New meroterpenoids and polyketides from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1 and their anti-inflammatory and SARS-CoV-2 Mpro inhibitory activities.

Seven new meroterpenoids, paraphaeones A-G (1-7), and two new polyketides, paraphaeones H-I (8-9), along with eight known compounds (10-17), were isolated from the endophytic fungus Paraphaeosphaeria sp. C-XB-J-1. The structures of 1-9 were identified through the analysis of 1H, 13C, and 2D NMR spectra, assisted by HR-ESI-MS data. Compounds 1 and 7 exhibited a dose-dependent decrease in lactate dehydrogenase levels, with IC50 values of 1.78 µM and 1.54 µM, respectively. Moreover, they inhibited the secretion of IL-1ß and CASP-1, resulting in a reduction in the activity levels of NLRP3 inflammasomes. Fluorescence microscopy results indicated that compound 7 concentration-dependently attenuated cell pyroptosis. Additionally, compounds 4 and 7 showed potential inhibitory effects on the severe acute respiratory syndrome coronavirus-2 main protease (SARS-CoV-2 Mpro), with IC50 values of 10.8 ± 0.9 µM and 12.9 ± 0.7 µM, respectively.

Bioactive constituents from Salacia cochinchinensis.

Two new isopimarane diterpenoids, named 1α-hydroxy-7-oxoisopimara-8, 15-diene (1), 11ß-hydroxy-7-oxoisopimara-8(14), 15-diene (2), together with six known compounds (3-8), were isolated from the medicinal plant Salacia cochinchinensis. All isolates were assayed for their cytotoxicity and α-glucosidase inhibitory activity. Results suggested compounds 1, 3 possessed significant cytotoxic activity against HepG2, HL60, and Hela cell lines with IC50 values ranging from 0.23 to 0.35 µM, and compounds 7, 8 exhibited noticeable α-glucosidase inhibitory ability with IC50 values of 0.25 and 0.31 µM, respectively.

[A new guaiane-type sesquiterpenoid from Croton yunnanensis].

Five sesquiterpenoids were isolated from 90% ethanol extract of Croton yunnanensis by silica gel,Sephadex LH-20 column chromatography,as well as prep-HPLC methods. Based on MS,1 D and 2 D NMR spectral analyses,the structures of the five compounds were identified as 11-methoxyl alismol(1),6ß,7ß-epoxy-4α-hydroxyguaian-10-ene(orientalol C,2),multisalactone D(3),arvestonol(4),and 4,5-dihydroblumenol A(5). Compound 1 was a new guaiane-type sesquiterpenoid. Compounds 2-4 were isolated from the Croton genus for the first time,and compound 5 was obtained from this plant for the first time.

Four new triterpene glucosides from Salacia cochinchinensis Lour.

Four new triterpene glucosides (1-4) were isolated from the 90% ethanol extract of Salacia cochinchinensis, together with five known compounds (5-9). The structures of the new compounds were elucidated by comprehensive spectroscopic analysis including HRESIMS, IR, 1 D and 2 D NMR analysis. All isolates were assayed for their α-glucosidase inhibitory activity. Compound 9 showed remarkable α-glucosidase inhibitory activity with an IC50 value of 0.31 µM, and the triterpene glycosides (1-5) exhibited moderate α-glucosidase inhibitory activity.

Bioactive glycosides from Salacia cochinchinensis.

Four new triterpene glycosides, named salaciacochinosides A-D (1-4) were isolated from the 90% ethanol extract of Salacia cochinchinensis, together with five known compounds 2α,3ß,23-trihydroxyurs-12,18-dien-28-oic acid 28-O-ß-d-glucopyranoside (5), racemiside (6), alangiplatanoside (7), acantrifoside E (8), and syringin (9). The structures of the four new triterpenoids were characterized by chemical methods and MS, IR, 1D and 2D NMR spectral analyses. The α-glucosidase inhibitory activities of the nine compounds were assessed, compounds 6 and 7 showed remarkable α-glucosidase inhibitory activities, with IC50 values of 0.44 and 0.75 µM, respectively. Compounds 1-5 exhibited moderate α-glucosidase inhibitory activities, and compounds 8 and 9 showed none α-glucosidase inhibitory activity in our current experiments.

Porphyromonas gingivalis infection accelerates intimal thickening in iliac arteries in a balloon-injured rabbit model.

BACKGROUND: Current epidemiologic data suggest that a localized infection (periodontitis) can disseminate into the distant tissues, and subgingival bacteria can migrate in the bloodstream, thereby contributing to independent systemic disease processes. To test this hypothesis, we investigated the effect of repeated systemic inoculations with Porphyromonas gingivalis (Pg) on intimal hyperplasia in iliac arteries in a rabbit model of balloon injury. METHODS: One week after single balloon injury to the iliac artery, 30 male New Zealand rabbits were randomly assigned to intravenous inoculation with 100 microl live Pg (10(7) colony-forming units; n = 15) or vehicle (n = 15) once weekly for 4, 8, or 12 consecutive weeks. Arteries were fixed by perfusion and removed for analysis of neointimal lesion formation. We measured intimal and medial lesion areas in iliac artery cross-sections as well as the intimal/medial ratio (I/M). We also analyzed Pg 16S ribosomal DNA amplification with polymerase chain reaction, systemic proinflammatory mediators with enzyme-linked immunosorbent assay, and immunolocalization of macrophages in the balloon-injured arteries. RESULTS: At 12 weeks, iliac intimal hyperplasia was accelerated, and I/M was significantly increased in Pg-inoculated animals (I/M 3.961 +/- 0.536 in the Pg group versus 3.585 +/- 0.353 in the control animals; P <0.01). Pg-inoculated animals also had significant increases in macrophage infiltration at 12 weeks, C-reactive protein levels at all time points, and interleukin-6 levels at 12 weeks. Moreover, Pg ribosomal DNA was found in the injured arteries of Pg-inoculated animals, but only after 12 weeks. CONCLUSION: Long-term systemic challenge with Pg, an oral pathogen, may accelerate intimal hyperplasia in balloon-injured iliac arteries.

Triterpenoid saponins from the roots of Cyathula officinalis and their inhibitory effects on nitric oxide production.

The present study was designed to investigate the chemical constituents of the roots of Cyathula officinalis. Compounds were isolated by silica gel, Sephadex LH-20, ODS column chromatography, and preparative HPLC. Their structures were determined on the basis of 1D and 2D NMR techniques, mass spectrometry, and chemical methods. One new oleanane-type triterpenoid saponin, 28-O-[α-L-rhamnopyranosyl-(1→3)-ß-D-glucuronopyranosyl-(1→3)-ß-D-glucopyranosyl] hederagenin (1), was isolated from the roots of Cyathula officinalis. The anti-inflammatory activities of the isolates were evaluated for their inhibitory effects against LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages cells. Compounds 2, 4, and 6 exhibited moderate anti-inflammatory activities.

Prevalence and quantification of the uncommon Archaea phylotype Thermoplasmata in chronic periodontitis.

OBJECTIVE: Chronic periodontitis is a chronic inflammatory disease of the periodontal tissues and is caused by invasion of certain types of bacteria and Archaea, with Methanobrevibacter oralis as the predominant archaeon. In this study, we investigated the prevalence and quantity of the newly discovered Archaea phylotype Thermoplasmata in patients with chronic periodontitis. METHODS: Subgingival plaque samples were obtained from 49 patients with chronic periodontitis and 45 periodontally healthy subjects. Qualitative analyses of Archaea and class Thermoplasmata were carried out by amplification of 16S rRNA genes in DNA extracts from plaque samples, and all the samples were quantitatively analyzed by real-time polymerase chain reaction (PCR). RESULTS: The prevalence of Archaea in patients with chronic periodontitis was 69.4% according to the conventional PCR results, but was 87.8% according to real-time PCR. In the control group, three samples were detected as positive, but none of these were confirmed in qualitative analyses. The prevalence of class Thermoplasmata was 18.4% by nested PCR and 24.5% by quantitative PCR in the chronic periodontitis group. The prevalence of Thermoplasmata was significantly lower than that of total Archaea. The relative abundances of Archaea and Thermoplasmata varied among samples. Thermoplasmata were not the predominant archaeons in the subgingival dental plaque. Among the clinical parameters of patients with periodontitis, probing depth was positively associated with Archaea detection. CONCLUSIONS: The existence of Archaea was correlated closely with the presence of chronic periodontitis. Thermoplasmata represented a minor archaeon in periodontal infection.

Exploring the dynamic core microbiome of plaque microbiota during head-and-neck radiotherapy using pyrosequencing.

Radiotherapy is the primary treatment modality used for patients with head-and-neck cancers, but inevitably causes microorganism-related oral complications. This study aims to explore the dynamic core microbiome of oral microbiota in supragingival plaque during the course of head-and-neck radiotherapy. Eight subjects aged 26 to 70 were recruited. Dental plaque samples were collected (over seven sampling time points for each patient) before and during radiotherapy. The V1-V3 hypervariable regions of bacterial 16S rRNA genes were amplified, and the high-throughput pyrosequencing was performed. A total of 140 genera belonging to 13 phyla were found. Four phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) and 11 genera (Streptococcus, Actinomyces, Veillonella, Capnocytophaga, Derxia, Neisseria, Rothia, Prevotella, Granulicatella, Luteococcus, and Gemella) were found in all subjects, supporting the concept of a core microbiome. Temporal variation of these major cores in relative abundance were observed, as well as a negative correlation between the number of OTUs and radiation dose. Moreover, an optimized conceptual framework was proposed for defining a dynamic core microbiome in extreme conditions such as radiotherapy. This study presents a theoretical foundation for exploring a core microbiome of communities from time series data, and may help predict community responses to perturbation as caused by exposure to ionizing radiation.

Characterization of oral bacterial diversity of irradiated patients by high-throughput sequencing.

The objective of this study was to investigate the compositional profiles and microbial shifts of oral microbiota during head-and-neck radiotherapy. Bioinformatic analysis based on 16S rRNA gene pyrosequencing was performed to assess the diversity and variation of oral microbiota of irradiated patients. Eight patients with head and neck cancers were involved in this study. For each patient, supragingival plaque samples were collected at seven time points before and during radiotherapy. A total of 147,232 qualified sequences were obtained through pyrosequencing and bioinformatic analysis, representing 3,460 species level operational taxonomic units (OTUs) and 140 genus level taxa. Temporal variations were observed across different time points and supported by cluster analysis based on weighted UniFrac metrics. Moreover, the low evenness of oral microbial communities in relative abundance was revealed by Lorenz curves. This study contributed to a better understanding of the detailed characterization of oral bacterial diversity of irradiated patients.

[The influence of different alkaline pH conditions on Enterococcus faecalis in planktonic and biofilm mode].

PURPOSE: To study the influence of different pH conditions on Enterococcus faecalis(E. faecalis) in planktonic and biofilm mode. METHODS: E. faecalis were prepared in planktonic and biofilm mode and cultured in TSB mediums 2 hours at pH 7,8,9,10,11 and 12. MTT assays were applied to evaluate the survival rate of bacterial cells in different pH value. SAS 6.12 software package was used for statistical analysis. RESULTS: Mild alkaline mediums (pH7-9) had no effect on cell vitality of E. faecalis and high alkaline condition (pH>10) led to significant declines of survival rate of cells. The biofilm cells of E. faecalis were more alkaline tolerant than corresponding planktonic cells. CONCLUSION: Biofilm formation is an important step in the development of alkaline tolerance of E. faecalis.

[Observation of genetic diversity in dental plaque of elder people with root caries].

PURPOSE: Bacterial community in dental plaque of elder people was analyzed to learn about the microhabitat composition and diversity. METHODS: Dental plaque samples were collected from 25 elders. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to evaluate the microbial diversity by displaying PCR-generated 16SrDNA fragments that migrate at different distances, reflecting the different sequence of fragment. SPSS12.0 software was used to analyze the variance of genotypes between different groups of bacteria. RESULTS: Genotypes of bacteria in dental plaques in the root caries group was significantly more than the other two groups. Crown caries group and caries-free group had no significant difference. CONCLUSIONS: The genetic diversity of the dental plaque microflora in the root caries group is significantly higher than coronal caries group and caries-free group.

[Analysis of community composition in dental plaque of elder people with root caries].

OBJECTIVE: To analyze the community in dental plaque of elder people with root caries. METHODS: Total DNAs were extracted from the root caries dental plaques of nine elders over 60 years of age. Polymerase chaid reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial composition, DGGE bands were excised from the gels for sequencing and identification. RESULTS: The dominant genus in root caries dental plaque of elder people were: Acinetobacte [0.9% (1/114)], Actinobaculum [1.8% (2/114)], Actinomyces [15.8% (18/114)], Aggregatibacter [0.9% (1/114)], Capnocytophaga [14.0% (16/114)], Corynebacterium [0.9% (1/114)], Haemophilus [0.9% (1/114)], Mobiluncus [0.9% (1/114)], Naxibacter [0.9% (1/114)], Neisseriaceae [10.5% (12/114)], Porphyromonas [0.9% (1/114)], Prevotella [12.3% (14/114)], Selenomonas [6.1% (7/114)], Staphylococcus [1.8% (2/114)], Oralis streptococcus [6.1% (7/114)], Mutans streptococcu [7.9% (9/114)], Tannerella [0.9% (1/114)], Treponema [1.8% (2/114)], Veillonella [10.5% (12/114)] and two uncultured unknown genus [1.8% (2/114)]. Uncultred genotypes accounted for 19.30% of the total. Gram-positive bacteria genotype accounted for 31.6% (36/114), and Gram-negative bacteria genotype accounted for 66.7% (76/114). CONCLUSIONS: There were many bacteria genotypes in root caries dental plaque in the elderly, which were widely distributed. Gram-negative bacteria accounted for the majority. Genotype-specific pathogenic bacteria were not found.

Effects of intensity-modulated radiotherapy on human oral microflora.

This study aimed to evaluate changes in the biodiversity of the oral microflora of patients with head and neck cancer treated with postoperative intensity-modulated radiotherapy (IMRT) or conventional radiotherapy (CRT). Pooled dental plaque samples were collected during the radiation treatment from patients receiving IMRT (n = 13) and CRT (n = 12). Denaturing gradient gel electrophoresis (DGGE) was used to analyze the temporal variation of these plaque samples. The stimulated and unstimulated salivary flow rates were also compared between IMRT and CRT patients. Reductions in the severity of hyposalivation were observed in IMRT patients compared with CRT patients. We also observed that the temporal stability of the oral ecosystem was significantly higher in the IMRT group (69.96 ± 7.82%) than in the CRT group (51.98 ± 10.45%) (P < 0.05). The findings of the present study suggest that IMRT is more conducive to maintaining the relative stability of the oral ecosystem than CRT.

[Nifedipine regulated expression of bcl-2 in human gingival epithelial cells in vitro].

PURPOSE: To investigate the effect of nifedipine(NIF) on transcription of bcl-2 in human gingival epithelial cells(HGECs) in vitro and to study the pathogenesis of epithelial thickening in drug-induced gingival overgrowth(DGO). METHODS: The gingival tissues obtained from periodontal surgeries were digested with enzyme and HGECs were cultured in vitro; HGECs were identified by immunohistochemistry; bcl-2 mRNA levels were quantitated by Real-time PCR 24 hours and 48 hours after cells stimulated by NIF with different concentration (0microg/ml, 1microg/ml,2microg/ml,3microg/ml), in which 0microg/ml NIF as blank control. The data was analyzed by one-way ANOVA using SPSS 11.0 software package. RESULTS: HGECs cultured in vitro showed keratin positive signal and vimentin negative signal; the level of bcl-2 mRNA increased with NIF 3microg/ml after 24 hours treatment, which appeared significant increase compared with blank control (P<0.05); after 48 hours treatment the level of bcl-2 mRNA in the groups of 2microg/ml and 3microg/ml showed significant increase compared with blank control (P<0.05). CONCLUSION: NIF regulates the level of bcl-2 mRNA.

Modeling of diffusion transport through oral biofilms with the inverse problem method.

AIM: The purpose of this study was to develop a mathematical model to quantitatively describe the passive transport of macromolecules within dental biofilms. METHODOLOGY: Fluorescently labeled dextrans with different molecular mass (3 kD, 10 kD, 40 kD, 70 kD, 2000 kD) were used as a series of diffusion probes. Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum were used as inocula for biofilm formation. The diffusion processes of different probes through the in vitro biofilm were recorded with a confocal laser microscope. RESULTS: Mathematical function of biofilm penetration was constructed on the basis of the inverse problem method. Based on this function, not only the relationship between average concentration of steady-state and molecule weights can be analyzed, but also that between penetrative time and molecule weights. CONCLUSION: This can be used to predict the effective concentration and the penetrative time of anti-biofilm medicines that can diffuse through oral biofilm. Furthermore, an improved model for large molecule is proposed by considering the exchange time at the upper boundary of the dental biofilm.

[Quantitative evaluation of residual endodontic microorganisms after mechanical root canal preparation different chemical preparations by real-time PCR].

PURPOSE: To establish a quick, sensitive method for quantifying root canal flora and investigate the effects of different root canal preparations on the pathogenic bacteria at RNA level. METHODS: A total of 24 single-rooted teeth with chronic apical periodontitis were selected and prepared using 3% H2O2 combined with 1% NaClO, EDTA combined with 3% H(2)O(2),1% NaClO, respectively,the samples were taken before and after root canal preparation. After isolation of total RNA from the root canal samples, cDNA was synthesized by reverse transcription, and detected by real-time PCR. The data were analyzed with SAS 6.12 software package. RESULTS: The number of bacteria in the root canal reduced dramatically after mechanical preparation and irrigated using 3% H(2)O(2) and 1% NaClO(P<0.01). Further combined with EDTA, its effect was better than that of simply irrigated using 3% H(2)O(2) and 1% NaClO(P<0.05). CONCLUSIONS: Real-time PCR can be employed in the identification of bacteria flora in the root canal, both methods of root canal preparation can effectively reduce the number of bacteria flora.

[Identification and quantitative analysis of Archaea involved in periodontal disease].

OBJECTIVE: To make qualitative and quantitative analysis of Archaea in subgingival plaque sample and to investigate the relationship between periodontal disease and Archaea. METHODS: Subgingival plaque was collected from 23 patients with aggressive periodontitis, 29 with chronic periodontitis, 35 with plaque-induced gingivitis and 38 healthy controls. Qualitative and quantitative analysis of methanogenic archaea was performed by amplification of the 16S rRNA genes in the DNA extracted from the plaque samples. RESULTS: Archaea were found in 65% of aggressive periodontitis patients, 72% of chronic periodontitis, 26% of gingivitis and zero of healthy subjects. Quantitative analysis showed the average abundance of archaeal 16S rRNA gene in Archaea-positive patients was different among the three groups. The average 16S rRNA gene copy number from per microg wet plaque was 6.66 x 10(6) in aggressive periodontitis sufferers, 4.47 x 10(6) in chronic periodontitis and 1.78 x 10(6) in gingivitis groups. The prevalence of Archaea and the average Archaea 16S rRNA gene numbers in periodontitis groups were higher than those in gingivitis group (P < 0.05). CONCLUSIONS: This suggests that Archaea may be implicated as causative agents for periodontitis.

[The impact of Yishenqinghuo recipe on alveolar bone reconstruction of rats with experimental periodontitis].

PURPOSE: To evaluate the impact of Yishenqinghuo recipe on alveolar bone reconstruction of rats with experimental periodontitis. METHODS: 12-months old Spague-Dawley rats were selected for the study. All rats were randomly divided into 4 groups. Group A: control group (with no periodontitis model, fed with the same dosage of saline as Group D); Group B: model group (with periodontitis model, fed with the same dosage of saline as Group D); Group C: high-dosage group (with periodontitis model, fed with doubling dosage of medicine as Group D); Group D: equivalent-dosage group (with periodontitis model, fed with clinical equivalent effective dosage of medicine). After being gavaged with medicine/saline for 3 months, the alveolar bone were collected to make undecalcified histological slices and evaluate the changes of alveolar bone histomorphometric parameters. All the results were analyzed by ANOVA, with the use of SAS 6.04 software package. RESULTS: Compared with group B, the formation of osteoid were increased and had less free ending trabecular in group C and D. The percent age of trabecular area (%Tb.Ar) and node-terminus ratio (NTR) of alveolar bone for this 4 groups were 75.24+/-3.82/1.49+/-0.12, 45.78+/-6.70/0.48+/-0.08, 73.33+/-4.20/1.33+/-0.06 and 67.69+/-2.83/1.26+/-0.10,respectively. This two parameters of group B were much lower than that in other three groups (P<0.01). At the same time, the NTR in group D was significantly lower than that in group A (P<0.01). The osteoid area (Os.Ar) of different groups were (88.44+/-7.52) microm(2), (145.37+/-13.91) microm(2), (211.10+/-22.96) microm(2) and (201.22+/-24.75) microm(2) (transformation variables), respectively. The Os.Ar of group A was lowest, and that in group B was lower than group C and D, there were significant differences(P<0.01). CONCLUSIONS: From this study, we conclude that Yishenqinghuo recipe is in favor of enhancing alveolar bone quantity, improving bone structure and reconstructing bone loss of rats with experimental periodontitis. Supported by National Key Technologies R&D Program (Grant No.2004BA726) and Natural Science Foundation of Shanghai Municipality (Grant No.03ZR14081).

[Comparison of the sucrose dependent cell adherence of Streptococcus mutans isolates from caries-active and caries-free children].

PURPOSE: To study the sucrose dependent cell adhesive ability of Streptococcus mutans islolated from the caries-active and caries-free children. METHODS: 60 isolated Streptococcus mutans strains were selected and identified from the dental plaque of 10 caries-active children and 10 caries-free children (3 to 5 years), in which 39 strains were from caries-active group(dmfs>or=6) and 21 strains from caries-free group(dmfs=0). With the use of ultraviolet spectrophotometer, the sucrose dependent cell adherence to glass wall of the sucrose-containing testing tubes was analyzed. One-way ANOVA was used by SPSS12.0 software package to determine the statistical difference of the adhesive ability between the two groups. RESULTS: The average adhesive ratio of the Streptococcus mutans strains isolated from caries-active group was 55.49%+26.16% in the 1% sucrose-containing culture medium, while the average adhesive ratio of the Streptococcus mutans strains isolated from caries-free group was 27.01%+18.39%. The difference between the two groups was statistically significant (P<0.01). CONCLUSION: In the sucrose-containing circumstance, the sucrose dependent cell adhesive ability of the Streptococcus mutans isolated from the caries-active children was significantly higher than that from the caries-free children. This indicated that the adhesive ability may be related to the caries-causing tendency.