Therapeutic efficacy of ethanolic extract of Aerva javanica aerial parts in the amelioration of CCl4-induced hepatotoxicity and oxidative damage in rats

Background Liver diseases, the fifth most common cause of global death, can be metabolic, toxin-induced, or infective. Though approximately 35 Saudi medicinal plants are traditionally used to treat liver disorders, the hepatoprotective potential of Aerva javanica has not been explored. Objective To investigate the antioxidative and hepatoprotective effect of Aerva javanica. Design Total ethanol extract of A. javanica aerial parts was prepared and tested on DCFH-toxicated HepG2 cells ex vivo, and in CCl4-injured Wistar rats in vivo. MTT assay was used to determine cell viability and the serum biochemical markers of liver injury as well as histopathology was performed. In vitro 1,1-diphenyl-2-picrylhydrazyl and β-carotene free-radical scavenging assay and phytochemical screening of the extract were done. Furthermore, A. javanica total extract was standardized and validated by high-performance thin layer chromatographic method. Results MTT assay showed that, while DCFH-injured cells were recovered to ~56.7% by 100 µg/ml of the extract, a 200 µg/ml dose resulted in hepatocytes recovery by ~90.2%. Oral administration of the extract (100 and 200 mg/kg.bw/day) significantly normalized the serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, gamma-glutamyl transferase, alkaline phosphatase, bilirubin, cholesterol, high-density lipoprotein, low-density lipoprotein, very-low-density lipoprotein, triglyceride, and malondialdehyde levels, including tissue nonprotein sulfhydryl and total protein in CCl4-injured rats. In addition, the histopathology of dissected liver also revealed that A. javanica cured the tissue lesion compared to silymarin treatment. In vitro assays revealed strong free-radical scavenging ability of the extract and presence of alkaloids, flavonoids, tannins, sterols, and saponins where rutin, a well-known antioxidant flavonoid, was identified. Conclusions Our findings demonstrate the potential of A. javanica in the attenuation of ex vivo and in vivo hepatotoxicity and oxidative damage. This further suggests its therapeutic value in various liver diseases. However, isolations of the active principles, their mechanisms of action, and other therapeutic contributions remain to be addressed.

L iver is one of the most vital organs of the body, considered to be the metabolic 'engine room'. Major functions of the liver include carbohydrate, protein, and fat metabolism as well as secretion of bile, and the storage and detoxification of several drugs and xenobiotics. Liver diseases can be classified as acute or chronic hepatitis (inflammatory liver diseases), hepatosis (non-inflammatory liver diseases), and cirrhosis. Liver disorders can be attributed to toxins like aflatoxin, chlorinated hydrocarbons, and certain drugs. Other factors include infectious pathogens such as hepatitis viruses (1) and alcohol consumption (2). Drug-induced liver toxicity accounts for approximately one-half of the cases of acute liver failure (3). Currently, there is a real need for effective therapeutic agents, especially natural products with a minimal incidence of toxic effects. Despite the  significant popularity of several mono-and poly-herbal preparations for liver diseases (4), only few (e.g., silymarin) have been approved for protection or treatment of liver disorders (5). Nevertheless, in recent years, many phytoproducts have been investigated against hepatotoxininduced liver damage (6Á10).
The genus Aerva belongs to the plant family Amaranthaceae, and there are approximately 28 species in this genus (11). Aerva javanica is an erect, muchbranched perennial herb that is a native of Africa and also distributed in various parts of the world. Different parts of the plant have wide applications in folk medicine, for example the whole plant is used for chest pain, diarrhea, and as a diuretic and demulcent. A decoction prepared from the aerial parts of A. javanica is used as a gargle to cure gum swelling and toothache. Though the seeds are used for headache relief, paste from the flowers and leaves is used externally to heal the wounds and the inflammation of joints (12). Also, it is used for dysentery, gonorrhea, and hyperglycemia (13). Previous biological screenings revealed the numerous biological activities of A. javanica. Its ethanolic and aqueous extracts showed significant anti-diarrheal activity at a dose level of 800 mg/kg in Wistar rats (14). In addition, A. javanica exhibited antibacterial, anti-fungal (15Á17), smooth muscle relaxant, and anti-ulcer activities (13). The alcoholic extract of root of A. javanica was shown to possess significant nephroprotective activity against cisplatin-and gentamycininduced renal toxicity in experimental animals (18).
A phytochemical analysis of A. javanica has revealed the presence of polyphenols, terpenoids, flavonoids, and alkaloids (11). Notably, four new ecdysteroids, aervecdysteroid AÁD (1Á4), and three new acylated flavone glycosides have been isolated from the flowers (19). In addition, heptacosane (3-allyl-6-methoxyphenol) and pentacosane were identified as the major components of the A. javanica seed oil (20). With this background information, the present study was intended to investigate the antioxidant and hepatoprotective activities of ethanolic extract of the aerial parts of A. javanica.

Plant materials
Aerial parts (leaves, stems, and inflorescences) of A. javanica (Family: Amaranthaceae) were collected from Taif, Kingdom of Saudi Arabia. The plant was authenticated by an expert plant taxonomist at the herbarium of College of Pharmacy, King Saud University, Riyadh, and a voucher specimen (No. 16281) was deposited.

Preparation of A. javanica total ethanolic extract
The shade-dried powdered plant material (400 g) were soaked in 70% aqueous ethanol (Merck, Germany) for 2 days at room temperature and filtered. The extraction process was repeated twice with the same solvent. Then, the extracts were evaporated using a rotary evaporator (Buchi, Switzerland) under reduced pressure at 408C. The obtained semi-solid extracts (37.68 g) were stored at (208C until used for further study. Human hepatoma cell culture and drugs Human hepatoma cell line (HepG2) was grown in RPMI-1640 medium, supplemented with 10% heat-inactivated bovine serum (Gibco, USA), 1) penicillinÁstreptomycin, and 1) sodium pyruvate streptomycin (HyClone Laboratories, USA) at 378C in a humified chamber with 5% CO 2 supply. 2,7-Dichlorofluorescein (DCFH; Sigma, USA) was used to induce cytotoxicity in cultured HepG2 cells. Silymarin (Sigma, USA) was used as standard hepatoprotective drug in rats.
Ex vivo hepatoprotective assay of A. javanica total ethanolic extract HepG2 cells were seeded (0.5 )10 5 cells/well, in triplicate) in a 96-well flat-bottom plate (Becton-Dickinson Labware, USA) and grown over night. The cytotoxicity of DCFH was determined by using MTT assay (MTT-Cell proliferation Assay Kit, Tervigen). The concentration of DCFH that caused a 50% inhibition of HepG2 cell proliferation (IC 50 : 100 mg/ml) was used as a cytotoxic dose. A. javanica total extract was dissolved in DMSO (100 mg/ml), followed by dilution with RPMI-1640 media to four doses (25, 50, 100, and 200 mg/ml). The final concentration of DMSO used never exceeded !0.1% and was therefore tolerated by cultured cells. The culture monolayer was replenished with RPMI-1640 containing 100 mg/ml DCFH plus a dose of A. javanica, including untreated as well as DCFH-treated controls. The treated cells were incubated for 48 h at 378C. Cells were treated with MTT reagent (10 ml/well) and further incubated for 3Á4 h. Upon the appearance of a purple color, detergent solution (100 ml) was added to each well and further incubated for 1.5 h. The optical density (OD) was recorded at 570 nm in a microplate reader (BioTek, ELx800). Nonlinear regression analysis was performed in Excel software to determine the cell survival (%) using the following equation: where OD(s), OD(b), and OD(c) are the absorbance of sample, blank, and negative control, respectively.

Microscopy
The morphological investigation of the cultured hepatoma cells was done under an inverted microscope (Optica, 40 ), and 100)) to observe any changes in the cells cultured with different concentrations of A. javanica total extract and/or DCFH at 24 and 48 h.

Determination of malondialdehyde (MDA)
The method reported by Utley et al. (31) was followed.
In brief, the liver tissues were removed homogenized in 0.15 M KCl (at 48C; Potter-Elvehjem type C homogenizer) to give a 10% w/v homogenate. The absorbance of the solution was then read at 532 nm. The content of MDA (nmol/g wet tissue) was then calculated, by reference to a standard curve of MDA solution.

Estimation of nonprotein sulfyhydryl (NP-SH)
Hepatic NP-SH was measured according to the method of Sedlak and Lindsay (32). The liver tissues were homogenized in ice-cold 0.02 mM ethylene diamine tetraacetic acid (EDTA). The absorbance was measured within 5 min of addition of 5,5?dithio-bis(2-nitrobenzoic acid) (DTNB) at 412 nm against a reagent blank.

Determination of total protein (TP)
Serum TP was estimated by the kit method (Crescent Diagnostics, Jeddah, Saudi Arabia). The absorbance (Abs) of the samples were recorded at 546 nm and concentration of serum TP was calculated using the following equation: ðSerum TP ¼ðAbs of sample=Abs of standardÞ)

Histopathological evaluation
The animals were sacrificed under ethyl ether anesthesia and livers were quickly removed. Liver tissues were fixed in 10% neutral buffered formalin for 24 h and processed overnight for dehydration, clearing, and paraffin impregnation using an automatic tissue processor (Sakura, Japan). The specimens were embedded in paraffin blocks using embedding station (Sakura, Japan) and sections of 4 micron thickness were cut using rotary microtome (Leica-RM2245, Germany) and stained with hematoxylin and eosin (H&E) stains using routine procedures (33). The stained sections were observed under light microscopy, and the required images were taken with digital microscopic mounted camera (OMX1200C, Nikon, Japan).
Antioxidant assay of A. javanica total extract 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay Antioxidant activity was estimated by free-radical scavenging ability of the total extract against DPPH according to the method described elsewhere (34) % Inhibition ¼ ½ðAsð120Þ À Acð120ÞÞ=ðAcð0Þ À Acð120ÞÞ Â 100 where As (120) and Ac (120) are the absorbance of the sample and control, respectively, at time 120 min, and AC(0) is the absorbance of the control at time 0 min.
In vitro phytochemical screening of ethanolic extract of A. javanica Qualitative phytochemical screening tests for major secondary metabolites, which included alkaloids, flavonoids, anthraquinones, tannins, saponins, and cardiac glycosides, were performed using standard procedures (36Á38).

Standardization of ethanolic extract of A. javanica by HPTLC method
A high-performance thin layer chromatography (HPTLC) method was used to standardize the 70% ethanol extract of A. javanica as described elsewhere (39). The chromatography was carried out on 10)10 cm precoated silica gel F 254 RP-HPTLC plate, using rutin as the reference standard. Several mobile phases were tried to get good resolution and separation of different compounds present in the A. javanica ethanol extract. Based on our observations, we selected acetonitrile and water in the ratio of 4:6 as suitable mobile phase to carry out the standardization of the extract. The standard along with samples was applied on the HPTLC plate by CAMAG Automatic TLC Sampler-4. The plate was developed under controlled condition in CAMAG Automated Developing Chamber-2 and scanned by CAMAG TLC Scanner-3 (l 0360 nm).

Statistical analysis
Results were expressed as mean9SEM. Total variation present in a set of data was estimated by one-way analysis of variance (ANOVA) followed by Dunnet's test. P B0.01 was considered significant.

Effect of A. javanica on cell morphology
Microscopic observations showed considerable cytotoxic effect of DCFH on the HepG2 cells as reflected by altered morphology compared to untreated cells. Interestingly, the DCFH-treated cells supplemented with 100 and 200 mg/ml of A. javanica extract were morphologically different from those treated with DCFH alone but comparable to untreated cells (data not shown).
Hepatoprotective and cell proliferative effect of A. javanica MTT test showed hepatoprotective effect of A. javanica ethanol extract in a dose-dependent manner against DCFH-toxicity. While DCFH-toxicated cells were recovered to about 56.7% after supplementation with 100 mg/ml of A. javanica extract, further doubling the dose (200 mg/ml) resulted in hepatocytes recovery by about 90.2% (Fig. 1).

In vivo effect of A. javanica extract on biochemical markers
Based on the ex vivo hepatoprotective activity on HepG2 cells, the effects of A. javanica extract were further examined in the experimental animal model. The administration of CCl 4 dramatically elevated the serum SGOT, SGPT, GGT ALP, and bilirubin levels compared to the normal control group (P B0.0001), indicating significant hepatotoxicity of CCl 4 treatment (Table 1). In contrast, oral administration of A. javanica extract significantly normalized the biochemical parameters in CCl 4 -treated rats, compared to the CCl 4 -treated group (Table 1). Moreover, CCl 4 -induced toxicity caused significant elevation in lipid profile including cholesterol, triglycerides, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) and a reduction in the HDL levels in serum.  significantly reduced the cholesterol, triglycerides, LDL, and VLDL levels and improved the HDL level in a dose-dependent manner ( Table 2). Silymarin, on the other hand, significantly prevented the CCl 4 -induced elevation in marker enzymes and lipid profile. Furthermore, our results indicated that treatment with A. javanica also resulted in a significant increase in MDA and decrease in NP-SH and TP concentration (Table 3). Oral supplementation of CCl 4 -treated rats with A. javanica extract dramatically diminished the levels of MDA and enhanced NP-SH and TP levels ( Table 3).

Histological improvement by A. javanica total extract
The histological examination of rat liver tissues revealed evidence of hepatic necrosis and fatty degenerative changes in CCl 4 -injured animals ( Fig. 2a and b). Compared to this, the A. javanica-treated (100 mg/kg.bw/day) animals exhibited congested central vein with mild necrosis and fatty changes (Fig. 2c). On the other hand, the higher dose (200 mg/kg.bw/day) of A. javanica showed normal hepatocytes and central vein with full recovery that was comparable to silymarin administration ( Fig. 2d and e). This finally confirmed the hepatoprotective efficacy of A. javanica total extract.

In vitro antioxidant activity of A. javanica
The total extract of A. javanica was able to reduce the stable free-radical DPPH to the yellow-colored DPPH (Fig. 3). At 500 mg/ml dose, the observed antioxidant activities were comparable to that of the positive control (ascorbic acid). In accordance with the DPPH radical scavenging assay results, A. javanica showed high antioxidant activity in b-caroteneÁlinoleic acid bleaching assay (Fig. 4).

Phytochemical screening of the total extract
The qualitative phytochemical screening of the A. javanica total extract showed the presence of alkaloids, flavonoids, tannins, sterols, and saponins and the absence of anthraquinones and cardiac glycosides.

Identification of rutin, a biflavonoid in A. javanica extract
The HPTLC analysis revealed the presence of rutin, a well-known antioxidant flavonoid in the total extract (Fig. 5a). Using acetonitrile and water as suitable mobile phase, the compact spot of rutin was found at R f 00.65 (Fig. 5b). Also, a good separation of different phytoconstituents present in the ethanol extract using the same mobile phase was achieved (Fig. 5c). The dried extract of A. javanica was found to contain 2.53 mg/mg of rutin.

Discussion
Among more than 100 species of medicinal plants documented in Saudi Arabia, approximately 35 plants are used in Saudi folk medicine for the treatment of liver disorders. However, most of them remain popular without scientific rationale. Of these, the hepatoprotective potential of Aerva javanica has been neither explored in traditional practices nor with experimental approaches. All values represent mean9SEM. *P B0.05; **P B0.01; ***P B0.001; ANOVA, followed by Dunnet's multiple comparison test. a As compared with normal group. b As compared with CCl 4 -only group. All values represent mean9SEM. *P B0.05; **P B0.01; ***P B0.001; ANOVA, followed by Dunnet's multiple comparison test. a As compared with normal group. b As compared with CCl 4 -only group. In this study, we have, therefore, evaluated the hepatoprotective and antioxidative potential of A. javanica total ethanolic extract against DCFH-induced cytotoxicity in HepG2 cells and CCl 4 -induced liver damage in rats, respectively. DCFH is generally used for the quantitative assessment of oxidative stress generated by free radicals All values represent mean9SEM. *P B0.05; **P B0.01; ***P B0.001; ANOVA, followed by Dunnet's multiple comparison test. a As compared with normal group. b As compared with CCl 4 -only group. through the principle of oxidation of DCFH to the fluorescent DCF (40). However, we used this agent because of its toxic effect on cultured cell, ex vivo. A. javanica total extract promoted HepG2 cell proliferation and recovery in a dose-dependent manner. These findings were further supported by microscopic examination of cell morphology and growth.

Treatment
To further confirm the ex vivo cell proliferative effects, the in vivo hepatoprotective potential activity of A. javanica total extract was examined in CCl 4 -injured rats. CCl 4 is a common hepatotoxin used in the experimental study of liver diseases that induces free-radical generation in liver tissues. In vivo, it is transformed to active free radicals that bind to macromolecules resulting in lipid peroxidation that causes cell injury (41,42). Clinical symptoms of CCl 4 -induced acute liver injury include jaundice and elevated levels of serum SGOT, SGPT, and ALP, indicators of liver necrosis and inflammation (43). In the present study, pretreatment with two doses of A. javanica total extract (100 and 200 mg/ kg.bw) showed its ability to reduce the SGOT, SGPT, GGT, ALP, and bilirubin levels significantly in a dosedependent manner. In addition, A. javanica extract was able to normalize the serum cholesterol and triglycerides and HDL levels in CCl 4 -injured rats. The hepatoprotective effect of A. javanica at 200 mg/kg.bw was comparable to standard drugs silymarin (10 mg/kg.bw). The rise of serum enzyme SGOT and SGPT levels had indicated that hepatocyte cells were injured where the leakage of cell membrane had contributed to the accumulation of these enzymes into the plasma (44). The significant reduction in the levels of LDL, and total cholesterol in the A. javanica-treated rats and an increase in the HDL level further indicated the hepatoprotective potential of A. javanica.
MDA is produced during the peroxidation of polyunsaturated fatty acids, and the amount of MDA is a marker for cell membrane lipid peroxidation and cell damage (45). Treatment with A. javanica extract or silymarin reduced the level of MDA in livers with CCl 4induced damage, which suggests that the tested extract was protective and curative against liver toxicity.
NP-SH plays an important role in the defense against oxidative cellular damage (46). In our study, the liver NP-SH level in the CCl 4 -treated group was markedly reduced when compared with the control group. Pretreatment of rats with A. javanica total extract or silymarin replenished NP-SH concentration as compared with CCl 4 only treated animals, suggesting free-radical scavenging activity of the tested extract.   The level of TP would be decreased in hepatotoxic conditions due to defective protein biosynthesis in liver. Thus, the reduction in TP level is a further indication of liver damage in CCl 4 -injured animals (47). In our study, the level of TP has been restored to the normal value, indicating the curative action of A. javanica total ethanolic extract. Moreover, most of the parameters in the animal group that received CCl 4 plus A. javanica extract had values comparable to those of the group receiving CCl 4 plus silymarin. This very clearly indicated that A. javanica was efficient to attenuate CCl 4 -induced hepatotoxicity in rats.
Conducting different assay methods for the evaluation of antioxidant activity (48,49) is now commonly recommended. In our in vitro assays, the antioxidant activity of A. javanica ethanolic extract revealed strong antioxidant activities in both DPPH and b-carotene methods. In addition, our preliminary phytochemical screening of the A. javanica total extract showed the presence of alkaloids, flavonoids, tannins, sterols, and saponins. Taken together, the antioxidant activity of the A. javanica extract could be attributed to the presence of antioxidant and freeradical scavenging phytoconstituents, such as polyphenol, flavonoids, and saponins, which are known to have hepatoprotective activities. Rutin (3,3?,4?,5,7-pentahydroxyflavone-3-rhamnoglucoside), a biflavonoid found in many plants, has a wide range of pharmacological properties against various metabolic and infectious diseases (50). The reactive-species-mediated in vivo hepatotoxicity can be effectively attenuated by rutin possessing antioxidant, free-radical scavenger, and anti-lipid per oxidant activities (51). Recently, hepatoprotective mechanisms of rutin in CCl 4 -intoxicated BALB/cN mice has also been demonstrated (52). Our identification of rutin in A. javanica extract by validated HPTLC method further supports its therapeutic use in the prevention and treatment of liver diseases.
Very recently, A. Javanica-derived aervecdysteroid AÁD (1Á4) and acylated flavone glycosides have been shown to inhibit enzymatic activities of lipoxygenase and butyrylcholinesterase (53). In mammals, lipoxygenases catalyze the formation of hydroperoxides as the first step in the biosynthesis of several inflammatory mediators (54). Butyrylcholinesterase, also known as serum or plasma cholinesterase, is synthesized in the liver that hydrolyzes many different choline-based esters. In clinical assays, serum butyrylcholinesterase activity is used as a liver function test for acute and chronic liver damage, including other inflammatory disorder (55). Nevertheless, further biological analysis of these compounds, including identification of other bioactive constituents of A. javanica with antioxidative and hepatoprotective activities, remains to be explored.

Conclusions
Our investigation of A. javanica total ethanol extract revealed its promising antioxidative and hepatoprotective potential against chemically induced liver injury ex vivo as well as in vivo. This was supported by the in vitro phytochemical analysis and identification of rutin, a well-known antioxidant flavonoid. Our finding therefore, suggests the therapeutic potentiality of A. javanica in various liver diseases. However, further studies on main active ingredients of the extract, their mechanism of action, and other therapeutic contribution of these interventions would be necessary.