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Phytochemical Appraisal and Effects of Aqueous Extract of Hypoestes Rosea on Haematological Parameters of Acetaminophen – Induced Toxicity in Albino Rats

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Phytochemical Appraisal and Effects of Aqueous Extract of Hypoestes Rosea on Haematological Parameters of Acetaminophen – Induced Toxicity in Albino Rats Phytochemical Appraisal and Effects of Aqueous Extract of Hypoestes rosea on Haematological parameters of acetaminophen – induced toxicity in albino rats. ABSTRACT Aim: The aim of this study was to conduct a phytochemical appraisal and evaluate the effects of aqueous extract of Hypoestes rosea on haematological parameters of acetaminophen – induced toxicity in albino rats. Study design: This study is an experimental study. Place and Duration of Study: This study was conducted at the Experimental Animal Unit of the Department of Human Physiology, University of Port- Harcourt, between June 2018 and December, 2019. Methodology: A total of 112 adult apparently healthy albino rats weighing (180-220g) were used for this study, the rats were divided into six experimental groups of extract control (EC), negative control (NC), positive control (PC), AEHr100mg/kg body weight (b w), AEHr 200mg/kg b w., and AEHr 300mg/kg b w. groups each of six rats. At the end of the study period, blood sample were taken through jugular vein under chloroform anaesthesia for the determination of haematological parameters (packed cell volume (PCV), haemoglobin concentration (HB), Red blood cell count (RBC), white blood cell count (WBC) and platelet count (PLT), using an auto-analyzer (Sysmex KX-21n Auto-analyzer, Kobe, Japan). Data were analyzed using SPSS version 23, and p-values less than 0.05 (P<.05) were considered statistically significant. Results: Results showed that phytochemical analyses, both qualitative and quantitative of Hypoestes rosea leaves revealed the presence of flavonoids, tannins, alkaloids, terpenoids/steroids, saponins, carbohydrates and cardiac glycosides. Acetaminophen induction in albino rats caused anaemia as evidenced by significantly reduced PCV, HB, RBC and increased WBC levels. P<.05 in the PC group when compared with other experimental groups. However, various concentrations of aqueous extract of Hypoestes rosea in a dose dependent pattern at the different treatment phases at acute and sub- chronic period was able to restore the anaemia caused by acetaminophen induction to normal. Conclusion: In conclusion, Hypoestes rosea leaves possessed active ingredients and constituents in its phytochemical responsible for disease prevention and promotion of health. Acetaminophen induced toxicity caused anaemia and consumption of various concentrations of aqueous extracts of Hypoestes rosea at different treatment phases, experimental groups and duration of exposure helped restore normalcy. Therefore, the results of this study suggest that Hypoestes rosea has erythropoietic effects in albino rats and should be subjected to further studies using higher mammals. Keywords: Phytochemical, Analysis, Hypoestes rosea Haematological parameters acetaminophen, toxicity rats. 1. INTRODUCTION Plants have been used as a folkloric source of medicinal agents since the beginning of mankind. Hypoestes rosea locally called ‘Ogbuchi’ in Etche -1, Rivers State, Nigeria and commonly called “Polka Dot plant’is from the phylum Tracheophyta, class; Magnoliopsida, order; Lamiales, family; Acanthaceaa, sub- family; Acanthoideae, Tribe; Ruellieae, sub- tribe; Justiciinae and genus Hypoestes. Hypoestes phyllostachya ‘rosea’ is found in most parts of West Africa and beyond used in treatment of fever, malaria and anaemic conditions. The reported medicinal uses of H. rosea by indigenous people in different parts of the world show considerable similarities. [1-2]. Therefore, Hypoestes rosea is one of such plants with acclaimed folk medicinal usage and reported to possess anti-inflammatory, anti-cancer, anti- malarial and antioxidant properties. [3-5]. The leaves are therefore medicinal plant products since it contains active organic ingredients employed in the treatment of diseases. Phytochemicals accumulate in different parts of plants, such as in the roots, stems, leaves, flowers, fruits or seeds [6]. It is well- known that plants produce these chemicals to protect themselves, but studies have demonstrated that many phytochemicals can also protect human against diseases [7]. These phytochemicals are biologically active, naturally occurring chemical compounds found in plants, which provide health benefits for humans further than those attributed to macronutrients and micronutrients [8]. Phytochemical appraisal or analysis is the extraction, screening and identification of medicinally active ingredients or substances found in plants. Acetaminophen is generally safe at recommended doses but because the drug is available without prescription, it is potentially more dangerous than other similar drugs when used in excess or overdose [9] and may be injurious and deleterious to vital organs of the body affecting their functions and activities such as the erythropoietic system. There are insufficient scientific data on phytochemical analysis of Hypoestes rosea and effects of aqueous extract of Hypoestes rosea on haematological parameters in acetaminophen -induced toxicity in albino rats. 2. MATERIALS AND METHODS 2.1 Plant Collection, Identification and Authentication Fresh Hypoestes rosea leaves were collected from Ulakwo -1 in Etche LGA (40 59’ 27.00’’ N, 70 03 16 00’’ E) Rivers state in Nigeria. It was identified by Dr. Osiyemi Seun 22/04/2019 with FHI no.: 112295 at the Taxonomy section of the Forest herbarium unit in the Forestry Research Institute of Nigeria, Ibadan. 2.1.1 Method of Extraction and Preparation of AEHr. The leaves of Hypoestes rosea was removed from the stem, washed and air dried under shade at room temperature for fourteen days (2 weeks) and then milled into powder. 450g of Hypoestes rosea powder were macerated in 1000 ml of water to dissolve for 48hr in a flask, the extract was decanted and then filtered through Whatman No. 1 filter paper to obtain a clear extract. The aqueous extract was further concentrated at 60°C using a rotary evaporator and dried using a freezer drier. The resulting crude extract which weighed 214 g was stored in a refrigerator maintained at 4-18°C until the analysis was over. The extracts were later weighed and reconstituted in distilled water to give the required doses of 100, 200 and 300 mg/kg body weight that were used in the study. 2.2 Collection of Experimental Animals and Acclimatization Albino rats were considered the animals of choice for this study because of its availability, cost, genetic makeup, its handling technique and the nature of the study. Adult apparently healthy albino rats weighing (180 – 220grams) were used. The rats were purchased from the Experimental Animal Unit of the Department of Human Physiology, University of Port- Harcourt. The rats were contained in conservative wire mesh cages under standard laboratory conditions. After the collection of the animals, they were weighed, identified and kept in wire gauge cages under favourable condition for two weeks. The animals were receiving food and water ad libitum and handled regularly so as to acclimatize with the environment. One hundred and fifty-six (156) albino rats 12 weeks’ old rats were used in this study. All animals handling protocols were in accordance with institutional guidelines for laboratory animals. (Ethic Reference Number PM/27/08/2011/MAA (R) and OECD guidelines. 2.3 Phytochemical Qualitative and Quantitative Analysis of Hypoestes rosea Leaf. Phytochemical analysis was carried out at the Plant Anatomy and Physiology Research Laboratory, University of Port Harcourt. 2.3.1 Pytochemical Qualitative Analysis of Flavonoids Into a clean test tube was 5ml of the methanolic, ethanolic and aqueous extracts Hypoestes rosea Leaf leaves separately pipetted with the further addition of 5ml of 10% of dilute ammonia solution into each tube. To the test sample, was the careful addition of 1ml of concentrated sulphuric acid., a yellowish coloration of the solution was observed which indicated the presence of flavonoid in the test sample. To indicate the presence of the severity of flavonoids, the below symbols were used: + → Mildly present ++ → Moderately present +++ → Highly present 2.3.2 Phytochemical Qualitative Analysis of Alkaloids using Wagner’s Reagent [10] 5ml of the three leaves extracts were pipetted into three dry clean test tubes. 3mls in drops of Wagner’s reagent was introduced into each test tube. Homogenity of the mixture was ensured as the test tubes were shaken thoroughly. A precipitate of the mixture was observed which indicated the presence of alkaloids. The severity of alkaloids was represented as described below: + → Mildly present ++ → Moderately present +++ → Highly present 2.3.3 Phytochemical Qualitative Analysis of Tannins using Folin-Denis’s Reagent 1ml each of the three leave extracts was pipetted into three clean test tubes. Into the test sample in the test tubes was a drop of sodium carbonate solution added, likewise was two drops of Folin’s Denis reagents added into the mixtures. The mixture in the test tubes were kept on standing for ten minutes for total colour development. A bluish colour of the mixtures indicated the presence of tannis. The severity of the presence of tannis was indicated with the symbol as shown below: + → Mildly present ++ → Moderately present +++ → Highly present 2.3.4 Phytochemical Qualitative Analysis of Saponins using Frothing’s Test [11] 5mls each of the three leave extracts was boiled in 20mls of distilled water in a water bath, after which it was then filtered. 10ml of the filtered was mixed with 5ml distilled water, shaken vigorously for the appearance of a stable persistent froth. The froth
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