Phenol-sulfuric Acid Method Research Articles

Nicotine promotes pathogenic bacterial growth and biofilm formation in peri-implant.

Introduction. Peri-implantitis is a plaque-associated disease that leads to implant loss and arises from bacterial biofilms on the surface of the implant. Smoking is a risk factor for peri-implantitis and impedes treatment effectiveness. Additionally, aryl hydrocarbon receptor (AHR), IL-6, and IL-22 levels are related to peri-implantitis.Aim. We aimed to investigate the effects of nicotine on inflammatory response, bacterial growth and biofilm formation.Hypothesis/Gap Statement. We hypothesized that nicotine promoted pathogenic bacterial growth and biofilm formation, thereby aggravating inflammation.Methodology. The expression of AHR, IL-6 and IL-22 was measured in peri-implant sulci fluid using quantitative PCR and Western blot analyses. The cementum was incubated with bacterial suspension including Porphyromonas gingivalis, Streptococcus sanguinis and Fusobacterium nucleatum and treated with 100, 200, 250 and 300 µg ml-1 nicotine, and then, the absorbance and number of colony-forming units were detected. Biofilm formation was evaluated using the tissue culture plate method and safranin O staining. Carbohydrates and proteins were measured by the phenol-sulfuric acid method and the bicinchoninic acid method, respectively.Results. The results indicated that smoking increased the levels of AHR, IL-6 and IL-22. Functionally, nicotine promoted the growth of P. gingivalis, S. sanguinis and F. nucleatum. Additionally, it promoted the biofilm formation of these bacteria and increased the contents of carbohydrates and proteins.Conclusion. Nicotine promoted bacterial growth and biofilm build-up, suggesting that smoking may aggravate the progression of peri-implantitis.

RNA-Seq-Based Transcriptome Analysis of Chinese Cordyceps Aqueous Extracts Protective Effect against Adriamycin-Induced mpc5 Cell Injury.

Pharmacogenomic analysis based on drug transcriptome characteristics is widely used to identify mechanisms of action. The purpose of this study was to elucidate the molecular mechanism of protective effect against adriamycin (ADM)-induced mpc5 cell injury of Chinese cordyceps aqueous extracts (WCCs) by a systematic transcriptomic analysis. The phytochemicals of WCCs were analyzed via the "phenol-sulfuric acid method", high-performance liquid chromatography (HPLC), and HPLC-mass spectrometry (MS). We analyzed the drug-reaction transcriptome profiles of mpc5 cell after treating them with WCCs. RNA-seq analysis revealed that WCCs alleviated ADM-induced mpc5 cell injury via restoring the expression of certain genes to normal level mainly in the one-carbon pool by the folate pathway, followed by the relaxin, apelin, PI3K-Akt, and nucleotide-binding, oligomerization domain (NOD)-like receptor signaling pathway, enhancing DNA synthesis and repair, cell proliferation, fibrosis reduction, and immune regulation. Otherwise, WCCs also modulated the proliferation and survival of the mpc5 cell by regulating metabolic pathways, and partially restores the expression of genes related to human disease pathways. These findings provide an innovative understanding of the molecular mechanism of the protective effect of WCCs on ADM-induced mpc5 cell injury at the molecular transcription level, and Mthfd2, Dhfr, Atf4, Creb5, Apln, and Serpine1, etc., may be potential novel targets for treating nephrotic syndrome.

Dellaglioa Algida Cell-Free Supernatant Inhibits Pseudomonas Fluorescence and Pseudomonas Fragi by Destroying Cell Membranes.

The aim of this study was to examine the components of the cell-free supernatant (CFS) derived from a novel strain of psychrophilic Lactobacillus, Dellaglioa algida, and to further elucidate the impact of this CFS on various cellular processes. Specifically, we sought to understand its effects on the cell membrane, protein and DNA release, protease activity, and metabolites of Pseudomonas fluorescens and Pseudomonas fragi, thereby clarifying the antibacterial mechanism involved. The CFS components were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS), the Coomassie Brilliant Blue method, and the phenol-sulfuric acid method. The inhibitory effect of the CFS on Pseudomonas fluorescens and Pseudomonas fragi was assessed using the ethidium bromide (EB) assay, Oxford cup assay, and ultramicroassay. Additionally, we analyzed the metabolites produced by Pseudomonas fluorescens and Pseudomonas fragi when treated with the CFS. The findings reveal that the CFS of Dellaglioa algida contains 94 volatile components, with protein and sugar concentrations of 32.857 ± 0.9705 mg/mL and 98.250 ± 4.210 mg/L, respectively. The CFS induces varying degrees of damage to the cell membranes of both Pseudomonas fluorescens and Pseudomonas fragi, leading to the release of intracellular proteins and DNA. Furthermore, the CFS reduced the protease activity and metabolic capacity of Pseudomonas fluorescens and Pseudomonas fragi. These results enhance our understanding of the mechanism by which psychrophilic Dellaglioa algida inhibits Pseudomonas fluorescens and Pseudomonas fragi, confirming that its inhibitory effect predominantly occurs through damage to the biological cell membranes of Pseudomonas. Dellaglioa algida is a newly identified cold-adapted inhibitor of Pseudomonas, indicating that its CFS is an effective microbial inhibitor in cold environments. This discovery suggests potential applications in inhibiting the growth and reproduction of Pseudomonas fluorescens and Pseudomonas fragi in food, pharmaceuticals, perfumes, and other chemicals, providing a valuable new reference for industrial preservation.

Implications of Ultrasonication-assisted Extraction with Response Surface Methodology on Phytochemical Compositions and Antioxidant Activity of Polysaccharide Extract from Phellinus rimosus (Berk.) Pilát Cultivated Mycelia in Northeastern Thailand

Background: Polysaccharides from the medicinal mushroom Phellinus rimosus (Berk.) Pilát (PR) are the major functional bioactive ingredients. However, there has been a marked natural decrease in the number of PR fruit bodies, leading to their increased cost. Moreover, the natural growth and development of mature PR fruit bodies takes several decades. Objective: The objective of this study was to produce a polysaccharide extract from cultured PR mycelia (PEPRM) by using ultrasonic-assisted extraction with response surface methodology (RSM), and determine its physicochemical composition and antioxidant potential. Methods: Polysaccharide and monosaccharide composition analyses were carried out by Fouriertransform infrared spectroscopy (FT-IR) and High-performance liquid chromatography (HPLC). Total contents of polysaccharides, beta-glucans, phenolic compounds, and flavonoids were investigated utilizing the phenol-sulfuric acid method, enzymatic-based commercial test kit, Folin-Ciocalteu method, and aluminium chloride colorimetric method, respectively. Antioxidant activity was determined by using 2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH) radical scavenging assay and 2,2- azino-bis (3-ethylbenzothiazol-6-sulfonic acid) (ABTS) radical cation decolorization assay. Results: Optimal conditions for the production of PEPRM included a ratio of 51.29 mL water to 1 g PR mycelia and an extraction time of 46.23 minutes, resulting in a total polysaccharide content of 577.5 mg/g of PEPRM. FT-IR spectra of PEPRM showed two broad bands at 3272.08 cm-1 and 2924.8 cm-1 in the carbohydrate region and the peaks at 1078.44, 1019.05, and 853.0 cm-1 indicated the presence of the pyranose ring skeleton, glycosidic linkage, and glucans. PEPRM had molar ratios of glucose: mannose: rhamnose: fucose, i.e., 21.86: 1.00: 2.08: 3.40, respectively. PEPRM had total contents of beta-glucans, phenolic compounds, and flavonoids as percentages of dry weight, i.e., 21.22, 2.51, and 5.71, respectively. PEPRM showed better inhibitory activity against ABTS radicals than DPPH radicals. result: Optimal conditions for the production of PEPRM were a ratio of 51.29 ml water to 1 g PR mycelia and extraction time of 46.23 min, yielding a total polysaccharide content of 577.5 mg/g of PEPRM. FT-IR spectra of PEPRM showed two broad bands at 3272.08 cm-1 and 2924.8 cm-1 in the carbohydrate region and the peaks at 1078.44, 1019.05, and 853.0 cm-1 indicated the presence of the pyranose ring skeleton, glycosidic linkage, and glucans. PEPRM had molar ratios of glucose: mannose: rhamnose: fucose, i.e., 21.86: 1.00: 2.08: 3.40, respectively. PEPRM had total contents of beta-glucan, phenolic compounds, and flavonoids as percentages of dry weight, i.e., 21.22, 2.51, and 5.71, respectively. PEPRM showed better inhibitory activity against ABTS radicals than DPPH radicals. Conclusion: This is the first finding to reveal that ultrasonic-assisted extraction with RSM was an environmentally friendly alternative to produce antioxidant polysaccharides from cultured PR mycelia. conclusion: This is the first finding to reveal that ultrasonic-assisted extraction with RSM was an environmentally friendly alternative to produce antioxidant polysaccharides from cultured PR mycelia.

Biological Characteristics, Artificial Domestication Conditions Optimization, and Bioactive Components of Beauveria caledonica.

In this study, one strain of Beauveria caledonica was isolated from wild fruiting bodies collected from Guizhou Province, China, and its species identification, biological characteristics, domestication, and cultivation methods were studied along with polysaccharide and adenosine content analysis. The mycelia were identified by ITS sequencing, and the fruiting bodies of B. caledonica were domestically cultivated for the first time using wheat and rice as basic cultivation media. The carbon sources, nitrogen sources, cultivation temperatures, and pH for mycelial growth were optimized through single-factor experiments and response surface methodology (RSM) experiments. The polysaccharide content was detected by the phenol-sulfuric acid method, and the adenosine content was measured by high-performance liquid chromatography (HPLC). The results confirmed that the identified mycelia were B. caledonica. The optimum medium for solid culture was 25.8 g/L glycerol, 10.9 g/L yeast extract, 1 g/L MgSO4·7H2O, 1 g/L KH2PO4, 10 mg/L vitamin B1, and 20 g/L agar; the optimum pH was 6.5, and the optimum culture temperature was 25 °C. The optimal liquid culture medium was 26.2 g/L glycerol, 11.1 g/L yeast extract, 1 g/L MgSO4·7H2O, 1 g/L KH2PO4, and 10 mg/L vitamin B1; the mycelia grew well at pH 6.6 and 25 °C. The average biological efficiencies of fruiting bodies on wheat and rice as culture media were 1.880% and 2.115%, respectively; the polysaccharide contents of fruiting bodies on the two media were 6.635% and 9.264%, respectively, while the adenosine contents were 0.145% and 0.150%, respectively. This study provides a valuable reference for further artificial cultivation and utilization of B. caledonica by investigating its biological characteristics, cultivation conditions for artificial domestication, and polysaccharide and adenosine contents in cultivated fruiting bodies.

Combined metabolomics and transcriptomics reveal the secondary metabolite networks in different growth stages of Bletilla striata (Thunb.) Reichb.f.

Bletilla striata (Thunb.) Reichb.f. (B. striata) is a traditional Chinese medicinal herb. B. striata polysaccharides (BSP), stilbenes and 2-isobutyl malic acid glucosoxy-benzyl ester compounds are the main active ingredients in B. striata. However, there is limited report on the changes of medicinal components and their biosynthesis regulation mechanisms in the tubers of B. striata at different stages. The tubers of B. striata were collected during the flowering period, fruiting period, and harvest period to determine the total polysaccharide content using the phenol sulfuric acid method. The changes in secondary metabolites in the tubers at these stages were analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS), and transcriptomics was conducted for further exploration of their biosynthetic pathways. The BSP content gradually increases from the flowering period to the fruiting period as the tubers develop, reaching its peak, but subsequently decreases at harvest time, which may be associated with the germination of B. striata buds in later stage. A total of 294 compounds were identified in this study. Among them, a majority of the compounds, such as 2-isobutyl malate gluconoxy-benzyl ester, exhibited high content during the fruit stage, while stilbenes like coelonin, 3'-O-methylbatatasin III, and blestriarene A accumulated during the harvesting period. The transcriptome data also revealed a substantial number of differentially expressed genes at various stages, providing a partial explanation for the complex changes in metabolites. We observed a correspondence between the expression pattern of GDP-Man biosynthesis-related enzyme genes and cumulative changes in BSP. And identified a positive correlation between 9 transcription factors and genes associated with polysaccharide biosynthesis, while 5 transcription factors were positively correlated with accumulation of 2-isobutyl malate gluconoxy-benzyl ester compounds and 5 transcription factors exhibited negative correlated with stilbene accumulation. It is imperative to determine the appropriate harvesting period based on the specific requirements of different active ingredients and the accumulation patterns of their metabolites. Considering the involvement of multiple transcription factors in the biosynthesis and accumulation of its active ingredients, a comprehensive investigation into the specific regulatory mechanisms that facilitate high-quality cultivation of B. striata is imperative.

Effects of honokiol combined with resveratrol on bacteria responsible for oral malodor and their biofilm

ABSTRACT Background This study aimed to investigate the effect of honokiol combined with resveratrol on bacteria responsible for oral malodor and their biofilm. Method This study investigated drug’s MIC, FICI and dynamic bactericidal susceptibility activities against Pg and Fn. The effects of drugs on biofilm metabolic activity, biofilm total amount, and biofilm microstructure were determined by CCK-8 experiment, semi-quantitative adhesion experiment and SEM, respectively. The effects of drugs on biofilm genes, extracellular polysaccharides, proteins and DNA content were determined by qRT-PCR, phenol-sulfuric acid method, BCA method and Nano Drop one C, respectively. Results The combination had synergistic antibacterial effect on Pg and Fn. 1/2×MIC and 1×MIC combination inhibit the whole process of Pg and Fn growth. The results showed that the combination effectively reduce biofilm metabolic activity and total amount, and destroy biofilm microstructure. The results showed that the combination downregulate the gene expression both Pg and Fn, reduce extracellular polysaccharides and DNA of Pg, and reduce extracellular proteins and DNA of Fn. Conclusion This study showed that the combination had a synergistic antibacterial effect on Pg and Fn, reduced the biofilm extracellular matrix, inhibited biofilm formation, and downregulated the expression of genes related to biofilm formation.

Study on the Skincare Effects of Red Rice Fermented by Aspergillus oryzae In Vitro.

Red rice, a variety of pigmented grain, serves dual purposes as both a food and medicinal resource. In recent years, we have witnessed an increasing interest in the dermatological benefits of fermented rice extracts, particularly their whitening and hydrating effects. However, data on the skincare advantages derived from fermenting red rice with Aspergillus oryzae remain sparse. This study utilized red rice as a substrate for fermentation by Aspergillus oryzae, producing a substance known as red rice Aspergillus oryzae fermentation (RRFA). We conducted a preliminary analysis of RRFA's composition followed by an evaluation of its skincare potential through various in vitro tests. Our objective was to develop a safe and highly effective skincare component for potential cosmetic applications. RRFA's constituents were assessed using high-performance liquid chromatography (HPLC), Kjeldahl nitrogen determination, the phenol-sulfuric acid method, and enzyme-linked immunosorbent assay (ELISA). We employed human dermal fibroblasts (FB) to assess RRFA's anti-aging and antioxidative properties, immortalized keratinocytes (HaCaT cells) and 3D epidermal models to examine its moisturizing and reparative capabilities, and human primary melanocytes (MCs) to study its effects on skin lightening. Our findings revealed that RRFA encompasses several bioactive compounds beneficial for skin health. RRFA can significantly promote the proliferation of FB cells. And it markedly enhances the mRNA expression of ECM-related anti-aging genes and reduces reactive oxygen species production. Furthermore, RRFA significantly boosts the expression of Aquaporin 3 (AQP3), Filaggrin (FLG), and Hyaluronan Synthase 1 (HAS1) mRNA, alongside elevating moisture levels in a 3D epidermal model. Increases were also observed in the mRNA expression of Claudin 1 (CLDN1), Involucrin (IVL), and Zonula Occludens-1 (ZO-1) in keratinocytes. Additionally, RRFA demonstrated an inhibitory effect on melanin synthesis. Collectively, RRFA contains diverse ingredients which are beneficial for skin health and showcases multifaceted skincare effects in terms of anti-aging, antioxidant, moisturizing, repairing, and whitening capabilities in vitro, highlighting its potential for future cosmetic applications.

Effects of different nitrogen application rates and picking batches on the nutritional components of Lycium barbarum L. fruits.

Lycium barbarum L., commonly known as wolfberry, is not only a traditional Chinese medicine but also a highly nutritious food. Its main nutrients include L. barbarum polysaccharide, flavonoid polyphenols, carotenoids, alkaloids, and other compounds, demonstrating its wide application value. This study investigated the effects of nitrogen application on the accumulation of the main nutrients and metabolites in wolfberry fruits under three different nitrogen application rates, namely, N1 (20% nitrogen (N) reduction, 540 kg·ha-2), N2 (medium N, 675 kg·ha-2), and N3 (20% nitrogen increase, 810 kg·ha-2,which is a local conventional nitrogen application amount.). Additionally, due to continuous branching, blossoming, and fruiting of wolfberry plants during the annual growth period, this research also explored the variation in nutritional composition among different harvesting batches. The contents of total sugar and polysaccharide in wolfberry fruit were determined by Fehling reagent method and phenol-sulfuric acid method, respectively;The content of betaine in fruit was determined by high-performance liquid chromatography,and the flavonoids and carotene in the wolfberry fruits were determined by spectrophotometry. Analysis of data over three consecutive years revealed that as nitrogen application increased, the total sugar content in wolfberry fruits initially decreased and then increased. The levels of L. barbarum polysaccharides, total flavonoids, and total carotenoids initially increased and then decreased, while the betaine content consistently increased. Different picking batches significantly impacted the nutrient content of wolfberry fruits. Generally, the first batch of summer wolfberry fruits had greater amounts of total sugar and flavonoids, whereas other nutrients peaked in the third batch. By employing a broadly targeted metabolomics approach, 926 different metabolites were identified. The top 20 differentially abundant metabolites were selected for heatmap generation, revealing that the contents of L-citrulline, 2-methylglutaric acid, and adipic acid increased proportionally to the nitrogen gradient. Conversely, the dibutyl phthalate and 2, 4-dihydroxyquinoline contents significantly decreased under high-nitrogen conditions. The remaining 15 differentially abundant metabolites, kaempferol-3-O-sophorosid-7-O-rhamnoside, trigonelline, and isorhamnosid-3-O-sophoroside, initially increased and then decreased with increasing nitrogen levels. Isofraxidin, a common differentially abundant metabolite across all treatments, is a coumarin that may serve as a potential biomarker for wolfberry fruit response to nitrogen. Differentially abundant metabolites were analyzed for GO pathway involvement, revealing significant enrichment in metabolic pathways and biosynthesis of secondary metabolites under different nitrogen treatments. In conclusion, a nitrogen application of 675 kg·ha-2, 20% less than the local farmers' actual application, was most beneficial for the quality of four-year-old Ningqi 7 wolfberry fruits. Consumers who purchase wolfberry-dried fruit for health benefits should not consider only the first batch of summer wolfberry fruits. These results offer a broader perspective for enhancing the quality and efficiency of the wolfberry industry.

Applicability of enzymatic and phenol–sulfuric acid methods for determination of cellulose nanofibers in ecotoxicity testing

In parallel with the development of novel materials, such as cellulose nanofibers (CNFs), their effects on aquatic organisms should be evaluated. In ecotoxicity testing, concentration measurements are important to obtain accurate exposure concentrations; however, few studies have measured the concentration and dispersion state of CNFs in test solutions. Therefore, we evaluated the applicability of enzymatic and phenol–sulfuric acid methods for CNF quantification in ecotoxicity testing. In the enzymatic method, CNF content was determined by quantifying the d-glucose produced from the CNFs using cellulase with a glucose assay kit. Three types of cellulases were purchased, and impurities such as d-glucose were identified and removed. The differences in CNF degradation efficiency among the cellulases were evaluated, and a cellulase suitable for CNF degradation was selected. In the phenol–sulfuric acid method, a microplate-based method was employed to handle a large number of samples. We demonstrated that both the enzymatic and phenol–sulfuric acid methods could quantify CNFs in media for ecotoxicity testing. The enzymatic method was able to measure lower CNF concentrations (~ 1 mg/L) than the phenol–sulfuric acid method (~ 10 mg/L) and had less interference with CNF quantification by algae and non-cellulosic polysaccharides.

Sugar content improvement by sonication in the pretreatment of empty fruit bunch hydrolysis

The empty palm fruit bunches (EFB) has great potential as an alternative feedstock for bioethanol production due to its high content of cellulose and hemicellulose. However, besides cellulose and hemicellulose, EFB also contains lignin, which can hinder the hydrolysis process and therefore requires delignification. This study aims to determine the effect of sonication in alkali delignification on the sugar content of hydrolysis. Ultrasonic in 37 KHz was performed at a temperature of 80 °C. Sonication process durations ranged from 30 minutes to 150 minutes using a 10 % (w/v) NaOH solvent. The hydrolysis of EFB fibers was carried out in a water bath at 80 °C using a 0.5 N sulfuric acid solvent in a ratio of 1:20 (w/v) for 2 hours. The sugar content was measured using the phenol-sulfuric acid method with UV-Visible spectrophotometry. In this study found that the ultrasonic irradiation time length gave good results at a time limit not exceeding 90 minutes due to hemicellulose characteristics . The highest sugar content was obtained at a sonication duration of 90 minutes, measuring 20.60 mg/L, which was 38.5 % higher than alkali delignification without sonication for 150 minutes. SEM analysis indicated that EFB had undergone changes in the surface morphology and structure. Qualitative FTIR analysis showed that the hydrolysis solution contained glucose and pentose, which are products of hydrolyzed cellulose and hemicellulose.

An improved method to quantify bulk carbohydrate in marine planktonic samples

AbstractThe TPTZ (2,4,6‐tripyridyl‐s‐triazine) method is used to detect monosaccharides from seawater and particulate matter samples because it is sensitive, precise, rapid and easy to perform. Contrary to mechanisms proposed in the literature, we provide evidence that the TPTZ method detects hydroxyl as well as aldehyde groups in monosaccharides when all reducing groups are fully deprotonated in alkaline medium. We use this insight to develop an optimized hydrolysis protocol to increase yields from polysaccharides while minimizing the dehydration of monosaccharides. Compared to the TPTZ method with commonly used hydrolysis protocols and the often‐used phenol–sulfuric acid method, our new optimized method detects a wider range of carbohydrates with a more consistent yield relative to glucose and much lower coefficient of variation. When applied to phytoplankton cultures and marine particulate samples, our new method achieves significantly higher bulk carbohydrate yields.

Heat-Killed Enterococcus faecium KU22001 Having Effective Anti-Cancer Effects on HeLa Cell Lines at a Lower Temperature.

The anti-cancer effects of heat-killed Enterococcus faecium KU22001 (KU22001), KU22002, and KU22005 isolated from human infant feces were investigated. The anti-proliferative activity of these strains against various cancer cell lines was evaluated using the MTT assay. To determine the production of exopolysaccharides (EPS) with potential anti-cancer effect, ethanol precipitation and phenol-sulfuric acid method was used with the cell free supernatant of strains grown at 25°C or 37°C. The EPS yield of E. faecium strains was higher at 25°C than at 37°C. Among these E. faecium strains, KU22001 grown at 25°C was associated with the highest bax/bcl-2 ratio, effective apoptosis rate, cell cycle arrest in the G0/G1 phase, and condensation of the nucleus in the cervical cancer HeLa cell line. In conclusion, these results suggest that KU22001 can be beneficial owing to the anti-cancer effects and production of functional materials, such as EPS.

Antioxidant activity and Cytotoxicity against the Cervical Epithelial Carcinoma (HeLa) Cell Line of Crude Ganoderma lucidum mycelial extracts

This study aimed to investigate the total polysaccharide content, antioxidant activity and cytotoxicity on the Cervical Epithelial Carcinoma (HeLa) cells of the ethanol and water reflux extracts from Ganoderma lucidum mycelia. The phenol-sulfuric acid method was used for the standard method of total polysaccharide analysis, the antioxidant was evaluated by DPPH scavenging assay, ABTS assay, and FRAP assay. The MTT assay was used to determine the cytotoxicity of the extracts. The results showed that the contents of total polysaccharide of the ethanol and water reflux extracts were 8.34 ± 0.47 and 207.81 ± 2.45 mg glucose g extract–1, respectively. The assays of antioxidant capacity against DPPH and ABTS demonstrated that the water reflux extract showed a strong radical scavenging effect than the ethanol extract. For the reducing capability of both extracts with FRAP method, they showed the reducing antioxidant activity and possessed concentration dependence by which the water reflux extract had a higher FRAP antioxidant activity than the ethanol extract. The cytotoxic assay of the extracts on normal mammalian cells (Vero cells) revealed that the ethanol extract of Ganoderma lucidum mycelia had a cytotoxic effect on normal mammalian cells at 62.5 – 1,000 mg mL–1, whereas the water reflux extract had no cytotoxicity to normal mammalian cells in all tested concentration. Then, the water reflux extract was selected to determine the cytotoxicity against the HeLa cancer cell line. The results indicated that the water reflux extract of Ganoderma lucidum mycelia exerted a significant cytotoxic effect on HeLa cancer cell line in possessed concentration dependence. The water reflux extract caused a 56.30% decrease in cervical epithelial carcinoma cell viability with concentration of 1 mg mL–1. Therefore, the results of this research demonstrated that the extract of Ganoderma lucidum mycelia provide both antioxidant and anti-cancer activities, which could be used as ingredients pharmaceutical products and functional foods. GRAPHICAL ABSTRACT HIGHLIGHTS Antioxidant activity and Cytotoxicity of Crude Ganoderma lucidum mycelial extracts

Quantification of Mixed-Linkage β-Glucan (MLG) in Bacteria.

Prokaryotes are known to produce and secrete a broad range of biopolymers with a high functional and structural heterogeneity, often with critical duties in the bacterial physiology and ecology. Among these, exopolysaccharides (EPS) play relevant roles in the interaction of bacteria with eukaryotic hosts. EPS can help to colonize the host and assist in bacterial survival, making this interaction more robust by facilitating the formation of structured biofilms. In addition, they are often key molecules in the specific recognition mechanisms involved in both beneficial and pathogenic bacteria-host interactions. A novel EPS known as MLG (Mixed-Linkage β-Glucan) was recently discovered in rhizobia, where it participates in bacterial aggregation and biofilm formation and is required for efficient attachment to the roots of their legume host plants. MLG is the first and, so far, the only reported linear Mixed-Linkage β-glucan in bacteria, containing a perfect alternation of β (1→3) and β (1→4) bonds. A phylogenetic study of MLG biosynthetic genes suggests that far from being exclusive of rhizobia, different soil and plant-associated bacteria likely produce MLG, adding this novel polymer to the plethora of surface polysaccharides that help bacteria thrive in the changing environment and to establish successful interactions with their hosts.In this work, a quantification method for MLG is proposed. It relays on the hydrolysis of MLG by a specific enzyme (lichenase), and the subsequent quantification of the released disaccharide (laminaribiose) by the phenol-sulfuric acid method. The protocol has been set up and optimized for its use in 96-well plates, which makes it suitable for high-throughput screening (HTS) approaches. This method stands out by its fast processing, technical simplicity, and capability to handle multiple samples and biological replicates at a time.

Quantification of antigen of Mycoplasma capricolum subsp. capripneumoniae by optical assay

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP) in goats. Inactivated vaccines and capsular polysaccharide (CPS) indirect hemagglutination reagents are available for prevention and serological detection, but high culture costs and complex antigen quantification have been plagued by production staff. In order to solve these problems in production practice, a sugar fermentation medium with an initial pH value of 7.8, which could improve the production of two antigens simultaneously, was screened out by changing the initial pH value based on previous Mccp metabolomics analysis. Since phenol red can be identified by UV absorption spectrum and cetyltrimethylammonium bromide (CTAB) can bind to anionic capsular polysaccharide, a UV spectrum measurement method for analyzing the culture stage reached by Mccp and a CTAB precipitation test for relative quantification of capsular polysaccharide antigen content in the fermentation broth were established. The UV spectrum observation method can guide the production of Mccp according to the growth curve of Mccp, which greatly reduces the monitoring time of the traditional CCU method and improves the accuracy of the original eye-observation method. The established CTAB precipitation test can complete the monitoring of CPS content within 5 hours, which greatly reduces the time required compared with the traditional differential technique, and its accuracy was verified by the phenol-sulfuric acid method. The optimized culture medium and the two correlation comparison methods established in this study can effectively reduce the production cost of Mccp and improve the production efficiency. The two assays have been used in the research at our laboratory, which provides experimental data for further improvement of the production process of CCPP inactivated vaccine and capsular polysaccharide as well as rapid quantification.

Effects of Hydrolysis Condition and Detection Method on the Monosaccharide Composition Analysis of Polysaccharides from Natural Sources

Monosaccharide composition analysis is essential to the structural characterization and research into the biological activity of polysaccharides. In this study, a systematic comparison was performed among commonly used monosaccharide composition analysis methods, including colorimetric and chromatographic methods. These were tested on 16 aldoses, ketoses, alditols, amino sugars, and uronic acids. Furthermore, the effect of hydrolysis methods was also investigated. The results showed that the phenol sulfuric acid method is greatly affected by the type of monosaccharide that is used as the reference substance. The determination of uronic acid using sulfuric acid carbazole is less affected by neutral sugars than that method using m-hydroxybiphenyl. The high-performance thin-layer chromatography (HPTLC) method can simultaneously analyze multiple samples and accurately determine the type of uronic acid. High-performance liquid chromatography (HPLC) can provide a good qualitative and quantitative analysis of aldose, amino sugars, and uronic acids, while gas chromatography–mass spectrometry (GC-MS) can detect aldose, ketose, and alditols. Fructose was detected in a large amount in inulin and Codonopsis pilosula after one-step hydrolysis, while it was totally destroyed in two-step hydrolysis. The release of galacturonic acid significantly increased after two-step hydrolysis in pectin and Lycium barbarum, which indicated that one-step hydrolysis is not enough for acidic polysaccharides. The results of this study are beneficial for selecting appropriate hydrolysis and analysis methods in order to accurately analyze the monosaccharide compositions of natural polysaccharides.

Sugar Substitute Stevia Inhibits Biofilm Formation, Exopolysaccharide Production, and Downregulates the Expression of Streptococcal Genes Involved in Exopolysaccharide Synthesis.

Acid production by sucrose fermentation disturbs the balance in dental plaque by lowering the oral pH. As a consequence of the profound effect of sucrose on caries initiation and progression, many studies have been directed towards finding non-cariogenic artificial sweeteners that can be used as a substitute to sucrose. Existing literature shows that dietary sucrose upregulates the expression of biofilm associated genes involved in exopolysaccharide (EPS) production. In this study, we aimed to investigate the effect of the sugar substitute stevia on biofilm formation, EPS secretion, and streptococcal genes encoding glucan-binding proteins (Gbps) and glucosyltransferases (Gtfs), which are essential for the synthesis of EPS. Streptococcus mutans and Streptococcus gordonii were grown as biofilm cultures with or without stevia and sucrose. Biomass was quantified for biofilm and EPS production by crystal violet staining and the phenol-sulfuric acid method, respectively. Expression of gtfB and gbpB genes was studied by RT-PCR. The quantities of biofilm were significantly lower when grown in the presence of stevia compared to sucrose in both species (p < 0.05). The proportion of EPS in the biofilm pellet decreased with increasing concentrations of stevia in both species but remained nearly unchanged with sucrose with respect to the control. In both streptococcal species, exposure of stevia decreased the expression of gtfB and gbpB genes compared to sucrose (p < 0.05). In comparison to the untreated control, the expression was decreased in the presence of stevia in both species, while it increased 2.5- to 4-fold in S. mutans and 1.5- to 2.5-fold in S. gordonii in the presence of sucrose. The ability of stevia to inhibit biofilm formation, reduce EPS production, and downregulate the expression of gtfB and gbpB genes in S. mutans and S. gordonii may have potential therapeutic applications in controlling dental plaques and caries.

Mulberry leaf multi-components exert hypoglycemic effects through regulation of the PI-3K/Akt insulin signaling pathway in type 2 diabetic rats

Ethnopharmacological relevancePhytochemicals have unique advantages in the treatment of diabetes due to their multi-target activity and low toxicity. Mulberry leaves, a traditional Chinese herbal medicine, have been used in the prevention and treatment of diabetes for centuries. The main active ingredients in mulberry leaves with regards to the hypoglycemic effect are 1-deoxynojirimycin, flavonoids, and polysaccharides. However, the combined hypoglycemic effects and mechanisms of mulberry leaf multi-components remain unclear. Aim of the studyThis study explored the anti-diabetic effects of mulberry leaf multi-components (MMC) and the role of the PI-3K/Akt insulin signalling pathway in improving insulin resistance. Materials and methodsThe main chemical components of MMC were analyzed using the phenol-sulfuric acid method, aluminum nitrate-sodium nitrite method, and HPLC-ultraviolet/fluorescence detection method. The T2DM rat model was created via feeding a high-fat diet and peritoneal injection of streptozotocin. T2DM rats were divided into four groups: model, model plus metformin, model plus low-dose, and model plus high-dose MMC groups (100 and 200 mg/kg body weight/day, respectively), and plus normal group for a total of five groups. MMC was administered by oral gavage for six weeks. Fasting blood glucose and serum lipid profiles were measured using a glucometer and an automatic biochemistry analyzer, respectively. Serum insulin and adipocytokine levels were analyzed by ELISA. Hepatic glucose metabolizing enzyme activity was evaluated by ELISA and the double antibody sandwich method. Expression of PI-3K/Akt signalling pathway proteins was analyzed by RT-PCR and Western blotting. ResultsExtracted 1-deoxynojirimycin, flavonoid, and polysaccharide purity was 70.40%, 52.34%, and 32.60%, respectively. These components were then mixed at a ratio of 1:6:8 to form MMC. MMC significantly reduced serum glucose, insulin, and lipid levels. In diabetic rats, MMC enhanced insulin sensitivity and alleviated inflammatory and oxidative damage by lowing adipocytokine levels and increasing anti-oxidative enzyme activity. Insulin resistance was also mitigated. MMC regulated the activity of key downstream enzymes of hepatic glucose metabolism via activating the expression of PI-3K, Akt, PDX-1, and GLUT4 at the mRNA and protein levels, thereby correcting hepatic glucolipid metabolism disorders and exerting a hypoglycemic effect. ConclusionMMC ameliorated hepatic glucolipid metabolism disorders and improved insulin resistance in T2DM rats by activating the PI-3K/Akt signaling pathway. These results highlight the multi-component, multi-target, and combined effects of MMC, and suggest it may be further developed as a hypoglycemic drug.

Inonotus obliquus polysaccharide are linear molecules that alter the abundance and composition of intestinal microbiota in Sprague Dawley rats.

The macromolecular polysaccharide Inonotus obliquus polysaccharide (IOP) is composed of various monosaccharides, and it could modulate the composition and diversity of intestinal flora. However, its impact on the intestinal flora in rats of different genders remains unclear. Therefore, this study aims to investigate the structural changes of IOP and its effects on the intestinal flora after administration in male and female rats. In this study, the molecular weight and purity of IOP were analyzed by high-performance gel permeation chromatography (HPGPC) and phenol sulfuric acid method, and NMR was used to confirm the chemical structure of IOP. Sex hormone [testosterone (T) and estradiol (E2)] levels and intestinal microbial changes were detected by enzyme-linked immunosorbent assay (ELISA) and 16S rRNA, respectively, after gavage of IOP (100 mg/kg) in male and female Sprague Dawley (SD) rats. HPGPC analysis showed that the average molecular weight (Mw) of IOP was 4,828 Da, and the total sugar content of the purified IOP was 96.2%, indicating that the polysaccharide is of high purity. NMR revealed that IOP is a linear macromolecule with an α-D-type glucose backbone. The results of ELISA and 16S rRNA showed that the IOP increased the abundance of beneficial bacteria, such as Clostridia_UCG-014 and Prevotellaceae_NK3B31, and reduced that of harmful bacteria, such as Colidextribacter and Desulfobacterota in the intestine of both male and female rats, and IOP changed the levels of sex hormones in male and female rats. Further analyses revealed that the increase in alpha diversity was higher in male than female rats. α diversity and β diversity revealed a significant difference in the composition of cecal microbiota between male and female rats in the control group, but IOP intake reduced this difference. Meanwhile, α analysis revealed a change in the composition of bacterial flora was more stable in male than female rats. This study enhances our comprehension of the IOP structure and elucidates the alterations in intestinal flora following IOP administration in rats of varying genders. Nonetheless, further investigation is warranted to explore the specific underlying reasons for these discrepancies.