The Impact of Plant Phytochemicals on the Gut Microbiota of Humans for a Balanced Life.

The gastrointestinal tract of humans is a complex microbial ecosystem known as gut microbiota. The microbiota is involved in several critical physiological processes such as digestion, absorption, and related physiological functions and plays a crucial role in determining the host's health. The habitual consumption of specific dietary components can impact beyond their nutritional benefits, altering gut microbiota diversity and function and could manipulate health. Phytochemicals are non-nutrient biologically active plant components that can modify the composition of gut microflora through selective stimulation of proliferation or inhibition of certain microbial communities in the intestine. Plants secrete these components, and they accumulate in the cell wall and cell sap compartments (body) for their development and survival. These compounds have low bioavailability and long time-retention in the intestine due to their poor absorption, resulting in beneficial impacts on gut microbiota population. Feeding diets containing phytochemicals to humans and animals may offer a path to improve the gut microbiome resulting in improved performance and/or health and wellbeing. This review discusses the effects of phytochemicals on the modulation of the gut microbiota environment and the resultant benefits to humans; however, the effect of phytochemicals on the gut microbiota of animals is also covered, in brief.

Present and future prospects of seaweeds in developing functional foods.

There has been a combined effort among scientists to explore and utilize varying food sources to develop functional foods to cater the ever-increasing demand from the consumers, who seek health-promoting roles of dietary compounds. Considering the diversity of biochemicals in seaweeds that are capable of exerting bioactivities, a growing trend is developing across globe to employ seaweeds in functional food development. Proteins, peptides, amino acids, polysaccharides, phenolics, lipids, vitamins, and minerals in seaweeds and their functional properties provide insights into the success of potential functional food products that can be developed utilizing seaweeds. However, several factors need to be taken into consideration in designing seaweed-based functional foods to obtain the market success. This chapter elaborates on the prospects of seaweeds in developing seaweed-based functional food products.

The inhibitory mechanism of a novel cationic glucosamine derivative against MMP-2 and MMP-9 expressions.

A number of recent researches have demonstrated the therapeutic effects of glucosamine (Glc) in a range of human diseases including arthritis. For the first time, we identified that a novel Glc derivative having quaternized amino functionality (QAGlc) suppresses MMP-9 and MMP-2, gelatinases in HT1080, human fibrosarcoma cells at 40microg/ml, following stimulation with PMA. Reporter gene assay results revealed that, the mechanism of suppression involves decreased transcriptional activation of MMP-9 and MMP-2 via transcription factors NF-kappaB and AP-1. However based on western blot results, QAGlc did not attenuate the nuclear translocation of both NF-kappaB and AP-1. Apparently, phorbol myristate acetate (PMA) stimulated expressions of ERK, JNK and p38 were altered in the presence of potent tumour inducer, phorbol myristate acetate QAGlc, suggesting their suppression also contributes to QAGlc-mediated inhibition of MMP-9 and MMP-2. Moreover, the ability of QAGlc to inhibit gelatinases was confirmed by its ability to act against invasiveness of HT1080 cells through extracellular matrix components.

Effect of spongin derived from Hymeniacidon sinapium on bone mineralization.

Marine sponges have been known to provide a source of novel bone and cartilage replacements because of their secondary metabolites and specific skeleton structures. In particular, it has been reported that spongin as a component of fibrous skeleton, pseudokeratin, neurokeratin, horny protein, and collagen-like protein in sponges can be used in several biomedical applications including osteoarthritis (OA). However, the pharmacological mechanism of action of spongin remains obscure. In this study, it was investigated whether spongin derived from Hymeniacidon sinapium can promote bone mineralization of osteoblast-like MG-63 cells. Our present study provides the first evidence that spongin is effective in activating bone mineralization. Furthermore, spongin increased ALP activity, collagen synthesis, and osteocalcin secretion in addition to bone mineralization in osteoblastic cells in vitro. In addition, it was demonstrated that spongin exerted the inhibitory effect on production of inflammatory mediators such as TNF-alpha, IL-1beta, and PGE(2) in macrophage, RAW264.7 cells. These results suggest that the anti-inflammatory effect of spongin derived from Hymeniacidon sinapium can play a critical role in bone mineralization of osteoblast-like MG-63 cells.

Suppression of cytokine production in lipopolysaccharide-stimulated mouse macrophages by novel cationic glucosamine derivative involves down-regulation of NF-kappaB and MAPK expressions.

Exposure of macrophages to bacterial lipopolysaccharide (LPS) induces release of proinflammatory cytokines that play crucial roles in chronic inflammation. Glucosamine has reported to possess anti-inflammatory properties and currently is the oral supplement of choice for the management of inflammation related complications including osteoarthritis. In this study, quaternized amino glucosamine (QAGlc), a newly synthesized cationic glucosamine (Glc) derivative was found to inhibit LPS-stimulated production of IL-1beta, IL-6, TNF-alpha, and PGE(2) in RAW264.7, mouse macrophages more potently than its starting material Glc. Since production of cytokines is regulated mainly via activation of NF-kappaB and regulation of mitogen-activated protein kinases (MAPKs), we examined if QAGlc could be responsible for the suppression of NF-kappaB pathway and MAPKs. We used reporter gene assay and Western blotting to examine the effects of QAGlc on activation and translocation of NF-kappaB. Further, QAGlc-mediated inhibition of NF-kappaB was accompanied with a suppression of its translocation. Apparently, QAGlc was shown to attenuate LPS-induced activation of p38 MAPK and JNK in RAW264.7 cells suggesting that inhibition of MAPK-mediated LPS signaling also contribute to suppression of cytokine production following stimulation of macrophages with LPS.

Laurencia okamurai extract containing laurinterol induces apoptosis in melanoma cells.

Laurinterol is a marine sesquiterpene that has been known to have antimicrobial activity. The purpose of this study is to investigate the effect of Laurencia okamurai extract containing laurinterol (LOEL) on induction of apoptosis in melanoma cells (B16F1). Anticancer activity of LOEL against melanoma cells was shown in a dose-dependent manner by the 1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. It was for the first time found that LOEL exhibited an excellent effect on the induction of apoptosis as determined by DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling assay, cell cycle analysis, and measurement of activities of several caspases in melanoma cells. It was also demonstrated that transcriptional activation of p53, a tumor suppressor gene, and activation of p21 promoter by LOEL were involved in the induction of apoptosis by reporter gene assay. In particular, western blot analysis confirmed that LOEL above 5 microg/mL significantly increased the expression level of phospho-p53, the active form. These results indicate that LOEL can induce apoptosis through a p53-dependent pathway in melanoma cells.

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 in lipopolysaccharide-stimulated RAW264.7 cells by carboxybutyrylated glucosamine takes place via down-regulation of mitogen-activated protein kinase-mediated nuclear factor-kappaB signaling.

Glucosamine (GlcN) has been reported to possess several biomedical properties, and currently a great deal of attention has been focused on improving the functional properties of GlcN for different applications. Therefore, this study was conducted to introduce a carboxybutyryl functional group to GlcN and to find out the inhibitory mechanism of a novel GlcN derivative, carboxybutyrylated GlcN (CGlcN), on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in bacterial lipopolysaccharide (LPS)-induced mouse macrophages (RAW264.7 cells). In the initial experiments, the production of NO and prostaglandin E(2) (PGE(2)) was inhibited by CGlcN pretreatment and suggested the possibility of down-regulating their respective genes, iNOS and COX-2. Reverse transcription-polymerase chain reaction and Western blot analysis revealed that CGlcN can affect both transcriptional and translational levels of iNOS and COX-2 expression. The data from the nuclear factor-kappaB (NF-kappaB) promoter gene transfection experiment supported the idea that inhibition of iNOS and COX-2 is caused by the down-regulation of their transcription factor, NF-kappaB. Following stimulation with LPS, p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) present upstream of NF-kappaB signaling were also inhibited by CGlcN treatment. However, the protein level of another MAPK, extracellular signal-regulated kinase (ERK), remained unaffected. Moreover, following treatment with CGlcN, the protein expression of I-kappaB kinase (IKK) clearly confirmed that its down-regulation directly inhibited the degradation of IkappaB and release of NF-kappaB. Therefore, it can be concluded that CGlcN is capable of inhibiting iNOS and COX-2 expression in LPS-induced RAW264.7 cells via attenuation of NF-kappaB signaling by p38 MAPK and JNK, but not by ERK.

Sulfated glucosamine inhibits oxidation of biomolecules in cells via a mechanism involving intracellular free radical scavenging.

Although, several effects of glucosamine and its sulfated form (sulfated glucosamine) have been proposed for the suppression of osteoarthritis, their exact mechanisms have not been completely elucidated. This study explains the novel possibility of involvement of sulfated glucosamine in improving cellular antioxidant potential and thereby controlling oxidative damage that could be effective for its therapeutic potential in osteoarthritis. Treatment with sulfated glucosamine to human chondrocytes and macrophages inhibited radical simulated oxidation of membrane lipids, proteins and DNA in a dose-dependent manner. Moreover, detection of reactive oxygen species by electron spin resonance (ESR) spectroscopy and 2',7'-dichlorodihydrofluororescein diacetate (DCFH-DA) fluorescence probe clearly confirmed effective radical scavenging potential of sulfated glucosamine in cellular and non-cellular systems. More importantly, NF-kappaB reporter gene assay and western blot analysis revealed that sulfated glucosamine inhibits radical mediated expression and activation of nuclear factor kappaB (NF-kappaB) proteins (transcription factor involves in expression of a number of genes related to osteoarthritis). Further, sulfated glucosamine enhanced reduced glutathione (GSH) level in oxidatively stressed human chondrocytes improving cellular redox balance. In conclusion, it is suggested that potential effects of sulfated glucosamine in controlling osteoarthritis might be partly via mechanisms involving direct scavenging of cellular radical species and alteration of oxidation mediated destructive events.

Sulfated glucosamine inhibits MMP-2 and MMP-9 expressions in human fibrosarcoma cells.

In the present study, sulfated glucosamine (SGlc) that has been reported to relieve joint pain and inflammation in many arthritis patients was studied for its inhibitory effects on MMP-2 and MMP-9 in human fibrosarcoma cells. Expression and activity of above MMPs studied using gelatin zymography suggested SGlc as a potent MMP inhibitor. Further, transfection of promoter genes of MMPs and their transcription factors clearly exhibited that inhibition of MMP-2 and MMP-9 was due to down-regulation of transcription factor, NF-kappaB. However expression of activator protein-1 (AP-1), another important transcription factor of MMPs, was not affected by SGlc treatment. In addition, protein expression results of Western blot analysis were also in agreement with the results of gene transfection experiments. Moreover, down-regulation of NF-kappaB resulted in production of low levels of both NF-kappaB p50 and p65 proteins and directly affected activation process of MMP-2 and MMP-9 expressions. Since MMPs involve in joint inflammation, it can be presumed that inhibition of MMP-2 and MMP-9 can be one of the mechanisms of SGlc to be an effective drug in relieving the symptoms of osteoarthritis.

An in vitro cellular analysis of the radical scavenging efficacy of chitooligosaccharides.

Despite extensive study on biological activities of chitosan and chitooligosaccharides (COS), there is no experimental evidence available as to COS mediated inhibition of free radical damage in cellular oxidizing systems. In this study, radical scavenging efficacies of different molecular weight bearing COS were assessed and their intracellular radical scavenging effects were tested employing B16F1, murine melanoma cell line. The results exhibited appreciable suppression in occurrence of intracellular radical species in the presence of low molecular weight bearing COS (<1 kDa) confirming low molecular weight is important for observed activities in biological systems. However, DNA oxidation carried out in the presence of COS clearly exhibited that COS exert protective effect on oxidative damage of purified genomic DNA regardless of molecular weight. Low molecular weight bearing COS was observed to be successively participated in suppression of NF-kappaB gene promoter activity suggesting its capability to prevent oxidative stress related disease complications. Moreover, induction of intracellular glutathione (GSH) level in the presence of COS promoted the effectiveness of COS to act against cellular oxidative stress.

Glucosamine sulfate promotes osteoblastic differentiation of MG-63 cells via anti-inflammatory effect.

Glucosamine sulfate (SGlc) has been known to be effective in controlling osteoarthritis (OA) symptoms in several clinical studies. However, the mechanisms of this positive effect of SGlc in human OA still remain elusive. Therefore, first, the effects of SGlc on the differentiation of osteoblast-like MG-63 cells were investigated. Our results demonstrate that SGlc can increase ALP activity, collagen synthesis, osteocalcin secretion, and mineralization in osteoblastic cells in vitro. Furthermore, it was observed that SGlc exhibited anti-inflammatory effect on production of TNF-alpha, IL-1beta, and PGE(2) in macrophage, RAW264.7 cells. In conclusion, these results suggest that SGlc can promote cell differentiation in cultured MG-63 osteoblast cells via anti-inflammatory effect.

Inhibition of free radical-mediated oxidation of cellular biomolecules by carboxylated chitooligosaccharides.

The objective of this study was to identify the cellular antioxidant effects of carboxylated chitooligosaccharides (CCOS), a chemically modified derivative of chitooligosaccharides (COS), by assessing oxidation inhibition potential on cellular biomolecules such as lipids, proteins, and direct scavenging of reactive oxygen species (ROS). Radical-mediated oxidation of cell membrane lipids and proteins was dose-dependently inhibited by CCOS, assessed by amount of lipid hydroperoxides and carbonyl carbon content in mouse macrophages, RAW264.7 cells. Further, CCOS inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL60) suggesting indirect possibility of inhibiting generation of reactive oxygen species (ROS) such as superoxide radicals, H(2)O(2) and HOCl. Direct radical scavenging studies carried out with DCFH-DA fluorescence probe concluded that CCOS can act as a potent radical scavenger in cells.

Carboxylated chitooligosaccharides (CCOS) inhibit MMP-9 expression in human fibrosarcoma cells via down-regulation of AP-1.

Matrix metalloproteinases (MMPs) play a fundamental role in invasion and metastasis of tumor and, recent advances in medicinal chemistry have approached designing of MMP inhibitors with desired structural properties, selectivity and bioavailability. In the present study, novel low-molecular-weight carboxylated chitooligosaccharides (CCOS) were evaluated for their MMP-9 inhibitory effect on human fibrosarcoma cell line (HT1080). In zymography experiments, a clear dose-dependent inhibition on MMP-9 mediated gelatinolytic activities were observed in HT1080 cells following treatment with CCOS. Further, transfection studies carried out with MMP-9 and AP-1 reporter constructs suggested that the observed reduction in MMP-9 expression was due to down-regulation of MMP-9 transcription that mediated via inhibition of AP-1. Moreover, expression of c-Fos protein levels in cytoplasm and nucleus confirmed that CCOS could inhibit AP-1 expression but not its translocation. However, in the presence of CCOS, NF-kappaB and TIMP-1 expression levels remained constant. More importantly, inhibition of MMP-9 expression clearly led to inhibit tumor invasiveness that was studied with reconstituted basement membrane matrix proteins coated synthetic membranes. Taken together, this study discusses MMP-9 inhibition potential of CCOS and their involvement to demote degradation and cellular invasion of extracellular matrix (ECM) and basement membrane. Thus, control of MMP-9 expression by CCOS has considerable significance for the regulation of tumor progression.

Isolation and characterization of visceral excitatory neuropeptides from striped mullet (Mugil cephalus L.) brain.

From a brain extract of the catadromous fish, striped mullet (Mugil cephalus), two visceral excitatory neuropeptides (Mvp-1 and Mvp-2) were isolated by means of reversed phase chromatography together with bioassay on fish hindgut. The primary structure of Mvp-1 was elucidated to be SGPAGVLamide by ESI-Q-TOF mass spectrometry. The threshold concentration of Mvp-1 that changes spontaneous contraction of mullet hindgut was between 10(-9) and 10(-8) M. In addition, Mvp-1 was found to exert excitatory activities on some other smooth muscle segments (oviduct and esophagus) of mullet but it did not show any effect on body wall muscle strips. Therefore, the present study suggests that Mvp-1 and Mvp-2 peptides act as factors that control visceral contractions of mullet gastrointestinal tract.

Phlorotannins in Ecklonia cava extract inhibit matrix metalloproteinase activity.

Matrix metalloproteinase (MMP) inhibitors have been identified as potential therapeutic candidates for metastasis, arthritis, chronic inflammation and wrinkle formation. For the first time here we report a detailed study on the inhibitory effects of phlorotannins in brown algae, Ecklonia cava (EC) on MMP activities in cultured human cell lines. A novel gelatin digestion assay could visualize complete inhibition of bacterial collagenase-1 activity at 20 microg/ml of EC extract during preliminary screening studies. Sensitive fluorometric assay revealed that EC extract can specifically inhibit both MMP-2 and MMP-9 activities significantly (P < 0.001) at 10 microg/ml. In addition, artificially induced activities of MMP-2 and MMP-9 in human dermal fibroblasts and HT1080 cells were inhibited by EC extract in a more or less similar manner to the positive control doxycycline. Even though the expression levels of MMPs differ from one cell type to the other, gelatin zymography clearly revealed that both MMP expression and activity in cells can be inhibited by EC extract. More interestingly, EC extract did not exert any cytotoxic effect even at 100 microg/ml anticipating its potential use as a safe MMP inhibitor.

Carboxy derivatized glucosamine is a potent inhibitor of matrix metalloproteinase-9 in HT1080 cells.

Experimental evidences have confirmed that matrix metalloproteinases (MMPs) play a fundamental role in a wide variety of pathologic conditions and recent advances in medicinal chemistry approach to the design of MMP inhibitors with desired structural and functional properties. Among MMPs, MMP-9 has demonstrated to play a major role in the establishment of metastases and it is substantially increased in the majority of malignant tumors. Inhibition of MMP-9 is thought to have a therapeutic benefit to cancer. Results of this study present a novel synthetic MMP-9 inhibitor that downregulates MMP-9 expression level in HT1080, human fibrosarcoma cells.

Strong electronic charge as an important factor for anticancer activity of chitooligosaccharides (COS).

Even though several studies report the importance of chitosan derivatives for their anticancer activity, no clear information is available to describe the relationship between their charge properties and observed activities. In this research, differently charged chitooligosaccharide (COS) derivatives were synthesized and their anticancer activities were studied using three cancer cell lines, HeLa, Hep3B and SW480. Neutral red and MTT cell viability studies revealed that, highly charged COS derivatives could significantly reduce cancer cell viability, regardless to the positive or negative charge. Further, fluorescence microscopic observations and DNA fragmentation studies confirmed that the anticancer effect of these highly charged COS derivatives were due to necrosis. However, the exact molecular mechanism for anticancer activity of strongly charged COS compared to their poorly charged counterparts is not clear.

Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems.

Low molecular weight peptides obtained from ultrafiltration (UF) of giant squid (Dosidicus gigas) muscle protein were studied for their antioxidative effects in different in vitro oxidative systems. The most potent two peptides, Asn-Ala-Asp-Phe-Gly-Leu-Asn-Gly-Leu-Glu-Gly-Leu-Ala (1307 Da) and Asn-Gly-Leu-Glu-Gly-Leu-Lys (747 Da), exhibited their antioxidant potential to act as chain-breaking antioxidants by inhibiting radical-mediated peroxidation of linoleic acid, and their activities were closer to highly active synthetic antioxidant, butylated hydroxytoluene. Addition of these peptides could enhance the viability of cytotoxic embryonic lung fibroblasts significantly (P<.05) at a low concentration of 50 microg/ml, and it was presumed due to the suppression of radical-induced oxidation of membrane lipids. Electron spin trapping studies revealed that the peptides were potent scavengers of free radicals in the order of carbon-centered (IC(50) 396.04 and 304.67 microM), hydroxyl (IC(50) 497.32 and 428.54 microM) and superoxide radicals (IC(50) 669.34 and 573.83 microM). Even though the exact molecular mechanism for scavenging of free radicals was unclear, unusually high hydrophobic amino acid composition (more than 75%) of giant squid muscle peptides was presumed to be involved in the observed activities.

Factors affecting the free radical scavenging behavior of chitosan sulfate.

Scavenging activity of hydroxyethyl chitosan sulfate (HCS) against 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl and carbon-centered radical species were studied using electron spin resonance (ESR) spectroscopy. In addition, its antioxidant activity to retard lipid peroxidation was also evaluated in a linoleic acid model system. HCS could scavenge DPPH (33.78%, 2.5 mg/mL) and carbon-centered radicals (67.74%, 0.25 mg/mL) effectively. However, chitosan sulfate did not exhibit any scavenging activity against hydroxyl radicals, but increased its generation. This was different from the published literature and was presumed due to the loss of chelating ability on Fe2+. This assumption could further confirm from the results obtained for Fe2+-ferrozine method that upon sulfation chitooligosaccharides lost its chelation properties. Therefore, HCS can be identified as antioxidant that effectively scavenges carbon centered radicals to retard lipid peroxidation.

Improvement of ACE inhibitory activity of chitooligosaccharides (COS) by carboxyl modification.

In the present research, chitooligosaccharides (COS) were carboxylated with -COCH(2)CH(2)COO(-) groups to obtain specific structural features similar to Captopril. Angiotensin I converting enzyme (ACE) inhibitory activity of carboxylated COS was studied and observed to enhance its activity with increased substitution degree. Further, Lineweaver-Burk plot analysis revealed that inhibition was competitive via obligatory binding site of the enzyme. This was accompanied with substitution of positively charged quarternized amino groups to COS with different substitution degrees, in which negative impact on ACE inhibition was observed.