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.

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.

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

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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The post Effect of Eugenol On Neurotrace Elements, and Histology of the Cerebral Cortex of Wistar Rats Exposed to Aluminium Chloride. appeared first on Nigerian Biomedical Science Journal.

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

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

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.

(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.
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The post Toxicity studies of extract of African Mistletoe: Agelanthus Dodoneifolius Polh and Wiens in Rats appeared first on Nigerian Biomedical Science Journal.

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.

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.

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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The post Toxicological Study of the Effect of Ethanol Leaf Extract of Pterocarpus santalinus Extract on Liver of Wister Rats appeared first on Nigerian Biomedical Science Journal.