UPLC–ESI-Q-TOF/MS based metabolic profiling of protosappanin B in rat plasma, bile, feces, urine and intestinal bacteria samples

Background: Caesalpinia sappan L. is a traditional medicinal plant that is used to promote blood circulation and treat stroke in China. Protosappanin B (PTB) is a unique homoisoflavone compound isolated from Sappan Lignum (the heartwood of Caesalpinia sappan L). In a previous study, the metabolic fate of PTB remained unknown. Objective: To explore whether PTB is extensively metabolized, the metabolites of PTB in bile, plasma, urine, feces, and intestinal bacteria samples in rats were investigated. Method: The biosamples were investigated by ultraperformance liquid chromatography combined with time-of-flight mass spectrometry (UPLC-TOF-MS/MS) with MetabolitePilot software. Result: 28 metabolites were identified in the biosamples: 18 metabolites in rat bile, 8 in plasma, 20 in feces, 7 in urine and 2 in intestinal bacteria samples. Both phase I and phase II metabolites were observed. Metabolite conversion occurred via 9 proposed pathways: sulfate conjugation, glucuronide conjugation, bis-glucuronide conjugation, glucose conjugation, dehydration, oxidation, hydrolysis, methylation and hydroxymethylene loss. The metabolic pathways differed among biosamples and exhibited different distributions. Among these pathways, the most important were sulfate and glucuronide conjugation. Conclusion: The results showed that the small intestinal and biliary routes play an important role in the clearance and excretion of PTB. The main sites of metabolism in the PTB chemical structure were the phenolic hydroxyl and the side-chains on the eight-element ring.

PSK signaling controls ABA homeostasis and promotes shoot growth under mannitol stress

We hypothesized that the growth-promoting activity of the peptide hormone phytosulfokine (PSK), may be important to maintain growth under abiotic stress. To test this hypothesis, we employed mannitol as a stressor and analyzed the involvement of abscisic acid (ABA) that mediates a subset of mannitol responses. Inhibition of seed germination by ABA or mannitol was independent of PSK receptor signaling whereas repression of cotyledon greening was partially dependent on PSKR signaling with receptor null pskr1-3 pskr2-1 seedlings showing enhanced greening. Mannitol led to dose-dependent shoot growth inhibition that was alleviated by PSKR signaling. With mannitol, pskr1-3 pskr2-1 seedlings had strongly reduced shoot fresh and dry weights incompared to wild type. Analysis of pskr1-3 and pskr2-1 single receptor loss-of-function lines revealed that signaling via PSKR1 is crucial for shoot growth promotion under mannitol stress. Mannitol and ABA induced expression of PSK3 and PSKR1 and ABA promoted expression of PSK2 and PSK4. In turn, PSKR signaling was required for mannitol-induced accumulation of ABA in the shoot revealing a regulatory feedback loop. PSKRs repressed expression of UGT71B6 encoding an ABA UDP-glucosyltransferase and maintained expression of BG1, an ABA glucosidase which may lead to reduced ABA-glucose conjugation and enhanced ABA remobilization. Marker gene expression analyses supported the conclusion that PSKR and mannitol signaling pathways crosstalk, a conclusion that was supported by the observation that PSK receptor signaling enhanced sensitivity to ABA in mannitol-stressed shoots. In conclusion, PSKR signaling maintains shoot growth under mannitol stress in part by interfering with the ABA pathway.One-sentence summaryPSK receptor signaling is required to delay cotyledon greening in response to mannitol and ABA nad it promotes shoot growth under mannitol stress by controlling ABA levels and ABA responsiveness.

Improved drug delivery system for cancer treatment by D-glucose conjugation with eugenol from natural product

Introduction: Bioconjugations are swiftly progressing and are being applied to solve several limitations of conventional drug delivery systems (DDS) such as lack of water solubility, non-specific, and poor bioavailability. The main goals of DDS are to achieve greater drug effectiveness and minimize toxicity to the healthy tissues. Objectives: In this study, D-glucose was conjugated with eugenol to target the cancer cells. To identify the implication of the anticancer effect, osteosarcoma (K7M2) cells were cultured and the anti-proliferative effect was performed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay] test in order to evaluate the viability and toxicity on cells with various concentrations of eugenol and D-glucose-eugenol conjugate in 24-hour incubation. Results: It was found that, the successful confirmation of the conjugation between D-glucose and eugenol was obtained by 1 H NMR spectroscopy. MTT assay showed inhibitory concentration (IC50 value) of D-glucose-eugenol was at 96.2 µg/ml and the decreased of osteosarcoma cell survival was 48%. Conclusion: These findings strongly indicate that K7M2 cells would be affected by toxicity of D-glucose-eugenol. Therefore, the present study suggests that D-glucose-eugenol has high potential to act as an anti-proliferative agent who may promise a new modality or approach as the drug delivery treatment for cancer or chemotherapeutic agent.

OsIAGT1 Is a Glucosyltransferase Gene Involved in the Glucose Conjugation of Auxins in Rice

Background In cereal crop rice, auxin is known as an important class of plant hormone that regulates a plethora of plant growth and development. Glycosylation of auxin is known to be one of the important mechanisms mediating auxin homeostasis. However, the relevant auxin glucosyltransferase (GT) in rice still remains largely unknown. Results In this study, using known auxin glucosyltransferases from other species as queries, twelve putative auxin UDP-glycosyltransferase (UGT) genes were cloned from rice and the one showing highest sequence similarity, named as OsIAGT1, was expressed as recombinant protein. In vitro enzymatic analysis showed that recombinant OsIAGT1 was capable of catalyzing glucosylation of IAA, IBA and other auxin analogs, and that OsIAGT1 is quite tolerant to a broad range of reaction conditions with peak activity at 30 °С and pH 8.0. OsIAGT1 showed favorite activity towards native auxins over artificially synthesized ones. Further study indicated that expression of OsIAGT1 can be upregulated by auxin in rice, and with OsIAGT1 overexpressing lines we confirmed that OsIAGT1 is indeed able to glucosylate IAA in vivo. Consistently, ectopic expression of OsIAGT1 leads to declined endogenous IAA content, as well as upregulated auxin synthesis genes and reduced expression of auxin-responsive genes, which likely leads to the reduced plant stature and root length in OsIAGT1 overexpression lines. Conclusion Our result indicated that OsIAGT1 plays an important role in mediating auxin homeostasis by catalyzing auxin glucosylation, and by which OsIAGT1 regulates growth and development in rice.

Successful Targeting of the Warburg Effect in Prostate Cancer by Glucose-Conjugated 1,4-Naphthoquinones

Treatment of castration-resistant prostate cancer (CRPC) remains challenging due to the development of drug resistance. The Warburg effect describes the ability of cancer cells to consume larger amounts of glucose compared to normal tissues. We identified derivatives of natural 1,4-naphthoquinones to be active in CRPC and further synthetically modified them via glucose conjugation to increase selectivity by Warburg effect targeting. Mechanisms of action were examined by quantitative proteomics followed by bioinformatical analysis and target validation. Four synthesized molecules revealed the highest selectivity towards human CRPC cells, which correlated with higher GLUT-1 activity and expression. The compounds were able to induce pro-apoptotic signs and to inhibit the pro-survival processes and mechanisms of drug resistance (i.e., AR-signaling and autophagy). Proteome analysis suggested a disruption of the mitochondria/oxidative phosphorylation, which was validated by further functional analysis: thus, mitochondria depolarization, elevated levels of cytotoxic ROS, an increase of Bax/Bcl-2 ratio as well as release of mitochondrial AIF and cytochrome C to cytoplasm were observed. In conclusion, glucose-conjugated 1,4-naphthoquinones show potent activity and selectivity in human CRPC exerted via mitochondrial targeting. The compounds can overcome drug resistance against current standard therapies and suppress pro-survival mechanisms. This unique combination of properties makes them new promising candidates for the treatment of CRPC.

A Complete Study of Farrerol Metabolites Produced in Vivo and in Vitro

Although farrerol, a characteristically bioactive constituent of Rhododendron dauricum L., exhibits extensive biological and pharmacological activities (e.g., anti-oxidant, anti-immunogenic, and anti-angiogenic) as well as a high drug development potential, its metabolism remains underexplored. Herein, we employed ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry coupled with multiple data post-processing techniques to rapidly identify farrerol metabolites produced in vivo (in rat blood, bile, urine and feces) and in vitro (in rat liver microsomes). As a result, 42 in vivo metabolites and 15 in vitro metabolites were detected, and farrerol shown to mainly undergo oxidation, reduction, (de)methylation, glucose conjugation, glucuronide conjugation, sulfate conjugation, N-acetylation and N-acetylcysteine conjugation. Thus, this work elaborates the metabolic pathways of farrerol and reveals the potential pharmacodynamics forms of farrerol.