be as a result of the presence of phytochemicals such as tannins, flavonoids, phenols, glycosides; same have been reported by (25) on other plants. Also, these phytochemicals that have the active compounds such as flavonoids and glycosides possess antidiabetic effect due to their antioxidant activity (26).

   The study showed that the extract does not alter the following blood parameters (RBC, Hb, PCV, MCV, and MCH) significantly, hence, these findings agree with the report by (27). The absence of any observed effect of C. albidum on the Hb and RBC may imply that there was no alteration in the oxygen levels in the blood and the quantity of oxygen that is been transported to the tissues was likely to be constant because Hb and RBC are relevant in conveying respiratory gases (28). Hence, the non-effect of C. albidum observed on RBC, HB, MCH, PCV and MCV may suggest that the extract does not have effect on the haemoglobin; the structure and osmotic brittleness of the red blood cells that was produced also did not change (29). According to this study, the methanolic root bark extract of C. albidum did not induce anaemia throughout the period of administration.

   However, increase in WBC reported in this study may imply that C. albidum enhances the body’s ability to respond to infection. Neutrophils are known to attack and also destroy bacteria in the blood while the immune system’s major effector’s cells are the lymphocytes (30). Hence, the significant rise in the percentage of neutrophils may be due to the ability of neutrophils to engulf and destroy foreign agents (31). The lack of change in the lymphocyte levels may imply that the lymphocytes that participate in immune responses were not suppressed since the body is not vulnerable to any present infections as at the time of the study.Hence, C. albidum extract may serve as a good immune system booster.

   The increase in the platelet activities observed in this research is contrary to the report by (27) in which a decrease in platelet count was observed. This disparity could be associated to the fact that different parts of the plant were used in both studies. Blood coagulation demands that the platelets ought to be of adequate number, size and function, therefore the increase observed in platelet could be due to stimulatory response on thrombopoietin (32, 33). The observed increase in platelets is an indicator that the root bark extract of C. albidum could induce the biosynthesis of clotting factors necessary for clotting, especially during severe bleeding (34).

	CONCLUSION

   The methanolic root bark extract of C. albidum does not have the potential to harm the haematological indices. Also, the extract possesses the ability to act as an oral hypoglycaemic agent.

	ACKNOWLEDGEMENT

   The authors are grateful to Mrs Chioma B. Ezimora and Ngozi Amuchie for their technical assistance.

	ABBREVIATIONS

RBC	red blood cell count
Hb	haemoglobin
PVC	packed cell volume
MCH	mean corpuscular haemoglobin
MCV	mean cell volume
WBC	white blood cell count
PLT	platelets
LD50	Lethal Dose

	CONFLICTS OF INTEREST

   The authors declare that no conflicting interests exist.

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