Hepatoprotective activity of aerial parts of Erythrina crista-galli

Hepatoprotective activity was measured for Erythrina crista-galli extract as well as fractions. Fractions II and III have shown a remarkable protective effect against CCl4-induced hepatocyte injury. This was evidenced by their ability to significantly ameliorate CCl4-induced elevation in ALT and AST levels. This is supported by the notion that pretreatment of hepatocytes with either Erythrina crista-galli extracts or fractions significantly alleviated CCl4induced GSH and SOD depletion and replenished CCL4 reduction of TAC. Hepatoprotective activity mechanism is attributed at least in part, to the free radical scavenging and antioxidant activity of the phenolic compounds present in the extract proved by the phytochemical screening of the fractions.


INTRODUCTION
Phenolic compounds are commonly found in both edible and non-edible plants and they have been reported to have multiple biological effects.Family Leguminosae which include genera that embrace phenolic-rich species is capable of synthesizing and accumulating the high percentage of phenolics particularly flavonoids [1].
The genus Erythrina (Leguminosae) contains more than 110 species with a broad distribution [2].These species have been widely used in indigenous traditional medicine [3] Erythrina has been used in folk medicine for treatment of insomnia malaria fever, venereal disease, asthma, and toothache.The alkaloid Erythroidine was used as a muscle relaxant.Haemoerythrina alkaloids were investigated for their anti-cancer activity [4].Erythrina crista-galli is widely distributed in subtropical and tropical regions.It is known as cockspur coral tree.It is also commonly called "Corticeira" in Brazil, and its bark is used for rheumatism, hepatitis, sedation, and hypnogenesis.Phytochemical studies on this plant showed the presence of Erythrina alkaloids, benzylisoquinoline alkaloids, isoflavonoids, and pterocarpans [31].

Studies on phytochemical of
Since only a few reports are available in the current literature about the hepatoprotection activity of the leaves of Erythrina crista-galli, it was, therefore, found interesting to subject the extracts and fractions of the leaves as a continuation to our previous study where we isolated polyphenols with a phytoestrogen activity, here we aim to assess the hepatoprotective activity for the extract and fractions.

Plant material
Fresh leaves of Erythrina crista-galli (Fabaceae) were collected from plants grown in El-Giza Zoo garden, Giza, on April (2011).They were kindly authenticated by Mrs. Tereize Labib, agricultural engineer, El-Orman botanical garden, Giza, Egypt.Voucher specimens of the authenticated plant were deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt.

Plant extraction and fractionation
Fresh leaves (1 kg) were exhaustively extracted with 10 L of aqueous methanol (75%).Leaves were boiled in double distilled water for exactly 2 h.The aqueous extract was dried by lyophilization.The dry lyophilized extract was then further extracted with methanol (HPLC grade) for 30 min at 40 °C to ensure complete extraction of phenolic components.This method helps to avoid extraction of other plant metabolites as carbohydrates, water-soluble protein, inorganics, lipids, or alkaloids.Finally, the extract was completely evaporated in vacuo at a low temperature until dryness.
Fractionation of the extract (40 g) on cellulose column, using water followed by water-methanol mixtures of decreasing polarities, yielded three main fractions (I, II and III).Two-dimensional PC of the extract and fractions II and III proved the presence of a high percentage of phenolic constituents (blue color reaction with ferric chloride TS and ammonia).

Assay for hepatoprotective activity
HepG2 monolayer culture after attachment was pretreated with the aqueous extract, aqueous methanol extract, fraction I, fraction II, and silymarin (100 μg/mL in phosphate-buffered saline) for one hour.An aliquot of 40 mM CCl 4 in 0.05% dimethyl sulfoxide (DMSO) was added and incubation was continued for another two hours.The supernatant medium and cell lysate were then collected and stored at -20 °C until analysis.Silymarin was used as a standard.The positive control was a set of cells maintained in culture medium and treated only with CCl 4 (40 mM); while the negative control was a set of cells maintained in phosphate-buffered saline.

AST and ALT measurement
Activities of the marker enzymes alanine transaminase (ALT) and aspartate transaminase (AST) were determined according to the method of Reitman and Frankel (1957) [32].

Glutathione reduced (GSH)
The level of GSH in cell culture supernatant was determined as protein-free sulfhydryl content using 5,5-dithiobis-(2-nitrobenzoic acid) (Ellman's reagent) that is reduced by the SHgroup in GSH to form 5-thio-2-nitrobenzoic acid, which has a stable yellow color measured colorimetrically at 412 nm as described by Ellman [34].In detail, protein precipitation was attained by mixing equal volumes of cell culture supernatant and 10% trichloroacetic acid-0.005M EDTA solution followed by centrifugation at 600g for 15 min.To 0.5 mL of the resulting supernatant, 0.85 mL phosphate buffer (0.1 M, pH 8) and 0.05 mL Ellman's reagent (10 mM) was added in a microcuvette and the optical density was measured at 412 nm.

Total antioxidant capacity (TAC)
The determination of the antioxidant capacity is performed by the reaction of the antioxidants in the sample with a defined amount of hydrogen peroxide (H 2 O 2 ).The antioxidants in the sample eliminate a certain amount of the provided H 2 O 2 .The residual H 2 O 2 is determined colorimetrically by the enzymatic reaction which involves the conversion of 3,5, dichloro-2hydroxybenzensulphonate to a colored product.

Statistical analysis
The statistical analysis was carried out by oneway analysis of variance (ANOVA).The values are represented as mean ± SEM the results were considered significant if P < 0.05.

Phytochemical screening
Phytochemical screening of powdered dried samples of the aerial parts of Erythrina cristagalli cultivated in Egypt showed that the plant is rich in flavonoids, coumarins, sterols and/or triterpenes, carbohydrates and/or glycosides, alkaloids, and anthraquinones.
Liver injuries induced by CCl 4 are the best characterized system of xenobiotic-induced hepatotoxicity and commonly used models for  The GSH system includes reduced glutathione, GPx and glutathione-S-transferase. GSH is involved in several defense processes against oxidative damage.It protects cells against free radicals, peroxides and other toxic compounds.Indeed, GSH depletion increases the sensitivity of cells to various aggressions and also has several metabolic effects.It is widely known that a deficiency of GSH within living organisms can lead to tissue disorder and injury [39].
SOD is a scavenger of peroxide anion radicals, which could inhibit the initiation of lipid peroxidation by free radicals [40].
Taking these together, the hepatoprotective effect of Erythrina crista-galli extracts and its farctions is at least partly due to antioxidant activity.

CONCLUSION
Phytochemical screening of the aqueous methanol extract of Erythrina crista-galli leaves indicated the presence of phenolic metabolites.The aim of the present study was to evaluate the potential hepatoprotective activity of Erythrina crista-galli extracts.
Erythrina crista-galli aqueous and aqueous methanol extracts as well as fraction II and III have shown a remarkable protective effect against CCl 4 -induced hepatocyte injury.This was evidenced by their ability to significantly ameliorate CCl 4 -induced elevation in ALT and AST levels.The observed hepatoprotective activity of the tested extracts and fractions can be, at least partly, attributed to their antioxidant Hepatoprotective activity mechanism is attributed at least in part, to the free radical scavenging and antioxidant activity of the phenolic compounds present in the extract.
Total antioxidant capacity = (AB-AS)*3.33mM H 2 O 2 Where: AB = the absorbance of the blank AS = the absorbance of the sample [35]

Fig. 1 Fig. 2
Fig.1The effect of the aqueous, aqueous methanol extracts and fractions of the aerial parts of the Erythrina crista-galli on ALT level.Data are mean ± SEM, (n=3).* Significantly different from CCl 4 group at p < 0.05

Fig. 3 Fig. 4 Fig. 5
Fig.3 The effect of the aqueous, aqueous methanol extracts and isolated fractions of the aerial parts of the Erythrina crista-galli on SOD activity.Data are mean ± SEM, (n=3).* Significantly different from CCl 4 group at p < 0.05 is supported by the notion that pretreatment of hepatocytes with either Erythrina crista-galli extracts or fractions significantly alleviated CCl 4 -induced GSH and SOD depletion and replinshed CCL 4 reduction of TAC.