Harzianic acids and oxazolidinone from the endophytic fungus Ilyonectria sp. and their cytotoxicity activity.

Four undescribed compounds including three harzianic acids (1, 3 and 4) and one oxazolidinone (2), along with three known ones (5-7) were isolated from the solid fermented product of endophytic fungus Ilyonectria sp., their structures were elucidated as 1-amino-harzianic acid (1), ilyonectria-oxazolidinone (2),10'-nor- isoharzianic acid (3), isohomoharzianic acid (4), harzianic acid (5), isoharzianic acid (6), homoharzianic acid (7) by means of detailed chemical evidences and spectroscopic data analysis. All the compounds were evaluated for cytotoxicity against SMMC-7721 human cancer cell lines by MTS assay. Among the seven tested compounds, 1-amino-harzianic acid (1) demonstrated well cytotoxic activity against SMMC-7721 with IC50 value of 26.84 µM. The results of molecular docking indicated that compound exhibited moderate anti-tumor activity may through binding to apoptosis related proteins.

Network Pharmacology Study on Herb Pair Bletilla striata-Galla chinensis in the Treatment of Chronic Skin Ulcers.

BACKGROUND: Herb pair Bletilla striata-Galla chinensis (BS-GC) is a classic combination of topical traditional Chinese medicine formulae in the treatment of chronic skin ulcers (CSUs). OBJECTIVE: The aim of this study is to explore the effective active ingredients of BS-GC, as well as the core targets and signal transduction pathways of its action on CSUs. METHODS: The ingredients of BS-GC were obtained from TCMSP and HERB databases. The targets of all active ingredients were retrieved from the SwissTargetPrediction database. The targets of CSUs were obtained from OMIM, GeneCards, Drugbank, and DisGeNET databases. A drug-disease target protein-protein interaction (PPI) network was constructed to select the most core targets, and an herb-ingredient-target network was built by utilizing Cytoscape 3.7.2. Furthermore, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes database (KEGG) analysis and verified the results of network pharmacology through molecular docking. RESULTS: A total of 40 active ingredients from the herb pair BS-GC were initially screened, and a total of 528 targets were retrieved. Meanwhile, the total number of CSU targets was 1032. Then, the number of common targets between BS-GC and CSUs was 107. The 13 core targets of herb pair BS-GC with CSUs were filtered out according to the PPI network, including AKT1, TNF, EGFR, BCL2, HIF1A, MMP-9, etc. The 5 main core active ingredients were 1-(4-Hydroxybenzyl)-2-methoxy-9,10-dihydrophenanthrene-4,7-diol, 1-(4- Hydroxybenzyl)-4-methoxy-9,10-dihydrophenanthrene-2,7-diol, physcion, dihydromyricetin, and myricetin. The main biological processes were inflammation, oxidative stress, and immune response, involving the AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, NF-κB signaling pathway, and calcium signaling pathway. Molecular docking results showed good binding activity between the 5 main core active ingredients and 13 core targets. CONCLUSION: This study predicted the core targets and signal transduction pathways in the treatment of CSUs to provide a reference for further molecular mechanism research.

[Effects of salt stress on bacterial community composition and diversity in rhizosphere soil of Bletilla striata]. / ç›èƒè¿«å¯¹ç™½åŠæ ¹é™ ç»†èŒç¾¤è½ç»„æˆåŠå¤šæ ·æ€§çš„å½±å“.

Salinization environment affects the normal growth and development of plants, as well as the microbial community in the rhizosphere. To explore the succession dynamics of bacterial communities in the rhizosphere soil of Bletilla striata under salt stress condition, we performed 16S rRNA high-throughput sequencing to determine the bacterial community composition and diversity of B. striata in the rhizosphere under different salt stress concentrations, measured the effects of salt stress on the growth and development of B. striata and soil physicochemical pro-perties, and analyzed the correlation between community composition of rhizosphere bacteria and the soil environmental factors. The results showed that compared with the control, salt stress reduced growth rate and health degree of B. striata, and significantly decreased the content of soil organic matter, nitrogen and phosphorus. Under the salt stress treatment, species diversity and evenness of the bacterial communities in the rhizosphere of B. striata showed a trend of first decreasing and then increasing. There were significant differences in the relative abundance and variation trends of the dominant bacterial taxa in the rhizosphere soil of B. striata at the phylum and class levels between the control and the salt stress treatments. Salt stress intensity and duration were important factors affecting bacterial community composition in the rhizosphere soil of B. striata. Soil organic matter, available nitrogen, and total phosphorus content were key environmental factors affecting the structure of rhizosphere bacterial community composition. Functional genes related to cytoskeleton, cell motility, substance metabolism and signal transduction mechanisms may be involved in the adaptation and stress response of bacterial communities to salt stress. This study would provide theoretical basis and reference for the cultivation management of B. striatain saline area.

A dual network cross-linked hydrogel with multifunctional Bletilla striata polysaccharide/gelatin/tea polyphenol for wound healing promotion.

Wound healing is a dynamic and complex biological process, and traditional biological excipients cannot meet the needs of the wound healing process, and there is an urgent need for a biological dressing with multifunctionality and the ability to participate in all stages of wound healing. This study developed tea polyphenol (TP) incorporated multifunctional hydrogel based on oxidized Bletilla striata polysaccharide (OBSP) and adipic acid dihydrazide modified gelatin (Gel-ADH) with antimicrobial, antioxidant hemostatic, and anti-inflammatory properties to promote wound healing. The composite OBSP, Gel-ADH, TP (OBGTP) hydrogels prepared by double crosslinking between OBSP, TP and Gel-ADH via Schiff base bonding and hydrogen bonding had good rheological and swelling properties. The introduction of TP provided the composite hydrogel with excellent antioxidant antibacterial activities against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coil). In the rat liver hemorrhage model and skin injury model, the OBGTP composite hydrogel had significant (p < 0.001) hemostatic ability, and had the ability to accelerate collagen deposition, reduce the expression of inflammatory factors, and promote rapid wound healing. In addition, OBGTP hydrogels had adhesive properties and good biocompatibility. In conclusion, OBGTP multifunctional composite hydrogels have great potential for wound healing applications.

Bletilla striata polysaccharides protect against ARDS by modulating the NLRP3/caspase1/GSDMD and HMGB1/TLR4 signaling pathways to improve pulmonary alveolar macrophage pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bletilla striata polysaccharides (BSP) extracted from the B. striata tuber, have been demonstrated to possess anti-inflammatory properties. However, their potential protective effect against ARDS and their role in regulating cell pyroptosis remained unexplored. AIM OF THE STUDY: The aim of this study was to investigate the therapeutic effect of BSP in the alleviation of lipopolysaccharide (LPS)-induced ARDS, and to explore its mechanism of action. METHODS: The effect of BSP was assessed by LPS injection into the intraperitoneal cavity in vivo; pathological changes of ARDS mice were gauged by immunohistochemical, hematoxylin and eosin staining, and immunofluorescence assays. MH-S cells were used to model the pyroptosis in vitro. Finally, the pyroptosis of alveolar macrophage was detected by western blots, qPCR, and flow cytometry for NLRP3/caspase1/GSDMD and HMGB1/TLR4 pathway-associated proteins and mRNA. RESULTS: BSP could significantly increase the weight and survival rate of mice with ARDS, alleviate the cytokine storm in the lungs, and reduce lung damage in vivo. BSP inhibited the inflammation caused by LPS/Nigericin significantly in vitro. Compared with the control group, there was a remarkable surge in the incidence of pyroptosis observed in ARDS lung tissue and alveolar macrophages, whereas BSP significantly diminished the pyroptosis ratio. Besides, BSP reduced NLRP3/caspase1/GSDMD and HMGB1/TLR4 levels in ARDS lung tissue and MH-S cells. CONCLUSIONS: These findings proved that BSP could improve LPS-induced ARDS via inhibiting pyroptosis, and this effect was mediated by NLRP3/caspase1/GSDMD and HMGB1/TLR4, suggesting a therapeutic potential of BSP as an anti-inflammatory agent for ARDS treatment.

Exploration of the anti-inflammatory, analgesic, and wound healing activities of Bletilla Striata polysaccharide.

Bletilla Striata (BS) Polysaccharide (BSP) is one of the main components of the traditional Chinese medicinal plant Bletilla striata Rchb. F. BSP has been widely used in antimicrobial and hemostasis treatments in clinics. Despite its use in skin disease treatment and cosmetology, the effects of BSP on wound healing remain unclear. Here we investigated the anti-inflammatory, antioxidant, and analgesic effects of BSP and explored its impact on morphological changes and inflammatory mediators during wound healing. A carrageenan-induced mouse paw edema model was established to evaluate the anti-inflammatory effect of BSP. Antioxidant indicators, including NO, SOD, and MDA, were measured in the blood and liver. The increased pain threshold induced by BSP was also determined using the hot plate test. A mouse excisional wound model was applied to evaluate the wound healing rate, and HE staining and Masson staining were used to detect tissue structure changes. In addition, ELISA was employed to detect the expression of pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß in serum. BSP significantly decreased the concentration of NO and MDA in serum and liver while increasing SOD activity. It exhibited a notable improvement in mouse paw edema induced by carrageenan. BSP dose-dependently delayed the appearance of licking behavior in mice, indicating its analgesic effect. Compared to the control group, the wound healing rate was significantly improved in the BSP treatment group. HE and Masson staining results showed that the BSP and 'Jingwanhong' ointment groups had slightly milder inflammatory responses and significantly promoted more new granulation tissue formation. The levels of serum inflammatory mediators TNF-α, IL-1ß, and IL-6 were reduced to varying degrees. The results demonstrated that BSP possesses anti-inflammatory, antioxidant, analgesic, and wound healing properties, and it may promote wound healing through inhibition of inflammatory cytokine synthesis and release.

The "crosstalk" between microbiota and metabolomic profile in high-fat-diet-induced obese mice supplemented with Bletilla striata polysaccharides and composite polysaccharides.

The potential prebiotic feature of Bletilla striata polysaccharides (BSP) has been widely accepted, while the beneficial effect of BSP on high-fat-diet-induced obesity is unclear. Moreover, the "crosstalk" between microbiota and metabolomic profile in high-fat-diet-induced obese mice supplemented with BSP still need to be further explored. The present study attempted to illustrate the effect of BSP and/or composite polysaccharides on high-fat-diet-induced obese mice by combining multi-matrix (feces, urine, liver) metabolomics and gut microbiome. The results showed that BSP and/or composite polysaccharides were able to reduce the abnormal weight gain induced by high-fat diet. A total of 175 molecules were characterized by proton nuclear magnetic resonance (1H NMR) in feces, urine and liver, suggesting that multi-matrix metabolomics could provide a comprehensive view of metabolic regulatory mechanism of BSP in high-fat-diet-induced obese mice. Several pathways were altered in response to BSP supplementation, mainly pertaining to amino acid, purine, pyrimidine, ascorbate and aldarate metabolisms. In addition, BSP ameliorated high-fat-diet-induced imbalanced gut microbiome, by lowering the ratio of Firmicutes/Bacteroidetes. Significant correlations were illustrated between particular microbiota's features and specific metabolites. Overall, the anti-obesity effect of BSP could be attributed to the amelioration of the disorders of gut microbiota and to the regulation of the "gut-liver axis" metabolism.

Effect fraction of Bletilla striata (Thunb.) Reichb.f. alleviates LPS-induced acute lung injury by inhibiting p47phox/NOX2 and promoting the Nrf2/HO-1 signaling pathway.

BACKGROUND & AIMS: The effect fraction of Bletilla striata (Thunb.) Reichb.f. (EFBS), a phenolic-rich extract, has significant protective effects on lipopolysaccharide (LPS)-induced acute lung injury (ALI), but its composition and molecular mechanisms are unclear. This study elucidated its chemical composition and possible protective mechanisms against LPS-induced ALI from an antioxidant perspective. METHODS: EFBS was prepared by ethanol extraction, enriched by polyamide column chromatography, and characterized using ultra-performance liquid chromatography/time-of-flight mass spectrometry. The LPS-induced ALI model and the RAW264.7 model were used to evaluate the regulatory effects of EFBS on oxidative stress, and transcriptome analysis was performed to explore its possible molecular mechanism. Then, the pathway by which EFBS regulates oxidative stress was validated through inhibitor intervention, flow cytometry, quantitative PCR, western blotting, and immunofluorescence techniques. RESULTS: A total of 22 compounds in EFBS were identified. The transcriptome analyses of RAW264.7 cells indicated that EFBS might reduce reactive oxygen species (ROS) production by inhibiting the p47phox/NADPH oxidase 2 (NOX2) pathway and upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Both in vitro and in vivo data confirmed that EFBS significantly inhibited the expression and phosphorylation of p47phox protein, thereby weakening the p47phox/NOX2 pathway and reducing ROS production. EFBS significantly increased the expression of Nrf2 in primary peritoneal macrophages and lung tissue and promoted its nuclear translocation, dose-dependent increase in HO-1 levels, and enhancement of antioxidant activity. In vitro, both Nrf2 and HO-1 inhibitors significantly reduced the scavenging effects of EFBS on ROS, further confirming that EFBS exerts antioxidant effects at least partially by upregulating the Nrf2/HO-1 pathway. CONCLUSIONS: EFBS contains abundant phenanthrenes and dibenzyl polyphenols, which can reduce ROS production by inhibiting the p47phox/NOX2 pathway and enhance ROS clearance activity by upregulating the Nrf2/HO-1 pathway, thereby exerting regulatory effects on oxidative stress and improving LPS-induced ALI.

Bletilla striata polysaccharide attenuated the progression of pulmonary fibrosis by inhibiting TGF-ß1/Smad signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Bletilla striata, a traditional medicinal plant, has been utilized as a folk medicine for many years because of its superior biological activity in China. However, Bletilla striata polysaccharide (BSP) has received less attention, and its specific mechanism for ameliorating pulmonary fibrosis is completely unclear. AIMS OF THE STUDY: In this study, we aim to assess BSP on the treatment of PF and explore potential mechanisms. MATERIALS AND METHODS: BSP was successfully extracted and purified from Bletilla striata. The mechanisms were assessed in bleomycin-induced pulmonary fibrosis model and lung fibroblasts activated by transforming growth factor-ß1 (TGF-ß1). Histological analysis, immunofluorescence, Western blot and flow cytometry were used to explore the alterations after BSP intervention. RESULTS: The results in vivo showed an anti-PF effect of BSP treatment, which reduced pathogenic damages. Furthermore, TGF-ß1-induced abnormal migration and upregulated expression of collagen I (COL1A1), vimentin and α-smooth muscle actin (α-SMA) were suppressed by BSP in L929 cells. Moreover, the abnormal proliferation was retarded by inhibiting the cell cycle of G1 to S phase. Immunofluorescence assay showed that BSP activated autophagy and played an antifibrotic role by inhibiting the expression of p62 and phospho-mammalian target of rapamycin (p-mTOR). Last but not least, the suppression of TGF-ß1/Smad signaling pathway was critical for BSP to perform therapeutic effects in vitro and in vivo. CONCLUSION: The possible mechanisms were involved in improving ECM deposition, regulating cell migration and proliferation, and promoting cellular autophagy. Briefly, all of the above revealed that BSP might be a novel therapy for treating pulmonary fibrosis.

A rapid-floating natural polysaccharide gel-raft with double-effect for the treatment of gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is a prevalent gastrointestinal condition characterized by regurgitating stomach contents into the esophagus, causing mucosal damage or erosion. Clinical physical protection treatment mainly relies on the use of floating rafts. Bletilla striata (BS) is widely regarded as the first-choice drug for treating digestive tract injuries in Chinese Medicine. The rapid-floating gel-raft (B-R) was prepared via a one-step swelling method using natural BS polysaccharide and glyceryl monooleate. Panax notoginseng saponins (PNS) were loaded to further prepare P/B-R according to clinical experience. Possessing hydrophobic dense, stratified porous structure and stable rheological properties, an outperforming floating performance of P/B-R was proven compared with Gaviscon® (alginate-antacid formulation) in vitro. In vivo imaging results showed that P/B-R can retain and adhere to the gastric mucosa of rats for up to 90 min, protecting and repairing the mucosa. Besides physical protection in situ, the systemic effects of antioxidant and anti-inflammatory actions for treating GERD were achieved through the intestinal release of PNS. Acid-labile PNS was protected by P/B-R against gastric acid, attaining the desired release and permeability. A significantly effective mucosa injury protective effect of P/B-R was found in ethanol-induced gastric damage model on rats. Moreover, P/B-R exhibits excellent biosafety at the cellular level.

Utilizing epoxy Bletilla striata polysaccharide collagen sponge for hemostatic care and wound healing.

Hemostatic materials that are lightweight and possess good blood absorption performance have been widely considered for use in modern wound care. Natural hemostatic ingredients derived from traditional Chinese medicine have also received extensive attention. Bletilla polysaccharides are valued by researchers for their excellent hemostatic performance and good reactivity. Collagen is favored by researchers due to its high biocompatibility and low immunogenicity. In this study, Bletilla striata polysaccharide, the main hemostatic component of Bletilla striata, was activated by epoxy groups, and epoxidized Bletilla striata polysaccharide (EBSP) was prepared. Then, EBSP was crosslinked with collagen under alkaline conditions, and a new hemostatic material that was an epoxidized Bletilla polysaccharide crosslinked collagen hemostatic sponge was prepared. We demonstrated that endowing collagen with better hemostatic performance, cytocompatibility, and blood compatibility does not destroy its original three-stranded helical structure. Compared with the medical gauze, hemostasis time was shorter (26.75 ±â€¯2.38 s), and blood loss was lower (0.088 ±â€¯0.051 g) in the rat liver injury hemostasis model. In the rat model of severed tail hemostasis, hemostasis time was also shorter (47.33 ±â€¯2.05 s), and the amount of blood loss was lower (0.330 ±â€¯0.122 g). The sponge possessed good hemostatic and healing performance.

Safe utilization evaluation of two typical traditional Chinese medicinal materials in Cd-contaminated soil based on the analysis of Cd transfer and AHP model.

Due to the increasing scarcity of wild resources, most traditional Chinese medicinal materials (TCMMs) in the market are produced via artificial cultivation. The widespread pollution of cadmium (Cd) in soil limits the safe cultivation and use of TCMMs. This study investigated Cd accumulation, distribution, and the medicinal component content under simulated field conditions to clarify the differences in the Cd absorption, transfer and detoxification mechanisms of Polygonatum cyrtonema Hua and Bletilla striata, and provide the preliminary safe utilization conditions of TCMMs based on the analytic hierarchy process (AHP). The results showed that the Cd content of P. cyrtonema Hua was lower than the safety threshold under a high soil Cd concentration of 0.91 mg/kg (Cd-L), while B. striata was safe only at a low Cd concentration of 0.25 mg/kg (CK). Cd at 0.91 mg/kg induced hormesis affecting the net increase in biomass and medicinal component content for both TCMMs, while P. cyrtonema Hua showed better potential for safe utilization. Additionally, P. cyrtonema Hua had stronger resistance to Cd stress, exhibiting superior characteristics for synergistic absorption of Cd with mineral elements, transfer to nonmedical part and safer fixation forms in subcellular components. In contrast, B. striata showed insufficient Cd tolerance, and Cd was easily accumulated in organelles to inhibit plant growth. Our findings may attract more attention to the safe cultivation of TCMMs and provide insight into guidance for the safe utilization of slightly Cd-contaminated soil.

Antimicrobial Activity of Stilbenes from Bletilla striata against Cutibacterium acnes and Its Effect on Cell Membrane.

The abnormal proliferation of Cutibacterium acnes is the main cause of acne vulgaris. Natural antibacterial plant extracts have gained great interest due to the efficacy and safety of their use in skin care products. Bletilla striata is a common externally used traditional Chinese medicine, and several of its isolated stilbenes were reported to exhibit good antibacterial activity. In this study, the antimicrobial activity of stilbenes from B. striata (BSS) against C. acnes and its potential effect on cell membrane were elucidated by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), bacterial growth curve, adenosine triphosphate (ATP) levels, membrane potential (MP), and the expression of genes related to fatty acid biosynthesis in the cell membrane. In addition, the morphological changes in C. acnes by BSS were observed using transmission electron microscopy (TEM). Experimentally, we verified that BSS possessed significant antibacterial activity against C. acnes, with an MIC and MBC of 15.62 µg/mL and 62.5 µg/mL, respectively. The growth curve indicated that BSS at 2 MIC, MIC, 1/2 MIC, and 1/4 MIC concentrations inhibited the growth of C. acnes. TEM images demonstrated that BSS at an MIC concentration disrupted the morphological structure and cell membrane in C. acnes. Furthermore, the BSS at the 2 MIC, MIC, and 1/2 MIC concentrations caused a decrease in the intracellular ATP levels and the depolarization of the cell membrane as well as BSS at an MIC concentration inhibited the expression of fatty acid biosynthesis-associated genes. In conclusion, BSS could exert good antimicrobial activity by interfering with cell membrane in C. acnes, which have the potential to be developed as a natural antiacne additive.

Bletilla Striata polysaccharides thermosensitive gel for photothermal treatment of bacterial infection.

Skin is the most important defense shield which touched external environment directly. Effectively clearing microbes in infected wound via non-antibiotic therapy is crucial for the promotion of recovery in complex biological environments, and the wound healing is a crucial process after sterilization to avoid superinfection. Herein, a kind of Prussian blue-based photothermal responsive gel, Bletilla striata polysaccharide-mingled, isatin-functionalized Prussian blue gel (PB-ISA/BSP gel) was reported for effective treatment of bacterial infection and wound healing. The introduction of effective components of traditional Chinese medicine (TCM), isatin (ISA), enhanced the efficiency of sterilization synergistically. Furthermore, the process of wound healing was promoted by Bletilla striata polysaccharides (BSP). PB-ISA@BSP had a considerable antibacterial rate with 98.5 % under an 808 nm laser for 10 min in vitro. Besides, PB-ISA/BSP gel showed an effective antibacterial efficacy in vivo and a fast wound healing rate as well. The as-prepared functional particles can invade and destroy bacteria membrane to kill microbes. This work highlights that PB-ISA/BSP gel is a promising antibacterial agent based on synergistically enhanced photothermal effect and wound healing promotion ability and provides inspiration for future therapy based on the synergy between photothermal agent and active components in TCM.

First report of Fusarium solani causing Fusarium wilt of Bletilla striata (hyacinth orchid) in Zhejiang province of China.

Bletilla striata, a member of the family Orchidaceae, is a perennial herbaceous plant used in Chinese medicine. It is a commonly cultivated economic crop in the Yangtze River Basin provinces of China, as its roots are used to treat bleeding and inflammation. In Zhejiang province, Bletilla striata has a planting area of 1400 hectares with a total production of approximately 2.6×106 kg. In October 2021, over 40% of B. striata plants showed severe wilt in a traditional Chinese medicine plantation (ca. 10 ha) in Xianju City, Zhejiang Province, China. In July, leaf curling, crinkling, and leaf-edge browning of the diseased plants were first noticed in the field. Then, necrotic streaks gradually spread to the roots. Stems displayed chlorosis and withering and when they were cut vertically, symptoms such as vascular bundle discoloration, appeared. After October, the individual plants slowly wilted and died, their aboveground parts became filamentous, and the epidermis detached from the corm's fibrous roots. Diseased plants were easily removed as the corm root had fractured. White mycelia were clearly seen in the stem. Three symptomatic leaves and three stems were cut, their surfaces disinfected, and plated on potato dextrose agar (PDA). Six strains were subsequently isolated from all samples. Fungal colonies with white to cream-colored mycelia from all tissues appeared after 3 d of incubation at 26 °C. Pure cultures obtained after monospore isolation were examined for their morphological characteristics. The colonies grew rapidly, were fluffy and appressed, and had cottony white to pale cream coloration. Microconidia were hyaline, oval to reniform, with zero or one-septate (4.0-12.0 × 1.0-5.5 µm), and usually formed on elongated monophialidic conidiogenous cells. Macroconidia were wide, fusiform, or slightly curved with one or three septa (23.0-36.0 × 4.5-7.0 µm). Chlamydospores were spherical and were abundant on carrot agar (CA) medium within 2 wk. Fresh mycelia and conidia that grew at 26 ℃ for 7 d were collected from PDA plates. Next, DNA was extracted using the Ezup Column Fungi Genomic DNA Purification kit (Sangon Biotech, Shanghai, China). We amplified a portion of RNA polymerase II second largest subunit gene (RPB2) using primers 5f2/7cr (O'Donnell et al. 2010), the internal transcribed spacer (ITS) region using primers ITS1F/ITS4 (White et al. 1990), and the partial translation elongation factor-1α gene using primers EF1/ EF2 (O'Donnell et al. 1998) from the genomic DNA and sent the PCR amplicons for sequencing at Tsingke Biotechnology Co., Ltd., Wuhan, China. A BLAST search of the obtained sequences (GenBank accessions OP743920, OP913183, and OP913180) showed 99-100% homology with the respective sequences of the Fusarium solani reference isolate NRRL46702 (O'Donnell et al. 2008). Based on the morphological and molecular characteristics and BLAST search, the fungus was identified as F. solani (Leslie and Summerell 2006). Pathogenicity of the purified F. solani isolate was assessed by inoculateing a F. solani spore suspension of 1×106 conidia/mL (20 mL per seedling) on corm wounds made with a toothpick. Four inoculated and three non-inoculated seedlings (sterilized water as a negative control) were grown in a greenhouse at 26 °C under natural sunlight and covered with plastic bags to maintain humidity for 72 h. After 15 d, leaf browning on leaf edges, new leaf bases, and corm epidermis was observed. Symptoms, similar to those detected in the original sample, developed on the inoculated leaves, whereas the controls remained asymptomatic. Fusarium solani was successfully re-isolated from all four inoculated seedlings, and their identity confirmed by generating partial Tef1 and RPB2 sequences, thereby fulfilling the Koch's postulate. To our knowledge, F. solani has not been previously reported as a pathogen of B. striata.

First report of Alternaria burnsii causing leaf spot on Bletilla striata in China.

Bletilla striata (named "Bai Ji" in Chinese) is a plant from the Orchidaceae family that has been employed in traditional Chinese medicine for thousands of years in China. Polysaccharides extracted from B. striata have been shown to have an effect on Alzheimer's disease (Lin et al. 2021). Since 2021, leaf spots have been observed in the B. striata plantation in Chongqing, China. Out of 200 plants, the disease incidence was estimated at 56%, and the disease index was estimated at 32%. The symptoms were necrotic lesions with brown edges and yellow halos; severe infection caused the infected leaves to become blighted, dry and fall off. To identify the causal agent, eighteen leaves with typical symptoms were collected from the B. striata plantation (30.60°N, 108.64°E). The margins of infected tissue areas were cut into small pieces (5×5 mm), surface sterilized with 70% ethanol for 1 min, and rinsed twice with sterile distilled water. The tissue was then surface sterilized in 3% sodium hypochlorite for 2 min, followed by three rinses with sterile water. The tissue was then placed onto potato dextrose agar (PDA) plates and incubated at 25°C for 3 days, pure cultures of fungal isolates were obtained by single-spore isolation, stored on PDA slants and maintained at 4°C. Colonies of the fungal isolates showed three color types, ranging from grayish white to green above with olive green on the reverse, but conidial characteristics were more similar and indicated this was a single fungus. Conidiophores were single, lateral from hyphae or terminal; straight or curved; smooth-walled with 1 to 8 septa; pale brown; usually with only one pigmented terminal conidiogenous site, sometimes with one additional lateral conidiogenous locus; sometimes slightly swollen at the apex; and 15 to 170 µm long, 2.5 to 4.5 µm wide. Conidia were in short or moderately long chains of 2-8 conidia normally, sometimes with more; rarely branched; normally 14.07 to 50 × 5.24 to 10 µm in size; ellipsoid, fusiform, long ellipsoid, obclavate or ovoid with 1 to 11 transverse septa and 2 to 4 longitudinal septa; beakless or with subcylindric or cylindric secondary conidiophores, analogous to the beak 4.25 to 58.6 µm long, 3.2 to 4.8 µm wide. The fungal isolates were tentatively identified as Alternaria sp. The representative isolate BJ8 was selected for the pathogenicity test. The leaves of six healthy plants of B. striata (two years old) grown in pots were washed with sterile water. Ten mL of conidial suspension (1×106 conidia mL-1) contained in 0.05% Tween 80 buffer was brushed onto upper and lower surfaces of all the leaves on three plants, while other plants were brushed with 10 mL 0.05% Tween 80 buffer to serve as controls. Plants were placed in a greenhouse at 25°C and 95±1% relative humidity after inoculation and observed for symptoms. The symptoms initially developed as irregular brown necrotic lesions on the inoculated leaves after 7 days, with a yellow halo around the lesions, consistent with the symptoms in the field. Leaves on the control plants did not produce any symptoms. For molecular identification, the genomic DNAs of representative isolates BJ5, BJ6, and BJ8 were extracted. The internal transcribed spacer (ITS) region and RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used for polymerase chain reaction (PCR), using primers ITS5/ITS4, GPD1/GPD2, EF-1F/EF-1B and RPB27cR/RPB25F2, respectively (White et al. 1990; Berbee and Pirseyedi et al. 1999; Carbone and Kohn 1999; Liu et al. 1999). The neighbor-joining tree revealed that these isolates are clustered together with the reference strain of A. burnsii. The sequences were deposited in NCBI GenBank BJ5 [ITS: OP897263; GAPDH: OQ544937; TEF1: OQ544941; RPB2: OQ544939], BJ6 [ITS: OP897262; GAPDH: OQ544938; TEF1: OQ544942; RPB2: OQ544940], and BJ8 [ITS: OK285209; GAPDH: OK340046; TEF1: OK340047; RPB2: OQ544936]. All three isolates showed 100% similarity with A. burnsii CBS 107.38 [ITS: KP124420; GAPDH: JQ646305; TEF1: KP125198; RPB2: JQ646457] ex-type sequence, thus the pathogen causing the leaf spot on B. striata was identified as A. burnsii. A. burnsii is an important pathogenic fungus causing blight of cumin (Shekhawat et al. 2013). Furthermore, Al-Nadabi et al. (2018) found that A. burnsii can cause leaf spots on wheat and date palms, and Sunapao et al. (2022) reported that A. burnsii can infect coconuts (Cocos nucifera), causing dirty panicle disease. This is the first report of A. burnsii causing leaf spot on B. striata in China. The new discovery shows that since A. burnsii can readily adapt to a variety of climatic conditions, controlling the fungus is crucial for the healthy growth of B. striata in the future. This study will provide a basis for further elucidating the pathogenic mechanism and development of effective control measures for this disease.

Neuroprotective neolignan glycosides from the pseudobulbs of Bletilla striata.

Four undescribed neolignan glycosides, bletineosides A-D (1-4) were isolated from the pseudobulbs of Bletilla striata. Their structures with absolute configurations were elucidated on the basis of spectroscopic analyses, along with acidic hydrolysis reactions and ECD experiments. All isolates were evaluated for their neuroprotective activities against glutamate-induced PC12 cell injury. Compound 3 and 4 showed significantly neuroprotective effects at the concentration of 10 µM when compared with the model group. Compounds 1-4 represented the first examples of neolignan glycosides from the genus Bletilla. This study disclosed the potency of Bletilla striata as a new source of anti-neurodegenerative agents.

First report of leaf spot caused by Botrytis cinerea on Bletilla striata in Anhui province, China.

Bletilla striata (Thunb.) is a perennial herb plant of the orchidaceous family and is used as an ornamental plant in Europe and the United States. Furthermore, it is important as traditional Chinese medicine (TCM) in East Asian countries, such as China, Japan, Korea, Mongolia, and Myanmar (Gou et al. 2022). In April 2023, a severe disease similar to gray mold occurred in a B. striata plantation in Anqing, Anhui province, China (N30°27'15″, E116°18'32″), causing disease on about 20% of the plants in the field. Early symptoms were characterized by brown spots or stripes on the leaves of B. striata, and as the disease progressed, large brown irregular spots appeared. Eventually disease spots coalesced, covering the entire leaf surface and causing leaf death. A gray mildew layer was observed on the senescent leaves. To investigate the causal agent, 10 plants with typical symptoms were collected from the field. Leaf pieces (5 × 5 mm) from the border of infected areas were soaked in 75% ethanol for 10 seconds, and then transferred into 0.1% mercury bichloride for three min, rinsed three times with sterile water, and transferred to PDA at 25 °C for three days. Pure cultures were obtained by single spore isolation, and the resulting colonies were morphologically similar, indicating a single pathogen, of which the representative BSFC-7 was selected for further study. BSFC-7 colonies were initially white to gray-brown, and cottony aerial hyphae grew over the entire petri dish after five days of incubation. Grayish, branched conidiophores and their terminal unicellular conidia were observed under a microscope after additional two days at 25 °C. Conidia were colorless or gray, elliptical or oval, and 7.06-12.54 × 8.33-13.55 µm (n=30). Sclerotia appeared in BSFC-7 culture up to about two weeks and were black, hard, and round or irregularly shaped (0.81-4.32 × 0.97-5.68 mm, n=20). The morphological characteristics fit the description of Botrytis cinerea (Li et al. 2016). To further identify the species, genomic DNA of BSFC-7 was extracted. PCR analysis was performed with species-specific primer pairs C729+/C729- and two nuclear genes G3PDH and RPB2 with their corresponding primer pairs G3PDH-F/G3PDH-R and RPB2-F/RPB2-R (Rigotti et al. 2002; Aktaruzzaman et al. 2018). The sequences for all three PCR products of C729, G3PDH, and RPB2 (GenBank accession nos. OR287069, OR255923, and OR255924 respectively) exhibited 99 to 100% similarity with other B. cinerea isolates. In the pathogenicity test, detached leaves of B. striata were inoculated with the BSFC-7 isolate. The leaves were soaked in sodium hypochlorite (1%) for two min, washed with sterile distilled water, and then inoculated with 10 µl of conidial suspension (106 conidia/ml). Sterile water was used as control and samples were incubated at 25 °C. After three days, all leaves inoculated with conidia showed dark brown water-soaked lesions similar to those observed in the field, while the control leaves remained healthy. The pathogen was re-isolated from the affected leaves, fulfilling Koch's postulates. B. cinerea is a common pathogen on a wide range of host plant species worldwide and has been reported to infect B. striata in Yunnan province, China (Romanazzi and Feliziani 2014; Zhang et al. 2020). To our knowledge, this is the first report of B. cinerea causing leaf spots on B. striata in Anhui province, China. This study will provide a basis for controlling the prevalence and economic losses of gray mold on B. striata.

Baicalin, silver titanate, Bletilla striata polysaccharide and carboxymethyl chitosan in a porous sponge dressing for burn wound healing.

OBJECTIVE: This study tests the efficacy of Bletilla striata polysaccharide (BSP), carboxymethyl chitosan (CMC), baicalin (BA) and silver titanate (ST) in a wound dressings to fight infection, promote healing and provide superior biocompatibility. METHODS: The antibacterial activity of BA and ST was evaluated in vitro using the inhibition zone method. BA/ST/BSP/CMC porous sponge dressings were prepared and characterized. The biocompatibility of BA/ST/BSP/CMC was assessed using the cell counting kit-8 assay. The therapeutic effect of BA/ST/BSP/CMC was further investigated using the dorsal skin burn model in Sprague-Dawley rats. RESULTS: The wound dressing had good antibacterial activity against Escherichia coli and Staphylococcus aureus through BA and ST, while the combination of BSP and CMC played an important role in promoting wound healing. The BA/ST/BSP/CMC porous sponge dressings were prepared using a freeze-drying method with the concentrations of BA and ST at 20 and 0.83 mg/mL, respectively, and the optimal ratio of 5% BSP to 4% CMC was 1:3. The average porosity, water absorption and air permeability of BA/ST/BSP/CMC porous sponge dressings were measured to be 90.43%, 746.1% and 66.60%, respectively. After treatment for 3 and 7 days, the healing rates of the BA/ST/BSP/CMC group and BA/BSP/CMC group were significantly higher than those of the normal saline (NS) group and silver sulfadiazine (SSD) group (P < 0.05). Interleukin-1ß expression in the BA/ST/BSP/CMC group at 1 and 3 days was significantly lower than that in the other three groups (P < 0.05). After being treated for 3 days, vascular endothelial growth factor expression in the BA/BSP/CMC group and BA/ST/BSP/CMC group was significantly higher than that in the NS group and SSD group (P < 0.05). Inspection of histological sections showed that the BA/ST/BSP/CMC group and BA/BSP/CMC group began to develop scabbing and peeling of damaged skin after 3 days of treatment, indicating accelerated healing relative to the NS group and SSD group. CONCLUSION: The optimized concentration of BA/ST/BSP/CMC dressing was as follows: 6 mg BSP, 14.4 mg CMC, 0.5 mg ST and 12 mg BA. The BA/ST/BSP/CMC dressing, containing antibacterial constituents, was non-cytotoxic and effective in accelerating the healing of burn wounds, making it a promising candidate for wound healing. Please cite this article as: Gong YR, Zhang C, Xiang X, Wang ZB, Wang YQ, Su YH, Zhang HQ. Baicalin, silver titanate, Bletilla striata polysaccharide and carboxymethyl chitosan in a porous sponge dressing for burn wound healing. J Integr Med. 2023; 21(5): 487-495.

Bletilla striata composite nanofibrous membranes prepared by emulsion electrospinning for enhanced healing of diabetic wounds.

Diabetic wounds impose enormous distress and financial burden on patients, and finding effective dressings to manage wounds is critical. As a Chinese herbal medicine with a long history of Clinical application, Bletilla striata has significant medicinal effects in the therapy of various wounds. In this study, PLA and the pharmacodynamic substances of Bletilla striata were prepared into fibrous scaffolds by emulsion electrospinning technology for the management of diabetic wounds in mice. The results of scanning electron microscopy showed that the core-shell structure fibre was successfully obtained by emulsion electrospinning. The fibre membrane exhibited excellent water absorption capability and water vapor transmission rate, could inhibit the growth of Staphylococcus aureus and Pseudomonas aeruginosa, had good compatibility, and achieved excellent healing effect on diabetic wounds. Especially in the in vivo wound healing experiment, the wound healing rate of composite fibre membrane treatment reached 98.587 ± 2.149% in 16 days. This work demonstrated the good therapeutic effect of the developed fibrous membrane to diabetic wound, and this membrane could be potentially applied to chronic wound healing.