Effect of aqueous Leaf extract of Scoparia dulcis – induced liver injury

Effect of aqueous Leaf extract of Scoparia dulcis on carbon tetrachloride induced liver injury in albino rats

Abstract

Hepatotoxicity is a worldwide problem usually caused by chemicals especially drugs. It can lead to jaundice, cirrhosis, hypoproteinemia and death. The effect of aqueous extract of the leaf of Scoparia dulcis on carbon tetrachloride (CCl4) induced liver injury in albino rats was investigated. The plant sample was collected and all procedures were carried out in 2015. Fifteen rats were randomly divided into three groups of five rats each. Hepatotoxicity was induced in experimental rats by administering CCl4 in olive oil at 120mg/kg body weight intra peritoneally for rats of groups II and III. Group III rats were orally administered 400mg/kg of the extract for two weeks. Serum markers of liver injury (Alanine aminotransferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP), albumine, and bilirubin (direct and total) were estimated at the end of the treatment. The results of the current study showed that there was significant (P<0.05) decrease in the activities of the enzymes across all the induced and treated rats in comparison with the induced non-treated rats. The results of the current study indicated that administration of the extract reduced levels of the enzymes and may mitigate hepatotoxicity.  Acute toxicity of the leaves extract was also investigated using Lorks method. The lethal dose (LD50) of the leaves extract was greater than 5000mg/kg.

Keywords: Scoparia dulcis, Carbon tetrachloride, liver injury, albino rats

Introduction

History of medicine can be traced back to the existence of human civilization and herbal medicine has been the basis of treatment of human ailments [1]. Nowadays, greater emphasis is again being laid to phytotherapy all over the world as management of all kinds of diseases is possible through plant drugs minus toxicity. A large proportion of the populations of developing countries use traditional medicines including medicinal plants to meet their primary health care needs probably due to their local availability, cultural importance and inexpensive obtaining [2]. According to the WHO statistics, about 80% of African inhabitants access their primary health care through traditional medicine and most of this therapy involves the use of plant extracts or their active components [2]. Worldwide considerable attention has been focused on herbal medicine which is based on the premise that these herbal plants may contain natural substances that can promote health and alleviate diseases. An estimated 60% of new drugs introduced between 1981 and 2002 are plants derived [3]. Herbal drugs play a major role in the treatment of hepatic disorders. A number of medicinal plants and their formulations are widely used for the treatment of these disorders [4]. In addition to the already existing medicinal plants, such as Cochlorspermum tinctorium and Psidium guajava, there are several unexplored medicinal plants that need to be studied for their therapeutic potential against liver disorders [5]. Scoparia dulcis linn or sweet broom in English (family: Scrophulariaceae) is a glabrous under shrub with small white flowers, commonly found on wastelands and fallow fields [6].This plant is widely used in the indigenous system of medicine for treating various diseases including liver ailments [7].

Phytochemical screening has revealed that the plant contains diterpenoids, flavonoids, tannins, alkaloids, triterpenes, hexacosonol, β-sitosterol, ketone-dulcitone and amellin, an antidiabetic compound [8]. Scoparia has long been recognized in herbal medicine in every tropical country where it grows, and its application by natives as traditional medicine is well acknowledged [9]. In Nigeria, the plant has been used in the management of sickle cell anaemia and typhoid fever [10]. Extracts of Scoparia dulcis L. have been shown to possess analgesic and anti-inflammatory properties due to the presence of flavonoid and glutinol [11].The diterpenoid, scoparic acid A isolated from the plant has been reported to be a potent β-glucuronidase inhibitor [12].

Liver diseases especially chemical-induced hepatotoxicity represent a major cause of morbidity and mortality in man [13] the liver is the organ for metabolism and detoxification of various components that enter the body, it is involved in wide range of functions and hence it is expose to toxic substance and drugs absorbed from the intestine. Apart from the toxins and drugs, viral infection (hepatitis A, B, C, D etc) and other microbial infections also cause damage to the hepatocytes [14]. Acute and chronic liver diseases constitute a global concern and medical treatments for these diseases are often difficult to handle and have limited efficiency [15]. Therefore, there has been considerable interest in role of complementary and alternative medicines for treatment of liver diseases. Developing therapeutically effective agents from natural products may reduce the risk of toxicity associated with synthetic drugs. The aim of this work is to carry out acute toxicity studies and evaluate the hepatocurative effect of the leaves of Scoparia dulcis.

Materials and Methods

Reagents/chemical

The reagents for the determination of albumin, bilirubin, and the kits for ALT, AST and ALP used were obtained from Randox, laboratories crumlin Co. Antrim, Northern Ireland UK. The carbon tetrachloride used was obtained from BDH, London.

Experimental animals                                           

Twenty seven albino rats were obtained from the Biological science department, Bayero University Kano and were fed ad libitum with vital commercial feed and water

Plant sample

Scoparia dulcis whole plant was collected in 2015 from Konduga LG area of Borno state Nigeria after which the leaves were separated for the studies.

Experimental Design:

Fifteen rats were randomly divided into three groups of five rats each for the hepatocurative studies while twelve rats were used for the acute toxicity studies of the leaves of the plant. Group 1 rats served as normal control and received distilled water. Groups II and III rats were induced by administering CCl4 in olive oil at 120mg/kg body weight intra peritoneally. Group III were treated with 400mg/kgbw of the aqueous leaves extracts orally for 21 days while group II were not treated. The effects of the leaves extracts on the liver parameters were assessed by assaying the levels of ALT, AST, ALP, TBL, DBL and albumin in the treated and untreated groups and comparing them.

Methodologies

Preparation of Plant Extract

Scoparia dulcis whole plants were collected in October from konduga local government area of Borno state The leaves were separated from the stems which were air dried and grinded to powder with pistle and mortar. The powder (500g) was then mixed in a container with 2.5L of distilled water and stirred. After 24 hours the residues were removed by filtration and the aqueous extracts were heated to dryness in ovum at 40 degree centigrade to get solid yield [16].

Acute toxicity studies

The acute toxicity studies of the leaf extracts was carried out by the method of [17]

Induction of liver injury

Liver injury was induced by administering CCl4 in olive oil at 120mg/kg body weight intra peritoneally for rats of groups II and III

Estimation of Biochemical Parameters:

At the end of the experimental period, animals were sacrificed by decapitation. Blood were collected, centrifuged and serum was separated for biochemical analysis.

Liver marker enzymes such as Aspartate amino transaminase (AST), Alanine amino transaminase (ALT) and alkaline phosphatase (ALP) were estimated. Also estimated are bilirubin and albumin.

Principles

Alanine Aminotransferase – ALT was measured by monitoring the concentration of pyruvate hydrazone formed with 2, 4-dinitrophenylhydrazine at 540nm [18].

α- oxoglutarate + L-alanine       =           L-glutamate + pyruvate

Aspartate Aminotransferase

AST was measured according to [18] by monitoring the concentration of oxaloacetic hydrazine formed with 2, 4-dinitrophenylhydrazine calorimetrically at 540nm.

α- oxoglutarate + L-aspartate     =            L-glutamate + oxaloacetate

Alkaline Phosphatase

ALP was assayed by hydrolysis of P-nitro phenyl phosphate as described by [19]. Phenol released by enzymatic hydrolysis from phenylphosphate at temperature of 37 and pH of 9.8 was estimated calorimetrically at 405nm.

P-nitro phenyl phosphate + H2O    =      Phosphate + p-nitrophenol

Albumin (Dye Binding Method)

The measurement of serum albumin was based on its quantitative binding to the indicator 3, 3, 5, 5’-tetrabromo-m cresol sulphonephthalein (bromocresol green, BCG). The albumin-BCG complex absorbs maximally at 578nm, the absorbance being propotional to the concentration of albumin in the sample.

Bilirubin

Bilirubin was measured by Colorimetric method described by [20]. Direct (conjugated) bilirubin reacts with diazotized sulphanilic acid in alkaline medium to form a blue coloured complex measured at 546nm. Total bilirubin is determined in the presence of caffeine, which releases albumin-bound bilirubin, by the reaction with diazotized sulphanilic acid.

Histopathological Studies

Anatomy of the liver was studied immediately after sacrificing the animals. A small portion of the liver was fixed in 10% formalin. Representative liver tissues were processed by the paraffin embedding technique. Sections of 5microns thickness were cut and stained with Haematoxylin and Eosin and histology was studied. [21]

Startistical analysis

Results were expressed as mean ± sd for five rats. All data were analyzed by one way analysis of variance (ANOVA).

RESULT

Acute toxicity studies of oral aqueous leaf extracts of S. dulcis were conducted on experimental rats. No death was observed up to a dose of 5000mg/Kg body weight (Table1).

The hepatocurative effect of aqueous leaf extracts of S. dulcis was tested on carbon tetrachloride induced liver injury in albino rats for a period of three weeks. The effect of aqueous leaf extract is presented in table 2. High activities of alanine aminotransferase, and aspartate aminotransferase observed in the rats treated with CCl₄ alone were significantly (p˂0.05) decreased (ALT. 19.0±0.28, AST 27.25±10.4) in the leaves extracts treated rats. There was a significant (p˂0.05) decrease in albumin level in the CCl₄ treated group compared to the normal rats. But the extracts administered rats have significantly (p˂0.05) increased concentrations of albumin compared to the CCl₄ alone treated group. While ALP, direct bilirubin and total bilirubin did not show significant changes comparing the normal and CCl4 administered groups.

Discussion

Scarcity of scientific data on the safety and efficacy of medicinal plants has raised concerns regarding toxicity. The acute toxicity test performed in this study has indicated that the leaves extract of S dulcis are nontoxic up to a dose of 5000mg/kg body weight. The toxicity study conducted suggests that the extract may be safe for medicinal purposes as evidenced by its high LD50 value

Carbon tetrachloride (CCl4) is one of the major contaminants of our environment that has been detected in air, water and land. It is one of the most commonly used environmental chemicals in the experimental study of liver injury since the changes associated with CCl -induced liver damage is similar to that of viral acute hepatitis [22].

CCl4 alkylates cellular macromolecules with a simultaneous oxidative degradation of membrane lipids of endoplasmic reticulum rich in poly unsaturated fatty acids. This leads to generation of lipid peroxides, which in turn causes release of products like malondialdehyde (MDA) that cause injury to the membrane [23]. An obvious sign of hepatotoxic injury is leakage of cellular enzymes into the plasma, when the liver cells are necrosed or damaged. A variety of enzymes, like AST, ALT and ALP are normally located in the cytosol and are released into bloodstream. It has been documented that increased serum activities of alanine aminotransferase, asparate aminotransferaseand alkaline phosphatase are biochemical markers of hepatic injury. Their estimation in the serum is a useful quantitative marker of the extent and the type of hepatocellular damage [24]. Hepatotoxicity of CCl4 is evidenced in these animals by increases in activities of serum alanine aminotransferase and asparate aminotransferase.

The results of this study obtained in table 2 showed elevated activities of alanine aminotransferase (ALT), and aspartate aminotransferase (AST), in the serum of the carbon tetrachloride alone treated rats compared with that of the normal rats. This may be due to the leakage of the biochemical markers in the serum as a result of the oxidative damage caused by carbon tetrachloride (CCl4) to the hepatocytes.  The substantial decrease in the levels of ALT and AST observed (at the dose of 400mg/kg) in the extract administered rats might be as a result of the decreased leakage of the enzymes in liver cells. This may suggests that the leaves extract of Scoparia dulcis could cure hepatic injury and or restore the cellular membrane integrity and permeability, thereby reducing the toxic effect and preventing enzyme leakage into the blood circulation. These hepatocurative activities of the extract may be attributed to the free radical scavenging potential of the flavonoid 7-O-methyl scutellarein present in the plant as reported [8]. The antioxidant effect of this plant may be attributed to this flavonoid. In addition, the plant contains scoparic acid A, a labdane type of diterpene acid which is a potent β–glucuronidase inhibitor [25]. The inhibition of β–glucuronidase may also contribute to the hepatocurative effect since it has been reported that liver microsomal β–glucuronidase is involved in the pathogenesis of liver injury [12]. [6] had earlier reported that the leaves of S. dulcis have high antioxidant and free radical scavenging activities. The significant decrease in the levels of these enzymes in the treated group could also be as a result of the anti-inflammatory effect of the plant since inflammation enhances oxidative stress through liberating reactive oxygen species by phagocytes [23]. This is supported by the fact that S dulcis is known to inhibit inflammation [7].

Alkaline phosphatase (ALP), which is a biomarker of cholestatic liver injury that reflects pathological alteration in biliary flow [26], did not show any increase confirming that CCl4 induced liver injury was not of the cholestatic type but is of the necrogenic type [27].The non-increased in the level of serum bilirubin is in line with this.

Albumin is the major secretory protein synthesized by the liver; hypoalbuminemia is often taken as an indication of reduced hepatic synthesis. The result of this study shows significant (p˂0.05) reduction from the mean value of the normal (52.33±10.97) compared to the mean value of the test control (30.66±2.08) at p˂0.05. An increase was shown by the extracts treated groups compared with the test control (p˂0.01). So the decreased level of the Serum albumin may be associated with reduced hepatic synthesis due to injury.

Rat liver of normal control group, revealed that the architecture of the lobule was intact, with hepatocytes arranged as radiating plates around the central vein. (fig.1a)  while CCl4 alone treated rat liver shows fibrosis (fig.1b) and marked lymphoplasmacytic infiltration (fig.1c) indicating inflammation probably due to the injury caused by CCl4. These lesions observed were markedly reversed in the livers of the treated rats as shown in (fig.1d) with moderate pericentral lymphoplasmacytic infiltration and mild fibrosis.

Conclusion

Conclusively, it can be suggested that the plant can ameliorate liver injury or may help in the treatment of hepatitis but further pharmacological studies are recommended. The plant can be safe to use in the field of ethno medicine considering its high LD50

References

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Table 1: Results of acute toxicity studies of the leaves extract of S. dulcis for determination of LD50

First phase
 

Dose (mg/kg)                               mortality  rate

10                                               0/3

100                                             0/3

1000                                           0/3

Second phase

Dose (mg/kg)                               mortality rate

1600                                             0/1

2900                                             0/1

5000                                             0/1

 

Table 2: The effects of 21 days treatments of aqueous leaf extracts of Scoparia dulcis on liver parameters of rats

Group Dose ALT (iU/L) AST (iU/L) ALP (iU/L) T.BIL (mg/dl) D.BIL (mg/dl) ALB. (g/L)
I _ 19.0±.28 27.25±10.4 51.16±2.00 1.46±0.50 0.82±0.40 52.33±10.97
II 120mg/kg 106.5±6.36⃰ 135.00±24.55⃰ 124.25±37.50 2.1±1.15 1.00± 0.4 30.66± 2.08⃰
III 400mg/kg 18.66± 2.08⃰ 92.00± 4.35⃰ 66.00± 23.58 1.0±0.00 0.8±0.28 37.33± 0.58⃰

Results are expressed as mean + standard deviation (n=3) Values with star in the same column are significant at p˂0.05 comparison were made between test control and normal, between test control and treated groups and between normal and treated groups Group i – Normal control not induced not treated; Group ii – Induced with CCl4 but not treated; Group iii – Induced with CCl4 and

treated with root extract

Lists of titles of figures.

Figure 1a – Photomicrograph of a liver section of group 1 (normal control) rats

Figure 1b – Photomicrograph of a liver section of group 2 (CCl4 alone treated group) rats

Figure 1c – Photomicrograph of a liver section of group 2 (CCl4 alone treated group) rats

Figure 1d – Photomicrograph of a liver section of group 3 (leaf extract treated group) rats

 

 

Fig-1a liver section of Group1(normal Control) animals which has normal architecture, where the central vein (arrow), portal tracts, (blue arrow ) hepatocytes and sinusoids appeared normal with no infiltration by inflammatory cells×10

 

 

Fig.1b group2 rat liver tissue with bridging fibrosis (arrow) ×40,

 

 

 

Fig. 1c Group 2 CCl4 treated rat liver tissue with marked pericentral lympho plasmacytic infiltrate (arrow) ×40

 

 

 

 

Fig.1d liver tissue of rat treated with leaf extract with mild lympho plasmacytic infiltration (arrow) ×100

 

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