You are on page 1of 5

Ode Okwoche J et al.

/ JPBMS, 2010, 8 (13)

Available online at www.jpbms.info

ISSN NO- 2230 - 7885

Original Research Article


JPBMS

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES The Haematological Effects of Chronic Toxicity with Cassia singueana Leaf Extract in Rats
1Ph.D,

Abstract: The methanol extract of Cassia singueana leaves was reported to have exhibited potent anti-ulcer effects when it significantly decreased stomach hydrochloric acid production and reduced gastric emptying in test rats. In most cases, ulcer patients require prolonged (4-8 weeks) therapy with anti-ulcer drugs for successful treatment. The long-term effects of C. singueana leaf extract on haematological parameters were investigated in rats. The extract was prepared by cold marceration using 80% methanol, it was then filtered and concentrated. Four groups (A-D) of albino wistar rats were subjected to chronic exposure by feeding the animal groups with normal diet or 0.25 g extract /kg feed, 0.5 g extract/kg feed and 1.0 g extract/kg feed respectively for 12 weeks. Haematological parameters were assessed on days 28, 56 and 84 using standard procedures. The data collected were statistically analyzed using oneway Analysis of variance (ANOVA) and LSD post hoc test. The results revealed that Cassia singueana extract (CSE) had no significant (P=0.05) effect on red blood cell (RBC) count, white blood cell (WBC) count, packed cell volume (PCV), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) of control rats relative to the separate rat groups that were given varying doses of CSE in the diet. The results of this study suggest that C. singueana extract does not possess haemotoxic activities that could limit its therapeutic use as an anti-ulcer agent.
Blood is the medium in which vital body nutrients, drugs, hormones and waste products of metabolism are conveyed. Erythropoietic organs such as bone marrow and spleen in young animals and rodents are responsible for blood formation. Blood contains erythrocytes or red blood cell (RBC), leucocytes or white blood cell (WBC) and platelets (thrombocytes) as formed elements suspended in plasma [1]. Red blood cells transport oxygen to the body tissues in exchange for carbondioxide which is taken for elimination out of the body. Leucocytes function in defense of the body while platelets serve for blood clotting. Plasma is the liquid intracellular material which imparts to the blood its fluid properties. Hypovolemic shock is associated with hemorrhage, trauma, loss of fluid in burns and major surgery [2]. There is marked reduction in blood volume from either loss of blood, plasma, fluid or internal hemorrhage. Different animal species have specific number of blood formed elements which could be described as normal range with respect to the specie. A marked decrease in the number of any of the blood formed elements becomes dangerous. Abnormally low packed cell volume (PCV), RBC count, or hemoglobin concentration infers anemia. Low WBC count presumes immunopathology while platelet counts of less than 40,000/l in dog and cats results in spontaneous hemorrhage [3].

Okwoche J.1, and Nwaehujor, Chinaka O.2 Department of Veterinary pharmacology and Toxicology, University of Abuja, PMB 117 Abuja, Nigeria. 2M.Sc, Department of Biochemistry, University of Nigeria, Nsukka, Enugu State.

*Ode,

Keyword: Bone marrow; Cassia singueana; chronic toxicity; Erythrocyte; Haematological parameters.
Introduction

Dr.Ode, Okwoche J., Ph.D, Department of Veterinary Toxicology, University of Abuja, PMB 117 Abuja, Nigeria. Contact no: +234 07036527917

*Corresponding Author

Some chemotherapeutic agents reportedly cause bone marrow toxicity resulting in anemia or blood dyscrasias e.g. chloramphenicol antibiotic [4], colchicine, an antiarthritis [5], oxyphenbutazole, analgesic [6], fluorocytosine, anti-fungi [7]. Immunosuppression is also remarkable in some disease conditions such as Human immunovirus (HIV) infections [8] , Feline panleucopenia virus (FPV), Feline immunodeficiency virus [9], and Infectious bursa disease (IBD) in chickens [10]. Drastic reduction in platelets causes disorder in blood clothing and hemorrhage. Non-steroidal antiinflammatory drugs e.g. aspirin, ibuprofen, naproxen, indomethacin, piroxicam inhibit thromboxane and platelet aggregation to cause prolonged bleeding time [11]. Poor blood clotting is encountered in sweet clover [12] and warfarin poisonings [13]. Cassia singueana belongs to the class Leguminosae and family Caesalpinioideae [14]. The local names in different languages are rumfu in Hausa, rumfuhi in Fulani, tugelele in Kanuri and Shadarat al bashini in Shuwa Arabic [15]. The root bark extract of the plant possessed sedative effects [16]. In Northern Nigeria, C. singueana is used for the treatment of acute malaria [17]. C. singueana extract exhibited profound anti-ulcer potentials [18] when total hydrochloric acid production and gastric emptying were decreased in experimental rats. The aim of this study was to evaluate the haematological effects of long-time exposure of rats to Cassia singueana leaf extract. Chemicals, reagents and Instruments: Freshly prepared solutions and analytical grade chemicals were used for the experiments. Methanol was obtained from Riedel-deHaen, Germany, ether (Sigma Aldrich, USA), microhaematocrit

Materials and Methods

Pharmacology

and

Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 08, Issue 08

Chronic toxicity test: The study took duration of 12 weeks and rats were given C. singueana extract at varying doses in feed, water was provided ad libitum. Fifty six (56) albino rats of both sexes were separated randomly into 4 groups (A-D) of 14 rats per group. Males and females were placed in different cages to avoid breeding. The animal feed was prepared and pelleted out into different rations as feed without extract, 0.25 g extract /kg feed, 0.5 g extract/kg feed and 1.0 g extract/kg feed. Group A (control) was given feed without extract, group B received low dose (0.25 g extract /kg feed), group C was fed with medium dose (0.5 g extract/kg feed) and group D received high dose (1.0 g extract/kg feed). The feeding of the animals followed the normal feed consumption of rats at 10 g feed/100 g b.w. /day [19]. Blood samples from four rats in each group were collected under light ether anaesthesia on days 28, 56 and 84 into dry bijour bottles containing ethylenediaminetetracetic acid (EDTA) anticoagulant and used for the assessment of the haematological parameters.

centrifuge and microhaematocrit reader (Hawksley, England). Animals: Inbred matured albino wistar rats of both sexes weighing 120-180 g, bred in the laboratory animal unit of the Faculty of Veterinary medicine, University of Nigeria, Nsukka were used in the experiments. Animals were allowed 7 days to acclimatize. They were given clean drinking water and fed standard feed (Grower mash pellets, Vital feed, Nigeria). Plant Extraction: Fresh leaves of C. singueana plant were dried under mild sunlight and pulverized into powder in a grinding machine. Cold extraction was performed using 80% methanol for 48 h with intermittent shaking at 2 h interval. The extract was concentrated by vacuum rotary evaporation and stored in a refrigerator at 40C. The concentration of the extract was determined.

Ode Okwoche J et al. et al. / JPBMS, 2010, 8 (13)

Experimental

was then collected with pipette and added to 4 ml of the red blood cell diluting fluid in a clean test tube to make a 1: 200 dilution of the blood sample. The diluted blood sample was loaded onto a Neubauer counting chamber and all red blood cells in the five groups of 16 small squares in the central area of the Neubauer chamber was counted using a light microscope (Leica Inc; USA) at x40 objective. The number of cells enumerated for each sample was multiplied by 10,000 to obtain the red blood cell count per microlitre of blood. (d) Haemoglobin concentration (Hb): The haemoglobin concentration of the blood samples was determined by the Cyanomethaemoglobin method [22]. Drabkins haemoglobin reagent (5 ml) was placed in 5 clean test tubes. Then 0.02 ml of the blood sample from individual rat in the different groups was added to the reagent in 4 of the test tubes and mixed properly. The mixture was allowed to react for 20 min, then transferred into cuvettes and the absorbance read at 540 nm against a reagent blank in the 5th test tube using spectrophotometer. Standards were also prepared and read as stated above. The hemoglobin concentration of the blood samples was obtained by multiplying the absorbance of the sample with a calibration factor (x36.8) derived from the absorbance and concentration of the standard. (e) Mean corpuscular volume (MCV): This was determined by dividing the PCV by erythrocyte count values determined as described above and then multiplied by a constant of 10. Values obtained were expressed as femtolitres: (f)
PCV% x 10 (in femtolitres). RBC Count

(a) Packed cell volume (PCV): PCV was determined by the microhaematocrit method [20]. 2 ml of blood was collected in an EDTA bijour bottle from each of the four rats per group. A microcapillary tube (Marienfeld, Germany) was nearly filled with the blood sample and sealed at one end. It was then centrifuged at 10,000 revolutions per minute for 5 minutes using a microhaematocrit centrifuge. PCV value was read with a microhaematocrit reader (Hawksley, England). (b) Total white blood cell count: Total leukocyte count was determined by Haemocytometer method. Blood (0.02 ml) was collected with pipette into a small test tube containing 0.38 ml of white blood cell diluting fluid to make a 1: 20 dilution of the blood sample. The diluted sample was loaded on to the Neubauer counting chamber, and all cells on the four corner squares were counted using a light microscope at x10 objective. The number of cells counted for each blood sample was multiplied by 50 to obtain the total white blood cell count per microlitre of blood [21]. (c) Red blood cell count: Erythrocyte count was determined using Haemocytometer method [21]. Blood was collected in an EDTA bottle from each of the four rats in the groups (A-D). 0.02 ml of the blood sample

Haematological Analysis

The values are expressed in picograms. (g) Mean corpuscular hemoglobin concentration: This was calculated by dividing hemoglobin concentration by the PCV value already obtained and then multiplied by 100. The values were expressed in grams per liter:
Hb (g/dl) x 10 RBC Count Hb (g/dl) x 100 PCV%

Mean corpuscular hemoglobin (MCH): This was calculated by dividing the hemoglobin concentration by the erythrocyte count, already determined and then multiplied by a factor of 10:

Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 08, Issue 08

Packed cell volume: The PVC results for the rats subjected to chronic exposure to graded concentrations of C. singueana extract for a period of 12 weeks is presented in Table 1. There was no significant (p>0.05) change between the hematocrit values of the control rats and the treatment groups (B, C and D) that were given extract.

The data collected from assessing all the hematological parameters were subjected to statistical analysis using one-way ANOVA and LSD post hoc test from Statistical Package for Social Sciences (SPSS) 16.0 software. Mean differences at P=0.05 were considered significant.

Statistical Analysis

Results

Erythrocyte counts: The results of the effect of C. singueana extract on erythrocyte counts for all the groups are presented in Table 2. There was no significant (p>0.05) change in the mean red blood cell counts of the treated rats when compared with control group throughout the duration of the study. Total leukocyte count: The results of the effect of C. singueana extract on total leukocyte count for all the groups are presented in Table 3. There were no significant (p>0.05) variations in the white blood cell count of control and extract-treated rat groups while the study lasted. Hemoglobin concentration: The mean Hb concentrations of the treatment groups B, C and D did not vary significantly (p>0.05) from the control (Table 4). Experimental period (days) 28 56 84

Ode Okwoche J et al. et al. / JPBMS, 2010, 8 (13)

Table 1: The mean packed cell volume (%) of the control and treatment groups B-D of rats (X + S.E.) Packed cell volume (%) Group A. control Group B 44.45 + 0.8 44.38 + 2.0 43.58 + 1.4 43.90 + 0.6 43.88 + 1.2 43.85 + 2.1 45.03 + 0.7 45.13 + 1.5 44.63 + 0.9 Group C

Mean corpuscular volume: Table 5 shows the results of the mean corpuscular volume for all the four groups. Treatment groups B, C and D revealed no significant (p>0.05) change in their mean MCV values different from the control. Mean Corpuscular hemoglobin: The results of the mean corpuscular hemoglobin for all the groups are presented in Table 6. Similarly, the MCH values did not change significantly (p>0.05) in all the treatment groups relative to the control. Mean corpuscular hemoglobin concentration: The results of the mean corpuscular hemoglobin concentration for all the groups are presented in Table 7. The MCHC did not differ significantly in all the rats that received C. singueana extract relative to the normal control rats. 44.80 + 0.8 44.00 + 0.5 44.80 + 1.0 Group D

Values on the same row with control are not significantly (p>0.05) different. B=0.25 g extract/kg feed, C=0.5 g extract/kg feed and C=0.1 g extract/kg feed.

Table 2: The mean erythrocyte counts of the control and treatment groups B-D of rats (X + S.E.) Experimental period (days) 28 56 84 Packed cell volume (%) Group A. control 6.04 + 0.1 6.58 + 0.1 7.06 + 0.0 6.27 + 0.1 6.43 + 0.2 6.86 + 0.3 Group B 6.26 + 0.1 6.41 + 0.2 6.88 + 0.2 Group C 6.03 + 0.1 6.48 + 0.2 6.99 + 0.4 Group D

Values on the same row are not significantly (p>0.05) different when compared with control. B=0.25 g extract/kg feed, C=0.5 g extract/kg feed and C=0.1 g extract/kg feed.

Table 3: The mean total leukocyte counts of the control and treatment groups B, C and D of rats (X + S.E.) Experimental period (days) 28 56 84 Packed cell volume (%) Group A. control Group B 12.98 + 1.2 12.58 + 0.8 13.11 + 0.9 12.23 + 1.3 12.01 + 0.3 14.05 + 0.7 12.68 + 0.5 12.05 + 0.9 13.96 + 0.6 Group C 12.64 + 1.2 12.98 + 1.0 14.05 + 0.8 Group D

Values on the same row and control are not significantly (p>0.05) different. B=0.25 g extract/kg feed, C=0.5 g extract/kg feed and C=0.1 g extract/kg feed.

Table 4: The mean hemoglobin concentration (g/dl) of the control and treatment groups (B-D) of rats (X + S.E.) Hemoglobin concentration (g/dl) Experimental period (days) 28 56 84 Packed cell volume (%) Group A. control Group B 15.03 + 0.5 15.08 + 0.3 14.96 + 0.5 15.36 + 0.3 14.39 + 0.3 15.12 + 0.7 15.52 + 0.4 14.86 + 0.9 14.89 + 0.7 Group C 15.43 + 0.2 14.64 + 0.4 14.51 + 0.3 Group D

Values on the same row with control are not significantly (p>0.05) different. B=0.25 g extract/kg feed, C=0.5 g extract/kg feed and C=0.1 g extract/kg feed.

Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 08, Issue 08

Ode Okwoche J et al. et al. / JPBMS, 2010, 8 (13)

Table 5: The mean corpuscular volume (ft) of the control and treatment groups of rats (X + S.E.) Mean corpuscular volume (ft) Packed cell volume (%) Experimental period (days) Group A. control Group B Group C Group D 28 56 84 73.59 + 1.8 67.45 + 2.0 61.72 + 2.0 70.02 + 1.9 68.24 + 1.2 63.92 + 2.3 71.93 + 0.6 70.41 + 2.8 64.87 + 1.7 74.30 + 0.7 67.90 + 1.4 64.09 + 1.5

Values on the same row and control are not significantly (p>0.05) different. B=0.25 g extract/kg feed, C=0.5 g extract/kg feed and C=0.1 g extract/kg feed.

Table 6: The mean corpuscular hemoglobin (pg) of the control and treatment groups B, C and D of rats (X + S.E.)Mean corpuscular hemoglobin concentration (pg)

Experimental period (days) 28 56 84

Packed cell volume (%) Group A. control Group B 24.88 + 0.8 22.92 + 0.6 21.12 + 0.7

Values on the same row with control are not significantly (p>0.05) different. B=0.25 g extract/kg feed, C=0.5 g extract/kg feed and C=0.1 g extract/kg feed.

24.50 + 0.3 22.38 + 0.8 22.04 + 0.3

24.79 + 0.7 23.18 + 0.7 21.64 + 1.0

Group C

25.59 + 0.4 22.59 + 0.9 20.76 + 0.7

Group D

Table 7: The mean corpuscular hemoglobin concentration of the control and treatment groups B, C and D of rats (X + S.E.) Mean corpuscular hemoglobin concentration (g/dl) Packed cell volume (%) Experimental period (days) Group A. control Group B Group C Group D 28 56 84 33.81 + 0.6 33.98 + 1.5 34.33 + 0.4 34.99 + 0.6 32.79 + 1.7 34.48 + 1.6 36.07 + 0.7 32.93 + 0.7 33.36 + 1.0

Values on the same row and control are not significantly (p>0.05) different. B=0.25 g extract/kg feed, C=0.5 g extract/kg feed and C=0.1 g extract/kg feed.

34.44 + 0.4 33.27 + 0.9 32.39 + 1.3

1.Ross MH, Reith EJ. Histology, A Text and Atlas, 1st edition. New York, Harper & Row Publishers, 1985;17297. 2.Akpavie SO. General Veterinary Pathology Notes for Undergraduates, 1st edition. Lagos, Stirling-Horden Publishers, 1985;46. 3.Kirk RW. Current Veterinary Therapy, Small Animal Practice, 1st edition. Philadelphia, W.B. Sanders Company, 1989;494-7.

The results of the hematological evaluation of rats exposed to chronic toxicity with C. singueana extract did not reveal any significant difference between the blood parameters (PCV, RBC count, WBC count, Hb, MCV, MCH and MCHC) of rats that were given the extract and the control. C. singueana extract induced no haemolysis or bone marrow depression; hence there were no signs of anemia in the test rats. The red blood cell mass can be measured by three methods: PCV, hemoglobin concentration and erythrocyte count [23]. Determination of erythrocytic indices such as MCV, MCH and MCHC are helpful in classifying certain anemia [23]. In the same manner, there were no significant changes in the total leukocyte counts of extract-treated and normal rats. However, differential leukocyte counts of the test rats were not investigated. Mature and immature neutrophils, lymphocytes,

Discussion

Conclusion

monocytes, eosinophils and basophils make up the leucocytes (WBC) found in the blood. Leukocytosis is a normal body response to an underlying pathophysiological condition. The immune system correlates with circulating white blood cells, all of which derive from a single precursor, the pluripotent hemapoietic stem cells [24]. Immunosuppression often followed contacts with toxicants [25].

References:-

4.Raef TA. Veterinarian Convicted on Chloramphenicol Charge. Journal of American Veterinary Medical Association 1991;198:1492. 5.Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology, 5th edition. London, Churchill Livingstone, 2003;225. 6. Webster CRL. Quick Look Series in Veterinary Medicine, Clinical Pharmacology, 1st edition. USA, Teton newmedia, 2001;14. 7. Raasch RH, Hopfer RL. Antifungal Agents. In, Munson PL, Mueller RA, Breese GR. (eds). Principles of Pharmacology:

The study has demonstrated that long-term exposure to C. singueana extract exerted no toxic effects on the hematological indices of rats. The use of the extract for therapeutic medication may not predispose people to blood-mediated health risks.

Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 08, Issue 08

Basic Concepts and Clinical Applications, 1st edition. USA, Chapman & Hall, 1401;1410. 8.Lackner AA, Veasey RS. Current Concepts in AIDS Pathogenesis: Insights from the SIV/Macaque Model. Annual Review of medicine 2007;58:461-76. 9.Duno S. Kiss Guide to Cat Care. London, Dorling Kindersley Limited, 2001;176. 10.Jordan FTW, Pattison M. Poultry Diseases 4th edition. Philadelphia, W.B. Sanders Company, 1996;200. 11.McNamara DB, Mayeux PR. Nonopiate Analgesics and Anti-inflammatory Drugs. In, Munson PL, Mueller RA, Breese GR (eds). Principles of Pharmacology: Basic Concepts and Clinical Applications. USA, Chapman & Hall, 1159;185. 12.McDonald GK. Moldy Sweet Clover Poisoning in a Horse. Can Vet J 1980;21:250-51. 13.Spahr JE, Maul JS, Rodgers GM. Superwafarin Poisoning: A Report of Two Cases and Review of the Literature. Am J Haematol 2007;82:656-60. 14.Keay RWJ, Onochie CFA, Stanfield DP. Nigerian trees. London, Macmillan, 1964;3-68. 15.Keay RWJ. Trees of Nigeria. London, Oxford University press, 1989;25. 16.Adzu B, Gamaniel K. Sedative Effects of Cassia singueana root bark. J Natural Remedies 2003;3:134-7. 17.Adzu B, Abbah J, Vongtau H, Gamaniel K. Studies on the Use of Cassia singueana in Malaria Ethnopharmacy. J Ethnopharmacol 2003;88:261-7.

Ode Okwoche J et al. et al. / JPBMS, 2010, 8 (13)

18.Ode OJ, Onakpa MM. Evaluation of Cassia singueana Extract on Stomach HCl Production and Gastric Emptying in Rats. International Journal of Applied Biology and Pharmaceutical Technology 2010;1:1352-8. 19.Hafez ESE. Reproductive and Breeding Techniques for Laboratory Animals. Philadelphia, Lea Fabiger, 1970;1031. 20.Coles EH. Determination of packed cell volume. In, Coles EH (ed). Veterinary Clinical Pathology. Philadelphia, W. B. Saunders Company, 1986;17-19. 21.Schalm OW, Jain NC, Carroll EJ. Determination of Total Leukocyte Count. Veterinary Haematology, 3rd edition. Philadelphia, Lea & Febiger, 1975;19-25. 22.Kachmar JF. Determination of Blood Haemoglobin by the Cyanomethaemoglobin Procedure. In, Tiez NW (ed). Fundamentals of Clinical Chemistry. Philadelphia, W. B. Sanders Company, 1970;268-9. 23.Pratt PW. Laboratory Procedures for Animal Health Techniques, 1st edition. Goleta, CA., American Veterinary Publications Inc, 1985;39-88. 24.Scott MA, Gordon MV. In Search of the Haemopoietic Stem Cell. Br J Haematol 1995;90: 738-43. 25.Reddy RV, Sharma RP, Taylor MJ. Dose and Time Related Response of Immunologic Functions to Aflatoxin B1 in Mice. In, Hayes AW, Schnell RC, Miya TS (eds). Development in the Science and Practice of Technology. Amsterdam, Elsevier, 1983;431-434.

Conflict of Interest: - None. Source of funding: - Not declared.

Journal of Pharmaceutical and Biomedical Sciences (JPBMS), Vol. 08, Issue 08