Ethanol extract of Bletilla striata has remarkable anti-inflammatory and anti-pulmonary fibrosis activities in the rat silicosis model. However, its active substances and molecular mechanism are still unclear. To uncover the active ingredients and potential molecular mechanism of the Bletilla striata extract, the lipopolysaccharide (LPS)-induced macrophage inflammation model and phospho antibody array were used. Coelonin, a dihydrophenanthrene compound was isolated and identified. It significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 μg/mL. The microarray data indicate that the phosphorylation levels of 32 proteins in the coelonin pre-treated group were significantly down-regulated. In particular, the phosphorylation levels of the key inflammatory regulators factor nuclear factor-kappa B (NF-κB) were significantly reduced, and the negative regulator phosphatase and tensin homologue on chromosome ten (PTEN) was reduced. Moreover, the phosphorylation level of cyclin dependent kinase inhibitor 1B (p27^Kip1), another downstream molecule regulated by PTEN was also reduced significantly. Western blot and confocal microscopy results confirmed that coelonin inhibited LPS-induced PTEN phosphorylation in a dose-dependent manner, then inhibited NF-κB activation and p27^Kip1 degradation by regulating the phosphatidylinositol-3-kinases/ v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway negatively. However, PTEN inhibitor co-treatment analysis indicated that the inhibition of IL-1β, IL-6 and TNF-α expression by coelonin was independent of PTEN, whereas the inhibition of p27^Kip1 degradation resulted in cell-cycle arrest in the G1 phase, which was dependent on PTEN. The anti-inflammatory activity of coelonin in vivo, which is one of the main active ingredients of Bletilla striata, deserves further study.

We have previously shown that ethanol or chloroform extracts of the leaves ofImpatiens balsamina (LIB) have anti-tumor activity against the human hepatocellularcarcinoma cell line HepG2. The ethanol extracts were separated into five fractionsaccording to polarity. An MTT assay indicated that two of the fractions had anti-tumoractivity and that the petroleum ether fraction (PEF) was the most active. But the availablequantities of both the PEF and chloroform fractions (CHF) were limited, precluding furtherstudy. The chloroform extract (CHE) shared almost all the same spots with the PEF andCHF and was plentiful enough to carry out further separations. Thus, the CHE was furtherseparated into six sub-fractions (CHE1~6) by column chromatography. A MTT assayshowed that only the CHE2 fraction had a strong tumor inhibition ratio (IC₅₀ = 6.47±0.05mg/L), which was superior to that of curcumin (IC₅₀ = 13.95±0.11 mg/L). However, TLCrevealed that CHE2 was not pure and still contained two more components. After furtherseparation and purification, followed by TLC and MTT assay confirmation, the final activecomponent was isolated and identified as 2-methoxy-1,4-naphthoquinone by m.p., UV, MSand ¹³C- and ¹H-NMR data. This is the first report demonstrating that2-methoxy-1,4-naphthoquinone has intensive in vitro anti-tumor activity against HepG2cells.

Studies on endophytes, a relatively under-explored group of microorganisms, are currently popular amongst biologists and natural product researchers. A fungal strain (ME4-2) was isolated from flower samples of mistletoe (Viscum coloratum) during a screening program for endophytes. As limited information on floral endophytes is available, the aim of the present study is to characterise fungal endophytes using their secondary metabolites. ME4-2 grew well in both natural and basic synthetic media but produced no conidia. Sequence analysis of its internal transcribed spacer rDNA demonstrated that ME4-2 forms a distinct branch within the genus Lasiodiplodia and is closely related to L. pseudotheobromae. This floral endophyte was thus identified as Lasiodiplodia sp. based on its molecular biological characteristics. Five aromatic compounds, including cyclo-(Trp-Ala), indole-3-carboxylic acid (ICA), indole-3-carbaldehyde, mellein and 2-phenylethanol, were found in the culture. The structures of these compounds were determined using spectroscopic methods combined with gas chromatography. To the best of our knowledge, our work is the first to report isolation of these aromatic metabolites from a floral endophyte. Interestingly, ICA, a major secondary metabolite produced by ME4-2, seemed to be biosynthesized via an unusual pathway. Furthermore, our results indicate that the fungus ME4-2 is a potent producer of 2-phenylethanol, which is a common component of floral essential oils. This study introduces a fungal strain producing several important aromatic metabolites with pharmaceutical or food applications and suggests that endophytic fungi isolated from plant flowers are promising natural sources of aromatic compounds.

Bletillae Rhizoma, the tuber of Bletilla striata, has been used in Chinese traditional medicine to treat infectious diseases. Chemical studies indicated that phenanthrene was one of the most important components of the herb, with a broad spectrum of antibiotic activity against Gram-positive bacteria. The objective of this study was to further characterize the antibacterial activity of the phenanthrene fraction from the fibrous root of the pseudobulb of B. striata. The phenanthrene fraction (EF60) from the ethanol extract of fibrous roots of Bletilla striata pseudobulbs was isolated using polyamide column chromatography. The antibacterial activity of the fraction was evaluated in vitro using a 96-well microtiter plate and microbroth dilution method. The cytotoxicity of EF60 against mammalian cells was tested by hemolysis and MTT assays. EF60 was obtained using alcohol extraction and polyamide column chromatography, with a yield of 14.9 g per 1 kg of the fibrous roots of B. striata. In vitro tests indicated that EF60 was active against all tested strains of Staphylococcus aureus, including clinical isolates and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration (MIC) values of EF60 against these pathogens ranged from 8 to 64 μg/mL. Minimum bactericidal concentration tests demonstrated that EF60 was bactericidal against S. aureus 3304 and ATCC 29213 and was bacteriostatic against S. aureus 3211, ATCC 25923, and ATCC 43300. Consistently, the time-kill assay indicated that EF60 could completely kill S. aureus ATCC 29213 at 2× the MIC within 3 h but could kill less than two logarithmic units of ATCC 43300, even at 4× the MIC within 24 h. The postantibiotic effects (PAE) of EF60 (4× MIC) against strains 29213 and 43300 were 2.0 and 0.38 h, respectively. Further studies indicated that EF60 (160 μg/mL) showed no cytotoxicity against human erythrocytes, and was minimally toxic to Human Umbilical Vein Endothelial Cells with an IC50 of 75 μg/mL. Our studies indicated that EF60 is worthy of further investigation as a potential phytotherapeutic agent for treating infections caused by S. aureus and MRSA.

Background: Bletilla striata (Thunb.) Reichb. f. (B. striata) is a perennial herbaceous plant in the Orchidaceae family known for its diverse pharmacological activities, such as promoting wound healing, hemostasis, anti-inflammatory effects, antioxidant properties, and immune regulation. Nevertheless, the microbe-plant-metabolite regulation patterns for B. striata remain largely undetermined, especially in the field of rhizosphere microbes. To elucidate the interrelationships between soil physics and chemistry and rhizosphere microbes and metabolites, a comprehensive approach combining metagenome analysis and targeted metabolomics was employed to investigate the rhizosphere soil and tubers from four provinces and eight production areas in China. Results: Our study reveals that the core rhizosphere microbiome of B. striata is predominantly comprised of Paraburkholderia, Methylibium, Bradyrhizobium, Chitinophaga, and Mycobacterium. These microbial species are recognized as potentially beneficial for plants health. Comprehensive analysis revealed a significant association between the accumulation of metabolites, such as militarine and polysaccharides in B. striata and the composition of rhizosphere microbes at the genus level. Furthermore, we found that the soil environment indirectly influenced the metabolite profile of B. striata by affecting the composition of rhizosphere microbes. Notably, our research identifies soil organic carbon as a primary driving factor influencing metabolite accumulation in B. striata. Conclusion: Our fndings contribute to an enhanced understanding of the comprehensive regulatory mechanism involving microbe-plant-metabolite interactions. This research provides a theoretical basis for the cultivation of high-quality traditional Chinese medicine B. striata.

Bletilla striata is a well‐known traditional Chinese herb with anti‐inflammatory properties that is widely used in the treatment of lung conditions such as silicosis, tuberculosis, and pneumogastric hemorrhage. However, little information on the anti‐inflammatory ingredients and their activities is available. In this study, an effect fraction of Bletilla striata (EFBS) was enriched, and its anti‐inflammatory activities and underlying mechanisms were investigated. EFBS was enriched by polyamide column chromatography and characterized by HPLC; an LPS‐induced acute lung injury model was used to evaluate the anti‐inflammatory activities of EFBS. Meanwhile, the main anti‐inflammation‐contributing ingredients and possible molecular mechanism of anti‐inflammatory activity in EFBS were verified by component‐knockout method combined with LPS‐induced RAW264.7 cell model. The EFBS mainly consisted of coelonin (15.88%), batatasin III (32.49%), 3 ^′‐O‐methylbatatasin III (6.96%), and 3‐hydroxy‐5‐methoxy bibenzyl (2.51%). Pretreatment with the EFBS (20 mg/kg and 60 mg/kg) for five days prior to the administration of LPS resulted in decreases in wet‐to‐dry lung weight ratio, neutrophil number, MPO activity, total protein concentration, NO level, and MDA level, as well as IL‐1β, IL‐6, MCP‐1, and TNF‐α concentrations in the bronchoalveolar lavage fluid. Western blot analysis demonstrated the increased expressions of iNOS, COX‐2, and NF‐κB p65 in the LPS treatment group, all of which were ameliorated by EFBS pretreatment. Histological examination confirmed the protective effect of the EFBS. Additionally, component‐knockout assay confirmed that these four quantitative components contributed significantly to the anti‐inflammatory effect of EFBS. Coelonin, batatasin III, 3 ^′‐O‐methylbatatasin III and 3‐hydroxy‐5‐methoxy bibenzyl were the main anti‐inflammatory components of EFBS and could regulate the expression of downstream inflammatory cytokines by inhibiting p65 nuclear translocation. These findings uncover, in part, the molecular basis underlying the anti‐inflammatory activity of Bletilla striata.