Hepatoprotective activities of two Ethiopian medicinal plants

Abstract

The present study evaluated the in vivo hepatoprotective activity of two medicinal plants, namely, Justicia schimperiana (Hochst. ex Nees) (Acanthaceae) and Verbascum sinaiticum Benth. (Scrophulariaceae) used in Ethiopian traditional medical practices for the treatment of liver diseases. The levels of hepatic marker enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were used to assess their hepatoprotective activity against carbon tetrachloride (CCl₄)-induced hepatotoxicity in Swiss albino mice. The results revealed that pretreating mice with the hydro-alcoholic extracts of both plants significantly suppressed the plasma AST ((P < 0.01) J. schimperiana; (P < 0.05) V. Sinaiticum) and ALT ((P < 0.05) J. schimperiana) activity when compared with the CCl₄ intoxicated control. Among the Soxhlet extracts of each of the plants, the methanol extract of J. schimperiana showed significant hepatoprotective activity. Further fractionation of this extract using solid phase extraction and testing them for bioactivity indicated that the fractions did not significantly reverse liver toxicity caused by CCl₄. However, the percentage hepatoprotection of the distilled water fraction was comparable with that of the standard drug silymarin at the same dose (50 mg/kg) as evidenced by biochemical parameters. Histopathological studies also supported these results. In vitro DPPH assay conducted on the water fraction of J. schimperiana and the Soxhlet methanol fraction of V. sinaiticum showed that they possess moderate radical scavenging activity (IC₅₀ = 51.2 and 41.7 μg/mL, respectively) which led to the conclusion that the hepatoprotective activity of the plants could be in part through their antioxidant action.

Keywords::

• Hepatic marker enzymes
• histopathological studies
• in vivo hepatoprotective activity
• Justicia schimperiana
• Verbascum sinaiticum
• radical scavenging activity

Introduction

Liver diseases remain serious health problems and are caused, among others, by drugs, chemicals, and alcohol. Chronic liver disease is a major cause of morbidity and mortality throughout the world. Conventional medical therapy for many common liver disorders, including non-alcoholic fatty liver disease and viral hepatitis, has limited efficacy and potentially life-threatening side effects. This has increased dependence on complementary and alternative medicine (CAM), especially herbal therapy. Various medicinal plants and their formulations are used in traditional medicine for their hepatoprotective effects and a number of herbal preparations are available on the market. The most common medicinal plants and herbal preparations used for the management of liver diseases include Phyllanthus spp. (Euphorbiaceae), Silybum marianum (L.) Gaertn (milk thistle) (Asteraceae), glycyrrhizin (licorice root extract), and Liv 52 (a mixture of herbs). Most of these herbal drugs were shown to speed up the natural healing process of the liver (Dhiman & Chawla, 2005; Mani Senthilkumar et al., 2005).

The biodiversity of the Ethiopian flora offers great possibilities in the search for natural products with novel structures that have a range of biological activities including treatment of various liver disorders (Asres et al., 2001a).

Justicia schimperiana (Hochst. ex Nees) (syn. Adhatoda schimperiana (Hochst. ex Nees) Andreson) (Acanthaceae), is an erect shrub up to 4 m high with woody and internodal stem (Abebe et al., 2003). The plant is very common in villages and towns as well as in cities growing on waste places or grown as a hedge and used as a boundary marker around native compounds. The plant is relatively fast growing and prefers altitudes of 2400 m or above (Getahun, 1976). In Ethiopian traditional medicine, the whole plant is used as a remedy for excessive pellagra, as a laxative (Getahun, 1976), as well as for the treatment of stomach complaints, hepatitis, venereal diseases, malaria, asthma, leishmaniasis and cough (Asres et al., 2001b); jaundice, epilepsy, mental illness and leprosy (Abebe & Ayehu, 1993). Geyid et al. (2005) reported the presence of polyphenols, unsaturated sterol/triterpenes and saponins in the methanol extract of the dried leaves of J. schimperiana.

Verbascum sinaiticum Benth (Scrophulariaceae) is an erect herb reaching up to 2 m high. The whole plant is densely shaggy, grayish and its leaves are simple and broadly ovate-oblong (Abebe et al., 2003). In Ethiopian traditional medicine, the plant is used in the treatment of syphilis, tumor, liver diseases, stomach troubles, diabetes, scabies, amoebiasis, diarrhea, epilepsy, and cough (Abebe et al., 2003). Afifi et al. (1993) reported the presence of two flavonolignans, hydrocarpin and sinaiticin, as well as two flavones, chrysoeriol and luteolin, in the leaves of V. sinaiticum. All these compounds were shown to have dose-dependent cytotoxic effect when tested against cultured P-388 cells (a murine leukemia cell line). Phytochemical and biological studies on V. sinaiticum growing in Egypt have revealed the presence two iridoids: ajugol and aucubin in the aerial parts of the plant. Moreover, investigation of the flavonoid constituents led to the isolation and identification of luteolin and chrysoeriol-7-glucoside. Both the alcohol and methylene chloride/methanol (1:1) extracts were shown to have hepatoprotective effect against carbon tetrachloride (CCl₄) induced cytotoxicity at 25 and 10 μg/mL for the alcoholic and CH₂Cl₂/MeOH extracts, respectively. The study attributed hepatoprotective activity to the presence of iridoid compounds which constitute the major components of the CH₂Cl₂-MeOH (1:1) extract (Mahmoud et al., 2007).

Despite the significant popularity of several herbal medicines in general, and those used for the management of liver diseases in particular, they have not yet become acceptable treatment modalities. The present study was therefore conducted to test the hepatoprotective activities of the aforementioned two medicinal plants used in Ethiopian traditional medical practices for the treatment of liver diseases.

Materials and methods

Plant materials and extraction

The leaves of J. schimperiana and V. sinaiticum were collected in October 2005 from Yeka subcity, around Bella, Addis Ababa, Ethiopia. The identity of each plant specimen was confirmed by Melaku Wondafrash at the National Herbarium, Department of Biology, Addis Ababa University, Ethiopia, where voucher specimens were deposited (J. schimperiana, US 001 and V. sinaiticum, US 004).

The required plant parts were shade-dried and powdered. The powder of each plant (100 g) was separately extracted with 80% methanol by maceration for 72 h (three times) with frequent agitation and the resulting liquid was filtered (Whatman No. 3 filter paper). The combined extracts were concentrated under reduced pressure at a temperature not exceeding 40°C. The concentrated extract was then dried in an oven at 40°C for about 48 h. The resulting dried mass of each plant (yield: 15.8% for J. schimperiana, and 21.1% for V. sinaiticum) was then powdered, packed into a glass vial, properly labeled and stored in a desiccator over silica gel until use.

J. schimperiana and V. sinaiticum powders (20 g each) were separately and sequentially extracted with petroleum ether, chloroform, acetone and methanol using Soxhlet apparatus. The solvents were evaporated under reduced pressure and the fractions were then placed in a vacuum oven at 30°C for about 24 h to remove any residual solvent. The resulting semisolid mass of each fraction was stored in a desiccator until use under the same conditions as the crude extracts.

Fractionation by solid-phase extraction

Further fractionation of the methanol extract of J. schimperiana was carried out by solid phase extraction on Isolute C₁₈ (10 g, IST, Hengoed, Wales) using MeOH-H₂O gradient. The methanol extract (500 mg) obtained from Soxhlet extraction was eluted first with 50 mL of distilled water followed by the same amount of 25%, 50%, and 75% methanol solutions in water and 100% methanol, successively to give fractions designated A₀, A₁, A₂, A₃ and A₄, respectively. Each fraction was concentrated and dried following the same procedure as in the crude extracts and kept in a desiccator for future use.

Animals

Swiss albino mice of either sex, weighing 22–30 g and aged 6–8 weeks were used for the experiments. The animals were purchased from the Ethiopian Health and Nutrition Research Institute (EHNRI), Addis Ababa, and the National Veterinary Institute, Debrezeit. All animals were maintained at a temperature of 22° ± 2°C and with a 12 h light/12 h dark cycle and given pellet diet and water ad libitum. The animals were acclimatized to their new environment for at least one week prior to the experiments. All procedures performed were reviewed and approved by the Institutional Review Board and conform to internationally accepted principles.

Acute toxicity testing

Thirty-five Swiss albino mice were divided into seven groups (I-VII) of five animals each. Group I received distilled water 10 mL/kg orally and animals from Groups II to VI were given hydro-alcoholic extracts of J. schimperiana at doses of 200, 300, 400, 500, 1000 mg/ kg orally, respectively. Group VII received 2000 mg/kg hydro-alcoholic extracts of V. sinaiticum p.o. Symptoms of toxicity and mortality were observed for 24 h. The behavioral and CNS profiles such as spontaneous rearing and grooming, evidence of calmness and sedation, and loss of writhing reflex were also observed.

In vivo hepatoprotective activity testing

In vivo hepatoprotective activity was evaluated on the basis of CCl₄-induced liver damage in mice as previously described by Mansour (2000). The animals were divided into six groups of six animals (Table 1). Group I was kept on normal diet and served as normal control and received distilled water (10 mL/kg, p.o.) daily for 5 days and then given olive oil (3 mL/kg, i.p.) on day 5, 30 min after receiving distilled water; Group II served as CCl₄ control group, and received distilled water (10 mL/kg, p.o.) daily for 5 days and then received CCl₄: olive oil (1:120; 3 ml/kg, i.p.) on day 5, 30 min after administration of distilled water. Groups III and IV, V and VI were treated with total extracts of the study plants at a dose of 200 mg/kg body weight daily for 5 days. Groups III and V further received olive oil (3 mL/kg, i.p.) on day 5, 30 min after receiving extract (Williamson et al., 1998) whereas Groups IV and VI, received CCl₄: olive oil (1:120; 3 mL/kg, i.p.) on day 5, 30 min after administration of the extract. All of the animals were restricted to water after administration of CCl₄ or olive oil on day 5. Twenty-four h after the i.p. administration of CCl₄ in olive oil or olive oil alone, the mice were anesthetized with ether, and their blood was collected directly from the heart and allowed to clot for 30 min at room temperature.

Table 1.  Effects of the hydro-alcoholic extracts of Justicia schimperiana and Verbascum sinaiticum at a dose of 200 mg/kg on activities of serum enzymes in mice injected with CCl₄ in olive oil (25 µL/kg), i.p.

Treatment	Serum enzyme activities (U/L)(Mean ± SEM)
	AST	ALT
Group I (Vehicle control)	40.3 ± 5.2	31.7 ± 3.8
Group II (CCl4 control)	160.2 ± 14.3	105.3 ± 11.7
Group III (J. schimperiana)	45.5 ± 3.5	35.9 ± 5.2
Group IV (J. schimperiana + CCl4)	106.3 ± 3.2(45.1)**	65.1 ± 8.5(54.5)*
Group V (V. sinaiticum )	41.2 ± 5.7	29.8 ± 4.1
Group VI (V. sinaiticum + CCl4)	110.2 ± 4.5(41.7)*	80.6 ± 8.1(33.8)

Six animals per group were used; *P < 0.05 and **P < 0.01 significantly different from CCl₄ control; numbers in parenthesis indicate percentage protection.

Biochemical assays

Serum was separated by centrifugation at 3000 rpm for 15 min and liver damage was assessed by estimation of serum activities of alanine aminotransferase (ALT) (Bergmeyer, 1980), aspartate aminotransferase (AST) (Bergmeyer et al., 1976) and alkaline phosphatase (ALP) (Tietz et al., 1983), using commercially available test kits and an automatic blood biochemical analyzer (Bioanalyzer, SABA PM 4000; Rome, Italy).

The chloroform (Group C & F), acetone (Group D & G) and methanol (Group E & H) extracts from the Soxhlet extraction of J. schimperiana and V. sinaiticum, at a dose of 200 mg/kg, and silymarin (Group I) (standard drug) at a dose of 50 mg/kg, were tested for hepatoprotective activity following the above procedure (Table 2). Similarly, the 100% water fraction of J. schimperiana at doses of 50 and 100 mg/kg; 25% methanol fraction at a dose of 50 mg/kg; 100% methanol fraction at a dose of 50 mg/kg; and silymarin at a dose of 50 mg/kg, were also tested for hepatoprotective activity following the above procedure. In addition, in this case, the livers were harvested, grossly examined and preserved in 10% formalin solution for histological examination.

Histopathological study

Small pieces of the liver, fixed in 10% buffered formalin, were processed for embedding in paraffin. Sections of 5–6 µm were cut and stained with hematoxylin and eosin (H&E) and examined under the microscope for histopathological changes. Images were captured using an Olympus DP12 CCD camera at original magnification of 10 × (Olympus DP12 Microsystems Digital Imaging Olympus, Japan).

Dose-dependent activity study

The methanol fraction obtained from the Soxhlet extraction of J. schimperiana was tested against CCl₄-induced liver injury in mice at dose levels of 100 (Group b), 200 (Group c), 300 (Group d), and 400 mg/kg (Group e) following the above procedure.

In vitro DPPH assay

The hydrogen-donating or radical-scavenging capacities of the 100% water fraction of J. schimperiana and the methanol fraction of V. sinaiticum were measured using the stable radical, DPPH. following the method given by Blois (1958). The samples were diluted with methanol to prepare solutions equivalent to 2,000, 1,000, 500, 250, and 125 μg of dried sample per mL of solutions. An aliquot of 0.004% DPPH solution (5 mL) in methanol was pipetted into a test tube followed by the addition of 50 μL of a sample solution. The mixture was incubated at 37°C for 30 min in the dark and its absorbance at 517 nm was measured (Jenway Model 6500 spectrophotometer). The percentage inhibition of DPPH radicals was calculated using the following equation:

where, A_o is absorbance of the control, and A_s is absorbance of the sample at 517 nm.

The IC₅₀ values were calculated by linear regression of the plots, where the x-axis represented the concentration of the test plant extracts and the y-axis represented the average percentage of radical-scavenging activity from three replicates.

Statistical analysis

All values are expressed as mean ± SEM. The data were analyzed by one-way analysis of variance (ANOVA) and Tukey post test. The hepatoprotective activity of the test material was calculated using the following formula:

where, a is the mean value of the marker produced by hepatotoxin; b is the mean value of the marker produced by toxin plus test material; and c is the mean value produced by the vehicle control (Garg et al., 1994). P-values < 0.05 were considered significant.

Results and discussion

CCl₄ is one of the oldest and most widely used toxins for experimental induction of liver fibrosis in laboratory animals. CCl₄ requires bioactivation by the cytochrome P450 system of phase I in liver and yields the reactive metabolite trichloromethyl radical (CCl₃˙) and peroxy trichloromethyl radical (CCl₃O˙) Peroxidation of membrane lipids secondary to the formation of the CCl˙ and/or CCl₃OO˙ radicals is believed to be the basis for the toxic effect of CCI₄ (Bahcecioğlu et al., 1999). Both trichloromethyl and its peroxy radical are capable of binding to proteins or lipids, or of abstracting hydrogen atoms from polyunsaturated fatty acid (PUFA), initiating lipid peroxidation thus causing damage to cell membrane, changing enzyme activity and finally inducing hepatic injury or necrosis (Kumar et al., 2005; Hung et al., 2006). The method was chosen for this study because CCl₄-induced liver injury resembles the damage caused by acute viral hepatitis, and also evaluation of the preventive action in liver damage induced by CCl₄ has been widely used for hepatoprotective drug screening. Damage to the structural integrity of the liver by CCl₄ is reflected by an increase in the activities of AST, ALT, ALP, and LDH, which are released into the circulation after cellular damage. CCl₄ also induces ample infiltration of the lymphocytes and Kupffer cells, massive centrilobular necrosis, sinusoidal dilatation and congestion (Galati et al., 2005).

Hepatoprotective activity of the hydro-alcoholic extracts

In the present study, treating the mice with CCl₄ at a dose of 25 µL/kg i.p. caused hepatocellular damage as indicated by an increase in plasma AST and ALT activity (Table 1). Pretreating mice with the hydro-alcoholic extracts of J. schimperiana significantly suppressed the plasma AST (P < 0.01) and ALT (P < 0.05) activity when compared with the CCl₄ intoxicated control. Similarly pretreating mice with the hydro-alcoholic extracts of V. sinaiticum also significantly suppressed the plasma AST (P < 0.05) activity when compared with the CCl₄-intoxicated control.

The results of the present study indicated that there were no significant changes in the activities of serum AST and ALT levels in mice which were given hydro-alcoholic extracts of J. schimperiana and V. sinaiticum, at a dose of 200 mg/kg p.o. for 5 days without the hepatotoxin CCl₄ (Table 1). However, a significant increase (P < 0.001) in the activities of serum enzymes occurred 24 h after exposure to CCl₄ in the hepatotoxin only groups, which was not observed when the total extracts of J. schimperiana and V. sinaiticum were administered. As shown in Table 1, the hydro-alcoholic extracts of J. schimperiana and V. sinaiticum displayed significant protective effects on CCl₄-induced liver damage in mice. Therefore, the total extracts of these plants were subjected to solvent fractionation by Soxhlet extraction.

Hepatoprotective activity of Soxhlet extracts

As shown in Table 2, mice treated with a single dose of CCl₄ in olive oil developed significant hepatic damage as observed from elevated serum levels of hepatospecific enzymes. However, pretreatment of mice with different Soxhlet extracts of J. schimperiana revealed that the methanol extract significantly (P < 0.05) suppressed the plasma AST, ALT and ALP activities when compared with the CCl₄-intoxicated control. Similar effects were observed for the methanol extract of V. sinaiticum on plasma AST level. It can, therefore, be said that the active constituents of the plants reside mainly in the methanol extract and that the compounds are polar in nature. Moreover, the application of heat might have facilitated the solubility of these compounds (Vontagu et al., 2004).

Table 2.  Effects of the different Soxhlet extracts of Justicia schimperiana and Verbascum sinaiticum at a dose of 200 mg/kg on activities of serum enzymes in mice injected with CCl₄ in olive oil (25 µL/kg), i.p.

Group	Serum enzyme activities (U/L)(Mean ± SEM)
	AST	ALT	ALP
A (vehicle control)	43.07 ± 2.3	33.5 ± 7.1	50.1 ± 6.7
B (CCl4 control)	152.2 ± 16.2	206.3 ± 40.8	160.9 ± 13.2
C (chloroform extract of V. sinaiticum)	130.4 ± 10.6(19.5)	174.8 ± 20.2(18.1)	142.8 ± 17.1(16.3)
D (acetone extract of V. sinaiticum)	116.3 ± 12.6(32.0)	160.8 ± 16.8(26.2)	137.9 ± 10.1(16.3)
E (methanol extract of V. sinaiticum)	82.4 ± 13.2(63.9)*	111.4 ± 18.1(54.9)	117.9 ± 9.7(38.8)
F (chloroform extract of J. schimperiana)	131.4 ± 17.2(18.6)	195.9 ± 19.7(8.9)	147.3 ± 12.3(18.1)
G (acetone extract of J. schimperiana)	143.8 ± 13.4(7.8)	196.1 ± 28.7(5.8)	148.7 ± 15.7(11.0)
H (methanol extract of J. schimperiana)	84.5 ± 12.7(61.5)*	98.3 ± 8.2(61.8)*	97.5 ± 13.4(57.2)*
I (silymarin: 50 mg/kg)	67.5 ± 19.8(72.9)**	87.6 ± 9.5(69.3)*	89.1 ± 10.2(64.8)**

Six animals per group were used; *P < 0.05 and **P < 0.01, significantly different from CCl₄ control; numbers in parenthesis indicate percentage protection.

As can be seen from the overall percentage hepatoprotection, the methanol extract of J. schimperiana has a statistically significant hepatoprotective activity compared to the methanol extract of V. sinaiticum. As shown in Table 2, the results of the present study revealed that the methanol extract of J. schimperiana possesses somewhat comparable hepatoprotective activity with that of the standard drug silymarin and was therefore, selected for further fractionation using solid phase extraction.

Hepatoprotective activity of the solid phase fractions of J. schimperiana

The different fractions obtained from solid phase extraction of J. schimperiana did not show significant protective activity compared to the CCl₄ control (Table 3). The results on the hepatoprotective activity of the fractions from solid phase extraction of J. schimperiana clearly showed that preadministration of A₀ (distilled water fraction) at doses of 50 and 100 mg/kg did not significantly reverse the increase in serum enzymes AST, ALT and ALP caused by administration of CCl₄. As can be seen from the percentage protection data, the plant extract displayed better activity at a dose of 50 mg/kg than at 100 mg/kg. This could be due to intrinsic toxicity of the compound(s) responsible for hepatoprotective activity at higher doses. A dose-dependent activity study also supported this finding. At an equivalent dose (50 mg/kg), the hepatoprotective activities of A₀ and silymarin were shown to be comparable.

Table 3.  Effects of fractions from the solid phase extraction of the methanol extract of Justicia schimperiana on activities of serum enzymes in mice injected with CCl₄ in olive oil (25 µL/kg), i.p.

Group	Serum enzyme activities (U/L)(Mean ± SEM)
	AST	ALT	ALP
I (vehicle control)	51.5 ± 35.3	36.7 ± 10.5	57.5 ± 25.8
II (fraction A0: 50 mg/kg)	141.5 ± 57.2(53.6)	90.3 ± 7.9(57.2)	161.9 ± 27.8(50.0)
III (fraction A0: 100 mg/kg)	148.2 ± 26.5(50.2)	94.1 ± 15.8(54.2)	180.2 ± 32.2(41.3)
IV (fraction A1: 50 mg/kg)	165.6 ± 27.6(41.2)	116.4 ± 47.4(36.3)	185.9 ± 76.1(38.5)
V (fraction A4: 50 mg/kg)	180.6 ± 63.0(33.5)	123.1 ± 18.3(30.9)	198.1 ± 33.3(32.7)
VI (silymarin, 50 mg/kg)	102.4 ± 41.0(73.8)	82.6 ± 16.6(63.3)*	135.1 ± 24.5(62.9)*
VII (CCl4 control)	245.6 ± 62.5	161.9 ± 27.3	266.34 ± 34.6

Numbers in parenthesis indicate percentage protection; six animals per group were used; *P < 0.05 significantly different from CCl₄ control.

Dose-dependent activity study

An experiment was carried out to check whether the hepatoprotective activity of J. schimperiana was dose-dependent or not. Thus, pretreatment of mice with different doses of the methanol fraction obtained by Soxhlet extraction showed that hepatoprotective activity decreased with increasing dose (Table 4). But statistical data revealed that there is no significant difference in the hepatoprotective activity among the test groups.

Table 4.  Effects of the methanol extracts of Justicia schimperiana on activities of serum enzymes in mice injected with CCl₄ in olive oil. (25 µL/kg), i.p.

Group	Serum enzyme activities (U/L)(Mean ± S.E.M)
	AST	ALT	ALP
a (vehicle control)	35.06 ± 6.02	41.06 ± 19.2	55.4 ± 7.6
b (100 mg/kg)	297.5 ± 36.2(56.2)***	230.6 ± 23.6(47.7)*	114.8 ± 6.8(50.2)
c (200 mg/kg)	337.8 ± 63.8(49.5)**	238.5 ± 27.4(45.5)	120.2 ± 15.7(45.6)
d (300 mg/kg)	363.5 ± 58.1(45.8)**	252.2 ± 20.2(41.7)	138.6 ± 9.6(30.2)
e (400 mg/kg)	404.3 ± 52.2(38.2)*	276.7 ± 66.7(34.9)	150.4 ± 25.1(20.4)
f (CCl4 treated control)	634.3 ± 33.2	403.5 ± 47.3	174.7 ± 36.7

Numbers in parenthesis indicate percentage protection; six animals per group were used; *P < 0.05, **P < 0.01 and ***P < 0.001, significantly different from CCl₄ control.

As shown in Table 4, the percent hepatoprotection is relatively low at higher doses. This could be due to inherent toxicity of some components which may be present together with the hepatoprotective principles or the hepatoprotective principles themselves might be toxic at higher doses.

Histopathological study

Histopathological assessment of liver damage was carried out by studying hematoxylin and eosin stained slides of liver tissue. Figure 1A shows the severe hepatic lesions caused by CCl₄. Major centrilobular and hepatocellular necrosis as well as major critical disorganization of the complete lobules accompanied by fatty changes and ballooning degeneration were observed in hepatocytes of the livers of mice treated with CCl₄ (Group VII). Moreover, the lipid peroxidation and degree of damage is very intense in this group of animals compared to the other groups. As is apparent in Figure 1B, the severe hepatic lesions induced by CCl₄ were remarkably lowered by the administration of A₀ (distilled water fraction of J. schimperiana, 50 mg/kg), which is in good agreement with the results of the biochemical analysis. The vehicle control group (Figure 1C) showed the normal parenchymal architecture with cords of hepatocytes, portal tracts and central veins without noticeable gross alterations except for light congestion of blood vessel compared to the CCl₄ control. The toxin mediated changes in livers of mice pretreated with plant extracts Group II (Figure 1B), Group III (Figure 1D) and the standard drug silymarin, Group VI (Figure 1E), 24 h after the administration of CCl₄, were much less in intensity than those observed in the livers of CCl₄-treated mice (Figure 1A). Though the extent of cellular necrosis was less pronounced compared to the CCl₄ group, increase in liver size, disorganization of the lobules, reactive inflammatory processes and necrosis with acidophilic cells, were observed in animals of Group III (Figure 1D), Group D (Figure 1F) and Group V (Figure 1G) compared to groups I, II and VI. In the liver of animals of Group V (Figure 1G) there were also intense tumefaction and fatty changes which were an indication of severe damage compared to the other groups with the exception of Group VII animals (Figure 1A).

Figure 1.  Representative microphotographs of H&E (×10)-stained histological sections of liver (1A) CCl₄ control; (1B) mice treated with fraction A₀ (distilled water fraction of Justicia schimperiana), 50 mg/ kg, p.o. and CCl₄ (25 µL/kg, i.p.); (1C) vehicle control; (1D) mice treated with A₀, 100 mg/kg and CCl₄ (25 µL/kg, i.p.); (1E) mice treated with silymarin 50 mg/kg, p.o. and CCl₄ (25 µL/kg, i.p.); (1F) mice treated with A₁ (25% methanol fraction of Justicia schimperiana), 50 mg/kg and CCl₄ (25 µL/kg, i.p.); (1G) mice treated with fraction A₄ (pure methanol fraction of Justicia schimperiana) 50 mg/kg, p.o. and CCl₄ (25 µL/kg, i.p.).

The results of these histopathological findings clearly indicated that the extent of cellular damage was less in animals of Group II and Group VI compared to the CCl₄ group and animals of Groups III, IV, and V. This indicates pre-administration of fraction A₀ or silymarin at 50 mg/kg has significantly protected the livers of the animals from CCl₄ induced damage. Necrosis, which is a more severe form of damage, was also markedly minimized by the pretreatment of the animals with 50 mg/kg of A₀ (Figure 1B). In addition, the results of histopathological studies also showed that the degree of protection of A₀ is much higher at 50 mg/kg than it is at a dose of 100 mg/kg, which is in good agreement with the results of the serum transferase activities.

In vitro DPPH Assay

Many hepatotoxicants, including CCl₄, nitrosamines, and polycyclic aromatic hydrocarbons require metabolic activation, particularly by the liver cytochrome P450 enzymes, to form reactive, toxic metabolites, which in turn cause liver injury in experimental animals and humans. Oxidative damage triggered by CCl₄ is believed to be due to the formation of reactive peroxy radicals that are initiators of lipid peroxidation chain reaction, which subsequently provoke inflammatory reactions and hence destruction and damage to cell membrane (Shahjahan et al., 2005). Thus, natural compounds that reduce the chemical activating enzymes, or that scavenge free radicals generated, might be good candidates for protection against chemically induced liver toxicity (Lee et al., 2004). Also, reports show that many compounds known to be effective against CCl₄-mediated liver injury exert their protective action either via a decreased production of CCl₄-derived free radicals or through antioxidant activity of the protecting agents themselves (Hewawasam et al., 2003).

Thus, in an attempt to delineate the possible mechanisms involved in the hepatoprotective action of J. schimperiana and V. sinaiticum against CCl₄-induced hepatotoxicity in mice, the antioxidant activity of the extracts was investigated using in vitro DPPH assay. The water fraction (A₀) obtained by solid phase extraction of J. schimperiana, and the methanol fraction obtained from Soxhlet extraction of V. sinaiticum, displayed dose-dependent free radical scavenging activity with IC₅₀ values of 51.2 and 41.7 µg/mL, respectively. Therefore, the hepatoprotection of J. schimperiana and V. sinaiticum against CCl₄-induced hepatotoxicity in mice, in part, could be attributed to the free radical scavenging activity of their extracts.

Acute toxicity study

The results of acute oral toxicity studies in mice indicated that the hydro-alcoholic extract of J. schimperiana cause no death or sign of toxicity up to a dose of 1 g/kg of body weight. One of the animals however, showed symptoms of excitation at a dose of 1 g/kg.

Conclusion

The present study suggests that the hydro-alcoholic extracts and methanol fractions of J. schimperiana and V. sinaiticum, at a dose of 200 mg/kg, possess potent hepatoprotective activity in CCl₄-induced liver injury in Swiss albino mice. The methanol extracts of J. schimperiana and V. sinaiticum possess antioxidant activities and may inhibit the deleterious effect of free radicals generated by CCl₄. These observations provide biochemical data supporting the traditional uses of J. schimperiana and V. sinaiticum in Ethiopia for the treatment of some hepatic disorders and suggest the possible utilization of these plants as a source of new drugs.

Acknowledgements

The authors would like to express their utmost gratitude to Professor Franklin Perry, Department of Anatomy and Histopathology, Faculty of Medicine, Addis Ababa University, for carrying out the histo-pathological analysis of the liver and to Melaku Wondafrash of the National Herbarium, Department of Biology, Addis Ababa University, for identification of the plant materials.

Declaration of interest

This work was supported by the Office of Graduate Studies and Research of Addis Ababa University.