Phytochemical Appraisal and Effects of Aqueous Extract of 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 ’is from the phylum Tracheophyta, class; Magnoliopsida,

order; , 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 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 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 formed was mixed with 3 drops of olive oil for each tube, which was again shaken vigorously for uniformity and the three tubes were observed for the formation of an emulsion. The concentration of the emulsion formed to show the presence of saponins was recorded with its severity as:

+ → Mildly present ++ → Moderately present +++ → Highly present

2.3.5 Phytochemical Quantitative Analysis of Alkaloids [12]

5g each of the aqueous, ethanolic and methanolic leave extracts of the sample was weighed and dispensed into three different 250ml beaker, to which 200mls of 10% acetic acetic in ethanol was added to each tube. The mixture was covered and allowed to stand for 4 hours after which the filterate that was filtered through a Whatman’s number 541 filter paper was concentrated on a water bath. To a one quarter of each of the leave extract sample of the original volume collected was the addition of concentrated ammonium hydroxide which was added in a drop wise volume to the filtrate which showed a complete precipitation process. The entire mixture of the solution was left on standing to settle while the precipitate formed was washed with ammonium hydroxide and again filtered. The residue on the filter paper was dried and weighed and calculated thus:

Weight of Alkaloid = Weight of filter paper + residue – Weight of empty filter paper Therefore, percentage yield of Alkaloid, = weight of filter paper + residue – weight of empty filter paper x 100 Weight of sample

2.3.6 Phytochemical Quantitative Analysis of Flavonoids [13] 10g each of the three leave extracts was extracted repeatedly with 100ml of 80% aqueous methanol at room temperature. The complete portion of this mixture was filtered through a Whatman’s number 42 filter paper. The filtrates obtained were then transferred into three crucibles and then subjected to a water bath for them to evaporate into dryness and further air dried in an air oven, cooled to room temperature in a desiccator and weighed in an analytical balance.

The calculation used to obtain the quantified flavonoid included: Weight of Flavonoids = Weight of Beaker x residue – weight of empty beaker % flavonoids = weight of flavonoid x 100 Weight of sample 1

2.3.7 Phytochemical Quantitative Analysis of Saponins

10g each of the three different leave extracts was weighed and transferred into three different 250ml conical flask. 20% in 100mls each of aqueous ethanol solution was added to the samples. The samples were subjected to heat on a water bath with series of stirring on a temperature maintained at 55oC for 4 hours. The mixtures were then filtered while the residues were re-extracted with 20% of a 200ml in portion of ethanol. The combined extracts were evaporated to 40ml over a temperature of 90oC over a water bath. The aqueous layer that was recovered during the process was kept while the ether layer was discarded. The recovered aqueous layer was purified with 60ml n-butanol. The combined n-butanol extracts were washed twice with 10ml of 55% aqueous solution of sodium chloride. The left-over solution was heated in a water bath and further left to air dry in an evaporator where its weight was obtained with the use of this formula:

Weight of Saponin = weight of flask x residue – weight of empty flask % Saponin = weight of saponin residue x 100 Weight of sample 1

2.3.8 Phytochemical Quantitative Analysis of Tannins [14]

0.1g each of the three leave extracts was weighed on a weighing scale and transferred into three 250ml conical flasks. 100ml of distilled water was added into the samples and boiled for 1 hour. The samples were allowed to cool at room temperature and diluted with 50ml of distilled water. 1ml each of the diluent was pipetted into three test tubes and 2 to 5mls of Folin-Denis’s reagent was added with 1ml of 17% sodium carbonate.

A blank test was prepared with 1ml distilled water and the reagents as earlier stated. The bluish colour formed in the test sample was read spectrophotometrically at 750nm wavelength using blank to calibrate the spectrophotometer.

0.1g of the tannic acid was dissolved into 100ml dissolved water to prepare the standard concentration to enable the dilutions of the working standards of choice to be plotted against the concentration.

A linear graph that passed through the margin was obtained. The concentrations of tannin in the three samples were extrapolated from the standard graph.

2.4 Experimental Design

2.4.1. Animal Grouping and Treatment Regimen.

A total of one hundred and twelve (112) adult albino rats were assigned by weight into eighteen (18) groups and allowed to acclimatize for (fourteen) 14 days (2 weeks). The duration of the study was fifteen (15) days acute and thirty (30) days sub-chronic study. Eight (8) albino rats each were assigned for the two (2) positive control groups and six (6) albino rats each were assigned to the other groups. The study groups comprised of two treatment phases each, (Pre-treatment and Post-treatment phases), duration of treatment (Acute and Sub-chronic) with six experimental groups in each of the phases. In the treatment phases, the albino rats are administered with AHEr extracts before acetaminophen induction, while in the post-treatment phases, the albino rats were treated with AHEr extract after acetaminophen induction.

The groups are as follows:

Group 1. Negative control (NC): Apparently healthy rats receiving de-ionized water and normal feed only.

Group 2. Positive control (PC): 500mg/kg b w. acetaminophen induced rats at 14th day in acute and 29th day in Sub-chronic study. Group 3. Extract Control (EC): Apparently healthy rats that received AHEr 100mg/kg b w. orally daily for fifteen (15) days and thirty (30) days. Group 4. Acetaminophen induced treatment group aqueous extract of Hypoestes rosea of 100 mg/kg b w. Group 5. Acetaminophen induced treatment group aqueous extract of Hypoestes rosea of 200 mg/kg b w. Group 6. Acetaminophen induced treatment group aqueous extract of Hypoestes rosea of 300 mg/kg b w.

2.5. Sample Collection and Analysis

Rats were anaesthetized using chloroform and were sacrificed on the 15th and the 30th days after an overnight fast. Blood samples were collected by puncture of the jugular vein and put into EDTA bottles for estimation of haematological parameters (PCV, HB, RBC, WBC and PLT) were assayed using an auto-analyzer (Sysmex KX-21n Auto-analyzer, Kobe, Japan). The laboratory analysis took place at the Research Laboratory of the departments of Biochemistry and Physiology, University of Port-Harcourt, Port-Harcourt, Nigeria.

2.6. Quality Control.

Quality control sera, standard operating procedures and good laboratory/ best practices were adhered.

2.7. Data Analysis

Data were analyzed using SPSS version 23, they were presented as Mean ± SEM. Variations between were determined using Analysis of variance (ANOVA) and Tukey Test of Multiple Comparison used to differentiate variations in means between groups. p-values less than 0.05 (P<.05) were considered statistically significant.

2. RESULTS AND DISCUSSION

The results of the qualitative semi-quantitative phytochemical analyses for fresh and dry Hypoestes rosea leaves are presented in Table 1. The qualitative results of the fresh leaf clearly showed that Hypoestes rosea yielded alkaloids; positive for Dragendorff’s, Mayer’s and Hager’s test, flavonoids; positive for Shinoda and lead acetate tests, tannins; positive for iron chloride test, triterpenoid/steroid; positive for Lieberman-Buchard and Salwaski test; fix oils; carbohydrates; positive for Molish and Fehling’s tests, cardenolide; positive for Keller Killani test. Phytochemical for dry leaf yielded alkaloids, carbohydrates, flavonoids, glycosides, tannins, terpenoid/steroid and saponin at different reference percentages.

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

The percentage in constituent in the quantitative phytochemical is presented in the Figure 1. The medicinal effects of Hypoestes rosea, like other medicinal plants are attributed to the presence of active bio-ingredients or phytochemicals in them which generally are responsible for disease prevention and promotion of health. [15]. Studies have reported it possesses anti – cancer, anti-inflammatory, anti-malaria and antioxidant effects. [3-5]. However, this present study reveals Hypoestes rosea has phytochemicals such as alkaloids, carbohydrates, flavonoids, glycosides, tannins, terpenoids/steroids, saponins and cardenolides (Table 1 and Fig. 1), which almost agrees with [16] earlier phytochemical study on Hypoestes rosea yielding positive for flavonoids, terpenes, sterols, balsam,

carbohydrates, monosaccharides reducing sugars, tannins and saponins while resins and glycosides were negative.

10.8

32.4 75.6

43.2 0

64.8 61.2

ALKALOIDS FLAVONOIDS 64.8 GLYCOSIDES TANINS TERPENOIDS/STEROIDS SAPONINS

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

Acetaminophen/ acetyl-para-aminophenol (APAP) is generally safe at recommended doses but since the drug is readily available without prescription, it is potentially more dangerous than other similar drugs when taken in excess or overdose [9] and may affect vital organs responsible for functions and routine homeostasis.

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 (x WBC (x Platelets (x Phase Group 10^12/L) 10^12/L) 10^9/L)

Mean ± Mean ± Mean ± Mean ± Mean ± SEM SEM SEM SEM SEM EC 44.33±1.33b 14.75±0.45b 5.70±0.20 5.82±0.38a 253.33±18.52 NC 41.83±1.87a 13.95±0.62a 5.30±0.18 8.85±0.57b 261.67±12.24 PC 39.50±1.23a 13.17±0.41a 5.18±0.23 10.08±0.27b 269.50±18.13 AEHr(100mg/kg) 44.50±1.06a 14.83±0.36b 5.50±0.13 6.67±0.55a 233.33±15.14 Pre-Treatment AEHr(200mg/kg) 46.67±1.26a 15.55±0.42b 5.13±0.16 7.73±0.49a 247.83±19.56 AEHr(300mg/kg) 49.17±1.08a 16.38±0.36b 5.75±0.39 7.45±0.61a 300.67±12.32 Test Statistics F-Ratio 6.589 6.572 1.303 9.691 2.001 P-value 0.0003*** 0.0003*** 0.2890ns <0.0001**** 0.7071ns EC 44.33±1.33 14.75±0.45 5.70±0.20 5.82±0.38a 253.33±18.52 NC 41.83±1.87 13.95±0.62 5.30±0.18 8.85±0.57b 261.67±12.24 PC 39.50±1.23 13.17±0.41 5.18±0.23 10.08±0.27b 269.50±18.13 Post-Treatment AEHr(100mg/kg) 40.17±1.01 13.38±0.34 4.95±0.13 6.92±0.47ac 257.67±16.68 AEHr(200mg/kg) 42.33±0.98 14.12±0.33 5.17±0.18 7.63±0.48ac 252.00±18.84 AEHr(300mg/kg) 44.67±0.88 14.88±0.29 5.87±0.28 7.50±0.50ac 317.17±6.87 Test Statistics F-Ratio 2.782 0.9149 2.898 10.85 2.426 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).

The acute and sub–chronic effects of various concentrations of aqueous extract of Hypoestes rosea on haematological parameters; haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC) white blood cell count (WBC) and platelets of acetaminophen induced toxicity in albino rats after 15 days exposure and 30 days exposure (Tables 2 and 3 respectively). The acute and sub-chronic study result shows that acetaminophen inducement at 500mg/kg.b. wt. slightly decreased PCV when compared with other experimental groups in the pre- treatment phase and post treatment phases. However, treatment with various concentrations of aqueous extract of Hypoestes rosea significantly increased the PCV to normal. P- value for PCV in the study was significant (P<.05).

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

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

The acute and sub-chronic study result shows that acetaminophen inducement at 500mg/kg.b. wt. causes increase in WBC when compared with other experimental groups in the pre- treatment phase and post treatment phases. However, treatment with various concentrations of aqueous extract of Hypoestes rosea significantly decreased WBC to normal. P- value for WBC in the study was significant (P<0.05).

The result shows that acetaminophen induction at 500mg/kg b. wt and various treatment concentrations with aqueous extract of Hypoestes rosea does not affect platelets and P- value is therefore, insignificant (P> 0.05).

The haematological parameters showed that treatment of rats with 500mg/kg b. wt. of acetaminophen caused significant decrease of PCV, Hb and RBC implicating the presence of anaemia. This anaemia may be attributed to destruction of RBCs due to increased oxidative damage and lipid peroxidation in cell membranes or to the effect of paracetamol on erythropoiesis by inhibiting erythropoietin release through direct effect on kidney [17].

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(x WBC(x Platelets(x Phase Group

10^12/L) 10^12/L) 10^9/L)

Mean ± Mean ± SEM Mean ± Mean ± Mean ± SEM SEM SEM SEM EC 44.67±1.12a 14.90±0.37a 6.02±0.27 6.07±0.35b 214.00±16.31 NC 42.83±0.60a 14.28±0.20a 6.00±0.27 7.62±0.57a 226.83±13.94 PC 41.83±1.08a 13.95±0.37b 5.48±0.16 9.50±0.34a 246.17±15.02 a a b Pre- AEHr(100mg/kg) 45.83±1.25 15.72±0.62 5.90±0.36 6.42±0.56 204.50±16.99 b a b Treatment AEHr(200mg/kg) 46.67±1.45 15.55±0.48 5.85±0.28 6.32±0.31 221.00±27.27 AEHr(300mg/kg) 49.17±0.79b 16.38±0.27a 6.10±0.31 6.53±0.43b 281.00±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 EC 44.67±1.12a 14.90±0.37bc 6.02±0.27 6.07±0.35 214.00±16.31 c NC 42.83±0.60a 14.28±0.20 c 6.00±0.27 7.62±0.57 226.83±13.94 c a cde bc PC 41.83±1.08 13.95±0.37 5.48±0.16 9.50±0.34 246.17±15.02 Post- b e ab Treatment AEHr(100mg/kg) 39.00±0.58 13.00±0.20 6.20±0.36 7.72±0.67 282.67±21.60 AEHr(200mg/kg) 39.83±0.70b 13.27±0.24 de 5.85±0.19 6.50±0.34 311.67±18.42a AEHr(300mg/kg) 42.50±0.62a 14.17±0.22 cd 6.05±0.28 8.13±0.38 304.83±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).

Also noticed in the haematological study were increase in WBC and slight increase in platelets even though its within normal limits. However, rats treated with aqueous extract of Hypoestes rosea in combination with acetaminophen either pre – treatment or post treatment showed parameters that were consistent with control values and normal ranges. These results further link that Hypoestes rosea may cause some protection against acetaminophen toxicity. This was similar to study of [18] and [19] who reported that Moringa has high iron content which enters in Hb synthesis and thus stimulating erythropoiesis.

4. 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.

ETHICAL APPROVAL

All authors hereby declare that Principles of laboratory animal care (NIH publication No. 85- 23, revised 1985) were followed, as well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee

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