The In Vitro Antimicrobial and Antibiofilm Activities of Lysozyme against Gram-Positive Bacteria.

Objective: To analyze the in vitro antibacterial and antibiofilm activities of lysozyme (LYS) and its combination with various drugs against Gram-positive bacteria (GPB, n = 9), thus to provide an exploration direction for drug development. Methods: The minimum inhibitory concentrations (MICs) of linezolid (LZD), amikacin (AMK), ceftriaxone/sulbactam (CRO/SBT), cefotaxime/sulbactam (CTX/SBT), piperacillin/sulbactam (PIP/SBT), doxycycline (DOX), levofloxacin (LVX), amoxicillin/clavulanate potassium (7 : 1, AK71), imipenem (IPM), azithromycin (AZM), and their combinations with LYS were determined with tuber twice dilution. The antimicrobial and antibiofilm activities of LYS, AZM, LVX, and their combinations with others were evaluated through MTT and crystal violet assay. Results: High-dose LYS (30 µg/mL) combined with PIP/SBT and AK71, respectively, showed synergistic antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), while it showed no synergistic activities when combined with other drugs. LYS and AZM inhibited the biofilm formation of one MRSA strain, but they and LVX had no similar activities against methicillin-resistant Staphylococcus epidermidis (MRSE) or vancomycin-resistant Enterococcus faecium (VREF). Particularly, LYS increased the permeability of biofilms of MRSA 33 and exhibited antibiofilm activities against MRSA 31 (inhibition rate = 38.1%) and MRSE 61 (inhibition rate = 46.6%). The combinations of PIP/SBT+LYS, AMK+LYS, and LZD+LYS showed stronger antibiofilm activities against MRSA 62, MRSE 62, MRSE 63, and VREF 11. Conclusion: The antimicrobial and antibiofilm activities of LYS against MRSA were better than AZM, while that of LYS against MRSE and VREF, respectively, was similar with AZM and LVX.

A homogeneous polysaccharide from Lycium barbarum: Structural characterizations, anti-obesity effects and impacts on gut microbiota.

Lycium barbarum polysaccharides (LBPs) are known for their beneficial effects on diabetes, NAFLD and related chronic metabolic diseases induced by high-fat diet (HFD). However, the relevant researches are mainly about the whole crude polysaccharides, the specific active ingredient of LBPs and its bioactivity have been rarely explored. Herein, a homogeneous polysaccharide (LBP-W) was isolated and purified from crude LBPs. Structure characterizations indicated that LBP-W contained a main chain consisting of a repeated unit of →6)-ß-Galp(1 â†’ residues with branches composed of α-Araf, ß-Galp and α-Rhap residues at position C-3. The objective of this study was to evaluate the anti-obesogenic effect of LBP-W and figure out the underlying mechanisms. In vivo efficacy trial illustrated that LBP-W supplements can alleviate HFD-induced mice obesity significantly. Gut microbiota analysis showed that LBP-W not only improved community diversity of intestinal flora, but also regulated their specific genera. Moreover, LBP-W can increase the content of short-chain fatty acids (SCFAs), a metabolite of the intestinal flora. In summary, all these results demonstrated that the homogeneous polysaccharide purified from L. barbarum could be used as a prebiotic agent to improve obesity by modulating the composition of intestinal flora and the metabolism of SCFAs.

Immunoregulation and antioxidant activities of a novel acidic polysaccharide from Radix Paeoniae Alba.

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, immunomodulatory, cancer, and other diseases. In this paper, a novel acidic polysaccharide RPAPS purified from Radix Paeoniae Alba was evaluated for its structural features and potential of immunomodulatory and antioxidant activities. RPAPS (molecular weight: 1.0× 105 Da) was mainly composed of α-(1 → 4)-Glcp, α-Arap, α-Galp, α-Rhap, ß-D-Glcp, α-(1 → 6)-linked Glcp and GalA. Immunological tests indicated that RPAPS could improve RAW264.7 phagocytic activity and LPS-induced splenocyte proliferation. For antioxidant activities, RPAPS showed reducing power and DPPH scavenging activity in dose dependent. Moreover, RPAPS could significantly protect the PC12 cells from H2O2 damage. These data implied polysaccharides RPAPS had the potential to be novel natural antioxidative and immunopotentiating agents for using in functional foods or medicine.

One water-soluble polysaccharide from Ginkgo biloba leaves with antidepressant activities via modulation of the gut microbiome.

Depression, a mental illness characterized by persistent feeling of sadness and loss of interest, has been a serious health problem worldwide. Manipulation of the microbiota by probiotics and prebiotics represents a novel emerging strategy for the treatment of various psychiatric disorders such as major depressive disorders. Here, we show that one water-soluble polysaccharide from Ginkgo biloba leaves (GPS) reduced stress-induced depression and reversed gut dysbiosis. Similar to the antidepressant paroxetine, GPS significantly reduced the immobility times in the tail suspension test (TST) and forced swimming test (FST) and anxiety-like behavior in the open field test (OFT). Consistent with the improvement of depression-like behavior above, GPS mice had elevated serotonin and dopamine levels in multiple brain regions including the hippocampus, cerebral cortex and olfactory bulb, relative to unpredictable chronic mild stress (UCMS) treatment mice. GPS treatment could alleviate the stress-induced reduction in the density of serotonin-positive and dopamine-positive cells. Fecal microbiome transplant (FMT) combined with antibiotic treatment showed that the anti-depressant activity of GPS had a causal relationship with intestinal microbes. By performing a pyrosequencing-based analysis of bacterial 16S rRNA (V3 + V4 region) in fecal of the mice, the results showed that GPS reversed depression-associated gut dysbiosis and increased the richness of Lactobacillus species which has been proven to be a path to relieve depression. Our results demonstrated that the polysaccharide from Ginkgo biloba leaves might be a promising pharmacotherapeutic candidate for treating depression.

Pathogen-targeting glycovesicles as a therapy for salmonellosis.

Antibiotic therapy is usually not recommended for salmonellosis, as it is associated with prolonged fecal carriage without reducing symptom duration or severity. Here we show that antibiotics encapsulated in hydrogen sulfide (H2S)-responsive glycovesicles may be potentially useful for the treatment of salmonellosis. The antibiotics are released in the presence of Salmonella, which is known to produce H2S. This approach prevents the quick absorption of antibiotics into the bloodstream, allows localized targeting of the pathogen in the gut, and alleviates disease symptoms in a mouse infection model. In addition, it reduces antibiotic-induced changes in the gut microbiota, and increases the abundance of potentially beneficial lactobacilli due to the release of prebiotic xylooligosaccharide analogs.

Protein-Bound ß-glucan from Coriolus Versicolor has Potential for Use Against Obesity.

SCOPE: The prevalence of obesity and related disorders has vastly increased throughout the world and prevention of such circumstances thus represents a major challenge. Here, it has been shown that one protein-bound ß-glucan (PBG) from the edible mushroom Coriolus versicolor can be a potent anti-obesity component. METHODS AND RESULTS: PBG can reduce obesity and metabolic inflammation in mice fed with a high-fat diet (HFD). Gut microbiota analysis reveals that PBG markedly increases the abundance of Akkermansia muciniphila, although it does not rescue HFD-induced change in the Firmicutes to Bacteroidetes ratio. It appears that PBG alters host physiology and creates an intestinal microenvironment favorable for A. muciniphila colonization. Fecal transplants from PBG-treated animals in part reduce obesity in recipient HFD-fed mice. Further, PBG is shown to upregulate expression of a set of genes related to host metabolism in microbiota-depleted mice. CONCLUSION: The data highlight that PBG may exert its anti-obesity effects through a mirobiota-dependent (richness of specific microbiota) and -independent (modulation of host metabolism) manner. The fact that C. versicolor PBGs are approved oral immune boosters in cancers and chronic hepatitis with well-established safety profiles may accelerate PBG as a novel use for obesity treatment.

Light controllable chitosan micelles with ROS generation and essential oil release for the treatment of bacterial biofilm.

Bacterial biofilms are widely associated with persistent infections and food contamination. High resistance to conventional antimicrobial agents resulted in an urgent need for novel formulation to eliminate these bacterial communities. Herein we fabricated light controllable chitosan micelles loading with thymol (T-TCP) for elimination of biofilm. Due to the exterior chitosan, T-TCP micelles easily bind to negative biofilm through electrostatic interaction and efficiently deliver the essential oil payloads. Under irradiation, T-TCP micelles generated ROS, which triggered simultaneous thymol release and also resulted in additional ROS-inducing bactericidal effects, both effectively eradicating biofilms of Listeria monocytogenes and Staphylococcus aureus. This formulation provided a platform for other water-insoluble antimicrobials and might be used as a potent and controllable solution to biofilm fighting.

Investigation of three lignin complexes with antioxidant and immunological capacities from Inonotus obliquus.

Mushroom Inonotus obliquus (I. obliquus), a folk medicine, has been widely used to treat several human malicious tumors since 16th century. In this study, three homogenous biomolecules (designated IOA1, IOA2 and IOA3) were prepared from the alkali extract of I. obliquus. Their molecular weights were measured to be 6.1 × 10(4), 2.9 × 10(4) and 3.5 × 10(4) g/mol respectively and all of them were characterized as lignin-carbohydrate complexes mainly comprised lignin as well as -25% carbohydrates. Antioxidant assays indicated that all of them exhibited pronounced reductive power and strong scavenging activities on DPPH and hydroxyl radicals in vitro. Immunological tests showed that they could also significantly stimulate nitric oxide production and phagocytic activity in RAW 264.7 macrophages. These results implied that the lignin-carbohydrate complexes extracted from I. obliquus might be used as novel natural antioxidants or immunostimulants in functional foods or pharmaceutical candidates.

Structural characterization and biological activities of two α-glucans from radix paeoniae alba.

Radix Paeoniae Alba is widely used in Chinese traditional medicine to treat various diseases such as gastrointestinal disorders, cancer, and other diseases. In this study, two polysaccharides RPAPW1 and RPAPW2 were isolated from Radix Paeoniae Alba by DEAE-52 cellulose chromatography and G-25 sephadex. According to physicochemical methods, NMR and methylation analysis, RPAPW1 and RPAPW2 were established to be α-glucans consisting of predominant 4-linked α- Glc residues branched at O-6 and contained trace amount of protein and uronic acid. Immunological tests indicated that RPAPW1, RPAPW2 and could promote splenocyte proliferation and RAW264.7 phagocytic activity. In vitro, RPAPW1 and RPAPW2 elicited a week reducing power, DPPH scavenging activity and could not protect the PC12 cells from H2O2 damage. These data implied polysaccharides RPAPW1 and RPAPW2 had the potential to be a natural immunopotentiating and antioxidant supplement for preparing functional foods and nutraceuticals.

Characterization of polysaccharides with antioxidant and immunological activities from Rhizoma Acori Tatarinowii.

Rhizoma Acori Tatarinowii is widely used in traditional Chinese medicine. In this study, three novel polysaccharides designated RATPW, RATPS1 and RATPS2 were isolated from Rhizoma Acori Tatarinowii by DEAE-52 cellulose chromatography. Their structures were characterized using physicochemical and spectral methods. Chemical analysis indicated that RATPW (6.5×10(3)Da) mainly composed of glucose and fructose. RATPS1 (1.5×10(5)Da) contained galactose and arabinose, while RATPS2 (5.3×10(4)Da) contained ∼49.5% galacturonic acid along with rhamnose, fructose, galactose, and arabinose. In vitro, RATPS2 showed the most significant scavenging activity on DPPH and hydroxyl radical. Three polysaccharides could protect the PC12 cells from H2O2-induced damage. Immunological tests indicated that both RATPW and RATPS2 significantly stimulated NO production and phagocytic activity in RAW264.7, and promoted splenocyte proliferation. These data suggested that polysaccharides RATPW and RATPS2 had the potential as novel natural sources of antioxidative and immunopotentiating agents.

Extraction optimization, characterization and immunity activity of polysaccharides from Fructus Jujubae.

The versatile Fructus Jujubae is widely used in Chinese and Korean traditional medicine. In this study, the extraction optimization, characterization and immunostimulatory activities of polysaccharides from Fructus Jujubae were investigated. Based on a four-variable-three-level Box-Behnken statistical design, the optimal extraction parameters were optimized as follows: extraction temperature 90 °C, extraction time 3.23 h, water to raw material ratio 33:1 and extraction 3 times. Under these conditions, the experimental yield of polysaccharides was 6.47 ± 0.26%, which was close to the predicted yield value (6.54%). The crude Fructus Jujubae polysaccharide was further purified by DEAE-cellulose chromatography repeatedly, and two homogenous fractions, designated as RQP1d and RQP2d with molecular weight of 83.8 and 123.0 kDa respectively, were obtained. Their structures were determined by chemical analysis, Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). Finally, preliminary immunological tests indicated that both RQP1d and RQP2d significantly stimulated NO production in RAW264.7 macrophages, and promoted LPS-induced splenocyte proliferation. These data implied Fructus Jujubae polysaccharides had the potential to be explored as novel natural immunostimulant for using in functional foods or medicine.

Homogalacturonans from preinfused green tea: structural characterization and anticomplementary activity of their sulfated derivatives.

Two homogeneous water-soluble polysaccharides (TPSR4-2B and TPSR4-2C) were obtained from preinfused green tea. Their average molecular weights were estimated to be 41 kDa and 28 kDa, respectively. A combination of composition, methylation, and configuration analysis, as well as NMR spectroscopy, indicated that both TPSR4-2B and TPSR4-2C were poly-(1-4)-α-d-galactopyranosyluronic acid in which 30.5 ± 0.3% and 28.3 ± 0.5%, respectively, of uronic acid existed as methyl ester. Two sulfated derivatives (Sul-R4-2B and Sul-R4-2C) from TPSR4-2B and TPSR4-2C were prepared after sulfation with a 2:1 chlorosulfonic acid-pyridine ratio. The anticomplementary assay showed that Sul-R4-2B and Sul-R4-2C demonstrated a stronger inhibitory effect on the complement activation through the classic pathway, compared to that of heparin. Preliminary mechanism studies by using complement component depleted-sera indicated that both Sul-R4-2B and Sul-R4-2C selectively interact with C1q, C1r, C1s, C2, C5, and C9 but not with C3 and C4. The relationship between DS and the anticomplementary activity of sulfated derivatives of homogalacturonans showed that low sulfated derivatives of homogalacturonans also exhibited potent anticomplementary effect, which might greatly reduce the side effects related to heparin and oversulfated chondroitin sulfate, such as anticoagulant activity and allergic-type reaction. These results suggested that sulfated derivatives of homogalacturonans might be promising drug candidates for therapeutic complement inhibition.

Variation in antioxidant activities of polysaccharides from Fructus Jujubae in South Xinjiang area.

The Fructus Jujubae has been widely used as favorable food and folk medicine in China and Russia. In this study, we compared the carbohydrate constituents and antioxidative effects of Fructus Jujubae polysaccharides from five different production areas in South Xinjiang. Results demonstrated that the average annual temperature (r=0.590) and frost-free period (r=0.779) were well correlated to the uronic acid content, while the neutral carbohydrate content showed negative correlation with precipitation amount (r=-0.567). Antioxidative tests indicated that Fructus Jujubae polysaccharides could scavenge chemicals-induced reactive oxygen species in a dose-dependent manner. In addition, these polysaccharides could rescue H2O2-induced HUVEC death. The antioxidative activity of polysaccharides from the Fructus Jujubae might contribute to their diverse medicinal and nutritional values.

Antioxidative properties of crude polysaccharides from Inonotus obliquus.

The mushroom Inonotus obliquus has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. In this study, water-soluble and alkali-soluble crude polysaccharides (IOW and IOA) were isolated from I. obliquus, and the carbohydrate-rich fractions IOW-1 and IOA-1 were obtained respectively after deproteination and depigmentation. Their contents, such as neutral carbohydrate, uronic acid and protein, were measured. Their antioxidant properties against chemicals-induced reactive species (ROS) including 1,1'-Diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide anion radical, as well as their protective effects on H(2)O(2)-induced PC12 cell death were investigated. Results showed that I. obliquus polysaccharides can scavenge all ROS tested above in a dose-dependent manner. IOA and its product IOA-1 could rescue PC12 cell viability from 38.6% to 79.8% and 83.0% at a concentration of 20µg/mL. Similarly, IOW and its product IOW-1 at the same dose, can also increase cell viability to 84.9% and 88.6% respectively. The antioxidative activities of water-soluble and alkali-soluble polysaccharide constituents from I. obliquus might contribute to diverse medicinal and nutritional values of this mushroom.

Structural characterization of a homogalacturonan from Capparis spinosa L. fruits and anti-complement activity of its sulfated derivative.

A water-soluble polysaccharide CSPS-2B-2 with a molecular mass of 8.8 kDa, was obtained from the fruits of Capparis spinosa L. Chemical and NMR spectral analysis verified CSPS-2B-2 was a linear poly-(1-4)-α-D-galactopyranosyluronic acid in which 12.9±0.4% of carboxyl groups existed as methyl ester and 2.6±0.1% of D-GalpA residues were acetylated. A sulfated derivative Sul-2B-2 with a sulfation degree of 0.88±0.02 was prepared via the substitution of C-2 and/or C-3 of GalpA residues in CSPS-2B-2. Bioassay on the complement and coagulation system demonstrated that Sul-2B-2 (CH(50): 3.5±0.2 µg/mL) had a stronger inhibitory effect on the activation of complement system through the classic pathway than that of heparin (CH(50): 8.9±0.3 µg/mL). Interestingly, Sul-2B-2 at low dose even middle dose (for example 52 µg/mL) had no effect on coagulation system, which was totally different from heparin. Thus, our observation indicated that Sul-2B-2 was more efficient than heparin in inhibiting the activation of the complement system through classical pathway and exhibiting a relatively less anti-coagulant activity. These results suggested that the sulfated derivative Sul-2B-2 prepared from the homogalacturonan in the fruits of Capparis spinosa L, might be a promising drug candidate in case of necessary therapeutic complement inhibition.

Characterization of a pectic polysaccharide from the leaves of Diospyros kaki and its modulating activity on lymphocyte proliferation.

Pectin is a group of carbohydrate polymers constructing the primary cell walls and the middle lamella of terrestrial plants. Herein, we demonstrated the structure and immunomodulatory activity of the major pectic polysaccharide DL-3B(2) isolated from the leaves of Diospyros kaki. Based on composition analysis, methylation analysis, two-step acid hydrolysis, lithium-mediated selective degradation, (13)C NMR spectroscopy, and electrospray ionization mass spectrometry, DL-3B(2) was found to contain an alpha-1, 4-linked galacturonic acid (GalA) backbone with some insertions of alpha-1, 2-linked rhamnose residues. The arabinan- and arabinogalactan-side chains were attached to O-4 of the rhamnose residues, whereas the linear arabinoxylan was probably linked to O-3 of the GalA residues. Immunological tests in vitro showed that DL-3B(2) could help stimulate lipopolysaccharide-induced B lymphocyte proliferation, but not ConA-induced T lymphocyte proliferation, and that the arabinose residues play a role in maintaining this immunological activity.

Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki.

A pectic polysaccharide DL-2A with a molar mass of 8.5 x 10(5), was obtained from the boiling water extract of Diospyros kaki leaves. It had [alpha]20D -21.8 degrees (c 0.22, H2O) and consisted of rhamnose, arabinose, galactose, xylose and galacturonic acid units in the molar ratio of 0.4:3.4:2.4:1.0:0.8, along with traces of glucuronic acid. About 16.7% of galacturonic acid existed as the methyl ester. A combination of linkage analyses, periodate oxidation, partial acid hydrolysis, selective lithium-degraded reaction, ESIMS, 1H- and 13C- NMR spectral analyses revealed its structural features. It was found that DL-2A possessed an alpha-(1-->4)-galacturonan backbone with some insertions of alpha-1,2-Rhap residues. The side-chains of arabino-3,6-galactan were attached to the backbone via O-4 of Rhap residues and O-3 of GalAp residues, while 4-linked xylose residues (forming short linear chains) were directly linked to O-4 of rhamnose residues, not as part of the xylogalacturonan. These novel structural features enlarge the knowledge on the fine structure of pectic substances in the plant kingdom.

Structural features of a pectic arabinogalactan with immunological activity from the leaves of Diospyros kaki.

A water-soluble acidic heteroglycan, DL-3Bb, isolated from the leaves of Diospyros kaki, had [alpha](D)(20) -19.9 degrees (c 0.30, water), and contained rhamnose, arabinose, xylose, galactose and galacturonic acid in the molar ratio of 1.0:4.5:0.7:1.5:1.0. About 44% of the galacturonic acid existed as its methyl ester, and O-acetyl groups (approx 5.7%) were also identified. Its molecular weight was determined to be 9.0x10(5) Da by high-performance gel-permeation chromatography. Its structural features were elucidated by a combination of methylation analysis, periodate oxidation, two steps of partial acid hydrolysis, and 1H and 13C NMR spectroscopy and ESI mass spectrometry. The data obtained indicated that DL-3Bb possessed a backbone of a disaccharide of [-->4)-alpha-GalAp-(1-->2)-alpha-Rhap-(1-->], with approx 58.7% substitution at O-4 of the rhamnopyranosyl residues by beta-(1-->4)-linked xylopyranosyl residues, and by beta-(1-->3) and beta-(1-->6)-linked galactopyranosyl (galactan) residues. The side chains were further substituted by arabinofuranosyl residues at O-2 by beta-(1-->4)-linked xylopyranosyl residues and at O-3 by beta-(1-->6)-linked galactopyranosyl residues. Preliminary tests in vitro revealed that it could stimulate LPS-induced B lymphocyte proliferation, but not for ConA-induced T lymphocyte proliferation. It was proposed that the acid-labile arabinofuranosyl residues in the side chains would not be needed for the expression of the enhancement of the immunological activity, and that the presence of GalAp in the backbone has an important, but not crucial effect on the expression of the activity.