J Complement Integr Med. 2015; 12(1): 53–56
Johnny Olufemi Olukunle*, Eniope Bamidele Jacobs, Olusola Lawrence Ajayi, Khalid Talha Biobaku and Mathew Oluwole Abatan
Toxicological evaluation of the aqueous extract of Acalypha wilkesiana in Wistar albino rats Abstract Background: Acalypha wilkesiana (Euphorbiaceae) is highly accepted for traditional treatment of human plasmodiasis in Africa. Methods: The toxicological effects of the aqueous leaf extract of A. wilkesiana were studied in 45 male and female Wistar albino rats. An acute toxicity testing was done using 21 rats divided into seven groups and LD50 determined. In the sub-chronic toxicity study, the extract was administered orally over a period of 28 days to rats in three groups with doses of 400 mg kg−1, 800 mg kg−1 and 1,600 mg kg−1, respectively, and the fourth group administered with water served as control. Blood samples were collected for hematological and serum biochemical analysis; organs of the animals were harvested for histopathological examination. Results: The acute toxicity testing showed that the extract was non-toxic at doses up to 3,000 mg kg−1 and the LD50 was calculated to be 2,828.34 mg kg−1. The study showed that at 1,600 mg kg−1 dose, the extract caused a decrease in the level of neutrophils (NEUT) while lymphocytes (LYMP) were statistically significantly increased. The administration of the extract also resulted in varying significant dose dependent increase in the levels of aspartate amino transferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). There were also significant increases in the level of total protein (TP), urea (URN) and albumin (GLB) especially at 1,600 mg kg−1 dosage. Histopathology showed that the extract caused mild to severe significant lesions that
are dose dependent in the liver and kidney when compared with the control group. Conclusions: Prolonged administration of high dose of A. wilkesiana extract has tendency to cause organ toxicity. Keywords: Acalypha wilkesiana, hematology, histopathology, serum biochemistry, toxicity DOI 10.1515/jcim-2013-0066 Received December 9, 2013; accepted October 7, 2014; previously published online November 8, 2014
Introduction In this study, close attention is paid to Acalypha wilkesiana (Euphorbiaceae) as it has been reputed for traditional treatment of human plasmodiasis in Africa [1] and listed by various author as an antibiotic [2], antifungal [3], antihypertensive [4], hemolytic agent [2] and as antitumor agent [4], and all these pharmacologic activities have been attributed to the leave of the plant; hence, this study is undertaking to assess the safety or otherwise of the plant extract in rats using hematology, serum biochemistry and histopathology as indices for toxicities.
Materials and methods Plant material
*Corresponding author: Johnny Olufemi Olukunle, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Federal University of Agriculture, PMB 2240, Abeokuta, Ogun State, Nigeria, E-mail: [email protected] Eniope Bamidele Jacobs, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria Olusola Lawrence Ajayi, Department of Veterinary Pathology, College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria Khalid Talha Biobaku, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Federal University of Agriculture, PMB 2240, Abeokuta, Ogun State, Nigeria Mathew Oluwole Abatan, Department of Veterinary Physiology Biochemistry and Pharmacology, Faculty of Veterinary Medicine, University of Ibadan, Oyo State, Nigeria
The fresh leaves of A. wilkesiana were obtained from a farm in Odeda, Abeokuta, the plant was authenticated and the leaves were air-dried, pulverized, finely sieved and the extract prepared as described by Olukunle et al. [5]. The dry extract was stored in a refrigerator at −4 °C until needed for experiments.
Experimental animals Forty-five, sexually matured, Wistar albino rats of both sexes weighing between 180 and 240 g were housed in the Experimental Animal Unit of the College of Veterinary Medicine, University of Agriculture, Abeokuta, Ogun State, Nigeria, in well-ventilated metal cages. They were allowed a 2-week acclimatization period prior to the study, fed standard ration (Vital feed Limited, Ibadan, Nigeria) and clean water ad-libitum. A period of 12 h light and 12 h darkness was maintained. The equipment,
Brought to you by | Karolinska Institute Authenticated Download Date | 5/25/15 10:22 AM
54
Olukunle et al.: Toxicological effect of Acalypha wilkesiana
handling and sacrificing of the animals were in accordance with the Animal Ethics Committee approval of the College of Veterinary Medicine, Federal University of Agriculture, Abeokuta.
Experimental procedure and design Acute toxicity testing and determination of median lethal dose (LD50): Twenty-one rats were used for the acute toxicity testing employing the method described by Lorke [6]. The rats were divided into seven groups of three animals each and six doses of 500 mg kg−1, 1,000 mg kg−1, 2,000 mg kg−1, 3,000 mg kg−1, 4,000 mg kg−1 and 5,000 mg kg−1, therefore a 20 % extract solution was administered slowly into groups I (1 mL), II (2 mL), III (4 mL), IV (6 mL), V (8 mL), VI (10 mL) respectively and group VII rats administered with 10 mL/kg distilled water served as control. The rats in the six groups were thereafter monitored for symptoms of acute toxicity and mortality. The median lethal dose (LD50) was calculated as the square root (√) of the product of the lowest lethal dose and the highest non-lethal dose. pffiffiffi pffiffiffi LD50 ¼ n L
symptoms of acute toxicity like lethargy, restlessness, incoordination, respiratory distress and weakness were observed in the group of rats dosed 3,000 mg kg−1 but mortality was only recorded in the group dosed 4,000 mg kg−1 when compared to the control group. The mortality recorded implies that the extract is toxic and lethal to rats at dose of 4,000 mg kg−1 and beyond. Thus the maximum safe dose of A. wilkesiana leaf extract is 2,000 mg kg−1 and the minimum lethal dose was 4,000 mg kg−1. LD50 ¼
pffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffi 2000 4000
LD50 ¼ 44:72 63:24 ¼ 2; 828:34 mg kg1 The median lethal dose of the aqueous leaf extract of A. wilkesiana is ¼ 2,828.34 mg kg−1.
where n ¼ highest non-lethal dose and L ¼ lowest lethal dose Sub-chronic toxicity testing: Twenty-four rats were randomly divided into four groups (I–IV). Aqueous extract of A. wilkesiana leaves at (400 mg kg−1, 800 mg kg−1 and 1,600 mg kg−1 body weight) was administered orally to groups A, B and C respectively for 28 days. The fourth group (Group D) received distilled water (10 mg kg−1) orally for 28 days and then euthanized. Prior to euthanasia, about 3 mL of blood was collected from the medial canthus of the right eye of each rat for hematology and serum chemistry. Thereafter, the rats were euthanized by placing them in a glass chamber containing cotton wool soaked in diethyl ether till they lost consciousness. Abdominal incision was then made and the kidneys, liver, heart and spleen of each rat were removed and stored in Bouin’s fluid. A 5 µm thickness of the tissues for histopathological evaluation was cut, processed, stained with H&E and read at 300 magnification. Hematological and serum biochemical examination: Hematology and serum biochemical studies were as described by Olukunle et al. [5].
Statistical analysis Results were expressed as mean standard error of mean and were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. Lesions observed in histopathology were scored as mild, moderate or severe. Results were considered statistically significant at 95% confidence interval (p < 0.05).
Results Acute toxicity testing of aqueous leaf extract of Acalypha wilkesiana in rat The groups of rats dosed 500–2,000 mg kg−1 (i.e. groups I–III) exhibited no signs of acute toxicity, whereas
Effect of aqueous leaf extract of Acalypha wilkesiana on the hematological parameters of Wistar rats A. wilkesiana at 400 and 800 mg kg−1 did not produce any statistically significant change (p < 0.05) in the hematological parameters of the rats but the group of rats administered with 1,600 mg kg−1 dose of A. wilkesiana exhibited a statistically significant (p < 0.05) reduction in neutrophil (29.40 1.9 103/mm3) while the lymphocytes value of (71.60 2.1 103/mm3) was significantly increased at (p < 0.05) when compared with the control (Table 1).
Effect of aqueous leaf extract of Acalypha wilkesiana on some selected serum biochemical parameters of Wistar rats The groups of rats dosed 400 and 800 mg kg−1 aqueous extract of A. wilkesiana produced statistically significant (p < 0.05) increase in the serum aspartate aminotransferase (AST) values of (76.07 5.1 U/L) and (84.96 9.7 U/L) respectively when compared to control group (Table 2). Whereas the group administered with 1,600 mg kg−1 dose of A. wilkesiana exhibited a statistically significant increase (p < 0.05) in the blood urea (UREA) level (48.80 2.7 U/L), total protein (TP) level (97.94 2.9 g/L), globulin (GLOB) (53.63 5.4 g/L), serum aspartate
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Olukunle et al.: Toxicological effect of Acalypha wilkesiana
Table 1
Effect of aqueous leaf extract of Acalypha wilkesiana on the hematology of rats.
Groups, n ¼ 6
Group Group Group Group
55
PCV, %
RBC count, 106/mm3
Hb, g/dL
A, 400 mg kg−1 28.4 1.6 28.4 1.5 B, 800 mg kg−1 C, 1,600 mg kg−1 25.2 1.0 D (Control) 27.0 1.5a
4.46 0.3 4.42 0.2 3.96 0.2 4.24 0.2
9.54 0.6 9.45 0.7 8.50 0.4 9.22 0.4
WBC count, 103/mm3
NEUT
LYMP
MONO
BASO
EOSINO
3.94 0.4 29.69 4.5 69.54 4.8 0.51 0.4 3.64 0.3 32.09 4.3 67.03 4.3 0.88 0.4 3.94 0.3 29.00 1.9a 71.00 2.1a 0.00 0.00 3.68 0.4 38.04 3.1 61.95 2.8 0.01 0.2
0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0
0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0
Values are mean standard error of mean. aSuperscripted items are statistically significant at p < 0.05. PCV, packed cell volume; Hb, hemoglobin concentration; RBC, red blood cell; WBC, white blood cell.
Table 2
Effect of aqueous leaf extracts of Acalypha wilkesiana on the serum biochemistry of Wistar rats.
Groups, n ¼ 6
Total protein,
Urea,
Creatinine,
g/L
mg/dL
mg/dL
Albumin, g/L
Globulin,
AST, U/L
ALP, U/L
ALT, U/L
g/L
Total bilirubin, mg/dL
Group A, 400 mg/kg
83.63 4.3
32.78 3.5
0.78 0.1
42.41 1.9
41.21 5.1
76.07 5.1a
45.12 8.3
118.98 7.6
0.75 0.2
Group B, 800 mg/kg
81.55 4.3
38.41 3.9
0.78 0.12
44.61 1.3
36.94 5.3
84.96 9.7a
50.19 4.6
126.40 5.1
0.69 0.3
Group C, 1,600 mg/kg
97.94 2.9a
48.80 2.7a
0.78 0.1
44.28 þ 4.1
53.63 5.4a
88.69 4.6a
57.96 4.9a
131.72 4.0a
1.16 0.2
Group D (Control)
83.50 4.9
36.23 3.8
0.86 010
40.97 1.7
42.14 5.7
37.27 4.3
47.92 9.5
124.00 6.4
1.08 0.3
Values are mean standard error of mean. aSuperscripted items are statistically significant at p < 0.05. AST, aspartate amino transferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase.
aminotransferase (AST) (88.69 4.6 U/L), alkaline phosphatase (ALP) level (57.96 4.9 U/L) and alanine aminotransferase (ALT) activity of (131.72 4.0 U/L) when compared with the values in the control group (Table 2).
Histopathology There were no significant lesions found in the organs of the groups of rats dosed 400 and 800 mg kg−1, but at the dose of 1,600 mg kg−1, the extract caused centrilobular degeneration and necrosis of the hepatocytes, mild tubular degeneration and necrosis of the kidney with evidence of regeneration. But there were no significant lesions found in the spleen and the heart of the rats at all the treatment doses.
Discussion and conclusions The oral acute toxicity study did not show toxicity symptoms at low doses of 400 and 800 mg kg−1 as compared to 1,600 mg kg−1 dose, Oladunmoye [2] reported a relatively close LD50 value of A. wilkesiana to be 2,197.72 mg kg−1 in mice, a value highly comparable to 2,828.34 mg kg−1 in rats as obtained in this study and also reported similar physical signs of acute toxicity.
In comparison with the administered 400 and 800 mg kg−1 doses, the high dose of 1,600 mg kg−1 elicited the observed significant increase in the serum enzymes AST, ALT, ALP of the liver, and TP and Urea of the kidney because it appears to be extra-pharmacological when administered over an extended duration of time. These increases in the biochemical parameters may have culminated in the observed pathology of the liver and the kidney as reported in this study [7, 8]. This may indicate that the extract of A. wilkesiana could be hepatotoxic at high doses when administered for a prolonged period of time. In conclusion, the extract of A. wilkesiana has been shown to be safe at doses of 400 and 800 mg kg−1 and may be organotoxic at doses higher than 800 mg kg−1 especially when administered for an extended period of time. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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56
Olukunle et al.: Toxicological effect of Acalypha wilkesiana
References 1. Udobang JA, Nwafor PA, Okokon JE. Analgesic and antimalarial activities of crude leaf extract and fractions of acalypha wilkesiana. J Ethnopharmacol 2010;127:373–8. 2. Oladunmoye MK. Comparative evaluation of antimicrobial activities and phytochemical screening of two varieties of acalypha wilkesiana. Trends Appl Sci Res 2006;1:538–41. 3. Akinde BE, Odeyemi OO. Extraction and microbiological evaluation of oils from the leaves of acalypha wilkesiana. Niger Med J 1987;38:147–58.
4. Kokko JP. Site and mechanism of action of diuretics. Am J Med 1984;77:11–17. 5. Olukunle JO, Ajayi OL, Adenubi OT, Okediran BS, Akinkuotu OA. Evaluation of the toxic effect of aqueous extract of the bark of morinda morindoides root in male Wistar rats. Niger J Anim Prod 2012;39:163–71. 6. Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol 1983;54:275–86. 7. Kaneko JJ, Cornelius CE. Clinical biochemistry of domestic animals. New York: Academic Press, 1980. 8. Edwards CRW, Bouchier IAD, Haslet C, Chilvers ER. Davidson’s principles and practice of medicine, 17th ed. Scotland: Churchill Livingstone, 1995:488–90.
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Johnny Olufemi Olukunle*, Eniope Bamidele Jacobs, Olusola Lawrence Ajayi, Khalid Talha Biobaku and Mathew Oluwole Abatan
Toxicological evaluation of the aqueous extract of Acalypha wilkesiana in Wistar albino rats Abstract Background: Acalypha wilkesiana (Euphorbiaceae) is highly accepted for traditional treatment of human plasmodiasis in Africa. Methods: The toxicological effects of the aqueous leaf extract of A. wilkesiana were studied in 45 male and female Wistar albino rats. An acute toxicity testing was done using 21 rats divided into seven groups and LD50 determined. In the sub-chronic toxicity study, the extract was administered orally over a period of 28 days to rats in three groups with doses of 400 mg kg−1, 800 mg kg−1 and 1,600 mg kg−1, respectively, and the fourth group administered with water served as control. Blood samples were collected for hematological and serum biochemical analysis; organs of the animals were harvested for histopathological examination. Results: The acute toxicity testing showed that the extract was non-toxic at doses up to 3,000 mg kg−1 and the LD50 was calculated to be 2,828.34 mg kg−1. The study showed that at 1,600 mg kg−1 dose, the extract caused a decrease in the level of neutrophils (NEUT) while lymphocytes (LYMP) were statistically significantly increased. The administration of the extract also resulted in varying significant dose dependent increase in the levels of aspartate amino transferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). There were also significant increases in the level of total protein (TP), urea (URN) and albumin (GLB) especially at 1,600 mg kg−1 dosage. Histopathology showed that the extract caused mild to severe significant lesions that
are dose dependent in the liver and kidney when compared with the control group. Conclusions: Prolonged administration of high dose of A. wilkesiana extract has tendency to cause organ toxicity. Keywords: Acalypha wilkesiana, hematology, histopathology, serum biochemistry, toxicity DOI 10.1515/jcim-2013-0066 Received December 9, 2013; accepted October 7, 2014; previously published online November 8, 2014
Introduction In this study, close attention is paid to Acalypha wilkesiana (Euphorbiaceae) as it has been reputed for traditional treatment of human plasmodiasis in Africa [1] and listed by various author as an antibiotic [2], antifungal [3], antihypertensive [4], hemolytic agent [2] and as antitumor agent [4], and all these pharmacologic activities have been attributed to the leave of the plant; hence, this study is undertaking to assess the safety or otherwise of the plant extract in rats using hematology, serum biochemistry and histopathology as indices for toxicities.
Materials and methods Plant material
*Corresponding author: Johnny Olufemi Olukunle, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Federal University of Agriculture, PMB 2240, Abeokuta, Ogun State, Nigeria, E-mail: [email protected] Eniope Bamidele Jacobs, Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria Olusola Lawrence Ajayi, Department of Veterinary Pathology, College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria Khalid Talha Biobaku, Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Federal University of Agriculture, PMB 2240, Abeokuta, Ogun State, Nigeria Mathew Oluwole Abatan, Department of Veterinary Physiology Biochemistry and Pharmacology, Faculty of Veterinary Medicine, University of Ibadan, Oyo State, Nigeria
The fresh leaves of A. wilkesiana were obtained from a farm in Odeda, Abeokuta, the plant was authenticated and the leaves were air-dried, pulverized, finely sieved and the extract prepared as described by Olukunle et al. [5]. The dry extract was stored in a refrigerator at −4 °C until needed for experiments.
Experimental animals Forty-five, sexually matured, Wistar albino rats of both sexes weighing between 180 and 240 g were housed in the Experimental Animal Unit of the College of Veterinary Medicine, University of Agriculture, Abeokuta, Ogun State, Nigeria, in well-ventilated metal cages. They were allowed a 2-week acclimatization period prior to the study, fed standard ration (Vital feed Limited, Ibadan, Nigeria) and clean water ad-libitum. A period of 12 h light and 12 h darkness was maintained. The equipment,
Brought to you by | Karolinska Institute Authenticated Download Date | 5/25/15 10:22 AM
54
Olukunle et al.: Toxicological effect of Acalypha wilkesiana
handling and sacrificing of the animals were in accordance with the Animal Ethics Committee approval of the College of Veterinary Medicine, Federal University of Agriculture, Abeokuta.
Experimental procedure and design Acute toxicity testing and determination of median lethal dose (LD50): Twenty-one rats were used for the acute toxicity testing employing the method described by Lorke [6]. The rats were divided into seven groups of three animals each and six doses of 500 mg kg−1, 1,000 mg kg−1, 2,000 mg kg−1, 3,000 mg kg−1, 4,000 mg kg−1 and 5,000 mg kg−1, therefore a 20 % extract solution was administered slowly into groups I (1 mL), II (2 mL), III (4 mL), IV (6 mL), V (8 mL), VI (10 mL) respectively and group VII rats administered with 10 mL/kg distilled water served as control. The rats in the six groups were thereafter monitored for symptoms of acute toxicity and mortality. The median lethal dose (LD50) was calculated as the square root (√) of the product of the lowest lethal dose and the highest non-lethal dose. pffiffiffi pffiffiffi LD50 ¼ n L
symptoms of acute toxicity like lethargy, restlessness, incoordination, respiratory distress and weakness were observed in the group of rats dosed 3,000 mg kg−1 but mortality was only recorded in the group dosed 4,000 mg kg−1 when compared to the control group. The mortality recorded implies that the extract is toxic and lethal to rats at dose of 4,000 mg kg−1 and beyond. Thus the maximum safe dose of A. wilkesiana leaf extract is 2,000 mg kg−1 and the minimum lethal dose was 4,000 mg kg−1. LD50 ¼
pffiffiffiffiffiffiffiffiffiffiffi pffiffiffiffiffiffiffiffiffiffiffi 2000 4000
LD50 ¼ 44:72 63:24 ¼ 2; 828:34 mg kg1 The median lethal dose of the aqueous leaf extract of A. wilkesiana is ¼ 2,828.34 mg kg−1.
where n ¼ highest non-lethal dose and L ¼ lowest lethal dose Sub-chronic toxicity testing: Twenty-four rats were randomly divided into four groups (I–IV). Aqueous extract of A. wilkesiana leaves at (400 mg kg−1, 800 mg kg−1 and 1,600 mg kg−1 body weight) was administered orally to groups A, B and C respectively for 28 days. The fourth group (Group D) received distilled water (10 mg kg−1) orally for 28 days and then euthanized. Prior to euthanasia, about 3 mL of blood was collected from the medial canthus of the right eye of each rat for hematology and serum chemistry. Thereafter, the rats were euthanized by placing them in a glass chamber containing cotton wool soaked in diethyl ether till they lost consciousness. Abdominal incision was then made and the kidneys, liver, heart and spleen of each rat were removed and stored in Bouin’s fluid. A 5 µm thickness of the tissues for histopathological evaluation was cut, processed, stained with H&E and read at 300 magnification. Hematological and serum biochemical examination: Hematology and serum biochemical studies were as described by Olukunle et al. [5].
Statistical analysis Results were expressed as mean standard error of mean and were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. Lesions observed in histopathology were scored as mild, moderate or severe. Results were considered statistically significant at 95% confidence interval (p < 0.05).
Results Acute toxicity testing of aqueous leaf extract of Acalypha wilkesiana in rat The groups of rats dosed 500–2,000 mg kg−1 (i.e. groups I–III) exhibited no signs of acute toxicity, whereas
Effect of aqueous leaf extract of Acalypha wilkesiana on the hematological parameters of Wistar rats A. wilkesiana at 400 and 800 mg kg−1 did not produce any statistically significant change (p < 0.05) in the hematological parameters of the rats but the group of rats administered with 1,600 mg kg−1 dose of A. wilkesiana exhibited a statistically significant (p < 0.05) reduction in neutrophil (29.40 1.9 103/mm3) while the lymphocytes value of (71.60 2.1 103/mm3) was significantly increased at (p < 0.05) when compared with the control (Table 1).
Effect of aqueous leaf extract of Acalypha wilkesiana on some selected serum biochemical parameters of Wistar rats The groups of rats dosed 400 and 800 mg kg−1 aqueous extract of A. wilkesiana produced statistically significant (p < 0.05) increase in the serum aspartate aminotransferase (AST) values of (76.07 5.1 U/L) and (84.96 9.7 U/L) respectively when compared to control group (Table 2). Whereas the group administered with 1,600 mg kg−1 dose of A. wilkesiana exhibited a statistically significant increase (p < 0.05) in the blood urea (UREA) level (48.80 2.7 U/L), total protein (TP) level (97.94 2.9 g/L), globulin (GLOB) (53.63 5.4 g/L), serum aspartate
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Olukunle et al.: Toxicological effect of Acalypha wilkesiana
Table 1
Effect of aqueous leaf extract of Acalypha wilkesiana on the hematology of rats.
Groups, n ¼ 6
Group Group Group Group
55
PCV, %
RBC count, 106/mm3
Hb, g/dL
A, 400 mg kg−1 28.4 1.6 28.4 1.5 B, 800 mg kg−1 C, 1,600 mg kg−1 25.2 1.0 D (Control) 27.0 1.5a
4.46 0.3 4.42 0.2 3.96 0.2 4.24 0.2
9.54 0.6 9.45 0.7 8.50 0.4 9.22 0.4
WBC count, 103/mm3
NEUT
LYMP
MONO
BASO
EOSINO
3.94 0.4 29.69 4.5 69.54 4.8 0.51 0.4 3.64 0.3 32.09 4.3 67.03 4.3 0.88 0.4 3.94 0.3 29.00 1.9a 71.00 2.1a 0.00 0.00 3.68 0.4 38.04 3.1 61.95 2.8 0.01 0.2
0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0
0.00 0.0 0.00 0.0 0.00 0.0 0.00 0.0
Values are mean standard error of mean. aSuperscripted items are statistically significant at p < 0.05. PCV, packed cell volume; Hb, hemoglobin concentration; RBC, red blood cell; WBC, white blood cell.
Table 2
Effect of aqueous leaf extracts of Acalypha wilkesiana on the serum biochemistry of Wistar rats.
Groups, n ¼ 6
Total protein,
Urea,
Creatinine,
g/L
mg/dL
mg/dL
Albumin, g/L
Globulin,
AST, U/L
ALP, U/L
ALT, U/L
g/L
Total bilirubin, mg/dL
Group A, 400 mg/kg
83.63 4.3
32.78 3.5
0.78 0.1
42.41 1.9
41.21 5.1
76.07 5.1a
45.12 8.3
118.98 7.6
0.75 0.2
Group B, 800 mg/kg
81.55 4.3
38.41 3.9
0.78 0.12
44.61 1.3
36.94 5.3
84.96 9.7a
50.19 4.6
126.40 5.1
0.69 0.3
Group C, 1,600 mg/kg
97.94 2.9a
48.80 2.7a
0.78 0.1
44.28 þ 4.1
53.63 5.4a
88.69 4.6a
57.96 4.9a
131.72 4.0a
1.16 0.2
Group D (Control)
83.50 4.9
36.23 3.8
0.86 010
40.97 1.7
42.14 5.7
37.27 4.3
47.92 9.5
124.00 6.4
1.08 0.3
Values are mean standard error of mean. aSuperscripted items are statistically significant at p < 0.05. AST, aspartate amino transferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase.
aminotransferase (AST) (88.69 4.6 U/L), alkaline phosphatase (ALP) level (57.96 4.9 U/L) and alanine aminotransferase (ALT) activity of (131.72 4.0 U/L) when compared with the values in the control group (Table 2).
Histopathology There were no significant lesions found in the organs of the groups of rats dosed 400 and 800 mg kg−1, but at the dose of 1,600 mg kg−1, the extract caused centrilobular degeneration and necrosis of the hepatocytes, mild tubular degeneration and necrosis of the kidney with evidence of regeneration. But there were no significant lesions found in the spleen and the heart of the rats at all the treatment doses.
Discussion and conclusions The oral acute toxicity study did not show toxicity symptoms at low doses of 400 and 800 mg kg−1 as compared to 1,600 mg kg−1 dose, Oladunmoye [2] reported a relatively close LD50 value of A. wilkesiana to be 2,197.72 mg kg−1 in mice, a value highly comparable to 2,828.34 mg kg−1 in rats as obtained in this study and also reported similar physical signs of acute toxicity.
In comparison with the administered 400 and 800 mg kg−1 doses, the high dose of 1,600 mg kg−1 elicited the observed significant increase in the serum enzymes AST, ALT, ALP of the liver, and TP and Urea of the kidney because it appears to be extra-pharmacological when administered over an extended duration of time. These increases in the biochemical parameters may have culminated in the observed pathology of the liver and the kidney as reported in this study [7, 8]. This may indicate that the extract of A. wilkesiana could be hepatotoxic at high doses when administered for a prolonged period of time. In conclusion, the extract of A. wilkesiana has been shown to be safe at doses of 400 and 800 mg kg−1 and may be organotoxic at doses higher than 800 mg kg−1 especially when administered for an extended period of time. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
Brought to you by | Karolinska Institute Authenticated Download Date | 5/25/15 10:22 AM
56
Olukunle et al.: Toxicological effect of Acalypha wilkesiana
References 1. Udobang JA, Nwafor PA, Okokon JE. Analgesic and antimalarial activities of crude leaf extract and fractions of acalypha wilkesiana. J Ethnopharmacol 2010;127:373–8. 2. Oladunmoye MK. Comparative evaluation of antimicrobial activities and phytochemical screening of two varieties of acalypha wilkesiana. Trends Appl Sci Res 2006;1:538–41. 3. Akinde BE, Odeyemi OO. Extraction and microbiological evaluation of oils from the leaves of acalypha wilkesiana. Niger Med J 1987;38:147–58.
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