Evaluation of the effects of Leech Salivary Extract (LSE) on Haematological parameters in Rats

The effects of Leech Salivary Extract (LSE) on some haematological, immunological and organ weight parameters in rats, during a twenty eight days oral administration of 25, 50 and 100 mg/kg body weight doses, was investigated. LD50 and sub chronic toxicity was determined using standard methods. The oral LD50 was above 5000mg/kgbw. Oral administration of LSE (25mg/kgbw, 50mg/kgbw, 100mg/kgbw) for 28days had no signifi cant (p>0.05) effect on the differential white blood cells (lymphocytes, monocytes, basophils, neutrophils, eosinophils), red blood cell indices (RBC count, PCV, HB, platelets, MCHC and MCH), feed intake, body weight gain and relative organ weight of lung, heart, liver, kidney, spleen and stomach of rats. However, the LSE evoked a signifi cant (p>0.05) increase in the level of MCV in treated rats compared to the control. These results, indicating low toxicity and no negative signifi cant effects of LSE on haemato-immunological indices in rats, suggest that the extract is safe for development and use as therapeutic for managing clinical conditions. Research Article Evaluation of the effects of Leech Salivary Extract (LSE) on Haematological parameters in Rats Hausatu Babayi1*, Ojo Olufunmilola Praise1, Labake Fadipe Ajoke2, Israel Kayode Olayemi3, Elisha Baba4 and Oladosun Oluwale Peter5 1Department of Microbiology, Federal University of Technology Minna, Niger 2Department of Chemistry, Federal University of Technology Minna, Niger 3Department of Biological Sciences, Federal University of Technology Minna, Niger, State. 4Clinic, National Institute for Pharmaceutical Research Development, Idu-Abuja, Nigeria. 5Department of Microbiology, National Institute for Pharmaceutical Research Development, Idu-Abuja, Nigeria *Address for Correspondence: Hausatu Babayi, Department of Microbiology, Federal University of Technology Minna, Niger, Tel: +2348033700406; Email: acadbabayi@yahoo.com Submitted: 09 January 2018 Approved: 18 January 2018 Published: 19 January 2018 Copyright: 2018 Babayi H, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited


Introduction
The studies of haematological parameters are of ecological and physiological interest that helps to understand the characteristics of blood in relation to the environment (Obel et al., 2016). The assessments of haematological and differential parameters reveal the effects of foreign compounds in blood constituents in living system. The evaluation of these parameters: Red blood cells (erythrocytes), White blood cells (leucocytes) and platelets (thrombocytes) give the avenue to investigate the presence of several constituents in the blood of animals which play important roles in nutritional, pathological and physiological status of animals [1]. Packed cell volume (PCV) is the percentage of red blood cells (RBC) in the blood circulated around the body. It is a point of reference of the capability of RBCs to deliver oxygen to tissues. Reference range for PCV is 34-57% [2]. Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV) and Mean Corpuscular Hemoglobin Concentration (MCHC) are haematological parameters which are related to individual RBC. MCV re lects the average size of RBC while MCH re lects the average weight of haemoglobin (HB) content of RBC and MCHC re lects the average concentration of haemoglobin content of the RBC. Haemoglobin on the other hand measures the total amount of the oxygen carrying protein in the blood which generally re lects the number of RBC in the blood. Platelets are determinants of blood viscosity which correlates positively to blood pressure. Basophils are the least

Extraction of leech saliva
Leech salivary extract (LSE) was collected without sacri icing the animal as described by Abdulkader et al., 2011). Starved leeches were transferred into a closed glass test tube immersed in an ice container. The set-up was then allowed to stand for 15 -20 minutes. The leeches at this point were completely paralyzed and forced to regurgitate their intestinal content that appeared a colourless, foamy salivary luis. The salivary extract was then aspirated using a sterile hypodermic needle and syringe and then transferred into a sterile container. Bloody salivary extract was discarded. The extract was then stored at -4°C.

Experimental animals
Thirty healthy Wister rats (Rattus novergicus) weighing an average of 170g were used in this study. The animals were obtained from the Animal House, Department of Biochemistry, Faculty of Natural Sciences, Ibrahim Badamasi Babangida University Lapai, and Nigeria. They were housed in stainless steel cages bedded with dried clean wood shavings, maintained at a temperature of 25±2 o C and observed under 12-hour light/dark cycle, in a well-ventilated room, for 2 weeks before the commencement of the experiment. They were fed with standard animal feeds (Bendel feeds and lour mills, Edo State, Nigeria) and tap water ad libitum. The cages were cleaned and disinfected every two days. Soiled wood shavings were replaced often. The feed, water containers were washed regularly. The animals were housed and cared for in accordance with Good Laboratory Practice (GLP) regulations of WHO (1998). The principles of Laboratory Animal Care [8], were also followed throughout the study.

Experimental design
The rats were randomly assigned into six groups with each group having ive rats. The groups were designated as 1-6. Group 1 was the Control group and received normal tap water, Groups 2, 3 and 4 were administered 25, 50 and 100mg/kgbw LSE, respectively. Groups 5 and 6 were administered Jobelyn (4.17mg/kgbw) and Dexamethasone (3mg/kgbw), respectively. The administration was done orally with the aid of an oropharyngeal cannula for 28 days. Each rat was marked at speci ic points on the body to distinguish it from the others. The volume of LSE to be administered according to the weight of the rat was calculated using the formula: Volume (ml) = Weight of animal (g) x Dose to administer (mg) Concentration (mg/ml) x 1000g [9] Acute toxicity studies (LD 50 ) of leech salivary extracts The method of Aniagu et al. [10], was employed in the acute toxicology studies (LD 50 ) of LSE. Healthy Wister rats were used for this study. The rats were fasted for 12hours before the administration of the LSE. The rats were weighed before administration of the extract. The acute toxicity study was conducted to observe the range of toxicity so the proper dose level could be established. The study was conducted in two phases. In the irst phase, nine rats were divided into 3 groups of 3 rats each. Group 1, 2 and 3 animals were administered with 10mg/kgbw, 100mg/kgbw and 1000mg/kg body weight of LSE, respectively, to possibly establish the range of dose producing toxic effect. In the second phase, the experiment was set up as the irst phase of three groups with 1 rat per group. Groups 1, 2 and 3 animals were administered with 1600mg/ kgbw, 2900mg/kgbw and 5000mg/kgbw of the LSE, respectively. Administration of the LSE was done orally with the use of oral cannula. The weights of the rats were taken again one week after administration of the LSE.

Weekly body weight and relative organ weight
The body weights of the rats were taken weekly in the course of the experiment and after the experiment. The weight gains were computed as follows: Weight gain= Final weight of rat (g)-Initial weight of rat (g) The relative organ weight (ROW) was calculated as follows:

ROW =
Weight of animal (g) x Dose to administer (mg) 100 Body Weight of Rat on Sacrifi ce Day (kg) 1X

Collection of blood samples
The collection of blood was done as described by Shittu et al. [11]. At the end of the 28days treatment, the animals were starved overnight but still had water ad libitum before they were sacri iced under diethyl ether anaesthesia. The whole blood was collected in heparin bottles.

Statistical Analysis
Results were expressed as mean value±standard error mean (SEM). Among groups, comparisons of means were performed by the Analysis of Variance (ANOVA) test, for statistical signi icance of differences, at p=0.05. The means were subsequently separated using Duncan Multiple Range Test (DMRT. All data analysis were done using the statistical package SPSS version 19.0.

Acute oral toxicity of leech salivary extract
There was no death recorded in the animals that received LSE from 10mg/kgbw to 5000 mg/kgbw (Table 1). Furthermore, the animals did not show any change in general behavior and other physiological activities such as dizziness, aggressiveness, tachypnoea (abnormal rapid breathing), or convulsion. From the above toxicity studies, LD 50 of the LSE is estimated to be greater than 5000mg/kgbw in rats.

Effect of leech salivary extract on body weight gain of the rats
As shown in table 2, there was an insigni icant (p˃0.05) increase in the initial and inal weights of the rats in all treated groups when compared with the Control. The rats gained signi icant (p<0.05) weight across the groups after 28days of LSE as well as dexamethasone and jobelyn administration though, weight gain observed in groups administered 50mg/kgbw LSE and dexamethasone were similar but low when compared with the other groups and Control.

Feed intake of rats fed with leech salivary extract over 28 days study period
The daily feed intake of the rats following oral administration of LSE is presented in table 3. There was no signi icant (p˃0.05) difference in the daily feed intake between the treated groups and the Control in the irst week of treatment. However, there were signi icant (p<0.05) increases in feed intake in the groups treated with 100mg/kgbw LSE and dexamethasone when compared with control. In the 3 rd week, however, there was a signi icant (p>0.05) reduction in feed intake of rats that received 50mg/kgbw LSE,

Relative organ weight of rats after oral administration of leech salivary extract
The computed liver, kidney, spleen, lungs, heart and stomach body weight ratios of the rats, as represented in table 4, were not signi icantly (p˃0.05) different across the LSE treatment groups, as well as, dexamethasone and jobelyn with respect to the Control. However, the group that received dexamethasone had a signi icant (p˂0.05) reduction in the relative organ weight of the lungs when compared with the Control. Table 5 illustrates the changes in haematological indices in rats, following chronic administration of LSE (25mg/kgbw, 50mg/kgbw and 100mg/kg). All the doses of LSE and jobelyn and dexamethasone administered did not signi icantly (P>0.05) affect the levels of PCV, HB, platelets and MCHC in the rats. There was an insigni icant (p˃0.05) decrease in the WBC count in groups treated with LSE as well as dexamethasone but a signi icant (p˂0.05) increase in the group treated with jobelyn compared with the Control. Also, at 25mg/kgbw and 50mg/kgbw LSE, there was a signi icant (p˂0.05) reduction in the RBC count of the rats. At 100mg/kgbw LSE, there was an insigni icant (p˃0.05) reduction in RBC count while in the dexamethasone and jobelyn groups, there was an insigni icant (p˃0.05) increase in RBC when compared with the Control but signi icantly higher than those of the LSE treated rats. However, while there was an insigni icant (p˃0.05) increase in the MCV level of the rats at 25mg/kgbw and 100mg/ kgbw LSE as well as dexamethasone, a signi icant (p˂0.05) increase in rats administered 50mg/kgbw LSE and a signi icant (p˂0.05) decrease in jobelyn group were recorded when compared with their respective Control. Finally, there was a non-signi icant  Effect of leech salivary extract on differential count of rats Table 6 illustrates the changes in differential counts of rats following chronic administration of LSE. The groups treated with LSE, as well as, jobelyn and dexamethasone had an insigni icant (p˃0.05) decrease in the neutrophil count, but insigni icant (p˃0.05) increase in the lymphocyte count. There was an insigni icant decrease in the monocyte count in all treatment groups when compared with their respective control. There was an insigni icant (p˃0.05) increase in eosinophil count for groups administered 25mg/kgbw LSE and dexamethasone. Also, for the groups administered 50mg/kgbw and 100mg/kgbw LSE as well as jobelyn, the eosinophil count decreased insigni icantly when compared with their respective control. The basophil counts of the LSE treated groups as well as dexamethasone and jobelyn were not signi icantly (p˃0.05) different when compared with the Control group rats.

Discussion
The median lethal dose (LD 50 ) is measured as the relative response of an experimental animal exposed to a test substance [12]. It is a standard measurement of acute toxicity stated in milligrams (mg) of the test substance, per kilogram (kg) of body weight of experimental animal. The value of LD 50 for a test substance is the dose required to kill half the number of a tested population after a speci ic test duration [13]. The results obtained from the present study showed that LSE did not cause rat mortality or other signs of toxicity even at a dose limit of 5000mg /kgbw. This result is in agreement with the reports of Kabiru et al. [14], and Atsafack et al. [15], that revealed that methanolic leaf extract of Eucalyptus camaldulensis exhibited LD 50 above 5000mg/kgbw and was, therefore, considered non-toxic acutely.
Changes in body weight of an animal serve as a sensitive indication of the general health status of the animal and also one of the irst critical signs of toxicity [16]. Increase in body weight may be presumed as normal while decreased body weight is seen as a sign of ill health. It was observed in this present study that the mean body weight of the animals in all experimental groups increased insigni icantly when compared with the control. The weight gain observed may be an indication that the extract did not hamper the growth of the animals as reported by Ezeja et al. [17], in the acute and sub-acute toxicity pro ile of methanol leaf extract of Gouania longipetala in rats.
In toxicological studies, an important requirement is the ability to assess the effects of chemical compounds on speci ic organs. Therefore, the analysis of organ weight is an integral component in the assessment of these compounds for their potential harmful effects on organs [18] (Sellers et al., 2007). Organ weight can be the most sensitive indicator of an effect exerted by an experimental substance on the experimental animal, as the substance may have delirious effects on the animal without showing any morphological changes [19]. According to Albert et al. [20], a variation in relative organ weight of an animal is a major index of toxicity. Result of the relative organ weight obtained from the present study showed that there was no signi icant (p˃0.05) difference in the relative weights of kidney, heart, liver, lung, spleen and stomach of rats administered LSE when compared with the Control. This observation corroborates the indings of Enechi and Ozougwu [21], who reported insigni icant change in the liver weight of rats administered ethanol leaf extract of Mucuna pruriens and deduced that the extract did not exert any gross pathological effect on the liver of the rats.
An increased PCV value means abnormal increase in RBC production while a low level of PCV depicts loss of RBC as a result of blood loss and/or failure of bone marrow production. In this study, the LSE did not interfere with the level of PCV of the animals since there was no signi icant difference between the test groups and Control. Since PCV level is indicative of oxygen uptake and transfer to tissues, the steady insigni icant increase in PCV values in this study showed that the extract did not have any effect on the PCV count of the rats. This is in accordance with the report of Olatunji et al. [22], who stated that administration of aqueous extract of Hibiscus sabdariffa calyx showed no signi icant difference on haematocrit (PCV) count of treated rats.
However the observed reduction in RBC level in rats administered with 25mg/ kgbw and 50mg/kgbw LSE should be taken with caution. This reduction could be due to the prolonged administration of the extract which may allow the extract to gradually manifest its adverse effect on the bone marrow of the groups of rats for that duration. The effect was observed by Olatunji et al. [22], in consumption of aqueous extract of Hibiscus sabdariffa for a long time which led to some level of anaemia despite its bene icial characteristics. Furthermore, at 100mg/kgbw LSE, dexamethasone and jobelyn, there was no signi icant effect exerted by the extract on RBC count. Therefore, the extract did not exhibit any toxic effect on oxygen uptake and transfer to the tissues of the rats at this concentration. The extract may contain elements that are capable of reducing the RBC count at low concentrations while stabilizing it at high concentration.
The white blood cell (WBC) count following administration of LSE, could be indicative of its ability to boost the immune system of the rats against invaders [23]. Since there was no signi icant difference in WBC count across all groups except jobelyn, it can be presumed that the production of WBC was constant in all treated groups but increased in the groups administered with jobelyn (being an immune stimulant). The stability of WBC count across LSE treated groups suggests the inability of the extract to produce any toxic effect that may interfere with the immune mechanism of the rats. Mbajiorgu et al. [24], observed elevated WBC count in rats fed low protein diets.
The results of the present study have shown that administration of LSE for 28days did not cause any signi icant toxic effect on the haemoglobin concentration, platelet count, MCH and MCHC. This means that neither the incorporation of haemoglobin into red blood cells nor the morphology and osmotic fragility of the red blood cells were altered as reported by Adebayo et al. [25], who observed that yoyo bitters did not exert any toxic effect on some of the haematological parameters in Wister rats.
Indices of white blood cells, i.e., the differential leucocytes, are the principal haematological parameters which confers immunity. They defend the body against foreign proteins. Results from this study showed that there was no signi icant alteration in the differential counts of the LSE treated animals compared with the Control. This suggests that the consumption of LSE did not induce toxic effects that may trigger immune response or allergy in the rats. Similar observation was made in previous reports of Sani et al. [26], in which prolonged administration of aqueous stem extract of Anisopus mannii at graded doses did not signi icantly affect the values of Hb and WBC when compared with their respective day zero values.

Conclusion
Oral LD 50 of LSE is above 5000mg/kgbw. Oral administration of LSE for 28 days did not cause signi icant variation in the red blood cell indices (PCV, MCH, MCHC, MCV, and HB), white blood cell and differential counts (basophils, neutrophils, eosinophils and monocytes and lymphocytes). It, also, increased the feed intake as treatment progressed leading to increased body weight in the rats. The LSE did not produce any toxic effect on the relative organ weight of the lungs, liver, heart, spleen, stomach and kidney of the rats. These indings, therefore, stand LSE in a good stead for development as a safe therapeutic agent for human ailments, where found to be potent.