Anti-Obesity Effect of Nostoc commune Ethanol Extract In Vitro and In Vivo.

Nostoc commune is an edible terrestrial blue-green alga. It has shown many beneficial effects on human health. This study aimed to investigate the phytochemical assay of N. commune ethanol extract (NEE) and its anti-obesity effects. The effect of a high-calorie diet on lipid accumulation in 3T3-L1 preadipocytes is investigated, and a Wistar rat model is used to demonstrate the anti-obesity effect of NEE and its mechanism. The results showed that the NEE has phytochemical compounds, such as total polyphenol, total flavonoids, and total terpenoids. NEE was also shown to suppress cell proliferation and lipid accumulation (26.9%) in 3T3-L1 preadipocytes. Furthermore, NEE reduced the body weight (13.5%), fat tissue weight (13.3%), and the serum FFA (19.4%), TG (14.2%), TC (11.8%), and LDL-C (16.4%) of rats. In histopathology, NEE was shown to diminish the size of adipocytes and hepatic lipid droplets. The NEE downregulated the mRNA expression of adipogenesis (PPAR-γ, SREBP-1c) and lipid lysis-related genes (ATGL, HSL) in epididymal adipose tissue. The NEE also upregulated the mRNA expression of ß-oxidation related genes (AMPK, CPT-1, PPAR-α) in the liver. Overall, this study suggests NEE has the potential to be developed as a functional food for anti-obesity.

Antioxidative and Antiglycative Properties of Mycosporine-Like Amino Acids-Containing Aqueous Extracts Derived from Edible Terrestrial Cyanobacteria.

The terrestrial filamentous cyanobacterium, Nostoc commune, has been used as a food source in many countries, especially countries in Asia. In this study, N. commune-derived aqueous extracts were evaluated with regard to their antioxidative and antiglycative properties. The antioxidative activity was significantly higher in N. commune colonies isolated from the field than in extracts from colonies cultured in the laboratory. The antioxidative compound content of extracts, including phenolic compounds and phycobiliproteins, was correlated with their antioxidative power. In addition, two mycosporine-like amino acids (MAAs), specifically detected in colonies isolated from the field, were purified. In addition to assessing their antioxidative properties, the antiglycative activity of these MAAs was also assessed. Their inhibitory effects on glycation-dependent protein cross-linking might contribute to the antiglycative power of the extract prepared from field colonies. Taken together, the results from this study revealed that N. commune may have beneficial properties for functional food applications, both by preventing oxidative stress and suppressing the formation of advanced glycation end-products.

Molecular α-relaxation process of exopolysaccharides extracted from Nostoc commune cyanobacteria.

Broadband dielectric spectroscopy was used to investigate the molecular α-relaxation of the exopolysaccharides (EPS) extracted from Nostoc commune cyanobacteria. The EPS were modified in different ways. EPS were carboxymethylated to obtain carboxymethyl-exopolysaccharides (CEPS). EPS and CEPS were doped with ammonium iodide and 1-butyl-3-methylimidazolium chloride. An α relaxation process was observed for all specimens. The temperature dependence of the relaxation times for pure and doped, EPS and CEPS polymers exhibited non-Arrhenius behavior. This relaxation process was associated with the glass transition of the complex heteropolysaccharides produced by the cyanobacteria. The molecular mobility at the glass transition, Tg, was affected by both the carboxymethylation treatment and the doping. The fragility index also decreased for the doped specimens, which may be attributed to an increase in the mobility of the polymer chains due to the plasticizing effect of the doping agents.

Kinetic modeling of the ultrasonic-assisted extraction of polysaccharide from Nostoc commune and physicochemical properties analysis.

Based on the theory of extraction and diffusion of Chinese herbal medicine, the dynamic model of ultrasonic-assisted extraction process of polysaccharide from Nostoc commune Vauch. was established according to the second law of Fick, and further verified at different solid-liquid ratio (1/40-1/80 g/mL), temperature (313.15-353.15 K), ultrasonic power (240-600 W) and extraction time (0-25 min), the dynamic parameters including rate constant and relative extraction rate were respectively analyzed. The rate constant (k) gradually increased with the increase of temperature at different solid-liquid ratio. The maximum concentration of polysaccharide (NCVP) from N. commune was obtained with an optimal extraction condition at solid-liquid ratio of 1:50, extraction temperature of 353.15 K, ultrasonic power of 540 W and extraction time of 25 min. NCVP, the non reducing sugar with typical infrared spectrum characteristics of polysaccharide, dissolves in water but not dissolved in ethanol, acetone and petroleum ether and displays a good stability and smooth surface. The results provide the basis for NCVP in depth theoretical study of polysaccharide extraction processing.

Migration inhibition of water stress proteins from Nostoc commune Vauch. via activation of autophagy in DLD-1 cells.

Water stress proteins (WSP1) from Nostoc commune Vauch. had been proven to selectively induce colon cancer cells apoptosis. In this study, the effect of WSP1 on migration of human colon cancer cells was investigated. It showed that WSP1 inhibited DLD-1 cell migration, but with an insignificant effect on normal human colon epithelial cells. The data further indicated that WSP1 activated autophagy through down regulation of PI3K/AKT/mTOR pathway. Meanwhile, ß­catenin was degraded by autophagy, which then restrained epithelial-mesenchymal transition (EMT) of DLD-1 cell and its migration was subsequently suppressed significantly. The same changes occurred in xenografted nude mice according to the obtained immunohistochemical results. Consistently, the application of autophagy inhibitor largely reversed the inhibited migration by WSP1 treatment. Taken together, WSP1 could suppress migration of DLD-1 cells by autophagy inhibited EMT. The results suggested that WSP1 possessed the potential as a selective therapeutic agent against metastatic colon cancer.

Characterization of extracellular matrix components from the desiccation-tolerant cyanobacterium Nostoc commune.

The terrestrial cyanobacterium Nostoc commune forms macroscopic colonies in its natural habitats, and these colonies consist of both cellular filaments and massive extracellular matrixes. In this study, the biochemical features of the extracellular matrix components were investigated. Naturally growing N. commune was tolerant to desiccation, and produced massive extracellular polysaccharides that contained both neutral sugars and glucuronic acid as constituent monosaccharides. The extracellular polysaccharide contents and desiccation tolerance were compared in laboratory culture strains of Nostoc species. The laboratory culture of N. commune strain KU002 was sensitive to desiccation and produced smaller amounts of extracellular polysaccharides, unlike the field-isolated naturally growing colonies. Nostoc punctiforme strain M-15, which is genetically closed to N. commune, was able to tolerate desiccation, although the other Nostoc strains were desiccation-sensitive. A laboratory culture strain of the aquatic cyanobacterium Nostoc sphaericum produced massive extracellular polysaccharides but was sensitive to desiccation, suggesting that extracellular matrix production is not enough to make this strain tolerant to desiccation. WspA (water stress protein) and SodF (superoxide dismutase) were found to be characteristic protein components of the extracellular matrix of N. commune. Because the WspA proteins were heterogeneous, the wspA genes were highly diverse among the different genotypes of N. commune, although the sodF gene was rather conservative. The heterogeneity of the WspA proteins suggests their complex roles in the environmental adaptation mechanism in N. commune.

Unusual reversible elastomeric gels from Nostoc commune.

Nostoc commune cyanobacteria grow in extreme conditions of desiccation and nutrient-poor soils. Their colonies form spherical gelatinous bodies are composed of a variety of polysaccharides that allow them to store water and nutrients. In this paper, we study this type of biological gel that shows characteristics of both chemical and physical gels. The structure of this gel was assessed by means of scanning electron microscopy, plate-plate rheometry, Fourier transform infrared spectroscopy and absorption/desorption tests. The storage modulus of this gel was found to be frequency independent, as is usual for chemical gels. The stress sweeps showed a reversible stress softening behaviour that was explained in terms of the physical nature of the interactions of this network. The high density of physical crosslinks probably allows this physical network to behave as a highly elastomeric chemical network, limiting the relaxation of individual chains. On the other hand, reversibility is associated with the physical nature of its bonds.

Migration Suppression of Small Cell Lung Cancer by Polysaccharides from Nostoc commune Vaucher.

Nostoc commune Vauch., classified into cyanobacteria, has been always well appreciated as a healthy food and medicine worldwide owing to its rich nutrition and potent bioactivities. Nevertheless, the inhibitory effect of polysaccharides from N. commune Vauch. (NVPS) against cancer cell progression and metastasis is still being unraveled. The results in this study showed that NVPS remarkably suppressed cell migration through blocking the epithelial-mesenchymal transition program in NCI-H446 and NCI-H1688 human small cell lung cancer cells. The inhibitory effects were attributed to the suppression of integrin ß1/FAK signaling through regulating cell-matrix adhesion. Furthermore, NVPS treatment could increase E-cadherin expression, but down-regulate N-cadherin, Vimentin, and MMP-9 expression, which resulted in the blockage of STAT3 nuclear translocation and JAK1 signaling. These findings suggest that NVPS may be a good candidate for development as a possible antitumor agent against small cell lung cancer.

Isolation and antitumor efficacy evaluation of a polysaccharide from Nostoc commune Vauch.

Nostoc commune Vauch. has been traditionally used as a healthy food and medicine for centuries especially in China. It has been demonstrated that the polysaccharides isolated from Nostoc commune Vauch. exhibit strong antimicrobial and antioxidant activities. However, little is known about their anticancer activities and the underlying mechanisms of action. Herein, we report the isolation of a polysaccharide from Nostoc commune Vauch. (NVPS), and its physicochemical properties were analyzed. In an attempt to demonstrate the potential application of NVPS in tumor chemotherapy, the in vitro antitumor activity was determined. NVPS significantly suppressed the growth and proliferation of MCF-7 and DLD1 cells. The molecular mechanism underlying this in vitro antitumor efficacy was elucidated, and the results indicated that NVPS simultaneously triggered intrinsic, extrinsic and endoplasmic reticulum stress (ERS)-mediated apoptotic signaling pathways. Collectively, these findings demonstrate that NVPS could be used as a novel promising source of natural antitumor agents.

Hypolipidemic Effect of a Blue-Green Alga (Nostoc commune) Is Attributed to Its Nonlipid Fraction by Decreasing Intestinal Cholesterol Absorption in C57BL/6J Mice.

We previously demonstrated that Nostoc commune var. sphaeroids Kützing (NO), a blue-green alga (BGA), exerts a hypolipidemic effect in vivo and its lipid extract regulates the expression of genes involved in cholesterol and lipid metabolism in vitro. The objective of this study was to investigate whether the hypolipidemic effect of NO is attributed to an algal lipid or a delipidated fraction in vivo compared with Spirulina platensis (SP). Male C57BL/6J mice were fed an AIN-93M diet containing 2.5% or 5% of BGA (w/w) or a lipid extract equivalent to 5% of BGA for 4 weeks to measure plasma and liver lipids, hepatic gene expression, intestinal cholesterol absorption, and fecal sterol excretion. Plasma total cholesterol (TC) was significantly lower in 2.5% and 5% NO-fed groups, while plasma triglyceride (TG) levels were decreased in the 5% NO group compared with controls. However, neither NO organic extract (NOE) nor SP-fed groups altered plasma lipids. Hepatic mRNA levels of sterol regulatory element-binding protein 2, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), carnitine palmitoyltransferase-1α, and acyl-CoA oxidase 1 were induced in 5% NO-fed mice, while there were no significant changes in hepatic lipogenic gene expression between groups. NO, but not NOE and SP groups, significantly decreased intestinal cholesterol absorption. When HepG2 cells and primary mouse hepatocytes were incubated with NOE and SP organic extract (SPE), there were marked decreases in protein levels of HMGR, low-density lipoprotein receptor, and fatty acid synthase. In conclusion, the nonlipid fraction of NO exerts TC and TG-lowering effects primarily by inhibiting intestinal cholesterol absorption and by increasing hepatic fatty acid oxidation, respectively.

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic cyanobacterium Nostoc commune.

A carbonic anhydrase (CA, EC 4.2.1.1) belonging to the γ-class has been cloned, purified and characterized from the Antarctic cyanobacterium Nostoc commune. The enzyme showed a good catalytic activity for the physiologic reaction (hydration of carbon dioxide to bicarbonate and a proton) with the following kinetic parameters, kcat of 9.5×10(5)s(-1) and kcat/KM of 8.3×10(7)M(-1)s(-1), being the γ-CA with the highest catalytic activity described so far. A range of aromatic/heterocyclic sulfonamides and one sulfamate were investigated as inhibitors of the new enzyme, denominated here NcoCA. The best NcoCA inhibitors were some sulfonylated sulfanilamide derivatives possessing elongated molecules, aminobenzolamide, acetazolamide, benzolamide, dorzolamide, brinzolamide and topiramate, which showed inhibition constants in the range of 40.3-92.3nM. As 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and γ-CAs are closely associated in carboxysomes of cyanobacteria for enhancing the affinity of RubisCO for CO2 and the efficiency of photosynthesis, investigation of this new enzyme and its affinity for modulators of its activity may bring new insights in these crucial processes.

Water stress proteins from Nostoc commune Vauch. exhibit anti-colon cancer activities in vitro and in vivo.

Nostoc commune has been traditionally used in China as a health food and medicine. The water stress proteins (WSP) of Nostoc commune are the major component of the extracellular matrix. This study purified and identified the water stress proteins (WSP1) from Nostoc commune Vauch., which could inhibit the proliferation of human colon cancer cell lines. The IC50 values of WSP1 against DLD1, HCT116, HT29, and SW480 cells were 0.19 ± 0.02, 0.21 ± 0.03, 0.39 ± 0.05, and 0.41 ± 0.01 µg/µL, respectively. Notably, it displayed very little effect on the normal human intestinal epithelial FHC cell line. The IC50 value of WSP1 against FHC cells was 0.67 ± 0.05 µg/µL. Moreover, the growth of DLD1 xenografted tumors in nude mice were significantly suppressed in the WSP1 treated group. Mechanistically, the cell-cycle analysis revealed that WSP1 induced growth inhibition by G1/S arrest. Meanwhile, Western blotting and immunohistochemistry assays showed WSP1 could activate caspase-8, -9, and -3, along with subsequent PARP cleavage. Furthermore, the pan-caspase inhibitor, z-VAD-FMK, partly reversed the effect caused by WSP1, confirming that WSP1 induced cell apoptosis through caspase-dependent pathway. Collectively, WSP1 has targeted inhibition for colon cancer proliferation both in vitro and in vivo and it is valuable for future exploitation and utilization as an antitumor agent.

A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt.

Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent.

Reduced scytonemin isolated from Nostoc commune suppresses LPS/IFNγ-induced NO production in murine macrophage RAW264 cells by inducing hemeoxygenase-1 expression via the Nrf2/ARE pathway.

Reduced scytonemin (R-scy) and scytonemin (Scy) isolated from Nostoc commune exhibit anti-tumor and ultraviolet-absorbing properties. In this study, we examined the effects of R-scy and Scy on the induction of nitric oxide (NO) production by lipopolysaccharide (LPS) and interferon-γ (IFNγ) in murine macrophage RAW264 cells. While both R-scy and Scy suppressed LPS/IFNγ-induced NO production, R-scy exhibited a stronger inhibitory effect compared with Scy. To further elucidate the mechanisms underlying the anti-inflammatory effects of R-scy, we examined the changes in the intracellular signaling cascade after LPS/IFNγ stimulation in cells. In addition to the attenuation of LPS/IFNγ-induced upregulation of the inducible isoform of NO synthase, R-scy decreased the activity of nuclear factor-κB, phosphatidylinositol 3-kinase (PI3K)/Akt, and mitogen-activated protein kinases (MAPKs) after LPS/IFNγ stimulation. R-scy treatment increased heme oxygenase-1 (HO-1) expression by increasing the intracellular levels of reactive oxygen species and thereby activating nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant response element signaling. The induction of HO-1 by R-scy was inhibited by pretreatment with an antioxidant, N-acetyl-cysteine (NAC), as well as SB203580 and LY294002, inhibitors for p38 MAPK and PI3K/Akt, respectively. Our findings suggest that the anti-inflammatory effects of R-scy could involve both the ROS/PI3K/Akt and the p38 MAPK/Nrf2 signaling pathways.

Antimicrobial and anti-inflammatory properties of nostocionone isolated from Nostoc commune Vauch and its derivatives against Propionibacterium acnes.

Propionibacterium acnes is the primary pathogenic agent responsible for acne vulgaris on the skin and hair follicles. Overgrowth of this bacterium inhibits growth and promotes follicular inflammation, with an associated increase in pro-inflammatory cytokine production. P. acnes has therefore been considered the main target for the prevention and medical treatment of acne vulgaris. The aim of this study was to evaluate the in vitro anti-P. acnes and anti-inflammatory properties of 6 compounds isolated from Nostoc commune. One of these compounds, nostocionone (Nost), and one of its derivatives, NostD3 [(1E,4E)-1-(3,4-dihydroxyphenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)penta-1,4-dien-3-one], significantly inhibited P. acnes growth. Furthermore, we investigated the effects of Nost and NostD3 on heat-killed (hk) P. acnes-induced inflammation in macrophages. Both Nost and NostD3 suppressed hk P. acnes-induced nitric oxide (NO) production through the suppression of inducible NO synthase expression, following inactivation of nuclear factor kappa B. Taken together, our findings suggested that both Nost and NostD3 were promising agents for the treatment of acne vulgaris, and that NostD3 showed higher efficacy than Nost.

Preparation of polysaccharides from cyanobacteria Nostoc commune and their antioxidant activities.

In this study, water soluble polysaccharides were prepared from cyanobacteria Nostoc commune by water extraction. Factors affecting the polysaccharide yields were investigated, and the optimum extraction conditions were determined as follows: time, 4h; temperature, 90 °C; the ratio of liquid to solid, 60:1 (v/w); and extraction times, 4. The extract was filtered, concentrated to ∼10% (w/v), precipitated with 3 volumes of ethanol, freeze-dried, and ground to yield a water soluble power. The polysaccharide content of the product was 96.7%, and the yield was 9.18% (w/w). Fourier transform infrared spectra demonstrated that the product samples were mainly composed of polysaccharides. The polysaccharides showed high hydroxyl radical scavenging activity (92.71%) and reducing capacity (0.445) at the concentration of 10 mg/mL.

Accumulation of radioactive cesium released from Fukushima Daiichi Nuclear Power Plant in terrestrial cyanobacteria Nostoc commune.

The Fukushima Daiichi Nuclear Power Plant accident released large amounts of radioactive substances into the environment and contaminated the soil of Tohoku and Kanto districts in Japan. Removal of radioactive material from the environment is an urgent problem, and soil purification using plants is being considered. In this study, we investigated the ability of 12 seed plant species and a cyanobacterium to accumulate radioactive material. The plants did not accumulate radioactive material at high levels, but high accumulation was observed in the terrestrial cyanobacterium Nostoc commune. In Nihonmatsu City, Fukushima Prefecture, N. commune accumulated 415,000 Bq/kg dry weight (134)Cs and 607,000 Bq kg(-1) dry weight (137)Cs. The concentration of cesium in N. commune tended to be high in areas where soil radioactivity was high. A cultivation experiment confirmed that N. commune absorbed radioactive cesium from polluted soil. These data demonstrated that radiological absorption using N. commune might be suitable for decontaminating polluted soil.

Glycosylated porphyra-334 and palythine-threonine from the terrestrial cyanobacterium Nostoc commune.

Mycosporine-like amino acids (MAAs) are water-soluble UV-absorbing pigments, and structurally different MAAs have been identified in eukaryotic algae and cyanobacteria. In this study novel glycosylated MAAs were found in the terrestrial cyanobacterium Nostoc commune (N. commune). An MAA with an absorption maximum at 334 nm was identified as a hexose-bound porphyra-334 derivative with a molecular mass of 508 Da. Another MAA with an absorption maximum at 322 nm was identified as a two hexose-bound palythine-threonine derivative with a molecular mass of 612 Da. These purified MAAs have radical scavenging activities in vitro, which suggests multifunctional roles as sunscreens and antioxidants. The 612-Da MAA accounted for approximately 60% of the total MAAs and contributed approximately 20% of the total radical scavenging activities in a water extract, indicating that it is the major water-soluble UV-protectant and radical scavenger component. The hexose-bound porphyra-334 derivative and the glycosylated palythine-threonine derivatives were found in a specific genotype of N. commune, suggesting that glycosylated MAA patterns could be a chemotaxonomic marker for the characterization of the morphologically indistinguishable N. commune. The glycosylation of porphyra-334 and palythine-threonine in N. commune suggests a unique adaptation for terrestrial environments that are drastically fluctuating in comparison to stable aquatic environments.

Reduced scytonemin isolated from Nostoc commune induces autophagic cell death in human T-lymphoid cell line Jurkat cells.

Nostoc commune is a terrestrial benthic blue-green alga that often forms an extended mucilaginous layer on the soil, accumulates on stones and mud in aquatic environments. Reduced-scytonemin (R-scy), isolated from N. commune Vaucher, has been shown to suppress the human T-lymphoid Jurkat cell growth. To reveal the mechanisms underlying the R-scy-mediated inhibition of Jurkat cell growth, we examined cell morphology, DNA fragmentation, and microtubule-associated protein light chain 3 (LC3) modification in these cells. We observed multiple vacuoles as well as the conversion of LC3-I to LC3-II in R-scy-treated cells. These results suggest that the R-scy induced Jurkat cell growth inhibition is attributable to the induction of type II programmed cell death (PCD II; autophagic cell death or autophagy). We further examined the mechanisms underlying R-scy-induced PCDII. The cells treated with R-scy produced large amounts of reactive oxygen species (ROS), leading to the induction of mitochondrial dysfunction. However, the elimination of R-scy-induced ROS by treatment with N-acetyl-L-cysteine (NAC) markedly opposed R-scy-induced PCDII. Based on these results, we conclude that ROS formation plays a critical role in R-scy-induced PCDII.

Structural characterisation of a complex heteroglycan from the cyanobacterium Nostoc commune.

An alkali-extractable O-methylated ribofuranose-containing heteroglycan, Nc-5-s, was isolated from wild-growing field colonies of the cyanobacterium Nostoc commune collected in Iceland, using ethanol fractionation and anion-exchange chromatography. The average molecular weight was estimated to be 1500 kDa. Structural characterisation of the heteroglycan was performed by high-field NMR spectroscopy (1D proton, 2D-COSY, 2D-NOESY, 2D-TOCSY, (1)H (13)C-HSQC, HMBC, H2BC and HSQC-NOESY) as well as monosaccharide analysis after methanolysis by GC and supported by linkage analysis by GC-MS. According to the data obtained, the structure of Nc-5-s is composed of repeating units of 1, 1a, 1b and 2 and 2a in approximate molar ratio of (10:25:50:5:10).