Serekara Gideon Christian, Ransom Baribefii Jacob; Moyosore Ruth Adedeji

Department of Medical Laboratory Science, Faculty of Science, Rivers State University, Nkpolu-Oroworukwo.

P.M.B. 5080, Port-Harcourt, Rivers State, Nigeria.

All Correspondences to: Serekara Gideon Christian E-mail: serekara.christian1@ust.edu.ng

ABSTRACT

The study aimed at evaluating the variability in some haemo-rheological parameters of individuals having different haemoglobin genotypes in Rumuche community, Emohua Local Government Area of Rivers state. A cross-sectional and comparative study design was applied. A total of eighty-seven subjects (sixty-nine AA and eighteen AS haemoglobin genotypes) aged between 20 – 90 years were recruited. Samples collected were analyzed for packed cell volume, erythrocyte sedimentation rate, haemoglobin and fibrinogen concentration using standard methods. Results for haemoglobin genotypes reveals that sixty-nine subjects were haeamoglobin AA (79.31 %), eighteen subjects were haemoglobin AS (20.69 %), no haemoglobin SS genotype was identified among the subjects. Also, the result showed a significant difference in haemoglobin concentration when haemoglobin AA were compared to AS genotypes (14.68 ± 2.600g/dl vs 12.76 ± 2.931g/dl; p = 0.0450). Packed cell volume, erythrocyte sedimentation rate and fibrinogen concentration showed no statistical difference despite variations upon comparison of haemoglobin AA and haemoglobin AS. Based on gender, the variations observed were not statistically significant in all parameters at p < 0.05. The study revealed a significant increase in haemoglobin concentration in favour of individuals with haemoglobin AA genotype. Therefore, a directly proportional difference in the blood viscosity of the different haemoglobin genotypes was observed and this can be a useful diagnostic tool alongside fibrinogen concentration and packed cell volume for predicting the occurrence of some diseases such as atherosclerosis, thrombotic disorders, cardiovascular diseases or haemorrhagic disorders. No variation was observed in packed cell volume, erythrocyte sedimentation rate, and fibrinogen.

Keywords: Haemoglobin Genotype; Haemo-rheological; Packed Cell Volume; Erythrocyte Sedimentation Rate; Fibrinogen; Rumuche Community; Emohua.

INTRODUCTION

Rumuche is one of the communities of Emohua Local Government Area, which was part of the Ikwerre ethnic group of  Rivers  State.  The community has an estimated population of about 5,000 persons as at 2016 [1, 2]. The indigenes of Rumuche are majorly farmers. Haemo-rheology consist of two main words namely,  “haemo”  which means blood and “rheology” which means the science of how blood is being deformed and how it flows. Rheology which includes blood and plasma viscosity is important in the investigation of some vascular disorders. Haemo-rheology is therefore defined as the study of blood flow in blood vasculatures, taking note of the behavior of red blood cells,  their interaction among themselves and also with the endothelial cells of the vascular wall [3]. Haemo-rheology is the study of flow properties of blood and its elements of plasma and cells. Proper tissue perfusion can occur only when the blood’s rheological properties are within certain levels [4].It includes the examination of visible blood properties by the use of rheometric experiments (such as packed cell volume  (PCV)  and erythrocyte aggregation,  plasma (PCV), erythrocyte sedimentation rate (ESR), haemoglobin concentration (Hb) and fibrinogen level of individuals having different haemoglobin genotypes in Rumuche community, Emohua Local Government Area (LGA), Rivers state. The knowledge gain in blood viscosity can be used as a diagnostic tool for the prediction of some diseases such as thrombotic disorders, cardiovascular diseases, atherosclerosis or haemorrhagic disorders, based on the individual’s haemoglobin genotype.

MATERIALS AND METHODS

2.1 Study Design

This is a cross sectional and comparative study which was aimed at evaluating variability in haemorheological parameters of individuals having haemoglobin genotypic differences in Rumuche community, Emohua Local Government Area of Rivers state of Nigeria. A total of eighty-seven subjects were recruited and blood samples were collected and analyzed.

2.2 Study Area

Rumuche is one of the communities of Emohua Local Government Area of Rivers State. Southern Nigeria. Rumuche is located in Nigeria about 470km south of Abuja, the country’s capital town. The Port Harcourt International Airport is about 19km northeast from the community. The community has an estimated population of about 5,000 persons as at 2016. The climate in Rumuche is usually rainy and dry seasons. Rumuche is situated at Latitude 4⁰52’31”N and Longitude 6⁰51’39”E. The indigenes of Rumuche are majorly farmers. The analyses were carried out at the Haematology Laboratory, Medical Laboratory Science Department, Rivers State University, Port Harcourt, Rivers State University Teaching Hospital, Port Harcourt, De-Integrated Medical Diagnostics and Research Laboratory, Ada-George, Port Harcourt and Professor Nimi Briggs Hospital, Rivers State University, Port Harcourt.

2.3 Study Population

A total of 87 subjects (32 males and 55 females) within the age range of 20-90 years were recruited randomly with a total of 69 subjects having AA genotype and 18 subjects having AS genotype. Non indigenes of Rumuche were excluded from the study and those who present signs and symptoms of illness were also excluded from the study. Only apparently healthy subjects were recruited for the study.

2.4 Collection of Blood Samples and Storage

5mL of venous blood was drawn from each subject and 3mL of blood was dispensed into labelled Tripotassium Ethylene Diamine Tetraacetic Acid (K3EDTA) anticoagulated bottle (at concentration of 1.2mg/ml) for haematological analysis while 2mL was dispensed into labelled plain bottles for fibrinogen estimation. Samples were kept in a box and transported to the laboratory at room temperature. The K3EDTA samples were analyzed within 6 hours of collection while the plain bottle samples were spun at 3000rpm for 5 minutes to obtain serum and stored at – 20⁰C until the analysis was done.

2.5 Methodology

2.5.1 Determination of Haemoglobin Genotype

Method: Cellulose acetate method as described by Wild and Bain [8].

Principle: Haemoglobin is a negatively charged protein which when subjected to electrophoresis, migrates toward the anode. Different haemoglobin variants have different migration rates. Due to structural variation in haemoglobin molecules, they possess different electrical charges and therefore separate into different variants during electrophoresis.

Procedure: The cellulose acetate membrane was prepared and 100ml of the Tris-EDTA-borate buffer was poured into the outer sections of the electrophoresis chamber. Two wicks were made wet in the buffer and draped over each support bridge avoiding air bubbles under the wick. The chamber was covered to prevent evaporation. 5 μL of each haemosylate sample was transferred into the well plate. Cellulose acetate membrane was placed in the Zip-Zone aligning plate and samples were applied using the applicator stick. The cellulose acetate membrane was immediately placed in the electrophoresis chamber with the cellulose acetate side down. The chamber was connected to power supply and electrophoresed for 20 minutes at 350 volts and 50 mA.

2.5.2 Determination of Packed Cell Volume

Method: Microhaematocrit method as described by Cheesbrough [9].

Principle: When anticoagulated whole blood is centrifuged in the microhaematocrit centrifuge at 12,000g for 5 minutes, the blood separates into three layers known as the plasma, buffy coat and red cells. The red cells occupy the lower part of the tube and the volume occupied is known as a percentage of the whole blood. This is measured in percentage (%) or litre/litre (L/L).

Procedure: Capillary tubes were filled by capillary action with whole blood up to three-quarter volume. The tubes were properly sealed with a plasticine at one end. The tubes were carefully placed in the numbered slots of the micro-haematocrit rotor with the sealed end against the rim gasket to prevent breakage. The inner lid was scrapped and then the top lid was covered. The samples were centrifuged for 5 minutes at 12,000g. The packed cell volume was read from the micro-haematocrit reader on the zero line and the top of the column on the 100 line and recorded in percentage.

2.5.3 Determination of Haemoglobin Concentration

Method: Cyanmethaemoglobin method as described by Ochei and Kolhatkar [10].

Principle: Haemoglobin is treated with a reagent containing potassium ferricyanide, potassium cyanide and potassium dihydrogen phosphate. The ferricyanide forms m e t h a e m o g l o b i n w h i c h i s c o n v e r t e d t o cyanmethaemoglobin by the cyanide. All forms of haemoglobin except sulfhaemoglobin are converted to cyanmethaemoglobin.

Procedure: 0.02ml of blood was added to 5ml of Cyanmethaemoglobin reagent (Drabkin’s solution) in a test tube (1:250 dilution). The tube was well mixed and allowed to stand for 10 minutes. Absorbance (A) was read in the spectrophotometer at 540nm, while zeroing the spectrophotometer with the blank (Drabkin’s solution).

Calculation of Results

	Absorbance of Standard
Concentration of Test =		x Concentration of Standard
	Absorbance of Test

Results were recorded in g/dL.

2.5.4 Determination of Erythrocyte Sedimentation

Rate

Method: Westergren method as described by Osei-Bimpong and Burthem [11].

Principle:

When citrated blood in a vertically positioned Westergren tube is left undisturbed, red cells aggregate, stack together to form rouleaux and sediment through the plasma.

Procedure:

0.4ml (1 part) of sodium citrate was added into a Westergren bucket and 1.6ml (4 parts) of well mixed whole blood was added directly into the bucket. A Westergren tube of 200mm was inserted into bucket with the diluted blood drawn up to the zero mark and mixed properly. The tube was vertically placed on the stand, away from direct sunlight and left undisturbed and free from vibrations for 1 hour. The result was read in mm/hr after 1 hour.

2.5.5 Determination of Fibrinogen Concentration Method: Sandwich enzyme-linked immunosorbent assay (ELISA) method. Using Fibrinogen Elisa Kit, Elabscience Biotech Co., Ltd, China. Lot No PHLR6HT9ME; Expiry Date: 2020/07/11

Principle:

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human fibrinogen. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human fibrinogen and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human fibrinogen, biotinylated detection antibody and Avidin-

HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human fibrinogen. You can calculate the concentration of Human fibrinogen in the samples by comparing the OD of the samples to the standard curve.

Procedure:

Standard working solution was added to the first two columns: Each concentration of the solution was added in duplicate, to one well each, side by side (100 uL for each well). Samples were added to the other wells (100 uL for each well). The plate was covered with the sealer provided in the kit and incubated for 90 min at 37⁰C. Note: Solutions were added to the bottom of the micro ELISA plate well, touching of the inside wall was avoided. The liquid was then removed out of each well. Washing was avoided. 100 μL of Biotinylated Detection Antibody working solution was immediately added to each well and covered with the Plate sealer. It was gently mixed and incubated for 1 hour at 37°C. The solution was decanted from each well, and 350 uL of wash buffer was added to each well and soaked for 1-2 min and the solution was decanted from each well and patted dry against clean absorbent paper. The wash step was repeated 3 times. 100 μL of HRP Conjugate working solution was added to each well and covered with the Plate sealer. It was incubated for 30 min at 37°C. The solution was decanted from each well and the wash process was repeated for five times. 90 μL of Substrate reagent was added to each well and covered with a new plate sealer. This was incubated for about 15 min at 37°C. The plate was protected from light. 50 μL of Stop Solution was added to each well. The optical density (OD value) of each well was determined at once with a micro-plate reader set to 450 nm. Calculation of results was performed using Beer-Lambert’s law.

Absorbance of Test

Conc.of Test = x Conc.of Standards nearest to test

Absorbance of Standards

2.6 Statistical Analysis

Statistical analysis was done using Graph-pad prism version 8.2.0 for both descriptive and inferential statistics for the different groups under study. The descriptive data was given as mean ± standard deviation (SD).

RESULTS

3.1 Demographic Profile of Participants in the Study population

A total of 87 subjects were recruited for the study between August 4th and August 20th, 2019. Eighteen (20.69%) of the total number were of AS haemoglobin genotype while 69 (79.31%) were of the AA haemoglobin genotype. Table 3.1 shows the demographic profile of the study population.

Table 3.1: Demographic Profile of Participants in the Study Population

Haemoglobin Genotype	Study Population
AA	69 (79.31 %)
AS	18 (20.69 %)

Evaluation of Haemo-rheological Variability…

3.2	Comparison of Some Haemo-rheological	value of 0.0450. There is a non-significant increase
	Parameters Based on Differences in	(p>0.05) in the PCV of AA individuals as compared to the
	Haemoglobin Genotypes	AS individuals with a p-value of 0.1013. A non-significant
		increase (p>0.05) in the ESR of AA and AS individuals
From table 3.2, there is a significant increase (p<0.05) in		with a P-value of 0.0649. A non-significant increase
the haemoglobin concentration of AA genotype		(p>0.05) in the fibrinogen level of AS individuals as
individuals as compared to the AS individuals with a p-		compared to AA individuals.

Table 3.2: Comparison of Some Haemo-rheological Parameters Based on Differences in Haemoglobin Genotypes

Parameters	AA	AS	p-value	Inference
	Mean ± SD	Mean ± SD
Haemoglobin (g/dl)	14.68 ± 2.600	12.76 ± 2.931	0.0450	S
PCV (%)	37.78 ± 4.466	35.11 ± 5.016	0.1013	NS
ESR (mm/hr)	23.94 ± 21.16	37.00 ± 19.88	0.0649	NS
Fibrinogen (ng/ml)	474.9±190.1	499.4±199.6	0.7086	NS

3.3 Comparison of Some Haemo-rheological

Parameters in Males Based on Differences in Haemoglobin Genotypes

From table 3.3, there is a non-significant increase (p>0.05) in the Hb of AA male individuals as compared to AS male individuals with a p-value of 0.7219. A non-significant increase (p>0.05) in the PCV of AA male individuals as compared to the AS male individuals wth a p-value of 0.7524. A non-significant increase (p>0.05) in the ESR of AS male individuals as compared to the AA male individuals with a p-value of 0.6068. A non-significant increase (p>0.05) in the fibrinogen level of AA males as compared to AS males with a p-value of 0.4450.

Table 3.3: Comparison of Some Haemo-rheological Parameters in Males Based on Differences in Haemoglobin Genotypes

Parameters	AA	AS	p-value	Inference
	Mean ± SD	Mean ± SD
Haemoglobin (g/dl)	14.13 ± 2.605	12.14 ± 3.048	0.0858	NS
PCV (%)	37.46 ± 4.294	34.31 ± 4.535	0.0811	NS
ESR (mm/hr)	26.54 ± 21.94	37.62 ± 23.33	0.2244	NS
Fibrinogen (ng/ml)	460.0 ± 174.0	495.2 ± 205.6	0.6420	NS

Key: S=Significant; NS=Non Significant; PCV= Packed Cell Volume; ESR=Erythrocyte Sedimentation Rate; Mean ± SD= Mean ± Standard Deviation. Note: The abbreviations are applicable to all tables

DISCUSSION

The normal haemoglobin usually found in adult humans is Hb A, although some other forms occur as a result of point mutation in the globin chains responsible for this formation. These include the Hb S, Hb C, Hb D, amongst many others. Haemoglobin genotypes are inherited characters determined by the different combinations of amino acid sequence found on the globin polypeptide chains. They include HbAA, HbAS, HbAC, HbSS, HbSC amongst others. Haemorheological tests are a group of tests used majorly to determine the blood rheological status of an individual. In course of this research work, two haemoglobin genotypes were considered, which are AA and AS genotypes [7, 12].

Haemoglobins found in the red blood cells are intracellular proteins which help in the transportation of oxygen from the lungs to the tissues. From the results obtained, haemoglobin concentration for Hb AA genotype 14.68 ± 2.600 was found to be significantly increased by p<0.05 when compared to that of the Hb AS genotype 12.76 ± 2.931. This result is in accordance with the study of Obeagu et al., [13] and the significant difference is as a result of Hb S seen in the Hb AS genotype which causes occlusion in blood vessels thereby reducing the oxygen carrying capacity of the blood and the circulating red blood cells due to their destruction by the spleen. The mean level of haemoglobin concentration in male subjects for AA genotype was 14.96 ± 2.876 g/dL while that of AS was 14.38 ± 2.024 g/dL. The mean level for AA females was found to be 14.13 ± 2.605 g/dL while that of AS was 12.14 3.048 g/dL. The values of haemoglobin concentration for haemoglobin genotype AA was within the normal reference values according to World Health Organization which is 13.0-18.0g/dL for males and 12.5-16.0g/dL for females which confirms the works of Bakare et al. [14] and Obeagu et al. [13] that a significant difference occurs when haemoglobin genotype AA is compared with AS.

The major determinant of blood viscosity is the packed cell volume and an increase in packed cell volume causes increase in blood viscosity. Also, reduction in packed cell volume will lead to reduction in the blood viscosity. Packed cell volume for both haemoglobin genotypes had mean values of 37.78 ± 4.466 % for Hb AA genotype and 35.11 ± 5.016 % for Hb AS genotype. There was a non-significant difference of p>0.05 seen and when both genotypes were compared. Also, when genders (males and females) of both genotypes were compared, the mean value was found to be 38.40 ± 5.459 % for AA genotype and 37.20 ± 6.140 % for AS genotype males, while the females had 37.46 ± 4.294 % for AA genotype and 34.31 ± 4.535 % for AS genotype. There was also no significant difference seen which contradicts the work of Obeagu et al, [13] that due to the destruction of sickle shaped cells before the normal life span of 120 days elapses in AS individuals.

There was a non-significant difference of p>0.05 in the comparison of the erythrocyte sedimentation rate of haemoglobin genotypes AA and AS individuals. Based on this, being AA or AS does not invariably affect erythrocyte sedimentation rate in apparently healthy individuals. Fibrinogen level for both haemoglobin genotypes indicated that there was a non-significant difference and when genders of both genotypes were compared, there was also no significant difference which was in accordance with the work of Ajayi and Uche [13] that there is no significant increase in the fibrinogen level of AA and AS genotypes.

CONCLUSION

From the study, haemoglobin concentration was significantly higher in AA individuals than in AS individuals. Packed cell volume showed no significant variation; erythrocyte sedimentation rate showed no significant variation due to the absence of sickle cell disease, inflammatory disease, infections, amongst other disease conditions. Also, fibrinogen level showed no significant variation because the clotting factors of these individuals seems normal at the time of the research.

As a result of the significant difference in the haemoglobin concentration, there will be a directly proportional difference in the blood viscosity of the different haemoglobin genotypes and this can be a useful diagnostic tool alongside fibrinogen concentration for predicting the occurrence of some diseases such as atherosclerosis, thrombotic disorders, cardiovascular diseases or haemorrhagic disorders.

LIMITATION OF THE STUDY

The study did not include subjects with haemoglobin SS and/or SC, due to the fact that Sicklers (those with Sickle Cell Anaemia) did not give their consent to be part of the study.

Serekara Gideon Christian

CONSENT

A written consent was obtained from each participant.

ETHICAL APPROVAL

Approval to conduct the research was granted by the Department of Medical Laboratory Science, Rivers State University, Nigeria.

COMPETING INTERESTS

Authors have declared that no competing interests exist.

REFERENCES

• World postal code (2019). Emohua post codes: Nigeria . Retrieved from https://www. worldpostalcode.com/nigeria/rivers/emohua. Accessed 11th July, 2019.
• Rumuche in Rivers state, Nigeria. Destination Guide. Retrieved from https://www.tripmondo. com/nigeria/rivers-state/emohua/rumuche. Accessed 28th July, 2019.
• Robertson A. M., Sequeira A., Kameneva M. V. (2008). Hemorheology. In: Hemodynamical Flows (pp 63-120). Oberwolfach Seminars, vol
  1. Birkhäuser Basel, Switzerland.
• Cokelet, G. R. & Meiselman, H. J. (2007). Basic aspects of haemorheology, in Baskurt, O. K., Hardeman, M. R., Rumping, M. W. & Meiselman, H. J. (Eds), Handbook of Haemorheology and Haemodynamics (7th ed; pp 21-33). The Netherlands; IOS Press.
• Ajayi, O. I., Famodu, A. A., Otakpor, A. N. & Idubor, J. F. (2005). Fibrinolytic risk of unmodified electroconvulsive therapy in Nigeria depressed patients, Haema, 8(3), 436-438.
• Hardisson, R. C. (2012). Evolution of haemoglobin and its genes, Cold Spring Habour Perspectives in Medicine, 2(12), 1-18.
• Buseri, F. I. & Okonkwo, C. (2014). Abnormal haemoglobin genotypes and rhesus blood groups associated with HIV infection among HIV – exposed infants in North-Western Nigeria, Dove Medical Press, 2014(6), 15-20.
• Wild, B. J. & Bain, B. J. (2012). Investigation of abnormal haemoglobins and thalassemia, in Bain, B. J., Bates, I., Laffan, M. A. & Lewis, S. M. (Eds), Dacie and Lewis practical haematology (11th ed; pp. 301-332). United Kingdom; Churchill Livingstone.
• Cheesbrough, M. (2006). Haematological tests, in Cheesbrough, M. (Ed), District Laboratory Practice in Tropical Countries (2nd ed; pp 268-347). New York, Cambridge University Press.
• Ochei, J. O. & Kolhatkar, A. A. (2007). The complete blood count, in Ochei, J. O. & Kolhatkar, A. A. (Eds), Medical Laboratory Science Theory and Practice (pp. 273-287). United Kingdom, McGraw-Hill publishing company limited.
• Osei-Bimpong, A. & Burthem, J. (2012). Supplementary techniques including blood parasites diagnosis, in Bain, B. J., Bates, I., Laffan, M. A. & Lewis, S. M. (Eds), Dacie and Lewis Practical Haematology (11th ed; pp 101-121). United Kingdom; Churchill Livingstone.
• Akhigbe, R. E., Ige, S. F., Afolabi, A. O., Azeez, O. M., Adegunlola, G. J. & Bamidele, J. O. (2009). Prevalence of haemoglobin variants, ABO and rhesus blood groups in Ladoke Akintola University of Technology, Ogbomoso, Nigeria, Trends in Medical Research, 4(2), 24-29.
• Obeagu, E. I., Okoroiwu, I. L., Daniel-Igwe, G. & Elemchukwu, Q. (2015). Comparison of some haematological profiles in genotype AA, AS and AC persons amongst Imo State University students, International Journal of Advanced Multidisciplinary Research, 2(12), 23-28.
• Bakare, A. A., Azeez, M. A. & Agboiade, J. O. (1997). Gene frequencies of ABO and Rhesus blood groups and haemoglobin variants in Ogbomoso, south-west, Nigeria, Global Journal of Medical Science, 3(3), 17-22.

Ajileye A. B.

Department of Biomedical Laboratory Science, College of Medicine, University of Ibadan. ayobless05@gmail.com

Esan E. O.

Department of Medical Laboratory Services, State General Hospital, Okitipupa, Ondo State.

Adeyemi O. A.

Department of Medical Laboratory Science, College of Medicine and Health Sciences, Afe Babalola University,

Ado Ekiti, Ekiti State. olkems145@gmail.com

Alade D. T.,

Department of Medical Laboratory Services, Ladoke Akintola University of Technology Teaching Hospital, Osogbo, Osun State. dupe401@gmail.com

All correspondence to: Ajileye A. B. Department of Biomedical Laboratory Science, College of Medicine, University of Ibadan. ayobless05@gmail.com

ABSTRACT

Cervical cancer is a type of cancer that develops in a woman’s cervix, it’s characterized by the growth of abnormal cells in the cervix. The main cause of cervical cancer is Human Papillomavirus (HPV). Others include; smoking, multiple sexual partners, use of oral contraceptives etc. Signs and symptoms include; increased vaginal discharge, pelvic pain, bleeding or pain after sexual intercourse etc. Screening tests include; Pap test, HPV test, Visual inspection by lugol’s iodine and acetic acid. The sample for screening is collected from the cervix using a cytobrush, while the patient lies in a lithotomy position. Further cytological tests are carried out to diagnose cervical cancer after screening like cone biopsy, colposcopy and pap smears. However these cytological tests have low specificity and significant variability in the diagnosis of cervical dysplastic lesions. Recently, the use of molecular-biology methods to detect the presence of Human Papillomavirus (HPV) has been employed. The molecular methods are more specific than the cytological methods. After the samples are collected, the DNAs are extracted using QIAamp DNA kit. The methods for detecting HPV are – Nucleic acid hybridization assays, Signal amplification assay, Nucleic-acids amplification assay. Molecular techniques are most commonly used for HPV testing, and are the gold standard for diagnosing this viral infection. In spite of their value, molecular techniques still must become more rapid, automated, and low-cost to be of practical use in low-income populations and countries.

KEYWORDS: Cervical-Cancer, Papillomavirus, Lithotomy, Molecular, Hybridization.

INTRODUCTION

Cervical growth is a malignancy emerging from the cervix. It is because of the irregular development of cells that can attack or spread to different parts of the body. From the get-go, commonly no indications are seen¹; later indications may incorporate unusual vaginal bleeding, pelvic pain, or torment amid sex. While bleeding after sex may not be serious, it may also indicate the presence of cervical cancer1. Human Papilloma Virus (HPV) as an important cause of cervical cancer, based on their association with cervical cancer and precursor lesions, HPVs can also be grouped to high-risk and low-risk HPV types. Low-risk HPV types include types 6, 11, 42, 43, and 44. High-risk HPV types include types 16, 18, 31, 33, 34, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, and 70². Included in the high-risk group are some HPV types that are less frequently found in cancers but are often found in squamous intraepithelial lesions (SILs). Some authors refer to these HPV types as intermediate-risk. Low-risk subtypes are also occasionally found in cervical carcinomas^2&3. HPV DNA testing may reduce costs by triaging patients into appropriate management strategies and reducing unnecessary colposcopy and less frequent screening in low-risk patients⁵.

Fig 1. ANATOMY OF THE CERVIX³

“The cervix is the lower part of the uterus in the human female reproductive system. The opening into the uterus is called the internal OS, and the opening into the vagina is called external os. The lower part of the cervix is known as the vaginal portion of the cervix or ectocervix, bulges into the top of the vagina” ⁵.

The cervical canal is lined with a single layer column shaped cells, while the ectocervix is covered with multiple layers of cells topped with flat cells. The two types of epithelia meet at the squamocolumnar junction. Infection with the human papillomavirus (HPV) can cause changes in the epithelium, which can lead to cancer of the cervix⁵.

Risk factors for contracting cervical cancer include

1. Human Papilloma Virus (HPV)
2. Smoking
3. Having a weakened immune system e.g HIV
4. Having more than one sexual partner
5. Using oral contraceptives for a long time⁶.

SIGNS AND SYMPTOMS OF CERVICAL CANCER

1. Bleeding that occurs between regular menstrual periods.
2. Bleeding after sexual intercourse.
3. Menstrual periods that last longer and are heavier than before.
4. Pelvic pain.
5. Bleeding after going through menopause.
6. Increased vaginal discharge⁷.

Cervical screening is the process of detecting and removing abnormal tissue or cells in the cervix before cervical cancer develops. By aiming to detect and treat cervical neoplasia early on, cervical cancer. Several screening methods for cervical cancer include:

1. Pap test ( also known as Pap smear or conventional cytology)
2. Liquid-based cytology
3. The HPV DNA testing
4. The visual inspection with acetic acid and lugols iodine⁸.

Pap test is a method of examining with a microscope a sample of superficial cells that line the inner wall of the uterine cervix to detect any abnormal cell for early diagnosis of uterine cancer⁹. The position for taking the pap. smear from the patient is known as lithotomy¹⁰. After the sample is collected from the patient, smears are made on clean slides, fixed immediately in 95% alcohol and then stain with Papanicolaou stain, mounted and then view under a microscope for analysis¹⁰.

Fig 2.0: A Lithotomy Position, Showing how Cervical Samples are Collected¹⁰

Colposcopy

A colposcopy is a special way of looking at the cervix. It uses a light and low powered microscope to make the cervix appear much larger¹¹. It helps in the diagnosis of premalignant and early malignant changes in the cervix ¹².

Cone Biopsy

A cone biopsy is a small operation to remove a cone shaped piece of tissue from the cervix and examined under a microscope¹¹. Cone biopsy removes abnormal tissue that is high in the cervical canal. Pap test and liquid-based cytology have been effective in diminishing incidence and mortality rates of cervical cancer in developed countries but not developing countries¹¹. Prospective screening methods that can be used in low-resource areas in the developing countries are the HPV DNA and the visual inspection⁸.

Conventional Cytology

In the conventional Pap smear, the cells collected are smeared on a microscopic slide and its fixed in a cytological fixative. The slide is then sent to a laboratory for evaluation. The report for the accuracy of conventional cytology shows 94% specificity and 72% sensitivity¹³.

Liquid Based Cytology

Liquid based cytology is a technique that enables cells to be suspended in a monolayer and thus making better morphological assessment possible with improved sensitivity and specificity¹⁰. Liquid-based cytology is a method of preparing samples for examination in cytopathology. The sample is collected, normally by a small brush, in the same way as for a conventional smear test, but rather than the smear being transferred directly to a microscope slide, the sample is deposited into a small bottle of preservative liquid. At the laboratory the liquid is treated to remove other elements such as mucus before a layer of cells is placed on a slide. The technique allows more accurate results¹³. For many years, efforts have been made to develop methods that would enhance the sensitivity and specificity of the Papanicolaou smear. Emphasis has been placed on creating automated screening machines whose success depends on a representative sampling of cells on standardized slides containing a monolayer of well-stained, well-preserved cells.

From recent research and development, liquid-based preparations outperform conventional smears because of improved fixation, decreased obscuring factors, and standardization of cell transfer. Proponents point out that, in direct smears, the cells are not transferred in a representative fashion and that up to 90% of the material scraped from the cervix may be discarded with the sampling device. With liquid-based collection, the sampling will be representative and operator-dependent variation will not occur since processing is controlled by the laboratory¹³.

Methods of Carrying Out liquid based cytology

There are several systems that are currently available. The most widely used are:

1. Sure Path (autocyte, TriPath Imaging)
2. Thin Prep (CYTYC)

The SurePath method, the sample is vortexed, strained, layered onto a density gradient, and centrifuged. Instruments required are a computer-controlled robotic pipette and a centrifuge. The cells form a circle 12.5 mm in diameter.

The ThinPrep method requires an instrument and special polycarbonate filters. After the instrument immerses the filter into the vial, the filter is rotated to homogenize the sample. Cells are collected on the surface of the filter when a vacuum is applied. The filter is then pressed against a slide to transfer the cells into a 20 mm diameter circle.

Both methods result in a well-preserved approximate monolayer of cells, with a background devoid of blood and mucus. Other methods not commonly being used in liquid based cytology include:

1. The cytoscreen method
2. The Labonard Easy Prep

The cytoscreen method is a manual process relying upon photometry to evaluate the cellularity of the cell suspension prior to centrifugation onto a glass slide.

The Labonard Easy Prep is another manual method, whereby an aliquot of sample fluid is loaded into a separation chamber attached to a glass slide, which contains absorbent Paper. The cells settle in a thin layer and the preparation is stained using normal laboratory procedures.

The Human Papilloma Virus (HPV) DNA Test

The test is done by looking for pieces of the DNA of the HPV carcinogenic genotypes in cervical cells. The test can be done at the same time as the Pap test, with the same swab or a second swab. The HPV DNA test is most often done and used in 2 situations:

• The HPV gene test can be used in combination with the Pap test to screen for cancer. It is recommended for women that are 30 years and above because women in their 20s who are sexually active are much likely to have an HPV infection that will go away on its own. For these younger women, results of this test are not as significant and may be more confusing
• The test can also be used in women who have slightly abnormal Pap test results (ASC-US) to find out if they might need more testing or treatment¹⁴. If the Pap test result is normal, the patient still tests positive for HPV, the main options are:

• Repeat co-testing (with a Pap test and HPV test) in one year

• Testing for HPV type 16 or 18 (this can often be done on the sample in the lab). If the test is positive for type 16 or 18, colposcopy would be recommended. If the test is negative, the co-testing is repeated in one year¹⁴.

Visual Inspection Methods

Visual inspection of the cervix after application of Lugol’s iodine, the first method used for cervical cancer screening, was introduced in the 1930s by Schiller. However, Schiller’s test has poor specificity and was almost replaced with the advent of cervical cytology.

Current cervical cancer screening protocols typically include a combination of cervical cytology and human papillomavirus testing. Visual inspection of the cervix has re-emerged as a screening tool for low-resource settings, despite its limited specificity, since it is economical and provides immediate results. Visual inspection can be performed with acetic acid (VIA) or Visual inspection with Lugol’s iodine (VILI).

Visual inspection is indicated for women for whom cervical cancer screening is recommended and for whom these methods are the best screening option i.e women who do not have access to cervical cytology and human papillomavirus testing.

There are no absolute contraindications to visual inspection of the cervix. Visual inspection with acetic acid (VIA), rather than visual inspection with Lugol’s iodine (VILI), should be performed in women with an allergy to iodine. Visual inspection can be performed during pregnancy, but cervical biopsies are relatively contraindicated in pregnant women unless invasive cancer is suspected¹⁶.

Molecular Methods over Cytology Methods in the Diagnosis Of Cervical Cancer

Although traditional cytology still has a place in the modern clinical laboratory, it is now starting to make way for techniques that utilizes the increased resolution, accuracy and speed offered by the molecular revolution¹⁶. Microarrays, next generation sequencing, and advances in automation all have the potential to further improve the accuracy and reliability of clinical research and diagnosis, and may eventually replace microscope-based methods¹⁶.

To date, HPV cannot be cultured in vitro, and immunological tests are inadequate to determine the presence of HPV cervical infection. Indirect evidence of anogenital HPV infection can be obtained through physical examination and by the presence of characteristic cellular changes associated with viral replication in Pap smear or biopsy specimens¹⁵. Alternatively, biopsies can be analyzed by nucleic acid hybridization to directly detect the presence of HPV DNA¹⁷.

Most adults have been infected with HPV at some time. An infection may go away on its own. But sometimes it can cause genital warts or lead to cervical cancer. That’s why it’s important for women to have regular Pap tests. Pap test can find changes in cervical cells before they turn malignant¹⁸. If you treat these cell changes, you may prevent cervical cancer.

Cytology-based nation-wide cervical screening has led to a substantial reduction of the incidence of cervical cancer in western countries. However, the sensitivity of cytology for the detection of high-grade precursor lesions or cervical

cancer is limited; therefore, repeated testing is necessary to achieve a very effective result. In addition to that, adenocarcinomas and its precursors are often missed by cytology¹⁹.

Consequently, there is need for a better screening test. The insight that infection with high risk human papillomavirus (hrHPV) is the causal agent of cervical cancer and its precursors has led to the development of molecular tests for the detection of high risk human papilloma virus (hrPV)¹⁹. Strong evidence now supports the use of hrHPV testing in the prevention of cervical cancer²⁰.

From a clinical point of view, testing for hrHPV is only useful when a positive hrHPV test result is informative about the presence or absence of CIN2+ (clinical sensitivity and specificity). Thus, in order to prevent excessive follow-up procedures for women with transient hrHPV infections or hrHPV-positive women without cervical lesions, candidate hrHPV tests to be used for cervical screening should be clinically validated¹⁹.

Pathogenesis

Transmission of HPV occurs primarily by skin-to-skin contact21. Basal cells of stratified squamous epithelium may be infected by HPV. Other cells types appear to be relatively resistant. It is assumed that the HPV replication cycle begins with entry of the virus into the cells of the basal layer of the epithelium¹⁹.

It is likely that HPV infection of the basal layer requires mild abrasion or microtrauma of the epidermis. Once inside the host cell, HPV DNA replicates progress to the surface of the epithelium. In the basal layer, viral replication is considered to be non-productive, and the virus establishes itself as a low-copy-number episome by using the host DNA replication machinery to synthesize its DNA on average once per cell cycle . In the differenciated keratinocytes of the suprabasallayer of the epithelium, the virus switches to a rolling-circle mode of DNA replication, amplifies its DNA to high copy number, synthesizes capsid proteins, and causes viral assembly²¹.

Molecular biology-based techniques

The human papillomavirus (HPV) is the causative agent of cervical cancer, but not all genotypes of HPV are causal factors, some cause genital warts. Out of the 100-200 different HPV genotypes, the human papillomavirus (HPV) genotypes most frequently indicated as the causal factor of cervical cancer are HPV16, 18, 31, 33, 35, 45, 52 and 58 which can also be reffered to as the high risk factor of Human Papilloma virus. The Pap smear unquestionably is a successful screening test for cervical cancer. However, recent advances in technology have raised questions regarding whether the conventional Pap smear is still the standard of care²². HPV cannot be propagated in tissue culture, and therefore, in most cases its accurate identification relies on molecular biology techniques. With a double-stranded DNA genome of about 8000 base pairs (bp) and a well-known physical structure and gene organization, the tests of choice for detecting HPV in clinical specimens are based on nucleic probe technology²³. The six main possible clinical applications of HPV DNA testing are:

1. triage of women with equivocal or low-grade cytological abnormalities;
2. follow-up of women with abnormal screening results who are negative at colposcopy/biopsy;
3. prediction of the therapeutic outcome after treatment of cervical intraepithelial neoplasia (CIN);
4. primary screening for HPV DNA testing, alone or in combination with a Pap smear, to detect cervical-cancer precursors.
5. gain valuable information on the persistence of certain HPV types
6. Investigation of regional and country-based prevalence of type-specific HPV, to provide baseline values against which the global impact of HPV vaccination can be assessed in the future.

Extraction of DNA from Cervical Smears or Tissue For Molecular Analysis

To carry out the molecular detection of HPV, the patient sits in a litothomy position, a sterile speculum is used to dilate the cervix and then with the use of a sterile swab or with the use of a cytobrush, cervical samples are collected at the squamo-columnar junction, the smears must be fixed immediately in a cytological fixative and thereafter, DNA extraction can be made and then used to analyse cervical cancer molecularly. DNA extraction can also be obtained from Cone biopsy formalin-fixed paraffin-embedded samples for the molecular analysis of cervical cancer. DNA extraction is carried out using;

QIAamp DNA kits – these kits use vacuum procedures or fast spin-column. The DNA binds to the silica-gel membrane while contaminants pass through. The DNA is purified after two efficient wash steps (that removes PCR inhibitors). The DNA is left to be eluted²³.

The presence of HPV can be inferred from morphological, serological and clinical findings. However, HPV diagnosis relies on molecular-biology techniques that allow its accurate detection and typing²⁴. These molecular-biology techniques are;

1. Nucleic acid-hybridization assays
2. Signal-amplification assays
3. Nucleic-acid amplification.

Nucleic-Acid Hybridization Assays This has 3 techniques;

• Southern blot
• In situ hybridization
• Dot blot hybridization

These techniques use radio-labelled nucleic acid hybridization assays to detect HPV infection in cervical samples. Although these techniques generated high-quality information, the disadvantages of these direct-probe approaches include low sensitivity, the need for relatively large amounts of purified DNA, and time-consuming procedures23. The southern blot is the gold standard for HPV genomic analysis; it’s a very good technique in the analysis of the presence of HPV in association with their morphology. But the disadvantage of this southern blot technique is that it is time consuming and with a low sensitivity. Southern blot and hybridization cannot use degared DNA²⁵.

B. Signal-Amplification Assays

This has 2 techniques;

• Digene HPV test using Hybrid Capture 2 (hc2) technology
• Cervista HPV HR assay

Hybrid Capture 2

The Hybrid Capture 2 system is a non-radioactive signal-amplification method based on the hybridization of the target HPV-DNA to labeled RNA probes in solution¹⁷. This test detects 13 HR-HPV types (-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59 and -68) or 5 LR-types (-6, -11, -42, -43, and -44)¹⁷.

This assay distinguishes between HR and LR groups, but was not designed for genotyping single HPV (Cuzick et al, 2008). This is a significant finding, since with persistent infection the risk of a precancerous lesion is between 10 and 15% with HPV types -16/18, and below 3% for all other HR types combined. Therefore, HPV genotyping is very important to identify single oncogenic HPV types and to provide more information regarding risk-stratification as well as persistence of infection ²⁶.

Cervista HPV

The Cervista HPV detects the presence of 14 HR-HPV types, consisting of -16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58,-59,-66 and -6826. This assay also utilizes a signal-amplification method for the detection of specific nucleic acids.

In comparison with HC2, the Cervista assay demonstrated 100% sensitivity in the detection of CIN III and 98% sensitivity in the detection of CIN II. In addition, this assay showed a lower false-positive rate, and high sensitivity and specificity to genotyping HPV -16/1826.

Nucleic Acid-Amplification Methods Microarray analysis

This method uses probe amplification, the PCR (Polymerase chain reaction) product is hybridized onto a chip, and after a washing step, hybridized signals are visualized with a DNA chip scanner. The microarray-based automated techniques allow for parallel analysis of multiple DNA samples. At present, the two major applications of DNA microarrays are gene-expression profiling and mutation analysis¹⁶.

Some studies have demonstrated that DNA microarray analysis coupled with PCR can be successfully applied to detection and genotyping of the HPV. The HPV DNA chip showed higher sensitivity and specificity than gel electrophoresis, and in some cases they produce better results than direct DNA sequencing²⁰.

PapilloCheck®

This assay detects and genotypes 24 HPV types in a single reaction (HPV -6, -11, -16, -18, -31, -33, -35, -39, -40, -42, – 43, -44, -45, -51, -52, -53, -55, -56, -58, -59, -66, -68, -70, – 73, and -82). The assay uses a multiplex PCR with fluorescent primers to amplify a 350 bp fragment of the E1 gene of HPV, comprising 28 probes, each in 5 replicate spots fixed on a DNA chip. Co-amplification of the human ADAT1 gene is used as internal control. The hybridization is performed on a microarray chip, which is automatically scanned and analyzed using the CheckScanner™ at both 532 and 635 nm, and the Check-Report™ software, respectively²⁷.

The main advantage of the PapilloCheck® assay (Greiner Bio-One GmbH, Frickenhausen, Germany) is HR/LR-HPV identification, and detection of multiple infections, and may be considered a reliable screening test. However, this assay does not amplify HPV -35 and -53, the cost is still relatively high, and it requires specific apparatus²⁷.

Polymerase chain reaction (PCR)

The PCR-based techniques are highly sensitive, specific, and widely used. In a conventional PCR, the thermostable DNA polymerase recognizes and extends a pair of oligonucleotide primers that flank the region of interest. In the final process, the PCR can generate one billion copies from a single double-stranded DNA molecule after 30 cycles of amplification²⁸.

The HPV-PCR protocols use consensus primers such as PGMY09/PGMY1 and GP5+/GP6+, which allow amplification of a large number of HPV genotypes in a single reaction. The primers target conserved regions of the HPV genome, such as the L1 capsid gene. After amplification, the HPV genotypes can be determined separately, using techniques such as restriction-fragment length polymorphism (RFLP), linear probe assays, direct sequencing, or genotype-specific primers. Some researchers have used a type-specific PCR, with primers that amplify the long control region L1 and E6/E7²⁹.

These PCR techniques also have some drawbacks, mainly in competition for reagents, leading to false negative results for multiple type infections that are contained in samples at lower copy numbers. Because of this problem, the PCR method may not detect all the HPV genotypes that are present in the sample. Another downside is that multiple infections are not uncommon²⁸. Amplification of samples containing DNA from more than one HPV genotype can lead to a much stronger amplification of one of the sequences present, which would complicate the detection of all genotypes in a sample with multiple infections. Sometimes, additional, labor-intensive procedures, such as sequencing or type-specific PCR, are required²⁸.

PCR-RFLP

Genotyping by PCR-RFLP allows the HPV to be typed, and is easier and less expensive than sequencing. The method is simple and robust, does not require sophisticated equipment, and is particularly suited to settings in which financial resources are limited²⁷. PCR-RFLP shows good discriminatory power by differentiating the virus in HR or LR, and it is possible to identify single or multiple infections. In this technique, the amplified DNA is digested by restriction enzymes, resulting in DNA fragments of various lengths. The commonest restriction enzymes are BamHI, Dd6eI, HaeIII, HinfI, PstI and RsaI. However, Santiago et al.,³⁰ used a single restriction enzyme, HpyCH4V, to detect 21 HR- and 31 LR-HPV genotypes ²⁷.

Real-time PCR

This assay is a reliable, sensitive, and specific diagnostic tool for detection and genotyping of targeted HPV genotypes in tissue specimens³¹ and cellular samples. The advantages of this method are: (i) ability to detect viral load; (ii) with the use of different fluorochromes that emit fluorescence, as the PCR reaction proceeds, the reactions can be performed in multiples and can amplify different nucleic-acid targets; (iii) nucleic acids can be detected even

in a very small concentrations, using a 7-log dynamic range to extrapolate the viral load/concentration over the standard curve; and finally, (iv) it is extremely reproducible, rapid, and applicable to clinical samples³².

Abbott real-time PCR

The Abbott Real-Time HR-HPV test is a novel assay based on concurrent individual genotyping for HPV-16 /18 and pooled detection of 12 HPV genotypes: 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68²³.

COBAS® 4800 HPV test

This test features automated sample preparation combined with Real-Time PCR technology to detect 14 HR-HPV. The PCR amplification and detection occur in a single tube, (i) HPV -16, (ii) HPV -18, (iii) 12 HR (-31, -33, -35, – 39, -45, -51, -52, -56, -58, -59, -66, and -68) as a pool, and

1. β-globin as the control for extraction and amplification adequacy¹⁴.

The agreement between COBAS® 4800 (Roche Molecular Systems, Pleasanton, CA, USA) and Real-Time PCR was strong in a study that determined the reproducibility, involving a sequence of several consecutive steps, both intra- and inter-laboratory. The assay is easy to use because it is adapted for primary specimens, and the results can be obtained approximately 4 hours after the sample is received. COBAS® 4800 fulfils all requirements as defined in the international guidelines to consider it clinically validated for screening, and is reliable in the detection of HR-HPV. This test has been clinically validated for ASC-US triage³².

HPV genome sequencing

The dideoxy chain-termination technique (Sanger technique) was first described for genome sequencing more than three decades ago. Fluorescently labeled nucleotides were incorporated into Sanger sequencing, and advances have led to increasing expansion and development of high-quality, thorough sequencing²⁰. However, it has not been validated for clinical use. Similar to dideoxy sequencing methods, pyrosequencing is applicable to any source of DNA or RNA that can be amplified by PCR (blood, saliva, cell line, plasma, serum, tissue, formalin-fixed paraffin-embedded samples, and whole genome-amplified DNA). The method is based on the detection of the pyrophosphate released during DNA synthesis, and has many advantages over dideoxy sequencing for a wide range of applications that require short-to medium-sequence stretches. The primary advantage is simplicity: the readout sequence itself is obtained, rather than a fluorescent signal that must be converted to a sequence. Secondly, it is faster and less expensive: savings result from its sequence-by-synthesis process where a DNA sequence is read in real time, and it is synthesized by addition of inexpensive, unlabeled nucleotides; and finally, the method is uniquely quantitative²².

CLART® human papillomavirus 2

The CLART® Human Papillomavirus 2 (Genomica, Madrid, Spain) methodology uses biotinylated primers that amplify a 450 bp fragment within the HPV L1 region. Co-amplification of an 892 bp region of the FTR gene and a 1.202 bp fragment of a transformed plasmid provides a control to ensure DNA extraction adequacy and PCR efficiency. Amplicons are detected by hybridization in a low-density microarray containing triplicate DNA probes specific for 35 HPV (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85 and 89). Semi-quantitative results can be obtained in an automatic reader with highly comparable outcomes, showing excellent sensitivity, specificity, and reproducibility³³.

INNO-LiPA

This assay genotypes all 14 HPV that are covered by Real-Time²¹. INNO-LiPA (LiPA HBV GT; Innogenetics N.V., Ghent, Belgium) is based on the co-amplification of the 65 bp region of the HPV L1 gene and the 270 bp of the human HLA-DP1 gene using SPF10 biotinylated primers, followed by genotyping. Some carcinogenic genotypes such as HPV 35, 39, 52, 56 and 66 were not covered by this method, and it was found to be the least effective genotyping for HPV 42 and 5916.

Although the majority of nucleic-acid amplification methods can reliably detect HPV in cervical-swab specimens, only a few, including Real-Time PCR, are potentially suitable for archival clinical specimens, since they target a relatively small portion of the HPV genome (less than 160 bp). Therefore, the observed differences in internal control amplification efficacy between Real-Time and INNO-LiPA can be attributed most reasonably to the differences in target amplicon length: 136 bp vs. 270 bp, respectively. This kit can be also used on samples taken with swabs, brushes, tampons, and lavage¹⁶.

The Linear array®

The Linear Array® HPV Genotyping (Roche Molecular Diagnostics, Pleasanton, CA, USA) is a PCR-based assay coupled with a reversed line blot hybridization. This assay allows the discrimination of 36 HPV, including 15 HR (-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68, -73 and -82), 3 probable HR (-26, -53 and -66), 10 LR (-6, -11, – 40, -42, -54, -61, -70,-72, -81 and -CP6108) and 9 genotypes for which the risk is still undetermined (-55, -62, -64, -67, -69, -71, -83, -84 and -IS39)³⁴.

The test uses biotinylated PGMY09/11 primers to amplify a 450 bp fragment within the polymorphic L1 region of the HPV genome. Co-amplification of the 268 bp region of the human β-globin gene provides a control to ensure DNA extraction adequacy and PCR efficiency. The hybridization and detection of the amplified product are performed with the Auto – LIPA™ instrument (Innogenetics, Ghent, Belgium), which can process up to 30 strips simultaneously in a perfectly standardized fashion. Colored signals on the strips are read by the naked eye and interpreted according to the Linear Array® reference guide. Equivocal results can be obtained for HPV 52 when HPV 33, 35 or 58 are also present, because it is detected through a cross-hybridization probe for these 4 HPV types. An additional, specific probe is present on the strip to confirm the detection of HPV 33, 35 and 58, but not of HPV 52³⁴.

Clinical arrays® HPV

This kit (Genomica SAU, Madrid, Spain) allows the detection and genotyping of HPV. The DNA extraction method is a modified procedure using absorption columns. The kit employs biotinylated primers to define a sequence of 451 nucleotides within the polymorphic L1 region of the HPV genome. A human cystic-fibrosis transmembrane conductance regulator (CFTR) gene and control plasmids are used in order to check both the PCR procedure and the integrity of the DNA²⁴. This also allows the detection of the 35 genotypes that are individually associated with HR ( 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 82 and 85) or LR-HPV ( 6, 11, 40, 42, 43, 44, 54, 61, 62, 71, 72, 81, 83, 84 and 89). It is possible to identify simple infections or co-infections²⁴.

Microplate colorimetric hybridization assay (MCHA)

The MCHA (Boehringer Mannheim, Germany) is a method for identifying six HR-HPV ( 16, 18, 31, 33, 39 and 45) and is based on the amplification by PCR of the 150 bp fragment within the L1 region by consensus primers GP5+/6+, followed by colorimetric hybridization to six type-specific probes on microwell plates (Immobilizer™ Amino Surface, Nunc, Roskilde, Denmark)²².

The MCHA showed very good agreement with PapilloCheck® for HPV 31, 33, 45 and higher sensitivity in identifying HPV 16 and 18, but poor agreement for HPV

To improve MCHA for detection of other genotypes, probes for HPV 35, 52, 56 and 58 should be included²².

Conclusion

Cervical cancer develops over a long period, through precursor lesions that may regress spontaneously without treatment. The challenge of cytological screening is to detect the lesions that have a high risk of progression. Consequently, various biomarkers associated with the risk of progression of this cancer have been investigated, and most are associated with high risk-HPV. Molecular techniques are most commonly used for HPV testing, and are the gold standard for diagnosing this viral infection. In spite of their value, molecular techniques still must become more rapid, automated, and low-cost to be of practical use in low-income populations and countries.

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Wazis Chama Haruna

Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Maiduguri, Bornu State, Nigeria.

Joseph Oyepata Simeon and Modupe Builders

Department of Pharmacology, Faculty of Pharmacy, Bingham University, Nasarawa, Nigeria

Joseph Opeyemi Tosin

Department of Pharmacology, Faculty of Basic Medical Sciences, University of Port Harcourt, Rivers State, Nigeria.

All Correspondences to: Joseph Oyepata Simeon E-mail: simeon4unme@yahoo.com

ABSTRACT

Aim/Introduction: plant has been the primary and initial source of drug development. Plant has been used for medicinal purposes long before prehistoric period. The aim of this study is to evaluate the effect of Pterocarpus santalinus on rat’s kidney over a period of 28 days. Method: A total of 24 rats of either sex were selected. Group 1 received distilled water (10 ml/kg), while group 2, 3 and 4 received Pterocarpus santalinus 100, 200 and 400 mg/kg respectively. Animals were kept in standard cages and given access to the extract, water and food orally for 28 days, after which they were weighed and sacrificed. Blood was collected by cardiac puncture and taken immediately for analysis. The histological effect of the plant on the kidney was also studied. Result: There was slightly Significant (P<0.05) decrease in RBC, HGB, MCV, while there was no change in the level of neutrophiles, basophiles, eosinophiles and platelets. Pterocarpus santalinus, slightly significantly (p<0.05) increased Na level at 300 mg/kg when compared to the control while other parameter (K, CL and Urea levels) remained relatively unchanged. Histological features agree with haematological parameter. Conclusion: The result of the study showed that the Pterocarpus santalinus has little to no toxicity potential on the kidney of rats, indicating that it may

be safe for human consumption

Keyword: Pterocarpus santalinus, rat, blood, kidney

INTRODUCTION

Most people have two kidneys1. They are bean-shaped organs located on both sides of the spine, behind the stomach. Each one is about the size of an adult fist1. Their main purpose is to keep the composition of blood in the body balanced to maintain good health. The kidneys filter extra water and toxins from the blood. The kidneys filter about 120 to 152 quarts (113 to 144 liters) of blood to create 1 to 2 quarts (0.94 to 1.8 l) of urine every day, according to the National Institutes of Health (NIH)2. They aren’t just one big filtering sponge, though. Each kidney is a system of millions of tiny filters called nephrons. A nephron has two parts. The glomerulus is the first part of the filter. It strains blood cells and large molecules from the toxins and fluid. The fluids and toxins that pass through then go through the tubule3. The tubule collects minerals that the body needs and puts them back into the bloodstream and filters out more toxins. While filtering, the kidneys produce urine to carry the toxins away. The urine is sent through two tubes called ureters down to the bladder, where the urine then leaves the body through the urethra4.

The therapeutic use of herbs is as old as human civilization and has evolved along with it. Local practitioners have used indigenous plants and herbs for centuries all over the world to treat a variety of ailments and these have exhibited clear pharmacological activities5. Historically, herbal drugs were used as tinctures, poultices, powders and teas followed by formulations, and lastly as pure compounds6. Across the cultures, knowledge about use of medicinal plants exists in the form of local folklore available with families, tribes and cultures, handed down from generation to generation. Medicinal plants or their extracts have been used by humans since time immemorial for different ailments and have provided valuable drugs such as analgesics (morphine), antitussives (codeine), antihypertensives (reserpine), cardiotonics (digoxin), antineoplastics (vinblastine and taxol) and antimalarials (quinine and artemisinin)7. Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer, malaria, cardiovascular diseases and neurological disorders8.

Pterocarpus santalinus is a light-demanding small tree, growing to 8 metres (26 ft) tall with a trunk 50–150 cm diameter. It is fast-growing when young, reaching 5 metres (16 ft) tall in three years, even on degraded soils. It is not frost tolerant, being killed by temperatures of −1 °C9. The leaves are alternate, 3–9 cm long, trifoliate with three leaflets. The flowers are produced in short racemes. The fruit is a pod 6–9 cm long containing one or two seeds9.

Pterocarpus santalinus is used in traditional herbal medicine as an antipyretic, anti-inflammatory, anthelmintic, tonic, hemorrhage, dysentery, aphrodisiac, anti-hyperglycaemic and diaphoretic. Pterocarpus santalinus (red sandalwood) is one of the medicinal plants used in traditional medicine, and is rich in flavonoids and phenols10.Many previous studies found that different plant extracts have significant antidiabetic effects8,9,10. The aim of this study is to evaluate the effect of Pterocarpus santalinus on rat’s kidney over a period of 28 days.

MATERIALS AND METHOD

Animals: A total of twenty four (24) male and female wister rats were obtained from Bingham University, Animal House. They were maintained on standard animal pellets and given water ad libitum. Permission and approval for animal studies were obtained from the College of Health Sciences Animal Ethics Committee of Bingham University.

Plant collection: Leaves of Pterocarpus santalinus were collected from its natural habitat from village in Karu, Nasarawa State, Nigeria. The plant was authenticated from Department of Botany, Bingham University, Nasarawa State Nigeria.

Plant extraction: The leaves were shadow dried for two weeks. The dried plant material was further reduced into small pieces and pulverized. The powdered material was macerated in 70% ethanol. The liquid filtrates were concentrated and evaporated to dryness at 40 C in vacuum using rotary evaporator. The ethanol extract was stored at – 4 C until used.

Animal study: Twenty four (24) rats of either sex (average weight of 240g) were selected and randomized into four groups of six rats per group. Group 1 served as the control and received normal saline (10ml/kg) while the rats in groups 2, 3 and 4 were giving 100, 200, and 400 mg/kg of extract respectively. The weights of the rats were recorded at the beginning of the experiment and at weekly intervals. The first day of dosing was taken as D0 while the day of sacrifice was designated as D29.

Haematological analysis: The rats were sacrificed on the 29th day of experiment. Blood samples were collected via cardiac puncture. One portion of the blood was collected into sample bottles containing EDTA for hematological analysis such as Hemoglobin concentration, white blood cell counts (WBC), differentials (neutrophils, eosinophils, basophils, lymphocyte and monocyte), red blood cell count (RBC), platelets and hemoglobin (Hb) concentration using automated Haematology machine (Cell-Dyn, Abbott, USA).

Kidney Function Test: Level of electrolytes (Na+, K+, Cl-, and HCO3-), creatinine and blood urea where determine using chemical pathology as markers of kidney function using diagnostic kits. The above parameters were determined at the Chemical Pathology Department of University of Jos Teaching Hospital. Kidney harvested were preserved in 10% formal saline solution, processed, sectioned and stained with Heamatoxylin and eosin (H&E) according to standard procedures at Department of Chemical Pathology, University of Jos Teaching Hospital, Jos.

Statistical analysis: Data were expressed as the Mean ±Standard Error of the Mean (SEM). Data were analyzed statistically using one-way Analysis of Variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons between the control and treated groups.

Values of P≤ 0.05 were considered significant.

RESULT

Effect of oral administration of Pterocarpus santalinus on hematological parametersin rats. Pterocarpus santalinus caused slightly significant (p<0.05) decrease in the level of red blood cell, hemoglobin, platelet etc. and significantly (p<0.05) caused an increase in mean corpuscular hemoglobin concentration in the rats at the dose level of 100 mg/kg compared to the control. The level of basophiles, neutrophiles, eosinophils and lymphocytes were however not significantly (p<0.05) affected by mean

Table 1: Effect of oral administration of Pterocarpus santalinus on hematological parameters in wistar rats.

	Hematological parameters	DW(10ml/kg)	Treatment (mg/kg)	200	400
			100
	WBC (×109/L)	8.21±0.772	6.74±1.32	7.71±0.71*	7.23±1.85
	RBC (×1012/L)	8.30±0.34	6.65±0.66*	8.11±0.57	7.78±0.56
	HGB (g/dL)	15.95±0.56	11.29±0.66*	14.33±0.96	14.62±0.11
	HCT (g/dL)	60.26±2.03	56.60±3.74	34.67±3.18	53.40±1.81
	MCV	66.62±0.93	60.40±1.44	57.17±0.31	69.60±1.72
	MCH	19.17±0.17	17.80±1.02	18.83±0.37	18.80±0.20
	MCHC (g/dL)	35.71±0.23	27.40±1.12	32.65±0.32	34.43±0.71
	PLT (×109/L)	683.83±40.35	471.00±23.12*	652.31±12.20	677.34±52.32
	LYM (%)	92.11±4.56	89.20±4.11	89.83±6.19	86.11±1.25
	NEUT (×109/L)	12.14±3.67	11.99±3.54	13.14±5.66	11.56±5.32
	EOSI (×109/L)	2.67±0.35	2.41±0.66	1.96±0.14	1.90±0.27
	BASO (×109/L)	1.88±0.28	2.00±0.59	2.13±1.70	2.31±2.11

Data presented as Mean ± SEM: n = 6, (WBC = white blood cells, RBC = red blood cells, HGB = hemoglobin, HCT = hematocrit, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, PLT = platelet, LYM = lymphocyte, NEUT = neutrophils, EOSI = eosinophils, BASO = basophils). * = P<0.05

c o r p u s c u l a r h e m o g l o b i n c o n c e n t r a t i o n Effect of oral administration of Pterocarpus santalinus on renal indices and electrolytes in Wistar rats.

Pterocarpus santalinus significantly (p<0.05) increased Na 100 mg/kg when compared to the control. Other parameters such as creatinine, K, CL, and Urea levels) were not significantly affected.

Histopathological Investigations of the effect of oral administration of Pterocarpus santalinus on renal indices and electrolytes in Wistar rats.

The kidney showed very slight tubular distortion and glomerular necrosis at 100 mg/kg. There was also, Slight tubular necrosis with lymphocyte hyperplasia at 100 mg/kg. Normal renal histological features were observed in the control group.

Table 2: Effect of oral administration of Pterocarpus santalinus on renal indices and electrolytes in wistar rats.

Renal indices and	DW(10ml/kg)	Treatment (mg/kg)
electrolytes		100	200	400
Potassium (mmol/L)	6.26±0.24	6.69±0.21	5.92±0.36	5.68±0.26
Sodium (mmol/L)	147.00±2.90	122.20±2.82*	147.00±1.95	144.25±1.88
Chloride (mmol/L)	110.15±5.83	101.87±6.34	107.32±2.36	101.50±2.67
Urea (mmol/L)	9.35±0.29	8.95±0.86	9.46±0.27	8.65±0.42
Creatinine (µmol/L)	69.47±9.65	72.23±15.11	64.33±15.21	64.54±6.10

Data presented as Mean ± SEM: n = 6, *significantly different from the distilled water (DW) control at p <0.05. SHBP = Safi® herbal blood purifier, DW = distilled water. * = P<0.05

Serum k

Doses of extract administered

Fig 1: graph showing effect of the ethanol leaf extract of Pterocarpus santalinus on serum potassium level in rats

Serum

sodium

Doses of extract administered

Fig 2: graph showing effect of the ethanol leaf extract of Pterocarpus santalinus on serum sodium level in rats

Serum

urea

Dose of extract administered

Fig 3: graph showing effect of the ethanol leaf extract of Pterocarpus santalinus on serum urea level in rats

Serum creatinine

Fig 4: graph showing effect of the ethanol leaf extract of Pterocarpus santalinus on serum creatinine level in rats

Plate 1: Histological sections of Kidneys of a) rats treated with Normal saline 10 ml/kg, (b) Pterocarpus santalinus 100 mg/kg (c), Pterocarpus santalinus 200 mg/kg bw (d) and Pterocarpus santalinus 400 mg/kg stained with H&E Technique.

DISCUSSION

Healthy kidneys filter about a half cup of blood every minute, removing wastes and extra water to make urine11. The urine flows from the kidneys to the bladder through two thin tubes of muscle called ureters, one on each side of your bladder. Traditionals, especially third world countries often consume medicinal plant for curative or preventive purposes. This could potentially endanger the functionality and intergrity of the kidney. This underscores the relevance of this work12. In the study, Wister rats were used to screen the effect of Pterocarpus santalinus at various dose level of the plant extract with hematological and biochemical estimation from blood and histopathology of kidney for 28 days.

The hemoglobin concentrations and heamatocrit are values revealing the degree of anemia while the MCHC is a useful index of the average haemoglobin concentrations of the red cells12. Generally, low readings for RBC, Hb and hematocrit indicate anemia. From the result obtained, at 200 and 400mg/kg dose all parameters studied were not significantly affected by Pterocarpus santalinus compared to the control group. Significant decrease in RBC, HGB, PLT and MCV at 100 mg/kg dose level indicate that Pterocarpus santalinus interferes with the normal production of haemoglobin and its concentration within RBCs and may thus possess the potential to cause anaemia at this dose level13. In addition, the significant (p<0.05) decrease in hemoglobin and hematocrit levels at 100 mg/kg body weight dose could be the optimal concentration of the product which may cause effect on the red blood cells indices. Some phytochemicals have been found to have effect on hematocrit. Saponins have been found to be cytolytic and can produce anemia14. Therefore, low red cells indices including hematocrit and hemoglobin observed may be attributed to presence of saponins found in some of the active ingredients in the product.

Serum creatinine, urea, uric acid and serum electrolytes are renal biochemical markers that are perturbed with the advent of nephrotoxicity, therefore, alterations in their levels connote impairment in the functional capacity of the kidney15,16,17. Result from the table above reveals their was significant increase in the serum sodium ion concentration following the administration of Pterocarpus santalinus, which suggests a possible effect on the pump that maintains the constancy of its extracellular concentration even though the serum potassium ion concentration is unaffected18,19,20,21. Although single-file destruction of nephrons does not occur in chronic renal disease, it is the rule rather than the exception that sodium balance is maintained until the vast majority of the original nephron population has been destroyed22,23,24. No significant increase in creatinine, urea, uric acid, K+, Cl-and HCO3 content of the serum following the administration of an extract or drug may suggest little to nocompromise of the renal functional capacity25,26,27. The plant extract may not have the ability to interfered with biochemical parameters such as creatinine metabolism leading to no difference in it synthesis and/or it may not compromise the functional capacity of the tissue28,29.

In the current study, the functional capacity of the kidney was not significantly affected in rat administered ethanol leaf extract of Pterocarpus santalinus due to no change in the level of serum levels of urea, uric acid, K+, Cl- and HCO3-, across most doses administered. There was also no change in histological features of the rat’s kidney of rat harvested and evaluated. This study agrees with hematological parameter that the plant may not affect the functionality and structural integrity of the kidney.

CONCLUSION

Result obtained from this study suggest that the ethanol plant extract of Pterocarpus santalinus had no effect on the hematological and histological parameters of rat kidney indicating that it may be safe for consumption even when used for a sustained period.

ACKNOWLEDGMENT

The authors wish to thank everyone who has contributed to the success of this research work.

REFERENCES

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Mesole S.B.

Department of Human Anatomy Texila American University Zambia.

Ibegbu A.O

Department of Anatomy Alex Ekwueme Federal University Ndufu-Alike, Nigeria

Mesole S.B, Musa S, Bauchi Z, Agbon A.N, Animoku AA and Kolawole OJ

Department of Anatomy, Faculty of Basic Medical Sciences, College of Medical Sciences, Ahmadu Bello University (A.B.U), Zaria, Nigeria.

Okpanachi A.O

Department of Physiology, Kampala International University, Uganda

Mesole S.B., Musa S,, Bauchi Z, Agbon A.N, Animoku AA and Kolawole OJ

Neuroanatomy and Neuroscience Unit, Department of Human Anatomy, A.B.U, Zaria.

Animoku AA

Department of Anatomy, Kogi State University Ayingba Nigeria.

All correspondence to: Mesole S.B e-mail: ms361450@gmail.com

ABSTRACT

Aluminium contamination can occurs via food, vaccines and water. The present study was Acarried out to study the effects of Eugenol on Brain neurotrace elements (Iron Fe; Manganese Mn: Magnesium Mg), cognition using morris water maze and histology of the cerebrum (Layer III and V) following administration of Aluminium chloride on Wistar rats. Materials and Methods. Thirty (30) adult Wistar rats were divided into six (6) groups with five (5) rats in each group. The rats were sacrificed 24 hours after administration of the last dose by 0.8ml/kg of ketamine as an anesthetic agent. Results: Aluminium chloride treatment of rats resulted in significant (p<0.05) elevation of manganese and Aluminium levels in the brain of rats. This is accompanied by a significant decrease (p<0.05) in brain levels of Iron (Fe) and Magnesium. Morris water maze test result revealed a significant (p<0.05) increase in latency time in the rats treated with aluminium chloride when pretreatment is compared to day-21 of treatment. However treatment with eugenol revealed a significant (p<0.05) reduction in latency time. Histological examination of the cerebral cortex Layer III and V using haematoxylin and Eosin revealed pyknosis perineuronal vacuolations of pyramidal cells of group-administered 100 mg/kg of aluminium chloride. However, treatment with Eugenol revealed an almost normal cytoarchitecture of the pyramidal cells of the cerebrum of the Wistar rats. Conclusions: Eugenol has the ability to protect rat brain from the deleterious effect of aluminium chloride on brain neurotrace elements, improve cognition and

preserve cytoarchitecture of the brain of rats.

Keywords: Prineuronal vacoulations, Pyknosis, Pyramidal cells and Anesthetic agent.

It is of interest to note that humans live in what is referred to as ”the Aluminium Age”.Objects made with the metal aluminium are strong, durable, light and corrosion-resistant (Hirata et al.,2011). Relative to bio-availability, the metal can be found in drinking water due to its property as a flocculant, it is a common additive to various processed foods, cosmetics of various types and pharmaceutical products (Tomljenovic and shaw, 2011).
Aluminium  mimics  physiological  elements  such  as magnesium (Mg), calcium (Ca), and iron (Fe) in the human body  hence  results  to  Physiological  alterations  and dysfunction of the body system (Hirata et al., 2011; Wu Zhihao  et  al.,  2012).  Aluminium  can  also  induce neurodegeneration, by increasing the accumulation of iron and  generation  of  reactive  oxygen  species  (ROS) production (Wu Zhihao et al., 2012; Maya et al., 2016).
The physical and chemical properties of aluminium allow it to effectively mimic the above-mentioned metals (Mg, Ca and Fe) in their respective biological functions and trigger a series of physiological abnormalities. Aluminium has been proven to replace Mg and bind to phosphate groups on the cell membrane (Kawahara and Kato., 2011; Singh et al., 2017). Eugenol (4-allyl-2-methoxyphenol), mainly exists in clove oil, camphorated oil, cinnamon leaf oil, and nutmeg oil. At normal temperatures, eugenol is a pale yellow viscous oily liquid with a strong clove flavor and a special hot taste or brown powder in the dried form (Chaieb et al., 2007). Eugenol, which is an active compound (nutraceuticals) in many spice plants such as clove, Ocimum sanctum and Ocimum gratissimum is a well-established antioxidant (Zoppi et al., 2006; Patra et al., 2018). This study was undertaken to assess the protective effect of eugenol on brain neurotrace elements (Mg, Mn and Fe), neurobehavioural (learning and memory) and the histology of the cerebral cortex (layers III and V) following aluminium induced neurotoxicity in rats.

MATERIALS AND METHODS

Chemicals:- Eugenol, a light brownish powdered substance used for this study was obtained from Wuhan JCJ Logis, China, manufactured by Yueyang Jiazhiyuan Biological Co Ltd china (#58-23-4). While aluminium chloride which was used as a neurotoxic agent was obtained from Guandong Guanghua Sci-Tech Co. Ltd China (#7446-70-0).

Animals: A total of thirty (30) apparently healthy Wistar rats of both sex (140 to 160 g) were obtained from the Animal House of the Department of Human Anatomy, Faculty of Basic Medical Sciences, Ahmadu Bello University, Zaria, Kaduna State Nigeria and housed in wired cages in the same facility to acclimatize for a weeks prior to the commencement of the experiments. Ethical approval was obtained from Department of Anatomy Postgraduate research committee Ahmadu Bello University Zaria Nigeria. All rats were given food purchased from Grand Cereals and Oil Mills Limited (GCOML) Jos, Plateau State, Nigeria and water ad libitum. Treatment groups were administered eugenol/aluminium Chloride in addition to water and rat chow.

Experimental Design: Each groups consisted of 5 rats each and all route of administration was via the oral route. E u g e n o l a n d a l u m i n i u m w e r e a d m i n i s t e r e d simultaneously. Group I rats received 300 mg/kg of eugenol (10% LD50) (LD50 3000mg/kg as provided on the safety data sheet), Group II received 150 mg/kg (Mesole et al; 2020) (5% LD50) of eugenol, Group III rats that received 300 mg/kg of eugenol and 100 mg/kg of aluminium chloride, Group IV rats that received 150 mg/kg of eugenol and 100mg/kg of aluminium, Group V rats that received 100 mg/kg of aluminium chloride (Anil et al; 2009; Mesole et al; 2020), Group VI rats served as control and was administered 2 ml/kg of distilled water as placebo. Duration of the entire treatment was for 21 days. Rats were humanely sacrificed 24 hours after the last administration with 0.8 ml/kg (Mesole et al; 2020) of ketamine as anesthesia (Table 1).

Table 1: Animal Grouping and treatment

Groups	Dose
Group I	300 mg/kg eugenol
Group II	150 mg/kg eugenol
Group III	300 mg/kg eugenol + 100 mg/kg
	aluminium chloride
Group IV	150 mg/kg eugenol + 100 mg/kg
	aluminium chloride
Group V	100 mg/kg of aluminium chloride
Group VI	2 ml distil water

Brain Trace Elements

At the end of the experiment, brains were dissected (under ketamine anaesthesia, the rats were decapitated and the skull was carefully opened to expose the brain) weighed and homogenized in 0.1M Phosphate buffer (pH 7.4) (1g tissue/ 4ml (Ige et al., 2011). The homogenate were then centrifuged and aliquots of the supernatant were obtained for analysis of brain trace elements.

Neurochemical analysis for (Fe, Mn, Mg. and Al) estimation in the in the tissue (brain) homogenate was conducted using atomic Absorption Spectrophotometer (AAS – AA240FS, Varian) at the Multiuser Laboratory, Department of Chemistry Ahmadu Bello University, Zaria. The analytical method for determining metals in biological tissues as reported by Environmental Monitoring Methods Index, EMMI (1997) was adopted and is summarized below:

Tissue preparation/digestion

Take 1 ml (0.25g) of sample (homogenized tissue; 1g in 4ml of phosphate buffer) into boiling tube and add 2 ml of concentrated HNO3. The sample is heated at100°C for 2 hours and allowed to cool. This is followed by addition 0.3 ml of 30% hydrogen peroxide (H2O2) to the already cooled sample. Heat again at 100°C for 1-2 hours and allow to cool. Filter using whatmann’s filter paper.

Dilution/ Deionization

Make volume (of digested sample) into 20ml using deionized water analyze using an atomic absorption spectrophotometry (AAS)

Neurobehavioral Studies:

This method as described by Morris et al., (1982), as reported by Drapeau et al., (2003), for spatial memory and learning was adopted for this study. Rats were tested in a Morris water maze (180 cm diameter, 60 cm height) filled with water. An escape platform was hidden 2cm below the surface of the water in a fixed location in one of the four quadrants halfway between the wall and the middle of the pool.

Procedure:

Before the commencement of the treatment, the rats were trained in the pool daily for four (4) days. During the training, animals were required to locate the submerged platform by using distal extra-maze cues. They were tested for four trials per day (90 seconds with an inter trial interval of 30 seconds and beginning from different start points that varied randomly each day). Rats were tested after seven

(7), fourteen (14) and twenty one (21) days of treatment. Preparation of tissue for microscopy: The brain was removed and fixed in formol saline and processed for microscopy. Tissues were processed to obtain 5 µm thick paraffin sections, stained with haematoxylin and eosin (Feldman and Wolfe, 2014) as outlined below:

Removal of wax with xylene (dewaxing I and II for 3 minutes) and followed by Hydration with graded alcohol; absolute alcohol for 1 minute; 95% alcohol for 1 minute, 70% alcohol for 2minutes, 50% alcohol for 2 minutes and 30% alcohol 1minute.

Staining: Haematoxylin for 10-20 minutes, distilled water (washing), 35% alcohol for 1 minute. Acid – alcohol for 30 seconds (for differentiation between nucleus and cytoplasm). Followed by Distilled water for 1 minute and Staining in 1 % eosin for 2 minutes.

Dehydration in alcohol (90% alcohol 10 – 15 seconds) followed by absolute Alcohol for 1 minute. Clearing in, Xylene-alcohol for 2 minutes, Pure xylene I for 3 minutes and pure xylene III for 3 minutes. Cleared tissue is mounted In DPX

Statistical analysis: Results obtained were analyzed using statistical software, statistical package for social sciences (IBM SPSS version 21.0, SPSS and Microsoft Office Excel 2007 for charts. Results were expressed as mean ± Standard error of mean (S.E.M) and presence of significant differences among means of the groups were determined using one way analysis of variance (ANOVA) with least significant difference (LSD) post hoc test for significance.

Values were considered significant when p≤0.05.

RESULTS

Eugenol treatment on brain neurotrace element (Iron) following aluminium chloride-induced neurotoxicity, revealed a significant (p< 0.01) reduction in brain iron levels in rats administered 100mg/kg of AlCl3 when compared to control. Treatment with Eugenol, however resulted in significant (p<0.001) elevated level of iron in rats administered 300 mg/kg (eugenol) + 100 mg/kg (AlCl3) and 150 mg/kg (eugenol) + 100 mg/kg (AlCl3) when compared to the group treated with 100 mg/kg AlCl3This elevation was found to be significant (p<0.001). But when comparison is made with the control, the reduced levels of Iron (Fe) in the brain which was observed in rats administered 300 mg/kg (eugenol) + 100 mg/kg AlCl3 and 150 mg/kg (eugenol) + 100 mg/kg AlCl3, was not significant (p>0.05). The Increase iron levels observed in rats administered 300 mg/kg and 150 mg/kg eugenol were found to be not significant (p>0.05) when compared to control. (Figure 1)

Figure 1: Effect of eugenol on neurotrace brain element (Iron Fe) following administration of aluminium chloride on Wistar rats.

n = 5; mean ± SEM One way ANOVA LSD post hoc test: q, s = p<0.01when compared to the AlCl3 y = p<0.001 when compared with control .Group I and II (Eugenol 300mg/kg and 150mg/kg respectively), Group V = (Aluminium chloride 100 mg/kg), Group VI = (Control 2.0ml/kg)

Figure 2: Shows the effect of Eugenol treatment on brain neurotrace element (Magnesium) following aluminium chloride-induced neurotoxicity. This result shows a significant (p<0.01) reduction in brain levels of magnesium in the AlCl3 treated group when compared to the control. Treatment with eugenol, however, revealed a significant (p<0.05) increase in the level of brain magnesium as observed in Groups III and IV when compared to V. However Groups I and II levels of brain magnesium revealed a non significant (p>0.05) difference when compared to control.

Figure 2: Effect of Eugenol on Neurotrace Brain element (Magnesium Mg) following administration of aluminium chloride on Wistar rats

n = 5; mean ± SEM One way ANOVA LSD post hoc test: q = p<0.05 when compared with the AlCl3 treated group; y= p<0.01; when compared with control group respectively. Groups I and II (Eugenol 300mg/kg; 150mg/kg), Group V (Aluminium chloride 100mg/kg), Group VI (Control 2.0ml/kg)

Figure 3: Shows the effect of Eugenol treatment on brain neurotrace element Manganese (Mn) following aluminium chloride-induced neurotoxicity. This result shows a significant (p<0.01) elevated level of brain Manganese in AlCl3 when compared to the control. Treatment with Eugenol however significantly (p<0.05) reduced the manganese level in Groups III and IV when compared to Group V. Comparison of Groups I and II to Group VI reveals a non-statistical significance (p>0.05) between the brain levels of manganese.

Figure 3: Effect of Eugenol on Neurotrace Brain element (Manganese Mn) following administration of aluminium chloride on Wistar rats.

Figure 4: Shows the level of aluminium in the brain following oral administration of aluminium chloride. The result shows a significant (p<0.01) elevation in brain Al levels when AlCl3 is compared to the control. It will be observed that the administration of Eugenol significantly reduced (p<0.05) the level of aluminium as observed in Groups III and IV when compared to Group V.

Figure 4: Effect of Eugenol on Aluminium Brain element following administration of aluminium chloride on Wistar rats.

n = 5; mean ± SEM One way ANOVA LSD post hoc test: q = p<0.05 when compared to the AlCl3 treated group y = p<0.001 AlCl3 treated group is compared to the control group. Groups I and II (Eugenol 300mg/kg; 150mg/kg), Group V (Aluminium chloride 100mg/kg), Group VI (Control 2.0 ml/kg)

Figure 5: Transfer Latency of Wistar rats on Morris water maze habituation.

Group I= Eugenol (300 mg/kg); Group II = Eugenol (150 mg/kg), Group V = Aluminium chloride (100 mg/kg) Group VI = Control (distilled water 2.0 ml/ kg).

Figure 5: shows training latency time in seconds from day 1 to day 4. On day one rats from all groups had an increased latency time when compared to day 2,3 and 4 where there was reduction in latency time.

Figure 6: Effect of Eugenol on Cognition (Morris water maze) following administration of aluminium chloride.

n = 5; mean ± SEM; Paired sample t-test, a,b,c = p<0.05; p<0.01; p<0.001 when comparison is made between pretreatment, day 7, day 14 and day 21 b = p<0.01; c = p<0.001. One way ANOVA LSD post hoc test, x = p<0.05 when comparison is made with the control group at day-14.

Group I = Eugenol (300 mg/kg); Group II = Eugenol (150 mg/kg), Group V = Aluminium chloride (100 mg/kg) Group VI = Control (distilled water 2.0 ml/ kg).

Figure 6: Shows a significant (p<0.05) increase in latency time on Day 14 in the group treated with AlCl3 when compared to pre-treatment and this increase in latency time is significant (p<0.001) when compared to the control (Grp VI) on day 14. Administration of eugenol however was able to reduce latency time this reduction was significant (p<0.05; p<0.001) in Group III and IV when pre-treatment is compared to day 21 .

A B

G

P

P

C

G

P

PV

E

PV

P

G

G

P

PV G

D

G

P

PV

F

PV

G

P

H

P ^G PV

I J

G

P

P G

K L

P

P

G

O

Figure 7 : Shows the micrograph of the section of the cerebral cortex (Layer III and V). A and B shows the histological features of the cerebral cortex of the control rat. C and D shows cerebral cortex (layer III and V) of Group V that was administered 100mg/kg aluminium chloride with perineuronal vacoulations (PV). E and F shows cerebral cortex (Layer III and V) of rats administered 300 mg/kg of eugenol and 100mg/kg aluminium chloride showing mild perineuronal vacoulations. G and H shows the cerebral cortex of rats (Layer III and V) administered 150mg/kg eugenol and 100mg/kg aluminium chloride showing very mild perineuronal vacoulations when compared to the group administered 100mg/kg of aluminium chloride only, I and J shows the cerebral cortex of rats (Layer III and V) administered 300mg/kg of eugenol showing normal histology of the cortex when compared to the control group, L and M shows the cerebral cortex of rats (Layer III and V) administered 150mg/kg eugenol showing a normal histology of the cerebral cortex when compared to the control (Pyramidal cell P, Glial cell G, Oligodendrocyte, O, Perineuronal vacoulations PV).

DISCUSSION

Oral administration of aluminium chloride resulted in extensive neuronal vacuolation and necrosis (neuro-degeneration) of the cerebral cortex of wistar rats (Buraimoh et al., 2012). These degenerative changes could occur in the following ways such as suppression of neuronal energy production (especially mitochondrial energy production) and greatly enhances excitotoxic sensitivity of neurons (Henneberry, 1989; Nicholls and Budd, 1998; Beal et al., 1993).

Aluminium is also known to inhibit or suppress cellular energy-producing enzymes, including mitochondrial electron transport enzymes (Blaylock and Ridgeland, 2004). The clinical importance of neuronal energy suppression by aluminium lies in the fact that mitochondrial energy suppression is intimately connected as an early event to neurodegenerative diseases such as Alzheimer’s dementia and Parkinson’s disease (Meltzer et al., 1996; Schapira et al., 1998). Hence neuronal energy suppression is one of the bases for cellular degeneration within the central nervous system (Gibson et al., 1999).

The main mechanism of aluminium toxicity involves the disruption of the homeostasis of metals, such as magnesium (Mg), calcium (Ca), and iron (Fe) manganese. The physical and chemical properties of aluminium allow it to effectively mimic these metals in their respective biological functions and trigger biochemical anomalies.

Aluminium has been shown to replace Mg and bind to phosphate groups on the cell membrane (Kawahara and Kato, 2011).

Oral exposure to aluminium results in accumulation within the cerebral cortex, cerebellum and hippocampus of the brain and thus affect some essential elements (Fe, Zn, Cu, Mn, and Mg) contents at varying levels (Kruck et al, 2004). Previous studies have correlated neurological disorders to the accumulation of aluminium chloride in the brain of Wistar rats (Mahmoud and Marwa, 2017; Sies and Jones, 2007)

Manganese is an essential mineral for maintaining brain function, manganese toxicity in humans is associated with Parkinsonian-like symptoms such as ataxia and altered balance may develop (Watts, 1990). Exposure to aluminium has been shown to induce changes in the cerebral, cerebellar and hippocampal levels of neurotrace elements (Mahmoud and Marwa, 2017).

In this study exposure to aluminium resulted in increased levels of manganese and this increase was higher than the control group. Increase in the levels of manganese within the brain also act as a prooxidant and hence a toxicant to the brain (elevated amounts) which is deleterious to neurons within the brain. However, administration of eugenol was able to lower brain manganese levels close to normal as observed in Group III and IV.

Magnesium (Mg) is known to play an important role in supporting brain plasticity, this primes the brain for maximal learning, memory and cognitive function. Increasing brain magnesium levels have been shown to restore critical brain Plasticity and thus improves cognition (Slutsky et al., 2010)

In this study, decreased Mg brain levels as observed in aluminium treated group. This is in tandem with the study of Slutsky et al., 2010. Eugenol was able to reverse the reduction in the Mg levels that were induced by aluminium resulting in an increase in Mg levels when compared to the control group. The groups administered eugenol only (Groups I and II) showed elevated brain Mg levels when compared to the control (Group VI). Eugenol’s ability to increase brain Mg levels might be responsible for its cognitive improving properties. In a Eugenol the salvaged groups (Group III and IV) was able to elevate magnesium close to Group VI.

Iron deficiency is not perceived as a life-threatening disorder. But lowered levels of Iron (Fe) has resulted in impaired behaviors including learning (Youdim, 2008).

Results from this study revealed reduced brain iron levels in Group V when compared to Group VI. Also, groups treated with eugenol (III and IV) showed an increase in Fe levels when compared to the aluminium treated group. Rats that received eugenol showed increased levels of Fe When compared to the control group. Reduced Fe levels in rat brains (Group V) might be responsible for cognitive deficits elicited by rats which might result in a defective dopaminergic interaction with the opiate system and cholinergic neurotransmission.

Elevated levels of aluminium in the brain have been associated with neurological diseases such as Alzheimer’s or Parkinsonism (Exley, 2004), which has been attributed to the accumulation of such metals in the brain of affected individuals (Walton, 2012).

Oral exposure to aluminium results in accumulation within the hippocampus of the brain and thus affect essential trace elements (Fe, Zn, Cu, Mn, and Mg) contents in the hippocampus at varying levels (Sies and Jones, 2007). Previous studies have correlated neurological disorders to the accumulation of aluminium chloride in the brain of Wistar rats (Mahmoud and Marwa, 2017). Aluminium has been revealed to affect the homeostasis of brain neurotrace elements which are essential for brain function.

Morris water maze is one of the most widely used tasks in behavioural neuroscience for studying the psychological process and neural mechanisms of spatial learning and memory (Brandies et al., 1989; He et al., 2011). Learning and memory of rats is reflected by escape latency compared to the performance at pre-treatment session.

Increased latency as observed with aluminium treatment at day-7, 14 and 21 is an indication of learning and memory impairment. Memory forms can be classified as declarative or explicit (ability to recall past events deliberately) and are hippocampus dependant; and non-declarative or procedural (implicit), defined by unconsciously performed skills (motor or cognitive) that are mainly dependent on the straitum and cerebellum (Packard and McGaugh, 199). Eugenol treatment showed a decrease in latency time when compared to the aluminium treated group, and administration of Eugenol especially at day-21 of treatment had a neuroprotective effect on aluminium intoxication by decreased latency. Zhibin et al. (2013) also reported that Eugenol can increase learning and memory, using MWM to assess learning and memory.

In this study, light microscopic examination of histological (Haematoxylin and Eosin H&E) sections routinely stained histological sections of the Cerebral cortex –layer III and V were conducted as shown in Figure 7. Neurodegeneration is a process involved in both neuropathological conditions and brain ageing (Kumar and Khanum, 2012). Histoarchitectural distortion of neural tissue manifesting as neuronal degenerative changes are indicative of neurotoxicity in the central nervous system (Nahla et al., 2011; Kalantariapour et al., 2012). Degenerative changes are observed as cortical neuronal shrinkage, perineuronal vacuolations, loss of pyramidal neurone process in sections of the brain studied regions of aluminium-treated rat compared to the control, indicates treatment (aluminium) related neurotoxicity and result obtained from the histological study is in agreement with the studies carried out by buraimoh et al., (2012). However treatment with eugenol was able to protect the histological features of the cerebrum and this is in agreement with the study carried out by Mahmoud and Marwa, 2017.

CONCLUSION

The present study concludes that Eugenol has the ability to protect and enhance brain function by restoring brain neurotrace elements (Iron, Magnesium and Manganese), improving cognitive deficits and preserving histoarchitecture of the cerebral cortex from histoarchitectural changes induced by aluminium.

ACKNOWLEDGMENT

This is to acknowledge Mr Peter Akpulu, chief technologist at the Department of Anatomy Ahmadu Bello University, Zaria Nigeria.

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Builder, M.I. and Joseph, S.O

Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Bingham University, Karu, Nasarawa, Nigeria

Olugbemi T.O

Department of Physiology, Faculty of Basic Medical Sciences, Edo University, Iyamho, Nigeria

Akande, T

Department of Medical Laboratory Sciences, Bingham University, Karu, Nigeria

All Correspondences to: Builders M.I. E-mail:modupebuilders@yahoo.com

ABSTRACT

gelathus dodoneifolius (AD) which is also known as African mistletoe is widely used to treat Adifferent diseases such as circulatory and respiratory diseases, malaria, diabetes, hypertension and sterility. The sub-acute toxicity studies of water extract of Agelathus dodoneifolius was undertaken to assess its safety and tolerability profile in long term treatment. Sub-acute toxicity (21-days) studies with Agelathus dodoneifolius were done on rats to determine its consequences on food and fluid intake, body weight, heamatological, biochemical, and mortality. Rats treated with the extracts had progressive decrease in food, fluid intake and body weight which was significantly (P< 0.05) and highly significant (P< 0.01) different from control. The water extract increased both haematological and liver function indices significantly compare to the control. The renal function parameters were not significantly different in all the groups. These preliminary results suggest that water extract of Agelanthus dodoneifolius was likely to be non toxic. However, increase in liver enzymes will require further histopathological and chronic toxicity evaluation to confirm its safety.

Keywords: Agelathus dodoneifolius, Subchronic toxicity, Haematological, Biochemical.

INTRODUCTION

n health care, medicinal plant plays an important role Iin Africa. However, these medicinal plants are not devoid of toxicity as well as unwanted side effects (Awodele et al., 2015). Agelanthus dodoneifolius, (synonyms – Tapinanthus dodoneifolius, DC Danser (Loranthaceae) is a ubiquist plant, especially parasitizing Mimosaceae which largely grow in West Africa (Boussim et al., 2004). The Loranthaceae constitutes the largest group of parasitic plants with about 950 plants distributed in 77 genera ( Engone and Salle, 2006). Loranthacean mistletoe, including A. dodoneifolius (DC) and other species are widely distributed in Nigeria and the plants are found on many host trees such as Mangifera indica, Phyllanthus niruri, Parkia biglobosa, Ziziphus spina-christi and Azadirachta indica trees ( Deeni and Sadiq, 2002).

African mistletoe (Agelanthus dodoneifolius [DC]) called ‘Kauchi’ in Hausa is a hemi-plant parasite used ethno medicinally by the Hausa and the Fulani tribes of Northern Nigeria as a remedy for several human and animal ailments that include stomach ache, diarrhoea, dysentery, wound and cancer (Deeni and Sadiq, 2002). The leaves and young twigs of the plants have been used in folklore medicine to treat different diseases such as circulatory and respiratory diseases, malaria, diabetes, hypertension and sterility (Efuntoye et al., 2010). Agelanthus dodoneifolius Polh and Wiens, had been shown to possess antiplasmodial activity (Builders et al., 2012a). The cardiovascular, spasmolytic and antiinflammatory activities of water extract of A. dodoneifolius have been reported ( Ouédraogo et al., 2005) Cepleanu et al., 1994 also reported the larvicidal and molluscicidal activities of this plant.

The present study was undertaken to determine the sub-acute toxicity profile of the water of the twigs of A. dodoneifolius parasitic on Parkia biglobosa.

MATERIALS AND METHODS
Plant collection and preparation

The twigs of A. dodoneifolius were collected from host plant P. biglobosa in the month of February, 2009 from Chaza village in Niger state of Nigeria. The plant was identified and authenticated and a voucher specimen (NIPRD/H/6543) was deposited at NIPRD Herbarium for future reference.

Extraction of plant materials:

The plant material was cleaned, air dried under shade and pounded into fine powder using a mortar and pestle. A 100 g quantity of the powder was boiled with 1 l of distilled water for 30 min. The decoction was decanted, centrifuged at 4500 rpm (Hamburg-Eppendorf, Germany) for 30 min and freeze-dried. The total yield of dark brown extract was 11.33% w/w of crude starting material. The freeze-dried powder was stored in an airtight container and used for the study.

Chemicals and Reagents

All chemicals were purchased from Sigma – Aldrich, USA. Phytochemical tests

The phytochemical screening of A.dodoneifolius twig extracts were carried out to determine the presence of the following compounds; alkaloid, flavonoids, tannins, anthraquinones cardiac glycosides, saponins, glycosides, sterols, resins, volatile oil, terpenes and phenols using standard procedures described by (Builders et al.,( 2011)

Animals

Forty (40) adult wistar rats (180-250 g) of either sex maintained at Animal Facility Centre (AFC) of the Department of Pharmacology and Therapeutics, Bingham University were used for the study. The animals were fed with commercial pellets with free access to purified drinking water ad libitum, standard conditions of 12h:12h light/dark cycle, and temperature (23˚C-25˚C). All of the applied protocols (BU/125/30) were approved by Bingham University Research Ethics Committee.

Sub Acute Toxicity Study

Twenty four (24) rats were selected by randomization and then divided into four groups of six each. The first group served as control while the remaining three groups were given 125, 250 and 500 mg/kg of A.dodoneifolius single oral dose for 21 days according to the oral median lethal dose (LD50) in mice which was estimated to be greater than 5000 mg/kg by Builders et al., 2012a. The first day of dosing was taken as D0 whereas the day of sacrifice was designated as D21. This was carried out according to the method of Orisakwe et al., ( 2003)

Metabolic cage study

Water and food intake were monitored daily for 21days.

Haematological methods

The rats were euthanized in an airtight glass chamber saturated with chloroform and after opening up the rats surgically after 21 days. Blood samples were collected by cardiac puncture into ethylene diamine tetraacetic acid (EDTA) bottles for the analysis of haematological parameters [white blood cell (WBC), packed cell volume (PCV), platelets (PLT) , neutrophils and lymphocytes (LMP)] using Sysmex KX-21N automated hematology a n a l y z e r ( S y s m e x A m e r i c a I n c , U S A ) . T h e microhaematocrit and cyanmethanemoglobin methods of ReyV ´ azquez and Guerrero, 2007 were used for the assay.

Biochemical analysis of serum

Blood collected into non heparinized tubes were then centrifuged at 3000 rpm for 10 min.

The serum separated was analysed to evaluate the liver enzymes [Aspartate aminotransferase (AST) and Alkaline phosphatase (ALP)], using the method of Pieme et al.,( 2006). Serum urea and creatinine were evaluated by the method of Aniagu et al., (2005).

Statistical analysis

The data were statistically evaluated by one way ANOVA. Comparison between treatment and control group were made by Student’s t- test then followed with Fisher’s exact. Differences between groups were considered significant at P<0.05 and highly significant at P<0.01

RESULTS

Table 1: Phytochemical Composition of water extracts of Agelathus dodoneifolius

Table 1 indicates the phytochemical analysis revealed the presence of anthraquinones, glycosides, phenols, saponins, steroids, tannins and terpenes while alkaloids and flavonoids were found to be absent.

Phytochemicals Remarks

_

Alkaloids

+

Anthraquinones

Flavonoids ^_

Glycosides ⁺

Phenols ⁺

Saponins +

Steroids +

Tannins ₊

Terpenes ₊

-Absence , + Presence

Effect of the extract on body weight

There were significant changes in the body weight of the treated rats compared to the control groups during the 21 days observation; this was highly significant from 250mg to 500mg extract /kg body weight as indicated in figure 1.

Figure 1 : Effect of Water extract of A.D on body weight

Effect of the extract on water intake

There were significant increases in water intake observed for all the treatment groups when

compared to the control group, this was highly significant after 21 days (P<0.01) as presented in figure 2.

Figure 2 : Effect of Water extract of A.D on water intake

Effect of the extract on food intake

There were significant increases in food intake with the extract treated groups

compared to the control group. This was highly significant after 21 days (P< 0.01) as shown in figure 3.

Figure 2 : Effect of Water extract of A.D on food intake

Effect of the extract on haematological parameters in rats

There were increase in white blood cell count, highly significant from 250mg/kg-500mg/kg (P<0.01). A non-

significant increase in packed cell volume was observed in all the treated groups compared to the control. There

were significant reductions in platelet count, significant increase in neutrophil and lymphocyte level and no

significant changes in monocytes, eosinophil and basophil level as indicated in table 2.

Table 2: Effect of water extract of A.D on haematological parameters

Parameters	Control		125mg/kg		250mg/kg		500mg/kg
WBC	4133.33 ± 0.05		4550.10 ± 1.12.		7000.01 ± 0.89**		7300.21 ± 1.23**
PCV	33.7	± 0.31	34.8	± 0.56	36.7	± 1.3	38.7 ± 1.21
Platelet	591 ± 1.11		268.8 ± 0.67**		484.8 ± 0.72*		114.0 ± 0.90**
Neutrophil	18.7 ± 1.21		15.7 ± 1.10		28.3 ± 0.55**		32.0 ± 0.42**
Lymphocyte	44.2 ± 0.35		75.5 ± 1.12**		68.8 ± 0.98**		120.2 ± 0.20**
Monocyte	5.3	± 1.00	7.7	± 0.60	5.5	± 1.35	5.3 ± 1.09
Eosinophil	2.7	± 0.44	2.7	± 1.33	2.7	± 0.86	2.7 ± 1.42
Basophil	2.5	± 0.69	1.5	± 1.37	2.3	± 1.17	2.0 ± 0.67

n = 6; *significantly different from the control at p<0.05; **significantly different from the control at P < 0.01.

Effect of the extract on biochemical parameters

There were highly dose dependent significant increases in alanine transferase and aspartase enzymes (P<0.01).

No significant changes in the level of urea and creatinine as illustrated in table 3.

Table 3: Effect of water extract of A.D on biochemical parameters

Parameters	Control		125mg/kg		250mg/kg		500mg/kg
ALT	60.1	± 1.24	130.7 ± 0.41**		104.7 ± 1.00**		314	± 0.98**
AST	196.7 ± 0.66		218.7 ± 1.20**		302.3 ± 0.45**		774.0 ± 1.11**
Urea	8.92	± 0.33	8.52 ±	0.86	8.71	± 1.32	8.50	± 0.49
Creatinine	41.1	± 1.12	39.1 ±	1.30	38.0	± 0.78	36.6	± 1.17

n = 6; *significantly different from the control at p<0.05; **significantly different from the control at P < 0.01.

DISCUSSION

Ethnopharmacological use of plants can therefore be a basis for phytochemical and phytopharmacological investigation (Kuria et al., 2001). The phytochemical tests revealed that the chemical composition of water extract of AD included anthraquinones and cardiac glycosides, these phytochemicals have protective /disease preventive properties.

The water extract of the twigs of A. doneifolius is acutely nontoxic according to the research conducted by Builders et al., (2012a) in which the LD50 of the water extract of the twig of A. dodoneifolius is greater than 5000 mg/ kg p.o. The high safety profile obtained may have been responsible for its wide spread use in different ethno-therapeutic interventions.

The increase in body weights of the treated rats is an indication of the improvement of the nutritional state of the animal which may be due to increase in food and water intake, this is similar to research conducted by Orisakwe et al.,( 2003) in which progressive increase body weight was also be attributed to growth response.

Increase in haematological parameters of the extract treated groups is an indication of the antianaemic activities of the extract. Study carried out by Onyenyili et al., (1998) showed that anaemia is as a result of breakdown of blood cells and or inhibition of blood cells synthesis.

The dose dependent elevation in white blood cells count implies that the extract has the potential to boost the activity of immune system, this in agreement to the research carried out by Aniagu and co-workers in 2005 ( Aniagu et al., (2005).

Specific immune response against pathogens is

lymphocytes while phagocytosis is carried out by neutrophils (Sacher and Mcpherson, 1991) . According to Muhi-eldeen et al., (2008), severe local inflammatory response in muscles is associated with significant increase in neutrophils and lymphocytes count this is in accordance to the findings of our study.

Haemostasis which is a process of reduction of blood loss and vascular injury repair is the responsibility of platelets ( Dahlback, 2007). The decrease in platelet number indicates that the extract has the ability to depress the biosynthesis of clotting factors by liver, therefore the extract has antiplatelet activities similar to many bioactive compounds such as garlic, vitamins, carotenoids ( Naidu, 2015; Bhowal and Mehta, 2017; Imran et al., 2012) .

Alanine amino transferase (ALT) and aspartate aminotransferase (AST) are markers of liver function; increase in these liver enzyme parameters is an indication of hepatic damage which is similar to study conducted by Builders et al., (2012b) in which the water extract of the parasitizing plant Parkia biglobosa caused severe histopathological changes in the liver.

The extract of Agelathus dodoneifolius did not interfere with renal function since the

blood urea and creatinine levels were normal; this shows that the renal integrity was preserved. This is similar to research conducted by Builders et al., 2012b in which the water extract of the parasitizing plant Parkia biglobosa did not affect the renal function.

CONCLUSION

These preliminary results suggest that the methanolic extract of Agelanthus dodoneifolius was non- toxic. However histopathological and chronic toxicity evaluations will be required to confirm its safety.

ACKNOWLEDGMENT

The authors gratefully acknowledge the technical support of the entire staff of the Animal Facility Centre of the Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Bingham University for providing enabling environment for this research.

REFERENCES

• Awodele, O., Amagon, K.I., Agbo, J., and Prasad, M.N. (2015). Toxicological evaluation of the aqueous stem bark extract of Bridelia Ferruginea (Euphobiaceae) in rodents. Interdiscip Toxicol. 8: 89–98.
• Boussim, I.J., Guinko, S., Tuquet, C., and Salle, G. (2004). Mistletoes of the agroforestry parklands of Burkina Faso. Agroforestry Syst 60:39–49.
• Engone Obiang, N.L., and Sallé, G. (2006). Is there any point to eradicate Phragmanthera capitata parasitizing African rubber trees? C R Biol. 3 :185–195.
• Deeni, Y.Y., and Sadiq, N.M. (2002). Antimicrobial properties and phytochemical constituents of the leaves of African mistletoe (Tapinanthus dodoneifolius (DC) Danser) (Loranthaceae): An ethnomedicinal plant of Hausaland, Northern Nigeria. J Ethnopharmacol. 83:235–240.
• Efuntoye, M.O., Ayodele, A.E., Thomas, B.T., and Ajayi, T.O. (2010). Does host plant affect the antibacterial activity of Tapinanthus bangwensis (Engl. and K. Krause) Danser (Loranthaceae)? J Med Plant Res. 4:1281–1284.
• Builders M.I., Uguru, M.O., and Aguiyi J.C. (2012a). Antiplasmodial potential of African mistletoe: Agelanthus dodoneifolius Polh and wiens. Indian J Pharm Res. 189-280.

• Ouédraogo, S., Aristide, T.N., Somea, M.L., Guisso, P.I., Bucher, S.C., and Andriantsihaina, R.(2005). Cardiovascular properties of aqueous extract from Tapinanthus dodoneifolius DC DANSER. Afr J Tradit Complement Altern Med. 1:25–30.
• Cepleanu, F., Hamburger, M.O., Sordat, B., Msonthi, J.D., Gupta, M.P., Saadou, M., and Hostettman, K. (1994). Screening of tropical medicinal plants for molluscicidal, larvicidal, fungicidal and cytotoxic activities and brine shrimp toxicity. Int J Pharmacol. 323:294–307.
• Builders, M.I., Wannang, N.N., Ajoku, G.A., Builders, P.F., Orishadipe, A., and Aguiyi,J.C. (2011). Evaluation of antimalarial potential of Vernonia ambigua. Int J Pharmacol. 1811: 1-10.
• Orisakwe, O.R., Afonne, O.J., Chude, M.A., Obi, E and Dioka, C.E. (2003). Sub chronic toxicity studies of the aqueous extract of Boerhavia diffusa leaves. J. Health. Sc . 49: 444-447.
• ReyV´ azquez, G., and Guerrero G.A. (2007). Characterization of blood cells and hematological parameters in Cichlasomadimerus (Teleostei, Perciformes). Tissue and Cel1 . 39: 151- 160.

12.	Pieme, C.A., Penlap, V.N., Nkegoum, B., Taziebou,
	C.L., Tekwu, E.M., Etoa, F.X., and Ngongang, J.

(2006). Evaluation of acute and subacute toxicities of aqueous ethanolic extract of leaves of Senna alata (L.) Roxb (Ceasalpiniaceae). Afr. J. Biotech 5: 283-289.

• Aniagu, S.O., Nwinyi, F.C., Akumka, D.D., Ajoku, G.A., Dzarma, S., Izebe, K.S., Ditse, M., Patrick, E., Nwaneri, C., Wambebe, C., and Gamaniel, K. (2005). Toxicity studies in rats fed nature cure bitters. Afr. J. Biotech 4: 72-78.
• Kuria, K.A., De coster, S., Muriuki, G., Masengo, W., Kibwage, I., and Hoogmartens, J. (2001) Anti – malarial activity of Ajuga remota Benth. (Labiatae) and Caesalpinia volkensii (Caesalpiniceae) in vitro confirmation of ethnopharmacological use. J Ethnopharmacol 74: 141-148.

• Onyeyilli ,P.A., Iwuoha, C.L., and Akinniyi, J.A.(1998). Chronic toxicity study of Fiscus platyphylla blume in rats. West African J Pharmacol Drug Res 14: 27-30.
• Sacher, R.A., and McPherson, R.A . (1991). Widmann’s Clinical interpretation of laboratory tests. 10th Ed. F. A. Davis, Philadelphia; pp. 1- 6.
• Muhideen, Z., Al-Shamma, K.J., Al-Hussany, T.M., Al-Kassi, E.N., Daraji, A.M., and Ibrahim, H. (2008). Acute toxicological studies on the extract of Iraqi Peganum Harmala in rats. European J Sc Res . 494-500.

• Dahlback, B ., (2007). Blood coagulation. Lancet; 355: 1627-1632. In: Bertram G. Katzung. Basic and Clinical Pharmacology. 10th Ed. Boston, USA; pp. 543-558.

• Naidu, J.R., Ismail, R., Kumar, P., Jothy, S., Chen, Y., and Sasidharan, S. (2015). Antiplatelet activity and quantification of polyphenol contents of methanol extract of Ocimum basilicum and Mentha spicata. Res J Pharm Biolog Chem Sc . 6: 1236-1243.
• Bhowal , M., and Mehta D.M. (2017). An overview of medicinal plants as potential anti-platelet agents. IOSR J Pharmacy Biolog Sc. 12: 17- 20.
• Imran, I., Hussain, L., Ahmed, S., Rasool, N., Rasool, S., Abbas, G., and Ali, M.Y. (2012). Antiplatelet activity of methanolic extract of Acacia leucophloea bark. J Med Plt Res. 6: 4185-4188.
• Builders, M.I., Isichie, C.O., and Aguiyi, J.C. (2012b). Toxicity studies of the extracts of Parkia biglobosa stem bark in rats. Br J Pharm Res . 2: 1-16.

 

Wazis Chama Haruna

Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Maiduguri, Bornu State, Nigeria.

Modupe Builders and Joseph Oyepata Simeon

Department of Pharmacology, Faculty of Pharmacy, Bingham University, Nasarawa, Nigeria

Joseph Opeyemi Tosin

Department of Pharmacology, Faculty of Basic Medical Sciences, University of Port Harcourt, Rivers State, Nigeria.

All Correspondences to: Joseph Oyepata Simeon E-mail: simeon4unme@yahoo.com

ABSTRACT

Introduction: The use of medicinal plants has attained a commanding role in health Asystem all over the world. Pterocarpus santalinus is a plant common in Asia and Africa has been used traditionally in management of several ailments. Due to the relevance of Pterocarpus santalinus in medicine, there is the need to establish the safety profile of this plant on various organs of the body. The aim of this study is to evaluate the effect of Pterocarpus santalinus on rat’s kidney over a period of 28 days. Method: Animals of either sex were selected. Group 1 received distilled water (10 ml/kg), while groups 2, 3, and 4 received Pterocarpus santalinus 100, 200 and 400 mg/kg respectively. Animals were kept in standard cages and given access to the extract, water and food orally for 28 days, after which they were weighed and sacrificed. Blood was collected by cardiac puncture and taken immediately for hematological and chemo pathological analysis. The histological toxic potential of the plant on the liver was studied using haematotoxylin and eosin (H&E) staining technique. Result: There was slightly Significant (P<0.05) decrease in RBC, HGB, MCV, while there was no change in the level of neutrophiles, basophiles, eosinophiles and platelets. Pterocarpus santalinus, slightly significantly (p<0.05) increased There were also no significant (P<0.05) increase in Alkaline phosphatase, level of bilirubin. Histological features agrees with other biomarkers. Conclusion: The result of the study showed that the Pterocarpus santalinus may be safe for human consumption, though with caution particularly at higher dose.

Keywords: Pterocarpus santalinus, rat, blood, liver.

INTRODUCTION

t would be difficult to overestimate the importance of Ithe liver to the healthy functioning of the human body1. It is a remarkable organ. The liver acts as a processing plant, a battery, a filter, a warehouse and a distribution centre all in one1. The immune system, digestive tract, kidney, brain and cardiovascular system all depend on a healthy and well-functioning liver. This is why liver diseases such as hepatitis C can have such varied symptoms1. Because a diseased liver can potentially affect all the body’s major systems and organs, it is very important to understand how it works and how to look after it. In most developing countries, the indigenous modes of herbal treatment are a part of the culture and the dominant method of healing therapy2. These remedies, with a considerable extent of effectiveness, are socially accepted, economically viable and, mostly, are the only available source3. Plants used in traditional medicine, therefore, have a critical role in the maintenance of health all over the world. The drugs of herbal, herbo-mineral, and animal origin have been used by the traditional healers to maintain health and treat diseases since antiquity. Such medicines are widely used in Africa and Asia, including India and China3,4. Due to the adverse side-effects, and also the development of resistance against synthetic drugs, the uses of plant-derived drugs are becoming popular in developed countries also5. The liver performs the normal metabolic homeostasis of the body as well as biotransformation, detoxification and excretion of many endogenous and exogenous compounds, including pharmaceutical and environmental chemicals. Drug induced hepatotoxicity is a major cause of iatrogenic diseases, accounting for one in 600 to one in 3500 of all hospital admissions6.

Medicinal plants or their extracts have been used by humans since time immemorial for different ailments and have provided valuable drugs such as analgesics (morphine), antitussives (codeine), antihypertensives (reserpine), cardiotonics (digoxin), antineoplastics (vinblastine and taxol) and antimalarials (quinine and artemisinin)7. Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer, malaria, cardiovascular diseases and neurological disorders8.

Pterocarpus santalinus is a light-demanding small tree, growing to 8 metres (26 ft) tall with a trunk 50–150 cm diameter. It is fast-growing when young, reaching 5 metres (16 ft) tall in three years, even on degraded soils. It is not frost tolerant, being killed by temperatures of −1 °C9. The leaves are alternate, 3–9 cm long, trifoliate with three leaflets. The flowers are produced in short racemes. The fruit is a pod 6–9 cm long containing one or two seeds9. Pterocarpus santalinus is used in traditional herbal medicine as an antipyretic, anti-inflammatory, anthelmintic, tonic, hemorrhage, dysentery, aphrodisiac, anti-hyperglycaemic and diaphoretic. Pterocarpus santalinus (red sandalwood) is one of the medicinal plants used in traditional medicine, and is rich in flavonoids and phenols10.Many previous studies found that different plant extracts have significant antidiabetic effects8,9,10. The aim of this study is to evaluate the effect of Pterocarpus santalinus on rat’s liver over a period of 28 days.

MATERIALS AND METHOD

Animals: A total of twenty four (24) male and female wister rats were obtained from Bingham University, Animal House. They were maintained on standard animal pellets and given water ad libitum. Permission and approval for animal studies were obtained from the College of Health Sciences Animal Ethics Committee of Bingham University.

Plant collection: Leaves of Pterocarpus santalinus were collected from its natural habitat from nearby Karu village, Nasarawa State, Nigeria. The plant was authenticated from Department of Botany, Bingham University, Nasarawa State Nigeria.

Plant extraction: The leaves were shadow dried for two weeks. The dried plant material was further reduced into small pieces and pulverized. The powdered material was macerated in 70% ethanol. The liquid filtrates were concentrated and evaporated to dryness at 40 C in vacuum using rotary evaporator. The ethanol extract was stored at – 4 C until used.

Animal study: Twenty four (24) rats of either sex (average weight of 240g) were selected and randomized into four groups of six rats per group. Group 1 served as the control and received normal saline (10ml/kg) while the rats in groups 2, 3 and 4 were giving 100, 200, and 400 mg/kg of extract respectively. The weights of the rats were recorded at the beginning of the experiment and at weekly intervals. The first day of dosing was taken as D0 while the day of sacrifice was designated as D29.

Haematological study: The rats were sacrificed on the 29th day of experiment. Blood samples were collected via cardiac puncture. One portion of the blood was collected into sample bottles containing EDTA for hematological analysis such as Hemoglobin concentration, white blood cell counts (WBC), differentials (neutrophils, eosinophils, basophils, lymphocyte and monocyte), red blood cell count (RBC), platelets and hemoglobin (Hb) concentration using automated Haematology machine (Cell-Dyn, Abbott, USA).

Biochemical analysis: A Portion of the blood was collected used to estimate biochemical parameters including liver enzymes: alanine amino transaminase(ALT), aspartate amino transaminase (AST), alkaline phosphatase (ALP),albumin (ALB), total protein (TP), conjugated bilirubin (BILD), unconjugated bilirubin(BILT) using a photoelectric method.

Histopathology: Tissues collected were preserved in 10% formal saline solution. Small block of the tissues were taken from liver and fixed in Bouin’s fluid for 16 to 24hours. Tissue were slices and processed according to the method described by (Lison,1960) and stained with haemotoxylin and eosin.

Statistical analysis: Data were expressed as the Mean ±Standard Error of the Mean (SEM). Data were analyzed statistically using one-way Analysis of Variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons between the control and treated groups.

Values of P≤ 0.05 were considered significant.

RESULT

Effect of oral administration of Pterocarpus santalinus on hematological parametersin rats. Pterocarpus santalinus caused slightly significant (p<0.05) decrease in the level of

Table 1: Effect of oral administration of Pterocarpus santalinus on hematological parameters in wistar rats.

Hematological		Treatment (mg/kg)
parameters	DW(10ml/kg)	100	200	400
WBC (×109/L)	8.21±0.772	6.74±1.32	7.71±0.71*	7.23±1.85
RBC (×1012/L)	8.30±0.34	6.65±0.66*	8.11±0.57	7.78±0.56
HGB (g/dL)	15.95±0.56	11.29±0.66*	14.33±0.96	14.62±0.11
HCT (g/dL)	60.26±2.03	56.60±3.74	34.67±3.18	53.40±1.81
MCV	66.62±0.93	60.40±1.44	57.17±0.31	69.60±1.72
MCH	19.17±0.17	17.80±1.02	18.83±0.37	18.80±0.20
MCHC (g/dL)	35.71±0.23	27.40±1.12	32.65±0.32	34.43±0.71
PLT (×109/L)	683.83±40.35	471.00±23.12*	652.31±12.20	677.34±52.32
LYM (%)	92.11±4.56	89.20±4.11	89.83±6.19	86.11±1.25
NEUT (×109/L)	12.14±3.67	11.99±3.54	13.14±5.66	11.56±5.32
EOSI (×109/L)	2.67±0.35	2.41±0.66	1.96±0.14	1.90±0.27
BASO (×109/L)	1.88±0.28	2.00±0.59	2.13±1.70	2.31±2.11

Data presented as Mean ± SEM: n = 6, (WBC = white blood cells, RBC = red blood cells, HGB = hemoglobin, HCT = hematocrit, MCV = mean corpuscular volume, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, PLT = platelet, LYM = lymphocyte, NEUT = neutrophils, EOSI = eosinophils, BASO = basophils).

38 Nigerian Biomedical Science Journal Vol. 17 No 1 2020

red blood cell, hemoglobin, platelet etc. and significantly (p<0.05) caused an increase in mean corpuscular hemoglobin concentration in the rats at the dose level of 100 mg/kg compared to the control. The level of basophiles, neutrophiles, eosinophils and lymphocytes were however not significantly (p<0.05) affected.

Effect oral administration of Pterocarpus santalinus on hepatic indices in rats.

At 100 mg/kg dose level, Ocimum canum produced significant (p<0.05) decrease in BILD concentration in the treated rats while at 100 mg/kg dose no significant

Joseph Oyepata Simeon

(p<0.05) increase was obtained in ALP levels, BILD and BILT concentrations when compared to the control (Table 2).

Effect of oral administration of ethanol leaf extract of Pterocarpus santalinus on histology Liver of rats.

The liver showed slight vascular congestion, slight hepatic necrosis and lymphocyte hyperplasia at 100 mg/kg and 200 mg/kg. There was slight Sinusoidal congestion observed at 400 mg/kg. However, there was no sign of damage to the liver of the rats in control group (Plate 1).

Table 2: Effect of sub-acute oral administration of Pterocarpus santalinus on hepatic indices in wistar rats.

Hepatic indices			Treatment (mg/kg)
		DW(10ml/kg)	100	200	400
ALB (g/L)		43.62±1.23	43.21±0.15	45.11±1.12	41.71±2.20
ALP (IU/L)		113.12±6.43	132.00±3.29	170.10±43.23	128.50±6.74
ALT (IU/L)	S	65.25±3.01	67.34±7.12	81.40±12.19	85.22±27.17
AST (IU/L)		300.30±79.90	299.20±57.65	278.21±35.18	253.00±11.75
BILD (µmol/L)		0.28±0.17	0.16±0.12*	0.57±0.19*	0.25±0.33
BILT (µmol/L)		2.65±0.51	2.66±0.22	3.46±0.76*	2.45±0.11
TP (g/L)		79.13±2.11	76.14±2.65	71.35±5.17	81.13±2.65

Data presented as Mean ± SEM: n = 6, *significantly different from the distilled water (DW) control at p <0.05.

DW = distilled water (ALB = albumin, ALP = alanine phosphatase, ALT = alanine transaminase,

BILD = unconjugated bilirubin, BILT = conjugated bilirubin, TP = total protein).

Fig 1: graph showing effect of the ethanol leaf extract of Pterocarpus santalinus on serum ALP level.

Fig 2: graph showing effect of the ethanol leaf extract of Pterocarpus santalinus on serum ALT level.

Nigerian Biomedical Science Journal Vol. 17 No 1 2020 39

Toxicological Study of the Effect of Ethanol Leaf…

Fig 3: graph showing effect of the ethanol leaf extract of Pterocarpus santalinus on serum AST level.

Plate 1:. figure of the liver (a) Control group, shows normal hepatocyte (H). (b) Pterocarpus santalinus 100 mg/kg (c) Pterocarpus santalinus 200 mg/kg,. d) 400 mg/kg Pterocarpus santalinsus

DISCUSSION

Herbal medicines proved to be the major remedy in traditional system of medicine. They have been used extensively in medical practices since ancient times11. There have been reports of accidental medicinal plant poisoning and over dose. In most cases this traditionally formulated drugs are consumed without appropriately establishing the dose that is safe for use. This has resulted into many untoward after effect12,13. Hematological parameters are useful indices that can be employed to assess the toxic potentials of plant extracts in living systems14,15,16. They can also be used to explain blood relating functions of chemical compound/plant extract15.

The hemoglobin concentrations and heamatocrit are values revealing the degree of anemia while the MCHC is a useful index of the average haemoglobin concentrations of the red cells17. Generally, low readings for RBC, Hb and hematocrit indicate anemia. At 200 and 400mg/kg dose all parameters studied were not significantly affected by

Pterocarpus santalinus compared to the control group. Significant decrease in RBC, HGB, PLT and MCV at 100 mg/kg dose level indicate that Pterocarpus santalinus interferes with the normal production of haemoglobin and its concentration within RBCs and may thus possess the potential to cause anaemia at this dose level18. In addition, the significant (p<0.05) decrease in hemoglobin and hematocrit levels at 100 mg/kg body weight dose could be the optimal concentration of the product which may cause effect on the red blood cells indices. Some phytochemicals have been found to have effect on hematocrit. Saponins have been found to be cytolytic and can produce anemia19,20. Therefore, low red cells indices including hematocrit and hemoglobin observed may be attributed to presence of saponins found in some of the active ingredients in the product.

Chemicals produce a wide variety of clinical and pathological hepatic injury. Biochemical markers (e.g. alanine transferase, alkaline phosphatase and bilirubin) are often used to indicate liver damage21. Liver injury is defined as a rise in either (a) ALT level more than three times of upper limit of normal (ULN), (b) ALP level more than twice ULN, or (c) total bilirubin level more than twice ULN when associated with increased ALT or ALP21,22,23. Liver damage is further characterized into hepatocellular (predominantly initial alanine transferase elevation) andcholestatic (initial alkaline phosphatase rise) types.However they are not mutually exclusive and mixed types of injuries are often encountered18,24.

The biochemical indices monitored in the liver is a useful ‘markers’ for assessment of tissue damage. The measurement of activities of various enzymes in the tissues and body fluids plays a significant role in disease investigation and diagnosis25, assault on the organs/tissues and to a reasonable extent the toxicity of the drug26. Tissue enzymes can also indicate tissue cellular damage caused by chemical compounds long before structural damage that can be picked by conventional histological techniques27. Alkaline phosphatase, a ‘marker’ enzyme for plasma and endoplasmic reticulum16,24,28, is often employed to assess the integrity of plasma membrane29. In this study there ethanol extract of Pterocarpus santalinus did not cause significant change in most of liver function test values. This indicates that though the plant is use regularly by locals in different countries to exploit it medicinal benefits, it may be safe for consumption. Histological evaluation cellular and tissue parameter also agrees with chemical-pathology evaluation.

CONCLUSION

Result from the study suggests that at the doses administered ethanol leaf extract of Pterocarpus santalinus may not affect the functionality and integrity of liver, because most biomarkers accessed were relatively not negatively affected. This may prove useful to traditional people that use it regularly in the management of different conditions.

ACKNOWLEDGMENT

The authors wish to thank everyone who has contributed to the success of this research work.

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