Therapeutic potential of Litsea cubeba essential oil in modulating inflammation and the gut microbiome.

Inflammation, a sophisticated and delicately balanced physiological mechanism, is paramount to the host's immunological defense against pathogens. However, unfettered and excessive inflammation can be instrumental in engendering a plethora of chronic ailments and detrimental health repercussions, notably within the gastrointestinal tract. Lipopolysaccharides (LPS) from bacteria are potent endotoxins capable of instigating intestinal inflammation through the disruption of the intestinal epithelial barrier and the stimulation of a pro-inflammatory immune response. In this study, we sought to investigate the influence of Litsea cubeba essential oil (LCEO) on LPS-induced intestinal inflammation and associated changes in the gut microbiota. We investigated the therapeutic potential of LCEO for gut health, with particular emphasis on its gut protective properties, anti-inflammatory properties and modulation of the gut microbiome. LCEO exhibited protective effects on colonic tissue by protecting crypts and maintaining epithelial integrity, and anti-inflammatory properties by reducing TNF-α, IL-6, and IL-1ß levels in the liver and intestine. Citral, a major component of LCEO, showed robust binding to IL-1ß, IL-6, and TNF-α, exerting anti-inflammatory effects through hydrogen bonding interactions. Using community barplot and LEfSe analyses, we detected significant variation in microbial composition, identified discrete biomarkers, and highlighted the influence of essential oils on gut microbial communities. Our research suggests that LCEO may be a promising natural compound for ameliorating diarrhea and intestinal inflammation, with potential implications for modulating the gut microbiome. These observations provide invaluable insight into the potential therapeutic role of LCEO as a natural anti-inflammatory agent for treating intestinal inflammatory disorders, particularly in the setting of a dysregulated immune response and altered gut microbiota. Furthermore, our findings highlight the need to understand the complex interplay between the host, the gut microbiome and natural products in the context of inflammatory diseases.

[Effects of Biochar on Antibiotic Environmental Behaviors in Soil: A Meta-analysis].

Biochar, as a soil amendment for synergizing the reduction of pollution and carbon emissions, shows great potential and future prospects in controlling antibiotic contamination. In order to research the effects of biochar on antibiotic behaviors in soil systematically, a Meta-analysis was conducted based on 20 studies published from 2011 to 2021. The results showed that the adsorption and degradation of antibiotics in the soil were significantly affected by the application rate and property of biochar. A 2% biochar application dose seemed to be the highest effect size (ES) of 0.19 on adsorption, while there was a significant effect (ES=0.23) on the degradation when the application rate was 5%. The specific surface area, polarity, stability, and aromaticity of biochar could increase the partition coefficient significantly, and the ES was 0.11, 0.13, 0.09, and 0.18, respectively, whereas the effects of antibiotic transport on the dose and property of biochar were insignificant. Biochar also indirectly controlled antibiotic behavior by altering the soil environment. However, the response of the coupling mechanism in antibiotic behaviors on biochar application into soil is still unclear. Moreover, the long-term and negative effects of biochar application in the field are still lacking basic data.

Effects of codon optimization, N-terminal truncation and gene dose on the heterologous expression of berberine bridge enzyme.

Morphine, sanguinarine and chelerythrine are benzylisoquinoline alkaloids (BIAs), and these compounds possess strong biological activities. (S)-scoulerine is a commonly shared precursor of these compounds, and berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the synthesis of (S)-scoulerine. We isolated the BBE gene from Macleaya cordata (McBBE) and used CEN.PK2-1C as a chassis strain. We compared the catalytic efficiency of five codon-optimized McBBE genes in Saccharomyces cerevisiae and finally obtained a yeast strain (YH03) that exhibited a 58-fold increase in yield (1.12 mg/L). Then, we truncated the N-terminus of McBBE by 8 and 22 amino acids and found that with the increase in the number of N-terminal truncated amino acids, the production of (S)-scoulerine gradually decreased. Additionally, we used CRISPR-Cas9 to integrate the McBBE gene at the delta site of the S. cerevisiae genome to achieve stable genetic inheritance and found that the yield of (S)-scoulerine was not significantly increased in the integrated strain. In conclusion, our work achieved high-efficiency expression of McBBE in S. cerevisiae, explored the influence of N-terminal truncation on the yield of (S)-scoulerine, and obtained a genetically stable S. cerevisiae strain with high McBBE expression. This study provides a reference for further complex metabolic engineering optimization and lays a foundation for the efficient biosynthesis of BIAs.

Influence of different elicitors on BIA production in Macleaya cordata.

Sanguinarine (SAN) and chelerythrine (CHE) have been widely used as substitutes for antibiotics for decades. For a long time, SAN and CHE have been extracted from mainly Macleaya cordata, a plant species that is a traditional herb in China and belongs to the Papaveraceae family. However, with the sharp increase in demand for SAN and CHE, it is necessary to develop a new method to enhance the supply of raw materials. Here, we used methyl jasmonate (MJ), salicylic acid (SA) and wounding alone and in combination to stimulate aseptic seedlings of M. cordata at 0 h, 24 h, 72 h and 120 h and then compared the differences in metabolic profiles and gene expression. Ultimately, we found that the effect of using MJ alone was the best treatment, with the contents of SAN and CHE increasing by 10- and 14-fold, respectively. However, the increased SAN and CHE contents in response to combined wounding and MJ were less than those for induced by the treatment with MJ alone. Additionally, after MJ treatment, SAN and CHE biosynthetic pathway genes, such as those encoding the protopine 6-hydroxylase and dihydrobenzophenanthridine oxidase enzymes, were highly expressed, which is consistent with the accumulation of SAN and CHE. At the same time, we have also studied the changes in the content of synthetic intermediates of SAN and CHE after elicitor induction. This study is the first systematic research report about using elicitors to increase the SAN and CHE in Macleaya cordata.

Modulation of benzylisoquinoline alkaloid biosynthesis by overexpression berberine bridge enzyme in Macleaya cordata.

Macleaya cordata produces a variety of benzylisoquinoline alkaloids (BIAs), such as sanguinarine, protopine, and berberine, which are potential anticancer drugs and natural growth promoters. The genes encoding the berberine bridge enzyme (BBE) were isolated from M. cordata and Papaver somniferum, and then the two genes were overexpressed in M. cordata. Through liquid chromatography with triple-quadrupole mass spectrometry analysis, it was determined that McBBE-OX caused higher levels of (S)-norcoclaurine, (S)-coclaurine, (S)-N-cis-methylcoclaurine, (S)-reticuline, (S)-tetrahydrocolumbamine, (S)-tetrahydroberberine, (S)-cheilanthifoline, and (S)-scoulerine than PsBBE-OX, empty vector or control treatments. qRT-PCR analysis demonstrated that the introduced genes in the transgenic lines were all highly expressed. However, the levels of sanguinarine (SAN) and chelerythrine (CHE) in all the transgenic lines were slightly lower than those in the wild-type lines, possibly because the overexpression of McBBE causes feedback-inhibition. This is the first report on the overexpression of potential key genes in M. cordata, and the findings are important for the design of metabolic engineering strategies that target BIAs biosynthesis.

Hairy root induction and benzylisoquinoline alkaloid production in Macleaya cordata.

Sanguinarine is currently widely used to replace antibiotic growth promoters in animal feeding and has demonstrated useful anticancer activity. Currently, the main source of sanguinarine is from an important medicinal plant, Macleaya cordata. To obtain a new source of sanguinarine production, we established hairy root cultures of M. cordata by co-cultivating leaf and stem explants with Agrobacterium rhizogenes. Except the co-cultivation medium, all growth media contained 200 mg/L timentin to eliminate A. rhizogenes. Through comparing the metabolic profiles and gene expression of hairy roots and wild-type roots sampled at five time points, we found that the sanguinarine and dihydrosanguinarine contents of hairy roots were far higher than those of wild-type roots, and we revealed the molecular mechanism that causes these metabolites to increase. Consequently, this study demonstrated that the hairy root system has further potential for bioengineering and sustainable production of sanguinarine on a commercial scale. To the best of our knowledge, this is the first efficient protocol reported for the establishment of hairy root cultures in M. cordata using A. rhizogenes.

Loss of large tumor suppressor 1 promotes growth and metastasis of gastric cancer cells through upregulation of the YAP signaling.

Accumulating evidence shows that large tumor suppressor 1 (LATS1) as a novel resident governor of cellular homeostasis is implicated in multiple tumorigenic properties including cell growth, apoptosis and metastasis. However, the contribution of LATS1 to gastric carcinoma (GC) remains unclear. The correlation of LATS1 expression with clinicopathologic characteristics, GC prognosis and recurrence was analyzed by immunohistochemistry, Univariate and Kaplan-Meier analysis. Functional experiments were performed to investigate biological behaviors of GC cells and underlying molecular mechanisms. Tumor growth and metastasis was assessed in vivo using orthotopic implantation GC models in severe combined immune deficiency (SCID) mice. Consequently, decreased LATS1 expression was significantly associated with the lymph node metastasis, poor prognosis and recurrence. Ectopic expression of LATS1 decreased GC cell proliferation and invasion in vitro and inhibited tumor growth and liver metastasis in vivo, but depletion of LATS1 expression restored the invasive phenotype. Further observation indicated that YAP pathway was required for LATS1-induced inhibition of cell growth and invasion, and LATS1 restrained nuclear transfer of YAP, downregulated YAP, PCNA, CTGF, MMP-2, MMP-9, Bcl-2 and CyclinD1 expression and upregulated p-YAP and Bax expression. Our findings suggest that LATS1 is a potential candidate tumor suppressor and inhibits the growth and metastasis of GC cells via downregulation of the YAP signaling.

[A new 6,6a-offbenzophenanthridine alkaloid from Macleaya cordata].

A new alkaloid was isolated from the leaves of Macleaya cordata with 95% ethanol extracted and its isolation was by column chromatography and preparation HPLC. The new structure was elucidated as 6'-hydroxy-2',3'-dimethoxyarnottianamide on the basis of its spectroscopic date.