EFFECT OF HOME MADE CAMEL MILK YOGHURT SUPPLEMENTED WITH FIG AND HONEY BEE IN RATS WITH INDUCED STEATOHEPATITIS

Introduction:

Fatty liver refers to a large spectrum of diseases characterized by excessive fat accumulation in the liver, which could be alcoholic or non-alcoholic in origin. Non-alcoholic fatty liver disease is clinically important because it affects 25% of the population, with widespread pathological changes in the liver that range from simple nonprogressive steatosis to non-alcoholic steatohepatitis (NASH). This can progress to cirrhosis, hepatocellular carcinoma, and liver failure with increased hepatic-related mortality (Brunt, 2004 and Soderberg et al., 2010). Clinically, NASH is linked to visceral obesity, insulin resistance, dyslipidemia, and type II diabetes mellitus (Yamaguchi et al., 2007 and Cusi, 2009).

Interest concerned about the use of alternative medicines for the treatment of hepatic disease has been arisen. Camel milk (Camelus dromedarius) is used in hot and arid regions as an essential nutritional source, and its high energy and vitamin contents are known to help immune-deficient patients as well as those recovering from diseases (Al-Awadi and Srikumar, 2001). Camel milk is different from other ruminant milk having low cholesterol, fat, sugar, high minerals (sodium, potassium, iron, manganese, magnesium, copper and zinc), and vitamins C and E and large concentrations of insulin (Zulueta et al., 2007 and Romero-Huelva et al., 2012). Camel milk whey protein is rich in cysteine (Girardet et al., 2000). The value of camel milk is due to its high concentrations of linoleic acid and polyunsaturated fatty acids which are essential for human nutrition (Agrawal et al., 2004).  

In addition to its specific composition, the digestion of camel milk in the gastrointestinal tract produces many bioactive compounds with antimicrobial, antioxidant, immunomodulatory, and hepatoprotective effects (Salami et al., 2010). Camel milk shows significant activity and biological effects, and it is also very effective and protective against toxicity from heavy metal (Al-Hashem, 2009) and viral and bacterial infections (El Agamyet al., 1992). Using the orange syrup at 15% in camel milk enhanced the flavor acceptability and it also leads to decreasing in microbial total counts; which could be beneficial in preservation of the product (Toloun et al., 2013).

  Figs fruit (Ficus carica Linn) are an excellent source of minerals, vitamins and dietary fibre; they are fat and cholesterol-free and contain a high number of amino acids (Slavin, 2006) and are good source of flavonoids and phenols (Vinson et al., 2005) which play a vital role in preventing numerable health disorders related to oxidative stress (Sirisha et al., 2010). Fig has been used for metabolic, cardiovascular, respiratory, antispasmodic and anti-inflammatory disorders (Patil et al., 2010). Honey a product of honey bees is used as a medicine in many culture times and is known to exhibit a broad spectrum of activities including antiviral, antibacterial, antioxidant activity and immune-stimulant properties (Aljadi and Kamaruddin, 2004 and Khadr, et al., 2007).

The aim of the current work was to evaluate the effect of homemade yoghurt produced from camel milk and supplemented with fig and honey bee against steatohepatitis induced in adult male albino rats fed on MCDD.

Materials and methods:

Material:

Rats: Forty eight adult male albino rats were obtained from Helwan Farm, Ministry of Health and Population, Cairo, Egypt. Diet: Casein, vitamins, cellulose, minerals, methionine and choline were obtained from Morgan Company for Chemicals, Cairo, Egypt. Chemicals: Chemical kits were purchased from Gama Trade Company for Chemicals, Cairo, Egypt.Fresh Camel milk (Camelus dromedarius), fresh fig (Ficus carica L.), honey bee and orange juice samples were obtainedfrom local market.    

Methods:

-    Drying of Figs:

            Fifty kg of Figs were chosen full maturity, washed well and put in boiling water for 30 seconds. Then they were cut into halves, flooded in a solution of citric acid (5 g/L) for 10 minutes and rinsed with cold water and left for 24 hours until dried completely. Figs were arranged in single layer on the drying tray. Drying was performed at 40°C by hybrid solar convective drying system belonging to solar energy department, National Research Center, Dokki, Giza. The dried figs were grounded and kept in tightly closed containers at room temperature to be ready for addition to the yoghurt (Dipersio et al., 2003).

-    Preparation of yoghurt:

Yoghurt was produced using fresh camel milk with adding lactic acid bacteria according to the method described by (Kavas, 2016), withsome modifications included additionwith orange juice (15%) to enhance flavor, then milk was divided into three treatments with addition 30% of fig or honey bee or both fig and honey bee (at ratio 1:1) respectively. The samples were homogenized with Blender then added into 200 g plastic cups and incubated for 12 hours at 50º C. Then the yoghurt transferred to refrigerator at 4ºC for 2 days. Dried fig fruits or honey bee were added to the camel milk at the level of 30%. Samples of yoghurt from camel's milk were prepared every week.

-    Experimental design:

The basal diet consisted of 100 g sucrose (g/kg diet), 200 g casein (> 80 % protein), 515 g corn starch, 40 g corn oil, 50 g cellulose, 35 g mineral mixture, 10 g vitamin mixture, 30 g DL-methionine, and 20 g choline bitartrate (Reeves et al., 1993). In this study 47 adult male albino rats, weighing (200 ± 10 g) were housed in well-aerated wire cages. All animals were kept under normal healthy condition and fed on basal diet for one week for adaptation. Rats were divided into two main groups, the first main group (n=7 rats), negative control, was fed on basal diet. The second main group (n=40 rats) were fed on basal diet with methionine- choline deficient diet to induce steatohepatitis in rats (Veteläinen et al., 2007). To ensure the occurrence of steatohepatitis, five rats from the 2^nd main group were sacrificed after one week and five weeks of feeding the MCDD, then liver obtained for histopathological examination. Hepatocytes screening showed that <30% the cells were affected without inflammation, while severe steatosis was reached after 5 weeks (> 60% of the hepatocytes were affected with inflammation). Rats (n=35 rats) with induced steatohepatitis were fed on MCDD and were divided into five subgroups as follow:

Subgroup (1): positive control was fed on a MCDD. Subgroup (2): was fed on a MCDD supplemented with yoghurt camel milk at the level of 30%.Subgroup 3: was fed on a MCDD supplemented with yoghurt camel milk containing fig at the level of 30%. Subgroup (4): was fed on a MCDD supplemented with yoghurt camel milk containing honey beeat the level of 30%. Subgroup (5): was fed on a MCDD supplemented with yoghurt camel milk containing fig and honey bee at the level of 30%.    

At the end of experimental period (8 weeks), rats were sacrificed after overnight fasting and blood of each rat was taken from the abdominal aorta under anesthesia by diethyl ether. The serum was separated by leaving the blood samples 15 minutes at room temperature then centrifuged at 3000 rpm for 20 minutes, then kept in plastic vials at -20°C until biochemical analysis. Rats’ livers were removed and cleaned for histopathological examination (Carleton, 1979).

-    Biochemical Analysis:

Serum liver enzymes aspartat aminotransaminase (AST), alanine aminotransaminase (ALT) andalkaline phosphatase (ALP) were determined according to the methods described by Reitman and Frankel, (1957). Serum total protein, albumin and globulin were estimated according to Weissmanet al., (1950).  Total cholesterol (TC), triglycerides (TG) high density lipoprotein cholesterol(HDL-c)were determined according to Richmond, (1973), Wahlefeld, (1974) and Albers et al., (1983) respectively. While, low density lipoprotein cholesterol(LDL-c) and very low density lipoprotein cholesterol(VLDL-c) levels were calculated according to Fridewald et al., (1972) respectively.  

Oxidative stress markers: Glutatione (GSH), Superoxide Dismutase (SOD) and malondialdehyde (MDA) were determined according to the methods described by Beutler et al., (1963), Kakkar et al., (1984) andDraper and Hadly, (1990)respectively. TG in tissue was determined according to Naito and David, (1984).

Sensory evaluation:

The yoghurt samples were evaluated by 20 trained panelists among the staff of Nutrition and Food Science Dept., Faculty of Home Economic, Helwan University for taste, flavor, texture and general acceptability as follows: scores were very good (9-10), good (7-8.9), acceptable (5-6.9), weak (3-4.9), very weak (1-2.9) according to Lanza, et al., (1995).

Statistical analysis:

         The obtained data was statistically analyzed using the Statistical Package for Social Science (SPSS) version 18.0. Values are represented as means with their standard errors. Un-paried analysis was conducted using one way analysis of variance (ANOVA) for continuous variables. P value of less than 0.05 was considered to indicate statistical significance (Snedecor and Cochran, 1980).

Results:

Results presented in Table (1) show the mean scores for the panel test of taste, flavor, texture and general acceptability for yoghurt samples produced from camel milk containing fig and honey bee. It was observed that yoghurt produced from camel milk significantly (P<0.05) had lower taste, flavor, texture and general acceptability compared to cow milk yoghurt. The addition of orange juice plus fig to camel milk yoghurt significantly (P<0.05) increased the score of taste, flavor, texture and the general acceptability of the produced yoghurt compared to the sample of camel milk yoghurt. In regarding to the taste, there were no changes of the produced yoghurt among the samples fortified with fig, honey bee or their combination. The yoghurt sample that fortified with honey bee had significantly (P<0.05) higher score of flavor compared to the camel milk yoghurt.  However, there was no change in the flavor between the yoghurt that fortified with honey bee and the yoghurt that fortified with both together. It was clear that, the general acceptability for the cow milk yoghurt had significantly (P<0.05) higher score compared to the yoghurt from camel milk. The score for the general acceptability for all yoghurt samples that contain fig, honey bee and their combination significantly increased (P<0.05) compared to the yoghurt from camel milk. Also, there were significant changes in the general acceptability among all tested yoghurt. Generally, it was observed that, orange juice with fig and honey bee supplementation to camel milk yoghurt samples increased the general acceptability.

Table (1): Sensory evaluation for yoghurt produced from camel milk and containing fig, honey and their combination compared with cow milk yogurt.

Values are expressed as means ± SE. 

Values at the same column with different letters are significantly different at P<0.05.

Table (2) illustrates the effect of camel milk yoghurt supplemented with fig and honey on liver enzymes (ALT, AST and ALP) in rats with induced steatohepatitis. The present data revealed that steatohepatitis significantly (P<0.05) increased the levels of liver enzymes in rats on the MCDD compared to the negative control group. Rats in groups fed on different diets supplemented with camel milk yoghurt, yoghurt camel milk containing fig and honey bee had lowered levels of serum AST, ALT and ALP that differ significantly (P<0.05) from those of the positive control group.  Also, considerable differences were observed between the group fed on basal diet supplemented with camel milk yoghurt containing fig or honey for ALT, AST and ALP. However, the mean value of ALT, AST and ALP were not significantly altered between the groups fed on camel milk yoghurt or the group fed on camel milk yoghurt containing honey bee. The highest reduction in liver enzymes was found in the group fed on camel milk yoghurt fortified with both fig and honey bee. The current results denote the effect of steatohepatitis on liver enzymes. Diets supplemented with either camel milk yoghurt only or containing fig and honey bee significantly (P<0.05) lowered the increments of liver enzymes levels.

Table (2): Effect of camel milk yoghurt supplemented with fig and honey on liver functions in rats with induced steatohepatitis

Values are expressed as means ± SE. 

Values at the same column with different letters are significantly different at P<0.05.

Table (3) presents the effect of camel milk yoghurt supplemented with fig and honey on serum protein parameters in rats with induced steatohepatitis. The data revealed that steatohepatitis induced significant (P<0.05) decrease in the levels of serum total protein, albumin and globulin in rats on the CMDD compared to the negative control group. Supplementation with camel milk yoghurt only or with fig and honey bee significantly (P<0.05) increased the levels of total protein, albumin and globulin compared to the positive control group. It was found that, there were significant (P<0.05) differences in serum protein parameters between the groups fed on yoghurt using camel milk and the group fed on camel milk yoghurt that fortified with fig or both fig and honey. However, there were no significant differences in serum protein parameters between the group fed on camel milk yoghurt and the group fed on camel milk yoghurt fortified with honey. The highest increase in serum protein parameters was recorded in the group fed on camel milk yoghurt containing fig and honey bee. 

 Table (3): Effect of camel milk yoghurt supplemented with fig and honey on serum protein parameters in rats with steatohepatitis

Values are expressed as means ± SE.

Values at the same column with different letters are significantly different at P<0.05.

Effect of camel milk yoghurt supplemented with fig and honey bee on oxidative stress in rats with induced steatohepatitis was shown in Table (4). Rats with steatohepatitis showed significant (P<0.05) increase in the level of MDA however, the mean value of GSH and SOD were significantly (P<0.05) decreased compared to the negative control group. Supplementation with camel milk yoghurt significantly (P<0.05) decreased the level of MDA, and significantly (P<0.05) increased the level of GSH and SOD compared to the positive control group. It was observed that, camel milk yoghurt containing fig and honey bee significantly (P<0.05) increased the level of GSH and SOD, however the level of MDA significantly (P<0.05) decreased compared to the positive control group. There were no significant differences in the level of GSH, SOD and MDA between the group fed on camel milk yoghurt containing fig or honey bee. The highest increase in the level of GSH and SOD was found in the group fed on camel milk yoghurt that fortified with both fig and honey compared to the other treated groups.

Table (4): Effect of camel milk yoghurt supplemented with fig and honey on oxidative stress in rats with induced steatohepatitis 

Values are expressed as means ± SE.

Values at the same column with different letters are significantly different at P<0.05.

Table (5) shows the effect of camel milk yoghurt supplemented with fig and honey bee on TG content in liver of rats with induced steatohepatitis. The presented data indicated that steatohepatitis significantly (P<0.05) increased the levels of tissue TG in rats on the CMDD compared to the negative control group. The elevated level of tissue TG were significantly decreased in the groups fed on diets supplemented with camel milk yoghurt or camel milk yoghurt containing fig and honey compared to the positive control group. There was no significant difference in the level of tissue TG between the groups fed on camel milk yoghurt containing fig or both fig and honey bee. The highest reduction in tissue TG was found in the group fed on camel milk yoghurt containing fig and honey bee compared to other treated groups.

Table (5): Effect of camel milk yoghurt supplemented with fig and honey bee on triglycerides content in liver rats with steatohepatitis

Values are expressed as means ± SE. 

Values at the same column with different letters are significantly different at P<0.05.

Table (6) presents the effect of camel milk yoghurt supplemented with fig and honey on lipid profile in rats with induced steatohepatitis. The results indicated that steatohepatitis significantly increased (P<0.05) the levels of TC, TG, VLDL-c, LDL-c, however HDL-c was significantly (P<0.05) decreased compared to the negative control group. The levels of TC, TG, VLDL-c were significantly (P<0.05) decreased in the groups that fed on camel milk yoghurt, camel milk yoghurt that containing fig and honey bee compared to the positive control group,  however  the level of HDL-c was significantly (P<0.05) increased for the same groups. There were no significant differences in the level of TG, VLDL-c and HDL-c between the groups fed on camel milk yoghurt and the group fed on camel milk yoghurt fortified with honey. Camel milk yoghurt fortified with fig significantly decreased the level of TC and LDL-c compared to the group fed on camel milk containing honey. The highest improvement in lipid profile was recorded in the group that fed on camel milk yoghurt containing fig and honey compared to the other treated groups.

Histopathological results: MCDD causedsteatosis of hepatocytes and focal hepatic necrosis associated with inflammatory cells infiltration. Yoghurt produced from camel milk supplemented with fig and honey bee protected against steatosis induced pathological changes in rats' liver.

Table (6): Effect of camel milk yoghurt supplemented with fig and honey bee on lipid profile in rats with induced steatohepatitis

Values are expressed as means ± SE.

Values at the same column with different letters are significantly different at P<0.05.

Histopathological results:

 Discussion:

The worldwide epidemic of obesity and diabetes is closely associated with the increased incidence of non-alcoholic fatty liver disease (Suanarunsawat et al., 2009). Life style modification and the acquisition of healthy food habits are the most clinically recognized and effective methods to control the disease and minimize the progression to NASH and cirrhosis (Xiao et al., 2013).

Interest concerned the use of alternative medicines for the treatment of hepatic disease has been arisen. Camel milk has gained a good reputation due to its health benefits stated by the folklore stories detailing its shielding effects against a wide range of diseases. The current results indicated that treatment with camel milk yoghurt containing fig and honey bee was found to suppress the increase of serum liver activities induced by steatohepatitis in rats. This finding implies that camel milk challenge to protect liver tissue from steatohepatitis injury.

Camel milk decreased CCl₄ induced elevated enzyme levels, indicating the protection of structural integrity of hepatocytes cell membrane or regeneration of damaged liver cells due to its antioxidant properties (Palanivel et al., 2008). The presence of peptides and proteins in camel’s milk exhibits its biological activities that have beneficial effect on many bioprocesses as digestion, absorption, growth and immunity (Yagil et al., 1984 andKorhonen and Pihlanto, 2001).

The reversal of increased serum enzymes in steatohepatitis-induced liver damage by camel milk may be due to the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and the regeneration of hepatocytes (Thabrew et al., 1987). In this respect, the protective effect of camel milk against steatohepatitis induced oxidative stress in the rats is due to its antioxidant properties, camel milk was found to contain high concentrations of vitamins A, B, C and E and is very rich in magnesium and other trace elements, these vitamins act as antioxidants and have been found to be useful in preventing toxicant-induced tissue injury (Yousef, 2004). Several studies by Al-Fartosi et al., (2012) and Hamad et al., (2011) have provided a considerablesupport for evidencing the protective effects of camel milk on liver damage. Moreover, camel milk (100mL/24h/cage) as their sole source of drinking water) treatment may play a protective role by improving the changes in histological structure against CCl₄-induced liver damages and enhancing liver enzyme activities in rats (Althnaian, 2012). 

The increase of albumin concentration after treatment with camel milk may be due to that camel milk could induce decrease in lipid peroxidation processes as well as increase in the activities of plasma protein thiols as albumin and other serum proteins in both animal and human (Al-Fartosi et al., 2012). Similarly, the healing of the hepatic parenchyma observed by microscopic examination in the camel milk was associated with the recovery of normal serum proteins and albumin levels and decreased serum transaminases and bilirubin levels. In this regard, vitamin E treatment was reported to decrease serum transaminases and hepatic steatosis (Yakaryilmaz et al., 2007), which supports that the high vitamins and trace elements content in camel milk protect hepatocytes integrity and prevents the release of transaminases into the blood.

            In this regard, Mohamed et al., (2016) showed that honey bee demonstrates good ameliorative and antioxidant capacity toward diethyl nitrosamine induced hepatocellular damage in rats. Similar results were reported by Bawazeer and Qahl, (2016) who mentioned that, chronic treatment with fig, olive green (2 ml/kg) and both of them orally for 16 weeks decreased hepatic liver enzymes and increased total plasma proteins and albumin in nonalcoholic fatty liver rats.

Camel milk has higher antibacterial and antiviral properties than cow milk. This is partially because of the higher concentration of lactoferrin in camel milk (220 mg/L) than in cow milk (110 mg/ L) and the higher concentration of lysozyme in camel milk (288 mg/100 mL) than in cow milk (13 mg/100 mL). In addition, camel milk has a higher level of lactoperoxidase, immunoglobulin G, and secretory immunoglobulin A with antimicrobial activity, and higher vitamin C content (Kamal et al., 2007 and Al-Hashem, 2009).

It was observed from the current results that, camel milk yoghurt with or without honey bee or fig supplementation significantly decreased the lipid profile in rats with induced steatohepatitis. Camel milk has a unique composition that is rich in minerals and vitamins and it increased HDL-c and ameliorated the biochemical and cellular features of non-alcoholic fatty liver disease (NAFLD) in rats that received a high-fat, cholesterol-rich diet (HCD). The antioxidant effect of camel milk is a likely mechanism for the altered metabolism and absorption of HCD in the presence of camel milk. Regular consumption of camel milk could provide a natural way to protect against NAFLD induced by a high-fat diet (Korish  and Arafah, 2013).

Phenols and flavonoid compounds have the feature of higher antioxidant than vitamins and that recognized when measured as a disincentive for the oxidation of low-density lipoprotein LDL, which is an indicator of atherosclerotic disease (Morenoet al., 2000). Fig antioxidants can enrich lipoproteins in plasma and protect them from subsequent oxidation (Vinson et al., 2005). In this respect, the analysed phenolics in fig were gallic acid (30.99 mg), followed by epigallocatechine (25.44 mg), caffeine (20.23 mg), catechine (13.88 mg), epicatechine (12.48 mg), rutin (3.26 mg) and epigallocatechine gallate (2.52 mg) (Tawfik  and Alhejy, 2014).

Both enzymatic and non-enzymatic antioxidant system are essential for cellular response in order to deal with oxidative stress under physiological condition. Therefore, antioxidant enzymes such as CAT, SOD, and GSH-Px and non-enzymatic electron receptors such as GSH are affected and used as indexes to evaluate the level of oxidative stress (Dey and Lakshmanan, 2013 and Mallikarjuna et al., 2010). The current results revealed that supplementation with camel milk yoghurt alone or with fig and honey bee significantly decreased the level of MDA, and significantly increased the level of GSH and SOD. The high magnesium and trace element contents in camel milk can protect against oxidative damage and help the absorption and metabolism of the antioxidant vitamins B, C, and E (Kamal et al., 2007). The antioxidant effect of camel milk plays a role in the reduction of hepatic fat accumulation and decreases systemic and hepatic oxidative stress (Barbagallo, 1999), as evidenced by increased GSH levels and CAT activity and decreased MDA production. Collostral and mature milk of camel contain very high quality of protective and bioactive proteins (lactoferrin) that play an important role in the immune system response and in protecting body against infections such as infection with Schistosoma mansoni (Maghraby et al., 2005).

Lattanzio, (2003) reported that besides antioxidant effects, phenolic compounds have a wide variety of biochemical properties and can also have a useful effect in preventing the development of ailment like cancer and cardiovascular diseases. There is linear correlation between the total content of phenolics and the antioxidant capacity (Cai et al., 2004 and kumaran and karunakaran, 2006).

Evidence suggested that honey can produce many powerful health effects as hepato-protective (Al-Waili et al., 2006) antioxidant (Erejuwa et al., 2010) and antiinflammatory (Kassim et al., 2010). In rats with severe steatosis, usually associated with increased hepatic ROS formation, oxidative stress and inflammation (Celebi et al., 2004), honey supplementation significantly reduced the levels of MDA and increased GSH content in the liver (Kilicoglu et al., 2008).Supplementation with honey and ginseng was reported to protect against CCl₄-induced hepatotoxicity in rats by reducing lipid peroxidation and enhancing antioxidant capacity (El Denshary et al., 2012).

It was concluded that, camel milk with orange juice may be used to produce yoghurt. Camel milk yoghurt containing fig and honey bee improved liver functions and reduced lipid profile in rats with induced steatohepatitis.