International Journal of Research and Reports in Hematology

3(1): 27-36, 2020; Article no.IJR2H.62051

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

I. E. Ogregade1*, F. U. Igwe2, T. G. Davies1 and E. S. Bartimaeus1

1Department of Medical Laboratory Science, Rivers State University, Port Harcourt, Nigeria. 2Department of Biochemistry, Rivers State University, Port Harcourt, Nigeria.

Authors’ contributions

This work was carried out in collaboration among all authors. Author ESB designed the study. Author FI wrote the protocol. Author TGD wrote the first draft of the manuscript. Author IEO managed the analyses of the study and the literature searches. All authors read and approved the final manuscript.

Article Information

Editor(s): (1) Dr. Dharmesh Chandra Sharma, G. R. Medical College & J. A. Hospital, India. Reviewers: (1) Albert Opoku, Nursing and Midwifery Training College, Kumasi, Ghana. (2) Szakacs Andrei-Radu, University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, Romania. Complete Peer review History: http://www.sdiarticle4.com/review-history/62051

Received 10 August 2020 Accepted 17 October 2020 Original Research Article Published 30 October 2020

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 (180-220 g) were used for this study, the rats were divided into six experimental groups of extract control (EC), negative control (NC), positive control (PC), AEHr100 mg/kg body weight (b w), AEHr 200 mg/kg b w., and AEHr 300 mg/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

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*Corresponding author: E-mail: [email protected];

Ogregade et al.; IJR2H, 3(1): 27-36, 2020; Article no.IJR2H.62051

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 , which provide health benefits for humans further than those attributed to macronutrients Plants have been used as a folkloric source of and micronutrients [8]. medicinal agents since the beginning of mankind. Hypoestes rosea locally called ‘Ogbuchi’ in Etche Phytochemical appraisal or analysis is the -1, Rivers State, Nigeria and commonly called extraction, screening and identification of “Polka Dot ’is from the phylum medicinally active ingredients or substances Tracheophyta, class; Magnoliopsida, order; found in plants. Acetaminophen is generally safe , family; Acanthaceaa, sub- family; at recommended doses but because the drug is , Tribe; Ruellieae, sub- tribe; available without prescription, it is potentially Justiciinae and genus Hypoestes. Hypoestes more dangerous than other similar drugs when phyllostachya ‘rosea’ is found in most parts of used in excess or overdose [9] and may be West Africa and beyond used in treatment of injurious and deleterious to vital organs of the fever, malaria and anaemic conditions. The body affecting their functions and activities such reported medicinal uses of H. rosea by as the erythropoietic system. There are indigenous people in different parts of the world insufficient scientific data on phytochemical show considerable similarities [1,2]. Therefore, analysis of Hypoestes rosea and effects of Hypoestes rosea is one of such plants with aqueous extract of Hypoestes rosea on acclaimed folk medicinal usage and reported to haematological parameters in acetaminophen - possess anti-inflammatory, anti-cancer, anti- induced toxicity in albino rats. malarial and antioxidant properties [3-5]. The leaves are therefore medicinal plant products 2. MATERIALS AND METHODS since it contains active organic ingredients employed in the treatment of diseases. Phytochemicals accumulate in different parts of 2.1 Plant Collection, Identification and plants, such as in the roots, stems, leaves, Authentication flowers, fruits or seeds [6]. It is well- known that Fresh Hypoestes rosea leaves were collected plants produce these chemicals to protect 0 0 themselves, but studies have demonstrated that from Ulakwo -1 in Etche LGA (4 59’ 27.00’’ N, 7 many phytochemicals can also protect human 03 16 00’’ E) Rivers state in Nigeria. It was against diseases [7]. identified by Dr. Osiyemi Seun 22/04/2019 with FHI no: 112295 at the Taxonomy section of the These phytochemicals are biologically active, Forest herbarium unit in the Forestry Research naturally occurring chemical compounds found in Institute of Nigeria, Ibadan.

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2.1.1 Method of extraction and preparation of addition of 5 ml of 10% of dilute ammonia AEHr solution into each tube. To the test sample, was the careful addition of 1 ml of concentrated The leaves of Hypoestes rosea was removed sulphuric acid, a yellowish coloration of the from the stem, washed and air dried under shade solution was observed which indicated the at room temperature for fourteen days (2 weeks) presence of flavonoid in the test sample. and then milled into powder. 450 g of Hypoestes rosea powder were macerated in 1000 ml of To indicate the presence of the severity of water to dissolve for 48 hr in a flask, the extract flavonoids, the below symbols were used: was decanted and then filtered through Whatman No. 1 filter paper to obtain a clear extract. The + → Mildly present aqueous extract was further concentrated at ++ → Moderately present 60°C using a rotary evaporator and dried using a +++ → Highly present freezer drier. The resulting crude extract which weighed 214 g was stored in a refrigerator 2.3.2 Phytochemical qualitative analysis of maintained at 4-18°C until the analysis was over. alkaloids using wagner’s reagent [10] The extracts were later weighed and reconstituted in distilled water to give the 5 ml of the three leaves extracts were pipetted required doses of 100, 200 and 300 mg/kg body into three dry clean test tubes. 3 mls in drops of weight that were used in the study. Wagner’s reagent was introduced into each test tube. Homogenity of the mixture was ensured as 2.2 Collection of Experimental Animals the test tubes were shaken thoroughly. A and Acclimatization precipitate of the mixture was observed which indicated the presence of alkaloids. The severity Albino rats were considered the animals of of alkaloids was represented as described below: choice for this study because of its availability, cost, genetic makeup, its handling technique and + → Mildly present the nature of the study. Adult apparently healthy ++ → Moderately present albino rats weighing (180–220 grams) were +++ →Highly present used. The rats were purchased from the Experimental Animal Unit of the Department of 2.3.3 Phytochemical qualitative analysis of Human Physiology, University of Port- Harcourt. tannins using folin-denis’s reagent The rats were contained in conservative wire mesh cages under standard laboratory 1 ml each of the three leave extracts was conditions. After the collection of the animals, pipetted into three clean test tubes. Into the test they were weighed, identified and kept in wire sample in the test tubes was a drop of sodium gauge cages under favourable condition for two carbonate solution added, likewise was two weeks. The animals were receiving food and drops of Folin’s Denis reagents added into the water ad libitum and handled regularly so as to mixtures. The mixture in the test tubes were kept acclimatize with the environment. One hundred on standing for ten minutes for total colour and fifty-six (156) albino rats 12 weeks’ old rats development. A bluish colour of the mixtures were used in this study. indicated the presence of tannis. The severity of the presence of tannis was indicated with the 2.3 Phytochemical Qualitative and symbol as shown below: Quantitative Analysis of Hypoestes rosea Leaf + → Mildly present ++ → Moderately present

+++ →Highly present Phytochemical analysis was carried out at the Plant Anatomy and Physiology Research 2.3.4 Phytochemical qualitative analysis of Laboratory, University of Port Harcourt. saponins using frothing’s test [11]

2.3.1 Pytochemical qualitative analysis of 5 mls each of the three leave extracts was boiled flavonoids in 20 mls of distilled water in a water bath, after which it was then filtered. 10 ml of the filtered Into a clean test tube was 5ml of the methanolic, was mixed with 5 ml distilled water, shaken ethanolic and aqueous extracts Hypoestes rosea vigorously for the appearance of a stable Leaf leaves separately pipetted with the further persistent froth. The froth formed was mixed with

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3 drops of olive oil for each tube, which was Weight of Flavonoids = Weight of Beaker x again shaken vigorously for uniformity and the residue – weight of empty beaker three tubes were observed for the formation of an emulsion. The concentration of the emulsion % flavonoids = weight of flavonoid x 100 formed to show the presence of saponins was Weight of sample 1 recorded with its severity as: 2.3.7 Phytochemical quantitative analysis of + → Mildly present saponins ++ → Moderately present +++ → Highly present 10 g each of the three different leave extracts was weighed and transferred into three different 2.3.5 Phytochemical quantitative analysis of 250 ml conical flask. 20% in 100 mls each of alkaloids [12] aqueous ethanol solution was added to the samples. The samples were subjected to heat on 5 g each of the aqueous, ethanolic and a water bath with series of stirring on a methanolic leave extracts of the sample was temperature maintained at 55°C for 4 hours. The weighed and dispensed into three different 250 mixtures were then filtered while the residues ml beaker, to which 200 mls of 10% acetic acetic were re-extracted with 20% of a 200 ml in portion in ethanol was added to each tube. The mixture of ethanol. The combined extracts were was covered and allowed to stand for 4 hours evaporated to 40 ml over a temperature of 90oC after which the filterate that was filtered through a over a water bath. The aqueous layer that was Whatman’s number 541 filter paper was recovered during the process was kept while the concentrated on a water bath. To a one quarter ether layer was discarded. The recovered of each of the leave extract sample of the original aqueous layer was purified with 60 ml n-butanol. volume collected was the addition of The combined n-butanol extracts were washed concentrated ammonium hydroxide which was twice with 10 ml of 55% aqueous solution of added in a drop wise volume to the filtrate which sodium chloride. The left-over solution was showed a complete precipitation process. The heated in a water bath and further left to air dry in entire mixture of the solution was left on standing an evaporator where its weight was obtained with to settle while the precipitate formed was washed the use of this formula: with ammonium hydroxide and again filtered. The residue on the filter paper was dried and weighed Weight of Saponin = weight of flask x residue and calculated thus: – weight of empty flask

Weight of Alkaloid = Weight of filter paper + % Saponin = weight of saponin residue x 100 residue – Weight of empty filter paper Weight of sample 1

2.3.8 Phytochemical quantitative analysis of Therefore, percentage yield of Alkaloid,= tannins [14] weight of filter paper + residue – weight of empty filter paper/ Weight of sample x 100 0.1 g each of the three leave extracts was weighed on a weighing scale and transferred into 2.3.6 Phytochemical quantitative analysis of three 250 ml conical flasks. 100 ml of distilled flavonoids [13] water was added into the samples and boiled for 1 hour. The samples were allowed to cool at 10 g each of the three leave extracts was room temperature and diluted with 50 ml of extracted repeatedly with 100 ml of 80% distilled water. 1 ml each of the diluent was aqueous methanol at room temperature. The pipetted into three test tubes and 2 to 5 mls of complete portion of this mixture was filtered Folin-Denis’s reagent was added with 1ml of through a Whatman’s number 42 filter paper. The 17% sodium carbonate. filtrates obtained were then transferred into three crucibles and then subjected to a water bath for A blank test was prepared with 1 ml distilled them to evaporate into dryness and further air water and the reagents as earlier stated. The dried in an air oven, cooled to room temperature bluish colour formed in the test sample was read in a desiccator and weighed in an analytical spectrophotometrically at 750nm wavelength balance. using blank to calibrate the spectrophotometer.

The calculation used to obtain the quantified 0.1 g of the tannic acid was dissolved into 100 ml flavonoid included: dissolved water to prepare the standard

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concentration to enable the dilutions of the after an overnight fast. Blood samples working standards of choice to be plotted against were collected by puncture of the jugular vein the concentration. and put into EDTA bottles for estimation of haematological parameters (PCV, HB, RBC, A linear graph that passed through the margin WBC and PLT) were assayed using an was obtained. The concentrations of tannin in the auto-analyzer (Sysmex KX-21n Auto- three samples were extrapolated from the analyzer, Kobe, Japan). The laboratory analysis standard graph. took place at the Research Laboratory of the departments of Biochemistry and Physiology, 2.4 Experimental Design University of Port-Harcourt, Port-Harcourt, Nigeria. 2.4.1 Animal grouping and treatment regimen 2.6 Quality Control A total of one hundred and twelve (112) adult albino rats were assigned by weight into eighteen Quality control sera, standard operating (18) groups and allowed to acclimatize for procedures and good laboratory/ best practices (fourteen) 14 days (2 weeks). The duration of the were adhered. study was fifteen (15) days acute and thirty (30) days sub-chronic study. Eight (8) albino rats 2.7 Data Analysis each were assigned for the two (2) positive control groups and six (6) albino rats each were Data were analyzed using SPSS version 23, they assigned to the other groups. The study groups were presented as Mean ± SEM. Variations comprised of two treatment phases each, (Pre- between were determined using Analysis of treatment and Post-treatment phases), duration variance (ANOVA) and Tukey Test of Multiple of treatment (Acute and Sub-chronic) with six Comparison used to differentiate variations in experimental groups in each of the phases. In means between groups. p-values less than the pre-treatment phases, the albino rats are 0.05 (P<.05) were considered statistically administered with AHEr extracts before significant. acetaminophen induction, while in the post- treatment phases, the albino rats were treated 3. RESULTS AND DISCUSSION with AHEr extract after acetaminophen induction.

The groups are as follows: The results of the qualitative and semi- quantitative phytochemical analyses for fresh 1. Negative control (NC): Apparently healthy and dry Hypoestes rosea leaves are presented rats receiving de-ionized water and normal in Table 1. The qualitative results of the fresh feed only. leaf clearly showed that Hypoestes rosea 2. Positive control (PC): 500 mg/kg b w. yielded alkaloids; positive for Dragendorff’s, acetaminophen induced rats at 14th day in Mayer’s and Hager’s test, flavonoids; positive acute and 29th day in Sub-chronic study. for Shinoda and lead acetate tests, tannins; 3. Extract Control (EC): Apparently healthy positive for iron chloride test, triterpenoid/steroid; rats that received AHEr 100 mg/kg b w. positive for Lieberman-Buchard and Salwaski orally daily for fifteen (15) days and thirty test; fix oils; carbohydrates; positive for (30) days. Molish and Fehling’s tests, cardenolide; 4. Acetaminophen induced treatment group positive for Keller Killani test. Phytochemical for aqueous extract of Hypoestes rosea of 100 dry leaf yielded alkaloids, carbohydrates, mg/kg b w. flavonoids, glycosides, tannins, terpenoid/ 5. Acetaminophen induced treatment group steroid and saponin at different reference aqueous extract of Hypoestes rosea of 200 percentages. mg/kg b w. 6. Acetaminophen induced treatment group The percentage in constituent in the quantitative aqueous extract of Hypoestes rosea of 300 phytochemical is presented in the Fig. 1. The mg/kg b w. medicinal effects of Hypoestes rosea, like other medicinal plants are attributed to the presence of 2.5 Sample Collection and Analysis active bio-ingredients or phytochemicals in them which generally are responsible for disease Rats were anaesthetized using chloroform and prevention and promotion of health [15]. Studies th th were sacrificed on the 15 and the 30 days have reported it possesses anti – cancer, anti-

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inflammatory, anti-malaria and antioxidant effects The acute and sub–chronic effects of various [3-5]. However, this present study reveals concentrations of aqueous extract of Hypoestes Hypoestes rosea has phytochemicals such as rosea on haematological parameters; alkaloids, carbohydrates, flavonoids, glycosides, haemoglobin (Hb), packed cell volume (PCV), tannins, terpenoids/steroids, saponins and red blood cell count (RBC) white blood cell count cardenolides (Table 1 and Fig. 1), which almost (WBC) and platelets of acetaminophen induced agrees with [16] earlier phytochemical study on toxicity in albino rats after 15 days exposure and Hypoestes rosea yielding positive for flavonoids, 30 days exposure (Tables 2 and 3 respectively). terpenes, sterols, balsam, carbohydrates, monosaccharides reducing sugars, tannins and The acute and sub-chronic study result shows saponins while resins and glycosides were that acetaminophen inducement at 500 mg/kg.b. negative. wt. slightly decreased PCV when compared with other experimental groups in the pre- treatment Acetaminophen/acetyl-para-aminophenol (APAP) phase and post treatment phases. However, is generally safe at recommended doses but treatment with various concentrations of aqueous since the drug is readily available without extract of Hypoestes rosea significantly prescription, it is potentially more dangerous than increased the PCV to normal. P- value for PCV other similar drugs when taken in excess or in the study was significant (P<.05). overdose [9] and may affect vital organs responsible for functions and routine Also, like PCV, the acute and sub-chronic study homeostasis. result shows that acetaminophen inducement at

Table 1. Result of phytochemical analysis of fresh and dry leaf of Hypoestes rosea

Constituents Qualitative result of fresh leaf Quantitative result of dry leaf in % Alkaloids + 21 Carbohydrates + 20 Flavonoids + 18 Glycosides + 17 Tannins + 12 Terpenoids/Steroids + 9 Saponins + 3 Cardenolides + - Key: + = present - = absent

10.8

32.4 75.6

43.2 0

64.8 61.2

ALKALOIDS FLAVONOIDS 64.8 GLYCOSIDES TANINS TERPENOIDS/STEROIDS SAPONINS

Fig. 1. Phytochemical constituents of Hypoestes rosea presented in a pie-chart

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500 mg/kg.b. wt. slightly decreased Hb when Likewise, the acute and sub-chronic study compared with other experimental groups in the result showed that acetaminophen inducement at pre- treatment phase and post treatment phases. 500 mg/kg.b. wt. slightly decreased RBC However, treatment with various concentrations when compared with other experimental groups of aqueous extract of Hypoestes rosea in the pre- treatment phase and post treatment significantly increased Hb levels to normal. P - phases. However, treatment with various value in the acute study pre- treatment P< .0003) concentrations of aqueous extract of and therefore was significant while P – value in Hypoestes rosea significantly increased the RBC acute study post treatment is (P < .4847) not to normal. P-value for RBC in the acute significant. P - value for Hb in the sub chronic study was significant (P<.05) while P>0.05 study for both pre-treatment and post treatment in the sub -chronic study and therefore not were significant P<.05. significant.

Table 2. Acute Effects of various concentrations of aqueous extract of Hypoestes rosea (AEHr) on haematological parameters (PCV, HB, RBC, WBC and platelets) of acetaminophen-induced albino rats by treatment phase and experimental group

Treatment Experimental PCV (%) HB (g/dl) RBC WBC Platelets Phase Group (x 10^12/L) (x 10^12/L) (x 10^9/L) Mean Mean Mean Mean Mean ±SEM ± SEM ± SEM ± SEM ± SEM Pre- EC 44.33 14.75 5.70 5.82 253.33 Treatment ±1.33b ±0.45b ±0.20 ±0.38a ±18.52 NC 41.83 13.95 5.30 8.85 261.67 ±1.87a ±0.62a ±0.18 ±0.57b ±12.24 PC 39.50 13.17 5.18 10.08 269.50 ±1.23a ±0.41a ±0.23 ±0.27b ±18.13 AEHr(100 mg/kg) 44.50 14.83 5.50 6.67 233.33 ±1.06a ±0.36b ±0.13 ±0.55a ±15.14 AEHr(200 mg/kg) 46.67 15.55 5.13 7.73 247.83 ±1.26a ±0.42b ±0.16 ±0.49a ±19.56 AEHr(300 mg/kg) 49.17 16.38 5.75 7.45 300.67 ±1.08a ±0.36b ±0.39 ±0.61a ±12.32 Test F-Ratio 6.589 6.572 1.303 9.691 2.001 Statistics P-value 0.0003*** 0.0003*** 0.2890ns <0.0001**** 0.7071ns Post- EC 44.33 14.75 5.70 5.82 253.33 Treatment ±1.33 ±0.45 ±0.20 ±0.38a ±18.52 NC 41.83 13.95 5.30 8.85 261.67 ±1.87 ±0.62 ±0.18 ±0.57b ±12.24 PC 39.50 13.17 5.18 10.08 269.50 ±1.23 ±0.41 ±0.23 ±0.27b ±18.13 AEHr(100 mg/kg) 40.17 13.38 4.95 6.92 257.67 ±1.01 ±0.34 ±0.13 ±0.47ac ±16.68 AEHr(200 mg/kg) 42.33 14.12 5.17 7.63 252.00 ±0.98 ±0.33 ±0.18 ±0.48ac ±18.84 AEHr(300 mg/kg) 44.67 14.88 5.87 7.50 317.17 ±0.88 ±0.29 ±0.28 ±0.50ac ±6.87 Test F-Ratio 2.782 0.9149 2.898 10.85 2.426 Statistics P-value 0.0352* 0.4847ns 0.0299ns <0.0001**** 0.0583ns Key: a, b, c, d mean significantly different from each other. Same number indicates no statistical difference in mean values. ** =p<0.01, ***=p<0.001 and ****=p<0.0001. Treatment Phases: Pre-Treatment, Post-Treatment. N for each level mean=12. Within and across treatment phases by experimental groups, each parameter means ± SEM are not significantly different (p>0.05). Significance Level: ns=Not Significant (p>0.05). Experimental Groups: Negative Control (NC), Positive Control (PC), Extract Control (EC), Aqueous Extract of Hypoestes rosea at 100 mg/kg (AEHR (100 mg/kg)), AEHR (200 mg/kg), AEHR (300 mg/kg)

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Table 3. Sub-chronic effects of various concentrations of aqueous extract of Hypoestes rosea (AEHR) on haematological parameters (PCV, HB, RBC, WBC and platelets of acetaminophen- induced albino rats by treatment phase and experimental group

Treatment Experimental PCV (%) HB (g/dl) RBC WBC Platelets Phase Group (x 10^ (x 10^ (x 10^ 12/L) 12/L) 9/L) Mean Mean Mean Mean Mean ± SEM ± SEM ± SEM ± SEM ± SEM Pre- EC 44.67 14.90 6.02 6.07 214.00 Treatment ±1.12a ±0.37a ±0.27 ±0.35b ±16.31 NC 42.83 14.28 6.00 7.62 226.83 ±0.60a ±0.20a ±0.27 ±0.57a ±13.94 PC 41.83 13.95 5.48 9.50 246.17 ±1.08a ±0.37b ±0.16 ±0.34a ±15.02 AEHr(100 mg/kg) 45.83 15.72 5.90 6.42 204.50 ±1.25a ±0.62a ±0.36 ±0.56b ±16.99 AEHr(200 mg/kg) 46.67 15.55 5.85 6.32 221.00 ±1.45b ±0.48a ±0.28 ±0.31b ±27.27 AEHr(300 mg/kg) 49.17 16.38 6.10 6.53 281.00 ±0.79b ±0.27a ±0.31 ±0.43b ±17.92 Test F-Ratio 6.028 5.071 0.6105 8.803 2.253 Statistics P-value 0.0006*** 0.0017** 0.6925ns <0.0001**** 0.0746ns Post- EC 44.67 14.90 6.02 6.07 214.00 Treatment ±1.12a ±0.37bc ±0.27 ±0.35 ±16.31 c NC 42.83 14.28 6.00 7.62 226.83 ±0.60a ±0.20 c ±0.27 ±0.57 ±13.94 c PC 41.83 13.95 5.48 9.50 246.17 ±1.08a ±0.37cde ±0.16 ±0.34 ±15.02bc AEHr(100 mg/kg) 39.00 13.00 6.20 7.72 282.67 ±0.58b ±0.20 e ±0.36 ±0.67 ±21.60ab AEHr(200 mg/kg) 39.83 13.27 5.85 6.50 311.67 ±0.70b ±0.24 de ±0.19 ±0.34 ±18.42a AEHr(300 mg/kg) 42.50 14.17 6.05 8.13 304.83 ±0.62a ±0.22 cd ±0.28 ±0.38 ±8.57 a Test F-Ratio 6.48 6.41 0.8881 7.079 6.507 Statistics P-value <0.0003*** <0.0004*** 0.5013 ns 0.0002*** 0.0003*** Key: a, b, c, d mean significantly different from each other. Same number indicates no statistical difference in mean values. ** =p<0.01, ***=p<0.001 and ****=p<0.0001. Treatment Phases: Pre-Treatment, Post-Treatment. N for each level mean=12. Within and across treatment phases by experimental groups, each parameter means ± SEM are not significantly different (p>0.05). Significance Level: ns=Not Significant (p>0.05). Experimental Groups: Negative Control (NC), Positive Control (PC), Extract Control (EC), Aqueous Extract of Hypoestes rosea at 100 mg/kg (AEHR (100 mg/kg)), AEHR (200 mg/kg), AEHR (300 mg/kg)

The acute and sub-chronic study result shows Hypoestes rosea does not affect platelets and P- that acetaminophen inducement at 500 mg/kg.b. value is therefore, insignificant (P> 0.05). wt. causes increase in WBC when compared with other experimental groups in the pre- The haematological parameters showed that treatment phase and post treatment phases. treatment of rats with 500 mg/kg b. wt. of However, treatment with various concentrations acetaminophen caused significant decrease of of aqueous extract of Hypoestes rosea PCV, Hb and RBC implicating the presence of significantly decreased WBC to normal. P-value anaemia. This anaemia may be attributed to for WBC in the study was significant (P<0.05). destruction of RBCs due to increased oxidative damage and lipid peroxidation in cell membranes The result shows that acetaminophen induction or to the effect of paracetamol on erythropoiesis at 500 mg/kg b. wt and various treatment by inhibiting erythropoietin release through direct concentrations with aqueous extract of effect on kidney [17].

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Also noticed in the haematological study were Elechi-Amadi for their efforts in statistical increase in WBC and slight increase in platelets analysis. even though its within normal limits. However, rats treated with aqueous extract of Hypoestes COMPETING INTERESTS rosea in combination with acetaminophen either pre – treatment or post treatment showed Authors have declared that no competing parameters that were consistent with control interests exist. values and normal ranges. REFERENCES These results further link that Hypoestes rosea may cause some protection against acetaminophen toxicity. This was similar to study 1. Al - Haidari, P. A review of traditional uses, of [18] and [19] who reported that Moringa has phytochemicals and bioactivities of the high iron content which enters in Hb synthesis genus Hypoestes. Afr J Trad Comp Alt and thus stimulating erythropoiesis. Med. 2018;15(3):1-17. 2. Haidan Y, Qiangian M, Guangchun, P. 4. CONCLUSION The traditional medicine and modern medicine from natural products. Mol. 2016;

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All authors hereby declare that Principles of cereal grains and other crops. in recent laboratory animal care (NIH publication No. 85- advances in phytochemistry, vol. 33, 23, revised 1985) were followed, as well as phytochemicals in human health specific national laws where applicable. All protection, nutrition, and plant defence, ed. experiments have been examined and approved JT Romeo, New York. 1999;67-87. by the appropriate ethics committee. 7. Narasinga R. Bioactive phytochemicals in Indian foods and their potential in health All animals handling protocols were in promotion and disease prevention. As Pac accordance with institutional guidelines for J Clin Nut. 2003;12(1):9-22. laboratory animals. (Ethic Reference Number 8. Hasler C, Blumberg J. Symposium on PM/27/08/2011/MAA (R) and OECD guidelines. phytochemicals: Biochemistry and physiology. J Nut. 129:756-7. ACKNOWLEDGEMENTS 9. Sharma A, Rathore H. Prevention of acetaminophen induced hepato-renal Authors are grateful to all that contributed in one damage in mice with rhizomes of way or the other to the success of the article. Glycyrriza Glabra A histological study. Anc Pastor Woy of University of Port Harcourt, helped Sci Life. 2011;30(3):72-7. in taking care of the laboratory animals, Mr Uche 10. Kokate CK, Khandelwal KR, Pawar AP, and Gbenga for their efforts in laboratory Gokhale SP. Practical Pharmacognosy, analysis and Dr. U.A. Obisike and Dr. K.N. Nirali Prakashan, Pune. 2000;1:45-6.

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