Hepatoprotective and antioxidant activity of Leucas aspera against d-galactosamine induced liver damage in rats

Abstract

Context: Whole plant of Leucas aspera (LA) Willd. (Labiatae) is traditionally used in Siddha medicine for hepatic ailments.

Objective: LA was investigated for its hepatoprotective, antioxidant, and protective effect on microsomal drug metabolizing enzymes (MDMEs).

Materials and methods: LA aqueous extract (200 and 400 mg/kg, p.o.) was evaluated for its hepatoprotective and antioxidant activity in d-galactosamine (d-GalN)-induced hepatotoxicity in rats. Biochemical and histopathological studies were performed to assess hepatoprotective activity. Hexobarbitone-induced sleeping time model was used to study the protective effect of LA on MDMEs.

Results: d-GalN administration induced hepatotoxicity in rats which was manifested by increased levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total cholesterol, triglycerides, total bilirubin and oxidative stress. Pretreatment with LA extract significantly protected the liver in d-GalN administered rats. LA extract significantly elevated antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and decreased lipid peroxidation levels in liver. The total phenolic and flavonoid content in LA aqueous extract was found to be 28.33 ± 0.19 gallic acid equivalents mg/g of extract and 3.96 ± 0.57 rutin equivalent mg/g of extract, respectively. LA extract (200 and 400 mg/Kg) treatment with CCl₄ decreased the hexobarbitone-induced sleeping time in mice by 56.67 and 71.30%, respectively, which indicated the protective effect of LA on hepatic MDMEs. Histological studies showed that LA at 400 mg/kg attenuated the hepatocellular necrosis in d-GalN intoxicated rats.

Conclusion: Our results contribute towards validation of the traditional use of LA in hepatic disorders.

Keywords::

• Phenol
• flavonoid
• gallic acid
• rutin

Introduction

Impairment of vital organs like liver leads to serious consequences for the health of an individual and, in the majority of cases, is life threatening. Management of liver diseases is still a challenge to the modern medicine. Leucas aspera (LA) Willd. (Labiatae) is commonly called thumbai in India and is found in open, dry, sandy soil, being a weed it is locally abundant (Meghashri et al., 2010). LA is traditionally used for coughs, cold, painful swelling, and in chronic skin eruptions (Kripa et al., 2011). LA is an ingredient of polyherbal liver-protective Siddha formulations (Siddhar Hepato capsule, Siddhar Pharma, India). Compounds isolated from LA include a hydroxyl tetra triacontan-4-one, aliphatic ketones, nicotine, α-farnesene, α-thujene, menthol from leaf volatiles and amyl propionate, isoamyl propionate from flower volatiles. Long chain aliphatic compounds, sterols, triterpenes, phenols, flavonids such as leucasin have been isolated from LA extract (Meghashri et al., 2010). To provide a scientific rationale of this plant, in this study LA extract was investigated for its hepatoprotective activity in d-galactosamine (d-GalN) induced hepatitis model.

Materials and methods

Materials used

All the materials used in this study were of analytical grade purchased from Himedia Lab Limited, India. Biochemical estimations were carried out using kits purchased from Ecoline Merck Limited, India.

Collection and extraction of Leucas aspera

Whole plant of LA was collected at 2010 during full blooming period and it was authenticated by Dr. P. Venu, Joint Director of Botanical Survey of India, Tamil Nadu Agriculture University, Coimbatore (Voucher specimen no. 864). Whole plant has kept for drying under room temperature, after drying the plant was made in to coarse powder. Aqueous extract of LA was prepared by defatting the whole plant in Soxhlet extractor using petroleum ether and then by soaking the extract in 1 L water for 8 h. This mixture was then dried till the water content reduced to one-fourth of the original mixture. The slurry was filtered and concentrated to obtain aqueous extract of LA. This extraction was carried out according to the procedure reported by Sathayea et al. (2011). The yield of the petroleum ether and aqueous extracts was found to be 2.6 and 8.4% (w/w), respectively.

Total phenolic and flavonoid estimations

Total phenolic and flavonoid content of the LA extract were determined using Folin–Ciocalteu reagent (Chandler & Dodds, 1983) and Dowd method (Arvouet-Grand et al., 1994), respectively.

Acute toxicity study

This study was carried out as per OECD test guideline 423 (OECD, 2001) in Wistar albino rats. The Animal Ethics Committee of the institution approved the study protocol (CPCSEA No. 870/03/C/CPCSEA). The extract fell under class 4 (LD₅₀ > 2000 mg/kg). One-tenth and one-fifth of this dose was selected as the therapeutic dose for the evaluation of hepatoprotective activity (Roy et al., 2010).

Hepatoprotective study

Five groups of adult Wistar albino female rats (200–220 g) containing six animals each was used for this study. Group I served as vehicle control (sodium carboxy methylcellulose 0.3%, 1 mL/kg, p.o.). Group II served as negative control received the vehicle. Group III served as standard received silymarin (100 mg/kg, p.o.) (Jaishree & Badami, 2010). Groups IV and V were treated with aqueous extract of LA at 200 and 400 mg/kg body weight, respectively. All these treatments were given orally for 21 days. On 21st day, the rats of groups II–V received a single dose of d-GalN in distilled water at 400 mg/kg body weight intraperitoneally after 1 h of the vehicle or silymarin or LA extract treatments. On day 22, all the animals were anaesthetized and blood samples were collected through retro-orbital sinus puncture (Najmi et al., 2005). Plasma parameters like alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) alkaline phosphatase (ALP), total cholesterol (TC), triglycerides (TGL), total bilirubin (TB) were estimated. On the same day, rats were sacrificed by cervical decapitation liver samples were dissected out and homogenates (10% w/v) were prepared and centrifuged. The obtained supernatant was used for the estimation of total protein (TP) (Lowry et al., 1951), superoxide dismutase (SOD) (Kakkar et al., 1984), catalase (CAT) (Beers & Sizer, 1952), glutathione peroxidase (GPx) (Paglia & Valentine, 1967) and lipid peroxidation (LPO) (Ohkawa et al., 1979). For histopathological studies, liver sections were prepared, stained with alum hematoxylin and eosin, examined microscopically for histopathological changes.

Hexobarbitone-induced sleeping time

This study was carried out in five groups (n = 6) of adult Swiss albino mice (27 ± 2 g) according to Suja et al. (2004). Group I served as normal control and received vehicle only. Groups II–V were administered CCl₄ (1.5 mL/kg, i.p.) and group II served as negative control. Group III received silymarin (100 mg/kg) and groups IV and V received LA extract (200 and 400 mg/kg, p.o.), respectively, after 1 h of CCl₄ administration. Hexobarbitone (60 mg/kg, i.p.) was administered to all the groups after 2 h of last administration. Time of onset of loss of reflex up to the recovery was taken in minutes as duration of sleep and protective activity of silymarin and LA was calculated by given formula:

Where T is the sleep time; c, d and n are CCl₄, drug (LA and silymarin) and normal groups, respectively.

Statistical analysis

Values are expressed in mean ± SD for six rats in each group. P value was calculated using one way ANOVA followed by Dunnett’s test for multiple comparisons. Values of p < 0.05 were considered significant in all cases.

Results

The total phenolic and flavonoid content in LA aqueous extract was found to be 28.33 ± 0.19 gallic acid equivalents mg/g of extract and 3.96 ± 0.57 rutin equivalent mg/g of extract, respectively. In the acute toxicity studies, LA extract did not show any toxic signs or mortality at 2000 mg/kg dose. The elevated levels of ASAT, ALAT, ALP, TGL, TC, TB in d-GalN intoxication were significantly reduced in the rats pre-treated with LA extract (Table 1). Pre-treatment with LA extract (400 mg/kg) exhibited significant hepatoprotective activity which was comparable with the standard drug silymarin.Table 1 also depicts there is a decrease in the levels of SOD, CAT, GPx and increase in LPO in d-GalN treated rats liver in comparison to vehicle treated group. LA extract pre-treatment dose dependently caused a significant increase in the levels of antioxidants and decreased the LPO levels in comparison to d-GalN treated rats. Histopathological results of d-GalN treated rats liver show the evidence of bridging necrosis and periportal inflammation. Inflammatory infiltrate is composed chiefly of lymphocytes and in some areas, eosinophils are found admixed. LA extract (200 mg/kg) pretreated rats showed low periportal infiltration by eosinophils and LA extract (400 mg/kg) pretreated rats showed no marked hepatocellular necrosis and only mild evidence of eosinophilic infiltration (see supplementary). LA extract treatment with CCl₄ decreased the hexobarbitone induced sleeping time in mice (Table 2).

Table 1.  Effect of Leucas aspera (LA) on biochemical and antioxidant parameters in d-GalN induced hepatotoxicity.

Parameters	ALAT(IU/L)	ASAT(IU/L)	ALP(IU/L)	TC(mg/dL)	TGL(mg/dL)	TB(mg/dL)	TP(g/dL)	SOD(U/mg protein)	CAT(U/mg protein)	GPx(U/mg protein)	LPO(nmol of MDA/mg protein)
Control	60.10 ± 5.06	146.81 ± 14.22	147.60 ± 38.17	34.40 ± 6.41	44.20 ± 10.53	0.47 ± 0.11	7.51 ± 0.62	4.96 ± 1.86	13.00 ± 1.80	10.73 ± 1.17	176.00 ± 22.61
d-GalN (400 mg/kg)	112.15 ± 30.61**	549.74 ± 64.70**	664.33 ± 79.70**	76.52 ± 18.85*	98.22 ± 18.38*	1.34 ± 0.30*	5.38 ± 0.41*	1.16 ± 0.30**	4.56 ± 0.92**	5.06 ± 0.85*	400.70 ± 70.87**
Silymarin (100 mg/kg)	66.83 ± 5.59^‡	174.86 ± 25.50^‡	249.08 ± 40.84^†	47.08 ± 4.03^†	49.17 ± 10.08^†	0.51 ± 0.17^†	6.97± 0.32^#	3.93 ± 0.38^†	10.70 ± 2.09^†	9.13 ± 1.81^†	216.30 ± 28.75^‡
LA (200 mg/kg)	91.45 ± 13.86^#	352.04 ± 37.03^†	350.87 ± 60.17^#	59.61 ± 17.48^#	56.17 ± 17.08^#	0.82 ± 0.13^#	6.01 ± 0.45	3.40 ± 0.44^†	9.83 ± 3.56^#	8.50 ± 1.24^#	286.30 ± 30.27^†
LA (400 mg/kg)	72.36 ± 8.71^†	214.84 ± 44.33^‡	302.00 ± 51.90^†	51.38 ± 10.05^#	45.25 ± 11.37^†	0.67 ± 0.14^#	6.74 ± 0.42^#	4.13 ± 0.78^†	11.53 ± 1.82^†	10.23 ± 1.02^†	198.70 ± 17.75^‡

Data were analysed by ANOVA followed by Dunnett’s test. Each value is the mean ± SD; n = 6.

ALAT, alanine aminotransferase; ALP, alkaline phosphatase; ASAT, aspartate aminotransferase; CAT, catalase; GPx, glutathione peroxidase; LPO, lipid peroxidation; MDA, Malondialdehyde;  SOD, superoxide dismutase; TB, total bilirubin; TC, total cholesterol; TGL, triglycerides; TP, total protein.

*p < 0.01 and **p < 0.001, when compared with control group.

^#p < 0.05, ^†p < 0.01, and ^‡p < 0.001, when compared with d-GalN group.

Table 2.  Effect of Leucas aspera (LA) aqueous extract on hexobarbitone-induced sleeping time in CCl4 intoxicated mice.

Groups	Sleeping time (minutes)	Hepatoprotective activity (%)
Normal control	24.00 ± 6.87	–
CCl4 control (1.5 mL/kg, i.p.)	124.30 ± 15.42*	–
Silymarin (100 mg/kg)	41.62 ± 7.84^‡	82.43
CCl4 + LA (200 mg/kg)	67.45 ± 10.34^†	56.67
CCl4 + LA (400 mg/kg)	52.78 ± 5.43^†	71.30

Data were analysed by ANOVA followed by Dunnett’s test.

Each value is the mean ± SD; n = 6.

*p < 0.001, when compared with control group.

^†p < 0.01 and ^‡p < 0.001 denotes, when compared with CCl₄ group.

Discussion

Elevated levels of ALAT, ASAT, ALP enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver (Jadon et al., 2007). Pre-treatment with LA extract decreased the level of hepatic enzymes towards their respective normal value is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage caused by d-GalN. The observed increase in cholesterol level might be due to the inability of the diseased liver to remove cholesterol from circulation. TGL accumulation in plasma of d-GalN administered rats might be from an imbalance between the rate of synthesis and the rate of release of TGL by the parenchymal cells into the systemic circulation (Pushpavalli et al., 2010). An increase in TB and ALP reflects the pathological alteration in biliary flow (Ravikumar et al., 2005). LA extract mediated suppression of the increased TB level suggests the possibility of the extract being able to stabilize biliary dysfunction. These biochemical findings were further substantiated by histopathological studies. Pre-treatment with LA extract caused an elevation of SOD, CAT, GPx and decreased LPO in liver suggesting that it restored antioxidant mechanisms in the damage caused by d-GalN. It has been found that there was a direct relationship between antioxidant activity and total flavonoid, phenolic content which was correlated to their chemical structures (Sun et al., 2002). Hexobarbitone is metabolized by hepatic microsomal drug metabolizing enzymes (MDMEs) and duration of hexobarbitone induced sleep in intact animals is considered as a reliable index for the activity of hepatic MDMEs. Prolongation in hexobarbitone induced sleep time after CCl₄ toxicity substantiated decreased availability of CYP2E1 contents (Sharma & Shukla, 2011). LA extract shortened this prolongation of hexobarbitone sleep time suggesting its protective effect on CYP2E1 system. The histopathological studies are direct means for assessing the protective effect of the drug. The groups received d-GalN alone, showed severe hepatotoxicity with heavy infiltration of inflammatory cells around portal tract and in the liver parenchymal cells. Pre-treatment with LA extract protected the rat livers from d-GalN induced necrosis and periportal inflammation. The results of the histopathological studies supported and well correlated with data obtained from evaluation of the biochemical parameters which indicates the hepatoprotective activity of LA aqueous extract. Since LA is rich in flavonoids and phenolics, the possibility of the mechanism of hepatoprotection of LA extract may be due to its antioxidant action.

Conclusion

The present study provides a scientific rationale for the traditional use of this plant in the management of liver disorders. The isolation and characterization of the extracts of LA and screening of isolated compounds against the liver damage has to be carried out to identify an efficient and potent hepatoprotective drug.

Declaration of interest

The authors report no conflicts of interest.