Insights into the mechanisms of microbiome and metabolome changes mediated by understory interplanting mode in Polygonatum sibiricum.

Background: Polygonatum sibiricum is an understory economic plant, and its dried rhizome is a traditional Chinese medicine. The purpose of this study was to connect the quality improvement of the understory plant P. sibiricum with specific microorganisms. Methods: Amplicon and metabolome sequencing were conducted for P. sibiricum interplanted under three types of trees and in the field, and the relationship between the microbiome and secondary metabolism was explored. Results: Principal component analysis (PCA) divided field cultivated and understory interplanted groups into two classes. A total of 95 different metabolites were found, with four expression patterns. The alpha diversity of rhizosphere bacteria and endosphere fungi in the understory interplanted group was significantly higher than that in the farmland cultivated group. There were 276 different rhizosphere microorganism genera among the four groups; however, only 33 different endosphere genera were observed, indicating that endophytic microbial diversity was relatively stable within the P. sibiricum rhizome, especially for endosphere bacteria. Cointertia analysis (CoIA) suggested that the metabolite changes in P. sibiricum induced by interplanting under different trees were more strongly affected by rhizosphere microorganisms than by endosphere microorganisms. In addition, the interactions between rhizosphere microorganisms and metabolites in the farmland group were weakened compared with those in the underplanted groups. Canonical correspondence analysis (CCA) showed that Aspergillus and Ellin6067 had the greatest influence on the metabolites. Myrmecridium, as a shared microbe in the rhizosphere and endosphere, had interaction effects with the largest number of microbes. Conclusion: This study revealed the interactions between the microbes and metabolites in P. sibiricum and systematically explored the mechanism underlying their correlation, which was mediated by the understory interplanting mode. This study provides feasible strategies for improving the medicinal value of P. sibiricum by regulating microorganisms.

Regulatory effects of nitric oxide on reproduction and melanin biosynthesis in onion pathogenic fungus Stemphylium eturmiunum.

The formation of propagules is the critical stage for transmission of the pathogenic fungus Stemphylium eturmiunum. However, how the development of these propagules is regulated remains to be fully understood. Here, we show that nitric oxide (NO) is necessary for reproduction in S. eturmiunum.Application of NO scavenger carboxy-CPTIO (cPTIO) or soluble guanylate cyclase (sGC) inhibitor NS-2028 abolishes propagules formation, which was increased by a supplement of sodium nitroprusside (SNP). SNP supplement also triggered increased biosynthesis of melanin, which can be inhibited upon the addition of arbutin or tricyclazole, the specific inhibitors for DOPA and DHN synthetic pathway, respectively. Intriguingly, enhanced melanin biosynthesis corelates with an increased propagules formation; The SNP-induced increment propagules formation can be also compromised upon the supplement of cPTIO or NS-2028. RT-PCR analysis showed that SNP promoted transcription of brlA, abA and wetA at 0.2 mmol/L, but inhibited at 2 mmol/L. In contrast, SNP increased transcription of mat1, and mat2, and the synthetic genes for DHN and DOPA melanins at 2 mmol/L. However, the increased transcription of these genes is down-regulated upon the supplement of cPTIO or NS-2028. Thus, NO regulates reproduction and melanin synthesis in S. eturmiunum possibly through the NO-sGC-GMP signaling pathway.

The effectiveness of traditional Chinese medicine fumigation and washing nursing care after arthroscopic debridement of Knee Osteoarthritis: A protocol for systematic review and meta-analysis.

BACKGROUND: Knee Osteoarthritis (KOA) is a degenerative osteoarthrosis with knee joint pain as the main symptom. In recent years, arthroscopic removal of loose body and repair of meniscus have become common methods for the treatment of KOA. However, postoperative pain, swelling and limited joint movement affect the functional recovery of knee joint and the effect of surgical treatment. Early postoperative control of pain and swelling is of great significance to improve the curative effect of arthroscopic debridement and promote the recovery of knee joint function. In recent years, many clinical studies have reported that the nursing method of fumigation and washing with Chinese medicine after arthroscopic debridement of KOA can relieve pain, promote the recovery of joint function and improve the clinical curative effect, but there is a lack of evidence-based medicine. The purpose of this study is to evaluate the efficacy and safety of fumigation and washing with traditional Chinese medicine after KOA arthroscopy. METHODS: Computer retrieval English database (PubMed, Embase, Web of Science, the Cochrane Library) and Chinese database (China National Knowledge Infrastructure, Wanfang, VIP Database for Chinese Technical Periodicals, China Biology Medicine disc), moreover manual retrieval academic, Google and baidu from building to since December 2020, traditional Chinese medicine fumigation applied to KOA arthroscopy postoperative nursing of randomized controlled clinical research, by two researchers independently evaluated the quality of the included study and extracted the data. Meta-analysis of the included literatures was performed using RevMan5.3 software. RESULTS: The main observation index of this study was the effective rate, and the secondary indexes included Visual Analogue Scale Score, the Western Ontario and McMaster university orthopedic index, Lysholms score and adverse reactions, so as to evaluate the efficacy and safety of traditional Chinese medicine fumigation nursing after KOA arthroscopy. CONCLUSION: This study will provide reliable evidence for the clinical application of Fumigation and washing nursing of traditional Chinese medicine after KOA arthroscopy. ETHICS AND DISSEMINATION: Private information from individuals will not be published. This systematic review also does not involve endangering participant rights. Ethical approval will not be required. The results may be published in a peer-reviewed journal or disseminated at relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/THZP4.

Deciphering the antitumoral potential of the bioactive metabolites from medicinal mushroom Inonotus obliquus.

ETHNOPHARMACOLOGICAL RELEVANCE: The crude extracts of the medicinal mushroom Inonotus obliquus have been used as an effective traditional medicine to treat malicious tumors, gastritis, gastric ulcers, and other inflammatory conditions in Russia and most Baltic countries. AIM OF THIS REVIEW: Deciphering the antitumoral potential of the bioactive metabolites from I. obliquus and addressing its possibility to be used as effective agents for tumor treatment, restoration of compromised immunity and protection of gastrointestinal damage caused by chemotherapy. MATERIALS AND METHODS: We analysed the current achievements and dilemma in tumor chemo- or immunotherapy. In this context, we searched the published literatures on I. obliquus covering from 1990 to 2020, and summarized the activities of antitumor, antioxidation, and immunomodulation by the polysaccharides, triterpenoids, small phenolic compounds, and hispidin polyphenols. By comparing the merits and shortcomings of current and traditional methodology for tumor treatment, we further addressed feasibility for the use of I. obliquus as an effective natural drug for tumor treatment and prevention. RESULTS: The diverse bioactive metabolites confer I. obliquus great potential to inhibit tumor growth and metastasis. Its antitumor activities are achieved either through suppressing multiple oncogenic signals including but not limited to the activation of NF-κB and FAK, and the expression of RhoA/MMP-9 via ERK1/2 and PI3K/Akt signaling pathway. The antitumor activities can also be achieved by inhibiting tyrosinase activity via PAK1-dependent signaling pathway or altering lysosomal membrane permeabilization through blocking tubulin polymerization and/or disturbing energy metabolism through LKB1/AMPK pathway. In addition, the metabolites from I. obliquus also harbour the potentials to reverse MDR either through selective inhibition on P-gp/ABCB1 or MRP1/ABCC1 proteins or the induction of G2/M checkpoint arrest in tumor cells of chemoresistant phenotypes mediated by Nox/ROS/NF-kB/STAT3 signaling pathway. In addition to the eminent effects in tumor inhibition, the metabolites in I. obliquus also exhibit immunomodulatory potential to restore the compromised immunity and protect against ulcerative damage of GI tract caused by chemotherapy. CONCLUSIONS: I. obliquus possesses the potential to reduce incidence of tumorigenesis in healthy people. For those whose complete remission has been achieved by chemotherapy, administration of the fungus will inhibit the activation of upstream oncogenic signals and thereby prevent metastasis; for those who are in the process of chemotherapy administration of the fungus will not only chemosensitize the tumor cells and thereby increasing the chemotherapeutic effects, but also help to restore the compromised immunity and protect against ulcerative GI tract damage and other side-effects induced by chemotherapy.

L-Arginine Supplementation Improves Vascular Endothelial Dysfunction Induced by High-Fat Diet in Rats Exposed to Hypoxia.

INTRODUCTION: Our previous study showed that high-fat diet inhibited the increase in nitric oxide and endothelial nitric oxide synthase expression in the aortic endothelium of rats exposed to hypoxia, and hypoxia plus a high-fat diet led to earlier and more severe vascular endothelial dysfunction (VED) than hypoxia alone. The purpose of the present study was to investigate the effects of L-arginine on high-fat diet-induced VED of rats in hypoxia. METHODS: Forty male Sprague-Dawley rats were randomly divided into 4 groups and treated with hypoxia (H group), hypoxia plus high-fat diet (H+HFD group), hypoxia plus L-arginine (H+L-Arg group), and hypoxia plus high-fat diet and L-arginine (H+HFD+L-Arg group) for 1 wk. Hypoxia was simulated in a hypobaric chamber with an altitude of 5000 m. Aortic morphology and endothelium-dependent vasorelaxation were used to assess VED. RESULTS: High-fat diet impaired vascular remodeling and reduced endothelium-dependent vasodilator response to acetylcholine in rats exposed to hypoxia, secondary to dysregulation of the nitric oxide pathway. L-arginine supplementation significantly increased plasma nitrates and nitrites and endothelial nitric oxide synthase mRNA levels and improved ultrastructural changes in aortic endothelium and endothelium-dependent vasodilator response. CONCLUSIONS: L-arginine prevents aortic ultrastructural changes and reverses VED induced by high-fat diet in rats exposed to hypoxia, which may have implications for VED induced by high-fat diet in high altitude dwellers.

[Application of Orbitrap high resolution mass spectrometry for the screening and identification of 15 adulterated weight loss compounds in dietary supplements].

A liquid chromatography-Orbitrap high resolution mass spectrometry (LC-HRMS) method and a TraceFinder database were developed for the screening and identification of 15 adulterated weight loss compounds in dietary supplements. The samples were extracted with methanol and filtered through a 0.22 µm microfiltration membrane prior to LC-HRMS analysis. The Full MS/dd-MS2 mode was utilized in both positive and negative ion modes and the collected data were imported into the TraceFinder screening software. The established compound database and screening method were used for rapid, automatic, and high-precision screening to determine if the weight loss compounds were adulterated. The method validation results indicated that all of the analytes showed excellent linear relationships with regression coefficients (r) above 0.998. The recoveries were in the range of 79.7%-95.4% while the precisions ranged from 3.3% to 8.7%. The method and database were used to screen weight loss adulterants in 29 batches of dietary supplements; six batches of samples tested positive for adulterants with the identification of four compounds including sibutramine. This method enables the automatic high-precision screening and identification of adulterants, providing a novel and powerful tool for combating the increasingly rampant occurrence of adulteration in dietary supplements.

Direct filtration for the treatment of the coagulated domestic sewage using flat-sheet ceramic membranes.

Direct membrane filtration (DMF) is considered as a promising technology for municipal wastewater treatment. We utilized an innovative application of flat-sheet ceramic membranes (FSCM) for DMF for the rapid treatment of domestic sewage. Coagulation was applied before FSCM filtration to increase the pollutant removal and to mitigate membrane fouling. This coagulation-FSCM filtration can significantly reduce the pollutant load on the downstream treatment and concentrate organics and nutrients into sludge to facilitate resource recovery. Using polyaluminum chloride (PACl) based FSCM filtration, approximately 90.0% of the chemical oxygen demand (COD) and 99.0% of the phosphorus (P) were removed from the sewage influent and retained in the concentrated sludge, with less than 25.0 mg/L COD left in the effluent. Long-term operation of the PACl-based FSCM filtration stably maintained a high flux of 41.7 L/m2-h (LMH, or 1.0 m/d). The fouled membranes were cleaned chemically every 3-5 d, and the membrane permeability could almost be completely recovered using chemical backwash for only 10 min with a diluted acidic, alkaline, chlorine or H2O2 solution. The novel FSCM process will fundamentally advance wastewater treatment technology. It can be readily modularized and installed as simple add-on units to upgrade and retrofit existing wastewater treatment systems.

Disturbance in biosynthesis of arachidonic acid impairs the sexual development of the onion blight pathogen Stemphylium eturmiunum.

The formation of sexual fruiting bodies for plant pathogenic fungi is a key strategy to propagate their progenies upon environmental stresses. Stemphylium eturmiunum is an opportunistic plant pathogen fungus causing blight in onion. This self-fertilizing filamentous ascomycete persists in the soil by forming pseudothecia, the sexual fruiting body which helps the fungus survive in harsh environments. However, the regulatory mechanism of pseudothecial formation remains unknown. To uncover the mechanism for pseudothecial formation so as to find a practical measure to control the propagation of this onion pathogen, we tentatively used DNA methyltransferase inhibitor 5-azacytidine (5-AC) to treat S. eturmiunum. 5-AC treatment silenced the gene-encoding monoacylglycerol lipase (magl) concomitant with the presence of the inheritable fluffy phenotype and defectiveness in pseudothecial development. Moreover, the silence of magl also resulted in a reduction of arachidonic acid (AA) formation from 27 ± 3.1 µg/g to 9.5 ± 1.5 µg/g. To correlate the biosynthesis of AA and pseudothecial formation, we created magl knockdown and overexpression strains. Knockdown of magl reduced AA to 11 ± 2.4 µg/g, which subsequently disabled pseudothecial formation. In parallel, overexpression of magl increased AA to 37 ± 3.4 µg/g, which also impaired pseudothecial formation. Furthermore, exogenous addition of AA to the culture of magl-silenced or magl knockdown strains rescued the pseudothecial formation but failed in the gpr1 knockdown strain of S. eturmiunum, which implicates the involvement of AA in signal transduction via a putative G protein-coupled receptor 1. Thus, AA at a cellular level of 27 ± 3.1 µg/g is essential for sexual development of S. eturmiunum. Disturbance in the biosynthesis of AA by up- and down-regulating the expression of magl disables the pseudothecial development. The specific requirement for AA in pseudothecial development by S. eturmiunum provides a hint to curb this onion pathogen: to impede pseudothecial formation by application of AA.

Regulation of Anticancer Styrylpyrone Biosynthesis in the Medicinal Mushroom Inonotus obliquus Requires Thioredoxin Mediated Transnitrosylation of S-nitrosoglutathione Reductase.

The medicinal macrofungus Inonotus obliquus widely utilized as folk medicine in Russia and Baltic countries is a source of phenylpropanoid-derived styrylpyrone polyphenols that can inhibit tumor proliferation. Insights into the regulatory machinery that controls I. obliquus styrylpyrone polyphenol biosynthesis will enable strategies to increase the production of these molecules. Here we show that Thioredoxin (Trx) mediated transnitrosylation of S-nitrosoglutathione reductase (GSNOR) underpins the regulation of styrylpyrone production, driven by nitric oxide (NO) synthesis triggered by P. morii coculture. NO accumulation results in the S-nitrosylation of PAL and 4CL required for the synthesis of precursor phenylpropanoids and styrylpyrone synthase (SPS), integral to the production of styrylpyrone, inhibiting their activities. These enzymes are targeted for denitrosylation by Trx proteins, which restore their activity. Further, this Trx S-nitrosothiol (SNO) reductase activity was potentiated following S-nitrosylation of Trx proteins at a non-catalytic cysteine (Cys) residue. Intriguingly, this process was counterbalanced by Trx denitrosylation, mediated by Trx-dependent transnitrosylation of GSNOR. Thus, unprecedented interplay between Trx and GSNOR oxidoreductases regulates the biosynthesis of styrylpyrone polyphenols in I. obliquus.

Simultaneous identification, confirmation and quantitation of illegal adulterated antidiabetics in herbal medicines and dietary supplements using high-resolution benchtop quadrupole-Orbitrap mass spectrometry.

This paper presents an application of ultrahigh-performance liquid chromatography and quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HR MS) for the screening, confirmation and quantification of 11 antidiabetics in herbal medicines and dietary supplements. The mass spectrometer was operated in Full MS/dd-MS(2) (data-dependent MS(2)) mode. The full MS scan acquired data for identification and quantification, and dd-MS(2) scan obtained product ion spectra for confirmation. UHPLC-Q-Orbitrap MS quantification was achieved using matrix-matched standard calibration curves with phenacetin as internal standard. The method validation that included selectivity, sensitivity, calibration curve, accuracy and precision, recovery, matrix effect and stability was evaluated. The response showed good linear relationship with the concentrations of analytes over wide ranges (e.g., 0.0004-1 µg/g for metformin) with all the coefficients of correlation (r(2)) >0.9991. The detection limits (LODs) were in the range of 0.05-0.5 ng/g for different analytes. The recoveries yielded results higher than 74.3% for all compounds. The accuracy was in the range of -6.75 to 3.85%, while the intra- and inter-day precision ranged from 0.048 to 11.5%. Among 63 batches of herbal medicines and 34 batches of dietary supplements samples, 7 batches of dietary supplements were positive, while all the herbal medicines were negative. Overall, the novel UHPLC-Q-Orbitrap has demonstrated great performance for identification, confirmation and quantification of antidiabetics in herbal medicines and dietary supplements, ensuring food safety and public health.

Application of a high resolution benchtop quadrupole-Orbitrap mass spectrometry for the rapid screening, confirmation and quantification of illegal adulterated phosphodiesterase-5 inhibitors in herbal medicines and dietary supplements.

In this study, the applicability of high resolution quadrupole-Orbitrap (Q-Orbitrap) mass spectrometry for the simultaneous qualitative and quantitative analysis of illegal adulterated phosphodiesterase-5 inhibitors (PDE-5 inhibitors) in herbal medicines and dietary supplements was investigated. The mass spectrometer was operated in full MS scan/dd-MS(2) (data-dependent MS(2)) mode. The use of 70,000 FWHM mass resolution and narrow mass windows (5 ppm) could effectively improve the selectivity of the method, increasing the signal-to-noise ratio for the analytes. The response showed good linear relationship with the analytes' concentrations over wide ranges (e.g., 0.05-10 µg/g for sildenafil) with all the coefficient of determinations (r(2)) >0.9996. The detection limits (LODs) were in the range of 1.0-5.0 ng/g for different analytes. The recoveries ranged from 85.4% to 96.7%. The intra- and inter-day accuracies were in the range of -6.6 to 10.1%, while the intra- and inter-day precision ranged from 0.0039% to 13.2%. Among 68 batches of herbal medicines and 20 batches of dietary supplements (including 83 capsules, 3 pellets and 2 liquid) samples, sildenafil was detected in 8 dietary supplements, while noracetildenafil was detected in only one dietary supplement. The novel Q-Orbitrap mass spectrometry has been proved to be a very promising and powerful tool for routine screening of illegal adulterate in herbal medicines and dietary supplements, ensuring food safety and public health.

Crocin Exhibits Antitumor Effects on Human Leukemia HL-60 Cells In Vitro and In Vivo.

Crocin is a carotenoid of the saffron extract that exhibits antitumor activity against many human tumors. However, the effects of crocin on HL-60 cells in vivo have not been evaluated. This study aimed to examine the effects of crocin on HL-60 cells in vitro and in vivo and investigate the underlying mechanisms. HL-60 cells were treated by crocin, and cell proliferation, apoptosis, and cell cycle profiles were examined by MTT assay, AO/EB staining, and flow cytometry, respectively. Furthermore, HL-60 cells were xenografted into nude mice and treated by crocin, the tumor weight and size were calculated, and the expression of Bcl-2 and Bax in xenografts was detected by immunohistochemical staining. The results showed that crocin (0.625-5 mg/mL) inhibited HL-60 cell proliferation and induced apoptosis and cell cycle arrest at G0/G1 phase, in a concentration and time-dependent manner. In addition, crocin (6.25, 25 mg/kg) inhibited the tumor weight and size of HL-60 xenografts in nude mice, inhibited Bcl-2 expression, and increased Bax expression in xenografts. In summary, crocin inhibits the proliferation and tumorigenicity of HL-60 cells, which may be mediated by the induction of apoptosis and cell cycle arrest and the regulation of Bcl-2 and Bax expression.

[Proliferative inhibition and apoptotic induction effects of crocin on human leukemia HL-60 cells and their mechanisms].

This study was to investigate the proliferative inhibition and apoptosis of human leukemia HL-60 cells induced by crocin and their possible mechanisms. The cell viability was tested by cell counting. The morphology of HL-60 cells was observed by fluorescence microscopy. The MTT assay was used to evaluate the inhibitory effect of crocin on the growth of HL-60 cells. Flow cytometry was used to measure the cell cycle. RT-PCR was used to detect bcl-2 and bax expression. The results indicated that the growth of HL-60 cells was inhibited remarkably in the dose and time dependent way. When the crocin concentration was higher than 5 mg/ml, the percentage of apoptotic HL-60 cells was not increased, on the contrary this percentage decreased, the cells manifested necrosis. Flow cytometry profiles revealed that cells were blocked in G0/G1 phase, the cell proliferation was inhibited obviously at 5 mg/ml. RT-PCR detection revealed that the expression of bcl-2 was down-regulated strikingly and bax was up-regulated. It is concluded that the crocin can inhibit the proliferation of HL-60 cells effectively, and block cells in G0/G1 phase. The mechanisms by which crocin induced apoptosis in HL-60 cells may be related to the inhibition of bcl-2 and activation of bax.

Chemical diversity of biologically active metabolites in the sclerotia of Inonotus obliquus and submerged culture strategies for up-regulating their production.

Inonotus obliquus (Fr.) Pilat is a white rot fungus belonging to the family Hymenochaetaceae in the Basidiomycota. In nature, this fungus rarely forms a fruiting body but usually an irregular shape of sclerotial conk called 'Chaga'. Characteristically, I. obliquus produces massive melanins released to the surface of Chaga. As early as in the sixteenth century, Chaga was used as an effective folk medicine in Russia and Northern Europe to treat several human malicious tumors and other diseases in the absence of any unacceptable toxic side effects. Chemical investigations show that I. obliquus produces a diverse range of secondary metabolites including phenolic compounds, melanins, and lanostane-type triterpenoids. Among these are the active components for antioxidant, antitumoral, and antiviral activities and for improving human immunity against infection of pathogenic microbes. Geographically, however, this fungus is restricted to very cold habitats and grows very slowly, suggesting that Chaga is not a reliable source of these bioactive compounds. Attempts for culturing this fungus axenically all resulted in a reduced production of bioactive metabolites. This review examines the current progress in the discovery of chemical diversity of Chaga and their biological activities and the strategies to modulate the expression of desired pathways to diversify and up-regulate the production of bioactive metabolites by the fungus grown in submerged cultures for possible drug discovery.

Phenolic acids analysis in ligusticum chuanxiong using HPLC.

A reversed-phase high-performance liquid chromatographic method with diode array UV detection is developed for the determination of five kinds of phenolic acids common in herbal medicines. Based on this method, ferulic acid and caffeic acid are found to be two main phenolic acids in Chuanxiong (one of the important crude drugs in traditional Chinese medicine). More important, ferulic acid is found to exist in free form, and caffeic acid--a previously unreported component--is found to exist in esterified or insoluble-bound form.