Natural Product BO-1 as an Inner Responsive Molecule Inhibits Antimicrobial-Resistant Staphylococcus aureus via Synergism.

Multidrug-resistant Staphylococcus aureus, a Gram-positive bacterium that causes several difficult-to-treat human infections, is a considerable threat to global healthcare. We hypothesize that there exist inner responsive molecules (IRMs) which can function synergistically with antibiotics to restore the sensitivity of resistant bacteria to existing antibiotics without inducing new antibiotic resistance. An investigation of the extracts of the Chinese medicinal herb Piper betle L. led to the isolation of six benzoate esters, BO-1-BO-6. Among these, BO-1 as a distinct IRM displayed considerable synergism by potentiating antibacterial activity against five antibiotic-resistant S. aureus strains. Mechanistic studies demonstrated that BO-1 acted as a suppressing drug resistance IRM via inhibiting efflux activity. A combination of BO-1 with ciprofloxacin significantly inhibited resistance to this antibiotic and reversed its resistance in the S. aureus strain. Furthermore, BO-1 effectively enhanced the activity of ciprofloxacin against the efflux fluoroquinolone-resistant S. aureus strain SA1199B that caused infection in two animal models and significantly decreased the inflammatory factors IL-6 and C-reactive protein of the infected mice, thereby showing the practice utility of this approach.

[Construction of cell factories for production of patchoulol in Saccharomyces cerevisiae].

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.

Antiliver Fibrosis Formula of Fuzheng Huayu Alleviates Inflammatory Response.

Fuzheng Huayu's (FZHY) formula ameliorated liver fibrosis in clinical and experimental practice. Based on the close link between fibrosis and inflammation, its anti-inflammatory effect and related mechanisms were explored in this present study. With the aid of the inflammatory macrophage model, FZHY significantly blocked nitrite accumulation without observable cytotoxicity due to its suppression of inducible nitric oxide synthase (iNOS) gene and protein expressions in a concentration-depended manner. Proinflammatory mediators including IL-6, CD86, and CD40 were also restrained by FZHY. Interestingly, FZHY induced anti-inflammatory mediators heme oxygenase 1 (HO-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) expressions simultaneously. Downregulation of iNOS and miR-155 and upregulation of PPAR-γ were also observed in CCl4-induced liver fibrosis mice upon FZHY administration. Mechanically, FZHY strikingly eliminated the phosphorylation of STAT1 and MAPK. Taken together, FZYH regulated the balance of proinflammatory and anti-inflammatory mediators partially via modulating STAT1/MAPK pathways and the miR-155/PPAR-γ axis.

[Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae].

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

[Construction of cell factories for high production of ginsenoside Rh_2 in Saccharomyces cerevisiae].

Ginsenoside Rh_2 is a rare active ingredient in precious Chinese medicinal materials such as Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Panacis Quinquefolii Radix. It has important pharmacological activities such as anti-cancer and improving human immunity. However, due to the extremely low content of ginsenoside Rh_2 in the source plants, the traditional way of obtaining it has limitations. This study intended to apply synthetic biological technology to develop a cell factory of Saccharomyces cerevisiae to produce Rh_2 by low-cost fermentation. First, we used the high protopanaxadiol(PPD)-yielding strain LPTA as the chassis strain, and inserted the Panax notoginseng enzyme gene Pn1-31, together with yeast UDP-glucose supply module genes[phosphoglucose mutase 1(PGM1), α-phosphoglucose mutase(PGM2), and uridine diphosphate glucose pyrophosphorylase(UGP1)], into the EGH1 locus of yeast chromosome. The engineered strain LPTA-RH2 produced 17.10 mg·g~(-1) ginsenoside Rh_2. This strain had low yield of Rh_2 while accumulated much precursor PPD, which severely restricted the application of this strain. In order to further improve the production of ginsenoside Rh_2, we strengthened the UDP glucose supply module and ginsenoside Rh_2 synthesis module by engineered strain LPTA-RH2-T. The shaking flask yield of ginsenoside Rh_2 was increased to 36.26 mg·g~(-1), which accounted for 3.63% of the dry weight of yeast cells. Compared with those of the original strain LPTA-RH2, the final production and the conversion efficiency of Rh_2 increased by 112.11% and 65.14%, respectively. This study provides an important basis for further obtaining the industrial-grade cell factory for the production of ginsenoside Rh_2.

Simultaneous determination of eight analytes of Fuzheng Huayu recipe in beagle dog plasma by UHPLC-Q/exactive Orbitrap HRMS and its application to toxicokinetics.

Fuzheng Huayu recipe (FZHY) is a Chinese patent medicine for the treatment of liver fibrosis. This study aimed to investigate the toxicokinetics of FZHY in beagle dogs after oral administration. Blood samples were collected on days 1, 15 and 28 after oral gavage of FZHY dosages of 400 or 1,200 mg/kg body weight once a day. A UHPLC-Q-Orbitrap method was developed and validated to simultaneously determine and quantify eight components of FZHY in beagle dog plasma. The times to peak concentration for eight components were18-120 min. The peak concentrations (Cmax ) of amygdalin, genistein, daidzein and 3,4-dihydroxybenzaldehyde were 1.43-43.50 ng/ml, the areas under the concentration-time curve (AUC(0-t) ) were 2.45-6,098.25 ng min/ml, and the apparent volumes of distribution (Vd ) were 0.05-131.23 × 104 ml/kg. The values of Cmax of prunasin, schisantherin A, schisandrin A and schisandrin were 7.35-1,450.73 ng/ml, the values of AUC(0-t) were 3,642.30-330,388.65 ng min/ml, and the values of Vd were 11.15-1,087.18 × 104 ml/kg. No obvious accumulation of the eight compounds was observed in beagle dogs. The results showed that the method is rapid, accurate and sensitive, and is suitable for detecting the eight analytes of FZHY. This study provides an important basis for the assessment of FZHY safety.

Comprehensive profiling and characterization of the absorbed components and metabolites in mice serum and tissues following oral administration of Qing-Fei-Pai-Du decoction by UHPLC-Q-Exactive-Orbitrap HRMS.

Qing-Fei-Pai-Du decoction (QFPDD) is a Chinese medicine compound formula recommended for combating corona virus disease 2019 (COVID-19) by National Health Commission of the People's Republic of China. The latest clinical study showed that early treatment with QFPDD was associated with favorable outcomes for patient recovery, viral shedding, hospital stay, and course of the disease. However, the effective constituents of QFPDD remain unclear. In this study, an UHPLC-Q-Orbitrap HRMS based method was developed to identify the chemical constituents in QFPDD and the absorbed prototypes as well as the metabolites in mice serum and tissues following oral administration of QFPDD. A total of 405 chemicals, including 40 kinds of alkaloids, 162 kinds of flavonoids, 44 kinds of organic acids, 71 kinds of triterpene saponins and 88 kinds of other compounds in the water extract of QFPDD were tentatively identified via comparison with the retention times and MS/MS spectra of the standards or refereed by literature. With the help of the standards and in vitro metabolites, 195 chemical components (including 104 prototypes and 91 metabolites) were identified in mice serum after oral administration of QFPDD. In addition, 165, 177, 112, 120, 44, 53 constituents were identified in the lung, liver, heart, kidney, brain, and spleen of QFPDD-treated mice, respectively. These findings provided key information and guidance for further investigation on the pharmacologically active substances and clinical applications of QFPDD.

Phenolic constituents with neuroprotective activities from Hypericum wightianum.

Five undescribed phenolic compounds, inclusing a depsidone derivative, hyperwightin A, a flavone derivative, hyperwightin B, and three benzophenone glycosides, hyperwightins C-E, along with four known ones were isolated from the 95% EtOH extract of the whole plants of Hypericum wightianum. Structures of the obtained compounds were elucidated by spectroscopic analyses. The protective effects of the isolates against corticosterone-induced PC12 cell injury were assessed. Hyperwightin E, petiolin G and hyperxanthone exhibited noticeable neuroprotection at 10 µM.

Nitrogen and phosphorus doped polymer carbon dots as a sensitive cellular mapping probe of nitrite.

Nitrite (NO2 -) is one of the important pollutants in food and the environment, which can seriously endanger the health of human beings. Therefore, detecting nitrite in food, environmental and biological samples is very significant for health monitoring. Herein, polymer carbon dots (PCDs) doped with nitrogen and phosphorus were prepared by polymerization of ascorbic acid (AA) and polyethylenimine (PEI) with phosphoric acid, and exhibited excellent stability, adjustable fluorescence emissions and good biocompatibility. It was found that the PCDs presented a sensitive response to nitrite (NO2 -), and they were successfully applied for NO2 - analysis in water and milk samples, and the dynamic monitoring of nitrite entry into Hep-2 cells.

[Studies on phenanthrene constituents from stems of Dendrobium candidum].

OBJECTIVE: To investigate chemical constituents from the stems of Dendrobium candidum. METHODS: The constituents were isolated by various column chromatography methods, and their structures were elucidated by spectral analysis (IR, UV, NMR and MS). RESULTS: Six compounds were isolated from ethanol extract and their structures were identified as 2,3,4,7-tetramethoxyphenanthrene (I), nakaharain (II), 2,5-dihydroxy-3,4-dimethoxyphenanthrene (III), confusarin (IV), nudol (V) and bulbophyllanthrin (VI). CONCLUSION: Among these compounds, compounds II, III, VI are isolated from Dendrobium candidum for the first time.