Hemato-Protective Effects of Ocimum Gratissimum Leaves Aqueous Extract in Adult Male Wistar Rats

The Lamiaceae family includes Ocimum gratissimum. It's known as 'alfavaca' because of its medicinal and culinary properties, and it's grown in countless farms throughout Nigeria's villages and huts. The hemato-protective effects of O. gratissimum leaf extract (aqueous) were examined in adult male wistar rats in this study. Thirty-five (35) adult male wistar rats weighing 150–200 g were obtained, acclimatized for two weeks, and then divided into seven (7) groups of five (5) rats each (n = 5). Groups II–IV received phenylhydrazine (hemolytic anemia induced, untreated – negative control), 100 mg/kg body weight (BW) of O. Gratissimum aqueous crude extract (after generating hemolytic anemia), and 5-Flou-rourasilday intraperitoneal injection for one week (Leucocytope-nia induced, untreated), respectively. Leucocytopenia (fed with O. Gratissimum aqueous leaf extract), Thrombocytopenia (untreated), and Thrombocytopenia (treated) were elicited in groups V-VII, re - spectively (extract treated). Blood samples were taken from animals (using a 2ml syringe) after two weeks of test chemical administration, preserved in EDTA, and sent to the lab for analysis. The results of group mean comparisons (using ANOVA) demonstrate that the extract treated group had a statistically significant increase (p 0.05) in RBC, PCV, and haemoglobin (HGB) concentration when compared to the control and untreated (but anemic) groups. Furthermore, neutrophil and WBC counts increased significantly (p 0.05) in extract-treated rats compared to control and leucocy-topenia-induced


Introduction
Hematology, which is the study and the use of blood cells; red cells (erythrocytes), white cells (leucocytes), and platelets (thrombocytes) is an important aspect of science that investigates the numbers and morphology of the cellular elements of blood in the diagnosis and control of diseases [1,2]. Hematological tests are valuable in the diagnosis and investigation of the degree of blood loss in many diseases [3,4]. To better explain the relationship between blood characteristics and the environment, hematological studies are of ecological and physiological importance [5] and could therefore be useful in the selection of animals that are genetically resistant to certain diseases and environmental conditions [6].
Good measures of the physiological state of animals are hematological parameters. These parameters are related to the organ formation of the blood [7] that serves as a pathological reflector of the status of toxicants and other conditions exposed to animals. As reported by Isaac et al, (2013) [8] animals with good blood composition are likely to demonstrate good performance. While laboratory blood tests are valuable tools in the animal or human body for diagnosing any deviation from normal [9], Blood testing allows the presence of certain metabolites and other constituents in the body of animals to be investigated; it thus plays a crucial role in the physiological, nutritional and pathological status of an organism [10]. Blood analysis of its components can provide useful information for the diagnosis and prognosis of animal diseases, according to Olafedehan et al (2010) [11]. In relation to physiological health conditions, blood constituents change [12]. In the evaluation of animal responses to different physiological conditions, these changes are significant and may be influenced by dietary. According to Afolabi et al., changes in hematological parameters are often used to assess the different state of the body and to classify stresses due to environmental, pathological and/or nutritional factors [13]. One of such often common, nutritious substance commonly referred in Africa is the scent leaf, a local traditional herb.
Groups V-VII were respectively induced with Leucocytopenia (fed with O. Gratissimum aqueous leaf extract), Thrombocytopenia (untreated) and Thrombocytopenia (extract treated). After period of administration of test substances (two weeks), blood samples were obtained from animals (using a 2ml syringe), stored in an EDTA, and passed on for laboratory assay. Results of comparisons between group means (using ANOVA) shows a statistically significant increase (p < 0.05) in RBC, PCV and haemoglobin (HGB) concentration of extract treated group as against the control and untreated (but anemic) group. Also, neutrophil and WBC counts showed a significant increase (p < 0.05) for extract treated than control and leucocytopenia induced (negative control II) animals; whereas, there was a significant increase in platelet count for extract treated than control and thrombocytopenia induced (untreated) rats. By implication, O gratissimum contains potent pharmacological ingredients that may be useful in management of hematological diseases. We recommend a study on the phytochemical ingredients in O gratissimum for publics' awareness.

Keywords
Blood; Ocimum gratissimum; Anemia; Leucocytopenia Ocimum Gratissimum (scent leaf) is a plant or shrub of the Lamiacea family. It is mostly found in Africa and Asian continent where majority of the plant exist [14]. Several medical uses of O. Gratissimum were reported by Mshana et al., (2000); one of it being described to be active against several species of fungi and bacteria [15]. Some recent findings have shown O. Gratissimum to be useful against gonorrheal infection, vaginitis and treatment of mental illness. Though a lot of literatures on the antibacterial and antifungul action of the plant are available, little information is known about its role in the managements of haematological disorders. Thus, this study was conducted to examine the hepatoprotective (blood protecting) activities of the aqueous extract of O. Gratissimum leaf adult male wistar rats.

Study area
The study was conducted in the animal house of the department of Human Physiology, Faculty of Basic Medical Sciences, College of Medicine, Ambrose Alli University (AAU), Ekpoma, Edo State.

Animal procurement
A total of thirty five (35) mature male wistar rats weighing between 150 -200 g were procured. They were first and acclimatized for (two weeks) within the animal house of the study institution (AAU), while delivering standard rat chow and clean water to them at liberty.

Study design
Study design was experimental in nature. A total of thirty five (35) adult male wistar rats were grouped into seven (7)  Gratissimum aqueous leaf extract after inducing thrombocytopenia (blood platelet disease)

Ethical Consent
Ethical consent was obtained from the research and ethics committee of the college of medicine, Ambrose Alli University, Ekpoma, Edo State. Also, before actual investigation, consent forms were administered to seek participants' permission.

Extract Preparation from O. Gratissimum Leaf
After obtaining fresh O. Gratissimum (scent) leaves, leaves were air-dried at ambient temperature (30 ± 2°C) for 10 days. They were thereafter pulverized with mechanical grinder, obtained fine powders were stored until further needed. About 50g of the powdered sample was then dissolved in 500 ml of distilled water (via maceration) for 48hrs in line with the procedure of Eno et al. (2001) [16]. Weighed samples (20 g in 10 ml distilled water) of the extract was then use to prepare a 500 mg/ml stock solution that was labelled appropriately and refrigerated at 4C O until required for use.

Determination of Ld 50
LD 50 for oral administration of aqueous O. Gratissimum leaves extract to Wistar rats as recommended by Mohammed et al. (2007) is 1264.9 mg/kg. Safe dose is 1/10 of LD 50 . Therefore, less than 10% doses of (126.49 mg/kg bwt) of the LD50 was used in this study.

Method of administration
O. Gratissimum leaves extract was orally administered to the rats using 1ml syringe with in-tube sterile cannula. The time of administration was between 8:00am and 10:00am daily as recommended by Ejebe et al. (2009) [17].

Inducing and Confirming Anemia
Repetitive doses of phenylhydrazine was administered to the animals in line with the methods of Crosby and Conrad, (1960) in order to induce haemolytic anaemia [18]. Blood sample collection was carried out at various times while examining the fragility profile and osmotic resistance. Animals had their baseline values measured prior to administration of phenylhydrazine.

Inducing and confirming leukocytopenia
Intraperitoneally injection of 20mg/kg body weight of 5-Flourourasilday for 2 weeks induced leukopenia in line with the procedures of Shun et al., (1996) and Jain, (1986) [19]. By this method, leukopenia was induced and confirmed (by WBC count) in animals prior to feeding with O. Gratissimum extract.

Induction of Thrombocytopenia Reitman and Frankel, (1957)
This was achieved by daily injection of low molecular weight Heparin subcutaneously to animals at the dose of 2000 IU/kg for a period of 10 days in accordance with the protocol spelt by Reitman and Frankel, (1957) [20]. Confirmation was followed with platelet count after 10 days of heparin administration.

Statistical Analysis
Results obtained from the study were expressed as Mean ± SEM (Standard Error of Mean), while considering p -value less than 0.05 (p < 0.05) was taken to be statistically significant, a one-way analysis of variance (ANOVA) was used to determine the mean differences between multiple groups. All statistical procedure was performed with the graph pad prism (version 8.0).   Above   Above table (table II) shows a comparative effects of O. gratissimum leaf extract on white blood cell (leucocytes). From the table, neutrophil and WBC counts returned a statistically significant decrease and increase in extract fed group as against control animals respectively. Whereas, monocyte, lymphocyte, basophil and eosinophil counts returned an insignificant increased (p > 0.05) with ANOVA upon comparison of extract and untreated groups with normal control

Discussion
Despite decades of investigations, blood disorders persist as some of the main health challenges globally, with numerous reported high death rates and associated complications. Although few systemic ailments are diagnosable in blood, in recent times however, serious attention has been focused on the effects of herbal plants on hematological health indicators [16]. While contradictory evidences surface on the matter, it has therefore become overbearing to make efforts at clarifying these reports; justifying the need for this study. Thirty five (35) mature male wistar rats of between 150 -200 g were procured, acclimatized for (two weeks) and grouped into seven (7) of five (5) rats per group (n = 5). The animals received 100 mg / kg body weight of O. Gratissimum leaf extract (aqueous) under different blood disease conditions, while testing against extract untreated (negative control) groups for the respective haematological diseases that were induced.  [16].
Again, Table 2 shows the comparative changes in WBCs for extract treated, Leucocytopenic induced wistar rats as against the control and untreated animals. From the table, one could observe that average values of basophil count increased in extract treated than untreated and control groups, although insignificantly (p > 0.05). This change was however noticed to decrease significantly for neutrophil counts in extract treated than untreated and control groups; implicative that O. Gratissimum leaf extract (aqueous) has the capability of decreasing Neutrophil activity in immune reactions. Functionally, Neutrophils are a type of white blood cell (WBC or granulocyte) that protect from infections, among other functions. They make up approximately 40% to 60% of the white blood cells in our bodies [21],and are the first cells to arrive on the scene when one experiences an infection. A normal (absolute) neutrophil count is between 2500 and 7500 neutrophils per microliter of blood [22], and may be high with infections, due to increased production in the bone marrow as with leukemia, or due to physical or emotional stress. A low number of neutrophils may also be a sign of disease in conditions such as leukemia, some infections, vitamin B12 deficiency, chemotherapy, and more. gratissimum) on thrombocytopenia induced rats caused a statistically significant increase (p < 0.05) in platelet levels of extract fed group as compared with control and non-treated animals. Since high platelet levels are implicated in increased blood clothing and thrombogenesis, the implication of this finding therefore is that O. gratissimum extract is a potent accelerator of blood clotting in experimental animals as seen in this table. One explanation for this could be traceable to the active ingredients in O. gratissimum, and the mechanism by which this is made possible; intrinsic or extrinsic, even though this is speculative. Also observed for extract treated group was thrombocytosis, which is an indicator that the extract also contains some thrombopoietin releasing compounds [23]. It is noteworthy to stress that platelets play an important role in the maintenance of homeostasis (blood clotting), which in turn is an indicator of proper platelet function, including platelet aggregation; release of thromboxane A 2 , platelet factor 4, and beta-thromboglobulin; and expression of glycogen 1b and glycogen IIb / IIIa receptors [24,25], though many studies have shown that increased platelet levels is a major risk factor for development of myocardial infarction, cerebral ischemia/transient ischemic attacks, and chronic vascular disease [26,27].

Conclusion
In this study, oral administration of O gratissimum notably caused an increase in RBC, PCV, hemoglobin level, Neutrophil, platelet and WBC counts, specifically significantly in extract treatments than untreated and control animals. All other haematological parameters assayed showed no significant change in O gratissimum administration as compared with normal rats. By implication, O gratissimum could contain potent pharmacological ingredients that may be useful in management of haematological diseases.