Anti-stress and Antioxidant Effects of Non Centrifuged Cane Sugar, Kokuto, in Restraint-Stressed Mice.

Stress is a part of everyday life, but excessive stress can be related to diverse diseases. Recently, oral intake of a non-centrifuged cane sugar, Kokuto, was reported to produce potential anti-stress effects in humans. However, the molecular components associated with the anti-stress property of Kokuto remain mostly unknown. Therefore, we focused on the non-sugar component (NSC) fractions of Kokuto, and investigated how serum corticosterone level (used as a stress marker) and antioxidant activity were affected in restraint-stressed mice treated with NSC fractions obtained from the elusion on HP-20 resin with 25%, 50%, 75%, and 100% aqueous methanol (MeOH) solutions. Among the four NSC fractions, the 50% MeOH fraction showed a high content of phenolic compounds and high antioxidant activity. Moreover, oral administration of the 50% MeOH fraction suppressed both corticosterone secretion into the serum and reduction of antioxidant activity in serum and liver in restraint-stressed mice. Component analysis of the 50% MeOH fraction identified five antioxidative phenolic compounds: p-hydroxybenzaldehyde, p-hydroxyacetophenone, schaftoside, isoschaftoside, and p-coumaric acid. Phenolic compounds detected in the NSC fractions of Kokuto might contribute to the anti-stress property of Kokuto. In addition, this research provides more understanding of potential health benefits offered by the constituents of Kokuto.

Protective effect of sugar cane extract against dextran sulfate sodium-induced colonic inflammation in mice.

Sugar cane extract (SCE) exhibits various biological effects and has been reported to enhance animal growth performance. However, the effect of SCE on inflammation in animals is still obscure. To study the effects and underlying mechanism of SCE on dextran sulfate sodium (DSS)-induced colonic inflammation, forty female ICR mice (26.63±0.19g, 6-week-old) were assigned into four groups: a control group (Cont), a DSS-challenged group (DSS), a SCE-supplemented group (SCE), and a DSS+SCE group (DSS+SCE). Mice in Cont group and DSS group were fed basic diet and other mice received 1% SCE supplemented in basic diet from 6-week to 8-week-old. Mice in DSS and DSS+SCE groups were also given a 4% DSS solution from 7-week to 8-week-old via drinking water to induce colonic inflammation. After 2 weeks, mice were sacrificed and samples were collected. The results showed that dietary SCE alleviated DSS induced growth suppression, splenic damage, colonic histological changes, colonic inflammation, oxidative stress, and colonic dysfunction of tight junctions. Meanwhile, the DSS exposure activated nuclear transcription factor kappa B p65 and inhibited nuclear factor E2-related factor 2 (Nrf2), while SCE markedly attenuated the DSS-promoted effect on the p65 nuclear accumulation and the DSS-inhibited effect on the Nrf2 nuclear accumulation. In conclusion, SCE conferred a protective role in the DSS-induced inflammation and the mechanism might be associated with the activated signals of the nuclear factor kappa B p65 and Nrf2.

Composition, Taste, Aroma, and Antioxidant Activity of Solidified Noncentrifugal Brown Sugars Prepared from Whole Stalk and Separated Pith of Sugarcane (Saccharum officinarum L.).

In this study, 2 types of solidified noncentrifugal brown sugars (W-NCS and P-NCS) were prepared from the whole stalk and separated pith, respectively, of raw sugarcane (Saccharum officinarum L.). These products were discriminated in terms of their quality attributes, including color, sugars and minerals composition, taste, aroma, and antioxidant activity. The brown color of P-NCS was clearly different compared with that of W-NCS with a color difference value (ΔE* ) of 9.36. There was no difference in the sugars and minerals composition between the 2 types of sugar, which led to very similar taste profiles. However, P-NCS had a weaker aroma intensity than W-NCS did. Moreover, P-NCS retained more than 60% of the antioxidant activity of W-NCS. The information gleaned from this study might be used to select appropriate end-uses for these 2 types of sugars.

DNA damage protection against free radicals of two antioxidant neolignan glucosides from sugarcane molasses.

BACKGROUND: Sugarcane molasses is a potential by-product of the sugarcane manufacturing industry that is rich in antioxidant materials. The present study aimed to obtain antioxidative compounds from sugarcane molasses and to evaluate their ability to protect DNA from oxidative damage. RESULTS: Two neolignan glucosides were isolated from sugarcane molasses using bioassay and UV spectra monitoring-guided fractionation. The compounds were elucidated as (7R,8S)-dehydrodiconiferyl alcohol-4-O-ß-d-glucoside (1) and (7S,8R)-simulanol-9'-O-ß-d-glucoside (2). Neolignan glucoside 2 protected against DNA damage caused by free radicals more effectively than did neolignan glucoside 1 (13.62 and 9.08 µmol L(-1) for peroxyl and hydroxyl radicals, respectively, compared to 48.07 and 14.42 µmol L(-1) ). Additionally, neolignan glucoside 2 exhibited superior DNA protection against free radicals compared with various known antioxidative compounds, including p-coumaric acid, ferulic acid, vanillic acid and epigallocatechin gallate. CONCLUSION: The isolated neolignan glucosides from sugarcane molasses are able to protect DNA from oxidative damage caused by free radicals. This is the first identification of these two compounds in sugarcane molasses. The sugarcane molasses can therefore be used as potential nutraceutical preventative agents, and the findings may foster the utilization of this by-product as a bioresource-based product. © 2015 Society of Chemical Industry.

Antioxidant activity of sugarcane molasses against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced peroxyl radicals.

Sugarcane molasses is a rich source of antioxidant materials with peroxyl radical scavenging effects. To explore the potent antioxidant activity of sugarcane molasses against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced peroxyl radicals, 7 methanolic fractions of sugarcane molasses (F1-F7) were separated via bioassay-guided fractionation and evaluated by oxygen radical absorbance capacity (ORAC), cellular antioxidant activity (CAA), and oxidative DNA damage protective activity assays. The ORAC values of sugarcane molasses fractions ranged from 4399 to 6,266 µmol TE/g, whilst the EC50 values for CAA ranged from 3.7 to 5.9 µg/ml. Moreover, it was found that sugarcane molasses fractions, particularly F6 and F7, could protect against oxidative DNA damage caused by peroxyl radicals at an effective concentration of 100 µg/ml. Ten phenolic constituents were identified in the fractions, including known antioxidative compounds, viz., schaftoside, isoschaftoside, ferulic acid, p-coumaric acid, and p-hydroxybenzaldehyde.

Galactose-PEG dual conjugation of beta-(1-->3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake.

Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000-6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery.

CpG DNA/zymosan complex to enhance cytokine secretion owing to the cocktail effect.

Zymosan, classified among beta-(1-->3)-d-glucans, is produced from the cell wall of yeast and well known to induce proinflammatory cytokines when ingested by immune cells. We found that zymosan forms a complex with immunostimulatory CpG DNA, where both zymosan and CpG DNA can induce cytokine secretion according to the different mechanisms (i.e., recognized by different receptors). The complex activated macrophages and induced cytokine secretion, more efficiently than separate administration of zymosan or CpG DNA. Microscopic observation showed that this increment of the cytokine secretion can be explained by the fact that zymosan and zymosan/CpG DNA complex are up-taken more than naked CpG DNA. Additionally, existence of two different immunostimulants in the same cells may enhance the immunoresponse. This report presents a new strategy to construct a delivering vehicle for CpG DNA and to enhance its activity with the 'cocktail effect' of the two immunostimulants.

Linear double-stranded DNA that mimics an infective tail of virus genome to enhance transfection.

Our previous work showed that a natural beta-(1-->3)-d-glucan schizophyllan (SPG) can form a stable complex with single-stranded oligonucleotides (ssODNs). When protein transduction peptides were attached to SPG and this modified SPG was complexed with ssODNs, the resultant complex could induce cellular transfection of the bound ODNs, without producing serious cytotoxicity. However, no technique was available to transfect double-stranded DNAs (dsDNA) or plasmid DNA using SPG. This paper presents a new approach to transfect dsDNA, showing preparation and transfection efficiency for a minimal-size gene having a loop-shaped poly(dA)(80) on both ends. This poly(dA) loops of dsDNA can form a complex with SPG. An siRNA-coding dsDNA with the poly(dA) loop was complexed with Tat-attached SPG to silence luciferase expression. When LTR-Luc-HeLa cells that can express luciferase under the control of the LTR promoter were exposed to this complex, the expression of luciferase was suppressed (i.e., RNAi effect was enhanced). Cytotoxicity studies showed that the Tat-SPG complex induced much less cell death compared to polyethylenimine, indicating that the proposed method caused less harm than the conventional method. The Tat-SPG/poly(dA) looped dsDNA complex had a structure similar to the viral genome in that the dsDNA ends were able to induce transfection and protection. The present work identifies the SPG and poly(dA) looped minimum-sized gene combination as a candidate for a non-toxic gene delivery system.

PEG-appended beta-(1-->3)-D-glucan schizophyllan to deliver antisense-oligonucleotides with avoiding lysosomal degradation.

Schizophyllan is a natural beta-(1-->3)-d-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO). As we already reported, when a homo-polynucleotide [e.g., poly(dA) or poly(C)] is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. One of the potential applications for this novel complex is an antisense-oligonucleotide (AS ODN) carrier. The present paper describes a modification technique that enabled us to introduce PEG only to the side chain of schizophyllan. This technique consisted of periodate oxidation of the glucose side chain and subsequent reaction between methoxypolyethylene glycol amine and the formyl terminate, followed by reduction with NaBH4. Subsequently, we made a complex from PEG-appended schizophyllan and an AS ODN sequence, and carried out an in vitro antisense assay, administrating the AS ODN complex to depress A375 c-myb mRNA of A375 melanoma cell lines. The PEG-SPG/AS ODN complex showed more enhanced antisnese effect than naked AS ODN dose, i.e., the same level as that of RGD-appended SPG. Here, the RGD system has been shown one on the most effective AS ODN carrier (Science 261 (1993) 1004-1012). When we added nigericin to the assay system, the antisense effect was not affected in the PEG-SPG system, on the other hand, it was almost eliminated in the RGD system. Nigericin is well known to interrupt transport from endosome to lysosome. Therefore, the difference between the PEG and RGD complexes indicates that, in the PEG system, AS ODN was able to escape from lysosomal degradation. The present work has thus proposed a new strategy to delivery AS ODN using schizophyllan as a new carrier.

Proposal of new modification technique for linear double-stranded DNAs using the polysaccharide schizopyllan.

A natural polysaccharide schizophyllan (SPG) has been known to form a stable complex with poly(dA). We attached a poly(dA)(80) tail to the both ends of a linear double-stranded DNA, which had been prepared from a plasmid DNA vector. The poly(dA) tailed DNA verified to form complex with SPG by gel electrophoresis and atomic force microscopy (AFM). AFM images indicated that the complexes exhibit a dumbbell-like architecture, that is, quite similar to that of adenovirus genome. The complex demonstrated excellent exonuclease resistance, probably because of the protection effect by SPG complexation.

Removal of the side-chain glucose groups from schizophyllan improves the thermal stability of the polycytidylic acid complexes under the physiological conditions.

Thermal stabilization of the complex between polycytidylic acid [poly(C)] and the modified schizophyllan (SPG) whose hydrophilic side-chain glucose groups are selectively removed utilizing mild Smith-degradation has been investigated. With the decrease in the side-chain glucose groups of schizophyllan, the complex with poly(C) can be considerably stabilized compared with unmodified SPG; for example, the T(m) value after the removal of the side-chain glucose groups from 33.3 (unmodified) to 1.0 is enhanced by 14 degrees C. In addition, the thermal stabilization effect is even operative under the physiological conditions ([NaCl] = 0.15 mol dm(-3)). This effect is exerted owing to the construction of the hydrophobic atmosphere around the complex. Although schizophyllan lost the side-chain glucose groups, it still kept the protection effect of the bound poly(C) chain against RNaseA-mediated hydrolysis as observed for unmodified schizophyllan. The assessment of the cytotoxicity for A375:human malignant melanoma, and HL60:human promyelocytic leukemia revealed that the modified schizophyllan scarcely increases the cytotoxicity. These results indicate that the present modification for schizophyllan is of great significance in a viewpoint to develop the practical gene carriers operative even under the physiological conditions.

A polysaccharide carrier for immunostimulatory CpG DNAs to enhance cytokine secretion.

A beta-(1 --> 3)-d-glucan schizophyllan (SPG) forms a stoichiometric complex with some polynucleotides. This communication describes our attempt to apply the SPG complex to deliver CpG DNA to endosomes to enhance cytokine secretion. To increase cellular uptake, we introduced spermine, arginine-glycine-aspartic acid tripeptide, octaarginine, or cholesterol to the SPG side chain. The chemically modified SPG showed essentially no cytotoxicity. When CpG DNA complex made therefrom was exposed to macrophages, dramatic enhancement in the cytokine secretion was observed. It increased 5-10 times from the naked dose and 100 times from the background. This performance promises that SPG can be an excellent carrier for CpG DNA.

Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency.

Schizophyllan is a natural beta-(1-->3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when some homo-phosphodiester polynucleotide (for example, poly(dA) or poly(C)) is added to the schizophyllan/DMSO solution and subsequently DMSO is exchanged for water, the single chain of schizophyllan forms a complex with the polynucleotide. Furthermore, we have already demonstrated that one of the potential applications of this novel complex is an antisense-oligonucleotide (AS ODN) carrier. This work describes a versatile and universal modification technique which enables us to introduce various functional groups only to the side chain of schizophyllan. This technique consists of periodate oxidation of the glucose side chain (it does not react with the main chain because of the absence of the 1,2-diol group in beta-(1-->3)-glucan) and subsequent introduction of the functional groups into the formyl terminate. In the present work, the introduced functional groups were spermine, octa-arginine (R8), arginine-glycine-aspartic acid tripeptide (RGD) and some amino or alpha-amino acid compounds. Using these compounds, we made the complexes and carried out an in vitro antisense assay for them, administrating a phosphorothioate AS ODN to the melanoma A375 or leukemia HL-60 cell lines to depress their c-myb mRNA. When we used the R8 or RGD modified schizophyllan as the antisense carrier, the antisense effect was most enhanced among others. Their superiority can be ascribed to enhancement of endocytosis due to these functional peptides. Furthermore, the cytotoxicity for these two modified schizophyllans was negligibly as small as the natural (unmodified) schizophyllan. One of the peculiar features of our system is that the complex (i.e., carrier+AS ODN) is charged negatively in total, which is different from the conventional systems. The present work has thus clarified that schizophyllan can act as a new potential candidate for AS ODN carriers.

First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan.

Schizophyllan is a natural beta-(1-->3)-D-glucan that exists as a triple helix in H(2)O and as a single chain in dimethylsulfoxide (DMSO) or basic solution (pH >13). As we have already reported, when a homo-polynucleotide (e.g., poly(dA), poly(A), or poly(C)) is added to a schizophyllan/DMSO solution, and, subsequently, DMSO is exchanged for H(2)O, the single chain of schizophyllan forms a complex with the polynucleotide. Since eukaryotic mRNAs have poly(A) tails, we hypothesized that schizophyllan can bind to mRNA by interacting with this tail. However, we have not yet observed complexation between schizophyllan and mRNA after exchanging DMSO for H(2)O. In this report, we show that the complexation can be accelerated when the solution pH is changed from 13 to 7-8 in the presence of schizophyllan and polynucleotides. By this approach, we found that schizophyllan forms a complex with a yeast mRNA.