INTRODUCTION

Liver, the largest organ in vertebrate body, is the major site of intense metabolic activities. Liver injury caused by toxic chemicals and certain drugs has been recognized as a toxicological problem. Ayurvedic system of medicine assigns much importance to the pharmacological aspects of many plants (Venukumar and Latha, 2002). The hepatoprotective activity of Justicia gendarussa (JG) has not been evaluated scientifically to substantiate its ancient literature regarding its usefulness as hepatoprotective herb (Pandey, 2005). So, this study was conceptualized to evaluate its hepatoprotective property.

Justicia gendarussa considered as native of China and it is frequently grown in Indian gardens as hedge or border plant; it is sometimes found as an escape. It is propagated by cuttings and grows quickly. It is hardy, withstands heavy rainfall and thrives in shade. It is found to be useful in bronchitis, inflammations, vaginal discharges, dyspepsia, tympanitis, eye diseases, fevers according to its ethnobotanical reports. Decoction of the root boiled in milk is given in rheumatism, dysentery and jaundice and the leaf and tender shoots are diaphoretic, given in the form of decoction in chronic rheumatism. Fresh leaves are used topically in edema and beriberi. It is reported that the flowering head along with the portion of leaf is used as demulcent and astringent. The leaves are reported to contain a bitter and slightly toxic alkaloid. A decoction of alcoholic extract of the roots produced slight paralysis in rats in doses of 1-2 g kg^-1 body weight. and in doses of 10-20 g kg^-1 it is antipyretic and depressant producing violent diarrhea eventually death (Chopra et al., 1956; Kirtikar and Basu, 2005; Publication and Information Directorate, 1959).

Chakravarty et al. (1982) isolated four simple o-disubstituted aromatic amines from the leaf of JG and characterized these constituents as 2-amino benzyl alcohol, 2-(2'-amino-benzylamino) benzyl alcohol and their respective 0-methyl ethers 1 and 2 from 'H NMR and mass spectral analyses of the bases and their acetates. Duke (2004) reported four compounds present in the plant namely 2-(2'-aminobenzylamino)-benzyl-alcohol, 2-(2'-aminobenzylamino)-benzyl-alcohol-o-methyl-ether, 2-amino-benzyl-alcohol, 2-amino-benzyl-alcohol-o-methyl-ether along with β-sitosterol. The various extracts of leaf were studied for it’s anti-inflammatory and antioxidant (Devprakash, 2000), reverse transcriptase inhibitory (Woradulayapinij et al., 2005), analgesic activities (Ratnasooriya et al., 2007) and antioxidant potential (Mruthunjaya and Hukkeri, 2007).

MATERIAL AND METHODS

Plant Material
Leaf samples were collected from the Veer Narmad South Gujarat University Campus, Surat, India, in the month of September 2007 and sample was identified and authenticated by Dr. Minu Parabia, Professor and Head, Bioscience Department. A specimen sample (KLKJG01) was deposited in the Department.

Preparation of the Extracts
Shade dried leaves of about 500 g was subjected for size reduction to coarse powder. The powder was extracted with 5 L methanol (analytical grade) using soxhlet apparatus till the exhaustion for about 24 h (MLE). Marc obtained after methanolic extraction was extracted with pure distilled water by reflex (WE). Both the extracts were concentrated under vacuum to get the residues. The percentage yield of methanolic leaf extract and water extract were found to be 12.72 and 1.83%.

In vitro Antioxidant Activities
The extracts were subjected for the preliminary phytochemical studies to know the phytochemical nature of the extracts (Finar, 2006; Khandelwal, 2006; Kokate et al., 1996; Onwukaeme et al., 2007; Evans, 2005).

Determination of Total Phenolics
The total phenolic content of both the extracts were determined by using Folin-Ciocalteu’s assay (Marinova et al., 2005). An aliquot (0.4 mL) of extract or standard solution of gallic acid (1, 5, 10, 15 and 20 μg mL^-1) was added to 10 mL volumetric flask, containing 3.6 mL of double distilled water. Reagent blank using double distilled water was prepared. Folin Ciocalteu’s phenol reagent (0.4 mL) was added to the mixture and shaken. After 5 min, 4 mL of 7% Na₂CO₃ solution was added to the mixture. The solution was diluted to volume (10 mL) with double distilled water and mixed. After incubation for 90 min at room temperature, the absorbance against reagent blank was determined at 750 nm with an UV-Visible spectrophotometer Shimadzu 1700. Total phenolic content of various extracts were expressed as mg Gallic Acid Equivalent (GAE)/100 g of extract. All samples were analyzed in triplicate.

Free Radical Scavenging Activity by DPPH Method
Free radical scavenging potentials of the extracts were tested against a methanolic solution of α, α-diphenyl-β-picryl hydrazyl using the method of Choi et al. (2002). Stock solutions (1 mg mL^-1) of both the extracts were diluted to final concentrations of 50 to 300 μg mL^-1 in case of MLE and 300 to 800 μg mL^-1 in case of WE in methanol. A total of 0.3 mM methanolic solution of DPPH was added to 2.5 mL of sample solution of different concentration and allowed to react at room temperature. After 30 min, the absorbance values were recorded at 518 nm and converted into the percentage of scavenging capacity using the following equation:

Methanol (1 mL) and extract solution (2.5 mL) was used as a blank, while DPPH solution plus methanol was used as a negative control. The positive controls were DPPH solution plus 1 mL of standard flavonoids (Ascorbic acid, Butylated Hydroxy Toluene and Gallic acid).

The EC₅₀ values were calculated by linear regression of plot, where the abscissa represents the concentration of tested plant extract or flavonoids and the ordinate represents the average percentage scavenging capacity from three replicates.

Reduction of Ferric Ions
The reaction mixture containing o-phenanthroline (0.5 mg), ferric chloride (0.2 mM) and test compounds (extracts/standard) dissolved in 0.2 mL ethanol in a final volume of 5 mL was incubated for 10 min at ambient temperature. The absorbance at 510 nm was measured. In another experiment, sodium dithionate (0.3 mM) was added instead of the test compound and the absorbance obtained was taken as equivalent to 100% reduction of all the ferric ions present (Rajakumar and Rao, 1993). All experiments were carried out in triplicate and percentage ferric ion reduction activities of various extracts were reported as Mean±SD.

The reduction of ferric ion in presence of EDTA also determined as the method of Mruthunjaya and Hukkeri (2007).

Hydrogen Peroxide Scavenging Activity
Hydrogen peroxide scavenging ability of extracts was determined by simple UV spectroscopic method. Different concentrations of extracts in the range of 50-200 μg mL^-1 were taken. Volume adjusted to 3 mL with phosphate buffer and 1 mL of 30 mM H₂O₂ was added. After 10 min, the absorbance value of the reaction mixture was recorded at 230 nm. Blank solution was containing the phosphate buffer without H₂O₂ (Kaur et al., 2006). The percentage of scavenging activity was calculated by using below formula:

where, A₀ is the absorbance of the control and A₁ is the absorbance of the sample.

Estimation of Total Flavonoids
Total flavonoid content present in both the extract was determined by the aluminium chloride colorimetric assay. An aliquot (0.5 mL) of extract or standard solution of quercetin was added to test tube containing 2 mL of double distilled water. To the test tube was added 0.15 mL of 5% Sodium nitrite. After 5 min, 0.15 mL of 10% aluminium chloride solution was added. At 6th min, 2 mL of 1 M Sodium hydroxide was added and the total volume was made to 5 mL with DD water. The solution was mixed well and the absorbance was measured against reagent blank at 510 nm. Total flavonoid content of the extracts was expressed as mg of quercetin equivalent to 1 g of the extract. All experiments were done in triplicate and total flavonoid content was reported as Mean±SD (Marinova et al., 2005).

Animal Studies
Experimental Animals
Thirty six of both sex and ten of female albino rats of Wistar strain weighing 150-220 g were procured from the Central Animal Facility Centre of JSS Medical College, Mysore, India,. The animals were housed in polypropylene cages and maintained in controlled temperature (27±2°C) and light cycle (12 h light and 12 h dark). They were fed with feed (Hindustan Animal Feeds) and water ad libitum. The animals were acclimatized with the laboratory conditions for a week period. Ethical clearance for the use of animals was obtained from the IAEC of JSS College of Pharmacy, Mysore, Karnataka, India (Proposal No. 029/2008, dated 25th July 2008).

Acute Toxicity Studies
Acute toxicity study of MLE of JG was done as per OECD guideline 425 using albino female rats. The animals were kept fasting for overnight providing only water, after which the extract was administered orally for one animal at the limit dose of 2000 mg kg^-1 and observed for 14 days (with special attention for the first 4 h of administration followed by the next 20 h). If the animal dies, the limit test was terminated and main test was conducted. If the animal survives, four additional animals were dosed sequentially so that five animals are tested. However, if three animals died, the limit test was terminated and the main test was performed. The LD₅₀ is greater than 2000 mg kg^-1 if three or more animals survived. If an animals unexpectedly dies late in the study and there are other survivors, it is appropriate to stop dosing and observing all animals to see if other animals will also die during a similar observation period. The same method was adopted at limit dose of 5000 mg kg^-1.

Hepatoprotective Activity
Rats were divided into six groups of six animals each as follows: Group 1 served as control and received oral administration of 2.5% gum acacia (vehicle) at the dose of 1 mL kg^-1 body weight. Group 2 is hepatotoxic rats and received oral administration of vehicle plus CCl₄ (1 mL/kg b.wt. in 1:2 Olive oil). Group 3 received silymarin at the dose of 100 mg kg^-1 body weight plus CCl₄ as group 2 animals and considered as standard drug treated animals. Group 4, 5 and 6 received the MLE at the three (I, II, III) dose level of 150, 300 and 500 mg kg^-1 body weight plus CCl₄ as group 2 animals. All treatments were given once daily for seven days. Samples were prepared as suspensions using 2.5% gum acacia.

On the 7th day, all group of animals except group 1 were given a single dose of CCl₄ (1 mL kg^-1 b.wt.) in 1:2 Olive oil after 6 h of last dose administration. The group 1 animals were given 2.5% gum acacia and olive oil in 1:2 ratio of 1 mL kg^-1. Animals were sacrificed 24 h after the last dose and blood was collected by carotid bleeding (Kaur et al., 2006). The liver was isolated and weighed for the weight variation studies among different groups.

Biochemical Estimation
Blood was centrifuged; serum was separated and used for estimation of biochemical parameters. Serum Glutamic Oxaloacetic Transaminase (SGOT) and Serum Glutamic Pyruvic Transaminase (SGPT) were estimated by Thefeld et al. (1974), method serum alkaline phosphatase (SALP), bilirubin by Winsten and Chelyk (1969) method and total protein by colour complexation with copper ion in an alkali solution (Gornall et al., 1949). All the determinations were carried out using standard diagnostic kits (Agappe Diagnostic Pvt. Ltd, Kerala, India) by using fully automated Biochemical Analyzer ChemWell, manufactured by Awareness Technologies Inc, USA.

The wet weight of liver of all animal were calculated and compared with that of vehicle and CCl₄ treated animal.

Statistical Analysis
All values are expressed as Mean±SEM. Statistical analysis were performed by one-way Analysis of Variance (ANOVA) and individual comparisons of the group mean values were done using Tukey's Multiple Comparison Test, with the help of Graph Pad prism 4.0 software. The value of P lower than 0.05 were considered as significant (P is probability).

RESULTS AND DISCUSSION

Preliminary phytochemical investigation has revealed the presence of flavonoids, phenolic compounds, cardiac glycosides, reducing sugars and terpenoids in MLE.

Total Phenolic Content
This is based on the principle that polyphenols when react with FOLIN reagent give blue colour chromogen in alkaline media, which can be measured at 760 nm. The concentration of polyphenols in extracts is calculated by using standard curve prepared with gallic acid. The total phenolic content of MLE of JG was found to be 164.16±3.00 mg gallic acid equivalent per g of extras. The value for WE was found to be 29.44±3.46 mg gallic acid equivalent per g of extract (Table 1), the amount of polyphenols present in the WE is very less when compared to the MLE.

Total Flavonoid Content
The total flavonoid content of MLE is more when compare to WE. The value of MLE was found to be 54.33±4.50 mg equivalent of quercetin per g of extract. Where as the values was found to be 26.17±0.94 mg equivalent of quercetin per g of extract for WE. The potent antioxidant activity of MLE may be due to the presence of more amount of flavonoid content (Table 1).

Free Radical Scavenging Activity by DPPH Method
As shown in the Table 2, MLE strongly scavenged DPPH radical with the IC₅₀ value of 222.68±1.26 μg mL^-1. The scavenging was found to be dose dependent. Where as ascorbic acid, gallic acid and Butylated Hydroxyl Toluene (BHT) used as standards were shown IC₅₀ value of 4.17±0.5, 1.86±0.0 and 29.08±0.52 μg mL^-1, respectively. 

Table 1:	Total phenolic and flavonoid content of JG leaf extracts
Values are Mean±SD

Table 2:	Percentage free radical scavenging activity of JG leaf extracts, Ascorbic acid, Gallic acid and BHT in DPPH Method
MLE: Methanolic leaf extract, WE: Aqueous extract, As: Acid-ascorbic acid, BHT: Butylated hydroxy toluene

DDPH radical react with suitable reducing agents then losing colour stiochiometrically with the number of electrons consumed which is measured spectrophotometrically at 517 nm. Ascorbic acid is a potent free radical scavenger and BHT is known antioxidant and is used as preservative (Singh et al., 2002; Mathew and Abraham, 2006). So, when compared to the pure compounds, IC₅₀ value of 222.68±1.26 of MLE of JG is moderately high and shows that JG is moderately potent DPPH free radical scavenger. Where as the DPPH free radical scavenging activity of WE was found to be very less (455.0±5.0 μg mL^-1), this may be due the presence of more phenolic/flavonoid content of the MLE than in the WE.

Ferric Ion Reduction Potential
Extracts react with Fe³⁺ to reduce and convert it to Fe²⁺. The degree of coloration indicates the reduction potential of the extracts. The change in the absorbance produced at 510 nm has been used as a measure of Ferric ions reducing activity. The reduction is measured taking Sodium dithionite instead of the extract and considered as equivalent to 100% reduction of all the ferric ions present. Fe²⁺ reacts rapidly with 1, 10- O-phenanthroline and forms orange coloured complex which is exceptionally stable. This complex has a strong absorption in the visible spectrum at a wavelength of 510 nm (Rajakumar and Rao, 1993). The reduction of ferric ion in presence of EDTA also determined by the method of Mruthunjaya and Hukkeri (2007). As shown in Table 3, in absence of EDTA, MLE has shown 50.92±0.23 % activity at 1000 μg concentration with IC₅₀ value of 983.86±1.27, where as WE has less % of reduction, 48.00±0.15% at the same concentration. IC₅₀ value of WE was not detected, where as IC₅₀ value of standards ascorbic acid and gallic acid were found to be 27.57±0.32 and 6.93±0.42 μg, respectively. But in the presence of EDTA neither the extract nor the standards were shown ferric ion reduction activity. These result indicates that the JG extracts were reduced the ferric ions moderately, when compared to the standard ascorbic acid and gallic acid.

Hydrogen Peroxide Scavenging Activity
H₂O₂ in phosphate buffer has the λ_max of 230 nm. In control tubes the absorbance will be only due to H₂O₂. In presence of extracts, the reduction of absorbance at 230 nm indicates scavenging or breakdown of H₂O₂. WhenH₂O₂ is scavenged or breakdown occurs the absorbance λ_max will be changed from 230 nm. As shown in the Table 4. MLE has scavenged the hydrogen peroxide to the extent of 35.92±1.55% at 200 μg mL^-1 concentration, where as WE has shown 27.18±0.88% at the same concentration. These results show that, JG extracts are mild hydrogen peroxide scavenger.

Table 3:	Percentage Ferric ion reduction activity of JG leaf extracts, ascorbic acidand gallic acid
Concentration is the total extract/standard present in the reaction mixture in μgs. ND: Not detected at the tested level. No activity was detected for the extracts/standards in presence of EDTA

Table 4:	Percentage of hydrogen peroxide scavenging activity of JG leaf extracts

Acute Toxicity Test
The MLE of JG extract has not shown any mortality at the limit dose of 2000 and 5000 mg kg^-1 b.wt. MLE was found to be safe even at the higher concentration, based on this the dose for the hepatoprotective activity were chosen.

CCl₄ Induced Hepatotoxicity
The ability of a hepatoprotective drug to reduce the injurious effects or to preserve the normal hepatic physiological mechanisms, which have been disturbed by a hepatotoxin, is the index of its protective effect. Protection of hepatic damage caused by carbontetrachloride administration has been widely used as an indicator of liver protective activity of drugs in general (Clauson, 1989). CCl₄ mediated hepatotoxicity was chosen as the experimental model. It has been established that, CCl₄ is accumulated in hepatic parenchyma cells and metabolically activated by cytochrome P₄₅₀ dependent monooxygenases to form a trichloromethyl radical (CCl₃). The CCl₃ radical alkylates cellular proteins and other macromolecules with a simultaneous attack on polyunsaturated fatty acids, in the presence of oxygen to produce lipid peroxides, leading to liver damage (Bishayee et al., 1995). Thus, antioxidant or free radical generation inhibition is important in protection against CCl₄ induced liver lesion (Castro et al., 1974). Serum Glutamic Oxaloacetic Transferase, Serum Glutamic Pyruvic Transaminase, alkaline phosphatase, total and direct bilirubin in plasma have been reported to be sensitive indicators of liver injury (Molander et al., 1955). The disturbance in the transport function of the hepatocytes as a result of hepatic injury, causes, the leakage of enzymes from cells due to altered permeability of membrane. This results in decreased levels of SGOT, SGPT, bilirubin and alkaline phosphatase in the hepatic cells and a raised level in serum. The present study revealed a significant increase in the activities of SGOT, SGPT, SALP and serum bilirubin after exposure to the CCl₄, indicating considerable hepatocellular injury.

The MLE has not shown any mortality at maximum acute dose of 5000 mg kg^-1 in the acute toxicity studies. The maximum dose selected for the in vivo hepatoprotective activity was one tenth (500 mg kg^-1) of the maximum acute dose tested.

The results obtained in the biological experiment are presented in Table 5 and 6. The effect of MLE of JG was assessed using in vivo model of CCl₄ induced hepatotoxicity by the acute oxidative stress. CCl₄ afflicts acute oxidative injury to the liver causing oxidative damage and other changes around the central vein in the liver that leads to the leakage of the marker enzymes such as SGOT and SGPT in the serum (Recknagel, 1967).

Table 5:	Show the hepatoprotective activity of MLE of JG leaf on CCl4 induced hepatotoxicity
Values are Mean±SEM. n = 6 animals in each group, *Highly significant reduction compared to hepatotoxin (CCl4) (p<0.001), **Significant reduction compared to hepatotoxin (CCl4) (p<0.05)

Table 6:	Shows the effect of JG leaf extract on wet liver weight on CCl4 induced hepatotoxicity
Values are Mean±SEM. n = 6 animals in each group, *Highly significant reduction compared to hepatotoxin (CCl4) (p<0.001), **Significant reduction compared to hepatotoxin (CCl4) (p<0.05)

The activities of SGOT and SGPT after 24 h of oral administration of CCl₄ at the dose of 1 mL kg^-1 body weight are depicted in Table 5 and 6. As can be seen, CCl₄ administration resulted in a significant elevation in the level of both SGOT (527.76% of the vehicle treated control) and SGPT (776.09% of the vehicle treated control). Administration of MLE at the 150 and 300 mg kg^-1 body weight for seven days prior to CCl₄administration dose dependently restored the activities of SGOT and SGPT (Fig. 1, 2). The maximum reduction in the elevated biomarker enzymes (SGOT/SGPT) were found to be at dose level II (300 mg kg^-1) and percentage of reduction was 58.45 and 30.71%, respectively. The extract at dose level of 500 mg kg^-1 body weight, shown the reduction in the hepatoprotective activity and further studies are required to elucidate the exact mechanism. These results indicate JG leaf possesses moderate antihepatotoxic activities at dose of 300 mg kg^-1. The total and direct bilirubin content present in the serum after 24 h of CCl₄ exposure is given in the Table 5. As shown in the Fig. 3 and 4 the MLE at the dose of 300 mg kg^-1 has shown maximum activity in reducing the amount of bilirubin in the serum. The percentage of reduction was found to be 73.71 and 31.70%, respectively for total and direct bilirubin, where as the value for the Silymarin were found to be 71.64 and 48.78%, respectively. The liver is the site for the production of various physiological proteins. On exposure to the hepatotoxin like CCl₄the amount of total protein present in the serum decreases due to less activity of the hepatocytes.

Fig. 1:	The effect of JG leaf extract on the serum glutamic oxaloacetic transferase on CCl4 induced hepatotoxicity

Fig. 2:	The effect of JG leaf extract on the serum glutamic pyruvic transferase on CCl4 induced hepatotoxicity

Fig. 3:	The effect of JG leaf extract on the total bilirubin on CCl4 induced hepatotoxicity

Fig. 4:	The effect of JG leaf extract on the direct bilirubin on CCl4 induced hepatotoxicity

Fig. 5:	The effect of JG leaf extract on the total protein on CCl4 induced hepatotoxicity

This can be seen from the Table 5, the total protein was decreased after CCl₄ administration to about 24.81% as compared to the vehicle treated animal group. The MLE has restored the protein content by maximum of 21.2% at dose level II (Fig. 5). The serum alkaline phosphatase (SALP) is the one more parameter used to assess the hepatoprotective activity of MLE.

Fig. 6:	Shows the effect of JG leaf extract on the serum alkaline phosphatase on CCl4 induced hepatotoxicity

Fig. 7:	Shows the effect of JG leaf extract on the wet liver weight on CCl4 induced hepatotoxicity

As given in the Table 5 the SALP has increased significantly in the CCl₄ treated group when compare to the vehicle treated group. The MLE has decreased the SALP level in the dose dependent manner at 150 and 300 mg kg^-1 dose level and maximum protective effect was found at 300 mg kg^-1 body weight and the effect was declined as the dose increased to 500 mg kg^-1 b.wt. (Fig. 6). All these studies show that, the leaf extract of JG has a moderate hepatoprotective activity, which can be correlated to its antioxidant and free radical scavenging activity.

To access the effect of MLE on the liver weight variation after CCl₄ intoxication, the wet liver weight was calculated for each group of animals soon after the sacrification. The results are given in Table 6. The effect of JG extract on the wet liver weight is shown in the Fig. 7.

CONCLUSION

By these studies it could be concluded that, JG leaf extract bear a moderate antioxidant activity. Their constituents scavenge free radical, reduce metal ions and exert protective effect against oxidative stress. JG extract also protects against oxidative injury induced by CCl₄ in vivo, the extract is capable of preventing the damage caused by the CCl₄ intoxication. The preliminary phytochemical studies of the extracts, has shown the presence of a number of polyphenols, which may be responsible for the antioxidant and hepatoprotective activities. Isolation of these compounds is in progress.

ACKNOWLEDGMENT

One of the authors Mr. KL Krishna is highly thankful to Dr. H.G. Shivakumar, Principal, JSS College of Pharmacy, for his constant encouragement and support and JSS Mahavidyapeetha, Mysore, for providing the required infrastructure to carryout the research activities.