Effects of sugar cane extract on steroidogenesis in testicular interstitial cells of male Japanese quail (Coturnix japonica).

Sugar cane extract (SCE) is the end product of glucose, fructose, and sucrose elimination in molasses. SCE has various biological effects, such as anti-inflammation and antioxidation, and it is commonly found in animal feed. The present research is aimed at investigating the reproductive endocrine influence of SCE in male Japanese quails (Coturnix japonica) by feeding SCE containing food. In addition, in vitro Leydig cell culture was conducted to clarify the mechanism of SCE's influence. Our results showed that SCE feed extended the latency to the first neck grab, decreased male quail testis and epididymis weights, cloaca gland size, and reduced serum testosterone concentrations. Steroidogenic enzymes 3ßHSD, 17ßHSD, P450c17, and P450scc gene expression in the testis were decreased in the SCE groups. Western blot analysis showed decreased 3ßHSD in the testis after feeding SCE. Isolated testicular interstitial cells cultured with SCE and ovine-LH suppressed testosterone secretion and 3ßHSD gene expression. In conclusion, SCE as a feed additive has an impact on the sexual behavior and reproductive function of male Japanese quail, with the suppression of steroidogenesis in the Leydig cell. Our results may provide beneficial information to the livestock management and the poultry industry.

Colonic fermentation of water soluble fiber fraction extracted from sugarcane (Sacchurum officinarum L.) bagasse in murine models.

Biochemical effects of the water soluble fiber fraction of sugarcane bagasse (BSF) fermented in the colon was examined to evaluate its potential health promoting effects. A feeding experiment involving Fischer 344 rats, was conducted with 3 experimental diets containing, cellulose (CON), a commercial xylo-oligosaccharide (XYO) and BSF (BGS). Cumulative feed intake was significantly lower in XYO group while cecal weight was significantly higher. Acetic and propionic acid contents in the cecal content were significantly higher in the BGS and XYO, respectively. Total short chain fatty acid content was significantly higher in BGS and XYO resulting significantly lower cecal pH. Beneficial bacteria such as Bifidobacterium, Blautia, Akkermansia and Roseburia abundance was significantly higher in the XYO and BGS groups. Further, mucin and immunoglobulin-A contents were significantly higher in BGS group compared to CON group. Thus, BSF exhibited its ability to enhance the intestinal and systemic health upon fermentation in the colon.

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.

Changes in the physicochemical characteristics, including flavour components and Maillard reaction products, of non-centrifugal cane brown sugar during storage.

Changes in the quality attributes of non-centrifugal cane brown sugar represented by physicochemical characteristics as well as flavour components and Maillard reaction products (MRPs) were monitored every 3 months over 1 year of storage. Stored cane brown sugar became darker, and its moisture content and water activity (a(w)) increased during storage. Fructose and glucose levels decreased as non-enzymatic browning via the Maillard reaction occurred in the stored sample, and a similar trend was also discovered in aconitic and acetic acids. Stored cane brown sugar lost its acidic and sulfuric odours (58.70-39.35% and 1.85-0.08%, respectively); subsequently, the nutty and roasted aroma increased from 26.52% to 38.59% due to the volatile MRPs. The browning rate of stored cane brown sugar was positively associated with the development of volatile MRPs (Pearson's coefficient = 0.860), whereas the amount of 3-deoxyglucosone, an intermediate product of the Maillard reaction, had a lower association with the brown colour due to its relatively slow degradation rate.

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.

Palatinose-blended sugar compared with sucrose: different effects on insulin sensitivity after 12 weeks supplementation in sedentary adults.

BACKGROUND: We investigated the effects of daily palatinose intake on the risk factors of metabolic syndrome in sedentary non-obese Japanese adults. METHODS: Japanese adults (40 females and 10 males, age: 53 +/- 9 years, range: 31-72 years old) were randomized into two groups for a double-blind, placebo-controlled intervention study and given either 40 g/day palatinose-blended sugar (PS group) or 40 g/day sucrose (S group) in their diet for 12 weeks. RESULTS: After the intervention, the insulin resistance index (HOMA-IR) had significantly decreased only in the PS group; the inter-group difference was significant at P = 0.006. Although the S group showed a significant increase in the leptin concentration and the systolic blood pressure, the PS group showed no significant changes; the inter-group differences were significant at P = 0.018 and P = 0.037, respectively. CONCLUSION: Palatinose intake possibly improves insulin sensitivity when compared with sucrose intake.

A polysaccharide carrier to effectively deliver native phosphodiester CpG DNA to antigen-presenting cells.

Oligodeoxynucleotides containing unmethylated CpG sequences (CpG DNAs) activate the vertebrate innate immune system via toll-like receptor 9 (TLR-9). Although CpG DNA is a promising immunotherapeutic agent, its short circulation time in biological fluids due to nuclease is the major drawback. This paper proposes that a natural polysaccharide called schizophyllan (SPG) can be used as an effective CpG DNA carrier because SPG can complex with CpG DNA and the resultant complex shows the nuclease resistance of the bound DNA. In order to increase cellular uptake in vitro, we chemically attached spermine, cholesterol, arginine octamer, or RGD peptide to SPG. The complexes made of the chemically modified SPG and CpG DNA having a phosphorothioate (PS) or phosphodiester (PO) backbone led to increased secretion of cytokines of about 4- to 15-fold, compared with the uncomplexed dose. Furthermore, when PO CpG DNA was complexed with unmodified SPG, the IL-12 level increased by almost 3- to 11-fold compared with the naked dose. The PO CpG DNA/unmodified SPG complex data suggested that unmodified SPG might effectively deliver PO in vivo due to the electrically neutral nature of unmodified SPG. When the complexed CpG DNAs were injected intraperitoneally, a large amount of IL-12 production was observed compared with the uncomplexed material. Both in vivo and vitro assays indicated that the SPG complex may be of use for CpG DNA therapy.

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.

Parallel vs. anti-parallel orientation in a curdlan/oligo(dA) complex as estimated by a FRET technique.

We already found that beta-1,3-glucan polysaccharides form polymeric complexes with certain polynucleotides, but the parallel vs. anti-parallel orientation in those complexes had remained unsolved. In this paper, this controversial problem has been discussed for curdlan/oligo(dA) complexes utilizing two different energy transfer techniques. The first system consists of a combination of fluorescein-labeled curdlan and 3'-(or 5'-)tetramethyl-rhodamine (TAMRA)-labeled oligo(dA). The second system utilizes gold nanoparticles: that is, two curdlan chains were linked by a disulfide bond and after complexation with oligo(dA), the complex was immobilized on gold nanoparticles. In this system, TAMRA was attached to the 3'(or 5') end of oligo(dA) and the gold particle acted as a fluorescence quencher (energy acceptor). These experiments have led us to conclude that in the curdlan/oligo(dA) complex, parallel orientation is more favourable than anti-parallel orientation. These findings have enabled us to envision a clearer image for the complexation mode between beta-1,3-glucan polysaccharides and polynucleotides.

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.

Schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier.

Schizophyllans carrying beta-lactoside and alpha-mannoside appendages were prepared from native schizophyllan through NaIO4 oxidation followed by reductive amination using aminoethyl-beta-lactoside and alpha-mannoside, respectively. The resulting schizophyllans form stable macromolecular complexes with polynucleotides, such as poly(C) and poly(dA). Specific affinity between these macromolecular complexes and saccharide-binding proteins was demonstrated by surface plasmon resonance and agarose gel staining assays. beta-lactoside-appended schizophyllan enhanced an antisense activity in hepatocytes which express lactoside-binding proteins on their cell surfaces.

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.

Long-term expression with a cationic polymer derived from a natural polysaccharide: schizophyllan.

Among the various synthetic gene carriers based on biomaterials, cationic polymers with polysaccharide backbones have long been studied as nonviral vectors due to their low immunogenicity and high water solubility. Schizophyllan, a beta-(1,3)-glucan, is one of the various polysaccharides that are clinically administered. Furthermore, its safety in the human body has already been confirmed. Various functional groups can be selectively introduced into the side chain, not into the main chain of schizophyllan. Therefore, we have synthesized various oligoamine conjugates from schizophyllan. It was confirmed that their in vitro transfection efficiencies are superior to that of polyethylenimine by adjusting the molecular weight and the degree of amination of cationic schizophyllan. While it was possible to reduce cytotoxicity by adjusting the amount of DNA complex per cell, as seen with poly-L-lysine, polyethylenimine, and chitosan, PEGylation was the most effective means of reducing toxicity. Furthermore, using cationic schizophyllan carriers, it was also possible to express a reporter protein for a long period of time due to a long residence time of plasmid DNA in cells.