Health properties of propolis, curcumin and their Mixtureson Hepatotoxicity Rats

Health properties of propolis, curcumin and their Mixtureson Hepatotoxicity Rats

 

Hany Hamdy Elgazzar

 

Nutrition and Food Science Department, Faculty of

SpecificEducation, Matrouh University.

 

Abstract

 

The present study aimed to evaluate the effect of propolis, curcumin and their mixtures on hepatotoxicity rats. Forty-eight adult male Sprague-Dawley rats weighing 150±10gm were divided into two main groups; the first main group (6 rats) was fed on basal diet as a negative control group. The second main group (42 rats) was fed on basal diet and injected with CCl4 (1 mL/kg b.w, 1:1 v/v mixture of CCl4 and liquid paraffin) every 72 h for 14 days to induce hepatic toxicity, then divided into 7 groups from group 2 to group 8. Group 2 was fed on basal diet as positive control group. Groups 3, 4, 5 were fed on basal diet supplemented with (1%) propolis, curcumin and their mixture from basal diet, respectively. Groups 6, 7, 8 were fed on basal diet supplemented with (2%) propolis, curcumin and their mixture from basal diet, respectively. At the end of the experimental period (4 weeks), rats were scarified and blood samples were taken for biochemical analyses. Results indicated that serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, total cholesterol, triglyceride, LDL-C and malondialdehyde (MDA) were significantly elevated (P<0.05) by CCl4 administration (positive control group) compared with negative control group, and significantly (P<0.05)decreased in HDL-C and glutathione peroxidase (GPX). It also indicated that supplemented diet with propolis and curcumin improved these changes that caused by CCl4 administration. We concluded that propolis and curcumin supplementation had beneficial effects on hepatotoxicity rats.

 

Key Words: carbon tetrachloride (CCl4), hepatotoxicity, rats, propolis and curcumin.

 

Introduction

 

Hepatotoxicity refers to liver dysfunction or liver damage that is associated with an overload of drugs or xenobiotics (Navarro and Senior, 2006).It is well known that carbon tetrachloride (CCl4)-induced acute liver injury in a murine model is a classical system for investigating potential hepato-protective agents, owing to the similarity of its molecular mechanism with acute chemical liver injury in humans (Niu et al., 2016).In recent years, many researchers have examined the effect of plants used traditionally to support function and treat diseases of the liver. Phytotherapy is the treatment and prevention of disease using plants, plant parts and preparations made from them. Plants traditionally used in phytotherapy are called medical plants (Weiss and Fintelman, 2000).

Propolis has been used extensively in folk medicine, due to its several pharmacological and biological properties such as antimicrobial, immunomodulatory, anticancer, anti-inflammatory, and antioxidant activities (Nakamura et al., 2010). Recent research has found that Caffeic acid phenethyl ester is one of major compounds in propolis that has immunomodulatory and hepatoprotective activities (Akyol et al., 2016). Propolis has free radical scavenging properties can induce the activation of the antioxidant enzyme system. These medicinal properties of propolis are directly related to its polyphenols which have been proved to scavenge free radicals and act as a strong cytoprotective natural product against any exogenous toxicity (Rizk et al., 2014 and Campos et al., 2015).

 

Curcumin is a hydrophobic polyphenol compound naturally concentrated in the rhizome of the herb Curcuma longa, commonly known as turmeric (Altenburg et al., 2011). Many studies reveal the protective or therapeutic effects of Curcumin in different forms of liver injury such as hepatitis, alcoholic and nonalcoholic fatty change, hepatocellular carcinoma and hepatotoxicity induced by drugs (Farombi et al., 2008 and Nabavi et al., 2014). The therapeutic potential of curcumin for treating hepatic disorders is well known. The beneficial effects of curcumin in liver diseases can be due to its anti-inflammatory, antioxidant effects and antifibrogenic properties (Ghosh et al., 2011). In addition, curcumin can decrease levels of thiobarbituric acid reactive substances (TBARS) and increase GSH and SOD levels in the liver homogenates from LPS-challenged rats supplemented with curcumin (Abdel-Daim and Abdou., 2015).

The present study aimed to evaluate the effect of propolis, curcumin and their mixtures on hepatotoxicity rats.

 

Materials and Methods

Materials:

Basal diet was prepared according to (Reeves et al., 1993). Propolisand curcumin powder have been purchased from local company for medicinal plants and herps, Cairo governorate, Egypt. Carbon tetrachloride Ccl₄, chemical kits for biochemical analysis wereobtained from EL-Gomhoryia pharmaceutical company, Cairo, Egypt. Forty-eight (48) albino rats, Sprague Dawley strain, weighing 140 ±5 g, were obtained from Serum and Vaccine Center-Cairo.

 

Experimental design:

Forty-eight (48) Sprague-Dawley white male albino rats, each weighing 140±5 g, were housed in group cages under conditions and fed on basal diet for adaptation for at least seven days before experiments. The rats were divided into eightmain groups (6 rats each) as follows: Group (1) Negative control group (-Ve): Fed on basal diet only.Forty-two (42) rats were treated subcutaneous injection with carbon tetrachloride Ccl₄mixed with an equal volume of paraffin oil and injected subcutaneously in a volume of 2 ml/kg BW twice weekly to induce liver dysfunction(Kanter et al., 2003).then divided into seven groups as follow: Group (2) Positive control group (+Ve): Fed on basal diet only.Group (3): Fed on basal diet plus 1% of propolis. Group (4): Fed on basal diet plus 1% of curcumin. Group (5): Fed on basal diet plus 1% ofequalized mixture of propolis and curcumin.Group (6): Fed on basal diet plus 2% of propolis. Group (7): Fed on basal diet plus 2% of curcumin. Group (8): Fed on basal diet plus 2% ofequalized mixture of propolis and curcumin.At the end of the experimental period (4 weeks), rats were scarified and blood samples were taken for biochemical analyses.

 

Biochemical analysis:

Blood samples were used for determination of the following parameters by commercially available (BioMerieux) kits: Determination Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP) and Total Bilirubin Concentration were determined according to the method described by Young, (2001),Malondialdehyde(MDA) according to the method of Ohkawa et al., (1979), Glutathione peroxidase (GPx) by Flohe and Gunzler, (1984),Total cholesterol (TC by Richmond, (1973), Triglyceride (TG)Wahlefeld, (1974),          Highdensity lipoprotein (HDL–c by Albers et al., (1983) and Low density lipoprotein (LDL–c by Friedewald et al., (1972).

 

Estimation of Total Phenolic:         

The qualitative and quantitative determination of phenolic compounds were carried out following a modified procedure of Croci et al., (2009).

 

Statistical analysis:

Statistical analysis was performed by using computer program statistical package for social science SPSS, 1998 and compared with each other using the suitable tests. The mean ± SDwere indicated. A paired T. test was used to evaluate differences between the groups of rats and its respective control. For statistical analysis of time course experiments, multiple measurements ANOVA were performed.

 

Results and Discussion

 

Phenolic compound of propolis and curcumin showed in table (1). Results reveled that propolis had high concentration of phenolic compounds compared with curcumin. Data showed that propolis had 428.7mg / 100ml of coffeic acid, while it was 0.0 mg / 100ml for curcumin. Concerning ellagic acid concentration it was 355.9mg / 100ml in propolis, while 0.2mg / 100ml in curcumin. Also, propolis had 182mg / 100ml, 114.5mg / 100ml and 8mg / 100ml from naringenin, querectin and vanillin respectively, while curcumin was 0.0 mg / 100ml. Furthermore, propolis contained ferulic acid and cinnamic acid in concentration 73.7mg / 100ml and 9.4mg / 100ml respectively, while curcumin contained 0.1mg / 100ml and 0.2mg / 100ml from these compounds. On the other hand, curcumin had concentration of gallic acid (1.4mg / 100ml) and catechin (0.6mg / 100ml) while propolis had 0.0 mg / 100ml from this compound. There are many limiting factors affecting on the concentration of phenolic compounds, type of solvents, extract temperature, stirring and the origin and source of the propolis (Salonen et al., 2012). The contents of total flavonoid and total polyphenol in propolis were 21.3 and 69.0mg/g of solid propolis, respectively. The contents of p-coumaric acid, kaempferol, chrysin, and artepillin C in the extract were 14.9, 6.75, 2.38, and 47.8mg/g of solid propolis, respectively. However, no chlorogenic acid, quercetin, and cinnamic acid were detected in the extract (Nakamura et al., 2013). In addition, (Huang et al., 2014) conclusion that propolis rich in phenylpropanoids including cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid and their derivatives.Nisar et al., (2015) studied the total phenolic content in turmeric extract and they found that Total phenolic content (TPC) means related to three solvents at their different concentrations i.e. Ethanol (60%), Ethanol (80%), methanol (60%), methanol (80%) and aqueous extract were 678.76 mg GAE/ 100 g, 745.76 mg GAE/ 100 g, 53.87 mg GAE/ 100 g, 682.43 mg GAE/ 100 g and 496.76 mg GAE/ 100 g respectively. Results showed that the highest value of total phenolic content (TPC) was recorded in ethanolic extract (80%) while the lowest for aqueous extract.

 

Results recorded in Table (2) showed that the mean value ± SE of serumconcentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubinwere significantly (P< 0.05) elevated by CCl4 administration (positive control group) compared with negative control group. It was observed significant (P< 0.05) reduce in serum AST, ALT, ALP and total bilirubin levels for all groups treated with propolis, curcumin and their mixtures compared to positive control group except in groups which fed on (1%) propolis or curcumin showed no significant difference in the serum concentration of ALT. The best decrease in the serum of ALT, AST, ALP and total bilirubin was observed at the group that fed with 2% (propolis + curcumin).The results refer to a significant increase in serum ALT, AST and ALP enzymes these increases may be due to the release of hepatocyte cytosolic enzymes such as ALT and AST in the blood Their appearance in blood does not necessarily indicate cell death and also that enzyme release during reversible cell damage occurs with an apparent lack of histological evidence of necrosis (Solter, 2005).The obtained results indicated that diet supplemented with propolis and curcumin at two levels significantly P<0.05 improved the mean levels of serum ALT, AST, ALP and total bilirubin in toxicants rats. In this respect, Saleh, (2012) Indicated that, AST, ALT, ALP, GGT and total bilirubin were significantly increased (P<0.05) in rats receiving 4-tert-OP when compared with the control group. Meanwhile, there was a significant decrease (P<0.05) in the level of the same parameter in propolis group compared to the control group. Also, Abdel Malak et al., (2015) showed that, the level of ALT, AST and total bilirubin in rats injected with CCl4 and treated with Honey and propolis. The levels were increased significantly after injection of rats with CCl4 compared to normal rats. But, after treatment with honey and propolis, the levels were decreased significantly (p<0.05).Other studies have found very high significant increase in the levels of AST, ALT, ALP, and total bilirubin in the Aflatoxin B1 (AFB1) treated group when compared with the control groups. The levels of AST, ALT, ALP and total bilirubin are significantly decreased (p < 0.05–0.001) in rats that were treated with AFB1 along with curcumin when compared with the AFB1- treated group (Alm-Eldeen et al., 2012). Kalantari et al., (2007)reported that the increase levels of marker enzymes such as AST, ALT, and ALP in serum is mitigated by treatment with curcumin by scavenging or neutralizing free radicals, inhibits peroxidation of membrane lipids and maintains cell membrane integrity and their function.

 

 

 

As seen in Table (3), serum malondialdehyde (MDA) level was significantly increased (P< 0.05) in the positive control group compared with the negative control group whereas all treated groups with propolis and curcumin significantly decreased (P< 0.05) compared to the positive control group. On the other hand, serum glutathione peroxidase (GPX) activity showed a significant decrease (P< 0.05) in the positive control group compared with the negative control group. It was clear that, there was significant (P< 0.05) increase in serum GPX activity for all treated groups with propolis and curcumin compared to the positive control group. Concerning to it was also observed that rats were fed on 2% Pro considered the best group for increasing the serum GPX and rats were fed on 2% Curcumin considered the best group for reducing serum MDA levels. Abdel Malak et al., (2015) whoshowed That Malondialdehyde (MDA) level was increased significantly in rats injected with CCl4 (P<0.05). On the other hand, the concentration of glutathione (GSH) and glutathione peroxidase (GSH-PX) was reduced significantly after injection of rats with CCl4 compared with that of the normal rats. Results of this study indicated that propolis and curcumin improved antioxidant enzyme levels. In this respect Gomaa et al., (2011) investigated that cypermethrin induced a significant increase in the mean value of MDA and a significant decrease in the mean values of antioxidant enzyme activities (CAT, SOD and GPx) in rats' liver as compared with control group. On the other hand, Propolis co-administration with cypermethrin induced a significant decrease in the mean value of MDA and a significant increase in the mean values of antioxidant enzyme activities (CAT, SOD and GPx) as compared with cypermethrin treated group. Also, this is confirmed by Yousef et al., (2019) showed that treatment with propolis lead to significant reduction in lipid peroxides levels and increase in antioxidant enzymes GPx, GST, CAT and SOD compared with control group, indicating the protection of the liver tissues from the damaging effect of MSG. Regarding curcumin effect, results showed that administration of CCl4 resulted in a significant increase in MDA production in liver and in serum. The treatment with curcumin normalized the increased MDA levels in serum and liver in rats, suggesting that curcumin reduced lipid peroxidation in hepatotoxicity (Hismiogullar et al., 2014). This is in agreements with the results of (Khorsandi et al., 2016) they demonstrated that the oral administration of Curcumin has hepatoprotective effects against NZnO(Zinc oxide nanoparticles) in rats. In Curcumin +NZnO treated rats, MDA level were lower than in the NZnO-intoxicated group. In the NZnO-intoxicated group, hepatic GPx and SOD activities were decreased. In Curcumin +NZnO treated rats, SOD and GPx activities were higher than the NZnO-intoxicatedrats.

 

Data in Table (4) revealed that serumtotal cholesterol (T.C), triglyceride (T.G) and LDL-C were significantly (P< 0.05) increased in the positive control group compared with the negative control group. Results also illustrated that all groups that were treated with Propolis and curcumin decreased significantly (P< 0.05) in serum TC, TG and LDL-C levels compared to the positive control group. Regarding serum HDL-C level, results showed a significant (P< 0.05) decrease in serum HDL-C level of the positive control group compared to the negative control group. There was a significant change in serum HDL-C level for all treated groups with Propolis and curcumin compared to negative control group. The highest improvement for lipid profile was observed at the group that fed on 2% (Propolis + Curcumin). The hyperlipidemic effect of CCl4 noticed in the serum of CCl4 intoxicated group, might suggest enhanced lipogenesis. Since cholesterol and triglycerides were increased significantly. The observed hyperlipidemia might reflect the impairment of liver cells to metabolize lipids and reduced transformation of cholesterol to bile acid and excretion (Deshpande et al., 1998). The results of the current study indicated that supplementation with propolis and curcumin significantly improved lipid profile in hepatotoxicity rats. (Nirala et al. 2008) which proved the modulating effect of propolis on total cholesterol and triglycerides levels with a significant increase in total proteins content after beryllium toxicity and the improvement of serum level of HDL-C by propolis in a dose-dependent manner. Furthermore, (Hassan et al., 2017)noted that there were significant differences between the positive control group, and all the affected groups of hepatitis, which have been treated with various doses of honeybee gum. Honeybee gum orally led to lower total cholesterol levels, triglycerides, and low and very low-density lipoproteins in the blood serum. In addition, these results are in agreement with those of (Gumaa et al., 2017) they revealed that serum cholesterol, triglycerides and LDL cholesterol increased while HDL cholesterol decreased in CCl4 intoxicated rats group compared with control group (P< 0.001) in the levels of the serum total lipids, total cholesterol, and triglycerides in the Aflatoxin B1(AFB1) treated group when compared with the control groups. The level of total lipids is significantly decreased (p < 0.05– 0.01) in rats that were treated with AFB1 along with curcumin. In addition, the levels of serum total cholesterol and triglycerides showed a very high significant decrease (p < 0.001) in rats that were treated with AFB1 along with curcumin when compared with the AFB1-treated group.

 

Conclusion

 

In conclusion, feeding on diets containing propolis, curcumin and their mixture improved the biological and biochemical parameters of hepatopathy rats, provided that best results obtained with their mixture diet.

 

Table (1): Active phenolic compounds content of propolis and curcumin extracts (mg / 100ml).

 

Table (2): Means ± SD values of liver functions in rats fed on basal diet and diet supplemented by propolis, curcumin and their mixtures.

Mean values are expressed as means ± SD.

Means with different superscript letters in the column are significantly different at P ≤ 0.05.

 

 

 

 

 

 

 

 

 

 

 

Table (3): Means ± SD values of GPX and MDA in rats fed on basal diet and diet supplemented by propolis, curcumin and their mixtures. 

 

Mean values are expressed as means ± SD.

 Means with different superscript letters in the column are significantly different at P ≤ 0.05

 

 

 

 

 

 

 

 

 

 

 

Table (4): Means ± SD values of lipids profile in rats fed on basal diet and diet supplemented by propolis, curcumin and their mixtures.

Mean values are expressed as means ± SD.

 Means with different superscript letters in the column are significantly different at P ≤ 0.05.

 

 

 

 

 

 

 

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الخصائص الصحيه للبروبوليس و الکرکم و خليطهما على الفئران المصابة بالسميه الکبديه

 

د/ هانى حمدى الجزار

 

کليه التربيه النوعيه – مطروح

 

الملخص العربى

 

هدفت الدراسة الحالية إلى تقييم تأثير البروبوليس والکرکم وخليطهما على اضطراب وظائف  الکبد الناجم عن رابع کلوريد الکربون في الجرذان البيضاء. تم تقسيم 48 ذکور فئران الألبينو البالغة وزنها 150 ± 10 جم إلى مجموعتين رئيسيتين. تم تغذية المجموعة الرئيسية الأولى (6 فئران) على الغذاء الأساسى کمجموعة ضابطة سلبية. تم تغذية المجموعة الرئيسية الثانية (42 جرذاً) على الغذائي الأساسي وحقنت برابع کلوريد الکربون (CCl4) (1 مل / کجم من وزن الجسم ، 1: 1 خليط من CCl4 والبارافين السائل) کل 72 ساعة لمدة 14 يومًا لأحداث سمية الکبد ، ثم قسمت إلى 7 مجموعات فرعيه من المجموعة 2 إلى المجموعة 8. تم تغذية المجموعة ( 2 ) : على النظام الغذائي الأساسي کمجموعة ضابطهموجبه. تم تغذية المجموعات 3 ، 4 ، 5 على الغذاءالأساسى المضاف إليه (1٪) من البروبوليس ، الکرکم وخليطهما من الغذاء الأساسي ، على التوالي. تم تغذية المجموعات 6 ، 7 ، 8 على الغذاء الأساسى المضاف إليه (2٪) من البروبوليس والکرکم وخليطهما من الغذاء الأساسى على التوالي. في نهاية الفترة التجريبية (4 أسابيع) تم تشريح الفئران وأخذ عينات من الدم للتحليل البيوکيميائي. أشارت النتائج إلى أن مستويات aminotransferase (AST) ، aminotransferase (ALT) ، (ALP) ، البيليروبين الکلي ، الکوليسترول الکلي ، الدهون الثلاثية ، LDL-C و malondialdehyde (MDA) ارتفعت بشکل ملحوظ (P <0.05) بواسطة CCl4. فى المجموعة الضابطه الموجبه مقارنة مع المجموعة الضابطهالسالبه ، وانخفاض معنوي (P <0.05) في HDL-C وجلوتاثيون بيروکسيديز (GPX). کما أشارت النتائج  إلى أن النظام الغذائي المدعم بالبروبوليس والکرکم يحسن هذه التغييرات التي سببها الحقن بCCl4.

الخلاصة:  أن تدعيم الأغذيه بالبروبوليس والکرکم و خليطهما  لهم آثار مفيدة على الفئران المصابه  باضطراب وظائف الکبد ( برابع کلوريد الکربون ).

 

 

 

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