Hepatoprotective Effect of Crude Extract and Isolated Lignans of Justicia simplex. Against CCl₄-Induced Hepatotoxicity

Abstract

The lignan-rich petroleum ether extract and the isolated lignans, simplexolin and sesamolin, of Justicia simplex. D. Don (Acanthaceae) were tested for protective effect on CCl₄-induced hepatotoxicity in rats. The induced CCl₄ toxicity elevated the levels of marker enzymes alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and stimulated lipid peroxidation along with decrease in triglycerides and glutathione content. The pretreatment with the extract and the isolated lignans at the dose of 100 mg/kg p.o. and 10 mg/kg p.o., respectively, normalized these toxic levels. The effect of the isolated lignans was comparable with the standard (Nirocil). This showed the hepatoprotective effect of petroleum other extract and lignans of J. simplex..

Keywords:

• CCl₄
• glutathione
• hepatoprotection
• Justicia simplex
• lignans
• LPO
• transaminases

Introduction

Justicia simplex. D. Don (Acanthaceae) is a well-known traditional medicinal plant, native to the Western Himalayas, and used as an antifatigue and stimulating agent. Our earlier studies revealed the presence of lignans and triterpenoid saponins as major chemical constituents of this plant (Ghosal et al., 1980). We have also reported that glucosylated furofurano lignans isolated from petroleum ether and ethanol extracts are responsible for the antistress activity exhibited by this plant. Justicisaponin-I, a triterpenoid saponin isolated from the ethanol extract of Justicia simplex., showed antispermicidal action (Ghosal et al., 1981). Lignans isolated from the Justicia. genus are well-known for their antitumor, antiplatelet, antiviral, and antidepressant activities (Rios et al., 2002). However, there exists no scientific investigation on the hepatoprotective effect of Justicia simplex.. In our continuing search to evaluate the medicinal uses of this plant, petroleum ether extract of Justicia simplex. whole plant and the isolated compounds were tested for protective action in CCl₄-induced liver damage in albino rats. CCl₄ toxicity is the model commonly used to study liver injury (Ko et al., 1993), and the intoxication induces necrosis and steatosis of the liver (Rubin et al., 1963). Liver is a vital organ involved in metabolism of foreign compounds entering the body. Human exposure to toxic substances and or intake of contaminated food causes liver toxicity. Conventional drugs used to cure liver toxicity are inefficient, and there is a need to find drugs that possess improved liver protection. The versatile nature of Justicia simplex. as a medicinal plant encouraged us to undertake the current study.

Materials and Methods

Plant material, extraction, and isolation

Justicia simplex. was collected from Ranikhet and was identified by the botanist from the Department of Botany, Banaras Hindu University, Varanasi, India. A voucher specimen has been preserved in the Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Banaras Hindu University. The whole plant (2 kg) was dried, milled, and continuously extracted with petroleum ether (fraction 60–80°C) in a Soxhlet apparatus for 48 h. The extract was evaporated until the solvent was completely removed. The extract gave a yellow gummy material (56 g). This extract was processed in the same way as previously reported for the isolation of simplexolin and sesamolin (Fig. 1), and the compounds were identified by direct comparison with the authentic sample (mmp and co-TLC) and spectroscopic data (MS, IR, and ¹H NMR) (Ghosal et al., 1979).

Figure 1 Structures of simplexolin and sesamolin.

Animals

Male albino rats (Charles foster strain) weighing 100–150 g were obtained from the Central Animal House (reg. no. 542/02/ab/CPCSEA), Institute of Medical Sciences, Banaras Hindu University. Three rats per cage were kept and maintained on a 12-h light/dark cycle at an ambient temperature. The animals had free access to standard pellet diet (M/s Hindustan Lever Ltd, Mumbai, India) and water ad libitum..

Chemicals

Ellman's reagent was obtained from Sisco Laborotaries (Varanasi, India). Thiobarbituric acid (TBA) and 1,1, 3,3-tetraethoxypropane (TEP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade. Span diagnostic kits were used for the spectrometric enzyme assays.

Experimental design

The experimental animals were divided into six groups of six animals each. Group I was marked as control. In group II (intoxicated), hepatotoxicity was induced by oral administration of CCl₄ (2 mL/kg in 50% paraffin) 48 h before decapitation. Group III served as standard and was given the herbal preparation Nirocil (Solumicks Division, Bombay, India) at the dose of 100 mg/kg, p.o. Groups IV, V, and VI were given petroleum ether extract (JSE; 100 mg/kg, p.o.), simplexolin (10 mg/kg, p.o.), and sesamolin (10 mg/kg, p.o.) as 0.3% suspension in carboxyl methyl cellulose (CMC), respectively, for 6 consecutive days. The dose of the extract was ascertained by pilot studies over a range of dosages varying from 50 to 200 mg/kg, and also treatment was done with the isolated compounds over the dosage range 5–20 mg/kg. The plant extract and the isolated compounds did not show any toxicity over the tested dosage range. On day 7, animals were sacrificed by decapitation. The blood was collected by cardiac puncture and centrifuged to separate the serum. Serum was used for the estimation of ALT (alanine aminotranerase) (Mohur & Cook, 1957), AST (aspartate aminotransferase) (Mohur & Cook, 1957), ALP (alkaline phosphatase), LPO (lipid peroxidation) (Ohkawa et al., 1979), total protein (Lowry et al., 1951), and TG (triglycerides) using the spectrometric diagnostic kits.

The liver tissue was excised immediately and washed with ice-cold saline. A portion of liver tissue was homogenized with potassium chloride (1.15% w/v), and the homogenate was used for the estimation of lipid peroxidation (Ohkawa et al., 1979) and hepatic GSH (glutathione) (Sedlak & Lindsay, 1986).

Statistical analysis

Statistical analysis was done by Student's t.-test. The results were considered significant if p < 0.05.

Results

Hepatic toxicity induced by CCl₄ treatment

The hepatic damage induced by CCl₄ was significant as evidenced from significant (p < 0.001) increase in serum levels of ALT, AST, ALP, and LPO compared with normal rats as given in Table 1. The levels of total protein and TG were also decreased significantly (p < 0.001) in intoxicated rats than in the normal rats (Table 1). CCl₄ toxicity significantly decreased (p < 0.001) the hepatic GSH level and showed an increase (p < 0.001) in LPO compared with the normal group (Table 2).

Table 1. Effect of the Justicia simplex. crude extract and isolated lignans on the biochemical parameters of CCl₄-intoxicated rats (n = 6, mean ± SEM)

Groups	Dose	ALT (U/L)	AST (U/L)	ALP (U/L)	LPO (nmol/mL)	TG (mg/dL)	Total protein (g/dL)
Control	—	20.61 ± 2.5	32.7 ± 3.2	22.56 ± 1.6	25.21 ± 2.1	52.43 ± 2.2	8.22 ± 0.31
CCl4	2 mL/kg	122.21 ± 5.2^a	152.18 ± 2.4^a	84.78 ± 1.8^a	42.31 ± 1.8^a	26.22 ± 1.8^a	5.23 ± 0.24^a
Std + CCl4	100 mg/kg	80.64 ± 3.2^b	120.24 ± 1.2^b	30.22 ± 1.8^b	23.42 ± 1.6^b	51.56 ± 1.4^b	7.92 ± 0.82^b
JSE + CCl4	100 mg/kg	72.63 ± 5.3^b	98.16 ± 3.5^b,c	30.19 ± 2.8^b	22.82 ± 1.2^b	50.32 ± 1.6^b	7.24 ± 0.12^b
Simplexolin + CCl4	10 mg/kg	68.23 ± 3.2^b,c	94.32 ± 2.1^b,c	28.02 ± 1.4^b	21.64 ± 0.8^b	49.32 ± 0.8^b	7.36 ± 0.18^b
Sesamolin + CCl4	10 mg/kg	64.5 ± 3.8^b,c	90.86 ± 2.1^b,c	24.43 ± 2.6^b	20.92 ± 1.8^b	47.22 ± 1.2^b	7.52 ± 0.09^b

a = p < 0.001 when compared with normal group; b = p < 0.001 when compared with CCl₄ groups; c = p < 0.001 when compared with standard.

Table 2. Effect of Justicia simplex. crude extract and isolated lignans on glutathione depletion and lipid peroxidation in CCl₄-intoxicated rats (n = 6, mean±SEM)

Groups	Dose	GSH (mmol/g tissue)	LPO (nmol/g of wet tissue)
Control	—	5.82 ± 2.4	362 ± 3.2
CCl4	2 mL	1.82 ± 1.2^a	594 ± 2.4^a
Std + CCl4	100 mg/kg	3.70 ± 2.0^b	372 ± 1.2^b
JSE + CCl4	100 mg/kg	2.80 ± 1.8^b,c	394 ± 3.5^b,c
Simplexolin + CCl4	10 mg/kg	2.40 ± 1.6^b,c	384 ± 2.1^b,c
Sesamolin + CCl4	10 mg/kg	2.21 ± 1.8^b,c	386 ± 2.1^b,c

a = p < 0.001 when compared with normal group; b = p < 0.001 when compared with CCl₄ groups; c = p < 0.001when compared with standard.

Protective effect of pretreatment with the lignans

Pretreatment of rats with JSE and isolated simplexolin and sesamolin decreased the levels of hepatic enzymes, serum and hepatic LPO and increased the levels of serum protein, TG, and hepatic GSH at a significant level (p < 0.001) indicating the protective effect of the lignans under study in the pretreated rats. (Tables 1 and 2).

Discussion

Administration of CCl₄ caused hepatic damage due to the free radical formed from CCl₄ by the activation of the NADPH-Cyt P450 system of liver endoplasmic reticulum (Reckangale et al., 1974). This led to functional and morphologic changes in the cell membrane and resulted in leakage of hepatic enzymes. When compared with other toxins, CCl₄ is known to cause hepatic damage with a marked increase in blood serum transaminases and phosphatase activity (Tiegs & Wendel, 1988; Dwivedi et al., 1990). The oxidation of fatty acids by free trichloromethyl radical () liberates lipid peroxides (Reckangale & Ghosal, 1966). The current study also proves these effects of CCl₄ toxicity by the marked increase in serum hepatic enzymes and LPO levels (Table 1). Impairment of hepatic antioxidant status by CCl₄ was evident by the increase in tissue LPO and decrease in GSH levels. Depletion of available GSH resulted in enhanced LPO and decreased GST activity (Anandan et al., 1999) as shown in Table 2.

Pretreatment with JSE (100 mg/kg) and simplexolin (10 mg/kg) and sesamolin (10 mg/kg) significantly (p < 0.001) normalized these enzyme levels compared with the intoxicated rats (Table 1). The pretreated rats showed significant (p < 0.001) hepatoprotective effect to CCl₄ toxicity by decreased tissue susceptibility to peroxide-induced toxicity as supported by decrease in tissue lipid peroxide and GSH levels (Table 2). It has been suggested that methylenedioxy group in certain lignans is a critical feature for antihepatotoxic activity and could act through inhibition of lipid peroxidation by a nonenzymatic step (Kiso et al., 1985). The lignans used in this study also contained methylenedioxy group, which could be responsible for the protective effect exerted by these lignans. Previous studies have revealed that furofuran lignans like sesamolinol and schisanhenol have exhibited antioxidant properties through their metabolites (Potterat, 1997). The in vivo. metabolites of lignans used in the current study might also be responsible for their hepatoprotection through their antioxidant potential. The exact mechanism and structural feature responsible for the hepatoprotection needs further investigation. There are many reports about hepatoprotective effect of lignans but this is the first report of hepatoprotection by an aryloxy furofuran lignan isolated from Justicia simplex..

Acknowledgment

We are thankful to UGC, New Delhi, India, for a Senior Research Fellowship awarded to Mrs. S. Jasemine.