Bee pollen has been used as an apitherapy agent for several centuries to treat burns, wounds, gastrointestinal disorders, and various other diseases. The aim of our study was to investigate the hepatoprotective effects of chestnut bee pollen against carbon tetrachloride (CCI4)-induced liver damage. Total phenolic content, flavonoid, ferric reducing/antioxidant power, and DPPH radical activity measurements were used as antioxidant capacity determinants of the pollen. The study was conducted in rats as seven groups. Two different concentrations of chestnut bee pollens (200 and 400 mg/kg/day) were given orally and one group was administered with silibinin (50 mg/kg/day, i.p.) for seven days to the rats following the CCI4 treatment. The protective effect of the bee pollen was monitored by aspartate transaminase (AST) and alanine transaminase (AST) activities, histopathological imaging, and antioxidant parameters from the blood and liver samples of the rats. The results were compared with the silibinin-treated and untreated groups. We detected that CCI4 treatment induced liver damage and both the bee pollen and silibinin-treated groups reversed the damage; however, silibinin caused significant weight loss and mortality due, severe diarrhea in the rats. The chestnut pollen had showed 28.87 mg GAE/g DW of total phenolic substance, 8.07 mg QUE/g DW of total flavonoid, 92.71 mg Cyn-3-glu/kg DW of total anthocyanins, and 9 mg
Apitherapy has been used in folk medicine since the early ages of human beings, and in the recent years, its application in the treatment of burns, wounds, gastrointestinal disorders, ulcers, and carcinogenesis has been increasing. Bee pollen is one of the richest and purest natural foods that way ever discovered; the tremendous nutritional and medicinal value of the pollen has been used for centuries. Bee pollen is a perfectly balanced food and is rich in amino acids, proteins, hormones, enzymes, carbohydrates, minerals, fats, a considerable amount of vitamins, phenolic substances, phytochemicals, and significant quantities of antioxidant agents [
Carbon tetrachloride (CCI4) is a hepatotoxic agent that enhances the formation of free radicals, which cause lipid peroxidation of cellular and organelle membranes [
It is recognized that hepatocyte damage is one of the serious pathological disorders for human. To measure aspartate transaminase (AST) and alanine transaminase (ALT) activities in the serum or plasma is the simplest method to diagnose hepatocyte injuries [
In this study, we determined the therapeutic effects of chestnut bee pollen on the CCI4-induced liver damage in the rat model. We analyzed the chemical and antioxidant properties of the chestnut pollen and determined that pollen supplementation recovered the body weight, AST and ALT enzyme levels, malondialdehyde (MDA), and superoxide dismutase (SOD) levels as well as decreased the histological damage and apoptosis at the hepatocytes following the CCI4 treatment. We detected that both the bee pollen- and silibinin-treated groups reversed the CCI4-induced hepatic damage; however, silibinin caused significant weight loss and mortality due to severe diarrhea in the rats. Our results suggest that chestnut pollen can be used as a safe alternative for the treatment of liver damaging diseases.
CCI4, silibinin, gallic acid, quercetin, ethyl alcohol, methanol, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), 2,4,6-tripyridyl-s-triazine (TPTZ), Folin-Ciocalteu’s phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), cyanidin-3-O-glucoside, TBA, 1,1,3,3-tetramethoxypropane,
Pollen samples were obtained from the expert beekeepers of Zonguldak, Turkey (Western Black Sea area) in 2008 flowering season. The samples were dried at 40°C oven and palynological identification was done by Dr. Sibel Silici of Erciyes University, Turkey. Nine families of pollen pellets were found in the sample: Fabaceae (
For the analysis of antioxidant potential of the pollen, the samples were prepared by mixing 1 g of dried powder of pollen sample with 10 mL methanol in a flask attached condenser, then sonicated in a sonicator apparatus (Elma Transsonic Digital, Germany). After 3 h sonication, the extract was used for antioxidant tests. Although we dissolved the pollen in sterile H2O to feed the animals by gavage, it has been shown that the antioxidant potential of the pollen is revealed with better percentage when it is extracted using methanol [
Chemical analysis of the bee pollen was performed according to the method described at AOAC [
Forty-nine Sprague-Dawley rats were studied, which were 12 weeks old and of 250–300 g approximate weight. Animals were fed with standard rat feed and allowed to drink water. Animals were kept in temperature controlled (20–25°C) cages with 12 h dark and 12 h light cycles.
Animals were divided into seven groups (see Supplementary Table 1 in Supplementary Material available online at
Plasma was obtained from the whole blood samples of the treated rats through centrifugation at 2000 ×g for 10 min and stored at −20°C until analysis. AST and ALT activities were measured by a Roche Diagnostics Modular Analyzer using the manufacturer’s commercial kits according to the instructions (Roche Diagnostics GmbH, D-68298, and Mannheim, Germany).
Reduction of nitroblue tetrazolium by xanthine-xanthine oxidase system was used to measure superoxide dismutase (SOD) activity in erythrocyte hemolysate of the rat blood samples. Formazan formation was examined at 560 nm using the spectrophotometer (Beckman-coulter, DU 530). The enzyme activity that causes 50% inhibition was regarded as one unit using bovine erythrocytes SOD as standard, and the results were read as U/g Hb [
MDA levels were measured with a colorimetric test with thiobarbituric acid (TBA) which is used to assess endogenous lipid [
For histological analysis, liver tissue samples were fixed immediately in 10% buffered formaldehyde, dehydrated with ethanol series, cleaned with xylene, embedded in paraffin, and sectioned as 5
For the detection of apoptotic cells within our groups (G1–G7), 4
The results were presented as mean values and standard deviations. Data and regression analyses were performed via Microsoft Office Excel 2003 (Microsoft Corporation, Redmond, WA). Data were tested using SPSS (version 9.0 for Windows 98, SPSS Inc.). Statistical analyses of the results were based on Kruskal-Wallis test and Pearson correlation analysis, which is a nonparametric test. The significance of the differences was statistically considered at the level of
This study investigated the hepatoprotective potential of aquatic extracts of chestnut bee pollen on CCI4-induced hepatic damages in rats. Before the treatments, some chemical and antioxidant properties of the pollen were investigated, which are summarized in Table
Chemical and antioxidant properties of the investigated honeybee pollen of chestnut.
Chemical properties | ||||
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Moistures % | Ash % | Protein % | Starch % | Fat % |
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Antioxidant properties | |||||
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Total phenolic content |
Total flavonoid |
Total anthocyanins |
Total carotenoid |
FRAP |
DPPH |
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The phenolic compounds of the pollens vary depending on the geographic characteristics and the flora of the region [
We measured the initial weight of the 49 rats that we studied in 7 groups (G1–G7) and compared their weights at the end of the 7th day (Table
Experimental results of transaminase enzymes and antioxidant parameters in the plasma
Groups | Weight gain %(1) | Plasma | Erythrocytes | Liver | |||||
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AST | ALT | MDA | SOD | MDA | SOD | MDA | SOD | ||
U/L | U/L | nmol/mL plasma | U/mg protein | nM/g Hb | U/g Hb | nmol/g tissue | U/g liver | ||
G1 (control) | +0.7 (±1.07)b |
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G2 (control) | +0.7 (±1.07)b |
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G3 (control) | −5.4 (±0.946)a |
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G4 |
−9.5 (±0.905)a |
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G5 (silibinin) | −8.2 (±0.918)a |
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G6 (low pollen) | −6.7 (±0.933)a |
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G7 (high pollen) | −1.6 (±0.984)ab |
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The activities of plasma AST and ALT enzymes were significantly elevated in rats treated with CCI4 due to toxic effect (Table
Histologic analysis and scoring of liver sections of the treated and untreated rats.
Groups | Hepatocyte degeneration | Vascular congestion | Sinusoidal dilatation | Congestion in enlarged sinusoids | Fatty |
Apoptosis |
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G1 (control) |
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G3 (control) |
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G4 (CCI4) |
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G5 (silibinin) |
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G6 (low pollen) |
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G7 (high pollen) |
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The histological score was calculated using a scale from 0 to 3, 0: none, 1: mild, 2: moderate, and 3: severe.
Among the apitherapy studies related to the prevention and protection of liver damage and toxicity, Türkez et al. [
Malondialdehyde (MDA), a lipid peroxidation product, was measured in plasma, erythrocyte, and liver tissues of the treated animals to analyze the damage caused by oxidative stress induced via CCI4 treatment. The amount of MDA in liver tissue serves as an indicator of lipid peroxidation, which is a well-known occurrence in the liver injury due to generation of reactive species [
Superoxide dismutase (SOD) is an important antioxidant enzyme that protects the organism from the harmful effects of superoxide radicals formed as a result of oxidative stress [
Microscopic examination showed liver parenchyma and sinusoids of the hepatocytes were healthy in the control groups (G1–G3) (Figure
Histopathological analysis of treated and untreated liver sections. (a) Normal hepatocytes (↑) and sinusoids (▲) in the control group, untreated CCI4 (H&E ×100). (b) Destroyed group with CCI4 (G4), increased fatty degeneration (↑). (c) Pollen-treated group (G6) with 200 mg/kg, decreased fatty degeneration and regeneration in hepatocytes (↑). (d) Pollen-treated group (G7) with 400 mg/kg pollen, fatty degeneration markedly decreased (↑).
Hepatocyte apoptosis was analyzed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay. The number of apoptotic nuclei was much higher in sick group (G4) than that of the control groups (Figures
Results on apoptosis analysis (TUNEL) of CCI4-induced apoptosis in primary rat hepatocytes, apoptotic hepatocytes (↑), and normal hepatocytes (▲) (×400). (a) Control group untreated with CCI4 (G1), brown labeled apoptotic hepatocytes (↑), and blue labeled normal hepatocytes (▲). (b) Destroyed with CCI4 (G4), apoptotic hepatocytes (↑). (c) Pollen-treated group (G6) with 200 mg/Kg. (d) Pollen-treated group (G7) with 400 mg/Kg.
Experimental liver damage model is mostly achieved by exposing rats to CCI4. It has been shown that lipid peroxidation increases and free oxygen radicals are released in the rats exposed to CCI4 [
CCI4 is a hepatotoxic agent that enhances the formation of free radicals, which cause lipid peroxidation of cellular and organ damages. The chestnut bee pollen contains substantial nutrients and possesses many phenolic compounds, which are the factors of high antioxidant properties. Our study clearly shows that the chestnut bee pollen exerts highly beneficial biological activities in the protection of hepatocytes from oxidative stress and toxicity induced by CCI4 exposure. Therefore, we conclude that the chestnut bee pollen could be safely included in the daily human diet as a food additive, which will enhance the inhibition of the oxidative stress. Chestnut bee pollen could be used as a suitable alternative to silibinin in the treatment of hepatocellular pathologies.
This study was supported by Research Fund of Karadeniz Technical University (Project no. 2009.111.002.5). Two of the authors, Oktay Yildiz and Huseyin Sahin, were funded by TUBITAK-BIDEB for their graduate studies.