Propolis Prevents Hepatorenal Injury Induced by  Chronic Exposure to Carbon Tetrachloride

Bhadauria, Monika

doi:https://doi.org/10.1155/2012/235358

Evidence-Based Complementary and Alternative Medicine

On this page

Abstract Introduction Methods Results Discussion References Copyright Related Articles

Research Article | Open Access

Volume 2012 | Article ID 235358 | https://doi.org/10.1155/2012/235358

Propolis Prevents Hepatorenal Injury Induced by Chronic Exposure to Carbon Tetrachloride

Monika Bhadauria¹

Academic Editor: Alvin J. Beitz

Received01 Feb 2011

Revised24 Apr 2011

Accepted05 May 2011

Published04 Aug 2011

Abstract

Carbon tetrachloride (CCl₄) is a well-known hepatotoxicant, and its exposure induces hepatorenal injury via oxidative stress and biochemical alterations. This study had been conducted to confirm the protective role of propolis extract on CCl₄-induced hepatorenal oxidative stress and resultant injury. Propolis extracts collected from Gwalior district and 24 female Sprague Dawley rats were used for experiment. Animals were exposed to CCl₄ (0.15 mL/kg, i.p.) for 12 weeks (5 days/week) followed by treatment with propolis extract (200 mg/kg, p.o.) for consecutive 2 weeks. CCl₄ exposure significantly depleted blood sugar and hemoglobin level and raised the level of transaminases, alkaline phosphatase, lactate dehydrogenase, protein, urea, albumin, bilirubin, creatinine, triglycerides, and cholesterol in serum. Lipid peroxidation was enhanced, whereas GSH was decreased significantly in liver and kidney in CCl₄-intoxicated group. Ethanolic extract of propolis successfully prevented these alterations in experimental animals. Activities of catalase, adenosine triphosphatase, glucose-6-phosphatase, acid, and alkaline phosphatase were also maintained towards normal with propolis therapy. Light microscopical studies showed considerable protection in liver and kidney with propolis treatment, thus, substantiated biochemical observations. This study confirmed hepatoprotective potential of propolis extract against chronic injury induced by CCl₄ by regulating antioxidative defense activities.

1. Introduction

The chronic liver diseases are common worldwide and are characterized by a progressive evolution from steatosis to chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma [1, 2]. Free radicals and reactive oxygen species (ROS) play a crucial role in development of liver diseases [3]. The liver is exposed to absorbed drugs or xenobiotics in concentrated form due to its unique vascular and metabolic features. Drug-metabolizing enzymes detoxify many xenobiotics but bioactivate the toxicity of others [4]. In case of bioactivation, liver is the first organ exposed to the damaging effects of newly formed toxic substance. Therefore, protective armaments for liver are of particular interest. Considerable efforts are being made to obtain useful herbal medicines from documented medicinal plants [5] for a wide variety of clinical conditions. Dietary antioxidants of natural products may serve as therapeutics to cope with liver damage [3] against free radicals and ROS-induced liver diseases pathology and progression. Natural antioxidants in complex mixtures if ingested with the diet are more efficacious than pure compounds in preventing oxidative stress-related pathologies due to particular interactions and synergisms [3] by modulating antioxidant, drug-metabolizing, and repairing enzymes along with acting as signaling molecules in important cascades for cell survival [6, 7].

Propolis is an adhesive, resinous substance collected, transformed and used by honeybees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of intruders. Honeybees collect the resin from cracks in the bark of trees and leaf buds. They bring propolis back to the hive, where it is modified and mixed with other substances, including bees’ own wax and salivary secretions [8]. Propolis has been used in folk medicine all over the world. It has anti-inflammatory, immunoregulatory, bacteriostatic, and antibacterial activities [9–11]. It provides excellent cartilage protection suggesting a potential application in joint disease [12]. Long-term administration of propolis does not induce any significant change in seric parameters; thus, it might not have any cardiac injury [13]. Propolis extracts present low toxicity to experimental animals, with LD₅₀ higher than 7 g/kg for mice [14, 15]. Previously we reported strong hepatoprotective effect of propolis against acute hepatic damage [16–21] and subchronic hepatic injury induced by CCl₄ [22] and acetaminophen [23]. In the present communication, an attempt has been made to explore potential of propolis in preventing chronic hepatorenal injury.

2. Material and Methods

2.1. Animals and Hepatorenal Injury by CCl₄

Female Sprague Dawley rats (130 ± 10 g) were kept (3/cage: 6/group) in the animal house under standard husbandry conditions (temp. 25 ± 2°C; relative humidity 60 ± 5%; 12 h light/dark cycle). The animals were fed on pelleted diet (Pranav Agro Industries Ltd., India) and drinking water ad libitum. Experiments were conducted in accordance with the guidelines set by the Institutional Animal Ethics Committee (501/01/A/CPCSEA) of Jiwaji University. Hepatorenal injury was induced by CCl₄ (0.15 mL/kg; diluted with liquid paraffin) that was administered for 12 weeks (5 days/week) [24].

2.2. Preparation and Administration of Propolis

Crude propolis from the hive of Apis mellifera was collected from apiaries nearby Gwalior district (MP) by Professor O. P. Agrawal, Senior Entomologist, School of Studies in Zoology, Jiwaji University, Gwalior (India). Propolis is a plant-derived product, and it has been proved that bees do not change its chemical composition [25]. It is reported that more than 300 compounds of different classes are present in propolis among which more than 100 are common worldwide. A lot of knowledge has already been gathered on active components, and one of the most important active principles was found to be caffeic acid phenethyl ester [26]. A 90% ethanolic extract was obtained as described previously [16, 17], yield of dried residue was about 61.4% (w/w) and kept at 4°C for further use. Aqueous suspension of propolis was prepared in 1% gum acacia suspension, and selected optimum dose was administered (200 mg/kg, p.o.) to the animals for 2 weeks on the basis of our previous studies [22, 23]. Silymarin was given as positive control in respect to propolis.

2.3. Experimental Design

Animals of group 1 received vehicle only and served as normal control. Animals of group 2–4 received CCl₄ for 12 weeks (5 days/week) and group 2 served as experimental control. Group 3 and 4 received treatments of propolis and silymarin, respectively, for 2 weeks (5 days/week). Blood was collected from retro-orbital venous plexus after 48 h of the last administration. The animals of entire groups were euthanized; liver and kidney were immediately excised and processed for biochemical analyses and histological preparations. Scheme of different treatments is given in Table 1.

2.4. Isolation of Serum and Homogenate Preparation

Serum was isolated after keeping the blood for 1 h at room temperature followed by centrifugation at 1000 g for 15 min and stored at −20°C until analyzed. Tissue samples of liver and kidney were homogenized with ice-cold 150 mM KCl for the determination of TBARS and CAT activity and in 1% sucrose for GSH determination. Homogenates of liver and kidney were prepared in chilled hypotonic solution (10% w/v) for other biochemical assays, that is, total protein, cholesterol, adenosine triphosphatase (ATPase), acid, and alkaline phosphatase (ACPase and ALPase).

2.5. Determination of Hepatorenal Marker Enzymes in Serum and Other Blood Biochemical Endpoints

Blood was immediately used for determination of hemoglobin [27] and blood sugar level [28]. Serum was used for the determination of aspartate aminotransferase (AST) [29], alanine aminotransferase (ALT) [29], alkaline phosphatase (SALP) [30], lactate dehydrogenase (LDH) [31], and serum protein contents [32]. Serum bilirubin, albumin, urea, creatinine, and triglycerides were determined by E-Merck’s kit according to the manufacturer’s instructions.

2.6. Assessment of Peroxidative Stress and Antioxidant Status

Lipid peroxidation (LPO) was measured using thiobarbituric acid (TBA) [33] and expressed as n moles TBARS/g tissue using an extinction coefficient of 1.56 × 105/M/cm. The GSH measurement was performed using dithionitrobenzoic acid [34]. The GSH level was calculated using an extinction coefficient of 13600/M/cm and expressed as μ moles GSH/g tissue. Catalase activity was determined as per method of Aebi [35]. Decomposition of H₂O₂ was monitored by decrease in the absorbance at λ 240 nm. H₂O₂ concentration was calculated using extinction coefficient of 0.0394/mM/cm and activity was expressed as n moles H₂O₂/min/mg protein.

2.7. Tissue Biochemical Assay

Fresh tissues of liver and kidney were immediately processed to determine glycogen by anthrone reagent method [36]. Total proteins were determined using bovine serum albumin as standard, and blue color was developed by the reaction of proteins and Folin-Ciocalteau reagent [32]. Hepatic and renal triglycerides were determined according to Neri and Frings [37]. Determination of enzymatic activities including ACPase [30], ALPase [30], and ATPase [38] was also performed in liver and kidney.

2.8. Histopathological Observations

For histological studies, samples from liver and kidney were fixed in Bouin’s fixative and processed routinely for embedding in paraffin. Tissue sections of 5 μm thickness were stained with hematoxylin and eosin (H&E) and examined under compound light microscope.

2.9. Statistics

Data are expressed as mean ± SE of six animals used in each group. Statistical analysis was carried out by one way analysis of variance (ANOVA) considered significant at followed by Student’s t-test [39].

3. Results

3.1. Hepatorenal Marker Enzymes in Serum and Other Blood Biochemical Endpoints

Chronic exposure to CCl₄ exhibited elevation in the leakage of AST, ALT, LDH, and SALP significantly () (Figure 1). Treatment of propolis extract significantly reduced the leakage of AST, ALT, LDH, and SALP in circulation (), thereby, confirming its protective effect in chronic injury. Chronic administration of CCl₄ decreased hemoglobin level, whereas significant rise was observed in blood sugar, serum triglycerides and cholesterol () (Figure 2). Propolis therapy significantly reversed the level of hemoglobin and blood sugar as well as serum triglycerides and cholesterol towards control (). CCl₄ exposure significantly increased serum albumin, bilirubin, urea, and creatinine () (Figure 3). Treatment with propolis for 2 weeks significantly decreased the level of these serological variables and brought the values very near to control group (). Efficacy of propolis was well compared with positive control silymarin, and percent protection clearly showed that propolis possesses almost equal degree of protection in all biochemical endpoints as silymarin-treated positive control.

(a)

(b)

(c)

(d)

Figure 1

Showing therapeutic potential of propolis on CCl₄-induced alteration in liver marker enzymes of serum. Values are mean ± SE of in each group. @Significant for ANOVA at 5% level. P value CCl₄ versus normal at ; P value treatment versus CCl₄ at for Student’s t-test; F variance for ANOVA of AST = 60.3^†, ALT = 145^†, LDH = 114^†, SALP = 204^†. Abbreviations: Control (C); Carbon tetrachloride (CCl₄); Propolis (P); Silymarin (S); Aspartate aminotransferase (AST); Alanine aminotransferase (ALT); Lactate dehydrogenase (LDH), and Alkaline phosphatase (SALP).

(a)

(b)

(c)

(d)

(a)

(b)

(c)

(d)

3.2. Peroxidative Stress and Antioxidant Status

Peroxidative stress and antioxidant status both in liver and kidney tissues were determined in terms of LPO, GSH, and catalase (Figures 4(a)–4(f)). CCl₄ administration enhanced the formation of TBARS in hepatic and renal tissues after its chronic exposure (). Therapy with propolis extract diminished the production of TBARS (), which ultimately reduced peroxidative stress up-to a considerable extent. The level of GSH and catalase was significantly decreased in liver and kidney after administration of CCl₄ (). Propolis extract was therapeutically effective in restoring GSH level and in maintaining CAT enzyme in liver and kidney in a significant manner (), which could help in mitigating oxidative stress.

(a)

(b)

(c)

(d)

(e)

(f)

Figure 4

Showing therapeutic potential of propolis on CCl₄-induced hepatorenal oxidative stress. Values are mean ± SE of in each group. @Significant for ANOVA at 5% level. P value CCl₄ versus normal at ; P value treatment versus CCl₄ at for Student’s t-test; F variance for ANOVA of hepatic LPO = 104^†, renal LPO = 45.2^†, hepatic GSH = 50.3^†, renal GSH = 27.7^†, hepatic CAT = 37.7^†, and renal CAT = 18.9^†. Abbreviations: Control (C); Carbon tetrachloride (CCl₄); Propolis (P); Silymarin (S).

3.3. Tissue Biochemical Assay

Significantly enhanced enzymatic activity of ACPase was found in liver and kidney after CCl₄ exposure (; Table 2). Propolis therapy was significantly effective in reducing elevated activity of ACPase in both tissues (). Significant fall was noticed in activities of ALPase and ATPase in liver and kidney after 12-week administration of CCl₄ (Table 2; ). Therapeutic effect of propolis extract was found to be significant in both organs, and altered enzymatic activities were reversed towards control (). Significant fall in total protein and glycogen contents was observed after chronic exposure to CCl₄, whereas triglycerides were increased significantly in liver and kidney (; Table 3). Therapy with propolis extract alleviated total protein and glycogen contents and regulated hepatic triglycerides towards control (). Propolis was found to be equally effective as ilymarin on the basis of percent protection.

3.4. Histopathological Observations

Liver of control rats depicted regular histoarchitecture (Figure 5(a)). Liver sections of chronic CCl₄ toxicity showed degeneration so liver cells were seen swollen. It is encountered simultaneously with ballooning degeneration and steatosis. Focal necrosis was seen clearly. Nuclear changes such as karyopyknosis and degeneration of the cell membrane indicated necrosis. These liver showed presence of fibrous septa with heterogenous population of nonparenchymal cells, foamy degeneration due to plenty of vacuolation and disturbed hepatic cord array (Figures 5(b), 5(c), and 5(d)). The protective effect of propolis against CCl₄-induced chronic liver damage and cirrhosis was confirmed by conventional histological examinations. Liver sections obtained from animals, those followed by propolis therapy, showed consistent reduction of liver necrosis and inflammation. Moreover, the cirrhosis process was seen reduced. Proper sinusoidal spaces and cord arrangement were clearly visible with binuclear hepatocytes indicating regenerative effects (Figure 5(e)). Silymarin treatment also improved histoarchitecture showing well-formed hepatic cord arrangements with clearly visible nuclei and sinusoidal spaces (Figure 5(f)).

(a)

(b)

(c)

(d)

(e)

(f)

Figure 5

(a) Photomicrograph of control liver with polygonal hepatocytes cordially arranged around hepatic central vein. Hepatic nuclei (N), uniform sinusoidal spaces (S) terminating into central vein (CV) were clearly visible (200x). (b) Liver sections of CCl₄ induced chronic hepatic injury with disturbed cord arrangement of hepatocytes, vacuolation (V) due to collapsed cellular membranes, high accumulation of nonparenchymal or inflammatory cells (NP) and irregular sinusoids (200x). (c and d) Liver sections of CCl₄ induced chronic hepatic injury with disturbed cord arrangement of hepatocytes and high degree of vacuolation (V) due to collapsed cellular membranes (400x). (e) Photomicrograph of liver after propolis therapy with minimal degree of vacuolation, better formed polygonal hepatocytes cordially arranged towards hepatic central vein (CV). Hepatic nuclei (N), uniform sinusoids (S) terminating into central vein were clearly visible (200x). (f) Silymarin therapy with better formed features of liver histology as comparable to experimental control (400x).

Kidney of control rat showed regular histological features (Figure 6(a)). Administration of CCl₄ for 12 weeks provoked histopathological lesions, including disruption in the epithelial cells of the tubules; proximal tubules showed hypertrophy in proximal tubules with debris in the lumen due to which the lumen was found to be obstructed. The glomeruli occupied whole Bowman’s capsule, medullary tubules showed degeneration, and nuclei showed apical position (Figures 6(b) and 6(c)). Propolis therapy made proximal tubules well organized (Figure 6(d)), glomeruli reversed to the regular shape leaving wider space between glomerulus and Bowman’s capsule wall (Figure 6(e)). Administration of silymarin improved histological features of kidney. Endothelial lining was improved, and cortical and medullary tubules were found to be well formed (Figure 6(f)).

(a)

(b)

(c)

(d)

(e)

(f)

Figure 6

(a) Photomicrograph of kidney of control rat showing well-formed Bowman’s capsule and normal glomeruli (G), uniform space between glomerulus and capsule wall, tubular lumen with basal and apical nuclei (200x). (b) CCl₄ administration caused swelling in glomerulus and tubular obstruction. loss of glomerulus space (thick arrows) and (200x). (c) CCl₄ exposure caused swelling in glomerulus so that space between glomerulus and capsule wall (curved arrow) was reduced (400x). (d): Administration of propolis retained the kidney histoarchitecture with opened lumen of tubules (T) and uniform space between glomerulus and capsule wall (curved arrow) (200x). (e) Propolis therapy maintained uniform space between glomerulus and capsule wall (curved arrow) (400x). (f) Silymarin therapy recovered the kidney histoarchitecture with wider lumen of tubules (T) (400x).

4. Discussion

Various environmental toxicants and clinically useful drugs can cause severe cellular damages in different organs through their metabolic activation. CCl₄ is one of such extensively studied toxicants that has been used to induce liver injury for evaluation and confirmation of hepatoprotective drugs [40]. The reactive metabolite trichloromethyl radicals (^•CCl₃) are formed from the metabolic conversion of CCl₄ by cytochrome P-450 [41]. As O₂ tension rises, greater fraction of ^•CCl₃ present in the system reacts rapidly with O₂, and many orders of magnitude of more reactive free radicals CCl₃OO^• are generated [42]. These free radicals initiate peroxidation of membrane polyunsaturated fatty acids [43] and covalently bind to microsomal lipids and proteins [44] resulting in ROS. Various enzymatic and nonenzymatic systems have been developed by the cell to cope with ROS and other free radicals. However, a condition of oxidative stress establishes due to insufficient defense capacities against ROS [45]. The ROS also affect antioxidant defense mechanisms, reduce intracellular concentration of GSH, decrease the activity of CAT, and cause organ injury and carcinogenesis [46].

Lipid peroxidations as well as altered levels of some endogenous scavengers are taken as reliable indices for oxidative stress [47, 48]. In the present study, administration of CCl₄ enhanced LPO in liver and kidney. Treatment of propolis extract inhibited the generation of TBARS in both tissues that confirmed its antilipid peroxidative effects in chronic condition of hepatorenal injury. GSH constitutes the first line of defense against free radicals [18]. Another defense mechanism involves antioxidant enzymes, including CAT that converts active oxygen molecules into nontoxic compounds. CCl₄ administration decreased the activity of CAT and reduced GSH concentration in the tissues, which is in agreement with earlier reports [19, 20]. Oral administration of propolis reactivated the activities of CAT and restored GSH level, which in turn increased the detoxification of active metabolites of CCl₄. Flavonoids and their esters in propolis are pharmacologically active molecules and have been hypothesized to influence the antioxidant activity of propolis [49, 50]. AST, ALT, SALP, and LDH are sensitive markers of liver injury, and several fold increase in the release of these enzymes indicated severity of damage in chronic study. Serum bilirubin, albumin, urea, creatinine, and triglycerides were also found to be abnormally higher, which indicated hepatic and renal damage. Administration of propolis extract brought these endpoints towards control indicating improvement in metabolic processes. In most of the parameters, efficacy of propolis was found to be the same as sylimarin treatment. Evidently, histopathological examinations of liver and kidney also supported propolis therapy as it helped in improving cellular architecture. This clearly indicated membrane stabilizing effect of propolis probably by scavenging free radicals.

GSH depletion increases the sensitivity of cells to various aggressions and also has several metabolic effects, for example, a decrease in the rate of gluconeogenesis or an increase in glycogenolysis [51]. Fall in protein and glycogen contents in liver and kidney tissues was observed in this investigation that might be associated with increased hepatorenal injury, which in turn resulted into decreased capacity of synthesize protein and glycogen. Propolis therapy augmented protein and glycogen contents in liver and kidney, which indicated that propolis prevented hepatorenal injury and improved physiology of these organs by modulating cellular metabolism and regeneration.

Activities of membranes enzymes ATPase and ALPase were decreased considerably in liver and kidney after CCl₄ exposure [19, 23, 24]. Free radicals are produced inside mitochondria and are frequently released into the cytosol. The production of ATP in the mitochondria involves the transport of protons across the inner mitochondrial membrane via the electron transport chain. Uncoupling of oxidative phosphorylation leads to fall in activity of ATPase. The use of ATPase level measurement was considered as an appropriate index of membrane damage. Significant recovery was found in these enzymes with the treatment of propolis. It may be suggested that impaired mitochondrial oxidative phosphorylation in liver is retained thereby preventing depletion of ATP energy stores [5]. Considerable reversal in ATPase, ALPase, and ACPase activities indicated membrane stabilizing effect of propolis extract. Propolis is also supposed to be helpful in absorption and utilization of various minerals due to the presence of organic acid derivatives in it, which in turn improved physiological functions by regulating the ion dependent enzymatic activities [52]. Efficacy of propolis was found to be the same as of silymarin on the basis of percent protection.

Since, liver and kidneys are closely related organs, which take part in different metabolic and excretion events. Any abnormality in liver may also lead to impairment in kidney functions. That is why both the organs had been taken into consideration in this study. In conclusion, results of this study validate the folklore use of propolis against various ailments. Propolis is interestingly effective in ameliorating acute, subchronic, and chronic injury to liver. It also has wider therapeutic index, and thus it may serve as clinically useful hepatoprotective natural product in future.

Acknowledgment

The author is thankful to ICMR, New Delhi and CSIR, New Delhi, India for providing financial assistance.

References

C. F. Lima, M. Fernandes-Ferreira, and C. Pereira-Wilson, “Drinking of Salvia officinalis tea increases CCl₄-induced hepatotoxicity in mice,” Food and Chemical Toxicology, vol. 45, no. 3, pp. 456–464, 2007.
View at: Publisher Site | Google Scholar
C. Loguercio and A. Federico, “Oxidative stress in viral and alcoholic hepatitis,” Free Radical Biology and Medicine, vol. 34, no. 1, pp. 1–10, 2003.
View at: Publisher Site | Google Scholar
P. Vitaglione, F. Morisco, N. Caporaso, and V. Fogliano, “Dietary antioxidant compounds and liver health,” Critical Reviews in Food Science and Nutrition, vol. 44, no. 7-8, pp. 575–586, 2004.
View at: Publisher Site | Google Scholar
H. Jaeschke, G. J. Gores, A. I. Cederbaum, J. A. Hinson, D. Pessayre, and J. J. Lemasters, “Mechanisms of hepatotoxicity,” Toxicological Sciences, vol. 65, no. 2, pp. 166–176, 2002.
View at: Publisher Site | Google Scholar
S. A. Tasduq, P. Kaiser, D. K. Gupta et al., “Protective effect of a 50% hydroalcoholic fruit extract of Emblica officinalis against anti-tuberculosis drugs induced liver toxicity,” Phytotherapy Research, vol. 19, no. 3, pp. 193–197, 2005.
View at: Google Scholar
L. R. Ferguson, M. Philpott, and N. Karunasinghe, “Dietary cancer and prevention using antimutagens,” Toxicology, vol. 198, no. 1–3, pp. 147–159, 2004.
View at: Publisher Site | Google Scholar
R. J. Williams, J. P. E. Spencer, and C. Rice-Evans, “Flavonoids: antioxidants or signalling molecules?” Free Radical Biology and Medicine, vol. 36, no. 7, pp. 838–849, 2004.
View at: Publisher Site | Google Scholar
E. Ghisalberti, “Propolis: a review,” Bee World, vol. 60, no. 2, pp. 59–84, 1979.
View at: Google Scholar
S. Ansorge, D. Reinhold, and U. Lendeckel, “Propolis and some of its constituents down-regulate DNA synthesis and inflammatory cytokine production but induce TGF-β1 production of human immune cells,” Zeitschrift für Naturforschung. Section C, vol. 58, no. 7-8, pp. 580–589, 2003.
View at: Google Scholar
E. A. Tosi, E. Ré, M. E. Ortega, and A. F. Cazzoli, “Food preservative based on propolis: bacteriostatic activity of propolis polyphenols and flavonoids upon Escherichia coli,” Food Chemistry, vol. 104, no. 3, pp. 1025–1029, 2007.
View at: Publisher Site | Google Scholar
L. C. Lu, Y. W. Chen, and C. C. Chou, “Antibacterial activity of propolis against Staphylococcus aureus,” International Journal of Food Microbiology, vol. 102, no. 2, pp. 213–220, 2005.
View at: Publisher Site | Google Scholar
V. Cardile, A. Panico, B. Gentile, F. Borrelli, and A. Russo, “Effect of propolis on human cartilage and chondrocytes,” Life Sciences, vol. 73, no. 8, pp. 1027–1035, 2003.
View at: Publisher Site | Google Scholar
F. Mani, H. C. R. Damasceno, E. L. B. Novelli, E. A. M. Martins, and J. M. Sforcin, “Propolis: effect of different concentrations, extracts and intake period on seric biochemical variables,” Journal of Ethnopharmacology, vol. 105, no. 1-2, pp. 95–98, 2006.
View at: Publisher Site | Google Scholar
A. Arvouet-Grand, B. Lejeune, P. Bastide, A. Pourrat, A. M. Privat, and P. Legret, “Propolis extract. I. Acute toxicity and determination of acute primary cutaneous irritation index,” Journal de Pharmacie de Belgique, vol. 48, pp. 165–170, 1993.
View at: Google Scholar
A. P. Dantas, B. P. Olivieri, F. H. M. Gomes, and S. L. De Castro, “Treatment of Trypanosoma cruzi-infected mice with propolis promotes changes in the immune response,” Journal of Ethnopharmacology, vol. 103, no. 2, pp. 187–193, 2006.
View at: Publisher Site | Google Scholar
S. Shukla, M. Bhadauria, and A. Jadon, “Effect of propolis extract on acute carbon tetrachloride induced hepatotoxicity,” Indian Journal of Experimental Biology, vol. 42, no. 10, pp. 993–997, 2004.
View at: Google Scholar
S. Shukla, M. Bhadauria, and A. Jadon, “Evaluation of hepatoprotective potential of propolis extract in carbon tetrachloride induced liver injury in rats,” Indian Journal of Biochemistry and Biophysics, vol. 42, no. 5, pp. 321–325, 2005.
View at: Google Scholar
M. Bhadauria, S. K. Nirala, and S. Shukla, “Propolis protects CYP 2E1 enzymatic activity and oxidative stress induced by carbon tetrachloride,” Molecular and Cellular Biochemistry, vol. 302, no. 1-2, pp. 215–224, 2007.
View at: Publisher Site | Google Scholar
M. Bhadauria, S. K. Nirala, and S. Shukla, “Duration-dependent hepatoprotective effects of propolis extract against carbon tetrachloride-induced acute liver damage in rats,” Advances in Therapy, vol. 24, no. 5, pp. 1136–1145, 2007.
View at: Publisher Site | Google Scholar
S. K. Nirala and M. Bhadauria, “Propolis reverses acetaminophen induced acute hepatorenal alterations: a biochemical and histopathological approach,” Archives of Pharmacal Research, vol. 31, no. 4, pp. 451–461, 2008.
View at: Publisher Site | Google Scholar
M. Bhadauria, S. K. Nirala, and S. Shukla, “Multiple treatment of propolis extract ameliorates carbon tetrachloride induced liver injury in rats,” Food and Chemical Toxicology, vol. 46, no. 8, pp. 2703–2712, 2008.
View at: Publisher Site | Google Scholar
M. Bhadauria, S. K. Nirala, and S. Shukla, “Hepatoprotective efficacy of propolis extract: a biochemical and histopathological approach,” Iranian Journal of Pharmacology and Therapeutics, vol. 6, no. 2, pp. 145–154, 2007.
View at: Google Scholar
M. Bhadauria and S. K. Nirala, “Reversal of acetaminophen induced sub chronic hepatorenal injury by propolis extract in rats,” Environmental Toxicology and Pharmacology, vol. 27, pp. 17–25, 2009.
View at: Google Scholar
M. Rojkind, “Inhibition of liver fibrosis by L azetidine 2 carboxylic acid in rats treated with carbon tetrachloride,” Journal of Clinical Investigation, vol. 52, no. 10, pp. 2451–2456, 1973.
View at: Google Scholar
V. S. Bankova, S. L. de Castro, and M. C. Marcucci, “Propolis: recent advances in chemistry and plant origin,” Apidologie, vol. 31, no. 1, pp. 3–15, 2000.
View at: Google Scholar
A. H. Banskota, Y. Tezuka, and S. Kadota, “Recent progress in pharmacological research of propolis,” Phytotherapy Research, vol. 15, no. 7, pp. 561–571, 2001.
View at: Publisher Site | Google Scholar
H. Swarup, S. Arora, and S. C. Pathak, “Sahli's acid haematin method for haemoglobin,” in Laboratory Techniques in Modern Biology, pp. 187–189, Kalyani Publishers, New Delhi, India, 1992.
View at: Google Scholar
A. M. Asatoor and E. J. King, “Simplified colorimetric blood sugar method,” Process Biochemistry, vol. 56, no. 325, 1954.
View at: Google Scholar
S. Reitman and S. Frankel, “A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases,” American Journal of Clinical Pathology, vol. 28, no. 1, pp. 56–63, 1957.
View at: Google Scholar
P. B. Halk, B. L. Oster, and W. H. Summerson, The Practical Physiological Chemistry, McGraw Hill, New York, NY, USA, 14th edition, 1954.
F. Wroblewski and J. S. La Due, “Colorimetric method for LDH,” in Microanalysis in Medical Biochemistry, I. D. P. Wootton, Ed., pp. 115–118, J and A Churchill, London, UK, 4th edition, 1955.
View at: Google Scholar
O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, “Protein measurement with the Folin phenol reagent,” The Journal of Biological Chemistry, vol. 193, no. 1, pp. 265–275, 1951.
View at: Google Scholar
S. K. Sharma and C. R. Krishnamurthy, “Production of lipidperoxides by brain,” Journal of Neurochemistry, vol. 15, no. 2, pp. 147–149, 1968.
View at: Google Scholar
J. E. Brehe and H. B. Burch, “Enzymatic assay for glutathione,” Analytical Biochemistry, vol. 74, no. 1, pp. 189–197, 1976.
View at: Google Scholar
H. Aebi, “[13] Catalase in vitro,” Methods in Enzymology, vol. 105, pp. 121–126, 1984.
View at: Publisher Site | Google Scholar
S. Seifter, S. Dayton, B. Novic, and E. Muintwyler, “The estimation of glycogen with anthrone reagent,” Archives of Biochemistry, vol. 25, no. 1, pp. 191–200, 1950.
View at: Google Scholar
B. P. Neri and C. S. Frings, “Improved method for determination of triglycerides in serum,” Clinical Chemistry, vol. 19, no. 10, pp. 1201–1202, 1973.
View at: Google Scholar
P. K. Seth and K. K. Tangri, “Biochemical effects of some newer salicylic acid congeners,” Journal of Pharmacy and Pharmacology, vol. 18, no. 12, pp. 831–833, 1966.
View at: Google Scholar
G. W. Snedecor and W. G. Cochran, Statistical Method, Iowa State University Press, Ames, Iowa, USA, 8th edition, 1994.
P. Manna, M. Sinha, and P. C. Sil, “Aqueous extract of Terminalia arjuna prevents carbon tetrachloride induced hepatic and renal disorders,” BMC Complementary and Alternative Medicine, vol. 6, article 33, 2006.
View at: Publisher Site | Google Scholar
T. Noguchi, K. L. Fong, E. K. Lai et al., “Specificity of aphenobarbital-induced cytochrome P450 for metabolism of carbontetrachloride to the trichloromethyl radical,” Biochemical Pharmacology, vol. 31, pp. 615–624, 1982.
View at: Google Scholar
J. E. Packer, T. F. Slater, and R. L. Willson, “Reactions of the carbon tetrachloride-related peroxy free radical ( ${CCl}_{3} O_{2}^{•}$ ) with amino acids: pulse radiolysis evidence,” Life Sciences, vol. 23, no. 26, pp. 2617–2620, 1978.
View at: Google Scholar
R. O. Recknagel, E. A. Glende Jr., J. A. Dolak, and R. L. Waller, “Mechanisms of carbon tetrachloride toxicity,” Pharmacology and Therapeutics, vol. 43, no. 1, pp. 139–154, 1989.
View at: Google Scholar
W. M. Tom, L. Y. Y. Fong, and D. Y. H. Woo, “Microsomal lipid peroxidation and oxidative metabolism in rat liver: influence of vitamin A intake,” Chemico-Biological Interactions, vol. 50, no. 3, pp. 361–366, 1984.
View at: Publisher Site | Google Scholar
B. Halliwell and J. M. C. Gutteridge, Free Radicals in Biology and Medicine, Oxford University Press, 2000.
P. Stal and J. Olson, “Ubiquinone: oxidative stress, and liver carcinogenesis,” in Coenzyme Q: Molecular Mechanisms in Health and Disease, V. E. Kagan and P. J. Quinn, Eds., pp. 317–329, CRC Press, Boca Raton, Fla, US, 2000.
View at: Google Scholar
A. S. El-Khatib, A. M. Moustafa, A.-A. Abdel-Aziz, O. A. Al-Shabanah, and H. A. El-Kashef, “Effects of aminoguanidine and desferrioxamine on some vascular and biochemical changes associated with streptozotocin-induced hyperglycaemia in rats,” Pharmacological Research, vol. 43, no. 3, pp. 233–240, 2001.
View at: Publisher Site | Google Scholar
N. Tirkey, S. Pilkhwal, A. Kuhad, and K. Chopra, “Hasperidin, a citrus bioflavonoid, decreases the oxidative stress produced by carbon tetrachloride in rat liver and kidney,” BMC Pharmacology, vol. 5, article 2, 2005.
View at: Publisher Site | Google Scholar
V. Bankova, R. Christov, A. Kujumgiev, M. C. Marcucci, and S. Popov, “Chemical composition and antibacterial activity of Brazilian propolis,” Zeitschrift für Naturforschung. Section C, vol. 50, no. 3-4, pp. 167–172, 1995.
View at: Google Scholar
S. K. Nirala and M. Bhadauria, “Synergistic effects of ferritin and propolis in modulation of beryllium induced toxicogenic alterations,” Food and Chemical Toxicology, vol. 46, no. 9, pp. 3069–3079, 2008.
View at: Publisher Site | Google Scholar
M. Gupta, U. K. Mazumder, T. S. Kumar, P. Gomathi, and R. S. Kumar, “Antioxidant and hepatoprotective effects of Bauhinia racemosa against paracetamol and carbon tetra-chloride induced liver damage in rats,” Iranian Journal of Pharmacology & Therapeutics, vol. 3, pp. 12–20, 2004.
View at: Google Scholar
A. Haro, I. López-Aliaga, F. Lisbona, M. Barrionuevo, M. J. M. Alférez, and M. S. Campos, “Beneficial effect of pollen and/or propolis on the metabolism of iron, calcium, phosphorus, and magnesium in rats with nutritional ferropenic anemia,” Journal of Agricultural and Food Chemistry, vol. 48, no. 11, pp. 5715–5722, 2000.
View at: Publisher Site | Google Scholar

Copyright

Copyright © 2012 Monika Bhadauria. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

2113

Downloads

1908

Citations

Evidence-Based Complementary and Alternative Medicine

Propolis Prevents Hepatorenal Injury Induced by Chronic Exposure to Carbon Tetrachloride

Abstract

1. Introduction

2. Material and Methods

2.1. Animals and Hepatorenal Injury by CCl4

2.2. Preparation and Administration of Propolis

2.3. Experimental Design

2.4. Isolation of Serum and Homogenate Preparation

2.5. Determination of Hepatorenal Marker Enzymes in Serum and Other Blood Biochemical Endpoints

2.6. Assessment of Peroxidative Stress and Antioxidant Status

2.7. Tissue Biochemical Assay

2.8. Histopathological Observations

2.9. Statistics

3. Results

3.1. Hepatorenal Marker Enzymes in Serum and Other Blood Biochemical Endpoints

3.2. Peroxidative Stress and Antioxidant Status

3.3. Tissue Biochemical Assay

3.4. Histopathological Observations

4. Discussion

Acknowledgment

References

Copyright

2.1. Animals and Hepatorenal Injury by CCl₄