Comparative assessment of hepatoprotective properties of Artesunate and flavonoids from Artemisia annua on acetaminophen and carbon tetrachloride-induced cytotoxicity in primary mice hepatocytes

Abstract

Background

Artesunate (ART) is a semi-synthetized molecule from Artemisinin, an active compound isolated from the medicinal plant Artemisia annua, widely used for the treatment of malaria. Previous studies reported that ART may exert a dual effect on the liver. Accordingly, this study investigated the potential protective action of ART against Acetaminophen (APAP) and Carbon tetrachloride (CCl₄)-induced hepatotoxicity in primary mice hepatocytes, in comparison to that of flavonoid extracted from A. annua (FAA). In addition, the antioxidant properties of FAA were also assessed.

Methods

The antioxidant activities of FAA and Ascorbic acid (ASC) (0.01–100 μg/mL) were assessed through inhibition of lipid peroxidation, reduction of ferric and phosphomolydenum, and hydroxyl and DPPH radicals scavenging assays. The hepatoprotective effects of FAA and ART (0.1–100 μg/mL) were evaluated against APAP (11 mM) or CCl4 (4 mM) induced oxidative damage in primary mouse hepatocytes. Biochemical parameters associated with hepatotoxicity assessed include cell viability, cell membrane integrity, cellular glutathione, and antioxidant enzyme activities.

Results

The obtained finding revealed FAA displayed a remarkable antioxidant activities as evidenced by the low IC₅₀/EC₅₀ values (3.85–19.32 μg/mL), comparable to that of ASC (3.26–18.04 μg/mL). When tested at 10 μg/mL, both FAA and ART significantly (p˂0.05) preserved cell viability, inhibited alanine aminotransferase leakage and lipid membrane peroxidation, and restored superoxide dismutase and catalase activities and glutathione content induced by APAP or CCl₄ in a similar way as Silymarin. However, ART showed a significant (p˂0.05) cytotoxic effect on hepatocytes at 100 and 1000 μg/mL and did not confer obvious protection at 100 μg/mL.

Conclusion

Overall, our data demonstrated that ART harms mice hepatocytes at high concentration while conferring relative protection against APAP and CCl₄-hepatotoxicity at low concentration. In contrast, FAA effectively protects liver cells without cytotoxicity effect, event at 100 μg/mL. Accordingly, ART should be given to the patient only under a medical prescription.

Abbreviations

ALT

Alanine aminotransferase

APAP

Acetaminophen

ART

Artesunate

ASC

Ascorbic acid

CAT

Catalase

CCl₄

Carbon tetrachloride

DPPH

2,2-Diphenyl-Picryl-Hydrazyl

FAA

Flavonoid extracted from A. annua

MDA

Malondialdehyde

Sil

Silymarin

SOD

Superoxide dismutase

1. Introduction

Artesunate (ART) is a molecule used in the treatment of malaria which is tirelessly the leading cause of mortality and morbidity in developing countries, especially in sub-Saharan region where malaria remains endemic and is considered as a major public health concern [1]. However, the uncontrolled use of Artesunate (ART) and other antimalarial drugs is becoming disturbing; although not documented, there is evidence of self-medication and free purchase of antimalarial drugs in these endemic areas, and such practices can contribute to the intoxication and injury of vital organs. For instance, toxicological investigations on ART reported acute nephrotoxicity [2], testis damage [3] neurotoxicity and hepatotoxicity [4]. Indeed, hepatotoxicity is the injury or liver damage caused by exposure to chemicals or drugs. Drug-induced hepatotoxicity is an adverse reaction that may be uncommon, but serious [5]. Drug-induced liver injury can be classified either as intrinsic, where the toxicity is dose-dependent, or idiosyncratic, which is more uncertain [6,7]. Drug-induced hepatotoxicity is one of the most frequent arguments raised for the non-approval of a new drug candidate or retraction of an approved drug from the market. Clinical symptoms of Drugs-induced hepatotoxicity are highly inconstant, fluctuating from asymptomatic elevation of liver function markers to fulminant hepatic failure [8,9].

Regarding ART, its effect on the liver, an important organ which plays a key role in the maintenance of body's homeostasis through its metabolic, digestive, biotransformation and excretory functions among others [10], is controversial. On one hand, it has been shown that sub-chronic administration of ART is associated with the abnormal elevated level of serum biomarkers of hepatotoxicity such alanine aminotransferase (ALT) and alkaline phosphatase [11,12]; while in other hands, the protective effect of ART against carbon tetrachloride (CCl₄)-induced liver damage has been reported [13].

It should be noted that ART itself is a semi-synthetic derivative of Artemisinin, an active compound isolated from the Chinese medicinal herb, Artemisia annua (Asteraceae), also used in traditional medicine in the treatment of malaria [14,15]. For the traditional application, A. annua is usually used for the treatment of intermittent fevers due to malaria, tuberculosis, lice, wounds, dysentery, pain and swelling around tooth, haemorrhoids … etc [16]. Pharmacological studies on A. annua reported several biological activities, including, but not limited to anti-parasitic, anti-bacterial, anti-fungal, antiviral, anti-inflammatory and antitumor effects [17,18]; and phytochemical investigations revealed it as a rich source of flavonoids, a class of secondary metabolites known in the literature as bearing antioxidant and hepatoprotective properties [19,20].

Considering the possible dual effect of ART on the liver, and its wide used in the general population in sub-Saharan countries, it is important to clarify whether ART is benefit for the liver or not. Accordingly, using acetaminophen (APAP) and CCl₄-induced oxidative damage in primary mice hepatocytes as models of hepatotoxicity, we aimed in this study at evaluating the hepatoprotective effect of ART, in comparison to that of flavonoids extracted from A. annua (FAA), in addition to the investigation of the chemical antioxidant properties of FAA.

2. Material and methods

2.1. Reagents

Collagenase type NB4 standard grade, Fetal bovine serum (FBS), Dulbecco's Modified Eagle's Medium (DMEM), Carbon tetrachloride (CCl4), Acetaminophen (APAP), Thiazolyl Blue Tetrazolium Bromide, Silymarin, Ascorbic acid (Vitamin C) and Quercetin were all purchased from Sigma-Aldrich (St Louis, USA). The others reagents were of analytic grade.

2.2. Animals and ethical considerations

Healthy adult Swiss albino mice of both sexes, weighing between 25 and 35g, were used. They were provided by the Animal House of the Laboratory of Pharmacology and Toxicology (University of Yaoundé I). They were maintained in a plastic cage with access to a standard diet and tap water ad libitum. All the procedures were in respect to the ARRIVE guidelines on animal care and approved by the Institutional Joint Review Board for Animals and Humans Bioethics of the University of Yaoundé I-Cameroon.

2.3. Extraction of flavonoids from A. annua (FAA)

The aerial parts (stems and leaves) of A. annua were collected in March 2019 in Banganté, Ndé Sub-Division, West Region-Cameroon and identified at the Cameroon national herbarium where voucher specimen is kept under the reference number 65647/HNC. The extraction of flavonoids was performed as described elsewhere [21] with slight modifications. In brief, the harvested plant sample was washed using tap water, air-dried, and ground. From the powder obtained, 500g were macerated at room temperature (25 °C) with 2 L of the solvent system ethanol-water (70:30, v/v) for 48 h. Whatman N°1 filter paper was then used to filter the mixture and the residue was re-extracted twice with the same volume of solvent. The collected filtrates were pooled and concentrated to yield an initial crude extract which was dissolved in distilled water at the density of 20% (w/v) and partitioned with an equal volume of petroleum ether. The aqueous phase was collected, filtered, and evaporated to yield the flavonoid extract (34g) of A. annua, designated as FAA, which was stored at −20 °C in a closed container till use.

Qualitative analysis to confirm the presence of flavonoids within the extract was performed as previously described [22] while quantitative analysis to determine the flavonoids content of the extract was assessed according to the aluminum chloride method as described by Ref. [23]. Quercetin, used as standard, enabled to draw a calibration curve and express the flavonoids content of the extract as milligram of quercetin equivalent (QE) per gram of extract, which was 99.36 ± 0.51 mg QE/g of extract.

2.4. Evaluation of the antioxidant activities of FAA

Investigation of the antioxidant activities of FAA was assessed through the following chemical antioxidant assays: in vitro inhibition of lipid peroxidation; hydroxyl (HO°) and 2,2-Diphenyl-Picryl-Hydrazyl (DPPH) free radical scavenging assays; and ferric and phosphomolydenum reducing antioxidant power, where FAA and ascorbic acid (reference antioxidant) were evaluated at the final concentrations of 0.01; 0.1; 1; 10 and 100 μg/mL.

2.4.1. In vitro inhibition assay of lipid peroxidation induced in mice liver homogenate

The assay was performed as previously described [24]. The assay was performed as previously described [24]. In brief, In brief, mice was anesthetized using petroleum ether, and sacrificed by cervical dislocation. Liver was excised and rinsed in 0.15 M KCl solution. Then, 10% (w/v) liver homogenate was prepared by weighting 5g of liver which were crushed using Potter homogenizer in ice-cold phosphate buffer (0.1 M; pH 7.4; final volume 50 mL). The homogenate was centrifuged (12000×g; 15 min; 4 °C) and the supernatant was collected and used as liposome for in vitro lipid peroxidation assay. Afterward, lipid peroxidation was induced in mice liver homogenate with FeCl₂–H₂O₂. 50 μL of the tested sample (FAA or ascorbic acid) was added 1 mL of mice liver homogenate (10%, w/v) and mixed; then, 50 μL of H₂O₂ (0.5 mM) and FeCl₂ (0.5 mM) each, were added. The mixture was incubated for 60 min at 37 °C before the addition of 1 mL of trichloroacetic acid (15%, w/v) and thiobarbituric acid (0.67%) respectively. The resulted mixture was heated for 15 min at 100 °C in a water bath, followed by centrifugation (3000 g, 5 min, 4 °C). Finally, the supernatant was collected and the absorbance read at 532 nm. The result expressed as the percentage of inhibition was determined with the formula (1).

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{o}\mathrm{f}\mathrm{l}\mathrm{i}\mathrm{p}\mathrm{i}\mathrm{d}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\%)=100\times (\left[{\mathrm{A}}_0–{\mathrm{A}}_1\right]/{\mathrm{A}}_0)$$ (1)

Where A₀ represents the absorbance of the control and A₁ represents the absorbance of the tested sample.

2.4.2. Hydroxyl radical scavenging assay

The procedure described by Su et al. [25], based on Fenton reaction through the generation of hydroxyl radical in vitro were used. In brief, in each test tube was successively added 444 μL of FeSO4 (3 mM), 635 μL of H2O2 (1 mM), 635 μL of distilled water, 32 μL of plant sample or standard to achieve the final desired concentration, and 254 μL of Sodium Salicylate (10 mM). The mixture was then incubated for 60 min at 37 °C and the absorbance read at 532 nm. The percentage of hydroxyl radical scavenging was determined using the formula (1).

2.4.3. DPPH radical scavenging assay

DPPH radical scavenging was determined according to the procedure described by Ref. [23]. Briefly, 3.1 mL of DPPH solution (40 μg/mL in methanol) was mixed with 50 μL of the tested sample. The absorbance was read 30 min later after incubation at 25 °C in the dark. The percentage of scavenging activity of DPPH radical was also determined using the formula (1).

2.4.4. Ferric reducing antioxidant power (FRAP) assay

FRAP assay was performed as outlined by Ref. [26] with slight modification. The FRAP mixture consisted of 2.2 mL phosphate buffer (0.2 M; pH 6.6) and 2 mL potassium ferrocyanate (0.25%, w/v). Then, 100 μL of FAA or ascorbic acid was added and the mixture incubated at 50 °C for 20 min. Then, 2 mL of trichloroacetic acid (10%, w/v) was added and the resulted mixture was centrifuged at 3000 g for 10 min. 2 mL of supernatant were mixed with 500 μL of freshly prepared FeCl3 (0.02%, w/v) and allowed to stand up for 10 min at 25 °C before reading the absorbance at 700 nm against the blank where the tested sample was replaced by distilled water. The percentage of reducing ability was calculated using the formula (2).

$$\mathrm{R}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}(\%)=100\times (\left[{\mathrm{A}}_1–{\mathrm{A}}_0\right]/{\mathrm{A}}_1)$$ (2)

Where A₁ is the absorbance of the tested sample while A₀ is the absorbance of control.

2.4.5. Phosphomolybdenum reducing assay (total antioxidant capacity)

Total antioxidant capacity of FAA was evaluated using the phosphomolybdenum assay as reported previously [24]. In test tube were successively added 300 μL of FAA or ascorbic acid, and 3 mL of reaction mixture containing 0.6 M sulphuric acid; 4 mM ammonium molybdate, and 28 mM sodium phosphate. The resulted mixture was heated at 95°Cfor 90 min. After cooling down, the absorbance of the solution was read at 695 nm. For the control, distilled water was used instead of FAA tested sample. The percentage of reduction was then determined using the formula (2).

2.4.6. Determination of half efficient/inhibitory (EC50/IC50) and correlations between the flavonoid content and the antioxidant activity

Different EC₅₀/IC₅₀ values were determined from the graph “Antioxidant Activity vs. Log [FAA or ascorbic acid]” using GraphPad Prism 5.03 software. Correlations between flavonoid content of FAA and the corresponding antioxidant activity were determined by linear regression. Four different solutions were prepared at the tested concentrations (0.1, 1, 10, and 100 μg/mL) and the flavonoid content of each solution was assayed. Then, for each antioxidant assay, the activity of FAA at each tested concentration was plotted against the flavonoid content using Microsoft Excel 2013, and the coefficient r² value was deduced from the graph.

2.5. Study of hepatoprotective properties of Flavonoid from A. annua (FAA) and Artesunate (ART) against APAP and CCl₄-induced toxicity in primary mice hepatocytes

2.5.1. Isolation of hepatocytes

Primary mice hepatocytes were isolated in situ by liver perfusion as previously described [27]. Briefly, ketamine (87.5 mg/kg body weight) was used for anesthetizing. Then, blood was washed from the liver by perfusion (flow rate: 10 mL/min; 10 min; 37 °C) with washing buffer (10 mM HEPES, 0.5 mM EDTA, 3 mM KCl, 130 mM NaCl, 1 mM NaH₂PO₄–H₂O, 10 mM glucose, pH 7.4). After removing blood, the liver was further perfused (flow rate: 7 mL/min; 10 min; 37 °C) with the digestion buffer (washing buffer containing 5 mM CaCl₂ and 0.65 mg/mL collagenase type NB4 standard grade). Then, the liver was excised, cut into pieces, suspended in digestion buffer without collagenase, and incubated at 37 °C. with gentle shaking for 5 min. Afterward, the suspension was filtered, centrifuged (600 g, 5 min, 4 °C). The resulted pellet was suspended in 50 mL 75% Percoll in PBS and centrifuged (600 g, 5 min, 4 °C). Finally, the cell pellet obtained was suspended in 5 mL DMEM containing 10% FBS, 100 UI/mL penicillin, and 100 μg/mL streptomycin. Cell viability (≥92%) was estimated immediately and cell density was adjusted to ≈ 1 × 10⁶ viable cells/mL and used throughout the next experiments.

2.5.2. General experimental design

FAA, ART, silymarin, APAP, and CCl₄ were dissolved in 20% DMSO. 1 mL of hepatocytes suspension (≈1 × 10⁶ viable cells/mL) was distributed in triplicate into a 12-well plate labeled as control, toxicant (APAP or CCl₄), and test (FAA, ART or silymarin + toxicant) and incubated for 30 min at 37 °C in an atmosphere of 5% CO2. After adherence, the medium was replaced with a fresh medium containing 0.2% DMSO (control group), APAP or CCl₄ (intoxicated group), or test + toxicant (intoxicated and treated group); and cells were incubated for 6 h followed by biochemical analysis.

2.5.2.1. Determination of toxic concentration of APAP and CCl₄ to be used

After adherence, hepatocytes were treated either with APAP or CCl₄ at the final concentration of 1.25; 2.5; 5; 10; 15; 20; 25, and 30 mM. At the end of incubation, cell viability and membrane integrity were evaluated respectively through MTT assay and measurement of the activity of the cytosolic enzyme alanine aminotransferase leakage into the incubation medium. Then, the half toxic concentration (TC₅₀) of each toxicant was determined from the respective concentration-response curve.

2.5.2.2. Evaluation of the effect of FAA and ART on the viability of primary mice hepatocytes

Freshly isolated hepatocytes were treated with FAA, ART, or silymarin at the final concentration of 0.1; 1; 10; 100 and 1000 μg/mL 6 h later, cells were used to evaluate the viability while an aliquot of cell culture supernatant served to assess membrane integrity.

2.5.2.3. Evaluation of the hepatoprotective activity of FAA and ART

In this assay, FAA, ART, and Silymarin (used as hepatoprotective reference substances) were tested at the final concentration of 0.1; 1; 10 and 100 μg/mL, and two sets of plates were constituted. Following cell adherence, the medium was changed and replaced with a fresh medium containing APAP or CCl4 at their respective predetermined CT₅₀, and FAA, ART, or silymarin at the desired concentration, and cells were incubated for 6h. At the end of incubation, the first set of plates was used for the assessment of cell viability and membrane integrity. Regarding the second set of plates, hepatocytes were harvested, lysed by sonication, centrifuged (12 000g, 20 min, 4 °C) and the supernatant was used to measured lipid peroxidation end products, antioxidant enzymes activity, and cellular glutathione content.

2.5.3. Biochemical analysis

2.5.3.1. Assessment of cell viability and membrane integrity

Cell viability was evaluated using 3-(4, 5-dimethylthiosol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide kit (MTT; Sigma-Aldrich) by following the manufacturer's instructions. The result was expressed as a percentage of control (untreated cell) where the viability is considered as 100%.

The leakage of liver function marker, enzyme alanine aminotransferase (ALT), into the incubation medium was used to evaluate cell membrane integrity. The activity of ALT was measured according to the reported method (Reitman and Frankel, 1957).

2.5.3.2. Assessment of lipid peroxidation

Lipid peroxidation, in terms of thiobarbituric acid reactive substances (TBARS) formation, was determined as previously described [28]. Briefly, 0.5 mL of cell supernatant was mixed with 1 mL TCA (20%, v/v) and 1 mL TBA (0.67%, v/v), and heated for 20 min at 100 °C. After cooling, the precipitate was removed by centrifugation (3000g, 15 min, 4 °C) and the absorbance of the supernatant was recorded at 535 nm against a blank containing all the reagents except cell supernatant, replaced by 0.5 mL PBS. The TBARS content was estimated in terms of malondialdehyde (MDA) and calculated using the extinction coefficient of MDA, which is 1.56 × 10⁵ M⁻¹.Cm⁻¹.

2.5.3.3. Assessment of antioxidant enzymes activity: superoxide dismutase (SOD) and catalase (CAT)

The activity of SOD in cell supernatant was measured as reported previously [29]. The reaction mixture included 1.2 mL sodium pyrophosphate buffer (50 mM; pH 8.3), 100 μL phenazine methosulfate (186 μM), 300 μL nitroblue tetrazolium (300 μM), 200 μL NADH (720 μM), the adjusted volume of cellular supernatant containing 10 μg of protein (protein concentration in cellular supernatant was measured using Bradford's reagent) and distilled water in a total volume of 3 mL. The assay started with the addition of NADH. After incubation at 30 °C during 90 s, the reaction was stopped by the addition of 1 mL glacial acetic acid; and the resulted mixture was stirred vigorously and shaken with 4 mL of n-butanol. The mixture was incubated for 10 min at 25 °C and centrifuged (3000g, 5min, 25 °C). Then, the absorbance of the chromogen in the butanol layer was recorded at 560 nm. One unit of enzyme activity was defined as enzyme concentration required to inhibit absorbance of chromogen production by 50% per minute and SOD activity was expressed as specific activity in Unit/min/mg protein.

CAT activity was evaluated according to the method described by Aebi, [30]. In brief, 1 mL phosphate buffer (50 mM; pH 7.2) was added to 990 μL H₂O₂ (10 mM) solution and 10 μL of cell supernatant was added. The decrease of the absorbance of H₂O₂ was monitored at 240 nm and recorded at 20 s and 80 s. The CAT activity was then calculated by using the following equation: CAT Activity (Unit/min/mg of protein) = (2.3033/ΔT) × (logA1/A2)/Q_protein where A1 is the absorbance at 20 s; A2 is the absorbance at 80 s; ΔT is the variation in time (1min) and Q_protein is the amount of protein (mg) in cell supernatant.

2.5.3.4. Assessment of reduced glutathione (GSH) content

The procedure described by Ellman [31], was used. 50 μL of cell supernatant was mixed to 3 mL Ellman's reagent (0.05 mM DTNB in phosphate buffer 0.1 M pH 6.5) and at 25 °C for 60 min and the optical density was recorded at 412 nm. Then, GSH content was calculated using its molar extinction coefficient (ε_GSH = 13,600 M⁻¹.Cm⁻¹).

2.6. Statistical analysis

The results were expressed as mean ± standard deviation (SD) of three independent assays in triplicate. The differences between the mean values of different groups were analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's post-test using GraphPad Prism 5.03 statistical software (Graph Pad Inc., USA). Differences between compared groups were considered significant for p < 0.05.

3. Results

3.1. Antioxidant properties of Flavonoids from A. annua (FAA)

The chemical antioxidant activities of FAA, along with that of Ascorbic acid (ASC) used as a reference antioxidant compound, are depicted in Fig. 1. Overall, FAA and ASC showed concentration-dependent activities for the five end-point chemical antioxidant models evaluated. Concerning the Inhibition of lipid peroxidation (Fig. 1A), Ferric reducing ability (Fig. 1C), and DPPH radical scavenging (Fig. 1E) assays, FAA displayed lower antioxidant activities with IC₅₀/EC₅₀ of 19.32 ± 3.21; 5.67 ± 1.21 and 8.83 ± 1.84 μg/mL respectively, compared to that of ASC with IC50/EC50 of 18.04 ± 2.56; 3.26 ± 1.49 and 7.19 ± 1.20 μg/mL respectively. In contrast, regarding the Total antioxidant capacity (Fig. 1B) and Hydroxyl radical scavenging (Fig. 1D) assays, FAA showed a lower EC₅₀ (3.85 ± 1.03 and 11.59 ± 1.44 μg/mL, respectively) than ASC (EC₅₀ of 6.60 ± 1.79 and 11.76 ± 1.82 μg/mL respectively). Furthermore, a positive correlation was observed between the flavonoid content of FAA and the five free-cell system antioxidant studied with r² (correlation coefficient) of 0.91; 0.86; 0.73; 0.83, and 0.76 respectively for Inhibition of lipid peroxidation (Fig. 1A), Total antioxidant capacity (Fig. 1B), Ferric reducing ability (Fig. 1C), Hydroxyl radical scavenging (Fig. 1D) and DPPH radical scavenging (Fig. 1E) assays.

Fig. 1.

Antioxidant properties of FAA. (A): Inhibition of rat liver peroxidation assay; (B): Total antioxidant capacity; (C): Ferric reducing ability; (D): Hydroxyl radical scavenging assay; (E): DPPH radical scavenging assay. Values are expressed as means ± SD of three independent experiments in triplicate. FAA: Flavonoids from A. annua; ASC: Ascorbic acid.

3.2. Cytotoxicity effect of APAP and CCl₄ on the viability and membrane integrity of primary mice hepatocytes

The cytotoxicity effect of APAP and CCl₄ on freshly isolated hepatocytes was determined through a dose-dependent assay with toxic concentrations ranging from 1.25 to 30 mM during 6 h. As presented in Fig. 2, incubation of primary hepatocytes in presence of APAP or CCl₄, significantly (p˂0.05) reduced cell viability (Fig. 2A) and disrupted cell membrane integrity through an increased level of extracellular ALT activity (Fig. 2B) found in the incubation medium, in a concentration-dependent manner. Half toxic concentrations (TC₅₀) were 11.42 ± 1.15 and 3.30 ± 1.36 mM respectively for APAP and CCl₄. Accordingly, 11 and 4 mM were chosen respectively as toxic concentrations for APAP and CCl₄ for the hepatoprotective studies.

Fig. 2.

Cytotoxicity effect of APAP, CCl₄, FAA and ART on the viability of primary mice hepatocytes. Primary mice hepatocytes were exposed to various concentration APAP or CCl₄ (1.25–30 mM); or FAA, ART or Sil (0.1–1000 μg/mL) for 6 h. (A) and (B): Effect of APAP and CCl4 on the viability of hepatocytes and activity of ALT released in the incubation medium respectively; (C) and (D): Effect of FAA, ART and CCl₄ on the viability of hepatocytes and activity of ALT released into the incubation medium respectively; Values are expressed as means ± SD of three independent experiments in triplicate. *P˂0.05, values significantly different when compared to control group (untreated cells). FAA: Flavonoids from A. annua; ART: Artesunate; Sil: Silymarin; ALT: Alanine aminotransferase; APAP: Acetaminophen; CCl₄: Carbon tetrachloride.

3.3. Effect of FAA and ART on the viability and membrane integrity of primary mice hepatocytes

Prior evaluation of the hepatoprotective properties of FAA and ART, their effect, along with that of silymarin (Sil), used as hepatoprotective reference compound, were evaluated on the viability and membrane integrity of primary mice hepatocytes via a concentration-dependent study (0.1–1000 μg/mL). Incubation of isolated hepatocytes for 6 h with FAA or Sil neither significantly (p > 0.05) affect the viability of cells (Fig. 2C) nor increase ALT activity in the incubation medium (Fig. 2D) at up to 1000 μg/mL, as compared to the untreated cells. However, a moderate, but significant (p˂0.05) toxicity was observed in cells treated with ART at 100 μg/mL (Fig. 2C and D). This toxicity was exacerbated when hepatocytes were treated with ART at 1000 μg/mL, with a significant (p˂0.05) reduction of cell viability to about 50% (Fig. 2C), and an elevated ALT activity (Fig. 2D) in the incubation medium, as compared to the untreated cells. Therefore, the concentration of 1000 μg/mL was excluded in the hepatoprotective study.

3.4. Effect of FAA and ART on the viability, membrane integrity, and lipid membrane peroxidation of primary mice hepatocytes exposed to APAP or CCl₄

Exposure of isolated hepatocytes to APAP (11 mM) or CCl₄ (4 mM) alone during 6 h significantly (p˂ 0.05) reduced cell viability (Fig. 3, Fig. 4A), increased ALT activity in the incubation medium (Fig. 3, Fig. 4B) and lipid membrane peroxidation through an elevated level of MDA content in the cell lysate (Fig. 3, Fig. 4C) respectively when compared to the non-treated cell. In co-treated cells with APAP or CCl₄, and FAA or Sil (0.1–100 μg/mL), a concentration-dependent protective and inhibitory effect was observed on cell viability (Fig. 3, Fig. 4A), ALT activity in the incubation medium (Fig. 3, Fig. 4B) and MDA formation (Fig. 3, Fig. 4C). These protective effects were significant (p˂ 0.05) when FAA or Sil was added at the final concentrations of 10 and 100 μg/mL as compared to the intoxicated and non-treated cells; and the optimum concentration being 100 μg/mL where cell viability was similar to that observed in control hepatocytes, with a lower level of ALT activity in the incubation medium and MDA content in cell lysate. In contrast, when hepatocytes were co-treated with ART (0.1–100 μg/mL), a significant (p˂ 0.05) protective and inhibitory effects on cell viability (Fig. 3, Fig. 4A), ALT activity in the incubation medium (Fig. 3, Fig. 4B) and MDA formation (Fig. 3, Fig. 4C) were obtained only at 10 μg/mL, as compared to APAP or CCl₄ intoxicated-cells, respectively.

Fig. 3.

Protective effect of FAA and ART against APAP-induced cytotoxicity in primary mice hepatocytes. Freshly isolated mice hepatocytes were incubated without (untreated cell), or with APAP (11 mM; intoxicated group), or simultaneously with APAP (11 mM) and FAA, ART and Sil (0.1–100 μg/mL). 6 h later, cell viability (A), ALT activity leakage in the incubation medium (B) and MDA content in the supernatant of lysed cells (C) were determined. Values are expressed as means ± SD of three independent experiments in triplicate. ^ΔP˂0.05, values significantly different when compared to control group (untreated cells). *P˂0.05, values significantly different when compared to APAP group (intoxicated and non-treated cells). FAA: Flavonoids from A. annua; ART: Artesunate; Sil: Silymarin; ALT: Alanine aminotransferase; MDA: Malondialdehyde; APAP: Acetaminophen.

Fig. 4.

Protective effect of FAA and ART against CCl₄-induced cytotoxicity in primary mice hepatocytes. Freshly isolated mice hepatocytes were incubated without (untreated cell), or with CCl₄ (4 mM; intoxicated group), or simultaneously with CCl₄ (4 mM) and FAA, ART and Sil (0.1–100 μg/mL). 6 h later, cell viability (A), ALT activity leakage in the incubation medium (B) and MDA content in the supernatant of lysed cells (C) were determined. Values are expressed as means ± SD of three independent experiments in triplicate. ^ΔP˂0.05, values significantly different when compared to control group (untreated cells). *P˂0.05, values significantly different when compared to CCl₄ group (intoxicated and non-treated cells). FAA: Flavonoids from A. annua; ART: Artesunate; Sil: Silymarin; ALT: Alanine aminotransferase; MDA: Malondialdehyde; CCl₄: Carbon tetrachloride.

3.5. Effect of FAA and ART on the activities of antioxidant enzymes (SOD and CAT) and cellular glutathione (GSH) content of primary mice hepatocytes exposed to APAP or CCl₄

Incubation of freshly isolated mice hepatocytes for 6 h in presence of APAP or CCl4 alone, resulted in a significant (p˂0.05) lowering of the activities of SOD (Fig. 5, Fig. 6A), CAT (Fig. 5, Fig. 6B), and GSH content (Fig. 5, Fig. 6C) respectively, as compared to the untreated cells. Co-treatment of cells with FAA at 10 and 100 μg/mL significantly (p˂0.05) restored the activities of SOD (Fig. 5, Fig. 6A), CAT (Fig. 5, Fig. 6B), and GSH content (Fig. 5, Fig. 6C) as compared to intoxicated and non-treated cells. These restorative effects were similar to that observed in Silymarin co-treated cells at the same concentrations. However, in ART co-treated hepatocytes, an obvious restorative effect on these parameters was obtained only at 10 μg/mL (Fig. 5, Fig. 6).

Fig. 5.

Effect of FAA and ART on the activities of antioxidant enzymes (SOD and CAT) and reduced glutathione (GSH) content in primary mice hepatocytes exposed to APAP. Primary mice hepatocytes were incubated without (untreated cell), or with APAP (11 mM; intoxicated group), or simultaneously with APAP (11 mM) and FAA, ART and Sil (0.1–100 μg/mL). After 6 h of incubation, cells were harvested and lysed, then activities of SOD (A), CAT (B) and GSH content (C) were determined in the cell supernatant. Values are expressed as means ± SD of three independent experiments in triplicate. ^ΔP˂0.05, values significantly different when compared to control group (untreated cells). *P˂0.05, values significantly different when compared to APAP group (intoxicated and non-treated cells). SOD: Superoxide Dismutase; CAT: Catalase; GSH: Reduced glutathione; FAA: Flavonoids from A. annua; ART: Artesunate; Sil: Silymarin; APAP: Acetaminophen.

Fig. 6.

Effect of FAA and ART on the activities of antioxidant enzymes (SOD and CAT) and reduced glutathione (GSH) content in primary mice hepatocytes exposed to CCl₄. Primary mice hepatocytes were incubated without (untreated cell), or with CCl₄ (4 mM; intoxicated group), or simultaneously with CCl₄ (4 mM) and FAA, ART and Sil (0.1–100 μg/mL). After 6 h of incubation, cells were harvested and lysed, then activities of SOD (A), CAT (B) and GSH content (C) were determined in the cell supernatant. Values are expressed as means ± SD of three independent experiments in triplicate. ^ΔP˂0.05, values significantly different when compared to control group (untreated cells). *P˂0.05, values significantly different when compared to CCl₄ group (intoxicated and non-treated cells). SOD: Superoxide Dismutase; CAT: Catalase; GSH: Reduced glutathione; FAA: Flavonoids from A. annua; ART: Artesunate; Sil: Silymarin; CCl₄: Carbon tetrachloride.

4. Discussion

In the present study, we evaluated and compared the protective effect of flavonoid extracts from Artemisia annua with that of Artesunate (ART), a popular anti-malarial drug often used in self-medication in the area where malaria remains endemic. Indeed, ART is derived from Artemisinin, an active compound isolated from A. annua, a medicinal plant also used for the treatment of malaria in folk medicine [14,15]. However, the impact of ART on the liver is still controversial, since some studies reported its toxicological effects on the liver, while others showed its protective role against xenobiotic-induced hepatotoxicity [[11], [12], [13]]. Therefore, taking into account ART is widely used in the general population without medical prescription, and its potential drawback on a vital organ as the liver, this study aim at investigating the protective action of ART, in comparison to that of flavonoid extracted from A. annua (FAA) in Acetaminophen (APAP) and Carbon tetrachloride (CCl₄)-induced hepatotoxicity in primary mice hepatocytes.

APAP and CCl₄ are two well-known models of drugs and chemicals-induced liver injury and continue to serve for the evaluation of the hepatoprotective effect of phytochemical and other natural derived compounds [32]. Both APAP and CCl₄ are metabolized through the cytochrome P450 system to initiate their toxicity. On one hand, hepatotoxicity mechanisms of CCl₄ involve its biotransformation to form the reactive metabolite trichloromethyl radicals, which bind to the polyunsaturated fatty acid and generate peroxy-radicals, leading to lipid peroxidation [33,34]. Subsequently, lipid peroxidation can therefore induce damage in the cell membrane, leakage of cytosolic enzymes, and finally cell necrosis [35]. On the other hand, APAP hepatotoxicity is mediated via oxidant stress through its toxic metabolite, N-acetyl-para-Benzoquinone-Imine, and an excessive production of reactive oxygen species (ROS) which bind to cellular proteins and DNA, to produce protein adducts and deplete cellular glutathione (GSH) pools, responsible to the amplification of the mitochondrial oxidant stress, mitochondrial dysfunction, and death of hepatocytes [[36], [37], [38]]. To face against negative effects of xenobiotics and their reactive metabolites, hepatocytes possess an endogenous antioxidant defense system that includes not only antioxidant enzymes such superoxide dismutase (SOD) and catalase (CAT), but also antioxidant molecule as GSH [39,40]. This endogenous antioxidant defense system is responsible for converting reactive metabolites resulting from xenobiotics biotransformation into less or non-toxic compounds, preventing oxidation of polyunsaturated fatty acid, scavenging ROS, and subsequently protects cells from oxidative injury (Mates et al., 1999). Unfortunately, these antioxidant enzymes are often inactivated under excessive oxidant stress conditions [39,41].

In the present study, APAP and CCl₄ were used at 11 mM and 4 mM respectively, to induce hepatotoxicity in primary mice hepatocytes. At these concentrations, the viability of hepatocytes decreased to about 50%, associated with elevated activity of ALT into the incubation medium (Fig. 2A and B). These observations can be interpreted as harmful consequences of APAP or CCl4 which were evidenced through a significant (p˂0.05) increase formation of MDA (Fig. 3, Fig. 4C), decrease of SOD and CAT activities, and depletion of cellular GSH content (Fig. 5, Fig. 6) observed in intoxicated and non-treated cells. However, co-treatment with FAA or Silymarin, used as hepatoprotective reference compound, at 10 and 100 μg/mL respectively, significantly (p˂0.05) preserved cell viability (Fig. 3, Fig. 4A) and inhibited ALT leakage into the incubation medium (Fig. 3, Fig. 4B). Likewise, co-treatment of cells with FAA or Silymarin at these protective concentrations significantly (p˂0.05) reversed APAP or CCl₄-induced overproduction of MDA (Fig. 3, Fig. 4C), a decrease of SOD and CAT activities, and depletion of cellular GSH content (Fig. 5, Fig. 6). As Silymarin is known to exert its hepatoprotective action through its ability to inhibit the reactive metabolites deriving from the biotransformation of xenobiotics such APPA and CCl₄, and its capacity to enhance hepatic glutathione and enzymatic antioxidant defense system [42], it can be suggested that the protective mechanisms of FAA against APAP or CCl₄ hepatotoxicity are comparable to that of Silymarin. These findings suggest the potential of FAA to interfere with the critical events involve in the pathological mechanism of APAP or CCl₄ hepatotoxicity and therefore protect hepatocytes from hepatocytes injury. These results are consistent with the findings of Choi et al. [43] who reported the anti-hepatotoxic effect of water extract of A. annua against ter-Butylhydroperoxide-induced oxidative damage in HepG2 cells (human hepatocarcinoma cell line). Similarly, our results corroborated previous finding showing the hepatoprotective effect of hydro-ethanolic leaf extract of A. annua on common carp (Cyprinus carpio) exposed to ambient ammonia [44]. In contrast, in ART-co-treated cells, relative protection against APAP or CCl₄ toxicity was noted only at 10 μg/mL (Fig. 3, Fig. 4, Fig. 5, Fig. 6). These observations corroborate previous findings that showed the protective effect of ART against CCl₄-induced hepatotoxicity in mouse [13]. However, ART itself appears to be toxic for the primary mice hepatocytes, as depicted in Fig. 2C and D, where administration of ART alone at 100 and 1000 μg/mL, reduced the viability of hepatocytes to about 74% and 53%, respectively. Ours findings are also consisted with previous studies reporting that the use of ART is associated with the abnormal elevated level of serum biomarkers of hepatotoxicity such alanine aminotransferase (ALT) and alkaline phosphatase [11,12]. These observations may justify why ART did not confer any protection against APAP or CCl₄ hepatotoxicity when administrated at 100 μg/mL. Based on these findings, we therefore suggest that ART should be used only under medical prescription since high doses appear to exert negative impacts on liver cells.

The implications of free radicals and oxidant stress in xenobiotics-induced hepatotoxicity as abovementioned suggest that plant extracts and their active ingredients possessing free radical scavenging and antioxidant activities may serve as promising candidates to develop hepato-protective phytomedicine. Medicinal plants bearing antioxidant activities have been shown to protect the liver against APAP or CCl₄-induced hepatotoxicity [23,27,28]. Accordingly, we assessed the antioxidant properties of FAA to investigate whether its protective effects observed against APAP and CCl₄ toxicity could be supported by its antioxidant activities. Hence, five end-point antioxidant assays in free cell-system, including inhibition of lipid peroxidation, scavenging of HO° and DPPH radicals, and ferric and phosphomolydenum reducing antioxidant power were used to analyze the antioxidant properties of FAA, in comparison to ascorbic acid (ASC), considered as a reference antioxidant molecule. Overall, FAA and ASC exhibited antioxidant effects with IC₅₀/EC₅₀ less than 20 μg/mL (Fig. 1). Interestingly, for each chemical antioxidant assay, there was no significant (p > 0.05) difference between IC₅₀/EC₅₀ of FAA and ASC, suggesting that FAA possesses strong antioxidant properties, comparable to that of ASC. Moreover, a strong and positive correlation was observed between the flavonoid content of FAA and the corresponding chemicals antioxidant activity. Excessive generation of ROS and lipid peroxidation being considered as critical events in CCl₄ or APAP-cell death mechanism [32,36,38,45], it can be suggested that the antioxidant properties displayed by FAA, may contribute to its protective action against CCl₄ and APAP-induced oxidative injury in primary mice hepatocytes.

5. Conclusion

Overall, the present study revealed on one hand, that flavonoid extracts from A. annua exhibit a hepato-protective action, comparable to that of Silymarin, and display strong antioxidant activities, similar to Ascorbic acid. On other hand, our findings indicated that Artesunate is toxic for liver cells at high concentrations and confers relative protection against APAP and CCl4-induced cytotoxicity at low doses. Although in-depth studies are necessary to clearly understand the toxico-pathological mechanism of Artesunate on the liver, we recommend its use only under medical prescription.

6. Limitations of the study

This study has potential limitations. The work has been carried out using only in vitro models of xenobiotics-induced liver injury based on freshly isolated mouse hepatocytes. Accordingly, it would be more interesting if the study was further sustained by animal models of hepatotoxicity. Furthermore, the in vivo models could offer a better approach to understand the molecular mechanism underlying the hepatoprotective properties of FAA.

Funding

The authors declare that there is no funding to report.

CRediT authorship contribution statement

Marie Ange Djeungoue Petga: Conceptualization, Investigation, Formal analysis, Writing – original draft, Writing – review & editing. Arnaud Fondjo Kouam: Conceptualization, Investigation, Methodology, Resources, Formal analysis, Validation, Writing – original draft, Writing – review & editing. Rosine Désirée Chougouo Kengne: Investigation, Formal analysis, Writing – review & editing. Boris Rosnay Galani Tietcheu: Investigation, Methodology, Formal analysis, Visualization, Writing – review & editing. Josué Simo Louokdom: Investigation, Visualization, Writing – review & editing. Claude Bérenger Ngantchouko Ngalemo: Investigation, Visualization, Writing – review & editing. Pascal Dieudonné Chuisseu Djamen: Conceptualization, Methodology, Resources, Supervision, Validation, Writing – review & editing. Paul Fewou Moundipa: Conceptualization, Resources, Supervision, Validation, Writing – review & editing.

Declaration of competing interest

The authors declare that no competing interests exist.