Chemical composition of propolis extract and its effects on epirubicin-induced hepatotoxicity in rats

ABSTRACT

Propolis is a natural substance, produced by honeybees from the resin of various plants. The purpose of this study was to determine the chemical composition and evaluate the hepatoprotective effect of ethyl acetate extract of propolis from Tigzirt, against the toxicity induced by epirubicin which is a anticancer agent, and belongs to the family of antracyclines. The study included thirty male Wistar albino rats divided into five groups. The rats received the extraction of propolis or the quercetin orally for 15 days. The hepatotoxicity was promoted by injection epirubicin (i.v.) with a cumulative dose of 9 mg/kg. Several biological parameters were measured. Oxidative status was also assessed by evaluating antioxidant enzyme and histological study of some organs. Epirubicin caused oxidative stress by a significant decrease in hepatic antioxidant enzymes (gluthation peroxidase, catalase, superoxide dismutase), increased malondialdehyde and liver parameters (ASAT, ALAT, γGT, ALP) compared to the control. The histological study revealed major damage to the liver. Perturbations in this liver function, antioxidant status and damage to the liver by epirubicin have been repaired by the administration of propolis. Furthermore, epirubicin showed inflammatory effects induced by an increase in TNF-α and PGE2. Pretreatment with propolis to rats restored these inflammatory parameters. The chemical identification of extract of propolis by HPLC/UV shows the presence of polyphenolic compounds and many flavonoids. The propolis extract showed a significant reduction in oxidative damage from oxidative stress and a very important protective effect against epirubicin-induced hepatotoxicity.

Keywords:
Propolis; Epirubicin; HPLC/UV; Hepatotoxicity; Liver stress oxydative

Introduction

The epirubicin (EPI) is one of the effective chemotherapeutic agents. It belongs to the anthracyclines family (Judson et al., 2014). It is widely used for the treatment of various breast cancers (Jing Wu et al., 2015). EPI is derived from the doxorubicine and it was metabolized in the liver to give metabolites such as the épirubicinol and EPI glucuronide (Weenen et al., 1984). It has been demonstrated that the doxorubicine and the epirubicin have high toxicity for the liver (Weenen, 1984; Le Bot et al., 1988). The mechanism of action of epirubicin appears to be related to its ability to bind to nucleic acids (Chang et al., 2014). It is fixed quickly on the nuclear structures of the cell by intercalating in the DNA between base pairs, blocking the synthesis of the DNA and RNA (Jain et al., 2005).

Several hypotheses were suggested regarding the mechanisms of toxicity induced by anthracyclines. It has been reported that the oxygen free radicals and lipid peroxidation play an essential role in the hepatic damages caused by the epirubicin (Singal and Iliskovic, 1998). In addition, it has been clearly shown that the culture of the hepatocytes in the presence of epirubicin decreases the cellular viability by disturbances of the cellular membranes. This process has been found to be related also to the generation of the oxygen free radicals produced during EPI metabolism (Germain et al., 2003). Moreover there is a correlation between EPI and the activity of serum transaminases among cancer patients having received this drug (Ganzina, 1983; Le Bot et al., 1988; Twelves et al., 1992).

Despite the discovery of new synthetic compounds that have antioxidant effects, natural sources remain the main supplier for obtaining antioxidant molecules which can be used to prevent the oxidative damages and the toxicity generated by chemotherapy. A large amount of researches therefore have been done on the natural products especially for those rich in polyphenols and flavonoids which have shown interesting antioxidant biological properties (Kurek-Górecka et al., 2014; Georgiev et al., 2014). In this context, many antioxidant molecules have been proposed as protective against oxidant toxicity (Du and Lou, 2008; Vincent et al., 2013).

The propolis is one of these antioxidant products (Segueni et al., 2016). It is a series of resinous, gummy and balsamic substances of viscous consistency collected by the bees on certain parts of plants (primarily buds and barks of certain trees) (Harfouch et al., 2016). Its extracts contain high amounts of polyphenols, flavonoids, and ascorbic acid. Thus it has potent antioxidant properties (Boufadi et al., 2014; Machado et al., 2016). Many studies have shown that propolis has a number of biological roles as analgesic-anesthetic activity (Orsatti and Sforcin, 2011), antiallergic activity (Mehmetn Yasar et al., 2016), antibacterial activity (Boufadi et al., 2016), anti-cancer activity (Zabaiou et al., 2017), immunomodulatory activity (Soltani et al., 2017), and anti-inflammatory activity (El-Guendouz et al., 2017).

The aim of this study is to evaluate the protective effect of ethyl acetate extract of propolis (EAP) obtained from Tigzirt (Tizi Ouzou, Algeria) by the method of grids on hepatotoxicity induced by the epirubicin in vivo.

Materials and methods

Reagents and chemicals

Epirubicin (EPI, 50 mg) was purchased from the laboratory Pharmazie Thymoorgan GmbH, Allmagne. The solvents CH₂Cl₂, MeOH, EtOH, EtOAc, and CHCl₃ were obtained from Sigma–Aldrich (St Louis, MO). formic acid, trifluoroacetic acid (TFA), acetonitrile, 1.1.3.3-tetramethoxypropane, thiobarbituric acid, Tris–HCl, EDTA, sucrose, bovin serum albumin, MnCl₂, mercaptoethanol, NADPH, Ellman's reagent – 5,5′-dithiobis(2-nitrobenzoic acid), MDA, SOD, H₂O₂, quercetin and formaldehyde is an aldehyde were also purchased from Sigma-Aldrich.

Collection and extraction of the propolis

Propolis was collected according to the method of grids in June 2015 from a region of Tigzirt (Tizi Ouzou, Algeria), and was stored at −18 °C. This propolis has antioxidant effects (Boufadi et al., 2014, 2017). It was made by a breed of bees (Apis mellifera intermissa).

The crude propolis (10 g) was divided into small pieces, crushed and extracted three times with ethanol 95% (100 ml) in ultrasonic water during 1.30 h. The suspension was then filtered on paper Whatman No. 1. The solvent was evaporated to dryness under reduced pressure at 60 °C. The filtrate represents the ethanolic extract of propolis (EEP).

Then the ethanolic dry extract (EEP) was suspended in 200 ml of distilled water and extracted with 200 ml of chloroform. The organic layer was eliminated and the aqueous phase was extracted with 200 ml of ethyl acetate (EtOAc) three times. The organic phase of EtOAc was after complete evaporation of solvent the ethyl acetate fraction was denominated EAP.

HPLC/UV analysis of phenolic compounds from extract propolis

Chromatographic analysis of ethyl acetate extract of propolis (EAP) was performed on a high performance liquid chromatography (Agilent 1100). Separation was done on an Agilent poroshell 120EC column (100 mm × 2.1 mm, 2.7 µm), using mobile phases: water/TFA/formic acid (99:0.25:0.75) (A) and acetonitrile (B). The elution was carried out at a flow rate of 0.6 ml/min with a 10 µl aliquot and at a temperature of 55 °C. Using a gradient method as following (t/min,% B): (0, 0), (1, 10), (2, 12.5), (3, 15), (9, 80), (10, 100), (11, 100), (14, 0) with post 5 min. Chromatograms were recorded at 270 and 320 nm.

The sample was prepared by diluting EAP with methanol 1:100 (v/v). The propolis components were identified by comparing their retention times and their UV spectra with different commercially phenolic standards (trans-cinnamic, gallic acid, benzoic acid, ferulic acid, m-coumaric acid, caffeic acid, rosmarinic acid, and ellagic acid), flavonoids (catechin, hesperidin, thymol, galangin, tectochrysin, pinocembrin, acacetin, rutin, chrysin, apigenin, kaempferol, and quercetin) and others compounds (ascorbic acid, menthol). The standards were dissolved in methanol to give stock solutions at 1 mg/ml. The quantification of constituents was determined using standard curves expressed in mg per 1 g of crude propolis.

Animals and accommodation conditions

Thirty male Wistar rats (100–150 g) (provided by the Pasteur Institute in Algeria) were used in the present experiment. The animals were housed in metallic cages and subjected to an adaptation period of two weeks at an ambient temperature and a photoperiod of 12/12 h. The rats have a free access to the food and water ad libitum. The protocol was consistent with the guidelines of the National Institutes of Health (NIH). All animal experiments were approved by the local ethical committee for animal care of the institution (University Abdelhamid Ibn Badis, Mostaganem) (rat/mouse 20% maintenance, RN-01-20K12; Carfil Quality).

Animal treatment protocol

After two weeks of acclimatization, the rats were divided into five groups, then they were treated daily by gastric gavage for 15 days as follows: the animals of group G1 and G2 received 1 ml of physiological saline, the rats of group G3 and G4 received 1 ml of 100 and 250 mg/kg of EAP respectively, and the group G5 received 50 mg/kg quercetin.

At day 15, G2–G5 rats received three intravenous injections (i.v.) of epirubicin at 48 h intervals for one week to reach a cumulative dose of 9 mg/kg (Dobbs et al., 2003). All animals were observed and body weights were recorded daily.

Twenty-four hours after the last epirubicin injection, the animals are fasted for 12 h before blood collection. In order to avoid any risk of changing the biochemical parameters by administering a general anesthesia, the animals were kept under light anesthesia chloroform just before the collection of blood samples by cardiac puncture protocol on either dry or heparinized tubes. Plasma and serum samples were obtained by centrifugation at 112 × g for 20 min and the remaining erythrocytes were washed with physiological saline three times, lysed by addition of ice-cold distilled water, and incubated for 15 min on ice. Cell debris was removed by centrifugation at 5000 t/min for 5 min, and the lysate was recovered to assay for antioxidant enzymes in erythrocytes. The peritoneal fluid was collected in sterile phosphate buffered saline (PBS). The liver was immediately removed, washed with saline and divided into two portions. One portion was stored in 10% formaldehyde at room temperature.

The hepatic tissue homogenate was prepared according to the method of Tang et al. (2012) with some modifications. The second part of liver pieces was homogenized in an ice-cold buffer (250 mM sucrose, 10 mM Tris–HCl, 0.5 mM EDTA and 0.5% bovin serum albumin, pH 7.4). The supernatant was recovered by centrifugation at 112 × g for 15 min at 4 °C. Washing and centrifugation were repeated twice, the mitochondria pellet was re-suspended in PBS buffer (0.1 ml of EDTA 10 mM, 0.1 ml of sodium azide 35%, pH 7.4), for various assays.

Assessment of hepatic function

The activity of aspartate aminotransferase (AST, EC 2.6.1.1) and alanine aminotransferase (ALT, EC 2.6.1.2) were determined with kits from bio-Merieux (bioMerieux SA France). Total bilirubin in plasma was measured using the method of Pearlman and Lee (1974). The activity of 5′ nucleotidase (5′NUC, EC 3.1.3.5) and alkaline phosphatase (ALP, EC 3.1.3.1) were determined with kits from BioVision (BioVision, USA). The activity of gamma-glutamyl transferase (γ-GT; EC 2.3.2.2) was determined with kits from GenWay Biotech (GenWay Biotech, USA).

Estimation of oxidative stress markers

The lipid peroxidation of the liver homogenate is measured by the method of Yagi et al. (1976), by measuring thiobarbituric acid reacting through spectrophotometric at 535 nm using 1,1,3,3-tetramethoxypropane as a standard. The results were expressed as equivalents of malondialdehyde (MDA) in tissue homogenate in nmol/g tissue.

The enzymatic activity of catalase in tissue homogenate from liver was determined by the method of Luck (1963) and Aebi (1974) which depends on evaluation of decomposition rate of hydrogen peroxide by a spectrophotometric at 240 nm. The results were expressed as unit/mg tissue. The enzymatic activity of superoxide dismutase was measured according to the method of Elstner et al. (1983), which is based on the chemical reaction that generates the superoxide ion (O²⁻) from molecular oxygen in the presence of EDTA, MnCl₂ and mercaptoethanol. The enzymatic activity of glutathione peroxidase (GPx) of liver was performed according to the method described by Paglia and Valentin (1967). The principle of the test is based on the conversion of NADPH + H⁺ into NADP⁺ as a result of a series of reactions. Results were given as U/mg tissue. The determination of the level of thiols in the liver was carried out according to the method of Riddles et al. (1979) using Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB), which is a chemical used to quantify the number or concentration of thiol groups in a sample. It was developed by George L. Ellman.

Assessment of inflammation markers

Prostaglandin E2 (PGE2) levels at the peritoneal fluid were quantified using the Prostaglandin E2 ELISA kit (Abcam Discover More, UK) according to the manufacturer's instructions. The level of TNF-α in the liver was evaluated using the ELISA kit (Thermo Fisher Scientific, USA).

Histological studies

Liver portions stored in 10% formaldehyde were embedded in paraffin, and cut with a rotating microtome (Leica, Germany) at 5 µm thickness, spread on microscope slides and stained with hematoxylin and eosin, and the slides were observed by light microscopy (Drury and Wallington, 1967).

Statistics

SigmaStat software (SPSS, 3.0, SPSS, Inc., Chicago, IL) was used for the analysis. Data was presented as mean ± SD and were evaluated by one-way ANOVA, with Dunnett's post hoc test. When appropriate, ANOVA on Rank with Dunn's post hoc test was used.

Results

EAP chemical composition

The HPLC chromatograms obtained at 270 and 320 nm for the propolis extract (EAP) from Tigzirt are shown in Fig. 1A and B, respectively. We were able to identify flavonoids: catechin (1), quercetin (2), rutin (3), acacetin (4), chlorogenic acid (5), apigenin (6), pinocembrin (7), chrysin (8), kaempferol (9), thymol (10); phenolic acids: ferulic acid (11), gallic acid (12), caffeic acid (13), ellagic acid (14), m-coumaric (15), rosmarinic acid (16), trans-cinnamic (17); and ascorbic acid (18). We have also been able to observe benzoic acid, galangin and tectochrysin in our propolis extract. The phenolic acid and flavonoid content of tigzirt propolis is shown in Table 1. The main phenolic acid found is caffeic acid (0.85 mg/g at 2.38 min), while pinocembrin is the main flavonoid (0.82 mg/g at 6.73 min). Rutin and apigenin was found in low levels.

Fig. 1
Chromatograms by HPLC/UV analysis of EAP at a wavelength of 270 (A) and 320 nm (B): 1, catechin; 2, quercetin; 3, rutin; 4, acacetin; 5, chlorogenic acid; 6, apigenin; 7, pinocembrin; 8, chrysin; 9, kaempferol; 10, thymol; 11, ferulic acid; 12, gallic acid; 13, caffeic acid; 14, ellagic acid; 15, m-coumaric acid; 16, rosmarinic acid; 17, trans-cinnamic acid; 18, ascorbic acid.

Table 1
Composition of propolis extracts from Tigzirt by HPLC/UV (mg/g).

Peak number	Compound	Retention time (min)	Amount (mg/g EAP)
1	Catechin	2.38	0.78
2	Quercetin	5.73	0.76
3	Rutin	4.58	0.01
4	Acacetin	5.25	0.17
5	Chlorogenic acid	5.66	0.2
6	Apigenin	6.08	0.01
7	Pinocembrin	6.73	0.82
8	Chrysin	7.25	0.55
9	Kaempferol	6.14	0.31
10	Thymol	7.85	0.21
11	Ferrulic acid	0.63	0.05
12	Gallic acid	2.13	0.29
13	Caffeic acid	2.38	0.85
14	Ellagic acid	3.75	0.6
15	m-Coumaric acid	4.02	0.41
16	Rosmarinic acid	2.13	0.18
17	trans-Cinnamic acid	5.66	0.34
18	Ascorbic acid	6.73	0.38

Rat body weight evolution and clinical observations

In this study, body weight was measured weekly (Table 2). No animal mortality was observed during the administration of the epirubicin dose. A very significant decrease in the weight of the rats that received only injections of epirubicin (G1) and those treated with quercetin (G5) was noted. Normal weight values were observed in rats treated with 100 and 250 mg/kg of EAP (G3 and G4).

Table 2
The body weight measured every three days of Wistar rats exposed to epirubicin (EPI).

Groups	Day 1	Day 3	Day 6	Day 9	Day 12	Day 15	Day 17	Day 19
G1	124 ± 1.54	129 ± 1.23	133 ± 1.84	136 ± 0.76	143 ± 1.63	148 ± 0.94	153 ± 1.45	159 ± 1.52
G2	123 ± 1.76a	132 ± 1.82a	137 ± 2.85	145 ± 1.54a	153 ± 1.95	156 ± 3.01b	144 ± 1.80	124 ± 2.69b
G3	117 ± 0.67	126 ± 1.34	131 ± 1.17	142 ± 1.02	156 ± 1.20	160 ± 1.36	163 ± 1.39a	163 ± 1.32
G4	119 ± 2.61b	130 ± 1.57	141 ± 2.34	148 ± 3.43b	160 ± 1.10	163 ± 1.18a	167 ± 1.00	169 ± 1.05
G5	125 ± 1.22	134 ± 1.93	143 ± 1.93	151 ± 1.43	154 ± 2.53	159 ± 1.77a	152 ± 1.79	146±1.61

• The values are expressed as mean ± SD (n = 5).
• ^a
  Significant difference from the control group (p < 0.05).
• ^b
  Significant difference from the EPI hepatotoxicity group (p < 0.05).

Clinical signs in rats in each group were followed continuously after injection of epirubicin, to detect the effects of epirubicin and the protective action of ethyl acetate propolis extract EAP.

The rats in G1 (control) and G4 (rats treated with 250 mg/kg EAP) did not show any change in the behavior indicating that propolis extract inhibited epirubicin hepatoxicity. However, there are serious changes in the physical activity and behavior of rats in G2 (which received only epirubicin) such as: drowsiness, hypoactivity, isolation, straightening of hair, tachycardia, difficulty breathing and loss of balance. Animals of G3 and G5 show only tachycardia and difficulty breathing.

Hepatic function

Several hepatic parameters were measured to evaluate the effect of Tigzirt propolis on the treatment of epirubicin-induced hepatotoxicity. Table 3 illustrates these hepatic parameters. Markers of liver function, AST, ALT, Bil T, ALP, 5′NUC and γGT were significantly elevated in the epirubicin-treated group compared with control. On the other hand, the group of rats treated with 250 mg/kg of EAP (G4) showed significant improvement in the hepatic status compared to G2. However, giving to rats (G5) one of the pure compounds orally (quercetin) had no significant hepatic function and antioxidant effect.

Table 3
Effect of ethyl acetate extract of propolis (EAP) from Tigzirt on hepatic function parameter values of Wistar rats exposed to epirubicin (EPI).

Parameter	G1	G2	G3	G4	G5
ALAT (UI/l)	40 ± 3.22	122.33 ± 6.28	50.66 ± 3.14	39 ± 3.57	61.66 ± 4.03
ASAT (UI/l)	38.33 ± 1.03	130 ± 10.31b	53.33 ± 2.87	35 ± 4.09	63.66 ± 9.39a
Bil T (mg/l)	05 ± 0.89	58 ± 6.75a	08.33 ± 0.51	06 ± 0.89a	15.33 ± 2.58
5'Nucléotidase (UI/l)	2.5 ± 0.89	12.33 ± 2.25a	04.50 ± 0.44	03 ± 0.44b	7.33 ± 1.12a
PAL (UI/l)	49 ± 3.57	288.33 ± 17.91b	73.33 ± 10.81b	50.33 ± 5.95a	114.66 ± 13.09a
γGT (UI/l)	27 ± 3.57	106 ± 16.17	40.33 ± 1.86	35 ± 2.68	55.33 ± 4.58

• The values are expressed as mean ± SD (n = 5).
• ^a
  Significant difference from the control group (p < 0.05).
• ^b
  Significant difference from the EPI hepatotoxicity group (p < 0.05).

Oxidative stress status

Table 4 demonstrates the level of parameters and enzymes related to oxidative stress status in the liver tissues of the different groups. These results indicate that epirubicin (G2) decreased antioxidant enzyme activities (SOD, CAT and GPx) and thiol-containing compounds in the liver. In addition, the level of lipid peroxidation in the liver was significantly increased (5.84 nmol/l) in the group that received only epirubicin (G2) compared with the control group. G4 did not show significant changes in oxidative parameters comparing to the control group. The rats in G3 (treated with 100 mg/kg EAP) and G5 (treated with 50 mg/kg quercetin) showed a reduction in hepatic antioxidant status parameters (SOD, CAT, GPx, and thiols) and increased MDA rate.

Table 4
Effect of ethyl acetate extract of propolis (EAP) from Tigzirt on malondialdehyde (MDA). superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), thiol and tumor necrosis factor alpha (TNF-α) in the liver of Wistar rats exposed to epirubicin (EPI).

Parameter	G1	G2	G3	G4	G5
MDA liver (nmol/ml)	0.96 ± 0.11	5.84 ± 0.99	2.20 ± 0.13b	0.89 ± 0.06	4.18 ± 0.11a
SOD liver (U/cg Hb)	48.80 ± 2.30	15.94 ± 2.33a	23.70 ± 1.93	50.74 ± 1.87b	20.76 ± 1.70
CAT liver (U/mg Hb)	90.38 ± 3.49	29.72 ± 1.9b	53.81 ± 1.87a	96.69 ± 1.6b	38.05 ± 1.24
GSH-Px liver (U/g Hb)	76.08 ± 2.42	12.78 ± 1.4b	46.04 ± 1.43a	86.51 ± 2.11a	36.26 ± 2.43
Thiol liver (mmol/g)	1.57 ± 0.16	0.25 ± 0.02b	1.27 ± 0.06	1.89 ± 0.07a	0.84 ± 0.04
TNF-α (ng/g)	63.66 ± 3.72	164 ± 7.79a	104.3± 5.81a	68.33 ± 2.73	127.66 ± 5.39

• The values are expressed as mean ± SD (n = 5).
• ^a
  Significant difference from the control group (p < 0.05).
• ^b
  Significant difference from the EPI hepatotoxicity group (p < 0.05).

Inflammation markers

We measured the concentration of PGE2 in peritoneal exudates of rats treated or not treated with EAP or quercetin and those suffering from toxicity caused by epirubicin. Administration of EAP at concentrations of 100 and 250 mg/kg (G3 and G4) respectively decreased PGE2 production by 37 and 69% (Fig. 2) compared to the control group. In G2 group, the level of PGE2 (2066.66 pg/ml) increased significantly in relation to G1 (750.3 pg/ml). In the same way, in G2 group (Table 4), the level of TNF-α in the liver (164 pg/g) increased significantly in relation to G1 (63.66 pg/g). This increase was reversed by pretreatment with 250 mg/kg of EAP (G4) which showed a significantly lower level of this parameter compared to the group G2 (the rats that were injected with epirubicin).

Fig. 2
Effects of treatment with EAP on Prostaglandin E2 in peritoneal exudates of rats. Bars represent the mean ± S.E.M. of five repitions. *Significant inhibition in relation to the control group, p < 0.05.

Histopathological studies

Histological studies of the liver of rats treated with propolis or not before induction of toxicity by epirubicin are illustrated in Fig. 3.

Fig. 3
Histological cuts of rat's liver of groups: G1 (A); G2 (B, C); G3 (D); G4 (E) and G5 (F). Abbreviations: CV, central vein; Cong, congestion; FN, focal necrosis; LII, lymphocytic inflammatory infiltrate; NHP, normal hepatic parenchyma; DS, dilation of sinusoids.

The rats in the control group (G1) show a normal histological structure of the hepatic parenchyma, with sinusoids and a centrilobular vein that are essentially normal (Fig. 3A). However, in the G2 group (rats treated with epirubicin only), the biopsy showed major damage in the liver, very severe cell necrosis with hepatic degeneration, dilation of sinusoids with a very dense lymphocyte inflammatory infiltrate, associated dilation and congestion of central veins filled with debris (Fig. 3B and C). The treatment of rats with 100 mg/kg of propolis shows a less severe focal necrosis localized around congestion of central veins (Fig. 3D).

The histological study of the liver (Fig. 3E) of rats received 250 mg/kg of EAP (G4) revealed architecture similar to that observed in the control group (G1). Fig. 3F shows that quercetin (G5) was unable to maintain a normal liver structure, where an injured parenchyma or degenerate hepatocytes responsible for focal necrosis, dilation of sinusoids, congestion of centrilobular veins surrounded by lymphocytic inflammatory infiltrate.

Discussion

The anthracyclines are chemotherapy drugs. They are used alone or in combination with many cancers' treatment. The doxorubicine and the epirubicin which belong to anthracyclines family are considered as the most toxic chemotherapy drugs for the liver (Weenen, 1984; Le Bot et al., 1988).

Our results revealed a very significant increase in the enzymatic activity of ALT, AST, ALP and γ-GT in the rats of the group G2 that received an injection of epirubicin (9 mg/kg). The increase in hepatic enzyme activity in the blood may be due to tissue damage in the liver, changes in membrane permeability, increased synthesis or decreased aminotransferase catabolism (Farag et al., 2010).

In addition, AST, ALT, and ALP are the primary enzymes used to assess the status of liver function (Wallace and Meyer, 2008). These are the most sensitive biomarkers, directly involved in the extent of cellular damage and toxicity, as they are cytoplasmic and released into the circulation after cell injury (Soudani et al., 2011).

Decrease in antioxidant activity of the tested polyphenols and flavonoids purs (curcumin, quercetin) in vivo could be explained by the poor absorption of these compounds from the gastric intestinal tract (Rasoanaivo et al., 2011). It has been shown that crude plant extracts sometimes have greater biological activity than isolated molecules at an equivalent dose (Rasoanaivo et al., 2011).

Thus, cell necrosis, destruction of the hepatic parenchyma, or increased membrane permeability of hepatocytes may cause these enzymes to flow into the bloodstream and thereby increase their serum levels (Adeneye et al., 2006; Jodynis-Liebert et al., 2010). On the other hand, ALP is a ubiquitous enzyme, especially in the liver, bile ducts, kidneys, bones and placenta, where it is found in high concentration. Their increase in circulation usually indicates intrahepatic biliary obstruction, primary biliary cirrhosis, or disruption of hepatic architecture (Epstein et al., 1986; Sharma et al., 2013).

Our study has shown that epirubicin causes severe liver damage agreeing with the results obtained by Iwakiri et al. (2007), Kebieche et al. (2009), Prado et al. (2010) and Hwan Shin et al. (2014) who also demonstrated the hepatotoxic effect of epirubicin.

The results also showed that the administration of Tigzirt propolis to rats exposed to epirubicin significantly reduced the enzymatic activity of ALT, AST and ALP. This reveals the propolis ability to ameliorate chemotherapy-induced liver injury (epirubicin) indicating that propolis is effective at preventing liver damage caused by epirubicin.

According to Boufadi et al. (2017), the extract propolis from Tigzirt has anti-oxidant and anti-radical effects. These authors confirmed that propolis can protect the body from damage caused by oxidative stress.

In the present study, epirubicin increased markers of oxidative stress in the liver (lipid peroxidation) while decreasing the level of endogenous antioxidative agents such as GSH, catalase and superoxide dismutase activity. Propolis from Tigzirt has successfully reversed this oxidative stress in liver tissues. This antioxidant effect is certainly due to the presence of phenolic acids and flavonoids in propolis, as well as their ability to trap free radicals. The oxidative stress induced by anthracyclines has been incriminated in the initiation of its multi-organ toxicity, because it undergoes a bio-reducing activation by redox cycle via its unique chemical structure favoring the formation of free radicals (Ravi and Das, 2004). Another previous study (Koka et al., 2010) suggested that the anticancer efficacy of epirubicin is related to its pro-oxidative properties. This fact can explain that the administration of antioxidants (such as propolis) does not affect the chemotherapeutic effectiveness of epirubicin (Koka et al., 2010). The protection provided by fruits and vegetables against degenerative diseases (cancer and cardiovascular diseases), has been attributed to various antioxidants contained in these foods, including flavonoids. Flavonoids, such as quercetin and kaempferol, cause damage to nuclear DNA and lipid peroxidation in the presence of transition metals (Rahal et al., 2014).

The lipid peroxidation of biological membranes may cause a loss of membrane fluidity as well as an increase in membrane permeability and an impairment of the function of the receivers (Nehru and Anand, 2005). The increase in lipid peroxidation is largely due to inhibition of the superoxide dismutase (SOD) and the catalase (Newairy et al., 2009).

It is also known that propolis generally acts by different mechanisms (Araujo et al., 2012) as inhibitor of cyclooxygenase, prostaglandins PGE2 and pro-inflammatory cytokines (Mirzoeva and Calder, 1996; Hu et al., 2005). It has been demonstrated that propolis extracts have high effect on inflammatory cell activity (cell migration, macrophage activation) (Bueno-Silva et al., 2013). Propolis from Tigzirt inhibited PGE2 and TNF-α production during epirubicin-induced toxicity. This may explain the anti-inflammatory effects of propolis extract, with the inhibition of neutrophil mobilization in the peritoneal cavity.

The source of propolis from Tigzirt is buds of poplar, eucalyptus, birch, willow, chestnut, and fruit trees, which are rich in flavonoids, phenolic acids and their esters. This propolis is dark brown and has been harvested in a wetland and a Mediterranean climate (Boufadi et al., 2014).

Conclusion

In our study, we have demonstrated that propolis extracts can prevent the toxic effects of epirubicin resulting from the oxidative damages induced by the chemotherapy by this agent. Our results have showed that propolis effects are not limited to the prevention of oxidative stress but also it can reverse the oxidative damages resulted from epirubicin. These founding make propolis a promise agent to treat and prevent the most important side effect of anthracyclines.

• Funding
  There has been no significant financial support for this work that could have influenced its outcome.

Acknowledgments

This study was supported by grants from the Abdelhamid Ibn Badis (Algeria) University and University ULB (Belgium).

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Publication Dates

• Publication in this collection
  26 Aug 2019
• Date of issue
  Mar-Apr 2019

History

• Received
  11 Sept 2018
• Accepted
  11 Jan 2019
• Published
  2 Mar 2019