Andriy Synytsya

Fourier transform Raman and infrared spectroscopy of pectins

Abstract The FT-Raman and FT-IR spectra of polygalacturonic (pectic) acid, potassium pectate and its derivatives, as well as commercial citrus and sugar beet pectins were measured and interpreted. Methyl and acetyl esters of potassium pectate derivatives have several characteristic Raman and IR bands that allow both this groups to be distinguished. The very intense Raman band at 857 cm −1 is sensitive to the state of uronic carboxyls and to O -acetylation. The wavenumber of this band decreases with methylation (min. 850 cm −1 ) and increases with acetylation (max. 862 cm −1 ). The acetylation of potassium pectate, as well as its acetylation together with methylation, causes drastic changes in the Raman spectra in the region below 700 cm −1 . Sugar beet pectin, but not citrus pectin, showed Raman bands at 1633 and 1602 cm −1 and IR band at 1518 cm −1 . All these bands rise from feruloyl groups and can be used for identification of pectins containing feruloyl groups.

Structural diversity of fungal glucans

Fungal glucans represent various structurally different d-glucose polymers with a large diversity of molecular mass and configuration. According to glucose anomeric structure, it is possible to distinguish α-D-glucans, β-D-glucans and mixed α,β-D-glucans. Further discrimination could be made on the basis of glycosidic bond position in a pyranoid ring, distribution of specific glycosidic bonds along a chain, branching and molecular mass. Fungal glucans can be chemically modified to obtain various derivatives of potential industrial or medicinal importance. NMR spectroscopy is a powerful tool in structural analysis of fungal glucans. Together with chemolytic methods like methylation analysis and periodate oxidation, NMR is able to determine exact structure of these polysaccharides. Fungal glucans or their derivatives exert various biological activities, which are usually linked to structure, molecular mass and substitution degree.

Purification, characterization and immunostimulating activity of water-soluble polysaccharide isolated from Capsosiphon fulvescens.

A water-soluble polysaccharide (SPS-CF) was isolated and purified from Korean Capsosiphon fulvescens by dilute acid extraction, ethanol precipitation, and DEAE-cellulose ion exchange chromatography. The purified SPS-CF was shown to be a glucuronogalactomannan with a molecular mass of 385 kDa and the monosaccharide composition of the SPS-CF was determined to be mannose (55.4% in mole percentage), galactose (25.3%), glucuronic acid (16.3%), and arabinose (0.8%). Fourier-transform infrared and elemental analysis indicated that the purified SPS-CF is a sulfated polysaccharide containing significant amount of sulfate esters (5.7% in mass). Enzyme Linked Immunosorbent Assay showed that the SPS-CF significantly stimulates the release of the pro-inflammatory cytokines, TNF-alpha and IL-6, in a dose-dependent manner. RT-PCR analysis demonstrated that the SPS-CF also induced a more than two-fold increase in the expression of iNOS and COX-2, responsible for the induction of NO and PGE2, respectively, at 5 microg/ml in RAW264.7 murine macrophages. These results suggest that the sulfated SPS-CF isolated from C. fulvescens has potent immunostimulating activity.

Purification, characterization and immunomodulating activity of a pectic polysaccharide isolated from Korean mulberry fruit Oddi (Morus alba L.).

A water-soluble polysaccharide (JS-MP-1) was isolated and purified from the Korean mulberry fruits Oddi (Morus alba L.) by crushing the fresh fruits then performing ethanol precipitation and DEAE-cellulose ion exchange chromatography. The neutral monosaccharide composition of the purified JS-MP-1 was determined to be composed mainly of galactose (37.6%, in mole percent), arabinose (36.3%), and rhamnose (18.4%), while other major sugars such as glucose, xylose, mannose, and fucose were present as minor components. HPLC analysis revealed that JS-MP-1 contains both galacturonic acid (GalA) and glucuronic acid (GlcA) at approximately 4:1 in mole percent. Monosaccharide composition, Fourier-transform infrared (FTIR) analysis, biochemical analysis, and elemental analysis suggested that JS-MP-1 is an acidic heteropolysaccharide, most likely a rhamnoarabinogalacturonan type plant pectic polysaccharide, with an apparent molecular mass of 1600 kDa containing no, or if any, negligible level of sulfate esters and proteins. Enzyme-Linked Immunosorbent Assay and RT-PCR analysis demonstrated that JS-MP-1 significantly stimulates murine RAW264.7 macrophage cells to release chemokines (RANTES and MIP-1α) and proinflammatory cytokines (TNF-α and IL-6) and to induce the expression of iNOS and COX-2, which are responsible for the production of NO and prostaglandin PGE2, respectively. These results suggest that the mulberry fruit-derived polysaccharide JS-MP-1 can act as a potent immunomodulator, and these observations may support the applicability of this polysaccharide as an immunotherapeutic adjuvant or the water extracts of the mulberry fruit as a beneficial health food.

Structural analysis of glucans

Glucans are most widespread polysaccharides in the nature. There is a large diversity in their molecular weight and configuration depending on the original source. According to the anomeric structure of glucose units it is possible to distinguish linear and branched α-, β- as well as mixed α,β-glucans with various glycoside bond positions and molecular masses. Isolation of glucans from raw sources needs removal of ballast compounds including proteins, lipids, polyphenols and other polysaccharides. Purity control of glucan fractions is necessary to evaluate the isolation and purification steps; more rigorous structural analyses of purified polysaccharides are required to clarify their structure. A set of spectroscopic, chemical and separation methods are used for this purpose. Among them, NMR spectroscopy is known as a powerful tool in structural analysis of glucans both in solution and in solid state. Along with chemolytic methods [methylation analysis (MA), periodate oxidation, partial chemical or enzymatic hydrolysis, etc.], correlation NMR experiments are able to determine the exact structure of tested polysaccharides. Vibration spectroscopic methods (FTIR, Raman) are sensitive to anomeric structure of glucans and can be used for purity control as well. Molecular weight distribution, homogeneity and branching of glucans can be estimated by size-exclusion chromatography (SEC), laser light scattering (LLS) and viscometry.

Anticoagulating activities of low-molecular weight fuco-oligosaccharides prepared by enzymatic digestion of fucoidan from the sporophyll of Korean Undaria pinnatifida.

In spite of their potential as biologically active compounds, the high molecular mass and viscous natures of fucoidans have hampered their applications especially as a therapeutic agent. Herein the fucoidan-degrading enzyme activities were partially purified from the cultured cells of Sphingomonas paucimobilis PF-1 mainly by ammonium sulfate precipitation. This enzyme preparation degraded fucoidans from the Korean Undaria pinnatifida sporophyll into several low-molecular weight fuco-oligosaccharides (LMFOs) with less than 3,749 Da. The FTIR spectra of intact fucoidan and mixture of LMFOs (1,389∼3,749 Da) showed no significant structural difference except for about 10% reduced level of sulfate esters in LMFOs. The LMFOs have exerted strong anticoagulating activities at which the activated partial thromboplastin time (APTT) and thrombin time (TT) were significantly prolonged, although 3∼20 times weaker activities were observed than those of intact fucoidan. In addition, unlike intact fucoidan, LMFOs did not affect significantly to the prothrombin time (PT). These results suggest that the partially purified fucoidan-degrading enzyme preparation is valuable for the production of fuco-oligosaccharides having anticoagulating activities, and that the molecular weight and/or sulfate content of the fucoidan from the Korean Undaria pinnatifida sporophyll could be important factors for its anticoagulating activity.

pH-Controlled Self-Assembling of meso-Tetrakis(4-sulfonatophenyl)porphyrin−Chitosan Complexes

Solid meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS(4))-chitosan supramolecular complexes were prepared by addition of porphyrin to an aqueous solution of chitosan at pH values. The precipitates obtained were assigned as 1 (pH 6.8) and 2 (pH 2.5) and characterized by spectroscopic, thermal, and microscopic methods. Spectroscopic investigation confirmed the presence of TPPS(4) and chitosan in both products and that the porphyrin is highly self-associated. H-type (stacked) of TPPS(4) aggregation was proposed for 1 and J-type (tilted) for 2. Thermal analysis revealed different pyrolysis routes of the complexes depending on their structural diversity. Light microscopic analysis indicated fibrous and lamellar microstructures, respectively, for 1 and 2. SEM and AFM analysis showed that both complexes consist of compact nanostructures; their size and interconnection is different for 1 and 2. Based on structural inferences, self-assembling hierarchy models were proposed for both of the TPPS(4)-chitosan supramolecular complexes.

Cell Wall Polysaccharides of Marine Algae

Marine algae are interesting as a plentiful source of many bioactive compounds, including polysaccharides, which represent various structurally different polymers of high diversity in monosaccharide composition, absolute and anomeric configuration, glycosidic linkages, molecular mass, and the presence and distribution of various functional groups. These polysaccharides are used by algae as cell wall structural components or food reserve. Algal polysaccharides are highly indicative for main algal taxa. Sulfated galactans of periodical unit/linkage sequence (agars, carrageenans) are typical for red algae, alginates, and fucoidans for brown algae; sulfated glucuronoxylorhamnans (ulvans) and other sulphated glycans for green algae. Algal cell wall polysaccharides are extracted from the raw material and further purified by preparative chromatography and/or chemical treatment. Many algal polysaccharides are assigned as phycocolloids due to their good solubility in water and their ability to create colloid systems, including gels and films. They also demonstrate various biological activities (immunomodulation, antitumor, anticoagulant, antiviral, and many other activities), which are prerequisites of pharmaceutical and medicinal applications. Finally, initially inactive or weakly active natural algal polysaccharides can be partially degraded or structurally modified to be fitted to various medicinal applications.

Structural analysis and anti-obesity effect of a pectic polysaccharide isolated from Korean mulberry fruit Oddi (Morus alba L.)

A water-soluble polysaccharide JS-MP-1 was isolated from Korean mulberry fruits Oddi (Morus alba L.). Sugar linkage analysis and NMR data confirmed that it is a rhamnogalacturonan type I (RG I) polymer carrying arabinan and arabinogalactan (AG II) side chains. JS-MP-1 reduced dose-dependently the viability of 3T3-L1 pre-adipocyte cells, significantly stimulated the cleavage of caspases 9 and 3 and poly (ADP-ribose) polymerase (PARP) and decreased the ratio of Bcl-2 to Bax expression level that led to mitochondrial dysfunction and apoptosis in pre-adipocyte cells. The apoptotic death was mediated by stimulation of MAPKs (ERK and p38) signalling pathway. These results suggest that JS-MP-1 is able to reduce the number of fat cells and the mass of adipose tissue via inhibition of pre-adipocyte proliferation and thus JS-MP-1 itself or a crude aqueous Oddi extract containing this polysaccharide can be used as functional ingredient of health-beneficial food supplements for the treatment or prevention of obesity disorders.

Modification of chitosan-methylcellulose composite films with meso-tetrakis(4-sulfonatophenyl)porphyrin.

Polysaccharide films containing chitosan, methylcellulose, and a mixture of these polysaccharides in various ratios were prepared and modified with meso-tetrakis(4-sulfonatophenyl)porphyrin in an aqueous medium at pH 7. The modified films were compared with the initial films using spectroscopic methods and microscopic imaging. Electronic (UV-vis absorption, electronic circular dichroism (ECD)) and vibrational (FTIR and Raman) spectra showed that the porphyrin macrocycles had a strong affinity toward chitosan and did not interact with the methylcellulose. The total porphyrin uptake depended on the chitosan: methylcellulose ratio and pure methylcellulose films did not retain porphyrin macrocycles. ECD measurements detected the presence of optically active porphyrin species bound to the films. SEM and AFM images confirmed that the porphyrin macrocycles caused structural changes on the film surface and within the film layer.