Research Articles

2020  |  Vol: 5(1)  |  Issue: 1 (January-February) | https://doi.org/10.31024/apj.2020.5.1.2
Toxicity study of aqueous extract of Acalypha wilkesiana Muell-Arg (Jacob’s coat, Copper leaf) leaves on some haematological parameters in mice

H. A. Madziga1*, M. Chiroma1, J. G. Usman2, N. Daniel3, I. J. Omeh1

1Department of Veterinary Physiology and Biochemistry, University of Maiduguri, Borno State, Nigeria

2Drug Development Division National Veterinary Research Institute, Vom, Plateau State, Nigeria

3Department of Veterinary Pharmacology and Toxicology, University of Maiduguri, Borno State, Nigeria

*Address for Corresponding Author

Hannah A. Madziga

Department of Veterinary Physiology and Biochemistry, University of Maiduguri, Borno State, Nigeria

 

Abstract

Objective: of present study was to evaluate the efficacy and safety of Acalypha wilkesiana on some haematological parameters in mice. Materials and Methods: The oral acute toxicity (LD50) was studied using the method of Kaber as modified by Aliyu and Nwude. The effects of the aqueous extract of Acalypha wilkesiana leaves on some haematological parameters in mice were studied. Results: The oral LD50 with 95% confidence interval of the aqueous leaf extract was estimated to be 1420mg/kg, indicating that the extract is not toxic. After prolonged oral treatment of mice with the leaf extract, significant increases in the levels of mean Hb concentration, RBC, and PCV were observed. Conclusion: This study suggests that the aqueous leaf extract of Acalypha wilkesiana contains some important chemical components which may have therapeutic values and have some beneficial effects on haematological parameters, suggesting that it could be used as an anti anaemic.

KeywordsAcalypha wilkesiana, acute toxicity, hematological parameter


Introduction                                                        

Acalypha wilkesiana belongs to the family Euphorbiacaece. The common names for this plant include, copper leaf, fire dragon, beef steak plant, Jacobs coat (Riley, 1963), and locally called eda or jiwanen. The plant is found all over the world, especially in tropic of Africa, America and Asia (Ogundaini, 2005). Traditional medicine has been practiced to some degree in all cultures. The traditional medical systems are based on experiments in using plant products in eradication of common ailments (Gupta, 1994). Thus, the World health Organization (WHO) has encouraged research on herbal medicines with a view to developing and incorporating the effective locally available substitutes/herbs remedies into the health care system (Abdulrahman et al. 2005). However, in traditional medicine, there is no proper dosage or quantity of the material given to recipients and the nature and type of active ingredient in the mixture may not be known. In such practices, the probability of administering toxic doses is high (Musa et al., 2005). Acute toxicity study is important since science requires the validation of drugs by medical practitioners and drug regulatory authorities demand that all potential drugs should pass through a rigorous series of study and scrutiny (Iwu, 1996; Abdulrahman, 2004). Recently some researchers confirmed some of the Folk use (Ogundaini 2005; Adesina et al., 2000) however there is not much study on this plant in the area acute toxicity and hematological parameters. Therefore, this study was design to conduct the acute toxicity studies of the leaf of the plant and to determine the effects of the extract on some haematological parameter in mice.

Materials and Methods

Plant collection and identification

Fresh leaves and stem of Acalypha wilkesiana were collected in the month of June from University of Maiduguri staff quarters. The plant was identified and authenticated by a botanist from the Department of Forestry, University of Makurdi, Benue state.

Extract preparation 

The fresh leaves were air-dried at room temperature (37oC) for two weeks. The dried leaves were then pulverized using a wooden mortar and pestle and then ground into powder form. Two hundred gram (200g) of the powdered form was poured into a round bottom flask and 1 liter (1000mls) of distilled water added after which the mixture was placed on a hot plate and allowed to boil for 1 hr. It was then allowed to cool and then filtered using Whatman filter paper No. 1, which was then evaporated to dryness using oven at 500C and was later stored at 40C until used. The concentration of the extract was 100 mg/ml and the yield was 22.85%W/W.

Experimental animals

A total of 105 adult mice were used for the experiments. They were obtained from the animal house of the Institute for Trypanosomiasis Research, Vom, Plateau State, Nigeria. They were housed in clean plastic cages in Veterinary Physiology, Pharmacology and Biochemistry Laboratory of the University of Maiduguri for two weeks for acclimatization, before the commencement of the experiments. They were fed with growers’ mash (Vital feedsR Nig. Ltd. Jos) and clean water ad libitum. All experiments on animals were carried out according to the biomedical principles involving animals (CIOMS and ICLAS, 2012).

Acute toxicity study

The arithmetic method of Karber (1931) as modified by Aliu and Nwude (1982) was used to evaluate the oral acute toxicity of the aqueous leaves extract of Acalypha wilkesiana.

In this method, twenty five (25) adult mice were selected at random and separated into 5 groups of 5 mice each. Groups were designated A, B, C, D and E. Group A, animals which served as the control were given distilled water while the other four groups (B, C, D and E) were given the aqueous leaf extract of Acalypha wilkesiana at different dosages 800, 1000, 12000 and 1600 mg/kg respectively and observed for clinical signs and death over a period of 24 hours.

Sub-acute toxicity studies

Eighty (80) adult female mice were selected and divided into four groups of 20 mice each. Group A was the control group. Groups B, C and D were treated orally with 100 mg/kg, 1200 mg/kg and 1600 mg/kg of aqueous extract of Acalypha wilkesiana, respectively for a period of 3 weeks (21days) while group A, (control) received distilled water only for the same period of time.

Haematological analysis

The estimates of the various haematological parameters were carried out on weekly basis for 3 weeks after the extract administration. At the end of each week, blood samples from 5 mice in each group were collected from the tail vein for determination of haematological parameters such as red blood cell counts (RBC), packed cell volume (PCV), mean haemoglobin concentrations (Hb) by haemocytometry, using standard procedures (Cole, 1974: Schalm et al., 1976; Brown, 1976; Dade and Lewis, 1984).

Statistical analysis

All values were expressed as Mean ± Standard Deviation (S.D). Analysis of variance (ANOVA) was used to analyze the extent of variations among groups and P values equal to or less than 0.05(P≤0.05) were considered significant. The computer statistical software GraphPadInstat (2003) was used to analyze the data.

Results

Acute toxicity study

The oral LD50 with 95% confidence limit was 1,420mg/kg. From the pilot study of the acute toxicity study done, 100 mg/ml stock concentration of the aqueous extract did not cause death.

Table 1. Calculation of LD50 using the Arithmetic method (Aliu and Nwude, 1982)

S.No.

Dosage

Dose Difference

Dead

Mean Dead

Mean dead X dose difference

1

800

 

0

 

 

 

 

200

 

0.5

            100

2

1000

 

1

 

 

 

 

200

 

1

            200

3

1200

 

1

 

 

 

 

400

 

1.5

            600

4

1600

 

2

 

 

LD50 = 1420mg/kg

 

 

Total 900


LD50= 1600-180

Effect of the extract on haematological parameters

The effect of prolonged oral administration of aqueous extract of Acalypha wilkesiana leaves on Red Blood Cell (RBC), Packed Cell Volume (PCV) and Haemoglobin (Hb) count of treated mice are presented in table 2.

At day 7 treatment with 800mg/kg, RBC values significantly (p<.0.05) increased from 745.20±34.40 to 843.20±68.90,similarly at 100mg/kg the RBC values increases to 951.60±41.60 as well as at dose 1200mg/kg with values of 977.20±27.80. Day 14 treatment at 800mg/kg indicated a significant (p<.0.05) increase from 691.40±26.30 to 1096.00±59.50, also at 100mg/kg and 1200mg/kg the RBC values increased to 1072.60±75.90and 1105.40±60.20 respectively when compared to 691.40±26.30which is the control. Day 21 treatment, RBC values also increased significantly (p<.0.05) from 643.20±36.40 to 940.40±46.80 at dose of 800mg/kg, 917.00±28.64 at dose 1000mg/kg and also 886.00±57.81at dose 1200mg/kg respectively. While day 7 and 14 of extract withdrawal, RBC values returned to non-significant (p>0.05) values when compared to control.

PCV values at day 7 of extract treatment with 800mg/kg indicated a significant (p<.0.05) increase, from 45.20 ±1.30 to 51.80 ±3.49,similarly at 1000mg/kg the values increased to 53.40 ±2.07 and also at 1200mg/kg PCV values increased to 53.00±2.55 when compared to the control. 

Day 14 treatment at 800mg/kg, 1000mg/kg and 1200mg/kg indicated a significant (p<.0.05) increase from 47.40 ±1.67 to 54.60 ±1.67, 53.60 ±1.67 and 53.00±3.16 respectively, so also day 21 treatment at 800mg/kg, 1000mg/kg and 1200mg/kg indicated a significant (p<.0.05) increase from 47.20 ±1.48 to 55.40 ±5.08,54.60 ±3.44 and 58.40±1.14 respectively.

Following withdrawal of extract at day 7 and 14, the PCV values returned to normal when compared to control.

Hb values at day 7 of extract treatment with 800mg/kg, 1000mg/kg and 1200mg/kg significant (p<.0.05) increased to 11.90 ±1.65, 11.70 ±1.44and 11.80 ±1.31, respectively when compared to the control at 9.90 ±0.36.Similarly at day 14 with the same dose of extract Hb values indicated a significant (p<.0.05) increase from 12.90 ±0.82 to 17.50 ±0.76, 18.10 ±0.37 and 18.10 ±0.40 respectively. Also day 21 treatment at 800mg/kg, 1000mg/kg and 1200mg/kg indicated a significant (p<.0.05) increase of 14.94 ±0.46, 14.32 ±0.33 and 14.32 ±0.33 respectively when compared to control 10.74 ±0.29.

Following extract withdrawal, the Hb values returned to non-significant (p>0.05) values at days 7 and 14.

Table 2. Effect of prolonged oral administration of aqueous extract of Acalypha wilkesiana leaves on Red Blood Cells, Haemoglobin Concentration and Packed Cell Volume in mice

Parameters

Extract treatment

Treatment Days

Days after withdrawal of treatment

7

14

 21

    7

21

Red Blood Cell (RBC) x106 mm3

Control (Distilled water)

745.20±34.40 a

691.40±26.30a

643.20±36.40a

637.60±41.60 a

634.60±15.40a

 

800 (mg/kg)

843.20±68.90b

1096.00±59.50b

940.40±46.80b

718.80±63.60a

671.60±24.90a

 

1000 (mg/kg)

951.60±41.60b

1072.60±75.90b

917.00±28.64b

675.40±53.90a

730.20±23.40a

 

1200 (mg/kg)

977.20±27.80b

1105.40±60.20b

886.00±57.81b

716.00±65.43a

874.00±74.80a

Packed Cell Volume. %

Control (Distilled water)

45.20 ±1.30a

47.40 ±1.67a

47.20 ±1.48a

45.40 ±0.55a

45.40 ±0.55a

 

800 (mg/kg)

51.80 ±3.49b

54.60 ±1.67b

55.40 ±5.08b

45.60 ±1.67a

44.60 ±0.55a

 

1000 (mg/kg)

53.40 ±2.07b

53.60 ±1.67b

54.60 ±3.44b

45.40 ±0.55a

45.80 ±0.84a

 

1200 (mg/kg)

53.00±2.55b

53.00±3.16b

58.40±1.14b

46.20±0.45a

45.20±0.45a

Haemoglobin g/dl.(Hb)

Control (Distilled water)

9.90 ±0.36a

12.90 ±0.82a

10.74 ±0.29a

10.80 ±0.31a

10.80 ±0.31a

 

800 (mg/kg)

11.90 ±1.65a

17.50 ±0.76b

14.94 ±0.46b

11.78 ±0.94a

11.06 ±0.26a

 

1000 (mg/kg)

11.70 ±1.44a

18.10 ±0.37b

14.32 ±0.33b

11.16 ±0.22a

11.28 ±0.33a

 

1200 (mg/kg)

11.80 ±1.31a

18.10 ±0.40b

14.92 ±0.38b

11.26 ±0.51a

11.42 ±0.23a

a, b,means with different superscript are significantly (P<0.05) different  (n=5)

Discussion

Previous studies on Phytochemical analysis of the aqueous leaf extract of Acalypha wilkesiana revealed high presence of carbohydrates, tannins and Flavonoid, and moderate, presence of phlobatannins, Alkaloid, cardiac glycosides, Terpene and steroids. Anthraquinone derivatives were not detected, while the elemental analysis showed the presence Cl-, Na+, K+, Ca2+, Fe2+, Mg2+, Zn2+, Cu2+ and Mn+ in varying quantities. Cd2+and Pb2+ were not found (Madziga et al., 2010).

The aqueous Leaves extract of Acalypha wilkesiana administered at the various doses appear to have some beneficial effects on the hematological indices given the observed increases in the values of RBC, Hb and PCV.   

The improvement of haematological parameters may be an indication of the possible usefulness of this extract as a haematinic and blood enhancer. According to (Brown, 1976, Jour, 2008), anti anaemic agents tend to stimulate production of RBC and improve the values of Hb and PCV. Presence of Carbohydrate may have contributed to its immune effect since carbohydrate is known to have numerous roles in living things, such as the storage and transport of energy and structural components (cellulose in plants, chitin in animals).  In addition carbohydrates and their derivatives play major roles in the working process of the immune system, fertilization pathogenesis, blood clotting and development (Maton et al, 1993., Patil and Muskan, 2009). Furthermore the Acalypha wilkesiana contained moderately high iron content; the presence of iron therefore could have contributed to the observed improvement of haematoglogical parameters. The increase in the haematological parameters in this study may also be due to the activities of the chemical constituents of the plant; saponins, as found in the extract, are known to hydrolyze and produce sapogenins which may be steroid or triterpene. Shapiro and Greenfield (1987) reported the stimulatory effects of steroid on bone marrow resulting in increased erythropoietin.

Conclusion

The aqueous leaf extract of Acalypha wilkesiana has oral LD50 1,420mg indicating that is not toxic. The leaf of Acalypha wilkesiana contains some important organic and chemical components which are pharmacologically active and may be beneficial and have therapeutic values, therefore the aqueous extract of this plant could be of immense medicinal value. The aqueous leaf extract of Acalypha wilkesiana increased haematological parameters, thus it appears to have some beneficial effect on haematological parameters of mice (RBC. Hb and PCV) suggesting that it could be used as an anti anaemic.

Acknowledgement

The authors gratefully acknowledge the technical assistance of Bitrus Wampana of Veterinary Physiology and Biochemistry, Isa of Veterinary Microbiology all of Faculty of Veterinary Medicine, University of Maiduguri and Fine Akawo of Chemistry Department.

References

Abdulrahman EI, Onyeyilli PA, Sanni S. 2005. Phytochemical screening and effect of aqueous root–bark extract of Terminalia avicennoides (Guill and pers) on haematological parameters in rats. Proc. Of the 28th Annual International conference of chemical society of Nigeria, Maiduguri, Nigeria pp 204-208.

Abdulrahman FI. 2004. Studies in the chemical contents and pharmacological activities of the root–bark extract of Vitex doniana (Black plum).Unpunished Ph.D Thesis, University of Maiduguri, Nigeria, pp.66

Adesina SK, Idowu O, Ogundaini AO, Oladimeyi H, Olugbade TA, Onawunmi GO, Pais M. 2000. Antimicrobial constituents of the leaves of Acalypha wilkesiana and Acalypha hispida. Phytotherapy Research Veterinary Science, (14): 371 – 374.

Aliu YO, Nwude N. 1982. Veterinary Pharmacology and Toxicology Experiments, 1st edition. Baraka Press and Publishers, Zaria, Nigeria, pp. 43-45, 104-109.

Brown BA. l976. Haematology, Principles and Procedures, 2nd ed. Lea and Fibiger, Philadelphia, USA. Pp.56-81.

CIOMS and ICLAS. 2012. Council for International Organisations of Medical Sciences and the International Council for Laboratory Animal Science. International Guiding Principles for Biomedical Research Involving Animal, December 2012.

Cole EH. 1974. Veterinary clinical Pathology, 2nd ed. W.B. Saunders C, Philadelphia, USA. pp. 110-116.

Dacie JV, Lewis SM. 1984. Practical Haemoatology, 6th ed. Churchill Livingstone, Edinburgh, UK. pp. 24-36.

GraphPad Instat 2003. GraphPad Instat version 3.2 for windows 95, graphpad software, San Diego, California, U.S.A. www.graphpad.com.

Gupta SS. 1994. Prospects and Prospective of Natural Plant products. Indian Journal of Pharmacology, 26:1-21.

Jour AD. Vovor GM, Eklu-G A, Aklikokou K, Agbonon K, Abena A, Souza A, Koffi C, Akpagana. 2008. Effect of Tectona grandis on phenylhydrazine-induced anaemia in rats. Fitoterapia 76:332.

Karber G. 1931. Beitrag zur kollaktiven Behandlung pharmnakohogischer Reihenversuche. Archiv Experimentelle  Pathologie, und Pharmakologie, 162: 480-487.

Madziga HA, Sanni S, Sandabe UK. 2010. Phytochemical and elemental analysis of Acalypha wilkesiana leaf. Journal of American Science, 6 (11):510-514.

Maton A, Jean CWM, Susan J, Maryanna QW, David L, Jill DW. 1993. Human Biology and Health. Englewood Cliffs, New Jersey, USA: Practice Hall, pp. 52-59.

Musa KY, Abdulrahman EM, Shok M, Argwa A, Musa H. 2005. Acute toxicity studies of ethanolic extract of dyschaiisite perrottetii NES (Acanthaceae) in Mice Nig. Journal of Pharmacology Research, 4(1):28-23.

Ogundainio AO. 2005. From Green into Medicine: Taking a lead from Nature. An inaugural lecture delivered at Oduduwa Hall, Obafemi Awolowo University, Ile Ife, Nkigeria, pp. 12 – 15.

Patil UK, Muskan K. 2009. Essential. ‎Biotechnology. books.google.com.ng  books pp, 66.

Riley HP. 1963. Families of flowering plants of southern Africa; University of Kenturky Press, USA, pp. 73.

Schalm OW, Jam NC, Carol EJ. 1976. Veterinary Haematologv. 3rd ed. Lea and Ferbinger. Philadelphia. USA. pp. 20-28.

Shapiro MF, Greenfield S. 1987. The complete blood count and leucocyte differential count. An approach to their application. Annals of Internal Medicine, 106: 65-74.

Manuscript Management System
Submit Article Subscribe Most Popular Articles Join as Reviewer Email Alerts Open Access