Hepatoprotective effect of aqeous methanolic extract of Rumex dentatus in paracetamol-induced hepatotoxicity in mice

Rumex dentatus is well known plant of Polygonaceae family which is widely used in traditional medicine to treat gastrointestinal tract. R. dentatus at doses 250 and 500 mg/kg significance decreased (p<0.001) the elevated level of ALT, AST, ALP and bilirubin induced by paracetamol and results are comparable (p<0.001) with silymarin. The results were supported by histopathological investigations, phytochemical screening and detection of hepatoprotective active constituents e.g quercetin, kaempferol, myricetin by HPLC. So, it is concluded that R. dentatus has hepatoprotective effect against paracetamol liver damage in mice.


Introduction
Liver is the one of the major and important organ of body.It has vital role in metabolism and elimination of various agents.All chemical agents first pass through liver prior to enter in blood circulation.So liver is at high risk of damage then other body organ (Samuel et al., 2012).The toxic agents which may damage the liver are drugs such as paracetamol, xenobiotics, viral infections, alcoholism and chemical agents (Ramchandara et al., 2007).That's why the liver injury is one of the major problems in World Wide Health.Synthetic preparations are available in the market to treat liver diseases but these synthetic are limited in number and also have side effects.So there is an urgent need to investigate the natural plants and isolate their constituents which can be beneficial to control liver diseases (Ravikumar et al., 2012).It has been seen that many herbal plants are found to be hepatoprotective such as Cichorium intybus (Heibatollah et al., 2008), Luminet racemosa (Gnanadesigan et al., 2011), Solanum nigrum (Raju et al., 2003), Hibiscus vitifolius (Samuel et al., 2012), Chenopodium murale (Saleem et al., 2014), Trichodesma sedgwickianum (Saboo et al., 2013), Oflpomoea staphylina (Bag and Mumtaz, 2013), Convolvulus arvensis (Ali et al., 2013), Suaeda fruticosa (Rehman et al., 2013) and Khamira Gaozaban Ambri Jadwar Ood Saleeb Wala (Akhtar et al., 2013).

Materials and Methods
Collection of plants: Fresh plants were collected from district Sialkot, Punjab Pakistan.Plant was identified by Dr. Mansoor Hameed, Department of Botany, University of Agriculture, Faisalabad.Vouchers No 270-13 can be used for future reference.
Preparation of plant extracts: Plant was washed, dried under shade and finally grounded to powder.The powdered plant was soaked in aqueous methanolic (30:70) solvent for 7 days with occasional shaking at regular intervals.The extract was filtered and evaporated by using rotary evaporator at 60°C.The residue was stored in amber coloured glass bottle at 4°C.
Experimental animals: Adult Swiss albino mice weighing about 22-34 g were used.All the animals were placed in the animal house of College of Pharmacy, GC University Faisalabad.The animals were allowed to acclimate under standard laboratory conditions prior to perform experiment.
Experimental protocol: All animals were divided into 5 groups containing 5 animals each.Group 1 served as a control group receiving distilled water only Group II served as paracetamol control group received paracetamol 250 mg/kg (p.o) 7 days (Sabir and Rocha, 2008).Group III was treated with silymarin 50 mg/kg (p.o) and followed by paracetamol administration 3 hours after silymarin for 7 days (Girish et al., 2009).Group IV was treated with aqueous methanolic extract of R. dentatus at dose of 250 mg/kg (p.o) followed by paracetamol administration 250 mg/kg (p.o) 3 hours after the extract dose for 7 days.Group V was treated with aqueous methanolic extract of R. dentatus at dose of 500 mg/kg (p.o) followed by paracetamol administration 250 mg/kg (p.o) 3 hours after the extract dose for 7 days (Sabir and Rocha, 2008).
Biochemical and histopathological investigations: On 8 th day, animals were sacrificed and blood was collected for serum separation.The change in aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase (ALP) and total bilirubin (TB) levels were measured for biochemical investigations.Histolopathological studies were also conducted to examine microscopic changes in liver architecture (Ali et al., 2013).
Histopathological examination: Histopathological study was carried out to see the microscopic changes in the hepatocytes.For this purpose the slides of the liver sections were prepared and were examined by microscope to see the changes in liver hepatocytes.

Preliminary phytochemical analysis
The phytochemical screening of the plant extracts were performed by standard procedures as given below: Test for alkaloids: 0.2 g of the extract was added in 2N HCl (5 mL) then heated on the boiling water bath.The mixture was filtered after cooling and the filtrate was divided in the two equal halves.Few drops of Mayer's reagent were added in one portion and the Dragendeoff's reagent was added in other portion.Turbidity of the formed precipitate in the both reagents indicates the presence of alkaloids (Mojab et al., 2003;Sharma et al., 2010) Test for tannins: 0.2 g extracts was dissolved in 10 mL of distilled water and warm on water bath.The mixture was filtered and 5% solution of ferric chloride was added to filtrate.The formation of dark green solution indicates the presence of tannins (Mojab et al., 2003).
Test for saponins: 0.2 g of extract was mixed in test tube with 5 mL of distilled water and warm on water bath until it begins to boil.Formation of foam that persists for 10 min indicates the presence of saponins (Mojab et al., 2003;Sofowora, 1993).
Test for terpenoids: 0.2 g of extract was shaken separately with 2 mL of chloroform (CHCl3), and then 3 mL of concentrated H2S04 was added carefully to form a layer.The formation of reddish brown color at inert face of the solution indicates the presence of terpenoids (Sofowora, 1993;Sharma et al., 2010).
Test for flavonoids: 0.2 g of extract was taken separately and dissolved in 5 mL diluted NaOH then 1M of 5 mL HCl was added.A yellow color solution that changes into colourless solution indicates the presence of flavonoids (Sofowora, 1993).
Test for steriods: Two milliliters of acetic anhydride was added to 0.5 g ethanolic extract of each sample with 2 mL H2S04.The colour changed from violet to blue or green in some samples indicating the presence of steroids.
Test for anthraquinones: 0.5 g of extract was boiled with 10% of HCl in water bath for few min and then filtered.The filtrate was cooled and then equal amount of CHCl3 was added in the filtrate.Few drops of 10%NH3 was added in the mixture and heated.The formation of rose -pink coloration indicates the presence of authraquinones (Mojab et al., 2003;Sharma et al., 2010).
Test for glycosides: 1.2 g of extract was hydrolyzed with 10 mL of 1% HCl and then neutralized by 10% of NaOH solution.Few drops of Fehling's solution A and B were poured in it.Formation of red color precipitates shows the presence of glycosides (Mojab et al., 2003;Sharma et al., 2010).

Identification of active constituents by HPLC:
Qualitative determination of active constituents having hepatoprotective activity was done by using HPLC.Sample was prepared by adding small amount of extract in 5 mL distilled water, 12 mL methanol and kept for 5 min.Then 6 mL distilled water was again added and kept for 5 min.10 mL of 15M HCl was added and placed in oven for 2 hours.Final solution was filtered with syringe filter.Phenolics were separated using a shim-pack CLC-ODS (C-18) column, 25 cm × 4.6 mm, 5 µm.Mobile phase used was isocratic: ACN: Dichloro-methane: methanol-60:20:20 at a flow rate of 1 mL/min.Samples were analysed using UV-Visible detector at 280 nm at room temperature while Keampferol was separated at 248 nm (Sultana et al., 2008).
Statistical analysis: Statistical analysis was done with one -way ANOVA (analysis of variance).Results were showed by mean ± SE.

Results
Hepatoprotective effect of aqueous methanolic extract at 250 mg/kg and 500 mg/kg was determined by estimating ALT, AST, ALP and total bilirubin (Table I).The average value of ALT of normal animals was 50.2 ± 3.0 U/L.Treatment with paracetamol raised this value to 172.4 ± 6.2 U/L.Administration of aqueous methanolic extract of R. dentatus at dose of 250 mg/kg and 500 mg/kg brought the level of this enzyme to 101.0 ± 16.4 and 86.6 ± 11.6 U/L respectively which is comparable to (p<0.01, p<0.001) standard hepatoprotective drug, silymarin 61.0 ± 6.1.U/L The average value of AST of normal animals was 57.8 ± 3.4 U/L.Treatment with paracetamol raised this value to 167.4 ± 6.5 U/L.Administration of aqueous methanolic extract of R. dentatus at dose of 250 mg/kg and 500 mg/kg brought the level of this enzyme to 103.2 ± 14.0 and 95.6 ± 12.4 U/L respectively which is comparable to (p<0.01, p<0.001) standard hepatoprotective drug, silymarin 68.8 ± 2.81 U/L.The average value of ALP of normal animals was 173.6 ± 16.2 U/L.Treatment with paracetamol raised this value to 407.6 ± 36.4 U/L.Administration of aqueous methanolic extract of R. dentatus at dose of 250 mg/kg and 500 mg/kg brought the level of this enzyme to 219.4 ± 39.0 and 243.6 ± 19.0 U/L respectively which is comparable to (p<0.01, p<0.001) standard hepatoprotective drug, silymarin 190.4 ± 12.4 U/L.Similarly the average value of total bilirubin of normal animals was 0.7 ± 0.0 mg/dL.Treatment with paracetamol raised this value to 2.0 ± 0.2 mg/dL.Administration of aqueous methanolic extract of R. dentatus at dose of 250 and 500 mg/kg brought the level of this enzyme to 1.0 ± 0.1 and 0.7 ± 0.0 mg/dL respectively which is comparable to (p<0.05, p<0.001) standard hepatoprotective drug, silymarin 0.8 ± 0.0 mg/dL.The phytochemical screening shows the presence of tannins, saponin, flavonoids (Table II).
Histopathology studies showed that the liver architecture was normal in the control group.Sections reveal unremarkable lines, parenchyma comprised of portal triads, central vein and hepatocytes are arranged in cord separated by sinusoidal spaces.While the paracetamol-treated group showed the sever periportal inflammation and sever tissue necrosis.There was also sever blooming and dilation in sinusoidal spaces.Silymarin treated group showed only mild inflammation  with no ballooning.Animals treated with aqueous methanolic extract of R. dentatus at 250 mg/kg showed mild inflammation with no ballooning while the animals treated at dose 500 mg/kg showed almost normal hepatocytes.Histopathological changes in liver architecture can be seen in Figure 1.
The HPLC chromatogram of R. dentatus showed the presence of chlorogenic acid, quercetin, myricitin, vitamin C and kaempferol (Figure 2 and 3; Table III).
The current HPLC of R. dentatus showed the presence of chlorogenic acid, quercetin, myricitin, vitamin C and kaempferol.Among these isolated constituents myrecitin (Rashid et al., 2013;Matic et al., 2013), quercetin (Janbaz et al., 2004), kaempherol (Song et al., 2003), have been reported to have hepatoprotective activity.In present study the R. dentatus significantly control the elevation of liver enzymes and were comparable to standard hepatoprotective drug, silymarin.So, this hepatoprotective action may be due to presence of above constituents.However, R. dentatus looks more potent at 500 mg/kg.It is concluded that R. dentatus have significant hepatoprotective activity in aqueous methanolic extract.species. Chem Nat Compd. 2003;39: 446-49.

Table I Effect of aqueous methanolic extract of Rumex dentatus at 250 mg/kg and 500 mg/kg on ALT, AST, ALP and total bilirubin
Mean ± SE. is used to show the results; The results are compared by one-way ANOVA (analysis of Variance); Significant a p<0.05, b p<0.01, c p<0.001