Multiple Fingerprint-Activity Relationship Assessment of Immunomodulatory Polysaccharides from Ganoderma lucidum Based on Chemometric Methods.

Polysaccharides with molecular weights ranging from 1.75 × 103 to 1.14 × 104 g/mol were obtained from the fruit bodies of Ganoderma lucidum. The multiple fingerprints and macrophage immunostimulatory activity of these fractions were analyzed as well as the fingerprint-activity relationship. The correlation analysis of molecular weight and immune activity demonstrated that polysaccharides with molecular weights of 4.27 × 103~5.27 × 103 and 1 × 104~1.14 × 104 g/mol were the main active fractions. Moreover, the results showed that galactose, mannose, and glucuronic acid were positively related to immunostimulatory activity. Additionally, partial least-squares regression and grey correlation degree analyses indicated that three peaks (P2, P3, P8) in the oligosaccharide fragment fingerprint significantly affected the immune activity of the polysaccharides. Hence, these ingredients associated with activity could be considered as markers to assess Ganoderma lucidum polysaccharides and their related products, and the study also provides a reference for research on the spectrum-effect relationship of polysaccharides in the future.

Rapid recognition of volatile organic compounds with colorimetric sensor arrays for lung cancer screening.

Volatile organic compounds (VOCs) in breath can be used as biomarkers to identify early stages of lung cancer. Herein, we report a disposable colorimetric array that has been constructed from diverse chemo-responsive colorants. Distinguishable difference maps were plotted within 4 min for specifically targeted VOCs. Through the consideration of various chemical interactions with VOCs, the arrays successfully discriminate between 20 different volatile organic compounds in breath that are related to lung cancer. VOCs were identified either with the visualized difference maps or through pattern recognition with an accuracy of at least 90%. No uncertainties or errors were observed in the hierarchical cluster analysis (HCA). Finally, good reproducibility and stability of the array was achieved against changes in humidity. Generally, this work provides fundamental support for construction of simple and rapid VOC sensors. More importantly, this approach provides a hypothesis-free array method for breath testing via VOC profiling. Therefore, this small, rapid, non-invasive, inexpensive, and visualized sensor array is a powerful and promising tool for early screening of lung cancer. Graphical abstract A disposable colorimetric array has been developed with broadly chemo-responsive dyes to incorporate various chemical interactions, through which the arrays successfully discriminate 20 VOCs that are related to lung cancer via difference maps alone or chemometrics within 4 min. The hydrophobic porous matrix provides good stability against changes in humidity.

Highly Sensitive Detection of Complicated Mutations of Drug Resistance in Mycobacterium tuberculosis Using a Simple, Accurate, Rapid, and Low-Cost Tailored-Design Competitive Wild-Type Blocking Assay.

Establishing simple, rapid, and highly sensitive molecular assays is crucial for timely diagnosis and effective treatment of drug-resistant tuberculosis. However, current genotypic drug susceptibility testing (DST) still encounters enormous challenges including lower sensitivity than phenotypic DST and insufficient accuracy. Herein, a simple, low-cost, multiplex real-time polymerase chain reaction-based assay is established to achieve highly sensitive detection of low-abundant mutants through competitive wild-type blocking (COWTB). Analytical performance of the COWTB assay can achieve 1% or even 0.1% mutants under background of 10 000 wild-type genomes/test. Furthermore, clinical practice feasibility is evaluated to identify resistance to rifampicin (RIF), isoniazid (INH), and streptomycin (SM) on 92 actual clinical samples, its sensitivity is 93.8% for RIF and 100% for INH and SM, and specificity is 100% each for RIF, INH, and SM when using DNA sequencing as the reference standard. In comparison, the sensitivity of reverse dot blotting assay commonly used in clinics is 93.8%, 90.0%, and 84.6%, and the specificity is 96.1%, 98.6%, and 100% for RIF, INH, and SM, respectively. Importantly, the COWTB assay can also be applicable for other drug-resistant mutations and pave a promising detection strategy to fill the gap between phenotypic and genotypic DST for detecting low-abundant drug-resistant M. tuberculosis.

Functional Components from the Liquid Fermentation of Edible and Medicinal Fungi and Their Food Applications in China.

Functional raw materials rich in various effective nutrients and active ingredients that are of stable quality can be obtained from the liquid fermentation of edible and medicinal fungi. In this review, we systematically summarize the main findings of this comparative study that compared the components and efficacy of liquid fermented products from edible and medicinal fungi with those from cultivated fruiting bodies. Additionally, we present the methods used in the study to obtain and analyze the liquid fermented products. The application of these liquid fermented products in the food industry is also discussed. With the potential breakthrough of liquid fermentation technology and the continued development of these products, our findings can serve as a reference for further utilization of liquid fermented products derived from edible and medicinal fungi. Further exploration of liquid fermentation technology is necessary to optimize the production of functional components from edible and medicinal fungi, and to enhance their bioactivity and safety. Investigation of the potential synergistic effects of combining liquid fermented products with other food ingredients is also necessary to enhance their nutritional values and health benefits.

Anti-inflammatory and intestinal microbiota modulation properties of Ganoderma lucidum ß-d-glucans with different molecular weight in an ulcerative colitis model.

This study systematically investigated the therapeutic effects and the corresponding mechanisms of ß-D-glucans from Ganoderma lucidum (G. lucidum) with different molecular weights (Mws) on ulcerative colitis (UC). Results showed that three ß-d-glucans (GLPS, GLPN and GLPW) from G. lucidum with different Mws exhibited the significant activities on the reduction of typical symptoms of UC by regulating inflammatory cytokine levels, modulating intestinal immunity, improving intestinal microbiota and metabolism of short-chain fatty acids (SCFAs) in the dextran sulfate sodium (DSS)-induced mice model. Among them, the effects of the microwave assisted degraded fraction (GLPW) mainly containing two fractions with smaller Mw (1.33 × 104 and 3.51 × 103 g/mol) on the regulation of inflammatory factors and SCFAs metabolism were found to be comparable to those of GLPN with medium Mw (3.49 × 104 g/mol), and superior to those of GLPS with large Mw (2.42 × 106 g/mol). The effect of GLPW on regulation of intestinal microbiota was even better than that of GLPN. These findings suggested that lowering Mw by means of physical degradation could improve the anti-inflammatory activities of G. lucidum ß-d-glucans. The analysis of anti-inflammatory mechanism also provided a feasible and theoretical basis for potential use of degraded ß-d-glucans in the prevention and treatment of UC.

Kaempferol inhibits renal fibrosis by suppression of the sonic hedgehog signaling pathway.

BACKGROUND: Most chronic kidney diseases (CKDs) develop to end-stage renal disease (ESRD), which is characterized by fibrosis and permanent tissue and function loss. As a result, better and more effective remedies are essential. Kaempferol (KAE) is a common flavonoid extracted from plants. It can control the progression of kidney fibrosis and the epithelial-to-mesenchymal transition (EMT) of the renal tubular system. PURPOSE: We aim to investigate the effect of KAE therapy on extracellular matrix deposition and stimulation of EMT in vitro and in vivo to elucidate the treatment mechanisms regulating these effects. STUDY DESIGN: Chronic hypertension-induced kidney fibrosis was studied in spontaneously hypertensive rats with chronic kidney disease. Biochemical analysis, histological staining, and the expression level of relative proteins were used to assess the effect of KAE on renal function and fibrosis. The direct impact of KAE on proliferation and migration was evaluated using human renal tubular epithelial cells (HK-2) induced by transforming growth factor-ß1 (TGF-ß1), which can then induce EMT. The molecular mechanism of KAE was verified using co-IP assay and immunofluorescence. RESULTS: KAE could reduce blood pressure and decrease the extracellular matrix (ECM) components (including collagen I and collagen Ш), TGF-ß1, and α-SMA in the kidneys of hypertension-induced rats with chronic kidney disease. Moreover, in HK-2 cell treated with TGF-ß1, KAE administration significantly suppressed proliferation, migration, and EMT via increasing the expression of E-cadherin, while reducing the N-cadherin and α-SMA. Sufu was exceedingly repressed in HK-2 cells treated with TGF-ß1. KAE inhibited the activation of Shh and Gli through increasing the expression of Sufu, thereby blocking the nuclear translocation of Gli1 in vitro. CONCLUSION: KAE ameliorated kidney fibrosis and EMT by inhibiting the sonic hedgehog signaling pathway, thereby to attenuate the pathological progression of hypertensive kidney fibrosis.

Structure-Activity Relationship of Novel ACE Inhibitory Undecapeptides from Stropharia rugosoannulata by Molecular Interactions and Activity Analyses.

Undecapeptide is the central peptide molecule in the peptide base material of Stropharia rugosoannulata, and angiotensin-converting enzyme (ACE) plays a crucial role in hypertension. To fully explore the interaction mechanism and ACE-inhibitory activity of long-chain peptides from Stropharia rugosoannulata, the binding conformations of twenty-seven undecapeptides with the ACE receptor were revealed by molecule docking. The undecapeptide GQEDYDRLRPL with better receptor binding capacity and higher secondary mass spectral abundance was screened. All amino acid residues except proline in GQEDYDRLRPL interacted with the ACE receptor. GQEDYDRLRPL interfered with the receptor's overall structure, with significant fluctuations in amino acid residues 340-355, including two residues in the receptor's active pockets. The binding constants of GQEDYDRLRPL to the ACE receptors were at the µM level, with a kinetic binding constant of 9.26 × 10-7 M, which is a strong binding, and a thermodynamic binding constant of 3.06 × 10-6 M. Intermolecular interaction were exothermic, enthalpy-driven, and specific binding reactions. GQEDYDRLRPL had an IC50 value of 164.41 µmol/L in vitro and superior antihypertensive effects at low-gavage administration in vivo. Obtaining information on the interaction mechanism of ACE-inhibitory undecapeptides from S. rugosoannulata with the ACE receptor will help to develop and utilize ACE inhibitors of natural origin.

Research on Extraction, Structure Characterization and Immunostimulatory Activity of Cell Wall Polysaccharides from Sparassis latifolia.

The cell wall polysaccharides were extracted from Sparassis latifolia fruit bodies by acid-alkali and superfine-grinding assisted methods, and the chemical characterization and in vitro immunity activities of these polysaccharide fractions were studied and compared. Results showed that superfine-grinding assisted extraction exhibited the highest yield of polysaccharides (SP, 20.80%) and low ß-glucan content (19.35%) compared with alkaline extracts. The results revealed that the 20% ethanol precipitated fraction (20E) from SP was mainly composed of ß-(1→3)-glucan and α-(1→4)-glucan. With the increase of ethanol precipitation, the fractions (30E, 40E, 50E) were identified as α-(1→4)-glucan with different molecular weights and conformations. Cell wall polysaccharides extracted through NaOH (NSP) and KOH (KSP) extraction had similar yields with 8.90% and 8.83%, respectively. Structural analysis indicated that the purified fraction from KSP (KSP-30E) was a ß-(1→3)-glucan backbone branched with ß-(1→6)-Glcp, while the purified fraction from NSP (NSP-30E) mainly contained ß-(1→3)-glucan with a small number of α-linked-Glcp. The two fractions both exhibited rigid chain conformation in aqueous solutions. All polysaccharide fractions exerted the activity of activating Dectin-1 receptor in vitro, and the KSP-30E mainly identified as ß-(1→3)-glucan with the terminal group via 1→6-linkage attached at every third residue exhibited a stronger enhancing effect than other fractions. Results suggested that KOH extraction could be efficient for the preparation of bioactive ß-(1→3, 1→6)-glucan as a food ingredient.

Effects of molecular weight on intestinal anti-inflammatory activities of ß-D-glucan from Ganoderma lucidum.

To investigate the influence of molecular weight (M w) on the anti-inflammatory activity of ß-D-glucan from Ganoderma lucidum, ultrasonic irradiation was applied to treat the ß-D-glucan (GLP, 2.42 × 106 g/mol) solution to obtain two degraded fractions with molecular weight of 6.53 × 105 g/mol (GLPC) and 3.49 × 104 g/mol (GLPN). Structural analysis proved that the degraded fractions possessed similar repeated units with the original ß-D-glucan. The in vitro anti-inflammatory activity studies showed that all fractions could significantly inhibit LPS-induced expression of cytokines including TNF-α, IL-8, MIF and MCP-1 in Caco-2 cells at certain concentrations. Moreover, GLPC and GLPN exhibited better anti-inflammatory activity than GLPC. The intestinal anti-inflammatory activity evaluated by dextran sulfate sodium (DSS)-induced colitis mice model showed that intragastric administration of GLPN (lower M w fraction) could significantly recover inflamed tissues of mice. Compared with GLP and GLPC, GLPN exhibited stronger ability to inhibit the secretion of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6). The results revealed that M w of ß-D-glucan influenced its anti-inflammatory activity and decreasing of M w would improve the activity, which provided evidence for the potential use of ß-D-glucan from G. lucidum as anti-colitis ingredients.

Effects of Oleic Acid Addition Methods on the Metabolic Flux Distribution of Ganoderic Acids R, S and T's Biosynthesis.

The effects of oleic acid addition methods on the metabolic flux distribution of ganoderic acids R, S and T's biosynthesis from Ganoderma lucidum were investigated. The results showed that adding filter-sterilized oleic acid in the process of submerged fermentation and static culture is of benefit to the synthesis of ganoderic acids R, S and T. The metabolic fluxes were increased by 97.48%, 78.42% and 43.39%, respectively. The content of ganoderic acids R, S and T were 3.11 times, 5.19 times and 1.44 times higher, respectively, than they were in the control group, which was without additional oleic acid. Ganoderic acids R, S and T's synthesis pathways (GAP), tricarboxylic acid cycles (TCA), pentose phosphate pathways (PP) and glycolysis pathways (EMP) were all enhanced in the process. Therefore, additional oleic acid can strengthen the overall metabolic flux distribution of G. lucidum in a submerged fermentation-static culture and it can reduce the accumulation of the by-product mycosterol. This study has laid an important foundation for improving the production of triterpenes in the submerged fermentation of G. lucidum.

Structure and chain conformation of bioactive ß-D-glucan purified from water extracts of Ganoderma lucidum unbroken spores.

Two polysaccharide fractions (GLSB50 and GLSB70) with total sugar content of 82.07 wt% and 53.79 wt%, respectively, were obtained from the water extracts of unbroken Ganoderma lucidum spores by sequential ethanol precipitation treatment. Compared with GLSB70, GLSB50 exhibited better activity on stimulation of humoral immune responses in immunosuppressed mice. A novel ß-D-glucan (GLSB50A-III-1) with weight average molecular weight (Mw) of 1.93 × 105 g/mol was purified from GLSB50 through chromatography separation. The exponent α value of Mark-Houwink-Sakurada equation was calculated to be 0.13, indicating that GLSB50A-III-1 presented globular spheres conformation in aqueous solution. Structural analysis showed that GLSB50A-III-1 mainly consisted of (1 â†’ 3), (1 â†’ 4), (1 â†’ 6)-linked ß-d-glucose residues in the backbone, with two single ß-D-Glcp attached at O-6 of ß-(1 â†’ 3) and ß-(1 â†’ 4)-linked residues separately as side chains. The repeat unit of GLSB50A-III-1 was deduced as follows.

A tailored LNA clamping design principle: Efficient, economized, specific and ultrasensitive for the detection of point mutations.

In the development of personalized medicine, the ultrasensitive detection of point mutations that correlate with diseases is important to improve the efficacy of treatment and guide clinical medication. In this study, locked nucleic acid (LNA) was introduced as an amplification suppressor of a massive number of wild-type alleles in an amplification refractory mutation system (ARMS) to achieve the detection of low-abundance mutations with high specificity and sensitivity of at least 0.1%. By integrating the length of clamp, base type, number and position of LNA modifications, we have established a "shortest length with the fewest LNA bases" principle from which each LNA base would play a key role in the affinity and the ability of single base discrimination could be improve. Finally, based on this LNA design guideline, a series of the most important single point mutation sites of epidermal growth factor receptor (EGFR) was verified to achieve the optimal amplification state which as low as 0.1% mutation gene amplification was not affected under the wild gene amplification was completely inhibited, demonstrating that the proposed design principle has good applicability and versatility and is of great significance for the detection of circulating tumor DNA.

The Combination of Scutellaria baicalensis Georgi and Sophora japonica L. ameliorate Renal Function by Regulating Gut Microbiota in Spontaneously Hypertensive Rats.

Chronic kidney disease (CKD) is becoming a notable health concern globally. The combination of Scutellaria baicalensis Georgi (SB) and Sophora japonica L. (SJ) has been demonstrated to have anti-hypertensive effects and improve kidney injury clinically. This study aimed to explore the renal protective effect of the combination of SB and SJ against CKD and clarify the potential mechanisms. Male spontaneously hypertensive rats (SHR) were used to induce hypertensive nephropathy and were treated with SB or SJ separately or in combination for 15 weeks, and an antibiotic group was used for a rescue experiment. Blood pressure, serum or urine biochemical markers, serum inflammation factors, short-chain fatty acids (SCFAs), indoxyl sulfate (IS), and oxidative stress indicators were assessed. Western blot analysis was performed to determine the expression of intestinal tight junction proteins, including occludin and ZO-1. The mRNA expression of the SCFAs receptors olfactory 78 (Olfr78) and G protein-coupled receptor 41 (GPR41) was determined by quantitative real-time PCR. Gut microbiota profiles were established via high-throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene. SB and SJ significantly ameliorated the severity of renal injury induced by hypertension. The combination also decreased the ratio of Firmicutes/Bacteroidetes, increased the relative abundance of Lactobacillus, and reduced that of Clostridiaceae. The intestinal barrier was improved, and the change in dominant bacteria reduced IS accumulation and further inhibited oxidative stress activation in kidneys. SB and SJ increased SCFAs production, inhibited inflammatory factor release, and regulated blood pressure by decreasing the expression of Olfr78 and increasing that of GPR41, then alleviated kidney damage. This research demonstrated the positive effects of SB and SJ in a rat model of hypertensive nephropathy, indicated that the treatment of SB and SJ by improving the intestinal barrier function, increasing SCFAs, reducing inflammation, decreasing IS, and inhibiting oxidative stress reactions.

Regioselective sulfation of ß-glucan from Ganoderma lucidum and structure-anticoagulant activity relationship of sulfated derivatives.

In the present study, regioselective sulfation of ß-glucan (GLP) from Ganoderma lucidum were firstly established by using 4,4'-dimethoxytrityl chloride and hexamethyldisilazane as protecting precursor. 2,4,6-O-sulfated, 6-O-sulfated and 2,4-O-sulfated GLP derivatives were prepared and the molecular weights (Mw) of derivatives were determined to range from 0.94 × 104 to 6.27 × 104 g/mol, while the degrees of sulfation (DS) were calculated to vary from 0.83 to 1.74. The regioselective sulfation of GLP was confirmed by FT-IR, 13C NMR spectroscopy and methylation analysis. Results indicated that the sulfated substitution sites were predominantly at C-6 in 6-O-sulfated GLP (S6-OGLP) and C-4 in 2,4-O-sulfated GLP (S2,4-OGLP), respectively. Clotting assays (APTT, PT and TT) in vitro showed that sulfate groups were essential for anticoagulant activity and S6-OGLP exhibited much higher than others. Meanwhile, sulfated GLP with higher DS and Mw showed stronger anticoagulant activity in the case of the same condition.

Structural characterization and immuno-enhancing activity of a highly branched water-soluble ß-glucan from the spores of Ganoderma lucidum.

A water soluble ß-glucan (GLSWA-I) with a weight average molecular weight ∼1.57×105g/mol, isolated from the aqueous extract of the broken cellular wall Ganoderma lucidum spores, was purified by anion-exchange and gel-permeation chromatography. The immunological activity test in vivo showed that GLSWA-I could significantly promote dinitrochlorobenzene (DNCB) induced delayed-type ear swelling in mice. Based on the monosaccharides composition analysis, methylation analysis, IR, 1D and 2D NMR spectroscopy, the repeating unit of polysaccharide GLSWA-I was elucidated as follows.

A novel Ganoderma lucidum G0119 fermentation strategy for enhanced triterpenes production by statistical process optimization and addition of oleic acid.

A novel enhanced triterpenes fermentation production process by Ganoderma lucidum G0119 with the addition of oleic acid in the medium has been developed and optimized. All of the six exogenous additives tested were found to exhibit stimulatory effect on mycelial growth and triterpenes biosynthesis by G. lucidum. The results show that oleic acid addition had significant role in promoting triterpenes production. The optimal concentration and time of oleic acid addition were determined to be 30 mL/L and 0 h, respectively. Furthermore, three significant factors influencing triterpenes production were identified as glucose, magnesium sulfate and temperature using the Plackett-Burman design. The optimized conditions by central composite design were 27.83 g/L glucose, 1.32 g/L magnesium sulfate, 26.2°C temperature. The triterpenes fermentation yield with the optimized medium based on actual confirmatory experimental data in 6 L fermentor was 1.076 g/L versus the statistical model predicted value of 1.080 g/L. Our innovatively developed triterpenes fermentation production technology and process has been proven to produce high triterpenes productivity and yield conceivably useful for industrial production.

Characterization of Polysaccharides from the Fruiting Bodies of Two Species of Genus Ganoderma (Agaricomycetes) and Determination of Water-Soluble ß-D-Glucan Using High-Performance Liquid Chromatography.

Molecular weight (Mw) distributions of polysaccharides from the fruiting bodies of different Ganoderma lucidum strains and G. sinense were investigated and compared using high-pressure size exclusion chromatography/multiangle laser light scattering/refractive index analysis. Results showed that there were big differences in the Mw distributions and characteristics of polysaccharides from 2 species of Ganoderma. All tested G. lucidum materials exhibited similar polysaccharide distributions and similar characteristics for each fraction. The fraction with highest Mw (peak 1) was identified as ß-(1→3)-linked D-glucan with (1→6)-ß-D-glucopyranosyl side branches. G. sinense fruiting bodies did not include the ß-D-glucan when compared with G. lucidum. A high-pressure size exclusion chromatography method was developed and applied to determine the amount of high-Mw ß-D-glucan in G. lucidum fruiting bodies. Results indicated that there was no obvious relationship between ß-D-glucan content and the genetic similarity of G. lucidum. The strain labeled "Longzhi no. 2" was determined to possess the largest amount of ß-D-glucan: 8.2 mg/mL based on the dry weight of fruiting bodies. The ß-D-glucan content in the hot water extract of Longzhi no. 2 reached 17.05%. For the "Hunong no. 1" strain, the ß-D-glucan content in log-cultivated fruiting bodies was much higher than that in bag-cultivated ones. This method could be used to improve quality control of polysaccharides in G. lucidum.

Biological Activities of Ethanol Extracts of Phellinus baumii (Higher Basidiomycetes) Obtained by Different Fermentation Methods.

Phellinus baumii was used for fermentation, and 3 corresponding ethanol extracts were obtained by 3 different methods: extract I, liquid fermentation; extract II, solid fermentation in polypropylene plastic bags with medium mainly consisting of sawdust and wheat bran; and extract III, solid fermentation in culture bottles with medium mainly consisting of rice. Ethanol extract I presented the best inhibition ability on HepG2 cell growth; inhibiting rates were 48.2% and 65.0% at doses of 10 and 100 µg/mL, respectively. Ethanol extracts II and III had a better regeneration effect on injured PC12 neural cells than extract I. The superoxide radical, hydrogen peroxide radical, and DPPH radical scavenging activities of ethanol extract III was better than those of the other 2 extracts.

Antitumor Compounds from the Stout Camphor Mushroom Taiwanofungus camphoratus (Higher Basidiomycetes) Spent Culture Broth.

A known compound, 5-(hydroxymethyl) furan-2-carbaldehyde, and a novel compound, 3-isobutyl-1-methoxy-4-(4'-(3-methylbut-2-enyloxy)phenyl)-1H-pyrrole-2,5-dione were isolated from spent broth from submerged cultures of Taiwanofungus camphoratus. Their structures were elucidated by nuclear magnetic resonance (1H, 13C, and 2D) and mass spectra. These compounds inhibited the proliferation of K562 and HepG2 tumor cells in vitro.