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ORIGINAL ARTICLE
Year : 2014  |  Volume : 2  |  Issue : 1  |  Page : 37-41

Serum aminotransferase activities and bilirubin levels in salt loaded experimental rabbits treated with aqueous and ethanol extracts of Acalypha wilkesiana


1 Department of Biochemistry, College of Basic Medical Sciences, Igbinedion University, Okada, Edo State, Nigeria
2 Department of Biochemistry, Faculty of Natural Sciences, Ambrose Alli University, Ekpoma, Edo State, Nigeria

Date of Web Publication1-Jul-2014

Correspondence Address:
Omage Kingsley
Department of Biochemistry, College of Basic Medical Sciences, Igbinedion University, Okada, Edo State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2348-0149.135728

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  Abstract 

Background: Acalypha wilkesiana, commonly called Irish petticoat, is native to the South Pacific Islands and belongs to the family Euphorbiaceae. In traditional medicine practice, the leaves of A. wilkesiana are eaten as vegetables for the management of hypertension, being a diuretic plant. Aim: This study was conducted to evaluate the effects of extracts of A. wilkesiana leaves on activities of serum aminotransferases and bilirubin levels, which are indicators of liver function, in salt loaded rabbits. Materials and Methods: A total of 30 rabbits were randomly divided into five Groups (A to E) of six rabbits each and treated with salt loaded diet, aqueous extract (Group B) and ethanol extract (Group C) of A. wilkesiana leaves; continuous salt loading (Group A); salt loaded and nontreated (D); nonloaded (with salt) and nontreated (with extract) (E). Results: Salt loading resulted in a significantly (P < 0.05) higher serum alanine aminotransferases (ALT) and aspartate aminotransferases (AST), and a significantly (P < 0.05) lower serum direct bilirubin, when compared with the control. Treatment with A. wilkesiana leaf extracts, at a dose of 300 mg/kg body weight, resulted in a nonsignificantly (P > 0.05) lower serum total bilirubin and direct bilirubin, as compared with the control, in the salt loaded rabbits. Discussion and Conclusion: The use of A. wilkesiana leaf could be relatively safe considering its effects on serum ALT, AST, total and direct bilirubin of the experimental animals. Thus, it may be useful in the management of any possible deleterious effect of salt load to the liver.

Keywords: Acalypha wilkesiana , alanine aminotransferase, aspartate aminotransferase, bilirubin, salt load


How to cite this article:
Kingsley O, Marshall AA. Serum aminotransferase activities and bilirubin levels in salt loaded experimental rabbits treated with aqueous and ethanol extracts of Acalypha wilkesiana. Niger J Exp Clin Biosci 2014;2:37-41

How to cite this URL:
Kingsley O, Marshall AA. Serum aminotransferase activities and bilirubin levels in salt loaded experimental rabbits treated with aqueous and ethanol extracts of Acalypha wilkesiana. Niger J Exp Clin Biosci [serial online] 2014 [cited 2019 Dec 10];2:37-41. Available from: http://www.njecbonline.org/text.asp?2014/2/1/37/135728


  Introduction Top


There are considerable human and animal experimental studies implicating excessive dietary salt intake in cardiovascular diseases especially hypertension. [1],[2] Evidence from many sources suggests a possible relationship between excess salt ingestion and human hypertension. [3],[4],[5] Increase in blood pressure leads to damages to the kidney, heart, blood vessels, brain, and the eyes. Salt excess also had deleterious renal effects in spontaneously hypertensive rat (SHR) reflected by a massive proteinuria after 8 week of 8% salt loading. [6] It has been demonstrated that salt loading enhanced ventricular and renal fibrosis in normotensive and SHR rats. [6],[7] Clinical studies have also provided evidence that a high-salt diet was an independent determinant of renal injury. [8],[9],[10] Increase in blood pressure leads to damages to the kidney, heart, blood vessels, brain, and the eyes. These deleterious effects of excess salt can be managed with the use of medications or medicinal herbs.

Medicinal herbs are plants, which contain active principles that are effective for therapeutic purposes and can serve as precursors for the synthesis of drugs. [11] Acalypha wilkesiana, also known as Irish petticoat or Jacob's coat, is indigenous to the South Pacific Islands and belongs to the family Euphorbiaceae. The plant has been reported to contain sesquiterpenes, monoterpenes, triterpenoids and polyphenols. [12] The leaves reportedly contain saponins, tannins, anthraquinones and glycosides. [13] The plant has antimicrobial and antifungal properties and in traditional medicine, the leaves are eaten as vegetables for the management of hypertension, being a diuretic plant. [13] The plant has been shown to be a potential source of useful drugs. [14]


  Materials and methods Top


Plant Materials

Fresh A. wilkesiana leaves were obtained from local gardens within Benin City and authenticated at the Department of Plant Biology and Biotechnology, University of Benin, Benin City. The leaves were properly washed, air-dried and ground into fine powder.

Preparation of Ethanol Extract

Portion (100 g) of the powdered leaves was soaked in 400 ml of ethanol (95%) for 72 h (3 days), with occasional stirring using a magnetic stirrer to ensure proper mixture of the vessel content. The content was then filtered using a sintered funnel, (which is equivalent to four-fold of bandage or sheet of cheese cloth). The extract (filtrate) was then concentrated using rotary evaporator and weighed.

Preparation of Aqueous Extract

Portion (100 g) of the powdered leaves was soaked in 400 ml of distilled water for 72 h (3 days), and treated as described above.

Preparation of Salt-Loaded Diet (Feed)

The salt-loaded diet (8% NaCl) was prepared by mixing 8 g of analytical NaCl (from BDH Chemicals, England) with 92 g of the feed. The mixture was then fed to the experimental animals as described in the experimental design below.

Experimental Animals

Thirty adult rabbits of the New Zealand strain, weighing between 0.9 and 1.5 kg, purchased from local dealers at Aduwawa Cattle Market Benin City, were used for the study. The animals were kept in the animal house of the Department of Biochemistry, University of Benin and maintained on a 12-h light and dark cycle in clean disinfected cages. They were allowed free access to feed (standard pelletized growers feed from UAC - Vital Feed, Jos, Plateau State) and water throughout the duration of the experiment. The animals were treated according to the International guidelines for the care and use of laboratory animals and allowed to acclimatize to the new environment for a period of 3 weeks. They were then randomized into five groups (Groups A-E) of six rabbits each.

Experimental Design

Groups A-E rabbits were treated as follows;

Group A: Continuous salt-loading

Group B: Salt-loaded and treated with aqueous extract

Group C: Salt-loaded and treated with ethanol extract

Group D: Salt-loaded and nontreated

Group E: Nonloaded (with salt) and nontreated (with extract) (control).

The animals in Groups A-D were fed with the salt-loaded diet for a period of 70 days, after which Groups B and C were treated with aqueous and ethanol extracts, respectively, for a period of 7 days. Group A animals were given the salt-loaded diet continuously until the 77 th day, while Group E animals were neither given salt-loaded diets nor treated with the extract (i.e., it served as control). The extracts were administered orally at a dose of 300 mg/kg body weight.

Administration of Extracts

Five grams of the dry mass (concentrated extracts) were suspended in distilled water for administration to the experimental animals. The extracts (aqueous or ethanol) were administered orally at a dose of 300 mg/kg body weight for a period of 7 days.

Collection of Blood

After 70 days of salt loaded diets and prior to treatment (day 71) with the extracts, blood samples were collected from the veins located on the dorsal side of the ear lobes of the experimental animals (rabbits), using sterilized hypodermic needles. At day 78 after treatment with the extracts, blood samples were also collected. Samples were collected into fluoride oxalate and plane universal bottles immersed in ice. Immediately after collection of blood, the tubes were centrifuged at 3,500 rpm for 10 min to obtain clear plasma (fluoride oxalate) and serum (plane bottles) for further analysis.

Assay Methods

Alanine aminotransferase (ALT) was by the method of Reitman and Frankel [15] where ALT was measured by monitoring the concentration of pyruvate hydrazone formed with 2,4-dinitrophenyl hydrazine at 546 nm. Aspartate aminotransferase (AST) was also by the method of Reitman and Frankel [15] where AST was measured by monitoring the concentration of oxaloacetate formed with 2,4-dimtrophenyl hydrazine at 546 nm. Bilirubin was determined by the method of Jendrassik and Grof. [16] The principle involves the reaction of direct (conjugated) bilirubin with diazotized sulfanilic acid in alkaline medium to form a blue colored complex. Total bilrubin was determined in the presence of caffeine, which releases albumin bound bilrubin, by the reaction with diazotized sulfanilic acid and absorbance was read at 578 nm.

Statistical Analysis

Data are presented as mean ± standard error of the mean (SEM) (n = 6). Significance of difference was tested by Student's t-test, ANOVA and Turkey-Kramer test, using the GraphPad Instat Version 3 (GraphPad Software Inc. San Diego, California USA). Statistical Significance was set at P < 0.05.


  Results Top


The effects of oral administration of extracts (aqueous and ethanol) of A. wilkesiana leaves on some serum parameters in salt loaded experimental rabbits, are as described below.

After 70 days of salt loading, serum ALT activity were shown to be significantly (P < 0.05) higher in all the salt loaded groups, as compared with the nonloaded group (control). This trend was maintained 1 week after [Table 1]. At day 78, Group A showed a significantly (P < 0.05) higher values, Groups C and D showed nonsignificantly (P > 0.05) higher value, while Group B showed nonsignificantly (P > 0.05) lower ALT activity (U/L).
Table 1: Serum ALT (U/L) of salt-loaded rabbits; treated with extracts of Acalypha wilkesiana leaves

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Aspartate aminotransferase activity were also significantly (P < 0.05) higher in all the groups given salt load as compared with the control [Table 2]. But at day 78, only Group A (given continuous salt load), maintained the increase (P > 0.05) as compared with the control. Other Groups (B-D) showed nonsignificantly (P > 0.05) lower AST activity. Still at day 78, after administration of the extracts (aqueous or ethanol), there were significantly (P < 0.05) lower AST activity in the treated groups (day 78 compared with day 71), as compared with that of the untreated group. The ethanol extract was, however, shown to be more effective.
Table 2: Serum AST (U/L) of salt-loaded rabbits; treated with extracts of Acalypha wilkesiana leaves

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As shown in [Table 3], salt loading and/or treatment with aqueous or ethanol extract of A. wilkesiana leaves had no effect on levels of serum total bilirubin of the experimental animals.
Table 3: Serum total bilirubin (mg/dl) of salt-loaded rabbits; treated with extracts of Acalypha wilkesiana leaves

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Serum direct bilirubin levels of the animals given salt load showed slight differences when compared with the control, after 70 days of salt load [Table 4]. Group B showed significantly (P > 0.05) higher levels of direct bilirubin while Group C showed a significantly (P < 0.05) lower levels as compared with that of Group E. However, at day 78, after 1 week of treatment, there was no significant difference (P > 0.05) in the levels of direct bilirubin in all the groups.
Table 4: Serum direct bilirubin (mg/dl) of salt-loaded rabbits; treated with extracts of Acalypha wilkesiana leaves

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  Discussion and conclusion Top


Aminotransferases are found in many tissues throughout the body, including the liver, heart, muscles, kidney, and brain. If any of these organs or tissues is affected by disease or injury, AST is released into the bloodstream. Serum aminotransferase activities are sensitive indicators of parenchymal liver damage. Both ALT and ASTs have been implicated in infective hepatitis, infectious mononucleosis, and hepatocellular damage. Exposure to toxic substances such as carbon tetrachloride poisoning, therapeutic substances such as chloramphenicol, cephalosporins, and paracetamol overdose have also resulted in elevated AST and ALT activities.

After 70 days of salt loading, serum ALT activities were shown to be significantly (P < 0.05) higher in all the salt loaded groups, as compared with the nonloaded group (control). This trend was maintained 1 week after [Table 1]. At day 78, Group A showed a significantly (P < 0.05) higher values, Groups C and D showed nonsignificantly (P > 0.05) higher values, while Group B showed nonsignificantly (P > 0.05) lower ALT activity (U/L).

This may portent the protective effect of the plant against the possible hepatocellular damage resulting from the salt load. AST activities were also significantly (P < 0.05) higher in all the groups given salt load as compared with the control [Table 2]. But at day 78, only Group A (given continuous salt load), maintained the increase (P > 0.05) as compared with the control. Other Groups (B-D) showed nonsignificantly (P > 0.05) lower AST activities. Still at day 78, after administration of the extracts (aqueous or ethanol), there were significantly (P < 0.05) lower AST activities in the treated groups (day 78 compared with day 71), as compared with that of the untreated group. The ethanol extract was, however, shown to be more effective. The sustained increase in AST activity of the group given continuous salt load as against the decrease in AST activity of the treated group is indicative of the possible adverse effect of the salt load on the functional status of the liver. This may be supported by the observed decrease in Group D after cessation of salt load. The decrease in AST activity in the treated groups, resulting from the extracts, is also indicative of the possible protective effect of the plant against the adverse effects of salt load on the liver.

As shown in [Table 3], salt loading and/or treatment with aqueous or ethanol extract of A. wilkesiana leaves resulted in nonsignificant (P > 0.05) effects in levels of serum total bilirubin of the experimental animals. Total bilirubin concentration reflects the levels of both the conjugated and unconjugated fractions of bilirubin. Total bilirubin levels are elevated in various forms of liver disease such as cirrhosis, hepatitis, and obstructions of the hepatobiliary system such as gallstones or tumors. Elevated total bilirubin levels are also observed in cases of intravascular hemolysis. [17] Serum direct bilirubin levels of the animals given salt load showed slight differences when compared with the control, after 70 days of salt load [Table 4]. Group B showed significantly (P > 0.05) higher levels of direct bilirubin while Group C showed a significantly (P < 0.05) lower levels as compared with that of Group E. However, at day 78, after 1 week of treatment, there was no significant difference (P > 0.05) in the levels of direct bilirubin in all the groups. The lower levels of direct (conjugated) bilirubin in some groups probably caused by salt load may be protective as lower levels indicate decrease hemoglobin lyses.

A. wilkesiana leaf can be seen to be relatively safe considering its effects on serum ALT, AST, total and direct bilirubin of the experimental animals.

 
  References Top

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2.Greaney JL, DuPont JJ, Lennon-Edwards SL, Sanders PW, Edwards DG, Farquhar WB. Dietary sodium loading impairs microvascular function independent of blood pressure in humans: Role of oxidative stress. J Physiol 2012;590:5519-28.  Back to cited text no. 2
    
3.Hummel SL, Seymour EM, Brook RD, Kolias TJ, Sheth SS, Rosenblum HR, et al. Low-sodium dietary approaches to stop hypertension diet reduces blood pressure, arterial stiffness, and oxidative stress in hypertensive heart failure with preserved ejection fraction. Hypertension 2012;60:1200-6.  Back to cited text no. 3
    
4.Pons H, Ferrebuz A, Quiroz Y, Romero-Vasquez F, Parra G, Johnson RJ, et al. Immune reactivity to heat shock protein 70 expressed in the kidney is cause of salt-sensitive hypertension. Am J Physiol Renal Physiol 2013;304:F289-99.  Back to cited text no. 4
    
5.Kelly TN, Rebholz CM, Gu D, Hixson JE, Rice TK, Cao J, et al. Analysis of sex hormone genes reveals gender differences in the genetic etiology of blood pressure salt sensitivity: The GenSalt study. Am J Hypertens 2013;26:191-200.  Back to cited text no. 5
    
6.Varagic J, Susic D, Matavelli LC, Frohlich ED. AT 1 receptor antagonism attenuates adverse cardiovascular and renal effects of salt excess in SHR without affecting arterial pressure. 59 th Annual Fall Conference and Scientific Sessions of the Council for High Blood Pressure Research in Association with the Council on the Kidney in Cardiovascular Disease (Abstract). Hypertension 2005;46:819.  Back to cited text no. 6
    
7.Varagic J, Frohlich ED, Díez J, Susic D, Ahn J, González A, et al. Myocardial fibrosis, impaired coronary hemodynamics, and biventricular dysfunction in salt-loaded SHR. Am J Physiol Heart Circ Physiol 2006;290:H1503-9.  Back to cited text no. 7
    
8.du Cailar G, Ribstein J, Mimran A. Dietary sodium and target organ damage in essential hypertension. Am J Hypertens 2002;15:222-9.  Back to cited text no. 8
    
9.Swift PA, Markandu ND, Sagnella GA, He FJ, MacGregor GA. Modest salt reduction reduces blood pressure and urine protein excretion in black hypertensives: A randomized control trial. Hypertension 2005;46:308-12.  Back to cited text no. 9
    
10.Verhave JC, Hillege HL, Burgerhof JG, Janssen WM, Gansevoort RT, Navis GJ, et al. Sodium intake affects urinary albumin excretion especially in overweight subjects. J Intern Med 2004;256:324-30.  Back to cited text no. 10
    
11.Sofowora A. African Medicinal Plants. Ile Ife, Nigeria: University of Ife Press; 1984. p. 104.  Back to cited text no. 11
    
12.Akinde BE. Phytochemicals and microbiological evaluation of the oils from the leaves of Acalypha wilkesiana. In: Sofowora A, editor. The State of Medicinal Plant Research in Nigeria. Nigeria: University of Ibadan Press; 1986. p. 362-3.  Back to cited text no. 12
    
13.Oladunmoye MK. Comparative evaluation of antimicrobial activities and phytochemical screening of two varieties of Acalypha wilkesiana. Trends Appl Sci Res 2006;206:538-41.  Back to cited text no. 13
    
14.Kingsley O, Marshall AA, Inegbenose II, Meg IA. Phytochemical, proximate and elemental analysis of Acalypha wilkesiana leaves. Sci J Pure Appl Sci 2013;2:323-31.  Back to cited text no. 14
    
15.Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957;28:56-63.  Back to cited text no. 15
    
16.Jendrassik L, Gróf P. Vereinfachte photometrische Methoden zur Bestimmung des Blutbilirubins. Biochem Z 1938;297:82-9.  Back to cited text no. 16
    
17.Burtis CA, Ashwood ER. Tietz Textbook of Clinical Chemistry and Laboratory Medicine. 3 rd ed. Philadelphia, PA: W.B. Saunders Company; 1999. p. 1915.  Back to cited text no. 17
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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