Cholesterol Degradation Effect Analyzed using Marine Cyanobacterial Species Spirulina subsalsa

In this study was designed to understand the mechanisms of cholesterol degradation by cyanobacterial species Spirulina subsalsa. We analyzed the degradation of cholesterol in albino mice using basal feed diet (control) and different concentration (1%, 2% and 4%) of cyanobacterial diet. Cholesterol was estimated from the control and cyanobacterial fed to the albino mice. Blood cholesterol level reduced 8.1, 24.3, 30.6 % in 1, 2 and 4% cyanobacterial diet respectively. Phycobiliproteins are the most abundant soluble protein complexes in cyanobacteria and phycocyanin have 75% in phycobiliprotein. We concluded, phycocyanin was major role for cholesterol degradation in albino mice. Cholesterol Degradation Effect Analyzed using Marine Cyanobacterial Species Spirulina subsalsa


Introduction
Cyanobacterium Spirulina is free-floating filamentous microalgae with spiral characteristics of its filaments with characteristic of photosynthetic capability [1,2]. Initially it was classified as plant kingdom because of plant pigments as well as photosynthesis ability. Later it was placed in bacteria kingdom based on new understanding on its genetics, physiology and biochemical properties [3]. Naturally it grows in high-salt alkaline water reservoirs in subtropical and tropical areas including America, Asian and Africa [3,4]. It is cultivated and commercialized worldwide due to its nutritional characteristics including high concentration of protein (~65%), vitamins and mineral salts [5,6]. Over 400 years ago, the Spirulina used as food by Mayas, Toltecs and Kanembu in Mexico during the Aztec civilization [7]. The Intergovernmental Institution for the use of Micro-algae Spirulina Against Malnutrition (IIMSAM) was launched in the middle 70's to promote as high nutritional food to fight against starvation and malnutrition in the world [8].
Cholesterol is a lipid, produced by the liver that is required for various functions, found in some foods. Cholesterol is parent compound of hormone, bile acid and vitamin D [9]. Cholesterol is circulated in the blood by transport protein, which is termed lipoproteins. Lowdensity lipoproteins (LDLs) carry cholesterol from the liver to various body cells, but if there is an excess beyond the cells' requirements, cholesterol can accumulate in the walls of arteries. LDL is therefore sometimes called "bad cholesterol". High-density lipoprotein (HDL), on the other hand, carries cholesterol away from cells to the liver, where it is broken down or treated as a waste product. This lipoprotein is referred to"good cholesterol". A normal or desirable cholesterol level is defined as less than 200 mg of cholesterol per deciliter of blood (mg/ dL). Blood cholesterol is considered to be borderline when it is in the range of 200 to 239 mg/dL. Elevated cholesterol level is 240 mg/dL or above is considered to be hypercholesterolemia. Hypercholesterolemia indicates that, HDL is decreased and LDL is increased [10]. Cholesterol circulates in the blood stream. It is an essential molecule for the human body. Cholesterol is a molecule from which hormones and steroids are made. It is also used maintain nerve cells. Between 75 and 80% of the cholesterol that circulates in a person's bloodstream is made in that person's liver. The remainder is acquired from outside source. Cholesterol is found in animal sources of food, not found in plants.
Excess of cholesterol is risk factor, cause of death worldwide, each year more than 17 million people or 30% of all death worldwide and 25 million death are expected in 2020 [11]. The presence of high levels of cholesterol in the blood called Hypercholesterolemia. The Spirulina used in the treatment of many diseases, including Cholesterol degradation [12,13], as well as to reduce body weight in humans [14]. The cholesterol lowering activity of Spirulina was first reported in albino rat [15], followed by in mice [16]. Dietary proteins were shown to influence serum cholesterol concentrations in many studies [17][18][19][20][21][22][23].
Reports pointed Spirulina an important breakthrough in trickling the problem of hypercholesterolemia and in animal experiments, it has proved that Spirulina had positive effects in reducing serum total cholesterol and elevating high density lipoprotein (HDL) cholesterol level [15,16,[24][25][26]. It contains protein, lipid, chlorophyll, carotenoids, vitamins, minerals, and phycobiliproteins such as C-phycocyanin. Phycocyanin which is anti-oxidizing and anti-inflammatory [27] and gamma linolenic acid (GLA) used for its cholesterol reduction effect [28]. The mechanism of degradation of cholesterol by cyanobacteria has yet to be identified. No researchers have identified any algal protein that exerts cholesterol effect. Therefore, this present study has been undertaken to find the effect of marine cyanobacteria, on body weight as well as blood cholesterol level of albino mice.

Feed for a mammalian system
The diets were prepared by supplementing a basal feed with Spirulina subsalsa dry powered. The dried material of cyanobacteria power was incorporated at 1, 2 and 4% of basal feed. This basal (control) feed was prepared using wheat flour (738 g), Bengal gram (122 g),

Mammalian feeding experiment
The albino mice used as a testing organisms of mammalian feeding experiment. Male mice of similar size were randomly divided into four groups, each group contain two mice. They were caged individually, and supplied with food and water ad lib. One group of mice was fed on basal diet and used as control. The other three groups were fed on diets of different concentrations (1, 2, 4%) of cyanobacterium. The diets were given twice per day at a rate of 20% of body weight of mice. The food pellets were kept on the feeding bars of the animal cage ( Figure. 2).

Determination of blood cholesterol
The blood samples were taken only one time from the albino mice at the end of the experiment (after 10 days) and cholesterol were estimated [29]. The blood sample was centrifuged and the serum was collected. In a test tube 0.1 ml of serum was taken, 5 ml of ferric chloride (0.05% in acetic acid) and 3 ml of concentrated sulphuric acid were added. After 20 minutes of incubation, absorbance of the reaction mixture was read at 560 nm against the reagent blank in spectrophotometer. The amount of cholesterol present in the sample was determined by using the standard cholesterol. The results expressed as microgram of cholesterol present in hundred micro litter of blood serum.

Effect of cyanobacterial diets on blood cholesterol of albino mice
The cyanobacteria Spirulina subsalsa was used as supplemented feed to albino mice for analysis the cholesterol reduced activity (hypercholestrolemic). The results shows, cholesterol level was gradually reduced when increase the percentage of cyanobacteria used as supplementary feed. In control diet without cyanobacteria, albino mice blood cholesterol level was assumed as 100% level. In the 1% cyanobacterial diet fed albino mice, the cholesterol level was reduced at 8.1% (Figure 3). Blood cholesterol level was reduced at 24.3% in the 2% cyanobacterial diet fed albino mice (Figure 4). The cholesterol level was reduced at 30.6% in the 4% cyanobacterial diet fed albino mice ( Figure 5). The blood cholesterol are statically significant at 5% level.
Similar results were reported by other authors. Cholesterol level found be reduced in rabbit by cyanobacterial diet at the rate of 63% level [30], at the rate of 57% level [31] and 69.5% level [23]. The addition of Spirulina in 5, 10 and 15% in mice diets caused significant inhibition in total cholesterol [32]. Four grams supplementation of  Spirulina recorded the maximum reduction of serum Cholesterol [13]. In the present study 2% Spirulina fed mice had more reduction of serum cholesterol.
Phycoyanins is the most abundant proteins found in cyanobacterium species of Spirulina. Phycocyanin was characterized as a major phycobiliprotein with a molecular weight of 44 kDa [33]. The relation between the serum cholesterol-lowering activity of dietary protein and the amino acid contents of protein was reported previously [34][35][36]. Phycocyanin contain a higher level of cystine than control diet, the differences in amino acid content may relate to the differences in serum cholesterol concentrations. Phycocyanin has antioxidant action [27,37] and confers cholesterol degradation effect against liver damage induced by CCl 4 [38]. In Spirulina antioxidant compounds like Phycocyanin, Poly-unsaturated fatty acid may be the reason to decrease of serum cholesterol level. The phycocyanin and proteic pigments extracted from Spirulina, caused hypocholesterolemic activity in rats [21]. Poly-unsaturated fatty acids demonstrated by many authors [22,39,40] compounds respect therapeutic properties such as its ability to decrease blood cholesterol levels [41,42].
This cholesterol degradation may be elicited by some factors(s) such as an enzyme i.e., anticholesterase or unsaponisiable sterols [43]. Thus it is clear that reduce the cholesterol of blood serum in albino mice was due to the diet of cyanobacterial species Spirulina subsalsa. Our conclusion with other authors suggestion is phycocyanin is major role to reduce the blood serum cholesterol of albino mice.