Biochemical and Safety Examination of Ethanol Extract of Justicia Carnae on PHZ -Produced Anaemia in Wistar Rats

ل لوناثيلإا صلختسمل يويحويميكلا صحفلا Justicia Carnae لع ى مدلا رقف ب ثدحتسملا PHZ ناذرجلا يف ةصلاخلا ةتبن قاروأ صحف مت Justicia carnae لصملا تاميزنإ ةطشنأ مادختساب ةيويحويميكلاو ةيمّدلا ةباجتسلاا ةسارد لجأ نم عون نم ناذرجلا يف Wistar . يف اهعيمجت متو ثحبلا اذه يف ًاذرج نيرشع مادختسا مت 4 نم 5 اهنم لكل ًاذرج . ىلولاا ةعومجملا تّدعُ تاعومجملا امأ ، طقف رطقملا ءاملاب تجلوعو ةيبلس ةرطيس ةعومجم 2 و 3 و 4 ـب تدوز ةجلاعم تاعومجم تّدعُ دقف 100 و 200 و 400 مغلم / صلختسم نم مسجلا نزو نم مغك J. carnae ، يلاوتلا ىلع . نيزارديه لينيف ةطساوب مدلا رقف ثادحتسا مت ( PHZ ) متو ، ةدمل ناذرجلا جلاع 14 ابتخلاا ءارجلإ بلقلا نم مدلا عمجو اهلتق تمت كلذ دعبو ، امًوي تار . صلختسم ريثأت صحف مت J. carnae ىلع لصملا تاميزنا ةطشنأو ةيمّدلا ريياعملا . ةيئاصحلإا ةيونعملا ىوتسمب ةرطيسلا ةعومجم عم جلاعلا تاعومجم يف جئاتنلا عيمج ةنراقم تمت (P<0.05) . ةرطيس ةعومجمك ةجلاعملا ريغ ةعومجملا ظفح مت . ريياعملل يجيردت عافترا كانه ناك ةيمّدلا نم صلختسملا ةعرج ةدايزب 100 ، 200 ىلإ ، 400 مغلم / مسجلا نزو نم مغك . ةيمّدلا ريياعملا ترهظأ : ةصوصرملا تايركلا مجح PCV رمحلا مدلا تايرك ددع ، RBC مدلا باضخو Hb ةيونعم ةدايز ىوتسمب (P<0.05) . للاخ نم اًيجيردت اعًافترا ضيب مدلا تايرك ددع يلامجإ رهظأ ً ايئاصحإ هتيمهأ مدع نم مغرلا ىلع صلختسملا . راشأ تايضمحلاو ةديحولا ايلاخلاو تايفمللا و تلادعلا دادعأ يف يونعم ريغ فيفط ضافخنا ىلإ يقيرفتلا ءاضيبلا مدلا ايلاخ دادعت . ترهظأ لصملا تاميزنإ نم ةيعيبط تايوتسم ةيريرسلا ةيويحويميكلا تاملعملا AST و ALT و ALP لا ىلع عم فيفط ضافخنا دوجو نم مغر ةدايز ةعرج صلختسم J. carnae . صلختسملا ةعرج ةدايز عم فيفط لكشب نيبوريليبلاو ايرويلاو يلكلا نيتوربلاو نينيتايركلا داز ةيئاصحإ ةللاد تاذ نكت مل يلاتلابو ةيعيبطلا تلادعملا نمض نكلو . ميق تراشأ MCV و MCH و MCHC ن نم مدلا رقف ةلاح ىلإ عو normocytic normochromic . صلختسم نا ةساردلا نم جتنتسي J. carnae ةعرجب ىلكلا تاميزنإو دبكلاو مدلا ايلاخل نمآ 400 مغلم / مسجلا نزو نم مغك .


Introduction
Plants have beneficial properties because they contain phytochemical compounds (1) and GCMS analysis has shown that phytochemicals are made up of primary and secondary metabolites that can protect the plants, human and animals against diseases (2)(3)(4). GC-MS analysis to identify phytochemicals present in Justicia carnae leaves was done by (5). Justicia of family, Acanthacea consists of about 600 species including herbs and shrubs, and are found plenty in Africa, (6) (7). Justicia carnae is a flowering plant abundantly distributed in different parts of Africa. Grown around homes and act as a fence and can be propagated from stem cutting (8).
The Igbo tribe in Nigeria calls the plant 'Ogwu obara' literally meaning drug for blood production. (9) reported its use in the control of inflammation, respiratory tract infection, gastrointestinal disorders, diabetes, diarrhoea, and liver diseases. It also possesses cardioprotective, antitumour, and antiviral activities(10), antioxidant activity (11). Anaemia is decreased below the normal range of red blood cells (RBCs), packed red cell volume (PCV) or haemoglobin concentration (Hb), in the blood. It is a sign of a disease and not a disease. It has three major categories of which include: hypoproliferation, maturation defects, and hemolysis/blood loss. It is a hidden epidemic worldwide and can have serious consequences if left untreated. The use of Phenylhydrazine (PHZ) and compounds related to it were used as an antipyretic agent and demonstrated toxicity to RBC on red blood cells (12). This compound was, however, found to be useful in experimental models, hence an approved method of inducing haemolytic anemia for the study haematinic properties of new agents, erythropoietin regenerative response of plant materials through clinical, pathological, and morphological studies (13) .
Phenylhydrazine administration has been shown to cause haematotoxicity which leads to haemolytic anaemia by altering iron metabolism, activating, and interferes with the binding of erythropoietin on its receptors and the formation of Heinz bodies in RBC as a side effect, (15). AST and ALT elevation in conditions of hepatocyte damage in the inflammatory condition of the liver, hypoxic states, hepatotoxicity by toxicants, trauma, and some plant extracts, (16). Liver ALP elevation also in hepatocyte and biliary epithelial damage. They could also be ALP elevation in osteoblast, intestinal epithelial, and corticosteroid stimulation when used for treatment (17) (18). Hyperproteinaemia is associated with dehydration occasioned by vomiting, diarrhoea, impaired renal concentration ability, excessive sweating or decreased water intake, (19). Elevated urea production is associated with intestinal haemorrhage, increased dietary urea or increased protein catabolism, (20). Elevated creatinine occure in pathological processes that cause a decrease in glomerular filtration rate which could be pre-renal, renal or post renal, (21). Hyperbilirubinaemia occur in diseases associated with haemolysis of blood as seen in babesiosis, anaplasmosis, trypanosomiasis, snake bite and some plant toxicants, (22). We designed this research to examine the safety of J. carnae extract after recovering from anaemia by examining its effect on blood cells; biochemical liver enzyme markers ALT, AST, and ALP. Kidney markers urea, creatinine, bilirubin, and total protein. To investigate the haematinic and haematopoetic potential of leaf extract of Justicia canae in phenyl-hydrazine-induced haemolytic anemia in albino Wistar rats.

Materials and methods Plant Materials
Fresh leaves of J. carnae were collected from the University environment in Vol.

Preparation of Plant Extract
The identified leaves of J. carnae were dried under shade for 10 days and grinned to a coarse powder using a manual grinder (Corona-Landers C 1A SA). Extraction was done by the Soxhlet method described by (23) and 35g of coarse powdered sample was introduced into the extraction chamber using ethanol as solvent. Throughout the extraction time of 48 hrs the temperature was kept at 70 0 C in the chamber. The extract was concentrated in an oven at 30 0 C and the dried extract weighed and kept in a labelled sterile specimen bottle for the work. Different doses of 100, 200 and 400 mg/kg body weight were prepared and administered to rats in groups 2, 3, and 4 respectively. These doses were calculated from a stock solution dissolved in distilled water.

Haematology and Biochemical Investigation
For Haematological screening, PCV and differential counts were measured by the micro-haematocrit method as described by (24). Haemoglobin concentrations was determined by cyanomethemoglobin method, Kachmar (25).
A biochemical investigation was performed using ELISA reagent kits. The measure included alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), determined by the method of (16). Using serum enzyme levels to determine liver and kidney state (17). Urea by (20) and Creatinine by (21). Total protein was determined by the Biuret method as described by (19). Samples were analyzed immediately to avoid artifactual changes (30).

Experimental Animals
Adult albino rats were purchased from University Farm. Approval was obtained from the College of Veterinary Medicine of the University in line with the guidelines for the care and use of laboratory animals provided by the National Research Council (31). The rats were acclimatized and fed ad libitum. hydrochloride at interval of 3 days, for the duration of the experiment.

Experimental design
Twenty-five rats were used for the research, they were grouped into 5 of 5 rats each. Group 1 was the normal control group and was administered distilled water orally. Groups 2 was the untreated anaemic group, 3, 4 and 5 were the treatment groups that received 100, 200 and 400 mg/kg body weight of the J. carnae extract respectively orally by intubation. The rats were treated for 14 days, thereafter they were sacrificed and blood collected from the heart for analysis. The effect of J. carnae extract was checked on haematological parameters and serum enzyme activities.

Statistical analysis
Analysis of statistical data was computed using Statistical Package for Social Sciences (SPSS) version 20. Values were expressed as mean ± Standard Error of Mean (SEM) and were further subjected to one -way analysis of variance (ANOVA) to compare doses with untreated anaemic group . Duncan posthoc test was used to separate the mean that showed significant difference. The statistical confidence was set at p<0.05.

Results and discussion
The result of Fig 1 shows   The graph in Fig 4 represents Fig 3 showed that liver enzymes were restored to the normal reference range. The mechanism of action of J. carnae may solely depend on the restoration of these hematological parameters thereby preventing damage to the liver and/or restoration of injured hepatocytes indicating that J. carnae extract could be hepatoprotective against hemolytic anemia and/or phenylhydrazine induced hepatotoxicity in rats. Our research agrees with (36) that J. carnae can revive anaemic condition. In this work also, the extract of J. carnae is safe to liver and kidney cells at dose <400 mg/kg body weight. Rats showed significant recovery at a higher dose of J. carnae. The significant recovery as shown in Fig 1-3 could be due to the administration of J. carnae extract, and not by the natural physiological compensation of the bone marrow.

Conclusion
Therefore, it can be concluded that J. carnae extract increased the PCV, Hb and RBC levels and caused a reduction in AST, ALT, and ALT with the restoration of hepatocytes after phenylhydrazine induced haemolytic anaemia. This suggests that the extract may be beneficial in the treatment of haemolytic anaemia induced by phenylhydrazine or haemolysis as a result of infectious agent.