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Introduction

The liver is a vital organ that serves a role in the metabolism of endogenous and exogenous substances where physiological imbalances cause cellular oxidative stress and the formation of toxic free radicals (1). Increased accumulation of intracellular reactive oxygen and nitrogen species together with decreased antioxidant defense results in hepatotoxicity that may progress to liver dysfunction, carcinoma and failure (1). Therefore, developing preventive therapeutic strategies against hepatic oxidative stress and toxicity remains an important issue. Plants contain a number of bioactive secondary metabolites, including flavonoids, polyphenols, alkaloids, saponins and terpenoids, which possess radical scavenging and hepatoprotective activities (2–6).

The genus Atriplex (subfamily Chenopodiaceae), commonly known as lagoon or sprawling saltbush are widely distributed in arid and semi-arid regions, including the Middle East (7). Globally, ~400 species of Atriplex herbs and shrubs have been recognized (7,8). Of these, the protein-rich shoots of A. halimus are an important fodder for sheep, goats and camels (9). In addition, the protein-rich leaves of A. lampa have been proposed as a potential dietary supplement for animals and humans (10). In traditional medicine, A. halimus decoction has been used to treat syphilis (11) and its leaves have been used to treat heart disease, diabetes and rheumatism in the Arabian Peninsula (12). In addition, methanol and hexane extracts of the aerial parts of A. halimus have been demonstrated to have antimicrobial activity (13). A previous study, in which phytochemical analysis was performed on the aerial parts of A. halimus, revealed the presence of myricetin, quercetin, isorhamnetin glycosides, phenolic acids and esters (14). Recently, triterpenoids isolated from A. laciniata demonstrated antibacterial, antioxidant and antiurease activities (15), including anticholinesterase effects against Alzheimer's and other neurological disorders (16). The fungicidal effects of A. semibaccata, A. portulacoides and A. infalata have been previously reported (17) and the molluscicidal and larvicidal activities of A. inflata have also been identified (18). Furthermore, Gođevac et al (19) revealed that flavonoid glycosides isolated from the aerial parts of A. littoralis exhibited protection against in vitro biochemical and cytogenetic damage to human lymphocytes (19).

In Saudi Arabia, of the 10 reported species of saltbush, A. coriacea, A. dimorphostegia, A. farinosa, A glauca, A. halimus, A. leucoclada and A. tatarica are native, whereas A. canescens, A. semibaccata and A. suberecta were introduced and naturalized (20). A. suberecta I. Verd is a herb with thin and narrow leaves, separate male and female flowers and capsulated fruits (20,21). Compared with other species, there have been few phytochemical and bioactivity studies on A. suberecta. To the best of our knowledge, the only previous study into A. suberecta leaf protein concentrate, suggested its nutritional value was due to its high lysine content (21). The aim of the present study was to investigate the in vitro and in vivo antioxidative and hepatoprotective potential of A. suberecta ethanol-extract (ASEE), including standardization and validation by chromatography.

Materials and methods

Collection of plant material and extract preparation

The clean and healthy aerial shoots of Atriplex suberecta I. Verd were collected from Jazan (Saudi Arabia) and authenticated (voucher specimen no. 16386) by a plant taxonomist at the College of Pharmacy, King Saud University (Riyadh, Saudi Arabia). Briefly, the air-dried leaf powder (300 g) was soaked in 70% ethanol (Merck KGaA, Darmstadt, Germany) for 2 days at room temperature and filtered (Whatman® Filter paper, grade 1; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The extraction process was repeated twice with the same solvent, followed by evaporation using a rotary evaporator (BÜCHI Labortechnik AG, Flawil, Switzerland) under reduced pressure at 40°C. The obtained semi-solid ASEE (31.5 g) was stored at −20°C prior to use.

Human hepatoblastoma cell cultures and drugs

The human hepatoblastoma cell line, HepG2 (22) was maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% heat-inactivated bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 1X penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and 1X sodium pyruvate (GE Healthcare Life Sciences, Logan, UT, USA) at 37°C in a humidified chamber containing 5% CO2. Silymarin, 2,7-dichlorofluorescein (DCFH), ascorbic acid (all Sigma-Aldrich; Merck KGaA) and gallic acid (Fluka; Honeywell International Inc., Morris Plains, NJ, USA) were also purchased.

Free-radical scavenging activity of ASEE

The free-radical scavenging ability of ASEE against 1,1-diphenyl-2-picrylhydrazyl (DPPH) was evaluated quantitatively as described previously (23) with minor modifications. In brief, 100 µl of different concentrations (31.25, 62.5, 125 and 250 µg/ml) of the ASEE was mixed with 40 µl DPPH (0.2 mM in methanol) in a 96-well microplate. The control was prepared using the solvent (methanol) only in addition to the same amount of DPPH reagent to remove any inherent solvent effect. Ascorbic acid was used as the standard. Following 30 min incubation at 25°C the decrease in absorbance (Abs) was measured at λ=517 nm using a microplate reader (ELx800; BioTek Instruments, Inc., Winooski, VT, USA). The experiment was performed in triplicate and the radical scavenging activity was calculated from the following equation: Percentage radical scavenging activity = [1 - (Abssample/Abscontrol)] × 100.

Lipid peroxidation assay

The lipid peroxidation activity of ASEE was evaluated using the β-carotene bleaching method as previously described (24) with minor modifications. Briefly, 0.25 mg β-carotene was dissolved in 0.5 ml chloroform and added to flasks containing 12.5 µg linoleic acid and 100 mg Tween-40. The chloroform was evaporated at 43°C using a Savant™ Universal SpeedVac™ Vacuum system concentrator (Thermo Fisher Scientific, Inc.). The resultant mixture was immediately diluted to 25 ml with distilled water and agitated vigorously for 2–3 min to form an emulsion. A 200 µl aliquot of the emulsion was added to a 96-well plate containing 50 µl ASEE or 500 µg/ml gallic acid (standard). A control containing solvent (emulsion) was also prepared. The test was performed in triplicate and the plate was incubated at 50°C for 2 h. The Abs was read at λ=470 nm at 30 min intervals using a microplate spectrophotometer. The antioxidant activity was estimated using two different methods; initially the kinetic curve was obtained by plotting Abs of each sample against time and then the antioxidant activity was expressed as percentage inhibition of lipid peroxidation using the following equation: Percentage inhibition = [(As120 - Ac120)/(Ac0 - Ac120)] × 100, where As120 and Ac120 are the Abs of the sample and control at 120 min, respectively, and Ac0 is the Abs of the control at 0 min.

In vitro hepatoprotection assay of ASEE

HepG2 cells were seeded in a 96-well flat-bottom plate (0.5×105 cells/well) and grown for 24 h as described above. Liver cytotoxicity was induced by DCFH (IC50 100 µg/ml) treatment as previously described (25). ASEE was initially dissolved in DMSO (200 mg/ml) and further diluted in RPMI-1640 medium to prepare four doses (25, 50, 100 and 200 µg/ml) and an untreated control containing DMSO only (volume equivalent to 200 µg/ml ASEE). As determined previously (data not shown), the final concentration of DMSO used never exceeded >0.1% and therefore was tolerated by the cultured cells. The culture monolayers were replaced with culture medium containing DCFH (100 µg/ml) and a dose of ASEE, including the untreated as well as the DCFH only-treated controls. The treated cells were incubated for 48 h at 37°C in a CO2 incubator, followed by an MTT assay (TACS MTT Cell Proliferation Assay kit, Trevigen, Gaithersburg, MD, USA) according to the manufacturer's instructions. Briefly, MTT reagent (10 µl/well) was added to the cells and incubated for 3 h. The lysis buffer (100 µl/well) was gently added and further incubated for ~1.5 h. The Abs was recorded at λ=570 nm by a microplate reader, and data was analyzed using non-linear regression (Excel software 2010; Microsoft Corporation, Redmond, WA, USA) to determine the percentage cell survival as follows: Percentage cell survival = [(Abssample - Absblank)/(Abscontrol - Absblank)] × 100.

Anti-apoptotic signaling assay of ASEE

To determine the anti-apoptotic effect of ASEE, caspase-3 and −7 activation was measured using an Apo-ONE® homogenous caspase-3/-7 assay kit (Promega Corporation, Madison, WI, USA) following the manufacturer's protocol. Briefly, HepG2 toxicity was induced with DCFH (100 µg/ml) and treated with ASEE (25, 50, 100 and 200 µg/m) for 48 h as described above. Caspase-3/-7 reagent (100 µl/per well) was added and mixed by gently rocking the culture plate. Treated cultures were incubated for 5–6 h in the dark at room temperature and the Abs was measured at λ=570 nm. Non-linear regression analysis was performed to determine the percentage cell proliferation and caspase activity as follows: Percentage cell proliferation = [(Abssample - Absblank)/(Abscontrol - Absblank)] × 100.

Animals and acute toxicity test

A total of 30 male Wistar rats (weight, 200–220 g; age, 8–9 weeks) received from the Experimental Animal Care Center, King Saud University (Riyadh, Saudi Arabia) were kept in polycarbonate cages in a sterile room under a controlled 12 h dark/light cycle at 25±2°C with 50–60% humidity. The animals were provided standard rodent chow diet (Grain Silos & Flour Mills Org., Riyadh, Saudi Arabia) and water ad libitum. The animals were divided into five test groups (n=6/group/cage) that were each fed different doses of ASEE (50, 100, 250 and 500 mg/kg.bw), including a control group that was fed normal saline instead of ASEE. All ASEE treated rats, including the control group were observed continuously and uninterruptedly for 1 h and then at 30 min intervals for 4 h for any gross behavioral change and general motor activities, including writhing, convulsion, response to tail pinching, gnawing, pupil size and feeding behavior, and additionally monitored for up to 72 h for any mortality. No behavioral change was observed in the treated or control rats. The present study was approved by the Ethics Committee of the Experimental Animal Care Society (King Saud University, Riyadh, Saudi Arabia) and adhered to its guidelines.

Experimental design and treatment

Upon acclimatization to the laboratory conditions for 1 week, the rats (n=30) were randomized and assorted into five groups (GI-GV) with 6 rats in each group. The GI group was fed orally with normal saline (1 ml) and served as the untreated control, the GII group received carbon tetrachloride (CCl4) in liquid paraffin (1:1) only, 1.25 m/kg intraperitoneally (IP). The GIII, GIV and GV groups also received CCl4 and the GIII and GIV were also treated with ASEE 100 and 200 mg/kg, respectively whereas GV was treated with silymarin (10 mg/kg) used as a comparison to the current experimental and clinical standard (23,26). All treatment was administered for 3 weeks

Rat sacrifice, blood collection and liver tissue preparation

Following 3 weeks of treatment, rats (weight, 200–220 g) were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally; Sigma-Aldrich; Merck KGaA) and sacrificed by cervical dislocation. Death was confirmed by monitoring the heartbeat, absence of withdrawal to paw pinch and non-response of pupils to light. While under anesthesia, rats blood was collected with a 23G needle via cardiac puncture and sera were separated at 1,000 × g for 10 min at 4°C, and stored at −20°C until biochemical analysis. The livers were quickly removed and fixed in 10% neutral buffered formalin (NBF) for 48 h at room temperature. The fixed specimens were processed overnight for dehydration, clearing and paraffin impregnation using an automatic tissue processor (Sakura Finetek Europe B.V., The Netherlands) and cut into 4-µm-thick sections using a rotary microtome (RM2245; Leica Microsystems GmbH, Wetzlar, Germany).

Estimation of serum marker enzymes, bilirubin, lipid profile and total protein

Serum glutamate oxaloacetate (SGOT) (Reflotron GOT, cat. no. 10745120; Roche Diagnostics GmbH, Mannheim, Germany), serum pyruvate transaminase (SGPT) (Reflotron GPT, cat. no. 10745138; Roche Diagnostics GmbH), alkaline phosphatase (ALP) (Reflotron ALP, cat. no. 11622773; Roche Diagnostics GmbH), γ-glutamyl transferase (GGT) (Reflotron GGT, cat. no. 10745081; Roche Diagnostics GmbH), bilirubin (Reflotron BIL, cat. no. 10905321; Roche Diagnostics GmbH), total cholesterol (TC) (Reflotron Cholesterol, cat. no. 10745065; Roche Diagnostics GmbH), triglycerides (TG) (Reflotron TG, cat. no. 10745049; Roche Diagnostics GmbH) and high-density lipoproteins (HDL) (Reflotron HDL Cholesterol, cat. no. 11208756; Roche Diagnostics GmbH) were estimated using test-specific commercial kits and a Reflotron Plus Analyzer (Woodley Equipment Co., Ltd., Horwich, Lancashire, UK). Very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) were calculated using the two following standard formulas: TG/5 and [Cholesterol-(VLDL+HDL)], respectively. The serum total protein (TP) was estimated using a kit (Crescent Diagnostics, Jeddah, Saudi Arabia) and the following equation: TP = (Abssample/Absstandard) × concentration of standard.

Determination of tissue malondialdehyde (MDA) and non-protein sulfhydryl (NP-SH)

For tissue MDA the method reported by Utley et al (27) was followed. Briefly, the liver tissues were homogenized in 0.15 M KCl at 40°C (Potter-Elvehjem type C homogenizer) to give a 10% w/v homogenate. The Abs of the solution was then read at λ=532 nm and the MDA content (nmol/g wet tissue) was calculated by reference to a standard curve of MDA solution. Hepatic NP-SH was measured according to the method of Sedlak and Lindsay (28). The tissues were homogenized in ice-cold 0.02 mM EDTA and the Abs (λ=412 nm) was measured following the addition of 5,5′dithio-bis(2-nitrobenzoic acid) against the control.

Microscopy and histopathological evaluation

Morphological investigation of the cultured HepG2 cells was performed under a microscope to investigate any changes in the cells cultured with different concentrations of ASEE and DCFH at 24 and 48 h post-treatment. The sections of liver tissues fixed in NBF (for 48 h at room temperature) were stained with hematoxylin and eosin for 2–3 min at room temperature as previously described (29). Tissue sections were histopathologically examined under a light microscope (OMX1200C; Nikon Corporation, Tokyo, Japan) and images (at magnifications ×200 and ×400) were captured using a mounted digital camera.

Qualitative phytochemical screening of ASEE

Phytochemical screening tests for major secondary metabolites, including alkaloids, flavonoids, tannins and saponins were performed using standard procedures as described previously (30–32). Briefly, for alkaloids 0.5 mg ASEE was dissolved in 2% hydrochloric acid (Sigma-Aldrich, Merck KGaA) and filtered. Fresh Mayer's reagent (0.68 g mercuric chloride and 2.5 g potassium iodide; Sigma-Aldrich; Merck KGaA) prepared in distilled water (50 ml, final volume) was added to the 3 ml ASEE solution in a test tube. The formation of a yellow precipitate confirmed the presence of alkaloids. For flavonoids, 5 ml ASEE solution was treated with several drops of 20% sodium hydroxide (Sigma-Aldrich; Merck KGaA) in a test tube. The appearance of an intense yellow color that turned colorless following the addition of diluted hydrochloric acid was indicative of flavonoids. For tannins, 0.25 mg ASEE was dissolved in 10 ml water in a test tube and several drops of 5% ferric chloride (Sigma-Aldrich; Merck KGaA) were added. The development of a brown-green or blue-black color indicated the presence of tannins. For saponins, 0.5 mg ASEE was dissolved in 10 ml water in a test tube and agitated vigorously to form a thick persistent froth, which represented a positive result for saponin.

Standardization of ASEE by the validated high-performance thin-layer chromatography (HPTLC) method

The reverse phase (RP)-HPTLC method was used to standardize the 70% ethanol extract of A. subrecta as described previously (33). The chromatography was performed on a 10×10 cm precoated silica gel F254 RP-HPTLC plate using rutin as the standard reference. Several mobile phases were tried to obtain a good resolution and separation of the different compounds present in the ASEE. Based on observations, acetonitrile and water were selected in the ratio of 4:6 as a suitable mobile phase to perform the standardization of ASEE. The standard and the samples were applied on the HPTLC plate by an Automatic TLC Sampler-4 (CAMAG Chemie-Erzeugnisse & Adsorptionstechnik AG, Muttenz, Switzerland). The plate was developed under controlled condition in an Automated Developing Chamber-2 and scanned by TLC Scanner-3 (λ=363 nm) (both CAMAG Chemie-Erzeugnisse & Adsorptionstechnik AG).

Statistical analysis

Data are presented as the mean ± standard error of three (in vitro) and six (in vivo) determinants. Total variation present in a set of data was estimated by one-way analysis of variance followed by Dunnett's post hoc test. Excel 2010 (Microsoft, Tulsa, OK, USA) was used to analyze the data. P<0.05 was considered to indicate a statistically significant difference.

Results

Antioxidant activity of ASEE

The DPPH radical scavenging activity of ASEE was observed to be dose-dependent and 15.9, 51.1, 65.9 and 71.5% at concentrations of 31.25, 62.5, 125 and 250 µg/ml, respectively, compared with ascorbic acid (Fig. 1). Similarly, in the β-carotene-linoleic acid assay ASEE significantly inhibited lipid peroxidation in a dose-dependent manner (P<0.01, P<0.001; Fig. 2) confirming its antioxidative potential.

Figure 1. In vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of different concentrations of ASEE and AA (standard control). Data are presented as the mean ± standard error of the mean (n=3). **P<0.01, ***P<0.001 vs. AA. ASEE, Atriplex suberecta ethanol-extract; AA, ascorbic acid.

Figure 2. In vitro anti-lipid peroxidation activity of ASEE assayed by the β-carotene-linoleic acid bleaching method. (A) β-carotene bleaching rate in the presence of 250 µg/ml ASEE, GA (standard) or BC. (B) Percentage inhibition of lipid peroxidation by different concentrations of ASEE and GA. Data are presented as the mean ± standard error of the mean (n=3). ##P<0.01, ###P<0.001 vs. BC. *P<0.05, **P<0.01, ***P<0.001 vs. GA. ASEE, Atriplex suberecta ethanol-extract; GA, gallic acid; BC, blank control; LPO, anti-lipid peroxidation activity.

In vitro cytoprotective and anti-apoptotic effect of ASEE on HepG2 cells

Visual observation under a microscope revealed the marked cytotoxic effect of DCFH on the HepG2 cells, which was indicated by apoptosis or altered morphology compared with the untreated cells (data not shown). However, treatment with ASEE resulted in morphological recovery against DCFH toxicity at 24 and 48 h (data not shown). An MTT assay revealed attenuation of the HepG2 cells toxicity by ASEE in a dose-dependent manner (Fig. 3A). Treatment with 50, 100 and 200 µg/ml ASEE significantly restored the cells proliferation to 68, 76 and 110%, respectively compared with the untreated cells (P<0.001; Fig. 3A). Furthermore, in the anti-apoptotic signaling assay, ASEE at doses of 50, 100 and 200 µg/ml significantly downregulated caspase-3/-7 activity to 76, 43 and 18%, respectively compared with the DCFH-only group (P<0.001; Fig. 3B).

Figure 3. In vitro hepatoprotection of HepG2 cells by ASEE. (A) MTT-cell proliferation assay demonstrating the attenuation of DCFH-induced oxidative damage. (B) Apoptotic assay demonstrating the inhibition of DCFH-induced caspase-3/-7 activation. ASEE, Atriplex suberecta ethanol-extract; DCFH, 2,7-dichlorofluorescein. Data are presented as the mean ± standard error of the mean (n=3). ###P<0.001 vs. untreated group; ***P<0.001 vs. DCFH-only group.

Normalization of liver biochemical markers by ASEE

The acute toxicity test revealed the toleration of ASEE (500 mg/kg.bw) and healthy survival of the animals (data not shown). Furthermore, the therapeutic potential of ASEE (100 and 200 mg/kg.bw) was examined against CCl4-induced in vivo hepatotoxicity. In CCl4-only treated rats, serum SGOT, SGPT, GGT ALP and bilirubin levels were significantly elevated compared with the control group (P<0.001; Table I), which demonstrated significant hepatotoxicity. By contrast, the administration of ASEE (200 mg/kg) significantly normalized these parameters in line with silymarin, compared with the control group (P<0.01 and P<0.001; Table I). The SGOT, SGPT, GGT ALP and bilirubin levels in the ASEE (200 mg/kg) +CCl4 groups were significantly reduced compared with the CCL4 only group (P<0.05, P<0.01 and P<0.001). In addition, in the CCl4-injured rats with altered serum lipid profiles, ASEE (200 mg/kg) treatment significantly reduced the cholesterol, TG and VLDL levels, and improved the HDL level, which was comparable to that of the silymarin treated group (P<0.01 and P<0.001; Table II). Furthermore, compared with the increase in MDA level, a decrease in NP-SH tissue and decrease in TP concentrations in the CCL4 only group was indicated. ASEE (200 mg/kg) significantly normalized these parameters in CCl4-injured rats (P<0.01 and P<0.001; Table III).

Table I. Effect of ASEE on CCl4-induced hepatotoxicity-associated parameters in rats.

Table I.

Effect of ASEE on CCl4-induced hepatotoxicity-associated parameters in rats.

Treatment group	Dose (mg/kg)	SGOT (U/l)	SGPT (U/l)	ALP (U/l)	GGT (U/l)	Bilirubin (mg/dl)
Control	0	107.45±5.31	28.83±2.2	321.66±13.88	4.06±0.32	0.54±0.01
CCl4	0	294.83±8.33b	230.83±9.62b	515.16±13.70b	12.85±0.98b	2.16±0.08b
ASEE + CCl4	100	309.33±5.93c	209±7.47c	509.66±12.48c	13±0.53c	2.08±0.05c
ASEE + CCl4	200	286±6.29c	193±6.52d	467±11.00c	10.66±0.38c	1.91±0.06a
Silymarin + CCl4	10	136.66±6.00e	85.66±4.31e	396.33±7.62e	5.58±0.28e	1.06±0.06e

{ label (or @symbol) needed for fn[@id='tfn1-etm-0-0-5919'] } All values represent mean ± standard error of the mean.

a P<0.01

b P<0.001 vs. the control group.

c P<0.05

d P<0.01

e P<0.001 vs. the CCl₄ only group. Analysis of variance, followed by Dunnett's multiple comparison test. SGOT, serum glutamate oxaloacetate; SGPT, serum pyruvate transaminase; ALP, alkaline phosphatase; GGT, γ-glutamyl transferase; CCl₄, carbon tetrachloride; ASEE, Atriplex suberecta ethanol-extract.

Table II. Effect of ASEE on CCl4-induced lipid profile change in rats.

Table II.

Effect of ASEE on CCl4-induced lipid profile change in rats.

Treatment group	Dose mg/kg	Cholesterol (mg/dl)	Triglycerides (mg/dl)	HDL (mg/dl)	LDL (mg/dl)	VLDL (mg/dl)
Control	0	109.83±3.94	59.01±2.74	55.18±2.4	42.84±3.22	11.8±0.54
CCl4	0	206±4.53a	151.16±4.61a	26.25±1.79a	149.51±4.28a	30.23±0.92a
ASEE + CCl4	100	192.33±7.03b	133.83±5.61c	26.91±1.3b	138.65±8.21b	26.76±1.12c
ASEE + CCl4	200	169.83±5.28d	120±6.67d	26.85±1.14b	118.98±5.94c	24±1.33d
Silymarin + CCl4	10	147.66±4.88d	110.16±5.26d	40.41±2.97c	85.21±5.98d	22.02±1.05d

{ label (or @symbol) needed for fn[@id='tfn7-etm-0-0-5919'] } All values represent mean ± standard error of the mean.

a P<0.001 vs. the control group.

b P<0.05

c P<0.01

d P<0.001 vs. the CCl₄ only group. Analysis of variance, followed by Dunnett's multiple comparison test. CCl₄, carbon tetrachloride; ASEE, Atriplex suberecta ethanol-extract; HDL, high-density lipoprotein; LDL, low-density lipoprotein; VLDL, very-low-density lipoprotein.

Table III. Effect of ASEE on biochemical parameters of liver tissues in CCl4-treated rats.

Table III.

Effect of ASEE on biochemical parameters of liver tissues in CCl4-treated rats.

Treatment group	Dose (mg/kg)	MDA (nmol/g)	NP-SH (nmol/g)	TP (g/l)
Control	0	0.50±0.02	7.39±0.53	113.76±2.81
CCl4	0	4.82±0.29b	3.86±0.44b	49.11±1.82a
ASEE + CCl4	100	4.63±0.21c	5.38±0.47d	46.30±2.59
ASEE + CCl4	200	3.54±0.24d	5.70±0.44d	65.06±2.58e
Silymarin + CCl4	10	1.37±0.16e	6.52±0.31e	91.81±4.08e

{ label (or @symbol) needed for fn[@id='tfn12-etm-0-0-5919'] } All values represent mean ± standard error of the mean.

a P<0.05

b P<0.001 vs. the control group.

c P<0.05

d P<0.01

e P<0.001 vs. the CCl₄ only group. Analysis of variance, followed by Dunnett's multiple comparison test. CCl₄, carbon tetrachloride; ASEE, Atriplex suberecta ethanol-extract; MDA, malondialdehyde; NP-SH, non-protein sulfhydryl; TP, total protein.

Histopathological improvement by ASEE

The rat liver histopathological analysis revealed CCl4-induced necrotic and fatty degenerative changes (panel GII; Fig. 4) as compared to the control group (panel GI; Fig. 4). In the ASEE group (100 mg/kg.bw/day), congested central vein with mild necrosis and fatty changes were observed (panel GIII; Fig. 4). In addition, the higher dose of ASEE (200 mg/kg.bw/day) normalized the hepatocyte lesion and resulted in a full recovery (panel GIV; Fig. 4), comparable to that observed in the silymarin group (panel GV; Fig. 4). The histopathological data therefore confirmed the in vivo hepatoprotective efficacy of ASEE.

Figure 4. Histopathology of rat livers from all groups. Histopathology analysis revealed rat livers with healthy tissues, normal hepatocytes and central vein (panel GI), CCl4-injured tissue with necrosis and fatty degenerative changes (panel GII), tissue with congested central vein with necrosis and fatty changes following ASEE supplementation in CCl4-treated animals (panels GIII and GIV), liver with normal hepatocytes and fully recovered central vein following silymarin supplementation in CCl4-treated animals (panel GV). CCl4, carbon tetrachloride; ASEE, Atriplex suberecta ethanol-extract.

Phytochemical screening of ASEE

The qualitative phytochemical screening revealed the presence of flavonoids, alkaloids, tannins and saponins in ASEE (data not shown).

Chromatographic quantification of rutin in ASEE

ASEE was further standardized by a validated RP-HPTLC method using rutin as an antioxidant biomarker. Of the various solvent combinations tested, acetonitrile and water (4:6; v/v) was indicated as the optimal mobile phase for the estimation of rutin in ASEE (Fig. 5A and B). A sharp and compact spot of rutin was identified at Rf=0.67 (Fig. 5C), with clear separation along with different phytoconstituents of ASEE (Fig. 5D) at the optimized mobile phase volume (20 ml) and saturation time (20 min). The estimated content of rutin in ASEE was 1.94 µg/mg (dry weight).

Figure 5. Quantification of rutin in ASEE by validated RP-HPTLC at λ=363 nm (mobile phase-acetonitrile: water, 4:6, v/v). (A) Pictogram of developed HPTLC plate. (B) 3-D display of all tracks. (C) Chromatogram of standard rutin (1 µg spot-1), peak 1 (Rf=0.67). (D) Chromatogram of ASEE containing rutin (peak 2; Rf=0.67). RP-HPTLC, reverse-phase high-performance thin-layer chromatography; ASEE, Atriplex suberecta ethanol-extract.

Discussion

Cellular oxidative stress is a process where reactive oxygen and nitrogen species, common toxic products of redox reactions, are increased (1). Oxidative stress is closely associated with the occurrence and development of various conditions, including cirrhosis and carcinoma, which are chronic liver diseases (34). The healthy body has a set of hepatic antioxidant enzymes to prevent and neutralize free-radical induced cellular damage (35). However, exposure to a hepatotoxic agent may cause the generation of free radicals to exceed the protective effects of the antioxidant enzymes (34,35). The effectiveness of hepatoprotective agents is therefore dependent on their ability to attenuate the harmful free radicals and to maintain normal liver functions (3,4,34). In the present study, the in vitro and in vivo antioxidative and hepatoprotective potential of ASEE was investigated.

DPPH is a molecule containing a stable free radical, which upon receiving an electron from antioxidant agents undergoes reduction in the intensity of its purple solution, and hence in absorbance (23). As it is recommended to conduct more than one in vitro assay (36), in the present study the antioxidant activity of ASEE was also confirmed by β-carotene bleaching. In the β-carotene bleaching method linoleic acid generated free radicals attack unsaturated β-carotene to undergo oxidation and subsequently cause it to lose its orange color. During in vitro DPPH free radical scavenging and β-carotene-linoleic acid bleaching assays, ASEE demonstrated antioxidant activity that appeared to be close to the levels of ascorbic and galic acids. Notably, flavonol glycosides from the aerial parts of A. halimus have been revealed to have a clear DPPH radical scavenging ability (37). In addition, septanosides isolated from A. portulacoides have recently been highlighted for their in vitro antioxidant activity using DPPH, ABTS+, Fe3+ and catalase assays (38).

DCFH is typically used to estimate in vitro oxidative stress generated by free-radicals through the oxidation of DCFH into the fluorescent DCF (39). In addition, it is also used as a potent cytotoxic agent against an array of human cell lines (25). In the in vitro hepatoblastoma cell culture model used in the present study, ASEE promoted HepG2 cell proliferation and recovery against DCFH-toxicity in a dose-dependent manner. Apoptotic cell death caused by reactive oxygen or nitrogen molecules is a well known phenomenon (34,35). In the present study the apoptotic-signaling assay revealed a dose-dependent inhibition of caspase-3/-7 activation by ASEE against DCFH-induced HepG2 cell death. In conclusion, ASEE exhibited a promising antioxidative and cytoprotective salutation against chemical-toxicity.

To further confirm the in vitro effects, the in vivo therapeutic potential of ASEE was examined in CCl4-injured livers of Wistar rats. CCl4 is a common hepatotoxin used in the experimental study of liver diseases that induces free-radical generation in liver tissues (23,26). Clinically, CCl4-induced acute hepatotoxicity manifests as jaundice and elevated levels of liver enzymes, followed by hepatic necrosis (40). In a previous study, A. lentiformis ethanol and n-butanol extracts were reported to have antioxidant activities, including normalization of liver functions by a significant increase in serum alanine transferase levels (41). In the present study, the significant elevation of serum SGOT, SGPT, GGT, ALP, bilirubin and TP was observed in CCl4-treated rats, which indicates damage to the hepatic tissues. Treatment with ASEE demonstrated its therapeutic ability to normalize the serum biomarkers via attenuation of CCl4 toxicity at a comparable level to treatment with silymarin. In addition to this, ASEE also normalized the serum cholesterol, TC, LDL and HDL levels in the CCl4 treated rats.

MDA is used as a marker for lipid peroxidation of the cell membrane, which may cause cell damage (42). The level of MDA was reduced in ASEE treated rats suggesting its cytoprotective and curative activities against CCl4. In addition, the liver NP-SH level in CCl4-treated animals was significantly diminished compared with the control group, suggesting oxidative hepatocellular damage. The administration of ASEE or silymarin replenished NP-SH in the CCl4-treated animals demonstrating its protective activity.

The histopathological changes observed in the liver tissues revealed that the administration of ASEE caused the recovery of hepatic damage. This was revealed by the presence of normal hepatic cords and the absence of necrosis and lesser fatty infiltration in CCl4-treated rats. These results indicate the in vivo hepatoprotective effects of ASEE by abating the chemical-induced oxidative and apoptotic pathways.

Furthermore, the antioxidative and hepatoprotective activities of ASEE may be attributed to the presence of antioxidant flavonoids, alkaloids, polyphenols and saponins as confirmed by the qualitative phytochemical screening. The hepatoprotective activity of flavonoids is due to their ability to scavenge and reduce cellular free radicals. Rutin, a natural bioflavonoid that is distributed in a range of medicinal plants, is known for its pharmacological properties, including strong its antioxidant and anti-lipid peroxidative activities (43,44). Previously, the in vivo hepatoprotective efficacy of rutin in CCl4-treated BALB/cN mice has been reported (45). The identification of rutin in A. subrecta by the validated HTPLC method is in agreement with previous findings and endorses its therapeutic attribution to the prevention and treatment of liver diseases. In conclusion, to the best of our knowledge this is the first investigation into the hepatoprotective effects of A. subrecta and it has revealed its promising antioxidative and hepatoprotective potential against chemical-induced in vitro and in vivo liver injury. These results were supported by the phytochemical analysis and identification of rutin, a well-known antioxidant flavonoid in the plant. Therefore, A. subrecta may be a valuable source of natural antioxidant or health protective agent to manage oxidative stress-associated diseases. However, further investigation into its phytochemical properties and active principles, including an assessment of any other therapeutic contributions is required.

Acknowledgements

The authors would like to thank Mr. Malik Saud from the College of Pharmacy, King Saud University (Riyadh, Saudi Arabia) for his assistance with animal care.

Funding

The study was supported by the Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia (grant no. RG-1435-053).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

MKP conceptualized and designed the study, participated in the in vitro and in vivo experiments, analyzed the data and wrote the manuscript. AHA performed the in vitro experiments, analyzed data and contributed to writing the paper. MSAD and AJAR collected and extracted the plant material and participated in phytochemical analysis. PA performed the HPTLC study and analyzed data. KEI performed the liver histopathology. MSA and SR participated in the in vivo study and data analysis. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the Experimental Animal Care Society (King Saud University, Riyadh, Saudi Arabia) and adhered to its guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References