الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Diabetes mellitus is a disease characterized by increased glucose level in blood due to insulin-resistance, (high glucose concentration lead to increases the oxidative stress. Peripheral and adipose tissues protect themselves from the damaging effects of oxidative stress producing resistance to the action of insulin by preventing the penetration of glucose into the cells), which represents the problem to type 2 diabetes and/or an impaired insulin secretion. Patients with type 2 diabetes taking oral hypoglycemic agents or insulin, which it possess various side effects on human health.
The aim of this study was to evaluate the effect of Spirulina platensis, its water and ethanolic extracts and bioactive compounds phycocyanin, phycocyanopeptide and phycocyanobilin which isolated from Spirulina platensis on diabetic rats. In addition to study the potential application of the most effective Spirulina platensis fractions as source of natural antioxidant and antidiabetic agents can be used as supplement to hypogylcemic drugs to decrease the side effects of these drugs
This study included two objectives:
First: In vitro experiment: including determination of chemical compostion of Spirulina platensis and its ethanolic and aqueous extracts, and seperation and identification of phenolic compounds in Spirulina platensis and its ethanolic and aqueous extracts by HPLC. Determination of antioxidant activity of Spirulina platensis and its two different extracts (ethanolic and aqueous). In addition to, extract and purify phycocyanin (PC) from Spirulina platensis. The process of extraction and purification of PC mainly included freezing and thawing, sonication, centrifugation, precipitation using ammonium sulfate, dialysis and gel filtration chromatography, then Breaking thioether bond and separation of two parts of phycocyanin (phycocyanobilin and phycocyanopeptide). In addition to, determination of antioxidant activities of phycocyanin, phycocyanobilin and phycocyanopeptide, and determination of selenium and chromium contents in phycocyanin, phycocyanobilin, phycocyanopeptide.
Second: In vivo experiment: was designed to biochemically and histologically evaluate of hypoglycemic and hypolipidemic activity of Spirulina platensis, its water and ethanolic extracts, phycocyanin, phycocyanopeptide and phycocyanobilin on male diabetic rats. The rats were equally divided into nine groups designated as normal control, diabetic control, diabetic + glibenclamide drug (600 µg/kg body weight), diabetic + Spirulina platensis biomass suspension (50 mg/mL/kg body weight), diabetic + ethanolic extract of Spirulina platensis (50 mg/kg body weight), diabetic + water extract of Spirulina platensis (50 mg/kg body weight), diabetic + phycocyanin (50 mg/kg body weight), diabetic +phycocyanopeptide (49 mg/kg body weight) and diabetic+ phycocyanobilin (982 µg/kg body weight). After 30 days of experiment, rats were fasted overnight and then blood samples were collected and centrifuged at 3000 rpm for 15 minutes at 4°C to separate the serum for biochemical analysis like fasting serum glucose, insulin, α-amylase , insulin resistance (HOMA-IR), β-cells function (HOMA-β), total cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, liver functions, kidney finctions, oxidative stress marker (MDA) and pancreatic tissue samples were carefully dissected out for histological studies.
The main results obtained from this study can be summarized follows:
( I ) In vitro experiment:
1- phycocyanin content in dried with Spirulina by different methods.
The results show that spray drying is the best method to dry Spirulina alga, which give the highest level of phycocyanin, and don’t cause denaturation of phycocyanin compared to other methods (sun, oven and drum drying methods).
2- Chemical composition of Spirulina platensis alga and its water and ethanolic extracts.
The results revealed that ethanolic extract of Spirulina platensis contained the highest total phenols and flavonoids content, and the lowest carbohydrate content, while water extract of Spirulina platensis showed the highest content of protein and phycocyanin, and the lowest content of total phenols and flavonoids. Also, the data showed that ethanolic extract of Spirulina platensis contained the highest carotenoids and chlorophyll-a, content while water extract of Spirulina platensis showed the lowest content of carotenoids and chlorophyll-a.
3- Identification and quantification of phenolic compounds of Spirulina platensis alga, the aqueous and ethanolic extracts of the alga by HPLC.
Ethanolic and aqueous extracts of Spirulina platensis were subjected to HPLC analysis to investigate the type of phenolic compounds responsible for their antioxidant and antidiabetic activity. Results showed that E-vanillic acid was identified as the major compound in ethanolic extract (18.20 %) and pyrogallol (12.33%) in aqueous extract. the major compound of flavonoid was hespirdin in ethanolic (1.639%), and aqueous (3.517%) extracts .
4- Antioxidant activity of Spirulina platensis alga and aqueous and ethanolic extracts of the alga by DPPH free radical scavenging assay.
The results showed that ethanolic extract of Spirulina platensis significantly exhibited the strongest antioxidant activity measured by DPPH assay. On the other hand aqueous extract had significantly low antioxidant activity compared to ethanolic extract.
5- Isolation and purification of phycocyanin from Spirulina platensis.
Data showed that ultrasonic homogenizer method is better than freezing and thawing method because it extracted the highest amount of phycocyanin. Purification using ammonium sulphate saturation at 40 % saturation showed that the highest phycocyanin content (0.431 mg/mL). The other fractions (20, 60 and 80 % ammonium sulphate saturation) gave relatively low phycocyanin contents. Fractionation of the phycocyanin produced by 40% saturation using gel filtration showed that number 10 and 11 which obtained from gel filtration chromatography has the highest phycocyanin content and purity ratio.
6- Separation of two parts of phycocyanin (phycocyanobilin and phycocyanopeptide).
The data showed that conformation of phycocyanin structure and antioxidant activity were not affected when cleavage thioester bond in phycocyanin via addition HCl.
7- Selenium and chromium content in phycocyanin, phycocyanope- ptide and phycocyanobilin.
The results showd that both selenium and chromium were bonded with phycocyanopeptide and phycocyanobilin. Selenium and chromium contents were higher in phycocyanopeptide (0.13 and 2.39 ppm) compared to phycocyanobilin (0.04 and 0.15 ppm), respectively.
( II ) In vivo experiment :-
(A) Biochemical analysis:
The obtained data could be summarized in the main following points:
1- Fasting serum glucose, insulin and α-amylase:
The results of biological experiment showed that administration of diabetic rats (induced by STZ) with Spirulina platensis biomass, its ethanolic and water extracts led to a significant decrease in fasting serum glucose level and activity of α-amylase compared with diabetic control. Administration of diabetic rats with Spirulina platensis suspension and its ethanolic and water extracts led to a significant increase in insulin level. Our results illustrated that the levels of fasting glucose and activity of α-amylase in serum diabetic rats treated with phycocyanin, phycocyanopeptide and phycocyanobilin were lower than rats treated with glibenclamide reference drug. The data also, showed that administration of diabetic rats with phycocyanopeptide led to a significant increase in insulin level compared to diabetic rats treated with phycocyanobilin. The maximum value of insulin level was recorded in diabetic rats treated with phycocyanopeptide followed by phycocyanin and glibenclamide drug.
2- β-cell function (HOMA-β score) and insulin resistance (HOMA-IR score):
Administration of diabetic rats with Spirulina platensis, its ethanolic and water extracts led to a significant decrease in insulin resistance (HOMA-IR score) and a significant increase in β-cell function (HOMA-β score) compared with diabetic control. Administration of rats with phycocyanin, phycocyanopeptide and phycocyanobilin led to a significant decrease in insulin resistance (HOMA-IR score) compared with diabetic control. The results showed that phycocyanin, phycocyanobilin and phycocyanopeptide treatments led to higher values of β-cell function (HOMA-β score) than glibenclamide drug.
3- Lipid profile (total cholesterol, triglycerides, HDL-cholesterol and LDL- cholesterol).
The results indicated that diabtic rats orally treated with Spirulina platensis, its ethanolic and aqueous extracts, phycocynin, phycocyanopeptide and phycocyanobilin led to a significant decrease in lipid profile levels compared with diabetic control. Adminstration diabetic rats with Spirulina platensis, its ethanolic and aqueous extracts, phycocynin, phycocyanopeptide and phycocyanobilin led to a significant decrease in triglycerides, total cholesterol, and LDL-cholesterol level compared with diabetic control. While, adminstration of diabetic rats with Spirulina platensis, its ethanolic and water extracts, phycocynin, phycocyanopeptide and phycocyanobilin led to a significant increase in HDL-cholesterol level compared with diabetic control.
4- Kidney functions (serum urea and creatinine levels).
Administration of diabetic rats with Spirulina platensis, its aqueous and ethanolic extracts, phycocynin, phycocyanopeptide and phycocyanobilin led to a significant decrease in serum urea and creatinine levels compared with diabetic control.
5- Liver functions (AST and ALT activities).
The results illustrated that orally administration of diabtic rats with Spirulina platensis, its aqueous and ethanolic extracts led to a significant decrease in the activitiy of AST and ALT enzymes compared with diabetic control. Orally administration of diabetic rats with phycocynin, phycocyanopeptide and phycocyanobilin led also to a significant decrease in serum AST and ALT activities compared with diabetic control.
6- Oxidative cell biomarker (lipid peroxidation).
In lipid peroxidation, data of serum MDA level showed that the groups which treated with with Spirulina platensis, its water and ethanolic extracts phycocynin, phycocyanopeptide and phycocyanobilin led to a significant decrease in serum MDA level (oxidative stress marker) compared with diabetic control. The reduction of MDA level in these treatments were higher than the reduction in diabetic rats treated with glibenclamide drug.
(B) Histopathological Results:
The obtained data could be summarized in the main following points:
Microscopical examinations of pancrease tissues of rats treated with ethanolic extract, phycocyanin, phycocyanobilin and phycocyanopeptide showed a slight improvement in pancreatic islets and an obvious regeneration in pancreatic cells with respects to diabetic rats.
In conclusion, Spirulina platensis, aqueous and ethanolic extracts, phycocyanin, phycocyanobilin and phycocyanopeptide acts as hypoglycemic, restoring relatively insulin level near the normal level, decrease blood glucose and α-amylase levels near to normal, and acts as hypolipidemic agent. The efficiency of Spirulina platensis, its extracts and the different component extracted from Spirulina platensis such as phycocyanin, phycocyanobilin and phycocyanopeptide differ relatively in their activities toward hypoglycemic actions.
The activities of Spirulina platensis and other component are due different factors:
1- Antioxidant compounds including phenolic compounds and flavonoids, phycocyanin, phycocyanopeptide (contaning selenium and (SH) group of cysteine residue have antioxidant activities) and phycocyanobilin (contaning selenium and open tetrapirol chain (chromophore)), which let to a remarkable reduction in insulin resistance and oxidative stress marker (MDA), and protect β-cells against apoptotic cell death by attenuating oxidative stress.
2- Chromium-binding phycocyanopeptide which act as an activator of insulin receptors.
3- Bioactive peptide can be released during gastrointestinal digestion of food proteins, and which possess antioxidant properties.
4- Essential amino acids, vitamins, especially vitamin B12, minerals and carotenoids.