Dose dependent effects of Spirulina Platensis on liver health in rats

Life quality is in linked with body weight, height, waist circumference, waist/height ratio and abdominal fat weight which also attributed with the liver health. The liver and body fat topography play an important role in organ metabolism and also provide the regulation of health. Especially liver examines as an end organ which has an interaction with viseral fat (Vague, 1956; Marchesini et al., 2001). It has been suggested that viseral fat accumulation in liver fatty is responsible for the metabolic syndrome’s pathophysiology (Chan et al., 2006). Besides that, the association of abdominal fat with adverse outcomes and metabolic diseases was indicated by some researchers (Vague, 1956; Klein et al., 2007). It was also reported that the truncal obesity was measured by waist circumference which is an important definition for metabolic syndrome (Grundy et al., 2005). Because of increase of these diseases, worlwide nutrition is being changed and healtier and natural foods are interested. A proper nutrition is neccessary to prevent some important metabolic disorders such as diabetes or its complication, as known.
       
Natural resources contains in a big sciencific family, but mostly the come from plant and plant derivates (Marhesini et al., 2001; Despres and Lemiwux, 2006). The utilization of these resources has been observed by researchers for many years, especially algae. The algae has an important protein source among all these supplements which have been used until now. Alga, according to their structural characteristics, are divided into two main groups as macroalgae and microalgae. The most popular algae among alternative medicinal foods and nutrition is Spirulina platensis. S. platensis is a microalgae which has been called as “Super food”  and an acceptable alternative health food by World Health Organization (Habib et al., 2008). This microalgae does not contain cellulose in its cell wall and so it can be digested by both human and animals. Notably, it contains proteins, essential amino acids, carotenoids, antioxidant pigments, gamma linolenic acid and phycocyanin which especially effective on growth, antioxidant, cholesterol and immunomodulatory mechanisms (Sotiroudis and Sotiroudis, 2013; Seyidoglu et al., 2017; Sivakumar et al., 2018). Belong its nutritional components, it was suggested that this microalgae supports a good health.
       
The potential effects of S.platensis have been contributed the literatures in both human and animals(Plaza et al., 2008; Heidarpour et al., 2011; Peipei and Sumin, 2018). It has a protective effect on lowering blood lipid and improvement immunity (Ruan et al., 1990). Also it was reported that Spirulina is one of the natural additive for regulation and reducing cholesterol and thereby effect on obesity, cardiovascular and liver problems (Becker et al.,  1986; Ferreira et al., 2010). A study with obese humans, it was observed that 2,8 g S.platensis additive has an impact effect on lowering cholesterol and body weight (Becker et al., 1986). On the other hand, beta carotene, phenolic acids and tocopherols contents of S.platensis is related to antioxidant activity of organism. As discussed with these contents, S.platensis has an important role on reduce hypercholesterol, obesity and liver diseases (Samuels et al., 2002; Klein et al., 2007; Karkos et al., 2011). Nowadays, for prevent of these disorders, natural foods are choosen, like as S.platensis. However, besides its nutritional ability, the prevent of health for metabolic disorders such as cardiovascular problems, obesity or hypertension are still in scientific discussion. This study is aimed to demonstrate a link between liver tissue health, body mass index and viseral fat with increasing amounts of S. platensis. Also, it is aimed to detect any importance of the histopathologic changes of liver tissue of rats in dose dependent manner.

Animals, groups and feeding
 
The experimental protocols were approved by the Animal Care and Use Committee of Namik Kemal University and are in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The study was carried out with the permission of Namik Kemal University Animal Experimentation Local Ethics Committee (Approval No: 2016-04/9).
       
In this trial, 30 male Wistar Albino rat, aged 7-8 weeks were randomly allocated on a weight basis to three groups: Control,  (basal diet), SP-1 (added 500 mg/kgbw S. platensis, daily) and SP-2 (added 1000 mg/kgbw S. platensis, daily). The rats were housed in purpose-built metal cages. Feed and water were offered ad libitum throughout the 45 day test. Basal diet was formulated to contain 2000-2500 kj ME/kg metabolize energy, 23% crude protein, 3% crude fat, 7% crude fiber, 8% crude ash, and was projected to take on maintenance requirements according to the NRC (NRC, 1995).  Basal diet was supplemented with S.platensis (Egert, Izmir-Turkey) and doses were provided and modified according to literature (Nagaoka et al., 2005; Moreira et al., 2011).
 
Measurement
 
The effects of different doses of S.platensis on body weight, height, abdominal fat, waist circumference (WC), body mass index (BMI) and waist /height ratio (WHtR) were determined at the begining and the end of the study. Blood samples were collected by tail venipucture on the 45th day from overnight-fasted rats. Serum total cholesterol was determined by spectrofotometrically, and the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by ELISA method. Besides that, the histopathologic changes in the liver tissue (balloon degeneration, sinusoidal congestion, active cupper cells, sinusoidal dilatation, karyolysis, karyorrhexis and caryopictypnosis) were recorded for observation of liver health(Nassar et al., 2010; Arsad et al., 2014).
 
Statistical analysis
 
Statistical analyses were performed with SPSS (Version 17.0). Data were tested for normality distribution and variance homogeneity assumptions. All the values were grouped and the means and standard errors were calculated. One-way ANOVA was applied to the all parameters to examine the difference between groups. Differences were considered significant at P<0.05. If the difference between groups was provided to be significant (P<0.05), differences evaluated by Tukey’s test (Dowdy and Wearden, 1981). On the other hand, in non-homogenous groups, differences between means were analyzed by Kruskal Wallis and following Mann Whitney U test between groups one by one(Dawson and Trapp, 2001). The correlation analysis were identified by Pearson Correlation method.

There is no statistical differences among all groups in body mass index parameters (Table 1, p>0.05), but positively and statististically significant correlations were observed. Especially in group SP-1, the lower dose, the positive correlations were determined between body weight and height (r: 0.821; p:0.004), body weight and BMI (r: 0.891; p:0.001), body weight and abdominal fat weight (r: 0.644; p:0.045), height and BMI(r: 0.891; p:0.001), height and abdominal fat weight(r:0.695; p:0.026), and WC and abdominal fat weight (r: 0.770; p:0.009). In group SP-2, the positively significantly correlation were found between body weight and heigth (r: 0,677; p: 0,045), and BMI and WHtR (r: 0,716; p:0,003). Nevertheless, there were no significant changes in the liver weight, serum cholesterol level, concentration of serum ALT and AST enzymes and ALT/ AST ratio as shown in Table 2 (p>0.05). Also, there were no statistically differences found in histopathologic changes of the liver tissue (balloon degeneration, sinusoidal congestion, active cupper cells, sinusoidal dilatation, karyolysis, karyorrhexis and caryopictypnosis; Table 3, p>0.05).
 

 

 

       
S.platensis have been known as an important source and a lot of studies have been performed about the effect on life quality (Connor et al., 2007; Park et al., 2008; Khanna et al.,  2016). For a good health, body weight and abdominal fat distribution are important as well as dietary fat composition. Also, WC and WHtR have shown better association between central obesity. This study was designed to investigate the association of different indices of metabolic adverse outcomes (BMI, WC and WHtR, abdominal fat) with natural additive S. platensis. Although there were no differences among all these parameters in all groups, the positive and statistically correlation were determined among all groups and parameters especially between body weight and abdominal fat weight in group SP-1, the lower dose. Both of the body weight and abdominal fat weight parameters were lowered when 500 mg/kg dose S.platensis given. Besides that, there were found the statistically positive corelation between WC and abdominal fat weight in group SP-1 and also WHtR and BMI in group SP-2. Belong to these results, if considered as the only indicator of body fat distrubiton parameters are WC and WHtR, either the high dose or low dose of S.platensis may affect on the life quality positively. This interesting result was pointed out that calibrating the dose of natural additives was assesing link between body condition and life quality. Also, it suggested that optimal dose of dietary requirement was important, and it was necessary to evaluate the aspect of S. platensis.
       
Nevertheless, studies demonstrated that there was a strong association between abdominal fat weight, liver injury and liver enzmyes and so cholesterol metabolism (Adams et al., 2005; Malhi and Gores, 2008). Todays, for this reason, researchers attended to study on protection and regulation effects of natural additives on health. S.platensis is one of the best natural foods which  is desired in life quality due to its inhibition effect on cholesterol metabolism (Nagaoka et al., 2005; Colla et al., 2008). S.platensis contents phycocyanin and polyunsaturated fatty acids which have an important role on serum lipid profiles (Kato et al., 1984; Colla et al., 2008; Seyidoglu and Galip 2014). Some researchers reported that diet with S.platensis had significant inhibiton for serum total cholesterol due to its effect on both jejeunal absorption and ileal reabsorption in rats (Kato et al., 1984; Nagaoka et al., 2005; Miridha et al., 2010). At the same time, average values of some liver enzmyes named ALT and AST were detected for cholesterol metabolism. Because these enzymes are located intracellulary in liver, kidney and heart. If there is a damage situation in these tissues, these enzmyes levels in the blood were increased. However, in our study, no differences were found in abdominal fat weight, serum cholesterol and liver enzmyes. It was suggested that both the doses of S.platensis have no adverse effect on the cell of vital organs and cholesterol metabolism. These results agreed with results of some researchers (Moreira et al., 2011; Sixabela et al., 2011).
       
The hepatoprotective effects of S.platensis were reported by some researchers(Pardhasaradhi et al., 2003; Karadeniz et al., 2009). Pardhasaradhi et al., (2003) determined the protective effect of S.platensis on induced rat with hepatic degeneration and fibrosis. Similary in our study, it was reported that daily 1500mg/kg oral dose of S.platensis has any toxic effect on hepatic tissue (Karadeniz et al., 2009). However, some researchers identified the negative effects of this natural additive in human health (Araújoet_al2003; Malhi and Gores, 2008). In these studies, it was suggested that more protein uptake due to S.platensis content causes gout or hepatic diseases. Although any negative effects were specified for S.platensis, there is not much information about the effects on systemic toxicity. In our study, no negative and unfavorable results were found when the histopathological staining of the liver was evaluated and there were no statisticaly differences among all groups.

In conclusion, we have demonstrated that weight gain, abdominal fat weight, metabolic adverse outcomes and serum cholesterol were associated with each other. Importantly, this correlation is connected with hepatoprotective effect and so life quality. It was suggested that in our study, parallel analyzes both in terms of metabolism and liver may contribute to the literature. Nevertheless, S. platensis have been known as an important source instead of medicine or a natural additive for centuries. The fact that supplementation with this natural additives may have a crucial role on the life quality. Nevertheless, it suggested that dietary requirement is based on optimal dose, and it is necessary to evaluate the aspect of S. platensis boosting the health status.