Alleviated Actions of Plantago albicans Extract on Lead Acetate-Produced Hepatic Damage in Rats Through Antioxidant and Free Radical Scavenging Capacities

Abstract

The aim of this study was to explore the possible protective mechanisms and to determine the antioxidant capacity of phenolic compounds extracted from Plantago albicans against lead acetate-induced hepatic injury. High performance liquid chromatography-photo diode array/electrospray ionization-mass spectrometry (HPLC-PDA/ESI-MS) assay was used to identify the P. albicans extract phenolic compounds. Animals received 100 mg of lead acetate/kg of body weight (bw) in the drinking water for a period of 30 days. The other groups of rats were orally administered with silymarin (300 mg/kg bw) or the P. albicans extract at two doses (100 and 300 mg/kg of bw), once daily, by gastric gavage for the same time. The P. albicans exhibited high total phenolic, flavonoid, and anthocyanin contents. The antioxidant in vitro activity demonstrated that the P. albicans exhibits an important effect against deleterious reactive species. The in vivo results showed that P. albicans prevented the lead acetate-induced significant changes on serum and liver lipid levels. In contrast, P. albicans succeeded in improving the biochemical parameters of serum and liver bringing them closer to the normal values of the control group. It also significantly promoted (P < .05) pro-inflammatory cytokines (TNF-α, IL-6, and NF-κB) in the liver of the experimental animals. The evaluated sample with HPLC-PDA/ESI-MS method showed to contain 10 dominant polyphenols, 2 hydroxycinnamic acids (p-coumaric acid and chlorogenic acids), 4 flavones (Apigenin, Luteolin, Cirsiliol, and Luteolin-7-O-rutinoside), and an anthocyanin (cyanidin-3-glucoside). Hence, it can be concluded that P. albicans could be a potent source of health-beneficial phytochemicals providing a novel therapy to protect liver against lead exposure.

Introduction

Lead (Pb) is an environmental contaminant that causes occupational health problems due to the toxicity arising from environmental pollution.¹ Pb is the most prevalent environmental poison inducing the majority of diseases in man and animals.² Lead is present in the atmosphere, water, and soil. Elevated lead concentrations in farmed fish may be extremely dangerous to human health. Many investigations have indicated that lead accumulation in tissues such as liver, kidney, and the brain impacts various biological activities at the molecular and cellular levels.^3,4

Some effects of lead acetate-induced liver injury are the increase of reactive oxygen species (ROS) production and a direct depletion of antioxidant reserves.⁵ This metal also causes oxidative damages through the leakage of marker enzymes in different organs.⁶ The massive generation of oxygen-derived free radicals in the lead acetate-induced organ also leads to the unavoidable production of lipid peroxidation on the cell membrane and alters enzyme activity.⁷ Moreover, these free radicals do not only affect lipids. Oxidative stress causes profound alterations in various biological structures, including the disruption of membrane fluidity, protein denaturation, and the destruction of DNA.⁸

Natural antioxidants, which are found in medicinal plants, have received great attention and have been studied extensively since they have a high free radical scavenging capacity related to various diseases. In living systems, various antioxidant mechanisms may act by raising the levels of endogenous defenses by upregulating the expression of genes encoding the enzymes and cytokines.⁹

Recently, some studies have confirmed that certain plantains reveal considerable bioactivity, which could be used as alternative therapy for prostatitis antispasmodic effects, including atherosclerosis and thrombosis, and bacterial and viral infections¹⁰ due to their natural constituents, the high bioactivity of this species, and its availability. We found it was worthwhile to investigate the phenolic profile and biological activity of endemic Plantago albicans since there is very little literature on its chemical constituents or biological potential. To the best of our knowledge there are no other data on the chemical profile of this species.

P. albicans L. (Plantaginaceae) is an herbaceous plant found naturally in regions of subtropical and temperate climate and it is easily cultivated in Tunisia. The extracts of Plantago species are often used in traditional medicine due to their hepatoprotective,^11
–13 anti-inflammatory, analgesic, and antipyretic properties.¹⁴ In addition, P. albicans induced structural and functional corrections in liver, as well as heart tissue.¹⁵ In fact, this genus contains a high amount of primary and secondary metabolites,¹⁶ which leads us to consider the high potential of this plant species as a source of biologically active compounds. Few previously published studies have dealt with the chemical composition of P. albicans and explored the mechanism by which it regulates the in vitro and in vivo antioxidant defense system of the aqueous extract of P. albicans.

The objective of the present study is to determine the phenolic compounds in the P. albicans aqueous extract by HPLC-PDA/ESI-MS and to explore its antioxidant properties against the toxic effects of lead acetate using biochemical and histopathological methods. The messenger RNA (mRNA) expressions of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), TNF-α, IL-6, and NF-κB in the liver were determined to explore the molecular mechanisms responsible for the preventive effects.

Materials and Methods

Chemicals

All chemicals were of the highest purity (99.0%). Lead acetate, trichloroacetic acid (TCA), bovine serum albumin (BSA), ferrozine, glacial acetic acid, FeCl₃, bovine CAT, epinephrine, malondialdehyde (MDA), catechin, thiobarbituric acid (TBA), 5,5-dithio bis 2-nitrobenzoic acid (DTNB), chloro-2,4-dinitrobenzene (CDNB), disodium salt of ethylene diamine tetraacetic acid (EDTA), and reduced glutathione (GSH) were obtained from Scharlau Chemie S.A. (Barcelona, Spain). Hydrogen peroxide (H₂O₂), sulfosalicylic acid (SSA), Nitro blue tetrazolium (NBT), HCl, the Folin–Ciocalteu reagent, catechin, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, sodium nitrite (NaNO₂), aluminum chloride [AlCl₃], potassium ferricyanide, sulfanilamide, N 1-naphthylethylenediamine, and orthophosphoric acid were purchased from Sigma Chemical Company (St. Louis, MO, USA). Sodium nitroprusside (SNP), NBT, riboflavin, deoxyribose, and silymarin were obtained from Sigma-Aldrich Co.. All other chemicals used were of analytical grade.

Plant material and preparation of the extracts

The plant material consists of the leaves collected from the Kasserine (region in the west-center of Tunisia) during March 2018. The botanical identification of P. albicans was carried out by Professor Ben Nasri-Ayachi, Sciences Faculty of Tunisia. Free phenols were extracted according to standard protocols¹⁷ with some modifications. Two hundred grams of air-dried powder was boiled on slow heat in distilled water for 30 min in an ultrasonic homogenizer. The extract was centrifuged at 4000 g for 10 min and filtered. The filtrates were collected and concentrated using a rotary evaporator until dryness. The percentage yields based on the dried starting material were 28.5% for dry aqueous extract (w/w). The resulting powder was stored at 4°C for their subsequent analysis.

Determination of total phenolic content

The total phenolic content (TPC) of the plants' extract was determined using the Folin–Ciocalteu colorimetric method described in Dewanto et al. ¹⁸ Sample solutions at different concentrations (125, 250, 500, 750, and 1000 μg/mL) were mixed with 1 mL of the Folin–Ciocalteu reagent. The mixture was shaken and allowed to stand for 6 min. After that, 0.8 mL of 75 g/L sodium carbonate aqueous solution was added. The absorbance of the resulting blue color was measured at 760 nm after 90 min. The TPC was expressed as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g DW) through the calibration curve with gallic acid. Triplicate measurements were taken and the mean values calculated.

Determination of total flavonoid content

The total flavonoid content was measured according to the AlCl₃ colorimetric method developed by Khan et al. ¹⁹ with some modifications, using (+) catechin as the reference standard. Appropriate dilutions of the sample or standard solution were reacted with NaNO₂ (solution), followed by the formation of a flavonoid–aluminum complex using AlCl₃. The absorbance at 510 nm was read against a blank prepared similarly without the plant extract. The flavonoid concentration was expressed as mg (+) of catechin equivalent per gram of dry weight (mg CE/g DW). The data are reported as the mean ± standard deviation (SD) for three replicates.

Polyphenolic profile characterization by HPLC-PDA/ESI-MS

The polyphenolic compounds in the aqueous extract of P. albicans were estimated with the HPLC-PDA/ESI-MS method. The extract was filtered through a 0.45-μm cellulose acetate membrane filter before injection. The HPLC-PDA/ESI-MS method HPLC analyses were performed on an Agilent Technologies (INRAP) modular model 1200 system, consisting of a vacuum degasser, a binary pump, an autosampler, and the thermostated column compartment, which was used for the separation of all analytes. Chromatographic separation was performed by a Zorbax Eclipse XDB-C18 RR 4.6 mm × 150 mm × 1.8 μm (Agilent Technologies) reverse-phase column held at 25°C.

The binary mobile phase consisted of acetonitrile water, and formic acid (0.1% and 0.05%) and acetic acid (0.1% and 0.05%) were tested to identify the optimal mobile phase. The mobile phase was composed of solvent A (acetonitrile with 0.05% acetic acid) and solvent B (water with 2% formic acid) as described previously.²⁰ Gradient elution was performed using the following solvent gradient: 0–2 min (10% B), 2–18 min (10–40% B), 18–20 min (40–90% B), 20–24 min (90% B), 24–25 min (90–10% B), and 25–30 min (10% B). The mobile phase rate was 0.8 mL/min, and each injection volume was set to 10 μL. Various phenolic compounds were identified by comparing their m/z values and ultraviolet (UV) spectra with authentic compounds and were detected using an external standard method.

In vitro antioxidant activity assays

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity

The antioxidant activity of the extract was evaluated using a DPPH assay, which was carried out according to the method of Shen et al. ²¹ Briefly, 50 μL of the extract was added to 1950 mL of DPPH (6 × 10⁻⁵, dissolved in 95% methanol). The mixture was vigorously shaken and maintained at room temperature for 30 min in the dark. The scavenging capacity was then measured by monitoring the decrease in absorbance at 515 nm. The antioxidant activity was expressed as a percentage of inhibition of the DPPH radical relative to the blank control and was determined using the following equation: $DPPH scavenging effect (%) = [A_{0} - A_{1} ∕ A_{0}] \times 100$

where A₀ was the absorbance of the control, and A₁ was the absorbance of the extract/standard. The scavenging capacity was expressed as IC₅₀, which was defined as the concentration of the tested sample required for the inhibition of the [DPPH^•] radical formation by 50%.

Ferric-reducing power

The reducing power of the extract was determined by the methods of Atmani et al. ²² with some modifications. Briefly, 0.2 μL of the P. albicans extract was mixed with 0.5 mL of phosphate buffer (0.2 M pH 6.6) and 0.01 g/ml potassium ferricyanide (0.5 mL). After 30 min of incubation at 50°C, 0.1 g/ml of TCA (0.5 mL) was added, and the mixture was centrifuged at 3000 g for 10 min. Finally, the supernatant from the sample (0.5 mL) was mixed with distilled water (0.5 mL) and 0.1 mL of ferric chloride (10⁻³ g/ml, in water). Then the absorbance was measured at 700 nm at a reaction time of 10 min. The obtained values were means of triplicate analyses.

Nitric oxide radical scavenging assay

The nitric oxide radical (^•NO) generated from SNP was assayed according to the method of Sreejayan.²³ Briefly, NO was measured using the Griess reagent (1% sulfanilamide, 0.1% N 1-naphthylethylenediamine, 2% orthophosphoric acid). The reaction mixture (3 mL) containing 10 mM of SNP in phosphate buffered saline (0.025 M, pH 7.4) and the fractions or the reference compound (ascorbic acid) at different concentrations (100–800 μg/mL) were incubated at 29°C for 180 min. Thereafter, 0.5 mL of incubation solution was removed and diluted with 0.5 mL Griess' reagent and allowed to react for 30 min. The absorbance of the chromophore (purple azo dye) formed was measured at 546 nm. Inhibition of the nitric oxide generated was measured by comparing the absorbance values of the control, the fractions, and the reference compound. The inhibition % was calculated using the formula and plotting a graph and was then compared to the standard. $\begin{matrix} Nitric oxide scavenged (%) = \\ (A c o n t r o l - A t e s t) ∕ A c o n t r o l \times 100 \end{matrix}$

where A control = absorbance of the control sample and A test = absorbance in the presence of the samples of extracts or standards.

Superoxide radical (O2^•−) scavenging assay

This assay was based on the reduction of NBT in the presence of riboflavin and methionine as previously published.²⁴ The 3 mL reaction mixture contained 2 μM of riboflavin, 13 mM of methionine, 100 μM of EDTA, NBT (75 μM), and 1.0 mL of test sample solutions. The tubes were kept in front of a fluorescent light (725 lumens, 34 W), and the absorbance change was recorded at 560 nm after 20 min. The mixture was maintained at room temperature for 30 min in the dark. Percent inhibition was calculated against a control without the extract. $(%) I n h i b i t i o n = [(A_{0} - A_{1}) ∕ A_{0}] \times 100$

where A ₀ is the absorbance of the control, and A ₁ is the absorbance of the tested sample. The IC₅₀ represents the concentration of the extract that inhibited 50% of the radical.

Deoxyribose degradation assay

The colorimetric deoxyribose (2-deoxy-d-ribose) method was used to determine the hydroxyl radical (OH^•) scavenging activity in an aqueous medium as described previously²⁵ with minor modifications. A measure of 500 μL of P. albicans extracts was mixed with 100 μL of deoxyribose (28 mM in 20 mM KH₂PO₄ buffer, pH 7.4), containing 100 μL of 1.0 mM H₂O₂. An aliquot of FeCl₃ (200 μM) premixed with 1.04 mM of EDTA was added, the mixture was vortexed, and the reaction was initiated by adding 0.1 mL of 2 mM ascorbic acid. The reaction mixtures were incubated at 37°C for 1 h, and 1 mL of 1% TBA and 1.0 mL of 2.8% TCA were added. Samples were heated on a water bath at 100°C for 20 min, cooled, and the absorbance at 532 nm was measured. The inhibition ratio of the herbal extract (%) was calculated using the following formula: $Inhibition ratio (%) = [A_{0} - A_{s}] \times 100 ∕ A_{0} .$

where A₀ is the absorbance of the control samples, and A_s is the absorbance of the test samples. The IC₅₀ represents the concentration of the extract that inhibited 50% of the radical.

Antioxidant activity in vivo

Animals and experimental design

Twenty male Wistar rats (10–12 weeks old) were obtained from the Pasteur Institute of Tunis animal laboratory to perform all the in vivo experiments. They were provided with a standard diet (EL BADR, Utique-Bizerte, Tunisia). The animals were fed with standard granulates with free access to water and kept at a standardized temperature and humidity (23°C ± 2°C, relative humidity 55% ± 0.5%, 12-h light/12-h dark cycle). This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals and was approved by the Institutional Animal Care and Use Committee of National Institute of Health.²⁶

The animals were divided into five groups, each consisting of 6 animals, which included: Control group (1) (normal): rats were given physiological saline (0.9% NaCl) by oral gavage during the whole course of the experiment. Animals of groups (2–5) received distilled water containing 100 mg of lead acetate/kg of body weight (bw) as the only drinking fluid for induction of oxidative stress once daily for 30 days.²⁷ For the preparation of the lead acetate solution, 0.1 mL of acetic acid was added to prevent the precipitation of Pb. Group (2) did not receive any treatment and served as the toxic control group (treated with physiological saline). Group (3) was orally administered silymarin (300 mg/kg bw) for the 30 days as per the method described in Mehana et al. ²⁸ Group (4) was orally administered the P. albicans extract in doses of 100 mg/kg of bw once daily for 4 weeks. Group (5) rats were treated with P. albicans extract (300 mg/kg, bw/daily) by gastric gavage for 4 weeks. Body weights were measured daily for 30 days. At the end of this experiment, animals were sacrificed. Blood and liver were immediately put in liquid nitrogen and stored at −80°C until further analysis.

Biochemical analysis

Animals were sacrificed by cervical dislocation. The blood samples were collected and centrifuged at 3000 g at −4°C for 10 min. The livers were excised, washed, and homogenized in ice-cold physiological saline to prepare a 0.1 g/ml homogenate, then they were centrifuged at 9000 g at 4°C for 20 min to purge cellular debris, and the supernatant was collected and stored at −80°C until the biochemical assays were performed.

Part of the organ was transferred into specimen bottles containing 10% formalin for histopathological examination. To determine the effect of the P. albicans extract on oxidative stress induced in the lead acetate model, the levels of MDA, GSH, SOD, CAT, and GPx were measured in the serum and liver. Briefly, to determine the extent of lipid peroxidation in sample homogenates, thiobarbituric acid-reactive substances (TBARS) were measured according to the procedure of Puntel et al. ²⁹ GSH was estimated using the method described by Sedlak and Lindsay.³⁰ The SOD activity was indirectly estimated by a colorimetric method based on the autoxidation of epinephrine to adrenochrome according to the method of Rtibi et al. ³¹ CAT activities were estimated by measuring the decrease in H₂O₂ concentration at 230 nm using the method of Livingstone.³² The levels of GSH (GPx) in the sample homogenates were determined using the method of Flohé and Günzler.³³

All activities are expressed as units per milligram of protein (U/mg protein) in the organ or units per milliliter (U/mL) in the serum. The protein content in the organ and blood supernatants was determined using the Bradford method and using BSA as the standard.³⁴

Atomic absorption spectrometry for the blood and liver Pb level analysis

To perform the lead level assay, tissue and blood samples were digested overnight with 4 mL of a nitric/perchloric acid mixture at room temperature, as described in Atteia et al. ³⁵ with minor modifications. Then, the samples were heated at 80°C for 2 h. After filtration, deionized water was added. The tissue and blood samples were then analyzed for Pb content using an atomic absorption spectrophotometer (PinAAcle 900; PerkinElmer, Bizerte, Tunisia). The suitable wavelength for the lead was 283.31 nm.

RNA isolation and reverse transcription–polymerase chain reaction (RT-PCR)

To determine the mRNA expression, the total RNA was purified from the liver using an RNA Mini-Prep Kit (Bio Basic) according to the manufacturer's instructions. The quantity and quality of the RNA were verified by measuring the A260/A280 ratio and by gel electrophoresis. The total RNA was transcribed into complementary DNA (cDNA) using the PrimeScript reverse transcriptase (TaKaRa Bio, Inc.) following the manufacturer's protocols. The resulting cDNAs were amplified using a RT-PCR. Target gene amplification was performed using specific oligonucleotide primers in a normal PCR system.

The sequences of primers used to specifically amplify the genes of interest were as follows: 5′-CCGACCAGGGCATCAAAA-3′ (forward) and 5′-GAGGCCATAATCCGGATCTTC-3′ (reverse) for CAT; 5′-AATGTGTCCATTGAAGATCGTGTGA-3′ (forward) and 5′-GCTTCCAGCATTTCCAGTCTTTGTA-3′ (reverse) for SOD; 5′-TGC AAT CAG TTC GGA CAT CA-3′ (forward) and 5′-ACC ATT CAC CTC GCACTT C-3′ (reverse) for GPx; 5′-GGGGCCACCACGCTCTTCTGTC-3′ (forward) and 5′-TGGGCTACGGGCTTGTCACTCG-3′ (reverse) for TNF-α; 5′-TGTGCAATGGCAATTCTGAT-3′ (forward) and 5′ GGAAATTGGGGTAGGAAGGA-3′ (reverse) for IL-6; and 5′-CAC TTA TGG ACA ACT ATG AGG TCT CTG G-3′ (forward) and 5′-CTG TCT TGT GGA CAA CGC AGT GGA ATT TTA GG-3′ (reverse) for NF-κB. B-actin was amplified as an internal control gene with the following primers: 5′-GGAGATTACTGCCCTGGCTCCTA-3′ (forward) and 5′-GACTCATCGTACTCCTGCTTGCTG-3′ (reverse).

PCR products were resolved on agarose (1.2%) gels and visualized with ethidium bromide staining. The band intensity was quantified by scanning with a gel documentation and gel doc analysis system (Bio-Rad).

Statistical analyses

All tests were conducted in triplicate, and the results were expressed as mean ± SD. Statistical analyses were performed using one-way analysis of variance followed by Tukey post hoc test (GraphPad Prism 5) for multiple comparisons with statistical significance of P < .05.

Results and Discussion

Phenolic and flavonoid contents

The total phenolic and flavonoid contents of the P. albicans aqueous extract were 592.75 ± 7.53 mg/g gallic acid equivalent and 116.7 ± 1.02 mg catechin equivalent/g DW, respectively. Results from the present study show a significantly higher concentration of total phenolic and flavonoid (Table 1). Polyphenol compounds widely distributed in plants have many benefits on human health as free radical scavengers and are mainly responsible for the antioxidant capacity of plants.³⁶ For this reason, we identify the composition of our extract by HPLC-PDA/ESI-MS. The richness of the P. albicans extracts in antioxidants might encourage the development of the function of antioxidant dietary food. Thus, samples with higher phenolic content might have lower ROS values and reduce the risk of many diseases.³⁷ Recently, it has been demonstrated that P. albicans extract contains polyphenolic compounds, which can be widely used as food preservatives.³⁸

Table 1.

Phenolic Properties and Antioxidant Capacity of Plantago albicans

Test substance	Folin–Ciocalteu (mg GAE/g DW)	(+)-Catechin (mg CE/g DW)	DPPH (μg/mL)	O₂ ^•− (μg/mL)	NO^• (μg/mL)	HO^• (μg/mL)
Test substance	Folin–Ciocalteu (mg GAE/g DW)	(+)-Catechin (mg CE/g DW)	In vitro methods IC₅₀ (μg/mL)
Extract	592.75 ± 7.53	116.45 ± 1.02	20.40 ± 0.56	21.05 ± 0.07	216.66 ± 5.58	73.52 ± 1.45
Standard (vitamin C)			22.45 ± 0.76	44.73 ± 0.07	268 ± 3.25	60.24 ± 0.89

Each value is expressed as mean ± standard deviation of three replicates.

CE, catechin equivalent; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DW, dry weight; GAE, gallic acid equivalent.

DPPH radical scavenging activity

The DPPH method uses one of the most extensively used and stable chromogenic compounds to test free-radical scavenging activity of natural antioxidants. In the DPPH assay, the antioxidants were able to reduce the stable DPPH radical (purple) to the nonradical form, DPPH-H (yellow).

The DPPH scavenging activity of an antioxidant is attributed to its hydrogen-donating ability. As shown in (Table 1), the concentration of P. albicans and vitamin C (Vc), exerting 50% DPPH scavenging potential and known as the IC₅₀ value, was found to be 20.40 ± 0.56 and 22.45 ± 0.76 μg/mL, respectively. There was a significant decrease in the concentration of the DPPH radical as the concentration of the P. albicans extract increased due to its ability to scavenge free radicals. At a concentration of 20 μg/mL, the DPPH scavenging activity of P. albicans and Vc was 44.60% and 39.90%, respectively. Therefore, the results indicated that aqueous extracts of the P. albicans had a strong DPPH radical scavenging activity. Our results are similar to those reported by previous works on the antioxidant activity of Plantago in Egypt.¹⁵

Superoxide radical scavenging activity

The superoxide radical generated from the photochemical reduction of riboflavin was readily scavenged by the extract. Inhibition of NBT reduction by superoxide in the presence of the test preparation increased with increasing concentrations. The IC₅₀ value of aqueous extracts of the P. albicans was 21.05 ± 0.072 μg/mL compared to the ascorbic acid 44.73 ± 0.34 μg/mL (Table 1). Superoxide anion has been involved in several pathophysiological processes owing to its transformation into a more reactive species such as H₂O₂, hydroxyl radical, and singlet oxygen, which can alter the structure and function of many cellular components.³⁹ The superoxide scavenging activity of P. albicans was investigated because the extract has the potential to scavenge superoxide anions.

Nitric oxide radical (NO^•) scavenging assay

Nitric oxide radical (NO^•) released from SNP has a strong NO⁺ character, which can alter the structure and function of many cellular components. In addition, the toxicity of NO increases when it reacts with superoxide to form the peroxynitrite anion (^•ONOO⁻), which is a potential strong oxidant that can decompose to produce NO₂.⁴⁰ The aqueous extract of P. albicans effectively decreased the amount of nitrite generated from the in vitro decomposition of SNP at 546 nm. The maximum free radical scavenging activity and potency were interpolated from Table 1. The extract exhibited a strong NO^· scavenging activity with an IC₅₀ of 216.66 ± 5.58 μg/mL, while that for standard Vc was found to be 268 ± 3.25 μg/mL. Our finding suggests that the phenolic components present in the P. albicans extract might be responsible for the nitric oxide scavenging effect. The presence of flavonoids could explain why P. albicans was more potent at quenching the NO radical than the Vc.⁴¹

Reducing power

The antioxidant power of phenols is usually evaluated by the ferric-reducing power assay in water solution. Determination of the reducing power is an important aspect for the estimation of the antioxidant potential of vegetables. In the present study, the presence of antioxidants in the samples causes the reduction of the ferric-ferric cyanide complex to the ferrous form (Fe²⁺), and Fe²⁺ can be monitored by measuring the formation of Perl's Prussian blue at 700 nm.⁴² The ferric reducing antioxidant power of P. albicans and Vc is shown in Table 2. The antioxidant activity exerted by the extract was by breaking the free radical chain through the contribution of a hydrogen atom. The reducing power increased with the increasing amount of the extract and it was higher than the standard Vc. The ferric reducing ability of the extract at 500–2000 μg was in the 0, 36–1, 98 range and that of the standard Vc was 0, 46–1, 3. These values suggested that aqueous P. albicans extracts had a higher ferric reducing antioxidant power, which coincides with other reported, Singh et al. ⁷

Table 2.

Reducing Power by Aqueous Extract of Plantago albicans

Test substance		Concentration (mg/mL)
	0.5	1	1.5	2
Extract	0.36 ± 0.14	1.03 ± 0.01	1.34 ± 0.2	1.89 ± 0.12
Vitamin C	0.46 ± 0.02	0.98 ± 0.10	1.17 ± 0.02	1.3 ± 0.15

Values are expressed as mean ± standard deviation (n = 3) indicate triplicate measurement.

Hydroxyl radical scavenging assay

Hydroxyl radical is a highly reactive oxygen free radical that is responsible for various types of cellular damage and lipid peroxidation in living organisms and foods.⁴³ In the present study, the oxygen-derived hydroxyl radicals along with the added transition metal ion (Fe²⁺) cause the degradation of deoxyribose into MDA, which forms a pink chromogen with thiobarbituric acid.⁴⁴ As shown in Table 1, all the fractions of P. albicans and the standard (Vc) inhibited the production of hydroxyl radicals. At a concentration of 50 μg/mL, the scavenging activity for P. albicans and Vc was 26.51% and 69%, respectively, and Vc showed a higher hydroxyl radical scavenging activity than aqueous extracts of P. albicans. The hydroxyl radical scavenging activity of the aqueous extract of P. albicans in terms of IC₅₀ was 73.52 ± 1.45 μg/mL, while that of Vc was 60.24 ± 0.89 μg/mL. The results indicate the scavenging potential of P. albicans against hydroxyl radicals.

HPLC-PDA/ESI-MS analysis of phenolic compounds

The electrospray ionizations of the phenolic compounds were characterized by the predominant formation of the [M ⁺ H] ⁺ pseudo molecular ion. These compounds had been identified according to their retention time, UV spectra, and then compared with standards and with the existing literature.^45
–47 The analyzed sample contained 10 dominant polyphenols, 2 hydroxycinnamic acids (p-coumaric acid and chlorogenic acids), 4 flavones (Apigenin, Luteolin, Cirsiliol, and Luteolin-7-O-rutinoside), and an anthocyanin (cyanidin-3-glucoside). The chromatographic separation of the aqueous extract is shown in Fig. 1. The identification of the compounds, including phenolic, flavonoids, and anthocyanin, based on the mass spectra obtained by HPLC-PDA/ESI-MS method is shown in Table 3. These phenolic profiles indicate that the aqueous extract of P. albicans is a rich source of the compounds, including both phenolic and flavonoids along with the anthocyanins. This result was in general in agreement with three previous reports.^48
–50 Accordingly, it has been shown that many of these compounds have been investigated for their biological and pharmacological properties. Namely, phenolic acids exert antioxidant and hepatoprotective activities,⁵¹ in addition to the anti-inflammatory and anticarcinogenic properties of flavonoids. For this reason we identified the composition of our extract by HPLC-PDA/ESI-MS. This analysis demonstrated that the aqueous extract of P. albicans contained flavonoids and phenolic acid compounds, which are well known for their strong antioxidant activities and reduction of the lead acetate induced. These findings were supported by the findings of Solà et al. ³⁸ and Khalil et al. ⁵²

FIG. 1.

Chromatograms of HPLC-PDA/ESI-MS in positive ionization modes for aqueous extract of Plantago albicans. For peak assignments, see Table 3. HPLC-PDA/ESI-MS, high performance liquid chromatography-photo diode array/electrospray ionization-mass spectrometry.

Table 3.

HPLC-PDA/ESI-MS Determination of Different Phenolic, Flavonoid, and Anthocyanin Compounds of Plantago albicans Extracts

Compound	Nominal mass	Phenolics ([M+H]+ion, fragments) peak	References
p-Coumaric acid	163	163.28	Bhat and Riar⁴⁵
(E)-coniferaldehyde	177	177.39	Bhat and Riar⁴⁵
Caffeic acid hexoside	242	242.52	Fang et al. ⁴⁸
Cyanidin	287	287.41	Smeriglio et al. ⁴⁶
Eriodictiol	288	288.34	Smeriglio et al. ⁴⁶
Luteolin-7-O-rutinoside	309	309.36	Bhat and Riar⁴⁵
Malvidin	331	331.38	Košir et al. ⁴⁷
Gallic acid hexoside	332	332.38	Fang et al. ⁴⁸
Chlorogenic acid	354	354.36	Lai et al. ⁴⁹
Chrysophanol 8-O-glucoside	415	415.39	Bhat and Riar⁴⁵
Apigenin	271	271.45	Lin and Harnly⁵⁰

Pb concentrations in the blood and liver

Lead acetate exposure significantly increased the levels of lead in blood and the liver tissue compared with the levels recorded in the control group (P < .05). Conversely, a significant reduction of lead levels from the lead group was recorded in the blood and liver of rats treated with P. albicans aqueous extract for 4 weeks relative to the lead-exposed group (P < .05) (Table 4). Lead is a pervasive substance that pollutes the environment and which has no beneficial biological role. Results from this study demonstrate that a lead acetate treatment triggers an oxidative stress situation in blood and liver, resulting in free radical generation and the damaging of various tissues.^53,54

Table 4.

Effect of Daily Oral Concurrent Administration of Plantago albicans (100 mg/kg Body Weight and 300 mg/kg Body Weight) for 4 Weeks on Blood and Liver Lead Levels in Lead Acetate (1 g/L)-Exposed Male Rats for 4 Weeks

Groups	Normal	PbAc	PbAc+silymarin	PbAc +100 mg/kg	PbAc +300 mg/kg
Blood lead level (μg/dL)	11.1 ± 0.12	23.8 ± 0.912^a	17.12 ± 0.24^ab	17.2 ± 0.172^ab	9.9 ± 0.014^ab
Liver lead level (ppm)	1.045 ± 0.003	1.2 ± 0.001^a	1.11 ± 0.10^b	1.045 ± 0.001^b	0.77 ± 0.02^ab

Lead was given at a daily dose of 1 g/L for 30 days. The reference group treated with physiological saline (Normal; Group 1), lead acetate (PbAc)+(0.9% NaCl) group, PbAc+Silymarin (300 mg/kg, bw) group, PbAc+P. albicans (100 mg/kg, bw) group, PbAc+P. albicans (300 mg/kg, bw) group. Six rats from each group were sacrificed 1 day after the treatment.

Significant variation from Control group at P < .05.

Significant variation from Pb acetate group at P < .05.

bw, body weight.

Rat blood Pb levels in the intoxicated group ranged between 9.9 and 23.8 μg/dL (Table 4) and are comparable to those found in liver with a severe overt toxicity. However, no significant change in the blood and liver tissue Pb levels is seen compared with the silymarin group of rats and the group treated with 100 mg/kg bw of P. albicans (P > .05) (Table 4). Significant reduction of lead levels from the lead group was recorded in the blood and liver of rats treated with P. albicans extract with doses of 300 mg/kg bw during the 4 weeks (P < .05). Furthermore, the decrease in the lead levels caused by the P. albicans extract was mostly related to the chelating capacity of P. albicans regarding heavy metals. In fact, phenolic compounds have a rapid lead adsorption rate and a high lead adsorption capacity and enhance the elimination of heavy metals from the body.

These results are in agreement with the results of Banji et al. ⁵⁵ and El-Nekeety et al. ⁵⁶ In this study, P. albicans has significantly decreased lead levels in the blood and liver tissue, which may partly explain its protective effect.

Effect of P. albicans on the antioxidant activity of serum and liver

P. albicans suppressed lead acetate induced oxidative stress and increased the MDA's concentration in the serum (Table 5) and in the liver tissues (Table 6) when treated with PbAc (P < .05), but the GSH levels and the activities of enzymic antioxidants (SOD, CAT, GPx) were significantly decreased by chronic exposure to lead acetate (P < .05) compared with the control group. The statistical analysis indicated a significant (P < .05) interaction between lead acetate and P. albicans in the serum and liver.

Table 5.

Measurement of the Antioxidant Activity and Lipid Peroxidation of the Blood

Groups	GSH mmol/mL	CAT (μmol) H₂O₂ (min/mL)	SOD (U/mL)	GPx (μmol) GSH (mL)	Lipid peroxidation (nmol) MDA (mL)
Normal	0.031 ± 0.026	23.66 ± 1.89	2 ± 0.5	8.44 ± 1.22	75.28 ± 1.54
Lead acetate	0.009^a ± 0.0001	2.91^a ± 0.62	0.12^a ± 0.025	2.00^a ± 0.02	133.87^a ± 4.61
Silymarin	0.032^b ± 0.001	32.36^ab ± 2.70	2.13^b ± 0.32	5.56^ab ± 0.66	77.54^b ± 1.22
100 mg/kg	0.033^b ± 0.001	23.82^b ± 0.75	1.86^b ± 0.74	4.75^ab ± 1.03	76.81^b ± 1.53
300 mg/kg	0.102^ab ± 0.015	39.82^ab ± 2.48	4.67^ab ± 0.76	8.05^b ± 1.23	70.37^ab ± 0.56

Values are mean ± SEM (n = 6). Control group (Normal), lead acetate+(0.9% NaCl) group, lead acetate+Silymarin (300 mg/kg, bw) group, lead acetate+P. albicans (100 mg/kg, bw) group, lead acetate+P. albicans (300 mg/kg, bw) group. Significant difference at P < .05 (ANOVA) compared with normal control and lead acetate treated group.

Significant compared to the reference normal group.

Significant compared to the reference lead acetate treated group.

ANOVA, analysis of variance; CAT, catalase; GPx, glutathione peroxidase; GSH, reduced glutathione; H₂O₂, hydrogen peroxide; MDA, malondialdehyde; SEM, standard error of the mean; SOD, superoxide dismutase.

Table 6.

Measurement of the Antioxidant Activity and Lipid Peroxidation of the Liver Homogenates

Groups	GSH (mmol/mg protein)	CAT (μmol) H₂O₂ (min/mg protein)	SOD (U/mg protein)	GPx (μmol) GSH (mg protein)	Lipid peroxidation (nmol) MDA (mg protein)
Normal	0.16 ± 0.026	66.18 ± 10.19	8.43 ± 0.6	34.74 ± 3.07	33.21 ± 0.18
Lead acetate	0.09^a ± 0.01	19.29^a ± 0.82	2.24^a ± 0.25	6.95^a ± 0.27	73.26^a ± 0.062
Silymarin	0.17^b ± 0.01	69.67^b ± 4.31	5.14^ab ± 0.15	15.68^ab ± 9.11	41.11^ab ± 3.74
100 mg/kg	0.18^b ± 0.01	61.89^b ± 11.95	5.17^ab ± 0.15	20.75^ab ± 8.75	40.66^ab ± 4.04
300 mg/kg	0.19^b ± 0.006	107.06^ab ± 11.98	9.0^b ± 1.11	46.943^ab ± 3.80	25.5^ab ± 0.5

Values are mean ± SEM (n = 6). The reference group treated with physiological saline (Normal; Group 1), lead acetate+(0.9% NaCl) group, lead acetate+silymarin (300 mg/kg, bw) group, lead acetate+P. albicans (100 mg/kg, bw) group, lead acetate+P. albicans (300 mg/kg, bw) group. Significant difference at P < .05 (ANOVA) compared with normal control and lead acetate treated group. ^aSignificant compared to the reference normal group.

Significant compared to the reference lead acetate treated group.

Silymarin and both doses of P. albicans extract significantly decreased (P < .05) the Pb content value shown in lead acetate-treated rats compared with the healthy control animals. Treatment with P. albicans extract with doses of 300 mg/kg bw (high dose) along with lead acetate, significantly enhanced the level of GSH and of SOD, CAT, and GPx activities (P < .05) and decreased the MDA content (two fold). The main outcome of this study was that the administration of lead acetate resulted in a significant oxidative stress-mediated damage in the serum and liver, which was inferred from the altered biochemical and histopathological indices under investigation. MDA is an oxidative stress marker and has been found to be elevated in various diseases related to free radical damage. Many studies have reported that lead exposure induces an increase of the MDA level in serum and liver.⁵⁷ GSH serves as a major intracellular reducing agent. GSH is consumed by detoxifying enzymes such as GPx in oxidative conditions and acts as a direct ROS scavenger.⁵⁸

Our results show a depletion of ROS after lead acetate administration, which was prevented by treatment with the two different doses of P. albicans. SOD, CAT, and GPx are the most important antioxidant enzymes that can also protect cellular compounds against damage induced by free radicals and are usually used as biomarkers to indicate ROS production.⁵⁹ Many studies have shown that lead can reduce enzyme antioxidants such as SOD, CAT, and GPx, because of its high affinity for sulfhydryl groups or metal cofactor in these enzymes and molecules.⁶⁰ Hence, these antioxidant enzymes are potential targets for lead toxicity, whose main biological role is to protect the host organism from oxidative damage.⁶¹ In the present study, lead acetate induces severe metabolic dysfunctions, lipid overloading, and a decrease in the activities of enzymes such as SOD, CAT, and GPx in Pb-intoxicated rats.⁶² These results suggest that the administration of P. albicans extract in high doses could alleviate the oxidative stress in the serum and liver of mice caused by lead acetate. We found that polyphenols markedly decreased the MDA level in lead-treated rat serum (Table 5) and liver (Table 6) in a previous study.⁶³ Similarly, in the current study, we have witnessed a significant increase in the GSH level and activities of enzymes such as SOD, CAT, and GPx with the P. albicans treatment suggesting that the free radical scavenging activity of P. albicans may facilitate the upregulation of antioxidant enzymes and maintenance of the thiol status.

These findings are similar to those in the previous study.⁶⁴ In addition, polyphenols, flavonoids, and anthocyanins are strong agents that might protect against the toxicity of lead acetate in serum and liver through the inhibition of oxidative damage.⁶⁵ This is confirmed by the data obtained in the present study, which demonstrated that treatment by P. albicans reduced the lead acetate induced and prevented the reduction in antioxidant indices.

Effects of P. albicans on the histological changes in the liver of PbAc-treated rats

To investigate the inhibitory effect of dietary P. albicans administration on hepatic lead acetate accumulation, we analyzed the liver tissue histology using hematoxylin and eosin staining. The histological tissue sections of the normal group showed normal hepatocytes and a central vein (Fig. 2A). The lead acetate-induced histopathological changes in the liver were observed with significant hepatocellular damage, necrosis, diffuse areas of congestion, and perivenular inflammatory infiltrates (Fig. 2B). The rats treated with 300 mg/kg bw of P. albicans significantly saw the liver damage in lead acetate-treated rats alleviated compared to the liver of control rats (Fig. 2E). In contrast, rats treated with silymarin (Fig. 2C) and 100 mg/kg bw of P. albicans (Fig. 2D) showed a mild-to-moderate degree of congestion. No detectable macroscopic changes showed in the control group except minute foci of congestion. The 300 mg/kg bw P. albicans group showed more normal tissues than the silymarin and the 100 mg/kg bw of P. albicans groups of rats.

FIG. 2.

Representative microphotographs from the liver showing the protective effect of Plantago albicans extract on lead acetate-induced hepatic injury in rats. (A) Showing NH and CV (H&E × 100). (B) Rats treated with lead acetate showing infiltrating leukocytes in veins, necrosis lacuna of hepatocytes (N), HB, areas of hemorrhage (H), DCCV, and RBCs pooling were also seen in the sinusoids (H&E × 100). Liver of lead acetate and Silymarin-administered rats showed restored normal hepatic architecture with mild congestion of CV, PF, and PI (C) (H&E × 100). (D) Rats treated with P. albicans 100 mg/kg and PbAc showed marked normal hepatocyte structure and lymphocyte aggregation (H&E × 100). (E) Rats treated with P. albicans (300 mg/kg) and lead acetate showed prominent improvement in hepatocytes with few areas of hemorrhages (circle) (H&E × 100). CV, central vein; DCCV, dilated and congested central vein; HB, hepatocyte ballooning; H&E, hematoxylin and eosin; NH, normal hepatocyte architecture; PF, portal fibrosis; PI, portal inflammation; RBCs, red blood cells.

The liver of the 300 mg/kg bw P. albicans and lead acetate-administered rats restored normal hepatic architecture that showed improvement in hepatocytes with a mild dilation of the central veins. The histological examination of the liver tissue of the animals treated with lead acetate revealed severe histopathological changes. Similar results were reported by Kubo et al. ⁶⁶ Moreover, recent studies have shown lead-induced liver hyperplasia followed by apoptosis mediated by oxidative stress in kupffer cells.⁶⁶ The group treated with P. albicans extract and lead acetate showed a mitigative protective effect against histopathological changes that occurred in hepatocytes without necrosis and binucleated cells that represent a good sign of regeneration. The aqueous P. albicans extract was found to protect against hepatotoxicity induced by lead acetate in rats as assessed by the histopathological examination, which also showed that P. albicans is rich in compounds known to be strong antioxidants and reduced liver damage induced by lead acetate.

This effect was the same as that of other phenolic compounds or antioxidants previously reported by Wei et al. ⁶⁸ and Clichici et al. ⁶⁹

Effects of P. albicans on mRNA expression levels of the antioxidant enzyme in the liver of PbAc rats

Lead acetate administration significantly decreased the relative gene expression levels of the SOD, GPx, and CAT transcription in Lead acetate intoxicated groups compared with their respective control values (P < .05) (Fig. 3). However, polyphenols notably promoted the lead-induced downregulation of the mRNA expression levels of antioxidant enzymes. The relative gene expression levels of SOD, CAT, and GPx in the group treated with 300 mg/kg bw of P. albicans extract increased by 39.55%, 43.42%, and 17.79%, respectively, compared with those of the PbAc group (P < .05) (Fig. 3). The relative gene expression of SOD, CAT, and GPx in the silymarin group increased by 53.53%, 51.55%, and 25.92% compared with the Lead acetate group, respectively (P < .05) (Fig. 3). However, no significant change in the mRNA expression levels of the antioxidant enzymes was observed in the liver tissues treated with 100 mg/kg bw P. albicans and silymarin. P. albicans plays an important role in the transcription of antioxidant enzymes. It was reported that P. albicans could improve the antioxidant potentials in cells by enhancing hepatic SOD, CAT, and the GPx mRNA expression.^70,71 In the present study, SOD, CAT, and the GPx mRNA expression levels are reduced by exposure to lead. Furthermore, our study has shown that the introduction of lead decreases the expression of antioxidant enzymes and its target genes and that the reduced expression is related to increased oxidative stress in the liver of rats (Fig. 3). This result is in agreement with studies on hepatocyte exposure to oxidants.⁷² This research indicates that P. albicans has a protective effect against lead-induced hepatotoxicity in rats through the renewal of the antioxidant enzyme activities and the restoration of the lead-induced downregulation of the gene expression levels of antioxidant enzymes.⁷³

FIG. 3.

Quantitative reverse transcription PCR analyses of mRNA abundance of selected ROS scavengers in liver tissues of rats. Control group (Normal), lead acetate (PbAc)+(0.9%NaCl) group, PbAc+Silymarin (300 mg/kg) group, PbAc+Plantago albicans (100 mg/kg) group, PbAc+P. albicans (300 mg/kg) group. Significant difference at P < .05 (ANOVA) compared with normal control. ^aIndicates P < .05 compared to the control group; ^bindicates P < .05 compared to PbAc group, n = 6/group. ANOVA, analysis of variance; CAT, catalase; GPx, glutathione peroxidase; mRNA, messenger RNA; PCR, polymerase chain reaction; ROS, reactive oxygen species; SOD, superoxide dismutase.

Effects of P. albicans on the mRNA expression of TNF-α, IL-6, and NF-κB in lead-induced rat liver disruption

To evaluate the molecular mechanism against lead-induced liver injury, the mRNA expression of the TNF-α, IL-6, and NF-κB was determined by RT-PCR. As shown in Fig. 4, the liver cytokine TNF-α and IL-6 levels in rats in the 100 and 300 mg/kg bw P. albicans-treated groups were significantly lower than those of the lead acetate group (P < .05). The relative gene expression levels of TNF-α and IL-6 in the lead control group increased by 42.85% and 20.70%, respectively, compared with those of the normal group (P < .05). The levels of this pro-inflammatory cytokine in rats treated with 100 mg/kg bw P. albicans and silymarin were similar to those of the normal group, and the effects in the 300 mg/kg bw P. albicans-treated rats were significantly better than those in the 100 mg/kg and silymarin treated rats (P < .05). In addition, to explore the harmful effect of lead acetate on liver function efficiency, we examined the expression of the NF-κB gene that is responsible for regulating the expression of genes required for cellular proliferation, inflammatory responses, and cell adhesion.⁷⁴ As shown in Fig. 4, lead acetate-induced rats showed an increase in the mRNA expression of NF-κB compared with the 100 mg/kg bw P. albicans and silymarin group (P < .05).

FIG. 4.

Effects of Plantago albicans on the mRNA expression levels of TNF-α, IL-6, and liver tissues of mice. Control group (Normal); lead acetate (PbAc)+(0.9%NaCl) group, Lead acetate (PbAc)+Silymarin (300 mg/kg) group; PbAc+P. albicans (100 mg/kg) group; and PbAc+P. albicans (300 mg/kg). Fold-ratio: gene expression/β-actin x control numerical value (control fold ratio: 1). Values are mean ± SD (n = 6), ANOVA followed by Tukey's test. ^aIndicates P < .05 compared to the control group, ^bindicates P < .05 compared to the PbAc group, n = 6/group. SD, standard deviation.

The relative gene expression of NF-κB in the lead acetate group increased by 58.64% compared with the normal group (P < .05). However, the treatment with a 300 mg/kg bw P. albicans dose of lead-intoxicated rats revealed a significant decrease in the NF-κB level compared to lead-treated animals (P < .05). IL-6 is responsible for stimulating acute phase protein synthesis, as well as the production of neutrophils in the bone marrow. It supports the growth of B cells and is antagonistic to regulatory T cells, particularly during tissue damage, which leads to inflammation. TNF-α is a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. The primary role of TNF-α is in the regulation of immune cells. TNF-α is able to induce fever, apoptotic cell death, inflammation and to inhibit tumor genesis and viral replication and respond to sepsis through IL-6 producing cells.⁷⁵ NF-κB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress.⁷⁶ NF-κB plays a key role in regulating the immune response to infection.⁷⁷ In response to environmental perturbation, NF-κB regulates a series of cytotoxic cytokines, including IL-6, participating in the chronic inflammation of the liver.⁷⁸

In the current study, lead acetate treatment significantly increased mRNA expressions of the inflammatory cytokines TNF-α and IL-6, which is consistent with a previous report.⁷⁹ In contrast, it is well known that lead reduces total hepatic RNA content indicating a lower rate of hepatic protein synthesis. It can also perturb protein synthesis in hepatocytes.⁸⁰ Furthermore, Shaban El-Neweshy and Said El-Sayed⁸¹ reported a decrease in total hepatic protein content in response to lead intoxication. However, P. albicans was associated with a reduction of the mRNA expression of pro-inflammatory cytokines TNF-α and IL-6 in the hepatocytes, an increase in the proliferative activity of cells of the main parenchymal tissue of the liver and a strengthening of antioxidant mechanisms.⁸² These findings indicate that P. albicans could prevent hepatic damage by suppressing the inflammatory cytokine and through downregulating NF-κB.⁸³ P. albicans also had stronger protective effects against liver damage than silymarin at equivalent doses.

Our study confirms previous findings according to which the oxidation of lipids, proteins, and DNA induced by lead decreases the antioxidant defense systems in the serum and liver. The protective effects of P. albicans on serum and hepatic damage were evaluated through various in vitro and in vivo models, including MDA, SOD, CAT, and GPx level assays, a histological test, and RT-PCR assays for the pro-inflammatory TNF-α, IL-6, and NF-κB related genes. Our data have clearly demonstrated that rats that were administered 300 mg/kg of P. albicans showed significantly decreased serum and hepatic levels of MDA and a notably increased level of SOD, GPx, and CAT compared with rats in the lead acetate group. The antioxidant defense system and gene expression were improved after oral gavage with 300 mg/kg P. albicans. These results suggest that P. albicans might participate in lowering the toxic heavy metal load in rats, indicating that P. albicans has a potent antioxidant activity and might be utilized as a novel natural antioxidant in food and therapeutics.

Footnotes

Acknowledgment

The authors thank Prof. Ben Attia Msaddek for his help and scientific discussion.

Author Disclosure Statement

The authors declare that they have no conflicts of interest.

Funding Information

This research was supported by the Tunisian Ministry of Higher Education and Scientific Research and Faculty of Sciences of Bizerte.

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Alleviated Actions of Plantago albicans Extract on Lead Acetate-Produced Hepatic Damage in Rats Through Antioxidant and Free Radical Scavenging Capacities

Abstract

Introduction

Materials and Methods

Chemicals

Plant material and preparation of the extracts

Determination of total phenolic content

Determination of total flavonoid content

Polyphenolic profile characterization by HPLC-PDA/ESI-MS

In vitro antioxidant activity assays

2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity

Ferric-reducing power

Nitric oxide radical scavenging assay

Superoxide radical (O2•−) scavenging assay

Deoxyribose degradation assay

Antioxidant activity in vivo

Animals and experimental design

Biochemical analysis

Atomic absorption spectrometry for the blood and liver Pb level analysis

RNA isolation and reverse transcription–polymerase chain reaction (RT-PCR)

Statistical analyses

Results and Discussion

Phenolic and flavonoid contents

DPPH radical scavenging activity

Superoxide radical scavenging activity

Nitric oxide radical (NO•) scavenging assay

Reducing power

Hydroxyl radical scavenging assay

HPLC-PDA/ESI-MS analysis of phenolic compounds

Pb concentrations in the blood and liver

Effect of P. albicans on the antioxidant activity of serum and liver

Effects of P. albicans on the histological changes in the liver of PbAc-treated rats

Effects of P. albicans on mRNA expression levels of the antioxidant enzyme in the liver of PbAc rats

Effects of P. albicans on the mRNA expression of TNF-α, IL-6, and NF-κB in lead-induced rat liver disruption

Footnotes

Acknowledgment

Author Disclosure Statement

Funding Information

References

Superoxide radical (O2^•−) scavenging assay

Nitric oxide radical (NO^•) scavenging assay