Preparation Of Series Alginate Oligosaccharides And The Study On Their Antioxidant-related Bioactivities (Protection Against Ethanol-induced Liver Injury And Prevention Of Parkinson’s Disease)

In recent years, with the rise of study on marine drugs, marine polysaccharides and oligosaccharides have become the hot research topics because of their unique chemical structures and excellent biological activities. Algae have been considered as the very important marine resources.Since it contains substantial polysaccharides and oligosaccharides with diverse biological activities. Alginate is a polyanionic polysaccharide derived from brown algae, which has been widely applied and studied in many research areas, including chemical industry, food engineering and medicinal technology. Furthermore, alginate oligosaccharides produced by a variety of degradation methods exhibit diverse pharmacological activities. Thusthe study on alginate oligosaccharides has attracted lots of attention from the academia recently. Since the worsening environmental pollution, the stress of fast-paced life, unhealthy lifestyles and lacking of nutrients, the oxidative stress has been found in the human body. Accumulating evidence strongly suggests that such stress play an important role in the development of cardiovascular disorder, autoimmune diseases, alcoholic liver disease, neurodegenerative disease and carcinogenesis. Moreover, many clinical and basic researches demonstrated that some compounds with antioxidant activities can improve the therapeutic effects on diseases related to oxidative stress. It has been reported that alginate oligosaccharide, as a natural, non-toxic, resource-rich oligosaccharide, exhibits good antioxidant activity. Therefore, it may have a strong capability to prevent the oxidative stress induced human disease. In addition, China is the largest country producing alginate products and providing over 70% of the total production in the world. Thus, whether from the economic perspective or medicinal value, it is important and worthwhile to study on the pharmaceutical activities of alginate oligosaccharides.Based on these reasons, the objective of this paper was to prepare series of alginate oligosaccharides using different degradation methods, as well as to systematicly study their antioxidant activities and the related pharmaceutical activities.1. Preparation and characterization of series alginate oligosaccharides. We obtained series alginate oligosaccharides with various Mw (weight average molecular weight) by acid degradation, oxidative degradation, enzymatic degradation combined with ultrafiltration and microwave-assistant degradation method. Importantly, it’s the first time to report that hydrolysis alginate without any additional acid by microwave-assistant degradation method. The fractions and oligosaccharide monomer were further characterized by UV, IR, ESI-MS,1H-NMR,13C-NMR, and 2D-NMR spectroscopy techniques. The results indicated that the oligosaccharides produced by different degradation methods have different chemical structures. The oligosaccharides produced by acid hydrolysis and microwave-assistant degradation method contained saturated non-reducing end residues. However, the oligosaccharides by enzymatic hydrolysis combined with ultrafiltration method contained unsaturated non-reducing end residues with a double bond in C4-C5. Besides, the oligosaccharides with carboxyl group on the reducing end C1 can be produced by hydrogen peroxide. Moreover, two series of alginate oligosaccharides (22 fractions) with different weight average molecular weights and structures (series mannuronic acid and guluronic acid series oligosaccharides) were obtained. In addition,15 of saturated alginate oligosaccharide monomers were also prepared. It provided the material basis for the biological activity study and structure-activity relationship of alginate oligosaccharides. Furthermore, compared with the traditional acid degradation, it is efficient and clean to prepare alginate oligosaccharides with low degree by microwave hydrolysis method. It was also a simple post-treatment (without desalination) and high yield (over 80%) method that could be applied in preparation of alginate oligosaccharide standard.2. Systematical comparisition of the antioxidant activities of series alginate oligosaccharides. We evaluated their antioxidant activities by the scavenging activities of DPPH, superoxide radicals and the chelating ability of ferrous ion. The results indicated that the Mw (degree of polymerization, DP), monosaccharide composition and structural differences of the terminal end of the sugar ring have certain effects on antioxidant activity of alginate oligosaccharides:①The oligosaccharides with lower Mw or smaller DP showed stronger antioxidant activitie.②The terminal end structure of the sugar ring affect the antioxidant activity of the oligosaccharides Saturated alginate oligosaccharides exhibited stronger DPPH and superoxide radical scavenging activities, and the changes in the structure of the terminal end of sugar ring may affect their hydrogen-donating poperties; The oligosaccharides containing carboxyl group at reducing end C1 showed stronger Fe2+ chelating ability, since it has two carboxyl groups;③Monosaccharide composition (M/G) of alginate oligosaccharides also affected the antioxidant activities. The superoxide radical scavenging activity of saturated M oligosaccharide was stronger than the saturated G oligosaccharide, However, the G oligosaccharide containg carboxyl group at reducing end C1 exhibited better ferrous ions chelating ability than M oligosaccharide, since it can form the stable "egg box" structure with metal ions.④Considering the above factors, the alginate oligosaccharides with Mw< 3kDa have good antioxidant activities. Thus, compared to Vc, resveratrol, α-lipoic acid, EDTA and other antioxidants, alginate oligosaccharides and monomers can be used more widely in various fields, because of its direct radical scavenging activity and indiect antioxidant activity.3. Study on the effect of series alginate oligosaccharides in mice acute alcohol-induced liver injury model. The results indicated that alginate oligosaccharides dramatically reduced the liver index, serum and hepatic TG levels, and attenuated the activity of ALT/AST in serum. The effect of alginate oligosaccharides increased with the decreasing Mw, M-3K and G-3K were optimal fractions with comparative activity, which indicated that the effect of alginate oligosaccharides on alcoholic liver injury was mainly related to the Mw of oligosaccharides.Further studies on the protective mechanism of alginate oligosaccharides (M-3K and G-3K) in alcohol liver injury showed that:alginate oligosaccharides could promote the levels of non-enzymitic antioxidants and the activity of antioxidant enzymes and inhibit lipid peroxidation in vivo. It also downregulated the expression of CYP1A2, CYP2E1 and CYP3A in hepatic tissue MEOS system. Therefore, alginate oligosaccharides can protect the liver by reducing the oxidative stress induced by the ethanol metabolic process. The alginate oligosaccharides can improve the activities of ADH and ALDH in liver, accelerate the metabolism of ethanol and its metabolite acetaldehyde, which can reduce their liver toxicity. Furthermore, the oligosaccharides can inhibit the secretion of cytokine TNF-a, IL-6, and increase the level of adiponectin that considered as an anti-inflammatory factor in serum. Thus, alginate oligosaccharides could antagonize the apoptosis of liver cell induced by inflammation and protect the liver tissue. In addition, alginate oligosaccharides activated p-AMPK and PPAR-α and inhibited the expression of mSREBP-1, which can result in reduction of the fat synthesis and acceleration of the β-oxidation of fatty acids, thereby further inhibited the fat accumulation in liver and improved the liver steatosis.4. The Evaluation of the alginate oligosaccharide neuroprotective effects of alginate oligosaccharides were evaluated on Parkinson’s disease using in an MPPf induced cellular model of Parkinson’s disease. The results showed that the alginate oligosaccharides with low Mw exhibited higher stronger neuroprotective activity, especially among them; the M-3K and G-3K have showed strongest neuroprotective activity. Moreover, pentosaccharides of both mannuronic acid oligosaccharide and gulunuronic acid oligosaccharide (M5/G5) exhibited the dramatically high strong activity among the monomers, and also, their effects are better than M-3K and G-3K. M5/G5 inhibited accumulated accumulation of MPP1-induced ROS, increased the intracellular levels of GSH and ATP, which can prevented against the SK-N-MC cell injure by inhibiting the oxidative stress. The further study indicated that M5/G5 protect the cell by enhancing the activity of mitochondrial complex I, increasing the expression of mitochondrial complex I,DJ-1 and PINK1 to antagonize MPP+-induced mitochondrial dysfunction. M5/G5 also can increase the expression of p-AMPK, which suggested that the oligosaccharides could activate mitophagy to prevent mitochondrial dysfunction.In summary, alginate oligosaccharides not only have good antioxidant activities in chemical molecular level, but also in the cellular and animal levels. Since its strong antioxidant activity, the oligosaccharide exhibited a preventive effect on oxidative stress-related diseases. Moreover, the strong preventive effects on oxidative stress might also be attributed by oligosaccharides-induced multi-routes and multi-targets. Thus, alginate oligosaccharide can be considered as a potential candidate for the therapy of oxidative stress-related diseases, and will be widely used in various research areas and industry because of its substantial resources and biosafety.