Advanced methods for natural product drug discovery in the field of nutraceuticals

Advances in analytical methods and bioassay development have helped to push forward the research in natural products. In plant extracts and nutraceuticals, bioactive compounds are part of a complex mixture. The development of high-resolution methods related to HPLC for both chemical and biological profiling has significantly increased the efficiency of classical bioactivity-guided fractionation procedures. Furthermore, the level of sensitivity obtained by these methods give the possibility to work with few micrograms of compound. This represents a key advantage for rapid localisation of the biological activity and subsequent identification of the compounds of interest. The same methods are also used to study the extracts from a metabolomic view point. The possibility to study them as a whole can highlight synergetic effects, which are likely to occur in plant extracts and nutraceuticals. In this paper, the main trends are summarised and the developments made in our laboratory on profiling crude extracts with UHPLC-TOF-MS, natural product identification at the microgram level using microflow NMR and integration of these methods with biological evaluation are highlighted.     

Bioactive superparamagnetic nanoparticles for multifunctional composite bone cements

Magnetite-based nanoparticles (NPs) were synthesized by co-precipitation process and coated with a thin layer of silica, eventually doped with calcium, by a modified Stöber method. The potential bioactive behavior of NPs was investigated by dipping samples in simulated body fluid (SBF) and analyzing them with Field-Emission Scanning Electron Microscope (FESEM) equipped with Energy Dispersive Spectroscopy (EDS). Silica-coated NPs displayed evidence of HAp grown on their surface and were then used as a filler for polymethyl methacrylate (PMMA)-based bone cement to impart bioactive and magnetic properties. The influence of the amount of magnetic NPs and the cement mixing method (manual or mechanical) were estimated in terms of NPs dispersion, compressive strength and bioactive behavior. The obtained data evidenced that both the NPs amount and the mixing method influenced the strength of the composites. A delay in the bioactivity was observed for manual mixed cement; moreover, mechanically mixed composites containing a low amount of NPs showed superparamagnetic behavior. These results suggest that the investigated composite bone cements are promising materials for the treatment of bone tumors and associated complications.     

Zebrafish Melanoma-Derived Interstitial EVs Are Carriers of ncRNAs That Induce Inflammation

Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRAS^V12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system.     

Rapid Discovery of Aspartyl Protease Inhibitors Using an Anchoring Approach

Pharmacophore searches that include anchors, fragments contributing above average to receptor binding, combined with one-step syntheses are a powerful approach for the fast discovery of novel bioactive molecules. Here, we are presenting a pipeline for the rapid and efficient discovery of aspartyl protease inhibitors. First, we hypothesized that hydrazine could be a multi-valent warhead to interact with the active site Asp carboxylic acids. We incorporated the hydrazine anchor in a multicomponent reaction and created a large virtual library of hydrazine derivatives synthetically accessible in one-step. Next, we performed anchor-based pharmacophore screening of the libraries and resynthesized top-ranked compounds. The inhibitory potency of the molecules was finally assessed by an enzyme activity assay and the binding mode confirmed by several soaked crystal structures supporting the validity of the hypothesis and approach. The herein reported pipeline of tools will be of general value for the rapid generation of receptor binders beyond Asp proteases.     

Generation of a Novel Transgenic Zebrafish for Studying Adipocyte Development and Metabolic Control

Zebrafish have become a popular animal model for studying various biological processes and human diseases. The metabolic pathways and players conserved among zebrafish and mammals facilitate the use of zebrafish to understand the pathological mechanisms underlying various metabolic disorders in humans. Adipocytes play an important role in metabolic homeostasis, and zebrafish adipocytes have been characterized. However, a versatile and reliable zebrafish model for long-term monitoring of adipose tissues has not been reported. In this study, we generated stable transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) in adipocytes. The transgenic zebrafish harbored adipose tissues that could be detected using GFP fluorescence and the morphology of single adipocyte could be investigated in vivo. In addition, we demonstrated the applicability of this model to the long-term in vivo imaging of adipose tissue development and regulation based on nutrition. The transgenic zebrafish established in this study may serve as an excellent tool to advance the characterization of white adipose tissue in zebrafish, thereby aiding the development of therapeutic interventions to treat metabolic diseases in humans.     

Anthratectone and Naphthotectone,Two Quinones from Bioactive Extracts of <Emphasis Type="Italic">Tectona grandis</Emphasis>

Two new quinones, (an isoprenoid quinone, and a dimeric anthraquinone) named naphthotectone and anthratectone, respectively, were isolated from bioactive leaf extracts from Tectona grandis. Their structures were determined by a combination of 1D and 2D NMR techniques. The bioactivity profile of naphthotectone was assessed using the etiolated wheat coleoptiles bioassay in aqueous solutions at concentrations ranging from 10⁻³ to 10⁻⁵M, as well as the standard target species lettuce, cress, tomato, and onion. Naphthotectone showed high level of activities in both bioassays. This fact, along with the presence of this compound as the major component in Tectona grandis, suggests that it may be involved in the allelopathic activity previously described for this species, and probably in other defense mechanisms.     

Antagonism of microRNA function in zebrafish embryos by using locked nucleic acid enzymes (LNAzymes)

MicroRNAs (miRNAs) have crucial functions in many cellular processes, such as differentiation, proliferation and apoptosis; aberrant expression of miRNAs has been linked to human diseases, including cancer. Tools that allow specific and efficient knockdown of miRNAs would be of immense importance for exploring miRNA function. Zebrafish serves as an excellent vertebrate model system to understand the functions of miRNAs involved in a variety of biological processes. We designed and employed a strategy based on locked nucleic acid enzymes (LNAzymes) for in vivo knockdown of miRNA in zebrafish embryos. We demonstrate that LNAzyme can efficiently knockdown miRNAs with minimal toxicity to the zebrafish embryos.     

New approaches for studying the chemical diversity of natural resources and the bioactivity of their constituents

Natural products (NPs) have historically been an important source of lead molecules in drug discovery. However, the interest that the pharmaceutical industry has had in NPs has declined in part because of the lack of compatibility of traditional natural-product extract libraries with high-throughput screenings and the low hit rate. Furthermore, in contrast to the synthetic libraries, compounds from natural sources are likely to have complex structures which slow down the identification process and contribute to problems related to supply and manufacturing. In this paper, we summarise some of the strategies that are being developed in our research unit to address these issues. On one hand, differential screening strategies were established with the aim of identifying dynamically induced NPs from silent biosynthetic pathways in plants and fungi that had been exposed to different stress situations. On the other hand, high-resolution HPLC techniques were optimised for biological and chemical profiling of crude extracts. This led to an integrated platform for rapid and efficient identification of new drug-leads and biomarkers of interest that were based on miniaturised technological approaches and metabolomics.     

一步法合成三羟甲基丙烷脂肪酸己二酸混合酯

采用红外光谱,核磁共振等分析手段研究了一步法合成三羟甲基丙烷脂肪酸己二酸混合酯,并考察了己二酸在一步法合成混合酯时的反应情况,结果表明:一步法合成三羟甲基丙烷脂肪酸己二酸混合酯酯化反应可达99.0%,己二酸在该体系中更易反应完全。并且与两步法合成的三羟甲基丙烷脂肪酸己二酸混合酯结构相近,性能相当。     

Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia

Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left–right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.     

Hemodynamic Forces Regulate Cardiac Regeneration-Responsive Enhancer Activity during Ventricle Regeneration

Cardiac regenerative capacity varies widely among vertebrates. Zebrafish can robustly regenerate injured hearts and are excellent models to study the mechanisms of heart regeneration. Recent studies have shown that enhancers are able to respond to injury and regulate the regeneration process. However, the mechanisms to activate these regeneration-responsive enhancers (RREs) remain poorly understood. Here, we utilized transient and transgenic analysis combined with a larval zebrafish ventricle ablation model to explore the activation and regulation of a representative RRE. lepb-linked enhancer sequence (LEN) directed enhanced green fluorescent protein (EGFP) expression in response to larval ventricle regeneration and such activation was attenuated by hemodynamic force alteration and mechanosensation pathway modulation. Further analysis revealed that Notch signaling influenced the endocardial LEN activity as well as endogenous lepb expression. Altogether, our work has established zebrafish models for rapid characterization of cardiac RREs in vivo and provides novel insights on the regulation of LEN by hemodynamic forces and other signaling pathways during heart regeneration.     

Genetic design of bioactive glass

This paper reviews the discovery that controlled release of biologically active Ca and Si ions from bioactive glasses leads to the up-regulation and activation of seven families of genes in osteoprogenitor cells that give rise to rapid bone regeneration. This finding offers the possibility of creating a new generation of gene activating glasses designed specially for tissue engineering and in situ regeneration of tissues. Recent findings also indicate that controlled release of lower concentrations of ionic dissolution products from bioactive glasses can be used to induce angiogenesis and thereby offer potential for design of gene activating glasses for soft tissue regeneration.     

药用植物内生真菌及其生物活性物质研究概况

综述药用植物内生真菌及其生物活性物质的研究现状。分析了内生真菌产生物活性物质的种类、方式与作用,介绍了内生真菌分离与活性物质的筛选及其在新药研发中的应用。     

Evaluation of ZnO-Vitamin B1 Nanoparticles on Bioactivity and Physiochemical Properties of the Polycaprolactone-Based Nanocomposites

In the present study, we investigated the use of thiamine chloride hydrochloride (vitamin B₁)-modified ZnO nanoparticles (ZnO-VB₁ NPs) to reinforce polycaprolactone matrix. The stable and bioactive PCL/ZnO-VB₁ nanocomposites were fabricated with the combination of ultrasonication and solution casting methods. Transmission electron microscope results indicated that the ZnO-VB₁ NPs were uniformly dispersed in the matrix. The nanocomposites showed high hydroxyapatite formation (high bioactivity) in the simulated body fluid. The nanocomposites with 2?wt% of the modified nanoparticles were found to have highest mechanical strength. The nanocomposites with more nanofiller concentrations exhibited high wettability.     

An Efficient Synthesis of 3-Alkylpyridine Alkaloids Enables Their Biological Evaluation

3-Alkylpyridine alkaloids (3-APAs) isolated from the arctic sponge Haliclona viscosa are a promising group of bioactive marine alkaloids. However, due to limited bioavailability, investigations of their bioactivity have been hampered. Additionally, synthesis of a common intermediate requires the use of protecting groups and harsh conditions. In this work, we developed a simple and concise two-step route to nine different natural and synthetic haliclocyclins. These compounds displayed modest antibiotic activity against several Gram-positive bacterial strains.     

Synthesis of Microcrystalline Wollastonite Bioceramics and Evolution of Bioactivity

Alpha and beta wollastonite can serve as alternative low-cost bioceramics for bone repair and drug delivery. However, it has been challenging to develop energy-saving, facile and rapid synthetic methodologies for bioactive wollastonite phases. The aim of this work was the rapid preparation and characterization of bioactive α- and β-wollastonite powders by a coprecipitation method and their in-vitro bioactivity evaluation in the SBF solution. The results revealed that heating of the reactant solution at 100 ^°C for 2 h before sintering induced rapid formation of pure α- and β-wollastonite powders with agglomerated particles size in the range 2–7 μm. In-vitro bioactivity testing showed that the prepared α- and β-Wollastonite powders exhibit excellent bioactivity performance. Therefore, this method is promising for preparing bioactive wollastonite structures for medical applications such as bone substitutes and drug carriers.     

Co-Formulation of Amphiphilic Cationic and Anionic Cyclodextrins Forming Nanoparticles for siRNA Delivery in the Treatment of Acute Myeloid Leukaemia

Non-viral delivery of therapeutic nucleic acids (NA), including siRNA, has potential in the treatment of diseases with high unmet clinical needs such as acute myeloid leukaemia (AML). While cationic biomaterials are frequently used to complex the nucleic acids into nanoparticles, attenuation of charge density is desirable to decrease in vivo toxicity. Here, an anionic amphiphilic CD was synthesised and the structure was confirmed by Fourier-transform infrared spectroscopy (FT-IR), Nuclear Magnetic Resonance (NMR), and high-resolution mass spectrometry (HRMS). A cationic amphiphilic cyclodextrin (CD) was initially used to complex the siRNA and then co-formulated with the anionic amphiphilic CD. Characterisation of the co-formulated NPs indicated a significant reduction in charge from 34 ± 7 mV to 24 ± 6 mV (p < 0.05) and polydispersity index 0.46 ± 0.1 to 0.16 ± 0.04 (p < 0.05), compared to the cationic CD NPs. Size was similar, 161–164 nm, for both formulations. FACS and confocal microscopy, using AML cells (HL-60), indicated a similar level of cellular uptake (60% after 6 h) followed by endosomal escape. The nano co-formulation significantly reduced the charge while maintaining gene silencing (21%). Results indicate that blending of anionic and cationic amphiphilic CDs can produce bespoke NPs with optimised physicochemical properties and potential for enhanced in vivo performance in cancer treatment.     

Molecular Cloning and Expression Analysis of P-Selectin from Zebrafish (Danio rerio)

The glycoprotein P-selectin belongs to the selectin family of cell adhesion molecules. In this study, we cloned the full-length cDNA of P-selectin from zebrafish (Danio rerio) by the method of rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Zebrafish P-selectin cDNA is 2,800 bp and encodes a putative 868 amino acid protein with a theoretical molecular weight of 122.36 kDa and isoelectric point of 6.27. A signal peptide of 25 amino acids is predicted at the N-terminus of the putative protein. All structural domains involved in P-selectin function are conserved in the putative protein. The amino acid sequence of zebrafish P-selectin is 37% to 39% identical to that of mammalian P-selectins. Real-time quantitative PCR and whole-mount in situ hybridization analysis revealed that P-selectin was expressed in early embryonic development, the expression increased from 0.2 hpf (1-cell stage) to 72 hpf, and the expression significantly upregulated within 30 minutes of ADP induction. The results indicate that the structure of P-selectin protein is highly conserved among species and zebrafish P-selectin plays an important role in early embryonic development and probably has similar biological function to mammalian P-selectins.     

Effect of Tocopherol/Sesamol on the Physical Stability and Bioactivity of Oil-in-Water Emulsions of Different Oil Types

Lipid-based delivery systems are widely used to incorporate chemicals with poor solubility. The influence of tocopherol and sesamol on emulsion delivery systems, including their bioactivity, is not well-known. Herein, the overall aim of this study is to understand the effect of tocopherol and sesamol on the physical stability and bioactivity of various emulsions comprised of oil (medium chain triglyceride, coconut oil, palm oil) with different carbon chain lengths using the C. elegans model. All emulsions show good physical stability; droplet size, polydispersity index, zeta potential, and microstructure are relatively constant over the incubation time. Tocopherol and sesamol show the greatest bioactivity (greater lipid-lowering effect and in vivo antioxidant activity) in emulsions fabricated with palm oil. Knowledge on emulsion stability and bioactivity is important for delivery systems in the food industry involving tocopherol, sesamol, and other related bioactive compounds.     

骨修复用生物玻璃研究进展

生物活性玻璃和生物微晶玻璃因其优异的生物活性及组分与性能可设计性而引起广泛关注,人们力图在其基础上研制出性能优良的骨修复材料.近来有报道发现特定组分的玻璃能激活基因从而促进骨组织再生,为生物玻璃的应用开拓了新的领域.本文综述了目前的生物玻璃及生物微晶玻璃体系、组分与制备工艺对其理化性能和生物活性的影响、生物活性的评价方式及其活性机理.