Danmalam , U.H.

Galadanchi, F.

Idris, Z.Y.

Jeremiah, C.

Sallau, A.B.

English

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Special Conference Edition, November, 2019Bayero Journal of Pure and Applied Sciences, 12(1):ISSN 2006 – 6996 TOXICITY STUDIES ON THE METHANOL LEAF EXTRACT OF Ziziphus mucronata *1Idris Z. Y., 1Danmalam U. H. and 1 Department of Pharmacognosy and Drug Development, Ahmadu Bello University Zaria2Department of Biochemistry, Ahmadu Bello University Zaria*Correspondent author: ABSTRACT Zizphus mucronata is a specie from the genus Ziziphus which is a member of the Rhamnaceae family. It is commonlykuura in Hausa language. It ailments including urinary, metabolicevaluating the effect of methanol extract of Ziziphus mucronata on toxicity sWister rats. The effect of methanol extract on percentage change in body weight and some biochemical parameters on wistar rats was carried out using the OECD guideline. The acute oral toxicity of the extracts on the wistar rats showed no sign of mortality at 5000 mg/kg after being administered and observed for 24 hours and subsequently for 48 hours. The effect of subchronic administration of methanol extract on the body weight of the wistar rats showed significant increase (and 4 when compared with the control (1ml/kg normal saline). The effect of oral daily administration of the methanol extract on liver function tests produced a significant decrease (p<0.01) in ALT level at the dose of 250, 500 and 1000mg/kg alsoof AST at the dose of 1000mg/kg (palkaline phosphatase (ALP), total bilirubin (TB) conjugated bilirubin (CB) showed no significance difference (p>0.05showed no significant difference (p>0.05)findings from this studies can be used to as a scientific basis for the traditional use as well as safety evaluation of the leaves of this plant. Keywords: Ziziphus mucronata, Percentage change in body weight, Biochemical studies INTRODUCTION Ziziphus mucronata is a small to mediumtree, 10-20 m high; with a spreading canopy. The main stem is green and hairy when young; year old branches often zigzag. Leaves are ovate to broadly ovate, shiny, densely hairy the strong thorns are in pairs from where the leaves arise between the two thorns. Flowers are small, inconspicious and yellow. Fruits are reddishbrown drupe (Orwa et al., 2009). A decoction of the glutinous roots is commonly administered as a painkiller for all sorts of pains as well as dysentery. A concoction of the bark and the leaves is used for respiratory ailments and other septic swellings of the skin. Pastes of the root and leaves can be applied to treat boils, swollen glands, wounds and sores. Steam baths from the bark are used to purify and improve the complexion (Palmer and Pitman 1972). In       545 - 550         WILLD  2A. B. Sallau, 1Jeremiah C., and Galadanchi, F. A.  - Nigeria idrisbella@gmail.com Tel:  +2347032737309  known as buffalo thorn and locally as is being used traditionally in the treatment of various  and infectious diseases. This study is aimed at p<0.01) at< 0.01) when compared to control. However, the ). The kidney function tests for urea and cre when compared to the control group. The  -sized -East Africa, roots are used for treating snake bites. All of the above can be attributed to the peptide alkaloids and antifungal properties isolated from the bark and leaves.(Hutchingsal., 1996)Its leaves and roots have been used totreat diarrhea, tumor, cough, chest complaints (Muhammed et al., 2012). They are also used for treatment of dysentery, sores, glandular swellings, skin diseases, open and swollen wounds, ear inflammation, asthma, syphilis, gonorrhea, lumbago, measles, as rheumatic pains and fever (Vhutshilo and P2015). Other reports include the use of the roots by the Bambara and Malinke tribes in Tanzania for the psychiatric treatment, and the use of the leaf juice to prevent abortion. The plant also has analgesic and anti-inflammatory activity and antibiotic activites (Abdullahi, 2008). http://dx.doi.org/10.4314/bajopas.v12i1.545 - Nigeria Magaryar tudies in toxicity and  week 1, 2  in the level atinine   et  well as eter,  83S Special Conference Edition, November, 2019 MATERIALS AND METHODS  Plant materials The fresh leaves and fruits of Z. mucronata was collected from the Kudingi forest in Sabon Gari Local Government Area of Kaduna State and was taken to the herbarium unit of Botany Department of Ahmadu Bello University Zaria, Kaduna State, Nigeria for identification by the taxonomist. The plant was identified and a voucher number of 1054 was deposited for future reference. Preparation of Plant Material A sufficient quantity of the fresh leaf was collected, washed and cleaned with water, air dried to remove all foreign matter removed. Fresh Z. mucronata leaves were used for macroscopic and microscopic evaluation. A portion of the plant collected was shade dried for a few days at room temperature. When the leaves were completely dry, it was pulverized then kept in an air-tight container for subsequent use. 3.9.1 Experimental Animals Healthy adult wistar rats of both sexes were obtained from the Animal House facility of the Department of Pharmacology and Therapeutics, Ahmadu Bello University, Zaria, Nigeria. The animals were kept under well–ventilated conditions, fed on standard laboratory animal feeds and had access to water ad libitum. The animals weighed before commencement of the experiment and at the end. Acute safety studies This study was carried out to determine the LD50 of the extracts using Organization for Economic Cooperation and Development (OECD) method Guideline 425 (OECD, 2001). Three mice were used to determine the LD50 of the methanol extract. All three mice were administered the extract at dose of 5000 mg/kg body weight one after the other. The animals were observed after at intervals for 14 days for the short-term outcome. Sub Chronic Toxicity A 28-days repeated-dose toxicity study of Z. mucronata methanol leaves extract was given to 20 rats randomly grouped into four groups containing five rats of both sexes orally based on OECD Test Guideline 425. The methanol leaf extract of Z. mucronata was tested at the selected oral doses of 250, 500 and 1000 mg/kg body weight. The selections of doses were based on 20% of LD50 of the oral acute toxicity study. The wistar rats were divided into four groups of five animals each. The experimental animals were housed in a cage, made of aluminum with the top covered iron mesh. The bedding was made of saw dust, maintained at room temperature and natural daylight/night conditions. The animals weighed at on the first day before administration of the extract and weekly with the 28 days.   Group I: Received 1 ml/kg of normal saline water which served as the control group. Group II: Received 250 mg/kg of methanol extract dissolved in distilled water. Group III: Received 500 mg/kg of methanol extract dissolved in distilled water. Group IV: Received 1000 mg/kg of methanol extract dissolved in distilled water.  Biochemical Studies At the end of the treatment period (28 days), the animals were euthanized using chloroform and blood samples were collected by puncturing the prominent jugular vein with syringe into anti-coagulated and ethylene-diamine-tetra-acetic acid (EDTA) anti-coagulated tubes. The anti-coagulated blood was centrifuged at 3,000 rpm for 10 min and serum were collected. The level of biochemical indices such as blood urea nitrogen (BUN), Creatinine and bilirubin were determined for the control group and the treated groups using an automated hematological machine (Jaijoy et al., 2011). Statistical Analysis The results were expressed as Mean ± Standard Error of Mean (SEM) and percentages except where otherwise stated. The data obtained were analyzed using Statistical Package for Social Sciences (SPSS), IBM version 20 and Microsoft Excel 2010. The data was statistically analyzed using One Way Analysis of Variance (ANOVA) followed by Tukey post hoc test for multiple comparisons. Values of p≤0.05 were considered statistically significant (Duncan, 1955; Okasha et al, 2012).  RESULTS  Acute safety margin of Z .mucronata Methanol leaves extract The acute fixed oral dose toxicity (LD50) of the methanol extract of Z. mucronata leaves was determined using (OECD, 2001) method via oral route on three (3) healthy Wistar rats for 14 days and showed that the LD50 is greater than 5000 mg/kg for  the extract. There was no mortality or any toxicity signs in the animals after the treatment. Subchronic Toxicity Effect of Weekly Administration of the Extract on the Body Weight of Wister Rats The effects of oral daily administration of methanol extract on the body weight of Wistar rats showed significant increase (p<0.01) at 250mg/kg in week 1, week 2 and week 4 respectively when compared with the control group (1 mg/kg normal saline) (Fig 1). 546 Special Conference Edition, November, 2019   Fig 1: Effect of sub chronic administration of methanol leaves extract of ziziphus mucronata on body weights of wistar rats  Values are Mean ± S.E.M., **= p<0.01, ***= p<0.001 compared control group (Normal saline); n=6, MEZM = Methanol extract of Ziziphus mucronata–Repeated measure ANOVA followed by Bonferroni post hoc test.   Table 1: Effect of 28 days Administration of Methanol Leaf Extract of Ziziphus mucronata on Relative organ body Weights of Wistar Rats  Treatment group Relative organ-body weight (%) Liver                                          Kidney Control   1 ml/kg 3.04 ± 0.53 0.70 ± 0.05 MEZM 250 mg/kg 5.42 ± 0.53 1.21 ± 0.10 MEZM 500 mg/kg 4.57 ± 0.43 0.87 ± 0.07 MEZM 1000 mg/kg 4.91 ± 0.75 1.01 ± 0.21 Values are Mean ± S.E.M., No significant difference between the groups - One way ANOVA, n=6, MEZM = Methanol extract of Ziziphus mucronata        051015202530354045Week 1 Week 2 Week 3 Week 4Percentage change in body weight (100%)Time (weeks)Control (1mg/kg)MEZM 250 (mg/kg)MEZM 500 (mg/kg)MEZM 1000(mg/kg)***547 Special Conference Edition, November, 2019  Table 2: Effect of 28 days Administration of Methanol Leaf Extract of Ziziphus mucronata on Biochemical Parameters in Wistar Rats Treatment groups ALT (U/L) AST (U/L) ALP (U/L) TB (U/L) CB (µmol/L) Urea (mmol/L) Creatinine (µmol/L) N/S  1 ml/kg 96.33±6.84 60.23±8.66 38.80±11.89 12.83±2.62 6.63±1.27 52.00±4.62 0.73±0.09 MEZM 250 mg/kg 63.00±3.21b 39.23±0.82 16.10±2.93 8.83±1.30 4.67±0.72 50.67±5.81 1.20±0.12 MEZM 500 mg/kg 60.00±7.02b 50.20±4.95 12.23±3.24 6.97±0.97 4.17±0.17 64.00±10.07 1.00±0.21 MEZM 1000 mg/kg 56.67±2.03b 26.10±3.67b 10.63±3.01 10.00±0.78 5.53±0.87 85.33±21.83 1.40±0.23 Values are Mean ± S.E.M., b = p<0.01 compared normal group – One way ANOVA followed by Tukey post hoc test, n=6, ALT= Alanine amino transferase, AST = Aspartate amino transferase, ALP = Alkaline phosphatase, TB = Total bilirubin, CB= Conjugated bilirubin, D/W = Distilled water, MEZM = Methanol extract of Ziziphus mucronata   DISCUSSION  The evaluation of herbal remedies for safety and efficacy is very necessary due to their growing use all over the world. Determination of median lethal dose value of traditional medicine using acute toxicity study is of paramount importance as it provides information regarding the safety margin of the plant.   The median lethal dosage was estimated to be greater than 5000mg/kg indicating that the extract did not produce any sign of toxicity and could be assumed practically safe and non-toxic which the study of Zezi et al., (2012) who carried out an acute toxicity on the liver and kidney and reported no sign of toxicity on both the liver and kidney function tests. This result is in agreement with Olorunisola et al. (2012); Parasuraman et al. (2014); Ugbogu et al. (2016) and Adesegun et al. (2016) that reported acute toxicity of plants could be considered practically nontoxic and safe above administration of 5000 mg/kg especially when administered orally. However, Oslon (2000); Pak et al. (2011); Maikai et al. (2008); Obidike and Salawu (2013) stated that, further toxicity assay should be carried out in order to revealed possible longer term toxicity effect on the physiology and organs for proper recommendation on its utilization. Changes in the body weight have been used as an indicator of adverse effects of drugs and chemicals (Nandy, and Datta 2012, Hayelom et al., 2012). Sub chronic toxicity studies on the extract on the mean body weight of the rats showed significant difference only in 250 mg/kg showed in week 1, 2, and 4 when compared to the control. From the result obtained from the percentage body weights calculated, there was no adverse effect on the body weight of the animals with an increasing time (Fig 1). This implies that the methanol extract may have no chemical effect on the appetite and food consumption of the Wistar rats (Grance et al, 2008; Nandy and Datta 2012). A similar result was reported by Sireeratawong et al., (2012) who conducted a chronic toxicity studies on toxicities of the water extract from Ziziphus attopensis pierre and found that there was no adverse effect on the weight of the animals used over the stipulated period of time and there was also no gross morphological change in the weight of the animals. The relative organ weight is fundamental to establish whether or not the organ was exposed to the injury. The administration of the methanol extract showed no significant change in the relative organ body weight on either the liver or kidney at all the doses administered to the animals (Table 1), this result means that there was no damage to the organs (liver and kidney).  548 Special Conference Edition, November, 2019 The result of the biochemical studies of the methanol extract of Ziziphus mucronata at all the doses tested showed that there was a significance decrease (p<0.01) in the level of alkaline amino(ALT) at 250, 500 and 1000 mg/kg body weight and aspartate aminotransferase (AST) at 1000mg/kg body weight (Table 2) when compared to the control group. Nevertheless, only a significant increase in the level of these enzymes is considered a sign of toxicity. Urea and creatinine usually determine the general function of the kidney while the electrolytes are determinants of the tubular function. These parameters: urea and creatinine which were analyzed in these rats were not statistically different from the control groups and the values are within the standard, suggesting that the methanol extract of Ziziphus mucronata is not nephron toxic in rats within the period of the study. A decrease in glomerular filtration rate as in renal dysfunction would result in an increase serum creatinine concentration. Therefore serum creatinine is a reliable index of renal dysfunction. The serum urea level follows similar pattern, but may be affected by some factors, such as increase protein catabolism and hydration status. The Kidney plays a vital role in detoxification of drugs and xenobiotics (Harizal et al., 2010). The kidney as the main excretory organ could be assessed for safety a drug (Gupta et al., 1994). The complexity of the liver structure and frequency of its exposure to drugs and foods that might cause harm therefore, make it susceptible to many kinds of diseases, including hepatitis, cirrhosis, fatty liver, liver cancers and genetic diseases. The liver has a unique ability to regenerate itself. Liver diseases are among the most serious ailments (Samir, 2001) and have become some of the major causes of morbidity and mortality in man and animals all over the globe and hepatotoxicity due to drugs appears to be the most common contributing factor (Nadeem et al., 1997).  CONCLUSION  The study has established: The acute toxicity profile LD50 of the methanol extract revealed that the median lethal of the extract was greater than 5000 mg/kg which shows that the extract was practically non-toxic. 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Toxicity studies on the methanol leaf extract of Ziziphus mucronata willd

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Toxicity studies on the methanol leaf extract of Ziziphus mucronata willd

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